Use of Isatuximab in Combination with Other Agents for the Treatment of Multiple Myeloma

Abstract

The present disclosure provides methods for treating multiple myeloma, comprising administering to an individual in need thereof an anti-CD38 antibody and an interleukin-2 analog, and optionally administering natural killer (NK) cells with reduced or knocked-out expression of CD38.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 317,253, filed March 7, 2022, the disclosure of which is incorporated by reference in its entirety herein.

[0002] Electronic Sequence Listing Reference The contents of the electronic sequence listing (183952034340SEQLIST.xml; size: 11,571 bytes; and creation date March 6, 2023) are incorporated herein by reference in their entirety.

[0003] The present disclosure relates to methods for treating multiple myeloma by administering an anti-CD38 antibody, such as isatuximab. [Background technology]

[0004] Multiple myeloma (MM) is a malignant plasma cell disorder characterized by clonal proliferation of plasma cells in the bone marrow (BM) and production of excessive amounts of monoclonal immunoglobulins (usually of the IgG or IgA type or free urinary light chains, i.e. paraproteins, M proteins or M components). Patients with MM may experience bone pain, fractures, fatigue, anemia, infections, hypercalcemia and kidney problems (Rollig et al. (2015) Lancet. 385(9983):2197-208). CD38 expression is particularly prominent in MM, with >98% of patients being positive for this protein (Goldmacher et al. (1994) Blood. 84(9):3017-25; Lin et al. (2004) Am J Clin Pathol. 121(4):482-8). The strong and uniform expression of CD38 on malignant clonal MM cells contrasts with its restricted expression pattern on normal cells, suggesting that this antigen may be useful for specific targeting of tumor cells.

[0005] Generally, MM patients will receive treatment regimens over their lifetime that include drugs such as proteasome inhibitors (e.g., bortezomib, ixazomib, and carfilzomib), immunomodulatory agents or "IMiDs®" (e.g., lenalidomide, pomalidomide, and thalidomide), monoclonal antibodies (e.g., elotuzumab), histone deacetylase (HDAC) inhibitors (e.g., panobinostat), alone or in combination.

[0006] Despite significant advances and increases in overall survival (OS), multiple myeloma (MM) remains incurable and the majority of patients will relapse and require additional treatment (Kumar SK, Rajkumar V, Kyle RA, et al. Multiple myeloma. Nat Rev Dis Primer. 2017;3:17046).

[0007] All references cited herein, including patent applications, patent publications, and UniProtKB / Swiss-Prot accession numbers, are hereby incorporated by reference in their entirety to the same extent as if each individual reference was specifically and individually indicated to be incorporated by reference. Summary of the Invention [Means for solving the problem]

[0008] Provided herein are anti-CD38 antibodies and interleukin-2 analogs (IL-2 analogs) for use in treating multiple myeloma. In some embodiments, the anti-CD38 antibodies and IL-2 analogs provided herein are combined with NK cells in which CD38 expression has been reduced or knocked out (NK-CD38KO cells).

[0009] Provided herein are methods for treating multiple myeloma, comprising administering to an individual an anti-CD38 antibody and an interleukin-2 analog (IL-2 analog), where the anti-CD38 antibody is administered on days 1, 8, 15, and 22 of a first 28 day cycle, then on days 1 and 15 of the 28 day cycle for at least one additional cycle, and administering to the individual an IL-2 analog, where the IL-2 analog is administered once every two weeks or once every three weeks. In some embodiments, the methods comprise administering NK-CD38KO cells in combination with an anti-CD38 antibody and / or an IL-2 analog.

[0010] Provided herein is a use of an anti-CD38 antibody for the treatment of multiple myeloma, wherein the anti-CD38 antibody is administered to an individual in combination with an interleukin-2 analog (IL-2 analog), the anti-CD38 antibody being administered on days 1, 8, 15, and 22 of a first 28 day cycle, then on days 1 and 15 of the 28 day cycle for at least one additional cycle, and the IL-2 analog being administered once every two weeks or once every three weeks. In some embodiments, the anti-CD38 antibody and the IL-2 analog are administered in combination with NK-CD38KO cells. [Brief description of the drawings]

[0011] [Figure 1] FIG. 1 is a bar graph showing that the fratricide effect is avoided in CD38 KO K-NK cells from BC50 samples compared to wild-type KNK cells in the presence of isatuximab (light grey) or isotype control (dark grey). [Diagram 2] FIG. 2 is a bar graph showing that the fratricide effect is avoided in CD38 KO K-NK cells from BC45 samples compared to wild-type KNK cells in the presence of isatuximab (light grey) or isotype control (dark grey). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] Overview The present disclosure provides anti-CD38 antibodies and interleukin-2 analogs (IL-2 analogs) for use in treating multiple myeloma. In some embodiments, the anti-CD38 antibodies and IL-2 analogs provided herein are combined with NK cells with reduced or knocked-out expression of CD38 (NK-CD38KO cells). Provided herein are methods for treating or delaying the progression of multiple myeloma (MM) in an individual. In some embodiments, the patient has not previously been treated for MM (e.g., newly diagnosed MM). In some embodiments, the individual has received one, two, three or more than three prior lines of treatment for MM. In some embodiments, the individual has received one or more prior lines of treatment including an anti-CD38 agent and an anti-BCMA agent.

[0013] Provided herein are methods for treating multiple myeloma, comprising administering to an individual an anti-CD38 antibody and an interleukin-2 analog (IL-2 analog). Provided herein are uses of anti-CD38 antibodies for treating multiple myeloma, wherein the anti-CD38 antibody is administered to an individual in combination with an IL-2 analog. In some embodiments of the methods and uses provided herein, the anti-CD38 antibody and the IL-2 analog are administered in combination with NK-CD38KO cells.

[0014] The method includes administering to the individual an effective amount of an anti-CD38 antibody (e.g., isatuximab) in combination with natural killer (NK) cells that have been "knocked out" of CD38 expression (e.g., the cells have been genetically modified to remove all or a portion of the CD38 gene such that CD38 is not expressed by the cells) or IL-15 or an analog thereof, or a combination thereof. In some embodiments, dexamethasone is also administered. In some embodiments, the treatment extends the progression-free survival (PFS) and / or overall survival (OS) of the individual. In some embodiments, the treatment extends the progression-free survival (PFS) and / or overall survival (OS) of the individual compared to an individual not receiving the treatment. In some embodiments, the individual is negative for minimal residual disease (MRD) after treatment (e.g., within 10 -4 Less than or equal to 10 -5 Less than or equal to 10 -6 below the threshold) (also called "MRD negative").

[0015] Anti-CD38 antibody In some embodiments, the anti-CD38 antibody binds to human CD38. In some embodiments, the anti-CD38 antibody is a human antibody, a humanized antibody, or a chimeric antibody. In some embodiments, the anti-CD38 antibody comprises: (a) a heavy chain variable domain (VH1) comprising the amino acid sequence DYWMQ (SEQ ID NO: 1), a CDR-H2 comprising the amino acid sequence TIYPGDGDTGYAQKFQG (SEQ ID NO: 2), and a CDR-H3 comprising the amino acid sequence GDYYGSNSLDY (SEQ ID NO: 3). H ) and (b) a light chain variable domain (V) comprising a CDR-L1 comprising the amino acid sequence KASQDVSTVVA (SEQ ID NO: 4), a CDR-L2 comprising the amino acid sequence SASYRYI (SEQ ID NO: 5), and a CDR-L3 comprising the amino acid sequence QQHYSPPYT (SEQ ID NO: 6). L In some embodiments, the anti-CD38 antibody comprises a heavy chain variable domain (V) comprising an amino acid sequence at least 90% identical (e.g., at least one of 91%, 92%, 94%, 95%, 96%, 97%, 98% or 99%, including any ranges between these values) to SEQ ID NO:7.H Additionally or alternatively, in some embodiments, the anti-CD38 antibody comprises a light chain variable domain (V) comprising an amino acid sequence that is at least 90% identical (e.g., at least any one of 91%, 92%, 94%, 95%, 96%, 97%, 98% or 99%, including any ranges between these values) to SEQ ID NO:8 or SEQ ID NO:9. L In some embodiments, the anti-CD38 antibody comprises a VH comprising SEQ ID NO:7 and a VH comprising SEQ ID NO:8 or SEQ ID NO:9. L Includes. QVQLVQSGAE VAKPGTSVKL SCKASGYTFT DYWMQWVKQR PGQGLEWIGT IYPGDGDTGY AQKFQGKATL TADKSSKTVY MHLSSLASED SAVYYCARGD YYGSNSLDYW GQGTSVTVSS (SEQ ID NO: 7) DIVMTQSHLS MSTSLGDPVS ITCKASQDVS TVVAWYQQKP GQSPRRLIYS ASYRYIGVPD RFTGSGAGTD FTFTISSVQA EDLAVYYCQQ HYSPPYTFGG GTKLEIKR (SEQ ID NO: 8) DIVMAQSHLS MSTSLGDPVS ITCKASQDVS TVVAWYQQKP GQSPRRLIYS ASYRYIGVPD RFTGSGAGTD FTFTISSVQA EDLAVYYCQQ HYSPPYTFGG GTKLEIKR (SEQ ID NO: 9)

[0016] In some embodiments, the anti-CD38 antibody is isatuximab (CAS Registry Number: 1461640-62-9). Isatuximab, also known as hu38SB19 and SAR650984, is an anti-CD38 antibody described in WO 2008 / 047242 and U.S. Patent No. 8,153,765, the contents of both of which are incorporated herein by reference in their entireties.

[0017] The heavy chain of isatuximab has the amino acid sequence: [ka] and the light chain of isatuximab comprises the amino acid sequence: [ka] Includes.

[0018] Anti-CD38 antibodies may be produced using recombinant methods. For recombinant production of anti-antigen antibodies, nucleic acid encoding the antibody is isolated and inserted into a replicable vector for further cloning (amplification of the DNA) or expression. DNA encoding the antibody may be easily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to genes encoding the heavy and light chains of the antibody). Many vectors are available. Vector components 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, an enhancer element, a promoter, and a transcription termination sequence. The vector is typically transformed into a host cell suitable for expression of the nucleic acid. In some embodiments, the host cell is a eukaryotic or prokaryotic cell. In some embodiments, the eukaryotic host cell is a mammalian cell. Examples of useful mammalian host cell lines are the SV40 transformed monkey kidney CV1 line (COS-7, ATCC CRL 1651); human embryonic kidney line (293 cells or 293 cells subcloned to grow in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocytes (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals NYAcad. Sci. 383:44-68 (1982)); MRC5 cells; FS4 cells; and a human hepatocyte 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. US A77:4216 (1980)); and myeloma cell lines, such as NSO and S P2 / 0. For a review of certain mammalian host cell lines suitable for antibody production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 255-268. Anti-CD38 antibodies prepared from the cells can be purified using, e.g., hydroxyapatite chromatography, hydrophobic interaction chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography typically being among the preferred purification steps. In general, various methodologies for preparing antibodies for use in research, testing and clinical applications are well established in the art and are consistent with the above-mentioned methodologies and / or deemed suitable by those of skill in the art.

[0019] In some embodiments, the IL-2 analog is PEGylated IL-2. In some embodiments, the IL-2 analog has (e.g., includes or comprises) an unnatural amino acid, such as N6-((2-azidoethoxy)-carbonyl)-L-lysine (AzK). In some embodiments, the unnatural amino acids are PEGylated one by one with a linear PEG group having an average molecular weight of 30 kDa. Suitable IL-2 analogs are described, for example, in WO 2020 / 163532, which is incorporated herein in its entirety.

[0020] In some embodiments, natural killer (NK) cells modified to reduce or knock out expression of CD38 (NK-CD38KO cells) are used in combination with isatuximab and / or IL-2 analogs for the treatment of multiple myeloma. Such NK-CD38KO cells can be produced, for example, by the methods described in WO2021087466, the teachings of which are incorporated in their entirety.

[0021] In some embodiments, the individual has received one or more lines of therapy for the treatment of multiple myeloma before receiving the first cycle of treatment with anti-CD38 antibody, IL-2 analog, and optionally NK-CD38KO cells. Anti-CD38 drugs include, for example, mono- or multi-specific binding agents capable of specifically binding to human CD38. Anti-BCMA drugs include, for example, mono- or multi-specific binding agents capable of specifically binding to human BCMA. The binding agent can be, for example, a monoclonal antibody, bispecific antibody, or trispecific antibody, or an antibody analog containing an antigen-specific region of a conventional antibody capable of specifically binding to CD38 or BCMA, respectively. The binding agent can also be an antibody-drug conjugate.

[0022] Pharmaceutical Compositions and Uses Also provided herein are pharmaceutical compositions and formulations for the treatment of multiple myeloma, including, for example, an anti-CD38 antibody (such as isatuximab), an IL-2 analog, NK-CD38KO cells, and / or dexamethasone. In some embodiments, each of the anti-CD38 antibody, the IL-2 analog, the NK-CD38KO cells, and optional dexamethasone is provided as a separate pharmaceutical composition. In some embodiments, the pharmaceutical compositions and formulations further include a pharma- ceutically acceptable carrier.

[0023] In some embodiments, the anti-CD38 antibody is in a formulation suitable for intravenous administration, e.g., about 20 mg / mL (500 mg / 25 mL) antibody, about 20 mM histidine, about 10% (w / v) sucrose, about 0.02% (w / v) polysorbate 80, pH 6.0. In some embodiments, the anti-CD38 antibody is in a formulation comprising about 20 mg / mL antibody, about 100 mg / mL sucrose, 2.22 mg / mL histidine hydrochloride monohydrate, about 1.46 mg / ml histidine, and about 0.2 mg / ml polysorbate 80. In some embodiments, the formulation comprises water for injection (WFI), such as sterile water for injection (SWFI). In some embodiments, the formulation is sterile. In some embodiments, a single-use formulation comprises 5 ml of the formulation (i.e., 100 mg anti-CD38 antibody). In some embodiments, the single-use 5 ml formulation is provided in, for example, a 16 ml clear, colorless glass vial with an elastomeric stopper. In some embodiments, the vial fill volume is scaled to ensure removal of 5 ml. In some embodiments, the fill volume is 5.4 ml. In some embodiments, the single-use formulation contains 25 ml of the formulation (i.e., 500 mg anti-CD38 antibody). In some embodiments, the single-use 25 ml formulation is provided in, for example, a 30 ml clear, colorless glass vial with an elastomeric stopper. In some embodiments, the vial fill volume is scaled to ensure removal of 25 ml. In some embodiments, the formulation is stable at temperatures between about 2° C. and about 8° C. for at least about 6, 12, 18, 24, 30, or 36 months, including any ranges between these values, protected from light. In some embodiments, the formulation is diluted for injection in 0.9% sodium chloride or 5% dextrose. In some embodiments, the diluted infusion solution is stable between about 2° C. and about 8° C. for up to about 6, 12, 18, 24, 30, 36, 42, or 48 hours, including any ranges between these values. In some embodiments, the diluted infusion solution is stable for an additional 8 hours (including the time of infusion) at room temperature after storage between about 2° C. and about 8° C. In some embodiments, the diluted infusion solution is stable in the presence of light.In some embodiments, the bag in which the dilution solution for infusion is stored is made from polyolefin (PO), polyethylene (PE), polypropylene (PP), polyvinyl chloride (PVC) with di(ethylhexyl)phthalate (DEHP) or ethylvinyl acetate (EVA). In some embodiments, the tubing used for infusion is made from PE, PVC (with or without DEHP), polybutyldiene (PBD) or polyurethane (PU) with an in-line filter (polyethersulfone (PES), polysulfone or nylon).

[0024] In some embodiments, the anti-CD38 antibody is provided in a formulation for subcutaneous administration. In some embodiments, the anti-CD38 antibody comprises isatuximab at a concentration of 140 mg / mL, 9 mM histidine, 110 mM Arg-Cl, 2% (w / v) sucrose, and 0.4% (w / v) poloxamer 188.

[0025] Use of anti-CD38 antibodies in combination with other agents for the treatment of multiple myeloma - Patent Application 20070123333 Provided herein are anti-CD38 antibodies for use in combination with one or more of an IL-2 analog, natural killer (NK) cells modified to reduce or knock out expression of CD38 (NK-CD38KO cells), and dexamethasone for the treatment of multiple myeloma in an individual (e.g., a human individual). In some embodiments, the use comprises administering to the individual an effective amount of an anti-CD38 antibody (e.g., (a) a heavy chain variable domain (V) comprising a CDR-H1 comprising the amino acid sequence DYWMQ (SEQ ID NO: 1), a CDR-H2 comprising the amino acid sequence TIYPGDGDTGYAQKFQG (SEQ ID NO: 2), and a CDR-H3 comprising the amino acid sequence GDYYGSNSLDY (SEQ ID NO: 3). H ) and (b) a light chain variable domain (V) comprising a CDR-L1 comprising the amino acid sequence KASQDVSTVVA (SEQ ID NO: 4), a CDR-L2 comprising the amino acid sequence SASYRYI (SEQ ID NO: 5), and a CDR-L3 comprising the amino acid sequence QQHYSPPYT (SEQ ID NO: 6). L In some embodiments, the anti-CD38 antibody is isatuximab.

[0026] In some embodiments, the methods include administering to the individual an anti-CD38 antibody (e.g., isatuximab) at a dose of 10 mg / kg on days 1, 8, 15, and 22 of a first 28-day cycle, and administering an anti-CD38 antibody at a dose of 10 mg / kg on days 1 and 15 of the 28-day cycle for at least one additional cycle.

[0027] In some embodiments, the methods include at least 11 cycles of administering an anti-CD38 antibody at a dose of 10 mg / kg on days 1 and 15 of a 28-day cycle, followed by administering an anti-CD38 antibody (e.g., isatuximab) at a dose of 10 mg / kg once every 28 days for one or more additional 28-day cycles.

[0028] In some embodiments, the methods include administering an anti-CD38 antibody (e.g., isatuximab) at a dose of 10 mg / kg once every 28 days for one or more additional 28-day cycles after the subject achieves at least a very good partial response (VGPR) while treated with the anti-CD38 antibody.

[0029] In some embodiments, the methods include administering an anti-CD38 antibody (e.g., isatuximab) at a dose of 10 mg / kg once every 28 days for one or more additional 28-day cycles after the subject achieves MRD negativity while being treated with the anti-CD38 antibody.

[0030] In some embodiments, the methods include administering an anti-CD38 antibody (e.g., isatuximab) at a dose of 10 mg / kg once every 28 days for one or more additional 28-day cycles after the subject achieves MRD negativity while being treated with the anti-CD38 antibody.

[0031] In some embodiments described herein, the IL-2 analog is IL-2 modified to include the unnatural amino acid, N6-((2-azidoethoxy)-carbonyl)-L-lysine (AzK), where AzK is pegylated. In some embodiments, the IL-2 analog is administered once every two weeks or once every three weeks, or a combination thereof. In some embodiments, the IL-2 analog is administered at a dose of 24 ug / kg. In some embodiments, the IL-2 analog is administered at a dose of 32 ug / kg. In some embodiments, the IL-2 analog is administered at a dose of 16 ug / kg.

[0032] In some embodiments, the therapeutic regimens described herein extend the progression-free survival (PFS) of an individual.

[0033] In some embodiments, the multiple myeloma is smoldering multiple myeloma (SMM). In some embodiments, the multiple myeloma is newly diagnosed multiple myeloma. In some embodiments, the multiple myeloma is relapsed and / or refractory multiple myeloma (RRMM). In some embodiments, the individual has had one, two or three prior therapies for multiple myeloma. In some embodiments, the individual has had more than three prior therapies for multiple myeloma. In some embodiments, the individual has had prior treatment with a proteasome inhibitor. In some embodiments, the individual has had prior treatment with an immunomodulatory agent. In certain embodiments, the subject has not had prior treatment with an anti-CD38 antibody.

[0034] In certain embodiments, the subject has been previously treated with an anti-CD38 antibody. In some embodiments, the prior anti-CD38 antibody is daratumumab. In some embodiments, the prior anti-CD38 antibody is isatuximab.

[0035] Product or kit In another embodiment of the invention, an article of manufacture or kit is provided that includes an anti-CD38 antibody (e.g., isatuximab). In some embodiments, the article of manufacture or kit further includes at least one additional agent (e.g., one or more IL-2 analogs, NK-CD38KO cells, or dexamethasone). In some embodiments, the article of manufacture or kit further includes a package insert that includes instructions for using the anti-CD38 antibody (e.g., isatuximab) and other agents according to the uses described herein to treat or delay the progression of multiple myeloma (e.g., smoldering multiple myeloma, newly diagnosed multiple myeloma, refractory multiple myeloma, or relapsed and refractory multiple myeloma).

[0036] definition As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a "molecule" optionally includes a combination of two or more such molecules, and so forth.

[0037] "Sustained response" refers to a sustained effect on preventing or delaying disease (e.g., multiple myeloma) progression and / or improving one or more response criteria after cessation of treatment. For example, response to treatment for multiple myeloma may be measured according to the criteria in Kumar et al. (2016) "International Myeloma Working Group consensus criteria for response and minimal residual disease assessment in multiple myeloma." Lancet Oncol. 17(8): e328-e346) and Durie et al. (2006) "International uniform response criteria for multiple myeloma. Leukemia. 20: 1467-1473 (see also Table A below). In some embodiments, the sustained response has a duration at least as long as the treatment duration, at least 1.5x, 2.0x, 2.5x, or 3.0x the length of the treatment duration.

[0038] [Table 1]

[0039] [Table 2]

[0040] Methods for measuring serum and urinary M protein levels are well known in the art and are described, for example, in Jenkins (2009) Clin Biochem Rev. 30(3):119-122; Leung, Nelson "Chapter 8: Clinical Tests for Monoclonal Proteins." Onco-Nephrology Curriculum, American Society of Nephrology 2016, pages 1-5).

[0041] In some embodiments, VGPR is assessed according to the criteria in Kumar et al. (2016) "International Myeloma Working Group consensus criteria for response and minimal residual disease assessment in multiple myeloma." Lancet Oncol. 17(8):e328-e346) and Durie et al. (2006) "International uniform response criteria for multiple myeloma. Leukemia. 20:1467-1473, the contents of which are incorporated herein by reference). (See also Table A).

[0042] The term "pharmaceutical formulation" refers to a preparation that is in a form that allows the biological activity of the active ingredient to be effective and does not contain additional ingredients that are unacceptably toxic to the subject to which the formulation is administered. Such formulations are sterile. "Pharmaceutically acceptable" excipients (vehicles, additives) are those that can be reasonably administered to a mammalian subject to provide an effective dose of the active ingredient employed.

[0043] As used herein, the term "treatment" refers to a clinical intervention designed to alter the natural course of the disease or cells (e.g., cancer cells) being treated while clinical symptoms are present. Desirable effects of treatment include a reduction in the rate of disease progression, reduction or alleviation of the disease state, and remission or improved prognosis. For example, an individual is "treated" successfully if one or more symptoms associated with cancer are alleviated or eliminated, including, but not limited to, a reduction (or destruction) in the proliferation of cancer cells, a reduction in symptoms caused by the disease, an improvement in the quality of life of people suffering from the disease, a reduction in the dose of other drugs required to treat the disease, and / or an increase in the survival time of the individual.

[0044] As used herein, "delaying disease progression" means to postpone, delay, slow, prevent, stabilize and / or postpone the onset of a disease (such as cancer). This delay can vary in length of time depending on the disease history and / or the individual being treated. As will be apparent to one of skill in the art, a sufficient or significant delay can, in effect, encompass prevention, in that the individual does not develop the disease. For example, the onset of late-stage cancer, such as metastasis, may be delayed.

[0045] "Effective amount" refers to the minimum amount required to bring about a measurable improvement or prevention of a particular disorder. Effective amounts herein may vary depending on factors such as the disease state, age, sex and weight of the individual / patient, and the ability of the antibody to elicit a desired response in the individual. An effective amount is also an amount in which any toxic or detrimental effects of the treatment are outweighed by the therapeutically beneficial effects. For prophylactic use, beneficial or desired results include results such as elimination or reduction of risk, lessening the severity or delaying the onset of the disease, including the biochemical, histological and / or behavioral symptoms of the disease, its complications, and intermediate pathological phenotypes that appear during the development of the disease. For therapeutic use, beneficial or desired results include clinical results such as reduction of one or more symptoms caused by the disease, improvement in the quality of life of people suffering from the disease, reduction in the dose of other drugs required to treat the disease, enhancement of the effect of another drug, such as through targeting, delaying the progression of the disease, and / or prolonging survival. In the case of cancer or tumors, an effective amount of a drug may have an effect in reducing the number of cancer cells, reducing tumor size, inhibiting (i.e., slowing to some extent, preferably stopping) cancer cell invasion into peripheral organs, inhibiting (i.e., slowing to some extent, preferably stopping) tumor metastasis, inhibiting to some extent tumor growth, and / or alleviating to some extent one or more symptoms associated with the disorder. An effective amount may be administered in one or more administrations. For purposes of the present invention, an effective amount of a drug, compound, or pharmaceutical composition is an amount sufficient to directly or indirectly effect prophylactic or therapeutic treatment. As will be understood in a clinical context, an effective amount of a drug, compound, or pharmaceutical composition may or may not be achieved in combination with another drug, compound, or pharmaceutical composition. Thus, an "effective amount" may be considered in the context of administering one or more therapeutic agents, and a single agent may be considered to be given in an effective amount if a desired result can be or is achieved in combination with one or more other agents.

[0046] As used herein, "in combination with" refers to the administration of one therapeutic method in addition to another therapeutic method. Thus, "in combination with" refers to the administration of one therapeutic method before, during or after the administration of the other therapeutic method to an individual.

[0047] A "subject" or "individual" for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sport or pet animals, such as dogs, horses, cats, cows, etc. Preferably, the mammal is a human.

[0048] The term "antibody" as used herein is used in the broadest sense and specifically encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired biological activity.

[0049] Human light chains are typically classified as kappa and lambda light chains, and human heavy chains are typically classified as mu, delta, gamma, alpha, or epsilon, defining the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subclasses, including, but not limited to, IgG1, IgG2, IgG3, and IgG4. IgM has subclasses, including, but not limited to, IgM1 and IgM2. IgA is similarly subdivided into subclasses, including, but not limited to, IgA1 and IgA2. Within full-length light and heavy chains, the variable and constant domains are typically joined by a "J" region of about 12 or more amino acids, with heavy chains also including a "D" region of about 10 or more amino acids. See, e.g., Fundamental Immunology (Paul, W., ed., Raven Press, 2nd ed., 1989), which is incorporated by reference in its entirety for any purpose. The variable regions of each light / heavy chain pair typically form the antigen-binding site. The variable domains of antibodies typically exhibit the same general structure, with relatively conserved framework regions (FR) connected by three hypervariable regions, also called complementarity determining regions or CDRs. The CDRs from the two chains of each pair are typically arranged with framework regions, which may enable binding to a specific epitope. From the amino terminus to the carboxyl terminus, both the light chain variable domain and the heavy chain variable domain typically comprise, in order, the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.

[0050] The term "CDR set" refers to a group of three CDRs present in a single variable region capable of binding to an antigen. The exact boundaries of these CDRs have been defined in various ways according to various schemes. The scheme described by Kabat (Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991)) not only provides a clear residue numbering scheme that can be applied to any variable region of an antibody, but also provides precise residue boundaries that define the three CDRs. These CDRs may be referred to as Kabat CDRs.

[0051] The term "Fc" as used herein refers to the sequence of a non-antigen binding fragment resulting from the digestion of an antibody or produced by other means, whether in monomeric or multimeric form, and may contain a hinge region. The original immunoglobulin source of native Fc is preferably of human origin and may be any of the immunoglobulins. Fc molecules are composed of monomeric polypeptides that may be linked by covalent (i.e., disulfide bonds) and non-covalent bonds into dimeric or multimeric forms. The number of intermolecular disulfide bonds between the monomeric subunits of native Fc molecules ranges from one to four depending on the class (e.g., IgG, IgA, and IgE) or subclass (e.g., IgG1, IgG2, IgG3, IgA1, IgGA2, and IgG4). One example of an Fc is a disulfide-bonded dimer resulting from papain digestion of IgG. The term "native Fc" as used herein can refer to any of the monomeric, dimeric, and multimeric forms.

[0052] As used herein, the term "overall response rate" or "ORR" refers to the proportion of individuals / patients with stringent complete response (sCR), complete response (CR), very good partial response (VGPR), and partial response (PR) as assessed by an IRC using the IMWG response criteria described in Kumar et al. (2016) "International Myeloma Working Group consensus criteria for response and minimal residual disease assessment in multiple myeloma." Lancet Oncol. 17(8):e328-e346 and Durie et al.. (2006) "International uniform response criteria for multiple myeloma. Leukemia. 20:1467-1473. See also Table A herein.

[0053] The specification is believed to be sufficient to enable one skilled in the art to practice the embodiments. Various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the above description and fall within the scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes. EXAMPLES

[0054] The present disclosure will be better understood by referring to the following examples. However, the following examples should not be interpreted as limiting the scope of the present invention. It is understood that the examples and embodiments described herein are for illustrative purposes only, and in light thereof, various modifications or changes will be suggested to those skilled in the art and are within the spirit and scope of the present application and the scope of the appended claims.

[0055] Example 1 Measurement of in vitro cytotoxicity Antibody-dependent cellular cytotoxicity (ADCC) Antibody-dependent cellular cytotoxicity (ADCC) against LP-1RFP multiple myeloma cells (target cells) using WT or CD38KO K-NK cells (effector cells) in combination with isatuximab and SAR444245 (a pegylated IL-2 analog) was measured longitudinally by the Incucyte® live-cell imaging and analysis system (Essen Bioscience).

[0056] LP-1 RFP cells were generated by infecting LP-1 cells (DSMZ) with Incucyte® Nuclight Red Lentivirus (Sartorius) to express red fluorescent protein (RFP). LP-1 RFP cells were maintained in IMDM medium (Gibco, #12440053) supplemented with 20% fetal bovine serum (FBS heat inactivated, Biowest, #S181H-100), 1% L-glutamine (Gibco, #25030-024) and incubated at 37°C, 5% CO2. LP-1RFP cells were centrifuged at 300g for 5 min and resuspended in RPMI1640 complete medium (RPMI1640 supplemented with 10% fetal bovine serum - FBS, Biowest, #S181H-100-, 1% L-glutamine - Gibco, #25030-024) before being added to Incucyte® plates (details below).

[0057] Natural killer (NK) cells with reduced or knocked out CD38 expression (CD38KO K-NK cells) were generated using peripheral blood NK cells (BC45 and BC50) isolated from two healthy donors and expanded using PM21 particle technology to generate highly activated K-NK cells. BC45 cells have a V / V phenotype for CD16, and BC50 cells have a F / V phenotype.

[0058] To generate CD38KO K-NK cells, they were subjected to CRISPR gene editing during NK cell expansion by electroporating Cas9 / RNP complexes targeting CD38. After expansion, WT and CD38KO K-NK cells were frozen and kept at -150°C. WT and CD38KO K-NK cells were thawed and seeded in RPMI1640 complete medium supplemented with 50U / mL IL-2 (Peprotech, #200-02) and incubated at 37°C 5% CO2 for 16-20 hours. Cells were then added to Incucyte® plates after centrifugation at 300g for 5 minutes.

[0059] Compounds and cells were added to appropriate wells of Incucyte® plates (96-well flat-bottom microplates CellCoat™ treated with poly-D-lysine, Greiner Bio-One; #655946) in the following order (final volume in each well: 200 μl):

[0060] Isatuximab (or isotype control) (pre-diluted in RPMI1640 complete medium) was added to appropriate wells to have a final concentration of either 0.1, 1 or 10 ng / ml (50 μl / well).

[0061] SAR444245 (pre-diluted in RPMI1640 complete medium) was then added to the appropriate wells (50 μL / well) to have a final concentration of 333.33 ng / ml.

[0062] LP-1RFP cells (target cells, T) were then added to each well (to have 20000 LP1-RFP cells / well) (50 μL / well).

[0063] WT or CD38KO K-NK cells (effector cells, E) were then added to the appropriate wells. To assess the various E:T ratios (1:1, 3:1 and 5:1 respectively), either 20,000, 60,000 or 100,000 cells were added (50 μL / well).

[0064] The Incucyte® plate was then centrifuged at 100g for 1 min before being placed in an Incucyte® (IncucyteS3, EssenBio) placed in a dedicated incubator at 37°C and 5% CO2. Images (4 images / well) were taken every 2 hours using a 10x objective and standard scan type, using phase and red channels. The growth of LP-1RFP target cells was monitored by fluorescence imaging for up to 90 hours, and the number of live target cells was quantified using IncucyteS3 software and normalized to the number of live target cells at zero time.

[0065] Experiments were performed at least twice with WT and CD38KO K-NK cells from two donors (BC45 and BC50) in duplicate for each condition.

[0066] The fratricide effect WT and CD38KO K-NK cells were thawed and seeded in RPMI1640 complete medium (RPMI1640 supplemented with 10% fetal bovine serum - FBS, Biowest, #S181H-100-, 1% L-glutamine - Gibco, #25030-024) supplemented with 50 U / mL IL-2 (Peprotech, #200-02) and incubated for 16-20 hours at 37 °C 5% CO2. Cells were then centrifuged at 300g for 5 min, counted, and resuspended in RPMI1640 complete medium to have 50000 K-NK cells / 50 μL.

[0067] Compounds and cells were added to appropriate wells of a Corning™ 96-Well Clear Ultra Low Attachment Microplate (Corning, #7007) in the following order (final volume in each well: 200 μl);

[0068] RPMI1640 complete medium was added to each well (100 μL / well).

[0069] Isatuximab (or isotype control) (pre-diluted in RPMI1640 complete medium) was added to appropriate wells (50 μL / well) to have a final concentration of 10 ng / ml.

[0070] WT or CD38KO K-NK cells were then added to the appropriate wells (to have 50,000 K-NK cells / well) (50 μL / well).

[0071] The plates were then centrifuged at 100 g for 1 minute and then placed in a 37° C., 5% CO 2 incubator for 4 hours.

[0072] To quantify fratricide, WT and CD38KO K-NK cells were stained with either Annexin V-FITC kit or DiOC6 / DRAQ7.

[0073] For labeling with Annexin V-FITC kit (Miltenyi Biotec; #130-092-052):

[0074] After 4 hours of incubation, the plates were centrifuged at 300 g for 5 minutes, the medium was removed, and the pellet was resuspended in 200 μL of Annexin V-FITC kit buffer solution. The plates were centrifuged again at 300 g for 5 minutes.

[0075] The buffer was removed and the pellet was then resuspended in 100 μL of Annexin V-FITC kit buffer solution and 5 μL of Annexin V-FITC was added to the appropriate wells.

[0076] The plate was incubated at room temperature in the dark for 15 minutes.

[0077] 100 μL of Annexin V-FITC kit buffer solution was added, the plate was centrifuged at 300 g for 5 min, and the buffer was removed.

[0078] The pellet was resuspended in 200 μL of Annexin V-FITC kit buffer solution and the plate was centrifuged again at 300 g for 5 min.

[0079] The buffer was removed and the pellet was resuspended in 100 μL of Annexin V-FITC kit buffer solution.

[0080] 1 μL of propidium iodide (PI) was added to the appropriate wells immediately prior to analysis.

[0081] Samples were then analyzed on a MACSQuant 16 flow cytometer (Miltenyi Biotec).

[0082] Data were analyzed with VenturiOne software using the following procedure: The K-NK cell population, excluding debris, was gated, followed by gating on single cells, from which viable PIneg and Annexin V-FITCneg cells were quantified.

[0083] For graphing, viable K-NK cells (Annexin Vneg / PIneg) were quantified as the percentage of viable cells in the presence of isatuximab normalized to the percentage of viable cells in the presence of isotype control (100%) (see Figure 1).

[0084] To label with DiOC6 / DRAQ7:

[0085] After 4 hours of incubation, 20 μL of 100 nM DiOC6 (3,3′-dihexyloxacarbocyanine iodide, ThermoFisher, #D273) was added to appropriate wells.

[0086] The plate was incubated for 20 minutes at 37° C., 5% CO2.

[0087] The plates were then centrifuged at 300 g for 5 minutes, the medium removed and the pellet resuspended in 100 μL of 3 μM DRAQ7 (BD Biosciences, #564904).

[0088] Samples were then analyzed on a MACSQuant 16 flow cytometer (Miltenyi Biotec).

[0089] Data were analyzed with VenturiOne software using the following procedure: The K-NK cell population, excluding debris, was gated, and then single cells were gated. From the single cells, viable DiOC6pos and DRAQ7neg cells were quantified.

[0090] For graphing, surviving K-NK cells (DiOC6pos / DRAQ7neg) were quantified as the percentage of viable cells in the presence of isatuximab normalized to the percentage of viable cells in the presence of isotype control (100%) (Figure 2).

[0091] Experiments were performed with WT and CD38KO K-NK cells (BC45 and BC50) from two donors. As shown in Figure 1, the fratricide effect is avoided in CD38 KO K-NK cells (BC50 with F / V CD16 phenotype) in the presence of isatuximab. As shown in Figure 2, the fratricide effect is avoided in CD38 KO K-NK cells (BC45 with V / V CD16 phenotype) in the presence of isatuximab. Furthermore, the presence of SAR444245 enhanced the cytotoxic activity of isatuximab against LP-1 cells in the presence of wild-type NK cells, and the cytotoxic activity of isatuximab was further enhanced in the presence of CD38 KO K-NK cells. The cytotoxic activity of CD38KO K-NK cells was sustained for a long time, up to 90 hours.

[0092] Each embodiment described herein may be combined with any other embodiment unless expressly indicated to the contrary. In particular, any feature or embodiment indicated as being preferred or advantageous may be combined with any other feature or embodiment indicated as being preferred or advantageous unless expressly indicated to the contrary.

[0093] All references cited in this application are expressly incorporated herein by reference.

Claims

1. A pharmaceutical composition comprising an anti-CD38 antibody for the treatment of multiple myeloma, The anti-CD38 antibody is administered to an individual in combination with an interleukin-2 analog (IL-2 analog), the anti-CD38 antibody is administered on days 1, 8, 15 and 22 of the first 28-day cycle, and then on days 1 and 15 of the 28-day cycle for at least one additional cycle. A pharmaceutical composition comprising administering the IL-2 analog to the individual, wherein the IL-2 analog is administered once every two weeks or once every three weeks.

2. The pharmaceutical composition according to claim 1, wherein the anti-CD38 antibody is isatuximab, and is administered intravenously at a dose of 10 mg / kg.

3. The pharmaceutical composition according to claim 1, wherein the CD-38 antibody is isatuximab, and is administered subcutaneously at a dose of 1400 mg.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the IL-2 analog comprises a non-natural amino acid, N6-((2-azidoethoxy)-carbonyl)-L-lysine (AzK), and the AzK is pegylated.

5. The pharmaceutical composition according to claim 1, wherein the IL-2 analog is administered in doses of 16 ug / kg, 24 ug / kg, or 32 ug / kg.

6. The pharmaceutical composition according to any one of claims 1 to 3 or 5, wherein the anti-CD38 antibody is administered on days 1 and 15 of a 28-day cycle for at least 11 cycles, and then administered on day 1 of a 28-day cycle for one or more additional cycles.

7. The pharmaceutical composition according to any one of claims 1 to 3 or 5, wherein the individual has received one or more lines of treatment for the treatment of multiple myeloma prior to receiving a first cycle of treatment, and the one or more prior lines of treatment comprise an anti-CD38 agent and an anti-BCMA agent.

8. A pharmaceutical composition comprising an anti-CD38 antibody for the treatment of multiple myeloma, The aforementioned anti-CD38 antibody is administered to an individual in combination with an interleukin-2 analog (IL-2 analog). The anti-CD38 antibody is isatuximab, and the IL-2 analog comprises the non-natural amino acid N6-((2-azidoethoxy)-carbonyl)-L-lysine (AzK), wherein the AzK is pegylated. The isatuximab is administered on days 1, 8, 15 and 22 of the first 28-day cycle, and then on days 1 and 15 of the 28-day cycle for at least one additional cycle. The isatuximab is administered intravenously at a dose of 10 mg / kg or subcutaneously at a dose of 1400 mg. The IL-2 analog is administered to the individual at a dose of 16 ug / kg, 24 ug / kg, or 32 ug / kg once every two weeks or once every three weeks. The individual has received one or more lines of treatment for multiple myeloma prior to receiving the first cycle of treatment, wherein the one or more prior lines of treatment are pharmaceutical compositions comprising an anti-CD38 drug and an anti-BCMA drug.

9. The pharmaceutical composition according to any one of claims 1 to 3, 5, or 8, wherein natural killer (NK) cells in which CD38 expression is reduced or knocked out (NK-CD38KO cells) are also administered to the individual.

10. The pharmaceutical composition according to claim 9, wherein the NK cells are isolated from a human sample.

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