A pharmaceutical combination for treating tumors, including anti-CD19 antibodies and natural killer cells.

Abstract

To provide a pharmaceutical combination that is capable of exhibiting synergistic therapeutic effects on malignant tumors of B-cell origin such as non-Hodgkin's lymphoma, chronic lymphocytic leukemia, and / or acute lymphoblastic leukemia.SOLUTION: The present invention provides a combination comprising an antibody specific for CD19 and a natural killer (NK) cell. It is preferable that the antibody comprises a heavy chain variable region comprising an HCDR1 region of SYVMH (SEQ ID NO: 1), an HCDR2 region of NPYNDG (SEQ ID NO: 2), and an HCDR3 region of GTYYYGTRVFDY (SEQ ID NO: 3).SELECTED DRAWING: Figure 5

Description

[Technical Field]

[0001] Embodiments of the present invention relate to a pharmaceutical combination comprising an anti-CD19 antibody and natural killer cells, further having a synergistic therapeutic effect against B-cell-derived malignancies such as non-Hodgkin lymphoma, chronic lymphocytic leukemia (CLL), and / or acute lymphoblastic leukemia (ALL). [Background technology]

[0002] Natural killer cells (NK cells) are lymphoid cells that recognize targets in a way that is not limited by MHC, unlike T cells, and play an important role in the innate immune response. Natural killer cells can exert antiviral and anticancer effects. Specifically, natural killer cells play a role in directly killing malignant tumors or eliminating abnormal cells that have developed or are developing tumors by activating dendritic cells or inducing tumor-specific cytotoxic T lymphocytes (CTLs). Furthermore, regarding the anticancer effects of natural killer cells, the use of allogeneic natural killer cells with a mismatch in killer cell immunoglobulin-like receptor (KIR)-ligand pair in the treatment of cancer patients has been proven to be far more effective and safer than the use of autologous natural killer cells.

[0003] On the other hand, the human CD19 molecule is a cell surface receptor expressed on the surface of human B cells, such as pre-B cells, immature B cells in the early stages of development, mature B cells, and malignant B cells. In fact, most B-cell lineage malignancies, such as non-Hodgkin lymphoma, chronic lymphocytic leukemia (CLL), and acute lymphoblastic leukemia (ALL), express CD19. Therefore, anti-CD19 antibodies can bind to the CD19 antigen as a target, meaning they can be used in the therapeutic immunotherapy of the aforementioned B-cell-derived malignancies.

[0004] Despite the discovery and development of several anticancer drugs in recent years, improved methods or therapeutic agents for treating many types of cancer, including CD19-expressing tumors, remain necessary due to their poor prognosis. Therefore, the inventors have confirmed that the combined administration of natural killer cells and CD19-specific antibodies has a synergistic effect on the treatment of B-cell-derived malignant lymphoma, and have completed the present invention. [Prior art documents] [Non-patent literature]

[0005] Nadler et al., J. Immunol., 131:244-250 (1983) [Overview of the project] [Problems that the invention aims to solve]

[0006] Disclosure of the invention The object of the present invention is to provide a combination therapy using a CD19-specific antibody and natural killer cells, or a combination thereof. [Means for solving the problem]

[0007] Embodiments of the present invention that achieve the above-mentioned objectives can provide a pharmaceutical combination for treating cancer, comprising a CD19-specific antibody and natural killer cells (NK cells).

[0008] The antibody may have a heavy chain variable region including the HCDR1 region of SYVMH (SEQ ID NO: 1), the HCDR2 region of NPYNDG (SEQ ID NO: 2), and the HCDR3 region of GTYYYGTRVFDY (SEQ ID NO: 3).

[0009] The antibody may have a light chain variable region including the LCDR1 region of RSSKSLQNVNGNTYLY (SEQ ID NO: 4), the LCDR2 region of RMSNLNS (SEQ ID NO: 5), and the LCDR3 region of MQHLEYPIT (SEQ ID NO: 6).

[0010] The antibody may include a heavy chain variable region containing the HCDR1 region of SYVMH (SEQ ID NO: 1), the HCDR2 region of NPYNDG (SEQ ID NO: 2), and the HCDR3 region of GTYYYGTRVFDY (SEQ ID NO: 3), and a light chain variable region containing the LCDR1 region of RSSKSLQNVNGNTYLY (SEQ ID NO: 4), the LCDR2 region of RMSNLNS (SEQ ID NO: 5), and the LCDR3 region of MQHLEYPIT (SEQ ID NO: 6).

[0011] The antibody may contain the heavy chain variable region of EVQLVESGGGLVKPGGSLKLSCAASGYTFTSYVMHWVRQAPGKGLEWIGYINPYNDGTKYNEKFQGRVTISSDKSISTAYMELSSLRSEDTAMYYCARGTYYYGTRVFDYWGQGTLVTVSS (SEQ ID NO: 8) and the light chain variable region of DIVMTQSPATLSLSPGERATLSCRSSKSLQNVNGNTYLYWFQQKPGQSPQLLIYRMSNLNSGVPDRFSGSGSGTEFTLTISSLEPEDFAVYYCMQHLEYPITFGAGTKLEIK (SEQ ID NO: 9).

[0012] The antibody may contain the heavy chain constant region of ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKALPAPEEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 10).

[0013] The antibody may include the light chain constant region of RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 11).

[0014] The NK cells can be prepared by a method including step (i) of statically culturing the NK cells in a culture solution containing an anti-CD3 antibody, cytokines and feeder cells to stimulate cell-cell contact, step (ii) of adding cytokines, an anti-CD3 antibody and feeder cells thereto for restimulation and inducing intracellular contact by performing static culture again, and step (iii) of adding a medium containing cytokines to the cells and performing static culture or suspension culture while maintaining the cell concentration and cytokine concentration constant.

[0015] The NK cells can be prepared by a method including the step of co-culturing CD4(+) T cells isolated ex vivo as feeder cells or CD4(+) T cells expanded ex vivo with seed cells.

[0016] The CD4(+) T cells may be a CD4(+) / CD1(-) T cell line.

[0017] The CD4(+) / CD1(-) T cell line may be the H9 cell line or the HuT78 cell line.

[0018] The seed cells may be one or more selected from the group consisting of peripheral blood-derived cells, peripheral blood leukocytes, peripheral blood mononuclear cells (PBMC), cord blood leukocytes, cord blood mononuclear cells, stem cell-derived natural killer cells, enriched natural killer cells and isolated natural killer cells.

[0019] In addition, the pharmaceutical combination may further contain albumin.

[0020] Cancer may be at least one of the following: gastric cancer, liver cancer, lung cancer, colorectal cancer, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, cervical cancer, thyroid cancer, laryngeal cancer, acute lymphoblastic leukemia, brain tumor, neuroblastoma, retinoblastoma, head and neck cancer, salivary gland cancer, and lymphoma.

[0021] Lymphoma can be a malignant tumor originating from B cells.

[0022] B-cell-derived malignancies may be at least one selected from the group consisting of non-Hodgkin lymphoma, chronic lymphocytic leukemia, and acute lymphoblastic leukemia.

[0023] CD19-specific antibodies and NK cells may be administered separately.

[0024] CD19-specific antibodies and NK cells may be administered simultaneously.

[0025] Another embodiment of the present invention may provide a kit for treating cancer, including the combination described above.

[0026] Yet another embodiment of the present invention may provide the use of the above-mentioned pharmaceutically acceptable combinations for preparing drugs to treat cancer.

[0027] Furthermore, yet another embodiment of the present invention may provide a method for treating cancer, comprising the step of administering to a subject a combination of the above-mentioned antibody specific to CD19 and the above-mentioned NK cells. [Effects of the Invention]

[0028] The pharmaceutical combinations comprising CD19-specific antibodies and natural killer cells according to embodiments of the present invention are useful for the treatment of cancer, particularly B-cell malignancies such as non-Hodgkin lymphoma, chronic lymphocytic leukemia, and / or acute lymphoblastic leukemia. In particular, the natural killer cells are manufactured using a method that is clinically easier to handle compared to conventional methods, possess high cytotoxicity, cell viability, and long-term storage capabilities, and further exhibit synergistic effects in tumor treatment when administered in combination with specific CD19-specific antibodies. [Brief explanation of the drawing]

[0029] [Figure 1a] The results obtained by culturing Raji, a Burkitt B-cell lymphoma, with various concentrations of monoclonal antibodies in the presence of NK cells are shown. [Figure 1b] We present another result obtained by culturing Raji, a Burkitt B-cell lymphoma, with various concentrations of monoclonal antibodies in the presence of NK cells. [Figure 1c] The results obtained by culturing Ramos, a Burkitt B-cell lymphoma, with various concentrations of monoclonal antibodies in the presence of NK cells are shown. [Figure 1d] We present another result obtained by culturing Ramos, a Burkitt B-cell lymphoma, with various concentrations of monoclonal antibodies in the presence of NK cells. [Figure 2] The results obtained by evaluating the compatibility of Ramos cell lines in SCID mouse models are shown. [Figure 3] The results obtained by administering various concentrations of MOR208 to a SCID mouse model group that received Ramos cells intravenously are shown. [Figure 4] The results obtained by administering various concentrations of MG4101 to a group of SCID mouse models that received intravenous administration of Ramos cells are shown. [Figure 5] The results obtained by administering control, MOR208, MG4101, and "MOR208 / MG4101" to SCID mouse models that were intravenously injected with Ramos cells are shown. [Modes for carrying out the invention]

[0030] While natural killer cells have the potential to be used as therapeutic agents for various diseases, their availability has been limited due to several constraints, including the somewhat restricted number of natural killer cells in peripheral blood, the difficulty of large-scale production of cytolytic natural killer cells in accordance with Good Manufacturing Practices (GMP), and the need for natural killer cells to be activated in order to induce cell-mediated killing.

[0031] In recent years, the inventors have established a simple and efficient method for large-scale proliferation and activation of natural killer cells derived from healthy donors under GMP conditions (KR10-1644984B1). Furthermore, natural killer cells that were proliferated ex vivo and highly activated using a large-scale culture method of NK cells from umbilical cord blood mononuclear cells were produced under GMP conditions and showed strong anticancer activity in vitro and in vivo in preclinical studies.

[0032] On the other hand, therapies using monoclonal antibodies specific to epitope molecules expressed in cancer cells are known as the most successful cancer immunotherapy to date. One of its mechanisms of action is antibody-dependent cell-mediated cytotoxicity (ADCC) mediated by natural killer cells. Since the natural killer cells proliferated by the embodiment of the present invention expressed high levels of CD16, an ADCC-mediated Fc receptor, the inventors attempted to confirm that its anticancer activity against B-cell lymphoma could be enhanced in the presence of an anti-CD19 antibody.

[0033] Surprisingly, the inventors found that when a specific antibody targeting CD19 is administered in combination with natural killer cells, it exhibits a synergistic effect in vitro and in vivo on the direct death of human B-cell malignancies mediated by ADCC, compared to when the antibody or natural killer cells are administered alone.

[0034] One aspect of the present invention provides a pharmaceutical combination for treating cancer, comprising a CD19-specific antibody and natural killer cells.

[0035] As used herein, the term "CD19" refers to the following synonyms, B4, B lymphocyte antigen CD19, B lymphocyte surface antigen B4, CVID3, differentiation antigen CD19, MGC12802, and the protein known as CD19 having the T cell surface antigen Leu-12. CD19 may be human-derived CD19, which may contain the amino acid sequence of SEQ ID NO: 7. CD19 is expressed in most B-cell malignancies and in cells and tissues with various diseases and lesions on normal B cells.

[0036] Furthermore, the term "antibody" as used herein refers to monoclonal antibodies containing any isotype, such as IgG, IgM, IgA, IgD, and IgE. An IgG antibody consists of two identical heavy chains and two identical light chains linked by disulfide bonds. The heavy and light chains each contain a constant region and a variable region. The variable region contains three segments called "complementarity-determining regions (CDRs)" or "hypervariable regions," which are primarily involved in binding to the antigen epitope. These are numbered sequentially from the N-terminus and referred to as CDR1, CDR2, and CDR3. The more highly conserved portion of the variable region outside the CDRs is called the "framework region."

[0037] In other words, an anti-CD19 antibody is an antibody that specifically binds to the CD19 antigen, and may include not only the complete antibody form but also its antigen-binding fragment.

[0038] Examples of CD19-specific antibodies according to embodiments of the present invention may include those described in US12 / 377,251 (Xencor), WO2005 / 012493, WO2010 / 053716 (lmmunomedics), WO2007 / 002223 (Medarex), WO2008 / 022152 (Xencor), WO2008 / 031056 (Medimmune), WO2007 / 076950 (Merck Patent GmbH), WO2009 / 052431 (Seattle Genetics), and WO2010 / 095031 (Glenmark Pharmaceuticals).

[0039] Furthermore, the CD19-specific antibody according to the embodiment of the present invention may include a heavy chain variable region comprising the HCDR1 region of SYVMH (SEQ ID NO: 1), the HCDR2 region of NPYNDG (SEQ ID NO: 2), and the HCDR3 region of GTYYYGTRVFDY (SEQ ID NO: 3). Furthermore, the CD19-specific antibody of the present invention may include a light chain variable region comprising the LCDR1 region of RSSKSLQNVNGNTYLY (SEQ ID NO: 4), the LCDR2 region of RMSNLNS (SEQ ID NO: 5), and the LCDR3 region of MQHLEYPIT (SEQ ID NO: 6).

[0040] A CD19-specific antibody according to another embodiment of the present invention may include an antibody that cross-competes with the antibody of the present invention as described above.

[0041] As used herein, the term "cross-competition" means the ability of an antibody or other binder to interfere with the binding of another antibody or binder to CD19 in a standard competitive binding assay. The ability or extent to which an antibody or other binder can interfere with the binding of another antibody or binder to CD19, i.e., whether it can be called cross-competition according to the present invention, can be determined using a standard competitive binding assay.

[0042] Furthermore, a CD19-specific antibody according to another embodiment of the present invention may include an antibody that binds to the same epitope as the antibody of the present invention described above.

[0043] Furthermore, the antibody may include the heavy chain variable region of EVQLVESGGGLVKPGGSLKLSCAASGYTFTSYVMHWVRQAPGKGLEWIGYINPYNDGTKYNEKFQGRVTISSDKSISTAYMELSSLRSEDTAMYYCARGTYYYGTRVFDYWGQGTLVTVSS (SEQ ID NO: 8) and the light chain variable region of DIVMTQSPATLSLSPGERATLSCRSSKSLQNVNGNTYLYWFQQKPGQSPQLLIYRMSNLNSGVPDRFSGSGSGTEFTLTISSLEPEDFAVYYCMQHLEYPITFGAGTKLEIK (SEQ ID NO: 9). In addition, the antibody may contain the heavy chain constant region of ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKALPAPEEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 10). In addition, the antibody may contain the light chain constant region of RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 11). The above-mentioned anti-CD19 antibody may be "MOR208". The MOR208 antibody may be the one disclosed in U.S. Patent Application No. 12 / 377,251.

[0044] As used herein, the term "natural killer cell (hereinafter referred to as NK cell)" refers to cytotoxic lymphocytes that play an important role in the body's immune system. For the purposes of the present invention, NK cells may be endogenous NK cells of the subject and / or NK cells from outside the body, specifically isolated NK cells administered from outside the subject, but not limited thereto. In addition, NK cells can be obtained by purchasing commercially available products or by conventional methods such as isolation from the subject or culture.

[0045] On the other hand, NK cells included in the pharmaceutical combination according to the embodiments of the present invention may be prepared by stimulating mononuclear cells from which T cells have been removed in a culture medium containing cytokines, together with anti-CD3 antibodies and feeder cells, stimulating intracellular contact by performing intracellular contact for several days, and further performing static or suspension culture of NK cells while maintaining a constant concentration of cells and cytokines.

[0046] To increase the volume of NK cells, stimulation and static culture can be repeated before subsequent static or suspension culture. NK cells prepared by the method of the present invention can be cultured ex vivo at high efficiency and concentration while exhibiting high cell viability and cytotoxicity, and can therefore be administered in combination with an anti-CD19 antibody to treat B cell-derived malignancies.

[0047] For example, NK cells may be prepared by a method comprising the following steps (i) to (iii).

[0048] In step (i), isolated NK cells are cultured statically in a culture medium containing anti-CD3 antibody, cytokines, and feeder cells to stimulate cell-cell contact.

[0049] In step (ii), cytokines, anti-CD3 antibodies, and feeder cells are added to restimulate the cells, and static culture is performed again to induce intracellular contact.

[0050] In step (iii), a culture medium containing cytokines is added to these cells, and static or suspension culture is performed while maintaining a constant concentration of cells and cytokines.

[0051] The isolated NK cells may also be obtained by isolating leukocytes and NK cells from human peripheral blood or umbilical cord blood.

[0052] Furthermore, static culture after initial stimulation may be carried out for approximately 2 to 15 days, specifically 5 to 10 days, and static culture after restimulation may be carried out for approximately 2 to 7 days, specifically 3 to 5 days. In addition, after the completion of static culture, static culture or suspension culture may be carried out in an incubator while maintaining a constant cytokine concentration. Moreover, when preparing NK cells, adding albumin to the composition containing NK cells can significantly increase the cytotoxic effect and cell viability of the NK cells. Specifically, NK cells prepared by the above method can be referred to as "MG4101". The method for preparing NK cells and the NK cells according to embodiments of the present invention may be those described in Korean Patent No. 10-1644984.

[0053] As used herein, the term "feeder cell" (also called a culture support cell) refers to a cell that does not have the ability to proliferate by division but possesses metabolic activity and thereby produces various metabolites that help the proliferation of target NK cells. Possible feeder cells include genetically modified animal cell lines, peripheral blood leukocytes (PBLs) treated with various cytokines or compounds, autologous or non-autologous peripheral blood leukocytes (PBLs), T cells, B cells, or monocytes. Specifically, autologous peripheral blood mononuclear cells are used, but are not limited to these.

[0054] Furthermore, autologous peripheral blood mononuclear cells used as feeder cells can be inactivated to ensure safety. Conventional methods well-known in the art can be used for inactivation, but gamma irradiation, for example, can also be used. These inactivated feeder cells include isolated T cells. As described above, the proliferation method using feeder cells involves isolating them purely and then proliferating NK cells, which has the advantage that only pure NK cells subsequently proliferate continuously.

[0055] As used herein, the term "anti-CD3 antibody" refers to an antibody that specifically binds to the CD3 antigen, a group of molecules that bind to the T cell receptor (TCR) to form an antigen recognition complex. The CD3 molecule binds to the TCR and transmits an antigen recognition signal to the cell. The anti-CD3 antibody that can be used in this invention is not limited as long as it is an antibody that has the property of binding to CD3. For example, the anti-CD3 antibody may be selected from the group consisting of OKT3, UCHT1, and HIT3a, but is not limited to these.

[0056] In the present invention, the cytokines that may be included in the culture medium may be one or more selected from interleukins. For example, one or more selected from the group consisting of interleukin-2 (IL-2), interleukin-12 (IL-12), interleukin-15 (IL-15), interleukin-18 (IL-18), and interleukin-21 (IL-21) may be used, but are not limited to these.

[0057] Furthermore, the concentration of anti-CD3 antibody in the culture medium, which can be used for static and suspension cultures, may be 0.1–1,000 ng / ml, 1–100 ng / ml, or 5–20 ng / ml, and the concentration of cytokines in the culture medium may be 10–2,000 IU, 100–1,000 IU, or approximately 200–700 IU.

[0058] As used herein, the term "stimulation" refers to inducing the proliferation of NK cells by adding feeder cells or the like, and it is possible to use anti-CD3 antibodies in conjunction with this. Furthermore, as used herein, the term "restimulation" refers to re-inducing the proliferation of NK cells by adding feeder cells and / or anti-CD3 antibodies to the culture medium again after a certain culture period has elapsed.

[0059] Common animal cell culture media such as CellGro medium (Cellgenix), AIM-V medium, RPMI1640 medium, and X-VIVO20 can be used as culture media for preparing NK cells according to embodiments of the present invention.

[0060] In particular, in NK cell preparation methods, the concentrations of cytokines and cells in the culture medium can be measured at regular time intervals to maintain a constant concentration of cells and cytokines during suspension culture. Based on these measurements, the cytokine-containing culture medium can be provided to adapt to the concentrations of cells and cytokines.

[0061] Furthermore, culturing can be carried out by adding serum or plasma and additional growth factors that support lymphocyte proliferation to the culture medium. The type of serum or plasma added to the medium is not particularly limited. Any commercially available animal-derived serum or plasma can be used, and it is possible to use human-derived serum from the same individual. For example, a combination of cytokines that promote lymphocyte proliferation from peripheral blood mononuclear cells and lectins that stimulate lymphocyte proliferation can be added.

[0062] In particular, adding albumin to a composition containing NK cells prepared by the method of the present invention can significantly enhance the cell-killing effect and cell viability of the NK cells, from the viewpoint of long-term storage. The amount of albumin to be added is not particularly limited. Specifically, albumin may be included in the overall composition in the range of 0.1% to 5% by weight, and more specifically, in the range of 0.5% to 2% by weight.

[0063] Furthermore, by applying a culture method that maintains a constant cell concentration to prepare NK cells, it is possible to prevent abnormal cell proliferation and thereby maintain the cells in an optimal state. In particular, even after freezing and thawing, the function of the cells is not impaired, and it is possible to maintain a high cell viability and cytotoxic effect. Therefore, there is the advantage that storage and supply can be easily carried out in liquid or frozen storage form without additional treatment.

[0064] On the other hand, the NK cells used in the present invention may be prepared, for example, by a method that includes a step of co-culturing seed cells with CD4(+) T cells isolated ex vivo as feeder cells, or CD4(+) T cells cultured ex vivo on an expanded scale.

[0065] Specifically, the CD4(+) T cells used in this invention may be CD4(+) / CD1(-) T cell lines, more specifically, H9 cell lines or HuT78 cell lines.

[0066] As used herein, the term "seed cells" refers to cells that are the starting material for obtaining target cells. The seed cells used in the present invention may be, but are not limited to, one or more selected from the group consisting of peripheral blood-derived cells, peripheral blood leukocytes, peripheral blood mononuclear cells (PBMCs), umbilical cord blood leukocytes, umbilical cord blood mononuclear cells, stem cell-derived natural killer cells, enriched natural killer cells, and isolated natural killer cells.

[0067] Regarding the method for preparing NK cells and the NK cells according to embodiments of the present invention, these may be described in Korean Patent No. 10-1697473 or No. 10-1799986.

[0068] This method for preparing NK cells allows for the large-scale, selective proliferation of NK cells from a small amount of seed cells, while also maintaining their high killing effect. Therefore, by including NK cells produced in this way, the pharmaceutically active ingredients according to the embodiments of the present invention exhibit extremely excellent cancer therapeutic effects, possessing high cell viability and cytotoxicity, as well as specific anti-CD19 antibodies.

[0069] Furthermore, as used herein, the term “combination” means a composition in which one or more active ingredients, such as antibodies and natural killer cells, are combined. Each active ingredient in the combination of the present invention may be administered to a subject simultaneously or separately at different times. Therefore, each active ingredient does not necessarily have to exist as a combination in the form of a single composition at the time of administration. The combinations according to the present invention relate to combinations, drugs and pharmaceutical compositions. In embodiments, the components of a combination are administered to a patient when all components (drugs) are active at the same time. The “synergistic effect” implies that all drugs are effective in the patient at the same time. In embodiments, the components of a combination are administered together, simultaneously, separately, or subsequently, either physically or temporally. In embodiments, the components of a combination are administered simultaneously.

[0070] The pharmaceutical combination is applicable to all types of tumors, including solid tumors and hematological malignancies. Unlike hematological malignancies, solid tumors refer to cancers that form as a mass within an organ. Most cancers that occur in most organs fall under the category of solid tumors. There are no particular limitations on the tumors that can be treated with the pharmaceutical combination according to the present invention. The pharmaceutical combination according to the present invention may have a synergistic therapeutic effect on at least one disease selected from the group consisting of, for example, gastric cancer, liver cancer, lung cancer, colorectal cancer, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, cervical cancer, thyroid cancer, laryngeal cancer, acute myeloid leukemia, brain tumors, neuroblastoma, retinoblastoma, head and neck cancer, salivary gland cancer, and lymphoma.

[0071] As used herein, the terms "synergy," "synergistic effect," and "synergistic action" mean exceeding the expected additive effect of the combination.

[0072] Furthermore, lymphoma can specifically be a malignant tumor of B cell origin, but it can be at least one selected from the group consisting of non-Hodgkin lymphoma, chronic lymphocytic leukemia, and acute lymphoblastic leukemia.

[0073] Non-Hodgkin lymphoma may be a lymphoma selected from the group consisting of follicular lymphoma, small lymphocytic lymphoma, mucosa-associated lymphoid tissue, marginal zone, diffuse large B cell, Burkitt, and mantle cell lymphomas.

[0074] The two components of the synergistic combination of the present invention, for example, a CD19-specific antibody and NK cells, may be administered together, simultaneously, separately, or sequentially, from a physical or temporal standpoint. As used herein, the terms “administered” or “to administer” are intended to include, but are not limited to, delivery in an injectable form, such as via intravenous, intramuscular, intradermal, or subcutaneous routes, or in an inhaled nasal spray or aerosol, or in an ingestible solution, capsule, or tablet. In addition, the pharmaceutically active combination may be administered in combination with another drug or bioactive substance whose therapeutic effect on the disease being treated is known, or may be formulated in the form of a combination formulation with another drug.

[0075] For example, NK cells may be administered before and / or separately from a CD19-specific antibody, an example of which is MOR208. When the two components are administered together, they may be formulated with a single pharmaceutical composition that may contain a pharmaceutically acceptable carrier or excipient. The two components may also be formulated in various pharmaceutical compositions. In this case, the two components may be administered simultaneously or separately. Specifically, when a CD19-specific antibody and NK cells are administered, these components may be activated simultaneously in the body of the receiving patient. For example, if MOR208 is administered weekly and NK cells are administered daily, the active ingredients of both drugs will be present in the patient simultaneously.

[0076] Furthermore, another aspect of the present invention may provide a kit for treating cancer, including the above-described combination. The type of kit is not particularly limited, and kits of a type commonly used in the art may be used.

[0077] The kit may be packaged in such a way that the CD19-specific antibody and NK cells described above are each contained in separate containers, or in such a way that the CD19-specific antibody and NK cells are contained in a single container divided into one or more compartments. The CD19-specific antibody and NK cells may each be packaged in single-dose units, but are not limited to this. The CD19-specific antibody and NK cells in the kit may be administered separately or in combination at appropriate times depending on the health condition of the recipient.

[0078] Another aspect of the present invention provides the use of the above-described pharmaceutically acceptable combinations for preparing drugs to treat cancer. In addition, the use of the above-described pharmaceutically acceptable combinations for treating cancerous diseases and similar lesions is provided.

[0079] Furthermore, yet another aspect of the present invention provides a method for treating cancer, comprising the steps of (a) preparing NK cells, (b) preparing a CD19-specific antibody comprising the HCDR1 region of SYVMH (SEQ ID NO: 1), the HCDR2 region of NPYNDG (SEQ ID NO: 2), the HCDR3 region of GTYYYGTRVFDY (SEQ ID NO: 3), the LCDR1 region of RSSKSLQNVNGNTYLY (SEQ ID NO: 4), the LCDR2 region of RMSNLNS (SEQ ID NO: 5), and the LCDR3 region of MQHLEYPIT (SEQ ID NO: 6), and (c) administering a therapeutically effective amount of the CD19-specific antibody and NK cells to a subject.

[0080] As used herein, the term "subject" means a mammal, including humans, having or suffering from a cancerous disease that can be mitigated, suppressed, or treated by administering a CD19-specific antibody and NK cells according to embodiments of the present invention. Furthermore, the subject may be a non-human animal, and the term "non-human animal" includes, for example, vertebrates such as non-human primates, mammals such as sheep, dogs, cats, horses, cattle, chickens, amphibians and reptiles, and non-mammals.

[0081] It is possible to provide a method for treating cancer, comprising the steps of (a) preparing NK cells, (b) preparing a CD19-specific antibody, and (c) administering a therapeutically effective amount of the CD19-specific antibody and NK cells to a target.

[0082] Since the steps for preparing NK cells (a) and the CD19-specific antibody in step (b) have already been described above, their explanations will be omitted to avoid excessive redundancy.

[0083] The administration step (c) may be carried out by administering CD19-specific antibodies and NK cells simultaneously, sequentially, or in reverse order.

[0084] One aspect of the present disclosure includes a synergistic combination of CD19-specific antibodies, including the HCDR1 region of sequence SYVMH (SEQ ID NO: 1), the HCDR2 region of sequence NPYNDG (SEQ ID NO: 2), the HCDR3 region of sequence GTYYYGTRVFDY (SEQ ID NO: 3), the LCDR1 region of sequence RSSKSLQNVNGNTYLY (SEQ ID NO: 4), the LCDR2 region of sequence RMSNLNS (SEQ ID NO: 5), and the LCDR3 region of sequence MQHLEYPIT (SEQ ID NO: 6), as well as NK cells for the treatment of non-Hodgkin lymphoma, chronic lymphocytic leukemia, and / or acute lymphoblastic leukemia. In one embodiment, non-Hodgkin lymphoma is selected from the group consisting of follicular lymphoma, small lymphocytic lymphoma, mucosa-associated lymphoid tissue, marginal zone, diffuse large B cells, Burkitt cells, and mantle cells. In one embodiment, the NK cells are MG4101.

[0085] One aspect of the present disclosure comprises a CD19-specific antibody comprising the HCDR1 region of sequence SYVMH (SEQ ID NO: 1), the HCDR2 region of sequence NPYNDG (SEQ ID NO: 2), the HCDR3 region of sequence GTYYYGTRVFDY (SEQ ID NO: 3), the LCDR1 region of sequence RSSKSLQNVNGNTYLY (SEQ ID NO: 4), the LCDR2 region of sequence RMSNLNS (SEQ ID NO: 5), and the LCDR3 region of sequence MQHLEYPIT (SEQ ID NO: 6) for the treatment of non-Hodgkin lymphoma, chronic lymphocytic leukemia, and / or acute lymphoblastic leukemia, wherein the CD19-specific antibody is used in combination with NK cells. In one embodiment, non-Hodgkin lymphoma is selected from the group consisting of follicular lymphoma, small lymphocytic lymphoma, mucosa-associated lymphoid tissue, marginal zone, diffuse large B cells, Burkitt cells, and mantle cells. In one embodiment, the NK cells are MG4101. In one embodiment, non-Hodgkin lymphoma is follicular lymphoma. In another embodiment, non-Hodgkin lymphoma is small lymphocytic lymphoma. In another embodiment, non-Hodgkin lymphoma is mucosa-associated lymphoid tissue. In another embodiment, non-Hodgkin lymphoma is marginal zone lymphoma. In another embodiment, non-Hodgkin lymphoma is diffuse large B-cell lymphoma. In another embodiment, non-Hodgkin lymphoma is Burkitt lymphoma. In another embodiment, non-Hodgkin lymphoma is mantle cell lymphoma.

[0086] Another embodiment includes a method for treating non-Hodgkin lymphoma, chronic lymphocytic leukemia and / or acute lymphoblastic leukemia in an individual requiring it, the method comprising administration of a CD19-specific antibody in combination with NK cells. In embodiments of this method, the CD19-specific antibody comprises the HCDR1 region of sequence SYVMH (SEQ ID NO: 1), the HCDR2 region of sequence NPYNDG (SEQ ID NO: 2), the HCDR3 region of sequence GTYYYGTRVFDY (SEQ ID NO: 3), the LCDR1 region of sequence RSSKSLQNVNGNTYLY (SEQ ID NO: 4), the LCDR2 region of sequence RMSNLNS (SEQ ID NO: 5), and the LCDR3 region of sequence MQHLEYPIT (SEQ ID NO: 6). In embodiments of this method, the antibody comprises an exemplary CD19-specific antibody. In embodiments of this method, the NK cells are MG4101.

[0087] Another embodiment involves the use of a CD19-specific antibody, which, in combination synergistically with NK cells, comprises the HCDR1 region of sequence SYVMH (SEQ ID NO: 1), the HCDR2 region of sequence NPYNDG (SEQ ID NO: 2), the HCDR3 region of sequence GTYYYGTRVFDY (SEQ ID NO: 3), the LCDR1 region of sequence RSSKSLQNVNGNTYLY (SEQ ID NO: 4), the LCDR2 region of sequence RMSNLNS (SEQ ID NO: 5), and the LCDR3 region of sequence MQHLEYPIT (SEQ ID NO: 6) when producing a drug for the treatment of non-Hodgkin lymphoma, chronic lymphocytic leukemia, and / or acute lymphoblastic leukemia. In one embodiment, the NK cells are MG4101.

[0088] Furthermore, the administration route, dosage, and frequency of CD19-specific antibodies and NK cells may vary depending on the patient's symptoms and the presence or absence of side effects; therefore, they can be administered to the target in various ways and in various amounts. The optimal method, dosage, and frequency of administration can be selected by those skilled in the art within a suitable range.

[0089] CD19-specific antibodies and NK cells can be administered parenterally, and this administration can be carried out by any preferred method, such as intratumoral, intraperitoneal, subcutaneous, intradermal, intranodal, and intravenous routes. Specifically, CD19-specific antibodies and NK cells can be administered intratumoral, intraperitoneal, or intravenously. On the other hand, the dosage of CD19-specific antibodies and NK cells can be determined based on the administration schedule, dosage, and the patient's health condition.

[0090] Furthermore, NK cells may be administered 2 to 5 times, with intervals of 1 to 30 days, 2 to 15 days, or 3 to 10 days between administrations.

[0091] CD19-specific antibodies may be administered 2 to 48 hours after NK cell administration. Specifically, CD19-specific antibodies may be administered once daily for 9 to 11 days, starting 2 hours after NK cell administration, for 3 to 5 days prior to NK cell administration. Details regarding cancer, a target disease that shows synergistic therapeutic effects with the above-described combination therapy, are as described in the Pharmacognostic Combinations section.

[0092] Another aspect of the present invention provides the use of a CD19-specific antibody for use in a pharmaceutically appropriate combination with natural killer cells for preparing a drug to treat cancer.

[0093] The CD19-specific antibody, natural killer cells, and their combined administration used in this invention have already been described above, so their explanation will be omitted to avoid excessive redundancy.

[0094] The present invention will be described in more detail below by the following examples. Here, the inventors intend to illustrate the synergistic effect of the combined administration of anti-CD19 antibodies and natural killer cells. However, the following examples are merely illustrative of the present invention, and the scope of the invention is not limited thereto.

[0095] [Table 1-1] [Table 1-2]

[0096] Mode of the invention Preparation Example 1: Cell line, primary cells, and culture conditions The Raji and Ramos cell lines (human Burkitt lymphoma cell lines) were obtained from the American Type Culture Collection (ATCC, USA). The Raji and Ramos cell lines were stored in RPMI1640 medium supplemented with 10% fetal bovine serum (FBS) and 2 mmol / L glutamine. Regarding NK cells, peripheral blood mononuclear cells (PBMCs) were randomly isolated from healthy donors, and the NK cells were cultured under Good Manufacturing Practice (GMP) conditions using the method described above (MG4101, Green Cross Labcell Corporation). In short, PBMCs with almost completely removed CD3+ T cells were cultured in 1% autologous plasma and irradiated with radiation (2,000 rad) at a rate of 5 × 10⁶ cells. 6 Autologous PBMCs at a concentration of cells / mL were cultured in CellGro SCGM serum-free medium (Cellgenix) containing 10 ng / mL of CD3 monoclonal antibody (OKT3, eBioscience) and 500 IU / mL of IL2 (Proleukin). During NK cell culture, 500 IU / mL of IL2 was added to the medium every 2-3 days.

[0097] Preparation Example 2: Monoclonal Antibody MOR208, a CD19-specific antibody, was obtained from MorphoSys AG (Germany). Rituximab (anti-CD20 antibody) was purchased from Roche. Antigen expression levels, determined as bound MOR208 or rituximab antibody per cell, were measured by flow cytometry (Quantibrite kit, BD Biosciences) according to the manufacturer's instructions. The number of MOR208 binding sites per cell in Raji and Ramos was 56,702 and 27,772, respectively. The number of rituximab binding sites per cell in Raji and Ramos was 59,974 and 168,253, respectively.

[0098] Preparation Example 3: Cytotoxicity assay by calcein release Target cells were labeled with 30 μM calcein-acetoxymethyl ester (calcein-AM, Molecular Probe) at 37°C for 1 hour. After washing, the labeled target cells were placed in 1 × 10⁶ wells. 4 Cells were dispensed into 96-well plates. Monoclonal antibodies (MOR208, rituximab, or an unrelated control antibody) were added to the target cells at concentrations between 0.001 ng / mL and 10 ug / mL. MG4101 was collected, washed, and added to the target cells in a 3:1 (E / T) ratio. After 2 hours, the plate was rotated at 2,000 rpm for 3 minutes, and 100 uL of supernatant was collected. Next, the fluorescence value (OD480 / 535 nm) was measured using a fluorescence microplate reader (Victor3, Perkin Elmer) to determine the amount of calcein released. The specific amount of calcein released was calculated using the following formula.

[0099] Specific dissolution percentage = (Test release value - Spontaneous release value) × 100 / (Maximum release value - Spontaneous release value) The highest solubility was achieved with 1% Triton X-100.

[0100] Preparation Example 4: Mouse Tumor Model Preparation Example 4.1 Establishment of a Ramos xenograft model in SCID mice Ramos cells (1x10 per mouse)5 , 5x10 5 or 10x10 5 ) was suspended in PBS and intravenously injected into SCID mice. The animals were monitored daily for signs of disease, hind limb paralysis or death.

[0101] Preparation Example 4.2 MOR208 Dosage Test for Ramos Xenograft Model Ramos cells (1×10 per mouse 6 ) were suspended in PBS and intravenously injected into SCID mice (day 0). MOR208 was administered by intravenous injection at various concentrations (20, 60, or 200 μg / 100 μL / head) on days 3, 6, 10, 13, 17, and 20. The animals were monitored daily for signs of disease, hind limb paralysis or death.

[0102] Preparation Example 4.3 MG4101 Dosage Test for Ramos Xenograft Model Ramos cells (1×10 per mouse 6 ) were suspended in PBS and intravenously injected into SCID mice (day 0). MG4101 was administered by intravenous injection at various concentrations (1, 2, or 5×10 7 / 400 μL / head) on days 4, 7, 11, 14, 18, and 21. The animals were monitored daily for signs of disease, hind limb paralysis or death.

[0103] Preparation Example 4.4 MG4101 + MOR208 Efficacy Test for Ramos Xenograft Model Ramos cells (1×10 per mouse 6 ) were suspended in PBS and intravenously injected into SCID mice (day 0). The mice were divided into 6 groups and adapted as follows.

[0104] i) The medium was frozen and hIgG (200 μg) was intravenously injected ii) Intravenous injection of MG4101 (2x10 7 / 400 μL / head) iii) Intravenous injection of MOR208 (60 μg / head) iv) Intravenous injection of MOR208 (200 μg / head) v) Intravenous injection of MG4101 (2 x 10) 7 Intravenous injection of MOR208 (400uL / head) and 60ug / head vi) Intravenous injection of MG4101 (2x10) 7 Intravenous injection of MOR208 (400uL / head) and 200ug / head

[0105] These administrations involved intravenous injections of various concentrations (60 or 200 ug / 100 uL / head) of hIgG or MOR208 twice weekly for three weeks on days 3, 6, 10, 13, 17, and 20.

[0106] Intravenous injection of MG4101 (2 x 10) on days 4, 7, 11, 14, 18, and 21. 7 The procedure was performed at a concentration of 400 μL / head. Animals were monitored daily for signs of disease, hind limb paralysis, or death.

[0107] Experimental Example 1. Confirmation of strong ADCC activity of MOR208 against malignant B cells in vitro. Since NK cell-mediated ADCC is crucial for the activity of monoclonal antibodies, the inventors initially measured the ADCC activity of MOR208 (anti-CD19 antibody) and MG4101 (NK cells) in Burkitt lymphoma cell lines Raji and Ramos compared to rituximab, a standard monoclonal antibody for lymphoma treatment. Here, rituximab is used as the positive control and reference value in this invention.

[0108] First, Raji and Ramos cell lines (Burkitt lymphoma, CD19+CD20+) were obtained from three healthy donors and cultured for 2 hours in the presence of activated and expanded liquid and frozen MG4101, with increasing concentrations of MOR208 or rituximab (E / T ratio 3:1).

[0109] The exact ADCC (%) is calculated using the following formula, and the results are shown in Figure 1 (in Figure 1, the graph shows the average ADCC (%) ± SD of three experimental values ​​obtained from one NK cell donor, representing two different donors).

[0110] (Sample emission value - Natural emission value) / (Maximum emission value - Natural emission value) x 100.

[0111] As shown in Figures 1a-1d, MOR208 exhibited significant in vitro ADCC activity against Raji and Ramos cell lines. Compared to controls co-treated with hIgG, both MOR208 and rituximab showed dose-dependent efficacy against both tumor cell lines, exhibiting activity at lower antibody concentrations and further increasing the maximum value of maximal tumor cell lysis. Considering that both MOR208 and rituximab showed comparable maximal killing effects compared to CD19 (Materials and Methods), as well as substantial levels of CD20, MOR208 showed overall lower EC compared to rituximab on various malignant B cells, including Raji and Ramos cells. 50 It has been demonstrated that MOR208 possesses this property. This indicates that MOR208 could be an interesting alternative treatment under conditions where rituximab is ineffective.

[0112] Experimental Example 2. Effect of MOR208 on lymphoma cell death in xenografted SCID mice. Experimental Example 2.1. Establishment of a Ramos xenotransplant model in SCID mouse models. For in vivo experiments with MOR208, a xenograft SCID mouse model was established using Ramos cells to evaluate the compatibility of the Ramos cell line. Six groups of SCID mice were given 1x10 5 , 5X10 5 , 10x10 5 Ramos cells or PBS were injected intravenously, and the mice were monitored daily for signs of disease. All mice died between day 26 and day 37 after Ramos injection.

[0113] Regarding Figure 2, intravenous injection of various concentrations of Ramos cells showed tumorigenic activity in mice characterized by progressive weight loss and hindlimb paralysis, and nearly 100% of the mice died between day 26 and day 37 after injection. For follow-up experiments, the optimal dose of the Ramos cell line was 1 × 10⁶ 6 I selected cells.

[0114] Experimental Example 2.2. MOR208 Dose Study in a Ramos Xenograft Model The efficacy of MOR208 against B lymphoma cell death is clinically important for its potential to deplete primary malignant B cells in patients. The inventors evaluated the antitumor activity of MOR208 in a proliferated lymphoma model administered with Ramos cells intravenously to SCID mice, as described in the "Preparation Example" above. In this model, disseminated Ramos cells infiltrate all mouse organs, including the central nervous system, thus reflecting diseases such as proliferated Burkitt lymphoma and acute lymphoblastic leukemia. In this regard, in a group of 10 SCID mice, 1 × 10⁶ cells were present. 6 Ramos cells were injected intravenously (day 0). On days 3, 6, 10, 13, 17, and 20, mice were treated with monoclonal antibodies (20, 60, or 200 mg / 100 mL / head) or hIgG (hIgG 200 mg / 100 mL / head) as negative controls, and signs of disease were observed daily.

[0115] As shown in Figure 3, treatment with 200 mg of MOR208 significantly extended the survival time of mice compared to animals treated with a control monoclonal antibody (P<.001). The median survival time of mice treated with 200 mg of MOR208 was 36 days, which was longer than the median survival time of 28.5 days for mice treated with the control antibody.

[0116] Experimental Example 2.3. Dose-test of MG4101 in a Ramos xenograft model SCID mice injected with Ramos cells were intravenously injected with MG4101 cells that had been activated and expanded in vitro from a healthy donor, as described in "Preparation Example." The addition of these MG4101 cells was intended to mimic the presence of tissue NK cells that may be present in the patient's organs. Specifically, 1 × 10⁶ cells were added to each of the 10 SCID mouse groups. 6 Ramos cells were injected intravenously (day 0). On days 4, 7, 11, 14, 18, and 21, MG4101 (1, 2, or 5 × 10) was administered. 7 Mice were administered either NK cell freezing medium (400 mL / head) or, as a control, only, and observed daily for signs of disease.

[0117] As shown in Figure 4, MG4101 is placed 5x10 per mouse. 7 The survival rate of mice treated with 5 × 10⁶ MG4101 doses was significantly higher compared to control mice (frozen medium) (P<.05). 7 The median survival time for mice treated with cells was 33 days, while the median survival time for mice treated with a control medium (frozen medium) was 28 days.

[0118] Experimental Example 2.4. Confirmation of the effect of combined administration of MG4101 + MOR208 in a Ramos xenograft model. Three days after seeding Ramos cells as described in "Preparation Example," mice injected with Ramos cells were randomly divided into several groups and administered either MOR208 or MG4101 alone twice, a combination of MOR208 and MG4101 twice, or, as a control, NK cell freezing medium containing hIgG. Specifically, in the group of 10 SCID mice, 1 × 10⁶ mice were treated. 6 Ramos cells were injected intravenously (day 0). On days 3, 6, 10, 13, 17, and 20, mice were treated with monoclonal antibody (60 or 200 mg / 100 mL / head). Mice were given MG4101 (2 × 10⁶). 7 Intravenous injections were administered at a concentration of 400 mL / head on days 4, 7, 11, 14, 18, and 21, and monitored daily.

[0119] As shown in Figure 5, mice treated with MOR208 showed a significantly increased protective effect compared to controls injected with hIgG in frozen medium. The group injected with MG4101 showed a significant improvement compared to the control. The median survival time was 30.5 days for the control group treated with frozen medium compared to mice treated with 200 mg of MOR208 (P<.0001) and 2X10. 7 In MG4101-treated mice (P<.001), the cells increased at 45.5 days and 38 days, respectively.

[0120] On the other hand, animals treated with a combination of MOR208 and MG4101 showed a synergistic increase in survival rate during the experimental period. Specifically, the median survival time was higher for animals treated with 2X10 co-injected drugs. 7 In a cellular background of MG4101, mice treated with 60 mg and 200 mg of MOR208 showed increased survival rates at 52 and 62 days, respectively (P<.0001). A fairly large synergistic effect of combined administration of MOR208 and MG4101 was also demonstrated in the analysis of increased survival rates. In particular, the mean increase in survival rates with each treatment of MOR208 or MG4101 was 11.5% (60 mg MOR208), 49.2% (200 mg MOR208), and 24.6% (2 × 10⁻⁶), respectively. 7 The cells were MG4101. In contrast, simultaneous treatment with MOR208 and MG4101 showed an effect of increasing survival rates, with MG4101 being administered simultaneously at 70.5% (60 mg of MOR208) and 103.3% (200 mg of MOR208), respectively. These results confirm that simultaneous treatment (combination) with MG4101 and MOR208 significantly extended the survival period of mice by twofold, especially when 60 mg of MOR208 was administered simultaneously, compared to animals injected with MOR208 or MG4101 separately.

[0121] In other words, both in vitro and in vivo data have revealed that the present invention, in combination with anti-CD19 antibodies and natural killer cells, that is, combination therapy of the two components, can be a therapeutic agent for human B lymphoma, leukemia, and other diseases, and furthermore, it has the potential to show remarkable therapeutic effects against various B-cell malignancies.

Claims

1. A pharmaceutical composition comprising a CD19-specific antibody for use in the treatment of cancer in human subjects, The antibody comprises a heavy chain variable region containing the amino acid sequence specified in SEQ ID NO: 8, a light chain variable region containing the amino acid sequence specified in SEQ ID NO: 9, a heavy chain constant region containing the amino acid sequence specified in SEQ ID NO: 10, and a light chain constant region containing the amino acid sequence specified in SEQ ID NO: 11, and The aforementioned antibody is administered in combination with natural killer cells (NK cells). Pharmaceutical composition.

2. A pharmaceutical composition according to claim 1, The NK cells are subjected to the following steps (i) to (iii): Step (i) involves stimulating cell-cell contact by performing static culture of isolated NK cells in a culture medium containing anti-CD3 antibody, cytokines, and feeder cells, Step (ii) involves adding cytokines, anti-CD3 antibodies, and feeder cells to restimulate them, and then performing static culture again to induce intracellular contact. Step (iii) involves adding a culture medium containing cytokines to the cells and performing static culture or suspension culture while maintaining a constant concentration of the cells and the cytokines. The pharmaceutical composition prepared by a method comprising the following:

3. A pharmaceutical composition according to claim 1, The pharmaceutical composition is prepared by a method comprising the step of co-culturing the NK cells with seed cells, using CD4(+) T cells isolated in ex vivo or CD4(+) T cells cultured in ex vivo as feeder cells.

4. A pharmaceutical composition according to claim 3, The pharmaceutical composition wherein the CD4(+) T cells are a CD4(+) / CD1(-) T cell line.

5. A pharmaceutical composition according to claim 4, The pharmaceutical composition wherein the CD4(+) / CD1(-) T cell line is an H9 cell line or a HuT78 cell line.

6. A pharmaceutical composition according to claim 3, The pharmaceutical composition wherein the seed cells are one or more selected from the group consisting of peripheral blood-derived cells, peripheral blood leukocytes, peripheral blood mononuclear cells (PBMCs), umbilical cord blood leukocytes, umbilical cord blood mononuclear cells, stem cell-derived natural killer cells, enriched natural killer cells, and isolated natural killer cells.

7. A pharmaceutical composition according to any one of claims 1 to 6, The pharmaceutical composition wherein the cancer is at least one selected from the group consisting of gastric cancer, liver cancer, lung cancer, colorectal cancer, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, cervical cancer, thyroid cancer, laryngeal cancer, acute lymphoblastic leukemia, brain tumor, neuroblastoma, retinoblastoma, head and neck cancer, salivary gland cancer, lymphoma, and B-cell derived malignant tumors.

8. A pharmaceutical composition according to any one of claims 1 to 6, The pharmaceutical composition wherein the cancer is non-Hodgkin lymphoma, chronic lymphocytic leukemia, or acute lymphoblastic leukemia.

9. A pharmaceutical composition according to claim 8, The pharmaceutical composition wherein the cancer is non-Hodgkin lymphoma.

10. A pharmaceutical composition according to claim 9, The pharmaceutical composition wherein the non-Hodgkin lymphoma is follicular lymphoma, small lymphocytic lymphoma, mucosa-associated lymphoid lymphoma, marginal zone lymphoma, diffuse large B-cell lymphoma, Burkitt lymphoma, or mantle cell lymphoma.

11. A pharmaceutical composition according to any one of claims 1 to 10, The pharmaceutical composition comprising the CD19-specific antibody and the NK cells administered separately.

12. A pharmaceutical composition according to any one of claims 1 to 10, The pharmaceutical composition comprising the administration of an antibody specific to CD19 and the NK cells simultaneously.

13. The use of a CD19-specific antibody in the manufacture of a pharmaceutical product for the treatment of cancer in human subjects, The antibody comprises a heavy chain variable region containing the amino acid sequence specified in SEQ ID NO: 8, a light chain variable region containing the amino acid sequence specified in SEQ ID NO: 9, a heavy chain constant region containing the amino acid sequence specified in SEQ ID NO: 10, and a light chain constant region containing the amino acid sequence specified in SEQ ID NO: 11, and The aforementioned CD19-specific antibody is administered in combination with natural killer cells (NK cells). use.

14. The use described in claim 13, The NK cells are subjected to the following steps (i) to (iii): Step (i) involves stimulating cell-cell contact by performing static culture of isolated NK cells in a culture medium containing anti-CD3 antibody, cytokines, and feeder cells, Step (ii) involves adding cytokines, anti-CD3 antibodies, and feeder cells to restimulate them, and then performing static culture again to induce intracellular contact. Step (iii) involves adding a culture medium containing cytokines to the cells and performing static culture or suspension culture while maintaining a constant concentration of the cells and the cytokines. The use described above is prepared by a method including the preparation of the use.

15. The use described in claim 13, The use is described above, wherein the NK cells are prepared by a method that includes the step of co-culturing seed cells with CD4(+) T cells isolated in ex vivo or CD4(+) T cells cultured in ex vivo, as feeder cells.

16. The use described in claim 15, The use described above, wherein the CD4(+) T cells are a CD4(+) / CD1(-) T cell line.

17. The use described in claim 16, The use described above, wherein the CD4(+) / CD1(-) T cell line is an H9 cell line or a HuT78 cell line.

18. The use described in claim 15, The use wherein the seed cells are one or more selected from the group consisting of peripheral blood-derived cells, peripheral blood leukocytes, peripheral blood mononuclear cells (PBMCs), umbilical cord blood leukocytes, umbilical cord blood mononuclear cells, stem cell-derived natural killer cells, enriched natural killer cells, and isolated natural killer cells.

19. A use according to any one of claims 13 to 18, The use thereof, wherein the cancer is at least one selected from the group consisting of gastric cancer, liver cancer, lung cancer, colorectal cancer, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, cervical cancer, thyroid cancer, laryngeal cancer, acute lymphoblastic leukemia, brain tumor, neuroblastoma, retinoblastoma, head and neck cancer, salivary gland cancer, lymphoma, and B-cell derived malignant tumors.

20. A use according to any one of claims 13 to 18, The use thereof, wherein the cancer is non-Hodgkin lymphoma, chronic lymphocytic leukemia, or acute lymphoblastic leukemia.

21. The use described in claim 20, The aforementioned use, wherein the cancer is non-Hodgkin lymphoma.

22. The use described in claim 21, The use described above, where non-Hodgkin lymphoma is follicular lymphoma, small lymphocytic lymphoma, mucosa-associated lymphoid tissue, marginal zone lymphoma, diffuse large B-cell lymphoma, Burkitt lymphoma, or mantle cell lymphoma.

23. A use according to any one of claims 13 to 22, The use is characterized in that the antibody specific to CD19 and the NK cells are administered separately.

24. A use according to any one of claims 13 to 22, The use is characterized in that the antibody specific to CD19 and the NK cells are administered simultaneously.

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