KR20230085116A - Pharmaceutical combinations for treating tumor comprising anti-cd19 antibody and natural killer cell - Google Patents

Abstract

The present invention provides a pharmaceutical combination containing a CD19-specific antibody and natural killer cells, and a treatment method using the same. The pharmaceutical combination can exhibit a synergistic therapeutic effect against malignancies of B cell origin, such as non-Hodgkin's lymphoma, chronic lymphocytic leukemia and/or acute lymphocytic leukemia.

Description

Pharmaceutical Combinations Comprising Anti-CD 19 Antibodies and Natural Killer Cells for Tumor Treatment

One embodiment of the present invention is anti-CD 19, which has a synergistic therapeutic effect against malignant tumors of B cell origin, such as non-Hodgkin's lymphoma, chronic lymphocytic leukemia (CLL) and/or acute lymphocytic leukemia (ALL). It relates to a pharmaceutical combination comprising an antibody and natural killer cells.

Natural killer cells (NK cells) are known to be lymphoid cells that play an important role in the innate immune response, recognizing targets in a non-MHC-restricted manner, unlike T cells. Natural killer cells can exert antiviral and anticancer effects, and specifically, directly kill malignant tumors or induce dendritic cell activity or tumor-specific cytotoxic T lymphocytes (CTLs), resulting in tumors or tumors. It plays a role in removing abnormal cells that are in progress. In addition, in relation to the anticancer efficacy of natural killer cells, the use of allogeneic natural killer cells that do not match the killer cell immunoglobulin-like receptor (KIR)-ligand pair in the treatment of cancer patients is the use of autologous natural killer cells. It has been proven to be more effective and safer than ever.

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 early development, mature B cells and malignant B cells. Indeed, the majority of malignancies of the B cell lineage express CD19, including non-Hodgkin's lymphoma, chronic lymphocytic leukemia (CLL) and acute lymphocytic leukemia (ALL). Therefore, since the anti-CD19 antibody can bind to the CD19 antigen as a target, it can be used in the above-mentioned therapeutic immunotherapy of malignant tumors of B cell origin.

Despite the recent discovery and development of several anti-cancer agents, many cancer types, including CD19 expressing tumors, have poor prognosis, so there is still a need for improved methods or therapeutics for treating these types of cancer. Accordingly, the present inventors confirmed that the combined administration of natural killer cells and CD19-specific antibodies had a synergistic effect in the treatment of malignant lymphoma of B cell origin, and completed the present invention.

Nadler et al, J. Immunol., 131 :244-250 (1983) Leukemia, Vol. 27, pages 1595-1598 (2013)

An object of the present invention is to provide a combination therapy using a CD19-specific antibody and natural killer cells or a combination thereof.

To achieve the above purpose,

One embodiment of the present invention may provide a pharmaceutical combination for cancer treatment comprising an antibody specific to CD19 and natural killer cells (NK cells).

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

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

The antibody comprises a heavy chain variable region comprising the HCDR1 region of SEQ ID NO: 1 (SYVMH), the HCDR2 region of SEQ ID NO: 2 (NPYNDG), and the HCDR3 region of SEQ ID NO: 3 (GTYYYGTRVFDY), the LCDR1 region of SEQ ID NO: 4 (RSSKSLQNVNGNTYLY), It may have a light chain variable region including the LCDR2 region of SEQ ID NO: 5 (RMSNLNS) and the LCDR3 region of SEQ ID NO: 6 (MQHLEYPIT).

The antibody has a heavy chain variable region of SEQ ID NO: 8 (EVQLVESGGGLVKPGGSLKLSCAASGYTFTSYVMHWVRQAPGKGLEWIGYINPYNDGTKYNEKFQGRVTISSDKSISTAYMELSSLRSEDTAMYYCARGTYYYGTRVFDYWGQGTLVTVSS) and SEQ ID NO: 9 (DIVMTQSPATLSLSPGERATLSCRSSKSLQNVNGNTYLYWFQQKPG QSPQLLIYRMSNLNSGVPDRFSGSGSGTEFTLTISSLEPEDFAVYYCMQHLEYPITFGAGTKLEIK).

The antibody has SEQ ID NO: 10 (ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPR EEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKALPAPEEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK) of the heavy chain constant region.

The antibody may comprise a light chain constant region of SEQ ID NO: 11 (RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC).

The NK cells are (i) stimulating cell-to-cell contact by statically culturing NK cells in a medium containing anti-CD3 antibody, cytokine and support cells, (ii) cytokine, anti-CD3 antibody and support cells Inducing cell-to-cell contact by adding re-stimulation and stationary culture again; and (iii) adding a medium containing cytokines to the cells and performing stationary or suspension culture while maintaining constant cell concentration and cytokine concentration. It can be prepared in a way that includes.

The NK cells are prepared by a method comprising the step of co-cultivating CD4(+) T cells isolated in vitro as feeder cells or CD4(+) T cells cultured in vitro and seed cells It can be.

The CD4(+) T cell may be a CD4(+)/CD1(-) T cell line.

The CD4(+)/CD1(-) T cell line may be an H9 or HuT78 cell line.

The source cells include peripheral blood-derived cells, peripheral blood leukocyte cells, PBMC (peripheral blood mononuclear cells), cord blood leukocyte cells, cord blood mononuclear cells, natural killer cells derived from stem cells, enriched natural killer cells, and isolated It may be one or more selected from the group consisting of natural killer cells.

In addition, the pharmaceutical combination may further include albumin.

The cancer is gastric cancer, liver cancer, lung cancer, colon cancer, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, cervical cancer, thyroid cancer, laryngeal cancer, acute lymphocytic leukemia, brain tumor, neuroblastoma, retinoblastoma, head and neck cancer, salivary gland cancer and lymphoma. It may be at least one selected from the group consisting of

The lymphoma may be a malignant tumor of B cell origin.

The malignant tumor of B cell origin may be at least one selected from the group consisting of non-Hodgkin's lymphoma, chronic lymphocytic leukemia and acute lymphocytic leukemia.

The CD19-specific antibody and NK cells may be administered separately.

The CD19-specific antibody and NK cells may be administered simultaneously.

Another embodiment of the present invention may provide a kit for treating cancer comprising the combination described above.

Another embodiment of the present invention may provide the use of the aforementioned pharmaceutical combination to prepare a medicament for the treatment of cancer.

In addition, another embodiment of the present invention may provide a method for treating cancer comprising administering the above-mentioned CD19-specific antibody and the above-mentioned NK cells in combination to a subject.

According to one embodiment of the present invention, the pharmaceutical combination comprising an antibody specific for CD19 and natural killer cells is a cancer, particularly a B cell malignancy such as non-Hodgkin's lymphoma, chronic lymphocytic leukemia and/or acute lymphocytic leukemia. useful for the treatment of In particular, the natural killer cells are produced in a clinically friendly method compared to conventional methods and have high cell killing ability, cell viability, and long-term storage, and show synergistic effects in tumor treatment when administered in combination with a specific antibody specific to CD19. can

1A to 1D are results of culturing Raji and Ramos Burkitt B-cell lymphomas with various concentrations of monoclonal antibodies in the presence of NK cells.
Figure 2 is a result of evaluating the suitability of Ramos cell line in the SCID mouse model.
Figure 3 shows the results of administration of MOR208 by concentration in the SCID mouse model group intravenously injected with Ramos cells.
Figure 4 shows the results of administration of MG4101 by concentration in the SCID mouse model group intravenously injected with Ramos cells.
Figure 5 shows the results of administering the control group, MOR208, MG4101, and "MOR208/MG4101" to the SCID mouse model group intravenously injected with Ramos cells.

Despite the possibility that natural killer cells can be applied as a treatment for various diseases, the rather limited number of natural killer cells in peripheral blood, related to large-scale production of lysed natural killer cells in compliance with good manufacturing practice (GMP) Access was not easy due to various limitations, such as difficulties and the need to activate natural killer cells in order to induce natural killer cell-mediated killing.

Recently, the present inventors have established a simple and efficient method for large-scale proliferation and activation of natural killer cells from healthy donors under GMP conditions (KR10-1644984 B1). In addition, through the method of mass-cultivating NK cells from mononuclear cells of umbilical cord blood, natural killer cells that were proliferated in vitro and highly activated were produced under GMP conditions, and showed strong anticancer activity in vitro and in vivo in preclinical studies.

On the other hand, a treatment using a monoclonal antibody specific for an epitope molecule expressed in cancer cells is known to be the most successful cancer immunotherapy to date. One of their mechanisms of action is natural killer cell-mediated antibody dependent cellular cytotoxicity (ADCC). Since natural killer cells proliferated according to one embodiment of the present invention expressed high levels of CD16, an Fc receptor that mediates ADCC, the present inventors found that anticancer activity against B cell lymphoma could be improved in the presence of anti-CD19 antibody. I wanted to check if it exists.

Surprisingly, the present inventors found that when a specific antibody specific to CD19 and natural killer cells are administered in combination, the direct killing of human B cell malignancies through ADCC in vitro and in vivo is synergistic compared to the antibody or natural killer cells alone. found to have an effect.

One aspect of the present invention provides a pharmaceutical combination for cancer treatment comprising an antibody specific to CD19 and natural killer cells.

As used herein, the term "CD19" refers to the protein known as CD19, which has the following synonyms: B4, B-lymphocyte antigen CD19, B-lymphocyte surface antigen B4, CVID3, differentiation antigen CD19, MGC12802, and T-cell surface antigen Leu. -12. The CD19 may be human-derived CD19, and human CD19 may include the amino acid sequence of SEQ ID NO: 7. CD19 is expressed by cells and tissues in a variety of diseases and conditions, including most B cell malignancies, and on normal B cells.

Also, the term "antibody" as used herein refers to a monoclonal antibody including any isotype such as IgG, IgM, IgA, IgD and IgE. IgG antibodies are composed of two identical heavy chains and two identical light chains linked by disulfide bonds. Each heavy and light chain comprises a constant region and a variable region. Each variable region contains three segments called "complementarity-determining regions" ("CDRs") or "hypervariable regions" that are primarily responsible for binding an antigen to an epitope. These are called CDR1, CDR2, and CDR3, numbering sequentially from the N-terminus. The more highly conserved portions of the variable regions outside of the CDRs are called "framework regions".

That is, the anti-CD19 antibody, which is an antibody specific to CD19, is an antibody that specifically binds to the CD19 antigen, and may include an antigen-binding fragment thereof as well as a complete antibody form.

Antibodies specific to CD19 according to one embodiment of the present invention are disclosed in, for example, US 12/377,251 (Xencor); WO 2005/012493, WO 2010/053716 (lmmunomedics); WO 2007/002223 (Medarex); WO 2008/022152 (Xencor); WO 2008/031056 (Medimmune); WO 2007/076950 (Merck Patent GmbH); WO 2009/052431 (Seattle Genetics); and WO 2010/095031 (Glenmark Pharmaceuticals).

In addition, the CD19-specific antibody according to one embodiment of the present invention comprises a heavy chain comprising the HCDR1 region of SEQ ID NO: 1 (SYVMH), the HCDR2 region of SEQ ID NO: 2 (NPYNDG), and the HCDR3 region of SEQ ID NO: 3 (GTYYYGTRVFDY) A variable region may be included. In addition, the CD19-specific antibody of the present invention may include a light chain variable region comprising an LCDR1 region of SEQ ID NO: 4 (RSSKSLQNVNGNTYLY), a LCDR2 region of SEQ ID NO: 5 (RMSNLNS), and a LCDR3 region of SEQ ID NO: 6 (MQHLEYPIT). can

Antibodies specific to CD19 according to another embodiment of the present invention may include antibodies that cross-compete with the above-described antibodies of the present invention.

As used herein, the term "cross-competition" refers to the ability of an antibody or other binding agent to interfere with the binding of another antibody or binding agent to CD19 in a standard competitive binding assay. The ability or extent of an antibody or other binding entity to interfere with the binding of another antibody or binding entity to CD19, and thus cross-competition according to the present invention, can be determined using standard competition binding assays. can

In addition, the CD19-specific antibody according to another embodiment of the present invention may include an antibody that binds to the same epitope as the above-described antibody of the present invention.

In addition, the antibody has a heavy chain variable region of SEQ ID NO: 8 (EVQLVESGGGLVKPGGSLKLSCAASGYTFTSYVMHWVRQAPGKGLEWIGYINPYNDGTKYNEKFQGRVTISSDKSISTAYMELSSLRSEDTAMYYCARGTYYYGTRVFDYWGQGTLVTVSS) and SEQ ID NO: 9 (DIVMTQSPATLSLSPGERATLSCRSSKSLQNVNGNTYLYWFQQ KPGQSPQLLIYRMSNLNSGVPDRFSGSGSGTEFTLTISSLEPEDFAVYYCMQHLEYPITFGAGTKLEIK). In addition, the antibody is SEQ ID NO: 10 (ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKT KPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKALPAPEEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK) of the heavy chain constant region. In addition, the antibody may include a light chain constant region of SEQ ID NO: 11 (RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC). The anti-CD19 antibody is "MOR208". The MOR208 antibody may be that disclosed in US Patent Application Serial No. 12/377,251.

As used herein, the term "Natural Killer Cell (hereinafter referred to as 'NK cell')" refers to a cytotoxic lymphocyte that plays an important role in the body's immune system. For the purposes of the present invention, NK cells may be endogenous NK cells and/or foreign NK cells of an individual, and may specifically be isolated NK cells administered outside the individual, but are not limited thereto. In addition, NK cells can be obtained by conventional methods such as purchasing commercially available cells, isolating them from individuals, or culturing them.

On the other hand, NK cells included in the pharmaceutical combination according to an embodiment of the present invention are stimulated with anti-CD3 antibody and support cells in a medium containing T cells in a medium containing T cells, and then stimulated for several days. During stationary culture to stimulate cell-to-cell contact, it may be prepared by stationary or suspension culture of NK cells in a reactor while maintaining a constant cell concentration and cytokine concentration.

In order to obtain an increased content of NK cells, stimulation and stationary culture can be repeated before subsequent stationary or suspension culture. Since the NK cells prepared by the method according to the present invention exhibit high cell viability and cell killing ability and can be cultured in vitro with high efficiency and high concentration, they can be administered in combination with the anti-CD19 antibody to treat malignant tumors of B cell origin. .

The NK cells, as an example, may be prepared by a method comprising steps (i) to (iii) below.

(i) statically culturing the isolated NK cells in a medium containing anti-CD3 antibody, cytokines and feeder cells to stimulate intercellular contact;

(ii) restimulation by adding cytokine, anti-CD3 antibody, and feeder cells, followed by static culture to induce cell-to-cell contact; and

(iii) adding a medium containing cytokines to the cells, and performing still or suspension culture while maintaining constant cell concentration and cytokine concentration.

In this case, the isolated NK cells may be obtained by separating leukocyte cells and NK cells from human peripheral blood or umbilical cord blood.

At this time, the political culture after the initial stimulation may be about 2 to 15 days, specifically 5 to 10 days, and the political culture after re-stimulation may be about 2 to 7 days, specifically 3 to 5 days. In addition, after the stationary culture is completed, stationary or floating culture may be performed while maintaining a constant concentration of cytokines in the incubator. In addition, as albumin is added to a composition containing the same during preparation of NK cells, the cell killing ability and cell viability of NK cells can be greatly improved. Specifically, NK cells prepared by the above method may be labeled as "MG4101". Regarding the manufacturing method of the NK cells and the corresponding NK cells according to one embodiment of the present invention, it may be described in Korean Registered Patent No. 10-1644984.

As used herein, the term "feeder cell (also referred to as feeder cell)" refers to a cell that does not have the ability to divide and proliferate, but has metabolic activity and thus produces various metabolites to help the proliferation of target NK cells. Support cells that can be used include animal cell lines into which genes have been introduced, peripheral blood leukocyte cells (PBL) treated with various cytokines or compounds, peripheral blood leukocyte cells (PBL) of one's own or others, T-cells, There are B-cells, monocytes, and the like, and specifically, autologous peripheral blood mononuclear cells may be used, but are not limited thereto.

In addition, safety can be secured by inactivating the autologous peripheral blood mononuclear cells used as the supporting cells. As a method of inactivating, a conventional method known in the art may be used, and for example, a method of irradiating gamma-rays may be used. Such inactivated feeder cells include isolated T-cells. The proliferation method using support cells as described above is a method of proliferating pure NK cells after isolating them, and has an advantage in that only pure NK cells continue to proliferate thereafter.

As used herein, the term "anti-CD3 antibody" refers to an antibody that specifically binds to the CD3 antigen, which is a group of molecules that bind to the T cell receptor (TCR) to form an antigen recognition complex, and the CD3 molecule binds to the TCR to signal an antigen recognition signal. is responsible for transporting it into cells. Anti-CD3 antibodies that can be used in the present invention can be used without limitation as long as they have CD3-binding properties, and may be selected from the group consisting of, for example, OKT3, UCHT1 and HIT3a, but are not limited thereto.

In the present invention, the cytokine that may be included in the medium may be one or more selected from interleukins. For example, it consists of interleukin-2 (IL-2), interleukin-12 (IL-12), interleukin-15 (IL-15), interleukin-18 (IL-18) and interleukin-21 (IL-21). One or more selected from the group may be used, but is not limited thereto.

In addition, the concentration of the anti-CD3 antibody used for stationary culture and suspension culture in the medium may be 0.1 to 1,000 ng/ml, 1 to 100 ng/ml, or 5 to 20 ng/ml, and the cytokine in the medium The concentration may be 10 to 2,000 IU, 100 to 1,000 IU, or about 200 to 700 IU.

As used herein, the term “stimulation” means inducing proliferation of NK cells by adding support cells or the like, and anti-CD3 antibodies may also be used together. In addition, the term "restimulation" as used herein means re-induction of NK cell proliferation by adding feeder cells and/or anti-CD3 antibody to the medium again after a certain culture time has elapsed.

As the medium for producing NK cells according to one embodiment of the present invention, CellGro medium (Cellgenix), AIM-V medium, RPMI1640 medium, and conventional animal cell culture medium such as X-VIVO 20 may be used. If necessary, one or more components selected from NK cells isolated from human peripheral blood, peripheral blood mononuclear cells, anti-CD3 antibodies, and interleukins may be added to the culture medium for animal cells and used.

In particular, in the method for producing NK cells, in order to maintain a constant cell concentration and cytokine concentration during suspension culture, the cytokine and cell concentrations in the medium are measured at regular time intervals, and cytokines are included according to the measured values The culture medium may be provided according to cell concentration and cytokine concentration.

In addition, additional growth factors supporting the proliferation of serum or plasma and lymphocytes may be added to the medium and cultured. The type of serum or plasma to be added to the medium is not particularly limited, and various commercially available animal-derived ones can be used, but human-derived ones derived from the subject can be used. For example, methods such as adding a combination of cytokines that stimulate lymphocyte proliferation from peripheral blood mononuclear cells or lectins that stimulate lymphocyte proliferation can be used.

In particular, by adding albumin to the composition containing NK cells prepared by the method of the present invention, cell killing ability and cell viability in terms of long-term storage stability of NK cells could be greatly improved. The amount of albumin added is not particularly limited, but may be specifically included in the range of 0.1 to 5% by weight, more specifically in the range of 0.5 to 2% by weight in the total composition.

In addition, by applying a culture method that keeps the cell concentration constant for the production of NK cells, overgrowth of cells can be prevented, so that cells can maintain optimal conditions. In particular, even when thawed again after freezing, cell functions are not damaged and high cell viability and cell killing ability can be maintained. Therefore, there is an advantage in that it is easy to store and supply in a liquid or frozen form without additional processing.

On the other hand, the NK cells used in the present invention, as an example, CD4 (+) T cells isolated in vitro as feeder cells or CD4 (+) T cells cultured in vitro and seed cells It may be prepared by a method comprising the step of co-cultivating.

Specifically, the CD4(+) T cell used in the present invention may be a CD4(+)/CD1(-) T cell line, and more specifically, an H9 or HuT78 cell line.

As used herein, the term "source cell" means a cell as a starting material for obtaining a target cell. Source cells used in the present invention are peripheral blood, peripheral blood leukocyte cells, PBMC (peripheral blood mononuclear cells), cord blood leukocyte cells, cord blood mononuclear cells, natural killer cells derived from stem cells, enriched natural killer cells and It may be one or more selected from the group consisting of isolated natural killer cells, but is not limited thereto.

Regarding the manufacturing method of NK cells and corresponding NK cells according to an embodiment of the present invention, it may be described in Korean Registered Patent No. 10-1697473 or 10-1799986.

This method of producing NK cells can selectively proliferate only NK cells in a large amount from a small amount of source cells and maintain high killing ability. Therefore, the pharmaceutical combination according to one embodiment of the present invention contains NK cells having high cell viability and cell killing ability and a specific anti-CD 19 antibody, and thus has excellent cancer treatment effect.

In addition, as used herein, the term "combination" means a composition in which more than one active ingredient, for example, an active ingredient such as an antibody and natural killer cells is combined. Each active ingredient included in the combination of the present invention may be administered to a subject simultaneously or individually with a time difference. Therefore, each active ingredient need not necessarily exist in one combination at the time of administration. Combinations according to the present invention relate to combinations, medicaments, and pharmaceutical compositions.

The combination of the present invention can be administered together so that the two active ingredients can simultaneously have activity in a subject. Since the combination of the present invention shows a synergistic action, it can be seen that the two active ingredients have simultaneous activity in the subject.

The pharmaceutical combination can be applied to all types of tumors including solid cancer and hematological cancer. Unlike blood cancer, solid cancer refers to cancer formed by forming a mass in an organ, and cancers occurring in most organs fall under this category. Tumors that can be treated using the pharmaceutical combination according to the present invention are not particularly limited. For example, stomach cancer, liver cancer, lung cancer, colon cancer, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, cervical cancer, thyroid cancer, laryngeal cancer, acute myelogenous leukemia, brain tumor, neuroblastoma, retinoblastoma, head and neck cancer, salivary gland cancer and lymphoma. It may have a synergistic therapeutic effect on at least one disease selected from the group formed.

As used herein, the terms "synergy", "synergy", and "synergism" mean that the combination exceeds the expected additive effect of the combination.

In addition, the lymphoma may specifically be a malignant tumor of B cell origin, and the malignant tumor of B cell origin may be at least one selected from the group consisting of non-Hodgkin's lymphoma, chronic lymphocytic leukemia, and acute lymphocytic leukemia.

The non-Hodgkin's lymphoma can be a lymphoma selected from the group consisting of follicular lymphoma, small lymphocytic lymphoma, mucosal-associated lymphoid tissue, marginal zone, diffuse large B cells, Burkitt's and mantle cells.

The two components of the synergistic combination of the present invention, eg, an antibody specific for CD19 and NK cells, may be administered physically or temporally together, simultaneously, separately or sequentially. As used herein, the term "administered" or "administration" refers to an injectable form, e.g., by the intravenous, intramuscular, intradermal or subcutaneous route, or as an ingestible solution, e.g., as a nasal spray or aerosol for inhalation; delivery by mucosal routes as capsules or tablets. In addition, the pharmaceutical combination may be administered in combination with other drugs or physiologically active substances known to have therapeutic effects on the disease to be treated, or formulated in combination with other drugs.

For example, NK cells may be administered prior to and/or separately from administration of an antibody specific for CD19, eg, MOR208. When the two components are administered together, they can be formulated together as one pharmaceutical composition that may include a pharmaceutically acceptable carrier or excipient. Alternatively, the two components may also be formulated in different pharmaceutical compositions. In this case, the two components may be administered simultaneously or separately. Specifically, when the CD19-specific antibody and NK cells are administered, they can simultaneously have activity in the body of the administered patient. For example, if MOR208 is administered weekly and NK cells are administered daily, the active ingredients of both drugs are present in the patient simultaneously.

In addition, another aspect of the present invention provides a kit for treating cancer comprising the above combination. The type of the kit is not particularly limited, and kits of a type commonly used in the art may be used.

The kit may be packaged in a form in which the above-described CD19-specific antibody and NK cells are contained in individual containers, or in a single container divided into one or more compartments, and the CD19-specific antibody and NK cells Each may be packaged in a unit dosage form of a single dose, but is not limited thereto. The CD19-specific antibody and NK cells in the kit may be individually and concurrently administered at an appropriate time according to the health condition of the subject to be administered.

Another aspect of the present invention provides the use of the aforementioned pharmaceutical combination for the manufacture of a medicament for the treatment of cancer. Also provided is the use of the foregoing pharmaceutical combinations for the treatment of cancer diseases and such conditions.

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

As used herein, the term "subject" includes a human suffering from or a condition in which cancer disease can be reduced, suppressed, or treated by administering an antibody specific to CD19 and NK cells according to one embodiment of the present invention. means mammal. In addition, the subject may be a non-human animal, and the term “non-human animal” refers to all vertebrates, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, mammals such as amphibians, reptiles, etc. Includes animals and non-mammals.

(a) preparing NK cells, (b) preparing an antibody specific for CD19, and (c) administering to a subject a therapeutically effective amount of the antibody specific for CD19 and NK cells in combination. treatment method can be provided.

Since the preparation of NK cells in (a) and the CD19-specific antibody in (b) have already been described in detail, descriptions thereof are omitted to avoid excessive redundancy.

In the step of administering (c), the CD19-specific antibody and NK cells may be administered simultaneously, sequentially, or in reverse order.

In addition, the administration route, dosage and frequency of administration of the CD19-specific antibody and NK cells can be administered to the subject in various ways and amounts depending on the condition of the patient and the presence or absence of side effects, and the optimal administration method, dosage and The frequency of administration can be selected within an appropriate range by a person skilled in the art.

The CD19-specific antibody and NK cells may be administered parenterally, intratumoral, intraperitoneal, sub-cutaneous, intradermal, intra-nodal ) and intra-venous, etc. may be administered by suitable methods. Specifically, the CD19-specific antibody and NK cells may be administered intratumorally, intraperitoneally, or intravenously. Meanwhile, the dosage of the CD19-specific antibody and NK cells may be determined according to the administration schedule, dosage, and the patient's health condition.

In addition, the NK cells may be administered 2 to 5 times, and may be administered to the subject at intervals of 1 day to 30 days, 2 days to 15 days, or 3 days to 10 days.

The CD19-specific antibody may be administered within 2 to 48 hours after administration of NK cells. Specifically, the CD19-specific antibody may be administered 3 to 5 days before NK cell administration and 2 hours after NK cell administration once a day for 9 to 11 days. Details related to cancer, which is a target disease having a synergistic therapeutic effect due to the combined administration, are as described in the pharmaceutical combination.

Another aspect of the present invention provides a use of the CD19-specific antibody of the present invention used in combination with the natural killer cells of the present invention for the production of a medicament for cancer treatment.

Since the CD19-specific antibodies and natural killer cells used in the present invention and their administration methods have already been described above, their descriptions are omitted to avoid excessive redundancy.

Hereinafter, the present invention will be described in more detail by the following examples. Here, we try to explain the synergistic effect of the combined administration of anti-CD19 antibody and natural killer cells. However, the following examples are only for exemplifying the present invention, and the scope of the present invention is not limited only to these.

antibody sequence number amino acid sequence MOR208




HCDR1 One SYVMH HCDR2 2 NPYNDG HCDR3 3 GTYYYGTRVFDY LCDR1 4 RSSKSLQNVNGNTYLY LCDR2 5 RMSNLNS LCDR3 6 MQHLEYPIT VH 8 EVQLVESGGGLVKPGGSLKLSCAASGYTFTSYVMHWVRQAPGKGLEWIGYINPYNDGTKYNEKFQGRVTISSDKSISTAYMELSSLRSEDTAMYYCARGTYYYYGTRVFDYWGQGTLVTVSS VL 9 DIVMTQSPATLSLSPGERATLSCRSSKSLQNVNGNTYLYWFQQKPGQSPQLLIYRMSNLNSGVPDRFSGSGSGTEFTLTISSLEPEDFAVYYCMQHLEYPITFGAGTKLEIK heavy chain
invariant region 10 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVS VLTVVHQDWLNGKEYKCKVSNKALPAPEEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK light chain constant region 11 RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC CD19 human CD19 7 MPPPRLLFFLLFLTPMEVRPEEPLVVKVEEGDNAVLQCLKGTSDGPTQQLTWSRESPLKPFLKLSLGLPGLGIHMRPLAIWLFIFNVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWNVSDLGGLGCGLKNRSSEGPSPSGKLMSPKLYVWAKDRPEIWEGEPPCLPPRDSLNQSLSQDLTMAPGSTLWLSCG VPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVMETGLLLPRATAQDAGKYYCHRGNLTMSFHLEITARPVLWHWLLRTGGWKVSAVTLAYLIFCLCSLVGILHLQRALVLRRKRKRMTDPRRFFKVTPPPGSGPQNQYGNVLSLPTPTSGLGRAQRWAAGLGGTAPSYGNPSSDVQADGGPALGSRSPPGVEEEEEGY EEPDSEEDSEFYENDSNLGQDQLSQDGSGYENPEDEPLGPEDEDSFSNAESYENEDEELTQPVARTMDFLSPHGSAWDPSREATSLGSQSYEDMRGILYAAPQLRSIRGQPGPNHEEDADSYENMDNPDGPDPAWGGGGRMGTWSTR

Preparation Example 1. Cell lines, primary cells and culture conditions

Raji and Ramos (human Burkitt's lymphoma cell line) cell lines were obtained from the American Type Culture collection (ATCC, USA). Raji and Ramos cell lines were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 2 mmol/L glutamine. NK cells, PBMC (peripheral blood mononuclear cells) were isolated from random healthy donors, and NK cells were expanded and cultured as described above under GMP conditions (Good Manufacturing Practice, Manufacturing Quality Control Standards) (MG4101, Green Cross Lab Cell). Briefly, PBMCs from which CD3+ T cells were almost eliminated were cultured in 1% autologous plasma, 5X10 ⁶ cells/mL of autologous PBMC irradiated with radiation (2,000 rad), and 10 ng/mL of single clone for CD3 (OKT3, eBioscience). The cells were expanded in CellGro SCGM serum-free medium (Cellgenix) containing antibody and 500 IU/mL of IL2 (Proleukin). While the NK cells were being cultured, 500 IU/mL of IL2 was supplied to a fresh medium every 2 to 3 days.

Preparation Example 2. Monoclonal Antibody

MOR208, an antibody specific for CD19, was purchased from MorphoSys Pharmaceuticals (Germany). Rituximab was purchased from Roche. The number of MOR208 and rituximab binding sites per cell was determined by flow cytometry (Quantibrite kit, BD Biosciences) according to the manufacturer's instructions. The number of MOR208 binding sites per Raji and Ramos cell lines is 56702 and 27772, respectively. The number of rituximab binding sites per cell line for Raji and Ramos is 59974 and 168253, respectively.

Preparation Example 3. Calcein release cytotoxicity assay

Target cells were labeled with 30 uM Calcein-acetoxymethyl ester (Calcein-AM; Molecular probes) for 1 hour at 37°C. After washing, the labeled target cells were distributed in 96-well plates at 1×10 ⁴ cells per well. Monoclonal antibodies (rituximab, MOR208 or control irrelevant antibody) were added to the target cells (concentrations between 0.001 ng/mL and 10 ug/mL). MG4101 was obtained, washed, and added to target cells in a 3:1 (E/T) ratio. After 2 hours, the plate was rotated at 2000 rpm for 3 minutes, and 100 uL of the supernatant was obtained, and then the fluorescence value (OD480/535 nm) was measured using a fluorescence microplate reader (Victor3, Perkin Elmer) to determine the amount of calcein released. decided. Specific calcein release was calculated using the formula:

Specific Dissolution % = (Test Release - Spontaneous Release) Х 100/(Maximum Release - Spontaneous Release)

Maximum lysis was achieved with 1% Triton X-100.

Preparation Example 4. Mouse tumor model

Preparation Example 4.1. Establishment of the Ramos xenograft model in SCID mice

Ramos cells (1 X 10 ⁵ , 5 X 10 ⁵ or 10 X 10 ⁵ per mouse) were suspended in PBS and injected intravenously into SCID mice. Animals were monitored daily for signs of illness, hind limb paralysis or death.

Preparation Example 4.2. MOR208 capacity testing in Ramos xenograft model

Ramos cells (1 X 10 ⁶ per mouse) were suspended in PBS and injected intravenously into SCID mice (day 0). On days 3, 6, 10, 13, 17 and 20, MOR208 was administered intravenously at various concentrations (20, 60 or 200 ug/100 uL/head). Animals were monitored daily for signs of illness, hind limb paralysis or death.

Preparation Example 4.3. MG4101 capacity testing in Ramos xenograft model

Ramos cells (1 X 10 ⁶ per mouse) were suspended in PBS and injected intravenously into SCID mice (day 0). On days 4, 7, 11, 14, 18, and 21, MG4101 was intravenously administered at various concentrations (1, 2, 5 x 10 ⁷ /400 uL/head). Animals were monitored daily for signs of illness, hind limb paralysis or death.

Preparation Example 4.4. MG4101 + MOR208 efficacy test in Ramos xenograft model

Ramos cells (1 X 10 ⁶ per mouse) were suspended in PBS and injected intravenously into SCID mice (day 0). Mice were divided into 6 groups and adjusted as follows:

i) Frozen medium and hIgG (200ug) intravenous injection

ii) MG4101 (2x10 ⁷ /400 uL/head) intravenous injection

iii) MOR208 (60 ug/head) intravenous injection

iv) MOR208 (200 ug/head) intravenous injection

v) MG4101 (2x10 ⁷ /400 uL/head) intravenous injection and MOR208 (60 ug/head) intravenous injection

vi) MG4101 (2x10 ⁷ /400 uL/head) intravenous and MOR208 (200 ug/head) intravenous.

This administration consisted of hIgG or MOR208 at various concentrations (60, or 200 ug/100 uL/head) for 3 weeks, twice a week, intravenously on days 3, 6, 10, 13, 17, and 20. Injected.

Intravenous injections of MG4101 (2x10 ⁷ /400 uL/head) were performed on days 4, 7, 11, 14, 18 and 21. Animals were monitored daily for signs of illness, hind limb paralysis or death.

Experimental Example 1. Strong MOR208 against malignant B cells in vitro ADCC active check

Since NK cell-mediated ADCC is very important for the activity of monoclonal antibodies, the present inventors first compared Burkitt's lymphoma cell line, Raji, with rituximab, which was used as a positive control and reference value in the present invention, as a standard monoclonal antibody for the treatment of lymphoma. and ADCC activity of MOR208 (anti-CD19 antibody) and MG4101 (NK cells) in Ramos.

First, Raji and Ramos cell lines (Burkitt's lymphomas, CD19 + CD20 +) were incubated for 2 hours with increasing concentrations of MOR208 or rituximab in the presence of activated and expanded liquid and frozen forms of MG4101 obtained from three healthy donors. cultured during (E/T ratio: 3).

The specific ADCC (%) was calculated by the following formula, and the results are shown in Figure 1 (the graph is the average ADCC (%) ± SD of 3 experimental values obtained from a representative of one NK cell donor of two different donors). represents).

(Sample Emission - Spontaneous Emission) / (Maximum Emission - Spontaneous Emission) x100.

As shown in FIGS. 1A to 1D , MOR208 exhibited significant in vitro ADCC activity against Raji and Ramos cell lines. Compared to the hIgG co-treatment control, both MOR208 and rituximab mediated the highest level of ADCC with a leftward shift in the curve and increased maximal lysis. Considering that MOR208 and rituximab show substantially higher levels on CD20 compared to CD19 as well as comparable maximal killing efficacy, various malignant B cells, including the Ramos cell line and the Raji cell line, show better ADCC activity at lower doses. , MOR208 was demonstrated to have an overall lower EC50 compared to rituximab. This teaches that MOR208 may be an interesting alternative treatment for conditions where rituximab is not effective.

Experimental Example 2. Lymphoma cell killing effect of MOR208 in xenograft SCID mice

Experimental Example 2.1. Establishment of the Ramos xenograft model in the SCID mouse model

Regarding MOR208 in vivo experiments, we established a xenograft SCID mouse model using Ramos cells to evaluate the suitability of the Ramos cell line. Groups of six SCID mice were intravenously injected with 1 X 10 ⁵ , 5 X 10 ⁵ , or 10 X 10 ⁵ Ramos cells and monitored daily for signs of disease. All controls died between 26 and 37 days after Ramos injection.

Referring to FIG. 2, intravenous injection of Ramos cells at different concentrations showed tumorigenic activity in mice characterized by symptoms including progressive weight loss and hind limb paralysis, and almost between 26 and 37 days after injection. 100% mice were killed. For further experiments below, the optimal dose of Ramos cell line was selected as 1 X 10 ⁶ cells.

Experimental Example 2.2. MOR208 capacity test in Ramos xenograft model

The efficacy of MOR208 on B lymphoma cell killing is of clinical importance for its potential to deplete primary malignant B cells in patients. We evaluated the anti-tumor activity of MOR208 in a disseminated lymphoma model in which Ramos cells were intravenously injected into SCID mice and MOR208 was administered as described in "Preparation" above. In this model, Ramos cells infiltrated all mouse organs, including the central nervous system and mimetic diseases such as disseminated Burkitt's lymphoma and acute lymphocytic leukemia. In this regard, a group of 10 SCID mice was intravenously injected with 1 X 10 ⁶ Ramos cells (day 0). On days 3, 6, 10, 13, 17 and 20, mice were treated with monoclonal antibody (20, 60 or 200 μg/100 μL/head) or hIgG as negative control (hIgG 200 μg/ 100 μL/head). and observed daily for signs of disease.

As shown in Figure 3, 200 μg of MOR208 treatment significantly prolonged the survival of mice compared to animals injected with the control monoclonal antibody (P <.001). It was confirmed that the median survival time of mice treated with 200 μg of MOR208 was 36 days compared to 28.5 days for mice treated with control antibody.

Experimental Example 2.3. MG4101 capacity test in Ramos xenograft model

SCID mice injected with Ramos cells were intravenously injected with in vitro expanded and activated MG4101 cells from healthy donors as described in “Preparation”. This MG4101 cell supplement aims to mimic the presence of tissue NK cells that will be present in the patient's organ. Specifically, a group of 10 SCID mice was intravenously injected with 1 X 10 ⁶ Ramos cells (day 0). On days 4, 7, 11, 14, 18, and 21, MG4101 (1, 2, 5 X 10 ⁷ /400 μL/head) or NK cell freezing medium alone was administered to mice, and signs of disease were observed daily. Observed.

As shown in FIG. 4 , compared to mice treated with the control group (freezing medium), the survival rate of mice administered with MG4101 at a dose of 5×10 ⁷ cells per mouse was significantly improved (P <.05). The median survival time of mice treated with 5 X 10 ⁷ cells of MG4101 was 33 days, and mice treated with the control freezing medium was 28 days.

Experimental Example 2.4. Confirmation of the effect of MG4101 + MOR208 combination administration in Ramos xenograft model

Mice injected with Ramos cells were randomly divided into several groups, and as described in "Preparation Example", on the third day after Ramos cell inoculation, two doses of MOR208, MG4101 alone, and two doses of MOR208 and MG4101 were administered. Treatment with freezing medium containing hIgG as a combination or control. Specifically, a group of 10 SCID mice was intravenously injected with 1 X 10 ⁶ Ramos cells (day 0). Mice were treated with monoclonal antibodies (60 or 200 μg/100 μL/head) on days 3, 6, 10, 13, 17 and 20. On days 4, 7, 11, 14, 18, and 21, MG4101 (concentration of 2 X 10 ⁷ /400 μL/head) was injected intravenously, and the mice were observed daily.

As shown in FIG. 5 , it was confirmed that the protective effect of mice treated with MOR208 was significantly improved compared to the control group injected with hIgG freezing medium. The MG4101 injection group also showed significant improvement compared to the control group. Median survival times for mice treated with 200 μg of MOR208 (P <.0001) and mice treated with 2 × 10 ⁷ cells of MG4101 (P <.001) were 45.5 and 45.5 days compared to 30.5 days for controls treated with freezing medium, respectively. increased to 38 days.

On the other hand, animals treated with the combination of MOR208 and MG4101 showed a synergistic increase in survival during the experimental period. Specifically, in a situation where 2 × 10 ⁷ cells of MG4101 were co-injected, the median survival times for mice treated with 60 μg and 200 μg of MOR208 increased to 52 and 62 days, respectively (P <.0001). A significant synergistic effect of the combined administration of MOR208 and MG4101 was also shown in the analysis of increased survival. In particular, the average increase in survival rate of MOR208 or MG4101 treatment was 11.5% (MOR208 60 μg), 49.2% (MOR208 200 μg), or 24.6% (2 × 10 ⁷ cells MG4101), respectively. On the other hand, co-treatment with MOR208 and MG4101 showed 70.5% (MOR208 60μg) and 103.3% (MOR208 200μg) increase in survival rates, respectively, in the MG4101 co-injection situation. These results confirm that co-treatment (combination) of MG4101 and MOR208 significantly prolongs survival of mice by a factor of two, especially in the case of co-injection of 60 μg of MOR208, compared to animals injected with either MOR208 or MG4101 individually.

That is, both the in vitro and in vivo data confirmed herein suggest that the anti-CD19 antibody and natural killer cell combination, that is, the combination therapy of the two components can be an effective treatment for human B lymphoma and leukemia, and various B cell malignancies. It also suggests the possibility of showing a significant therapeutic effect on

<110> GC Cell Corporation MORPHOSYS AG <120> PHARMACEUTICAL COMBINATIONS FOR TREATING TUMOR COMPRISING ANTI-CD19 ANTIBODY AND NATURAL KILLER CELL <130> PDPB204189d01d01 <160> 11 <170> KoPatentIn 3.0 <210> 1 <211> 5 <212> PRT <213> artificial sequence <220> <223> Artificial Sequence <400> 1 Ser Tyr Val Met His 1 5 <210> 2 <211> 6 <212> PRT <213> artificial sequence <220> <223> Artificial Sequence <400> 2 Asn Pro Tyr Asn Asp Gly 1 5 <210> 3 <211> 12 <212> PRT <213> artificial sequence <220> <223> Artificial Sequence <400> 3 Gly Thr Tyr Tyr Tyr Gly Thr Arg Val Phe Asp Tyr 1 5 10 <210> 4 <211> 16 <212> PRT <213> artificial sequence <220> <223> Artificial Sequence <400> 4 Arg Ser Ser Lys Ser Leu Gln Asn Val Asn Gly Asn Thr Tyr Leu Tyr 1 5 10 15 <210> 5 <211> 7 <212> PRT <213> artificial sequence <220> <223> Artificial Sequence <400> 5 Arg Met Ser Asn Leu Asn Ser 1 5 <210> 6 <211> 9 <212> PRT <213> artificial sequence <220> <223> Artificial Sequence <400> 6 Met Gln His Leu Glu Tyr Pro Ile Thr 1 5 <210> 7 <211> 556 <212> PRT <213> artificial sequence <220> <223> Artificial Sequence <400> 7 Met Pro Pro Pro Arg Leu Leu Phe Phe Leu Leu Phe Leu Thr Pro Met 1 5 10 15 Glu Val Arg Pro Glu Glu Pro Leu Val Val Lys Val Glu Glu Gly Asp 20 25 30 Asn Ala Val Leu Gln Cys Leu Lys Gly Thr Ser Asp Gly Pro Thr Gln 35 40 45 Gln Leu Thr Trp Ser Arg Glu Ser Pro Leu Lys Pro Phe Leu Lys Leu 50 55 60 Ser Leu Gly Leu Pro Gly Leu Gly Ile His Met Arg Pro Leu Ala Ile 65 70 75 80 Trp Leu Phe Ile Phe Asn Val Ser Gln Gln Met Gly Gly Phe Tyr Leu 85 90 95 Cys Gln Pro Gly Pro Pro Ser Glu Lys Ala Trp Gln Pro Gly Trp Thr 100 105 110 Val Asn Val Glu Gly Ser Gly Glu Leu Phe Arg Trp Asn Val Ser Asp 115 120 125 Leu Gly Gly Leu Gly Cys Gly Leu Lys Asn Arg Ser Ser Glu Gly Pro 130 135 140 Ser Ser Pro Ser Gly Lys Leu Met Ser Pro Lys Leu Tyr Val Trp Ala 145 150 155 160 Lys Asp Arg Pro Glu Ile Trp Glu Gly Glu Pro Pro Cys Leu Pro Pro 165 170 175 Arg Asp Ser Leu Asn Gln Ser Leu Ser Gln Asp Leu Thr Met Ala Pro 180 185 190 Gly Ser Thr Leu Trp Leu Ser Cys Gly Val Pro Pro Asp Ser Val Ser 195 200 205 Arg Gly Pro Leu Ser Trp Thr His Val His Pro Lys Gly Pro Lys Ser 210 215 220 Leu Leu Ser Leu Glu Leu Lys Asp Asp Arg Pro Ala Arg Asp Met Trp 225 230 235 240 Val Met Glu Thr Gly Leu Leu Leu Pro Arg Ala Thr Ala Gln Asp Ala 245 250 255 Gly Lys Tyr Tyr Cys His Arg Gly Asn Leu Thr Met Ser Phe His Leu 260 265 270 Glu Ile Thr Ala Arg Pro Val Leu Trp His Trp Leu Leu Arg Thr Gly 275 280 285 Gly Trp Lys Val Ser Ala Val Thr Leu Ala Tyr Leu Ile Phe Cys Leu 290 295 300 Cys Ser Leu Val Gly Ile Leu His Leu Gln Arg Ala Leu Val Leu Arg 305 310 315 320 Arg Lys Arg Lys Arg Met Thr Asp Pro Thr Arg Arg Phe Phe Lys Val 325 330 335 Thr Pro Pro Pro Gly Ser Gly Pro Gln Asn Gln Tyr Gly Asn Val Leu 340 345 350 Ser Leu Pro Thr Pro Thr Ser Gly Leu Gly Arg Ala Gln Arg Trp Ala 355 360 365 Ala Gly Leu Gly Gly Thr Ala Pro Ser Tyr Gly Asn Pro Ser Ser Asp 370 375 380 Val Gln Ala Asp Gly Ala Leu Gly Ser Arg Ser Pro Pro Gly Val Gly 385 390 395 400 Pro Glu Glu Glu Glu Gly Glu Gly Tyr Glu Glu Pro Asp Ser Glu Glu 405 410 415 Asp Ser Glu Phe Tyr Glu Asn Asp Ser Asn Leu Gly Gln Asp Gln Leu 420 425 430 Ser Gln Asp Gly Ser Gly Tyr Glu Asn Pro Glu Asp Glu Pro Leu Gly 435 440 445 Pro Glu Asp Glu Asp Ser Phe Ser Asn Ala Glu Ser Tyr Glu Asn Glu 450 455 460 Asp Glu Glu Leu Thr Gln Pro Val Ala Arg Thr Met Asp Phe Leu Ser 465 470 475 480 Pro His Gly Ser Ala Trp Asp Pro Ser Arg Glu Ala Thr Ser Leu Gly 485 490 495 Ser Gln Ser Tyr Glu Asp Met Arg Gly Ile Leu Tyr Ala Ala Pro Gln 500 505 510 Leu Arg Ser Ile Arg Gly Gln Pro Gly Pro Asn His Glu Glu Asp Ala 515 520 525 Asp Ser Tyr Glu Asn Met Asp Asn Pro Asp Gly Pro Asp Pro Ala Trp 530 535 540 Gly Gly Gly Gly Arg Met Gly Thr Trp Ser Thr Arg 545 550 555 <210> 8 <211> 121 <212> PRT <213> artificial sequence <220> <223> Artificial Sequence <400> 8 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Val Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Ser Ser Asp Lys Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Gly Thr Tyr Tyr Tyr Gly Thr Arg Val Phe Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 9 <211> 112 <212> PRT <213> artificial sequence <220> <223> Artificial Sequence <400> 9 Asp Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ser Ser Lys Ser Leu Gln Asn Val 20 25 30 Asn Gly Asn Thr Tyr Leu Tyr Trp Phe Gln Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Arg Met Ser Asn Leu Asn Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile 65 70 75 80 Ser Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Met Gln His 85 90 95 Leu Glu Tyr Pro Ile Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 10 <211> 329 <212> PRT <213> artificial sequence <220> <223> Artificial Sequence <400> 10 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Trp Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr 65 70 75 80 Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys 85 90 95 Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro 100 105 110 Ala Pro Glu Leu Leu Gly Gly Pro Asp Val Phe Leu Phe Pro Pro Lys 115 120 125 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 130 135 140 Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr 145 150 155 160 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 165 170 175 Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His 180 185 190 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 195 200 205 Ala Leu Pro Ala Pro Glu Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln 210 215 220 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met 225 230 235 240 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 245 250 255 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 260 265 270 Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu 275 280 285 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 290 295 300 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 305 310 315 320 Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 <210> 11 <211> 107 <212> PRT <213> artificial sequence <220> <223> Artificial Sequence <400> 11 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105

Claims (15)

A pharmaceutical combination for the treatment of cancer, comprising an antibody specific to CD19 and natural killer cells (NK cells).
According to claim 1,
The antibody comprises a heavy chain variable region comprising a HCDR1 region of SEQ ID NO: 1 (SYVMH), a HCDR2 region of SEQ ID NO: 2 (NPYNDG), and a HCDR3 region of SEQ ID NO: 3 (GTYYYGTRVFDY). Pharmaceutical combination.
According to claim 1,
The antibody comprises a light chain variable region comprising an LCDR1 region of SEQ ID NO: 4 (RSSKSLQNVNGNTYLY), a LCDR2 region of SEQ ID NO: 5 (RMSNLNS), and a LCDR3 region of SEQ ID NO: 6 (MQHLEYPIT) Pharmaceutical combination.
According to claim 1,
The antibody has a heavy chain variable region of SEQ ID NO: 8 (EVQLVESGGGLVKPGGSLKLSCAASGYTFTSYVMHWVRQAPGKGLEWIGYINPYNDGTKYNEKFQGRVTISSDKSISTAYMELSSLRSEDTAMYYCARGTYYYGTRVFDYWGQGTLVTVSS) and SEQ ID NO: 9 (DIVMTQSPATLSLSPGERATLSCRSSKSLQNVNGNTYLYWFQQKPG A pharmaceutical combination comprising the light chain variable region of QSPQLLIYRMSNLNSGVPDRFSGSGSGTEFTLTISSLEPEDFAVYYCMQHLEYPITFGAGTKLEIK).
According to claim 1,
The antibody has SEQ ID NO: 10 (ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPR EEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKALPAPEEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK).
According to claim 1,
wherein the antibody comprises the light chain constant region of SEQ ID NO: 11 (RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC).
According to any one of claims 1 to 6,
The pharmaceutical combination, wherein the NK cells are prepared by a method comprising the following steps (i) to (iii):
(i) stimulating cell-to-cell contact by statically culturing the isolated NK cells in a medium containing anti-CD3 antibody, cytokines and feeder cells;
(ii) restimulation by adding cytokine, anti-CD3 antibody, and feeder cells, followed by static culture to induce cell-to-cell contact; and
(iii) adding a medium containing cytokines to the cells, and performing still or suspension culture while maintaining constant cell concentration and cytokine concentration.
According to any one of claims 1 to 6,
The NK cells are prepared by a method comprising the step of co-cultivating CD4(+) T cells isolated in vitro as feeder cells or CD4(+) T cells cultured in vitro and seed cells that is, a pharmaceutical combination.
The pharmaceutical combination according to claim 8, wherein the CD4(+) T cells are a CD4(+)/CD1(-) T cell line.
The pharmaceutical combination according to claim 9, wherein the CD4(+)/CD1(-) T cell line is an H9 or HuT78 cell line.
The method of claim 8, wherein the source cells are peripheral blood, peripheral blood leukocyte cells, PBMC (peripheral blood mononuclear cells), cord blood leukocyte cells, cord blood mononuclear cells, natural killer cells derived from stem cells, and enriched natural killer cells. A pharmaceutical combination of at least one selected from the group consisting of cells and isolated natural killer cells.
According to claim 1,
The cancers include gastric cancer, liver cancer, lung cancer, colon cancer, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, cervical cancer, thyroid cancer, laryngeal cancer, acute myelogenous leukemia, brain tumor, neuroblastoma, retinoblastoma, head and neck cancer, salivary gland cancer, lymphoma, and B A pharmaceutical combination, which is at least one selected from the group consisting of cell-origin malignancies.
According to claim 1,
A pharmaceutical combination wherein the CD19-specific antibody and NK cells are administered separately.
According to claim 1,
The pharmaceutical combination, wherein the CD19-specific antibody and NK cells are administered simultaneously.
A kit for the treatment of cancer comprising a combination according to any one of claims 1 to 6.

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