Cell therapeutic agent with improved in-vivo persistence and antitumor effect, and method for preparing same

Abstract

The present invention relates to a cell therapeutic agent with improved proliferation rate, in-vivo persistence, and antitumor effect, and a method for preparing same. Conventional γδ T cell therapeutic agents have failed to overcome immunosuppression, and thus cannot exhibit significant therapeutic effects due to short in-vivo persistence and reduced antitumor immune responses. However, a specific culture method or γδ T cells produced by genetic engineering, of the present invention, exhibit significantly improved proliferation rate, in-vivo persistence, and antitumor effect, and thus, are expected to greatly improve the survival rate of subjects with cancer when used alone or in combination with a conventional cancer immunotherapeutic agent.

Description

Cell therapy agent with improved in vivo persistence and antitumor effect, and method for manufacturing the same

[0001] The present invention relates to a γδ T cell therapeutic agent with improved proliferation rate, persistence in vivo, and antitumor effect, and a method for manufacturing the same.

[0002] Cancer is one of the most common causes of death worldwide. Approximately 10 million new cases occur annually, accounting for about 12% of all deaths and making it the third leading cause of death. Consequently, while continuous efforts have been made to develop effective anticancer drugs, the development of effective treatments remains very insufficient for brain cancer, particularly glioblastoma, to the extent that the survival rate is still below 10%. Even cancer immunotherapy, which utilizes the body's immune system to attack cancer cells and is touted as having opened new horizons in cancer treatment, is not effective in glioblastoma. This is because immunosuppressive mechanisms that inhibit immune cells are highly active in brain cancer, and the presence of the Blood-Brain Barrier, a characteristic of the brain, makes it difficult to activate the immune system and facilitate treatment. In fact, PD-1 inhibitors, one of the representative immune checkpoint blockers, recently failed in Phase 3 clinical trials for brain cancer treatment, and the development of CAR T-cell therapies is also facing difficulties.

[0003] Meanwhile, γδ T cells are important cells associated with a good prognosis in brain tumor patients, and unlike existing personalized cell therapies, they have the significant advantage of being allogeneic transplantable; therefore, attempts have been made to apply them for anticancer purposes as off-the-shelf cell therapies. However, existing γδ T cell therapies have failed to overcome immunosuppression, resulting in short in vivo persistence and reduced anti-tumor immune response, which have prevented significant therapeutic effects. Therefore, in order to actively utilize γδ T cells as a treatment for intractable brain cancer, there was a challenge to improve the in vivo persistence and anti-tumor immune response of γδ T cells.

[0004] The present invention is designed to solve the above-mentioned problems and relates to a γδ T cell therapy agent with improved proliferation rate, in vivo persistence, and antitumor effect, and a method for manufacturing the same. The researchers of the present invention have succeeded in producing γδ T cells with significantly improved proliferation rate, in vivo persistence, and antitumor effect by culturing γδ T cells in a special way or by genetically modifying them. Since the γδ T cells of the present invention are expected to significantly improve the survival rate of cancer subjects when used alone or in combination with conventional immunotherapies, they may be widely utilized in the health and medical fields.

[0005] One objective of the present invention is to provide a cell therapy agent for the prevention or treatment of cancer.

[0006] Another objective of the present invention is to provide a method for manufacturing the cell therapy agent.

[0007] Another objective of the present invention is to provide a pharmaceutical composition for the prevention or treatment of cancer comprising the cell therapy agent.

[0008] The cell therapy agent of the present invention is characterized by increased expression of Batf3 (Basic Leucine Zipper ATF-Like Transcription Factor 3), which improves the proliferation rate, increases persistence in the body, or enhances the anti-tumor effect.

[0009] However, the technical problems that the present invention aims to solve are not limited to those mentioned above, and other unmentioned problems will be clearly understood by those skilled in the art from the description below.

[0010] Various embodiments described herein are described with reference to the drawings. In the following description, various specific details, such as specific forms, compositions, and processes, are described for a complete understanding of the invention. However, specific embodiments may be practiced without one or more of these specific details, or in combination with other known methods and forms. In other examples, known processes and manufacturing techniques are not described as specific details so as not to unnecessarily obscure the invention. Reference throughout this specification to "one embodiment" or "an embodiment" implies that the particular features, forms, compositions, or characteristics described in association with the embodiment are included in one or more embodiments of the invention. Accordingly, the context of "in one embodiment" or "an embodiment" expressed at various locations throughout this specification does not necessarily represent the same embodiment of the invention. Additionally, particular features, forms, compositions, or characteristics may be combined in any suitable manner in one or more embodiments.

[0011] Unless otherwise specifically defined in the specification, all scientific and technical terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention pertains.

[0012] In the present invention, "γδ T cells" refers to a type of T cell that expresses a gamma-delta (γδ) T cell antigen receptor (TCR). T cells, a type of lymphocyte, are a key component of adaptive immunity along with B cells. T cells are generated through a process in which precursors produced from hematopoietic stem cells mature in the thymus. T cells consist of a majority of αβ T cells expressing alpha-beta (αβ) T cell receptors that circulate in the thymus and peripheral lymphoid tissues, and a small number of γδ T cells expressing gamma-delta (γδ) T cell receptors. γδ T cells are the first T cells produced in the thymus to migrate to other parts of the body, mostly moving to the skin, intestines, lungs, and epithelial tissues prior to birth. Although γδ T cells do not possess diversity in T-cell receptors, they recognize and respond to various biological damages inflicted on epithelial tissues; however, little is known about the autoantigens that induce these responses. γδ T cells exist in human peripheral blood at a very low rate of approximately 0.5–5% and are functionally involved in both adaptive and innate immune responses. Human γδ T cells exist in two major subsets, with TCR chains existing as either Vδ1 or Vδ2; however, most γδ T cells in the blood possess the Vδ2 chain and exist in pairs with the Vγ9 chain. γδ T cells develop in the thymus before αβ T cells and possess limited TCR variability against several antigens, including phosphoantigens; however, they trigger an immune response more rapidly than αβ T cells, inducing an innate-like immune response.γδ T cells, like αβ T cells, possess a more potent anticancer effect through cytolysis; however, unlike αβ T cells, which are the primary cause of graft-versus-host disease (GVHD) in cells expressing non-self MHC, γδ T cells are known to have no MHC restriction and do not cause GVHD. This implies that γδ T cells can serve as a promising off-the-shelf cell therapy for anticancer immunotherapy. Therefore, various technologies have been devised to secure a sufficient number of γδ T cells as an off-the-shelf cell therapy. For example, it has been revealed that γδ T cells specifically proliferate when treated with Interleukin-2 (IL-2), Interleukin-5 (IL-5), Zoledronic acid (ZA, ZOL), PHA (phytohaemagglutinin), Transforming growth factor beta (TGF-β), Concanavalin A, and Amphotericin B. Consequently, numerous preclinical and clinical trials have been attempted to apply γδ T cells obtained by these methods as therapeutic agents for various cancers. However, it has been confirmed that γδ T cells proliferated in vitro die within a few days when transplanted into an individual. Therefore, maintaining the persistence of γδ T cells in vivo and, furthermore, improving the rate of proliferation within the body remains a technical challenge.

[0013] Accordingly, the present invention provides a γδ T cell therapeutic agent that significantly improves proliferation rate, in vivo persistence, and antitumor effect by increasing the expression of Batf3 (Basic Leucine Zipper ATF-Like Transcription Factor 3) in γδ T cells as a solution to the above technical problem.

[0014] In the present invention, "cell therapy" refers to a pharmaceutical product (according to U.S. FDA regulations) used for the purposes of treatment, diagnosis, and prevention, consisting of cells and tissues produced through isolation, culture, and special manipulation from an individual. It refers to a pharmaceutical product used for the purposes of treatment, diagnosis, and prevention through a series of actions such as proliferating / selecting living autologous, allogenic, or xenogenic cells in vitro to restore the function of cells and tissues, or altering the biological characteristics of cells by other methods. The United States has managed cell therapy products as pharmaceutical products since 1993, and Korea since 2002. Such cell therapy products can be broadly classified into two categories: the first is stem cell therapy for tissue regeneration or organ function recovery, and the second is immunocell therapy for regulating immune responses, such as suppressing or enhancing immune responses within the body. In the present invention, the cell therapy agent is an immune cell therapy agent derived from γδ T cells, which are immune cells, and specifically, is characterized by being a γδ T cell with increased expression of Batf3 (Basic Leucine Zipper ATF-Like Transcription Factor 3). The cell therapy agent of the present invention can be usefully utilized for the treatment of cancer, particularly brain cancer, and a synergistic effect can be expected by using it in combination with conventional anticancer agents, immunotherapies, or immune checkpoint inhibitors.

[0015] In this invention, "brain cancer" is used interchangeably with "brain tumor" and refers to tumors formed within the skull, broadly encompassing tumors within the brain or spinal canal, as well as tumors occurring in the cerebral tissue and meninges. It encompasses tumors grown from brain cells, as well as all neoplasms that originate from blood vessels, nerves, meninges, and develop within the skull. Brain cancer possesses characteristics distinct from general cancer, stemming from the fact that the brain has a tissue structure completely different from other organs. Specifically, the brain's blood vessels feature a tight boundary known as the "Blood-Brain Barrier (BBB)," which prevents tumors from easily metastasizing to other organs via the blood vessels even if they develop within the brain. This also contributes to the difficulty of anticancer drugs being effectively delivered to brain cancer. Furthermore, unlike other cancers that are typically classified by stage, brain cancer is classified by grade. The grades are determined based on factors such as the rate of tumor cell division; generally, Grade 1 is considered benign, Grade 2 is borderline, and Grades 3–4 are malignant. However, glioblastoma may be classified as clinically malignant depending on the case, even if it is grade 1 or 2. The brain cancer mentioned above may be at least one selected from the group consisting of glioblastoma, malignant glioma, lymphoma, germ cell tumor, and metastatic tumor, but is not limited thereto.

[0016] In the present invention, "immune checkpoint inhibitor" refers to a drug that activates T cells to attack cancer cells by blocking the activity of immune checkpoint proteins involved in T cell suppression. That is, cancer cells have an evasion mechanism to avoid attack by expressing self-tolerance proteins that evade attacks by immune cells, thereby causing them to be recognized as themselves; the drug suppresses immune response evasion signals, thereby causing immune cells to attack cancer cells and enabling the elimination of cancer cells. Novel drugs developed to neutralize the immune system evasion response of cancer cells typically utilize substances that specifically bind to proteins by recognizing sites such as CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, TIM-3, BTLA, or KIR as targets. Here, the term "substance" in the above context refers to a broad concept including antibodies, but is not limited thereto. In the present invention, the immune checkpoint inhibitor may comprise at least one selected from the group consisting of CTLA-4 receptor inhibitors, PD-1 receptor inhibitors, PD-L1 ligand inhibitors, PD-L2 ligand inhibitors, LAG-3 receptor inhibitors, TIM-3 receptor inhibitors, BTLA receptor inhibitors, and KIR receptor inhibitors. Although the immune checkpoint inhibitor is a fourth-generation anticancer agent utilizing the immune system of the individual with cancer and has reported successful treatment results in various types of cancer, it has struggled specifically against brain cancer. Recently, there have been several clinical attempts to introduce immune checkpoint inhibitors for the treatment of glioblastoma, but they have not yet achieved significant results compared to existing treatments. Therefore, the treatment of brain tumors using such immune checkpoint inhibitors requires an additional breakthrough.

[0017] In the present invention, "prevention" may include, without limitation, any act of suppressing the occurrence of cancer itself, blocking symptoms caused by cancer, or suppressing or delaying such symptoms using a composition containing the active ingredient of the present invention.

[0018] In the present invention, "treatment" or "improvement" may be included without limitation as long as it is any act that improves or benefits symptoms caused by the occurrence or invasion of cancer by using a composition containing the active ingredient of the present invention.

[0019] Unlike anticancer agents that generally affect cancer cells directly, the pharmaceutical composition of the present invention is a therapeutic agent that enhances autoimmunity through the activation of the human immune system, thereby enabling the immune cells inherent to the human body to attack cancer cells. For the purposes of the present invention, the pharmaceutical composition refers to a therapeutic agent capable of enhancing the function and activity of T cells present in a cancerous individual.The pharmaceutical composition of the present invention may be additionally used in combination with a conventional anticancer agent other than an immune checkpoint inhibitor or an immunotherapeutic agent, wherein the conventional anticancer agent is a chemical anticancer agent such as nitrogen mustard, imatinib, oxaliplatin, rituximab, erlotinib, neratinib, lapatinib, gefitinib, vandetanib, nirotinib, cemasanib, bosutinib, axitinib, cediranib, restaurtinib, trastuzumab, gefitinib, bortezomib, sunitinib, carboplatin, sorafenib, bevacizumab, cisplatin, cetuximab, viscolumabum, asparaginase, tretinoin, hydroxycarbamide, dasatinib, estramustine, gemtuzumab ozogamicin, ibritumomab tussetane, heptaplatin, methylaminolevulinic acid, amsacrin, Alemtuzumab, Procarbazine, Alprostadil, Holmium Nitrate Chitosan, Gemcitabine, Doxifluridine, Pemetrexed, Tegafur, Capecitabine, Gimeracin, Oteracil, Azacitidine, Methotrexate, Uracil, Cytarabine, Fluorouracil, Fludabin, Enositabine, Flutamide, Capecitabine, Decitabine, Mercaptopurine, Thioguanine, Cladribine, Carmoper, Raltitrexed, Docetaxel, Paclitaxel, Irinotecan, Belotecan, Topotecan, Vinorelbine, Etoposide, Vinblastine, Idarubicin, Mitomycin, Bleromycin, Dactinomycin, Pirarubicin, Aclarubicin, Pepromycin, Temsirolimus, Temozolomide, Busulfan, It may comprise at least one selected from the group consisting of ifosfamide, cyclophosphamide, melphalan, altretmin, dacarbazine, thiotepa, nimustine, chlorambucil, mitolactol, leucovorin, tretonin, exemestane, aminoglutesimide, anagrelide, olaparib, navelbine, padrazol, tamoxifen, toremifene, testolactone, anastrozole, letrozole, borozol, bicalutamide, lomustine, 5FU, vorinostat, entinostet, and carmustine. The immunotherapeutic agent may be at least one selected from the group consisting of pembrolizumab, ipilimumab, nivolumab, atezolizumab, and durvalumab, but is not limited thereto.

[0020] The pharmaceutical composition of the present invention may be manufactured in the form of an injection, capsule, tablet, granule, powder, or beverage, and may be administered to humans.

[0021] In the present invention, the composition is most preferably in the form of an injectable formulation, but may be formulated and used in the form of oral formulations such as powders, granules, capsules, tablets, and aqueous suspensions, as well as topical preparations, suppositories, and sterile injectable solutions according to other conventional methods. The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier. For oral administration, the pharmaceutically acceptable carrier may use a binder, lubricant, disintegrant, excipient, solubilizer, dispersant, stabilizer, suspending agent, colorant, flavoring agent, etc.; for injectables, it may use a mixture of a buffer, preservative, analgesic, solubilizer, isotonic agent, stabilizer, etc.; and for topical administration, a base, excipient, lubricant, preservative, etc. Examples of carriers, excipients, and diluents suitable for the formulation of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, or mineral oil. Additionally, fillers, anticoagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives, etc. may be further included.

[0022] When the composition of the present invention is used for therapeutic purposes, it may vary depending on various factors including the age, weight, general health, gender, diet, time of administration, route of administration, elimination rate, drug combination, and the severity of the specific disease to be prevented or treated. The dosage of the pharmaceutical composition may vary depending on the patient's condition, weight, degree of disease, form of medication, route of administration, and duration, but can be appropriately selected by a person skilled in the art. The administration may be performed once a day or divided into several doses. The dosage does not limit the scope of the present invention in any way.

[0023] The cell therapy agent of the present invention, intended to enhance cancer therapeutic activity, may be administered by any device capable of moving to target cells or target tissues, and may be included in an effective amount for the treatment of a target disease. The therapeutically effective amount refers to the amount of an active ingredient or pharmaceutical composition that induces a biological or medical response in a tissue system, animal, or human as conceived by a researcher, veterinarian, physician, or other clinician, and includes an amount that induces the alleviation of symptoms of the disease or disorder being treated. It is obvious to those skilled in the art that the cell therapy agent included in the composition of the present invention will vary according to the desired effect. Therefore, the optimal cell therapy agent content can be easily determined by those skilled in the art and may be adjusted according to various factors including the type of disease, the severity of the disease, the content of other ingredients contained in the composition, the type of formulation, and the patient's age, weight, general health condition, gender, diet, time of administration, route of administration and secretion rate of the composition, duration of treatment, and concurrently used drugs. Considering all of the above factors, an amount that can obtain maximum effect with a minimum amount without side effects may be included.

[0024] As one embodiment of the present invention, the present invention provides a method for producing a γδ T cell therapeutic agent with increased proliferation rate or persistence in vivo, comprising the step of stimulating the γδ TCR (T cell antigen receptor) of γδ T cells isolated from an individual. The method of producing the present invention may further comprise, following the step of stimulating the γδ TCR (T cell antigen receptor) of γδ T cells isolated from an individual, the step of re-stimulating the γδ TCR of the γδ T cells; or the step of overexpressing Batf3 (Basic Leucine Zipper ATF-Like Transcription Factor 3). At this time, the γδ TCR stimulation or γδ TCR restimulation may be performed by treating with a composition containing an anti-γδ TCR antibody, and the composition containing the anti-γδ TCR antibody may further comprise one or more selected from the group consisting of an anti-CD28 antibody, Interleukin-2, Interleukin-5, Zoledronic acid, PHA (phytohaemagglutinin), Transforming growth factor beta, Concanavalin A, and Amphotericin B, and the step of restimulating the γδ TCR of the γδ T cell; is performed 3 to 7 days after, 4 to 6 days after, 3 to 6 days after, 4 to 7 days after, or 3 to 7 days after, from the step of stimulating the γδ TCR (T cell antigen receptor) of the γδ T cell isolated from the individual, or It may be performed after 4.5 to 5.5 days, preferably after 5 days, but is not limited thereto.In addition, the step of overexpressing the above Batf3 (Basic Leucine Zipper ATF-Like Transcription Factor 3) may be performed 3 to 72 hours, 3 to 60 hours, 3 to 48 hours, 3 to 24 hours, 6 to 72 hours, 12 to 72 hours, 24 to 72 hours, 48 ​​to 72 hours, 6 to 60 hours, 9 to 48 hours, 12 to 36 hours, 18 to 30 hours after the step of stimulating the γδ TCR (T cell antigen receptor) of γδ T cells isolated from an individual; and preferably may be performed 20 to 28 hours after, but is not limited thereto. In addition, the step of overexpressing Batf3 (Basic Leucine Zipper ATF-Like Transcription Factor 3) in the manufacturing method of the present invention may involve transforming into a vector containing a PGK promoter (phosphoglycerate kinase 1 promoter) and a Batf3 gene, but is not limited thereto, and it will be obvious that any method known to those skilled in the art to which the present invention belongs to overexpress Batf3 can be used without limitation.

[0025] In another aspect of the present invention, the present invention provides a method for improving the proliferation rate or persistence in vivo of γδ T cells, comprising the step of stimulating the γδ TCR (T cell antigen receptor) of γδ T cells isolated from an individual. The method of the present invention may further comprise, following the step of stimulating the γδ TCR (T cell antigen receptor) of γδ T cells isolated from an individual, the step of restimulating the γδ TCR of the γδ T cells; or the step of overexpressing Batf3 (Basic Leucine Zipper ATF-Like Transcription Factor 3). At this time, the γδ TCR stimulation or γδ TCR restimulation may be performed by treating with a composition containing an anti-γδ TCR antibody, and the composition containing the anti-γδ TCR antibody may further comprise one or more selected from the group consisting of an anti-CD28 antibody, Interleukin-2, Interleukin-5, Zoledronic acid, PHA (phytohaemagglutinin), Transforming growth factor beta, Concanavalin A, and Amphotericin B, and the step of restimulating the γδ TCR of the γδ T cell; is performed 3 to 7 days after, 4 to 6 days after, 3 to 6 days after, 4 to 7 days after, or 3 to 7 days after, from the step of stimulating the γδ TCR (T cell antigen receptor) of the γδ T cell isolated from the individual, or It may be performed after 4.5 to 5.5 days, preferably after 5 days, but is not limited thereto.In addition, the step of overexpressing the above Batf3 (Basic Leucine Zipper ATF-Like Transcription Factor 3) may be performed 3 to 72 hours, 3 to 60 hours, 3 to 48 hours, 3 to 24 hours, 6 to 72 hours, 12 to 72 hours, 24 to 72 hours, 48 ​​to 72 hours, 6 to 60 hours, 9 to 48 hours, 12 to 36 hours, 18 to 30 hours after the step of stimulating the γδ TCR (T cell antigen receptor) of γδ T cells isolated from an individual; and preferably may be performed 20 to 28 hours after, but is not limited thereto. In addition, the step of overexpressing Batf3 (Basic Leucine Zipper ATF-Like Transcription Factor 3) in the method of the present invention may involve transforming into a vector containing a PGK promoter (phosphoglycerate kinase 1 promoter) and the Batf3 gene, but is not limited thereto, and it will be obvious that any method known to those skilled in the art to which the present invention belongs to overexpress Batf3 may be used without limitation.

[0026] In another aspect of the present invention, the present invention provides a γδ T cell therapy product prepared by the method for preparing a γδ T cell therapy product having increased proliferation rate or persistence in the body as described above. The γδ T cell therapy product may be used for the treatment of cancer, and the cancer may be one or more selected from the group consisting of brain cancer, breast cancer, cervical cancer, melanoma, lung cancer, bladder cancer, prostate cancer, leukemia, kidney cancer, liver cancer, colorectal cancer, pancreatic cancer, stomach cancer, gallbladder cancer, ovarian cancer, lymphoma, osteosarcoma, uterine cancer, oral cancer, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, skin cancer, blood cancer, thyroid cancer, parathyroid cancer, ureteral cancer, adenocarcinoma, and thymic cancer, and preferably may be brain cancer, but is not limited thereto.

[0027] In another aspect of the present invention, the present invention provides a pharmaceutical composition for the prevention or treatment of cancer comprising, as an active ingredient, a γδ T cell therapy prepared by the method for preparing a γδ T cell therapy with increased proliferation rate or persistence in the body described above. The cancer may be one or more selected from the group consisting of brain cancer, breast cancer, cervical cancer, melanoma, lung cancer, bladder cancer, prostate cancer, leukemia, kidney cancer, liver cancer, colorectal cancer, pancreatic cancer, stomach cancer, gallbladder cancer, ovarian cancer, lymphoma, osteosarcoma, uterine cancer, oral cancer, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, skin cancer, blood cancer, thyroid cancer, parathyroid cancer, ureteral cancer, adenocarcinoma, and thymic cancer, and preferably may be brain cancer, but is not limited thereto. In addition, the pharmaceutical composition of the present invention may further include a chemical anticancer agent, an immunotherapeutic agent, or an immune checkpoint inhibitor as a conventional anticancer agent, or may be used in combination with a chemical anticancer agent, an immunotherapeutic agent, or an immune checkpoint inhibitor. At this time, the above chemical anticancer drugs are nitrogen mustard, imatinib, oxaliplatin, rituximab, erlotinib, neratinib, lapatinib, gefitinib, vandetanib, nirotinib, cemasanib, bosutinib, axitinib, cediranib, restaurtinib, trastuzumab, gefitinib, bortezomib, sunitinib, carboplatin, sorafenib, bevacizumab, cisplatin, cetuximab, viscolumabum, asparaginase, tretinoin, hydroxycarbamide, dasatinib, estramustine, gemtuzumab ozogamicin, ibritumomab tussetane, heptaplatin, methylaminolevulinic acid, amsacrin, alemtuzumab, procarbazine, alprostadil, holmium nitrate chitosan, gemcitabine, doxifluridine, Pemetrexed, Tegafur, Capecitabine, Gimeracin, Oteracil, Azacitidine, Methotrexate, Uracil, Cytarabine, Fluorouracil, Fludabin, Enositabine, Flutamide, Capecitabine, Decitabine, Mercaptopurine, Thioguanine, Cladribine, Carmoper, Raltitrexed, Docetaxel, Paclitaxel, Irinotecan, Belotecan, Topotecan, Vinorelbine, Etoposide, Vinblastine, Idarubicin, Mitomycin,The immunotherapeutic agent may be one or more selected from the group consisting of bleromycin, dactinomycin, pirarubicin, aclarubicin, pepromycin, temsirolimus, temozolomide, busulfan, ifosfamide, cyclophosphamide, melphalan, altretmin, dacarbazine, thiotepa, nimustine, chlorambucil, mitolactol, leucovorin, tretonin, exemestane, aminoglutesimide, anagrelide, olaparib, navelbine, padrazol, tamoxifen, toremifene, testolactone, anastrozole, letrozole, vorozol, bicalutamide, lomustine, 5FU, vorinostat, entinostet, and carmustine, and the immunotherapeutic agent may consist of pembrolizumab, ipilimumab, nivolumab, atezolizumab, and durvalumab. It may be one or more selected from the group, but is not limited thereto; it will be obvious to those skilled in the art to which the present invention pertains that any function as a chemical anticancer agent or an immunotherapeutic agent is known to them without limitation. Due to the characteristics of the present invention, the pharmaceutical composition of the present invention has excellent efficacy in overcoming immunosuppression; therefore, when used together with immune checkpoint inhibitors, a significant synergistic effect for cancer treatment is expected. The above immune checkpoint inhibitor may be one or more selected from the group consisting of PD-1 receptor inhibitors, PD-L1 ligand inhibitors, PD-L2 ligand inhibitors, CTLA-4 receptor inhibitors, LAG-3 receptor inhibitors, TIM-3 receptor inhibitors, BTLA receptor inhibitors, and KIR receptor inhibitors, and may specifically bind to at least one protein selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4, LAG-3, TIM-3, BTLA, and KIR, but is not limited thereto, and it will be obvious to those skilled in the art to which the present invention pertains that any agent with a function as an immune checkpoint inhibitor may be used without limitation.

[0028] In another aspect of the present invention, the present invention provides a method for preventing or treating cancer by administering to an individual a γδ T cell therapy product prepared by the method for preparing a γδ T cell therapy product with increased proliferation rate or persistence in the body described above.

[0029] In another aspect of the present invention, the present invention provides a use for cancer prevention or treatment of a γδ T cell therapy produced by the method for producing a γδ T cell therapy with increased proliferation rate or persistence in the body described above.

[0030] The γδ T cells produced by the method of the present invention have significantly improved proliferation rate, persistence in the body, and antitumor effect, and are expected to greatly improve the survival rate of cancer subjects when used alone or in combination with conventional immunotherapies.

[0031] Figure 1 is the result of comparing the cell proliferation rates of restimulated γδ T cells and a control group according to one embodiment of the present invention.

[0032] Figure 2 is a result of comparing the cell viability of restimulated γδ T cells and a control group according to one embodiment of the present invention.

[0033] Figure 3 is a result comparing the persistence of cells according to a mixed culture of restimulated γδ T cells and a control group, according to one embodiment of the present invention.

[0034] Figure 4 is the result of confirming the mRNA change of restimulated γδ T cells by Bulk RNA sequencing according to one embodiment of the present invention.

[0035] Figure 5 is the result of confirming whether the persistence of cells decreased following mixed culture of restimulated Batf3 knockout γδ T cells according to one embodiment of the present invention.

[0036] Figure 6 is the result of confirming whether the persistence of restimulated Batf3 knockout γδ T cells in vivo is reduced according to one embodiment of the present invention.

[0037] Figure 7 is the result of confirming the change in the mRNA expression level of Batf3 over time in restimulated γδ T cells according to one embodiment of the present invention.

[0038] FIG. 8 shows a plasmid map for producing Batf3 overexpressing γδ T cells according to one embodiment of the present invention.

[0039] Figure 9 is the result of confirming whether the Batf3 expression rate in γδ T cells is sustained following Batf3 overexpression according to one embodiment of the present invention.

[0040] Figure 10 is the result of confirming the change in proliferation rate during in vitro culture of Batf3 overexpressing γδ T cells according to one embodiment of the present invention.

[0041] Figure 11 is the result of confirming the change in cell cycle rate during in vitro culture of Batf3 overexpressing γδ T cells according to one embodiment of the present invention.

[0042] Figure 12 is the result of confirming whether the in vivo persistence of the Batf3 overexpressing γδ T cell therapy increases according to one embodiment of the present invention.

[0043] Figure 13 is the result of confirming the anti-tumor cytokine production and degranulation ability of Batf3 overexpressing γδ T cells according to one embodiment of the present invention.

[0044] Figure 14 is the result of confirming the change in survival rate when a Batf3 overexpressing γδ T cell therapy agent is administered to a brain tumor individual according to one embodiment of the present invention.

[0045] To confirm the efficacy of Batf3-overexpressing γδ T cells as a treatment for brain tumors, changes in survival rates after treatment were compared and analyzed. As a result, it was found that survival rates were significantly improved upon treatment with the Batf3-overexpressing γδ T cell therapy. In contrast, there was no change in survival rates upon treatment with the control group, the Empty Vector γδ T cell therapy. The above results indicate that the Batf3-overexpressing γδ T cell therapy, which enhances the efficacy of conventional γδ T cell therapies, can be used as a treatment for malignant brain tumors that have been difficult to treat until now.

[0046] The present invention will be described in more detail below through examples. These examples are intended solely to explain the present invention more specifically, and it will be obvious to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the invention.

[0047] Examples

[0048] [Experimental Method]

[0049] 1. Brain tumor mouse model

[0050] For the studies on Batf3 knockout and the in vivo persistence of Batf3-overexpressing γδ T cells, 8-week-old male C57BL / 6 mice were used based on the date of cancer cell transplantation, and 1 x 10⁶ mouse-derived brain tumor cell lines (GL261 cell line) 5 The cells were injected intracranially into the mice. In the therapeutic efficacy experiment of γδ T cells, 12-week-old male C57BL / 6 mice were used based on the date of cancer cell transplantation, and 1 x 10⁶ mouse-derived brain tumor cell lines (GL261-mCherry cell line) 5 The dog was injected into the skulls of mice. Subsequently, the survival rate was observed.

[0051] 2. In Vitro Culture and Transduction Methods for γδ T Cell Therapy

[0052] γδ T cells were isolated from the spleen of wild-type mice using magnetic cell separation technology, and then stimulated using anti-γδ TCR antibodies, anti-CD28 antibodies, and interleukin-2 (IL-2). For restimulation, cells were stimulated under the same conditions 5 days after the initial stimulation. Since a large number of cells were required to confirm the therapeutic efficacy of Batf3-overexpressing γδ T cells, TCR β knockout mice were used to amplify γδ T cells by stimulating them in the same manner as described above. For transduction, a control group or a retrovirus containing the Batf3-overexpression plasmid was applied to γδ T cells that had not undergone restimulation, and subsequently, γδ T cells expressing Thy1.1, a selection marker for transduction, were classified using magnetic cell separation technology. Twenty-four hours prior to flow cytometry analysis of in vitro γδ T cells, the cells were cultured in an IL-2-free medium to better simulate the in vivo environment. Also, for the same reason, γδ T cells were cultured in a medium without IL-2 during the mixed culture process.

[0053] 3. Treatment with γδ T cell therapeutic agent

[0054] To confirm the in vivo persistence of the γδ T cell therapy, γδ T cells were injected intravenously 7 days after the transplantation of the brain tumor cell line. After 5 days, the brain tumor was isolated, and the proportion and number of γδ T cells injected into the tumor microenvironment were confirmed using a flow cytometer.

[0055] In addition, to confirm the therapeutic efficacy of γδ T cell therapy, transgenic γδ T cells were injected intravenously at 7, 13, or 19 days after brain tumor cell line transplantation. DPBS was injected as a control.

[0056] [Experimental Results]

[0057] 1. Confirmation of increased in vivo persistence of γδ T cell therapy through restimulation culture

[0058] The restimulation culture method used in this invention is a novel culture method for the in vitro proliferation of γδ T cells, in which the γδ TCR is stimulated once and then restimulated after 5 days. As a result of confirming the proliferation rate and apoptosis of γδ T cells after the restimulation culture method, the proliferation rate and cell viability of the restimulated γδ T cells were significantly increased compared to the non-restimulated control group (Figs. 1 and 2). Furthermore, when the restimulated γδ T cells were mixed and cultured with the non-restimulated γδ T cells in a 1:1 ratio, the persistence of the restimulated γδ T cells was significantly increased compared to the control group (Fig. 3). This also indicates that the changes in γδ T cells caused by restimulation are attributed to intracellular factors. The above results imply that the short persistence, which is one of the key limitations of γδ T cells, can be overcome by the restimulation culture method.

[0059] 2. Confirmation of an increase in Batf3 due to restimulation and a decrease in Batf3 expression over time.

[0060] To investigate the key mechanism by which restimulation affects γδ T cells, changes in mRNA of restimulated γδ T cells were examined using bulk RNA sequencing. As a result, among the genes that increased statistically significantly due to restimulation compared to the control group, the expression of Batf3 was found to have increased the most (Fig. 4). Therefore, to determine whether Batf3 plays a key role in the increased persistence of γδ T cells caused by restimulation, restimulated Batf3 knockout γδ T cells were co-cultured with a control group. Consequently, it was confirmed that the persistence of restimulated Batf3 knockout γδ T cells was negligible compared to the control group (Fig. 5). Furthermore, when the control group and restimulated Batf3 knockout γδ T cells were simultaneously transplanted into a mouse model and the in vivo kinetics were analyzed, it was confirmed that the in vivo persistence of Batf3 knockout γδ T cells was significantly reduced compared to the control group (Fig. 6). This implies that increased Batf3 expression plays a key role in the in vivo persistence of restimulated γδ T cells.

[0061] Additionally, real-time polymerase chain reaction (qRT-PCR) was used to determine the mRNA expression levels of Batf3 in γδ T cells, and it was confirmed that unrestimulated γδ T cells expressed almost no Batf3. Furthermore, the Batf3 mRNA levels increased by restimulation were found to decrease over time (Fig. 7). This indicates that restimulation increases Batf3 in γδ T cells, but this effect is transient.

[0062] Therefore, the above results suggest that long-term Batf3 overexpression improves the persistence of γδ T cells in the body and has the potential to be utilized as a brain tumor treatment.

[0063] 3. Production and verification of Batf3 overexpressing γδ T cells

[0064] To construct Batf3-overexpressing γδ T cells, a plasmid containing the gene sequences of Batf3 and Thy1.1, an overexpression screening marker, was constructed (Fig. 8). After translating this into γδ T cells, the presence of Batf3 overexpression was confirmed by verifying the degree of Batf3 expression in the cells using qRT-PCR. As a result, it was confirmed that unlike restimulated γδ T cells, where the Batf3 expression rate almost disappeared over time, Batf3-overexpressing γδ T cells maintained a very high Batf3 expression rate regardless of restimulation (Fig. 9).

[0065] 4. Confirmation of in vitro proliferation and increased in vivo persistence of unrestimulated Batf3-overexpressing γδ T cells

[0066] The proliferation rate and changes in the cell cycle of Batf3-overexpressing γδ T cells during in vitro culture were confirmed using a flow cytometer. As a result, it was confirmed that the proliferation rate of Batf3-overexpressing γδ T cells was enhanced, and the cell cycle speed was accelerated through an increase in the proportion of BrdU+ cells labeled for the S phase (Figs. 10 and 11). This result is consistent with the improvement in the proliferation rate of Batf3-overexpressing γδ T cells. In addition, when the in vivo kinetics were analyzed by simultaneously transplanting a control group and Batf3-overexpressing γδ T cells into a mouse model, it was confirmed that the in vivo persistence of Batf3-overexpressing γδ T cells significantly increased compared to the control group (Fig. 12). This confirmed that the in vivo persistence of the γδ T cell therapeutic agent is enhanced due to Batf3 overexpression.

[0067] 5. Confirmation of increased anti-tumor cytokine production and degranulation ability of unrestimulated Batf3-overexpressing γδ T cells

[0068] To confirm changes in the anti-tumor immune response of Batf3-overexpressing γδ T cells, cytokine production and degranulation ability were analyzed using a flow cytometer. As a result, the production ability of IFN-γ and TNF-α, representative anti-tumor cytokines, was found to be significantly enhanced compared to the control group. Additionally, it was confirmed that the expression of CD107a, an indicator of degranulation ability, was also increased compared to the control group (Fig. 13). This implies that Batf3 overexpression enhances not only the production but also the secretion ability of γδ T cells of anti-tumor cytokines. Thus, it was confirmed that the anti-tumor immune response of γδ T cell therapies can be enhanced due to Batf3 overexpression.

[0069] 6. Confirmation of increased survival rate after administration of non-restimulated Batf3 overexpressing γδ T cell therapy in a mouse brain tumor model

[0070] Finally, to confirm the efficacy of Batf3 overexpressing γδ T cells as a treatment for brain tumors, changes in survival rates after treatment were compared and analyzed. As a result, it was found that survival rates were significantly improved upon treatment with the Batf3 overexpressing γδ T cell therapy. In contrast, there was no change in survival rates upon treatment with the control group, the Empty Vector γδ T cell therapy (Fig. 14). The above results indicate that the Batf3 overexpressing γδ T cell therapy, which enhances the efficacy of conventional γδ T cell therapies, can be used as a treatment for malignant brain tumors that have been difficult to treat until now.

[0071] The research results of the present invention present a new method to overcome the limitations of γδ T cell therapies, which have previously been impossible to apply clinically due to short in vivo persistence and reduced anti-tumor immune response. Specifically, by restimulating γδ T cells after primary stimulation of the γδ TCR or / and overexpressing Batf3 during cell culture, the proliferation rate and in vivo persistence can be significantly increased, and the anti-tumor immune response can be enhanced by overcoming immunosuppression in the body. The γδ T cell therapy cultured using the new method of the present invention can be effectively applied to patients with glioblastoma, which is classified as an intractable disease.

[0072] Foregoing, specific parts of the present invention have been described in detail. It is evident to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. Accordingly, the actual scope of the invention is defined by the appended claims and their equivalents.

[0073] The γδ T cells of the present invention are expected to significantly improve the survival rate of cancer patients when used alone or in combination with conventional immunotherapies, so they may be widely used in the health and medical fields.

Claims

1. A pharmaceutical composition for the prevention or treatment of cancer comprising γδ T cells with enhanced expression of Batf3 (Basic Leucine Zipper ATF-Like Transcription Factor 3) as an active ingredient.

2. In Paragraph 1, The above pharmaceutical composition is a pharmaceutical composition that exhibits a significant synergistic effect when used in combination with a chemical anticancer agent, an immunotherapeutic agent, or an immune checkpoint inhibitor.

3. A method for manufacturing a γδ T cell therapeutic agent with increased proliferation rate or persistence in vivo, comprising the step of stimulating the γδ TCR (T cell antigen receptor) of γδ T cells isolated from an individual.

4. In Paragraph 3, The above method is, A method further comprising the step of restimulating the γδ TCR of the γδ T cells; or the step of overexpressing Batf3 (Basic Leucine Zipper ATF-Like Transcription Factor 3).

5. In any one of paragraphs 3 to 4, A method wherein the above γδ TCR stimulation or γδ TCR restimulation is performed by treating with a composition comprising an anti-γδ TCR antibody.

6. In Paragraph 5, The above composition further comprises one or more selected from the group consisting of anti-CD28 antibody, interleukin-2, interleukin-5, zoledronic acid, phytohaemagglutinin (PHA), transforming growth factor beta, concanavalin A, and amphotericin B.

7. In Paragraph 4, A method wherein the step of restimulating the γδ TCR of the above γδ T cells is performed 3 to 7 days after the step of stimulating the γδ TCR (T cell antigen receptor) of the γδ T cells isolated from the individual.

8. In Paragraph 4, A method wherein the step of overexpressing the above Batf3 (Basic Leucine Zipper ATF-Like Transcription Factor 3) is performed 3 to 72 hours after the step of stimulating the γδ TCR (T cell antigen receptor) of γδ T cells isolated from an individual.

9. In Paragraph 4, The step of overexpressing the above Batf3 (Basic Leucine Zipper ATF-Like Transcription Factor 3) is a method comprising transforming into a vector containing a PGK promoter (phosphoglycerate kinase 1 promoter) and the Batf3 gene.

10. A method for improving the proliferation rate or persistence in vivo of γδ T cells, comprising the step of stimulating the γδ TCR (T cell antigen receptor) of γδ T cells isolated from an individual.

11. A γδ T cell therapy agent manufactured by the manufacturing method of paragraph 3.

12. In Paragraph 11, The above γδ T cell therapy is a cell therapy for cancer treatment.

13. In Paragraph 12, A cell therapy agent in which the above cancer is one or more selected from the group consisting of brain cancer, breast cancer, cervical cancer, melanoma, lung cancer, bladder cancer, prostate cancer, leukemia, kidney cancer, liver cancer, colorectal cancer, pancreatic cancer, stomach cancer, gallbladder cancer, ovarian cancer, lymphoma, osteosarcoma, uterine cancer, oral cancer, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, skin cancer, blood cancer, thyroid cancer, parathyroid cancer, ureteral cancer, adenocarcinoma, and thymic cancer.

14. A pharmaceutical composition for the prevention or treatment of cancer comprising the γδ T cell therapy agent of claim 11 as an active ingredient.

15. In Paragraph 14, The above pharmaceutical composition is a pharmaceutical composition used in combination with a chemical anticancer agent, an immunotherapeutic agent, or an immune checkpoint inhibitor.

16. In Paragraph 15, The above chemical anticancer drugs are nitrogen mustard, imatinib, oxaliplatin, rituximab, erlotinib, neratinib, lapatinib, gefitinib, vandetanib, nirotinib, semasanib, bosutinib, axitinib, cediranib, restaurtinib, trastuzumab, gefitinib, bortezomib, sunitinib, carboplatin, sorafenib, bevacizumab, cisplatin, cetuximab, viscolumabum, asparaginase, tretinoin, hydroxycarbamide, dasatinib, estramustine, gemtuzumab ozogamicin, ibritumomab tussetane, heptaplatin, methylaminolevulinic acid, amsacrin, alemtuzumab, procarbazine, alprostadil, holmium nitrate chitosan, gemcitabine, doxifluridine, Pemetrexed, Tegafur, Capecitabine, Gimeracin, Oteracil, Azacitidine, Methotrexate, Uracil, Cytarabine, Fluorouracil, Fludagabine, Enositabine, Flutamide, Capecitabine, Decitabine, Mercaptopurine, Thioguanine, Cladribine, Carmoper, Raltitrexed, Docetaxel, Paclitaxel, Irinotecan, Belotecan, Topotecan, Vinorelbine, Etoposide, Vinblastine, Idarubicin, Mitomycin, Bleromycin, Dactinomycin, Pirarubicin, Aclarubicin, Pepromycin, Temsirolimus, Temozolomide, Busulfan, Ifosfamide, Cyclophosphamide, Melphalan, Altretmine, Dacarbazine, Thiotepa, Nimustine, A pharmaceutical composition comprising one or more selected from the group consisting of chlorambucil, mitrolactol, leucovorin, tretonin, exemestane, aminoglutesimide, anagrelide, olaparib, nabelbine, padrazol, tamoxifen, toremifene, testolactone, anastrozole, letrozole, borozol, bicalutamide, lomustine, 5FU, vorinostat, entinostet, and carmustine.

17. In Paragraph 15, A pharmaceutical composition wherein the above-mentioned immunotherapeutic agent is one or more selected from the group consisting of pembrolizumab, ipilimumab, nivolumab, atezolizumab, and durvalumab.

18. In Paragraph 15, A pharmaceutical composition wherein the immune checkpoint inhibitor is one or more selected from the group consisting of PD-1 receptor inhibitors, PD-L1 ligand inhibitors, PD-L2 ligand inhibitors, CTLA-4 receptor inhibitors, LAG-3 receptor inhibitors, TIM-3 receptor inhibitors, BTLA receptor inhibitors, and KIR receptor inhibitors.

19. In Paragraph 15, The above immune checkpoint inhibitor is a pharmaceutical composition that specifically binds to at least one protein selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4, LAG-3, TIM-3, BTLA, and KIR.

20. A method of preventing or treating cancer by administering the γδ T cell therapy agent of claim 11 to an individual.

21. Use of the γδ T cell therapy of Paragraph 11 for the prevention or treatment of cancer.

Images