Pharmaceutical compositions comprising modified natural killer cells and antigen-specific T cells, methods for making same, and methods for using same

Abstract

To provide a pharmaceutical composition comprising a modified natural killer cell and an antigen-specific T cell, a manufacturing method thereof, and a method of using the same.SOLUTION: This disclosure provides a method of manufacturing a pharmaceutical composition for treating cancer, which provides great cell yield via not performing negative selection on CD3-CD19-CD14-. By administration of the pharmaceutical composition, cancer cells in a subject may be effectively inhibited via cell-mediated immunity.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present disclosure relates to pharmaceutical compositions comprising engineered natural killer (NK) cells and antigen-specific T cells. Methods for culturing the engineered NK cells and antigen-specific T cells are also provided. [Background technology]

[0002] Immune surveillance plays a key role in cancer and represents a very attractive therapeutic approach, especially considering the many shortcomings of traditional surgery, radiation, and chemotherapy in cancer management.

[0003] The human body's first line of defense against cancer is CD3 - CD14 - CD19 - CD56 + CD16 + NGK2D + CD11c dim HLA-DR - CD86 - CD83 - The phenotype of NK cells is that they are natural killer (NK) cells. NK cells are cytotoxic lymphocytes that actively scan the body for abnormal cells and destroy them before they can develop into actual cancer cells. As NK cells patrol the body, they interact with many types of cells using their array of activating and inhibitory surface receptors. Most cancer cells engage with NK cell activating receptors, which triggers their natural killing response.

[0004] US Patent Application Publication No. 20210260115 discloses modified NK cells with both NK cell and dendritic cell functions, as well as a method for culturing the same. When the modified NK cells are administered to a subject with cancer, the modified NK cells kill tumor cells in vivo, present tumor-associated antigens (TAA) to naive T cells, and induce a T cell response (Figure 1). However, prior to the culturing process, CD3 - CD14 - CD19 -A step of negative selection of mononuclear cells is required, which leads to problems of high cost and complicated manufacturing process due to low cell yield. Nevertheless, there is also uncertainty about the efficacy of TAA-specific CD8 T cells induced by engineered NK cells in vivo. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] U.S. Patent Application Publication No. 20210260115 [Non-patent literature]

[0006] [Non-Patent Document 1] Remington's Pharmaceutical Science, 21st Edition, Mack Publishing Company, Easton, PA [Non-patent document 2] Sonderstrup, Springer, Sem. Immunopathol. 25:35-45, 2003. [Non-patent document 3] Nikula et al., Inhal. Toxicol. 4(12j:123-53, 2000) [Non-patent document 4] PLoS ONE, 2017, DOI: 10.1371 / journal.pone.0186998 [Non-patent document 5] Sig Transduct Target Ther, 2021, DOI: 10.1038 / s41392-021-00589-1 [Non-patent document 6] Med Microbiol Immunol, 2019, DOI: 10.1007 / s00430-019-00608-7 Summary of the Invention [Problem to be solved by the invention]

[0007] Because there remains an unmet need for effective treatment and / or prevention of cancer, these findings support the rationale for developing NK-based therapies against cancer cells and culture methods for generating large numbers of therapeutically competent NK cells for clinical application. The present disclosure provides pharmaceutical compositions comprising engineered natural killer cells and antigen-specific T cells, as well as methods for their production. [Means for solving the problem]

[0008] In view of the urgent need in the art, provided herein are methods for producing pharmaceutical compositions comprising modified natural killer (NK) cells and antigen-specific T cells that are safe and effective in the treatment of cancer. In addition, the modified NK cells and antigen-specific T cells are easy to produce at low cost and with high yields.

[0009] In one embodiment, the present disclosure provides a method of manufacturing a pharmaceutical composition for treating cancer, comprising: obtaining mononuclear cells from a subject with cancer; contacting the mononuclear cells with a first culture medium containing IL-15, IL-12, and IL-18 for about 1 to 6 days to obtain a cultured cell population; and After contacting with the first culture medium, contacting the cultured cell population with a second culture medium containing IL-15 and IL-12 for about 1 to 6 days. Contains CD3 - CD19 - CD14 - wherein no negative selection is performed on said mononuclear cells prior to said contacting step.

[0010] In one embodiment, the present disclosure provides a method for detecting CD45 + CD3 - CD19 - CD14 - Modified NK cells with the phenotype of 4-1BB + CD8 +and a pharmaceutically acceptable carrier or excipient. In a preferred embodiment, the modified NK cells are antigen-specific T cells having a phenotype of CD56 hi CD16 dim NKG2D + CD11c + CD86 + HLA-DR + CD83 - HLA-ABC + The modified NK cells can further comprise a phenotype of CD45 + CD3 - CD19 - CD14 - CD56 hi CD16 dim NKG2D + CD11c + CD86 + HLA-DR + CD83 - HLA-ABC + In embodiments, the antigen-specific T cells may comprise a phenotype of CD25 hi CD27 - CD28 - In another embodiment, the antigen-specific T cells may further comprise a phenotype of perforin. dim Granzyme B dim CD107a + TNF-alpha + IFN-γ dim In a preferred embodiment, the modified NK cells can comprise a phenotype of CD25 hi CD27 - CD28 - 4-1BB + Perforin dim Granzyme B dim CD107a + TNF-alpha + IFN-γ dim The phenotype may further include:

[0011] Some embodiments provide pharmaceutical compositions produced by the methods described herein.

[0012] Some embodiments provide a method of treating cancer cells, comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition described herein.

[0013] In a preferred embodiment, the effective amount is about 1×10 per dose. 3 ~About l×10 9 It may be a single cell.

[0014] In preferred embodiments, the engineered NK cells or antigen-specific T cells can be autologous or allogeneic.

[0015] In preferred embodiments, the modified NK cells or antigen-specific T cells can be derived from peripheral blood, umbilical cord blood, or bone marrow.

[0016] In a preferred embodiment, the method can further comprise expanding the modified NK cells in vitro with antigen-specific T cells.

[0017] In a preferred embodiment, the first culture medium and the second culture medium may further comprise a hematopoietic cell medium, preferably an AIM-V medium.

[0018] In a preferred embodiment, the first culture medium and the second culture medium may further comprise a serum protein, preferably human platelet lysate.

[0019] The culturing methods described herein allow for the isolation of large numbers of modified NK cells from a fixed volume of sample, for example, 10 mL of blood.

[0020] Illustrative embodiments of the present application are described in detail below with reference to the following drawings: [Brief explanation of the drawings]

[0021] [Figure 1]Figure 1 illustrates that tumor-associated antigens (TAA) were presented to CD8 T cells by engineered NK cells in vivo, as in the prior art, U.S. Patent Application Publication No. 20210260115. Adoptive cell transfer of the engineered NK cells mediates NK-dependent cytotoxicity to kill tumor cells, resulting in the release of tumor-associated antigens (TAA). Simultaneously, the engineered NK cells also acquire tumor antigens and present the full spectrum of TAAs to CD8 T cells, resulting in the induction of multi-epitope tumor-specific CD8 T cell proliferation and activation. These tumor-specific T cells can then migrate to the tumor bed site in vivo to initiate long-term anti-tumor T cell responses. [Figure 2] Figure 2 illustrates that tumor-associated antigens (TAA) are presented to CD8+ T cells by engineered NK cells in vivo, and the antigen-specific CD8+ T cells are expanded in vitro and adoptively transferred back into the patient to be treated. The pharmaceutical composition comprises engineered NK cells and tumor-specific CD8+ T cells, i.e., antigen-specific T cells. The engineered NK cells can activate tumor-specific CD8+ T cells in vivo through antigen-presenting cell activity, which can be further expanded to produce engineered NK cells. Compared to that described in Figure 1 , the adoptive cell transfer of the present disclosure retains the original properties of the engineered NK cells to drive multi-epitope tumor-specific CD8+ T cell activation. In addition, it also delivers robust anti-tumor cytotoxic T lymphocyte (CTL) cell responses through the engineered NK cell-driven CD8+ T cells expanded in vitro. Repeated adoptive cell transfer of this novel pharmaceutical composition can deliver advanced anti-tumor immune responses. [Figure 3] FIG. 3 illustrates the absolute cell numbers of modified NK cells cultured according to prior art methods and the methods of the present disclosure. [Figure 4] FIG. 4 illustrates that the methods of the present disclosure significantly expanded CD8 T cell populations compared to fresh peripheral blood mononuclear cells (PBMCs). [Figure 5A] FIG. 5A illustrates that the methods of the present disclosure significantly expanded the CD8 T cell population and increased the CD8 to CD4 ratio. [Figure 5B] FIG. 5B illustrates that the methods of the present disclosure significantly expanded the CD8 T cell population and increased the CD8 to CD4 ratio. [Figure 6] FIG. 6 illustrates that the methods of the present disclosure induced antigen (Ag)-specific activation-inducible marker (AIM)+ CD8 T cells. [Figure 7] FIG. 7 illustrates that the methods of the present disclosure induced CD8 T cells expressing an Ag-specific T cell phenotype. [Figure 8] FIG. 8 illustrates that the methods of the present disclosure induced degranulation of CD8 T cells. [Figure 9] FIG. 9 illustrates that the methods of the present disclosure induced CD8 T cells to deliver degranulation molecules including perforin, granzyme B, CD107a, TNF-α, and IFN-γ. [Figure 10] FIG. 10 illustrates that pharmaceutical compositions prepared according to the methods of the present disclosure exhibited tumoricidal activity against primary ovarian epithelial cancer (EOC) cells. [Figure 11] FIG. 11 illustrates that pharmaceutical compositions prepared according to the methods of the present disclosure exhibited tumoricidal activity against primary ovarian epithelial cancer (EOC) cells. [Figure 12] FIG. 12 is a flow chart according to an embodiment of a method for producing a pharmaceutical composition. DETAILED DESCRIPTION OF THE INVENTION

[0022] The foregoing and other aspects of the present disclosure will now be described in more detail with reference to other embodiments described herein. It should be understood that the present invention may be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

[0023] The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used in the description of the invention and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly dictates otherwise.

[0024] As used herein, the terms "comprises," "comprising," "includes," "including," "has," "having," "contains," "containing," "characterized by," or any other variation thereof, are intended to cover a non-exclusive inclusion, subject to any limitations expressly indicated. For example, a composition, mixture, process, or method that includes a list of elements is not necessarily limited to only those elements, but may include other elements not expressly listed or inherent to such composition, mixture, process, or method.

[0025] The transitional phrase "consisting of" excludes any element, step, or ingredient not specified. In a claim, such a transitional phrase would close the claim to the inclusion of materials other than those recited, except for impurities originally associated with them. When the phrase "consisting of" appears in a clause in the body of a claim rather than immediately following the preamble, it limits only the elements recited in that clause; other elements are not excluded from the claim as a whole.

[0026] It is to be understood that where applicant defines the invention or any portion thereof with open-ended terms such as "comprising," the description shall be construed as also describing such invention using the term "consisting of" (unless otherwise specified).

[0027] All numbers herein may be understood to be modified by "about." As used herein, the term "about" is used to indicate that a value includes, for example, the inherent variation of error for the measurement device, the method used to determine the value, or the variation that exists between study subjects. Typically, the term is meant to encompass a variability of approximately 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%, or less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%, as appropriate.

[0028] Although the use of the term "or" in the claims is used to mean "and / or" unless expressly indicated to refer to alternatives only or that the alternatives are mutually exclusive, the present disclosure supports the definition to refer to alternatives only and "and / or."

[0029] "Subject," as used herein, refers to animals, including, for example, mammalian subjects who have cancer or have been diagnosed with or are suspected of having cancer. In embodiments, the term "subject" can refer to a vertebrate with cancer or who is considered to be in need of cancer treatment. Subjects include warm-blooded animals, such as mammals, such as primates, more preferably humans. Non-human primates are also subjects. The term subject includes domestic animals, such as cats, dogs, apes, etc., livestock (e.g., cows, horses, pigs, sheep, goats, etc.), and laboratory animals (e.g., mice, rabbits, rats, gerbils, guinea pigs, etc.). As such, veterinary uses and medical preparations are contemplated herein.

[0030] "Administering" or "administration," as used herein, refers to providing the NK cells or pharmaceutical compositions of the present application to a subject. By way of example, and not limitation, administration can be parenteral, subcutaneous, intramuscular, intravenous, intra-articular, intrabronchial, intraperitoneal, intracapsular, intrachondral, intracavity, intracavity, intracerebellar, intraventricular, intracolonic, intracervical, intragastric, intrahepatic, intramyocardial, intraosseous, intrapelvic, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical, bolus, vaginal, rectal, buccal, sublingual, intranasal, and transdermal. For example, injection can be by intravenous (iv), subcutaneous (sc), intradermal (id), intraperitoneal (ip), or intramuscular (im) injection. One or more such routes can be used. Parenteral administration can be, for example, by bolus injection or by gradual perfusion over time. Alternatively, or concurrently, administration can be by the oral route.

[0031] The use of the term "treat" or "treatment" herein refers to the administration of NK cells or pharmaceutical compositions to a subject for the purpose of curing, alleviating, ameliorating, correcting, preventing, or ameliorating a disorder, a symptom of a disorder, a disease state secondary to a disorder, or a predisposition to a disorder. The terms "inhibit," "reduce," or "prevent," or any variation of these terms, when used in the claims and / or specification, include any measurable decrease or complete inhibition to achieve the desired result.

[0032] "Cancers" that can be treated by the pharmaceutical compositions of the present application include cancers of the oral cavity and pharynx (lips, tongue, salivary glands, floor of the mouth, gums and other oral cavity, nasopharynx, tonsils, oropharynx, hypopharynx, other oral / pharyngeal); cancers of the digestive system (esophagus; stomach; small intestine; colon and rectum; anus, anal canal and anorectum; liver; intrahepatic bile duct; gallbladder; other bile ducts; pancreas; retroperitoneum; peritoneum, omentum and mesentery; other digestive tract); cancers of the respiratory system (nasal cavity, middle ear and sinuses; larynx; lungs and bronchi; pleura; trachea, mediastinum and other respiratory tract); mesothelioma; cancers of soft tissues, including bone and joints; and heart; skin cancers, including melanoma and other non-epithelial skin cancers; Kaposi's disease Cancers classified by site include sarcoma and breast cancer; cancers of the female reproductive system (cervix; uterus; uterus, ovaries; vagina; vulva; and other female reproductive organs); cancers of the male reproductive system (prostate; testes; penis; and other male reproductive organs); cancers of the urinary system (bladder; kidneys and renal pelvis; ureters; and other urinary organs); cancers of the eye and orbit; cancers of the brain and nervous system (brain; and other nervous systems); cancers of the endocrine system (thyroid and other endocrine glands, including the thymus); lymphomas (Hodgkin's disease and non-Hodgkin's lymphoma), multiple myeloma, and leukemias (lymphocytic leukemia; myeloid leukemia; monocytic leukemia; and other leukemias).

[0033] Other cancers classified by histological type that may be suitable targets for therapeutic compositions according to the present application include, but are not limited to, malignant neoplasms; carcinoma, not otherwise specified (NOS); undifferentiated carcinoma, NOS; giant cell and spindle cell carcinoma; small cell carcinoma, NOS; papillary carcinoma, NOS; squamous cell carcinoma, NOS; lymphoepithelial carcinoma; basal cell carcinoma, NOS; pyromatrix carcinoma; transitional cell carcinoma, NOS; papillary transitional cell carcinoma; adenocarcinoma, NOS; malignant gastrinoma; bile duct Cell carcinoma; Hepatocellular carcinoma, NOS; Mixed hepatocellular carcinoma and cholangiocarcinoma; Trabecular adenocarcinoma; Adenoid cystic carcinoma; Adenocarcinoma in adenomatous polyps; Adenocarcinoma in familial polyposis coli; Solid carcinoma, NOS; Malignant carcinoid tumor; Bronchioloalveolar adenocarcinoma; Papillary adenocarcinoma, NOS; Chromophobe carcinoma; Eosinophilic carcinoma; Eosinophilic adenocarcinoma; Basophilic carcinoma; Clear cell adenocarcinoma, NOS; Granular cell carcinoma; Follicular adenocarcinoma, NOS; Papillary and follicular adenocarcinoma; Non-encapsulated sclerosing carcinoma; Adrenal cortical carcinoma; Endometrioid carcinoma carcinoma); skin adnexal carcinoma; apocrine gland carcinoma; sebaceous gland carcinoma; ear canal adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma, NOS; papillary cystadenocarcinoma, NOS; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma, NOS; mucinous adenocarcinoma; signet ring cell carcinoma; invasive ductal carcinoma; medullary carcinoma, NOS; lobular carcinoma; inflammatory breast carcinoma; Paget's disease of the breast; acinic cell carcinoma; adenosquamous carcinoma; adenocarcinoma with squamous metaplasia; malignant thymoma; malignant ovarian stromal tumor; malignant thecoma; malignant granulosa cell tumor; malignant Sertoli stromal cell tumor; Sertoli cell carcinoma; malignant Leydig cell tumor; malignant lipid cell tumor; malignant paraganglioma; malignant extramammary paraganglioma; pheochromocytoma; hemangiosarcoma; malignant melanoma, NOS; amelanotic melanoma; superficial Spreading melanoma;Malignant melanoma in giant pigmented nevus;Epithelioid cell melanoma;Malignant blue nevus;Sarcoma, NOS;Fibrosarcoma, NOS;Malignant fibrous histiocytoma;Myxosarcoma;Liposarcoma, NOS;Leiomyosarcoma, NOS;Rhabdomyosarcoma, NOS;Embryonal rhabdomyosarcoma;Alveolar rhabdomyosarcoma;Stromatous sarcoma, NOS;Malignant mixed tumor, NOS;Müllerian mixed tumor;Nephroblastoma;Hepatoblastoma;Carcinosarcoma, NOS;Malignant mesenchymoma;Malignant Brenner tumor;Malignant phyllodes tumor;Synovial sarcoma, NOS;Malignant mesothelioma;Dysgerminoma;Embryonal carcinoma, NOS;Malignant teratoma, NOS;Malignant ovarian goiter;Choriocoma;Malignant mesonephroma;Angiosarcoma;Malignant hemangioendothelioma;Kaposi's sarcoma;Malignant hemangiopericytoma;Lymphangiosarcoma;Osteosarcoma, NOS; Parosteal Osteosarcoma; Chondrosarcoma, NOS; Malignant Chondroblastoma; Mesenchymal Chondrosarcoma; Giant Cell Tumor of Bone; Ewing's Sarcoma; Malignant Odontogenic Tumor; Ameloblastic Sarcoma; Malignant Ameloblastoma; Ameloblastic Fibrosarcoma; Malignant Pinealoma; Chordoma; Malignant Glioma; Ependymoma, NOS; Astrocytoma, NOS; Protoplasmic Astrocytoma; Fibrous Astrocytoma; Astroblastoma; Glioblastoma, NOS; Oligodendroglioma, NOS; Oligodendroglioma; Primitive Neuroectodermal Tumor; Cerebellar Sarcoma, NOS; Ganglioneuroblastoma; Neuroblastoma, NOS; Retinoblastoma, NOS; Olfactory Neurogenic Tumor; Malignant Meningioma; Neurofibrosarcoma; Malignant Schwannoma Malignant granular cell tumor; malignant lymphoma (NOS); Hodgkin's disease (NOS); Hodgkin's granuloma (NOS); small lymphocytic lymphoma; diffuse large cell lymphoma; follicular lymphoma (NOS); mycosis fungoides; other specific non-Hodgkin's lymphoma; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia (NOS); lymphocytic leukemia (NOS); plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia (NOS); basophilic leukemia; eosinophilic leukemia; monocytic leukemia (NOS); mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia.

[0034] "Effective amount," as used herein, refers to a dose of modified NK cells or pharmaceutical composition sufficient to reduce the symptoms and signs of cancer, including, but not limited to, weight loss, pain, or tumor burden detectable clinically as a palpable mass or radiologically by various imaging means.

[0035] In certain embodiments, it is desirable to limit, reduce, or reverse the size of a tumor or cancerous lesion. The route of administration will, of course, vary depending on the location and nature of the lesion or site being targeted, and includes, for example, local, parenteral, intravenous, intramuscular, and / or systemic administration and formulations. Direct injection, or injection into and from and into the vasculature or blood vessels within an organ or tissue, is specifically contemplated for the target area. Local, regional, or systemic administration may also be appropriate.

[0036] In this disclosure, the expression levels or surface densities of cell surface antigens using FACS / flow cytometry analysis are defined in Table 1. The interpretations for the various expression levels in Table 1 are examples of defining the expression levels of cell surface antigens. It should be noted that the intensity of the signal level in flow cytometry may vary depending on the following factors: flow cytometry, software, and different batches of antibodies used.

[0037] [Table 1]

[0038] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications, patent applications, patents, and other references cited herein are incorporated by reference in their entirety for the teachings related to the sentence and / or paragraph in which the reference is presented.

[0039] Modified NK cells and antigen-specific T cells Naturally occurring or conventional NK cells express CD16 + CD56 + In one embodiment, naturally occurring or conventional NK cells have a phenotype of CD3 - CD14 - CD19 - CD56 + CD16 + NKG2D + CD11c dim Naturally occurring or conventional T cells have the phenotype CD3 + CD56 + In one embodiment, naturally occurring or conventional T cells have a phenotype of CD3 + CD14 - CD19 - CD56 +have a phenotype.

[0040] In one embodiment, the present application provides a method for detecting CD3 - CD19 - CD14 - CD56 hi CD16 dim NKG2D + CD11c + CD86 + HLA-DR + CD83 - The present application provides modified NK cells comprising a cell phenotype that does not occur in nature. The modified NK cells comprise one or more fully activated dendritic (DC) cell surface antigens (e.g., HLA-DR and CD86) and possess both NK cell function and DC cell function, as well as enhanced anti-cancer activity.

[0041] [Table 2]

[0042] In one embodiment, the present application provides a method for detecting CD3 as described in Table 3. + CD14 - CD19 - CD56 + CD25 hi CD27 - CD28 - 4-1BB + Perforin dim Granzyme B dim CD107a + TNF-alpha + IFN-γ dim The present application provides antigen-specific T cells comprising a cell phenotype. The antigen-specific T cells of the present application are non-naturally occurring. The antigen-specific T cells comprise one or more markers that are not expressed on naturally occurring T cells, such as 4-1BB or CD25.

[0043] [Table 3]

[0044] Specifically, the present application relates to CD16 dim CD56 hi 1. A modified NK cell comprising a CD16 phenotype, dim CD56 hi NK cells do not contain the CD83 cell surface antigen (CD16 dim CD56 hi CD83 - The present invention provides modified NK cells having a phenotype of

[0045] In one embodiment, the expression level or surface density of a cell surface antigen is quantified by exposing the engineered NK cells or antigen-specific T cells to a specific fluorochrome-tagged anti-human monoclonal antibody (e.g., CD86-PE (Beckman Coulter; Cat. No. IM2729U) or anti-human CD83-PE-Cy5 (BioLegend; Cat. No. 305310)), followed by sorting the engineered NK cells or antigen-specific T cells using a flow cytometer (e.g., Navios, commercially available from Beckman Coulter, Inc., USA).

[0046] The engineered NK cells or antigen-specific T cells can be generated from a single individual, for example, autologous or allogeneic.

[0047] Pharmaceutical Composition In one embodiment, the present application provides a pharmaceutical composition comprising the modified NK cells and antigen-specific T cells described herein and a pharmaceutically acceptable carrier or excipient. In one embodiment, the modified NK cells and antigen-specific T cells are produced together from mononuclear cells, e.g., peripheral blood mononuclear cells (PBMCs).

[0048] The present application also provides a method for inhibiting cancer cells by administering the pharmaceutical composition to a subject in need thereof in an amount effective to inhibit cancer cells. Without being bound by any particular theory, it is believed that the pharmaceutical composition inhibits cancer cells by antigen-specific T cells and engineered NK cells having one or more of the following functions: enhancing cytotoxicity, stimulating cancer-specific T lymphocyte proliferation, or IFN-γ secretion.

[0049] Routes of administration of the pharmaceutical compositions or modified NK cells include, but are not limited to, intravenous, intramuscular, subcutaneous, oral, topical, intradermal, transdermal, subcutaneous, parenteral, rectal, spinal, or epidermal administration. In one embodiment, the modified NK cells are administered by intravenous injection or infusion.

[0050] The pharmaceutical compositions of the present application may be prepared as injectables, either as liquid solutions or suspensions, or as solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection.

[0051] The modified NK cells and antigen-specific T cells are formulated into pharmaceutical compositions for delivery to mammalian subjects. The pharmaceutical compositions are administered alone and / or mixed with a pharmaceutically acceptable vehicle, excipient, or carrier. Suitable vehicles include, for example, saline (e.g., normal saline), dextrose, glycerol, platelet-rich plasma (PRP), and the like, and combinations thereof. In addition, the vehicle may contain minor amounts of auxiliary substances, such as wetting or emulsifying agents, pH buffering agents, or adjuvants. The pharmaceutically acceptable carrier may contain a physiologically acceptable compound that acts, for example, to stabilize, or increase or decrease the absorption or clearance rate of the pharmaceutical composition of the present application. Physiologically acceptable compounds may include, for example, carbohydrates such as glucose, sucrose, or dextran; antioxidants such as ascorbic acid or glutathione; chelating agents; low-molecular-weight proteins; surfactants; liposome carriers; or excipients; or other stabilizers and / or buffers. Other physiologically acceptable compounds include wetting agents, emulsifying agents, dispersing agents, or preservatives. See, e.g., Remington's Pharmaceutical Sciences, 21st Edition, Mack Publishing Company, Easton, Pa. ("Remington's"). The pharmaceutical compositions of the present application may also include accessory substances, such as pharmacological agents, cytokines, or other biological response modifiers.

[0052] Actual methods for preparing such dosage forms will be known or apparent to those skilled in the art. See, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania, 21st Edition.

[0053] Whether the pharmaceutical composition is used for prophylactic or curative purposes, etc., it can be administered as a single dose treatment or a multiple dose treatment on a schedule and for a predetermined period of time appropriate to the age, weight, and condition of the subject, the particular composition used, and the route of administration. For example, in one embodiment, the pharmaceutical composition according to the present application is administered once a month, twice a month, three times a month, every other week (qow), once a week (qw), twice a week (biw), three times a week (tiw), four times a week (qiw), five times a week, six times a week, every other day (qod), daily (qd), twice a day (bid), three times a day (tid), or four times a day (qid).

[0054] The duration of treatment with a pharmaceutical composition according to the present application, e.g., the period over which the pharmaceutical composition is administered, can vary depending on any of a variety of factors, e.g., the subject's response, etc. For example, the pharmaceutical composition can be administered for a period ranging from about 1 second or more to 1 minute or more, from 1 hour or more to 1 day to about 1 week, from about 2 weeks to about 4 weeks, from about 1 month to about 2 months, from about 2 months to about 4 months, from about 4 months to about 6 months, from about 6 months to about 8 months, from about 8 months to about 1 year, from about 1 year to about 2 years, or from about 2 years to about 4 years, or for a longer period.

[0055] For ease of administration and uniformity of dosage, it is advantageous to formulate parenteral pharmaceutical compositions in dosage unit form. As used herein, dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated, each unit containing a predetermined quantity of modified NK cells and antigen-specific T cells calculated to produce the desired therapeutic effect in association with the necessary pharmaceutical carrier.

[0056] The data obtained from cell culture assays and animal studies can be used to formulate a range of dosages for use in humans. In one embodiment, the dosage of such pharmaceutical compositions is administered at an ED level with little or no toxicity. 50The therapeutically effective dose is within a circulating concentration range comprising the IC50, IC60, IC70, IC80, IC90, IC10, IC11, IC12, IC13, IC14, IC15, IC26, IC27, IC28, IC16, IC29, IC29, IC30, IC29, IC31, IC41, IC52, IC61, IC29, IC29, IC29, IC29, IC31 ... 50 The compounds can be formulated in animal models to achieve a circulating plasma concentration range that includes (i.e., the concentration of modified NK cells and antigen-specific T cells that achieves half-maximal inhibition of symptoms). Sonderstrup, Springer, Sem. Immunopathol. 25:35-45, 2003. Nikula et al., Inhal. Toxicol. 4(12j:123-53, 2000).

[0057] The pharmaceutical compositions are formulated to contain effective amounts of modified NK cells and antigen-specific T cells, where the amounts depend on the animal being treated and the condition being treated. The specific dose level for any particular subject will depend on a variety of factors, including the activity of the specific modified NK cells and antigen-specific T cells, age, body weight, general health, sex, diet, number of doses, route of administration, and rate of excretion, drug combination, and the severity of the particular disease being treated. An exemplary, non-limiting range for a therapeutically or prophylactically effective amount of the pharmaceutical compositions of the present application is at least about 1 x 10 per dose. 3 Approximately 1 x 10 cells per dose 9 The dose is, but is not limited to, 1 x 10 cells per dose. 4 , 1×10 5 , 1×10 6 , 1×10 7 , 1×10 8 or 1 x 10 9 Other dosages, including individual cells, are possible.

[0058] The pharmaceutical composition can be administered alone or in combination with another therapeutic agent, for example, chemotherapy, radiation therapy or targeted therapy, or a cancer vaccine.

[0059] Methods for producing pharmaceutical compositions In one embodiment, a method for producing modified NK cells and antigen-specific T cells is illustrated in Figure 12. Briefly, the method includes at least the steps of obtaining mononuclear cells; contacting the mononuclear cells with a first culture medium comprising IL-15, IL-12, and IL-18 for about 1-6 days to obtain a cultured cell population; and, after contact with the first culture medium, contacting the cultured cell population with a second culture medium comprising IL-15 and IL-12 for about 1-6 days, wherein the second culture medium comprises CD3 - CD19 - CD14 - No negative selection is performed on the mononuclear cells prior to the contacting step.

[0060] Preferably, the mononuclear cells used herein for the selection or production of modified NK cells and antigen-specific T cells are CD3+ mononuclear cells as the initial cell population for expansion culture. - CD14 - CD19 - Negative selection of cells has not been performed; they are mononuclear cells.

[0061] One embodiment of the present disclosure provides a method for identifying early cells from a sample that does not include removing one or more of the following cell surface antigens from mononuclear cells in the sample: CD3, CD14, or CD19.

[0062] The duration of culture of a composition comprising IL-12 can be important for the function of modified NK cells. In embodiments, mononuclear cells can be cultured with IL-12, e.g., human IL-12, for 1 to 12 days, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 days, to generate modified NK cells. In embodiments, mononuclear cells cultured with IL-12 for 3, 6, 9, or 12 days produce modified NK cells with similar cell yields and phenotypic patterns. In another embodiment, prolonged exposure to IL-12, e.g., 12 days, increased the cytotoxicity and antigen-presenting cell activity of the cultured cells compared to cells exposed to IL-12 for a shorter period of time, e.g., 9 days.

[0063] In one embodiment, the composition for culturing cells further comprises IL-18. In one embodiment, the effective concentration of IL-18 is about 1 to 300 ng / mL, e.g., 50, 100, 150, 200, 250, 300 ng / mL, etc. In another embodiment, the effective concentration of IL-18 is about 10 to about 250 ng / mL, or any value or range of values ​​therebetween in increments of 10 ng / mL (e.g., about 30 ng / mL, about 220 ng / mL, etc.).

[0064] In another embodiment, IL-18 has a reduced effect on the phenotypic pattern and functional activity of modified NK cells. Longer-term IL-18 exposure may have adverse effects on cell size, phenotype, and functional activity. In an embodiment, mononuclear cells can be cultured with IL-18, e.g., human IL-18, for 1 to 12 days, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 days, to generate modified NK cells. In an embodiment, mononuclear cells cultured with IL-18 for 3, 6, 9, or 12 days produce modified NK cells with various phenotypes. In another embodiment, sufficient exposure to IL-18, e.g., 6 days, may have adverse effects on the cell size, phenotype, and function of modified NK cells compared to cells exposed to IL-18 for a shorter period of time, e.g., 6 days.

[0065] In one embodiment, mononuclear cells are contacted with a culture composition comprising IL-18, IL-15, and / or IL-12. In another embodiment, mononuclear cells can be mixed with a composition consisting essentially of hematopoietic cell medium (e.g., X-vivo 20), IL-18, IL-15, IL-12, and serum proteins (e.g., human platelet lysate). In yet another embodiment, mononuclear cells can be mixed with a composition consisting essentially of X-vivo 20, IL-18, IL-15, IL-12, and human platelet lysate.

[0066] In another embodiment, the composition further comprises a hematopoietic cell culture medium, non-limiting examples of which include X-vivo 10, X-vivo 15, X-vivo 20 (commercially available from Lonza, Switzerland), and AIM-V (commercially available from ThermoFisher Scientific, USA).

[0067] In yet another embodiment, the composition further comprises a serum protein, e.g., human platelet lysate. In this application, a "serum protein" is a protein present in blood or plasma that performs many different functions, including regulating transport and cellular activity. Non-limiting examples of serum proteins include enzymes, complement components, protease inhibitors, kinin precursors, serum albumin, globulins, and fibrinogen.

[0068] Non-limiting examples of compositions for culturing cells include: (a) hematopoietic cell medium + IL-15 + IL-18; (b) hematopoietic cell medium + IL-15 + IL-18; (c) hematopoietic cell medium + IL-15 + IL-12 + IL-18; (d) X-vivo 20 + IL-15 + IL-18; (e) X-vivo 20 + IL-12 + IL-18; (f) X-vivo 20+IL-15+IL-12+IL-18;(g)AIM-V+IL-15+IL-18;(h)AIM-V+IL-12+IL-18;(i)AIM-V+IL-15+IL-12+IL-18;(j)Hematopoietic cells Medium+IL-15+IL-18+serum protein;(k)Hematopoietic cell medium+IL-12+IL-18+serum protein;(l)Hematopoietic cell medium+IL-15+IL-12+IL-18+serum protein;(m)X-vivo 20+IL-15+IL-18+serum protein;(n)X-vivo 20+IL-12+IL-18+serum protein;(o)X-vivo (p) AIM-V + IL-15 + IL-12 + IL-18 + serum protein; (q) AIM-V + IL-12 + IL-18 + serum protein; and (r) AIM-V + IL-15 + IL-12 + IL-18 + serum protein.

[0069] In one embodiment, the culture medium is used to culture the modified NK cells and antigen-specific T cells at 37° C. in the presence of 5% CO 2 .

[0070] CD3 - CD14 - CD19 - Methods according to some embodiments of the present application that do not involve negative selection enhance the yield of modified NK cells and antigen-specific T cells. The yield of modified NK cells was determined by mononuclear cell purity by FACS and viable cell counting. Other assays for cell proliferation, such as clonogenic assays, metabolic assays, and direct proliferation assays, are well known in the art.

[0071] Exemplary, non-limiting ranges for the contact time between the mononuclear cells and the culture medium are about 1 minute to about 1 hour, about 1 hour to about 24 hours, about 1 day to about 3 days, about 1 day to about 6 days, about 1 day to about 9 days, about 1 day to about 12 days, about 3 days to about 6 days, about 3 days to about 9 days, about 3 days to about 12 days, about 6 days to about 9 days, about 6 days to about 12 days, or at least 1 day. In one embodiment, the contact time is about 3 days. In another embodiment, the contact time is about 6 days.

[0072] In one embodiment, the mononuclear cells are contacted with a first culture medium comprising IL-15, IL-12, and IL-18; and subsequently with a second culture medium comprising IL-15 and IL-12. In another embodiment, the first and second culture media further comprise a hematopoietic cell medium such as AIM-V or X-vivo and / or a serum protein such as human platelet lysate.

[0073] In one embodiment, the modified NK cells and antigen-specific T cells can be produced by the following steps: (a) On day 0, l×l0 6 contacting mononuclear cells with a composition comprising AIM-V medium, HPL (concentration: 4% w / w), IL-15 (concentration: 30 ng / mL), IL-12 (concentration: 3 ng / mL), and IL-18 (concentration: 50 ng / mL); (b) on day 6, harvesting and centrifuging half of the initial cells in step (a), followed by resuspending the cell pellet in a composition comprising AIM-V medium, HPL, IL-15, and IL-12; (c) On day 12, harvesting all of the cultured cells in step (b).

[0074] Optionally, the composition for culturing the cells may be replaced, for example, on days 3 and 9 with fresh medium having the same components as on days 0 and 6, respectively, and the initial cells may be cultured as follows: (a) On day 0, l×l0 6 contacting mononuclear cells with a composition comprising AIM-V medium, HPL (concentration: 4% w / w), IL-15 (concentration: 30 ng / mL), IL-12 (concentration: 3 ng / mL), and IL-18 (concentration: 50 ng / mL); (b) on day 3, replacing the medium with a composition comprising AIM-V medium, HPL, IL-15, IL-12, and IL-18; (c) on day 6, harvesting and centrifuging half of the cultured cells in step (b), followed by resuspending the cell pellet in a composition comprising AIM-V medium, 4% w / w HPL, 30 ng / mL IL-15, and 3 ng / mL IL-12; (d) on day 9, replacing the medium with a composition comprising AIM-V medium, HPL, IL-15, and IL-12; (e) On day 12, harvesting all of the cultured cells in step (d).

[0075] Alternatively, the composition for culturing the cells may be replaced, for example, on days 3 and 9 with fresh medium having the same components as on days 0 and 6, respectively, and the initial cells may be cultured as follows: (a) On day 0, l×l0 6 contacting mononuclear cells with a composition comprising AIM-V medium, HPL (concentration: 4% w / w), IL-15 (concentration: 30 ng / mL), IL-12 (concentration: 3 ng / mL), and IL-18 (concentration: 50 ng / mL); (b) on day 3, replacing the medium with a composition comprising AIM-V medium, HPL, IL-15, IL-12, and IL-18; (c) on day 6, collecting and centrifuging half of the cultured cells in step (b), followed by resuspending the cell pellet in a composition comprising AIM-V medium, 4% w / w HPL, 30 ng / mL IL-15, 3 ng / mL IL-12, and 50 ng / mL IL-18; (d) on day 9, replacing the medium with a composition comprising AIM-V medium, HPL, IL-15, IL-12, and IL-18; (e) On day 12, harvesting all of the cultured cells in step (d).

[0076] The following examples of specific modes for carrying out the present invention are provided for illustrative purposes only and are not intended to limit the scope of the invention in any way. [Example]

[0077] Example 1 Preparation of initial cells 40 mL of peripheral blood was collected from healthy volunteers into vacuum tubes containing K2EDTA. The blood sample was mixed with an equal volume of pre-warmed phosphate-buffered saline (PBS) (Biological Industries, Israel). 40 mL of diluted peripheral blood aliquot was placed in a 50 mL centrifuge tube, and 10 mL of pre-warmed Ficoll-Paque™ PREMIUM was added. The centrifuge tube was centrifuged at 2000 rpm at room temperature for 30 minutes. Mononuclear cells at the interface layer were collected and washed once with PBS. The cell pellet was collected in 10 mL of PBS. 6 Resuspended to a density of 100 cells / 100 mL MACS buffer.

[0078] Example 2 Cultivation of engineered NK cells and antigen-specific T cells Mononuclear cells from Example 1 were cultured for 12 days in AIM-V medium containing 4% HPL, hIL-15 (days 0, 3, 6, 9), 3 ng / ml hIL-12 (days 0, 3, 6, 9), and hIL-18 (days 0, 3, 6). Total cell count and viability were determined by trypan blue exclusion. Absolute cell numbers were calculated using the following formula:

[0079] Results: As shown in Table 3 and Figure 3, the enrichment step in the prior art significantly reduced the number of initial cells for production. Although the purity of the modified NK cells was higher, the absolute cell number of the modified NK cells of the prior art was only one-fifth that of the modified NK cells of the present disclosure. On the other hand, with the method of the present disclosure, production significantly expanded the CD8 T cell population, as shown in Figures 4, 5A, and 5B.

[0080] [Table 4]

[0081] Example 3 Ag-specific CD8 T cells PBMCs were cultured for 12 days in AIM-V medium containing 4% HPL, 30 ng / ml hIL-15 (days 0, 3, 6, and 9), 3 ng / ml hIL-12 (days 0, 3, 6, and 9), and 50 ng / ml hIL-18 (days 0, 3, and 6). The preparations were stained with mAbs against CD45-ECD, CD56-APC-Alexa Flour 700, CD3-APC-Alexa Flour 750, CD14-APC-Alexa Flour 750, CD19-APC-Alexa Flour 750, CD8-Krome Orange (Beckman Coulter), TCRαβ-FITC, 4-1BB-PE, CD25-PE, CD28-PerCp-Cy5.5, CD4-PE-Cy7, TCRγδ-APC, and CD27-Pacific Blue (Biolegend). Sample acquisition was performed using a Navios flow cytometer and data analysis was performed using Kaluza software (Beckman Coulter). Statistical analysis was performed using Student's t-test, and results were considered significant if P<0.05. ns: not significant. *: P<0.05, **: P<0.01, ***: P<0.001.

[0082] Results: Activation-induced markers (AIMs), including CD25 (PLoS ONE, 2017, DOI: 10.1371 / journal.pone.0186998), CD69, 4-1BB (CD137), and IFN-γ upregulation (Sig Transduct Target Ther, 2021, DOI: 10.1038 / s41392-021-00589-1), or CD27 and CD28 downregulation (Med Microbiol Immunol, 2019, DOI: 10.1007 / s00430-019-00608-7), are used to identify antigen-specific T cells. As shown in Figures 6 and 7, in the expanded CD8 T cell populations prepared according to the present disclosure, CD8 T cells expressed Ag-specific T cell phenotypes, including upregulation of activation-induced markers 4-1BB and CD25 and downregulation of activation-induced markers CD27 and CD28, indicative of CD8 T cell activation. AIM expression was approximately 4-170-fold higher compared to fresh T cells in PBMCs.

[0083] Example 4 Degranulation of CD8 T cells PBMCs were cultured for 12 days in AIM-V medium containing 4% HPL, 30 ng / ml hIL-15 (days 0, 3, 6, and 9), 3 ng / ml hIL-12 (days 0, 3, 6, and 9), and 50 ng / ml hIL-18 (days 0, 3, and 6). Products of the present disclosure were stained with mAbs against CD56-APC-Alexa Flour 700, CD14-APC-Alexa Flour 750, CD19-APC-Alexa Flour 750 (Beckman Coulter), perforin-FITC, 4-1BB-PE, CD107a-PE / Dazzle 594, IFN-γ-PerCp-Cy5.5, TNF-α-PE-Cy7, granzyme B-Pacific Blue, and CD3-BV510 (Biolegend). Sample acquisition was performed using a Navios flow cytometer and data analysis was performed using Kaluza software (Beckman Coulter). Statistical analysis was performed using the Student's t-test, and results were considered significant if P<0.05. ns: not significant. *: P<0.05, **: P<0.01, ***: P<0.001.

[0084] Results: As shown in Figures 8 and 9, in the preparations described in this disclosure, CD8 T cells were induced to deliver approximately 60-fold more degranulation molecules, such as perforin, granzyme B, CD107a, TNF-α, and IFN-γ, compared to fresh T cells in PBMCs.

[0085] Example 5 The pharmaceutical composition performed antitumor activity Modified NK cells and antigen-specific T cells were isolated using magnetic beads, respectively, and then subjected to CD3 + Cells and CD56 +The cells were selected from the product by removing them. The cytotoxicity of the modified NK cells and antigen-specific T cells was evaluated using the PanToxilux kit (OncoImmunin, Inc.). Human ovarian epithelial carcinoma (EOC) cells served as target cells and were stained with TFL-4 at an optimal concentration for 50 minutes. TFL-4-labeled target cells and modified NK cells or antigen-specific T cells were co-incubated with a caspase substrate at 37°C for 60 minutes. The cells were harvested and stained with TFL-4. + substrate + Cell death was analyzed by determining the signal of β-glucan by flow cytometry. Statistical analysis was performed using Student's t-test, and results were considered significant if P<0.05. ns: not significant. *: P<0.05, **: P<0.01, ***: P<0.001.

[0086] Results: As shown in Figures 10 and 11, the percentage of caspase-positive target cells (for the modified NK cells alone or the Ag-specific T cells alone) was 9.63% and 10.14%, respectively, while the percentage of caspase-positive target cells (for the pharmaceutical composition of the present disclosure) was 12.45%, an increase of at least 22.7% compared to the modified NK cells alone or the Ag-specific T cells alone. This result indicates that the pharmaceutical composition of the present disclosure, generated from a patient, exhibited tumoricidal activity against primary tumor cells derived from the same patient.

[0087] [Table 5A]

[0088] [Table 5B]

[0089] [Table 5C]

[0090] While specific embodiments of the present invention have been described and illustrated, such embodiments should be considered merely as illustrative of the present invention and not limiting of the invention as interpreted according to the appended claims. All publications and patent applications cited herein are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference for all purposes. While the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those skilled in the art that certain changes and modifications can be made thereto in light of the teachings of the present invention without departing from the spirit or scope of the appended claims.

[0091] Without further elaboration, it is believed that one skilled in the art can, based on the preceding description, utilize the present invention to its fullest extent. The following specific examples, therefore, are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. All publications cited herein are incorporated by reference.

Claims

1. 1. A method for producing a pharmaceutical composition for treating cancer, comprising: i) contacting mononuclear cells from a subject with cancer with a first culture medium containing IL-15, IL-12, and IL-18 for about 1 to 6 days to obtain a cultured cell population; and ii) after contacting with the first culture medium, contacting the cultured cell population with a second culture medium comprising IL-15 and IL-12 for about 1 to 6 days. Contains CD3 - CD19 - CD14 - is not performed on said mononuclear cells prior to said contacting steps i) and ii).

2. The method of claim 1, wherein the mononuclear cells are derived from peripheral blood, umbilical cord blood, or bone marrow.

3. 10. The method of claim 1, wherein the first culture medium and the second culture medium further comprise a hematopoietic cell medium.

4. The method of claim 3, wherein the hematopoietic cell medium comprises AIM-V medium.

5. 4. The method of claim 3, wherein the first culture medium further comprises a serum protein.

6. The method of claim 5, wherein the serum protein comprises human platelet lysate.

7. (a) CD3 - CD19 - CD14 - CD56 hi CD16 dim NKG2D + CD11c + CD86 + HLA-DR + CD83 - modified natural killer (NK) cells with a phenotype of; (b) antigen-specific T cells with the phenotype CD3 + CD14 − CD19 − CD56 + CD25 hi CD27 − CD28 − 4-1BB + Perforin dim Ganzyme B dim CD107a + TNF-α + IFN-γ dim; and (c) a pharmaceutically acceptable carrier or excipient A pharmaceutical composition for treating cancer, comprising:

8. The pharmaceutical composition according to claim 7, wherein the NK cells and antigen-specific T cells are produced by the method according to any one of claims 1 to 6.

9. 10. Use of the pharmaceutical composition of claim 7 in the manufacture of a medicament for the treatment of cancer.

10. The effective dose is approximately 1 × 10 3 ~About l×10 9 The use according to claim 9, wherein the cell is a cell.

11. 10. The use of claim 9, wherein the modified NK cells or antigen-specific T cells are autologous or allogeneic.

12. 10. The use of claim 9, wherein the modified NK cells or antigen-specific T cells are derived from peripheral blood, umbilical cord blood, or bone marrow.

13. Cancers include cancers of the oral cavity and pharynx (lips, tongue, salivary glands, floor of the mouth, gums and other parts of the mouth, nasopharynx, tonsils, oropharynx, hypopharynx, other parts of the mouth / pharynx); cancers of the digestive system (esophagus; stomach; small intestine; colon and rectum; anus, anal canal and anorectum; liver; intrahepatic bile duct; gallbladder; other bile ducts; pancreas; retroperitoneum; peritoneum, omentum and mesentery; other digestive tract); cancers of the respiratory system (nasal cavity, middle ear and sinuses; larynx; lungs and bronchi; pleura; trachea, mediastinum and other respiratory tract); mesothelioma; cancers of soft tissue, including bones and joints, and the heart; skin cancer, including melanoma and other non-epithelial skin cancers; Kaposi's sarcoma and breast cancer; female reproductive health 10. The use of claim 9, including cancer of the reproductive system (cervix; uterus; uterus, ovaries; vagina; vulva; and other female reproductive organs); cancer of the male reproductive system (prostate; testes; penis; and other male reproductive organs); cancer of the urinary system (bladder; kidneys and renal pelvis; ureters; and other urinary organs); cancer of the eye and orbit; cancer of the brain and nervous system (brain; and other nervous systems); cancer of the endocrine system (thyroid and other endocrine glands including the thymus); lymphoma (Hodgkin's disease and non-Hodgkin's lymphoma), multiple myeloma, and / or leukemia (lymphocytic leukemia; myeloid leukemia; monocytic leukemia; and other leukemias).

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