Modified natural killer cells, pharmaceutical compositions, methods for preparing the same, and methods for using the same

Abstract

The present disclosure provides modified natural killer cells having natural killer (NK) cell function and dendritic cell function, and a method for culturing the same. By administering the modified natural killer cells, cell-regulated immunization can effectively inhibit cancer cells in a subject.

Description

Technical Field

[0001] The present disclosure relates to modified natural killer (NK) cells and pharmaceutical compositions comprising the modified natural killer cells. Methods for identifying the modified natural killer cells and culturing the modified natural killer cells are also provided. Background Art

[0002] Immune surveillance plays a quite important role in cancer and represents a very attractive treatment method, especially in view of the many drawbacks of traditional surgery, radiotherapy, and chemotherapy in cancer management.

[0003] The first line of defense of the human body against cancer is natural killer (NK) cells, which have a phenotype of CD3 - CD14 - CD19 - CD56 + CD16 + NGK2D + CD11c dim HLA-DR - CD86 - CD83 - . Natural killer cells are cytotoxic lymphocytes that can actively scan abnormal cells in the human body and destroy them before they develop into true cancer cells. When natural killer cells patrol the human body, they use their series of activating and inhibitory surface receptors to interact with various types of cells. Most cancer cells bind to the activating receptors of natural killer cells, thereby triggering their natural killer response.

[0004] Since the need for effective treatment and / or prevention of cancer remains unmet, these findings provide a theoretical basis for the development of natural killer cell-based therapies against cancer cells and culturing methods for generating more therapeutically capable natural killer cells for clinical applications. The present disclosure provides modified natural killer cells with a unique phenotype to meet these and other needs. The cells can be used for autologous therapy or allogeneic therapy. Summary of the Invention

[0005] In view of the urgent need in the art, provided herein are modified natural killer (NK) cells that are safe and effective in cancer treatment and pharmaceutical compositions comprising the cells.

[0006] In one embodiment, the present disclosure provides a composition comprising CD45 + CD3 - CD19 - CD14 -Modified natural killer cells of a phenotype. In a preferred embodiment, the modified natural killer cells may further comprise CD56 hi CD16 dim NKG2D + CD11c + CD86 + HLA-DR + CD83 - HLA-ABC + of a phenotype, that is, the modified natural killer cells may comprise CD45 + CD3-CD19-CD14-CD56 hi CD16 dim NKG2D + CD11c + CD86 + HLA-DR + CD83 - HLA-ABC + of a phenotype.

[0007] Certain embodiments provide a pharmaceutical composition comprising the modified natural killer cells described herein and a pharmaceutically acceptable carrier or excipient.

[0008] Certain embodiments provide a method of treating cancer cells, comprising administering to a subject in need thereof an effective amount of the modified natural killer cells or pharmaceutical composition described herein.

[0009] Certain embodiments provide the use of the modified natural killer cells or pharmaceutical composition in the preparation of a medicament for treating cancer cells, wherein the treatment comprises administering to a subject in need thereof a medicament comprising an effective amount of the modified natural killer cells or pharmaceutical composition described herein.

[0010] In a preferred embodiment, the effective amount may be from about 1×10 3 to about 1×10 9 cells per dose.

[0011] In a preferred embodiment, the modified natural killer cells may be autologous or allogeneic.

[0012] In a preferred embodiment, the modified natural killer cells may be derived from peripheral blood, cord blood, or bone marrow.

[0013] In a preferred embodiment, the method may further comprise expanding the modified natural killer cells in vitro.

[0014] Other embodiments provide a method of culturing modified natural killer cells having natural killer cell function and dendritic cell function, comprising

[0015] obtaining a body fluid containing monocytes;

[0016] contacting the monocytes with a first culture medium containing IL-15, IL-12, and IL-18 to obtain a cultured cell population; and

[0017] isolating from the cultured cell population modified natural killer cells having a CD3 - CD19 - CD14 - CD56 hi CD16 dim NKG2D + CD11c + CD86 + HLA-DR + CD83 - phenotype.

[0018] In a preferred embodiment, the monocytes may be derived from peripheral blood, cord blood, or bone marrow.

[0019] In a preferred embodiment, the first culture medium may further comprise: a hematopoietic cell culture medium, preferably, AIM-V medium.

[0020] In a preferred embodiment, the first culture medium may further comprise: a serum protein, preferably, human platelet lysate.

[0021] In a preferred embodiment, the monocytes may be contacted with the first culture medium for about 1 to 6 days.

[0022] In a preferred embodiment, the method may further comprise, after contacting with the first culture medium, contacting the cultured cell population with a second culture medium containing IL-15 and IL-12.

[0023] In a preferred embodiment, the second culture medium may further comprise: a hematopoietic cell culture medium, preferably, AIM-V medium.

[0024] In a preferred embodiment, the second culture medium may further comprise: a serum protein, preferably, human platelet lysate.

[0025] In a preferred embodiment, the cultured cell population may be contacted with the second culture medium for about 1 to 6 days.

[0026] In a preferred embodiment, the method may further comprise, prior to contacting with the first culture medium, negatively selecting monocytes from cells having a CD3 - CD14 - CD19 - phenotype.

[0027] The culturing method described herein allows for the isolation of a greater number of modified natural killer cells from a fixed amount of sample, such as 10 mL of blood. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The following is a detailed description of each exemplary embodiment of the present disclosure, and reference is made to the following drawings:

[0029] Figure 1A Illustrate the cell numbers of modified natural killer cells cultured in various culture media. Figure 1B And 1C Illustrate a collection of flow cytometry analysis images of modified natural killer cells cultured in various culture media for the expression of NKG2D, CD45, CD16, CD56, CD3, CD14, CD19, CD86, CD83, CD11c, and HLA-ABC.

[0030] Figure 2A Illustrate a collection of flow cytometry analysis images of modified natural killer cells cultured from various initial cells for the expression of CD45, CD3, CD14, and CD19. Figure 2B And 2C Illustrate a collection of flow cytometry analysis images of modified natural killer cells cultured from various initial cells for the NK phenotype and the DC phenotype, respectively.

[0031] Figure 3A Illustrate a collection of flow cytometry analysis images of modified natural killer cells cultured in the presence or absence of IFN-γ for the expression of CD45, CD3, CD14, and CD19. Figure 3B And 3C Illustrate a collection of flow cytometry analysis images of modified natural killer cells cultured for the NK phenotype and the DC phenotype in the presence or absence of IFN-γ, respectively.

[0032] Figure 4A Illustrate the cell numbers of modified natural killer cells cultured at various IL-12 exposure times. Figure 4B And 4C Illustrate a collection of flow cytometry analysis images of modified natural killer cells cultured at various IL-12 exposure times for the NK phenotype and the dendritic cell phenotype, respectively. Figure 4DA collection of flow cytometer images demonstrating the cytotoxicity of the modified natural killer cells. Figure 4E A collection of flow cytometer analyses of cell division demonstrating the antigen presenting cell (APC) activity of the modified natural killer cells on the proliferation of T lymphocytes.

[0033] Figure 5A Illustrating the cell numbers of the modified natural killer cells cultured at various IL-18 exposure concentrations. Figure 5B A collection of flow cytometer analysis images demonstrating the upregulation of CD25, HLA-DR, and CD86 expression of the modified natural killer cells on day 3. Figure 5C A collection of flow cytometer analysis images demonstrating the downregulation of HLA-DR and CD86 of the modified natural killer cells on day 12. Figure 5D A collection of flow cytometer images demonstrating the cytotoxicity of the modified natural killer cells cultured with IL-18. Figure 5E A collection of flow cytometer analyses of cell division demonstrating the APC activity of the modified natural killer cells cultured with IL-18 on the proliferation of T lymphocytes.

[0034] Figure 6A Illustrating the cell numbers of the modified natural killer cells cultured at various IL-18 exposure times. Figure 6B A collection of flow cytometer images demonstrating the cytotoxicity of the modified natural killer cells cultured at various IL-18 exposure times. Figure 6C A collection of flow cytometer analyses of cell division demonstrating the APC activity of the modified natural killer cells cultured at various IL-18 exposure times on the proliferation of T lymphocytes.

[0035] Figure 7 A flowchart of an embodiment of a method for culturing modified natural killer cells.

[0036] Figure 8 A phenotypic analysis of the modified natural killer cells according to an embodiment of the present invention.

[0037] Figure 9A Illustrating the cytotoxicity regulated by the modified natural killer cells according to an embodiment of the present invention. Figure 9B Illustrating the APC activity regulated by the modified natural killer cells according to an embodiment of the present invention. Detailed Description of the Invention

[0038] The foregoing and other aspects of the present disclosure will now be described in more detail with respect 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 to which this invention pertains.

[0039] The terms used in the description of the present disclosure are for the purpose of describing particular embodiments only and are not intended to limit the invention. As used in the specification and claims of the present disclosure, the singular forms "a", "an", and "the" are also intended to include the plural forms unless the context clearly dictates otherwise.

[0040] As used herein, the term "comprising", "including", "having", "containing", "characterized by", or any other variation thereof, is intended to cover a non-exclusive inclusion, unless otherwise explicitly stated. For example, a composition, mixture, process, or method that comprises a series of elements is not necessarily limited to those elements, but may include other elements not expressly listed or inherent to such composition, mixture, process, or method.

[0041] The connecting phrase "consisting of" does not include any unspecified elements, steps, or ingredients. If used in a claim, this will limit the scope of the claim to the recited materials, excluding only impurities ordinarily associated therewith. When the phrase "consisting of" appears in the body of a claim, rather than immediately following the preamble, it limits only the elements listed in that clause; overall, other elements are not excluded from the scope of the claim.

[0042] When the applicant has defined the invention or a part thereof using an open-ended term such as "comprising", it should be readily understood (unless otherwise stated) that the description should be interpreted as also using the term "consisting of" to describe the invention.

[0043] All numbers herein can be understood to be modified by the term "about". As used herein, the term "about" is used to indicate that a numerical value includes the inherent variations of, for example, the measuring device, the method for determining the value, or the variations that exist among the subjects under study. Generally, depending on the circumstances, this term is intended to cover variations of approximately or less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%.

[0044] Unless explicitly stated to refer to only the alternative or the alternatives are mutually exclusive, the use of the term "or" in a claim is meant to be "and / or", although the present disclosure supports definitions that refer only to the recited alternatives as well as "and / or".

[0045] As used herein, "subject" refers to an animal, including, for example, mammalian subjects diagnosed with, suspected of having, or developing cancer. In one embodiment, the term "subject" can refer to a vertebrate with cancer or a vertebrate considered in need of cancer treatment. Subjects include warm-blooded animals such as mammals, such as primates, and preferably humans. Non-human primates are also subjects. The term subject includes domestic animals such as cats, dogs, apes, etc., livestock (such as cows, horses, pigs, sheep, goats, etc.), and laboratory animals (such as mice, rabbits, rats, gerbils, guinea pigs, etc.). Thus, veterinary uses and pharmaceutical formulations are covered herein.

[0046] As used herein, "administering" refers to providing a natural killer cell or pharmaceutical composition of the present invention to a subject. By way of example and not limitation, administration can be by parenteral, subcutaneous, intramuscular, intravenous, intra-articular, intratracheal, intraperitoneal, intracapsular, intrachondral, intracavitary, intracranial, intracerebellar, intraventricular, intracolonic, endocervical, 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 (i.v.) injection, subcutaneous (s.c.) injection, intradermal (i.d.) injection, intraperitoneal (i.p.) injection, or intramuscular (i.m.) injection. One or more such routes can be employed. Parenteral administration can be, for example, by bolus injection or infusion over time. Alternatively or concurrently, administration can be by the oral route.

[0047] The use of the term "treating" herein refers to administering a natural killer cell or pharmaceutical composition to a subject for the purpose of curing, alleviating, mitigating, remedying, preventing, or ameliorating a disease, the symptoms of the disease, a disease state secondary to the disease, or susceptibility to the disease. When used in the claims and / or the specification, the terms "inhibit", "reduce", or "prevent" or any variation of these terms includes any measurable decrease or complete inhibition to achieve the desired result.

[0048] "Cancers" treatable by the natural killer cells or pharmaceutical compositions of the present invention include those classified by location, including cancers of the oral cavity and pharynx (lips, tongue, salivary glands, floor of the mouth, gums and other mouth, nasopharynx, tonsils, oropharynx, hypopharynx, other oral / pharynx); cancers of the digestive system (esophagus; stomach; small intestine; colon and rectum; anus, anal canal, and anorectum; liver; intrahepatic bile ducts; gallbladder; other biliary tract; pancreas; retroperitoneum; peritoneum, omentum, and mesentery; other digestive organs); cancers of the respiratory system (nasal cavity, middle ear, and paranasal sinuses; larynx; lung and bronchi; pleura; trachea, mediastinum, and other respiratory tract); cancers of mesothelioma; bones and joints; soft tissues, including the heart; skin cancers, including melanoma and other non-epithelial skin cancers; Kaposi's sarcoma and breast cancer; cancers of the female reproductive system (cervix; corpus uteri; uterus, ovaries; vagina; vulva; and other female genitalia); cancers of the male reproductive system (prostate, testis, penis, and other male genitalia); cancers of the urinary system (bladder; kidney and renal pelvis; ureters; and other urinary tract); eye and orbital cancers; cancers of the brain and nervous system (brain; and other nervous system); cancers of the endocrine system (thyroid and other endocrine, including thymus); lymphomas (Hodgkin's disease and non-Hodgkin's lymphoma), multiple myeloma, and leukemias (lymphocytic leukemia; myelogenous leukemia; monocytic leukemia; and other leukemias).

[0049] Other cancers classified by histological type that can be suitable targets for the therapeutic compositions according to the present invention include, but are not limited to, malignant tumors; carcinoma, not otherwise specified (NOS); undifferentiated carcinoma, not otherwise specified (NOS); giant cell and spindle cell carcinoma; small cell carcinoma, not otherwise specified (NOS); papillary carcinoma, not otherwise specified (NOS); squamous cell carcinoma, not otherwise specified (NOS); lymphoepithelioma; basal cell carcinoma, not otherwise specified (NOS); carcinoma of the labia; transitional cell carcinoma, not otherwise specified (NOS); papillary transitional cell carcinoma; adenocarcinoma, not otherwise specified (NOS); gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma, not otherwise specified (NOS); combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyps; adenocarcinoma, familial polyposis coli; solid carcinoma, not otherwise specified (NOS); carcinoid, malignant; bronchioloalveolar adenocarcinoma; papillary adenocarcinoma, not otherwise specified (NOS); chromophobe carcinoma; oxyphilic carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma, not otherwise specified (NOS); granular cell carcinoma; follicular adenocarcinoma, not otherwise specified (NOS); papillary and follicular adenocarcinoma; non-encapsulated sclerosing carcinoma; adrenocortical carcinoma; endometrial carcinoma; carcinoma of skin appendages; apocrine adenocarcinoma; sebaceous gland carcinoma; ceruminous gland adenocarcinoma; mucoepidermoid carcinoma of meninges; cystadenocarcinoma, not otherwise specified (NOS); papillary cystadenocarcinoma, not otherwise specified (NOS); papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma, not otherwise specified (NOS); mucinous adenocarcinoma; signet ring cell carcinoma; invasive ductal carcinoma; medullary carcinoma, not otherwise specified (NOS); lobular carcinoma; inflammatory carcinoma; Paget's disease of the breast; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma with squamous metastases; thymoma, malignant; ovarian stromal tumor, malignant; theca cell tumor, malignant; granulosa cell tumor, malignant; arrhenoblastoma, malignant; Sertoli cell carcinoma of the testis; Leydig cell tumor of the testis, malignant; lipoid cell tumor, malignant; paraganglioma, malignant; paraganglioma of breast, malignant; pheochromocytoma; angiosarcoma; malignant melanoma, not otherwise specified (NOS); non-melanoma; superficial spreading melanoma; Marjolin melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma, not otherwise specified (NOS); fibrosarcoma, not otherwise specified (NOS); malignant fibrous histiocytoma; myxosarcoma; liposarcoma, not otherwise specified (NOS); leiomyosarcoma, not otherwise specified (NOS); rhabdomyosarcoma, not otherwise specified (NOS); embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma, not otherwise specified (NOS); mixed tumor, malignant, not otherwise specified (NOS); Mullerian mixed tumor; Wilms' tumor; hepatoblastoma: carcinosarcoma, not otherwise specified (NOS); mesotheliosarcoma, malignant; testicular tubulo-ovarian adenoma, malignant; phyllodes tumor, malignant; synovial sarcoma, not otherwise specified (NOS); mesothelioma, malignant;Seminoma; embryonal carcinoma, not otherwise specified (NOS); teratoma, malignant, not otherwise specified (NOS); struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; angiosarcoma; hemangioendothelioma, malignant; Kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma, not otherwise specified (NOS); juxtacortical osteosarcoma; chondrosarcoma, not otherwise specified (NOS); chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; Ewing's sarcoma; odontogenic tumor, malignant; ameloblastic osteosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma, not otherwise specified (NOS); astrocytoma, not otherwise specified (NOS); protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma, not otherwise specified (NOS); oligodendroglioma, not otherwise specified (NOS); oligodendroglioblastoma; primitive neuroectoderm; cerebellar sarcoma, not otherwise specified (NOS); ganglioneuroblastoma; neuroblastoma, not otherwise specified (NOS); retinoblastoma, not otherwise specified (NOS); olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neuroma, malignant; granulosa cell tumor, malignant; malignant lymphoma, not otherwise specified (NOS); Hodgkin's disease, not otherwise specified (NOS); Hodgkin's granuloma, not otherwise specified (NOS); malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular, not otherwise specified (NOS); mycosis fungoides; other specified non-Hodgkin's lymphoma; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia, not otherwise specified (NOS); lymphocytic leukemia, not otherwise specified (NOS); plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia, not otherwise specified (NOS); basophilic leukemia; eosinophilic leukemia; monocytic leukemia, not otherwise specified (NOS); mast cell leukemia; megakaryocytic leukemia; myeloid sarcoma; and hairy cell leukemia.;

[0050] As used herein, "effective amount" refers to the dose of the modified natural killer cells or pharmaceutical composition sufficient to reduce the symptoms and signs of cancer, the symptoms and signs including but not limited to, weight loss, pain or tumor mass, which is a clinically palpable mass or radiologically detectable by various imaging methods.

[0051] In certain embodiments, it is desirable to limit, reduce, or ameliorate the size of a tumor or cancerous lesion. The route of administration will naturally vary with the location and nature of the lesion or site to be targeted and includes, for example, local, parenteral, intravenous, intramuscular, and / or systemic administration and formulations. For the target area, particular consideration is given to direct injection into an organ or tissue or from an organ or tissue or into the vasculature or blood vessels therefrom. Local, regional, or systemic administration may also be appropriate.

[0052] In the present disclosure, the expression level or surface density of cell surface antigens analyzed using FACS / flow cytometry is defined in Table 1. The interpretation of the various expression levels in Table 1 is an example of defining the cell surface antigen expression level. It should be noted that the flow cytometry signal intensity varies with the following factors: the flow cytometer, the software, and the different batches of antibodies used.

[0053] Table 1

[0054] Symbol Explanation - <![CDATA[The signal intensity of the flow cytometer is less than or equal to 10 0 (i.e., 1)]]> +(dim) <![CDATA[The signal intensity of the flow cytometer ranges from 10 0 to 10 1 in between]]> + <![CDATA[The signal intensity of the flow cytometer is between 10 1 and 10 2 > +(hi) <![CDATA[Flow cytometry signal intensity is greater than or equal to 10 2 >

[0055] 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 relevant to the sentence and / or paragraph in which the reference is present.

[0056] Modified natural killer cells

[0057] Naturally occurring or conventional natural killer cells have a CD16 + CD56 + phenotype. In one embodiment, naturally occurring or conventional natural killer cells have a CD3 - CD14 - CD19 - CD56 + CD16 + NKG2D + CD11c dim phenotype, as shown in Table 2.

[0058] In one embodiment, the present disclosure provides a modified natural killer cell comprising a CD3 - CD19 - CD14 - CD56 hi CD16 dim NKG2D + CD11c + CD86 + HLA-DR + CD83- Cell phenotypes, as shown in Table 2. The modified natural killer cells of the present invention are not naturally occurring. The modified natural killer cells comprise one or more fully activated dendritic (DC) cell surface antigens (e.g., HLA-DR and CD86), and have natural killer cell function, dendritic cell function, and enhanced anti-cancer activity.

[0059] Table 2. Comparison of phenotypes between conventional natural killer cells and modified natural killer cells

[0060] Conventional natural killer cell Modified natural killer cell CD3 - - CD14 - - CD19 - - CD56 + hi CD16 + dim NKG2D + + CD11c dim + HLA-DR - + CD86 - + CD83 - -

[0061] Specifically, the present disclosure provides a modified natural killer cell comprising a CD16 dim CD56 hi phenotype, wherein the CD16 dim CD56 hi natural killer cell does not contain the CD83 cell surface antigen (having the CD16 dim CD56 hi CD83 - phenotype).

[0062] WO2015 / 100495 discloses a modified natural killer cell comprising a CD3 - CD19 - CD14 - CD56 + CD16 + NKG2D + CD11c - CD86 + HLA-DR + CD83 + phenotype. Compared with the modified natural killer cells of WO2015 / 100495, the modified natural killer cells of the present disclosure have the general dendritic cell surface antigen CD11c, but lack the fully activated dendritic cell surface antigen CD83. Nevertheless, the modified natural killer cells of the present disclosure have higher and stronger dendritic cell function, e.g., antigen presentation, which is 66% higher than that of the modified natural killer cells of WO2015 / 100495.

[0063] In one embodiment, the expression level or surface density of cell surface antigens is quantified by exposing the modified natural killer cells to a fluorescent dye-labeled specific anti-human monoclonal antibody (e.g., CD86-PE (Beckman Coulter; catalog number: IM2729U) or anti-human CD83-PE-Cy5 (BioLegend; catalog number: 305310)), and then the modified natural killer cells are sorted using a flow cytometer (e.g., Navios, available from Beckman Coulter, USA).

[0064] The modified natural killer cells can be generated from a single individual, e.g., autologous or allogeneic.

[0065] Pharmaceutical composition

[0066] In one embodiment, the present disclosure provides a pharmaceutical composition comprising the modified natural killer cells described herein and a pharmaceutically acceptable carrier or excipient.

[0067] The present disclosure also provides a method of inhibiting cancer cells by administering to a subject in need thereof an effective amount of the modified natural killer cells of the present invention or the pharmaceutical composition of the present invention that inhibits cancer cells. Without being bound by any particular theory, it is believed that the modified natural killer cells inhibit cancer cells through one or more natural killer cell / dendritic cell functions: enhancing cytotoxicity, stimulating the proliferation of cancer-specific T lymphocytes, or IFN-γ secretion.

[0068] The administration routes of the pharmaceutical composition or the modified natural killer cells include but are not limited to intravenous, intramuscular, subcutaneous, oral, topical, intradermal, transdermal, subepidermal, parenteral, rectal, spinal or epidermal administration. In one embodiment, the modified natural killer cells are administered by intravenous injection or infusion.

[0069] The pharmaceutical composition of the present invention can be prepared as an injection, which can be a liquid solution or suspension, or a solid form suitable for dissolving or suspending in a liquid carrier before injection.

[0070] The modified natural killer cells of the present invention are formulated into a pharmaceutical composition for delivery to a mammalian subject. The pharmaceutical composition can be administered alone and / or mixed with a pharmaceutically acceptable carrier, excipient, or vehicle. Suitable carriers are, for example, saline (e.g., physiological saline), dextrose, glycerol, platelet-rich plasma (PRP), etc., and combinations thereof. In addition, the carrier may contain small amounts of auxiliary substances, such as wetting agents or emulsifiers, pH buffers, or adjuvants. The pharmaceutically acceptable vehicle may contain physiologically acceptable compounds that have, for example, the effect of stabilizing or increasing or decreasing the absorption or clearance rate of the pharmaceutical composition of the present invention. 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, detergents, liposome carriers, or excipients or other stabilizers and / or buffers. Other physiologically acceptable compounds include wetting agents, emulsifiers, dispersants, or preservatives. See, for example, Remington's Pharmaceutical Science, 21st Edition, Mack Publishing Company, Easton, PA ("Remington's"). The pharmaceutical composition of the present invention may also contain auxiliary substances, such as pharmacological agents, cytokines, or other biological response modifiers.

[0071] The actual methods for preparing such dosage forms are known or obvious to those of ordinary skill in the art to which the present invention pertains. See, for example, Remington's Pharmaceutical Science, 21st Edition, Mack Publishing Company, Easton, PA.

[0072] The modified natural killer cells or pharmaceutical compositions of the present invention can be administered as a single-dose treatment or a multi-dose treatment according to a schedule and for a period of time suitable for the age, weight, and condition of the subject, the specific composition used, and the route of administration, whether the modified natural killer cells or pharmaceutical compositions of the present invention are for prophylactic or therapeutic purposes, etc. For example, in one embodiment, the modified natural killer cells or pharmaceutical compositions according to the present invention are 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), once a day (qd), twice a day (bid), three times a day (tid), or four times a day (qid).

[0073] The duration of treatment with the modified natural killer cells or pharmaceutical compositions according to the present invention, for example, the period of time during which the modified natural killer cells or pharmaceutical compositions are administered, can vary depending on any of a variety of factors, such as, for example, the response of the subject, etc. For example, the modified natural killer cells or pharmaceutical compositions can be administered for about one or several seconds to one or several minutes, one or several hours to one day to about one week, from about two weeks to about four weeks, from about one month to about two months, from about two months to about four months, from about four months to about six months, from about six months to about eight months, from about eight months to about one year, from about 1 year to about 2 years, or from about 2 years to about 4 years, or for an even longer period of time.

[0074] It is advantageous to formulate parenteral pharmaceutical compositions or modified natural killer cells in unit dosage form for ease of administration and uniformity of dosage. As used herein, unit dosage form refers to physically discrete units suitable as unit doses for the subjects to be treated; each unit contains a predetermined amount of modified natural killer cells calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.

[0075] Data obtained from cell culture assays and animal studies can be used to formulate a dosage range for humans. In one embodiment, the dose of such natural killer cells is in the ED that contains little or no toxicity 50within the cyclic concentration range. The dosage can vary within this range, depending on the dosage form employed and the route of administration utilized. In another embodiment, a therapeutically effective dosage can initially be estimated from cell culture assays. The dosage can be formulated in an animal model to achieve a circulating plasma concentration range that includes the IC 50 (i.e., the concentration of modified natural killer cells that achieves half the maximal suppression of symptoms) as described in Sonderstrup, Springer, Sem. Immunopathol 25:35-45, 2003. Nikula et al., Inhal. Toxicol. 4(12):123-53, 2000.

[0076] The pharmaceutical composition is formulated to contain an effective amount of modified natural killer cells, where the amount depends on the animal to be treated and the condition to be treated. The specific dosage for any particular subject depends on a variety of factors, including the activity of the specific modified natural killer cells, age, body weight, general health, gender, diet, time of administration, route of administration, and rate of excretion, drug combination, and the severity of the particular disease being treated. Exemplary non-limiting ranges for a therapeutically or prophylactically effective amount of modified natural killer cells are at least about 1×10 3 cells per dose to about 1×10 9 cells per dose. Other dosages are possible, including, but not limited to, 1×10 4 , 1×10 5 , 1×10 6 , 1×10 7 , 1×10 8 , or 1×10 9 cells per dose.

[0077] The modified natural killer cells or the pharmaceutical composition can be administered alone or in combination with another therapeutic agent, such as chemotherapy, radiotherapy, or targeted therapy, or a cancer vaccine.

[0078] Methods for identifying and culturing modified natural killer cells

[0079] In one embodiment, a method for identifying primary natural killer cells and culturing modified natural killer cells is as Figure 7 shown. Briefly, the method includes at least the following steps: obtaining a body fluid containing monocytes; contacting the monocytes with a first culture medium containing IL-15, IL-12, and IL-18 to obtain a cultured cell population; and labeling the cells with CD3 - CD19 - CD14 - CD56 hi CD16 dimNKG2D + CD11c + CD86 + HLA-DR + CD83 - Natural killer cells with both natural killer cell function and dendritic cell function are isolated from the cultured cell population.

[0080] Preferably, the monocytes used herein for screening or generating modified natural killer cells are purified CD3 - CD14 - CD19 - monocytes as the initial cell population for expansion culture.

[0081] In one embodiment, highly purified CD3 - CD14 - CD19 - The identification / removal steps of the monocyte portion are as follows:

[0082] (a) A sample is collected from a subject. The sample includes, but is not limited to, any body fluid containing one or more monocytes, such as peripheral blood, cord blood, or bone marrow samples.

[0083] (b) By centrifugation (Ficoll-Paque TM PREMIUM, GE Healthcare, USA), the monocytes in the sample in step (a) are separated from other types of blood cells. Other methods for separating monocytes are known or obvious to those of ordinary skill in the art to which the present invention pertains.

[0084] (c) In step (b), one or more monocytes containing CD3 cell surface antigens (e.g., monocyte phenotype CD3 + CD56 - and CD3 + CD56 + ), CD14 cell surface antigens, or CD19 cell surface antigens are depleted from other monocytes, for example, by MACS sorting (Mitenyi Biotec, Germany).

[0085] (d) The CD3 - CD14 - CD19 - monocytes collected in step (c) are cultured.

[0086] One embodiment of the present invention provides a method for identifying naive cells from a sample, comprising removing monocytes expressing one or more of the following cell surface antigens from the sample: CD3, CD14, or CD19, wherein the naive cells are substantially free of one or more cell surface antigens selected from CD3, CD14, and CD19.

[0087] In one embodiment, monocytes expressing CD3 in step (b) are removed from the sample. In another embodiment, monocytes expressing CD14 in step (b) are removed from the sample. In yet another embodiment, monocytes expressing CD19 in step (b) are removed from the sample. In yet another embodiment, one or more monocytes expressing CD14 and one or more monocytes expressing CD3 in step (b) are removed from the sample. In yet another embodiment, one or more monocytes expressing CD14 and one or more monocytes expressing CD19 in step (b) are removed from the sample. In yet another embodiment, one or more monocytes expressing CD19 and one or more monocytes expressing CD3 in step (b) are removed from the sample. In yet another embodiment, one or more monocytes expressing CD19, one or more monocytes expressing CD3, and one or more monocytes expressing CD14 in step (b) are removed from the sample.

[0088] In one embodiment, substantially all monocytes expressing CD3 are removed from the sample. In another embodiment, substantially all monocytes expressing CD14 are removed from the sample. In yet another embodiment, substantially all monocytes expressing CD19 are removed from the sample.

[0089] In one embodiment, the composition for culturing cells further comprises IFN-γ. In an alternative embodiment, the composition for culturing cells does not comprise IFN-γ. In this embodiment, IFN-γ has little effect on the generation of the modified natural killer cells disclosed herein.

[0090] The culture time of the composition containing IL-12 may be crucial for the function of modified natural killer cells. In one embodiment, monocytes can be cultured with IL-12, such as human IL-12, for 1 to 12 days, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 days, to produce modified natural killer cells. In one embodiment, monocytes cultured with IL-12 for 3, 6, 9, or 12 days produce modified natural killer cells with similar cell yields and phenotypic patterns. In another embodiment, extending the exposure time of IL-12, such as 12 days, increases the cytotoxicity and antigen-presenting cell activity of the cultured cells compared to cells with a shorter exposure time to IL-12, such as 9 days.

[0091] In one embodiment, the composition for culturing cells further contains IL-18. In one embodiment, the effective concentration of IL-18 is about 1 to 300 ng / mL, such as 50, 100, 150, 200, 250, 300 ng / mL. In another embodiment, the effective concentration of IL-18 is about 10 to about 250 ng / mL, or any value between the two or a numerical range with an increment of 10 ng / mL (e.g., about 30 ng / mL, about 220 ng / mL, etc.).

[0092] In another embodiment, IL-18 has an impact on the phenotype and functional activity of modified natural killer cells. Long-term exposure to IL-18 may have opposite effects on cell size, phenotype, and functional activity. In one embodiment, monocytes can be cultured with IL-18, such as human IL-18, for 1 to 12 days, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 days, to produce modified natural killer cells. In one embodiment, monocytes cultured with IL-18 for 3, 6, 9, or 12 days produce modified natural killer cells with various phenotypes. In another embodiment, sufficient exposure to IL-18, such as 6 days, may have opposite effects on the cell size, phenotype, and function of the modified natural killer cells compared to cells with a shorter exposure time to IL-18, such as 3 days.

[0093] In one embodiment, highly purified CD3 - CD14 - CD19 - A portion of the monocytes is contacted with a culture composition containing IL-18, IL-15, and / or IL-12. In another embodiment, the CD3 in step (c) - CD14 - CD19 -Monocytes are mixed with a composition consisting essentially of a hematopoietic cell culture medium (e.g., X-vivo 20), IL-18, IL-15, IL-12, and a serum protein (e.g., human platelet lysate). In another embodiment, CD3 in step (c) - CD14 - CD19 - Monocytes are mixed with a composition consisting essentially of X-vivo 20, IL-18, IL-15, IL-12, and human platelet lysate.

[0094] In another embodiment, the composition further comprises a hematopoietic cell culture medium. Non-limiting examples of the hematopoietic cell culture medium include X-vivo 10, X-vivo 15, X-vivo 20 (available from Lonza, Switzerland), and AIM-V (available from ThermoFisher Scientific, USA).

[0095] In another embodiment, the composition further comprises a serum protein, such as human platelet lysate. In the present invention, "serum protein" refers to a protein present in blood or plasma, which has many different functions, including transporting and regulating cell activities. Non-limiting examples of serum proteins include enzymes, complement components, protease inhibitors, kinin precursors, serum albumin, globulins, and fibrinogen, etc.

[0096] Non-limiting examples of the composition for culturing cells include (a) hematopoietic cell medium + IL-15 + IL-18; (b) hematopoietic cell medium + IL-12 + IL-18; (c) hematopoietic cell medium + IL-15 + IL-12 + IL-18; (d) X-vivo20 + 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 cell 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 20 + IL-15 + IL-12 + IL-18 + serum protein; (p) AIM-V + IL-15 + 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.

[0097] In one embodiment, the composition is used to culture the modified natural killer cells at 37 °C and 5% CO 2 in the presence.

[0098] The composition according to certain embodiments of the present disclosure enhances the proliferation of the modified natural killer cells. In one embodiment, the composition substantially enhances the expression of the CD11c cell surface antigen on the modified natural killer cells. In another embodiment, the composition enhances the expression of fully activated dendritic cell markers such as HLA-DR and CD86 cell surface antigens on the modified natural killer cells, but does not enhance the expression of the CD83 cell surface antigen. Via FACS by CD3 - CD14 - CD19 - The purity and viable cell count of the cells determine the proliferation rate of the modified natural killer cells. Other assays for cell proliferation are well known in the art, such as the clonogenic assay, metabolic assay, and direct proliferation assay.

[0099] CD3 - CD14 - CD19- Exemplary non-limiting ranges for the contact time of monocytes with the composition are from about 1 minute to about 1 hour, from about 1 hour to about 24 hours, from about 1 day to about 3 days, from about 1 day to about 6 days, from about 1 day to about 9 days, from about 1 day to about 12 days, from about 3 days to about 6 days, from about 3 days to about 9 days, from about 3 days to about 12 days, from about 6 days to about 9 days, from 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.

[0100] In one embodiment, the CD3 - CD14 - CD19 - Monocytes are contacted with a first composition comprising IL-15, IL-12, and IL-18; and then with a second composition comprising IL-15 and IL-12. In another embodiment, the first and second compositions further comprise a hematopoietic cell culture medium (such as AIM-V or X-vivo) and / or a serum protein (such as human platelet lysate).

[0101] The following embodiments for practicing aspects of the present invention are provided for illustrative purposes only and are not intended to limit the scope of the present invention in any way. Examples

[0102] Example 1: Selection of Culture Medium

[0103] The CD3 - CD14 - CD19 -Monocytes were cultured for 15 days in AIM-V medium or X-vivo 20 medium containing 30 ng / mL of human recombinant IL-15 (hIL-15), 3 ng / mL of human recombinant IL-12 (hIL-12), 60 ng / mL of human recombinant IL-2 (hIL-2), 37.5 ng / mL of human recombinant IL-18 (hIL-18), and optionally 4% (w / w) human platelet lysate (HPL). The cell number of each group was counted by trypan blue dye exclusion. In addition, the cells were subsequently stained with monoclonal antibodies (mAbs) NKG2D-PE, CD45-ECD, CD16-PE-Cy7, CD56-APC-Alexa Flour 700, CD3-APC-Alexa Flour 750, CD14-APC-Alexa Flour750, CD19-APC-Alexa Flour 750 (Beckman Coulter), CD86-Alexa488, CD83-PE-Cy5, CD11c-APC, and HLA-ABC-Pacific Blue (Biolegend). Samples were acquired and analyzed by a Navios flow cytometer, and data analysis was performed by Kaluza software (Beckman Coulter).

[0104] As Figure 1A shown, the cell number of cells cultured in AIM-V medium supplemented with human platelet lysate (HPL) was higher than the yield of cell culture obtained by culturing in X-vivo 20, indicating that AIM-V medium with human platelet lysate (HPL) has the potential to increase cell yield. In addition, the cells cultured in AIM-V medium supplemented with human platelet lysate (HPL) produced a phenotype similar to that of cells cultured in X-vivo 20( Figure 1B - 1C ).

[0105] Example 2: Selection of initial monocytes

[0106] CD3 - CD14 - CD19 - (TN1) or CD25 - CD14 - CD19 -(TN2) Monocytes were cultured for 9 days in AIM-V medium containing 30 ng / mL hIL-15, 3 ng / mL hIL-12, 45 ng / mL human recombinant IFN-γ (hIFN-γ), and 4% (w / w) human platelet lysate (HPL). Cultured cells were counted by using trypan blue dye exclusion method and stained with monoclonal antibodies (mAb) CD45-ECD, CD3-APC-Alexa Flour 750, CD14-APC-Alexa Flour 750, and CD19-APC-Alexa Flour 750 (Beckman Coulter). Samples were acquired and analyzed by Navios flow cytometer, and data analysis was performed by Kaluza software (Beckman Coulter).

[0107] As Figure 2A shown in Table 3, TN1 initial cells provided target cells with high purity at a higher yield compared to those using TN2 initial cells.

[0108] Table 3

[0109] Cell type Purity Yield (number of cells) TN1 91.62％ <![CDATA[1.22×10 7 > TN2 22.88％ <![CDATA[6.2×10 6 >

[0110] On the other hand, cultured cells were stained with monoclonal antibodies NKG2D-PE, CD45-ECD, CD16-PE-Cy7, CD56-APC-Alexa Flour 700, CD3-APC-Alexa Flour 750, CD14-APC-Alexa Flour 750, CD19-APC-Alexa Flour 750 (Beckman Coulter), CD86-Alexa488, CD83-PE-Cy5, CD11c-APC, and HLA-ABC-Pacific Blue (Biolegend) to analyze their phenotypes. Samples were acquired and analyzed by Navios flow cytometer, and data analysis was performed by Kaluza software (Beckman Coulter).

[0111] As Figure 2B and 2C shown, phenotypic analysis showed similar phenotypes of cultured cells generated from TN1 and TN2 initial cells.

[0112] Therefore, although cultured cells generated from TN1 initial cells expressed natural killer cell and dendritic cell phenotypes similar to those of CD3 - CD14 - CD19- CD56 hi CD16 dim NKG2D + CD11c + HLA-DR + CD86 + CD83 - Similar, but the highly purified cultured cells generated from TN1 naive cells (CD3 - CD14 - CD19 - ) have a higher yield than those generated using TN2 naive cells (CD25 - CD14 - CD19 - ).

[0113] Example 3: The important role of IFN-γ

[0114] WO2015 / 100495 discloses that IFN-γ is quite important for the generation of modified natural killer cells with natural killer cell and dendritic cell functions. However, we unexpectedly found that IFN-γ has little role in the generation of the modified natural killer cells disclosed herein.

[0115] Cultured 1×10 6 cells / mL of CD3 - CD14 - CD19 - monocytes for 9 days in the presence of 30 ng / mL hIL-15, 3 ng / mL hIL-12, and with or without 45 ng / mL hIFN-γ. The cultured cells were stained with monoclonal antibodies CD45-ECD, CD3-APC-AlexaFlour750, CD14-APC-Alexa Flour 750, and CD19-APC-Alexa Flour 750 (Beckman Coulter). Samples were collected and analyzed by Navios flow cytometer, and data analysis was performed by Kaluza software (Beckman Coulter).

[0116] As Figure 3A and shown in Table 4, the cells cultured with or without IFN-γ produced substantially the same purity and yield, indicating that the cultured cells emerging from TN1 naive cells emerged in an IFN-γ-independent manner. In addition, phenotypic analysis showed a similar pattern of the cultured cells with or without IFN-γ( Figure 3B and 3C ).

[0117] Table 4

[0118] Cell type Purity Yield (number of cells) Without IFN-γ 91.82％ <![CDATA[1.22×10 7 > With IFN-γ 91.61％ <![CDATA[1.22×10 7 >

[0119] Therefore, in terms of cell purity, the yield and acquisition of natural killer cell and dendritic cell phenotypes, IFN-γ has little impact on the emergence of the cultured cells disclosed herein.

[0120] Example 4: Treatment period of IL-12

[0121] CD3 - CD14 - CD19 - Monocytes were cultured with 30 ng / mL hIL-15 for 12 days and with 3 ng / mL hIL-12 for 9 or 12 days. The cells were subcultured on day 6, and the culture medium was changed every three days. The cultured cells were counted by using the trypan blue dye exclusion method. In addition, the cultured cells were stained with monoclonal antibodies NKG2D-PE, CD45-ECD, CD16-PE-Cy7, CD56-APC-Alexa Flour 700, CD3-APC-Alexa Flour 750, CD14-APC-AlexaFlour 750, CD19-APC-Alexa Flour 750 (Beckman Coulter), CD86-Alexa488, CD83-PE-Cy5, CD11c-APC, and HLA-ABC-Pacific Blue (Biolegend). Samples were collected and analyzed by a Navios flow cytometer, and data analysis was performed by using Kaluza software (Beckman Coulter).

[0122] As Figure 4A shown, compared with the cells exposed to IL-12 for 9 days, extending the exposure to IL-12 (12 days) has less impact on the cell yield of the cultured cells. Similarly, phenotypic analysis shows that the cultured cells exposed to IL-12 for 9 or 12 days have similar phenotypes ( Figure 4B and 4C ).

[0123] On the other hand, functional assays were also performed to evaluate cytotoxicity and antigen-presenting activity. The cytotoxicity of the modified natural killer cells was evaluated using the PanToxilux kit (OncoImmunin). The human chronic myelogenous leukemia (CML) cell line K562 was used as the target cells and stained with TFL4 at the optimal concentration for 50 minutes. The TFL4-labeled target cells and the cultured cells were co-cultured with the caspase substrate at 37 °C for 20 minutes. The cells were harvested and the signal of TFL-4 was analyzed by flow cytometry. + substrate + The antigen-presenting activity of the cultured cells was evaluated by the mixed lymphocyte reaction (MLR). The responder cells (CD25 - PBMC) were enriched and stained with the CellTrace TM CFSE Cell Proliferation Kit (Invitrogen). The CSFE-labeled CD25 - PBMC were co-cultured with the modified natural killer cells at 37 °C for 5 days. hIL-2 and hIL-15 were added on day 1 and day 3 to lower the threshold of TCR involvement. The cells were harvested and the pattern of CFSE dilution was analyzed by flow cytometry.

[0124] As Figure 4D and 4E shown, compared with the cells exposed to IL-12 for 9 days, extended exposure to IL-12 (for 12 days in total) enhanced the cytotoxicity and antigen-presenting cell activity of the cultured cells.

[0125] Therefore, although extended exposure to IL-12 had little effect on the emergence of the cultured cells disclosed herein in terms of cell purity, the acquisition and yield of natural killer cell and dendritic cell phenotypes, extending the exposure to IL-12 to 12 days did enhance the cytotoxicity and antigen-presenting cell activity of the modified natural killer cells.

[0126] Example 5: Effect of IL-18 on the emergence of modified natural killer cells

[0127] In the presence of 30 ng / mL hIL-15, 3 ng / mL hIL-12, and 0, 50, 100, or 200 ng / mL hIL-18, CD3 - CD14 - CD19 -Monocytes were cultured for 12 days. The cells were subcultured on day 6, and the culture medium was changed every three days. The cultured cells were harvested on days 3, 6, 9, and 12 and counted by trypan blue dye exclusion. In addition, the cultured cells were harvested on days 3 and 12 and stained with monoclonal antibodies NKG2D-PE, CD45-ECD, CD16-PE-Cy7, CD56-APC-AlexaFlour 700, CD3-APC-Alexa Flour 750, CD14-APC-Alexa Flour 750, CD19-APC-AlexaFlour 750 (Beckman Coulter), CD86-Alexa488, CD83-PE-Cy5, CD25-PerCP / Cyanine5.5 CD11c-APC, and HLA-ABC-Pacific Blue (Biolegend). Samples were collected and analyzed by Navios flow cytometer, and data analysis was performed by Kaluza software (Beckman Coulter).

[0128] As shown in Figure 5A , the addition of IL-18 increased the expansion of the cells cultured from day 6 to day 12. Surprisingly, phenotypic analysis showed that the addition of IL-18 upregulated the expression of CD25, HLA-DR, and CD86 on day 3( Figure 5B ). However, the expression of HLA-DR and CD86 was downregulated on day 12( Figure 5C ).

[0129] Similarly, functional assays were also performed to evaluate the cytotoxicity and antigen-presenting activity of the cultured cells treated with different doses of IL-18. According to Figure 5D and 5E , the addition of IL-18 on day 12 negatively regulated the cytotoxicity and antigen-presenting cell activity of the cultured cells.

[0130] Therefore, the addition of 50 ng / mL IL-18 on day 3 enhanced the expression of CD25, HLA-DR, and CD86 of the modified natural killer cells, and the cell number of the modified natural killer cells continued to expand until day 12. However, long-term exposure to IL-18 may have the opposite effect on the cell size, phenotype, and function of the modified natural killer cells. Therefore, exposure to IL-18 for a sufficient time may be quite important for the generation of the modified natural killer cells.

[0131] Example 6: Treatment period of IL-18

[0132] In the presence of 30 ng / mL hIL-15 and 3 ng / mL hIL-12, CD3 - CD14 - CD19 - monocytes were cultured for 12 days. They were dynamically exposed to 50 ng / mL hIL-18 on days 0, 3, and 6. On day 6, the cells were subcultured, and the culture medium was changed every three days. The cultured cells were harvested on days 3, 6, 9, 12, and 15 and counted by trypan blue dye exclusion method.

[0133] Similarly, functional assays were also performed to evaluate the cytotoxicity and antigen-presenting activity of the cultured cells treated with IL-18 at different time periods.

[0134] As Figure 6A shown, compared with the cells to which IL-18 was added on day 0 (i.e., exposed for 3 days) or on days 0, 3, and 6 (i.e., exposed for 9 days), the addition of IL-18 on days 0 and 3 (i.e., exposed for 6 days) promoted the optimal expansion of the cultured cells on day 15. For cytotoxicity, compared with exposure to IL-18 for 3 days or 9 days, exposure to IL-18 for 6 days promoted the optimal cytotoxicity of the cultured cells on day 9 of culture ( Figure 6B ). For antigen-presenting activity, compared with exposure to IL-18 for 3 days or 9 days, exposure to IL-18 for 6 days increased the optimal antigen-presenting cell activity of the cultured cells ( Figure 6C ).

[0135] Most importantly, the antigen-presenting activity of the cultured cells exposed to IL-18 for 6 days reached the highest level on day 12 and then decreased significantly on day 15. Therefore, in the current cytokine environment, the optimal culture time for the cultured cells is less than 15 days.

[0136] Example 7: Preparation of initial cells

[0137] 40 mL of peripheral blood was collected from healthy volunteers and placed in a vacuum tube containing K 2 EDTA. The blood sample was mixed with an equal volume of pre-warmed phosphate-buffered saline (PBS) (Biological Industries, Israel). 40 mL aliquots of the diluted peripheral blood were placed in 50 mL centrifuge tubes and loaded with 10 mL of pre-warmed Ficoll-Paque TM PREMIUM. The centrifuge tubes were centrifuged at 2000 rpm for 30 minutes at room temperature. The monocytes in the interface layer were collected and washed once in PBS. The cell pellet was resuspended in MACS buffer at a density of 10 6 cells / 100 mL.

[0138] To remove CD14 + cells, CD19 + cells, and CD3 + cells, monocytes were immunomagnetically separated using a "QuadroMACS Separator" (Miltenyi Biotec Bergisch, Gladbach, Germany) according to the manufacturer's instructions. Briefly, monocytes were reacted with biotin - anti - CD14 antibody, biotin - anti - CD19 antibody, and biotin - anti - CD3 antibody, separated with a magnetic separator, and then CD14 - , CD19 - and CD3 - cell fractions were purified from unbound cells by washing. The enriched monocyte fraction was substantially free of CD14 + cells, CD19 + cells, and CD3 + cells.

[0139] Example 8: Cultivation of Modified Natural Killer Cells

[0140] After negative depletion, the purified CD14 - , CD19 - and CD3 - natural killer cell fraction from Example 7 was cultured as follows:

[0141] (a) On day 0, 1×10 6 cells / mL of CD14 - CD19 - CD3 - initial cells were contacted 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).

[0142] (b) Half of the initial cells were harvested and centrifuged in step (a), and then the cell pellet was resuspended on day 6 in a composition comprising AIM - V medium, HPL, IL - 15, and IL - 12.

[0143] (c) All the cultured cells from step (b) were collected on day 12.

[0144] Optionally, the composition used for culturing the cells can be replaced with fresh medium, and the same - composition media as on day 0 and day 6 are replaced on day 3 and day 9, respectively. For example, the initial cells can be cultured as follows:

[0145] (a) On day 0, 1×10 6 cells / mL of CD14 - CD19 - CD3 - natural killer cells are contacted 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).

[0146] (b) On day 3, the medium is replaced with a composition comprising AIM-V medium, HPL, IL-15, IL-12, and IL-18.

[0147] (c) On day 6, half of the cultured cells in step (b) are harvested and centrifuged, and then the cell pellet is resuspended in a composition comprising AIM-V medium, 4% w / w HPL, 30 ng / mL IL-15, and 3 ng / mL IL-12.

[0148] (d) On day 9, the medium is replaced with a composition comprising AIM-V medium, HPL, IL-15, and IL-12.

[0149] (e) On day 12, all the cultured cells in step (d) are collected.

[0150] Alternatively, fresh medium can be used instead of the composition for culturing cells, and the medium with the same components as on day 0 and day 6 is replaced on day 3 and day 9, respectively. For example, the initial cells can be cultured as follows:

[0151] (a) On day 0, 1×10 6 cells / mL of CD14 - CD19 - CD3 - natural killer cells are contacted 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).

[0152] (b) On day 3, the medium is replaced with a composition comprising AIM-V medium, HPL, IL-15, IL-12, and IL-18.

[0153] (c) On day 6, half of the cultured cells in step (b) are harvested and centrifuged, and then the cell pellet is resuspended 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.

[0154] (d) On day 9, replace the culture medium with a composition containing AIM-V medium, HPL, IL-15, IL-12, and IL-18.

[0155] (e) Collect all the cultured cells in step (d) on day 12.

[0156] Analyze the phenotype of the cultured cells in step (e) using a Navios flow cytometer (10 COLORS / 3 LASERS, serial number: AW40325, Beckman Coulter, USA), Kazula software version 2.1 (Beckman Coulter, USA), and the antibodies listed in Table 5.

[0157] Table 5. Reagents for phenotypic analysis of cultured functional assays and modified natural killer cells

[0158]

[0159]

[0160] Results: As Figure 8 shown, the obtained modified natural killer cells have natural killer cell and dendritic cell-related phenotypes of CD3 - CD19 - CD14 - CD56 hi CD16 dim NKG2D + CD11c + CD86 + HLA-DR + CD83 - .

[0161] Example 9: Determination of natural killer cell function

[0162] "Kill" assay

[0163] Evaluate the cytotoxicity of the modified natural killer cells of Example 8 using the PanToxilux kit (OncoImmunin). Use the human chronic myelogenous leukemia (CML) cell line K562 as the target cells and stain with TFL4 at the optimal concentration for 50 minutes. Co-culture the TFL-4-labeled target cells and the modified natural killer cells with the caspase substrate at 37 °C for 20 minutes. Harvest the cells and analyze the signal of TFL-4 + substrate + by flow cytometry. Caspase-positive cells represent cytotoxicity.

[0164] Results: AsFigure 9A As shown, the percentage of caspase-positive target cells (unmodified natural killer cells) was 0.77%, while the percentage of caspase-positive target cells (modified natural killer cells) was 40.2%. This result shows that the modified natural killer cells have cytotoxic effects on leukemia target cells K562.

[0165] Determination of Antigen Presentation Activity

[0166] The antigen presentation activity of the modified natural killer cells of Example 8 was evaluated by a mixed lymphocyte reaction (MLR). Enrich the responder cells (CD25 - PBMC), and stain them with the CellTrace TM CFSE Cell Proliferation Kit (Invitrogen). CSFE-labeled CD25 - PBMC and the modified natural killer cells were co-cultured at 37 °C for 5 days. hIL-2 and hIL-15 were added on the 1st and 3rd days to lower the threshold of TCR participation. Subsequently, the cells were collected, and the pattern of CFSE dilution was analyzed by flow cytometry.

[0167] Results: As Figure 9B shown, as identified by flow cytometry analysis, 4.32% of the responder cells were dividing. However, in the presence of the modified natural killer cells of Example 8, 24.71% of the cells were dividing. This result shows that the modified natural killer cells have antigen presentation activity on CD25 - PBMCs responder cells.

[0168] Although specific aspects of the invention have been described and illustrated, these aspects should be considered illustrative of the invention only and not limiting of the invention as construed in accordance with the claims. For all purposes, all publications and patent applications cited in this specification are incorporated herein by reference in their entirety as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference for all purposes. Although the above invention has been described in detail by way of the presentation of the drawings and examples for purposes of clear understanding, it will be apparent to those of ordinary skill in the art that certain changes and modifications can be made thereto without departing from the spirit or scope of the claims.

[0169] Without further elaboration, it is believed that those of ordinary skill in the art to which this invention pertains can make the fullest use of the invention based on the above description. Accordingly, the above embodiments should be construed as illustrative only and not in any way limiting the remainder of the invention. All publications cited herein are incorporated by reference.

Claims

1. A modified natural killer cell, comprising CD3 - CD19 - CD14 - CD56 hi CD16 dim NKG2D + CD11c + CD86 + HLA-DR + CD83 - with a phenotype, wherein the modified natural killer cell is cultured by the following method: Obtain a body fluid containing monocytes; Contact the monocytes with a first culture medium composed of IL-15, IL-12, IL-18, hematopoietic cell medium, and human platelet lysate for 3 to 6 days to obtain a first cultured cell population; After contact with the first culture medium, contact the first cultured cell population with a second culture medium composed of IL-15, IL-12, hematopoietic cell medium, and human platelet lysate for 3 to 6 days to obtain a second cultured cell population; and Isolate modified natural killer cells with a phenotype of CD3 - CD19 - CD14 - CD56 hi CD16 dim NKG2D + CD11c + CD86 + HLA-DR + CD83 - from the second cultured cell population.

2. A pharmaceutical composition comprising (a) the modified natural killer cells as claimed in claim 1; and (b) a pharmaceutically acceptable carrier or excipient.

3. Use of the modified natural killer cells as claimed in claim 1 in the preparation of a medicament for the treatment of cancer, said treatment comprising administering an effective amount of a medicament comprising the modified natural killer cells to a subject in need thereof.

4. The use according to claim 3, wherein the effective amount is 1×10 3 to 1×10 9 cells per dose.

5. The use as claimed in claim 3, wherein the modified natural killer cells are autologous or allogeneic.

6. The use as claimed in claim 3, wherein the modified natural killer cells are derived from peripheral blood, cord blood, or bone marrow.

7. The use as claimed in claim 3, further comprising in vitro expansion of the modified natural killer cells.

8. A method for culturing modified natural killer cells, comprising Obtain a body fluid containing monocytes; Contact the monocytes with a first culture medium composed of IL-15, IL-12, IL-18, hematopoietic cell medium, and human platelet lysate for 3 to 6 days to obtain a first cultured cell population; After contact with the first culture medium, contact the first cultured cell population with a second culture medium composed of IL-15, IL-12, hematopoietic cell medium, and human platelet lysate for 3 to 6 days to obtain a second cultured cell population; and Isolate modified natural killer cells having a phenotype of CD3 - CD19 - CD14 - CD56 hi CD16 dim NKG2D + CD11c + CD86 + HLA-DR + CD83 - from the second cultured cell population.

9. The method as claimed in claim 8, wherein the monocytes are derived from peripheral blood, cord blood, or bone marrow.

10. The method as claimed in claim 8, wherein the hematopoietic cell medium is AIM-V medium.

11. The method according to claim 8, wherein prior to contact with the first culture medium, cells having a CD3 - CD14 - CD19 - phenotype are selected as the monocytes.

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