CN116024169A - Method for culturing tumor-infiltrating lymphocytes - Google Patents

Abstract

The invention relates to a method for culturing tumor-infiltrating lymphocytes and a culture medium thereof. The invention provides tumor-infiltrating lymphocyte culture media comprising cytokines, stimulatory antibodies and cell culture components, wherein the cytokines comprise IL-21 and IL-15 and the stimulatory antibodies comprise CD28 antibodies and/or CD137 antibodies. The tumor infiltration NK cells obtained by the culture method have high activation degree and good potential tumor killing effect.

Description

Method for culturing tumor-infiltrating lymphocytes

Technical Field

The invention relates to the technical field of biology, in particular to a method for culturing tumor-infiltrating lymphocytes.

Background

Natural Killer (NK) cells are cytotoxic, lymphoid-derived Natural immune cells that are thought to account for about 5-20% of peripheral blood lymphocytes, with markers predominantly CD3-CD56+CD16+. In recent years, NK cells have received increasing attention for tumor immunotherapy. At present, it is common in clinic to add different combinations of cytokines to isolated peripheral blood PBMC of a patient for culture expansion. In order to obtain NK cells with tumor killing effect that can be used in clinical grade, the number of NK cells required is generally large. The collection of a large volume of blood from a patient is often difficult and the proportion of NK cells in the blood is also often low (5-20%), the treatment being focused on how to effectively expand NK cells. It has been reported that PBMC, CD3-CD56+ cells, CD56+ cells and the like are used as starting cells, and cytokines such as IL-2, IL-12, IL-15 or IL-21 and LPS or CD3 antibodies (such as OKT-3), CD16 antibodies and the like are added to conduct in vitro expansion culture of NK cells. However, the expansion of NK cells by isolated culture with cytokines, antibodies and/or LPS alone still does not satisfy the amount required for clinical treatment. Based on this, the use of cells expressing NK cell activating ligands on the membrane surface or cell lines genetically engineered to express cytokines on the cell membrane surface as feeder cells for large-scale expansion of NK cells in vitro after irradiation treatment has become a common means for in vitro culture of NK cells. Chinese patent CN107002039B describes a method for culturing NK cells using T cells, which uses cd4+ T cells as feeder cells to enhance NK cell proliferation, the cd4+ T cells used being shown as H9 or HuT78 cell lines. Chinese patent CN104321425B describes a method of inducing and expanding NK cells derived from PBMCs using irradiated Jurkat cells and irradiated continuous lymphocyte cell lines transformed by epstein barr virus (EBV-LCL) as feeder cells for co-culture with PBMCs. US20180155690A1 describes a high purity NK cell culture method using irradiated PBMCs as feeder cells and NK cells stimulated with CD16 antibodies.

In addition to blood PBMCs, infiltrated NK cells are also present in tumor tissue. Infiltration of NK may be predictive of a good prognosis for some neoplastic species such as breast, kidney, lung and head and neck squamous cell carcinomas. NK cells in the tumor bed may control tumor growth through direct interactions with tumor cells or through interactions with other immune cells. In solid tumors, especially highly advanced solid tumor tissues, NK cells are generally less invasive and infiltrated NK cells are generally inhibited by multiple pathways in the Tumor Microenvironment (TME).

Currently, NK cell immunotherapy has been successfully applied to partial hematological tumors, such as acute myeloid leukemia (acute myeloid leukemia, AML), but the clinical application of NK cell immunotherapy against solid tumors is not great, and the therapeutic clinical effect of NK therapy for solid tumors that has been applied is not ideal. Autologous or allogeneic peripheral blood PBMCs are now the most common NK cell source in the most common NK cell therapies. Compared with tumor infiltrating NK cells, peripheral blood NK cells lack chemokine receptors (such as CXCR3, CCR5, CCR7, etc.) that express tumor homing, resulting in difficulty in homing and infiltration of peripheral blood-derived NK cells into solid tumors, and very limited killing effect on solid tumors. But the number of infiltrated NK cells in the natural state in solid tumors is small, and the NK cells are also commonly in a state of being immunosuppressed by tumor microenvironment. Therefore, if tumor infiltrating NK cells with enough clinical treatment dosage can be obtained from solid tumor tissues through in vitro amplification culture and are reconverted from an intratumoral immunosuppressed state to an activated state, a brand new choice can be provided for NK cells to be used for tumor immune clinical treatment. However, there is still a lack of methods for efficient ex vivo expansion of cultured tumor infiltrating NK cells.

Disclosure of Invention

In a first aspect, the invention provides a tumor-infiltrating lymphocyte culture medium comprising a cytokine, a stimulatory antibody and a cell culture composition, wherein the cytokine comprises IL-21 and IL-15, and the stimulatory antibody comprises CD28 antibody and/or CD137 antibody.

In one or more embodiments, the tumor-infiltrating lymphocyte medium is a tumor-infiltrating NK cell medium.

In one or more embodiments, the tumor-infiltrating lymphocyte medium is used to culture tumor-infiltrating NK cells. Preferably, the tumor-infiltrating NK cells are derived from tumor tissue.

In one or more embodiments, the tumor-infiltrating lymphocyte medium is used to culture a mixture of tumor-infiltrating NK cells and T cells. Preferably, the tumor infiltrating NK cells and T cells are from the same tumor tissue or tumor body fluid.

In one or more embodiments, the medium is a serum medium or a serum-free medium. In one or more embodiments, the medium further comprises serum. Preferably, the serum concentration is 0-10 (v/v)%, more preferably 3-5 (v/v)%.

In one or more embodiments, the cell culture composition is a lymphocyte basal medium.

In one or more embodiments, the concentration of IL-21 in the medium is 50-4000U/mL, preferably 100-3000U/mL, more preferably 200-2000U/mL, more preferably 500-1000U/mL.

In one or more embodiments, the concentration of IL-15 in the medium is 500-4000U/mL, preferably 1000-3000U/mL, more preferably 1000-2000U/mL.

In one or more embodiments, the concentration of CD28 antibody in the medium is 0-200. Mu.g/mL, preferably 5-100. Mu.g/mL, more preferably 10-50. Mu.g/mL.

In one or more embodiments, the concentration of CD137 antibodies in the medium is 0-200 μg/mL, preferably 10-100 μg/mL, more preferably 10-25 μg/mL.

In one or more embodiments, the tumor-infiltrating lymphocyte medium further comprises an activated antibody comprising any one, two, or three selected from the group consisting of a CD40 antibody, an OX-40 antibody, and a CD16 antibody.

In one or more embodiments, the activating antibodies include (1) CD16 antibodies, (2) CD40 antibodies and CD16 antibodies, (3) OX-40 antibodies and CD16 antibodies.

In one or more embodiments, the concentration of CD40 antibody in the medium is 0-200. Mu.g/mL, preferably 10-100. Mu.g/mL, more preferably 10-50. Mu.g/mL.

In one or more embodiments, the concentration of OX-40 antibody in the medium is 0-200 μg/mL, preferably 10-50 μg/mL, more preferably 25 μg/mL.

In one or more embodiments, the concentration of CD16 antibody in the medium is 0-200. Mu.g/mL, preferably 10-100. Mu.g/mL, more preferably 10-25. Mu.g/mL.

In one or more embodiments, the cytokine further comprises any one or more selected from the group consisting of IL-7, IL-12, IL-18, and TNF alpha. In one or more embodiments, the cytokines further include IL-7 and any one or more selected from the group consisting of IL-12, IL-18, and TNF alpha. Preferably, the cytokines further include (1) IL-7, (2) IL-7 and IL-12, (3) IL-7 and IL-18, (4) IL-7, IL-18 and TNF alpha, (5) IL-7, IL-12 and IL-18, or (6) IL-7, IL-12, IL-18 and TNF alpha.

In one or more embodiments, the concentration of IL-7 in the medium is from 0 to 1500U/mL, preferably from 100 to 1000U/mL, more preferably from 200 to 500U/mL.

In one or more embodiments, the medium in IL-12 concentration of 0-1500U/mL, preferably 100-1000U/mL, more preferably 100-200U/mL.

In one or more embodiments, the concentration of IL-18 in the medium is from 0 to 1500U/mL, preferably from 200 to 1000U/mL, more preferably from 200 to 500U/mL.

In one or more embodiments, the concentration of TNFα in the medium is from 0 to 500pg/mL, preferably from 10 to 100pg/mL, more preferably from 10 to 50pg/mL.

In one or more embodiments, the medium further comprises an immune checkpoint inhibitor.

In one or more embodiments, the immune checkpoint inhibitor includes any one or more selected from the group consisting of PD-1 antibodies, CTLA-4 antibodies, TIM3 antibodies, lag3 antibodies, and TIGIT antibodies.

In one or more embodiments, the concentration of PD-1 antibodies in the medium is 0-200. Mu.g/mL, preferably 10-100. Mu.g/mL, more preferably 10-30. Mu.g/mL.

In one or more embodiments, the concentration of CTLA-4 antibodies in the medium is from 0 to 200. Mu.g/mL, preferably from 10 to 100. Mu.g/mL, more preferably from 10 to 30. Mu.g/mL.

In one or more embodiments, the concentration of TIM3 antibody in the medium is 0-200. Mu.g/mL, preferably 10-100. Mu.g/mL, more preferably 10-30. Mu.g/mL.

In one or more embodiments, the concentration of Lag3 antibody in the medium is 0-200. Mu.g/mL, preferably 10-100. Mu.g/mL, more preferably 10-30. Mu.g/mL.

In one or more embodiments, the concentration of TIGIT antibodies in the medium is in the range of 0-200 μg/mL, preferably 10-100 μg/mL, more preferably 10-30 μg/mL.

In a second aspect, there is provided a tumor-infiltrating lymphocyte medium comprising a stimulating factor, comprising a CD3 antibody, a stimulating factor and a cell culture composition, optionally further comprising a cytokine, wherein the stimulating factor comprises a CD28 antibody, and the cytokine comprises IL-2 and/or IL-15.

In one or more embodiments, the concentration of CD3 antibody in the medium is 0-200. Mu.g/mL, preferably 10-100. Mu.g/mL, more preferably 10-30. Mu.g/mL.

In one or more embodiments, the concentration of IL-2 in the medium is from 0 to 2000. Mu.g/mL, preferably from 0 to 1000. Mu.g/mL, more preferably from 0 to 500. Mu.g/mL.

In one or more embodiments, the concentration of IL-15 in the medium is from 0 to 2000. Mu.g/mL, preferably from 0 to 1000. Mu.g/mL, more preferably from 0 to 500. Mu.g/mL.

In one or more embodiments, the CD3 antibody is OKT3.

In one or more embodiments, the medium is a serum medium or a serum-free medium. In one or more embodiments, the medium further comprises serum. Preferably, the serum concentration is 0-10 (v/v)%, more preferably 3-5 (v/v)%.

In one or more embodiments, the tumor-infiltrating lymphocyte medium containing a stimulating factor is used to culture a mixture of tumor-infiltrating NK cells and T cells. Preferably, the tumor infiltrating NK cells and T cells are from the same tumor tissue or tumor body fluid.

In one or more embodiments, the stimulating factor further comprises a ligand molecule for an NK cell activating receptor; preferably, the NK cell activating receptor comprises any one or more of NKG2D, NKp, NKp44, NKp30, NKG2C, DNAM-1 and 2B 4; preferably, the NK cell activating receptor comprises NKG2D and the ligand molecule of NKG2D comprises MICA and/or MICB.

In one or more embodiments, the stimulating factor comprises (1) a CD28 antibody and (2) MICA and/or MICB.

In one or more embodiments, the stimulating factor is a stimulating factor in solution or immobilized (e.g., coupled) on a substrate (e.g., coated on a solid support or coupled to magnetic beads). Preferably, the stimulating factor is coupled to a substrate, e.g. coated on the wall of the culture vessel.

In one or more embodiments, the coating is applied at a concentration of each stimulating factor in the coating solution of 0-10. Mu.g/mL, preferably 1-5. Mu.g/mL.

In one or more embodiments, the concentration of each stimulating factor in the solution is from 0 to 500. Mu.g/mL, preferably from 1 to 100. Mu.g/mL.

In a third aspect, the present disclosure provides a method of culturing tumor-infiltrating NK cells, comprising the steps of:

(1) Culturing tumor tissue or tumor body fluid to obtain a first cell population,

(2) Culturing the first cell population in the presence of a stimulating factor to obtain a second cell population comprising tumor-infiltrating NK cells, the stimulating factor promoting proliferation of the tumor-infiltrating NK cells,

optionally (3) sorting tumor infiltrating NK cells, the sorting labels being CD3-CD56+ and/or CD3-CD16+.

In one or more embodiments, tumor-infiltrating T cells are present in the tumor tissue and/or tumor body fluid, preferably, the tumor-infiltrating T cells are from the same tumor tissue and/or tumor body fluid as the tumor-infiltrating NK cells.

In one or more embodiments, the culturing is ex vivo.

In one or more embodiments, the culturing of step (1) uses the tumor-infiltrating lymphocyte medium described in the first aspect herein.

In one or more embodiments, the tumor tissue is fragmented tumor tissue or enzymatically digested tumor tissue.

In one or more embodiments, the tumor body fluid is pleural effusion or peritoneal effusion of a tumor patient.

In one or more embodiments, the tumor is a solid tumor.

In one or more embodiments, the tumor comprises a respiratory tumor, a digestive tumor, a urinary tumor, a nervous system tumor, a reproductive tumor, a skin tumor; preferably comprising one or more selected from the group consisting of: liver cancer, gastrointestinal cancer, lung cancer, pancreatic cancer, ovarian cancer, stomach cancer, colon cancer, melanoma, endometrial cancer, cervical cancer, uterine sarcoma, vulval cancer, breast cancer, glioma, prostate cancer, fallopian tube cancer, laryngeal cancer, thyroid cancer, gall bladder cancer, kidney cancer, bladder cancer and brain cancer.

In one or more embodiments, the tumor is a metastatic cancer or a recurrent cancer.

In one or more embodiments, step (1) comprises: culturing a tumor tissue or tumor fluid with a tumor-infiltrating lymphocyte medium as described in the first aspect herein to obtain a first population of cells.

In one or more embodiments, the stimulating factor comprises a CD28 antibody. In one or more embodiments, the stimulating factor further comprises a ligand molecule for an NK cell activating receptor; preferably, the NK cell activating receptor comprises any one or more of NKG2D, NKp, NKp44, NKp30, NKG2C, DNAM-1 and 2B 4; preferably, the NK cell activating receptor comprises NKG2D and the ligand molecule of NKG2D comprises MICA and/or MICB.

In one or more embodiments, the stimulating factor comprises (1) a CD28 antibody, and (2) MICA and/or MICB.

In one or more embodiments, the stimulating factor is a stimulating factor in solution or immobilized (e.g., coupled) on a substrate (e.g., coated on a solid support or coupled to magnetic beads). Preferably, the stimulating factor is coupled to a substrate, e.g. coated on the wall of the culture vessel.

In one or more embodiments, the coating is applied at a concentration of each stimulating factor in the coating solution of 0-10. Mu.g/mL, preferably 1-5. Mu.g/mL.

In one or more embodiments, the concentration of each stimulating factor in the solution is from 0 to 500. Mu.g/mL, preferably from 1 to 100. Mu.g/mL.

In one or more embodiments, the culturing of step (2) uses a tumor infiltrating lymphocyte medium comprising a stimulating factor as described in the second aspect herein.

Also provided herein is a method of culturing tumor infiltrating NK cells comprising the steps of:

(1) Culturing tumor-infiltrating NK cells in the presence of tumor-infiltrating T cells to obtain a first cell population, preferably, the tumor-infiltrating T cells and the tumor-infiltrating NK cells are from the same tumor tissue and/or tumor body fluid,

(2) Culturing the first cell population in the presence of a stimulating factor to obtain a second cell population comprising tumor-infiltrating NK cells, the stimulating factor promoting proliferation of the tumor-infiltrating NK cells,

optionally (3) sorting tumor infiltrating NK cells, the sorting labels being CD3-CD56+ and/or CD3-CD16+.

Other features of the method are as described in the culture method of the third aspect herein.

The invention also provides tumor-infiltrating NK cells obtained by culturing the tumor-infiltrating lymphocyte medium of any embodiment herein or by the culture method of any embodiment herein.

The invention also provides a pharmaceutical composition comprising tumor-infiltrating NK cells obtained by culturing the tumor-infiltrating lymphocyte culture medium according to any embodiment herein and/or by the culture method according to any embodiment herein and pharmaceutically acceptable excipients.

The invention also provides application of the tumor-infiltrating NK cells obtained by the culture medium of the tumor-infiltrating lymphocytes in any embodiment or the culture method of any embodiment and/or the pharmaceutical composition containing the tumor-infiltrating NK cells in preparation of antitumor drugs.

In one or more embodiments, the tumor is a solid tumor.

In one or more embodiments, the tumor comprises a respiratory tumor, a digestive tumor, a urinary tumor, a nervous system tumor, a reproductive tumor, a skin tumor; preferably comprising one or more selected from the group consisting of: liver cancer, gastrointestinal cancer, lung cancer, pancreatic cancer, ovarian cancer, stomach cancer, colon cancer, melanoma, endometrial cancer, cervical cancer, uterine sarcoma, vulval cancer, breast cancer, glioma, prostate cancer, fallopian tube cancer, laryngeal cancer, thyroid cancer, gall bladder cancer, kidney cancer, bladder cancer and brain cancer.

The invention also provides application of the tumor-infiltrating T cells in culturing tumor-infiltrating NK cells. Preferably, the tumor-infiltrating T cells and the tumor-infiltrating NK cells are from the same tumor tissue and/or tumor body fluid.

In one or more embodiments, the culturing uses the tumor-infiltrating lymphocyte medium described in any embodiment herein or uses the culturing method described in any embodiment herein.

Advantages of the invention

1) According to the culture method and the tumor infiltrating NK cells cultured by the culture medium, the NK cells with the magnitude of billions can be obtained without adding feeder cells, and the process is greatly simplified compared with the traditional blood-derived NK cell culture;

2) The tumor infiltration NK cells obtained by the culture method have high activation degree, and the proportion of the CD8+ NK cells to the memory-like NK cells is high, so that the method has a good potential tumor killing effect.

3) The culture medium of the invention can be free of or less of IL-2, thereby greatly reducing the dependence of cultured NK cells on IL-2, reducing the use or non-use of IL-2 injection after cell reinfusion in potential clinical application, and reducing adverse reaction caused by the use of IL-2 in clinical application.

4) The invention takes tumor tissues (obtained by means of operation, puncture and the like) of tumor patients as raw materials, and skillfully utilizes cells inherent in the tumor tissues, in particular T cells, as feeder cells. By using a culture medium with a well-designed combination factor for culture, the infiltrated NK cells and the T cells in the tumor tissue are simultaneously cultured, then the T cells and the NK cells are stimulated comprehensively, and the infiltrated NK cells of the tumor are greatly amplified under the stimulation of the activated T cells and other stimulating factors to reach the billion order. The tumor infiltrating T cells used as feeder cells are autologous cells, rejection reaction possibly caused by allogeneic feeder cells does not exist, the cell population to be obtained is not required to be removed to be used as a feedback cell population, and the cell population expressing NK cell markers can be enriched through special sorting. The number of tumor-infiltrating NK cells obtained by the culture medium and the culture method can reach billions, and the inhibition of tumor microenvironment is better removed through multiple factor stimulation under the in-vitro condition, so that the tumor-infiltrating NK cells are fully activated. Meanwhile, the re-activated tumor infiltration NK cells naturally have higher tumor homing capability, can effectively home to tumor parts, and greatly improve the tumor killing effect.

Drawings

Fig. 1: flow cell phenotype of TIL cells comprising TINK cells obtained from culture in endometrial cancer.

Fig. 2: flow cell phenotype of TIL cells comprising TINK cells obtained from culture in breast cancer.

Fig. 3: flow cell phenotype of TIL cells comprising TINK cells obtained from culture in breast cancer.

Fig. 4: flow cell phenotype of TIL cells comprising TINK cells obtained from ovarian cancer culture.

Fig. 5: flow cell phenotype of TIL cells comprising TINK cells obtained from cervical cancer culture.

Fig. 6: flow cell phenotype of TIL cells comprising TINK cells obtained from culture in endometrial cancer.

Fig. 7: flow cell phenotype of TIL cells comprising TINK cells obtained from cervical cancer culture.

Fig. 8: flow cell phenotype of TIL cells comprising TINK cells obtained from culture in endometrial cancer.

Fig. 9: and the killing effect of PBNK and TINK of cervical cancer patients on RTCA of tumor cells.

Fig. 10: the effect of PBNK and TINK of endometrial cancer on RTCA killing of tumor cells.

Fig. 11: and the killing effect of PBNK and TINK of cervical cancer patients on RTCA of tumor cells.

Detailed Description

The inventors have found that a large number of infiltrating NK cells can be cultured from tumor tissue or tumor body fluid containing infiltrating T cells and infiltrating NK cells using the tumor infiltrating lymphocyte culture medium described herein.

Thus, the invention comprises the use of tumor-infiltrating T cells derived from the same tumor tissue and/or tumor body fluid as said tumor-infiltrating NK cells in the culture of tumor-infiltrating NK cells.

The tumor described herein may be any tumor, preferably a solid tumor. Exemplary solid tumors include, but are not limited to: respiratory system tumors, digestive system tumors, urinary system tumors, nervous system tumors, reproductive system tumors, skin tumors; preferably comprising one or more selected from the group consisting of: liver cancer, gastrointestinal cancer, lung cancer, pancreatic cancer, ovarian cancer, stomach cancer, colon cancer, melanoma, endometrial cancer, cervical cancer, uterine sarcoma, vulval cancer, breast cancer, glioma, prostate cancer, fallopian tube cancer, laryngeal cancer, thyroid cancer, gall bladder cancer, kidney cancer, bladder cancer and brain cancer. Prior to culturing, the tumor tissue may be treated, for example, by cutting into pieces or by enzymatic digestion. Tumor fluids include pleural or peritoneal effusions of tumor patients or any other fluid of tumor patients containing infiltrating T cells and infiltrating NK cells.

The invention provides a tumor infiltrating lymphocyte culture medium which comprises cytokines, a stimulating antibody and cell culture components. The invention also includes tumor-infiltrating NK cells obtained from the culture of any of the tumor-infiltrating lymphocyte culture media described herein.

Cytokines in the tumor-infiltrating lymphocyte medium include IL-21 and IL-15.IL-21 concentrations were 0, 1, 100, 200, 250, 500, 1000, 2000, 3000U/mL, or a range between any two of the above. IL-15 concentrations were 0, 1, 100, 200, 250, 500, 1000, 1500, 2000, 3000U/mL, or a range between any two of the above.

The cytokine may also include any one or more selected from the group consisting of IL-7, IL-12, IL-18, and TNF alpha. Preferably, the cytokines include, in addition to IL-2 and IL-15, IL-7 and any one or more selected from the group consisting of IL-12, IL-18 and TNF alpha. Illustratively, the cytokines include, in addition to IL-2 and IL-15, (1) IL-7, (2) IL-7 and IL-12, (3) IL-7 and IL-18, (4) IL-7, IL-18 and TNF alpha, (5) IL-7, IL-12 and IL-18, or (6) IL-7, IL-12, IL-18 and TNF alpha. If the cytokine is contained, the concentration is as follows: IL-7: 0. 1, 100, 200, 250, 500, 1000, 1500, 2000U/mL, or a range between any two of the foregoing values; IL-12: 0. 1, 100, 200, 250, 500, 1000, 1500, 2000U/mL, or a range between any two of the foregoing values; IL-18: 0. 1, 100, 200, 250, 500, 1000, 1500, 2000U/mL, or a range between any two of the foregoing values; tnfα: 0. 1, 50, 100, 200, 250, 500, 1000pg/mL, or a range between any two of the foregoing values.

The stimulating antibodies in the tumor-infiltrating lymphocyte medium comprise CD28 antibodies and/or CD137 antibodies. When included, CD28 antibody concentrations were: 1. 5, 10, 15, 20, 25, 30, 50, 100, 500, 1000 μg/mL, or a range between any two of the foregoing values; the CD137 antibody concentration was: 1. 5, 10, 15, 20, 25, 30, 50, 100, 500, 1000 μg/mL, or a range between any two of the foregoing values. The CD28 and CD137 antibodies may be any activated antibody known in the art that recognizes CD28 and CD 137.

The tumor-infiltrating lymphocyte medium optionally further comprises an activating antibody, including any one, two or three selected from the group consisting of CD40 antibody, OX-40 antibody and CD16 antibody, preferably including (1) CD16 antibody, (2) CD40 antibody and CD16 antibody, (3) OX-40 antibody and CD16 antibody. These antibody concentrations are each independently 0, 1, 5, 10, 15, 20, 25, 30, 50, 100, 500, 1000 μg/mL, or a range between any two of the above. The CD40 antibody, OX-40 antibody and CD16 antibody may be any antibody known in the art that recognizes CD40, OX-40 and CD 16.

The tumor-infiltrating lymphocyte medium further comprises an immune checkpoint inhibitor. The immune checkpoint inhibitor includes any one or more selected from the group consisting of PD-1 antibodies, CTLA-4 antibodies, TIM3 antibodies, lag3 antibodies, and TIGIT antibodies. The antibody may be any antibody known in the art that recognizes PD-1, CTLA-4, TIM3, lag3 and TIGIT. The concentration of these immune checkpoint inhibitors is each independently 0, 1, 5, 10, 15, 20, 25, 30, 50, 100, 500, 1000 μg/mL, or a range between any two of the above.

Herein, the term "antibody" includes monoclonal antibodies (mabs) (including full length antibodies, which have an immunoglobulin Fc region), antibody compositions having multi-epitope specificity, multi-specific antibodies (e.g., bispecific antibodies), diabodies and single chain molecules, and antibody fragments, particularly antigen-binding fragments, e.g., fab, F (ab') 2, and Fv.

In a specific embodiment, the tumor-infiltrating lymphocyte medium comprises IL-15, IL-21, CD137 antibodies, and cell culture components. Illustratively, the medium is as set forth in medium No. 1 of table 1. According to the method of the present invention, the medium can be used to culture tumor-infiltrating NK cells from vulvar cancer tissue.

In another specific embodiment, the tumor-infiltrating lymphocyte medium comprises IL-15, IL-21, CD28 antibodies, and cell culture components. Illustratively, the medium is as set forth in medium No. 2 of table 1. According to the method of the invention, the medium can be used to culture tumor-infiltrating NK cells from ovarian cancer tissue.

In another specific embodiment, the tumor-infiltrating lymphocyte medium comprises IL-15, IL-21, CD28 antibodies, and cell culture components. Illustratively, the medium is as set forth in medium No. 3 of table 1. According to the method of the present invention, the above-described medium can be used for culturing tumor-infiltrating NK cells from ovarian cancer tissue.

In another specific embodiment, the tumor-infiltrating lymphocyte medium comprises IL-15, IL-21, CD28 antibodies, CD137 antibodies, and cell culture components. Illustratively, the medium is as set forth in medium No. 4 of table 1. According to the method of the present invention, the above-described medium can be used for culturing tumor-infiltrating NK cells from endometrial cancer tissue.

In another specific embodiment, the tumor-infiltrating lymphocyte medium comprises IL-15, IL-21, CD28 antibodies, CD137 antibodies, CD16 antibodies, and cell culture components. Illustratively, the medium is as set forth in medium No. 5 of table 1. According to the method of the present invention, the above-described medium can be used for culturing tumor-infiltrating NK cells from breast cancer tissue.

In another specific embodiment, the tumor-infiltrating lymphocyte medium comprises IL-15, IL-21, CD28 antibody, CD137 antibody, CD16 antibody, CD40 antibody, and cell culture components. Illustratively, the medium is as set forth in medium No. 6 of table 1. According to the method of the present invention, the above-described medium can be used for culturing tumor-infiltrating NK cells from cervical cancer tissue.

In another specific embodiment, the tumor-infiltrating lymphocyte medium comprises IL-15, IL-21, CD28 antibody, CD137 antibody, CD16 antibody, OX-40 antibody, and a cell culture composition. Illustratively, the medium is as set forth in medium No. 7 of table 1. According to the method of the present invention, the above-described medium can be used for culturing tumor-infiltrating NK cells from ovarian cancer tissue.

In another specific embodiment, the tumor-infiltrating lymphocyte medium comprises IL-7, IL-15, IL-21, CD28 antibodies, CD137 antibodies, CD16 antibodies, CD40 antibodies, and cell culture components. Illustratively, the medium is as set forth in medium No. 8 of table 1. According to the method of the present invention, the above-described medium can be used for culturing tumor-infiltrating NK cells from cervical cancer tissue.

In another specific embodiment, the tumor-infiltrating lymphocyte medium comprises IL-7, IL-12, IL-15, IL-21, CD28 antibody, CD137 antibody, CD16 antibody, OX-40 antibody, and cell culture composition. Illustratively, the medium is as set forth in medium No. 9 of table 1. According to the method of the present invention, the above-described medium can be used for culturing tumor-infiltrating NK cells from ovarian cancer tissue.

In another specific embodiment, the tumor-infiltrating lymphocyte medium comprises IL-7, IL-15, IL-18, IL-21, CD28 antibody, CD137 antibody, CD16 antibody, CD40 antibody, and cell culture components. Illustratively, the medium is as set forth in medium No. 10 of table 1. According to the method of the present invention, the above-described medium can be used for culturing tumor-infiltrating NK cells from breast cancer tissue.

In another specific embodiment, the tumor-infiltrating lymphocyte medium comprises IL-7, IL-15, IL-21, CD28 antibody, CD137 antibody, CD16 antibody, CD40 antibody, PD-1 antibody, and cell culture components. Illustratively, the medium is as set forth in medium No. 11 of table 1. According to the method of the present invention, the above-described medium can be used for culturing tumor-infiltrating NK cells from ovarian cancer tissue.

In another specific embodiment, the tumor-infiltrating lymphocyte medium comprises IL-7, IL-15, IL-21, CD28 antibody, CD137 antibody, CD16 antibody, CD40 antibody, LAG-3 antibody, and cell culture components. Illustratively, the medium is as set forth in medium No. 12 of table 1. According to the method of the present invention, the above-described medium can be used for culturing tumor-infiltrating NK cells from lung cancer tissue.

In another specific embodiment, the tumor-infiltrating lymphocyte medium comprises IL-7, IL-15, IL-18, IL-21, TNF- α, CD28 antibody, CD137 antibody, CD16 antibody, CD40 antibody, CTLA-4 antibody, and cell culture components. Illustratively, the medium is as set forth in medium No. 13 of table 1. According to the method of the invention, the culture medium can be used for culturing tumor-infiltrating NK cells from brain glioma tissues.

In another specific embodiment, the tumor-infiltrating lymphocyte medium comprises IL-7, IL-12, IL-15, IL-18, IL-21, CD28 antibodies, CD137 antibodies, CD16 antibodies, CD40 antibodies, LAG-3 antibodies, TIGIT antibodies, and cell culture components. Illustratively, the medium is as set forth in medium No. 14 of table 1. According to the method of the present invention, the above-described medium can be used for culturing tumor-infiltrating NK cells from ovarian cancer tissue.

In another specific embodiment, the tumor-infiltrating lymphocyte medium comprises IL-7, IL-12, IL-15, IL-18, IL-21, TNF- α, CD28 antibody, CD137 antibody, CD16 antibody, CD40 antibody, LAG-3 antibody, CTLA-4 antibody, PD-1 antibody, and cell culture composition. Illustratively, the medium is as set forth in medium No. 15 of table 1. According to the method of the present invention, the above-described medium can be used for culturing tumor-infiltrating NK cells from cervical cancer tissue.

Also provided herein is a tumor-infiltrating lymphocyte medium comprising a stimulating factor, comprising a CD3 antibody, a stimulating factor, and a cell culture composition. The concentration of CD3 antibody in the medium is 0, 1, 5, 10, 15, 20, 25, 30, 50, 100, 500, 1000 μg/mL, or a range between any two of the above. The CD3 antibody may be any antibody known in the art that recognizes CD3, such as OKT3.

The stimulating factor includes a CD28 antibody. The stimulating factor may also include ligand molecules for NK cell activating receptors; preferably, the NK cell activating receptor comprises any one or more of NKG2D, NKp, NKp44, NKp30, NKG2C, DNAM-1 and 2B 4; preferably, the NK cell activating receptor comprises NKG2D and the ligand molecule of NKG2D comprises MICA and/or MICB. Specifically, the stimulating factor comprises (1) a CD28 antibody and (2) MICA and/or MICB. The stimulating factor may be immobilized (e.g., coupled) on a substrate (e.g., coated on a solid support or coupled to magnetic beads) in a medium. Preferably, the stimulating factor is coupled to a substrate, e.g. coated on the wall of the culture vessel. The concentration of the stimulating factors in the solution is respectively and independently: 0. 5, 10, 15, 20, 25, 30, 50, 100, 500 μg/mL, or a range between any two of the foregoing values.

The tumor-infiltrating lymphocyte medium containing a stimulating factor described herein optionally further comprises a cytokine. The cytokines mainly include IL-2 and/or IL-15. The concentration of IL-2 and IL-15 are each independently 0, 5, 10, 15, 20, 25, 30, 50, 100, 500, 1000U/mL, or a range between any two of the foregoing values.

In specific embodiments, the tumor-infiltrating lymphocyte medium containing a stimulating factor contains IL-2, CD3 antibodies, CD28 antibodies, and MICA and/or MICB; or IL-15, CD3 antibodies, CD28 antibodies, MICA and/or MICB; or IL-2, IL-15, CD3 antibodies, CD28 antibodies, MICA and/or MICB. Exemplary tumor infiltrating lymphocyte culture media containing stimulatory factors are shown in tables 2 and 3.

Herein, the cell culture component in the tumor-infiltrating lymphocyte medium is a lymphocyte basal medium. The lymphocyte basal medium described herein contains substances and energy required to satisfy biochemical reactions such as new lymphocyte synthesis, lymphocyte metabolism, etc., including but not limited to: water, energy and carbon sources, nitrogen sources, vitamins, inorganic salts.

Energy and carbon sources are used to sustain cell life and support cell growth, and mainly include sugars, products of glycolysis, and glutamine, with other amino acids being secondary energy and carbon source materials. The saccharides which can be utilized by the cells are mainly hexoses, and at present, most of the saccharides are mainly used as main carbon sources and energy sources of the cells in vitro culture, so that the cell culture medium basically contains the saccharides, and the content of the saccharides is generally 5-25 mmol/L.

The nitrogen source includes amino acids, especially essential amino acids.

Vitamins are classified into two types, namely water-soluble vitamins and fat-soluble vitamins, wherein the water-soluble vitamins mainly comprise pantothenic acid, vitamin B12, folic acid, nicotinamide, pyridoxal, thiamine, riboflavin, vitamin C, choline, inositol and the like; the fat-soluble vitamins mainly comprise vitamins A, D, E, K and the like; ATP and coenzyme A are also directly adopted in some culture solutions; biotin is also present in most media.

Inorganic salt is essential nutrient component for maintaining life activity of cells, and is mainly Na ⁺ 、K ⁺ 、Ca ²⁺ 、Mg ²⁺ 、Cl ^- 、PO ₄ ^3- 、SO ₄ ^2- 、HCO ₃ ^- And the like, which mainly serve to maintain the osmotic pressure balance of the cell culture fluid and participate in the metabolic activity of cells. By providing sodium, K ⁺ And Ca ²⁺ Helping cells regulate cell membrane function. Ca (Ca) ²⁺ And Mg (magnesium) ²⁺ Mainly participate in signal transduction, energy metabolism, fatty acid synthesis, ribosome stabilization, protein synthesis and other physiological functions. PO (Positive oxide) ₄ ^3- 、SO ₄ ^2- 、HCO ₃ ^- Is an anion required for the matrix and is also a regulator of intracellular charge. Phosphorus plays an important role in the growth, metabolism and regulation of cells, and phosphorus-containing compounds such as nucleic acids, phospholipids, proteins are essential components constituting cells, and ATP and ADP are indispensable compounds for energy generation, storage and utilization. In addition to ensuring that the concentration of the ions in the medium meets the requirements, the balance of the species and the proportion among the ions is ensured. In addition, trace elements such as iron, cobalt, nickel, selenium, iodine, copper, zinc, manganese, chromium, molybdenum, fluorine, etc. have promoting effects on cell growth metabolism and product synthesis. Iron is involved in oxygen transport in cells; cobalt is a component of vitamin B12 and participates in the synthesis of folic acid and fatty acid; nickel activates enzymes having important functions in cells, such as deoxyribonuclease and acetyl-CoA synthetase, and also has a function of stabilizing the nucleic acid structure; selenium in sodium selenite is taken as prosthetic group of glutathione peroxidase, has anti-peroxide capability, participates in eliminating fatty acid peroxide in cells, and improves the growth rate and activity of the cells.

Exemplary lymphocyte basal medium includes AIM-V, X-VIVO 15, CTS ^TM OpTmizer ^TM 。

The tumor-infiltrating lymphocyte culture medium described herein may or may not contain serum or serum replacement components. Serum replacement ingredients can be routinely selected by those skilled in the art, for example, commercially available serum replacement. Serum may be included in the basal medium at a final concentration of 0-10 (v/v)% in the tumor-infiltrating lymphocyte medium; the corresponding concentration of the serum replacement may be determined by one skilled in the art, for example, in accordance with the instructions for the serum replacement.

Also provided herein is a method of culturing tumor infiltrating NK cells comprising the steps of: (1) Culturing a tumor tissue or tumor body fluid to obtain a first cell population, (2) culturing the first cell population in the presence of a stimulating factor that stimulates proliferation of tumor infiltrating NK cells to obtain a second cell population comprising tumor infiltrating NK cells. Herein, tumor-infiltrating T cells are present in the tumor tissue and/or tumor body fluid, said tumor-infiltrating NK cells being cultured in the presence of tumor-infiltrating T cells; preferably, the tumor-infiltrating T cells and the tumor-infiltrating NK cells are from the same tumor tissue and/or tumor body fluid. The invention also includes tumor-infiltrating NK cells obtained by culture in the culture methods described herein.

In one or more embodiments, the method for culturing tumor-infiltrating NK cells from vulvar cancer tissue, comprising the steps of: (1) Culturing vulvar cancer tissue using an X-VIVO 15 medium comprising IL-15, IL-21, CD137 antibody and human AB serum to obtain a first cell population, (2) culturing the first cell population with an X-VIVO 15 medium comprising IL-2, CD3 antibody in the presence of CD28 antibody to obtain a second cell population comprising tumor infiltrating NK cells. Illustratively, the media of steps (1) and (2) are as shown in the T001 sample of Table 3.

In one or more embodiments, the method is for culturing tumor-infiltrating NK cells from ovarian cancer tissue, comprising the steps of: (1) Culturing ovarian cancer tissue using AIM-V medium comprising IL-15, IL-21, CD28 antibody and human AB serum to obtain a first cell population, (2) culturing the first cell population in the presence of CD28 antibody, MICA with X-VIVO 15 medium comprising IL-2, CD3 antibody to obtain a second cell population comprising tumor infiltrating NK cells. Illustratively, the media of steps (1) and (2) are as shown in the T002 sample of Table 3.

In one or more embodiments, the method is for culturing tumor-infiltrating NK cells from ovarian cancer tissue, comprising the steps of: (1) Culturing ovarian cancer tissue using AIM-V medium comprising IL-15, IL-21, CD28 antibody and human AB serum to obtain a first cell population, (2) culturing the first cell population in the presence of CD28 antibody, MICB with X-VIVO 15 medium comprising IL-15, CD3 antibody to obtain a second cell population comprising tumor infiltrating NK cells. Illustratively, the media of steps (1) and (2) are as shown in the T003 samples of Table 3.

In one or more embodiments, the method for culturing tumor-infiltrating NK cells from endometrial cancer tissue comprises the steps of: (1) CTS Using serum comprising IL-15, IL-21, CD28 antibody, CD137 antibody and human AB ^TM OpTmizer ^TM Culturing endometrial cancer tissue in a medium to obtain a first cell population, (2) culturing the first cell population in the presence of a CD28 antibody, MICA, MICB with an X-VIVO 15 medium comprising an IL-15, CD3 antibody to obtain a second cell population comprising tumor infiltrating NK cells. Illustratively, the media of steps (1) and (2) are as shown in the T004 sample of Table 3.

In one or more embodiments, the method is for culturing tumor-infiltrating NK cells from breast cancer tissue, comprising the steps of: (1) Culturing breast cancer tissue using an X-VIVO 15 medium comprising IL-15, IL-21, CD28 antibody, CD137 antibody, CD16 antibody and human AB serum to obtain a first population of cells, (2) culturing said first population of cells in the presence of CD28 antibody with an X-VIVO 15 medium comprising IL-2, IL-15, CD3 antibody to obtain a second population of cells comprising tumor infiltrating NK cells. Illustratively, the media of steps (1) and (2) are as shown in the T005 sample of Table 3.

In one or more embodiments, the method for culturing tumor-infiltrating NK cells from cervical cancer tissue comprises the steps of: (1) Culturing cervical cancer tissue using an X-VIVO 15 medium comprising IL-15, IL-21, CD28 antibody, CD137 antibody, CD16 antibody, CD40 antibody and human AB serum to obtain a first population of cells, (2) culturing said first population of cells in the presence of CD28 antibody, MICA, MICB with an X-VIVO 15 medium comprising IL-2, IL-15, CD3 antibody to obtain a second population of cells comprising tumor infiltrating NK cells. Illustratively, the media of steps (1) and (2) are as shown in the T006 samples of Table 3.

In one or more embodiments, the method is for culturing tumor-infiltrating NK cells from ovarian cancer tissue, comprising the steps of: (1) Culturing ovarian cancer tissue using an X-VIVO 15 medium comprising IL-15, IL-21, CD28 antibody, CD137 antibody, CD16 antibody, OX-40 antibody and human AB serum to obtain a first population of cells, (2) culturing said first population of cells in the presence of CD28 antibody, MICA with an X-VIVO 15 medium comprising IL-2, IL-15, CD3 antibody to obtain a second population of cells comprising tumor infiltrating NK cells. Illustratively, the media of steps (1) and (2) are as shown in the T007 samples of Table 3.

In one or more embodiments, the method for culturing tumor-infiltrating NK cells from cervical cancer tissue comprises the steps of: (1) CTS Using serum comprising IL-7, IL-15, IL-21, CD28 antibody, CD137 antibody, CD16 antibody, CD40 antibody and human AB ^TM OpTmizer ^TM Culturing cervical cancer tissue in a medium to obtain a first cell population, (2) culturing the first cell population in the presence of a CD28 antibody, MICA with an X-VIVO 15 medium comprising IL-2, IL-15, CD3 antibodies to obtain a second cell population comprising tumor infiltrating NK cells. Illustratively, the media of steps (1) and (2) are as shown in the T008 sample of Table 3.

In one or more embodiments, the method is for culturing tumor-infiltrating NK cells from ovarian cancer tissue, comprising the steps of: (1) CTS Using serum comprising IL-7, IL-12, IL-15, IL-21, CD28 antibody, CD137 antibody, CD16 antibody, OX-40 antibody and human AB ^TM OpTmizer ^TM Culturing ovarian cancer tissue in a medium to obtain a first cell population, (2) culturing the first cell population in the presence of CD28 antibody, MICA with X-VIVO 15 medium comprising IL-2, IL-15, CD3 antibody to obtain a second cell population comprising tumor infiltrating NK cells. Illustratively, the media of steps (1) and (2) are as shown in the T009 sample of table 3.

In one or more embodiments, the method is for culturing tumor-infiltrating NK cells from breast cancer tissue, comprising the steps of: (1) Culturing breast cancer tissue using an X-VIVO 15 medium comprising IL-7, IL-15, IL-18, IL-21, CD28 antibody, CD137 antibody, CD16 antibody, CD40 antibody and human AB serum to obtain a first population of cells, (2) culturing said first population of cells in the presence of CD28 antibody, MICA, MICB with an X-VIVO 15 medium comprising IL-2, IL-15, CD3 antibody to obtain a second population of cells comprising tumor infiltrating NK cells. Illustratively, the media of steps (1) and (2) are as shown in the T010 sample of Table 3.

In one or more embodiments, the method is for culturing tumor-infiltrating NK cells from ovarian cancer tissue, comprising the steps of: (1) Culturing ovarian cancer tissue using an X-VIVO 15 medium comprising IL-7, IL-15, IL-21, CD28 antibody, CD137 antibody, CD16 antibody, CD40 antibody, PD-1 antibody and human AB serum to obtain a first population of cells, (2) culturing said first population of cells in the presence of CD28 antibody, MICA, MICB with an X-VIVO 15 medium comprising IL-2, IL-15, CD3 antibody to obtain a second population of cells comprising tumor infiltrating NK cells. Illustratively, the media of steps (1) and (2) are as shown in the T011 sample in Table 3.

In one or more embodiments, the method is for culturing tumor-infiltrating NK cells from lung cancer tissue, comprising the steps of: (1) Culturing lung cancer tissue using an X-VIVO 15 medium comprising IL-7, IL-15, IL-21, CD28 antibody, CD137 antibody, CD16 antibody, CD40 antibody, LAG-3 antibody and human AB serum to obtain a first population of cells, (2) culturing said first population of cells in the presence of CD28 antibody, MICA with an X-VIVO 15 medium comprising IL-2, IL-15, CD3 antibody to obtain a second population of cells comprising tumor infiltrating NK cells. Illustratively, the media of steps (1) and (2) are as shown in the T012 samples in Table 3.

In one or more embodiments, the method for culturing tumor-infiltrating NK cells from brain glioma tissue comprises the steps of: (1) Culturing a brain glioma tissue using an X-VIVO 15 medium comprising IL-7, IL-15, IL-18, IL-21, TNF- α, CD28 antibody, CD137 antibody, CD16 antibody, CD40 antibody, CTLA-4 antibody and human AB serum to obtain a first cell population, (2) culturing said first cell population in the presence of CD28 antibody, MICA with an X-VIVO 15 medium comprising IL-2, IL-15, CD3 antibody to obtain a second cell population comprising tumor infiltrating NK cells. Illustratively, the media of steps (1) and (2) are as shown in the T013 sample of Table 3.

In one or more embodiments, the method is for culturing tumor-infiltrating NK cells from ovarian cancer tissue, comprising the steps of: (1) Culturing ovarian cancer tissue using an X-VIVO 15 medium comprising IL-7, IL-12, IL-15, IL-18, IL-21, CD28 antibody, CD137 antibody, CD16 antibody, CD40 antibody, LAG-3 antibody, TIGIT antibody, and human AB serum to obtain a first population of cells, (2) culturing the first population of cells in the presence of CD28 antibody, MICA with an X-VIVO 15 medium comprising IL-2, IL-15, CD3 antibody to obtain a second population of cells comprising tumor infiltrating NK cells. Illustratively, the media of steps (1) and (2) are as shown in the T014 samples of table 3.

In one or more embodiments, the method for culturing tumor-infiltrating NK cells from cervical cancer tissue comprises the steps of: (1) Culturing cervical cancer tissue using an X-VIVO 15 medium comprising IL-7, IL-12, IL-15, IL-18, IL-21, TNF- α, CD28 antibody, CD137 antibody, CD16 antibody, CD40 antibody, LAG-3 antibody, CTLA-4 antibody, PD-1 antibody and human AB serum to obtain a first population of cells, (2) culturing said first population of cells in the presence of CD28 antibody, MICA, MICB with an X-VIVO 15 medium comprising IL-2, IL-15, CD3 antibody to obtain a second population of cells comprising tumor infiltrating NK cells. Illustratively, the media of steps (1) and (2) are as shown in the T015 samples of Table 3.

In one or more embodiments, the method for culturing tumor-infiltrating NK cells from cervical cancer tissue comprises the steps of: (1) Culturing cervical cancer tissue using an X-VIVO 15 medium comprising IL-7, IL-12, IL-15, IL-18, IL-21, CD28 antibody, CD137 antibody, CD16 antibody, CD40 antibody, LAG-3 antibody, and TIGIT antibody to obtain a first population of cells, (2) culturing the first population of cells in the presence of CD28 antibody, MICA, MICB with an X-VIVO 15 medium comprising IL-2, IL-15, CD3 antibody to obtain a second population of cells comprising tumor infiltrating NK cells. Illustratively, the media of steps (1) and (2) are as shown in the T016 sample of table 3.

In one or more embodiments, the method for culturing tumor-infiltrating NK cells from cervical cancer tissue comprises the steps of: (1) Culturing cervical cancer tissue using an X-VIVO 15 medium comprising IL-7, IL-12, IL-15, IL-18, IL-21, TNF- α, CD28 antibody, CD137 antibody, CD16 antibody, CD40 antibody, LAG-3 antibody, CTLA-4 antibody, PD-1 antibody to obtain a first population of cells, (2) culturing said first population of cells in the presence of CD28 antibody, MICA, MICB with an X-VIVO 15 medium comprising IL-15, CD3 antibody to obtain a second population of cells comprising tumor infiltrating NK cells. Illustratively, the media of steps (1) and (2) are as shown in the T017 samples of Table 3.

In one or more embodiments, the method for culturing tumor-infiltrating NK cells from cervical cancer tissue comprises the steps of: (1) Culturing lung cancer tissue using an X-VIVO 15 medium comprising IL-7, IL-15, IL-21, CD28, CD137, CD16, CD40, and LAG-3 antibodies to obtain a first population of cells, (2) culturing the first population of cells in the presence of CD28, MICA with an X-VIVO 15 medium comprising IL-2, IL-15, CD3 antibodies to obtain a second population of cells comprising tumor infiltrating NK cells. Illustratively, the media of steps (1) and (2) are as shown in the T018 sample of Table 3.

The invention also provides a pharmaceutical composition comprising tumor-infiltrating NK cells obtained by culturing the tumor-infiltrating lymphocyte culture medium described herein or by culturing the tumor-infiltrating NK cells obtained by the culture method described herein, and pharmaceutically acceptable excipients. Pharmaceutically acceptable excipients herein refer to carriers and/or excipients that are pharmacologically and/or physiologically compatible with the subject and active ingredient, and in certain embodiments, acceptable excipients in pharmaceutical compositions and the like are preferably non-toxic to the recipient at the dosages and concentrations employed. In certain embodiments, the pharmaceutical compositions may contain such materials for improving, maintaining, or retaining, for example, pH, permeability, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, absorption or permeation of the composition. Such materials are known in the art and include, but are not limited to: pH adjusters, surfactants, ionic strength enhancers, diluents, carriers, solubilizers, emulsifiers, preservatives and/or adjuvants. More specifically, suitable pharmaceutically acceptable excipients may be those commonly used in the art for administration of immune cells, particularly tumor infiltrating NK cells. See, e.g., REMINGTON' S PHARMACEUTICAL SCIENCES, 18 th edition, a.r. genrmo, code 1990,Mack Publishing Company. The optimal pharmaceutical composition can be determined depending on the intended route of administration, the mode of delivery and the dosage required.

The pharmaceutical compositions of the present invention may be selected for parenteral delivery. Alternatively, the composition may be selected for inhalation or delivery through the digestive tract (such as orally). The preparation of such pharmaceutically acceptable compositions is within the skill of the art.

Other pharmaceutical compositions will be apparent to those skilled in the art, including formulations comprising immune cells, particularly tumor-infiltrating NK cells, in a sustained or controlled release delivery formulation. Techniques for formulating a variety of other sustained or controlled delivery means, such as liposome carriers, bioerodible particles or porous beads, and depot injections, are also known to those skilled in the art.

Pharmaceutical compositions for in vivo administration are generally provided in the form of sterile formulations. Sterilization is achieved by filtration through sterile filtration membranes. Compositions for parenteral administration may be stored in lyophilized form or in solution (e.g., lyophilized formulations). Parenteral compositions are typically placed in a container having a sterile access port, such as an intravenous solution tape or vial having a stopper pierceable by a hypodermic injection needle.

Once formulated, the pharmaceutical compositions are stored in sterile vials as solutions, suspensions, gels, emulsions, solids, crystals, freezers, or as dehydrated or lyophilized powders. The pharmaceutical formulation (e.g., a lyophilized formulation) may be stored in a ready-to-use form or in a form that is further formulated prior to administration. For example, a pharmaceutical composition suitable for delivery of tumor-infiltrating NKT cells as described herein may be a cryopreserved formulation, which can withstand long-distance transport without damaging the cells. In addition to the cells themselves, cryopreservation formulations typically include components such as cell cryopreservation solution, human Serum Albumin (HSA), and the like. Prior to administration (e.g., intravenous infusion), the cryopreserved pharmaceutical composition is stored (e.g., in liquid nitrogen). The frozen preparation can be directly infused into a patient or formulated as an infusion composition after thawing. The composition and concentration of conventional frozen stock solutions are known to those skilled in the art. For example, the frozen stock solution or infusion composition may further comprise sodium chloride, glucose, sodium acetate, potassium chloride, magnesium chloride, or the like, the concentration of which may be determined by one of skill in the art (e.g., an experienced physician) based on the condition of the cell, disease, patient, or the like. The invention also provides kits for producing single dose administration units. The kits of the invention may each contain a first container with a cryopreservation solution or a tumor-containing infiltrating NKT cell cryopreservation formulation and optionally a second container with an infusion formulation. In certain embodiments of the invention, kits are provided that contain single-lumen and multi-lumen prefilled infusers (e.g., liquid infusers).

The invention also provides a method of treating a patient by administering an immune cell, particularly an NK cell, or pharmaceutical composition thereof in accordance with any of the embodiments of the invention.

The terms "patient," "individual," "subject" are used interchangeably herein to include any organism, preferably an animal, more preferably a mammal (e.g., rat, mouse, dog, cat, rabbit, etc.), and most preferably a human. "treating" refers to a subject employing a treatment regimen described herein to achieve at least one positive therapeutic effect (e.g., reduced number of cancer cells, reduced tumor volume, reduced rate of infiltration of cancer cells into peripheral organs, or reduced rate of tumor metastasis or tumor growth). Typically, the pharmaceutical composition contains a therapeutically effective amount of cells. A therapeutically effective amount refers to a dose that achieves treatment, prevention, alleviation and/or relief of a disease or condition in a subject. The therapeutically effective amount may be determined by factors such as the age, sex, severity of the condition, other physical condition of the patient, and the like. Herein, a subject or patient refers generally to a mammal, particularly a human.

The therapeutically effective amount of the pharmaceutical composition comprising the immune cells of the invention, particularly tumor infiltrating NK cells, to be employed will depend, for example, on the extent of treatment and the goal. Those of skill in the art will appreciate that the therapeutic regimen effective to treat a patient will vary depending in part on the molecule delivered, the indication, the route of administration, and the size (body weight, body surface or organ size) and/or condition (age, general health, ability of the therapy to elicit an anti-cancer response in the subject) of the patient. In certain embodiments, the clinician may titrate the dose and alter the route of administration to obtain the optimal therapeutic effect.

The frequency of administration will depend on the pharmacokinetic parameters of the immune cells, particularly tumor infiltrating NK cells, in the formulation used. The clinician typically administers the composition until a dose is reached that achieves the desired effect. The composition may thus be administered as a single dose, or over time as two or more doses (which may or may not contain the same amount of the desired molecule), or as a continuous infusion through an implanted device or catheter.

The route of administration of the pharmaceutical composition is according to known methods, for example, by oral, intravenous, intraperitoneal, intracerebral (intraparenchymal), intracerebroventricular, intramuscular, intraocular, intraarterial, portal or intralesional route injection; either by a sustained release system or by an implanted device.

The invention also comprises application of tumor-infiltrating NK cells obtained by culturing the tumor-infiltrating lymphocyte culture medium or tumor-infiltrating NK cells obtained by culturing the tumor-infiltrating lymphocyte culture medium and/or a pharmaceutical composition containing the tumor-infiltrating NK cells in preparation of anti-tumor drugs. The tumors are as described elsewhere herein. The preparation may be carried out using methods and reagents conventional in the art for preparing antitumor drugs.

The invention will be illustrated by way of specific examples. It should be understood that these examples are illustrative only and are not intended to limit the scope of the invention. The methods and materials used in the examples are those conventional in the art, unless otherwise indicated.

Examples

The sources of the components used in the media of the examples herein are shown below.

EXAMPLE 1 preparation of TIL cell culture Medium

Different TIL cell culture media were formulated as in tables 1 and 2. The basal medium can be stored at 4 ℃ for no more than 1 month after serum and the diabody are proportionally added. The culture medium is taken out from 4 ℃ before use, placed in a water bath at 37 ℃ for preheating, and then added with other components (various cytokines and various antibodies) to working concentration before use for TIL cell culture.

Table 1: component concentration of TIL cell Medium 1-15

FV: make up to the final volume

Table 2: component concentration of TIL cell Medium 16-18

FV: make up to the final volume

Example 2 treatment of solid tumor samples (surgically resected tissue) and tumor infiltrating NK (Tumor Infiltrating NK, TINK) cell culture

1) Preparing physiological saline containing 100U/mL penicillin, 100 mug/mL streptomycin and 50 mug/mL gentamicin for later use;

2) Placing the obtained freshly isolated tumor tissue sample in a 10cm culture dish added with 30mL of the physiological saline prepared in the step 1) in a sterile environment in a secondary biosafety cabinet for washing, transferring the freshly isolated tumor tissue sample into a 10cm culture dish added with 30mL of the physiological saline prepared in the step 1) for washing, and repeating the washing for 3 times;

3) Removing fat tissue and necrotic tissue with a sterile scalpel, cutting tumor tissue becomes a diameter of 3X 3mm ³ 12 randomly selected pieces of tumor tissue were placed in each G-REX10 culture tank (from Wilsonwolf), each G-REX10 culture tank corresponding to a TIL cell culture medium of the specific formulation of example 1 of the present invention; the redundant tumor tissue blocks are frozen and stored by a cryo-Stor 10 (purchased from BioLifeSolons) frozen solution through a program cooling instrument liquid nitrogen;

4) Each group of TIL cell culture media from Table 1 of example 1 was added to a separate G-REX10 culture tank, 40mL each, and the tumor tissue mass was treated with 5% CO at 37 ℃ ₂ Culturing, during which cytokines and antibodies (if present) are fed once every 4 days, and harvesting the first cell population on day 12;

5) The plates were coated overnight at 4℃with a coating solution containing 5. Mu.g/mL of each stimulating factor, and the TIL cell culture medium described in Table 2 of example 1 was added and preheated at 37 ℃. Centrifuging 300g of the first cell population obtained in 4) for 10min, re-suspending the cell pellet with 37 deg.C pre-heating medium, adding into a plate coated with stimulating factor at 37 deg.C 5% CO ₂ The culture was continued with cytokine and antibody (if present) supplementation every 4 days, and after 12 days of continued culture a second cell population containing TINK cells was harvested and the phenotype of the harvested cell population was flow tested.

The medium numbers used for culturing the first cell population and the second cell population (corresponding to the medium numbers in tables 1 and 2) for each tumor tissue sample and the stimulating factors used for culturing the second cell population package plate are shown in table 3 below.

The respective tumor tissues treated in the examples of the present invention, the TIL cell culture medium used for culturing each tumor sample and the stimulating factors used for the coated well plate are shown in table 3:

TABLE 3 Table 3

Note that: the first cell population medium in the TINK cultures of T016, T017 and T018 corresponds to the medium numbered 14, 15 and 12 in Table 1 respectively but without serum, the other components being identical to the medium 14, 15 and 12 in Table 1.

The total number and the activation rate of the cells were counted with a cytometer for the second cell population obtained by the culture, and the phenotype of the cells was examined with a flow cytometer, and IFN-gamma secretion levels were examined according to the methods described in the specification with HTRF IFN-gamma detection kit (Cisbio Human IFN gamma kit cat# 62 HIFUNGPET). The culture results are shown in Table 4 and FIGS. 1 to 8. Table 4 shows the results of TIL cell culture of T001-T015 containing TINK cells; FIGS. 1-8 are flow cell phenotypes of TINK cells cultured from T004, T005, T010, T011, T015, T016, T017 and T018 tumor tissues, respectively.

Table 4: T001-T015 tumor tissue TIL cell culture results

Table 4 shows that at least 10 can be harvested after twenty-four days of culture using the TIL cell culture media of the invention for different solid tumor tissues ¹⁰ -10 ¹¹ On the order of the total number of cells. The cell activity rate obtained by different tumor tissue culture is above 90%, and the cell activity rate of part of samples is close to 100%. All harvested cells have higher IFN-gamma secretion level, the IFN-gamma secretion level is more than 9000pg/mL, the highest value is more than 15000pg/mL, and NK cells (CD 56 ⁺ CD3 ^- ) The proportion of CD45+ cells is mostly above 30%, and the highest proportion reaches 99.20%. The flow phenotype of NK cells harvested after a portion of tumor tissue in Table 3 was cultured with the TIL medium of the present invention is shown in FIGS. 1-8. Wherein the results of FIGS. 1-4 show the CD8+ NK cell ratio. FIG. 1-FIG. 4 shows that CD8+ NK cells account for nearly 24% of the highest NK cells obtained by sample culture. CD8+ NK cells are generally more killing NK cellsThe proportion of cells, but in blood, cd8+ NK cells is generally low.

The above results show that 10 can be obtained from different solid tumor tissues by culturing using the TIL cell culture medium of the invention ¹⁰ -10 ¹¹ And the obtained NK cells have stronger activity.

Example 3 detection of tumor cell killing Effect of TINK and peripheral blood NK

Mononuclear Cells (PBMC) were isolated from peripheral blood of three patients T015, T016 and T017 by Ficoll isolation, and Peripheral Blood NK (PBNK) cells were obtained by culture expansion from PBMC of three patients T015, T016 and T017 according to NK cell culture kit (CellXVivo Human NK Cell Expansion Kit, biotechne Catalog # CDK 015) instructions.

Cervical cancer cell line HT-3 (purchased from American type culture Collection ATCC, cat: HTB-32) was selected as a target cell, and the in vitro killing activity of T015, T016 and T017 PBNK cells prepared by the above method and T015, T016 and T017 TINK cells prepared in example 2 were examined using a real-time label-free cell function analyzer (RTCA) of the Eisen company, as follows:

(1) Zeroing: adding 50 mu L of DMEM or 1640 culture solution into each hole, placing into an instrument, selecting step 1, and zeroing;

(2) Target cell plating: HT-3 cells per well 10 ⁴ Spreading the cells/50 mu L in a plate containing a detection electrode, standing for several minutes, placing the cells in an instrument after the cells are stabilized, and starting step 2 to culture the cells;

(3) Adding effector cells: after the target cells were cultured for 20-24 hours until the Cell Index (Cell Index) reached 1.0, step 2 was suspended, 50. Mu.L of effector cells were added per well, TINK or PBNK of effector cells were added at different target ratios, step 3 was started, co-culture was continued for more than 120 hours, and then the Cell proliferation curve was observed.

The results are shown in FIGS. 9-11. FIGS. 9, 10 and 11 show the killing effect of PBNK and TINK on cervical cancer cells HT-3 derived from T015, T016 and T017 patients, respectively. Compared with the PBNK of the peripheral blood source, the killing level of the TINK of the tumor tissue source of 3 different patients on the tumor cells is obviously higher, and the killing level of each TINK on the tumor cells is further improved along with the improvement of the effective target ratio.

Although specific embodiments of the invention have been described in detail, those skilled in the art will appreciate. Numerous modifications and substitutions of details are possible in light of all the teachings disclosed, and such modifications are contemplated as falling within the scope of the present invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

Claims (10)

1. A tumor infiltrating lymphocyte culture medium comprises cytokines, a stimulating antibody and a cell culture component, wherein the cytokines comprise IL-21 and IL-15, the stimulating antibody comprises CD28 antibody and/or CD137 antibody,

Preferably, the medium further has one or more characteristics selected from the group consisting of:

the tumor-infiltrating lymphocyte culture medium is a tumor-infiltrating NK cell culture medium,

the cell culture component is lymphocyte basic culture medium,

the concentration of IL-21 in the medium is 50-4000U/mL, preferably 100-3000U/mL,

the concentration of IL-15 in the medium is 500-4000U/mL, preferably 1000-3000U/mL,

the concentration of CD28 antibody in the medium is 0-200. Mu.g/mL, preferably 5-100. Mu.g/mL,

the concentration of CD137 antibody in the culture medium is 0-200. Mu.g/mL, preferably 10-100. Mu.g/mL,

the medium may or may not contain serum or serum replacement components.

2. The tumor-infiltrating lymphocyte culture medium according to claim 1, further comprising an activating antibody comprising any one, two or three selected from the group consisting of a CD40 antibody, an OX-40 antibody and a CD16 antibody,

preferably, the method comprises the steps of,

the concentration of CD40 antibody in the medium is 0-200. Mu.g/mL, preferably 10-100. Mu.g/mL, and/or

The concentration of the OX-40 antibody in the medium is 0-200. Mu.g/mL, preferably 10-50. Mu.g/mL, and/or

The concentration of CD16 antibody in the medium is 0-200. Mu.g/mL, preferably 10-100. Mu.g/mL.

3. The tumor-infiltrating lymphocyte medium according to claim 1 or 2, wherein the cytokine further comprises any one or more selected from the group consisting of IL-7, IL-12, IL-18 and TNF alpha,

preferably, the method comprises the steps of,

the concentration of IL-7 in the medium is 0-1500U/mL, preferably 100-1000U/mL,

the concentration of IL-12 in the medium is 0-1500U/mL, preferably 100-1000U/mL,

the concentration of IL-18 in the medium is 0-1500U/mL, preferably 200-1000U/mL,

the concentration of TNFα in the medium is 0-500pg/mL, preferably 10-100pg/mL.

4. The tumor-infiltrating lymphocyte culture medium according to claim 1 or 2, wherein the medium further comprises an immune checkpoint inhibitor; preferably, the immune checkpoint inhibitor comprises any one or more selected from the group consisting of PD-1 antibodies, CTLA-4 antibodies, TIM3 antibodies, lag3 antibodies and TIGIT antibodies.

5. The tumor-infiltrating lymphocyte culture medium according to claim 4, wherein,

the PD-1 antibody concentration in the medium is 0-200. Mu.g/mL, preferably 10-100. Mu.g/mL, and/or

The concentration of CTLA-4 antibodies in the medium is 0-200. Mu.g/mL, preferably 10-100. Mu.g/mL, and/or

The concentration of TIM3 antibody in the medium is 0-200. Mu.g/mL, preferably 10-100. Mu.g/mL, and/or

The concentration of Lag3 antibody in the medium is 0-200. Mu.g/mL, preferably 10-100. Mu.g/mL, and/or

The concentration of TIGIT antibody in the medium is 0-200. Mu.g/mL, preferably 10-100. Mu.g/mL.

6. A method for culturing tumor infiltrating NK cells, comprising the steps of:

(1) Culturing tumor tissue or tumor body fluid to obtain a first cell population,

(2) Culturing the first cell population in the presence of a stimulating factor to obtain a second cell population comprising tumor-infiltrating NK cells, the stimulating factor promoting proliferation of the tumor-infiltrating NK cells,

optionally (3) sorting tumor infiltrating NK cells, sorting markers CD3-CD56+ and/or CD3-CD16+,

preferably, the method comprises the steps of,

the presence of tumor infiltrating T cells in the tumor tissue and/or tumor body fluid; more preferably, the tumor-infiltrating T cells and the tumor-infiltrating NK cells are from the same tumor tissue and/or tumor body fluid, and/or

Step (1) of culturing using the tumor-infiltrating lymphocyte medium according to any one of claims 1-5, and/or

Step (2) the culturing is performed using a tumor-infiltrating lymphocyte medium containing a stimulating factor, the tumor-infiltrating lymphocyte medium containing a stimulating factor further comprising a CD3 antibody and a cell culture component and optionally further comprising a cytokine, preferably the CD3 antibody is OKT-3; wherein the stimulating factor comprises a CD28 antibody, the cytokine comprises IL-2 and/or IL-15, and/or

The tumor comprises a respiratory system tumor, a digestive system tumor, a urinary system tumor, a nervous system tumor, a reproductive system tumor and a skin tumor; more preferably comprises one or more selected from the group consisting of: liver cancer, gastrointestinal cancer, lung cancer, pancreatic cancer, ovarian cancer, stomach cancer, colon cancer, melanoma, endometrial cancer, cervical cancer, uterine sarcoma, vulval cancer, breast cancer, glioma, prostate cancer, fallopian tube cancer, laryngeal cancer, thyroid cancer, gall bladder cancer, renal cancer, bladder cancer and brain cancer, and/or

The stimulating factor also includes ligand molecules of NK cell activating receptors; preferably, the NK cell activating receptor comprises any one or more of NKG2D, NKp, NKp44, NKp30, NKG2C, DNAM-1 and 2B 4; preferably, the NK cell activating receptor comprises NKG2D, the ligand molecule of the NKG2D comprises MICA and/or MICB, and/or

The stimulating factor is a stimulating factor in solution or is fixed on a substrate.

7. Tumor-infiltrating NK cells obtained by culturing the tumor-infiltrating lymphocyte medium of any one of claims 1-5 or by the culture method of claim 6.

8. A pharmaceutical composition comprising tumor-infiltrating NK cells obtained by the tumor-infiltrating lymphocyte culture medium according to any one of claims 1-5 and/or by the culture method according to claim 6, and a pharmaceutically acceptable adjuvant.

9. Use of tumor-infiltrating NK cells obtained by the culture medium for tumor-infiltrating lymphocytes according to any one of claims 1 to 5 or the culture method according to claim 6 and/or a pharmaceutical composition containing the tumor-infiltrating NK cells in the preparation of a medicament for preventing and/or treating an antitumor,

preferably, the tumor comprises a respiratory system tumor, a digestive system tumor, a urinary system tumor, a nervous system tumor, a reproductive system tumor, a skin tumor; preferably comprising one or more selected from the group consisting of: respiratory system tumors, digestive system tumors, urinary system tumors, nervous system tumors, reproductive system tumors, skin tumors; more preferably comprises one or more selected from the group consisting of: liver cancer, gastrointestinal cancer, lung cancer, pancreatic cancer, ovarian cancer, stomach cancer, colon cancer, melanoma, endometrial cancer, cervical cancer, uterine sarcoma, vulval cancer, breast cancer, glioma, prostate cancer, fallopian tube cancer, laryngeal cancer, thyroid cancer, gall bladder cancer, renal cancer, bladder cancer, intestinal cancer and brain cancer.

10. The application of tumor-infiltrating T cells in culturing tumor-infiltrating NK cells,

preferably, the tumor-infiltrating T cells and the tumor-infiltrating NK cells are from the same tumor tissue and/or tumor body fluid, and/or

The culturing is performed using the tumor-infiltrating lymphocyte medium of any one of claims 1-5 or using the culturing method of claim 6.

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