# US 12,110,506 B2 · Method for culturing tumor infiltrating lymphocytes > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 12,110,506 B2 - Document ID: US12110506B2 - Status: Granted - Kind: B2 - Application number: 18/319,901 - Filed: 2023-05-18 - Published: 2023-09-28 - Granted: 2024-10-08 - Earliest claimed: 2020-11-19 - Assignees: Suzhou Grit Biotechnology Co., Ltd.; Shanghai Grit Biotechnology Co., Ltd.; Zhuhai Tuoyu Biotechnology Co., Ltd.; Shenzhen Grit Biotechnology Co., Ltd. - Applicants: Suzhou Grit Biotechnology Co., Ltd.; Shanghai Grit Biotechnology Co., Ltd.; Zhuhai Tuoyu Biotechnology Co., Ltd.; Shenzhen Grit Biotechnology Co., Ltd. - Inventors: Yarong Liu; Peipei Zhao - Web page: https://patentgrep.com/patent/US12110506B2 ## CPC - C12N 5/0636: {T lymphocytes} - A61K 35/17: Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes (when activated by a specific antigen A61K39/00) - A61K 40/11: T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells - A61K 40/42: Cancer antigens - A61P 35/00: Antineoplastic agents - A61K 2239/31: characterized by the route of administration - C12N 2501/2302: Interleukin-2 (IL-2) - C12N 2501/515: CD3, T-cell receptor complex - C12N 2502/11: blood or immune system cells - C12N 2502/1121: Dendritic cells - C12N 2502/1157: Monocytes, macrophages - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D) - A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS ## Abstract A method for culturing tumor infiltrating lymphocytes (TIL), the method comprising co-culturing the expanded TIL with feeder cells after contacting the TIL with a T cell co-stimulatory molecule and/or a T cell growth factor for a period of time. In addition, further provided is a method for preventing and/or treating tumors by means of using the tumor infiltrating lymphocytes of the present application. ## Claims ### Claim 1 A method for culturing tumor infiltrating lymphocytes (TILs), wherein the method comprises subjecting TILs that are derived from tumor tissues and not expanded in vitro to at least one stage of in vitro expansion, and wherein a single stage of the in vitro expansion comprises co-culturing non-in vitro-expanded or in vitro-expanded TILs with feeder cells after contacting the non-in vitro-expanded or in vitro-expanded TILs with one or more T cell co-stimulatory molecules and one or more T cell growth factors for a period of time, wherein the period of time is 12 hours or more. ### Claim 2 (depends on 1) The method according to claim 1, wherein the method comprises subjecting the TILs that are derived from the tumor tissues and not expanded in vitro to at least two stages of the in vitro expansion, and wherein the second stage of the in vitro expansion comprises co-culturing in vitro-expanded TILs with the feeder cells after contacting the in vitro-expanded TILs with the one or more T cell co-stimulatory molecules and the one or more T cell growth factors for the period of time. ### Claim 3 (depends on 1) The method according to claim 1, wherein the period of time is about 12 to 72 hours. ### Claim 4 (depends on 1) The method according to claim 1, wherein the period of time is about 12 to 48 hours. ### Claim 5 (depends on 1) The method according to claim 1, wherein the period of time is about 12, 24, 48 or 72 hours. ### Claim 6 (depends on 1) The method according to claim 1, wherein the one or more T cell co-stimulatory molecules are one or more of the molecules selected from the group consisting of: CD80, CD86, B7-H3, 4-1BBL, CD27, CD30, CD134, B7h, CD40, LIGHT, an antibody that specifically binds to CD3, an antibody that specifically binds to CD28, an antibody that specifically binds to HVEM, an antibody that specifically binds to CD40L, an antibody that specifically binds to OX40, and an antibody that specifically binds to 4-1BB. ### Claim 7 (depends on 1) The method according to claim 1, wherein the one or more T cell co-stimulatory molecules comprise an antibody and/or an antigen-binding fragment thereof that specifically binds to CD3. ### Claim 8 (depends on 1) The method according to claim 1, wherein the one or more T cell growth factors are one or more of the factors selected from the group consisting of: IL-2, IL-4, IL-7, IL-10, IL-12, IL-15, IL-21, and interferon gamma. ### Claim 9 (depends on 1) The method according to claim 1, wherein the one or more T cell growth factors comprise IL-2 and/or a functionally active fragment thereof. ### Claim 10 (depends on 9) The method according to claim 9, wherein the one or more T cell growth factors comprise an engineered cell expressing the IL-2 and/or functionally active fragment thereof, a nanoparticle comprising the IL-2 and/or functionally active fragment thereof, or a polymer comprising the IL-2 and/or functionally active fragment thereof. ### Claim 11 (depends on 9) The method according to claim 9, wherein the one or more T cell growth factors comprise IL-2 and the initial concentration of IL-2 in the cell culture medium of the TILs is at least 1000 IU/mL. ### Claim 12 (depends on 1) The method according to claim 1, wherein the one or more T cell co-stimulatory molecules comprise an antibody and/or an antigen-binding fragment thereof that specifically binds to CD3, and the one or more T cell growth factors comprise IL-2 and/or a functionally active fragment thereof. ### Claim 13 (depends on 12) The method according to claim 12, wherein the one or more T cell growth factors comprise an engineered cell expressing the IL-2 and/or functionally active fragment thereof, a nanoparticle comprising the IL-2 and/or functionally active fragment thereof, or a polymer comprising the IL-2 and/or functionally active fragment thereof. ### Claim 14 (depends on 12) The method according to claim 12, wherein the one or more T cell growth factors comprise IL-2 and the initial concentration of IL-2 in the cell culture medium of the TILs is at least 1000 IU/mL. ### Claim 15 (depends on 1) The method according to claim 1, wherein the feeder cells comprise antigen presenting cells. ### Claim 16 (depends on 1) The method according to claim 1, wherein the feeder cells are peripheral mononuclear cells. ### Claim 17 (depends on 1) The method according to claim 1, wherein the feeder cells are dendritic cells or artificial antigen presenting cells. ### Claim 18 (depends on 1) The method according to claim 1, wherein the feeder cells are irradiated feeder cells. ### Claim 19 (depends on 1) The method according to claim 1, wherein the step of co-culturing comprises adding the feeder cells into the cell culture medium of the TILs. ### Claim 20 (depends on 1) The method according to claim 1, wherein the period of time is about 24 hours. ### Claim 21 (depends on 1) The method according to claim 1, wherein the period of time is about 48 hours. ### Claim 22 (depends on 1) The method according to claim 1, wherein the period of time is about 72 hours. ### Claim 23 (depends on 1) The method according to claim 1, wherein the period of time is about 4 days. ### Claim 24 (depends on 1) The method according to claim 1, wherein the period of time is about 5 days.