# US 11,304,979 B2 · Processes for production of tumor infiltrating lymphocytes and uses of same in immunotherapy > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 11,304,979 B2 - Document ID: US11304979B2 - Status: Granted - Kind: B2 - Application number: 17/383,280 - Filed: 2021-07-22 - Published: 2021-11-25 - Granted: 2022-04-19 - Earliest claimed: 2017-03-29 - Assignees: Iovance Biotherapeutics, Inc. - Applicants: Iovance Biotherapeutics, Inc. - Inventors: Seth Wardell; James Bender; Michael T. Lotze - Web page: https://patentgrep.com/patent/US11304979B2 ## CPC - 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) - A01N 1/162: Temperature processes, e.g. following predefined temperature changes over time - A61K 9/0019: {Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner (non-active ingredients are additionally classified in A61K47/00)} - A61K 31/675: having nitrogen as a ring hetero atom, e.g. pyridoxal phosphate - A61K 31/7076: containing purines, e.g. adenosine, adenylic acid - A61K 38/2013: {IL-2} - A61K 40/11: T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells - A61K 40/428: {Undefined tumor antigens, e.g. tumor lysate or antigens targeted by cells isolated from tumor} - A61P 35/00: Antineoplastic agents - C12N 5/0634: {Cells from the blood or the immune system} - C12N 5/0636: {T lymphocytes} - C12N 5/0638: {Cytotoxic T lymphocytes [CTL] or lymphokine activated killer cells [LAK]} - A61K 38/217: {IFN-gamma} - A61K 39/0011: {Cancer antigens} - A61K 40/50: characterised by the use of allogeneic cells - A61K 2039/5154: {Antigen presenting cells [APCs], e.g. dendritic cells or macrophages} - A61K 2039/5156: {expressing foreign proteins} - A61K 2039/5158: {Antigen-pulsed cells, e.g. T-cells} - A61K 2039/55533: {IL-2} - C12N 2500/90: Serum-free medium, which may still contain naturally-sourced components - C12N 2501/04: Immunosuppressors, e.g. cyclosporin, tacrolimus - C12N 2501/2302: Interleukin-2 (IL-2) - C12N 2501/2315: Interleukin-15 (IL-15) - C12N 2501/2321: Interleukin-21 (IL-21) - C12N 2501/24: Interferons [IFN] - C12N 2501/603: Oct-3/4 - C12N 2502/11: blood or immune system cells - C12N 2506/30: from cancer cells, e.g. reversion of tumour cells - 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) - A01N: PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF (preservation of food or foodstuff A23); BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES (preparations for medical, dental or toiletry purposes which kill or prevent the growth or proliferation of unwanted organisms A61K); PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS - A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) ## Abstract The present invention provides improved and/or shortened methods for expanding TILs and producing therapeutic populations of TILs, including novel methods for expanding TIL populations in a closed system that lead to improved efficacy, improved phenotype, and increased metabolic health of the TILs in a shorter time period, while allowing for reduced microbial contamination as well as decreased costs. Such TILs find use in therapeutic treatment regimens. ## Claims ### Claim 1 A method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic population of TILs, the method comprising: (a) performing a first expansion by culturing a first population of TILs from tumor fragments or a tumor digest obtained from a tumor resected from a subject in a cell culture medium comprising IL-2 to produce a second population of TILs, wherein the first expansion is performed in a closed container providing a first gas-permeable surface area, wherein the first expansion is performed for a first period of about 3 to 11 days to obtain the second population of TILs; (b) performing a second expansion by supplementing the cell culture medium with additional IL-2, OKT-3, and antigen presenting cells (APCs), to produce a third population of TILs, wherein the second expansion is performed for a second period of about 7 to 11 days in order to obtain the third population of TILs, and wherein the third population of TILs is a therapeutic population of TILs; (c) harvesting the therapeutic population of TILs obtained from step (b); (d) transferring the harvested therapeutic population of TILs from step (c) into an infusion bag; and (e) cryopreserving the infusion bag comprising the harvested TIL population from step (d) using a cryopreservation process. ### Claim 2 (depends on 1) The method according to claim 1, wherein the tumor digest in step (a) was prepared by incubating a sample of the tumor that was resected from the subject in an enzymatic media. ### Claim 3 (depends on 2) The method according to claim 2, further comprising disrupting the tumor sample mechanically so as to dissociate the tumor sample. ### Claim 4 (depends on 3) The method according to claim 3, further comprising purifying the disassociated tumor sample using a density gradient separation. ### Claim 5 (depends on 2) The method according to claim 2, wherein the enzymatic media comprises DNase. ### Claim 6 (depends on 5) The method according to claim 5, wherein the enzymatic media comprises 30 units/mL of DNase. ### Claim 7 (depends on 2) The method according to claim 2, wherein the enzymatic media comprises collagenase. ### Claim 8 (depends on 7) The method according to claim 7, wherein the enzymatic media comprises 1.0 mg/mL of collagenase. ### Claim 9 (depends on 1) The method according to claim 1, wherein the medium in the first expansion and/or the second expansion is free of human serum. ### Claim 10 (depends on 1) The method according to claim 1, wherein the therapeutic population of TILs harvested in step (c) comprises sufficient TILs for use in administering a therapeutically effective dosage to a subject. ### Claim 11 (depends on 10) The method according to claim 10, wherein the number of TILs sufficient for administering a therapeutically effective dosage is from about 1×109 to about 9×1010. ### Claim 12 (depends on 1) The method according to claim 1, wherein the APCs are peripheral blood mononuclear cells (PBMCs). ### Claim 13 (depends on 12) The method according to claim 12, wherein the PBMCs are supplemented at a ratio of about 1:25 TIL:PBMCs. ### Claim 14 (depends on 1) The method according to claim 1, wherein the therapeutic population of TILs harvested in step (d) exhibits an increased subpopulation of CD8+ cells relative to the first and/or second population of TILs. ### Claim 15 (depends on 1) The method according to claim 1, wherein the first expansion in step (a) and the second expansion in step (b) are each individually performed within a period of 11 days. ### Claim 16 (depends on 1) The method according to claim 1, wherein steps (a) through (d) are performed in about 10 days to about 22 days. ### Claim 17 (depends on 1) The method according to claim 1, wherein steps (a) through (d) are performed in about 15 days to about 22 days. ### Claim 18 (depends on 1) The method according to claim 1, wherein steps (a) through (d) are performed in about 20 days to about 22 days. ### Claim 19 (depends on 1) The method of claim 1, wherein the tumor digest is a cryopreserved tumor digest.