Methods of generating tils products enriched for tumor antigen-specific t cells

Abstract

The present invention provides improved and / or shortened processes and methods of reprogramming TILs to make therapeutic TIL populations with increased therapeutic efficacy. Such reprogrammed TILs can be used in therapeutic regimens.

Description

[0001] This application is a divisional application of the invention patent application filed on January 8, 2019, with application number 201980017442.8 (international application number PCT / US2019 / 012729) entitled "Method for producing TIL products rich in tumor antigen-specific T cells".

[0002] Related applications

[0003] This application claims priority to U.S. Provisional Patent Application No. 62 / 614,887, filed January 8, 2018; U.S. Provisional Patent Application No. 62 / 664,034, filed April 27, 2018; U.S. Provisional Patent Application No. 62 / 669,319, filed May 9, 2018; U.S. Provisional Patent Application No. 62 / 697,921, filed July 13, 2018; U.S. Provisional Patent Application No. 62 / 734,868, filed September 21, 2018; and U.S. Provisional Patent Application No. 62 / 773,715, filed November 30, 2018, the entire contents of which are incorporated herein by reference.

[0004] sequence list

[0005] This application contains a sequence list, which has been electronically submitted in ASCII format, the entire contents of which are incorporated herein by reference. The ASCII copy was created on January 7, 2019, and is named 116983-5034-WO_ST25.txt, with a size of 122KB. Background Technology

[0006] Using adoptive metastatic tumor-infiltrating lymphocytes (TILs) to treat large, refractory cancers represents an effective approach for treating patients with poor prognoses. (Gattinoni et al., Nat. Rev. Immunol., 2006, 6, 383-393). Successful immunotherapy requires large quantities of TILs, and commercialization necessitates robust and reliable methods. This presents a challenge due to technical, logistical, and regulatory issues surrounding cell expansion. Due to its speed and efficiency, IL-2-based TIL expansion followed by a rapid expansion process (REP) has become the preferred method for TIL expansion. Dudley et al., Science, 2002, 298, 850-54; Dudley et al., J. Clin. Oncol., 2005, 23, 2346-57; Dudley et al., J. Clin. Oncol., 2008, 26, 5233-39; Riddell et al., Science 1992, 257, 238-41; Dudley et al., J. I. M. M. Oncol., 2003, 26, 332-42. REP can amplify TILs by 1000-fold within 14 days, although it requires a large excess (e.g., 200-fold) of irradiated allogeneic peripheral blood mononuclear cells (PBMCs, also known as monocytes (MNCs)) as feeder cells, usually from multiple donors, as well as anti-CD3 antibody (OKT3) and high doses of IL-2. Dudley et al. J. Immunother. 2003, 26, 332-42. TILs undergoing the REP procedure yielded successful adoptive cell therapy in melanoma patients following host immunosuppression.

[0007] There is an urgent need for more potent or efficient methods for producing TILs and therapies based on such methods, suitable for commercial-scale production and regulatory approval for use in human patients across multiple clinical centers. This invention addresses this need by providing a transient genetic alteration method for reprogramming TILs to prepare a population of therapeutically potent TILs. Summary of the Invention

[0008] This invention provides an improved and / or shortened method for amplifying TILs and generating therapeutic TIL populations.

[0009] This invention provides a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, the method comprising: (i) obtaining a first TIL population from a tumor resected from a patient; (ii) performing a first expansion by culturing the first TIL population in a cell culture medium containing IL-2 and optionally OKT-3 to generate a second TIL population; (iii) performing a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3 and antigen-presenting cells (APCs) to generate a third TIL population; wherein the number of the third TIL population is at least 100 times greater than the number of the second TIL population; wherein the second expansion is performed for at least 14 days to obtain the third TIL population; wherein the third TIL population is a therapeutic TIL population; and (iv) exposing the second TIL population and / or the third TIL population to transcription factors (TFs) and / or other molecules capable of transiently altering protein expression; wherein the TFs and / or other molecules capable of transiently altering protein expression provide alterations in tumor antigen expression and / or the number of tumor antigen-specific T cells in the therapeutic TIL population.

[0010] In some embodiments, the method further includes exposing a second TIL population and / or a third TIL population to transcription factors (TFs) and / or other molecules capable of transiently altering protein expression; wherein the TFs and / or other molecules capable of transiently altering protein expression provide alterations in tumor antigen expression and / or the number of tumor antigen-specific T cells in the therapeutic TIL population.

[0011] The present invention also provides a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, the method comprising:

[0012] (a) Obtaining the first TIL group from the tumor removed from the patient by processing a tumor sample obtained from the patient into multiple tumor fragments;

[0013] (b) Adding tumor fragments to the closed system;

[0014] (c) A first amplification is performed by culturing a first TIL population in a cell culture medium containing IL-2 to generate a second TIL population; wherein the first amplification is performed in a closed container providing a first ventilated surface area; wherein the first amplification is performed for approximately 3 to 14 days to obtain the second TIL population; wherein the number of the second TIL population is at least 50 times greater than the number of the first TIL population; wherein the transition from step (b) to step (c) occurs without opening the system;

[0015] (d) A second expansion is performed by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, and antigen-presenting cells (APCs) to generate a third TIL population; wherein the second expansion is performed for approximately 7 to 14 days to obtain the third TIL population; wherein the third TIL population is a therapeutic TIL population; wherein the second expansion is performed in a closed container providing a second breathable surface area; wherein the transition from step (c) to step (d) occurs without opening the system;

[0016] (e) Exposing the second and / or third TIL groups to transcription factors (TFs) and / or other molecules capable of transiently altering protein expression; wherein the TFs and / or other molecules capable of transiently altering protein expression provide alterations in tumor antigen expression and / or the number of tumor antigen-specific T cells in the therapeutic TIL groups.

[0017] (f) Harvest the therapeutic TIL cluster obtained from step (d); wherein the transition from step (d) to step (e) occurs without opening the system; and

[0018] (g) The TIL clusters harvested in step (e) are transferred to an infusion bag; wherein the transition from step (e) to step (f) occurs without opening the system.

[0019] In some embodiments, the method further includes: performing a second expansion before or after step (iv) by supplementing the cell culture medium of the third TIL population with additional IL-2, additional OKT-3 and additional APC; wherein the second expansion is performed for at least 14 days to obtain a larger therapeutic TIL population than that obtained in step (iii); wherein the larger therapeutic TIL population exhibits an alteration in the number of tumor antigen-specific T cells.

[0020] In some embodiments, the method further includes the step of: cryopreserving the infusion bag containing the TIL clusters harvested from step (f) using a cryopreservation method.

[0021] In some implementations, the cryopreservation method uses a 1:1 ratio of harvested TILs to the cryopreservation medium.

[0022] In some embodiments, the antigen-presenting cells are peripheral blood mononuclear cells (PBMCs). In some embodiments, the PBMCs are irradiated and allogeneic. In some embodiments, the PBMCs are added to the cell culture on any day from day 9 to day 14 of step (d). In some embodiments, the antigen-presenting cells are artificial antigen-presenting cells.

[0023] In some implementations, a membrane-based cell processing system is used to perform the harvest in step (e).

[0024] In some implementations, the LOVO cell processing system is used to perform the harvest in step (e).

[0025] In some embodiments, the plurality of fragments comprises about 4 to about 50 fragments, each fragment having a volume of about 27 mm. 3 .

[0026] In some embodiments, the multiple fragments comprise approximately 30 to approximately 60 fragments, with a total volume of approximately 1300 mm. 3 Approximately 1500mm 3 .

[0027] In some embodiments, the multiple fragments include approximately 50 fragments with a total volume of approximately 1350 mm. 3 .

[0028] In some implementations, the multiple fragments include about 50 fragments with a total mass of about 1 gram to about 1.5 grams.

[0029] In some embodiments, the cell culture medium is provided in a container selected from G containers and Xuri cell bags.

[0030] In some embodiments, the cell culture medium in step (d) further contains IL-15 and / or IL-21.

[0031] In some implementations, the IL-2 concentration is from about 10,000 IU / mL to about 5,000 IU / mL.

[0032] In some implementations, the IL-15 concentration is from about 500 IU / mL to about 100 IU / mL.

[0033] In some implementations, the IL-21 concentration is from about 20 IU / mL to about 0.5 IU / mL.

[0034] In some implementations, the infusion bag in step (f) is an infusion bag containing HypoThermosol.

[0035] In some embodiments, the cryopreservation medium contains dimethyl sulfoxide (DMSO). In some embodiments, the cryopreservation medium contains 7% to 10% dimethyl sulfoxide (DMSO).

[0036] In some implementations, the first stage in step (c) and the second stage in step (e) are performed for 10 days, 11 days, or 12 days, respectively.

[0037] In some implementations, the first stage in step (c) and the second stage in step (e) each last for 11 days.

[0038] In some implementations, steps (a) through (f) take approximately 10 to approximately 22 days.

[0039] In some implementations, steps (a) through (f) take approximately 20 to approximately 22 days.

[0040] In some implementations, steps (a) through (f) take approximately 15 to approximately 20 days.

[0041] In some implementations, steps (a) through (f) take approximately 10 to approximately 20 days.

[0042] In some implementations, steps (a) through (f) take approximately 10 to 15 days.

[0043] In some implementations, steps (a) through (f) are performed for less than 22 days.

[0044] In some implementations, steps (a) through (f) are performed for less than 20 days.

[0045] In some implementations, steps (a) through (f) are performed for less than 15 days.

[0046] In some implementations, steps (a) through (f) are performed for less than 10 days.

[0047] In some implementations, steps (a) through (f) and cryopreservation are performed for less than 22 days.

[0048] In some implementations, the therapeutic TIL clusters harvested in step (e) contain sufficient TILs for a therapeutically effective dose of TILs.

[0049] In some implementations, the number of TILs sufficient for a therapeutically effective dose is approximately 2.3 × 10⁻⁶. 10 Approximately 13.7 × 10 10 .

[0050] In some implementations, steps (b) through (e) are performed in a single container; wherein performing steps (b) through (e) in a single container increases the TIL yield per resected tumor compared to performing steps (b) through (e) in more than one container.

[0051] In some implementations, during the second phase of step (d), antigen-presenting cells are added to the TIL without opening the system.

[0052] In some implementations, when administered to a subject, the third TIL group in step (d) provides at least 5 times the interferon-γ production.

[0053] In some implementations, the risk of microbial contamination is reduced compared to open systems.

[0054] In some implementations, the TIL from step (f) or step (g) is injected into the patient.

[0055] In some implementations, the multiple fragments include approximately four fragments.

[0056] The present invention also provides a method for treating a subject suffering from cancer, the method comprising administering expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

[0057] (a) First TIL clusters are obtained by processing tumor samples obtained from patients into multiple tumor fragments and tumors removed by the subject;

[0058] (b) Adding tumor fragments to the closed system;

[0059] (c) A first amplification is performed by culturing a first TIL population in a cell culture medium containing IL-2 to generate a second TIL population; wherein the first amplification is performed in a closed container providing a first ventilated surface area; wherein the first amplification is performed for approximately 3 to 14 days to obtain the second TIL population; wherein the number of the second TIL population is at least 50 times greater than the number of the first TIL population; wherein the transition from step (b) to step (c) occurs without opening the system;

[0060] (d) A second expansion is performed by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, and antigen-presenting cells (APCs) to generate a third TIL population; wherein the second expansion is performed for approximately 7 to 14 days to obtain the third TIL population; wherein the third TIL population is a therapeutic TIL population; wherein the second expansion is performed in a closed container providing a second breathable surface area; wherein the transition from step (c) to step (d) occurs without opening the system;

[0061] (e) Exposing second and / or third TIL groups to transcription factors (TFs) and / or other molecules capable of transiently altering protein expression; wherein TFs and / or other molecules capable of transiently altering protein expression provide an increase in tumor antigen expression and / or an increase in the number of tumor antigen-specific T cells in the therapeutic TIL group;

[0062] (f) Harvest the therapeutic TIL cluster obtained from step (d); wherein the transition from step (d) to step (e) occurs without opening the system; and

[0063] (g) The TIL clusters harvested in step (e) are transferred to an infusion bag; wherein the transition from step (e) to step (f) occurs without opening the system;

[0064] (h) Optionally, the infusion bag containing the TIL clusters harvested from step (f) is cryopreserved using a cryopreservation method; and

[0065] (i) Administer the therapeutically effective dose of the third TIL group in the infusion bag of step (g) to the patient.

[0066] In some implementations, the therapeutic TIL cluster harvested in step (f) contains sufficient TILs for administering a therapeutically effective dose of TILs in step (h).

[0067] In some embodiments, the amount of TIL sufficient for administering a therapeutically effective dose in step (h) is approximately 2.3 × 10⁻⁶. 10 Approximately 13.7 × 10 10 .

[0068] In some implementations, antigen-presenting cells (APCs) are PBMCs.

[0069] In some implementations, PBMCs are added to the cell culture on any day from day 9 to day 14 of step (d).

[0070] In some implementations, a non-myeloablative lymphodepletion regimen has been administered to the patient prior to the administration of a therapeutically effective dose of TIL cells in step (h).

[0071] In some implementations, the non-myeloablative lymphocyte depletion regimen includes a dose of 60 mg / m². 2 Cyclophosphamide was administered at a daily dose for 2 days, followed by a dose of 25 mg / m². 2 The procedure involves administering fludarabine at a daily dose for 5 days.

[0072] In some embodiments, the method further includes the step of treating the patient with a high-dose IL-2 regimen starting the day after administering TIL cells to the patient in step (h).

[0073] In some implementations, high-dose IL-2 regimens include administering 600,000 or 720,000 IU / kg via 15-minute intravenous infusion every 8 hours until tolerated.

[0074] In some implementations, the cancer is selected from: melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, cancer caused by human papillomavirus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), kidney cancer, and renal epithelial cell carcinoma.

[0075] In some implementations, the cancer is selected from melanoma, HNSCC, cervical cancer, and NSCLC.

[0076] In some implementations, the cancer is melanoma.

[0077] In some implementations, the cancer is HNSCC.

[0078] In some implementations, the cancer is cervical cancer.

[0079] In some implementations, the cancer is NSCLC.

[0080] The present invention also provides a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, the method comprising:

[0081] (a) Processed tumor fragments from a patient’s excised tumor are added to a closed system to obtain the first TIL group;

[0082] (b) A first amplification is performed by culturing a first TIL population in a cell culture medium containing IL-2 to generate a second TIL population; wherein the first amplification is performed in a closed container providing a first ventilated surface area; wherein the first amplification is performed for approximately 3 to 14 days to obtain the second TIL population; wherein the number of the second TIL population is at least 50 times greater than the number of the first TIL population; wherein the transition from step (a) to step (b) occurs without opening the system;

[0083] (c) A second expansion is performed by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3 and antigen-presenting cells (APCs) to generate a third TIL population; wherein the second expansion is performed for approximately 7 to 14 days to obtain the third TIL population; wherein the third TIL population is a therapeutic TIL population; wherein the second expansion is performed in a closed container providing a second breathable surface area; wherein the transition from step (b) to step (c) occurs without opening the system;

[0084] (d) Harvest the therapeutic TIL cluster obtained from step (c); wherein the transition from step (c) to step (d) occurs without opening the system; and

[0085] (e) The TIL clusters harvested in step (d) are transferred to an infusion bag; wherein the transfer from step (d) to step (e) occurs without opening the system.

[0086] In some implementations, the therapeutic TIL clusters harvested in step (d) contain sufficient TILs for a therapeutically effective dose of TILs.

[0087] In some implementations, the number of TILs sufficient for a therapeutically effective dose is approximately 2.3 × 10⁻⁶. 10 Approximately 13.7 × 10 10 .

[0088] In some embodiments, the method further includes the step of cryopreserving the infusion bag containing the harvested TIL clusters using a cryopreservation method.

[0089] In some implementations, the cryopreservation method uses a 1:1 ratio of harvested TILs to the cryopreservation medium.

[0090] In some implementations, the antigen-presenting cells are peripheral blood mononuclear cells (PBMCs).

[0091] In some implementations, the PBMC is an irradiated and allogeneic organism.

[0092] According to the method of claim 68, PBMCs are added to the cell culture on any one of days 9 to 14 of step (c).

[0093] In some implementations, the antigen-presenting cells are artificial antigen-presenting cells.

[0094] In some implementations, the LOVO cell processing system is used for harvesting in step (d).

[0095] In some embodiments, the plurality of fragments comprises approximately 4 to approximately 50 fragments, wherein each fragment has a volume of approximately 27 mm. 3 .

[0096] In some embodiments, the multiple fragments comprise approximately 30 to approximately 60 fragments, with a total volume of approximately 1300 mm. 3 Approximately 1500mm 3 .

[0097] In some embodiments, the multiple fragments include approximately 50 fragments with a total volume of approximately 1350 mm. 3 .

[0098] In some implementations, the multiple fragments include about 50 fragments with a total mass of about 1 gram to about 1.5 grams.

[0099] In some implementations, the multiple fragments include approximately four fragments.

[0100] In some embodiments, the second cell culture medium is provided in a container selected from G containers and Xuri cell bags.

[0101] In some implementations, the infusion bag in step (e) is an infusion bag containing HypoThermosol.

[0102] In some implementations, the first stage in step (b) and the second stage in step (c) are performed for 10 days, 11 days, or 12 days, respectively.

[0103] In some implementations, the first stage in step (b) and the second stage in step (c) each last for 11 days.

[0104] In some implementations, steps (a) through (e) take approximately 10 to approximately 22 days.

[0105] In some implementations, steps (a) through (e) take approximately 10 to approximately 20 days.

[0106] In some implementations, steps (a) through (e) take approximately 10 to 15 days.

[0107] In some implementations, steps (a) through (e) are performed for less than 22 days.

[0108] In some implementations, steps (a) through (e) and cryopreservation are performed for less than 22 days.

[0109] In some implementations, steps (b) through (e) are performed in a single container; wherein performing steps (b) through (e) in a single container increases the TIL yield per resected tumor compared to performing steps (b) through (e) in more than one container.

[0110] In some implementations, antigen-presenting cells are added to the TIL during the second phase of step (c) without opening the system.

[0111] In some implementations, the risk of microbial contamination is reduced compared to open systems.

[0112] In some implementations, the TIL from step (e) is injected into the patient.

[0113] In some implementations, the closed container comprises a single bioreactor.

[0114] In some implementations, the sealed container includes G-REX-10.

[0115] In some implementations, the sealed container includes G-REX-100.

[0116] In some embodiments, in step (d), antigen-presenting cells (APCs) are added to the cell culture of the second TIL population at an APC:TIL ratio of 25:1 to 100:1.

[0117] In some embodiments, the cell culture has a density of 2.5 × 10⁻⁶. 9 APC ratio 100×10 6 The ratio of TIL.

[0118] In some embodiments, in step (c), antigen-presenting cells (APCs) are added to the cell culture of the second TIL population at an APC:TIL ratio of 25:1 to 100:1.

[0119] In some embodiments, the cell culture has a density of 2.5 × 10⁻⁶. 9 APC and 100×10 6 The ratio of TIL.

[0120] The present invention also provides an expanded TIL population for treating subjects with cancer, wherein the expanded TIL population is a third TIL population obtained by a method comprising the following steps:

[0121] (a) First TIL clusters are obtained by processing tumor samples obtained from patients into multiple tumor fragments and tumors removed by the subject;

[0122] (b) Adding tumor fragments to the closed system;

[0123] (c) A first amplification is performed by culturing a first TIL population in a cell culture medium containing IL-2 to generate a second TIL population; wherein the first amplification is performed in a closed container providing a first ventilated surface area; wherein the first amplification is performed for approximately 3 to 14 days to obtain the second TIL population; wherein the number of the second TIL population is at least 50 times greater than the number of the first TIL population; wherein the transition from step (b) to step (c) occurs without opening the system;

[0124] (d) A second expansion is performed by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, and antigen-presenting cells (APCs) to generate a third TIL population; wherein the second expansion is performed for approximately 7 to 14 days to obtain the third TIL population; wherein the third TIL population is a therapeutic TIL population; wherein the second expansion is performed in a closed container providing a second breathable surface area; wherein the transition from step (c) to step (d) occurs without opening the system;

[0125] (e) Exposing second and / or third TIL groups to transcription factors (TFs) and / or other molecules capable of transiently altering protein expression; wherein TFs and / or other molecules capable of transiently altering protein expression provide an increase in tumor antigen expression and / or an increase in the number of tumor antigen-specific T cells in the therapeutic TIL group;

[0126] (f) Harvest the therapeutic TIL cluster obtained from step (d); wherein the transition from step (d) to step (e) occurs without opening the system; and

[0127] (g) The TIL clusters harvested in step (e) are transferred to an infusion bag; wherein the transition from step (e) to step (f) occurs without opening the system; and

[0128] (h) Optionally, the infusion bag containing the TIL clusters harvested from step (f) is cryopreserved using a cryopreservation method.

[0129] In some implementations, the TIL group is used to treat a subject with cancer according to the methods described above and herein, wherein the method further includes one or more of the features described above and herein.

[0130] This invention also provides a method for determining TIL activity. This disclosure provides a method for determining TIL activity by expanding tumor-infiltrating lymphocytes (TILs) into a larger TIL population, comprising:

[0131] (i) Obtain the previously expanded first TIL population;

[0132] (ii) A second TIL population was generated by first expansion through culturing the first TIL population in a cell culture medium containing IL-2; and

[0133] (iii) A second expansion was performed by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3 and antigen-presenting cells (APC) to generate a third TIL population; wherein the number of the third TIL population was at least 100 times greater than that of the second TIL population; wherein the second expansion was performed for at least 14 days to obtain the third TIL population; wherein the activity of the third TIL population was further analyzed.

[0134] In some implementations, the method further includes:

[0135] (iv) A second amplification was performed by supplementing the cell culture medium of the third TIL population with additional IL-2, additional OKT-3 and additional APC; wherein the second amplification was performed for at least 14 days to obtain a larger TIL population than that obtained in step (iii); wherein the activity of the third TIL population was further analyzed.

[0136] In some implementations, the cells are cryopreserved prior to step (i).

[0137] In some implementations, the cells are thawed before step (i).

[0138] In some implementations, step (iv) 1 to 4 is repeated to obtain sufficient TIL for analysis.

[0139] In some implementations, steps (i) through (iii) or (iv) are performed for approximately 40 to approximately 50 days.

[0140] In some implementations, steps (i) through (iii) or (iv) are performed for approximately 42 to approximately 48 days.

[0141] In some implementations, steps (i) through (iii) or (iv) take approximately 42 to approximately 45 days.

[0142] In some implementations, steps (i) through (iii) or (iv) take approximately 44 days.

[0143] In some implementations, cells from step (iii) or (iv) express similar levels of CD4, CD8, and TCRαβ as freshly harvested cells.

[0144] In some implementations, the antigen-presenting cells are peripheral blood mononuclear cells (PBMCs).

[0145] In some implementations, PBMCs are added to the cell culture on any day from day 9 to day 17 of step (iii).

[0146] In some implementations, the APC is an artificial APC (aAPC).

[0147] In some embodiments, the method further includes the step of transducing a first TIL population with an expression vector containing nucleic acid encoding a high-affinity T cell receptor.

[0148] In some implementations, the transduction step occurs before step (i).

[0149] In some embodiments, the method further includes the step of transducing a first TIL group with an expression vector containing nucleic acid encoding a chimeric antigen receptor (CAR) comprising a single-chain variable fragment antibody fused to at least one intracellular domain of a T cell signaling molecule.

[0150] In some implementations, the transduction step occurs before step (i).

[0151] In some implementations, the activity of TIL is measured.

[0152] In some implementations, the activity of the TIL is determined after cryopreservation.

[0153] In some implementations, the activity of TILs is determined after cryopreservation and after step (iv).

[0154] The present invention also provides a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, the method comprising exposing TILs to transcription factors (TFs) and / or other molecules capable of transiently altering protein expression to generate a therapeutic TIL population; wherein the TFs and / or other molecules capable of transiently altering protein expression provide an increase in tumor antigen expression and / or an increase in the number of tumor antigen-specific T cells in the therapeutic TIL population.

[0155] In some implementations, transient changes in protein expression lead to induced protein expression.

[0156] In some implementations, transient changes in protein expression lead to a decrease in protein expression.

[0157] In some implementations, more than one sd-RNA is used to reduce transient protein expression.

[0158] The present invention also provides a method for evaluating transcription factors (TFs) and / or other molecules capable of transiently altering protein expression; wherein the method comprises expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, exposing the TILs to transcription factors (TFs) and / or other molecules capable of transiently altering protein expression, thereby generating a therapeutic TIL population; wherein the TFs and / or other molecules capable of transiently altering protein expression provide alterations in tumor antigen expression and / or the number of tumor antigen-specific T cells in the therapeutic TIL population.

[0159] In some embodiments, transient alterations in protein expression target genes selected from the group consisting of: PD-1, TGFBR2, CBLB (CBL-B), CISH, CCR (chimeric co-stimulatory receptor), IL-2, IL-12, IL-15, IL-21, NOTCH 1 / 2ICD, TIM3, LAG3, TIGIT, TGFβ, CCR2, CCR4, CCR5, CXCR1, CXCR2, CSCR3, CCL2 (MCP-1), CCL3 (MIP-1α), CCL4 (MIP1-β), CCL5 (RANTES), CXCL1 / CXCL8, CCL22, CCL17, CXCL1 / CXCL8, VHL, CD44, PIK3CD, SOCS1, and cAMP protein kinase A (PKA).

[0160] In some embodiments, the present invention provides a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, the method comprising:

[0161] (a) Obtaining the first TIL group from the tumor removed from the patient by processing a tumor sample obtained from the patient into multiple tumor fragments;

[0162] (b) Adding tumor fragments to the closed system;

[0163] (c) The first expansion was performed by culturing the first TIL population in a cell culture medium containing IL-2 and optionally containing a 4-1BB agonist antibody for about 2 to 5 days;

[0164] (d) Contact the first TIL group with at least one sd-RNA; wherein the sd-RNA is added at the following concentrations: 0.1 μM sd-RNA / 10,000 TIL / 100 μL medium, 0.5 μM sd-RNA / 10,000 TIL / 100 μL medium, 0.75 μM sd-RNA / 10,000 TIL / 100 μL medium, 1 μM sd-RNA / 10,000 TIL / 100 μL medium, 1.25 μM sd-RNA / 10,000 TIL / 100 μL medium, 1.5 μM sd-RNA / 10,000 TIL / 100 μL medium, 2 μM sd-RNA / 10,000 TIL / 100 μL medium, 5 μM sd-RNA / 10,000 TIL / 100 μL medium, or 10 μM sd-RNA / 10,000 TIL / 100 μL medium. sd-RNA / 10,000 TIL / 100 μL culture medium; wherein, sd-RNA is used to inhibit the expression of molecules selected from PD-1, LAG-3, TIM-3, CISH and CBLB and combinations thereof;

[0165] (e) Optionally, the first TIL group is subjected to a sterile electroporation step; wherein the sterile electroporation step mediates the transfer of at least one sd-RNA;

[0166] (f) Adding OKT-3 to generate a second TIL population; wherein the first amplification is performed in a closed container providing a first ventilated surface area; wherein the first amplification is performed for approximately 1 to 3 days to obtain the second TIL population; wherein the number of the second TIL population is at least 50 times greater than the number of the first TIL population; wherein the transition from step (c) to step (f) occurs without opening the system.

[0167] (g) Let the second TIL group stand for about 1 day;

[0168] (h) A second expansion is performed by supplementing the cell culture medium of the second TIL population with additional IL-2, optional OKT-3 antibody, optional OX40 antibody, and antigen-presenting cells (APCs) to generate a third TIL population; wherein the second expansion is performed for approximately 7 to 11 days to obtain the third TIL population; wherein the second expansion is performed in a closed container providing a second breathable surface area; wherein the transition from step (c) to step (h) occurs without opening the system;

[0169] (i) Harvest the therapeutic TIL clusters obtained from step (h), providing the harvested TIL clusters; wherein the transition from step (h) to step (i) occurs without opening the system; wherein the harvested TIL clusters are therapeutic TIL clusters;

[0170] (j) The TIL cluster harvested in step (i) is transferred to an infusion bag; wherein the transfer from step (i) to step (j) occurs without opening the system; and

[0171] (k) Cryopreservation of harvested TIL groups using dimethyl sulfoxide-based cryopreservation medium.

[0172] In some embodiments, the present invention provides a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, the method comprising:

[0173] (a) Obtaining the first TIL group from the tumor removed from the patient by processing a tumor sample obtained from the patient into multiple tumor fragments;

[0174] (b) Adding tumor fragments to the closed system;

[0175] (c) The first expansion was performed by culturing the first TIL population in a cell culture medium containing IL-2 and optionally containing a 4-1BB agonist antibody for about 2 to 5 days;

[0176] (d) Contact the first TIL group with at least one sd-RNA, wherein the sd-RNA is added at the following concentrations: 0.1 μM sd-RNA / 10,000 TIL, 0.5 μM sd-RNA / 10,000 TIL, 0.75 μM sd-RNA / 10,000 TIL, 1 μM sd-RNA / 10,000 TIL, 1.25 μM sd-RNA / 10,000 TIL, 1.5 μM sd-RNA / 10,000 TIL, 2 μM sd-RNA / 10,000 TIL, 5 μM sd-RNA / 10,000 TIL, or 10 μM sd-RNA / 10,000 TIL; wherein the sd-RNA is used to inhibit the expression of a molecule selected from PD-1, LAG-3, TIM-3, CISH, and CBLB, and combinations thereof;

[0177] (e) Optionally, a sterile electroporation step is performed on the first TIL group; wherein the sterile electroporation step mediates the transfer of at least one sd-RNA;

[0178] (f) Adding OKT-3 to generate a second TIL population; wherein the first amplification is performed in a closed container providing a first ventilated surface area; wherein the first amplification is performed for approximately 1 to 3 days to obtain the second TIL population; wherein the number of the second TIL population is at least 50 times greater than the number of the first TIL population; wherein the transition from step (c) to step (f) occurs without opening the system.

[0179] (g) Let the second TIL group stand for about 1 day;

[0180] (h) A second expansion is performed by supplementing the cell culture medium of the second TIL population with additional IL-2, optional OKT-3 antibody, optional OX40 antibody, and antigen-presenting cells (APCs) to generate a third TIL population; wherein the second expansion is performed for approximately 7 to 11 days to obtain the third TIL population; wherein the second expansion is performed in a closed container providing a second breathable surface area; wherein the transition from step (g) to step (h) occurs without opening the system;

[0181] (i) Harvest the therapeutic TIL clusters obtained from step (h), providing the harvested TIL clusters; wherein the transition from step (h) to step (i) occurs without opening the system; wherein the harvested TIL clusters are therapeutic TIL clusters;

[0182] (j) The TIL cluster harvested in step (i) is transferred to an infusion bag; wherein the transfer from step (i) to step (j) occurs without opening the system; and

[0183] (k) Cryopreservation of harvested TIL groups using dimethyl sulfoxide-based cryopreservation medium.

[0184] In some embodiments, the present invention provides a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, the method comprising:

[0185] (a) Obtaining the first TIL group from the tumor removed from the patient by processing a tumor sample obtained from the patient into multiple tumor fragments;

[0186] (b) Adding tumor fragments to the closed system;

[0187] (c) The first expansion was performed by culturing the first TIL population in a cell culture medium containing IL-2 and optionally containing a 4-1BB agonist antibody for about 2 to 5 days;

[0188] (d) Add OKT-3 to generate a second TIL population; wherein the first amplification is performed in a closed container providing a first ventilated surface area; wherein the first amplification is performed for approximately 1 to 3 days to obtain the second TIL population; wherein the number of the second TIL population is at least 50 times greater than the number of the first TIL population; wherein the transition from step (c) to step (d) occurs without opening the system.

[0189] (e) Let the second TIL group stand for about 1 day;

[0190] (f) A second expansion is performed by supplementing the cell culture medium of the second TIL population with additional IL-2, optional OKT-3 antibody, optional OX40 antibody and antigen-presenting cells (APC) to generate a third TIL population; wherein the second expansion is performed for approximately 7 to 11 days to obtain the third TIL population; wherein the second expansion is performed in a closed container providing a second breathable surface area; wherein the transition from step (c) to step (f) occurs without opening the system;

[0191] (g) During any of steps (d), (e), and / or (f), the second TIL group is contacted with at least one sd-RNA; wherein the sd-RNA is added at the following concentrations: 0.1 μM sd-RNA / 10,000 TIL / 100 μL medium, 0.5 μM sd-RNA / 10,000 TIL / 100 μL medium, 0.75 μM sd-RNA / 10,000 TIL / 100 μL medium, 1 μM sd-RNA / 10,000 TIL / 100 μL medium, 1.25 μM sd-RNA / 10,000 TIL / 100 μL medium, 1.5 μM sd-RNA / 10,000 TIL / 100 μL medium, 2 μM sd-RNA / 10,000 TIL / 100 μL medium, 5 μM sd-RNA / 10,000 TIL / 100 μL medium or 10 μM sd-RNA / 10,000 TIL / 100 μL medium; wherein, sd-RNA is used to inhibit the expression of molecules selected from PD-1, LAG-3, TIM-3, CISH and CBLB and combinations thereof;

[0192] (h) Optionally, a sterile electroporation step is performed on the second TIL group; wherein the sterile electroporation step mediates the transfer of at least one sd-RNA;

[0193] (i) Harvest the therapeutic TIL clusters obtained from step (g) or (h), and provide the harvested TIL clusters; wherein the transition from step (g) to step (i) occurs without opening the system; wherein the harvested TIL clusters are therapeutic TIL clusters;

[0194] (j) The TIL cluster harvested in step (i) is transferred to an infusion bag; wherein the transfer from step (i) to step (j) occurs without opening the system; and

[0195] (k) Cryopreservation of harvested TIL groups using dimethyl sulfoxide-based cryopreservation medium.

[0196] In some embodiments, the present invention provides a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, the method comprising:

[0197] (a) Obtaining the first TIL group from the tumor removed from the patient by processing a tumor sample obtained from the patient into multiple tumor fragments;

[0198] (b) Adding tumor fragments to the closed system;

[0199] (c) The first expansion was performed by culturing the first TIL population in a cell culture medium containing IL-2 and optionally containing a 4-1BB agonist antibody for about 2 to 5 days;

[0200] (d) Add OKT-3 to generate a second TIL population; wherein the first amplification is performed in a closed container providing a first ventilated surface area; wherein the first amplification is performed for approximately 1 to 3 days to obtain the second TIL population; wherein the number of the second TIL population is at least 50 times greater than the number of the first TIL population; wherein the transition from step (c) to step (d) occurs without opening the system.

[0201] (e) Let the second TIL group stand for about 1 day;

[0202] (f) A second expansion is performed by supplementing the cell culture medium of the second TIL population with additional IL-2, optional OKT-3 antibody, optional OX40 antibody, and antigen-presenting cells (APCs) to generate a third TIL population; wherein the second expansion is performed for approximately 7 to 11 days to obtain the third TIL population; wherein the second expansion is performed in a closed container providing a second breathable surface area; wherein the transition from step (e) to step (f) occurs without opening the system;

[0203] (g) During any of steps (d), (e), and / or (f), the second TIL group is contacted with at least one sd-RNA; wherein the sd-RNA is added at the following concentrations: 0.1 μM sd-RNA / 10,000 TIL, 0.5 μM sd-RNA / 10,000 TIL, 0.75 μM sd-RNA / 10,000 TIL, 1 μM sd-RNA / 10,000 TIL, 1.25 μM sd-RNA / 10,000 TIL, 1.5 μM sd-RNA / 10,000 TIL, 2 μM sd-RNA / 10,000 TIL, 5 μM sd-RNA / 10,000 TIL or 10 μM sd-RNA / 10,000 TIL; wherein, sd-RNA is used to inhibit the expression of molecules selected from PD-1, LAG-3, TIM-3, CISH and CBLB and combinations thereof;

[0204] (h) Optionally, a sterile electroporation step is performed on the second TIL group; wherein the sterile electroporation step mediates the transfer of at least one sd-RNA;

[0205] (i) Harvest the therapeutic TIL clusters obtained from step (g) or (h), providing the harvested TIL clusters; wherein the transition from step (e) to step (h) occurs without opening the system; wherein the harvested TIL clusters are therapeutic TIL clusters;

[0206] (j) The TIL cluster harvested in step (i) is transferred to an infusion bag; wherein the transfer from step (h) to step (i) occurs without opening the system; and

[0207] (k) Cryopreservation of harvested TIL groups using dimethyl sulfoxide-based cryopreservation medium.

[0208] In some implementations, during the first amplification period, sdRNA is added to the first cell population twice a day, once a day, once every two days, once every three days, once every four days, once every five days, once every six days, or once every seven days.

[0209] In some implementations, during the first amplification period, sdRNA is added to the second cell population twice a day, once a day, once every two days, once every three days, once every four days, once every five days, once every six days, or once every seven days.

[0210] In some implementations, two sdRNAs are added to suppress the expression of two molecules selected from PD-1, LAG-3, TIM-3, CISH, and CBLB.

[0211] In some implementations, two sdRNAs are added to suppress the expression of two molecules, wherein the two molecules are selected from:

[0212] i.PD-1 and LAG-3;

[0213] ii. PD-1 and TIM-3;

[0214] iii. PD-1 and CISH;

[0215] iv. PD-1 and CBLB;

[0216] v.LAG-3 and TIM-3;

[0217] vi. LAG-3 and CISH;

[0218] vii. LAG-3 and CBLB;

[0219] viii.TIM-3 and CISH;

[0220] ix.TIM-3 and CBLB; and

[0221] X.CISH and CBLB.

[0222] In some implementations, more than two sd-RNAs are added to suppress the expression of more than two molecules selected from PD-1, LAG-3, TIM-3, CISH, and CBLB.

[0223] In some embodiments, in TILs that have been in contact with at least one sd-RNA, the expression of at least one molecule is reduced by at least 80%, 85%, 90%, or 95%, said at least one molecule being selected from PD-1, LAG-3, TIM-3, CISH, and CBLB.

[0224] In some embodiments, in TILs in contact with at least one sd-RNA, the expression of at least one molecule is reduced by at least 80%, 85%, 90%, or 95% for at least 12 hours, at least 24 hours, or at least 48 hours, said at least one molecule being selected from PD-1, LAG-3, TIM-3, CISH, and CBLB. Attached Figure Description

[0225] Figure 1 The diagram shows an implementation of process 2A (a 22-day process for producing TIL).

[0226] Figure 2 This section compares the implementation methods of process 1C and process 2A for producing TILs.

[0227] Figure 3 This displays the timeline of the 1C process.

[0228] Figure 4 This document illustrates the implementation process of a TIL therapy (including administration and combination therapy steps) using process 2A to produce TILs with high cell counts.

[0229] Figure 5 This document illustrates the process of implementing a TIL therapy (including administration and combination therapy steps) using procedure 2A to produce TILs when cell counts are low.

[0230] Figure 6 : This shows a detailed schematic diagram illustrating the implementation of the 2A process.

[0231] Figures 7A-7C The main steps of an implementation of process 2A (including the cryopreservation step) are described.

[0232] Figure 8 Example process diagram 2A provides an overview of steps A through F.

[0233] Figure 9 : Flowchart of the process for the data collection plan in Process 2A.

[0234] Figure 10The following is an exemplary implementation of the Rapid Expansion Protocol (REP). Upon tumor arrival, the tumor is lysed and placed in a G-Rex flask containing IL-2 for TIL amplification (pre-REP amplification) for 11 days. For triplet studies, IL-2 / IL-15 / IL-21 are added at the start of pre-REP. For the Rapid Expansion Protocol (REP), TILs are cultured with feeder cells and OKT3 for an additional 11 days for REP amplification.

[0235] Figure 11 Example production process of cryopreserved TIL (approximately 22 days).

[0236] Figure 12 The diagram shows an implementation of process 2A (a 22-day process for producing TIL).

[0237] Figure 13 Comparison table of steps A to F of exemplary embodiments of processes 1C and 2A.

[0238] Figure 14 A detailed comparison of the implementation methods of process 1C and process 2A.

[0239] Figure 15 Description of an implementation method for the cryopreservation of TIL production process (22 days).

[0240] Figure 16 Table of process improvements from Gen 1 to Gen 2.

[0241] Figure 17 : A scheme for the production process of Gen 2 cryopreserved LN-144.

[0242] Figure 18 The diagram shows an implementation of process 2A (a 22-day process for producing TIL).

[0243] Figure 19 The diagram shows a schematic of aseptically welding (see process note 5.11 of Example 16) the TIL suspension transfer pack to the bottom (single line) of the gravity blood filter.

[0244] Figure 20 The diagram shows a schematic of the process of aseptically welding the red culture medium removal tubing from the GRex100MCS (see process note 5.11 in Example 16) to the "supernatant" transfer pack.

[0245] Figure 21The diagram shows a schematic of welding a 4S-4M60 (see process note 5.11 of Example 16) to a CC2 CellConnect, where the single tip of the CellConnect device (B) is replaced at (G) with the four spike ends of the 4S-4M60 manifold.

[0246] Figure 22 The diagram shows a schematic of welding a repeater fluid transfer set (see process note 5.11 of Example 16) to one of the male Luer interface terminals of the 4S-4M60.

[0247] Figure 23 The diagram shows a schematic of aseptically welding the long end of a gravity blood filter (see process note 5.11 of Example 16) to a LOVO source bag.

[0248] Figure 24 The diagram shows a schematic of aseptically welding one of the two source lines of the filter (see process note 5.11 of Example 16) to the “combined TIL suspension” collection bag.

[0249] Figure 25 The diagram shows a 4S-4M60 aseptically soldered (see process note 5.11 of Example 16) to the CC2CellConnect, with the four tips of the 4S-4M60 manifold replacing the single tip of the Cell Connect device (B) at (G).

[0250] Figure 26 The diagram shows the aseptic welding of the CS750 cryopreservation bag (see process note 5.11 of Example 16) to the harness prepared in step 8.14.8, replacing one of the four male Luer interface ends (E) with the bag.

[0251] Figure 27 The diagram shows a schematic of welding a CS-10 bag (see process note 5.11 of Example 16) to the nozzle of a 4S-4M60.

[0252] Figure 28 The diagram shows the welding of the "Formulated TIL" bag (see process note 5.11 of Example 16) to the remaining tip (A) on the device prepared in step 8.14.10.

[0253] Figure 29 The diagram shows a heat seal at point F (see process note 5.12 in Example 16), with the empty retentate bag and CS-10 bag removed.

[0254] Figure 30 This diagram illustrates an implementation of the TIL process for transient gene editing.

[0255] Figure 31 This diagram illustrates an implementation of the TIL process for transient gene editing.

[0256] Figure 32 This diagram illustrates the incorporation of the RNA transfer step into the TIL process for the purpose of transient gene reprogramming.

[0257] Figure 33 This section provides an overview of the proposed genetic engineering approach for transiently altering gene expression in TILs.

[0258] Figure 34 This section presents an overview of chemokines and chemokine receptors, and how transient changes in the gene expression of these chemokines and receptors can be used to improve TIL transport to tumor sites.

[0259] Figure 35 This presents a second profile of chemokines and chemokine receptors, whose transient alterations in gene expression can be used to improve TIL transport to tumor sites.

[0260] Figure 36 This shows a schematic structural representation of an exemplary self-delivered ribonucleic acid (sdRNA) implementation. See Ligtenberg et al., Mol. Therapy, 2018.

[0261] Figure 37 This illustrates a schematic structural representation of an exemplary sd-RNA implementation. See U.S. Patent Publication No. 2016 / 0304873.

[0262] Figure 38 This illustrates an exemplary scheme for mRNA synthesis using a DNA template obtained by PCR using specially designed primers. The forward primer contains a phage promoter suitable for in vitro transcription, and the reverse primer contains a polyT stretch. The PCR product is an expression cassette suitable for in vitro transcription. The polyadenylated nucleotide at the 3' end of the nascent mRNA prevents runoff RNA synthesis and the production of double-stranded RNA products. After transcription, the polyA tail can be further extended using poly(A) polymerase (see U.S. Patent No. 8,859,229).

[0263] Figure 39The diagram shows Sd-rxRNA-mediated silencing of PDCD1, TIM3, CBLB, LAG3, and CISH.

[0264] Figure 40 Sd-rxRNA-mediated gene silencing in TILs; exemplary protocol. Exemplary tumors include melanoma (fresh or frozen; n=6), breast tumors (fresh or frozen; n=5), lung tumors (n=1), sarcomas (n=1), and / or ovarian cancer (n=1).

[0265] Figure 41 Reduced protein expression was detected in 4 out of 5 targets. PD1: n=9, TIM3: n=8, LAG3 / CISH: n=2, Cbl-b: n=2. Preparation from pre-REP melanoma and fresh breast cancer TILs (prep), 2 uM sd-rxRNA. Calculated as KD% (100 - (100 * (target gene / NTC))).

[0266] Figure 42 Sd-rxRNA-induced KD decreased with time and stimulation. n=3, preparation derived from pre-REP melanoma TIL, 2uM sd-rxRNA.

[0267] Figure 43 PDCD1 sd-rxRNA slightly affected TIL activity. PD1,TIM3 n>6, derived from TILs of pre-REP melanoma / fresh breast cancer. LAG3,CISH n=2, pre-REP melanoma and breast cancer TILs, 2uM sd-rxRNA.

[0268] Figure 44 PD1 and TIM3 Sd-rxRNA-mediated KD is associated with phenotypic alterations (indicating TIL activation). n=3, preparation from pre-REP melanoma TIL, 2 uM sd-rxRNA.

[0269] Figure 45A and Figure 45B PD1 and TIM3 knockout by sd-rxRNA did not affect the expression of other inhibition / exhaustion markers. A) and B) n=3, TIM3: n=2, formulation from pre-REP melanoma TIL, 2uM sd-rxRNA.

[0270] Figure 46 PD1 and TIM3 KD did not significantly improve IFNγ secretion. n=3, preparation derived from pre-REP melanoma TIL, 2 uM sd-rxRNA.

[0271] Figures 47A-47F: CD107a mobilization is unaffected by any sd-rxRNA. A) n=6, formulation from pre-REP melanoma TILs, 2 uM sd-rxRNA. B) n=2, formulation from pre-REP melanoma and breast cancer TILs, 2 uM sd-rxRNA. C) n=3, frozen melanoma and fresh breast cancer TILs. D) n=3, frozen melanoma and fresh breast and lung cancer TILs. E) and F) n=3, fresh formulation from breast cancer tumors.

[0272] Figure 48 xCELLigence Real-Time Cell Analysis (RTCA)

[0273] Figure 49A and Figure 49B PD1 KD TILs induce greater killing efficiency. A) Representative graph of killing efficiency. B) Representative graph of n=3, melanoma TILs, 2uM sd-rxRNA.

[0274] Figure 50 A and Figure 50 B: Sd-rxRNA dose-response assay. A) n=3, fresh formulation from breast cancer tumors. B) n=3, formulation from pre-REP melanoma TILs.

[0275] Figure 51: Sd-rxRNA-mediated CBLB knockout could not be detected. A) Graph. B) Flow cytometry analysis. n=2, formulations from pre-REP melanoma and fresh breast cancer TILs. CBLB mRNA levels were unchanged compared to NTC. Cbl-b protein levels were unchanged as determined by flow cytometry.

[0276] Figure 52A and Figure 52B This study demonstrates the detection of sd-rxRNA-mediated gene silencing during TIL production at Iovance, assessing the TIL phenotype. sd-rxRNA-mediated PD-1 knockout was associated with phenotypic alterations (indicating TIL activation). PD-1, n>6, was derived from a formulation of pre-REP melanoma / fresh breast cancer TILs, 2 uM sd-rxRNA. A) CD25, CCR7, CD27, CD28, CD56, CD95, 4-1BB, and OX40. B) CD25, CD56, CCR7, 4-1BB, and OX40. N=12, fresh and frozen TILs; breast, melanoma, ovary, and lung.

[0277] Figure 53 A and Figure 53B: Addition of PD1 sd-rxRNA significantly reduced cell growth but did not reduce TIL activity. A) Fold expansion. B) Cell viability. n=7, breast TIL, sarcoma TIL, and lung TIL.

[0278] Figure 54 A and Figure 54 B: In response to nonspecific stimulation, PD1 KD did not improve CD107a mobilization and IFNγ secretion. A) Percentage of CD8 cells expressing CD107a before and after stimulation. B) IFNγ secretion before and after stimulation. n=6, melanoma TIL.

[0279] Figure 55 The experimental design of Example 13 is shown.

[0280] Sequence List Description

[0281] SEQ ID NO: 1 is the amino acid sequence of the heavy chain of muromonab.

[0282] SEQ ID NO: 2 is the amino acid sequence of the light chain of moromumab.

[0283] SEQ ID NO: 3 is the amino acid sequence of recombinant human IL-2 protein.

[0284] SEQ ID NO: 4 is the amino acid sequence of adefovir.

[0285] SEQ ID NO: 5 is the amino acid sequence of recombinant human IL-4 protein.

[0286] SEQ ID NO: 6 is the amino acid sequence of recombinant human IL-7 protein.

[0287] SEQ ID NO: 7 is the amino acid sequence of recombinant human IL-15 protein.

[0288] SEQ ID NO: 8 is the amino acid sequence of recombinant human IL-21 protein.

[0289] SEQ ID NO: 9 is the amino acid sequence of human 4-1BB.

[0290] SEQ ID NO: 10 is the amino acid sequence of mouse 4-1BB.

[0291] SEQ ID NO: 11 is the heavy chain of utomilumab (PF-05082566), a monoclonal antibody that acts as a 4-1BB agonist.

[0292] SEQ ID NO: 12 is the light chain of utolumab (PF-05082566), a monoclonal antibody that acts as a 4-1BB agonist.

[0293] SEQ ID NO: 13 is the heavy chain variable region (V) of utolumab (PF-05082566), a 4-1BB agonist monoclonal antibody. H ).

[0294] SEQ ID NO: 14 is the light chain variable region (V) of utolumab (PF-05082566), a 4-1BB agonist monoclonal antibody. L ).

[0295] SEQ ID NO: 15 is the heavy chain CDR1 of utolumab (PF-05082566), a monoclonal antibody that acts as a 4-1BB agonist.

[0296] SEQ ID NO: 16 is the heavy chain CDR2 of utolumab (PF-05082566), a monoclonal antibody that acts as a 4-1BB agonist.

[0297] SEQ ID NO: 17 is the heavy chain CDR3 of utolumab (PF-05082566), a monoclonal antibody that acts as a 4-1BB agonist.

[0298] SEQ ID NO: 18 is the light chain CDR1 of utolumab (PF-05082566), a monoclonal antibody that acts as a 4-1BB agonist.

[0299] SEQ ID NO: 19 is the light chain CDR2 of utolumab (PF-05082566), a monoclonal antibody that acts as a 4-1BB agonist.

[0300] SEQ ID NO: 20 is the light chain CDR3 of utolumab (PF-05082566), a monoclonal antibody that acts as a 4-1BB agonist.

[0301] SEQ ID NO: 21 is the heavy chain of urelumab (BMS-663513), a monoclonal antibody that acts as a 4-1BB agonist.

[0302] SEQ ID NO: 22 is the light chain of urilumab (BMS-663513), a monoclonal antibody that acts as a 4-1BB agonist.

[0303] SEQ ID NO: 23 is the heavy chain variable region (V) of urilumab (BMS-663513), a 4-1BB agonist monoclonal antibody. H ).

[0304] SEQ ID NO: 24 is the light chain variable region (V) of urilumab (BMS-663513), a 4-1BB agonist monoclonal antibody. L ).

[0305] SEQ ID NO: 25 is the heavy chain CDR1 of urilumab (BMS-663513), a monoclonal antibody that acts as a 4-1BB agonist.

[0306] SEQ ID NO: 26 is the heavy chain CDR2 of urilumab (BMS-663513), a monoclonal antibody that acts as a 4-1BB agonist.

[0307] SEQ ID NO: 27 is the heavy chain CDR3 of urilumab (BMS-663513), a monoclonal antibody that acts as a 4-1BB agonist.

[0308] SEQ ID NO: 28 is the light chain CDR1 of urilumab (BMS-663513), a monoclonal antibody that acts as a 4-1BB agonist.

[0309] SEQ ID NO: 29 is the light chain CDR2 of urilumab (BMS-663513), a monoclonal antibody that acts as a 4-1BB agonist.

[0310] SEQ ID NO: 30 is the light chain CDR3 of urilumab (BMS-663513), a monoclonal antibody that acts as a 4-1BB agonist.

[0311] SEQ ID NO: 31 is the Fc domain of the TNFRSF agonist fusion protein.

[0312] SEQ ID NO: 32 is a linker for the TNFRSF agonist fusion protein.

[0313] SEQ ID NO: 33 is a linker for the TNFRSF agonist fusion protein.

[0314] SEQ ID NO: 34 is a linker for the TNFRSF agonist fusion protein.

[0315] SEQ ID NO: 35 is a linker for the TNFRSF agonist fusion protein.

[0316] SEQ ID NO: 36 is a linker for the TNFRSF agonist fusion protein.

[0317] SEQ ID NO: 37 is a linker for the TNFRSF agonist fusion protein.

[0318] SEQ ID NO: 38 is a linker for the TNFRSF agonist fusion protein.

[0319] SEQ ID NO: 39 is a linker for the TNFRSF agonist fusion protein.

[0320] SEQ ID NO: 40 is a linker for the TNFRSF agonist fusion protein.

[0321] SEQ ID NO: 41 is a linker for the TNFRSF agonist fusion protein.

[0322] SEQ ID NO: 42 is the Fc domain of the TNFRSF agonist fusion protein.

[0323] SEQ ID NO: 43 is a linker for the TNFRSF agonist fusion protein.

[0324] SEQ ID NO: 44 is a linker for the TNFRSF agonist fusion protein.

[0325] SEQ ID NO: 45 is a linker for the TNFRSF agonist fusion protein.

[0326] SEQ ID NO: 46 is the amino acid sequence of the 4-1BB ligand (4-1BBL).

[0327] SEQ ID NO: 47 is the soluble portion of the 4-1BBL polypeptide.

[0328] SEQ ID NO: 48 is the heavy chain variable region (V) of the 4-1BB agonist antibody 4B4-1-1 version 1. H ).

[0329] SEQ ID NO: 49 is the light chain variable region (V) of the 4-1BB agonist antibody 4B4-1-1 version 1. L ).

[0330] SEQ ID NO: 50 is the heavy chain variable region (V) of the 4-1BB agonist antibody 4B4-1-1 version 2. H ).

[0331] SEQ ID NO: 51 is the light chain variable region (V) of the 4-1BB agonist antibody 4B4-1-1 version 2. L ).

[0332] SEQ ID NO: 52 is the heavy chain variable region (V) of the 4-1BB agonist antibody H39E3-2. H ).

[0333] SEQ ID NO: 53 is the light chain variable region (V) of the 4-1BB agonist antibody H39E3-2. L ).

[0334] SEQ ID NO: 54 is the amino acid sequence of human OX40.

[0335] SEQ ID NO: 55 is the amino acid sequence of mouse OX40.

[0336] SEQ ID NO: 56 is the heavy chain of tavolixizumab (MEDI-0562), an OX40 agonist monoclonal antibody.

[0337] SEQ ID NO: 57 is the light chain of talixizumab (MEDI-0562), an OX40 agonist monoclonal antibody.

[0338] SEQ ID NO: 58 is the heavy chain variable region (V) of the OX40 agonist monoclonal antibody talixizumab (MEDI-0562). H ).

[0339] SEQ ID NO: 59 is the light chain variable region (V) of taliximab (MEDI-0562), an OX40 agonist monoclonal antibody. L ).

[0340] SEQ ID NO: 60 is the heavy chain CDR1 of talixizumab (MEDI-0562), an OX40 agonist monoclonal antibody.

[0341] SEQ ID NO: 61 is the heavy chain CDR2 of talixizumab (MEDI-0562), an OX40 agonist monoclonal antibody.

[0342] SEQ ID NO: 62 is the heavy chain CDR3 of talixizumab (MEDI-0562), an OX40 agonist monoclonal antibody.

[0343] SEQ ID NO: 63 is the light chain CDR1 of talixizumab (MEDI-0562), an OX40 agonist monoclonal antibody.

[0344] SEQ ID NO: 64 is the light chain CDR2 of talixizumab (MEDI-0562), an OX40 agonist monoclonal antibody.

[0345] SEQ ID NO: 65 is the light chain CDR3 of talixizumab (MEDI-0562), an OX40 agonist monoclonal antibody.

[0346] SEQ ID NO: 66 is the heavy chain of the OX40 agonist monoclonal antibody 11D4.

[0347] SEQ ID NO: 67 is the light chain of the OX40 agonist monoclonal antibody 11D4.

[0348] SEQ ID NO: 68 is the heavy chain variable region (V) of the OX40 agonist monoclonal antibody 11D4. H ).

[0349] SEQ ID NO: 69 is the light chain variable region (V) of the OX40 agonist monoclonal antibody 11D4. L ).

[0350] SEQ ID NO: 70 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody 11D4.

[0351] SEQ ID NO: 71 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody 11D4.

[0352] SEQ ID NO: 72 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody 11D4.

[0353] SEQ ID NO: 73 is the light chain CDR1 of the OX40 agonist monoclonal antibody 11D4.

[0354] SEQ ID NO: 74 is the light chain CDR2 of the OX40 agonist monoclonal antibody 11D4.

[0355] SEQ ID NO: 75 is the light chain CDR3 of the OX40 agonist monoclonal antibody 11D4.

[0356] SEQ ID NO: 76 is the heavy chain of 18D8, an OX40 agonist monoclonal antibody.

[0357] SEQ ID NO: 77 is the light chain of 18D8, an OX40 agonist monoclonal antibody.

[0358] SEQ ID NO: 78 is the heavy chain variable region (V) of the OX40 agonist monoclonal antibody 18D8. H ).

[0359] SEQ ID NO: 79 is the light chain variable region (V) of the OX40 agonist monoclonal antibody 18D8. L ).

[0360] SEQ ID NO: 80 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody 18D8.

[0361] SEQ ID NO: 81 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody 18D8.

[0362] SEQ ID NO: 82 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody 18D8.

[0363] SEQ ID NO: 83 is the light chain CDR1 of the OX40 agonist monoclonal antibody 18D8.

[0364] SEQ ID NO: 84 is the light chain CDR2 of the OX40 agonist monoclonal antibody 18D8.

[0365] SEQ ID NO: 85 is the light chain CDR3 of the OX40 agonist monoclonal antibody 18D8.

[0366] SEQ ID NO: 86 is the heavy chain variable region (V) of the OX40 agonist monoclonal antibody Hu119-122. H ).

[0367] SEQ ID NO: 87 is the light chain variable region (V) of the OX40 agonist monoclonal antibody Hu119-122. L ).

[0368] SEQ ID NO: 88 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody Hu119-122.

[0369] SEQ ID NO: 89 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody Hu119-122.

[0370] SEQ ID NO: 90 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody Hu119-122.

[0371] SEQ ID NO: 91 is the light chain CDR1 of the OX40 agonist monoclonal antibody Hu119-122.

[0372] SEQ ID NO: 92 is the light chain CDR2 of the OX40 agonist monoclonal antibody Hu119-122.

[0373] SEQ ID NO: 93 is the light chain CDR3 of the OX40 agonist monoclonal antibody Hu119-122.

[0374] SEQ ID NO: 94 is the heavy chain variable region (V) of the OX40 agonist monoclonal antibody Hu106-222. H ).

[0375] SEQ ID NO: 95 is the light chain variable region (V) of the OX40 agonist monoclonal antibody Hu106-222. L ).

[0376] SEQ ID NO: 96 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody Hu106-222.

[0377] SEQ ID NO: 97 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody Hu106-222.

[0378] SEQ ID NO: 98 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody Hu106-222.

[0379] SEQ ID NO: 99 is the light chain CDR1 of the OX40 agonist monoclonal antibody Hu106-222.

[0380] SEQ ID NO: 100 is the light chain CDR2 of the OX40 agonist monoclonal antibody Hu106-222.

[0381] SEQ ID NO: 101 is the light chain CDR3 of the OX40 agonist monoclonal antibody Hu106-222.

[0382] SEQ ID NO: 102 is the amino acid sequence of the OX40 ligand (OX40L).

[0383] SEQ ID NO: 103 is the soluble portion of the OX40L polypeptide.

[0384] SEQ ID NO: 104 is an alternative soluble fraction of the OX40L polypeptide.

[0385] SEQ ID NO: 105 is the heavy chain variable region (V) of the OX40 agonist monoclonal antibody 008. H ).

[0386] SEQ ID NO: 106 is the light chain variable region (V) of the OX40 agonist monoclonal antibody 008. L ).

[0387] SEQ ID NO: 107 is the heavy chain variable region (V) of the OX40 agonist monoclonal antibody 011. H ).

[0388] SEQ ID NO: 108 is the light chain variable region (V) of the OX40 agonist monoclonal antibody 011. L ).

[0389] SEQ ID NO: 109 is the heavy chain variable region (V) of the OX40 agonist monoclonal antibody 021. H ).

[0390] SEQ ID NO: 110 is the light chain variable region (V) of the OX40 agonist monoclonal antibody 021. L ).

[0391] SEQ ID NO: 111 is the heavy chain variable region (V) of the OX40 agonist monoclonal antibody 023. H ).

[0392] SEQ ID NO: 112 is the light chain variable region (V) of the OX40 agonist monoclonal antibody 023. L ).

[0393] SEQ ID NO: 113 is the heavy chain variable region (V) of the OX40 agonist monoclonal antibody. H ).

[0394] SEQ ID NO: 114 is the light chain variable region (V) of the OX40 agonist monoclonal antibody. L ).

[0395] SEQ ID NO: 115 is the heavy chain variable region (V) of the OX40 agonist monoclonal antibody. H ).

[0396] SEQ ID NO: 116 is the light chain variable region (V) of the OX40 agonist monoclonal antibody. L ).

[0397] SEQ ID NO: 117 is the heavy chain variable region (V) of a humanized OX40 agonist monoclonal antibody. H ).

[0398] SEQ ID NO: 118 is the heavy chain variable region (V) of a humanized OX40 agonist monoclonal antibody. H ).

[0399] SEQ ID NO: 119 is the light chain variable region (V) of a humanized OX40 agonist monoclonal antibody. L ).

[0400] SEQ ID NO: 120 is the light chain variable region (V) of a humanized OX40 agonist monoclonal antibody. L ).

[0401] SEQ ID NO: 121 is the heavy chain variable region (V) of a humanized OX40 agonist monoclonal antibody. H ).

[0402] SEQ ID NO: 122 is the heavy chain variable region (V) of a humanized OX40 agonist monoclonal antibody. H ).

[0403] SEQ ID NO: 123 is the light chain variable region (V) of a humanized OX40 agonist monoclonal antibody. L ).

[0404] SEQ ID NO: 124 is the light chain variable region (V) of a humanized OX40 agonist monoclonal antibody. L ).

[0405] SEQ ID NO: 125 is the heavy chain variable region (V) of the OX40 agonist monoclonal antibody. H ).

[0406] SEQ ID NO: 126 is the light chain variable region (V) of the OX40 agonist monoclonal antibody. L ). Detailed Implementation

[0407] I. Introduction

[0408] Adoptive cell therapy using TILs cultured in vitro via a rapid expansion protocol (REP) has yielded successful adoptive cell treatments in melanoma patients following host immunosuppression. Current infusion acceptance parameters depend on the TIL composition readings (e.g., CD28, CD8, or CD4 positive) and the fold increase and viability of the REP product.

[0409] Current REP protocols rarely consider the health status of the TILs to be injected into the patient. T cells undergo a profound metabolic transformation during their maturation from naive T cells to effector T cells (see Chang et al., Nat. Immunol. 2016, 17, 364, explicitly incorporated herein in its entirety, particularly the discussion and markers of anaerobic and aerobic metabolism). For example, naive T cells rely on mitochondrial respiration to produce ATP, while mature, healthy effector T cells such as TILs are highly glycolytic, relying on aerobic glycolysis to provide the bioenergetic substrates required for their proliferation, migration, activation, and antitumor efficacy.

[0410] Current TIL manufacturing processes are limited by length, cost, aseptic issues, and other factors described herein, severely restricting the commercialization potential of such methods; for these and other reasons, no commercially available methods are currently available. This invention provides a TIL manufacturing method and a therapy based on such a method, the TIL manufacturing method utilizing a transient protein expression alteration approach, which is suitable for commercial-scale production and regulatory approval for use in human patients at multiple clinical centers. This invention also provides a transient gene alteration method for reprogramming TILs to prepare a therapeutic TIL population with enhanced therapeutic efficacy.

[0411] II. Definition

[0412] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. All patents and publications cited herein are incorporated herein by reference in their entirety.

[0413] The term "in vivo" refers to events that occur within the body of a subject.

[0414] The term "in vitro" refers to events that occur outside the body of a subject. In vitro assays include cell-based assays using live or dead cells, and may also include cell-free assays that do not use intact cells.

[0415] The term "ex vivo" refers to an event involving the processing or procedure of cells, tissues, and / or organs that have been removed from a subject's body. Appropriately, these cells, tissues, and / or organs may be returned to the subject during surgery or treatment.

[0416] The term "rapid amplification" refers to an increase in the number of antigen-specific TILs by at least approximately 3 times (or 4, 5, 6, 7, 8, or 9 times) within one week, more preferably by at least approximately 10 times (or 20, 30, 40, 50, 60, 70, 80, or 90 times) within one week, or most preferably by at least approximately 100 times within one week. Some rapid amplification protocols are outlined below.

[0417] In this article, "tumor-infiltrating lymphocytes" or "TILs" refers to a cell population initially acquired as leukocytes that has left the subject's bloodstream and migrated to the tumor. TILs include, but are not limited to, CD8+. + Cytotoxic T cells (lymphocytes), Th1 and Th17 CD4 + T cells, natural killer cells, dendritic cells, and M1 macrophages. TILs include primary TILs and secondary TILs. “Primary TILs” are those obtained from patient tissue samples as described herein (sometimes referred to as “freshly harvested”), and “secondary TILs” are any population of TIL cells that has expanded or proliferated as discussed herein, including but not limited to bulk TILs and expanded TILs (“REP TILs” or “post-REP TILs”). TIL cell populations may include genetically modified TILs.

[0418] In this article, a "cell population" (including TILs) refers to a group of cells sharing common characteristics. Typically, the population size is 1 × 10⁻⁶. 6 Up to 1×10 10 Different TIL groups contain different amounts. For example, in the presence of IL-2, the initial growth of primary TILs produces approximately 1 × 10⁻⁶. 8A large population of TILs per cell. Typically, REP amplification is performed to provide 1.5 × 10⁶ cells. 9 Up to 1.5×10 10 A group of cells is used for infusion.

[0419] As used herein, “cryopreserved TIL” refers to primary, large-volume, or expanded TILs (REP TILs) that have been processed and stored at approximately -150°C to -60°C. General methods for cryopreservation are also described elsewhere herein, including in the examples. For clarity, “cryopreserved TIL” can be distinguished from frozen tissue samples that can be used as a source of primary TILs.

[0420] In this article, "thawed cryopreserved TILs" refers to a group of TILs that were previously cryopreserved and then processed to be restored to a temperature above room temperature (including but not limited to cell culture temperature or temperature at which TILs can be applied to patients).

[0421] Tumor-associated lymphoid tissue (TILs) can typically be defined biochemically using cell surface markers or functionally by their ability to infiltrate tumors and influence treatment. TILs are generally classified by expressing one or more of the following biomarkers: CD4, CD8, TCRαβ, CD27, CD28, CD56, CCR7, CD45Ra, CD95, PD-1, and CD25. Alternatively, TILs can be functionally defined by their ability to infiltrate solid tumors upon reintroduction into the patient.

[0422] The term "cryopreservation media" or "cryopreservation medium" refers to any culture medium that can be used for the cryopreservation of cells. Such media may include those containing 7% to 10% DMSO. Exemplary media include CryoStor CS10, Hyperthermasol, and combinations thereof. The term "CS10" refers to a commercially available cryopreservation medium from Stemcell Technologies or Biolife Solutions. CS10 medium may be marketed under the trade name "Cryopreservation Medium". The term "CS10" is used to refer to this medium. CS10 medium is a serum-free and animal-free medium containing DMSO.

[0423] The term "central memory T cells" refers to human cells that are CD45R0+ and constitutively express CCR7 (CCR7). 高 ) and CD62L (CD62 高The T cell subsets of central memory T cells. The surface phenotype of central memory T cells also includes TCR, CD3, CD127 (IL-7R), and IL-15R. Transcription factors for central memory T cells include BCL-6, BCL-6B, MBD2, and BMI1. Upon TCR triggering, central memory T cells primarily secrete IL-2 and CD40L as effector molecules. Central memory T cells are predominantly found in the CD4 compartment in the blood and are proportionally enriched in lymph nodes and tonsils in the human body.

[0424] The term "effective memory T cells" refers to a subset of human or mammalian T cells that, like central memory T cells, are CD45R0+ but lack constitutive expression of CCR7 (CCR7). 低 ), and heterogeneity or low expression of CD62L (CD62L) 低 The surface phenotype of central memory T cells also includes TCR, CD3, CD127 (IL-7R), and IL-15R. Transcription factors for central memory T cells include BLIMP1. Effector memory T cells rapidly secrete high levels of inflammatory cytokines, including interferon-γ, IL-4, and IL-5, upon antigen stimulation. Effector memory T cells are predominantly found in the CD8 compartment of the blood and are proportionally enriched in the lungs, liver, and intestines in the human body. CD8 + Effector memory T cells carry a large amount of perforin.

[0425] The term "closed system" refers to a system that is closed to the external environment. Any closed system suitable for cell culture methods can be used in the methods of this invention. Closed systems include, for example, but not limited to, closed G-containers. Once tumor fragments are added to the closed system, the system is not opened to the external environment until TIL is ready for administration to the patient.

[0426] The terms “fragmenting,” “fragment,” and “fragmented” used in this article to describe methods of destroying tumors include mechanical fragmentation methods, such as breaking, slicing, dividing, and pulverizing tumor tissue, as well as any other methods that destroy the physical structure of tumor tissue.

[0427] The terms "peripheral blood mononuclear cells" and "PBMCs" refer to peripheral blood cells with round nuclei, including lymphocytes (T cells, B cells, NK cells) and monocytes. Preferably, the peripheral blood mononuclear cells are irradiated allogeneic peripheral blood mononuclear cells. PBMCs are antigen-presenting cells.

[0428] The term "anti-CD3 antibody" refers to an antibody or its variants (such as monoclonal antibodies), including human, humanized, chimeric, or mouse antibodies against the CD3 receptor in the T-cell antigen receptor of mature T cells. Anti-CD3 antibodies include OKT-3, also known as moromuzumab. Anti-CD3 antibodies also include the UHCT1 clone, also known as T3 and CD3ε. Other anti-CD3 antibodies include, for example, otelixizumab, teplizumab, and visilizumab.

[0429] The term “OKT-3” (also referred to herein as “OKT3”) refers to a monoclonal antibody or its biosimilar or variant, including human, humanized, chimeric, or murine antibodies against the CD3 receptor in the T-cell antigen receptor of mature T cells, as well as commercially available forms such as OKT-3 (30 ng / mL, pure MACS GMP CD3, Miltenyi Biotech, Inc., San Diego, California, USA) and moromumab or its variants, conserved amino acid substitutions, glycoforms, or biosimilars. The amino acid sequences of the heavy and light chains of moromumab are given in Table 1 (SEQ ID NO: 1 and SEQ ID NO: 2). Hybridomas capable of generating OKT-3 are deposited at the American Type Culture Collection (ATCC) with the assigned ATCC accession number CRL 8001. Hybridomas capable of producing OKT-3 are also deposited at the European Collection of Authenticated Cell Cultures (ECACC), with the catalog number 86022706.

[0430] Table 1: Amino acid sequence of moromuzumab

[0431]

[0432] The term "IL-2" (also referred to herein as "IL2") refers to the T cell growth factor known as interleukin-2, including all forms of IL-2, including human and mammalian forms, conserved amino acid substitutions, glycoforms, biosimilars, and variants thereof. IL-2 is described, for example, in Nelson, J. I. mmunol. 2004, 172, 3983-88 and Malek, Annu. Rev. I. mmunol. 2008, 26, 453-79, the disclosures of which are incorporated herein by reference. The amino acid sequence of recombinant human IL-2 suitable for use in this invention is given in Table 2 (SEQ ID NO: 3). For example, the term IL-2 includes recombinant human IL-2, such as des-alanyl-1 (proleukin, commercially available from multiple suppliers, 22 million IU vials per individual use), and other commercially available recombinant IL-2 from CellGenix, Inc. (CELLGRO GMP) of Portsmouth, New Hampshire, USA, or ProSpec-Tany TechnoGene Ltd. (catalog number CYT-209-b) of East Brunswick, New Jersey, USA, and other commercial equivalents from other suppliers. Des-alanyl-1 is a non-glycosylated recombinant human form of IL-2 with a molecular weight of about 15 kDa. The amino acid sequence of the des-alanyl-1 suitable for use in this invention is given in Table 2 (SEQ ID NO: 4). The term IL-2 also includes PEGylated forms of IL-2 as described herein, including the PEGylated IL-2 prodrug NKTR-214, commercially available from Nektar Therapeutics, Inc., South San Francisco, California, USA. NKTR-214 and PEGylated IL-2 suitable for use in this invention are described in U.S. Patent Application Publication No. US2014 / 0328791A1 and International Patent Application Publication No. WO2012 / 065086A1, the disclosures of which are incorporated herein by reference. Alternative forms of conjugated IL-2 suitable for use in this invention are described in U.S. Patents 4,766,106, 5,206,344, 5,089,261, and 4,902,502, the disclosures of which are incorporated herein by reference. IL-2 formulations suitable for use in this invention are described in U.S. Patent No. 6,706,289, the disclosure of which is incorporated herein by reference.

[0433] Table 2: Amino acid sequence of interleukins

[0434]

[0435] The term “IL-4” (also referred to herein as “IL4”) refers to a cytokine called interleukin-4, which is produced by Th2 T cells and eosinophils, basophils, and mast cells. IL-4 regulates the differentiation of naive helper T cells (Th0 cells) into Th2 T cells. Steinke and Borish, Respir. Res. 2001, 2, 66-70. Upon activation by IL-4, Th2 T cells subsequently produce additional IL-4 in a positive feedback loop. IL-4 also stimulates B cell proliferation and class II MHC expression, and induces B cells to convert to IgE and IgG1 expression. The recombinant human IL-4 suitable for use in this invention is commercially available from several suppliers, including ProSpec-Tany TechnoGene Ltd. of East Brunswick, NJ (catalog number CYT-211) and ThermoFisher Scientific, Inc. of Waltham, MA (recombinant human IL-15 protein, catalog number Gibco CTP0043). The amino acid sequence of recombinant human IL-4 suitable for this invention is given in Table 2 (SEQ ID NO: 5).

[0436] The term “IL-7” (also referred to herein as “IL7”) refers to a glycosylated tissue-derived cytokine called interleukin-7, which is available from stromal cells, epithelial cells, and dendritic cells. Fry and Mackall, Blood 2002, 99, 3892-904. IL-7 can stimulate T cell growth. IL-7 binds to the IL-7 receptor, a heterodimer composed of the IL-7 receptor α and a common gamma chain receptor, which plays an important role in the thymic development and peripheral survival of T cells in a series of signaling pathways. The recombinant human IL-7 suitable for use in this invention is commercially available from several suppliers, including ProSpec-Tany TechnoGene Ltd. of East Brunswick, NJ (catalog number CYT-254) and ThermoFisher Scientific, Inc. of Waltham, MA (recombinant human IL-15 protein, catalog number GibcoPHC0071). The amino acid sequence of recombinant human IL-7 suitable for this invention is given in Table 2 (SEQ ID NO: 6).

[0437] The term "IL-15" (also referred to herein as "IL15") refers to the T-cell growth factor called interleukin-15 and includes all forms of IL-2, including human and mammalian forms, conserved amino acid substitutions, glycoforms, biosimilars, and variants thereof. IL-15 is described, for example, in Fehniger and Caligiuri, Blood 2001, 97, 14-32, the disclosure of which is incorporated herein by reference. IL-15 shares β and γ signaling receptor subunits with IL-2. Recombinant human IL-15 is a non-glycosylated polypeptide single chain containing 114 amino acids (and an N-terminal methionine) with a molecular weight of 12.8 kDa. Recombinant human IL-15 is commercially available from several suppliers, including ProSpec-Tany TechnoGene Ltd. (catalog number CYT-230-b) in East Brunswick, New Jersey, USA, and ThermoFisher Scientific, Inc. (recombinant human IL-15 protein, catalog number 34-8159-82) in Waltham, MA, USA. The amino acid sequence of the recombinant human IL-15 suitable for use in this invention is given in Table 2 (SEQ ID NO: 7).

[0438] The term "IL-21" (also referred to herein as "IL21") refers to a pleiotropic cytokine protein called interleukin-21 and includes all forms of IL-21, including human and mammalian forms, conserved amino acid substitutions, glycoforms, biosimilars, and variants thereof. IL-21 is described, for example, in Spolski and Leonard, Nat. Rev. Drug. Disc. 2014, 13, 379-95, the disclosure of which is incorporated herein by reference. IL-21 is primarily produced by natural killer T cells and activated human CD4+. + T cells produce this compound. Recombinant human IL-21 is a non-glycosylated polypeptide single chain containing 132 amino acids with a molecular weight of 15.4 kDa. Recombinant human IL-21 is commercially available from several suppliers, including ProSpec-Tany TechnoGene Ltd. (catalog number CYT-408-b) in East Brunswick, New Jersey, USA, and ThermoFisher Scientific, Inc. (recombinant human IL-21 protein, catalog number 14-8219-80) in Waltham, MA, USA. The amino acid sequence of the recombinant human IL-21 suitable for use in this invention is given in Table 2 (SEQ ID NO: 8).

[0439] When indicated as "effective antitumor dose," "effective tumor-suppressive dose," or "therapeutic dose," the precise amount of the composition of the invention to be administered can be determined by a physician, taking into account individual differences in age, weight, tumor size, degree of infection or metastasis, and the patient's (subject's) condition. Generally speaking, a pharmaceutical composition comprising tumor-infiltrating lymphocytes (e.g., second-generation TILs or genetically modified cytotoxic lymphocytes) as described herein can be 10 4 Up to 10 11 Cells / kg body weight (e.g., 10) 5 Up to 10 6 10 5 Up to 10 10 10 5 Up to 10 11 10 6 Up to 10 10 10 6 Up to 10 11 10 7 Up to 10 11 10 7 Up to 10 10 10 8 Up to 10 11 10 8 Up to 10 10 10 9 Up to 10 11 Or 10 9 Up to 10 10 Doses are administered in units of cells per kg of body weight, including all integer values ​​within these ranges. Compositions of tumor-infiltrating lymphocytes (including, in some cases, genetically modified cytotoxic lymphocytes) may also be administered in these doses multiple times. Tumor-infiltrating lymphocytes (including, in some cases, genetically modified ones) can be administered using infusion techniques commonly known in immunotherapy (see, for example, Rosenberg et al., “New”, Eng. J. of Med. 319: 1676, 1988). By monitoring the patient’s disease signs and adjusting treatment accordingly, medical professionals can readily determine the optimal dosage and treatment regimen for a particular patient.

[0440] The term "hematologic malignancies" refers to cancers and tumors of mammals affecting hematopoietic and lymphatic tissues (including but not limited to blood, bone marrow, lymph nodes, and tissues of the lymphatic system). Hematologic malignancies are also known as "liquid tumors." Hematologic malignancies include, but are not limited to, acute lymphoblastic leukemia (ALL), chronic lymphocytic lymphoma (CLL), small lymphocytic lymphoma (SLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute monocytic leukemia (AMoL), Hodgkin's lymphoma, and non-Hodgkin's lymphoma. The term "B-cell hematologic malignancies" refers to hematologic malignancies affecting B cells.

[0441] The term "solid tumor" refers to an abnormal mass of tissue that does not typically contain cysts or fluid-filled areas. Solid tumors can be benign or malignant. The term "cancer solid tumor" refers to a malignant, neoplastic, or cancerous solid tumor. Cancer solid tumors include, but are not limited to, sarcomas, malignant epithelial tumors, and lymphomas, such as lung cancer, breast cancer, prostate cancer, colon cancer, rectal cancer, and bladder cancer. The histological structure of a solid tumor consists of interdependent tissue compartments, including the parenchyma (cancer cells) and supporting stromal cells (the microenvironment in which cancer cells are dispersed and can provide support).

[0442] The term "liquid tumor" refers to an abnormal cluster of cells that is essentially a fluid. Liquid tumor cancers include, but are not limited to, leukemia, myeloma, and lymphoma, as well as other hematologic malignancies. TILs obtained from liquid tumors may also be referred to as bone marrow infiltrating lymphocytes (MILs) in this article.

[0443] As used herein, the term “microenvironment” can refer to the entire solid tumor or hematologic malignancy microenvironment, or to a single cell subpopulation within the microenvironment. As used herein, the tumor microenvironment refers to a complex mixture of “cells, soluble factors, signaling molecules, extracellular matrix, and mechanistic clues that promote tumor transformation, support tumor growth and invasion, protect tumors from host immunity, promote resistance to treatment, and provide a niche for the proliferation of overt metastases,” as described by Swartz et al., Cancer Res., 2012, 72, 2473. Although tumors express antigens that should be recognized by T cells, the immune system rarely clears tumors due to immunosuppression of the microenvironment.

[0444] In one embodiment, the present invention includes a method of treating cancer with a group of triglycerides (TILs); wherein the patient is pretreated with non-myeloablative chemotherapy prior to infusion of the TILs of the present invention. In some embodiments, a group of TILs may be provided; wherein the patient is pretreated with non-myeloablative chemotherapy prior to infusion of the TILs of the present invention. In one embodiment, the non-myeloablative chemotherapy is cyclophosphamide 60 mg / kg / day for 2 days (days 27 and 26 prior to TIL infusion) and fludarabine 25 mg / m². 2 / day, for 5 days (days 27 to 23 before TIL infusion). In one embodiment, following non-myeloablative chemotherapy according to the invention and TIL infusion (on day 0), the patient receives an intravenous infusion of IL-2 at 720,000 IU / kg every 8 hours until physiologically tolerated.

[0445] Experimental findings have shown that lymphocyte depletion prior to adoptive metastasis of tumor-specific T lymphocytes plays a key role in enhancing therapeutic efficacy by eliminating competing components of the regulatory T cells and the immune system (“cytokine precipitation”). Therefore, some embodiments of the present invention employ a lymphocyte depletion step (sometimes referred to as “immunosuppressive modulation”) on the patient prior to the introduction of the rTIL of the present invention.

[0446] As used herein, the terms “co-administered,” “combined with,” “in combination with,” “simultaneously,” and “co-” encompass administering two or more active pharmaceutical ingredients (in a preferred embodiment of the invention, for example, at least one potassium channel agonist in combination with multiple TILs) to a subject such that the active pharmaceutical ingredients and / or their metabolites are simultaneously present in the subject. Co-administration includes: administering different compositions simultaneously, administering different compositions at different times, or administering a composition in which two or more active pharmaceutical ingredients are present. Simultaneous administration of different compositions and administration of compositions in which two pharmaceutical ingredients are present are preferred.

[0447] The term "effective amount" or "therapeutic effective amount" refers to an amount of a compound or combination of compounds as described herein that is sufficient to achieve the intended application, including but not limited to the treatment of a disease. Therapeutic effective amounts can vary depending on the intended application (in vitro or in vivo), the subject being treated and the disease condition (e.g., the subject's weight, age, and sex), the severity of the disease condition, or the method of administration. This term also applies to doses that induce a specific response in target cells (e.g., a reduction in platelet adhesion and / or cell migration). The specific dose will depend on the specific compound chosen, the administration regimen to be followed, whether the compound is administered in combination with other compounds, the time of administration, the tissue of administration, and the physical delivery system carrying the compound.

[0448] The terms “treatment,” “treating,” “treat,” etc., refer to achieving the desired pharmacological and / or physiological effect. This effect may be preventative in relation to the complete or partial prevention of a disease or its symptoms, and / or therapeutic in relation to the partial or complete cure of a disease and / or adverse reactions caused by the disease. As used herein, “treatment” includes any treatment of a disease in mammals, particularly humans, including: (a) preventing the onset of the disease in subjects who may be susceptible to it but have not yet been diagnosed with it; (b) suppressing the disease, i.e., preventing its development or progression; and (c) alleviating the disease, i.e., causing disease remission and / or alleviating one or more disease symptoms. “Treatment” also means including the delivery of agents to provide a pharmacological effect, even in the absence of disease or symptom. For example, “treatment” includes the delivery of compositions that can elicit an immune response or confer immunity in the absence of disease, such as in the case of vaccines.

[0449] When referring to nucleic acid or protein components, the term "heterologous" means that the nucleic acid or protein contains two or more subsequences that are not naturally identical to each other. For example, nucleic acids are often produced through recombination and have two or more sequences from unrelated genes that are arranged to produce new functional nucleic acids, such as a promoter from one source and a coding region from another, or coding regions from different sources. Similarly, a heterologous protein means that the protein contains two or more subsequences that are not naturally identical to each other (e.g., a fusion protein).

[0450] In the case of two or more nucleic acids or peptides, the terms "sequence identity," "percentage identity," and "sequence percentage identity" (or their synonyms, such as "99% identical") refer to two or more sequences or subsequences that are identical or have a specified percentage of identical nucleotide or amino acid residues when compared and aligned (introducing vacancies if necessary) to obtain maximum correspondence, regardless of any conserved amino acid substitutions as part of sequence identity. The percentage of identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software are known in the art for obtaining alignments of amino acid or nucleotide sequences. Suitable procedures for determining the percentage of sequence identity include, for example, the BLAST program suite, available from the National Center for Biotechnology Information (NCBI) BLAST website of the U.S. government. The BLASTN or BLASTP algorithms can be used for comparisons between two sequences. BLASTN is used for comparing nucleic acid sequences, while BLASTP is used for comparing amino acid sequences. ALIGN, ALIGN-2 (Genentech, South San Francisco, California), or MegAlign, available from DNASTAR, are other publicly available software programs that can be used for sequence alignment. Those skilled in the art can determine appropriate parameters for maximizing alignment using specific alignment software. In some implementations, the default parameters of the comparison software are used.

[0451] As used herein, the term "variant" includes, but is not limited to, antibodies or fusion proteins that contain an amino acid sequence different from that of a reference antibody by means of one or more substitutions, deletions, and / or additions at positions within or near the amino acid sequence of the reference antibody. A variant may contain one or more conserved substitutions in its amino acid sequence compared to that of the reference antibody. Conserved substitutions may involve, for example, substituting similar charged or uncharged amino acids. The variant retains the ability to specifically bind to the antigen of the reference antibody. The term "variant" also includes polyethylene glycol-modified antibodies or proteins.

[0452] In this article, "tumor-infiltrating lymphocytes" or "TILs" refers to a cell population initially acquired as leukocytes that has left the subject's bloodstream and migrated to the tumor. TILs include, but are not limited to, CD8+. + Cytotoxic T cells (lymphocytes), Th1 and Th17 CD4 + T cells, natural killer cells, dendritic cells, and M1 macrophages. TILs include primary TILs and secondary TILs. "Primary TILs" are those obtained from patient tissue samples as described herein (sometimes referred to as "freshly harvested"), and "secondary TILs" are any populations of TIL cells that have expanded or proliferated as discussed herein, including but not limited to large numbers of TILs, expanded TILs ("REP TILs"), and "reREP TILs." For example, reREP TILs may include second-amplified TILs or additional second-amplified TILs (e.g., Figure 8 Those described in step D include a TIL called reREP TIL.

[0453] Tumor-associated lymphoid tissue (TILs) can generally be defined biochemically using cell surface markers or functionally by their ability to infiltrate tumors and influence treatment. TILs are typically classified by expressing one or more of the following biomarkers: CD4, CD8, TCRαβ, CD27, CD28, CD56, CCR7, CD45Ra, CD95, PD-1, and CD25. Alternatively, TILs can be functionally defined by their ability to infiltrate solid tumors after reintroduction into the patient. TILs can also be characterized by potency—for example, TILs can be considered effective if, for example, the release of interferon (IFN) is greater than about 50 pg / mL, greater than about 100 pg / mL, greater than about 150 pg / mL, or greater than about 200 pg / mL.

[0454] The term "deoxyribonucleic acid" encompasses both natural and synthetic unmodified and modified deoxyribonucleic acids. Modifications include changes to the sugar moiety, base moiety, and / or bonds between deoxyribonucleic acids in oligonucleotides.

[0455] The term "RNA" defines a molecule containing at least one ribonucleotide residue. The term "ribonucleotide" defines a nucleotide having a hydroxyl group at the 2' position of the bD-ribofuranosyl moiety. The term "RNA" includes double-stranded RNA, single-stranded RNA, isolated RNA (e.g., partially purified RNA, substantially pure RNA, synthetic RNA, recombinant RNA, and altered RNA that differs from naturally occurring RNA by the addition, deletion, substitution, and / or modification of more than one nucleotide). The nucleotides in the RNA molecules described herein may also include non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs may be referred to as analogs or analogs of naturally occurring RNA.

[0456] The term "modified nucleotide" refers to a nucleotide that has undergone one or more modifications to its nucleoside, nucleotide base, pentose ring, or phosphate group. For example, modified nucleotides do not include ribonucleotides containing adenosine monophosphate, guanosine monophosphate, uridine monophosphate, and cytidine monophosphate, nor deoxyribonucleotides containing deoxyadenosine monophosphate, deoxyguanosine monophosphate, deoxythymidine monophosphate, and deoxycytidine monophosphate. Modifications include those naturally occurring due to modifications by enzymes that modify nucleotides (e.g., methyltransferases).

[0457] Modified nucleotides also include synthetic nucleotides or nucleotides that are not naturally occurring. Synthetic or non-natural modifications in nucleotides include modifications having a 2' modification (e.g., 2'-O-methyl, 2'-methoxyethoxy, 2'-fluorine, 2'-allyl, 2'-O-[2-(methylamino)-2-oxoethyl], 4'-thio, 4'-CH2-O-2'-bridge, 4'-(CH2)2-O-2'-bridge, 2'-LNA, and 2'-O--(N-carbamate)) or those containing base analogues. With respect to 2'-modified nucleotides as described in this disclosure, "amino" refers to modified or unmodified 2'-NH2 or 2'-O--NH2. Such modifying groups are described, for example, in U.S. Patent Nos. 5,672,695 and 6,248,878, which are incorporated herein by reference.

[0458] The term "microRNA" or "miRNA" refers to a nucleic acid that forms a single-stranded RNA. When the miRNA is expressed in the same cell as a gene or target gene, the single-stranded RNA has the ability to alter the expression of the gene or target gene (reduce or suppress expression; regulate expression; directly or indirectly enhance expression). In one embodiment, miRNA refers to a nucleic acid that is substantially or completely identical to a target gene and forms a single-stranded miRNA. In some embodiments, miRNA may be in the form of pre-miRNA, wherein the pre-miRNA is a double-stranded RNA. The sequence of the miRNA may correspond to the full-length target gene or its subsequence. Typically, the length of miRNA is at least about 15 to 50 nucleotides (e.g., each sequence of a single-stranded miRNA is 15 to 50 nucleotides long, and the length of a double-stranded pre-miRNA is about 15 to 50 base pairs). In some embodiments, miRNA is 20 to 30 base nucleotides long. In some embodiments, miRNA is 20 to 25 nucleotides long. In some implementations, the miRNA is 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length.

[0459] The term “target gene” includes genes known or identified as regulating the expression of genes involved in immune resistance mechanisms. These genes may be one of several groups of genes, such as inhibitory receptors like CTLA4 and PD1; cytokine receptors that inactivate immune cells, such as TGF-β receptor, LAG3 and / or TIM3, and combinations thereof. In some embodiments, the target genes include one or more of PD-1, TGFBR2, CBLB (CBL-B), CISH, CCR (chimeric co-stimulatory receptor), IL-2, IL-4, IL-7, IL-10, IL-12, IL-15, IL-21, NOTCH 1 / 2 intracellular domain (ICD), NOTCH ligand mDLL1, TIM3, LAG3, TIGIT, TGFβ, CCR2, CCR4, CCR5, CXCR1, CXCR2, CSCR3, CCL2 (MCP-1), CCL3 (MIP-1α), CCL4 (MIP1-β), CCL5 (RANTES), CXCL1 / CXCL8, CCL22, CCL17, CXCL1 / CXCL8, VHL, CD44, PIK3CD, SOCS1, and / or cAMP protein kinase A (PKA).

[0460] The terms “small interfering RNA” or “siRNA” or “short interfering RNA” or “silent RNA” define a group of double-stranded RNA molecules, consisting of a sense RNA strand and an antisense RNA strand, each typically about 1022 nucleotides in length, optionally containing a 3' overhang of 1 to 3 nucleotides. siRNAs are active in the RNA interference (RNAi) pathway and interfere with the expression of specific target genes through complementary nucleotide sequences.

[0461] The term sd-RNA refers to a "self-delivering" RNAi reagent that forms an asymmetric double-stranded RNA-antisense oligonucleotide hybrid. The double-stranded RNA comprises a lead (sense) strand of approximately 19 to 25 nucleotides and a follower (antisense) strand of approximately 10 to 19 nucleotides, forming a double helix that results in a single-stranded phosphorothiolated tail of approximately 5 to 9 nucleotides. In some embodiments, the RNA sequence can be modified with stabilizing and hydrophobic modifications (e.g., sterols, such as cholesterol, vitamin D, naphthyl, isobutyl, benzyl, indole, tryptophan, and phenyl) that provide stability and efficient cellular uptake in the absence of any transfection reagents or formulations. In some embodiments, IFN-induced protein immunoreactivity assays have shown that sd-RNA produces reduced immunostimulation compared to other RNAi reagents. See, for example, Byrne et al., December 2013, J. Ocular Pharmacology and Therapeutics, 29(10): 855-864, which is incorporated herein by reference. In some embodiments, the sd-RNA described herein is available from Advirna LLC (Worcester, MA, USA).

[0462] The terms "pharmaceutically acceptable carrier" or "pharmaceuticalally acceptable excipient" are intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption-delaying agents, and inert components. The use of such pharmaceutically acceptable carriers or excipients for the active pharmaceutical ingredient is well known in the art. Unless any conventional pharmaceutically acceptable carrier or excipient is incompatible with the active pharmaceutical ingredient, its use in the therapeutic compositions of the present invention is contemplated. Other active pharmaceutical ingredients (e.g., other drugs) may also be incorporated into the described compositions and methods.

[0463] The terms “about” and “approximately” indicate a statistically significant range of values. Such a range may be within an order of magnitude of a given value or range, preferably within 50%, more preferably within 20%, more preferably within 10%, and even more preferably within 5%. The permissible variation included in the terms “about” or “approximately” depends on the specific system under study and will be readily understood by those skilled in the art. Furthermore, as used herein, the terms “about” and “approximately” mean that size, dimension, formulation, parameter, shape, and other properties and characteristics are not, and need not be, precise, but may be approximate and / or larger or smaller as needed, reflecting tolerances, conversion factors, rounding, measurement errors, and other factors known to those skilled in the art. Generally, size, dimension, formulation, parameter, shape, or other properties or characteristics are “about” or “approximate”, whether explicitly stated or not. It should be noted that embodiments with very different sizes, shapes, and dimensions may employ the aforementioned arrangements.

[0464] When used in the appended claims in both original and modified forms, the transitional terms “comprising,” “substantially consisting of,” and “consisting of” define the scope of the claims with respect to any additional, unlisted claim elements or steps (if any) excluded from the scope of the claims. The term “comprising” is intended to be inclusive or open-ended and does not exclude any additional, unlisted elements, methods, steps, or materials. The term “consisting of” excludes any elements, steps, or materials other than those described in the claims, and in the case of materials, the term “consisting of” excludes common impurities associated with the specified material. The term “substantially consisting of” limits the scope of the claims to the specified elements, steps, or materials and those that do not substantially affect the essential and novel features of the claimed invention. In alternative embodiments, all compositions, methods, and kits embodying the invention described herein may be more specifically defined by any of the transitional terms “comprising,” “substantially consisting of,” and “consisting of.”

[0465] III. Methods for transiently altering protein expression in TILs

[0466] In some embodiments, the amplified TILs of the present invention are further manipulated to alter protein expression before, during, or after the amplification step (including during the closed aseptic production method as described herein). In some embodiments, the transiently altered protein expression is due to transient gene editing. In some embodiments, the amplified TILs of the present invention are treated with transcription factors (TFs) and / or other molecules capable of transiently altering protein expression in TILs. In some embodiments, TFs and / or other molecules capable of transiently altering protein expression provide alterations in tumor antigen expression and / or the number of tumor antigen-specific T cells in the TIL population.

[0467] In some embodiments, the present invention includes gene editing via nucleotide insertion (e.g., via ribonucleic acid (RNA) insertion, including messenger RNA (mRNA) or small (or short) interfering RNA (siRNA) insertion) TIL groups to promote or inhibit the expression of one or more proteins, as well as combinations of simultaneously promoting one group of proteins and inhibiting another group of proteins.

[0468] In some embodiments, the amplified TILs of the present invention undergo a transient change in protein expression. In some embodiments, the transient change in protein expression occurs in a large population of TILs prior to the first amplification, including, for example, those derived from… Figure 8 The TIL population obtained in step A is shown, for example. In some embodiments, transient changes in protein expression occur during the first amplification, including, for example... Figure 8 The TIL population shown is, for example, the TIL population amplified in step B. In some embodiments, transient changes in protein expression occur after the first amplification, including, for example, the transitional TIL population between the first and second amplifications, by... Figure 8 The example shown is the TIL population obtained in step B and included in step C. In some embodiments, transient changes in protein expression occur in a large TIL population prior to the second amplification, including, for example, those obtained by… Figure 8 The TIL population shown is, for example, obtained in step C and prior to amplification in step D. In some embodiments, transient changes in protein expression occur during the second amplification, including, for example, in... Figure 8 The example shown is the TIL population amplified in step D. In some embodiments, transient changes in protein expression occur after the second amplification, including, for example, by... Figure 8 The TIL group is obtained by amplification, for example, in step D.

[0469] In one embodiment, the method for transiently altering protein expression in a TIL population includes an electroporation step. In one embodiment, according to... Figure 30 and Figure 31The method shown is a method for transiently altering protein expression in a TIL population. Electroporation is a method known in the art and described, for example, in Tsong, Biophys. J. 1991, 60, 297-306 and U.S. Patent Application Publication No. 2014 / 0227237A1, the disclosures of which are incorporated herein by reference. In one embodiment, the method for transiently altering protein expression in a TIL population includes a calcium phosphate transfection step. Calcium phosphate transfection methods (calcium phosphate DNA precipitation, cell surface coating, and endocytosis) are known in the art and described in Graham and van der Eb, Virology 1973, 52, 456-467; Wigler et al., Proc. Natl. Acad. Sci. 1979, 76, 1373-1376; Chen and Okayarea, Mol. Cell. Biol. 1987, 7, 2745-2752; and U.S. Patent No. 5,593,875, the disclosures of which are incorporated herein by reference. In one embodiment, a method for transiently altering protein expression in a TIL population includes a liposome transfection step. Liposome transfection methods, such as those using a 1:1 (w / w) lipid formulation of the cationic lipid N-[1-(2,3-dioleoyloxy)propyl]-n,n,n-trimethylammonium chloride (DOTMA) and dioleoylphosphatidylethanolamine (DOPE) in filtered water, are known in the art and described in Rose et al., Biotechniques 1991, 10, 520-525 and Felgner et al., Proc. Natl. Acad. Sci. U.S., 1987, 84, 7413-7417, and U.S. Patent Nos. 5,279,833; 5,908,635; 6,056,938; 6,110,490; 6,534,484 and 7,687,070, the disclosures of which are incorporated herein by reference. In one embodiment, a method for transiently altering protein expression in a TIL population includes a transfection step using the methods described in U.S. Patent Nos. 5,766,902; 6,025,337; 6,410,517; 6,475,994 and 7,189,705, the disclosures of which are incorporated herein by reference.

[0470] In some embodiments, transient changes in protein expression lead to an increase in T memory stem cells (TSCMs). TSCMs are early progenitor cells of central memory T cells that have experienced antigens. TSCMs typically exhibit defining stem cell long-term survival, self-renewal, and pluripotency, often required to produce potent TIL products. TSCMs have shown enhanced antitumor activity compared to other T cell subsets in mouse models of adoptive cell transfer (Gattinoni et al., Nat Med 2009, 2011; Gattinoni, Nature Rev. Cancer, 2012; Cieri et al., Blood 2013). In some embodiments, transient changes in protein expression result in a TIL population comprising a high proportion of TSCMs. In some embodiments, the transient change in protein expression causes an increase in the percentage of TSCM of at least 5%, at least 10%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%. In some embodiments, the transient change in protein expression causes an increase in TSCM of at least 1, 2, 3, 4, 5, or 10 times in the TIL population. In some embodiments, the transient change in protein expression results in a TSCM of at least 5%, at least 10%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% for the TIL population. In some embodiments, the transient change in protein expression results in a TSCM of at least 5%, at least 10%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% for the therapeutic TIL population.

[0471] In some embodiments, transient changes in protein expression cause rejuvenation of T cells that have experienced an antigen. In some embodiments, rejuvenation includes, for example, increased proliferation, increased T cell activation, and / or increased antigen recognition.

[0472] In some embodiments, transient changes in protein expression alter expression in most T cells to preserve the tumor-derived TCR repertoire. In some embodiments, transient changes in protein expression do not alter the tumor-derived TCR repertoire. In some embodiments, transient changes in protein expression maintain the tumor-derived TCR repertoire.

[0473] In some embodiments, transient changes in protein expression lead to alterations in the expression of specific genes. In some embodiments, the genes targeted by these transient changes in protein expression include, but are not limited to, PD-1 (also known as PDCD1 or CC279), TGFBR2, CCR4 / 5, CBLB (CBL-B), CISH, CCR (chimeric co-stimulatory receptor), IL-2, IL-12, IL-15, IL-21, NOTCH 1 / 2 ICD, TIM3, LAG3, TIGIT, TGFβ, CCR2, CCR4, CCR5, CXCR1, CXCR2, CSCR3, CCL2 (MCP-1), CCL3 (MIP-1α), CCL4 (MIP1-β), CCL5 (RANTES), CXCL1 / CXCL8, CCL22, CCL17, CXCL1 / CXCL8, VHL, CD44, PIK3CD, SOCS1, and / or cAMP protein kinase A (PKA). In some embodiments, the transient alteration of protein expression targets genes selected from PD-1, TGFBR2, CCR4 / 5, CBLB (CBL-B), CISH, CCR (chimeric co-stimulatory receptor), IL-2, IL-12, IL-15, IL-21, NOTCH 1 / 2 ICD, TIM3, LAG3, TIGIT, TGFβ, CCR2, CCR4, CCR5, CXCR1, CXCR2, CSCR3, CCL2 (MCP-1), CCL3 (MIP-1α), CCL4 (MIP1-β), CCL5 (RANTES), CXCL1 / CXCL8, CCL22, CCL17, CXCL1 / CXCL8, VHL, CD44, PIK3CD, SOCS1, and / or cAMP protein kinase A (PKA). In some embodiments, the transient alteration of protein expression targets PD-1. In some embodiments, the transient alteration of protein expression targets TGFBR2. In some embodiments, transient changes in protein expression target CCR4 / 5. In some embodiments, transient changes in protein expression target CBLB. In some embodiments, transient changes in protein expression target CISH. In some embodiments, transient changes in protein expression target CCR (chimeric co-stimulatory receptor). In some embodiments, transient changes in protein expression target IL-2. In some embodiments, transient changes in protein expression target IL-12. In some embodiments, transient changes in protein expression target IL-15. In some embodiments, transient changes in protein expression target IL-21. In some embodiments, transient changes in protein expression target NOTCH 1 / 2 ICD.

[0474] In some implementations, transient alterations in protein expression target the NOTCH signaling pathway, for example, via NOTCH1 / 2 ICD and / or via other NOTCH ligands, such as mDLL1 (see, for example, Kondo, T. et al., NOTCH-mediated conversion of activated T cells into stem cell memory-like T cells for adaptive immunotherapy, Nature Communications, Vol. 8, Article No. 15338 (2017), the entire contents of which are incorporated herein by reference).

[0475] In some embodiments, transient changes in protein expression target TIM3. In some embodiments, transient changes in protein expression target LAG3. In some embodiments, transient changes in protein expression target TIGIT. In some embodiments, transient changes in protein expression target TGFβ. In some embodiments, transient changes in protein expression target CCR1. In some embodiments, transient changes in protein expression target CCR2. In some embodiments, transient changes in protein expression target CCR4. In some embodiments, transient changes in protein expression target CCR5. In some embodiments, transient changes in protein expression target CXCR1. In some embodiments, transient changes in protein expression target CXCR2. In some embodiments, transient changes in protein expression target CSCR3. In some embodiments, transient changes in protein expression target CCL2 (MCP-1). In some embodiments, transient changes in protein expression target CCL3 (MIP-1α). In some embodiments, transient changes in protein expression target CCL4 (MIP1-β). In some embodiments, transient changes in protein expression target CCL5 (RANTES). In some embodiments, transient changes in protein expression target CXCL1. In some embodiments, transient changes in protein expression target CXCL8. In some embodiments, transient changes in protein expression target CCL22. In some embodiments, transient changes in protein expression target CCL17. In some embodiments, transient changes in protein expression target VHL. In some embodiments, transient changes in protein expression target CD44. In some embodiments, transient changes in protein expression target PIK3CD. In some embodiments, transient changes in protein expression target SOCS1. In some embodiments, transient changes in protein expression target cAMP protein kinase A (PKA).

[0476] In some embodiments, transient changes in protein expression result in an increase and / or overexpression of chemokine receptors. In some embodiments, the chemokine receptors overexpressed by transient protein expression include receptors with ligands, including but not limited to CCL2 (MCP-1), CCL3 (MIP-1α), CCL4 (MIP1-β), CCL5 (RANTES), CXCL1, CXCL8, CCL22, and / or CCL17. In some embodiments, the chemokine receptors overexpressed by transient protein expression include receptors with ligands, including but not limited to IL-2, IL-7, IL-10, IL-15, and IL-21, and the NOTCH1 / 2 intracellular domain (ICD). In some implementations, transient alterations in protein expression target the NOTCH signaling pathway, for example, via NOTCH 1 / 2 ICD and / or via other NOTCH ligands, such as mDLL1 (see, for example, Kondo, T. et al., NOTCH-mediated conversion of activated T cells into stem cell memory-like T cells for adoptive immunotherapy, Nature Communications, Vol. 8, Article No. 15338 (2017), the entire contents of which are incorporated herein by reference).

[0477] In some embodiments, transient changes in protein expression result in decreased and / or reduced expression of PD-1, CTLA-4, TIM-3, LAG-3, TIGIT, TGFβR2, and / or TGFβ (including, for example, TGFβ pathway blockade). In some embodiments, transient changes in protein expression result in decreased and / or reduced CBLB (CBL-B) expression. In some embodiments, transient changes in protein expression result in decreased and / or reduced CISH expression.

[0478] In some embodiments, transient changes in protein expression lead to an increase and / or overexpression of chemokine receptors, thereby, for example, improving TIL transport or motility to tumor sites. In some embodiments, transient changes in protein expression lead to an increase and / or overexpression of CCRs (chimeric co-stimulatory receptors). In some embodiments, transient changes in protein expression lead to an increase and / or overexpression of chemokine receptors selected from CCR1, CCR2, CCR4, CCR5, CXCR1, CXCR2, and / or CSCR3.

[0479] In some embodiments, transient changes in protein expression lead to an increase and / or overexpression of interleukins. In some embodiments, transient changes in protein expression lead to an increase and / or overexpression of interleukins selected from IL-2, IL-12, IL-15, and / or IL-21.

[0480] In some embodiments, transient alterations in protein expression target the NOTCH signaling pathway, for example, via NOTCH1 / 2 ICD and / or via other NOTCH ligands, such as mDLL1 (see, for example, Kondo, T. et al., NOTCH-mediated conversion of activated T cells into stem cell memory-like T cells for adaptive immunotherapy, Nature Communications, Vol. 8, Article No. 15338 (2017), the entire contents of which are incorporated herein by reference). In some embodiments, transient alterations in protein expression result in increased and / or overexpression of NOTCH 1 / 2 ICD. In some embodiments, transient alterations in protein expression result in increased and / or overexpression of NOTCH ligands (e.g., mDLL1). In some embodiments, transient alterations in protein expression result in increased and / or overexpression of VHL. In some embodiments, transient alterations in protein expression result in increased and / or overexpression of CD44. In some embodiments, transient alterations in protein expression result in increased and / or overexpression of PIK3CD. In some implementations, transient changes in protein expression lead to an increase and / or overexpression of SOCS1.

[0481] In some implementations, transient changes in protein expression lead to a decrease and / or reduction in cAMP protein kinase A (PKA) expression.

[0482] In some embodiments, the transient change in protein expression results in a decrease and / or reduction in the expression of one molecule selected from PD-1, LAG3, TIM3, CTLA-4, TIGIT, CISH, TGFβR2, PKA, CBLB, BAFF (BR3), and combinations thereof. In some embodiments, the transient change in protein expression results in a decrease and / or reduction in the expression of two molecules selected from PD-1, LAG3, TIM3, CTLA-4, TIGIT, CISH, TGFβR2, PKA, CBLB, BAFF (BR3), and combinations thereof. In some embodiments, the transient change in protein expression results in a decrease and / or reduction in the expression of PD-1 and one molecule selected from LAG3, TIM3, CTLA-4, TIGIT, CISH, TGFβR2, PKA, CBLB, BAFF (BR3), and combinations thereof. In some embodiments, the transient change in protein expression results in a decrease and / or reduction in the expression of PD-1, LAG-3, CISH, CBLB, TIM3, and combinations thereof. In some embodiments, transient changes in protein expression result in a decrease and / or reduction in the expression of PD-1 and one of LAG3, CISH, CBLB, TIM3, and combinations thereof. In some embodiments, transient changes in protein expression result in a decrease and / or reduction in the expression of PD-1 and LAG3. In some embodiments, transient changes in protein expression result in a decrease and / or reduction in the expression of PD-1 and CISH. In some embodiments, transient changes in protein expression result in a decrease and / or reduction in the expression of PD-1 and CBLB. In some embodiments, transient changes in protein expression result in a decrease and / or reduction in the expression of LAG3 and CISH. In some embodiments, transient changes in protein expression result in a decrease and / or reduction in the expression of LAG3 and CBLB. In some embodiments, transient changes in protein expression result in a decrease and / or reduction in the expression of CISH and CBLB. In some embodiments, transient changes in protein expression result in a decrease and / or reduction in the expression of TIM3 and PD-1. In some embodiments, transient changes in protein expression result in a decrease and / or reduction in the expression of TIM3 and LAG3. In some embodiments, transient changes in protein expression result in a decrease and / or reduction in the expression of TIM3 and CISH. In some embodiments, transient changes in protein expression result in a decrease and / or reduction in the expression of TIM3 and CBLB.

[0483] In some implementations, adhesion molecules selected from CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, and combinations thereof are inserted into a first TIL group, a second TIL group, or a harvested TIL group via gamma-retroviral or lentiviral methods (e.g., increased expression of adhesion molecules).

[0484] In some embodiments, transient changes in protein expression result in decreased and / or reduced expression of molecules selected from PD-1, LAG3, TIM3, CTLA-4, TIGIT, CISH, TGFβR2, PKA, CBLB, BAFF (BR3), and combinations thereof, and increased and / or enhanced expression of CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, and combinations thereof. In some embodiments, transient changes in protein expression result in decreased and / or reduced expression of molecules selected from PD-1, LAG3, TIM3, CISH, CBLB, and combinations thereof, and increased and / or enhanced expression of CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, and combinations thereof.

[0485] In some embodiments, expression is reduced by about 5%, about 10%, about 10%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, expression is reduced by at least about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, expression is reduced by at least about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, expression is reduced by at least about 80%, about 85%, about 90%, or about 95%. In some embodiments, expression is reduced by at least about 85%, about 90%, or about 95%. In some embodiments, expression is reduced by at least about 80%. In some embodiments, expression is reduced by at least about 85%. In some implementations, expression is reduced by at least about 90%. In some implementations, expression is reduced by at least about 95%. In some implementations, expression is reduced by at least about 99%.

[0486] In some embodiments, expression is increased by about 5%, about 10%, about 10%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, expression is increased by at least about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, expression is increased by at least about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, expression is increased by at least about 80%, about 85%, about 90%, or about 95%. In some embodiments, expression is increased by at least about 85%, about 90%, or about 95%. In some embodiments, expression is increased by at least about 80%. In some embodiments, expression is increased by at least about 85%. In some implementations, expression is increased by at least about 90%. In some implementations, expression is increased by at least about 95%. In some implementations, expression is increased by at least about 99%.

[0487] In some embodiments, transient alterations in protein expression are induced by treating the TIL with transcription factors (TFs) and / or other molecules capable of transiently altering protein expression in the TIL. In some embodiments, transcription factors (TFs) and / or other molecules capable of transiently altering protein expression are delivered intracellularly using a microfluidic platform without the SQZ carrier. Such methods, demonstrating the ability to deliver proteins (including transcription factors) to a variety of primary human cells (including T cells), have been described (Sharei et al., PNAS 2013, and Sharei et al., PLOS ONE 2015 and Greisbeck et al., J. Immunology 2015, Vol. 195), including rapid methods that use microfluidic constriction to deform cells, thereby allowing TF or other molecules to enter the cells; see, for example, International Patent Application Publication Nos. WO2013 / 059343A1, WO2017 / 008063A1 or WO2017 / 123663A1 or U.S. Patent Application Publication Nos. US2014 / 0287509A1, US2018 / 0201889A1 or US2018 / 0245089A1, all of which are incorporated herein by reference in their entirety. The methods described in International Patent Application Publications WO2013 / 059343A1, WO2017 / 008063A1, or WO2017 / 123663A1, or U.S. Patent Application Publications US2014 / 0287509A1, US2018 / 0201889A1, or US2018 / 0245089A1, can be used in conjunction with this invention to expose TIL populations to transcription factors (TFs) and / or other molecules capable of inducing transient protein expression, wherein the TFs and / or other molecules capable of inducing transient protein expression provide an increase in tumor antigen expression and / or an increase in the number of tumor antigen-specific T cells in the TIL populations, thereby resulting in the reprogramming of the TIL populations, and the reprogrammed TIL populations exhibit improved therapeutic efficacy compared to unreprogrammed TIL populations. In some embodiments, as described herein, reprogramming results in an increase in subsets of effector T cells and / or central memory T cells relative to the initial TIL population or the prior (i.e., prior to reprogramming) TIL populations.

[0488] In some embodiments, transcription factors (TFs) include, but are not limited to, TCF-1, NOTCH 1 / 2 ICD, and / or MYB. In some embodiments, the transcription factor (TF) is TCF-1. In some embodiments, the transcription factor (TF) is NOTCH 1 / 2 ICD. In some embodiments, the transcription factor (TF) is MYB. In some embodiments, the transcription factor (TF) is administered together with induced pluripotent stem cell cultures (iPSCs) (e.g., a commercially available KNOCKOUT serum alternative (Gibco / ThermoFisher)) to induce additional TIL reprogramming. In some embodiments, the transcription factor (TF) is administered together with a mixture of iPSCs to induce additional TIL reprogramming. In some embodiments, the transcription factor (TF) is administered without a mixture of iPSCs. In some embodiments, reprogramming results in an increase in the percentage of TSCM. In some implementations, reprogramming results in an increase in the TSCM percentage of approximately 5%, approximately 10%, approximately 10%, approximately 20%, approximately 25%, approximately 30%, approximately 35%, approximately 40%, approximately 45%, approximately 50%, approximately 55%, approximately 60%, approximately 65%, approximately 70%, approximately 75%, approximately 80%, approximately 85%, approximately 90%, or approximately 95%.

[0489] In some embodiments, methods for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population include:

[0490] (i) Obtaining the first TIL group from the tumor removed from the patient;

[0491] (ii) A second TIL population is generated by first expansion of the first TIL population by culturing the first TIL population in a cell culture medium containing IL-2 and optional OKT-3;

[0492] (iii) A second expansion is performed by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, and antigen-presenting cells (APCs) to generate a third TIL population; wherein the number of the third TIL population is at least 100 times greater than that of the second TIL population; wherein the second expansion is performed for at least 14 days to obtain the third TIL population; wherein the third TIL population is a therapeutic TIL population; and

[0493] (iv) Expose the second and / or third TIL groups to transcription factors (TFs) and / or other molecules capable of transiently altering protein expression; wherein the TFs and / or other molecules capable of transiently altering protein expression provide alterations in tumor antigen expression and / or the number of tumor antigen-specific T cells in the therapeutic TIL groups.

[0494] In one embodiment, a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population includes:

[0495] (a) Obtaining the first TIL group from the tumor removed from the patient by processing a tumor sample obtained from the patient into multiple tumor fragments;

[0496] (b) Adding tumor fragments to the closed system;

[0497] (c) The first expansion was performed by culturing the first TIL population in a cell culture medium containing IL-2 and optionally containing a 4-1BB agonist antibody for about 2 to 5 days;

[0498] (d) Add OKT-3 to generate a second TIL population; wherein the first amplification is performed in a closed container providing a first ventilated surface area; wherein the first amplification is performed for approximately 1 to 3 days to obtain the second TIL population; wherein the number of the second TIL population is at least 50 times greater than the number of the first TIL population; wherein the transition from step (c) to step (d) occurs without opening the system.

[0499] (e) Performing a sterile electroporation step on the second TIL group; wherein the sterile electroporation step mediates the transfer of at least one short interfering RNA or a messenger RNA;

[0500] (f) Let the second TIL group stand for about 1 day;

[0501] (g) A second expansion is performed by supplementing the cell culture medium of the second TIL population with additional IL-2, optional OKT-3 antibody, optional OX40 antibody and antigen-presenting cells (APC) to generate a third TIL population; wherein the second expansion is performed for approximately 7 to 11 days to obtain the third TIL population; wherein the second expansion is performed in a closed container providing a second breathable surface area; wherein the transition from step (f) to step (g) occurs without opening the system;

[0502] (h) Harvest the therapeutic TIL clusters obtained from step (g), providing the harvested TIL clusters; wherein the transition from step (g) to step (h) occurs without opening the system; wherein the harvested TIL clusters are therapeutic TIL clusters;

[0503] (i) The TIL clusters harvested in step (e) are transferred to an infusion bag; wherein the transition from step (e) to step (f) occurs without opening the system; and

[0504] (j) Cryopreservation of harvested TIL populations using a dimethyl sulfoxide-based cryopreservation medium;

[0505] The aseptic electroporation step includes delivering short interfering RNA to inhibit the expression of molecules selected from PD-1, LAG-3, TIM-3, CTLA-4, TIGIT, CISH, TGFβR2, PKA, CBLB, BAFF(BR3), and combinations thereof.

[0506] According to one embodiment, a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population includes:

[0507] (a) Obtaining the first TIL group from the tumor removed from the patient by processing a tumor sample obtained from the patient into multiple tumor fragments;

[0508] (b) Adding tumor fragments to the closed system;

[0509] (c) The first expansion was performed by culturing the first TIL population in a cell culture medium containing IL-2 and optionally containing a 4-1BB agonist antibody for about 2 to 5 days;

[0510] (d) Add OKT-3 to generate a second TIL population; wherein the first amplification is performed in a closed container providing a first ventilated surface area; wherein the first amplification is performed for approximately 1 to 3 days to obtain the second TIL population; wherein the number of the second TIL population is at least 50 times greater than the number of the first TIL population; wherein the transition from step (c) to step (d) occurs without opening the system.

[0511] (e) Performing an SQZ microfluidic membrane disruption step on the second TIL group; wherein the SQZ microfluidic membrane disruption step mediates the transfer of at least one short interfering RNA or a messenger RNA;

[0512] (f) Let the second TIL group stand for about 1 day;

[0513] (g) A second expansion is performed by supplementing the cell culture medium of the second TIL population with additional IL-2, optional OKT-3 antibody, optional OX40 antibody and antigen-presenting cells (APC) to generate a third TIL population; wherein the second expansion is performed for approximately 7 to 11 days to obtain the third TIL population; wherein the second expansion is performed in a closed container providing a second breathable surface area; wherein the transition from step (f) to step (g) occurs without opening the system;

[0514] (h) Harvest the therapeutic TIL clusters obtained from step (g), providing the harvested TIL clusters; wherein the transition from step (g) to step (h) occurs without opening the system; wherein the harvested TIL clusters are therapeutic TIL clusters;

[0515] (i) The TIL clusters harvested in step (e) are transferred to an infusion bag; wherein the transition from step (e) to step (f) occurs without opening the system; and

[0516] (j) Cryopreservation of harvested TIL populations using a dimethyl sulfoxide-based cryopreservation medium;

[0517] The SQZ microfluidic membrane disruption step includes delivering short interfering RNA to inhibit the expression of molecules selected from PD-1, LAG-3, TIM-3, CTLA-4, TIGIT, CISH, TGFβR2, PKA, CBLB, BAFF(BR3), and combinations thereof.

[0518] In some embodiments, the method for transiently altering protein expression as described above can be combined with a method for genetically modifying TIL groups, including a step of stably incorporating a gene for producing more than one protein. In one embodiment, the method for genetically modifying TIL groups includes a retroviral transduction step. In one embodiment, the method for genetically modifying TIL groups includes a lentiviral transduction step. Lentiviral transduction systems are known in the art and described, for example, in Levine et al., Proc. Nat'l Acad. Sci. 2006, 103, 17372-77; Zufferey et al., Nat. Biotechnol. 1997, 15, 871-75; Dull et al., J. Virology 1998, 72, 8463-71 and U.S. Patent No. 6,627,442, the contents of which are incorporated herein by reference. In one embodiment, the method for genetically modifying TIL groups includes a γ-retroviral transduction step. Gamma-retroviral transduction systems are known in the art and described, for example, by Cepko and Pear, Cur. Prot. Mol. Biol. 1996, 9.9.1-9.9.16, the disclosure of which is incorporated herein by reference. In one embodiment, a method for genetically modifying the TIL group includes a transposon-mediated gene transfer step. Transposon-mediated gene transfer systems are known in the art and include systems in which the transposase is provided in the form of a DNA expression vector or an expressible RNA or protein such that the transposase is not expressed long-term in transgenic cells, for example, a transposase provided in the form of mRNA (e.g., mRNA containing a cap and a poly-A tail). Suitable transposon-mediated gene transfer systems, including salmonid-type Tel-like transposases (SB or Sleeping Beauty transposases), such as SB10, SB11, and SB100x, as well as engineered enzymes with enhanced enzyme activity, are described, for example, in Hackett et al., Mol. Therapy 2010, 18, 674-83 and U.S. Patent No. 6,489,458, the contents of which are incorporated herein by reference.

[0519] In one embodiment, a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population includes:

[0520] (a) Obtaining the first TIL group from the tumor removed from the patient by processing a tumor sample obtained from the patient into multiple tumor fragments;

[0521] (b) Adding tumor fragments to the closed system;

[0522] (c) The first expansion was performed by culturing the first TIL population in a cell culture medium containing IL-2 and optionally containing a 4-1BB agonist antibody for about 2 to 5 days;

[0523] (d) Add OKT-3 to generate a second TIL population; wherein the first amplification is performed in a closed container providing a first ventilated surface area; wherein the first amplification is performed for approximately 1 to 3 days to obtain the second TIL population; wherein the number of the second TIL population is at least 50 times greater than the number of the first TIL population; wherein the transition from step (c) to step (d) occurs without opening the system.

[0524] (e) Performing a sterile electroporation step or an SQZ microfluidic membrane disruption step on the second TIL group; wherein the sterile electroporation step or the SQZ microfluidic membrane disruption step mediates the transfer of at least one short interfering RNA or a messenger RNA.

[0525] (f) Let the second TIL group stand for about 1 day;

[0526] (g) A second expansion is performed by supplementing the cell culture medium of the second TIL population with additional IL-2, optional OKT-3 antibody, optional OX40 antibody and antigen-presenting cells (APC) to generate a third TIL population; wherein the second expansion is performed for approximately 7 to 11 days to obtain the third TIL population; wherein the second expansion is performed in a closed container providing a second breathable surface area; wherein the transition from step (f) to step (g) occurs without opening the system;

[0527] (h) Harvest the therapeutic TIL clusters obtained from step (g), providing the harvested TIL clusters; wherein the transition from step (g) to step (h) occurs without opening the system; wherein the harvested TIL clusters are therapeutic TIL clusters;

[0528] (i) The TIL clusters harvested in step (e) are transferred to an infusion bag; wherein the transition from step (e) to step (f) occurs without opening the system; and

[0529] (j) Cryopreservation of harvested TIL populations using a dimethyl sulfoxide-based cryopreservation medium;

[0530] The electroporation step includes delivering short interfering RNA to inhibit the expression of a molecule selected from PD-1, LAG-3, TIM-3, CTLA-4, TIGIT, CISH, TGFβR2, PKA, CBLB, BAFF(BR3), and combinations thereof; further, the adhesion molecule is inserted into a first TIL group, a second TIL group, or a harvested TIL group via a γ-retroviral or lentiviral method, the adhesion molecule being selected from CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, and combinations thereof.

[0531] In some embodiments, the transient change in protein expression is induced by a decrease in expression due to self-delivering RNA interference (sd-RNA), which is a chemically synthesized asymmetric siRNA duplex with a high percentage of 2'-OH substitutions (typically fluorine or -OCH3). The sd-RNA comprises a 20-nucleotide sense (guide) strand and a 13- to 15-base sense (lackey) strand conjugated to cholesterol at its 3' end using a tetraethyl ethylene glycol (TEG) linker. Methods using sd-RNA have been described in Khvorova and Watts, Nat. Biotechnol. 2017, 35, 238–248; Byrne et al., J. Ocul. Pharmacol. Ther. 2013, 29, 855-864; and Ligtenberg et al., Mol. Therapy, 2018, in print, the disclosures of which are incorporated herein by reference. In one embodiment, the delivery of sd-RNA to TILs is accomplished not by electroporation, SQZ, or other methods, but by the following steps: exposing TILs to sd-RNA at a concentration of 1 μM / 10,000 TILs in a culture medium for 1 to 3 days. In some embodiments, the delivery of sd-RNA to TILs is accomplished by the following steps: exposing TILs to sd-RNA at a concentration of 10 μM / 10,000 TILs in a culture medium for 1 to 3 days. In one embodiment, the delivery of sd-RNA to TILs is accomplished by the following steps: exposing TILs to sd-RNA at a concentration of 50 μM / 10,000 TILs in a culture medium for 1 to 3 days. In one embodiment, the delivery of sd-RNA to TILs is accomplished by the following steps: exposing TILs to sd-RNA at concentrations ranging from 0.1 μM / 10,000 TILs to 50 μM / 10,000 TILs in a culture medium for 1 to 3 days. In one embodiment, the delivery of sd-RNA to TIL clusters is accomplished using the following steps: exposing TIL clusters to sd-RNA at concentrations of 0.1 μM / 10,000 TILs to 50 μM / 10,000 TILs in a culture medium for 1 to 3 days; wherein the exposure to sd-RNA is performed two, three, four, or five times by adding fresh sd-RNA to the culture medium. Other suitable methods are described, for example, in U.S. Patent Application Publications US2011 / 0039914A1, US2013 / 0131141A1, and US2013 / 0131142A1 and U.S. Patent No. 9,080,171, the disclosures of which are incorporated herein by reference.

[0532] In some implementations, using Figure 32The method involves inserting sd-RNA into the TIL group during the production process. In some embodiments, the sd-RNA encodes RNA that interferes with NOTCH 1 / 2 ICD, NOTCH ligand mDLL1, PD-1, CTLA-4TIM-3, LAG-3, TIGIT, TGFβ, TGFBR2, cAMP protein kinase A (PKA), BAFF BR3, CISH, and / or CBLB. In some embodiments, the reduction in expression is determined based on the percentage of gene silencing, for example, by flow cytometry and / or qPCR. In some embodiments, expression is reduced by approximately 5%, approximately 10%, approximately 10%, approximately 20%, approximately 25%, approximately 30%, approximately 35%, approximately 40%, approximately 45%, approximately 50%, approximately 55%, approximately 60%, approximately 65%, approximately 70%, approximately 75%, approximately 80%, approximately 85%, approximately 90%, or approximately 95%. In some embodiments, expression is reduced by at least about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, expression is reduced by at least about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, expression is reduced by at least about 80%, about 85%, about 90%, or about 95%. In some embodiments, expression is reduced by at least about 85%, about 90%, or about 95%. In some embodiments, expression is reduced by at least about 80%. In some embodiments, expression is reduced by at least about 85%. In some embodiments, expression is reduced by at least about 90%. In some embodiments, expression is reduced by at least about 95%. In some embodiments, expression is reduced by at least about 99%.

[0533] In one embodiment, a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population includes:

[0534] (a) Obtaining the first TIL group from the tumor removed from the patient by processing a tumor sample obtained from the patient into multiple tumor fragments;

[0535] (b) Adding tumor fragments to the closed system;

[0536] (c) The first expansion was performed by culturing the first TIL population in a cell culture medium containing IL-2 and optionally containing a 4-1BB agonist antibody for about 2 to 5 days;

[0537] (d) Add OKT-3 to generate a second TIL population; wherein the first amplification is performed in a closed container providing a first ventilated surface area; wherein the first amplification is performed for approximately 1 to 3 days to obtain the second TIL population; wherein the number of the second TIL population is at least 50 times greater than the number of the first TIL population; wherein the transition from step (c) to step (d) occurs without opening the system.

[0538] (e) A second expansion is performed by supplementing the cell culture medium of the second TIL population with additional IL-2, one or more self-delivering RNAs (sd-RNAs), optional OKT-3 antibody, optional OX40 antibody, and antigen-presenting cells (APCs) to generate a third TIL population; wherein the second expansion is performed for approximately 7 to 11 days to obtain the third TIL population; wherein the second expansion is performed in a closed container providing a second breathable surface area; wherein the transition from step (f) to step (g) occurs without opening the system;

[0539] (h) Harvest the therapeutic TIL clusters obtained from step (g), providing the harvested TIL clusters; wherein the transition from step (g) to step (h) occurs without opening the system; wherein the harvested TIL clusters are therapeutic TIL clusters;

[0540] (i) The TIL clusters harvested in step (e) are transferred to an infusion bag; wherein the transition from step (e) to step (f) occurs without opening the system; and

[0541] (j) Cryopreservation of harvested TIL populations using a dimethyl sulfoxide-based cryopreservation medium;

[0542] Among them, one or more sd-RNA transiently inhibits the expression of molecules selected from PD-1, LAG-3, TIM-3, CTLA-4, TIGIT, CISH, TGFβR2, PKA, CBLB, BAFF(BR3) and combinations thereof.

[0543] In one embodiment, a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population includes:

[0544] (a) Obtaining the first TIL group from the tumor removed from the patient by processing a tumor sample obtained from the patient into multiple tumor fragments;

[0545] (b) Adding tumor fragments to the closed system;

[0546] (c) The first expansion was performed by culturing the first TIL population in a cell culture medium containing IL-2 and optionally containing a 4-1BB agonist antibody for about 2 to 5 days;

[0547] (d) Add OKT-3 to generate a second TIL population; wherein the first amplification is performed in a closed container providing a first ventilated surface area; wherein the first amplification is performed for approximately 1 to 3 days to obtain the second TIL population; wherein the number of the second TIL population is at least 50 times greater than the number of the first TIL population; wherein the transition from step (c) to step (d) occurs without opening the system.

[0548] (e) A second expansion is performed by supplementing the cell culture medium of the second TIL population with additional IL-2, one or more self-delivering RNAs (sd-RNAs), optional OKT-3 antibody, optional OX40 antibody, and antigen-presenting cells (APCs) to generate a third TIL population; wherein the second expansion is performed for approximately 7 to 11 days to obtain the third TIL population; wherein the second expansion is performed in a closed container providing a second breathable surface area; wherein the transition from step (f) to step (g) occurs without opening the system;

[0549] (h) Harvest the therapeutic TIL clusters obtained from step (g), providing the harvested TIL clusters; wherein the transition from step (g) to step (h) occurs without opening the system; wherein the harvested TIL clusters are therapeutic TIL clusters;

[0550] (i) The TIL clusters harvested in step (e) are transferred to an infusion bag; wherein the transition from step (e) to step (f) occurs without opening the system; and

[0551] (j) Cryopreservation of harvested TIL populations using a dimethyl sulfoxide-based cryopreservation medium;

[0552] The expression of one or more sd-RNA transient inhibitory molecules is selected from PD-1, LAG-3, TIM-3, CTLA-4, TIGIT, CISH, TGFβR2, PKA, CBLB, BAFF(BR3), and combinations thereof; and the adhesion molecules are inserted into the first TIL group, the second TIL group, or the harvested TIL group by a γ-retroviral or lentiviral method, and the adhesion molecules are selected from CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, and combinations thereof.

[0553] A. sdRNA method

[0554] Chemically modified siRNA-based self-delivery RNAi technology can be used in conjunction with the methods of this invention to successfully deliver sd-RNA to the TIL described herein. Backbone modification involves the binding of asymmetric siRNA structures and hydrophobic ligands (see, for example, Ligtenberg et al., Mol. Therapy, 2018 and US20160304873, and the methods described herein). Figure 36 and 37This allows sd-RNA to penetrate cultured mammalian cells simply by being added to the culture medium, without the need for other formulations or methods, leveraging its nuclease stability. This stability supports constant levels of RNAi-mediated reduction in target gene activity solely by maintaining the active concentration of sd-RNA in the medium. Unbound by theory, the backbone stabilization of sd-RNA can prolong the reduction in gene expression effects, potentially lasting for months in non-dividing cells.

[0555] In one embodiment, the sd-RNA targeting the gene disclosed herein has... Figure 36 or Figure 37 The structure shown.

[0556] In some embodiments, TIL transfection efficiency exceeding 95% and target expression of various specific sd-RNAs are reduced. In some embodiments, sd-RNAs containing several unmodified ribose residues are replaced with fully modified sequences to increase the titer and / or lifetime of RNAi action. In some embodiments, the reduction in expression effect is maintained for 12 hours, 24 hours, 36 hours, 48 ​​hours, 5 days, 6 days, 7 days, or 8 days or longer. In some embodiments, the expression reduction effect decreases more than 10 days after TIL sd-RNA treatment. In some embodiments, the reduction in expression maintaining target expression exceeds 70%. In some embodiments, the reduction in expression maintaining TIL target expression exceeds 70%. In some embodiments, the reduction in expression in the PD-1 / PD-L1 pathway allows TILs to exhibit more effective in vivo action, which in some embodiments is due to the avoidance of PD-1 / PD-L1 pathway inhibition. In some embodiments, the reduction in PD-1 expression via sd-RNA leads to increased TIL proliferation.

[0557] 1. Selection and characteristics of sdRNA

[0558] a. sd-RNA oligonucleotide structure

[0559] Small interfering RNA (siRNA), sometimes called short interfering RNA or silent RNA, is a double-stranded RNA molecule that is typically 19 to 25 base pairs in length. siRNA is used for RNA interference (RNAi), in which it interferes with the expression of specific genes that have complementary nucleotide sequences.

[0560] Double-stranded DNA (dsRNA) is generally used to define any molecule containing a pair of complementary RNA strands, typically a sense (lagging) and an antisense (leading) strand, and may include single-stranded overhang regions. In contrast to siRNA, the term dsRNA typically refers to a precursor molecule containing the siRNA molecular sequence, which is released from a larger dsRNA molecule by the action of a cleavage enzyme system, including Dicer.

[0561] sd-RNA (self-delivered RNA) is a novel class of covalently modified RNAi compounds that do not require a delivery vector for cell entry and exhibit improved pharmacology compared to conventional siRNA. "Self-delivered RNA" or "sd-RNA" is a hydrophobically modified RNA interference-antisense hybrid that has been shown to be highly efficient in vitro in primary cells and in vivo upon topical administration. Robust uptake and / or silencing without toxicity have been demonstrated. sd-RNA is typically an asymmetrically modified nucleic acid molecule with a minimal double-stranded region. sd-RNA molecules generally contain both single-stranded and double-stranded regions and can incorporate various chemical modifications within these regions. Furthermore, as described herein, sd-RNA molecules can be linked to hydrophobic conjugates, such as conventional and higher sterol-type molecules. sd-RNA (and / or RNA that can be used in a manner similar to sd-RNA) and related methods for preparing such sd-RNA have been extensively described in, for example, U.S. Patent Publication No. US2016 / 0304873, International Patent Application Publication No. WO2010 / 033246, International Patent Application Publication No. WO2017 / 070151, International Patent Application Publication No. WO2009 / 102427, International Patent Application Publication No. WO201 / 1119887, International Patent Application Publication No. WO2010 / 033247, International Patent Application Publication No. WO2009045457, International Patent Application Publication No. WO2011 / 119852, International Patent Application Publication No. WO2011 / 119871, U.S. Patent Publication No. US2011 / 0263680, International Patent Application Publication No. WO2010 / 033248, International Patent Application Publication No. WO2010 / 078536, and other international patent applications. Patent Publication No. WO2010 / 090762, US Patent Publication No. US20110039914, International Publication No. WO2011 / 109698, International Patent Application Publication No. WO2010 / 090762, US Patent No. US8,815,818, International Patent Application Publication No. WO2016 / 094845, International Patent Application Publication No. WO2017 / 193053, US Patent Publication No. US2006 / 0276635 International Patent Application Publication No. WO2001 / 009312, US Patent Publication No. US2017 / 0043024, US Patent Publication No. US2017 / 0312367, US Patent Publication No. US2016 / 0319278, US Patent Publication No. US2017 / 0369882, US Patent No. US8,501,706, US Patent No. US2004 / 0224405, US Patent No. US8,252,755, US Patent No. US2007 / 0031844, US Patent No. US2007 / 0039072, US Patent Publication No. US2007 / 0207974, US Patent Publication No. US2007 / 0213520, US Patent Publication No. US2007 / 0213521, US Patent Publication No. US2007 / 0219362, US Patent Publication No. US2007 / 0238868, US Patent Publication No. US2014 / 0148362, US Patent Publication No. US2016 / 0193242, US Patent The entire contents of the following patent publications are incorporated herein by reference for all purposes: US2016 / 01946461, US2016 / 0201058, US2016 / 0201065, US2017 / 0349904, US2018 / 0119144, US7,834,170, US8,090,542, and US2012 / 0052487; sd-RNA is also commercially available from Advirna LLC (Worcester, MA, USA). To optimize the structure, chemical properties, target location, and sequence preference of sd-RNA, proprietary algorithms have been developed and used for sd-RNA potency prediction (see, for example, US20160304873). Based on these analyses, a functional sd-RNA sequence is generally defined as one whose expression is reduced by more than 70% at a concentration of 1 μM with a probability exceeding 40%. ,

[0562] b. sd-RNA oligonucleotide structure

[0563] In some embodiments, more than one sd-RNA for use in this invention can be generated from a linear double-stranded DNA template. In some embodiments, the linear double-stranded DNA template used to generate more than one sd-RNA is a template as described in U.S. Patent No. 8,859,229 and hereinafter.

[0564] In some embodiments, the linear double-stranded DNA template obtained by polymerase chain reaction (PCR) and suitable for in vitro transcription of mRNA comprises, from 5' to 3': an RNA polymerase promoter on the coding strand of the double-stranded DNA, a 5' untranslated region (less than 3,000 nucleotides in length, capable of efficiently translating mRNA into a detectable polypeptide after transfection into eukaryotic cells), and an open reading frame encoding the polypeptide; wherein the polypeptide is heterologous to the cells to be transfected; wherein the polypeptide is selected from: ligands or receptors of immune cells, polypeptides that stimulate or inhibit the function of the immune system, polypeptides that inhibit the function of oncogenic polypeptides, a 3' untranslated region capable of efficiently translating mRNA into a detectable polypeptide after transfection into eukaryotic cells, and a poly(A) segment of 50 to 5,000 nucleotides on the coding strand of the double-stranded DNA; wherein the promoter and the open reading frame are heterologous; wherein the DNA template is not contained within a DNA vector and terminates at the 3' end of the poly(A) segment. In some embodiments, the RNA polymerase promoter contains a common binding sequence for the RNA polymerase, which is selected from T7, T3, or SP6 RNA polymerases. In some embodiments, the open reading frame encodes a fusion polypeptide. In some embodiments, the open reading frame encodes a polypeptide selected from the group consisting of: PD-1, TGFBR2, CBLB (CBL-B), CISH, CCR (chimeric co-stimulatory receptor), IL-2, IL-12, IL-15, IL-21, NOTCH 1 / 2ICD, TIM3, LAG3, TIGIT, TGFβ, CCR2, CCR4, CCR5, CXCR1, CXCR2, CSCR3, CCL2 (MCP-1), CCL3 (MIP-1α), CCL4 (MIP1-β), CCL5 (RANTES), CXCL1 / CXCL8, CCL22, CCL17, CXCL1 / CXCL8, VHL, CD44, PIK3CD, SOCS1, cAMP protein kinase A (PKA), and combinations thereof. In some embodiments, the open reading frame encodes a polypeptide selected from the group consisting of PD-1, LAG-3, TIM-3, CTLA-4, TIGIT, CISH, TGFβR2, PKA, CBLB, BAFF(BR3), and combinations thereof. In some embodiments, the linear double-stranded DNA template further includes an internal ribosome entry site. In some embodiments, the poly(A) segment is 300-400 nucleotides in length.

[0565] In some embodiments, the linear double-stranded DNA template of claim 1 is comprising, from 5' to 3', an RNA polymerase promoter on the coding strand of the double-stranded DNA, a 5' untranslated region (less than 3,000 nucleotides in length, capable of efficiently translating mRNA into a detectable polypeptide after transfection into eukaryotic cells), and an open reading frame encoding the polypeptide; wherein the polypeptide is heterologous to the cells to be transfected; wherein the polypeptide is selected from: ligands or receptors of immune cells, polypeptides that stimulate or inhibit the function of the immune system, polypeptides that inhibit the function of oncogenic polypeptides, a 3' untranslated region capable of efficiently translating mRNA into a detectable polypeptide after transfection into eukaryotic cells, and a poly(A) segment of 50 to 5,000 nucleotides on the coding strand of the double-stranded DNA; wherein the promoter and the open reading frame are heterologous; wherein the DNA template is not contained within a DNA vector and terminates at the 3' end of the poly(A) segment. In some embodiments, the 3' untranslated region is at least 100 nucleotides in length.

[0566] In some embodiments, the present invention provides a method for generating the above-described linear double-stranded DNA template; wherein the method includes: generating a forward primer and a reverse primer; wherein the forward primer comprises: a plurality of nucleotides substantially complementary to the non-coding strand of the target double-stranded DNA, and a plurality of nucleotides serving as an RNA polymerase binding site; wherein the reverse primer comprises: a plurality of nucleotides substantially complementary to the coding strand of the target double-stranded DNA, and a plurality of deoxythymidine nucleotides; and, performing a polymerase chain reaction amplification on the target DNA using the forward primer and the reverse primer to form a linear double-stranded DNA template. In some embodiments, the present invention provides a method for generating the above-described linear double-stranded DNA template, wherein the method includes: generating a forward primer and a reverse primer; wherein the forward primer comprises a plurality of nucleotides substantially complementary to a nucleotide region directly upstream of the target double-stranded DNA; wherein the reverse primer comprises a plurality of nucleotides substantially complementary to a nucleotide region directly downstream of the target double-stranded DNA; and, performing a polymerase chain reaction amplification on the target DNA using the forward primer and the reverse primer to form a linear double-stranded DNA template. In some embodiments, the primers comprise nucleotide sequences substantially complementary to the stretches of nucleotides in the 5' and 3' untranslated regions of the target double-stranded DNA. In some embodiments, the primers comprise nucleotide sequences substantially complementary to the stretches of nucleotides within the open reading frame of the target double-stranded DNA. In some embodiments, the primers comprise nucleotide sequences substantially complementary to the stretches of nucleotides within the open reading frame of the target double-stranded DNA; wherein the primers also comprise nucleotide stretches containing both the 5' and 3' untranslated regions; wherein the nucleotide stretch in the forward primer containing the 5' untranslated region is between a nucleotide containing the RNA polymerase promoter and a nucleotide substantially complementary to the non-coding strand of the target double-stranded DNA; wherein the nucleotide stretch in the reverse primer containing the 3' untranslated region is between a plurality of deoxythymidine nucleotides and a nucleotide substantially complementary to the coding strand of the target double-stranded DNA. In some embodiments, the forward primer and the open reading frame comprise a shared Kozak sequence.

[0567] In some embodiments, the present invention provides a method for generating one or more RNAs for transfecting cells, the method comprising in vitro transcription from a linear double-stranded DNA template. In some embodiments, the method further comprises using a poly(A) polymerase to extend the poly(A) tail of the RNA with one or more adenine nucleotides or their analogues. In some embodiments, the method further comprises adding a nucleotide during transcription, the nucleotide acting as a 5' cap on the transcribed RNA. In some embodiments, RNA targets peptides selected from the group consisting of: PD-1, TGFBR2, CBLB (CBL-B), CISH, CCR (chimeric co-stimulatory receptor), IL-2, IL-12, IL-15, IL-21, NOTCH 1 / 2ICD, TIM3, LAG3, TIGIT, TGFβ, CCR2, CCR4, CCR5, CXCR1, CXCR2, CSCR3, CCL2 (MCP-1), CCL3 (MIP-1α), CCL4 (MIP1-β), CCL5 (RANTES), CXCL1 / CXCL8, CCL22, CCL17, CXCL1 / CXCL8, VHL, CD44, PIK3CD, SOCS1, cAMP protein kinase A (PKA), and combinations thereof. In some embodiments, RNA targets peptides selected from the group consisting of PD-1, LAG-3, TIM-3, CTLA-4, TIGIT, CISH, TGFβR2, PKA, CBLB, BAFF(BR3), and combinations thereof.

[0568] In some embodiments, the invention utilizes more than one isolated RNA, said isolated RNA being generated from a linear double-stranded DNA template and containing more than one open reading frame. In some embodiments, the invention provides a method for expressing more than one RNA in cells, the method comprising contacting the cells with more than one RNA generated from a linear double-stranded DNA template. In some embodiments, RNA is present in the cells in unequal molar amounts to provide different levels of RNA expression. In some embodiments, one or more RNAs target peptides selected from the group consisting of: PD-1, TGFBR2, CBLB (CBL-B), CISH, CCRs (chimeric co-stimulatory receptors), IL-2, IL-12, IL-15, IL-21, NOTCH 1 / 2ICD, TIM3, LAG3, TIGIT, TGFβ, CCR2, CCR4, CCR5, CXCR1, CXCR2, CSCR3, CCL2 (MCP-1), CCL3 (MIP-1α), CCL4 (MIP1-β), CCL5 (RANTES), CXCL1 / CXCL8, CCL22, CCL17, CXCL1 / CXCL8, VHL, CD44, PIK3CD, SOCS1, cAMP protein kinase A (PKA), and combinations thereof. In some embodiments, one or more RNA targets a polypeptide selected from the group consisting of PD-1, LAG-3, TIM-3, CTLA-4, TIGIT, CISH, TGFβR2, PKA, CBLB, BAFF(BR3), and combinations thereof.

[0569] (i) Non-translation area

[0570] Chemical structures that promote stability and / or translation efficiency may also be used. RNA preferably has a 5'UTR and a 3'UTR. The examples below show that including a 44-base-pair 5'UTR in a PCR template results in higher translation efficiency of transcribed CFP RNA compared to a PCR template containing only a 6-base-pair 5'UTR. The examples also show that adding a 113-base-pair 3'UTR results in higher translation efficiency of transcribed GFP RNA compared to a PCR template containing only an 11-base-pair 3'UTR. Typically, the 3'UTR is longer than 100 nucleotides, therefore a 3'UTR longer than 100 nucleotides is preferred. In one embodiment, the 3'UTR sequence is 100 to 5000 nucleotides. The length of the 5'UTR is less important than the length of the 3'UTR, and the 5'UTR can be shorter. In one embodiment, the 5'UTR is 0 to 3000 nucleotides long. The lengths of the 5'UTR and 3'UTR sequences to be added to the coding region can be varied using different methods, including, but not limited to, designing PCR primers for annealing to different UTR regions. Using this method, those skilled in the art can modify the desired 5'UTR and 3'UTR lengths to achieve optimal translation efficiency after transfection of the transcribed RNA.

[0571] The 5'UTR and 3'UTR can be naturally occurring endogenous 5'UTR and 3'UTR of the target gene. Alternatively, non-endogenous UTR sequences of the target gene can be added by incorporating the UTR sequence into the forward and reverse primers or through any other modification of the template. The use of non-endogenous UTR sequences of the target gene can be used to alter RNA stability and / or translation efficiency. For example, it is known that AU-rich elements in the 3'UTR sequence can reduce mRNA stability. Therefore, based on the characteristics of UTRs well known in the art, 3'UTRs can be selected or designed to increase the stability of transcribed RNA.

[0572] In one embodiment, the 5'UTR may contain the Kozak sequence of an endogenous gene. Alternatively, when a non-endogenous 5'UTR of the target gene is added via PCR as described above, the shared Kozak sequence can be redesigned by adding a 5'UTR sequence. Kozak sequences can improve the translation efficiency of some RNA transcripts, but it appears that not all RNAs require a Kozak sequence for efficient translation. The requirement for a Kozak sequence in many mRNAs is known in the art. In other embodiments, the 5'UTR may be derived from an RNA virus whose RNA genome is stable in the cell. In other embodiments, various nucleotide analogs may be used in the 3'UTR or 5'UTR to prevent exonuclease degradation of the mRNA.

[0573] (ii) RNA polymerase promoter

[0574] To synthesize RNA from a DNA template without gene cloning, a transcription promoter should be ligated to the DNA template upstream of the sequence to be transcribed. A phage RNA polymerase promoter sequence can be ligated to a St UTR using various genetic engineering methods (e.g., DNA ligation), or the phage RNA polymerase promoter sequence can be added to a forward primer (5') of a sequence substantially complementary to the target DNA. When the sequence acting as the RNA polymerase promoter is added to the 5' end of the forward primer, the RNA polymerase promoter is incorporated into the PCR product upstream of the open reading frame to be transcribed. In a preferred embodiment, the promoter is the T7 polymerase promoter as described above. Other useful promoters include, but are not limited to, the T3 and SP6 RNA polymerase promoters. The common nucleotide sequences of the T7, T3, and SP6 promoters are known in the art.

[0575] (iii) Poly(A) tail and 5' cap

[0576] In a preferred embodiment, the mRNA has caps at both the 5' end and the 3' poly(A) tail, which determine ribosome binding, translation initiation, and mRNA stability in the cell. On a circular DNA template (e.g., plasmid DNA), RNA polymerase produces a long concatameric product unsuitable for expression in eukaryotic cells. Transcription of plasmid DNA linearized at the 3' UTR end produces a normal-sized mRNA that, even if polyadenylated post-transcriptionally, is ineffective in eukaryotic transfection.

[0577] On a linear DNA template, phage T7 RNA polymerase can extend the 3' end of the transcript beyond the last base of the template (Schenborn and Mierendorf, Nuc. Acids Res., 13:6223-36 (1985); Nacheva and Berzal-Herranz, Eur. J. Biochem., 270:1485-65 (2003)). This can lead to runoff transcript bending, which then exchanges templates with the second DNA strand or the RNA itself (Triana-Alonso et al., J. Biol. Chem., 270:6298-307 (1995); Dunn and Studier, J. Mol. Biol., 166:477-535 (1983); Arnaud-Barbe et al., Nuc. Acids Res., 26:3550-54 (1998); Macdonald et al., 1993), which then leads to reverse aberrant transcription and accumulation of double-stranded RNA, thereby suppressing gene expression. DNA linearization alone is insufficient for proper transcription (Triana-Alonso et al., J. Biol. Chem., 270:6298-307 (1995); Dunn and Studier, J. Mol. Biol., 166:477-535 (1983); Arnaud-Barbe et al., 1998 Nuc. Acids). Res., 26:3550-54 (1998); Macdonald et al., J.Mol.Biol., 232:1030-47 (1993); Nakano et al., Biotechnol.Bioeng., 64:194-99 (1999)), linearized plasmid DNA downstream of a poly(A / T) segment of 64 to 100 nucleotides produces a good template (Saeboe-Larssen et al., J.Immunol.Meth., 259:191-203 (2002); Boczkowski et al., Cancer Res., 60:1028-34 (2000); Elango et al., Biochem Riophys Res Commun., 330:958-966 2005). The endogenous termination signal of T7 RNA polymerase encodes RNA that can fold into a stem-loop structure after uridine residue trace (Dunn and J.Mol.Biol., 166:477-535 (1983); Arnaud-Barbe et al., 1998 Nuc.Acids Res., 26:3550-54 (1998)).Even without hairpins, traces of synthetic uridine can weaken transcription (Kiyama and Oishi, Nuc. Acids Res., 24:4577-4583 (1996)). It is hypothesized that plasmid DNA linearization downstream of the poly(A / T) segment may form a “dynamic” terminator to prevent potentially aberrant transcription: 3' extension of the RNA transcript at the poly(A / T) segment and reverse transcription will generate a continuously growing terminal-like signal—an extended poly(U) segment and a poly(A / U) hairpin. Therefore, reverse PCR primers were designed with a 3' anchoring sequence downstream of the GFP gene and a 5' () of the 100-base segment of the poly(T). Figure 38 ).

[0578] The conventional method for integrating a polyA / T segment into a DNA template is molecular cloning. However, the polyA / T sequence integrated into plasmid DNA leads to plasmid instability, which is why plasmid DNA templates obtained from bacterial cells are often highly contaminated with deletions and other aberrations. This makes the cloning process not only time-consuming and laborious but also often unreliable. This is why there is a strong need for a method that can construct DNA templates with a polyA / T 3' segment without cloning.

[0579] The poly(A) / T region of the transcribed DNA template can be generated during PCR using a reverse primer containing a poly(T) tail (e.g., a 100T tail, the size of which can be 50T to 5000T)), or generated after PCR by any other method, including but not limited to DNA ligation or in vitro recombination. The poly(A) tail can also provide stability to the RNA and reduce RNA degradation. Generally, the length of the poly(A) tail is positively correlated with the stability of the transcribed RNA. In one embodiment, the poly(A) tail is 100 to 5000 adenosine. The examples below show that a 100-base-pair poly(A) segment is sufficient for efficient translation of RNA transcripts.

[0580] Following in vitro transcription, the poly(A) tail of the RNA can be further extended using a poly(A) polymerase (e.g., E. coli polyA polymerase (E-PAP)). The examples below demonstrate that increasing the length of the poly(A) tail from 100 nucleotides to 300-400 nucleotides improves RNA translation efficiency by approximately two-fold. Furthermore, attaching different chemical groups to the 3' end can increase mRNA stability. This attachment can include modified / artificial nucleotides, aptamers, and other compounds. For example, ATP analogs can be added to the poly(A) tail using a poly(A) polymerase. ATP analogs can further enhance RNA stability. Suitable ATP analogs include, but are not limited to, cordiocipin and 8-azaadenosine.

[0581] The 5' cap also provides stability to the RNA molecule. In a preferred embodiment, the RNA produced by the methods disclosed herein includes a 5' cap. For example, the 5' cap may be m 7 G(5')ppp(5')G、m 7 G(5')ppp(5')A, G(5')ppp(5')G, or G(5')ppp(5')A' cap analogs are commercially available. The 5' cap may also be an anti-reverse-cap analog (ARCA, anti-reverse-cap-analog) (see Stepinski et al., RNA, 7:1468-95 (2001)) or any other suitable analog. 5' caps are provided using techniques known in the art and described herein (Cougot et al., Trends in Biochem. Sci., 29:436-444 (2001); Stepinski et al., RNA, 7:1468-95 (2001); Elango et al., Biochim. Biophys. Res. Commun., 330:958-966 (2005)).

[0582] The RNA generated by the methods disclosed herein may also contain an internal ribosome entry site (IRES) sequence. The IRES sequence can be any viral, chromosomal, or artificially designed sequence that initiates cap-independent ribosome binding to mRNA and promotes the initiation of translation. It may contain any solute suitable for cell electroporation, including factors that promote cell permeability and viability, such as carbohydrates, peptides, lipids, proteins, antioxidants, and surfactants.

[0583] In some embodiments, the sd-RNA sequence used in this invention shows a 70% reduction in target gene expression. In some embodiments, the sd-RNA sequence used in this invention shows a 75% reduction in target gene expression. In some embodiments, the sd-RNA sequence used in this invention shows an 80% reduction in target gene expression. In some embodiments, the sd-RNA sequence used in this invention shows an 85% reduction in target gene expression. In some embodiments, the sd-RNA sequence used in this invention shows a 90% reduction in target gene expression. In some embodiments, the sd-RNA sequence used in this invention shows a 95% reduction in target gene expression. In some embodiments, the sd-RNA sequence used in this invention shows a 99% reduction in target gene expression. In some embodiments, when delivered at a concentration of about 0.25 μM to about 10 μM (in some embodiments, about 0.25 μM to about 4 μM), the sd-RNA sequence used in this invention shows a reduction in target gene expression. In some embodiments, when delivered at a concentration of about 0.25 μM, the sd-RNA sequence used in this invention shows a reduction in target gene expression. In some embodiments, when delivered at a concentration of about 0.5 μM, the sd-RNA sequence used in this invention shows a reduction in target gene expression. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 0.75 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 1.0 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 1.25 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 1.5 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 1.75 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 2.0 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 2.25 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 2.5 μM. In some embodiments, the sd-RNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 2.75 μM. In some embodiments, the sd-RNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 3.0 μM. In some embodiments, the sd-RNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 3.25 μM.In some embodiments, when delivered at a concentration of about 3.5 μM, the sd-RNA sequence used in this invention shows reduced target gene expression. In some embodiments, when delivered at a concentration of about 3.75 μM, the sd-RNA sequence used in this invention shows reduced target gene expression. In some embodiments, when delivered at a concentration of about 4.0 μM, the sd-RNA sequence used in this invention shows reduced target gene expression. In some embodiments, when delivered at a concentration of about 5.0 μM, the sd-RNA sequence used in this invention shows reduced target gene expression. In some embodiments, when delivered at a concentration of about 6.0 μM, the sd-RNA sequence used in this invention shows reduced target gene expression. In some embodiments, when delivered at a concentration of about 7.0 μM, the sd-RNA sequence used in this invention shows reduced target gene expression. In some embodiments, when delivered at a concentration of about 8.0 μM, the sd-RNA sequence used in this invention shows reduced target gene expression. In some embodiments, when delivered at a concentration of about 9.0 μM, the sd-RNA sequence used in this invention shows reduced target gene expression. In some embodiments, the sd-RNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 10.0 μM. In some embodiments, the sd-RNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 0.25 μM / 10,000 TIL to about 10 μM / 10,000 TIL or about 0.25 μM / 10,000 TIL to about 4 μM / 10,000 TIL. In some embodiments, the sd-RNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 0.25 μM / 10,000 TIL. In some embodiments, the sd-RNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 0.5 μM / 10,000 TIL. In some embodiments, the sd-RNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 0.75 μM / 10,000 TIL. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 1.0 μM / 10,000 TIL. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 1.25 μM / 10,000 TIL. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 1.5 μM / 10,000 TIL. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 1.75 μM / 10,000 TIL.In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 2.0 μM / 10,000 TIL. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 2.25 μM / 10,000 TIL. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 2.5 μM / 10,000 TIL. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 2.75 μM / 10,000 TIL. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 3.0 μM / 10,000 TIL. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 3.25 μM / 10,000 TIL. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 3.5 μM / 10,000 TIL. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 3.75 μM / 10,000 TIL. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 4.0 μM / 10,000 TIL. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 5.0 μM / 10,000 TIL. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 6.0 μM / 10,000 TIL. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 7.0 μM / 10,000 TIL. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 8.0 μM / 10,000 TIL. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 9.0 μM / 10,000 TIL. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 10.0 μM / 10,000 TIL. The sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 0.25 μM / 10,000 TIL / 100 μL of culture medium.When delivered at a concentration of approximately 0.5 μM / 10,000 TIL / 100 μL of culture medium, the sd-RNA sequence used in this invention showed decreased target gene expression. When delivered at a concentration of approximately 0.75 μM / 10,000 TIL / 100 μL of culture medium, the sd-RNA sequence used in this invention showed decreased target gene expression. When delivered at a concentration of approximately 1.0 μM / 10,000 TIL / 100 μL of culture medium, the sd-RNA sequence used in this invention showed decreased target gene expression. When delivered at a concentration of approximately 1.25 μM / 10,000 TIL / 100 μL of culture medium, the sd-RNA sequence used in this invention showed decreased target gene expression. When delivered at a concentration of approximately 1.5 μM / 10,000 TIL / 100 μL of culture medium, the sd-RNA sequence used in this invention showed decreased target gene expression. When delivered at a concentration of approximately 1.75 μM / 10,000 TIL / 100 μL of culture medium, the sd-RNA sequence used in this invention showed decreased target gene expression. When delivered at a concentration of approximately 2.0 μM / 10,000 TIL / 100 μL of culture medium, the sd-RNA sequence used in this invention showed decreased target gene expression. When delivered at a concentration of approximately 2.25 μM / 10,000 TIL / 100 μL of culture medium, the sd-RNA sequence used in this invention showed decreased target gene expression. When delivered at a concentration of approximately 2.5 μM / 10,000 TIL / 100 μL of culture medium, the sd-RNA sequence used in this invention showed decreased target gene expression. When delivered at a concentration of approximately 2.75 μM / 10,000 TIL / 100 μL of culture medium, the sd-RNA sequence used in this invention showed decreased target gene expression. When delivered at a concentration of approximately 3.0 μM / 10,000 TIL / 100 μL of culture medium, the sd-RNA sequence used in this invention shows decreased target gene expression. In some embodiments, when delivered at a concentration of approximately 3.25 μM / 10,000 TIL / 100 μL of culture medium, the sd-RNA sequence used in this invention shows decreased target gene expression. In some embodiments, when delivered at a concentration of approximately 3.5 μM / 10,000 TIL / 100 μL of culture medium, the sd-RNA sequence used in this invention shows decreased target gene expression. In some embodiments, when delivered at a concentration of approximately 3.75 μM / 10,000 TIL / 100 μL of culture medium, the sd-RNA sequence used in this invention shows decreased target gene expression. In some embodiments, when delivered at a concentration of approximately 4.0 μM / 10,000 TIL / 100 μL of culture medium, the sd-RNA sequence used in this invention shows decreased target gene expression. In some embodiments, when delivered at a concentration of about 5.0 μM / 10,000 TIL / 100 μL of culture medium, the sd-RNA sequence used in this invention shows reduced expression of the target gene.In some embodiments, the sd-RNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 6.0 μM / 10,000 TIL / 100 μL of culture medium. In some embodiments, the sd-RNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 7.0 μM / 10,000 TIL / 100 μL of culture medium. In some embodiments, the sd-RNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 8.0 μM / 10,000 TIL / 100 μL of culture medium. In some embodiments, the sd-RNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 9.0 μM / 10,000 TIL / 100 μL of culture medium. In some embodiments, the sd-RNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 10.0 μM / 10,000 TIL / 100 μL of culture medium.

[0584] c. sd-RNA modification

[0585] In some embodiments, the oligonucleotide reagent comprises more than one modification to increase the stability and / or efficacy of the therapeutic agent and to enable efficient delivery of the oligonucleotide to the cells or tissues to be treated. Such modifications may include 2'-O-methyl modifications, 2'-O-fluoro modifications, dithiophosphate modifications, 2'F-modified nucleotides, 2'-O-methyl-modified nucleotides, and / or 2'-deoxynucleotides. In some embodiments, the oligonucleotide is modified to include more than one hydrophobic modification, including, for example, sterols, cholesterol, vitamin D, naphthyl, isobutyl, benzyl, indole, tryptophan, and / or phenyl. In another specific embodiment, the chemically modified nucleotide is a combination of thiophosphate, 2'-O-methyl, 2'-deoxy, hydrophobic modifications, and thiophosphate. In some embodiments, the sugar may be modified, including but not limited to D-ribose, 2'-O-alkyl (including 2'-O-methyl and 2'-O-ethyl), i.e., 2'-alkoxy, 2'-amino, 2'-S-alkyl, 2'-halogen (including 2'-fluorine), T-methoxyethoxy, 2'-allyloxy (-OCH2CH=CH2), 2'-propynyl, 2'-propyl, ethynyl, vinyl, propenyl, and cyano. In one embodiment, the sugar moiety may be a hexose and incorporated into an oligonucleotide as described (Augustyns, K. et al., Nucl. Acids. Res. 18: 4711 (1992)).

[0586] In some embodiments, the double-stranded oligonucleotides of the present invention are double-stranded throughout their entire length, i.e., without any protruding single-stranded sequences at either end of the molecule, i.e., blunt-ended. In some embodiments, the individual nucleic acid molecules may have different lengths. In other words, the double-stranded oligonucleotides of the present invention are not double-stranded throughout their entire length. For example, when using two separate nucleic acid molecules, one of the molecules (e.g., the first molecule containing the antisense sequence) may be longer than the second molecule with which it hybridizes (resulting in a portion of the molecule being single-stranded). In some embodiments, when using a single nucleic acid molecule, a portion of that molecule may remain single-stranded at either end.

[0587] In some embodiments, the double-stranded oligonucleotides of the present invention contain mismatches and / or loops or protrusions, but at least about 70% of the length of the oligonucleotide is double-stranded. In some embodiments, the double-stranded oligonucleotides of the present invention are double-stranded at least about 80% of their length. In another embodiment, the double-stranded oligonucleotides of the present invention are double-stranded at least about 90%-95% of their length. In some embodiments, the double-stranded oligonucleotides of the present invention are double-stranded at least about 96%-98% of their length. In some embodiments, the double-stranded oligonucleotides of the present invention contain at least or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mismatches.

[0588] In some embodiments, oligonucleotides can be protected substantially from nucleases, for example, by modifying the 3' or 5' bond (e.g., U.S. Patent Nos. 5,849,902 and WO98 / 13526). For example, oligonucleotides can be made resistant by including a "blocking group." As used herein, the term "blocking group" refers to a substituent (e.g., a substituent other than an OH group) that can be attached to the oligonucleotide or nucleomonomer as a protecting group or coupling group for synthesis (e.g., FITC, propyl (CH2-CH2-CH3), ethylene glycol (-O-CH2-CH2-O-), phosphate (PO3)). 2+ ( ), phosphonate or phosphoramide) "blocking group" may also include "terminal blocking group" or "exonuclease blocking group" that protects the 5' and 3' ends of oligonucleotides, including modified nucleotide and non-nucleotide exonuclease resistant structures.

[0589] In some implementations, at least a portion of the continuous polynucleotides within the sd-RNA are linked by substitutional bonds (e.g., phosphate thioester bonds).

[0590] In some embodiments, the chemical modification can lead to enhanced cellular uptake by at least 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, and 500. In some embodiments, at least one of the C or U residues includes a hydrophobic modification. In some embodiments, multiple C and U contain hydrophobic modifications. In some embodiments, at least 10%, 15%, 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or at least 95% of C and U may contain hydrophobic modifications. In some embodiments, all C and U contain hydrophobic modifications.

[0591] In some embodiments, sd-RNA or sd-rxRNA exhibits enhanced endosome release of the sd-rxRNA molecule by incorporating a protonable amine. In some embodiments, a protonable amine is incorporated into the sense strand (in a portion of the molecule discarded after RISC loading). In some embodiments, the sd-RNA compounds of the present invention comprise an asymmetric compound comprising a double-stranded region (required for a valid RISC entry of 10-15 bases) and a single-stranded region of 4-12 nucleotides; a double-stranded region having 13 nucleotides. In some embodiments, a single-stranded region of 6 nucleotides is employed. In some embodiments, the single-stranded region of the sd-RNA comprises 2-12 phosphate-thioester nucleotide inter-bonds (referred to as phosphate-thioester modification). In some embodiments, 6-8 phosphate-thioester nucleotide inter-bonds are employed. In some embodiments, the sd-RNA compounds of the present invention also include a unique chemical modification pattern that provides stability and compatibility with RISC entries.

[0592] For example, the guide chain can also be modified by any chemical modification that enhances stability without interfering with the RISC entry. In some embodiments, the chemical modification pattern in the guide chain includes: most of the C and U nucleotides being 2'F modified and the 5' end being phosphorylated.

[0593] In some embodiments, at least 30% of the nucleotides in the sd-RNA or sd-rxRNA are modified. In some embodiments, at least 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62% of the nucleotides in the sd-RNA or sd-rxRNA are modified. % , 63% , 64% , 65% , 66% , 67% , 68% , 69% , 70% , 71% , 72% , 73% , 74% , 75% , 76% , 77% , 78% , 79% , 80% , 81% , 82% , 83% , 84% , 85% , 86% , 87% , 88% , 89% , 90% , 91% , 92% , 93% , 94% , 95% , 96% , 97% , 98% , or 99% of the nucleotides are modified. In some embodiments, 100% of the nucleotides in the sd-RNA or sd-rxRNA are modified.

[0594] In some embodiments, the sd-RNA molecule has a minimal double-stranded region. In some embodiments, the double-stranded region of the molecule is 8-15 nucleotides long. In some embodiments, the double-stranded region of the molecule is 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides long. In some embodiments, the double-stranded region is 13 nucleotides long. The leader and lagging strands may have 100% complementarity, or there may be more than one mismatch between the leader and lagging strands. In some embodiments, at one end of the double-stranded molecule, the molecule is blunt-ended or has a single nucleotide overhang. In some embodiments, the single-stranded region of the molecule is 4-12 nucleotides long. In some embodiments, the single-stranded region may be 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides long. In some embodiments, the single-stranded region may also be less than 4 nucleotides or greater than 12 nucleotides long. In some embodiments, the single-stranded region is 6 or 7 nucleotides long.

[0595] In some embodiments, the sd-RNA molecules exhibit increased stability. In certain cases, the chemically modified sd-RNA or sd-rxRNA molecules have a half-life in culture medium longer than 1, 2, 3, 4, 5, 6, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 hours or more, including any intermediate values. In some embodiments, the sd-rxRNA has a half-life in culture medium longer than 12 hours.

[0596] In some embodiments, sd-RNA is optimized to improve potency and / or reduce toxicity. In some embodiments, the nucleotide length of the guide and / or lagging strands and / or the amount of phosphate thioester modifications in the guide and / or lagging strands can affect the potency of the RNA molecule in some ways, while replacing 2'-fluoro(2'F) modifications with 2'-O-methyl (2'OMe) modifications can affect the toxicity of the molecule in some ways. In some embodiments, a reduction in the 2'F content in the molecule is expected to reduce the toxicity of the molecule. In some embodiments, the amount of phosphate thioester modifications in the RNA molecule can affect the molecule's uptake into cells, for example, the efficiency of passive uptake into cells. In some embodiments, sd-RNA does not have 2'F modifications but has equal efficacy in cellular uptake and tissue penetration.

[0597] In some embodiments, the guide chain is approximately 18 to 19 nucleotides in length and has approximately 2 to 14 phosphate ester modifications. For example, the guide chain may contain 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more than 14 phosphate-modified nucleotides. The guide chain may contain more than one modification that imparts increased stability without interfering with RISC entries. Phosphate-modified nucleotides, such as thiophosphate-modified nucleotides, may be located at the 3' end, the 5' end, or throughout the entire guide chain. In some embodiments, the 3' terminal 10 nucleotides of the guide chain contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 thiophosphate-modified nucleotides. The guide chain may also contain 2'F and / or 2'OMe modifications, which may be located throughout the molecule. In some embodiments, the first nucleotide of the guide chain (the nucleotide at the 5' position of the guide chain) is 2'OMe modified and / or phosphorylated. The C and U nucleotides within the guide chain may be 2'F modified. For example, the C and U nucleotides at positions 2-10 (or corresponding positions in guide strands of different lengths) of a 19nt leader strand may be 2'F modified. The C and U nucleotides within the leader strand may also be 2'OMe modified. For example, the C and U nucleotides at positions 11-18 (or corresponding positions in guide strands of different lengths) of a 19nt leader strand may be 2'OMe modified. In some embodiments, the nucleotide at the 3' end of the leader strand is unmodified. In some embodiments, most of the C and U nucleotides within the leader strand are 2'F modified, and the 5' end of the leader strand is phosphorylated. In other embodiments, the C or U at positions 1 and 11-18 are 2'OMe modified, and the 5' end of the leader strand is phosphorylated. In other embodiments, the C or U at positions 1 and 11-18 are 2'OMe modified, the 5' end of the leader strand is phosphorylated, and the C or U at positions 2-10 are 2'F modified.

[0598] d.sdRNA delivery

[0599] Self-delivered RNAi technology provides a method for directly transfecting cells with RNAi agents without the need for additional formulations or techniques. The ability to transfect difficult-to-transfect cell lines, high in vivo activity, and ease of use are characteristics of compositions and methods, offering significant functional advantages compared to conventional siRNA-based technologies. Therefore, the sd-RNA method has been employed in some embodiments relating to methods for reducing target gene expression in TILs of the present invention. The sd-RNAi method can directly deliver chemically synthesized compounds to a variety of primary cells and tissues in vivo and in vitro. The sd-RNAs described in some embodiments of the present invention are available from Advirna LLC (Worcester, MA, USA).

[0600] The general structure of an sdRNA molecule is as follows: Figure 36 As shown. The sd-RNA is formed as a hydrophobically modified siRNA-antisense oligonucleotide hybrid structure, as disclosed in, for example, Byrne et al., December 2013, J. Ocular Pharmacology and Therapeutics, 29(10): 855-864, the contents of which are incorporated herein by reference.

[0601] In some implementations, sterile electroporation can be used to deliver sd-RNA oligonucleotides to the TIL described herein.

[0602] In some embodiments, the oligonucleotide may be bound to a transmembrane delivery system for delivery to cells. In some embodiments, the transmembrane delivery system comprises lipids, viral vectors, etc. In some embodiments, the oligonucleotide agent is a self-delivering RNAi agent that requires no delivery agent.

[0603] In implementation, various methods can be used to introduce oligonucleotides (such as RNA or sd-RNA as described herein) into target cells. These methods include, for example, commercially available methods, including but not limited to, electroporation (Amaxa Nucleofector-II (Amaxa Biosystems, Cologne, Germany)), (ECM 830 (BTX) (Harvard Instruments, Boston, Massachusetts), Neon... TMTransfection Systems (available from ThermoFisher Scientific, Waltham, MIT) and / or Gene Pulser II (BioRad, Denver, Colorado), Multiporator (Eppendort, Hamburg, Germany), cationic liposome-mediated transfection using lipid transfection, polymer encapsulation, peptide-mediated transfection, or bioprojectile particle delivery systems, such as “gene guns” (see, for example, Nishikawa et al., Hum Gene Ther., 12(8): 861-70 (2001), the entire contents of which are incorporated herein by reference. See also U.S. Patent No. 8,859,229, U.S. Patent Application No. 2016 / 0230188, and Amaxa The II manual (available at http: / / icob.sinica.edu.tw / pubweb / bio-chem / Core%20Facilities / Data / R401-core / Nucleofector_Manual_II_Apr06.pdf)

[0604] In some embodiments, electroporation can be performed using Amaxa NUCLEOFECTOR.TM.-II as recommended by the manufacturer. In some embodiments, TILs can be transfected using NUCLEOFECTOR.TM.-II solution V and a set of recommended electroporation protocols. In some embodiments, TILs can be transfected using solutions V, T, and R, as well as different electroporation protocols. In some embodiments, TILs can be transfected using T cell NUCLEOFECTOR.TM.-II solution and different electroporation protocols. Alternative methods of nucleic acid delivery may also be used for transfection of the oligonucleotides described herein: cationic liposome-mediated transfection using LIPOFECTIN or LIPOFECTAMIN (Invitrogen). ECM 830 (BTX) (Harvard Instruments, Boston, MA), Gene Pulser II (BioRad, Denver, Colorado), Multiporator (Eppendorf, Hamburg, Germany) and / or Neon can also be used. TMElectroporation is performed using a Transfection System (available from ThermoFisher Scientific, Waltham, MIT). In some embodiments, pmaxGFP plasmid DNA (Amaxa Biosystems) can be used as a DNA control. In some embodiments, transfection efficiency (ET) can be determined by fluorescence-activated cell sorting (FACS) at approximately 3, 6, 9, 12, 15, and / or 18 hours post-transfection. In some experiments, transfectants can be further analyzed every 12 to 24 hours until GFP is undetectable in the GFP control. In some embodiments, cell viability can be determined by trypan blue exclusion.

[0605] Oligonucleotides and oligonucleotide compositions are contacted (e.g., contacted with, also referred to herein as being administered or delivered to) and absorbed by the TIL as described herein, including passive uptake via the TIL. sd-RNA may be added to the TIL as described herein during the first amplification (e.g., step B), after the first amplification (e.g., during step C), before or during the second amplification (e.g., before or during step D, after step D and before harvest in step E, during or after harvest in step F, before or during final formulation and / or transfer to the infusion bag in step F, and before any optional cryopreservation step in step F). Furthermore, sd-RNA may be added after thawing in any cryopreservation step in step F. In one embodiment, one or more sdRNAs targeting the genes described herein (including PD-1, LAG-3, TIM-3, CISH, and CBLB) may be added to a cell culture medium containing TIL and other reagents at concentrations of 100 nM to 20 mM, 200 nM to 10 mM, 500 nM to 1 mM, 1 μM to 100 μM, and 1 μM to 100 μM. In one embodiment, one or more sd-RNAs targeting the genes described herein (including PD-1, LAG-3, TIM-3, CISH, and CBLB) may be added to a cell culture medium containing TIL and other reagents in amounts selected from the group consisting of: 0.1 μM sd-RNA / 10,000 TIL / 100 μL medium, 0.5 μM sd-RNA / 10,000 TIL / 100 μL medium, 0.75 μM sd-RNA / 10,000 TIL / 100 μL medium, 1 μM sd-RNA / 10,000 TIL / 100 μL medium, 1.25 μM sd-RNA / 10,000 TIL / 100 μL medium, 1.5 μM sd-RNA / 10,000 TIL / 100 μL medium, 2 μM sd-RNA / 10,000 TIL / 100 μL medium, 5 ... sd-RNA / 10,000 TIL / 100 μL medium or 10 μM sd-RNA / 10,000 TIL / 100 μL medium. In one embodiment, during the pre-REP or REP phase, one or more sd-RNAs targeting the genes described herein (including PD-1, LAG-3, TIM-3, CISH, and CBLB) may be added to the TIL culture twice daily, once daily, once every two days, once every three days, once every four days, once every five days, once every six days, or once every seven days.In one embodiment, one or more sd-RNAs targeting the genes described herein (including PD-1, LAG-3, TIM-3, CISH, and CBLB) may be added to a cell culture medium containing TIL and other reagents in amounts selected from the group consisting of: 0.1 μM sd-RNA / 10,000 TIL / 100 μL medium, 0.5 μM sd-RNA / 10,000 TIL / 100 μL medium, 0.75 μM sd-RNA / 10,000 TIL / 100 μL medium, 1 μM sd-RNA / 10,000 TIL / 100 μL medium, 1.25 μM sd-RNA / 10,000 TIL / 100 μL medium, 1.5 μM sd-RNA / 10,000 TIL / 100 μL medium, 2 μM sd-RNA / 10,000 TIL / 100 μL medium, 5 ... sd-RNA / 10,000TIL / 100μL medium or 10μM sd-RNA / 10,000TIL / 100μL medium. In one embodiment, during the first amplification, second amplification, and / or additional amplification phases, one or more sd-RNAs targeting the genes described herein (including PD-1, LAG-3, TIM-3, CISH, and CBLB) may be added to the TIL culture twice daily, once daily, once every two days, once every three days, once every four days, once every five days, once every six days, or once every seven days.

[0606] The oligonucleotide compositions comprising sd-RNA of the present invention can be contacted with TILs during amplification as described herein, for example by dissolving a high concentration of sd-RNA in cell culture medium and allowing sufficient time for passive uptake. In some embodiments, the high concentration includes 0.1 μM sd-RNA / 10,000 TILs, 0.5 μM sd-RNA / 10,000 TILs, 0.75 μM sd-RNA / 10,000 TILs, 1 μM sd-RNA / 10,000 TILs, 1.25 μM sd-RNA / 10,000 TILs, 1.5 μM sd-RNA / 10,000 TILs, 2 μM sd-RNA / 10,000 TILs, 5 μM sd-RNA / 10,000 TILs, or 10 μM sd-RNA / 10,000 TILs. In some embodiments, high concentrations include 2 μM sd-RNA / 10,000 TIL, 5 μM sd-RNA / 10,000 TIL, or 10 μM sd-RNA / 10,000 TIL. In some embodiments, high concentrations include 5 μM sd-RNA / 10,000 TIL or up to 10 μM sd-RNA / 10,000 TIL.

[0607] In some embodiments, methods known in the art (see, for example, WO90 / 14074; WO91 / 16024; WO91 / 17424; U.S. Patent No. 4,897,355; Bergan et al., Nucleic Acids Research 1993, 21:3567) can be used to enhance the delivery of oligonucleotides into cells by suitable, art-recognized methods (including calcium phosphate, DMSO, glycerol or dextran, electroporation or by transfection, such as using cationic, anionic or neutral lipid compositions or liposomes).

[0608] e.sd-RNA combination

[0609] In some embodiments, more than one sd-RNA is used to reduce the expression of a target gene. In some embodiments, one or more sd-RNAs targeting PD-1, TIM-3, CBLB, LAG3, and / or CISH are used together. In some embodiments, PD-1 sd-RNA is used with one or more of TIM-3, CBLB, LAG3, and / or CISH to reduce the expression of more than one gene target. In some embodiments, LAG3 sd-RNA is used in combination with CISH-targeting sd-RNA to reduce the expression of two target genes. In some embodiments, the sd-RNAs targeting one or more of PD-1, TIM-3, CBLB, LAG3, and / or CISH described herein are commercially available from Advirna LLC (Worcester, MA, USA). In some embodiments, the sd-RNAs targeting one or more of PD-1, TIM-3, CBLB, LAG3, and / or CISH have Figure 36 or Figure 37 The structure shown.

[0610] In some embodiments, the sdRNA targets genes selected from PD-1, LAG3, TIM3, CTLA-4, TIGIT, CISH, TGFβR2, PKA, CBLB, BAFF (BR3), and combinations thereof. In some embodiments, the sdRNA targets genes selected from PD-1, LAG3, TIM3, CTLA-4, TIGIT, CISH, TGFβR2, PKA, CBLB, BAFF (BR3), and combinations thereof. In some embodiments, one sdRNA targets PD-1, and another sdRNA targets genes selected from LAG3, TIM3, CTLA-4, TIGIT, CISH, TGFβR2, PKA, CBLB, BAFF (BR3), and combinations thereof. In some embodiments, the sdRNA targets genes selected from PD-1, LAG-3, CISH, CBLB, TIM3, and combinations thereof. In some embodiments, the sdRNA targets genes selected from PD-1 and LAG3, CISH, CBLB, TIM3, and combinations thereof. In some embodiments, one sdRNA targets PD-1, and another targets LAG3. In some embodiments, one sdRNA targets PD-1, and another targets CISH. In some embodiments, one sdRNA targets PD-1, and another targets CBLB. In some embodiments, one sdRNA targets LAG3, and another targets CISH. In some embodiments, one sdRNA targets LAG3, and another targets CBLB. In some embodiments, one sdRNA targets CISH, and another targets CBLB. In some embodiments, one sdRNA targets TIM3, and another targets PD-1. In some embodiments, one sdRNA targets TIM3, and another targets LAG3. In some embodiments, one sdRNA targets TIM3, and another targets CISH. In some implementations, one sd-RNA targets TIM3, and another sd-RNA targets CBLB.

[0611] f. Overexpression of co-stimulatory receptors or adhesion molecules

[0612] According to other embodiments, altering the protein expression of TILs during the TIL amplification method may also allow for enhanced expression of more than one immune checkpoint gene in at least a subset of therapeutic TIL populations. For example, altering protein expression may result in enhanced expression of stimulatory receptors, meaning that the stimulatory receptor is overexpressed compared to its unmodified form. Non-limiting examples of immune checkpoint genes that can exhibit enhanced expression by transiently altering protein expression in the TILs of the present invention include certain chemokine receptors and interleukins, such as CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, IL-2, IL-4, IL-7, IL-10, IL-15, IL-21, the NOTCH 1 / 2 intracellular domain (ICD), and / or the NOTCH ligand mDLL1.

[0613] (i)CCR and CCL

[0614] For adoptive T-cell immunotherapy to be effective, T cells need to be appropriately transported to the tumor via chemokines. The matching between chemokines secreted by tumor cells, surrounding chemokines, and chemokine receptors expressed by T cells is crucial for the successful delivery of T cells to the tumor bed.

[0615] According to certain embodiments, the method of altering protein expression of the present invention can be used to increase the expression of certain chemokine receptors in TILs, such as one or more of CCR2, CCR4, CCR5, CXCR2, CXCR3, and / or CX3CR1. Following adoptive transfer, overexpression of CCRs may contribute to promoting effector function and TIL proliferation. In some embodiments, the method of altering protein expression of the present invention can be used to increase the expression of CCL2 (MCP-1), CCL3 (MIP-1α), CCL4 (MIP1-β), CCL5 (RANTES), CXCL1, CXCL8, CCL22, and / or CCL17 in TILs.

[0616] According to specific embodiments, the compositions and methods of the present invention enhance the expression of one or more of CCR2, CCR4, CCR5, CXCR2, CXCR3, and / or CX3CR1 in TIL. For example, any embodiment of the method described herein (e.g., process 2A or...) may be used. Figure 20 and 21The method described herein is a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, wherein the method includes gene editing of at least a portion of the TILs by enhancing the expression of one or more of CCR2, CCR4, CCR5, CXCR2, CXCR3, and / or CX3CR1. As described in more detail below, the gene editing process may include the use of a programmable nuclease that mediates double-strand or single-strand breaks in a chemokine receptor gene. For example, CRISPR, TALE, or zinc finger methods may be used to enhance the expression of certain chemokine receptors in TILs.

[0617] In one embodiment, CCR4 and / or CCR5 adhesion molecules are inserted into the TIL group using the γ-retroviral or lentiviral methods described herein. In one embodiment, CXCR2 adhesion molecules are inserted into the TIL group using the γ-retroviral or lentiviral methods described by Forget et al. (Frontiers Immunology 2017, 8, 908 or Peng et al., Clin. Cancer Res. 2010, 16, 5458, the disclosure of which is incorporated herein by reference).

[0618] In some embodiments, the present invention provides a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, the method comprising:

[0619] (i) Obtaining the first TIL group from the tumor removed from the patient;

[0620] (ii) A second TIL population is generated by first expansion of the first TIL population by culturing the first TIL population in a cell culture medium containing IL-2 and optional OKT-3;

[0621] (iii) A second expansion is performed by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, and antigen-presenting cells (APCs) to generate a third TIL population; wherein the number of the third TIL population is at least 100 times greater than that of the second TIL population; wherein the second expansion is performed for at least 14 days to obtain the third TIL population; wherein the third TIL population is a therapeutic TIL population; and

[0622] (iv) Expose the second and / or third TIL groups to transcription factors (TFs) and / or other molecules capable of transiently altering protein expression; wherein the TFs and / or other molecules capable of transiently altering protein expression provide alterations in tumor antigen expression and / or the number of tumor antigen-specific T cells in the therapeutic TIL groups; wherein the alteration in expression is an increase in the expression of one or more of CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, CCL2 (MCP-1), CCL3 (MIP-1α), CCL4 (MIP1-β), CCL5 (RANTES), CXCL1, CXCL8 and / or CCL22.

[0623] (ii) Interleukins and others

[0624] According to another embodiment, the gene editing method of the present invention can be used to increase the expression of certain interleukins (e.g., one or more of IL-2, IL-4, IL-7, IL-10, IL-15, and IL-21) and the intracellular domain (ICD) of NOTCH 1 / 2. Some interleukins have been shown to enhance T cell effector function and regulate tumor control.

[0625] According to specific embodiments, the compositions and methods of the present invention enhance the expression of one or more of IL-2, IL-4, IL-7, IL-10, IL-15, and IL-21, as well as the NOTCH 1 / 2 intracellular domain (ICD), in tumor-infiltrating lymphocytes (TILs). In some embodiments, the present invention provides a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, the method comprising:

[0626] (i) Obtaining the first TIL group from the tumor removed from the patient;

[0627] (ii) A second TIL population is generated by first expansion of the first TIL population by culturing the first TIL population in a cell culture medium containing IL-2 and optional OKT-3;

[0628] (iii) A second expansion is performed by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, and antigen-presenting cells (APCs) to generate a third TIL population; wherein the number of the third TIL population is at least 100 times greater than that of the second TIL population; wherein the second expansion is performed for at least 14 days to obtain the third TIL population; wherein the third TIL population is a therapeutic TIL population; and

[0629] (iv) Expose the second and / or third TIL groups to transcription factors (TFs) and / or other molecules capable of transiently altering protein expression; wherein the TFs and / or other molecules capable of transiently altering protein expression provide alterations in tumor antigen expression and / or the number of tumor antigen-specific T cells in the therapeutic TIL groups; wherein the alteration in expression is an increase in the expression of one or more of IL-2, IL-4, IL-7, IL-10, IL-15 and IL-21 and the intracellular domain (ICD) of NOTCH 1 / 2.

[0630] IV. TIL Production Process

[0631] Figure 1 An exemplary TIL procedure (referred to as procedure 2A) that includes some of these features is described. Figure 2 Some advantages of this embodiment of the invention relative to process 1C are described, such as Figure 13 and Figure 14 As shown. Figure 3 Process 1C is shown for comparison. Figure 4 (high cell count) and Figure 5 (Low cell count) shows two alternative timelines for TIL treatment based on process 2A. Figure 6 as well as Figure 8 An implementation of process 2A is shown. Figure 13 and Figure 14 An exemplary 2A process is also provided, compared to the exemplary 1C process.

[0632] As described herein, the present invention may include steps related to the restimulation of cryopreserved TILs to increase their metabolic activity and thus relative health prior to transplantation into a patient, and methods for detecting said metabolic health. As generally summarized herein, TILs are typically obtained from patient samples and manipulated to amplify their quantity prior to transplantation into a patient. In some embodiments, optionally, TILs may be genetically manipulated as described below.

[0633] In some implementations, TILs can be cryopreserved. Once thawed, they can also be restimulated to increase their metabolism before being infused into the patient.

[0634] In some implementations, as detailed below and as described in the examples and figures, the first amplification (including a process called pre-REP and...) Figure 8 The process shown as step A in the diagram is shortened to 3 to 14 days, and the second amplification (including a process called REP) is also shortened. Figure 8 The process (shown as step B in the text) is shortened to 7 to 14 days. In some implementations, as described in the examples and as... Figure 4 , Figure 5 , Figure 6 As shown in Figure 7, the first amplification (e.g.) Figure 8 The amplification described in step B is shortened to 11 days, and the second amplification (e.g.) Figure 8 The amplification described in step D is shortened to 11 days. In some embodiments, as detailed below and as illustrated in the examples and figures, the first amplification and the second amplification (e.g. Figure 8 The total amplification time described in steps B and D is shortened to 22 days.

[0635] The following "steps" marked A, B, C, etc. are for reference. Figure 8 and some of the embodiments described herein. The following and Figure 8 The order of the steps is exemplary, and any combination or order of steps, as well as additional steps, repetitions of steps, and / or omissions of steps, are contemplated in this application and the methods disclosed herein.

[0636] Step A: Obtaining a tumor sample from the patient.

[0637] Typically, TILs are initially obtained from patient tumor samples (“primary TILs”), and then expanded into a larger population for further manipulation, optional cryopreservation, restimulation as described herein, and optional assessment of phenotypic and metabolic parameters as indicators of TIL health.

[0638] Patient tumor samples can be obtained using methods known in the art, typically through surgical resection, needle aspiration biopsy, or other means of obtaining a sample containing a mixture of tumor and TIL cells. Generally, tumor samples can be derived from any solid tumor, including primary, invasive, or metastatic tumors. Tumor samples can also be liquid tumors, such as those obtained from hematologic malignancies. Solid tumors can be any type of cancer, including but not limited to breast cancer, pancreatic cancer, prostate cancer, colorectal cancer, lung cancer, brain cancer, kidney cancer, gastric cancer, and skin cancer (including but not limited to squamous cell carcinoma, basal cell carcinoma, and melanoma). In some embodiments, useful TILs are obtained from malignant melanoma tumors because they are reported to have particularly high levels of TILs.

[0639] The term "solid tumor" refers to an abnormal mass of tissue that does not typically contain cysts or fluid-filled areas. Solid tumors can be benign or malignant. The term "solid tumor carcinoma" refers to a malignant, neoplastic, or cancerous solid tumor. Solid tumor carcinomas include, but are not limited to, sarcomas, malignant epithelial tumors (carcinomas), and lymphomas, such as lung cancer, breast cancer, triple-negative breast cancer, prostate cancer, colon cancer, rectal cancer, and bladder cancer. In some embodiments, the cancer is selected from cervical cancer, head and neck cancer (including, for example, head and neck squamous cell carcinoma (HNSCC)), glioblastoma, ovarian cancer, sarcoma, pancreatic cancer, bladder cancer, breast cancer, triple-negative breast cancer, and non-small cell lung cancer. The histological structure of a solid tumor includes interdependent tissue compartments, including the parenchyma (cancer cells) and supporting stromal cells (the microenvironment in which cancer cells are dispersed and can provide support).

[0640] The term "hematologic malignancies" refers to cancers and tumors of the hematopoietic and lymphatic tissues of mammals, including but not limited to tissues of the blood, bone marrow, lymph nodes, and lymphatic system. Hematologic malignancies are also known as "liquid tumors." Hematologic malignancies include, but are not limited to: acute lymphoblastic leukemia (ALL), chronic lymphocytic lymphoma (CLL), small lymphocytic lymphoma (SLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute monocytic leukemia (AMoL), Hodgkin's lymphoma, and non-Hodgkin's lymphoma. The term "B-cell hematologic malignancies" refers to hematologic malignancies affecting B cells.

[0641] Once obtained, tumor samples are typically broken into 1mm pieces using a sharp dissection technique. 3 Approximately 8mm 3 Small pieces, approximately 2mm in size 3 Up to 3mm 3This is particularly useful. Enzymatic tumor digests are used to culture TILs from these fragments. Such tumor digests can be generated by incubation in an enzyme medium (e.g., Roswell Park Memorial Institute 1640 buffer, 2 mM glutamate, 10 mcg / mL gentamicin, 30 U / mL DNase, and 1.0 mg / mL collagenase) followed by mechanical dissociation (e.g., using a tissue dissociator). Tumor digests can be generated by placing the tumor in the enzyme medium, mechanically dissociating the tumor for approximately 1 minute, then incubating at 37°C with 5% CO2 for 30 minutes, and then repeating the mechanical dissociation and incubation under the above conditions until only small tissue fragments remain. At the end of this process, if the cell suspension contains a large number of red blood cells or dead cells, these cells can be removed by density gradient separation using FICOLL branched hydrophilic polysaccharides. Alternative methods known in the art, such as those described in U.S. Patent Application Publication No. 2012 / 0244133A1, the disclosure of which is incorporated herein by reference, can be used. Any of the foregoing methods may be used in any implementation of the methods for amplifying TILs or treating cancer described herein.

[0642] Typically, the harvested cell suspension is referred to as a "primary cell population" or a "freshly harvested" cell population.

[0643] In some embodiments, fragmentation includes physical fragmentation, including, for example, dissection and digestion. In some embodiments, fragmentation is physical fragmentation. In some embodiments, fragmentation is dissection. In some embodiments, fragmentation is by digestion. In some embodiments, TILs may be initially cultured from enzymatically digested tumor digests and tumor fragments obtained from the patient. In one embodiment, TILs may be initially cultured from enzymatically digested tumor digests and tumor fragments obtained from the patient.

[0644] In some implementations, when the tumor is a solid tumor, after obtaining a tumor sample in step A, for example, the tumor is physically fragmented (e.g., Figure 8 (As shown). In some embodiments, fragmentation occurs before cryopreservation. In some embodiments, fragmentation occurs after cryopreservation. In some embodiments, fragmentation occurs after obtaining the tumor without any cryopreservation. In some embodiments, the tumor is fragmented, and 10, 20, 30, 40 or more fragments or pieces are placed in each container for the first amplification. In some embodiments, the tumor is fragmented, and 30 or 40 fragments or pieces are placed in each container for the first amplification. In some embodiments, the tumor is fragmented, and 40 fragments or pieces are placed in each container for the first amplification. In some embodiments, the multiple fragments comprise about 4 to about 50 fragments, wherein each fragment has a volume of about 27 mm. 3In some embodiments, the multiple fragments comprise approximately 30 to approximately 60 fragments, with a total volume of approximately 1300 mm². 3 Approximately 1500mm 3 In some embodiments, the multiple fragments comprise approximately 50 fragments with a total volume of approximately 1350 mm². 3 In some embodiments, the plurality of fragments comprises about 50 fragments with a total mass of about 1 gram to about 1.5 grams. In some embodiments, the plurality of fragments comprises about 4 fragments.

[0645] In some embodiments, TILs are obtained from tumor fragments. In some embodiments, the tumor fragments are obtained through sharp dissection. In some embodiments, the tumor fragments are approximately 1 mm in size. 3 Up to 10mm 3 In some implementations, the tumor fragments are approximately 1 mm in size. 3 Up to 8mm 3 In some implementations, the tumor fragments are approximately 1 mm in size. 3 In some implementations, the tumor fragments are approximately 2 mm in size. 3 In some implementations, the tumor fragments are approximately 3 mm in size. 3 In some implementations, the tumor fragments are approximately 4 mm in size. 3 In some implementations, the tumor fragments are approximately 5 mm in size. 3 In some implementations, the tumor fragments are approximately 6 mm in size. 3 In some implementations, the tumor fragment is approximately 7 mm in size. 3 In some implementations, the tumor fragments are approximately 8 mm in size. 3 In some implementations, the tumor fragments are approximately 9 mm in size. 3 In some implementations, the tumor fragments are approximately 10 mm in size. 3 In some embodiments, the tumor is 1 mm to 4 mm × 1 mm to 4 mm × 1 mm to 4 mm. In some embodiments, the tumor is 1 mm × 1 mm × 1 mm. In some embodiments, the tumor is 2 mm × 2 mm × 2 mm. In some embodiments, the tumor is 3 mm × 3 mm × 3 mm. In some embodiments, the tumor is 4 mm × 4 mm × 4 mm.

[0646] In some embodiments, the tumor is removed to minimize the amount of hemorrhagic tissue, necrotic tissue, and / or adipose tissue in each small area. In some embodiments, the tumor is removed to minimize the amount of hemorrhagic tissue in each small area. In some embodiments, the tumor is removed to minimize the amount of necrotic tissue in each small area. In some embodiments, the tumor is removed to minimize the amount of adipose tissue in each small area.

[0647] In some embodiments, tumor fragmentation is performed to preserve the internal structure of the tumor. In some embodiments, tumor fragmentation is performed without a scalpel-like sawing motion. In some embodiments, TIL is obtained from tumor digests. In some embodiments, tumor digests are generated by incubation in an enzyme medium (e.g., but not limited to RPMI 1640, 2 mm GlutaMAX, 10 mg / mL gentamicin, 30 U / mL DNase, and 1.0 mg / mL collagenase) followed by mechanical dissociation (GentleMACS, Miltenyi Biotec, Aubum, California). After placing the tumor in the enzyme medium, it can be mechanically dissociated for approximately 1 minute. The solution is then incubated at 37°C and 5% CO2 for 30 minutes, followed by mechanical fragmentation again for approximately 1 minute. After incubation at 37°C and 5% CO2 for another 30 minutes, the tumor can be mechanically fragmented a third time for approximately 1 minute. In some embodiments, after the third mechanical disruption, if large tissue fragments are present, the sample is subjected to one or two additional mechanical dissociations, with or without an additional 30-minute incubation at 37°C and 5% CO2. In some embodiments, at the end of the final incubation, if the cell suspension contains a large number of red blood cells or dead cells, Ficoll density gradient separation can be used to remove these cells.

[0648] In some implementations, the cell suspension harvested prior to the first amplification step is referred to as the “primary cell population” or “freshly harvested” cell population.

[0649] In some embodiments, optionally, the cells may be frozen after sample harvesting and cryopreserved before proceeding to the amplification described in step B, which will be described in further detail below. Figure 8 The following is an example illustration.

[0650] B. Step B: First amplification

[0651] In some embodiments, the method of the present invention provides the acquisition of young TILs, which, compared to older TILs (i.e., TILs that have undergone more rounds of replication prior to administration to the subject / patient), can increase the replication cycle after administration to the subject / patient, and thus provide additional therapeutic benefits. The characteristics of young TILs have been described in the literature, for example, Donia et al., Scandinavian Journal of Immunology, 75:157-167 (2012); Dudley et al., Clin Cancer Res, 16:6122-6131 (2010); Huang et al., J Immunother, 28(3):258-267 (2005); Besser et al., Clin Cancer Res, 19(17):OF1-OF9 (2013); Besser et al., J Immunother, 32:415-423 (2009); Bunds et al., J Immunother, 32:415-423 (2009); Robbins et al., J Immunol, 2004; 173:7125-7130; Shen et al., J Immunother, 30:123-129 (2007); Zhou et al., J Immunother, 28:53-62 (2005); and Tran et al., J Immunother, 31:742-751 (2008), the entire contents of which are incorporated herein by reference.

[0652] Multiple antigen receptors on T and B lymphocytes are generated through somatic recombination of a limited but large number of gene fragments. These gene fragments—V (variable segment), D (diverse segment), J (connector segment), and C (constant segment)—determine the binding specificity of immunoglobulins and T cell receptors (TCRs) and their downstream applications. This invention provides a method for generating TILs that exhibit and increase the diversity of the T-cell repertoire. In some embodiments, TILs obtained by this method exhibit an increase in T-cell repertoire diversity. In some embodiments, freshly harvested TILs are used and / or other methods besides those provided herein (including, for example, those other than…) Figure 8 Compared to TILs prepared by methods other than those shown in the present invention, TILs obtained by the method of the present invention show increased T cell repertoire diversity. In some embodiments, compared to freshly harvested TILs and / or those prepared using methods such as... Figure 13Compared to TILs prepared by the method shown, referred to as Process 1C, TILs obtained by this method exhibit increased T cell repertoire diversity. In some embodiments, the TILs obtained from the first amplification show increased T cell repertoire diversity. In some embodiments, the increase in diversity is an increase in immunoglobulin diversity and / or T cell receptor diversity. In some embodiments, immunoglobulin diversity is immunoglobulin heavy chain diversity. In some embodiments, immunoglobulin diversity is immunoglobulin light chain diversity. In some embodiments, the diversity is T cell receptor diversity. In some embodiments, the diversity is the diversity of T cell receptors selected from one of α, β, γ, and δ receptors. In some embodiments, the expression of T cell receptor (TCR) α and / or β is increased. In some embodiments, the expression of T cell receptor (TCR) α is increased. In some embodiments, the expression of T cell receptor (TCR) β is increased. In some embodiments, the expression of TCRab (i.e., TCRα / β) is increased.

[0653] After dissecting or digesting tumor fragments (e.g., as Figure 8 As described in step A), the cells are cultured in serum containing IL-2 under conditions more favorable to TIL growth compared to tumor cells and other cells. In some embodiments, tumor digests are incubated in 2 mL wells in a medium containing inactivated human AB serum and 6000 IU / mL IL-2. This primary cell population is cultured for a period of time, typically 3 to 14 days, producing a large population of TILs, typically about 1 × 10⁻⁶. 8 A large number of TIL cells. In some embodiments, this primary cell population is cultured for 7 to 14 days to produce a large TIL population, typically about 1 × 10⁻⁶. 8 A large number of TIL cells. In some embodiments, this primary cell population is cultured for 10 to 14 days, producing a large TIL population, typically about 1 × 10⁶. 8 A large number of TIL cells. In some embodiments, this primary cell population is cultured for about 11 days, producing a large TIL population, typically about 1 × 10⁶. 8 A large number of TIL cells.

[0654] In a preferred embodiment, TIL amplification can be performed as follows: using the initial large-scale TIL amplification steps described below and herein (e.g., as... Figure 8The steps described in step B, which may include a process called pre-REP, are followed by a second amplification as described below and herein (step D, which includes a process called the Rapid Amplification Protocol (REP) step), subsequently optionally cryopreserved, and then a second step D as described below and herein (including a process called the Restimulation REP step). Optionally, the TILs obtained from this process can be characterized for phenotypic features and metabolic parameters as described herein.

[0655] In an embodiment of initiating TIL culture using a 24-well plate, for example, using a Costar 24-well cell culture plate, flat bottom (Corning, New York), 1 × 10⁶ cells can be seeded in each well with 2 mL of complete IL-2 medium (CM) (6000 IU / mL; Chiron Corp., Emeryville, CA). 6 A single tumor cell or a single tumor fragment. In some embodiments, the tumor fragment is approximately 1 mm in size. 3 Up to 10mm 3 .

[0656] In some embodiments, the first amplification medium is referred to as "CM," where CM is an abbreviation for culture medium. In some embodiments, the CM in step B consists of RPMI 1640 containing GlutaMAX, supplemented with 10% human AB serum, 25 mmHEPES, and 10 mg / mL gentamicin. The culture medium has a volume of 40 mL and a 10 cm aerated silica substrate. 2 In an implementation method that begins culturing in a permeable flask (e.g., G-Rex10; Wilson Wolf Manufacturing, New Brighton, MN) Figure 1 Each flask contained 10×10⁻⁶ mmol / L of CM containing IL-2 in 10 to 40 mL. 6 Up to 40×10 6 One live tumor cell or 5 to 30 tumor fragments were digested. G-Rex10 and 24-well plates were incubated in a humidified incubator at 37°C and 5% CO2. After 5 days of culture, half of the medium was removed and replaced with fresh CM and IL-2. After day 5, half of the medium was replaced every 2 to 3 days.

[0657] After preparing tumor fragments, the resulting cells (i.e., fragments) are cultured in serum containing IL-2 under conditions more favorable to TIL growth compared to tumor cells and other cells. In some embodiments, the tumor fragments are incubated in 2 mL wells of medium containing inactivated human AB serum (or, in some cases, as described herein, in the presence of an aAPC cell population) and 6000 IU / mL IL-2. The primary cell population is then cultured for a period of time, typically 10 to 14 days, to produce a large population of TILs, typically approximately 1 × 10⁻⁶. 8 A large number of TIL cells. In some embodiments, the growth medium during the initial expansion contains IL-2 or a variant thereof. In some embodiments, the IL is recombinant human IL-2 (rhIL-2). In some embodiments, the 1 mg vial of IL-2 stock solution has 20 × 10⁻⁶ cells. 6 IU / mg up to 30×10 6 Specific activity of IU / mg. In some embodiments, the 1 mg vial of IL-2 stock solution has a specific activity of 20 × 10⁻⁶ IU / mg. 6 IU / mg up to 30×10 6 Specific activity of IU / mg. In some embodiments, the 1 mg vial of IL-2 stock solution has a specific activity of 25 × 10⁻⁶ IU / mg. 6 Specific activity of IU / mg. In some embodiments, the 1 mg vial of IL-2 stock solution has a specific activity of 30 × 10⁻⁶ IU / mg. 6 Specific activity of IL-2 (IU / mg). In some embodiments, the final concentration of the IL-2 stock solution is 4 × 10⁻⁶. 6 IU / mg up to 8×10 6 IU / mg IL-2. In some embodiments, the final concentration of the IL-2 stock solution is 5 × 10⁻⁶. 6 IU / mg up to 7×10 6 IU / mg IL-2. In some embodiments, the final concentration of the IL-2 stock solution is 6 × 10⁻⁶. 6IU / mg IL-2. In some embodiments, an IL-2 stock solution is prepared as described in Example 4. In some embodiments, the first amplification medium contains about 10,000 IU / mL IL-2, about 9,000 IU / mL IL-2, about 8,000 IU / mL IL-2, about 7,000 IU / mL IL-2, about 6,000 IU / mL IL-2, or about 5,000 IU / mL IL-2. In some embodiments, the first amplification medium contains about 9,000 IU / mL to about 5,000 IU / mL IL-2. In some embodiments, the first amplification medium contains about 8,000 IU / mL to about 6,000 IU / mL IL-2. In some embodiments, the first amplification medium contains about 7,000 IU / mL to about 6,000 IU / mL IL-2. In some embodiments, the first amplification medium contains about 6,000 IU / mL IL-2. In one embodiment, the cell culture medium further comprises IL-2. In some embodiments, the cell culture medium comprises about 3000 IU / mL IL-2. In one embodiment, the cell culture medium further comprises IL-2. In a preferred embodiment, the cell culture medium comprises about 3000 IU / mL IL-2. In one embodiment, the cell culture medium comprises about 1000 IU / mL, about 1500 IU / mL, about 2000 IU / mL, about 2500 IU / mL, about 3000 IU / mL, about 3500 IU / mL, about 4000 IU / mL, about 4500 IU / mL, about 5000 IU / mL, about 5500 IU / mL, about 6000 IU / mL, about 6500 IU / mL, about 7000 IU / mL, about 7500 IU / mL, or about 8000 IU / mL IL-2. In one embodiment, the cell culture medium contains 1000 to 2000 IU / mL, 2000 to 3000 IU / mL, 3000 to 4000 IU / mL, 4000 to 5000 IU / mL, 5000 to 6000 IU / mL, 6000 to 7000 IU / mL, 7000 to 8000 IU / mL, or about 8000 IU / mL IL-2.

[0658] In some embodiments, the first amplification medium contains approximately 500 IU / mL IL-15, approximately 400 IU / mL IL-15, approximately 300 IU / mL IL-15, approximately 200 IU / mL IL-15, approximately 300 IU / mL IL-15, 180 IU / mL IL-15, approximately 160 IU / mL IL-15, approximately 140 IU / mL IL-15, approximately 120 IU / mL IL-15, or approximately 100 IU / mL IL-15. In some embodiments, the first amplification medium contains approximately 500 IU / mL IL-15 to approximately 100 IU / mL IL-15. In some embodiments, the first amplification medium contains approximately 400 IU / mL IL-15 to approximately 100 IU / mL IL-15. In some embodiments, the first amplification medium contains about 300 IU / mL IL-15 to about 100 IU / mL IL-15. In some embodiments, the first amplification medium contains about 200 IU / mL IL-15. In some embodiments, the cell culture medium contains about 180 IU / mL IL-15. In one embodiment, the cell culture medium also contains IL-15. In a preferred embodiment, the cell culture medium contains about 180 IU / mL IL-15.

[0659] In some embodiments, the first amplification medium contains about 20 IU / mL IL-21, about 15 IU / mL IL-21, about 12 IU / mL IL-21, about 10 IU / mL IL-21, about 5 IU / mL IL-21, about 4 IU / mL IL-21, about 3 IU / mL IL-21, about 2 IU / mL IL-21, about 1 IU / mL IL-21, or about 0.5 IU / mL IL-21. In some embodiments, the first amplification medium contains about 20 IU / mL IL-21 to about 0.5 IU / mL IL-21. In some embodiments, the first amplification medium contains about 15 IU / mL IL-21 to about 0.5 IU / mL IL-21. In some embodiments, the first amplification medium contains about 12 IU / mL IL-21 to about 0.5 IU / mL IL-21. In some embodiments, the first amplification medium contains about 10 IU / mL IL-21 to about 0.5 IU / mL IL-21. In some embodiments, the first amplification medium contains about 5 IU / mL IL-21 to about 1 IU / mL IL-21. In some embodiments, the first amplification medium contains about 2 IU / mL IL-21. In some embodiments, the cell culture medium contains about 1 IU / mL IL-21. In some embodiments, the cell culture medium contains about 0.5 IU / mL IL-21. In one embodiment, the cell culture medium also contains IL-21. In a preferred embodiment, the cell culture medium contains about 1 IU / mL IL-21.

[0660] In one embodiment, the cell culture medium contains OKT-3 antibody. In a preferred embodiment, the cell culture medium contains about 30 ng / mL of OKT3 antibody. In one embodiment, the cell culture medium contains about 0.1 ng / mL, about 0.5 ng / mL, about 1 ng / mL, about 2.5 ng / mL, about 5 ng / mL, about 7.5 ng / mL, about 10 ng / mL, about 15 ng / mL, about 20 ng / mL, about 25 ng / mL, about 30 ng / mL, about 35 ng / mL, about 40 ng / mL, about 50 ng / mL, about 60 ng / mL, about 70 ng / mL, about 80 ng / mL, about 90 ng / mL, about 100 ng / mL, about 200 ng / mL, about 500 ng / mL, and about 1 μg / mL of OKT3 antibody. In one embodiment, the cell culture medium contains OKT3 antibody at concentrations of 0.1 ng / mL to 1 ng / mL, 1 ng / mL to 5 ng / mL, 5 ng / mL to 10 ng / mL, 10 ng / mL to 20 ng / mL, 20 ng / mL to 30 ng / mL, 30 ng / mL to 40 ng / mL, 40 ng / mL to 50 ng / mL, or 50 ng / mL to 100 ng / mL. In some embodiments, the cell culture medium does not contain OKT-3 antibody.

[0661] In some embodiments, the cell culture medium contains more than one TNFRSF agonist. In some embodiments, the TNFRSF agonist includes a 4-1BB agonist. In some embodiments, the TNFRSF agonist is a 4-1BB agonist selected from urogenumab, utorumab, EU-101, fusion proteins, and fragments, derivatives, variants, biosimilars, and combinations thereof. In some embodiments, the TNFRSF agonist is added at a concentration sufficient to achieve 0.1 μg / mL to 100 μg / mL in the cell culture medium. In some embodiments, the TNFRSF agonist is added at a concentration sufficient to achieve 20 μg / mL to 40 μg / mL in the cell culture medium.

[0662] In some embodiments, in addition to one or more TNFRSF agonists, the cell culture medium also contains IL-2 at an initial concentration of about 3000 IU / mL and OKT-3 antibody at an initial concentration of about 30 ng / mL, wherein one or more TNFRSF agonists includes a 4-1BB agonist.

[0663] In some embodiments, the first amplification medium is referred to as "CM" (abbreviation for culture medium). In some embodiments, it is referred to as CM1 (culture medium 1). In some embodiments, CM consists of RPMI 1640 and GlutaMAX, supplemented with 10% human AB serum, 25 mM Hepes, and 10 mg / mL gentamicin. In some embodiments, a medium with a volume of 40 mL and 10 cm 2 Cultures were initiated in permeable flasks with permeable silica bottoms (e.g., G-Rex10; Wilson Wolf Manufacturing, New Brighton, MN), each flask containing 10 mL to 40 mL of CM supplemented with IL-2, the CM containing 10 × 10⁻⁶ ppm. 6 Up to 40×10 6 One live tumor cell or 5 to 30 tumor fragments. G-Rex10 and 24-well plates were incubated in a humidified incubator at 37°C and 5% CO2. Culture was carried out for 5 days after the start of culture, with half of the culture medium removed and replaced with fresh CM and IL-2. After day 5, half of the culture medium was replaced every 2 to 3 days. In some embodiments, CM is CM1 as described in the examples, see Example 5. In some embodiments, the first expansion occurs in the initial cell culture medium or first cell culture medium. In some embodiments, the initial cell culture medium or first cell culture medium contains IL-2.

[0664] In some implementations, as described in the embodiments and figures, the first amplification (including, for example) Figure 8 The processes described in step B, which may include those sometimes referred to as pre-REP, are shortened to 3 to 14 days. In some implementations, as described in the examples and Figure 4 and 5 As shown, and including, for example Figure 8 The amplification described in step B, the first amplification (including, for example) Figure 8 Those described in step B, which may include those sometimes referred to as pre-REP, are shortened to 7 to 14 days. In some implementations, as described in the embodiments and Figure 4 and 5 As shown, the first amplification in step B is shortened to 10 to 14 days. In some embodiments, in the first embodiment, as described in the examples and Figure 4 and 5 As shown, and including, for example Figure 8 The amplification process described in step B involves shortening the first amplification period to 11 days.

[0665] In some embodiments, the first TIL amplification can be performed for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days. In some embodiments, the first TIL amplification can be performed for 1 to 14 days. In some embodiments, the first TIL amplification can be performed for 2 to 14 days. In some embodiments, the first TIL amplification can be performed for 3 to 14 days. In some embodiments, the first TIL amplification can be performed for 4 to 14 days. In some embodiments, the first TIL amplification can be performed for 5 to 14 days. In some embodiments, the first TIL amplification can be performed for 6 to 14 days. In some embodiments, the first TIL amplification can be performed for 7 to 14 days. In some embodiments, the first TIL amplification can be performed for 8 to 14 days. In some embodiments, the first TIL amplification can be performed for 9 to 14 days. In some embodiments, the first TIL amplification can be performed for 10 to 14 days. In some embodiments, the first TIL amplification can be performed for 11 to 14 days. In some embodiments, the first TIL amplification may take 12 to 14 days. In some embodiments, the first TIL amplification may take 13 to 14 days. In some embodiments, the first TIL amplification may take 14 days. In some embodiments, the first TIL amplification may take 1 to 11 days. In some embodiments, the first TIL amplification may take 2 to 11 days. In some embodiments, the first TIL amplification may take 3 to 11 days. In some embodiments, the first TIL amplification may take 4 to 11 days. In some embodiments, the first TIL amplification may take 5 to 11 days. In some embodiments, the first TIL amplification may take 6 to 11 days. In some embodiments, the first TIL amplification may take 7 to 11 days. In some embodiments, the first TIL amplification may take 8 to 11 days. In some embodiments, the first TIL amplification may take 9 to 11 days. In some embodiments, the first TIL amplification may take 10 to 11 days. In some embodiments, the first TIL amplification may take 11 days.

[0666] In some embodiments, a combination of IL-2, IL-7, IL-15, and / or IL-21 is used as a combination during the first amplification. In some embodiments, the first amplification includes, for example, according to... Figure 8 During step B, as described herein, IL-2, IL-7, IL-15, and / or IL-21, and any combination thereof, may be included. In some embodiments, a combination of IL-2, IL-15, and IL-21 is used as a combination during the first amplification. In some embodiments, according to... Figure 8During step B, as described herein, IL-2, IL-15, and IL-21, and any combination thereof, may be included.

[0667] In some implementations, as described in the embodiments and Figure 4 and 5 As shown, the first amplification (including a process called pre-REP; for example, according to...) Figure 8 Step B) is shortened to 3 to 14 days. In some implementations, as described in the examples and Figure 4 and 5 As shown, the first amplification in step B is shortened to 7 to 14 days. In some implementations, as described in the examples and Figure 4 , Figure 5 , Figure 6 As shown in Figure 7, the first amplification in step B is shortened to 10 to 14 days. In some embodiments, as described in the examples and Figure 4 , Figure 5 , Figure 6 As shown in Figure 7, the first amplification period was shortened to 11 days.

[0668] In some implementations, the first amplification (e.g., according to...) Figure 8 Step B) is carried out in a closed-system bioreactor. In some embodiments, as described herein, a closed system is used for TIL amplification. In some embodiments, a single bioreactor is used. In some embodiments, for example, the single bioreactor used is a G-REX-10 or a G-REX-100. In some embodiments, the closed-system bioreactor is a single bioreactor.

[0669] In some implementations, during the first amplification (e.g., according to...) Figure 8Step B) involves adding one or more sd-RNAs targeting the genes described herein (including PD-1, LAG-3, TIM-3, CISH, and CBLB) to a cell culture medium containing amounts of TIL and other reagents selected from the group consisting of: 0.1 μM sd-RNA / 10,000 TIL / 100 μL medium, 0.5 μM sd-RNA / 10,000 TIL / 100 μL medium, 0.75 μM sd-RNA / 10,000 TIL / 100 μL medium, 1 μM sd-RNA / 10,000 TIL / 100 μL medium, 1.25 μM sd-RNA / 10,000 TIL / 100 μL medium, 1.5 μM sd-RNA / 10,000 TIL / 100 μL medium, 2 μM sd-RNA / 10,000 TIL / 100 μL medium, 5 μM sd-RNA / 10,000 TIL / 100 μL medium, or 10 μM sd-RNA / 10,000 TIL / 100 μL medium. In some embodiments, during the first amplification (e.g., according to...), Figure 8 Step B) may involve adding one or more sdRNAs targeting the genes described herein (including PD-1, LAG-3, TIM-3, CISH, and CBLB) to the TIL culture twice daily, once daily, once every two days, once every three days, once every four days, once every five days, once every six days, or once every seven days. In one embodiment, during the first amplification (e.g., according to...), Figure 8 In step B), one or more sd-RNAs targeting the genes described herein (including PD-1, LAG-3, TIM-3, CISH, and CBLB) may be added to a cell culture medium containing amounts of TIL and other reagents selected from the group consisting of: 0.1 μM sd-RNA / 10,000 TIL, 0.5 μM sd-RNA / 10,000 TIL, 0.75 μM sd-RNA / 10,000 TIL, 1 μM sd-RNA / 10,000 TIL, 1.25 μM sd-RNA / 10,000 TIL, 1.5 μM sd-RNA / 10,000 TIL, 2 μM sd-RNA / 10,000 TIL, 5 μM sd-RNA / 10,000 TIL, or 10 μM sd-RNA / 10,000 TIL. In one embodiment, during the first amplification (e.g., according to...), Figure 8 Step B) may involve adding one or more sdRNAs targeting the genes described herein (including PD-1, LAG-3, TIM-3, CISH, and CBLB) to TIL cultures twice a day, once a day, once every two days, once every three days, once every four days, once every five days, once every six days, or once every seven days.

[0670] C. Step C: Transition from the first amplification to the second amplification

[0671] In some cases, the schemes discussed below can be used to immediately cryopreserve large populations of TILs obtained from the first amplification, including, for example, from, for example Figure 8 The TIL population obtained in step B is shown. Optionally, the TIL population harvested from the first amplification (referred to as the second TIL population) may be amplified a second time (which may include amplification sometimes referred to as REP), and then cryopreserved as described below. Similarly, in cases where the genetically modified TILs will be used for treatment, the first TIL population (sometimes referred to as the large TIL population) or the second TIL population (which in some embodiments may include a population referred to as the REP TIL population) may be genetically modified before amplification or after the first amplification and before the second amplification for suitable treatment.

[0672] In some embodiments, the TIL obtained from the first amplification (e.g., step B as shown in FIG. 7) is stored until phenotypic selection is performed. In some embodiments, the TIL obtained from the first amplification (e.g., step B as shown in FIG. 7) is not stored but is directly subjected to a second amplification. In some embodiments, the TIL obtained from the first amplification is not cryopreserved after the first amplification and before the second amplification. In some embodiments, the transition from the first amplification to the second amplification occurs approximately 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days after fragmentation. In some embodiments, the transition from the first amplification to the second amplification occurs approximately 3 to 14 days after fragmentation. In some embodiments, the transition from the first amplification to the second amplification occurs approximately 4 to 14 days after fragmentation. In some embodiments, the transition from the first amplification to the second amplification occurs approximately 4 to 10 days after fragmentation. In some embodiments, the transition from the first amplification to the second amplification occurs approximately 7 to 14 days after fragmentation. In some implementations, the transition from the first amplification to the second amplification occurs approximately 14 days after fragmentation begins.

[0673] In some embodiments, the transition from the first amplification to the second amplification occurs approximately 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days after fragmentation. In some embodiments, the transition occurs from 1 to 14 days after fragmentation. In some embodiments, the first TIL amplification may take 2 to 14 days. In some embodiments, the transition occurs from 3 to 14 days after fragmentation. In some embodiments, the transition occurs from 4 to 14 days after fragmentation. In some embodiments, the transition occurs from 5 to 14 days after fragmentation. In some embodiments, the transition occurs from 6 to 14 days after fragmentation. In some embodiments, the transition occurs from 7 to 14 days after fragmentation. In some embodiments, the transition from the first amplification to the second amplification occurs 8 to 14 days after fragmentation. In some embodiments, the transition occurs 9 to 14 days after fragmentation. In some embodiments, the transition occurs 10 to 14 days after fragmentation. In some embodiments, the transition occurs 11 to 14 days after fragmentation. In some embodiments, the transition occurs 12 to 14 days after fragmentation. In some embodiments, the transition occurs 13 to 14 days after fragmentation. In some embodiments, the transition occurs 14 days after fragmentation. In some embodiments, the transition occurs 1 to 11 days after fragmentation. In some embodiments, the transition occurs 2 to 11 days after fragmentation. In some embodiments, the transition from the first amplification to the second amplification occurs 3 to 11 days after fragmentation. In some embodiments, the transition occurs 8 to 11 days after fragmentation. In some embodiments, the transition occurs 9 to 11 days after fragmentation. In some embodiments, the transition occurs 10 to 11 days after fragmentation. In some embodiments, the transition occurs 11 days after fragmentation.

[0674] In some implementations, TILs are not stored after the first amplification and before the second amplification, and the TILs are directly used for the second amplification (e.g., in some implementations, such as...). Figure 8 As shown, no storage occurs during the transition from step B to step D. In some embodiments, as described herein, the transition occurs in a closed system. In some embodiments, the TIL from the first amplification (i.e., the second TIL group) enters the second amplification directly without undergoing a transition period.

[0675] In some implementations, the transition from the first amplification to the second amplification is carried out in a closed-system bioreactor (e.g., according to...). Figure 8 Step C). In some embodiments, as described herein, a closed system is used for TIL amplification. In some embodiments, a single bioreactor is used. In some embodiments, for example, the single bioreactor used is a G-REX-10 or a G-REX-100. In some embodiments, the closed system bioreactor is a single bioreactor.

[0676] In some implementations, the transition from the first amplification to the second amplification (e.g., according to...) Figure 8 During step C), one or more sd-RNAs targeting the genes described herein (including PD-1, LAG-3, TIM-3, CISH, and CBLB) may be added to a cell culture medium containing amounts of TIL and other reagents selected from the group consisting of: 0.1 μM sd-RNA / 10,000 TIL / 100 μL medium, 0.5 μM sd-RNA / 10,000 TIL / 100 μL medium, 0.75 μM sd-RNA / 10,000 TIL / 100 μL medium, 1 μM sd-RNA / 10,000 TIL / 100 μL medium, 1.25 μM sd-RNA / 10,000 TIL / 100 μL medium, 1.5 μM sd-RNA / 10,000 TIL / 100 μL medium, 2 μM sd-RNA / 10,000 TIL / 100 μL medium, 5 μM sd-RNA / 10,000 TIL / 100 μL medium, or 10 μM sd-RNA / 10,000 TIL / 100 μL medium. In some embodiments, there is a transition from the first amplification to the second amplification (e.g., according to...). Figure 8 During step C), one or more sdRNAs targeting the genes described herein (including PD-1, LAG-3, TIM-3, CISH, and CBLB) may be added to the TIL culture twice daily, once daily, once every two days, once every three days, once every four days, once every five days, once every six days, or once every seven days. In one embodiment, during the transition from the first amplification to the second amplification (e.g., according to...), Figure 8 During step C), one or more sd-RNAs targeting the genes described herein (including PD-1, LAG-3, TIM-3, CISH, and CBLB) may be added to a cell culture medium containing amounts of TIL and other reagents selected from the group consisting of: 0.1 μM sd-RNA / 10,000 TIL, 0.5 μM sd-RNA / 10,000 TIL, 0.75 μM sd-RNA / 10,000 TIL, 1 μM sd-RNA / 10,000 TIL, 1.25 μM sd-RNA / 10,000 TIL, 1.5 μM sd-RNA / 10,000 TIL, 2 μM sd-RNA / 10,000 TIL, 5 μM sd-RNA / 10,000 TIL, or 10 μM sd-RNA / 10,000 TIL. In some implementations, the transition from the first amplification to the second amplification (e.g., according to...) Figure 8 During step C), one or more sd-RNAs targeting the genes described herein (including PD-1, LAG-3, TIM-3, CISH, and CBLB) may be added to TIL cultures twice a day, once a day, once every two days, once every three days, once every four days, once every five days, once every six days, or once every seven days.

[0677] 1. Cytokines

[0678] As is known in the art, the amplification methods described herein typically use culture media containing high doses of cytokines (particularly IL-2).

[0679] Alternatively, combinations of cytokines can be used for rapid expansion and / or secondary expansion of TILs; wherein combinations of two or more of IL-2, IL-15, and IL-21 are as outlined in International Publications WO2015 / 189356 and WO2015 / 189357, the entire contents of which are expressly incorporated herein by reference. Thus, possible combinations include IL-2 and IL-15, IL-2 and IL-21, IL-15 and IL-21, and IL-2, IL-15, and IL-21, the latter being found particularly useful in many embodiments. The use of combinations of cytokines is particularly beneficial for the production of lymphocytes (especially T cells as described herein).

[0680] Step D: Second amplification

[0681] In some implementations, after harvesting and initial mass treatment, such as after steps A and B, the number of TIL cells increases; this transition is referred to as step C. Figure 8As shown. This further amplification is referred to herein as a second amplification, which may include what is commonly known in the art as a rapid amplification process (REP); and Figure 8 The amplification process (as shown in step D) is typically performed using a culture medium containing multiple components, including feeder cells, a cytokine source, and an anti-CD3 antibody, in a permeable container.

[0682] In some implementations, a second amplification or a second TIL amplification (which may include amplification sometimes referred to as REP); and Figure 8 The process shown in step D can be performed using any TIL flask or container known to those skilled in the art. In some embodiments, the second TIL amplification can be performed for 10, 11, 12, 13, or 14 days. In some embodiments, the second TIL amplification can be performed for about 7 to about 14 days. In some embodiments, the second TIL amplification can be performed for about 8 to about 14 days. In some embodiments, the second TIL amplification can be performed for about 9 to about 14 days. In some embodiments, the second TIL amplification can be performed for about 10 to about 14 days. In some embodiments, the second TIL amplification can be performed for about 11 to about 14 days. In some embodiments, the second TIL amplification can be performed for about 12 to about 14 days. In some embodiments, the second TIL amplification can be performed for about 13 to about 14 days. In some embodiments, the second TIL amplification can be performed for about 14 days.

[0683] In one embodiment, the second amplification can be performed in a breathable container using the methods of this disclosure (including, for example, an amplification referred to as REP; and the process shown in step D of FIG7). For example, TILs can be rapidly amplified using nonspecific T cell receptor stimulation in the presence of interleukin-2 (IL-2) or interleukin-15 (IL-15). Nonspecific T cell receptor stimulation may include, for example, an anti-CD3 antibody, such as about 30 ng / mL of OKT3, a mouse monoclonal anti-CD3 antibody (commercially available from Ortho-McNeil, Raritan, NJ or Miltenyi Biotech, Auburn, CA) or UHCT-1 (commercially available from BioLegend, San Diego, CA, USA). During the second expansion, TILs can be rapidly expanded in vitro by further stimulation with a cancer antigen (including its antigenic motif, such as an epitope) that induces TILs. This antigen may optionally be expressed by a vector, such as a human leukocyte antigen A2 (HLA-A2) binding peptide, for example, 0.3 μM MART-L26-35 (27L) or gp 100:209-217 (210M). Other suitable antigens may include, for example, NY-ESO-1, TRP-1, TRP-2, tyrosinase carcinoma antigen, MAGE-A3, SSX-2, and VEGFR2, or their antigenic motifs. TILs can also be rapidly expanded by restimulating them with the same cancer antigen pulsed onto HLA-A2-expressing antigen-presenting cells. Alternatively, TILs can be further restimulated with, for example, irradiated autologous lymphocytes or irradiated HLA-A2+ allogeneic lymphocytes and IL-2. In some embodiments, restimulation occurs as part of the second expansion. In some implementations, a second expansion occurs in the presence of irradiated autologous lymphocytes or irradiated HLA-A2+ allogeneic lymphocytes and IL-2.

[0684] In one embodiment, the cell culture medium further comprises IL-2. In some embodiments, the cell culture medium comprises about 3000 IU / mL IL-2. In one embodiment, the cell culture medium comprises about 1000 IU / mL, about 1500 IU / mL, about 2000 IU / mL, about 2500 IU / mL, about 3000 IU / mL, about 3500 IU / mL, about 4000 IU / mL, about 4500 IU / mL, about 5000 IU / mL, about 5500 IU / mL, about 6000 IU / mL, about 6500 IU / mL, about 7000 IU / mL, about 7500 IU / mL, or about 8000 IU / mL IL-2. In one embodiment, the cell culture medium comprises 1000 to 2000 IU / mL, 2000 to 3000 IU / mL, 3000 to 4000 IU / mL, 4000 to 5000 IU / mL, 5000 to 6000 IU / mL, 6000 to 7000 IU / mL, 7000 to 8000 IU / mL, or 8000 IU / mL IL-2.

[0685] In one embodiment, the cell culture medium contains OKT3 antibody. In some embodiments, the cell culture medium contains about 30 ng / mL of OKT3 antibody. In one embodiment, the cell culture medium contains about 0.1 ng / mL, about 0.5 ng / mL, about 1 ng / mL, about 2.5 ng / mL, about 5 ng / mL, about 7.5 ng / mL, about 10 ng / mL, about 15 ng / mL, about 20 ng / mL, about 25 ng / mL, about 30 ng / mL, about 35 ng / mL, about 40 ng / mL, about 50 ng / mL, about 60 ng / mL, about 70 ng / mL, about 80 ng / mL, about 90 ng / mL, about 100 ng / mL, about 200 ng / mL, about 500 ng / mL, and about 1 μg / mL of OKT3 antibody. In one embodiment, the cell culture medium contains OKT3 antibody at concentrations of 0.1 ng / mL to 1 ng / mL, 1 ng / mL to 5 ng / mL, 5 ng / mL to 10 ng / mL, 10 ng / mL to 20 ng / mL, 20 ng / mL to 30 ng / mL, 30 ng / mL to 40 ng / mL, 40 ng / mL to 50 ng / mL, or 50 ng / mL to 100 ng / mL. In some embodiments, the cell culture medium does not contain OKT-3 antibody.

[0686] In some embodiments, the initial amplification cell culture medium contains one or more TNFRSF agonists. In some embodiments, the TNFRSF agonist includes a 4-1BB agonist. In some embodiments, the TNFRSF agonist is a 4-1BB agonist selected from: urogenumab, utorumab, EU-101, fusion proteins, and fragments, derivatives, variants, biosimilars, and combinations thereof. In some embodiments, the TNFRSF agonist is added at a concentration sufficient to achieve 0.1 μg / mL to 100 μg / mL in the cell culture medium. In some embodiments, the TNFRSF agonist is added at a concentration sufficient to achieve 20 μg / mL to 40 μg / mL in the cell culture medium.

[0687] In some embodiments, in addition to one or more TNFRSF agonists, the first amplification cell culture medium for initiating the first amplification also contains IL-2 at an initial concentration of about 3000 IU / mL and OKT-3 antibody at an initial concentration of about 30 IU / mL, wherein one or more TNFRSF agonists includes a 4-1BB agonist.

[0688] In some embodiments, a combination of IL-2, IL-7, IL-15, and / or IL-21 is used as a combination during the second amplification. In some embodiments, the second amplification (including, for example, according to...) Figure 8 Step D of the process (as described herein) may include IL-2, IL-7, IL-15, and / or IL-21, and any combination thereof. In some embodiments, a combination of IL-2, IL-15, and IL-21 is used as a combination during the second amplification. In some embodiments, according to... Figure 8 In step D, as described herein, IL-2, IL-15, and IL-21, and any combination thereof, may be included.

[0689] In some embodiments, the second expansion can be performed in a supplemental cell culture medium containing IL-2, OKT-3, antigen-presenting feeder cells, and optionally a TNFRSF agonist. In some embodiments, the second expansion occurs in a supplemental cell culture medium. In some embodiments, the supplemental cell culture medium contains IL-2, OKT-3, and antigen-presenting feeder cells. In some embodiments, the second cell culture medium contains IL-2, OKT-3, and antigen-presenting cells (APCs; also known as antigen-presenting feeder cells). In some embodiments, the second expansion occurs in a cell culture medium containing IL-2, OKT-3, and antigen-presenting feeder cells (i.e., antigen-presenting cells).

[0690] In some embodiments, the second amplification medium contains about 500 IU / mL of IL-15, about 400 IU / mL of IL-15, about 300 IU / mL of IL-15, about 200 IU / mL of IL-15, about 180 IU / mL of IL-15, about 160 IU / mL of IL-15, about 140 IU / mL of IL-15, about 120 IU / mL of IL-15, or about 100 IU / mL of IL-15. In some embodiments, the second amplification medium contains about 500 IU / mL of IL-15 to about 100 IU / mL of IL-15. In some embodiments, the second amplification medium contains about 400 IU / mL of IL-15 to about 100 IU / mL of IL-15. In some embodiments, the second amplification medium contains about 300 IU / mL of IL-15 to about 100 IU / mL of IL-15. In some embodiments, the second amplification medium contains about 200 IU / mL of IL-15. In some embodiments, the cell culture medium contains about 180 IU / mL of IL-15. In one embodiment, the cell culture medium also contains IL-15. In a preferred embodiment, the cell culture medium contains about 180 IU / mL of IL-15.

[0691] In some embodiments, the second amplification medium contains about 20 IU / mL of IL-21, about 15 IU / mL of IL-21, about 12 IU / mL of IL-21, about 10 IU / mL of IL-21, about 5 IU / mL of IL-21, about 4 IU / mL of IL-21, about 3 IU / mL of IL-21, about 2 IU / mL of IL-21, about 1 IU / mL of IL-21, or about 0.5 IU / mL of IL-21. In some embodiments, the second amplification medium contains about 20 IU / mL of IL-21 to about 0.5 IU / mL of IL-21. In some embodiments, the second amplification medium contains about 15 IU / mL of IL-21 to about 0.5 IU / mL of IL-21. In some embodiments, the second amplification medium contains about 12 IU / mL of IL-21 to about 0.5 IU / mL of IL-21. In some embodiments, the second amplification medium contains about 10 IU / mL of IL-21 to about 0.5 IU / mL of IL-21. In some embodiments, the second amplification medium contains about 5 IU / mL of IL-21 to about 1 IU / mL of IL-21. In some embodiments, the second amplification medium contains about 2 IU / mL of IL-21. In some embodiments, the cell culture medium contains about 1 IU / mL of IL-21. In some embodiments, the cell culture medium contains about 0.5 IU / mL of IL-21. In one embodiment, the cell culture medium further contains IL-21. In a preferred embodiment, the cell culture medium contains about 1 IU / mL of IL-21.

[0692] In some embodiments, the antigen-presenting feeder cells (APCs) are PBMCs. In one embodiment, during rapid expansion and / or secondary expansion, the ratio of TILs to PBMCs and / or antigen-presenting cells is about 1:25, about 1:50, about 1:100, about 1:125, about 1:150, about 1:175, about 1:200, about 1:225, about 1:250, about 1:275, about 1:300, about 1:325, about 1:350, about 1:375, about 1:400, or about 1:500. In one embodiment, during rapid expansion and / or secondary expansion, the ratio of TILs to PBMCs is from 1:50 to 1:300. In one embodiment, during rapid expansion and / or secondary expansion, the ratio of TILs to PBMCs is from 1:100 to 1:200.

[0693] In one embodiment, the REP and / or second amplification are performed in a flask; wherein a large amount of TIL is mixed with 100 or 200 times excess of inactivated feeder cells, 30 mg / mL OKT3 anti-CD3 antibody, and 3000 IU / mL IL-2 in 150 mL of culture medium. The culture medium is replaced (typically by aspirating fresh medium to replace 2 / 3 of the medium) until the cells are transferred to a replacement growth chamber. As discussed more fully below, the replacement growth chamber comprises a G-REX flask and a ventilated container.

[0694] In some implementations, as described in the examples and figures, the second amplification (which may include a process referred to as the REP process) is shortened to 7 to 14 days. In some implementations, the second amplification is shortened to 11 days.

[0695] In one embodiment, the REP and / or second amplification can be performed using T-175 flasks and aeration bags as described above (Tran et al., J. I. M ... 6 TIL was added to each T-175 flask. TIL was cultured in a 1:1 mixture of CM and AFM-V media supplemented with 3000 IU / mL IL-2 and 30 ng / mL anti-CD3. The T-175 flasks were incubated at 37°C and 5% CO2. On day 5, half of the medium was replaced with a 50 / 50 medium containing 3000 IU / mL IL-2. In some embodiments, on day 7, cells from two T-175 flasks were combined into a 3L bag, and 300 mL of AFM-V containing 5% human AB serum and 3000 IU / mL IL-2 was added to 300 mL of the TIL suspension. Cell counts were performed daily or every two days in each bag, and fresh medium was added to maintain a cell count of 0.5 × 10⁻⁶. 6 Up to 2.0×10 6 Cells / mL.

[0696] In one implementation, the second amplification (which may include amplification referred to as REP, as well as those mentioned in step D of FIG7) may be performed in a 500 mL volume chamber with a 100 cm³ permeable silicon substrate. 2 The experiment was conducted in a permeable flask (G-Rex 100, available from WilsonWolf Manufacturing Corporation, New Brighton, MN, USA), 5 × 106 Or 10×10 6 TILs can be cultured with PBMCs in 400 mL of 50 / 50 medium supplemented with 5% human AB serum, 3000 IU / mL IL-2, and 30 ng / mL anti-CD3 (OKT3). G-REX 100 flasks can be incubated at 37°C in 5% CO2. On day 5, 250 mL of the supernatant can be collected and centrifuged at 1500 rpm (491 × g) for 10 minutes. The TIL precipitate can be resuspended in 150 mL of fresh medium containing 5% human AB serum and 3000 IU / mL IL-2, and then added back to the original G-REX 100 flask. When TILs are serially amplified in G-REX 100 flasks, on day 7, the TILs from each G-REX 100 flask can be resuspended in 300 mL of culture medium in each flask, and the cell suspension can be aliquoted into three 100 mL aliquots for inoculating three G-REX 100 flasks. Then, 150 mL of AIM-V containing 5% human AB serum and 3000 IU / mL IL-2 can be added to each flask. The G-REX 100 flasks can be incubated at 37°C and 5% CO2 for 4 days. After that, 150 mL of AIM-V containing 3000 IU / mL IL-2 can be added to each G-REX 100 flask. Cells can be harvested on day 14 of culture.

[0697] In one embodiment, the second amplification (including an amplification termed REP) is performed in a flask; wherein a large amount of TIL is mixed with 100 or 200 times excess of inactivated feeder cells, 30 mg / mL OKT3 anti-CD3 antibody, and 3000 IU / mL IL-2 in 150 mL of culture medium. In some embodiments, the culture medium is replaced until the cells are transferred to a replacement growth chamber. In some embodiments, two-thirds of the culture medium is replaced by respiration with fresh culture medium. In some embodiments, as discussed more fully below, the replacement growth chamber comprises a G-REX flask and a vented container.

[0698] In one embodiment, a second amplification (including an amplification termed REP) is performed, and the process further includes a step of selecting tumor-responsive TILs. Any selection method known in the art can be used. For example, the method described in U.S. Patent Application Publication No. 2016 / 0010058A1 (the disclosure of which is incorporated herein by reference) can be used to select tumor-responsive TILs.

[0699] Optionally, standard detection methods known in the art can be used to determine cell viability after the second amplification (including amplification known as REP amplification). For example, a trypanblue exclusion assay can be performed on a large number of TIL samples, which selectively labels dead cells and allows for viability assessment. In some embodiments, the TIL samples can be counted and viability detected using a Cellometer K2 automated cell counter (Nexcelom Bioscience, Lawrence, MA). In some embodiments, viability is determined according to, for example, the Cellometer K2 ImageCytometer Automatic Cell Counter protocol described in Example 15.

[0700] In some embodiments, the second amplification of TIL (including amplification referred to as REP) can be performed using T-175 flasks and permeable bags as described above (Tran KQ, Zhou J, Durflinger KH et al., 2008, J. Immunother., 31: 742-751 and Dudley ME, Wunderlich JR, Shelton TE et al., 2003, J. Immunother., 26: 332-342) or permeable G-REX flasks. In some embodiments, the second amplification is performed using flasks. In some embodiments, the second amplification is performed using permeable G-REX flasks. In some embodiments, the second amplification is performed in T-175 flasks, with approximately 1 × 10⁻⁶ TILs applied. 6 TIL was suspended in approximately 150 mL of culture medium and added to each T-175 flask. The TIL was co-cultured with irradiated (50 Gy) allogeneic PBMCs (as "feeder" cells) at a 1:100 ratio in a 1:1 mixture of CM and AIM-V medium (50 / 50 medium), supplemented with 3000 IU / mL IL-2 and 30 ng / mL anti-CD3. The T-175 flasks were incubated at 37°C in 5% CO2. In some embodiments, on day 5, half of the medium was replaced with 50 / 50 medium containing 3000 IU / mL IL-2. In some embodiments, on day 7, cells from two T-175 flasks were combined into a 3 L bag, and 300 mL of AIM-V containing 5% human AB serum and 3000 IU / mL IL-2 was added to 300 mL of the TIL suspension. The cell count in each bag can be counted daily or every two days, and fresh culture medium can be added to maintain the cell count at approximately 0.5 × 10⁻⁶. 6 From approximately 2.0 × 106 Cells / mL.

[0701] In some implementations, the second amplification (including an amplification referred to as REP) is performed at a depth of 100 cm. 2 The experiment was conducted in a 500 mL volumetric flask (G-REX 100, Wilson Wolf) with a permeable silica bottom. Figure 1 Approximately 5 × 10⁻⁶ 6 Or 10×10 6 TILs were cultured with irradiated allogeneic PBMCs at a ratio of 1:100 in 400 mL of 50 / 50 medium supplemented with 3000 IU / mL IL-2 and 30 ng / mL anti-CD3. The G-REX 100 flasks were incubated at 37°C in 5% CO2. In some embodiments, on day 5, 250 mL of the supernatant was collected and centrifuged at 1500 rpm (491 g) for 10 minutes. The TIL precipitate was then resuspended in 150 mL of fresh 50 / 50 medium containing 3000 IU / mL IL-2 and added back to the original G-REX 100 flask. In the embodiment of continuous TIL amplification in G-REX 100 flasks, on day 7, the TIL from each G-REX 100 flask was resuspended in 300 mL of culture medium present in each flask. The cell suspension was divided into three 100 mL aliquots for inoculating three G-REX 100 flasks. Then, 150 mL of AIM-V containing 5% human AB serum and 3000 IU / mL IL-2 was added to each flask. The G-REX 100 flasks were incubated at 37°C and 5% CO2 for 4 days. After that, 150 mL of AIM-V containing 3000 IU / mL IL-2 was added to each G-REX 100 flask. Cells were harvested on day 14 of culture.

[0702] Multiple antigen receptors on T and B lymphocytes are generated through somatic recombination of a limited but large number of gene fragments. These gene fragments—V (variable region), D (multivariable region), J (connector region), and C (constant region)—determine the binding specificity and downstream applications of immunoglobulins and T cell receptors (TCRs). This invention provides a method for generating TILs that demonstrate and increase the diversity of the T cell repertoire. In some embodiments, TILs obtained by this method demonstrate an increase in T cell repertoire diversity. In some embodiments, TILs obtained through a second amplification demonstrate an increase in T cell repertoire diversity. In some embodiments, the increase in diversity is an increase in immunoglobulin diversity and / or T cell receptor diversity. In some embodiments, immunoglobulin diversity is immunoglobulin heavy chain diversity. In some embodiments, immunoglobulin diversity is immunoglobulin light chain diversity. In some embodiments, diversity is T cell receptor diversity. In some embodiments, diversity is the diversity of T cell receptors selected from one of α, β, γ, and δ receptors. In some embodiments, the expression of T cell receptor (TCR) α and / or β is increased. In some embodiments, the expression of T cell receptor (TCR) α is increased. In some implementations, the expression of T-cell receptor (TCR)β is increased. In some implementations, the expression of TCRab (i.e., TCRα / β) is increased.

[0703] In some implementations, the second amplification medium (e.g., sometimes referred to as CM2 or second cell culture medium) contains IL-2, OKT-3, and antigen-presenting feeder cells (APCs), as discussed in more detail below.

[0704] In some implementations, the second amplification (e.g., according to...) Figure 8 Step D) is carried out in a closed-system bioreactor. In some embodiments, as described herein, a closed system is used for TIL amplification. In some embodiments, a single bioreactor is used. In some embodiments, for example, the single bioreactor used is a G-REX-10 or a G-REX-100. In some embodiments, the closed-system bioreactor is a single bioreactor.

[0705] In some implementations, during the second amplification (e.g., according to...) Figure 8During step D), one or more sd-RNAs targeting the genes described herein (including PD-1, LAG-3, TIM-3, CISH, and CBLB) may be added to a cell culture medium containing amounts of TIL and other reagents selected from the group consisting of: 0.1 μM sd-RNA / 10,000 TIL / 100 μL medium, 0.5 μM sd-RNA / 10,000 TIL / 100 μL medium, 0.75 μM sd-RNA / 10,000 TIL / 100 μL medium, 1 μM sd-RNA / 10,000 TIL / 100 μL medium, 1.25 μM sd-RNA / 10,000 TIL / 100 μL medium, 1.5 μM sd-RNA / 10,000 TIL / 100 μL medium, 2 μM sd-RNA / 10,000 TIL / 100 μL medium, 5 μM sd-RNA / 10,000 TIL / 100 μL medium, or 10 μM sd-RNA / 10,000 TIL / 100 μL medium. In some embodiments, during the second amplification (e.g., according to...), Figure 8 During step D), one or more sdRNAs targeting the genes described herein (including PD-1, LAG-3, TIM-3, CISH, and CBLB) may be added to the TIL culture twice daily, once daily, once every two days, once every three days, once every four days, once every five days, once every six days, or once every seven days. In one embodiment, during the second amplification (e.g., according to...), Figure 8 During step D), one or more sd-RNAs targeting the genes described herein (including PD-1, LAG-3, TIM-3, CISH, and CBLB) may be added to a cell culture medium containing amounts of TIL and other reagents selected from the group consisting of: 0.1 μM sd-RNA / 10,000 TIL, 0.5 μM sd-RNA / 10,000 TIL, 0.75 μM sd-RNA / 10,000 TIL, 1 μM sd-RNA / 10,000 TIL, 1.25 μM sd-RNA / 10,000 TIL, 1.5 μM sd-RNA / 10,000 TIL, 2 μM sd-RNA / 10,000 TIL, 5 μM sd-RNA / 10,000 TIL, or 10 μM sd-RNA / 10,000 TIL. In some embodiments, during a second amplification (e.g., according to…), Figure 8During step D), one or more sdRNAs targeting the genes described herein (including PD-1, LAG-3, TIM-3, CISH, and CBLB) may be added to TIL cultures twice a day, once a day, once every two days, once every three days, once every four days, once every five days, once every six days, or once every seven days.

[0706] 1. Feeder cells and antigen-presenting cells

[0707] In one implementation, during REP TIL amplification and / or during the second amplification, the second amplification step described herein (e.g., including, for example) Figure 8 The steps described in step D, as well as those amplifications referred to as REP, require an excess of feeder cells. In many embodiments, the feeder cells are peripheral blood mononuclear cells (PBMCs) obtained from standard whole blood units from healthy blood donors. PBMCs are obtained using standard methods such as Ficoll-Paque gradient separation.

[0708] Typically, allogeneic PBMCs are inactivated by irradiation or heat treatment and used in the REP step, as described in the embodiments, which provides an exemplary scheme for evaluating the replication incompetence of irradiated allogeneic PBMCs.

[0709] In some implementations, if the total number of viable cells on day 14 is less than the initial number of viable cells introduced into culture on day 0 of the REP and / or day 0 of the second expansion (i.e., the start date of the second expansion), the PBMCs are considered to be incompletely replicating and are accepted for use in the TIL expansion step described herein.

[0710] In some embodiments, if the total number of live cells cultured on days 7 and 14 in the presence of OKT3 and IL-2 does not increase compared to the initial number of live cells introduced into culture on day 0 of REP and / or day 0 of the second amplification (i.e., the start date of the second amplification), then PBMCs are considered to have impaired replication function and are accepted for the TIL amplification step described herein. In some embodiments, PBMCs are cultured in the presence of 30 ng / mL OKT3 antibody and 3000 IU / mL IL-2.

[0711] In some embodiments, if the total number of viable cells cultured on days 7 and 14 in the presence of OKT3 and IL-2 does not increase compared to the initial number of viable cells introduced into culture on day 0 of REP and / or day 0 of the second amplification (i.e., the start date of the second amplification), then PBMCs are considered to have impaired replication function and are accepted for the TIL amplification step described herein. In some embodiments, PBMCs are cultured in the presence of 5 ng / mL to 60 ng / mL OKT3 antibody and 1000 IU / mL to 6000 IU / mL IL-2. In some embodiments, PBMCs are cultured in the presence of 10 ng / mL to 50 ng / mL OKT3 antibody and 2000 IU / mL to 5000 IU / mL IL-2. In some embodiments, PBMCs are cultured in the presence of 20 ng / mL to 40 ng / mL OKT3 antibody and 2000 IU / mL to 4000 IU / mL IL-2. In some implementations, PBMCs are cultured in the presence of 25 ng / mL to 35 ng / mL OKT3 antibody and 2500 IU / mL to 3500 IU / mL IL-2.

[0712] In some embodiments, the antigen-presenting feeder cells are PBMCs. In some embodiments, the antigen-presenting feeder cells are artificial antigen-presenting feeder cells. In one embodiment, the ratio of TILs to antigen-presenting feeder cells in the second expansion is about 1:25, about 1:50, about 1:100, about 1:125, about 1:150, about 1:175, about 1:200, about 1:225, about 1:250, about 1:275, about 1:300, about 1:325, about 1:350, about 1:375, about 1:400, or about 1:500. In one embodiment, the ratio of TILs to antigen-presenting feeder cells in the second expansion is from 1:50 to 1:300. In one embodiment, the ratio of TILs to antigen-presenting feeder cells in the second expansion is from 1:100 to 1:200.

[0713] In one implementation, the second amplification procedure described herein requires approximately 2.5 × 10⁻⁶. 9 Feeder cell ratio approximately 100 × 10 6 The ratio of TILs. In another embodiment, the second amplification procedure described herein requires approximately 2.5 × 10⁻⁶. 9 Feeder cell ratio approximately 50 × 10 6 The ratio of TILs. In yet another embodiment, the second amplification procedure described herein requires approximately 2.5 × 10⁻⁶. 9 Feeding cells to approximately 25 × 10 6 The ratio of TIL.

[0714] In one embodiment, the second amplification step described herein requires an excess of feeder cells during the second amplification. In many embodiments, the feeder cells are peripheral blood mononuclear cells (PBMCs) obtained from standard whole blood units from healthy blood donors. PBMCs are obtained using standard methods such as Ficoll-Paque gradient separation. In one embodiment, artificial antigen-presenting (aAPC) cells are used instead of PBMCs.

[0715] Typically, allogeneic PBMCs are inactivated by irradiation or heat treatment and used in the TIL amplification steps described herein, including, for example... Figure 4 , 5 The exemplary steps described in 6 and 7.

[0716] In one implementation, artificial antigen-presenting cells are used in the second expansion, either in place of PBMCs or in combination with PBMCs.

[0717] 2. Cytokines

[0718] As is known in the art, the amplification methods described herein typically use culture media containing high doses of cytokines (particularly IL-2).

[0719] Alternatively, combinations of cytokines can be used for rapid expansion and / or secondary expansion of TILs; wherein combinations of two or more of IL-2, IL-15, and IL-21 are as outlined in International Publications WO2015 / 189356 and WO2015 / 189357, the entire contents of which are expressly incorporated herein by reference. Thus, possible combinations include IL-2 and IL-15, IL-2 and IL-21, IL-15 and IL-21, and IL-2, IL-15, and IL-21, the latter being found particularly useful in many embodiments. The use of combinations of cytokines is particularly beneficial for the production of lymphocytes (especially T cells as described herein).

[0720] Step E: Harvesting TIL

[0721] After the second amplification step, cells can be harvested. In some implementations, for example, in... Figure 8 TILs are harvested after one, two, three, four, or more amplification steps as provided. In some implementations, for example, in... Figure 8 TILs are harvested after the two amplification steps provided in the paper.

[0722] TILs can be harvested in any suitable and aseptic manner, including, for example, by centrifugation. Methods for harvesting TILs are well known in the art, and any such known methods can be used in conjunction with the method of the present invention. In some embodiments, an automated system is used to harvest TILs.

[0723] Cell collectors and / or cell processing systems are commercially available from a variety of sources, including, for example, Fresenius Kabi, Tomtec Life Science, Perkin Elmer, and Inotech Biosystems International, Inc. Any cell-based collector can be used in the methods of this invention. In some embodiments, the cell collector and / or cell processing system is a membrane-based cell collector. In some embodiments, cells are harvested via a cell processing system, such as the LOVO system (manufactured by Fresenius Kabi). The term "LOVO cell processing system" also refers to any instrument or device manufactured by any supplier that can pump a cell-containing solution through a membrane or filter (such as a rotating membrane or rotary filter) in a sterile and / or closed system environment, allowing for continuous flow and cell processing to remove supernatant or cell culture medium without precipitate. In some embodiments, the cell collector and / or cell processing system can perform cell separation, washing, fluid exchange, concentration, and / or other cell processing steps in a closed, sterile system.

[0724] In some implementations, the harvest (e.g., according to) Figure 8 Step E) is carried out in a closed-system bioreactor. In some embodiments, as described herein, a closed system is used for TIL amplification. In some embodiments, a single bioreactor is used. In some embodiments, for example, the single bioreactor used is a G-REX-10 or a G-REX-100. In some embodiments, the closed-system bioreactor is a single bioreactor.

[0725] In some implementations, the process described in Example 16 is followed. Figure 8 Step E. In some embodiments, to maintain the sterility and closure of the system, the closed system is introduced under sterile conditions using a syringe. In some embodiments, a closed system as described in Example 16 is employed.

[0726] In some embodiments, TIL is harvested according to the method described in Example 16. In some embodiments, TIL from day 1 to day 11 is harvested using the method described in Section 8.5 (referred to as: Day 11 TIL Harvest in Example 16). In some embodiments, TIL from day 12 to day 22 is harvested using the method described in Section 8.12 (referred to as: Day 22 TIL Harvest in Example 16).

[0727] Step F: Final formulation / transfer to infusion bag

[0728] Following steps A through E as exemplarily provided in Figure 7 and as outlined above and in detail herein, the cells are transferred to a container for administration to a patient. In some embodiments, once a sufficient number of therapeutically adequate TILs have been obtained using the amplification method described above, they are transferred to a container for administration to a patient.

[0729] In one embodiment, TILs amplified by APCs of this disclosure are administered to a patient as a pharmaceutical composition. In one embodiment, the pharmaceutical composition is a suspension of TILs in a sterile buffer. TILs amplified by PBMCs of this disclosure can be administered via any suitable route known in the art. In some embodiments, T cells are administered via a single intra-arterial or intravenous infusion, preferably lasting about 30 to 60 minutes. Other suitable routes of administration include intraperitoneal, intrathecal, and intralymphatic administration.

[0730] 1. Drug composition, dosage and administration regimen

[0731] In one embodiment, TILs amplified using the methods of this disclosure are administered to a patient as a pharmaceutical composition. In one embodiment, the pharmaceutical composition is a suspension of TILs in a sterile buffer. TILs amplified using PBMCs of this disclosure can be administered via any suitable route known in the art. In some embodiments, T cells are administered via a single intra-arterial or intravenous infusion, preferably lasting about 30 to 60 minutes. Other suitable routes of administration include intraperitoneal, intrathecal, and intralymphatic administration.

[0732] Any suitable dose of TIL can be administered. In some implementations, particularly when the cancer is melanoma, approximately 2.3 × 10⁻⁶ is administered. 10 Approximately 13.7 × 10 10 TIL group, averaging approximately 7.8 × 10⁻⁶ 10 TIL group. In one implementation, approximately 1.2 × 10⁻⁶ is applied. 10 Approximately 4.3 × 10 10 TIL group. In some implementations, approximately 3 × 10⁻⁶ is applied. 10 Approximately 12×10 10 TIL group. In some implementations, approximately 4 × 10⁻⁶ is applied. 10 Approximately 10×10 10 TIL group. In some implementations, approximately 5 × 10⁻⁶ is applied. 10 Approximately 8×10 10 TIL group. In some implementations, approximately 6 × 10⁶ is applied. 10 Approximately 8×10 10 TIL group. In some implementations, approximately 7 × 10⁻⁶ is applied. 10 Approximately 8×10 10TIL group. In some implementations, the effective therapeutic dose is approximately 2.3 × 10⁻⁶. 10 Approximately 13.7 × 10 10 In some implementations, particularly when the cancer is melanoma, the effective therapeutic dose is approximately 7.8 × 10⁻⁶. 10 TIL group. In some implementations, the effective therapeutic dose is approximately 1.2 × 10⁻⁶. 10 Approximately 4.3 × 10 10 TIL group. In some implementations, the effective therapeutic dose is approximately 3 × 10⁻⁶. 10 Approximately 12×10 10 TIL group. In some implementations, the effective therapeutic dose is approximately 4 × 10⁻⁶. 10 Approximately 10×10 10 TIL group. In some implementations, the effective therapeutic dose is approximately 5 × 10⁻⁶. 10 Approximately 8×10 10 TIL group. In some implementations, the effective therapeutic dose is approximately 6 × 10⁻⁶. 10 Approximately 8×10 10 TIL group. In some implementations, the effective therapeutic dose is approximately 7 × 10⁻⁶. 10 Approximately 8×10 10 TIL group.

[0733] In some embodiments, the amount of TIL provided in the pharmaceutical composition of the present invention is about 1 × 10⁻⁶. 6 2×10 6 3×10 6 4×10 6 5×10 6 6×10 6 7×10 6 8×10 6 9×10 6 1×10 7 2×10 7 3×10 7 4×10 7 5×10 7 6×10 7 7×10 7 8×10 7 9×10 7 1×10 8 2×10 8 3×10 8 4×10 8 5×10 8 6×10 8 7×10 8 8×10 8 9×108 1×10 9 2×10 9 3×10 9 4×10 9 5×10 9 6×10 9 7×10 9 8×10 9 9×10 9 1×10 10 2×10 10 3×10 10 4×10 10 5×10 10 6×10 10 7×10 10 8×10 10 9×10 10 1×10 11 2×10 11 3×10 11 4×10 11 5×10 11 6×10 11 7×10 11 8×10 11 9×10 11 1×10 12 2×10 12 3×10 12 4×10 12 5×10 12 6×10 12 7×10 12 8×10 12 9×10 12 1×10 13 2×10 13 3×10 13 4×10 13 5×10 13 6×10 13 7×10 13 8×10 13 and 9×10 13 In one embodiment, the amount of TIL provided in the pharmaceutical composition of the present invention ranges from 1 × 10⁻⁶. 6 Up to 5×10 6 5×10 6 Up to 1×10 7 1×10 7 Up to 5×10 7 5×10 7 Up to 1×10 81×10 8 Up to 5×10 8 5×10 8 Up to 1×10 9 1×10 9 Up to 5×10 9 5×10 9 Up to 1×10 10 1×10 10 Up to 5×10 10 5×10 10 Up to 1×10 11 5×10 11 Up to 1×10 12 1×10 12 Up to 5×10 12 and 5×10 12 Up to 1×10 13 .

[0734] In some embodiments, the concentration of TIL provided in the pharmaceutical composition of the present invention is less than, for example, 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, or 0.07% of the pharmaceutical c...

Claims

1. A method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, the method comprising: (a) Obtaining the first TIL group from the tumor removed from the patient; (b) The first TIL population was expanded by culturing the first TIL population in a cell culture medium containing IL-2 for about 3-12 days to generate the second TIL population; (c) A second expansion was performed by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, and antigen-presenting cells (APCs) to generate a third TIL population. This second expansion took approximately 7-12 days to obtain the third TIL population, which is a therapeutic TIL population; and (d) Harvesting therapeutic TILs; Specifically, before step (d), the second TIL group before step (c) or the third TIL group after step (c) is exposed to one or more PD-1 specific sd-RNAs. The sd-RNA knocks out PD-1 expression in the exposed population, thereby causing a transient change in the expression of more than one protein in the exposed population; Steps (b) through (d) are carried out over approximately 22 days.

2. The method according to claim 1, wherein, The transient changes in expression include an increase in the expression of more than one protein.

3. The method according to claim 1, wherein, The one or more proteins mentioned include one or more of IL-2, IL-7, IL-10, IL-12, IL-15 and IL-21.

4. The method according to claim 3, wherein, The one or more proteins mentioned include one or more of IL-2, IL-15 and IL-21.

5. The method according to claim 4, wherein, The one or more proteins mentioned include one or more of IL-15 and IL-21.

6. The method according to claim 4, wherein, The one or more proteins include one or more of the membrane-bound forms of IL-2, IL-15, and IL-21.

7. The method according to claim 6, wherein, The one or more proteins include IL-15 in a membrane-bound form.

8. The method according to claim 3, wherein, The one or more proteins mentioned include one or more of IL-2 and IL-12.

9. The method according to claim 8, wherein, The one or more proteins mentioned include IL-2.

10. The method according to claim 8, wherein, The one or more proteins mentioned include IL-12.

11. The method according to claim 8, wherein, The one or more proteins include one or more of membrane-bound IL-2 and membrane-bound IL-12.

12. The method according to claim 1, wherein, The first amplification takes approximately 3 to 11 days.

13. The method according to claim 12, further comprising the following steps: (e) The TIL clusters harvested in step (d) are transferred to an infusion bag.

14. The method according to claim 1, wherein, The second amplification takes place over a period of approximately 7 to 11 days.

15. The method according to claim 1, wherein, The first amplification and the second amplification were each performed separately over a period of approximately 11 days.

16. The method according to claim 13, further comprising the following steps: (f) Cryopreservation of the infusion bag from step (e).

17. The method according to claim 1, wherein, Steps (b), (c), and (d) are performed in a closed system.

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