Tumor-infiltrating lymphocyte (TIL) transduction methods

Abstract

Provided herein are methods for preparing therapeutic populations tumor infiltrating lymphocytes (TILs) by lentiviral transduction. Such methods can comprise: (a) obtaining processed resected tumor product comprising TILs; (b) performing a first expansion by culturing the processed resected tumor product comprising TILs in a first cell culture medium comprising IL-2 to produce a first population of TILs; (c) transducing the TILs with lentivirus; (d) performing a second expansion by culturing the TILs in a second cell culture medium with IL-2, a CD3 agonist, and / or antigen presenting cells (APCs), to produce a second population of TILs; and (d) harvesting the TILs. Also provided are therapeutic populations of TILs obtained by such methods, and methods of treating a subject having a cancer comprising administering such therapeutic populations of TILs to the subject.

Description

TUMOR-INFILTRATING LYMPHOCYTE (TIL) TRANSDUCTION METHODSCROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 509,334, filed June 21, 2023, which is incorporated by reference in its entirety for all purposes.BACKGROUND

[0002] Adoptive T cell therapy utilizing tumor-specific autologous T cells has shown promising results for cancer treatment. Several general approaches have been taken such as the use of naturally occurring tumor reactive or tumor infiltrating lymphocytes (TILs) expanded ex vivo. While TILs utilize their endogenous TCRs to mediate tumor recognition, they can be subject to substantial limitations as tumor cells express very few costimulatory ligands. The ability to induce targeted costimulation of TILs through lentiviral transduction of engineered receptors is beneficial. Improved methods of lentiviral transduction to achieve higher transduction efficiency are needed.SUMMARY

[0003] Provided are methods for preparing therapeutic populations of tumor infiltrating lymphocytes (TILs), therapeutic populations of TILs obtained by such methods, and methods of treating a subject having a cancer.

[0004] In one aspect, provided are methods for preparing a therapeutic population of TILs. Some such methods comprise (a) obtaining a processed resected tumor product comprising TILs; (b) performing a first expansion by culturing the processed resected tumor product comprising TILs in a first cell culture medium comprising IL-2 to produce a first population of TILs; (c) transducing the TILs with lentivirus; (d) performing a second expansion by culturing the TILs in a second cell culture medium with IL-2, a CD3 agonist, and / or antigen presenting cells (APCs), to produce a second population of TILs; and (e) harvesting the TILs.

[0005] In some such methods, the TILs are not treated with an activating agent in step (c) or prior to step (c). In some such methods, the TILs are not treated with an activating agent comprising CD3 and / or CD28 agonists in step (c) or prior to step (c). In some such methods, the TILs are not treated with an activating agent comprising CD3 and CD28 agonists in step (c) orprior to step (c). In some such methods, step (c) does not comprise spinoculation. In some such methods, the TILs are not treated with an enhancer of viral-mediated transduction in step (c) or prior to step (c). In some such methods, the TILs are not treated with a recombinant human fibronectin fragment that is an enhancer of viral-mediated transduction in step (c) or prior to step (c).

[0006] In some such methods, the TILs are transduced about 2 to about 7 days after the start of the first expansion. In some such methods, the TILs are transduced about 2 days to about 3 days after the start of the first expansion. In some such methods, the TILs are transduced about 3 days to about 4 days after the start of the first expansion. In some such methods, the transduction in step (c) is performed at a multiplicity of infection (MOI) of no more than about 5. In some such methods, the transduction in step (c) is performed at a multiplicity of infection (MOI) of about 5 to about 20. In some such methods, the transduction in step (c) is performed at a multiplicity of infection (MOI) of about 5. In some such methods, the TILs are transduced about 2 days to about 3 days after the start of the first expansion or about 3 days to about 4 days after the start of the first expansion, wherein the TILs are not treated with an activating agent in step (c) or prior to step (c), wherein step (c) does not comprise spinoculation, and wherein the TILs are not treated with an enhancer of viral-mediated transduction in step (c) or prior to step (c). In some such methods, the TILs are transduced about 2 days to about 3 days after the start of the first expansion or about 3 days to about 4 days after the start of the first expansion, wherein the TILs are not treated with an activating agent comprising CD3 and CD28 agonists in step (c) or prior to step (c), wherein step (c) does not comprise spinoculation, and wherein the TILs are not treated with a recombinant human fibronectin fragment that is an enhancer of viral-mediated transduction in step (c) or prior to step (c).

[0007] In some such methods, the first expansion and the transducing occurs in an open system, optionally wherein the first expansion and the transducing occurs in 6-well plates or a gas permeable rapid cell expansion system. In some such methods, the first expansion occurs in adherent bags. In some such methods, the adherent bags in the first expansion utilize a treated form of fluorinated ethylene propylene (FEP) film to promote cell and protein adhesion to the surface of the bag. In some such methods, steps (b)-(d) or steps (b)-(e) are in a closed system.

[0008] In some such methods, the lentivirus encodes an exogenous protein, whereby the exogenous protein is expressed in the TILs following transduction. In some such methods, thelentivirus comprises a coding sequence for the exogenous protein operably linked to a myeloproliferative sarcoma virus enhancer, negative control region deleted (MND) promoter. In some such methods, the exogenous protein comprises an exogenous costimulatory receptor. In some such methods, the exogenous costimulatory receptor comprises a chimeric costimulatory antigen receptor (CoStAR) comprising an extracellular binding domain specific for a tumor- associated antigen operatively linked to a transmembrane domain and first and second signaling domains. In some such methods, (i) the first and second signaling domains comprise a CD28 signaling domain, a CD40 signaling domain, or both the CD28 signaling domain and the CD40 signaling domain; or (ii) the first and second signaling domains comprise a ICOS signaling domain, a CD40 signaling domain, or both the ICOS signaling domain and the CD40 signaling domain. In some such methods, the extracellular binding domain comprises an scFv. In some such methods, (1) the tumor-associated antigen comprises folate receptor alpha; (2) the tumor- associated antigen comprises carcinoembryonic antigen (CEA); or (3) the tumor-associated antigen comprises mesothelin. In some such methods, the method further comprises enriching for TILs expressing the exogenous protein.

[0009] In some such methods, step (a) comprises: (i) cry opreserving a resected tumor and disaggregating the cryopreserved tumor after thawing; (ii) disaggregating a resected tumor and cry opreserving the disaggregated tumor; (iii) cryopreserving a resected tumor and processing the tumor into multiple tumor fragments after thawing; or (iv) processing a resected tumor into multiple tumor fragments and cry opreserving the tumor fragments. In some such methods, the method further comprises a step of thawing and washing the processed resected tumor product prior to step (b). In some such methods, the processed resected tumor product is a fresh processed resected tumor product that has not been cryopreserved. In some such methods, step (a) comprises aseptically disaggregating a tumor resected from a subject to prepare the processed resected tumor product, wherein the disaggregation comprise repeated physical pressure applied 120 to 360 times per minute at up to 6 N / cm2in the presence of an enzyme solution, wherein the tumor is disaggregated into a cell suspension so that the processed resected tumor product can be subjected to a cell culture process. In some such methods, the resected tumor is not fragmented prior to disaggregation. In some such methods, the enzyme solution comprises DNase and collagenase. In some such methods, the disaggregation period is 90 minutes or less. In some such methods, the processed resected tumor product is filtered prior to the first expansion, optionallywherein the filtered processed resected tumor product constituents have an average size of less than 200 pm or less than 170 pm.

[0010] In some such methods, the tumor is from a melanoma, a cervical cancer, a non-small cell lung cancer (NSCLC), a head and neck squamous cell carcinoma (HNSCC), a cutaneous squamous cell carcinoma (cSCC), a renal cancer, an ovarian cancer, a colorectal cancer, a gastric cancer, a breast cancer, or a mesothelioma.

[0011] In some such methods, the first cell culture medium comprises between about 300 lU / mL IL -2 and about 3000 HJ / mL IL-2, or wherein the first cell culture medium comprises between about 2000 lU / mL IL-2 and about 3000 lU / mL IL-2, or wherein the first cell culture medium comprises about 3000 lU / mL IL -2. In some such methods, the first cell culture medium comprises fetal bovine serum (FBS). In some such methods, the first cell culture medium does not comprise fetal bovine serum (FBS). In some such methods, the first cell culture medium comprises human AB serum. In some such methods, the TILs are cultured in steps (b) and (c) for from about 10 days to about 13 days or from about 11 days to about 13 days.

[0012] In some such methods, the CD3 agonist is a CD3 agonist antibody, optionally wherein the CD3 agonist antibody is OKT-3. In some such methods, the second cell culture medium comprises between about 300 lU / mL IL-2 and about 3000 lU / mL IL-2, or wherein the second cell culture medium comprises between about 2000 lU / mL IL-2 and about 3000 lU / mL IL-2, or wherein the second cell culture medium comprises about 3000 lU / mL IL-2. In some such methods, the second cell culture medium does not comprise fetal bovine serum (FBS). In some such methods, the second cell culture medium comprises human AB serum. In some such methods, the APCs are artificial APCs or wherein the APCs: (i) are obtained by apheresis; and / or (ii) comprise peripheral blood mononuclear cells (PBMCs). In some such methods, the APCs comprise: (i) fresh PBMCs or cryopreserved PBMCs; and / or (iii) PBMCs from 2 to 10 donors, from 2 to 5 donors, from 3 to 4 donors, or from 3 donors.

[0013] In some such methods, the second expansion comprises a static expansion followed by a dynamic expansion. In some such methods, the static expansion is for about 5 days to about 7 days, and wherein the dynamic expansion is for about 7 days to about 9 days. In some such methods, the static expansion is performed in a working volume of about 1500 mL to about 2500 mL or about 1750 mL to about 2250 mL if the first population of TILs is greater than or equal to about 3xl06cells or between about 3xl06cells and about 17.5xl06cells, and wherein the staticexpansion is performed in a working volume of about 500 mL to about 750 mb if the first population of TILs is less than or equal to about 3xl06cells or between about 0.75xl06cells and about 3xl06cells, optionally wherein the cells that are counted are CD3+ cells. In some such methods, the static expansion is performed in a working volume of about 2000 mL if the first population of TILs is greater than or equal to about 3xl06cells or between about 3xl06cells and about 17.5xl06cells, and wherein the static expansion is performed in a working volume of about 625 mL if the first population of TILs is less than or equal to about 3xl06cells or between about 0.75xl06cells and about 3xl06cells, optionally wherein the cells that are counted are CD3+ cells. In some such methods, the dynamic expansion is performed in a working volume of about 2400 mL to about 4000 mL or about 2800 mL to about 3600 mL if the first population of TILs is greater than or equal to about 3xl06cells or between about 3xl06cells and about 17.5xl06cells, and wherein the dynamic expansion is performed in a working volume of about 500 mL to about 1500 mL or about 750 mL to about 1250 mL if the first population of TILs is less than or equal to about 3xl06cells or between about 0.75xl06cells and about 3xl06cells, optionally wherein the cells that are counted are CD3+ cells. In some such methods, the dynamic expansion is performed in a working volume of about 3200 mL if the first population of TILs is greater than or equal to about 3xl06cells or between about 3xl06cells and about 17.5xl06cells, and wherein the dynamic expansion is performed in a working volume of about 1000 mL if the first population of TILs is less than or equal to about 3xl06cells or between about 0.75xl06cells and about 3xl06cells, optionally wherein the cells that are counted are CD3+ cells. In some such methods, the dynamic expansion comprises perfusion steps at first, second, and third time points, optionally wherein the perfusion comprises removing spent cell culture medium while simultaneously adding fresh cell culture medium in equal parts to maintain a constant culture volume. In some such methods, the first time point is days 16-18, wherein the second time point is days 18-20, and wherein the third time point is days 20-21. In some such methods, the perfusion at the first time point is about 0.6 to about 1 L / day, the perfusion at the second time point is about 1.4 to about 1.8 L / day, and the perfusion at the third time point is about 3 to about 3.4 L / day if the first population of TILs is greater than or equal to about 3x 106cells or between about 3xl06cells and about 17.5xl06cells, and the perfusion at the first time point is about 0.2 to about 0.3 L / day, the perfusion at the second time point is about 0.4 to about 0.6 L / day, and the perfusion at the third time point is about 0.8 to about 1.2 L / day if the first population of TILs isless than or equal to about 3x106cells or between about 0.75x106cells and about 3x106cells, optionally wherein the cells that are counted are CD3+ cells. In some such methods, the perfusion at the first time point is about 0.8 L / day, the perfusion at the second time point is about 1.6 L / day, and the perfusion at the third time point is about 3.2 L / day if the first population of TILs is greater than or equal to about 3xl06cells or between about 3xl06cells and about 17.5xl06cells, and the perfusion at the first time point is about 0.25 L / day, the perfusion at the second time point is about 0.5 L / day, and the perfusion at the third time point is about 1 L / day if the first population of TILs is less than or equal to about 3xl06cells or between about 0.75xl06cells and about 3xl06cells, optionally wherein the cells that are counted are CD3+ cells. In some such methods, the dynamic expansion comprises rocking the cells, optionally at a rocking angle of about 8 degrees.

[0014] In some such methods, the harvesting is performed using a harvest medium comprising at least 2%, at least 3%, at least 4%, or at least 5% human serum albumin (HSA) and PBS, optionally wherein the harvesting is performed using a harvest medium comprising about 5% human serum albumin (HSA) and PBS. In some such methods, step (e) further comprises formulating the TILs with HSA and DMSO, optionally wherein the TIL formulation in step (e) comprises no more than 8%, no more than 7%, no more than 6%, no more than 5%, no more than 4%, no more than 3%, or no more than 2.5% HSA and no more than 9%, no more than 8%, no more than 7%, no more than 6%, or no more than 5% DMSO. In some such methods, the TIL formulation in step (e) comprises about 2.5% HSA and about 5% DMSO. In some such methods, the formulating comprises adding HSA and DMSO to the second population of TILs in a closed system. In some such methods, the harvested TILs are cryopreserved.

[0015] In some such methods, the method further comprises assessing the potency of the harvested TILs. In some such methods, assessing the potency of the harvested TILs comprises: (i) co-culturing a subpopulation of the harvested TILs with engineered cells that activate the subpopulation of the harvested TILs via CD3 or co-culturing the subpopulation of the harvested TILs with autologous tumor cells; (ii) detecting the presence or absence of viable CD2+ T-cells or viable CD3+ T-cells that express one or both of IFN-y and CD107a in the activated TILs; and (iii) determining the percent potency based on the percentage of TILs that express one or both of IFN-y and CD 107a. In some such methods, the detecting comprises flow cytometry gated on viable CD3+ TILs to measure the frequency of expression of one or both of IFN-y and CD107a.In some such methods, the detecting comprises flow cytometry gated on viable CD2+ TILs to measure the frequency of expression of one or both of IFN-y and CD 107a.

[0016] In another aspect, provided are therapeutic populations of TILs obtained by any of the above methods. Some such populations can be cryopreserved.

[0017] In another aspect, provided are methods of treating a subject having a cancer, comprising administering a therapeutic population of TILs obtained by any of the above methods to the subject. In some such methods, the cancer is melanoma, cervical cancer, non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC), cutaneous squamous cell carcinoma (cSCC), renal cancer, ovarian cancer, colorectal cancer, gastric cancer, breast cancer, or mesothelioma.BRIEF DESCRIPTION OF THE FIGURES

[0018] Figure 1 depicts the original ITIL-306 process and process design options for an altered activation / late transduction process and an altered no activation / early transduction process.

[0019] Figure 2 shows the ITIL-306 manufacturing process up to harvest day.

[0020] Figure 3 shows the starting material procurement process.

[0021] Figure 4 shows the ITIL-306 manufacturing process.

[0022] Figure 5 shows a schematic diagram of the TIL production process for frozen donor samples.

[0023] Figure 6 shows the numbers of live, live non-apoptotic, viable CD45+, and viable CD3+ cells within each sample on day 1 (DI) as assessed by flow cytometry.

[0024] Figures 7A-7B show the percentages of CD3+ and CD3-CD56+ cells at D10 of outgrowth. CD3+ cells, expressed as a percentage of CD45+ cells, were consistent among all tested groups in both 6-well plates and G-REX® vessels on D10 of outgrowth, as shown in Figure 7A. Also consistent among all groups were a small percentage of CD3-CD56+ NK cells, shown in Figure 7B.

[0025] Figure 8 shows the percentage of CoStAR+ transduced cells on D10 of outgrowth.

[0026] Figures 9A-9C show the percentage of transduced CD3+, CD4+, and CD8+ cells, respectively, on D10 of outgrowth. The percentage of transduced CD3+, CD4+, and CD8+ cells, respectively, was generally consistent across T cell type, with some variability restricted to thedonor 9834 sample, where more CD4+ cells were transduced than CD8+ cells (Figures 5B and 5C, respectively).

[0027] Figures 10A-10D show the percentage of CD4+, CD8+, CD4+CD8+ double positive (DP), and CD4-CD8- double negative (DN) cells in each transduction and outgrowth condition at DIO of outgrowth. There was no difference between the percentages of CD4+, CD8+, DP, and DN within each donor population for any of the transduction or outgrowth conditions.

[0028] Figure 11 shows a plot of the percentage of CoStAR+CD3+ cells versus the fold expansion of CD3+ cells for all experimental groups and samples. Regression analysis indicated there was a positive correlation between the percentage of transduced CoStAR+CD3+ cells with the fold expansion of CD3+ cells across all donors and experimental groups.

[0029] Figure 12 shows a schematic diagram of the TIL production process for fresh lung and renal donor samples.

[0030] Figures 13A-13C show CD3+ cell counts throughout the TIL production process. Figure 13A shows the cell counts during outgrowth and indicates the number of cells used in each experimental group for subsequent experiments. Figures 13B and 13C show the increase in CD3+ cells from pre-rapid expansion (REP) to post-REP.

[0031] Figures 14A-14C show flow cytometric analysis of CoStAR+ cells pre-REP (D13) and post-REP (D25). CoStAR+CD3+ cells are shown in Figure 14A, CoStAR+CD4+ cells are shown in Figure 14B, and CoStAR+CD8+ cells are shown in Figure 14C.

[0032] Figure 15 shows stability of transduction from pre-REP to post-REP by flow cytometric analysis of CoStAR+CD3+ in both lung and renal cells.

[0033] Figure 16 shows a comparison of the percentage of transduced cells with viral copy number (VCN) post-REP.

[0034] Figures 17A-17D show the percentage of CD4+, CD8+, CD4+CD8+ double positive (DP), and CD4-CD8- double negative (DN) cells in each MOI group. There were no differences in the proportion of CD4+, CD8+, DP, and DN cells between the various MOI groups pre- or post-REP.

[0035] Figures 18A-18D show the percentage of CD3+ and CD3- cells in lung and renal samples pre- and post-REP. A significant reduction was observed in the percentage of CD3- cells post-REP in the renal samples (Figure 18D vs. 18C). No difference in the percentage of CD3- cells post-REP was observed in the lung samples (Figures 18A and 148).

[0036] Figure 19 shows contour plots of the FOLR1 expression as detected by flow cytometry for the lung (left) and renal (right) samples. Anti-FOLRl antibody conjugated with PE was used to stain the cryopreserved digest samples. Both samples had detectable levels of FORL1 in the CD45- cell population of the digest ranging from 17-44% suggesting that recognition of the FORL1 on the tumor cells by the CoStAR will likely result in enhanced CoStAR activity given that the TILs recognize the tumor cells through their endogenous TCRs.

[0037] Figures 20A-20B show the functionality of the lentivirally transduced cells in cocultures with autologous digest as detected by the measurement of secreted IFNy. Nontransduced and varying MOI transduced CoStAR TILs were cocultured with autologous tumor digest for 24 hours, following which supernatants were collected and analyzed for cytokine secretion using an MSD immunoassay. TILs and digest alone conditions were used as negative controls. The graphs represent measured concentrations of IFNy in coculture supernatants for the lung (Figure 20A) and renal (Figure 20B) samples. A significant enhancement of the anti-tumor reactivity was observed in the cells transduced with CoStAR when compared to the MOCK transduced cells. The increasing MOI did not further result in additional enhancement of responsiveness.

[0038] Figure 21 shows the effect of 2d vs Id transduction on % transduced CD3+ cells at harvest (rapid expansion phase day 15 (REPD15)). Each point on the graph represents an individual donor and is an average of N=2 biological replicate cultures per donor per culture condition. Gray-filled circles represent Id lentiviral transduction, and black-filled circles represent 2d lentiviral transduction.

[0039] Figures 22A-22B show the effect of activation on culture viability. In Figure 22A and 22B, circle symbols represent Not Activated Culture and triangle symbols represent Activated Culture. Figure 22A shows the effect of activation on culture viability at the end of outgrowth (OG) (donors 1-3). Each point represents an average of N=2 biological replicate cultures per donor per condition. Analysis performed - two-way ANOVA with multiple comparisons; matched by donor. Line at mean. Open symbols (Donor 1), gray-filled symbols (Donor 2), and black-filled symbols (Donor 3). Figure 22B shows the effect of activation on culture viability during OG (donors 4-7). Each point represents an average of N=2 biological replicate cultures per donor per condition. Analysis performed - two-way ANOVA with multiplecomparisons; matched by donor. Line at mean +SD. Open symbols (Donor 4), light gray-fdled symbols (Donor 5), dark-gray-fdled symbols (Donor 6) and black-filled symbols (Donor 7).

[0040] Figures 23A-23B show the effect of activation on culture growth during OG. In Figure 23A-23B, circle symbols represent Not Activated Culture and triangle symbols represent Activated Culture. Figure 23A shows the effect of activation on culture growth (fold expansion (FE)) during OG (donors 1-3). Fold expansion was calculated by dividing the total number of cells in each culture on outgrowth day 15 (OGD15) by the number of cells seeded into each culture on outgrowth day 1 (OGD1). Each point represents an average of N=2 biological replicate cultures per donor per condition. Analysis performed - two-way ANOVA with multiple comparisons; matched by donor. Line at mean. Open symbols (Donor 1), gray-filled symbols (Donor 2), and black-filled symbols (Donor 3). Figure 23B shows the effect of activation on culture growth (FE) during OG (donors 4-7). Fold expansion was calculated by dividing the total number of cells in each culture on OGD15 by the number of cells seeded into each culture on OGD1. Each point represents an average of N=2 biological replicate cultures per donor per condition. Analysis performed - two-way ANOVA with multiple comparisons; matched by donor. Line at mean +SD. Open symbols (Donor 4), light gray-fdled symbols (Donor 5), dark- gray-fdled symbols (Donor 6) and black-filled symbols (Donor 7).

[0041] Figures 24A-24B show the effect of activation on culture growth (FE) during REP. In Figure 24A-24B, circle symbols represent Not Activated Culture and triangle symbols represent Activated Culture. Figure 24A shows the effect of activation on culture growth (FE) during REP (donors 1-3). Fold expansion was calculated by dividing the total number of cells in each culture on REPD15 by the number of cells seeded into each culture on rapid expansion phase day 1 (REPD1). Each point represents an average of N=2 biological replicate cultures per donor per condition. Analysis performed - two-way ANOVA with multiple comparisons; matched by donor. Line at mean. Open symbols (Donor 1), gray-fdled symbols (Donor 2), and black-fdled symbols (Donor 3). Figure 24B shows the effect of activation on culture growth (FE) during REP (donors 4,6,7). Fold expansion was calculated by dividing the total number of cells in each culture on REPD15 by the number of cells seeded into each culture on REPD1. Each point represents an average of N=2 biological replicate cultures per donor per condition. Analysis performed - paired two-tailed t-test. Open symbols (Donor 4), gray-fdled symbols (Donor 6), and black-fdled symbols (Donor 7).

[0042] Figures 25A-25B show the effect of activation on transduction (TD). Tn Figure 25A- 25B, circle symbols represent Not Activated Culture and triangle symbols represent Activated Culture. Figure 25A shows effect of activation on TD (donors 1-3). Each point represents an average of N=2 biological replicate cultures per donor per condition. Analysis performed - two- way ANOVA with multiple comparisons; matched by donor. Line at mean. Open symbols (Donor 1), gray-filled symbols (Donor 2), and black-filled symbols (Donor 3). Figure 25B shows the effect of activation on TD (donors 4,6,7). Each point represents an average of N=2 biological replicate cultures per donor per condition. Analysis performed - paired two-tailed t- test. Open symbols (Donor 4), gray-filled symbols (Donor 6), and black-filled symbols (Donor 7).DEFINITIONS

[0043] Compositions or methods “comprising” or “including” one or more recited elements may include other elements not specifically recited. For example, a composition that “comprises” or “includes” a protein may contain the protein alone or in combination with other ingredients. The transitional phrase “consisting essentially of’ means that the scope of a claim is to be interpreted to encompass the specified elements recited in the claim and those that do not materially affect the basic and novel characteristic(s) of the claimed invention. Thus, the term “consisting essentially of’ when used in a claim of this invention is not intended to be interpreted to be equivalent to “comprising.”

[0044] “Optional” or “optionally” means that the subsequently described event or circumstance may or may not occur and that the description includes instances in which the event or circumstance occurs and instances in which the event or circumstance does not.

[0045] Designation of a range of values includes all integers within or defining the range, and all subranges defined by integers within the range. For example, 5-10 nucleotides is understood as 5, 6, 7, 8, 9, or 10 nucleotides, whereas 5-10% is understood to contain 5% and all possible values through 10%.

[0046] When “at least,” “up to,” “more than,” “no more than,” “less than,” or other similar language modifies a number, it can be understood to modify each number in the series.

[0047] As used herein, “no more than” or “less than” is understood as the value adjacent to the phrase and logical lower values or integers, as logical from context, to zero. When “no morethan” or “less than” is present before a series of numbers or a range, it is understood that each of the numbers in the series or range is modified.

[0048] Unless otherwise apparent from the context, the term “about” encompasses values ± 5% of a stated value. In certain embodiments, the term “about” is understood to encompass tolerated variation or error within the art, e.g., 2 standard deviations from the mean, or the sensitivity of the method used to take a measurement, or a percent of a value as tolerated in the art, e.g., with age. When “about” is present before the first value of a series, it can be understood to modify each value in the series.

[0049] The term “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).

[0050] The term “or” refers to any one member of a particular list and also includes any combination of members of that list.

[0051] The singular forms of the articles “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a protein” or “at least one protein” can include a plurality of proteins, including mixtures thereof.

[0052] Statistically significant means p <0.05.

[0053] ‘Media” or “medium” means various solutions known in the art of cell culturing, cell handling and stabilization used to reduce cell death, including but not limited to one or more of the following media Organ Preservation Solutions , selective lysis solutions, PBS, DMEM, HBSS, DPBS, RPMI, Iscove’s medium, X-VIVO™, Lactated Ringer's solution, Ringer's acetate, saline, PLASMALYTE™ solution, crystalloid solutions and IV fluids, colloid solutions and IV fluids, five percent dextrose in water (D5W), Hartmann’s Solution. The media can be standard cell media like the above mentioned-media or special media for primary human cell culture (e.g., for endothelia cells, hepatocytes, or keratinocytes) or stem cells (e.g., dendritic cell maturation, hematopoietic expansion, keratinocytes, mesenchymal stem cells or T cell expansion). The media may have supplements or reagents well known in the art, such as albumins and transport proteins, amino acids and vitamins, antibiotics, attachments factors, growth factors and cytokines, hormones, metabolic inhibitors or solubilizing agents. Various media are commercially available e. g. from ThermoFisher Scientific or Sigma-Aldrich.

[0054] By ‘ ‘tumor infiltrating lymphocytes” or “TILs” is meant a population of cells originally obtained as white blood cells that have left the bloodstream of a subject and migrated into a tumor. TILs include, but are not limited to, CD8+cytotoxic T cells (lymphocytes), Thl and Thl7 CD4+T cells, natural killer cells, dendritic cells, and Ml macrophages. TILs include both primary and secondary TILs. “Primary TILs” are those that are obtained from patient tissue samples as outlined herein (sometimes referred to as “freshly harvested”), and “secondary TILs” are any TIL cell populations that have been 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 can include genetically modified TILs. TILs can generally be defined either biochemically, using cell surface markers, or functionally, by their ability to infiltrate tumors and effect treatment. TILs can be generally categorized by expressing one or more of the following biomarkers: CD4, CD8, TCR a£, CD27, CD28, CD56, CCR7, CD45Ra, CD62L, CD95, PD-1, and CD25. Additionally, and alternatively, TILs can be functionally defined by their ability to infiltrate solid tumors upon reintroduction into a patient. TILs may further be characterized by potency. For example, TILs may be considered potent or functional if in response to TCR engagement they produce, for example, interferon (IFN) release 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. Potency can also be assessed by intracellular staining for CD107a and INF-y or CD137, CD107a, INF-y, and TNF-a following TCR-induced stimulation by flow cytometry. See, e.g., WO 2022 / 271881, herein incorporated by reference in its entirety for all purposes.DETAILED DESCRIPTIONI. Overview

[0055] Major limiting factors for development of cell therapy modalities in solid tumors include clonal heterogeneity and the related lack of universally expressed tumor-specific antigens. As the only truly polyclonal cell product in advanced development, tumor-infiltrating lymphocytes (TILs) encompass the high diversity available with an unrestricted T-cell receptor repertoire (TCR), thereby offering broad diversity of anti-tumor reactivity and demonstrate effective clinical efficacy. The suppressive tumor microenvironment is characterized by high expression of coinhibitory receptors and poor costimulation. To overcome poor endogenous costimulation, we have developed the chimeric costimulatory antigen receptor (CoStAR)platform which provides costimulation via a chimeric CD28.CD40 costimulatory module upon engagement with tumour associated antigens such as human folate receptor 1, which is overexpressed in several malignancies including lung, ovarian, and renal cancer. See, e.g., WO 2020 / 152451, WO 2022 / 016112, and WO 2022 / 016114, each of which is herein incorporated by reference in its entirety for all purposes.

[0056] Here, we provide methods for efficient viral (e.g., lentiviral) transduction during the process of preparing therapeutic populations of TILs from resected tumor products (see, e.g., WO 2021 / 123832 and WO 2022 / 271847, each of which is herein incorporated by reference in its entirety for all purposes). In some such methods, a high viral or lentiviral transduction efficiency is achieved despite not treating the TILs with an activating agent (e.g., TransAct or Dynabeads) prior to or during transduction, not using spinoculation during transduction, and / or not treating the TILs with an enhancer of viral-mediated transduction (e.g., RetroNectin) prior to or during transduction.II. Methods of Preparing Therapeutic Populations of Immune Cells or TILs

[0057] Provided herein are methods for preparing therapeutic populations of immune cells such as tumor infiltrating lymphocytes (TILs). Such methods can comprise, for example: (a) transducing the immune cells with a virus (e.g., lentivirus); (b) culturing the immune cells; and (c) harvesting the cultured immune cells. For TILs, such methods can comprise, for example: (a) obtaining a processed resected tumor product comprising TILs; (b) performing a first expansion by culturing the processed resected tumor product comprising TILs in a first cell culture medium comprising IL-2 to produce a first population of TILs; (c) transducing the TILs with virus (e.g., lentivirus); (d) performing a second expansion by culturing the TILs in a second cell culture medium with IL-2, a CD3 agonist, and / or antigen presenting cells (APCs), to produce a second population of TILs; and (e) harvesting the TILs.

[0058] The immune cells can be any suitable type of immune cells. In some embodiments, the immune cells can be lymphocytes (B cells or T cells), natural killer (NK) cells, dendritic cells, macrophages, monocytes, mast cells, basophils, eosinophils, or neutrophils. In some embodiments, the immune cells are TILs. In some embodiments, the immune cells are T cells. In some embodiments, the immune cells are NK cells.

[0059] The virus or lentivirus can be, for example, a viral or lentiviral vector encoding an exogenous receptor. The exogenous receptor can be any suitable exogenous receptor. In some embodiments, the exogenous receptor is an exogenous T cell receptor (TCR) as described elsewhere herein. In some embodiments, the exogenous receptor is a chimeric antigen receptor (CAR) as described elsewhere herein. In some embodiments, the exogenous receptor is an exogenous costimulatory receptor (CoStAR) as described elsewhere herein.

[0060] Also provided are therapeutic populations of immune cells (e.g., TILs) obtained by such methods, and methods of treating a subject having a cancer comprising administering such therapeutic populations of immune cells (e.g., TILs) to the subject.A. Obtaining Immune Cells or Obtaining Processed Resected Tumor Product Comprising TILs

[0061] The methods described herein can begin with procurement of immune cells. The immune cells can be autologous (e.g., from the subject who will be treated with the immune cells) or can be from another donor (allogeneic). The immune cells can be from any suitable sources. The immune cells can be, for example, from blood (e.g., peripheral blood or umbilical cord blood) from a subject. In the case of TILs, the methods described herein can begin with tumor procurement. Following resection of the tumor at a clinical site, the starting material can undergo digestion and filtration steps. The tumor digest may either be cryopreserved or processed fresh.

[0062] In some embodiments, obtaining a processed resected tumor product comprising TILs can comprise cryopreserving a resected tumor and disaggregating the cryopreserved tumor (e.g., after thawing). In other embodiments, obtaining a processed resected tumor product comprising TILs can comprise disaggregating a resected tumor and cry opreserving the disaggregated tumor. In other embodiments, obtaining a processed resected tumor product comprising TILs can comprise cry opreserving a resected tumor and processing the tumor into multiple tumor fragments (e.g., after thawing). In other embodiments, obtaining a processed resected tumor product comprising TILs can comprise processing a resected tumor into multiple tumor fragments and cry opreserving the tumor fragments. Obtaining a processed resected tumor product comprising TILs can also comprise a step of thawing and washing the processed resected tumor product. In some embodiments, the method further comprises a step of thawing thecryopreserved resected tumor or cryopreserved resected tumor product before subsequent processing. In some embodiments, the method further comprises a step of washing the resected tumor or resected tumor product before subsequent processing. In some embodiments, the processed resected tumor product is a fresh processed resected tumor product that has not been cryopreserved.

[0063] Obtaining a processed resected tumor product comprising TILs can in some embodiments comprise aseptically disaggregating a tumor resected from a subject to prepare the processed resected tumor product. The disaggregation can comprise, for example, repeated physical pressure (e.g., applied 120 to 360 times per minute at up to 6 N / cm2) in the presence of an enzyme solution, wherein the tumor is disaggregated into a cell suspension so that the processed resected tumor product can be subjected to a cell culture process. In some methods, the enzyme solution comprises DNase and collagenase. In some methods, the disaggregation period can be, for example, 90 minutes or less.

[0064] In some embodiments, the resected tumor is not fragmented prior to disaggregation. In some methods, the processed resected tumor product is filtered prior to the first expansion. For example, the filtered processed resected tumor product constituents can, in some embodiments, have an average size of less than 200 pm or less than 170 pm. The tumor can be from any type of cancer. In some embodiments, the tumor is from a melanoma, a cervical cancer, a non-small cell lung cancer (NSCLC), a head and neck squamous cell carcinoma (HNSCC), a cutaneous squamous cell carcinoma (cSCC), a renal cancer, an ovarian cancer, a colorectal cancer, a gastric cancer, a breast cancer, or a mesothelioma.

[0065] Tumors can be surgically resected and then trimmed to remove one or more of: visibly necrotic tissue, visibly heathy or noncancerous tissue, and excess blood. The trimmed tumor can then be weighed, placed into a sterile bag, and then heat sealed.

[0066] A patient tumor sample may be obtained using methods known in the art, generally via surgical resection, needle biopsy, or other means for obtaining a sample that contains a mixture of tumor and TIL cells. In general, the tumor sample may be from any solid tumor, including primary tumors, invasive tumors or metastatic tumors. “Solid tumor” refers to an abnormal mass of tissue that usually does not contain cysts or liquid areas. Solid tumors may be benign or malignant. The term “solid tumor cancer” refers to malignant, neoplastic, or cancerous solid tumors. Solid tumor cancers include, but are not limited to, sarcomas, carcinomas, andlymphomas, such as cancers of the lung, breast, prostate, colon, rectum, and bladder. The tissue structure of solid tumors includes interdependent tissue compartments including the parenchyma (cancer cells) and the supporting stromal cells in which the cancer cells are dispersed and which may provide a supporting microenvironment. 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 carcinoma. The solid tumor may be of any cancer type, including, but not limited to, breast, ovary, cervical, pancreatic, prostate, colorectal, lung, brain, renal, stomach, and skin (including but not limited to squamous cell carcinoma, basal cell carcinoma, and melanoma). In some embodiments, TILs are obtained from melanoma tumors, as these have been reported to have particularly high levels of TILs. In some embodiments, TILs are obtained from cervical cancer, non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC), cutaneous squamous cell carcinoma (cSCC), renal cancer, ovarian cancer, colorectal cancer, gastric cancer, breast cancer, or mesothelioma tumors. The tumor sample may also be a liquid tumor, such as a tumor obtained from a hematological malignancy. The term “hematological malignancy” refers to mammalian cancers and tumors of the hematopoietic and lymphoid tissues, including but not limited to tissues of the blood, bone marrow, lymph nodes, and lymphatic system. Hematological malignancies are also referred to as “liquid tumors.” Hematological malignancies include, but are not limited to, acute lymphoblastic leukemia (ALL), chronic lymphocytic lymphoma (CLL), small lymphocytic lymphoma (SLL), acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), acute monocytic leukemia (AMoL), Hodgkin’s lymphoma, and non-Hodgkin’s lymphomas. The term “B cell hematological malignancy” refers to hematological malignancies that affect B cells. In some embodiments, the term “liquid tumor” refers to an abnormal mass of cells that is fluid in nature. Liquid tumor cancers include, but are not limited to, leukemias, myelomas, and lymphomas, as well as other hematological malignancies. TILs obtained from liquid tumors may also be referred to herein as marrow infdtrating lymphocytes (MILs).

[0067] Tumor material (e.g., metastatic tumor material) can be surgically removed using standard surgical practice within a surgical operating room. Prior to disaggregation, extraneous material can be removed (i.e., non-tumor material as defined macroscopically). The tumor material can be transferred into a sterile bag. Methods may further comprise tumor startingmaterial acceptance testing, which may involve one or more of the following processes. First, the source tissue can be confirmed to be tumor material. Second, a representative sample of the disaggregated tissue can be assessed for microbial load and where present antibiotic sensitivities defined (manufacturing may be performed at risk with antibiotics) but final material should be negative for microbial growth. Third, quantity and viability of TIL and tumor cells can be assessed by flow cytometry.

[0068] In some embodiments, the methods described herein can comprise aseptically disaggregating a tumor resected from a subject thereby producing a disaggregated tumor, wherein the resected tumor is sufficiently disaggregated if it can be cryopreserved without cell damage. In some embodiments, a programmable processor of a semi-automatic device may control disaggregation enabling the surfaces within disaggregation flexible containers to mechanically crush and shear the solid tissue (see, e.g., WO 2018 / 130845, herein incorporated by reference in its entirety for all purposes). Disaggregation surfaces may be controlled, for example, by mechanical pistons. In some embodiments, disaggregation can comprise mechanical disaggregation and / or enzymatic digestion.

[0069] For enzymatic digestion, enzymes such as DNase I or collagenase can be used. In a specific example, an enzyme digest medium is composed of PBS containing calcium chloride, DNase I, and collagenase. In some embodiments, the tumor and digest media are subjected to controlled, mechanical compression to facilitate mechanical disaggregation and / or enzymatic digestion. In some embodiments, the tumor is digested to generate a homogeneous cell suspension. In some embodiments, the tumor digest material can then be filtered.

[0070] For enzymatic digestion, a cell suspension (e.g., containing both T cells and tumor cells) can be generated from the resected tumor using an enzyme mixture of DNase I and Collagenase (Type IV). The combination of the repeated mechanical compression can expose additional surfaces for the enzymes to access and for the enzymatic reaction to speed up the process of turning a solid tissue into a cell suspension prior to optional cry opreservation. In some embodiments, upon completion of the disaggregation step, a DMSO based cryoprotectant can be added just prior to a controlled rate freezing cycle. In some embodiments, the enzymatic breakdown of the solid tissue may be by the selection and provision of one or more media enzyme solutions such as collagenase, trypsin, lipase, hyaluronidase, deoxyribonuclease,Liberase Hl, pepsin, or any mixture thereof. Enzymatic digestion of the resected tumor can occur in the disaggregation flexible containers of a semi-automatic device.

[0071] Where the disaggregation process is being supplemented with enzymatic digestion, the media formulation for enzymatic digestion can be supplemented with enzymes that aid in protein breakdown causing the cell to cell boundaries to break down. Various liquid formulations known in the art of cell culturing or cell handling can be used as the liquid formulation used for cell disaggregation and enzymatic digestion of solid tissues, including but not limited to one or more of the following media: Organ Preservation Solutions, selective lysis solutions, PBS, DMEM, HBSS, DPBS, RPMI, Iscove’s medium, XVIVO™, AIM-V™, Lactated Ringer’s solution, Ringer’s acetate, saline, PLASMALYTE™ solution, crystalloid solutions and IV fluids, colloid solutions and IV fluids, five percent dextrose in water (D5W), Hartmann’s Solution, DMEM, HBSS, DPBS, RPMI, AIM-V™, Iscove’s medium, XVIVO™, each of which can be optionally supplemented with one or more additional cell supporting factors, e.g., fetal calf serum, human serum, serum substitutes, or other nutrients or cytokines to aid in cell recovery, survival, and / or depletion of specific cell types. The media can be standard cell media like the above-mentioned media or special media, for example, media for culture of primary human cells (e.g., for endothelia cells, hepatocytes or keratinocytes) or stem cells (e.g., dendritic cell maturation, hematopoietic expansion, keratinocytes, mesenchymal stem cells or T cells). The media may have supplements or reagents well known in the art, e.g., albumins, transport proteins, amino acids and vitamins, metal-ion(s), antibiotics, attachments factors, de-attachment factors, surfactants, growth factors and cytokines, hormones, and / or solubilizing agents. Various media are commercially available from ThermoFisher, Lonza, or Sigma-Aldrich or similar media manufacturers and suppliers.

[0072] Processing of tumor material using dissection, enzymatic digestion, and homogenization can produce a single cell suspension of TILs which can be directly cryopreserved to stabilize the starting material for subsequent processing via the first expansion of the cell suspension of TILs in interleukin-2 (IL-2) to obtain a first population of TILs.

[0073] The methods disclosed herein can also comprise the step of cry opreserving the disaggregated tumor (e.g., the cell suspension). Cryopreserving the disaggregated tumor can be carried out on the same day as carrying out the step of aseptically disaggregating a tumor resected from a subject thereby producing a disaggregated tumor, wherein the resected tumor issufficiently disaggregated if it can be cryopreserved without cell damage. For example, cryopreserving can be carried out about 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, or 22 hours following the step of disaggregating the tumor. Cryopreservation of the disaggregated tumor (e.g., as a single cell suspension obtained from the enzymatic disaggregation in the disaggregation module of the semi-automatic device) can be carried out by cooling or maintaining the suspension at a temperature between 8 °C and at least -80 °C. Disaggregation can be as quick as 5 minutes or 45 minutes to 1 hour, and the cryopreservation can be as quick as 60 minutes or up to 150 minutes. In some embodiment, the methods include storing the cryopreserved disaggregated tumor.

[0074] Depending on whether disaggregation includes enzymatic disaggregation, and what is the optimal temperature of enzymatic digestion for a given enzyme, enzyme concentration and tissue type, temperatures at the start of cryopreservation include, without limitation, 40°C, 39°C, 38°C, 37°C, 36°C, 35°C, 34°C, 33°C, 32°C, 31°C, 30°C, 29°C, 28°C, 27°C, 26°C, 25°C, 24°C, 23°C, 22°C, 21°C, and 20°C, i.e., temperatures ranging from a mammalian body temperature to room temperature, and further include temperatures below room temperature, including but not limited to refrigeration temperatures such as, without limitation, 19°C, 18°C, 17°C, 16°C, 15°C, 14°C, 13°C, 12°C, 11°C, 10°C, 9°C, 8°C, 7°C, 6°C, 5°C, 4°C, 3°C, and 2°C. Target temperatures for cryogenic cooling include, without limitation, -60°C, -65°C, -70°C, -75°C, -80°C, -85°C, - 90°C, and temperatures in between as well as colder temperatures down to the temperature of liquid nitrogen vapor storage (-195.79°C). In some embodiments, the methods described herein are designed or programmed to minimize the time from physiological temperature or digestion temperature to cryostorage temperature. Exemplary programmed cooling rates include, without limitation, -0.5°C / min, -l°C / min, -1.5°C / min, -2°C / min, or -2.5°C / min. The cooling rates are program targets and may vary over a cooling cycle. The cooling rates may vary, for example by ± 0.1°C / min, ± 0.2°C / min, ± 0.3°C / min, ± 0.4°C / min, or ± 0.5°C / min. In some embodiments, the cry opreservation temperature is -80°C ± 10°C and the device is programmed to reduce temperature by l°C / min or 1.5°C / min or 2°C / min or l°C / min ± 0.5°C / min or 1.5°C / min ± 0.5°C / min or 2°C / min ± 0.5°C / min.

[0075] Cryopreservation may be employed throughout TIL manufacture including but not limited to, (i) cryopreservation of a processed tumor sample for use at a later time by thawing and TIL expansion, (ii) cryopreservation of a processed tumor sample for use at a later time by thawing and use of tumor cells, (iii) cry opreservation of a processed tumor sample for lateranalysis, (iv) cryopreservation of a pre-REP expansion culture for use at a later time by thawing and REP expansion, (v) cryopreservation of a portion of a pre-REP expansion culture (such as but not limited to a predetermined portion or to excess cells above a predetermined amount from a pre-REP culture) for use at a later time by thawing and REP expansion, (vi) cry opreservation of a post-REP culture for use at a later time in a subsequent pre-REP expansion or REP, or (vii) cry opreservation of a post-REP culture for use at a later time by thawing and administering to a subject.

[0076] Cryopreserved TIL intermediates, products, and samples may be washed upon thawing prior to subsequent processes or prior to use. In some embodiments, cryopreserved tumor digests are thawed, diluted in growth media, and washed one or more times. In some embodiments, washing comprises centrifugation and growth media change. In some embodiments, washing comprises fdtration and growth media change. In some embodiments, wash media is mixed into then withdrawn from a closed TIL container, such as a bag or dish and replaced by fresh media. The wash may be automated in a closed system or containers for TILs, comprising wash media, and other components interconnected by tubes and valves.

[0077] In certain embodiments, to increase proportions of TILs, TIL subsets, TIL viability, and / or TIL potency, upon thawing, dilution, and optional wash, cryopreserved TILs can be held in culture prior to outgrowth (i.e., pre-REP expansion). In certain embodiments, the hold time is chosen to maximize total viable cells or fold expansion measured by CD3. In certain embodiments, the hold time may comprise or consist of from 2 to 4 hours or from 4 to 6 hours or from 6 to 9 hours or from 9 to 12 hours or from 12 to 18 hours or from 18 to 24 hours.B. Outgrowth of Immune Cells or TILs

[0078] The methods disclosed herein can comprise a step of culturing the immune cells. For example, the methods disclosed herein can also comprise the step of performing a first expansion by culturing the immune cells or disaggregated tumor in a cell culture medium (e.g., comprising IL-2) to produce a first population of immune cells (e.g., TILs). The cells resulting from the steps described above can be cultured in medium containing IL-2 under conditions that favor the growth of immune cells (e.g., TILs) over tumor and other cells. In some embodiments, the medium can comprise human AB serum. This primary cell population can be cultured for a period of days, generally from 3 to 14 days, resulting in a bulk immune cell (e.g., TIL)population (e.g., about 1x108bulk immune cells (e.g., TILs)). In some embodiments, this primary cell population is cultured for a period of 7 to 14 days, resulting in a bulk immune cell (e.g., TIL) population. In some embodiments, this primary cell population is cultured for a period of 10 to 14 days, resulting in a bulk immune cell (e.g., TIL) population. In some embodiments, this primary cell population is cultured for a period of about 11 days, resulting in a bulk immune cell (e.g., TIL) population.

[0079] In some embodiments, the culture medium comprises between about 300 lU / mL and about 3000 lU / mL of IL-2, or the culture medium comprises between about 2000 lU / mL and about 3000 lU / mL IL-2. In some methods, the culture medium comprises fetal bovine serum (FBS). In some methods, the culture medium does not comprise FBS. In some methods, the culture medium comprises human AB serum. In some such methods, the outgrowth / expansion is from about 10 to about 13 days or from about 11 to about 13 days.

[0080] In some embodiments, expansion of immune cells (e g., TILs) may be performed using an initial bulk immune cell (e.g., TIL) expansion step as described herein, followed by a second expansion (including rapid expansion protocol (REP) steps and followed by restimulation REP steps) as described below.

[0081] In some embodiments, resuspended cryopreserved disaggregated tumor tissue is cultured in serum containing IL -2 under conditions that favor the growth of immune cells (e.g., TILs) over tumor and other cells. In some embodiments, the tumor digests are incubated in medium comprising inactivated human AB serum (or, in some cases, as outlined herein, in the presence of an artificial antigen-presenting (aAPC) cell population). In some embodiments, the culture medium comprises IL-2 or a variant thereof. In some embodiments, the IL is recombinant human IL-2 (rhIL-2). In some embodiments, the culture medium comprises about 10,000 lU / mL of IL-2, about 9,000 lU / mL of IL-2, about 8,000 lU / mL of IL-2, about 7,000 lU / mL of IL-2, about 6000 lU / mL of IL-2, or about 5,000 lU / mL of IL-2. In some embodiments, the culture medium comprises about 2,000 lU / mL to about 3,000 lU / mL of IL-2. In some embodiments, the culture medium comprises about 3000 lU / mL of IL-2. In some embodiments, the culture medium comprises about 2000 lU / mL of IL-2. In an embodiment, the culture medium comprises about 1000 lU / mL, about 1500 lU / mL, about 2000 lU / mL, about 2500 lU / mL, about 3000 lU / mL, about 3500 lU / mL, about 4000 lU / mL, about 4500 lU / mL, about 5000 lU / mL, about 5500 lU / mL, about 6000 lU / mL, about 6500 lU / mL, about 7000 lU / mL, about 7500 lU / mL, or about8000 lU / mL of IL-2. In some embodiments, the culture medium comprises between 1000 lU / mL and 2000 lU / mL or between 2000 lU / mL and 3000 lU / mL of IL-2. The term “IL-2” (also referred to herein as “IL2”) refers to the T cell growth factor known as interleukin-2, and includes all forms of IL-2 including human and mammalian forms, conservative amino acid substitutions, glycoforms, biosimilars, and variants thereof. IL-2 is described, e.g., in Nelson (2004) Immunol. 172:3983-88 and Malek (2008) Annu. Rev. Immunol. 26:453-79, each of which is herein incorporated by reference in its entirety for all purposes. For example, the term IL-2 encompasses human, recombinant forms of IL-2 such as aldesleukin (PROLEUKIN, available commercially from multiple suppliers in 22 million IU per single use vials), as well as the form of recombinant IL-2 commercially supplied by CellGenix, Inc., Portsmouth, N.H., USA (CELLGRO GMP) or ProSpec-Tany TechnoGene Ltd., East Brunswick, N.J., USA (Cat. No. CYT-209-b) and other commercial equivalents from other vendors. Aldesleukin (des-alanyl- 1, serine-125 human IL-2) is a nonglycosylated human recombinant form of IL-2 with a molecular weight of approximately 15 kDa. The term IL-2 also encompasses pegylated forms of IL-2, as described herein, including the pegylated IL2 prodrug NKTR-214, available from Nektar Therapeutics, South San Francisco, Calif., USA. NKTR-214 and pegylated IL-2 suitable for use is described in US 2014 / 0328791 Al and WO 2012 / 065086 Al, each of which is herein incorporated by reference in its entirety for all purposes. Alternative forms of conjugated IL-2 suitable for use are described in US Pat. Nos. 4,766,106, 5,206,344, 5,089,261 and 4,902,502, each of which is herein incorporated by reference in its entirety for all purposes. Formulations of IL-2 suitable for use are described in US 6,706,289, herein incorporated by reference in its entirety for all purposes.

[0082] In some embodiments, the culture medium comprises IL-12. In some embodiments, the culture medium comprises about 500 lU / mL of IL-12, about 400 lU / mL of IL-12, about 300 lU / mL of IL- 12, about 200 lU / mL of IL- 12, about 180 lU / mL of IL- 12, about 160 lU / mL of IL- 12, about 140 lU / mL of IL- 12, about 120 lU / mL of IL- 12, or about 100 lU / mL of IL- 12. In some embodiments, the first expansion culture medium comprises about 500 lU / mL of IL-12 to about 100 lU / mL of IL-12. In some embodiments, the culture medium comprises about 400 lU / mL of IL-12 to about 100 lU / mL of IL-12. In some embodiments, the culture medium comprises about 300 lU / mL of IL- 12 to about 100 lU / mL of IL- 12. In some embodiments, the culture medium comprises about 200 lU / mL of IL-12. In some embodiments, the culture medium comprisesabout 180 lU / mL of IL-12. In an embodiment, the culture medium further comprises IL-12. In a preferred embodiment, the culture medium comprises about 180 lU / mL of IL-12. The term “IL- 12” (also referred to herein as “IL12”) refers to the T cell growth factor known as interleukin- 12. Interleukin (IL)-12 is a secreted heterodimeric cytokine comprised of 2 disulfide-linked glycosylated protein subunits, designated p35 and p40 for their approximate molecular weights. IL- 12 is produced primarily by antigen-presenting cells and drives cell-mediated immunity by binding to a two-chain receptor complex that is expressed on the surface of T cells or natural killer (NK) cells. The IL-12 receptor beta- 1 (IL-12R.pi) chain binds to the p40 subunit of IL-12, providing the primary interaction between IL-12 and its receptor. However, it is IL-12p35 ligation of the second receptor chain, IL-12RP2, that confers intracellular signaling. IL- 12 signaling concurrent with antigen presentation is thought to invoke T cell differentiation towards the T helper 1 (Thl) phenotype, characterized by interferon gamma (IFNy) production. Thl cells are believed to promote immunity to some intracellular pathogens, generate complement-fixing antibody isotypes, and contribute to tumor immunosurveillance. Thus, IL- 12 is thought to be a significant component to host defense immune mechanisms. IL-12 is part of the IL-12 family of cytokines which also includes IL-23, IL-27, IL-35, IL-39.

[0083] In some embodiments, the culture medium comprises IL-15. In some embodiments, the culture medium comprises about 500 lU / mL of IL-15, about 400 lU / mL of IL-15, about 300 lU / mL of IL-15, about 200 lU / mL of IL-15, about 180 lU / mL of IL-15, about 160 lU / mL of IL- 15, about 140 lU / mL of IL-15, about 120 HJ / mL of IL-15, or about 100 lU / mL of IL-15. In some embodiments, the culture medium comprises about 500 lU / mL of IL-15 to about 100 lU / mL of IL-15. In some embodiments, the culture medium comprises about 400 lU / mL of IL- 15 to about 100 lU / mL of IL-15. In some embodiments, the culture medium comprises about 300 lU / mL of IL-15 to about 100 lU / mL of IL-15. In some embodiments, the culture medium comprises about 200 lU / mL of IL-15. In some embodiments, the culture medium comprises about 180 lU / mL of IL-15. In an embodiment, the culture medium further comprises IL-15. In a preferred embodiment, the culture medium comprises about 180 lU / mL of IL-15. The term “IL- 15” (also referred to herein as “IL15”) refers to the T cell growth factor known as interleukin- 15, and includes all forms of IL- 15 including human and mammalian forms, conservative amino acid substitutions, glycoforms, biosimilars, and variants thereof. IL-15 is described, e.g., in Fehniger and Caligiuri (2001) Bloodherein incorporated by reference in its entirety for allpurposes. IL-15 shares P and y signaling receptor subunits with IL-2. Recombinant human IL-15 is a single, non-glycosylated polypeptide chain containing 114 amino acids (and an N-terminal methionine) with a molecular mass of 12.8 kDa. Recombinant human IL-15 is commercially available from multiple suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick,N.J., USA (Cat. No. CYT-230-b) and ThermoFisher Scientific, Inc., Waltham, Mass., USA (human IL- 15 recombinant protein, Cat. No. 34-8159-82).

[0084] In some embodiments, the culture medium comprises IL-18. In some embodiments, the culture medium comprises about 500 lU / mL of IL-18, about 400 lU / mL of IL-18, about 300 lU / mL of IL- 18, about 200 lU / mL of IL- 18, about 180 lU / mL of IL- 18, about 160 lU / mL of IL- 18, about 140 lU / mL of IL-18, about 120 lU / mL of IL-18, or about 100 lU / mL of IL-18. In some embodiments, the culture medium comprises about 500 lU / mL of IL-18 to about 100 lU / mL of IL-18. In some embodiments, the culture medium comprises about 400 lU / mL of IL-18 to about 100 lU / mL of IL-18. In some embodiments, the culture medium comprises about 300 lU / mL of IL-18 to about 100 lU / mL of IL-18. In some embodiments, the culture medium comprises about 200 lU / mL of IL-18. In some embodiments, the culture medium comprises about 180 lU / mL of IL- 18. In an embodiment, the culture medium further comprises IL- 18. In a preferred embodiment, the culture medium comprises about 180 lU / mL of IL-18. The term “IL-18” (also referred to herein as “IL18”) refers to the T cell growth factor known as interleukin-15.Interleukin- 18 (IL-18) is a proinflammatory cytokine that belongs to the IL-1 cytokine family, due to its structure, receptor family and signal transduction pathways. Related cytokines include IL-36, IL-37, IL-38.

[0085] In some embodiments, the culture medium comprises IL-21. In some embodiments, the culture medium comprises about 20 lU / mL of IL-21, about 15 lU / mL of IL-21, about 12 lU / mL of IL-21, about 10 lU / mL of IL-21, about 5 lU / mL of IL-21, about 4 lU / mL of IL-21, about 3 lU / mL of IL-21, about 2 lU / mL of IL-21, about 1 lU / mL of IL-21, or about 0.5 lU / mL of IL-21. In some embodiments, the culture medium comprises about 20 lU / mL of IL-21 to aboutO.5 lU / mL of IL-21. In some embodiments, the culture medium comprises about 15 lU / mL of IL- 21 to about 0.5 lU / mL of IL-21. In some embodiments, the culture medium comprises about 12 lU / mL of IL-21 to about 0.5 lU / mL of IL-2L In some embodiments, the culture medium comprises about 10 lU / mL of IL-21 to about 0.5 lU / mL of IL-21. In some embodiments, the culture medium comprises about 5 lU / mL of IL-21 to about 1 lU / mL of IL-2L In someembodiments, the culture medium comprises about 2 TU / mL of IL-21 . In some embodiments, the culture medium comprises about 1 lU / mL of IL-21. In some embodiments, the culture medium comprises about 0.5 lU / mL of IL-21. In an embodiment, the culture medium further comprises IL-21. In a preferred embodiment, the cell culture medium comprises about 1 lU / mL of IL-21. The term “IL-21” (also referred to herein as “ZL21”) refers to the pleiotropic cytokine protein known as interleukin-21, and includes all forms of IL-21 including human and mammalian forms, conservative amino acid substitutions, glycoforms, biosimilars, and variants thereof. IL- 21 is described, e.g., in Spolski and Leonard (2014) Nat. Rev. Drug. Disc. 13:379-95, herein incorporated by reference in its entirety for all purposes. IL-21 is primarily produced by natural killer T cells and activated human CD4+T cells. Recombinant human IL-21 is a single, nonglycosylated polypeptide chain containing 132 amino acids with a molecular mass of 15.4 kDa. Recombinant human IL-21 is commercially available from multiple suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ., USA (Cat. No. CYT-408-b) and ThermoFisher Scientific, Inc., Waltham, Mass., USA (human IL-21 recombinant protein, Cat. No. 14-8219-80).

[0086] Also contemplated for inclusion in the culture medium are combinations of interleukins, such as but not limited to, two or more of IL-2, IL-12, IL-15, IL-18, and / or IL-2L Other cytokines are also contemplated, such as IL-23, IL -27, IL-35, IL-39, IL-18, IL-36, IL-37, IL-38, IFN-alpha, IFN-beta, IFN-gamma, or a combination thereof along with IL-2, IL-12, IL- 15, IL- 18, and IL-2L Antibodies, such as Th2 blocking reagents, are also contemplated, such as but not limited to, IL-4 (aIL4), anti-IL-4 (aIL4R), anti-IL-5R (aIL5R), anti-IL-5 (aIL5), anti- IL13R (aIL13R), or anti-IL13 (aIL13).

[0087] In some embodiments, the first immune cell (e.g., TIL) expansion can proceed for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days. In some embodiments, the first immune cell (e.g., TIL) expansion can proceed for 1 day to 14 days. In some embodiments, the first immune cell (e.g., TIL) expansion can proceed for 2 days to 14 days. In some embodiments, the first immune cell (e.g., TIL) expansion can proceed for 3 days to 14 days. In some embodiments, the first immune cell (e.g., TIL) expansion can proceed for 4 days to 14 days. In some embodiments, the first immune cell (e.g., TIL) expansion can proceed for 5 days to 14 days. In some embodiments, the first immune cell (e.g., TIL) expansion can proceed for 6 days to 14 days. In some embodiments, the first immunecell (e.g., TIL) expansion can proceed for 7 days to 14 days. In some embodiments, the first immune cell (e.g., TIL) expansion can proceed for 8 days to 14 days. In some embodiments, the first immune cell (e.g., TIL) expansion can proceed for 9 days to 14 days. In some embodiments, the first immune cell (e.g., TIL) expansion can proceed for 10 days to 14 days. In some embodiments, the first immune cell (e.g., TIL) expansion can proceed for 11 days to 14 days. In some embodiments, the first immune cell (e.g., TIL) expansion can proceed for 12 days to 14 days. In some embodiments, the first immune cell (e.g., TIL) expansion can proceed for 13 days to 14 days. In some embodiments, the first immune cell (e.g., TIL) expansion can proceed for 14 days. In some embodiments, the first immune cell (e.g., TIL) expansion can proceed for 1 day to 13 days. In some embodiments, the first immune cell (e.g., TIL) expansion can proceed for 2 days to 13 days. In some embodiments, the first immune cell (e.g., TIL) expansion can proceed for 3 days to 13 days. In some embodiments, the first immune cell (e.g., TIL) expansion can proceed for 4 days to 13 days. In some embodiments, the first immune cell (e.g., TIL) expansion can proceed for 5 days to 13 days. In some embodiments, the first immune cell (e.g., TIL) expansion can proceed for 6 days to 13 days. In some embodiments, the first immune cell (e.g., TIL) expansion can proceed for 7 days to 13 days. In some embodiments, the first immune cell (e.g., TIL) expansion can proceed for 8 days to 13 days. In some embodiments, the first immune cell (e.g., TIL) expansion can proceed for 9 days to 13 days. In some embodiments, the first immune cell (e.g., TIL) expansion can proceed for 10 days to 13 days. In some embodiments, the first immune cell (e.g., TIL) expansion can proceed for 11 days to 13 days. In some embodiments, the first immune cell (e.g., TIL) expansion can proceed for 12 days to 13 days. In some embodiments, the first immune cell (e.g., TIL) expansion can proceed for 1 day to 12 days. In some embodiments, the first immune cell (e.g., TIL) expansion can proceed for 2 days to 12 days. In some embodiments, the first immune cell (e.g., TIL) expansion can proceed for 3 days to 12 days. In some embodiments, the first immune cell (e.g., TIL) expansion can proceed for 4 days to 12 days. In some embodiments, the first immune cell (e.g., TIL) expansion can proceed for 5 days to 12 days. In some embodiments, the first immune cell (e.g., TIL) expansion can proceed for 6 days to 12 days. In some embodiments, the first immune cell (e.g., TIL) expansion can proceed for 7 days to 12 days. In some embodiments, the first immune cell (e.g., TIL) expansion can proceed for 8 days to 12 days. In some embodiments, the first immune cell (e.g., TIL) expansion can proceed for 9 days to 12 days. In some embodiments, the first immune cell(e g., TIL) expansion can proceed for 10 days to 12 days. In some embodiments, the first immune cell (e.g., TIL) expansion can proceed for 11 days to 12 days. In some embodiments, the first immune cell (e.g., TIL) expansion can proceed for 1 day to 11 days. In some embodiments, the first immune cell (e.g., TIL) expansion can proceed for 2 days to 11 days. In some embodiments, the first immune cell (e.g., TIL) expansion can proceed for 3 days to 11 days. In some embodiments, the first immune cell (e.g., TIL) expansion can proceed for 4 days to 11 days. In some embodiments, the first immune cell (e.g., TIL) expansion can proceed for 5 days to 11 days. In some embodiments, the first immune cell (e.g., TIL) expansion can proceed for 6 days to 11 days. In some embodiments, the first immune cell (e.g., TIL) expansion can proceed for 7 days to 11 days. In some embodiments, the first immune cell (e.g., TIL) expansion can proceed for 8 days to 11 days. In some embodiments, the first immune cell (e.g., TIL) expansion can proceed for 9 days to 11 days. In some embodiments, the first immune cell (e.g., TIL) expansion can proceed for 10 days to 11 days. In some embodiments, the first immune cell (e.g., TIL) expansion can proceed for 11 days.

[0088] In some embodiments, a combination of IL-2, IL-7, IL-15, and / or IL-21 is employed as a combination during the first expansion. In some embodiments, IL-2, IL-7, IL- 15, and / or IL- 21 as well as any combinations thereof can be included during the first expansion. In some embodiments, a combination of IL-2, IL-15, and IL-21 are employed as a combination during the first expansion. The term “IL-7” (also referred to herein as “IL7”) refers to a glycosylated tissue- derived cytokine known as interleukin 7, which may be obtained from stromal and epithelial cells, as well as from dendritic cells. Fry and Mackall (2002) Blood 99:3892-904, herein incorporated by reference in its entirety for all purposes. IL-7 can stimulate the development of T cells. IL-7 binds to the IL-7 receptor, a heterodimer consisting of IL-7 receptor alpha and common gamma chain receptor, which in a series of signals important for T cell development within the thymus and survival within the periphery. Recombinant human IL-7 suitable for use is commercially available from multiple suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ., USA (Cat. No. CYT-254) and ThermoFisher Scientific, Inc., Waltham, Mass., USA (human IL-15 recombinant protein, Cat. No. Gibco PHC0071).

[0089] In some embodiments, the first expansion is performed in an open system. For example, the first expansion can be performed in 6-well plates. In another example, the firstexpansion can be performed in a gas permeable rapid cell expansion system (e g., a G-REX 10M open system).

[0090] In some embodiments, the first expansion is performed in a closed system bioreactor. “Closed system” as used herein refers to a system that is closed to the outside environment. Any closed system appropriate for cell culture methods can be employed with the methods disclosed herein. Closed systems include, for example, but are not limited to closed G-REX containers or cell culture bags. In some embodiments, once a tumor segment is added to the closed system, the system is not open to the outside environment until the immune cells (e.g., TILs) are ready to be administered to the patient. In some embodiments, the closed system is the system disclosed in WO 2018 / 130845, herein incorporated by reference in its entirety for all purposes. In some embodiments, a closed system is employed for the immune cell (e.g., TIL) expansion, as described herein. In some embodiments, a single bioreactor is employed. In some embodiments, the single bioreactor employed is example a G-REX-10 or a G-REX-100 or the device of WO 2018 / 130845, herein incorporated by reference in its entirety for all purposes. In some embodiments, the closed system bioreactor is a single bioreactor.

[0091] The immune cell (e.g., TIL) population obtained from the first expansion can be subjected to a second expansion (which can include expansions sometimes referred to as REP). Similarly, in the case where genetically modified immune cells (e.g., TILs) will be used in therapy, the immune cells (e.g., TILs) can be subjected to genetic modifications for suitable treatments prior to expansion or after the first expansion and / or prior to the second expansion.C. Viral Transduction

[0092] The methods herein comprise a step of viral transduction. In particular embodiments, the transduction is lentiviral transduction. In some embodiments, the transduction is performed in an open system. For example, the transduction can be performed in 6-well plates. In another example, the transduction can be performed in a gas permeable rapid cell expansion system (e.g., a G-REX 10M open system).

[0093] In some embodiments, the transduction is performed in a closed system bioreactor. “Closed system” as used herein refers to a system that is closed to the outside environment. Any closed system appropriate for cell culture methods can be employed with the methods disclosed herein. Closed systems include, for example, but are not limited to closed G-REX containers orcell culture bags. In some embodiments, once a tumor segment is added to the closed system, the system is not open to the outside environment until the immune cells (e.g., TILs) are ready to be administered to the patient. In some embodiments, the closed system is the system disclosed in WO 2018 / 130845, herein incorporated by reference in its entirety for all purposes. In some embodiments, a closed system is employed for the immune cell (e.g., TIL) expansion, as described herein. In some embodiments, a single bioreactor is employed. In some embodiments, the single bioreactor employed is example a G-REX-10 or a G-REX-100 or the device of WO 2018 / 130845, herein incorporated by reference in its entirety for all purposes. In some embodiments, the closed system bioreactor is a single bioreactor.

[0094] The transduction in the methods described herein can comprise introducing a vector encoding an exogenous receptor (e.g., exogenous costimulatory receptor) into the immune cells, whereby the exogenous receptor is expressed in the immune cells. For example, in some embodiments, the methods described herein comprise introducing a vector encoding an exogenous receptor (e.g., exogenous costimulatory receptor) into the TILs in the processed resected tumor product, whereby the exogenous receptor (e.g., exogenous costimulatory receptor) is expressed in the TILs. The exogenous receptor (e.g., exogenous costimulatory receptor) can, for example, comprise an extracellular binding domain specific for a tumor- associated antigen.

[0095] The exogenous receptor can be any suitable exogenous receptor. In some embodiments, the exogenous receptor is an exogenous T cell receptor (TCR). The term “T cell receptor,” or “TCR,” refers to a heterodimeric receptor composed of aP or y8 chains that pair on the surface of a T cell. Each a, , y, and 6 chain is composed of two Ig-like domains: a variable domain (V) that confers antigen recognition through the complementarity determining regions (CDR), followed by a constant domain (C) that is anchored to cell membrane by a connecting peptide and a transmembrane (TM) region. The TM region associates with the invariant subunits of the CD3 signaling apparatus. Each of the V domains has three CDRs. These CDRs interact with a complex between an antigenic peptide bound to a protein encoded by the major histocompatibility complex (pMHC).

[0096] In some embodiments, the exogenous receptor is a chimeric antigen receptor (CAR). See, e.g., Sub kl ewe et al. (2019) Transfus. Med. Hemother. 46(1): 15-24, herein incorporated by reference in its entirety for all purposes. Generally, CARs are engineered receptor proteins toengineer cells to target cancer cell-specific antigen proteins. CARs can be composed of an intracellular activating signaling domain, a transmembrane region, and an extracellular antigen binding domain. The intracellular activating signaling domains (e.g., CD137 (4-1BB), CD28) mainly mediate the activation and cytotoxicity, while the extracellular antigen binding domains (e g., single-chain variable fragments (scFv)) recognize the specific antigen of tumor cells. In some embodiments, the CAR comprises a CD3^ chain linked to an scFv. For example, first generation CARs can comprise a single-chain variable region from a monoclonal antibody paired with an intracellular signaling domain, the CD3 zeta chain from the CD3 TCR or FcR gamma. Second and third generation CARs include an additional one or two co-stimulating domains (e.g., CD28, CD137 (4-1BB), OX-40, or others) that increase signal strength and persistence, with increased proliferation and cytokine production. In some embodiments, the CAR further comprises one or more costimulatory signaling domains (e.g., CD28, CD137 (4-1BB), 0X40, or others). In some embodiments, the CAR further comprises two or more costimulatory signaling domains (e.g., CD28, CD137 (4-1BB), 0X40, or others).

[0097] In some methods, the exogenous receptor (e.g., exogenous costimulatory receptor) comprises a chimeric costimulatory antigen receptor (CoStAR) comprising an extracellular binding domain specific for a tumor-associated antigen operatively linked to a transmembrane domain and one or more signaling domains (e.g., first and second signaling domains). See, e.g., WO 2020 / 152451, WO 2022 / 016112, and WO 2022 / 016114, each of which is herein incorporated by reference in its entirety for all purposes. Such CoStARs can provide a costimulatory signal to T-cells upon engagement with a defined tumor-associated antigen.

[0098] Each of the signaling domains can be, for example, a signaling domain or signaling fragment of a receptor, such as a tumor necrosis factor receptor superfamily (TNFRSF) receptor, including but not limited to CD2, CD9, CD26, CD27, CD28, CD29, CD38, CD40, CD43, CD46, CD49d, CD55, CD73, CD81, CD82, CD99, CD100, CD134 (0X40), CD137 (4 IBB), CD150 (SLAM), CD270 (HVEM), CD278 (ICOS), CD357 (GITR), or EphB6. In some CoStARs, the first and second signaling domains comprise a CD28 signaling domain, a CD40 signaling domain, or both the CD28 signaling domain and the CD40 signaling domain. In some CoStARs, the first and second signaling domains comprise a ICOS signaling domain, a CD40 signaling domain, or both the ICOS signaling domain and the CD40 signaling domain. In some embodiments, the first and second intracellular signaling domains are the same. In otherembodiments, the first and second intracellular signaling domains are different. In some embodiments, the costimulatory receptor is capable of dimerization. In some embodiments, dimerization or association with accessory molecules is assisted by costimulatory receptor interactions in the intracellular portion, and / or the extracellular portion of the costimulatory receptor.

[0099] CD40 is a member of the tumor necrosis factor receptor (TNFR) superfamily, and several isoforms are generated by alternative splicing. Its ligand, CD 154 (also called CD40L), is a protein that is primarily expressed on activated T cells. For reference, the human CD40 isoform 1 protein sequence is set forth in GenBank accession No. NP 001241.1, including signal peptide (amino acids 1-20), transmembrane domain (amino acids 194-215), and cytoplasmic domain (amino acids 216-277). CD40 receptor signaling involves adaptor proteins including but not limited to TNF receptor-associated factors (TRAF), and the CD40 cytoplasmic domain comprises signaling components, including amino acid sequences fitting an SH3 motif, TRAF 2 motif, TRAF 6 motif, and PKA motif

[0100] The human CD28 protein sequence is set forth in GenBank accession No. NP 006130.1, including signal peptide (amino acids 1-18), extracellular domain (amino acids 19- 152), transmembrane domain (amino acids 153-179) and cytoplasmic domain (amino acids 180- 200). The extracellular domain includes an immunoglobulin type domain (amino acids 21-136) which contains amino acids with compose the antigen-binding site and amino acids that form the homodimer interface. The extracellular domain includes several asparagine residues which may be glycosylated, and the intracellular domain comprises serine and tyrosine residues, which may be phosphorylated.

[0101] CD278 or ICOS (Inducible T-cell COStimulator) is a CD28-superfamily costimulatory molecule that is expressed on activated T cells. ICOS is assigned UniProt Accession No. Q53QY6.

[0102] The extracellular binding domain of the exogenous receptor (e.g., exogenous costimulatory receptor) can be one that binds any suitable tumor-associated antigen (TAA). Antigens have been identified in most of the human cancers, including Burkitt lymphoma, neuroblastoma, melanoma, osteosarcoma, renal cell carcinoma, breast cancer, prostate cancer, lung carcinoma, and colon cancer. TAAs include, without limitation, CD 19, CD20, CD22, CD24, CD33, CD38, CD123, CD228, CD138, BCMA, GPC3, CEA, folate receptor alpha (FRa,F0LR1), mesothelin (MSLN, MPF), CD276, gplOO, 5T4, GD2, EGFR, MUC-1 , PSMA, EpCAM, MCSP, SM5-1, MICA, MICB, ULBP, and HER-2. TAAs further include neoantigens, peptide / MHC complexes, and HSP / peptide complexes. In some embodiments, the extracellular binding domain binds to CD70, CD146, folate receptor alpha (FRa, FOLR1), carcinoembryonic antigen (CEA), 5T4, mellanotransferrin (CD228), Her2, EGFR, GPC3, melanoma-associated chondroitin sulphate proteoglycan (MCSP / CSPG4), CD71, EPCAM, SM5-1, CA125, PDL-1, CD155 PD-1, mesothelin, or a tumor specific peptide (p)-major histocompatibility (MHC) complex, or a tumor specific pMHC complex antigen specific single chain T-cell receptor (scTCR), or transferrin, or an antibody or antigen-binding protein. In some embodiments, the tumor-associated antigen comprises folate receptor alpha. In some embodiments, the tumor- associated antigen comprises carcinoembryonic antigen (CEA). In some embodiments, the tumor-associated antigen comprises mesothelin.

[0103] Folate receptor alpha (FRa, FOLR1, FOLR, folate receptor) binds to folate and reduced folic acid derivatives and mediates delivery of 5-methyltetrahydrofolate and folate analogs into the interior of cells. Human folate receptor alpha is assigned UniProt Accession No. P15328. This receptor is an attractive anticancer drug target owing to the overexpression of FRa in a range of solid tumors, including ovarian, lung and breast cancers.

[0104] Mesothelin (MSLN, MPF) is assigned UniProt Accession No. Q13421. Mesothelin is a tumor differentiation antigen that is normally present on the mesothelial cells lining the pleura, peritoneum and pericardium. It is however highly expressed in several human cancers including malignant mesothelioma, pancreatic, ovarian and lung adenocarcinoma.

[0105] Carcinoembryonic antigen (CEA, carcinoembryonic antigen-related cell adhesion molecule 5, CEACAM5, CD66e, meconium antigen 100) is a protein normally found in very low levels in the blood of adults. The CEA blood level may be increased in certain types of cancer and non-cancerous (benign) conditions. Human CEA is assigned UniProt Accession No. P06731.

[0106] The extracellular binding domain can comprise, for example, an antigen-binding domain, an scFv, a peptide, an antigen-binding portion of an antibody, an antibody heavy-chain, a ligand of a target receptor, or a ligand binding portion of a receptor. The term “antigen-binding domain” as used herein can refer to an antibody fragment including, but not limited to, a diabody, a Fab, a Fab’, a F(ab’)2, an Fv fragment, a disulfide stabilized Fv fragment (dsFv), a (dsFv)2, a bispecific dsFv (dsFv-dsFv1), a disulfide stabilized diabody (ds diabody), a single-chain antibody molecule (scFv), an scFv dimer (bivalent diabody), a multispecific antibody formed from a portion of an antibody comprising one or more CDRs, a camelized single domain antibody, a nanobody, a domain antibody, a bivalent domain antibody, or any other antibody fragment that binds to an antigen but does not comprise a complete antibody structure. An antigen-binding domain is capable of binding to the same antigen to which the parent antibody or a parent antibody fragment (e.g., a parent scFv) binds. In some embodiments, an antigen-binding fragment may comprise one or more complementarity determining regions (CDRs) from a particular human antibody grafted to frameworks (FRs) from one or more different human antibodies.

[0107] In some embodiments, the antigen-binding domain comprises a T-cell receptor or binding fragment thereof that binds to a defined tumor specific peptide-MHC complex.

[0108] In some embodiments, the antigen-binding domain comprises a natural ligand of a tumor expressed protein or tumor-binding fragment thereof

[0109] In some exogenous receptors (e.g., exogenous costimulatory receptors), the extracellular binding domain comprises an scFv. In some embodiments, the exogenous costimulatory receptor is a CoStAR that comprises an scFv that binds to carcinoembryonic antigen (CEA), a spacer and transmembrane sequence of CD28, a CD28 signaling domain, and a CD40 signaling domain. In some embodiments, the exogenous costimulatory receptor is a CoStAR that comprises an scFv that binds to folate receptor alpha, a spacer and transmembrane sequence of CD28, a CD28 signaling domain, and a CD40 signaling domain.

[0110] Exogenous receptors (e.g., exogenous costimulatory receptors) can be introduced by any suitable means, including using viral and non-viral-based tools. Retrovirus-based gene delivery is a mature, well-characterized technology, which has been used to permanently integrate CARs into the host cell genome. See, e.g., Scholler (2012) Sci. Transl. Med. 4:132ra53; Rosenberg et al. (1990) N. Engl. J. Med. 323:570-578, each of which is herein incorporated by reference in its entirety for all purposes. Non-viral DNA transfection methods can also be used, such as the Sleeping Beauty (SB) transposon system or the piggyBac transposon system. See, e.g., Singh et al. (2008) 68:2961-2971 and Morita et al. (2017) Mol. Then Methods Clin. Dev. 8: 131-140, each of which is herein incorporated by reference in its entirety for all purposes.

[0111] Vectors derived from retroviruses, such as the lentivirus, are suitable tools to achieve long-term gene transfer since they allow long-term, stable integration of a transgene ortransgenes and its propagation in daughter cells. The vector may be capable of transfecting or transducing a lymphocyte including a TIL. The expression of a nucleic acid encoding an exogenous receptor (e.g., exogenous costimulatory receptor) can be achieved by operably linking a nucleic acid encoding the exogenous receptor (e.g., exogenous costimulatory receptor) to one or more promoters, and incorporating the construct into an expression vector. Additional regulatory elements, such as enhancers, regulate the frequency of transcriptional initiation. One example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operatively linked thereto. Another example of a suitable promoter is Elongation Growth Factor-la (EF-la). However, other constitutive promoter sequences may also be used, including, but not limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, MSCV promoter, MND promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, as well as human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the hemoglobin promoter, and the creatine kinase promoter. In order to assess the expression of the exogenous receptor (e.g., exogenous costimulatory receptor), the expression vector to be introduced into a cell can also contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or transduced through viral vectors.

[0112] A vector encoding an exogenous receptor (e.g., exogenous costimulatory receptor) can be introduced by any suitable means. In some methods, the vector is introduced by viral transduction. For example, the vector can be introduced by lentiviral transduction (e.g., at a target MOI of 5). The transduction with virus such as lentivirus can be performed at an MOI of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 (e.g., an MOI of about 5, about 10, or about 20) based on the total number of live cells. In one example, the transduction with virus such as lentivirus can be performed at an MOI of, for example, no more than about 5 based on the total number of live cells. In another example, the transduction with virus such as lentivirus can be performed at an MOI of, for example, about 5 to about 20 based on the total number of live cells. In another example, the transduction with virus such as lentivirus can be performed at an MOI of, for example, about 5 based on the total number of live cells.

[0113] Lentiviruses are efficient gene transfer vehicles due to their ability to transduce both dividing and nondividing cells. While the most thoroughly investigated of the lentiviral gene therapy vectors are derived from human immunodeficiency virus (HIV) type 1, gene therapy vectors based on other primate and non-primate lentiviruses have also been developed, including, HIV-2, SIV, feline immunodeficiency virus (FIV), equine infectious anemia virus (EIAV), caprine arthritis encephalitis virus (CAEV), visna virus, and Jembrana disease virus (JDV).

[0114] Replication-deficient viral vectors can be used to prevent infection of a patient with a potentially deadly virus. Lentiviral vectors have been developed to become safer and more efficient. Recent third-generation vectors removed all accessory genes that aid in virulence and pathogenicity while splitting the remaining genes, which are vital for expression of a transgene across three plasmids. See, e.g, US 2006 / 0024274, herein incorporated by reference in its entirety for all purposes.

[0115] In some methods, the immune cells (e.g., TILs) are not treated with an activating agent such as TransAct™ or Dynabeads Human T-Activator CD3 / CD28 prior to or during viral (e.g., lentiviral) transduction. For example, the immune cells (e.g., TILs) in some methods are not treated with an activating agent comprising CD3 and / or CD28 agonists prior to or during viral (e.g., lentiviral) transduction, or the immune cells (e.g., TILs) in some methods are not treated with an activating agent comprising CD3 and CD28 agonists prior to or during viral (e.g., lentiviral) transduction. In other methods, the immune cells (e.g., TILs) are treated with an activating agent prior to or during viral (e.g., lentiviral) transduction. For example, the immune cells (e.g., TILs) in some methods are treated with an activating agent comprising CD3 and / or CD28 agonists prior to or during viral (e.g., lentiviral) transduction, or the immune cells (e.g., TILs) in some methods are treated with an activating agent comprising CD3 and CD28 agonists prior to or during viral (e.g., lentiviral) transduction. An example of such an activating agent is TransAct™, which is colloidal, polymeric nanomatrix, conjugated to recombinant humanized CD3 and CD28 agonists. See, e.g., WO 2015 / 162211, herein incorporated by reference in its entirety for all purposes. If the immune cells (e.g., TILs) are treated with an activating agent prior to or during viral (e.g., lentiviral) transduction, the treatment can be at any suitable time. For example, the treatment with the activating agent can be about 1 to about 4 days after a processed resected tumor product comprising immune cells (e.g., TILs) is obtained, or after cryopreserved immune cells (e.g., TILs) are thawed, or after the first expansion in step (b) isstarted, or after step (a). If the immune cells (e.g., TILs) are treated with an activating agent prior to or during viral (e.g., lentiviral) transduction, the length of time between treatment with the activating agent and the viral (e.g., lentiviral) transduction can be any suitable length of time. For example, there can be about a 2 or about a 3 day interval between treatment with the activating agent and transducing the immune cells (e.g., TILs) with virus (e.g., lentivirus).

[0116] In some methods, the immune cells (e.g., TILs) are not treated with an enhancer of viral-mediated transduction such as RetroNectin (rFN-CH-296) or LentiBOOST® prior to or during viral (e.g., lentiviral) transduction. For example, the immune cells (e.g., TILs) in some methods are not treated with a recombinant human fibronectin fragment that is an enhancer of viral-mediated transduction prior to or during viral (e.g., lentiviral) transduction. In other methods, the immune cells (e.g., TILs) are treated with an enhancer of viral mediated- transduction (e.g., a recombinant human fibronectin fragment that is an enhancer of viral- mediated transduction) prior to or during viral (e.g., lentiviral) transduction. For example, the immune cells (e.g., TILs) in some methods are treated with a recombinant human fibronectin fragment that is an enhancer of viral-mediated transduction (e.g., RetroNectin or LentiBOOST®) prior to or during or during viral (e.g., lentiviral) transduction. An example of such an enhancer is RetroNectin (rFN-CH-296), which is a 63 kD fragment of recombinant human fibronectin fragment (also referred to as rFN-CH-296) that enhances the efficiency of lentiviral- and retroviral-mediated gene transductions. Specifically, it enhances lentiviral- and retroviral- mediated gene transduction by aiding the colocalization of target cells and viral particles. Specifically, virus particles bind RetroNectin reagent via interaction with the H-domain, and target cells bind mainly through the interaction of cell surface integrin receptors VLA-5 and / or VLA-4 with the fibronectin C-domain and CS-1 sites, respectively. By facilitating close physical proximity, RetroNectin reagent can enhance viral-mediated gene transfer to target cells expressing integrin receptors VLA-4 and / or VLA-5. LentiBOOST® is a highly effective, non- cytotoxic transduction enhancer, originally developed for preclinical and clinical application of lentiviral vectors. For in vivo transduction, LentiBOOST® consists of the poloxamer 338. For in vitro use, the Poloxamer 388 (LentiBOOST®) is combined with polybrene, a polycationic substance that is used to enhance transduction efficiency. Both substances act synergistically on different pathways and in distinctive modes of action in enhancing transduction efficiency ex vivo. Polybrene reduces the charge-repulsion between the vector and the cell surface byinteracting with the negatively charged cellular membrane surface, while poloxamers interact with the lipid bilayer and change their physiochemical properties. If the immune cells (e.g., TILs) are treated with an enhancer prior to or during viral (e.g., lentiviral) transduction, the treatment can be at any suitable time. For example, the treatment with the enhancer can be about1 to about 4 days after a processed resected tumor product comprising immune cells (e.g., TILs) is obtained, or after cryopreserved immune cells (e.g., TILs) are thawed, or after the first expansion in step (b) is started, or after step (a). If the immune cells (e.g., TILs) are treated with an enhancer prior to or during viral (e.g., lentiviral) transduction, the length of time between treatment with the enhancer and the viral (e.g., lentiviral) transduction can be any suitable length of time. For example, there can be about a 2 or about a 3 day interval between treatment with the enhancer and transducing the immune cells (e.g., TILs) with virus (e.g., lentivirus).

[0117] In some methods, spinoculation (also called spin infection or spin-fection) is not used during viral (e.g., lentiviral) transduction. During the process of spinoculation, both the viral vector and the cells are centrifuged together to aid viral binding to the target cells. In other methods, spinoculation is used during viral (e.g., lentiviral) transduction.

[0118] In some methods, the immune cells (e.g., TILs) are not treated with an activating agent such as TransAct™ or Dynabeads Human T-Activator CD3 / CD28 or an enhancer of viral- mediated transduction such as RetroNectin (rFN-CH-296) prior to or during viral (e.g., lentiviral) transduction, and spinoculation is not used during viral (e.g., lentiviral) transduction.

[0119] Immune cells (e.g., TILs) can be transduced with virus (e.g., lentivirus) at any suitable time. For example, in some methods, the immune cells (e.g., TILs) are transduced about2 to about 7 days after the start of the first expansion. In other methods, the immune cells (e.g., TILs) are transduced about 2 to about 3 days after the start of the first expansion. In other methods, the immune cells (e g., TILs) are transduced about 3 to about 4 days after the start of the first expansion. In other methods, transduction is prior to or at the beginning of the outgrowth of immune cells (e.g., TILs) from the disaggregated tumor in the first expansion step. In other methods, transduction is in the middle of the first expansion step. In other methods, transduction is after or at the end of the first expansion step.D. Activation and Expansion of Immune Cells or TILs

[0120] The methods described herein can comprise performing a second expansion by culturing the first population of immune cells (e.g., TILs) in a second cell culture medium (e.g., with IL-2, a CD3 agonist (such as a CD3 agonist antibody), and antigen presenting cells (APCs)), to produce a second population of immune cells (e.g., TILs). Following the immune cell (e.g., TIL) outgrowth phase, the immune cells (e.g., TILs) can be activated and further expanded. In a specific example, polyclonal immune cell (e.g., TIL) activation can be mediated using anti-CD3 and irradiated allogeneic PBMCs to provide primary and co-stimulation for T- cell activation. The terms “peripheral blood mononuclear cells” and “PBMCs” refer to a peripheral blood cell having a round nucleus, including lymphocytes (T cells, B cells, NK cells) and monocytes. In some embodiments, the peripheral blood mononuclear cells are irradiated allogeneic peripheral blood mononuclear cells. PBMCs are a type of antigen-presenting cell. The immune cells (e.g., TILs) can be cultured, for example, in medium can be supplemented, for example, with human AB serum and IL-2 (e.g., anti-CD3 antibody, human AB serum, and IL-2). The immune cells (e.g., TILs) can further be expanded in medium comprising IL-2 (e.g., medium comprising human AB serum and IL-2).

[0121] In some embodiments, the methods disclosed herein can comprise a step of performing a second expansion by culturing the first population of immune cells (e.g., TILs) with additional IL-2, CD3 agonist (such as a CD3 agonist antibody) (e.g., anti-CD3 antibody or OKT-3), and antigen presenting cells (APCs), to produce a second population of immune cells (e.g., TILs). In some embodiments, the CD3 agonist is a CD3 agonist antibody (e.g., anti-CD3 antibody such as OKT-3). The term “anti-CD3 antibody” refers to an antibody or variant thereof (e g., a monoclonal antibody) and including human, humanized, chimeric, murine, or mammalian antibodies which are directed against the CD3 receptor in the T cell antigen receptor of mature human T cells. Anti-CD3 antibodies include OKT-3, also known as muromonab. Anti-CD3 antibodies also include the UHCT1 clone, also known as T3 and CD3. epsilon. Other anti-CD3 antibodies include, for example, otelixizumab, teplizumab, and visilizumab. “OKT-3” (also referred to herein as “OKT3”) refers to a monoclonal antibody or biosimilar or variant thereof, including human, humanized, chimeric, or murine antibodies, directed against the CD3 receptor in the T cell antigen receptor of mature T cells, and includes commercially-available forms such as OKT-3 (30 ng / mL, MACS GMP CD3 pure, Miltenyi Biotech, Inc., San Diego, Calif, USA)and muromonab or variants, conservative amino acid substitutions, glycoforms, or biosimilars thereof.

[0122] In some embodiments, the immune cells (e.g., TILs) obtained from the first expansion are not cryopreserved after the first expansion and prior to the second expansion. In some embodiments, the transition from the first expansion to the second expansion occurs at about 3 days, 4, days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days after the cryopreserved 11 days after the cryopreserved disaggregated tumor tissue is thawed. In some embodiments, the transition from the first expansion to the second expansion occurs at about 3 days to 21 days after the cryopreserved disaggregated tumor tissue is thawed. In some embodiments, the transition from the first expansion to the second expansion occurs at about 4 days to 14 days after the cryopreserved disaggregated tumor tissue is thawed. In some embodiments, the transition from the first expansion to the second expansion occurs at about 4 days to 10 days after the cryopreserved disaggregated tumor tissue is thawed. In some embodiments, the transition from the first expansion to the second expansion occurs at about 7 days to 14 days after the cryopreserved disaggregated tumor tissue is thawed. In some embodiments, the transition from the first expansion to the second expansion occurs at about 14 days after the cryopreserved disaggregated tumor tissue is thawed. In some embodiments the seeding of the REP culture occurs 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days after the cryopreserved disaggregated tumor tissue is thawed.

[0123] In some methods, the CD3 agonist antibody is OKT-3. In some methods, the culture medium comprises between about 300 HJ / mL and about 3000 lU / mL of IL-2, or the culture medium comprises between about 2000 lU / mL of IL-2 and about 3000 lU / mL IL-2. In some methods, the culture medium comprises fetal bovine serum (FBS). In some methods, the culture medium does not comprise FBS. In some methods, the culture medium comprises human AB serum. In some methods, the APCs are artificial APCs. In some methods, the APCs are obtained by apheresis. In some methods, the APCs comprise peripheral blood mononuclear cells (PBMCs). In some methods, the APCs comprise PBMCs from 2 to 10 donors, from 2-5 donors, from 3-4 donors, or from 3 donors.

[0124] In some embodiments, the transition from the first expansion to the second expansion occurs at 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days after the cryopreserved disaggregated tumor tissue is thawed. Insome embodiments, the transition from the first expansion to the second expansion occurs 1 day to 14 days after the cryopreserved disaggregated tumor tissue is thawed. In some embodiments, the first immune cell (e.g., TIL) expansion can proceed for 2 days to 14 days. In some embodiments, the transition from the first expansion to the second expansion occurs 3 days to 14 days after the cryopreserved disaggregated tumor tissue is thawed. In some embodiments, the transition from the first expansion to the second expansion occurs 4 days to 14 days after the cryopreserved disaggregated tumor tissue is thawed. In some embodiments, the transition from the first expansion to the second expansion occurs 5 days to 14 days after the cryopreserved disaggregated tumor tissue is thawed. In some embodiments, the transition from the first expansion to the second expansion occurs 6 days to 14 days after the cryopreserved disaggregated tumor tissue is thawed. In some embodiments, the transition from the first expansion to the second expansion occurs 7 days to 14 days after the cryopreserved disaggregated tumor tissue is thawed. In some embodiments, the transition from the first expansion to the second expansion occurs 8 days to 14 days after the cryopreserved disaggregated tumor tissue is thawed. In some embodiments, the transition from the first expansion to the second expansion occurs 9 days to 14 days after the cryopreserved disaggregated tumor tissue is thawed. In some embodiments, the transition from the first expansion to the second expansion occurs 10 days to 14 days after the cryopreserved disaggregated tumor tissue is thawed. In some embodiments, the transition from the first expansion to the second expansion occurs 11 days to 14 days after the cryopreserved disaggregated tumor tissue is thawed. In some embodiments, the transition from the first expansion to the second expansion occurs 12 days to 14 days after the cryopreserved disaggregated tumor tissue is thawed. In some embodiments, the transition from the first expansion to the second expansion occurs 13 days to 14 days after the cryopreserved disaggregated tumor tissue is thawed. In some embodiments, the transition from the first expansion to the second expansion occurs 14 days after the cryopreserved disaggregated tumor tissue is thawed. In some embodiments, the transition from the first expansion to the second expansion occurs 1 day to 11 days after the cryopreserved disaggregated tumor tissue is thawed. In some embodiments, the transition from the first expansion to the second expansion occurs 2 days to 11 days after the cryopreserved disaggregated tumor tissue is thawed. In some embodiments, the transition from the first expansion to the second expansion occurs 3 days to 11days after the cryopreserved disaggregated tumor tissue is thawed. In some embodiments, the transition from the first expansion to the second expansion occurs 4 days to 11 days after the cryopreserved disaggregated tumor tissue is thawed. In some embodiments, the transition from the first expansion to the second expansion occurs 5 days to 11 days after the cryopreserved disaggregated tumor tissue is thawed. In some embodiments, the transition from the first expansion to the second expansion occurs 6 days to 11 days after the cryopreserved disaggregated tumor tissue is thawed. In some embodiments, the transition from the first expansion to the second expansion occurs 7 days to 11 days after the cryopreserved disaggregated tumor tissue is thawed. In some embodiments, the transition from the first expansion to the second expansion occurs 8 days to 11 days after the cryopreserved disaggregated tumor tissue is thawed. In some embodiments, the transition from the first expansion to the second expansion occurs 9 days to 11 days after the cryopreserved disaggregated tumor tissue is thawed. In some embodiments, the transition from the first expansion to the second expansion occurs 10 days to 11 days after the cryopreserved disaggregated tumor tissue is thawed. In some embodiments, the transition from the first expansion to the second expansion occurs 11 days after the cryopreserved disaggregated tumor tissue is thawed.

[0125] In some embodiments, the immune cells (e.g., TILs) or a portion of the immune cells (e.g., TILs) from the first expansion are cryopreserved. In some embodiments, the immune cells (e g., TILs) are divided in two or more portions, with one or more portion proceeding to the second expansion, and one or more portion cryopreserved to be used in a later second expansion. In some embodiments, the number of cells at the end of the first expansion is determined and the culture is divided accordingly. In some embodiments, the average potency of the immune cells (e g., TILs) from the first expansion is determined and the culture is divided accordingly. In some embodiments, a predetermined minimum number or optimal number of immune cells (e.g., TILs) proceeds to the second expansion and the remaining immune cells (e.g., TILs) are cryopreserved, and later thawed and used in a further second expansion. In some embodiments, depending on the number and / or activity of left-over immune cells (e.g., TILs), the cryopreserved immune cells (e.g., TILs), can alternatively be used in a first expansion followed by a second expansion.

[0126] In some embodiments, the immune cells (e.g., TILs) are not stored after the first expansion and prior to the second expansion, and the immune cells (e.g., TILs) proceed directlyto the second expansion. In some embodiments, the transition occurs in closed system, as described herein. In some embodiments, the immune cells (e.g., TILs) from the first expansion, the second population of immune cells (e.g., TILs), and proceeds directly into the second expansion with no transition period.

[0127] In some embodiments, the transition from the first expansion to the second expansion is performed in a closed system bioreactor. In some embodiments, a closed system is employed for the immune cell (e.g., TIL) expansion, as described herein. In some embodiments, a single bioreactor is employed. In some embodiments, the single bioreactor employed is for example a G-REX-10 or a G-REX-100 or Xuri WAVE bioreactor. In some embodiments, the closed system bioreactor is a single bioreactor.

[0128] In some embodiments, the immune cell (e.g., TIL) cell population is expanded in number after harvest and initial bulk processing. This further expansion is referred to herein as the second expansion, which can include expansion processes generally referred to in the art as a rapid expansion process (REP). The second expansion is generally accomplished using a culture media comprising a number of components, including feeder cells, a cytokine source, and a CD3 agonist (e.g., a CD3 agonist antibody or an anti-CD3 antibody), in a gas-permeable or gas exchanging container.

[0129] In some embodiments, the second expansion or second immune cell (e.g., TIL) expansion of immune cells (e.g., TILs) can be performed using any immune cell (e.g., TIL) culture flasks or containers known by those of skill in the art. In some embodiments, the second immune cell (e.g., TIL) expansion can proceed for 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days. In some embodiments, the second immune cell (e.g., TIL) expansion can proceed for about 7 days to about 14 days. In some embodiments, the second immune cell (e.g., TIL) expansion can proceed for about 8 days to about 14 days. In some embodiments, the second immune cell (e.g., TIL) expansion can proceed for about 9 days to about 14 days. In some embodiments, the second immune cell (e.g., TIL) expansion can proceed for about 10 days to about 14 days. In some embodiments, the second immune cell (e.g., TIL) expansion can proceed for about 11 days to about 14 days. In some embodiments, the second immune cell (e.g., TIL) expansion can proceed for about 12 days to about 14 days. In some embodiments, the second immune cell (e.g., TIL) expansion can proceed for about 13 days to about 14 days. In some embodiments, the second immune cell (e.g., TIL) expansion can proceed for about 14 days. Insome embodiments, the second immune cell (e.g., TIL) expansion can proceed for about 7 days to about 13 days. In some embodiments, the second immune cell (e.g., TIL) expansion can proceed for about 8 days to about 13 days. In some embodiments, the second immune cell (e.g., TIL) expansion can proceed for about 9 days to about 13 days. In some embodiments, the second immune cell (e.g., TIL) expansion can proceed for about 10 days to about 13 days. In some embodiments, the second immune cell (e.g., TIL) expansion can proceed for about 11 days to about 13 days. In some embodiments, the second immune cell (e.g., TIL) expansion can proceed for about 12 days to about 13 days. In some embodiments, the second immune cell (e.g., TIL) expansion can proceed for about 7 days to about 12 days. In some embodiments, the second immune cell (e.g., TIL) expansion can proceed for about 8 days to about 12 days. In some embodiments, the second immune cell (e.g., TIL) expansion can proceed for about 9 days to about 12 days. In some embodiments, the second immune cell (e.g., TIL) expansion can proceed for about 10 days to about 12 days. In some embodiments, the second immune cell (e.g., TIL) expansion can proceed for about 11 days to about 12 days. In some embodiments, the second immune cell (e.g., TIL) expansion can proceed for about 12 days. In some embodiments, the second immune cell (e.g., TIL) expansion can proceed for about 13 days. In some embodiments, the second immune cell (e.g., TIL) expansion can proceed for about 14 days.

[0130] In an embodiment, the second expansion can be performed in a gas permeable container using the methods of the present disclosure. For example, immune cells (e.g., TILs) can be rapidly expanded using non-specific T-cell receptor stimulation in the presence of IL-2, IL-7, IL-15 and / or IL-12. The non-specific T-cell receptor stimulus can 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, N.J. or Miltenyi Biotech, Auburn, Calif.) or UHCT-1 (commercially available from BioLegend, San Diego, Calif., USA). The immune cell (e.g., TIL) expansion process can further comprise further stimulation of the immune cells (e.g., TILs) in vitro by including one or more antigens during the second expansion, wherein the antigen can include antigenic portions thereof, such as epitope(s), of the cancer, which can be optionally expressed from a vector, such as a human leukocyte antigen A2 (HLA-A2) binding peptide, e.g., 0.3 pM MART-1 :26-35 (27 L) or gpl00:209-217 (210M), optionally in the presence of a T-cell growth factor, such as 300 lU / mL IL-2 or IL- 15. Other suitable antigens may include, e.g., NY-ESO-1, TRP-1, TRP-2, tyrosinase cancer antigen, MAGE-A3, SSX-2, andVEGFR2, or antigenic portions thereof. Immune cells (e.g., TILs) may also be rapidly expanded by re-stimulation with the same antigen(s) of the cancer pulsed onto HLA-A2-expressing antigen-presenting cells. Alternatively, the immune cells (e.g., TILs) can be further re-stimulated with, e.g., example, irradiated, autologous lymphocytes or with irradiated HLA-A2+ allogeneic lymphocytes and IL-2. In some embodiments, the re-stimulation occurs as part of the second expansion. In some embodiments, the second expansion occurs in the presence of irradiated, autologous lymphocytes or with irradiated HLA-A2+ allogeneic lymphocytes and IL-2.

[0131] In some embodiments, the culture medium includes a CD3 agonist (such as a CD3 agonist antibody) (e.g., an anti-CD3 antibody). An anti-CD3 antibody in combination with IL-2 induces T cell activation and cell division in the immune cell (e.g., TIL) population. This effect can be seen with full length antibodies as well as Fab and F(ab')2 fragments, with the former being generally preferred; see, e.g., Tsoukas et al. (1985) J. Immunol. 135: 1719, herein incorporated by reference in its entirety for all purposes.

[0132] There are a number of suitable anti-human CD3 antibodies that can be used, including anti-human CD3 polyclonal and monoclonal antibodies from various mammals, including, but not limited to, murine, human, primate, rat, and canine antibodies. In particular embodiments, the OKT3 anti-CD3 antibody is used (commercially available from Ortho-McNeil, Raritan, N.J. or Miltenyi Biotech, Auburn, Calif.).

[0133] In some embodiments, the culture medium comprises IL-2 or a variant thereof. In some embodiments, the IL is recombinant human IL-2 (rhIL-2). In some embodiments, the culture medium comprises about 10,000 lU / mL of IL-2, about 9,000 lU / mL of IL-2, about 8,000 lU / mL of IL-2, about 7,000 lU / mL of IL-2, about 6000 lU / mL of IL-2, or about 5,000 lU / mL of IL-2. In some embodiments, the culture medium comprises about 2,000 lU / mL of IL-2 to about 3,000 lU / mL of IL-2. In some embodiments, the culture medium comprises about 3000 lU / mL of IL-2. In some embodiments, the culture medium comprises about 2000 lU / mL of IL-2. In an embodiment, the culture medium comprises about 1000 lU / mL, about 1500 lU / mL, about 2000 lU / mL, about 2500 lU / mL, about 3000 lU / mL, about 3500 lU / mL, about 4000 lU / mL, about 4500 lU / mL, about 5000 lU / mL, about 5500 lU / mL, about 6000 lU / mL, about 6500 lU / mL, about 7000 lU / mL, about 7500 lU / mL, or about 8000 lU / mL of IL-2. In some embodiments, the culture medium comprises between 1000 and 2000 lU / mL or between 2000 and 3000 lU / mL of IL-2. In some embodiments, the cell culture medium comprises a CD3 agonist (such as a CD3agonist antibody) (e.g., an 0KT3 antibody). In some embodiments, the cell culture medium comprises about 30 ng / mL of CD3 agonist (such as a CD3 agonist antibody) (e.g., OKT3 antibody). In some embodiments, the cell culture medium comprises 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 pg / mL of CD3 agonist (such as a CD3 agonist antibody) (e.g., OKT3 antibody). In some embodiments, the cell culture medium comprises between 0.1 ng / mL and 1 ng / mL, between 1 ng / mL and 5 ng / mL, between 5 ng / mL and 10 ng / mL, between 10 ng / mL and 20 ng / mL, between 20 ng / mL and 30 ng / mL, between 30 ng / mL and 40 ng / mL, between 40 ng / mL and 50 ng / mL, and between 50 ng / mL and 100 ng / mL of CD3 agonist (such as a CD3 agonist antibody) (e.g., OKT3 antibody).

[0134] In some embodiments, a combination of IL-2, IL-7, IL-15, and / or IL-21 are employed as a combination during the second expansion. In some embodiments, IL-2, IL-7, IL- 15, and / or IL-21 as well as any combinations thereof can be included during the second expansion. In some embodiments, a combination of IL-2, IL-15, and IL-21 are employed as a combination during the second expansion. In some embodiments, IL-2, IL-15, and IL-21 as well as any combinations thereof can be included.

[0135] In some embodiments, the culture medium comprises IL-12. In some embodiments, the culture medium comprises about 500 lU / mL of IL-12, about 400 lU / mL of IL-12, about 300 lU / mL of IL- 12, about 200 lU / mL of IL- 12, about 180 lU / mL of IL- 12, about 160 lU / mL of IL- 12, about 140 lU / mL of IL- 12, about 120 lU / mL of IL- 12, or about 100 lU / mL of IL- 12. In some embodiments, the first expansion culture medium comprises about 500 lU / mL of IL- 12 to about 100 lU / mL of IL-12. In some embodiments, the culture medium comprises about 400 lU / mL of IL-12 to about 100 lU / mL of IL-12. In some embodiments, the culture medium comprises about 300 lU / mL of IL- 12 to about 100 lU / mL of IL- 12. In some embodiments, the culture medium comprises about 200 lU / mL of IL-12. In some embodiments, the culture medium comprises about 180 lU / mL of IL-12. In an embodiment, the culture medium further comprises IL-12. In a preferred embodiment, the culture medium comprises about 180 lU / mL of IL-12.

[0136] In some embodiments, the culture medium comprises IL-15. In some embodiments, the culture medium comprises about 500 lU / mL of IL-15, about 400 lU / mL of IL-15, about 300lU / mL of IL-15, about 200 lU / mL of IL-15, about 180 lU / mL of IL-15, about 160 lU / mL of IL- 15, about 140 lU / mL of IL-15, about 120 lU / mL of IL-15, or about 100 lU / mL of IL-15. In some embodiments, the culture medium comprises about 500 lU / mL of IL-15 to about 100 lU / mL of IL-15. In some embodiments, the culture medium comprises about 400 lU / mL of IL- 15 to about 100 lU / mL of IL-15. In some embodiments, the culture medium comprises about 300 lU / mL of IL-15 to about 100 lU / mL of IL-15. In some embodiments, the culture medium comprises about 200 lU / mL of IL-15. In some embodiments, the culture medium comprises about 180 lU / mL of IL-15. In an embodiment, the culture medium further comprises IL-15. In a preferred embodiment, the culture medium comprises about 180 lU / mL of IL-15.

[0137] In some embodiments, the culture medium comprises IL-18. In some embodiments, the culture medium comprises about 500 lU / mL of IL-18, about 400 lU / mL of IL-18, about 300 lU / mL of IL- 18, about 200 lU / mL of IL- 18, about 180 lU / mL of IL- 18, about 160 lU / mL of IL- 18, about 140 lU / mL of IL-18, about 120 lU / mL of IL-18, or about 100 lU / mL of IL-18. In some embodiments, the culture medium comprises about 500 lU / mL of IL- 18 to about 100 lU / mL of IL-18. In some embodiments, the culture medium comprises about 400 lU / mL of IL-18 to about 100 lU / mL of IL-18. In some embodiments, the culture medium comprises about 300 lU / mL of IL-18 to about 100 lU / mL of IL-18. In some embodiments, the culture medium comprises about 200 lU / mL of IL-18. In some embodiments, the culture medium comprises about 180 lU / mL of IL- 18. In an embodiment, the culture medium further comprises IL- 18. In a preferred embodiment, the culture medium comprises about 180 lU / mL of IL-18.

[0138] In some embodiments, the culture medium comprises IL -21. In some embodiments, the culture medium comprises about 20 lU / mL of IL-21, about 15 lU / mL of IL-21, about 12 lU / mL of IL-21, about 10 lU / mL of IL-21, about 5 lU / mL of IL-21, about 4 lU / mL of IL-21, about 3 lU / mL of IL-21, about 2 lU / mL of IL-21, about 1 lU / mL of IL-21, or about 0.5 lU / mL of IL-21. In some embodiments, the culture medium comprises about 20 lU / mL of IL-21 to about 0.5 lU / mL of IL-21. In some embodiments, the culture medium comprises about 15 lU / mL of IL- 21 to about 0.5 lU / mL of IL -21. In some embodiments, the culture medium comprises about 12 lU / mL of IL-21 to about 0.5 lU / mL of IL-2L In some embodiments, the culture medium comprises about 10 lU / mL of IL-21 to about 0.5 lU / mL of IL -21. In some embodiments, the culture medium comprises about 5 lU / mL of IL-21 to about 1 lU / mL of IL-21. In some embodiments, the culture medium comprises about 2 lU / mL of IL-21. In some embodiments, theculture medium comprises about 1 TU / mL of IL-21 . In some embodiments, the culture medium comprises about 0.5 lU / mL of IL-21. In an embodiment, the culture medium further comprises IL-21. In a preferred embodiment, the cell culture medium comprises about 1 lU / mL of IL-21.

[0139] Also contemplated for the culture medium are combinations of interleukins, such as but not limited to, IL-2, IL-12, IL-15, IL-18, and / or IL-2L Other cytokines are also contemplated, such as IL-23, IL-27, IL-35, IL-39, IL-18, IL-36, IL-37, IL-38, IFN-alpha, IFN- beta, IFN-gamma, or a combination thereof along with IL-2, IL-12, IL-15, IL-18, and / or IL-21. Antibodies, such as Th2 blocking reagents, are also contemplated, such as but not limited to, IL- 4 (aIL4), anti-IL-4 (aIL4R), anti-IL-5R (aIL5R), anti-IL-5 (aIL5), anti-IL13R (aIL13R), or anti- IL13 (aIL13).

[0140] In some embodiments, the antigen-presenting feeder cells (APCs) are PBMCs. In some embodiments, the ratio of immune cells (e.g., TILs) to PBMCs and / or APCs in the rapid expansion and / or the second expansion is about 1 to 25, about 1 to 50, about 1 to 100, about 1 to 125, about 1 to 150, about 1 to 175, about 1 to 200, about 1 to 225, about 1 to 250, about 1 to 275, about 1 to 300, about 1 to 325, about 1 to 350, about 1 to 375, about 1 to 400, or about 1 to 500. In some embodiments, the ratio of immune cells (e.g., TILs) to PBMCs or APCs in the rapid expansion and / or the second expansion is between 1 to 50 and 1 to 300. In some embodiments, the ratio of immune cells (e.g., TILs) to PBMCs in the rapid expansion and / or the second expansion is between 1 to 100 and 1 to 200.

[0141] For example, the feeder cells can comprise a pool of PBMCs from multiple donors. In some embodiments, PBMCs comprise buffy coat cells (white blood cells) obtained by Ficoll density gradient centrifugation of a blood samples of multiple donors. In some embodiments, PBMCs comprising buffy coat cells of multiple donors are pooled. In some embodiments, the number of donor preparations to be pooled can be from 2-15 or more, from 5 to 10, from 10 to!5, from 8 to 12, or from 9 to 11. In some embodiments, PBMCs from 10 donors are pooled.

[0142] It has been found using an automated apheresis process to obtain PBMCs that recovery and viability of PBMCs is improved and the number of donors can be reduced. In some embodiments, PBMCs comprise white blood cells from apheresis. An exemplary, non-limiting system to produce a suitable apheresis product is the Sefia Cell Processing System (Cytiva). In some embodiments, the PBMCs are obtained commercially. In some embodiments, PBMCs comprising apheresis products of multiple donors are pooled. In some embodiments, the numberof donor products to be pooled can be from 2-15 or more. In some embodiments, the number of donors is from 2 to 8 or from 2 to 6 or from 2 to 4 donors. In some embodiments, PBMCs comprise apheresis products from 3 donors.

[0143] In some embodiments, the PBMCs are cryopreserved. Cryopreservation enables prescreening and PBMC inventory maintenance and reduces the number of donors needed for immune cell (e.g., TIL) manufacture.

[0144] In some embodiments, REP or the second expansion is performed in flasks with the bulk immune cells (e.g., TILs) being mixed with a 100- or 200-fold excess of inactivated feeder cells, 30 mg / mL OKT3 anti-CD3 antibody, and 3000 lU / mL IL-2. Media replacement can be done (generally 2 / 3 media replacement via respiration with fresh media) until the cells are transferred to an alternative growth chamber. Alternative growth chambers include G-REX flasks and gas permeable containers as more fully discussed below.

[0145] In some embodiments, the second expansion (which can include processes referred to as the REP process) is shortened to 7-14 days. In some embodiments, the second expansion is shortened to 11 days.

[0146] In some embodiments, REP and / or the second expansion may be performed using T- 175 flasks and gas permeable bags as previously described (Tran et al. (2008) J.Inimunother.3 .742-5V., Dudley et al. (2003) J. Immunother 26:332-42, each of which is herein incorporated by reference in its entirety for all purposes) or gas permeable cultureware (G-Rex flasks). In some embodiments, the second expansion (including expansions referred to as rapid expansions) is performed in T-175 flasks, and about IxlO6immune cells (e.g., TILs) suspended in 150 mL of media may be added to each T-175 flask. The immune cells (e.g., TILs) may be cultured in a 1 to 1 mixture of CM and AIM-V medium, supplemented with 3000 IU per mL of IL-2 and 30 ng per ml of anti-CD3. The T-175 flasks may be incubated at 37° C. in 5% CO2.Half the media may be exchanged on day 5 using 50 / 50 medium with 3000 IU per mL of IL-2. In some embodiments, on day 7 cells from two T-175 flasks may be combined in a 3 L bag and 300 mL of AIM V with 5% human AB serum and 3000 IU per mL of IL-2 was added to the 300 ml of immune cell (e.g., TIL) suspension. The number of cells in each bag can be counted every day or two and fresh media can be added to keep the cell count between 0.5 and 2.0xl06cells / mL.

[0147] In some embodiments, the second expansion may be performed in 500 mL capacity gas permeable flasks with 100 cm gas-permeable silicon bottoms (G-Rex 100, commerciallyavailable from Wilson Wolf Manufacturing Corporation, New Brighton, Minn., USA), 5xl06or 10xl06immune cells (e.g., TILs) may be cultured with PBMCs in 400 mL of 50 / 50 medium, supplemented with 5% human AB serum, 3000 IU per mL of IL-2 and 30 ng per mL of anti-CD3 (OKT3). The G-Rex 100 flasks may be incubated at 37°C. in 5% CO2. On day 5, 250 mL of supernatant may be removed and placed into centrifuge bottles and centrifuged at 1500 rpm (491xg) for 10 minutes. The immune cell (e.g., TIL) pellets may be re-suspended with 150 mL of fresh medium with 5% human AB serum, 3000 IU per mL of IL-2, and added back to the original G-Rex 100 flasks. When immune cells (e.g., TILs) are expanded serially in G-Rex 100 flasks, on day 7 the immune cells (e.g., TILs) in each G-Rex 100 may be suspended in the 300 mL of media present in each flask and the cell suspension may be divided into 3 100 mL aliquots that may be used to seed 3 G-Rex 100 flasks. Then 150 mL of AIM-V with 5% human AB serum and 3000 IU per mL of IL-2 may be added to each flask. The G-Rex 100 flasks may be incubated at 37° C. in 5% CO2 and after 4 days 150 mL of AIM-V with 3000 IU per mL of IL-2 may be added to each G-REX 100 flask. The cells may be harvested on day 14 of culture.

[0148] In some embodiments, the second expansion (including expansions referred to as REP) is performed in flasks with the bulk immune cells (e.g., TILs) being mixed with a 100- or 200-fold excess of inactivated feeder cells, 30 mg / mL OKT3 anti-CD3 antibody and 3000 lU / mL IL-2 in 150 mL media. In some embodiments, media replacement is done until the cells are transferred to an alternative growth chamber. In some embodiments, 2 / 3 of the media is replaced by respiration with fresh media. In some embodiments, alternative growth chambers include G- REX flasks and gas permeable containers as more fully discussed below.

[0149] In some embodiments, the second expansion (including expansions referred to as REP) is performed and further comprises a step wherein immune cells (e.g., TILs) are selected for superior tumor reactivity. Any selection method known in the art may be used. For example, the methods described in US 2016 / 0010058 Al, herein incorporated by reference in its entirety for all purposes, may be used for selection of immune cells (e.g., TILs) for superior tumor reactivity.

[0150] Optionally, a cell viability assay can be performed after the second expansion (including expansions referred to as the REP expansion), using standard assays known in the art. For example, a trypan blue exclusion assay can be done on a sample of the bulk immune cells (e.g., TILs), which selectively labels dead cells and allows a viability assessment. In someembodiments, immune cell (e.g., TIL) samples can be counted and viability determined using a Cellometer K2 automated cell counter (Nexcelom Bioscience, Lawrence, Mass.).

[0151] In some embodiments, the second expansion (including expansions referred to as REP) of immune cells (e.g., TILs) can be performed using T-175 flasks and gas-permeable bags as previously described (Tran et al. (2008) J. Immunother. 31 :742-751, and Dudley et al. (2003) J. Immunother. 26:332-342, each of which is herein incorporated by reference in its entirety for all purposes) or gas-permeable G-Rex flasks. In some embodiments, the second expansion is performed using flasks. In some embodiments, the second expansion is performed using gas- permeable G-Rex flasks. In some embodiments, the second expansion is performed in T-175 flasks, and about IxlO6immune cells (e.g., TILs) are suspended in about 150 mL of media and this is added to each T-175 flask. The immune cells (e.g., TILs) may be cultured with irradiated (50 Gy) allogeneic PBMC as feeder cells at a ratio of 1 to 100 and the cells may be cultured in a 1 to 1 mixture of CM and AIM-V medium (50 / 50 medium) and supplemented with 3000 lU / mL of IL-2 and 30 ng / mL of anti-CD3. The T-175 flasks may be incubated at 37° C. in 5% CO2. In some embodiments, half the media may be changed on day 5 using 50 / 50 medium with 3000 lU / mL of IL-2. In some embodiments, on day 7, cells from 2 T-175 flasks may be combined in a 3 L bag and 300 mL of AIM-V with 5% human AB serum and 3000 lU / mL of IL-2 is added to the 300 mL of immune cell (e.g., TIL) suspension. The number of cells in each bag may be counted every day or two and fresh media can be added to keep the cell count between about 0.5 and about 2.0xl06cells / mL.

[0152] In some embodiments, the second expansion (including expansions referred to as REP) are performed in 500 mL capacity flasks with 100 cm2gas-permeable silicon bottoms (G- Rex 100, Wilson Wolf), about 5xl06or 10xl06immune cells (e.g., TILs) may be cultured with irradiated allogeneic PBMC at a ratio of 1 to 100 in 400 mL of 50 / 50 medium, supplemented with 3000 lU / mL of IL-2 and 30 ng / mL of anti-CD3. The G-Rex 100 flasks are incubated at 37° C. in 5% CO2. In some embodiments, on day 5, 250 mL of supernatant is removed and placed into centrifuge bottles and centrifuged at 1500 rpm (491 g) for 10 minutes. The immune cell (e.g., TIL) pellets can then be resuspended with 150 mL of fresh 50 / 50 medium with 3000 lU / mL of IL-2 and added back to the original G-Rex 100 flasks. In embodiments where immune cells (e.g., TILs) are expanded serially in G-Rex 100 flasks, on day 7 the immune cells (e.g., TILs) in each G-Rex 100 may be suspended in the 300 mL of media present in each flask and the cellsuspension may be divided into three 100 mL aliquots that are used to seed 3 G-Rex 100 flasks. Then 150 mL of AIM-V with 5% human AB serum and 3000 lU / mL of IL-2 may be added to each flask. The G-Rex 100 flasks may be incubated at 37° C. in 5% CO2 and after 4 days 150 mL of AIM-V with 3000 lU / mL of IL-2 may be added to each G-Rex 100 flask. The cells may be harvested on day 14 of culture.

[0153] In some embodiments, the second expansion is performed in a closed system bioreactor. In some embodiments, a closed system is employed for the immune cell (e.g., TIL) expansion, as described herein. In some embodiments, a single bioreactor is employed. In some embodiments, the single bioreactor employed is for example G-REX- 10 or a G-REX- 100 or the device of WO 2018 / 130845. In some embodiments, the closed system bioreactor is a single bioreactor.

[0154] In some embodiments, the second expansion procedures described herein, as well as those referred to as REP use an excess of feeder cells during REP immune cell (e.g., TIL) expansion or during the second expansion. In many embodiments, the feeder cells may be peripheral blood mononuclear cells (PBMCs) obtained from standard whole blood units from healthy blood donors. The PBMCs may be obtained using standard methods such as Ficoll-Paque gradient separation.

[0155] In some embodiments, the PBMCs are allogenic, wherein the allogenic PBMCs can be inactivated, either via irradiation or heat treatment, and used in the REP procedures.

[0156] In some embodiments, PBMCs are considered replication incompetent and accepted for use in the immune cell (e.g., TIL) expansion procedures described herein if the total number of viable cells on day 14 is less than the initial viable cell number put into culture on day 0 of the REP and / or day 0 of the second expansion (i.e., the start day of the second expansion).

[0157] In some embodiments, PBMCs are considered replication incompetent and accepted for use in the immune cell (e.g., TIL) expansion procedures described herein if the total number of viable cells, cultured in the presence of OKT3 and IL-2, on day 7 and day 14 has not increased from the initial viable cell number put into culture on day 0 of the REP and / or day 0 of the second expansion (i.e., the start day of the second expansion). In some embodiments, the PBMCs are cultured in the presence of 30 ng / mL OKT3 antibody and 3000 lU / mL IL-2.

[0158] In some embodiments, PBMCs are considered replication incompetent and accepted for use in the immune cell (e.g., TIL) expansion procedures described herein if the total numberof viable cells, cultured in the presence of 0KT3 and IL-2, on day 7 and day 14 has not increased from the initial viable cell number put into culture on day 0 of the REP and / or day 0 of the second expansion (i.e., the start day of the second expansion). In some embodiments, the PBMCs may be cultured in the presence of 5-60 ng / mL OKT3 antibody and 1000-6000 lU / mL IL-2. In some embodiments, the PBMCs may be cultured in the presence of 10-50 ng / mL OKT3 antibody and 2000-5000 lU / mL IL-2. In some embodiments, the PBMCs may be cultured in the presence of 20-40 ng / mL OKT3 antibody and 2000-4000 lU / ml IL-2. In some embodiments, the PBMCs may be cultured in the presence of 25-35 ng / mL OKT3 antibody and 2500-3500 lU / mL IL-2.

[0159] 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 some embodiments, the ratio of immune cells (e.g., TILs) to antigen-presenting feeder cells in the second expansion is about 1 to 25, about 1 to 50, about 1 to 100, about 1 to 125, about 1 to 150, about 1 to 175, about 1 to 200, about 1 to 225, about 1 to 250, about 1 to 275, about 1 to 300, about 1 to 325, about 1 to 350, about 1 to 375, about 1 to 400, or about 1 to 500. In some embodiments, the ratio of immune cells (e.g., TILs) to antigen-presenting feeder cells in the second expansion is between 1 to 50 and 1 to 300. In some embodiments, the ratio of immune cells (e.g., TILs) to antigen-presenting feeder cells in the second expansion is between 1 to 100 and 1 to 200.

[0160] In some embodiments, the second expansion procedures described herein use a ratio of about 2.5xl09feeder cells to about lOOxlO6immune cells (e.g., TILs). In some embodiments, the second expansion procedures described herein use a ratio of about 2.5xl09feeder cells to about 50x106immune cells (e.g., TILs). In some embodiments, the second expansion procedures described herein use about 2.5xl09feeder cells to about 25xl06immune cells (e.g., TILs).

[0161] In some embodiments, the second expansion procedures described herein use an excess of feeder cells during the second expansion. In some embodiments, the feeder cells are peripheral blood mononuclear cells (PBMCs) obtained from standard whole blood units from healthy blood donors. The PBMCs can be obtained using standard methods such as FicolLPaque gradient separation. In an embodiment, artificial antigen-presenting (aAPC) cells are used in place of PBMCs.

[0162] In some embodiments, the allogenic PBMCs are inactivated, either via irradiation or heat treatment, and used in the immune cell (e.g., TIL) expansion procedure. In someembodiments, artificial antigen presenting cells are used in the second expansion as a replacement for, or in combination with, PBMCs.

[0163] Alternatively, using combinations of cytokines for the rapid expansion and or second expansion of immune cells (e.g., TILs) is additionally possible, with combinations of two or more of IL-2, IL-15, and IL-21 as is generally outlined in WO 2015 / 189356 and WO 2015 / 189357, each of which is herein incorporated by reference in its entirety for all purposes. 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, with the latter finding particular use in many embodiments. The use of combinations of cytokines specifically favors the generation of lymphocytes, and in particular T- cells as described therein.

[0164] In some embodiments, the second expansion can comprise a static expansion followed by a dynamic expansion. The static expansion can be for any suitable amount of time. Likewise, the dynamic expansion can be for any suitable amount of time. In some methods, the static expansion is for about 5 to about 7 days, about 4 to about 7 days, about 3 to about 7 days, about 2 to about 7 days, about 4 to about 8 days, about 3 to about 9 days, about 2 to about 10 days, about 5 to about 8 days, or about 5 to about 9 days, or about 5 to about 10 days. In some methods, the dynamic expansion is for about 7 to about 9 days, about 6 to about 9 days, about 5 to about 9 days, about 4 to about 9 days, about 3 to about 9 days, about 2 to about 9 days, about 6 to about 10 days, about 5 to about 11 days, about 4 to about 12 days, about 7 to about 10 days, about 7 to about 11 days, or about 7 to about 12 days. In some such methods, the dynamic expansion comprises rocking the first cells. The rocking can be at any suitable rocking angle and any suitable rocking speed. See, e.g., WO 2022 / 271847, herein incorporated by reference in its entirety for all purposes. In one example, the rocking angle can be about 8 degrees.

[0165] The static expansion can be in any suitable working volume of cell culture medium as disclosed in more detail elsewhere herein. Likewise, the dynamic expansion can be in any suitable volume of cell culture medium as disclosed in more detail elsewhere herein. For example, the static expansion can be performed in a working volume of about 500 mb to about 2500 mL, about 500 mL to about 2250mL, about 625 mL to about 2000 mL, about 1500 mL to about 2500 mL, about 1500 mL to about 2250 mL, about 1750 mL to about 2250 mL, about 1750 mL to about 2500 mL, or about 500 mL to about 750 mL. In some methods, the static expansion is performed in a working volume of about 1500 mL to about 2500 mL or about 1750mL to about 2250 mL if the first population of TILs is greater than about 3xl06cells or greater than or equal to about 3xl06cells or between about 3xl06cells and about 20xl06cells or between about 3xl06cells and about 17.5xl06cells. In some methods, the static expansion is performed in a working volume of about 500 mL to about 750 mL if the first population of TILs is less than about 3xl06cells or less than or equal to about 3xl06cells or between about 0.75xl06cells and about 3xl06cells. In some methods, the static expansion is performed in a working volume of about 2000 mL if the first population of TILs is greater than about 3xl06cells or greater than or equal to about 3xl06cells or between about 3xl06cells and about 20xl06cells or between about 3xl06cells and about 17.5xl06cells. In some methods, the static expansion is performed in a working volume of about 625 mL if the first population of TILs is less than about 3xl06cells or less than or equal to about 3xl06cells or between about 0.75xl06cells and about 3x106cells. In some methods, the cells that are counted are CD3+ cells.

[0166] The dynamic expansion can be in any suitable working volume of cell culture medium as disclosed in more detail elsewhere herein. For example, the dynamic expansion can be performed in a working volume of about 500 mL to about 4000 mL, about 500 mL to about 3600 mL, about 750 mL to about 4000 mL, about 750 mL to about 3600 mL, about 2400 mL to about 4000 mL, about 2400 mL to about 3600 mL, about 2800 mL to about 3600 mL, about 2800 mL to about 4000 mL, about 500 mL to about 1500 mL, about 500 mL to about 1250 mL, about 750 mL to about 1250 mL, or about 750 mL to about 1500 mL. In some methods, the dynamic expansion is performed in a working volume of about 2400 mL to about 4000 mL or about 2800 mL to about 3600 mL if the first population of TILs is greater than about 3xl06cells or greater than or equal to about 3xl06cells or between about 3xl06cells and about 20xl06cells or between about 3xl06cells and about 17.5xl06cells. In some methods, the static expansion is performed in a working volume of about 500 mL to about 1500 mL or about 750 mL to about 1250 mL if the first population of TILs is less than about 3xl06cells or less than or equal to about 3xl06cells or between about 0.75xl06cells and about 3xl06cells. In some methods, the dynamic expansion is performed in a working volume of about 3200 mL if the first population of TILs is greater than about 3xl06cells or greater than or equal to about 3xl06cells or between about 3xl06cells and about 20xl06cells or between about 3xl06cells and about 17.5xl06cells.In some methods, the static expansion is performed in a working volume of about 1000 mL if the first population of TILs is less than about 3xl06cells or less than or equal to about 3xl06cells orbetween about 0.75x106cells and about 3x106cells. In some methods, the cells that are counted are CD3+ cells.

[0167] In some methods, the dynamic expansion comprises perfusion steps as disclosed in more detail elsewhere herein. For example, the perfusion steps can be at fourth, fifth, and / or sixth time points, wherein the perfusion comprises removing spent cell culture medium while simultaneously adding fresh cell culture medium in equal parts to maintain a constant culture volume. In some methods, the fourth time point is days 16-18, the fifth time point is days 18-20, and the sixth time point is days 20-21 (e.g., where day 15 is the start of dynamic expansion, and / or where day 0 is tumor digest and / or day 1 is retrieving the tumor digest). In some methods, the fourth time point is days 20-21, the fifth time point is days 22-23, and the sixth time point is days 24-30. In some methods, the perfusion at the fourth time point is about 0.6 to about 1 L / day, the perfusion at the fifth time point is about 1.4 to about 1.8 L / day, and the perfusion at the sixth time point is about 3 to about 3.4 L / day if the first population of TILs is greater than about 3xl06cells or greater than or equal to about 3xl06cells or between about 3xl06cells and about 20x106cells or between about 3xl06cells and about 17.5xl06cells. In some methods, wherein the perfusion at the fourth time point is about 0.2 to about 0.3 L / day, the perfusion at the fifth time point is about 0.4 to about 0.6 L / day, and the perfusion at the sixth time point is about 0.8 to about 1.2 L / day if the first population of TILs is less than about 3xl06cells or less than or equal to about 3xl06cells or between about 0.75xl06cells and about 3xl06cells. In some methods, the perfusion at the fourth time point is about 0.8 L / day, the perfusion at the fifth time point is about 1.6 L / day, and the perfusion at the sixth time point is about 3.2 L / day if the first population of TILs is greater than about 3xl06cells or greater than or equal to about 3xl06cells or between about 3xl06cells and about 20xl06cells or between about 3xl06cells and about 17.5xl06cells. In some methods, the perfusion at the fourth time point is about 0.25 L / day, the perfusion at the fifth time point is about 0.5 L / day, and the perfusion at the sixth time point is about 1 L / day if the first population of TILs is less than about 3xl06cells or less than or equal to about 3xl06cells or between about 0.75xl06cells and about 3xl06cells. In some methods, the cells that are counted are CD3+ cells.E. Harvesting of Immune Cells or TILs

[0168] After expansion, the cells can be harvested. In some embodiments the immune cells (e.g., TILs) are harvested after one, two, three, four or more expansion steps. In some embodiments the immune cells (e.g., TILs) are harvested after two expansion steps. The harvested immune cells (e.g., TILs) can also be filtered and / or washed to reduce process-related impurities. For example, the cells can be washed multiple times using phosphate-buffered saline (PBS) supplemented with 5% albumin (human) (HSA) using an automated cell-processing system (Sefia™ from Cytiva LifeSciences). The cells can then be concentrated and resuspended in PBS supplemented with 5% HSA, in preparation for formulation.

[0169] In some methods, the harvesting is performed using a harvest medium comprising at least 2%, at least 3%, at least 4%, or at least 5% human serum albumin (HSA) and PBS. For example, the harvesting can be performed using a harvest medium comprising about 5% human serum albumin (HSA) and PBS. In some methods, harvesting further comprises formulating the immune cells (e.g., TILs) with HSA and DMSO. For example, the immune cell (e.g., TIL) formulation can, in some embodiments, comprise no more than 8%, no more than 7%, no more than 6%, no more than 5%, no more than 4%, no more than 3%, or no more than 2.5% HSA and no more than 9%, no more than 8%, no more than 7%, no more than 6%, or no more than 5% DMSO. In some methods, the immune cell (e.g., TIL) formulation comprises about 2.5% HSA and about 5% DMSO. In some methods, the formulating comprises adding HSA and DMSO to the immune cells (e.g., TILs) in a closed system. The harvested immune cells (e.g., TILs) can, in some embodiments, be cryopreserved.

[0170] In some methods, the method further comprises assessing the potency of the harvested immune cells (e.g., TILs). See, e.g., WO 2022 / 271881, herein incorporated by reference in its entirety for all purposes. In certain embodiments, the TIL product will have been assayed for the presence of and will contain T-cells expressing a combination of markers, the combination comprising two or more of: CD107a; CD137; TNF-a; and IFN-y. In certain embodiments, the combination comprises: CD107a and CD137; or CD107a and TNF-a; or CD107a and IFN-y; or CD137 and TNF-a; or CD137 and IFN-y; or TNF-a and IFN-y. In certain embodiments, the combination comprises three or more of: CD107a; CD137; TNF-a; and IFN-y. In certain embodiments, the combination comprises: CD107a, CD137, and TNF-a; or CD107a,CD137, and IFN-y; or CD107a, TNF-a, and IFN-y; or CD137, TNF-a, and IFN-y. In certain embodiments, the combination comprises each of CD107a, CD137, TNF-a, and IFN-y.

[0171] In certain embodiments, the TIL product will have been assayed for the presence of and will contain CD2+ T-cells expressing a combination of markers, the combination comprising two or more of: a T-cell expressing CD107a; a T-cell expressing CD137; a T-cell expressing TNF-a; and a T-cell expressing IFN-y. In some embodiments, the TIL product will have been assayed for the presence of and will contain CD3+ T-cells expressing a combination of markers, the combination comprising two or more of: a T-cell expressing CD107a; a T-cell expressing CD137; a T-cell expressing TNF-a; and a T-cell expressing IFN-y. In certain embodiments, the combination comprises: CD107a and CD137, or CD107a and TNF-a, or CD107a and IFN-y, or CD137 and TNF-a, or CD137 and IFN-y, or TNF-a and IFN-y. In certain embodiments, the combination comprises three or more of: CD107a; CD137; TNF-a; and IFN-y. In certain embodiments, the combination comprises: CD107a, CD137 and TNF-a; or CD107a, CD137 and IFN-y; or CD107a, TNF-a and IFN-y; or CD137, TNF-a and IFN-y. In certain embodiments, the combination comprises each of CD107a, CD137, TNF-a, and IFN-y.

[0172] In some embodiments, the TIL product will have been assayed for the presence of and will contain CD2+ T-cells expressing a combination of markers, the combination comprising CD 107a and IFN-y. In some embodiments, the TIL product will have been assayed for the presence of and will contain CD3+ T-cells expressing a combination of markers, the combination comprising CD107a and IFN-y.

[0173] Immune cells (e.g., TILs) can be harvested in any appropriate and sterile manner, including for example by centrifugation. Methods for immune cell (e.g., TIL) harvesting are well known in the art and any such know methods can be employed with the present process. In some embodiments, immune cells (e.g., TILs) are harvested using an automated system.

[0174] Cell harvesters 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 harvester can be employed with the present methods. In some embodiments, the cell harvester and / or cell processing systems is a membrane-based cell harvester. In some embodiments, cell harvesting is 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 vendor that canpump a solution comprising cells through a membrane or filter such as a spinning membrane or spinning filter in a sterile and / or closed system environment, allowing for continuous flow and cell processing to remove supernatant or cell culture media without pelletization. In some embodiments, the cell harvester and / or cell processing system can perform cell separation, washing, fluid-exchange, concentration, and / or other cell processing steps in a closed, sterile system.

[0175] In some embodiments, the harvest is performed from a closed system bioreactor. In some embodiments, a closed system is employed for the immune cell (e.g., TIL) expansion, as described herein. In some embodiments, a single bioreactor is employed. In some embodiments, the single bioreactor employed is for example G-REX-10 or a G-REX-100 or the device of WO 2018 / 130845, herein incorporated by reference in its entirety for all purposes. In some embodiments, the closed system bioreactor is a single bioreactor.

[0176] Harvested cells can be transferred to a container for use in administration to a patient. In some embodiments, once a therapeutically sufficient number of immune cells (e.g., TILs) are obtained using the expansion methods described above, they are transferred to a container for use in administration to a patient.III. Therapeutic Populations of Immune Cells or TILs and Methods of Treating a Subject Having Cancer

[0177] Provided herein are therapeutic populations of immune cells (e.g., TILs) obtained by the methods disclosed herein. The immune cells (e.g., TILs) produced by the methods described herein can be used in methods of treating a subject having a cancer, comprising administering a therapeutic population of cells (e.g., TILs) obtained by any of the above methods to the subject. The cancer can be any type of cancer. For example, the cancer can be melanoma, cervical cancer, non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC), cutaneous squamous cell carcinoma (cSCC), renal cancer, ovarian cancer, colorectal cancer, gastric cancer, breast cancer, or mesothelioma.

[0178] The terms “treatment,” “treating,” “treat,” and the like, refer to obtaining a desired pharmacologic and / or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and / or may be therapeutic in terms of a partial or complete cure for a disease and / or adverse effect attributable to the disease.“Treatment,” as used herein, covers any treatment of a disease in a mammal, particularly in a human, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development or progression; and (c) relieving the disease (i.e., causing regression of the disease and / or relieving one or more disease symptoms). “Treatment” is also meant to encompass delivery of an agent in order to provide for a pharmacologic effect, even in the absence of a disease or condition.

[0179] With TILs, the core element of treatment contributing to therapeutic activity can be the TILs themselves, which can target and eliminate tumor cells by a variety of methods utilized by T cells as a part of their normal function. These methods include direct methods (e.g., perforin-mediated cytotoxicity) and indirect methods (e.g., cytokine production). Other elements that can contribute to the therapy are pre-conditioning chemotherapy and optionally high dose intravenous IL-2. These elements are thought to act by supporting engraftment of T cells in the patient after infusion: initially through conditioning chemotherapy which removes competing and regulating immune cells; followed by the IL-2 component which supports survival of T cells.

[0180] As described above, the structure of the cell therapy product can be created by growing the immune cells (e.g., TILs) directly out of a digested tumor mass by means of growth supporting cell culture media and a T cell supporting growth factor IL-2. This enables tumor specific T cells to selectively survive and grow out of the tumor cell mixture, while T cells that do not recognize tumor antigens will not be stimulated and be selectively lost. The product can comprise an autologous T-cell based product where the T cells have been derived from a patient’s own cancer tissue and rapidly expanded to form a pure T cell population and T cells as defined by CD3 surface marker.

[0181] In some embodiments, immune cells (e.g., TILs) expanded using the methods described herein are administered to a patient as a pharmaceutical composition. In an embodiment, the pharmaceutical composition is a suspension of immune cells (e.g., TILs) in a sterile buffer. The immune cells (e.g., TILs) may be administered by any suitable route as known in the art. In some embodiments, the immune cells (e.g., TILs) are administered as a single intraarterial or intravenous infusion. Other suitable routes of administration include intraperitoneal, intrathecal, and intralymphatic.

[0182] Any suitable dose of immune cells (e.g., TILs) can be administered. In some embodiments, from about 2.3xlO10to about 13.7xlO10immune cells (e.g., TILs) are administered, with an average of around 7.8xlO10immune cells (e.g., TILs), particularly if the cancer is melanoma. In some embodiments, about 1.2xlO10to about 4.3xlO10of immune cells (e g., TILs) are administered. In some embodiments, about 3xlO10to about 12xlO10immune cells (e.g., TILs) are administered. In some embodiments, about 4xlO10to about lOxlO10immune cells (e.g., TILs) are administered. In some embodiments, about 5xlO10to about 8xlO10immune cells (e g., TILs) are administered. In some embodiments, about 6xlO10to about 8xlO10immune cells (e.g., TILs) are administered. In some embodiments, about 7xlO10to about 8xlO10immune cells (e.g., TILs) are administered. In some embodiments, the therapeutically effective dosage is about 2.3xlO10to about 13.7xlO10. In some embodiments, the therapeutically effective dosage is about 7.8xlO10immune cells (e.g., TILs), particularly of the cancer is melanoma. In some embodiments, the therapeutically effective dosage is about 1.2xlO10to about 4.3xlO10of immune cells (e.g., TILs). In some embodiments, the therapeutically effective dosage is about 3xlO10to about 12xlO10immune cells (e.g., TILs). In some embodiments, the therapeutically effective dosage is about 4xlO10to about lOxlO10immune cells (e.g., TILs). In some embodiments, the therapeutically effective dosage is about 5xlO10to about 8xlO10immune cells (e.g., TILs). In some embodiments, the therapeutically effective dosage is about 6xlO10to about 8xlO10immune cells (e.g., TILs). In some embodiments, the therapeutically effective dosage is about 7xlO10to about 8xlO10immune cells (e.g., TILs).

[0183] In some embodiments, the number of the immune cells (e.g., TILs) provided in the pharmaceutical compositions is about IxlO6, 2xl06, 3xl06, 4xl06, 5xl06, 6xl06, 7xl068xl06, 9xl06, IxlO7, 2xl07, 3xl07, 4xl07, 5xl07, 6xl07, 7xl07, 8xl07, 9xl07, IxlO8, 2xl08, 3xl08, 4xl08, 5xl08, 6xl08, 7xl08, 8xl08, 9xl08, IxlO9, 2xl09, 3xl09, 4xl09, 5xl09, 6xl09, 7xl09, 8xl09, 9xl09, IxlO102xlO10, 3xlO10, 4xlO10, 5xlO10, 6xlO10, 7xlO10, 8xlO10, 9xlO10, IxlO11, 2xlOn, 3xl0n, 4xlOn, 5xl0n, 6xlOn, 7xlOn, 8xl0n, 9xlOn, IxlO12, 2xl012, 3xl012, 4xl012, 5xl012, 6xl012, 7xl012, 8xl012, 9xl012, IxlO13, 2xl013, 3xl013, 4xl013, 5xl013, 6xl013, 7xl013, 8xl013, or 9xl013. In some embodiments, the number of the immune cells (e.g., TILs) provided in the pharmaceutical compositions is in the range of IxlO6to 5xl06, 5xl06to IxlO7, IxlO7to 5xl07, 5xl07to IxlO8, IxlO8to 5xl08, 5xl08to IxlO9, IxlO9to 5xl09, 5xl09to IxlO10, IxlO10to 5xlO10, 5xlO10to IxlO11, 5xl0nto IxlO12, IxlO12to 5xl012, or 5xl012to IxlO13.

[0184] In some embodiments, the concentration of the immune cells (e g., TILs) provided in the pharmaceutical compositions 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%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002%, or 0.0001% w / w, w / v or v / v of the pharmaceutical composition.

[0185] In some embodiments, the concentration of the immune cells (e g., TILs) provided in the pharmaceutical compositions is greater than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19.75%, 19.50%, 19.25% 19%, 18.75%, 18.50%, 18.25% 18%, 17.75%, 17.50%, 17.25% 17%,16.75%, 16.50%, 16.25% 16%, 15.75%, 15.50%, 15.25% 15%, 14.75%, 14.50%, 14.25% 14%,13.75%, 13.50%, 13.25% 13%, 12.75%, 12.50%, 12.25% 12%, 11.75%, 11.50%, 11.25% 11%,10.75%, 10.50%, 10.25% 10%, 9.75%, 9.50%, 9.25% 9%, 8.75%, 8.50%, 8.25% 8%, 7.75%,7.50%, 7.25% 7%, 6.75%, 6.50%, 6.25% 6%, 5.75%, 5.50%, 5.25% 5%, 4.75%, 4.50%, 4.25%, 4%, 3.75%, 3.50%, 3.25%, 3%, 2.75%, 2.50%, 2.25%, 2%, 1.75%, 1.50%, 125%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002%, or 0.0001% w / w, w / v, or v / v of the pharmaceutical composition.

[0186] In some embodiments, the concentration of the immune cells (e g., TILs) provided in the pharmaceutical compositions is in the range from about 0.0001% to about 50%, about 0.001% to about 40%, about 0.01% to about 30%, about 0.02% to about 29%, about 0.03% to about 28%, about 0.04% to about 27%, about 0.05% to about 26%, about 0.06% to about 25%, about 0.07% to about 24%, about 0.08% to about 23%, about 0.09% to about 22%, about 0.1% to about 21%, about 0.2% to about 20%, about 0.3% to about 19%, about 0.4% to about 18%, about 0.5% to about 17%, about 0.6% to about 16%, about 0.7% to about 15%, about 0.8% to about 14%, about 0.9% to about 12%, or about 1% to about 10% w / w, w / v or v / v of the pharmaceutical composition.

[0187] In some embodiments, the concentration of the immune cells (e.g., TILs) provided in the pharmaceutical compositions is in the range from about 0.001% to about 10%, about 0.01% to about 5%, about 0.02% to about 4.5%, about 0.03% to about 4%, about 0.04% to about 3.5%,about 0.05% to about 3%, about 0.06% to about 2.5%, about 0.07% to about 2%, about 0.08% to about 1.5%, about 0.09% to about 1%, or about 0.1% to about 0.9% w / w, w / v or v / v of the pharmaceutical composition.

[0188] In some embodiments, the amount of the immune cells (e.g., TILs) provided in the pharmaceutical compositions is equal to or less than 10 g, 9.5 g, 9.0 g, 8.5 g, 8.0 g, 7.5 g, 7.0 g, 6.5 g, 6.0 g, 5.5 g, 5.0 g, 4.5 g, 4.0 g, 3.5 g, 3.0 g, 2.5 g, 2.0 g, 1.5 g, 1.0 g, 0.95 g, 0.9 g, 0.85 g, 0.8 g, 0.75 g, 0.7 g, 0.65 g, 0.6 g, 0.55 g, 0.5 g, 0.45 g, 0.4 g, 0.35 g, 0.3 g, 0.25 g, 0.2 g, 0.15 g, 0.1 g, 0.09 g, 0.08 g, 0.07 g, 0.06 g, 0.05 g, 0.04 g, 0.03 g, 0.02 g, 0.01 g, 0.009 g, 0.008 g, 0.007 g, 0.006 g, 0.005 g, 0.004 g, 0.003 g, 0.002 g, 0.001 g, 0.0009 g, 0.0008 g, 0.0007 g, 0.0006 g, 0.0005 g, 0.0004 g, 0.0003 g, 0.0002 g, or 0.0001 g.

[0189] In some embodiments, the amount of the immune cells (e.g., TILs) provided in the pharmaceutical compositions is more than 0.0001 g, 0.0002 g, 0.0003 g, 0.0004 g, 0.0005 g, 0.0006 g, 0.0007 g, 0.0008 g, 0.0009 g, 0.001 g, 0.0015 g, 0.002 g, 0.0025 g, 0.003 g, 0.0035 g,0.004 g, 0.0045 g, 0.005 g, 0.0055 g, 0.006 g, 0.0065 g, 0.007 g, 0.0075 g, 0.008 g, 0.0085 g,0.009 g, 0.0095 g, 0.01 g, 0.015 g, 0.02 g, 0.025 g, 0.03 g, 0.035 g, 0.04 g, 0.045 g, 0.05 g, 0.055 g, 0.06 g, 0.065 g, 0.07 g, 0.075 g, 0.08 g, 0.085 g, 0.09 g, 0.095 g, 0.1 g, 0.15 g, 0.2 g, 0.25 g,0.3 g, 0.35 g, 0.4 g, 0.45 g, 0.5 g, 0.55 g, 0.6 g, 0.65 g, 0.7 g, 0.75 g, 0.8 g, 0.85 g, 0.9 g, 0.95 g, 1 g, 1.5 g, 2 g, 2.5, 3 g, 3.5, 4 g, 4.5 g, 5 g, 5.5 g, 6 g, 6.5 g, 7 g, 7.5 g, 8 g, 8.5 g, 9 g, 9.5 g, or 10 g-

[0190] The immune cells (e.g., TILs) provided in the pharmaceutical compositions can be effective over a wide dosage range. The exact dosage will depend upon the route of administration, the form in which the compound is administered, the gender and age of the subject to be treated, the body weight of the subject to be treated, and the preference and experience of the attending physician. The clinically established dosages of the immune cells (e.g., TILs) may also be used if appropriate. The amounts of the pharmaceutical compositions administered using the methods herein, such as the dosages of immune cells (e.g., TILs), will be dependent on the human or mammal being treated, the severity of the disorder or condition, the rate of administration, the disposition of the active pharmaceutical ingredients and the discretion of the prescribing physician.

[0191] In some embodiments, immune cells (e.g., TILs) may be administered in a single dose. Such administration may be by injection, e.g., intravenous injection. In some embodiments,immune cells (e.g., TILs) may be administered in multiple doses. Dosing may be once, twice, three times, four times, five times, six times, or more than six times per year. Dosing may be once a month, once every two weeks, once a week, or once every other day. Administration of immune cells (e.g., TILs) may continue as long as necessary.

[0192] In some embodiments, an effective dosage of immune cells (e g., TILs) is about IxlO6, 2xl06, 3xl06, 4xl06, 5xl06, 6xl06, 7xl06, 8xl06, 9xl06, IxlO7, 2xl07, 3xl07, 4xl07, 5xl07, 6xl07, 7xl07, 8xl07, 9xl07, IxlO8, 2xl08, 3xl08, 4xl08, 5xl08, 6xl08, 7xl08, 8xl08, 9xl08, IxlO9, 2xl09, 3xl09, 4xl09, 5xl09, 6xl09, 7xl09, 8xl09, 9xl09, IxlO10, 2xlO10, 3xlO10, 4xlO10, 5xlO10, 6xlO10, 7xlO108xlO10, 9xlO10, IxlO11, 2xlOn, 3xl0n, 4xlOn, 5xlOn, 6xlOn, 7xlOn, 8xl0n, 9xlOn, IxlO12, 2xl012, 3xl012, 4xl012, 5xl012, 6xl012, 7xl012, 8xl012, 9xl012, IxlO13, 2xl013, 3xl013, 4xl013, 5xl013, 6xl013, 7xl013, 8xl013, or 9xl013. In some embodiments, an effective dosage of immune cells (e g., TILs) is in the range of IxlO6to 5xl06, 5xl06to IxlO7, IxlO7to 5xl07, 5xl07to IxlO8, IxlO8to 5xl08, 5xl08to IxlO9, IxlO9to 5xl09, 5xl09to IxlO10, IxlO10to 5xlO10, 5xl010to IxlO11, 5xlOnto IxlO12, IxlO12to 5xl012, or 5xl012to IxlO13.

[0193] In some embodiments, an effective dosage of immune cells (e.g., TILs) is in the range of about 0.01 mg / kg to about 4.3 mg / kg, about 0.15 mg / kg to about 3.6 mg / kg, about 0.3 mg / kg to about 3.2 mg / kg, about 0.35 mg / kg to about 2.85 mg / kg, about 0.15 mg / kg to about 2.85 mg / kg, about 0.3 mg to about 2.15 mg / kg, about 0.45 mg / kg to about 1.7 mg / kg, about 0.15 mg / kg to about 1.3 mg / kg, about 0.3 mg / kg to about 1.15 mg / kg, about 0.45 mg / kg to about 1 mg / kg, about 0.55 mg / kg to about 0.85 mg / kg, about 0.65 mg / kg to about 0.8 mg / kg, about 0.7 mg / kg to about 0.75 mg / kg, about 0.7 mg / kg to about 2.15 mg / kg, about 0.85 mg / kg to about 2 mg / kg, about 1 mg / kg to about 1.85 mg / kg, about 1.15 mg / kg to about 1.7 mg / kg, about 1.3 mg / kg mg to about 1.6 mg / kg, about 1.35 mg / kg to about 1.5 mg / kg, about 2.15 mg / kg to about 3.6 mg / kg, about 2.3 mg / kg to about 3.4 mg / kg, about 2.4 mg / kg to about 3.3 mg / kg, about 2.6 mg / kg to about 3.15 mg / kg, about 2.7 mg / kg to about 3 mg / kg, about 2.8 mg / kg to about 3 mg / kg, or about 2.85 mg / kg to about 2.95 mg / kg.

[0194] In some embodiments, an effective dosage of immune cells (e.g., TILs) is in the range of about 1 mg to about 500 mg, about 10 mg to about 300 mg, about 20 mg to about 250 mg, about 25 mg to about 200 mg, about 1 mg to about 50 mg, about 5 mg to about 45 mg, about 10 mg to about 40 mg, about 15 mg to about 35 mg, about 20 mg to about 30 mg, about 23 mg toabout 28 mg, about 50 mg to about 150 mg, about 60 mg to about 140 mg, about 70 mg to about 130 mg, about 80 mg to about 120 mg, about 90 mg to about 110 mg, or about 95 mg to about 105 mg, about 98 mg to about 102 mg, about 150 mg to about 250 mg, about 160 mg to about 240 mg, about 170 mg to about 230 mg, about 180 mg to about 220 mg, about 190 mg to about 210 mg, about 195 mg to about 205 mg, or about 198 to about 207 mg.

[0195] An effective amount (e.g., an anti -tumor effective amount or a tumor-inhibiting effective amount or a therapeutic amount) of the immune cells (e.g., TILs) may be administered in either single or multiple doses by any of the accepted modes of administration of agents having similar utilities, including intranasal and transdermal routes, by intra-arterial injection, intravenously, intraperitoneally, parenterally, intramuscularly, subcutaneously, topically, by transplantation, or by inhalation. When “an anti-tumor effective amount,” “a tumor-inhibiting effective amount,” or “therapeutic amount” is indicated, the precise amount of the compositions to be administered can be determined by a physician with consideration of individual differences, for example, in age, weight, tumor size, extent of metastasis, and condition of the patient (subject). It can generally be stated that a pharmaceutical composition comprising the immune cells (e.g., TILs) (e.g. secondary TILs or genetically modified cytotoxic lymphocytes) described herein may be administered at a dosage of 104to 1011cells / kg body weight (e.g., 103to 106, 103to 1010, 105to 1011, 106to 1010, 106to 1011, 107to 1011, 107to 1010, 108to 1011, 108to 1010, 109to 1011, or 109to 1010cells / kg body weight), including all integer values within those ranges.Immune cells (e.g., TILs) (including in some cases, genetically modified cytotoxic lymphocytes) compositions may also be administered multiple times at these dosages. The immune cells (e.g., TILs) can be administered by using infusion techniques that are commonly known in immunotherapy (see, e.g., Rosenberg et al., (1988) New Eng. J. of Med. 319: 1676, herein incorporated by reference in its entirety for all purposes). The optimal dosage and treatment regime for a particular patient can readily be determined by one skilled in the art of medicine by monitoring the patient for signs of disease and adjusting the treatment accordingly. The term “effective amount” or “therapeutically effective amount” refers to that amount of a compound or combination of compounds as described herein that is sufficient to effect the intended application including, but not limited to, disease treatment. A therapeutically effective amount may vary depending upon the intended application (in vitro or in vivo), or the subject and disease condition being treated (e.g., the weight, age and gender of the subject), the severity of the diseasecondition, or the manner of administration. The term also applies to a dose that will induce a particular response in target cells (e.g., the reduction of platelet adhesion and / or cell migration). The specific dose will vary depending on the particular compounds chosen, the dosing regimen to be followed, whether the compound is administered in combination with other compounds, timing of administration, the tissue to which it is administered, and the physical delivery system in which the compound is carried.

[0196] All patent filings, websites, other publications, accession numbers and the like cited above or below are incorporated by reference in their entirety for all purposes to the same extent as if each individual item were specifically and individually indicated to be so incorporated by reference. If different versions of a sequence are associated with an accession number at different times, the version associated with the accession number at the effective filing date of this application is meant. The effective filing date means the earlier of the actual filing date or filing date of a priority application referring to the accession number if applicable. Likewise, if different versions of a publication, website or the like are published at different times, the version most recently published at the effective filing date of the application is meant unless otherwise indicated. Any feature, step, element, embodiment, or aspect of the invention can be used in combination with any other unless specifically indicated otherwise. Although the present invention has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be apparent that certain changes and modifications may be practiced within the scope of the appended claims.EXAMPLESExample 1. ITIL-306 Drug Substance and Drug Product Manufacturing Process.

[0197] ITIL-306 refers to a TIL product that has been genetically modified with an engineered costimulatory antigen receptor (“CoStAR”). W02022 / 016112A1, herein incorporated by reference in its entirety for all purposes, describes chimeric costimulatory antigen receptors (CoStAR) useful in adoptive cell therapy (ACT), and cells comprising the CoStAR. The CoStAR can act as a modulator of cellular activity enhancing responses to defined antigens.

[0198] ITIL-306 is an autologous product, in which each lot is manufactured from an individual patient’s resected tumor material. The starting material for ITIL-306 manufacturing is the digested cell suspension containing autologous TILs from resected tumor material. The ITIL-306 process is continuous, from drug substance to drug product, with no distinct drug substance intermediate. An overview of the manufacturing process is provided in Figure 2, and an exemplary procedure is as follows.I. Starting Material Procurement

[0199] The ITIL-306 manufacturing process begins with tumor procurement. Following resection and trimming of the tumor at the clinical site, the starting material undergoes the digestion and fdtration steps at the Tumor Hub Facility (also referred to as the Tumor Hub) as shown in Figure 3. The tumor digest may either be cryopreserved or processed fresh through the ITIL-306 manufacturing process.

[0200] Briefly, at the clinical site, the tumor is surgically resected and then trimmed to remove visibly necrotic tissue, visibly heathy or noncancerous tissue, and excess blood. The trimmed tumor is weighed, placed into a sterile bag, and then heat sealed. If a tumor is greater than 7 grams in weight, it may be split among multiple bags.

[0201] The trimmed tumor material is then prepared for transportation by introducing phosphate-buffered saline (PBS) containing human serum albumin (HSA), gentamicin, amphotericin B, and vancomycin by gravity draining it through a closed tubing connection. The bag is then shipped to the tumor hub at 1°C to 10°C (using the NanoCool™ shipper). Data supporting the shipment and storage conditions for fresh tumor (up to 54 hours at 1°C to 10°C) are not shown. The tumor bag is inspected to ensure bag integrity and confirm COI upon receipt at the tumor hub.

[0202] For the tumor digestion step, enzyme digest media (EDM) is added to the bag containing the tumor. EDM is composed of PBS containing calcium chloride, DNase 1 (dornase alfa), and collagenase. The bag containing resected tumor and digest media is then subjected to controlled, mechanical compression at a target temperature of 35°C ± 5°C using an automated device (VIA Extractor™ connected to the VIA Freeze™ from Cytiva LifeSciences), thereby facilitating mechanical and enzymatic digestion. The tumor is digested to generate a homogeneous cell suspension. The tumor digest material is then filtered using a blood filtration set in a closed system.

[0203] For shipment of fresh tumor digest, a Nanocool™ shipper is used to maintain a controlled environment. Data supporting the shipment and storage conditions of fresh tumor digest (up to 18 hours at room temperature) are not shown.

[0204] If the tumor digest material is intended to be cryopreserved, CryoStor® CS10 is added at 1 : 1 ratio to the tumor digest following the filtration step, resulting in a final concentration of approximately 5% dimethyl sulfoxide (DMSO) in the cryopreservation medium. The tumor digest is cryopreserved using a defined cooling program. The cryopreserved cell suspension is stored in the vapor-phase of liquid nitrogen (LN2) at <-130°C and transported to the GMP manufacturing site in a qualified shipper that maintains the cryopreserved cell suspension at <-130°C. Cryopreserved tumor digest is shipped using a LN2 dry vapor shipper; data supporting shipment and storage conditions of the cryopreserved tumor digest (up to 34 days at <-130°C) are not shown.2. Tumor Digest Receipt

[0205] Tumor digest is received at the Instil manufacturing facility and placed in an access- controlled room where it undergoes the receipt and inspection process to confirm COI and ensure bag integrity.3. Tumor Digest Dilution and TIL Outgrowth Seeding

[0206] For cryopreserved tumor digest, the bag is thawed using an automated thawing device before dilution. Tumor digest dilution is designed to transfer the cells out of EDM and EDM with DMSO, if cryopreserved. The cell suspension is first diluted in T cell media (TCM) supplemented with irradiated FBS, amphotericin B, gentamicin, vancomycin, and IL-2, then washed and resuspended in approximately 30 mL of the same media using an automated cellprocessing system (Sefia™ from Cytiva LifeSciences). Following resuspension, the post-wash solution is seeded, and incubated in a single-use culture bag under standard cell culture conditions (37°C, 5% CO2). Viable cell count and viability of the cell suspension are monitored during this process step.4. TIL Transduction

[0207] On Day 3 of the ITIL-306 manufacturing process, the cells are counted, and on Days 3 and 4 a controlled volume of anti-FOLRl-CoStAR Vector is added to the cell culture at a target MOI of 5.5. TIL Outgrowth

[0208] Following TIL transduction, the TIL outgrowth culture is monitored for T cell count and viability and diluted with TCM supplemented with irradiated FBS, amphotericin B, gentamicin, vancomycin, and IL-2 as part of culture maintenance.

[0209] If the resulting viable total T cell count is between 0.5 x 106to 20 x 106, the entire TIL outgrowth cell suspension is transferred to the subsequent process step. If the viable T cell count is >20 x 106, the excess T cells may be cryopreserved in CryoStor® CS10 and stored as reserve material, if needed for future processing.6. TIL Activation

[0210] Following the TIL outgrowth phase, polyclonal TIL activation is mediated using anti-CD3 and irradiated allogeneic PBMCs to provide primary and co-stimulation for T-cell activation.

[0211] For activation seeding, PBMCs are added to the viable T cells to achieve an approximate ratio of 1 : 100 to 1 :200 viable T cells to irradiated PBMCs in TCM supplemented with anti-CD3 antibody, irradiated human AB serum, and IL-2. Furthermore, a specified volume of irradiated PBMCs from the same material preparation is removed and transferred into a parallel control culture bag. The control culture bag is maintained in similar culture conditions parallel with the ITIL-306 culture. The control bag is monitored using flow cytometry to demonstrate the allogeneic PBMCs are not expanding post-irradiation. The TIL activation culture is incubated for 5 days under standard cell culture conditions (37°C, 5% CO2) and monitored for viable T cell count and viability.

[0212] When excess cryopreserved T cells from TIL outgrowth process step are used in the TIL activation process step, the cell suspension will be thawed and washed using an automated cell-processing system (Sefia™ from Cytiva LifeSciences) resuspended in TCM supplemented with irradiated human AB serum and IL-2. The cells are then seeded into a single use culture bag allowed to recover for up to 3 days under standard cell culture conditions (37°C, 5% CO2) prior to being processed through the TIL activation step.7. TIL Expansion

[0213] The activated TILs are transferred aseptically into a single-use culture bag in a functionally closed bioreactor (Xuri™ from Cytiva LifeSciences) and cultured under controlled and monitored cell culture conditions (37°C, 5% CO2). The cell suspension is provided a semi-continuous feed of TCM supplemented with IL-2 and is routinely monitored for viable T cell count and viability. The automated TIL expansion step is designed to achieve high cell densities and minimize operator variability.

[0214] TIL expansion is conducted for a minimum of 6 days. Cell growth rate varies from subject lot to subject lot; therefore, if the minimum viable T cell count of anti-FOLRl CoStAR+cells is not achieved, the TIL expansion step can be extended up to 2 additional days to ensure the final ITIL-306 dose requirements are met.8. Wash and Concentration

[0215] Following the TIL expansion step, cells are harvested, filtered through a single-use disposable blood filtration set, washed to reduce process-related impurities, and concentrated. The cells are washed multiple times using phosphate-buffered saline (PBS) supplemented with 5% albumin (human) (HSA) using an automated cell-processing system (Sefia™ from Cytiva LifeSciences). The cells are then concentrated and resuspended in PBS supplemented with 5% HSA, in preparation for the formulation step detailed in Example 2.Example 2. ITIL-306 Formulation, Cryopreservation, Storage, and Transportation Process.

[0216] Following the manufacture of the ITIL-306 drug product as described in Example 20 above, an overview of the formulation, cryopreservation, storage, and transportation process is provided in Figure 4, and an exemplary procedure is as follows.1. Formulation

[0217] Following the wash and concentration process step, CryoStor® CS10 (containing 10% DMSO) is added to the cell suspension in PBS and 5% HSA at a 1 : 1 volume ratio to formulate the ITIL-306 final product. Based on the amount of anti-FOLRl CoStAR+cells required for dose, the final product is formulated to 170mL ± 20mL in the final product bag, resulting in a final formulation of 5% DMSO and 2.5% HSA in PBS. Table 1 provides the ITIL- 306 final product composition.

[0218] Table 1. ITIL-306 Final Product Composition. Quantity at a final product volume of 170 Target final product excipient mL concentrations 0.5 x 109to 6.0 x 109viable CoStAR+T cells NA 85 mL perbag 5% DMSO 64 mL per bag Approximately 0.4X21 mL per bag 2.5% HSAAbbreviations: DMSO, dimethyl sulfoxide; HSA, human serum albumin; NA, not applicable; PBS, phosphate- buffered saline.2. Cryopreservation

[0219] Following the formulation step, the final product bag containing the formulated ITIL-306 final product is visually inspected and transferred into a control led-rate freezer. The final product is cryopreserved using a freezing rate of -l°C / minute until -80°C is reached.3. Storage and Transportation

[0220] Following the cry opreservation step, the ITIL-306 final product bag inside the cassette is transferred to vapor-phase LN2 at <-130°C for storage. ITIL-306 is maintained cryopreserved in storage (<-130°C) until release. When ready to ship, the final product bag in the cassette is shipped to the treatment site at <-130°C using a validated LN2 shipper.Example 3. Comparison of 6-Well R+D Method to Use of G-REXS10M Vessel.

[0221] In an effort to improve and streamline the TIL production process (i.e., improve transduction efficiency while minimizing any necessary manipulations of TILs), we compared our established 6-well R+D method of TIL production from frozen samples (Figure 5) with a similar method developed around the use of G-REX® vessels. The R+D method involves seeding of IxlO6cells / mL on DI in a 6-well plate up to maximum 5e6 / plate, collecting the cells on D3, centrifuging at 400xg for 5 minutes, resuspending in 1 mL for counts to calculate the vector requirements for MOI5, and then transducing the cells in 2mL of final volume of cells, medium, and viral vector. On D4, the cells are collected, centrifuged at 400xg for 5 minutes, and resuspended in 2 mL of final volume of cells, medium .and viral vector. On D5 the cells are collected, centrifuged at 400xg for 5 minutes, counted, and placed at a concentration of IxlO6cells / mL for outgrowth continuation until D13, on which day they are placed into a REP. The G-REX® system provides inherent benefits, including significantly larger media volume in a gaspermeable chamber that requires no feeding (i.e., media changes), thereby simplifying the logistics of producing TILs and reducing the number of possible opportunities for introduction of contaminants during the outgrowth and expansion of TILs. Such additional volume, however, requires re-evaluation of transduction procedures typically performed in the smaller volumes of 6-well tissue culture plates during the production of genetically modified TILs.

[0222] In addition to evaluating the G-REX® format, we also sought to examine the effect of eliminating growth factors, other compounds, and additional cell manipulations that are typically used to enhance transduction efficiency during TIL production. Minimizing the number of added reagents and manipulations during the production of an adoptive cell therapy is highly favorable from a regulatory perspective.

[0223] Frozen lung samples from four donors were used for the experiment , with cells divided into mock-transduced control groups or costimulatory antigen receptor (CoStAR) transduced groups for 6-well or G-REX® vessels. Cells were seeded at the same number across conditions (depending on cell availability) among all groups per donor. The DI volumes for the 6-well conditions for MOCK, CoStAR 2 mL, CoStAR 5 mL and CoStAR 10 m L were 5, 5, 5, and 10 mL, respectively. The DI volumes for the G-REX® conditions for MOCK, CoStAR 5, 10, 20, and 40 mL, were 40, 5, 10, 20, and 40 mL, respectively. On transduction day 3 (days of outgrowth), half of the media from each well of a 6-well plate or G-REX® vessel was removed, without disturbing the cells at the bottom of the vessels to ensure no cell losses. In the case of the 6-well 2 mL CoStAR transduction condition, 3.5 mL were removed carefully without disturbing the cell pellet. On D3, the remaining media on each vessel was resuspended to ensure the cells were mixed, and cells were counted to calculate the amount of the viral vector required for the transduction using a MOI of 5 across all groups. The cells were then transduced with an antifolate receptor alpha (FOLR1) CoStAR lentivirus by adding fresh media equivalent to the volume removed from each condition containing the viral vector. For the 6-well 2 mL transduction condition, the volume was 0.5 mL including the viral vector to obtain a 2 mL transduction condition. For the MOCK transduced cells only, fresh media was added to each well without viral vector. On transduction D4, half of the media from each well of a 6-well plate or G-REX® vessel was removed, without disturbing the cells at the bottom of the vessels to ensure no cell losses. In the case of the 6-well 2 mL CoStAR transduction condition, 0.5 mL were removed carefully without disturbing the cell pellet. No cell counts were performed, and the viralvector volumes were added as per the D3 transduction using the D3 counts. The cells were then transduced with a FOLR1 CoStAR lentivirus by adding fresh media equivalent to the volume removed from each condition containing the viral vector. For the 6-well 2 mb transduction condition, this was 0.5 mb including the viral vector. For the MOCK transduced cells only, fresh media was added to each well without viral vector. On D5 of outgrowth, the cells were collected from each vessel and washed to remove viral particles. The cells were then seeded in the appropriate media volumes for the rest of the outgrowth as per the DI seeding above. Unlike previous methods of transducing TILs, the transduction procedures in this experiment did not involve (i) TIL activation; (ii) transduction enhancers (e.g., retronectin); or (iii) spinoculation (i.e., centrifugation of TILs and virus to enhance contact and transduction).

[0224] Figure 6 illustrates the numbers of live, live non-apoptotic, viable CD45+, and viable CD3+ cells within each sample on day 1 (DI) as assessed by flow cytometry. 50 pL of each sample were transferred into 96-well U bottom plates in duplicates for flow cytometric analysis of the cells. A master mix containing antibodies against CD3, CD45, and annexin V was prepared and 50 pL were added to each sample. Following a 20 minute incubation at RT, 100 pL of a DRAQ7 master mix was added to each sample prior to immediate acquisition. The population of live cells from each donor was divided into the 9 experimental groups, as described above, to progress through the outgrowth, transduction, and rapid expansion process illustrated in Figure 5. As shown in Figure 7A, CD3+ cells, expressed as a percentage of CD45+ cells, were consistent among all tested groups in both 6-well plates and G-REX® vessels on D10 of outgrowth. Also consistent among all groups were a small percentage of CD3-CD56+ NK cells, as depicted in Figure 7B. We also assessed the percentage of transduced cells on D10 of outgrowth. As shown in Figure 8, despite an expected degree of variability between donors, the average percentage of CoStAR+CD3+ cells was similar among all transduction volumes tested in 6-well plates at approximately 70%, ranging from approximately 40% to 90% across donor samples. Likewise, Figure 8 shows that transduction in G-REX® vessels was also consistent across the 5 mL, 10 mL, and 2 OmL transduction volumes tested in the experiment with an average of approximately 50% CoStAR+CD3+ cells, ranging from approximately 40% to 80% across donor samples. However, there did appear to be a slight decline in the average number of CoStAR+CD3+ cells in the 40 mL transduction volume in the G-REX® vessel, suggesting that it may be ideal not to exceed a 20 mL transduction volume in G-REX® vessels. Surprisingly, iwithout using any activators, enhancers, or spinoculation techniques, all groups noted above achieved transduction efficiency that ranged from 40% to 90% of cells transduced. This is significantly greater transduction efficiency than published reports in which polyclonal TIL populations, treated with IL-2 and transduced with lentivirus using the Polybrene spinfection technique, resulted in approximately 25% of cells transduced (Jones et al. (2009) Hum. Gene Ther. 20(6): 630-640). Thus, the current method of full volume transduction on both days 3 and 4 of TIL outgrowth appears to be highly effective in the G-REX® vessels, with transduction efficiency comparable to that seen in our 6-well plate format TIL production process.

[0225] We next confirmed that transduction was consistent across all T cell types in the TIL population at D10 of outgrowth by flow cytometric analysis of CoStAR+CD3+, CoStAR+CD4+, and CoStAR+CD8+ populations. TILs were then assessed for transduction efficiency by staining 1 x 10?cells of each sample with antibodies against CD3, CD4, CD8, anti-FOLRl CoStAR molecule and a viability stain. As shown in Figures 9A-9C, the percentage of transduced CD3+, CD4+, and CD8+ cells, respectively, was generally consistent across T cell type, with some variability restricted to the donor 9834 sample, where more CD4+ cells were transduced than CD8+ cells. We also examined whether any of the transduction or outgrowth conditions altered the CD4::CD8 ratio by flow cytometric analysis to identify the percentage of CD4+, CD8+, CD4+CD8+ double positive (DP), and CD4-CD8- double negative (DN) cells in each transduction and outgrowth condition. Although there was expected variation between donor samples in these D10 cell populations, Figures 10A-10D show that there was no effect of transduction or outgrowth conditions on the percentages of CD4+, CD8+, DP, and DN within each donor population.

[0226] Finally, we observed a positive correlation between the percentage of transduced CoStAR+CD3+ cells with the fold expansion of CD3+ cells across all donors and experimental groups, as shown in Figure 11. This finding is consistent with other evidence in the art that more rapidly dividing cell populations are more amenable to viral transduction. Overall, the results of these experiments confirm that transduction on days 3 and 4 of TIL outgrowth, without activation, the use of enhancers, or the use of spinoculation techniques, is successful across all conditions tested for CD3+, CD4+, and CD8+ cells. Moreover, the methods are comparable between the 6-well plate format and the G-REX® system vessels, although transducing in theG-REX® vessel should ideally be performed in not higher than 20mL of media to avoid any drop-off in transduction efficiency.Example 4. Optimization of Viral Multiplicity of Infection (MOI) in the G-REX® TIL Production Process Using Fresh Samples from Different Tumor Sites.

[0227] Having established results in the G-REX® format for TIL production, we next sought to optimize the transduction process and examine whether the process methodology would be effective in (i) fresh tumor digests and (ii) tumor digests from disparate sites, as the ability to obtain reactive TILs from various tumor types and sites besides melanoma has been historically poor. Thus, we utilized a fresh lung adenocarcinoma donor sample (W8490) and a fresh renal cell carcinoma donor sample (H3702) for the following experiments.

[0228] The TIL production process for the following experiments was performed as depicted in the schematic shown in Figure 12. Briefly, the lung and renal cells were each placed into G-REX® 10M plates for outgrowth immediately following manual digestion of the donor sample. Outgrowth, transduction on D3 and D4, and REP procedures were performed as described above in Example 3, with the exception that all transduction volumes were 20 ml, and experimental groups were transduced with CoStAR lentivirus at an MOI of 5, 10, or 20, plus a mock transduction control. CD3+ cell counts throughout the TIL production process are shown in Figures 13A-13C.

[0229] As illustrated in Figures 14A-14C, there was minimal additional benefit of transduction with higher MOIs in both the fresh lung and renal samples when assessed by flow cytometric analysis of CoStAR+ cells at D13 of outgrowth (Pre-REP panels). The MOI20 renal sample was lost due to contamination observed at the end of outgrowth. Importantly, CoStAR transduction was consistent among CD3+, CD4+, and CD8+ cells, and this transduction remained stable post-REP at D25, as shown in the post-REP panels of Figures 14A-14C and Figure 15. Transduction efficiency was considerably higher in the lung samples compared to the renal samples (Figures 14A-14C and also illustrated by comparison with viral copy number (VCN) post-REP in Figure 16), possibly a result of high numbers of red blood cells in the renal donor sample, which may affect T cell growth and / or directly affect transduction efficiency.

[0230] Flow cytometric analysis was performed to identify the percentage of CD4+, CD8+, CD4+CD8+ double positive (DP), and CD4-CD8- double negative (DN) cells across the variousMOI groups in the lung and renal samples, both pre- and post-REP (DI 3 and D25, respectively). As illustrated in Figures 17A-17D, there were no differences in the proportion of CD4+, CD8+, DP, and DN cells between the various MOI groups pre- or post-REP. Likewise, there was little change in the proportion of CD4+, CD8+, DP, and DN cells when comparing post-REP to pre- REP. However, we did observe a significant reduction in the percentage of CD3- cells post-REP in the renal samples (Figure 18D vs. 18C). No difference in the percentage of CD3- cells post- REP was observed in the lung samples (Figures 18A and 18B), and the average post-REP percentage of CD3- cells was comparable between the lung and renal samples (4% and 12%, respectively).

[0231] Finally, we examined TIL tumor reactivity. Initially we assessed the FOLR1 expression on the cryopreserved tumor digest to be used for the autologous coculture assessment (Figure 19). Anti-FOLRl and anti-CD45 antibodies were used to detect the FOLRl+ve cells in the CD45- population, which should contain the tumor cells. Both samples had detectable levels of FORL1 in the CD45- cell population of the digest ranging from 17-44%, suggesting that recognition of the FORL1 on the tumor cells by the CoStAR will likely result in enhanced CoStAR activity given that the TILs recognize the tumor cells through their endogenous TCRs. To assess this, non-transduced mock cells and varying MOI transduced CoStAR TILs were cocultured with autologous tumor digest for 24 hours, following which supernatants were collected and analyzed for cytokine secretion using an MSD immunoassay. TILs and digest alone conditions were used as negative controls. The graphs represent concentrations of IFNy in coculture supernatants for the lung (Figure 20A) and renal (Figure 20B) samples. Indeed, both samples had measurable tumor recognition in cocultures with autologous digest as measured in the mock transduced TILs. A significant enhancement of the anti-tumor reactivity was observed in the cells transduced with CoStAR when compared to the MOCK transduced cells. This enhancement was significantly higher in the lung TILs (13.8-21.2-fold over the mock), potentially due to the higher FOLR1 expression in the tumor, when compared to the renal TIL (0.8-1.9 fold over the mock). The increasing MOI did not further result in significant enhancement of responsiveness.

[0232] The results of these experiments confirm that transduction of fresh samples can be successfully accomplished using the G-REX® format process as described in Example 3, and that the G-REX® 10M and 6-well formats are comparable. Fresh renal samples contained asignificant CD3- cell population that we have previously observed in frozen renal samples, although post-REP renal TIL populations are within spec regarding CD3+ cell purity. Finally, Autologous tumor reactivity was detected in the final TIL products from both fresh lung and renal samples, although CoStAR enhancement was observed to be significantly higher in lung samples than renal samples.Example 5. Comparison of 1-Day Lentiviral Transduction Versus 2-Day Lentiviral Transduction

[0233] Experiments were performed to assess effect of 1-day lentiviral transduction vs. 2-day lentiviral transduction.

[0234] Method - Id vs 2d Lentiviral Transduction1. On day 1 (‘outgrowth Day 1’ or ‘0GD1 ’), digested tumour material from N=3 donors was thawed, washed and plated into InstilBio (IB) outgrowth (OG) medium (“iCMT”: 90% proprietary T-cell medium (TCM) +10% irradiated Fetal Bovine serum (iFBS) +6000IU / mL pro-leukin (IL-2) +10pg / mL gentamycin +0.25pg / mL amphotericin +50pg / mL vancomycin).2. On OGD2, cells were counted (Total Viable Cell (TVC) and leukocyte (CD45+) counts performed using flow cytometer) and varying proportions of cell suspension assigned to either EARLY or LATE Id vs 2d transduction (TD) study arms. Cultures within LATE arm were taken through standard InstilBio (IB) outgrowth (OG) process consisting of medium changes and cell counts performed every 2-3 days.3. On OGD3, TVC and CD45+ counts were performed using a flow cytometer. Lentiviral vector encoding CoStAR (CoStAR LVV) was added to all EARLY cultures at an MOI of 10 (based on TVC count).4. On OGD4, CoStAR LVV was added to EARLY cultures in the 2 day (2d) transduction (TD) condition at an MOI of 10 (based on OGD3 TVC count). There was no intervention to cultures in the Id TD condition.5. On OGD5, all EARLY cultures, regardless of TD status (Id TD, 2d TD) were harvested, washed with iCMT and replated into fresh iCMT. Thereafter, cultures were'lltaken through standard InstilBio (IB) outgrowth (OG) process consisting of medium changes and cell counts performed every 2-3 days.6. On 0GD11, TVC and CD45+ counts were performed using a flow cytometer. CoStAR LVV was added to all LATE cultures at an MOI of 10 (based on TVC count).7. On OGD12, CoStAR LVV was added to LATE cultures in the 2 day (2d) transduction (TD) condition at an MOI of 10 (based on OGD3 TVC count). There was no intervention to cultures in the Id TD condition.8. On OGD13, all LATE cultures, regardless of TD status (Id TD, 2d TD) were harvested, washed with iCMT, replated into fresh iCMT and rested for 24-48HR.9. On OGD15, all cultures, regardless of timing of activation / TD or TD status (EARLY, LATE, Id TD, 2d TD) were harvested, counted and washed with Whole T-Cell Medium (WTCM: 92% proprietary T-cell medium (TCM) +8% irradiated Human AB Serum (HABS) +3000IU / mL IL-2). For each condition, 5E4 - 1E5 CD3+ cells were taken through standard IB Rapid Expansion Phase (REP). Briefly, REP consists of seven days of co-culture with cell-cycle arrested peripheral blood mononuclear cells (PBMCs) (from N=3 donors) a T-cell activating agent (OKT3) and 3000IU / mL IL-2. On REP day 8 (REPD8) perfusion (a stepped exchange of culture medium with 100% TCM +3000IU / mL IL-2) is initiated and continues until cells are harvested on REPD15.10. Following REPD15 harvest, the impact of Id vs 2d TD on %TD was assessed.

[0235] Results — Id vs 2d Lentiviral Transduction

[0236] Figure 21 shows % transduced CD3+ cells at harvest (REPD15) for Id vs 2d lentiviral transduction. Mean %TD was increased in 2d TD condition relative to Id TD condition, regardless of timing of TD. This increase was not statistically significant.

[0237] Table 2 shows % TD at harvest for Id vs 2d Lentiviral Transduction. Each number is derived from the average of N=2 biological replicate cultures per donor per culture condition. Id and 2d transduction resulted in an average of 11.63 - 20.42% and 14.03 - 25.17% CD3+CoStAR+ cells at harvest; on average, 2 days TD relative to Id TD resulted in an increase in CD3+CoStAR+ cells of 2.4 - 4.75 percentage points.

[0238] Table 2. %TD at harvest.

[0239] Summary

[0240] 2d TD resulted in an increase in %CD3+CoStAR+ cells of 2.4 - 4.75 percentage points relative to Id TD.Example 6. Comparison of Activation Versus No Activation

[0241] Experiments were performed to assess effect of activation vs. no activation prior to lentiviral transduction.

[0242] Method — Activation vs No Activation Donors 1-31. On OGD1, digested tumour material from N=3 donors was thawed, washed and plated into iCMT.2. On OGD2, cells were counted (TVC and CD45+ counts performed using flow cytometer) and varying proportions of cell suspension assigned to either EARLY or LATE study arms. Cultures within LATE arm were taken through standard InstilBio (IB) outgrowth (OG) process consisting of medium changes and cell counts performed every 2-3 days.3. EARLY cultures were resuspended to a density of 1.0E05 - 1.5E06 TVC / mL (target density of 1E06 TVC / mL; in some cultures, there were insufficient cells to achieve this density) in 2-2.5mL of fresh medium in a 24-well GREX plate and either treated with 10 pL of TransAct per 1E06 cells (ACTIVATED condition) or not treated (NOT ACTIVATED condition).4. On OGD3, TVC and CD45+ counts were performed using a flow cytometer. CoStAR LVV was added to all EARLY cultures (regardless of activation status) at an MOI of 10 (based on TVC count).5. On OGD4, CoStAR LVV was added to EARLY cultures in the 2 day (2d) transduction (TD) condition at an MOI of 10 (based on OGD3 TVC count). There was no intervention to cultures in the Id TD condition.6. On OGD5, all EARLY cultures, regardless of activation or TD status (ACTIVATED, NOT ACTIVATED, Id TD, 2d TD) were harvested, washed with iCMT and replated into fresh iCMT. Thereafter cultures were taken through standard InstilBio (IB) outgrowth (OG) process consisting of medium changes and cell counts performed every 2-3 days.7. On OGDIO, cells in all LATE cultures were counted (TVC and CD3+ counts performed using flow cytometer) and resuspended to a density of 1.0E05 - 1.5E06 TVC / mL (target density of 1E06 TVC / mL; in some cultures, there were insufficient cells to achieve this density) in 2-2.5 mL of fresh medium in a 24 well GREX plate. 10 pL of TransAct per 1E06 cells were added to each well in the ACTIVATED condition; there was no intervention in the NON-ACTIVATED condition.8. On OGD11, TVC and CD45+ counts were performed using a flow cytometer. CoStAR LVV was added to all LATE cultures (regardless of activation status) at an MOI of 10 (based on TVC count).9. On OGD12, CoStAR LVV was added to LATE cultures in the 2 day (2d) transduction (TD) condition at an MOI of 10 (based on OGD3 TVC count). There was no intervention to cultures in the Id TD condition.10. On OGD13, all LATE cultures, regardless of activation or TD status (ACTIVATED, NOT ACTIVATED, Id TD, 2d TD) were harvested, washed with iCMT, replated into fresh iCMT and rested for 24-48 hours.11. On OGD15, all cultures, regardless of timing of activation / TD, activation or TD status (EARLY, LATE, ACTIVATED, NOT ACTIVATED, Id TD, 2d TD) were harvested, counted and washed with Whole T-Cell Medium (WTCM: 92% proprietary T- cell medium (TCM) +8% irradiated Human AB Serum (HABS) +3000IU / mL IL-2). For each condition, 5E4 - 1E5 CD3+ cells were taken through standard IB Rapid Expansion Phase (REP). Briefly, REP consists of seven days of co-culture with cell-cycle arrested peripheral blood mononuclear cells (PBMCs) (from N=3 donors) a T-cell activating agent (0KT3) and 3000 lU / mL IL-2. On REP day 8 (REPD8) perfusion (a stepped exchange of culture medium with 100% TCM +3000 lU / mL IL-2) is initiated and continues until cells are harvested on REPD15.12. Following REPD15 harvest, the impact of Id vs 2d TD on %TD was assessed.

[0243] Method - Activation vs No Activation Donors 4-7

[0244] A further N=4 donors were assessed for impact of OGD10 activation and OGD11 TD against a control arm (standard IB outgrowth and TD process consisting of NO activation, TD on OGD3 + OGD4). Briefly, the method was as follows:1. On OGD1, digested tumour material from N=4 donors was thawed, washed and plated into iCMT.2. On OGD3, cells were counted (Total Viable Cell (TVC) and T-lymphocyte (CD2+) counts performed using flow cytometer) and each culture divided into two arms (NOT ACTIVATED vs ACTIVATED; 2 biological replicate cultures per donor per arm), with each arm consisting of an equal proportion of OGD3 material.3. On OGD3, TVC and CD2+ counts were performed using a flow cytometer. CoStAR LVV was added to all NOT ACTIVATED cultures at an MOI of 10 (based on TVC count).4. On OGD4, CoStAR LVV was added to all NOT ACTIVATED cultures at an MOI of 10 (based on OGD3 TVC count).5. On OGD5 all NOT ACTIVATED cultures were harvested, washed with iCMT and replated into fresh iCMT. Thereafter cultures were taken through standard IB OG process consisting of medium changes and cell counts performed every 2-3 days. Medium exchange was performed for all ACTIVATED cultures.6. On OGDIO, all ACTIVATED cultures were counted (TVC and CD2+ counts performed using flow cytometer) and resuspended to a density of 1.0E05 - 1.5E06 TVC / mL (target density of 1E06 TVC / mL; in some cultures, there were insufficient cells to achieve this density) in 2-2.5 mL of fresh medium in a 24 well GREX plate. 10 pL of TransAct per 1E06 cells were added to each well in the ACTIVATED condition.7. On OGD11, TVC and CD2+ counts were performed using a flow cytometer. CoStAR LVV was added to all ACTIVATED cultures at an MOI of 10 (based on TVC count).8. On OGD12, all cultures, regardless of activation status (ACTIVATED, NOT ACTIVATED) were harvested, counted and washed with WTCM. For each condition, 5E4 - 1E5 CD3+ cells were taken through standard IB Rapid Expansion Phase (REP). Briefly, REP consists of seven days of co-culture with cell-cycle arrested peripheral blood mononuclear cells (PBMCs) (from N=3 donors) a T-cell activating agent (OKT3; added to NOT ACTIVATED arms only) and 3000 lU / mL IL-2. On REP day 8 (REPD8) perfusion (a stepped exchange of culture medium with 100% TCM +3000 lU / mL IL-2) is initiated and continues until cells are harvested on REPD15.9. Following REPD15 harvest, the impact of process changes within the ACTIVATED arm relative to the IB control (NOT ACTIVATED) arm on critical product attributes was assessed.

[0245] Results - Activation vs No Activation

[0246] Owing to substantial differences in methodology between studies, data generated for donors 1-3 and donors 4-7 are presented separately.

[0247] Figures 22A-22B show the effect of activation on culture viability. Figure 22A shows the effect of activation on culture viability at end of OG (donors 1-3). Activation on0GD2 resulted in increased CD45+ viability on OGD15 (statistically significant for D3+4 TD condition only). Activation on OGDIO resulted in significantly decreased CD45+ viability on 0GD15. Figure 22B shows the effect of activation on culture viability during OG (donors 4-7). Activation on OGDIO resulted in significantly decreased CD2+ viability on 0GD11 and 0GD12.

[0248] Figures 23A-23B show the effect of activation on culture growth (fold expansion, FE) during OG. Figure 23A shows the effect of activation on culture growth (fold expansion (FE)) during OG (donors 1-3). Activation on OGD2 resulted in a statistically significant increase in CD45+ FE (D3 and D3+4 TD conditions). Activation on OGDIO resulted in decreased CD45+ FE relative to not activated condition (not statistically significant). Figure 23B shows the effect of activation on culture growth (FE) during OG (donors 4-7). Activation on OGDIO resulted in significantly decreased CD3+ FE relative to not activated condition. In one instance (donor 5), the decline in CD3+ cell number following activation was so substantial that there were insufficient cells to proceed into REP.

[0249] Table 3 shows the total number of CD45+ (donor 1-3) or CD3+ cells (donor 4-7) in not activated and activated cultures at the end of OG. For donors 1-3, only data from cultures activated or not activated on OGDIO are shown (i.e., data from cultures activated or not activated on OGD2 are not shown). In one instance (donor 5), the decline in CD3+ cell number following activation was so substantial that there were insufficient cells to proceed into REP.

[0250] Table 3. Total Number of CD45+ (Donor 1-3) or CD3+ Cells (Donor 4-7) in NotActivated and Activated Cultures at the End of OG.

[0251] Figures 24A-24B show the effect of activation on culture growth (FE) during RFP.Figure 24A shows the effect of activation on culture growth (FE) during REP (donors 1-3).Across all conditions, activation resulted in a statistically significant increase in CD3+ FE atREP harvest. Figure 24B shows the effect of activation on culture growth (FE) during REP (donors 4,6,7). Activation on OGDIO did not result in a significant change in CD3+ FE during REP.

[0252] Figures 25A-25B show the effect of effect of activation on TD. Figure 25A shows the effect of activation on TD (donors 1-3). Activation resulted in a statistically significant increase in TD (early TD conditions only). Figure 25B shows the effect of activation on TD (donors 4,6,7). Activation on OGDIO did not result in a significant change in TD at harvest.

[0253] Summary1. Activation resulted in decreased viability (in the days subsequent to activation).2. Activation resulted in decreased cell number (in the days subsequent to activation).3. In one study (n=3 donors; donors 1-3), activation resulted in increased FE during REP.4. In one study (n=3 donors; donors 4,6,7), changes resulting from activation seen during OG were eliminated during REP.5. In one study (n=3 donors; donors 1-3), activation resulted in increased %TD (in early TD conditions only).6. In one study (n=3 donors; donors 4,6,7), activation did not result in a change in %TD.

Claims

We claim:

1. A method for preparing a therapeutic population of tumor infiltrating lymphocytes (TILs), comprising:(a) obtaining a processed resected tumor product comprising TILs;(b) performing a first expansion by culturing the processed resected tumor product comprising TILs in a first cell culture medium comprising IL-2 to produce a first population of TILs;(c) transducing the TILs with lentivirus;(d) performing a second expansion by culturing the TILs in a second cell culture medium with IL-2, a CD3 agonist, and / or antigen presenting cells (APCs), to produce a second population of TILs; and(e) harvesting the TILs.

2. The method of claim 1, wherein the TILs are not treated with an activating agent in step (c) or prior to step (c).

3. The method of claim 1, wherein the TILs are not treated with an activating agent comprising CD3 and / or CD28 agonists in step (c) or prior to step (c).

4. The method of claim 1 , wherein the TILs are not treated with an activating agent comprising CD3 and CD28 agonists in step (c) or prior to step (c).

5. The method of any preceding claim, wherein step (c) does not comprise spinoculation.

6. The method of any preceding claim, wherein the TILs are not treated with an enhancer of viral-mediated transduction in step (c) or prior to step (c).

7. The method of any one of claims 1-5, wherein the TILs are not treated with a recombinant human fibronectin fragment that is an enhancer of viral-mediated transduction in step (c) or prior to step (c).

8. The method of any preceding claim, wherein the TILs are transduced about 2 to about 7 days after the start of the first expansion.

9. The method of claim 8, wherein the TILs are transduced about 2 days to about 3 days after the start of the first expansion or about 3 days to about 4 days after the start of the first expansion.

10. The method of any preceding claim, wherein the transduction in step (c) is performed at a multiplicity of infection (MOI) of no more than about 5.

11. The method of any one of claims 1-9, wherein the transduction in step (c) is performed at a multiplicity of infection (MOI) of about 5 to about 20.

12. The method of any preceding claim, wherein the transduction in step (c) is performed at a multiplicity of infection (MOI) of about 5.

13. The method of any preceding claim, wherein the TILs are transduced about 2 days to about 3 days after the start of the first expansion or about 3 days to about 4 days after the start of the first expansion, wherein the TILs are not treated with an activating agent in step (c) or prior to step (c), wherein step (c) does not comprise spinoculation, and wherein the TILs are not treated with an enhancer of viral -mediated transduction in step (c) or prior to step (c).

14. The method of any one of claims 1-12, wherein the TILs are transduced about 2 days to about 3 days after the start of the first expansion or about 3 days to about 4 days after the start of the first expansion, wherein the TILs are not treated with an activating agent comprising CD3 and CD28 agonists in step (c) or prior to step (c), wherein step (c) does not comprise spinoculation, and wherein the TILs are not treated with a recombinant human fibronectin fragment that is an enhancer of viral-mediated transduction in step (c) or prior to step (c).

15. The method of any preceding claim, wherein the first expansion and the transducing occurs in an open system, optionally wherein the first expansion and the transducing occurs in 6-well plates or a gas permeable rapid cell expansion system.

16. The method of any one of claims 1-15, wherein the first expansion occurs in adherent bags.

17. The method of claim 16, wherein the adherent bags in the first expansion utilize a treated form of fluorinated ethylene propylene (FEP) film to promote cell and protein adhesion to the surface of the bag.

18. The method of any one of claims 1-14, 16, and 17, wherein steps (b)-(d) or steps (b)-(e) are in a closed system.

19. The method of any preceding claim, wherein the lentivirus encodes an exogenous protein, whereby the exogenous protein is expressed in the TILs following transduction.

20. The method of claim 19, wherein the lentivirus comprises a coding sequence for the exogenous protein operably linked to a myeloproliferative sarcoma virus enhancer, negative control region deleted (MND) promoter.

21. The method of claim 19 or 20, wherein the exogenous protein comprises an exogenous costimulatory receptor.

22. The method of claim 21, wherein the exogenous costimulatory receptor comprises a chimeric costimulatory antigen receptor (CoStAR) comprising an extracellular binding domain specific for a tumor-associated antigen operatively linked to a transmembrane domain and first and second signaling domains.

23. The method of claim 22, wherein:(i) the first and second signaling domains comprise a CD28 signaling domain, a CD40 signaling domain, or both the CD28 signaling domain and the CD40 signaling domain; or(ii) the first and second signaling domains comprise a ICOS signaling domain, a CD40 signaling domain, or both the ICOS signaling domain and the CD40 signaling domain.

24. The method of claim 22 or 23, wherein the extracellular binding domain comprises an scFv.

25. The method of any one of claims 22-24, wherein:(1) the tumor-associated antigen comprises folate receptor alpha;(2) the tumor-associated antigen comprises carcinoembryonic antigen (CEA); or(3) the tumor-associated antigen comprises mesothelin.

26. The method of any one of claims 19-25, further comprising enriching for TILs expressing the exogenous protein.

27. The method of any preceding claim, wherein step (a) comprises:(i) cryopreserving a resected tumor and disaggregating the cryopreserved tumor after thawing;(ii) disaggregating a resected tumor and cry opreserving the disaggregated tumor;(iii) cryopreserving a resected tumor and processing the tumor into multiple tumor fragments after thawing; or(iv) processing a resected tumor into multiple tumor fragments and cry opreserving the tumor fragments.

28. The method of claim 27, further comprising a step of thawing and washing the processed resected tumor product prior to step (b).

29. The method of any one of claims 1-26, wherein the processed resected tumor product is a fresh processed resected tumor product that has not been cryopreserved.

30. The method of any preceding claim, wherein step (a) comprises aseptically disaggregating a tumor resected from a subject to prepare the processed resected tumor product, wherein the disaggregation comprise repeated physical pressure applied 120 to 360 times per minute at up to 6 N / cm2in the presence of an enzyme solution, wherein the tumor is disaggregated into a cell suspension so that the processed resected tumor product can be subjected to a cell culture process.

31. The method of claim 30, wherein the resected tumor is not fragmented prior to disaggregation.

32. The method of claim 30 or 31 , wherein the enzyme solution comprisesDNase and collagenase.

33. The method of any one of claims 30-32, wherein the disaggregation period is 90 minutes or less.

34. The method of any one of claims 30-33, wherein the processed resected tumor product is filtered prior to the first expansion, optionally wherein the filtered processed resected tumor product constituents have an average size of less than 200 pm or less than 170 pm.

35. The method of any preceding claim, wherein the tumor is from a melanoma, a cervical cancer, a non-small cell lung cancer (NSCLC), a head and neck squamous cell carcinoma (HNSCC), a cutaneous squamous cell carcinoma (cSCC), a renal cancer, an ovarian cancer, a colorectal cancer, a gastric cancer, a breast cancer, or a mesothelioma.

36. The method of any preceding claim, wherein the first cell culture medium comprises between about 300 lU / mL IL-2 and about 3000 lU / mL IL-2, or wherein the first cell culture medium comprises between about 2000 lU / mL IL-2 and about 3000 lU / mL IL-2, or wherein the first cell culture medium comprises about 3000 lU / mL IL-2.

37. The method of any preceding claim, wherein the first cell culture medium comprises fetal bovine serum (FBS).

38. The method of any one of claims 1-36, wherein the first cell culture medium does not comprise fetal bovine serum (FBS).

39. The method of any preceding claim, wherein the first cell culture medium comprises human AB serum.

40. The method of any preceding claim, wherein the TILs are cultured in steps (b) and (c) for from about 10 days to about 13 days or from about 11 days to about 13 days.

41. The method of any preceding claim, wherein the CD3 agonist is a CD3 agonist antibody, optionally wherein the CD3 agonist antibody is OKT-3.

42. The method of any preceding claim, wherein the second cell culture medium comprises between about 300 lU / mL IL-2 and about 3000 lU / mL IL-2, or wherein the second cell culture medium comprises between about 2000 lU / mL IL-2 and about 3000 lU / mL IL-2, or wherein the second cell culture medium comprises about 3000 lU / mL IL-2.

43. The method of any preceding claim, wherein the second cell culture medium does not comprise fetal bovine serum (FBS).

44. The method of any preceding claim, wherein the second cell culture medium comprises human AB serum.

45. The method of any preceding claim, wherein the APCs are artificial APCs or wherein the APCs: (i) are obtained by apheresis; and / or (ii) comprise peripheral blood mononuclear cells (PBMCs).

46. The method of claim 45, wherein the APCs comprise: (i) fresh PBMCs or cryopreserved PBMCs; and / or (iii) PBMCs from 2 to 10 donors, from 2 to 5 donors, from 3 to 4 donors, or from 3 donors.

47. The method of any preceding claim, wherein the second expansion comprises a static expansion followed by a dynamic expansion.

48. The method of claim 47, wherein the static expansion is for about 5 days to about 7 days, and wherein the dynamic expansion is for about 7 days to about 9 days.

49. The method of claim 47 or 48, wherein the static expansion is performed in a working volume of about 1500 mL to about 2500 mL or about 1750 mL to about 2250 mL if the first population of TILs is greater than or equal to about 3xl06cells or between about 3xl06cells and about 17.5xl06cells, and wherein the static expansion is performed in a working volume of about 500 mL to about 750 mL if the first population of TILs is less than or equal to about 3xl06cells or between about 0.75xl06cells and about 3xl06cells, optionally wherein the cells that are counted are CD3+ cells.

50. The method of any one of claims 47-49, wherein the static expansion is performed in a working volume of about 2000 mL if the first population of TILs is greater thanor equal to about 3xl06cells or between about 3xl06cells and about 17.5xl06cells, and wherein the static expansion is performed in a working volume of about 625 mL if the first population of TILs is less than or equal to about 3xl06cells or between about 0.75xl06cells and about 3xl06cells, optionally wherein the cells that are counted are CD3+ cells.

51. The method of any one of claims 47-50, wherein the dynamic expansion is performed in a working volume of about 2400 mL to about 4000 mb or about 2800 mb to about 3600 mL if the first population of TILs is greater than or equal to about 3xl06cells or between about 3xl06cells and about 17.5xl06cells, and wherein the dynamic expansion is performed in a working volume of about 500 mL to about 1500 mL or about 750 mL to about 1250 mL if the first population of TILs is less than or equal to about 3xl06cells or between about 0.75xl06cells and about 3xl06cells, optionally wherein the cells that are counted are CD3+ cells.

52. The method of any one of claims 47-51, wherein the dynamic expansion is performed in a working volume of about 3200 mL if the first population of TILs is greater than or equal to about 3xl06cells or between about 3xl06cells and about 17.5xl06cells, and wherein the dynamic expansion is performed in a working volume of about 1000 mL if the first population of TILs is less than or equal to about 3xl06cells or between about 0.75xl06cells and about 3xl06cells, optionally wherein the cells that are counted are CD3+ cells.

53. The method of any one of claims 47-52, wherein the dynamic expansion comprises perfusion steps at first, second, and third time points, optionally wherein the perfusion comprises removing spent cell culture medium while simultaneously adding fresh cell culture medium in equal parts to maintain a constant culture volume.

54. The method of claim 53, wherein the first time point is days 16-18, wherein the second time point is days 18-20, and wherein the third time point is days 20-21.

55. The method of claim 53 or 54, wherein the perfusion at the first time point is about 0.6 to about 1 L / day, the perfusion at the second time point is about 1.4 to about 1.8 L / day, and the perfusion at the third time point is about 3 to about 3.4 L / day if the first population of TILs is greater than or equal to about 3xl06cells or between about 3xl06cells and about 17.5xl06cells, andwherein the perfusion at the first time point is about 0.2 to about 0.3 L / day, the perfusion at the second time point is about 0.4 to about 0.6 L / day, and the perfusion at the third time point is about 0.8 to about 1.2 L / day if the first population of TILs is less than or equal to about 3xl06cells or between about 0.75xl06cells and about 3xl06cells, optionally wherein the cells that are counted are CD3+ cells.

56. The method of any one of claims 53-55, wherein the perfusion at the first time point is about 0.8 L / day, the perfusion at the second time point is about 1.6 L / day, and the perfusion at the third time point is about 3.2 L / day if the first population of TILs is greater than or equal to about 3xl06cells or between about 3xl06cells and about 17.5xl06cells, and wherein the perfusion at the first time point is about 0.25 L / day, the perfusion at the second time point is about 0.5 L / day, and the perfusion at the third time point is about 1 L / day if the first population of TILs is less than or equal to about 3xl06cells or between about 0.75xl06cells and about 3xl06cells, optionally wherein the cells that are counted are CD3+ cells.

57. The method of any one of claims 47-56, wherein the dynamic expansion comprises rocking the cells, optionally at a rocking angle of about 8 degrees.

58. The method of any preceding claim, wherein the harvesting is performed using a harvest medium comprising at least 2%, at least 3%, at least 4%, or at least 5% human serum albumin (HSA) and PBS, optionally wherein the harvesting is performed using a harvest medium comprising about 5% human serum albumin (HSA) and PBS.

59. The method of any preceding claim, wherein step (e) further comprises formulating the TILs with HSA and DMSO, optionally wherein the TIL formulation in step (e) comprises no more than 8%, no more than 7%, no more than 6%, no more than 5%, no more than 4%, no more than 3%, or no more than 2.5% HSA and no more than 9%, no more than 8%, no more than 7%, no more than 6%, or no more than 5% DMSO.

60. The method of claim 59, wherein the TIL formulation in step (e) comprises about 2.5% HSA and about 5% DMSO.61 . The method of claim 59 or 60, wherein the formulating comprises adding HSA and DMSO to the second population of TILs in a closed system.

62. The method of any preceding claim, wherein the harvested TILs are cryopreserved.

63. The method of any preceding claim, further comprising assessing the potency of the harvested TILs.

64. The method of claim 63, wherein assessing the potency of the harvested TILs comprises:(i) co-culturing a subpopulation of the harvested TILs with engineered cells that activate the subpopulation of the harvested TILs via CD3 or co-culturing the subpopulation of the harvested TILs with autologous tumor cells;(ii) detecting the presence or absence of viable CD2+ T-cells or viable CD3+ T-cells that express one or both of IFN-y and CD 107a in the activated TILs; and(iii) determining the percent potency based on the percentage of TILs that express one or both of IFN-y and CD 107a.

65. The method of claim 64, wherein the detecting comprises flow cytometry gated on viable CD3+ TILs to measure the frequency of expression of one or both of IFN-y and CD 107a.

66. The method of claim 64, wherein the detecting comprises flow cytometry gated on viable CD2+ TILs to measure the frequency of expression of one or both of IFN-y and CD 107a.

67. A therapeutic population of TILs obtained by method of any preceding claim.

68. The therapeutic population of TILs of claim 67, wherein the TILs are cryopreserved.

69. A method of treating a subject having a cancer, comprising administering a therapeutic population of TILs obtained by the method of any one of claims 1-66 to the subject.

70. The method of claim 69, wherein the cancer is melanoma, cervical cancer, non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC), cutaneous squamous cell carcinoma (cSCC), renal cancer, ovarian cancer, colorectal cancer, gastric cancer, breast cancer, or mesothelioma.

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