Methods and Compositions for T Cell Co-Culture Potency Assays and Use with Cell Therapy Products

Abstract

The present invention provides novel processes, compositions, and methods for analyzing or assaying the efficacy and / or functionality of tumor infiltrating lymphocyte (TIL) products for use in therapeutic methods, including human cancer therapy, as well as for analyzing or assaying the efficacy and / or functionality of other polyclonal products, such as bone marrow infiltrating lymphocyte (MIL) products and peripheral blood lymphocyte (PBL) products. Compositions, methods, and kits prepared using the TIL, MIL, and PBL products to treat cancer are also provided.

Description

[Background technology]

[0001] The treatment of bulky, refractory cancers using adoptive autologous transfer of tumor-infiltrating lymphocytes (TILs) represents a powerful approach to treating patients with poor prognosis. Gattinoni, et al., Nat. Rev. Immunol. 2006, 6, 383-393. IL-2-based TIL expansion followed by the "rapid expansion process" (REP) has become the preferred method of TIL expansion due to its speed and efficiency. Dudley, et al., Science 2002, 298, 850-54; Dudley, et al., J. Clin. Oncol. 2005, 23, 2346-57; Dudley, et al., J. Clin. Oncol. 2008, 26, 5233-39; Riddell, et al., Science 1992, 257, 238-41; Dudley, et al., J. Immunother. 2003, 26, 332-42. Many approaches to improving response to TIL therapy in melanoma and extending TIL therapy to other tumor types have met with limited success, and the field remains challenging. Goff et al., J. Clin. Oncol. 2016, 34, 2389-97; Dudley et al., J. Clin. Oncol. 2008, 26, 5233-39; Rosenberg et al., Clin. Cancer Res. 2011, 17, 4550-57. Combination studies with single immune checkpoint inhibitors have also been described, but further research is ongoing and additional treatment options are needed (Kverneland et al., Oncotarget, 2020, 11(22), 2092-2105).

[0002] Furthermore, current TIL production and treatment processes are limited by length, cost, sterility concerns, and other factors described herein, severely limiting the potential to treat patients who are refractory to other checkpoint inhibitor therapies. There is an urgent need to provide TIL production processes and quality control processes for therapies based on such processes that are suitable for use in treating patients who have few or no remaining viable treatment options. The present invention fills this need by providing an abbreviated manufacturing process for use in generating TILs, with additional steps that provide a mechanism for assessing the efficacy and / or functionality of the expanded TILs.

[0003] The present invention provides improved processes, methods, and compositions for preparing and assessing the potency and / or functionality of T cells, including TILs, to prepare therapeutic TIL populations with increased therapeutic efficacy, for example, for the treatment of cancer. These potency assays can offer improvements over assays known in the art, including, among other things, superior performance, better control over the potency of T cell products, and increased biological relevance. Manufacturing processes, administration methods, and pharmaceutical compositions using the potency assays are also described. These processes and methods can additionally be used in conjunction with the administration of TILs in combination with CTLA-4 and PD-1 and / or PD-L1 inhibitors described herein. Summary of the Invention

[0004] Provided herein are methods for assessing the efficacy and / or functionality of expanded TILs and other polyclonal T cell products, including bone marrow infiltrating lymphocytes (MILs) and peripheral blood lymphocytes (PBLs), which can be used to treat cancer by administering TILs, MILs, PBLs, or other polyclonal T cell products assessed by these methods.

[0005] In one aspect, there is provided a method of determining the potency of a T cell product, comprising: co-culturing the target cells with the aT cell product cells for a first period of time; b. Obtaining a harvest from the co-culture; c. assessing the harvest (1) for expression of one or more markers on the T cell product cells, or (2) for one or more analytes secreted from the T cell product cells to obtain one or more observations and determine the potency of the T cell product.

[0006] In some embodiments, the method comprises: an additional step of a second co-culture of (i) the cells or (ii) a negative control comprising a human leukocyte antigen (HLA) blocking antibody and target cells with the dT cell product cells for a second period of time; e. the additional step of obtaining a second harvest from the second co-culture; f. the additional step of assessing the second harvest for (1) expression of one or more markers on the T cell product cells, or (2) one or more analytes secreted from the T cell product cells to obtain one or more control values; g. The additional step of comparing one or more observed values ​​from step c to one or more control values ​​from step f to determine the potency of the T cell product, wherein each observed value is compared to a corresponding control value.

[0007] In some embodiments, the T cell product is selected from the group consisting of a tumor infiltrating lymphocyte (TIL) product, a bone marrow infiltrating lymphocyte (MIL) product, or a peripheral blood lymphocyte (PBL) product.

[0008] In some embodiments, the T cell product is a human-derived TIL product, wherein the TIL product is obtained by tumor resection or tumor fragmentation or digestion and is manufactured by a TIL expansion process that includes a rapid expansion protocol step.

[0009] In some embodiments, the method includes releasing the T cell product for use in treating a human patient.

[0010] In some embodiments, the target cells are irradiated Raji cells or derivatives, variants, modifications, or progeny thereof.

[0011] In some embodiments, the negative control cells lack MHC or HLA class I and MHC or HLA class II expression.

[0012] In some embodiments, the negative control cells are irradiated K562 cells or derivatives, variants, modifications, or progeny thereof.

[0013] In some embodiments, the HLA blocking antibody comprises an HLA-I blocking antibody, an HLA-II blocking antibody, or a combination thereof.

[0014] In some embodiments, the ratio of the number of TIL product cells to the number of target cells is 5:1 to 1:5.

[0015] In some embodiments, the ratio of the number of TIL product cells to the number of negative control cells is 5:1 to 1:5.

[0016] In some embodiments, the ratio of the number of TIL product cells to the number of target cells is 3:1 to 1:3.

[0017] In some embodiments, the ratio of the number of TIL product cells to the number of negative control cells is 3:1 to 1:3.

[0018] In some embodiments, the ratio between the number of TIL product cells and the number of target cells is 1:2 to 1:4.

[0019] In some embodiments, the ratio of the number of TIL product cells to the number of negative control cells is 1:2 to 1:4.

[0020] In some embodiments, the ratio of the number of TIL product cells to the number of target cells is 1:25 to 1:35.

[0021] In some embodiments, the ratio of the number of TIL product cells to the number of negative control cells is 1:25 to 1:35.

[0022] In some embodiments, the ratio of the number of TIL product cells to the number of target cells is about 1:2.

[0023] In some embodiments, the ratio of the number of TIL product cells to the number of negative control cells is about 1:2.

[0024] In some embodiments, the ratio of the number of TIL product cells to the number of target cells is about 1:3.

[0025] In some embodiments, the ratio of the number of TIL product cells to the number of negative control cells is about 1:3.

[0026] In some embodiments, the ratio of the number of TIL product cells to the number of target cells is about 1:4.

[0027] In some embodiments, the ratio of the number of TIL product cells to the number of negative control cells is about 1:4.

[0028] In some embodiments, the co-culture is performed after thawing a cryopreserved TIL, MIL, or PBL product and allowing the thawed TIL, MIL, or PBL product to recover under incubation for a period selected from the group consisting of 12 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, and 72 hours.

[0029] In some embodiments, the first period of time is from about 6 hours to about 48 hours.

[0030] In some embodiments, the second period of time is from about 6 hours to about 48 hours.

[0031] In some embodiments, the first period of time is selected from the group consisting of about 12 hours, about 18 hours, and about 24 hours.

[0032] In some embodiments, the second period of time is selected from the group consisting of about 12 hours, about 18 hours, and about 24 hours.

[0033] In some embodiments, the first period of time and the second period of time are the same period of time.

[0034] In some embodiments, the one or more markers on the T cell product are selected from the group consisting of CD25, CD69, CD134, CD137, CD150, KLRG1, or a combination thereof.

[0035] In some embodiments, the one or more analytes secreted from the T cell product are selected from the group consisting of IFN-α, IFN-β, IFN-γ, granzyme B, perforin, TNF-α, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-9, IL-10, IL-13, IL-14, IL-16, IL-17, IL-18, IL-22, IL-25, IL-26, MIP-1β, and combinations thereof.

[0036] In some embodiments, the one or more analytes secreted from the TIL product are selected from the group consisting of IFN-α, IFN-β, IFN-γ, granzyme B, perforin, TNF-α, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-9, IL-10, IL-13, IL-14, IL-16, IL-17, IL-18, IL-22, IL-25, IL-26, MIP-1β, and combinations thereof, and the amount of the observed value is normalized to the amount of a control value for each of the one or more analytes, and the increase in the observed value over the control value for each of the one or more analytes is selected from the group consisting of at least 1-fold, at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, and at least 5-fold.

[0037] In some embodiments, the TIL product is manufactured from a tumor obtained by surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining a sample containing a mixture of tumor cells and TIL cells from a human patient.

[0038] In another aspect, the present invention provides a method of treating cancer with a population of tumor infiltrating lymphocytes (TILs) in a patient in need thereof, comprising: (a) obtaining and / or receiving a first population of TILs from a tumor excised from a patient by surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means by processing a tumor sample obtained from the patient into (i) multiple tumor fragments or (ii) tumor digests; (b) adding the first TIL population to the closed system; (c) performing a first expansion by culturing the first TIL population in a cell culture medium containing IL-2 to produce a second TIL population, wherein the first expansion is performed in a sealed container that provides a first gas permeable surface area, and the first expansion is performed for about 3 to 14 days to obtain the second TIL population, and the transition from step (b) to step (c) occurs without opening the system; (d) performing a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, and antigen-presenting cells (APCs) to produce a third TIL population, wherein the second expansion is performed for about 7 to 14 days to obtain the third TIL population, the third TIL population being a therapeutic TIL population, and wherein the second expansion is performed in a sealed container that provides a second gas-permeable surface area, and the transition from step (c) to step (d) occurs without opening the system. (e) harvesting the therapeutic TIL population obtained from step (d), wherein the transition from step (d) to step (e) occurs without opening the system; (f) determining the efficacy of the therapeutic TIL population, i. co-culturing target cells with a portion of the therapeutic TIL population for a first period of time; ii. Obtaining a harvest from the co-culture; iii. assessing the harvest (1) for expression of one or more markers on the portion of the therapeutic TIL population, or (2) for one or more analytes secreted from the portion of the therapeutic TIL population to obtain one or more observations to determine efficacy of the therapeutic TIL population; iv. A second co-culture of the negative control cells or (i) the negative control cells or (ii) the human leukocyte antigen-blocking antibody with a second portion of the therapeutic TIL population for a second period of time; v. obtaining a second harvest from the second co-culture; vi. assessing the second harvest for (1) expression of one or more markers on the second portion of the therapeutic TIL population, or (2) one or more analytes secreted from the second portion of the therapeutic TIL population to obtain one or more control values; vii. determining the efficacy of the therapeutic TIL population, comprising comparing one or more observed values ​​to one or more control values ​​to determine the efficacy of the therapeutic TIL population, wherein each observed value is compared to its corresponding control value; (g) transferring the therapeutic TIL population from step (e) into an infusion bag, wherein the transition from step (e) to (g) occurs without opening the system; (h) optionally, cryopreserving the infusion bag containing the therapeutic TIL population from step (g) using a cryopreservation process; (i) if the therapeutic TIL population is determined to be effective, administering a therapeutically effective dose of the therapeutic TIL population to the patient from the infusion bag of step (g) or (h).

[0039] In some embodiments, testing the potency and / or functionality of the harvested TILs is performed after cryopreservation, or optionally before or after the cryopreservation step.

[0040] In some embodiments, the patient has a tumor that is unresectable, metastatic, resistant, or refractory to a CTLA-4 inhibitor, a PD-1 inhibitor, or a PD-L1 inhibitor, and optionally, the patient has been previously treated with a CTLA-4 inhibitor, a PD-1 inhibitor, or a PD-L1 inhibitor.

[0041] In some embodiments, the second population of TILs in step (c) is at least 50-fold more numerous than the first population of TILs.

[0042] In some embodiments, the first expansion is carried out over a period of about 10 to about 12 days.

[0043] In some embodiments, the second expansion is carried out over a period of about 10 to about 12 days.

[0044] In some embodiments, the first expansion is carried out over a period of about 11 days.

[0045] In some embodiments, the second expansion is carried out over a period of about 11 days.

[0046] In some embodiments, IL-2 is present in the cell culture medium at an initial concentration of 1000 IU / mL to 6000 IU / mL in the first expansion.

[0047] In some embodiments, in the second expansion step, IL-2 is present at an initial concentration of 1000 IU / mL to 6000 IU / mL and the OKT-3 antibody is present at an initial concentration of about 30 ng / mL.

[0048] In some embodiments, the method further comprises treating the patient with a non-myeloablative lymphodepletion regimen prior to administering the TILs to the patient.

[0049] In some embodiments, the non-myeloablative lymphodepletion regimen includes cyclophosphamide at 60 mg / m 2 / day for 2 days, followed by fludarabine at 25 mg / m2 / day for 5 days.

[0050] In some embodiments, the non-myeloablative lymphodepletion regimen includes cyclophosphamide at 60 mg / m 2 Fludarabine at a dose of 25 mg / m 2 / day for 2 days, followed by fludarabine at 25 mg / m 2 / day for 3 days.

[0051] In some embodiments, the method further comprises treating the patient with an IL-2 regimen starting the day after administering the therapeutic TIL population to the patient.

[0052] In some embodiments, the method further comprises treating the patient with an IL-2 regimen beginning on the same day that the therapeutic TIL population is administered to the patient.

[0053] In some embodiments, the IL-2 regimen is administered about 3 to about 24 hours after completion of administration of the therapeutic TIL population to the patient.

[0054] In some embodiments, the IL-2 regimen is a high-dose IL-2 regimen comprising 600,000 or 720,000 IU / kg aldesleukin, or a biosimilar or variant thereof, administered as a 15-minute bolus intravenous infusion every 8 hours until tolerated.

[0055] In some embodiments, processing the tumor sample obtained from the patient in step (a) into a tumor digest further comprises incubating the tumor sample in an enzyme medium.

[0056] In some embodiments, processing the tumor sample obtained from the patient in step (a) into a tumor digest further comprises mechanically disrupting the tumor sample to dissociate the tumor sample.

[0057] In some embodiments, processing the tumor sample obtained from the patient into a tumor digest in step (a) further comprises purifying the dissociated tumor sample using density gradient separation.

[0058] In some embodiments, the enzyme medium comprises DNase.

[0059] In some embodiments, the enzyme medium contains 30 units / mL of DNase.

[0060] In some embodiments, the enzyme medium comprises collagenase.

[0061] In some embodiments, the enzyme medium comprises about 1.0 mg / mL of collagenase.

[0062] In some embodiments, the cancer is selected from the group consisting of melanoma, ovarian cancer, pancreatic cancer, endometrial cancer, thyroid cancer, cervical cancer, non-small cell lung cancer, small cell lung cancer, bladder cancer, breast cancer, head and neck cancer, glioblastoma, gastrointestinal cancer, kidney cancer, sarcoma, and renal cell carcinoma.

[0063] In some embodiments, the method comprises administering to the patient a PD-1 inhibitor, a PD-L1 inhibitor, or a CTLA-4 inhibitor.

[0064] In another aspect, there is provided a method of determining the potency of a T cell product, comprising: performing at least three co-cultures of target cells with aT cell product cells at different target cell concentrations; performing at least three co-cultures of target cells with bT cell reference standard cells at different target cell concentrations; c. extracting the supernatant from each of the co-cultures; d. assessing the supernatant for cytokines secreted by the T cell product cells and the T cell reference standard cells to obtain dose concentrations for determining the potency of the T cell product; The method is provided wherein the target cells are monocyte cells.

[0065] In some embodiments, the T cell product is a tumor infiltrating lymphocyte (TIL) product, a bone marrow infiltrating lymphocyte (MIL) product, or a peripheral blood lymphocyte (PBL) product.

[0066] In some embodiments, the monocytic cells are U937 or Thp1 cells or derivatives, variants, modifications, or progeny thereof, and the cytokine is interferon-γ.

[0067] In some embodiments, the co-culturing is carried out for a period selected from the group consisting of about 6 hours, about 12 hours, about 18 hours, about 24 hours, about 30 hours, about 36 hours, about 42 hours, and about 48 hours.

[0068] In some embodiments, the T cell product is a TIL product and has about 4 x 10 cells per well. 5 , about 2×10 5 , about 1×10 5 , and approximately 0.5 × 10 5 Four target cell dose concentrations of target cells, as well as approximately 1.5 x 10 per well 6 A single TIL cell concentration of TILs is used.

[0069] In some embodiments, at least four co-cultures of target cells with T cell product cells and at least four co-cultures of target cells with T cell reference standard cells are used, a parallel line analysis is performed, and one outlier target cell dose concentration is discarded.

[0070] In some embodiments, the method is a component of a potency assay matrix.

[0071] In some embodiments, the potency assay matrix may be a bead or plate-based assay using CD3, CD28, and / or CD137 stimulating and reporting interferon-γ, granzyme B, or tumor necrosis factor-α; an assay for total viable cells; an assay for percentage viable cells; CD4 + Assay for cell content, CD8 +Assay for cell content, T EM Assay for cell content, T CM Assay for cell content, LAG3 + Assays for cellular content and KLRG1 + Assay for cell content, CD101 + Assay for cell content, CD69 + Assay for cell content, T SCM Assay for cell content, T EMRA Assay for cell content, T reg Assay for cellular content, PD-1 + Assay for cellular content, TIM3 + Assay for cell content, CD25 + Assay for cell content, CD27 + Assay for cell content, CD28 + Assay for cell content, CD56 + Assay for cellular content, CTLA-4 + Assay for cell content, TIGIT + Assays for cell content and CD57 + The assays include one or more assays selected from the group consisting of assays for cellular content.

[0072] In another aspect, the present invention provides a method for treating a subject having cancer, comprising: (a) adding a tumor digest or tumor fragment to the closed system, the tumor digest or tumor fragment comprising a first TIL population and obtained from a tumor resected from the subject; (b) performing a first expansion by culturing the first TIL population in a cell culture medium containing IL-2 to produce a second TIL population, wherein the first expansion is performed in a sealed container that provides a first gas permeable surface area, and the first expansion is performed for about 3 to 11 days to obtain the second TIL population, and the transition from step (b) to step (c) occurs without opening the system; (c) performing a second expansion by supplementing the cell culture medium with additional cell culture medium containing IL-2, OKT-3, and antigen-presenting cells (APCs) to produce a third TIL population, wherein the second expansion is performed for about 7 to 11 days to obtain the third TIL population, and the second expansion is performed in a sealed container that provides a second gas-permeable surface area, and the transition from step (b) to step (c) occurs without opening the system. (d) harvesting the third population of TILs obtained from step (c), wherein the transition from step (c) to step (d) occurs without opening the system; and (e) transferring the harvested third population of TILs from step (d) to an infusion bag, wherein the transition from step (d) to (e) occurs without opening the system; (f) cryopreserving the infusion bag containing the harvested TIL population from step (e) using a cryopreservation process; (g) administering a therapeutically effective dose of a third population of TILs to the subject from the infusion bag in step (f); The method is provided wherein the APC is selected from the group consisting of Raji, Ramos, Daudi, U937, or Thp1 cells, or derivatives, variants, modifications, or progeny thereof.

[0073] In some embodiments, the cancer is selected from the group consisting of melanoma (including metastatic melanoma and uveal melanoma), ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), small cell lung cancer, bladder cancer, breast cancer, cancer caused by human papillomavirus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), esophageal cancer, esophagogastric junction cancer, gastric cancer, gastrointestinal cancer, renal cancer, and renal cell carcinoma.

[0074] In some embodiments, the first expansion in step (a) and the second expansion in step (b) are each performed separately within a period of 11 days.

[0075] In some embodiments, steps (a) through (d) are carried out in about 10 days to about 22 days.

[0076] In some embodiments, the efficacy of the TILs is determined using the methods described herein.

[0077] In some embodiments, the efficacy of the TILs is determined using the methods described herein. [Brief explanation of the drawings]

[0078] [Figure 1] An exemplary Gen2 (Process 2A) chart providing an overview of steps A-F. [Figure 2A] 1 is a process flow diagram of an embodiment of Gen2 (Process 2A) for TIL fabrication. [Figure 2B] 1 is a process flow diagram of an embodiment of Gen2 (Process 2A) for TIL fabrication. [Figure 2C] 1 is a process flow diagram of an embodiment of Gen2 (Process 2A) for TIL fabrication. [Figure 3] FIG. 1 shows a diagram of an embodiment of an exemplary manufacturing process (approximately 22 days) for cryopreserved TILs. [Figure 4] FIG. 1 shows a diagram of an embodiment of Gen2 (Process 2A), a 22-day process for TIL fabrication. [Figure 5] 1 is a comparison table of steps A-F from exemplary embodiments of Process 1C and Gen2 (Process 2A) for TIL fabrication. [Figure 6] Detailed comparison of Process 1C and Gen2 (Process 2A) embodiments for TIL fabrication. [Figure 7] 1. Exemplary Gen3 TIL fabrication process. [Figure 8A] A comparison of embodiments of the 2A process (an approximately 22 day process) and the Gen3 process (an approximately 14-16 day process) for TIL fabrication is shown. [Figure 8B]An exemplary Process Gen3 chart providing an overview of steps A-F (approximately a 14-16 day process). [Figure 8C] A chart providing three exemplary Gen3 processes along with an overview of steps A-F (approximately 14- to 16-day processes) for each of the three process variations. [Figure 8D] An exemplary modified Gen2-like process (approximately a 22-day process) providing an overview of steps A-F. [Figure 9] 1 provides an experimental flow chart for the comparison between Gen2 (Process 2A) and Gen3 processes. [Figure 10] 1 shows a comparison of various Gen2 (Process 2A) and Gen3.1 process embodiments. [Figure 11] 1 is a table illustrating various features of embodiments of the Gen2, Gen2.1, and Gen3.0 processes. [Figure 12] Summary of media conditions for an embodiment of the Gen3 process, designated Gen3.1. [Figure 13] 1 is a table illustrating various features of embodiments of the Gen2, Gen2.1, and Gen3.0 processes. [Figure 14] 1 is a table comparing various features of embodiments of the Gen2 and Gen3.0 processes. [Figure 15] 1 is a table providing media use in various embodiments of the described expansion process. [Figure 16] Schematic of an exemplary embodiment of the Gen3 process (16-day process). [Figure 17] Schematic of an exemplary embodiment of a method for expanding T cells from hematopoietic malignancies using the Gen3 expansion platform. [Figure 18]Structures IA and IB are provided. The cylinders refer to individual polypeptide binding domains. Structures IA and IB comprise three linearly linked TNFRSF-binding domains derived from antibodies that bind, for example, to 4-1BBL or 4-1BB, that fold to form a trivalent protein, which is then linked to a second trivalent protein via IgG1-Fc (comprising the CH3 and CH2 domains), which is then used to link two of the trivalent proteins together via disulfide bonds (small oblong ellipses), stabilizing the structure and providing an agonist that can bring together the six receptor and intracellular signaling domains of the signaling protein to form a signaling complex. The TNFRSF-binding domains shown as cylinders can be, for example, scFv domains comprising VH and VL chains connected by a linker that may contain hydrophilic residues and Gly and Ser sequences for flexibility, and Glu and Lys for solubility. [Figure 19] Schematic of an exemplary embodiment of the Gen3 process (16-day process). [Figure 20] 1 provides a process overview of an exemplary embodiment of the Gen3.1 process (16-day process). [Figure 21] Schematic of an exemplary embodiment of the Gen3.1 testing process (16-17 day process). [Figure 22] Schematic of an exemplary embodiment of the Gen3 process (16-day process). [Figure 23] 1 is a comparison table of an exemplary Gen2 process and an exemplary Gen3 process. [Figure 24] Schematic of an exemplary embodiment of the preparation timeline for the Gen3 process (16-17 day process). [Figure 25] Schematic of an exemplary embodiment of the Gen3 process (14-16 day process). [Figure 26A] Schematic of an exemplary embodiment of the Gen3 process (16-day process). [Figure 26B] Schematic of an exemplary embodiment of the Gen3 process (16-day process). [Figure 27]Schematic of an exemplary embodiment of the Gen3 process (16-day process). [Figure 28] Comparison of Gen2, Gen2.1, and Gen3 process (16-day process) embodiments. [Figure 29] Comparison of Gen2, Gen2.1, and Gen3 process (16-day process) embodiments. [Figure 30] Components of a Gen3 embodiment. [Figure 31] Flowchart comparison of Gen3 embodiments (Gen3.0, Gen3.1 control, Gen3.1 test). [Figure 32] Components of an exemplary embodiment of the Gen3 process (16-17 day process) are shown. [Figure 33] Approval criteria table. [Figure 34] FIG. 10. Diagram of an embodiment of a TIL-Raji co-culture assay with a TIL-K562 negative control, and MLR (patient-matched PBMC) and bead-based positive controls. [Figure 35] CD25 levels observed by flow cytometry (% of CD3) for different co-culture periods and TIL:target ratios for lung tumors (L4224) and melanoma tumors (M1152) using the TIL-Raji co-culture assay. [Figure 36] CD69 levels observed by flow cytometry (% of CD3) for different co-culture periods and TIL:target ratios for lung tumors (L4224) and melanoma tumors (M1152) using the TIL-Raji co-culture assay. [Figure 37] CD137 (4-1BB) levels observed by flow cytometry (% of CD3) for different coculture periods and TIL:target ratios for lung tumors (L4224) and melanoma tumors (M1152) using the TIL-Raji coculture assay. [Figure 38] CD134 (OX40) levels observed by flow cytometry (% of CD3) for different coculture periods and TIL:target ratios for lung tumors (L4224) and melanoma tumors (M1152) using the TIL-Raji coculture assay. [Figure 39] IFN-γ secretion at different co-culture periods and TIL:target ratios for lung tumor (L4224) and melanoma tumor (M1152) using the TIL-Raji co-culture assay. [Figure 40] CCL4 secretion at different co-culture periods and TIL:target ratios for lung tumors (L4224) and melanoma tumors (M1152) using the TIL-Raji co-culture assay. [Figure 41] Granzyme B secretion at different co-culture periods and TIL:target ratios for lung tumors (L4224) and melanoma tumors (M1152) using the TIL-Raji co-culture assay. [Figure 42] FIG. 10 is a diagram of an embodiment of a TIL-Raji co-culture assay with a TIL-K562 negative control. [Figure 43] Co-culture experimental setup for an exemplary embodiment of a TIL-Raji cell-based potency assay. [Figure 44] Phenotypic expression of activation markers in TIL-Raji coculture assays. [Figure 45] Phenotypic expression of CD69 (Gen2 TIL:301-001) in TIL-Raji cocultures. [Figure 46] IFN-γ secretion levels (pg / mL). [Figure 47] Granzyme B secretion levels (pg / mL). [Figure 48] IFN-γ and granzyme B secretion levels. [Figure 49] Fold change in IFN-γ release: TIL+ cell lines / TIL alone. [Figure 50] Granzyme fold change: TIL+ cell lines / TIL alone. [Figure 51] Fold change in cytokine release: TIL+ cell lines / TIL alone. [Figure 52] Fold change in IFN-γ release: TIL+ cell lines / TIL+K562. [Figure 53] Fold change in granzyme B release: TIL+ cell lines / TIL+K562. [Figure 54]Fold change in cytokine release: TIL+ cell lines / TIL+K562. [Figure 55] Summary table of IFN-γ and granzyme B secretion. [Figure 56] Schematic of an embodiment of a TIL-Raji cell-based potency assay showing TIL activation by dominant MHC recognition. [Figure 57] Co-culture experimental plate setup for an exemplary embodiment of a TIL-Raji cell-based potency assay. [Figure 58] FIG. 10 is a diagram of an embodiment of a TIL-Raji co-culture assay with an optional TIL-K562 negative control. [Figure 59] IFN-γ secretion (pg / mL) of melanoma TIL lots at TIL:Raji or TIL:K562 ratios of 3:1, 1:1, and 1:3. Secretion levels (pg / mL) are shown on the y-axis and TIL:target ratios are shown on the x-axis. [Figure 60] IFN-γ secretion (pg / mL) of melanoma TIL lots at TIL:Raji or TIL:K562 ratios of 3:1, 1:1, and 1:3. Secretion levels (pg / mL) are shown on the y-axis and TIL:target ratios are shown on the x-axis. [Figure 61] IFN-γ secretion (pg / mL) of melanoma TIL lots at TIL:Raji or TIL:K562 ratios of 3:1, 1:1, and 1:3. Secretion levels (pg / mL) are shown on the y-axis and TIL:target ratios are shown on the x-axis. [Figure 62] Figure 1 shows the fold change in IFN-γ release for TILs plus Raji or K562 cells relative to IFN-γ release from TILs alone for melanoma TIL lots at TIL:Raji or TIL:K562 ratios of 3:1, 1:1, and 1:3. Fold change is shown on the y-axis, and TIL:target ratios are shown on the x-axis. [Figure 63] Figure 1 shows the fold change in IFN-γ release for TILs plus Raji or K562 cells relative to IFN-γ release from TILs alone for melanoma TIL lots at TIL:Raji or TIL:K562 ratios of 3:1, 1:1, and 1:3. Fold change is shown on the y-axis, and TIL:target ratios are shown on the x-axis. [Figure 64] Figure 1 shows the fold change in IFN-γ release for TILs plus Raji or K562 cells relative to IFN-γ release from TILs alone for melanoma TIL lots at TIL:Raji or TIL:K562 ratios of 3:1, 1:1, and 1:3. Fold change is shown on the y-axis, and TIL:target ratios are shown on the x-axis. [Figure 65] Figure 1 shows the fold change in IFN-γ release for TIL+Raji or K562 cells relative to IFN-γ release from TIL+K562 cells for melanoma TIL lots at TIL:Raji or TIL:K562 ratios of 3:1, 1:1, and 1:3. Fold change is shown on the y-axis, and TIL:target ratios are shown on the x-axis. [Figure 66] Figure 1 shows the fold change in IFN-γ release for TIL+Raji or K562 cells relative to IFN-γ release from TIL+K562 cells for melanoma TIL lots at TIL:Raji or TIL:K562 ratios of 3:1, 1:1, and 1:3. Fold change is shown on the y-axis, and TIL:target ratios are shown on the x-axis. [Figure 67] Figure 1 shows the fold change in IFN-γ release for TIL+Raji or K562 cells relative to IFN-γ release from TIL+K562 cells for melanoma TIL lots at TIL:Raji or TIL:K562 ratios of 3:1, 1:1, and 1:3. Fold change is shown on the y-axis, and TIL:target ratios are shown on the x-axis. [Figure 68] Fold change in IFN-γ release for TIL+Raji or K562 cells relative to TIL alone at 12 and 18 hour incubation times, showing cumulative dataset at 1:3 TIL:Raji or TIL:K562 cell ratio; * indicates p-value less than or equal to 0.05, ** indicates p-value less than or equal to 0.01, and *** indicates p-value less than or equal to 0.001. [Figure 69]Fold change in IFN-γ release for TIL+Raji or K562 cells relative to TIL alone or K562 cells at 12 and 18 hour incubation times, showing cumulative dataset at 1:3 TIL:Raji or TIL:K562 cell ratio; * indicates p-value less than or equal to 0.05, ** indicates p-value less than or equal to 0.01, and *** indicates p-value less than or equal to 0.001. [Figure 70] Fold change in IFN-γ release for TIL+K562 cells or TILs versus TIL+Raji cells at 12 and 18 h incubation times, showing cumulative dataset at 1:3 TIL:Raji or TIL:K562 cell ratios. * indicates p-value less than or equal to 0.05, NS indicates not significant at full scale. [Figure 71] Changes in IFN-γ release for TIL+K562 cells or TILs versus TIL+Raji cells at 12 and 18 h incubation times, showing cumulative data at a 1:3 TIL:Raji or TIL:K562 cell ratio and expanded to show details at lower fold change levels. [Figure 72] Granzyme B secretion (pg / mL) of melanoma TIL lots at TIL:Raji or TIL:K562 ratios of 3:1, 1:1, and 1:3. Secretion levels (pg / mL) are shown on the y-axis and TIL:target ratios are shown on the x-axis. [Figure 73] Granzyme B secretion (pg / mL) of melanoma TIL lots at TIL:Raji or TIL:K562 ratios of 3:1, 1:1, and 1:3. Secretion levels (pg / mL) are shown on the y-axis and TIL:target ratios are shown on the x-axis. [Figure 74] Granzyme B secretion (pg / mL) of melanoma TIL lots at TIL:Raji or TIL:K562 ratios of 3:1, 1:1, and 1:3. Secretion levels (pg / mL) are shown on the y-axis and TIL:target ratios are shown on the x-axis. [Figure 75]Figure 1 shows the fold change in granzyme B for TIL + Raji or K562 cells relative to granzyme B release from TIL alone for melanoma TIL lots at TIL:Raji or TIL:K562 ratios of 3:1, 1:1, and 1:3. Fold change is shown on the y-axis, and TIL:target ratios are shown on the x-axis. [Figure 76] Figure 1 shows the fold change in granzyme B release for TILs + Raji or K562 cells relative to that from TILs alone for melanoma TIL lots at TIL:Raji or TIL:K562 ratios of 3:1, 1:1, and 1:3. Fold change is shown on the y-axis, and TIL:target ratios are shown on the x-axis. [Figure 77] Figure 1 shows the fold change in granzyme B for TIL + Raji or K562 cells relative to granzyme B release from TIL alone for melanoma TIL lots at TIL:Raji or TIL:K562 ratios of 3:1, 1:1, and 1:3. Fold change is shown on the y-axis, and TIL:target ratios are shown on the x-axis. [Figure 78] Figure 1 shows the fold change in granzyme B release from TIL+Raji or K562 cells relative to that from TIL+K562 cells for melanoma TIL lots at TIL:Raji or TIL:K562 ratios of 3:1, 1:1, and 1:3. Fold change is shown on the y-axis, and TIL:target ratios are shown on the x-axis. [Figure 79] Figure 1 shows the fold change in granzyme B for TIL+Raji or K562 cells relative to granzyme B release from TIL+K562 cells for melanoma TIL lots at TIL:Raji or TIL:K562 ratios of 3:1, 1:1, and 1:3. Fold change is shown on the y-axis, and TIL:target ratios are shown on the x-axis. [Figure 80] Figure 1 shows the fold change in granzyme B release from TIL+Raji or K562 cells relative to that from TIL+K562 cells for melanoma TIL lots at TIL:Raji or TIL:K562 ratios of 3:1, 1:1, and 1:3. Fold change is shown on the y-axis, and TIL:target ratios are shown on the x-axis. [Figure 81]Granzyme B release (pg / mL / TIL) of TILs at 12 and 18 h incubation times. [Figure 82] Granzyme B release for TIL+Raji or K562 cells versus TIL alone at 12 and 18 hour incubation times. Cumulative data set at 1:3 TIL:Raji or TIL:K562 cell ratios shown. *** indicates p-value less than or equal to 0.001, **** indicates p-value less than or equal to 0.0001. [Figure 83] Granzyme B release for TIL+Raji or K562 cells relative to K562 cells at 12 and 18 hour incubation times. Cumulative data set at 1:3 TIL:Raji or TIL:K562 cell ratios is shown. **** indicates a p-value of 0.0001 or less. [Figure 84] Fold change in granzyme B release for TIL+K562 cells or TILs versus TIL+Raji cells at 12 and 18 hour incubation times, showing cumulative dataset at 1:3 TIL:Raji or TIL:K562 cell ratio; ** indicates p-value less than or equal to 0.01, NS indicates not significant. [Figure 85] TNF-α secretion (pg / mL) of melanoma TIL lots at TIL:Raji or TIL:K562 ratios of 3:1, 1:1, and 1:3. Secretion levels (pg / mL) are shown on the y-axis and TIL:target ratios are shown on the x-axis. [Figure 86] TNF-α secretion (pg / mL) of melanoma TIL lots at TIL:Raji or TIL:K562 ratios of 3:1, 1:1, and 1:3. Secretion levels (pg / mL) are shown on the y-axis and TIL:target ratios are shown on the x-axis. [Figure 87] TNF-α secretion (pg / mL) of melanoma TIL lots at TIL:Raji or TIL:K562 ratios of 3:1, 1:1, and 1:3. Secretion levels (pg / mL) are shown on the y-axis and TIL:target ratios are shown on the x-axis. [Figure 88]Figure 1 shows the fold change in TNF-α release for TILs + Raji or K562 cells relative to granzyme B release from TILs alone for melanoma TIL lots at TIL:Raji or TIL:K562 ratios of 3:1, 1:1, and 1:3. Fold change is shown on the y-axis, and TIL:target ratios are shown on the x-axis. [Figure 89] Figure 1 shows the fold change in TNF-α release for TILs + Raji or K562 cells relative to granzyme B release from TILs alone for melanoma TIL lots at TIL:Raji or TIL:K562 ratios of 3:1, 1:1, and 1:3. Fold change is shown on the y-axis, and TIL:target ratios are shown on the x-axis. [Figure 90] Figure 1 shows the fold change in TNF-α for TIL+Raji or K562 cells relative to granzyme B release from TIL alone for melanoma TIL lots at TIL:Raji or TIL:K562 ratios of 3:1, 1:1, and 1:3. Fold change is shown on the y-axis and TIL:target ratio is shown on the x-axis. [Figure 91] Figure 1 shows the fold change in TNF-α release for TIL+Raji or K562 cells relative to granzyme B release from TIL+K562 cells for melanoma TIL lots at TIL:Raji or TIL:K562 ratios of 3:1, 1:1, and 1:3. Fold change is shown on the y-axis and TIL:target ratio is shown on the x-axis. [Figure 92] Figure 1 shows the fold change in TNF-α release for TIL+Raji or K562 cells relative to granzyme B release from TIL+K562 cells for melanoma TIL lots at TIL:Raji or TIL:K562 ratios of 3:1, 1:1, and 1:3. Fold change is shown on the y-axis and TIL:target ratio is shown on the x-axis. [Figure 93] Figure 1 shows the fold change in TNF-α release for TIL+Raji or K562 cells relative to granzyme B release from TIL+K562 cells for melanoma TIL lots at TIL:Raji or TIL:K562 ratios of 3:1, 1:1, and 1:3. Fold change is shown on the y-axis and TIL:target ratio is shown on the x-axis. [Figure 94]Fold change of TNF-α for TIL+Raji or K562 cells relative to TIL alone at an 18-hour incubation time, showing the cumulative dataset at a TIL:Raji or TIL:K562 cell ratio of 1:3; **** indicates a p-value of 0.0001 or less. [Figure 95] Figure 1. Biolaster of TNF-α release for TIL+Raji or K562 cells relative to K562 cells at an 18-hour incubation time. Cumulative data set shown at a TIL:Raji or TIL:K562 cell ratio of 1:3. **** indicates a p-value of 0.0001 or less. [Figure 96] Fold change in TNF-α release for TIL+Raji cells versus TIL alone or TIL+K562 cells at an 18-hour incubation time, showing the cumulative dataset at a TIL:Raji or TIL:K562 cell ratio of 1:3; **** indicates a p-value of 0.0001 or less. [Figure 97] 1 is a summary table of Granzyme B and IFN-γ results for 22 samples tested using an embodiment of the Raji cell co-culture assay. [Figure 98] FIG. 10 is a diagram of an embodiment of a TIL-Raji co-culture assay with an optional TIL-K562 negative control. [Figure 99] Assay results for TIL lot M1173. [Figure 100] Assay results for TIL lot M1152. [Figure 101] Assay results for TIL lot M1187. [Figure 102] Assay results for TIL lot M1179. [Figure 103] Assay results for TIL lot M1183. [Figure 104] Cytokine secretion results of IFN-γ showing the fold change of TIL+K562 or Raji cells over TIL alone at different post-thaw recovery times. [Figure 105]Cytokine secretion results of IFN-γ showing the fold change of TIL+K562 or Raji cells relative to TIL+K562 cells at different post-thaw recovery times. [Figure 106] Granzyme B cytokine secretion results showing fold change of TIL+K562 or Raji cells over TIL alone at different post-thaw recovery times. [Figure 107] Cytokine secretion results of granzyme B showing the fold change of TIL+K562 or Raji cells relative to TIL+K562 cells at different post-thaw recovery times. [Figure 108] Allogeneic Recognition Assay Embodiments. [Figure 109] Allogeneic Recognition Assay Detection Method Embodiments. [Figure 110] Results for post-thaw condition 1 (18-24 hours overnight after thawing). The effector:target (E:T) ratios tested ranged from 35:1 to 2.5:1, and the co-culture durations tested were 4, 8, 16, and 36 hours. [Figure 111] Results from post-thaw condition 2 (no post-thaw settling). The effector:target (E:T) ratios tested ranged from 10:1 to 1:10, and the co-culture duration was overnight (16-24 hours). TILs generated from ovarian cancer tumors (OV8178) and melanoma tumors (M1203) were tested. [Figure 112] 1. An exemplary embodiment of a TIL-Raji cell-based potency assay. [Figure 113] Total viable cells (TVC, upper plot) and % viability (lower plot) versus resting time. [Figure 114] Results of Raji and K562 co-culture experiments using two TIL cell lines with or without 300 IU / mL IL-2, showing IFN-γ secretion (pg / mL). [Figure 115] Results of Raji and K562 co-culture experiments using two TIL cell lines with or without 300 IU / mL IL-2, showing IFN-γ secretion (units of fold change). [Figure 116]Results of Raji and K562 co-culture experiments with two TIL cell lines under different co-culture conditions, showing IFN-γ secretion (pg / mL). [Figure 117] Results of Raji and K562 co-culture experiments using two TIL cell lines under different co-culture conditions, showing IFN-γ secretion (units of fold change). [Figure 118] Results of co-culture experiments with two TIL cell lines, Raji and K562, under different co-culture conditions to evaluate cytokine secretion effects as the recovery period after TIL thawing is extended. IFN-γ secretion (pg / mL) is shown. [Figure 119] 1 shows the results of Raji and K562 co-culture experiments with two TIL cell lines under different co-culture conditions, an embodiment of the present invention, to evaluate the cytokine secretion effect as the recovery period after TIL thawing is extended, showing IFN-γ secretion (units of fold change). [Figure 120] Flow cytometry analysis of residual cell populations of the parental organism population of TIL line M1179 in two embodiments of co-culture conditions over a 72-hour static period. [Figure 121] Growth profile of the Raji cell line. [Figure 122] Growth profile of the K562 cell line. [Figure 123] FIG. 1 is a diagram of the experimental design for a TIL:tumor cell line co-culture assay, an embodiment of the present invention. [Figure 124] IFN-γ secretion (pg / mL, absolute values) of tested target (Raji, Ramos, Thp1, and U937) and negative control (K562) cell lines at three TIL:target cell ratios (3:1, 1:1, and 1:3), an embodiment of the present invention. [Figure 125] IFN-γ secretion of tested target (Raji, Ramos, Thp1, and U937) and negative control (K562) cell lines (fold change of [TIL + target] / [TIL alone]) at three TIL:target cell ratios (3:1, 1:1, and 1:3), an embodiment of the present invention. [Figure 126]IFN-γ secretion of tested target (Raji, Ramos, Thp1, and U937) and negative control (K562) cell lines ([TIL+target] / [TIL+K562]) at three TIL:target cell ratios (3:1, 1:1, and 1:3), an embodiment of the present invention. [Figure 127] Figure 1. TNF-α secretion (pg / mL, absolute values) of tested target (Raji, Ramos, Thp1, and U937) and negative control (K562) cell lines at three TIL:target cell ratios (3:1, 1:1, and 1:3), an embodiment of the present invention. [Figure 128] Figure 1 shows TNF-α secretion in tested target (Raji, Ramos, Thp1, and U937) and negative control (K562) cell lines (fold change of [TIL + target] / [TIL alone]) at three TIL:target cell ratios (3:1, 1:1, and 1:3), an embodiment of the present invention. [Figure 129] Figure 1 shows TNF-α secretion of tested target (Raji, Ramos, Thp1, and U937) and negative control (K562) cell lines ([TIL+target] / [TIL+K562]) at three TIL:target cell ratios (3:1, 1:1, and 1:3), an embodiment of the present invention. [Figure 130] FIG. 1 is a diagram of the experimental design for a TIL:tumor cell line co-culture assay, an embodiment of the present invention. [Figure 131] IFN-γ secretion (pg / mL) of the target and negative control (K562) cell lines tested, an embodiment of the present invention. [Figure 132] IFN-γ secretion (fold change of [TIL+target] / [TIL alone]) of tested target and negative control (K562) cell lines, an embodiment of the present invention. [Figure 133] IFN-γ secretion of tested target and negative control (K562) cell lines (fold change of [TIL+target] / [TIL+K562]), an embodiment of the present invention. [Figure 134] Mean and range of fold change of TIL+target cell line (or combination) versus TIL alone (left panel), and TIL+target cell line (or combination) versus TIL+K562 (right panel). [Figure 135] TNF-α secretion (pg / mL) of tested target and negative control cell lines, including combination target cell lines, for melanoma TIL lines M1152, M1187, M1198, and M1200. [Figure 136] TNF-α secretion (fold change of [TIL + tumor cell line or combination] / [TIL alone]) of tested target and negative control cell lines, including combination target cell lines, for melanoma TIL lines M1152, M1187, M1198, and M1200. [Figure 137] TNF-α secretion (fold change of [TIL + tumor cell line or combination] / [TIL + K562]) of tested target and negative control cell lines, including combination target cell lines, for melanoma TIL lines M1152, M1187, M1198, and M1200. [Figure 138] Granzyme B secretion (pg / mL) of tested target and negative control cell lines, including combination target cell lines, for melanoma TIL lines M1152, M1187, M1198, and M1200. [Figure 139] Granzyme B secretion (fold change of [TIL + tumor cell line or combination] / [TIL alone]) of tested target and negative control cell lines, including combination target cell lines, for melanoma TIL lines M1152, M1187, M1198, and M1200. [Figure 140] Granzyme B secretion (fold change of [TIL + tumor cell line or combination] / [TIL + K562]) of tested target and negative control cell lines, including combination target cell lines, for melanoma TIL lines M1152, M1187, M1198, and M1200. [Figure 141] FIG. 1 is a diagram of the experimental design for a TIL:tumor cell line co-culture assay, an embodiment of the present invention. [Figure 142] IFN-γ secretion (pg / mL) of tested target and negative control cell lines, including combination target cell lines, for the study NSCLC TIL lines L4253, L4254, L4262, and L4270. Black dotted line: 3:1 TIL alone. Gray dotted line: 1:1 TIL alone. [Figure 143]Fold IFN-γ secretion of tested targets and NSCLC cell lines (TILs + tumor cell line / TILs alone), including combination target cell lines, for the studied NSCLC TIL lines L4253, L4254, L4262, and L4270. [Figure 144] Fold IFN-γ secretion of tested targets and NSCLC cell lines, including combination target cell lines, for the studied NSCLC TIL lines L4253, L4254, L4262, and L4270 (TIL+tumor cell line / [TIL+K562]). [Figure 145] IFN-γ secretion (pg / mL) of tested target and negative control cell lines, including combination target cell lines, for clinical NSCLC TIL lines 1013-304, 1036-307, 1057-304, 1004-303, 1057-302, and 1088-303. Black dotted line: TIL alone. Data were acquired at a 1:1 TIL:target ratio.

[0079] [Figure 146] Fold IFN-γ secretion of tested target and negative control cell lines, including combination target cell lines, for clinical NSCLC TIL lines 1013-304, 1036-307, 1057-304, 1004-303, 1057-302, and 1088-303 (TIL + tumor cell line / TIL alone). Black dotted line: TIL alone. Data were acquired at a 1:1 TIL:target ratio. [Figure 147] Fold IFN-γ secretion (TIL + tumor cell line / [TIL + K562]) of tested target and negative control cell lines, including combination target cell lines, for clinical NSCLC TIL lines 1013-304, 1036-307, 1057-304, 1004-303, 1057-302, and 1088-303. Black dotted line: TIL alone. Data were acquired at a 1:1 TIL:target ratio. [Figure 148]Summary of fold change in IFN-γ secretion (TIL + tumor cell line / TIL alone) of tested target and negative control cell lines for clinical NSCLC TIL lines 1013-304, 1036-307, 1057-304, 1004-303, 1057-302, and 1088-303. Filled red symbols represent clinical partial responses, and remaining symbols represent clinical stable disease responses. [Figure 149] Summary of fold change in IFN-γ secretion (TIL+tumor cell line / [TIL+K562]) of tested target and negative control cell lines for clinical NSCLC TIL lines 1013-304, 1036-307, 1057-304, 1004-303, 1057-302, and 1088-303. Filled red symbols represent clinical partial responses, and remaining symbols represent clinical stable disease responses. [Figure 150] Summary of fold change in IFN-γ secretion (TIL + tumor cell line / TIL alone) for tested target and negative control cell lines for 19 research and clinical melanoma and NSCLC TIL lines. Boxes represent one standard deviation on either side of the mean, which is indicated by a line. [Figure 151] Summary of fold change in IFN-γ secretion (TIL+tumor cell line / [TIL+K562]) for tested target and negative control cell lines for 19 research and clinical melanoma and NSCLC TIL lines. Boxes represent one standard deviation on either side of the mean, which is indicated by a line. [Figure 152] Effect of irradiation on proliferation (total viable cells, TVC) of Raji and K562 cells during 24 h of co-culture. [Figure 153] Concentrations of secreted IFN-γ for two cervical cancer TIL lots upon co-culture with non-irradiated and irradiated Raji cells. [Fig. 154] Surface marker expression on K562 and Raji cells determined by flow cytometry for three markers with irradiation ("Irr.") and without irradiation ("Non-irr."). [Figure 155]IFN-γ fold change (triplicate samples) versus assay co-culture duration (hours) relative to TIL alone using three melanoma, two cervical, and one NSCLC TIL lines. [Figure 156] FIG. 1 is a diagram of the experimental design for a TIL:tumor cell line co-culture assay, an embodiment of the present invention. [Figure 157] Average fold change of tumor cell line IFN-γ+TILs relative to TILs alone at 1.0×10 TVC / well for 12 melanoma TIL lines. Boxes represent one standard deviation on either side of the mean, indicated by the line. [Figure 158] Mean fold change in IFN-γ of tumor cell line + TILs relative to TILs alone at 2.0 x 10 TVC / well for 12 melanoma TIL lines. Boxes represent one standard deviation on either side of the mean, indicated by the line. [Figure 159] Mean fold change in IFN-γ of tumor cell line + TILs relative to K562 negative control cells at 1.0 x 10 TVC / well for 12 melanoma TIL lines. Boxes represent one standard deviation on either side of the mean, indicated by the line. [Figure 160] Mean fold change in IFN-γ of tumor cell lines + TILs relative to K562 negative control cells at 2.0 x 10 TVC / well for 12 melanoma TIL lines. Boxes represent one standard deviation on either side of the mean, indicated by the line. [Figure 161] 1 is a diagram of the experimental design for the TIL:tumor cell line co-culture assay for two types of Thp1 (THP-1) monocytic cell lines, wild-type (WT) and genetically modified, which is also an embodiment of the present invention. [Figure 162] Comparison of IFN-γ values ​​(pg / mL) obtained for WT and genetically modified Thp1 target cells. [Figure 163] Comparison of IFN-γ fold change relative to TIL alone values ​​obtained for WT and gene-modified Thp1 target cells. [Fig. 164] FIG. 1 shows HLA blocking, a negative control for the assay of the present invention in certain embodiments. [Figure 165]Effect of HLA-I blockade on Thp1 (THP-1) and U937 monocytic cell lines at different antibody concentrations, as measured by percent viability for each monocytic cell line. [Figure 166] Effect of HLA-II blockade on Thp1 (THP-1) and U937 monocytic cell lines at different antibody concentrations, as measured by percent viability for each monocytic cell line. [Figure 167] Effect of HLA-II blockade on Raji cells at different antibody concentrations as measured by percent viability of Raji cells and genetically modified Raji cells (B2M KO). [Figure 168] Effect of HLA-I (aMHC I) and HLA-II (aMHC II) antibody blockade on the TIL cell line M1213. [Figure 169] Effect of HLA-I (aMHC I) and HLA-II (aMHC II) antibody blockade on the TIL cell line M1214. [Figure 170] Effect of HLA-I (aMHC I) and HLA-II (aMHC II) antibody blockade on the TIL cell line PD-LIM-20-08. [Figure 171] HLA (MHC) Class I and II Antibody Dose Titration Results. HLA Blocking Negative Control Method, Embodiment I, was performed using 20 μg / mL of HLA-I blocking antibody and 10 μg / mL of HLA-II blocking antibody. HLA Blocking Negative Control Method, Embodiment II, was performed using 10 μg / mL of HLA-I blocking antibody and 5 μg / mL of HLA-II blocking antibody. [Fig. 172] Results of background IFN-γ secretion from 10 TIL lines upon addition of anti-HLA-I antibody (designated αMHC I) and anti-HLA-II antibody (designated αMHC II), each performed in triplicate. Embodiment I and Embodiment II refer to experimental embodiments shown in Figure 173 and Figure 184, respectively, and described elsewhere herein, including the use of 20 μg / mL of HLA-I blocking antibody and 10 μg / mL of HLA-II blocking antibody for Embodiment I, and 10 μg / mL of HLA-I blocking antibody and 5 μg / mL of HLA-II blocking antibody for Embodiment II. [Figure 173] FIG. 1 is a diagram of the experimental design for TIL:tumor cell line co-culture assays using HLA-blocking antibodies as negative controls in place of negative control cell lines; these assays are collectively referred to herein as "Embodiment I," where applicable, and are also embodiments of the present invention. [Fig. 174] Results of an HLA-blocking negative control experiment of melanoma TIL line M1152 co-cultured with target cells and K562 cells as a control, reported as pg / mL of IFN-γ. [Figure 175] Results of an HLA-blocking negative control experiment of melanoma TIL line M1187 co-cultured with target cells and K562 cells as a control, reported as pg / mL of IFN-γ. [Figure 176] Results of an HLA-blocking negative control experiment of melanoma TIL line M1164 co-cultured with target cells and K562 cells as a control, reported as pg / mL of IFN-γ. [Figure 177] Results of HLA-blocking negative control experiments of melanoma TIL line M1213 co-cultured with target cells and K562 cells as a control, reported as pg / mL of IFN-γ. [Figure 178] Results of an HLA-blocking negative control experiment of melanoma TIL line M1214 co-cultured with target cells and K562 cells as a control, reported as pg / mL of IFN-γ. [Figure 179] Results of an HLA-blocked negative control experiment of the melanoma TIL line M1152 co-cultured with target cells and K562 cells as a control, reported as fold enhancement of IFN-γ release over TIL alone. [Figure 180] Results of an HLA-blocked negative control experiment of the melanoma TIL line M1187 co-cultured with target cells and K562 cells as a control, reported as fold enhancement of IFN-γ release over TIL alone. [Figure 181] Results of an HLA-blocked negative control experiment of the melanoma TIL line M1164 co-cultured with target cells and K562 cells as a control, reported as fold enhancement of IFN-γ release over TIL alone. [Figure 182]Results of an HLA-blocked negative control experiment of the melanoma TIL line M1213 co-cultured with target cells and K562 cells as a control, reported as fold enhancement of IFN-γ release over TIL alone. [Figure 183] Results of an HLA-blocked negative control experiment of the melanoma TIL line M1214 co-cultured with target cells and K562 cells as a control, reported as fold enhancement of IFN-γ release over TIL alone. [Figure 184] FIG. 1 is a diagram of the experimental design for TIL:tumor cell line co-culture assays using HLA-blocking antibodies as negative controls in place of negative control cell lines; these assays are collectively referred to herein as "Embodiment II," where applicable, and are also embodiments of the present invention. [Figure 185] Results of an HLA-blocking negative control experiment of the melanoma TIL line M1161 co-cultured with target cells and K562 cells as a control, reported as pg / mL of IFN-γ. The CD4+ and CD8+ populations of M1161 were determined by flow cytometry to be 1.4% and 97.6%, respectively. [Figure 186] Results of an HLA-blocked negative control experiment of the melanoma TIL line M1163 co-cultured with target cells and K562 cells as a control, reported as pg / mL of IFN-γ. The CD4+ and CD8+ populations of M1163 were determined by flow cytometry to be 60.6% and 34.9%, respectively. [Figure 187] Results of an HLA-blocked negative control experiment of the melanoma TIL line M1172 co-cultured with target cells and K562 cells as a control, reported as pg / mL of IFN-γ. The CD4+ and CD8+ populations of M1172 were determined by flow cytometry to be 71.8% and 22.2%, respectively. [Figure 188] Results of an HLA-blocking negative control experiment of the melanoma TIL line M1173 co-cultured with target cells and K562 cells as a control, reported as pg / mL of IFN-γ. The CD4+ and CD8+ populations of M1173 were determined by flow cytometry to be 13.4% and 81.6%, respectively. [Figure 189] Results of an HLA-blocking negative control experiment of the melanoma TIL line M1174 co-cultured with target cells and K562 cells as a control, reported as pg / mL of IFN-γ. The CD4+ and CD8+ populations of M1174 were determined by flow cytometry to be 92.3% and 6.2%, respectively. [Figure 190] Results of an HLA-blocked negative control experiment of the melanoma TIL line M1161 co-cultured with target cells and K562 cells as a control, reported as fold-enhancement of IFN-γ release relative to TIL alone. The CD4+ and CD8+ populations of M1161 were determined by flow cytometry to be 1.4% and 97.6%, respectively. [Figure 191] Results of an HLA-blocked negative control experiment of the melanoma TIL line M1163 co-cultured with target cells and K562 cells as a control, reported as fold-enhancement of IFN-γ release over TIL alone. The CD4+ and CD8+ populations of M1163 were determined by flow cytometry to be 60.6% and 34.9%, respectively. [Figure 192] Results of an HLA-blocked negative control experiment of the melanoma TIL line M1172 co-cultured with target cells and K562 cells as a control, reported as fold-enhancement of IFN-γ release relative to TIL alone. The CD4+ and CD8+ populations of M1172 were determined by flow cytometry to be 71.8% and 22.2%, respectively. [Figure 193] Results of an HLA-blocked negative control experiment of the melanoma TIL line M1173 co-cultured with target cells and K562 cells as a control, reported as fold-enhancement of IFN-γ release over TIL alone. The CD4+ and CD8+ populations of M1173 were determined by flow cytometry to be 13.4% and 81.6%, respectively. [Figure 194]Results of an HLA-blocked negative control experiment of the melanoma TIL line M1174 co-cultured with target cells and K562 cells as a control, reported as fold-enhancement of IFN-γ release relative to TIL alone. The CD4+ and CD8+ populations of M1174 were determined by flow cytometry to be 92.3% and 6.2%, respectively. [Figure 195] 1 is a diagram of the experimental design for TIL:tumor cell line co-culture assays using HLA-blocking antibodies as negative controls in place of negative control cell lines or in addition to TIL-alone control experiments; all of these assays are also embodiments of the present invention. 15 melanoma cell lines are used. [Figure 196] Results of HLA-blocking negative control experiments of 15 melanoma TIL lines cocultured with U937 target cells. [Figure 197] Results of an HLA-blocked negative control experiment of 15 melanoma TIL lines cocultured with Thp1 (THP-1) target cells. [Figure 198] Determination of effective TIL concentration for Thp1 cells. Stars indicate a 3:1 TIL:Thp1 ratio at 2 x 106 TVC. [Figure 199] Determination of effective TIL concentration on U937 cells. Stars indicate a 3:1 TIL:U937 ratio at 2x106 TVC. [Figure 200] Dose-response curves of three TIL lots (M1164, M1163, and M1169) using Thp1 and U937 target cells. [Figure 201] Dose-response curves of three additional TIL lots (M1218, M1174, and M1150A) using Thp1 and U937 target cells. [Figure 202] Parallel line analysis of six TIL lots using U937 target cells. All six TILs show a clear dose response. [Figure 203] Parallel line analysis of six TIL lots using Thp1 target cells. TIL M1173 and TIL M1213 show a clear dose response. [Figure 204]Dose-response curves for three TIL lots (M1145, M1161, and M1173) at a concentration of 1.5 x 10 TILs with or without HLA-blocking antibodies using U937 target cells. All three TIL lots show a clear dose response and complete inhibition of signal (specific inhibition) by antibody treatment. [Figure 205] Dose-response curves for three TIL lots (M1197, M1213, and M1214) at a concentration of 1.5 x 10 TILs with or without HLA-blocking antibodies using U937 target cells. All three TIL lots show a clear dose response and complete inhibition of signal (specific inhibition) by antibody treatment. [Figure 206] Histogram of LAG3 expression by flow cytometry for melanoma TIL clinical samples. [Figure 207] Histogram of KLRG1 expression by flow cytometry for melanoma TIL clinical samples. [Figure 208] Logarithmic distribution assessment of histograms of relative potency for 28 historical clinical lots using the U937 alloreactive co-culture assay. When the relative potency measurements calculated for each TIL lot were log-transformed, the resulting distribution was normal, indicating that the underlying distribution was lognormal. [Figure 209] Expansion of TILs incubated with U937 cells or allogeneic PBMCs during REP. [Figure 210] IFN-γ secretion in TILs stimulated with anti-CD3 and anti-CD28 beads after REP with U937 cells or allogeneic PBMCs. [Figure 211] Comparison of an embodiment of the alloreactive co-culture assay of the present invention (denoted "allo-pMHC-TCR interaction-induced T cell") with a conventional antibody bead-based assay for T cell potency testing.

[0080] Brief description of the sequence listing SEQ ID NO: 1 is the amino acid sequence of the heavy chain of muromonab.

[0081] SEQ ID NO: 2 is the amino acid sequence of the light chain of muromonab.

[0082] SEQ ID NO: 3 is the amino acid sequence of recombinant human IL-2 protein.

[0083] SEQ ID NO: 4 is the amino acid sequence of aldesleukin.

[0084] SEQ ID NO: 5 is an IL-2 form.

[0085] SEQ ID NO: 6 is an IL-2 form.

[0086] SEQ ID NO: 7 is an IL-2 form.

[0087] SEQ ID NO: 8 is a mucin domain polypeptide.

[0088] SEQ ID NO: 9 is the amino acid sequence of recombinant human IL-4 protein.

[0089] SEQ ID NO: 10 is the amino acid sequence of recombinant human IL-7 protein.

[0090] SEQ ID NO: 11 is the amino acid sequence of recombinant human IL-15 protein.

[0091] SEQ ID NO: 12 is the amino acid sequence of recombinant human IL-21 protein.

[0092] SEQ ID NO: 13 is the IL-2 sequence.

[0093] SEQ ID NO: 14 is the IL-2 mutein sequence.

[0094] SEQ ID NO: 15 is the sequence of an IL-2 mutein.

[0095] SEQ ID NO: 16 is HCDR1_IL-2 of IgG.IL2R67A.H1.

[0096] SEQ ID NO: 17 is HCDR2 of IgG.IL2R67A.H1.

[0097] SEQ ID NO: 18 is the HCDR3 of IgG.IL2R67A.H1.

[0098] SEQ ID NO: 19 is HCDR1_IL-2 Kabat of IgG.IL2R67A.H1.

[0099] SEQ ID NO: 20 is the HCDR2 Kabat of IgG.IL2R67A.H1.

[0100] SEQ ID NO: 21 is the HCDR3 Kabat of IgG.IL2R67A.H1.

[0101] SEQ ID NO: 22 is the HCDR1_IL-2 clotia of IgG.IL2R67A.H1.

[0102] SEQ ID NO: 23 is the HCDR2 clone of IgG.IL2R67A.H1.

[0103] SEQ ID NO: 24 is the HCDR3 clone of IgG.IL2R67A.H1.

[0104] SEQ ID NO: 25 is HCDR1_IL-2 IMGT of IgG.IL2R67A.H1.

[0105] SEQ ID NO: 26 is HCDR2 IMGT of IgG.IL2R67A.H1.

[0106] SEQ ID NO: 27 is the HCDR3 IMGT of IgG.IL2R67A.H1.

[0107] SEQ ID NO: 28 is the V H It's a chain.

[0108] SEQ ID NO: 29 is the heavy chain of IgG.IL2R67A.H1.

[0109] SEQ ID NO: 30 is the LCDR1 Kabat of IgG.IL2R67A.H1.

[0110] SEQ ID NO: 31 is the LCDR2 Kabat of IgG.IL2R67A.H1.

[0111] SEQ ID NO: 32 is the LCDR3 Kabat of IgG.IL2R67A.H1.

[0112] SEQ ID NO: 33 is the LCDR1 chothia of IgG.IL2R67A.H1.

[0113] SEQ ID NO: 34 is the LCDR2 chothia of IgG.IL2R67A.H1.

[0114] SEQ ID NO: 35 is the LCDR3 chothia of IgG.IL2R67A.H1.

[0115] SEQ ID NO: 36 is V L It's a chain.

[0116] SEQ ID NO: 37 is the light chain.

[0117] SEQ ID NO: 38 is the light chain.

[0118] SEQ ID NO: 39 is the light chain.

[0119] SEQ ID NO: 40 is the amino acid sequence of human 4-1BB.

[0120] SEQ ID NO: 41 is the amino acid sequence of mouse 4-1BB.

[0121] SEQ ID NO: 42 is the heavy chain of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0122] SEQ ID NO: 43 is the light chain of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0123] SEQ ID NO: 44 represents the heavy chain variable region (V) of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566). H )

[0124] SEQ ID NO: 45 is the light chain variable region (V) of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566). L )

[0125] SEQ ID NO: 46 is the heavy chain CDR1 of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0126] SEQ ID NO: 47 is the heavy chain CDR2 of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0127] SEQ ID NO: 48 is the heavy chain CDR3 of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0128] SEQ ID NO: 49 is the light chain CDR1 of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0129] SEQ ID NO: 50 is the light chain CDR2 of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0130] SEQ ID NO: 51 is the light chain CDR3 of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0131] SEQ ID NO: 52 is the heavy chain of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0132] SEQ ID NO: 53 is the light chain of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0133] SEQ ID NO: 54 is the heavy chain variable region (VH) of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0134] SEQ ID NO: 55 is the light chain variable region (VL) of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0135] SEQ ID NO: 56 is the heavy chain CDR1 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0136] SEQ ID NO: 57 is the heavy chain CDR2 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0137] SEQ ID NO: 58 is the heavy chain CDR3 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0138] SEQ ID NO: 59 is the light chain CDR1 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0139] SEQ ID NO: 60 is the light chain CDR2 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0140] SEQ ID NO: 61 is the light chain CDR3 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0141] SEQ ID NO: 62 is the Fc domain of the TNFRSF agonist fusion protein.

[0142] SEQ ID NO: 63 is the linker of the TNFRSF agonist fusion protein.

[0143] SEQ ID NO: 64 is the linker of the TNFRSF agonist fusion protein.

[0144] SEQ ID NO: 65 is the linker of the TNFRSF agonist fusion protein.

[0145] SEQ ID NO: 66 is the linker of the TNFRSF agonist fusion protein.

[0146] SEQ ID NO: 67 is the linker for the TNFRSF agonist fusion protein.

[0147] SEQ ID NO: 68 is the linker for the TNFRSF agonist fusion protein.

[0148] SEQ ID NO: 69 is the linker for the TNFRSF agonist fusion protein.

[0149] SEQ ID NO: 70 is the linker of the TNFRSF agonist fusion protein.

[0150] SEQ ID NO: 71 is the linker for the TNFRSF agonist fusion protein.

[0151] SEQ ID NO: 72 is the linker for the TNFRSF agonist fusion protein.

[0152] SEQ ID NO: 73 is the Fc domain of the TNFRSF agonist fusion protein.

[0153] SEQ ID NO: 74 is the linker for the TNFRSF agonist fusion protein.

[0154] SEQ ID NO: 75 is the linker for the TNFRSF agonist fusion protein.

[0155] SEQ ID NO: 76 is the linker for the TNFRSF agonist fusion protein.

[0156] SEQ ID NO: 77 is the 4-1BB ligand (4-1BBL) amino acid sequence.

[0157] SEQ ID NO: 78 is the soluble portion of the 4-1BBL polypeptide.

[0158] SEQ ID NO: 79 is the heavy chain variable region (V) of 4-1BB agonist antibody 4B4-1-1 version 1 H )

[0159] SEQ ID NO: 80 represents the light chain variable region (V) of 4-1BB agonist antibody 4B4-1-1 version 1 L )

[0160] SEQ ID NO: 81 is the heavy chain variable region (V H )

[0161] SEQ ID NO: 82 is the light chain variable region (V) of 4-1BB agonist antibody 4B4-1-1 version 2 L )

[0162] SEQ ID NO: 83 identifies the heavy chain variable region (V H )

[0163] SEQ ID NO: 84 identifies the light chain variable region (V L )

[0164] SEQ ID NO: 85 is the amino acid sequence of human OX40.

[0165] SEQ ID NO: 86 is the amino acid sequence of mouse OX40.

[0166] SEQ ID NO: 87 is the heavy chain of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0167] SEQ ID NO: 88 is the light chain of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0168] SEQ ID NO: 89 is the heavy chain variable region (V) of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562). H)

[0169] SEQ ID NO: 90 is the light chain variable region (V) of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562). L )

[0170] SEQ ID NO: 91 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0171] SEQ ID NO: 92 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0172] SEQ ID NO: 93 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0173] SEQ ID NO: 94 is the light chain CDR1 of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0174] SEQ ID NO: 95 is the light chain CDR2 of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0175] SEQ ID NO: 96 is the light chain CDR3 of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0176] SEQ ID NO: 97 is the heavy chain of the OX40 agonist monoclonal antibody 11D4.

[0177] SEQ ID NO: 98 is the light chain of the OX40 agonist monoclonal antibody 11D4.

[0178] SEQ ID NO: 99 is the heavy chain variable region (V) of the OX40 agonist monoclonal antibody 11D4. H )

[0179] SEQ ID NO: 100 is the light chain variable region (V) of the OX40 agonist monoclonal antibody 11D4. L )

[0180] SEQ ID NO: 101 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody 11D4.

[0181] SEQ ID NO: 102 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody 11D4.

[0182] SEQ ID NO: 103 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody 11D4.

[0183] SEQ ID NO: 104 is the light chain CDR1 of the OX40 agonist monoclonal antibody 11D4.

[0184] SEQ ID NO: 105 is the light chain CDR2 of the OX40 agonist monoclonal antibody 11D4.

[0185] SEQ ID NO: 106 is the light chain CDR3 of the OX40 agonist monoclonal antibody 11D4.

[0186] SEQ ID NO: 107 is the heavy chain of the OX40 agonist monoclonal antibody 18D8.

[0187] SEQ ID NO: 108 is the light chain of the OX40 agonist monoclonal antibody 18D8.

[0188] SEQ ID NO: 109 is the heavy chain variable region (V) of the OX40 agonist monoclonal antibody 18D8. H )

[0189] SEQ ID NO: 110 is the light chain variable region (V) of the OX40 agonist monoclonal antibody 18D8. L )

[0190] SEQ ID NO: 111 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody 18D8.

[0191] SEQ ID NO: 112 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody 18D8.

[0192] SEQ ID NO: 113 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody 18D8.

[0193] SEQ ID NO: 114 is the light chain CDR1 of the OX40 agonist monoclonal antibody 18D8.

[0194] SEQ ID NO: 115 is the light chain CDR2 of the OX40 agonist monoclonal antibody 18D8.

[0195] SEQ ID NO: 116 is the light chain CDR3 of the OX40 agonist monoclonal antibody 18D8.

[0196] SEQ ID NO: 117 is the heavy chain variable region (V) of the OX40 agonist monoclonal antibody Hu119-122. H )

[0197] SEQ ID NO: 118 is the light chain variable region (V) of the OX40 agonist monoclonal antibody Hu119-122. L )

[0198] SEQ ID NO: 119 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody Hu119-122.

[0199] SEQ ID NO: 120 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody Hu119-122.

[0200] SEQ ID NO: 121 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody Hu119-122.

[0201] SEQ ID NO: 122 is the light chain CDR1 of the OX40 agonist monoclonal antibody Hu119-122.

[0202] SEQ ID NO: 123 is the light chain CDR2 of the OX40 agonist monoclonal antibody Hu119-122.

[0203] SEQ ID NO: 124 is the light chain CDR3 of the OX40 agonist monoclonal antibody Hu119-122.

[0204] SEQ ID NO: 125 is the heavy chain variable region (V) of the OX40 agonist monoclonal antibody Hu106-222. H )

[0205] SEQ ID NO: 126 is the light chain variable region (V) of the OX40 agonist monoclonal antibody Hu106-222. L )

[0206] SEQ ID NO: 127 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody Hu106-222.

[0207] SEQ ID NO: 128 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody Hu106-222.

[0208] SEQ ID NO: 129 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody Hu106-222.

[0209] SEQ ID NO: 130 is the light chain CDR1 of the OX40 agonist monoclonal antibody Hu106-222.

[0210] SEQ ID NO: 131 is the light chain CDR2 of the OX40 agonist monoclonal antibody Hu106-222.

[0211] SEQ ID NO: 132 is the light chain CDR3 of the OX40 agonist monoclonal antibody Hu106-222.

[0212] SEQ ID NO: 133 is the OX40 ligand (OX40L) amino acid sequence.

[0213] SEQ ID NO: 134 is the soluble portion of the OX40L polypeptide.

[0214] SEQ ID NO: 135 is an alternative soluble portion of the OX40L polypeptide.

[0215] SEQ ID NO: 136 is the heavy chain variable region (V H )

[0216] SEQ ID NO: 137 is the light chain variable region (V) of OX40 agonist monoclonal antibody 008. L )

[0217] SEQ ID NO: 138 is the heavy chain variable region (V) of OX40 agonist monoclonal antibody 011. H )

[0218] SEQ ID NO: 139 is the light chain variable region (V) of the OX40 agonist monoclonal antibody 011. L )

[0219] SEQ ID NO: 140 is the heavy chain variable region (V) of the OX40 agonist monoclonal antibody 021. H )

[0220] SEQ ID NO: 141 is the light chain variable region (V) of the OX40 agonist monoclonal antibody 021. L )

[0221] SEQ ID NO: 142 is the heavy chain variable region (V) of the OX40 agonist monoclonal antibody 023. H )

[0222] SEQ ID NO: 143 is the light chain variable region (V) of the OX40 agonist monoclonal antibody 023. L )

[0223] SEQ ID NO: 144 is the heavy chain variable region (V H )

[0224] SEQ ID NO: 145 is the light chain variable region (V) of the OX40 agonist monoclonal antibody L )

[0225] SEQ ID NO: 146 is the heavy chain variable region (V H )

[0226] SEQ ID NO: 147 is the light chain variable region (V) of the OX40 agonist monoclonal antibody L )

[0227] SEQ ID NO: 148 is the heavy chain variable region (V H )

[0228] SEQ ID NO: 149 is the heavy chain variable region (V) of a humanized OX40 agonist monoclonal antibody. H )

[0229] SEQ ID NO: 150 is the light chain variable region (V) of a humanized OX40 agonist monoclonal antibody. L )

[0230] SEQ ID NO: 151 is the light chain variable region (V) of a humanized OX40 agonist monoclonal antibody. L )

[0231] SEQ ID NO: 152 is the heavy chain variable region (V H )

[0232] SEQ ID NO: 153 is the heavy chain variable region (V) of a humanized OX40 agonist monoclonal antibody. H )

[0233] SEQ ID NO: 154 is the light chain variable region (V) of a humanized OX40 agonist monoclonal antibody. L )

[0234] SEQ ID NO: 155 is the light chain variable region (V) of a humanized OX40 agonist monoclonal antibody. L)

[0235] SEQ ID NO: 156 is the heavy chain variable region (V H )

[0236] SEQ ID NO: 157 is the light chain variable region (V) of the OX40 agonist monoclonal antibody L )

[0237] SEQ ID NO: 158 is the heavy chain amino acid sequence of the PD-1 inhibitor nivolumab.

[0238] SEQ ID NO: 159 is the light chain amino acid sequence of the PD-1 inhibitor nivolumab.

[0239] SEQ ID NO: 160 represents the heavy chain variable region (V) of the PD-1 inhibitor nivolumab H ) amino acid sequence.

[0240] SEQ ID NO: 161 represents the light chain variable region (V) of the PD-1 inhibitor nivolumab L ) amino acid sequence.

[0241] SEQ ID NO: 162 is the heavy chain CDR1 amino acid sequence of the PD-1 inhibitor nivolumab.

[0242] SEQ ID NO: 163 is the heavy chain CDR2 amino acid sequence of the PD-1 inhibitor nivolumab.

[0243] SEQ ID NO: 164 is the heavy chain CDR3 amino acid sequence of the PD-1 inhibitor nivolumab.

[0244] SEQ ID NO: 165 is the light chain CDR1 amino acid sequence of the PD-1 inhibitor nivolumab.

[0245] SEQ ID NO: 166 is the light chain CDR2 amino acid sequence of the PD-1 inhibitor nivolumab.

[0246] SEQ ID NO: 167 is the light chain CDR3 amino acid sequence of the PD-1 inhibitor nivolumab.

[0247] SEQ ID NO: 168 is the heavy chain amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0248] SEQ ID NO: 169 is the light chain amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0249] SEQ ID NO: 170 represents the heavy chain variable region (V) of the PD-1 inhibitor pembrolizumab H ) amino acid sequence.

[0250] SEQ ID NO: 171 is the light chain variable region (V) of the PD-1 inhibitor pembrolizumab L ) amino acid sequence.

[0251] SEQ ID NO: 172 is the heavy chain CDR1 amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0252] SEQ ID NO: 173 is the heavy chain CDR2 amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0253] SEQ ID NO: 174 is the heavy chain CDR3 amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0254] SEQ ID NO: 175 is the light chain CDR1 amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0255] SEQ ID NO: 176 is the light chain CDR2 amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0256] SEQ ID NO: 177 is the light chain CDR3 amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0257] SEQ ID NO: 178 is the heavy chain amino acid sequence of the PD-L1 inhibitor durvalumab.

[0258] SEQ ID NO: 179 is the light chain amino acid sequence of the PD-L1 inhibitor durvalumab.

[0259] SEQ ID NO: 180 represents the heavy chain variable region (V) of the PD-L1 inhibitor durvalumab H ) amino acid sequence.

[0260] SEQ ID NO: 181 represents the light chain variable region (V) of the PD-L1 inhibitor durvalumab L ) amino acid sequence.

[0261] SEQ ID NO: 182 is the heavy chain CDR1 amino acid sequence of the PD-L1 inhibitor durvalumab.

[0262] SEQ ID NO: 183 is the heavy chain CDR2 amino acid sequence of the PD-L1 inhibitor durvalumab.

[0263] SEQ ID NO: 184 is the heavy chain CDR3 amino acid sequence of the PD-L1 inhibitor durvalumab.

[0264] SEQ ID NO: 185 is the light chain CDR1 amino acid sequence of the PD-L1 inhibitor durvalumab.

[0265] SEQ ID NO: 186 is the light chain CDR2 amino acid sequence of the PD-L1 inhibitor durvalumab.

[0266] SEQ ID NO: 187 is the light chain CDR3 amino acid sequence of the PD-L1 inhibitor durvalumab.

[0267] SEQ ID NO: 188 is the heavy chain amino acid sequence of the PD-L1 inhibitor avelumab.

[0268] SEQ ID NO: 189 is the light chain amino acid sequence of the PD-L1 inhibitor avelumab.

[0269] SEQ ID NO: 190 represents the heavy chain variable region (V) of the PD-L1 inhibitor avelumab H ) amino acid sequence.

[0270] SEQ ID NO: 191 is the light chain variable region (V) of the PD-L1 inhibitor avelumab L ) amino acid sequence.

[0271] SEQ ID NO: 192 is the heavy chain CDR1 amino acid sequence of the PD-L1 inhibitor avelumab.

[0272] SEQ ID NO: 193 is the heavy chain CDR2 amino acid sequence of the PD-L1 inhibitor avelumab.

[0273] SEQ ID NO: 194 is the heavy chain CDR3 amino acid sequence of the PD-L1 inhibitor avelumab.

[0274] SEQ ID NO: 195 is the light chain CDR1 amino acid sequence of the PD-L1 inhibitor avelumab.

[0275] SEQ ID NO: 196 is the light chain CDR2 amino acid sequence of the PD-L1 inhibitor avelumab.

[0276] SEQ ID NO: 197 is the light chain CDR3 amino acid sequence of the PD-L1 inhibitor avelumab.

[0277] SEQ ID NO: 198 is the heavy chain amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0278] SEQ ID NO: 199 is the light chain amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0279] SEQ ID NO: 200 represents the heavy chain variable region (V) of the PD-L1 inhibitor atezolizumab H ) amino acid sequence.

[0280] SEQ ID NO: 201 is the light chain variable region (V) of the PD-L1 inhibitor atezolizumab L ) amino acid sequence.

[0281] SEQ ID NO: 202 is the heavy chain CDR1 amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0282] SEQ ID NO: 203 is the heavy chain CDR2 amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0283] SEQ ID NO: 204 is the heavy chain CDR3 amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0284] SEQ ID NO: 205 is the light chain CDR1 amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0285] SEQ ID NO: 206 is the light chain CDR2 amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0286] SEQ ID NO: 207 is the light chain CDR3 amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0287] SEQ ID NO: 208 is the heavy chain amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0288] SEQ ID NO: 209 is the light chain amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0289] SEQ ID NO: 210 represents the heavy chain variable region (V) of the CTLA-4 inhibitor ipilimumab H ) amino acid sequence.

[0290] SEQ ID NO: 211 represents the light chain variable region (V) of the CTLA-4 inhibitor ipilimumab L ) amino acid sequence.

[0291] SEQ ID NO: 212 is the heavy chain CDR1 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0292] SEQ ID NO: 213 is the heavy chain CDR2 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0293] SEQ ID NO: 214 is the heavy chain CDR3 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0294] SEQ ID NO: 215 is the light chain CDR1 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0295] SEQ ID NO: 216 is the light chain CDR2 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0296] SEQ ID NO: 217 is the light chain CDR3 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0297] SEQ ID NO: 218 is the heavy chain amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0298] SEQ ID NO: 219 is the light chain amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0299] SEQ ID NO: 220 represents the heavy chain variable region (V) of the CTLA-4 inhibitor tremelimumab H ) amino acid sequence.

[0300] SEQ ID NO: 221 represents the light chain variable region (V) of the CTLA-4 inhibitor tremelimumab L ) amino acid sequence.

[0301] SEQ ID NO: 222 is the heavy chain CDR1 amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0302] SEQ ID NO: 223 is the heavy chain CDR2 amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0303] SEQ ID NO: 224 is the heavy chain CDR3 amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0304] SEQ ID NO: 225 is the light chain CDR1 amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0305] SEQ ID NO: 226 is the light chain CDR2 amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0306] SEQ ID NO: 227 is the light chain CDR3 amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0307] SEQ ID NO: 228 is the heavy chain amino acid sequence of the CTLA-4 inhibitor zalifrelimab.

[0308] SEQ ID NO: 229 is the light chain amino acid sequence of the CTLA-4 inhibitor zalifrelimab.

[0309] SEQ ID NO: 230 is the heavy chain variable region (V) of the CTLA-4 inhibitor zalifrelimab H ) amino acid sequence.

[0310] SEQ ID NO: 231 is the light chain variable region (V) of the CTLA-4 inhibitor zalifrelimab L ) amino acid sequence.

[0311] SEQ ID NO: 232 is the heavy chain CDR1 amino acid sequence of the CTLA-4 inhibitor zalifrelimab.

[0312] SEQ ID NO: 233 is the heavy chain CDR2 amino acid sequence of the CTLA-4 inhibitor zalifrelimab.

[0313] SEQ ID NO: 234 is the heavy chain CDR3 amino acid sequence of the CTLA-4 inhibitor zalifrelimab.

[0314] SEQ ID NO: 235 is the light chain CDR1 amino acid sequence of the CTLA-4 inhibitor zalifrelimab.

[0315] SEQ ID NO: 236 is the light chain CDR2 amino acid sequence of the CTLA-4 inhibitor zalifrelimab.

[0316] SEQ ID NO: 237 is the light chain CDR3 amino acid sequence of the CTLA-4 inhibitor zalifrelimab. DETAILED DESCRIPTION OF THE INVENTION

[0317] I. Introduction Adoptive cell therapy utilizing TILs cultured ex vivo by rapid expansion protocols (REPs) has been successful in patients with cancers such as melanoma following host immunosuppression. Current infusion acceptance parameters rely on readouts of TIL composition (e.g., CD28, CD8, or CD4 positive), expression of various cytokines and other markers upon stimulation in bead-based potency assays (e.g., IFN-γ), and numerical fold expansion and viability of the REP product. Described herein are potency assays that can offer improvements over assays known in the art, including, among other things, superior performance, better control over the potency of T cell products, and increased biological relevance.

[0318] II. Definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All patents and publications referenced herein are incorporated by reference in their entirety.

[0319] As used herein, the terms "co-administration," "co-administering," "administered in combination," "administering in combination," "simultaneous," and "concurrent" encompass administration of two or more active pharmaceutical ingredients (e.g., multiple TILs) to a subject or patient such that both active pharmaceutical ingredients and / or their metabolites are present in the subject or patient at the same time. Simultaneous administration includes simultaneous administration in separate compositions, administration at different times in separate compositions, or administration in a composition in which two or more active pharmaceutical ingredients are present. Concurrent administration in separate compositions and administration in a composition in which both agents are present are preferred.

[0320] The term "in vivo" refers to events that take place inside a subject's body or patient's body.

[0321] The term "in vitro" refers to events that occur outside a subject's or patient's body. In vitro assays include cell-based assays in which living or dead cells are used, and can also include cell-free assays in which no intact cells are used.

[0322] The term "ex vivo" refers to events involving performing a treatment or procedure on cells, tissues, and / or organs that have been removed from a subject's or patient's body. Suitably, the cells, tissues, and / or organs may be returned to the subject's or patient's body in a surgical or therapeutic manner.

[0323] The term "rapid expansion" refers to an increase in the number of antigen-specific TILs by at least about 3-fold (or 4, 5, 6, 7, 8, or 9-fold) over a one-week period, more preferably by at least about 10-fold (or 20, 30, 40, 50, 60, 70, 80, or 90-fold) over a one-week period, or most preferably by at least about 100-fold over a one-week period. Several rapid expansion protocols are described herein.

[0324] As used herein, "tumor-infiltrating lymphocytes" or "TILs" refer to a population of cells originally acquired as leukocytes that leave a subject's bloodstream and migrate to a tumor. TILs include, but are not limited to, CD8+ cytotoxic T cells (lymphocytes), Th1 and Th17 CD4+ T cells, natural killer cells, dendritic cells, and M1 macrophages. TILs include both primary and secondary TILs. "Primary TILs" are those obtained from a patient tissue sample as outlined herein (sometimes referred to as "freshly harvested"), and "secondary TILs" are any expanded or propagated TIL cell populations discussed herein, including, but not limited to, bulk TILs and expanded TILs ("REP TILs" or "post-REP TILs"). TIL cell populations may include genetically modified TILs.

[0325] As used herein, a "population of cells" (including TILs) refers to several cells that share a common trait. Generally, a population is roughly 1 x 10 6 ~1×10 10 The number of TILs ranges from approximately 1 x 10 to 1 x 10, with different TIL populations containing different numbers. For example, the initial growth of primary TILs in the presence of IL-2 is approximately 1 x 10 8 REP expansion typically yields a bulk TIL population of 1.5 x 10 cells for injection. 9 ~1.5×10 10 This is done to provide a population of cells.

[0326] As used herein, "cryopreserved TILs" refers to TILs, either primary, bulk, or expanded (REP TILs), that are processed and stored at temperatures ranging from approximately -150°C to -60°C. General methods for cryopreservation are described elsewhere herein, including in the Examples. For clarity, "cryopreserved TILs" can be distinguished from frozen tissue samples that may be used as a source of primary TILs.

[0327] By "thawed cryopreserved TILs" herein is meant a population of TILs that have been previously cryopreserved and then processed to return to room temperature or above, including but not limited to, cell culture temperature or a temperature at which the TILs can be administered to a patient.

[0328] TILs can generally be defined either biochemically using cell surface markers or functionally by their ability to infiltrate tumors and achieve therapy. TILs can generally be classified by expression of one or more of the following biomarkers: CD4, CD8, TCRαβ, CD27, CD28, CD56, CCR7, CD45Ra, CD95, PD-1, and CD25. Additionally and alternatively, TILs can be functionally defined by their ability to infiltrate solid tumors upon reintroduction into patients.

[0329] The term "cryopreservation media" or "cryopreservation medium" refers to any medium that can be used for cryopreserving cells. Such media can include media containing 7% to 10% DMSO. Exemplary media include CryoStor CS10, Hyperthermasol, and combinations thereof. The term "CS10" refers to cryopreservation media obtained from Stemcell Technologies or Biolife Solutions. CS10 medium may be referred to by the trade name "CryoStor® CS10." CS10 medium is a serum-free, animal-component-free medium that contains DMSO.

[0330] The term "central memory T cells" refers to cells that are CD45R0+ and CCR7 (CCR7hi or CCR7) in humans. + ) and CD62L (CD62hi). The surface phenotype of central memory T cells also includes TCR, CD3, CD127 (IL-7R), and IL-15R. Transcription factors on central memory T cells include BCL-6, BCL-6B, MBD2, and BMI1. Central memory T cells primarily secrete IL-2 and CD40L as effector molecules after TCR triggering. Central memory T cells predominate in the CD4 compartment in the blood and are proportionally enriched in lymph nodes and tonsils in humans. Self-renewing stem memory T cells, or "T SCM " is T CM or T EM This is a subset that can differentiate into cells, as described in Gattinoni, et al., Nature Med. 2011, 17, 1290-97.

[0331] "Effector memory T cells," "effector memory T cells," or "T EM The term "CD45R0+" refers to cells that, like central memory T cells, are CD45R0+ but contain CCR7 (CCR7lo or CCR -This refers to a subset of human or mammalian T cells that have lost constitutive expression of CD62L (CD62Llo) and have heterogeneous or low CD62L expression (CD62Llo). The surface phenotype of central memory T cells also includes TCR, CD3, CD127 (IL-7R), and IL-15R. Transcription factors on central memory T cells include BLIMP1. Effector memory T cells rapidly secrete high levels of inflammatory cytokines after antigen stimulation, including interferon-γ, IL-4, and IL-5. Effector memory T cells predominate in the CD8 compartment in the blood and are proportionally enriched in the lung, liver, and intestine in humans. CD8 + Effector memory T cells carry large amounts of perforin. Terminally differentiated T cells re-express the CD45RA marker. EM The cells are called "T EMRA " or "TEMRA" cells.

[0332] The term "closed system" refers to a system that is closed to the external environment. Any closed system suitable for cell culture methods can be used in the methods of the present invention. Closed systems include, but are not limited to, sealed G containers. Once tumor segments are added to the closed system, the system is not opened to the external environment until the TILs are ready to be administered to a patient.

[0333] The terms "fragmenting," "fragments," and "fragmented" as used herein to describe processes for destroying tumors include mechanical fragmentation methods such as crushing, slicing, dividing, and mincing tumor tissue, as well as any other method for disrupting the physical structure of tumor tissue.

[0334] The terms "peripheral blood mononuclear cells" and "PBMCs" refer to peripheral blood cells with round nuclei, including lymphocytes (T cells, B cells, NK cells) and monocytes. When used as antigen-presenting cells (PBMCs are a type of antigen-presenting cell), the peripheral blood mononuclear cells are preferably irradiated allogeneic peripheral blood mononuclear cells.

[0335] The terms "peripheral blood lymphocytes" and "PBLs" refer to T cells expanded from peripheral blood. In some embodiments, PBLs are isolated from whole blood or apheresis products from a donor. In some embodiments, PBLs are isolated from whole blood or apheresis products from a donor by positive or negative selection of a T cell phenotype, such as a CD3+CD45+ T cell phenotype.

[0336] The term "major histocompatibility complex" or "MHC" refers to a large genetic locus on vertebrate DNA that contains a series of closely linked, polymorphic genes that encode cell surface proteins known as MHC molecules, which are essential for the function of the adaptive immune system. MHC genes are highly polymorphic and result in two major products: MHC class I and MHC class II molecules. MHC class I proteins present endogenous antigens that arise from the cytoplasm. MHC class II proteins present exogenous antigens that arise extracellularly from foreign bodies, such as bacteria.

[0337] The term "MHC-dominant recognition" refers to the allogeneic interaction between the TCR complex of a T cell and the HLA-peptide complex of a target cell. MHC-dominant recognition is described in Felix and Allen, Nat. Rev. Immunol., 2007, 7(12), 942-53; Matzinger and Bevan, Cell Immunol., 1977, 29(1), 1-5; and Janeway, The Major Histocompatibility Complex and Its Functions in Immunobiology: The Immune System in Health and Disease, 5th edition, Garland Science, 2001, the disclosures of each of which are incorporated herein by reference. In MHC-dominant recognition, the allogeneic MHC molecule is better suited to the T cell receptor, which may result in strong binding that is less dependent on the peptide bound to the MHC molecule.

[0338] The term "human leukocyte antigen" or "HLA" or "HLA complex" refers to MHC molecules expressed on the surface of human cells. Human leukocyte antigens are encoded by the MHC gene complex in humans.

[0339] The terms "target cell" and "target cell line" refer to cells that are assay targets for T cells, such as TILs, MILs, or PBLs. In some embodiments, target cells express MHC class I and / or class II. In some embodiments, target cells include Raji cells and their derivatives, variants, modifications, and progeny, as well as other cells that express MHC class I and / or class II. In some embodiments, target cells include Thp1 cells and their derivatives, variants, modifications, and progeny. In some embodiments, target cells include Ramos cells and their derivatives, variants, modifications, and progeny. In some embodiments, target cells include U937 cells and their derivatives, variants, modifications, and progeny. In some embodiments, target cells include Daudi cells and their derivatives, variants, modifications, and progeny. In some embodiments, target cells are irradiated. In some embodiments, target cells are not irradiated. In some embodiments, the target cell line is a combination of any two of the Raji cell line, the Thp1 cell line, the Ramos cell line, the U937 cell line, and the Daudi cell line. In some embodiments, the target cell line is a combination of any three of the Raji cell line, the Thp1 cell line, the Ramos cell line, the U937 cell line, and the Daudi cell line. In some embodiments, the target cell line is a combination of any four of the Raji cell line, the Thp1 cell line, the Ramos cell line, the U937 cell line, and the Daudi cell line. In some embodiments, the target cell line is a combination of the Raji cell line, the Thp1 cell line, the Ramos cell line, the U937 cell line, and the Daudi cell line. In some embodiments, the target cell line is a mixed tumor target cell line. In some embodiments, the target cell line is an alloreactive target cell line. In some embodiments, the target cell line is a mixed tumor alloreactive target cell line. In one embodiment, the target cell line is a mixed tumor alloreactive target cell line comprising a combination of at least two of the following cell lines: Raji cell line, Ramos cell line, Thp1 cell line, U937 cell line, and Daudi cell line.

[0340] The term "Raji cells" or "Raji cell line" refers to a Burkitt's lymphoma cell line with B cell characteristics, available from multiple vendors, including the American Type Culture Collection (Manassas, VA, USA), as well as its derivatives, variants, modifications, and progeny. Raji cells are described in Theofilopoulos, et al., J. Clin. Invest. 1976, 57, 169-182; Sobel and Bokisch, Fed. Proc. 1975, 34, 965; and Theofilopoulos, et al., J. Exp. Med. 1974, 140, 1230-1244, the disclosures of each of which are incorporated herein by reference. Raji cells lack membrane-bound immunoglobulins but possess receptors for IgG Fc, C3b, C3d, and Clq. In one embodiment, Raji cells, or their derivatives, variants, modifications, or progeny, are target cells. In some embodiments, Raji cells are genetically modified to express a fluorescent, phosphorescent, chemiluminescent, or bioluminescent label, such that upon disruption of the cell membrane, the fluorescent, phosphorescent, chemiluminescent, or bioluminescent label is released into the medium for potential use in detection. For example, the genetic modification approach for bioluminescent detection described in U.S. Patent No. 10,415,015 (the disclosure of which is incorporated herein by reference) can be used to modify Raji cells.

[0341] The term "Thp1 cells" or "Thp1 cell line," also known as "THP-1 cells" or "THP-1 cell line," refers to a human acute monocytic leukemia cell line available from multiple vendors, including the American Type Culture Collection (Manassas, VA, USA), as well as its derivatives, variants, modifications, and progeny. The Thp1 cell line is described in Tsuchiya, et al., Int. J. Cancer 1980, 26, 171-176 and Bosshart and Heinzelmann, Ann. Transl. Med. 2016, 4(21), 438, the disclosures of each of which are incorporated herein by reference. In some embodiments, Thp1 cells or their derivatives, variants, modifications, or progeny are target cells. In some embodiments, Thp1 cells are genetically modified to express a fluorescent, phosphorescent, chemiluminescent, or bioluminescent label, such that upon disruption of their cell membrane, the fluorescent, phosphorescent, chemiluminescent, or bioluminescent label is released into the medium for potential use in detection. For example, the genetic modification approach for bioluminescent detection described in US Pat. No. 10,415,015 (the disclosure of which is incorporated herein by reference) can be used to modify Thp1 cells.

[0342] The term "Ramos cells" or "Ramos cell line" refers to a human Burkitt's lymphoma cell line, available from multiple vendors, including the American Type Culture Collection (Manassas, VA, USA), as well as its derivatives, variants, modifications, and progeny. The Ramos cell line is described in Benjamin, et al. J. Immunol. 1982, 129, 1336-1342, the disclosure of which is incorporated herein by reference. In some embodiments, Ramos cells or their derivatives, variants, modifications, or progeny are target cells. In some embodiments, Ramos cells are genetically modified to express a fluorescent, phosphorescent, chemiluminescent, or bioluminescent label, such that upon disruption of the cell membrane, the fluorescent, phosphorescent, chemiluminescent, or bioluminescent label is released into the medium for potential use in detection. For example, the genetic modification approach for bioluminescent detection described in U.S. Pat. No. 10,415,015 (the disclosure of which is incorporated herein by reference) can be used to modify Ramos cells.

[0343] The term "U937 cells" or "U937 cell line" refers to a human histiocytic lymphoma cell line, available from multiple vendors, including the American Type Culture Collection (Manassas, VA, USA), as well as its derivatives, variants, modifications, and progeny. The U937 cell line is described in Kraus, et al., J. Clin. Microbiol. 2007, 45, 3777-3780, the disclosure of which is incorporated herein by reference. In some embodiments, U937 cells or their derivatives, variants, modifications, or progeny are target cells. In some embodiments, U937 cells are genetically modified to express a fluorescent, phosphorescent, chemiluminescent, or bioluminescent label, such that upon disruption of the cell membrane, the fluorescent, phosphorescent, chemiluminescent, or bioluminescent label is released into the medium for potential use in detection. For example, the genetic modification approach for bioluminescent detection described in US Pat. No. 10,415,015 (the disclosure of which is incorporated herein by reference) can be used to modify U937 cells.

[0344] The term "Daudi cells" or "Daudi cell line" refers to a human Burkitt's lymphoma cell line, available from multiple vendors, including the American Type Culture Collection (Manassas, VA, USA), as well as its derivatives, variants, modifications, and progeny. The Daudi cell line is described in Gao, et al., J. Virol. 1997, 71, 84-94, the disclosure of which is incorporated herein by reference. In some embodiments, Daudi cells or their derivatives, variants, modifications, or progeny are target cells. In some embodiments, Daudi cells are genetically modified to express a fluorescent, phosphorescent, chemiluminescent, or bioluminescent label, such that upon disruption of the cell membrane, the fluorescent, phosphorescent, chemiluminescent, or bioluminescent label is released into the medium for potential use in detection. For example, the genetic modification approach for bioluminescent detection described in U.S. Pat. No. 10,415,015, the disclosure of which is incorporated herein by reference, can be used to modify Daudi cells.

[0345] The terms "negative control," "negative control cells," and "negative control cell line" refer to cells used as a negative control for T cell assays, including TIL, MIL, or PBL assays. In some embodiments, the target cells lack MHC class I and class II expression. In some embodiments, the target cells lack MHC class I expression. In some embodiments, the target cells lack MHC or HLA class I expression. In some embodiments, the target cells express minimal levels of MHC or HLA class I and / or class II.

[0346] The term "K562 cells" or "K562 cell line" refers to a human Caucasian chronic myeloid leukemia cell line with lymphoblastoid morphology available from multiple vendors, including the American Type Culture Collection (Manassas, VA, USA). K562 cells are described in Lozzio and Lozzio, Blood, 1975, 45, 321-34, and in certain embodiments, negative control cells include K562 cells and their derivatives, variants, modifications, and progeny, as well as other cells that do not express MHC or HLA class I or class II.

[0347] The term "anti-CD3 antibody" refers to an antibody or variant thereof, e.g., a monoclonal antibody, including a human, humanized, chimeric, or murine antibody, directed against the CD3 receptor in the T cell antigen receptor of mature 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 CD3ε. Other anti-CD3 antibodies include, for example, otelixizumab, teplizumab, and visilizumab.

[0348] The term "OKT-3" (also referred to herein as "OKT3") refers to a monoclonal antibody, including a human, humanized, chimeric, or murine antibody against the CD3 receptor in the T cell antigen receptor of mature T cells, or a biosimilar or variant thereof, including commercially available forms such as OKT-3 (30 ng / mL, MACS GMP CD3 pure, Miltenyi Biotech, Inc., San Diego, CA, USA) and muromonab, or variants, conservative amino acid substitutions, glycoforms, or biosimilars thereof. The amino acid sequences of the heavy and light chains of muromonab are shown in Table 1 (SEQ ID NO: 1 and SEQ ID NO: 2). A hybridoma capable of producing OKT-3 has been deposited with the American Type Culture Collection and assigned ATCC accession number CRL8001. A hybridoma capable of producing OKT-3 has also been deposited with the European Collection of Authenticated Cell Cultures (ECACC) and assigned catalog number 86022706. [Table 1]

[0349] 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. IL-2 is described, for example, in Nelson, J. Immunol. 2004, 172, 3983-88 and Malek, Annu. Rev. Immunol. 2008, 26, 453-79, the disclosures of which are incorporated herein by reference. The amino acid sequence of recombinant human IL-2 suitable for use in the present invention is shown in Table 2 (SEQ ID NO: 3). For example, the term IL-2 encompasses human recombinant IL-2 forms such as aldesleukin (PROLEUKIN, commercially available from multiple suppliers at 22 million IU per single-use vial), as well as the recombinant IL-2 form (catalog number CYT-209-b) marketed by CellGenix, Inc., Portsmouth, NH, USA (CELLGRO GMP) or ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA, and other commercial equivalents from other vendors. Aldesleukin (des-alanyl-1, serine-125 human IL-2) is a non-glycosylated human recombinant IL-2 form with a molecular weight of approximately 15 kDa. The amino acid sequence of aldesleukin suitable for use in the present invention is shown in Table 2 (SEQ ID NO: 4). The term IL-2 also encompasses pegylated forms of IL-2 described herein, including the pegylated IL-2 prodrug NKTR-214 available from Nektar Therapeutics (South San Francisco, CA, USA). NKTR-214 and pegylated IL-2 suitable for use in the present invention are described in U.S. Patent Application Publication No. US2014 / 0328791A1 and International Patent Application Publication No. WO2012 / 065086A1, the disclosures of which are incorporated herein by reference. Alternative forms of conjugated IL-2 suitable for use in the present invention are described in U.S. Patent Nos. 4,766,106, 5,206,344, 5,089,261, and 4,902,502, the disclosures of which are incorporated herein by reference.Formulations of IL-2 suitable for use in the present invention are described in US Pat. No. 6,706,289, the disclosure of which is incorporated herein by reference.

[0350] In some embodiments, a suitable form of IL-2 for use in the present invention is THOR-707. Additional alternative forms of IL-2 suitable for use in the present invention are described in U.S. Patent Application Publication Nos. US2020 / 0181220A1 and US2020 / 0330601A1, the disclosures of which are incorporated herein by reference. In some embodiments, a suitable form of IL-2 for use in the present invention is ALKS-4230. Additional alternative forms of IL-2 suitable for use in the present invention are described in U.S. Patent Application Publication Nos. US2021 / 0038684A1 and U.S. Patent No. 10,183,979, the disclosures of which are incorporated herein by reference. In some embodiments, an IL-2 form suitable for use in the present invention is an interleukin-2 (IL-2) conjugate comprising an isolated and purified IL-2 polypeptide and a conjugation moiety that binds to the isolated and purified IL-2 polypeptide at an amino acid position selected from K35, T37, R38, T41, F42, K43, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107, where the numbering of the amino acid residues corresponds to SEQ ID NO: 1 in U.S. Patent Application Publication No. US2020 / 018120. In some embodiments, the amino acid position is selected from T37, R38, T41, F42, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107. In some embodiments, the amino acid position is selected from T37, R38, T41, F42, F44, Y45, E61, E62, E68, P65, V69, L72, and Y107. In some embodiments, the amino acid position is selected from T37, T41, F42, F44, Y45, P65, V69, L72, and Y107. In some embodiments, the amino acid position is selected from R38 and K64. In some embodiments, the amino acid position is selected from E61, E62, and E68. In some embodiments, the amino acid position is E62. In some embodiments, an amino acid residue selected from K35, T37, R38, T41, F42, K43, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107 is further mutated to lysine, cysteine, or histidine.In some embodiments, the amino acid residue is mutated to cysteine. In some embodiments, the amino acid residue is mutated to lysine. In some embodiments, an amino acid residue selected from K35, T37, R38, T41, F42, K43, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107 is further mutated to a non-naturally occurring amino acid. In some embodiments, the unnatural amino acid is N6-azidoethoxy-L-lysine (AzK), N6-propargylethoxy-L-lysine (PraK), BCN-L-lysine, norbornene lysine, TCO-lysine, methyltetrazine lysine, allyloxycarbonyl lysine, 2-amino-8-oxononanoic acid, 2-amino-8-oxooctanoic acid, p-acetyl-L-phenylalanine, p-azidomethyl-L-phenylalanine (pAMF), p-iodo-L-phenylalanine, m-acetylphenylalanine, 2-amino-8-oxononanoic acid, p-propargyloxyphenylalanine, p-propargyl-phenylalanine, 3-methyl-phenylalanine, L-dopa, fluorinated phenylalanine, isopropyl and selenocysteine, or selenocysteine. In some embodiments, the IL-2 conjugate has a reduced affinity for the IL-2 receptor alpha (IL-2Rα) subunit compared to the wild-type IL-2 polypeptide.In some embodiments, the decreased affinity is about a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or greater than 99% decrease in binding affinity to IL-2Rα compared to the wild-type IL-2 polypeptide. In some embodiments, the decreased affinity is about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 30-fold, 50-fold, 100-fold, 200-fold, 300-fold, 500-fold, 1000-fold, or greater, compared to the wild-type IL-2 polypeptide. In some embodiments, the conjugation moiety impairs or blocks binding of IL-2 to IL-2Rα. In some embodiments, the conjugation moiety comprises a water-soluble polymer. In some embodiments, an additional conjugation moiety comprises a water-soluble polymer. In some embodiments, each of the water-soluble polymers independently comprises polyethylene glycol (PEG), poly(propylene glycol) (PPG), copolymers of ethylene glycol and propylene glycol, poly(oxyethylated polyol), poly(olefinic alcohol), poly(vinylpyrrolidone), poly(hydroxyalkyl methacrylamide), poly(hydroxyalkyl methacrylate), poly(saccharide), poly(α-hydroxy acid), poly(vinyl alcohol), polyphosphazene, polyoxazoline (POZ), poly(N-acryloylmorpholine), or a combination thereof. In some embodiments, each of the water-soluble polymers independently comprises PEG. In some embodiments, the PEG is linear PEG or branched PEG. In some embodiments, each of the water-soluble polymers independently comprises a polysaccharide. In some embodiments, the polysaccharide comprises dextran, polysialic acid (PSA), hyaluronic acid (HA), amylose, heparin, heparan sulfate (HS), dextrin, or hydroxyethyl starch (HES). In some embodiments, each of the water-soluble polymers independently comprises a glycan. In some embodiments, each of the water-soluble polymers independently comprises a polyamine. In some embodiments, the conjugated moiety comprises a protein. In some embodiments, the additional conjugated moiety comprises a protein.In some embodiments, each of the proteins independently comprises albumin, transferrin, or transthyretin. In some embodiments, each of the proteins independently comprises an Fc portion. In some embodiments, each of the proteins independently comprises an Fc portion of IgG. In some embodiments, the conjugation moiety comprises a polypeptide. In some embodiments, the additional conjugation moiety comprises a polypeptide. In some embodiments, each of the proteins independently comprises an XTEN peptide, a glycine-rich homoamino acid polymer (HAP), a PAS polypeptide, an elastin-like polypeptide (ELP), a CTP peptide, or a gelatin-like protein (GLK) polymer. In some embodiments, the isolated and purified IL-2 polypeptide is modified by glutamylation. In some embodiments, the conjugation moiety is directly attached to the isolated and purified IL-2 polypeptide. In some embodiments, the conjugation moiety is indirectly attached to the isolated and purified IL-2 polypeptide via a linker. In some embodiments, the linker comprises a homobifunctional linker.In some embodiments, the homobifunctional linker is selected from the group consisting of the Romant reagents dithiobis(succinimidyl propionate) DSP, 3′3′-dithiobis(sulfosuccinimidyl propionate) (DTSSP), disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl)suberate (BS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo-DST), ethylene glycobis(succinimidyl succinate) (EGS), disuccinimidyl glutarate (DSG), N,N′-disuccinimidyl carbonate (DSC), dimethyl adipimidate (DMA), dimethyl pimelimidate (DMP), dimethyl suberimidate (DMS), dimethyl-3,3′-dithiobispropionimidate (DTBP), 1,4-di-(3′-(2′-pyridyldithio)bis(2 ... (e)propionamido)butane (DPDPB), bismaleimidohexane (BMH), halogenated aryl-containing compounds (DFDNB), such as 1,5-difluoro-2,4-dinitrobenzene or 1,3-difluoro-4,6-dinitrobenzene, 4,4'-difluoro-3,3'-dinitrophenyl sulfone (DFDNPS), bis-[β-(4-azidosalicylamido)ethyl]disulfide (BASED), formaldehyde, glutaraldehyde, 1,4-butanediol diglycidyl ether, adipic acid dihydrazide, carbohydrazide, o-toluidine, 3,3'-dimethylbenzidine, benzidine, α,α'-p-diaminodiphenyl, diiodo-p-xylenesulfonic acid, N,N'-ethylene-bis(iodoacetamide), or N,N'-hexamethylenebis(iodoacetamide). In some embodiments, the linker comprises a heterobifunctional linker.In some embodiments, the heterobifunctional linker is N-succinimidyl 3-(2-pyridyldithio)propionate (sPDP), long-chain N-succinimidyl 3-(2-pyridyldithio)propionate (LC-sPDP), water-soluble long-chain N-succinimidyl 3-(2-pyridyldithio)propionate (sulfo-LC-sPDP), succinimidyloxycarbonyl-α-methyl-α-(2-pyridyldithio)toluene (sMPT), sulfosuccinimidyl-6-[α-methyl-α-(2-pyridyldithio)toluamido]hexanoate (sMPT), or sulfosuccinimidyl-6-[α-methyl-α-(2-pyridyldithio)toluamido]hexanoate (sMPT). sulfo-LC-sMPT), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sMCC), sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-sMCC), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MB), m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (sulfo-MB), N-succinimidyl (4-iodoacetyl)aminobenzoate (sIAB), sulfosuccinimidyl (4-iodoacetyl)aminobenzoate (sIAB), cetyl)aminobenzoate (sulfo-sIAB), succinimidyl-4-(p-maleimidophenyl)butyrate (sMPB), sulfosuccinimidyl-4-(p-maleimidophenyl)butyrate (sulfo-sMPB), N-(γ-maleimidobutyryloxy)succinimide ester (GMB), N-(γ-maleimidobutyryloxy)sulfosuccinimide ester (sulfo-GMB), succinimidyl 6-((iodoacetyl)amino)hexanoate (sIAX), succinimidyl 6-[6-(((iodoacetyl)amino)hexanoate

[0033] succinimidyl 4-(((iodoacetyl)amino)methyl)cyclohexane-1-carboxylate (sIAC), succinimidyl 6-(((((4-iodoacetyl)amino)methyl)cyclohexane-1-carbonyl)amino)hexanoate (sIACX), p-nitrophenyl iodoacetate (NPIA), carbonyl-reactive and sulfhydryl-reactive crosslinkers, such as 4-(4-N-maleimidophenyl)butyric acid hydrazide (MPBH), 4-(N-maleimidomethyl)cyclohexanoate. Benzene-1-carboxyl-hydrazide-8 (M2C2H), 3-(2-pyridyldithio)propionylhydrazide (PDPH), N-hydroxysuccinimidyl-4-azidosalicylate (NHs-AsA), N-hydroxysulfosuccinimidyl-4-azidosalicylate (sulfo-NHs-AsA), sulfosuccinimidyl-(4-azidosalicylamido)hexanoate (sulfo-NHs-LC-AsA), sulfosuccinimidyl-2-(p-azidosalicylamido)ethyl-1,3'-dithiopropionate (sAsD), N-hydroxysuccinimidyl-4-azidosalicylate (NHs-AsA), sulfosuccinimidyl-4-azidosalicylamido ...4-azidosalicylamido-1,3'- Succinimidyl-4-azidobenzoate (HsAB), N-hydroxysulfosuccinimidyl-4-azidobenzoate (sulfo-HsAB), N-succinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate (sANPAH), sulfosuccinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate (sulfo-sANPAH), N-5-azido-2-nitrobenzoyloxysuccinimide (ANB-NO), sulfosuccinimidyl-2-(m-azido-o-nitrobenzamido)-ethyl-1,3 '-dithiopropionate (sAND), N-succinimidyl-4(4-azidophenyl)-1,3'-dithiopropionate (sADP), N-sulfosuccinimidyl (4-azidophenyl)-1,3'-dithiopropionate (sulfo-sADP), sulfosuccinimidyl 4-(p-azidophenyl)butyrate (sulfo-sAPB), sulfosuccinimidyl 2-(7-azido-4-methylcoumarin-3-acetamido)ethyl-1,3'-dithiopropionate (sAED), sulfosuccinimidyl 7-azido-4-methylcoumarin-3-acetamido acetate (sulfo-sAMCA), p-nitrophenyl diazopyruvate (pNPDP), p-nitrophenyl-2-diazo-3,3,3-trifluoropropionate (PNP-DTP), 1-(ρ-azidosalicylamido)-4-(iodoacetamido)butane (AsIB), N-[4-(ρ-azidosalicylamido)butyl]-3'-(2'-pyridyldithio)propionamide (APDP), benzophenone-4-iodoacetamide, p-azidobenzoylhydrazide (ABH), 4-(ρ-azidosalicylamido)butylamine (AsBA),or p-azidophenylglyoxal (APG). In some embodiments, the linker comprises a cleavable linker, optionally including a dipeptide linker. In some embodiments, the dipeptide linker comprises Val-Cit, Phe-Lys, Val-Ala, or Val-Lys. In some embodiments, the linker comprises a non-cleavable linker. In some embodiments, the linker optionally comprises a maleimide group, including maleimidocaproyl (mc), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sMCC), or sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-sMCC). In some embodiments, the linker further comprises a spacer. In some embodiments, the spacer comprises p-aminobenzyl alcohol (PAB), p-aminobenzyloxycarbonyl (PABC), a derivative, or an analog thereof. In some embodiments, the conjugated moiety is capable of extending the serum half-life of the IL-2 conjugate. In some embodiments, the additional conjugated moiety is capable of extending the serum half-life of the IL-2 conjugate. In some embodiments, the IL-2 form suitable for use in the present invention is a fragment of any of the IL-2 forms described herein. In some embodiments, the IL-2 form suitable for use in the present invention is pegylated as disclosed in U.S. Patent Application Publication Nos. US2020 / 0181220A1 and US2020 / 0330601A1. In some embodiments, a suitable IL-2 form for use in the present invention is an IL-2 conjugate comprising an IL-2 polypeptide comprising N6-azidoethoxy-L-lysine (AzK) covalently attached to a conjugation moiety comprising polyethylene glycol (PEG), wherein the IL-2 polypeptide comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 1 in U.S. Patent Application Publication No. 2020 / 0330601 (listed herein in Table 2 as SEQ ID NO: 5), and wherein AzK is at positions K35, F42, F44, F50, F52, F54, F56, F58, F59, F60, F61, F62, F63, F64, F65, F66, F67, F68, F69, F69, F70, F71, F72, F73, F74, F75, F76, F77, F78, F79, F80, F81, F82, F83, F84, F85, F86, F87, F88, F89, F90, F91, F92, F93, F94, F95, F96, F97, F98, F99, F99, F100, F101, F102, F103, F104, F105, F106, F107, F108, F109, F110, F111, F112, F113, F114, F115, F116, F117, F118, F119, F120, F121, F122, F123, F124, F125, F126, F127, F128, F129, F130, F131The amino acid substitution is at positions K43, E62, P65, R38, T41, E68, Y45, V69, or L72. In some embodiments, the IL-2 polypeptide comprises a one-residue N-terminal deletion relative to SEQ ID NO: 1 in U.S. Patent Publication No. US2020 / 0330601 (listed herein as SEQ ID NO: 5 in Table 2). In some embodiments, an IL-2 form suitable for use in the present invention lacks IL-2R alpha chain association but retains normal binding to the intermediate affinity IL-2R beta-gamma signaling complex. In some embodiments, an IL-2 form suitable for use in the present invention is ALKS-4230. A form of IL-2 suitable for use in the present invention is set forth as SEQ ID NO: 1 in U.S. Patent Publication No. US2021 / 0038684A1 (listed herein as SEQ ID NO: 6 in Table 2). In some embodiments, a suitable IL-2 form for use in the present invention is a fusion protein comprising amino acids 24-452 of SEQ ID NO:2 in U.S. Patent No. 10,183,979 (SEQ ID NO:2 in U.S. Patent No. 10,183,979, listed herein as SEQ ID NO:7 in Table 2). In some embodiments, a suitable IL-2 form for use in the present invention is a fusion protein comprising amino acids 24-452 of SEQ ID NO:2 in U.S. Patent No. 10,183,979, or an amino acid sequence homologous to amino acids 24-452 of SEQ ID NO:2 in U.S. Patent No. 10,183,979, having at least 98% amino acid sequence identity over the entire length of amino acids 24-452 of SEQ ID NO:2 in U.S. Patent No. 10,183,979, and having the receptor antagonist activity of amino acids 24-452 of SEQ ID NO:2 in U.S. Patent No. 10,183,979. Optionally, in some embodiments, a form of IL-2 suitable for use in the present invention is a fusion protein comprising a first fusion partner linked to a second fusion partner by a mucin domain polypeptide linker, wherein the first fusion partner is IL-1Rα or a protein having at least 98% amino acid sequence identity to IL-1Rα and having receptor antagonist activity of IL-1Rα, and the second fusion partner comprises all or a portion of an immunoglobulin comprising an Fc region, and the mucin domain polypeptide linker is10,183,979 (listed herein as SEQ ID NO:8 in Table 2) or an amino acid sequence having at least 90% sequence identity to SEQ ID NO:14 in U.S. Pat. No. 10,183,979 (listed herein as SEQ ID NO:8 in Table 2), wherein the half-life of the fusion protein is greater than or equal to the half-life of the first fusion partner with the second fusion partner in the absence of a mucin domain polypeptide linker; [Table 2]

[0351] In some embodiments, IL-2 forms suitable for use in the present invention comprise a heavy chain variable region (V) comprising complementarity determining regions HCDR1, HCDR2, and HCDR3. H ) and a light chain variable region (V L ) and V H or V L and an IL-2 molecule or a fragment thereof grafted onto the CDR of a heavy chain variable region (V) comprising complementarity determining regions HCDR1, HCDR2, and HCDR3. H ) and a light chain variable region (V L ) and V H or V L and an IL-2 molecule or fragment thereof grafted onto the CDRs of:H or V L and an IL-2 molecule or a fragment thereof grafted onto the CDR of the IL-2 molecule, wherein the IL-2 molecule is a mutein, and the antibody cytokine grafted protein preferentially expands T effector cells over regulatory T cells, and the antibody comprises an IgG class light chain comprising SEQ ID NO: 69 in U.S. Patent Application Publication No. US2020 / 0270334A1 and an IgG class heavy chain comprising SEQ ID NO: 53 in U.S. Patent Application Publication No. US2020 / 0270334A1, an IgG class light chain comprising SEQ ID NO: 37 in U.S. Patent Application Publication No. US2020 / 0270334A1 and an IgG class heavy chain comprising SEQ ID NO: 54 in U.S. Patent Application Publication No. US2020 / 0270334A1. The antibody further comprises an IgG class heavy chain and an IgG class light chain selected from the group consisting of an IgG class heavy chain comprising SEQ ID NO: 21 in U.S. Patent Application Publication No. US2020 / 0270334A1, an IgG class light chain comprising SEQ ID NO: 69 in U.S. Patent Application Publication No. US2020 / 0270334A1 and an IgG class heavy chain comprising SEQ ID NO: 21 in U.S. Patent Application Publication No. US2020 / 0270334A1, and an IgG class light chain comprising SEQ ID NO: 37 and an IgG class heavy chain comprising SEQ ID NO: 53 in U.S. Patent Application Publication No. US2020 / 0270334A1.

[0352] In one embodiment, an IL-2 molecule or a fragment thereof is grafted into HCDR1 of VH, and the IL-2 molecule is a mutein. In one embodiment, an IL-2 molecule or a fragment thereof is grafted into HCDR2 of VH, and the IL-2 molecule is a mutein. In one embodiment, an IL-2 molecule or a fragment thereof is grafted into HCDR3 of VH, and the IL-2 molecule is a mutein. In one embodiment, an IL-2 molecule or a fragment thereof is grafted into LCDR1 of VL, and the IL-2 molecule is a mutein. In one embodiment, an IL-2 molecule or a fragment thereof is grafted into LCDR2 of VL, and the IL-2 molecule is a mutein. In one embodiment, an IL-2 molecule or a fragment thereof is grafted into LCDR3 of VL, and the IL-2 molecule is a mutein.

[0353] The insertion of the IL-2 molecule can be at or near the N-terminal region of the CDR, the middle region of the CDR, or at or near the C-terminal region of the CDR. In some embodiments, the antibody cytokine graft protein comprises an IL-2 molecule incorporated into the CDR, where the IL2 sequence does not frameshift the CDR sequence. In some embodiments, the antibody cytokine graft protein comprises an IL-2 molecule incorporated into the CDR, where the IL-2 sequence replaces all or part of the CDR sequence. The replacement with the IL-2 molecule can be at or near the N-terminal region of the CDR, the middle region of the CDR, or the C-terminal region of the CDR. The replacement with the IL-2 molecule can be as little as one or two amino acids of the CDR sequence, or the entire CDR sequence.

[0354] In some embodiments, the IL-2 molecule is directly grafted onto the CDR without a peptide linker and without additional amino acids between the CDR sequence and the IL-2 sequence. In some embodiments, the IL-2 molecule is indirectly grafted onto the CDR using a peptide linker with one or more additional amino acids between the CDR sequence and the IL-2 sequence.

[0355] In some embodiments, the IL-2 molecule described herein is an IL-2 mutein. In some cases, the IL-2 mutein comprises an R67A substitution. In some embodiments, the IL-2 mutein comprises the amino acid sequence SEQ ID NO:4 or SEQ ID NO:6 of U.S. Patent Application Publication No. 2020 / 0270334A1. In some embodiments, the IL-2 mutein comprises the amino acid sequence of Table 1 of U.S. Patent Application Publication No. US2020 / 0270334A1, the disclosure of which is incorporated herein by reference.

[0356] In some embodiments, the antibody cytokine transplant protein comprises an HCDR1 selected from the group consisting of SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:13, and SEQ ID NO:16 of U.S. Patent Application Publication No. US2020 / 0270334A1. In some embodiments, the antibody cytokine transplant protein comprises an HCDR1 selected from the group consisting of SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:13, and SEQ ID NO:16 of U.S. Patent Application Publication No. US2020 / 0270334A1, and an HCDR2 selected from the group consisting of SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, and SEQ ID NO:17 of U.S. Patent Application Publication No. US2020 / 0270334A1. In some embodiments, the antibody cytokine transplant protein comprises an HCDR1 selected from the group consisting of SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:13, and SEQ ID NO:16, an HCDR2 selected from the group consisting of SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, and SEQ ID NO:17 of U.S. Patent Application Publication No. US2020 / 0270334A1, and an HCDR3 selected from the group consisting of SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:15, and SEQ ID NO:18. In some embodiments, the antibody cytokine transplant protein comprises a VH region comprising the amino acid sequence of SEQ ID NO: 19 of U.S. Patent Application Publication No. US2020 / 0270334A1. In some embodiments, the antibody cytokine transplant protein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 21 of U.S. Patent Application Publication No. US2020 / 0270334A1. In some embodiments, the antibody cytokine transplant protein comprises a VL region comprising the amino acid sequence of SEQ ID NO: 35 of U.S. Patent Application Publication No. US2020 / 0270334A1. In some embodiments, the antibody cytokine transplant protein comprises a light chain comprising the amino acid sequence of SEQ ID NO: 37 of U.S. Patent Application Publication No. US2020 / 0270334A1. In some embodiments, the antibody cytokine transplant protein comprises a VH region comprising the amino acid sequence of SEQ ID NO: 19 of U.S. Patent Application Publication No. US2020 / 0270334A1, and a VL region comprising the amino acid sequence of SEQ ID NO: 35. In one embodiment, the antibody cytokine transplant protein comprises a heavy chain region comprising the amino acid sequence of SEQ ID NO: 21 of U.S. Patent Application Publication No. US2020 / 0270334A1, and a light chain region comprising the amino acid sequence of SEQ ID NO: 37.In some embodiments, the antibody cytokine transplant protein comprises IgG.IL2R67A.H1 of U.S. Patent Application Publication No. 2020 / 0270334A1. In some embodiments, the antibody component of the antibody cytokine transplant protein described herein comprises immunoglobulin sequences, framework sequences, or CDR sequences of palivizumab.

[0357] In some embodiments, the antibody cytokine transplant proteins described herein have a longer serum half-life than wild-type IL-2 molecules, such as, but not limited to, aldesleukin (Proleukin®) or equivalent molecules.

[0358] In certain embodiments, the antibody cytokine transplant proteins described herein have the sequences set forth in Table 3. [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4]

[0359] The term "IL-4" (also referred to herein as "IL4") refers to the cytokine known as interleukin 4, which is produced by Th2 T cells, as well as eosinophils, basophils, and mast cells. IL-4 regulates the differentiation of naive helper T cells (Th0 cells) into Th2 T cells. Steinke and Borish, Respir. Res. 2001, 2, 66-70. Upon activation by IL-4, Th2 T cells subsequently produce additional IL-4 in a positive feedback loop. IL-4 also stimulates B cell proliferation and class II MHC expression, inducing class switching from B cells to IgE and IgG1 expression. Recombinant human IL-4 suitable for use in the present invention is commercially available from several suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (catalog number CYT-211) and ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-15 recombinant protein, catalog number Gibco CTP0043). The amino acid sequence of recombinant human IL-4 suitable for use in the present invention is shown in Table 2 (SEQ ID NO: 9).

[0360] The term "IL-7" (also referred to herein as "IL7") refers to a glycosylated tissue-derived cytokine known as interleukin-7, which can be obtained from stromal and epithelial cells, as well as dendritic cells. Fry and Mackall, Blood 2002, 99, 3892-904. IL-7 can stimulate T cell development. IL-7 binds to the IL-7 receptor, a heterodimer consisting of the IL-7 receptor alpha and the common gamma chain receptor, which is a series of signals important for T cell development in the thymus and survival in the periphery. Recombinant human IL-7 suitable for use in the present invention is commercially available from several suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (catalog number CYT-254) and ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-15 recombinant protein, catalog number Gibco PHC0071). The amino acid sequence of a recombinant human IL-7 suitable for use in the present invention is shown in Table 2 (SEQ ID NO: 10).

[0361] 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-2, including human and mammalian forms, conservative amino acid substitutions, glycoforms, biosimilars, and variants. IL-15 is described, for example, in Fehniger and Caligiuri, Blood 2001, 97, 14-32, the disclosure of which is incorporated herein by reference. IL-15 shares β and γ signaling receptor subunits with IL-2. Recombinant human IL-15 is a single, non-glycosylated polypeptide chain containing 114 amino acids (and an N-terminal methionine) with a molecular weight of 12.8 kDa. Recombinant human IL-15 is commercially available from several suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (catalog number CYT-230-b) and ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-15 recombinant protein, catalog number 34-8159-82). The amino acid sequence of recombinant human IL-15 suitable for use in the present invention is shown in Table 2 (SEQ ID NO: 11).

[0362] The term "IL-21" (also referred to herein as "IL21") 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. IL-21 is described, for example, in Spolski and Leonard, Nat. Rev. Drug. Disc. 2014, 13, 379-95, the disclosure of which is incorporated herein by reference. IL-21 primarily stimulates natural killer T cells and activated human CD4 +It is produced by T cells. Recombinant human IL-21 is a single, non-glycosylated polypeptide chain containing 132 amino acids with a molecular weight of 15.4 kDa. Recombinant human IL-21 is commercially available from several suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (catalog number CYT-408-b) and ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-21 recombinant protein, catalog number 14-8219-80). The amino acid sequence of recombinant human IL-21 suitable for use in the present invention is shown in Table 2 (SEQ ID NO: 12).

[0363] When an "antitumor effective amount," "tumor inhibiting effective amount," or "therapeutic amount" is indicated, the exact amount of the composition of the present invention to be administered can be determined by a physician, taking into account individual differences in the age, weight, tumor size, extent of infection or metastasis, and health status of the patient (subject). Generally, the tumor-infiltrating lymphocytes (e.g., secondary TILs or genetically modified cytotoxic lymphocytes) described herein are administered at a dose of 10 per kg of body weight. 4 ~10 11 cells (e.g., 10 per kg body weight) 5 ~10 6 , 10 5 ~10 10 , 10 5 ~10 11 , 10 6 ~10 10 , 10 6 ~10 11 , 10 7 ~10 11 , 10 7 ~10 10 , 10 8 ~10 11 , 10 8 ~10 10 , 10 9 ~10 11 , or 10 9 ~10 10The tumor-infiltrating lymphocyte (optionally including genetically modified cytotoxic lymphocytes) compositions may also be administered at multiple doses of 1000-150 ...

[0364] The terms "hematological malignancies," "blood system malignancies," or terms of related meaning, refer to cancers and tumors of mammalian hematopoietic and lymphatic tissues, including, but not limited to, blood, bone marrow, lymph nodes, and lymphatic tissues. Hematological malignancies are also referred to as "liquid tumors." Hematological malignancies may include, but are not limited to, acute lymphoblastic leukemia (ALL), chronic lymphocytic lymphoma (CLL), small lymphocytic lymphoma (SLL), acute myeloid leukemia (AML), chronic myelogenous leukemia (CML), acute monocytic leukemia (AMoL), Hodgkin's lymphoma, and non-Hodgkin's lymphoma. The term "B-cell hematological malignancies" refers to hematological malignancies affecting B cells.

[0365] 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, leukemia, myeloma, and lymphoma, as well as other hematological malignancies. TILs obtained from liquid tumors may also be referred to herein as bone marrow-infiltrating lymphocytes (MILs). TILs obtained from liquid tumors, including those circulating in peripheral blood, may also be referred to herein as PBLs. The terms MILs, TILs, and PBLs are used interchangeably herein and differ only based on the tissue type from which the cells are derived.

[0366] The term "microenvironment" as used herein may refer to the solid or hematological tumor microenvironment as a whole, or to individual subsets of cells within the microenvironment. As used herein, the tumor microenvironment refers to a complex mixture of "cells, soluble factors, signaling molecules, extracellular matrix, and mechanical cues that promote neoplastic transformation, support tumor growth and invasion, protect tumors from host immunity, foster therapeutic resistance, and provide a niche for successful and dominant metastasis," as described in Swartz, et al., Cancer Res., 2012, 72, 2473. Tumors express antigens that are recognized by T cells, but tumor clearance by the immune system is rare due to immunosuppression by the microenvironment.

[0367] In one embodiment, the present invention includes a method of treating cancer with a TIL population, wherein the patient is pretreated with non-myeloablative chemotherapy prior to infusion of TILs according to the present invention. In some embodiments, a TIL population can be provided, and the patient is pretreated with non-myeloablative chemotherapy prior to infusion of TILs according to the present invention. In one embodiment, the non-myeloablative chemotherapy is cyclophosphamide 60 mg / kg / day for two days (27 and 26 days before TIL infusion) and fludarabine 25 mg / m2 / day for five days (27-23 days before TIL infusion). In one embodiment, after non-myeloablative chemotherapy and TIL infusion according to the present invention (day 0), the patient receives an intravenous infusion of IL-2 at 720,000 IU / kg every 8 hours to physiological tolerance.

[0368] Experimental findings indicate that lymphodepletion prior to adoptive transfer of tumor-specific T lymphocytes plays an important role in enhancing therapeutic efficacy by eliminating regulatory T cells and competing elements of the immune system ("cytokine sinks"). Accordingly, some embodiments of the present invention utilize a lymphodepletion step (sometimes referred to as "immunosuppressive conditioning") on patients prior to introducing the TILs of the present invention.

[0369] The term "effective amount" or "therapeutically effective amount" refers to an amount of a compound or combination of compounds described herein sufficient to achieve the intended use, including, but not limited to, disease treatment. A therapeutically effective amount may vary depending on the intended use (in vitro or in vivo), the subject and condition being treated (e.g., the subject's weight, age, and sex), the severity of the condition, or the method of administration. The term also applies to a dose that induces a specific response in target cells (e.g., reduced platelet adhesion and / or cell migration). The specific dose will vary depending on the particular compound selected, the dosing regimen to be followed, whether the compound is administered in combination with other compounds, the timing of administration, the tissue to which it is administered, and the physical delivery system by which the compound is delivered.

[0370] The terms "treatment," "treating," "treating," and the like refer to obtaining a desired pharmacological and / or physiological effect. The effect can be preventative, in terms of completely or partially preventing the disease or condition, and / or therapeutic, in terms of partially or completely curing the disease and / or side effects caused by the disease. "Treatment," as used herein, encompasses any treatment of disease in a mammal, particularly a human, and includes (a) preventing the disease from occurring in a subject who may be susceptible to the disease but has not yet been diagnosed with it; (b) suppressing the disease, i.e., arresting its onset or progression; and (c) palliating the disease, i.e., causing regression of the disease and / or alleviating one or more disease symptoms. "Treatment" is also intended to encompass the delivery of an agent to provide a pharmacological effect even in the absence of a disease or condition. For example, "treatment" encompasses the delivery of a composition capable of eliciting an immune response or conferring immunity in the absence of a pathology, e.g., in the case of a vaccine.

[0371] The term "heterologous," when used with reference to portions of a nucleic acid or protein, indicates that the nucleic acid or protein comprises two or more subsequences that are not found in the same relationship to each other in nature. For example, nucleic acids are typically produced recombinantly, with two or more sequences from unrelated genes arranged to create a new functional nucleic acid, e.g., a promoter from one source and a coding region from another source, or coding regions from different sources. Similarly, a heterologous protein indicates that the protein comprises two or more subsequences that are not found in the same relationship to each other in nature (e.g., a fusion protein).

[0372] The terms "sequence identity," "percent identity," and "percent sequence identity" (or their synonyms, e.g., "99% identical") in the context of two or more nucleic acids or polypeptides refer to two or more sequences or subsequences that are the same or have a specified percentage of the same nucleotide or amino acid residues when compared and aligned for maximum correspondence (introducing gaps, if necessary), without considering any conservative amino acid substitutions as part of the sequence identity. Percent identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software that can be used to obtain alignment of amino acid or nucleotide sequences are known in the art. Suitable programs for determining percent sequence identity include, for example, the BLAST suite of programs available from the BLAST website of the U.S. government's National Center for Biotechnology Information. Comparison between two sequences can be performed using either the BLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acid sequences, and BLASTP is used to compare amino acid sequences. ALIGN, ALIGN-2 (Genentech, South San Francisco, California), or MegAlign, available from DNASTAR, are additional publicly available software programs that can be used to align sequences. Those skilled in the art can determine the appropriate parameters for maximum alignment depending on the particular alignment software. In certain embodiments, the default parameters of the alignment software are used.

[0373] As used herein, the term "variant" includes, but is not limited to, an antibody or fusion protein comprising an amino acid sequence that differs from the amino acid sequence of a reference antibody by one or more substitutions, deletions, and / or additions at specific positions within or adjacent to the amino acid sequence of the reference antibody. A variant may contain one or more conservative substitutions in its amino acid sequence compared to the amino acid sequence of the reference antibody. Conservative substitutions may include, for example, substitutions of similarly charged or uncharged amino acids. A variant retains the ability of the reference antibody to specifically bind to an antigen. The term variant also includes pegylated antibodies or proteins.

[0374] As used herein, "tumor infiltrating lymphocytes" or "TILs" refers to a population of cells that are initially acquired as leukocytes that have left a subject's bloodstream and migrated to a tumor. TILs include CD8 + Cytotoxic T cells (lymphocytes), Th1 and Th17 CD4 + These include, but are not limited to, T cells, natural killer cells, dendritic cells, and M1 macrophages. TILs include both primary and secondary TILs. "Primary TILs" are those obtained from a patient tissue sample as outlined herein (sometimes referred to as "freshly harvested"). "Secondary TILs" are any TIL cell populations that have been expanded or propagated as discussed herein, including, but not limited to, bulk TILs, expanded TILs ("REP TILs"), and "reREP TILs" as discussed herein. reREP TILs can include, for example, second expanded TILs or second additional expanded TILs (e.g., those described in step D of FIG. 8, including TILs designated as reREP TILs).

[0375] TILs can generally be defined either biochemically using cell surface markers or functionally by their ability to infiltrate tumors and achieve therapy. TILs can generally be classified by the expression of one or more of the following biomarkers: CD4, CD8, TCRαβ, CD27, CD28, CD56, CCR7, CD45Ra, CD95, PD-1, and CD25. Additionally or alternatively, TILs can be functionally defined by their ability to infiltrate solid tumors upon reintroduction into patients. TILs can be further characterized by efficacy; for example, TILs can be considered potent if their 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.

[0376] The term "deoxyribonucleotide" encompasses natural and synthetic, unmodified and modified deoxyribonucleotides. Modifications include changes to the sugar moiety, the base moiety, and / or the linkages between deoxyribonucleotides in an oligonucleotide.

[0377] The term "RNA" defines a molecule containing at least one ribonucleotide residue. The term "ribonucleotide" defines a nucleotide having a hydroxyl group at the 2' position of a bD-ribofuranose moiety. The term RNA includes double-stranded RNA, single-stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, and altered RNA that differs from naturally occurring RNA by the addition, deletion, substitution, and / or alteration of one or more nucleotides. The nucleotides of the RNA molecules described herein can also include non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs may be referred to as analogs or analogs of naturally occurring RNA.

[0378] The term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and inactive ingredients. The use of such pharmaceutically acceptable carriers or pharmaceutically acceptable excipients for active pharmaceutical ingredients is well known in the art. Except insofar as any conventional pharmaceutically acceptable carrier or pharmaceutically acceptable excipient is incompatible with the active pharmaceutical ingredient, its use in the therapeutic compositions of the invention is contemplated. Additional active pharmaceutical ingredients, such as other drugs, can also be incorporated into the compositions and methods described.

[0379] The terms "about" and "approximately" mean within a statistically significant range of values. Such a range may be within one order of magnitude of a given value or range, preferably within 50%, more preferably within 20%, even more preferably within 10%, and even more preferably within 5%. The acceptable deviation encompassed by the term "about" or "approximately" depends on the particular system under study and can be readily understood by one of ordinary skill in the art. Furthermore, as used herein, the terms "about" and "approximately" mean that dimensions, sizes, formulations, parameters, shapes, and other quantities and characteristics are not, and need not be, exact, but may be approximate and / or larger or smaller, as appropriate, reflecting tolerances, conversion factors, rounding, measurement errors, etc., and other factors known to those of ordinary skill in the art. In general, a dimension, size, formulation, parameter, shape, or other quantity or characteristic is "about" or "approximately" whether or not expressly stated as such. It should be noted that embodiments of widely different sizes, shapes, and dimensions may employ the described configurations.

[0380] When used in the appended claims, the transitional terms "comprising," "consisting essentially of," and "consisting of" define the claim in its original and amended form, in terms of whether additional, unrecited claim elements or steps, if any, are excluded from the claim(s). The term "comprising" is intended to be inclusive or open-ended and does not exclude any additional, unrecited elements, methods, steps, or materials. The term "consisting of" excludes any element, step, or material other than those specified in the claim, and in the latter case, also excludes impurities normally associated with the specified material(s). The term "consisting essentially of" limits the claim to the specified element, step, or material(s) and does not materially affect the basic and novel feature(s) of the claimed invention. All compositions, methods, and kits described herein embodying the present invention may, in alternative embodiments, be more specifically defined by any of the transitional terms "comprising," "consisting essentially of," and "consisting of."

[0381] The terms "antibody" and its plural "antibodies" refer to whole immunoglobulins and any antigen-binding fragment ("antigen-binding portion") or single chains thereof. "Antibody" also refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or antigen-binding portions thereof. Each heavy chain contains a heavy chain variable region (referred to herein as V H Each light chain is composed of a light chain variable region (abbreviated herein as V) and a heavy chain constant region. The heavy chain constant region is composed of three domains: CH1, CH2, and CH3. L The light chain constant region consists of one domain, C L The V of the antibody H and V L The regions can be further subdivided into regions of hypervariability, termed complementarity-determining regions (CDRs) or hypervariable regions (HVRs), which may be interspersed with more conserved regions called framework regions (FRs). H and VL is composed of three CDRs and four FRs arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with one or more antigen epitopes. The constant region of the antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0382] The term "antigen" refers to a substance that induces an immune response. In some embodiments, an antigen is a molecule that can be bound by an antibody or TCR when presented by a major histocompatibility complex (MHC) molecule. As used herein, the term "antigen" also encompasses T cell epitopes. An antigen can additionally be recognized by the immune system. In some embodiments, an antigen can induce a humoral or cellular immune response, leading to the activation of B and / or T lymphocytes. In some cases, this may require that the antigen contain or be bound by a Th cell epitope. An antigen may also have one or more epitopes (e.g., B- and T-epitopes). In some embodiments, an antigen preferably reacts with a corresponding antibody or TCR, typically in a highly specific and selective manner, and not with many other antibodies or TCRs that may be induced by other antigens.

[0383] The terms "monoclonal antibody," "mAb," "monoclonal antibody composition," or their plurals, refer to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition exhibits a single binding specificity and affinity for a particular epitope. Monoclonal antibodies specific for a particular receptor can be produced using knowledge and techniques in the art by injecting a test subject with an appropriate antigen and then isolating hybridomas expressing antibodies with the desired sequence or functional characteristics. DNA encoding the monoclonal antibody is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to genes encoding the heavy and light chains of the monoclonal antibody). Hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA can be placed into an expression vector and then transfected into host cells such as E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not produce immunoglobulin protein to obtain the synthesis of the monoclonal antibody in the recombinant host cells. Recombinant production of antibodies is described in more detail below.

[0384] As used herein, the term "antigen-binding portion" or "antigen-binding fragment" of an antibody (or simply "antibody portion" or "fragment") refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed by the term "antigen-binding portion" of an antibody include: (i) V L , V H , C L (ii) a F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; and (iii) a V H and an Fd fragment consisting of the CH1 domain, (iv) a V of a single arm of an antibody L and V H Fv fragment consisting of domains, (v) V H or VL (v) domain antibody (dAb) fragments, which may consist of two domains, V and VD (Ward, et al., Nature, 1989, 341, 544-546), and (vi) isolated complementarity-determining regions (CDRs). L and V H are encoded by separate genes, which can be synthesized using recombinant methods. L and V H The regions can be joined by a synthetic linker that allows them to pair as a single protein chain to form a monovalent molecule known as a single-chain Fv (scFv); see, e.g., Bird, et al., Science 1988, 242, 423-426, and Huston, et al., Proc. Natl. Acad. Sci. USA 1988, 85, 5879-5883. Such scFv antibodies are also intended to be encompassed by the term "antigen-binding portion" or "antigen-binding fragment" of an antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as intact antibodies.

[0385] As used herein, the term "human antibody" is intended to include antibodies having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region also is derived from human germline immunoglobulin sequences. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). As used herein, the term "human antibody" is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0386] The term "human monoclonal antibody" refers to antibodies displaying a single binding specificity which have variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. In certain embodiments, human monoclonal antibodies are produced by hybridomas comprising B cells obtained from a transgenic non-human animal, e.g., a transgenic mouse, whose genome comprises human heavy chain and light chain transgenes fused to an immortalized cell.

[0387] As used herein, the term "recombinant human antibody" includes all human antibodies prepared, expressed, created, or isolated by recombinant means, such as (a) antibodies isolated from animals (such as mice) that are transgenic or transchromosomal for human immunoglobulin genes or hybridomas prepared therefrom (described further below); (b) antibodies isolated from host cells transformed to express human antibodies, e.g., from transfectomas; (c) antibodies isolated from recombinant combinatorial human antibody libraries; and (d) antibodies prepared, expressed, created, or isolated by any other means, including splicing human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable regions in which the framework and CDR regions are derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies may be subjected to in vitro mutagenesis (or, when animals transgenic for human Ig sequences are used, in vivo somatic mutagenesis) to thereby modify the V sequences of the recombinant antibodies. H and V L The amino acid sequence of the region is human germline V H and V L These are sequences that are derived from and related to sequences, but may not naturally occur within the human antibody germline repertoire in vivo.

[0388] As used herein, "isotype" refers to the antibody class (e.g., IgM or IgG1) that is encoded by heavy chain constant region genes.

[0389] The phrases "an antibody that recognizes an antigen" and "an antibody that is specific for an antigen" are used interchangeably herein with the term "an antibody that specifically binds to an antigen."

[0390] The term "human antibody derivative" refers to any modified form of a human antibody, including a conjugate of the antibody with another active pharmaceutical ingredient or antibody. The terms "conjugate," "antibody drug conjugate," "ADC," or "immunoconjugate" refer to an antibody or fragment thereof conjugated to another therapeutic moiety, which can be conjugated to the antibodies described herein using methods available in the art.

[0391] The terms "humanized antibody," "humanized antibodies," and "humanization" are intended to refer to antibodies in which CDR sequences derived from the germline of another mammalian species, such as mouse, have been grafted onto human framework sequences. Additional framework region modifications can be made within the human framework sequences. Humanized forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. In most cases, humanized antibodies are human immunoglobulins (recipient antibodies) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate having the desired specificity, affinity, and capacity. In some cases, Fv framework (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may contain residues that are not found in the recipient or donor antibody. These modifications are made to further refine antibody performance. Generally, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence. The humanized antibody will also optionally comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones, et al., Nature 1986, 321, 522-525; Riechmann, et al., Nature 1988, 332, 323-329; and Presta, Curr. Op. Struct. Biol. 1992, 2, 593-596. The antibodies described herein can also be modified to use any Fc variant known to confer improved (e.g., reduced) effector function and / or FcR binding.Fc variants are described in, for example, International Patent Application Publication Nos. WO1988 / 07089A1, WO1996 / 14339A1, WO1998 / 05787A1, WO1998 / 23289A1, WO1999 / 51642A1, WO99 / 58572A1, WO2000 / 09560A2, WO2000 / 32767A1, WO2000 / 42072A2, and WO2002 / 4 4215A2, WO2002 / 060919A2, WO2003 / 074569A2, WO2004 / 016750A2, WO2004 / 029207A2, WO2004 / 03 5752A2, WO2004 / 063351A2, WO2004 / 074455A2, WO2004 / 099249A2, WO2005 / 040217A2, WO2005 / 07 0963A1, WO2005 / 077981A2, WO2005 / 092925A2, WO2005 / 123780A2, WO2006 / 019447A1, WO2006 / 047350A2, and WO2006 / 085967A2, as well as U.S. Pat. Nos. 5,648,260, 5,739,277, 5,834,250, 5,869,046, 6,096 ,871, 6,121,022, 6,194,551, 6,242,195, 6,277,375, 6,528,624, 6,538,124, 6,737,056, 6,821,505, 6,998,253, and 7,083,784 (the disclosures of which are incorporated herein by reference).

[0392] The term "chimeric antibody" is intended to refer to an antibody in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, e.g., an antibody in which the variable region sequences are derived from a murine antibody and the constant region sequences are derived from a human antibody.

[0393] A "diabody" is a small antibody fragment that has two antigen-binding sites. The fragments bind to the same polypeptide chain (VH -V L or V L -V H ) in the light chain variable domain (V L ) connected to the heavy chain variable domain (V H ). When a linker that is too short to pair the two domains on the same chain is used, the domains are forced to pair with complementary domains on another chain and create two antigen-binding sites. Bispecific antibodies are more fully described, for example, in European Patent No. EP 404,097, International Patent Publication No. WO 93 / 11161, and Bolliger, et al., Proc. Natl. Acad. Sci. USA 1993, 90, 6444-6448.

[0394] The term "glycosylation" refers to modified derivatives of antibodies. An aglycosylated antibody lacks glycosylation. Glycosylation can be altered to, for example, increase the affinity of an antibody for an antigen. Such carbohydrate modifications can be achieved, for example, by altering one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions can be made that result in the elimination of one or more variable region framework glycosylation sites, thereby eliminating glycosylation at that site. As described in U.S. Pat. Nos. 5,714,350 and 6,350,861, aglycosylation may increase the affinity of an antibody for an antigen. Additionally or alternatively, antibodies can be generated with altered types of glycosylation, such as hypofucosylated antibodies with reduced amounts of fucosyl residues or antibodies with increased bisecting GlcNac structures. Such altered glycosylation patterns have been demonstrated to increase antibody potency. Such carbohydrate modifications can be achieved, for example, by expressing the antibody in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells to express the recombinant antibodies of the present invention, thereby producing antibodies with altered glycosylation. For example, the cell lines Ms704, Ms705, and Ms709 lack the fucosyltransferase gene FUT8 (alpha(1,6) fucosyltransferase), such that antibodies expressed in these cell lines lack fucose on their carbohydrates. The Ms704, Ms705, and Ms709 FUT8- / - cell lines were generated by targeted disruption of the FUT8 gene in CHO / DG44 cells using two replacement vectors (see, e.g., U.S. Patent Publication No. 2004 / 0110704 or Yamane-Ohnuki, et al., Biotechnol. Bioeng., 2004, 87, 614-622).As another example, European Patent No. EP 1,176,195 describes cell lines with a functionally disrupted FUT8 gene encoding a fucosyltransferase, thereby resulting in antibodies expressed in such cell lines exhibiting hypofucosylation by reducing or eliminating alpha-1,6 bond-related enzymes. It also describes cell lines with reduced or no enzymatic activity for adding fucose to N-acetylglucosamine linked to the Fc region of antibodies, such as the rat myeloma cell line YB2 / 0 (ATCC CRL 1662). International Patent Publication No. WO 03 / 035835 describes a variant CHO cell line, Lec13 cells, that has a reduced ability to attach fucose to Asn(297)-linked carbohydrates, which also results in hypofucosylation of antibodies expressed in the host cells (see also Shields, et al., J. Biol. Chem. 2002, 277, 26733-26740). International Patent Publication No. WO 99 / 54342 describes cell lines engineered to express glycoprotein-modifying glycosyltransferases (e.g., beta(1,4)-N-acetylglucosaminyltransferase III (GnTIII)), such that antibodies expressed in the engineered cell lines exhibit increased bisecting GlcNac structures, resulting in increased ADCC activity of the antibodies (see also Umana, et al., Nat. Biotech. 1999, 17, 176-180). Alternatively, fucosidase enzymes can be used to cleave fucose residues from antibodies. For example, the fucosidase alpha-L-fucosidase removes fucosyl residues from antibodies, as described in Tarentino, et al., Biochem. 1975, 14, 5516-5523.

[0395] "PEGylation" refers to a modified antibody or fragment thereof that has been reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions that result in one or more PEG groups being attached to the antibody or antibody fragment. PEGylation can, for example, increase the biological (e.g., serum) half-life of the antibody. Preferably, PEGylation is carried out via an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer). As used herein, the term "polyethylene glycol" refers to a mono (C1-C 10 PEG is intended to encompass any of the forms of PEG that have been used to derivatize other proteins, such as alkoxy- or aryloxy-polyethylene glycol, or polyethylene glycol-maleimide. The antibody to be pegylated may be an aglycosylated antibody. Methods for pegylation are known in the art and can be applied to the antibodies of the invention, for example, as described in European Patent Nos. EP 0 154 316 and EP 0 401 384, and U.S. Pat. No. 5,824,778 (the disclosures of each of which are incorporated herein by reference).

[0396] The term "biosimilar" refers to a biological product that is highly similar to a reference biological product approved in the United States, despite minor differences in clinically inactive components, including monoclonal antibodies or proteins, and that has no clinically meaningful differences between the biological product and the reference product in terms of product safety, purity, and potency. Furthermore, a similar biological or "biosimilar" drug is a biological product similar to another biological product already approved for use by the European Medicines Agency. The term "biosimilar" is also used interchangeably by regulatory agencies in other countries and regions. Biological products or biological products are medicines made by or derived from biological sources, such as bacteria or yeast. They can consist of relatively small molecules, such as human insulin or erythropoietin, or complex molecules, such as monoclonal antibodies. For example, if the reference IL-2 protein is aldesleukin (proleukin), a protein approved by a drug regulatory agency for aldesleukin is a "biosimilar" of aldesleukin or a "biosimilar of" aldesleukin. In Europe, a similar biological or "biosimilar" medicinal product is a biological product similar to another biological product already authorized for use by the European Medicines Agency (EMA). The legal basis for similar biological uses in Europe is Article 6 of Regulation (EC) No. 726 / 2004, as amended, and Article 10(4) of Directive 2001 / 83 / EC. Therefore, in Europe, biosimilars may be authorized or approved for authorization or licensing purposes under Article 6 of Regulation (EC) No. 726 / 2004 and Article 10(4) of Directive 2001 / 83 / EC. The original biological product already authorized is sometimes referred to as the "reference medicinal product" in Europe. Some of the requirements for a product to be considered a biosimilar are outlined in the CHMP guideline on biosimilar medicinal products. Additionally, product-specific guidelines, including those related to monoclonal antibody biosimilars, are provided by the EMA on a product-by-product basis and are available on its website.Biosimilars described herein may be similar to the reference medicinal product in terms of quality characteristics, biological activity, mechanism of action, safety profile, and / or efficacy. Furthermore, biosimilars may be used or intended for use to treat the same condition as the reference medicinal product. Thus, biosimilars described herein may be considered to have similar or very similar quality characteristics to the reference medicinal product. Alternatively, or in addition, biosimilars described herein may be considered to have similar or very similar biological activity to the reference medicinal product. Alternatively, or in addition, biosimilars described herein may be considered to have a similar or very similar safety profile to the reference medicinal product. Alternatively, or in addition, biosimilars described herein may be considered to have similar or very similar efficacy to the reference medicinal product. As described herein, biosimilars in Europe are compared to reference medicinal products authorized by the EMA. However, in some cases, biosimilars may be compared in specific studies to biopharmaceuticals authorized outside the European Economic Area (non-EEA-authorized "comparators"). Such studies include, for example, specific clinical studies and in vivo nonclinical studies. As used herein, the term "biosimilar" also refers to a biopharmaceutical that has been or can be compared to a non-EEA-approved comparator. Particular biosimilars are proteins, such as antibodies, antibody fragments (e.g., antigen-binding portions), and fusion proteins. Protein biosimilars may have amino acid sequences with minor modifications to the amino acid structure (e.g., including amino acid deletions, additions, and / or substitutions) that do not significantly affect the function of the polypeptide. A biosimilar may have an amino acid sequence that has 97% or more sequence identity, e.g., 97%, 98%, 99%, or 100%, to the amino acid sequence of its reference pharmaceutical. A biosimilar may include one or more post-translational modifications, such as, but not limited to, glycosylation, oxidation, deamidation, and / or cleavage, that differ from the post-translational modifications of the reference pharmaceutical, provided that the differences do not result in a change in the safety and / or efficacy of the pharmaceutical. A biosimilar may have the same or a different glycosylation pattern as the reference pharmaceutical, provided that the differences do not result in a change in the safety and / or efficacy of the pharmaceutical.In particular, but not exclusively, biosimilars may have different glycosylation patterns if the differences address or are intended to address safety concerns associated with the reference drug. Additionally, biosimilars may deviate from the reference drug, for example, in its strength, dosage form, formulation, excipients, and / or presentation, provided that the drug's safety and efficacy are not compromised. Biosimilars may contain differences, for example, in their pharmacokinetic (PK) and / or pharmacodynamic (PD) profiles, compared to the reference drug, but are still considered sufficiently similar to the reference drug to be approved or deemed suitable for approval. In certain circumstances, biosimilars exhibit different binding characteristics compared to the reference drug, and these different binding characteristics are not considered by regulatory authorities, such as the EMA, to be a barrier to approval as a similar biological product. The term "biosimilar" is also used interchangeably by regulatory agencies in other countries and regions.

[0397] III. Potency Assay Methods and Compositions Without being limited to any particular theory, commercially viable TIL and T cell potency assays are believed to be limited by the unavailability of neoantigen-expressing target cells, the lack of overlap between neoantigens expressed by patients and their tumors, and the inability to identify all relevant neoantigens in time to construct an assay. Consequently, unlike commercially available and clinical T cell therapies using transduced chimeric antigen receptors, which have in some cases received regulatory approval using target-based assays, target-based assays are not feasible on the time scale required for effective cancer therapy for polyclonal T cell products, such as TIL, MIL, or PBL therapies or products. At the same time, bead-based assays are limited by their inability to recapitulate the natural activation and killing capacity of TIL or T cells. For example, bead- or plate-based assays utilizing anti-CD3 coatings bind to the CD3ε chain on TIL or T cells that is noncovalently associated with the TCR, when binding to the TCR itself would be more preferable. Therefore, a drawback of bead-based assays using CD3 is that they do not utilize the alpha and beta components of the TCR for their interaction. The presence of other costimulatory molecules, such as CD28 or CD137 (4-1BB), on beads also results in unnatural and less favorable interactions, at least because activation of these costimulatory molecules results in assays that are less selective for tumor-resident cells, such as TILs. The present invention provides a solution to these problems. Without being limited to any particular theory, it is believed that the interaction of MHC or HLA on target cells with the TCR of T cells, including TILs, MILs, or PBLs, produces an analyte that can be compared to an analyte produced by T cells, including TILs, MILs, or PBLs, co-cultured with an MHC-negative control to evaluate one or more properties of the T cells, including TILs, MILs, or PBLs, such as their potency, identity, or other useful properties for assisting in the manufacture or delivery of a therapy for cancer.Furthermore, without being limited to any particular theory, it is believed that because T cells such as TILs and MILs are found at the site of a tumor or because PBLs have previously been activated by tumor exposure, such T cells, including TILs, MILs, and PBLs, are already cross-primed or primed T cells, and CD28 and 4-1BB are already activated.

[0398] In one embodiment, the present invention includes a potency assay composition comprising Raji cells and T cells, such as TILs, co-cultured in medium. In one embodiment, the present invention includes a potency assay composition comprising Thp1 cells and T cells, such as TILs, co-cultured in medium. In one embodiment, the present invention includes a potency assay composition comprising Ramos cells and T cells, such as TILs, co-cultured in medium. In one embodiment, the present invention includes a potency assay composition comprising U937 cells and T cells, such as TILs, co-cultured in medium. In one embodiment, the present invention includes a potency assay composition comprising Daudi cells and T cells, such as TILs, co-cultured in medium. In one embodiment, the potency assay composition includes K562 cells and T cells, such as TILs, co-cultured in medium and used as a negative control. In one embodiment, the potency assay composition includes peripheral blood mononuclear cells (PBMCs) and T cells, such as TILs, co-cultured in medium and used as a mixed lymphocyte reaction (MLR) positive control. In one embodiment, the TILs and PBMCs in the foregoing embodiments are from the same patient. In some embodiments, the TILs are produced using the Gen2 or Gen3 process, or other manufacturing processes described herein. In some embodiments, the TILs are produced using at least one REP step. In some embodiments, a potency assay is performed using TILs, MILs, or PBLs produced using the expansion or manufacturing processes described herein. In some embodiments, the TILs are produced using the methods described in U.S. Patent Application Publication No. US2018 / 0282694A1 or U.S. Patent Nos. 10,130,659, 10,166,257, 10,272,113, 10,363,273, 10,398,734, 10,420,799, 10,463,697, and 10,537,599. Nos. 10,646,517, 10,653,723, 10,693,330, 10,695,372, 10,894,063, and 10,905,718 (the disclosures of each of which are incorporated herein by reference) and then tested in the potency assays described herein.In some embodiments, the TILs are produced using the process described in U.S. Patent No. 10,918,666 (the disclosure of which is incorporated herein by reference) and then tested in the potency assays described herein. In some embodiments, the TILs are produced using the process described in U.S. Patent Application Publication No. 2020 / 0277573A1 (the disclosure of which is incorporated herein by reference) and then tested in the potency assays described herein. In some embodiments, the TILs are produced using the process described in International Patent Application Publication No. 2019 / 210131A1 (the disclosure of which is incorporated herein by reference) and then tested in the potency assays described herein. In some embodiments, the TILs are produced using the process described in International Patent Application Publication No. 2019 / 136456A1 (the disclosure of which is incorporated herein by reference) and then tested in the potency assays described herein. In some embodiments, TILs are produced using the process described in International Patent Application Publication No. 2021 / 123832A1 (the disclosure of which is incorporated herein by reference) and then tested in the potency assays described herein. In some embodiments, MILs and PBLs are produced using the process described in U.S. Patent Application Publication No. 2020 / 0224161A1 (the disclosure of which is incorporated herein by reference) and then tested in the potency assays described herein. In some embodiments, TILs are produced using the process described in International Patent Application Publication No. 2019 / 145711A1 (the disclosure of which is incorporated herein by reference) and then tested in the potency assays described herein. In some embodiments, TILs are produced using the process described in International Patent Application Publication No. 2020 / 152451A1 (the disclosure of which is incorporated herein by reference) and then tested in the potency assays described herein.

[0399] A. Assay Methods In one embodiment, the present invention includes a method for determining the potency of a T cell product using target cells capable of binding to a T cell receptor. In one embodiment, the present invention includes an assay based on allogeneic interaction (also referred to as MHC-dominant recognition) of a T cell or TIL, MIL, or PBL TCR complex with an HLA-peptide complex of the target cell. In one embodiment, the present invention includes an assay that utilizes MHC-dominant recognition of a T cell or TIL, MIL, or PBL TCR complex with a target cell to produce an analyte that is compared to an analyte produced by co-culture of a T cell or TIL with an MHC-negative control. In one embodiment, binding of a T cell or TIL, MIL, or PBL TCR complex to a target cell occurs via its alpha (α) and beta (β) chains. In one embodiment, the target cell expresses HLA. In one embodiment, the target cell is a B cell. In one embodiment, the target cell is a B cell lymphoblast or a B lymphoblast. In one embodiment, the target cell is a Burkitt's lymphoma cell. In one embodiment, the target cell is a myeloid cell. In some embodiments, the target cell is a monocyte. In some embodiments, the target cell is an acute monocytic leukemia cell. In some embodiments, the target cell is an M5 subtype acute monocytic leukemia cell. In some embodiments, the target cell is a Raji cell, or a derivative, variant, modification, or progeny thereof. In some embodiments, the target cell is a Thp1 cell, or a derivative, variant, modification, or progeny thereof. In some embodiments, the target cell is a Ramos cell, or a derivative, variant, modification, or progeny thereof. In some embodiments, the target cell is a U937 cell, or a derivative, variant, modification, or progeny thereof. In some embodiments, the target cell is a Daudi cell, or a derivative, variant, modification, or progeny thereof. In some embodiments, the target cell is a melanocyte cell. In some embodiments, the target cell is an HLA-A-02-positive melanocyte cell. In some embodiments, the target cell is a melanoma cell. In some embodiments, the target cell is an HLA-A-02-positive melanoma cell.In some embodiments, the target cells are melanoma cells selected from the group consisting of Sk-MEL-5, Malme-3M, SK-MEL-28, SK-MEL-3, SH-4, SK-MEL-24, RPMI-7951, SK-MEL-1, A375, G-361, and combinations thereof.

[0400] In some embodiments, the present invention includes assays based on alloreactive TCR or HLA recognition between tumor cells and T cells, such as TILs, MILs, or PBLs, produced using the expansion or manufacturing processes described herein. In some embodiments, the present invention includes assays based on alloreactive TCR or HLA recognition between Raji, Thp1, Ramos, U937, or Daudi cells, or derivatives, variants, modifications, or progeny thereof, and T cells, such as TILs, MILs, or PBLs, produced using the expansion or manufacturing processes described herein. In some embodiments, the present invention includes the use of the foregoing embodiments in combination with a negative control, as described herein. In some embodiments, the negative control serves as a basal or baseline activity level of T cells, such as TILs, MILs, or PBLs, for comparison with T cell assays based on MHC dominant recognition. In some embodiments, the negative control is a cell line lacking HLA class I and / or HLA class II expression. In some embodiments, the negative control is a cell line lacking MHC class I and / or MHC class II expression. In one embodiment, the negative control is a cell line lacking HLA or MHC to control for assay variability, hi one embodiment, the negative control is a cell line lacking HLA or MHC to demonstrate that MHC dominant recognition is the primary variable in the potency assay.

[0401] In one embodiment, the invention includes the use of K562 cells, or derivatives, variants, modifications, or progeny thereof, as a negative control as described herein by co-culturing a T cell product, such as a TIL product, MIL product, or PBL product, with K562 cells. The use of K562 cells as a negative control can be used in conjunction with the assays described herein, as well as with bead- and plate-bound antibody stimulation bioassays, such as the anti-CD3 plate assay for IFN-γ.

[0402] In one embodiment, the invention involves the use of one or more HLA-blocking antibodies, or fragments, derivatives, variants, or modifications thereof, in co-culture of a T cell product, such as a TIL product, MIL product, or PBL product, with a target cell or combination of target cells as a negative control. The use of an HLA-blocking antibody or multiple HLA-blocking antibodies as a negative control can be used in conjunction with the assays described herein.

[0403] In some embodiments, the potency and / or functionality of expanded TILs produced by methods described herein or known in the art are examined. Examining the potency and / or functionality of expanded TILs allows for characterization of clinical TIL product lots. In certain embodiments, TIL potency is defined as increased expression of select surface markers, optionally expressed as the percentage of marker-positive cells or mean fluorescence intensity (MFI), upon co-culture with Raji cells compared to K562 cells. Alternatively, Raji cells, K562 cells, and TILs may be cultured alone. In some embodiments, PBMCs from the same patient from which TILs or MILs are obtained are used as a control. In some embodiments, bead-based CD3 / CD28 stimulation can also be used as a control. In some embodiments, bead-based CD3 / CD28 / CD137 stimulation can also be used as a control. In some embodiments, the addition of a mouse monoclonal antibody against CD28 is used to ensure additional costimulation to amplify T cell responses. In some embodiments, PBMCs can be co-cultured with each TIL lot as a positive control for MLR responses.

[0404] In certain embodiments, the potency assay comprises a co-cultivation step, wherein the co-cultivation is carried out for a period of time selected from the group consisting of 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 25 hours, 26 hours, 27 hours, 28 hours, 29 hours, 30 hours, 31 hours, 32 hours, 33 hours, 34 hours, 35 hours, 36 hours, 37 hours, 38 hours, 39 hours, 40 hours, 41 hours, 42 hours, 43 hours, 44 hours, 45 hours, 46 hours, 47 hours, 48 ​​hours, 49 hours, and 50 hours. In certain embodiments, the potency assay comprises a co-cultivation step, wherein the co-cultivation is carried out for a period selected from the group consisting of 6 hours, 12 hours, 18 hours, 24 hours, and 32 hours.

[0405] In one embodiment, the potency assay comprises a step of co-culturing target cells and T cells (comprising TILs, MILs, or PBLs), wherein the co-culturing is carried out for a period of time selected from the group consisting of 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 25 hours, 26 hours, 27 hours, 28 hours, 29 hours, 30 hours, 31 hours, 32 hours, 33 hours, 34 hours, 35 hours, 36 hours, 37 hours, 38 hours, 39 hours, 40 hours, 41 hours, 42 hours, 43 hours, 44 hours, 45 hours, 46 hours, 47 hours, 48 ​​hours, 49 hours, and 50 hours. In certain embodiments, the potency assay comprises a co-cultivation step, wherein the co-cultivation is carried out for a period selected from the group consisting of 6 hours, 12 hours, 18 hours, 24 hours, and 32 hours.

[0406] In one embodiment, the potency assay comprises a step of co-culturing negative control cells and T cells (comprising TILs, MILs, or PBLs), wherein the co-culturing is carried out for a period of time selected from the group consisting of 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 25 hours, 26 hours, 27 hours, 28 hours, 29 hours, 30 hours, 31 hours, 32 hours, 33 hours, 34 hours, 35 hours, 36 hours, 37 hours, 38 hours, 39 hours, 40 hours, 41 hours, 42 hours, 43 hours, 44 hours, 45 hours, 46 hours, 47 hours, 48 ​​hours, 49 hours, and 50 hours. In certain embodiments, the potency assay comprises a co-cultivation step, wherein the co-cultivation is carried out for a period selected from the group consisting of 6 hours, 12 hours, 18 hours, 24 hours, and 32 hours.

[0407] In one embodiment, the present invention includes a potency assay method for determining the efficacy of a TIL, MIL, PBL, or other T cell product in relation to clinical benefit. Clinical benefit can be measured in multiple ways, including, but not limited to, demonstration of cell function in vitro, disease control rate, overall response rate, duration of response, or other scientific measure of clinical benefit or potential clinical benefit.

[0408] In some embodiments, the potency of expanded or formulated TILs, MILs, and PBLs is determined by the coculture assay described herein. Determining the potency of expanded or formulated TILs, MILs, and PBLs allows for the characterization of TIL, MIL, or PBL product lots. Potency of TILs, MILs, or PBLs, also referred to as activation, is defined as increased expression of select surface markers, expressed as the percentage of marker-positive cells or mean fluorescence intensity (MFI), when TILs, MILs, or PBLs are cocultured with Raji cells or their derivatives, variants, modifications, or progeny, compared to when TILs, MILs, or PBLs are cocultured with K562 cells.

[0409] In one embodiment, the present invention includes a method for determining the potency of a T cell product using target cells capable of binding to a T cell receptor, wherein the method for determining potency is an allorecognition assay. In one embodiment, the present invention includes a method for determining the potency of a T cell product using target cells capable of binding to a T cell receptor, wherein the method for determining potency is an allorecognition assay. In one embodiment, the present invention includes a method for determining the potency of a T cell product using Raji cells, or derivatives, variants, modifications, or progeny thereof, capable of binding to a T cell receptor, wherein the method for determining potency is an allorecognition assay. In one embodiment, the present invention includes a method for determining the potency of a T cell product using Thp1 cells, or derivatives, variants, modifications, or progeny thereof, capable of binding to a T cell receptor, wherein the method for determining potency is an allorecognition assay. In one embodiment, the present invention includes a method for determining the potency of a T cell product using Ramos cells, or derivatives, variants, modifications, or progeny thereof, capable of binding to a T cell receptor, wherein the method for determining potency is an allorecognition assay. In one embodiment, the invention includes a method for determining the potency of a T cell product using U937 cells, or derivatives, variants, modifications, or progeny thereof, capable of binding to a T cell receptor, wherein the method for determining potency is an allorecognition assay. In one embodiment, the invention includes a method for determining the potency of a T cell product using Daudi cells, or derivatives, variants, modifications, or progeny thereof, capable of binding to a T cell receptor, wherein the method for determining potency is an allorecognition assay.

[0410] In one embodiment, the present invention includes a method for determining the potency of a T cell product using target cells capable of binding to a T cell receptor, wherein protein secretion or expression is detected using an enzyme-linked immunosorbent assay (ELISA) detection method. In a further embodiment, the ELISA method is automated using an automated or robotic system, such as the ELLA system (available from the Protein Simple division of BioTechne, Inc., San Jose, CA, USA), the ISOPLEXIS system (available from Isoplexis, Inc., Branford, CT, USA), or the LUNARIS system (available from AYOXXA Biosystems GmbH, Cologne, Germany). In one embodiment, potency assay detection is performed using a multiplexed assay detection method, such as the LUMINEX system (available from the R&D Systems division of BioTechne, Inc., Minnesota, MN, USA). In one embodiment, the present invention includes a potency assay method using a flow cytometry detection method. In one embodiment, the present invention includes a potency assay method for secreted proteins. In one embodiment, the present invention includes a potency assay method for cell surface or binding proteins. In one embodiment, the present invention includes a potency assay method using a binding assay detection method. In one embodiment, the ELLA system measures IFN-γ concentrations after activation of the co-culture assay described herein, and supernatants collected from the co-culture are screened for IFN-γ production in each multi-well (e.g., 72-well) commercially validated cartridge. In one embodiment, the ELLA automated system operates down to sub-picogram levels of sensitivity, including 4 logs of sensitivity within the dynamic range. In one embodiment, assay repeatability and precision are increased by minimizing procedural errors using the ELLA system compared to traditional ELISA methods.

[0411] In one embodiment, the invention includes a method for determining the potency of a T cell product using target cells capable of binding to a T cell receptor, wherein the secretion or expression of an analyte is detected using a detection method. In one embodiment, the detected analyte is a protein. In one embodiment, the detected analyte is a cytokine. In one embodiment, the detected analyte is an interferon. In one embodiment, the detected analyte is interferon-alpha, also known as IFN-α or IFNα. In one embodiment, the detected analyte is interferon-alpha, also known as IFN-β or IFNβ. In one embodiment, the detected analyte is interferon-gamma, also known as IFN-γ or IFNγ. In one embodiment, the detected analyte is granzyme B, also known as GzmB. In one embodiment, the detected analyte is perforin. In one embodiment, the detected analyte is tumor necrosis factor alpha, also known as TNF-α or TNFα. In one embodiment, the detected analyte is an interleukin. In some embodiments, the analyte detected is IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-9, IL-10, IL-13, IL-14, IL-16, IL-17, IL-18, IL-22, IL-25, or IL-26. In some embodiments, the analyte detected is CD25. In some embodiments, the analyte detected is CD69. In some embodiments, the analyte detected is CD137 (4-1BB). In some embodiments, the analyte detected is CD134 (OX40). In some embodiments, the analyte detected is CCL4. In some embodiments, the analyte detected is CD150, also known as SLAM, SLAM F1, or signal lymphocyte activation molecule 1. In some embodiments, the analyte detected is KLRG1. In some embodiments, the analyte detected is KLRG1. In one embodiment, the analyte to be detected is secreted MIP-1β.

[0412] In one embodiment, the invention includes a method for determining the potency of a T cell product using target cells capable of binding to a T cell receptor, wherein the secretion or expression of an analyte is detected using a detection method, wherein the T cell product is a TIL product and the analyte is a cell surface marker selected from the group consisting of CD25, CD69, CD134, CD137, and CD150.

[0413] In one embodiment, the invention includes a method for determining the potency of a T cell product using cells capable of allogeneic binding to a T cell receptor, wherein a negative control comprising cells lacking MHC I and MHC II expression is used in comparison of a T cell product (such as a TIL product, MIL product, or PBL product) to co-culture with target cells. In one embodiment, the invention includes a method for determining the potency of a TIL product using Raji cells capable of allogeneic binding to a T cell receptor, wherein a negative control comprising K562 cells lacking MHC I and MHC II expression is used in comparison of a TIL product to co-culture with Raji cells.

[0414] In some embodiments, the increase in expression of selective surface markers, expressed as the percentage of marker-positive cells or mean fluorescence intensity (MFI), upon co-culture with Raji cells is at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% increase compared to K562 cells.

[0415] In some embodiments, the increase in expression of selective surface markers, expressed as the percentage of marker-positive cells or mean fluorescence intensity (MFI), upon co-culture with Raji cells is at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% increase compared to K562 cells.

[0416] In some embodiments, supernatants from the co-culture methods described herein are tested immediately after removal from the co-culture. In some embodiments, supernatants from the co-culture methods described herein are tested 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 10 hours, 12 hours, 18 hours, 24 hours, or 48 hours after removal from the co-culture. In some embodiments, supernatants from the co-culture methods described herein are frozen after removal from the co-culture and later thawed for testing. In some embodiments, supernatants from the co-culture methods described herein are evaluated for IFN-γ, granzyme B, TNF-α, CCL4, and / or MIP-1β secretion.

[0417] In some embodiments, the invention provides methods for assaying TIL activity, comprising: (a) irradiating Raji cells and K562 cells, optionally with an agonistic or superagonistic anti-CD28 antibody; (b) co-culturing the irradiated Raji cells and irradiated K562 cells with TILs at one or more of the following target ratios: 50:1, 25:1, 12.5:1, 6.25:1, 3:1, 1:1, 1:3, 1:6.25, 1:12.5, 1:25; (c) after 6 to 24 hours, collecting supernatant from the co-cultured cells in step (b); (d) harvesting the cells from step (b) and measuring expression of surface markers of T cell activation; and (e) assaying the supernatant from the co-cultured cells collected in step (c) for markers of T cell activation.

[0418] In some embodiments, the potency and / or functionality of the expanded TILs is examined prior to cryopreservation of the TILs. In some embodiments, the potency and / or functionality of the expanded TILs is examined prior to cryopreservation. In certain embodiments, the potency of the TIL, MIL, or PBL product is examined as part of a pharmaceutical release study. In certain embodiments, the potency of the TIL, MIL, or PBL product is examined as part of a pharmaceutical stability study, for example, after thawing a cryopreserved product or after storage of a product that has previously been thawed or never frozen for a defined period of time under different environmental conditions.

[0419] In some embodiments, the increase in expression of selective surface markers, expressed as the percentage of marker-positive cells or mean fluorescence intensity (MFI), upon co-culture with Raji cells is at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% increase compared to K562 cells.

[0420] In some embodiments, the increase in expression of selective surface markers, expressed as the percentage of marker-positive cells or mean fluorescence intensity (MFI), upon co-culture with Raji cells compared to K562 cells is at least 1 fold, at least 1.5 fold, at least 2 fold, at least 2.5 fold, at least 3 fold, at least 3.5 fold, at least 4 fold, at least 4.5 fold, at least 5 fold, at least 5.5 fold, at least 6 fold, at least 6.5 fold, at least 7 fold, at least 7.5 fold, at least 8 fold, at least 8.5 fold, At least a 9-fold, at least 9.5-fold, at least 10-fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 16-fold, at least 17-fold, at least 18-fold, at least 19-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold, at least 55-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, or at least 100-fold increase.

[0421] In some embodiments, the increase in CD25 expression, expressed as the percentage of marker-positive cells or mean fluorescence intensity (MFI), upon co-culture with Raji cells is at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% increase compared to K562 cells.

[0422] In some embodiments, the increase in CD25 expression, expressed as the percentage of marker-positive cells or mean fluorescence intensity (MFI), upon co-culture with Raji cells compared to K562 cells is at least 1 fold, at least 1.5 fold, at least 2 fold, at least 2.5 fold, at least 3 fold, at least 3.5 fold, at least 4 fold, at least 4.5 fold, at least 5 fold, at least 5.5 fold, at least 6 fold, at least 6.5 fold, at least 7 fold, at least 7.5 fold, at least 8 fold, at least 8.5 fold, or less. at least a 9-fold, at least 9.5-fold, at least 10-fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 16-fold, at least 17-fold, at least 18-fold, at least 19-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold, at least 55-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, or at least 100-fold increase.

[0423] In some embodiments, the increase in CD69 expression, expressed as the percentage of marker-positive cells or mean fluorescence intensity (MFI), upon co-culture with Raji cells is at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% increase compared to K562 cells.

[0424] In some embodiments, the increase in CD69 expression, expressed as the percentage of marker-positive cells or mean fluorescence intensity (MFI), upon co-culture with Raji cells compared to K562 cells is at least 1 fold, at least 1.5 fold, at least 2 fold, at least 2.5 fold, at least 3 fold, at least 3.5 fold, at least 4 fold, at least 4.5 fold, at least 5 fold, at least 5.5 fold, at least 6 fold, at least 6.5 fold, at least 7 fold, at least 7.5 fold, at least 8 fold, at least 8.5 fold, or less. at least a 9-fold, at least 9.5-fold, at least 10-fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 16-fold, at least 17-fold, at least 18-fold, at least 19-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold, at least 55-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, or at least 100-fold increase.

[0425] In some embodiments, the increase in CD134 expression, expressed as the percentage of marker-positive cells or mean fluorescence intensity (MFI), upon co-culture with Raji cells is at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% increase compared to K562 cells.

[0426] In some embodiments, the increase in CD134 expression, expressed as the percentage of marker-positive cells or mean fluorescence intensity (MFI), upon co-culture with Raji cells compared to K562 cells is at least 1 fold, at least 1.5 fold, at least 2 fold, at least 2.5 fold, at least 3 fold, at least 3.5 fold, at least 4 fold, at least 4.5 fold, at least 5 fold, at least 5.5 fold, at least 6 fold, at least 6.5 fold, at least 7 fold, at least 7.5 fold, at least 8 fold, at least 8.5 fold, or less. at least a 9-fold, at least 9.5-fold, at least 10-fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 16-fold, at least 17-fold, at least 18-fold, at least 19-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold, at least 55-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, or at least 100-fold increase.

[0427] In some embodiments, the increase in CD137 expression, expressed as the percentage of marker-positive cells or mean fluorescence intensity (MFI), upon co-culture with Raji cells is at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% increase compared to K562 cells.

[0428] In some embodiments, the increase in CD137 expression, expressed as the percentage of marker-positive cells or mean fluorescence intensity (MFI), upon co-culture with Raji cells compared to K562 cells is at least 1 fold, at least 1.5 fold, at least 2 fold, at least 2.5 fold, at least 3 fold, at least 3.5 fold, at least 4 fold, at least 4.5 fold, at least 5 fold, at least 5.5 fold, at least 6 fold, at least 6.5 fold, at least 7 fold, at least 7.5 fold, at least 8 fold, at least 8.5 fold, or less. at least a 9-fold, at least 9.5-fold, at least 10-fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 16-fold, at least 17-fold, at least 18-fold, at least 19-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold, at least 55-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, or at least 100-fold increase.

[0429] In some embodiments, the increase in CD150 expression, expressed as the percentage of marker-positive cells or mean fluorescence intensity (MFI), upon co-culture with Raji cells is at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% increase compared to K562 cells.

[0430] In some embodiments, the increase in CD150 expression, expressed as the percentage of marker-positive cells or mean fluorescence intensity (MFI), upon co-culture with Raji cells compared to K562 cells is at least 1 fold, at least 1.5 fold, at least 2 fold, at least 2.5 fold, at least 3 fold, at least 3.5 fold, at least 4 fold, at least 4.5 fold, at least 5 fold, at least 5.5 fold, at least 6 fold, at least 6.5 fold, at least 7 fold, at least 7.5 fold, at least 8 fold, at least 8.5 fold, or less. at least a 9-fold, at least 9.5-fold, at least 10-fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 16-fold, at least 17-fold, at least 18-fold, at least 19-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold, at least 55-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, or at least 100-fold increase.

[0431] In some embodiments, the increase in expression of secreted Granzyme B, expressed as an ELISA detection analyte or mean fluorescence intensity (MFI), upon co-culture with Raji cells is at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% increase compared to K562 cells.

[0432] In some embodiments, the increase in expression of secreted Granzyme B, expressed as a percentage of an ELISA-detected analyte or mean fluorescence intensity (MFI), upon co-culture with Raji cells compared to K562 cells is at least 1 fold, at least 1.5 fold, at least 2 fold, at least 2.5 fold, at least 3 fold, at least 3.5 fold, at least 4 fold, at least 4.5 fold, at least 5 fold, at least 5.5 fold, at least 6 fold, at least 6.5 fold, at least 7 fold, at least 7.5 fold, at least 8 fold, at least An increase of 8.5 fold, at least 9 fold, at least 9.5 fold, at least 10 fold, at least 11 fold, at least 12 fold, at least 13 fold, at least 14 fold, at least 15 fold, at least 16 fold, at least 17 fold, at least 18 fold, at least 19 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 35 fold, at least 40 fold, at least 45 fold, at least 50 fold, at least 55 fold, at least 60 fold, at least 70 fold, at least 80 fold, at least 90 fold, or at least 100 fold.

[0433] In some embodiments, the increase in expression of secreted IFN-γ, expressed as a percentage or mean fluorescence intensity (MFI) of an ELISA-detected analyte, upon co-culture with Raji cells is at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% increase compared to K562 cells.

[0434] In some embodiments, the increase in expression of secreted IFN-γ, expressed as a percentage of an ELISA-detected analyte or mean fluorescence intensity (MFI), upon co-culture with Raji cells compared to K562 cells is at least 1-fold, at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, at least 5-fold, at least 5.5-fold, at least 6-fold, at least 6.5-fold, at least 7-fold, at least 7.5-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 16-fold, at least 17-fold, at least 18-fold, at least 19-fold, at least 20-fold, at least 21-fold, at least 22-fold, at least 23-fold, at least 24-fold, at least 25-fold, at least 26-fold, at least 27-fold, at least 28-fold, at least 29-fold, at least 30-fold, at least 31-fold, at least 32-fold, at least 33-fold, at least 34-fold, at least 35-fold, at least 36-fold, at least 37-fold, at least 38-fold, at least 39-fold, at least 40-fold, at least 41-fold, at least 42-fold, at least 43-fold, at least 44-fold, at least 45-fold, at least 46-fold, at least 47-fold, at least 48-fold, at least 49-fold, at least 50-fold, at 0.5-fold, at least 9-fold, at least 9.5-fold, at least 10-fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 16-fold, at least 17-fold, at least 18-fold, at least 19-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold, at least 55-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, or at least 100-fold increase.

[0435] In some embodiments, the increase in expression of TNF-α, expressed as an ELISA detection analyte or mean fluorescence intensity (MFI) or secreted, upon co-culture with Raji cells is at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% increase compared to K562 cells.

[0436] In some embodiments, the increase in expression of secreted TNF-α, expressed as a percentage of an ELISA-detected analyte or mean fluorescence intensity (MFI), upon co-culture with Raji cells compared to K562 cells is at least 1-fold, at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, at least 5-fold, at least 5.5-fold, at least 6-fold, at least 6.5-fold, at least 7-fold, at least 7.5-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 16-fold, at least 17-fold, at least 18-fold, at least 19-fold, at least 20-fold, at least 21-fold, at least 22-fold, at least 23-fold, at least 24-fold, at least 25-fold, at least 26-fold, at least 27-fold, at least 28-fold, at least 29-fold, at least 30-fold, at least 31-fold, at least 32-fold, at least 33-fold, at least 34-fold, at least 35-fold, at least 36-fold, at least 37-fold, at least 38-fold, at least 39-fold, at least 40-fold, at least 41-fold, at least 42-fold, at least 43-fold, at least 44-fold, at least 45-fold, at least 46-fold, at least 47-fold, at least 48-fold, at least 49-fold, at least 50-fold, at least 0.5-fold, at least 9-fold, at least 9.5-fold, at least 10-fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 16-fold, at least 17-fold, at least 18-fold, at least 19-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold, at least 55-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, or at least 100-fold increase.

[0437] In some embodiments, the increase in expression of secreted perforin, expressed as a percentage or mean fluorescence intensity (MFI) of an ELISA-detected analyte upon co-culture with Raji cells, is at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% increase compared to K562 cells.

[0438] In some embodiments, the increase in expression of secreted perforin, expressed as a percentage of an ELISA-detected analyte or mean fluorescence intensity (MFI), upon co-culture with Raji cells compared to K562 cells is at least 1-fold, at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, at least 5-fold, at least 5.5-fold, at least 6-fold, at least 6.5-fold, at least 7-fold, at least 7.5-fold, at least 8-fold, at least An increase of 8.5 fold, at least 9 fold, at least 9.5 fold, at least 10 fold, at least 11 fold, at least 12 fold, at least 13 fold, at least 14 fold, at least 15 fold, at least 16 fold, at least 17 fold, at least 18 fold, at least 19 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 35 fold, at least 40 fold, at least 45 fold, at least 50 fold, at least 55 fold, at least 60 fold, at least 70 fold, at least 80 fold, at least 90 fold, or at least 100 fold.

[0439] In some embodiments, the increase in expression of secreted CCL4, expressed as an ELISA detection analyte or mean fluorescence intensity (MFI), upon co-culture with Raji cells is at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% increase compared to K562 cells.

[0440] In some embodiments, the increase in expression of secreted CCL4, expressed as a percentage of an ELISA-detected analyte or mean fluorescence intensity (MFI), upon co-culture with Raji cells compared to K562 cells is at least 1-fold, at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, at least 5-fold, at least 5.5-fold, at least 6-fold, at least 6.5-fold, at least 7-fold, at least 7.5-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 16-fold, at least 17-fold, at least 18-fold, at least 0.5-fold, at least 9-fold, at least 9.5-fold, at least 10-fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 16-fold, at least 17-fold, at least 18-fold, at least 19-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold, at least 55-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, or at least 100-fold increase.

[0441] In some embodiments, the increase in expression of secreted MIP-1β, expressed as a percentage or mean fluorescence intensity (MFI) of an ELISA-detected analyte upon co-culture with Raji cells, is at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% increase compared to K562 cells.

[0442] In some embodiments, the increase in expression of secreted MIP-1β, expressed as a percentage of an ELISA-detected analyte or mean fluorescence intensity (MFI), upon co-culture with Raji cells compared to K562 cells is at least 1-fold, at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, at least 5-fold, at least 5.5-fold, at least 6-fold, at least 6.5-fold, at least 7-fold, at least 7.5-fold, at least 8-fold, at least An increase of 8.5 fold, at least 9 fold, at least 9.5 fold, at least 10 fold, at least 11 fold, at least 12 fold, at least 13 fold, at least 14 fold, at least 15 fold, at least 16 fold, at least 17 fold, at least 18 fold, at least 19 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 35 fold, at least 40 fold, at least 45 fold, at least 50 fold, at least 55 fold, at least 60 fold, at least 70 fold, at least 80 fold, at least 90 fold, or at least 100 fold.

[0443] In some embodiments, the amount of Granzyme B secreted upon co-culture with Raji cells is at least 20 pg / mL, at least 30 pg / mL, at least 40 pg / mL, at least 50 pg / mL, at least 60 pg / mL, at least 70 pg / mL, at least 80 pg / mL, at least 90 pg / mL, at least 100 pg / mL, at least 150 pg / mL, at least 200 pg / mL, at least 250 pg / mL, at least 300 pg / mL, at least 350 pg / mL, at least 400 pg / mL, at least 450 pg / mL, at least 500 pg / mL, at least 600 pg / mL, at least 700 pg / mL, at least 800 pg / mL, at least 900 pg / mL, at least 1000 pg / mL, at least 1100 pg / mL, or at least 1200 pg / mL, and each mL of test sample is 1 x 10 5 TIL, 2 × 10 5 TIL, 3 × 10 5 TIL, 4 × 10 5 TIL, 5 × 105 TIL, 6 × 10 5 TIL, 7×10 5 TIL, 8×10 5 TIL, 9×10 5 TIL, or 10x10 5 Contains TILs.

[0444] In some embodiments, the amount of IFN-γ secreted upon co-culture with Raji cells is at least 20 pg / mL, at least 30 pg / mL, at least 40 pg / mL, at least 50 pg / mL, at least 60 pg / mL, at least 70 pg / mL, at least 80 pg / mL, at least 90 pg / mL, at least 100 pg / mL, at least 150 pg / mL, at least 200 pg / mL, at least 250 pg / mL, at least 300 pg / mL, at least 350 pg / mL, at least 400 pg / mL, at least 450 pg / mL, at least 500 pg / mL, at least 600 pg / mL, at least 700 pg / mL, at least 800 pg / mL, at least 900 pg / mL, at least 1000 pg / mL, at least 1100 pg / mL, or at least 1200 pg / mL, and each mL of test sample is 1 x 10 5 TIL, 2 × 10 5 TIL, 3 × 10 5 TIL, 4 × 10 5 TIL, 5 × 10 5 TIL, 6 × 10 5 TIL, 7×10 5 TIL, 8×10 5 TIL, 9×10 5 TIL, or 10x10 5 Contains TILs.

[0445] In some embodiments, the amount of TNF-α secreted upon co-culture with Raji cells is at least 20 pg / mL, at least 30 pg / mL, at least 40 pg / mL, at least 50 pg / mL, at least 60 pg / mL, at least 70 pg / mL, at least 80 pg / mL, at least 90 pg / mL, at least 100 pg / mL, at least 150 pg / mL, at least 200 pg / mL, at least 250 pg / mL, at least 300 pg / mL, at least 350 pg / mL, at least 400 pg / mL, at least 450 pg / mL, at least 500 pg / mL, at least 600 pg / mL, at least 700 pg / mL, at least 800 pg / mL, at least 900 pg / mL, at least 1000 pg / mL, at least 1100 pg / mL, or at least 1200 pg / mL, and each mL of test sample is 1 x 10 5 TIL, 2 × 10 5 TIL, 3 × 10 5 TIL, 4 × 10 5 TIL, 5 × 10 5 TIL, 6 × 10 5 TIL, 7×10 5 TIL, 8×10 5 TIL, 9×10 5 TIL, or 10x10 5 Contains TILs.

[0446] In some embodiments, the amount of perforin secreted upon co-culture with Raji cells is at least 20 pg / mL, at least 30 pg / mL, at least 40 pg / mL, at least 50 pg / mL, at least 60 pg / mL, at least 70 pg / mL, at least 80 pg / mL, at least 90 pg / mL, at least 100 pg / mL, at least 150 pg / mL, at least 200 pg / mL, at least 250 pg / mL, at least 300 pg / mL, at least 350 pg / mL, at least 400 pg / mL, at least 450 pg / mL, at least 500 pg / mL, at least 600 pg / mL, at least 700 pg / mL, at least 800 pg / mL, at least 900 pg / mL, at least 1000 pg / mL, at least 1100 pg / mL, or at least 1200 pg / mL, and each mL of test sample is 1 x 10 5 TIL, 2 × 10 5 TIL, 3 × 10 5 TIL, 4 × 10 5 TIL, 5 × 10 5 TIL, 6 × 10 5 TIL, 7×10 5 TIL, 8×10 5 TIL, 9×10 5 TIL, or 10x10 5 Contains TILs.

[0447] In some embodiments, the amount of CCL4 secreted upon co-culture with Raji cells is at least 20 pg / mL, at least 30 pg / mL, at least 40 pg / mL, at least 50 pg / mL, at least 60 pg / mL, at least 70 pg / mL, at least 80 pg / mL, at least 90 pg / mL, at least 100 pg / mL, at least 150 pg / mL, at least 200 pg / mL, at least 250 pg / mL, at least 300 pg / mL, at least 350 pg / mL, at least 400 pg / mL, at least 450 pg / mL, at least 500 pg / mL, at least 600 pg / mL, at least 700 pg / mL, at least 800 pg / mL, at least 900 pg / mL, at least 1000 pg / mL, at least 1100 pg / mL, or at least 1200 pg / mL, and each mL of test sample is 1 x 10 5 TIL, 2 × 10 5 TIL, 3 × 10 5 TIL, 4 × 10 5 TIL, 5 × 10 5 TIL, 6 × 10 5 TIL, 7×10 5 TIL, 8×10 5 TIL, 9×10 5 TIL, or 10x10 5 Contains TILs.

[0448] In some embodiments, the amount of MIP-1β secreted upon co-culture with Raji cells is at least 20 pg / mL, at least 30 pg / mL, at least 40 pg / mL, at least 50 pg / mL, at least 60 pg / mL, at least 70 pg / mL, at least 80 pg / mL, at least 90 pg / mL, at least 100 pg / mL, at least 150 pg / mL, at least 200 pg / mL, at least 250 pg / mL, at least 300 pg / mL, at least 350 pg / mL, at least 400 pg / mL, at least 450 pg / mL, at least 500 pg / mL, at least 600 pg / mL, at least 700 pg / mL, at least 800 pg / mL, at least 900 pg / mL, at least 1000 pg / mL, at least 1100 pg / mL, or at least 1200 pg / mL, and each mL of test sample is 1 x 10 5 TIL, 2 × 10 5 TIL, 3 × 10 5 TIL, 4 × 10 5 TIL, 5 × 10 5 TIL, 6 × 10 5 TIL, 7×10 5 TIL, 8×10 5 TIL, 9×10 5 TIL, or 10x10 5 Contains TILs.

[0449] In certain embodiments, the ratio of target cells to T cells (including TILs, MILs, or PBLs) is about 1:1.1, about 1:1.2, about 1:1.3, about 1:1.4, about 1:1.5, about 1:1.6, about 1:1.7, about 1:1.8, about 1:1.9, about 1:2, about 1:2.1, about 1:2.2, about 1:2.3, about 1:2.4, about 1:2.5, about 1:2.6, about 1:2.7, about 1:2.8, about 1:2.9, about 1:3, about 1:3.1, about 1:3.2, about 1:3.3, about 1:3.4 , approximately 1:3.5, approximately 1:3.6, approximately 1:3.7, approximately 1:3.8, approximately 1:3.9, approximately 1:4, approximately 1:4.1, approximately 1:4.2, approximately 1:4.3, approximately 1:4.4, approximately 1:4.5, approximately 1:4.6, approximately 1:4.7, approximately 1:4.8, approximately 1:4.9, approximately 1:5, approximately 1:5.1, approximately 1:5.2, approximately 1:5.3, approximately 1:5.4, approximately 1:5.5, approximately 1:5.6, approximately 1:5.7, approximately 1:5.8, approximately 1:5.9, approximately 1:6, approximately 1:6.1, approximately 1:6.2, approximately 1:6.25, approximately 1:6 .3, approximately 1:6.4, approximately 1:6.5, approximately 1:6.6, approximately 1:6.7, approximately 1:6.8, approximately 1:6.9, approximately 1:7, approximately 1:7.1, approximately 1:7.2, approximately 1:7.3, approximately 1:7.4, approximately 1:7.5, approximately 1:7.6, approximately 1:7.7, approximately 1:7.8, approximately 1:7.9, approximately 1:8, approximately 1:8.1, approximately 1:8.2, approximately 1:8.3, approximately 1:8.4, approximately 1:8.5, approximately 1:8.6, approximately 1:8.7, approximately 1:8.8, approximately 1:8.9, approximately 1:9, approximately 1:9.1, approximately 1:9.2, approximately 1 about 1:9.3, about 1:9.4, about 1:9.5, about 1:9.6, about 1:9.7, about 1:9.8, about 1:9.9, about 1:10, about 1:11, about 1:12, about 1:12.5, about 1:13, about 1:14, about 1:15, about 1:16, about 1:17, about 1:18, about 1:19, about 1:20, about 1:25, about 1:30, about 1:35, about 1:40, about 1:45, about 1:50, about 1:60, about 1:70, about 1:80, about 1:90, or about 1:100.

[0450] In certain embodiments, the ratio of T cells (including TILs, MILs, or PBLs) to target cells is about 1:1.1, about 1:1.2, about 1:1.3, about 1:1.4, about 1:1.5, about 1:1.6, about 1:1.7, about 1:1.8, about 1:1.9, about 1:2, about 1:2.1, about 1:2.2, about 1:2.3, about 1:2.4, about 1:2.5, about 1:2.6, about 1:2.7, about 1:2.8, about 1:2.9, about 1:3, about 1:3.1, about 1:3.2, about 1:3.3, about 1:3.4 , approximately 1:3.5, approximately 1:3.6, approximately 1:3.7, approximately 1:3.8, approximately 1:3.9, approximately 1:4, approximately 1:4.1, approximately 1:4.2, approximately 1:4.3, approximately 1:4.4, approximately 1:4.5, approximately 1:4.6, approximately 1:4.7, approximately 1:4.8, approximately 1:4.9, approximately 1:5, approximately 1:5.1, approximately 1:5.2, approximately 1:5.3, approximately 1:5.4, approximately 1:5.5, approximately 1:5.6, approximately 1:5.7, approximately 1:5.8, approximately 1:5.9, approximately 1:6, approximately 1:6.1, approximately 1:6.2, approximately 1:6.25, approximately 1:6 .3, approximately 1:6.4, approximately 1:6.5, approximately 1:6.6, approximately 1:6.7, approximately 1:6.8, approximately 1:6.9, approximately 1:7, approximately 1:7.1, approximately 1:7.2, approximately 1:7.3, approximately 1:7.4, approximately 1:7.5, approximately 1:7.6, approximately 1:7.7, approximately 1:7.8, approximately 1:7.9, approximately 1:8, approximately 1:8.1, approximately 1:8.2, approximately 1:8.3, approximately 1:8.4, approximately 1:8.5, approximately 1:8.6, approximately 1:8.7, approximately 1:8.8, approximately 1:8.9, approximately 1:9, approximately 1:9.1, approximately 1:9.2, approximately 1 about 1:9.3, about 1:9.4, about 1:9.5, about 1:9.6, about 1:9.7, about 1:9.8, about 1:9.9, about 1:10, about 1:11, about 1:12, about 1:12.5, about 1:13, about 1:14, about 1:15, about 1:16, about 1:17, about 1:18, about 1:19, about 1:20, about 1:25, about 1:30, about 1:35, about 1:40, about 1:45, about 1:50, about 1:60, about 1:70, about 1:80, about 1:90, or about 1:100.

[0451] In certain embodiments, the ratio of Raji cells to T cells (including TILs, MILs, or PBLs) is about 1:1.1, about 1:1.2, about 1:1.3, about 1:1.4, about 1:1.5, about 1:1.6, about 1:1.7, about 1:1.8, about 1:1.9, about 1:2, about 1:2.1, about 1:2.2, about 1:2.3, about 1:2.4, about 1:2.5, about 1:2.6, about 1:2.7, about 1:2.8, about 1:2.9, about 1:3, about 1:3.1, about 1:3.2, about 1:3.3, or about 1:3.4. 4, approximately 1:3.5, approximately 1:3.6, approximately 1:3.7, approximately 1:3.8, approximately 1:3.9, approximately 1:4, approximately 1:4.1, approximately 1:4.2, approximately 1:4.3, approximately 1:4.4, approximately 1:4.5, approximately 1:4.6, approximately 1:4.7, approximately 1:4.8, approximately 1:4.9, approximately 1:5, approximately 1:5.1, approximately 1:5.2, approximately 1:5.3, approximately 1:5.4, approximately 1:5.5, approximately 1:5.6, approximately 1:5.7, approximately 1:5.8, approximately 1:5.9, approximately 1:6, approximately 1:6.1, approximately 1:6.2, approximately 1:6.25, approximately 1: 6.3, approximately 1:6.4, approximately 1:6.5, approximately 1:6.6, approximately 1:6.7, approximately 1:6.8, approximately 1:6.9, approximately 1:7, approximately 1:7.1, approximately 1:7.2, approximately 1:7.3, approximately 1:7.4, approximately 1:7.5, approximately 1:7.6, approximately 1:7.7, approximately 1:7.8, approximately 1:7.9, approximately 1:8, approximately 1:8.1, approximately 1:8.2, approximately 1:8.3, approximately 1:8.4, approximately 1:8.5, approximately 1:8.6, approximately 1:8.7, approximately 1:8.8, approximately 1:8.9, approximately 1:9, approximately 1:9.1, approximately 1:9.2, approximately 1 about 1:9.3, about 1:9.4, about 1:9.5, about 1:9.6, about 1:9.7, about 1:9.8, about 1:9.9, about 1:10, about 1:11, about 1:12, about 1:12.5, about 1:13, about 1:14, about 1:15, about 1:16, about 1:17, about 1:18, about 1:19, about 1:20, about 1:25, about 1:30, about 1:35, about 1:40, about 1:45, about 1:50, about 1:60, about 1:70, about 1:80, about 1:90, or about 1:100.

[0452] In certain embodiments, the ratio of T cells (including TILs, MILs, or PBLs) to Raji cells is about 1:1.1, about 1:1.2, about 1:1.3, about 1:1.4, about 1:1.5, about 1:1.6, about 1:1.7, about 1:1.8, about 1:1.9, about 1:2, about 1:2.1, about 1:2.2, about 1:2.3, about 1:2.4, about 1:2.5, about 1:2.6, about 1:2.7, about 1:2.8, about 1:2.9, about 1:3, about 1:3.1, about 1:3.2, about 1:3.3, or about 1:3.4. 4, approximately 1:3.5, approximately 1:3.6, approximately 1:3.7, approximately 1:3.8, approximately 1:3.9, approximately 1:4, approximately 1:4.1, approximately 1:4.2, approximately 1:4.3, approximately 1:4.4, approximately 1:4.5, approximately 1:4.6, approximately 1:4.7, approximately 1:4.8, approximately 1:4.9, approximately 1:5, approximately 1:5.1, approximately 1:5.2, approximately 1:5.3, approximately 1:5.4, approximately 1:5.5, approximately 1:5.6, approximately 1:5.7, approximately 1:5.8, approximately 1:5.9, approximately 1:6, approximately 1:6.1, approximately 1:6.2, approximately 1:6.25, approximately 1: 6.3, approximately 1:6.4, approximately 1:6.5, approximately 1:6.6, approximately 1:6.7, approximately 1:6.8, approximately 1:6.9, approximately 1:7, approximately 1:7.1, approximately 1:7.2, approximately 1:7.3, approximately 1:7.4, approximately 1:7.5, approximately 1:7.6, approximately 1:7.7, approximately 1:7.8, approximately 1:7.9, approximately 1:8, approximately 1:8.1, approximately 1:8.2, approximately 1:8.3, approximately 1:8.4, approximately 1:8.5, approximately 1:8.6, approximately 1:8.7, approximately 1:8.8, approximately 1:8.9, approximately 1:9, approximately 1:9.1, approximately 1:9.2, approximately 1 about 1:9.3, about 1:9.4, about 1:9.5, about 1:9.6, about 1:9.7, about 1:9.8, about 1:9.9, about 1:10, about 1:11, about 1:12, about 1:12.5, about 1:13, about 1:14, about 1:15, about 1:16, about 1:17, about 1:18, about 1:19, about 1:20, about 1:25, about 1:30, about 1:35, about 1:40, about 1:45, about 1:50, about 1:60, about 1:70, about 1:80, about 1:90, or about 1:100.

[0453] In certain embodiments, the ratio of negative control cells to T cells (including TILs, MILs, or PBLs) is about 1:1.1, about 1:1.2, about 1:1.3, about 1:1.4, about 1:1.5, about 1:1.6, about 1:1.7, about 1:1.8, about 1:1.9, about 1:2, about 1:2.1, about 1:2.2, about 1:2.3, about 1:2.4, about 1:2.5, about 1:2.6, about 1:2.7, about 1:2.8, about 1:2.9, about 1:3, about 1:3.1, about 1:3.2, about 1:3.3, or about 1:3.4. 4, approximately 1:3.5, approximately 1:3.6, approximately 1:3.7, approximately 1:3.8, approximately 1:3.9, approximately 1:4, approximately 1:4.1, approximately 1:4.2, approximately 1:4.3, approximately 1:4.4, approximately 1:4.5, approximately 1:4.6, approximately 1:4.7, approximately 1:4.8, approximately 1:4.9, approximately 1:5, approximately 1:5.1, approximately 1:5.2, approximately 1:5.3, approximately 1:5.4, approximately 1:5.5, approximately 1:5.6, approximately 1:5.7, approximately 1:5.8, approximately 1:5.9, approximately 1:6, approximately 1:6.1, approximately 1:6.2, approximately 1:6.25, approximately 1: 6.3, approximately 1:6.4, approximately 1:6.5, approximately 1:6.6, approximately 1:6.7, approximately 1:6.8, approximately 1:6.9, approximately 1:7, approximately 1:7.1, approximately 1:7.2, approximately 1:7.3, approximately 1:7.4, approximately 1:7.5, approximately 1:7.6, approximately 1:7.7, approximately 1:7.8, approximately 1:7.9, approximately 1:8, approximately 1:8.1, approximately 1:8.2, approximately 1:8.3, approximately 1:8.4, approximately 1:8.5, approximately 1:8.6, approximately 1:8.7, approximately 1:8.8, approximately 1:8.9, approximately 1:9, approximately 1:9.1, approximately 1:9.2, approximately 1 about 1:9.3, about 1:9.4, about 1:9.5, about 1:9.6, about 1:9.7, about 1:9.8, about 1:9.9, about 1:10, about 1:11, about 1:12, about 1:12.5, about 1:13, about 1:14, about 1:15, about 1:16, about 1:17, about 1:18, about 1:19, about 1:20, about 1:25, about 1:30, about 1:35, about 1:40, about 1:45, about 1:50, about 1:60, about 1:70, about 1:80, about 1:90, or about 1:100.

[0454] In certain embodiments, the ratio of T cells (including TILs, MILs, or PBLs) to negative control cells is about 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:2.1, 1:2.2, 1:2.3, 1:2.4, 1:2.5, 1:2.6, 1:2.7, 1:2.8, 1:2.9, 1:3, 1:3.1, 1:3.2, 1:3.3 , 1:3.4, 1:3.5, 1:3.6, 1:3.7, 1:3.8, 1:3.9, 1:4, 1:4.1, 1:4.2, 1:4.3, 1:4.4, 1:4.5, 1:4.6, 1:4.7, 1:4.8, 1:4.9, 1:5, 1:5.1, 1:5.2, 1:5.3, 1:5.4, 1:5.5, 1:5.6, 1:5.7, 1:5.8, 1:5.9, 1:6, 1:6.1, 1:6.2, 1:6.25 ,1:6.3,1:6.4,1:6.5,1:6.6,1:6.7,1:6.8,1:6.9,1:7,1:7.1,1:7.2,1:7.3,1:7.4,1:7.5,1:7.6,1:7.7,1:7.8,1:7.9,1:8,1:8.1,1:8.2,1:8.3,1:8.4,1:8.5,1:8.6,1:8.7,1:8.8,1:8.9,1:9,1:9.1,1:9.2, 1:9.3, 1:9.4, 1:9.5, 1:9.6, 1:9.7, 1:9.8, 1:9.9, 1:10, 1:11, 1:12, 1:12.5, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:60, 1:70, 1:80, 1:90, or 1:100.

[0455] In certain embodiments, the ratio of K562 cells to T cells (including TILs, MILs, or PBLs) is about 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:2.1, 1:2.2, 1:2.3, 1:2.4, 1:2.5, 1:2.6, 1:2.7, 1:2.8, 1:2.9, 1:3, 1:3.1, 1:3.2, 1:3.3 , 1:3.4, 1:3.5, 1:3.6, 1:3.7, 1:3.8, 1:3.9, 1:4, 1:4.1, 1:4.2, 1:4.3, 1:4.4, 1:4.5, 1:4.6, 1:4.7, 1:4.8, 1:4.9, 1:5, 1:5.1, 1:5.2, 1:5.3, 1:5.4, 1:5.5, 1:5.6, 1:5.7, 1:5.8, 1:5.9, 1:6, 1:6.1, 1:6.2, 1:6.25 ,1:6.3,1:6.4,1:6.5,1:6.6,1:6.7,1:6.8,1:6.9,1:7,1:7.1,1:7.2,1:7.3,1:7.4,1:7.5,1:7.6,1:7.7,1:7.8,1:7.9,1:8,1:8.1,1:8.2,1:8.3,1:8.4,1:8.5,1:8.6,1:8.7,1:8.8,1:8.9,1:9,1:9.1,1:9.2, 1:9.3, 1:9.4, 1:9.5, 1:9.6, 1:9.7, 1:9.8, 1:9.9, 1:10, 1:11, 1:12, 1:12.5, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:60, 1:70, 1:80, 1:90, or 1:100.

[0456] In certain embodiments, the ratio of T cells (including TILs, MILs, or PBLs) to K562 cells is about 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:2.1, 1:2.2, 1:2.3, 1:2.4, 1:2.5, 1:2.6, 1:2.7, 1:2.8, 1:2.9, 1:3, 1:3.1, 1:3.2, 1:3.3 , 1:3.4, 1:3.5, 1:3.6, 1:3.7, 1:3.8, 1:3.9, 1:4, 1:4.1, 1:4.2, 1:4.3, 1:4.4, 1:4.5, 1:4.6, 1:4.7, 1:4.8, 1:4.9, 1:5, 1:5.1, 1:5.2, 1:5.3, 1:5.4, 1:5.5, 1:5.6, 1:5.7, 1:5.8, 1:5.9, 1:6, 1:6.1, 1:6.2, 1:6.25 ,1:6.3,1:6.4,1:6.5,1:6.6,1:6.7,1:6.8,1:6.9,1:7,1:7.1,1:7.2,1:7.3,1:7.4,1:7.5,1:7.6,1:7.7,1:7.8,1:7.9,1:8,1:8.1,1:8.2,1:8.3,1:8.4,1:8.5,1:8.6,1:8.7,1:8.8,1:8.9,1:9,1:9.1,1:9.2, 1:9.3, 1:9.4, 1:9.5, 1:9.6, 1:9.7, 1:9.8, 1:9.9, 1:10, 1:11, 1:12, 1:12.5, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:60, 1:70, 1:80, 1:90, or 1:100.

[0457] In certain embodiments, the ratio of target cells to T cells (including TILs, MILs, or PBLs) is 1:1 to 1:2, 1:2 to 1:3, 1:3 to 1:4, 1:4 to 1:5, 1:5 to 1:6, 1:6 to 1:7, 1:7 to 1:8, 1:8 to 1:9, 1:9 to 1:10, 1:10 to 1:11, 1:11 to 1:12, 1:12 to 1:13, 1:13 to 1:14, 1:1 4~1:15, 1:15~1:16, 1:16~1:17, 1:17~1:18, 1:18~1:19, 1:19~1:20, 1:20~1:25, 1:25~1:30, 1:30~1:35, 1:35~1:40, 1:40~1:45, 1:45~1:50, 1:50~1:55, 1:55~1:60, 1:60~1:70, 1:70~1:8 0, 1:80-1:90, or 1:90-1:100, or alternatively 1:0.5-1:100, 1:0.5-1:50, 1:0.5-1:40, 1:0.5-1:30, 1:0.5-1:25, 1:0.5-1:20, 1:0.5-1:15, 1:0.5-1:10, 1:0.5-1:5, 1:0.75-1:50, 1:0.75-1: 40, 1:0.75-1:30, 1:0.75-1:25, 1:0.75-1:20, 1:0.75-1:15, 1:0.75-1:10, 1:0.75-1:5, 1:0.5-1:2.5, 1:1-1:20, 1:1-1:10, 1:1-1:5, 1:1-1:2.5, 1:2-1:5, 1:2.5-1:5, or 1:3-1:5.

[0458] In certain embodiments, the ratio of T cells (including TILs, MILs, or PBLs) to target cells is 1:1 to 1:2, 1:2 to 1:3, 1:3 to 1:4, 1:4 to 1:5, 1:5 to 1:6, 1:6 to 1:7, 1:7 to 1:8, 1:8 to 1:9, 1:9 to 1:10, 1:10 to 1:11, 1:11 to 1:12, 1:12 to 1:13, 1:13 to 1:14, 1:1 4~1:15, 1:15~1:16, 1:16~1:17, 1:17~1:18, 1:18~1:19, 1:19~1:20, 1:20~1:25, 1:25~1:30, 1:30~1:35, 1:35~1:40, 1:40~1:45, 1:45~1:50, 1:50~1:55, 1:55~1:60, 1:60~1:70, 1:70~1:8 0, 1:80-1:90, or 1:90-1:100, or alternatively 1:0.5-1:100, 1:0.5-1:50, 1:0.5-1:40, 1:0.5-1:30, 1:0.5-1:25, 1:0.5-1:20, 1:0.5-1:15, 1:0.5-1:10, 1:0.5-1:5, 1:0.75-1:50, 1:0.75-1: 40, 1:0.75-1:30, 1:0.75-1:25, 1:0.75-1:20, 1:0.75-1:15, 1:0.75-1:10, 1:0.75-1:5, 1:0.5-1:2.5, 1:1-1:20, 1:1-1:10, 1:1-1:5, 1:1-1:2.5, 1:2-1:5, 1:2.5-1:5, or 1:3-1:5.

[0459] In certain embodiments, the ratio of Raji cells to T cells (including TILs, MILs, or PBLs) is 1:1 to 1:2, 1:2 to 1:3, 1:3 to 1:4, 1:4 to 1:5, 1:5 to 1:6, 1:6 to 1:7, 1:7 to 1:8, 1:8 to 1:9, 1:9 to 1:10, 1:10 to 1:11, 1:11 to 1:12, 1:12 to 1:13, 1:13 to 1:14, 1:15 to 1:16, 1:17 to 1:18, 1:19 to 1:20, 1:21 to 1:22, 1:23 to 1:24, 1:25 to 1:26, 1:27 to 1:28, 1:29 to 1:30, 1:31 to 1:32, 1:33 to 1:34, 1:35 to 1:35, 1:36 to 1:37, 1:38 to 1:39, 1:40 to 1:41, 1:42 to 1:43, 1:43 to 1:44, 1:45 to 1:45, 1:46 to 1:47, 1:48 to 1:50, 1:49 to 1:51, 1:52 to 1:53, 1:54 to 1:55, 1:56 to 1:57, 1:58 to 1:59, 1:60 to 1:61, 1:62 to 1:63, 1:63 to 1:64, 1:64 to 1:65, 1:65 to :14~1:15, 1:15~1:16, 1:16~1:17, 1:17~1:18, 1:18~1:19, 1:19~1:20, 1:20~1:25, 1:25~1:30, 1:30~1:35, 1:35~1:40, 1:40~1:45, 1:45~1:50, 1:50~1:55, 1:55~1:60, 1:60~1:70, 1:70~1: 80, 1:80 to 1:90, or 1:90 to 1:100, or alternatively 1:0.5 to 1:100, 1:0.5 to 1:50, 1:0.5 to 1:40, 1:0.5 to 1:30, 1:0.5 to 1:25, 1:0.5 to 1:20, 1:0.5 to 1:15, 1:0.5 to 1:10, 1:0.5 to 1:5, 1:0.75 to 1:50, 1:0.75 to 1:100 :40, 1:0.75-1:30, 1:0.75-1:25, 1:0.75-1:20, 1:0.75-1:15, 1:0.75-1:10, 1:0.75-1:5, 1:0.5-1:2.5, 1:1-1:20, 1:1-1:10, 1:1-1:5, 1:1-1:2.5, 1:2-1:5, 1:2.5-1:5, or 1:3-1:5.

[0460] In certain embodiments, the ratio of T cells (including TILs, MILs, or PBLs) to Raji cells is 1:1 to 1:2, 1:2 to 1:3, 1:3 to 1:4, 1:4 to 1:5, 1:5 to 1:6, 1:6 to 1:7, 1:7 to 1:8, 1:8 to 1:9, 1:9 to 1:10, 1:10 to 1:11, 1:11 to 1:12, 1:12 to 1:13, 1:13 to 1:14, 1:15 to 1:16, 1:17 to 1:18, 1:19 to 1:20, 1:21 to 1:22, 1:23 to 1:24, 1:25 to 1:26, 1:27 to 1:28, 1:29 to 1:30, 1:31 to 1:32, 1:33 to 1:34, 1:35 to 1:35, 1:36 to 1:37, 1:38 to 1:39, 1:40 to 1:41, 1:42 to 1:43, 1:43 to 1:44, 1:45 to 1:45, 1:46 to 1:47, 1:48 to 1:50, 1:49 to 1:51, 1:52 to 1:53, 1:54 to 1:55, 1:56 to 1:57, 1:58 to 1:59, 1:60 to 1:61, 1:62 to 1:63, 1:63 to 1:64, 1:64 to 1:65, 1:65 to :14~1:15, 1:15~1:16, 1:16~1:17, 1:17~1:18, 1:18~1:19, 1:19~1:20, 1:20~1:25, 1:25~1:30, 1:30~1:35, 1:35~1:40, 1:40~1:45, 1:45~1:50, 1:50~1:55, 1:55~1:60, 1:60~1:70, 1:70~1: 80, 1:80 to 1:90, or 1:90 to 1:100, or alternatively 1:0.5 to 1:100, 1:0.5 to 1:50, 1:0.5 to 1:40, 1:0.5 to 1:30, 1:0.5 to 1:25, 1:0.5 to 1:20, 1:0.5 to 1:15, 1:0.5 to 1:10, 1:0.5 to 1:5, 1:0.75 to 1:50, 1:0.75 to 1:100 :40, 1:0.75-1:30, 1:0.75-1:25, 1:0.75-1:20, 1:0.75-1:15, 1:0.75-1:10, 1:0.75-1:5, 1:0.5-1:2.5, 1:1-1:20, 1:1-1:10, 1:1-1:5, 1:1-1:2.5, 1:2-1:5, 1:2.5-1:5, or 1:3-1:5.

[0461] In certain embodiments, the ratio of negative control cells to T cells (including TILs, MILs, or PBLs) is 1:1 to 1:2, 1:2 to 1:3, 1:3 to 1:4, 1:4 to 1:5, 1:5 to 1:6, 1:6 to 1:7, 1:7 to 1:8, 1:8 to 1:9, 1:9 to 1:10, 1:10 to 1:11, 1:11 to 1:12, 1:12 to 1:13, 1:13 to 1:14, 1:15 to 1:16, 1:17 to 1:18, 1:19 to 1:20, 1:21 to 1:22, 1:23 to 1:24, 1:25 to 1:26, 1:27 to 1:28, 1:29 to 1:30, 1:31 to 1:32, 1:33 to 1:34, 1:35 to 1:35, 1:36 to 1:37, 1:38 to 1:39, 1:40 to 1:41, 1:42 to 1:43, 1:43 to 1:44, 1:44 to 1:50, 1:45 to 1:51, 1:46 to 1:47, 1:48 to 1:52, 1:49 to 1:53, 1:54 to 1:55, 1:55 to 1:60, 1:56 to 1:61, 1:57 to 1:58, 1:59 to 1:62, 1:63 to 1:64, 1:65 to 1:65, 1:66 to :14~1:15, 1:15~1:16, 1:16~1:17, 1:17~1:18, 1:18~1:19, 1:19~1:20, 1:20~1:25, 1:25~1:30, 1:30~1:35, 1:35~1:40, 1:40~1:45, 1:45~1:50, 1:50~1:55, 1:55~1:60, 1:60~1:70, 1:70~1: 80, 1:80 to 1:90, or 1:90 to 1:100, or alternatively 1:0.5 to 1:100, 1:0.5 to 1:50, 1:0.5 to 1:40, 1:0.5 to 1:30, 1:0.5 to 1:25, 1:0.5 to 1:20, 1:0.5 to 1:15, 1:0.5 to 1:10, 1:0.5 to 1:5, 1:0.75 to 1:50, 1:0.75 to 1:100 :40, 1:0.75-1:30, 1:0.75-1:25, 1:0.75-1:20, 1:0.75-1:15, 1:0.75-1:10, 1:0.75-1:5, 1:0.5-1:2.5, 1:1-1:20, 1:1-1:10, 1:1-1:5, 1:1-1:2.5, 1:2-1:5, 1:2.5-1:5, or 1:3-1:5.

[0462] In certain embodiments, the ratio of T cells (including TILs, MILs, or PBLs) to negative control cells is 1:1 to 1:2, 1:2 to 1:3, 1:3 to 1:4, 1:4 to 1:5, 1:5 to 1:6, 1:6 to 1:7, 1:7 to 1:8, 1:8 to 1:9, 1:9 to 1:10, 1:10 to 1:11, 1:11 to 1:12, 1:12 to 1:13, 1:13 to 1:14, 1:15 to 1:16, 1:17 to 1:18, 1:19 to 1:20, 1:21 to 1:22, 1:23 to 1:24, 1:25 to 1:26, 1:27 to 1:28, 1:29 to 1:30, 1:31 to 1:32, 1:33 to 1:34, 1:35 to 1:35, 1:36 to 1:37, 1:38 to 1:39, 1:40 to 1:41, 1:42 to 1:43, 1:43 to 1:44, 1:45 to 1:45, 1:46 to 1:47, 1:48 to 1:50, 1:49 to 1:51, 1:52 to 1:53, 1:54 to 1:55, 1:56 to 1:57, 1:58 to 1:59, 1:60 to 1:61, 1:62 to 1:63, 1:63 to 1:64, 1:64 to 1:65, 1:65 to :14~1:15, 1:15~1:16, 1:16~1:17, 1:17~1:18, 1:18~1:19, 1:19~1:20, 1:20~1:25, 1:25~1:30, 1:30~1:35, 1:35~1:40, 1:40~1:45, 1:45~1:50, 1:50~1:55, 1:55~1:60, 1:60~1:70, 1:70~1: 80, 1:80 to 1:90, or 1:90 to 1:100, or alternatively 1:0.5 to 1:100, 1:0.5 to 1:50, 1:0.5 to 1:40, 1:0.5 to 1:30, 1:0.5 to 1:25, 1:0.5 to 1:20, 1:0.5 to 1:15, 1:0.5 to 1:10, 1:0.5 to 1:5, 1:0.75 to 1:50, 1:0.75 to 1:100 :40, 1:0.75-1:30, 1:0.75-1:25, 1:0.75-1:20, 1:0.75-1:15, 1:0.75-1:10, 1:0.75-1:5, 1:0.5-1:2.5, 1:1-1:20, 1:1-1:10, 1:1-1:5, 1:1-1:2.5, 1:2-1:5, 1:2.5-1:5, or 1:3-1:5.

[0463] In certain embodiments, the ratio of K562 cells to T cells (including TILs, MILs, or PBLs) is 1:1 to 1:2, 1:2 to 1:3, 1:3 to 1:4, 1:4 to 1:5, 1:5 to 1:6, 1:6 to 1:7, 1:7 to 1:8, 1:8 to 1:9, 1:9 to 1:10, 1:10 to 1:11, 1:11 to 1:12, 1:12 to 1:13, 1:13 to 1:14, 1:15 to 1:16, 1:17 to 1:18, 1:19 to 1:20, 1:21 to 1:22, 1:23 to 1:24, 1:25 to 1:26, 1:27 to 1:28, 1:29 to 1:30, 1:31 to 1:32, 1:33 to 1:34, 1:35 to 1:35, 1:36 to 1:37, 1:38 to 1:39, 1:40 to 1:41, 1:42 to 1:43, 1:43 to 1:44, 1:44 to 1:50, 1:45 to 1:51, 1:46 to 1:47, 1:48 to 1:52, 1:49 to 1:53, 1:54 to 1:55, 1:55 to 1:60, 1:61 to 1:62, 1:63 to 1:64, 1:65 to 1:66, 1:66 to 1:70, 1:71 to 1:80, 1:82 :14~1:15, 1:15~1:16, 1:16~1:17, 1:17~1:18, 1:18~1:19, 1:19~1:20, 1:20~1:25, 1:25~1:30, 1:30~1:35, 1:35~1:40, 1:40~1:45, 1:45~1:50, 1:50~1:55, 1:55~1:60, 1:60~1:70, 1:70~1: 80, 1:80 to 1:90, or 1:90 to 1:100, or alternatively 1:0.5 to 1:100, 1:0.5 to 1:50, 1:0.5 to 1:40, 1:0.5 to 1:30, 1:0.5 to 1:25, 1:0.5 to 1:20, 1:0.5 to 1:15, 1:0.5 to 1:10, 1:0.5 to 1:5, 1:0.75 to 1:50, 1:0.75 to 1:100 :40, 1:0.75-1:30, 1:0.75-1:25, 1:0.75-1:20, 1:0.75-1:15, 1:0.75-1:10, 1:0.75-1:5, 1:0.5-1:2.5, 1:1-1:20, 1:1-1:10, 1:1-1:5, 1:1-1:2.5, 1:2-1:5, 1:2.5-1:5, or 1:3-1:5.

[0464] In certain embodiments, the ratio of T cells (including TILs, MILs, or PBLs) to K562 cells is 1:1 to 1:2, 1:2 to 1:3, 1:3 to 1:4, 1:4 to 1:5, 1:5 to 1:6, 1:6 to 1:7, 1:7 to 1:8, 1:8 to 1:9, 1:9 to 1:10, 1:10 to 1:11, 1:11 to 1:12, 1:12 to 1:13, 1:13 to 1:14, 1:15 to 1:16, 1:17 to 1:18, 1:19 to 1:20, 1:21 to 1:22, 1:23 to 1:24, 1:25 to 1:26, 1:27 to 1:28, 1:29 to 1:30, 1:31 to 1:32, 1:33 to 1:34, 1:35 to 1:35, 1:36 to 1:37, 1:38 to 1:39, 1:40 to 1:41, 1:42 to 1:43, 1:43 to 1:44, 1:45 to 1:45, 1:46 to 1:47, 1:48 to 1:50, 1:49 to 1:51, 1:52 to 1:53, 1:54 to 1:55, 1:56 to 1:57, 1:58 to 1:59, 1:60 to 1:61, 1:62 to 1:63, 1:63 to 1:64, 1:64 to 1:65, 1:65 :14~1:15, 1:15~1:16, 1:16~1:17, 1:17~1:18, 1:18~1:19, 1:19~1:20, 1:20~1:25, 1:25~1:30, 1:30~1:35, 1:35~1:40, 1:40~1:45, 1:45~1:50, 1:50~1:55, 1:55~1:60, 1:60~1:70, 1:70~1: 80, 1:80 to 1:90, or 1:90 to 1:100, or alternatively 1:0.5 to 1:100, 1:0.5 to 1:50, 1:0.5 to 1:40, 1:0.5 to 1:30, 1:0.5 to 1:25, 1:0.5 to 1:20, 1:0.5 to 1:15, 1:0.5 to 1:10, 1:0.5 to 1:5, 1:0.75 to 1:50, 1:0.75 to 1:100 :40, 1:0.75-1:30, 1:0.75-1:25, 1:0.75-1:20, 1:0.75-1:15, 1:0.75-1:10, 1:0.75-1:5, 1:0.5-1:2.5, 1:1-1:20, 1:1-1:10, 1:1-1:5, 1:1-1:2.5, 1:2-1:5, 1:2.5-1:5, or 1:3-1:5.

[0465] In certain embodiments, the ratio of TILs to Raji cells in the target cell co-culture is selected from the group consisting of about 15:1, about 14:1, about 13:1, about 12:1, about 11:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4.5:1, about 4:1, about 3.5:1, about 3:1, about 2.5:1, about 2:1, about 1.5:1, about 1:1, about 1:1.5, about 1:2, about 1:2.5, about 1:3, about 1:3.5, about 1:4, about 1:4.5, about 1:5, about 1:6, about 1.7, about 1:8, about 1:9, about 1:10, about 1:11, about 1:12, about 1:13, about 1:14, and about 1:15.

[0466] In certain embodiments, the ratio of TILs to K562 cells in the target cell co-culture is selected from the group consisting of about 15:1, about 14:1, about 13:1, about 12:1, about 11:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4.5:1, about 4:1, about 3.5:1, about 3:1, about 2.5:1, about 2:1, about 1.5:1, about 1:1, about 1:1.5, about 1:2, about 1:2.5, about 1:3, about 1:3.5, about 1:4, about 1:4.5, about 1:5, about 1:6, about 1.7, about 1:8, about 1:9, about 1:10, about 1:11, about 1:12, about 1:13, about 1:14, and about 1:15.

[0467] In one embodiment, about 1×10 5 ~10×10 5 TILs, approximately 1 x 10 5 ~10×10 5 In one embodiment, approximately 3 x 10 Raji cells are co-cultured. 5 ~7×10 5 TILs, approximately 1 x 10 5 ~3×10 5 In one embodiment, approximately 5 x 10 Raji cells are co-cultured. 5 ~6×10 5 TILs, approximately 3 x 10 5 ~7×10 5 In one embodiment, approximately 4 x 10 Raji cells are co-cultured. 5 TILs, approximately 5 x 10 5 In one embodiment, approximately 5 x 10 Raji cells are co-cultured. 5 TILs, approximately 5 x 10 5 In one embodiment, approximately 5 x 10 Raji cells are co-cultured. 5 TILs, approximately 4 x 10 5 In one embodiment, approximately 3 x 10 Raji cells are co-cultured. 5 ~7×10 5 TILs, approximately 7 x 10 5 ~20×10 5 In one embodiment, approximately 4 x 10 Raji cells are co-cultured. 5 ~4×10 5 TILs, approximately 10 x 10 5 ~15×10 5In one embodiment, approximately 5 x 10 Raji cells are co-cultured. 5 TIL, approximately 15 x 10 5 Co-culture with Raji cells.

[0468] In some embodiments, the invention provides a method for assaying TIL activity, comprising: (a) irradiating Raji cells and K562 cells; (b) co-culturing the irradiated Raji cells and irradiated K562 cells with TILs at a target ratio selected from the group consisting of 50:1, 25:1, 12.5:1, 6.25:1, 1:1, 1:6.25, 1:12.5, and 1:25), wherein the co-culturing is optionally performed using an anti-CD28 antibody; (c) after 6 to 24 hours, collecting supernatant from the co-cultured cells in step (b); (d) harvesting the cells from step (b) and measuring expression of surface markers of T cell activation; and (e) assaying the supernatant from the co-cultured cells collected in step (c) for markers of T cell activation.

[0469] In one embodiment, the potency assay involves thawing a TIL cryopreserved product, a MIL cryopreserved product, or a PBL cryopreserved product and measuring the percentage of the total number of cells per 100 cells in the culture medium at 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 25 hours, 26 hours, 27 hours, 28 hours, 29 hours, 30 hours, 31 hours, 32 hours, 33 hours, 34 hours, 35 hours, 36 hours, 37 hours, 38 hours, 39 hours, 40 hours, 41 hours, 42 hours, 43 hours, 44 hours, 45 hours, 46 hours, 47 hours, 48 ​​hours, 49 hours, 50 hours, 51 hours, 52 hours, 53 hours, 54 hours, 55 hours, 56 hours, 57 hours, 58 hours, 59 hours, 60 hours, 61 hours, 62 hours, 63 hours, 64 hours, 65 hours, 66 hours, 67 hours, 68 hours, 69 hours, 70 hours, 71 hours, 72 hours, 73 hours, 74 hours, 75 hours, 76 hours, 77 hours, 78 hours, 79 hours, 80 hours, 81 hours, 82 hours, 83 hours, 84 hours, 85 hours, 86 hours, 87 hours, 88 hours, 89 hours, 90 hours, 91 hours, 92 hours, 93 hours, 94 , 46 hours, 47 hours, 48 ​​hours, 49 hours, 50 hours, 51 hours, 52 hours, 53 hours, 54 hours, 55 hours, 56 hours, 57 hours, 58 hours, 59 hours, 60 hours, 61 hours, 62 hours, 63 hours, 64 hours, 65 hours, 66 hours, 67 hours, 68 hours, 69 hours, 70 hours, 71 hours, 72 hours, 73 hours, 74 hours, 75 hours, 76 hours, 77 hours, 78 hours, 79 hours, 80 hours, 85 hours, 90 hours, 95 hours, 100 hours, 110 hours, and 120 hours. In certain embodiments, the potency assay includes a recovery step in which the TIL, MIL, or PBL cryopreserved product is thawed and allowed to recover at ambient or refrigerated temperature for a period of time selected from the group consisting of about 12 hours, about 24 hours, about 48 hours, about 72 hours, and about 96 hours, wherein the aforementioned duration can be measured from the completion of the thawing process or from the start of the thawing process.

[0470] In one embodiment, the invention includes a method for determining the potency of a T cell product, the method comprising: co-culturing the target cells with the aT cell product cells for a first period of time; b. Obtaining a harvest from the co-culture; c. assessing the collection (1) for expression of one or more markers on the T cell product cells, or (2) for one or more analytes secreted from the T cell product cells to obtain one or more observations and determine the potency of the T cell product.

[0471] In one embodiment, the invention includes a method for determining the potency of a T cell product, the method comprising: co-culturing the target cells with the aT cell product cells for a first period of time; b. Obtaining a harvest from the co-culture; c. assessing the harvest (1) for expression of one or more markers on the T cell product cells, or (2) for one or more analytes secreted from the T cell product cells to obtain one or more observations and determine the potency of the T cell product; The aforementioned methods include CD3, CD28, and / or CD137 stimulation and reporting, bead or plate-based assays using interferon-γ, granzyme B, or tumor necrosis factor-α, assays for total viable cells, assays for percentage viable cells, CD4 + Assay for cell content, CD8 + Assay for cell content, T EM Assay for cell content, T CM Assay for cell content, LAG3 + Assays for cellular content and KLRG1 + Assay for cell content, CD101 + Assay for cell content, CD69 + Assay for cell content, T SCM Assay for cell content, T EMRA Assay for cell content, T reg Assay for cellular content, PD-1 + Assay for cellular content, TIM3 + Assay for cell content, CD25 + Assay for cell content, CD27 + Assay for cell content, CD28 + Assay for cell content, CD56 + Assay for cellular content, CTLA-4 + Assay for cell content, TIGIT + Assays for cell content and CD57+ In one embodiment, the assay is a flow cytometry assay.

[0472] In one embodiment, the invention includes a method for determining the potency of a T cell product, the method comprising: co-culturing the target cells with the aT cell product cells for a first period of time; b. Obtaining a harvest from the co-culture; c. assessing the harvest (1) for expression of one or more markers on the T cell product cells, or (2) for one or more analytes secreted from the T cell product cells, to obtain one or more observations and determine the potency of the T cell product; conducting a second co-culture of the negative control cells with the dT cell product cells for a second period of time; e. obtaining a second harvest from the second co-culture; f. assessing the second harvest for (1) expression of one or more markers on the T cell product cells, or (2) one or more analytes secreted from the T cell product cells to obtain one or more control values; g. Comparing one or more observed values ​​from step c to one or more control values ​​from step f, where each observed value is compared to a corresponding control value, to determine the potency of the T cell product.

[0473] In one embodiment, the invention includes a method for determining the potency of a T cell product, the method comprising: co-culturing the target cells with the aT cell product cells for a first period of time; b. Obtaining a harvest from the co-culture; c. assessing the harvest (1) for expression of one or more markers on the T cell product cells, or (2) for one or more analytes secreted from the T cell product cells, to obtain one or more observations and determine the potency of the T cell product; conducting a second co-culture of the negative control cells with the dT cell product cells for a second period of time; e. obtaining a second harvest from the second co-culture; f. assessing the second harvest for (1) expression of one or more markers on the T cell product cells, or (2) one or more analytes secreted from the T cell product cells to obtain one or more control values; g. comparing one or more observed values ​​from step c to one or more control values ​​from step f, where each observed value is compared to a corresponding control value, to determine the potency of the T cell product; The T cell product is selected from the group consisting of a tumor infiltrating lymphocyte (TIL) product, a bone marrow infiltrating lymphocyte (MIL) product, or a peripheral blood lymphocyte (PBL) product.

[0474] In one embodiment, the invention includes a method for determining the potency of a T cell product, the method comprising: co-culturing the target cells with the aT cell product cells for a first period of time; b. Obtaining a harvest from the co-culture; c. assessing the harvest (1) for expression of one or more markers on the T cell product cells, or (2) for one or more analytes secreted from the T cell product cells, to obtain one or more observations and determine the potency of the T cell product; conducting a second co-culture of the negative control cells with the dT cell product cells for a second period of time; e. obtaining a second harvest from the second co-culture; f. assessing the second harvest for (1) expression of one or more markers on the T cell product cells, or (2) one or more analytes secreted from the T cell product cells to obtain one or more control values; g. comparing one or more observed values ​​from step c to one or more control values ​​from step f, where each observed value is compared to a corresponding control value, to determine the potency of the T cell product; The T cell product is a human-derived TIL product, which is obtained by tumor resection or tumor fragmentation or digestion and is produced by a TIL expansion process that includes a rapid expansion protocol step.

[0475] In one embodiment, the invention includes a method for determining the potency of a T cell product, the method comprising: co-culturing the target cells with the aT cell product cells for a first period of time; b. Obtaining a harvest from the co-culture; c. assessing the harvest (1) for expression of one or more markers on the T cell product cells, or (2) for one or more analytes secreted from the T cell product cells, to obtain one or more observations and determine the potency of the T cell product; conducting a second co-culture of the negative control cells with the dT cell product cells for a second period of time; e. obtaining a second harvest from the second co-culture; f. assessing the second harvest for (1) expression of one or more markers on the T cell product cells, or (2) one or more analytes secreted from the T cell product cells to obtain one or more control values; g. Comparing one or more observed values ​​from step c to one or more control values ​​from step f, where each observed value is compared to a corresponding control value, to determine the potency of the T cell product; h. releasing the T cell product for use in treating human patients; The T cell product is a human-derived TIL product, which is obtained by tumor resection or tumor fragmentation or digestion and is produced by a TIL expansion process that includes a rapid expansion protocol step.

[0476] In one embodiment, the invention includes a method for determining the potency of a T cell product, the method comprising: co-culturing the target cells with the aT cell product cells for a first period of time; b. Obtaining a harvest from the co-culture; c. assessing the harvest (1) for expression of one or more markers on the T cell product cells, or (2) for one or more analytes secreted from the T cell product cells, to obtain one or more observations and determine the potency of the T cell product; conducting a second co-culture of the negative control cells with the dT cell product cells for a second period of time; e. obtaining a second harvest from the second co-culture; f. assessing the second harvest for (1) expression of one or more markers on the T cell product cells, or (2) one or more analytes secreted from the T cell product cells to obtain one or more control values; g. Comparing one or more observed values ​​from step c to one or more control values ​​from step f, where each observed value is compared to a corresponding control value, to determine the potency of the T cell product; h. releasing the T cell product for use in treating human patients; The T cell product is a human-derived TIL product, wherein the TIL product is obtained by tumor resection or tumor fragmentation or digestion and is produced by a TIL expansion process that includes a rapid expansion protocol step, and the target cells are irradiated Raji cells, Ramos cells, Daudi cells, U937 cells, or Thp1 cells, or derivatives, variants, modifications, or progeny thereof.

[0477] In one embodiment, the invention includes a method for determining the potency of a T cell product, the method comprising: co-culturing the target cells with the aT cell product cells for a first period of time; b. Obtaining a harvest from the co-culture; c. assessing the harvest (1) for expression of one or more markers on the T cell product cells, or (2) for one or more analytes secreted from the T cell product cells, to obtain one or more observations and determine the potency of the T cell product; conducting a second co-culture of the negative control cells with the dT cell product cells for a second period of time; e. obtaining a second harvest from the second co-culture; f. assessing the second harvest for (1) expression of one or more markers on the T cell product cells, or (2) one or more analytes secreted from the T cell product cells to obtain one or more control values; g. Comparing one or more observed values ​​from step c to one or more control values ​​from step f, where each observed value is compared to a corresponding control value, to determine the potency of the T cell product; h. releasing the T cell product for use in treating human patients; The T cell product is a human-derived TIL product, the TIL product being obtained by tumor resection or tumor fragmentation or digestion and produced by a TIL expansion process including a rapid expansion protocol step, the target cells being irradiated Raji cells, Ramos cells, Daudi cells, U937 cells, or Thp1 cells, or derivatives, variants, modifications, or progeny thereof, and the negative control cells lacking MHC class I and MHC class II expression.

[0478] In one embodiment, the invention includes a method for determining the potency of a T cell product, the method comprising: co-culturing the target cells with the aT cell product cells for a first period of time; b. Obtaining a harvest from the co-culture; c. assessing the harvest (1) for expression of one or more markers on the T cell product cells, or (2) for one or more analytes secreted from the T cell product cells, to obtain one or more observations and determine the potency of the T cell product; conducting a second co-culture of the negative control cells with the dT cell product cells for a second period of time; e. obtaining a second harvest from the second co-culture; f. assessing the second harvest for (1) expression of one or more markers on the T cell product cells, or (2) one or more analytes secreted from the T cell product cells to obtain one or more control values; g. Comparing one or more observed values ​​from step c to one or more control values ​​from step f, where each observed value is compared to a corresponding control value, to determine the potency of the T cell product; h. releasing the T cell product for use in treating human patients; The T cell product is a human-derived TIL product, the TIL product being obtained by tumor resection or tumor fragmentation or digestion and produced by a TIL expansion process including a rapid expansion protocol step, the target cells being irradiated Raji cells, Ramos cells, Daudi cells, U937 cells, or Thp1 cells, or derivatives, variants, modifications, or progeny thereof, and the negative control cells being irradiated K562 cells or derivatives, variants, modifications, or progeny thereof.

[0479] In one embodiment, the invention includes a method for determining the potency of a T cell product, the method comprising: co-culturing the target cells with the aT cell product cells for a first period of time; b. Obtaining a harvest from the co-culture; c. assessing the harvest (1) for expression of one or more markers on the T cell product cells, or (2) for one or more analytes secreted from the T cell product cells, to obtain one or more observations and determine the potency of the T cell product; conducting a second co-culture of the negative control cells with the dT cell product cells for a second period of time; e. obtaining a second harvest from the second co-culture; f. assessing the second harvest for (1) expression of one or more markers on the T cell product cells, or (2) one or more analytes secreted from the T cell product cells to obtain one or more control values; g. Comparing one or more observed values ​​from step c to one or more control values ​​from step f, where each observed value is compared to a corresponding control value, to determine the potency of the T cell product; h. releasing the T cell product for use in treating human patients; The T cell product is a human-derived TIL product, obtained by tumor resection or tumor fragmentation or digestion and produced by a TIL expansion process including a rapid expansion protocol step, the target cells are irradiated Raji cells, Ramos cells, Daudi cells, U937 cells, or Thp1 cells, or their derivatives, variants, modifications, or progeny, the negative control cells are irradiated K562 cells or their derivatives, variants, modifications, or progeny, and the ratio of the number of TIL product cells to the number of target cells is 5:1 to 1:5.

[0480] In one embodiment, the invention includes a method for determining the potency of a T cell product, the method comprising: co-culturing the target cells with the aT cell product cells for a first period of time; b. Obtaining a harvest from the co-culture; c. assessing the harvest (1) for expression of one or more markers on the T cell product cells, or (2) for one or more analytes secreted from the T cell product cells, to obtain one or more observations and determine the potency of the T cell product; conducting a second co-culture of the negative control cells with the dT cell product cells for a second period of time; e. obtaining a second harvest from the second co-culture; f. assessing the second harvest for (1) expression of one or more markers on the T cell product cells, or (2) one or more analytes secreted from the T cell product cells to obtain one or more control values; g. Comparing one or more observed values ​​from step c to one or more control values ​​from step f, where each observed value is compared to a corresponding control value, to determine the potency of the T cell product; h. releasing the T cell product for use in treating human patients; The T cell product is a human-derived TIL product, the TIL product being obtained by tumor resection or tumor fragmentation or digestion and produced by a TIL expansion process including a rapid expansion protocol step, the target cells being irradiated Raji cells, Ramos cells, Daudi cells, U937 cells, or Thp1 cells, or their derivatives, variants, modifications, or progeny, the negative control cells being irradiated K562 cells or their derivatives, variants, modifications, or progeny, the ratio of the number of TIL product cells to the number of target cells being 5:1 to 1:5, and the ratio of the number of TIL product cells to the number of negative control cells being 5:1 to 1:5.

[0481] In one embodiment, the invention includes a method for determining the potency of a T cell product, the method comprising: co-culturing the target cells with the aT cell product cells for a first period of time; b. Obtaining a harvest from the co-culture; c. assessing the harvest (1) for expression of one or more markers on the T cell product cells, or (2) for one or more analytes secreted from the T cell product cells, to obtain one or more observations and determine the potency of the T cell product; conducting a second co-culture of the negative control cells with the dT cell product cells for a second period of time; e. obtaining a second harvest from the second co-culture; f. assessing the second harvest for (1) expression of one or more markers on the T cell product cells, or (2) one or more analytes secreted from the T cell product cells to obtain one or more control values; g. Comparing one or more observed values ​​from step c to one or more control values ​​from step f, where each observed value is compared to a corresponding control value, to determine the potency of the T cell product; h. releasing the T cell product for use in treating human patients; The T cell product is a human-derived TIL product, the TIL product being obtained by tumor resection or tumor fragmentation or digestion and produced by a TIL expansion process including a rapid expansion protocol step, the target cells being irradiated Raji cells, Ramos cells, Daudi cells, U937 cells, or Thp1 cells, or derivatives, variants, modifications, or progeny thereof, the negative control cells being irradiated K562 cells or derivatives, variants, modifications, or progeny thereof, the first period of time being about 6 hours to about 48 hours, and the second period of time being about 6 hours to about 48 hours.

[0482] In one embodiment, the invention includes a method for determining the potency of a T cell product, the method comprising: co-culturing the target cells with the aT cell product cells for a first period of time; b. Obtaining a harvest from the co-culture; c. assessing the harvest (1) for expression of one or more markers on the T cell product cells, or (2) for one or more analytes secreted from the T cell product cells, to obtain one or more observations and determine the potency of the T cell product; conducting a second co-culture of the negative control cells with the dT cell product cells for a second period of time; e. obtaining a second harvest from the second co-culture; f. assessing the second harvest for (1) expression of one or more markers on the T cell product cells, or (2) one or more analytes secreted from the T cell product cells to obtain one or more control values; g. Comparing one or more observed values ​​from step c to one or more control values ​​from step f, where each observed value is compared to a corresponding control value, to determine the potency of the T cell product; h. releasing the T cell product for use in treating human patients; The T cell product is a human-derived TIL product, the TIL product being obtained by tumor resection or tumor fragmentation or digestion and produced by a TIL expansion process including a rapid expansion protocol step, the target cells being irradiated Raji cells, Ramos cells, Daudi cells, U937 cells, or Thp1 cells, or derivatives, variants, modifications, or progeny thereof, the negative control cells being irradiated K562 cells or derivatives, variants, modifications, or progeny thereof, the first period of time being selected from the group consisting of about 12 hours, about 18 hours, and about 24 hours, and the second period of time being selected from the group consisting of about 12 hours, about 18 hours, and about 24 hours.

[0483] In one embodiment, the invention includes a method for determining the potency of a T cell product, the method comprising: co-culturing the target cells with the aT cell product cells for a first period of time; b. Obtaining a harvest from the co-culture; c. assessing the harvest (1) for expression of one or more markers on the T cell product cells, or (2) for one or more analytes secreted from the T cell product cells, to obtain one or more observations and determine the potency of the T cell product; conducting a second co-culture of the negative control cells with the dT cell product cells for a second period of time; e. obtaining a second harvest from the second co-culture; f. assessing the second harvest for (1) expression of one or more markers on the T cell product cells, or (2) one or more analytes secreted from the T cell product cells to obtain one or more control values; g. Comparing one or more observed values ​​from step c to one or more control values ​​from step f, where each observed value is compared to a corresponding control value, to determine the potency of the T cell product; h. releasing the T cell product for use in treating human patients; The T cell product is a human-derived TIL product, the TIL product being obtained by tumor resection or tumor fragmentation or digestion and produced by a TIL expansion process including a rapid expansion protocol step, the target cells being irradiated Raji cells, Ramos cells, Daudi cells, U937 cells, or Thp1 cells, or derivatives, variants, modifications, or progeny thereof, the negative control cells being irradiated K562 cells or derivatives, variants, modifications, or progeny thereof, and the one or more markers on the T cell product are selected from the group consisting of CD25, CD69, CD134, CD137, CD150, KLRG1, or a combination thereof.

[0484] In one embodiment, the invention includes a method for determining the potency of a T cell product, the method comprising: co-culturing the target cells with the aT cell product cells for a first period of time; b. Obtaining a harvest from the co-culture; c. assessing the harvest (1) for expression of one or more markers on the T cell product cells, or (2) for one or more analytes secreted from the T cell product cells, to obtain one or more observations and determine the potency of the T cell product; conducting a second co-culture of the negative control cells with the dT cell product cells for a second period of time; e. obtaining a second harvest from the second co-culture; f. assessing the second harvest for (1) expression of one or more markers on the T cell product cells, or (2) one or more analytes secreted from the T cell product cells to obtain one or more control values; g. Comparing one or more observed values ​​from step c to one or more control values ​​from step f, where each observed value is compared to a corresponding control value, to determine the potency of the T cell product; h. releasing the T cell product for use in treating human patients; The T cell product is a human-derived TIL product, the TIL product being obtained by tumor resection or tumor fragmentation or digestion and produced by a TIL expansion process including a rapid expansion protocol step, the target cells being irradiated Raji cells, Ramos cells, Daudi cells, U937 cells, or Thp1 cells, or derivatives, variants, modifications, or progeny thereof, and the negative control cells being irradiated K562 cells or derivatives, variants, modifications, or progeny thereof. , modified, or progeny thereof, wherein the one or more analytes selected from the T cell product are selected from the group consisting of IFN-α, IFN-β, IFN-γ, granzyme B, perforin, TNF-α, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-9, IL-10, IL-13, IL-14, IL-16, IL-17, IL-18, IL-22, IL-25, IL-26, MIP-1β, and combinations thereof.

[0485] In one embodiment, the invention includes a method for determining the potency of a T cell product, the method comprising: co-culturing the target cells with the aT cell product cells for a first period of time; b. Obtaining a harvest from the co-culture; c. assessing the harvest (1) for expression of one or more markers on the T cell product cells, or (2) for one or more analytes secreted from the T cell product cells, to obtain one or more observations and determine the potency of the T cell product; conducting a second co-culture of the negative control cells with the dT cell product cells for a second period of time; e. obtaining a second harvest from the second co-culture; f. assessing the second harvest for (1) expression of one or more markers on the T cell product cells, or (2) one or more analytes secreted from the T cell product cells to obtain one or more control values; g. Comparing one or more observed values ​​from step c to one or more control values ​​from step f, where each observed value is compared to a corresponding control value, to determine the potency of the T cell product; h. releasing the T cell product for use in treating human patients; The T cell product is a human-derived TIL product, the TIL product being obtained by tumor resection or tumor fragmentation or digestion and produced by a TIL expansion process including a rapid expansion protocol step, the target cells being irradiated Raji cells, Ramos cells, Daudi cells, U937 cells, or Thp1 cells, or derivatives, variants, modifications, or progeny thereof, the negative control cells being irradiated K562 cells, or derivatives, variants, modifications, or progeny thereof, and the one or more analytes secreted from the T cell product are IFN-α, IFN-β, IFN-γ, granzyme B, perforin, TNF-α, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-9, IL-10, IL-13, IL-14, IL-16, IL-17, IL-18, IL-22, IL-25, IL-26 , MIP-1β, and combinations thereof; and the one or more analytes secreted from the TIL product are selected from the group consisting of IFN-α, IFN-β, IFN-γ, granzyme B, perforin, TNF-α, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-9, IL-10, IL-13, IL-14, IL-16, IL-17, IL-18, IL-22, IL-25, IL-26, MIP-1β, and combinations thereof; the amount of the observed values ​​is normalized to the amount of a control value for each of the one or more analytes; and the increase in the observed values ​​over the control value for each of the one or more analytes is selected from the group consisting of at least 1-fold, at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, and at least 5-fold.

[0486] In any of the foregoing embodiments, tumor digestion can be performed by the methods described herein or by methods known in the art. In any of the foregoing embodiments, tumor digestion is performed according to the methods or using the compositions or devices described in International Patent Publication No. WO2021 / 123832A1 (the disclosure of which is incorporated herein by reference).

[0487] In some embodiments, the present invention provides methods for assaying TIL polyfunctional activity to evaluate the efficacy and / or functionality of expanded TILs, and other polyclonal T cell products, including MILs and PBLs, that can be used in the treatment of cancer by administering TILs, MILs, PBLs, or other polyclonal T cell products being evaluated, the methods comprising: co-culturing the target cells with the aT cell product cells for a first period of time; b. Obtaining a harvest from the co-culture; c. assessing the collection (1) for expression of one or more markers on the T cell product cells, or (2) for two or more analytes secreted from the T cell product cells to obtain one or more observations and determine the potency of the T cell product.

[0488] In some embodiments, the method comprises: an additional step of performing a second co-culture of the negative control cells with the dT cell product cells for a second period of time; e. the additional step of obtaining a second harvest from the second co-culture; f. the additional step of assessing the second harvest for (1) expression of one or more markers on the T cell product cells, or (2) two or more analytes secreted from the T cell product cells to obtain one or more control values; g. the additional step of comparing one or more observed values ​​from step c to one or more control values ​​from step f, where each observed value is compared to a corresponding control value, to determine the potency of the T cell product; the two or more analytes secreted from the TIL product are selected from the group consisting of IFN-α, IFN-β, IFN-γ, granzyme B, perforin, TNF-α, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-9, IL-10, IL-13, IL-14, IL-16, IL-17, IL-18, IL-22, IL-25, IL-26, MIP-1β, and combinations thereof, and the amount of the observed value is normalized to the amount of a control value for each of the two or more analytes, and the increase in the observed value over the control value for each of the two or more analytes is selected from the group consisting of at least 1-fold, at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, and at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, and at least 10-fold.

[0489] In some embodiments, the present invention provides methods for assaying TIL polyfunctional activity to evaluate the efficacy and / or functionality of expanded TILs, and other polyclonal T cell products, including MILs and PBLs, that can be used in the treatment of cancer by administering TILs, MILs, PBLs, or other polyclonal T cell products being evaluated, the methods comprising: co-culturing the target cells with the aT cell product cells for a first period of time; b. Obtaining a harvest from the co-culture; c. assessing the collection (1) for expression of one or more markers on the T cell product cells, or (2) for three or more analytes secreted from the T cell product cells to obtain one or more observations and determine the potency of the T cell product.

[0490] In some embodiments, the method comprises: an additional step of performing a second co-culture of the negative control cells with the dT cell product cells for a second period of time; e. the additional step of obtaining a second harvest from the second co-culture; f. the additional step of assessing the second harvest for (1) expression of one or more markers on the T cell product cells, or (2) three or more analytes secreted from the T cell product cells to obtain one or more control values; g. the additional step of comparing one or more observed values ​​from step c to one or more control values ​​from step f, where each observed value is compared to a corresponding control value, to determine the potency of the T cell product; the three or more analytes secreted from the TIL product are selected from the group consisting of IFN-α, IFN-β, IFN-γ, granzyme B, perforin, TNF-α, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-9, IL-10, IL-13, IL-14, IL-16, IL-17, IL-18, IL-22, IL-25, IL-26, MIP-1β, and combinations thereof, and the amounts of the observed values ​​are normalized to the amount of a control value for each of the three or more analytes, and the increase in the observed values ​​over the control value for each of the three or more analytes is selected from the group consisting of at least 1-fold, at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, and at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, and at least 10-fold.

[0491] In some embodiments, the present invention provides methods for assaying TIL polyfunctional activity to evaluate the efficacy and / or functionality of expanded TILs, and other polyclonal T cell products, including MILs and PBLs, that can be used in the treatment of cancer by administering TILs, MILs, PBLs, or other polyclonal T cell products being evaluated, the methods comprising: a. co-culturing a plurality of target cells with a plurality of T cell product cells for a first period of time; b. Obtaining a harvest from the co-culture; c. assessing the collection (1) for expression of one or more markers on the T cell product cells, or (2) for one or more analytes secreted from the T cell product cells to obtain one or more observations and determine the potency of the T cell product.

[0492] In some embodiments, the method comprises: an additional step of performing a second co-culture of the plurality of negative control cells with the dT cell product cells for a second period of time; e. the additional step of obtaining a second harvest from the second co-culture; f. the additional step of assessing the second harvest for (1) expression of one or more markers on the T cell product cells, or (2) one or more analytes secreted from the T cell product cells to obtain one or more control values; g. the additional step of comparing one or more observed values ​​from step c to one or more control values ​​from step f, where each observed value is compared to a corresponding control value, to determine the potency of the T cell product; the one or more analytes secreted from the TIL product are selected from the group consisting of IFN-α, IFN-β, IFN-γ, granzyme B, perforin, TNF-α, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-9, IL-10, IL-13, IL-14, IL-16, IL-17, IL-18, IL-22, IL-25, IL-26, MIP-1β, and combinations thereof, and the amount of the observed value is normalized to the amount of a control value for each of the one or more analytes, and the increase in the observed value over the control value for each of the one or more analytes is selected from the group consisting of at least 1-fold, at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, and at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, and at least 10-fold.

[0493] In one embodiment, the invention includes the above-described method, wherein the target cells are selected from the group consisting of Raji cells, Thp1 cells, Ramos cells, U937 cells, Daudi cells, and combinations thereof. In one embodiment, the target cells are a combination of two or more cell lines. In one embodiment, the target cells are a 1:1 combination of two or more cell lines, wherein the two or more cell lines are different and each independently selected from the group consisting of Raji cells, Thp1 cells, Ramos cells, U937 cells, and Daudi cells. In one embodiment, the target cells are a 1:1:1 combination of three or more cell lines, wherein the three or more cell lines are different and each independently selected from the group consisting of Raji cells, Thp1 cells, Ramos cells, U937 cells, and Daudi cells. In some embodiments, the target cells are a 1:1:1:1 combination of four or more cell lines, each of which is different and independently selected from the group consisting of Raji cells, Thp1 cells, Ramos cells, U937 cells, and Daudi cells. In some embodiments, the target cells are a 1:1:1:1:1 combination of two or more cell lines, each of which is different and independently selected from the group consisting of Raji cells, Thp1 cells, Ramos cells, U937 cells, and Daudi cells.

[0494] In one embodiment, the target cells are a combination of Raji cells and Thp1 cells, wherein the ratio of Raji cells to Thp1 cells is selected from the group consisting of 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, and 1:5. In one embodiment, the target cells are a combination of Raji cells and Ramos cells, wherein the ratio of Raji cells to Ramos cells is selected from the group consisting of 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, and 1:5. In one embodiment, the target cells are a combination of Raji cells and U937 cells, wherein the ratio of Raji cells to U937 cells is selected from the group consisting of 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, and 1:5. In one embodiment, the target cells are a combination of Raji cells and Daudi cells, and the ratio of Raji cells to Daudi cells is selected from the group consisting of 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, and 1:5.

[0495] In one embodiment, the invention includes the aforementioned method, wherein the co-culture comprises cell culture medium. In one embodiment, the invention includes the aforementioned method, wherein the co-culture comprises CM1 medium. In one embodiment, the invention includes the aforementioned method, wherein the co-culture comprises AIM-V medium (L-glutamine, 50 μM streptomycin sulfate, and 10 μM gentamicin sulfate), also known as AIM-V medium, commercially available from Invitrogen (Carlsbad, CA). In one embodiment, the invention includes the aforementioned method, wherein the co-culture and the second co-culture comprise cell culture medium. In one embodiment, the invention includes the aforementioned method, wherein the co-culture and the second co-culture each comprise CM1 medium. In one embodiment, the invention includes the aforementioned method, wherein the co-culture and the second co-culture each comprise AIM-V medium. In one embodiment, the co-culture comprises IL-2, which is continuously added during the co-culture and maintained at a concentration of 50 IU / mL to 1000 IU / mL during the co-culture. In some embodiments, the co-culture comprises IL-2, which is added continuously during the co-culture and maintained at a concentration of 100 IU / mL to 500 IU / mL during the co-culture. In some embodiments, the co-culture comprises IL-2, which is added continuously during the co-culture and maintained at a concentration of 200 IU / mL to 400 IU / mL during the co-culture. In some embodiments, the co-culture comprises IL-2, which is added continuously during the co-culture and maintained at a concentration selected from the group consisting of about 50 IU / mL, about 100 IU / mL, about 150 IU / mL, about 200 IU / mL, about 250 IU / mL, about 300 IU / mL, about 350 IU / mL, about 400 IU / mL, about 450 IU / mL, and about 500 IU / mL.

[0496] In one embodiment, the invention includes a method for determining the potency of a T cell product, the method comprising: co-culturing the target cells with the aT cell product cells for a first period of time; b. Obtaining a harvest from the co-culture; c. assessing the harvest (1) for expression of one or more markers on the T cell product cells, or (2) for one or more analytes secreted from the T cell product cells to obtain one or more observations and determine the potency of the T cell product; IL-2 is added continuously during the co-culture, and IL-2 is maintained at a concentration in the co-culture selected from the group consisting of about 50 IU / mL, about 100 IU / mL, about 150 IU / mL, about 200 IU / mL, about 250 IU / mL, about 300 IU / mL, about 350 IU / mL, about 400 IU / mL, about 450 IU / mL, and about 500 IU / mL.

[0497] In one embodiment, the invention includes a method for determining the potency of a T cell product, the method comprising: co-culturing the target cells with the aT cell product cells for a first period of time; b. Obtaining a harvest from the co-culture; c. assessing the harvest (1) for expression of one or more markers on the T cell product cells, or (2) for one or more analytes secreted from the T cell product cells, to obtain one or more observations and determine the potency of the T cell product; an additional step of performing a second co-culture of the negative control cells with the dT cell product cells for a second period of time; e. the additional step of obtaining a second harvest from the second co-culture; f. the additional step of assessing the second harvest for (1) expression of one or more markers on the T cell product cells, or (2) one or more analytes secreted from the T cell product cells to obtain one or more control values; g. the additional step of comparing one or more observed values ​​from step c to one or more control values ​​from step f to determine the potency of the T cell product, wherein each observed value is compared to a corresponding control value; IL-2 is continuously added during the co-culture and the second co-culture, and IL-2 is maintained at a concentration in each of the co-culture and the second co-culture selected from the group consisting of about 50 IU / mL, about 100 IU / mL, about 150 IU / mL, about 200 IU / mL, about 250 IU / mL, about 300 IU / mL, about 350 IU / mL, about 400 IU / mL, about 450 IU / mL, and about 500 IU / mL.

[0498] In one embodiment, the invention includes a method for determining the potency of a T cell product, the method comprising: co-culturing the target cells with the aT cell product cells for a first period of time; b. Obtaining a harvest from the co-culture; c. assessing the harvest (1) for expression of one or more markers on the T cell product cells, or (2) for one or more analytes secreted from the T cell product cells to obtain one or more observations and determine the potency of the T cell product; IL-2 is added continuously during the co-culture, and IL-2 is maintained at a concentration in the co-culture of 50 IU / mL to 1000 IU / mL.

[0499] In one embodiment, the invention includes a method for determining the potency of a T cell product, the method comprising: co-culturing the target cells with the aT cell product cells for a first period of time; b. Obtaining a harvest from the co-culture; c. assessing the harvest (1) for expression of one or more markers on the T cell product cells, or (2) for one or more analytes secreted from the T cell product cells, to obtain one or more observations and determine the potency of the T cell product; conducting a second co-culture of the negative control cells with the dT cell product cells for a second period of time; e. the additional step of obtaining a second harvest from the second co-culture; f. the additional step of assessing the second harvest for (1) expression of one or more markers on the T cell product cells, or (2) one or more analytes secreted from the T cell product cells to obtain one or more control values; g. the additional step of comparing one or more observed values ​​from step c to one or more control values ​​from step f, where each observed value is compared to a corresponding control value, to determine the potency of the T cell product; IL-2 is continuously added during the co-culture and the second co-culture, and IL-2 is maintained at a concentration of 50 IU / mL to 1000 IU / mL in each of the co-culture and the second co-culture.

[0500] In some embodiments, the present invention provides methods for assaying TIL, MIL, or PBL activity, where a killing assay is used. In some embodiments, the present invention provides methods for assaying TIL, MIL, or PBL activity, where a killing assay is used and a cytolytic endpoint is detected. In some embodiments, the present invention provides methods for assaying TIL, MIL, or PBL activity, where a killing assay is used. In some embodiments, the present invention provides methods for assaying TIL, MIL, or PBL activity, where a killing assay is used and a cytolytic endpoint is detected by genetic modification of the target cell line to express a fluorescent, chemiluminescent, or bioluminescent protein when lysed. Suitable genetic modifications and assays include luciferase assays, such as those described in U.S. Patent No. 9,631,225, the disclosure of which is incorporated herein by reference. For example, the genetic modification approach described for bioluminescence detection in U.S. Pat. No. 10,415,015 (the disclosure of which is incorporated herein by reference) can be used to modify target cells such as Raji cells, K562 cells, Daudi cells, Ramos cells, U937 cells, or Thp1 cells.

[0501] In certain embodiments, the invention provides methods for assaying the efficacy of a T cell product, further comprising pre-irradiating target cells to arrest proliferation. In certain embodiments, the invention provides methods for assaying the efficacy of a T cell product, further comprising using target cells that have not been irradiated to arrest proliferation. In certain embodiments, the invention provides methods for assaying the efficacy of a T cell product, further comprising chemically or biologically treating target cells to arrest proliferation.

[0502] In certain embodiments, the invention provides methods for assaying the efficacy of a T cell product, further comprising pre-irradiating negative control cells to arrest proliferation. In certain embodiments, the invention provides methods for assaying the efficacy of a T cell product, further comprising using negative control cells that are not irradiated to arrest proliferation. In certain embodiments, the invention provides methods for assaying the efficacy of a T cell product, further comprising chemically or biologically treating the negative control cells to arrest proliferation.

[0503] In certain embodiments, the target cells are irradiated using X-rays or gamma rays with a total absorbed dose of about 5 Gy, 10 Gy, 15 Gy, 20 Gy, 25 Gy, 30 Gy, 35 Gy, 40 Gy, 45 Gy, 50 Gy, 55 Gy, 60 Gy, 65 Gy, 70 Gy, 75 Gy, 80 Gy, 85 Gy, 90 Gy, 95 Gy, 100 Gy, 105 Gy, 110 Gy, 115 Gy, 120 Gy, 125 Gy, 130 Gy, 135 Gy, 140 Gy, 145 Gy, 150 Gy, 155 Gy, 160 Gy, 165 Gy, 170 Gy, 175 Gy, 180 Gy, 185 Gy, 190 Gy, or 200 Gy.

[0504] In some embodiments, the target cells are irradiated with about 100 rads, 200 rads, 300 rads, 400 rads, 500 rads, 600 rads, 700 rads, 800 rads, 900 rads, 1000 rads, 1100 rads, 1200 rads, 1300 rads, 1400 rads, 1500 rads, 1600 rads, 1700 rads, 1800 rads, or 1900 rads using X-rays or gamma rays. ads, 1900rads, 2000rads, 2100rads, 2200rads, 2300rads, 2400rads, 2500rads, 2600rads, 2700rads, 2800ra ds, 2900rads, 3000rads, 3100rads, 3200rads, 3300rads, 3400rads, 3500rads, 3600rads, 3700rads, 3800rad s, 3900rads, 4000rads, 4100rads, 4200rads, 4300rads, 4400rads, 4500rads, 4600rads, 4700rads, 4800rads , 4900rads, 5000rads, 5100rads, 5200rads, 5300rads, 5400rads, 5500rads, 5600rads, 5700rads, 5800rads, A total absorbed dose of radiation of 5900 rads, 6000 rads, 6100 rads, 6200 rads, 6300 rads, 6400 rads, 6500 rads, 6600 rads, 6700 rads, 6800 rads, 6900 rads, 7000 rads, 7500 rads, 8000 rads, 8500 rads, 9000 rads, 9500 rads, or 10000 rads is administered.

[0505] In certain embodiments, target cells are irradiated using X-rays or gamma rays at a rate of about 20 rads / min, 40 rads / min, 60 rads / min, 80 rads / min, 100 rads / min, 120 rads / min, 140 rads / min, 160 rads / min, 180 rads / min, 200 rads / min, 220 rads / min, 240 rads / min, 260 rads / min, 280 rads / min, 300 rads / min, 320 rads / min, 340 rads / min, 360 rads / min, 380 rads / min, 400 rads / min, 420 rads / min, 440 rads / min, 460 rads / min, 480 rads / min, 490 rads / min, 500 rads / min, 510 rads / min, 520 rads / min, 530 rads / min, 540 rads / min, 550 rads / min, 560 rads / min, ...

Claims

1. A potency and identity assay matrix for determining the potency and / or functionality of a tumor infiltrating lymphocyte (TIL) product, the potency and identity assay matrix comprising at least one potency assay and at least one identity assay; the at least one potency assay or the at least one identity assay is selected from the group consisting of a co-culture assay, a bead-based assay, a plate-based assay, and a flow cytometry assay; The potency and identity assay matrices include a co-culture assay for IFN-γ release, a bead-based assay for IFN-γ release, a flow cytometry assay for LAG3 + cell content, a flow cytometry assay for KLRG1 + cell content, a flow cytometry assay for combined CD4 + and CD8 + cell content, a flow cytometry assay for total viable T CM and T EM cell populations, and a flow cytometry assay for CD45 + / CD3 + cell content. Assay matrix.

2. The co-culture assay includes co-culturing TIL cells from the TIL product with target cells and detecting IFN-γ released by the TIL cells; 2. The assay matrix of claim 1, comprising:

3. A bead-based or plate-based assay comprising stimulating TIL cells from a TIL preparation with anti-CD3, anti-CD28, and / or anti-CD137 antibodies and detecting IFN-γ released from the TIL cells; 2. The assay matrix of claim 1, comprising:

4. The co-culture assay, a) co-culturing target cells with TIL cells; b) extracting the supernatant from the co-culture; c) assessing the supernatant for IFN-γ; The assay matrix of claim 2 , comprising:

5. The assay matrix described in claim 2, wherein the target cells are monocyte cells.

6. The assay matrix of claim 5, wherein the monocytic cells are U937 cells or derivatives, variants, modifications, or progeny thereof.

7. The assay matrix of claim 4, wherein the co-culture is performed for a period selected from the group consisting of about 6 hours, about 12 hours, about 18 hours, about 24 hours, about 30 hours, about 36 hours, about 42 hours, and about 48 hours.

8. The assay matrix of claim 4, wherein the ratio of TIL cells:target cells is about 15:4, about 15:2, about 15:1, and / or about 30:

1.

9. The assay matrix of claim 4, wherein the co-culture is performed in a cell culture medium containing IL-2.

10. The assay matrix of claim 9, wherein the cell culture medium contains IL-2 at a concentration of about 300 IU / mL.

11. The assay matrix of claim 4, wherein an HLA blocking antibody is added during co-culture as a negative control.

12. The assay matrix of claim 11, wherein the HLA blocking antibody is used at a concentration of about 1 to 20 μg / mL.

13. The TIL product, (a) performing a first expansion by culturing the first TIL population in a cell culture medium containing IL-2 to produce a second TIL population, wherein the first expansion is performed for about 3-14 days to obtain the second TIL population; (b) performing a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, and antigen presenting cells (APCs) to produce a third TIL population, wherein the second expansion is performed for about 7-14 days to obtain a third TIL population, the third TIL population being a therapeutic TIL population; (c) harvesting the therapeutic TIL population obtained from step (b); (d) transferring the therapeutic TIL population of step (c) into an infusion bag; and (e) storing the infusion bag frozen; 2. The assay matrix of claim 1 produced by a method comprising:

14. The assay matrix of claim 13, wherein the potency and identity assay is performed after frozen storage.

15. The assay matrix of claim 1, wherein the TIL product comprises CD8+ TILs and CD4+ TILs.

16. The assay matrix of claim 4, wherein anti-CD3 antibody is not added to the co-culture during the co-culture assay.

17. A co-culture assay for IFN-γ release comprising: a) performing at least three different co-cultures of target cells with TIL cells at different target cell concentrations; b) extracting the supernatant from each of the co-cultures; and c) assessing the supernatants from each of the co-cultures for IFN-γ secreted by the TIL cells to determine the potency of the TIL product; The assay matrix of claim 2 , comprising: