Methods and compositions for T-cell co-culture efficacy assays, and their 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 large, refractory cancers using adoptive autotransfer of tumor-infiltrating lymphocytes (TILs) presents a powerful approach to treating patients with poor prognoses. Gattinoni, et al., Nat. Rev. Immunol. 2006, 6, 383-393. IL-2-based TIL dilation followed by a “rapid dilation process” (REP) has become the preferred method of TIL dilation 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 improve the response to TIL therapy in melanoma and extend TIL therapy to other tumor types have achieved limited success, and this area 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. While combination studies with single immune checkpoint inhibitors are described, further research is ongoing and additional treatment options are needed (Kverneland, et al., Oncotarget, 2020, 11(22), 2092-2105).

[0002] Furthermore, current TIL manufacturing and treatment processes are limited by length, cost, sterility concerns, and other factors described herein, severely limiting the possibility of treating patients who are refractory to other checkpoint inhibitor therapies. There is an urgent need to provide a quality control process for such process-based therapies suitable for use in treating patients for whom few or no viable treatment options remain. The present invention satisfies this need by providing a shortened manufacturing process for use in generating TILs, with an additional step that provides a mechanism for evaluating the potency and / or functionality of extended TILs.

[0003] The present invention provides improved processes, methods, and compositions for preparing and evaluating the potency and / or functionality of T cells containing TILs, for example, to prepare a therapeutic TIL population having increased therapeutic efficacy 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, in addition, be used with the administration of TILs in combination with CTLA-4 and PD-1 inhibitors and / or PD-L1 inhibitors as described herein. [Overview of the project]

[0004] Methods for evaluating the efficacy and / or functionality of extended TILs, as well as other polyclonal T cell products including bone marrow-infiltrating lymphocytes (MILs) and peripheral blood lymphocytes (PBLs), which can be used in the treatment of cancer by administering TILs, MILs, PBLs, or other polyclonal T cell products evaluated by these methods.

[0005] In one embodiment, a method for determining the efficacy of a T cell product, The first step involves co-culturing target cells with aT cell product cells, b. Steps to obtain samples from co-culture, c. A method is provided which includes the steps of (1) evaluating the expression of one or more markers on T cell product cells, or (2) evaluating one or more analytes secreted from T cell product cells to obtain one or more observations and determine the potency of the T cell product.

[0006] In some embodiments, this method is An additional step is to perform a second co-culture of (i) cells or (ii) a negative control containing human leukocyte antigen (HLA) blocking antibodies and target cells with dT cell product cells for a second period, e. An additional step of obtaining a second sample from the second co-culture, f. The second sample is subjected to an additional step of obtaining one or more control values ​​by (1) evaluating the expression of one or more markers on T cell product cells, or (2) evaluating one or more analytes secreted from T cell product cells. g. An additional step of determining the potency of a T cell product, wherein each observation from step c is compared to one or more control values ​​from step f, the additional step of determining the potency of a T cell product, wherein each observation is compared to a corresponding control value.

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

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

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

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

[0011] In some embodiments, 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 their derivatives, variants, modifications, or offspring.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0028] In some embodiments, co-culturing is performed after thawing the cryopreserved TIL product, MIL product, or PBL product and allowing the thawed TIL product, MIL product, 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 is approximately 6 to 48 hours.

[0030] In some embodiments, the second period is approximately 6 to 48 hours.

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

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

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

[0034] In some embodiments, 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, 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, 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 value is normalized for each of the one or more analytes relative to the amount of the control value, and the increase in the observed value above the control value for each of the one or more analytes is selected from the group consisting of at least 1x, at least 1.5x, at least 2x, at least 2.5x, at least 3x, at least 3.5x, at least 4x, at least 4.5x, and at least 5x.

[0037] In some embodiments, the TIL product is manufactured from tumors obtained by surgical excision, needle biopsy, core biopsy, microbiopsy, 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 relates to a method for treating a population of tumor-infiltrating lymphocytes (TILs) in a patient requiring treatment for cancer, (a) obtaining and / or receiving a first TIL population from a tumor resected from a patient by surgical resection, needle biopsy, core biopsy, microbiopsy, or other means by processing a tumor sample obtained from the patient into (i) multiple tumor fragments or (ii) tumor digests, (b) The step of adding the first TIL group to the closed system, (c) A step of producing a second TIL population by performing a first expansion by culturing a first TIL population in a cell culture medium containing IL-2, wherein the first expansion is performed in a sealed container providing a first gas-permeable surface area, the first expansion is performed for approximately 3 to 14 days to obtain a second TIL population, and the transition from step (b) to step (c) occurs without opening the system, (d) A step of producing a third TIL population by 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), wherein the second expansion is carried out for approximately 7 to 14 days to obtain the third TIL population, the third TIL population being a therapeutic TIL population, the second expansion being carried out in a sealed container providing a second gas-permeable surface area, and the transition from step (c) to step (d) occurring without opening the system, (e) A step of collecting the therapeutic TIL population obtained from step (d), wherein the transition from step (d) to step (e) occurs without opening the system, (f) A step in determining the efficacy of the therapeutic TIL population, i. Co-culture of target cells with a portion of the therapeutic TIL population for the first period, ii. Obtaining samples from co-culture, iii. The efficacy of the therapeutic TIL population is determined by (1) evaluating the expression of one or more markers on a portion of the therapeutic TIL population, or (2) evaluating one or more analytes secreted from a portion of the therapeutic TIL population, thereby obtaining one or more observational values. iv. A second co-culture of negative control cells or (i) negative control cells or (ii) human leukocyte antigen blocking antibodies with a second portion of the therapeutic TIL population for a second period. v. Obtaining a second sample from the second co-culture, vi. The second sample is evaluated by (1) the expression of one or more markers on the second portion of the therapeutic TIL population, or (2) the evaluation of 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 a therapeutic TIL population, which includes determining the efficacy of a therapeutic TIL population by comparing one or more observations with one or more control values, wherein each observation is compared with its corresponding control value. (g) A step of transferring the therapeutic TIL population from step (e) to an infusion bag, wherein the transition from step (e) to (g) occurs without opening the system, (h) Optionally, cryopreserve the infusion bag containing the therapeutic TIL population from step (g) using a cryopreservation process. The present invention provides a method comprising (i) if it is determined that the therapeutic TIL population is 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, the potency and / or functionality of the collected TIL is examined 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, PD-1 inhibitor, or PD-L1 inhibitor, and optionally, the patient has been previously treated with a CTLA-4 inhibitor, PD-1 inhibitor, or PD-L1 inhibitor.

[0041] In some embodiments, the second TIL group in step (c) is at least 50 times larger in number than the first TIL group.

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

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

[0047] In some embodiments, during 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 approximately 30 ng / mL.

[0048] In some embodiments, the method further includes the step of treating the patient with a non-myeloablative lymphocyte depletion regimen before administering TIL to the patient.

[0049] In some embodiments, a non-myeloablative lymphocyte depletion regimen involves cyclophosphamide at 60 mg / m². 2 Administer at a daily dose for 2 days, followed by fludarabine at 25 mg / m².2 The step includes administering the drug at a daily dose for 5 days.

[0050] In some embodiments, a non-myeloablative lymphocyte depletion regimen involves cyclophosphamide at 60 mg / m². 2 The daily dose is 25 mg / m² of fludarabine. 2 Administer at a daily dose for 2 days, followed by fludarabine at 25 mg / m². 2 The step includes administering the drug at a daily dose for 3 days.

[0051] In some embodiments, the method further includes the step of treating the patient with an IL-2 regimen, which is initiated the day after the patient is administered a therapeutic TIL population.

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

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

[0054] In some embodiments, the IL-2 regimen is a high-dose IL-2 regimen containing 600,000 or 720,000 IU / kg of aldesleukin, or its biosimilar or variant, administered as a 15-minute bolus intravenous infusion every 8 hours until an acceptable level is reached.

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

[0056] In some embodiments, processing the tumor sample obtained from the patient in step (a) into tumor digests further includes mechanically destroying the tumor sample to dissociate it.

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

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

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

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

[0061] In some embodiments, the enzyme medium contains approximately 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 includes the step of administering a PD-1 inhibitor, a PD-L1 inhibitor, or a CTLA-4 inhibitor to a patient.

[0064] In another embodiment, a method for determining the efficacy of a T cell product, The steps include co-culturing at least three target cells with aT cell product cells at different target cell concentrations, The steps include co-culturing at least three target cells with bT cell reference standard cells at different target cell concentrations, c. The step of extracting the supernatant from each of the co-cultures, d. The step of evaluating the supernatant for cytokines secreted from T cell product cells and T cell reference standard cells to obtain a dose concentration for determining the potency of the T cell product, This method provides a method in which the target cell is a monocyte.

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

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

[0067] In some embodiments, 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.

[0068] In some embodiments, the T cell product is a TIL product, and about 4×10 5 , about 2×10 5 , about 1×10 5 , and about 0.5×10 5 of four target cell dose concentrations of target cells, and a single TIL cell concentration of about 1.5×10 6 of TIL per well are 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, 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 is a bead or plate-based assay using CD3, CD28, and / or CD137 stimulation and reporting interferon-γ, granzyme B, or tumor necrosis factor-α, an assay for total viable cells, an assay for viable cell percentage, an assay for CD4 + cell content, an assay for CD8 +Assay on cell content, T EM Assay on cell content, T CM Assay on cell content, LAG3 + Assay on cell content, and KLRG1 + Assay on cell content, CD101 + Assay on cell content, CD69 + Assay on cell content, T SCM Assay on cell content, T EMRA Assay on cell content, T reg Assay on cell content, PD-1 + Assay on cell content, TIM3 + Assay on cell content, CD25 + Assay on cell content, CD27 + Assay on cell content, CD28 + Assay on cell content, CD56 + Assay on cell content, CTLA-4 + Cell content assay, TIGIT + Assay on cell content, and CD57 + It includes one or more assays selected from the group consisting of assays for cell content.

[0072] In another aspect, the present invention relates to a method for treating a subject having cancer, (a) Adding tumor digests or tumor fragments to a closed system, wherein the tumor digests or tumor fragments include a first TIL population and are obtained from tumors resected from the subject. (b) Performing a first expansion by culturing a 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 providing a first gas-permeable surface area, the first expansion is performed for approximately 3 to 11 days to obtain a second TIL population, and the transition from step (b) to step (c) occurs without opening the system to produce a second TIL population. (c) Performing a second expansion by supplementing 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 approximately 7–11 days to obtain the third TIL population, and the second expansion is performed in a sealed container providing a second gas-permeable surface area, and the transition from step (b) to step (c) occurs without opening the system, thereby producing a third TIL population. (d) Collecting a third TIL population obtained from step (c), wherein the transition from step (c) to step (d) occurs without opening the system. (e) Transferring the third TIL population collected from step (d) to an injection bag, wherein the transition from step (d) to (e) occurs without opening the system. (f) Using a cryopreservation process, the infusion bag containing the TIL population taken from step (e) is cryopreserved, (g) administering expanded tumor-infiltrating lymphocytes (TILs), including administering a therapeutically effective dose of a third TIL population to the subject from the infusion bag in step (f), The present invention provides a method for selecting an APC from the group consisting of Raji, Ramos, Daudi, U937, or Thp1 cells, or their derivatives, variants, modifications, or offspring.

[0073] In some embodiments, 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 carried out separately within a period of 11 days.

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

[0076] In some embodiments, the effect of TIL is determined using the method described herein.

[0077] In some embodiments, the effect of TIL is determined using the method described herein. [Brief explanation of the drawing]

[0078] [Figure 1] An example Gen2 (Process 2A) chart providing an overview of steps A through F. [Figure 2A] Process flowchart of a Gen2 (Process 2A) embodiment for TIL manufacturing. [Figure 2B] Process flowchart of a Gen2 (Process 2A) embodiment for TIL manufacturing. [Figure 2C] Process flowchart of a Gen2 (Process 2A) embodiment for TIL manufacturing. [Figure 3] A diagram illustrating an exemplary manufacturing process (approximately 22 days) for cryopreserved TIL is shown. [Figure 4] This diagram shows an embodiment of Gen2 (Process 2A), a 22-day process for TIL manufacturing. [Figure 5] A comparison table of steps A to F from exemplary embodiments of Process 1C and Gen2 (Process 2A) for TIL manufacturing. [Figure 6] A detailed comparison between the Process 1C embodiment and the Gen2 (Process 2A) embodiment for TIL manufacturing. [Figure 7] An exemplary Gen3 TIL manufacturing process. [Figure 8A] This document compares embodiments of the 2A process (approximately a 22-day process) and the Gen3 process (approximately a 14-16 day process) for TIL manufacturing. [Figure 8B]An exemplary Gen3 process chart (approximately a 14-16 day process) providing an overview of steps A-F. [Figure 8C] A chart providing three exemplary Gen3 processes for each of the three process variations, along with an overview of steps A through F (approximately a 14-16 day process). [Figure 8D] An example of a modified Gen2-like process (approximately a 22-day process) providing an overview of steps A-F. [Figure 9] This document provides an experimental flowchart for comparing the Gen2 (Process 2A) and Gen3 processes. [Figure 10] This section shows a comparison of various Gen2 (Process 2A) and Gen3.1 process embodiments. [Figure 11] A table illustrating the various characteristics of the Gen2, Gen2.1, and Gen3.0 process embodiments. [Figure 12] An overview of the culture medium conditions for an embodiment of the Gen3 process, referred to as Gen3.1. [Figure 13] A table illustrating the various characteristics of the Gen2, Gen2.1, and Gen3.0 process embodiments. [Figure 14] A table comparing various features of the Gen2 and Gen3.0 process embodiments. [Figure 15] A table providing the use of culture media in various embodiments of the described expansion process. [Figure 16] A schematic diagram of an exemplary embodiment of the Gen3 process (16-day process). [Figure 17] A schematic diagram 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 provide an agonist that comprises, for example, three linearly bound TNFRSF-binding domains derived from an antibody bound to 4-1BBL or 4-1BB, which are folded to form a trivalent protein, which is then bound to a second trivalent protein by IgG1-Fc (containing CH3 and CH2 domains), and which is then used to bind two of the trivalent proteins together by a disulfide bond (small oval), thereby stabilizing the structure and bringing together the intracellular signaling domains of six receptor and signaling proteins to form a signaling complex. The TNFRSF-binding domain, shown as a cylinder, may be an scFv domain containing VH and VL chains bound by a linker which may contain, for example, Gly and Ser sequences for hydrophilic residues and flexibility, as well as Glu and Lys for solubility. [Figure 19] A schematic diagram of an exemplary embodiment of the Gen3 process (16-day process). [Figure 20] This document provides a process overview of an exemplary embodiment of the Gen3.1 process (16-day process). [Figure 21] A schematic diagram of an exemplary embodiment of the Gen3.1 test process (16-17 day process). [Figure 22] A schematic diagram of an exemplary embodiment of the Gen3 process (16-day process). [Figure 23] A comparison table of exemplary Gen2 and exemplary Gen3 processes. [Figure 24] A schematic diagram of an exemplary embodiment of the preparation timeline for the Gen3 process (16-17 day process). [Figure 25] A schematic diagram of an exemplary embodiment of the Gen3 process (14-16 day process). [Figure 26A] A schematic diagram of an exemplary embodiment of the Gen3 process (16-day process). [Figure 26B] A schematic diagram of an exemplary embodiment of the Gen3 process (16-day process). [Figure 27]A schematic diagram of an exemplary embodiment of the Gen3 process (16-day process). [Figure 28] Comparison of the Gen2, Gen2.1, and Gen3 process (16-day process) embodiments. [Figure 29] Comparison of the Gen2, Gen2.1, and Gen3 process (16-day process) embodiments. [Figure 30] Components of the 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] Figure illustrating an embodiment of the TIL-Raji co-culture assay using a TIL-K562 negative control and positive controls using MLR (Patient-Specific Personalized Microconcentrates) and bead-based controls. [Figure 35] CD25 levels were observed by flow cytometry (%) of CD3 for lung tumors (L4224) and melanoma tumors (M1152) with different co-culture periods and TIL:target ratios using the TIL-Raji co-culture assay. [Figure 36] CD69 levels were observed by flow cytometry (%) of CD3 for lung tumors (L4224) and melanoma tumors (M1152) with different co-culture periods and TIL:target ratios using the TIL-Raji co-culture assay. [Figure 37] CD137 (4-1BB) levels were observed by flow cytometry (%) of CD3 for lung tumors (L4224) and melanoma tumors (M1152) with different co-culture periods and TIL:target ratios using the TIL-Raji co-culture assay. [Figure 38] CD134 (OX40) levels were observed by flow cytometry (%) of CD3 for lung tumors (L4224) and melanoma tumors (M1152) with different co-culture periods and TIL:target ratios using the TIL-Raji co-culture assay. [Figure 39] Using the TIL-Raji co-culture assay, IFN-γ secretion was analyzed for lung tumors (L4224) and melanoma tumors (M1152) at different co-culture periods and TIL:target ratios. [Figure 40] Using the TIL-Raji co-culture assay, CCL4 secretion was analyzed for different co-culture periods and TIL:target ratios in lung tumors (L4224) and melanoma tumors (M1152). [Figure 41] The TIL-Raji co-culture assay was used to study granzyme B secretion in lung tumors (L4224) and melanoma tumors (M1152) at different co-culture periods and TIL:target ratios. [Figure 42] A diagram illustrating an embodiment of the TIL-Raji co-culture assay using a TIL-K562 negative control. [Figure 43] Co-culture experimental setup for an exemplary embodiment of a TIL-Raji cell-based efficacy assay. [Figure 44] Phenotypic expression of activation markers in the TIL-Raji co-culture assay. [Figure 45] Phenotypic expression of CD69 (Gen2 TIL:301-001) in TIL-Raji co-culture. [Figure 46] IFN-γ secretion level (pg / mL). [Figure 47] Granzyme B secretion level (pg / mL). [Figure 48] IFN-γ and granzyme B secretion levels. [Figure 49] IFN-γ release ratio change: TIL+ cell line / TIL alone. [Figure 50] Granzyme magnification change: TIL + cell line / TIL alone. [Figure 51] Cytokine release ratio change: TIL+ cell line / TIL alone. [Figure 52] IFN-γ release ratio change: TIL+ cell line / TIL+K562. [Figure 53] Changes in the ratio of granzyme B release: TIL+ cell line / TIL+K562. [Figure 54]Cytokine release ratio change: TIL+ cell line / TIL+K562. [Figure 55] Summary table of IFN-γ and granzyme B secretion. [Figure 56] A schematic diagram illustrating an embodiment of a TIL-Raji cell-based efficacy assay demonstrating TIL activation mediated by MHC dominance recognition. [Figure 57] Co-culture experimental plate setup for an exemplary embodiment of a TIL-Raji cell-based efficacy assay. [Figure 58] A diagram illustrating an embodiment of the TIL-Raji co-culture assay using an optional TIL-K562 negative control. [Figure 59] IFN-γ secretion (pg / mL) from melanoma TIL lots with 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 the TIL:target ratio is shown on the x-axis. [Figure 60] IFN-γ secretion (pg / mL) from melanoma TIL lots with 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 the TIL:target ratio is shown on the x-axis. [Figure 61] IFN-γ secretion (pg / mL) from melanoma TIL lots with 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 the TIL:target ratio is shown on the x-axis. [Figure 62] Calculation changes in IFN-γ release from TIL+Raji or K562 cells compared to TIL alone, for melanoma TIL lots with TIL:Raji or TIL:K562 ratios of 3:1, 1:1, and 1:3. Calculation changes are shown on the y-axis, and the TIL:target ratio is shown on the x-axis. [Figure 63] Calculation changes in IFN-γ release from TIL+Raji or K562 cells compared to TIL alone, for melanoma TIL lots with TIL:Raji or TIL:K562 ratios of 3:1, 1:1, and 1:3. Calculation changes are shown on the y-axis, and the TIL:target ratio is shown on the x-axis. [Figure 64] Calculation changes in IFN-γ release from TIL+Raji or K562 cells compared to TIL alone, for melanoma TIL lots with TIL:Raji or TIL:K562 ratios of 3:1, 1:1, and 1:3. Calculation changes are shown on the y-axis, and the TIL:target ratio is shown on the x-axis. [Figure 65] Calculation changes in IFN-γ release from TIL+Raji or K562 cells compared to IFN-γ release from TIL+K562 cells in melanoma TIL lots with TIL:Raji or TIL:K562 ratios of 3:1, 1:1, and 1:3. Calculation changes are shown on the y-axis, and the TIL:target ratio is shown on the x-axis. [Figure 66] Calculation changes in IFN-γ release from TIL+Raji or K562 cells compared to IFN-γ release from TIL+K562 cells in melanoma TIL lots with TIL:Raji or TIL:K562 ratios of 3:1, 1:1, and 1:3. Calculation changes are shown on the y-axis, and the TIL:target ratio is shown on the x-axis. [Figure 67] Calculation changes in IFN-γ release from TIL+Raji or K562 cells compared to IFN-γ release from TIL+K562 cells in melanoma TIL lots with TIL:Raji or TIL:K562 ratios of 3:1, 1:1, and 1:3. Calculation changes are shown on the y-axis, and the TIL:target ratio is shown on the x-axis. [Figure 68] The fold change in IFN-γ release for TIL+Raji or K562 cells compared to TIL alone during incubation times of 12 and 18 hours, with cumulative datasets shown for a 1:3 TIL:Raji or TIL:K562 cell ratio. * indicates a p-value of ≤0.05, ** indicates a p-value of ≤0.01, and *** indicates a p-value of ≤0.001. [Figure 69]The graphs show the multiplier changes in IFN-γ release for TIL + Raji or K562 cells compared to TIL alone or K562 cells over 12 and 18-hour incubation periods, with cumulative datasets for a 1:3 TIL:Raji or TIL:K562 cell ratio. * indicates a p-value of ≤0.05, ** indicates a p-value of ≤0.01, and *** indicates a p-value of ≤0.001. [Figure 70] The data shows the multiplicative change in IFN-γ release for TIL+K562 cells or TIL+Raji cells relative to TIL over incubation times of 12 and 18 hours, with cumulative datasets for a 1:3 TIL:Raji or TIL:K562 cell ratio. * indicates a p-value of ≤0.05, and NS indicates not statistically significant at full scale. [Figure 71] Changes in IFN-γ release for TIL+K562 cells or TIL+Raji cells compared to TIL during 12 and 18-hour incubation periods. Cumulative data are shown for a 1:3 TIL:Raji or TIL:K562 cell ratio and have been expanded to show details at lower magnification levels of change. [Figure 72] Granzyme B secretion (pg / mL) from melanoma TIL lots with 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 the TIL:target ratio is shown on the x-axis. [Figure 73] Granzyme B secretion (pg / mL) from melanoma TIL lots with 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 the TIL:target ratio is shown on the x-axis. [Figure 74] Granzyme B secretion (pg / mL) from melanoma TIL lots with 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 the TIL:target ratio is shown on the x-axis. [Figure 75]Magnification changes in granzyme B release from melanoma TIL lots with TIL:Raji or TIL:K562 ratios of 3:1, 1:1, and 1:3 compared to TIL alone, compared to TIL + Raji or K562 cells. Magnification changes are shown on the y-axis, and the TIL:target ratio is shown on the x-axis. [Figure 76] Diagrams showing the granzyme B release from melanoma TIL lots with TIL:Raji or TIL:K562 ratios of 3:1, 1:1, and 1:3, compared to granzyme B release from TIL alone, for TIL + Raji or K562 cells. The magnification change is shown on the y-axis, and the TIL:target ratio is shown on the x-axis. [Figure 77] Magnification changes in granzyme B release from melanoma TIL lots with TIL:Raji or TIL:K562 ratios of 3:1, 1:1, and 1:3 compared to TIL alone, compared to TIL + Raji or K562 cells. Magnification changes are shown on the y-axis, and the TIL:target ratio is shown on the x-axis. [Figure 78] Magnification changes in granzyme B release from TIL+Raji or K562 cells compared to TIL+K562 cells in melanoma TIL lots with TIL:Raji or TIL:K562 ratios of 3:1, 1:1, and 1:3. Magnification changes are shown on the y-axis, and the TIL:target ratio is shown on the x-axis. [Figure 79] Magnification changes in granzyme B release from TIL+Raji or K562 cells compared to TIL+K562 cells in melanoma TIL lots with TIL:Raji or TIL:K562 ratios of 3:1, 1:1, and 1:3. Magnification changes are shown on the y-axis, and the TIL:target ratio is shown on the x-axis. [Figure 80] Magnification changes in granzyme B release from TIL+Raji or K562 cells compared to TIL+K562 cells in melanoma TIL lots with TIL:Raji or TIL:K562 ratios of 3:1, 1:1, and 1:3. Magnification changes are shown on the y-axis, and the TIL:target ratio is shown on the x-axis. [Figure 81]Granzyme B release of TILs (pg / mL / TIL) during 12 and 18-hour incubation periods. [Figure 82] Granzyme B release for TIL+Raji or K562 cells compared to TIL alone during incubation periods of 12 and 18 hours. Cumulative datasets are shown for TIL:Raji or TIL:K562 cell ratios of 1:3, where *** indicates a p-value of ≤0.001 and **** indicates a p-value of ≤0.0001. [Figure 83] The data shows the release of granzyme B from TIL+Raji or K562 cells at incubation times of 12 and 18 hours, with cumulative datasets for a 1:3 TIL:Raji or TIL:K562 cell ratio, where **** indicates a p-value less than 0.0001. [Figure 84] The reticular change in granzyme B release for TIL+K562 cells or TIL+Raji cells relative to TIL during incubation times of 12 and 18 hours, with cumulative datasets shown for a 1:3 TIL:Raji or TIL:K562 cell ratio. ** indicates a p-value of 0.01 or less, and NS indicates not significant. [Figure 85] TNF-α secretion (pg / mL) from melanoma TIL lots with 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 the TIL:target ratio is shown on the x-axis. [Figure 86] TNF-α secretion (pg / mL) from melanoma TIL lots with 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 the TIL:target ratio is shown on the x-axis. [Figure 87] TNF-α secretion (pg / mL) from melanoma TIL lots with 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 the TIL:target ratio is shown on the x-axis. [Figure 88]The magnitude changes in TNF-α release from melanoma TIL lots with TIL:Raji or TIL:K562 ratios of 3:1, 1:1, and 1:3 compared to granzyme B release from TIL alone, compared to TIL + Raji or K562 cells. Magnitude changes are shown on the y-axis, and the TIL:target ratio is shown on the x-axis. [Figure 89] The magnitude changes in TNF-α release from melanoma TIL lots with TIL:Raji or TIL:K562 ratios of 3:1, 1:1, and 1:3 compared to granzyme B release from TIL alone, compared to TIL + Raji or K562 cells. Magnitude changes are shown on the y-axis, and the TIL:target ratio is shown on the x-axis. [Figure 90] Factor-to-factor ratios of TIL:Raji or TIL:K562 cells to granzyme B release from TIL alone, for melanoma TIL lots with TIL:Raji or TIL:K562 ratios of 3:1, 1:1, and 1:3. Factor-to-factor ratios are shown on the y-axis, and the TIL:target ratio is shown on the x-axis. [Figure 91] The magnitude changes in TNF-α release from TIL+Raji or K562 cells compared to granzyme B release from TIL+K562 cells in melanoma TIL lots with TIL:Raji or TIL:K562 ratios of 3:1, 1:1, and 1:3. Magnitude changes are shown on the y-axis, and the TIL:target ratio is shown on the x-axis. [Figure 92] The magnitude changes in TNF-α release from TIL+Raji or K562 cells compared to granzyme B release from TIL+K562 cells in melanoma TIL lots with TIL:Raji or TIL:K562 ratios of 3:1, 1:1, and 1:3. Magnitude changes are shown on the y-axis, and the TIL:target ratio is shown on the x-axis. [Figure 93] The magnitude changes in TNF-α release from TIL+Raji or K562 cells compared to granzyme B release from TIL+K562 cells in melanoma TIL lots with TIL:Raji or TIL:K562 ratios of 3:1, 1:1, and 1:3. Magnitude changes are shown on the y-axis, and the TIL:target ratio is shown on the x-axis. [Figure 94]The metric change in TNF-α for TIL+Raji or K562 cells compared to TIL alone during an 18-hour incubation period is shown, with cumulative datasets for a 1:3 TIL:Raji or TIL:K562 cell ratio, where **** indicates a p-value less than 0.0001. [Figure 95] This is a bioluster of TIL+Raji or TNF-α release for K562 cells over an 18-hour incubation period, showing cumulative datasets for 1:3 TIL:Raji or TIL:K562 cell ratios, where **** indicates a p-value less than 0.0001. [Figure 96] The metric change in TNF-α release for TIL+Raji cells compared to TIL+K562 cells over an 18-hour incubation period is shown, with cumulative datasets for a 1:3 TIL:Raji or TIL:K562 cell ratio. **** indicates a p-value less than 0.0001. [Figure 97] 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] A diagram illustrating an embodiment of the TIL-Raji co-culture assay using 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] IFN-γ cytokine secretion results showing the fold change in TIL+K562 or Raji cells compared to TIL alone at different post-thaw recovery times. [Figure 105]IFN-γ cytokine secretion results showing the fold change in TIL+K562 or Raji cells compared to TIL+K562 cells at different post-thawing recovery times. [Figure 106] Cytokine secretion results of granzyme B, showing the fold change in TIL+K562 or Raji cells compared to TIL alone at different post-thaw recovery times. [Figure 107] Cytokine secretion results of granzyme B, showing the fold change in TIL+K562 or Raji cells compared to TIL+K562 cells at different post-thawing recovery times. [Figure 108] Embodiments of an allogeneic recognition assay. [Figure 109] Embodiment of a detection method for a homogeneous, heterogeneous recognition assay. [Figure 110] Results for state 1 after thawing (left to stand overnight for 18-24 hours after thawing). The tested effector:target (E:T) ratio ranged from 35:1 to 2.5:1, and the tested co-culture durations were 4 hours, 8 hours, 16 hours, and 36 hours. [Figure 111] Results for post-thawing condition 2 (no standing period after thawing). The tested effector:target (E:T) ratio ranged from 10:1 to 1:10, and the co-culture duration was overnight (16-24 hours). TILs produced from ovarian cancer tumors (OV8178) and melanoma tumors (M1203) were tested. [Figure 112] An exemplary embodiment of a TIL-Raji cell-based efficacy assay. [Figure 113] Total viable cells (TVC, upper plot) and survival rate % (lower plot) versus standing time. [Figure 114] These are the results of co-culture experiments using two TIL cell lines, Raji and K562, regardless of the presence or absence of IL-2 at 300 IU / mL, and show IFN-γ secretion (pg / mL). [Figure 115] These are the results of co-culture experiments using two TIL cell lines, Raji and K562, with and without the presence of 300 IU / mL of IL-2, and show IFN-γ secretion (unit of magnification change). [Figure 116]These are the results of co-culture experiments with two TIL cell lines, Raji and K562, under different co-culture conditions, showing IFN-γ secretion (pg / mL). [Figure 117] These are the results of co-culture experiments using two TIL cell lines, Raji and K562, under different co-culture conditions, and show IFN-γ secretion (unit of magnification change). [Figure 118] These are the results of co-culture experiments using two TIL cell lines, Raji and K562, under different co-culture conditions to evaluate the cytokine secretion effect as the recovery period after TIL thawing is extended, and show IFN-γ secretion (pg / mL). [Figure 119] This is an embodiment of the present invention for evaluating the cytokine secretion effect as the recovery period after TIL thawing is extended, showing the results of co-culture experiments with Raji and K562 cell lines under different co-culture conditions, and indicating IFN-γ secretion (unit of multiplicative change). [Figure 120] Flow cytometry analysis of residual cell populations of the parental population of TIL strain M1179 in two embodiments of co-culture conditions during a 72-hour standing period. [Figure 121] Growth profile of the Raji cell line. [Figure 122] Growth profile of the K562 cell line. [Figure 123] A diagram of the experimental design for the TIL: tumor cell line co-culture assay, which is also an embodiment of the present invention. [Figure 124] Embodiments of the present invention include IFN-γ secretion (pg / mL, absolute value) from target cell lines (Raji, Ramos, Thp1, and U937) and negative control (K562) cell lines tested at three TIL:target cell ratios (3:1, 1:1, and 1:3). [Figure 125] Embodiments of the present invention include IFN-γ secretion from target cell lines (Raji, Ramos, Thp1, and U937) and negative control (K562) cell lines ([TIL + target] / [TIL alone] ratio changes) tested at three TIL:target cell ratios (3:1, 1:1, and 1:3). [Figure 126]Embodiments of the present invention include IFN-γ secretion from target cell lines (Raji, Ramos, Thp1, and U937) and negative control (K562) cell lines ([TIL + target] / [TIL + K562]) tested at three TIL:target cell ratios (3:1, 1:1, and 1:3). [Figure 127] Embodiments of the present invention include TNF-α secretion (pg / mL, absolute value) from target cell lines (Raji, Ramos, Thp1, and U937) and negative control (K562) cell lines tested at three TIL:target cell ratios (3:1, 1:1, and 1:3). [Figure 128] Embodiments of the present invention include TNF-α secretion from target cell lines (Raji, Ramos, Thp1, and U937) and negative control (K562) cell lines tested at three TIL:target cell ratios (3:1, 1:1, and 1:3) ([TIL + target] / [TIL alone] ratio changes). [Figure 129] Embodiments of the present invention include TNF-α secretion from target cell lines (Raji, Ramos, Thp1, and U937) and negative control (K562) cell lines ([TIL + target] / [TIL + K562]) tested at three TIL:target cell ratios (3:1, 1:1, and 1:3). [Figure 130] A diagram of the experimental design for the TIL: tumor cell line co-culture assay, which is also an embodiment of the present invention. [Figure 131] An embodiment of the present invention is the IFN-γ secretion (pg / mL) of the tested target and negative control (K562) cell lines. [Figure 132] An embodiment of the present invention shows the IFN-γ secretion (magnitude change between [TIL + target] and [TIL alone]) of tested target and negative control (K562) cell lines. [Figure 133] An embodiment of the present invention is the IFN-γ secretion of tested target and negative control (K562) cell lines (multiple changes in [TIL+target] / [TIL+K562]). [Figure 134] Mean and range of magnification changes for TIL + target cell lines (or combinations) compared to TIL alone (left panel), and for TIL + K562 (right panel) compared to TIL + target cell lines (or combinations). [Figure 135] TNF-α secretion (pg / mL) of tested target and negative control cell lines, including combined target cell lines, for melanoma TIL strains M1152, M1187, M1198, and M1200. [Figure 136] TNF-α secretion (magnitude change between [TIL + tumor cell line or combination] / [TIL alone]) of tested target and negative control cell lines, including combined target cell lines, for melanoma TIL strains M1152, M1187, M1198, and M1200. [Figure 137] TNF-α secretion (magnitude change of [TIL + tumor cell line or combination] / [TIL + K562]) of tested target and negative control cell lines, including combined 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 combined target cell lines, for melanoma TIL cell lines M1152, M1187, M1198, and M1200. [Figure 139] Granzyme B secretion (magnitude change between [TIL + tumor cell line or combination] / [TIL alone]) of tested target and negative control cell lines, including combined target cell lines, for melanoma TIL strains M1152, M1187, M1198, and M1200. [Figure 140] Granzyme B secretion (magnitude change of [TIL + tumor cell line or combination] / [TIL + K562]) of tested target and negative control cell lines, including combined target cell lines, for melanoma TIL lines M1152, M1187, M1198, and M1200. [Figure 141] A diagram of the experimental design for the TIL: tumor cell line co-culture assay, which is also an embodiment of the present invention. [Figure 142] IFN-γ secretion (pg / mL) of tested target and negative control cell lines, including combined target cell lines, for the research NSCLC TIL strains L4253, L4254, L4262, and L4270. Black dotted line: 3:1 TIL alone. Gray dotted line: 1:1 TIL alone. [Figure 143]The study examined the IFN-γ secretion ratio of target and NSCLC cell lines (TIL + tumor cell line / TIL alone) for NSCLC TIL strains L4253, L4254, L4262, and L4270, including combined target cell lines. [Figure 144] The study examined the IFN-γ secretion ratio of target and NSCLC cell lines (TIL + tumor cell line / [TIL + K562]) for NSCLC TIL strains L4253, L4254, L4262, and L4270, including combined target cell lines. [Figure 145] IFN-γ secretion (pg / mL) of tested target and negative control cell lines, including combined target cell lines, for clinical NSCLC TIL strains 1013-304, 1036-307, 1057-304, 1004-303, 1057-302, and 1088-303. Black dotted line: TIL alone. Data were obtained at a 1:1 TIL:target ratio.

[0079] [Figure 146] Dilution of IFN-γ secretion (TIL + tumor cell line / TIL alone) of tested target and negative control cell lines, including combined target cell lines, for clinical NSCLC TIL strains 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 147] Dilution of IFN-γ secretion (TIL + tumor cell line / [TIL + K562]) of tested target and negative control cell lines, including combined target cell lines, for clinical NSCLC TIL strains 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 changes in IFN-γ secretion (TIL + tumor cell line / TIL alone) of tested target and negative control cell lines for clinical NSCLC TIL strains 1013-304, 1036-307, 1057-304, 1004-303, 1057-302, and 1088-303. Filled red symbols represent clinical partial responses, and remaining symbols represent clinically stable disease responses. [Figure 149] Summary of fold changes in IFN-γ secretion (TIL+ tumor cell line / [TIL+K562]) of tested target and negative control cell lines for clinical NSCLC TIL strains 1013-304, 1036-307, 1057-304, 1004-303, 1057-302, and 1088-303. Filled red symbols represent clinical partial responses, and remaining symbols represent clinically stable disease responses. [Figure 150] Summary of 19 studies and clinical analyses of fold changes in IFN-γ secretion (TIL + tumor cell line / TIL alone) for tested target and negative control cell lines for melanoma and NSCLC TIL strains. Each box represents one standard deviation on either side of the mean, which is indicated by a line. [Figure 151] Summary of 19 studies and clinically tested target and negative control cell lines for melanoma and NSCLC TIL strains, showing the fold change in IFN-γ secretion (TIL+ tumor cell line / [TIL+K562]). Each box represents one standard deviation on either side of the mean, which is indicated by a line. [Figure 152] Effects of irradiation on the proliferation (total viable cells, TVC) of Raji cells and K562 cells during 24-hour co-culture. [Figure 153] Concentrations of secreted IFN-γ during co-culture of unirradiated and irradiated Raji cells in two cervical cancer TIL lots. [Figure 154] Surface marker expression on K562 and Raji cells, determined by flow cytometry, for three markers: those with irradiation ("Irr.") and those without irradiation ("Non-irr."). [Figure 155]Changes in IFN-γ magnification (triple samples) versus assay co-culture period (hours) for TIL alone, using three melanomas, two cervical samples, and one NSCLC TIL strain. [Figure 156] A diagram of the experimental design for the TIL: tumor cell line co-culture assay, which is also an embodiment of the present invention. [Figure 157] Mean magnification change of tumor cell lines with IFN-γ+ TILs compared to TILs alone at 1.0 × 10⁶ TVC / well for 12 melanoma TIL lines. The boxes represent one standard deviation on either side of the mean, which is shown by the line. [Figure 158] Mean IFN-γ scaling change of tumor cell lines + TILs compared to TILs alone for 12 melanoma TIL lines at 2.0 × 10⁶ TVC / well. The boxes represent one standard deviation on either side of the mean, which is shown by the line. [Figure 159] Mean magnification change of tumor cell lines + TILs compared to K562-negative control cells in 1.0 × 10⁶ TVC / well for 12 melanoma TIL lines. The boxes represent one standard deviation on either side of the mean, which is shown by the line. [Figure 160] Mean magnification change of tumor cell lines + TILs compared to K562-negative control cells in 2.0 × 10⁶ TVC / well for 12 melanoma TIL lines. The boxes represent one standard deviation on either side of the mean, which is shown by the line. [Figure 161] A diagram illustrating the experimental design for a TIL: tumor cell line co-culture assay using two types of Thp1 (THP-1) monocyte cell lines, wild-type (WT) and genetically modified, which is also an embodiment of the present invention. [Figure 162] Comparison of IFN-γ levels (pg / mL) obtained from WT and genetically modified Thp1 target cells. [Figure 163] Comparison of IFN-γ ratio changes relative to TIL alone obtained in WT and genetically modified Thp1 target cells. [Figure 164] A figure showing HLA blockade, which is a negative control of the assay of the present invention in a particular embodiment. [Figure 165]The effect of HLA-I blockade on Thp1 (THP-1) and U937 monocyte cell lines at different antibody concentrations, measured by the survival percentage for each monocyte cell line. [Figure 166] The effect of HLA-II blockade on Thp1 (THP-1) and U937 monocyte cell lines at different antibody concentrations, measured by the survival percentage for each monocyte cell line. [Figure 167] The effect of HLA-II blockade on Raji cells at different antibody concentrations is measured by the survival percentage 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 TIL cell line M1213. [Figure 169] Efficacy of HLA-I (aMHC I) and HLA-II (aMHC II) antibody blockade against 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] Results of dose titration of HLA (MHC) class I and II antibodies. Embodiment I of the HLA blockade-negative control method was performed using 20 μg / mL of HLA-I blocking antibody and 10 μg / mL of HLA-II blocking antibody. Embodiment II of the HLA blockade-negative control method was performed using 10 μg / mL of HLA-I blocking antibody and 5 μg / mL of HLA-II blocking antibody. [Figure 172] Results of background IFN-γ secretion from 10 TIL strains upon addition of anti-HLA-I antibody (denoted as αMHC I) and anti-HLA-II antibody (denoted as αMHC II), each performed three times. Embodiments I and II refer to experimental embodiments shown in Figures 173 and 184, respectively, and described elsewhere in this specification, with Embodiment I including the use of 20 μg / mL HLA-I blocking antibody and 10 μg / mL HLA-II blocking antibody, and Embodiment II including the use of 10 μg / mL HLA-I blocking antibody and 5 μg / mL HLA-II blocking antibody. [Figure 173] This is a diagram of an experimental design for a TIL: tumor cell line co-culture assay, in which an HLA-blocking antibody is used as a negative control instead of a negative control cell line. These assays are collectively referred to herein as "Embodiment I" where applicable and are also embodiments of the present invention. [Figure 174] Results of HLA-blocking-negative control experiments of melanoma TIL strain M1152 co-cultured with target cells and control K562 cells, as reported in IFN-γ pg / mL. [Figure 175] Results of HLA-blocking negative control experiments of melanoma TIL strain M1187 co-cultured with target cells and control K562 cells, as reported in IFN-γ pg / mL. [Figure 176] Results of HLA-blocking-negative control experiments of melanoma TIL strain M1164 co-cultured with target cells and control K562 cells, as reported in IFN-γ pg / mL. [Figure 177] Results of HLA-blocking-negative control experiments of melanoma TIL strain M1213 co-cultured with target cells and control K562 cells, as reported in IFN-γ pg / mL. [Figure 178] Results of HLA-blocking negative control experiments of melanoma TIL strain M1214 co-cultured with target cells and control K562 cells, as reported in IFN-γ pg / mL. [Figure 179] Results of HLA-blocking-negative control experiments of melanoma TIL strain M1152 co-cultured with target cells and control K562 cells, reported as a fulcrum enhancement of IFN-γ release compared to TIL alone. [Figure 180] Results of HLA-blocking-negative control experiments of melanoma TIL strain M1187 co-cultured with target cells and control K562 cells, reported as a fulcrum enhancement of IFN-γ release compared to TIL alone. [Figure 181] Results of HLA-blocking-negative control experiments of melanoma TIL strain M1164 co-cultured with target cells and control K562 cells, reported as a fulcrum enhancement of IFN-γ release compared to TIL alone. [Figure 182]Results of HLA-blocking-negative control experiments of melanoma TIL strain M1213 co-cultured with target cells and control K562 cells, reported as a fulcrum enhancement of IFN-γ release compared to TIL alone. [Figure 183] Results of HLA-blocking-negative control experiments of melanoma TIL strain M1214 co-cultured with target cells and control K562 cells, reported as a fulcrum enhancement of IFN-γ release compared to TIL alone. [Figure 184] This is a diagram of an experimental design for a TIL: tumor cell line co-culture assay, in which an HLA-blocking antibody is used as a negative control instead of a negative control cell line. These assays are collectively referred to herein as "Embodiment II" where applicable and are also embodiments of the present invention. [Figure 185] Results of HLA-blocking-negative control experiments of melanoma TIL strain M1161 co-cultured with target cells and control K562 cells, reported as IFN-γ pg / mL. The CD4+ and CD8+ populations of M1161 were determined to be 1.4% and 97.6%, respectively, by flow cytometry. [Figure 186] Results of an HLA-blocking negative control experiment of melanoma TIL strain M1163 co-cultured with target cells and control K562 cells, reported as IFN-γ pg / mL. The CD4+ and CD8+ populations of M1163 were determined to be 60.6% and 34.9%, respectively, by flow cytometry. [Figure 187] Results of HLA-blocking-negative control experiments of melanoma TIL strain M1172 co-cultured with target cells and control K562 cells, reported as IFN-γ pg / mL. The CD4+ and CD8+ populations of M1172 were determined to be 71.8% and 22.2%, respectively, by flow cytometry. [Figure 188] Results of an HLA-blocking negative control experiment of melanoma TIL strain M1173 co-cultured with target cells and control K562 cells, reported as IFN-γ pg / mL. The CD4+ and CD8+ populations of M1173 were determined to be 13.4% and 81.6%, respectively, by flow cytometry. [Figure 189] Results of HLA-blocking-negative control experiments of melanoma TIL strain M1174 co-cultured with target cells and control K562 cells, reported as IFN-γ pg / mL. The CD4+ and CD8+ populations of M1174 were determined to be 92.3% and 6.2%, respectively, by flow cytometry. [Figure 190] Results of HLA-blocking-negative control experiments of melanoma TIL strain M1161 co-cultured with target cells and control K562 cells, reported as a fulcrum enhancement of IFN-γ release compared to TIL alone. The CD4+ and CD8+ populations of M1161 were determined to be 1.4% and 97.6%, respectively, by flow cytometry. [Figure 191] Results of an HLA-blocking-negative control experiment of melanoma TIL strain M1163 co-cultured with target cells and control cells (K562 cells), reported as a factorial enhancement of IFN-γ release compared to TIL alone. The CD4+ and CD8+ populations of M1163 were determined to be 60.6% and 34.9%, respectively, by flow cytometry. [Figure 192] Results of HLA-blocking-negative control experiments of melanoma TIL strain M1172 co-cultured with target cells and control cells (K562 cells), reported as a fulcrum enhancement of IFN-γ release compared to TIL alone. The CD4+ and CD8+ populations of M1172 were determined to be 71.8% and 22.2%, respectively, by flow cytometry. [Figure 193] Results of an HLA-blocking-negative control experiment of melanoma TIL strain M1173 co-cultured with target cells and control K562 cells, reported as a factorial enhancement of IFN-γ release compared to TIL alone. The CD4+ and CD8+ populations of M1173 were determined to be 13.4% and 81.6%, respectively, by flow cytometry. [Figure 194]Results of HLA-blocking-negative control experiments of melanoma TIL strain M1174 co-cultured with target cells and control K562 cells, reported as a fulcrum enhancement of IFN-γ release compared to TIL alone. The CD4+ and CD8+ populations of M1174 were determined to be 92.3% and 6.2%, respectively, by flow cytometry. [Figure 195] This is a diagram of the experimental design for a TIL:tumor cell line co-culture assay, using an HLA-blocking antibody as a negative control instead of a negative control cell line, or in addition to a TIL-only control experiment, and all of these assays are also embodiments of the present invention. Fifteen melanoma cell lines are used. [Figure 196] Results of an HLA-blocking-negative control experiment using 15 melanoma TIL strains co-cultured with U937 target cells. [Figure 197] Results of an HLA-blocking-negative control experiment using 15 melanoma TIL strains co-cultured with Thp1 (THP-1) target cells. [Figure 198] Determination of effective TIL concentration for Thp1 cells. The stars indicate a 3:1 TIL:Thp1 ratio in 2 × 10⁶ TVC cells. [Figure 199] Determination of effective TIL concentration for U937 cells. The star indicates a 3:1 TIL:U937 ratio at 2 × 10⁶ TVCs. [Figure 200] Dose-response curves of three TIL lots (M1164, M1163, and M1169) using Thp1 and U937 target cells. [Figure 201] Dose-response curves for 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 showed a clear dose-response. [Figure 203] Parallel line analysis of six TIL lots using Thp1-targeted cells. TIL M1173 and TIL M1213 show clear dose-response. [Figure 204]Dose-response curves of three TIL lots (M1145, M1161, and M1173) at a TIL concentration of 1.5 × 10⁶ with and without HLA-blocking antibody, using U937 target cells. All three TIL lots showed a clear dose-response and complete inhibition of the signal by antibody treatment (specific inhibition). [Figure 205] Dose-response curves of three TIL lots (M1197, M1213, and M1214) at a TIL concentration of 1.5 × 10⁶ with and without HLA-blocking antibody, using U937 target cells. All three TIL lots showed a clear dose-response and complete inhibition of the signal by antibody treatment (specific inhibition). [Figure 206] Histogram of LAG3 expression by flow cytometry in melanoma TIL clinical samples. [Figure 207] Histogram of KLRG1 expression by flow cytometry in melanoma TIL clinical samples. [Figure 208] Logarithmic distribution evaluation of histograms of relative potency for 28 past clinical lots using the U937 alloreactive co-culture assay. Logarithmically transforming the relative potency measurements calculated for each TIL lot resulted in a normal distribution, indicating that the underlying distribution is log-normal. [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 using U937 cells or allogeneic PBMCs. [Figure 211] An embodiment of the alloreactive co-culture assay of the present invention (referred to as "allo-pMHC-TCR interaction-induced T cells") for T cell efficacy testing, and a comparison with a conventional antibody bead-based assay.

[0080] A brief explanation of sequence listings Sequence ID 1 is the amino acid sequence of the heavy chain of muromonab.

[0081] Sequence ID 2 is the amino acid sequence of the light chain of muromonab.

[0082] Sequence ID 3 is the amino acid sequence of recombinant human IL-2 protein.

[0083] Sequence ID 4 is the amino acid sequence of aldethleukin.

[0084] Sequence ID 5 is the IL-2 form.

[0085] Sequence ID 6 is the IL-2 form.

[0086] Sequence ID 7 is the IL-2 form.

[0087] Sequence ID 8 is a mucin domain polypeptide.

[0088] Sequence ID 9 is the amino acid sequence of recombinant human IL-4 protein.

[0089] Sequence ID 10 is the amino acid sequence of recombinant human IL-7 protein.

[0090] Sequence ID 11 is the amino acid sequence of recombinant human IL-15 protein.

[0091] Sequence ID 12 is the amino acid sequence of recombinant human IL-21 protein.

[0092] Sequence ID 13 is the IL-2 sequence.

[0093] Sequence ID 14 is the IL-2 mutain sequence.

[0094] Sequence ID 15 is the IL-2 mutain sequence.

[0095] Sequence ID 16 is IgG.IL2R67A.H1, which is HCDR1_IL-2.

[0096] Sequence ID 17 is HClR2 of IgG.IL2R67A.H1.

[0097] Sequence ID 18 is IgG.IL2R67A.H1 HCDR3.

[0098] Sequence ID 19 is the IgG.IL2R67A.H1 HCDR1_IL-2 kabat.

[0099] Sequence ID 20 is the HCl2 kabat for IgG.IL2R67A.H1.

[0100] Sequence ID 21 is the HCl3 kabat for IgG.IL2R67A.H1.

[0101] Sequence ID 22 is IgG.IL2R67A.H1, which is HCDR1_IL-2 Clothia.

[0102] Sequence ID 23 is IgG.IL2R67A.H1, an HCDR2 Clothia.

[0103] Sequence ID 24 is IgG.IL2R67A.H1, an HCDR3 Clothia.

[0104] Sequence ID 25 is IgG.IL2R67A.H1 HCDR1_IL-2 IMGT.

[0105] Sequence ID 26 is the HCl2 IMGT of IgG.IL2R67A.H1.

[0106] Sequence ID 27 is the HCl3 IMGT of IgG.IL2R67A.H1.

[0107] Sequence ID 28 is V of IgG.IL2R67A.H1 H It is a chain.

[0108] Sequence ID 29 is the heavy chain of IgG.IL2R67A.H1.

[0109] Sequence ID 30 is the LCDR1 kabat for IgG.IL2R67A.H1.

[0110] Sequence ID 31 is the LCDR2 kabat for IgG.IL2R67A.H1.

[0111] Sequence ID 32 is the LCDR3 kabat for IgG.IL2R67A.H1.

[0112] Sequence ID 33 is LCDR1 chothia of IgG.IL2R67A.H1.

[0113] Sequence ID 34 is LCDR2 chothia of IgG.IL2R67A.H1.

[0114] Sequence ID 35 is LCDR3 chothia of IgG.IL2R67A.H1.

[0115] Sequence ID 36 is V L It is a chain.

[0116] Sequence ID 37 is a light chain.

[0117] Sequence ID 38 is a light chain.

[0118] Sequence ID 39 is a light chain.

[0119] Sequence ID No. 40 is the amino acid sequence of human 4-1BB.

[0120] Sequence ID 41 is the amino acid sequence of mouse 4-1BB.

[0121] Sequence ID 42 is the heavy chain of the 4-1BB agonist monoclonal antibody utomirumab (PF-05082566).

[0122] Sequence ID 43 is the light chain of the 4-1BB agonist monoclonal antibody utomirumab (PF-05082566).

[0123] Sequence ID 44 is the heavy chain variable region (V) of the 4-1BB agonist monoclonal antibody utomirumab (PF-05082566). H )

[0124] Sequence ID 45 is the light chain variable region (V) of the 4-1BB agonist monoclonal antibody utomirumab (PF-05082566). L )

[0125] Sequence ID 46 is the heavy chain CDR1 of the 4-1BB agonist monoclonal antibody utomirumab (PF-05082566).

[0126] Sequence ID 47 is the heavy chain CDR2 of the 4-1BB agonist monoclonal antibody utomirumab (PF-05082566).

[0127] Sequence ID 48 is the heavy chain CDR3 of the 4-1BB agonist monoclonal antibody utomirumab (PF-05082566).

[0128] Sequence ID 49 is the light chain CDR1 of the 4-1BB agonist monoclonal antibody utomirumab (PF-05082566).

[0129] Sequence ID 50 is the light chain CDR2 of the 4-1BB agonist monoclonal antibody utomirumab (PF-05082566).

[0130] Sequence ID 51 is the light chain CDR3 of the 4-1BB agonist monoclonal antibody utomirumab (PF-05082566).

[0131] Sequence ID 52 is the heavy chain of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0132] Sequence ID 53 is the light chain of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0133] Sequence ID 54 is the heavy chain variable region (VH) of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0134] Sequence ID 55 is the light chain variable region (VL) of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0135] Sequence ID 56 is the heavy chain CDR1 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0136] Sequence ID 57 is the heavy chain CDR2 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0137] Sequence ID 58 is the heavy chain CDR3 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0138] Sequence ID 59 is the light chain CDR1 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0139] Sequence ID 60 is the light chain CDR2 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0140] Sequence ID 61 is the light chain CDR3 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0141] Sequence ID 62 is the Fc domain of the TNFRSF agonist fusion protein.

[0142] Sequence ID 63 is the linker for the TNFRSF agonist fusion protein.

[0143] Sequence ID 64 is the linker for the TNFRSF agonist fusion protein.

[0144] Sequence ID 65 is the linker for the TNFRSF agonist fusion protein.

[0145] Sequence ID 66 is the linker for the TNFRSF agonist fusion protein.

[0146] Sequence ID 67 is the linker for the TNFRSF agonist fusion protein.

[0147] Sequence ID 68 is the linker for the TNFRSF agonist fusion protein.

[0148] Sequence ID 69 is the linker for the TNFRSF agonist fusion protein.

[0149] Sequence ID 70 is the linker for the TNFRSF agonist fusion protein.

[0150] Sequence ID 71 is the linker for the TNFRSF agonist fusion protein.

[0151] Sequence ID 72 is the linker for the TNFRSF agonist fusion protein.

[0152] Sequence ID 73 is the Fc domain of the TNFRSF agonist fusion protein.

[0153] Sequence ID 74 is the linker for the TNFRSF agonist fusion protein.

[0154] Sequence ID 75 is the linker for the TNFRSF agonist fusion protein.

[0155] Sequence ID 76 is the linker for the TNFRSF agonist fusion protein.

[0156] Sequence ID 77 is the amino acid sequence of the 4-1BB ligand (4-1BBL).

[0157] Sequence ID 78 is the soluble portion of the 4-1BBL polypeptide.

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

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

[0160] Sequence ID 81 is the heavy chain variable region (V) of the 4-1BB agonist antibody 4B4-1-1 version 2. H )

[0161] Sequence ID 82 is the light chain variable region (V) of the 4-1BB agonist antibody 4B4-1-1 version 2. L )

[0162] Sequence ID 83 is the heavy chain variable region (V) of the 4-1BB agonist antibody H39E3-2. H )

[0163] Sequence ID 84 is the light chain variable region (V) of the 4-1BB agonist antibody H39E3-2. L )

[0164] Sequence ID 85 is the amino acid sequence of human OX40.

[0165] Sequence ID 86 is the amino acid sequence of mouse OX40.

[0166] Sequence ID 87 is the heavy chain of the OX40 agonist monoclonal antibody tavorixizumab (MEDI-0562).

[0167] Sequence ID 88 is the light chain of the OX40 agonist monoclonal antibody tavorixizumab (MEDI-0562).

[0168] Sequence ID 89 is the heavy chain variable region (V) of the OX40 agonist monoclonal antibody tavorixizumab (MEDI-0562). H)

[0169] Sequence ID 90 is the light chain variable region (V) of the OX40 agonist monoclonal antibody tavorixizumab (MEDI-0562). L )

[0170] Sequence ID 91 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody tavorixizumab (MEDI-0562).

[0171] Sequence ID 92 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody tavorixizumab (MEDI-0562).

[0172] Sequence ID 93 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody tavorixizumab (MEDI-0562).

[0173] Sequence ID 94 is the light chain CDR1 of the OX40 agonist monoclonal antibody tavorixizumab (MEDI-0562).

[0174] Sequence ID 95 is the light chain CDR2 of the OX40 agonist monoclonal antibody tavorixizumab (MEDI-0562).

[0175] Sequence ID 96 is the light chain CDR3 of the OX40 agonist monoclonal antibody tavorixizumab (MEDI-0562).

[0176] Sequence ID 97 is the heavy chain of the OX40 agonist monoclonal antibody 11D4.

[0177] Sequence ID 98 is the light chain of the OX40 agonist monoclonal antibody 11D4.

[0178] Sequence ID 99 is the heavy chain variable region (V) of the OX40 agonist monoclonal antibody 11D4. H )

[0179] Sequence ID 100 is the light chain variable region (V) of the OX40 agonist monoclonal antibody 11D4. L )

[0180] Sequence ID 101 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody 11D4.

[0181] Sequence ID No. 102 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody 11D4.

[0182] Sequence ID 103 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody 11D4.

[0183] Sequence ID 104 is the light chain CDR1 of the OX40 agonist monoclonal antibody 11D4.

[0184] Sequence ID No. 105 is the light chain CDR2 of the OX40 agonist monoclonal antibody 11D4.

[0185] Sequence ID No. 106 is the light chain CDR3 of the OX40 agonist monoclonal antibody 11D4.

[0186] Sequence ID No. 107 is the heavy chain of the OX40 agonist monoclonal antibody 18D8.

[0187] Sequence ID 108 is the light chain of the OX40 agonist monoclonal antibody 18D8.

[0188] Sequence ID 109 is the heavy chain variable region (V) of the OX40 agonist monoclonal antibody 18D8. H )

[0189] Sequence ID 110 is the light chain variable region (V) of the OX40 agonist monoclonal antibody 18D8. L )

[0190] Sequence ID 111 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody 18D8.

[0191] Sequence ID No. 112 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody 18D8.

[0192] Sequence ID No. 113 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody 18D8.

[0193] Sequence ID No. 114 is the light chain CDR1 of the OX40 agonist monoclonal antibody 18D8.

[0194] Sequence ID No. 115 is the light chain CDR2 of the OX40 agonist monoclonal antibody 18D8.

[0195] Sequence ID No. 116 is the light chain CDR3 of the OX40 agonist monoclonal antibody 18D8.

[0196] Sequence ID 117 is the heavy chain variable region (V) of the OX40 agonist monoclonal antibody Hu119-122. H )

[0197] Sequence ID 118 is the light chain variable region (V) of the OX40 agonist monoclonal antibody Hu119-122. L )

[0198] Sequence ID 119 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody Hu119-122.

[0199] Sequence ID 120 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody Hu119-122.

[0200] Sequence ID 121 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody Hu119-122.

[0201] Sequence ID 122 is the light chain CDR1 of the OX40 agonist monoclonal antibody Hu119-122.

[0202] Sequence ID 123 is the light chain CDR2 of the OX40 agonist monoclonal antibody Hu119-122.

[0203] Sequence ID 124 is the light chain CDR3 of the OX40 agonist monoclonal antibody Hu119-122.

[0204] Sequence ID 125 is the heavy chain variable region (V) of the OX40 agonist monoclonal antibody Hu106-222. H )

[0205] Sequence ID 126 is the light chain variable region (V) of the OX40 agonist monoclonal antibody Hu106-222. L )

[0206] Sequence ID 127 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody Hu106-222.

[0207] Sequence ID 128 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody Hu106-222.

[0208] Sequence ID 129 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody Hu106-222.

[0209] Sequence ID No. 130 is the light chain CDR1 of the OX40 agonist monoclonal antibody Hu106-222.

[0210] Sequence ID 131 is the light chain CDR2 of the OX40 agonist monoclonal antibody Hu106-222.

[0211] Sequence ID 132 is the light chain CDR3 of the OX40 agonist monoclonal antibody Hu106-222.

[0212] Sequence ID 133 is the amino acid sequence of the OX40 ligand (OX40L).

[0213] Sequence ID No. 134 is the soluble portion of the OX40L polypeptide.

[0214] Sequence ID No. 135 is the alternative soluble moiety of the OX40L polypeptide.

[0215] Sequence ID 136 is the heavy chain variable region (V) of OX40 agonist monoclonal antibody 008. H )

[0216] Sequence ID 137 is the light chain variable region (V) of the OX40 agonist monoclonal antibody 008. L )

[0217] Sequence ID 138 is the heavy chain variable region (V) of OX40 agonist monoclonal antibody 011. H )

[0218] Sequence ID 139 is the light chain variable region (V) of OX40 agonist monoclonal antibody 011. L )

[0219] Sequence ID 140 is the heavy chain variable region (V) of the OX40 agonist monoclonal antibody 021. H )

[0220] Sequence ID 141 is the light chain variable region (V) of the OX40 agonist monoclonal antibody 021. L )

[0221] Sequence ID 142 is the heavy chain variable region (V) of the OX40 agonist monoclonal antibody 023. H )

[0222] Sequence ID 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) of the OX40 agonist monoclonal antibody. H )

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

[0225] Sequence ID 146 is the heavy chain variable region (V) of the OX40 agonist monoclonal antibody. H )

[0226] Sequence ID 147 is the light chain variable region (V) of the OX40 agonist monoclonal antibody. L )

[0227] Sequence ID 148 is the heavy chain variable region (V) of a humanized OX40 agonist monoclonal antibody. H )

[0228] Sequence ID 149 is the heavy chain variable region (V) of a humanized OX40 agonist monoclonal antibody. H )

[0229] Sequence ID 150 is the light chain variable region (V) of a humanized OX40 agonist monoclonal antibody. L )

[0230] Sequence ID 151 is the light chain variable region (V) of a humanized OX40 agonist monoclonal antibody. L )

[0231] Sequence ID 152 is the heavy chain variable region (V) of a humanized OX40 agonist monoclonal antibody. H )

[0232] Sequence ID 153 is the heavy chain variable region (V) of a humanized OX40 agonist monoclonal antibody. H )

[0233] Sequence ID 154 is the light chain variable region (V) of a humanized OX40 agonist monoclonal antibody. L )

[0234] Sequence ID 155 is the light chain variable region (V) of a humanized OX40 agonist monoclonal antibody. L)

[0235] Sequence ID 156 is the heavy chain variable region (V) of the OX40 agonist monoclonal antibody. H )

[0236] Sequence ID 157 is the light chain variable region (V) of the OX40 agonist monoclonal antibody. L )

[0237] Sequence ID 158 is the heavy chain amino acid sequence of the PD-1 inhibitor nivolumab.

[0238] Sequence ID 159 is the light chain amino acid sequence of the PD-1 inhibitor nivolumab.

[0239] Sequence ID 160 is the heavy chain variable region (V) of the PD-1 inhibitor nivolumab. H ) This is the amino acid sequence.

[0240] Sequence ID 161 is the light chain variable region (V) of the PD-1 inhibitor nivolumab. L ) This is the amino acid sequence.

[0241] Sequence ID 162 is the heavy chain CDR1 amino acid sequence of the PD-1 inhibitor nivolumab.

[0242] Sequence ID 163 is the heavy chain CDR2 amino acid sequence of the PD-1 inhibitor nivolumab.

[0243] Sequence ID 164 is the heavy chain CDR3 amino acid sequence of the PD-1 inhibitor nivolumab.

[0244] Sequence ID 165 is the CDR1 amino acid sequence of the light chain of the PD-1 inhibitor nivolumab.

[0245] Sequence ID 166 is the CDR2 amino acid sequence of the light chain of the PD-1 inhibitor nivolumab.

[0246] Sequence ID 167 is the CDR3 amino acid sequence of the light chain of the PD-1 inhibitor nivolumab.

[0247] Sequence ID 168 is the heavy chain amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0248] Sequence ID 169 is the light chain amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0249] Sequence ID 170 is the heavy chain variable region (V) of the PD-1 inhibitor pembrolizumab. H ) This is the amino acid sequence.

[0250] Sequence ID 171 is the light chain variable region (V) of the PD-1 inhibitor pembrolizumab. L ) This is the amino acid sequence.

[0251] Sequence ID 172 is the heavy chain CDR1 amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0252] Sequence ID 173 is the heavy chain CDR2 amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0253] Sequence ID 174 is the heavy chain CDR3 amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0254] Sequence ID 175 is the CDR1 amino acid sequence of the light chain of the PD-1 inhibitor pembrolizumab.

[0255] Sequence ID 176 is the CDR2 amino acid sequence of the light chain of the PD-1 inhibitor pembrolizumab.

[0256] Sequence ID 177 is the CDR3 amino acid sequence of the light chain of the PD-1 inhibitor pembrolizumab.

[0257] Sequence ID 178 is the heavy chain amino acid sequence of the PD-L1 inhibitor durvalumab.

[0258] Sequence ID 179 is the light chain amino acid sequence of the PD-L1 inhibitor durvalumab.

[0259] Sequence ID 180 is the heavy chain variable region (V) of the PD-L1 inhibitor durvalumab. H ) This is the amino acid sequence.

[0260] Sequence ID 181 is the light chain variable region (V) of the PD-L1 inhibitor durvalumab. L ) This is the amino acid sequence.

[0261] Sequence ID 182 is the heavy chain CDR1 amino acid sequence of the PD-L1 inhibitor durvalumab.

[0262] Sequence ID 183 is the heavy chain CDR2 amino acid sequence of the PD-L1 inhibitor durvalumab.

[0263] Sequence ID 184 is the heavy chain CDR3 amino acid sequence of the PD-L1 inhibitor durvalumab.

[0264] Sequence ID 185 is the CDR1 amino acid sequence of the light chain of the PD-L1 inhibitor durvalumab.

[0265] Sequence ID 186 is the CDR2 amino acid sequence of the light chain of the PD-L1 inhibitor durvalumab.

[0266] Sequence ID 187 is the CDR3 amino acid sequence of the light chain of the PD-L1 inhibitor durvalumab.

[0267] Sequence ID 188 is the heavy chain amino acid sequence of the PD-L1 inhibitor avelumab.

[0268] Sequence ID 189 is the light chain amino acid sequence of the PD-L1 inhibitor avelumab.

[0269] Sequence ID 190 is the heavy chain variable region (V) of the PD-L1 inhibitor avelumab. H ) This is the amino acid sequence.

[0270] Sequence ID 191 is the light chain variable region (V) of the PD-L1 inhibitor avelumab. L ) This is the amino acid sequence.

[0271] Sequence ID 192 is the heavy chain CDR1 amino acid sequence of the PD-L1 inhibitor avelumab.

[0272] Sequence ID 193 is the heavy chain CDR2 amino acid sequence of the PD-L1 inhibitor avelumab.

[0273] Sequence ID 194 is the heavy chain CDR3 amino acid sequence of the PD-L1 inhibitor avelumab.

[0274] Sequence ID 195 is the CDR1 amino acid sequence of the light chain of the PD-L1 inhibitor avelumab.

[0275] Sequence ID 196 is the CDR2 amino acid sequence of the light chain of the PD-L1 inhibitor avelumab.

[0276] Sequence ID 197 is the CDR3 amino acid sequence of the light chain of the PD-L1 inhibitor avelumab.

[0277] Sequence ID 198 is the heavy chain amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0278] Sequence ID 199 is the light chain amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0279] Sequence ID 200 is the heavy chain variable region (V) of the PD-L1 inhibitor atezolizumab. H ) This is the amino acid sequence.

[0280] Sequence ID 201 is the light chain variable region (V) of the PD-L1 inhibitor atezolizumab. L ) This is the amino acid sequence.

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

[0282] Sequence ID 203 is the heavy chain CDR2 amino acid sequence of the PD-L1 inhibitor atezolizumab.

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

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

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

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

[0287] Sequence ID 208 is the heavy chain amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0288] Sequence ID 209 is the light chain amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0289] Sequence ID 210 is the heavy chain variable region (V) of the CTLA-4 inhibitor ipilimumab. H ) This is the amino acid sequence.

[0290] Sequence ID 211 is the light chain variable region (V) of the CTLA-4 inhibitor ipilimumab. L ) This is the amino acid sequence.

[0291] Sequence ID 212 is the heavy chain CDR1 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0292] Sequence ID 213 is the heavy chain CDR2 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0293] Sequence ID 214 is the heavy chain CDR3 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0294] Sequence ID 215 is the CDR1 amino acid sequence of the light chain of the CTLA-4 inhibitor ipilimumab.

[0295] Sequence ID 216 is the CDR2 amino acid sequence of the light chain of the CTLA-4 inhibitor ipilimumab.

[0296] Sequence ID 217 is the CDR3 amino acid sequence of the light chain of the CTLA-4 inhibitor ipilimumab.

[0297] Sequence ID 218 is the heavy chain amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0298] Sequence ID 219 is the light chain amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0299] Sequence ID 220 is the heavy chain variable region (V) of the CTLA-4 inhibitor tremelimumab. H ) This is the amino acid sequence.

[0300] Sequence ID 221 is the light chain variable region (V) of the CTLA-4 inhibitor tremelimumab. L ) This is the amino acid sequence.

[0301] Sequence ID 222 is the heavy chain CDR1 amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0302] Sequence ID 223 is the heavy chain CDR2 amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0303] Sequence ID 224 is the heavy chain CDR3 amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0304] Sequence ID 225 is the CDR1 amino acid sequence of the light chain of the CTLA-4 inhibitor tremelimumab.

[0305] Sequence ID 226 is the CDR2 amino acid sequence of the light chain of the CTLA-4 inhibitor tremelimumab.

[0306] Sequence ID 227 is the CDR3 amino acid sequence of the light chain of the CTLA-4 inhibitor tremelimumab.

[0307] Sequence ID 228 is the heavy chain amino acid sequence of the CTLA-4 inhibitor zarifremab.

[0308] Sequence ID 229 is the light chain amino acid sequence of the CTLA-4 inhibitor zarifremab.

[0309] Sequence ID 230 is the heavy chain variable region (V) of the CTLA-4 inhibitor zarifremab. H ) This is the amino acid sequence.

[0310] Sequence ID 231 is the light chain variable region (V) of the CTLA-4 inhibitor zarifremab. L ) This is the amino acid sequence.

[0311] Sequence ID 232 is the heavy chain CDR1 amino acid sequence of the CTLA-4 inhibitor zarifremab.

[0312] Sequence ID 233 is the heavy chain CDR2 amino acid sequence of the CTLA-4 inhibitor zarifremab.

[0313] Sequence ID 234 is the heavy chain CDR3 amino acid sequence of the CTLA-4 inhibitor zarifremab.

[0314] Sequence ID 235 is the CDR1 amino acid sequence of the light chain of the CTLA-4 inhibitor zarifremab.

[0315] Sequence ID 236 is the CDR2 amino acid sequence of the light chain of the CTLA-4 inhibitor zarifremab.

[0316] Sequence ID 237 is the CDR3 amino acid sequence of the light chain of the CTLA-4 inhibitor zarifremab. [Modes for carrying out the invention]

[0317] I. Introduction Adoptive cell therapy using TILs cultured ex vivo by the Rapid Expansion Protocol (REP) has been successful in post-host immunosuppression in patients with cancers such as melanoma. Current infusion receptor parameters depend on the compositional readings of the TILs (e.g., CD28, CD8, or CD4 positive), the expression of various cytokines and other markers upon stimulation in bead-based potency assays (e.g., IFN-γ), and the numerical multipliers of expansion and viability of the REP product. Compared with assays known in the art, a potency assay is described herein that can offer improvements, in particular, superior performance, better control of the potency of the T cell product, and increased biological relevance.

[0318] II. Definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art in which the present invention pertains. All patents and publications referenced herein are incorporated in their entirety by reference.

[0319] As used herein, the terms “simultaneous administration,” “administering simultaneously,” “administered in combination,” “administering in combination,” “simultaneous,” and “concurrent” encompass the administration of two or more pharmacokinetic ingredients (for example, multiple TILs in a preferred embodiment of the present invention) to a subject or patient, such that both the pharmacokinetic ingredients and / or their metabolites are present simultaneously in the subject or patient. Simultaneous administration includes simultaneous administration in separate compositions, administration in separate compositions at different times, or administration in a composition containing two or more pharmacokinetic ingredients. Simultaneous administration in separate compositions and administration in a composition containing both drugs are preferred.

[0320] The term "in vivo" refers to events that occur within the body of a subject or patient.

[0321] The term "in vitro" refers to an event that occurs outside the body of the subject or patient. In vitro assays include cell-based assays that use living or dead cells, and may also include cell-free assays that do not use intact cells.

[0322] The term "ex vivo" refers to an event involving the treatment or procedure of cells, tissues, and / or organs removed from the subject's body or the patient's body. Appropriately, the cells, tissues, and / or organs may be returned to the subject's body or the patient's body by surgical or therapeutic means.

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

[0324] In this specification, “tumor-infiltrating lymphocytes” or “TILs” means the population of cells initially obtained as leukocytes that have migrated from the bloodstream of the subject to the tumor. TILs include, but are not limited to, CD8+ cytotoxic T cells (lymphocytes), Th1 and Th17 CD4+ T cells, natural killer cells, dendritic cells, and M1 macrophages. TILs include both primary and secondary TILs. “Primary TILs” are obtained from patient tissue samples as outlined herein (sometimes referred to as “freshly collected”), and “secondary TILs” are any expanded or proliferated TIL cell populations considered 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 TIL) means several cells sharing a common trait. Generally, the population is generally in the range of 1×10 6 to 1×10 10 , and different TIL populations contain different numbers. For example, the initial growth of primary TIL in the presence of IL-2 results in a population of bulk TIL of approximately 1×10 8 cells. REP expansion is generally performed to provide a population of 1.5×10 9 to 1.5×10 10 cells for injection.

[0326] As used herein, "cryopreserved TIL" means that TIL of any of primary, bulk, or expanded (REP TIL) is processed and stored in the range of about -150°C to -60°C. General methods for cryopreservation are also described elsewhere in this specification, including in the examples. For clarity, "cryopreserved TIL" can be distinguished from cryopreserved tissue samples that may be used as a source of primary TIL.

[0327] [[ID=***]] As used herein, "thawed cryopreserved TIL" means a population of TIL that was previously cryopreserved and then processed to return to a temperature above room temperature, including but not limited to cell culture temperature or a temperature at which the TIL can be administered to a patient.

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

[0329] a It should be noted that there seems to be an error in the original text where the tag appears twice in the translation section. I have translated it as per the instructions while highlighting this potential issue. If this is a specific formatting or intended repetition in the original, it may need to be adjusted according to the actual requirements.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% - 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 media may be referred to by the trade name "CryoStor® CS10". CS10 media is a serum-free and animal component-free medium containing DMSO.

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

[0331] The term "effector memory T cells", "effector memory T cells" or "T EM " are CD45R0+ like central memory T cells, but CCR7 (CCR7lo or CCR -This refers to a subset of human or mammalian T cells that have lost constitutive expression of CD62L 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. The transcription factor of central memory T cells includes BLIMP1. Effector memory T cells rapidly secrete high levels of inflammatory cytokines, including interferon-γ, IL-4, and IL-5, after antigen stimulation. Effector memory T cells are dominant in the CD8 compartment in the blood and are proportionally enriched in the lungs, liver, and intestines in humans. + Effector memory T cells carry large amounts of perforin. Terminally differentiated T cells reexpress the CD45RA marker. EM The cell is "T EMRA These are also called "TEMRA" cells.

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

[0333] As used herein to describe the process of destroying a tumor, the terms “fragmentation,” “fragment,” and “fragmented” include mechanical fragmentation methods such as crushing, slicing, splitting, and cutting tumor tissue, as well as any other method for destroying 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), peripheral blood mononuclear cells are preferably irradiated allogeneic peripheral blood mononuclear cells.

[0335] The terms “peripheral blood lymphocytes” and “PBL” refer to expanded T cells 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 donor-derived whole blood or apheresis products by positive or negative selection of T cell phenotypes, such as CD3+CD45+ T cell phenotypes.

[0336] The term "major histocompatibility complex" or "MHC" refers to a large locus on vertebrate DNA containing a set 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, yielding two major products: MHC class I and MHC class II molecules. MHC class I proteins present endogenous antigens originating from the cytoplasm. MHC class II proteins present exogenous antigens originating extracellularly from foreign substances 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 these are incorporated herein by reference. In MHC dominant recognition, allogeneic MHC molecules may be more compatible with the T cell receptor and result in strong binding with less dependence on peptides that bind to the MHC molecule.

[0338] The terms "human leukocyte antigen," "HLA," or "HLA complex" refer 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 cells” and “target cell lines” refer to cells that are the target of T cell assays, 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 expressing 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 one embodiment, the target cell line is any two combinations of the Raji cell line, Thp1 cell line, Ramos cell line, U937 cell line, and Daudi cell line. In one embodiment, the target cell line is any three combinations of the Raji cell line, Thp1 cell line, Ramos cell line, U937 cell line, and Daudi cell line. In one embodiment, the target cell line is any four combinations of the Raji cell line, Thp1 cell line, Ramos cell line, U937 cell line, and Daudi cell line. In one embodiment, the target cell line is a combination of the Raji cell line, Thp1 cell line, Ramos cell line, U937 cell line, and Daudi cell line. In one embodiment, the target cell line is a mixed tumor target cell line. In one embodiment, the target cell line is an alloreactive target cell line. In one embodiment, 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 at least two combinations of the following cell lines: Raji cell line, Ramos cell line, Thp1 cell line, U937 cell line, and Daudi cell line.

[0340] The terms “Raji cells” or “Raji cell line” refer to Burkitt lymphoma cell lines possessing B-cell characteristics, available from multiple vendors including the American Type Culture Collection (Manassas, VA, USA), as well as their 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 respective disclosures of which are incorporated herein by reference. Raji cells lack membrane-bound immunoglobulins but possess receptors for IgG Fc, C3b, C3d, and Clq. In some embodiments, Raji cells or their derivatives, variants, modifications, or progeny are target cells. In one embodiment, Raji cells are genetically modified to express a fluorescent, phosphorescent, chemiluminescent, or bioluminescent label, thereby disrupting their cell membrane, which releases the fluorescent, phosphorescent, chemiluminescent, or bioluminescent label into the culture medium for potential use in detection. For example, the genetic modification approach for bioluminescent detection described in U.S. Patent No. 10,415,015 (whose disclosure is incorporated herein by reference) may be used to modify Raji cells.

[0341] The terms “Thp1 cells” or “Thp1 cell line” are also known as “THP-1 cells” or “THP-1 cell line” and refer to a human acute monocytic leukemia cell line, as well as its derivatives, variants, modifications, and progeny, available from multiple vendors, including the American Type Culture Collection (Manassas, VA, USA). Thp1 cell lines are described in Tsuchiya, et al., Int. J. Cancer 1980, 26, 171-176 and Bosshart and Heinzelmann, Ann. Transl. Med. 2016, 4(21), 438, and each of these disclosures is 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, thereby releasing the fluorescent, phosphorescent, chemiluminescent, or bioluminescent label into the culture medium for potential use in detection. For example, the gene modification approach for bioluminescence detection described in U.S. Patent No. 10,415,015 (the disclosure thereof is incorporated herein by reference) may be used to modify Thp1 cells.

[0342] The terms “Ramos cells” or “Ramos cell lines” refer to human Burkitt lymphoma cell lines, as well as their derivatives, variants, modifications, and progeny, available from multiple vendors, including American Type Culture Collection (Manassas, VA, USA). Ramos cell lines are 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, thereby disrupting their cell membrane, which releases the fluorescent, phosphorescent, chemiluminescent, or bioluminescent label into the culture 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) may be used to modify Ramos cells.

[0343] The terms “U937 cells” or “U937 cell line” refer to a human histiocytic lymphoma cell line, as well as its derivatives, variants, modifications, and progeny, available from multiple vendors, including the American Type Culture Collection (Manassas, VA, USA). The U937 cell line is described in Kraus, et al., J. Clin. Microbiol. 2007, 45, 3777-3780, and its disclosure 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, which, upon disruption of their cell membrane, is released into the culture medium for potential use in detection. For example, the gene modification approach for bioluminescence detection described in U.S. Patent No. 10,415,015 (the disclosure thereof is incorporated herein by reference) may be used to modify U937 cells.

[0344] The terms “Daudi cells” or “Daudi cell lines” refer to human Burkitt lymphoma cell lines, as well as their derivatives, variants, modifications, and progeny, available from multiple vendors, including American Type Culture Collection (Manassas, VA, USA). Daudi cell lines are 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, thereby disrupting their cell membrane, which releases the fluorescent, phosphorescent, chemiluminescent, or bioluminescent label into the culture 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) may be used to modify Daudi cells.

[0345] The terms “negative control,” “negative control cells,” and “negative control cell line” refer to cells used as negative controls in T cell assays, including TIL, MIL, or PBL assays. In some embodiments, target cells lack MHC class I and class II expression. In some embodiments, target cells lack MHC class I expression. In some embodiments, target cells lack MHC or HLA class I expression. In some embodiments, target cells express MHC or HLA class I and / or class II at minimal levels.

[0346] The terms “K562 cells” or “K562 cell line” refer to a human Caucasian chronic myeloid leukemia cell line having a lymphoblastic 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 some 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 its variant, such as a monoclonal antibody, including human, humanized, chimeric, or mouse antibodies against the CD3 receptor on 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, teprizumab, and bicilizumab.

[0348] The term "OKT-3" (also referred to herein as "OKT3") refers to a monoclonal antibody or its biosimilar or variant, including human, humanized, chimeric, or mouse antibodies against the CD3 receptor at the T cell antigen receptor of mature T cells, and includes commercially available forms such as OKT-3 (30 ng / mL, MACS GMP CD3 pure, Miltenyi Biotech, Inc., San Diego, CA, USA) and muromonab, or their variants, conserved amino acid substitutions, glycoforms, or biosimilars. 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). Hybridomas capable of producing OKT-3 are deposited in the American Type Culture Collection and assigned ATCC accession number CRL8001. Hybridomas capable of producing OKT-3 are also deposited in 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, conserved 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. Table 2 shows the amino acid sequence of recombinant human IL-2 suitable for use in the present invention (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 recombinant IL-2 forms commercially available from CellGenix, Inc., Portsmouth, NH, USA (CELLGRO GMP) or ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (catalog number CYT-209-b) and other commercial equivalents from other vendors. Aldesleukin (des-alanil-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 this invention is shown in Table 2 (SEQ ID NO: 4). The term IL-2 also encompasses the pegylated IL-2 forms 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 US2014 / 0328791A1 and International Patent Application Publication 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. Patents 4,766,106, 5,206,344, 5,089,261, and 4902,502, the disclosures of which are incorporated herein by reference.Formulations of IL-2 suitable for use in the present invention are described in U.S. Patent No. 6,706,289, the disclosure of which is incorporated herein by reference.

[0350] In some embodiments, the IL-2 form preferred for use in the present invention is THOR-707. Additional alternative forms of IL-2 preferred for use in the present invention are described in U.S. Patent Application Publication US2020 / 0181220A1 and U.S. Patent Application Publication US2020 / 0330601A1, the disclosures of which are incorporated herein by reference. In some embodiments, the IL-2 form preferred for use in the present invention is ALKS-4230. Additional alternative forms of IL-2 preferred for use in the present invention are described in U.S. Patent Application Publication US2021 / 0038684A1 and U.S. Patent No. 10,183,979, the disclosures of which are incorporated herein by reference. In some embodiments, the IL-2 form preferred for use in the present invention is an interleukin-2 (IL-2) conjugate comprising an isolated and purified IL-2 polypeptide and a complex moiety bound 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 amino acid residue numbering corresponds to Sequence ID No. 1 in U.S. Patent Application Publication 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-natural amino acid. In some embodiments, the non-natural amino acids are N6-azidoethoxy-L-lysine (AzK), N6-propargylethoxy-L-lysine (PraK), BCN-L-lysine, norbornenyllysine, TCO-lysine, methyltetrazinyllysine, allyloxycarbonyllysine, 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-methylphenylalanine, L-DOPA, fluorinated phenylalanine, isopropyl Contains L-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, O-allyl-tyrosine, O-methyl-L-tyrosine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, phosphonotyrosine, tri-O-acetyl-GlcNAcp-serine, L-phosphoserine, phosphonoserine, L-3-(2-naphthyl)alanine, 2-amino-3-((2-((3-(benzyloxy)-3-oxopropyl)amino)ethyl)ceranyl)propanoic acid, 2-amino-3-(phenylceranyl)propanoic acid, or selenocysteine. In some embodiments, the IL-2 conjugate has reduced affinity for the IL-2 receptor α (IL-2Rα) subunit compared to the wild-type IL-2 polypeptide.In some embodiments, the reduced affinity is a reduction of approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more than 99% of the binding affinity to IL-2Rα compared to the wild-type IL-2 polypeptide. In some embodiments, the reduced affinity is approximately 1x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 30x, 50x, 100x, 200x, 300x, 500x, or more than 1000x compared to the wild-type IL-2 polypeptide. In some embodiments, the composite moiety impairs or blocks the binding between IL-2 and IL-2Rα. In some embodiments, the composite moiety comprises a water-soluble polymer. In some embodiments, an additional composite moiety comprises a water-soluble polymer. In some embodiments, each of the water-soluble polymers independently includes polyethylene glycol (PEG), poly(propylene glycol) (PPG), copolymers of ethylene glycol and propylene glycol, poly(oxyethylated polyol), poly(olefin 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 combinations thereof. In some embodiments, each of the water-soluble polymers independently includes PEG. In some embodiments, PEG is linear PEG or branched PEG. In some embodiments, each of the water-soluble polymers independently includes 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 complex portion comprises a protein. In some embodiments, an additional complex portion comprises a protein.In some embodiments, each protein independently comprises albumin, transferrin, or transthyretin. In some embodiments, each protein independently comprises an Fc moiety. In some embodiments, each protein independently comprises an Fc moiety of IgG. In some embodiments, the complex moiety comprises a polypeptide. In some embodiments, an additional complex moiety comprises a polypeptide. In some embodiments, each protein 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 complex moiety is directly conjugated to the isolated and purified IL-2 polypeptide. In some embodiments, the complex moiety is indirectly conjugated 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 a Romant reagent dithiobis(succinimidylpropionate) DSP, 3'3'-dithiobis(sulfosuccinimidylpropionate) (DTSSP), disuccinimidylsverate (DSS), bis(sulfosuccinimidyl)sverate (BS), disuccinimidyl tartarate (DST), disulfosuccinimidyl tartarate (sulfoDST), ethylene glycobis(succinimidylsuccinate) (EGS), disuccinimidylglutarate (DSG), N,N'-disuccinimidylcarbonate (DSC), dimethyladipimidate (DMA), dimethylpimerimidate (DMP), dimethylsverimidate (DMS), dimethyl-3,3'-dithiobispropionimidate (DTBP), 1,4-di-(3'-(2'-pyridyldithio This includes (O) propionamide) butane (DPDPB), bismaleimide hexane (BMH), aryl halide-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 includes 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)toluamide]hexanoate (s (Luho-LC-sMPT), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sMCC), sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-sMCC), m-maleimidobenzoyl-N-hydroxysuccinimidate (MB), m-maleimidobenzoyl-N-hydroxysulfosuccinimidate (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)succinimidide ester (GMB), N-(γ-maleimidobutyryloxy)sulfosuccinimidide ester (sulfo-GMB), succinimidyl 6-((iodoacetyl)amino)hexanoate (sIAX), succinimidyl 6-[6-(((iodoacetyl)amino)hexanoate Noyl)amino]hexanoate (slAXX), succinimidyl 4-(((iodoacetyl)amino)methyl)cyclohexane-1-carboxylate (sIAC), succinimidyl 6-(((((4-iodoacetyl)amino)methyl)cyclohexane-1-carbonyl)amino)hexanoate (sIACX), p-nitrophenyliodoacetate (NPIA), carbonyl-reactive and sulfhydryl-reactive crosslinking agents, e.g., 4-(4-N-maleimidophenyl)butyrate hydrazide (MPBH), 4-(N-maleimidomethyl)cyclohexanoate. N-1-carboxyl-hydrazide-8(M2C2H), 3-(2-pyridyldithio)propionylhydrazide(PDPH), N-hydroxysuccinimidyl-4-azidosalicylic acid(NHs-AsA), N-hydroxysulfosuccinimidyl-4-azidosalicylic acid(sulfo-NHs-AsA), sulfosuccinimidyl-(4-azidosalicylamide)hexanoate(sulfo-NHs-LC-AsA), sulfosuccinimidyl-2-(p-azidosalicylamide)ethyl-1,3'-dithiopropionate(sAsD), N-hydroxysucci Imidyl-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-nitrobenzamide)-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 Cete (sulfo-sAMCA), p-nitrophenyl diazopirubate (pNPDP), p-nitrophenyl-2-diazo-3,3,3-trifluoropropionate (PNP-DTP), 1-(ρ-azidosalicylamide)-4-(iodoacetamide)butane (AsIB), N-[4-(ρ-azidosalicylamide)butyl]-3'-(2'-pyridyldithio)propionamide (APDP), benzophenone-4-iodoacetamide, p-azidobenzoylhydrazide (ABH), 4-(ρ-azidosalicylamide)butylamine (AsBA),or comprising p-azidophenylglyoxal (APG). In some embodiments, the linker comprises a cleavable linker, optionally comprising 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 an incleavable linker. In some embodiments, the linker comprises a maleimide group, optionally comprising 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), its derivatives, or analogs. In some embodiments, the composite portion can extend the serum half-life of the IL-2 conjugate. In some embodiments, an additional composite portion can extend the serum half-life of the IL-2 conjugate. In some embodiments, a suitable IL-2 form for use in the present invention is a fragment of any of the IL-2 forms described herein. In some embodiments, a suitable IL-2 form for use in the present invention is pegged as disclosed in U.S. Patent Application Publication US2020 / 0181220A1 and U.S. Patent Application Publication US2020 / 0330601A1. In some embodiments, a suitable form of IL-2 for use in the present invention is an IL-2 conjugate comprising an IL-2 polypeptide comprising N6-azidoethoxy-L-lysine (AzK) covalently bonded to a polyethylene glycol (PEG) complex, 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 2020 / 0330601 (listed herein as SEQ ID NO: 5 in Table 2), and AzK has amino acid positions K35, F42, F44,The amino acid is substituted at positions K43, E62, P65, R38, T41, E68, Y45, V69, or L72. In some embodiments, the IL-2 polypeptide contains a one-residue N-terminal deletion compared 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, the IL-2 form preferred 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, the IL-2 form preferred for use in the present invention is ALKS-4230. The IL-2 form preferred for use in the present invention is described 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 containing amino acids 24-452 of Sequence ID No. 2 in U.S. Patent No. 10,183,979 (listed herein as Sequence ID No. 7 in Table 2, U.S. Patent No. 10,183,979). In some embodiments, a suitable IL-2 form for use in the present invention is a fusion protein containing amino acids 24-452 of Sequence ID No. 2 in U.S. Patent No. 10,183,979, or an amino acid sequence homologous to amino acids 24-452 of Sequence 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 Sequence ID No. 2 in U.S. Patent No. 10,183,979, and having receptor antagonist activity of amino acids 24-452 of Sequence ID No. 2 in U.S. Patent No. 10,183,979. In some optional embodiments, a suitable IL-2 form 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 with IL-1Rα and possessing receptor antagonist activity of IL-Rα, the second fusion partner comprises all or part of an immunoglobulin including an Fc region, and the mucin domain polypeptide linker isThe fusion protein comprises an amino acid sequence having at least 90% sequence identity to Sequence ID No. 14 in U.S. Patent No. 10,183,979 (listed herein as Sequence ID No. 8 in Table 2) or Sequence ID No. 14 in U.S. Patent No. 10,183,979 (listed herein as Sequence ID No. 8 in Table 2), and the half-life of the fusion protein is such that the first fusion partner has no second fusion partner in the absence of a mucin domain polypeptide linker. [Table 2]

[0351] In some embodiments, a suitable IL-2 configuration for use in the present invention includes a heavy chain variable region (V) which comprises complementarity-determining regions HCDR1, HCDR2, and HCDR3. H ) and the light chain variable region (V) including LCDR1, LCDR2, and LCDR3 L ) and V H or V L The antibody cytokine transplantation protein comprises an IL-2 molecule or fragment thereof transplanted into the CDR, and the antibody cytokine transplantation protein preferentially expands T effector cells over regulatory T cells. In one embodiment, the antibody cytokine transplantation protein comprises a heavy chain variable region (V) including complementarity-determining regions HCDR1, HCDR2, and HCDR3. H ) and the light chain variable region (V) including LCDR1, LCDR2, and LCDR3 L ) and V H or V L The regimen comprises an IL-2 molecule or fragment thereof transplanted into a CDR, the IL-2 molecule being mutain, and the antibody cytokine transplant protein preferentially expands T effector cells over regulatory T cells. In one embodiment, the IL-2 regimen comprises administration of an antibody described in U.S. Patent Application Publication No. 2020 / 0270334A1, the disclosure of which is incorporated herein by reference. In one embodiment, the antibody cytokine transplant protein comprises a heavy chain variable region (VH) containing complementarity-determining regions HCDR1, HCDR2, HCDR3, and a light chain variable region (VL) containing LCDR1, LCDR2, LCDR3, and VH or V L The CDR is implanted with an IL-2 molecule or fragment thereof, the IL-2 molecule is mutein, the antibody cytokine transplant protein preferentially expands T effector cells over regulatory T cells, the antibody is an IgG class light chain including SEQ ID NO. 69 in U.S. Patent Publication US2020 / 0270334A1, an IgG class heavy chain including SEQ ID NO. 53 in U.S. Patent Publication US2020 / 0270334A1, an IgG class light chain including SEQ ID NO. 37 in U.S. Patent Publication US2020 / 0270334A1 and U.S. The present invention further includes IgG class heavy chains and IgG class light chains selected from the group consisting of an IgG class heavy chain containing SEQ ID NO. 21 in U.S. Patent Publication No. US2020 / 0270334A1, an IgG class light chain containing SEQ ID NO. 69 in U.S. Patent Publication No. US2020 / 0270334A1, an IgG class heavy chain containing SEQ ID NO. 21 in U.S. Patent Publication No. US2020 / 0270334A1, and an IgG class light chain containing SEQ ID NO. 37 and an IgG class heavy chain containing SEQ ID NO. 53 in U.S. Patent Publication No. US2020 / 0270334A1.

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

[0353] The insertion of the IL-2 molecule may occur in or near the N-terminal region of the CDR, in the middle region of the CDR, or in or near the C-terminal region of the CDR. In some embodiments, the antibody-cytokine transplant protein contains an IL-2 molecule incorporated into the CDR, and the IL-2 sequence does not frameshift the CDR sequence. In some embodiments, the antibody-cytokine transplant protein contains an IL-2 molecule incorporated into the CDR, and the IL-2 sequence replaces all or part of the CDR sequence. The replacement by the IL-2 molecule may occur in the N-terminal region of the CDR, in the middle region of the CDR, or in or near the C-terminal region of the CDR. The replacement by the IL-2 molecule may occur in just one or two amino acids of the CDR sequence, or in the entire CDR sequence.

[0354] In some embodiments, the IL-2 molecule is directly transplanted into 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 transplanted into 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 IL-2 mutein. In some cases, IL-2 mutein includes the R67A substitution. In some embodiments, IL-2 mutein includes 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, IL-2 mutein includes 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 one embodiment, the antibody cytokine transplant protein includes HCDR1 selected from the group consisting of SEQ ID NOs. 7, 10, 13, and 16 of U.S. Patent Application Publication US2020 / 0270334A1. In another embodiment, the antibody cytokine transplant protein includes HCDR1 selected from the group consisting of SEQ ID NOs. 7, 10, 13, and 16 of U.S. Patent Application Publication 2020 / 0270334A1, and HCDR2 selected from the group consisting of SEQ ID NOs. 8, 11, 14, and 17. In yet another embodiment, the antibody cytokine transplant protein includes HCDR1 selected from the group consisting of SEQ ID NOs. 7, 10, 13, and 16 of U.S. Patent Application Publication US2020 / 0270334A1, HCDR2 selected from the group consisting of SEQ ID NOs. 8, 11, 14, and 17, and HCDR3 selected from the group consisting of SEQ ID NOs. 9, 12, 15, and 18. In one embodiment, the antibody cytokine transplant protein includes a VH region containing the amino acid sequence of SEQ ID NO: 19 of U.S. Patent Application Publication US2020 / 0270334A1. In another embodiment, the antibody cytokine transplant protein includes a heavy chain containing the amino acid sequence of SEQ ID NO: 21 of U.S. Patent Application Publication US2020 / 0270334A1. In yet another embodiment, the antibody cytokine transplant protein includes a VL region containing the amino acid sequence of SEQ ID NO: 35 of U.S. Patent Application Publication US2020 / 0270334A1. In yet another embodiment, the antibody cytokine transplant protein includes a light chain containing the amino acid sequence of SEQ ID NO: 37 of U.S. Patent Application Publication US2020 / 0270334A1. In yet another embodiment, the antibody cytokine transplant protein includes a VH region containing the amino acid sequence of SEQ ID NO: 19 of U.S. Patent Application Publication US2020 / 0270334A1, and a VL region containing the amino acid sequence of SEQ ID NO: 35. In one embodiment, the antibody cytokine transplant protein includes a heavy chain region containing the amino acid sequence of SEQ ID NO: 21 of U.S. Patent Application Publication US2020 / 0270334A1, and a light chain region containing the amino acid sequence of SEQ ID NO: 37.In one embodiment, the antibody cytokine transplant protein comprises IgG.IL2R67A.H1 of U.S. Patent Application Publication No. 2020 / 0270334A1. In another embodiment, the antibody component of the antibody cytokine transplant protein described herein comprises an immunoglobulin sequence, a framework sequence, or a CDR sequence of palivizumab.

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

[0358] In one embodiment, the antibody cytokine transplant protein described herein has the sequence shown 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. When activated 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 this 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 this 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 set of signals important for T cell development in the thymus and survival in the periphery. Recombinant human IL-7 suitable for use in this invention is commercially available from several suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (catalog no. CYT-254) and ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-15 recombinant protein, catalog no. Gibco PHC0071). Table 2 shows the amino acid sequence of recombinant human IL-7 suitable for use in the present invention (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, conserved 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 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 (recombinant human IL-15 protein, catalog number 34-8159-82). Table 2 shows the amino acid sequence of recombinant human IL-15 suitable for use in the present invention (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, conserved 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 is primarily used in 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 no. CYT-408-b) and ThermoFisher Scientific, Inc., Waltham, MA, USA (recombinant human IL-21 protein, catalog no. 14-8219-80). Table 2 shows the amino acid sequence of recombinant human IL-21 suitable for use in this invention (SEQ ID NO: 12).

[0363] Where “antitumor effective dose,” “tumor inhibitory effective dose,” or “therapeutic dose” is indicated, the exact amount of the composition of the present invention administered may be determined by a physician, taking into account individual differences in the patient’s age, weight, tumor size, degree of infection or metastasis, and health status. Generally, tumor-infiltrating lymphocytes (e.g., secondary TILs or genetically modified cytotoxic lymphocytes) described herein are 10 per kg of body weight. 4 ~10 11 Cells (for example, 10 per kg of 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 cells may be administered in doses (including all integer values ​​within their range). Tumor-infiltrating lymphocytes (including, in some cases, genetically modified cytotoxic lymphocytes) compositions may also be administered multiple times in these doses. Tumor-infiltrating lymphocytes (including, in some cases, genetically modified ones) may be administered by using infusion techniques commonly known in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). The optimal dosage and treatment regimen for a particular patient can be readily determined by medical professionals by monitoring the patient for signs of the disease and adjusting treatment as appropriate.

[0364] The terms “hematological malignancies” or “hematological malignancies” or related terms refer to cancers and tumors of hematopoietic and lymphoid tissues in mammals, including but not limited to the blood, bone marrow, lymph nodes, and lymphoid tissues. Hematological malignancies are also called “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 myeloid leukemia (CML), acute monocytic leukemia (AMoL), Hodgkin lymphoma, and non-Hodgkin lymphoma. The term “B-cell hematological malignancies” refers to hematological malignancies that affect B cells.

[0365] The term "liquid tumor" refers to an abnormal mass of cells that is naturally fluid. 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 liquid tumors circulating in the peripheral blood, may also be referred to herein as PBLs. The terms MIL, TIL, and PBL are used interchangeably herein and differ only based on the tissue type from which the cells originate.

[0366] As used herein, the term “microenvironment” 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 the tumor from host immunity, promote treatment resistance, and provide a niche for successful dominant metastasis.” Although tumors express antigens that should be recognized by T cells, tumor clearance by the immune system is rare due to immunosuppression by the microenvironment.

[0367] In some embodiments, the present invention comprises a method for treating cancer with a TIL population, wherein the patient is pre-treated with non-myeloablative chemotherapy prior to the infusion of the TIL according to the present invention. In some embodiments, a TIL population may be provided, and the patient is pre-treated with non-myeloablative chemotherapy prior to the infusion of the TIL according to the present invention. In some embodiments, the non-myeloablative chemotherapy is cyclophosphamide 60 mg / kg / day for 2 days (27 and 26 days before TIL infusion) and fludarabine 25 mg / m2 / day for 5 days (27 to 23 days before TIL infusion). In some embodiments, after the non-myeloablative chemotherapy according to the present invention and TIL infusion (day 0), the patient receives intravenous infusion of IL-2 at 720,000 IU / kg every 8 hours until physiological tolerance is reached.

[0368] Experimental findings indicate that lymphocyte depletion prior to adoptive transfer of tumor-specific T lymphocytes plays a crucial role in enhancing therapeutic efficacy by eliminating regulatory T cells and competing elements of the immune system ("cytokine sinks"). Therefore, some embodiments of the present invention utilize a lymphocyte depletion step (sometimes referred to as "immunosuppressive conditioning") in the patient before introducing the TIL of the present invention.

[0369] The terms “effective dose” or “therapeutic effective dose” refer to the amount of a compound or combination of compounds described herein that is sufficient to achieve the intended use, including but not limited to disease treatment. The therapeutic effective dose 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 the 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 specific 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 through which the compound is carried.

[0370] Terms such as “treatment,” “to treat,” and “to heal” refer to obtaining a desired pharmacological and / or physiological effect. Such effect may be preventive in terms of completely or partially preventing the disease or its symptoms, and / or therapeutic in terms of partial or complete cure of the disease and / or side effects resulting from it. “Treatment,” as used herein, encompasses any treatment of a disease in mammals, particularly humans, including (a) preventing the development of the disease in a subject susceptible to the disease but not yet diagnosed with the disease, (b) suppressing the disease, i.e., inhibiting its onset or progression, and (c) mitigating the disease, i.e., causing regression of the disease and / or reducing one or more symptoms of the disease. “Treatment” is also intended to encompass the delivery of a drug to provide a pharmacological effect even in the absence of a disease or condition. For example, “treatment” includes the delivery of a composition that can induce an immune response or provide immunity in the absence of a medical condition, for example, in the case of a vaccine.

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

[0372] In the context of two or more nucleic acids or polypeptides, the terms “sequence identity,” “identity percentage,” and “sequence identity percentage” (or their synonyms, e.g., “99% identical”) refer to two or more sequences or subsequences that are identical or, when compared and aligned for maximum match (with gaps introduced as necessary), without considering any conserved amino acid substitutions as part of sequence identity, have a certain percentage of identical nucleotide or amino acid residues. The identity percentage can be measured using sequence comparison software or algorithms, or by visual inspection. Various algorithms and software that can be used to obtain amino acid or nucleotide sequence alignments are known in the art. Suitable programs for determining the sequence identity percentage include, for example, the BLAST program suite available from the BLAST website of the U.S. National Center for Biotechnology Information. Comparisons 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 using specific 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, antibodies or fusion proteins having an amino acid sequence different from that of a reference antibody due to one or more substitutions, deletions, and / or additions within or adjacent to the amino acid sequence of the reference antibody. A variant may have one or more conservative substitutions in its amino acid sequence compared to that of the reference antibody. Conservative substitutions may include, for example, substitutions of similarly charged or uncharged amino acids. A variant retains the ability to specifically bind to the antigen of the reference antibody. The term “variant” also includes pegylated antibodies or proteins.

[0374] In this specification, “tumor-infiltrating lymphocytes” or “TIL” means the population of cells initially obtained as leukocytes that have left the bloodstream and migrated to the tumor. TILs include CD8 + Cytotoxic T cells (lymphocytes), Th1 and Th17 CD4 + TILs include, but are not limited to, T cells, natural killer cells, dendritic cells, and M1 macrophages. TILs include both primary and secondary TILs. A “primary TIL” is obtained from a patient tissue sample as outlined herein (sometimes referred to as “freshly collected”), and a “secondary TIL” is any expanded or proliferated TIL cell population as considered herein, and includes, but is not limited to, bulk TILs, expanded TILs (“REP TILs”), and “reREP TILs” as considered herein. A reREP TIL may include, for example, a second expanded TIL or a second additional expanded TIL (such as the one described in step D of Figure 8, which includes a TIL referred to as a reREP TIL).

[0375] TILs can generally be defined either biochemically using cell surface markers or functionally by their ability to invade tumors and achieve therapeutic effects. TILs can generally be classified by the expression of one or more biomarkers from among CD4, CD8, TCRαβ, CD27, CD28, CD56, CCR7, CD45Ra, CD95, PD-1, and CD25. Furthermore, and alternatively, TILs can be functionally defined by their ability to invade solid tumors upon reintroduction into a patient. TILs can be further characterized by their potency; for example, a TIL may be considered potent if its interferon (IFN) release exceeds approximately 50 pg / mL, approximately 100 pg / mL, approximately 150 pg / mL, or approximately 200 pg / mL.

[0376] The term "deoxyribonucleotide" encompasses both natural and synthetic, unmodified and modified deoxyribonucleotides. Modifications include alterations to the sugar moiety, base moiety, and / or the bonding between deoxyribonucleotides in oligonucleotides.

[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 the bD-ribofuranose moiety. The term RNA includes isolated RNA such as double-stranded RNA, single-stranded RNA, and partially purified RNA, as well as essentially pure RNA, synthetic RNA, recombinantly produced RNA, and modified RNA that differs from naturally occurring RNA due to the addition, deletion, substitution, and / or alteration of one or more nucleotides. The nucleotides of RNA molecules described herein may also include non-standard nucleotides such as unnaturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These modified RNAs may be referred to as analogs or analogs of naturally occurring RNA.

[0378] The terms “pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” are intended to include all solvents, dispersions, coatings, antibacterial and antifungal agents, isotonic and absorption retardants, and inactive components. The use of such pharmaceutically acceptable carriers or excipients for pharmaceutically active ingredients is well known in the art. Unless any conventional pharmaceutically acceptable carrier or excipient is incompatible with the pharmaceutically active ingredient, its use in the therapeutic compositions of the present invention is intended. Additional pharmaceutically active ingredients, such as other drugs, may 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 decimal place 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 tolerances encompassed by the terms “about” or “approximately” depend on the specific system under study and will be readily understood by those skilled in the art. Furthermore, as used herein, the terms “about” and “approximately” mean that dimensions, sizes, formulations, parameters, shapes, and other quantities and features are not and do not need to be precise, but may be approximate and / or greater or less, as needed, to reflect tolerances, conversion factors, rounding, measurement errors, and other factors known to those skilled in the art. In general, dimensions, sizes, formulations, parameters, shapes, or other quantities or features are “about” or “approximately,” whether so explicitly stated. Note that configurations describing very different embodiments of size, shape, and dimension may be adopted.

[0380] When used in the attached claims, the transitional terms “including,” “essentially consisting of,” and “consisting of,” in their original and amended forms, define the claims in terms of whether any additional unenumerated claim elements or steps, if present, are excluded from the claims. The term “including” is intended to be inclusive or free form and does not exclude any additional unenumerated elements, methods, steps, or materials. The term “consisting of” excludes any elements, steps, or materials other than those specified in the claims, and in the latter case, also excludes any impurities typically associated with the specified material(s). The term “essentially consisting of” limits the claims to such an extent that it does not substantially affect the specific elements, steps, or materials(s) and the basic and novel features(s) of the claimed invention. All compositions, methods, and kits described herein that embody the invention may be more specifically defined in alternative embodiments by any of the transitional terms “including,” “essentially consisting of,” and “consisting of.”

[0381] The terms “antibody” and its plural form “antibody” refer to the entire immunoglobulin and any antigen-binding fragment (“antigen-binding region”) or a single chain thereof. “Antibody” further refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or its antigen-binding region. Each heavy chain comprises a heavy chain variable region (V in this specification). H It consists of a light chain variable region (abbreviated as V in this specification) and a heavy chain steady region. The heavy chain steady region consists of three domains: CH1, CH2, and CH3. Each light chain is composed of a light chain variable region (V in this specification). L It consists of a (abbreviated as) and a light chain steady region. The light chain steady region consists of one domain, C L It is composed of the antibody V H and V L The region is called the Complementarity Determination Region (CDR) or Hypervariability Region (HVR), and can be further subdivided into hypervariability regions, which may contain more conserved regions called framework regions (FR). H and VL It consists of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxyl terminus: 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 can mediate the binding of immunoglobulins to various cells of the immune system (e.g., effector cells) and to host tissues or factors including 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, if it is presented by a major histocompatibility complex (MHC) molecule. As used herein, the term “antigen” also encompasses T cell epitopes. Antigens can, in addition, 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 lymphocytes and / or T lymphocytes. In some cases, this may require the antigen to contain or be bound to 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 the corresponding antibody or TCR in a typically highly specific and selective manner, and does not react with a number of other antibodies or TCRs that may be induced by other antigens.

[0383] The terms “monoclonal antibody,” “mAb,” and “monoclonal antibody composition,” or their plural forms, refer to preparations of antibody molecules with a single molecular composition. Monoclonal antibody compositions exhibit a single binding specificity and affinity for a particular epitope. Monoclonal antibodies specific to a particular receptor can be produced using knowledge and techniques in the art, which involve injecting a suitable antigen for the test subject and then isolating hybridomas that express antibodies having the desired sequence or functional characteristics. The DNA encoding the monoclonal antibody can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to the 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 proteins to obtain the synthesis of monoclonal antibodies in recombinant host cells. Recombinant antibody production will be explained in more detail below.

[0384] As used herein, the terms “antigen-binding moiety” or “antigen-binding fragment” (or simply “antibody moiety” or “fragment”) of an antibody refer to one or more fragments of an antibody that possess the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody may be performed by fragments of a full-length antibody. Examples of binding fragments encompassed by the term “antigen-binding moiety” of an antibody include (i)V L , V H , C L (ii) a monovalent fragment consisting of the CH1 domain, a Fab fragment; (ii) a bivalent fragment containing two Fab fragments linked by disulfide crosslinks in the hinge region, a F(ab')2 fragment; (iii) V H and an Fd fragment consisting of the CH1 domain, (iv) a single arm of the antibody V L and V H Fv fragment consisting of domains, (v)V H or VL The fragment may consist of domain antibodies (dAb) fragments (Ward, et al., Nature, 1989, 341, 544-546), and (vi) isolated complementarity-determining regions (CDRs). The two domains of the Fv fragment, V L and V H These are encoded by separate genes, but these can be recombined using a recombination method. L and V H The regions can be joined by synthetic linkers, which allow them to be constructed as a single protein chain forming a monovalent molecule known as a single-stranded Fv (scFv), see, for example, 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 terms “antigen-binding portion” or “antigen-binding fragment” of the 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 in which both the framework and CDR regions have variable regions derived from human germline immunoglobulin sequences. Furthermore, if the antibody includes a constant region, the constant region also originates from a human germline immunoglobulin sequence. The human antibodies of the present invention may include amino acid residues not encoded by the human germline immunoglobulin sequence (e.g., mutations introduced by random or site-directed mutagenesis in vitro, or by somatic mutation in vivo). As used herein, the term “human antibody” is not intended to include antibodies in which a CDR sequence derived from the germline of another mammalian species, such as mouse, is transplanted onto a human framework sequence.

[0386] The term "human monoclonal antibody" refers to a single binding-specific antibody having variable regions in both its framework region and CDR region derived from human germline immunoglobulin sequences. In one embodiment, the human monoclonal antibody is produced by a hybridoma containing B cells obtained from a transgenic non-human animal, such as a transgenic mouse, which has a genome containing human heavy chain and light chain transgenes fused to immortalized cells.

[0387] As used herein, the term “recombinant human antibody” includes all human antibodies prepared, expressed, created, or isolated by recombinant means, for example: (a) antibodies isolated from animals (such as mice) that are transgenic or transchromosomal with respect to human immunoglobulin genes or hybridomas prepared therefrom (as further described below); (b) antibodies isolated from host cells transformed to express human antibodies, for example, 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 with other DNA sequences. Such recombinant human antibodies have variable regions in which the framework and CDR region are derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies may be subjected to in vitro mutagenesis (or, if transgenic animals with respect to human Ig sequences are used, in vivo somatic mutagenesis), and thus the V of recombinant antibodies H and V L The amino acid sequence of the region is human germline V H and V L These sequences are derived from and related to other sequences, but may not naturally exist within the human antibody germline repertoire in vivo.

[0388] As used herein, “isotype” refers to the antibody class (e.g., IgM or IgG1) encoded by a heavy chain constant region gene.

[0389] The phrases "antibody that recognizes an antigen" and "antibody that is specific to an antigen" are interchangeable in this specification with the term "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 an antibody with another active pharmaceutical ingredient or antibody. The terms “conjugate,” “antibody-drug conjugate,” “ADC,” or “immune conjugate” refer to an antibody or fragment thereof conjugated with another therapeutic portion, which may be conjugated to the antibodies described herein using methods available in the art.

[0391] The terms "humanized antibody," "humanized antibody(s)," and "humanized" are intended to refer to antibodies in which a CDR sequence derived from the germline of another mammalian species, such as mouse, has been transplanted into a human framework sequence. Additional framework region modifications can be made within the human framework sequence. The humanized form of a non-human (e.g., mouse) antibody is a chimeric antibody containing a minimal sequence derived from a non-human immunoglobulin. In most cases, a humanized antibody is a human immunoglobulin (recipient antibody), in which residues from the recipient's hypervariable region are replaced by residues from the 15 hypervariable regions of a non-human species (donor antibody) or non-human primate, such as mouse, rat, or rabbit, that possess the desired specificity, affinity, and capabilities. In some cases, Fv framework (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may contain residues not found in the recipient or donor antibody. These modifications are made to further improve the performance of the antibody. Generally, humanized antibodies will contain at least one, typically two, substantially all of the variable domains, where all or substantially all of the hypervariable loop corresponds to that of a non-human immunoglobulin, and all or substantially all of the FR region corresponds to that of a human immunoglobulin sequence. Humanized antibodies will also optionally contain the immunoglobulin constant region (Fc), typically at least a portion of the constant region 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 may also be modified to use any Fc variant known to provide effector function and / or improved (e.g., reduced) FcR binding.Fc variants include, for example, International Patent Application Publications 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 Patent Nos. 0963A1, WO2005 / 077981A2, WO2005 / 092925A2, WO2005 / 123780A2, WO2006 / 019447A1, WO2006 / 047350A2, and WO2006 / 085967A2, as well as U.S. Patent Nos. 5,648,260, 5,739,277, 5,834,250, 5,869,046, and 6,096 This may include any one of the amino acid substitutions disclosed in Nos. 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 (their disclosures are incorporated herein by reference).

[0392] The term "chimeric antibody" is intended to refer to an antibody in which the variable region sequence originates from one species and the constant region sequence originates from another species, for example, an antibody in which the variable region sequence originates from a mouse antibody and the constant region sequence originates from a human antibody.

[0393] A "bispecific antibody (diabody)" is a small antibody fragment that has two antigen-binding sites. The fragment is the same polypeptide chain (VH -V L or V L -V H ) contains a light chain variable domain (V L ) connected to the heavy chain variable domain (V H ) includes. If a linker that is too short to pair two domains on the same chain is used, the domains are paired with complementary domains on another chain, creating two antigen-binding sites. Bispecific antibodies are described more fully, for example, in European Patent No. EP404,097, International Patent Publication No. WO93 / 11161, and in Bolliger, et al., Proc. Natl. Acad. Sci. USA 1993, 90, 6444-6448.

[0394] The term "glycosylation" refers to modified derivatives of antibodies. Non-glycosylated antibodies lack glycosylation. Glycosylation can be altered, for example, to increase the affinity of an antibody to an antigen. Such carbohydrate modifications can be achieved, for example, by altering one or more glycosylation sites in the antibody sequence. For example, one or more amino acid substitutions can be made, resulting in the exclusion of glycosylation sites in one or more variable region frameworks, thereby eliminating glycosylation at those sites. As described in U.S. Patents 5,714,350 and 6,350,861, non-glycosylation can increase the affinity of an antibody to an antigen. In addition or alternatively, antibodies with modified types of glycosylation can be produced, for example, low-fucosylated antibodies with a reduced amount of fucosyl residues or antibodies with increased bipartite GlcNac structures. Such alterations in glycosylation patterns have been demonstrated to increase the capacity of antibodies. Such carbohydrate modifications can be achieved, for example, by expressing the antibody in host cells in which the glycosylation mechanism has been altered. Cells with altered glycosylation mechanisms 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), so that antibodies expressed in the Ms704, Ms705, and Ms709 cell lines lack fucose on their carbohydrates. The Ms704, Ms705, and Ms709 FUT8- / - cell lines were created by targeted disruption of the FUT8 gene in CHO / DG44 cells using two substitution vectors (see, for example, U.S. Patent Publication No. 2004 / 0110704 or Yamane-Ohnuki, et al., Biotechnol. Bioeng., 2004, 87, 614-622).As another example, European Patent EP1,176,195 describes a cell line having a functionally disrupted FUT8 gene that encodes a fucosyltransferase, thereby causing antibodies expressed in such cell lines to exhibit hypofucosylation by reducing or eliminating alpha-1,6 linkage-related enzymes, and also describes cell lines with low or no enzymatic activity for attaching fucose to N-acetylglucosamine bound to the Fc region of antibodies, such as the rat myeloma cell line YB2 / 0 (ATCC CRL 1662). International Patent Publication WO03 / 035835 describes the Lec13 cell line, a variant CHO cell line that has reduced ability to attach fucose to Asn(297)-linked carbohydrates and also results in hypofucosylation of antibodies expressed in its host cells (see also Shields, et al., J. Biol. Chem. 2002, 277, 26733-26740). International Patent Publication WO99 / 54342 describes a cell line engineered to express a glycoprotein-modifying glycosyltransferase (e.g., beta(1,4)-N-acetylglucosaminyltransferase III (GnTIII)), in which the antibody expressed in the engineered cell line exhibits an increase in bifacial GlcNac structure, resulting in increased ADCC activity of the antibody (see also Umana, et al., Nat. Biotech. 1999, 17, 176-180). Alternatively, fucosidase enzymes can be used to cleave fucose residues in antibodies. For example, fucosidase alpha-L-fucosidase removes fucosyl residues from antibodies, as described in Tarentino, et al., Biochem. 1975, 14, 5516-5523.

[0395] "Pegation" refers to a modified antibody or antibody fragment that is reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions that one or more PEG groups adhere to the antibody or antibody fragment. Pegylation can, for example, increase the biological (e.g., serum) half-life of an antibody. Preferably, pegylation is carried out via an acylation or alkylation reaction with a reactive PEG molecule (or a similar reactive water-soluble polymer). As used herein, the term "polyethylene glycol" means mono(C1-C1) 10 ) is intended to encompass any of the forms of PEG used to derivatize other proteins, such as alkoxy- or aryloxy-polyethylene glycol, or polyethylene glycol-maleimide. The antibody to be pegylated may be a non-glycosylated antibody. Methods for pegylation are known in the art and can be applied to the antibody of the present invention as described, for example, in European Patent Nos. EP0154316 and EP0401384, and U.S. Patent No. 5,824,778 (each disclosure incorporated herein by reference).

[0396] The term "biosimilar" refers to a bioproduct that is very similar to a reference bioproduct approved in the United States, despite minor differences in clinically inactive components, including monoclonal antibodies or proteins, and where there are no clinically significant differences between the bioproduct and the reference product in terms of safety, purity, and potency. Furthermore, a similar bio or "biosimilar" drug is a biopharmaceutical that is similar to another biopharmaceutical already authorized for use by the European Medicines Agency. The term "biosimilar" is also used synonymously by regulatory bodies in other countries and regions. A bioproduct or biopharmaceutical is a medicine made from or derived from a biological source, 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), then a protein approved by a drug regulatory authority for aldesleukin is either a "biosimilar" of aldesleukin or "its biosimilar" of aldesleukin. In Europe, a similar biopharmaceutical or "biosimilar" drug is a biopharmaceutical that is similar to another biopharmaceutical that is already authorized for use by the European Medicines Agency (EMA). The legal basis for similar biological uses in Europe is Article 6 of the amended Regulation (EC) No 726 / 2004 and Article 10(4) of Directive 2001 / 83 / EC, and therefore in Europe, biosimilars may be authorized or approved for authorization or application under Article 6 of Regulation (EC) No 726 / 2004 and Article 10(4) of Directive 2001 / 83 / EC. The original biopharmaceutical that is already authorized is sometimes referred to as a "reference drug" in Europe. Some of the requirements for products considered biosimilars are outlined in the CHMP guidelines on biosimilar drugs. In addition, product-specific guidelines, including those related to monoclonal antibody biosimilars, are provided by the EMA on a product-by-product basis and are published on its website.The biosimilars described herein may be similar to the reference drug in terms of quality characteristics, bioactivity, mechanism of action, safety profile, and / or efficacy. Furthermore, the biosimilars may be used or intended to be used to treat the same conditions as the reference drug. Therefore, the biosimilars described herein may be considered to have similar or very similar quality characteristics to the reference drug. Alternatively, or further, the biosimilars described herein may be considered to have similar or very similar bioactivity to the reference drug. Alternatively, or further, the biosimilars described herein may be considered to have similar or very similar safety profiles to the reference drug. Alternatively, or further, the biosimilars described herein may be considered to have similar or very similar potency to the reference drug. As described herein, biosimilars in Europe are compared to reference drugs approved by the EMA. However, in some cases, biosimilars may be compared to biopharmaceuticals approved outside the European Economic Area in specific studies ("comparators" not approved by the EEA). Such studies include, for example, certain clinical studies and in vivo nonclinical studies. As used herein, the term “biosimilar” also refers to biopharmaceuticals that have been compared to, or may be compared to, a comparator not approved by the EEA. Certain biosimilars are proteins, such as antibodies, antibody fragments (e.g., antigen-binding moieties), 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. Biosimilars may contain amino acid sequences with 97% or more, e.g., 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of their reference drug. Biosimilars may contain one or more post-translational modifications, e.g., glycosylation, oxidation, deamidation, and / or cleavage, which differ from the post-translational modifications of the reference drug, provided that the differences do not result in an alteration of the drug’s safety and / or efficacy. Biosimilars may have the same or different glycosylation patterns as the reference drug.In particular, biosimilars may have different glycosylation patterns if the differences address or are intended to address safety concerns associated with the reference drug, although this is not exclusive. In addition, biosimilars may deviate from the reference drug in terms of, for example, their potency, dosage form, formulation, excipients, and / or presentation, provided that the safety and efficacy of the drug are not compromised. Biosimilars may include differences in, for example, 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 may exhibit different binding characteristics compared to the reference drug, and these different binding characteristics are not considered barriers to approval as a similar bioproduct by regulatory authorities such as the EMA. The term “biosimilar” is also used synonymously by regulatory bodies in other countries and regions.

[0397] III. Efficacy assay method and composition Without being limited to any particular theory, commercially viable TIL and T cell efficacy assays are considered 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 a timely manner to construct an assay. Consequently, unlike commercially available and clinical T cell therapies using transduced chimeric antigen receptors that have received regulatory approval using target-based assays in some cases, target-based assays are not viable on the timescale required for effective cancer therapy for polyclonal T cell products such as TILs, MILs, or PBL therapies or products. At the same time, bead-based assays are limited by their inability to replicate the innate activating and killing capabilities of TILs or T cells. For example, bead-based or plate-based assays utilizing anti-CD3 coatings bind to the CD3ε chain on TILs or T cells that are non-covalently associated with the TCR, when it is more preferable for CD3 to bind to the TCR itself. Thus, 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 co-stimulatory molecules, such as CD28 or CD137(4-1BB), on the beads also leads to unnatural and less desirable interactions, at least because the activation of these co-stimulatory molecules results in assays with low selectivity for cells present in tumors, such as TILs. This invention provides solutions to these problems. Without being limited to any particular theory, it is thought that the interaction of MHC or HLA on target cells with the TCR of T cells containing TILs, MILs, or PBLs produces an analyte that can be compared to an analyte produced by T cells containing TILs, MILs, or PBLs co-cultured with an MHC-negative control to evaluate one or more properties of T cells containing TILs, MILs, or PBLs, such as their potency, identity, or other useful properties, in order to assist in the manufacture or delivery of therapies for cancer.Furthermore, without being limited to any particular theory, since T cells such as TILs and MILs are found at tumor sites, or because PBLs have been previously activated by tumor exposure, such T cells, including TILs, MILs, and PBLs, are thought to be already cross-primed or primed T cells with already activated CD28 and 4-1BB.

[0398] In some embodiments, the present invention comprises a efficacy assay composition comprising Raji cells and T cells such as TILs co-cultured in a culture medium. In some embodiments, the present invention comprises a efficacy assay composition comprising Thp1 cells and T cells such as TILs co-cultured in a culture medium. In some embodiments, the present invention comprises a efficacy assay composition comprising Ramos cells and T cells such as TILs co-cultured in a culture medium. In some embodiments, the present invention comprises a efficacy assay composition comprising U937 cells and T cells such as TILs co-cultured in a culture medium. In some embodiments, the present invention comprises a efficacy assay composition comprising Daudi cells and T cells such as TILs co-cultured in a culture medium. In some embodiments, the efficacy assay composition comprises K562 cells and T cells such as TILs co-cultured in a culture medium and used as a negative control. In some embodiments, the efficacy assay composition comprises peripheral blood mononuclear cells (PBMCs) and T cells such as TILs co-cultured in a culture medium and used as a mixed lymphocyte reaction (MLR) positive control. In some embodiments, the TILs and PBMCs in the above embodiments are derived from the same patient. In some embodiments, the TIL is manufactured using a Gen2 or Gen3 process, or other manufacturing processes described herein. In some embodiments, the TIL is manufactured using at least one REP step. In some embodiments, the efficacy assay is performed using the TIL, MIL, or PBL produced using the extension or manufacturing processes described herein. In some embodiments, the TIL is manufactured using U.S. Patent Application Publication No. US2018 / 0282694A1 or U.S. Patents No. 10,130,659, 10,166,257, 10,272,113, 10,363,273, 10,398,734, 10,420,799, 10,463,697, and 10,537,59 After being manufactured using the processes described in No. 5, No. 10,646,517, No. 10,653,723, No. 10,693,330, No. 10,695,372, No. 10,894,063, and No. 10,905,718 (each of which disclosures is incorporated herein by reference), the product is tested using the efficacy assay described herein.In some embodiments, the TIL is manufactured using the process described in U.S. Patent No. 10,918,666 (the disclosure of which is incorporated herein by reference) and then tested with the efficacy assay described herein. In some embodiments, the TIL is manufactured 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 with the efficacy assay described herein. In some embodiments, the TIL is manufactured using the process described in International Patent Application Publication No. 2019 / 210131A1 (the disclosure of which is incorporated herein by reference) and then tested with the efficacy assay described herein. In some embodiments, the TIL is manufactured using the process described in International Patent Application Publication No. 2019 / 136456A1 (the disclosure of which is incorporated herein by reference) and then tested with the efficacy assay described herein. In some embodiments, TIL is manufactured using the process described in International Patent Application Publication No. 2021 / 123832A1 (the disclosure of which is incorporated herein by reference) and then tested with the efficacy assay described herein. In some embodiments, MIL and PBL are manufactured 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 with the efficacy assay described herein. In some embodiments, TIL is manufactured using the process described in International Patent Application Publication No. 2019 / 145711A1 (the disclosure of which is incorporated herein by reference) and then tested with the efficacy assay described herein. In some embodiments, TIL is manufactured using the process described in International Patent Application Publication No. 2020 / 152451A1 (the disclosure of which is incorporated herein by reference) and then tested with the efficacy assay described herein.

[0399] A. Assay Method In some embodiments, the present invention includes a method for determining the potency of a T cell product using target cells capable of binding to T cell receptors. In some embodiments, the present invention includes an assay based on allogeneic interaction (also referred to as MHC dominant recognition) between T cells or TIL, MIL, or PBL TCR complexes and the HLA-peptide complex of target cells. In some embodiments, the present invention includes an assay that utilizes MHC dominant recognition between T cells or TIL, MIL, or PBL TCR complexes and target cells to produce an analyte, which is compared to an analyte produced by co-culturing T cells or TILs with an MHC-negative control. In some embodiments, the binding of T cells or TIL, MIL, or PBL TCR complexes to target cells occurs via their alpha (α) and beta (β) chains. In some embodiments, the target cells express HLA. In some embodiments, the target cells are B cells. In some embodiments, the target cells are B cell lymphoblasts or B lymphoblasts. In some embodiments, the target cells are Burkitt lymphoma cells. In some embodiments, the target cells are myeloid cells. In some embodiments, the target cells are monocytes. In some embodiments, the target cells are acute monocytic leukemia cells. In some embodiments, the target cells are M5 subtype acute monocytic leukemia cells. In some embodiments, the target cells are Raji cells or their derivatives, variants, modifications, or offspring. In some embodiments, the target cells are Thp1 cells or their derivatives, variants, modifications, or offspring. In some embodiments, the target cells are Ramos cells or their derivatives, variants, modifications, or offspring. In some embodiments, the target cells are U937 cells or their derivatives, variants, modifications, or offspring. In some embodiments, the target cells are Daudi cells or their derivatives, variants, modifications, or offspring. In some embodiments, the target cells are melanocyte cells. In some embodiments, the target cells are HLA-A-02 positive melanocyte cells. In some embodiments, the target cells are melanoma cells. In some embodiments, the target cells are HLA-A-02 positive melanoma cells.In one embodiment, 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 an assay based on alloreactive TCR or HLA recognition occurring between tumor cells and T cells such as TIL, MIL, or PBL produced using the expansion or manufacturing process described herein. In some embodiments, the present invention includes an assay based on alloreactive TCR or HLA recognition occurring between Raji, Thp1, Ramos, U937, or Daudi cells, or their derivatives, variants, modifications, or progeny, and T cells such as TIL, MIL, or PBL produced using the expansion or manufacturing process described herein. In some embodiments, the present invention includes the use of the above embodiments in combination with a negative control described herein. In some embodiments, the negative control serves as a basal or baseline activity level of T cells such as TIL, MIL, or PBL for comparison with an MHC-dominant recognition-based T cell assay. 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 assay variability. In another embodiment, the negative control is a cell line lacking HLA or MHC to demonstrate that MHC dominance recognition is a primary variable in the efficacy assay.

[0401] In some embodiments, the present invention includes the use of K562 cells, or their derivatives, variants, modifications, or offspring, as a negative control as described herein, by co-culturing a T cell product such as a TIL product, a MIL product, or a 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-conjugated antibody-stimulated bioassays such as the anti-CD3 plate assay against IFN-γ.

[0402] In some embodiments, the present invention includes 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, a MIL product, or a PBL product, with target cells or a combination of target cells as a negative control. The use of HLA-blocking antibodies 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 extended TILs produced by methods described herein or known in the art is investigated. Investigating the potency and / or functionality of extended TILs allows for the characterization of clinical TIL product lots. In some embodiments, TIL potency is optionally defined as increased expression of a selective surface marker, expressed as the percentage of marker-positive cells or mean fluorescence intensity (MFI) when co-cultured with Raji cells compared to K562 cells. Alternatively, Raji cells, K562, and TILs may be cultured alone. In some embodiments, PBMCs derived from the same patient from which the TILs or MILs are obtained are used as a control. In some embodiments, bead-based CD3 / CD28 stimulation may also be used as a control. In some embodiments, bead-based CD3 / CD28 / CD137 stimulation may also be used as a control. In some embodiments, the addition of a mouse monoclonal antibody against CD28 is used to ensure additional co-stimulation for amplifying the T cell response. In some embodiments, PBMCs may be co-cultured with each TIL lot as a positive control for the MLR response.

[0404] In one embodiment, the efficacy assay includes a co-culture step in which the co-culture is carried out over a period 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 one embodiment, the efficacy assay includes a co-culture step in which the co-culture is performed over a period selected from the group consisting of 6 hours, 12 hours, 18 hours, 24 hours, and 32 hours.

[0405] In one embodiment, the efficacy assay includes a co-culture step of target cells and T cells (including TILs, MILs, or PBLs) over a period 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 one embodiment, the efficacy assay includes a co-culture step in which the co-culture is performed over a period selected from the group consisting of 6 hours, 12 hours, 18 hours, 24 hours, and 32 hours.

[0406] In one embodiment, the efficacy assay includes a co-culture step of negative control cells and T cells (including TILs, MILs, or PBLs) over a period 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 one embodiment, the efficacy assay includes a co-culture step in which the co-culture is performed over 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 TIL, MIL, PBL or other T cell products in relation to clinical benefit. Clinical benefit can be measured by multiple methods, including, but not limited to, demonstration of cell function in vitro, disease control rate, overall response rate, duration of response, or other scientific measures of clinical benefit or potential clinical benefit.

[0408] In some embodiments, the potency of expanded or formulated TIL, MIL, and PBL is investigated by co-culture assays described herein. Investigating the potency of expanded or formulated TIL, MIL, and PBL allows for the characterization of TIL product lots, MIL product lots, or PBL product lots. The potency of TIL, MIL, or PBL, also referred to as activation, is defined as the increased expression of a selective surface marker, expressed as the percentage of marker-positive cells or mean fluorescence intensity (MFI), when TIL, MIL, or PBL is co-cultured with Raji cells or their derivatives, variants, modifications, or progeny, compared to co-cultured with K562 cells.

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

[0410] In some embodiments, the present invention includes a method for determining the potency of a T cell product using target cells capable of binding to T cell receptors, wherein protein secretion or expression is detected using an enzyme-linked immunosorbent assay (ELISA) detection method. In further embodiments, the ELISA method is automated using an automated or robotic system such as the ELLA system (available from the ProteinSimple 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 some embodiments, the potency assay detection is performed using a multiplex assay detection system such as the LUMINEX system (available from the R&D Systems division of BioTechne, Inc., Minnesota, MN, USA). In some embodiments, the present invention includes a potency assay method using a flow cytometry detection method. In some embodiments, the present invention includes a potency assay method for secreted proteins. In some embodiments, 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 the IFN-γ concentration after activation of the co-culture assay described herein, and the supernatant collected from the co-culture is screened for IFN-γ production in each multi-well (e.g., 72-well) commercially validated cartridge. In one embodiment, the ELLA automated system performs to sub-picogram level sensitivity, including four logs of sensitivity within a dynamic range. In one embodiment, assay repeatability and accuracy are increased compared to conventional ELISA methods by minimizing procedural errors using the ELLA system.

[0411] In some embodiments, the present invention includes a method for determining the potency of a T cell product using target cells capable of binding to T cell receptors, wherein the secretion or expression of an analyte is detected using a detection method. In some embodiments, the detected analyte is a protein. In some embodiments, the detected analyte is a cytokine. In some embodiments, the detected analyte is an interferon. In some embodiments, the detected analyte is interferon-alpha, also known as IFN-α or IFNα. In some embodiments, the detected analyte is interferon-alpha, also known as IFN-β or IFNβ. In some embodiments, the detected analyte is interferon-gamma, also known as IFN-γ or IFNγ. In some embodiments, the detected analyte is granzyme B, also known as GzmB. In some embodiments, the detected analyte is perforin. In some embodiments, the detected analyte is tumor necrosis factor alpha, also known as TNF-α or TNFα. In some embodiments, the detected analyte is interleukin. In some embodiments, the detected analytes are 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 detected analyte is CD25. In some embodiments, the detected analyte is CD69. In some embodiments, the detected analyte is CD137(4-1BB). In some embodiments, the detected analyte is CD134(OX40). In some embodiments, the detected analyte is CCL4. In some embodiments, the detected analyte is CD150, also known as SLAM, SLAM F1, or signaling lymphocyte activating molecule 1. In some embodiments, the detected analyte is KLRG1. In some embodiments, the detected analyte is KLRG1. In one embodiment, the analyte detected is secreted MIP-1β.

[0412] 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 secretion or expression of an analyte is detected using a detection method, 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 present invention includes a method for determining the potency of a T cell product using cells capable of allogeneic binding to the T cell receptor, wherein a negative control, including cells lacking MHC I and MHC II expression, is used to compare the T cell product (such as a TIL product, MIL product, or PBL product) with a co-culture with target cells. In another embodiment, the present invention includes a method for determining the potency of a TIL product using Raji cells capable of allogeneic binding to the T cell receptor, wherein a negative control, including K562 cells lacking MHC I and MHC II expression, is used to compare the TIL product with a co-culture with Raji cells.

[0414] In some embodiments, the increase in the expression of selective surface markers, expressed as the percentage of marker-positive cells or mean fluorescence intensity (MFI) when co-cultured 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% compared to K562 cells.

[0415] In some embodiments, the increase in the expression of selective surface markers, expressed as the percentage of marker-positive cells or mean fluorescence intensity (MFI) when co-cultured 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% compared to K562 cells.

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

[0417] In some embodiments, the present invention provides a method for assaying TIL activity, comprising the steps of: a) irradiating Raji cells and K562 cells with an agonist or superagonist anti-CD28 antibody (optionally); (b) co-culturing the irradiated Raji cells and K562 cells with TIL 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) collecting the supernatant from the co-cultured cells in step (b) after 6 to 24 hours; (d) collecting cells from step (b) and measuring the 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 extended TIL is examined before the TIL is cryopreserved. In some embodiments, the potency of the TIL product, MIL product, or PBL product is examined as part of the drug release test. In some embodiments, the potency of the TIL product, MIL product, or PBL product is examined as part of the drug stability test, for example, after thawing a cryopreserved product, or after storage of a product that has been previously thawed or has never been frozen for a defined period of time under different environmental conditions.

[0419] In some embodiments, the increase in the expression of selective surface markers, expressed as the percentage of marker-positive cells or mean fluorescence intensity (MFI) when co-cultured 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% compared to K562 cells.

[0420] In some embodiments, the increase in the expression of selective surface markers, expressed as the percentage of marker-positive cells or mean fluorescence intensity (MFI) when co-cultured with Raji cells, was 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, compared to K562 cells. An increase of at least 9 times, at least 9.5 times, at least 10 times, at least 11 times, at least 12 times, at least 13 times, at least 14 times, at least 15 times, at least 16 times, at least 17 times, at least 18 times, at least 19 times, at least 20 times, at least 25 times, at least 30 times, at least 35 times, at least 40 times, at least 45 times, at least 50 times, at least 55 times, at least 60 times, at least 70 times, at least 80 times, at least 90 times, or at least 100 times.

[0421] In some embodiments, the increase in CD25 expression, expressed as the percentage of marker-positive cells or mean fluorescence intensity (MFI) when co-cultured 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% 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) when co-cultured with Raji cells, was 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, and less than that of K562 cells. The increase is at least 9 times, at least 9.5 times, at least 10 times, at least 11 times, at least 12 times, at least 13 times, at least 14 times, at least 15 times, at least 16 times, at least 17 times, at least 18 times, at least 19 times, at least 20 times, at least 25 times, at least 30 times, at least 35 times, at least 40 times, at least 45 times, at least 50 times, at least 55 times, at least 60 times, at least 70 times, at least 80 times, at least 90 times, or at least 100 times.

[0423] In some embodiments, the increase in CD69 expression, expressed as the percentage of marker-positive cells or mean fluorescence intensity (MFI) when co-cultured 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% 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) when co-cultured with Raji cells, was 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, and less than that of K562 cells. The increase is at least 9 times, at least 9.5 times, at least 10 times, at least 11 times, at least 12 times, at least 13 times, at least 14 times, at least 15 times, at least 16 times, at least 17 times, at least 18 times, at least 19 times, at least 20 times, at least 25 times, at least 30 times, at least 35 times, at least 40 times, at least 45 times, at least 50 times, at least 55 times, at least 60 times, at least 70 times, at least 80 times, at least 90 times, or at least 100 times.

[0425] In some embodiments, the increase in CD134 expression, expressed as a percentage of marker-positive cells or mean fluorescence intensity (MFI) when co-cultured 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% 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) when co-cultured with Raji cells, was 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, and less than that of K562 cells. The increase is at least 9 times, at least 9.5 times, at least 10 times, at least 11 times, at least 12 times, at least 13 times, at least 14 times, at least 15 times, at least 16 times, at least 17 times, at least 18 times, at least 19 times, at least 20 times, at least 25 times, at least 30 times, at least 35 times, at least 40 times, at least 45 times, at least 50 times, at least 55 times, at least 60 times, at least 70 times, at least 80 times, at least 90 times, or at least 100 times.

[0427] In some embodiments, the increase in CD137 expression, expressed as a percentage of marker-positive cells or mean fluorescence intensity (MFI) when co-cultured 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% 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) when co-cultured with Raji cells, was 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, and less than that of K562 cells. The increase is at least 9 times, at least 9.5 times, at least 10 times, at least 11 times, at least 12 times, at least 13 times, at least 14 times, at least 15 times, at least 16 times, at least 17 times, at least 18 times, at least 19 times, at least 20 times, at least 25 times, at least 30 times, at least 35 times, at least 40 times, at least 45 times, at least 50 times, at least 55 times, at least 60 times, at least 70 times, at least 80 times, at least 90 times, or at least 100 times.

[0429] In some embodiments, the increase in CD150 expression, expressed as the percentage of marker-positive cells or mean fluorescence intensity (MFI) when co-cultured 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% 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) when co-cultured with Raji cells, was 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, and less than that of K562 cells. The increase is at least 9 times, at least 9.5 times, at least 10 times, at least 11 times, at least 12 times, at least 13 times, at least 14 times, at least 15 times, at least 16 times, at least 17 times, at least 18 times, at least 19 times, at least 20 times, at least 25 times, at least 30 times, at least 35 times, at least 40 times, at least 45 times, at least 50 times, at least 55 times, at least 60 times, at least 70 times, at least 80 times, at least 90 times, or at least 100 times.

[0431] In some embodiments, the increased expression of granzyme B, expressed as an ELISA detection analyte or mean fluorescence intensity (MFI), or secreted, during 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% compared to K562 cells.

[0432] In some embodiments, the increased expression of secreted granzyme B, expressed as a percentage of ELISA detection analytes or mean fluorescence intensity (MFI) during co-culture with Raji 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, and at least An increase of 8.5 times, at least 9 times, at least 9.5 times, at least 10 times, at least 11 times, at least 12 times, at least 13 times, at least 14 times, at least 15 times, at least 16 times, at least 17 times, at least 18 times, at least 19 times, at least 20 times, at least 25 times, at least 30 times, at least 35 times, at least 40 times, at least 45 times, at least 50 times, at least 55 times, at least 60 times, at least 70 times, at least 80 times, at least 90 times, or at least 100 times.

[0433] In some embodiments, the increase in IFN-γ expression, expressed as a percentage of the ELISA detection analyte or mean fluorescence intensity (MFI) or secreted IFN-γ expression during 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% compared to K562 cells.

[0434] In some embodiments, the increase in IFN-γ expression, expressed as a percentage of the ELISA detection analyte or mean fluorescence intensity (MFI) during co-culture with Raji cells, or the increase in secreted IFN-γ, 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, and at least 8-fold compared to K562 cells. An increase of 0.5 times, at least 9 times, at least 9.5 times, at least 10 times, at least 11 times, at least 12 times, at least 13 times, at least 14 times, at least 15 times, at least 16 times, at least 17 times, at least 18 times, at least 19 times, at least 20 times, at least 25 times, at least 30 times, at least 35 times, at least 40 times, at least 45 times, at least 50 times, at least 55 times, at least 60 times, at least 70 times, at least 80 times, at least 90 times, or at least 100 times.

[0435] In some embodiments, the increase in TNF-α expression, expressed as an ELISA detection analyte or mean fluorescence intensity (MFI), or secreted, during 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% compared to K562 cells.

[0436] In some embodiments, the increase in TNF-α expression, expressed as a percentage of ELISA detection analytes or mean fluorescence intensity (MFI) during co-culture with Raji cells, or the increase in secreted TNF-α, 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, and at least 8-fold compared to K562 cells. An increase of 0.5 times, at least 9 times, at least 9.5 times, at least 10 times, at least 11 times, at least 12 times, at least 13 times, at least 14 times, at least 15 times, at least 16 times, at least 17 times, at least 18 times, at least 19 times, at least 20 times, at least 25 times, at least 30 times, at least 35 times, at least 40 times, at least 45 times, at least 50 times, at least 55 times, at least 60 times, at least 70 times, at least 80 times, at least 90 times, or at least 100 times.

[0437] In some embodiments, the increase in perforin expression, expressed as a percentage of the ELISA detection analyte or mean fluorescence intensity (MFI) or secreted perforin when co-cultured 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% compared to K562 cells.

[0438] In some embodiments, the increased expression of perforin secreted during co-culture with Raji cells, expressed as a percentage of the ELISA detection analyte or mean fluorescence intensity (MFI), 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, and at least An increase of 8.5 times, at least 9 times, at least 9.5 times, at least 10 times, at least 11 times, at least 12 times, at least 13 times, at least 14 times, at least 15 times, at least 16 times, at least 17 times, at least 18 times, at least 19 times, at least 20 times, at least 25 times, at least 30 times, at least 35 times, at least 40 times, at least 45 times, at least 50 times, at least 55 times, at least 60 times, at least 70 times, at least 80 times, at least 90 times, or at least 100 times.

[0439] In some embodiments, the increase in CCL4 expression, expressed as an ELISA detection analyte or mean fluorescence intensity (MFI), or secreted during 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% compared to K562 cells.

[0440] In some embodiments, the increased expression of secreted CCL4, expressed as a percentage of ELISA detection analytes or mean fluorescence intensity (MFI) during co-culture with Raji 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-fold compared to K562 cells. An increase of 0.5 times, at least 9 times, at least 9.5 times, at least 10 times, at least 11 times, at least 12 times, at least 13 times, at least 14 times, at least 15 times, at least 16 times, at least 17 times, at least 18 times, at least 19 times, at least 20 times, at least 25 times, at least 30 times, at least 35 times, at least 40 times, at least 45 times, at least 50 times, at least 55 times, at least 60 times, at least 70 times, at least 80 times, at least 90 times, or at least 100 times.

[0441] In some embodiments, the increase in MIP-1β expression, expressed as a percentage of the ELISA detection analyte or mean fluorescence intensity (MFI) or secreted during 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% compared to K562 cells.

[0442] In some embodiments, the increased expression of MIP-1β, expressed as a percentage of the ELISA detection analyte or mean fluorescence intensity (MFI) when co-cultured with Raji cells, or secreted, 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, and at least An increase of 8.5 times, at least 9 times, at least 9.5 times, at least 10 times, at least 11 times, at least 12 times, at least 13 times, at least 14 times, at least 15 times, at least 16 times, at least 17 times, at least 18 times, at least 19 times, at least 20 times, at least 25 times, at least 30 times, at least 35 times, at least 40 times, at least 45 times, at least 50 times, at least 55 times, at least 60 times, at least 70 times, at least 80 times, at least 90 times, or at least 100 times.

[0443] In some embodiments, the amount of granzyme B secreted during 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 the test sample contains 1 × 10⁶ 5 TIL, 2×10 5 TIL, 3×10 5 TIL, 4x10 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 TIL

[0444] In some embodiments, the amount of IFN-γ secreted during 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 the test sample is 1 × 10⁶ 5 TIL, 2×10 5 TIL, 3×10 5 TIL, 4x10 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 TIL

[0445] In some embodiments, the amount of TNF-α secreted during 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 the test sample contains 1 × 10⁶ 5 TIL, 2×10 5 TIL, 3×10 5 TIL, 4x10 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 TIL

[0446] In some embodiments, the amount of perforin secreted during 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 the test sample is 1 × 10⁶ 5 TIL, 2×10 5 TIL, 3×10 5 TIL, 4x10 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 TIL

[0447] In some embodiments, the amount of CCL4 secreted during 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 the test sample is 1 × 10⁶ 5 TIL, 2×10 5 TIL, 3×10 5 TIL, 4x10 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 TIL

[0448] In some embodiments, the amount of MIP-1β secreted during 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 the test sample is 1 × 10⁶ 5 TIL, 2×10 5 TIL, 3×10 5 TIL, 4x10 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 TIL

[0449] In one embodiment, the target cell to T cell (including TIL, MIL, or PBL) ratios are approximately 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, and 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 0.3, 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 The group is selected from the following: 9.3, approximately 1:9.4, approximately 1:9.5, approximately 1:9.6, approximately 1:9.7, approximately 1:9.8, approximately 1:9.9, approximately 1:10, approximately 1:11, approximately 1:12, approximately 1:12.5, approximately 1:13, approximately 1:14, approximately 1:15, approximately 1:16, approximately 1:17, approximately 1:18, approximately 1:19, approximately 1:20, approximately 1:25, approximately 1:30, approximately 1:35, approximately 1:40, approximately 1:45, approximately 1:50, approximately 1:60, approximately 1:70, approximately 1:80, approximately 1:90, or approximately 1:100.

[0450] In one embodiment, the ratio of T cells (including TILs, MILs, or PBLs) to target cells is approximately 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, and 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 0.3, 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 The group is selected from the following: 9.3, approximately 1:9.4, approximately 1:9.5, approximately 1:9.6, approximately 1:9.7, approximately 1:9.8, approximately 1:9.9, approximately 1:10, approximately 1:11, approximately 1:12, approximately 1:12.5, approximately 1:13, approximately 1:14, approximately 1:15, approximately 1:16, approximately 1:17, approximately 1:18, approximately 1:19, approximately 1:20, approximately 1:25, approximately 1:30, approximately 1:35, approximately 1:40, approximately 1:45, approximately 1:50, approximately 1:60, approximately 1:70, approximately 1:80, approximately 1:90, or approximately 1:100.

[0451] In one embodiment, the ratio of Raji cells to T cells (including TILs, MILs, or PBLs) is approximately 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, and 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 The group is selected from the following: 9.3, approximately 1:9.4, approximately 1:9.5, approximately 1:9.6, approximately 1:9.7, approximately 1:9.8, approximately 1:9.9, approximately 1:10, approximately 1:11, approximately 1:12, approximately 1:12.5, approximately 1:13, approximately 1:14, approximately 1:15, approximately 1:16, approximately 1:17, approximately 1:18, approximately 1:19, approximately 1:20, approximately 1:25, approximately 1:30, approximately 1:35, approximately 1:40, approximately 1:45, approximately 1:50, approximately 1:60, approximately 1:70, approximately 1:80, approximately 1:90, or approximately 1:100.

[0452] In one embodiment, the ratio of T cells (including TILs, MILs, or PBLs) to Raji cells is approximately 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, and 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 The group is selected from the following: 9.3, approximately 1:9.4, approximately 1:9.5, approximately 1:9.6, approximately 1:9.7, approximately 1:9.8, approximately 1:9.9, approximately 1:10, approximately 1:11, approximately 1:12, approximately 1:12.5, approximately 1:13, approximately 1:14, approximately 1:15, approximately 1:16, approximately 1:17, approximately 1:18, approximately 1:19, approximately 1:20, approximately 1:25, approximately 1:30, approximately 1:35, approximately 1:40, approximately 1:45, approximately 1:50, approximately 1:60, approximately 1:70, approximately 1:80, approximately 1:90, or approximately 1:100.

[0453] In one embodiment, the ratio of negative control cells to T cells (including TILs, MILs, or PBLs) is approximately 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, and 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 The group is selected from the following: 9.3, approximately 1:9.4, approximately 1:9.5, approximately 1:9.6, approximately 1:9.7, approximately 1:9.8, approximately 1:9.9, approximately 1:10, approximately 1:11, approximately 1:12, approximately 1:12.5, approximately 1:13, approximately 1:14, approximately 1:15, approximately 1:16, approximately 1:17, approximately 1:18, approximately 1:19, approximately 1:20, approximately 1:25, approximately 1:30, approximately 1:35, approximately 1:40, approximately 1:45, approximately 1:50, approximately 1:60, approximately 1:70, approximately 1:80, approximately 1:90, or approximately 1:100.

[0454] In one embodiment, the ratio of T cells (including TILs, MILs, or PBLs) to negative control cells is approximately 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, The group is selected from the following: 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 one embodiment, the ratio of K562 cells to T cells (including TILs, MILs, or PBLs) is approximately 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, and 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, The group is selected from the following: 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 one embodiment, the ratio of T cells (including TIL, MIL, or PBL) to K562 cells is approximately 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, and 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, The group is selected from the following: 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 one embodiment, the target cell to T cell (including TIL, MIL, or PBL) ratio is 1:1-1:2, 1:2-1:3, 1:3-1:4, 1:4-1:5, 1:5-1:6, 1:6-1:7, 1:7-1:8, 1:8-1:9, 1:9-1:10, 1:10-1:11, 1:11-1:12, 1:12-1:13, 1:13-1:14, 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: The time intervals are 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 one embodiment, the ratio of T cells (including TILs, MILs, or PBLs) to target cells is 1:1-1:2, 1:2-1:3, 1:3-1:4, 1:4-1:5, 1:5-1:6, 1:6-1:7, 1:7-1:8, 1:8-1:9, 1:9-1:10, 1:10-1:11, 1:11-1:12, 1:12-1:13, 1:13-1:14, 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: The time intervals are 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 one embodiment, the ratio of Raji cells to T cells (including TILs, MILs, or PBLs) is 1:1-1:2, 1:2-1:3, 1:3-1:4, 1:4-1:5, 1:5-1:6, 1:6-1:7, 1:7-1:8, 1:8-1:9, 1:9-1:10, 1:10-1:11, 1:11-1:12, 1:12-1:13, 1:13-1:14, 1 :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, 1:80~1:90, 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 The time intervals are 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 one embodiment, the ratio of T cells (including TILs, MILs, or PBLs) to Raji cells is 1:1-1:2, 1:2-1:3, 1:3-1:4, 1:4-1:5, 1:5-1:6, 1:6-1:7, 1:7-1:8, 1:8-1:9, 1:9-1:10, 1:10-1:11, 1:11-1:12, 1:12-1:13, 1:13-1:14, 1 :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, 1:80~1:90, 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 The time intervals are 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 one embodiment, the ratio of negative control cells to T cells (including TILs, MILs, or PBLs) is 1:1-1:2, 1:2-1:3, 1:3-1:4, 1:4-1:5, 1:5-1:6, 1:6-1:7, 1:7-1:8, 1:8-1:9, 1:9-1:10, 1:10-1:11, 1:11-1:12, 1:12-1:13, 1:13-1:14, 1 :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, 1:80~1:90, 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 The time intervals are 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 one embodiment, the ratio of T cells (including TIL, MIL, or PBL) to negative control cells is 1:1-1:2, 1:2-1:3, 1:3-1:4, 1:4-1:5, 1:5-1:6, 1:6-1:7, 1:7-1:8, 1:8-1:9, 1:9-1:10, 1:10-1:11, 1:11-1:12, 1:12-1:13, 1:13-1:14, 1 :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, 1:80~1:90, 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 The time intervals are 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 one embodiment, the ratio of K562 cells to T cells (including TILs, MILs, or PBLs) is 1:1-1:2, 1:2-1:3, 1:3-1:4, 1:4-1:5, 1:5-1:6, 1:6-1:7, 1:7-1:8, 1:8-1:9, 1:9-1:10, 1:10-1:11, 1:11-1:12, 1:12-1:13, 1:13-1:14, 1 :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, 1:80~1:90, 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 The time intervals are 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 one embodiment, the ratio of T cells (including TIL, MIL, or PBL) to K562 cells is 1:1-1:2, 1:2-1:3, 1:3-1:4, 1:4-1:5, 1:5-1:6, 1:6-1:7, 1:7-1:8, 1:8-1:9, 1:9-1:10, 1:10-1:11, 1:11-1:12, 1:12-1:13, 1:13-1:14, 1 :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, 1:80~1:90, 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 The time intervals are 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 one embodiment, the TIL to Raji cell ratio in target cell co-culture is selected from the group consisting of approximately 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4.5:1, 4:1, 3.5:1, 3:1, 2.5:1, 2:1, 1.5:1, 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, 1:5, 1:6, 1.7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, and 1:15.

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

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

[0468] In some embodiments, the present 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 TIL 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 such co-culturing is optionally performed using an anti-CD28 antibody; (c) collecting supernatant from the co-cultured cells in step (b) after 6 to 24 hours; (d) taking cells from step (b) and measuring the 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 efficacy assay thaws the TIL cryopreserved product, MIL cryopreserved product, or PBL cryopreserved product and performs the assay for 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, and 45 hours. The process includes a recovery step that allows recovery at ambient temperature or refrigerated temperature for a period selected from the group consisting of 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 one embodiment, the potency assay includes a recovery step in which the TIL cryopreserved product, MIL cryopreserved product, or PBL cryopreserved product is thawed and allowed to recover at ambient or refrigerated temperature for a period selected from the group consisting of about 12 hours, about 24 hours, about 48 hours, about 72 hours, and about 96 hours. The aforementioned duration may be measured from the completion of the thawing process or from the start of the thawing process.

[0470] In one embodiment, the present invention includes a method for determining the potency of a T cell product, the method being: The first step involves co-culturing target cells with aT cell product cells, b. Steps to obtain samples from co-culture, c. The procedure includes the steps of (1) evaluating the expression of one or more markers on T cell product cells, or (2) evaluating one or more analytes secreted from T cell product cells to obtain one or more observations and determine the potency of the T cell product.

[0471] In one embodiment, the present invention includes a method for determining the potency of a T cell product, the method being: The first step involves co-culturing target cells with aT cell product cells, b. Steps to obtain samples from co-culture, c. The steps include: (1) evaluating the expression of one or more markers on T cell product cells, or (2) evaluating one or more analytes secreted from T cell product cells to obtain one or more observations and determine the potency of the T cell product; The aforementioned methods include bead or plate-based assays using CD3, CD28, and / or CD137 stimulation and reporting interferon-γ, granzyme B, or tumor necrosis factor-α, assays for total viable cells, assays for viable cell percentage, and CD4 + Assay on cell content, CD8 + Assay on cell content, T EM Assay on cell content, T CM Assay on cell content, LAG3 + Assay on cell content, and KLRG1 + Assay on cell content, CD101 + Assay on cell content, CD69 + Assay on cell content, T SCM Assay on cell content, T EMRA Assay on cell content, T reg Assay on cell content, PD-1 + Assay on cell content, TIM3 + Assay on cell content, CD25 + Assay on cell content, CD27 + Assay on cell content, CD28 + Assay on cell content, CD56 + Assay on cell content, CTLA-4 + Cell content assay, TIGIT + Assay on cell content, and CD57+ The components of the efficacy assay matrix include at least one other assay selected from the group consisting of assays for cell content. In one embodiment, the assay is a flow cytometry assay.

[0472] In one embodiment, the present invention includes a method for determining the potency of a T cell product, the method being: The first step involves co-culturing target cells with aT cell product cells, b. Steps to obtain samples from co-culture, c. The steps of (1) evaluating the expression of one or more markers on T cell product cells, or (2) evaluating one or more analytes secreted from T cell product cells, obtaining one or more observational values, and determining the potency of the T cell product, The second step involves co-culturing negative control cells with dT cell product cells for a second period, e. The step of obtaining a second sample from the second co-culture, f. The second sample is evaluated by (1) the expression of one or more markers on T cell product cells, or (2) the evaluation of one or more analytes secreted from T cell product cells, to obtain one or more control values. g. The step of determining the potency of the T cell product by comparing one or more observations from step c to one or more control values ​​from step f, where each observation is compared to a corresponding control value.

[0473] In one embodiment, the present invention includes a method for determining the potency of a T cell product, the method being: The first step involves co-culturing target cells with aT cell product cells, b. Steps to obtain samples from co-culture, c. The steps of (1) evaluating the expression of one or more markers on T cell product cells, or (2) evaluating one or more analytes secreted from T cell product cells, obtaining one or more observational values, and determining the potency of the T cell product, The second step involves co-culturing negative control cells with dT cell product cells for a second period, e. The step of obtaining a second sample from the second co-culture, f. The second sample is evaluated by (1) the expression of one or more markers on T cell product cells, or (2) the evaluation of one or more analytes secreted from T cell product cells, to obtain one or more control values. g. The step of determining the potency of the T cell product by comparing one or more observations from step c to one or more control values ​​from step f, where each observation is compared to a corresponding control value, T-cell products are selected from a group consisting of tumor-infiltrating lymphocyte (TIL) products, bone marrow-infiltrating lymphocyte (MIL) products, or peripheral blood lymphocyte (PBL) products.

[0474] In one embodiment, the present invention includes a method for determining the potency of a T cell product, the method being: The first step involves co-culturing target cells with aT cell product cells, b. Steps to obtain samples from co-culture, c. The steps of (1) evaluating the expression of one or more markers on T cell product cells, or (2) evaluating one or more analytes secreted from T cell product cells, obtaining one or more observational values, and determining the potency of the T cell product, The second step involves co-culturing negative control cells with dT cell product cells for a second period, e. The step of obtaining a second sample from the second co-culture, f. The second sample is evaluated by (1) the expression of one or more markers on T cell product cells, or (2) the evaluation of one or more analytes secreted from T cell product cells, to obtain one or more control values. g. The step of determining the potency of the T cell product by comparing one or more observations from step c to one or more control values ​​from step f, where each observation is compared to a corresponding control value, The T-cell product is a human-derived TIL product, which is obtained by tumor resection or tumor fragmentation or digestion, and manufactured by a TIL expansion process that includes a rapid expansion protocol step.

[0475] In one embodiment, the present invention includes a method for determining the potency of a T cell product, the method being: The first step involves co-culturing target cells with aT cell product cells, b. Steps to obtain samples from co-culture, c. The steps of (1) evaluating the expression of one or more markers on T cell product cells, or (2) evaluating one or more analytes secreted from T cell product cells, obtaining one or more observational values, and determining the potency of the T cell product, The second step involves co-culturing negative control cells with dT cell product cells for a second period, e. The step of obtaining a second sample from the second co-culture, f. The second sample is evaluated by (1) the expression of one or more markers on T cell product cells, or (2) the evaluation of one or more analytes secreted from T cell product cells, to obtain one or more control values. g. A step in which the potency of the T cell product is determined by comparing one or more observations from step c with one or more control values ​​from step f, where each observation is compared with a corresponding control value. h. Including releasing a T cell product for use in the treatment of human patients, The T-cell product is a human-derived TIL product, which is obtained by tumor resection or tumor fragmentation or digestion, and manufactured by a TIL expansion process that includes a rapid expansion protocol step.

[0476] In one embodiment, the present invention includes a method for determining the potency of a T cell product, the method being: The first step involves co-culturing target cells with aT cell product cells, b. Steps to obtain samples from co-culture, c. The steps of (1) evaluating the expression of one or more markers on T cell product cells, or (2) evaluating one or more analytes secreted from T cell product cells, obtaining one or more observational values, and determining the potency of the T cell product, The second step involves co-culturing negative control cells with dT cell product cells for a second period, e. The step of obtaining a second sample from the second co-culture, f. The second sample is evaluated by (1) the expression of one or more markers on T cell product cells, or (2) the evaluation of one or more analytes secreted from T cell product cells, to obtain one or more control values. g. A step in which the potency of the T cell product is determined by comparing one or more observations from step c with one or more control values ​​from step f, where each observation is compared with a corresponding control value. h. Including releasing a T cell product for use in the treatment of human patients, The T-cell product is a human-derived TIL product, which is obtained by tumor excision or tumor fragmentation or digestion, and manufactured 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 their derivatives, variants, modifications, or offspring.

[0477] In one embodiment, the present invention includes a method for determining the potency of a T cell product, the method being: The first step involves co-culturing target cells with aT cell product cells, b. Steps to obtain samples from co-culture, c. The steps of (1) evaluating the expression of one or more markers on T cell product cells, or (2) evaluating one or more analytes secreted from T cell product cells, obtaining one or more observational values, and determining the potency of the T cell product, The second step involves co-culturing negative control cells with dT cell product cells for a second period, e. The step of obtaining a second sample from the second co-culture, f. The second sample is evaluated by (1) the expression of one or more markers on T cell product cells, or (2) the evaluation of one or more analytes secreted from T cell product cells, to obtain one or more control values. g. A step in which the potency of the T cell product is determined by comparing one or more observations from step c with one or more control values ​​from step f, where each observation is compared with a corresponding control value. h. Including releasing a T cell product for use in the treatment of human patients, The T-cell product is a human-derived TIL product, obtained by tumor resection, or tumor fragmentation or digestion, and manufactured by a TIL expansion process including a rapid expansion protocol step, with target cells being irradiated Raji cells, Ramos cells, Daudi cells, U937 cells, or Thp1 cells, or their derivatives, variants, modifications, or progeny, and negative control cells lacking MHC class I and MHC class II expression.

[0478] In one embodiment, the present invention includes a method for determining the potency of a T cell product, the method being: The first step involves co-culturing target cells with aT cell product cells, b. Steps to obtain samples from co-culture, c. The steps of (1) evaluating the expression of one or more markers on T cell product cells, or (2) evaluating one or more analytes secreted from T cell product cells, obtaining one or more observational values, and determining the potency of the T cell product, The second step involves co-culturing negative control cells with dT cell product cells for a second period, e. The step of obtaining a second sample from the second co-culture, f. The second sample is evaluated by (1) the expression of one or more markers on T cell product cells, or (2) the evaluation of one or more analytes secreted from T cell product cells, to obtain one or more control values. g. A step in which the potency of the T cell product is determined by comparing one or more observations from step c with one or more control values ​​from step f, where each observation is compared with a corresponding control value. h. Including releasing a T cell product for use in the treatment of human patients, The T-cell product is a human-derived TIL product, which is obtained by tumor excision or tumor fragmentation or digestion, and manufactured by a TIL expansion process including a rapid expansion protocol step, with target cells being irradiated Raji cells, Ramos cells, Daudi cells, U937 cells, or Thp1 cells, or their derivatives, variants, modifications, or offspring, and negative control cells being irradiated K562 cells or their derivatives, variants, modifications, or offspring.

[0479] In one embodiment, the present invention includes a method for determining the potency of a T cell product, the method being: The first step involves co-culturing target cells with aT cell product cells, b. Steps to obtain samples from co-culture, c. The steps of (1) evaluating the expression of one or more markers on T cell product cells, or (2) evaluating one or more analytes secreted from T cell product cells, obtaining one or more observational values, and determining the potency of the T cell product, The second step involves co-culturing negative control cells with dT cell product cells for a second period, e. The step of obtaining a second sample from the second co-culture, f. The second sample is evaluated by (1) the expression of one or more markers on T cell product cells, or (2) the evaluation of one or more analytes secreted from T cell product cells, to obtain one or more control values. g. A step in which the potency of the T cell product is determined by comparing one or more observations from step c with one or more control values ​​from step f, where each observation is compared with a corresponding control value. h. Including releasing a T cell product for use in the treatment of human patients, The T-cell product is a human-derived TIL product, obtained by tumor excision or tumor fragmentation or digestion, and manufactured 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 TIL product cells to target cells is 5:1 to 1:5.

[0480] In one embodiment, the present invention includes a method for determining the potency of a T cell product, the method being: The first step involves co-culturing target cells with aT cell product cells, b. Steps to obtain samples from co-culture, c. The steps of (1) evaluating the expression of one or more markers on T cell product cells, or (2) evaluating one or more analytes secreted from T cell product cells, obtaining one or more observational values, and determining the potency of the T cell product, The second step involves co-culturing negative control cells with dT cell product cells for a second period, e. The step of obtaining a second sample from the second co-culture, f. The second sample is evaluated by (1) the expression of one or more markers on T cell product cells, or (2) the evaluation of one or more analytes secreted from T cell product cells, to obtain one or more control values. g. A step in which the potency of the T cell product is determined by comparing one or more observations from step c with one or more control values ​​from step f, where each observation is compared with a corresponding control value. h. Including releasing a T cell product for use in the treatment of human patients, The T-cell product is a human-derived TIL product, obtained by tumor excision or tumor fragmentation or digestion, manufactured 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 offspring, the negative control cells are irradiated K562 cells or their derivatives, variants, modifications, or offspring, the ratio of TIL product cells to target cells is 5:1 to 1:5, and the ratio of TIL product cells to negative control cells is 5:1 to 1:5.

[0481] In one embodiment, the present invention includes a method for determining the potency of a T cell product, the method being: The first step involves co-culturing target cells with aT cell product cells, b. Steps to obtain samples from co-culture, c. The steps of (1) evaluating the expression of one or more markers on T cell product cells, or (2) evaluating one or more analytes secreted from T cell product cells, obtaining one or more observational values, and determining the potency of the T cell product, The second step involves co-culturing negative control cells with dT cell product cells for a second period, e. The step of obtaining a second sample from the second co-culture, f. The second sample is evaluated by (1) the expression of one or more markers on T cell product cells, or (2) the evaluation of one or more analytes secreted from T cell product cells, to obtain one or more control values. g. A step in which the potency of the T cell product is determined by comparing one or more observations from step c with one or more control values ​​from step f, where each observation is compared with a corresponding control value. h. Including releasing a T cell product for use in the treatment of human patients, The T-cell product is a human-derived TIL product, obtained by tumor excision or tumor fragmentation or digestion, and manufactured 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 offspring, the negative control cells being irradiated K562 cells or their derivatives, variants, modifications, or offspring, the first period being approximately 6 to 48 hours, and the second period being approximately 6 to 48 hours.

[0482] In one embodiment, the present invention includes a method for determining the potency of a T cell product, the method being: The first step involves co-culturing target cells with aT cell product cells, b. Steps to obtain samples from co-culture, c. The steps of (1) evaluating the expression of one or more markers on T cell product cells, or (2) evaluating one or more analytes secreted from T cell product cells, obtaining one or more observational values, and determining the potency of the T cell product, The second step involves co-culturing negative control cells with dT cell product cells for a second period, e. The step of obtaining a second sample from the second co-culture, f. The second sample is evaluated by (1) the expression of one or more markers on T cell product cells, or (2) the evaluation of one or more analytes secreted from T cell product cells, to obtain one or more control values. g. A step in which the potency of the T cell product is determined by comparing one or more observations from step c with one or more control values ​​from step f, where each observation is compared with a corresponding control value. h. Including releasing a T cell product for use in the treatment of human patients, The T-cell product is a human-derived TIL product, which is obtained by tumor resection, or tumor fragmentation or digestion, and manufactured 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 offspring, the negative control cells being irradiated K562 cells or their derivatives, variants, modifications, or offspring, the first period being selected from groups of approximately 12 hours, approximately 18 hours, and approximately 24 hours, and the second period being selected from groups of approximately 12 hours, approximately 18 hours, and approximately 24 hours.

[0483] In one embodiment, the present invention includes a method for determining the potency of a T cell product, the method being: The first step involves co-culturing target cells with aT cell product cells, b. Steps to obtain samples from co-culture, c. The steps of (1) evaluating the expression of one or more markers on T cell product cells, or (2) evaluating one or more analytes secreted from T cell product cells, obtaining one or more observational values, and determining the potency of the T cell product, The second step involves co-culturing negative control cells with dT cell product cells for a second period, e. The step of obtaining a second sample from the second co-culture, f. The second sample is evaluated by (1) the expression of one or more markers on T cell product cells, or (2) the evaluation of one or more analytes secreted from T cell product cells, to obtain one or more control values. g. A step in which the potency of the T cell product is determined by comparing one or more observations from step c with one or more control values ​​from step f, where each observation is compared with a corresponding control value. h. Including releasing a T cell product for use in the treatment of human patients, The T cell product is a human-derived TIL product, obtained by tumor excision or tumor fragmentation or digestion, manufactured 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 offspring, the negative control cells are irradiated K562 cells or their derivatives, variants, modifications, or offspring, and one or more markers on the T cell product are selected from the group consisting of CD25, CD69, CD134, CD137, CD150, KLRG1, or combinations thereof.

[0484] In one embodiment, the present invention includes a method for determining the potency of a T cell product, the method being: The first step involves co-culturing target cells with aT cell product cells, b. Steps to obtain samples from co-culture, c. The steps of (1) evaluating the expression of one or more markers on T cell product cells, or (2) evaluating one or more analytes secreted from T cell product cells, obtaining one or more observational values, and determining the potency of the T cell product, The second step involves co-culturing negative control cells with dT cell product cells for a second period, e. The step of obtaining a second sample from the second co-culture, f. The second sample is evaluated by (1) the expression of one or more markers on T cell product cells, or (2) the evaluation of one or more analytes secreted from T cell product cells, to obtain one or more control values. g. A step in which the potency of the T cell product is determined by comparing one or more observations from step c with one or more control values ​​from step f, where each observation is compared with a corresponding control value. h. Including releasing a T cell product for use in the treatment of human patients, The T-cell product is a human-derived TIL product, obtained by tumor excision or tumor fragmentation or digestion, manufactured 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 offspring, and the negative control cells are irradiated K562 cells or their derivatives, variants. One or more analytes selected from the T cell product, which are modified or offspring, 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 present invention includes a method for determining the potency of a T cell product, the method being: The first step involves co-culturing target cells with aT cell product cells, b. Steps to obtain samples from co-culture, c. The steps of (1) evaluating the expression of one or more markers on T cell product cells, or (2) evaluating one or more analytes secreted from T cell product cells, obtaining one or more observational values, and determining the potency of the T cell product, The second step involves co-culturing negative control cells with dT cell product cells for a second period, e. The step of obtaining a second sample from the second co-culture, f. The second sample is evaluated by (1) the expression of one or more markers on T cell product cells, or (2) the evaluation of one or more analytes secreted from T cell product cells, to obtain one or more control values. g. A step in which the potency of the T cell product is determined by comparing one or more observations from step c with one or more control values ​​from step f, where each observation is compared with a corresponding control value. h. Including releasing a T cell product for use in the treatment of human patients, The T cell product is a human-derived TIL product, obtained by tumor resection, or tumor fragmentation or digestion, and manufactured 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. 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, and IL-26. The group consists 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 the control value for each of the one or more analytes, and the increase in the observed value above the control value for each of the one or more analytes is selected from the group consisting of at least 1x, at least 1.5x, at least 2x, at least 2.5x, at least 3x, at least 3.5x, at least 4x, at least 4.5x, and at least 5x.

[0486] In any of the embodiments described above, tumor digestion may be carried out by the methods described herein or by methods known in the art. In any of the embodiments described above, tumor digestion may be carried out in accordance with the method described herein, or by using the composition or device thereof, as described in International Patent Publication WO2021 / 123832A1 (the disclosure of which is incorporated herein by reference).

[0487] In some embodiments, the present invention provides a method for assaying the multifunctional activity of extended TILs, as well as other polyclonal T cell products including MILs and PBLs, for evaluating the potency and / or functionality of these products, which can be used in the treatment of cancer by administering TILs, MILs, PBLs, or other polyclonal T cell products being evaluated. The first step involves co-culturing target cells with aT cell product cells, b. Steps to obtain samples from co-culture, c. The procedure includes the steps of (1) evaluating the expression of one or more markers on T cell product cells, or (2) evaluating two or more analytes secreted from T cell product cells to obtain one or more observations and determine the potency of the T cell product.

[0488] In some embodiments, this method is An additional step is to perform a second co-culture of negative control cells with dT cell product cells for a second period, e. An additional step of obtaining a second sample from the second co-culture, f. The second sample is subjected to an additional step of (1) evaluating the expression of one or more markers on T cell product cells, or (2) evaluating two or more analytes secreted from T cell product cells to obtain one or more control values. g. The additional step of determining the potency of the T cell product by comparing one or more observations from step c to one or more control values ​​from step f, where each observation is compared to a corresponding control value, 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. The amount of the observed value is normalized for each of the two or more analytes relative to the amount of the control value. For each of the two or more analytes, the increase in the observed value above the control value is selected from the group consisting of at least 1x, at least 1.5x, at least 2x, at least 2.5x, at least 3x, at least 3.5x, at least 4x, at least 4.5x, and at least 5x, at least 6x, at least 7x, at least 8x, at least 9x, and at least 10x.

[0489] In some embodiments, the present invention provides a method for assaying the multifunctional activity of extended TILs, as well as other polyclonal T cell products including MILs and PBLs, for evaluating the potency and / or functionality of these products, which can be used in the treatment of cancer by administering TILs, MILs, PBLs, or other polyclonal T cell products being evaluated. The first step involves co-culturing target cells with aT cell product cells, b. Steps to obtain samples from co-culture, c. The procedure includes the steps of (1) evaluating the expression of one or more markers on T cell product cells, or (2) evaluating three or more analytes secreted from T cell product cells to obtain one or more observations and determine the potency of the T cell product.

[0490] In some embodiments, this method is An additional step is to perform a second co-culture of negative control cells with dT cell product cells for a second period, e. An additional step of obtaining a second sample from the second co-culture, f. The second sample is subjected to an additional step of either (1) evaluating the expression of one or more markers on T cell product cells, or (2) evaluating three or more analytes secreted from T cell product cells to obtain one or more control values. g. The additional step of determining the potency of the T cell product by comparing one or more observations from step c to one or more control values ​​from step f, where each observation is compared to a corresponding control value, 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. The amount of the observed value is normalized for each of the three or more analytes relative to the amount of the control value. For each of the three or more analytes, the increase in the observed value above the control value is selected from the group consisting of at least 1x, at least 1.5x, at least 2x, at least 2.5x, at least 3x, at least 3.5x, at least 4x, at least 4.5x, and at least 5x, at least 6x, at least 7x, at least 8x, at least 9x, and at least 10x.

[0491] In some embodiments, the present invention provides a method for assaying the multifunctional activity of extended TILs, as well as other polyclonal T cell products including MILs and PBLs, for evaluating the potency and / or functionality of these products, which can be used in the treatment of cancer by administering TILs, MILs, PBLs, or other polyclonal T cell products being evaluated. a. A step of co-culturing multiple target cells with multiple T cell product cells for a first period, b. Steps to obtain samples from co-culture, c. The procedure includes the steps of (1) evaluating the expression of one or more markers on T cell product cells, or (2) evaluating one or more analytes secreted from T cell product cells to obtain one or more observations and determine the potency of the T cell product.

[0492] In some embodiments, this method is An additional step is to perform a second co-culture of multiple negative control cells with dT cell product cells for a second period, e. An additional step of obtaining a second sample from the second co-culture, f. The second sample is subjected to an additional step of obtaining one or more control values ​​by (1) evaluating the expression of one or more markers on T cell product cells, or (2) evaluating one or more analytes secreted from T cell product cells. g. The additional step of determining the potency of the T cell product by comparing one or more observations from step c to one or more control values ​​from step f, where each observation is compared to a corresponding control value, 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 value is normalized for each of the one or more analytes relative to the amount of the control value. For each of the one or more analytes, the increase in the observed value above the control value is selected from the group consisting of at least 1x, at least 1.5x, at least 2x, at least 2.5x, at least 3x, at least 3.5x, at least 4x, at least 4.5x, and at least 5x, at least 6x, at least 7x, at least 8x, at least 9x, and at least 10x.

[0493] In one embodiment, the present invention includes the above 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. In one embodiment, the target cells are a 1:1 combination of two or more cell lines, where the two or more cell lines are different and each is 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, where the three or more cell lines are different and each is 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 1:1:1:1 combination of four or more cell lines, where the four or more cell lines are distinct and each is independently selected from the group consisting of Raji cells, Thp1 cells, Ramos cells, U937 cells, and Daudi cells. In another embodiment, the target cells are a 1:1:1:1:1 combination of two or more cell lines, where the two or more cell lines are distinct and each is 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, and 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 another embodiment, the target cells are a combination of Raji cells and Ramos cells, and 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 yet another embodiment, the target cells are a combination of Raji cells and U937 cells, and 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 some embodiments, the present invention includes the method described above, and the co-culture includes a cell culture medium. In some embodiments, the present invention includes the method described above, and the co-culture includes CM1 medium. In some embodiments, the present invention includes the method described above, and the co-culture includes AIM-V medium (L-glutamine, 50 μM streptomycin sulfate, and 10 μM gentamicin sulfate), which is also referred to as AIM V medium commercially available from Invitrogen (Carlsbad, CA). In some embodiments, the present invention includes the method described above, and the co-culture and the second co-culture include a cell culture medium. In some embodiments, the present invention includes the method described above, and the co-culture and the second co-culture each include CM1 medium. In some embodiments, the present invention includes the method described above, and the co-culture and the second co-culture each include AIM-V medium. In some embodiments, the co-culture includes IL-2, which is continuously added during the co-culture, and the concentration of IL-2 is maintained at 50 IU / mL to 1000 IU / mL during the co-culture. In one embodiment, the co-culture contains IL-2, which is continuously added during the co-culture and maintained at a concentration of 100 IU / mL to 500 IU / mL during the co-culture. In another embodiment, the co-culture contains IL-2, which is continuously added during the co-culture and maintained at a concentration of 200 IU / mL to 400 IU / mL during the co-culture. In yet another embodiment, the co-culture contains IL-2, which is continuously added during the co-culture and maintained at a concentration selected from the group consisting of approximately 50 IU / mL, approximately 100 IU / mL, approximately 150 IU / mL, approximately 200 IU / mL, approximately 250 IU / mL, approximately 300 IU / mL, approximately 350 IU / mL, approximately 400 IU / mL, approximately 450 IU / mL, and approximately 500 IU / mL.

[0496] In one embodiment, the present invention includes a method for determining the potency of a T cell product, the method being: The first step involves co-culturing target cells with aT cell product cells, b. Steps to obtain samples from co-culture, c. The steps include: (1) evaluating the expression of one or more markers on T cell product cells, or (2) evaluating one or more analytes secreted from T cell product cells to obtain one or more observations and determine the potency of the T cell product; IL-2 is continuously added during co-culture, and the IL-2 concentration is maintained during co-culture at concentrations selected from the group consisting of approximately 50 IU / mL, 100 IU / mL, 150 IU / mL, 200 IU / mL, 250 IU / mL, 300 IU / mL, 350 IU / mL, 400 IU / mL, 450 IU / mL, and 500 IU / mL.

[0497] In one embodiment, the present invention includes a method for determining the potency of a T cell product, the method being: The first step involves co-culturing target cells with aT cell product cells, b. Steps to obtain samples from co-culture, c. The steps of (1) evaluating the expression of one or more markers on T cell product cells, or (2) evaluating one or more analytes secreted from T cell product cells, obtaining one or more observational values, and determining the potency of the T cell product, An additional step is to perform a second co-culture of negative control cells with dT cell product cells for a second period, e. An additional step of obtaining a second sample from the second co-culture, f. The second sample is subjected to an additional step of obtaining one or more control values ​​by (1) evaluating the expression of one or more markers on T cell product cells, or (2) evaluating one or more analytes secreted from T cell product cells. g. An additional step of determining the potency of a T cell product by comparing one or more observations from step c with one or more control values ​​from step f, wherein each observation is compared with a corresponding control value, IL-2 is continuously added during the co-culture and the second co-culture, and the IL-2 concentration is maintained at concentrations selected from the group consisting of approximately 50 IU / mL, approximately 100 IU / mL, approximately 150 IU / mL, approximately 200 IU / mL, approximately 250 IU / mL, approximately 300 IU / mL, approximately 350 IU / mL, approximately 400 IU / mL, approximately 450 IU / mL, and approximately 500 IU / mL, at concentrations in each of the co-cultures.

[0498] In one embodiment, the present invention includes a method for determining the potency of a T cell product, the method being: The first step involves co-culturing target cells with aT cell product cells, b. Steps to obtain samples from co-culture, c. The steps include: (1) evaluating the expression of one or more markers on T cell product cells, or (2) evaluating one or more analytes secreted from T cell product cells to obtain one or more observations and determine the potency of the T cell product; IL-2 is continuously added during co-culture, and the IL-2 concentration is maintained at a co-culture concentration of 50 IU / mL to 1000 IU / mL.

[0499] In one embodiment, the present invention includes a method for determining the potency of a T cell product, the method being: The first step involves co-culturing target cells with aT cell product cells, b. Steps to obtain samples from co-culture, c. The steps of (1) evaluating the expression of one or more markers on T cell product cells, or (2) evaluating one or more analytes secreted from T cell product cells, obtaining one or more observational values, and determining the potency of the T cell product, The second step involves co-culturing negative control cells with dT cell product cells for a second period, e. An additional step of obtaining a second sample from the second co-culture, f. The second sample is subjected to an additional step of obtaining one or more control values ​​by (1) evaluating the expression of one or more markers on T cell product cells, or (2) evaluating one or more analytes secreted from T cell product cells. g. The additional step of determining the potency of the T cell product by comparing one or more observations from step c to one or more control values ​​from step f, where each observation is compared to a corresponding control value, IL-2 is continuously added during the co-culture and the second co-culture, and the concentration of IL-2 is maintained at 50 IU / mL to 1000 IU / mL in each of the co-cultures.

[0500] In some embodiments, the present invention provides a method for assaying TIL activity, MIL activity, or PBL activity, and a toxic assay is used. In some embodiments, the present invention provides a method for assaying TIL activity, MIL activity, or PBL activity, and a toxic assay is used, and a cell lysis endpoint is detected. In some embodiments, the present invention provides a method for assaying TIL activity, MIL activity, or PBL activity, and a toxic assay is used. In some embodiments, the present invention provides a method for assaying TIL activity, MIL activity, or PBL activity, and a toxic assay is used, and a cell lysis endpoint is detected by gene modification of the target cell line to express a fluorescent, chemiluminescent, or bioluminescent protein when lysed. Suitable gene 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 gene modification approach described for bioluminescence detection in U.S. Patent No. 10,415,015 (the disclosure of which is incorporated herein by reference) may be used to modify target cells such as Raji cells, K562 cells, Daudi cells, Ramos cells, U937 cells, or Thp1 cells.

[0501] In one embodiment, the present invention provides a method for assaying the potency of a T cell product, the method further comprising the step of pre-irradiating target cells to halt their proliferation. In another embodiment, the present invention provides a method for assaying the potency of a T cell product, the method comprising the use of unirradiated target cells to halt their proliferation. In yet another embodiment, the present invention provides a method for assaying the potency of a T cell product, the method further comprising the step of chemically or biologically treating target cells to halt their proliferation.

[0502] In one embodiment, the present invention provides a method for assaying the potency of a T cell product, the method further comprising the step of pre-irradiating negative control cells to halt their proliferation. In another embodiment, the present invention provides a method for assaying the potency of a T cell product, the method comprising the use of unirradiated negative control cells to halt their proliferation. In yet another embodiment, the present invention provides a method for assaying the potency of a T cell product, the method further comprising the step of chemically or biologically treating negative control cells to halt their proliferation.

[0503] In one embodiment, target cells are irradiated with radiation using X-rays or gamma rays to a total absorbed dose of approximately 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 one embodiment, target cells are exposed to X-rays or gamma rays at approximately 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, and 1800 rads. 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, Radiation is administered with a total absorbed dose 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.

[0505] In one embodiment, target cells are exposed to X-rays or gamma rays at approximately 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, and 36 The subject is irradiated with radiation at an absorbed dose rate of 0 rads / min, 380 rads / min, 400 rads / min, 420 rads / min, 440 rads / min, 460 rads / min, 480 rads / min, 500 rads / min, 520 rads / min, 540 rads / min, 560 rads / min, 580 rads / min, 600 rads / min, 620 rads / min, 640 rads / min, 660 rads / min, 680 rads / min, or 700 rads / min. In one embodiment, target cells are exposed to X-rays or gamma rays at approximately 50 rads / min, 100 rads / min, 150 rads / min, 200 rads / min, 250 rads / min, 300 rads / min, 350 rads / min, 400 rads / min, 450 rads / min, 500 rads / min, 550 rads / min, 600 rads / min, 650 rads / min, 700 rads / min, 750 rads / min, 800 rads / min, 850 rads / min, 900 rads / min, 950 rads / min, 1000 rads / min, and 1050 rads / min. The radiation is irradiated at an absorbed dose rate of rads / min, 1100 rads / min, 1150 rads / min, 1200 rads / min, 1250 rads / min, 1300 rads / min, 1350 rads / min, 1400 rads / min, 1450 rads / min, 1500 rads / min, 1550 rads / min, 1600 rads / min, 1650 rads / min, 1700 rads / min, 1750 rads / min, 1800 rads / min, 1850 rads / min, 1900 rads / min, 1950 rads / min, or 2000 rads / min.

[0506] In one embodiment, the present invention includes ...

Claims

1. A method for determining the efficacy and / or functionality of a TIL product containing tumor-infiltrating lymphocytes (TILs), wherein the method is (a) A step of measuring IFN-γ release from the TIL product by co-culture assay, wherein the co-culture assay comprises (i) co-culturing TIL cells derived from the TIL product with U937 target cells, and (ii) detecting IFN-γ released from the TIL cells, wherein the U937 target cells express major histocompatibility complex (MHC), the TIL cells express T cell receptor (TCR), and the TIL cells are activated during the co-culture assay via allogeneic MHC-TCR engagement between the MHC of the U937 target cells and the TCR of the TIL cells; (b) A step of measuring IFN-γ release from the TIL product by a bead-based assay or a plate-based assay; and (c) Using a flow cytometry assay, the TIL product LAG3 ＋ Cell content, KLRG1 ＋ Cell content, CD4 ＋ Cell content and CD8 ＋ Total cell content, total viability T ＣＭ and T ＥＭ The procedure includes the step of measuring the cell population and the CD45 + CD3 + cell content, method.

2. A bead-based or plate-based assay stimulates TIL cells from a TIL product with anti-CD3 antibody, anti-CD28 antibody, and / or anti-CD137 antibody, and detects IFN-γ released from the TIL cells. The method according to claim 1, including the method described in claim 1.

3. The method according to claim 1, wherein the co-culturing in step (a) 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.

4. The method according to claim 1, wherein the ratio of TIL cells to U937 target cells is approximately 15:4, approximately 15:2, approximately 15:1, and / or approximately 30:

1.

5. The method according to any one of claims 1 to 4, wherein the co-culture in step (a) is carried out in a cell culture medium containing IL-2.

6. The method according to claim 5, wherein the cell culture medium contains IL-2 at a concentration of about 300 IU / mL.

7. The method according to any one of claims 1 to 4, wherein an HLA-blocking antibody is added to the co-culture as a negative control.

8. The method according to claim 7, wherein the HLA blocking antibody is used at a concentration of about 1 to 20 μg / mL.

9. TIL products, (a) A first expansion is performed by culturing a first TIL population in a cell culture medium containing IL-2 to produce a second TIL population, wherein the first expansion is performed for approximately 3 to 14 days to obtain the second TIL population; (b) A second expansion is performed by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, and antigen-presenting cells (APCs) to produce a third TIL population, where the second expansion is performed for approximately 7 to 14 days to obtain the third TIL population, which is the therapeutic TIL population; (c) A step of collecting the therapeutic TIL population obtained from step (b), (d) The step of transferring the therapeutic TIL population from step (c) to an infusion bag, and (e) Step of freezing the infusion bag, The method according to any one of claims 1 to 4, produced by a method comprising:

10. The method according to claim 9, wherein the method is performed after step (e).

11. The method according to any one of claims 1 to 4, wherein the TIL product includes CD8+TIL and CD4+TIL.

12. The method according to any one of claims 1 to 4, wherein the anti-CD3 antibody is not added to the co-culture during the co-culture assay.

13. A co-culture assay for IFN-γ release is a) Co-culture TIL cells and U937 target cells at at least three different U937 target cell concentrations. b) Extract the supernatant from each of the co-cultures, and c) Evaluate the supernatant from each co-culture for IFN-γ secreted from TIL cells and determine the efficacy of the TIL product. The method according to any one of claims 1 to 4, including the method described in any one of claims 1 to 4.

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