Systems and methods for coordinating the production of cells for patient-specific immunotherapy

Abstract

A method for coordinating the manufacture of an expanded cell therapy product for a patient may include receiving a cell order request to expand a cell therapy product for a patient, generating a patient-specific identifier or cell order identifier associated with the cell order request, and initiating a process to expand the cell therapy product from at least a portion of a solid tumor obtained from the patient. If approval parameters of the expanded cell therapy product do not meet certain approval criteria at a second time point subsequent to the first time point of the expansion process, determining whether it is possible to re-perform the expansion of the cell therapy product using a cell expansion technique from the first time point based on the approval parameters at the second time point. If it is possible to re-perform such expansion, the patient treatment event using the expanded cell therapy product is rescheduled.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No. 63 / 272,660, filed October 27, 2021, which is incorporated herein by reference in its entirety for all purposes. [Background technology]

[0002] The treatment of bulky, refractory cancers using adoptive transfer of tumor-infiltrating lymphocytes (TILs) offers a powerful approach for treating patients with poor prognosis. (Gattinoni, et al., Nat. Rev. Immunol. 2006, 6, 383-393) Successful immunotherapy requires large numbers of TILs, and commercialization requires a robust and reliable process. This has been difficult to achieve due to technical, logistical, and regulatory challenges associated with cell expansion. IL-2-based TIL expansion followed by the "rapid expansion process" (REP) has become the preferred method of TIL expansion due to its speed and efficiency. Dudley, et al., Science 2002, 298, 850-54; Dudley, et al., J. Clin. Oncol. 2005, 23, 2346-57; Dudley, et al., J. Clin. Oncol. 2008, 26, 5233-39; Riddell, et al., Science 1992, 257, 238-41; Dudley, et al., J. Immunother. 2003, 26, 332-42. REP requires a large excess (e.g., 200-fold) of irradiated allogeneic peripheral blood mononuclear cells (PBMCs, also known as mononuclear cells (MNCs)) often derived from multiple donors as feeder cells, as well as anti-CD3 antibodies (OKT3) and high doses of IL-2, but can result in a 1,000-fold expansion of TILs over 14 days. Dudley, et al., J. Immunother. 2003, 26, 332-42. TILs subjected to the REP procedure were used successfully in adoptive cell therapy following host immunosuppression in patients with melanoma. [Brief explanation of the drawings]

[0003] [Figure 1] An exemplary Gen2 (Process 2A) chart providing an overview of steps A-F. [Figure 2A] 1 is a process flow chart of several embodiments of Gen2 (Process 2A) for TIL fabrication. [Figure 2B] 1 is a process flow chart of several embodiments of Gen2 (Process 2A) for TIL fabrication. [Figure 2C] 1 is a process flow chart of several embodiments of Gen2 (Process 2A) for TIL fabrication. [Figure 3] 1 shows diagrams of several embodiments of an exemplary manufacturing process (approximately 22 days) for cryopreserved TILs. [Figure 4] 1 shows diagrams of several embodiments of Gen2 (Process 2A), a 22-day process for TIL fabrication. [Figure 5] 1 is a comparison table of steps A-F from exemplary embodiments of Process 1C and Gen2 (Process 2A) for TIL fabrication. [Figure 6] Detailed comparison of some embodiments of Process 1C with some embodiments of Gen2 (Process 2A) for TIL fabrication. [Figure 7] An exemplary GEN3 TIL manufacturing process. [Figure 8A] 1 shows a comparison of several embodiments of the 2A process (an approximately 22 day process) and the Gen3 process (an approximately 14-16 day process) for TIL fabrication. [Figure 8B] 1 illustrates an exemplary Process Gen3 chart providing an overview of steps A-F (approximately a 14-16 day process). [Figure 8C] A chart is shown providing three exemplary Gen3 processes along with an overview of steps A-F (approximately 14-16 day processes) for each of the three process variations. [Figure 8D]1 illustrates an exemplary modified Gen2-like process providing an overview of steps A-F (approximately a 22 day process). [Figure 9] 1 provides an experimental flow chart for the comparison between Gen2 (Process 2A) and Gen3 processes. [Figure 10] 1 shows a comparison of various Gen2 (Process 2A) and Gen3.1 process embodiments. [Figure 11] 1 is a table illustrating various features of embodiments of the Gen2, Gen2.1, and Gen3.0 processes. [Figure 12] Summary of media conditions for several embodiments of the Gen3 process, referred to as Gen3.1. [Figure 13] 1 is a table illustrating various features of embodiments of the Gen2, Gen2.1, and Gen3.1 processes. [Figure 14] 1 is a table comparing various features of embodiments of the Gen2 and Gen3.0 processes. [Figure 15] 1 is a table providing media use in various embodiments of the described expansion process. [Figure 16] Schematic of an exemplary embodiment of the Gen3 process (16-day process). [Figure 17] Schematic of an exemplary embodiment of a method for expanding T cells from hematopoietic malignancies using the Gen3 expansion platform. [Figure 18]Structures IA and IB are provided. The cylinders refer to individual polypeptide binding domains. Structures IA and IB comprise three linearly linked TNFRSF-binding domains derived from antibodies that bind, for example, to 4-1BBL or 4-1BB, that fold to form a trivalent protein, which is then linked to a second trivalent protein via IgG1-Fc (comprising the CH3 and CH2 domains), which is then used to link two of the trivalent proteins together via disulfide bonds (small oblong ellipses), stabilizing the structure and providing an agonist that can bring together the six receptor and intracellular signaling domains of the signaling protein to form a signaling complex. The TNFRSF-binding domains shown as cylinders can be, for example, scFv domains comprising VH and VL chains connected by a linker that may contain hydrophilic residues and Gly and Ser sequences for flexibility, and Glu and Lys for solubility. [Figure 19] Schematic of an exemplary embodiment of the Gen3 process (16-day process). [Figure 20] 1 provides a process overview of an exemplary embodiment of the Gen3.1 process (16-day process). [Figure 21] Schematic of an exemplary embodiment of the Gen3.1 Test (Optimized Gen3.1) process (16-17 day process). [Figure 22] Schematic of an exemplary embodiment of the Gen3 process (16-day process). [Figure 23] 1 is a comparison table of an exemplary Gen2 process and an exemplary Gen3 process, with exemplary differences highlighted. [Figure 24] Schematic of an exemplary embodiment of the preparation timeline for the Gen3 process (16-17 day process). [Figure 25] Schematic of an exemplary embodiment of the Gen3 process (14-16 day process). [Figure 26A] Schematic of an exemplary embodiment of the Gen3 process (16-day process). [Figure 26B] Schematic of an exemplary embodiment of the Gen3 process (16-day process). [Figure 27] Schematic of an exemplary embodiment of the Gen3 process (16-day process). [Figure 28] Comparison of several embodiments of the Gen2, Gen2.1, and Gen3 processes (16-day processes). [Figure 29] Comparison of several embodiments of the Gen2, Gen2.1, and Gen3 processes (16-day processes). [Figure 30] Components of a Gen3 embodiment. [Figure 31] Flowchart comparison of Gen3 embodiments (Gen3.0, Gen3.1 control, Gen3.1 test). [Figure 32] The components of an exemplary embodiment of the Gen3 process (Gen3-Optimized, 16-17 day process) are shown. [Figure 33] Approval criteria table. [Figure 34] FIG. 1 shows a block diagram of a system for tracking patient-specific immunotherapy data, according to some embodiments. [Figure 35A] 1 shows a block diagram of a system for coordinating the production of TILs in a patient. [Figure 35B] 3 illustrates the object schema of the components of system 300 as suitably modified or built on commercially available software platforms, in addition to standards within those platforms, according to some embodiments. [Figure 35C] 1A-1C illustrate schematically tracking biomaterials through a manufacturing process at a manufacturing facility, according to some embodiments. [Figure 35D] 1A-1C illustrate schematically tracking biomaterials through a manufacturing process at a manufacturing facility, according to some embodiments. [Figure 35E] 1A-1C illustrate schematically tracking biomaterials through a manufacturing process at a manufacturing facility, according to some embodiments. [Figure 35F]1A-1C illustrate a schematic diagram of a process for maintaining COC and COI throughout the journey of a cell therapy product from acquisition of a solid tumor through a manufacturing process to infusion into a patient, according to some embodiments of the manufacturing process (e.g., the GEN3 process). [Figure 35G] 1 is a representative image of a label for a patient tumor specimen, according to some embodiments. [Figure 35H] 1 is a table illustrating various types of labels generated during the manufacturing process of a cell therapy product, according to some embodiments. [Figure 35I] 1 is a representative image of a label on a finished product, according to some embodiments. [Figure 35J] 1 is a representative image of a label on a finished product, according to some embodiments. [Figure 35K] 1 is a representative screenshot image of a tumor procurement configuration, according to some embodiments. [Figure 35L] 1 is a representative screenshot image of a tumor procurement configuration, according to some embodiments. [Figure 35M] 1 is a representative screenshot image of a tumor procurement configuration, according to some embodiments. [Figure 35N] 1 is a representative screenshot image of a tumor procurement configuration, according to some embodiments. [Figure 35O] 1 is a representative screenshot image of a tumor procurement configuration, according to some embodiments. [Figure 35P] 1 is a representative screenshot image of a tumor procurement configuration, according to some embodiments. [Figure 36A] 1 shows a flow chart for determining the schedule of patient treatment events based on the success of the TIL manufacturing process. [Figure 36B] 1 shows a flow chart for determining the schedule of patient treatment events based on the success of the TIL manufacturing process. [Figure 36C] 10 shows a flowchart of an alternative embodiment for determining the schedule of patient treatment events based on the success of the TIL manufacturing process. [Figure 37A] 1 illustrates an exemplary UI for updating patient registration data and submitting tumor specimen procurement orders by a hospital user (e.g., a hospital). [Figure 37B] 1 illustrates an exemplary UI for updating patient registration data and submitting tumor specimen procurement orders by a hospital user (e.g., a hospital). [Figure 37C] 1 illustrates an exemplary UI for updating patient registration data and submitting tumor specimen procurement orders by a hospital user (e.g., a hospital). [Figure 37D] 1 illustrates an exemplary UI for updating patient registration data and submitting tumor specimen procurement orders by a hospital user (e.g., a hospital). [Figure 37E] 1 illustrates an exemplary UI for updating patient registration data and submitting tumor specimen procurement orders by a hospital user (e.g., a hospital). [Figure 37F] 1 illustrates an exemplary UI for updating patient registration data and submitting tumor specimen procurement orders by a hospital user (e.g., a hospital). [Figure 37G] 1 illustrates an exemplary UI for updating patient registration data and submitting tumor specimen procurement orders by a hospital user (e.g., a hospital). [Figure 37H] 1 illustrates an exemplary UI for updating patient registration data and submitting tumor specimen procurement orders by a hospital user (e.g., a hospital). [Figure 38A] 10 illustrates an exemplary UI for approving a tumor specimen procurement order by a case manager user and generating a requested lot number based on the approved order. [Figure 38B] 10 illustrates an exemplary UI for approving a tumor specimen procurement order by a case manager user and generating a requested lot number based on the approved order. [Figure 38C] 10 illustrates an exemplary UI for approving a tumor specimen procurement order by a case manager user and generating a requested lot number based on the approved order. [Figure 38D]10 illustrates an exemplary UI for approving a tumor specimen procurement order by a case manager user and generating a requested lot number based on the approved order. [Figure 39A] 38A-38D illustrate an exemplary UI for a manufacturing facility user to assign requested lot numbers and verify assigned requested lot orders generated by the case manager user of FIGS. 38A-38D. [Figure 39B] 38A-38D illustrate an exemplary UI for a manufacturing facility user to assign requested lot numbers and verify assigned requested lot orders generated by the case manager user of FIGS. 38A-38D. [Figure 39C] 38A-38D illustrate an exemplary UI for a manufacturing facility user to assign requested lot numbers and verify assigned requested lot orders generated by the case manager user of FIGS. 38A-38D. [Figure 39D] 38A-38D illustrate an exemplary UI for a manufacturing facility user to assign requested lot numbers and verify assigned requested lot orders generated by the case manager user of FIGS. 38A-38D. [Figure 39E] 38A-38D illustrate an exemplary UI for a manufacturing facility user to assign requested lot numbers and verify assigned requested lot orders generated by the case manager user of FIGS. 38A-38D. [Figure 40A] 1 illustrates an exemplary UI for a care facility user to track the chain of custody before, during, and after surgery. [Figure 40B] 1 illustrates an exemplary UI for a care facility user to track the chain of custody before, during, and after surgery. [Figure 40C] 1 illustrates an exemplary UI for a care facility user to track the chain of custody before, during, and after surgery. [Figure 40D] 1 illustrates an exemplary UI for a care facility user to track the chain of custody before, during, and after surgery. [Figure 40E] 1 illustrates an exemplary UI for a care facility user to track the chain of custody before, during, and after surgery. [Figure 40F] 1 illustrates an exemplary UI for a care facility user to track the chain of custody before, during, and after surgery. [Figure 40G] 1 illustrates an exemplary UI for a care facility user to track the chain of custody before, during, and after surgery. [Figure 40H] 1 illustrates an exemplary UI for a care facility user to track the chain of custody before, during, and after surgery. [Figure 40I] 1 illustrates an exemplary UI for a care facility user to track the chain of custody before, during, and after surgery. [Figure 40J] 1 illustrates an exemplary UI for a care facility user to track the chain of custody before, during, and after surgery. [Figure 40K] 1 illustrates an exemplary UI for a care facility user to track the chain of custody before, during, and after surgery. [Figure 41A] 1 illustrates an exemplary UI for surgical documentation at a treatment facility, according to some embodiments. [Figure 41B] 1 illustrates an exemplary UI for surgical documentation at a treatment facility, according to some embodiments. [Figure 41C] 1 illustrates an exemplary UI for surgical documentation at a treatment facility, according to some embodiments. [Figure 42] 1 illustrates an exemplary post-operative UI for packaging documentation at a treatment facility, according to some embodiments. [Figure 43] 43 illustrates an exemplary generated waybill label based on the packaging and documentation steps described in FIG. 42 , according to some embodiments. [Figure 44] 9 illustrates an exemplary COI and COC report UI 900 from the system end-to-end, according to some embodiments. [Figure 45A]1 illustrates an exemplary UI for a manufacturing facility UI upon receipt of a tumor specimen at the manufacturing facility, according to some embodiments. [Figure 45B] 1 illustrates an exemplary UI for a manufacturing facility UI upon receipt of a tumor specimen at the manufacturing facility, according to some embodiments. [Figure 45C] 1 illustrates an exemplary UI for a manufacturing facility UI upon receipt of a tumor specimen at the manufacturing facility, according to some embodiments. [Figure 46] 1 illustrates a tumor specimen scan recorded on the backend, according to some embodiments. [Figure 47] Components of an exemplary embodiment of the Gen3 process (16-17 day process) are shown. [Figure 48] Approval criteria table. [Figure 49] Experimental flow diagram of the full-scale PD-1 KO TIL TALEN process. [Figure 50] Experimental flow diagram of the full-scale PD-1 KO TIL TALEN process. [Figure 51] AD: Schematic of an exemplary embodiment of the KO TIL TALEN process. [Figure 52] Schematic of an exemplary embodiment of the process described in Example 12. [Figure 53] A-B: In vivo efficacy of PDCD-1 KO TILs. A) Efficacy of PDCD-1 KO assessed by flow cytometry. B) hIL-2 NOG mice (n=14 per treatment group) engrafted with melanoma tumor cells were adoptively transferred with PDCD-1 KO or mock TILs. Anti-PD-1 antibody treatment in combination with mock TILs was included as a control for PD-1 / PD-L1 blockade. Statistical significance is indicated by *p<0.05, **p<0.01, and ****p<0.0001. [Figure 54] A-E: Analysis of TIL products. A) viable cell dose, B) purity, C) identity, D) potency, and E) PDCD-1 KO efficiency of TIL products. [Figure 55] A-B: Analysis of TIL products. A) TIL differentiation, and B) TIL memory. [Figure 56] A-B: Expression of activation- and inhibition-related markers on PDCD-1 KO TILs. [Figure 57] A-B: IL-2-independent proliferation assay of PDCD-1 KO TIL products. [Figure 58] Summary of karyotyping results from PDCD-1 KO TIL products. [Figure 59] A-B: Cell viability (A) and fold recovery (B) of cells before electroporation. [Figure 60] A-B: Magnification recovery of cells after electroporation (A) and cell viability (B). [Figure 61] A-C: Knockout efficiency in CD3+ (A), CD8+ (B), and CD4+ (C) cells. [Figure 62] A-B: Magnification recovery of cells after electroporation (A) and cell viability (B). [Figure 63] A-B: Fold recovery of cells (A) and cell viability (B) after electroporation using 6000 IU / mL of IL-2. [Figure 64] A-B: Fold recovery of cells after electroporation using various conditions (A) and cell viability (B). [Figure 65] A-C: Knockout efficiency in CD3+ (A), CD8+ (B), and CD4+ (C) cells. [Figure 66] Cell viability before electroporation. [Figure 67] Magnification recovery of cells before electroporation. [Figure 68] A-B: Magnification recovery of cells after electroporation (A) and cell viability (B). [Figure 69A] Knockout efficiency in CD3+ cells. [Figure 69B] Knockout efficiency in CD8+ cells. [Figure 69C] Knockout efficiency in CD4+ cells. [Figure 70]A-B: Cell number (A) and viability (B) after various washing steps. [Figure 71] A-B: Cell counts after various spin conditions using a PBS wash (A) or a Cyto wash (B). [Figure 72] A-B: Cell viability after various spin conditions using a PBS wash (A) or a Cyto wash (B). [Figure 73] A-B: Total spin-comparative cell number (A) and total spin-comparative cell viability (B) of cells after various spin conditions. [Figure 74] Total spin comparative cell loss rate after various spin conditions. [Figure 75] AC: Cell loss and viability during electroporation, specifically, cell loss during the washing step (A), cell loss after electroporation (B), and cell viability after electroporation (C). [Figure 76] A-C: Knockout efficiency in CD3+ (A), CD8+ (B), and CD4+ (C) cells. [Figure 77] A-B: Cell viability (A) and fold expansion (B) of REP harvests. [Figure 78] A-B: Cell loss rate (A) and cell viability (B) after electroporation. [Figure 79] A-C: Knockout efficiency in CD3+ (A), CD4+ (B), and CD8+ (C) cells. [Figure 80] A-B: Fold expansion (A) and cell viability (B) of REP harvest. [Figure 81] AC: Cell proliferation (A), first electroporation knockout efficiency (B), and second electroporation knockout efficiency (C). [Figure 82] Growth rate during 3 days of standing. [Figure 83] A–C: PD-1 knockout efficiency. [Figure 84] PDCD1 gene modification by NGS. [Figure 85]A-B: Distribution of TCR Vβ subtypes in bulk PD-1 KO TIL preparations and distribution of NE TILs in the CD3+PD-1 subset. [Figure 86] A-B: PD-1 KO TIL effector function as measured by MLR (A) and polyfunctionality (B). [Figure 87] In vivo antitumor activity of M1152 PD-1 KO TIL products. [Figure 88] A-B: Persistence of TALEN proteins in autologous TILs as a function of time measured by Western blot. [Figure 89A] Illustrative TIL fabrication process. [Figure 89B] Illustrative TIL fabrication process. [Figure 89C] Illustrative TIL fabrication process. [Figure 89D] Illustrative TIL fabrication process. [Figure 89E] Illustrative TIL fabrication process. [Figure 89F] Illustrative TIL fabrication process. [Figure 90A] Schema of the Phase 1 / 2 study described in Example 22. [Figure 90B] Schema of the Phase 1 / 2 study described in Example 22. [Figure 91] Summary of data described in Example 23. [Figure 92] A to D: Results of the demo day experiment for Example 23. [Figure 93] A-C: Results from Neon Exp 1 in Example 23. [Figure 94] AC: Results from Xenon Exp 1 in Example 23. [Figure 95] A to B: Results of Xenon Exp 3 in Example 23. [Figure 96] A to C: Results for Xenon Exp 4 in Example 23. DETAILED DESCRIPTION OF THE INVENTION

[0004] I. Introduction A. Adoptive Cell Transfer Adoptive cell therapy using TILs cultured ex vivo by rapid expansion protocols (REP) has been successful in patients with cancers such as melanoma after host immunosuppression. Current infusion acceptance parameters rely on TIL composition (e.g., CD28, CD8, or CD4 positivity) as a readout, as well as the numerical fold expansion and viability of the REP product. While TILs can be reactivated and expanded ex vivo, their epigenetic programming in the suppressive tumor microenvironment can maintain TILs in a more differentiated and less functional state when administered.

[0005] The present invention relates to the use of epigenetic reprogramming agents in the cell culture medium during ex vivo expansion of TILs to counter the effects of the suppressive tumor microenvironment and improve the quality of the expanded TILs with respect to persistence, functionality, and anti-tumor potential.

[0006] II. Definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All patents and publications referenced herein are incorporated by reference in their entirety.

[0007] As used herein, the terms "co-administration," "co-administering," "administered in combination," "administering in combination," "simultaneous," and "concurrent" encompass administration of two or more active pharmaceutical ingredients (e.g., multiple TILs in preferred embodiments of the present invention) to a subject such that both active pharmaceutical ingredients and / or their metabolites are present in the subject at the same time. Simultaneous administration includes simultaneous administration in separate compositions, administration at different times in separate compositions, or administration in a composition in which two or more active pharmaceutical ingredients are present. Concurrent administration in separate compositions and administration in a composition in which both agents are present are preferred.

[0008] The term "in vivo" refers to events that take place inside a subject's body.

[0009] The term "in vitro" refers to events that occur outside a subject's body. In vitro assays include cell-based assays, in which living or dead cells are used, and can also include cell-free assays, in which no intact cells are used.

[0010] The term "ex vivo" refers to events involving the administration of a therapy or treatment to cells, tissues, and / or organs that have been removed from a subject's body. Suitably, the cells, tissues, and / or organs may be returned to the subject's body in a surgical or therapeutic manner.

[0011] The term "rapid expansion" refers to an increase in the number of antigen-specific TILs of at least about 3-fold (or 4, 5, 6, 7, 8, or 9-fold) over a one-week period, more preferably at least about 10-fold (or 20, 30, 40, 50, 60, 70, 80, or 90-fold) over a one-week period, or most preferably at least about 100-fold over a one-week period. Several rapid expansion protocols are described herein.

[0012] As used herein, "tumor infiltrating lymphocytes" or "TILs" refers to a population of cells that are initially acquired as leukocytes that have left a subject's bloodstream and migrated to a tumor. TILs include CD8 + Cytotoxic T cells (lymphocytes), Th1 and Th17 CD4 +These include, but are not limited to, T cells, natural killer cells, dendritic cells, and M1 macrophages. TILs include both primary and secondary TILs. "Primary TILs" are those obtained from a patient tissue sample as outlined herein (sometimes referred to as "freshly harvested"), and "secondary TILs" are any expanded or propagated TIL cell populations discussed herein, including, but not limited to, bulk TILs and expanded TILs ("REP TILs" or "post-REP TILs"). TIL cell populations may include genetically modified TILs.

[0013] As used herein, a "population of cells" (including TILs) refers to several cells that share a common trait. Generally, a population is roughly 1 x 10 6 ~1×10 10 The number of TILs ranges from 1 x 10 to 1 x 10, with different TIL populations containing different numbers. For example, the initial growth of primary TILs in the presence of IL-2 is approximately 1 x 10 8 REP expansion typically yields a bulk TIL population of 1.5 x 10 cells for injection. 9 ~1.5×10 10 This is done to provide a population of cells.

[0014] As used herein, "cryopreserved TILs" refers to TILs, either primary, bulk, or expanded (REP TILs), that are processed and stored at temperatures ranging from approximately -150°C to -60°C. General methods for cryopreservation are described elsewhere herein, including in the Examples. For clarity, "cryopreserved TILs" can be distinguished from frozen tissue samples that may be used as a source of primary TILs.

[0015] As used herein, "thawed cryopreserved TILs" refers to a population of TILs that have been previously cryopreserved and then processed to return to room temperature or above, including but not limited to, cell culture temperature or a temperature at which the TILs can be administered to a patient.

[0016] TILs can generally be defined either biochemically using cell surface markers or functionally by their ability to infiltrate tumors and achieve therapy. TILs can generally be classified by expression of one or more of the following biomarkers: CD4, CD8, TCRαβ, CD27, CD28, CD56, CCR7, CD45Ra, CD95, PD-1, and CD25. Additionally and alternatively, TILs can be functionally defined by their ability to infiltrate solid tumors upon reintroduction into patients.

[0017] The term "cryopreservation media" or "cryopreservation medium" refers to any medium that can be used for cryopreserving cells. Such media can include media containing 7% to 10% DMSO. Exemplary media include CryoStor CS10, Hyperthermasol, and combinations thereof. The term "CS10" refers to cryopreservation media obtained from Stemcell Technologies or Biolife Solutions. CS10 medium may be referred to by the trade name "CryoStor® CS10." CS10 medium is a serum-free, animal-component-free medium that contains DMSO.

[0018] The term "central memory T cells" refers to cells that are CD45R0+ and CCR7 (CCR7 高 ) and CD62L (CD62 高 ) is a subset of T cells that constitutively express the CD4 receptor. The surface phenotype of central memory T cells also includes TCR, CD3, CD127 (IL-7R), and IL-15R. Transcription factors of central memory T cells include BCL-6, BCL-6B, MBD2, and BMI1. After TCR triggering, central memory T cells primarily secrete IL-2 and CD40L as effector molecules. Central memory T cells predominate in the CD4 compartment in the blood and are proportionally enriched in lymph nodes and tonsils in humans.

[0019] The term "effector memory T cells" refers to T cells that are CD45R0+ like central memory T cells, but have lost constitutive expression of CCR7 (CCR7 低 ), heterogeneous or low CD62L expression (CD62L 低 ), refers to a subset of human or mammalian T cells. The surface phenotype of central memory T cells also includes TCR, CD3, CD127 (IL-7R), and IL-15R. Transcription factors of central memory T cells include BLIMP1. Effector memory T cells rapidly secrete high levels of inflammatory cytokines after antigen stimulation, including interferon gamma, IL-4, and IL-5. Effector memory T cells predominate in the CD8 compartment in the blood and are proportionally enriched in the lung, liver, and intestine in humans. CD8+ effector memory T cells carry large amounts of perforin.

[0020] The term "closed system" refers to a system that is closed to the external environment. Any closed system suitable for cell culture methods can be used in the methods of the present invention. Closed systems include, but are not limited to, sealed G containers. Once tumor segments are added to the closed system, the system is not opened to the external environment until the TILs are ready to be administered to a patient.

[0021] The terms "fragmenting," "fragments," and "fragmented" as used herein to describe processes for destroying tumors include mechanical fragmentation methods such as crushing, slicing, dividing, and mincing tumor tissue, as well as any other method for disrupting the physical structure of tumor tissue.

[0022] The terms "peripheral blood mononuclear cells" and "PBMCs" refer to peripheral blood cells with round nuclei, including lymphocytes (T cells, B cells, NK cells) and monocytes. When used as antigen-presenting cells (PBMCs are a type of antigen-presenting cell), the peripheral blood mononuclear cells are preferably irradiated allogeneic peripheral blood mononuclear cells.

[0023] The terms "peripheral blood lymphocytes" and "PBLs" refer to T cells expanded from peripheral blood. In some embodiments, PBLs are isolated from whole blood or apheresis products from a donor. In some embodiments, PBLs are isolated from whole blood or apheresis products from a donor by positive or negative selection of a T cell phenotype, such as a CD3+CD45+ T cell phenotype.

[0024] The term "anti-CD3 antibody" refers to an antibody or variant thereof, e.g., a monoclonal antibody, including a human, humanized, chimeric, or murine antibody, directed against the CD3 receptor in the T cell antigen receptor of mature T cells. Anti-CD3 antibodies include OKT-3, also known as muromonab. Anti-CD3 antibodies also include the UHCT1 clone, also known as T3 and CD3ε. Other anti-CD3 antibodies include, for example, otelixizumab, teplizumab, and visilizumab.

[0025] The term "OKT-3" (also referred to herein as "OKT3") refers to a monoclonal antibody, including a human, humanized, chimeric, or murine antibody against the CD3 receptor in the T cell antigen receptor of mature T cells, or a biosimilar or variant thereof, including commercially available forms such as OKT-3 (30 ng / mL, MACS GMP CD3 pure, Miltenyi Biotech, Inc., San Diego, CA, USA) and muromonab, or variants, conservative amino acid substitutions, glycoforms, or biosimilars thereof. The amino acid sequences of the heavy and light chains of muromonab are shown in Table 1 (SEQ ID NO: 1 and SEQ ID NO: 2). A hybridoma capable of producing OKT-3 has been deposited with the American Type Culture Collection and assigned ATCC accession number CRL8001. A hybridoma capable of producing OKT-3 has also been deposited with the European Collection of Authenticated Cell Cultures (ECACC) and assigned catalog number 86022706.

[0026] [Table 1]

[0027] The term "IL-2" (also referred to herein as "IL2") refers to the T cell growth factor known as interleukin-2 and includes all forms of IL-2, including human and mammalian forms, conservative amino acid substitutions, glycoforms, biosimilars, and variants. IL-2 is described, for example, in Nelson, J. Immunol. 2004, 172, 3983-88 and Malek, Annu. Rev. Immunol. 2008, 26, 453-79, the disclosures of which are incorporated herein by reference. The amino acid sequence of recombinant human IL-2 suitable for use in the present invention is shown in Table 2 (SEQ ID NO: 3). For example, the term IL-2 encompasses human recombinant IL-2 forms such as aldesleukin (PROLEUKIN, commercially available from multiple suppliers at 22 million IU per single-use vial), as well as the recombinant IL-2 form (catalog number CYT-209-b) marketed by CellGenix, Inc., Portsmouth, NH, USA (CELLGRO GMP) or ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA, and other commercial equivalents from other vendors. Aldesleukin (des-alanyl-1, serine-125 human IL-2) is a non-glycosylated human recombinant IL-2 form with a molecular weight of approximately 15 kDa. The amino acid sequence of aldesleukin suitable for use in the present invention is shown in Table 2 (SEQ ID NO: 4). The term IL-2 also refers to the pegylated IL-2 prodrug bempegaldesleukin (NKTR-214, an IL-2 prodrug in which an average of six lysine residues are replaced with [(2,7-bis{[methylpoly(oxyethylene)]carbamoyl}-9H-fluoren-9-yl)methoxy]carbonyl). 6The present invention also encompasses pegylated forms of IL-2 described herein, including pegylated human recombinant IL-2 such as SEQ ID NO: 4, which is available from Nektar Therapeutics (South San Francisco, CA, USA) or can be prepared by methods known in the art, such as the method described in Example 19 of International Patent Application Publication No. WO2018 / 132496 A1 or Example 1 of U.S. Patent Application Publication No. US2019 / 0275133 A1, the disclosures of which are incorporated herein by reference. Benpegaldesleukin (NKTR-214) and other pegylated IL-2 molecules suitable for use in the present invention are described in U.S. Patent Application Publication No. US2014 / 0328791 A1 and International Patent Application Publication No. WO2012 / 065086 A1, the disclosures of which are incorporated herein by reference. Alternative forms of conjugated IL-2 suitable for use in the present invention are described in U.S. Patent Nos. 4,766,106, 5,206,344, 5,089,261, and 4,902,502, the disclosures of which are incorporated herein by reference. Formulations of IL-2 suitable for use in the present invention are described in U.S. Patent No. 6,706,289, the disclosure of which is incorporated herein by reference.

[0028] In some embodiments, a suitable IL-2 form for use in the present invention is THOR-707, available from Synthorx, Inc. The preparation and properties of THOR-707 and additional alternative forms of IL-2 suitable for use in the present invention are described in U.S. Patent Application Publication Nos. US2020 / 0181220 A1 and US2020 / 0330601 A1, the disclosures of which are incorporated herein by reference. In some embodiments, a suitable IL-2 form for use in the present invention is an interleukin-2 (IL-2) complex comprising an isolated and purified IL-2 polypeptide and a conjugation moiety that binds to the isolated and purified IL-2 polypeptide at an amino acid position selected from K35, T37, R38, T41, F42, K43, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107, wherein the numbering of the amino acid residues corresponds to SEQ ID NO:5. 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, K35, T37, R38, T41, F42, K43, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107 are ...and Y107 are further mutated to an unnatural amino acid. In some embodiments, the unnatural amino acid is selected from N6-azidoethoxy-L-lysine (AzK), N6-propargylethoxy-L-lysine (PraK), BCN-L-lysine, norbornene lysine, TCO-lysine, methyltetrazine lysine, allyloxycarbonyl lysine, 2-amino-8-oxononanoic acid, 2-amino-8-oxooctanoic acid, p-acetyl-L-phenylalanine, p-azidomethyl-L-phenylalanine (pAMF), p-iodo-L-phenylalanine, m-acetylphenylalanine, 2-amino-8-oxononanoic acid, p-propargyloxyphenylalanine, p-propargyl-phenylalanine, 3-methyl-phenylalanine, L-dopa, fluorinated phenylalanine, isopropyl and selenocysteine, or selenocysteine. In some embodiments, the IL-2 complex has a reduced affinity for the IL-2 receptor alpha (IL-2Rα) subunit compared to a wild-type IL-2 polypeptide. In some embodiments, the reduced affinity is about a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or greater than a 99% reduction in binding affinity for IL-2Rα compared to a wild-type IL-2 polypeptide. In some embodiments, the reduced affinity is about a 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 30-fold, 50-fold, 100-fold, 200-fold, 300-fold, 500-fold, or greater reduction in binding affinity for IL-2Rα compared to a wild-type IL-2 polypeptide.1000-fold or more. In some embodiments, the conjugated moiety impairs or blocks the binding of IL-2 to IL-2Rα. In some embodiments, the conjugated moiety comprises a water-soluble polymer. In some embodiments, the additional conjugated moiety comprises a water-soluble polymer. In some embodiments, each of the water-soluble polymers independently comprises polyethylene glycol (PEG), poly(propylene glycol) (PPG), copolymers of ethylene glycol and propylene glycol, poly(oxyethylated polyol), poly(olefinic alcohol), poly(vinylpyrrolidone), poly(hydroxyalkyl methacrylamide), poly(hydroxyalkyl methacrylate), poly(saccharide), poly(α-hydroxy acid), poly(vinyl alcohol), polyphosphazene, polyoxazoline (POZ), poly(N-acryloylmorpholine), or a combination thereof. In some embodiments, each of the water-soluble polymers independently comprises PEG. In some embodiments, the PEG is linear PEG or branched PEG. In some embodiments, each of the water-soluble polymers independently comprises a polysaccharide. In some embodiments, the polysaccharide comprises dextran, polysialic acid (PSA), hyaluronic acid (HA), amylose, heparin, heparan sulfate (HS), dextrin, or hydroxyethyl starch (HES). In some embodiments, each of the water soluble polymers independently comprises a glycan. In some embodiments, each of the water soluble polymers independently comprises a polyamine. In some embodiments, the conjugation moiety comprises a protein. In some embodiments, the additional conjugation moieties comprise a protein. In some embodiments, each of the proteins independently comprises albumin, transferrin, or transthyretin. In some embodiments, each of the proteins independently comprises an Fc portion. In some embodiments, each of the proteins independently comprises an Fc portion of an IgG. In some embodiments, the conjugation moiety comprises a polypeptide. In some embodiments, the additional conjugation moieties comprise a polypeptide. In some embodiments, each of the proteins independently comprises an XTEN peptide, a glycine-rich homoamino acid polymer (HAP),The conjugated moiety may comprise 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 conjugated moiety is directly attached to the isolated and purified IL-2 polypeptide. In some embodiments, the conjugated moiety is indirectly attached to the isolated and purified IL-2 polypeptide via a linker. In some embodiments, the linker comprises a homobifunctional linker. In some embodiments, the homobifunctional linker is selected from the group consisting of the Romant reagents dithiobis(succinimidyl propionate) DSP, 3'3'-dithiobis(sulfosuccinimidyl propionate) (DTSSP), disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl) suberate (BS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfoDS T), ethylene glycobis(succinimidyl succinate) (EGS), disuccinimidyl glutarate (DSG), N,N'-disuccinimidyl carbonate (DSC), dimethyl adipimidate (DMA), dimethyl pimelimidate (DMP), dimethyl suberimidate (DMS), dimethyl-3,3'-dithiobispropionimidate (DTBP), 1,4-di-(3'-(2'-pyridyldithio) (e) propionamido) butane (DPDPB), bismaleimidohexane (BMH), halogenated aryl-containing compounds (DFDNB), such as 1,5-difluoro-2,4-dinitrobenzene or 1,3-difluoro-4,6-dinitrobenzene, 4,4'-difluoro-3,3'-dinitrophenyl sulfone (DFDNPS), bis-[β-(4-azidosalicylamido)ethyl] disulfide (BASED), formaldehyde, glutaraldehyde, 1,4-butanediol diglycidyl ether, adipic acid dihydrazide, carbohydrazide, o-toluidine, 3,3'-dimethylbenzidine, benzidine, α,α'-p-diaminodiphenyl, diiodo-p-xylene sulfonic acid, N,N'-ethylene-bis(iodoacetamide), or N,N'-hexamethylenebis(iodoacetamide). In some embodiments, the linker isIn some embodiments, the heterobifunctional linker includes 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]hexamethyl. Succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-LC-sMPT), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sMCC), sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-sMCC), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MB), m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (sulfo-MB), N-succinimidyl (4-iodoacetyl)aminobenzoate (sIAB), sulfosuccinimide Succinimidyl (4-iodoacetyl)aminobenzoate (sulfo-sIAB), succinimidyl-4-(p-maleimidophenyl)butyrate (sMPB), sulfosuccinimidyl-4-(p-maleimidophenyl)butyrate (sulfo-sMPB), N-(γ-maleimidobutyryloxy)succinimide ester (GMB), N-(γ-maleimidobutyryloxy)sulfosuccinimide ester (sulfo-GMB), succinimidyl 6-((iodoacetyl)amino)hexanoate (sIAX), succinimidyl 6-[6-(( (iodoacetyl)amino)hexanoyl)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-nitrophenyl iodoacetate (NPIA), carbonyl-reactive and sulfhydryl-reactive crosslinkers, such as 4-(4-N-maleimidophenyl)butyric acid hydrazide (MPBH),4-(N-maleimidomethyl)cyclohexane-1-carboxyl-hydrazide-8 (M2C2H), 3-(2-pyridyldithio)propionylhydrazide (PDPH), N-hydroxysuccinimidyl-4-azidosalicylate (NHs-AsA), N-hydroxysulfosuccinimidyl-4-azidosalicylate (sulfo-NHs-AsA), sulfosuccinimidyl-(4, -azidosalicylamido)hexanoate (sulfo-NHs-LC-AsA), sulfosuccinimidyl-2-(p-azidosalicylamido)ethyl-1,3'-dithiopropionate (sAsD), N-hydroxysuccinimidyl-4-azidobenzoate (HsAB), N-hydroxysulfosuccinimidyl-4-azidobenzoate (sulfo-HsAB), N-succinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate (sANPAH), sulfo Succinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate (sulfo-sANPAH), N-5-azido-2-nitrobenzoyloxysuccinimide (ANB-NO), sulfosuccinimidyl-2-(m-azido-o-nitrobenzamido)-ethyl-1,3'-dithiopropionate (sAND), N-succinimidyl-4(4-azidophenyl)1,3'-dithiopropionate (sADP), N-sulfosuccinimidyl(4-azidophenyl)- 1,3'-dithiopropionate (sulfo-sADP), sulfosuccinimidyl 4-(ρ-azidophenyl)butyrate (sulfo-sAPB), sulfosuccinimidyl 2-(7-azido-4-methylcoumarin-3-acetamido)ethyl-1,3'-dithiopropionate (sAED), sulfosuccinimidyl 7-azido-4-methylcoumarin-3-acetate (sulfo-sAMCA), p-nitrophenyl diazopyruvate (pNPDP), p-nitrophenyl diazo-3 ,3,3-trifluoropropionate (PNP-DTP), 1-(ρ-azidosalicylamido)-4-(iodoacetamido)butane (AsIB), N-[4-(ρ-azidosalicylamido)butyl]-3'-(2'-pyridyldithio)propionamide (APDP), benzophenone-4-iodoacetamide, p-azidobenzoylhydrazide (ABH), 4-(ρ-azidosalicylamido)butylamine (AsBA), or p-azidophenylglyoxal (APG). In some embodiments, the linker comprises a cleavable linker, optionally including a dipeptide linker. In some embodiments, the dipeptide linker comprises Val-Cit, Phe-Lys, Val-Ala, or Val-Lys.In some embodiments, the linker comprises a non-cleavable linker. In some embodiments, the linker optionally comprises a maleimide group, including maleimidocaproyl (mc), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sMCC), or sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-sMCC). In some embodiments, the linker further comprises a spacer. In some embodiments, the spacer comprises p-aminobenzyl alcohol (PAB), p-aminobenzyloxycarbonyl (PABC), a derivative, or an analog thereof. In some embodiments, the conjugated moiety is capable of extending the serum half-life of the IL-2 conjugate. In some embodiments, the additional conjugated moiety is capable of extending the serum half-life of the IL-2 conjugate. In some embodiments, the IL-2 form suitable for use in the present invention is a fragment of any of the IL-2 forms described herein. In some embodiments, IL-2 forms suitable for use in the present invention are pegylated as disclosed in U.S. Patent Application Publication Nos. US2020 / 0181220 A1 and US2020 / 0330601 A1. In some embodiments, IL-2 forms suitable for use in the present invention are IL-2 conjugates comprising an IL-2 polypeptide comprising N6-azidoethoxy-L-lysine (AzK) covalently attached to a conjugation moiety comprising polyethylene glycol (PEG), wherein the IL-2 polypeptide comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO:5, and wherein AzK substitutes an amino acid at position K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69, or L72 relative to amino acid position within SEQ ID NO:5. In some embodiments, the IL-2 polypeptide comprises an N-terminal deletion of one residue relative to SEQ ID NO:5. In some embodiments, forms of IL-2 suitable for use in the present invention lack IL-2R alpha chain association but retain normal binding to the intermediate affinity IL-2R beta-gamma signaling complex.In some embodiments, a suitable IL-2 form for use in the present invention is an IL-2 conjugate comprising an IL-2 polypeptide comprising N6-azidoethoxy-L-lysine (AzK) covalently attached to a conjugation moiety comprising polyethylene glycol (PEG), wherein the IL-2 polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:5, and wherein AzK substitutes an amino acid at position K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69, or L72 relative to amino acid position in SEQ ID NO:5. In some embodiments, a suitable IL-2 form for use in the present invention is an IL-2 conjugate comprising an IL-2 polypeptide comprising N6-azidoethoxy-L-lysine (AzK) covalently attached to a conjugation moiety comprising polyethylene glycol (PEG), wherein the IL-2 polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:5, and wherein AzK substitutes an amino acid at position K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69, or L72 relative to amino acid position in SEQ ID NO:5. In some embodiments, a suitable IL-2 form for use in the present invention is an IL-2 conjugate comprising an IL-2 polypeptide comprising N6-azidoethoxy-L-lysine (AzK) covalently attached to a conjugation moiety comprising polyethylene glycol (PEG), wherein the IL-2 polypeptide comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO:5, and wherein AzK substitutes an amino acid at position K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69, or L72 relative to amino acid position in SEQ ID NO:5.

[0029] In some embodiments, a form of IL-2 suitable for use in the present invention is nembareukin alfa, also known as ALKS-4230 (SEQ ID NO: 6), available from Alkermes, Inc. Nembareukin alfa is a nucleotide analogue of IL-2 that is linked via a peptidyl linker ( 60 GG 61 ) and fused to human interleukin-2 fragment (62-132) via a peptidyl linker ( 133 GSGGGS138 Human interleukin-2 receptor α-chain fragment (139-303) fused via a nucleotide sequence (Cys), produced in Chinese hamster ovary (CHO) cells, and glycosylated. 125 >Ser 51 ; human interleukin-2 (IL-2) (4-74)-peptide (62-132) fused via a G2 peptide linker (60-61) and human interleukin-2 receptor alpha chain (IL2R subunit alpha, IL2Rα, IL2RA) (1-165)-peptide (139-303) fused via a GSG3S peptide linker (133-138), produced in Chinese hamster ovary (CHO) cells, and alpha-glycosylated human interleukin-2 (IL-2) (75-133)-peptide [Cys 125(51)>Ser]-mutant (1-59). The amino acid sequence of nemvaleukin alpha is set forth in SEQ ID NO: 6. In some embodiments, nemvaleukin alpha exhibits the following post-translational modifications: disulfide bridges at the following positions: 31-116, 141-285, 184-242, 269-301, 166-197, or 166-199, 168-199, or 168-197 (using the numbering of SEQ ID NO: 6), and glycosylation sites at the following positions: N187, N206, T212, using the numbering of SEQ ID NO: 6. The preparation and properties of nemvaleukin alpha, as well as additional alternative forms of IL-2 suitable for use in the present invention, are described in U.S. Patent Application Publication No. US2021 / 0038684 A1 and U.S. Patent No. 10,183,979, the disclosures of which are incorporated herein by reference. In some embodiments, an IL-2 form suitable for use in the present invention is a protein having at least 80%, at least 90%, at least 95%, or at least 90% sequence identity to SEQ ID NO:6. In some embodiments, an IL-2 form suitable for use in the present invention has the amino acid sequence set forth in SEQ ID NO:6 or conservative amino acid substitutions thereof. In some embodiments, an IL-2 form suitable for use in the present invention is a fusion protein comprising amino acids 24-452 of SEQ ID NO:7, or a variant, fragment, or derivative thereof. In some embodiments, an IL-2 form suitable for use in the present invention is a fusion protein comprising an amino acid sequence having at least 80%, at least 90%, at least 95%, or at least 90% sequence identity to amino acids 24-452 of SEQ ID NO:7, or a variant, fragment, or derivative thereof. Other IL-2 forms suitable for use in the present invention are described in U.S. Pat. No. 10,183,979, the disclosure of which is incorporated herein by reference.Optionally, in some embodiments, a form of IL-2 suitable for use in the present invention is a fusion protein comprising a first fusion partner linked to a second fusion partner by a mucin domain polypeptide linker, wherein the first fusion partner is IL-1Rα or a protein having at least 98% amino acid sequence identity to IL-1Rα and having receptor antagonist activity for IL-Rα, the second fusion partner comprises all or a portion of an immunoglobulin comprising an Fc region, and the mucin domain polypeptide linker comprises SEQ ID NO:8 or an amino acid sequence having at least 90% sequence identity to SEQ ID NO:8, and wherein the half-life of the fusion protein is improved compared to the fusion of the first fusion partner with the second fusion partner in the absence of the mucin domain polypeptide linker.

[0030] [Table 2]

[0031] In some embodiments, IL-2 forms suitable for use in the present invention comprise an antibody cytokine graft protein comprising a heavy chain variable region (VH) comprising complementarity determining regions HCDR1, HCDR2, and HCDR3, a light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3, and an IL-2 molecule or a fragment thereof grafted into the CDRs of the VH or VL, wherein the antibody cytokine graft protein preferentially expands T effector cells over regulatory T cells. In some embodiments, the antibody cytokine graft protein comprises a heavy chain variable region (VH) comprising complementarity determining regions HCDR1, HCDR2, and HCDR3, a light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3, and an IL-2 molecule or a fragment thereof grafted into the CDRs of the VH or VL, wherein the IL-2 molecule is a mutein, and the antibody cytokine graft protein preferentially expands T effector cells over regulatory T cells. In some embodiments, the IL-2 regimen comprises administration of an antibody described in U.S. Patent Application Publication No. 2020 / 0270334 A1, the disclosure of which is incorporated herein by reference. In some embodiments, the antibody cytokine graft protein comprises a heavy chain variable region (VH) comprising complementarity determining regions HCDR1, HCDR2, and HCDR3, a light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3, and an IL-2 molecule or a fragment thereof grafted into the CDRs of the VH or VL, wherein the IL-2 molecule is a mutein, and the antibody cytokine graft protein preferentially expands T effector cells over regulatory T cells, and the antibody further comprises an IgG class heavy chain and an IgG class light chain selected from the group consisting of an IgG class light chain comprising SEQ ID NO: 39 and an IgG class heavy chain comprising SEQ ID NO: 38, an IgG class light chain comprising SEQ ID NO: 37 and an IgG class heavy chain comprising SEQ ID NO: 29, an IgG class light chain comprising SEQ ID NO: 39 and an IgG class heavy chain comprising SEQ ID NO: 29, an IgG class light chain comprising SEQ ID NO: 37 and an IgG class heavy chain comprising SEQ ID NO: 38.

[0032] In some embodiments, an IL-2 molecule or a fragment thereof is grafted into HCDR1 of VH and the IL-2 molecule is a mutein. In some embodiments, an IL-2 molecule or a fragment thereof is grafted into HCDR2 of VH and the IL-2 molecule is a mutein. In some embodiments, an IL-2 molecule or a fragment thereof is grafted into HCDR3 of VH and the IL-2 molecule is a mutein. In some embodiments, an IL-2 molecule or a fragment thereof is grafted into LCDR1 of VL and the IL-2 molecule is a mutein. In some embodiments, an IL-2 molecule or a fragment thereof is grafted into LCDR2 of VL and the IL-2 molecule is a mutein. In some embodiments, an IL-2 molecule or a fragment thereof is grafted into LCDR3 of VL and the IL-2 molecule is a mutein.

[0033] The insertion of the IL-2 molecule can be at or near the N-terminal region of the CDR, the middle region of the CDR, or at or near the C-terminal region of the CDR. In some embodiments, the antibody cytokine graft protein comprises an IL-2 molecule incorporated into the CDR, where the IL2 sequence does not frameshift the CDR sequence. In some embodiments, the antibody cytokine graft protein comprises an IL-2 molecule incorporated into the CDR, where the IL-2 sequence replaces all or part of the CDR sequence. The replacement with the IL-2 molecule can be at or near the N-terminal region of the CDR, the middle region of the CDR, or the C-terminal region of the CDR. The replacement with the IL-2 molecule can be as little as one or two amino acids of the CDR sequence, or the entire CDR sequence.

[0034] In some embodiments, the IL-2 molecule is directly grafted onto the CDR without a peptide linker and without additional amino acids between the CDR sequence and the IL-2 sequence. In some embodiments, the IL-2 molecule is indirectly grafted onto the CDR using a peptide linker with one or more additional amino acids between the CDR sequence and the IL-2 sequence.

[0035] In some embodiments, the IL-2 molecule described herein is an IL-2 mutein. In some cases, the IL-2 mutein comprises an R67A substitution. In some embodiments, the IL-2 mutein comprises the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 15. In some embodiments, the IL-2 mutein comprises the amino acid sequence of Table 1 of U.S. Patent Application Publication No. US2020 / 0270334 A1, the disclosure of which is incorporated herein by reference.

[0036] In some embodiments, the antibody cytokine transplant protein comprises an HCDR1 selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22, and SEQ ID NO: 25. In some embodiments, the antibody cytokine transplant protein comprises an HCDR1 selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 13, and SEQ ID NO: 16. In some embodiments, the antibody cytokine transplant protein comprises an HCDR2 selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 20, SEQ ID NO: 23, and SEQ ID NO: 26. In some embodiments, the antibody cytokine transplant protein comprises an HCDR3 selected from the group consisting of SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO: 24, and SEQ ID NO: 27. In some embodiments, the antibody cytokine transplant protein comprises a VH region comprising the amino acid sequence of SEQ ID NO: 28. In some embodiments, the antibody cytokine transplant protein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 29. In some embodiments, the antibody cytokine transplant protein comprises a VL region comprising the amino acid sequence of SEQ ID NO: 36. In some embodiments, the antibody cytokine transplant protein comprises a light chain comprising the amino acid sequence of SEQ ID NO: 37. In some embodiments, the antibody cytokine transplant protein comprises a VH region comprising the amino acid sequence of SEQ ID NO:28 and a VL region comprising the amino acid sequence of SEQ ID NO:36. In some embodiments, the antibody cytokine transplant protein comprises a heavy chain region comprising the amino acid sequence of SEQ ID NO:29 and a light chain region comprising the amino acid sequence of SEQ ID NO:37. In some embodiments, the antibody cytokine transplant protein comprises a heavy chain region comprising the amino acid sequence of SEQ ID NO:29 and a light chain region comprising the amino acid sequence of SEQ ID NO:39. In some embodiments, the antibody cytokine transplant protein comprises a heavy chain region comprising the amino acid sequence of SEQ ID NO:38 and a light chain region comprising the amino acid sequence of SEQ ID NO:37. In some embodiments, the antibody cytokine transplant protein comprises a heavy chain region comprising the amino acid sequence of SEQ ID NO:38 and a light chain region comprising the amino acid sequence of SEQ ID NO:39.In some embodiments, the antibody cytokine transplant protein comprises IgG.IL2F71A.H1 or IgG.IL2R67A.H1 of U.S. Patent Application Publication No. 2020 / 0270334 A1, or a variant, derivative, or fragment thereof, or a conservative amino acid substitution thereof, or a protein having at least 80%, at least 90%, at least 95%, or at least 98% sequence identity thereto. In some embodiments, the antibody component of the antibody cytokine transplant protein described herein comprises an immunoglobulin sequence, framework sequence, or CDR sequence of palivizumab. In some embodiments, the antibody cytokine transplant protein described herein has a longer serum half-life than a wild-type IL-2 molecule, such as, but not limited to, aldesleukin or an equivalent molecule. In some embodiments, the antibody cytokine transplant protein described herein has a sequence set forth in Table 3.

[0037] [Table 3-1] [Table 3-2]

[0038] The term "IL-4" (also referred to herein as "IL4") refers to the cytokine known as interleukin 4, which is produced by Th2 T cells, as well as eosinophils, basophils, and mast cells. IL-4 regulates the differentiation of naive helper T cells (Th0 cells) into Th2 T cells. Steinke and Borish, Respir. Res. 2001, 2, 66-70. Upon activation by IL-4, Th2 T cells subsequently produce additional IL-4 in a positive feedback loop. IL-4 also stimulates B cell proliferation and class II MHC expression, inducing class switching from B cells to IgE and IgG1 expression. Recombinant human IL-4 suitable for use in the present invention is commercially available from several suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (catalog number CYT-211) and ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-15 recombinant protein, catalog number Gibco CTP0043). The amino acid sequence of recombinant human IL-4 suitable for use in the present invention is shown in Table 2 (SEQ ID NO: 9).

[0039] The term "IL-7" (also referred to herein as "IL7") refers to a glycosylated tissue-derived cytokine known as interleukin-7, which can be obtained from stromal and epithelial cells, as well as dendritic cells. Fry and Mackall, Blood 2002, 99, 3892-904. IL-7 can stimulate T cell development. IL-7 binds to the IL-7 receptor, a heterodimer consisting of the IL-7 receptor alpha and the common gamma chain receptor, which is a series of signals important for T cell development in the thymus and survival in the periphery. Recombinant human IL-7 suitable for use in the present invention is commercially available from several suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (catalog number CYT-254) and ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-15 recombinant protein, catalog number Gibco PHC0071). The amino acid sequence of a recombinant human IL-7 suitable for use in the present invention is shown in Table 2 (SEQ ID NO: 10).

[0040] The term "IL-15" (also referred to herein as "IL15") refers to the T-cell growth factor known as interleukin-15 and includes all forms of IL-2, including human and mammalian forms, conservative amino acid substitutions, glycoforms, biosimilars, and variants. IL-15 is described, for example, in Fehniger and Caligiuri, Blood 2001, 97, 14-32, the disclosure of which is incorporated herein by reference. IL-15 shares β and γ signaling receptor subunits with IL-2. Recombinant human IL-15 is a single, non-glycosylated polypeptide chain containing 114 amino acids (and an N-terminal methionine) with a molecular weight of 12.8 kDa. Recombinant human IL-15 is commercially available from several suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (catalog number CYT-230-b) and ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-15 recombinant protein, catalog number 34-8159-82). The amino acid sequence of recombinant human IL-15 suitable for use in the present invention is shown in Table 2 (SEQ ID NO: 11).

[0041] The term "IL-21" (also referred to herein as "IL21") refers to the pleiotropic cytokine protein known as interleukin-21, and includes all forms of IL-21, including human and mammalian forms, conservative amino acid substitutions, glycoforms, biosimilars, and variants. IL-21 is described, for example, in Spolski and Leonard, Nat. Rev. Drug. Disc. 2014, 13, 379-95, the disclosure of which is incorporated herein by reference. IL-21 primarily stimulates natural killer T cells and activated human CD4 +It is produced by T cells. Recombinant human IL-21 is a single, non-glycosylated polypeptide chain containing 132 amino acids with a molecular weight of 15.4 kDa. Recombinant human IL-21 is commercially available from several suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (catalog number CYT-408-b) and ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-21 recombinant protein, catalog number 14-8219-80). The amino acid sequence of recombinant human IL-21 suitable for use in the present invention is shown in Table 2 (SEQ ID NO: 21).

[0042] When an "antitumor effective amount," "tumor inhibiting effective amount," or "therapeutic amount" is indicated, the exact amount of the composition of the present invention to be administered can be determined by a physician, taking into account individual differences in the age, weight, tumor size, extent of infection or metastasis, and health status of the patient (subject). Generally, the tumor-infiltrating lymphocytes (e.g., secondary TILs or genetically modified cytotoxic lymphocytes) described herein are administered at a dose of 10 per kg of body weight. 4 ~10 11 cells (e.g., 10 per kg body weight) 5 ~10 6 , 10 5 ~10 10 , 10 5 ~10 11 , 10 6 ~10 10 , 10 6 ~10 11 , 10 7 ~10 11 , 10 7 ~10 10 , 10 8 ~10 11 , 10 8 ~10 10 , 10 9 ~10 11 , or 10 9 ~10 10The TIL (optionally including genetically engineered TIL) compositions may be administered at doses of 1000-15000 cells (including all integer values ​​within those ranges). TIL (optionally including genetically modified cytotoxic lymphocytes) compositions may also be administered multiple times at these doses. TIL (optionally including genetically engineered TIL) can be administered by using injection techniques commonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 1988, 319, 1676). Optimal dosages and treatment regimes for a particular patient can be readily determined by one skilled in the medical arts by monitoring the patient for signs of disease and adjusting treatment accordingly.

[0043] The terms "hematological malignancies," "blood system malignancies," or terms of related meaning, refer to cancers and tumors of mammalian hematopoietic and lymphatic tissues, including, but not limited to, blood, bone marrow, lymph nodes, and lymphatic tissues. Hematological malignancies are also referred to as "liquid tumors." Hematological malignancies may include, but are not limited to, acute lymphoblastic leukemia (ALL), chronic lymphocytic lymphoma (CLL), small lymphocytic lymphoma (SLL), acute myeloid leukemia (AML), chronic myelogenous leukemia (CML), multiple myeloma, acute monocytic leukemia (AMoL), Hodgkin's lymphoma, and non-Hodgkin's lymphoma. The term "B-cell hematological malignancies" refers to hematological malignancies affecting B cells.

[0044] The term "liquid tumor" refers to an abnormal mass of cells that is fluid in nature. Liquid tumor cancers include, but are not limited to, leukemia, myeloma, and lymphoma, as well as other hematological malignancies. TILs obtained from liquid tumors may also be referred to herein as bone marrow-infiltrating lymphocytes (MILs). TILs obtained from liquid tumors, including those circulating in peripheral blood, may also be referred to herein as PBLs. The terms MILs, TILs, and PBLs are used interchangeably herein and differ only based on the tissue type from which the cells are derived.

[0045] The term "microenvironment" as used herein may refer to the solid or hematological tumor microenvironment as a whole, or to individual subsets of cells within the microenvironment. As used herein, the tumor microenvironment refers to a complex mixture of "cells, soluble factors, signaling molecules, extracellular matrix, and mechanical cues that promote neoplastic transformation, support tumor growth and invasion, protect tumors from host immunity, foster therapeutic resistance, and provide a niche for successful and dominant metastasis," as described in Swartz, et al., Cancer Res., 2012, 72, 2473. Tumors express antigens that are recognized by T cells, but tumor clearance by the immune system is rare due to immunosuppression by the microenvironment.

[0046] In some embodiments, the invention includes methods of treating cancer with a TIL population, wherein the patient is pretreated with non-myeloablative chemotherapy prior to infusion of TILs according to the invention. In some embodiments, a TIL population may be provided, wherein the patient is pretreated with non-myeloablative chemotherapy prior to infusion of TILs according to the 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 (27-23 days before TIL infusion). In some embodiments, after non-myeloablative chemotherapy and TIL infusion according to the invention (day 0), the patient receives an intravenous infusion of IL-2 at 720,000 IU / kg every 8 hours to physiological tolerance.

[0047] Experimental findings indicate that lymphodepletion prior to adoptive transfer of tumor-specific T lymphocytes plays an important role in enhancing therapeutic efficacy by eliminating regulatory T cells and competing elements of the immune system ("cytokine sinks"). Accordingly, some embodiments of the present invention utilize a lymphodepletion step (sometimes referred to as "immunosuppressive conditioning") on patients prior to introducing the TILs of the present invention.

[0048] The term "effective amount" or "therapeutically effective amount" refers to an amount of a compound or combination of compounds described herein sufficient to achieve the intended use, including, but not limited to, disease treatment. A therapeutically effective amount may vary depending on the intended use (in vitro or in vivo), the subject and condition being treated (e.g., the subject's weight, age, and sex), the severity of the condition, or the method of administration. The term also applies to a dose that induces a specific response in target cells (e.g., reduced platelet adhesion and / or cell migration). The specific dose will vary depending on the particular compound selected, the dosing regimen to be followed, whether the compound is administered in combination with other compounds, the timing of administration, the tissue to which it is administered, and the physical delivery system by which the compound is delivered.

[0049] The terms "treatment," "treating," "treating," and the like refer to obtaining a desired pharmacological and / or physiological effect. The effect can be preventative, in terms of completely or partially preventing the disease or condition, and / or therapeutic, in terms of partially or completely curing the disease and / or side effects caused by the disease. "Treatment," as used herein, encompasses any treatment of disease in a mammal, particularly a human, and includes (a) preventing the disease from occurring in a subject who may be susceptible to the disease but has not yet been diagnosed with it; (b) suppressing the disease, i.e., arresting its onset or progression; and (c) palliating the disease, i.e., causing regression of the disease and / or alleviating one or more disease symptoms. "Treatment" is also intended to encompass the delivery of an agent to provide a pharmacological effect even in the absence of a disease or condition. For example, "treatment" encompasses the delivery of a composition capable of eliciting an immune response or conferring immunity in the absence of a pathology, e.g., in the case of a vaccine.

[0050] The term "heterologous," when used with reference to portions of a nucleic acid or protein, indicates that the nucleic acid or protein comprises two or more subsequences that are not found in the same relationship to each other in nature. For example, nucleic acids are typically produced recombinantly, with two or more sequences from unrelated genes arranged to create a new functional nucleic acid, e.g., a promoter from one source and a coding region from another source, or coding regions from different sources. Similarly, a heterologous protein indicates that the protein comprises two or more subsequences that are not found in the same relationship to each other in nature (e.g., a fusion protein).

[0051] The terms "sequence identity," "percent identity," and "percent sequence identity" (or their synonyms, e.g., "99% identical") in the context of two or more nucleic acids or polypeptides refer to two or more sequences or subsequences that are the same or have a specified percentage of the same nucleotide or amino acid residues when compared and aligned for maximum correspondence (introducing gaps, if necessary), without considering any conservative amino acid substitutions as part of the sequence identity. Percent identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software that can be used to obtain alignment of amino acid or nucleotide sequences are known in the art. Suitable programs for determining percent sequence identity include, for example, the BLAST suite of programs available from the BLAST website of the U.S. government's National Center for Biotechnology Information. Comparison between two sequences can be performed using either the BLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acid sequences, and BLASTP is used to compare amino acid sequences. ALIGN, ALIGN-2 (Genentech, South San Francisco, California), or MegAlign, available from DNASTAR, are additional publicly available software programs that can be used to align sequences. Those skilled in the art can determine the appropriate parameters for maximum alignment depending on the particular alignment software. In certain embodiments, the default parameters of the alignment software are used.

[0052] As used herein, the term "variant" includes, but is not limited to, an antibody or fusion protein comprising an amino acid sequence that differs from the amino acid sequence of a reference antibody by one or more substitutions, deletions, and / or additions at specific positions within or adjacent to the amino acid sequence of the reference antibody. A variant may contain one or more conservative substitutions in its amino acid sequence compared to the amino acid sequence of the reference antibody. Conservative substitutions may include, for example, substitutions of similarly charged or uncharged amino acids. A variant retains the ability of the reference antibody to specifically bind to an antigen. The term variant also includes pegylated antibodies or proteins.

[0053] As used herein, "tumor infiltrating lymphocytes" or "TILs" refers to a population of cells that are initially acquired as leukocytes that have left a subject's bloodstream and migrated to a tumor. TILs include CD8 + Cytotoxic T cells (lymphocytes), Th1 and Th17 CD4 + These include, but are not limited to, T cells, natural killer cells, dendritic cells, and M1 macrophages. TILs include both primary and secondary TILs. "Primary TILs" are those obtained from a patient tissue sample as outlined herein (sometimes referred to as "freshly harvested"). "Secondary TILs" are any TIL cell populations that have been expanded or propagated as discussed herein, including, but not limited to, bulk TILs, expanded TILs ("REP TILs"), and "reREP TILs" as discussed herein. reREP TILs can include, for example, second expanded TILs or second additional expanded TILs (e.g., those described in step D of FIG. 8, including TILs designated as reREP TILs).

[0054] TILs can generally be defined either biochemically using cell surface markers or functionally by their ability to infiltrate tumors and achieve therapy. TILs can generally be classified by expression of one or more of the following biomarkers: CD4, CD8, TCRαβ, CD27, CD28, CD56, CCR7, CD45Ra, CD95, PD-1, and CD25. Additionally and alternatively, TILs can be functionally defined by their ability to infiltrate solid tumors upon reintroduction into patients. TILs can be further characterized by potency; for example, TILs can be considered potent if their interferon (IFN) release is greater than about 50 pg / mL, greater than about 100 pg / mL, greater than about 150 pg / mL, or greater than about 200 pg / mL. For example, TILs can be considered potent if interferon (IFNγ) release is greater than about 50 pg / mL, greater than about 100 pg / mL, greater than about 150 pg / mL, or greater than about 200 pg / mL, greater than about 300 pg / mL, greater than about 400 pg / mL, greater than about 500 pg / mL, greater than about 600 pg / mL, greater than about 700 pg / mL, greater than about 800 pg / mL, greater than about 900 pg / mL, or greater than about 1000 pg / mL.

[0055] The term "deoxyribonucleotide" encompasses natural and synthetic, unmodified and modified deoxyribonucleotides. Modifications include changes to the sugar moiety, the base moiety, and / or the linkages between deoxyribonucleotides in an oligonucleotide.

[0056] The term "RNA" defines a molecule containing at least one ribonucleotide residue. The term "ribonucleotide" defines a nucleotide having a hydroxyl group at the 2' position of a bD-ribofuranose moiety. The term RNA includes double-stranded RNA, single-stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, and altered RNA that differs from naturally occurring RNA by the addition, deletion, substitution, and / or alteration of one or more nucleotides. The nucleotides of the RNA molecules described herein can also include non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs may be referred to as analogs or analogs of naturally occurring RNA.

[0057] The term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and inactive ingredients. The use of such pharmaceutically acceptable carriers or pharmaceutically acceptable excipients for active pharmaceutical ingredients is well known in the art. Except insofar as any conventional pharmaceutically acceptable carrier or pharmaceutically acceptable excipient is incompatible with the active pharmaceutical ingredient, its use in the therapeutic compositions of the invention is contemplated. Additional active pharmaceutical ingredients, such as other drugs, can also be incorporated into the compositions and methods described.

[0058] The terms "about" and "approximately" mean within a statistically significant range of values. Such a range may be within one order of magnitude of a given value or range, preferably within 50%, more preferably within 20%, even more preferably within 10%, and even more preferably within 5%. The acceptable deviation encompassed by the term "about" or "approximately" depends on the particular system under study and can be readily understood by one of ordinary skill in the art. Furthermore, as used herein, the terms "about" and "approximately" mean that dimensions, sizes, formulations, parameters, shapes, and other quantities and characteristics are not, and need not be, exact, but may be approximate and / or larger or smaller, as appropriate, reflecting tolerances, conversion factors, rounding, measurement errors, etc., and other factors known to those of ordinary skill in the art. In general, a dimension, size, formulation, parameter, shape, or other quantity or characteristic is "about" or "approximately" whether or not expressly stated as such. It should be noted that embodiments of widely different sizes, shapes, and dimensions may employ the described configurations.

[0059] When used in the appended claims, the transitional terms "comprising," "consisting essentially of," and "consisting of" define the claim in its original and amended form, in terms of whether additional, unrecited claim elements or steps, if any, are excluded from the scope of the claim(s). The term "comprising" is intended to be inclusive or open-ended and does not exclude any additional, unrecited elements, methods, steps, or materials. The term "consisting of" excludes any element, step, or material other than those specified in the claim, and in the latter case, also excludes impurities normally associated with the specified material(s). The term "consisting essentially of" limits the claim to the specified element, step, or material(s) and does not materially affect the basic and novel feature(s) of the claimed invention. All compositions, methods, and kits described herein embodying the present invention may, in alternative embodiments, be more specifically defined by any of the transitional terms "comprising," "consisting essentially of," and "consisting of."

[0060] The terms "antibody" and its plural "antibodies" refer to whole immunoglobulins and any antigen-binding fragment ("antigen-binding portion") or single chains thereof. "Antibody" also refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or antigen-binding portions thereof. Each heavy chain contains a heavy chain variable region (referred to herein as V H Each light chain is composed of a light chain variable region (abbreviated herein as V) and a heavy chain constant region. The heavy chain constant region is composed of three domains: CH1, CH2, and CH3. L The light chain constant region consists of one domain, C L The V of the antibody H and V L The regions can be further subdivided into regions of hypervariability, called complementarity-determining regions (CDRs) or hypervariable regions (HVRs), which may be interspersed with more conserved regions called framework regions (FRs). H and VL is composed of three CDRs and four FRs arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with one or more antigen epitopes. The constant region of the antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0061] The term "antigen" refers to a substance that induces an immune response. In some embodiments, an antigen is a molecule that can be bound by an antibody or TCR when presented by a major histocompatibility complex (MHC) molecule. As used herein, the term "antigen" also encompasses T cell epitopes. An antigen can additionally be recognized by the immune system. In some embodiments, an antigen can induce a humoral or cellular immune response, leading to the activation of B and / or T lymphocytes. In some cases, this may require that the antigen contain or be bound 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 a corresponding antibody or TCR, typically in a highly specific and selective manner, and not with many other antibodies or TCRs that may be induced by other antigens.

[0062] The terms "monoclonal antibody," "mAb," "monoclonal antibody composition," or their plurals, refer to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition exhibits a single binding specificity and affinity for a particular epitope. Monoclonal antibodies specific for a particular receptor can be produced using knowledge and techniques in the art by injecting a test subject with an appropriate antigen and then isolating hybridomas expressing antibodies with the desired sequence or functional characteristics. DNA encoding the monoclonal antibody is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to genes encoding the heavy and light chains of the monoclonal antibody). Hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA can be placed into an expression vector and then transfected into host cells such as E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not produce immunoglobulin protein to obtain the synthesis of the monoclonal antibody in the recombinant host cells. Recombinant production of antibodies is described in more detail below.

[0063] As used herein, the term "antigen-binding portion" or "antigen-binding fragment" of an antibody (or simply "antibody portion" or "fragment") refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed by the term "antigen-binding portion" of an antibody include: (i) V L , V H , C L (ii) a Fab fragment, which is a monovalent fragment consisting of two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a V H and an Fd fragment consisting of the CH1 domain, (iv) a V of a single arm of an antibody L and V H Fv fragment consisting of domains, (v) V H or VL and (v) isolated complementarity-determining regions (CDRs). L and V H are encoded by separate genes, which can be synthesized using recombinant methods. L and V H The regions may be joined by a synthetic linker that allows them to pair as a single protein chain to form a monovalent molecule known as a single-chain Fv (scFv) (see, e.g., Bird, et al., Science 1988, 242, 423-426, and Huston, et al., Proc. Natl. Acad. Sci. USA 1988, 85, 5879-5883). Such scFv antibodies are also intended to be encompassed by the term "antigen-binding portion" or "antigen-binding fragment" of an antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as intact antibodies. In some embodiments, the scFv protein domain comprises a V H Part and V L The scFv molecule contains a V L If the domain is the N-terminal portion of the scFv molecule, V L -LV H , or V H If the domain is the N-terminal portion of the scFv molecule, V H -LV L Methods for producing scFv molecules and designing suitable peptide linkers are described in U.S. Pat. Nos. 4,704,692, 4,946,778, R. Raag and M. Whitlow, "Single Chain Fvs," FASEB Vol. 9:73-80 (1995), and RE Bird and BW Walker, Single Chain Antibody Variable Regions, TIBTECH, Vol. 9:132-137 (1991), the disclosures of which are incorporated herein by reference.

[0064] As used herein, the term "human antibody" is intended to include antibodies having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region also is derived from human germline immunoglobulin sequences. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). As used herein, the term "human antibody" is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0065] The term "human monoclonal antibody" refers to antibodies displaying a single binding specificity which have variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. In some embodiments, human monoclonal antibodies are produced by hybridomas comprising B cells obtained from a transgenic non-human animal, e.g., a transgenic mouse, whose genome comprises human heavy chain and light chain transgenes fused to an immortalized cell.

[0066] As used herein, the term "recombinant human antibody" includes all human antibodies prepared, expressed, created, or isolated by recombinant means, such as (a) antibodies isolated from animals (such as mice) that are transgenic or transchromosomal for human immunoglobulin genes or hybridomas prepared therefrom (described further below); (b) antibodies isolated from host cells transformed to express human antibodies, e.g., from transfectomas; (c) antibodies isolated from recombinant combinatorial human antibody libraries; and (d) antibodies prepared, expressed, created, or isolated by any other means, including splicing human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable regions in which the framework and CDR regions are derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies may be subjected to in vitro mutagenesis (or, when animals transgenic for human Ig sequences are used, in vivo somatic mutagenesis) to thereby modify the V sequences of the recombinant antibodies. H and V L The amino acid sequence of the region is human germline V H and V L These are sequences that are derived from and related to sequences, but may not naturally occur within the human antibody germline repertoire in vivo.

[0067] As used herein, "isotype" refers to the antibody class (e.g., IgM or IgG1) that is encoded by heavy chain constant region genes.

[0068] The phrases "an antibody that recognizes an antigen" and "an antibody that is specific for an antigen" are used interchangeably herein with the term "an antibody that specifically binds to an antigen."

[0069] The term "human antibody derivative" refers to any modified form of a human antibody, including a conjugate of the antibody with another active pharmaceutical ingredient or antibody. The terms "conjugate," "antibody drug conjugate," "ADC," or "immunoconjugate" refer to an antibody or fragment thereof conjugated to another therapeutic moiety, which can be conjugated to the antibodies described herein using methods available in the art.

[0070] The terms "humanized antibody," "humanized antibodies," and "humanization" are intended to refer to antibodies in which CDR sequences derived from the germline of another mammalian species, such as mouse, have been grafted onto human framework sequences. Additional framework region modifications can be made within the human framework sequences. Humanized forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. In most cases, humanized antibodies are human immunoglobulins (recipient antibodies) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate having the desired specificity, affinity, and capacity. In some instances, Fv framework (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may contain residues that are not found in the recipient or donor antibody. These modifications are made to further refine antibody performance. Generally, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence. The humanized antibody will also optionally comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones, et al., Nature 1986, 321, 522-525; Riechmann, et al., Nature 1988, 332, 323-329; and Presta, Curr. Op. Struct. Biol. 1992, 2, 593-596. The antibodies described herein can also be modified to use any Fc variant known to confer improved (e.g., reduced) effector function and / or FcR binding.Fc variants are described in, for example, International Patent Application Publication Nos. WO1988 / 07089A1, WO1996 / 14339A1, WO1998 / 05787A1, WO1998 / 23289A1, WO1999 / 51642A1, WO99 / 58572A1, WO2000 / 09560A2, WO2000 / 32767A1, WO2000 / 42072A2, and WO2002 / 4 4215A2, WO2002 / 060919A2, WO2003 / 074569A2, WO2004 / 016750A2, WO2004 / 029207A2, WO2004 / 03 5752A2, WO2004 / 063351A2, WO2004 / 074455A2, WO2004 / 099249A2, WO2005 / 040217A2, WO2005 / 07 0963A1, WO2005 / 077981A2, WO2005 / 092925A2, WO2005 / 123780A2, WO2006 / 019447A1, WO2006 / 047350A2, and WO2006 / 085967A2, as well as U.S. Pat. Nos. 5,648,260, 5,739,277, 5,834,250, 5,869,046, 6,096 ,871, 6,121,022, 6,194,551, 6,242,195, 6,277,375, 6,528,624, 6,538,124, 6,737,056, 6,821,505, 6,998,253, and 7,083,784 (the disclosures of which are incorporated herein by reference).

[0071] The term "chimeric antibody" is intended to refer to an antibody in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, e.g., an antibody in which the variable region sequences are derived from a murine antibody and the constant region sequences are derived from a human antibody.

[0072] A "diabody" is a small antibody fragment that has two antigen-binding sites. The fragments bind to the same polypeptide chain (VH -V L or V L -V H ) in the light chain variable domain (V L ) connected to the heavy chain variable domain (V H ). When a linker that is too short to pair the two domains on the same chain is used, the domains are forced to pair with complementary domains on another chain and create two antigen-binding sites. Bispecific antibodies are more fully described, for example, in European Patent No. EP 404,097, International Patent Publication No. WO 93 / 11161, and Bolliger, et al., Proc. Natl. Acad. Sci. USA 1993, 90, 6444-6448.

[0073] The term "glycosylation" refers to modified derivatives of antibodies. An aglycosylated antibody lacks glycosylation. Glycosylation can be altered to, for example, increase the affinity of an antibody for an antigen. Such carbohydrate modifications can be achieved, for example, by altering one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions can be made that result in the elimination of one or more variable region framework glycosylation sites, thereby eliminating glycosylation at those sites. As described in U.S. Pat. Nos. 5,714,350 and 6,350,861, aglycosylation can increase the affinity of an antibody for an antigen. Additionally or alternatively, antibodies can be generated with altered types of glycosylation, such as hypofucosylated antibodies with reduced amounts of fucosyl residues or antibodies with increased bisecting GlcNac structures. Such altered glycosylation patterns have been demonstrated to increase antibody potency. Such carbohydrate modifications can be achieved, for example, by expressing the antibody in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells to express the recombinant antibodies of the present invention, thereby producing antibodies with altered glycosylation. For example, the cell lines Ms704, Ms705, and Ms709 lack the fucosyltransferase gene FUT8 (alpha(1,6) fucosyltransferase), such that antibodies expressed in these cell lines lack fucose on their carbohydrates. The Ms704, Ms705, and Ms709 FUT8- / - cell lines were generated by targeted disruption of the FUT8 gene in CHO / DG44 cells using two replacement vectors (see, e.g., U.S. Patent Publication No. 2004 / 0110704 or Yamane-Ohnuki, et al., Biotechnol. Bioeng., 2004, 87, 614-622).As another example, European Patent No. EP 1,176,195 describes cell lines with a functionally disrupted FUT8 gene encoding a fucosyltransferase, thereby resulting in antibodies expressed in such cell lines exhibiting hypofucosylation by reducing or eliminating alpha-1,6 bond-related enzymes. It also describes cell lines with reduced or no enzymatic activity for adding fucose to N-acetylglucosamine linked to the Fc region of antibodies, such as the rat myeloma cell line YB2 / 0 (ATCC CRL 1662). International Patent Publication No. WO 03 / 035835 describes a variant CHO cell line, Lec13 cells, that has a reduced ability to attach fucose to Asn(297)-linked carbohydrates, which also results in hypofucosylation of antibodies expressed in the host cells (see also Shields, et al., J. Biol. Chem. 2002, 277, 26733-26740). International Patent Publication No. WO 99 / 54342 describes cell lines engineered to express glycoprotein-modifying glycosyltransferases (e.g., beta(1,4)-N-acetylglucosaminyltransferase III (GnTIII)), such that antibodies expressed in the engineered cell lines exhibit increased bisecting GlcNac structures, resulting in increased ADCC activity of the antibodies (see also Umana, et al., Nat. Biotech. 1999, 17, 176-180). Alternatively, fucosidase enzymes can be used to cleave fucose residues from antibodies. For example, the fucosidase alpha-L-fucosidase removes fucosyl residues from antibodies, as described in Tarentino, et al., Biochem. 1975, 14, 5516-5523.

[0074] "PEGylation" refers to a modified antibody or fragment thereof that has been reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions that result in one or more PEG groups being attached to the antibody or antibody fragment. PEGylation can, for example, increase the biological (e.g., serum) half-life of the antibody. Preferably, PEGylation is carried out via an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer). As used herein, the term "polyethylene glycol" refers to a mono (C1-C 10 PEG is intended to encompass any of the forms of PEG that have been used to derivatize other proteins, such as alkoxy- or aryloxy-polyethylene glycol, or polyethylene glycol-maleimide. The antibody to be pegylated may be an aglycosylated antibody. Methods for pegylation are known in the art and can be applied to the antibodies of the invention, for example, as described in European Patent Nos. EP 0 154 316 and EP 0 401 384, and U.S. Pat. No. 5,824,778 (the disclosures of each of which are incorporated herein by reference).

[0075] The term "biosimilar" refers to a biological product that is highly similar to a reference biological product approved in the United States, despite minor differences in clinically inactive components, including monoclonal antibodies or proteins, and that has no clinically meaningful differences between the biological product and the reference product in terms of product safety, purity, and potency. Furthermore, a similar biological or "biosimilar" drug is a biological product similar to another biological product already approved for use by the European Medicines Agency. The term "biosimilar" is also used interchangeably by regulatory agencies in other countries and regions. Biological products or biological products are medicines made by or derived from biological sources, such as bacteria or yeast. They can consist of relatively small molecules, such as human insulin or erythropoietin, or complex molecules, such as monoclonal antibodies. For example, if the reference IL-2 protein is aldesleukin (proleukin), a protein approved by a drug regulatory agency for aldesleukin is a "biosimilar" of aldesleukin or a "biosimilar of" aldesleukin. In Europe, a similar biological or "biosimilar" medicinal product is a biological product similar to another biological product already authorized for use by the European Medicines Agency (EMA). The legal basis for similar biological uses in Europe is Article 6 of Regulation (EC) No. 726 / 2004, as amended, and Article 10(4) of Directive 2001 / 83 / EC. Therefore, in Europe, biosimilars may be authorized or approved for authorisation or licensing under Article 6 of Regulation (EC) No. 726 / 2004 and Article 10(4) of Directive 2001 / 83 / EC. The original biological product already authorized is sometimes referred to as the "reference medicinal product" in Europe. Some of the requirements for a product to be considered a biosimilar are outlined in the CHMP guideline on biosimilar medicinal products. Additionally, product-specific guidelines, including those related to monoclonal antibody biosimilars, are provided by the EMA on a product-by-product basis and are available on its website.Biosimilars described herein may be similar to the reference medicinal product in terms of quality characteristics, biological activity, mechanism of action, safety profile, and / or efficacy. Furthermore, biosimilars may be used or intended for use to treat the same condition as the reference medicinal product. Thus, biosimilars described herein may be considered to have similar or very similar quality characteristics to the reference medicinal product. Alternatively, or in addition, biosimilars described herein may be considered to have similar or very similar biological activity to the reference medicinal product. Alternatively, or in addition, biosimilars described herein may be considered to have a similar or very similar safety profile to the reference medicinal product. Alternatively, or in addition, biosimilars described herein may be considered to have similar or very similar efficacy to the reference medicinal product. As described herein, biosimilars in Europe are compared to reference medicinal products authorized by the EMA. However, in some cases, biosimilars may be compared in specific studies to biopharmaceuticals authorized outside the European Economic Area (non-EEA authorized "comparators"). Such studies include, for example, specific clinical studies and in vivo non-clinical studies. As used herein, the term "biosimilar" also refers to a biopharmaceutical that has been or can be compared to a non-EEA-approved comparator. Particular biosimilars are proteins, such as antibodies, antibody fragments (e.g., antigen-binding portions), and fusion proteins. Protein biosimilars may have amino acid sequences with minor modifications to the amino acid structure (e.g., including amino acid deletions, additions, and / or substitutions) that do not significantly affect the function of the polypeptide. A biosimilar may have an amino acid sequence that has 97% or more sequence identity, e.g., 97%, 98%, 99%, or 100%, to the amino acid sequence of its reference pharmaceutical. A biosimilar may include one or more post-translational modifications, such as, but not limited to, glycosylation, oxidation, deamidation, and / or cleavage, that differ from the post-translational modifications of the reference pharmaceutical, provided that the differences do not result in a change in the safety and / or efficacy of the pharmaceutical. A biosimilar may have the same or a different glycosylation pattern as the reference pharmaceutical, provided that the differences do not result in a change in the safety and / or efficacy of the pharmaceutical.In particular, but not exclusively, biosimilars may have different glycosylation patterns if the differences address or are intended to address safety concerns associated with the reference drug. Additionally, biosimilars may deviate from the reference drug, for example, in its strength, dosage form, formulation, excipients, and / or presentation, provided that the drug's safety and efficacy are not compromised. Biosimilars may contain differences, for example, in their pharmacokinetic (PK) and / or pharmacodynamic (PD) profiles compared to the reference drug, but are still considered sufficiently similar to the reference drug to be approved or deemed suitable for approval. In certain circumstances, biosimilars exhibit different binding characteristics compared to the reference drug, and these different binding characteristics are not considered by regulatory authorities, such as the EMA, to be a barrier to approval as a similar biological product. The term "biosimilar" is also used interchangeably by regulatory agencies in other countries and regions.

[0076] III. Gen2 TIL Manufacturing Process An exemplary family of TIL processes known as Gen2 (also known as Process 2A) that includes some of these features is shown in Figures 1 and 2. An embodiment of Gen2 is shown in Figure 2.

[0077] As discussed herein, the present invention may include steps related to restimulating cryopreserved TILs prior to transplantation into a patient to improve their metabolic activity, and thus their relative health, and methods for testing said metabolic health. As generally outlined herein, TILs are generally harvested from a patient sample and manipulated to expand their numbers prior to transplantation into a patient. In some embodiments, TILs may optionally be genetically engineered, as discussed below.

[0078] In some embodiments, TILs can be cryopreserved, and upon thawing, they can be restimulated to enhance their metabolism before infusion into patients.

[0079] In some embodiments, as discussed in detail below and in the Examples and Figures, the first expansion (including the process referred to as pre-REP and the process shown as step A in FIG. 1 ) is shortened to 3-14 days, and the second expansion (including the process referred to as REP and the process shown as step B in FIG. 1 ) is shortened to 7-14 days. In some embodiments, the first expansion (e.g., the expansion described as step B in FIG. 1 ) is shortened to 11 days, and the second expansion (e.g., the expansion described as step D in FIG. 1 ) is shortened to 11 days. In some embodiments, as discussed in detail below and in the Examples and Figures, the combined first and second expansion (e.g., the expansion described as steps B and D in FIG. 1 ) is shortened to 22 days.

[0080] The "step" designations A, B, C, etc. below refer to Figure 1 and to certain specific embodiments described herein. The order of steps below and in Figure 1 is exemplary, and any combination or order of steps, as well as additional steps, repeated steps, and / or omission of steps, are contemplated by the present application and methods disclosed herein.

[0081] A. Step A: Obtaining a patient tumor sample Generally, TILs are initially obtained from a patient tumor sample, then expanded into larger populations for further manipulation as described herein, optionally cryopreserved, restimulated as outlined herein, and optionally assessed for phenotypic and metabolic parameters as indicators of TIL health.

[0082] Patient tumor samples can be obtained using methods known in the art, generally via surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining a sample containing a mixture of tumor cells and TIL cells. In some embodiments, multi-lesion sampling is used. In some embodiments, surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining a sample containing a mixture of tumor cells and TIL cells includes multi-lesion sampling (i.e., obtaining samples from one or more tumor sites and / or locations in a patient, as well as one or more tumors at the same or adjacent locations). Generally, tumor samples can be derived from any solid tumor, including primary tumors, invasive tumors, or metastatic tumors. Tumor samples can be liquid tumors, such as tumors obtained from hematological malignancies. Solid tumors can be of lung tissue. In some embodiments, useful TILs are obtained from non-small cell lung cancer (NSCLC). Solid tumors can be of skin tissue. In some embodiments, useful TILs are obtained from melanoma.

[0083] Once obtained, tumor samples are generally fragmented into pieces measuring 1 to approximately 8 mm using sharp dissection, with pieces measuring approximately 2 to 3 mm being particularly useful. In some embodiments, TILs are cultured from these fragments using an enzymatic tumor digest. Such tumor digests can be produced by incubation in an enzymatic medium (e.g., Roswell Park Memorial Institute (RPMI) 1640 buffer, 2 mM glutamate, 10 mcg / mL gentamicin, 30 units / mL DNase, and 1.0 mg / mL collagenase) followed by mechanical dissociation (e.g., using a tissue dissociator). Tumor digests can be produced by placing the tumor in the enzymatic medium, mechanically dissociating the tumor for approximately 1 minute, followed by incubation at 37°C in 5% CO for 30 minutes, and then repeating cycles of mechanical dissociation and incubation under the aforementioned conditions until only small tissue fragments are present. At the end of this process, if the cell suspension contains a large number of red blood cells or dead cells, density gradient separation using FICOLL branched hydrophilic polysaccharides can be performed to remove these cells. Alternative methods known in the art can be used, such as those described in U.S. Patent Application Publication No. 2012 / 0244133A1 (the disclosure of which is incorporated herein by reference). Any of the foregoing methods can be used in any of the embodiments described herein for methods of expanding TILs or methods of treating cancer.

[0084] The tumor dissociation enzyme mixture can include one or more dissociation (digestion) enzymes, such as, but not limited to, collagenase (including any blend or type of collagenase), Accutase™, Accumax™, hyaluronidase, neutral protease (dispase), chymotrypsin, chymopapain, trypsin, caseinase, elastase, papain, type XIV protease (pronase), deoxyribonuclease I (DNase), trypsin inhibitor, any other dissociation enzyme or proteolytic enzyme, and any combination thereof.

[0085] In some embodiments, the dissociation enzyme is reconstituted from a lyophilized enzyme, hi some embodiments, the lyophilized enzyme is reconstituted with an amount of a sterile buffer, such as HBSS.

[0086] In some cases, collagenase (such as animal-free type 1 collagenase) is reconstituted in 10 mL of sterile HBSS or another buffer. Lyophilized stock enzyme may be at a concentration of 2892 PZ U / vial. In some embodiments, collagenase is reconstituted in 5 mL to 15 mL of buffer. In some embodiments, the collagenase stock after reconstitution is in the range of about 100 U / mL to about 400 U / mL, e.g., about 100 U / mL to about 400 U / mL, about 100 U / mL to about 350 U / mL, about 100 U / mL to about 300 U / mL, about 150 U / mL to about 400 U / mL, about 100 U / mL, about 150 U / mL, about 200 U / mL, about 210 U / mL, about 220 U / mL, about 230 U / mL, about 240 U / mL, about 250 U / mL, about 260 U / mL, about 270 U / mL, about 280 U / mL, about 289.2 U / mL, about 300 U / mL, U / mL, about 350 PZ U / mL, or about 400 PZ U / mL.

[0087] In some embodiments, the neutral protease is reconstituted in 1 mL of sterile HBSS or another buffer. The lyophilized stock enzyme may be at a concentration of 175 DMC U / vial. In some embodiments, the neutral protease stock after reconstitution may range from about 100 DMC / mL to about 400 DMC / mL, e.g., from about 100 DMC / mL to about 400 DMC / mL, from about 100 DMC / mL to about 350 DMC / mL, from about 100 DMC / mL to about 300 DMC / mL, from about 150 DMC / mL to about 400 DMC / mL, from about 100 DMC / mL, or about 110 DMC / mL. C / mL, about 120 DMC / mL, about 130 DMC / mL, about 140 DMC / mL, about 150 DMC / mL, about 160 DMC / mL, about 170 DMC / mL, about 175 DMC / mL, about 180 DMC / mL, about 190 DMC / mL, about 200 DMC / mL, about 250 DMC / mL, about 300 DMC / mL, about 350 DMC / mL, or about 400 DMC / mL.

[0088] In some embodiments, DNAse I is reconstituted in 1 mL of sterile HBSS or another buffer. The lyophilized stock enzyme was at a concentration of 4 KU / vial. In some embodiments, the reconstituted DNase I stock ranges from about 1 KU / mL to 10 KU / mL, e.g., about 1 KU / mL, about 2 KU / mL, about 3 KU / mL, about 4 KU / mL, about 5 KU / mL, about 6 KU / mL, about 7 KU / mL, about 8 KU / mL, about 9 KU / mL, or about 10 KU / mL.

[0089] In some embodiments, enzyme stocks are variable and concentrations may need to be determined. In some embodiments, the concentration of the lyophilized stock can be verified. In some embodiments, the final amount of enzyme added to the digestion cocktail is adjusted based on the determined stock concentration.

[0090] In some embodiments, the enzyme mixture comprises about 10.2 ul of neutral protease (0.36 DMC U / mL), 21.3 μL of collagenase (1.2 PZ / mL), and 250 ul of DNAse I (200 U / mL) in about 4.7 mL of sterile HBSS.

[0091] As noted above, in some embodiments, the TILs are derived from solid tumors. In some embodiments, the solid tumor is not fragmented. In some embodiments, the solid tumor is not fragmented and is subjected to enzymatic digestion as a whole tumor. In some embodiments, the tumor is digested in an enzyme mixture comprising collagenase, DNase, and hyaluronidase. In some embodiments, the tumor is digested in an enzyme mixture comprising collagenase, DNase, and hyaluronidase for 1-2 hours. In some embodiments, the tumor is digested in an enzyme mixture comprising collagenase, DNase, and hyaluronidase for 1-2 hours at 37°C and 5% CO2. In some embodiments, the tumor is digested in an enzyme mixture comprising collagenase, DNase, and hyaluronidase for 1-2 hours at 37°C and 5% CO2 with rotation. In some embodiments, the tumor is digested overnight with constant rotation. In some embodiments, the tumor is digested overnight at 37°C and 5% CO2 with constant rotation. In some embodiments, the whole tumor is combined with the enzymes to form a tumor digestion reaction mixture.

[0092] In some embodiments, the tumor is reconstituted with lyophilized enzyme in a sterile buffer. In some embodiments, the buffer is sterile HBSS.

[0093] In some embodiments, the enzyme mixture includes collagenase. In some embodiments, the collagenase is collagenase IV. In some embodiments, the working stock of collagenase is a 10x working stock of 100 mg / mL.

[0094] In some embodiments, the enzyme mixture comprises DNAse, hi some embodiments, the working stock of DNAse is a 10x working stock of 10,000 IU / mL.

[0095] In some embodiments, the enzyme mixture comprises hyaluronidase. In some embodiments, the working stock of hyaluronidase is a 10x working stock of 10 mg / mL.

[0096] In some embodiments, the enzyme mixture comprises 10 mg / mL collagenase, 1000 IU / mL DNAse, and 1 mg / mL hyaluronidase.

[0097] In some embodiments, the enzyme mixture comprises 10 mg / mL collagenase, 500 IU / mL DNAse, and 1 mg / mL hyaluronidase.

[0098] Generally, the harvested cell suspension is referred to as a "primary cell population" or "freshly harvested" cell population.

[0099] In some embodiments, fragmentation comprises physical fragmentation, including, for example, dissection and digestion. In some embodiments, fragmentation is physical fragmentation. In some embodiments, fragmentation is dissection. In some embodiments, fragmentation is by digestion. In some embodiments, TILs may be initially cultured from enzymatic tumor digests and tumor fragments obtained from digesting or fragmenting a tumor sample obtained from a patient.

[0100] In some embodiments where the tumor is a solid tumor, after a tumor sample is obtained, for example, in step A (provided in FIG. 1), the tumor undergoes physical fragmentation. In some embodiments, fragmentation occurs before cryopreservation. In some embodiments, fragmentation occurs after cryopreservation. In some embodiments, fragmentation occurs after obtaining the tumor in the absence of any cryopreservation. In some embodiments, the tumor is fragmented and 10, 20, 30, 40, or more fragments or pieces are placed in each container for the first expansion. In some embodiments, the tumor is fragmented and 30 or 40 fragments or pieces are placed in each container for the first expansion. In some embodiments, the tumor is fragmented and 40 fragments or pieces are placed in each container for the first expansion. In some embodiments, the plurality of fragments comprises about 4 to about 50 fragments, each fragment having a volume of about 27 mm. In some embodiments, the plurality of fragments comprises about 30 to about 60 fragments with a total volume of about 1300 mm to about 1500 mm. In some embodiments, the plurality of pieces comprises about 50 pieces with a total volume of about 1350 mm. In some embodiments, the plurality of pieces comprises about 50 pieces with a total mass of about 1 gram to about 1.5 grams. In some embodiments, the plurality of pieces comprises about 4 pieces.

[0101] In some embodiments, the TILs are obtained from tumor fragments. In some embodiments, the tumor fragments are obtained by sharp dissection. In some embodiments, the tumor fragments are about 1 mm to 10 mm. In some embodiments, the tumor fragments are about 1 mm to 8 mm. In some embodiments, the tumor fragments are about 1 mm. In some embodiments, the tumor fragments are about 2 mm. In some embodiments, the tumor fragments are about 3 mm. In some embodiments, the tumor fragments are about 4 mm. In some embodiments, the tumor fragments are about 5 mm. In some embodiments, the tumor fragments are about 6 mm. In some embodiments, the tumor fragments are about 7 mm. In some embodiments, the tumor fragments are about 8 mm. In some embodiments, the tumor fragments are about 9 mm. In some embodiments, the tumor fragments are about 10 mm. In some embodiments, the tumor is 1-4 mm x 1-4 mm x 1-4 mm. In some embodiments, the tumor is 1 mm x 1 mm x 1 mm. In some embodiments, the tumor is 2 mm x 2 mm x 2 mm. In some embodiments, the tumor is 3 mm x 3 mm x 3 mm. In some embodiments, the tumor is 4 mm x 4 mm x 4 mm.

[0102] In some embodiments, the tumor is resected to minimize the amount of hemorrhagic, necrotic, and / or fatty tissue on each piece. In some embodiments, the tumor is resected to minimize the amount of hemorrhagic tissue on each piece. In some embodiments, the tumor is resected to minimize the amount of necrotic tissue on each piece. In some embodiments, the tumor is resected to minimize the amount of fatty tissue on each piece.

[0103] In some embodiments, tumor fragmentation is performed to maintain the internal structure of the tumor. In some embodiments, tumor fragmentation is performed without sawing with a scalpel. In some embodiments, TILs are obtained from tumor digests. In some embodiments, tumor digests are generated by incubation in an enzyme medium, such as, but not limited to, RPMI 1640 (2 mM GlutaMAX, 10 mg / mL gentamicin, 30 U / mL DNase, and 1.0 mg / mL collagenase), followed by mechanical dissociation (GentleMACS, Miltenyi Biotec, Auburn, CA). After placing the tumor in the enzyme medium, the tumor may be mechanically dissociated for approximately 1 minute. The solution may then be incubated at 37°C in 5% CO2 for 30 minutes, after which it may be mechanically disrupted again for approximately 1 minute. After again incubating at 37°C in 5% CO2 for 30 minutes, the tumor may be mechanically disrupted a third time for approximately 1 minute. In some embodiments, if large tissue debris was present, one or two additional rounds of mechanical dissociation were applied to the sample after the third mechanical disruption, with incubation for an additional 30 minutes at 37° C. in 5% CO In some embodiments, if the cell suspension contains a large number of red blood cells or dead cells, density gradient separation using Ficoll can be performed at the end of the final incubation to remove these cells.

[0104] In some embodiments, the cell suspension harvested prior to the first expansion step is referred to as a "primary cell population" or "freshly harvested" cell population.

[0105] In some embodiments, the cells may be optionally frozen after sampling and cryopreserved before undergoing expansion as described in step B, which is described in more detail below and also illustrated in Figures 1 and 8.

[0106] 1. Pleural fluid T cells and TILs In some embodiments, the sample is a pleural fluid sample. In some embodiments, the source of T cells or TILs for expansion by the processes described herein is a pleural fluid sample. In some embodiments, the sample is a pleural fluid-derived sample. In some embodiments, the source of TILs for expansion by the processes described herein is a pleural fluid-derived sample. See, e.g., the methods described in U.S. Patent Publication No. US2014 / 0295426, which is incorporated by reference in its entirety for all purposes.

[0107] In some embodiments, any pleural fluid or pleural effusion that appears to be and / or contains TILs can be utilized. Such samples can be derived from primary or metastatic lung cancer, such as NSCLC or SCLC. In some embodiments, samples can be derived from secondary metastatic cancer cells from another organ, such as the breast, ovary, colon, or prostate. In some embodiments, the sample used in the expansion methods described herein is a pleural effusion. In some embodiments, the sample used in the expansion methods described herein is a pleural transudate. Other biological samples can include other serous fluids containing TILs, including, for example, ascites from the abdomen or pancreatic cyst fluid. Ascites and pleural fluids have very similar chemical systems, and both the abdomen and lungs have mesothelial lines and fluid forms in the pleural and abdominal spaces of the same material in malignant tumors; in some embodiments, such fluids contain TILs. In some embodiments in which the disclosed methods utilize pleural effusion, the same methods can be performed with similar results using other cyst fluids containing TILs.

[0108] In some embodiments, the pleural fluid is in an unprocessed form removed directly from the patient. In some embodiments, the unprocessed pleural fluid is placed in a standard collection tube, such as an EDTA or heparin tube, before further processing steps. In some embodiments, the unprocessed pleural fluid is placed in a standard CellSave® tube (Veridex) before further processing steps. In some embodiments, the sample is placed in a CellSave tube immediately after collection from the patient to avoid a decrease in the number of viable TILs. The number of viable TILs can decrease significantly within 24 hours if left in unprocessed pleural fluid, even at 4°C. In some embodiments, the sample is placed in an appropriate collection tube within 1 hour, 5 hours, 10 hours, 15 hours, or up to 24 hours after removal from the patient. In some embodiments, the sample is placed in an appropriate collection tube at 4°C within 1 hour, 5 hours, 10 hours, 15 hours, or up to 24 hours after removal from the patient.

[0109] In some embodiments, the pleural fluid sample from a selected subject may be diluted. In some embodiments, the dilution is 1:10 pleural fluid to diluent. In some embodiments, the dilution is 1:9 pleural fluid to diluent. In some embodiments, the dilution is 1:8 pleural fluid to diluent. In some embodiments, the dilution is 1:5 pleural fluid to diluent. In some embodiments, the dilution is 1:2 pleural fluid to diluent. In some embodiments, the dilution is 1:1 pleural fluid to diluent. In some embodiments, the diluent includes saline, phosphate-buffered saline, another buffer, or a physiologically acceptable diluent. In some embodiments, the sample is placed into a CellSave tube immediately after collection from the patient and diluted to avoid a significant loss of viable TILs, which can occur within 24 to 48 hours if left in unprocessed pleural fluid, even at 4°C. In some embodiments, the pleural fluid sample is placed into a suitable collection tube within 1 hour, 5 hours, 10 hours, 15 hours, 24 hours, 36 hours, or up to 48 hours after removal from the patient and dilution. In some embodiments, the pleural fluid sample is placed into a suitable collection tube within 1 hour, 5 hours, 10 hours, 15 hours, 24 hours, 36 hours, or up to 48 hours after removal from the patient and dilution at 4° C.

[0110] In yet other embodiments, the pleural fluid sample is concentrated by conventional means prior to further processing steps. In some embodiments, this pretreatment of the pleural fluid is preferred in situations where the pleural fluid must be frozen for transport to the laboratory where the method will be performed or for subsequent analysis (e.g., more than 24-48 hours after collection). In some embodiments, the pleural fluid sample is prepared by centrifuging the pleural fluid sample after it is collected from the subject and resuspending the centrate or pellet in a buffer. In some embodiments, the pleural fluid sample is subjected to multiple centrifugations and resuspensions before being frozen for transport or subsequent analysis and / or processing.

[0111] In some embodiments, the pleural fluid sample is concentrated prior to further processing steps by using a filtration method. In some embodiments, the pleural fluid sample used in further processing is prepared by filtering the fluid through a filter containing a known, essentially uniform pore size that allows the pleural fluid to pass through the membrane but retains tumor cells. In some embodiments, the membrane pore diameter can be at least 4 μM. In other embodiments, the pore diameter can be 5 μM or greater, and in other embodiments, 6, 7, 8, 9, or 10 μM. After filtration, the TIL-containing cells retained by the membrane can be rinsed from the membrane into an appropriate physiologically acceptable buffer. The thus-enriched TIL-containing cells can then be used in further processing steps of the method.

[0112] In some embodiments, a pleural fluid sample (e.g., including unprocessed pleural fluid), diluted pleural fluid, or a resuspended cell pellet is contacted with a lysis reagent that specifically lyses nonnucleated red blood cells present in the sample. In some embodiments, this step is performed before further processing steps in situations where the pleural fluid contains a significant number of RBCs. Suitable lysis reagents include a single lysis reagent or a lysis reagent and a quenching reagent, or a lysis agent, a quenching reagent, and a fixation reagent. Suitable lysis systems are commercially available and include the BD Pharm Lyse™ system (Becton Dickenson). Other lysis systems include the Versalyse™ system, the FACSlyse™ system (Becton Dickenson), the Immunoprep™ system, or the Erythrolyse II system (Beckman Coulter, Inc.), or an ammonium chloride system. In some embodiments, the lysis reagent can vary depending on key requirements, such as efficient lysis of red blood cells and preservation of TILs and their phenotypic characteristics in the pleural fluid. In addition to utilizing a single reagent for lysis, lysis systems useful in the methods described herein can include a second reagent, e.g., one that quenches or delays the effect of the lysis reagent during the remaining steps of the method, such as Stabilyse™ reagent (Beckman Coulter, Inc.) Depending on the choice of lysis reagent or the preferred implementation of the method, conventional fixative reagents can also be used.

[0113] In some embodiments, pleural fluid samples that have been unprocessed, diluted, or multiple centrifuged or processed as described herein are stored frozen at a temperature of about -140°C before being further processed and / or expanded as provided herein.

[0114] B. Step B: First Expansion In some embodiments, the methods provide for obtaining young TILs that are capable of increasing their replication cycle upon administration to a subject / patient and thus may provide additional therapeutic benefit over older TILs (i.e., TILs that have undergone more rounds of replication prior to administration to a subject / patient). The characteristics of young TILs are described in the literature, for example, Donia, et al., Scand. J. Immunol. 2012, 75, 157-167, Dudley, et al., Clin. al.,Clin.Cancer Res.2013,19,OF1-OF9, Besser,et al.,J.Immunother.2009,32:415-423,Robbins,et al.,J.Immunol.2004,173,7125-7130,Shen,et al. al.,J.Immunother.,2007,30,123-129, Zhou,et al., J. Immunother. 2005, 28, 53-62, and Tran, et al., J. Immunother., 2008, 31, 742-751, the disclosures of each of which are incorporated herein by reference.

[0115] The diverse antigen receptors of T and B lymphocytes are produced by somatic recombination of a limited number of gene segments. These gene segments: V (variable), D (diversity), J (joining), and C (constant) determine the binding specificity and downstream applications of immunoglobulins and T cell receptors (TCRs). The present invention provides methods for generating TILs that exhibit and increase T cell repertoire diversity. In some embodiments, TILs obtained by the present methods exhibit increased T cell repertoire diversity. In some embodiments, TILs obtained by the present methods exhibit increased T cell repertoire diversity compared to freshly harvested TILs and / or TILs prepared using methods other than those provided herein, including, for example, methods other than those embodied in FIG. 1. In some embodiments, TILs obtained by the present methods exhibit increased T cell repertoire diversity compared to freshly harvested TILs and / or TILs prepared using a method designated Process 1C as illustrated in FIG. 5 and / or FIG. 6. In some embodiments, the TILs obtained in the first expansion exhibit increased T cell repertoire diversity. In some embodiments, the increased diversity is increased immunoglobulin diversity and / or T cell receptor diversity. In some embodiments, the diversity is in immunoglobulins and in immunoglobulin heavy chains. In some embodiments, the diversity is in immunoglobulins and in immunoglobulin light chains. In some embodiments, the diversity is in T cell receptors. In some embodiments, the diversity is in one of the T cell receptors selected from the group consisting of alpha, beta, gamma, and delta receptors. In some embodiments, expression of T cell receptor (TCR) alpha and / or beta is increased. In some embodiments, expression of T cell receptor (TCR) alpha is increased. In some embodiments, expression of T cell receptor (TCR) beta is increased. In some embodiments, expression of TCRab (i.e., TCRα / β) is increased.

[0116] For example, after dissection or digestion of tumor fragments, such as those described in step A of Figure 1, the resulting cells are cultured in serum containing IL-2 under conditions that favor the growth of TILs over tumor and other cells. In some embodiments, tumor digests are incubated in 2 mL wells in medium containing inactivated human AB serum with 6000 IU / mL of IL-2. This primary cell population is cultured for several days, generally 3-14 days, resulting in a bulk TIL population, generally about 1 x 10 8 In some embodiments, this primary cell population is cultured for 7-14 days, resulting in a bulk TIL population, generally about 1 x 10 8 In some embodiments, this primary cell population is cultured for 10-14 days, resulting in a bulk TIL population, generally about 1 x 10 8 In some embodiments, this primary cell population is cultured for about 11 days, resulting in a bulk TIL population, generally about 1 x 10 8 Generate bulk TIL cells.

[0117] In a preferred embodiment, TIL expansion can be performed using an initial bulk TIL expansion step (such as that described in step B of FIG. 1, which may include a process referred to as pre-REP) as described below and herein, followed by step D below and a second expansion as described herein (including step D, a process referred to as the rapid expansion protocol (REP) step), followed by optional cryopreservation, followed by a second step D below and described herein (including a process referred to as the restimulation REP step). TILs obtained from this process can optionally be characterized for phenotypic characteristics and metabolic parameters as described herein.

[0118] In embodiments in which TIL cultures are initiated in 24-well plates, e.g., using Costar 24-well cell culture clusters, flat bottom (Corning Incorporated, Corning, NY), 1 x 10 cells are cultured in 2 mL of complete medium (CM) containing IL-2 (6000 IU / mL, Chiron Corp., Emeryville, CA). 6 Tumor digested cells or one tumor fragment can be seeded into each well. In some embodiments, the tumor fragment is approximately 1 mm 3 ~10mm 3 is.

[0119] In some embodiments, the first expansion culture medium is referred to as "CM," an abbreviation for culture medium. In some embodiments, the CM in step B consists of RPMI 1640 with GlutaMAX, supplemented with 10% human AB serum, 25 mM Hepes, and 10 mg / mL gentamicin. When the culture is in a 40 mL volume and 10 cm 2 In embodiments initiated in gas-permeable flasks with gas-permeable silicon bottoms (e.g., G-REX10, Wilson Wolf Manufacturing, New Brighton, MN), each flask contains 10-40 x 10 cells in 10-40 mL of CM with IL-2. 6 Live tumor cells or 5–30 tumor fragments were loaded. Both G-REX10 and 24-well plates were incubated in a humidified incubator at 37°C with 5% CO2. Five days after the start of culture, half of the medium was removed and replaced with fresh CM and IL-2. After five days, half of the medium was replaced every 2–3 days.

[0120] In some embodiments, the medium used in the expansion processes disclosed herein is a serum-free or synthetic medium. In some embodiments, the serum-free or synthetic medium comprises a basal cell culture medium and a serum supplement and / or serum replacement. In some embodiments, the serum-free or synthetic medium is used to prevent and / or reduce experimental variation due in part to lot-to-lot variation in serum-containing medium.

[0121] In some embodiments, the serum-free or defined medium comprises a basal cell culture medium and a serum supplement and / or serum replacement. In some embodiments, the basal cell culture medium includes, but is not limited to, CTS™ OpTmizer™ T-cell Expansion Basal Medium, CTS™ OpTmizer™ T-Cell Expansion SFM, CTS™ AIM-V Medium, CTS™ AIM-V SFM, LymphoONE™ T-Cell Expansion Xeno-Free Medium, Dulbecco's Modified Eagle's Medium (DMEM), Minimum Essential Medium (MEM), Basal Eagle's Medium (BME), RPMI 1640, F-10, F-12, Minimum Essential Medium (αMEM), Glasgow Minimum Essential Medium (G-MEM), RPMI Growth Medium, and Iscove's Modified Dulbecco's Medium.

[0122] In some embodiments, the serum supplement or serum replacement includes, but is not limited to, CTS™ OpTmizer T-Cell Expansion Serum Supplement, CTS™ Immune Cell Serum Replacement, one or more albumins or albumin substitutes, one or more amino acids, one or more vitamins, one or more transferrins or transferrin substitutes, one or more antioxidants, one or more insulins or insulin substitutes, one or more collagen precursors, one or more antibiotics, and one or more trace elements. In some embodiments, the synthetic medium contains albumin and one or more of glycine, L-histidine, L-isoleucine, L-methionine, L-phenylalanine, L-proline, L-hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, thiamine, reduced glutathione, L-ascorbic acid-2-phosphate, iron-saturated transferrin, insulin, and trace element moieties. + , Al 3+ , Ba 2+ , Cd 2+ , Co 2+ , Cr 3+ , Ge4+ , Se 4+ , Br, T, Mn 2+ , P, Si 4+ , V 5+ , Mo 6+ , Ni 2+ , Rb + , Sn 2+ , and Zr 4+ and one or more components selected from the group consisting of compounds containing: In some embodiments, the synthetic medium further comprises L-glutamine, sodium bicarbonate, and / or 2-mercaptoethanol.

[0123] In some embodiments, CTS™ OpTmizer™ T-cell Immune Cell Serum Replacement is used with conventional growth media, including, but not limited to, CTS™ OpTmizer™ T-cell Expansion Basal Medium, CTS™ OpTmizer™ T-cell Expansion SFM, CTS™ AIM-V Medium, CST™ AIM-V SFM, LymphoONE™ T-Cell Expansion Xeno-Free Medium, Dulbecco's Modified Eagle's Medium (DMEM), Minimum Essential Medium (MEM), Basal Eagle's Medium (BME), RPMI 1640, F-10, F-12, Minimum Essential Medium (αMEM), Glasgow Minimum Essential Medium (G-MEM), RPMI Growth Medium, and Iscove's Modified Dulbecco's Medium.

[0124] In some embodiments, the total serum replacement concentration (vol%) in the serum-free or synthetic medium is about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% by volume of the total serum-free or synthetic medium. In some embodiments, the total serum replacement concentration is about 3% of the total volume of the serum-free or synthetic medium. In some embodiments, the total serum replacement concentration is about 5% of the total volume of the serum-free or synthetic medium. In some embodiments, the total serum replacement concentration is about 10% of the total volume of the serum-free or synthetic medium.

[0125] In some embodiments, the serum-free or defined medium is CTS™ OpTmizer™ T-cell Expansion SFM (ThermoFisher Scientific). Any formulation of CTS™ OpTmizer™ is useful in the present invention. CTS™ OpTmizer™ T-cell Expansion SFM is a combination of 1 L of CTS™ OpTmizer™ T-cell Expansion Basal Medium and 26 mL of CTS™ OpTmizer™ T-Cell Expansion Supplement, which are mixed together before use. In some embodiments, CTS™ OpTmizer™ T-cell Expansion SFM is supplemented with approximately 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific). In some embodiments, CTS™ OpTmizer™ T-cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) along with 55 mM 2-mercaptoethanol. In some embodiments, CTS™ OpTmizer™ T-cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and the final concentration of 2-mercaptoethanol in the medium is 55 μM.

[0126] In some embodiments, the defined medium is CTS™ OpTmizer™ T-cell Expansion SFM (ThermoFisher Scientific). Any formulation of CTS™ OpTmizer™ is useful in the present invention. CTS™ OpTmizer™ T-cell Expansion SFM is a combination of 1 L of CTS™ OpTmizer™ T-cell Expansion Basal Medium and 26 mL of CTS™ OpTmizer™ T-Cell Expansion Supplement, which are mixed together before use. In some embodiments, CTS™ OpTmizer™ T-cell Expansion SFM is supplemented with approximately 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) along with 55 mM 2-mercaptoethanol. In some embodiments, CTS™ OpTmizer™ T-cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific), 55 mM 2-mercaptoethanol, and 2 mM L-glutamine. In some embodiments, CTS™ OpTmizer™ T-cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific), 55 mM 2-mercaptoethanol, and 2 mM L-glutamine, and further comprises about 1000 IU / mL to about 8000 IU / mL of IL-2. In some embodiments, CTS™ OpTmizer™ T-cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific), 55 mM 2-mercaptoethanol, and 2 mM L-glutamine, and further contains about 3000 IU / mL of IL-2.In some embodiments, CTS™ OpTmizer™ T-cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific), 55 mM 2-mercaptoethanol, and 2 mM L-glutamine, and further comprises about 6000 IU / mL of IL-2. In some embodiments, CTS™ OpTmizer™ T-cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and 55 mM 2-mercaptoethanol, and further comprises about 1000 IU / mL to about 8000 IU / mL of IL-2. In some embodiments, CTS™ OpTmizer™ T-cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and 55 mM 2-mercaptoethanol, and further comprises about 3000 IU / mL of IL-2. In some embodiments, CTS™ OpTmizer™ T-cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and 55 mM 2-mercaptoethanol, and further comprises about 1000 IU / mL to about 6000 IU / mL of IL-2. In some embodiments, CTS™ OpTmizer™ T-cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and about 2 mM glutamine, and further contains about 1000 IU / mL to about 8000 IU / mL of IL-2.In some embodiments, the CTS™ OpTmizer™ T-cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and about 2 mM glutamine, and further comprises about 3000 IU / mL of IL-2. In some embodiments, the CTS™ OpTmizer™ T-cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and about 2 mM glutamine, and further comprises about 6000 IU / mL of IL-2. In some embodiments, the CTS™ OpTmizer™ T-cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific), and the final concentration of 2-mercaptoethanol in the medium is 55 μM.

[0127] In some embodiments, serum-free or synthetic media is supplemented with glutamine (i.e., GlutaMAX®) at a concentration of about 0.1 mM to about 10 mM, 0.5 mM to about 9 mM, 1 mM to about 8 mM, 2 mM to about 7 mM, 3 mM to about 6 mM, or 4 mM to about 5 mM. In some embodiments, serum-free or synthetic media is supplemented with glutamine (i.e., GlutaMAX®) at a concentration of about 2 mM.

[0128] In some embodiments, serum-free or synthetic medium is supplemented with 2-mercaptoethanol at a concentration of about 5 mM to about 150 mM, 10 mM to about 140 mM, 15 mM to about 130 mM, 20 mM to about 120 mM, 25 mM to about 110 mM, 30 mM to about 100 mM, 35 mM to about 95 mM, 40 mM to about 90 mM, 45 mM to about 85 mM, 50 mM to about 80 mM, 55 mM to about 75 mM, 60 mM to about 70 mM, or about 65 mM. In some embodiments, serum-free or synthetic medium is supplemented with 2-mercaptoethanol at a concentration of about 55 mM. In some embodiments, the final concentration of 2-mercaptoethanol in the medium is 55 μM.

[0129] In some embodiments, the synthetic media described in International PCT Publication No. WO / 1998 / 030679, incorporated herein by reference, are useful in the present invention. That publication describes serum-free eukaryotic cell culture media. The serum-free eukaryotic cell culture media include basal cell culture media supplemented with serum-free supplements capable of supporting cell growth in serum-free culture. The serum-free eukaryotic cell culture medium supplement comprises, or is obtained by combining, one or more components selected from the group consisting of one or more albumins or albumin substitutes, one or more amino acids, one or more vitamins, one or more transferrins or transferrin substitutes, one or more antioxidants, one or more insulins or insulin substitutes, one or more collagen precursors, one or more trace elements, and one or more antibiotics. In some embodiments, the synthetic media further comprise L-glutamine, sodium bicarbonate, and / or beta-mercaptoethanol. In some embodiments, the synthetic medium comprises albumin or an albumin substitute and one or more components selected from the group consisting of one or more amino acids, one or more vitamins, one or more transferrin or transferrin substitutes, one or more antioxidants, one or more insulin or insulin substitutes, one or more collagen precursors, and one or more trace elements. In some embodiments, the synthetic medium comprises albumin and one or more components selected from the group consisting of glycine, L-histidine, L-isoleucine, L-methionine, L-phenylalanine, L-proline, L-hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, thiamine, reduced glutathione, L-ascorbic acid-2-phosphate, iron-saturated transferrin, insulin, and the trace element moiety Ag. + , Al 3+ , Ba 2+ , Cd 2+ , Co 2+ , Cr 3+ , Ge 4+ , Se 4+ , Br, T, Mn 2+ , P, Si 4+ , V 5+ , Mo 6+ , Ni 2+ , Rb+ , Sn 2+ , and Zr 4+ and one or more components selected from the group consisting of compounds containing: In some embodiments, the basal cell culture medium is selected from the group consisting of Dulbecco's Modified Eagle's Medium (DMEM), Minimum Essential Medium (MEM), Basal Eagle's Medium (BME), RPMI 1640, F-10, F-12, Minimum Essential Medium (αMEM), Glasgow Minimum Essential Medium (G-MEM), RPMI Growth Medium, and Iscove's Modified Dulbecco's Medium.

[0130] In some embodiments, the concentration of glycine in the synthetic medium ranges from about 5 to 200 mg / L, the concentration of L-histidine from about 5 to 250 mg / L, the concentration of L-isoleucine from about 5 to 300 mg / L, the concentration of L-methionine from about 5 to 200 mg / L, the concentration of L-phenylalanine from about 5 to 400 mg / L, the concentration of L-proline from about 1 to 1000 mg / L, the concentration of L-hydroxyproline from about 1 to 45 mg / L, the concentration of L-serine from about 1 to 250 mg / L, the concentration of L-threonine from about 10 to 500 mg / L, and the concentration of L-tryptophan from about 2 to 110 mg / L. / L, the concentration of L-tyrosine is about 3 to 175 mg / L, the concentration of L-valine is about 5 to 500 mg / L, the concentration of thiamine is about 1 to 20 mg / L, the concentration of reduced glutathione is about 1 to 20 mg / L, the concentration of L-ascorbic acid 2-phosphate is about 1 to 200 mg / L, the concentration of iron-saturated transferrin is about 1 to 50 mg / L, the concentration of insulin is about 1 to 100 mg / L, the concentration of sodium selenite is about 0.000001 to 0.0001 mg / L, and the concentration of albumin (e.g., AlbuMAX® I) is about 5,000 to 50,000 mg / L.

[0131] In some embodiments, the non-trace element components in the synthetic medium are present at the concentration ranges listed in the column under the heading "Concentration Ranges in 1x Medium" in Table 4 below. In other embodiments, the non-trace element components in the synthetic medium are present at the final concentrations listed in the column under the heading "Preferred Embodiments of 1x Medium" in Table 4 below. In other embodiments, the synthetic medium is a basal cell culture medium that includes a serum-free supplement. In some of these embodiments, the serum-free supplement includes non-trace element components of the type and concentration listed in the column under the heading "Preferred Embodiments of Supplement" in Table 4 below.

[0132] [Table 4]

[0133] In some embodiments, the osmolality of the synthetic medium is about 260-350 mOsmol. In some embodiments, the osmolality is about 280-310 mOsmol. In some embodiments, the synthetic medium is supplemented with sodium bicarbonate at up to about 3.7 g / L, or about 2.2 g / L. The synthetic medium may be further supplemented with L-glutamine (final concentration about 2 mM), one or more antibiotics, non-essential amino acids (NEAA, final concentration about 100 μM), and 2-mercaptoethanol (final concentration about 100 μM).

[0134] In some embodiments, the defined media described in Smith, et al., Clin Transl Immunology, 4(1) 2015 (doi:10.1038 / cti.2014.31) are useful in the present invention. Briefly, RPMI or CTS™ OpTmizer™ was used as the basal cell culture medium, supplemented with either 0, 2%, 5%, or 10% CTS™ Immune Cell Serum Replacement.

[0135] In some embodiments, the cell culture medium in the first and / or second gas-permeable container is unfiltered. The use of unfiltered cell culture medium can simplify the procedures required to expand the number of cells. In some embodiments, the cell culture medium in the first and / or second gas-permeable container lacks beta-mercaptoethanol (BME or βME, also known as 2-mercaptoethanol, CAS 60-24-2).

[0136] After preparation of tumor fragments, the resulting cells (i.e., fragments) are cultured in serum containing IL-2 under conditions that favor the growth of TILs over tumor and other cells. In some embodiments, tumor digests are incubated in 2 mL wells (or in some cases, in the presence of an APC cell population, as outlined herein) in medium containing inactivated human AB serum with 6000 IU / mL of IL-2. This primary cell population is cultured for several days, generally 10-14 days, resulting in a bulk TIL population, generally approximately 1 x 10 bulk TIL cells. In some embodiments, the growth medium during the first expansion includes IL-2 or a variant thereof. In some embodiments, the IL is recombinant human IL-2 (rhIL-2). In some embodiments, the IL-2 stock solution has a specific activity of 20-30 x 10 IU / mg per 1 mg vial. In some embodiments, the IL-2 stock solution has a specific activity of 20 x 10 IU / mg per 1 mg vial. In some embodiments, the IL-2 stock solution has a specific activity of 25×10 IU / mg per 1 mg vial. In some embodiments, the IL-2 stock solution has a specific activity of 30×10 IU / mg per 1 mg vial. In some embodiments, the IL-2 stock solution has a final concentration of IL-2 of 4-8×10 IU / mg. In some embodiments, the IL-2 stock solution has a final concentration of IL-2 of 5-7×10 IU / mg. In some embodiments, the IL-2 stock solution has a final concentration of IL-2 of 6×10 IU / mg. In some embodiments, the IL-2 stock solution is prepared as described in Example 5. In some embodiments, the first expansion culture medium contains about 10,000 IU / mL IL-2, about 9,000 IU / mL IL-2, about 8,000 IU / mL IL-2, about 7,000 IU / mL IL-2, about 6,000 IU / mL IL-2, or about 5,000 IU / mL IL-2. In some embodiments, the first expansion culture medium contains about 9,000 IU / mL to about 5,000 IU / mL IL-2. In some embodiments, the first expansion culture medium contains about 8,000 IU / mL to about 6,000 IU / mL IL-2.In some embodiments, the first expansion culture medium comprises about 7,000 IU / mL to about 6,000 IU / mL of IL-2. In some embodiments, the first expansion culture medium comprises about 6,000 IU / mL of IL-2. In some embodiments, the cell culture medium further comprises IL-2. In some embodiments, the cell culture medium comprises about 3000 IU / mL of IL-2. In some embodiments, the cell culture medium further comprises IL-2. In some embodiments, the cell culture medium comprises about 3000 IU / mL of IL-2. In some embodiments, the cell culture medium comprises about 1000 IU / mL, about 1500 IU / mL, about 2000 IU / mL, about 2500 IU / mL, about 3000 IU / mL, about 3500 IU / mL, about 4000 IU / mL, about 4500 IU / mL, about 5000 IU / mL, about 5500 IU / mL, about 6000 IU / mL, about 6500 IU / mL, about 7000 IU / mL, about 7500 IU / mL, or about 8000 IU / mL of IL-2. In some embodiments, the cell culture medium contains 1000-2000 IU / mL, 2000-3000 IU / mL, 3000-4000 IU / mL, 4000-5000 IU / mL, 5000-6000 IU / mL, 6000-7000 IU / mL, 7000-8000 IU / mL, or about 8000 IU / mL of IL-2.

[0137] In some embodiments, the first expansion culture medium contains about 500 IU / mL IL-15, about 400 IU / mL IL-15, about 300 IU / mL IL-15, about 200 IU / mL IL-15, about 180 IU / mL IL-15, about 160 IU / mL IL-15, about 140 IU / mL IL-15, about 120 IU / mL IL-15, or about 100 IU / mL IL-15. In some embodiments, the first expansion culture medium contains about 500 IU / mL to about 100 IU / mL IL-15. In some embodiments, the first expansion culture medium contains about 400 IU / mL to about 100 IU / mL IL-15. In some embodiments, the first expansion culture medium comprises about 300 IU / mL to about 100 IU / mL of IL-15. In some embodiments, the first expansion culture medium comprises about 200 IU / mL of IL-15. In some embodiments, the cell culture medium comprises about 180 IU / mL of IL-15. In some embodiments, the cell culture medium further comprises IL-15. In some embodiments, the cell culture medium comprises about 180 IU / mL of IL-15.

[0138] In some embodiments, the first expansion culture medium comprises about 20 IU / mL IL-21, about 15 IU / mL IL-21, about 12 IU / mL IL-21, about 10 IU / mL IL-21, about 5 IU / mL IL-21, about 4 IU / mL IL-21, about 3 IU / mL IL-21, about 2 IU / mL IL-21, about 1 IU / mL IL-21, or about 0.5 IU / mL IL-21. In some embodiments, the first expansion culture medium comprises about 20 IU / mL to about 0.5 IU / mL IL-21. In some embodiments, the first expansion culture medium comprises about 15 IU / mL to about 0.5 IU / mL IL-21. In some embodiments, the first expansion culture medium comprises about 12 IU / mL to about 0.5 IU / mL of IL-21. In some embodiments, the first expansion culture medium comprises about 10 IU / mL to about 0.5 IU / mL of IL-21. In some embodiments, the first expansion culture medium comprises about 5 IU / mL to about 1 IU / mL of IL-21. In some embodiments, the first expansion culture medium comprises about 2 IU / mL of IL-21. In some embodiments, the cell culture medium comprises about 1 IU / mL of IL-21. In some embodiments, the cell culture medium comprises about 0.5 IU / mL of IL-21. In some embodiments, the cell culture medium further comprises IL-21. In some embodiments, the cell culture medium comprises about 1 IU / mL of IL-21.

[0139] In some embodiments, the cell culture medium comprises an anti-CD3 agonist antibody, such as an OKT-3 antibody. In some embodiments, the cell culture medium comprises about 30 ng / mL of the OKT-3 antibody. In some embodiments, the cell culture medium comprises about 0.1 ng / mL, about 0.5 ng / mL, about 1 ng / mL, about 2.5 ng / mL, about 5 ng / mL, about 7.5 ng / mL, about 10 ng / mL, about 15 ng / mL, about 20 ng / mL, about 25 ng / mL, about 30 ng / mL, about 35 ng / mL, about 40 ng / mL, about 50 ng / mL, about 60 ng / mL, about 70 ng / mL, about 80 ng / mL, about 90 ng / mL, about 100 ng / mL, about 200 ng / mL, about 500 ng / mL, or about 1 μg / mL of the OKT-3 antibody. In some embodiments, the cell culture medium comprises OKT-3 antibody at concentrations of 0.1 ng / mL to 1 ng / mL, 1 ng / mL to 5 ng / mL, 5 ng / mL to 10 ng / mL, 10 ng / mL to 20 ng / mL, 20 ng / mL to 30 ng / mL, 30 ng / mL to 40 ng / mL, 40 ng / mL to 50 ng / mL, and 50 ng / mL to 100 ng / mL. In some embodiments, the cell culture medium does not contain OKT-3 antibody. In some embodiments, the OKT-3 antibody is muromonab. See, for example, Table 1.

[0140] In some embodiments, the cell culture medium comprises one or more TNFRSF agonists in the cell culture medium. In some embodiments, the TNFRSF agonist comprises a 4-1BB agonist. In some embodiments, the TNFRSF agonist is a 4-1BB agonist, and the 4-1BB agonist is selected from the group consisting of urelumab, utomilumab, EU-101, fusion proteins, and fragments, derivatives, variants, biosimilars, and combinations thereof. In some embodiments, the TNFRSF agonist is added at a concentration sufficient to achieve a concentration in the cell culture medium of 0.1 μg / mL to 100 μg / mL. In some embodiments, the TNFRSF agonist is added at a concentration sufficient to achieve a concentration in the cell culture medium of 20 μg / mL to 40 μg / mL.

[0141] In some embodiments, in addition to the one or more TNFRSF agonists, the cell culture medium further comprises IL-2 at an initial concentration of about 3000 IU / mL and OKT-3 at an initial concentration of about 30 ng / mL, and the one or more TNFRSF agonists comprise a 4-1BB agonist.

[0142] In some embodiments, the first expansion culture medium is referred to as "CM," an abbreviation for culture medium. In some embodiments, it is referred to as CM1 (culture medium 1). In some embodiments, CM consists of RPMI 1640 with GlutaMAX, supplemented with 10% human AB serum, 25 mM Hepes, and 10 mg / mL gentamicin. In embodiments where cultures are initiated in gas-permeable flasks with a 40 mL volume and a 10 cm gas-permeable silicone bottom (e.g., G-REX10, Wilson Wolf Manufacturing, New Brighton, MN), 10-40 x 10 viable tumor-digested cells or 5-30 tumor fragments in 10-40 mL of CM containing IL-2 are placed in each flask. Both the G-REX10 and 24-well plates were incubated in a humidified incubator at 37°C with 5% CO2. Five days after the initiation of culture, half of the medium was removed and replaced with fresh CM and IL-2. After five days, half of the medium was replaced every two to three days. In some embodiments, the CM is CM1 as described in the Examples, see Example 1. In some embodiments, the first expansion occurs in the initial cell culture medium or the first cell culture medium. In some embodiments, the initial cell culture medium or the first cell culture medium contains IL-2.

[0143] In some embodiments, as discussed in the Examples and Figures, the first expansion (including processes such as those described in step B of FIG. 1, which may include what may be referred to as pre-REP) process is shortened to 3-14 days. In some embodiments, as discussed in the Examples and shown in FIGS. 4 and 5, the first expansion (including processes such as those described in step B of FIG. 1, which may include what may be referred to as pre-REP), including the expansion described in step B of FIG. 1, is shortened to 7-14 days. In some embodiments, the first expansion of step B is shortened to 10-14 days. In some embodiments, the first expansion is shortened to 11 days, as discussed in the expansion described in step B of FIG. 1, for example.

[0144] In some embodiments, the first TIL expansion can continue for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days. In some embodiments, the first TIL expansion can continue for 1 day to 14 days. In some embodiments, the first TIL expansion can continue for 2 days to 14 days. In some embodiments, the first TIL expansion can continue for 3 days to 14 days. In some embodiments, the first TIL expansion can continue for 4 days to 14 days. In some embodiments, the first TIL expansion can continue for 5 days to 14 days. In some embodiments, the first TIL expansion can continue for 6 days to 14 days. In some embodiments, the first TIL expansion can continue for 7 days to 14 days. In some embodiments, the first TIL expansion can continue for 8 days to 14 days. In some embodiments, the first TIL expansion can continue for 9 days to 14 days. In some embodiments, the first TIL expansion can continue for 10 to 14 days. In some embodiments, the first TIL expansion can continue for 11 to 14 days. In some embodiments, the first TIL expansion can continue for 12 to 14 days. In some embodiments, the first TIL expansion can continue for 13 to 14 days. In some embodiments, the first TIL expansion can continue for 14 days. In some embodiments, the first TIL expansion can continue for 1 to 11 days. In some embodiments, the first TIL expansion can continue for 2 to 11 days. In some embodiments, the first TIL expansion can continue for 3 to 11 days. In some embodiments, the first TIL expansion can continue for 4 to 11 days. In some embodiments, the first TIL expansion can continue for 5 to 11 days. In some embodiments, the first TIL expansion can continue for 6 to 11 days. In some embodiments, the first TIL expansion can continue for 7 to 11 days. In some embodiments, the first TIL expansion can continue for 8 to 11 days.In some embodiments, the first TIL expansion can continue for 9 to 11 days. In some embodiments, the first TIL expansion can continue for 10 to 11 days. In some embodiments, the first TIL expansion can continue for 11 days.

[0145] In some embodiments, a combination of IL-2, IL-7, IL-15, and / or IL-21 is used as a combination during the first expansion. In some embodiments, IL-2, IL-7, IL-15, and / or IL-21, and any combination thereof, may be included during the first expansion, including, for example, during step B of the process according to Figure 1 and described herein. In some embodiments, a combination of IL-2, IL-15, and IL-21 is used as a combination during the first expansion. In some embodiments, IL-2, IL-15, and IL-21, and any combination thereof, may be included during step B of the process according to Figure 1 and described herein.

[0146] In some embodiments, as discussed in the Examples and Figures, the first expansion (including the process referred to as Pre-REP, e.g., step B according to Figure 1) process is shortened to 3-14 days. In some embodiments, the first expansion of step B is shortened to 7-14 days. In some embodiments, the first expansion of step B is shortened to 10-14 days. In some embodiments, the first expansion is shortened to 11 days.

[0147] In some embodiments, the first expansion, e.g., step B according to FIG. 1, is performed in a closed system bioreactor. In some embodiments, a closed system is used for TIL expansion as described herein. In some embodiments, a single bioreactor is used. In some embodiments, the single bioreactor used is, for example, a G-REX-10 or G-REX-100. In some embodiments, the closed system bioreactor is a single bioreactor.

[0148] 1. Cytokines and other additives The expansion methods described herein generally use culture media containing high doses of cytokines, particularly IL-2, as known in the art.

[0149] Alternatively, the use of cytokine combinations for rapid and / or secondary expansion of TILs is additionally possible using combinations of two or more of IL-2, IL-15, and IL-21, as described in U.S. Patent Application Publication No. US2017 / 0107490A1 (the disclosure of which is incorporated herein by reference). Thus, possible combinations include IL-2 and IL-15, IL-2 and IL-21, IL-15 and IL-21, and IL-2 or IL-15 and IL-21, the latter finding particular use in many embodiments. The use of cytokine combinations is particularly advantageous for the generation of lymphocytes, particularly T cells as described therein.

[0150] In some embodiments, step B may also include the addition of an OKT-3 antibody or muromonab to the culture medium, as described elsewhere herein. In some embodiments, step B may also include the addition of a 4-1BB agonist to the culture medium, as described elsewhere herein. In some embodiments, step B may also include the addition of an OX-40 agonist to the culture medium, as described elsewhere herein. In other embodiments, additives such as peroxisome proliferator-activated receptor gamma coactivator I-alpha agonists, including proliferator-activated receptor (PPAR)-gamma agonists such as thiazolidinedione compounds, may be used in the culture medium during step B, as described in U.S. Patent Application Publication No. US2019 / 0307796 A1 (the disclosure of which is incorporated herein by reference).

[0151] C. Step C: Moving from the first expansion to the second expansion In some cases, the bulk TIL population obtained from the first expansion, including, for example, the TIL population obtained from step B shown in Figure 1, can be immediately cryopreserved using the protocols discussed below. Alternatively, the TIL population obtained from the first expansion, referred to as the second TIL population, can be subjected to a second expansion (which may include an expansion sometimes referred to as REP), as discussed below, and then cryopreserved. Similarly, when genetically modified TILs are used for therapy, the first TIL population (which may be referred to as the bulk TIL population) or the second TIL population (which in some embodiments may include a population referred to as the REP TIL population) can be subjected to genetic modification for the appropriate therapy before expansion or after the first expansion and before the second expansion.

[0152] In some embodiments, TILs obtained from the first expansion (e.g., from step B shown in FIG. 1 ) are stored until phenotyping for selection. In some embodiments, TILs obtained from the first expansion (e.g., from step B shown in FIG. 1 ) are not stored and proceed directly to the second expansion. In some embodiments, TILs obtained from the first expansion are not cryopreserved after the first expansion and before the second expansion. In some embodiments, the transition from the first expansion to the second expansion occurs about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs about 3-14 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs about 4-14 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs about 4-10 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs about 7-14 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs about 14 days after fragmentation occurs.

[0153] In some embodiments, the transition from the first expansion to the second expansion occurs 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 1 to 14 days after fragmentation occurs. In some embodiments, the first TIL expansion can continue for 2 to 14 days. In some embodiments, the transition from the first expansion to the second expansion occurs 3 to 14 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 4 to 14 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 5 to 14 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 6 to 14 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 7 to 14 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 8 to 14 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 9 to 14 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 10 to 14 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 11 to 14 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 12 to 14 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 13 to 14 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 14 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 1 to 11 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 2 to 11 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 3 to 11 days after fragmentation occurs.In some embodiments, the transition from the first expansion to the second expansion occurs 4 to 11 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 5 to 11 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 6 to 11 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 7 to 11 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 8 to 11 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 9 to 11 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 10 to 11 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 11 days after fragmentation occurs.

[0154] In some embodiments, the TILs are not stored after the first expansion and before the second expansion, and the TILs proceed directly to the second expansion (e.g., in some embodiments, they are not stored during the transition from step B to step D shown in FIG. 1). In some embodiments, the transfer occurs in a closed system as described herein. In some embodiments, the second TIL population, the TILs from the first expansion, proceed directly to the second expansion without a transfer period.

[0155] In some embodiments, the transition from the first expansion to the second expansion, e.g., step C according to FIG. 1, is performed in a closed system bioreactor. In some embodiments, a closed system is used for TIL expansion as described herein. In some embodiments, a single bioreactor is used. In some embodiments, the single bioreactor used is, for example, a G-REX-10 or G-REX-100 bioreactor. In some embodiments, the closed system bioreactor is a single bioreactor.

[0156] D. Step D: Second Expansion In some embodiments, the TIL cell population is expanded in number after harvesting and initial bulk processing, e.g., after steps A and B, and the transition referred to as step C, as shown in Figure 1. This further expansion is referred to herein as secondary expansion, which may include an expansion process commonly referred to in the art as a rapid expansion process (REP), as well as the process shown in Figure 1 at step D. Secondary expansion is generally accomplished in a gas-permeable container using culture medium containing several components, including feeder cells, a cytokine source, and an anti-CD3 antibody.

[0157] In some embodiments, the second expansion or second TIL expansion (which may include expansion sometimes referred to as REP, and the process shown in step D of FIG. 1 ) may be performed using any TIL flask or vessel known to one of skill in the art. In some embodiments, the second TIL expansion may proceed for 7, 8, 9, 10, 11, 12, 13, or 14 days. In some embodiments, the second TIL expansion may proceed for about 7 days to about 14 days. In some embodiments, the second TIL expansion may proceed for about 8 days to about 14 days. In some embodiments, the second TIL expansion may proceed for about 9 days to about 14 days. In some embodiments, the second TIL expansion may proceed for about 10 days to about 14 days. In some embodiments, the second TIL expansion may proceed for about 11 days to about 14 days. In some embodiments, the second TIL expansion may proceed for about 12 days to about 14 days. In some embodiments, the second TIL expansion can proceed for about 13 to about 14 days. In some embodiments, the second TIL expansion can proceed for about 14 days.

[0158] In some embodiments, the second expansion can be performed in a gas-permeable container using the methods of the present disclosure (e.g., expansion referred to as REP, including the process shown in step D of FIG. 1). For example, TILs can be rapidly expanded using nonspecific T cell receptor stimulation in the presence of interleukin-2 (IL-2) or interleukin-15 (IL-15). Nonspecific T cell receptor stimulation can include, for example, an anti-CD3 antibody, such as about 30 ng / mL OKT3, a mouse monoclonal anti-CD3 antibody (commercially available from Ortho-McNeil, Raritan, NJ or Miltenyi Biotech, Auburn, CA), or UHCT-1 (commercially available from BioLegend, San Diego, CA, USA). TILs can be expanded in vitro to induce further stimulation of TILs by including one or more antigens during the second expansion, including an antigenic moiety, such as a cancer epitope(s), which can be expressed from a vector, e.g., a human leukocyte antigen A2 (HLA-A2)-binding peptide, e.g., 0.3 μM MART-1:26-35(27L) or gpl 00:209-217(210M), optionally in the presence of a T cell growth factor, such as 300 IU / mL IL-2 or IL-15. Other suitable antigens can include, for example, NY-ESO-1, TRP-1, TRP-2, tyrosinase cancer antigen, MAGE-A3, SSX-2, and VEGFR2, or antigenic moieties thereof. TILs can also be rapidly expanded by restimulating with the same cancer antigen(s) pulsed onto antigen-presenting cells expressing HLA-A2. Alternatively, TILs can be further restimulated, e.g., with, for example, irradiated autologous lymphocytes or irradiated HLA-A2+ allogeneic lymphocytes and IL-2. In some embodiments, the restimulation occurs as part of a second expansion. In some embodiments, the second expansion occurs in the presence of irradiated autologous lymphocytes or with irradiated HLA-A2+ allogeneic lymphocytes and IL-2.

[0159] In some embodiments, the cell culture medium further comprises IL-2. In some embodiments, the cell culture medium comprises about 3000 IU / mL of IL-2. In some embodiments, the cell culture medium comprises about 1000 IU / mL, about 1500 IU / mL, about 2000 IU / mL, about 2500 IU / mL, about 3000 IU / mL, about 3500 IU / mL, about 4000 IU / mL, about 4500 IU / mL, about 5000 IU / mL, about 5500 IU / mL, about 6000 IU / mL, about 6500 IU / mL, about 7000 IU / mL, about 7500 IU / mL, or about 8000 IU / mL of IL-2. In some embodiments, the cell culture medium contains 1000-2000 IU / mL, 2000-3000 IU / mL, 3000-4000 IU / mL, 4000-5000 IU / mL, 5000-6000 IU / mL, 6000-7000 IU / mL, 7000-8000 IU / mL, or 8000 IU / mL of IL-2.

[0160] In some embodiments, the cell culture medium comprises an OKT-3 antibody. In some embodiments, the cell culture medium comprises about 30 ng / mL of the OKT-3 antibody. In some embodiments, the cell culture medium comprises about 0.1 ng / mL, about 0.5 ng / mL, about 1 ng / mL, about 2.5 ng / mL, about 5 ng / mL, about 7.5 ng / mL, about 10 ng / mL, about 15 ng / mL, about 20 ng / mL, about 25 ng / mL, about 30 ng / mL, about 35 ng / mL, about 40 ng / mL, about 50 ng / mL, about 60 ng / mL, about 70 ng / mL, about 80 ng / mL, about 90 ng / mL, about 100 ng / mL, about 200 ng / mL, about 500 ng / mL, or about 1 μg / mL of the OKT-3 antibody. In some embodiments, the cell culture medium comprises an OKT-3 antibody at a concentration of 0.1 ng / mL to 1 ng / mL, 1 ng / mL to 5 ng / mL, 5 ng / mL to 10 ng / mL, 10 ng / mL to 20 ng / mL, 20 ng / mL to 30 ng / mL, 30 ng / mL to 40 ng / mL, 40 ng / mL to 50 ng / mL, or 50 ng / mL to 100 ng / mL. In some embodiments, the cell culture medium does not contain an OKT-3 antibody. In some embodiments, the OKT-3 antibody is muromonab.

[0161] In some embodiments, the cell culture medium comprises one or more TNFRSF agonists in the cell culture medium. In some embodiments, the TNFRSF agonist comprises a 4-1BB agonist. In some embodiments, the TNFRSF agonist is a 4-1BB agonist, and the 4-1BB agonist is selected from the group consisting of urelumab, utomilumab, EU-101, fusion proteins, and fragments, derivatives, variants, biosimilars, and combinations thereof. In some embodiments, the TNFRSF agonist is added at a concentration sufficient to achieve a concentration in the cell culture medium of 0.1 μg / mL to 100 μg / mL. In some embodiments, the TNFRSF agonist is added at a concentration sufficient to achieve a concentration in the cell culture medium of 20 μg / mL to 40 μg / mL.

[0162] In some embodiments, in addition to the one or more TNFRSF agonists, the cell culture medium further comprises IL-2 at an initial concentration of about 3000 IU / mL and OKT-3 at an initial concentration of about 30 ng / mL, and the one or more TNFRSF agonists comprise a 4-1BB agonist.

[0163] In some embodiments, a combination of IL-2, IL-7, IL-15, and / or IL-21 is used as a combination during the second expansion. In some embodiments, IL-2, IL-7, IL-15, and / or IL-21, and any combination thereof, may be included during the second expansion, including, for example, during step D of the process according to Figure 1 and described herein. In some embodiments, a combination of IL-2, IL-15, and IL-21 is used as a combination during the second expansion. In some embodiments, IL-2, IL-15, and IL-21, and any combination thereof, may be included during the process according to Figure 1 and described herein.

[0164] In some embodiments, the second expansion may be performed in a supplemented cell culture medium comprising IL-2, OKT-3, antigen-presenting feeder cells, and optionally a TNFRSF agonist. In some embodiments, the second expansion occurs in a supplemented cell culture medium. In some embodiments, the supplemented cell culture medium comprises IL-2, OKT-3, and antigen-presenting feeder cells. In some embodiments, the second cell culture medium comprises IL-2, OKT-3, and antigen-presenting cells (APCs, also referred to as antigen-presenting feeder cells). In some embodiments, the second expansion occurs in a cell culture medium comprising IL-2, OKT-3, and antigen-presenting feeder cells (i.e., antigen-presenting cells).

[0165] In some embodiments, the second expansion culture medium contains about 500 IU / mL IL-15, about 400 IU / mL IL-15, about 300 IU / mL IL-15, about 200 IU / mL IL-15, about 180 IU / mL IL-15, about 160 IU / mL IL-15, about 140 IU / mL IL-15, about 120 IU / mL IL-15, or about 100 IU / mL IL-15. In some embodiments, the second expansion culture medium contains about 500 IU / mL to about 100 IU / mL IL-15. In some embodiments, the second expansion culture medium contains about 400 IU / mL to about 100 IU / mL IL-15. In some embodiments, the second expansion culture medium comprises about 300 IU / mL to about 100 IU / mL of IL-15. In some embodiments, the second expansion culture medium comprises about 200 IU / mL of IL-15. In some embodiments, the cell culture medium comprises about 180 IU / mL of IL-15. In some embodiments, the cell culture medium further comprises IL-15. In some embodiments, the cell culture medium comprises about 180 IU / mL of IL-15.

[0166] In some embodiments, the second expansion culture medium comprises about 20 IU / mL IL-21, about 15 IU / mL IL-21, about 12 IU / mL IL-21, about 10 IU / mL IL-21, about 5 IU / mL IL-21, about 4 IU / mL IL-21, about 3 IU / mL IL-21, about 2 IU / mL IL-21, about 1 IU / mL IL-21, or about 0.5 IU / mL IL-21. In some embodiments, the second expansion culture medium comprises about 20 IU / mL to about 0.5 IU / mL IL-21. In some embodiments, the second expansion culture medium comprises about 15 IU / mL to about 0.5 IU / mL IL-21. In some embodiments, the second expansion culture medium comprises about 12 IU / mL to about 0.5 IU / mL of IL-21. In some embodiments, the second expansion culture medium comprises about 10 IU / mL to about 0.5 IU / mL of IL-21. In some embodiments, the second expansion culture medium comprises about 5 IU / mL to about 1 IU / mL of IL-21. In some embodiments, the second expansion culture medium comprises about 2 IU / mL of IL-21. In some embodiments, the cell culture medium comprises about 1 IU / mL of IL-21. In some embodiments, the cell culture medium comprises about 0.5 IU / mL of IL-21. In some embodiments, the cell culture medium further comprises IL-21. In some embodiments, the cell culture medium comprises about 1 IU / mL of IL-21.

[0167] In some embodiments, the antigen-presenting feeder cells (APCs) are PBMCs. In some embodiments, the ratio of TILs to PBMCs and / or antigen-presenting cells during rapid expansion and / or secondary expansion is about 1:25, about 1:50, about 1:100, about 1:125, about 1:150, about 1:175, about 1:200, about 1:225, about 1:250, about 1:275, about 1:300, about 1:325, about 1:350, about 1:375, about 1:400, or about 1:500. In some embodiments, the ratio of TILs to PBMCs during rapid expansion and / or secondary expansion is 1:50 to 1:300. In some embodiments, the ratio of TILs to PBMCs during rapid expansion and / or secondary expansion is 1:100 to 1:200.

[0168] In some embodiments, REP and / or secondary expansion is performed in flasks in which bulk TILs are mixed with a 100-fold or 200-fold excess of inactivated feeder cells, 30 mg / mL OKT3 anti-CD3 antibody, and 3000 IU / mL IL-2 in 150 mL of medium. Medium changes are performed (typically a two-thirds medium change by breathing in fresh medium) until the cells are transferred to an alternative growth chamber. Alternative growth chambers include G-REX flasks and gas-permeable vessels, as discussed more fully below.

[0169] In some embodiments, as discussed in the Examples and Figures, the second expansion (which may include a process referred to as the REP process) is shortened to 7-14 days, hi some embodiments, the second expansion is shortened to 11 days.

[0170] In some embodiments, REP and / or second expansion may be performed using previously described T-175 flasks and gas-permeable bags (Tran, et al., J. Immunother. 2008, 31, 742-51; Dudley, et al., J. Immunother. 2003, 26, 332-42) or gas-permeable cultureware (G-REX flasks). In some embodiments, second expansion (including expansion referred to as rapid expansion) is performed in T-175 flasks, containing approximately 1 x 10 cells suspended in 150 mL of medium. 6 TILs can be added to each T-175 flask. TILs can be cultured in a 1:1 mixture of CM and AIM-V medium supplemented with 3000 IU / mL IL-2 and 30 ng / mL anti-CD3. T-175 flasks can be incubated at 37°C in 5% CO2. Half of the medium can be replaced on day 5 using 50 / 50 medium containing 3000 IU / mL IL-2. In some embodiments, on day 7, cells from two T-175 flasks can be combined in a 3L bag, and 300 mL of AIM-V containing 5% human AB serum and 3000 IU / mL IL-2 was added to the 300 mL TIL suspension. The number of cells in each bag can be counted daily or every other day, and fresh medium can be added to increase the number of cells to 0.5-2.0 x 10. 6 The cell count was maintained between 100 and 150 cells / mL.

[0171] In some embodiments, the second expansion (which may include the expansion referred to as REP, as well as the expansion referenced in step D of FIG. 1) may be performed in a 500 mL gas-permeable flask with a 100 cm gas-permeable silicone bottom (G-REX-100, commercially available from Wilson Wolf Manufacturing Corporation, New Brighton, MN, USA). 6 or 10 x 10 6TILs can be cultured with PBMCs in 400 mL of 50 / 50 medium supplemented with 5% human AB serum, 3000 IU / mL IL-2, and 30 ng / mL anti-CD3 (OKT3). The G-REX-100 flask can be incubated at 37°C in 5% CO2. On day 5, 250 mL of supernatant can be removed, placed in a centrifuge bottle, and centrifuged at 1500 rpm (491 x g) for 10 minutes. The TIL pellet can be resuspended in 150 mL of fresh medium containing 5% human AB serum, 3000 IU / mL IL-2, and added back to the original G-REX-100 flask. If TILs are continuously expanded in G-REX-100 flasks, on day 7, the TILs in each G-REX-100 flask can be suspended in 300 mL of medium present in each flask, and the cell suspension can be divided into three 100 mL aliquots, which can be used to seed three G-REX-100 flasks. Then, 150 mL of AIM-V containing 5% human AB serum and 3000 IU / mL IL-2 can be added to each flask. The G-REX-100 flasks can be incubated at 37°C in 5% CO2, and after 4 days, 150 mL of AIM-V containing 3000 IU / mL IL-2 can be added to each G-REX-100 flask. On day 14 of culture, the cells can be harvested.

[0172] In some embodiments, the second expansion (including expansion referred to as REP) is performed in flasks where bulk TILs are mixed with a 100-fold or 200-fold excess of inactivated feeder cells, 30 mg / mL OKT3 anti-CD3 antibody, and 3000 IU / mL IL-2 in 150 mL of medium. In some embodiments, medium changes are performed until the cells are transferred to an alternative growth chamber. In some embodiments, two-thirds of the medium is exchanged with fresh medium by respiration. In some embodiments, the alternative growth chamber includes G-REX flasks and gas-permeable vessels, as discussed more fully below.

[0173] In some embodiments, a second expansion (including expansion referred to as REP) is performed, further comprising selecting TILs for superior tumor reactivity. Any selection method known in the art may be used. For example, the method described in U.S. Patent Application Publication No. 2016 / 0010058A1 (the disclosure of which is incorporated herein by reference) may be used to select TILs for superior tumor reactivity.

[0174] Optionally, a cell viability assay can be performed after the second expansion (including expansion referred to as REP expansion) using standard assays known in the art. For example, a trypan blue exclusion assay, which selectively labels dead cells and allows for assessment of viability, can be performed on a sample of bulk TILs. In some embodiments, TIL samples can be counted using a Cellometer K2 Automatic Cell Counter (Nexcelom Bioscience, Lawrence, MA) to determine viability. In some embodiments, viability is determined according to a standard Cellometer K2 Image Cytometer Automatic Cell Counter protocol.

[0175] In some embodiments, the second expansion of TILs (including expansion referred to as REP) can be performed using T-175 flasks and gas-permeable bags as previously described (Tran, et al., 2008, J Immunother., 31:742-751 and Dudley, et al. 2003, J Immunother., 26:332-342) or gas-permeable G-REX flasks. In some embodiments, the second expansion is performed using flasks. In some embodiments, the second expansion is performed using gas-permeable G-REX flasks. In some embodiments, the second expansion is performed in T-175 flasks and approximately 1 x 10 6TILs are suspended in approximately 150 mL of medium, which is added to each T-175 flask. The TILs are cultured with irradiated (50 Gy) allogeneic PBMCs at a 1:100 ratio as "feeder" cells, and the cells are cultured in a 1:1 mixture of CM and AIM-V medium (50 / 50 medium) supplemented with 3000 IU / mL IL-2 and 30 ng / mL anti-CD3. The T-175 flasks are incubated at 37°C in 5% CO. In some embodiments, half of the medium is replaced on day 5 using 50 / 50 medium containing 3000 IU / mL IL-2. In some embodiments, on day 7, cells from two T-175 flasks are combined in a 3 L bag, and 300 mL of AIM-V containing 5% human AB serum and 3000 IU / mL IL-2 is added to the 300 mL TIL suspension. The number of cells in each bag can be counted daily or every other day, and fresh medium can be added to obtain a cell count of about 0.5 to about 2.0 x 10 6 The cell count can be maintained at 100 cells / mL.

[0176] In some embodiments, the second extension (including the extension referred to as REP) is 100 cm 2 The experiment was carried out in a 500 mL flask with a gas-permeable silicon bottom (G-REX-100, Wilson Wolf), and approximately 5 × 10 6 or 10 x 10 6TILs are cultured with irradiated allogeneic PBMCs at a 1:100 ratio in 400 mL of 50 / 50 medium supplemented with 3000 IU / mL IL-2 and 30 ng / mL anti-CD3. The G-REX-100 flask is incubated at 37°C in 5% CO2. In some embodiments, on day 5, 250 mL of supernatant is removed, placed in a centrifuge bottle, and centrifuged at 1500 rpm (491 g) for 10 minutes. The TIL pellet can then be resuspended in 150 mL of fresh 50 / 50 medium containing 3000 IU / mL IL-2 and added back to the original G-REX-100 flask. In an embodiment in which TILs are continuously expanded in G-REX-100 flasks, on day 7, the TILs in each G-REX-100 flask are suspended in 300 mL of medium present in each flask, and the cell suspension is divided into three 100 mL aliquots that are used to seed three G-REX-100 flasks. Then, 150 mL of AIM-V containing 5% human AB serum and 3000 IU / mL of IL-2 is added to each flask. The G-REX-100 flasks are incubated at 37°C in 5% CO2, and after four days, 150 mL of AIM-V containing 3000 IU / mL of IL-2 is added to each G-REX-100 flask. On day 14 of culture, the cells are harvested.

[0177] The diverse antigen receptors of T and B lymphocytes are produced by somatic recombination of a limited number of gene segments. These gene segments: V (variable), D (diversity), J (joining), and C (constant) determine the binding specificity and downstream applications of immunoglobulins and T cell receptors (TCRs). The present invention provides methods for generating TILs that exhibit and increase T cell repertoire diversity. In some embodiments, TILs obtained by the methods exhibit increased T cell repertoire diversity. In some embodiments, TILs obtained in the second expansion exhibit increased T cell repertoire diversity. In some embodiments, the increased diversity is increased immunoglobulin diversity and / or T cell receptor diversity. In some embodiments, the diversity is in immunoglobulins and in immunoglobulin heavy chains. In some embodiments, the diversity is in immunoglobulins and in immunoglobulin light chains. In some embodiments, the diversity is in T cell receptors. In some embodiments, the diversity is in one of the T cell receptors selected from the group consisting of alpha, beta, gamma, and delta receptors. In some embodiments, expression of T cell receptor (TCR) alpha and / or beta is increased. In some embodiments, expression of T cell receptor (TCR) alpha is increased. In some embodiments, expression of T cell receptor (TCR) beta is increased. In some embodiments, expression of TCRab (i.e., TCRα / β) is increased.

[0178] In some embodiments, the second expansion culture medium (e.g., sometimes referred to as CM2 or second cell culture medium) comprises IL-2, OKT-3, and antigen-presenting feeder cells (APCs), as discussed in more detail below.

[0179] In some embodiments, the medium used in the expansion processes disclosed herein is a serum-free or synthetic medium. In some embodiments, the serum-free or synthetic medium comprises a basal cell culture medium and a serum supplement and / or serum replacement. In some embodiments, the serum-free or synthetic medium is used to prevent and / or reduce experimental variation due in part to lot-to-lot variation in serum-containing medium.

[0180] In some embodiments, the serum-free or defined medium comprises a basal cell culture medium and a serum supplement and / or serum replacement. In some embodiments, the basal cell culture medium includes, but is not limited to, CTS™ OpTmizer™ T-cell Expansion Basal Medium, CTS™ OpTmizer™ T-Cell Expansion SFM, CTS™ AIM-V Medium, CTS™ AIM-V SFM, LymphoONE™ T-Cell Expansion Xeno-Free Medium, Dulbecco's Modified Eagle's Medium (DMEM), Minimum Essential Medium (MEM), Basal Eagle's Medium (BME), RPMI 1640, F-10, F-12, Minimum Essential Medium (αMEM), Glasgow Minimum Essential Medium (G-MEM), RPMI Growth Medium, and Iscove's Modified Dulbecco's Medium.

[0181] In some embodiments, the serum supplement or serum replacement includes, but is not limited to, CTS™ OpTmizer T-Cell Expansion Serum Supplement, CTS™ Immune Cell Serum Replacement, one or more albumins or albumin substitutes, one or more amino acids, one or more vitamins, one or more transferrins or transferrin substitutes, one or more antioxidants, one or more insulins or insulin substitutes, one or more collagen precursors, one or more antibiotics, and one or more trace elements. In some embodiments, the synthetic medium contains albumin and one or more of glycine, L-histidine, L-isoleucine, L-methionine, L-phenylalanine, L-proline, L-hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, thiamine, reduced glutathione, L-ascorbic acid-2-phosphate, iron-saturated transferrin, insulin, and trace element moieties. + , Al 3+ , Ba 2+ , Cd 2+ , Co 2+ , Cr 3+ , Ge 4+ , Se 4+ , Br, T, Mn 2+ , P, Si 4+ , V 5+ , Mo 6+ , Ni 2+ , Rb + , Sn 2+ , and Zr 4+ and one or more components selected from the group consisting of compounds containing: In some embodiments, the synthetic medium further comprises L-glutamine, sodium bicarbonate, and / or 2-mercaptoethanol.

[0182] In some embodiments, CTS™ OpTmizer™ T-cell Immune Cell Serum Replacement is used with conventional growth media, including, but not limited to, CTS™ OpTmizer™ T-cell Expansion Basal Medium, CTS™ OpTmizer™ T-cell Expansion SFM, CTS™ AIM-V Medium, CST™ AIM-V SFM, LymphoONE™ T-Cell Expansion Xeno-Free Medium, Dulbecco's Modified Eagle's Medium (DMEM), Minimum Essential Medium (MEM), Basal Eagle's Medium (BME), RPMI 1640, F-10, F-12, Minimum Essential Medium (αMEM), Glasgow Minimum Essential Medium (G-MEM), RPMI Growth Medium, and Iscove's Modified Dulbecco's Medium.

[0183] In some embodiments, the total serum replacement concentration (vol%) in the serum-free or synthetic medium is about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% by volume of the total serum-free or synthetic medium. In some embodiments, the total serum replacement concentration is about 3% of the total volume of the serum-free or synthetic medium. In some embodiments, the total serum replacement concentration is about 5% of the total volume of the serum-free or synthetic medium. In some embodiments, the total serum replacement concentration is about 10% of the total volume of the serum-free or synthetic medium.

[0184] In some embodiments, the serum-free or defined medium is CTS™ OpTmizer™ T-cell Expansion SFM (ThermoFisher Scientific). Any formulation of CTS™ OpTmizer™ is useful in the present invention. CTS™ OpTmizer™ T-cell Expansion SFM is a combination of 1 L of CTS™ OpTmizer™ T-cell Expansion Basal Medium and 26 mL of CTS™ OpTmizer™ T-Cell Expansion Supplement, which are mixed together before use. In some embodiments, CTS™ OpTmizer™ T-cell Expansion SFM is supplemented with approximately 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific). In some embodiments, CTS™ OpTmizer™ T-cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) along with 55 mM 2-mercaptoethanol. In some embodiments, CTS™ OpTmizer™ T-cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and the final concentration of 2-mercaptoethanol in the medium is 55 μM.

[0185] In some embodiments, the defined medium is CTS™ OpTmizer™ T-cell Expansion SFM (ThermoFisher Scientific). Any formulation of CTS™ OpTmizer™ is useful in the present invention. CTS™ OpTmizer™ T-cell Expansion SFM is a combination of 1 L of CTS™ OpTmizer™ T-cell Expansion Basal Medium and 26 mL of CTS™ OpTmizer™ T-Cell Expansion Supplement, which are mixed together before use. In some embodiments, CTS™ OpTmizer™ T-cell Expansion SFM is supplemented with approximately 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) along with 55 mM 2-mercaptoethanol. In some embodiments, CTS™ OpTmizer™ T-cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific), 55 mM 2-mercaptoethanol, and 2 mM L-glutamine. In some embodiments, CTS™ OpTmizer™ T-cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific), 55 mM 2-mercaptoethanol, and 2 mM L-glutamine, and further comprises about 1000 IU / mL to about 8000 IU / mL of IL-2. In some embodiments, CTS™ OpTmizer™ T-cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific), 55 mM 2-mercaptoethanol, and 2 mM L-glutamine, and further contains about 3000 IU / mL of IL-2.In some embodiments, CTS™ OpTmizer™ T-cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific), 55 mM 2-mercaptoethanol, and 2 mM L-glutamine, and further comprises about 6000 IU / mL of IL-2. In some embodiments, CTS™ OpTmizer™ T-cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and 55 mM 2-mercaptoethanol, and further comprises about 1000 IU / mL to about 8000 IU / mL of IL-2. In some embodiments, CTS™ OpTmizer™ T-cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and 55 mM 2-mercaptoethanol, and further comprises about 3000 IU / mL of IL-2. In some embodiments, CTS™ OpTmizer™ T-cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and 55 mM 2-mercaptoethanol, and further comprises about 1000 IU / mL to about 6000 IU / mL of IL-2. In some embodiments, CTS™ OpTmizer™ T-cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and about 2 mM glutamine, and further contains about 1000 IU / mL to about 8000 IU / mL of IL-2.In some embodiments, the CTS™ OpTmizer™ T-cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and about 2 mM glutamine, and further comprises about 3000 IU / mL of IL-2. In some embodiments, the CTS™ OpTmizer™ T-cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and about 2 mM glutamine, and further comprises about 6000 IU / mL of IL-2. In some embodiments, the CTS™ OpTmizer™ T-cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific), and the final concentration of 2-mercaptoethanol in the medium is 55 μM.

[0186] In some embodiments, serum-free or synthetic media is supplemented with glutamine (i.e., GlutaMAX®) at a concentration of about 0.1 mM to about 10 mM, 0.5 mM to about 9 mM, 1 mM to about 8 mM, 2 mM to about 7 mM, 3 mM to about 6 mM, or 4 mM to about 5 mM. In some embodiments, serum-free or synthetic media is supplemented with glutamine (i.e., GlutaMAX®) at a concentration of about 2 mM.

[0187] In some embodiments, serum-free or synthetic medium is supplemented with 2-mercaptoethanol at a concentration of about 5 mM to about 150 mM, 10 mM to about 140 mM, 15 mM to about 130 mM, 20 mM to about 120 mM, 25 mM to about 110 mM, 30 mM to about 100 mM, 35 mM to about 95 mM, 40 mM to about 90 mM, 45 mM to about 85 mM, 50 mM to about 80 mM, 55 mM to about 75 mM, 60 mM to about 70 mM, or about 65 mM. In some embodiments, serum-free or synthetic medium is supplemented with 2-mercaptoethanol at a concentration of about 55 mM. In some embodiments, the final concentration of 2-mercaptoethanol in the medium is 55 μM.

[0188] In some embodiments, the synthetic media described in International PCT Publication No. WO / 1998 / 030679, incorporated herein by reference, are useful in the present invention. That publication describes serum-free eukaryotic cell culture media. The serum-free eukaryotic cell culture media include basal cell culture media supplemented with serum-free supplements capable of supporting cell growth in serum-free culture. The serum-free eukaryotic cell culture medium supplement comprises, or is obtained by combining, one or more components selected from the group consisting of one or more albumins or albumin substitutes, one or more amino acids, one or more vitamins, one or more transferrins or transferrin substitutes, one or more antioxidants, one or more insulins or insulin substitutes, one or more collagen precursors, one or more trace elements, and one or more antibiotics. In some embodiments, the synthetic media further comprise L-glutamine, sodium bicarbonate, and / or beta-mercaptoethanol. In some embodiments, the synthetic medium comprises albumin or an albumin substitute and one or more components selected from the group consisting of one or more amino acids, one or more vitamins, one or more transferrin or transferrin substitutes, one or more antioxidants, one or more insulin or insulin substitutes, one or more collagen precursors, and one or more trace elements. In some embodiments, the synthetic medium comprises albumin and one or more components selected from the group consisting of glycine, L-histidine, L-isoleucine, L-methionine, L-phenylalanine, L-proline, L-hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, thiamine, reduced glutathione, L-ascorbic acid-2-phosphate, iron-saturated transferrin, insulin, and the trace element moiety Ag. + , Al 3+ , Ba 2+ , Cd 2+ , Co 2+ , Cr 3+ , Ge 4+ , Se 4+ , Br, T, Mn 2+ , P, Si 4+ , V 5+ , Mo 6+ , Ni 2+ , Rb+ , Sn 2+ , and Zr 4+ and one or more components selected from the group consisting of compounds containing: In some embodiments, the basal cell culture medium is selected from the group consisting of Dulbecco's Modified Eagle's Medium (DMEM), Minimum Essential Medium (MEM), Basal Eagle's Medium (BME), RPMI 1640, F-10, F-12, Minimum Essential Medium (αMEM), Glasgow Minimum Essential Medium (G-MEM), RPMI Growth Medium, and Iscove's Modified Dulbecco's Medium.

[0189] In some embodiments, the concentration of glycine in the synthetic medium ranges from about 5 to 200 mg / L, the concentration of L-histidine from about 5 to 250 mg / L, the concentration of L-isoleucine from about 5 to 300 mg / L, the concentration of L-methionine from about 5 to 200 mg / L, the concentration of L-phenylalanine from about 5 to 400 mg / L, the concentration of L-proline from about 1 to 1000 mg / L, the concentration of L-hydroxyproline from about 1 to 45 mg / L, the concentration of L-serine from about 1 to 250 mg / L, the concentration of L-threonine from about 10 to 500 mg / L, and the concentration of L-tryptophan from about 2 to 110 mg / L. / L, the concentration of L-tyrosine is about 3 to 175 mg / L, the concentration of L-valine is about 5 to 500 mg / L, the concentration of thiamine is about 1 to 20 mg / L, the concentration of reduced glutathione is about 1 to 20 mg / L, the concentration of L-ascorbic acid 2-phosphate is about 1 to 200 mg / L, the concentration of iron-saturated transferrin is about 1 to 50 mg / L, the concentration of insulin is about 1 to 100 mg / L, the concentration of sodium selenite is about 0.000001 to 0.0001 mg / L, and the concentration of albumin (e.g., AlbuMAX® I) is about 5,000 to 50,000 mg / L.

[0190] In some embodiments, the non-trace element components in the defined medium are present in the concentration ranges listed in the column under the heading "Concentration Ranges in 1x Medium" in Table 4. In other embodiments, the non-trace element components in the defined medium are present at the final concentrations listed in the column under the heading "Preferred Embodiments of 1x Medium" in Table 4. In other embodiments, the defined medium is a basal cell culture medium that includes a serum-free supplement. In some of these embodiments, the serum-free supplement includes non-trace element components of the type and concentration listed in the column under the heading "Preferred Embodiments of Supplement" in Table 4.

[0191] In some embodiments, the osmolality of the synthetic medium is about 260-350 mOsmol. In some embodiments, the osmolality is about 280-310 mOsmol. In some embodiments, the synthetic medium is supplemented with sodium bicarbonate at up to about 3.7 g / L, or about 2.2 g / L. The synthetic medium may be further supplemented with L-glutamine (final concentration about 2 mM), one or more antibiotics, non-essential amino acids (NEAA, final concentration about 100 μM), and 2-mercaptoethanol (final concentration about 100 μM).

[0192] In some embodiments, the defined media described in Smith, et al., Clin Transl Immunology, 4(1) 2015 (doi:10.1038 / cti.2014.31) are useful in the present invention. Briefly, RPMI or CTS™ OpTmizer™ was used as the basal cell culture medium, supplemented with either 0, 2%, 5%, or 10% CTS™ Immune Cell Serum Replacement.

[0193] In some embodiments, the cell culture medium in the first and / or second gas-permeable container is unfiltered. The use of unfiltered cell culture medium can simplify the procedures required to expand the number of cells. In some embodiments, the cell culture medium in the first and / or second gas-permeable container lacks beta-mercaptoethanol (BME or βME, also known as 2-mercaptoethanol, CAS 60-24-2).

[0194] In some embodiments, the second expansion, e.g., step D according to FIG. 1, is performed in a closed system bioreactor. In some embodiments, a closed system is used for TIL expansion as described herein. In some embodiments, a single bioreactor is used. In some embodiments, the single bioreactor used is, for example, a G-REX-10 or G-REX-100. In some embodiments, the closed system bioreactor is a single bioreactor.

[0195] In some embodiments, the rapid or secondary expansion step is divided into multiple steps, and the scale-up of the culture is achieved by (a) performing rapid or secondary expansion by culturing the TILs in small-scale culture in a first vessel, e.g., a G-REX-100 MCS vessel, for about 3-7 days, and then (b) transferring the TILs in the small-scale culture to a second vessel, e.g., a G-REX-500-MCS vessel, that is larger than the first vessel, and culturing the TILs from the small-scale culture in a larger culture in the second vessel for about 4-7 days.

[0196] In some embodiments, the rapid or second expansion step is divided into multiple steps, and the rapid or second expansion step is achieved by (a) culturing the TILs in a first small-scale culture in a first vessel, e.g., a G-REX-100MCS vessel, for about 3-7 days, and then (b) transferring and distributing the TILs from the first small-scale culture into at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 second vessels equal in size to the first vessel, and in each second vessel, a portion of the transferred TILs from the first small-scale culture are cultured in a second small-scale culture for about 4-7 days.

[0197] In some embodiments, the first small-scale TIL culture is distributed into a plurality of about 2-5 subpopulations of TILs.

[0198] In some embodiments, the rapid or secondary expansion step is divided into multiple steps to achieve scale-out and scale-up of the culture by (a) culturing the TILs in small-scale culture in a first vessel, e.g., a G-REX-100MCS vessel, for about 3-7 days, and then (b) transferring and distributing the TILs from the small-scale culture into at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 second vessels, e.g., a G-REX-500MCS vessel, that are larger in size than the first vessel, and culturing a portion of the TILs from the small-scale culture transferred to such second vessel in a larger scale for about 4-7 days in each second vessel.

[0199] In some embodiments, the rapid or secondary expansion step is divided into multiple steps, and the rapid or secondary expansion step is achieved by (a) culturing the TILs in a small-scale culture in a first vessel, e.g., a G-REX-100MCS vessel, for about 5 days, and then (b) transferring and distributing the TILs from the small-scale culture into two, three, or four second vessels, e.g., a G-REX-500MCS vessel, that are larger in size than the first vessel, thereby achieving scale-out and scale-up of the culture, and in each second vessel, a portion of the TILs transferred from the small-scale culture to such second vessel are cultured in a larger culture for about 6 days.

[0200] In some embodiments, upon division of the rapid or second expansion, each second container contains at least 10 8 In some embodiments, upon division of the rapid or second expansion, each second vessel contains at least 10 TILs. 8 TIL, at least 10 9 TIL, or at least 10 10 In one exemplary embodiment, each second container contains at least 10 TILs. 10 Contains TILs.

[0201] In some embodiments, the first small-scale TIL culture is allocated into a plurality of subpopulations. In some embodiments, the first small-scale TIL culture is allocated into a plurality of about 2-5 subpopulations. In some embodiments, the first small-scale TIL culture is allocated into a plurality of about 2, 3, 4, or 5 subpopulations.

[0202] In some embodiments, after completion of rapid or secondary expansion, the plurality of subpopulations comprises a therapeutically effective amount of TILs. In some embodiments, after completion of rapid or secondary expansion, one or more subpopulations of TILs are pooled together to produce a therapeutically effective amount of TILs. In some embodiments, after completion of rapid expansion, each subpopulation of TILs comprises a therapeutically effective amount of TILs.

[0203] In some embodiments, the rapid or secondary expansion is carried out for about 3-7 days before being divided into multiple steps. In some embodiments, the division of the rapid or secondary expansion occurs on about day 3, day 4, day 5, day 6, or day 7 after the initiation of the rapid or secondary expansion.

[0204] In some embodiments, the division of the rapid or second expansion occurs on about day 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16, 17, or 18 after the initiation of the first expansion (i.e., pre-REP expansion). In one exemplary embodiment, the division of the rapid or second expansion occurs on about day 16 after the initiation of the first expansion.

[0205] In some embodiments, the rapid or secondary expansion is carried out for an additional 7-11 days after division, hi some embodiments, the rapid or secondary expansion is carried out for an additional 5, 6, 7, 8, 9, 10, or 11 days after division.

[0206] In some embodiments, the cell culture medium used for the rapid or second expansion before division contains the same components as the cell culture medium used for the rapid or second expansion after division. In some embodiments, the cell culture medium used for the rapid or second expansion before division contains different components than the cell culture medium used for the rapid or second expansion after division.

[0207] In some embodiments, the cell culture medium used for rapid or second expansion before division comprises IL-2, optionally OKT-3, and further optionally APC. In some embodiments, the cell culture medium used for rapid or second expansion before division comprises IL-2, OKT-3, and further optionally APC. In some embodiments, the cell culture medium used for rapid or second expansion before division comprises IL-2, OKT-3, and APC.

[0208] In some embodiments, cell culture medium used for rapid or second expansion before division is produced by supplementing cell culture medium during first expansion with fresh culture medium containing IL-2, optionally OKT-3, and further optionally APCs. In some embodiments, cell culture medium used for rapid or second expansion before division is produced by supplementing cell culture medium during first expansion with fresh culture medium containing IL-2, OKT-3, and APCs. In some embodiments, cell culture medium used for rapid or second expansion before division is produced by replacing cell culture medium during first expansion with fresh culture medium containing IL-2, optionally OKT-3, and further optionally APCs. In some embodiments, cell culture medium used for rapid or second expansion before division is produced by replacing cell culture medium during first expansion with fresh cell culture medium containing IL-2, OKT-3, and further optionally APCs.

[0209] In some embodiments, the cell culture medium used for rapid or second expansion after division comprises IL-2 and optionally OKT-3. In some embodiments, the cell culture medium used for rapid or second expansion after division comprises IL-2 and OKT-3. In some embodiments, the cell culture medium used for rapid or second expansion after division is generated by replacing the cell culture medium used for rapid or second expansion before division with fresh culture medium comprising IL-2 and optionally OKT-3. In some embodiments, the cell culture medium used for rapid or second expansion after division is generated by replacing the cell culture medium used for rapid or second expansion before division with fresh culture medium comprising IL-2 and OKT-3.

[0210] In some embodiments, the rapid expansion disruption occurs in a closed system.

[0211] In some embodiments, scaling up TIL cultures during rapid or secondary expansion involves adding fresh cell culture medium to the TIL culture (also referred to as feeding the TILs). In some embodiments, feeding involves frequently adding fresh cell culture medium to the TIL culture. In some embodiments, feeding involves adding fresh cell culture medium to the TIL culture at regular intervals. In some embodiments, fresh cell culture medium is supplied to the TILs via a constant flow. In some embodiments, an automated cell expansion system, such as the Xuri W25, is used for rapid expansion and feeding.

[0212] 1. Feeder cells and antigen-presenting cells In some embodiments, the second expansion procedures described herein (including, for example, expansion as described in step D of FIG. 1, and referred to as REP) require excess feeder cells during REP TIL expansion and / or during the second expansion. In many embodiments, the feeder cells are peripheral blood mononuclear cells (PBMCs) obtained from a standard whole blood unit from a healthy blood donor. PBMCs are obtained using standard methods, such as Ficoll-Paque gradient separation.

[0213] Generally, allogeneic PBMCs are inactivated by either irradiation or heat treatment and used in the REP procedure, as described in the Examples, which provide an exemplary protocol for assessing the replicative incompetence of irradiated allogeneic PBMCs.

[0214] In some embodiments, if the total number of viable cells on day 14 is less than the initial number of viable cells cultured on day 0 of REP and / or day 0 of second expansion (i.e., the start day of second expansion), the PBMCs are considered replication-incompetent and are approved for use in the TIL expansion procedures described herein.

[0215] In some embodiments, if the total number of viable cells cultured in the presence of OKT3 and IL-2 on days 7 and 14 does not increase from the initial number of viable cells cultured on REP day 0 and / or second expansion day 0 (i.e., the start day of second expansion), the PBMCs are considered replication-incompetent and are approved for use in the TIL expansion procedures described herein. In some embodiments, PBMCs are cultured in the presence of 30 ng / mL OKT3 antibody and 3000 IU / mL IL-2.

[0216] In some embodiments, if the total number of viable cells cultured in the presence of OKT3 and IL-2 on days 7 and 14 does not increase from the initial number of viable cells cultured on day 0 of REP and / or day 0 of second expansion (i.e., the start day of second expansion), the PBMCs are considered replication-incompetent and are approved for use in the TIL expansion procedures described herein. In some embodiments, PBMCs are cultured in the presence of 5-60 ng / mL OKT3 antibody and 1000-6000 IU / mL IL-2. In some embodiments, PBMCs are cultured in the presence of 10-50 ng / mL OKT3 antibody and 2000-5000 IU / mL IL-2. In some embodiments, PBMCs are cultured in the presence of 20-40 ng / mL OKT3 antibody and 2000-4000 IU / mL IL-2. In some embodiments, PBMCs are cultured in the presence of 25 to 35 ng / mL of OKT3 antibody and 2500 to 3500 IU / mL of IL-2.

[0217] In some embodiments, the antigen-presenting feeder cells are PBMCs. In some embodiments, the antigen-presenting feeder cells are artificial antigen-presenting feeder cells. In some embodiments, the ratio of TILs to antigen-presenting feeder cells in the second expansion is about 1:25, about 1:50, about 1:100, about 1:125, about 1:150, about 1:175, about 1:200, about 1:225, about 1:250, about 1:275, about 1:300, about 1:325, about 1:350, about 1:375, about 1:400, or about 1:500. In some embodiments, the ratio of TILs to antigen-presenting feeder cells in the second expansion is 1:50 to 1:300. In some embodiments, the ratio of TILs to antigen-presenting feeder cells in the second expansion is 1:100 to 1:200.

[0218] In some embodiments, the second expansion procedure described herein is performed at a concentration of about 2.5×10 9 Feeder cells: approximately 100 x 10 6 In other embodiments, the second expansion procedure described herein requires a ratio of about 2.5 x 10 TILs. 9 Feeder cells: approximately 50 x 10 6 In yet another embodiment, the second expansion procedure described herein requires a ratio of about 2.5 x 10 TILs. 9 Feeder cells: approximately 25 x 10 6 Requires TIL.

[0219] In some embodiments, the second expansion procedure described herein requires excess feeder cells during the second expansion. In many embodiments, the feeder cells are peripheral blood mononuclear cells (PBMCs) obtained from a standard whole blood unit from a healthy blood donor. PBMCs are obtained using standard methods, such as Ficoll-Paque gradient separation. In some embodiments, artificial antigen-presenting (aAPC) cells are used in place of PBMCs.

[0220] Generally, allogeneic PBMCs are inactivated by either irradiation or heat treatment and used in the TIL expansion procedures described herein, including the exemplary procedures described in the figures and examples.

[0221] In some embodiments, artificial antigen-presenting cells are used in the second expansion as a replacement for or in combination with PBMCs.

[0222] 2. Cytokines and other additives The expansion methods described herein generally use culture media containing high doses of cytokines, particularly IL-2, as known in the art.

[0223] Alternatively, the use of cytokine combinations for rapid and / or secondary expansion of TILs is additionally possible using combinations of two or more of IL-2, IL-15, and IL-21, as described in U.S. Patent Application Publication No. US2017 / 0107490A1 (the disclosure of which is incorporated herein by reference). Thus, possible combinations include IL-2 and IL-15, IL-2 and IL-21, IL-15 and IL-21, and IL-2, IL-15, and IL-21, the latter of which finds particular use in many embodiments. The use of cytokine combinations is particularly advantageous for the generation of lymphocytes, particularly T cells as described therein.

[0224] In some embodiments, step D may also include the addition of an OKT-3 antibody or muromonab to the culture medium, as described elsewhere herein. In some embodiments, step D may also include the addition of a 4-1BB agonist to the culture medium, as described elsewhere herein. In some embodiments, step D may also include the addition of an OX-40 agonist to the culture medium, as described elsewhere herein. In addition, additives such as peroxisome proliferator-activated receptor gamma coactivator I-alpha agonists, including proliferator-activated receptor (PPAR)-gamma agonists such as thiazolidinedione compounds, may be used in the culture medium during step D, as described in U.S. Patent Application Publication No. US2019 / 0307796 A1 (the disclosure of which is incorporated herein by reference).

[0225] E. Step E: Harvesting TILs After the second expansion step, the cells may be harvested. In some embodiments, the TILs are harvested after one, two, three, four, or more expansion steps, for example, as provided in Figure 1. In some embodiments, the TILs are harvested after two expansion steps, for example, as provided in Figure 1.

[0226] TILs can be collected by any suitable sterilization method, including, for example, centrifugation.The method for collecting TILs is well known in the art, and any such known method can be used in this process.In some embodiments, TILs are collected using an automated system.

[0227] Cell harvesters and / or cell processing systems are commercially available from a variety of sources, including, for example, Fresenius Kabi, Tomtec Life Science, Perkin Elmer, and Inotech Biosystems International, Inc. Any cell-based harvester can be used with the present methods. In some embodiments, the cell harvester and / or cell processing system is a membrane-based cell harvester. In some embodiments, cell harvesting is by a cell processing system such as the LOVO system (manufactured by Fresenius Kabi). The term "LOVO cell processing system" also refers to any device or apparatus manufactured by any vendor that pumps a cell-containing solution through a membrane or filter, such as a spinning membrane or spinning filter, in a sterile and / or closed environment, allowing continuous flow, processes the cells, and removes the supernatant or cell culture medium without pelleting. In some embodiments, the cell harvester and / or cell processing system can perform cell separation, washing, fluid exchange, concentration, and / or other cell processing steps within a sterile, closed system.

[0228] In some embodiments, harvesting, e.g., step E according to FIG. 1, is performed from a closed system bioreactor. In some embodiments, a closed system is used for TIL expansion as described herein. In some embodiments, a single bioreactor is used. In some embodiments, the single bioreactor used is, for example, a G-REX-10 or G-REX-100. In some embodiments, the closed system bioreactor is a single bioreactor.

[0229] In some embodiments, step E according to Figure 1 is performed according to a process described herein. In some embodiments, the closed system is accessed via a syringe under sterile conditions to maintain the sterility and closure of the closed system. In some embodiments, the closed system described in the Examples is used.

[0230] In some embodiments, TILs are harvested according to the methods described in the Examples. In some embodiments, TILs between days 1 and 11 are harvested using the methods described in the steps referred to herein, such as harvesting TILs on day 11 in the Examples. In some embodiments, TILs between days 12 and 24 are harvested using the methods described in the steps referred to herein, such as harvesting TILs on day 22 in the Examples. In some embodiments, TILs between days 12 and 22 are harvested using the methods described in the steps referred to herein, such as harvesting TILs on day 22 in the Examples.

[0231] F. Step F: Final formulation and transfer to infusion containers After completing steps A-E, provided in exemplary order in Figure 1 and outlined in detail above and herein, the cells are transferred to a container for use in patient administration, such as an infusion bag or sterile vial. In some embodiments, once therapeutically sufficient numbers of TILs are obtained using the expansion methods described above, they are transferred to a container for use in patient administration.

[0232] In some embodiments, TILs expanded using APCs of the present disclosure are administered to a patient as a pharmaceutical composition. In some embodiments, the pharmaceutical composition is a TIL suspension in a sterile buffer. TILs expanded using PBMCs of the present disclosure can be administered by any suitable route known in the art. In some embodiments, T cells are administered as a single intra-arterial or intravenous infusion, preferably lasting approximately 30-60 minutes. Other suitable routes of administration include intraperitoneal, intrathecal, and intralymphatic administration.

[0233] IV. Gen3 TIL Manufacturing Process Without being limited to any particular theory, it is believed that the first expansion by priming, which primes T cells for activation, followed by a second rapid expansion that promotes T cell activation, as described in the methods of the present invention, allows for the preparation of expanded T cells that retain a "younger" phenotype, and therefore, it is expected that the expanded T cells of the present invention will exhibit greater cytotoxicity against cancer cells than T cells expanded by other methods. In particular, it is believed that the activation of T cells primed by exposure to an anti-CD3 antibody (e.g., OKT-3), IL-2, and optionally antigen-presenting cells (APCs), followed by subsequent exposure to additional anti-CD3 antibody (e.g., OKT-3), IL-2, and APCs, as taught by the methods of the present invention, limits or avoids T cell maturation in culture, producing a T cell population with a less mature phenotype that is less exhausted by expansion in culture and exhibits greater cytotoxicity against cancer cells. In some embodiments, the rapid second expansion step is divided into multiple steps, and the scale-up of the culture is achieved by (a) performing the rapid second expansion by culturing the T cells in a small-scale culture in a first vessel, e.g., a G-REX-100 MCS vessel, for about 3-4 days, and then (b) transferring the T cells in the small-scale culture to a second vessel, e.g., a G-REX-500 MCS vessel, that is larger than the first vessel, and culturing the T cells from the small-scale culture in a larger culture in the second vessel for about 4-7 days. In some embodiments, the rapid expansion step is divided into multiple steps to achieve scale-out of the culture by (a) performing a rapid second expansion by culturing T cells in a first small-scale culture in a first container, e.g., a G-REX-100 MCS container, for about 3-4 days, and then (b) transferring and distributing T cells from the first small-scale culture into at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 second containers equal in size to the first container, wherein a portion of the transferred T cells from the first small-scale culture are cultured in the second small-scale culture for about 4-7 days.In some embodiments, the rapid expansion step is divided into multiple steps to achieve scale-out and scale-up of the culture by (a) performing a rapid secondary expansion by culturing T cells in a small-scale culture in a first vessel, e.g., a G-REX-100 MCS vessel, for about 3-4 days, and then (b) transferring and distributing the T cells from the small-scale culture into at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 second vessels, e.g., a G-REX-500 MCS vessel, that are larger in size than the first vessel, wherein a portion of the transferred T cells from the small-scale culture are cultured in a larger culture for about 4-7 days in each second vessel. In some embodiments, the rapid expansion step is divided into multiple steps to achieve scale-out and scale-up of the culture by (a) performing a rapid second expansion by culturing T cells in a small-scale culture in a first vessel, e.g., a G-REX-100 MCS vessel, for about 4 days, and then (b) transferring and distributing the T cells from the small-scale culture into two, three, or four second vessels, e.g., a G-REX-500 MCS vessel, that are larger in size than the first vessel, in each of which a portion of the transferred T cells from the small-scale culture are cultured in larger culture for about 5 days.

[0234] In some embodiments, upon division of the rapid expansion, each second container contains at least 10 8 In some embodiments, upon division of rapid expansion, each second vessel contains at least 10 TILs. 8 TIL, at least 10 9 TIL, or at least 10 10 In one exemplary embodiment, each second container contains at least 10 TILs. 10 Contains TILs.

[0235] In some embodiments, the first small-scale TIL culture is allocated into a plurality of subpopulations. In some embodiments, the first small-scale TIL culture is allocated into a plurality of about 2-5 subpopulations. In some embodiments, the first small-scale TIL culture is allocated into a plurality of about 2, 3, 4, or 5 subpopulations.

[0236] In some embodiments, after completion of rapid expansion, the plurality of subpopulations comprises a therapeutically effective amount of TILs. In some embodiments, after completion of rapid expansion, one or more subpopulations of TILs are pooled together to produce a therapeutically effective amount of TILs. In some embodiments, after completion of rapid expansion, each subpopulation of TILs comprises a therapeutically effective amount of TILs.

[0237] In some embodiments, rapid expansion occurs for about 1-5 days before being split into multiple steps, hi some embodiments, the splitting of rapid expansion occurs on about day 1, day 2, day 3, day 4, or day 5 after the initiation of rapid expansion.

[0238] In some embodiments, the splitting off of rapid expansion occurs about 8, 9, 10, 11, 12, or 13 days after the initiation of the first expansion (i.e., pre-REP expansion). In one exemplary embodiment, the splitting off of rapid expansion occurs about 10 days after the initiation of the first expansion by priming. In one exemplary embodiment, the splitting off of rapid expansion occurs about 11 days after the initiation of the first expansion.

[0239] In some embodiments, rapid expansion is continued for about 4 to 11 days after cleavage, hi some embodiments, rapid expansion is continued for about 3, 4, 5, 6, 7, 8, 9, 10, or 11 days after cleavage.

[0240] In some embodiments, the cell culture medium used for rapid expansion before division comprises the same components as the cell culture medium used for rapid expansion after division, hi some embodiments, the cell culture medium used for rapid expansion before division comprises different components than the cell culture medium used for rapid expansion after division.

[0241] In some embodiments, the cell culture medium used for pre-mitotic rapid expansion comprises IL-2, optionally OKT-3, and further optionally APC. In some embodiments, the cell culture medium used for pre-mitotic rapid expansion comprises IL-2, OKT-3, and further optionally APC. In some embodiments, the cell culture medium used for pre-mitotic rapid expansion comprises IL-2, OKT-3, and APC.

[0242] In some embodiments, the cell culture medium used for rapid expansion before division is generated by supplementing the cell culture medium during the first expansion with fresh culture medium comprising IL-2, optionally OKT-3, and further optionally APCs. In some embodiments, the cell culture medium used for rapid expansion before division is generated by supplementing the cell culture medium during the first expansion with fresh culture medium comprising IL-2, OKT-3, and APCs. In some embodiments, the cell culture medium used for rapid expansion before division is generated by replacing the cell culture medium during the first expansion with fresh culture medium comprising IL-2, optionally OKT-3, and further optionally APCs. In some embodiments, the cell culture medium used for rapid expansion before division is generated by replacing the cell culture medium during the first expansion with fresh cell culture medium comprising IL-2, OKT-3, and further optionally APCs.

[0243] In some embodiments, the cell culture medium used for rapid expansion after division comprises IL-2, and optionally OKT-3. In some embodiments, the cell culture medium used for rapid expansion after division comprises IL-2 and OKT-3. In some embodiments, the cell culture medium used for rapid expansion after division is generated by replacing the cell culture medium used for rapid expansion before division with fresh culture medium comprising IL-2 and optionally OKT-3. In some embodiments, the cell culture medium used for rapid expansion after division is generated by replacing the cell culture medium used for rapid expansion before division with fresh culture medium comprising IL-2 and OKT-3.

[0244] In some embodiments, the rapid expansion disruption occurs in a closed system.

[0245] In some embodiments, scaling up TIL cultures during rapid expansion involves adding fresh cell culture medium to the TIL culture (also referred to as feeding the TILs). In some embodiments, feeding involves frequently adding fresh cell culture medium to the TIL culture. In some embodiments, feeding involves adding fresh cell culture medium to the TIL culture at regular intervals. In some embodiments, fresh cell culture medium is supplied to the TILs via a constant flow. In some embodiments, an automated cell expansion system, such as the Xuri W25, is used for rapid expansion and feeding.

[0246] In some embodiments, the rapid second expansion occurs after the T cell activation resulting from the first expansion by priming has begun to decrease, diminish, wane, or subside.

[0247] In some embodiments, the rapid second expansion occurs after the activation of T cells resulting from the first expansion by priming has occurred at or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 , 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or after a 100% reduction.

[0248] In some embodiments, the rapid second expansion is performed after the activation of T cells resulting from the first expansion by priming has decreased by a percentage ranging from exactly or about 1% to 100%.

[0249] In some embodiments, the rapid second expansion is performed after the activation of T cells resulting from the first expansion by priming has decreased by a percentage in the range of exactly or about 1%-10%, 10%-20%, 20%-30%, 30%-40%, 40%-50%, 50%-60%, 60%-70%, 70%-80%, 80%-90%, or 90%-100%.

[0250] In some embodiments, the rapid second expansion is performed after the activation of T cells resulting from the first expansion by priming has progressed to at least exactly or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% reduction.

[0251] In some embodiments, the rapid second expansion is achieved by priming the activation of T cells resulting from the first expansion, but not by more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or after a 100% reduction.

[0252] In some embodiments, the decreased activation of T cells resulting from the first expansion by priming is determined by a decrease in the amount of interferon gamma released by the T cells in response to stimulation with an antigen.

[0253] In some embodiments, the first expansion by priming of T cells occurs over a period of up to or about 7 days or about 8 days.

[0254] In some embodiments, the first expansion by priming of T cells occurs over a period of at most about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or 8 days.

[0255] In some embodiments, the first expansion by priming of T cells occurs over a period of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or 8 days.

[0256] In some embodiments, the rapid secondary expansion of T cells occurs over a period of up to exactly or about 11 days.

[0257] In some embodiments, the rapid secondary expansion of T cells occurs over a period of at most about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, or 11 days.

[0258] In some embodiments, the rapid second expansion of T cells occurs over a period of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, or 11 days.

[0259] In some embodiments, the first expansion by priming of T cells occurs over a period of exactly or about 1 day to exactly or about 7 days, and the rapid second expansion of T cells occurs over a period of exactly or about 1 day to exactly or about 11 days.

[0260] In some embodiments, the priming first expansion of T cells occurs over a period of up to exactly or about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or 8 days, and the rapid second expansion of T cells occurs over a period of up to exactly or about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, or 11 days.

[0261] In some embodiments, the first expansion by priming of T cells occurs over a period of exactly or about 1 day to exactly or about 8 days, and the second rapid expansion of T cells occurs over a period of exactly or about 1 day to exactly or about 9 days.

[0262] In some embodiments, the first expansion by priming of T cells occurs over an 8 day period, and the second rapid expansion of T cells occurs over a 9 day period.

[0263] In some embodiments, the first expansion by priming of T cells occurs over a period of exactly or about 1 day to exactly or about 7 days, and the rapid second expansion of T cells occurs over a period of exactly or about 1 day to exactly or about 9 days.

[0264] In some embodiments, the first expansion by priming of T cells occurs over a 7 day period, and the second rapid expansion of T cells occurs over a 9 day period.

[0265] In some embodiments, the T cells are tumor infiltrating lymphocytes (TILs).

[0266] In some embodiments, the T cells are bone marrow infiltrating lymphocytes (MIL).

[0267] In some embodiments, the T cells are peripheral blood lymphocytes (PBLs).

[0268] In some embodiments, the T cells are obtained from a donor suffering from cancer.

[0269] In some embodiments, the T cells are TILs obtained from a tumor resected from a patient suffering from cancer.

[0270] In some embodiments, the T cells are MILs obtained from the bone marrow of a patient suffering from a hematological malignancy.

[0271] In some embodiments, the T cells are PBLs obtained from peripheral blood mononuclear cells (PBMCs) from a donor. In some embodiments, the donor is afflicted with cancer. In some embodiments, the cancer is a cancer selected from the group consisting of melanoma, ovarian cancer, endometrial cancer, thyroid cancer, cervical cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, cancer caused by human papillomavirus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), glioblastoma (including GBM), gastrointestinal cancer, renal cancer, and renal cell carcinoma. In some embodiments, the cancer is selected from the group consisting of melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, cancer caused by human papillomavirus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), glioblastoma (including GBM), gastrointestinal cancer, renal cancer, and renal cell carcinoma. In some embodiments, the donor is afflicted with a tumor. In some embodiments, the tumor is a liquid tumor. In some embodiments, the tumor is a solid tumor. In some embodiments, the donor is afflicted with a hematological malignancy.

[0272] In certain aspects of the present disclosure, immune effector cells, e.g., T cells, can be obtained from a unit of blood collected from a subject using any number of techniques known to those skilled in the art, such as FICOLL separation. In a preferred aspect, cells from an individual's circulating blood are obtained by apheresis. The apheresis product typically includes lymphocytes, e.g., T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In one aspect, cells collected by apheresis are washed to remove the plasma fraction, and optionally, the cells can be placed in an appropriate buffer or medium for subsequent processing steps. In some embodiments, the cells are washed with phosphate-buffered saline (PBS). In alternative embodiments, the wash solution may lack calcium, magnesium, or many, but not all, divalent cations. In one embodiment, T cells are isolated from peripheral blood lymphocytes by lysing red blood cells and depleting monocytes, for example, by centrifugation through a PERCOLL gradient or by counterflow centrifugal elutriation.

[0273] In some embodiments, the T cells are PBLs isolated from lymphocyte-enriched whole blood or apheresis products from a donor. In some embodiments, the donor is afflicted with cancer. In some embodiments, the cancer is selected from the group consisting of melanoma, ovarian cancer, endometrial cancer, thyroid cancer, cervical cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, cancer caused by human papillomavirus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), glioblastoma (including GBM), gastrointestinal cancer, renal cancer, and renal cell carcinoma. In some embodiments, the cancer is selected from the group consisting of melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, cancer caused by human papillomavirus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), glioblastoma (including GBM), gastrointestinal cancer, renal cancer, and renal cell carcinoma. In some embodiments, the donor is afflicted with a tumor. In some embodiments, the tumor is a liquid tumor. In some embodiments, the tumor is a solid tumor. In some embodiments, the donor is afflicted with a hematological malignancy. In some embodiments, PBLs are isolated from lymphocyte-enriched whole blood or apheresis products by using positive or negative selection methods, i.e., by removing PBLs using marker(s) for T cell phenotype, e.g., CD3+CD45+, or by removing non-T cell phenotype cells, leaving PBLs. In other embodiments, PBLs are isolated by gradient centrifugation. Upon isolation of PBLs from donor tissue, a first priming expansion of PBLs can be performed according to the first priming expansion step of any of the methods described herein to obtain a suitable number of isolated PBLs (in some embodiments, approximately 1 x 10 7 The cells can be initiated by seeding with pluripotent stem (PBLs).

[0274] An exemplary TIL process known as Process 3 (also referred to herein as Gen3), including some of these features, is shown in FIG. 8 (particularly, e.g., FIG. 8B and / or FIG. 8C and / or FIG. 8D), and some of the advantages of this embodiment of the invention over Gen2 are described in FIGS. 1, 2, 8, 30, and 31 (particularly, e.g., FIG. 8A and / or FIG. 8B and / or FIG. 8C and / or FIG. 8D). A Gen3 embodiment is shown in FIGS. 1, 8, and 30 (particularly, FIG. 8A and / or FIG. 8B and / or FIG. 8C and / or FIG. 8D). Process 2A or Gen2 or Gen2A is also described in U.S. Patent Publication No. 2018 / 0280436, which is incorporated herein by reference in its entirety. The Gen3 process is also described in International Patent Publication No. WO2020 / 096988.

[0275] As discussed herein and generally outlined, TILs are harvested from patient samples and engineered to expand their numbers before transplantation into the patient using the TIL expansion process described herein and referred to as Gen3. In some embodiments, TILs may optionally be genetically engineered, as discussed below. In some embodiments, TILs may be cryopreserved before or after expansion. Upon thawing, they may be restimulated to enhance their metabolism before infusion into the patient.

[0276] In some embodiments, as discussed in detail below and in the Examples and Figures, the first expansion with priming (including the process referred to herein as pre-rapid expansion (pre-REP) and the process shown as step B in FIG. 8 (particularly, e.g., FIG. 8A and / or FIG. 8B and / or FIG. 8C and / or FIG. 8D)) is shortened to 1 to 8 days, and the second rapid expansion (including the process referred to herein as rapid expansion protocol (REP) and the process shown as step D in FIG. 8 (particularly, e.g., FIG. 8A and / or FIG. 8B and / or FIG. 8C and / or FIG. 8D)) is shortened to 1 to 9 days. In some embodiments, as discussed in detail below and in the Examples and Figures, the first expansion with priming (including the process referred to herein as pre-rapid expansion (pre-REP) and the process shown as step B in FIG. 8 (particularly, e.g., FIG. 8A and / or FIG. 8B and / or FIG. 8C and / or FIG. 8D)) is shortened to 1 to 8 days, and the second rapid expansion (including the process referred to herein as rapid expansion protocol (REP) and the process shown as step D in FIG. 8 (particularly, e.g., FIG. 8A and / or FIG. 8B and / or FIG. 8C and / or FIG. 8D)) is shortened to 1 to 8 days. In some embodiments, as discussed in detail below and in the Examples and Figures, the first expansion with priming (including the process referred to herein as pre-rapid expansion (pre-REP) and the process shown as step B in FIG. 8 (particularly, e.g., FIG. 8A and / or FIG. 8B and / or FIG. 8C and / or FIG. 8D)) is shortened to 1 to 7 days, and the second rapid expansion (including the process referred to herein as rapid expansion protocol (REP) and the process shown as step D in FIG. 8 (particularly, e.g., FIG. 8A and / or FIG. 8B and / or FIG. 8C and / or FIG. 8D)) is shortened to 1 to 9 days.In some embodiments, as discussed in detail below and in the Examples and Figures, the first expansion by priming (including the process referred to herein as pre-rapid expansion (pre-REP) and the process shown as step B in FIG. 8 (particularly, e.g., FIG. 1B and / or FIG. 8C)) is shortened to 1-7 days, and the second rapid expansion (including the process referred to herein as rapid expansion protocol (REP) and the process shown as step D in FIG. 8 (particularly, e.g., FIG. 8A and / or FIG. 8B and / or FIG. 8C and / or FIG. 8D)) is 1-10 days. In some embodiments, the first expansion by priming (e.g., the expansion described as step B in FIG. 8 (particularly, e.g., FIG. 8A and / or FIG. 8B and / or FIG. 8C and / or FIG. 8D)) is shortened to 8 days, and the second rapid expansion (e.g., the expansion described as step D in FIG. 8 (particularly, e.g., FIG. 8A and / or FIG. 8B and / or FIG. 8C and / or FIG. 8D)) is 7-9 days. In some embodiments, the first priming expansion (e.g., the expansion described as step B in FIG. 8 (particularly, e.g., FIG. 8A and / or FIG. 8B and / or FIG. 8C and / or FIG. 8D)) is 8 days, and the rapid second expansion (e.g., the expansion described as step D in FIG. 8 (particularly, e.g., FIG. 8A and / or FIG. 8B and / or FIG. 8C and / or FIG. 8D)) is 8-9 days. In some embodiments, the first priming expansion (e.g., the expansion described as step B in FIG. 8 (particularly, e.g., FIG. 8A and / or FIG. 8B and / or FIG. 8C and / or FIG. 8D)) is shortened to 7 days, and the rapid second expansion (e.g., the expansion described as step D in FIG. 8 (particularly, e.g., FIG. 8A and / or FIG. 8B and / or FIG. 8C and / or FIG. 8D)) is 7-8 days. In some embodiments, the first priming expansion (e.g., the expansion described as step B in FIG. 8 (particularly, e.g., FIG. 8A and / or FIG. 8B and / or FIG. 8C and / or FIG. 8D)) is shortened to 8 days, and the rapid second expansion (e.g., the expansion described as step D in FIG. 8 (particularly, e.g., FIG. 8A and / or FIG. 8B and / or FIG. 8C and / or FIG. 8D)) is 8 days.In some embodiments, the first expansion by priming (e.g., the expansion described as step B in FIG. 8 (particularly, e.g., FIG. 8A and / or FIG. 8B and / or FIG. 8C and / or FIG. 8D)) is 8 days, and the second rapid expansion (e.g., the expansion described as step D in FIG. 8 (particularly, e.g., FIG. 8A and / or FIG. 8B and / or FIG. 8C and / or FIG. 8D)) is 9 days. In some embodiments, the first expansion by priming (e.g., the expansion described as step B in FIG. 8 (particularly, e.g., FIG. 8A and / or FIG. 8B and / or FIG. 8C and / or FIG. 8D)) is 8 days, and the second rapid expansion (e.g., the expansion described as step D in FIG. 8 (particularly, e.g., FIG. 8A and / or FIG. 8B and / or FIG. 8C and / or FIG. 8D)) is 10 days. In some embodiments, the first expansion by priming (e.g., the expansion described as step B in FIG. 8 (particularly, e.g., FIG. 8A and / or FIG. 8B and / or FIG. 8C and / or FIG. 8D)) is 7 days, and the second rapid expansion (e.g., the expansion described as step D in FIG. 8 (particularly, e.g., FIG. 8A and / or FIG. 8B and / or FIG. 8C and / or FIG. 8D)) is 7 to 10 days. In some embodiments, the first expansion by priming (e.g., the expansion described as step B in FIG. 8 (particularly, e.g., FIG. 8A and / or FIG. 8B and / or FIG. 8C and / or FIG. 8D)) is 7 days, and the second rapid expansion (e.g., the expansion described as step D in FIG. 8 (particularly, e.g., FIG. 8A and / or FIG. 8B and / or FIG. 8C and / or FIG. 8D)) is 8 to 10 days. In some embodiments, the first priming expansion (e.g., the expansion described as step B in FIG. 8 (particularly, e.g., FIG. 8A and / or FIG. 8B and / or FIG. 8C and / or FIG. 8D)) is 7 days, and the rapid second expansion (e.g., the expansion described as step D in FIG. 8 (particularly, e.g., FIG. 8A and / or FIG. 8B and / or FIG. 8C and / or FIG. 8D)) is 9 to 10 days.In some embodiments, the first priming expansion (e.g., the expansion described as step B in FIG. 8 (particularly, e.g., FIG. 8A and / or FIG. 8B and / or FIG. 8C and / or FIG. 8D)) is shortened to 7 days, and the rapid second expansion (e.g., the expansion described as step D in FIG. 8 (particularly, e.g., FIG. 8A and / or FIG. 8B and / or FIG. 8C and / or FIG. 8D)) is 7-9 days. In some embodiments, the combination of the first priming expansion and the rapid second expansion (e.g., the expansion described as steps B and D in FIG. 8 (particularly, e.g., FIG. 1B and / or FIG. 8C)) is 14-16 days, as discussed in detail below and in the Examples and Figures. In particular, it is contemplated that certain embodiments of the present invention include a first priming expansion step in which TILs are activated by exposure to an anti-CD3 antibody, e.g., OKT-3, in the presence of IL-2, or by exposure to an antigen in the presence of at least IL-2 and an anti-CD3 antibody, e.g., OKT-3. In certain embodiments, the TILs activated in the first expansion step by priming as described above are a first TIL population, i.e., a primary cell population.

[0277] The "Step" designations A, B, C, etc. below refer to the non-limiting examples in Figure 8 (particularly, e.g., Figures 8A and / or 8B and / or 8C and / or 8D) and to certain non-limiting embodiments described herein. The order of steps below and in Figure 8 (particularly, e.g., Figures 8A and / or 8B and / or 8C and / or 8D) is exemplary, and any combination or order of steps, as well as additional steps, repeated steps, and / or omission of steps, is contemplated by the present application and methods disclosed herein.

[0278] A. Step A: Obtaining a patient tumor sample Generally, TILs are initially obtained from a patient's tumor sample ("primary TILs") or from circulating lymphocytes, such as peripheral blood lymphocytes, including those with TIL-like characteristics, and then expanded into larger populations for further manipulation, optionally cryopreserved, and optionally evaluated for phenotypic and metabolic parameters as indicators of TIL health, as described herein.

[0279] A patient's tumor sample can generally be obtained by surgical resection, needle biopsy, or other means for obtaining a sample containing a mixture of tumor cells and TIL cells, using methods known in the art. Generally, the tumor sample can be from any solid tumor, including a primary tumor, an invasive tumor, or a metastatic tumor. The tumor sample can also be from a liquid tumor, such as a tumor obtained from a hematological malignancy. The solid tumor can be any type of cancer, including, but not limited to, breast cancer, pancreatic cancer, prostate cancer, colorectal cancer, lung cancer, brain cancer, renal cancer, gastric cancer, and skin cancer (including, but not limited to, squamous cell carcinoma, basal cell carcinoma, and melanoma). In some embodiments, the cancer is selected from cervical cancer, head and neck cancer (including, for example, head and neck squamous cell carcinoma (HNSCC)), glioblastoma (GBM), gastrointestinal cancer, ovarian cancer, sarcoma, pancreatic cancer, bladder cancer, breast cancer, triple-negative breast cancer, and non-small cell lung cancer. In some embodiments, the cancer is melanoma. In some embodiments, useful TILs are obtained from melanoma tumors, as these tumors have been reported to have particularly high levels of TILs.

[0280] Once obtained, tumor samples are generally cut into sections of 1 to approximately 8 mm using sharp dissection. 3 fragmented into small pieces of about 2-3 mm 3are particularly useful. TILs are cultured from these fragments using enzymatic tumor digests. Such tumor digests can be produced by incubation in an enzymatic medium (e.g., Roswell Park Memorial Institute (RPMI) 1640 buffer, 2 mM glutamic acid, 10 mcg / mL gentamicin, 30 units / mL DNase, and 1.0 mg / mL collagenase) followed by mechanical dissociation (e.g., using a tissue dissociator). Tumor digests can be produced by placing the tumor in the enzymatic medium, mechanically dissociating the tumor for approximately 1 minute, followed by incubation at 37°C in 5% CO2 for 30 minutes, and then repeating cycles of mechanical dissociation and incubation under the aforementioned conditions until only small tissue fragments are present. At the end of this process, if the cell suspension contains a large number of red blood cells or dead cells, density gradient separation using FICOLL branched hydrophilic polysaccharides can be performed to remove these cells. Alternative methods known in the art can be used, such as those described in U.S. Patent Application Publication No. 2012 / 0244133A1, the disclosure of which is incorporated herein by reference. Any of the foregoing methods can be used in any of the embodiments described herein for methods of expanding TILs or methods of treating cancer.

[0281] As noted above, in some embodiments, the TILs are derived from solid tumors. In some embodiments, the solid tumor is not fragmented. In some embodiments, the solid tumor is not fragmented and is subjected to enzymatic digestion as a whole tumor. In some embodiments, the tumor is digested in an enzyme mixture comprising collagenase, DNase, and hyaluronidase. In some embodiments, the tumor is digested in an enzyme mixture comprising collagenase, DNase, and hyaluronidase for 1-2 hours. In some embodiments, the tumor is digested in an enzyme mixture comprising collagenase, DNase, and hyaluronidase for 1-2 hours at 37°C and 5% CO2. In some embodiments, the tumor is digested in an enzyme mixture comprising collagenase, DNase, and hyaluronidase for 1-2 hours at 37°C and 5% CO2 with rotation. In some embodiments, the tumor is digested overnight with constant rotation. In some embodiments, the tumor is digested overnight at 37°C and 5% CO2 with constant rotation. In some embodiments, the whole tumor is combined with the enzymes to form a tumor digestion reaction mixture.

[0282] In some embodiments, the tumor is reconstituted with lyophilized enzyme in a sterile buffer. In some embodiments, the buffer is sterile HBSS.

[0283] In some embodiments, the enzyme mixture includes collagenase. In some embodiments, the collagenase is collagenase IV. In some embodiments, the working stock of collagenase is a 10x working stock of 100 mg / mL.

[0284] In some embodiments, the enzyme mixture comprises DNAse, hi some embodiments, the working stock of DNAse is a 10x working stock of 10,000 IU / mL.

[0285] In some embodiments, the enzyme mixture comprises hyaluronidase. In some embodiments, the working stock of hyaluronidase is a 10x working stock of 10 mg / mL.

[0286] In some embodiments, the enzyme mixture comprises 10 mg / mL collagenase, 1000 IU / mL DNAse, and 1 mg / mL hyaluronidase.

[0287] In some embodiments, the enzyme mixture comprises 10 mg / mL collagenase, 500 IU / mL DNAse, and 1 mg / mL hyaluronidase.

[0288] Generally, cell suspensions obtained from tumors are referred to as "primary cell populations" or "freshly obtained" or "freshly isolated" cell populations. In certain embodiments, freshly obtained TIL cell populations are exposed to cell culture medium comprising antigen-presenting cells, IL-12, and OKT-3.

[0289] In some embodiments, fragmentation includes physical fragmentation, including, for example, dissection and digestion. In some embodiments, the fragmentation is physical fragmentation. In some embodiments, the fragmentation is by dissection. In some embodiments, the fragmentation is by digestion. In some embodiments, TILs can be initially cultured from enzymatic tumor digests and tumor fragments obtained from a patient. In some embodiments, TILs can be initially cultured from enzymatic tumor digests and tumor fragments obtained from a patient.

[0290] In some embodiments, where the tumor is a solid tumor, after a tumor sample is obtained, e.g., in step A (provided in FIG. 8 (particularly, e.g., FIG. 8A and / or FIG. 8B and / or FIG. 8C and / or FIG. 8D)), the tumor undergoes physical fragmentation. In some embodiments, fragmentation occurs before cryopreservation. In some embodiments, fragmentation occurs after cryopreservation. In some embodiments, fragmentation occurs after obtaining the tumor, in the absence of any cryopreservation. In some embodiments, the fragmentation step is an in vitro or ex vivo process. In some embodiments, the tumor is fragmented and 10, 20, 30, 40, or more fragments or pieces are placed in each container for the first expansion by priming. In some embodiments, the tumor is fragmented and 30 or 40 fragments or pieces are placed in each container for the first expansion by priming. In some embodiments, the tumor is fragmented and 40 fragments or pieces are placed in each container for the first expansion by priming. In some embodiments, the plurality of fragments comprises about 4 to about 50 fragments, each fragment having a length of about 27 mm. 3 In some embodiments, the plurality of pieces has a volume of about 1300 mm 3 ~approx. 1500mm 3 In some embodiments, the plurality of pieces comprises about 30 to about 60 pieces with a total volume of about 1350 mm 3 In some embodiments, the plurality of fragments comprises about 50 fragments with a total mass of about 1 gram to about 1.5 grams. In some embodiments, the plurality of fragments comprises about 4 fragments.

[0291] In some embodiments, the TILs are obtained from tumor fragments. In some embodiments, the tumor fragments are obtained by sharp dissection. In some embodiments, the tumor fragments are approximately 1 mm 3 ~10mm 3 In some embodiments, the tumor fragment is about 1 mm 3 ~8mm 3 In some embodiments, the tumor fragment is about 1 mm 3In some embodiments, the tumor fragment is about 2 mm 3 In some embodiments, the tumor fragment is about 3 mm 3 In some embodiments, the tumor fragment is about 4 mm 3 In some embodiments, the tumor fragment is about 5 mm 3 In some embodiments, the tumor fragment is about 6 mm 3 In some embodiments, the tumor fragment is about 7 mm 3 In some embodiments, the tumor fragment is about 8 mm 3 In some embodiments, the tumor fragment is about 9 mm 3 In some embodiments, the tumor fragment is about 10 mm 3 In some embodiments, the tumor fragment is 1-4 mm x 1-4 mm x 1-4 mm. In some embodiments, the tumor fragment is 1 mm x 1 mm x 1 mm. In some embodiments, the tumor fragment is 2 mm x 2 mm x 2 mm. In some embodiments, the tumor fragment is 3 mm x 3 mm x 3 mm. In some embodiments, the tumor fragment is 4 mm x 4 mm x 4 mm.

[0292] In some embodiments, the tumor is fragmented to minimize the amount of hemorrhagic, necrotic, and / or fatty tissue on each piece. In some embodiments, the tumor is fragmented to minimize the amount of hemorrhagic tissue on each piece. In some embodiments, the tumor is fragmented to minimize the amount of necrotic tissue on each piece. In some embodiments, the tumor is fragmented to minimize the amount of fatty tissue on each piece. In certain embodiments, the tumor fragmentation step is an in vitro or ex vivo method.

[0293] In some embodiments, tumor fragmentation is performed to maintain the internal structure of the tumor. In some embodiments, tumor fragmentation is performed without sawing with a scalpel. In some embodiments, TILs are obtained from tumor digests. In some embodiments, tumor digests are generated by incubation in an enzyme medium, such as, but not limited to, RPMI 1640 (2 mM GlutaMAX, 10 mg / mL gentamicin, 30 U / mL DNase, and 1.0 mg / mL collagenase), followed by mechanical dissociation (GentleMACS, Miltenyi Biotec, Auburn, CA). After placing the tumor in the enzyme medium, the tumor may be mechanically dissociated for approximately 1 minute. The solution may then be incubated at 37°C in 5% CO2 for 30 minutes, after which it may be mechanically disrupted again for approximately 1 minute. After again incubating at 37°C in 5% CO2 for 30 minutes, the tumor may be mechanically disrupted a third time for approximately 1 minute. In some embodiments, if large tissue debris was present, one or two additional rounds of mechanical dissociation were applied to the sample after the third mechanical disruption, with incubation for an additional 30 minutes at 37° C. in 5% CO In some embodiments, if the cell suspension contains a large number of red blood cells or dead cells, density gradient separation using Ficoll can be performed at the end of the final incubation to remove these cells.

[0294] In some embodiments, the cell suspension before the first expansion step by priming is referred to as a "primary cell population" or a "freshly obtained" or "freshly isolated" cell population.

[0295] In some embodiments, the cells may optionally be frozen after sample isolation (e.g., after obtaining the tumor sample and / or after obtaining a cell suspension from the tumor sample) and cryopreserved prior to expansion as described in step B, which is described in further detail below and illustrated in Figure 8 (particularly, e.g., Figure 8B).

[0296] 1. Core / small biopsy-derived TILs In some embodiments, TILs are initially obtained from patient tumor samples obtained by core biopsy or similar procedures ("primary TILs"), and then expanded into larger populations for further manipulation, optionally cryopreserved, and optionally evaluated for phenotypic and metabolic parameters, as described herein.

[0297] In some embodiments, a patient tumor sample can be obtained using methods known in the art, generally by mini-biopsy, core biopsy, needle biopsy, or other means for obtaining a sample containing a mixture of tumor cells and TIL cells. Generally, the tumor sample can be from any solid tumor, including a primary tumor, an invasive tumor, or a metastatic tumor. The tumor sample can be from a liquid tumor, such as a tumor obtained from a hematological malignancy. In some embodiments, the sample can be from multiple small tumor samples or biopsies. In some embodiments, the sample can include multiple tumor samples from a single tumor from the same patient. In some embodiments, the sample can include multiple tumor samples from one, two, three, or four tumors from the same patient. In some embodiments, the sample can include multiple tumor samples from multiple tumors from the same patient. The solid tumor can be lung cancer and / or non-small cell lung cancer (NSCLC).

[0298] Generally, cell suspensions obtained from tumor cores or fragments are referred to as "primary cell populations" or "freshly obtained" or "freshly isolated" cell populations. In certain embodiments, the freshly obtained TIL cell population is exposed to cell culture medium comprising antigen-presenting cells, IL-2, and OKT-3.

[0299] In some embodiments, if the tumor is metastatic and the primary lesion has previously been effectively treated / removed, removal of one of the metastatic lesions may be required. In some embodiments, the least invasive approach is to remove the skin lesion or, if available, lymph nodes in the neck or axillary region. In some embodiments, the skin lesion is removed or a small biopsy thereof is removed. In some embodiments, the lymph node or a small biopsy thereof is removed. In some embodiments, the tumor is melanoma. In some embodiments, a small biopsy of the melanoma includes a mole or a portion thereof.

[0300] In some embodiments, the small biopsy is a punch biopsy. In some embodiments, the punch biopsy is obtained with a circular blade pressed into the skin. In some embodiments, the punch biopsy is obtained with a circular blade pressed into the skin around the suspicious mole. In some embodiments, the punch biopsy is obtained with a circular blade pressed into the skin to remove a round piece of skin. In some embodiments, the small biopsy is a punch biopsy to remove a round portion of the tumor.

[0301] In some embodiments, the small biopsy is an excision biopsy. In some embodiments, the small biopsy is an excision biopsy, where the entire mole or growth is removed. In some embodiments, the small biopsy is an excision biopsy, where the entire mole or growth is removed along with a small margin of normal-appearing skin.

[0302] In some embodiments, the mini-biopsy is an incisional biopsy. In some embodiments, the mini-biopsy is an incisional biopsy, where only the most irregular part of the mole or growth is taken. In some embodiments, the mini-biopsy is an incisional biopsy, where an incisional biopsy is used when other techniques cannot be achieved, such as when the suspicious mole is very large.

[0303] In some embodiments, the small biopsy is a lung biopsy. In some embodiments, the small biopsy is obtained by bronchoscopy. Generally, in bronchoscopy, the patient is anesthetized and a small instrument is passed through the nose or mouth and down the throat into the bronchial passages, where the small instrument is used to remove some tissue. In some embodiments, where a tumor or growth cannot be reached by bronchoscopy, a transthoracic needle biopsy may be used. Generally, with a transthoracic needle biopsy, the patient is also anesthetized and a needle is inserted directly through the skin into the suspected location to remove a small tissue sample. In some embodiments, a transthoracic needle biopsy may require interventional radiology (e.g., the use of an X-ray or CT scan to guide the needle). In some embodiments, the small biopsy is obtained by needle biopsy. In some embodiments, the small biopsy is obtained by endoscopic ultrasound (e.g., an endoscope with a light and placed through the mouth into the esophagus). In some embodiments, the small biopsy is obtained surgically.

[0304] In some embodiments, the minibiopsy is a head and neck biopsy. In some embodiments, the minibiopsy is an incisional biopsy. In some embodiments, the minibiopsy is an incisional biopsy, where a small piece of tissue is cut from the area that looks abnormal. In some embodiments, if the abnormal area is easily accessible, the sample can be taken without hospitalization. In some embodiments, if the tumor is deeper in the mouth or throat, the biopsy may need to be done in an operating room using general anesthesia. In some embodiments, the minibiopsy is an excisional biopsy. In some embodiments, the minibiopsy is an excisional biopsy, where the entire area is removed. In some embodiments, the minibiopsy is a fine needle aspiration (FNA). In some embodiments, the minibiopsy is a fine needle aspiration (FNA), where a very thin needle attached to a syringe is used to extract (aspirate) cells from a tumor or mass. In some embodiments, the minibiopsy is a punch biopsy. In some embodiments, the minibiopsy is a punch biopsy, where a piece of suspicious area is taken using punch forceps.

[0305] In some embodiments, the mini-biopsy is a cervical biopsy. In some embodiments, the mini-biopsy is obtained by colposcopy. Generally, colposcopy employs the use of a lighted magnifying instrument (a colposcope) attached to magnifying binoculars, which is then used to biopsy a small section of the surface of the cervix. In some embodiments, the mini-biopsy is a cone biopsy. In some embodiments, the mini-biopsy is a cone biopsy, which may require outpatient surgery to remove a larger piece of tissue from the cervix. In some embodiments, a cone biopsy, in addition to helping confirm a diagnosis, may be an initial treatment.

[0306] The term "solid tumor" refers to an abnormal mass of tissue that usually does not contain cysts or liquid areas. Solid tumors can be benign or malignant. The term "solid tumor cancer" refers to a malignant, neoplastic, or cancerous solid tumor. Solid tumor cancers include cancer of the lung. In some embodiments, the cancer is melanoma. In some embodiments, the cancer is non-small cell lung cancer (NSCLC). The histology of a solid tumor includes interdependent tissue compartments, including the parenchyma (cancer cells), and supporting stromal cells, in which the cancer cells are dispersed and which may provide a supportive microenvironment.

[0307] In some embodiments, the sample from the tumor is obtained as a fine needle aspirate (FNA), core biopsy, or mini-biopsy (including, for example, a punch biopsy). In some embodiments, the sample is first placed in a G-REX-10. In some embodiments, if one or two core biopsy and / or mini-biopsy samples are present, the sample is first placed in a G-REX-10. In some embodiments, if three, four, five, six, eight, nine, or ten or more core biopsy and / or mini-biopsy samples are present, the sample is first placed in a G-REX-100. In some embodiments, if three, four, five, six, eight, nine, or ten or more core biopsy and / or mini-biopsy samples are present, the sample is first placed in a G-REX-500.

[0308] FNAs can be obtained from skin tumors, including, for example, melanoma. In some embodiments, FNAs are obtained from skin tumors, such as skin tumors from patients with metastatic melanoma. In some cases, patients with melanoma have previously undergone surgical treatment.

[0309] FNAs can be obtained from lung tumors, including, for example, NSCLC. In some embodiments, FNAs are obtained from lung tumors, such as lung tumors from patients with non-small cell lung cancer (NSCLC). In some cases, patients with NSCLC have previously undergone surgical treatment.

[0310] The TILs described herein can be obtained from FNA samples. In some cases, FNA samples are obtained or isolated from patients using a fine gauge needle, ranging from an 18-gauge needle to a 25-gauge needle. The fine gauge needle can be 18-gauge, 19-gauge, 20-gauge, 21-gauge, 22-gauge, 23-gauge, 24-gauge, or 25-gauge. In some embodiments, an FNA sample from a patient may contain at least 400,000 TILs, e.g., 400,000 TILs, 450,000 TILs, 500,000 TILs, 550,000 TILs, 600,000 TILs, 650,000 TILs, 700,000 TILs, 750,000 TILs, 800,000 TILs, 850,000 TILs, 900,000 TILs, 950,000 TILs, or more.

[0311] In some cases, the TILs described herein are obtained from a core biopsy sample. In some cases, the core biopsy sample is obtained or isolated from a patient using a surgical or medical needle ranging from an 11-gauge needle to a 16-gauge needle. The needle can be 11-gauge, 12-gauge, 13-gauge, 14-gauge, 15-gauge, or 16-gauge. In some embodiments, a core biopsy sample from a patient may contain at least 400,000 TILs, e.g., 400,000 TILs, 450,000 TILs, 500,000 TILs, 550,000 TILs, 600,000 TILs, 650,000 TILs, 700,000 TILs, 750,000 TILs, 800,000 TILs, 850,000 TILs, 900,000 TILs, 950,000 TILs, or more.

[0312] Generally, the harvested cell suspension is referred to as a "primary cell population" or "freshly harvested" cell population.

[0313] In some embodiments, the TILs are not obtained from a tumor digest, hi some embodiments, the solid tumor core is not fragmented.

[0314] In some embodiments, TILs are obtained from tumor digests. In some embodiments, tumor digests are generated by incubation in an enzyme medium, such as, but not limited to, RPMI 1640 (2 mM GlutaMAX, 10 mg / mL gentamicin, 30 U / mL DNase, and 1.0 mg / mL collagenase), followed by mechanical dissociation (GentleMACS, Miltenyi Biotec, Auburn, CA). After placing the tumor in the enzyme medium, the tumor may be mechanically dissociated for approximately 1 minute. The solution may then be incubated for 30 minutes at 37°C in 5% CO2, after which it may be mechanically disrupted again for approximately 1 minute. After incubating again for 30 minutes at 37°C in 5% CO2, the tumor may be mechanically disrupted a third time for approximately 1 minute. In some embodiments, if large tissue fragments are present, one or two additional rounds of mechanical dissociation are applied to the sample after the third mechanical disruption, regardless of whether it is incubated for an additional 30 minutes at 37°C in 5% CO2. In some embodiments, if the cell suspension contains a large number of red blood cells or dead cells, density gradient separation using Ficoll can be performed at the end of the final incubation to remove these cells.

[0315] In some embodiments, obtaining the first population of TILs comprises a multi-lesion sampling method.

[0316] The tumor dissociation enzyme mixture can include one or more dissociation (digestion) enzymes, such as, but not limited to, collagenase (including any blend or type of collagenase), Accutase™, Accumax™, hyaluronidase, neutral protease (dispase), chymotrypsin, chymopapain, trypsin, caseinase, elastase, papain, type XIV protease (pronase), deoxyribonuclease I (DNase), trypsin inhibitor, any other dissociation enzyme or proteolytic enzyme, and any combination thereof.

[0317] In some embodiments, the dissociation enzyme is reconstituted from a lyophilized enzyme, hi some embodiments, the lyophilized enzyme is reconstituted with an amount of a sterile buffer, such as Hank's Balanced Salt Solution (HBSS).

[0318] In some cases, collagenase (such as animal-free type 1 collagenase) is reconstituted in 10 mL of sterile HBSS or another buffer. Lyophilized stock enzyme may be at a concentration of 2892 PZ U / vial. In some embodiments, collagenase is reconstituted in 5 mL to 15 mL of buffer. In some embodiments, the collagenase stock after reconstitution is in the range of about 100 U / mL to about 400 U / mL, e.g., about 100 U / mL to about 400 U / mL, about 100 U / mL to about 350 U / mL, about 100 U / mL to about 300 U / mL, about 150 U / mL to about 400 U / mL, about 100 U / mL, about 150 U / mL, about 200 U / mL, about 210 U / mL, about 220 U / mL, about 230 U / mL, about 240 U / mL, about 250 U / mL, about 260 U / mL, about 270 U / mL, about 280 U / mL, about 289.2 U / mL, about 300 U / mL, U / mL, about 350 PZ U / mL, or about 400 PZ U / mL.

[0319] In some embodiments, the neutral protease is reconstituted in 1 mL of sterile HBSS or another buffer. The lyophilized stock enzyme may be at a concentration of 175 DMC U / vial. In some embodiments, the neutral protease stock after reconstitution may range from about 100 DMC / mL to about 400 DMC / mL, e.g., from about 100 DMC / mL to about 400 DMC / mL, from about 100 DMC / mL to about 350 DMC / mL, from about 100 DMC / mL to about 300 DMC / mL, from about 150 DMC / mL to about 400 DMC / mL, from about 100 DMC / mL, or about 110 DMC / mL. C / mL, about 120 DMC / mL, about 130 DMC / mL, about 140 DMC / mL, about 150 DMC / mL, about 160 DMC / mL, about 170 DMC / mL, about 175 DMC / mL, about 180 DMC / mL, about 190 DMC / mL, about 200 DMC / mL, about 250 DMC / mL, about 300 DMC / mL, about 350 DMC / mL, or about 400 DMC / mL.

[0320] In some embodiments, DNAse I is reconstituted in 1 mL of sterile HBSS or another buffer. The lyophilized stock enzyme was at a concentration of 4 KU / vial. In some embodiments, the reconstituted DNase I stock ranges from about 1 KU / mL to 10 KU / mL, e.g., about 1 KU / mL, about 2 KU / mL, about 3 KU / mL, about 4 KU / mL, about 5 KU / mL, about 6 KU / mL, about 7 KU / mL, about 8 KU / mL, about 9 KU / mL, or about 10 KU / mL.

[0321] In some embodiments, since enzyme stocks may vary, check the concentration of the lyophilized stock and modify the final amount of enzyme added to the digestion cocktail accordingly.

[0322] In some embodiments, the enzyme mixture comprises about 10.2 ul of neutral protease (0.36 DMC U / mL), 21.3 ul of collagenase (1.2 PZ / mL), and 250 ul of DNAse I (200 U / mL) in about 4.7 mL of sterile HBSS.

[0323] 2. Pleural fluid T cells and TILs In some embodiments, the sample is a pleural fluid sample. In some embodiments, the source of T cells or TILs for expansion by the processes described herein is a pleural fluid sample. In some embodiments, the sample is a pleural fluid-derived sample. In some embodiments, the source of TILs for expansion by the processes described herein is a pleural fluid-derived sample. See, e.g., the methods described in U.S. Patent Publication No. US2014 / 0295426, which is incorporated by reference in its entirety for all purposes.

[0324] In some embodiments, any pleural fluid or effusion that appears to be and / or contains TILs can be utilized. Such samples can be derived from primary or metastatic lung cancer, such as NSCLC or SCLC. In some embodiments, the sample can be secondary metastatic cancer cells from another organ, such as the breast, ovary, colon, or prostate. In some embodiments, the sample used in the expansion methods described herein is a pleural effusion. In some embodiments, the sample used in the expansion methods described herein is a pleural transudate. Other biological samples can include other serous fluids containing TILs, including, for example, ascites from the abdomen or pancreatic cyst fluid. Ascites and pleural fluids have very similar chemical systems; both the abdomen and lungs have mesothelial lines and fluid forms in the pleural and abdominal spaces of the same substance in malignant tumors; in some embodiments, such fluids contain TILs. In some embodiments, where the present disclosure exemplifies pleural effusion, the same method can be performed with similar results using pleural fluid or other cyst fluids containing TILs.

[0325] In some embodiments, the pleural fluid is in an unprocessed form removed directly from the patient. In some embodiments, the unprocessed pleural fluid is placed in a standard collection tube, such as an EDTA or heparin tube, before the contacting step. In some embodiments, the unprocessed pleural fluid is placed in a standard CellSave® tube (Veridex) before the contacting step. In some embodiments, the sample is placed in a CellSave tube immediately after collection from the patient to avoid a decrease in the number of viable TILs. The number of viable TILs can decrease significantly within 24 hours if left in unprocessed pleural fluid, even at 4°C. In some embodiments, the sample is placed in an appropriate collection tube within 1 hour, 5 hours, 10 hours, 15 hours, or up to 24 hours after removal from the patient. In some embodiments, the sample is placed in an appropriate collection tube at 4°C within 1 hour, 5 hours, 10 hours, 15 hours, or up to 24 hours after removal from the patient.

[0326] In some embodiments, the pleural fluid sample from a selected subject may be diluted. In some embodiments, the dilution is 1:10 pleural fluid to diluent. In some embodiments, the dilution is 1:9 pleural fluid to diluent. In some embodiments, the dilution is 1:8 pleural fluid to diluent. In some embodiments, the dilution is 1:5 pleural fluid to diluent. In some embodiments, the dilution is 1:2 pleural fluid to diluent. In some embodiments, the dilution is 1:1 pleural fluid to diluent. In some embodiments, the diluent includes saline, phosphate-buffered saline, another buffer, or a physiologically acceptable diluent. In some embodiments, the sample is placed into a CellSave tube immediately after collection from the patient and diluted to avoid a significant loss of viable TILs, which can occur within 24 to 48 hours if left in unprocessed pleural fluid, even at 4°C. In some embodiments, the pleural fluid sample is placed into a suitable collection tube within 1 hour, 5 hours, 10 hours, 15 hours, 24 hours, 36 hours, or up to 48 hours after removal from the patient and dilution. In some embodiments, the pleural fluid sample is placed into a suitable collection tube within 1 hour, 5 hours, 10 hours, 15 hours, 24 hours, 36 hours, or up to 48 hours after removal from the patient and dilution at 4° C.

[0327] In yet other embodiments, the pleural fluid sample is concentrated by conventional means prior to further processing steps. In some embodiments, this pretreatment of the pleural fluid is preferred in situations where the pleural fluid must be frozen for transport to the laboratory where the method will be performed or for subsequent analysis (e.g., more than 24-48 hours after collection). In some embodiments, the pleural fluid sample is prepared by centrifuging the pleural fluid sample after it is collected from the subject and resuspending the centrate or pellet in a buffer. In some embodiments, the pleural fluid sample is subjected to multiple centrifugations and resuspensions before being frozen for transport or subsequent analysis and / or processing.

[0328] In some embodiments, the pleural fluid sample is concentrated prior to further processing steps by using a filtration method. In some embodiments, the pleural fluid sample used in the contacting step is prepared by filtering the fluid through a filter containing a known, essentially uniform pore size that allows pleural fluid to pass through the membrane but retains tumor cells. In some embodiments, the membrane pore diameter can be at least 4 μM. In other embodiments, the pore diameter can be 5 μM or greater, and in other embodiments, 6, 7, 8, 9, or 10 μM. After filtration, the TIL-containing cells retained by the membrane can be rinsed from the membrane into an appropriate physiologically acceptable buffer. The thus-enriched TIL-containing cells can then be used in the contacting step of the method.

[0329] In some embodiments, a pleural fluid sample (e.g., including unprocessed pleural fluid), diluted pleural fluid, or a resuspended cell pellet is contacted with a lysis reagent that specifically lyses nonnucleated red blood cells present in the sample. In some embodiments, this step is performed before further processing steps in situations where the pleural fluid contains a significant number of RBCs. Suitable lysis reagents include a single lysis reagent or a lysis reagent and a quenching reagent, or a lysis agent, a quenching reagent, and a fixation reagent. Suitable lysis systems are commercially available and include the BD Pharm Lyse™ system (Becton Dickenson). Other lysis systems include the Versalyse™ system, the FACSlyse™ system (Becton Dickenson), the Immunoprep™ system, or the Erythrolyse II system (Beckman Coulter, Inc.), or an ammonium chloride system. In some embodiments, the lysis reagent can vary depending on key requirements, such as efficient lysis of red blood cells and preservation of TILs and their phenotypic characteristics in the pleural fluid. In addition to utilizing a single reagent for lysis, lysis systems useful in the methods described herein can include a second reagent, e.g., one that quenches or delays the effect of the lysis reagent during the remaining steps of the method, such as Stabilyse™ reagent (Beckman Coulter, Inc.) Depending on the choice of lysis reagent or the preferred implementation of the method, conventional fixative reagents can also be used.

[0330] In some embodiments, pleural fluid samples that have been unprocessed, diluted, or multiple centrifuged or processed as described herein are stored frozen at a temperature of about -140°C before being further processed and / or expanded as provided herein.

[0331] 3. How to expand peripheral blood lymphocytes (PBLs) from peripheral blood PBL Method 1. In some embodiments of the invention, PBLs are expanded using the processes described herein. In some embodiments of the invention, the method includes obtaining a PBMC sample from whole blood. In some embodiments, the method includes enriching T cells by isolating pure T cells from the PBMCs using negative selection of the non-CD19+ fraction. In some embodiments, the method includes enriching T cells by isolating pure T cells from the PBMCs using magnetic bead-based negative selection of the non-CD19+ fraction.

[0332] In some embodiments of the invention, PBL Method 1 is performed as follows: On day 0, cryopreserved PBMC samples are thawed and PBMCs are counted. T cells are isolated using a human Pan T cell isolation kit and LS columns (Miltenyi Biotec).

[0333] PBL Method 2. In some embodiments of the invention, PBLs are expanded using PBL Method 2, which involves obtaining a PBMC sample from whole blood. PBMC-derived T cells are enriched by incubating the PBMCs at 37° C. for at least 3 hours, followed by isolation of non-adherent cells.

[0334] In some embodiments of the present invention, PBL Method 2 is performed as follows: On day 0, cryopreserved PBMC samples are thawed and PBMC cells are seeded at 6 million cells / well in 6-well plates in CM-2 medium and incubated for 3 hours at 37° C. After 3 hours, non-adherent cells, which are PBLs, are removed and counted.

[0335] PBL Method 3. In some embodiments of the invention, PBLs are expanded using PBL Method 3, which involves obtaining a PBMC sample from peripheral blood. B cells are isolated using CD19+ selection and T cells are selected using negative selection of the non-CD19+ fraction of the PBMC sample.

[0336] In some embodiments of the present invention, PBL Method 3 is performed as follows: On day 0, cryopreserved PBMCs obtained from peripheral blood are thawed and counted. CD19+ B cells are sorted using a CD19 Multisort Kit, Human (Miltenyi Biotec). Of the non-CD19+ cell fraction, T cells are purified using a Human Pan T Cell Isolation Kit and an LS column (Miltenyi Biotec).

[0337] In some embodiments, PBMCs are isolated from a whole blood sample. In some embodiments, the PBMC sample is used as a starting material for expanding PBLs. In some embodiments, the sample is cryopreserved prior to the expansion process. In other embodiments, a fresh sample is used as a starting material for expanding PBLs. In some embodiments of the invention, T cells are isolated from PBMCs using methods known in the art. In some embodiments, T cells are isolated using a human T pan-cell isolation kit and an LS column. In some embodiments of the invention, T cells are isolated from PBMCs using antibody selection methods known in the art, such as CD19 negative selection.

[0338] In some embodiments of the invention, the PBMC sample is incubated at a desired temperature for a period of time effective to identify non-adherent cells. In some embodiments of the invention, the incubation time is about 3 hours. In some embodiments of the invention, the temperature is about 37°C. The non-adherent cells are then expanded using the process described above.

[0339] In some embodiments, the PBMC sample is from a subject or patient who has been optionally pretreated with a regimen comprising a kinase inhibitor or an ITK inhibitor. In some embodiments, the tumor sample is from a subject or patient who has been pretreated with a regimen comprising a kinase inhibitor or an ITK inhibitor. In some embodiments, the PBMC sample is from a subject or patient who has been pretreated with a regimen comprising a kinase inhibitor or an ITK inhibitor and has been treated for at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, or a year or more. In other embodiments, the PBMCs are from a patient currently receiving an ITK inhibitor regimen, such as ibrutinib.

[0340] In some embodiments, the PBMC sample is from a subject or patient who has been pre-treated with a regimen comprising a kinase inhibitor or an ITK inhibitor and is refractory to treatment with a kinase inhibitor or an ITK inhibitor, such as ibrutinib.

[0341] In some embodiments, the PBMC sample is from a subject or patient who was previously treated with a regimen containing a kinase inhibitor or an ITK inhibitor but is no longer receiving treatment with the kinase inhibitor or ITK inhibitor. In some embodiments, the PBMC sample is from a subject or patient who was previously treated with a regimen containing a kinase inhibitor or an ITK inhibitor but is no longer receiving treatment with the kinase inhibitor or ITK inhibitor and has not been treated with the kinase inhibitor or ITK inhibitor for at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, or at least 1 year, or longer. In other embodiments, the PBMCs are from a patient who was previously exposed to an ITK inhibitor but has not been treated within at least 3 months, at least 6 months, at least 9 months, or at least 1 year.

[0342] In some embodiments of the invention, on day 0, cells are selected for CD19+ and sorted accordingly. In some embodiments of the invention, selection is performed using antibody-coupled beads. In some embodiments of the invention, pure T cells are isolated from PBMCs on day 0.

[0343] In some embodiments of the invention, for patients not pretreated with ibrutinib or other ITK inhibitors, 10-15 mL of buffy coat contains approximately 5 x 10 9 This produced approximately 5.5 x 10 PBMCs, which were then 7 Produces PBLs.

[0344] In some embodiments of the present invention, for patients pretreated with ibrutinib or other ITK inhibitors, the expansion process is approximately 20×10 9 In some embodiments of the invention, 40.3 x 10 PBLs are produced. 6 PBMCs are approximately 4.7 x 10 5 Produces PBLs.

[0345] In any of the foregoing embodiments, the PBMCs may be obtained from a whole blood sample, by apheresis, from a buffy coat, or from any other method known in the art for obtaining PBMCs.

[0346] In some embodiments, the PBLs are prepared using the methods described in U.S. Patent Application Publication No. US2020 / 0347350 A1, the disclosure of which is incorporated herein by reference.

[0347] 4. How to expand bone marrow-infiltrating lymphocytes (MIL) from bone marrow-derived PBMCs MIL Method 3. In some embodiments of the invention, the method involves obtaining PBMCs from bone marrow. On day 0, the PBMCs are selected and sorted for CD3+ / CD33+ / CD20+ / CD14+, the non-CD3+ / CD33+ / CD20+ / CD14+ cell fraction is sonicated, and a portion of the sonicated cell fraction is added back to the selected cell fraction.

[0348] In some embodiments of the invention, MIL method 3 is performed as follows: On day 0, cryopreserved PBMC samples are thawed and PBMCs are counted. Cells are stained with CD3, CD33, CD20, and CD14 antibodies and sorted using sorted S3e cells (Bio-Rad). Cells are sorted into two fractions: the immune cell fraction (or MIL fraction) (CD3+CD33+CD20+CD14+) and the AML blast fraction (non-CD3+CD33+CD20+CD14+).

[0349] In some embodiments of the present invention, PBMCs are obtained from bone marrow. In some embodiments, PBMCs are obtained from bone marrow by apheresis, aspiration, needle biopsy, or other similar means known in the art. In some embodiments, the PBMCs are fresh. In other embodiments, the PBMCs are cryopreserved.

[0350] In some embodiments of the invention, the MIL is expanded from 10 to 50 mL of bone marrow aspirate. In some embodiments of the invention, 10 mL of bone marrow aspirate is obtained from a patient. In other embodiments, 20 mL of bone marrow aspirate is obtained from a patient. In other embodiments, 30 mL of bone marrow aspirate is obtained from a patient. In other embodiments, 40 mL of bone marrow aspirate is obtained from a patient. In other embodiments, 50 mL of bone marrow aspirate is obtained from a patient.

[0351] In some embodiments of the present invention, the number of PBMCs generated from about 10-50 mL of bone marrow aspirate is about 5 x 10 7 ~About 10×10 7 In other embodiments, the number of PBMCs produced is about 7 x 10 7 PBMC.

[0352] In some embodiments of the invention, about 5×10 7 ~About 10×10 7 PBMCs, approximately 0.5 x 10 6 ~Approx. 1.5×10 6In some embodiments of the present invention, about 1 x 10 6 MIL is produced.

[0353] In some embodiments of the present invention, 12×10 derived from bone marrow aspirate 6 PBMCs are approximately 1.4 × 10 5 Produces MIL.

[0354] In any of the foregoing embodiments, the PBMCs may be obtained from a whole blood sample, from bone marrow, by apheresis, from buffy coat, or any other method known in the art for obtaining PBMCs.

[0355] In some embodiments, the MIL is prepared using the methods described in U.S. Patent Application Publication No. US2020 / 0347350 A1, the disclosure of which is incorporated herein by reference.

[0356] B. Step B: First Expansion by Priming In some embodiments, the methods provide younger TILs, which may provide additional therapeutic benefit over older TILs (i.e., TILs that have undergone more rounds of replication before administration to a subject / patient). The characteristics of young TILs are described in the literature, for example, Donia, et al., Scand. J. Immunol. 2012, 75, 157-167, Dudley, et al., Clin. al.,Clin.Cancer Res.2013,19,OF1-OF9, Besser,et al.,J.Immunother.2009,32,415-423,Robbins,et al.,J.Immunol.2004,173,7125-7130,Shen,et al. al.,J.Immunother.,2007,30,123-129, Zhou,et al., J. Immunother. 2005, 28, 53-62, and Tran, et al., J. Immunother., 2008, 31, 742-751, the disclosures of each of which are incorporated herein by reference.

[0357] For example, after dissection or digestion of tumor fragments and / or tumor fragments, such as those described in step A of FIG. 8 (particularly, e.g., FIG. 8A and / or FIG. 8B and / or FIG. 8C), the resulting cells are cultured in serum with IL-2, OKT-3, and feeder cells (e.g., antigen-presenting feeder cells) under conditions that favor the growth of TILs over tumor and other cells. In some embodiments, IL-2, OKT-3, and feeder cells are added at the initiation of culture (e.g., day 0) along with the tumor digest and / or tumor fragments. In some embodiments, the tumor digest and / or tumor fragments are incubated in a vessel containing up to 60 fragments per vessel and 6000 IU / mL of IL-2. In some embodiments, this primary cell population is cultured for several days, generally 1-8 days, resulting in a bulk TIL population, generally about 1×10 8In some embodiments, this primary cell population is cultured for several days, generally 1-7 days, resulting in a bulk TIL population, generally about 1 x 10 8 In some embodiments, the first expansion by priming occurs over 1-8 days, resulting in a bulk TIL population, generally about 1 x 10 8 In some embodiments, the first expansion by priming occurs over 1-7 days, resulting in a bulk TIL population, generally about 1 x 10 8 In some embodiments, this first expansion by priming occurs over a period of 5-8 days, resulting in a bulk TIL population, generally about 1 x 10 8 In some embodiments, this first expansion by priming occurs over a period of 5-7 days, resulting in a bulk TIL population, generally about 1 x 10 8 In some embodiments, this first expansion by priming occurs over about 6-8 days, resulting in a bulk TIL population, generally about 1 x 10 8 In some embodiments, this first expansion by priming occurs over about 6-7 days, resulting in a bulk TIL population, generally about 1 x 10 8 In some embodiments, this first expansion by priming occurs over about 7-8 days, resulting in a bulk TIL population, generally about 1 x 10 8 In some embodiments, this first expansion by priming occurs over about 7 days, resulting in a bulk TIL population, generally about 1 x 10 8 In some embodiments, this first expansion by priming occurs over about 8 days, resulting in a bulk TIL population, generally about 1 x 10 8 yielding bulk TIL cells.

[0358] In some embodiments, expansion of TILs can be performed using a first expansion step by priming as described below and herein (e.g., such as that described in step B of FIG. 8 (particularly, e.g., FIG. 8A and / or FIG. 8B and / or FIG. 8C and / or FIG. 8D), which may include a process referred to as pre-REP or priming REP and contains feeder cells from day 0 and / or culture initiation), followed by step D below and a rapid second expansion as described herein (including step D, a process referred to as the rapid expansion protocol (REP) step), followed by optional cryopreservation, followed by a second step D below and described herein (including a process referred to as the restimulation REP step). TILs obtained from this process can optionally be characterized for phenotypic characteristics and metabolic parameters as described herein. In some embodiments, tumor fragments are approximately 1 mm 3 ~10mm 3 is.

[0359] In some embodiments, the first expansion culture medium is referred to as "CM," an abbreviation for culture medium. In some embodiments, the CM of Step B consists of RPMI 1640 with GlutaMAX, supplemented with 10% human AB serum, 25 mM Hepes, and 10 mg / mL gentamicin.

[0360] In some embodiments, there are 240 or fewer tumor fragments. In some embodiments, there are 240 or fewer tumor fragments placed in four or fewer containers. In some embodiments, the containers are GREX100 MCS flasks. In some embodiments, 60 or fewer tumor fragments are placed in one container. In some embodiments, each container contains 500 mL or fewer of medium per container. In some embodiments, the medium contains IL-2. In some embodiments, the medium contains 6000 IU / mL of IL-2. In some embodiments, the medium contains antigen-presenting feeder cells (also referred to herein as "antigen-presenting cells"). In some embodiments, the medium contains 2.5 x 10 tumor fragments per container. 8In some embodiments, the medium comprises OKT-3. In some embodiments, the medium comprises 30 ng / mL OKT-3 per vessel. In some embodiments, the vessel is a GREX100 MCS flask. In some embodiments, the medium comprises 6000 IU / mL IL-2, 30 ng OKT-3, and 2.5 x 10 8 In some embodiments, the medium contains 6000 IU / mL of IL-2, 30 ng / mL of OKT-3, and 2.5 x 10 antigen-presenting feeder cells per vessel. 8 The antigen-presenting feeder cells are included.

[0361] After preparation of the tumor fragments, the resulting cells (i.e., the fragments, which are the primary cell population) are cultured in medium containing IL-2, antigen-presenting feeder cells, and OKT-3 under conditions that favor the growth of TILs over tumor and other cells and allow for TIL priming and accelerated growth starting from culture initiation on day 0. In some embodiments, tumor digests and / or tumor fragments are incubated with 6000 IU / mL of IL-2, antigen-presenting feeder cells, and OKT-3. This primary cell population is cultured for several days, generally 1-8 days, resulting in a bulk TIL population, generally about 1 x 10 8 In some embodiments, the growth medium during the first expansion by priming comprises IL-2 or a variant thereof, as well as antigen-presenting feeder cells and OKT-3. In some embodiments, this primary cell population is cultured for several days, generally 1-7 days, resulting in a bulk TIL population, generally about 1 x 10 8 In some embodiments, the growth medium during the first expansion by priming contains IL-2 or a variant thereof, as well as antigen-presenting feeder cel...

Claims

1. 1. A method for modulating the production of expanded T cells for treating cancer in a patient, said method comprising: and manufacturing the cell therapy product by expanding a population of cells obtained from a tumor from the patient into the cell therapy product, said manufacturing comprising: providing a patient registration portal that enables hospital personnel at a hospital facility, via a computing device, to securely register the patient, assign a unique patient identifier to the patient and / or submit a product order request including an order identifier associated with the patient identifier, select a manufacturing facility for manufacturing the cell therapy product from the patient's biological sample, and have information about the patient and the product order request stored in a central database; providing, via the computing device, a tumor procurement portal including a smart checklist configured to facilitate the hospital personnel securely extracting the biological specimen from the patient, associating the biological specimen with the order identifier, and creating a record of the procedure for extracting the biological specimen including a record of the chain of custody of the biological specimen and an inventory of materials used during the procedure, wherein the tumor procurement portal allows the hospital personnel to generate labels for containers for the extracted biological specimens, the labels including the order identifier, and wherein data entered into the smart checklist by the hospital personnel when performing the procedure is stored and / or updated in the central database; enabling, via the computing device, maintaining the record of the chain of custody while securely receiving the biological specimen shipped from the hospital facility; enabling automation of a manufacturing process for the cell therapy product from the biological sample via the computing device; expanding the cell therapy product from at least some of the population of cells contained in the biological sample obtained from the patient using a cell expansion technique, and determining acceptance parameters of the expanded cell population based on one or more quality control assays at a first time point and a second time point subsequent to the first time point; enabling, via the computing device, manufacturing personnel to record data relating to the manufacturing process and the quality control assays, including the record of the chain of custody, to the central database via a manufacturing facility portal, the data relating to the quality control assays including approval parameters obtained at the first time point and the second time point, the approval parameters including one or more of viability, sterility, cell count, mycoplasma count, CD3+ cell count, endotoxin assay results, and Gram stain assay results; generating, via the computing device, labels for containers to be used during the process for manufacturing the cell therapy product and for containers for shipping the manufactured cell therapy product to the hospital facility; Coordinating manufacturing schedules and shipping schedules and exchanging chain of custody and chain of identity records via said computing device; via the computing device, coordinating the scheduling of the shipment and maintaining the record of the chain of custody during the shipment of the patient's biological sample and the manufactured cell therapy product; via the computing device, conducting a manufacturing quality review and generating a preliminary schedule of the patient treatment events that will occur upon receipt of the cell therapy product from the manufacturing facility based on the time required to release the cell therapy product, the time required for shipment to and from the selected manufacturing facility, and time schedules of different patient treatment events, generating a delivery schedule and automatically ordering corresponding pickups and receipts; generating, via the computing device, a report about the end-to-end process from extraction of the biological sample from the patient to infusion of the manufactured cell therapy product into the patient, the report including the record of the chain of custody.

2. i) providing a third user interface configured to enable a third party, including the patient (or their representative), the hospital facility (or its staff), or an administrator of the computing device, to access information regarding the schedule of patient treatment events and / or to securely edit information regarding the patient; ii) communicating with a customer relationship management (CRM) database storing information regarding personnel qualified to interact with the patient to perform tasks related to the patient's treatment with the cell therapy product, the CRM database including the training status of the personnel qualified to interact with the patient; iii) enabling modification of the preliminary schedule of patient treatment events based on outcomes or results during the manufacturing process and approval parameters obtained at one or more points during the manufacturing process to generate a modified schedule of the patient treatment events, and modifying the dispatch schedule according to the modified schedule of the patient treatment events; iv) enabling the hospital personnel, the patient, or the patient's representative to coordinate patient support services, including activities associated with insurance coverage and reimbursement, patient transportation, and / or financial assistance for the patient, while maintaining compliance with HIPAA regulations; v) allowing a limited view of the manufacturing process and / or movement of the biological samples between and / or within the hospital facility and the manufacturing facility; vi) using the smart checklist to limit the display of certain information based on data stored in the central database and data provided by the hospital personnel performing the extraction of the biological sample; vii) using the smart checklist to limit data entry for a subsequent process step during the extraction of the biological sample in response to a failure to match information printed on the label of a container used in the subsequent process with information entered during the current process step, wherein the information includes one or more parameters including an order identifier and the type of reagent or material used in the subsequent process step, the expiration date of a reagent or material used in the subsequent process step, and the identification and training status of hospital personnel corresponding to the subsequent process step; vii) enabling information regarding active and available production slots to be updated to the central database and enabling display of the active and available production slots to enable determination of production capacity and inventory; ix) updating information regarding material inventory associated with said manufacturing process; x) verifying and reconciling a number of labels printed for each of the various process steps performed during the manufacturing of the cell therapy product by matching the labels and information printed thereon with corresponding information on the central database; xi) determining a change in the production schedule based on quality information obtained at one or more points during the production process to determine a modified production schedule; xii) enabling an authorized user of said computing device to reschedule one or more of said patient treatment events in response to said modified production schedule; xiii) enabling a logistics provider to generate a shipping label that includes an order identifier, a lot number, and information regarding a handler that will handle the shipping container during intermediate shipping and / or transportation steps, thereby enabling the logistics provider to include the intermediate shipping and / or transportation steps while maintaining the record of the chain of custody; The method of claim 1 further comprising:

3. 10. The method of claim 1, wherein the patient registration portal further enables the hospital personnel to review, adjust, and approve the product order request, and the computing device is further configured to generate a purchase order and generate a lot number for manufacturing the cell therapy product based on the order identifier and the patient identifier.

4. 10. The method of claim 1, wherein the product order includes one or more of the order identifier, patient identification, a preliminary schedule for manufacturing, information regarding the expected manufacturing process, and information regarding the hospital facility and hospital facility personnel, and information regarding expected quality control parameters of the cell therapy product.

5. 10. The method of claim 1, wherein generating the preliminary schedule of the patient treatment events is further based on availability of manufacturing slots at the manufacturing facility and schedules of the hospital personnel associated with various treatment processes.

6. 10. The method of claim 1, wherein selecting the manufacturing facility for manufacturing the cell therapy product is based on availability of manufacturing slots, geographic location of the manufacturing facility, and availability of a desired process for manufacturing the cell therapy product.

7. The method of claim 1 , wherein the label on the container includes the order identifier, information about the hospital personnel performing the current process step, and usability information for objects used during the current process step.

8. 10. The method of claim 1, wherein verifying the information printed on the label comprises scanning the label and using a machine-readable algorithm to extract the information printed on the label.

9. 10. The method of claim 1, wherein the tumor procurement portal further enables generation and printing of a shipping label for shipping a shipping container containing the biological sample to the manufacturing facility, the shipping label including at least the order identifier and one or more of information regarding the hospital staff handing over the container to delivery personnel, information regarding the delivery personnel, parameters associated with the shipping container, and proof of handoff between the hospital staff and the delivery personnel.

10. the manufacturing facility portal: i) to enable verification of the training status of manufacturing personnel receiving shipping containers containing said biological samples of said patients from said hospital facility; ii) upon receipt of the shipping container containing the patient's biological sample from the hospital facility, allowing input of one or more parameters associated with the shipping container and the quality of the biological sample contained therein, and matching the one or more parameters with corresponding data stored in the central database to verify that the chain of custody, the patient's identity, and the desired quality of the biological sample correspond to the product order; and / or iii) configured to enable automation of manufacturing processes by displaying the status of the cell therapy product in real time, wherein the status includes quality control information for a current process, a process immediately preceding the current process, and an expected time to complete the current process; The method of claim 1 further comprising:

11. 10. The method of claim 1, wherein the label on a container used during the process to manufacture the cell therapy product includes at least one of the order identifier and a quality control report, information about manufacturing personnel handling the container, information about the process step in which the container is used, and a reason code corresponding to the reason the label was printed.

12. 10. The method of claim 1, wherein at each process step, information printed on the label is matched with corresponding information in the central database entered during or upon completion of the immediately preceding process step.

13. 10. The method of claim 1, wherein some or all of the information printed on the label is coded using a one-dimensional or two-dimensional machine-readable code.

14. 10. The method of claim 1, wherein verifying the information printed on the label comprises scanning the label and using a machine-readable algorithm to extract the information printed on the label.

15. 2. The method of claim 1, wherein information extracted from the label is recorded in the central database to enable the generation of a report of the chain of custody and / or the chain of identity.

16. 10. The method of claim 1, wherein the manufacturing facility portal further enables generation and printing of a shipping label for shipping a shipping container containing the manufactured cell therapy product to the hospital facility, the shipping label including at least the order identifier and one or more of information regarding the manufacturing personnel handing over the container to the delivery personnel, information regarding the delivery personnel, parameters associated with the shipping container, and proof of handoff between the manufacturing personnel and the delivery personnel.

17. A method for producing expanded T cells for treating cancer in a patient, said method comprising: and manufacturing the cell therapy product by expanding a population of cells obtained from a tumor from the patient into the cell therapy product, said manufacturing comprising: providing a patient registration portal that enables hospital personnel at a hospital facility, via a computing device, to securely register the patient, assign a unique patient identifier to the patient and / or submit a product order request including an order identifier associated with the patient identifier, select a manufacturing facility for manufacturing the cell therapy product from the patient's biological sample, and have information about the patient and the product order request stored in a central database; providing, via the computing device, a tumor procurement portal including a smart checklist configured to facilitate the hospital personnel securely extracting the biological specimen from the patient, associating the biological specimen with the order identifier, and creating a record of the procedure for extracting the biological specimen including a record of the chain of custody of the biological specimen and an inventory of materials used during the procedure, wherein the tumor procurement portal allows the hospital personnel to generate labels for containers for the extracted biological specimens, the labels including the order identifier, and wherein data entered into the smart checklist by the hospital personnel when performing the procedure is stored and / or updated in the central database; maintaining, via the computing device, the record of the chain of custody while securely receiving the biological specimen shipped from the hospital facility; expanding at least a portion of the cell population by a multi-step cell expansion process conducted in a closed bioreactor at said manufacturing facility to obtain a cell therapy product; performing one or more quality control assays on at least a portion of the cell therapy product at a first time point during the multi-step cell expansion process and at a second time point subsequent to the first time point to obtain approval parameters for the expanded cell population at corresponding time points, the approval parameters comprising one or more of viability, sterility, cell count, mycoplasma count, CD3+ cell count, endotoxin assay results, and Gram stain assay results; recording, via the computing device, via a manufacturing facility portal, data relating to the manufacturing process and the quality control assays, including the record of the chain of custody, into the central database, the data relating to the quality control assays including approval parameters obtained at the first time point and the second time point; automatically generating, via the computing device, labels for containers used during the process for manufacturing the cell therapy product and for shipping the manufactured cell therapy product to the hospital facility, the labels including the unique patient identifier and the order identifier; generating, via said computing device, a production schedule and a shipping schedule and exchanging said chain of custody and chain of identity records; via the computing device, generating a schedule of the shipment and maintaining the record of the chain of custody during the shipment of the patient's biological sample and the manufactured cell therapy product; via the computing device, conducting a manufacturing quality review and generating a preliminary schedule of the patient treatment events to occur before and upon receipt of the cell therapy product from the manufacturing facility based on the time required to release the cell therapy product, the time required for shipment to and from the selected manufacturing facility, and time schedules of different patient treatment events, generating a delivery schedule and automatically ordering corresponding pickups and receipts; providing a user interface configured to allow parties, including the patient (or their representative), the hospital facility (or its staff), or an administrator of the computing device, via a computing device, to selectively remotely access information regarding the schedule of patient treatment events over a network and / or securely edit information regarding the patient; generating, via the computing device, a report about the end-to-end process from extraction of the biological sample from the patient to infusion of the manufactured cell therapy product into the patient, the report including the record of the chain of custody; The method determining modifications to the manufacturing schedule based on changes in the approved parameters of the expanded cell population obtained at one or more time points during the manufacturing process; generating a report, further comprising: enabling an authorized user of the computing device to reschedule one or more patient treatment events in response to the modified production schedule; The method comprising:

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