Compositions and methods for the expansion of T cells and tumor-infiltrating lymphocytes

Abstract

The present disclosure provides compositions and methods for expanding T cells or tumor infiltrating lymphocytes (TILs) in vitro. K562 feeder cells engineered to express costimulatory molecules (e.g., 41BB ligand (41BBL)) and either interleukin 21 (IL21) or interleukin 7 (IL7) can be used in a rapid expansion protocol (REP) step to expand T cells or TILs. Thus, provided herein is a culture comprising T cells or TILs and engineered K562 feeder cells. T cells can be engineered to express chimeric antigen receptors (CARs) or T cell receptors (TCRs), or TILs can be engineered to express membrane-bound IL15 (mbIL15). T cells or TILs can be expanded in vitro using REP without exogenous interleukin 2 (IL2), and the expanded cells can be used in adoptive cell therapy for the treatment of cancer without the concomitant use of exogenous cytokines such as IL2.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Application No. 63 / 139,305 filed on 19 January 2021, U.S. Provisional Application No. 63 / 153,367 filed on 24 February 2021, U.S. Provisional Application No. 63 / 226,114 filed on 27 July 2021, and U.S. Provisional Application No. 63 / 244,166 filed on 14 September 2021, which are incorporated herein by reference in their entirety for all purposes. [Background technology]

[0002] Adoptive cell therapy (ACT) using T cells or tumor infiltrating lymphocytes (TILs) has emerged as a clinical tool for the treatment of immunocompromised or cancer subjects. T cells prepared from peripheral blood mononuclear cells (PBMCs) or TILs prepared from tumors have been used with varying degrees of success in ACT. Typically, in vitro expansion of the cell population is required before injection into the subject. Conventional expansion involves a two-step process, with the first step referred to as the pre-rapid expansion protocol (pre-REP) step and the second step referred to as the rapid expansion protocol (REP) step. The pre-REP step requires that the isolated cells be cultured in the presence of interleukin 2 (IL2) but in the absence of feeder cells, while the REP step typically requires feeder cells (e.g., peripheral blood mononuclear cells (PBMCs)), high-dose IL2, and optionally an anti-CD3 antibody (OKT3). Conventional expansion works for most T cells or TILs, but genetically modified cells may require a modified expansion process to selectively expand the population of modified T cells or TILs. Additionally, expanded T cells or TILs typically receive co-administration of IL2 when administered to a patient to activate and expand the T cell or TIL population in vitro. However, IL2 exhibits dose-dependent toxicity, which can manifest in multiple organ systems, most notably the heart, lungs, kidneys, and central nervous system. The most common sign of IL-2 toxicity is capillary leak syndrome, which results in hypovolemia and fluid accumulation in the extravascular space. A significant number of patients cannot tolerate adjuvant IL2 therapy and must be excluded from TIL treatment. In order to make ACT a safer and more effective therapeutic tool, in situ improvements are needed. (Background Art)

Summary of the Invention

Means for Solving the Problems

[0003] This disclosure relates to compositions and methods for expanding modified T cells or tumor infiltrating lymphocytes (TILs) in vitro. K562 feeder cells engineered to express either a costimulatory molecule (e.g., 41BB ligand (41BBL)) and interleukin 21 (IL21) or interleukin 7 (IL7) are used at the REP stage to expand modified T cells or TILs, particularly T cells or TILs that do not expand well under conventional REP conditions. Accordingly, cultures containing modified T cells or TILs and modified K562 feeder cells are provided herein. The K562 feeder cells optionally are replication-incompetent and contain a first exogenous nucleic acid sequence encoding a costimulatory molecule selected from the tumor necrosis factor superfamily (e.g., 41BBL) and a second exogenous nucleic acid sequence encoding IL21 or IL7. The T cells or TILs are modified T cells or TILs. As an example, the culture can contain T cells or TILs modified to express a cytokine (e.g., IL15 or membrane-bound IL15 (mbIL15)), a chimeric antigen receptor (CAR), and / or a T cell receptor (TCR). Optionally, one or more of the CAR, TCR, or mbIL15 is operably linked, optionally, to a drug-responsive domain (DRD), and the K562 feeder cells optionally express 41BBL and IL21 (e.g., membrane-bound IL21).

[0004] The DRD is a polypeptide that can control the abundance and / or activity of a payload, such as mbIL15, upon binding to a ligand. Multiple DRDs can regulate a single payload, for example, in series. One or more DRDs are operably linked to a mbIL15, CAR, or TCR payload, and interaction of the DRD with an effective amount of a ligand under appropriate conditions results in modification of the biological activity or amount of the payload.

[0005] Also provided is a method for expanding modified T cells or TILs by culturing them in the presence of a population of modified K562 feeder cells, wherein the modified K562 feeder cells comprise a first exogenous nucleic acid sequence encoding a costimulatory molecule selected from the tumor necrosis factor superfamily (e.g., 41BBL) and a second exogenous nucleic acid sequence encoding IL21 or IL7. This method allows the REP step to proceed in the absence of exogenous IL2. The T cells or TILs expanded by this method are modified (manipulated). For example, this method can be used to expand TILs modified to express membrane-bound IL15 which can be operably linked to a DRD, or to expand T cells modified to express cytokines, CARs, and / or TCRs, any of which can be operably linked to a DRD.

[0006] In a method for expanding modified T cells or TILs, modified K562 feeder cells may be non-reproducible and can be engineered to express IL21 (e.g., secreted IL21 or membrane-bound IL21). Furthermore, K562 feeder cells can be engineered to express 41BBL.

[0007] Furthermore, this specification also provides a method for expanding TILs engineered to express mbIL15 by culturing the TILs in the presence of modified K562 feeder cells. TILs engineered to express mbIL15 expand in the absence of exogenous cytokines (e.g., exogenous interleukins such as IL2, IL7, IL15, or their variants). Modified K562 feeder cells are non-replicating and can be modified to express a costimulatory molecule selected from the tumor necrosis factor superfamily (e.g., 41BBL), and / or to express IL21 or IL7. Optionally, in the method for expanding TILs, K562 feeder cells are modified to express mbIL21.

[0008] An expanded population of T cells or TILs is provided. The population of modified T cells or TILs is expanded in the presence of K562 feeder cells that are non-replicating and modified to express a costimulatory molecule selected from the tumor necrosis factor superfamily (e.g., 41BBL), and / or modified to express IL21 (e.g., mbIL21) or IL7 (e.g., mbIL7). Modified T cells and TILs modified by the method herein have certain advantageous properties. The absence of IL2 in the REP step has the advantage of producing TILs or T cells that differentiate or are effluxed less than unmanipulated TILs or T cells expanded in the presence of IL2. Furthermore, TILs produced according to the method herein can be administered to patients in need without requiring concomitant IL2 therapy, which is toxic to many patients and also effluxes T cells or TILs. For example, expanded modified T cells or TILs survive (persist) more than unmanipulated cells. The expanded T cell or TIL population has a higher proportion of CD8+ cells and a lower proportion of CD4+ cells compared to the control population of non-expanded T cells or TILs. Therefore, the expanded T cell or TIL population has a lower CD4:CD8 ratio than the control population of expanded non-expanded TILs or TILs or T cells on allogeneic PBMCs. Furthermore, the expanded mbIL15 T cell or TIL population has a lower CD4:CD8 ratio than the control population of T cells or TILs on allogeneic PBMCs. reg Compared to the proportion of cells, T regThe proportion of cells is low. The population of expanded T cells or TILs also has a lower proportion of PD1+ cells compared to the proportion of PD1+ cells in the control population of unexpanded T cells or TILs or the control population of expanded TILs or T cells on PBMCs. The population of expanded T cells or TILs described herein also has a higher proportion of polyfunctional cells producing tumor necrosis factor α (TNFα) and interferon γ (IFNγ) compared to the proportion of T cells and TILs producing TNFα and IFNγ in the control population of unexpanded TILs. Furthermore, modified T cells (e.g., T cells modified to express cytokines, CARs, or TCRs) or modified TILs (e.g., TILs modified to express mbIL15) expand more than unmodified TILs in the presence of K562 feeder cells, 41BB ligand (41BBL), and interleukin 21 (IL21, secreted or membrane-bound to K562 feeder cells). The preferential expansion of modified T cells or TILs occurs in the absence of exogenous cytokines such as IL2.

[0009] This specification provides a method for treating cancer in a subject by administering an expanded population of modified T cells or modified TILs, expanded according to the method described herein. Optionally, T cells or TILs are expanded in the presence of K562 feeder cells that are non-replicating and modified to express a costimulatory molecule selected from the tumor necrosis factor superfamily (e.g., 41BBL), and / or modified to express IL21 (e.g., mbIL21) or IL7 (e.g., mbIL7). The expansion step is optionally performed in the absence of IL2. Furthermore, the expanded cells can be administered to a subject without the administration of exogenous IL2. Systemic administration of IL2 to cancer patients in combination with or after immunotherapy often causes toxicity in patients who are already medically vulnerable. Many patients suffer severe, life-threatening side effects after IL2 administration, including hypotension and shock due to capillary leak syndrome. TIL therapy with low-dose combination IL2 has been attempted, but immunotherapy has been less effective than when IL2 is administered at high doses. Therefore, modified T cells or modified TILs expanded according to the methods described herein can be used in therapeutic regimens that demonstrate less toxicity to subjects with cancer than current therapeutic regimens that require the use of exogenous IL2. T cells or TILs expanded according to the methods taught herein and administered to a subject may be further engineered such that the T cells are engineered to express cytokines (e.g., IL15), CARs, or TCRs operably linked to one or more DRDs, or the TILs are engineered to express mbIL15 operably linked to one or more DRDs.

[0010] The identified embodiments are illustrative only and therefore non-limiting. Details of one or more non-limiting embodiments of the present invention are described in the accompanying drawings and the following description. Other embodiments of the present invention should be apparent to those skilled in the art after considering this disclosure. [Brief explanation of the drawing]

[0011] [Figure 1]The frequency of CD3+ T cells in CD45+ cells (left) and CD45+ cells (right) in fresh tumor digests and 3 weeks after pre-REP TIL culture. [Figure 2] This shows the transduction efficiency of IL15-293 constructs in two melanoma TIL donors, measured by flow cytometry 5 days after transduction. [Figure 3] This shows antigen- and IL2-independent expansion and survival of TILs expressing mbIL15. A shows TIL donor 006 cells (TIL006) transduced with constitutive mbIL15 or GFP and expanded in REP for 12 days with or without 6000 IU / mL IL2. B shows TIL006 transduced with constitutive mbIL15 (without IL2 and expanded in REP) or GFP (expanded in REP with 6000 IU / mL IL2) and enumerated in an antigen-independent survival assay for 14 days with or without 6000 IU / mL IL2. [Figure 4] This shows antigen-independent TIL expansion after the rapid expansion protocol (REP). After REP, unprocessed and mbIL15-processed TILs (constitutive or modified mbIL15) were plated with or without exogenous IL2 or acetazoleamide (ACZ), and new wells were collected every 3 days for cell enumeration and phenotypic evaluation. [Figure 5] This study demonstrates TIL expansion in antigen-dependent settings. Following a rapid expansion protocol (REP), unmodified and mbIL15-modified TILs were plated with HLA-matched mitomycin C-treated melanoma cells in a TIL:tumor co-culture assay, with or without exogenous IL2, acetazoleamide, or vehicle (DMSO). Wells were collected every three days, and cell enumeration and phenotypic evaluation were performed. [Figure 6]This shows the tumor reactivity of TILs after the rapid expansion protocol (REP). A shows TIL006 and TIL005, both transduced with controlled mbIL15 and an uncontrolled control, and co-cultured for 24 hours with HLA-matched mitomycin C-treated melanoma cells. IFNγ in the supernatant was measured by the MSD assay. B shows the cytotoxicity of TILs in co-culture, measured by loss of luminescence in luciferase-tagged HLA-matched melanoma cells. [Figure 7] This shows the TIL expansion and transduction efficiency before injection into animals for in vivo adoptive cell therapy experiments. A shows the cell expansion of TIL donor 006 used for in vivo adoptive cell transfer (ACT) of unmanipulated and mbIL15-manipulated TILs. B shows the transduction efficiency after the rapid expansion protocol (REP). Transduction efficiency was evaluated for IL15 and IL15RaFc expression in unmanipulated and mbIL15-manipulated TILs. [Figure 8-1] This paper presents a list of TILs and an analysis of IL15 expression for in vivo adoptive cell therapy experiments. Adoptive-transferred, unmodified, and mbIL15-modified TILs are listed by flow cytometry from peripheral blood samples. TILs were identified as live human CD3+ mouse CD45- cells in submandibular vein blood samples. [Figure 8-2] This paper presents the enumeration of TILs and analysis of IL15 expression for in vivo adoptive cell therapy experiments. It shows the enumeration of TILs (hCD3+mCD45-) and IL15 expression (IL15+IL15RaFc+) in spleen and bone marrow samples isolated 14 or 53 days after ACT. [Figure 8-3] This paper presents the enumeration of TILs and analysis of IL15 expression for in vivo adoptive cell therapy experiments. It shows the enumeration of TILs (hCD3+mCD45-) and IL15 expression (IL15+IL15RaFc+) in spleen and bone marrow samples isolated 14 or 53 days after ACT. [Figure 9]This study demonstrates that acetazoleamide (ACZ) regulation of IL15 expression and signaling in cryopreserved regulated mbIL15 TILs occurs in a dose-dependent manner. Regulated mbIL15 TILs from four patients (patients 1-4) were thawed, left in ACZ-free medium for 24 hours, and then regulated in 0.1, 1, 2.5, 5, 10, 25, and 100 μM ACZ for 18 hours. Regulated mbIL15 TILs were then collected using a phosphoflow cytometry-based assay and analyzed for IL15 expression and signaling. A shows the frequency of IL15+ TILs as a percentage of CD3+ cells. B-E show the results for each patient. Here, cells were further gated on IL15+, and the geometric mean fluorescence intensity of each pSTAT5 (outlined square) and pS6 (closed circle) was calculated. The values ​​shown are set relative to the vehicle control. N=4 human donors. [Figure 10] The mean fluorescence intensity (MFI) of pSTAT5 and pS6 for patients 1-4 is shown. A shows the MFI of pSTAT5. B shows the MFI of pS6. [Figure 11] This study demonstrates that constitutive mbIL15 expression and ACZ regulation of regulated mbIL15 TILs are involved in the IL15 signaling pathway. Here, unmanipulated and regulated mbIL15 TILs from patients 1–3 were thawed, left in ACZ-free medium for 24 hours, and then regulated with IL2 or ACZ for 18 hours. Cells were then collected using phosphoflow cytometry-based assays and analyzed for IL15 expression and signaling. Unmanipulated TILs and regulated mbIL15 TIL+ vehicles were gated on live cells, then on singlets, then on CD3+. Constitutive IL15 TILs and regulated mbIL15 TIL+ ACZ conditions were further gated on IL15+ staining. Geometric mean fluorescence intensities for each pSTAT5 and pS6 were calculated. N=3 human donors. [Figure 12]This study demonstrates that modified mbIL5-modified TILs, free from exogenous cytokines, exhibit greater polyfunctionality than unmodified TILs + IL2. Unmodified TILs and modified mbIL15 TILs were thawed and left in ACZ-free medium for 24 hours. Unmodified TILs were then treated with IL2 at the following concentrations: 20, 200, 1000, and 6000 IU / mL or vehicle, and modified mbIL15 TILs were treated with ACZ at the following concentrations: 0.1, 1, 5, 10, 25, and 100 μM ACZ or vehicle. The treatment lasted 18 hours. Cells were stimulated with PMA and ionomycin for 6 hours in the presence of brefeldin A and monensin. Unstimulated TILs were used as a control (data omitted). After stimulation, cells were analyzed for IL15 and intracellular TNFα and IFNγ expression using flow cytometry-based assays. TILs were gated with live cells, then singlets, then CD3+, and modified mbIL15 TILs were further gated with IL15+. A shows the TNFα and IFNγ double-positive population for unmodified TILs with IL2 and modified mbIL15 TILs with ACZ. B shows IL15 expression in modified mbIL15 TIL cultures. C shows a comparison of selected IL2 (200 IU / mL) and ACZ (25 μM) doses. [Figure 13] The results of a patient-derived xenograft (PDX) efficiency model are shown. At the end of the rapid expansion protocol (REP), unmanipulated TILs and modified mbIL15 TILs (+ / - acetazoleamide (ACZ)) were adopted into mice with human melanoma PDX. Mean tumor volume was assessed (+ / - SEM). A shows the mean tumor volume of a given treatment in days after adoptive cell transfer (ACT). B shows the tumor volume in days after ACT for no TIL (top left), unmanipulated TIL + IL2 (top right), modified mbIL15 TIL + vehicle (bottom left), and modified mbIL15 TIL + ACZ (bottom right). Here, modified mbIL15 TIL + ACZ showed significantly superior antitumor efficacy compared to unmanipulated TIL + IL2 (*p<0.05, Mann U Whitney test). [Figure 14] The results for the SK-MEL-1 xenograft cancer model are shown. At the end of the rapid expansion protocol (REP), unmanipulated TILs and modified mbIL15 TILs (+ / - acetazoleamide (ACZ)) were adopted into mice with SK-MEL-1 tumors. Mean tumor volume was assessed (+ / - SEM). A shows the mean tumor volume of a given treatment in days after adoptive cell transfer (ACT). B shows the tumor volume in days after ACT for no TIL (top left), unmanipulated TIL + IL2 (top right), modified mbIL15 TIL + vehicle (bottom left), and modified mbIL15 TIL + ACZ (bottom right). Here, modified mbIL15 TIL + ACZ showed significantly superior antitumor efficacy compared to unmanipulated TIL + IL2 (*p<0.05, Mann U Whitney test). [Figure 15] This study demonstrates that modified mbIL15 TILs achieve enhanced MHC-I-dependent cytotoxicity against melanoma in vitro. Here, unmodified and modified mbIL15 TILs were cryopreserved at the end of a rapid expansion protocol (REP). The cryopreserved TILs were thawed, left overnight under cytokine-free conditions, and then co-cultured with cell-trace violet-labeled melanoma cells (SK-MEL-1) in 1:1 and 5:1 effector-to-target (TIL:melanoma) ratios. To control MHC-1-dependent cytotoxicity, melanoma cells were pre-treated with 80 μg / mL of HLA ABC MHC blocking antibody for 2 hours prior to the assay. After 3 hours of co-culture, SK-MEL-1 cells were evaluated for intracellular cleavage caspase 3 expression (a marker of irreversible commitment to cell death) by flow cytometry. Quantified cleavage caspase 3 was normalized to that of target cells alone (spontaneous or background release). The bar graph shows the expression of cleavage capsase-3 on target tumor cells when co-cultured with TILs from six individual patients. [Figure 16]This graph shows that maximum TIL dilation occurs in REP when mbIL15 TIL (constitutive) is generated using K562 feeder cells that have both IL21 and 41BBL-mediated co-stimulation. [Figure 17] This graph shows that maximal TIL expansion occurs in REP when unmanipulated TILs are generated in pooled PBMC feeders or K562 feeder cells expressing membrane-bound IL21 and 41BBL. [Figure 18] This study demonstrates that TILs containing mbIL15 (constitutive) are generated in K562 feeder cells and that maximum dilation of IL15+ TILs occurs in REP when co-stimulated via both IL21 and 41BBL. The results for feeder cells on days 8, 11, 15, and 18 are shown from left to right. These are PBMC feeders, K562 parental feeders, K562+41BBL, K562+41BBL feeders with recombinant human IL21, K562+mbIL21 feeders, and K562+41BBL+mbIL21 feeders. [Figure 19] This graph shows that IL15 expression is generated in K562 feeder cells and enriched through the REP process of mbIL15 TIL (constitutive) that receives both IL21 and 41BBL-mediated co-stimulation. [Figure 20]This graph shows that TILs enlarged with mbIL15 generated in K562 feeder cells with both IL21 and 41BBL-mediated co-stimulation exhibit a reduced CD4:CD8 ratio via REP. Therefore, TILs with enlarged mbIL15 in the presence of K562 feeder cells with both IL21 and 41BBL stimulation are enriched in CD8+ cytotoxic effector cells, in contrast to enlarged TILs with mbIL15 generated in pooled PBMC feeders, unmodified K562 feeders, or K562 feeders expressing 41BBL in the absence of IL21. The CD4:CD8 ratio is shown from left to right on days 8, 11, 15, and 18. These are PBMC feeders, K562 parent feeders, K562+41BBL feeders, recombinant human IL21 K562+41BBL feeders, K562+mbIL21 feeders, and K562+41BBL+mbIL21 feeders. [Figure 21] This graph shows that the proportion of TNFα+ interferon-γ+ cells is higher in expanded mbIL15 TILs generated from K562 feeder cells expressing both mbIL21 and 41BBL compared to mbIL15 TILs generated from PBMC feeder cells or unmodified K562 feeder cells. The higher proportion of TNFα+ interferon-γ+ TILs indicates improved polyfunctionality in expanded mbIL15 TILs generated from K562 feeder cells expressing both mbIL21 and 41BBL. [Figure 22]This graph shows the results of a 10-day survival assay for mbIL15 TILs generated from PBMC feeder cells, unmodified K562 feeder cells, K562 feeder cells expressing only mb41BBL, K562 feeder cells expressing only mbIL21, K562 feeder cells expressing both 41BBL and mbIL21, and K562 feeder cells expressing 41BBL in the presence of recombinant human IL21. Extended mbIL15 TILs generated from K562 feeder cells and co-stimulated via both IL21 and 41BBL demonstrated improved antigen-independent survival after REP compared to mbIL15 TILs generated from PBMC feeder cells or K562 feeder cells that were either unmodified or modified to independently express mbIL21 or 41BBL. [Figure 23] This shows the relative proportions of the TCRVβ subfamily in unmanipulated TILs and mbIL15 TILs expanded using PBMC feeders, K562 feeders, K562+mbIL21 feeders, K562+41BBL feeders, K562+41BBL+mbIL21 feeders, or K562+41BBL+rhIL21 feeders. Expanded mbIL15 TILs and unmanipulated TILs maintain a diverse subfamily distribution regardless of feeder cell type or conditions. [Figure 24] The images show PD1 expression on the surface of mbIL15 TILs, gated on live CD3+ cells from left to right, in non-extended and extended TILs generated using PBMC feeders, K562 parental feeders, K562+41BBL feeders, recombinant human IL21 K562+41BBL feeders, K562+mbIL21 feeders, and K562+41BBL+mbIL21 feeders. PD1 expression was highest in non-extended mbIL15 TILs, and extension of mbIL15 TILs with both 41BBL and IL21-mediated signaling generated TILs with near-baseline PD1 expression. [Figure 25]This section shows phenotypic determination comparing pre-REP TILs (described in Example 1) with manipulated mbIL15 TILs (described in Example 3). Pre-REP and post-REP TILs were phenotyped by flow cytometry using antibodies against CD3, CD4, CD8, and PD1, as described in Example 13. As shown in A, compared to the corresponding pre-REP TILs from the same TIL donor, the post-REP mbIL15 TILs have a higher frequency of CD8+ T cells and a lower frequency of CD4+ T cells. In B, the post-REP mbIL15 TILs express lower levels of PD1 than the corresponding pre-REP TILs from the same TIL donor. C shows the proportion of regulatory T cell populations in mbIL15 TILs identified as CD3+ T cells, which are gated as CD4+ and further classified as CD25 and FoxP3 double-positive cells. The proportion of regulatory T cells in mbIL15 TILs is reduced compared to the pre-REP TILs before the manipulation step. [Figure 26] As determined by flow cytometry, the expression of conserved melanoma-associated antigens MART-1 and gp100 is shown on the A375 melanoma cell line and patient-derived xenograft (PDX) cells (PDX163A as described in Example 11). [Figure 27] The proportions of MART-1 tetramer-positive TILs and gp100-tetramer-positive TILs are shown in mbIL15 TILs derived from four different TIL donors whose HLA matches PDX 163A. The tetramer-positive population indicates that the TILs contain a subset of cells that respond to the corresponding melanoma-associated antigen via the HLA:A2:01 locus. Donors marked with * were used in the PDX efficacy study, as shown in Figure 30. [Figure 28] TILs demonstrate interferon-gamma (IFNγ) production after co-culture of tumor cells and accurately predict TIL donors that respond to PDX. This in vitro assay demonstrates that TIL donors 006, 39A, and 41A are the donors that produce the highest amount of IFNγ in response to PDX, and therefore supports their candidate status as donors for investigating in vitro efficacy, as described in Example 15. [Figure 29] This is a schematic diagram showing an exemplary xenograft model derived from a melanoma patient treated with an extended TIL expressing mbIL15 operably linked to CA2 DRD and CA2 ligand ACZ. [Figure 30] Figure 29 shows that treatment of patient-derived xenograft models with the treatment paradigm presented yields superior antitumor efficacy compared to treatment with unmanipulated TILs and combination IL2 therapy. At the end of the rapid expansion protocol (REP), unmanipulated TILs and modified mbIL15 TILs (+ / - acetazoleamide (ACZ)) were adopted into mice with human melanoma PDX. Mean tumor volume was assessed (+ / - SEM). [Figure 31] This shows that TILs expressing mbIL15 operably linked to CA2 DRDs exhibit significantly more intratumoral invasion than unmanipulated TILs + IL2. A is a micrograph of tumor sections immunostained for human CD3, showing intratumoral invasion of TILs in animals treated with unmanipulated TILs and IL2, in the presence and absence of CA2 ligand ACZ, and in animals treated with TILs expressing mbIL15 operably linked to CA2 DRDs. B is a graph showing the number of TILs in stroma + tumor, stroma only, and tumor only. [Modes for carrying out the invention]

[0012] Current processes for TIL and T cell expansion require interleukin-2 (IL2)-based TIL expansion (pre-rapid expansion protocol or pre-REP), followed by a rapid expansion protocol (REP). The pre-REP stage involves culturing TILs or T cells with exogenous IL2, with tumor antigens present in the dissociated tumor tissue clumps. Therefore, pre-REP requires IL2 in the absence of feeder cells. The REP step typically requires additional feeder cells to support rapid TIL or T cell expansion. REP feeder cells and TIL or T cell stimulation are typically irradiated peripheral blood mononuclear cells (PBMCs) and high doses of IL2. However, IL2 during REP tends to efflux cells, reducing the intensity of TIL or T cell products. After in vitro REP using the current process, expanded TILs or T cells are administered to the patient with IL2, which may be administered before, during, and / or after TIL / T cell administration, again potentially depleting cells. Current general protocols for TIL therapy require high-dose IL2 administration initiated on the same day or the day after TIL infusion. For example, a high-dose IL2 regimen may consist of bolus intravenous infusions every 8 hours until tolerable, up to 14 doses, a 9-day rest, and another 14 doses. Other IL2 regimens may consist of 4-day IL2 administration cycles repeated up to 4 times every 28 days, or PEGylated IL2 regimens lasting up to 21 days.

[0013] In addition to promoting TIL or T cell depletion, high doses of IL-2 can cause serious side effects in cancer patients and are often unacceptable for patients requiring ACT. This composition and method provides a TIL or T cell therapy that, optionally, does not require the administration of exogenous cytokines such as interleukins like IL-2 before, during, or after TIL or T cell administration. In other words, this method does not require the combined use of TIL or T cell infusion and interleukin therapy. For example, optionally, subjects do not require the administration of exogenous IL-2 before TIL or T cell infusion or for 5, 7, 10, 14, 21, or 28 days after TIL infusion. Similarly, this method eliminates the need for the infusion of modified IL-2 or other modified cytokines (such as modified IL-7 or IL-15). As an example, modified interleukins retain the function of one or more of IL-2, IL-7, or IL-15, but CD4+T reg These may be mutants or fragments of IL2, IL7, or IL15 that reduce binding affinity to specific receptors, such as receptors, which can promote cell proliferation (for example, by reducing affinity).

[0014] As used herein, expansion refers to an increase in number or quantity. When the term “expanded” is used herein in relation to a population or subpopulation of T cells or TILs, the term refers to the population of cells after REP. The size of the population (i.e., the number of T cells or TILs after REP) is greater than the unexpanded population (i.e., the number of T cells or TILs before REP, or the number of T cells or TILs after an unsuccessful REP resulting in the absence of functional expansion of the cells). When used in relation to cells such as expanded T cells or expanded TILs, it refers to T cells or TILs that are products or results of REP (i.e., a culture with feeder cells and selected stimulants) that resulted in functional expansion of the TIL population. Thus, as used herein, expanded T cells or TILs are descendants of T cells or TILs (e.g., modified T cells or modified TILs) that have been cultured under REP and resulted in functional expansion. Similarly, as used herein, unexpanded T cells or TILs refer to T cells or TILs that have not yet undergone REP. However, such non-expanded T cells or TILs may have gone through the initial IL2 pre-REP step or an unsuccessful REP that results in the absence of functional expansion of the cell.

[0015] Where used herein, the term “expansion” may be used quantitatively, such as greater expansion, less expansion, larger expansion, smaller expansion, etc. Such relative terms generally refer to an increase in T cells or TILs in a population or subpopulation from a larger multiple to a smaller multiple compared to a different population or subpopulation (e.g., expansion of modified T cells or TILs compared to expansion of an unmodified subpopulation). Thus, for example, a larger expansion of a subpopulation of modified T cells or TILs compared to an unmodified T cell or TIL means a larger multiple increase of modified T cells or TILs compared to an unmodified T cell or TIL, e.g., a 1.5x increase compared to a 1.25x increase, a 2x increase compared to a 1.5x increase, a 5x increase compared to a 2x increase, a 10x increase compared to a 5x increase, a 40x increase compared to a 10x increase, etc.

[0016] A method for expanding modified T cells or tumor-infiltrating lymphocytes using K562 cells. Methods for expanding modified T cells or TILs in the presence of engineered feeder cells are provided herein. More specifically, methods are provided for expanding T cells or TILs engineered to express cytokines (e.g., IL15), CARs, and / or TCRs in the presence of modified K562 feeder cells, optionally, in the absence of exogenous cytokines (e.g., interleukins such as IL2) during the REP step.

[0017] The modified feeder cells are K562 feeder cells that can be modified to include a first exogenous nucleic acid sequence encoding a co-stimulatory molecule selected from the tumor necrosis factor superfamily and a second exogenous nucleic acid sequence encoding IL21 or IL7. Parental K562 feeder cells obtained from the American Type Culture Collection (ATCC) can be modified by transduction with one or more heterologous polynucleotides. In some embodiments, the feeder cells are modified by introduction of polypeptides by viral or non-viral vector delivery methods. As a non-limiting example, a vector containing one or more heterologous polynucleotides may be introduced into TILs or T cells by physical methods such as needles, electroporation, sonoporation, hydroporation, inorganic particles (e.g., calcium phosphate, silica, gold) and / or chemical carriers. In some embodiments, synthetic or naturally occurring biodegradable agents may be used for delivery of cationic lipids, lipid nanoemulsions, nanoparticles, peptide-based vectors, or polymer-based vectors. For example, feeder cells can be transduced by viral (e.g., retrovirus or lentivirus) transduction with nucleic acid encoding 41BBL(CD137L). Transduced feeder cells can be further transduced with a nucleic acid sequence encoding mbIL21, for example, a Sleeping Beauty transposon system expressing mbIL21. For example, a transposon may include a first nucleic acid sequence encoding IL21 and a second nucleic acid sequence encoding a transmembrane domain. The transmembrane domain may optionally include an IL21 receptor, an MHC1 transmembrane domain, a CD8 transmembrane domain, a B7-1 transmembrane domain, a CD4 transmembrane domain, a CD28 transmembrane domain, a CTLA-4 transmembrane domain, a PD-1 transmembrane domain, or a human IgG4 transmembrane domain. IL21 may be directly linked to the transmembrane domain or linked via a linker or hinge. Therefore, when the nucleic acid sequence is expressed, IL21 is directly or indirectly ligated to the transmembrane domain that binds IL21 to the membrane of transduced K562 cells.By choice, feeder cells are not modified to express the Fc-γ receptor CD32.

[0018] Numerous linker arrays (linkers) are known in the art. Examples of linkers include, but are not limited to, GS linkers, GSG linkers, and GGSG linkers. These linkers consist of one or more repetitions of a subunit. Therefore, a GS linker is a GS linker. n It is a linker, and n is a number that is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more. Similarly, a GSG linker is a GS n It is a linker, and n is a number that is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more. The GGSG linker is GGSG n It is a linker, and n is a number that is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or greater.

[0019] A hinge sequence is a short sequence of amino acids that facilitates flexibility between connected components. A hinge sequence may originate from any suitable molecule or be any suitable sequence obtained from any suitable molecule. A hinge sequence may originate from an immunoglobulin (e.g., IgG1, IgG2, IgG3, IgG4) hinge region, i.e., the sequence between the CH1 and CH2 domains of an immunoglobulin (e.g., IgG4 Fc hinge), or from all or part of the extracellular region of a type 1 membrane protein such as CD8α CD4, CD28, and CD7, which may be the wild-type sequence or a derivative thereof. Some hinge regions include the immunoglobulin CH3 domain or both the CH3 and CH2 domains. In some embodiments, the hinge originates from a transmembrane domain.

[0020] If K562 feeder cells are not modified to express a costimulatory molecule selected from the tumor necrosis factor superfamily and / or IL21 or IL7, exogenous costimulatory molecules and / or interleukins may be added to the culture medium. However, modifying the feeder cells to express these molecules eliminates or reduces the need to add exogenous costimulatory molecules or interleukins during the REP phase.

[0021] Nucleic acid sequences encoding IL21 or IL7, or 41BBL, or both, may include a signal sequence or a leader sequence. For example, the signal sequence could be the GM-CSF signal peptide (SEQ ID NO: 33).

[0022] Before feeder cells are used in the method of the present invention, they are first rendered incapable of replication. Various means of treating feeder cells are known in the art. Such methods include irradiation (e.g., by gamma rays), mitomycin C treatment, electrical pulses, mild chemical fixation (e.g., by formaldehyde or glutaraldehyde), or transduction of feeder cells by suicide genes. In some embodiments, the feeder cells are human cells. For example, irradiation may be 25-300 Gy delivered by, for example, a cesium source or an X-ray source.

[0023] In particular, methods for expanding modified T cells or TILs can occur in the absence of exogenous cytokines (e.g., IL2) during the REP phase. T cells or TILs expanded according to the methods described herein are modified. For example, T cells or TILs can be modified to express mbIL15, CAR, or TCR. The expressed cytokine (e.g., mbIL15), CAR, or TCR can be operably linked to the DRD.

[0024] The expansion achieved by this method will, at an optional rate, result in at least a 10-fold expansion within 7 to 21 days, but may result in expansions exceeding 40-fold, 75-fold, or 100-fold. For example, modified T cells or TILs will expand by 500 to 2000-fold, or any amount in between, within 14 days. The expansion ratio can be calculated by dividing the number of target cells cultured at the end of the REP by the number of target cells cultured at the start of the REP.

[0025] cell culture K562 feeder cells are provided herein in a culture comprising modified T cells or TILs and modified K562 feeder cells, wherein the K562 feeder cells comprise a first exogenous nucleic acid sequence encoding a costimulatory molecule selected from the tumor necrosis factor superfamily, and a second exogenous nucleic acid sequence encoding IL21 or IL7. The T cells or TILs may be prepared from a subject and engineered to express cytokines, CARs, and / or TCRs. For example, the T cells or TILs may be engineered to express IL15 (e.g., mbIL15), which can be operably linked to DRD.

[0026] Modified K562 feeder cells in culture are non-replicable and engineered to express 41BBL and, optionally, IL21. Optionally, modified K562 feeder cells express 41BBL and IL21 is present in the culture medium. Optionally, modified K562 feeder cells express 41BBL and mbIL21 or secreted IL21 polypeptide (e.g., UniProtKB-A0A224B028_HUMAN). In one embodiment, IL21 comprises an amino acid sequence corresponding to SEQ ID NO: 35, or a polypeptide having at least 85, 90, 95, or 99% identity with SEQ ID NO: 35, which retains one or more IL21 functions (e.g., promoting the expansion of modified T cells or TILs in vitro). Optionally, IL21 directly or indirectly binds to the transmembrane domain that binds IL21 to the membrane of K562 feeder cells. Optionally, IL21 is SEQ ID NO: 35. Optionally, modified K562 feeder cells express 41BBL (e.g., SEQ ID NO: 47). Exemplary IL21-41BBL inserts are shown in Table 3. For example, see SEQ ID NOs: 46 and 47 for nucleic acid and amino acid sequences. Optionally, K562 cells are not modified to express the Fc-γ receptor CD32.

[0027] The culture may further contain additional components, such as a defined medium for conditions that allow for rapid expansion of modified T cells or TILs. Optionally, exogenous cytokines are not added to the culture. For example, the culture may optionally be exogenous IL2-free. Optionally, the initial ratio of feeder cells to T cells or TILs is in the range of 1:1 to 200:1 or any ratio in between. In some embodiments, the initial ratio of feeder cells to TILs is 5:1. However, upon completion of the REP step, the number of expanded T cells or TILs significantly exceeds the number of feeder cells, especially when non-replicating feeder cells are used.

[0028] Modified tumor-infiltrating lymphocytes (TILs) TILs include T cells, NK cells, B cells, and NKT cells, but in at least certain tumor types, they primarily include T cells (e.g., cytotoxic T cells that are CD8+ and helper T cells that are CD4+). The TILs described herein are engineered to express mbIL15. Thus, the modified TILs include an exogenous nucleic acid sequence encoding IL15, an exogenous nucleic acid sequence encoding a transmembrane domain, and optionally an exogenous nucleic acid sequence encoding a linker or hinge. Since IL15 is not generally expressed as a membrane-bound molecule, in order to express mbIL15, IL15 must associate with its transmembrane domain. As used herein, IL15 refers to the IL15 polypeptide (e.g., UniProtKB-P40933(IL15_HUMAN)). In one embodiment, the IL15 payload comprises an amino acid sequence provided in Table 2 (SEQ ID NO: 12), or a polypeptide having at least 85, 90, 95, or 99% identity to SEQ ID NO: 12 that retains one or more IL15 functions (e.g., promoting in vivo expansion of modified TILs and promoting cytotoxicity of T cells and NK cells).

[0029] Exemplary transmembrane domains include the MHC1 transmembrane domain, CD8 transmembrane domain, B7-1 (CD80) transmembrane domain, CD4 transmembrane domain, CD28 transmembrane domain, CTLA-4 transmembrane domain, PD-1 transmembrane domain, human IgG4 transmembrane domain, or IL15 receptor subunit (e.g., IL15αR). IL15 may be directly ligated to the transmembrane domain, or it may be ligated via a linker or hinge.

[0030] The modified TILs described herein optionally further comprise an exogenous nucleic acid sequence encoding an intracellular (cytoplasmic) tail. The intracellular tail may be, for example, the B7-1(CD80) intracellular tail.

[0031] The modified TILs described herein optionally further include an exogenous nucleic acid sequence encoding a signal sequence (leader sequence). Representative leader sequences include MDMRVPAQLLGLLLLWLSGARC (SEQ ID NO: 10), MDWTWILFLVAAATRVHS (IgEss, SEQ ID NO: 58), MRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEA (natural IL15LS, SEQ ID NO: 59), and MGLVRRGARAGPRMPRGWTALCLLSLLPSGFMA (CD34: SEQ ID NO: 60).

[0032] In addition, certain TILs further contain an exogenous nucleic acid sequence encoding a DRD. While IL15 is important for T cell and NK cell proliferation, continuous exposure to high levels of IL15 can lead to the depletion of these cells in vivo, which would reduce the efficacy of IL15-expressing TILs. Therefore, in certain embodiments, the DRD is operably linked to mbIL15 to provide regulation of IL15 activity or abundance during TIL immunotherapy.

[0033] A drug-responsive domain (DRD) is a polypeptide that modulates the expression or activity level of a payload. Although referred to as a drug-responsive domain, the ligand to which a DRD is responsive does not need to be a drug. More specifically, a DRD interacts with a ligand in such a way that, when the DRD is operably ligated to the payload, it provides ligand-dependent, reversible modulation of the payload's characteristics (e.g., activity or expression level). U.S. Patents 9,487,787 and 10,137,180, U.S. Publications 2019 / 0192691, 2020 / 0101142, 2020 / 0172879, and 2021 / 0069248, and U.S. Patent Applications 17 / 251,635 and 17 / 288,373 (the contents of which are incorporated herein by reference in their entirety) provide examples of DRDs (and their paired ligands) as provided in this disclosure. Some of these DRDs and other exemplary DRDs provided herein are also provided elsewhere in this Spec. A DRD can be selected from an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with respect to the DRD functional portion of FKBP (SEQ ID NO: 4), ecDHFR (SEQ ID NO: 1), hDHFR (SEQ ID NO: 2), ER (SEQ ID NO: 9), PDE5 full length (SEQ ID NO: 6), PDE5 ligand-binding domain (SEQ ID NO: 5), and CA2 (SEQ ID NO: 7), or any of the aforementioned portions that maintain DRD function, or SEQ ID NOs: 1, 2, 4, 5, 6, 7, or 9 or their DRD functional portion. For example, one or more mutations (including cleavage, substitution, and deletion) in the amino acid sequences of FKBP, ecDHFR, hDHFR, ER, PDE5, and CA2 may be advantageous in further destabilizing the DRD. Suitable DRDs, which may be referred to as destabilizing domains or ligand-binding domains, are known in the art.For example, WO2018 / 161000, WO2018 / 231759, WO2019 / 241315, US8,173,792, US8,530,636, WO201 8 / 237323, WO2017 / 181119, US2017 / 0114346, US2019 / 0300864, ​​WO2017 / 156238, Miyazaki et al.,J Am Chem Soc,134:3942(2012);Banaszynski et al.(2006)Cell 126:995-1004;Stankunas,K.et al.(2003)Mol.Cell 12:1615-1624;Banaszynski et al. See also al. (2008) Nat. Med. 14:1123-1127; Iwamoto et al. (2010) Chem. Biol. 17:981-988; Armstrong et al. (2007) Nat. Methods 4:1007-1009; Madeira da Silva et al. (2009) Proc. Natl. Acad. Sci. USA 106:7583-7588; Pruett-Miller et al. (2009) PLoS Genet. 5:e1000376, and Feng et al. (2015) Elife 4:e10606 (the contents of each are incorporated herein by reference in their entirety).

[0034] While not limited by theory, DRDs are thought to be unstable polypeptides that degrade in the absence of their corresponding stabilizing ligand (also referred to as the paired ligand or ligand), but whose stability is saved by binding to the stabilizing ligand. Since the binding of the ligand to the DRD is reversible, if the ligand is later removed, the DRD unfolds, becomes unstable, and is ultimately tagged for degradation by the ubiquitin-proteasome system ("UPS"). Therefore, if the DRD is operably ligated to a payload such as mbIL15, the entire construct (i.e., DRD + IL15) itself is thought to become unstable and degraded by the UPS. However, in the presence of the paired ligand, the construct is stabilized, and the mbIL15 payload remains usable. Furthermore, the conditional nature of DRD stability is thought to allow for a rapid and non-perturbative switch from a stable protein to an unstable UPS substrate, which may facilitate the regulation or modulation of the payload's activity level, and / or modulation of the payload's activity level.

[0035] Since the abundance of a payload (e.g., mbIL15) is related to the activity of the payload, unless otherwise explicitly stated or meaningless in context, the level of abundance and the level of activity, or abundance and activity, are referred to herein as activity or activity level. Furthermore, measurements of abundance may be used as a surrogate for and to reflect the activity level. Thus, a change in the abundance of a payload in the presence of an effective amount of ligand, compared to the absence of the ligand, can optionally serve as a surrogate for measuring a change in the activity level. Therefore, abundance level and activity level are used interchangeably throughout this disclosure.

[0036] Numerous DRDs are described herein, but those skilled in the art can identify additional DRDs. For example, DRDs can be identified using library screening and structural induction engineering to select the optimal DRD mutant that has sufficient instability in the absence of a ligand and sufficient stability in the presence of a ligand. A mutant library can be generated using random mutagenesis screening by transducing cells (e.g., Jurkat cells) with candidate mutant DRDs. To generate an enriched library, cells with the desired characteristics (low basal activity / expression and high dynamic range) are then selected by testing the expression of the reported gene over a range of ligand concentrations. Single-cell clones are then constructed and characterized to identify candidate DRDs. DRDs can affect the characteristics of the payload to which they are operably linked, e.g., abundance or activity level. Furthermore, one or more DRDs interact with a ligand to provide ligand-dependent reversible regulation of the payload characteristics.

[0037] The DRDs described herein respond to a paired ligand. Optionally, the DRD responds to a paired ligand that is a small molecule drug, such as an FDA-approved small molecule. However, those skilled in the art can select the DRD and its paired ligand to meet the specific needs of the system. Stabilizing ligands for specific DRDs described herein and examples of their use are shown in Table 1 and in U.S. Patent No. 9,487,787 filed March 22, 2012, U.S. Patent No. 10,137,180 filed September 6, 2013, PCT application PCT / US2018 / 037005 filed June 12, 2018, PCT application PCT / US2019 / 036654 filed June 12, 2019, PCT application PCT / US2019 / 057698 filed October 23, 2019, PCT application PCT / US2020 / 021596 filed March 6, 2020, and U.S. Patent Application No. 16 / 558,224 filed September 2, 2019, all disclosures of the aforementioned applications are incorporated herein by reference in their entirety. [Table 1]

[0038] Optionally, the DRDs of this disclosure may be derived from human carbon anhydrase 2 (hCA2), a member of the carbonic anhydrase superfamily of metalloenzymes. The DRDs of this disclosure may be derived from amino acids 1-260 of CA2 (Uniprot ID: P00918) (SEQ ID NO: 7). Optionally, the DRDs may be derived from CA2 containing amino acids 2-260 (e.g., amino acids 2-260) of the parent CA2 sequence. This is referred to herein as the CA2 M1del mutation (CA2, SEQ ID NO: 55). Optionally, the DRDs of this disclosure may include a region or the whole of human carbonic anhydrase 2 and further include one or more mutations in the full-length sequence selected from M1del, L156H, and S56N. Optionally, the DRDs may be selected from the group consisting of SEQ ID NOs: 7, 26, 55, 56, and 57.

[0039] For example, a modified TIL may include a nucleic acid sequence encoding a transmembrane domain that is C-terminus of the IL15 polypeptide component and an intracellular tail that is C-terminus of the transmembrane domain.

[0040] Table 2 shows non-limiting examples of modified TIL constructs and construct components. Constructs designated as OT-IL15-292 consist of a signal sequence, IL15, (GS) from the N-terminus. 15 It includes the linker, hinge region, transmembrane region, and intracellular tail. Constructs designated as OT-IL15-293 include a DRD (specifically, CA2 DRD(M1del, L156H)) at the C-terminus. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4]

[0041] To generate membrane-bound cytokines such as IL15 operably linked to a DRD with a sufficient dynamic range (i.e., an acceptable activity range corresponding to ligand fluctuations from zero to maximum saturation), the polypeptide optionally comprises a payload (IL15), a linker, a hinge, a transmembrane region, a tail, and a DRD from the N-terminus. The tail and / or linker, as well as the length of the tail and linker, may affect the activity in the absence of ligand, and in some embodiments, specific tails and / or linkers and lengths are selected to maximize the on-state (e.g., maximum activity level) while maintaining low basal activity levels and ligand responsiveness. Specific hinges may allow conformational changes, thereby affecting ligand responsiveness over a sufficient dynamic range.

[0042] The modified TIL expressing mbIL15 described herein has several advantages. Firstly, the modified TIL can be expanded in vitro in the presence of feeder cells (K562 feeder cells expressing 41BBL, and IL21, etc.). Importantly, following the REP pre-step, the modified TIL can be expanded in vitro in the absence of exogenous cytokines, and the expanded TIL can be activated and further expanded in vivo without administration of exogenous cytokines such as IL2.

[0043] A population of TILs containing multiple modified TILs can include a subpopulation of expanded TILs (i.e., REPs having feeder cells and stimulatory molecules such as IL12 and 4BB1). Expanded TILs demonstrate several advantages. For example, modified TILs that have received REPs can then survive in cultures lacking feeder cells. More specifically, TILs engineered to express mbIL15 can survive longer than unengineered cells in the absence of exogenous cytokines such as interleukin-2. TILs engineered to express mbIL15 operably linked to DRD have a higher survival rate in the absence of exogenous cytokines than unengineered TILs or mbIL15 TILs regulated in the absence of the ligand, although in the presence of the ligand. Furthermore, the population of expanded TILs shows preferential expansion of specific TILs and thus indicates that there are fewer or more subtypes of TILs compared to a control population of non-expanded TILs. As used herein, the control population of non-expanded TILs refers to TILs that have been modified in the same manner as the expanded TILs but have not received REPs.

[0044] The population of expanded TILs, for example, has a higher proportion of CD8+ cells, a lower proportion of CD4+ cells, and a lower CD4+:CD8+ ratio compared to a control population of non-expanded TILs. CD8+ TILs are thought to play an important role in killing cancer cells by releasing cytotoxic molecules and cytokines, and the number of CD8+ TILs compared to the number of CD4+ TILs in the tumor (i.e., the CD4+:CD8+ ratio) has been found to correlate with a positive outcome.

[0045] Furthermore, in certain embodiments, the population of expanded TILs has a lower proportion of CD4 T reg cells compared to the proportion of Treg cells in pre-REP TILs prior to the manipulation and expansion in the REP. CD4 T reg cells play a role in immune tolerance and immune homeostasis by suppressing the immune response. Thus, in immunotherapies such as ACT using TILs, a lower proportion of T reg cells is desirable.

[0046] The expanded TIL population also exhibits fewer depleted TILs and more polyfunctional TILs. The expanded TIL population has a lower proportion of PD1+ cells compared to the non-expanded TIL control population. Furthermore, the expanded TIL population has a higher proportion of cells producing both tumor necrosis factor α (TNFα) and interferon-γ (IFNγ) compared to the non-expanded TIL control population where TILs produce both TNFα and IFNγ.

[0047] This specification provides a mixed population of TILs, comprising a subpopulation of unmodified TILs and a subpopulation of modified TILs, which optionally include mbIL15 operably linked to a DRD. The subpopulation of modified TILs expands in the presence of K562 feeder cells, 41BBL, and IL21. The subpopulation of modified TILs expands more than the subpopulation of unmodified TILs in the presence of K562 feeder cells, 41BBL, and IL21. This preferential expansion of modified TILs occurs in the absence of exogenous IL2 during REP.

[0048] modified T cells Modified T cells that can be expanded according to the methods described herein include, for example, activated T cells or T cells manipulated to express cytokines, CARs, or TCRs. Methods for activating and manipulating T cells are known in the art. T cells can be manipulated to express cytokines, such as IL15, or more specifically, mbIL15, according to the methods for TILs described above. T cells can be manipulated to express CARs or TCRs, and optionally, cytokines (such as membrane-bound cytokines).

[0049] CARs containing an extracellular targeting domain (e.g., an scFv that recognizes a specific tumor antigen or other tumor cell surface molecule), a transmembrane domain / region, and an intracellular signaling / activation domain (e.g., a signaling region of CD3ζ and / or one or more co-stimulatory signaling domains, e.g., signaling domains derived from CD28, 4-1BB (CD137), and OX-40 (CD134)) can be expressed by modified T cells.

[0050] Method for creating modified TIL TILs can be prepared from a tumor or a biopsy using methods known in the art. For example, a tumor fragment (e.g., 1–5 mm in size) is subjected to enzymatic digestion (e.g., collagenase (e.g., 0.5–5 mg / mL), DNAse, hyaluronidase) or mechanical dissociation. The dissociated cells are incubated in cell culture medium under conditions that favor the growth of TILs over other cells (i.e., in the presence of IL-2). This step is the pre-REP step. In the pre-REP step, cells can be cultured in the presence of 2000–8000 IU / mL of IL-2 (e.g., 6000 IU / mL) and, optionally, in the presence of inactivated human AB serum (e.g., 3–28 days). In some embodiments, the cells are cultured for a period of time, generally 3–28 days. In some embodiments, this pre-REP cell population is cultured for 7–21 days.

[0051] TILs or T cells prior to REP can be cryopreserved. The cryopreserved cells are thawed and left to stand before activation. The cells can be activated using, for example, plate-bound OKT3, soluble OKT3, costimulatory antibody (e.g., antibody against CD28 or 41BB) + OKT3, or anti-CD3 and anti-CD28 antibodies conjugated to beads or fragments. The activation step may take 1-2 days or longer. After activation, one or more TILs are then manipulated to express membrane-bound interleukin 15 (mbIL15) by transduction using a vector having a first nucleic acid sequence encoding IL15 and a second nucleic acid sequence encoding a transmembrane domain. Optionally, the vector further includes one or more nucleic acid sequences encoding a signal peptide, linker, hinge, intracellular tail, or DRD. The vector can be constructed in any number of ways to achieve the desired mbIL15. Exemplary nucleic acid constructs include nucleic acid sequences encoding OT-IL15-293 and OT-IL15-292, respectively, with or without DRD. Therefore, the vector optionally includes sequence numbers 29, 31, 53, or 54. The vector includes or encodes additional elements such as promoter sequences and other regulatory elements (enhancers, translation-controlling elements (e.g., IRES), and half-life-controlling elements). The vector optionally includes or may include nucleic acid sequences encoding translation-controlling elements (e.g., IRES, WPRE, etc.).

[0052] The vector can be selected from viral vectors, plasmids, cosmids, and artificial chromosomes. For example, the vector may be a viral vector, such as a lentiviral vector or a retroviral vector. Examples of viral vectors include a baboon endogenous retroviral envelope (BaEV) pseudolentiviral vector or a Gibbonian monkey leukemia virus (GALV) envelope pseudogammaretroviral vector, which contain a nucleic acid sequence encoding IL15 and a nucleic acid sequence encoding a transmembrane domain. Upon expression, IL15 associates with the transmembrane domain and is membrane-bound by the transmembrane domain.

[0053] The vectors are optionally introduced into cells by non-viral methods such as needles, electroporation, sonoporation, hydroporation, chemical carriers (e.g., inorganic particles (e.g., calcium phosphate, silica, gold)), and / or chemical methods. In some embodiments, synthetic or naturally occurring biodegradable agents are used for delivery of cationic lipids, lipid nanoemulsions, nanoparticles, peptide-based vectors, or polymer-based vectors.

[0054] The nucleic acid sequence encoding IL15 can be genomic or non-genomic nucleic acid. That is, the delivery system used to deliver the IL15-coding nucleic acid can be integrated into the TIL genome, or it can be non-integrated (i.e., episome), or it can be transferred into the cytoplasm as RNA using an RNA vector.

[0055] TILs containing mbIL15 are expanded during the REP phase (e.g., any amount in between, including 5–21 days or 7–14 days) without IL2. As further described in the examples, modified TILs expressing IL15 are expanded in the presence of K562 feeder cells as well as 41BBL and IL21. In certain embodiments, K562 feeder cells are engineered to express 41BBL and IL21, which may be membrane-bound IL21 (mbIL21), thus reducing or eliminating the need for exogenous cytokines such as IL2, IL7, or IL15 during REP. In some embodiments, modified TILs are cultured in K562 cells (TIL: feeder cells) modified to express mbIL21 and 41BBL in ratios of 1:1–100:1, 1:1–50:1, 1:1–20:1, 1:1–10:1, or 2:1–5:1.

[0056] Before feeder cells are used in the method of the present invention, they are first rendered incapable of replication. Various means of treating feeder cells are known in the art. Such methods include irradiation (e.g., by gamma rays or X-rays), mitomycin C treatment, electrical pulses, mild chemical fixation (e.g., by formaldehyde or glutaraldehyde), or transduction of feeder cells by suicide genes. In some embodiments, the feeder cells are human cells. For example, irradiation may be 25-100 Gy delivered by, for example, a cesium source or an X-ray source.

[0057] Following expansion on feeder cells, TILs are optionally isolated from the feeder cells. As used herein, the term isolation does not imply that TILs are completely free of other components such as feeder cells, but merely that TILs are relatively free of feeder cells.

[0058] This specification provides methods for producing TILs containing mbIL15, populations of TILs, or subpopulations of TILs. Also provided are nucleic acid sequences encoding mbIL15, vectors containing nucleic acid sequences encoding mbIL15, non-replicating K562 feeder cells modified to express 41BBL and IL21, and TILs produced by the methods described herein.

[0059] This specification provides methods for producing TILs containing mbIL15, populations of TILs, or subpopulations of TILs. Also provided are nucleic acid sequences encoding mbIL15, vectors containing nucleic acid sequences encoding mbIL15, non-replicating K562 feeder cells modified to express 41BBL and IL21, and TILs produced by the methods described herein.

[0060] Method for creating modified T cells Primary human T cells can be derived from human peripheral blood mononuclear cells (PBMCs), bone marrow, or umbilical cord, and can be collected after stimulation with G-CSF. Nucleic acid constructs designed for cistron or multicistron expression are introduced into the T cells using any of the delivery techniques described above. Optionally, T cells are transduced with cytokines, e.g., membrane-bound cytokines, or cytokines and receptors (e.g., CARs or TCRs). For example, T cells can be transduced with one or more nucleic acids encoding IL15, and when IL15 is expressed, it is ligated to a transmembrane domain, CAR, or TCR. For example, T cells can be transduced with a first vector encoding mbIL15, and a second vector encoding one or more components of the CAR, e.g., an extracellular targeting domain (e.g., scFv that recognizes a specific tumor antigen or other tumor cell surface molecule), a transmembrane domain / region, and an intracellular signaling / activation domain (e.g., the signaling region of CD3ζ, and / or one or more co-stimulatory signaling domains, e.g., signaling domains derived from CD28, 4-1BB (CD137), and OX-40 (CD134)). Optionally, T cells may be transduced with a third vector encoding any component of the CAR not encoded by the second vector.

[0061] Pharmaceutical composition This specification provides pharmaceutical compositions suitable for use in ACT. The pharmaceutical compositions may include modified T cells or TILs, such as expanded T cells or TILs, and a pharmaceutically acceptable carrier. The population of modified T cells or TILs in the pharmaceutical composition is optionally a mixed population including subpopulations of modified T cells (e.g., CAR+ T cells) or modified TILs (e.g., TILs engineered to express mbIL15) and unmodified T cells or TILs (i.e., unengineered T cells or TILs).

[0062] The term “carrier” means a compound, composition, substance, or structure that, when combined with a compound or cell, assists or promotes the preparation, storage, administration, delivery, efficacy, selectivity, or any other characteristics of the compound or cell for its intended use or purpose. For example, a carrier may be selected to minimize any degradation of TILs and to minimize any adverse side effects in the subject. Such pharmaceutically acceptable carriers include, but are not limited to, sterile, biocompatible pharmaceutically acceptable carriers, including saline, buffered saline, artificial cerebrospinal fluid, dextrose, and water. Pharmacologically acceptable means a biologically or otherwise undesirable substance that can be administered to an individual together with a population of T cells or TILs without causing an unacceptable biological effect or without interacting with T cells or TILs in an adverse manner.

[0063] Optionally, the pharmaceutical composition further comprises a cryoprotective agent (cryopreservative). Such a cryoprotective agent helps prevent unacceptable cell lysis or damage if the TIL is frozen for future use. Cryoprotective agents are known in the art. Such a cryoprotective agent may be selected from glycerol, ethylene glycol, propylene glycol, or dimethyl sulfoxide (DMSO).

[0064] The pharmaceutical compositions described herein optionally further comprise one or more pharmaceutically acceptable excipients (e.g., human serum albumin or polymer materials (e.g., PEG)).

[0065] The compositions of this disclosure can be formulated in any manner suitable for delivery. T cells or TILs may be administered, for example, in the form of nanoparticles, poly(coglycolic acid lactate) (PLGA) microspheres, lipids, lipoplexes, liposomes, polymers, carbohydrates (including monosaccharides), cationic lipids, or combinations thereof.

[0066] The descriptions of pharmaceutical compositions provided herein primarily concern pharmaceutical compositions suitable for administration to humans, but it should be understood by those skilled in the art that such compositions are generally suitable for administration to any other animal, such as non-human mammals. Subjects to which the pharmaceutical compositions are intended include, but are not limited to, agricultural animals such as cattle, horses, chickens, and pigs, livestock such as cats and dogs, or research animals such as mice, rats, rabbits, dogs, and non-human primates.

[0067] Treatment method A method for treating cancer in a subject (i.e., a recipient subject) is provided herein by administering to the subject a modified T cell or TIL population expanded according to the method described herein. Thus, modified T cells or TILs expanded in the presence of K562 cells, e.g., K562 cells modified to express the TNF superfamily (e.g., 41BBL) and / or IL21 or IL7 costimulatory molecules, are then administered to a subject having cancer, optionally, in the form of a pharmaceutical composition. Cancer may be, but is not limited to, melanoma, uveal (ocular) melanoma, cervical cancer, ovarian cancer, head and neck cancer, non-small cell lung cancer (NSCLC), bladder cancer, breast cancer, renal cell carcinoma, pancreatic cancer, prostate cancer, central nervous system cancer, or gastrointestinal cancer (e.g., colorectal cancer).

[0068] Conventional T cell or TIL therapies often require the simultaneous administration of T cells or TILs, and subsequently, the co-administration of high doses of IL-2. However, unlike conventional therapies with T cells or TILs, this method does not require the administration of IL-2. Rather, modified TILs or T cells, by expressing mbIL15, provide a sufficient cytokine source to stimulate TIL proliferation and activity, and expansion on modified K562 cells in the absence of IL-2 can enable preferential expansion of the modified cells.

[0069] A method for treating cancer may further include isolating one or more T cells from a subject or isolating TILs from a tumor described herein, and introducing a nucleic acid sequence expressing mbIL15, optionally CAR, or TCR into one or more T cells or TILs. The T cells or TILs may be derived from the recipient subject (autogenous source). The tumor from which the TILs are derived may be a primary tumor or a metastatic tumor. Therefore, if TILs or a portion of a primary tumor derived from a biopsy are cryopreserved, they can be thawed and used later to treat the resulting metastatic tumor or a different primary tumor. Alternatively, the T cells or TILs may be derived from a donor subject (allogeneic source), and the donor subject is not the recipient subject. TILs derived from the same tumor being treated have the advantage of possessing neoantigens and heterogeneity comparable to those of the tumor. TILs derived from different tumors of the same subject or from tumors of different donors may be selected for their reactivity with cancer antigens present in the recipient subject tumor by methods known in the art, such as tetramer staining of TCRs. If the T cells or TILs originate from a donor subject (who is not the recipient subject), the method may further include selecting a donor subject that is HLA-compatible with the recipient subject in order to reduce the graft-versus-host response.

[0070] T cells may be isolated from PBMCs, while TILs can be obtained from surgical excision, tissue biopsy, needle biopsy, or other means as a first step of tumor specimens. The T cells or TILs are then transduced or modified as described herein and then expanded in vitro to provide a larger population of expanded cells for ACT.

[0071] Administration of K562 cells with expanded modified T cells or TILs results in approximately 1,000 cells / injection to a maximum of approximately 10 billion cells / injection, e.g., 2 × 10⁶ 11 , 1 x 10 11 , 1 x 10 10 , 1 x 10 9 , 1 x 10 8 , 1 x 10 7, 5×10 7 , 1 x 10 6 , 5×10 6 , 1 x 10 5 , 5×10 5 , 1 x 10 4 , 5×10 4 , 1 x 10 3 , or 5×10 3 The quantity may include individual cells / injections, or a range between any two numbers including the endpoints. Optionally, 1 × 10 8 ~2×10 11 It is administered to individual cells.

[0072] The modified T cells or TILs of this disclosure can be administered by any preferred route. In some embodiments, the cells are administered by intravenous infusion, intra-arterial infusion, intraperitoneal, subarachnoid, or intralymphatic administration. Optionally, the modified T cells or TILs are administered locally, for example, directly to a tumor or the blood vessel supplying the tumor.

[0073] Modified T cells or TILs can be administered in single doses, but in certain cases, they may be administered in multiple doses.

[0074] The treatment method may further include lymphatic depletion of the recipient before administration of modified T cells or TILs. Studies in human and mouse models of melanoma have shown that lymphatic depletion can suppress regulatory T(T) proliferation. reg This suggests that it depletes negative regulatory cells, including T cells and peripheral bone marrow-derived suppressor cells. Therefore, lymphocyte depletion promotes the proliferation of adoptively transferred T cells or TILs. A lymphocyte depletion conditioning regimen may include, for example, pretreatment of receptor targets with whole-body irradiation and / or lymphocyte depletion agents before adoptive transfer of T cells or TILs. This preconditioning promotes the proliferation of T cells or TILs. reg This can be expanded by eliminating cells and removing potential cytokine sinks where normal cells compete with newly injected T cells or TILs.

[0075] An example of a lymphocyte depletion agent is fludarabine (e.g., in doses of 0.5 μg / ml to 10 μg / ml). In some embodiments, fludarabine is administered daily at a concentration of 1 μg / ml for 1 to 7 days prior to the administration of modified T cells or TILs. In some embodiments, fludarabine is administered in doses of 10 mg / kg / day, 15 mg / kg / day, 20 mg / kg / day, 25 mg / kg / day, 30 mg / kg / day, 35 mg / kg / day, 40 mg / kg / day, or 45 mg / kg / day. In some embodiments, the fludarabine treatment is administered at 35 mg / kg / day for 2 to 7 days. In some embodiments, the fludarabine treatment is administered at 35 mg / kg / day for 4 to 5 days. In some embodiments, the fludarabine treatment is administered at 25 mg / kg / day for 4 to 5 days.

[0076] In some embodiments, cyclophosphamide is administered at concentrations of 0.5 μg / mL to 10 μg / mL to provide its active form, maphosphamide. In some embodiments, cyclophosphamide is administered daily at a concentration of 1 μg / mL for 1 to 7 days prior to TIL administration to provide maphosphamide. In some embodiments, cyclophosphamide is administered at a concentration of 50 mg / m². 2 / day, 75mg / m 2 / day, 100mg / m 2 / day, 150mg / m 2 / day, 175mg / m 2 / day, 200mg / m 2 / day, 225mg / m 2 / day, 250mg / m 2 / day, 275mg / m 2 / day, or 300mg / m² 2 It is administered at a daily dose. In some embodiments, cyclophosphamide is administered intravenously (iv). In some embodiments, the cyclophosphamide treatment is administered intravenously (iv) at 35 mg / kg / day for 2 to 7 days. In some embodiments, the cyclophosphamide treatment is administered at 250 mg / m². 2It is administered intravenously (iv) at a dose of 250 mg / m² per day for 4-5 days. In some embodiments, the cyclophosphamide treatment is 250 mg / m². 2 It is administered intravenously (IV) at a rate of / day for 4 days.

[0077] In a specific embodiment, lymphocyte depletion was induced with cyclophosphamide 60 mg / kg for 2 days and fludarabine 25 mg / m². 2 or cyclophosphamide 250 mg / m² for 5 days 2 / day for 4 days and fludarabine 25 mg / m² 2 This includes administration of a combination of lymphocyte-depleting agents for 4 days or more.

[0078] If IL15, CAR, or TCR expressed by T cells or TILs are operably linked to the DRD, the method may further include administering to a recipient subject a second agent that binds to the DRD in an amount effective in increasing the payload activity of the T cells or TILs. The ligand may be administered using a dosing regimen that provides the subject with a selected amount of payload activity. The ligand may be delivered to achieve continuous or intermittent payload activity in the subject. Determining the frequency and duration of administration to the subject is determined by those skilled in the art, for example, by considering higher doses or longer durations of ligand administration when more payload activity is desired and ligand administration is reduced or eliminated when less activity is desired. The dose and duration of ligand administration, as well as the resulting payload activity, are also selected to avoid unacceptable side effects or toxicity in the subject. Thus, the subject is administered an effective amount of ligand to achieve an effective payload. The term effective amount is defined as any amount required to produce the desired physiological response. The effective dose and schedule for administering the ligand can be empirically determined by those skilled in the art based on the amount of payload obtained, the activity of the payload, or one or more indicators of the effect of the payload activity. The dosage range of the ligand is from zero to the saturated dose, and the resulting payload activity is from the baseline to the maximum level, and optionally has a sufficient dynamic range to allow small changes in the amount of ligand to result in small changes in the amount of payload or payload activity. The dose or frequency of ligand administration, as well as the resulting amount and activity of the payload, should not be so large as to cause unacceptable side effects and will vary depending on the patient's age, condition, sex, the type and stage of cancer being treated, and whether other therapeutic agents are included in the treatment regimen. Guidance on appropriate doses for a given class of ligands can be found in the literature.

[0079] In some embodiments, modified T cells or TILs having a configurable payload (i.e., a payload operably linked to a DRD) can be administered in combination with one or more immune checkpoint regulators. Checkpoint inhibitors include, but are not limited to, antibodies that target PD-1 or inhibit the binding of PD-1 to PD-L1, such as nivolumab (BMS-936558, Bristol-Myers Squibb, Opdivo®), pembrolizumab (lambrolizumab, MK03475 or MK-3475, Merck, Keytruda®), humanized anti-PD-1 antibody JS001 (ShangHai JunShi), monoclonal anti-PD-1 antibody TSR-042 (Tesaro, Inc.), pidilizumab (anti-PD-1 mAb CT-011, Medivation), anti-PD-1 monoclonal antibody BGB-A317 (BeiGene), and / or anti-PD-1 antibody SHR-1210 (ShangHai This includes HengRui, human monoclonal antibody REGN2810 (Regeneron), human monoclonal antibody MDX-1106 (Bristol-Myers Squibb), and / or humanized anti-PD-1 IgG4 antibody PDR001 (Novartis).

[0080] Subjects are optionally monitored for treatment outcomes. Therefore, for example, the number of malignant cells in a sample, circulating tumor DNA in a sample, or the size of a solid tumor at the time of imaging can be detected. If a desired endpoint is achieved (e.g., demonstrating success in cancer treatment), the ligand can be reduced or discontinued to reduce or eliminate the payload. Similarly, if a subject develops a cytokine storm, allergic reaction, or other adverse effects from the payload, the ligand can be reduced or discontinued.

[0081] definition When the terms "approximately" and "approximately" are used to refer to measurable values ​​such as volume, concentration, dose, time, temperature, activity, level, number, frequency, percentage, dimension, size, weight, location, or length, they mean that the specified volume, concentration, dose, time, temperature, activity, level, number, frequency, percentage, dimension, size, weight, location, or length may include variations due to experimental error of ±15%, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1%. All measurements or numerical values ​​are implicitly understood to be modified by the word "approximately," even if the measurement or numerical value is not explicitly modified by the word "approximately." When the terms "approximately" and "approximately" are used in relation to the arrangement or location of a region within a reference polypeptide, these terms include variations of up to ±20 amino acid residues, up to ±15 amino acid residues, up to ±10 amino acid residues, up to ±5 amino acid residues, up to ±4 amino acid residues, up to ±3 amino acid residues, up to ±2 amino acid residues, or even up to ±1 amino acid residue.

[0082] As used herein, operably coupled means that, in the presence of a paired ligand, a DRD is directly or indirectly coupled to a payload to alter a measurable property of the payload (e.g., altering the activity level of the payload compared to the activity level in the absence of the paired ligand). In some embodiments, the volume and / or activity level of the payload increases in the presence of an effective amount of ligand compared to the expression or activity level in the absence of the ligand. The effective amount of ligand means the amount of ligand required to see an increase in the volume or activity measurement of the payload. In some embodiments, the effective amount is not large enough to produce unacceptable toxicity or off-target effects. Optionally, the measurable property is a therapeutic outcome, the volume of payload in a sample, or the biological activity level of the payload (measuring the volume of the payload can serve as a surrogate for this).

[0083] Where linked or combined phrases are used, unless otherwise explicitly stated or meaningless in context, the phrase is understood to follow directly or indirectly. Therefore, any reference to a DRD linked, combined, or associated with a payload means, in each case, that the DRD is directly or indirectly linked to the payload.

[0084] As used herein, the terms TIL or T cell survival and TIL or T cell persistence are used interchangeably. Survival is determined based on the persistence of the TIL or T cell.

[0085] As used herein, expansion is used to refer to the functional increase in cell number that occurs during functional REP. Functional REP results in an expanded cell population that provides a sufficient number of cells for therapeutic purposes. Conversely, failure of REP will result in a lack of a functional multiplier increase in cell number. Unexpanded cells include cells before REP and cells that did not undergo functional expansion during REP, compared to the expanded cell population. Non-functional expansion includes expansion of 10% or less of the expanded cell population. For example, a TIL population that expanded 100-fold over a given period can be compared to an unexpanded population that expanded 10-fold or less. Thus, as used herein, expanded cells or expanded cell population refers to cells or a population of cells that underwent functional REP. Unexpanded cells or a population of cells refers to cells or a population of cells before or after REP that did not result in a functional expansion of the cell population. As an example, certain modified TILs expand on modified K562 feeder cells, but the expansion multiplier on PBMCs is less than 10% of the expansion multiplier on modified K562 feeder cells. Therefore, an unextended TIL could be a pre-REP modified TIL or a modified TIL after REP on PBMC.

[0086] The term "identity" as known in the art refers to the relationship between two or more sequences, determined by comparing them. In the art, identity also means the degree of sequence relevance between sequences, determined by the number of matching residues (amino acids or nucleic acids) between the chains of two or more sequences. Identity measures the degree of identical matching between two or more sequences that have gap alignments (if any) addressed by a specific mathematical model or computer program (i.e., an algorithm). The identity of related sequences can be readily calculated by known methods. Such methods include, but are not limited to, those described in Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, AM and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M. Stockton Press, New York, 1991; and Carillo et al., SIAM J. Applied Math. 48, 1073 (1988). Generally, variants of any particular polynucleotide or polypeptide in this disclosure have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, but less than 100% sequence identity with respect to their particular reference polynucleotide or polypeptide, as determined by sequence alignment programs and parameters described herein and known to those skilled in the art.Such tools for alignment include those in the BLAST suite (Stephen F. Altschul, Thomas L. Madren, Alejandro A. Schaffer, Jinghui Zhang, Zheng Zhang, Webb Miller, and David J. Lipman (1997), "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res. 25: 3389-3402).

[0087] As used herein, the term “feeder cell” refers to a cell that supports the expansion of TILs or T cells in a culture, such as by secreting into the culture or by presenting on feeder cell membrane growth or survival factors. In some embodiments, the feeder cells are arsenal (i.e., non-reproducible).

[0088] As used herein, "subject" and "patient" are synonymous and are not limited to human subjects or patients.

[0089] As used herein, the treatment refers to the reduction or delay of one or more signs or symptoms of cancer. For example, a favorable change of at least 10%, preferably at least 20%, 30%, 40%, 50%, or more, in a measurable parameter of the disease may indicate an effective treatment. Thus, the effectiveness of treating or improving a disease can be evaluated, for example, by measuring disease progression, disease remission, symptom severity, pain reduction, quality of life, the dose of drug required to maintain the therapeutic effect, levels of disease markers, or any other measurable parameter appropriate for a given disease being treated or targeted for treatment. In relation to the administration of the compositions of this disclosure, an effective dose of cancer treatment indicates that administration in a clinically appropriate manner results in at least a statistically significant beneficial effect on a subset of patients, such as improvement of symptoms, cure, reduction of disease burden, reduction of tumor mass or cell number, extension of lifespan, improvement of quality of life, or other effects generally recognized as good by physicians familiar with the treatment of a particular type of cancer.

[0090] If a range is specified, the endpoints are included. Furthermore, unless otherwise indicated or is evident from the context and understanding of those skilled in the art, values ​​expressed as a range may, in different embodiments of this disclosure, be assumed to be any specific value or subrange within the described range, up to one-tenth of the lower limit unit of the range, unless otherwise explicitly indicated in the context.

[0091] Details of one or more embodiments of this disclosure are described in the description and accompanying drawings. It should be understood that other embodiments may be utilized and structural or process modifications may be made without departing from the scope of this disclosure. In other words, exemplary embodiments and aspects are described. However, it should be understood that in developing such actual embodiments, numerous implementation-specific decisions may be made to achieve the developer's specific goals, such as compliance with clinically relevant constraints, and these may vary from implementation to implementation. Furthermore, it should be understood that while such development efforts may be complex and time-consuming, they are nevertheless routine work for those skilled in the art who are interested in this disclosure.

[0092] Publications cited herein and materials from which they are cited are incorporated herein by reference in their entirety.

[0093] The following examples are intended to further illustrate specific embodiments of the methods and compositions described herein and are not intended to limit the scope of the claims. [Examples]

[0094] Example 1: Isolation and expansion of TILs from patient tumor samples (REP pre-culture) Melanoma and head and neck tumor samples were obtained from the Cooperative Human Tissue Network. Tumor samples were cut into 1-3 mm fragments in Hanks equilibrium salt solution (HBSS) buffer, and each fragment was placed in a multi-well plate, one fragment per well, in 2 mL of TIL medium (RPMI-1640 supplemented with GlutaMAX (Thermo Fisher), 1% penicillin / streptomycin, 1 mM sodium pyruvate, 1% HEPES, 50 μM 2-mercaptoethanol (Invitrogen), and 10% thermo-inactivated human AB serum (Valley Bio)) containing 6000 IU / mL of IL2 (Peprotech) and 0.1 mg / mL of Normocin (InvivoGen). Half of the medium was replaced with fresh medium containing IL2 from day 5, and the cells were divided into multiple wells or flasks and allowed to become confluent for 3 weeks. This culture process is referred to as pre-rapid expansion protocol (pre-REP). After pre-REP, the TILs were divided and placed in cell freezing medium (Bambanker, Bulldog). The samples were frozen in Bio (or Cryostor-10, STEMCELL Technologies) and stored for an extended period in liquid nitrogen.

[0095] To determine the change in T cell frequency before and after pre-culture of tumor cells (REP), a portion of the tumor tissue was digested with collagenase and DNase I before pre-culture to create a single-cell suspension, which was then compared with cells obtained after pre-culture. T cell frequency was analyzed by flow cytometry using fluorescent dye-complex anti-CD45 and anti-CD3 antibodies. As shown in Figure 1, nearly half (44.29 ± 21.67%) of the cells in the pre-culture tumor cell suspension were CD45+, and of these, only about 40% (~39.85 ± 23.69%) were CD3+ T cells. After 3 weeks of culture in the presence of IL2 (pre-REP), the majority of cells were CD45+ (90.35 ± 7.28%), indicating hematopoietic cell enrichment, and CD3+ (80.64 ± 15.19%), indicating T cell enrichment.

[0096] TILs of other human tumor types, including melanoma and malignant tumors, were isolated from the breast, lung, kidney, endometrium, liver, pancreas, and ovaries using the same method as described above.

[0097] Example 2: Generation of K562 cells expressing membrane-bound IL21 and 4-1BBL Membrane-bound IL21 and 4-1BBL vector construct assembly The IL21-41BBL-001 insert contains a leader sequence, membrane-bound IL21 (mbIL21), a P2A sequence, and a nucleic acid sequence encoding 4-1BBL. The mbIL21 nucleic acid sequence sequentially encodes the IL21 sequence, IgG hinge, IgG4 chain, CD4 transmembrane domain, and Glycine-Serine (GS) linker (see Table 3). OT-IL21-41BBL-001, containing the IL21-41BBL-001 insert, was constructed in a pELNS vector (third-generation self-inactivating lentiviral expression vector) using standard molecular biology techniques. The gene fragment (Gblock) was inserted into the pELNS vector and placed under the control of the EF1a promoter using Gibson assembly (NEBuilder Hifi). The assembled plasmid was transformed into E. coli (NEB stable) for amplification and sequenced before proceeding with viral production. [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] [Table 3-6] [Table 3-7] [Table 3-8] [Table 3-9] [Table 3-10] [Table 3-11]

[0098] Table 3 shows the nucleic acid and amino acid sequences of the domains of the mbIL21-41BBL constructs disclosed herein. The OT-IL12-(241-262) and OT-CD19-IL12-(297-316,319-332) plasmids were constructed in pELNS vectors (third-generation self-inactivating lentiviral expression vectors) using standard molecular biology techniques. The gene fragment encoding IL12 (Gblocks or strand DNA), glycineserine linker, various hinges, transmembrane domains, and cytoplasmic tail were purchased from Integrated DNA Technologies or Thermo-fisher Scientific. The gene fragments were inserted into pELNS vectors and placed under the control of the EF1a promoter using Gibson assembly (NEBuilder Hifi). The assembled plasmids were transformed into E. coli (NEB stable) for amplification and sequence verification before proceeding with viral production.

[0099] OTLV-IL21-41BBL-001 lentivirus production On the day of transfection, HEK293T cells were transferred to 15 × 10¹¹ cells in 20 mL total volume of growth medium (Dulbecco's Modified Eagle Medium (DMEM), 5% fetal bovine serum (FBS), and 1% penicillin / streptomycin). 6 Cells were seeded in collagen-coated tissue culture flasks containing the cells / flask. One hour before transfection, the growth medium was replaced with warmed SFM4Transfx-293. Cells were transfected with OT-IL21-41BBL-001 and the packaging plasmids pRSV.Rev, pMDLg / pRRE, and pMD2.G (Addgene#122590) using Lipofectamine3000 transfection reagent and P3000 enhancer reagent in Opti-MEM medium. The medium was replaced with SFM4Transfx-293 6-8 hours after transfection. The supernatant containing OTLV-IL21-41BBL-001 was collected 24 hours after transfection, fresh medium was added, and the supernatant was collected again 48 hours after transfection. The viral supernatant was filtered to remove debris and concentrated by ultracentrifugation at 25,000 g for 2 hours at 4°C. The lentivirus OTLV-IL21-41BBL-001 was resuspended, divided into equal parts, and stored at -80°C.

[0100] Transduction of K562 cells by OTLV-IL21-41BBL-001 lentivirus K562 cells were cultured in growth medium containing RPMI-1640 (complete RPMI, Thermo Fisher) with 2 mM L-glutamine and 10% FBS. On the day of transfection, K562 cells were transfected in 500 μL of K562 cell growth medium, 1.5 × 10⁶ cells. 5Cells were seeded in multi-well plates at a cell / well ratio. Cells were transduced with the OTLV-IL21-41BBL-001 lentivirus and then centrifuged at 800 g at 32°C for 1 hour. Cells were incubated for 24–48 hours, and then the viability and expression of IL21 and 4-1BBL were evaluated by flow cytometry using the antibodies eFluor 780 (Thermo Fisher, 1:1000), 4-1BBL phycoerythrin (1:50), and IL21 allophycocyanin (1:50). Transduced K562 cells were expanded in complete RPMI for 17 days, then fractionated, frozen using cell freezing medium (Bambanker, Bulldog Bio), and stored for extended periods in liquid nitrogen. These transduced K562 cells are referred to herein as K562-IL21-41BBL.

[0101] K562-IL21-41BBL cells were irradiated or treated with mitomycin C and then used as feeder cells in the TIL REP process. For irradiation, K562-IL21-41BBL cells were harvested from fresh cell cultures, centrifuged, and divided into 5-20 × 10⁶ cells. 6 The cells were resuspended in complete RPMI at a concentration of cells / mL. The resuspended cells were exposed to 50-200 Gy in an X-ray irradiator, then washed, and 3 × 10⁶ cells were prepared for immediate use in the REP process. 6 The cells were resuspended at a concentration of cells / mL. For mitomycin C treatment, the cells were thawed, centrifuged, and placed in TIL medium at a concentration of 5 × 10⁶. 6 The cells were resuspended at a concentration of cells / mL. 10 μg / mL of mitomycin-C was added to the cells, and the cells were incubated at 37°C for 30 minutes. Subsequently, the cells were washed three times with 50 mL of TIL medium, and 3 × 10⁶ cells were removed. 6 The cells were resuspended at a concentration of 1 / mL and immediately used in the REP process.

[0102] Example 3. Transduction of TIL using a lentiviral vector IL15 Vector Construct Assembly OT-IL15-292 and OT-IL15-293 (sequences below) were constructed in pELNS vectors (third-generation self-inactivating lentiviral expression vectors) using standard molecular biology techniques. Gene fragments (Gblocks) encoding codon-optimized IL15, GS linker, B7-1 hinge, transmembrane domain, and cytoplasmic tail were purchased from Integrated DNA Technologies, Inc. (IDT, Coralville, Iowa). The gene fragments were inserted into pELNS vectors and placed under the control of the EF1a promoter using Gibson assembly (NEBuilder Hifi). The assembled plasmids were transformed into E. coli (NEB stable) for amplification and sequence verification before proceeding with virus production.

[0103] Tables 1 and 2 (provided above) show the nucleic acid and amino acid sequences of the components of the constitutive mbIL15 construct (OT-IL15-292) and the ACZ-controlled mbIL15 construct (OT-IL15-293) disclosed herein. Construct OT-IL15-293 contains a destabilizing domain labeled as CA2(M1del, L156H) in Table 1.

[0104] Table 2 (provided above) also shows the nucleic acid and amino acid sequences of the constitutive IL15 (IL15-292) and ACZ-regulated IL15 (IL15-293) constructs disclosed herein.

[0105] BaEV-like lentivirus production HEK293T cells were seeded on collagen-coated tissue culture plates until 70% confluence. Cells were transfected with constitutive (IL15-292) or modified (IL15-293) IL15 constructs, as well as pELNS transfer vectors carrying the packaging plasmids pRSV.Rev (Addgene#12253), pMDLg / pRRE (Addgene#12251), and OT-BaEVg-002 (SEQ ID NO: XX), using Lipofectamine 3000 transfection reagent and P3000 enhancer reagent (Thermo Fisher) in Opti-MEM medium (Thermo Fisher). The medium was replaced with serum-free medium (SFM4Transfx-293, Cytiva) 6-8 hours after transfection. The supernatant containing the virus was collected 24 hours after transfection, fresh medium was added, and the supernatant was collected again 48 hours after transfection. The viral supernatant was filtered to remove debris and concentrated by low-speed ultracentrifugation. The virus was resuspended, divided into equal parts, and stored at -80°C.

[0106] Transduction of TIL using the Rapid Expansion Protocol (REP) and BaEV-pseudo-lentiviral vectors TILs generated from head and neck tumor samples prepared as described in Example 1 were manipulated 3 weeks after pre-culture in REP. The TILs were thawed with 6000 IU / mL human IL2 and left overnight in TIL medium. Next, the TILs were activated for 24 hours in a 24-well plate with anti-CD3 / CD28 beads (Dynabeads, Thermo Fisher) in a 3:1 bead-to-TIL ratio, or with plate-bound OKT3 at 3 μg / mL (Ultra-LEAF purified anti-human CD3 antibody, Biolegend) and 6000 IU / mL human IL2. A 96-well uncoated cell culture plate was coated overnight at 4°C with RetroNectin (30 μg / mL). The following day, RetroNectin was removed, the plate was blocked with 2% bovine serum albumin (BSA) in PBS, and then the plate was washed with PBS. The BaEV pseudo-lentivirus supernatant prepared as described above was diluted in TIL medium and added at a total volume of 100-200 μL per well for an MOI of 1-4 TU / cell. The plate containing the viral vector was centrifuged at 1400 g for 2 hours at 32°C, and then the supernatant was removed. After supernatant removal, 1.5 × 10⁶ per well was obtained. 5One activated TIL was transferred with IL-2 at 0–6000 IU / mL and incubated overnight at 37°C. Cells were similarly treated without virus addition and used as a negative control ("unprocessed"). 24 hours after transduction, the TILs were transferred to 6M GREX well plates (Wilson Wolf) in a total of 16–40 mL of TIL medium (RPMI-1640 supplemented with GlutaMAX (Thermo Fisher), 1% penicillin / streptomycin, 1 mM sodium pyruvate, 1% HEPES, 50 μM 2-mercaptoethanol (Invitrogen), and 10% thermo-inactivated human AB serum (Valley Bio)). Irradiated or mitomycin-C treated K562 feeder cells transduced in 41BBL and mbIL21 were added to the cultures in a 2:1 or 5:1 ratio of K562 to TIL, as described in Example 2. TILs transduced with a modified mbIL15 construct received 25 μM acetazolamide (SelleckChem), while untransduced TILs received 6000 IU / mL of IL2. Cells were grown in GREX plates for 14 days using the “rapid expansion protocol” or REP, with medium added or replaced as needed. During expansion, each GREX well was resuspended and thoroughly mixed, and aliquots were taken for cell counting (Cellaca Cell Counter, Nexcelom) and flow cytometry staining. Samples were stained with the antibodies CD3-BUV395 (BD), CD56-BV711 (Biolegend), CD4-BV605 (Biolegend), CD8-Alexa Fluor 700 (Biolegend), IL15-DyL650 (LakePharma, in-house conjugated), IL15RaFc-Biotin (ACRO Biosystems), secondary streptavidin-BV421 (Biolegend), and the fixable viability dye eFluor 780 (Thermo Fisher). Samples were flowed on a BD Fortessa flow cytometer and analyzed using Flow Jo V10.7.1. Transduction efficiency was determined by the percentage of cells stained double-positive for IL15-DyL650 and IL15RaFc-Biotin within the live CD3-positive cell population (Figure 2).

[0107] TILs transduced with a lentivirus containing the nucleic acid sequence encoding mbIL15 described herein may be referred to as "mbIL15 TILs" in the following examples. TILs transduced with a lentivirus containing the nucleic acid sequence encoding a modified mbIL15, such as OT-IL15-293, may also be referred to as "modified mbIL15 TILs" in the following examples. TILs transduced with a lentivirus containing the nucleic acid sequence encoding a constitutive mbIL15, such as OT-IL15-292, may also be referred to as "constitutive mbIL15 TILs" in the following examples.

[0108] Example 4. TIL extension of rapid expansion protocol TILs and feeder cells were generated as described in Examples 1-3 above. Briefly, three weeks after REP pre-culture, cryopreserved TILs were thawed and left overnight in 6000 IU / mL human IL-2. The TILs were then activated for 24 hours on multi-well plates coated with anti-CD3 / CD28 Dynabeads or OKT3, after which they were transduced with constitutive mbIL15 (OT-IL15-292) or GFP (OT-EGFP-001) lentiviral vectors, or left unmodified in an unprocessed state. 24 hours after transduction, the TILs were expanded with K562-IL21-41BBL feeder cells (feeder cell:TIL ratio 2:1) in GREX 6M well plates (Wilson Wolf), and 6000 IU / mL of I-2 was added to both unprocessed TILs and experimental "+IL2" conditions. Cells were grown in GREX plates for 12 days using the “rapid expansion protocol” or REP, with medium added or replaced as needed. Each GREX well was resuspended on days 5, 8, and 12 post-transduction. Aliquots were taken for flow cytometry staining to quantify the number of IL15+ or GFP+ cells as described in Example 3, using the antibodies CD3-BUV395 (BD), CD56-BV711 (Biolegend), CD4-BV605 (Biolegend), CD8-Alexa Fluor 700 (Biolegend), IL15-DyL650 (LakePharma, in-house conjugated), IL15RaFc-Biotin (ACRO Biosystems) and secondary streptavidin-BV421 (Biolegend), and the fixable viability dye eFluor 780 (Thermo Fisher). GFP-expressing TILs require exogenous IL2 for expansion in REP, while constitutive mb-IL15-expressing TILs expand in the absence of IL2 (Figure 3A).

[0109] Example 5. Expansion and survival in antigen-independent settings Next, TILs after REP were evaluated for their ability to persist or expand in association with an in vitro antigen-independent survival assay. Twelve days after REP expansion, mbIL15 transdextrinsed cells expanded without cytokines and GFP cells expanded with 6000 IU / mL IL2 were debeaded, washed, and left overnight without cytokines. The following day, TILs were expanded in TIL medium at a rate of 5 × 10⁴ 5 Cells were plated in 48-well plates at a cell / well rate. Cells were divided or culture medium was added every two days for a total of 10 days. For flow cytometry staining, aliquots were also taken every two days, and the number of IL15+ or GFP+ cells was quantified as described in Example 3. Constitutive mbIL15 TILs expanded during the 14-day survival assay, with or without exogenous IL2, while GFP TILs required IL2 for expansion (Figure 3B).

[0110] In a novel study involving modified mbIL15-expressing TILs, TILs and feeder cells were generated as described in Examples 1-3 above. Briefly, three weeks after pre-culture in REP, cryopreserved TILs were thawed and left overnight in 6000 IU / mL human IL2. The TILs were then activated for 24 hours on multi-well plates coated with anti-CD3 / CD28 Dynabeads or OKT3, and subsequently transduced with constitutive mbIL15 (OT-IL15-292) or modified mbIL15 (OT-IL15-293) lentiviral vectors, or transduced with unmodified vectors. Twenty-four hours after transduction, 6000 IU / mL of IL-2 was added to UT TILs, and the TILs were expanded in K562-IL21-41BBL feeder cells (feeder cell:TIL ratio 5:1) in GREX 6M well plates (Wilson Wolf) with 25 μM acetazoleamide (SelleckChem) added to the modified mbIL15 TILs. After 14 days of expansion, the TILs were isolated and refractory to 5 × 10⁶ cells in TIL medium, regardless of whether IL-2 (200 IU / mL, Peprotech) or acetazoleamide (25 μM, SelleckChem) was added.5 Cells / well were plated into multi-well plates. Entire wells were collected for cell expansion by cell counting (Celleca Cell Counter, Nexelom) and phenotypic analysis by flow cytometry (BD Fortessa), and fresh cytokines / ligands were added every 3 days. As demonstrated in Figure 4, over 15 days of this assay, unmodified TILs did not expand without exogenous cytokines (0.07±0.03-fold expansion), but were able to expand more than 20-fold (27.8±0.25-fold expansion) with exogenous IL2 (200 IU / mL). In contrast, modified TILs expanded significantly without the addition of exogenous cytokines; after 15 days, constitutive mbIL15 TILs expanded 8-fold (8.28±1.9-fold expansion), and modified mbIL15 TILs given 25 μM acetazoleamide expanded 17-fold (17.3±0.82-fold expansion). Without the addition of acetazoleamide, regulated mbIL15 TILs expanded four-fold less than their ligand counterparts (4.52 ± 0.48-fold expansion), highlighting the role of acetazoleamide in regulating the survival of regulated mbIL15 TILs.

[0111] Example 6. Expansion and survival in antigen-dependent settings TILs and feeder cells were generated as described in Examples 1-3 above. Briefly, three weeks after pre-culture in REP, cryopreserved TILs were thawed and left overnight in 6000 IU / mL human IL-2. The TILs were then activated for 24 hours in multi-well plates coated with anti-CD3 / CD28 Dynabeads or OKT3, and subsequently transduced with a modified mbIL15 (OT-IL15-293) lentiviral vector or transduced in an unmodified form. 24 hours after transduction, 6000 IU / mL of IL-2 was added to the UT TILs, and the TILs were expanded with K562-IL21-41BBL feeder cells (feeder cell:TIL ratio 5:1) in GREX 6M well plates (Wilson Wolf) to which 25 μM acetazoleamide (SelleckChem) was added to the modified mbIL15 TILs. After 14 days of expansion, TILs were cryopreserved in Bambanker freezing medium (Bulldog Bio). Subsequently, the cryopreserved TILs were thawed and left overnight in TIL medium with 200 IU / mL of IL2 (unmodified TILs) or in TIL medium with 25 μM of acetazoleamide (modified mbIL15 TILs). After overnight, the TILs were plated in a 1:1 ratio with mitomycin C-treated melanoma cells in a TIL:tumor co-culture assay, with or without the addition of IL2 (200 IU / mL, Peprotech) or acetazoleamide (25 μM, SelleckChem), and the assay was maintained for a total of 27 days. A vehicle-only control was included in the acetazoleamide group, and the same volume of DMSO was added to the vehicle control group. Melanoma cells were derived from the A375 cell line (ATCC) modified with a puromycin-dependent luciferase vector, and were treated with 10 μg / mL mitomycin C as described above (Example 3) to prevent proliferation of these tumor cells. Every three days, the wells of this co-culture assay were mixed, and aliquots were isolated for cell expansion by cell counting (Celleca Cell Counter, Nexelom) and phenotypic analysis by flow cytometry (BD Fortessa).A375 melanoma cells treated with fresh mitomycin C and fresh cytokines / ligands in TIL medium were added every 3 days. As demonstrated in Figure 5, acetazoleamide-modulated mbIL15 TILs established stable dilation kinetics, and transduced TILs persisted even in this antigen-dependent setting where chronic stimulation would rapidly efflux TILs and reduce cell number. In the assay, unmodified TILs did not dilate without exogenous cytokines (0.46 ± 0.02-fold dilation from day 1 to day 27), but with exogenous IL2 (200 IU / mL), dilation of over 25-fold (25.4 ± 4.06-fold dilation from day 1 to day 27) was possible. In contrast, modified TILs dilated without the addition of any exogenous cytokines, and a particularly modified mbIL15 TIL given 25 μM acetazoleamide dilated 12-fold (12.2 ± 0.10-fold dilation) from day 1 to day 27. Without the addition of acetazoleamide (with vehicle control only), regulated mbIL15 TILs expanded four times less than ligand (2.68 ± 0.42-fold expansion from day 1 to day 27), highlighting the role of acetazoleamide in regulating the survival of regulated mbIL15 TILs.

[0112] Example 7. Tumor response of fresh TIL after REP TILs derived from two melanoma donors were generated as described in Examples 1-3. Briefly, three weeks after pre-culture in REP, the cryopreserved TILs were thawed and left overnight in 6000 IU / mL human IL2. The TILs were then activated for 24 hours in multi-well plates coated with anti-CD3 / CD28 Dynabeads or OKT3, and subsequently transduced with a modified mbIL15 (OT-IL15-293) lentiviral vector or transduced in an unmodified form. 24 hours after transduction, the TILs were expanded with K562-IL21-41BBL feeder cells (feeder cell:TIL ratio 5:1) in GREX 6M well plates (Wilson Wolf), 6000 IU / mL IL2 was added to the unmodified TILs, and 25 μM acetazoleamide (SelleckChem) was added to the modified mbIL15 TILs. After 14 days of dilation, TILs were harvested, debeaded, and left overnight with or without IL2 and acetazoleamide. Melanoma cell lines expressing luciferase and A375-FLuc-Puro (ATCC) were selected in 5 × 10⁻⁶ samples. 6 The cells were resuspended in TIL medium at a concentration of cells / mL. 10 μg / mL of mitomycin-C was added to the cells, and then incubated at 37°C for 30 minutes. The cells were then washed three times with 50 mL of TIL medium. 1 × 10⁶ cells per well. 5A375 cells were added to a 96-well flat-bottom tissue culture plate. In some wells, 80 μg / mL of HLA-ABC (Biolegend) blocking antibody was added to block MHC class I on target cells. TILs, left overnight, were added in TIL:A375 ratios of 1:1 or 3:1, with a total volume of 200 μL per well. At 48 hours, the supernatant was saved from each well, and the IFNγ concentration was assayed by MSD. Lysis of tumor cells was analyzed using the CellTiterGlo Luminescent Cell Viability Assay (Promega) according to the manufacturer's protocol. The lysis percentage was calculated by dividing the luminescence in the co-culture well minus the background fluorescence by the luminescence in the control well containing only A375 minus the background fluorescence. Both untransduced TILs cultured with IL2 and regulated mbIL15 TILs expanded during REP in the absence of IL2 produced increased IFNγ in co-culture with the A375 melanoma cell line compared to TIL alone (Figure 6A). In addition, there was specific lysis of tumor cells under the co-culture conditions, as measured by a decrease in the luminescence of the target cell line (Figure 6B). Under co-culture conditions with an MHC class I blocking antibody, both the specific lysis percentage and IFNγ production decreased, indicating that the cytotoxicity of TILs against this tumor cell line is MHC class I dependent. This result was repeated in two melanoma donors.

[0113] Example 8.mbIL15 TIL is long-lasting in vivo without IL2. TILs were generated from one donor and feeder cell as described in Examples 1-3 above. Briefly, three weeks after pre-culture in REP, the cryopreserved TILs were thawed and left overnight in 6000 IU / mL human IL2. The TILs were then activated with anti-CD3 / CD28 Dynabeads for 24 hours, and subsequently transduced with constitutive mbIL15 (OT-IL15-292) or modified mbIL15 (OT-IL15-293) lentiviral vectors or unmodified versions. Twenty-four hours after transduction, TILs were expanded with K562-IL21-41BBL feeder cells (feeder cell:TIL ratio 5:1) in GREX 6M well plates (Wilson Wolf). 6000 IU / mL IL2 was added to the unmodified TILs, and 25 μM acetazoleamide (SelleckChem) was added to the modified mbIL15 TILs. After 14 days of expansion, the TILs were harvested, debeaded, and prepared for adoptive cell transfer. Unmodified TILs expanded 612-fold, constitutive mbIL5 TILs expanded 1080-fold, and modified mbIL15 TILs expanded 450-fold (Figure 7A).

[0114] NSG (NOD.Cg-PrkdcscidIl2rgtm1Wjl / SzJ) mice were purchased from Jackson Laboratories. Female mice aged 6-8 weeks were given 10 × 10¹⁶ doses, with or without exogenous IL2 (Proleukin) or clinical-grade acetazoleamide or vehicle, as described in Table 4. 6 TIL / mouse was injected systemically. [Table 4]

[0115] TILs were evaluated for IL15 expression on the day of adoptive cell therapy. Constitutive mbIL15-transduced TILs showed slightly higher levels of mbIL15 transduction (30.2 ± 0.46% IL15 + IL15RaFc +) than controlled mbIL15-transduced TILs (23.6 ± 1.1% IL15 + IL15RaFc +), but both transduction populations were acceptable for adoptive cell transfer (Figure 7B). IL15 expression or transduction efficiency was evaluated by flow cytometry, with cells incubated in Fc blocks and stained first with DyL650 (Lake Pharma, in-house conjugated) and then with IL15 conjugated to biotinylated IL15RaFc (ACROBiosystems). After incubation at room temperature in the dark for 25 minutes, the cells were washed in FACS buffer, centrifuged, and resuspended in FACS buffer containing streptavidin conjugated to BV421 (Biolegend). After incubation at 4°C in the dark for 20 minutes, the cells were washed in FACS buffer, centrifuged, resuspended in FACS buffer, and the samples were run on a BD Fortessa flow cytometer. Analysis was performed using FlowJo V10.7.1.

[0116] On days 7, 14, 21, 32, 39, 46, and 53 after adoptive cell therapy, 75 μL of systemic blood was isolated via submandibular vein collection in EDTA-containing tubes and processed for TIL enumeration. Blood samples were received in 1–3 mL of ACK lysis buffer (Gibco) and incubated for 10–20 minutes to lyse red blood cells (RBCs). After RBC lysis, the samples were filtered through a 70 μm cell strainer, centrifuged, and resuspended in FACS buffer. Aliquots of each sample were isolated for cell counting (Celleca Cell Counter, Nexelom) analysis, and the remainder was used for phenotypic evaluation by flow cytometry (BD Fortessa). To evaluate phenotypic characteristics, blood samples were stained with antibodies specific to CD3 (BD), mouse CD45, CD25 (BD), FoxP3, CD4, CD8, IL15 (Lake Pharma, in-house conjugated), KLRG1, CD127, CD45RA, CD45RO, CD95, CD69, CCR7, CD56, and biotinylated IL15RaFc (ACROBiosystems). The antibodies were conjugated with FITC, PE, PE-Cy5, PE-Cy7, PerCP-Cy5.5, DyL650, APC-Cy7, BUV395, BUV737, BV421, BV510, BV605, BV711, or BV786 (all anti-human antibodies for Biolegend unless otherwise specified). In addition, viability dye (e780 fixable viability dye, Invitrogen) was included in all samples. Samples were flowed on a BD Fortessa flow cytometer and analyzed using Flow Jo V10.7.1. To enumerate TILs throughout the study, TILs were gated as live cells, followed by lymphocytes, followed by human CD3+ and mouse CD45- cells. As shown in Figure 8A, unmanipulated TILs rapidly decreased in vivo and reached undetectable levels by 53 days post-injection. Unmanipulated TILs treated with exogenous IL2 performed better, but their persistence was lower by 53 days post-injection, with quantified TILs accounting for 0.64 ± 0.17%. In contrast, transduced TILs remained systemically detectable by 53 days post-injection, with constitutive mbI15 TILs accounting for 5.73 ± 1.2%.The mbIL15 TIL+ACZ was then adjusted to 10.2±2.0%. By day 53 post-infusion, the mbIL15 TIL+vehicle was adjusted to 2.94±0.36%, which was almost undetectable, demonstrating the in vivo modulating effect of acetazoleamide.

[0117] On days 14 and 53 after adoptive cell therapy, a cohort of five animals per experimental group was sacrificed for end-stage collection. From these animals, 200 μL of systemic blood was collected by cardiac puncture, the spleen was isolated, and bone marrow was collected from one femur. The blood was processed as described above. The spleen was mechanically ruptured through a 70 μm cell strainer, subjected to ACK lysis for 3 minutes to lyse RBCs, and collected again through a 70 μm cell strainer. Bone marrow (BM) was flushed through one femur and collected through a 70 μm cell strainer. Aliquots of each processed tissue suspension were isolated for cell counting analysis (Celleca Cell Counter, Nexelom), and the remainder was used for phenotypic evaluation by flow cytometry (BD Fortessa). For phenotypic evaluation, samples were stained with antibodies specific to CD3 (BD), mouse CD45, CD25 (BD), FoxP3, CD4, CD8, IL15 (Lake Pharma), KLRG1, CD127, CD45RA, CD45RO, CD95, CD69, CCR7, CD56, and biotinylated IL15RaFc (ACROBiosystems). The antibodies were conjugated with FITC, PE, PE-Cy5, PE-Cy7, PerCP-Cy5.5, DyL650, APC-Cy7, BUV395, BUV737, BV421, BV510, BV605, BV711, or BV786 (all anti-human antibodies for Biolegend unless otherwise specified). In addition, viability dye (e780 fixable viability dye, Invitrogen) was included in all samples. Samples were flowed onto a BD Fortessa flow cytometer and analyzed using Flow Jo V10.7.1. To enumerate TILs throughout the study, TILs were gated as live cells, followed by lymphocytes, and then human CD3+ and mouse CD45- cells. As demonstrated in Figures 8B and 8C, transduced TILs were identified at high levels in peripheral lymphoid organs at 14 and 53 days post-infusion, and ACZ-treated modified mbIL15 TILs demonstrated significantly higher persistence than their vehicle-treated counterparts (p<0.005).

[0118] Table 5 shows the viral vector sequences of various constructs described herein. [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4] [Table 5-5] [Table 5-6] [Table 5-7] [Table 5-8] [Table 5-9] [Table 5-10]

[0119] Example 9. Rapid Expansion Protocol Using TIL Retrovirus Transfection Pre-REP TIL was prepared in the same manner as in Example 1. Briefly, melanoma and head and neck tumor samples were obtained from the Cooperative Human Tissue Network. The tumor samples were cut into 1-3 mm fragments in Hanks equilibrium salt solution (HBSS) buffer, and the fragments were placed in a Grex container with 1-10 fragments per flask in TIL medium (RPMI-1640 supplemented with GlutaMAX (Thermo Fisher), 1% penicillin / streptomycin, 1 mM sodium pyruvate, 1% HEPES, 50 μM 2-mercaptoethanol (Invitrogen), and 10% thermo-inactivated human AB serum (Valley Bio)) containing 6000 IU / mL IL2 (Peprotech), 10 μg / mL 41BB antibody (Creative BioLabs), 30 ng / mL CD3 antibody (OKT3, Biolegend), and 0.1 mg / mL Normocin (InvivoGen). When nutrient depletion was identified, the cells were regularly supplied to the blood vessels approximately every 3-4 days. This culture process is referred to as pre-rapid expansion protocol (pre-REP). After pre-REP, the TILs were divided into equal portions, frozen in cell freezing media (Bambanker, Bulldog Bio, or Cryostor-10, STEMCELL Technologies), and stored for extended periods in liquid nitrogen.

[0120] These pre-REP TILs were thawed and left overnight in TIL medium containing 6000 IU / mL human IL-2. The TILs were then activated for 24 hours in 24-well plates coated with 3 ug / mL (Ultra-LEAF purified anti-human CD3 antibody, Biolegend) OKT3 and 6000 IU / mL human IL-2. The 24-well non-tissue culture plates were coated overnight at 4°C with RetroNectin (30 μg / mL). The following day, RetroNectin was removed, the plates were blocked with 2% bovine serum albumin (BSA) in PBS, and then washed with PBS. Supernatants of a Gibbon's monkey leukemia virus (GALV) pseudo-gammaretrovirus vector (mbIL15-CA2 DRD expression under the control of a mouse leukemia virus LTR-derived promoter) were prepared from stable producing cell lines. The retroviral vector supernatant was diluted in TIL medium and added at a total volume of 500 μL per well to obtain an approximate MOI of 16–80. The plates containing the viral vector were centrifuged at 1400 × g for 2 hours at 32°C, and then the supernatant was removed. After supernatant removal, 1.0 × 10⁶ per well was obtained. 6 Individual activated TIL cells were transferred with 100 IU / mL of IL-2 and incubated overnight at 37°C. Cells were similarly treated without virus addition and used as a negative control ("unprocessed"). 24 hours after transduction, 5 × 10⁶ cells were observed. 5TILs were transferred to each well of a 6M GREX well plate (Wilson Wolf) using a total of 60 mL of TIL medium per well (RPMI-1640 supplemented with GlutaMAX (Thermo Fisher), 1% penicillin / streptomycin, 1 mM sodium pyruvate, 1% HEPES, 50 μM 2-mercaptoethanol (Invitrogen), and 10% thermoinactivated human AB serum (Valley Biomed)). Irradiated K562 feeder cells (transduced with 4-1BBL and mbIL21 and irradiated at 100 Gy) or irradiated PBMC feeder cells (irradiated at 25 Gy) were thawed and added to the cultures in a ratio of 50:1 K562:TIL or 200:1 PBMC:TIL, respectively. TILs transduced with a modified mbIL15 construct received 25 μM acetazoleamide (SelleckChem), while untransduced TILs received 6000 IU / mL of IL2. Cells were grown in GREX plates for 14 days using the “rapid expansion protocol” or REP, with medium added or replaced as needed.

[0121] Example 10. Modified mbIL15-modified TIL: signaling and polyfunctionality ACZ modulates IL15 expression and signaling in regulated mbIL15 TILs in a dose-dependent manner. TILs prior to REP were prepared in the same manner as in Examples 1-3 and 9, and unprocessed and mbIL15 TILs were generated accordingly as described in Examples 1-3 and 9. Engagement of the IL15 signaling pathway results in downstream phosphorylation of signaling molecules, including the transcription factor protein STAT5 and the ribosomal protein S6. To demonstrate that ACZ-controlled mbIL15 expression results in IL15 signaling in controlled mbIL15 TILs, a phosphoflow cytometry-based assay was used as follows: Frozen-stored controlled mbIL15 TILs obtained from four human donors (patients 1-4) were thawed and then left in ACZ-free medium for 24 hours. Next, the controlled mbIL15 TILs were controlled for 18 hours in the presence of a series of ACZ concentrations, including 0.1, 1, 2.5, 5, 10, 25, and 100 μM, as well as a vehicle control. The controlled mbIL15 TILs were then collected for staining and FACS analysis.

[0122] In short, cells were stained with antibodies against CD3, CD4, CD8, and IL15, as well as live / dead markers. The cells were then fixed in 2% formaldehyde (BD Cytofix), permeabilized with methanol-based buffer (BD Phospho Perm III Buffer), and stained with antibodies specific to phosphorylated STAT5 (Biolegend) and S6 (Cell Signaling Technology). Cells were acquired on BD Symphony and analyzed using FlowJo software.

[0123] As the concentration of ACZ expression in mbIL15 increases, it plateaus around 10–25 μM of ACZ (Figure 9A). Similarly, the staining intensity of pSTAT5 and pS6 increases with higher concentrations of ACZ in controlled mbIL15 TILs, indicating a greater degree of IL15 signaling. These results demonstrate a dose-dependent relationship between ACZ and IL15 expression and signaling (Figures 9B–E and 10).

[0124] Regulated mbIL15 TIL constitutive mbIL15 expression and ACZ regulation are involved in the IL15 signaling pathway. To compare different strategies for IL15 expression, we used mbIL15 and modified mbIL15 TILs constitutively expressing TILs. Cryopreserved unmanipulated TILs, constitutive mbIL15 TILs, and modified mbIL15 TILs from three human donors were thawed and then left in ACZ-free medium for 24 hours. Next, the aforementioned TILs were prepared in medium for 18 hours as follows: (1) 200 IU / mL of IL2 (Peprotech) was added to the unmanipulated TILs, and (2) 25 μM ACZ was added to the modified mbIL15 TIL cultures. A vehicle was added under control conditions. After 18 hours of treatment, cells were stained with antibodies against CD3, CD4, CD8, IL15, and Live / Dead markers. Next, cells were fixed in 2% formaldehyde (BD Cytofix) and permeabilized with methanol-based buffer (BD Phospho Perm III Buffer) before staining with antibodies specific to phosphorylated STAT5 (Biolegend) and S6 (Cell Signaling Technology). Cells were acquired on BD Fortessa and analyzed using FlowJo software.

[0125] As shown in Figure 11, IL2 shares signaling pathways that overlap with IL15, including signaling via STAT5 and S6. Unmanipulated TILs cultured in IL2 showed increased involvement of signaling pathways compared to the corresponding vehicle state. Figure 11. Similarly, both constitutive mbIL15 expression and regulated mbIL15 TIL+ACZ showed increased phosphorylation of STAT5 and S6 compared to regulated mbIL15 TIL+ vehicle controls. Figure 11.

[0126] The modified mbIL15 TIL demonstrates greater polyfunctionality than the unmodified TIL+IL2. Polyfunctional T cells have the ability to simultaneously produce multiple effector molecules in response to stimulation. In addition, polyfunctionality correlates with T cell efficacy. To compare the polyfunctionality of unmanipulated TILs with regulated mbIL15 TILs, cryopreserved cells were thawed and left in a medium without IL2 and ACZ for 24 hours. Next, cell regulation occurred as follows: Unmanipulated TILs were regulated for 18 hours in the presence of IL2 concentrations ranging from 20, 200, 1000, and 6000 IU / mL, or vehicle. Regulated mbIL15 TILs were regulated for 18 hours in the presence of ACZ (0.1, 1, 5, 10, 25, 100 μM ACZ, or vehicle). Next, cells were stimulated for 6 hours with phorbol 12-myristate 13-acetate (PMA) and ionomycin (Biolegend) in the presence of brefelzin A (Biolegend) and monensin (Life Technologies Corporation). Unstimulated, unmanipulated TILs and unstimulated, modified mbIL15 TILs were used as controls. After stimulation, cells were collected for staining and FACS analysis.

[0127] In short, cells were stained with antibodies against CD3, CD4, CD8, and IL15, as well as viability dyes. The cells were then fixed and permeabilized with formaldehyde (BD Cytofix / Cytoperm kit), and subsequently stained with antibodies against TNFα and IFNγ (Biolegend). Cells were acquired on BD Fortessa and analyzed using FlowJo software. Cells that were double-positive for TNFα and IFNγ expression were considered polyfunctional.

[0128] As shown in Figure 12, all culture conditions contained several polyfunctional populations, but polyfunctionality in regulated mbIL15 TILs increased with higher concentrations of ACZ. Figures 12A, 12B. Furthermore, regulated mbIL15 TILs were more polyfunctional than unregulated TILs + IL2 from the same donor. Figures 12A, 12C. The proportion of regulated mbIL15 TILs expressing mbIL15 also showed a dose-response relationship with ACZ dose.

[0129] Example 11. In vivo efficacy of modified IL15 TIL PDX163A Effectiveness A patient-derived xenograft (PDX) model was generated from a fresh primary melanoma sample (patient tumor number M1200163A) obtained from the Cooperative Human Tissue Network (CHTN) tumor bank. A mouse model using NSG female mice was established (Jackson Laboratory, catalog number 000557). Once the model was established, cryopreserved tumor sections were aseptically transplanted into isoflurane-anesthetized, immunodeficient mice (NSG female mice, Jackson Laboratory, catalog number 000557). The tumor was placed at approximately 1000 mm². 3 ~2000mm 3 The tumors were allowed to grow until they reached a certain size, after which the mice were euthanized. The tumors were aseptically harvested, sectioned into approximately 100 mg sections, and transplanted into a larger cohort of mice that had been grown for 13 days. After 13 days, the tumors were measured and the mice were randomly assigned to their respective treatment groups (50 mm). 3 ~100mm 3 The following day, 10 million (10M) TILs were introduced intravenously. The TILs were generated according to the rapid dilation protocol (REP) described above.

[0130] The treatment groups were as follows: (1) unmanipulated TILs treated with IL2, and (2) modified mbIL15 TILs treated with acetazoleamide (ACZ). Mice treated with unmanipulated TILs were administered 50,000 international units (IU) of IL2 twice daily for 5 days. Mice treated with modified mbIL15 TILs were administered either a vehicle or 200 mg / kg of acetazoleamide (ACZ) daily throughout the study. Tumors and body weight were collected twice weekly.

[0131] Figure 13 shows the results of a patient-derived xenograft (PDX) model. At the end of the rapid expansion protocol (REP), unmanipulated TILs and modified mbIL15 TILs (+ / - acetazoleamide (ACZ)) were adopted into mice with human melanoma PDX. Mean tumor volume was assessed (+ / - SEM). Figure 13A shows the mean tumor volume of the given treatment in days after adoptive cell transfer (ACT). Figure 13B shows the tumor volume in days after ACT for no TIL (top left), unmanipulated TIL + IL2 (top right), modified mbIL15 TIL + vehicle (bottom left), and modified mbIL15 TIL + ACZ (bottom right). As shown in Figure 13, modified mbIL15 TIL + ACZ has significantly better antitumor efficacy compared to unmanipulated TIL + IL2.

[0132] SK-MEL-1 effectiveness To evaluate the modified mbIL15 TIL of the present invention, an SK-MEL-1 xenograft cancer model was created. The model was created using cells obtained from the thoracic tube of a patient with extensive and rapidly progressing malignant melanoma (ATCC catalog number HTB-67). NSG female mice (Jackson Laboratory, catalog number 000557) were used to accept the cancer cells. Briefly, low-passage cells were thawed and grown to maintain a viable subfluidic culture. On the day of injection, cells were counted, washed, and 30 × 10⁶ cells were prepared. 6 Cells / mL (3 per 100 μL injection) 6 The cells were resuspended in sterile PBS at the appropriate concentration. Each mouse was administered 100 μL of cells subcutaneously to its shaved right side using a BD tuberculin syringe containing a 27-gauge, 1 / 2-inch needle. The tumors were allowed to expand for 9 days, then measured and randomly assigned to their respective treatment groups (50 mm). 3 ~100mm 3 The following day, 10 million (10M) TILs were introduced intravenously. The TILs were generated according to the rapid dilation protocol (REP) described above.

[0133] The treatment groups were as follows: (1) unmanipulated TILs treated with IL2, and (2) modified mbIL15 TILs treated with acetazoleamide (ACZ). Mice treated with unmanipulated TILs were administered 50,000 international units (IU) of IL2 twice daily for 5 days. Mice treated with modified mbIL15 TILs were administered either a vehicle or 200 mg / kg of acetazoleamide (ACZ) daily throughout the study. Tumors and body weight were collected twice weekly.

[0134] Figure 14 shows the results from the SK-MEL-1 xenograft cancer model. At the end of the rapid expansion protocol (REP), unmanipulated TILs and modified mbIL15 TILs (+ / - acetazoleamide (ACZ)) were adopted into mice with SK-MEL-1 tumors. Mean tumor volume was assessed (+ / - SEM). Figure 14A shows the mean tumor volume of the given treatment in days after adoptive cell transfer (ACT). Figure 14B shows the tumor volume in days after ACT for no TIL (top left), unmanipulated TIL + IL2 (top right), modified mbIL15 TIL + vehicle (bottom left), and modified mbIL15 TIL + ACZ (bottom right). As shown in Figure 14, the results demonstrate that modified mbIL15 TIL + ACZ shows significantly superior antitumor efficacy compared to unmanipulated TIL + IL2.

[0135] Example 12. In vitro cytotoxicity with modified mbIL15 TIL TILs prior to REP were prepared in the same manner as in Examples 1-3 and 9, and unprocessed and mbIL15 TILs were generated according to the methods of Examples 1-3 and 9. To evaluate the potential antitumor cytotoxicity of the modified mbIL15 TILs, tumor-TIL co-culture assays were performed using the HLA-matched tumor cell line SK-MEL-1 (ATCC) and six different patient TIL samples. The same experiments were also set up using PDX cells. The patient TIL samples evaluated were either expanded unprocessed TILs or expanded modified mbIL15 TILs. Modified mbIL15 TILs were prepared according to the above REP protocol (Examples 1-9) and then cryopreserved. Then, unprocessed and modified mbIL15 TILs from the six patients were thawed, counted, and measured 7.5 × 10⁶. 5 Cells were left in medium supplemented with one of the following for 24 hours at a cell density of cells / mL: + / - 6000 IU / mL IL2 for unmanipulated TILs or vehicle (DMSO), or 25 μM ACZ for modified mbIL15 TILs. The following day, HLA-matched SK-MEL-1 cells were harvested from the in vitro culture and labeled with Cell Trace Far Red according to the manufacturer's protocol. In addition, PDX cells were obtained from fresh or cryopreserved masses and digested with GentleMACs (Miltenyi) according to the manufacturer's protocol.

[0136] Next, TILs were co-cultured with melanoma cells labeled under the same supplemental IL2 or ACZ conditions as described above, in 5:1 and 1:1 (TIL effector: tumor target) ratios, with or without MHC class I blocking reagents (tumor cells alone were cultured with 80 μg / mL anti-human HLA ABC for 2 hours, followed by co-culture with TILs). Additional controls of unlabeled and labeled melanoma cells alone were included to evaluate background caspase-3 activity in the co-culture system. This TIL tumor cell co-culture was incubated for 3 hours, after which the cells were fixed, permeabilized, and stained for intracellular cleavage caspase-3 (a marker of irreversible commitment to cell death within tumor cells).

[0137] Samples were acquired using a BD Fortessa flow cytometer with analysis performed using Flow Jo V10.7.1. Here, cytotoxicity was determined by the percentage of cells positively stained for cleavage caspase-3 within a population of living Cell Trace Far Red-positive cells (after subtracting background caspase-3 positivity).

[0138] As shown in Figure 15, in this evaluation of the antitumor cytotoxicity of TIL tumor pairs, modified mbIL15 TILs showed superior antitumor cytotoxic activity across all six donors compared to unmodified TILs + IL2. Figure 15.

[0139] Example 13: Generation of unmanipulated and mbIL15 TILs with distinct feeder cells Pre-REP TILs generated from tumor samples were prepared as described in Examples 1 and 9. The pre-REP TILs were thawed and incubated for 48 hours in TIL medium (RPMI-1640 supplemented with 6000 IU / mL human IL2 (Peprotech), GlutaMAX (Thermo Fisher), 1% HEPES, 50 μM 2-mercaptoethanol (Invitrogen), and 10% thermo-inactivated human AB serum (Valley Bio)). The TILs were then activated for 24 hours in 24-well NUNC plates coated with 3 μg / L of anti-CD3 (OKT3, Miltenyi Biotec) and 6000 IU / mL soluble human IL2. The 24-well uncoated cell culture plates were coated overnight at 4°C with RetroNectin (30 μg / mL). The following day, RetroNectin was removed, the plate was blocked with 2.5% human serum albumin (HSA) in PBS, and then the plate was washed with PBS. The BaEV pseudo-lentivirus supernatant prepared as described in Example 9 was diluted in TIL medium and added to each well to achieve an MOI of 0.01–0.6. The plate containing the viral vector was centrifuged at 1400 g for 2 hours at 32°C, and then the supernatant was removed. After supernatant removal, 1 × 10⁶ per well was used. 6One activated TIL was transferred with 0–100 IU / mL of IL-2 and incubated overnight at 37°C. Cells were similarly treated without adding the virus to the TIL medium and used as a negative control ("unmanipulated"). 24 hours after transduction, TIL was transferred to a 6M GREX flask (Wilson Wolf) with a total of 40 mL of TIL REP medium (50% TIL medium and 50% AIM-V medium (Gibco) as described above). Feeder cells with impaired growth (irradiated or treated with mitomycin-C) (pooled PBMCs, unmodified K562 feeders, K562 modified to express membrane-bound IL-21, K562 modified to express 41BBL, and K562 modified to express 41BBL and membrane-bound IL-21) were added to the culture in a K562 to TIL ratio of 50:1. The group designated to receive exogenous IL21 was administered 50 ng / mL of recombinant human IL21. TIL constructs transduced with a modified mbIL15 construct received 25 μM acetazoleamide (Hikma), while unmodified TILs received 3000 IU / mL of IL2. Cells were expanded in GREX plates for 14 days using the “rapid expansion protocol” or REP, with culture medium added as needed.

[0140] Evaluation of TIL extensions in REP During expansion, each GREX well was periodically resuspended and thoroughly mixed, and aliquots were taken for cell counting using Acridine Orange / Propidium Iodide viability dye (Cellaca Cell Counter, Nexcelom) and flow cytometry staining. Samples were stained with antibodies CD3-BUV395 (BD), CD56-BV711 (Biolegend), CD4-BV605 (Biolegend), CD8-Alexa Fluor 700 (Biolegend), IL15RaFc-Biotin (ACRO Biosystems) with secondary streptavidin-BV421 (Biolegend), and the fixable viability dye eFluor 780 (Thermo Fisher). Samples were flowed onto a BD Symphony flow cytometer and analyzed using Flow Jo V10.7.1.

[0141] Total TIL expansion was determined by obtaining the total viable cell count at a specific point in time through REP. Figure 16 shows that for mbIL15 TILs, both the use of K562 feeder cells and co-stimulation via IL-21 and 41BBL supported only suboptimal levels of TIL expansion in REP and PBMC feeder cells, as well as in K562 feeder cells without 41BBL, with the use of K562 feeder cells. In contrast, unmanipulated TILs expanded in the presence of IL-2 using either feeder cell, but PBMC feeder cells promoted maximum expansion of unmanipulated TILs during REP.

[0142] IL15 expression was determined by the percentage of cells staining positive for BV421-streptavidin within a population of living cells, CD3-positive cells, and CD56-negative cells. In mbIL15 TILs generated in K562 feeder cells and co-stimulated both via IL-21 and 41BBL, the frequency of mbIL15+ TILs increased throughout the REP process, suggesting enrichment of the mbIL15 transduction subset in the manipulated TIL cell culture (Figure 18). Similarly, the maximum expansion of mbIL15+ TILs in REP occurred when generating either constitutive or regulated mbIL15+ TILs using K562 feeder cells with co-stimulation both via IL-21 and 41BBL (Figure 19).

[0143] The CD4:CD8 ratio was determined by the ratio of the proportion of cells staining CD4-positive (live, CD3-positive, and CD56-negative cells) to the proportion of cells staining CD8-positive (live, CD3-positive, and CD56-negative cells). Extended mbIL15 TILs generated in K562 feeder cells and co-stimulated both via IL-21 and 41BBL were enriched in CD8+ cytotoxic effector cells. This is indicated by a decrease in the CD4:CD8 ratio through REP (Figure 20). In contrast, the CD4:CD8 ratio of mbIL15 TILs generated in pooled PBMC feeders, unmodified K562 feeders, or K562 feeders expressing 41BBL alone did not decrease during REP.

[0144] To evaluate multifunctionality, untreated and mbIL15 TIL samples at the end of REP were co-cultured in 96-well tissue culture-treated round-bottom plates using Immunocult CD3 / CD28 stimulation (Stem Cell Technologies), according to the manufacturer's protocol. After 1 hour of incubation, 1000x transport inhibitors (Monensin from eBiosciences, Brefeldin A from Biolegend) were added, and the co-cultures were incubated at 37°C for a further 5 hours. After incubation, the samples were stained with the antibodies mentioned above, and then fixed and permeabilized using Cytofix / Cytoperm reagent (BD Biosciences). Intracellular staining was performed using antibodies against IL2-BV737 (BD), IFNγ-FITC (Biolegend), Perforin-PerCPCy5.5 (Biolegend), TNFα-PECF594 (Biolegend), and granzymeB-Alexa Fluor 700 (Biolegend). Samples were flowed onto a BD Symphony flow cytometer and analyzed using Flow Jo V10.7.1. Polyfunctionality was determined as the percentage of TNFα and IFNγ double-positive cells among living lymphocytes. mbIL15 TILs generated from K562 feeder cells expressing both membrane-bound IL-21 and 41BBL showed improved polyfunctionality at the end of the REP compared to mbIL15 TILs generated from PBMC feeder cells or unmodified K562 feeder cells (Figure 21).

[0145] Evaluation of in vitro TIL persistence in antigen-independent survival assays TILs after repetition therapy (REP) were evaluated for in vitro persistence in antigen-independent survival assays. At the end of REP, unmodified and mbIL15 TILs were left for 24 hours without supplementation. The following day, unmodified cells were placed in 24-well GREX plates at a rate of 1 × 10⁶ cells, either without cytokine support or with 6000 IU / mL of IL2. 6Cells were double-cultured in wells, and mbIL15 TILs were cultured at the same density in either 25 μM ACZ or the same volume of vehicle (DMSO). On day 0, 100 μL of each well was sampled for TIL enumeration and phenotypic characterization, which was performed by cell counting and antibody staining as described above. On day 4, the cells were resuspended, 500 μL of cells were removed, and 500 μL of medium + treatment was added to each well to increase the culture volume to 1000 μL. On day 6, the cells were resuspended, 100 μL aliquots were sampled for phenotyping, 400 μL of cells were removed, and 500 μL of medium + treatment was added to each well to increase the culture volume to 1000 μL. On day 8, the cells were resuspended, 500 μL of cells were removed, and 500 μL of medium + treatment was added to each well to increase the culture volume to 1000 μL. On day 10, the cells were resuspended, 100 μL aliquots were sampled to determine the phenotype, and the culture was terminated. The samples were flowed on a BD Symphony flow cytometer and analyzed using Flow Jo V10.7.1. Extended mbIL15 TILs generated in K562 feeder cells and co-stimulated both via IL-21 and 41BBL showed improved persistence in the 10-day survival assay compared to mbIL15 TILs generated in PBMC feeder cells or unmodified K562 feeder cells independently expressing mbIL-21 and 41BBL (Figure 22).

[0146] Assessment of TCR diversity To measure the diversity of the TCRVβ subfamily, unprocessed and mbIL15 TILs at the end of the repertoire pulp (REP) were stained for flow cytometry using the Beta Mark TCR Vbeta Repertoire Kit (Beckman Coulter) according to the manufacturer's protocol. Samples were flowed on a BD Symphony flow cytometer and analyzed using Flow Jo V10.7.1 to assess the TCRVβ subfamily distribution by evaluating the positive rate of each subfamily and displaying the data as a set of all subfamilies of interest. Both unprocessed and mbIL15 TILs maintained a diverse TCRVβ subfamily distribution regardless of the feeder cells for expansion in the REP (Figure 23).

[0147] PD1 expression in mbIL15 TILs that have both 41BBL and IL21-mediated signaling To assess the level of TIL depletion, PD1 expression was determined. Samples were stained using the antibodies CD3-BUV395 (BD), CD56-BV711 (Biolegend), CD4-BV605 (Biolegend), CD8-Alexa Fluor 700 (Biolegend), PD1-PECy7 (Biolegend), CD25-BUV737 (Biolegend), IL15RaFc-Biotin (ACRO Biosystems) with secondary streptavidin-BV421 (Biolegend), and the fixable viability dye eFluor 780 (Thermo Fisher). For intracellular staining, cells were first stained with the above surface antibodies, then fixed, and permeabilized using the BD Cytofix / Cytoperm manufacturer's protocol. Next, permeable cells were stained with the antibody FoxP3-FITC (Biolegend), and the samples were flowed on a BD Symphony flow cytometer and analyzed using Flow Jo V10.7.1. PD1 expression was determined by the percentage of cells staining PD1 positively within a population of living cells, CD3-positive cells, and CD56-negative cells. As shown in Figure 25, PD1 expression was highest in unexpanded mbIL15 TILs, and expansion of mbIL15 TILs with both 41BBL and IL21-mediated signaling generates TILs with near-baseline PD1 expression.

[0148] Example 14: Phenotypic changes of mbIL15 TILs during manipulation and expansion compared to TILs (frequency of CD8+, CD4+, PD1+, and regulatory T cells) before REP. Phenotyping was performed to compare pre-REP TILs (as described in Example 1) with manipulated mbIL15 TILs (as described in Example 3). Pre-REP and post-REP TILs were phenotyped by flow cytometry using antibodies against CD3, CD4, CD8, and PD1, as described in Example 13. As shown in Figure 25A, the frequency of CD8+ T cells was higher in post-REP mbIL15 TILs compared to the corresponding pre-REP TILs from the same TIL donor, and the frequency of CD4+ T cells was lower, which is consistent with the results shown in Figure 20 from Example 13. This increase in CD8+ T cells reflects an increase in cytotoxic effector cells, as discussed and evaluated in Example 13. Similarly, as shown in Figure 25B, post-REP mbIL15 TILs expressed lower levels of PD1 than the corresponding pre-REP TILs from the same TIL donor, which is consistent with the results shown in Figure 24 from Example 13.

[0149] Regulatory T cells (T) in an expanded TIL population regTo detect cells, samples were stained using the antibodies CD3-BUV395 (BD), CD56-BV711 (Biolegend), CD4-BV605 (Biolegend), CD8-Alexa Fluor 700 (Biolegend), PD1-PECy7 (Biolegend), CD25-BUV737 (Biologend), IL15RaFc-Biotin (ACRO Biosystems) with secondary streptavidin-BV421 (Biolegend), and the fixable viability dye eFluor 780 (Thermo Fisher). For intracellular staining, cells were first stained with the above surface antibodies, then fixed, and permeabilized using the BD Cytofix / Cytoperm manufacturer's protocol. Permeabilized cells were then stained with the antibody FoxP3-FITC (Biolegend), and the samples were flowed on a BD Fortessa flow cytometer and analyzed using Flow Jo V10.7.1. Regulatory T cells were gated as CD4+ and further identified as CD3+ T cells, which are classified as CD25 and FoxP3 double-positive cells. As shown in Figure 25C, expanded mbIL15 TILs have a reduced proportion of regulatory T cells compared to pre-REP TILs before the manipulation step.

[0150] Example 15: Treatment using patient-derived xenograft (PDX) models and manipulated TILs Establishment of patient-derived xenograft (PDX) models A patient-derived xenograft (PDX) model (PDX 163A) was prepared from fresh primary melanoma samples obtained from a tumor bank, as described in Example 11. Once the model was established, cryopreserved tumor sections were aseptically transplanted into immunodeficient mice under isoflurane anesthesia. The tumors measured approximately 1000 mm² at the time of euthanasia. 3 ~2000mm 3 The tumors grew and were subsequently passaged to subsequent animals to maintain PDX tumor growth and establish an animal cohort for efficacy testing (described below).

[0151] PDX163A tumors excised from tumor-carrying animals were also evaluated for the expression of shared melanoma tumor antigens using flow cytometry. To assess the levels of conserved melanoma antigens on melanoma cells, the melanoma cell lines A375 and melanoma PDX described herein were assayed by flow cytometry. Tumor masses(s) derived from melanoma PDX as described in Example 11 were obtained fresh or from cryopreservation and digested using GentleMACs (Miltenyi) according to the manufacturer's protocol to obtain viable single-cell suspensions. Samples were stained and blocked with Fc blocking reagents using antibodies against MART-1 (Biolegend), gp100 (Biolegend), and the fixable viability dye eFluor 780 (Thermo Fisher). Samples were flowed on a BD Symphony flow cytometer and analyzed using Flow Jo V10.7.1. The frequency of melanoma-associated antigen-expressing tumor cells was determined by the proportion of cells staining positive for either MART-1 or gp100 within a living cell population. Figure 26 shows that both the conserved melanoma-associated antigens MART-1 and gp100 were expressed on PDX tumors selected for TIL efficacy modeling, as described in this embodiment (below).

[0152] Donor selection for allogeneic gene efficacy modeling TILs were generated from eight melanoma donors as described in Examples 1-3 or 9. Briefly, three weeks after pre-culture in REP, the cryopreserved TILs were thawed and left overnight in 6000 IU / mL human IL-2. The TILs were then activated for 24 hours in anti-CD3 / CD28 Dynabeads or OKT3 coated multi-well plates, after which they were transduced with a modified mbIL15 vector or transduced in an unmodified form. 24 hours after transduction, the TILs were expanded with K562-IL21-41BBL feeder cells in GREX 6M well plates (Wilson Wolf), with 6000 IU / mL of IL-2 added to the unmodified TILs and 25 μM acetazoleamide (SelleckChem or Hikma) added to the modified mbIL15 TILs. After 14 days of dilation, the TIL was collected, the beads were removed, and it was left overnight with or without IL2 and acetazoleamide.

[0153] Tetramer staining was used to determine which TIL donors reacted to the shared melanoma antigen, MART-1, and gp100. Flow cytometry was performed to assess the frequency of tetramer-reactive cells in order to evaluate the level of antigen-reactive TILs. Samples were blocked with Fc blocking reagents and stained with the antibodies CD3-BUV395 (BD), CD4-BV605 (Biolegend), CD8-Alexa Fluor 700 (Biolegend), HLA-A2:01-MART-1 tetramer (MBL International), HLA-A2:01-gp100 (MBL International), IL15RaFc-Biotin (ACRO Biosystems) with secondary streptavidin-BV421 (Biolegend), and the fixable viability dye eFluor 780 (Thermo Fisher). Samples were flowed on a BD Symphony flow cytometer and analyzed using Flow Jo V10.7.1. The frequency of antigen-reactive TILs was independently determined by the proportion of cells that stained positively for each of the two tetramers within a population of living, CD3-positive, and CD8-positive cells. As shown in Figure X, all four donors tested reacted to the MART-1 antigen, and three of the four donors tested reacted to the gp100 antigen. The tetramer-positive population indicates that TILs contain a subset of cells that react to the corresponding melanoma-associated antigens via the HLA:A2:01 locus. In Figure 27, donors marked with * were used in the PDX efficacy study as shown in this example (below).

[0154] Tumor masses (or multiple masses) derived from melanoma PDX as described in Example 11 were obtained fresh or from cryopreservation, and digested with GentleMACs (Miltenyi) according to the manufacturer's protocol to obtain viable single-cell suspensions. The PDX cells were then refueled in TIL medium at a rate of 5 × 10⁻⁶ cells. 6 The cells were resuspended at a concentration of cells / mL. 10 μg / mL of mitomycin-C was added to the cells, and then incubated at 37°C for 30 minutes. The cells were then washed three times with 50 mL of TIL medium. 1 × 10⁶ cells per well. 5PDX cells were added to 96-well flat-bottom tissue culture-treated plates. In some wells, 80 μg / mL of HLA-ABC (Biolegend) blocking antibody was added to block MHC class I on target cells. TILs, left overnight, were added in a TIL:PDX ratio of 1:1, with a total volume of 200 μL per well. As a positive control, TILs were co-cultured with PMA / ionomycin at 1:1000 to induce maximal IFNγ secretion. As a negative control, TILs were co-cultured without additional reagents or cells and identified as "unstimulated" TILs. At 24 hours, the supernatant was saved from each well and the IFNγ concentration was assayed by MSD.

[0155] Figure 28 shows that interferon-gamma (IFNγ) production after TIL:tumor cell co-culture can be used to predict TIL donors that will respond to PDX tumors. This in vitro assay shows that TIL donors 006, 39A, and 41A are the donors that produce the highest amounts of IFNγ in response to PDX, thus supporting their candidate status as donors for investigating in vivo efficacy, as described in this example (below).

[0156] Use of patient-derived xenograft (PDX) models for TIL efficacy trials Tumors from PDx tumor-carrying mice (passaged as described above) were aseptically harvested, sectioned into approximately 100 mg sections, then transplanted into a larger mouse cohort, grown for 13 days, measured, and randomly assigned to their respective treatment groups (50 mm). 3 ~100mm 3The following day, 10M TILs were introduced intravenously. Mice receiving unmanipulated TILs were administered 600,000 international units (IU) of IL2 daily for 4 days. Mice receiving mbIL15 products, in which mbIL15 was operably linked to CA2, were administered 200 mg / kg of acetazoleamide (ACZ) daily throughout the study. Tumors and body weight were collected twice weekly. The treatment paradigm is shown in Figure 29. As shown in Figure 30, manipulated TILs + ACZ showed superior antitumor effects compared to unmanipulated TILs + IL2. In addition, manipulated TILs, particularly in the presence of ACZ, showed better tumor infiltration, as shown in Figure 31A, and more tumors in both the stroma and tumor compartment, as shown in Figure 31B. The present invention provides, for example, the following items: (Item 1) A method for expanding tumor-infiltrating lymphocytes (TILs) that have been engineered to express membrane-bound IL15, comprising culturing the TILs in the presence of modified K562 feeder cells. (Item 2) The method according to item 1, wherein the expansion occurs in the absence of exogenous IL2. (Item 3) The method according to item 1 or 2, wherein the modified K562 feeder cells are unable to replicate. (Item 4) The method according to any one of items 1 to 3, wherein the modified K562 feeder cells express a costimulatory molecule selected from the tumor necrosis factor superfamily. (Item 5) The method according to item 3, wherein the co-stimulatory molecule selected from the tumor necrosis factor superfamily is 41BBL. (Item 6) The method according to any one of items 1 to 5, wherein the K562 feeder cells express IL21 or IL7. (Item 7) The method according to item 6, wherein the K562 feeder cells express membrane-bound IL21. (Item 8) A cultured product, (a) T cells or tumor-infiltrating lymphocytes, (b) The culture comprising modified K562 feeder cells, wherein the K562 feeder cells comprise a first exogenous nucleic acid sequence encoding a costimulatory molecule selected from the tumor necrosis factor superfamily, and a second exogenous nucleic acid sequence encoding IL21 or IL7. (Item 9) The culture described in item 8, wherein the culture contains modified tumor-infiltrating lymphocytes (TILs). (Item 10) The culture according to item 9, wherein the TIL is modified to express membrane-bound IL15. (Item 11) The culture according to item 10, wherein the expressed membrane-bound IL15 is operably linked to a drug-responsive domain. (Item 12) The culture described in any one of items 8 to 11, wherein the modified K562 feeder cells are unable to replicate. (Item 13) The culture described in any one of items 8 to 12, wherein the modified K562 feeder cells express membrane-bound IL21. (Item 14) A culture according to any one of items 8 to 13, wherein the co-stimulatory molecule selected from the tumor necrosis factor superfamily is 41BBL. (Item 15) A method for expanding T cells or tumor-infiltrating lymphocytes (TILs), comprising culturing modified T cells or TILs in the presence of a population of modified K562 feeder cells, wherein the modified K562 feeder cells support the expansion of T cells or TILs in the absence of IL2. (Item 16) The method according to item 15, wherein the modified K562 feeder cells include a first exogenous nucleic acid sequence encoding a costimulatory molecule selected from the tumor necrosis factor superfamily and a second exogenous nucleic acid sequence encoding IL21 or IL7. (Item 17) The method described in item 15 or 16, which extends the aforementioned modified TIL. (Item 18) The method according to item 17, wherein the TIL is modified to express membrane-bound IL15. (Item 19) The method according to item 18, wherein the expressed membrane-bound IL15 is operably linked to a drug-responsive domain. (Item 20) The method according to any one of items 15-19, wherein the modified K562 feeder cells are unable to replicate. (Item 21) The method according to any one of items 15 to 20, wherein the modified K562 feeder cells express membrane-bound IL21. (Item 22) The method according to any one of items 15 to 21, wherein the co-stimulatory molecule selected from the tumor necrosis factor superfamily is 41BBL. (Item 23) An expanded, modified T cell population prepared by the method described in any one of items 15-22. (Item 24) An extended TIL population prepared by the method described in any one of items 1-7. (Item 25) A method for treating cancer in a subject, comprising administering to the subject an expanded modified TIL population, wherein the TILs are expanded according to the method described in any one of items 1 to 7. (Item 26) A method for treating the cancer described in item 25, in which the subject is not administered exogenous IL2.

Claims

1. A method for expanding tumor-infiltrating lymphocytes (TILs), the method comprising culturing the TILs in the presence of modified K562 feeder cells, wherein the TILs are configured to express membrane-bound IL15 (mbIL15), and the mbIL15 is operably linked to a carbon dehydration enzyme 2 (CA2) drug-responsive domain (DRD).

2. The method according to claim 1, wherein the expansion occurs in the absence of exogenous IL2.

3. The method according to claim 1 or 2, wherein the modified K562 feeder cells are non-replicable.

4. The method according to any one of claims 1 to 3, wherein the modified K562 feeder cells express a costimulatory molecule selected from the tumor necrosis factor superfamily.

5. The method according to claim 4, wherein the co-stimulatory molecule selected from the tumor necrosis factor superfamily is 41BBL.

6. The method according to any one of claims 1 to 5, wherein the K562 feeder cells express IL21 or IL7.

7. The method according to claim 6, wherein the K562 feeder cells express membrane-bound IL21.

8. A cultured product, (a) T cells or tumor-infiltrating lymphocytes (TILs), wherein the T cells or TILs are engineered to express membrane-bound IL-15 (mbIL-15), and the mbIL-15 is operably linked to a carbon dehydration enzyme 2 (CA2) drug-responsive domain (DRD), (b) The culture comprising modified K562 feeder cells, wherein the K562 feeder cells comprise a first exogenous nucleic acid sequence encoding a costimulatory molecule selected from the tumor necrosis factor superfamily, and a second exogenous nucleic acid sequence encoding IL21 or IL7.

9. The culture according to claim 8, wherein the modified K562 feeder cells are incapable of replication.

10. The culture according to claim 8 or 9, wherein the modified K562 feeder cells express membrane-bound IL21.

11. The culture according to any one of claims 8 to 10, wherein the co-stimulatory molecule selected from the tumor necrosis factor superfamily is 41BBL.

12. A method for expanding T cells, comprising culturing the T cells in the presence of a population of modified K562 feeder cells, wherein the T cells are configured to express membrane-bound IL15 (mbIL15), the mbIL15 is operably linked to a carbon dehydration enzyme 2 (CA2) drug-responsive domain (DRD), and the modified K562 feeder cells support the expansion of the T cells in the absence of IL2.

13. The method according to claim 12, wherein the modified K562 feeder cell comprises a first exogenous nucleic acid sequence encoding a costimulatory molecule selected from the tumor necrosis factor superfamily, and a second exogenous nucleic acid sequence encoding IL21 or IL7.

14. The method according to claim 12 or 13, wherein the T cells are expanded.

15. The method according to any one of claims 12 to 14, wherein the modified K562 feeder cells are non-replicable.

16. The method according to any one of claims 12 to 15, wherein the modified K562 feeder cells express membrane-bound IL21.

17. The method according to any one of claims 12 to 16, wherein the co-stimulatory molecule selected from the tumor necrosis factor superfamily is 41BBL.

18. An expanded T cell population prepared by the method described in any one of claims 12 to 17.

19. An extended TIL group produced by the method described in any one of claims 1 to 7.

20. A composition for treating cancer in a subject, wherein the composition comprises an expanded TIL population, the TIL being expanded according to the method of any one of claims 1 to 7.

21. The composition for treating cancer according to claim 20, wherein the subject is not administered exogenous IL2.

22. A composition for treating cancer in a subject, wherein the composition comprises an expanded T cell population, the T cells being expanded according to the method of any one of claims 12 to 17.

23. The composition for treating cancer according to claim 22, wherein the subject is not administered exogenous IL2.

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