Improved selection of tumor-reactive T cells

Abstract

The present invention provides methods for preselecting TILs based on PD-1, CD39, CD38, CD103, CD101, LAG3, TIM3, and / or TIGIT expression, as well as methods for expanding those preselected PD-1, CD39, CD38, CD103, CD101, LAG3, TIM3, and / or TIGIT positive TILs to produce therapeutic TIL populations with enhanced tumor-specific killing capacity (e.g., enhanced cytotoxicity).

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 019,907, filed on 4 May 2020, and U.S. Provisional Patent Application No. 63 / 146,400, filed on 5 February 2021, the disclosures of which are incorporated herein by reference in their entirety for all purposes. [Background technology]

[0002] The treatment of large, refractory cancers using adoptive transfer of tumor-infiltrating lymphocytes (TILs) offers a powerful approach to treating patients with poor prognoses. Gattinoni, et al., Nat. Rev. Immunol. 2006, 6, 383-393. A large number of TILs are required for successful immunotherapy, and commercialization necessitates a robust and reliable process. This has been difficult to achieve due to technical, logistical, and regulatory issues related to cell proliferation. IL-2 systemic TIL proliferation and the subsequent "rapid proliferation process" (REP) have become the preferred method of TIL proliferation due to their speed and efficiency. Dudley, et al., Science 2002, 298, 850-54; Dudley, et al., J. Clin. Oncol. 2005, 23, 2346-57; Dudley, et al., J. Clin. Oncol. 2008, 26, 5233-39; Riddell, et al., Science 1992, 257, 238-41; Dudley, et al., J. Immunother. 2003, 26, 332-42. REP requires a large oversupply (e.g., 200-fold) of irradiated allogeneic peripheral blood mononuclear cells (PBMCs, also known as mononuclear cells (MNCs)), often from multiple donors, as feeder cells, as well as anti-CD3 antibody (OKT3) and high doses of IL-2, which can result in 1,000-fold proliferation of TIL over 14 days. Dudley, et al., J. Immunother. 2003, 26, 332-42. TILs treated with the REP procedure successfully underwent adoptive cell therapy following host immunosuppression in patients with melanoma. Current infusion acceptance parameters depend on the TIL composition reading (e.g., CD28, CD8, or CD4 positive), as well as the fold expansion and viability of the REP product.

[0003] Current TIL manufacturing processes are limited by length, cost, sterility concerns, and other factors described herein, severely restricting the potential for commercializing such processes. For example, while TILs have been characterized, they have been shown to express a variety of receptors, including inhibitory receptor programmed cell death 1 (PD-1, also known as CD279) (see Gros, A., et al., Clin Invest. 124(5):2246-2259 (2014)), and the usefulness of this information in the development of a therapeutic TIL population has not yet been fully realized. There is an urgent need to provide a TIL manufacturing process and a therapeutic approach based on such a process that is suitable for commercial-scale manufacturing and regulatory approval for use in human patients at multiple clinical centers. The present invention satisfies this need by providing a method for pre-selecting TILs based on PD-1, CD39, CD38, CD103, CD101, LAG3, TIM3, and / or TIGIT expression in order to obtain TILs having enhanced tumor-specific lethality (e.g., enhanced cytotoxicity). [Overview of the Initiative]

[0004] The present invention provides a method for expanding TILs and producing a therapeutic TIL population, comprising a pre-selection step of PD-1+, CD39+, CD103+, CD101+, LAG3+, TIM3+, and / or TIGIT+ states.

[0005] In some embodiments, the present invention is a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, (a) Obtaining and / or receiving a first TIL population from tumors excised from subjects by processing tumor samples obtained from subjects into multiple tumor fragments, (b) Select PD-1, CD39, CD38, CD103, CD101, LAG3, TIM3, and / or TIGIT-positive TILs from the first TIL population in (a) to obtain a TIL population rich in PD-1, CD39, CD38, CD103, CD101, LAG3, TIM3, and / or TIGIT, (c) A TIL population rich in PD-1, CD39, CD38, CD103, CD101, LAG3, TIM3, and / or TIGIT is cultured in a cell culture medium containing IL-2, OKT-3, and antigen-presenting cells (APCs) to produce a second TIL population by priming, wherein the first priming growth is carried out in a container having a first gas-permeable surface area, and the first priming growth is carried out for a first period of approximately 1 to 7, 8, 9, 10, or 11 days, thereby obtaining a second TIL population, the second TIL population being produced in a larger number than the first TIL population. (d) Producing a third TIL population by rapidly growing a second TIL population by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, and APC, wherein the number of APCs added to the rapid second growth is at least twice the number of APCs added in step (c), the rapid second growth is carried out for a second period of approximately 1 to 11 days, and a third TIL population is obtained, the third TIL population being a therapeutic TIL population, and the rapid second growth is carried out in a container having a second gas-permeable surface area. (e) Collect the therapeutic TIL population obtained from step (d), The present invention provides a method comprising (f) transferring the TIL population collected from step (e) into an injection bag.

[0006] In some embodiments, the present invention is a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, a) Obtaining and / or receiving a first TIL population from tumors excised from subjects by processing tumor samples obtained from subjects into multiple tumor fragments, (b) Select PD-1, CD39, CD38, CD103, CD101, LAG3, TIM3, and / or TIGIT-positive TILs from the first TIL population in (a) to obtain a TIL population rich in PD-1, CD39, CD38, CD103, CD101, LAG3, TIM3, and / or TIGIT, c) Producing a second TIL population by priming a TIL population rich in PD-1, CD39, CD38, CD103, CD101, LAG3, TIM3, and / or TIGIT in a cell culture medium containing IL-2, OKT-3, and optionally antigen-presenting cells (APCs), wherein the first priming growth is carried out for a first period of approximately 1 to 7, 8, 9, 10, or 11 days, yielding a second TIL population, and the second TIL population is produced in greater numbers than the first TIL population. d) Rapid growth of a second TIL population by contacting it with a cell culture medium containing IL-2, OKT-3, and APC to produce a third TIL population, wherein the rapid second growth takes place over a second period of approximately 1 to 11 days, yielding a third TIL population, which is the therapeutic TIL population. The present invention provides a method comprising e) collecting a therapeutic TIL population obtained from step (d).

[0007] In some embodiments, in step (c), the cell culture medium further comprises antigen-presenting cells (APCs), and the number of APCs in the culture medium in step (d) is greater than the number of APCs in the culture medium in step (c).

[0008] In some embodiments, in step (c), the cell culture medium further comprises antigen-presenting cells (APCs), and the number of APCs in the culture medium in step (d) is equal to the number of APCs in the culture medium in step (c).

[0009] In some embodiments, PD-1, CD39, CD38, CD103, CD101, LAG3, TIM3, and / or TIGIT-positive TILs are high in PD-1, high / low in CD39, low in CD38, high / low in CD103, low in CD101, high in LAG3, high in TIM3, and / or high in TIGIT.

[0010] In some embodiments, the present invention is a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, (a) A first TIL population selected to be PD-1, CD39, CD38, CD103, CD101, LAG3, TIM3, and / or TIGIT positive, obtained by processing a tumor sample from a subject by tumor digestion and selecting PD-1, CD39, CD38, CD103, CD101, LAG3, TIM3, and / or TIGIT positive TILs, and a second TIL population produced by priming a first proliferation by culturing the first TIL population in a cell culture medium containing IL-2, OKT-3, and antigen-presenting cells (APCs), wherein the priming a first proliferation is carried out in a container having a first gas-permeable surface area, the priming a first proliferation is carried out for a first period of approximately 1 to 7, 8, 9, 10, or 11 days, and a second TIL population is obtained, wherein the second TIL population is produced in a larger number than the first TIL population. (b) Producing a third TIL population by rapidly growing the second TIL population by contacting the second TIL population with a cell culture medium containing additional IL-2, OKT-3, and APCs, wherein the number of APCs in the rapid second growth is at least twice the number of APCs in step (a), the rapid second growth is carried out for a second period of approximately 1 to 11 days, and a third TIL population is obtained, the third TIL population being a therapeutic TIL population, and the rapid second growth is carried out in a container having a second gas-permeable surface area. The present invention provides a method comprising (c) collecting a therapeutic TIL population obtained from step (b).

[0011] In some embodiments, the present invention is a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, (a) Producing a second TIL population by culturing a first TIL population in a cell culture medium containing IL-2, OKT-3, and optionally antigen-presenting cells (APCs), thereby performing a first priming-induced proliferation of the first TIL population selected to be positive for PD-1, CD39, CD38, CD103, CD101, LAG3, TIM3, and / or TIGIT, wherein the first priming-induced proliferation is performed for a first period of approximately 1 to 7, 8, 9, 10, or 11 days, thereby obtaining a second TIL population, the second TIL population being produced in a larger number than the first TIL population. (b) Rapid growth of a second TIL population by contacting it with a cell culture medium containing IL-2, OKT-3, and APC to produce a third TIL population, wherein the rapid second growth takes place over a second period of approximately 1 to 11 days, yielding a third TIL population, the third TIL population being a therapeutic TIL population. The present invention provides a method comprising (c) collecting a therapeutic TIL population obtained from step (b).

[0012] In some embodiments, in step (a), the cell culture medium further comprises antigen-presenting cells (APCs), and the number of APCs in the culture medium in step (b) is greater than the number of APCs in the culture medium in step (a).

[0013] In some embodiments, in step (a), the cell culture medium further comprises antigen-presenting cells (APCs), and the number of APCs in the culture medium in step (b) is equal to the number of APCs in the culture medium in step (a).

[0014] In some embodiments, PD-1, CD39, CD38, CD103, CD101, LAG3, TIM3, and / or TIGIT-positive TILs are high in PD-1, high in CD39, low in CD38, high in CD103, low in CD101, high in LAG3, high in TIM3, and / or high in TIGIT.

[0015] In some embodiments, the selection in step (b) or step (a) includes a selection method selected from the group consisting of flow cytometry (including, for example, FACS), antibody-based bead selection, and antibody-based magnetic bead selection.

[0016] In some embodiments, the selection of step (b) or step (a) includes flow cytometry (including, for example, FACS).

[0017] In some embodiments, the selection of step (b) or step (a) includes (i) exposing a first TIL population to an excess of monoclonal anti-PD-1 IgG4 antibody that binds to PD-1 by the N-terminal loop outside the IgV domain of PD-1; (ii) adding the excess anti-IgG4 antibody conjugated to a fluorophore; and (iii) performing fluid cell sorting based on the fluorophore to obtain a PD-1-rich TIL population.

[0018] In some embodiments, the selection of PD-1, CD39, CD38, CD103, CD101, LAG3, TIM3, and / or TIGIT-positive TILs occurs until at least 1 × 10⁶ PD-1, CD39, CD38, CD103, CD101, LAG3, TIM3, and / or TIGIT-positive TILs are present.

[0019] In some embodiments, the cell culture medium for culturing the first TIL population contains 2-mercaptoethanol.

[0020] In some embodiments, the cell culture medium for culturing the second TIL population contains 2-mercaptoethanol.

[0021] In some embodiments, the cell culture medium for culturing the first TIL population and the second TIL population contains 2-mercaptoethanol.

[0022] In some embodiments, PD-1, CD39, CD38, CD103, CD101, LAG3, TIM3, and / or TIGIT-positive TILs are selected using anti-PD-1, anti-CD39, anti-CD38, anti-CD103, anti-CD101, anti-LAG3, anti-TIM3, and / or anti-TIGIT antibody composite beads, respectively.

[0023] In some embodiments, PD-1, CD39, CD38, CD103, CD101, LAG3, TIM3, and / or TIGIT-positive TILs are selected using anti-PD-1, anti-CD39, anti-CD38, anti-CD103, anti-CD101, anti-LAG3, anti-TIM3, and / or anti-TIGIT antibody composite magnetic beads, respectively.

[0024] In some embodiments, PD-1, CD39, CD38, CD103, CD101, LAG3, TIM3, and / or TIGIT-positive TILs bind to anti-PD-1, anti-CD39, anti-CD38, anti-CD103, anti-CD101, anti-LAG3, anti-TIM3, and / or anti-TIGIT antibody complex beads, respectively, while PD-1, CD39, CD38, CD103, CD101, LAG3, TIM3, and / or TIGIT-negative TILs do not bind to anti-PD-1, anti-CD39, anti-CD38, anti-CD103, anti-CD101, anti-LAG3, anti-TIM3, and / or anti-TIGIT antibody complex beads, respectively.

[0025] In some embodiments, the monoclonal anti-PD-1 IgG4 antibody is nivolumab, or a variant, fragment, or complex thereof.

[0026] In some embodiments, the anti-IgG4 antibody is clone anti-human IgG4, clone HP6023.

[0027] In some embodiments, the ratio of the number of APCs in the first growth by priming to the number of APCs in the rapid second growth is a ratio selected from the range of about 1.5:1 to about 20:1.

[0028] In some embodiments, the ratio is selected from the range of about 1.5:1 to about 10:1.

[0029] In some embodiments, the ratio is selected from the range of about 2:1 to about 5:1.

[0030] In some embodiments, the ratio is selected from the range of about 2:1 to about 3:1.

[0031] In some embodiments, the ratio is about 2:1.

[0032] In some embodiments, the number of APCs in the first growth by priming is about 1x10 8 APC to about 3.5x10 8 selected from the range of APCs, and the number of APCs in the rapid second growth is about 3.5x10 8 APC to about 1x10 9 selected from the range of APCs.

[0033] In some embodiments, the number of APCs in the first growth by priming is about 1.5x10 8 APC to about 3x10 8 selected from the range of APCs, and the number of APCs in the rapid second growth is about 4x10 8 APC to about 7.5x10 8 selected from the range of APCs.

[0034] In some embodiments, the number of APCs in the first growth by priming is about 2x10 8 APC to about 2.5x10 8 selected from the range of APCs, and the number of APCs in the rapid second growth is about 4.5x10 8 APC to about 5.5x1o 8 selected from the range of APCs.

[0035] In some embodiments, approximately 2.5 x 10 8 APC is added to the first propagation by priming, 5x10 8 APC is added to the rapid second growth.

[0036] In some embodiments, the ratio of the number of TILs in the second TIL group to the number of TILs in the first TIL group is approximately 1.5:1 to approximately 100:1.

[0037] In some embodiments, the ratio of the number of TILs in the second TIL group to the number of TILs in the first TIL group is approximately 50:1.

[0038] In some embodiments, the ratio of the number of TILs in the second TIL group to the number of TILs in the first TIL group is approximately 25:1.

[0039] In some embodiments, the ratio of the number of TILs in the second TIL group to the number of TILs in the first TIL group is approximately 20:1.

[0040] In some embodiments, the ratio of the number of TILs in the second TIL group to the number of TILs in the first TIL group is approximately 10:1.

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

[0042] In some embodiments, the method, after the step of collecting a therapeutic TIL population,

[0043] This includes the additional step of transferring the collected therapeutic TIL population to an infusion bag.

[0044] In some embodiments, a first growth by priming is carried out in a plurality of separate containers, in each of the separate containers a second TIL population is obtained from the first TIL population in the first growth by priming step, a third TIL population is obtained from the second TIL population in the rapid second growth step, and the therapeutic TIL population obtained from the third TIL population is collected from each of the plurality of containers and combined to produce a harvested TIL population.

[0045] In some embodiments, the plurality of separate containers include at least two separate containers.

[0046] In some embodiments, the multiple separate containers include 2 to 20 separate containers.

[0047] In some embodiments, the multiple separate containers include 2 to 10 separate containers.

[0048] In some embodiments, the multiple separate containers include 2 to 5 separate containers.

[0049] In some embodiments, each of the separate containers includes a first gas-permeable surface area.

[0050] In some embodiments, the first growth step, priming, is performed in a single container.

[0051] In some embodiments, a single container includes a first gas-permeable surface area.

[0052] In some embodiments, during a first proliferation step by priming, the cell culture medium contains antigen-presenting cells (APCs), which are layered on a first gas-permeable surface area with an average thickness of about 1 to 3 cell layers.

[0053] In some embodiments, during the first proliferation step by priming, the APCs are layered on a first gas-permeable surface area with an average thickness of about 1.5 to 2.5 cell layers.

[0054] In some embodiments, during the first proliferation step by priming, the APCs are layered on a first gas-permeable surface area with an average thickness of about two cell layers.

[0055] In some embodiments, during a rapid second proliferation step, the APCs are layered on the first gas-permeable surface area to a thickness of approximately 3 to 5 cell layers.

[0056] In some embodiments, during a rapid second proliferation step, the APCs are layered on the first gas-permeable surface area to a thickness of approximately 3.5 to 4.5 cell layers.

[0057] In some embodiments, during a rapid second proliferation step, the APCs are layered on the first gas-permeable surface area to a thickness of approximately 4 cell layers.

[0058] In some embodiments, in a first growth step by priming, the first growth by priming is carried out in a first container having a first gas-permeable surface area, and in a rapid second growth step, the rapid second growth is carried out in a second container having a second gas-permeable surface area.

[0059] In some embodiments, the second container is larger than the first container.

[0060] In some embodiments, during a first proliferation step by priming, the cell culture medium contains antigen-presenting cells (APCs), which are layered on a first gas-permeable surface area with an average thickness of about 1 to 3 cell layers.

[0061] In some embodiments, during the first proliferation step by priming, the APCs are layered on a first gas-permeable surface area with an average thickness of about 1.5 to 2.5 cell layers.

[0062] In some embodiments, during the first proliferation step by priming, the APCs are layered on a first gas-permeable surface area with an average thickness of about two cell layers.

[0063] In some embodiments, during a rapid second proliferation step, the APCs are layered on a second gas-permeable surface area with an average thickness of about 3 to 5 cell layers.

[0064] In some embodiments, during a rapid second proliferation step, the APCs are layered on a second gas-permeable surface area with an average thickness of approximately 3.5 to 4.5 cell layers.

[0065] In some embodiments, during a rapid second proliferation step, the APCs are layered on a second gas-permeable surface area with an average thickness of approximately four cell layers.

[0066] In some embodiments, for each container in which a first growth by priming is performed on a first TIL population, a rapid second growth is performed in the same container on a second TIL population produced from such first TIL population.

[0067] In some embodiments, each container includes a first gas-permeable surface area.

[0068] In some embodiments, during a first proliferation step by priming, the cell culture medium contains antigen-presenting cells (APCs), which are layered on a first gas-permeable surface area with an average thickness of about 1 to 3 cell layers.

[0069] In some embodiments, during the first proliferation step by priming, the APCs are layered on a first gas-permeable surface area with an average thickness of about 1.5 to 2.5 cell layers.

[0070] In some embodiments, during the first proliferation step by priming, the APCs are layered on a first gas-permeable surface area with an average thickness of about two cell layers.

[0071] In some embodiments, during a rapid second proliferation step, the APCs are layered on a first gas-permeable surface area with an average thickness of about 3 to 5 cell layers.

[0072] In some embodiments, during a rapid second proliferation step, the APCs are layered on the first gas-permeable surface area with an average thickness of about 3.5 to 4.5 cell layers.

[0073] In some embodiments, during a rapid second proliferation step, the APCs are layered on a first gas-permeable surface area with an average thickness of approximately 4 cell layers.

[0074] In some embodiments, in the first growth step by priming, for each container in which the first growth by priming is performed on a first TIL population, the container comprises a first gas-permeable surface area, the cell culture medium comprises antigen-presenting cells (APCs), the APCs are layered on the first gas-permeable surface area, and the ratio of the average number of layers of APCs layered in the first growth step by priming to the average number of layers of APCs layered in the rapid second growth step is selected from the range of approximately 1:1.1 to approximately 1:10.

[0075] In some embodiments, the ratio of the average number of layers of APCs stratified in the first growth step by priming to the average number of layers of APCs stratified in the rapid second growth step is selected from a range of about 1:1.2 to about 1:8.

[0076] In some embodiments, the ratio of the average number of layers of APCs stratified in the first growth step by priming to the average number of layers of APCs stratified in the second rapid growth step is selected from a range of about 1:1.3 to about 1:7.

[0077] In some embodiments, the ratio of the average number of layers of APCs stratified in the first growth step by priming to the average number of layers of APCs stratified in the rapid second growth step is selected from a range of about 1:1.4 to about 1:6.

[0078] In some embodiments, the ratio of the average number of layers of APCs stratified in the first growth step by priming to the average number of layers of APCs stratified in the rapid second growth step is selected from a range of about 1:1.5 to about 1:5.

[0079] In some embodiments, the ratio of the average number of layers of APC stratified in the first growth step by priming to the average number of layers of APC stratified in the rapid second growth step is selected from a range of about 1:1.6 to about 1:4.

[0080] In some embodiments, the ratio of the average number of layers of APCs stratified in the first growth step by priming to the average number of layers of APCs stratified in the rapid second growth step is selected from a range of about 1:1.7 to about 1:3.5.

[0081] In some embodiments, the ratio of the average number of layers of APC stratified in the first growth step by priming to the average number of layers of APC stratified in the rapid second growth step is selected from a range of approximately 1:1.8 to approximately 1:3.

[0082] In some embodiments, the ratio of the average number of layers of APCs stratified in the first growth step by priming to the average number of layers of APCs stratified in the rapid second growth step is selected from a range of about 1:1.9 to about 1:2.5.

[0083] In some embodiments, the ratio of the average number of layers of APCs stratified in the first growth step by priming to the average number of layers of APCs stratified in the second rapid growth step is approximately 1:2.

[0084] In some embodiments, after a rapid second growth step of 2-3 days, additional IL-2 is added to the cell culture medium.

[0085] In some embodiments, the method further includes, in the step of collecting a therapeutic TIL population using a cryopreservation process, cryopreserving the collected TIL population.

[0086] In some embodiments, the method further includes the step of freezing the injection bag.

[0087] In some embodiments, the cryopreservation process is carried out using a 1:1 ratio of the collected TIL population to the cryopreservation medium.

[0088] In some embodiments, the antigen-presenting cells are peripheral blood mononuclear cells (PBMCs).

[0089] In some embodiments, the PBMCs are irradiated and are homogeneous.

[0090] In some embodiments, in the first growth step by priming, the cell culture medium contains peripheral blood mononuclear cells (PBMCs), and the total number of PBMCs in the cell culture medium in the first growth step by priming is 2.5 × 10⁻⁶. 8 That is the case.

[0091] In some embodiments, during the rapid second growth step, the antigen-presenting cells (APCs) in the cell culture medium are peripheral blood mononuclear cells (PBMCs), and the total number of PBMCs added to the cell culture medium during the rapid second growth step is 5 × 10⁻⁶. 8 That is the case.

[0092] In some embodiments, the antigen-presenting cells are artificial antigen-presenting cells.

[0093] In some embodiments, the sampling in the step of collecting therapeutic TIL populations is performed using a membrane-based cell processing system.

[0094] In some embodiments, the harvesting in step (d) is performed using a LOVO cell processing system.

[0095] In some embodiments, the multiple fragments include about 60 fragments per container in the first growth step by priming, and each fragment is about 27 mm 3 It has the volume of .

[0096] In some embodiments, multiple pieces are approximately 1300 mm 3 ~about 1500mm 3 It contains approximately 30 to 60 fragments, totaling a substantial volume.

[0097] In some embodiments, multiple pieces are approximately 1350 mm 3 It contains approximately 50 fragments with a total volume.

[0098] In some embodiments, the multiple fragments include about 50 fragments with a total mass of about 1 gram to about 1.5 grams.

[0099] In some embodiments, the cell culture medium is provided in a container selected from the group consisting of G containers and Xuri cell bags.

[0100] In some embodiments, 2-3 days after step (d), the cell culture medium is supplemented with additional IL-2.

[0101] In some embodiments, the IL-2 concentration is approximately 10,000 IU / mL to approximately 5,000 IU / mL.

[0102] In some embodiments, the IL-2 concentration is approximately 6,000 IU / mL.

[0103] In some embodiments, the infusion bag used in the step of transferring the collected therapeutic TIL population to an infusion bag is an infusion bag containing HypoThermosol.

[0104] In some embodiments, the cryopreservation medium contains dimethyl sulfoxide (DMSO).

[0105] In some embodiments, the cryopreservation medium contains 7% to 10% DMSO.

[0106] In some embodiments, the first period in the first growth step by priming and the second period in the rapid second growth step are each carried out individually within a period of 5, 6, 7, 8, 9, 10, or 11 days.

[0107] In some embodiments, the first period of the first growth step by priming is carried out within a period of 5, 6, or 7 days.

[0108] In some embodiments, the first period of the first growth step by priming is carried out within a period of 8, 9, 10, or 11 days.

[0109] In some embodiments, the second period of the rapid second growth step is carried out within a period of 7, 8, or 9 days.

[0110] In some embodiments, the second period of the rapid second growth step is carried out within a period of 10 or 11 days.

[0111] In some embodiments, the first period of the first growth step by priming and the second period of the rapid second growth step are each carried out individually within a period of 7 days.

[0112] In some embodiments, the first period of the first growth step by priming and the second period of the rapid second growth step are each carried out individually within a period of 11 days.

[0113] In some embodiments, the first growth step, priming through the collection of a therapeutic TIL population, is carried out within a period of approximately 14 to 16 days.

[0114] In some embodiments, the first growth step, priming through the collection of a therapeutic TIL population, is carried out within a period of approximately 15 to 16 days.

[0115] In some embodiments, the first growth step, involving priming through the collection of a therapeutic TIL population, is carried out within a period of approximately 14 days.

[0116] In some embodiments, the first growth step, involving priming through the collection of a therapeutic TIL population, is carried out within a period of approximately 15 days.

[0117] In some embodiments, the first growth step, priming through the collection of a therapeutic TIL population, is carried out within a period of approximately 16 days.

[0118] In some embodiments, the method further includes the step of cryopreserving a collected therapeutic TIL population using a cryopreservation process, wherein the first step of priming and cryopreservation through the collection of the therapeutic TIL population is performed within 16 days or less.

[0119] In some embodiments, the therapeutic TIL population collected in the step of collecting the therapeutic TIL population contains enough TILs to constitute a therapeutically effective dose of TILs.

[0120] In some embodiments, the number of TILs sufficient for a therapeutically effective dose is approximately 2.3 × 10⁶ 10 ~Approx. 13.7×10 10 That is the case.

[0121] In some embodiments, the third TIL population in a rapid second growth step provides increased efficacy, increased interferon-gamma production, and / or increased polyclonality.

[0122] In some embodiments, the third TIL population in a rapid second growth step provides at least 1 to 5 times greater interferon-gamma production compared to TILs prepared by a process longer than 16 days.

[0123] In some embodiments, effector T cells and / or central memory T cells obtained from the third TIL population in a rapid second proliferation step show increased CD8 and CD28 expression compared to effector T cells and / or central memory T cells obtained from the second TIL population in a first proliferation step by priming.

[0124] In some embodiments, the therapeutic TIL population is injected into the patient following the step of collecting the therapeutic TIL population.

[0125] In some embodiments, the method further includes the step of cryopreserving an infusion bag containing a collected TIL population using a cryopreservation process.

[0126] In some embodiments, the cryopreservation process is carried out using a 1:1 ratio of the collected TIL population to the cryopreservation medium.

[0127] In some embodiments, the antigen-presenting cells are peripheral blood mononuclear cells (PBMCs).

[0128] In some embodiments, the PBMCs are irradiated and are homogeneous.

[0129] In some embodiments, the antigen-presenting cells are artificial antigen-presenting cells.

[0130] In some embodiments, the harvesting step is performed using a membrane-based cell processing system.

[0131] In some embodiments, the harvesting step is performed using a LOVO cell processing system.

[0132] In some embodiments, the multiple fragments include approximately 60 fragments, each fragment being approximately 27 mm in diameter. 3 It has the volume of .

[0133] In some embodiments, multiple pieces are approximately 1300 mm 3 ~about 1500mm 3 It contains approximately 30 to 60 fragments, totaling a substantial volume.

[0134] In some embodiments, multiple pieces are approximately 1350 mm 3 It contains approximately 50 fragments with a total volume.

[0135] In some embodiments, the multiple fragments include about 50 fragments with a total mass of about 1 gram to about 1.5 grams.

[0136] In some embodiments, the cell culture medium is provided in a container selected from the group consisting of G containers and Xuri cell bags.

[0137] In some embodiments, the IL-2 concentration is approximately 10,000 IU / mL to approximately 5,000 IU / mL.

[0138] In some embodiments, the IL-2 concentration is approximately 6,000 IU / mL.

[0139] In some embodiments, the infusion bag in step (d) is an infusion bag containing HypoThermosol.

[0140] In some embodiments, the cryopreservation medium contains dimethyl sulfoxide (DMSO).

[0141] In some embodiments, the cryopreservation medium contains 7% to 10% DMSO.

[0142] In some embodiments, the first and second periods in step (c) are each carried out individually within a period of 5, 6, or 7 days.

[0143] In some embodiments, the first period is carried out within a period of 5, 6, or 7 days.

[0144] In some embodiments, the second period is carried out within a period of 10 or 11 days.

[0145] In some embodiments, the first period and the second period are each carried out separately within a period of 7 days.

[0146] In some embodiments, all steps are carried out within a period of approximately 14 to 22 days.

[0147] In some embodiments, all steps are carried out within a period of approximately 14 to 21 days.

[0148] In some embodiments, all steps are carried out within a period of approximately 14 to 20 days.

[0149] In some embodiments, all steps are carried out within a period of approximately 14 to 19 days.

[0150] In some embodiments, all steps are carried out within a period of approximately 14 to 18 days.

[0151] In some embodiments, all steps are carried out within a period of approximately 14 to 17 days.

[0152] In some embodiments, all steps are carried out within a period of approximately 14 to 16 days.

[0153] In some embodiments, all steps are carried out within a period of approximately 15 to 16 days.

[0154] In some embodiments, all steps are performed within a period of approximately 14 days.

[0155] In some embodiments, all steps are performed within a period of approximately 15 days.

[0156] In some embodiments, all steps are performed within a period of approximately 16 days.

[0157] In some embodiments, all steps and cryopreservation are carried out within 16 days.

[0158] In some embodiments, the collected therapeutic TIL population contains enough TILs to constitute a therapeutically effective dose of TILs.

[0159] In some embodiments, the number of TILs sufficient for a therapeutically effective dose is approximately 2.3 × 10⁶ 10 ~Approx. 13.7×10 10 That is the case.

[0160] In some embodiments, the container in the first growth step by priming is larger than the container in the second rapid growth step.

[0161] In some embodiments, the third TIL population provides increased efficacy, increased interferon-gamma production, and / or increased polyclonality.

[0162] In some embodiments, the third TIL population provides at least 1 to 5 times greater interferon-gamma production compared to TILs prepared by processes longer than 16 days.

[0163] In some embodiments, effector T cells and / or central memory T cells obtained from a third TIL population exhibit increased CD8 and CD28 expression compared to effector T cells and / or central memory T cells obtained from a second cell population.

[0164] In some embodiments, the collected TIL is injected into the patient.

[0165] In some embodiments, the present invention is a method for treating a subject having cancer, (a) Obtaining and / or receiving a first TIL population from tumors excised from subjects by processing tumor samples obtained from subjects into multiple tumor fragments, (b) Select PD-1, CD39, CD38, CD103, CD101, LAG3, TIM3, and / or TIGIT-positive TILs from the first TIL population in (a) to obtain a TIL population rich in PD-1, CD39, CD38, CD103, CD101, LAG3, TIM3, and / or TIGIT, (c) A TIL population rich in PD-1, CD39, CD38, CD103, CD101, LAG3, TIM3, and / or TIGIT is cultured in a cell culture medium containing IL-2, OKT-3, and antigen-presenting cells (APCs) to produce a second TIL population by priming a first growth, wherein the first growth by priming is carried out in a container having a first gas-permeable surface area, and the first growth by priming is carried out for approximately 1 to 7, 8, 9, 10, or 11 days to obtain and produce the second TIL population. (d) Producing a third TIL population by rapidly growing a second TIL population by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, and APC, wherein the number of APCs added to the rapid second growth is at least twice the number of APCs added in step (b), the rapid second growth is carried out for approximately 1 to 11 days, and a third TIL population is obtained, the third TIL population being a therapeutic TIL population, and the rapid second growth is carried out in a container containing a second gas-permeable surface area. (e) Collect the therapeutic TIL population obtained from step (c), (f) Transfer the TIL sample collected from step (d) to an injection bag, The present invention provides a method comprising administering expanded tumor-infiltrating lymphocytes (TILs), including (g) administering them to TILs from step (e) a therapeutically effective dose.

[0166] In some embodiments, the number of TILs sufficient to administer a therapeutically effective dose in step (g) is approximately 2.3 × 10⁻⁶. 10 ~Approx. 13.7×10 10 That is the case.

[0167] In some embodiments, PD-1, CD39, CD38, CD103, CD101, LAG3, TIM3, and / or TIGIT-positive TILs are high in PD-1, high in CD39, low in CD38, high / low in CD103, low in CD101, high in LAG3, high in TIM3, and / or high in TIGIT.

[0168] In some embodiments, the selection in step (b) includes (i) exposing a first TIL population to an excess of monoclonal anti-PD-1 IgG4 antibody that binds to PD-1 by the N-terminal loop outside the IgV domain of PD-1; (ii) adding the excess anti-IgG4 antibody conjugated to a fluorophore; and (iii) performing fluid cell sorting based on the fluorophore to obtain a PD-1-rich TIL population.

[0169] In some embodiments, the monoclonal anti-PD-1 IgG4 antibody is nivolumab, or a variant, fragment, or complex thereof.

[0170] In some embodiments, the anti-IgG4 antibody is clone anti-human IgG4, clone HP6023.

[0171] In some embodiments, antigen-presenting cells (APCs) are PBMCs.

[0172] In some embodiments, a non-myeloablative lymphocyte depletion regimen is administered to the subject before administering a therapeutically effective dose of TIL cells in step (g).

[0173] In some embodiments, a non-myeloablative lymphocyte depletion regimen involves cyclophosphamide at 60 mg / m². 2 Administer at a daily dose for 2 days, followed by fludarabine at 25 mg / m². 2 The step includes administering the drug at a daily dose for 5 days.

[0174] In some embodiments, the method further includes the step of treating the patient with a high-dose IL-2 regimen, which is initiated the day after the administration of TIL cells to the subject in step (g).

[0175] In some embodiments, the high-dose IL-2 regimen includes 600,000 or 720,000 IU / kg, administered as a 15-minute bolus intravenous infusion every 8 hours until an acceptable level is reached.

[0176] In some embodiments, the third TIL population in step (c) provides increased efficacy, increased interferon-gamma production, and / or increased polyclonality.

[0177] In some embodiments, the third TIL population in step (d) provides at least 1 to 5 times greater interferon-gamma production compared to TILs prepared by a process longer than 16 days.

[0178] In some embodiments, effector T cells and / or central memory T cells obtained from a third TIL population exhibit increased CD8 and CD28 expression compared to effector T cells and / or central memory T cells obtained from a second TIL population.

[0179] In some embodiments, cancer is selected from the group consisting of melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, triple-negative breast cancer, cancer, cancer caused by human papillomavirus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), glioblastoma (including GBM), gastrointestinal cancer, kidney cancer, and renal cell carcinoma.

[0180] In some embodiments, the cancer is selected from the group consisting of melanoma, HNSCC, cervical cancer, NSCLC, glioblastoma (including GBM), and gastrointestinal cancer.

[0181] In some embodiments, the cancer is melanoma.

[0182] In some embodiments, the cancer is HNSCC.

[0183] In some embodiments, the cancer is cervical cancer.

[0184] In some embodiments, the cancer is NSCLC.

[0185] In some embodiments, the cancer is glioblastoma (including GBM).

[0186] In some embodiments, the cancer is gastrointestinal cancer.

[0187] In some embodiments, the cancer is a highly mutated cancer.

[0188] In some embodiments, the cancer is a childhood high-frequency mutation cancer.

[0189] In some embodiments, a first growth by priming is carried out in a first container, and a rapid second growth is carried out in a second container, with each of the first and second containers being GREX-10.

[0190] In some embodiments, a first growth by priming is carried out in a first sealed container, and a rapid second growth is carried out in a second sealed container, each of the first and second sealed containers being GREX-100.

[0191] In some embodiments, a first growth by priming is carried out in a first sealed container, and a rapid second growth is carried out in a second sealed container, each of the first and second sealed containers being GREX-500.

[0192] In some embodiments, the subjects have been previously treated with an anti-PD-1 antibody.

[0193] In some embodiments, the subjects have not been previously treated with anti-PD-1 antibodies.

[0194] In some embodiments, a first proliferation step by priming is performed on a first TIL population to be selected or enriched for PD-1-positive TILs by contacting the first TIL population with an anti-PD-1 antibody to form a first complex of the anti-PD-1 antibody and TIL cells in the first TIL population, and then isolating the first complex to obtain a first TIL population to be selected or enriched for PD-1-positive TILs.

[0195] In some embodiments, the anti-PD-1 antibody comprises an Fc region, and after the step of forming a first complex and before the step of isolating the first complex, the method further includes the step of contacting the first complex with an anti-Fc antibody bound to the Fc region of the anti-PD-1 antibody to form a second complex of the anti-Fc antibody and the first complex, and the step of isolating the first complex is performed by isolating the second complex.

[0196] In some embodiments, the anti-PD-1 antibody is EH12.2H7, PD1.3.1, SYM021, M1H4, A17188B, nivolumab (BMS-936558, Bristol-Myers Squibb; Opdivo®), pembrolizumab (lambrolizumab, MK03475 or MK-3475, Merck; Keytruda®), H12.1, PD1.3.1, NAT 105, humanized anti-PD-1 antibody JS001 (ShangHai JunShi), monoclonal anti-PD-1 antibody TSR-042 (Tesaro, Inc.), or pizilizumab (anti-PD-1 mAb The antibody is selected from the group consisting of CT-011 (Medivation), anti-PD-1 monoclonal antibody BGB-A317 (BeiGene), and / or anti-PD-1 antibody SHR-1210 (ShangHai HengRui), human monoclonal antibody REGN2810 (Regeneron), human monoclonal antibody MDX-1106 (Bristol-Myers Squibb), humanized anti-PD-1 IgG4 antibody PDR001 (Novartis), and RMP1-14 (rat IgG)-BioXcell catalog number BP0146.

[0197] In some embodiments, the anti-PD-1 antibody is EH12.2H7.

[0198] In some embodiments, the anti-PD-1 antibody binds to an epitope different from that of nivolumab or pembrolizumab.

[0199] In some embodiments, the PD-1 antibody binds to the same epitope as EH12.2H7 or nivolumab.

[0200] In some embodiments, the anti-PD-1 antibody is nivolumab.

[0201] In some embodiments, the subjects have been previously treated with a first anti-PD1 antibody, and a first proliferation step by priming is performed on a first TIL population selected or enriched for PD-1 positive TILs by contacting a first TIL population with a second anti-PD-1 antibody to form a first complex of the second anti-PD-1 antibody and TIL cells in the first TIL population, and then isolating the first complex to obtain a first TIL population selected or enriched for PD-1 positive TILs, wherein the second anti-PD-1 antibody is not blocked from binding to the first TIL population by the first anti-PD-1 antibody which has been insolubilized in the first TIL population.

[0202] In some embodiments, the subjects have been previously treated with a first anti-PD1 antibody, and a first proliferation step by priming is performed on a first TIL population selected or enriched for PD-1 positive TILs by contacting a first TIL population with a second anti-PD-1 antibody to form a first complex of the second anti-PD-1 antibody and TIL cells in the first TIL population, and then isolating the first complex to obtain a first TIL population selected or enriched for PD-1 positive TILs, wherein the second anti-PD-1 antibody is blocked from binding to the first TIL population by the first anti-PD-1 antibody which has been insoluble in the first TIL population.

[0203] In some embodiments, the first anti-PD-1 antibody and the second anti-PD-1 antibody include an Fc region, and after the step of forming the first complex and before the step of isolating the first complex, the method further includes the step of contacting the first complex with an anti-Fc antibody bound to the Fc region of the first anti-PD-1 antibody and the Fc region of the second anti-PD-1 antibody to form a second complex of the anti-Fc antibody and the first complex, and the step of isolating the first complex is performed by isolating the second complex.

[0204] In some embodiments, the subject has been previously treated with a first anti-PD1 antibody, and the first proliferation step by priming is to (i) contact a first TIL population with a second anti-PD-1 antibody to form a first complex of the second anti-PD-1 antibody and the first TIL population, wherein the second anti-PD-1 antibody is blocked from binding to PD-1-positive TILs by the first anti-PD-1 antibody insolubilized by the first TIL population, and the first anti-PD-1 antibody and the second anti-PD-1 antibody form a complex including an Fc region, and (ii) the first complex is to the second (iii) Contacting the anti-PD-1 antibody with an anti-Fc antibody that binds to the Fc region of the anti-PD-1 antibody to form a second complex of the anti-Fc antibody and the first complex; contacting the first anti-PD-1 antibody, which has been insoluble in the first TIL population, with the anti-Fc antibody to form a third complex of the anti-Fc antibody and the first anti-PD-1 antibody, which has been insoluble in the first TIL population; and (iii) separating the second and third complexes to obtain a first TIL population that is selected or enriched for PD-1 positive TILs.

[0205] In some embodiments, the present invention provides a therapeutic tumor-infiltrating lymphocyte (TIL) population prepared from PD-1, LAG3, TIM3, and / or TIGIT-positive cells selected from the digest of a tumor tissue sample obtained from a patient, wherein the therapeutic TIL population provides increased efficacy and / or increased interferon-gamma production.

[0206] In some embodiments, the therapeutic TIL population provides increased interferon-gamma production.

[0207] In some embodiments, the therapeutic TIL population provides increased efficacy.

[0208] In some embodiments, the therapeutic TIL population is capable of producing at least one-fold more interferon-gamma compared to TILs prepared by processes longer than 16 days.

[0209] In some embodiments, the therapeutic TIL population is capable of producing at least 1 times more interferon-gamma compared to TILs prepared by processes longer than 16–22 days.

[0210] In some embodiments, a first proliferation step by priming is performed on a first TIL population selected or enriched for PD-1, LAG3, TIM3, and / or TIGIT-positive TILs, with at least 11.27% to 74.4% being PD-1-positive TILs.

[0211] In some embodiments, the first propagation step is priming. (i) Exposing a first TIL population and a PBMC population to an excess of monoclonal anti-PD-1 IgG4 antibody that binds to PD-1 by the N-terminal loop outside the IgV domain of PD-1, (ii) The step of adding an excess anti-IgG4 antibody conjugated to the fluorophore, (iii) A step of obtaining a first TIL population that is selected or enriched for PD-1-positive TILs based on the intensity of the fluorophores of PD-1-positive TILs in the first TIL population compared to the intensity of the PBMC population as performed by fluorescence-activated cell sorting (FACS), and performed on the first TIL population that is selected or enriched for PD-1-positive TILs by the first TIL population.

[0212] In some embodiments, the fluorophore intensity in both the first TIL population and the PBMC population is used to set up FACS gates to establish low, medium, and high intensity levels corresponding to PD-1-negative TILs, PD-1-intermediate TILs, and PD-1-positive TILs, respectively.

[0213] In some embodiments, the FACS gate is set after step (a).

[0214] In some embodiments, PD-1, LAG3, TIM3, and / or TIGIT-positive TILs are PD-1 high, LAG3 high, TIM3 high, and / or TIGIT high TILs.

[0215] In some embodiments, at least 80% of a first TIL population selected or enriched for PD-1-positive TILs are PD-1-positive TILs, at least 80% of a first TIL population selected or enriched for LAG3-positive TILs are LAG3-positive TILs, at least 80% of a first TIL population selected or enriched for TIM3-positive TILs are TIM3-positive TILs, and / or at least 80% of a first TIL population selected or enriched for TIGIT-positive TILs are TIGIT-positive TILs.

[0216] In some embodiments, the present invention is a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, (a) Obtaining and / or receiving a first TIL population from tumors excised from subjects by processing tumor samples obtained from subjects into multiple tumor fragments, (b) Selecting PD-1, CD39, CD38, CD103, CD101, LAG3, TIM3, and / or TIGIT-positive TILs from the first TIL population of (a) to obtain a TIL population rich in PD-1, CD39, CD38, CD103, CD101, LAG3, TIM3, and / or TIGIT, wherein at least 10% to 80% of the first TIL population are PD-1, CD39, CD38, CD103, CD101, LAG3, TIM3, and / or TIGIT-positive TILs. (c) A TIL population rich in PD-1, CD39, CD38, CD103, CD101, LAG3, TIM3, and / or TIGIT is cultured in a cell culture medium containing IL-2, OKT-3, and antigen-presenting cells (APCs) to produce a second TIL population by priming, wherein the first priming growth is carried out in a container having a first gas-permeable surface area, and the first priming growth is carried out for a first period of approximately 1 to 7, 8, 9, 10, or 11 days, thereby obtaining a second TIL population, the second TIL population being produced in a larger number than the first TIL population. (d) Producing a third TIL population by rapidly growing a second TIL population by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, and APC, wherein the number of APCs added to the rapid second growth is at least twice the number of APCs added in step (c), the rapid second growth is carried out for a second period of approximately 1 to 11 days, and a third TIL population is obtained, the third TIL population being a therapeutic TIL population, and the rapid second growth is carried out in a container having a second gas-permeable surface area. (e) Collect the therapeutic TIL population obtained from step (d), The present invention provides a method comprising (f) transferring the TIL population collected from step (e) into an injection bag.

[0217] In some embodiments, the selection of step (b) is (i) Exposing a first TIL population and a PBMC population to an excess of monoclonal anti-PD-1 IgG4 antibody that binds to PD-1 by the N-terminal loop outside the IgV domain of PD-1, (ii) The step of adding an excess anti-IgG4 antibody conjugated to the fluorophore, (iii) The step of obtaining a PD-1-rich TIL population based on the intensity of the fluorophores of PD-1-positive TILs in a first TIL population compared to the intensity of the PBMC population when performed by fluorescence-activated cell sorting (FACS).

[0218] In some embodiments, the intensity of fluorophores in both the first population and the PBMC population is used, i) Each of these is classified as PD-1 negative TIL, PD-1 low TIL, PD-1 intermediate TIL, and PD-1 positive TIL, ii) CD39-negative TILs, CD39-low TILs, CD39-intermediate TILs, and CD39-positive TILs, respectively iii) CD38-negative TILs, CD38-low TILs, CD38-intermediate TILs, and CD38-positive TILs, respectively. iv) CD103-negative TIL, CD103-low TIL, CD103-intermediate TIL, and CD103-positive TIL, respectively. v) CD101-negative TIL, CD101-low TIL, CD101-intermediate TIL, and CD101-positive TIL, respectively. vi) LAG3-negative TILs, LAG3-low TILs, LAG3-intermediate TILs, and LAG3-positive TILs, respectively. vii) TIM3-negative TILs, TIM3-low TILs, TIM3-intermediate TILs, and TIM3-positive TILs, and / or viii) Set up FACS gates to establish low, medium, and high intensity levels corresponding to TIGIT-negative TILs, low TIGIT TILs, intermediate TILs, and TIGIT-positive TILs, respectively.

[0219] In some embodiments, the FACS gate is set after step (a).

[0220] In some embodiments, PD-1, LAG3, TIM3, and / or TIGIT-positive TILs are PD-1 high, LAG3 high, TIM3 high, and / or TIGIT high TILs.

[0221] In some embodiments, at least 80% of a TIL population rich in PD-1, LAG3, TIM3, and / or TIGIT are PD-1, LAG3, TIM3, and / or TIGIT-positive TILs.

[0222] In some embodiments, the third TIL group contains at least about 1 × 10⁸ TILs in the container.

[0223] In some embodiments, the third TIL group contains at least about 1 × 10⁹ TILs in the container.

[0224] In some embodiments, the number of PD-1, LAG3, TIM3, and / or TIGIT-rich TILs in the first proliferation by priming is approximately 1 × 10⁻⁶ 4 ~Approx. 1×10 6 That is the case.

[0225] In some embodiments, the number of PD-1, LAG3, TIM3, and / or TIGIT-rich TILs in the first proliferation by priming is approximately 5 × 10 4 ~Approx. 1×10 6 That is the case.

[0226] In some embodiments, the number of PD-1, LAG3, TIM3, and / or TIGIT-rich TILs in the first proliferation by priming is approximately 2 × 10⁻⁶ 5 ~Approx. 1×10 6 That is the case.

[0227] In some embodiments, the method further includes the step of cryopreserving a first population of TILs from tumors excised from the subject before performing step (a).

[0228] In some embodiments, the present invention is a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, (a) Obtaining and / or receiving a first TIL population from tumors excised from subjects by processing tumor samples obtained from subjects into multiple tumor fragments, (b) Select PD-1, CD39, CD38, CD103, CD101, LAG3, TIM3, and / or TIGIT-positive TILs from the first TIL population in (a) to obtain a TIL population rich in PD-1, CD39, CD38, CD103, CD101, LAG3, TIM3, and / or TIGIT, (c) First cell culture medium, IL-2, and i) A first culture supernatant obtained from a first culture of antigen-presenting feeder cells (APCs), wherein the first culture supernatant contains OKT-3, or ii) Performing a first proliferation by priming by culturing a first TIL population in a first TIL cell culture containing either APC or OKT-3, The first proliferation by priming is performed by culturing the first TIL cell culture in a first container containing a first gas-permeable surface area for a first period of approximately 1 to 7, 8, 9, 10, or 11 days, thereby obtaining a second TIL population. The first proliferation by priming is performed when the second TIL population is larger than the first TIL population. (d) Second cell culture medium, IL-2, and i) A second culture supernatant obtained from a second culture of APC, wherein the second culture supernatant contains OKT-3, or ii) Rapid second proliferation is performed by transferring the first TIL cell culture to a second container having a second gas-permeable surface area supplemented with either APC or OKT-3. The process involves forming a second TIL cell culture, where rapid second proliferation is achieved by culturing the second TIL cell culture for a second period of approximately 1 to 11 days, thereby obtaining a third TIL population, the third TIL population being the therapeutic TIL population, the first TIL cell culture not containing either the first culture supernatant or APC, and the second TIL cell culture not containing either the second culture supernatant or supplemental APC. (e) Collect the therapeutic TIL population obtained from step (d), The present invention provides a method comprising (f) transferring the TIL population collected from step (e) into an injection bag.

[0229] In some embodiments, in the first proliferation by priming in step (c), the first TIL cell culture contains the first culture supernatant, and in the rapid second proliferation in step (d), the first TIL cell culture is supplemented with OKT-3 and APC to form a second TIL cell culture.

[0230] In some embodiments, in the first proliferation by priming in step (c), the first TIL cell culture contains OKT-3 and APC, and in the rapid second proliferation in step (d), the first TIL cell culture is supplemented with a second culture supernatant to form a second TIL cell culture.

[0231] In some embodiments, in the first proliferation by priming in step (c), the first TIL cell culture contains the first culture supernatant, and in the rapid second proliferation in step (d), the first TIL cell culture is supplemented with the second culture supernatant to form a second TIL cell culture.

[0232] In some embodiments, a first culture supernatant for use in step (c) is obtained. 1) To provide an APC cell culture medium containing IL-2 and OKT-3, 2)1) In the APC cell culture medium, at least about 5 × 10 8The APC is cultured for approximately 3-4 days to produce the first culture supernatant, 3) Includes collecting a first culture supernatant from the cell culture of 2).

[0233] In some embodiments, a second culture supernatant is obtained for use in step (d). 1) To provide an APC cell culture medium containing IL-2 and OKT-3, 2)1) In the APC cell culture medium, at least about 1 × 10 7 The APC is cultured for approximately 3-4 days to produce a second culture supernatant, 3) Includes collecting a second culture supernatant from the cell culture of 2).

[0234] In some embodiments, the rapid second growth in step (d) i) The step further includes supplementing the second TIL cell culture with additional IL-2 approximately 3 or 4 days after the start of the second period in step (d).

[0235] In some embodiments, the APC is exogenous to the subject.

[0236] In some embodiments, APCs are peripheral blood mononuclear cells (PBMCs).

[0237] In some embodiments, the rapid second growth in step (d) i) At the start of the second period, or after approximately 3 or 4 days, the second TIL cell culture is transferred from the second container to several third containers, and a subculture of the second TIL cell culture is formed in each of the several third containers. ii) The step of culturing subcultures of the second TIL cell culture in each of a plurality of third containers for the remainder of the second period.

[0238] In some embodiments, in step i), an equal volume of the second TIL cell culture is transferred to a plurality of third containers.

[0239] In some embodiments, each of the third containers is the same size as the second container.

[0240] In some embodiments, each of the third containers is larger than the second container.

[0241] In some embodiments, the third container is of equal size.

[0242] In some embodiments, the third container is larger than the second container.

[0243] In some embodiments, the third container is smaller than the second container.

[0244] In some embodiments, the second container is a G-Rex 100M flask.

[0245] In some embodiments, the second container is a G-Rex 100M flask, and each of the plurality of third containers is a G-Rex 100M flask.

[0246] In some embodiments, a plurality of third containers are selected from the group consisting of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 second containers.

[0247] In some embodiments, the multiple second containers are two third containers.

[0248] In some embodiments, the method further includes supplementing each passage of the second TIL cell culture with additional IL-2 prior to step ii).

[0249] In some embodiments, prior to step ii), the method further includes supplementing each passage of the second TIL cell culture with the second cell culture medium and IL-2.

[0250] In some embodiments, the first cell culture medium and the second cell culture medium are the same.

[0251] In some embodiments, the first cell culture medium and the second cell culture medium are different.

[0252] In some embodiments, the first cell culture medium is DM1, and the second cell culture medium is DM2.

[0253] In some embodiments, the TIL is selected as PD-1 positive (PD-1+), LAG3 positive (LAG3+ positive), CD38 positive (CD38+), and CD101 positive (CD101+).

[0254] In some embodiments, TIL is selected as PD-1 high, LAG3 high, CD38 low, and CD101 low.

[0255] In some embodiments, TILs are selected as PD-1 positive (PD-1+), LAG3 positive (LAG3+ positive), and CD38 positive (CD38+).

[0256] In some embodiments, TIL is selected as PD-1 high, LAG3 high, and CD38 low.

[0257] In some embodiments, TILs are selected as PD-1 positive (PD-1+), LAG3 positive (LAG3+ positive), and CD101 positive (CD101+).

[0258] In some embodiments, TIL is selected as PD-1 high, LAG-3 high, and CD101 low.

[0259] In some embodiments, the TIL is selected as PD-1 positive (PD-1+) and CD38 positive (CD38+).

[0260] In some embodiments, TIL is selected as PD-1 high and CD38 low.

[0261] In some embodiments, the TIL is selected as PD-1 positive (PD-1+) and CD101 positive (CD101+).

[0262] In some embodiments, TIL is selected as PD-1 high and CD101 low.

[0263] In some embodiments, the selection includes a selection method that is selected from the group consisting of flow cytometry (including, for example, FACS), antibody-based bead selection, and antibody-based magnetic bead selection.

[0264] In some embodiments, the selection method includes flow cytometry (e.g., FACS).

[0265] In some embodiments, the selection method includes antibody bead selection.

[0266] In some embodiments, the selection includes antibody-based magnetic bead selection.

[0267] In some embodiments, the selection includes a two-step selection. i) A first selection step including a method for selecting PD-1+, LAG3+, TIM3+, and / or TIGIT+, ii) a second selection step including a method for selecting CD38+ and / or CD101+.

[0268] In some embodiments, the first selection step includes a method for selecting PD-1 high, LAG3 high, TIM3 high, and / or TIGIT high.

[0269] In some embodiments, the first selection step includes a method for selecting either PD-1 high or LAG3 high.

[0270] In some embodiments, the second selection step includes a method for selecting CD38 low and / or CD101 low.

[0271] In some embodiments, the first selection step includes flow cytometry (including, for example, FACS), and the second selection step includes flow cytometry (including, for example, FACS).

[0272] In some embodiments, the first selection step includes antibody bead selection, and the second selection step includes flow cytometry (including, for example, FACS).

[0273] In some embodiments, the first selection step includes antibody-based magnetic bead selection, and the second selection step includes flow cytometry (including, for example, FACS).

[0274] In some embodiments, the first selection step includes antibody-based bead selection or antibody-based magnetic bead selection, and the second selection step includes antibody-based bead selection or antibody-based magnetic bead selection.

[0275] In some embodiments, the beads used for selecting PD-1+, LAG3+, TIM3+, and / or TIGIT+TIL antibody-based beads are anti-PD-1, anti-LAG3, anti-TIM3, and / or anti-TIGIT antibody-complex beads, respectively.

[0276] In some embodiments, the beads used for selecting CD38+ or CD101+ TIL antibody-based beads are anti-CD38 or anti-CD101 antibody-complex beads, respectively.

[0277] In some embodiments, the beads used for selecting PD-1+, LAG3+, TIM3+, and / or TIGIT+TIL antibody-based magnetic beads are anti-PD-1, anti-LAG3, anti-TIM3, and / or anti-TIGIT antibody-complex magnetic beads, respectively.

[0278] In some embodiments, the beads used for selecting CD38+ or CD101+ TIL antibody-based magnetic beads are anti-CD38 or anti-CD101 antibody-composite magnetic beads, respectively.

[0279] In some embodiments, PD-1+, LAG3+, TIM3+, and TIGIT+ TILs bind to anti-PD-1, anti-LAG3, anti-TIM3, and / or anti-TIGIT antibody complex beads, respectively, while PD-1, LAG3, TIM3, and / or TIGIT-negative TILs do not bind to anti-PD-1, anti-LAG3, anti-TIM3, and / or anti-TIGIT antibody complex beads, respectively.

[0280] In some embodiments, a first growth step by priming is performed on a first TIL population selected or concentrated from a digest of a tumor sample obtained from a patient or subject.

[0281] In some embodiments, digestion is carried out using a mixture of enzymes.

[0282] In some embodiments, the enzyme mixture includes a neutral protease, a collagenase, and a DNase. [Brief explanation of the drawing]

[0283] [Figure 1A] This shows a comparison between the 2A process (approximately a 22-day process) and the PD-1 Gen3 process (approximately a 14-22 day process) for TIL manufacturing. [Figure 1B] An example process chart for PD-1 Gen3, providing an overview of steps A-F (a process lasting approximately 14-22 days). [Figure 1C] A diagram illustrating the exemplary process PD-1 Gen3, providing an overview of steps A through F (a process lasting approximately 14 to 22 days). [Figure 1D] A chart providing three exemplary Gen3 processes for each of the three process variations, along with an overview of steps A through F (approximately 14-18 day processes). [Figure 1E]A chart providing three exemplary PD-1 Gen 3 processes for each of the three process variations, along with an overview of steps A-F (approximately 14-18 day processes). [Figure 1F] A chart providing an example of the PD-1 Gen 3 process, along with an overview of steps A through F (a process lasting approximately 14 to 18 days). [Figure 1G] A chart providing an example of the PD-1 Gen 3 process, along with an overview of steps A through F (a process lasting approximately 14 to 18 days). [Figure 1H] A chart providing an example of the PD-1 Gen 3 process, along with an overview of steps A through F (a process lasting approximately 14 to 18 days). [Figure 2] This document provides an experimental flowchart regarding the comparability between GEN2 (Process 2A) and PD-1 GEN3. [Figure 3] This document shows a comparison of various Gen2 (2A process) and Gen3.1 process embodiments. [Figure 4] A table illustrating the various characteristics of the Gen2, Gen2.1, and Gen3.0 process embodiments. [Figure 5] An overview of the culture medium conditions for an embodiment of the Gen3 process, referred to as Gen3.1. [Figure 6] A schematic diagram of an exemplary embodiment of the Gen3 process (16-day process). [Figure 7] A schematic diagram of PD-1 selection before proliferation. [Figure 8] The binding structure of nivolumab to PD-1. See Figure 5 from Tan, S. et al. (Tan, S. et al., Nature Communications, 8:14369|DOI:10.1038 / ncomms14369(2017)). [Figure 9] The binding structure of pembrolizumab to PD-1. See Figure 5 from Tan, S. et al. (Tan, S. et al., Nature Communications, 8:14369 | DOI: 10.1038 / ncomms14369 (2017)). [Figure 10] A streamlined protocol was developed to expand PD1+TILs to a clinically appropriate level. Tumors are excised from patients and transported to the laboratory. Upon arrival, the tumors are digested, and the single-cell suspension is stained for CD3 and PD1. PD1+TILs are sorted by FACS using an FX500 instrument (Sony). The PD1+ cell fraction is placed in a flask containing anti-human CD3 antibody (OKT3, 30 ng / ml), and allogeneic PBMCs (feeders) are irradiated at a ratio of 1:100 (TIL:feeder) and rapidly expanded for 22 days (REP). [Figure 11] Identifying methods for digesting tumor tissue. [Figure 12] A schematic diagram of an exemplary embodiment of the steps of tumor digestion and PD-1+ selection, including high PD-1 selectivity. [Figure 13] A schematic diagram of an exemplary embodiment of a modified Gen2 process developed for the TIL selected in PD1. [Figure 14] A schematic diagram of an exemplary embodiment of a modified growth process developed for TIL selected in PD1. [Figure 15] A schematic diagram of an exemplary embodiment of a modified growth process developed for TIL selected in PD1. [Figure 16] Schematic diagram of a full-scale process embodiment for PD1 TIL culture. [Figure 17] Small-scale process embodiments: PD1-A is the condition using the nivolumab staining procedure outlined in this protocol. PD1-B is the condition using the anti-PD1-PE (clonal number EH12.2H7) staining method. The bulk condition serves as a control. [Figure 18] Overview of an embodiment of the PD-1+ high Gen-2 process. [Figure 19] Exemplary embodiment of the PD-1+TIL culture process (Research / PD-1+Gen2 / Synthetic medium / Initial REP) [Figure 20]Structures IA and IB are provided, where the cylinders refer to the individual polypeptide-binding domains. Structures IA and IB provide an agonist that can fold three linearly bound TNFRSF-binding domains derived from, for example, an antibody bound to 4-1BBL or 4-1BB to form a trivalent protein, which is then bound to a second trivalent protein by IgG1-Fc (containing CH3 and CH2 domains), and then used to bind two of the trivalent proteins together by disulfide bonds (small oval) to stabilize the structure and bring together the intracellular signaling domains of six receptor and signaling proteins to form a signaling complex. The TNFRSF-binding domain shown as a cylinder may be an scFv domain containing VH and VL chains bound by a linker which may contain, for example, Gly and Ser sequences for hydrophilic residues and flexibility, as well as Glu and Lys for solubility. [Figure 21] Identification of PD-1-positive TILs in multiple cancer types and proliferation of PD-1-selective TILs. [Figure 22] Ex vivo-expanded PD-1 selective TILs exhibiting autotumor reactivity. [Figure 23] Ex vivo-extended PD1+ TILs showed effector activity in several in vitro assays. The data suggest that TILs selected for PD1+ are antigen-specific and possess greater effector function. [Figure 24] Schematic diagram of an exemplary selection and propagation protocol. [Figure 25] Data showing the phenotypic characteristics of CD39+ positive cells. [Figure 25] Data illustrating the characteristics of CD39+ positive cells. [Figure 26] Data showing the results of phenotypic evaluation of PD-1-selective TILs in tumor digestive tissue. [Figure 27] Data showing the results of phenotypic evaluation of PD-1-selective TILs in tumor digestive tissue. [Figure 28] Overview of the PD1+ Selective Gen-2 process. [Figure 29]Phase 1 Experiment Overview. [Figure 30] Experimental overview of the Phase-2 full-scale PD1+ selected Gen-2 process. [Figure 31] Data showing cell population gating in Example 7. [Figure 32] Data showing cell population gating in Example 7. [Figure 33] Schematic diagrams of two exemplary PD-1 selection methods. [Figure 34] Anti-PD-1 microbead composite and detection. [Figure 35] Data showing >85% purity were obtained during magnetic selection of PD-1+TILs using anti-PD-1 (EH12.2H7). 2e6 REP TILs were added to 10 μl of cocktail (EH12.2H7) + 5 μl of microbeads, incubated at RT for 15 minutes, incubated in magnetic field at RT for 1 minute, and positive selection was performed using magnetic field twice. Selected cells were stained with secondary mIgG1-PE and aCD3 FITC. [Figure 36] When using anti-PD-1 (M1H4) in the magnetic selection of PD-1+TIL, data showing a purity of >85% was obtained. [Figure 37] Experimental design. Comparison of two exemplary selected embodiments: fluid separation vs. magnetic separation. [Figure 38] The TVC yield after sorting using the magnetic method (EH12.2H7) was higher than that obtained using the fluid sorting method. [Figure 39] The growth characteristics, identity, and function of magnetically selected TILs were equivalent to those of flow-sorted TILs. [Figure 40] Differentiation, activation, and depletion (CD4+) phenotypic markers were comparable. [Figure 41] Differentiation, activation, and depletion (CD8+) phenotypic markers were comparable. [Figure 42] >99% of TCR Vbeta clones of the flow-separated PD-1 selected TIL were present during magnetic separation (EH12.H7). Data show the intrinsic CDR3 count and Shannon Diversity Index for all test samples. [Figure 43] >99% of TCR Vbeta clones of the PD-1 selected TIL were present during magnetic separation (EH12.H7). The data shows overlapping uCDR3 samples between test samples. [Figure 44] Pre-selection was performed based on CD39. PD1 hyperCD39- cells were predominantly composed of CD4+ cells. PD1 hyperCD39+ cells had significantly reduced CD69 levels compared to unselected TILs. PD-1 hyperCD39+ cells, when stimulated with anti-CD3 / anti-CD28 / anti-41BB, had significantly reduced IFNγ secretion compared to both unselected and PD-1 hyperCD39- TILs. IFNγ secretion in response to autologous tumors was detected in PD-1 hyperCD39+ TILs in 4 out of 6 evaluable tumors (2 tumors were not evaluated).

[0284] A brief explanation of sequence listings Sequence ID 1 is the amino acid sequence of the heavy chain of muromonab.

[0285] Sequence ID 2 is the amino acid sequence of the light chain of muromonab.

[0286] Sequence ID 3 is the amino acid sequence of recombinant human IL-2 protein.

[0287] Sequence ID 4 is the amino acid sequence of aldethleukin.

[0288] Sequence ID 5 is the amino acid sequence of recombinant human IL-4 protein.

[0289] Sequence ID 6 is the amino acid sequence of recombinant human IL-7 protein.

[0290] Sequence ID 7 is the amino acid sequence of recombinant human IL-15 protein.

[0291] Sequence ID 8 is the amino acid sequence of recombinant human IL-21 protein.

[0292] Sequence ID 9 is the amino acid sequence of human 4-1BB.

[0293] Sequence ID 10 is the amino acid sequence of mouse 4-1BB.

[0294] Sequence ID 11 is the heavy chain of the 4-1BB agonist monoclonal antibody utomirumab (PF-05082566).

[0295] Sequence ID 12 is the light chain of the 4-1BB agonist monoclonal antibody utomirumab (PF-05082566).

[0296] Sequence ID 13 is the heavy chain variable region (VH) of the 4-1BB agonist monoclonal antibody utomirumab (PF-05082566).

[0297] Sequence ID 14 is the light chain variable region (VL) of the 4-1BB agonist monoclonal antibody utomirumab (PF-05082566).

[0298] Sequence ID 15 is the heavy chain CDR1 of the 4-1BB agonist monoclonal antibody utomirumab (PF-05082566).

[0299] Sequence ID 16 is the heavy chain CDR2 of the 4-1BB agonist monoclonal antibody utomirumab (PF-05082566).

[0300] Sequence ID No. 17 is the heavy chain CDR3 of the 4-1BB agonist monoclonal antibody utomirumab (PF-05082566).

[0301] Sequence ID No. 18 is the light chain CDR1 of the 4-1BB agonist monoclonal antibody utomirumab (PF-05082566).

[0302] Sequence ID 19 is the light chain CDR2 of the 4-1BB agonist monoclonal antibody utomirumab (PF-05082566).

[0303] Sequence ID No. 20 is the light chain CDR3 of the 4-1BB agonist monoclonal antibody utomirumab (PF-05082566).

[0304] Sequence ID 21 is the heavy chain of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0305] Sequence ID 22 is the light chain of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0306] Sequence ID 23 is the heavy chain variable region (VH) of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0307] Sequence ID No. 24 is the light chain variable region (VL) of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0308] Sequence ID 25 is the heavy chain CDR1 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0309] Sequence ID No. 26 is the heavy chain CDR2 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0310] Sequence ID No. 27 is the heavy chain CDR3 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0311] Sequence ID No. 28 is the light chain CDR1 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0312] Sequence ID 29 is the light chain CDR2 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0313] Sequence ID No. 30 is the light chain CDR3 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0314] Sequence ID 31 is the Fc domain of the TNFRSF agonist fusion protein.

[0315] Sequence ID 32 is the linker for the TNFRSF agonist fusion protein.

[0316] Sequence ID 33 is the linker for the TNFRSF agonist fusion protein.

[0317] Sequence ID 34 is the linker for the TNFRSF agonist fusion protein.

[0318] Sequence ID 35 is the linker for the TNFRSF agonist fusion protein.

[0319] Sequence ID 36 is the linker for the TNFRSF agonist fusion protein.

[0320] Sequence ID 37 is the linker for the TNFRSF agonist fusion protein.

[0321] Sequence ID 38 is the linker for the TNFRSF agonist fusion protein.

[0322] Sequence ID 39 is the linker for the TNFRSF agonist fusion protein.

[0323] Sequence ID 40 is the linker for the TNFRSF agonist fusion protein.

[0324] Sequence ID 41 is the linker for the TNFRSF agonist fusion protein.

[0325] Sequence ID 42 is the Fc domain of the TNFRSF agonist fusion protein.

[0326] Sequence ID 43 is the linker for the TNFRSF agonist fusion protein.

[0327] Sequence ID 44 is the linker for the TNFRSF agonist fusion protein.

[0328] Sequence ID 45 is the linker for the TNFRSF agonist fusion protein.

[0329] Sequence ID 46 is the amino acid sequence of the 4-1BB ligand (4-1BBL).

[0330] Sequence ID No. 47 is the soluble portion of the 4-1BBL polypeptide.

[0331] Sequence ID 48 is the heavy chain variable region (VH) of the 4-1BB agonist antibody 4B4-1-1 version 1.

[0332] Sequence ID 49 is the light chain variable region (VL) of the 4-1BB agonist antibody 4B4-1-1 version 1.

[0333] Sequence ID 50 is the heavy chain variable region (VH) of the 4-1BB agonist antibody 4B4-1-1 version 2.

[0334] Sequence ID 51 is the light chain variable region (VL) of the 4-1BB agonist antibody 4B4-1-1 version 2.

[0335] Sequence ID 52 is the heavy chain variable region (VH) of the 4-1BB agonist antibody H39E3-2.

[0336] Sequence ID 53 is the light chain variable region (VL) of the 4-1BB agonist antibody H39E3-2.

[0337] Sequence ID 54 is the amino acid sequence of human OX40.

[0338] Sequence ID 55 is the amino acid sequence of mouse OX40.

[0339] Sequence ID 56 is the heavy chain of the OX40 agonist monoclonal antibody tavorixizumab (MEDI-0562).

[0340] Sequence ID 57 is the light chain of the OX40 agonist monoclonal antibody tavorixizumab (MEDI-0562).

[0341] Sequence ID 58 is the heavy chain variable region (VH) of the OX40 agonist monoclonal antibody tavorixizumab (MEDI-0562).

[0342] Sequence ID 59 is the light chain variable region (VL) of the OX40 agonist monoclonal antibody tavorixizumab (MEDI-0562).

[0343] Sequence ID 60 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody tavorixizumab (MEDI-0562).

[0344] Sequence ID 61 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody tavorixizumab (MEDI-0562).

[0345] Sequence ID 62 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody tavorixizumab (MEDI-0562).

[0346] Sequence ID 63 is the light chain CDR1 of the OX40 agonist monoclonal antibody tavorixizumab (MEDI-0562).

[0347] Sequence ID 64 is the light chain CDR2 of the OX40 agonist monoclonal antibody tavorixizumab (MEDI-0562).

[0348] Sequence ID 65 is the light chain CDR3 of the OX40 agonist monoclonal antibody tavorixizumab (MEDI-0562).

[0349] Sequence ID 66 is the heavy chain of the OX40 agonist monoclonal antibody 11D4.

[0350] Sequence ID 67 is the light chain of the OX40 agonist monoclonal antibody 11D4.

[0351] Sequence ID 68 is the heavy chain variable region (VH) of the OX40 agonist monoclonal antibody 11D4.

[0352] Sequence ID 69 is the light chain variable region (VL) of the OX40 agonist monoclonal antibody 11D4.

[0353] Sequence ID 70 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody 11D4.

[0354] Sequence ID 71 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody 11D4.

[0355] Sequence ID 72 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody 11D4.

[0356] Sequence ID 73 is the light chain CDR1 of the OX40 agonist monoclonal antibody 11D4.

[0357] Sequence ID 74 is the light chain CDR2 of the OX40 agonist monoclonal antibody 11D4.

[0358] Sequence ID 75 is the light chain CDR3 of the OX40 agonist monoclonal antibody 11D4.

[0359] Sequence ID 76 is the heavy chain of the OX40 agonist monoclonal antibody 18D8.

[0360] Sequence ID 77 is the light chain of the OX40 agonist monoclonal antibody 18D8.

[0361] Sequence ID 78 is the heavy chain variable region (VH) of the OX40 agonist monoclonal antibody 18D8.

[0362] Sequence ID 79 is the light chain variable region (VL) of the OX40 agonist monoclonal antibody 18D8.

[0363] Sequence ID 80 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody 18D8.

[0364] Sequence ID No. 81 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody 18D8.

[0365] Sequence ID No. 82 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody 18D8.

[0366] Sequence ID No. 83 is the light chain CDR1 of the OX40 agonist monoclonal antibody 18D8.

[0367] Sequence ID 84 is the light chain CDR2 of the OX40 agonist monoclonal antibody 18D8.

[0368] Sequence ID No. 85 is the light chain CDR3 of the OX40 agonist monoclonal antibody 18D8.

[0369] Sequence ID 86 is the heavy chain variable region (VH) of the OX40 agonist monoclonal antibody Hu119-122.

[0370] Sequence ID 87 is the light chain variable region (VL) of the OX40 agonist monoclonal antibody Hu119-122.

[0371] Sequence ID 88 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody Hu119-122.

[0372] Sequence ID 89 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody Hu119-122.

[0373] Sequence ID 90 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody Hu119-122.

[0374] Sequence ID 91 is the light chain CDR1 of the OX40 agonist monoclonal antibody Hu119-122.

[0375] Sequence ID 92 is the light chain CDR2 of the OX40 agonist monoclonal antibody Hu119-122.

[0376] Sequence ID 93 is the light chain CDR3 of the OX40 agonist monoclonal antibody Hu119-122.

[0377] Sequence ID 94 is the heavy chain variable region (VH) of the OX40 agonist monoclonal antibody Hu106-222.

[0378] Sequence ID 95 is the light chain variable region (VL) of the OX40 agonist monoclonal antibody Hu106-222.

[0379] Sequence ID 96 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody Hu106-222.

[0380] Sequence ID 97 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody Hu106-222.

[0381] Sequence ID 98 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody Hu106-222.

[0382] Sequence ID 99 is the light chain CDR1 of the OX40 agonist monoclonal antibody Hu106-222.

[0383] Sequence ID No. 100 is the light chain CDR2 of the OX40 agonist monoclonal antibody Hu106-222.

[0384] Sequence ID 101 is the light chain CDR3 of the OX40 agonist monoclonal antibody Hu106-222.

[0385] Sequence ID 102 is the amino acid sequence of the OX40 ligand (OX40L).

[0386] Sequence ID No. 103 is the soluble portion of the OX40L polypeptide.

[0387] Sequence ID No. 104 is the alternative soluble moiety of the OX40L polypeptide.

[0388] Sequence ID 105 is the heavy chain variable region (VH) of the OX40 agonist monoclonal antibody 008.

[0389] Sequence ID 106 is the light chain variable region (VL) of the OX40 agonist monoclonal antibody 008.

[0390] Sequence ID 107 is the heavy chain variable region (VH) of the OX40 agonist monoclonal antibody 011.

[0391] Sequence ID 108 is the light chain variable region (VL) of the OX40 agonist monoclonal antibody 011.

[0392] Sequence ID 109 is the heavy chain variable region (VH) of the OX40 agonist monoclonal antibody 021.

[0393] Sequence ID No. 110 is the light chain variable region (VL) of the OX40 agonist monoclonal antibody 021.

[0394] Sequence ID 111 is the heavy chain variable region (VH) of the OX40 agonist monoclonal antibody 023.

[0395] Sequence ID 112 is the light chain variable region (VL) of the OX40 agonist monoclonal antibody 023.

[0396] Sequence ID 113 is the heavy chain variable region (VH) of an OX40 agonist monoclonal antibody.

[0397] Sequence ID No. 114 is the light chain variable region (VL) of an OX40 agonist monoclonal antibody.

[0398] Sequence ID No. 115 is the heavy chain variable region (VH) of an OX40 agonist monoclonal antibody.

[0399] Sequence ID 116 is the light chain variable region (VL) of an OX40 agonist monoclonal antibody.

[0400] Sequence ID No. 117 is the heavy chain variable region (VH) of a humanized OX40 agonist monoclonal antibody.

[0401] Sequence ID 118 is the heavy chain variable region (VH) of a humanized OX40 agonist monoclonal antibody.

[0402] Sequence ID No. 119 is the light chain variable region (VL) of a humanized OX40 agonist monoclonal antibody.

[0403] Sequence ID No. 120 is the light chain variable region (VL) of a humanized OX40 agonist monoclonal antibody.

[0404] Sequence ID 121 is the heavy chain variable region (VH) of a humanized OX40 agonist monoclonal antibody.

[0405] Sequence ID 122 is the heavy chain variable region (VH) of a humanized OX40 agonist monoclonal antibody.

[0406] Sequence ID 123 is the light chain variable region (VL) of a humanized OX40 agonist monoclonal antibody.

[0407] Sequence ID No. 124 is the light chain variable region (VL) of a humanized OX40 agonist monoclonal antibody.

[0408] Sequence ID No. 125 is the heavy chain variable region (VH) of an OX40 agonist monoclonal antibody.

[0409] Sequence ID No. 126 is the light chain variable region (VL) of an OX40 agonist monoclonal antibody.

[0410] Sequence numbers 127-462 are currently unassigned.

[0411] Sequence ID 463 is the heavy chain amino acid sequence of the PD-1 inhibitor nivolumab.

[0412] Sequence ID 464 is the light chain amino acid sequence of the PD-1 inhibitor nivolumab.

[0413] Sequence ID 465 is the amino acid sequence of the heavy chain variable region (VH) of the PD-1 inhibitor nivolumab.

[0414] Sequence ID 466 is the amino acid sequence of the light chain variable region (VL) of the PD-1 inhibitor nivolumab.

[0415] Sequence ID 467 is the heavy chain CDR1 amino acid sequence of the PD-1 inhibitor nivolumab.

[0416] Sequence ID 468 is the heavy chain CDR2 amino acid sequence of the PD-1 inhibitor nivolumab.

[0417] Sequence ID 469 is the heavy chain CDR3 amino acid sequence of the PD-1 inhibitor nivolumab.

[0418] Sequence ID 470 is the CDR1 amino acid sequence of the light chain of the PD-1 inhibitor nivolumab.

[0419] Sequence ID 471 is the CDR2 amino acid sequence of the light chain of the PD-1 inhibitor nivolumab.

[0420] Sequence ID 472 is the CDR3 amino acid sequence of the light chain of the PD-1 inhibitor nivolumab.

[0421] Sequence ID 473 is the heavy chain amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0422] Sequence ID 474 is the light chain amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0423] Sequence ID 475 is the amino acid sequence of the heavy chain variable region (VH) of the PD-1 inhibitor pembrolizumab.

[0424] Sequence ID 476 is the amino acid sequence of the light chain variable region (VL) of the PD-1 inhibitor pembrolizumab.

[0425] Sequence ID 477 is the heavy chain CDR1 amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0426] Sequence ID 478 is the heavy chain CDR2 amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0427] Sequence ID 479 is the heavy chain CDR3 amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0428] Sequence ID 480 is the CDR1 amino acid sequence of the light chain of the PD-1 inhibitor pembrolizumab.

[0429] Sequence ID 481 is the CDR2 amino acid sequence of the light chain of the PD-1 inhibitor pembrolizumab.

[0430] Sequence ID 482 is the CDR3 amino acid sequence of the light chain of the PD-1 inhibitor pembrolizumab.

[0431] Sequence ID 483 is the heavy chain amino acid sequence of the PD-L1 inhibitor durvalumab.

[0432] Sequence ID 484 is the light chain amino acid sequence of the PD-L1 inhibitor durvalumab.

[0433] Sequence ID 485 is the heavy chain variable region (VH) amino acid sequence of the PD-L1 inhibitor durvalumab.

[0434] Sequence ID 486 is the amino acid sequence of the light chain variable region (VL) of the PD-L1 inhibitor durvalumab.

[0435] Sequence ID 487 is the heavy chain CDR1 amino acid sequence of the PD-L1 inhibitor durvalumab.

[0436] Sequence ID 488 is the heavy chain CDR2 amino acid sequence of the PD-L1 inhibitor durvalumab.

[0437] Sequence ID 489 is the heavy chain CDR3 amino acid sequence of the PD-L1 inhibitor durvalumab.

[0438] Sequence ID 490 is the CDR1 amino acid sequence of the light chain of the PD-L1 inhibitor durvalumab.

[0439] Sequence ID 491 is the CDR2 amino acid sequence of the light chain of the PD-L1 inhibitor durvalumab.

[0440] Sequence ID 492 is the CDR3 amino acid sequence of the light chain of the PD-L1 inhibitor durvalumab.

[0441] Sequence ID 493 is the heavy chain amino acid sequence of the PD-L1 inhibitor avelumab.

[0442] Sequence ID 494 is the light chain amino acid sequence of the PD-L1 inhibitor avelumab.

[0443] Sequence ID 495 is the amino acid sequence of the heavy chain variable region (VH) of the PD-L1 inhibitor avelumab.

[0444] Sequence ID 496 is the amino acid sequence of the light chain variable region (VL) of the PD-L1 inhibitor avelumab.

[0445] Sequence ID 497 is the heavy chain CDR1 amino acid sequence of the PD-L1 inhibitor avelumab.

[0446] Sequence ID 498 is the heavy chain CDR2 amino acid sequence of the PD-L1 inhibitor avelumab.

[0447] Sequence ID 499 is the heavy chain CDR3 amino acid sequence of the PD-L1 inhibitor avelumab.

[0448] Sequence ID 500 is the CDR1 amino acid sequence of the light chain of the PD-L1 inhibitor avelumab.

[0449] Sequence ID 501 is the CDR2 amino acid sequence of the light chain of the PD-L1 inhibitor avelumab.

[0450] Sequence ID 502 is the CDR3 amino acid sequence of the light chain of the PD-L1 inhibitor avelumab.

[0451] Sequence ID 503 is the heavy chain amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0452] Sequence ID 504 is the light chain amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0453] Sequence ID 505 is the heavy chain variable region (VH) amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0454] Sequence ID 506 is the amino acid sequence of the light chain variable region (VL) of the PD-L1 inhibitor atezolizumab.

[0455] Sequence ID 507 is the heavy chain CDR1 amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0456] Sequence ID 508 is the heavy chain CDR2 amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0457] Sequence ID 509 is the heavy chain CDR3 amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0458] Sequence ID 510 is the CDR1 amino acid sequence of the light chain of the PD-L1 inhibitor atezolizumab.

[0459] Sequence ID 511 is the CDR2 amino acid sequence of the light chain of the PD-L1 inhibitor atezolizumab.

[0460] Sequence ID 512 is the CDR3 amino acid sequence of the light chain of the PD-L1 inhibitor atezolizumab.

[0461] Sequence ID 513 is the heavy chain amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0462] Sequence ID 514 is the light chain amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0463] Sequence ID 515 is the amino acid sequence of the heavy chain variable region (VH) of the CTLA-4 inhibitor ipilimumab.

[0464] Sequence ID 516 is the amino acid sequence of the light chain variable region (VL) of the CTLA-4 inhibitor ipilimumab.

[0465] Sequence ID 517 is the heavy chain CDR1 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0466] Sequence ID 518 is the heavy chain CDR2 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0467] Sequence ID 519 is the heavy chain CDR3 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0468] Sequence ID 520 is the CDR1 amino acid sequence of the light chain of the CTLA-4 inhibitor ipilimumab.

[0469] Sequence ID 521 is the CDR2 amino acid sequence of the light chain of the CTLA-4 inhibitor ipilimumab.

[0470] Sequence ID 522 is the CDR3 amino acid sequence of the light chain of the CTLA-4 inhibitor ipilimumab.

[0471] Sequence ID 523 is the heavy chain amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0472] Sequence ID 524 is the light chain amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0473] Sequence ID 525 is the amino acid sequence of the heavy chain variable region (VH) of the CTLA-4 inhibitor tremelimumab.

[0474] Sequence ID 526 is the amino acid sequence of the light chain variable region (VL) of the CTLA-4 inhibitor tremelimumab.

[0475] Sequence ID 527 is the heavy chain CDR1 amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0476] Sequence ID 528 is the heavy chain CDR2 amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0477] Sequence ID 529 is the heavy chain CDR3 amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0478] Sequence ID 530 is the CDR1 amino acid sequence of the light chain of the CTLA-4 inhibitor tremelimumab.

[0479] Sequence ID 531 is the CDR2 amino acid sequence of the light chain of the CTLA-4 inhibitor tremelimumab.

[0480] Sequence ID 532 is the CDR3 amino acid sequence of the light chain of the CTLA-4 inhibitor tremelimumab.

[0481] Sequence ID 533 is the heavy chain amino acid sequence of the CTLA-4 inhibitor zarifremab.

[0482] Sequence ID 534 is the light chain amino acid sequence of the CTLA-4 inhibitor zarifremab.

[0483] Sequence ID 535 is the heavy chain variable region (VH) amino acid sequence of the CTLA-4 inhibitor zarifremab.

[0484] Sequence ID 536 is the amino acid sequence of the light chain variable region (VL) of the CTLA-4 inhibitor zarifremab.

[0485] Sequence ID 537 is the heavy chain CDR1 amino acid sequence of the CTLA-4 inhibitor zarifremab.

[0486] Sequence ID 538 is the heavy chain CDR2 amino acid sequence of the CTLA-4 inhibitor zarifremab.

[0487] Sequence ID 539 is the heavy chain CDR3 amino acid sequence of the CTLA-4 inhibitor zarifremab.

[0488] Sequence ID 540 is the CDR1 amino acid sequence of the light chain of the CTLA-4 inhibitor zarifremab.

[0489] Sequence ID 541 is the CDR2 amino acid sequence of the light chain of the CTLA-4 inhibitor zarifremab.

[0490] Sequence ID 542 is the CDR3 amino acid sequence of the light chain of the CTLA-4 inhibitor zarifremab.

[0491] Sequence ID 543 is the IL-2 sequence.

[0492] Sequence ID 544 is the IL-2 mutain sequence.

[0493] Sequence ID 545 is the IL-2 mutain sequence.

[0494] Sequence ID 546 is IgG.IL2R67A.H1, which is HCDR1_IL-2.

[0495] Sequence ID 547 is HClR2 of IgG.IL2R67A.H1.

[0496] Sequence ID 548 is IgG.IL2R67A.H1 HCDR3.

[0497] Sequence ID 549 is the HCl1_IL-2 kabat of IgG.IL2R67A.H1.

[0498] Sequence ID 550 is the HCl2 kabat for IgG.IL2R67A.H1.

[0499] Sequence ID 551 is the HCDR3 kabat for IgG.IL2R67A.H1.

[0500] Sequence ID 552 is IgG.IL2R67A.H1, an HCDR1_IL-2 Clothia.

[0501] Sequence ID 553 is HClB2 Clothia with IgG.IL2R67A.H1.

[0502] Sequence ID 554 is HClR3 Clothia with IgG.IL2R67A.H1.

[0503] Sequence ID 555 is IgG.IL2R67A.H1 HCDR1_IL-2 IMGT.

[0504] Sequence ID 556 is IgG.IL2R67A.H1 HCDR2 IMGT.

[0505] Sequence ID 557 is IgG.IL2R67A.H1 HCDR3 IMGT.

[0506] Sequence ID 558 is the VH chain of IgG.IL2R67A.H1.

[0507] Sequence ID 559 is the heavy chain of IgG.IL2R67A.H1.

[0508] Sequence ID 560 is the LCDR1 kabat for IgG.IL2R67A.H1.

[0509] Sequence ID 561 is the LCDR2 kabat for IgG.IL2R67A.H1.

[0510] Sequence ID 562 is the LCDR3 kabat for IgG.IL2R67A.H1.

[0511] Sequence ID 563 is LCDR1 Clothia with IgG.IL2R67A.H1.

[0512] Sequence ID 564 is LCDR2 Clothia with IgG.IL2R67A.H1.

[0513] Sequence ID 565 is IgG.IL2R67A.H1 LCDR3 Clothia.

[0514] Sequence ID 566 is a VL chain.

[0515] Sequence ID 567 is a light chain.

[0516] Sequence ID 568 is a light chain.

[0517] Sequence ID 569 is a light chain.

[0518] I. Definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art in which the present invention pertains. All patents and publications referenced herein are incorporated in their entirety by reference.

[0519] The term "in vivo" refers to events that occur within the subject's body.

[0520] The term "in vitro" refers to events that occur outside the body of the subject. In vitro assays include cell-based assays that use living or dead cells, and may also include cell-free assays that do not use intact cells.

[0521] The term "ex vivo" refers to an event involving the treatment or procedure of cells, tissues, and / or organs removed from the subject's body. Appropriately, the cells, tissues, and / or organs may be returned to the subject's body by surgical or therapeutic means.

[0522] The term "rapid proliferation" means an increase in the number of antigen-specific TILs of at least approximately 3 times (or 4, 5, 6, 7, 8, or 9 times) over a one-week period, more preferably at least approximately 10 times (or 20, 30, 40, 50, 60, 70, 80, or 90 times) over a one-week period, or most preferably at least approximately 100 times over a one-week period. Several rapid proliferation protocols are outlined below.

[0523] In this specification, “tumor-infiltrating lymphocytes” or “TIL” means the population of cells initially obtained as leukocytes that have left the bloodstream and migrated to the tumor. TILs include CD8 + Cytotoxic T cells (lymphocytes), Th1 and Th17 CD4 + TILs include, but are not limited to, T cells, natural killer cells, dendritic cells, and M1 macrophages. TILs include both primary and secondary TILs. "Primary TILs" are those obtained from patient tissue samples as outlined herein (sometimes referred to as "newly acquired" or "newly isolated"), and "secondary TILs" are any expanded or proliferated TIL cell populations considered herein, including, but not limited to, bulk TILs and expanded TILs ("REP TILs" or "post-REP TILs"). TIL cell populations may include genetically modified TILs.

[0524] In this specification, “cell population” (including TIL) means several cells that share common traits. Generally, a population is approximately 1 × 10⁶ 6 ~1 × 10 10 This range includes different TIL populations, each containing a different number. For example, the initial growth of primary TILs in the presence of IL-2 is approximately 1 × 10⁻⁶. 8 This results in a population of bulk TILs in cells. REP proliferation is generally performed with 1.5 × 10⁶ cells for injection. 9 ~1.5×10 10 This is done to provide a population of cells.

[0525] In this specification, “cryopreserved TIL” means primary, bulk, or expanded (REP TIL) tissue that has been processed and stored at a temperature range of approximately -150°C to -60°C. General methods for cryopreservation are also described elsewhere in this specification, including in the examples. For clarity, “cryopreserved TIL” can be distinguished from frozen tissue samples that may be used as a source for primary TIL.

[0526] In this specification, “thawed cryopreserved TILs” means a group of TILs that have been previously cryopreserved and subsequently treated to return to room temperature or above, including but not limited to cell culture temperatures or temperatures at which the TILs may be administered to a patient.

[0527] TILs can generally be defined either biochemically using cell surface markers or functionally by their ability to invade tumors and achieve therapeutic effects. TILs can generally be classified by the expression of one or more biomarkers from among CD4, CD8, TCRαβ, CD27, CD28, CD56, CCR7, CD45Ra, CD95, PD-1, and CD25. Furthermore, and alternatively, TILs can be functionally defined by their ability to invade solid tumors upon reintroduction into a patient.

[0528] The term "cryopreservation media" or "cryopreservation medium" refers to any medium that can be used for the cryopreservation of cells. Such media may include media containing 7% to 10% DMSO. Exemplary media include CryoStor CS10, Hyperthermasol, and combinations thereof. The term "CS10" refers to cryopreservation media obtained from Stemcell Technologies or Biolife Solutions. CS10 medium may be referred to by the trade name "CryoStor® CS10". CS10 medium is a serum-free, animal component-free medium containing DMSO.

[0529] The term "central memory T cell" refers to CD45R0+ cells in humans, and CCR7(CCR7) 高 ) and CD62L (CD62 高 This refers to a subset of T cells that constitutively express IL-2. The surface phenotype of central memory T cells also includes TCR, CD3, CD127 (IL-7R), and IL-15R. The transcription factors of central memory T cells include BCL-6, BCL-6B, MBD2, and BMI1. After TCR triggering, central memory T cells primarily secrete IL-2 and CD40L as effector molecules. Central memory T cells are dominant in the CD4 compartment of the blood and are proportionally concentrated in the lymph nodes and tonsils in humans.

[0530] The term "effector memory T cells" refers to cells that are CD45R0+ like central memory T cells, but have lost constitutive expression of CCR7 (CCR7 低 ), CD62L expression is heterogeneous or low (CD62L 低 Central memory T cells refer to a subset of human or mammalian T cells. The surface phenotype of central memory T cells also includes TCR, CD3, CD127 (IL-7R), and IL-15R. The transcription factor of central memory T cells includes BLIMP1. Effector memory T cells rapidly secrete high levels of inflammatory cytokines after antigen stimulation, including interferon-gamma, IL-4, and IL-5. Effector memory T cells are dominant in the CD8 compartment of the blood and are proportionally enriched in the lungs, liver, and intestines in humans. CD8+ effector memory T cells carry large amounts of perforin.

[0531] The term "closed system" refers to a system that is closed off from the external environment. Any closed system suitable for cell culture can be used in the method of the present invention. Closed systems include, but are not limited to, sealed G containers. Once the tumor segment is added to the closed system, the system is not opened to the external environment until it is ready to administer the TIL to the patient.

[0532] As used herein to describe the process of destroying a tumor, the terms “fragmentation,” “fragment,” and “fragmented” include mechanical fragmentation methods such as crushing, slicing, splitting, and cutting tumor tissue, as well as any other method for destroying the physical structure of tumor tissue.

[0533] The terms "peripheral blood mononuclear cells" and "PBMCs" refer to peripheral blood cells with round nuclei, including lymphocytes (T cells, B cells, NK cells) and monocytes. When used as antigen-presenting cells (PBMCs are a type of antigen-presenting cell), peripheral blood mononuclear cells are preferably irradiated allogeneic peripheral blood mononuclear cells.

[0534] The terms “peripheral blood lymphocytes” and “PBL” refer to expanded T cells from peripheral blood. In some embodiments, PBLs are isolated from whole blood or apheresis products from a donor. In some embodiments, PBLs are isolated from donor-derived whole blood or apheresis products by positive or negative selection of T cell phenotypes, such as CD3+CD45+ T cell phenotypes.

[0535] The term "anti-CD3 antibody" refers to an antibody or its variant, such as a monoclonal antibody, including human, humanized, chimeric, or mouse antibodies against the CD3 receptor on the T cell antigen receptor of mature T cells. Anti-CD3 antibodies include OKT-3, also known as muromonab. Anti-CD3 antibodies also include the UHCT1 clone, also known as T3 and CD3ε. Other anti-CD3 antibodies include, for example, otelixizumab, teprizumab, and bicilizumab.

[0536] The term "OKT-3" (also referred to herein as "OKT3") refers to a monoclonal antibody or its biosimilar or variant, including human, humanized, chimeric, or mouse antibodies against the CD3 receptor on the T cell antigen receptor of mature T cells, and includes commercially available forms such as OKT-3 (30 ng / mL, MACS GMP CD3 pure, Miltenyi Biotech, Inc., San Diego, CA, USA) and muromonab, or their variants, conserved amino acid substitutions, glycoforms, or biosimilars. The amino acid sequences of the heavy and light chains of muromonab are shown in Table 1 (SEQ ID NO: 1 and SEQ ID NO: 2). Hybridomas capable of producing OKT-3 are deposited in the American Type Culture Collection and assigned ATCC accession number CRL8001. Hybridomas capable of producing OKT-3 are also deposited in the European Collection of Authenticated Cell Cultures (ECACC) and assigned catalog number 86022706. [Table 1]

[0537] The term "IL-2" (also referred to herein as "IL2") refers to the T cell growth factor known as interleukin-2 and includes all forms of IL-2, including human and mammalian forms, conserved amino acid substitutions, glycoforms, biosimilars, and variants. IL-2 is described, for example, in Nelson, J. Immunol. 2004, 172, 3983-88 and Malek, Annu. Rev. Immunol. 2008, 26, 453-79, the disclosures of which are incorporated herein by reference. Table 2 shows the amino acid sequence of recombinant human IL-2 suitable for use in the present invention (SEQ ID NO: 3). For example, the term IL-2 encompasses human recombinant IL-2 forms such as aldesleukin (PROLEUKIN, commercially available from multiple suppliers at 22 million IU per single-use vial), as well as recombinant IL-2 forms commercially available from CellGenix, Inc., Portsmouth, NH, USA (CELLGRO GMP) or ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (catalog number CYT-209-b) and other commercial equivalents from other vendors. Aldesleukin (des-alanil-1, serine-125 human IL-2) is a non-glycosylated human recombinant IL-2 form with a molecular weight of approximately 15 kDa. The amino acid sequence of aldesleukin suitable for use in this invention is shown in Table 2 (SEQ ID NO: 4). The term IL-2 also refers to the pegylated IL-2 prodrug benpegaldesleukin (NKTR-214, in which an average of 6 lysine residues are substituted with [(2,7-bis{[methylpoly(oxyethylene)]carbamoyl}-9H-fluoren-9-yl)methoxy]carbonyl). 6This invention also includes pegylated forms of IL-2 described herein, such as pegylated human recombinant IL-2 (e.g., SEQ ID NO: 4), which are available from Nektar Therapeutics, South San Francisco, CA, USA, or can be prepared by methods known in the Art, such as the method described in Example 19 of International Patent Application Publication WO2018 / 132496 A1, or the method described in Example 1 of U.S. Patent Application Publication US2019 / 0275133 A1, the disclosures thereof being incorporated herein by reference. Benpegaldesleukine (NKTR-214) and other pegylated IL-2 molecules suitable for use in the present invention are described in U.S. Patent Application Publication US2014 / 0328791 A1 and International Patent Application Publication WO2012 / 065086 A1, the disclosures thereof being incorporated herein by reference. Suitable alternative forms of compounded IL-2 for use in the present invention are described in U.S. Patents No. 4,766,106, No. 5,206,344, No. 5,089,261, and No. 4,902,502, the disclosures of which are incorporated herein by reference. Suitable formulations of IL-2 for use in the present invention are described in U.S. Patent No. 6,706,289, the disclosures of which are incorporated herein by reference.

[0538] In some embodiments, the preferred IL-2 form for use in the present invention is THOR-707, available from Synthorx, Inc. Preparations and characterizations of THOR-707 and additional alternative forms of IL-2 preferred for use in the present invention are described in U.S. Patent Application Publications US2020 / 0181220 A1 and US2020 / 0330601 A1, and their disclosures are incorporated herein by reference. In some embodiments, the preferred IL-2 form for use in the present invention is an interleukin-2 (IL-2) complex comprising an isolated and purified IL-2 polypeptide and a complex moiety bound to the isolated and purified IL-2 polypeptide at amino acid positions selected from K35, T37, R38, T41, F42, K43, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107, where the amino acid residue numbering corresponds to Sequence ID No. 5. In some embodiments, the amino acid position is selected from T37, R38, T41, F42, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107. In some embodiments, the amino acid position is selected from T37, R38, T41, F42, F44, Y45, E61, E62, E68, P65, V69, L72, and Y107. In some embodiments, the amino acid position is selected from T37, T41, F42, F44, Y45, P65, V69, L72, and Y107. In some embodiments, the amino acid position is selected from R38 and K64. In some embodiments, the amino acid position is selected from E61, E62, and E68. In some embodiments, the amino acid position is E62. In some embodiments, an amino acid residue selected from K35, T37, R38, T41, F42, K43, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107 is further mutated to lysine, cysteine, or histidine. In some embodiments, the amino acid residue is mutated to cysteine. In some embodiments, the amino acid residue is mutated to lysine. In some embodiments, K35, T37, R38, T41, F42, K43, F44, Y45, E61, E62, E68, K64, P65, V69, L72,The amino acid residues selected from and Y107 are further mutated into non-natural amino acids. In some embodiments, the non-natural amino acids are N6-azidoethoxy-L-lysine (AzK), N6-propargylethoxy-L-lysine (PraK), BCN-L-lysine, norbornenyllysine, TCO-lysine, methyltetrazinyllysine, allyloxycarbonyllysine, 2-amino-8-oxononanoic acid, 2-amino-8-oxooctanoic acid, p-acetyl-L-phenylalanine, p-azidomethyl-L-phenylalanine (pAMF), p-iodo-L-phenylalanine, m-acetylphenylalanine, 2-amino-8-oxononanoic acid, p-propargyloxyphenylalanine, p-propargyl-phenylalanine, 3-methylphenylalanine, L-DOPA, fluorinated phenylalanine, isopropyl Contains L-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, O-allyl-tyrosine, O-methyl-L-tyrosine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, phosphonotyrosine, tri-O-acetyl-GlcNAcp-serine, L-phosphoserine, phosphonoserine, L-3-(2-naphthyl)alanine, 2-amino-3-((2-((3-(benzyloxy)-3-oxopropyl)amino)ethyl)ceranyl)propanoic acid, 2-amino-3-(phenylceranyl)propanoic acid, or selenocysteine. In some embodiments, the IL-2 complex has a reduced affinity for the IL-2 receptor α (IL-2Rα) subunit compared to the wild-type IL-2 polypeptide. In some embodiments, the reduced affinity is a reduction of approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more than 99% of the binding affinity to IL-2Rα compared to the wild-type IL-2 polypeptide. In some embodiments, the reduced affinity is approximately 1x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 30x, 50x, 100x, 200x, 300x, 500x compared to the wild-type IL-2 polypeptide.The ratio is 1000 times or more. In some embodiments, the composite portion impairs or blocks the bond between IL-2 and IL-2Rα. In some embodiments, the composite portion includes a water-soluble polymer. In some embodiments, an additional composite portion includes a water-soluble polymer. In some embodiments, each of the water-soluble polymers independently includes polyethylene glycol (PEG), poly(propylene glycol) (PPG), copolymers of ethylene glycol and propylene glycol, poly(oxyethylated polyol), poly(olefin alcohol), poly(vinylpyrrolidone), poly(hydroxyalkyl methacrylamide), poly(hydroxyalkyl methacrylate), poly(saccharide), poly(α-hydroxy acid), poly(vinyl alcohol), polyphosphazene, polyoxazoline (POZ), poly(N-acryloylmorpholine), or a combination thereof. In some embodiments, each of the water-soluble polymers independently includes PEG. In some embodiments, PEG is linear PEG or branched PEG. In some embodiments, each of the water-soluble polymers independently includes polysaccharide. In some embodiments, the polysaccharide comprises dextran, polysialic acid (PSA), hyaluronic acid (HA), amylose, heparin, heparan sulfate (HS), dextrin, or hydroxyethyl starch (HES). In some embodiments, each of the water-soluble polymers independently comprises a glycan. In some embodiments, each of the water-soluble polymers independently comprises a polyamine. In some embodiments, the complex portion comprises a protein. In some embodiments, an additional complex portion comprises a protein. In some embodiments, each of the proteins independently comprises albumin, transferrin, or transthyretin. In some embodiments, each of the proteins independently comprises an Fc moiety. In some embodiments, each of the proteins independently comprises an Fc moiety of IgG. In some embodiments, the complex portion comprises a polypeptide. In some embodiments, an additional complex portion comprises a polypeptide. In some embodiments, each of the proteins independently comprises XTEN peptide, glycine-rich homoamino acid polymer (HAP),The compound comprises a PAS polypeptide, an elastin-like polypeptide (ELP), a CTP peptide, or a gelatin-like protein (GLK) polymer. In some embodiments, the isolated and purified IL-2 polypeptide is modified by glutamylation. In some embodiments, the complex is directly conjugated to the isolated and purified IL-2 polypeptide. In some embodiments, the complex is indirectly conjugated to the isolated and purified IL-2 polypeptide via a linker. In some embodiments, the linker comprises a homobifunctional linker. In some embodiments, the homobifunctional linker is a Romant reagent dithiobis(succinimidylpropionate) DSP, 3′3′-dithiobis(sulfosuccinimidylpropionate) (DTSSP), disuccinimidyl sverat (DSS), bis(sulfosuccinimidyl) sverat (BS), disuccinimidyl tartarate (DST), disulfosuccinimidyl tartarate (sulfoDS T), ethylene glycobis(succinimidyl succinate) (EGS), disuccinimidyl glutarate (DSG), N,N'-disuccinimidyl carbonate (DSC), dimethyl adipimidate (DMA), dimethyl pimelimidate (DMP), dimethyl spelmidate (DMS), dimethyl-3,3'-dithiobispropionimidate (DTBP), 1,4-di-(3'-(2'-pyridyldithio This includes propionamide butane (DPDPB), bismaleimide hexane (BMH), aryl halide-containing compounds (DFDNB), such as 1,5-difluoro-2,4-dinitrobenzene or 1,3-difluoro-4,6-dinitrobenzene, 4,4′-difluoro-3,3′-dinitrophenyl sulfone (DFDNPS), bis-[β-(4-azidosalicylamido)ethyl]disulfide (BASED), formaldehyde, glutaraldehyde, 1,4-butanediol diglycidyl ether, adipic acid dihydrazide, carbohydrazide, o-toluidine, 3,3′-dimethylbenzidine, benzidine, α,α′-p-diaminodiphenyl, diiodo-p-xylenesulfonic acid, N,N′-ethylene-bis(iodoacetamide), or N,N′-hexamethylenebis(iodoacetamide). In some embodiments, the linker isThe heterobifunctional linker is included. In some embodiments, the heterobifunctional linker is N-succinimidyl 3-(2-pyridyldithio)propionate (sPDP), long-chain N-succinimidyl 3-(2-pyridyldithio)propionate (LC-sPDP), water-soluble long-chain N-succinimidyl 3-(2-pyridyldithio)propionate (sulfo-LC-sPDP), succinimidyloxycarbonyl-α-methyl-α-(2-pyridyldithio)toluene (sMPT), sulfosuccinimidyl-6-[α-methyl-α-(2-pyridyldithio)toluamide]hexanoate (sulfo-LC-sMPT), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sMCC), sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane- 1-Carboxylate (sulfo-sMCC), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MB), m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (sulfo-MB), N-succinimidyl (4-iodoacetyl)aminobenzoate (sIAB), sulfosuccinimidyl (4-iodoacetyl)aminobenzoate (sulfo-sIAB), succinimidyl-4-(p-maleimidophenyl)butyrate (sMPB), sulfosuccinimidyl-4-(p-maleimidophenyl)butyrate (sulfo-sMPB), N-(γ-maleimidobutyryloxy)succinimide ester (GMB), N-(γ-maleimidobutyryloxy)sulfosuccinimide ester (sulfo-GMB), succinimidyl 6-((iodoacetyl)amino)hexanoate (sIAX), succinimidyl 6-[6-(((iodoacetyl)amino)hexanoyl)amino]hexanoate (slAXX), succinimidyl 4-(((iodoacetyl)amino)methyl)cyclohexane-1-carboxylate (sIAC), succinimidyl 6-(((((4-iodoacetyl)amino)methyl)cyclohexane-1-carbonyl)amino)hexanoate (sIACX), p-nitrophenyl iodoacetate (NPIA), carbonyl-reactive and sulfhydryl-reactive crosslinking agents, e.g., 4-(4-N-maleimidophenyl)butyrate hydrazide (MPBH),4-(N-maleimidomethyl)cyclohexane-1-carboxyl-hydrazide-8(M2C2H), 3-(2-pyridyldithio)propionylhydrazide(PDPH), N-hydroxysuccinimidyl-4-azidosalicylic acid(NHs-AsA), N-hydroxysulfosuccinimidyl-4-azidosalicylic acid(sulfo-NHs-AsA), sulfosuccini, Midyl-(4-azidosalicylamide)hexanoate (sulfo-NHs-LC-AsA), sulfosuccinimidyl-2-(p-azidosalicylamide)ethyl-1,3'-dithiopropionate (sAsD), N-hydroxysuccinimidyl-4-azidobenzoate (HsAB), N-hydroxysulfosuccinimidyl-4-azidobenzoate (sulfo-HsAB), N-succinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate (sANPAH), sulfosuccinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate Noate (sulfo-sANPAH), N-5-azido-2-nitrobenzoyloxysuccinimide (ANB-NO), sulfosuccinimidyl-2-(m-azido-o-nitrobenzamide)-ethyl-1,3'-dithiopropionate (sAND), N-succinimidyl-4(4-azidophenyl)1,3'-dithiopropionate (sADP), N-sulfosuccinimidyl(4-azidophenyl)-1,3'-dithiopropionate (sulfo-sADP), sulfosuccinimidyl-4-(p-azidophenyl)butyrate (sulfo-sAPB), sulfosuccinimidyl 2-(7-azido-4-methylcoumarin-3-acetamido)ethyl-1,3'-dithiopropionate (sAED), sulfosuccinimidyl 7-azido-4-methylcoumarin-3-acetate (sulfo-sAMCA), p-nitrophenyl diazopirubate (pNPDP), p-nitrophenyl-2-diazo-3,3,3-trifluoropropionate (PNP-DTP), 1-(ρ-azidosalicylamide)-4-(iodoacetamide)butane (AsIB), N-[4-(ρ-azidosalicylamide)butyl]-3'-(2'-pyridyldithio)propionamide (APDP), benzophenone-4-iodoacetamide, p-azidobenzoyl hydrazide (ABH), 4-(p-azidosalicylamide)butylamine (AsBA), or p-azidophenyl Contains glyoxal (APG). In some embodiments, the linker includes a cleavable linker, which optionally includes a dipeptide linker. In some embodiments, the dipeptide linker includes Val-Cit, Phe-Lys, Val-Ala, or Val-Lys.In some embodiments, the linker comprises an uncleavable linker. In some embodiments, the linker optionally comprises a maleimide group comprising maleimidocaproyl (mc), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sMCC), or sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-sMCC). In some embodiments, the linker further comprises a spacer. In some embodiments, the spacer comprises p-aminobenzyl alcohol (PAB), p-aminobenzyloxycarbonyl (PABC), its derivatives, or analogs. In some embodiments, the complex portion can extend the serum half-life of the IL-2 complex. In some embodiments, additional complex portions can extend the serum half-life of the IL-2 complex. In some embodiments, a suitable IL-2 form for use in the present invention is any fragment of the IL-2 forms described herein. In some embodiments, the IL-2 form preferred for use in the present invention is pegylated, as disclosed in U.S. Patent Application Publication US2020 / 0181220 A1 and U.S. Patent Application Publication US2020 / 0330601 A1. In some embodiments, the IL-2 form preferred for use in the present invention is an IL-2 complex comprising an IL-2 polypeptide containing N6-azidoethoxy-L-lysine (AzK) covalently bonded to a polyethylene glycol (PEG) complex, wherein the IL-2 polypeptide comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 5, and AzK substitutes an amino acid at the K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69, or L72 positions with respect to the amino acid positions in SEQ ID NO: 5. In some embodiments, the IL-2 polypeptide comprises a one-residue N-terminal deletion compared to SEQ ID NO: 5. In some embodiments, the IL-2 form suitable for use in the present invention lacks IL-2R alpha chain association but retains normal binding to the intermediate affinity IL-2R beta-gamma signaling complex.In some embodiments, a suitable form of IL-2 for use in the present invention is an IL-2 complex comprising an IL-2 polypeptide containing N6-azidoethoxy-L-lysine (AzK) covalently bonded to a polyethylene glycol (PEG) complex, wherein the IL-2 polypeptide comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 5, and AzK substitutes an amino acid at the K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69, or L72 positions in SEQ ID NO: 5. In some embodiments, a suitable form of IL-2 for use in the present invention is an IL-2 complex comprising an IL-2 polypeptide containing N6-azidoethoxy-L-lysine (AzK) covalently bonded to a polyethylene glycol (PEG) complex, wherein the IL-2 polypeptide comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 5, and AzK substitutes an amino acid at the K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69, or L72 positions with respect to the amino acid positions in SEQ ID NO: 5. In some embodiments, a suitable form of IL-2 for use in the present invention is an IL-2 complex comprising an IL-2 polypeptide containing N6-azidoethoxy-L-lysine (AzK) covalently bonded to a polyethylene glycol (PEG) complex, wherein the IL-2 polypeptide comprises an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 570, and AzK substitutes an amino acid at the K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69, or L72 positions with respect to the amino acid positions in SEQ ID NO: 570.

[0539] In some embodiments, a suitable IL-2 form for use in the present invention is nembalquin alfa, also known as ALKS-4230 (SEQ ID NO: 571), which is available from Alkermes, Inc. Nembalquin alfa is a peptidyl linker ( 60 GG 61 It fuses with human interleukin 2 fragment (62-132) via ) and peptidyl linker ( 133 GSGGGS138 Human interleukin-2 receptor α chain fragments (139-303) fuse via () and are produced in Chinese hamster ovary (CHO) cells, and are glycosylated, human interleukin-2 fragments (1-59), variant (Cys 125 >Ser 51 );Fusion with human interleukin-2 (IL-2)(4-74)-peptide(62-132) via G2 peptide linker(60-61), and fusion with human interleukin-2 receptor α chain (IL2R subunit alpha, IL2Rα, IL2RA)(1-165)-peptide(139-303) via GSG3S peptide linker(133-138), resulting in human interleukin-2 (IL-2)(75-133)-peptide[Cys], which is produced in Chinese hamster ovary (CHO) cells and alpha-glycosylated. 125Also known as (51)>Ser]-mutants (1-59). The amino acid sequence of nembalukin alpha is shown in SEQ ID NO: 571. In some embodiments, nembalukin alpha exhibits the following post-translational modifications: disulfide crosslinks at the following positions: 31-116, 141-285, 184-242, 269-301, 166-197, or 166-199, 168-199, or 168-197 (using the numbering in SEQ ID NO: 571), and glycosylation sites at the following positions: N187, N206, T212 (using the numbering in SEQ ID NO: 571). The preparation and characterization of nembalukin alpha, as well as additional alternative forms of IL-2 suitable for use in the present invention, are described in U.S. Patent Application Publication No. 2021 / 0038684 A1 and U.S. Patent No. 10,183,979, the disclosures of which are incorporated herein by reference. In some embodiments, a suitable IL-2 form for use in the present invention is a protein having at least 80%, at least 90%, at least 95%, or at least 90% sequence identity with SEQ ID NO: 571. In some embodiments, a suitable IL-2 form for use in the present invention has the amino acid sequence shown in SEQ ID NO: 571 or its conserved amino acid substitutions. In some embodiments, a suitable IL-2 form for use in the present invention is a fusion protein comprising amino acids 24-452 of SEQ ID NO: 572, or its variants, fragments, or derivatives. In some embodiments, a suitable IL-2 form for use in the present invention is a fusion protein comprising an amino acid sequence having at least 80%, at least 90%, at least 95%, or at least 90% sequence identity with amino acids 24-452 of SEQ ID NO: 572, or its variants, fragments, or derivatives. Other suitable IL-2 forms for use in the present invention are described in U.S. Patent No. 10,183,979, the disclosure of which is incorporated herein by reference.Optionally, in some embodiments, a suitable IL-2 form for use in the present invention is a fusion protein comprising a first fusion partner linked to a second fusion partner by a mucin domain polypeptide linker, wherein the first fusion partner is IL-1Rα, or a protein having at least 98% amino acid sequence identity with IL-1Rα and possessing receptor antagonist activity of IL-Rα, the second fusion partner comprises all or part of an immunoglobulin including an Fc region, the mucin domain polypeptide linker comprises SEQ ID NO: 573, or an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 573, and the half-life of the fusion protein is improved compared to the fusion of the first fusion partner with the second fusion partner in the absence of the mucin domain polypeptide linker. [Table 2]

[0540] In some embodiments, a suitable IL-2 configuration for use in the present invention includes a heavy chain variable region (V) which comprises complementarity-determining regions HCDR1, HCDR2, and HCDR3. H ) and the light chain variable region (V) including LCDR1, LCDR2, and LCDR3 L ) and V H or V L The antibody cytokine transplantation protein includes an IL-2 molecule or fragment thereof transplanted into the CDR, and the antibody cytokine transplantation protein preferentially expands T effector cells over regulatory T cells. In some embodiments, the antibody cytokine transplantation protein includes a heavy chain variable region (V) which contains complementarity-determining regions HCDR1, HCDR2, and HCDR3. H ) and the light chain variable region (V) including LCDR1, LCDR2, and LCDR3 L ) and V H or V LThe regime comprises an IL-2 molecule or fragment thereof transplanted into a CDR, the IL-2 molecule being mutain, and the antibody cytokine transplant protein preferentially expands T effector cells over regulatory T cells. In some embodiments, the IL-2 regime comprises administration of an antibody described in U.S. Patent Application Publication 2020 / 0270334 A1, the disclosure of which is incorporated herein by reference. In some embodiments, the antibody cytokine transplant protein comprises a heavy chain variable region (VH) containing complementarity-determining regions HCDR1, HCDR2, HCDR3, and a light chain variable region (VL) containing LCDR1, LCDR2, LCDR3, and V H or V L The antibody comprises an IL-2 molecule or fragment thereof transplanted into a CDR, wherein the IL-2 molecule is mutain, the antibody cytokine transplant protein preferentially expands T effector cells over regulatory T cells, and the antibody further comprises an IgG class heavy chain and an IgG class light chain selected from the group consisting of an IgG class light chain containing SEQ ID NO: 569 and an IgG class heavy chain containing SEQ ID NO: 568, an IgG class light chain containing SEQ ID NO: 567 and an IgG class heavy chain containing SEQ ID NO: 559, an IgG class light chain containing SEQ ID NO: 37 and an IgG class heavy chain containing SEQ ID NO: 568.

[0541] In some embodiments, the IL-2 molecule or a fragment thereof is V H The IL-2 molecule is implanted in HCDR1 and is mutain. In some embodiments, the IL-2 molecule or a fragment thereof is V H The IL-2 molecule is implanted in HCDR2 and is mutain. In some embodiments, the IL-2 molecule or a fragment thereof is V H The IL-2 molecule is implanted in HCDR3 and is mutain. In some embodiments, the IL-2 molecule or a fragment thereof is V L The IL-2 molecule is implanted in LCDR1, and the IL-2 molecule is mutain. In some embodiments, the IL-2 molecule or a fragment thereof is V LThe IL-2 molecule is implanted in LCDR2 and is mutain. In some embodiments, the IL-2 molecule or a fragment thereof is V L It is transplanted into LCDR3, and the IL-2 molecule is mutain.

[0542] The insertion of the IL-2 molecule may occur in or near the N-terminal region of the CDR, in the middle region of the CDR, or in or near the C-terminal region of the CDR. In some embodiments, the antibody-cytokine transplant protein contains an IL-2 molecule incorporated into the CDR, and the IL-2 sequence does not frameshift the CDR sequence. In some embodiments, the antibody-cytokine transplant protein contains an IL-2 molecule incorporated into the CDR, and the IL-2 sequence replaces all or part of the CDR sequence. The replacement by the IL-2 molecule may occur in the N-terminal region of the CDR, in the middle region of the CDR, or in or near the C-terminal region of the CDR. The replacement by the IL-2 molecule may occur in just one or two amino acids of the CDR sequence, or in the entire CDR sequence.

[0543] In some embodiments, the IL-2 molecule is directly transplanted into the CDR without a peptide linker and without additional amino acids between the CDR sequence and the IL-2 sequence. In some embodiments, the IL-2 molecule is indirectly transplanted into the CDR using a peptide linker, with one or more additional amino acids between the CDR sequence and the IL-2 sequence.

[0544] In some embodiments, the IL-2 molecule described herein is IL-2 mutein. In some cases, IL-2 mutein includes the R67A substitution. In some embodiments, IL-2 mutein includes the amino acid sequence of SEQ ID NO: 544 or SEQ ID NO: 545. In some embodiments, IL-2 mutein includes the amino acid sequence of Table 1 of U.S. Patent Application Publication US2020 / 0270334 A1, the disclosure of which is incorporated herein by reference.

[0545] In some embodiments, the antibody cytokine transplant protein includes HCDR1 selected from the group consisting of SEQ ID NO: 546, SEQ ID NO: 549, SEQ ID NO: 552, and SEQ ID NO: 555. In some embodiments, the antibody cytokine transplant protein includes HCDR1 selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 543, and SEQ ID NO: 546. In some embodiments, the antibody cytokine transplant protein includes HCDR1 selected from the group consisting of HCDR2 selected from the group consisting of SEQ ID NO: 547, SEQ ID NO: 550, SEQ ID NO: 553, and SEQ ID NO: 556. In some embodiments, the antibody cytokine transplant protein includes HCDR3 selected from the group consisting of SEQ ID NO: 548, SEQ ID NO: 551, SEQ ID NO: 554, and SEQ ID NO: 557. In some embodiments, the antibody cytokine transplant protein includes V containing the amino acid sequence of SEQ ID NO: 558. H Includes a region. In some embodiments, the antibody cytokine transplant protein includes a heavy chain containing the amino acid sequence of SEQ ID NO: 559. In some embodiments, the antibody cytokine transplant protein includes a V containing the amino acid sequence of SEQ ID NO: 566. L Includes a region. In some embodiments, the antibody cytokine transplant protein includes a light chain containing the amino acid sequence of SEQ ID NO: 567. In some embodiments, the antibody cytokine transplant protein includes a V containing the amino acid sequence of SEQ ID NO: 28. H V containing the region and the amino acid sequence of SEQ ID NO: 566 LThe antibody cytokine transplant protein includes a heavy chain region containing the amino acid sequence of SEQ ID NO: 559 and a light chain region containing the amino acid sequence of SEQ ID NO: 567. In some embodiments, the antibody cytokine transplant protein includes a heavy chain region containing the amino acid sequence of SEQ ID NO: 559 and a light chain region containing the amino acid sequence of SEQ ID NO: 569. In some embodiments, the antibody cytokine transplant protein includes a heavy chain region containing the amino acid sequence of SEQ ID NO: 568 and a light chain region containing the amino acid sequence of SEQ ID NO: 567. In some embodiments, the antibody cytokine transplant protein includes a heavy chain region containing the amino acid sequence of SEQ ID NO: 568 and a light chain region containing the amino acid sequence of SEQ ID NO: 569. In some embodiments, the antibody cytokine transplant protein includes IgG.IL2F71A.H1 or IgG.IL2R67A.H1 of U.S. Patent Application Publication No. 2020 / 0270334 A1, or a variant, derivative, or fragment thereof, or a conserved amino acid substitution thereof, or a protein having at least 80%, at least 90%, at least 95%, or at least 98% sequence identity thereto. In some embodiments, the antibody component of the antibody-cytokine transplant protein described herein comprises an immunoglobulin sequence, a framework sequence, or a CDR sequence of palivizumab. In some embodiments, the antibody-cytokine transplant protein described herein has a longer serum half-life than a wild-type IL-2 molecule, such as but not limited to aldethleukin or an equivalent molecule. [Table 3-1] [Table 3-2] [Table 3-3]

[0546] The term "IL-4" (also referred to herein as "IL4") refers to the cytokine known as interleukin 4, which is produced by Th2 T cells, as well as eosinophils, basophils, and mast cells. IL-4 regulates the differentiation of naive helper T cells (Th0 cells) into Th2 T cells. Steinke and Borish, Respir. Res. 2001, 2, 66-70. When activated by IL-4, Th2 T cells subsequently produce additional IL-4 in a positive feedback loop. IL-4 also stimulates B cell proliferation and class II MHC expression, inducing class switching from B cells to IgE and IgG1 expression. Recombinant human IL-4 suitable for use in this invention is commercially available from several suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (catalog number CYT-211) and ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-15 recombinant protein, catalog number Gibco CTP0043). The amino acid sequence of recombinant human IL-4 suitable for use in this invention is shown in Table 2 (SEQ ID NO: 5).

[0547] The term "IL-7" (also referred to herein as "IL7") refers to a glycosylated tissue-derived cytokine known as interleukin-7, which can be obtained from stromal and epithelial cells, as well as dendritic cells. Fry and Mackall, Blood 2002, 99, 3892-904. IL-7 can stimulate T cell development. IL-7 binds to the IL-7 receptor, a heterodimer consisting of the IL-7 receptor α and the common gamma chain receptor, which is a set of signals important for T cell development in the thymus and survival in the periphery. Recombinant human IL-7 suitable for use in this invention is commercially available from several suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (catalog no. CYT-254) and ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-15 recombinant protein, catalog no. Gibco PHC0071). Table 2 shows the amino acid sequence of recombinant human IL-7 suitable for use in the present invention (SEQ ID NO: 6).

[0548] The term "IL-15" (also referred to herein as "IL15") refers to the T cell growth factor known as interleukin-15 and includes all forms of IL-2, including human and mammalian forms, conserved amino acid substitutions, glycoforms, biosimilars, and variants. IL-15 is described, for example, in Fehniger and Caligiuri, Blood 2001, 97, 14-32, the disclosure of which is incorporated herein by reference. IL-15 shares β and γ signaling receptor subunits with IL-2. Recombinant human IL-15 is a single non-glycosylated polypeptide chain containing 114 amino acids (and N-terminal methionine) with a molecular weight of 12.8 kDa. Recombinant human IL-15 is commercially available from several suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (catalog number CYT-230-b) and ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-15 recombinant protein, catalog number 34-8159-82). Table 2 shows the amino acid sequence of recombinant human IL-15 suitable for use in the present invention (SEQ ID NO: 7).

[0549] The term "IL-21" (also referred to herein as "IL21") refers to the pleiotropic cytokine protein known as interleukin-21 and includes all forms of IL-21, including human and mammalian forms, conserved amino acid substitutions, glycoforms, biosimilars, and variants. IL-21 is described, for example, in Spolski and Leonard, Nat. Rev. Drug. Disc. 2014, 13, 379-95, the disclosure of which is incorporated herein by reference. IL-21 is primarily used in natural killer T cells and activated human CD4 +It is produced by T cells. Recombinant human IL-21 is a single nonglycosylated polypeptide chain containing 132 amino acids with a molecular weight of 15.4 kDa. Recombinant human IL-21 is commercially available from multiple suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (Catalog No. CYT-408-b) and ThermoFisher Scientific, Inc., Waltham, MA, USA (Human IL-21 Recombinant Protein, Catalog No. 14-8219-80). The amino acid sequence of recombinant human IL-21 suitable for use in the present invention is shown in Table 2 (SEQ ID NO: 8).

[0550] When an "anti-tumor effective amount", "tumor inhibitory effective amount", or "therapeutic amount" is indicated, the exact amount of the composition of the present invention to be administered can be determined by a physician taking into account the age, weight, tumor size, degree of infection or metastasis, and individual differences in condition of the patient (subject). A pharmaceutical composition containing tumor infiltrating lymphocytes (e.g., secondary TIL or genetically modified cytotoxic lymphocytes) described herein is 10 4 ~10 11 cells / kg body weight (e.g., 10 5 ~10 6 、10 5 ~10 10 、10 5 ~10 11 、10 6 ~10 10 、10 6 ~10 11 、10 7 ~10 11 、10 7 ~10 10 、10 8 ~10 11 、10 8 ~10 10 、10 9 ~10 11 、or 10 9 ~10 10It may be outlined that the drug may be administered in doses of cells / kg body weight (including all integer values ​​within those ranges). Tumor-infiltrating lymphocytes (including, in some cases, genetically modified cytotoxic lymphocytes) compositions may also be administered multiple times in these doses. Tumor-infiltrating lymphocytes (including, in some cases, genetically modified ones) may be administered by using infusion techniques commonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). The optimal dosage and treatment regimen for a particular patient can be readily determined by medical professionals by monitoring the patient for signs of disease and adjusting treatment as appropriate.

[0551] The terms “hematological malignancies,” “hematological malignancies,” or related terms refer to cancers and tumors of mammalian hematopoietic and lymphoid tissues, including but not limited to the blood, bone marrow, lymph nodes, and lymphoid tissues. Hematological malignancies are also called “liquid tumors.” Hematological malignancies may include, but are not limited to, acute lymphoblastic leukemia (ALL), chronic lymphocytic lymphoma (CLL), small lymphocytic lymphoma (SLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute monocytic leukemia (AMoL), Hodgkin lymphoma, and non-Hodgkin lymphoma. The term “B-cell hematological malignancies” refers to hematological malignancies that affect B cells.

[0552] The term "solid tumor" typically refers to an abnormal mass of tissue that does not contain cysts or fluid areas. Solid tumors can be benign or malignant. The term "solid tumor carcinoma" refers to a malignant, neoplastic, or cancerous solid tumor. Solid tumor carcinomas include, but are not limited to, sarcomas, carcinomas, and lymphomas, such as lung cancer, breast cancer, prostate cancer, colon cancer, rectal cancer, and bladder cancer. The histological structure of a solid tumor includes interdependent tissue compartments containing parenchyma (cancer cells) and supporting stromal cells that can provide a supporting microenvironment in which the cancer cells are dispersed.

[0553] The term "liquid tumor" refers to an abnormal mass of cells that is naturally fluid. Liquid tumor cancers include, but are not limited to, leukemia, myeloma, and lymphoma, as well as other hematological malignancies. TILs obtained from liquid tumors may also be referred to herein as bone marrow-infiltrating lymphocytes (MILs). TILs obtained from liquid tumors, including liquid tumors circulating in the peripheral blood, may also be referred to herein as PBLs. The terms MIL, TIL, and PBL are used interchangeably herein and differ only based on the tissue type from which the cells originate.

[0554] As used herein, the term “microenvironment” may refer to the solid or hematological tumor microenvironment as a whole, or to individual subsets of cells within the microenvironment. As used herein, the tumor microenvironment refers to a complex mixture of “cells, soluble factors, signaling molecules, extracellular matrix, and mechanical cues that promote neoplastic transformation, support tumor growth and invasion, protect the tumor from host immunity, promote treatment resistance, and provide a niche for successful dominant metastasis.” Although tumors express antigens that should be recognized by T cells, tumor clearance by the immune system is rare due to immunosuppression by the microenvironment.

[0555] In some embodiments, the present invention includes a method for treating cancer in a TIL population, wherein the patient is pre-treated with non-myeloablative chemotherapy before infusing the TIL according to the present invention. In some embodiments, a TIL population may be provided, and the patient is pre-treated with non-myeloablative chemotherapy before infusing the TIL according to the present invention. In some embodiments, the non-myeloablative chemotherapy is cyclophosphamide 60 mg / kg / day for 2 days (27 and 26 days before TIL infusion) and fludarabine 25 mg / m2 / day for 5 days (27 to 23 days before TIL infusion). In some embodiments, the non-myeloablative chemotherapy is cyclophosphamide 60 mg / kg / day for 2 days (27 and 26 days before TIL infusion) and fludarabine 25 mg / m2 / day for 3 days (27 to 25 days before TIL infusion). In some embodiments, non-myeloablative chemotherapy consists of cyclophosphamide 60 mg / kg / day for 2 days (27 and 26 days before TIL infusion), followed by fludarabine 25 mg / m2 / day for 3 days (25 to 23 days before TIL infusion). In some embodiments, after non-myeloablative chemotherapy according to the present invention and TIL infusion (day 0), the patient receives intravenous infusion of IL-2 at 720,000 IU / kg every 8 hours until physiological tolerance is reached.

[0556] Experimental findings indicate that lymphocyte depletion prior to adoptive transfer of tumor-specific T lymphocytes plays a crucial role in enhancing therapeutic efficacy by eliminating regulatory T cells and competing elements of the immune system ("cytokine sinks"). Therefore, some embodiments of the present invention utilize a lymphocyte depletion step (sometimes referred to as "immunosuppressive conditioning") in the patient prior to the introduction of the rTIL of the present invention.

[0557] As used herein, the terms “simultaneous administration,” “administering simultaneously,” “administered in combination,” “administered in combination,” “simultaneous,” and “concurrent” encompass the administration of two or more pharmacokinetic ingredients (in preferred embodiments of the present invention, for example, at least one potassium channel agonist in combination with multiple TILs) to a subject, thereby resulting in both the pharmacokinetic ingredients and / or their metabolites being present in the subject simultaneously. Simultaneous administration includes simultaneous administration in separate compositions, administration in separate compositions at different times, or administration in a composition containing two or more pharmacokinetic ingredients. Simultaneous administration in separate compositions and administration in a composition containing both drugs are preferred.

[0558] The terms “effective dose” or “therapeutic effective dose” refer to the amount of a compound or combination of compounds described herein that is sufficient to achieve the intended use, including but not limited to disease treatment. The therapeutic effective dose may vary depending on the intended use (in vitro or in vivo), the subject and condition being treated (e.g., the subject’s weight, age, and sex), the severity of the condition, or the method of administration. The term also applies to the dose that induces a specific response in target cells (e.g., reduced platelet adhesion and / or cell migration). The specific dose will vary depending on the specific compound selected, the dosing regimen to be followed, whether the compound is administered in combination with other compounds, the timing of administration, the tissue to which it is administered, and the physical delivery system through which the compound is carried.

[0559] Terms such as “treatment,” “to treat,” and “to heal” refer to obtaining a desired pharmacological and / or physiological effect. This effect may be preventive in the sense of completely or partially preventing the disease or its symptoms, and / or therapeutic in the sense of partial or complete cure of the disease and / or side effects resulting from it. As used herein, “treatment” encompasses all treatments of disease in mammals, particularly humans, and includes (a) preventing the development of the disease in a subject susceptible to the disease but not yet diagnosed with the disease, (b) suppressing the disease, i.e., inhibiting its onset or progression, and (c) mitigating the disease, i.e., causing regression of the disease and / or reducing one or more symptoms of the disease. “Treatment” is also intended to encompass the delivery of a drug to provide a pharmacological effect even in the absence of a disease or condition. For example, “treatment” includes the delivery of a composition that can induce an immune response or provide immunity in the absence of a disease, e.g., in the case of a vaccine.

[0560] The term "heterogeneous," when used in reference to a nucleic acid or protein, indicates that the nucleic acid or protein contains two or more subsequences that are not found in nature in the same relation to each other. For example, a nucleic acid typically has two or more sequences from unrelated genes that are recombinantly produced and arranged to create a new functional nucleic acid, e.g., a promoter from one source and a coding region from another, or a coding region from different sources. Similarly, a heterogeneous protein indicates that the protein contains two or more subsequences that are not found in nature in the same relation to each other (e.g., a fusion protein).

[0561] In the context of two or more nucleic acids or polypeptides, the terms “sequence identity,” “identity percentage,” and “sequence identity percentage” (or their synonyms, e.g., “99% identical”) refer to two or more sequences or subsequences that are identical or, when compared and aligned for maximum match (with gaps introduced as necessary), without considering any conserved amino acid substitutions as part of sequence identity, have a certain percentage of identical nucleotide or amino acid residues. The identity percentage can be measured using sequence comparison software or algorithms, or by visual inspection. Various algorithms and software that can be used to obtain amino acid or nucleotide sequence alignments are known in the art. Suitable programs for determining the sequence identity percentage include, for example, the BLAST program suite available from the BLAST website of the U.S. National Center for Biotechnology Information. Comparisons between two sequences can be performed using either the BLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acid sequences, and BLASTP is used to compare amino acid sequences. ALIGN, ALIGN-2 (Genentech, South San Francisco, California), or MegAlign, available from DNASTAR, are additional publicly available software programs that can be used to align sequences. Those skilled in the art can determine the appropriate parameters for maximum alignment using specific alignment software. In certain embodiments, the default parameters of the alignment software are used.

[0562] As used herein, the term “variant” includes, but is not limited to, antibodies or fusion proteins having an amino acid sequence different from that of a reference antibody due to one or more substitutions, deletions, and / or additions within or adjacent to the amino acid sequence of the reference antibody. A variant may have one or more conservative substitutions in its amino acid sequence compared to that of the reference antibody. Conservative substitutions may include, for example, substitutions of similarly charged or uncharged amino acids. A variant retains the ability to specifically bind to the antigen of the reference antibody. The term “variant” also includes pegylated antibodies or proteins.

[0563] In this specification, “tumor-infiltrating lymphocytes” or “TIL” means the population of cells initially obtained as leukocytes that have left the bloodstream and migrated to the tumor. TILs include CD8 + Cytotoxic T cells (lymphocytes), Th1 and Th17 CD4 + TILs include, but are not limited to, T cells, natural killer cells, dendritic cells, and M1 macrophages. TILs include both primary and secondary TILs. A “primary TIL” is obtained from a patient tissue sample as outlined herein (sometimes referred to as “newly acquired” or “newly isolated”), and a “secondary TIL” is any expanded or proliferated TIL cell population as considered herein, and includes, but is not limited to, bulk TILs, expanded TILs (“REP TILs”), and “reREP TILs” as considered herein. A reREP TIL may include, for example, a second proliferating TIL or a second additional proliferating TIL (e.g., one described in step D of Figure 27, which includes a TIL referred to as a reREP TIL).

[0564] TILs can generally be defined either biochemically using cell surface markers or functionally by their ability to invade tumors and achieve therapeutic effects. TILs can generally be classified by the expression of one or more biomarkers from among CD4, CD8, TCRαβ, CD27, CD28, CD56, CCR7, CD45Ra, CD95, PD-1, and CD25. Furthermore, and alternatively, TILs can be functionally defined by their ability to invade solid tumors upon reintroduction into a patient. TILs can be further characterized by their potency; for example, a TIL may be considered potent if its interferon (IFN) release exceeds approximately 50 pg / mL, approximately 100 pg / mL, approximately 150 pg / mL, or approximately 200 pg / mL. For example, a TIL may be considered potent if the interferon (IFNγ) release exceeds approximately 50 pg / mL, 100 pg / mL, 150 pg / mL, 200 pg / mL, 300 pg / mL, 400 pg / mL, 500 pg / mL, 600 pg / mL, 700 pg / mL, 800 pg / mL, 900 pg / mL, or 1000 pg / mL.

[0565] The terms “pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” are intended to include all solvents, dispersions, coatings, antibacterial and antifungal agents, isotonic and absorption retardants, and inactive components. The use of such pharmaceutically acceptable carriers or excipients for pharmaceutically active ingredients is well known in the art. Unless any conventional pharmaceutically acceptable carrier or excipient is incompatible with the pharmaceutically active ingredient, its use in the therapeutic compositions of the present invention is intended. Additional pharmaceutically active ingredients, such as other drugs, may also be incorporated into the compositions and methods described.

[0566] The terms “about” and “approximately” mean within a statistically significant range of values. Such a range may be within one decimal place of a given value or range, preferably within 50%, more preferably within 20%, even more preferably within 10%, and even more preferably within 5%. The tolerances encompassed by the terms “about” or “approximately” depend on the specific system under study and will be readily understood by those skilled in the art. Furthermore, as used herein, the terms “about” and “approximately” mean that dimensions, sizes, formulations, parameters, shapes, and other quantities and features are not and do not need to be precise, but may be approximate and / or greater or less, as needed, to reflect tolerances, conversion factors, rounding, measurement errors, and other factors known to those skilled in the art. In general, dimensions, sizes, formulations, parameters, shapes, or other quantities or features are “about” or “approximately,” whether so explicitly stated. Note that configurations describing very different embodiments of size, shape, and dimension may be adopted.

[0567] Where used in the attached claims, the transitional terms “including,” “essentially consisting of,” and “consisting of,” in their original and amended forms, define the claims in terms of whether any additional unlisted claim elements or steps, if present, are excluded from the scope of the claim. The term “including” is intended to be inclusive or free form and does not exclude any additional unlisted elements, methods, steps, or materials. The term “consisting of” excludes any elements, steps, or materials other than those specified in the claims, and in the latter case, also excludes any impurities typically associated with the specified material(s). The term “essentially consisting of” limits the claims to such an extent that it does not substantially affect any particular element, step, or material(s) and the basic and novel features(s) of the claimed invention. All compositions, methods, and kits described herein that embody the invention may be more specifically defined in alternative embodiments by any of the transitional terms “including,” “essentially consisting of,” and “consisting of.”

[0568] "PD-1 high" or "PD-1 high" or "PD-1 高 (PD-1 high The term "PD-1 high TILs" refers to high levels of PD-1 protein expression in cells, including but not limited to tumor-infiltrating lymphocytes or T cells, compared to control cells from healthy subjects. In some embodiments, the level of PD-1 expression is determined using standard methods known to those skilled in the art for measuring the level of protein present on cells, such as flow cytometry, fluorescence-activated cell sorting (FACS), and immunocytochemistry. In some cases, PD-1 high TILs express higher levels of PD-1 compared to immune cells from healthy subjects. In some cases, PD-1 high TIL populations express higher levels of PD-1 compared to immune cell populations (e.g., peripheral blood mononuclear cells) from healthy subjects or groups of healthy subjects. PD-1 high cells may be referred to as PD-1 clear cells.

[0569] "PD-1 intermediate (PD-1 intermediate)" or "PD-1 intermediate (PD-1int)" or "PD-1 intermediate (PD-1 int The term "PD-1-positive TILs" refers to intermediate or moderate levels of PD-1 protein expression in cells, including but not limited to tumor-infiltrating lymphocytes or T cells, compared to control cells from a healthy subject. For example, PD-1 intermediate T cells express PD-1 protein at levels or ranges similar to or substantially equivalent to the highest range of PD-1 protein expressed by control cells from a healthy subject (e.g., peripheral blood mononuclear cells). In other words, PD-1 intermediate TILs have PD-1 expression levels similar to or substantially equivalent to the background levels of PD-1 expression in control immune cells from a healthy subject. PD-1 intermediate cells may be referred to as PD-1dim cells. Those skilled in the art will recognize that PD-1-positive TILs may be PD-1-high TILs or PD-1 intermediate TILs.

[0570] "PD-1 negative" or "PD-1 neg" or "PD-1 陰性 (PD-1 neg The term "PD-1-negative lymphocytes" refers to negative or low levels of PD-1 protein expression in cells, including but not limited to tumor-infiltrating lymphocytes or T cells, compared to control cells from a healthy subject. For example, PD-1-negative T cells do not express PD-1 protein. In some cases, PD-1-negative T cells express PD-1 protein at levels similar to or substantially equivalent to the lowest levels expressed by control cells from a healthy subject (e.g., peripheral blood mononuclear cells). PD-1-negative lymphocytes may express PD-1 at the same levels or to the same extent as the majority of lymphocytes in the control population.

[0571] PD-1 high, PD-1 intermediate, and PD-1 negative TILs are distinct and represent different subsets of TILs that have been expanded ex vivo according to the methods described herein. In some embodiments, the ex vivo expanded TIL population includes PD-1 high TILs, PD-1 intermediate TILs, and PD-1 negative TILs.

[0572] II. TIL Manufacturing Process (Gen3 Process Embodiment, optionally including synthetic culture medium) In addition to the methods described herein, International Application No. PCT / US2019 / 059716 is incorporated herein by reference in its entirety for all purposes. Without limiting to any particular theory, it is believed that the methods of the present invention, which involve a first proliferation by priming to prime T cells for activation and a subsequent rapid second proliferation to promote T cell activation, enable the preparation of extended T cells that retain a “younger” phenotype, and therefore, it is expected that the extended T cells of the present invention will exhibit higher cytotoxicity against cancer cells than T cells extended by other methods. In particular, the activation of T cells, which is primed by exposure to an anti-CD3 antibody (e.g., OKT-3), IL-2, and optionally antigen-presenting cells (APCs), as taught by the method of the present invention, and subsequently promoted by additional exposure to anti-CD3 antibody (e.g., OKT-3), IL-2, and APCs, is thought to limit or avoid T cell maturation in culture, producing a population of T cells with a less mature phenotype, which are less exhausted by proliferation in culture and exhibit higher cytotoxicity against cancer cells. In some embodiments, the rapid second proliferation step is divided into several steps: (a) rapidly second proliferation is performed by culturing T cells in a small-scale culture in a first container, e.g., a G-REX 100MCS container, for about 3-4 days; and then (b) the T cells from the small-scale culture are transferred to a larger second container, e.g., a G-REX 500MCS container, and the T cells derived from the small-scale culture are cultured in the second container on a larger scale for about 4-7 days to achieve a scale-up of the culture.In some embodiments, the rapid proliferation step is divided into multiple steps to achieve a scale-out of the culture by (a) rapidly growing T cells in a first small culture for 3-4 days in a first container, e.g., a G-REX 100MCS container, and then (b) transferring and distributing the T cells from the first small culture to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 second containers of the same size as the first container, and in each second container, a portion of the T cells from the first small culture transferred to the second container are cultured in the second small culture for about 4-7 days. In some embodiments, the rapid proliferation step is divided into multiple steps to achieve a rapid second proliferation by (a) culturing T cells in a small-scale culture in a first container, e.g., a G-REX 100MCS container, for about 3-4 days, and then (b) transferring and distributing the T cells derived from the small-scale culture into at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 second containers, e.g., G-REX 500MCS containers, which are larger than the first container, thereby achieving a scale-out and scale-up of the culture, in which a portion of the T cells derived from the small-scale culture transferred to the second container are cultured in a larger-scale culture for about 4-7 days. In some embodiments, the rapid proliferation step is divided into multiple steps: (a) rapidly proliferating the T cells by culturing them in a small-scale culture in a first container, e.g., a G-REX 100MCS container, for about 4 days; and then (b) scaling out and scaling up the culture by transferring and distributing the T cells derived from the small-scale culture into two, three, or four second containers larger than the first container, e.g., G-REX 500MCS containers, in which case a portion of the T cells derived from the small-scale culture transferred to each second container are cultured in a larger-scale culture for about 5 days.

[0573] In some embodiments, rapid second proliferation occurs after the T cell activation brought about by the first proliferation by priming has begun to decrease, be reduced, decline, or subside.

[0574] In some embodiments, rapid second proliferation occurs when the activation of T cells brought about by the first proliferation by priming occurs precisely or approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 , performed after a decrease of 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%.

[0575] In some embodiments, the rapid second proliferation occurs after the T cell activation resulting from the first proliferation by priming has decreased by exactly or by a percentage ranging from approximately 1% to 100%.

[0576] In some embodiments, the rapid second proliferation occurs after the T cell activation resulting from the first proliferation by priming has decreased by exactly or by a percentage in the range of 1%–10%, 10%–20%, 20%–30%, 30%–40%, 40%–50%, 50%–60%, 60%–70%, 70%–80%, 80%–90%, or 90%–100%.

[0577] In some embodiments, rapid second proliferation occurs when the activation of T cells brought about by the first proliferation by priming occurs at least exactly or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, This is done after a decrease of 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%.

[0578] In some embodiments, rapid second proliferation occurs when the activation of T cells brought about by the first proliferation by priming is at most exactly or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, This is done after a decrease of 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%.

[0579] In some embodiments, the reduction in T cell activation resulting from the first proliferation by priming is determined by a decrease in the amount of interferon-gamma released by T cells in response to antigen stimulation.

[0580] In some embodiments, the first proliferation of T cells due to priming occurs over a period of up to exactly 7 or about 8 days.

[0581] In some embodiments, the first proliferation of T cells due to priming occurs over a period of up to exactly 1, 2, 3, 4, 5, 6, 7, or 8 days.

[0582] In some embodiments, the first proliferation of T cells due to priming occurs over a period of 1, 2, 3, 4, 5, 6, 7, or 8 days.

[0583] In some embodiments, the rapid second proliferation of T cells occurs within a period of up to exactly 11 days or approximately 11 days.

[0584] In some embodiments, the rapid second proliferation of T cells occurs over a period of up to exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 days.

[0585] In some embodiments, the rapid second proliferation of T cells occurs during a period of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 days.

[0586] In some embodiments, the first proliferation of T cells due to priming occurs within a period of exactly or about 1 day to exactly or about 7 days, and the rapid second proliferation of T cells occurs within a period of exactly or about 1 day to exactly or about 11 days.

[0587] In some embodiments, the first proliferation of T cells due to priming occurs over a period of up to exactly or about 1, 2, 3, 4, 5, 6, 7, or 8 days, and the rapid second proliferation of T cells occurs over a period of up to exactly or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 days.

[0588] In some embodiments, the first proliferation of T cells due to priming occurs within a period of exactly or about 1 day to exactly or about 8 days, and the rapid second proliferation of T cells occurs within a period of exactly or about 1 day to exactly or about 9 days.

[0589] In some embodiments, the first proliferation of T cells due to priming occurs during a period of 8 days, and the rapid second proliferation of T cells occurs during a period of 9 days.

[0590] In some embodiments, the first proliferation of T cells due to priming occurs within a period of exactly or about 1 day to exactly or about 7 days, and the rapid second proliferation of T cells occurs within a period of exactly or about 1 day to exactly or about 9 days.

[0591] In some embodiments, the first proliferation of T cells due to priming occurs over a 7-day period, and the second rapid proliferation of T cells occurs over a 9-day period.

[0592] In some embodiments, T cells are tumor-infiltrating lymphocytes (TILs).

[0593] In some embodiments, T cells are bone marrow-infiltrating lymphocytes (MILs).

[0594] In some embodiments, T cells are peripheral blood lymphocytes (PBLs).

[0595] In some embodiments, T cells are obtained from a donor who has cancer.

[0596] In some embodiments, the T cells are TILs obtained from tumors excised from patients with cancer.

[0597] In some embodiments, the T cells are MILs obtained from the bone marrow of patients with hematological malignancies.

[0598] In some embodiments, the T cells are PBLs obtained from donor-derived peripheral blood mononuclear cells (PBMCs). In some embodiments, the donor has cancer. In some embodiments, the donor has hematological malignancies.

[0599] In certain embodiments of this disclosure, immunoeffector cells, such as T cells, can be obtained from units of blood collected from a subject using any number of techniques known to those skilled in the art, such as FICOLL isolation. In one preferred embodiment, cells derived from the circulating blood of an individual are obtained by apheresis. Apheresis products typically include lymphocytes, such as T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes, and platelets. In one embodiment, cells collected by apheresis are washed to remove the plasma fraction, and optionally, the cells may be placed in a suitable buffer or medium for subsequent processing steps. In some embodiments, the cells are washed with phosphate-buffered saline (PBS). In alternative embodiments, the washing solution may be calcium-deficient, magnesium-deficient, or many but not all divalent cations. In one embodiment, T cells are isolated from peripheral blood lymphocytes by lysing red blood cells and depleting monocytes, for example, by centrifugation using a PERCOLL gradient or by countercurrent centrifugation.

[0600] In some embodiments, the T cells are PBLs isolated from donor-derived lymphocyte-enriched whole blood or apheresis products. In some embodiments, the donor has cancer. In some embodiments, the donor has cancer. In some embodiments, the cancer is selected from the group consisting of melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, triple-negative breast cancer, cancer, cancer caused by human papillomavirus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), glioblastoma (including GBM), gastrointestinal cancer, kidney cancer, and renal cell carcinoma. In some embodiments, the cancer is selected from the group consisting of melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, cancer caused by human papillomavirus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), glioblastoma (including GBM), gastrointestinal cancer, kidney cancer, and renal cell carcinoma. In some embodiments, the donor has a tumor. In some embodiments, the tumor is a liquid tumor. In some embodiments, the tumor is a solid tumor. In some embodiments, the donor has a hematological malignancy.

[0601] In certain embodiments of this disclosure, immunoeffector cells, such as T cells, can be obtained from units of blood collected from a subject using any number of techniques known to those skilled in the art, such as FICOLL isolation. In one preferred embodiment, cells derived from the circulating blood of an individual are obtained by apheresis. Apheresis products typically include lymphocytes, such as T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes, and platelets. In one embodiment, cells collected by apheresis are washed to remove the plasma fraction, and optionally, the cells may be placed in a suitable buffer or medium for subsequent processing steps. In some embodiments, the cells are washed with phosphate-buffered saline (PBS). In alternative embodiments, the washing solution may be calcium-deficient, magnesium-deficient, or many but not all divalent cations. In one embodiment, T cells are isolated from peripheral blood lymphocytes by lysing red blood cells and depleting monocytes, for example, by centrifugation using a PERCOLL gradient or by countercurrent centrifugation.

[0602] In some embodiments, the T cells are PBLs isolated from donor-derived lymphocyte-enriched whole blood or apheresis products. In some embodiments, the donor has cancer. In some embodiments, the cancer is selected from the group consisting of melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, triple-negative breast cancer, cancer caused by human papillomavirus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), glioblastoma (including GBM), gastrointestinal cancer, kidney cancer, and renal cell carcinoma. In some embodiments, the donor has a tumor. In some embodiments, the tumor is a liquid tumor. In some embodiments, the tumor is a solid tumor. In some embodiments, the donor has a hematological malignancy. In some embodiments, PBLs are isolated from lymphocyte-enriched whole blood or apheresis products by using a positive or negative selection method, i.e., by removing PBLs using a marker(s), e.g., CD3+CD45+, for T cell phenotype, or by removing non-T cell phenotyped cells and leaving PBLs. In other embodiments, PBLs are isolated by gradient centrifugation. Once PBLs have been isolated from the donor tissue, a first proliferation by priming of PBLs is performed according to a first proliferation by priming step of any of the methods described herein, with a suitable number of isolated PBLs (in some embodiments, approximately 1 × 10⁶) under first proliferation by priming culture. 7 This can be initiated by sowing PBL (Project-Based Learning).

[0603] An exemplary TIL process known as Process 3 (also referred to herein as GEN3), which includes some of these features, is shown in Figure 1 (in particular, e.g., Figure 1B), and some of the advantages of this embodiment of the present invention relating to Process 2A are described in Figures 1, 2, 30, and 31 (in particular, e.g., Figure 1B). Two embodiments of Process 3 are shown in Figures 1 and 30 (in particular, e.g., Figure 1B). Process 2A, or Gen2, is also described in U.S. Patent Publication 2018 / 0280436, which is incorporated herein by reference in its entirety. The Gen3 process is also described in USSN 62 / 755,954 (116983-5045-PR), filed November 5, 2018.

[0604] As discussed herein and generally outlined, TILs are collected from patient samples and manipulated to increase their number before being implanted in the patient using a TIL proliferation process described herein and referred to as Gen3. In some embodiments, TILs may be optionally genetically engineered, as discussed below. In some embodiments, TILs may be cryopreserved before or after proliferation. Upon thawing, they can be restimulated to increase their metabolism before injection into the patient.

[0605] In some embodiments, as will be discussed in detail below and in the examples and figures, the first growth by priming (a process referred herein as pre-rapid growth (pre-REP), and including the process shown as step B in Figure 1 (in particular, e.g., Figure 1B and / or Figure 1C and / or Figure 1D and / or Figure 1E and / or Figure 1F and / or Figure 1G and / or Figure 1H)) is shortened to 1 to 8 days, and the rapid second growth (a process referred herein as the rapid growth protocol (REP), and including the process shown as step D in Figure 1 (in particular, e.g., Figure 1B and / or Figure 1C and / or Figure 1D and / or Figure 1E and / or Figure 1F and / or Figure 1G and / or Figure 1H)) is shortened to 1 to 9 days. In some embodiments, as will be discussed in detail below and in the examples and figures, the first growth by priming (a process referred herein as pre-rapid growth (pre-REP), and including the process shown as step B in Figure 1 (in particular, e.g., Figure 1B and / or Figure 1C and / or Figure 1D and / or Figure 1E and / or Figure 1F and / or Figure 1G and / or Figure 1H)) is shortened to 1 to 8 days, and the rapid second growth (a process referred herein as the rapid growth protocol (REP), and including the process shown as step D in Figure 1 (in particular, e.g., Figure 1B and / or Figure 1C and / or Figure 1D and / or Figure 1E and / or Figure 1F and / or Figure 1G and / or Figure 1H)) is shortened to 1 to 8 days. In some embodiments, as will be discussed in detail below and in the examples and figures, the first growth by priming (a process referred herein as pre-rapid growth (pre-REP), and including the process shown as step B in Figure 1 (in particular, e.g., Figure 1B and / or Figure 1C and / or Figure 1D and / or Figure 1E and / or Figure 1F and / or Figure 1G and / or Figure 1H)) is shortened to 1 to 7 days, and the rapid second growth (a process referred herein as the rapid growth protocol (REP), and including the process shown as step D in Figure 1 (in particular, e.g., Figure 1B and / or Figure 1C and / or Figure 1D and / or Figure 1E and / or Figure 1F and / or Figure 1G and / or Figure 1H)) is shortened to 1 to 9 days.In some embodiments, as will be discussed in detail below and in the examples and figures, the first growth by priming (a process referred herein as pre-rapid growth (pre-REP), and including the process shown as step B in Figure 1 (in particular, e.g., Figure 1B and / or Figure 1C and / or Figure 1D and / or Figure 1E and / or Figure 1F and / or Figure 1G and / or Figure 1H)) is 1 to 7 days, and the rapid second growth (a process referred herein as the rapid growth protocol (REP), and including the process shown as step D in Figure 1 (in particular, e.g., Figure 1B and / or Figure 1C and / or Figure 1D and / or Figure 1E and / or Figure 1F and / or Figure 1G and / or Figure 1H)) is 1 to 10 days. In some embodiments, the first growth by priming (e.g., the growth described as step B in Figure 1 (e.g., Figures 1B and / or 1C and / or 1D and / or 1E and / or 1F and / or 1G and / or 1H)) is shortened to 8 days, and the rapid second growth (e.g., the growth described as step D in Figure 1 (e.g., Figures 1B and / or 1C and / or 1D and / or 1E and / or 1F and / or 1G and / or 1H)) is 7 to 9 days. In some embodiments, the first growth by priming (e.g., the growth described as step B in Figure 1 (e.g., Figure 1B and / or Figure 1C and / or Figure 1D and / or Figure 1E and / or Figure 1F and / or Figure 1G and / or Figure 1H)) is 8 days, and the rapid second growth (e.g., the growth described as step D in Figure 1 (e.g., Figure 1B and / or Figure 1C and / or Figure 1D and / or Figure 1E and / or Figure 1F and / or Figure 1G and / or Figure 1H)) is 8 to 9 days.In some embodiments, the first growth by priming (e.g., the growth described as step B in Figure 1 (especially, e.g., Figures 1B and / or 1C and / or 1D and / or 1E and / or 1F and / or 1G and / or 1H)) is shortened to 7 days, and the rapid second growth (e.g., the growth described as step D in Figure 1 (especially, e.g., Figures 1B and / or 1C and / or 1D and / or 1E and / or 1F and / or 1G and / or 1H)) is 7 to 8 days. In some embodiments, the first growth by priming (e.g., the growth described as step B in Figure 1 (e.g., Figure 1B and / or Figure 1C and / or Figure 1D and / or Figure 1E and / or Figure 1F and / or Figure 1G and / or Figure 1H)) is shortened to 8 days, and the rapid second growth (e.g., the growth described as step D in Figure 1 (e.g., Figure 1B and / or Figure 1C and / or Figure 1D and / or Figure 1E and / or Figure 1F and / or Figure 1G and / or Figure 1H)) is 8 days. In some embodiments, the first growth by priming (e.g., the growth described as step B in Figure 1 (e.g., Figures 1B and / or 1C and / or 1D and / or 1E and / or 1F and / or 1G and / or 1H)) is 8 days, and the rapid second growth (e.g., the growth described as step D in Figure 1 (e.g., Figures 1B and / or 1C and / or 1D and / or 1E and / or 1F and / or 1G and / or 1H)) is 9 days. In some embodiments, the first growth by priming (e.g., the growth described as step B in Figure 1 (e.g., Figure 1B and / or Figure 1C and / or Figure 1D and / or Figure 1E and / or Figure 1F and / or Figure 1G and / or Figure 1H)) is 8 days, and the rapid second growth (e.g., the growth described as step D in Figure 1 (e.g., Figure 1B and / or Figure 1C and / or Figure 1D and / or Figure 1E and / or Figure 1F and / or Figure 1G and / or Figure 1H)) is 10 days.In some embodiments, the first growth by priming (e.g., the growth described as step B in Figure 1 (e.g., Figure 1B and / or Figure 1C and / or Figure 1D and / or Figure 1E and / or Figure 1F and / or Figure 1G and / or Figure 1H)) is 7 days, and the rapid second growth (e.g., the growth described as step D in Figure 1 (e.g., Figure 1B and / or Figure 1C and / or Figure 1D and / or Figure 1E and / or Figure 1F and / or Figure 1G and / or Figure 1H)) is 7 to 10 days. In some embodiments, the first growth by priming (e.g., the growth described as step B in Figure 1 (e.g., Figure 1B and / or Figure 1C and / or Figure 1D and / or Figure 1E and / or Figure 1F and / or Figure 1G and / or Figure 1H)) is 7 days, and the rapid second growth (e.g., the growth described as step D in Figure 1 (e.g., Figure 1B and / or Figure 1C and / or Figure 1D and / or Figure 1E and / or Figure 1F and / or Figure 1G and / or Figure 1H)) is 8 to 10 days. In some embodiments, the first growth by priming (e.g., the growth described as step B in Figure 1 (e.g., Figure 1B and / or Figure 1C and / or Figure 1D and / or Figure 1E and / or Figure 1F and / or Figure 1G and / or Figure 1H)) is 7 days, and the rapid second growth (e.g., the growth described as step D in Figure 1 (e.g., Figure 1B and / or Figure 1C and / or Figure 1D and / or Figure 1E and / or Figure 1F and / or Figure 1G and / or Figure 1H)) is 9 to 10 days. In some embodiments, the first growth by priming (e.g., the growth described as step B in Figure 1 (e.g., Figure 1B and / or Figure 1C and / or Figure 1D and / or Figure 1E and / or Figure 1F and / or Figure 1G and / or Figure 1H)) is shortened to 7 days, and the rapid second growth (e.g., the growth described as step D in Figure 1 (e.g., Figure 1B and / or Figure 1C and / or Figure 1D and / or Figure 1E and / or Figure 1F and / or Figure 1G and / or Figure 1H)) is 7 to 9 days.In some embodiments, as will be discussed in detail below and in the examples and figures, the combination of a first growth by priming and a rapid second growth (e.g., the growth described as steps B and D in Figure 1 (especially, for example, Figures 1B and / or 1C and / or 1D and / or 1E and / or 1F and / or 1G and / or 1H)) is 14 to 16 days. In particular, certain embodiments of the present invention are thought to include a first growth by priming step in which TILs are activated by exposure to an anti-CD3 antibody, e.g., OKT-3, in the presence of IL-2, or by exposure to an antigen in the presence of at least IL-2 and an anti-CD3 antibody, e.g., OKT-3. In certain embodiments, the TILs activated in the first growth by priming step as described above are a first TIL population, i.e., a primary cell population.

[0606] The following “Step” notations A, B, C, etc. refer to non-limiting examples in Figure 1 (especially, for example, Figure 1B and / or Figure 1C and / or Figure 1D and / or Figure 1E and / or Figure 1F and / or Figure 1G and / or Figure 1H) and refer to certain non-limiting embodiments described herein. The order of steps below and in Figure 1 (especially, for example, Figure 1B and / or Figure 1C and / or Figure 1D and / or Figure 1E and / or Figure 1F and / or Figure 1G and / or Figure 1H) are illustrative, and any combination or order of steps, as well as additional steps, repetition of steps, and / or omission of steps, are contemplated by the methods disclosed herein and herein.

[0607] A. Step A: Obtain a tumor sample from the patient. Generally, TILs are initially obtained from a patient's tumor sample ("primary TIL") or from circulating lymphocytes such as peripheral blood lymphocytes containing TIL-like features, and then expanded to a larger population for further processing as described herein, optionally cryopreserved, and optionally evaluated for phenotypic and metabolic parameters as indicators of TIL health.

[0608] Patient tumor samples can generally be obtained using methods known in the art, such as surgical excision, needle biopsy, or other means for obtaining a sample containing a mixture of tumor cells and TIL cells. Generally, tumor samples may originate from any solid tumor, including primary tumors, invasive tumors, or metastatic tumors. Tumor samples may also be liquid tumors, such as tumors obtained from hematological malignancies. Solid tumors can be any type of cancer, including but not limited to breast cancer, pancreatic cancer, prostate cancer, colorectal cancer, lung cancer, brain cancer, kidney cancer, gastric cancer, and skin cancer (including but not limited to squamous cell carcinoma, basal cell carcinoma, and melanoma). In some embodiments, cancer is selected from cervical cancer, head and neck cancer (including, for example, head and neck squamous cell carcinoma (HNSCC)), glioblastoma (GBM), gastrointestinal cancer, ovarian cancer, sarcoma, pancreatic cancer, bladder cancer, breast cancer, triple-negative breast cancer, and non-small cell lung cancer. In some embodiments, malignant melanoma tumors have been reported to have particularly high levels of TILs; therefore, useful TILs are obtained from malignant melanoma tumors.

[0609] At the time of acquisition, tumor specimens are generally 1 to approximately 8 mm in size, using sharp dissection. 3 It is fragmented into small pieces, about 2-3 mm in size. 3This is particularly useful. TILs are cultured from these fragments using enzymatic tumor digests. Such tumor digests can be produced by incubation in an enzymatic medium (e.g., Roswell Park Memorial Institute (RPMI) 1640 buffer, 2 mM glutamate, 10 mcg / mL gentamicin, 30 units / mL DNase, and 1.0 mg / mL collagenase), followed by mechanical dissociation (e.g., using a tissue dissociator). Tumor digests can be produced by placing the tumor in an enzymatic medium, mechanically dissociating the tumor for approximately 1 minute, then incubating at 37°C in 5% CO2 for 30 minutes, followed by repeating the mechanical dissociation and incubation cycle under the aforementioned conditions until only small tissue fragments remain. At the end of this process, if the cell suspension contains a large number of erythrocytes or dead cells, these cells can be removed by density gradient separation using FICOLL branched hydrophilic polysaccharides. Alternative methods known in the art, such as those described in U.S. Patent Application Publication No. 2012 / 0244133A1 (the disclosure thereof is incorporated herein by reference), may be used. Any of the aforementioned methods may be used in any of the embodiments described herein for methods of expanding TIL or for treating cancer.

[0610] Tumor dissociation enzyme mixtures may include one or more dissociation (digestion) enzymes, such as, but not limited to, collagenase (including any blend or type of collagenase), Accutase®, Accumax®, hyaluronidase, neutral protease (dispase), chymotrypsin, chymopapain, trypsin, caseinase, elastase, papain, type XIV protease (pronase), deoxyribonuclease I (DNase), trypsin inhibitors, any other dissociation enzyme or proteolytic enzyme, and any combination thereof.

[0611] In some embodiments, the dissociated enzyme is reconstituted from a lyophilized enzyme. In some embodiments, the lyophilized enzyme is reconstituted in an amount of sterile buffer such as HBSS.

[0612] In some cases, collagenase (such as animal-free type 1 collagenase) is reconstituted in 10 ml of sterile HBSS or another buffer. The lyophilized stock enzyme may be at a concentration of 2892 PZ U / vial. In some embodiments, the collagenase is reconstituted in 5 ml to 15 ml of buffer. In some embodiments, the reconstituted collagenase stock is in the range of approximately 100 PZ U / ml to approximately 400 PZ U / ml, for example, approximately 100 PZ U / ml to approximately 400 PZ U / ml, approximately 100 PZ U / ml to approximately 350 PZ U / ml, approximately 100 PZ U / ml to approximately 300 PZ U / ml, approximately 150 PZ U / ml to approximately 400 PZ U / ml, approximately 100 PZ U / ml, approximately 150 PZ U / ml, approximately 200 PZ U / ml, approximately 210 PZ U / ml, approximately 220 PZ U / ml, approximately 230 PZ U / ml, approximately 240 PZ U / ml, approximately 250 PZ U / ml, approximately 260 PZ U / ml, approximately 270 PZ U / ml, approximately 280 PZ U / ml, approximately 289.2 PZ U / ml, and approximately 300 PZ U / ml. This is U / ml, approximately 350 PZ U / ml, or approximately 400 PZ U / ml.

[0613] In some embodiments, the neutral protease is reconstituted in 1 ml of sterile HBSS or another buffer. The lyophilized stock enzyme may be at a concentration of 175 DMCU / vial. In some embodiments, the reconstituted neutral protease stock may be in the range of about 100 DMC / ml to about 400 DMC / ml, for example, about 100 DMC / ml to about 400 DMC / ml, about 100 DMC / ml to about 350 DMC / ml, about 100 DMC / ml to about 300 DMC / ml, about 150 DMC / ml to about 400 DMC / ml, about 100 DMC / ml, about 110 DM These are approximately C / ml, 120 DMC / ml, 130 DMC / ml, 140 DMC / ml, 150 DMC / ml, 160 DMC / ml, 170 DMC / ml, 175 DMC / ml, 180 DMC / ml, 190 DMC / ml, 200 DMC / ml, 250 DMC / ml, 300 DMC / ml, 350 DMC / ml, or 400 DMC / ml.

[0614] In some embodiments, DNase I is reconstituted in 1 ml of sterile HBSS or another buffer. The lyophilized stock enzyme was at a concentration of 4 KU / vial. In some embodiments, the reconstituted DNase I stock is in the range of approximately 1 KU / ml to 10 KU / ml, for example, approximately 1 KU / ml, approximately 2 KU / ml, approximately 3 KU / ml, approximately 4 KU / ml, approximately 5 KU / ml, approximately 6 KU / ml, approximately 7 KU / ml, approximately 8 KU / ml, approximately 9 KU / ml, or approximately 10 KU / ml.

[0615] In some embodiments, the enzyme stock may vary, so it should be noted that the concentration of the freeze-dried stock should be checked and the final amount of enzyme added to the digestion cocktail adjusted accordingly.

[0616] In some embodiments, the enzyme mixture comprises a neutral protease, a DNase, and a collagenase.

[0617] In some embodiments, the enzyme mixture contains approximately 10.2 μl of neutral protease (0.36 DMCU / ml), 21.3 μl of collagenase (1.2 PZ / ml), and 250 μl of DNAseI (200 U / ml) in approximately 4.7 ml of sterile HBSS.

[0618] As shown above, in some embodiments, the TIL is derived from a solid tumor. In some embodiments, the solid tumor is not fragmented. In some embodiments, the solid tumor is not fragmented and is subjected to enzymatic digestion as a whole tumor. In some embodiments, the tumor is digested in an enzyme mixture containing collagenase, DNase, and hyaluronidase. In some embodiments, the tumor is digested in an enzyme mixture containing collagenase, DNase, and hyaluronidase for 1-2 hours. In some embodiments, the tumor is digested in an enzyme mixture containing collagenase, DNase, and hyaluronidase for 1-2 hours at 37°C and 5% CO2 with rotation. In some embodiments, the tumor is digested in an enzyme mixture containing collagenase, DNase, and hyaluronidase for 1-2 hours at 37°C and 5% CO2 with rotation. In some embodiments, the tumor is digested overnight with constant rotation. In some embodiments, the tumor is digested overnight at 37°C and 5% CO2 with constant rotation. In some embodiments, the entire tumor is combined with an enzyme to form a tumor digestion reaction mixture. In some embodiments, the tumor is digested and then frozen before continuing the selection process. In some embodiments, the tumor is digested and then frozen before continuing the growth process.

[0619] In some embodiments, the tumor is reconstituted with lyophilized enzymes in a sterile buffer. In some embodiments, the buffer is sterile HBSS.

[0620] In some embodiments, the enzyme mixture contains collagenase. In some embodiments, the collagenase is collagenase IV. In some embodiments, the working stock of collagenase is a 10x working stock of 100 mg / ml.

[0621] In some embodiments, the enzyme mixture contains DNAse. In some embodiments, the working stock of DNAse is a 10x working stock of 10,000 IU / ml.

[0622] In some embodiments, the enzyme mixture contains hyaluronidase. In some embodiments, the working stock of hyaluronidase is a 10x working stock of 10 mg / ml.

[0623] In some embodiments, the enzyme mixture comprises 10 mg / ml collagenase, 1000 IU / ml DNAse, and 1 mg / mL hyaluronidase.

[0624] In some embodiments, the enzyme mixture comprises 10 mg / ml collagenase, 500 IU / ml DNAse, and 1 mg / ml hyaluronidase.

[0625] In some embodiments, the enzyme mixture contains about 10 mg / ml of collagenase, about 1000 IU / ml of DNAse, and about 1 mg / ml of hyaluronidase.

[0626] Generally, cell suspensions obtained from tumors are referred to as “primary cell populations” or “newly obtained” or “newly isolated” cell populations. In certain embodiments, newly obtained TIL cell populations are exposed to a cell culture medium containing antigen-presenting cells, IL-12, and OKT-3.

[0627] In some embodiments, the digest can be frozen before proceeding with the first growth by priming, for example, step B of Figure 1 (in particular, for example, Figures 1B and / or 1C and / or 1D and / or 1E and / or 1F and / or 1G and / or 1H).

[0628] In some embodiments, fragmentation includes physical fragmentation, such as dissection and digestion. In some embodiments, fragmentation is physical fragmentation. In some embodiments, fragmentation is dissection. In some embodiments, fragmentation is by digestion. In some embodiments, TILs may be cultured from enzyme-induced tumor digests and tumor fragments obtained from a patient first. In some embodiments, TILs may be cultured from enzyme-induced tumor digests and tumor fragments obtained from a patient first.

[0629] In some embodiments where the tumor is a solid tumor, the tumor sample is obtained, for example, in step A (provided in Figure 1 (in particular, for example, Figures 1B and / or 1C and / or 1D and / or 1E and / or 1F and / or 1G and / or 1H)), after which the tumor undergoes physical fragmentation. In some embodiments, fragmentation occurs before cryopreservation. In some embodiments, fragmentation occurs after cryopreservation. In some embodiments, fragmentation occurs after obtaining the tumor if there is no cryopreservation. In some embodiments, the fragmentation step is an in vitro or ex vivo process. In some embodiments, the tumor is fragmented, and 10, 20, 30, 40, or more fragments or pieces are placed in each container for a first growth by priming. In some embodiments, the tumor is fragmented, and 30 or 40 fragments or pieces are placed in each container for a first growth by priming. In some embodiments, the tumor is fragmented, and 40 fragments or pieces are placed in each container for a first growth by priming. In some embodiments, the fragments consist of approximately 4 to 50 fragments, each fragment measuring approximately 27 mm. 3 It has a volume of approximately 1300 mm. In some embodiments, the multiple pieces are approximately 1300 mm 3 ~about 1500mm 3 It includes approximately 30 to 60 fragments with a total volume of approximately 1350 mm². In some embodiments, the fragments comprise approximately 1350 mm². 3It contains about 50 fragments with a total volume. In some embodiments, the multiple fragments consist of about 50 fragments with a total mass of about 1 gram to about 1.5 grams. In some embodiments, the multiple fragments consist of about 4 fragments.

[0630] In some embodiments, TILs are obtained from tumor fragments. In some embodiments, tumor fragments are obtained by sharp dissection. In some embodiments, tumor fragments are approximately 1 mm in size. 3 ~10mm 3 In some embodiments, the tumor fragment is approximately 1 mm 3 ~8mm 3 In some embodiments, the tumor fragment is approximately 1 mm 3 In some embodiments, the tumor fragment is approximately 2 mm in size. 3 In some embodiments, the tumor fragment is approximately 3 mm in size. 3 In some embodiments, the tumor fragment is approximately 4 mm 3 In some embodiments, the tumor fragment is approximately 5 mm in size. 3 In some embodiments, the tumor fragment is approximately 6 mm. 3 In some embodiments, the tumor fragment is approximately 7 mm 3 In some embodiments, the tumor fragment is approximately 8 mm in size. 3 In some embodiments, the tumor fragment is approximately 9 mm 3 In some embodiments, the tumor fragment is approximately 10 mm. 3 In some embodiments, the tumor fragment is 1-4 mm × 1-4 mm × 1-4 mm. In some embodiments, the tumor fragment is 1 mm × 1 mm × 1 mm. In some embodiments, the tumor fragment is 2 mm × 2 mm × 2 mm. In some embodiments, the tumor fragment is 3 mm × 3 mm × 3 mm. In some embodiments, the tumor fragment is 4 mm × 4 mm × 4 mm.

[0631] In some embodiments, the tumor is fragmented to minimize the amount of hemorrhagic, necrotic, and / or fatty tissue on each fragment. In some embodiments, the tumor is fragmented to minimize the amount of hemorrhagic tissue on each fragment. In some embodiments, the tumor is fragmented to minimize the amount of necrotic tissue on each fragment. In some embodiments, the tumor is fragmented to minimize the amount of fatty tissue on each fragment. In certain embodiments, the tumor fragmentation step is performed by an in vitro or ex vivo method.

[0632] In some embodiments, tumor fragmentation is performed while preserving the internal structure of the tumor. In some embodiments, tumor fragmentation is performed without performing a scalpel cutting action. In some embodiments, TIL is obtained from tumor digestate. In some embodiments, tumor digestate is produced by incubation in an enzyme medium, for example, RPMI 1640 (2 mM GlutaMAX, 10 mg / mL gentamicin, 30 U / mL DNase, and 1.0 mg / mL collagenase), followed by mechanical dissociation (GentleMACS, Miltenyi Biotec, Auburn, CA). After the tumor is placed in the enzyme medium, the tumor may be mechanically dissociated for approximately 1 minute. The solution may then be incubated at 37°C in 5% CO2 for 30 minutes, and then mechanically dissociated again for approximately 1 minute. After incubation again at 37°C in 5% CO2 for 30 minutes, the tumor may undergo a third mechanical dissociation for approximately 1 minute. In some embodiments, if large tissue fragments were present, one or two additional mechanical dissociations were applied to the sample after the third mechanical disruption, or after further incubation at 37°C in 5% CO2 for another 30 minutes. In some embodiments, if the cell suspension contained a large number of erythrocytes or dead cells, density gradient separation using Ficoll could be performed at the end of the final incubation to remove these cells.

[0633] In some embodiments, the cell suspension prior to the first proliferation step by priming is referred to as the “primary cell population” or the “newly acquired” or “newly isolated” cell population. In some embodiments, cells may be optionally frozen after sample isolation (e.g., after obtaining a tumor sample and / or a cell suspension from the tumor sample) and cryopreserved before proceeding to the proliferation described in step B, which is described in more detail below and is also illustrated in Figure 1 (in particular, for example, Figures 1B and / or 1C and / or 1D and / or 1E and / or 1F and / or 1G and / or 1H).

[0634] 1. TILs originating from core / small biopsy In some embodiments, TILs are first obtained from patient tumor samples ("primary TILs") acquired by core biopsy or a similar procedure, then expanded to a larger population for further processing as described herein, optionally cryopreserved, and optionally evaluated for phenotypic and metabolic parameters.

[0635] In some embodiments, a patient's tumor sample can be obtained using methods known in the art, generally by small biopsy, core biopsy, needle biopsy, or other means for obtaining a sample containing a mixture of tumor cells and TIL cells. Generally, a tumor sample may originate from any solid tumor, including primary tumors, invasive tumors, or metastatic tumors. A tumor sample may be a liquid tumor, such as a tumor obtained from a hematological malignancy. In some embodiments, a sample may originate from multiple small tumor samples or biopsies. In some embodiments, a sample may include multiple tumor samples from a single tumor from the same patient. In some embodiments, a sample may include multiple tumor samples from one, two, three, or four tumors from the same patient. In some embodiments, a sample may include multiple tumor samples from multiple tumors from the same patient. Solid tumors may include, but are not limited to, breast cancer, pancreatic cancer, prostate cancer, colorectal cancer, lung cancer, brain cancer, kidney cancer, gastric cancer, and skin cancer (including, but not limited to, squamous cell carcinoma, basal cell carcinoma, and melanoma). In some embodiments, the cancer is selected from cervical cancer, head and neck cancer (including, for example, head and neck squamous cell carcinoma (HNSCC)), glioblastoma (GBM), gastrointestinal cancer, ovarian cancer, sarcoma, pancreatic cancer, bladder cancer, breast cancer, triple-negative breast cancer, and non-small cell lung cancer (NSCLC). In some embodiments, useful TILs are obtained from malignant melanoma tumors, as malignant melanoma tumors have been reported to have particularly high levels of TILs.

[0636] Generally, cell suspensions obtained from tumor cores or fragments are referred to as “primary cell populations” or “newly acquired” or “newly isolated” cell populations. In certain embodiments, the newly acquired TIL cell population is exposed to a cell culture medium containing antigen-presenting cells, IL-2, and OKT-3.

[0637] In some embodiments, if the tumor is metastatic and the primary lesion has been successfully treated / removed in the past, removal of one of the metastatic lesions may be required. In some embodiments, the least invasive approach is to remove a skin lesion or, if available, lymph nodes in the neck or axilla region. In some embodiments, a skin lesion is removed or a biopsy thereof is removed. In some embodiments, a lymph node or a biopsy thereof is removed. In some embodiments, a lung or liver metastatic lesion, or intraperitoneal or thoracic lymph nodes or biopsies thereof may be used.

[0638] In some embodiments, the tumor is a melanoma. In some embodiments, a small biopsy of the melanoma includes a mole or a portion thereof.

[0639] In some embodiments, the small biopsy is a punch biopsy. In some embodiments, the punch biopsy is taken with a circular blade pressed into the skin. In some embodiments, the punch biopsy is taken with a circular blade pressed into the skin around a suspected mole. In some embodiments, the punch biopsy is taken with a circular blade pressed into the skin, and a round piece of skin is collected. In some embodiments, the small biopsy is a punch biopsy, and a round portion of the tumor is collected.

[0640] In some embodiments, the biopsy is an excisional biopsy. In some embodiments, the biopsy is an excisional biopsy in which the entire mole or growth is removed. In some embodiments, the biopsy is an excisional biopsy in which the entire mole or growth is removed along with a small border of normal-looking skin.

[0641] In some embodiments, the small biopsy is an incisional biopsy. In some embodiments, the small biopsy is an incisional biopsy in which only the most irregular portion of the mole or proliferation is taken. In some embodiments, the small biopsy is an incisional biopsy, which is used when other techniques cannot be achieved, such as when the suspected mole is very large.

[0642] In some embodiments, the small biopsy is a lung biopsy. In some embodiments, the small biopsy is obtained by bronchoscopy. Generally, in bronchoscopy, the patient is anesthetized, and a small instrument is inserted through the nose or mouth, down the throat, into the bronchial passages, where the small instrument is used to collect some tissue. In some embodiments where the tumor or growth cannot be reached by bronchoscopy, transthoracic needle biopsy may be used. Generally, in the case of transthoracic needle biopsy, the patient is also anesthetized, and a needle is inserted directly through the skin to the suspected site to collect a small tissue sample. In some embodiments, transthoracic needle biopsy may require interventional radiology (e.g., the use of X-ray or CT scanning to guide the needle). In some embodiments, the small biopsy is obtained by needle biopsy. In some embodiments, the small biopsy is obtained by endoscopic ultrasound (e.g., an endoscope with illumination, inserted through the mouth into the esophagus). In some embodiments, the small biopsy is obtained surgically.

[0643] In some embodiments, the biopsy is a head and neck biopsy. In some embodiments, the biopsy is an incisional biopsy. In some embodiments, the biopsy is an incisional biopsy in which a small piece of tissue is excised from the area that appears abnormal. In some embodiments, if the abnormal area is easily accessible, the sample may be taken without hospitalization. In some embodiments, if the tumor is located deeper in the mouth or throat, the biopsy may need to be performed in an operating room under general anesthesia. In some embodiments, the biopsy is an excisional biopsy. In some embodiments, the biopsy is an excisional biopsy in which the entire area is removed. In some embodiments, the biopsy is a fine-needle aspiration (FNA). In some embodiments, the biopsy is a fine-needle aspiration (FNA) in which cells are extracted (aspirated) from the tumor or mass using a very fine needle attached to a syringe. In some embodiments, the biopsy is a punch biopsy. In some embodiments, the biopsy is a punch biopsy in which a piece of the suspicious area is taken using punch forceps.

[0644] In some embodiments, the small biopsy is a cervical biopsy. In some embodiments, the small biopsy is obtained by speculum examination. Generally, speculum examination employs the use of an illuminated magnifying instrument (vaginal speculum) attached to magnifying binoculars, which is then used to biopsy a small section of the surface of the cervix. In some embodiments, the small biopsy is a cone biopsy. In some embodiments, the small biopsy is a cone biopsy, which may require outpatient surgery to obtain a larger tissue sample from the cervix. In some embodiments, a cone biopsy can be an initial treatment, in addition to helping confirm the diagnosis.

[0645] The term “solid tumor” typically refers to an abnormal mass of tissue that does not contain cysts or fluid areas. Solid tumors can be benign or malignant. The term “solid tumor carcinoma” refers to a malignant, neoplastic, or cancerous solid tumor. Solid tumor carcinomas include, but are not limited to, sarcomas, carcinomas, and lymphomas, such as lung cancer, breast cancer, triple-negative breast cancer, prostate cancer, colon cancer, rectal cancer, and bladder cancer. In some embodiments, cancers are selected from cervical cancer, head and neck cancer, glioblastoma, ovarian cancer, sarcoma, pancreatic cancer, bladder cancer, breast cancer, triple-negative breast cancer, and non-small cell lung cancer. The tissue structure of a solid tumor includes interdependent tissue compartments containing parenchyma (cancer cells) and supporting stromal cells that can provide a supporting microenvironment in which the cancer cells are dispersed.

[0646] In some embodiments, tumor-derived specimens are obtained as fine-needle aspiration (FNA), core biopsies, or microbiops (including, for example, punch biopsies). In some embodiments, the specimen is first placed in the G-Rex 10. In some embodiments, if there are one or two core biopsy and / or microbiopsy specimens, the specimen is first placed in the G-Rex 10. In some embodiments, if there are three, four, five, six, eight, nine, or more than ten core biopsy and / or microbiopsy specimens, the specimen is first placed in the G-Rex 100. In some embodiments, if there are three, four, five, six, eight, nine, or more than ten core biopsy and / or microbiopsy specimens, the specimen is first placed in the G-Rex 500.

[0647] FNA can be obtained from tumors selected from the group consisting of lung, melanoma, head and neck, cervix, ovary, pancreas, glioblastoma, colorectal, and sarcoma. In some embodiments, FNA is obtained from lung tumors, such as lung tumors from patients with non-small cell lung cancer (NSCLC). In some cases, patients with NSCLC have previously undergone surgical treatment.

[0648] The TILs described herein may be obtained from FNA specimens. In some cases, FNA specimens are obtained or isolated from patients using fine-gauge needles ranging from 18-gauge to 25-gauge. Fine-gauge needles may be 18-gauge, 19-gauge, 20-gauge, 21-gauge, 22-gauge, 23-gauge, 24-gauge, or 25-gauge. In some embodiments, a patient-derived FNA sample may contain at least 400,000 TILs, for example, 400,000 TILs, 450,000 TILs, 500,000 TILs, 550,000 TILs, 600,000 TILs, 650,000 TILs, 700,000 TILs, 750,000 TILs, 800,000 TILs, 850,000 TILs, 900,000 TILs, 950,000 TILs, or more.

[0649] In some cases, the TILs described herein are obtained from core biopsy specimens. In some cases, core biopsy specimens are obtained from or isolated from a patient using surgical or medical needles ranging from 11-gauge to 16-gauge. The needles may be 11-gauge, 12-gauge, 13-gauge, 14-gauge, 15-gauge, or 16-gauge. In some embodiments, the patient-derived core biopsy sample may contain at least 400,000 TILs, for example, 400,000 TILs, 450,000 TILs, 500,000 TILs, 550,000 TILs, 600,000 TILs, 650,000 TILs, 700,000 TILs, 750,000 TILs, 800,000 TILs, 850,000 TILs, 900,000 TILs, 950,000 TILs, or more.

[0650] Generally, a collected cell suspension is called a "primary cell population" or a "freshly collected" cell population.

[0651] In some embodiments, TIL is not obtained from tumor digests. In some embodiments, the solid tumor core is not fragmented.

[0652] In some embodiments, TIL is obtained from tumor digestate. In some embodiments, tumor digestate is produced by incubation in an enzyme medium, such as RPMI 1640 (2 mM GlutaMAX, 10 mg / mL gentamicin, 30 U / mL DNase, and 1.0 mg / mL collagenase), followed by mechanical dissociation (GentleMACS, Miltenyi Biotec, Auburn, CA). After the tumor is placed in the enzyme medium, it can be mechanically dissociated for approximately 1 minute. The solution can then be incubated at 37°C in 5% CO2 for 30 minutes, and then mechanically dissociated again for approximately 1 minute. After incubation again at 37°C in 5% CO2 for 30 minutes, the tumor can undergo a third mechanical dissociation for approximately 1 minute. In some embodiments, if large tissue fragments are present, one or two additional mechanical dissociations were applied to the sample after the third mechanical dissociation, with or without further incubation at 37°C in 5% CO2 for 30 minutes. In some embodiments, if the cell suspension contains a large number of red blood cells or dead cells, density gradient separation using Ficoll can be performed at the end of the final incubation to remove these cells.

[0653] In some embodiments, obtaining a first TIL population involves a multi-lesion sampling method.

[0654] Tumor dissociation enzyme mixtures may include one or more dissociation (digestion) enzymes, such as, but not limited to, collagenase (including any blend or type of collagenase), Accutase®, Accumax®, hyaluronidase, neutral protease (dispase), chymotrypsin, chymopapain, trypsin, caseinase, elastase, papain, type XIV protease (pronase), deoxyribonuclease I (DNase), trypsin inhibitors, any other dissociation enzyme or proteolytic enzyme, and any combination thereof.

[0655] In some embodiments, the dissociated enzyme is reconstituted from a lyophilized enzyme. In some embodiments, the lyophilized enzyme is reconstituted in an amount of sterile buffer such as HBSS.

[0656] In some cases, collagenase (such as animal-free type 1 collagenase) is reconstituted in 10 ml of sterile HBSS or another buffer. The lyophilized stock enzyme may be at a concentration of 2892 PZ U / vial. In some embodiments, the collagenase is reconstituted in 5 ml to 15 ml of buffer. In some embodiments, the reconstituted collagenase stock is in the range of approximately 100 PZ U / ml to approximately 400 PZ U / ml, for example, approximately 100 PZ U / ml to approximately 400 PZ U / ml, approximately 100 PZ U / ml to approximately 350 PZ U / ml, approximately 100 PZ U / ml to approximately 300 PZ U / ml, approximately 150 PZ U / ml to approximately 400 PZ U / ml, approximately 100 PZ U / ml, approximately 150 PZ U / ml, approximately 200 PZ U / ml, approximately 210 PZ U / ml, approximately 220 PZ U / ml, approximately 230 PZ U / ml, approximately 240 PZ U / ml, approximately 250 PZ U / ml, approximately 260 PZ U / ml, approximately 270 PZ U / ml, approximately 280 PZ U / ml, approximately 289.2 PZ U / ml, and approximately 300 PZ U / ml. This is U / ml, approximately 350 PZ U / ml, or approximately 400 PZ U / ml.

[0657] In some embodiments, the neutral protease is reconstituted in 1 ml of sterile HBSS or another buffer. The lyophilized stock enzyme may be at a concentration of 175 DMCU / vial. In some embodiments, the reconstituted neutral protease stock may be in the range of about 100 DMC / ml to about 400 DMC / ml, for example, about 100 DMC / ml to about 400 DMC / ml, about 100 DMC / ml to about 350 DMC / ml, about 100 DMC / ml to about 300 DMC / ml, about 150 DMC / ml to about 400 DMC / ml, about 100 DMC / ml, about 110 DM These are approximately C / ml, 120 DMC / ml, 130 DMC / ml, 140 DMC / ml, 150 DMC / ml, 160 DMC / ml, 170 DMC / ml, 175 DMC / ml, 180 DMC / ml, 190 DMC / ml, 200 DMC / ml, 250 DMC / ml, 300 DMC / ml, 350 DMC / ml, or 400 DMC / ml.

[0658] In some embodiments, DNase I is reconstituted in 1 ml of sterile HBSS or another buffer. The lyophilized stock enzyme was at a concentration of 4 KU / vial. In some embodiments, the reconstituted DNase I stock is in the range of approximately 1 KU / ml to 10 KU / ml, for example, approximately 1 KU / ml, approximately 2 KU / ml, approximately 3 KU / ml, approximately 4 KU / ml, approximately 5 KU / ml, approximately 6 KU / ml, approximately 7 KU / ml, approximately 8 KU / ml, approximately 9 KU / ml, or approximately 10 KU / ml.

[0659] In some embodiments, the enzyme stock may vary, so it should be noted that the concentration of the freeze-dried stock should be checked and the final amount of enzyme added to the digestion cocktail adjusted accordingly.

[0660] In some embodiments, the enzyme mixture includes a neutral protease, collagenase, and DNase.

[0661] In some embodiments, the enzyme mixture contains approximately 10.2 μl of neutral protease (0.36 DMCU / ml), 21.3 μl of collagenase (1.2 PZ / ml), and 250 μl of DNAseI (200 U / ml) in approximately 4.7 ml of sterile HBSS.

[0662] 2. Pleural effusion TIL In some embodiments, the sample is an intrapleural fluid sample. In some embodiments, the source of TILs for growth by the process described herein is an intrapleural fluid sample. In some embodiments, the sample is a pleural fluid-derived sample. In some embodiments, the source of TILs for growth by the process described herein is a pleural fluid-derived sample. See, for example, the method described in U.S. Patent Publication 2014 / 0295426, which is incorporated herein by reference in its entirety for all purposes.

[0663] In some embodiments, any intrapleural fluid or pleural effusion that appears to be and / or contains TILs can be utilized. Such samples may originate from primary or metastatic lung cancer such as NSCLC or SCLC. In some embodiments, the sample may be secondary metastatic cancer cells originating from another organ, e.g., the breast, ovary, colon, or prostate. In some embodiments, the sample used in the growth method described herein is pleural exudate. In some embodiments, the sample used in the growth method described herein is pleural transfusion. Other biological samples may include other serous fluids containing TILs, such as ascites from the abdomen or pancreatic cystic fluid. Ascites and intrapleural fluids have very similar chemical systems, and both the abdomen and lungs have mesothelial glands and fluid forms in the pleural and abdominal spaces of the same substance in malignant tumors, and in some embodiments, such fluids contain TILs. In some embodiments in which this disclosure exemplified pleural fluid, the same method may be performed with similar results using intrapleural fluid or other cystic fluids containing TILs.

[0664] In some embodiments, the intrapleural fluid is in an untreated form, directly removed from the patient. In some embodiments, the untreated intrapleural fluid is placed in a standard blood collection tube, such as an EDTA or heparin tube, before the contact step. In some embodiments, the untreated intrapleural fluid is placed in a standard CellSave® tube (Veridex) before the contact step. In some embodiments, the sample is placed in a CellSave tube immediately after collection from the patient to avoid a reduction in the number of viable TILs. The number of viable TILs can decrease significantly within 24 hours if left in untreated intrapleural fluid, even at 4°C. In some embodiments, the sample is placed in a suitable collection tube within 1, 5, 10, 15, or up to 24 hours after removal from the patient. In some embodiments, the sample is placed in a suitable collection tube at 4°C within 1, 5, 10, 15, or up to 24 hours after removal from the patient.

[0665] In some embodiments, intrapleural fluid samples from selected subjects may be diluted. In some embodiments, the dilution is 1:10 intrapleural fluid to diluent. In other embodiments, the dilution is 1:9 intrapleural fluid to diluent. In other embodiments, the dilution is 1:8 intrapleural fluid to diluent. In other embodiments, the dilution is 1:5 intrapleural fluid to diluent. In other embodiments, the dilution is 1:2 intrapleural fluid to diluent. In other embodiments, the dilution is 1:1 intrapleural fluid to diluent. In some embodiments, the diluent includes saline, phosphate-buffered saline, another buffer, or a physiologically acceptable diluent. In some embodiments, the sample is placed in a CellSave tube immediately after collection and dilution from the patient to avoid a reduction in viable TILs, which can occur significantly within 24-48 hours if left in untreated intrapleural fluid, even at 4°C. In some embodiments, the intrapleural fluid sample is placed in a suitable collection tube within 1 hour, 5 hours, 10 hours, 15 hours, 24 hours, 36 hours, and up to 48 hours after removal from the patient and dilution. In some embodiments, the intrapleural fluid sample is placed in a suitable collection tube at 4°C within 1 hour, 5 hours, 10 hours, 15 hours, 24 hours, 36 hours, and up to 48 hours after removal from the patient and dilution.

[0666] In yet another embodiment, the intrapleural fluid sample is concentrated by conventional means prior to further processing steps. In some embodiments, this pretreatment of the intrapleural fluid is preferred in situations where the pleural fluid must be cryopreserved for transport to the laboratory where the method is performed or for subsequent analysis (e.g., more than 24-48 hours after collection). In some embodiments, the intrapleural fluid sample is prepared by centrifuging the intrapleural fluid sample after it has been collected from the subject and resuspending the centrifugated material or pellet in a buffer. In some embodiments, the intrapleural fluid sample is subjected to multiple centrifugations and resuspensions before being cryopreserved for transport or subsequent analysis and / or processing.

[0667] In some embodiments, the intrapleural fluid sample is concentrated before further processing steps by using a filtration method. In some embodiments, the intrapleural fluid sample used in the contact step is prepared by filtering the fluid through a filter having a known and essentially uniform pore size that allows the intrapleural fluid to pass through a membrane but retains tumor cells. In some embodiments, the pore diameter of the membrane may be at least 4 μM. In other embodiments, the pore diameter may be 5 μM or greater, and in other embodiments, it may be any of 6, 7, 8, 9, or 10 μM. After filtration, the cells containing the TILs retained by the membrane may be rinsed off the membrane in a suitable physiologically acceptable buffer. The cells containing the TILs thus concentrated can then be used in the contact step of the method.

[0668] In some embodiments, an intrapleural fluid sample (e.g., including untreated intrapleural fluid), diluted pleural fluid, or resuspended cell pellet is brought into contact with a lysis reagent that specifically lyses anucleated red blood cells present in the sample. In some embodiments, this step is performed before further processing steps, in situations where the intrapleural fluid contains a significant number of RBCs. Suitable lysis reagents include a single lysis reagent or a lysis reagent and quench reagent, or a lysis reagent, quench reagent and fixation reagent. Suitable lysis systems are commercially available and include the BD Pharm Lyse® system (Becton Dickenson). Other lysis systems include the Versalyse® system, the FACSlyse® system (Becton Dickenson), the Immunoprep® system, or the Erythrolyse II system (Beckman Coulter, Inc.), or an ammonium chloride system. In some embodiments, the lysis reagent may differ depending on key requirements such as efficient lysis of red blood cells, as well as preservation of TILs and their phenotypic properties in the pleural fluid. In addition to utilizing a single reagent for dissolution, dissolution systems useful for the methods described herein may include a second reagent, for example, one that quenches or delays the effect of the dissolving reagent during the remaining steps of the method, such as Stabilyse® reagent (Beckman Coulter, Inc.). Depending on the choice of dissolving reagent or preferred implementation of the method, conventional fixation reagents may also be used.

[0669] In some embodiments, as described herein, untreated, diluted, or multi-centrifugated or treated intrapleural fluid samples are frozen and stored at a temperature of about -140°C before being further processed and / or expanded as provided herein.

[0670] 3. Methods for expanding peripheral blood lymphocytes (PBL) from peripheral blood. PBL Method 1. In some embodiments of the present invention, PBL is extended using the processes described herein. In some embodiments of the present invention, the method comprises obtaining a PBMC sample from whole blood. In some embodiments, the method comprises enriching T cells by isolating pure T cells from PBMCs using negative selection of the non-CD19+ fraction. In some embodiments, the method comprises enriching T cells by isolating pure T cells from PBMCs using magnetic bead-based negative selection of the non-CD19+ fraction.

[0671] In some embodiments of the present invention, PBL method 1 is performed as follows: On day 0, the cryopreserved PBMC sample is thawed and the PBMCs are counted. T cells are isolated using a human Pan T cell isolation kit and an LS column (Miltenyi Biotec).

[0672] PBL Method 2. In some embodiments of the present invention, PBL is extended using PBL Method 2, which includes obtaining a PBMC sample from whole blood. T cells derived from PBMCs are enriched by incubating the PBMCs at 37°C for at least 3 hours, and then isolating non-adherent cells.

[0673] In some embodiments of the present invention, PBL method 2 is performed as follows: On day 0, the cryopreserved PMBC sample is thawed, and PBMC cells are seeded at a rate of 6 million cells / well in a 6-well plate in CM-2 medium and incubated at 37°C for 3 hours. After 3 hours, non-adherent cells, which are PBL, are removed and counted.

[0674] PBL Method 3. In some embodiments of the present invention, PBL is extended using PBL Method 3, which includes obtaining a PBMC sample from peripheral blood. B cells are isolated using CD19+ selection, and T cells are selected using negative selection of the non-CD19+ fraction of the PBMC sample.

[0675] In some embodiments of the present invention, PBL method 3 is performed as follows: On day 0, cryopreserved PBMCs obtained from peripheral blood are thawed and counted. CD19+ B cells are sorted using the CD19 Multisort Kit, Human (Miltenyi Biotec). From the non-CD19+ cell fraction, T cells are purified using the Human Pan T Cell Isolation Kit and LS column (Miltenyi Biotec).

[0676] In some embodiments, PBMCs are isolated from a whole blood sample. In some embodiments, the PBMC sample is used as a starting material for expanding the PBL. In some embodiments, the sample is cryopreserved before the growth process. In other embodiments, a fresh sample is used as a starting material for expanding the PBL. In some embodiments of the present invention, T cells are isolated from PBMCs using methods known in the art. In some embodiments, T cells are isolated using a human T pancell isolation kit and an LS column. In some embodiments of the present invention, T cells are isolated from PBMCs using antibody selection methods known in the art, for example, negative selection of CD19.

[0677] In some embodiments of the present invention, the PBMC sample is incubated for a period of time at a desired temperature effective for identifying non-adherent cells. In some embodiments of the present invention, the incubation time is about 3 hours. In some embodiments of the present invention, the temperature is about 37°C. The non-adherent cells are then expanded using the process described above.

[0678] In some embodiments, PBMC samples are derived from subjects or patients who have been optionally prior-treated with a regimen containing a kinase inhibitor or ITK inhibitor. In some embodiments, tumor samples are derived from subjects or patients who have been prior-treated with a regimen containing a kinase inhibitor or ITK inhibitor. In some embodiments, PBMC samples are derived from subjects or patients who have been prior-treated with a regimen containing a kinase inhibitor or ITK inhibitor for at least one month, at least two months, at least three months, at least four months, at least five months, at least six months, or one year or longer. In other embodiments, PBMCs are derived from patients currently receiving an ITK inhibitor regimen such as ibrutinib.

[0679] In some embodiments, the PBMC samples are derived from subjects or patients who have been previously treated with a regimen containing a kinase inhibitor or an ITK inhibitor and are refractory to treatment with the kinase inhibitor or an ITK inhibitor such as ibrutinib.

[0680] In some embodiments, PBMC samples are derived from subjects or patients who have been previously treated with a regimen containing a kinase inhibitor or ITK inhibitor but are no longer receiving treatment with a kinase inhibitor or ITK inhibitor. In some embodiments, PBMC samples are derived from subjects or patients who have been previously treated with a regimen containing a kinase inhibitor or ITK inhibitor but are no longer receiving treatment with a kinase inhibitor or ITK inhibitor and have not received treatment for at least one month, at least two months, at least three months, at least four months, at least five months, at least six months, or at least one year or longer. In other embodiments, PBMCs are derived from patients who have been previously exposed to an ITK inhibitor but have not been treated within at least three months, at least six months, at least nine months, or at least one year.

[0681] In some embodiments of the present invention, on day 0, cells are selected for CD19+ and sorted accordingly. In some embodiments of the present invention, the selection is performed using antibody-conjugated beads. In some embodiments of the present invention, pure T cells are isolated from PBMCs on day 0.

[0682] In some embodiments of the present invention, for patients who have not been previously treated with ibrutinib or other ITK inhibitors, 10-15 ml of buffycoat is used for approximately 5 × 10 9 It produces PBMCs, which then produce approximately 5.5 × 10⁻⁶ PBMCs. 7 This produces PBL (Problem-Based Learning).

[0683] In some embodiments of the present invention, in patients previously treated with ibrutinib or other ITK inhibitors, the proliferation process is approximately 20 × 10 9 It produces PBL. In some embodiments of the present invention, 40.3 × 10 6 The PBMC is approximately 4.7 x 10 5 This produces PBL (Problem-Based Learning).

[0684] In any of the embodiments described above, PBMCs may be obtained from a whole blood sample, by apheresis, from a buffy coat, or by any other method known in the art for obtaining PBMCs.

[0685] 4. Method for expanding myeloinfiltrating lymphocytes (MILs) from bone marrow-derived PBMCs MIL Method 3. In some embodiments of the present invention, the method includes obtaining PBMCs from bone marrow. On day 0, PBMCs are selected and sorted for CD3+ / CD33+ / CD20+ / CD14+, the non-CD3+ / CD33+ / CD20+ / CD14+ cell fraction is sonicated, and a portion of the sonicated cell fraction is returned and added to the selected cell fraction.

[0686] In some embodiments of the present invention, the MIL method 3 is performed as follows: On day 0, the cryopreserved PBMC sample is thawed and the PBMCs are counted. The cells are stained with CD3, CD33, CD20, and CD14 antibodies and sorted using sorted S3e cells (Bio-Rad). The cells are sorted into two fractions: an immune cell fraction (or MIL fraction) (CD3+CD33+CD20+CD14+) and an AML blast cell fraction (non-CD3+CD33+CD20+CD14+).

[0687] In some embodiments of the present invention, PBMCs are obtained from bone marrow. In some embodiments, PBMCs are obtained from bone marrow by apheresis, aspiration, needle biopsy, or other similar means known in the art. In some embodiments, the PBMCs are fresh. In other embodiments, the PBMCs are cryopreserved.

[0688] In some embodiments of the present invention, MIL is expanded from 10 to 50 ml of bone marrow aspirate. In some embodiments of the present invention, 10 ml of bone marrow aspirate is obtained from the patient. In other embodiments, 20 ml of bone marrow aspirate is obtained from the patient. In other embodiments, 30 ml of bone marrow aspirate is obtained from the patient. In other embodiments, 40 ml of bone marrow aspirate is obtained from the patient. In other embodiments, 50 ml of bone marrow aspirate is obtained from the patient.

[0689] In some embodiments of the present invention, the number of PBMCs produced from approximately 10-50 ml of bone marrow aspirate is approximately 5 × 10 7 ~About 10×10 7 This is a PBBC. In other embodiments, the number of PMBCs produced is approximately 7 × 10 7 It is a PBMC.

[0690] In some embodiments of the present invention, about 5 × 10 7 ~About 10×10 7 The PBMC is approximately 0.5 x 10 6 ~Approx. 1.5×10 6 It produces MIL. In some embodiments of the present invention, about 1 × 10 6MIL is produced.

[0691] In some embodiments of the present invention, 12 × 10⁶ units obtained from bone marrow aspirate are used. 6 The PBMC is approximately 1.4 × 10 5 It produces MIL.

[0692] In any of the embodiments described above, PBMCs may be obtained from a whole blood sample, from bone marrow, by apheresis, from a buffy coat, or by any other method known in the art for obtaining PBMCs.

[0693] 5. Pre-selection of PD-1-PD-1 (as exemplified in step A2 of Figure 1) According to the method of the present invention, TILs are pre-selected to be PD-1 positive (PD-1+) before the first proliferation by priming.

[0694] In some embodiments, a minimum of 3,000 TILs are required to sow in the first propagation. In some embodiments, the pre-selection step yields a minimum of 3,000 TILs. In some embodiments, a minimum of 4,000 TILs are required to sow in the first propagation. In some embodiments, the pre-selection step yields a minimum of 4,000 TILs. In some embodiments, a minimum of 5,000 TILs are required to sow in the first propagation. In some embodiments, the pre-selection step yields a minimum of 5,000 TILs. In some embodiments, a minimum of 6,000 TILs are required to sow in the first propagation. In some embodiments, the pre-selection step yields a minimum of 6,000 TILs. In some embodiments, a minimum of 7,000 TILs are required to sow in the first propagation. In some embodiments, the pre-selection step yields a minimum of 7,000 TILs. In some embodiments, a minimum of 8,000 TILs are required to sow in the first propagation. In some embodiments, the pre-selection step yields a minimum of 8,000 TILs. In some embodiments, a minimum of 9,000 TILs are required to seed for a first growth. In some embodiments, the pre-selection step yields a minimum of 9,000 TILs. In some embodiments, a minimum of 10,000 TILs are required to seed for a first growth. In some embodiments, the pre-selection step yields a minimum of 10,000 TILs. In some embodiments, the cells are grown or expanded to a density of 200,000. In some embodiments, the cells are grown or expanded to a density of 200,000 to provide about 2e8 TILs to initiate a rapid second growth. In some embodiments, the cells are grown or expanded to a density of 150,000. In some embodiments, the cells are grown or expanded to a density of 150,000 to provide about 2e8 TILs to initiate a rapid second growth. In some embodiments, the cells are grown or expanded to a density of 250,000.In some embodiments, cells are grown or expanded to a density of 250,000 to provide approximately 2e8 TIL for initiating rapid second proliferation. In some embodiments, the minimum cell density is 10,000 cells to give 10e6 for initiating rapid second proliferation. In some embodiments, a seeding density of 10e6 for initiating rapid second proliferation may result in a TIL of more than 1e9.

[0695] In some embodiments, the TIL for use in the first proliferation by priming is PD-1 positive (PD-1+) (e.g., after pre-selection and before the first proliferation by priming). In some embodiments, the TIL for use in the first proliferation by priming is at least 75% PD-1 positive, at least 80% PD-1 positive, at least 85% PD-1 positive, at least 90% PD-1 positive, at least 95% PD-1 positive, at least 98% PD-1 positive, or at least 99% PD-1 positive (e.g., after pre-selection and before the first proliferation by priming). In some embodiments, the PD-1 population is PD-1 high. In some embodiments, the TIL for use in the first growth by priming is at least 25% PD-1 high, at least 30% PD-1 high, at least 35% PD-1 high, at least 40% PD-1 high, at least 45% PD-1 high, at least 50% PD-1 high, at least 55% PD-1 high, at least 60% PD-1 high, at least 65% PD-1 high, at least 70% PD-1 high, at least 75% PD-1 high, at least 80% PD-1 high, at least 85% PD-1 high, at least 90% PD-1 high, at least 95% PD-1 high, at least 98% PD-1 high, or at least 99% PD-1 high (e.g., after pre-selection and before the first growth by priming).

[0696] In some embodiments, high PD-1 is indicated by a TIL population that is at least 75% PD-1 positive, at least 80% PD-1 positive, at least 85% PD-1 positive, at least 90% PD-1 positive, at least 95% PD-1 positive, at least 98% PD-1 positive, or at least 99% PD-1 positive, or 100% PD-1 positive. In some embodiments, high PD-1 is indicated by a TIL population that is at least 80% PD-1 positive. In some embodiments, high PD-1 is indicated by a TIL population that is at least 85% PD-1 positive. In some embodiments, high PD-1 is indicated by a TIL population that is at least 90% PD-1 positive. In some embodiments, high PD-1 is indicated by a TIL population that is at least 95% PD-1 positive. In some embodiments, high PD-1 is indicated by a TIL population that is at least 98% PD-1 positive. In some embodiments, high PD-1 is indicated by a TIL population that is at least 99% PD-1 positive. In some embodiments, high PD-1 levels are indicated by a TIL population that is 100% PD-1 positive.

[0697] In some embodiments, high PD-1 levels are defined as TIL expressing PD-1 at least 25% higher than control or baseline PD-1 levels, expressing PD-1 at least 30% higher than control or baseline PD-1 levels, expressing PD-1 at least 35% higher than control or baseline PD-1 levels, expressing PD-1 at least 40% higher than control or baseline PD-1 levels, expressing PD-1 at least 45% higher than control or baseline PD-1 levels, expressing PD-1 at least 50% higher than control or baseline PD-1 levels, expressing PD-1 at least 55% higher than control or baseline PD-1 levels, expressing PD-1 at least 60% higher than control or baseline PD-1 levels, and control or baseline PD-1 This is indicated by the TIL population, which expresses PD-1 at least 65% higher than the level, at least 70% higher than the control or baseline PD-1 level, at least 75% higher than the control or baseline PD-1 level, at least 80% higher than the control or baseline PD-1 level, at least 85% higher than the control or baseline PD-1 level, at least 90% higher than the control or baseline PD-1 level, at least 95% higher than the control or baseline PD-1 level, at least 99% higher than the control or baseline PD-1 level, or at least 100% higher than the control or baseline PD-1 level.

[0698] In some embodiments, PD-1 elevated levels are indicated by TIL populations where TILs express PD-1 at a level of 1 or more higher than control or baseline PD-1 levels. In some embodiments, PD-1 elevated levels are indicated by TIL populations where TILs express PD-1 at a level of 1, 2, 3, 4, 5, or 10 or more higher than control or baseline PD-1 levels. In some embodiments, PD-1 elevated levels are indicated by TIL populations where TILs express PD-1 at a level of 1 or more higher than control or baseline PD-1 levels. In some embodiments, PD-1 elevated levels are indicated by TIL populations where TILs express PD-1 at a level of 2 or more higher than control or baseline PD-1 levels. In some embodiments, PD-1 elevated levels are indicated by TIL populations where TILs express PD-1 at a level of 3 or more higher than control or baseline PD-1 levels. In some embodiments, PD-1 elevated levels are indicated by TIL populations where TILs express PD-1 at a level of 4 or more higher than control or baseline PD-1 levels. In some embodiments, PD-1 elevated levels are indicated by a TIL population in which the TILs express PD-1 at a level 5 or higher than the control or baseline PD-1 levels. In some embodiments, PD-1 elevated levels are indicated by a TIL population in which the TILs express PD-1 at a level 10 or higher than the control or baseline PD-1 levels.

[0699] In some embodiments, pre-selection of PD-1-positive TILs is performed by staining the primary cell population, whole tumor digest, and / or whole tumor cell suspension TILs with an anti-PD-1 antibody. In some embodiments, the anti-PD-1 antibody is a polyclonal antibody, e.g., mouse anti-human PD-1 polyclonal antibody, goat anti-human PD-1 polyclonal antibody, etc. In some embodiments, the anti-PD-1 antibody is a monoclonal antibody. In some embodiments, the anti-PD-1 antibody may be, for example, EH12.2H7, PD1.3.1, SYM021, M1H4, A17188B, nivolumab (BMS-936558, Bristol-Myers Squibb; Opdivo®), pembrolizumab (lambrolizumab, MK03475 or MK-3475, Merck; Keytruda®), H12.1, PD1.3.1, NAT 105, humanized anti-PD-1 antibody JS001 (ShangHai JunShi), monoclonal anti-PD-1 antibody TSR-042 (Tesaro, Inc.), or pizilizumab (anti-PD-1 mAb This includes, but is not limited to, CT-011 (Medivation), anti-PD-1 monoclonal antibody BGB-A317 (BeiGene), and / or anti-PD-1 antibody SHR-1210 (ShangHai HengRui), human monoclonal antibody REGN2810 (Regeneron), human monoclonal antibody MDX-1106 (Bristol-Myers Squibb), and / or humanized anti-PD-1 IgG4 antibody PDR001 (Novartis). In some embodiments, the PD-1 antibody is derived from clone: ​​RMP1-14 (rat IgG)-BioXcell catalog number BP0146. Other suitable antibodies for use in the pre-selection of PD-1 positive TILs for use in the proliferation of TILs by the method of the present invention, as exemplified by steps A-F as described herein, are the anti-PD-1 antibodies disclosed in U.S. Patent No. 8,008,449, incorporated herein by reference.In some embodiments, the anti-PD-1 antibody used in pre-selection binds to a different epitope than nivolumab (BMS-936558, Bristol-Myers Squibb; Opdivo®). In some embodiments, the anti-PD-1 antibody used in pre-selection binds to a different epitope than pembrolizumab (lambrolizumab, MK03475 or MK-3475, Merck; Keytruda®). In some embodiments, the anti-PD-1 antibody used in pre-selection binds to a different epitope than the humanized anti-PD-1 antibody JS001 (ShangHai JunShi). In some embodiments, the anti-PD-1 antibody used in pre-selection binds to a different epitope than the monoclonal anti-PD-1 antibody TSR-042 (Tesaro, Inc.). In some embodiments, the anti-PD-1 antibody used for pre-selection binds to a different epitope than pidilizumab (anti-PD-1 mAb CT-011, Medivation). In some embodiments, the anti-PD-1 antibody used for pre-selection binds to a different epitope than the anti-PD-1 monoclonal antibody BGB-A317 (BeiGene). In some embodiments, the anti-PD-1 antibody used for pre-selection binds to a different epitope than the anti-PD-1 antibody SHR-1210 (ShangHai HengRui). In some embodiments, the anti-PD-1 antibody used for pre-selection binds to a different epitope than the human monoclonal antibody REGN2810 (Regeneron). In some embodiments, the anti-PD-1 antibody used for pre-selection binds to a different epitope than the human monoclonal antibody MDX-1106 (Bristol-Myers Squibb). In some embodiments, the anti-PD-1 antibody used in pre-selection binds to a different epitope than the humanized anti-PD-1 IgG4 antibody PDR001 (Novartis). In some embodiments, the anti-PD-1 antibody used in pre-selection binds to a different epitope than RMP1-14 (rat IgG)-BioXcell catalog number #BP0146.The structures of nivolumab and pembrolizumab binding to PD-1 are known and are described, for example, in Tan, S. et al. (Tan, S. et al., Nature Communications, 8:14369 | DOI:10.1038 / ncomms14369 (2017), the whole of which is incorporated herein by reference for all purposes). In some embodiments, the anti-PD-1 antibody is EH12.2H7. In some embodiments, the anti-PD-1 antibody is PD1.3.1. In some embodiments, the anti-PD-1 antibody is not PD1.3.1. In some embodiments, the anti-PD-1 antibody is M1H4. In some embodiments, the anti-PD-1 antibody is not M1H4.

[0700] In some embodiments, the anti-PD-1 antibody for use in pre-selection binds to at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% of cells expressing PD-1.

[0701] In some embodiments, the patient is being treated with an anti-PD-1 antibody. In some embodiments, the subject is anti-PD-1 antibody treatment naive. In some embodiments, the subject is not being treated with an anti-PD-1 antibody. In some embodiments, the subject has been previously treated with a chemotherapeutic agent. In some embodiments, the subject has been previously treated with a chemotherapeutic agent but is no longer being treated with a chemotherapeutic agent. In some embodiments, the subject is post-chemotherapeutic or post-anti-PD-1 antibody treatment. In some embodiments, the subject is post-chemotherapeutic and post-anti-PD-1 antibody treatment. In some embodiments, the patient is anti-PD-1 antibody treatment naive. In some embodiments, the subject has treatment-naive cancer or is post-chemotherapeutic but is anti-PD-1 antibody treatment naive. In some embodiments, the subject is treatment-naive and post-chemotherapeutic but is anti-PD-1 antibody treatment naive.

[0702] In some embodiments where the patient has been previously treated with a first anti-PD-1 antibody, pre-selection is performed by staining the primary cell population, whole tumor digest, and / or whole tumor cell suspension TILs with a second anti-PD-1 antibody that is not blocked by the first anti-PD-1 antibody from binding to PD-1 on the surface of the primary TIL cell population.

[0703] In some embodiments where the patient has been previously treated with an anti-PD-1 antibody, pre-selection is performed by staining the primary TIL cell population with an antibody that binds to the Fc region of the anti-PD-1 antibody insoluble on the surface of the primary TIL cell population ("anti-Fc antibody"). In some embodiments, the anti-Fc antibody is a polyclonal antibody, e.g., mouse anti-human Fc polyclonal antibody, goat anti-human Fc polyclonal antibody, etc. In some embodiments, the anti-Fc antibody is a monoclonal antibody. In some embodiments where the patient has been previously treated with an anti-PD-1 human or humanized IgG antibody, the primary TIL cell population is stained with an anti-human IgG antibody. In some embodiments where the patient has been previously treated with an anti-PD-1 human or humanized IgG1 antibody, the primary TIL cell population is stained with an anti-human IgG1 antibody. In some embodiments where the patient has been previously treated with an anti-PD-1 human or humanized IgG2 antibody, the primary TIL cell population is stained with an anti-human IgG2 antibody. In some embodiments where the patient has been previously treated with anti-PD-1 human or humanized IgG3 antibody, the primary TIL cell population is stained with anti-human IgG3 antibody. In some embodiments where the patient has been previously treated with anti-PD-1 human or humanized IgG4 antibody, the primary TIL cell population is stained with anti-human IgG4 antibody.

[0704] In some embodiments where the patient has been previously treated with an anti-PD-1 antibody, pre-selection is performed by contacting the primary TIL cell population with the same anti-PD-1 antibody, and then staining the primary TIL cell population with an anti-Fc antibody that binds to the Fc region of the anti-PD-1 antibody insoluble on the surface of the primary TIL cell population.

[0705] In some embodiments, pre-selection is performed using a cell sorting method. In some embodiments, the cell sorting method is a flow cytometry method, such as flow-activated cell sorting (FACS). In some embodiments, the intensity of fluorophores in both the first population and the PBMC population is used to set up FACS gates to establish low, medium, and high intensity levels corresponding to PD-1 negative TILs, PD-1 intermediate TILs, and PD-1 positive TILs, respectively. In some embodiments, the cell sorting method is performed to distinguish three populations using PBMCs, an FMO control, and the sample itself, so that the gates are set to high, medium (also referred to as intermediate), and low (also referred to as negative). In some embodiments, PBMCs are used as a gating control. In some embodiments, the PD-1 high population is defined as the cell population that is positive for PD-1 above what is observed in the PBMCs. In some embodiments, the PD-1+ intermediate population of TILs includes PD-1+ cells in the PBMCs. In some embodiments, negative is gated based on FMO. In some embodiments, the FACS gate is set after the step of obtaining and / or receiving a first TIL population from the tumor excised from the subject by processing the tumor sample obtained from the subject into multiple tumor fragments. In some embodiments, gating is set for each sorting. In some embodiments, gating is set for each PBMC sample. In some embodiments, gating is set for each PBMC sample. In some embodiments, a gating template is set from the PBMC every 10, 20, 30, 40, 50, or 60 days. In some embodiments, a gating template is set from the PBMC every 60 days. In some embodiments, a gating template is set for each PBMC sample every 10, 20, 30, 40, 50, or 60 days. In some embodiments, a gating template is set for each PBMC sample every 60 days.

[0706] In some embodiments, the PD-1 pre-selection gating is fixed for each pre-selection procedure. In some embodiments, the fixed gating procedure is a CD3+ gating procedure. In some embodiments, the gating procedure is not fixed but is determined based on the population acquired during each selection. In some embodiments, the gating procedure is not fixed but is determined based on the population acquired during each selection event being a CD3+ gating procedure.

[0707] In some embodiments, the gating and correction of the mean fluorescence intensity (MFI) is in the range of approximately 0.5% ± 0.25%. In some embodiments, the gating and correction of the mean fluorescence intensity (MFI) is in the range of approximately 1.75% ± 0.25%. In some embodiments, the gating and correction of the mean fluorescence intensity (MFI) is in the range of approximately 1.75% ± 0.25% when PD-1 high gating is set in PBMCs. In some embodiments, the MFI calculation uses the average value measured from 1, 2, 3, or 4 or more lots or batches of PBMCs. In some embodiments, the MFI calculation uses the median value measured from 1, 2, 3, or 4 or more lots or batches of PBMCs.

[0708] In some embodiments, the FACS sorting and correction method used to determine the mean fluorescence intensity (MFI) is adjusted so that the MFI of PD-1 relative to control PBMCs (e.g., PBMCs from healthy donors) is in the range of approximately 0.5% to 2.0% (e.g., approximately 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, 0.95%, 1.0%, 1.05%, 1.1%, 1.15%, 1.2%, 1.25%, 1.3%, 1.35%, 1.4%, 1.45%, 1.5%, 1.55%, 1.6%, 1.65%, 1.7%, 1.75%, 1.8%, 1.85%, 1.9%, 1.95%, or 2.0%) relative to high PD-1 gate. In some embodiments, the MFI calculation is the mean value measured using at least two samples of PBMC. In some embodiments, the MFI calculation is the median value measured using at least two samples of PBMC.

[0709] In some embodiments, PD-1-positive (PD-1+) cells are sorted by FAC and / or other flow cytometry methods. In some embodiments, PD-1-positive TILs are PD-1 high TILs. In some embodiments, PD-1-positive TILs are PD-1 intermediate TILs. In some embodiments, PD-1+ cells are sorted by utilizing bead selection. In some embodiments, PD-1+ cells are sorted by utilizing magnetic bead selection. In some embodiments, bead selection utilizes antibody-conjugated beads, such as commercially available beads, including but not limited to Miltenyi or Fisher, for selection. In some embodiments, PD-1+ high cells are sorted by utilizing bead selection. In some embodiments, PD-1+ high cells are sorted by utilizing magnetic bead selection. In some embodiments, bead selection utilizes antibody-conjugated beads, such as commercially available beads, including but not limited to Miltenyi or Fisher, for selection. In some embodiments, an anti-PD-1 antibody is directly or indirectly conjugated with the beads. In some embodiments, the bead selection process selects both PD-1+ and CD3+TIL beads.In some embodiments, anti-PD-1 antibodies used in the bead selection method include, for example, EH12.2H7, PD1.3.1, SYM021, M1H4, A17188B, nivolumab (BMS-936558, Bristol-Myers Squibb; Opdivo®), pembrolizumab (lambrolizumab, MK03475 or MK-3475, Merck; Keytruda®), H12.1, PD1.3.1, NAT 105, humanized anti-PD-1 antibody JS001 (ShangHai JunShi), monoclonal anti-PD-1 antibody TSR-042 (Tesaro, Inc.), and pizilizumab (anti-PD-1 mAb This includes, but is not limited to, CT-011 (Medivation), anti-PD-1 monoclonal antibody BGB-A317 (BeiGene), and / or anti-PD-1 antibody SHR-1210 (ShangHai HengRui), human monoclonal antibody REGN2810 (Regeneron), human monoclonal antibody MDX-1106 (Bristol-Myers Squibb), and / or humanized anti-PD-1 IgG4 antibody PDR001 (Novartis). In some embodiments, the PD-1 antibody is derived from clone: ​​RMP1-14 (rat IgG)-BioXcell catalog number BP0146. Other suitable antibodies for use in the pre-selection of PD-1 positive TILs for use in the proliferation of TILs by the method of the present invention, as exemplified by steps A-F as described herein, are the anti-PD-1 antibodies disclosed in U.S. Patent No. 8,008,449, incorporated herein by reference. In some embodiments, the anti-PD-1 antibody used in pre-selection binds to a different epitope than nivolumab (BMS-936558, Bristol-Myers Squibb; Opdivo®). In some embodiments, the anti-PD-1 antibody used in pre-selection binds to a different epitope than pembrolizumab (lambrolizumab, MK03475 or MK-3475, Merck; Keytruda®).In some embodiments, the anti-PD-1 antibody used in pre-selection binds to a different epitope than the humanized anti-PD-1 antibody JS001 (ShangHai JunShi). In some embodiments, the anti-PD-1 antibody used in pre-selection binds to a different epitope than the monoclonal anti-PD-1 antibody TSR-042 (Tesaro, Inc.). In some embodiments, the anti-PD-1 antibody used in pre-selection binds to a different epitope than pidilizumab (anti-PD-1 mAb CT-011, Medivation). In some embodiments, the anti-PD-1 antibody used in pre-selection binds to a different epitope than the anti-PD-1 monoclonal antibody BGB-A317 (BeiGene). In some embodiments, the anti-PD-1 antibody used in pre-selection binds to a different epitope than the anti-PD-1 antibody SHR-1210 (ShangHai HengRui). In some embodiments, the anti-PD-1 antibody used in pre-selection binds to a different epitope than the human monoclonal antibody REGN2810 (Regeneron). In some embodiments, the anti-PD-1 antibody used in pre-selection binds to a different epitope than the human monoclonal antibody MDX-1106 (Bristol-Myers Squibb). In some embodiments, the anti-PD-1 antibody used in pre-selection binds to a different epitope than the humanized anti-PD-1 IgG4 antibody PDR001 (Novartis). In some embodiments, the anti-PD-1 antibody used in pre-selection binds to a different epitope than RMP1-14 (rat IgG)-BioXcell catalog number #BP0146. The structures of nivolumab and pembrolizumab binding to PD-1 are known and are described, for example, in Tan, S. et al. (Tan, S. et al., Nature Communications, 8:14369|DOI:10.1038 / ncomms14369(2017), the whole of which is incorporated herein by reference for all purposes). In some embodiments, the anti-PD-1 antibody is EH12.2H7.In some embodiments, the anti-PD-1 antibody is PD1.3.1. In some embodiments, the anti-PD-1 antibody is not PD1.3.1. In some embodiments, the anti-PD-1 antibody is M1H4. In some embodiments, the anti-PD-1 antibody is not M1H4.

[0710] In some embodiments, the collection buffer used to collect PD-1+ cells and / or PD-1-negative cells does not contain serum. In some embodiments, the collection buffer used to collect PD-1+ cells and / or PD-1-negative cells contains serum. In some embodiments, the collection buffer used to collect PD-1+ cells and / or PD-1-negative cells contains a component that reduces or minimizes the viscosity difference between the sorting buffer and the downstream buffer and / or culture medium. In some embodiments, the collection buffer used to collect PD-1+ cells and / or PD-1-negative cells contains only human serum albumin (HSA). In some embodiments, the collection buffer used to collect PD-1+ cells and / or PD-1-negative cells contains equal volumes of HSA and PBS / EDTA buffer. In some embodiments, the collection buffer used to collect PD-1+ cells and / or PD-1-negative cells contains HSA and PBS / EDTA buffer in a ratio of 1:1, 1:2, 1:3, 1:4, 2:1, 3:1, or 4:1.

[0711] In some embodiments, pre-selection involves selecting PD-1-positive TILs from a first TIL population to obtain a PD-1-rich TIL population, and includes selecting a TIL population from the first TIL population in which at least 11.27% to 74.4% are PD-1-positive TILs. In some embodiments, the first TIL population is at least 20-80% PD-1-positive TILs, at least 20-80% PD-1-positive TILs, at least 30-80% PD-1-positive TILs, at least 40-80% PD-1-positive TILs, at least 50-80% PD-1-positive TILs, at least 10-70% PD-1-positive TILs, at least 20-70% PD-1-positive TILs, at least 30-70% PD-1-positive TILs, or at least 40-70% PD-1-positive TILs.

[0712] In some embodiments, the selection step (e.g., pre-selection and / or selection of PD-1 positive cells) is performed. (i) Exposing a first TIL population and a PBMC population to an excess of monoclonal anti-PD-1 IgG4 antibody that binds to PD-1 by the N-terminal loop outside the IgV domain of PD-1, (ii) The step of adding an excess anti-IgG4 antibody conjugated to the fluorophore, (iii) The step of obtaining a PD-1-rich TIL population based on the intensity of the fluorophores of PD-1-positive TILs in a first TIL population compared to the intensity of the PBMC population when performed by fluorescence-activated cell sorting (FACS).

[0713] In some embodiments, PD-1-positive TILs are PD-1-high TILs.

[0714] In some embodiments, high PD-1 expression is determined by flow cytometry using a minimum normalized fluorescence intensity cutoff selected from the group consisting of approximately 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10%.

[0715] In some embodiments, at least 70% of a PD-1-rich TIL population are PD-1 positive TILs. In some embodiments, at least 80% of a PD-1-rich TIL population are PD-1 positive TILs. In some embodiments, at least 90% of a PD-1-rich TIL population are PD-1 positive TILs. In some embodiments, at least 95% of a PD-1-rich TIL population are PD-1 positive TILs. In some embodiments, at least 99% of a PD-1-rich TIL population are PD-1 positive TILs. In some embodiments, 100% of a PD-1-rich TIL population are PD-1 positive TILs.

[0716] In some embodiments, the selection of PD-1 positive TILs is at least 1 × 10⁻⁶ 4 PD-1 positive TILs, at least 1 × 10⁶ 5 PD-1 positive TILs, at least 1 × 10⁶ 6 PD-1 positive TILs, at least 1 × 10⁶ 7 PD-1 positive TILs, at least 1 × 10⁶ 8 The process continues until PD-1 positive TILs are found. In some embodiments, the selection of PD-1 positive TILs is performed until at least 1 × 10⁶ TILs are found. 6 The procedure continues until PD-1-positive TILs are found.

[0717] Different anti-PD-1 antibodies exhibit different binding characteristics to different epitopes within PD-1. In some embodiments, the anti-PD-1 antibody binds to an epitope different from pembrolizumab. In some embodiments, the anti-PD1 antibody binds to an epitope in the N-terminal loop outside the IgV domain of PD-1. In some embodiments, the anti-PD1 antibody binds via the N-terminal loop outside the IgV domain of PD-1. In some embodiments, the anti-PD-1 antibody is an anti-PD-1 antibody that binds to PD-1 via the N-terminal loop outside the IgV domain of PD-1. In some embodiments, the anti-PD-1 antibody is a monoclonal anti-PD-1 antibody that binds to PD-1 via the N-terminal loop outside the IgV domain of PD-1. In some embodiments, the monoclonal anti-PD-1 antibody is an anti-PD-1 IgG4 antibody that binds to PD-1 via the N-terminal loop outside the IgV domain of PD-1. For example, see Tan, S. Nature Comm. Vol 8, Argicle 14369:1-10 (2017).

[0718] In some embodiments, the selection step, exemplified as step A2 in Figure 1, includes (i) exposing a first TIL population to an excess of monoclonal anti-PD-1 IgG4 antibody that binds to PD-1 by the N-terminal loop outside the IgV domain of PD-1; (ii) adding the excess anti-IgG4 antibody conjugated to a fluorophore; and (iii) performing fluid cell sorting based on the fluorophore to obtain a PD-1-rich TIL population. In some embodiments, the monoclonal anti-PD-1 IgG4 antibody is nivolumab, or a variant, fragment, or complex thereof. In some embodiments, the anti-IgG4 antibody is clone anti-human IgG4, clone HP6023. In some embodiments, the anti-PD-1 antibody for use in the selection in step (b) binds to the same epitope as EH12.2H7 or nivolumab.

[0719] In some embodiments, the PD-1 gating method of WO2019156568 is used. To determine whether TILs derived from tumor samples are PD-1 high, those skilled in the art can utilize a reference value corresponding to the PD-1 expression level in peripheral T cells obtained from blood samples from one or more healthy human subjects. PD-1 positive cells in the reference sample can be defined using a control with one fluorescence minus a corresponding isotype control. In some embodiments, the PD-1 expression level is measured in CD3+ / PD-1+ peripheral T cells from healthy subjects (e.g., reference cells) and used to establish a threshold or cutoff value for the PD-1 immunostaining intensity in TILs obtained from tumors. The threshold can be defined as the minimum intensity of PD-1 immunostaining in PD-1 high T cells. Thus, TILs with PD-1 expression equal to or exceeding the threshold can be considered PD-1 high cells. In some cases, PD-1 high TILs represent those with the highest PD-1 immunostaining intensity, which corresponds to up to 1% or less of all CD3+ cells. In other cases, high PD-1 TILs represent the highest intensity of PD-1 immunostaining corresponding to up to 0.75% of all CD3+ cells. In some cases, high PD-1 TILs represent the highest intensity of PD-1 immunostaining corresponding to up to 0.50% of all CD3+ cells. In one case, high PD-1 TILs represent the highest intensity of PD-1 immunostaining corresponding to up to 0.25% of all CD3+ cells.

[0720] NSCLC patients. In short, PD-1 high, PD-1 intermediate, and PD-1 negative subsets can be identified based on their measured fluorescence intensity.

[0721] In some embodiments, selected PD-1-positive (PD-1+) cells can be frozen before proceeding with the first proliferation by priming, for example, step B of Figure 1 (in particular, for example, Figures 1B and / or 1C and / or 1D and / or 1E and / or 1F and / or 1G and / or 1H).

[0722] a. Fluorophores In some embodiments, the primary TIL cell population is stained with a cocktail containing fluorophore-conjugated anti-PD-1 antibody and fluorophore-conjugated anti-CD3 antibody. In some embodiments, the primary TIL cell population is stained with a cocktail containing fluorophore-conjugated anti-PD-1 antibody (e.g., PE, live / dead violet) and anti-CD3-FITC. In some embodiments, the primary TIL cell population is stained with a cocktail containing anti-PD-1-PE, anti-CD3-FITC, and live / dead blue stain (ThermoFisher, MA, catalog no. L23105). In some embodiments, after incubation with anti-PD1 antibody, PD-1 positive cells are selected for proliferation by a first proliferation by priming in step B, as described herein.

[0723] In some embodiments, the fluorophore includes, but is not limited to, PE (phycoerythrin), APC (allophycocyanin), PerCP (peridinine chlorophyll protein), DyLight 405, Alexa Fluor 405, Pacific Blue, Alexa Fluor 488, FITC (fluorescein isothiocyanate), DyLight 550, Alexa Fluor 647, DyLight 650, and Alexa Fluor 700. In some embodiments, the fluorophore includes, but is not limited to, PE-Alexa Fluor® 647, PE-Cy5, PerCP-Cy5.5, PE-Cy5.5, PE-Alexa Fluor® 750, PE-Cy7, and APC-Cy7. In some embodiments, the fluorophore includes, but is not limited to, fluorescein dyes. Examples of fluorescein dyes include, but are not limited to, 5-carboxyfluorescein, fluorescein-5-isothiocyanate and 6-carboxyfluorescein, 5,6-dicarboxyfluorescein, 5-(and 6)-sulfofluorescein, sulfonefluorescein, succinylfluorescein, 5-(and 6)-carboxySNARF-1, carboxyfluorescein sulfonate, carboxyfluorescein zwitterion, carboxyfluorescein quaternary ammonium, carboxyfluorescein phosphonate, carboxyfluorescein GABA, 5'(6')-carboxyfluorescein, carboxyfluorescein-cys-Cy5, and fluorescein glutathione. In some embodiments, the fluorescent moiety is a rhodamine dye. Examples of rhodamine dyes include, but are not limited to, tetramethylrhodamine-6-isothiocyanate, 5-carboxytetramethylrhodamine, 5-carboxylodol derivatives, carboxyrhodamine 110, tetramethyl and tetraethylrhodamine, diphenyldimethyl and diphenyldiethylrhodamine, dinaphthylrhodamine, and rhodamine 101 sulfonyl chloride (marketed under the trade name TEXAS RED®). In some embodiments, the fluorescent portion is a cyanine dye.Examples of cyanine pigments include, but are not limited to, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, and Cy7.

[0724] 6. Pre-selection of CD39-CD39 (as illustrated in step A2 of Figure 1) According to the method of the present invention, TILs are pre-selected to be CD39-positive (CD39+) before the first proliferation by priming.

[0725] In some embodiments, the TILs of the present invention are pre-selected for a depletion marker such as CD39 (see, for example, Canale, FP, et al. Cancer Res. 78:115-128 (2018) and / or Duhne, T., et al., Nat Commun. 9:2724 (2018)). According to the method of the present invention, the TILs are pre-selected to be CD39-positive (CD39+) before the first proliferation by priming.

[0726] In some embodiments, a minimum of 3,000 TILs are required to sow in the first propagation. In some embodiments, the pre-selection step yields a minimum of 3,000 TILs. In some embodiments, a minimum of 4,000 TILs are required to sow in the first propagation. In some embodiments, the pre-selection step yields a minimum of 4,000 TILs. In some embodiments, a minimum of 5,000 TILs are required to sow in the first propagation. In some embodiments, the pre-selection step yields a minimum of 5,000 TILs. In some embodiments, a minimum of 6,000 TILs are required to sow in the first propagation. In some embodiments, the pre-selection step yields a minimum of 6,000 TILs. In some embodiments, a minimum of 7,000 TILs are required to sow in the first propagation. In some embodiments, the pre-selection step yields a minimum of 7,000 TILs. In some embodiments, a minimum of 8,000 TILs are required to sow in the first propagation. In some embodiments, the pre-selection step yields a minimum of 8,000 TILs. In some embodiments, a minimum of 9,000 TILs are required to seed for a first growth. In some embodiments, the pre-selection step yields a minimum of 9,000 TILs. In some embodiments, a minimum of 10,000 TILs are required to seed for a first growth. In some embodiments, the pre-selection step yields a minimum of 10,000 TILs. In some embodiments, the cells are grown or expanded to a density of 200,000. In some embodiments, the cells are grown or expanded to a density of 200,000 to provide about 2e8 TILs to initiate a rapid second growth. In some embodiments, the cells are grown or expanded to a density of 150,000. In some embodiments, the cells are grown or expanded to a density of 150,000 to provide about 2e8 TILs to initiate a rapid second growth. In some embodiments, the cells are grown or expanded to a density of 250,000.In some embodiments, cells are grown or expanded to a density of 250,000 to provide approximately 2e8 TIL for initiating rapid second proliferation. In some embodiments, the minimum cell density is 10,000 cells to give 10e6 for initiating rapid second proliferation. In some embodiments, a seeding density of 10e6 for initiating rapid second proliferation may result in a TIL of more than 1e9.

[0727] In some embodiments, the TILs for use in the first proliferation by priming are CD39-positive (CD39+) (e.g., after pre-selection and before the first proliferation by priming). In some embodiments, the TILs for use in the first proliferation by priming are at least 75% CD39-positive, at least 80% CD39-positive, at least 85% CD39-positive, at least 90% CD39-positive, at least 95% CD39-positive, at least 98% CD39-positive, or at least 99% CD39-positive (e.g., after pre-selection and before the first proliferation by priming). In some embodiments, the CD39 population is CD39-high. In some embodiments, the TIL for use in the first growth by priming is at least 25% CD39 high, at least 30% CD39 high, at least 35% CD39 high, at least 40% CD39 high, at least 45% CD39 high, at least 50% CD39 high, at least 55% CD39 high, at least 60% CD39 high, at least 65% CD39 high, at least 70% CD39 high, at least 75% CD39 high, at least 80% CD39 high, at least 85% CD39 high, at least 90% CD39 high, at least 95% CD39 high, at least 98% CD39 high, or at least 99% CD39 high (e.g., after pre-selection and before the first growth by priming).

[0728] In some embodiments, high CD39 is represented by a TIL population that is at least 75% CD39 positive, at least 80% CD39 positive, at least 85% CD39 positive, at least 90% CD39 positive, at least 95% CD39 positive, at least 98% CD39 positive, at least 99% CD39 positive, or 100% CD39 positive. In some embodiments, high CD39 is represented by a TIL population that is at least 80% CD39 positive. In some embodiments, high CD39 is represented by a TIL population that is at least 85% CD39 positive. In some embodiments, high CD39 is represented by a TIL population that is at least 90% CD39 positive. In some embodiments, high CD39 is represented by a TIL population that is at least 95% CD39 positive. In some embodiments, high CD39 is represented by a TIL population that is at least 98% CD39 positive. In some embodiments, high CD39 is represented by a TIL population that is at least 99% CD39 positive. In some embodiments, high CD39 is indicated by a TIL population that is 100% CD39 positive.

[0729] In some embodiments, high CD39 means that TIL expresses CD39 at least 25% higher than control or baseline CD39 levels, expresses CD39 at least 30% higher than control or baseline CD39 levels, expresses CD39 at least 35% higher than control or baseline CD39 levels, expresses CD39 at least 40% higher than control or baseline CD39 levels, expresses CD39 at least 45% higher than control or baseline CD39 levels, expresses CD39 at least 50% higher than control or baseline CD39 levels, expresses CD39 at least 55% higher than control or baseline CD39 levels, expresses CD39 at least 60% higher than control or baseline CD39 levels, and This is demonstrated by the TIL population, which expresses CD39 at least 65% higher than the level, at least 70% higher than the control or baseline CD39 level, at least 75% higher than the control or baseline CD39 level, at least 80% higher than the control or baseline CD39 level, at least 85% higher than the control or baseline CD39 level, at least 90% higher than the control or baseline CD39 level, at least 95% higher than the control or baseline CD39 level, at least 99% higher than the control or baseline CD39 level, or at least 100% higher than the control or baseline CD39 level.

[0730] In some embodiments, high CD39 is indicated by a TIL population in which the TIL expresses 1-fold or more CD39 than the control or baseline CD39 level. In some embodiments, high CD39 is indicated by a TIL population in which the TIL expresses 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, or 10-fold or more CD39 than the control or baseline CD39 level. In some embodiments, high CD39 is indicated by a TIL population in which the TIL expresses 1-fold or more CD39 than the control or baseline CD39 level. In some embodiments, high CD39 is indicated by a TIL population in which the TIL expresses 2-fold or more CD39 than the control or baseline CD39 level. In some embodiments, high CD39 is indicated by a TIL population in which the TIL expresses 3-fold or more CD39 than the control or baseline CD39 level. In some embodiments, high CD39 is indicated by a TIL population in which the TIL expresses 4-fold or more CD39 than the control or baseline CD39 level. In some embodiments, high CD39 is represented by a TIL population in which the TILs express more than 5 times the CD39 level compared to the control or baseline CD39 level. In some embodiments, high CD39 is represented by a TIL population in which the TILs express more than 10 times the CD39 level compared to the control or baseline CD39 level.

[0731] In some embodiments, pre-selection of CD39-positive TILs is performed by staining the primary cell population, whole tumor digest, and / or whole tumor cell suspension TILs with an anti-CD39 antibody. In some embodiments, the anti-CD39 antibody is a polyclonal antibody, e.g., mouse anti-human CD39 polyclonal antibody, goat anti-human CD39 polyclonal antibody, etc. In some embodiments, the anti-CD39 antibody is a monoclonal antibody. In some embodiments, the anti-CD39 antibody includes, but is not limited to, BY40 (Nikolova, M., et al. PLoS Pathog. 7, e1002110 (2011)), IPH5201, TTX-0303, SRF617, and / or 5F2.

[0732] In some embodiments, the anti-CD39 antibody for use in pre-selection binds to at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% of the cells expressing CD39.

[0733] In some embodiments, the patient is being treated with an anti-CD39 antibody. In some embodiments, the subject is anti-CD39 antibody treatment naive. In some embodiments, the subject is not being treated with an anti-CD39 antibody. In some embodiments, the subject has been previously treated with a chemotherapeutic agent. In some embodiments, the subject has been previously treated with a chemotherapeutic agent but is no longer being treated with a chemotherapeutic agent. In some embodiments, the subject is post-chemotherapeutic or post-anti-CD39 antibody treatment. In some embodiments, the subject is post-chemotherapeutic and post-anti-CD39 antibody treatment. In some embodiments, the patient is anti-CD39 antibody treatment naive. In some embodiments, the subject has treatment-naive cancer or is post-chemotherapeutic but is anti-CD39 antibody treatment naive. In some embodiments, the subject is treatment-naive and post-chemotherapeutic but is anti-CD39 antibody treatment naive.

[0734] In some embodiments where the patient has been previously treated with a first anti-CD39 antibody, pre-selection is performed by staining the primary cell population, whole tumor digest, and / or whole tumor cell suspension TILs with a second anti-CD39 antibody that is not blocked by the first anti-CD39 antibody from binding to CD39 on the surface of the primary TIL cell population.

[0735] In some embodiments where the patient has been previously treated with an anti-CD39 antibody, pre-selection is performed by staining the primary TIL cell population with an antibody that binds to the Fc region of the anti-CD39 antibody insoluble on the surface of the primary TIL cell population ("anti-Fc antibody"). In some embodiments, the anti-Fc antibody is a polyclonal antibody, e.g., mouse anti-human Fc polyclonal antibody, goat anti-human Fc polyclonal antibody, etc. In some embodiments, the anti-Fc antibody is a monoclonal antibody. In some embodiments where the patient has been previously treated with an anti-CD39 human or humanized IgG antibody, the primary TIL cell population is stained with an anti-human IgG antibody. In some embodiments where the patient has been previously treated with an anti-CD39 human or humanized IgG1 antibody, the primary TIL cell population is stained with an anti-human IgG1 antibody. In some embodiments where the patient has been previously treated with an anti-CD39 human or humanized IgG2 antibody, the primary TIL cell population is stained with an anti-human IgG2 antibody. In some embodiments where the patient has been previously treated with anti-CD39 human or humanized IgG3 antibody, the primary TIL cell population is stained with anti-human IgG3 antibody. In some embodiments where the patient has been previously treated with anti-CD39 human or humanized IgG4 antibody, the primary TIL cell population is stained with anti-human IgG4 antibody.

[0736] In some embodiments where the patient has been previously treated with an anti-CD39 antibody, pre-selection is performed by contacting the primary TIL cell population with the same anti-CD39 antibody, and then staining the primary TIL cell population with an anti-Fc antibody that binds to the Fc region of the anti-CD39 antibody insoluble on the surface of the primary TIL cell population.

[0737] In some embodiments, pre-selection is performed using a cell sorting method. In some embodiments, the cell sorting method is a flow cytometry method, such as flow-activated cell sorting (FACS). In some embodiments, the intensity of fluorophores in both the first population and the PBMC population is used to set up FACS gates to establish low, medium, and high intensity levels corresponding to CD39-negative TILs, CD39-intermediate TILs, and CD39-positive TILs, respectively. In some embodiments, the cell sorting method is performed to distinguish three populations using PBMCs, an FMO control, and the sample itself, so that the gates are set to high, medium (also referred to as intermediate), and low (also referred to as negative). In some embodiments, PBMCs are used as a gating control. In some embodiments, the CD39-high population is defined as the cell population that is positive for CD39 above what is observed in the PBMCs. In some embodiments, the CD39+ intermediate population of TILs includes CD39+ cells in the PBMCs. In some embodiments, negative is gated based on FMO. In some embodiments, the FACS gate is set after the step of obtaining and / or receiving a first TIL population from the tumor excised from the subject by processing the tumor sample obtained from the subject into multiple tumor fragments. In some embodiments, gating is set for each sorting. In some embodiments, gating is set for each PBMC sample. In some embodiments, gating is set for each PBMC sample. In some embodiments, a gating template is set from the PBMC every 10, 20, 30, 40, 50, or 60 days. In some embodiments, a gating template is set from the PBMC every 60 days. In some embodiments, a gating template is set for each PBMC sample every 10, 20, 30, 40, 50, or 60 days. In some embodiments, a gating template is set for each PBMC sample every 60 days.

[0738] In some embodiments, the gating for CD39 preselection is fixed for each preselection procedure. In some embodiments, the fixed gating procedure is a CD3+ gating procedure. In some embodiments, the gating procedure is not fixed but is determined based on the population acquired during each selection. In some embodiments, the gating procedure is not fixed but is determined based on the population acquired during each selection event being a CD3+ gating procedure.

[0739] In some embodiments, the gating and correction of the mean fluorescence intensity (MFI) is in the range of approximately 0.5% ± 0.25%. In some embodiments, the gating and correction of the mean fluorescence intensity (MFI) is in the range of approximately 1.75% ± 0.25%. In some embodiments, the gating and correction of the mean fluorescence intensity (MFI) is in the range of approximately 1.75% ± 0.25% when CD39 high gate is set for PBMCs. In some embodiments, the MFI calculation uses the average value measured from 1, 2, 3, or 4 or more lots or batches of PBMCs. In some embodiments, the MFI calculation uses the median value measured from 1, 2, 3, or 4 or more lots or batches of PBMCs.

[0740] In some embodiments, the FACS sorting and correction method used to determine the mean fluorescence intensity (MFI) is adjusted so that the MFI of CD39 relative to control PBMCs (e.g., PBMCs from healthy donors) is in the range of approximately 0.5% to 2.0% (e.g., approximately 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, 0.95%, 1.0%, 1.05%, 1.1%, 1.15%, 1.2%, 1.25%, 1.3%, 1.35%, 1.4%, 1.45%, 1.5%, 1.55%, 1.6%, 1.65%, 1.7%, 1.75%, 1.8%, 1.85%, 1.9%, 1.95%, or 2.0%). In some embodiments, the MFI calculation is the mean value measured using at least two samples of PBMC. In some embodiments, the MFI calculation is the median value measured using at least two samples of PBMC.

[0741] In some embodiments, CD39-positive (CD39+) cells are sorted by FAC and / or other flow cytometry methods. In some embodiments, CD39-positive TILs are CD39-high TILs. In some embodiments, CD39-positive TILs are CD39-intermediate TILs. In some embodiments, CD39+ cells are sorted by utilizing bead selection. In some embodiments, CD39+ cells are sorted by utilizing magnetic bead selection. In some embodiments, bead selection utilizes antibody-conjugated beads, such as commercially available beads, including but not limited to Miltenyi or Fisher, for selection. In some embodiments, CD39+-high cells are sorted by utilizing bead selection. In some embodiments, CD39+-high cells are sorted by utilizing magnetic bead selection. In some embodiments, bead selection utilizes antibody-conjugated beads, such as commercially available beads, including but not limited to Miltenyi or Fisher, for selection. In some embodiments, an anti-CD39 antibody is directly or indirectly conjugated with the beads. In some embodiments, the bead selection process selects both CD39+ and CD3+ TILs. In some embodiments, the anti-CD39 antibodies used in the bead selection method include, but are not limited to, BY40 (Nikolova, M., et al. PLoS Pathog. 7, e1002110 (2011)), IPH5201, TTX-0303, SRF617, and / or 5F2.

[0742] In some embodiments, the collection buffer used to collect CD39+ cells and / or CD39-negative cells does not contain serum. In some embodiments, the collection buffer used to collect CD39+ cells and / or CD39-negative cells contains serum. In some embodiments, the collection buffer used to collect CD39+ cells and / or CD39-negative cells contains components that reduce or lessen the viscosity difference between the sorting buffer and the downstream buffer and / or culture medium. In some embodiments, the collection buffer used to collect CD39+ cells and / or CD39-negative cells contains only human serum albumin (HSA). In some embodiments, the collection buffer used to collect CD39+ cells and / or CD39-negative cells contains equal volumes of HSA and PBS / EDTA buffer. In some embodiments, the collection buffer used to collect CD39+ cells and / or CD39-negative cells contains HSA and PBS / EDTA buffer in a ratio of 1:1, 1:2, 1:3, 1:4, 2:1, 3:1, or 4:1.

[0743] In some embodiments, pre-selection involves selecting CD39-positive TILs from a first TIL population to obtain a CD39-rich TIL population, and includes selecting a TIL population from the first TIL population in which at least 11.27% to 74.4% are CD39-positive TILs. In some embodiments, the first TIL population is at least 20-80% CD39-positive TILs, at least 20-80% CD39-positive TILs, at least 30-80% CD39-positive TILs, at least 40-80% CD39-positive TILs, at least 50-80% CD39-positive TILs, at least 10-70% CD39-positive TILs, at least 20-70% CD39-positive TILs, at least 30-70% CD39-positive TILs, or at least 40-70% CD39-positive TILs.

[0744] In some embodiments, the selection step (e.g., pre-selection and / or selection of CD39-positive cells) is performed. (i) Exposing a first TIL population and PBMC population to an excess monoclonal anti-CD39 IgG4 antibody that binds to CD39 by the N-terminal loop outside the IgV domain of CD39, (ii) The step of adding an excess anti-IgG4 antibody conjugated to the fluorophore, (iii) The step of obtaining a CD39-rich TIL population based on the intensity of the fluorophores of CD39-positive TILs in a first TIL population compared to the intensity of the PBMC population when performed by fluorescence-activated cell sorting (FACS).

[0745] In some embodiments, CD39-positive TILs are CD39-high TILs.

[0746] In some embodiments, at least 70% of a CD39-rich TIL population are CD39-positive TILs. In some embodiments, at least 80% of a CD39-rich TIL population are CD39-positive TILs. In some embodiments, at least 90% of a CD39-rich TIL population are CD39-positive TILs. In some embodiments, at least 95% of a CD39-rich TIL population are CD39-positive TILs. In some embodiments, at least 99% of a CD39-rich TIL population are CD39-positive TILs. In some embodiments, 100% of a CD39-rich TIL population are CD39-positive TILs.

[0747] In some embodiments, the selection of CD39-positive TILs is at least 1 × 10 4 CD39-positive TILs, at least 1 × 10⁶ 5 CD39-positive TILs, at least 1 × 10⁶ 6 CD39-positive TILs, at least 1 × 10⁶ 7 CD39-positive TILs, at least 1 × 10⁶ 8 The selection of CD39-positive TILs is carried out until CD39-positive TILs are found. In some embodiments, the selection of CD39-positive TILs is at least 1 × 10⁶ 6 This process continues until CD39-positive TILs are found.

[0748] In some embodiments, the selection step, exemplified as step A2 in Figure 1, includes (i) exposing a first TIL population to an excess of monoclonal anti-CD39 IgG4 antibody bound to CD39 by the N-terminal loop outside the IgV domain of CD39; (ii) adding the excess anti-IgG4 antibody conjugated to a fluorophore; and (iii) performing fluid cell sorting based on the fluorophore to obtain a CD39-rich TIL population. In some embodiments, the monoclonal anti-CD39 IgG4 antibody is nivolumab, or a variant, fragment, or complex thereof. In some embodiments, the anti-IgG4 antibody is clone anti-human IgG4, clone HP6023. In some embodiments, the anti-CD39 antibody for use in the selection in step (b) is EH12.2H7 or conjugates to the same epitope as nivolumab.

[0749] To determine whether TILs derived from tumor samples are CD39-high, those skilled in the art can utilize a reference value corresponding to the CD39 expression level in peripheral T cells obtained from blood samples from one or more healthy human subjects. CD39-positive cells in the reference sample can be defined using a control with one fluorescence minus a corresponding isotype control. In some embodiments, the CD39 expression level is measured in CD3+ / CD39+ peripheral T cells from healthy subjects (e.g., reference cells) and used to establish a threshold or cutoff value for the CD39 immunostaining intensity in TILs obtained from tumors. The threshold can be defined as the minimum CD39 immunostaining intensity of CD39-high T cells. Thus, TILs with CD39 expression equal to or exceeding the threshold can be considered CD39-high cells. In some cases, CD39-high TILs represent those with the highest CD39 immunostaining intensity, which corresponds to up to 1% or less of all CD3+ cells. In other cases, high CD39 TIL represents the highest intensity of CD39 immunostaining corresponding to up to 0.75% of all CD3+ cells. In some cases, high CD39 TIL represents the highest intensity of CD39 immunostaining corresponding to up to 0.50% of all CD3+ cells. In one case, high CD39 TIL represents the highest intensity of CD39 immunostaining corresponding to up to 0.25% of all CD3+ cells.

[0750] In some embodiments, the PD-1 gating method of WO2019156568 is used for CD39. To determine whether TILs derived from tumor samples are CD39-high, those skilled in the art can utilize a reference value corresponding to the CD39 expression level in peripheral T cells obtained from blood samples from one or more healthy human subjects. CD39-positive cells in the reference sample can be defined using a control with one fluorescence minus a corresponding isotype control. In some embodiments, the CD39 expression level is measured in CD3+ / CD39+ peripheral T cells from healthy subjects (e.g., reference cells) and used to establish a threshold or cutoff value for the CD39 immunostaining intensity in TILs obtained from tumors. The threshold can be defined as the minimum intensity of CD39 immunostaining in CD39-high T cells. Thus, TILs with CD39 expression equal to or exceeding the threshold can be considered CD39-high cells. In some cases, CD39-high TILs represent those with the highest CD39 immunostaining intensity, which corresponds to up to 1% or less of all CD3+ cells. In other cases, high CD39 TIL represents the highest intensity of CD39 immunostaining corresponding to up to 0.75% of all CD3+ cells. In some cases, high CD39 TIL represents the highest intensity of CD39 immunostaining corresponding to up to 0.50% of all CD3+ cells. In one case, high CD39 TIL represents the highest intensity of CD39 immunostaining corresponding to up to 0.25% of all CD3+ cells.

[0751] In some embodiments, selected CD39-positive (CD39+) cells can be frozen before proceeding with the first proliferation by priming, for example, step B of Figure 1 (in particular, for example, Figures 1B and / or 1C and / or 1D and / or 1E and / or 1F and / or 1G and / or 1H).

[0752] a. Fluorophores In some embodiments, the primary TIL cell population is stained with a cocktail containing fluorophore-conjugated anti-CD39 antibody and fluorophore-conjugated anti-CD3 antibody. In some embodiments, the primary TIL cell population is stained with a cocktail containing fluorophore-conjugated anti-CD39 antibody (e.g., PE, live / dead violet) and anti-CD3-FITC. In some embodiments, the primary TIL cell population is stained with a cocktail containing anti-CD39-PE, anti-CD3-FITC, and live / dead blue stain (ThermoFisher, MA, catalog no. L23105). In some embodiments, after incubation with anti-PD1 antibody, CD39-positive cells are selected for proliferation by a first proliferation by priming in step B, as described herein.

[0753] In some embodiments, the fluorophore includes, but is not limited to, PE (phycoerythrin), APC (allophycocyanin), PerCP (peridinine chlorophyll protein), DyLight 405, Alexa Fluor 405, Pacific Blue, Alexa Fluor 488, FITC (fluorescein isothiocyanate), DyLight 550, Alexa Fluor 647, DyLight 650, and Alexa Fluor 700. In some embodiments, the fluorophore includes, but is not limited to, PE-Alexa Fluor® 647, PE-Cy5, PerCP-Cy5.5, PE-Cy5.5, PE-Alexa Fluor® 750, PE-Cy7, and APC-Cy7. In some embodiments, the fluorophore includes, but is not limited to, fluorescein dyes. Examples of fluorescein dyes include, but are not limited to, 5-carboxyfluorescein, fluorescein-5-isothiocyanate and 6-carboxyfluorescein, 5,6-dicarboxyfluorescein, 5-(and 6)-sulfofluorescein, sulfonefluorescein, succinylfluorescein, 5-(and 6)-carboxySNARF-1, carboxyfluorescein sulfonate, carboxyfluorescein zwitterion, carboxyfluorescein quaternary ammonium, carboxyfluorescein phosphonate, carboxyfluorescein GABA, 5'(6')-carboxyfluorescein, carboxyfluorescein-cys-Cy5, and fluorescein glutathione. In some embodiments, the fluorescent moiety is a rhodamine dye. Examples of rhodamine dyes include, but are not limited to, tetramethylrhodamine-6-isothiocyanate, 5-carboxytetramethylrhodamine, 5-carboxylodol derivatives, carboxyrhodamine 110, tetramethyl and tetraethylrhodamine, diphenyldimethyl and diphenyldiethylrhodamine, dinaphthylrhodamine, and rhodamine 101 sulfonyl chloride (marketed under the trade name TEXAS RED®). In some embodiments, the fluorescent portion is a cyanine dye.Examples of cyanine pigments include, but are not limited to, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, and Cy7.

[0754] 7. Pre-selection of CD38-CD38 (as illustrated in step A2 of Figure 1) According to the method of the present invention, TILs are pre-selected to be CD38-positive (CD38+) before the first proliferation by priming.

[0755] In some embodiments, the TILs of the present invention are pre-selected for a depletion marker such as CD38 (see, for example, Canale, FP, et al. Cancer Res. 78:115-128 (2018) and / or Duhne, T., et al., Nat Commun. 9:2724 (2018)). According to the method of the present invention, the TILs are pre-selected to be CD38-positive (CD38+) before the first proliferation by priming.

[0756] In some embodiments, a minimum of 3,000 TILs are required to sow in the first propagation. In some embodiments, the pre-selection step yields a minimum of 3,000 TILs. In some embodiments, a minimum of 4,000 TILs are required to sow in the first propagation. In some embodiments, the pre-selection step yields a minimum of 4,000 TILs. In some embodiments, a minimum of 5,000 TILs are required to sow in the first propagation. In some embodiments, the pre-selection step yields a minimum of 5,000 TILs. In some embodiments, a minimum of 6,000 TILs are required to sow in the first propagation. In some embodiments, the pre-selection step yields a minimum of 6,000 TILs. In some embodiments, a minimum of 7,000 TILs are required to sow in the first propagation. In some embodiments, the pre-selection step yields a minimum of 7,000 TILs. In some embodiments, a minimum of 8,000 TILs are required to sow in the first propagation. In some embodiments, the pre-selection step yields a minimum of 8,000 TILs. In some embodiments, a minimum of 9,000 TILs are required to seed for a first growth. In some embodiments, the pre-selection step yields a minimum of 9,000 TILs. In some embodiments, a minimum of 10,000 TILs are required to seed for a first growth. In some embodiments, the pre-selection step yields a minimum of 10,000 TILs. In some embodiments, the cells are grown or expanded to a density of 200,000. In some embodiments, the cells are grown or expanded to a density of 200,000 to provide about 2e8 TILs to initiate a rapid second growth. In some embodiments, the cells are grown or expanded to a density of 150,000. In some embodiments, the cells are grown or expanded to a density of 150,000 to provide about 2e8 TILs to initiate a rapid second growth. In some embodiments, the cells are grown or expanded to a density of 250,000.In some embodiments, cells are grown or expanded to a density of 250,000 to provide approximately 2e8 TIL for initiating rapid second proliferation. In some embodiments, the minimum cell density is 10,000 cells to give 10e6 for initiating rapid second proliferation. In some embodiments, a seeding density of 10e6 for initiating rapid second proliferation may result in a TIL of more than 1e9.

[0757] In some embodiments, the TILs for use in the first proliferation by priming are CD38-positive (CD38+) (e.g., after pre-selection and before the first proliferation by priming). In some embodiments, the TILs for use in the first proliferation by priming are at least 75% CD38-positive, at least 80% CD38-positive, at least 85% CD38-positive, at least 90% CD38-positive, at least 95% CD38-positive, at least 98% CD38-positive, or at least 99% CD38-positive (e.g., after pre-selection and before the first proliferation by priming). In some embodiments, the CD38 population is CD38-low (CD38-low). In some embodiments, the TIL for use in the first growth by priming is at least 25% CD38 low, at least 30% CD38 low, at least 35% CD38 low, at least 40% CD38 low, at least 45% CD38 low, at least 50% CD38 low, at least 55% CD38 low, at least 60% CD38 low, at least 65% CD38 low, at least 70% CD38 low, at least 75% CD38 low, at least 80% CD38 low, at least 85% CD38 low, at least 90% CD38 low, at least 95% CD38 low, at least 98% CD38 low, or at least 99% CD38 low (e.g., after pre-selection and before the first growth by priming).

[0758] In some embodiments, low CD38 is represented by TIL populations with ≤5% CD38 positivity, ≤10% CD38 positivity, ≤15% CD38 positivity, ≤20% CD38 positivity, ≤25% CD38 positivity, ≤30% CD38 positivity, ≤35% CD38 positivity, ≤40% CD38 positivity, ≤45% CD38 positivity, ≤50% CD38 positivity, ≤55% CD38 positivity, and ≤60% CD38 positivity. In some embodiments, low CD38 is represented by TIL populations with ≤5% CD38 positivity. In some embodiments, low CD38 is represented by TIL populations with ≤10% CD38 positivity. In some embodiments, low CD38 is represented by TIL populations with ≤15% CD38 positivity. In some embodiments, low CD38 is represented by TIL populations with ≤20% CD38 positivity. In some embodiments, low CD38 is represented by TIL populations with ≤25% CD38 positivity. In some embodiments, CD38 low is represented by a TIL population with less than 30% CD38 positivity.

[0759] In some embodiments, CD38 low means that TIL expresses CD38 that is 25% lower than control or baseline CD38 levels, 30% lower than control or baseline CD38 levels, 35% lower than control or baseline CD38 levels, 40% lower than control or baseline CD38 levels, 45% lower than control or baseline CD38 levels, 50% lower than control or baseline CD38 levels, 55% lower than control or baseline CD38 levels, or 60% lower than control or baseline CD38 levels. The TIL population exhibits CD38 levels that are 65% lower than the control or baseline CD38 level, 70% lower than the control or baseline CD38 level, 75% lower than the control or baseline CD38 level, 80% lower than the control or baseline CD38 level, 85% lower than the control or baseline CD38 level, 90% lower than the control or baseline CD38 level, 95% lower than the control or baseline CD38 level, or 99% lower than the control or baseline CD38 level.

[0760] In some embodiments, low CD38 is represented by a TIL population in which TILs express CD38 at a level 1 or less higher than the control or baseline CD38 level. In some embodiments, low CD38 is represented by a TIL population in which TILs express CD38 at a level 1, 2, 3, 4, 5, or 10 times higher than the control or baseline CD38 level. In some embodiments...

Claims

1. A method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, (a) To provide a first TIL population obtained from and / or received from tumors excised from the subject by processing tumor samples obtained from the subject into multiple tumor fragments, (b) Select PD-1, CD39, CD38, CD103, CD101, LAG3, TIM3, and / or TIGIT-positive TILs from the first TIL population of step (a) to obtain a TIL population rich in PD-1, CD39, CD38, CD103, CD101, LAG3, TIM3, and / or TIGIT, (c) Producing a second TIL population by performing a first proliferation by priming, wherein the TIL population rich in PD-1, CD39, CD38, CD103, CD101, LAG3, TIM3, and / or TIGIT is cultured in a cell culture medium containing IL-2, OKT-3, and antigen-presenting cells (APC), wherein the first proliferation by priming is performed in a container having a first gas-permeable surface area, the first proliferation by priming is performed for a first period of 1 to 11 days, and the second TIL population is obtained. (d) Producing a third TIL population by rapidly growing the second TIL population in the cell culture medium of the second TIL population by supplementing it with additional IL-2, OKT-3, and APC, wherein the number of APCs added to the rapid second growth is at least twice the number of APCs added in step (c), the rapid second growth is carried out for a second period of 1 to 11 days, obtaining the third TIL population, the third TIL population being a therapeutic TIL population, and the rapid second growth being carried out in a container having a second gas-permeable surface area. (e) Collecting the therapeutic TIL population obtained from step (d), (f) Transferring the TIL sample collected from step (e) to an injection bag, The method, including the method described above.

2. A method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, a) To provide a first TIL population obtained and / or received from tumors excised from the subject by processing tumor samples obtained from the subject into multiple tumor fragments, b) Selecting PD-1, CD39, CD38, CD103, CD101, LAG3, TIM3, and / or TIGIT-positive TILs from the first TIL population of step (a) to obtain a TIL population rich in PD-1, CD39, CD38, CD103, CD101, LAG3, TIM3, and / or TIGIT, c1) Producing a second TIL population by priming a TIL population rich in PD-1, CD39, CD38, CD103, CD101, LAG3, TIM3, and / or TIGIT in a cell culture medium containing IL-2 and OKT-3, wherein the first proliferation by priming is carried out for a first period of 1 to 11 days, thereby obtaining the second TIL population, or producing the second TIL population. c2) Producing a second TIL population by priming a TIL population rich in PD-1, CD39, CD38, CD103, CD101, LAG3, TIM3, and / or TIGIT in a cell culture medium containing IL-2, OKT-3, and antigen-presenting cells (APCs), wherein the first proliferation by priming is carried out for a first period of 1 to 7, 8, 9, 10, or 11 days, thereby obtaining the second TIL population, and producing the second TIL population. d) Producing a third TIL population by rapidly growing the second TIL population in contact with a cell culture medium containing IL-2, OKT-3, and APC, wherein the rapid second growth is carried out over a second period of 1 to 11 days, thereby obtaining the third TIL population, and the third TIL population is a therapeutic TIL population. e) The method comprising collecting the therapeutic TIL population obtained from step (d).

3. The method according to claim 2, wherein the number of APCs in the culture medium in step (d) is greater than the number of APCs in the culture medium in step (c2), or the number of APCs in the culture medium in step (d) is equal to the number of APCs in the culture medium in step (c2).

4. A method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, (a) A first TIL population selected to be PD-1, CD39, CD38, CD103, CD101, LAG3, TIM3, and / or TIGIT positive, wherein the first TIL population, which can be obtained by processing a tumor sample from a subject by tumor digestion and selecting the PD-1, CD39, CD38, CD103, CD101, LAG3, TIM3, and / or TIGIT positive TILs, is cultured in a cell culture medium containing IL-2, OKT-3, and antigen-presenting cells (APCs) to produce a second TIL population by performing a first proliferation by priming, wherein the first proliferation by priming is performed in a container having a first gas-permeable surface area, the first proliferation by priming is performed for a first period of 1 to 11 days, and the second TIL population is obtained, and the production is performed. (b) Producing a third TIL population by rapidly growing it second by contacting the second TIL population with a cell culture medium of the second TIL population containing additional IL-2, OKT-3, and APC, wherein the number of APCs in the rapid second growth is at least twice the number of APCs in step (a), the rapid second growth is carried out for a second period of 1 to 11 days, obtaining the third TIL population, the third TIL population being a therapeutic TIL population, and the rapid second growth being carried out in a container having a second gas-permeable surface area. (c) The method comprising taking the therapeutic TIL population obtained from step (b).

5. A method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, (a1) Producing a second TIL population by priming a first TIL population selected to be PD-1 positive, wherein the first priming growth is carried out for a first period of 1 to 7, 8, 9, 10, or 11 days, thereby obtaining the second TIL population, or producing the second TIL population, or (a2) Producing a second TIL population by culturing a first TIL population in a cell culture medium containing IL-2, OKT-3, and antigen-presenting cells (APCs), thereby performing a first proliferation by priming the first TIL population, which has been selected to be PD-1, CD39, CD38, CD103, CD101, LAG3, TIM3, and / or TIGIT positive, wherein the first proliferation by priming is performed for a first period of 1 to 7, 8, 9, 10, or 11 days, thereby obtaining the second TIL population, and producing the second TIL population. (b) Rapid second growth of the second TIL population by contacting it with a cell culture medium containing IL-2, OKT-3, and APC to produce a third TIL population, wherein the rapid second growth is carried out over a second period of 1 to 11 days, thereby obtaining the third TIL population, and the third TIL population is a therapeutic TIL population. (c) The method comprising taking the therapeutic TIL population obtained from step (b).

6. The method according to claim 5, wherein the number of APCs in the culture medium in step (b) is greater than the number of APCs in the culture medium in step (a2), or the number of APCs in the culture medium in step (b) is equal to the number of APCs in the culture medium in step (a2).

7. The method according to any one of claims 1 to 6, wherein the PD-1, CD39, CD38, CD103, CD101, LAG3, TIM3, and / or TIGIT-positive TILs are high PD-1 corresponding to a TIL population in which at least 75% are PD-1 positive, high CD39 corresponding to a TIL population in which at least 75% are CD39 positive, low CD38 corresponding to a TIL population in which 30% or less are CD38 positive, high CD103 corresponding to a TIL population in which at least 75% are CD103 positive, low CD101 corresponding to a TIL population in which 60% or less are CD101 positive, high LAG3 corresponding to a TIL population in which at least 75% are LAG3 positive, high TIM3 corresponding to a TIL population in which at least 75% are TIM3 positive, and / or high TIGIT corresponding to a TIL population in which at least 75% are TIGIT positive.

8. The method according to any one of claims 1 to 6, wherein the selection of the PD-1 positive TILs includes a selection method selected from the group consisting of flow cytometry, FACS, antibody-based bead selection, and antibody-based magnetic bead selection, the selection method comprising: (i) exposing the first TIL population to an excess of monoclonal anti-PD-1 IgG4 antibody bound to PD-1 by the N-terminal loop outside the IgV domain of PD-1; (ii) adding an excess of anti-IgG4 antibody compounded with a fluorophore; and (iii) performing flow cell sorting based on the fluorophore to obtain a PD-1 rich TIL population.

9. The method according to claim 8, wherein the monoclonal anti-PD-1 IgG4 antibody is nivolumab, or a variant, fragment, or complex thereof, and the anti-IgG4 antibody is clone anti-human IgG4, clone HP6023.

10. (i) The selection of PD-1, CD39, CD38, CD103, CD101, LAG3, TIM3, and / or TIGIT-positive TILs continues until at least 1 × 10⁶ PD-1, CD39, CD38, CD103, CD101, LAG3, TIM3, and / or TIGIT-positive TILs are present, and / or (ii) The cell culture medium for culturing the first TIL population contains 2-mercaptoethanol, or the cell culture medium for culturing the second TIL population contains 2-mercaptoethanol, or the cell culture medium for culturing the first TIL population and the second TIL population contains 2-mercaptoethanol, and / or (iii) The PD-1, CD39, CD38, CD103, CD101, LAG3, TIM3, and / or TIGIT-positive TILs are selected using anti-PD-1, anti-CD39, anti-CD38, anti-CD103, anti-CD101, anti-LAG3, anti-TIM3, and / or anti-TIGIT antibody composite beads, and / or the PD-1, CD39, CD38, CD103, CD101, LAG3, TIM3, and / or TIGIT-positive TILs are selected using anti-PD-1, anti-CD39, anti-CD38, anti-CD103, anti-CD101, anti-LAG3, anti-TIM3, and / or anti-TIGIT antibody composite magnetic beads, respectively. Selected, and / or the PD-1, CD39, CD38, CD103, CD101, LAG3, TIM3, and / or TIGIT-positive TILs each bind to anti-PD-1, anti-CD39, anti-CD38, anti-CD103, anti-CD101, anti-LAG3, anti-TIM3, and / or anti-TIGIT antibody complex beads, and the PD-1, CD39, CD38, CD103, CD101, LAG3, TIM3, and / or TIGIT-negative TILs each do not bind to anti-PD-1, anti-CD39, anti-CD38, anti-CD103, anti-CD101, anti-LAG3, anti-TIM3, and / or anti-TIGIT antibody complex beads, and / or (iv) The ratio of the number of APCs in the rapid second growth to the number of APCs in the first growth by priming is selected from the range of 1.5:1 to 20:1, and / or (v) The number of APCs in the first proliferation by priming is 1 × 10 ８ APC ~ 3.5 x 10 ８ Selected from the range of APCs, the number of APCs in the rapid second growth is 3.5 × 10 ８ APC ~ 1 x 10 ９ The method according to any one of claims 1 to 6, selected from the range of APC.

11. The method, after the step of collecting the therapeutic TIL population, The method according to any one of claims 2 to 6, further comprising the additional step of transferring the collected therapeutic TIL population to an infusion bag.

12. (i) The first growth by priming is carried out in a plurality of separate containers, in each of the separate containers, the second TIL population is obtained from the first TIL population in the first growth step by priming, the third TIL population is obtained from the second TIL population in the rapid second growth step, and the therapeutic TIL population obtained from the third TIL population is collected from each of the plurality of containers and compounded to produce the collected TIL population, wherein the plurality of separate containers comprises 2 to 20 separate containers, each of the separate containers comprises a first gas permeable surface area, or (ii) The method according to any one of claims 1 to 6, wherein the first growth by priming is carried out in a single container, the single container comprises a first gas permeable surface area, in the step of the first growth by priming, the cell culture medium comprises antigen-presenting cells (APCs), the APCs are layered on the first gas permeable surface area with an average thickness of 1 to 3 cell layers, and in the step of rapid second growth, the APCs are layered on the first gas permeable surface area with a thickness of 3 to 5 cell layers.

13. The method according to any one of claims 2 to 6, wherein in the first growth step by priming, the first growth by priming is carried out in a first container having a first gas-permeable surface area, and in the rapid second growth step, the rapid second growth is carried out in a second container having a second gas-permeable surface area, the second container being larger than the first container, and in the first growth step by priming, the cell culture medium contains antigen-presenting cells (APCs), and the APCs are layered on the first gas-permeable surface area with an average thickness of 1 to 3 cell layers, or in the first growth step by priming, the APCs are layered on the first gas-permeable surface area with an average thickness of about 1.5 to 2.5 cell layers, and in the rapid second growth step, the APCs are layered on the second gas-permeable surface area with an average thickness of 3 to 5 cell layers.

14. The method according to any one of claims 1 to 6, wherein, for each container in which the first proliferation by priming is performed on a first TIL population, the rapid second proliferation is performed in the same container on the second TIL population produced from the first TIL population, each container includes a first gas-permeable surface area, in the step of the first proliferation by priming, the cell culture medium includes antigen-presenting cells (APCs), and the APCs are layered on the first gas-permeable surface area with an average thickness of 1 to 3 cell layers, and in the step of the rapid second proliferation, the APCs are layered on the first gas-permeable surface area with an average thickness of 3 to 5 cell layers.

15. The method according to any one of claims 1 to 6, wherein, in the first proliferation by priming step, for each container in which the first TIL population is subjected to the first proliferation by priming step, the container includes a first gas-permeable surface area, the cell culture medium includes antigen-presenting cells (APCs), the APCs are layered on the first gas-permeable surface area, and the ratio of the average number of layers of the APCs layered in the first proliferation step by priming to the average number of layers of the APCs layered in the rapid second proliferation step is selected from the range of 1:1.1 to 1:

10.

16. (i) After the rapid second growth step of 2-3 days, the cell culture medium is supplemented with additional IL-2, and / or (ii) The method according to any one of claims 1 to 6, wherein the method comprises a step of collecting the therapeutic TIL population using a cryopreservation process which includes the step of cryopreserving the infusion bag, the cryopreservation process which is carried out using a 1:1 ratio of the collected TIL population to a cryopreservation medium containing 7% to 10% dimethyl sulfoxide (DMSO).

17. (i) The antigen-presenting cells (APCs) are peripheral blood mononuclear cells (PBMCs), the PBMCs are irradiated, allogeneic, and / or (ii) In the first proliferation step by priming, the cell culture medium contains peripheral blood mononuclear cells (PBMCs), and the total number of PBMCs in the cell culture medium in the first proliferation step by priming is 2.5 × 10 ８ is, and / or (iii) In the rapid second growth step, the antigen-presenting cells (APCs) in the cell culture medium are peripheral blood mononuclear cells (PBMCs), and the total number of PBMCs added to the cell culture medium in the rapid second growth step is 5 × 10 ８ is, and / or (iv) The step of collecting the therapeutic TIL population is performed using a membrane cell processing system or a LOVO cell processing system, and / or (v) The cell culture medium is provided in a container selected from the group consisting of G containers and Xuri cell bags, and / or (vi) Two to three days after the rapid second growth step, the cell culture medium is supplemented with additional IL-2, with an IL-2 concentration of 10,000 IU / mL to 5,000 IU / mL and / or (vii) The infusion bag in the step of transferring the collected therapeutic TIL population to an infusion bag is an infusion bag containing HypoThermosol, and / or (viiii) The plurality of tumor fragments, a) In the first propagation step by priming, each container contains 60 fragments, each fragment measuring 27 mm ３ Having a volume of, b) 1300 mm ３ to 1500 mm ３ comprising 30 to 60 fragments with a total volume of, or c) The method according to any one of claims 1 to 6, comprising about 50 fragments with a total mass of 1 gram to 1.5 grams.

18. The method according to any one of claims 1 to 6, wherein the first growth step by priming and the rapid second growth step are each carried out individually within a period of 5, 6, 7, 8, 9, 10, or 11 days.

19. (i) The first step of priming the therapeutic TIL population through the collection of the therapeutic TIL population is performed within a period of 14 to 16 days, and further comprises the step of cryopreserving the collected therapeutic TIL population using a cryopreservation process, and / or (ii) The therapeutic TIL population collected in the step of collecting the therapeutic TIL population contains a sufficient number of TILs to constitute a therapeutically effective dose of TILs, and the number of TILs sufficient to constitute a therapeutically effective dose is 2.3 × 10 １０ ~13.7 x 10 １０ The method according to any one of claims 1 to 6.

20. The method according to claim 1 or 11, further comprising the step of cryopreserving the infusion bag containing the collected TIL population using a cryopreservation process using a 1:1 ratio of the collected TIL population to a cryopreservation medium.

21. The method according to claims 1, 2, 4, and 5, wherein all steps are performed within a period of 14 to 22 days.

22. (i) The collected therapeutic TIL population contains enough TILs to make up a therapeutically effective dose of TILs, and the number of TILs to make up a therapeutically effective dose is 2.3 × 10 １０ ~13.7 x 10 １０ is, and / or (ii) The container in the first growth step by priming is larger than the container in the rapid second growth step, and / or (iii) The method according to any one of claims 1 to 6, wherein effector T cells and / or central memory T cells obtained from the third TIL population exhibit increased CD8 and CD28 expression compared to effector T cells and / or central memory T cells obtained from the second cell population.

23. A therapeutically effective dose of TIL is used to treat the target cancer, Prior to administering the therapeutically effective dose of TIL, a non-myeloablative lymphocyte depletion regimen has been administered to the subject, the non-myeloablative lymphocyte depletion regimen includes the step of administering cyclophosphamide at a dose of 60 mg / m² / day for 2 days, followed by fludarabine at a dose of 25 mg / m² / day for 5 days, and / or The method according to any one of claims 1 to 6, wherein the patient is further treated with a high-dose IL-2 regimen initiated the day following the administration of the TIL to the subject, the high-dose IL-2 regimen comprising 600,000 or 720,000 IU / kg administered as a 15-minute bolus intravenous infusion every 8 hours until an acceptable level is reached.

24. (i) The cancer is selected from the group consisting of melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, triple-negative breast cancer, cancer caused by human papillomavirus, head and neck cancer (head and neck squamous cell carcinoma (HNSCC)), glioblastoma (GBM), gastrointestinal cancer, kidney cancer, and renal cell carcinoma, and / or (ii) The first growth by priming is carried out in a first container, the rapid second growth is carried out in a second container, and each of the first and second containers is GREX-10, GREX-100, or GREX-500, and / or (iii) The subject has been previously treated with an anti-PD-1 antibody, or the subject has not been previously treated with an anti-PD-1 antibody, and / or (iv) The first proliferation step by priming is performed on the first TIL population that is selected or enriched for PD-1 positive TILs by contacting the first TIL population with an anti-PD-1 antibody to form a first complex of the anti-PD-1 antibody and TIL cells in the first TIL population, and then isolating the first complex to obtain the first TIL population that is selected or enriched for PD-1 positive TILs, wherein the anti-PD-1 antibody is performed on the first TIL population that is selected or enriched for PD-1 positive TILs, (a) comprising an Fc region, wherein after the step of forming the first complex and before the step of isolating the first complex, the method further includes the step of contacting the first complex with an anti-Fc antibody bound to the Fc region of the anti-PD-1 antibody to form a second complex of the anti-Fc antibody and the first complex, wherein the step of isolating the first complex is performed by isolating the second complex, or (b) EH12.2H7, PD1.3.1, SYM021, M1H4, A17188B, nivolumab (BMS-936558, Bristol-Myers Squibb; Opdivo®), pembrolizumab (lambrolizumab, MK03475 or MK-3475, Merck; Keytruda®), H12.1, PD1.3.1, NAT 105, humanized anti-PD-1 antibody JS001 (ShangHai JunShi), monoclonal anti-PD-1 antibody TSR-042 (Tesaro, Inc.), pizilizumab (anti-PD-1 mAb The method according to claim 23, comprising an antibody selected from the group consisting of CT-011 (Medivation), anti-PD-1 monoclonal antibody BGB-A317 (BeiGene), anti-PD-1 antibody SHR-1210 (ShangHai HengRui), human monoclonal antibody REGN2810 (Regeneron), human monoclonal antibody MDX-1106 (Bristol-Myers Squibb), humanized anti-PD-1 IgG4 antibody PDR001 (Novartis), and RMP1-14 (rat IgG) - BioXcell catalog number BP0146.

25. The subject is (i) previously treated with a first anti-PD1 antibody, and the first proliferation step by priming is performed on the first TIL population selected or enriched for PD-1 positive TILs by contacting the first TIL population with a second anti-PD-1 antibody to form a first complex of the second anti-PD-1 antibody and TILs in the first TIL population, and then isolating the first complex to obtain the first TIL population selected or enriched for PD-1 positive TILs, wherein the second anti-PD-1 antibody is not blocked from binding to the first TIL population by the first anti-PD-1 antibody insolubilized in the first TIL population, or (ii) having been previously treated with a first anti-PD1 antibody, the first proliferation step by priming is performed on the first TIL population selected or enriched for PD-1 positive TILs by contacting the first TIL population with a second anti-PD-1 antibody to form a first complex of the second anti-PD-1 antibody and TILs in the first TIL population, then isolating the first complex and obtaining the first TIL population selected or enriched for PD-1 positive TILs, wherein the second anti-PD-1 antibody is blocked from binding to the first TIL population by the first anti-PD-1 antibody which has been insolubilized in the first TIL population. The first anti-PD-1 antibody and the second anti-PD-1 antibody include an Fc region, and after the step of forming the first complex and before the step of isolating the first complex, the method is performed by (a) contacting the first complex with an anti-Fc antibody bound to the Fc region of the first anti-PD-1 antibody and the Fc region of the second anti-PD-1 antibody to form a second complex of the anti-Fc antibody and the first complex, and isolating the first complex by isolating the second complex, or (b) the first complex The method according to claim 24, further comprising the steps of contacting the conjugate with an anti-Fc antibody that binds to the Fc region of the second anti-PD-1 antibody to form a second complex of the anti-Fc antibody and the first complex; contacting the first anti-PD-1 antibody, insoluble in the first TIL population, with the anti-Fc antibody to form a third complex of the anti-Fc antibody and the first anti-PD-1 antibody, insoluble in the first TIL population; and separating the second and third complexes to obtain the first TIL population that is selected or concentrated for PD-1 positive TILs.

26. (i) The first proliferation step by priming is performed on a first TIL population selected or enriched for PD-1, LAG3, TIM3, and / or TIGIT-positive TILs, with at least 11.27% to 74.4% being PD-1-positive TILs, and / or (ii) The first propagation step by priming is (a) Exposing the first TIL population and PBMC population to an excess monoclonal anti-PD-1 IgG4 antibody that binds to PD-1 by the N-terminal loop outside the IgV domain of PD-1, (b) The step of adding an excess anti-IgG4 antibody conjugated to the fluorophore, (c) A step of obtaining a first TIL population that is selected or enriched for PD-1 positive TILs based on the intensity of the fluorophores of the PD-1 positive TILs in the first TIL population compared with the intensity of the PBMC population when performed by fluorescence-activated cell sorting (FACS), and a step of performing on the first TIL population that is selected or enriched for PD-1 positive TILs by the above. and / or (iii) The method according to any one of claims 1 to 6, wherein at least 80% of the first TIL population selected or concentrated for PD-1 positive TILs are PD-1 positive TILs, at least 80% of the first TIL population selected or concentrated for LAG3 positive TILs are LAG3 positive TILs, at least 80% of the first TIL population selected or concentrated for TIM3 positive TILs are TIM3 positive TILs, and / or at least 80% of the first TIL population selected or concentrated for TIGIT positive TILs are TIGIT positive TILs.

27. (i) The TIL is selected as PD-1 positive (PD-1+), LAG3 positive (LAG3+ positive), CD38 positive (CD38+), and CD101 positive (CD101+), or the TIL is selected as high PD-1, high LAG3, low CD38, and low CD101, or the TIL is selected as PD-1 positive (PD-1+), LAG3 positive (LAG3+ positive), and CD38 positive (CD38+), or the TIL is selected as high PD-1, high LAG3, and low CD38, or the TIL is selected as PD-1 positive (PD-1+), LAG3 positive (LAG3+ positive), and CD101 positive (CD101+), or the TIL is selected as high PD-1, high LAG3, and CD10 The method according to any one of claims 1 to 6, wherein the TIL is selected as low, or the TIL is selected as PD-1 positive (PD-1+) and CD38 positive (CD38+), or the TIL is selected as high PD-1 and low CD38, or the TIL is selected as PD-1 positive (PD-1+) and CD101 positive (CD101+), or the TIL is selected as high PD-1 and low CD101, with high PD-1 corresponding to a TIL population where at least 75% are PD-1 positive, high LAG3 corresponding to a TIL population where at least 75% are LAG3 positive, low CD38 corresponding to a TIL population where 30% or less are CD38 positive, and low CD101 corresponding to a TIL population where 60% or less are CD101 positive.

28. (i) The selection includes a selection method in which the selection is made from the group consisting of flow cytometry, FACS, antibody-based bead selection, and antibody-based magnetic bead selection, and / or (ii) The selection includes a two-step selection, (a) A first selection step including a method for selecting PD-1+, LAG3+, TIM3+, and / or TIGIT+, (b) a second selection step including a method for selecting CD38+ and / or CD101+, The method according to any one of claims 1 to 6.

29. (i) The beads used for selecting the antibody-based beads for PD-1+, LAG3+, TIM3+, and / or TIGIT+TIL are anti-PD-1, anti-LAG3, anti-TIM3, and / or anti-TIGIT antibody composite beads, respectively, and / or (ii) The beads used for selecting the antibody-based beads of the CD38+ or CD101+ TIL are anti-CD38 or anti-CD101 antibody-complex beads, respectively, and / or (iii) The beads used for selecting the antibody-based magnetic beads for PD-1+, LAG3+, TIM3+, and / or TIGIT+TIL are, respectively, anti-PD-1, anti-LAG3, anti-TIM3, and / or anti-TIGIT antibody composite magnetic beads, and / or (iv) The beads used in the selection of the antibody-based magnetic beads for CD38+ or CD101+ TIL are anti-CD38 or anti-CD101 antibody-composite magnetic beads, respectively, and / or (v) The method according to claim 25, wherein the PD-1+, LAG3+, TIM3+, and TIGIT+ TILs each bind to anti-PD-1, anti-LAG3, anti-TIM3, and / or anti-TIGIT antibody composite beads, and the PD-1, LAG3, TIM3, and / or TIGIT-negative TILs each do not bind to the anti-PD-1, anti-LAG3, anti-TIM3, and / or anti-TIGIT antibody composite beads.

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