Improved selection of tumor-reactive T cells

Abstract

The present invention provides methods for preselecting TILs based on PD-1 expression, as well as methods for expanding those preselected PD-1-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. 62 / 756,006 filed on 5 November 2018, U.S. Provisional Patent Application No. 62 / 826,831 filed on 29 March 2019, U.S. Provisional Patent Application No. 62 / 903,629 filed on 20 September 2019, and U.S. Provisional Patent Application No. 62 / 924,602 filed on 22 October 2019, which are incorporated herein by reference in their entirety. [Background technology]

[0002] The treatment of large, refractory cancers using adoptive transfer of tumor-infiltrating lymphocytes (TILs) represents 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 challenging to achieve due to technical, logistical, and regulatory issues related to cell proliferation. IL-2-based TIL proliferation and subsequent “rapid proliferation processes” (REP) have become the preferred method for 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 excess (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 a 14-day period. Dudley, et al., J. Immunother. 2003, 26, 332-42. TILs undergoing the REP procedure have successfully enabled adoptive cell therapy in patients with melanoma following host immunosuppression. Current infusion receptor parameters depend on the TIL composition reading (e.g., CD28, CD8, or CD4 positive), as well as the reticular proliferation and viability of the REP product.

[0003] Current TIL manufacturing processes are limited by length, cost, sterility concerns, and other factors described herein, thus 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 therapeutic TIL populations 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 expression to obtain TILs with enhanced tumor-specific lethality (e.g., enhanced cytotoxicity). [Overview of the Initiative]

[0004] The present invention provides a method for growing TILs and producing a therapeutic TIL population, the method comprising a PD-1 status pre-selection step.

[0005] In some embodiments, the present invention provides a method for growing tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, the method being (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-positive TILs from the first TIL population in (a) to obtain a TIL population rich in PD-1, (c) A PD-1-rich TIL population is cultured in a cell culture medium containing IL-2, OKT-3, and antigen-presenting cells (APCs) to perform a first proliferation by priming and produce a second TIL population, 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 approximately 1 to 7 / 8 days, and a second TIL population is obtained, and the second TIL population is produced in a larger number than the first TIL population. (d) Producing a third TIL population by rapidly growing a second population of cells 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 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), (f) Transferring the TIL population collected from step (e) to an injection bag.

[0006] In some embodiments, the present invention provides a method for growing tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, the method being 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-positive TILs from the first TIL population in (a) to obtain a TIL population rich in PD-1, c) A PD-1-rich TIL population is cultured in a cell culture medium containing IL-2, OKT-3, and optionally antigen-presenting cells (APCs) to perform a first proliferation by priming and produce a second TIL population, wherein the first proliferation by priming takes place over a first period of approximately 1 to 7 / 8 days, yielding a second TIL population, and the second TIL population is produced in a larger number than the first TIL population. d) Rapid growth of a second TIL population by contacting the second TIL population 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. e) Collecting a therapeutic TIL population obtained from step (d), including:

[0007] In some embodiments, “acquiring” indicates that the TIL used in the method and / or process may be directly derived from the specimen (including surgical excision, needle biopsy, core biopsy, micro-biopsy, or other specimen) as part of a step in the method and / or process. In some embodiments, “receiving” indicates that the TIL used in the method and / or process may be indirectly derived from the specimen (including surgical excision, needle biopsy, core biopsy, micro-biopsy, or other specimen) and then used in the method and / or process (for example, if step (a) begins with a TIL that has already been derived from a specimen by a separate process not included in part (a), such TIL may be referred to as “receiving”).

[0008] In some embodiments, in step (b), the cell culture medium further contains antigen-presenting cells (APCs), and the number of APCs in the culture medium in step (c) is greater than the number of APCs in the culture medium in step (b).

[0009] In some embodiments, in step (b), the cell culture medium further comprises antigen-presenting cells (APCs), and the number of APCs in the culture medium in step (c) is equal to the number of APCs in the culture medium in step (b).

[0010] In some embodiments, PD-1-positive TILs are PD-1-high TILs.

[0011] In some embodiments, the present invention provides a method for growing tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, the method being (a) A first TIL population selected to be PD-1 positive, obtained by processing tumor samples from subjects by tumor digestion and selecting PD-1 positive TILs, and a second TIL population produced by performing a first proliferation by priming in a cell culture medium containing IL-2, OKT-3, and antigen-presenting cells (APCs), 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 approximately 1 to 7 / 8 days, and a second TIL population is obtained, and the second TIL population is produced in a larger number than the first TIL population. (b) Producing a third TIL population by rapidly growing a second TIL population by contacting a 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. (c) Collecting a therapeutic TIL population obtained from step (b), which includes:

[0012] In some embodiments, the present invention provides a method for growing tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, the method being (a) A first TIL population is cultured in a cell culture medium containing IL-2, OKT-3, and optionally antigen-presenting cells (APCs) to produce a second TIL population by priming and proliferation of TILs selected to be PD-1 positive, wherein the first priming and proliferation takes place over a first period of approximately 1 to 7 / 8 days, yielding a second TIL population, and the second TIL population is produced in a larger number than the first TIL population. (b) Rapid growth of a second TIL population by contacting the second TIL population 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. (c) Collecting a therapeutic TIL population obtained from step (c), which includes:

[0013] In some embodiments, in step (b), the cell culture medium further contains antigen-presenting cells (APCs), and the number of APCs in the culture medium in step (c) is greater than the number of APCs in the culture medium in step (b).

[0014] In some embodiments, in step (b), the cell culture medium further comprises antigen-presenting cells (APCs), and the number of APCs in the culture medium in step (c) is equal to the number of APCs in the culture medium in step (b).

[0015] In some embodiments, PD-1-positive TILs are PD-1-high TILs.

[0016] In some embodiments, the selection of 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 an excess of anti-IgG4 antibody conjugated to a fluorophore; and (iii) performing flow-based cell sorting based on the fluorophore to obtain a PD-1-rich TIL population.

[0017] In some embodiments, the monoclonal anti-PD-1 IgG4 antibody is nivolumab, or a variant, fragment, or conjugate thereof. In some embodiments, the anti-IgG4 antibody is clone anti-human IgG4, clone HP6023.

[0018] In some embodiments, the ratio of the number of APCs in rapid second growth to the number of APCs in priming first growth is selected from a range of about 1.5:1 to about 20:1.

[0019] In some embodiments, the ratio is selected from a range of approximately 1.5:1 to approximately 10:1.

[0020] In some embodiments, the ratio is selected from a range of approximately 2:1 to approximately 5:1.

[0021] In some embodiments, the ratio is selected from a range of approximately 2:1 to approximately 3:1.

[0022] In some embodiments, the ratio is approximately 2:1.

[0023] In some embodiments, the number of APCs in the first proliferation by priming is approximately 1 × 10⁻⁶. 8 Individual APCs ~ approximately 3.5 × 10 8 Selected from a range of APCs, the number of APCs in rapid second proliferation is approximately 3.5 × 10⁻⁶. 8 Each APC is approximately 1 x 10⁻⁶ 9 Selected from the range of individual APCs.

[0024] In some embodiments, the number of APCs in the first proliferation by priming is selected from the range of about 1.5×10 8 APCs to about 3×10 8 APCs, and the number of APCs in the rapid second proliferation is selected from the range of about 4×10 8 APCs to about 7.5×10 8 APCs.

[0025] In some embodiments, the number of APCs in the first proliferation by priming is selected from the range of about 2×10 8 APCs to about 2.5×10 8 APCs, and the number of APCs in the rapid second proliferation is selected from the range of about 4.5×10 8 APCs to about 5.5×10 8 APCs.

[0026] In some embodiments, about 2.5×10<� 8 APCs are added for the first proliferation by priming, and 5×10 8 APCs are added for the rapid second proliferation.

[0027] In some embodiments, the ratio of the number of TILs in the second TIL population to the number of TILs in the first TIL population is from about 1.5:1 to about 100:1.

[0028] In some embodiments, the ratio of the number of TILs in the second TIL population to the number of TILs in the first TIL population is about 50:1.

[0029] In some embodiments, the ratio of the number of TILs in the second TIL population to the number of TILs in the first TIL population is about 25:1.

[0030] In some embodiments, the ratio of the number of TILs in the second TIL population to the number of TILs in the first TIL population is about 20:1.

[0031] 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.

[0032] In some embodiments, the second TIL group is at least 50 times larger than the first TIL group.

[0033] In some embodiments, the method includes an additional step of transferring the collected therapeutic TIL population to an infusion bag after the step of collecting the therapeutic TIL population.

[0034] In some embodiments, multiple tumor fragments are distributed in multiple separate containers, in each of the separate containers a second TIL population is obtained from the first TIL population in a first growth step by priming, a third TIL population is obtained from the second TIL population in a rapid second growth step, and a therapeutic TIL population obtained from the third TIL population is collected from each of the multiple containers and combined to result in a harvested TIL population.

[0035] In some embodiments, the plurality of separate containers include at least two separate containers.

[0036] In some embodiments, the multiple separate containers include 2 to 20 separate containers.

[0037] In some embodiments, the multiple separate containers include 2 to 10 separate containers.

[0038] In some embodiments, the multiple separate containers include 2 to 5 separate containers.

[0039] In some embodiments, each of the separate containers includes a first gas-permeable surface area.

[0040] In some embodiments, multiple tumor fragments are distributed within a single container.

[0041] In some embodiments, a single container includes a first gas-permeable surface area.

[0042] In some embodiments, during the 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.

[0043] 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.

[0044] 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.

[0045] 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.

[0046] 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.

[0047] 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.

[0048] 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.

[0049] In some embodiments, the second container is larger than the first container.

[0050] In some embodiments, during the 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.

[0051] 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.

[0052] 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.

[0053] 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.

[0054] 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.

[0055] 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 4 cell layers.

[0056] 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.

[0057] In some embodiments, each container includes a first gas-permeable surface area.

[0058] In some embodiments, during the 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.

[0059] 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.

[0060] 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.

[0061] 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 to 5 cell layers.

[0062] 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.

[0063] 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 4 cell layers.

[0064] 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 first container comprises a first surface area, the cell culture medium comprises antigen-presenting cells (APCs), the APCs are layered on a 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 a range of about 1:1.1 to about 1:10.

[0065] In some embodiments, 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 second rapid growth step is selected from a range of approximately 1:1.2 to approximately 1:8.

[0066] In some embodiments, 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 second rapid growth step is selected from a range of about 1:1.3 to about 1:7.

[0067] In some embodiments, 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 second rapid growth step is selected from a range of approximately 1:1.4 to approximately 1:6.

[0068] In some embodiments, 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 second rapid growth step is selected from a range of approximately 1:1.5 to approximately 1:5.

[0069] In some embodiments, 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 second rapid growth step is selected from a range of approximately 1:1.6 to approximately 1:4.

[0070] In some embodiments, 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 second rapid growth step is selected from a range of approximately 1:1.7 to approximately 1:3.5.

[0071] In some embodiments, 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 second rapid growth step is selected from a range of approximately 1:1.8 to approximately 1:3.

[0072] In some embodiments, 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 second rapid growth step is selected from a range of approximately 1:1.9 to approximately 1:2.5.

[0073] In some embodiments, 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 second rapid growth step is approximately 1:2.

[0074] In some embodiments, 2-3 days after the rapid second growth step, the cell culture medium is supplemented with additional IL-2.

[0075] In some embodiments, the method further includes cryopreserving the collected TIL population in the step of collecting a therapeutic TIL population using a cryopreservation process.

[0076] In some embodiments, this method further includes the step of cryopreserving the infusion bags.

[0077] In some embodiments, the cryopreservation process is carried out using a 1:1 ratio of collected TIL populations and cryopreservation medium.

[0078] In some embodiments, the antigen-presenting cells are peripheral blood mononuclear cells (PBMCs).

[0079] In some embodiments, the PBMCs are irradiated and are homogeneous.

[0080] 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 It is an individual.

[0081] 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 It is an individual.

[0082] In some embodiments, the antigen-presenting cells are artificial antigen-presenting cells.

[0083] In some embodiments, the sampling in the step of collecting therapeutic TIL populations is performed using a membrane-based cell processing system.

[0084] In some embodiments, the harvesting in step (d) is performed using a LOVO cell processing system.

[0085] In some embodiments, the multiple fragments include approximately 60 fragments per container in the first propagation step by priming, with each fragment being approximately 27 mm in size. 3 It has the volume of .

[0086] In some embodiments, multiple pieces are approximately 1300 mm 3 ~approx. 1500mm 3 It contains approximately 30 to 60 fragments with a total volume.

[0087] In some embodiments, multiple pieces are approximately 1350 mm 3 It contains approximately 50 fragments with a total volume.

[0088] In some embodiments, the multiple fragments include about 50 fragments having a total mass of about 1 gram to about 1.5 grams.

[0089] In some embodiments, the cell culture medium is provided in a container selected from the group consisting of G containers and Xuri cell bags.

[0090] In some embodiments, 2–3 days after step (d), the cell culture medium is supplemented with additional IL-2.

[0091] In some embodiments, the IL-2 concentration is approximately 10,000 IU / mL to approximately 5,000 IU / mL.

[0092] In some embodiments, the IL-2 concentration is approximately 6,000 IU / mL.

[0093] 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.

[0094] In some embodiments, the cryopreservation medium contains dimethyl sulfoxide (DMSO).

[0095] In some embodiments, the cryopreservation medium contains 7% to 10% DMSO.

[0096] 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.

[0097] In some embodiments, the first period in the first growth step by priming is carried out within a period of 5, 6, or 7 days.

[0098] In some embodiments, the second period in the rapid second growth step is carried out within a period of 7, 8, or 9 days.

[0099] 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 7 days.

[0100] In some embodiments, the first growth step, which involves priming by collecting a therapeutic TIL population, is performed within a period of approximately 14 to 16 days.

[0101] In some embodiments, the first growth step, which involves priming by collecting a therapeutic TIL population, is carried out within a period of approximately 15 to 16 days.

[0102] In some embodiments, the first growth step, which involves priming by collecting a therapeutic TIL population, is carried out within a period of approximately 14 days.

[0103] In some embodiments, the first growth step, which involves priming by collecting a therapeutic TIL population, is carried out within a period of approximately 15 days.

[0104] In some embodiments, the first growth step, which involves priming by collecting a therapeutic TIL population, is carried out within a period of approximately 16 days.

[0105] In some embodiments, the method further includes the step of cryopreserving the collected therapeutic TIL population using a cryopreservation process, wherein the first step of priming by collection and cryopreservation of the therapeutic TIL population is carried out within 16 days.

[0106] 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.

[0107] In some embodiments, the number of TILs sufficient for a therapeutically effective dose is approximately 2.3 × 10⁶ 10 ~Approx. 13.7×10 10 It is an individual.

[0108] In some embodiments, the third TIL population in a rapid second growth step provides increased potency, increased interferon-γ production, and / or increased polyclonality.

[0109] In some embodiments, the third TIL population in a rapid second growth step provides at least 1 to 5 times greater interferon-γ production compared to TILs prepared by a process longer than 16 days.

[0110] In some embodiments, effector T cells and / or central memory T cells obtained from a 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 a second TIL population in a first proliferation step by priming.

[0111] In some embodiments, the therapeutic TIL population, obtained from the step of collecting the therapeutic TIL population, is injected into the patient.

[0112] In some embodiments, this method further includes the step of cryopreserving the injection bag containing the TIL population collected in step (f) using a cryopreservation process.

[0113] In some embodiments, the cryopreservation process is carried out using a 1:1 ratio of collected TIL populations and cryopreservation medium.

[0114] In some embodiments, the antigen-presenting cells are peripheral blood mononuclear cells (PBMCs).

[0115] In some embodiments, the PBMCs are irradiated and are homogeneous.

[0116] In some embodiments, the antigen-presenting cells are artificial antigen-presenting cells.

[0117] In some embodiments, the harvesting in step (e) is performed using a membrane-based cell processing system.

[0118] In some embodiments, the harvesting in step (e) is performed using a LOVO cell processing system.

[0119] In some embodiments, the multiple fragments include about 60 fragments per first gas permeable surface area in step (c), and each fragment is about 27 mm 3 It has the volume of .

[0120] In some embodiments, multiple pieces are approximately 1300 mm 3 ~approx. 1500mm 3 It contains approximately 30 to 60 fragments with a total volume.

[0121] In some embodiments, multiple pieces are approximately 1350 mm 3 It contains approximately 50 fragments with a total volume.

[0122] In some embodiments, the multiple fragments include about 50 fragments having a total mass of about 1 gram to about 1.5 grams.

[0123] In some embodiments, the cell culture medium is provided in a container selected from the group consisting of G containers and Xuri cell bags.

[0124] In some embodiments, the IL-2 concentration is approximately 10,000 IU / mL to approximately 5,000 IU / mL.

[0125] In some embodiments, the IL-2 concentration is approximately 6,000 IU / mL.

[0126] In some embodiments, the infusion bag in step (d) is an infusion bag containing HypoThermosol.

[0127] In some embodiments, the cryopreservation medium contains dimethyl sulfoxide (DMSO).

[0128] In some embodiments, the cryopreservation medium contains 7% to 10% DMSO.

[0129] In some embodiments, the first period in step (c) and the second period in step (c) are each carried out individually within a period of 5, 6, or 7 days.

[0130] In some embodiments, the first period in step (c) is carried out within a period of 5, 6, or 7 days.

[0131] In some embodiments, the second period in step (d) is within a period of 7, 8, or 9 days.

[0132] In some embodiments, the first period in step (c) and the second period in step (c) are each carried out individually within a period of 7 days.

[0133] In some embodiments, steps (a) to (f) are carried out over a period of approximately 14 to 16 days.

[0134] In some embodiments, steps (a) to (f) are carried out over a period of approximately 15 to 16 days.

[0135] In some embodiments, steps (a) to (f) are carried out over a period of approximately 14 days.

[0136] In some embodiments, steps (a) to (f) are carried out over a period of approximately 15 days.

[0137] In some embodiments, steps (a) to (f) are carried out over a period of approximately 16 days.

[0138] In some embodiments, steps (a) to (f) and cryopreservation are carried out within 16 days.

[0139] In some embodiments, the therapeutic TIL population collected in step (f) contains enough TILs to constitute a therapeutically effective dose of TILs.

[0140] In some embodiments, the number of TILs sufficient for a therapeutically effective dose is approximately 2.3 × 10⁶ 10 ~Approx. 13.7×10 10 It is an individual.

[0141] In some embodiments, the container in step (c) is larger than the container in step (b).

[0142] In some embodiments, the third TIL population in step (d) provides increased potency, increased interferon-γ production, and / or increased polyclonality.

[0143] In some embodiments, the third TIL population in step (d) provides at least 1 to 5 times greater interferon-γ production compared to TILs prepared by a process longer than 16 days.

[0144] In some embodiments, effector T cells and / or central memory T cells obtained from the third TIL population in step (d) show increased CD8 and CD28 expression compared to effector T cells and / or central memory T cells obtained from the second cell population in step (c).

[0145] In some embodiments, the TIL from step (f) is injected into the patient.

[0146] In some embodiments, the present invention provides a method for treating a subject having cancer, and this method is (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-positive TILs from the first TIL population in (a) to obtain a TIL population rich in PD-1, (c) A PD-1-rich TIL population is cultured in a cell culture medium containing IL-2, OKT-3, and antigen-presenting cells (APCs) to perform a first proliferation by priming and produce a second TIL population, 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 approximately 1 to 7 days, and the second TIL population is obtained, and the second TIL population is produced in a quantity at least 50 times greater than the first TIL population. (d) Producing a third TIL population by rapidly growing a second 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, (g) administering proliferated tumor-infiltrating lymphocytes (TILs) to the TILs from step (e) of a therapeutically effective dose.

[0147] 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 It is an individual.

[0148] In some embodiments, PD-1-positive TILs are PD-1-high TILs.

[0149] In some embodiments, the selection of 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 an excess of anti-IgG4 antibody conjugated to a fluorophore; and (iii) performing flow-based cell sorting based on the fluorophore to obtain a PD-1-rich TIL population.

[0150] In some embodiments, the monoclonal anti-PD-1 IgG4 antibody is nivolumab, or a variant, fragment, or conjugate thereof.

[0151] In some embodiments, the anti-IgG4 antibody is clone anti-human IgG4, clone HP6023.

[0152] In some embodiments, antigen-presenting cells (APCs) are PBMCs.

[0153] In some embodiments, a non-myeloablative lymphocyte depletion regimen is administered to the patient before administering a therapeutically effective dose of TIL cells in step (g).

[0154] In some embodiments, the non-myeloablative lymphocyte depletion regimen involves cyclophosphamide at 60 mg / m². 2 The procedure involves administering a daily dose of fludarabine for two days, followed by a 25 mg / m² dose of fludarabine. 2 The step includes administering the drug at a daily dose for 5 days.

[0155] 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 patient in step (g).

[0156] 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.

[0157] In some embodiments, the third TIL population in step (c) provides increased potency, increased interferon-γ production, and / or increased polyclonality.

[0158] In some embodiments, the third TIL population in step (d) provides at least 1 to 5 times greater interferon-γ production compared to TILs prepared by a process longer than 16 days.

[0159] In some embodiments, effector T cells and / or central memory T cells obtained from the third TIL population in step (d) show increased CD8 and CD28 expression compared to effector T cells and / or central memory T cells obtained from the second cell population in step (c).

[0160] 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, 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.

[0161] In some embodiments, the cancer is selected from the group consisting of melanoma, HNSCC, cervical cancer, NSCLC, glioblastoma (including GBM), and gastrointestinal cancer.

[0162] In some embodiments, the cancer is melanoma.

[0163] In some embodiments, the cancer is HNSCC.

[0164] In some embodiments, the cancer is cervical cancer.

[0165] In some embodiments, the cancer is NSCLC.

[0166] In some embodiments, the cancer is glioblastoma (including GBM).

[0167] In some embodiments, the cancer is gastrointestinal cancer.

[0168] In some embodiments, the cancer is a hypermutated cancer.

[0169] In some embodiments, the cancer is a hypermutated cancer in children.

[0170] In some embodiments, the container is GREX-10.

[0171] In some embodiments, the sealed container includes GREX-100.

[0172] In some embodiments, the sealed container includes GREX-500.

[0173] In some embodiments, the subjects have been previously treated with an anti-PD-1 antibody.

[0174] In some embodiments, the subjects have not been previously treated with anti-PD-1 antibodies.

[0175] In some embodiments, in step (b), PD-1-positive TILs are selected from a first TIL population by contacting the first TIL population with an anti-PD-1 antibody to form a first complex between the anti-PD-1 antibody and TIL cells in the first TIL population, and then isolating the first complex to obtain a PD-1-rich TIL population.

[0176] In some embodiments, the anti-PD-1 antibody includes 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.

[0177] In some embodiments, the anti-PD-1 antibodies used in the selection in step (b) include EH12.2H7, PD1.3.1, M1H4, 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 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.

[0178] In some embodiments, the anti-PD-1 antibody used in selection is EH12.2H7.

[0179] In some embodiments, the anti-PD-1 antibody used in the selection in step (b) binds to an epitope different from nivolumab or pembrolizumab.

[0180] In some embodiments, the anti-PD-1 antibody used in the selection in step (b) binds to the same epitope as EH12.2H7 or nivolumab.

[0181] In some embodiments, the anti-PD-1 antibody to be used in the selection in step (b) is nivolumab.

[0182] In some embodiments, the subjects have been previously treated with a first anti-PD-1 antibody, and in step (b), PD-1-positive TILs are selected by contacting the first TIL population with a second anti-PD-1 antibody, 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 on the first TIL population.

[0183] In some embodiments, the subjects have been previously treated with a first anti-PD1 antibody, and in step (b), PD-1-positive TILs are selected by contacting the first TIL population with a second anti-PD-1 antibody, the second anti-PD-1 antibody being blocked from binding to the first TIL population by the first anti-PD-1 antibody which has been insolubilized on the first TIL population.

[0184] In some embodiments, the subjects have been previously treated with a first anti-PD1 antibody, and in step (b), PD-1-positive TILs are selected 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 the first TIL population, 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 immobilized on the first TIL population, and then by isolating the first complex to obtain a PD-1-rich TIL population.

[0185] 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 that binds to the Fc region of the first anti-PD-1 antibody and the Fc region of the second anti-PD-1 antibody to form the second anti-Fc antibody and the first complex, and the step of isolating the first complex is performed by isolating the second complex.

[0186] In some embodiments, the second anti-PD-1 antibody comprises an Fc region, the subject has been previously treated with a first anti-PD-1 antibody, and in step (b), the PD-1-positive TILs are selected by the step of contacting the first TIL population with the 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 not blocked from binding to the first TIL population by the first anti-PD-1 antibody which has been immobilized on the first TIL population, and after the step of forming the first complex, the method further comprises the step of contacting the first complex with an anti-Fc antibody which 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, and then the step of isolating the second complex to obtain a PD-1-rich TIL population.

[0187] In some embodiments, the subject has been previously treated with a first anti-PD1 antibody, and in step (b), the PD-1 positive TILs undergo a step of 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 the first TIL population, wherein the second anti-PD-1 antibody is blocked from binding to the PD-1 positive TILs by the first anti-PD-1 antibody which has been insolubilized on the first TIL population, and the first anti-PD-1 antibody and the second anti-PD-1 antibody include an Fc region, after the step of forming the first complex, The method is selected by further comprising the steps of contacting the first complex with an anti-Fc antibody bound to the Fc region of a 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, which has been immobilized on 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 immobilized on the first TIL population, and isolating the second and third complexes to obtain a PD-1-rich TIL population.

[0188] In some embodiments, PD-1-positive TILs are PD-1-high TILs.

[0189] In some embodiments, the present invention provides a therapeutic tumor-infiltrating lymphocyte (TIL) population prepared from PD-1-positive cells selected from a patient's tumor tissue, the therapeutic TIL population providing increased potency and / or increased interferon-γ production.

[0190] In some embodiments, the present invention provides a therapeutic tumor-infiltrating lymphocyte (TIL) population prepared from PD-1-positive cells selected from a patient's tumor tissue, the therapeutic TIL population providing increased potency and / or increased interferon-γ production.

[0191] In some embodiments, the present invention provides a therapeutic tumor-infiltrating lymphocyte (TIL) population prepared from PD-1-positive cells selected from a patient's tumor tissue, the therapeutic TIL population providing increased interferon-γ production.

[0192] In some embodiments, the present invention provides a therapeutic tumor-infiltrating lymphocyte (TIL) population prepared from PD-1-positive cells selected from a patient's tumor tissue, the therapeutic TIL population providing increased efficacy.

[0193] In some embodiments, the present invention provides a therapeutic tumor-infiltrating lymphocyte (TIL) population prepared from PD-1-positive cells selected from a patient's tumor tissue, the therapeutic TIL population capable of producing at least 1-fold more interferon-γ compared to TILs prepared by a process longer than 16 days.

[0194] In some embodiments, the present invention provides a therapeutic tumor-infiltrating lymphocyte (TIL) population prepared from PD-1-positive cells selected from a patient's tumor tissue, the therapeutic TIL population capable of producing at least 1-fold more interferon-γ compared to TILs prepared by a process longer than 16–22 days.

[0195] In some embodiments, selecting PD-1-positive TILs from a first TIL population to obtain a PD-1-rich TIL population includes selecting a TIL population from a first TIL population in which at least 11.27% to 74.4% are PD-1-positive TILs.

[0196] In some embodiments, the selection of steps is: (i) Exposing a first TIL population and 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) Adding an excess of anti-IgG4 antibody conjugated to a 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).

[0197] In some embodiments, the fluorophore intensity 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.

[0198] In some embodiments, the FACS gate is set after step (a).

[0199] In some embodiments, PD-1-positive TILs are PD-1-high TILs.

[0200] In some embodiments, at least 80% of the PD-1-rich TIL population are PD-1-positive TILs.

[0201] The present invention also provides a method for growing tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, the method being (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-positive TILs from the first TIL population in (a) to obtain a PD-1-rich TIL population, wherein at least 10% to 80% of the first TIL population are PD-1-positive TILs. (c) A PD-1-rich TIL population is cultured in a cell culture medium containing IL-2, OKT-3, and antigen-presenting cells (APCs) to perform a first proliferation by priming and produce a second TIL population, 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 approximately 1 to 7 / 8 days, and a second TIL population is obtained, and the second TIL population is produced in a larger number than the first TIL population. (d) Producing a third TIL population by rapidly growing a second population of cells 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 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), (f) Transferring the TIL population collected from step (e) to an injection bag.

[0202] In some embodiments, the selection of step (b) is (i) Exposing a first TIL population and 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) Adding an excess of anti-IgG4 antibody conjugated to a 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).

[0203] In some embodiments, the fluorophore intensity 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.

[0204] In some embodiments, the FACS gate is set after step (a).

[0205] In some embodiments, PD-1-positive TILs are PD-1-high TILs.

[0206] In some embodiments, at least 80% of the PD-1-rich TIL population are PD-1-positive TILs.

[0207] In some embodiments, the third TIL group is at least about 1 × 10 in the container. 8 Includes individual TILs.

[0208] In some embodiments, the third TIL group is at least about 1 × 10 in the container. 9 Includes individual TILs.

[0209] In some embodiments, the number of PD-1-rich TILs in the first proliferation by priming is approximately 1 × 10⁻⁶ 4 ~Approx. 1×10 6 It is an individual.

[0210] In some embodiments, the number of PD-1-rich TILs in the first proliferation by priming is approximately 5 × 10⁻⁶. 4 ~Approx. 1×10 6 It is an individual.

[0211] In some embodiments, the number of PD-1-rich TILs in the first proliferation by priming is approximately 2 × 10⁻⁶ 5 ~Approx. 1×10 6 It is an individual.

[0212] 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). [Brief explanation of the drawing]

[0213] [Figure 1A-1B]A) A comparison of embodiments of the 2A process (approximately 22-day process) and the Gen3 process (approximately 14-16 day process) for TIL manufacturing. B) A diagram of exemplary process PD-1 Gen3 providing an overview of steps A-F (approximately 14-16 day process). C) A diagram providing three exemplary Gen3 processes for each of the three process variations, along with an overview of steps A-F (approximately 14-16 day process). [Figure 2] This document provides an experimental flowchart regarding the comparability between GEN2 (Process 2A) and PD-1 GEN3. [Figure 3A-3C] A) Phenotypic characterization of TIL products in L4054-Gen2 and Gen3 processes. B) Phenotypic characterization of TIL products in L4055-Gen2 and Gen3 processes. C) Phenotypic characterization of TIL products in M1085T-Gen2 and Gen3 processes. [Figure 4A-4C] A) Memory marker analysis of TIL products from L4054-Gen2 and Gen3 processes. B) Memory marker analysis of TIL products from L4055-Gen2 and Gen3 processes. C) Memory marker analysis of TIL products from M1085T-Gen2 and Gen3 processes. [Figure 5] L4054 activation and exhaustion markers: (A) gated with CD4+, (B) gated with CD8+. [Figure 6] L4055 activation and exhaustion markers: (A) gated with CD4+, (B) gated with CD8+. [Figure 7] IFNγ production (pg / mL): (A) L4054, (B) L4055, and (C) M1085T for Gen2 and Gen3 processes. Each bar in this figure represents the mean + SEM optical density of stimulated, unstimulated, and medium control IFNγ levels measured at 450 nm. [Figure 8]ELISA analysis of IL-2 concentration in cell culture supernatant: (A) L4054 and (B) L4055. Each bar in this figure represents the mean + SEM optical density of IL-2 levels in used culture medium measured at 450 nm. [Figure 9] Quantification of glucose and lactate (g / L) in used medium: (A) glucose and (B) lactate: A decrease in glucose was observed throughout REP growth in both tumor lines and processes. Conversely, as expected, an increase in lactate was observed. Both the decrease in glucose and the increase in lactate were comparable between the Gen2 and Gen3 processes. [Figure 10] A) Quantification of L-glutamine in used L4054 and L4055 media. B) Quantification of Glutamax in used L4054 and L4055 media. C) Quantification of ammonia in used L4054 and L4055 media. [Figure 11] Telomere length analysis: The above RTL values ​​represent the average telomere fluorescence per chromosome / genome in the Gen2 and Gen3 processes of telomere fluorescence per chromosome / genome in a control cell line (1301 leukemia cell line) using the DAKO kit. [Figure 12] Intrinsic CDR3 sequence analysis of TIL end products in L4054 and L4055 under Gen2 and Gen3 processes. Columns show the number of intrinsic TCR B chronotypes identified from 1 × 10⁶ cells collected on the day of collection for Gen2 (e.g., day 22) and Gen3 processes (e.g., days 14–16). Gen3 exhibits higher clonal diversity compared to Gen2, based on the number of intrinsic peptide CDRs in the sample. [Figure 13] Frequency of unique CDR3 sequences in L4054 IL-collected final cell products (Gen2 (e.g., day 22) and Gen3 processes (e.g., days 14-16)). [Figure 14] Frequency of unique CDR3 sequences in L4055 TIL-collected final cell products (Gen2 (e.g., day 22) and Gen3 processes (e.g., days 14-16)). [Figure 15] Diversity index of TIL end products in L4054 and L4055 under Gen2 and Gen3 processes. The Shannon entropy diversity index is a more reliable general metric for comparison. Gen3 L4054 and L4055 showed slightly higher diversity than Gen2. [Figure 16] Table 22 shows the raw cell count data for day 7 - Gen3 REP initiation (see Example 5 below). [Figure 17] Table 22 shows the raw cell count data for day 11-Gen2 REP initiation and Gen3 scale-up (see Example 5 below). [Figure 18] Table 23 shows the raw cell count data for day 16-Gen2 scale-up and Gen3 harvesting (e.g., day 16) (see Example 5 below). [Figure 19] Table 23 shows the raw cell count data for day 22-Gen2 collection (e.g., day 22) (see Example 5 below). For L4054 Gen2, since this was the total number for the study, the count after LOVO was extrapolated to four flasks. One flask was contaminated, and extrapolation was performed for a total of 6.67E+10. [Figure 20] Raw data from flow cytometry results, shown in Figures 3A, 4A, and 4B. [Figure 21] Raw data from flow cytometry results, shown in Figures 3C and 4C. [Figure 22] Raw data from flow cytometry results, shown in Figures 5 and 6. [Figure 23] Raw data from the IFNγ production assay results for the L4054 sample shown in Figure 7. [Figure 24] Raw data from the IFNγ production assay of the L4055 sample shown in Figure 7. [Figure 25] Raw data from the IFNγ production assay of the M1085T sample shown in Figure 7. [Figure 26] Raw data from the IL-2 ELISA assay, shown in Figure 8. [Figure 27]Raw data of metabolic substrates and metabolic analysis results presented in Figures 9 and 10. [Figure 28] Raw data of the relative telomere length analysis results presented in Figure 11. [Figure 29] Raw data of unique CD3 sequences and clonal diversity analysis results presented in Figures 12 and 15. [Figure 30] This document shows a comparison between various Gen2 (2A process) embodiments and Gen3.1 process embodiments. [Figure 31] A table illustrating the various characteristics of the Gen2, Gen2.1, and Gen3.0 process embodiments. [Figure 32] An overview of the culture medium conditions for an embodiment of the Gen3 process, referred to as Gen3.1. [Figure 33] A table illustrating the various characteristics of the Gen2, Gen2.1, and Gen3.0 process embodiments. [Figure 34] A table comparing the various features of the Gen2 and Gen3.0 process embodiments. [Figure 35] A table providing the use of culture media in various embodiments of the described growth process. [Figure 36] Phenotypic comparison: The Gen3.0 and Gen3.1 embodiments of the process showed equivalent CD28, CD27, and CD57 expression. [Figure 37] Higher IFNγ production in the Gen3 final product. IFNγ analysis (by ELISA) was evaluated in the culture supernatant to compare both processes. For each tumor, fresh TIL product was used in each Gen2 (e.g., day 22) and Gen3 process (e.g., day 16) and stimulated overnight in coated anti-CD3 plates. Each bar in this figure represents the IFNγ levels in stimulated, unstimulated, and medium control. [Figure 38]Top: Intrinsic CDR3 sequence analysis of TIL final product: Columns show the number of intrinsic TCR B chronotypes identified from 1 × 10⁶ cells collected in Gen2 (e.g., day 22) and Gen3 processes (e.g., days 14–16). Gen3 shows higher clonal diversity compared to Gen2, based on the number of intrinsic peptide CDRs in the sample. Bottom: Diversity index of TIL final product: The Shannon entropy diversity index is a more reliable general metric for comparison. Gen3 showed slightly higher diversity than Gen2. [Figure 39] The 199 sequence is shared between the Gen3 final product and the Gen2 final product, representing 97.07% of the top 80% of unique CDR3 sequences from Gen2 that are shared with the Gen3 final product. [Figure 40] Sequence 1833 is shared between the Gen3 final product and the Gen2 final product, and represents 99.45% of the top 80% of unique CDR3 sequences from Gen2 that are shared with the Gen3 final product. [Figure 41] A schematic diagram of an exemplary embodiment of the Gen3 process (16-day process). [Figure 42] A schematic diagram of PD-1 selection before proliferation. [Figure 43] 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 44] 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 45]A streamlined protocol was developed to grow PD1+ TILs to clinically appropriate levels. Tumors were excised from patients and transported to the laboratory. Upon arrival, the tumors were digested, and the single-cell suspension was stained for CD3 and PD1. PD1+ TILs were sorted by FACS using an FX500 instrument (Sony). The PD1+ cell fraction was placed in a flask containing anti-human CD3 antibody (OKT3, 30 ng / ml), and allogeneic PBMCs (feeders) were irradiated at a ratio of 1:100 (TIL:feeder) to rapidly grow for 22 days (REP). [Figure 46] The frequency of PD1+ TILs varied among tumor samples, but the in vitro growth process reliably yielded over one billion TILs. Selected TILs and bulk TILs were grown from melanoma (n=6), lung cancer (n=7), breast cancer (n=6), and sarcoma (n=3). (A) The frequency of PD1+ cells in fresh tumor digests is shown for each individual sample. Horizontal and vertical lines represent the mean and standard error, respectively. (B) Cells sorted for PD1+ and PD1-, and bulk digests, were grown as shown in Figure 1. Cells were counted at the completion of REP, and the growth rate (final cell count / disseminated cell count) used to extrapolate the total cell count was calculated. For bulk TILs, the disseminated cell count was estimated using the percentage of T cells in the tumor digest. Means are plotted as bars, and standard errors are shown as vertical lines. [Figure 47]PD1+ TILs exhibit a different phenotypic profile compared to PD1- TILs. Digested tumors from melanoma (n=2), lung (n=2), and breast (n=2) were phenotypically evaluated by flow cytometry before screening. (A) Representative plot of surface marker expression in TILs from digested melanoma tumors. Specimens were first gated with CD3 and plotted biaxially against positive and negative PD1 events. The two fractions were then subjected to unsupervised viSNE clustering. The upper row contains PD1-positive events, and the lower row contains PD1-negative events. (B-C) Live lymphocytes were gated with CD3+ cells and evaluated for PD1+ and PD1-. PD1+ and PD1- populations were evaluated for (B) activation and (C) exhaustion marker cell surface expression. Mean values ​​are plotted as bars, and standard errors are shown as vertical lines. Statistical significance was evaluated using paired Student's t-tests (****P<0.0001, *p<0.05). [Figure 48] PD1 expression decreases during in vitro proliferation of PD1+ TILs. Cell surface expression of T cell markers was evaluated by flow cytometry in PD1+ pre-selected TILs and in vitro-proliferated PD1+ TILs (PD1+-derived TILs) from melanoma (n=1), lung (n=4), and breast (n=2). Bars represent the mean percentage of each subset in the two TIL preparations, and vertical lines represent the standard error. Statistical significance was assessed by paired Student's t-tests (***P<0.001, **p<0.01). [Figure 49]PD1+ TILs grown in vitro are phenotypically similar to bulk TILs. PD1+-derived TILs, PD1-derived TILs, and bulk TILs from melanoma (n=5), lung (n=7), breast (n=6), and sarcoma (n=3) were phenotypically evaluated by flow cytometry for the cell surface expression of T cell markers. (A) Four effector / memory subsets were identified based on the levels of (CD45RA and CCR7) in CD3+ cells: TEM = effector memory (CD45RA-, CCR7-), TCM = central memory (CD45RA-, CCR7+), TSCM = stem cell memory (CD45RA+, CCR7+), TEMRA = effector T cells (CD45RA+, CCR7-). (B) Markers of differentiation, (C) exhaustion, and (D) activation were also evaluated. The bars represent the average percentage of each subset in the three TIL preparations, and the vertical lines represent the standard error. [Figure 50]The proliferated PD1+ TILs are oligoclonal and contain the clonal fraction present in bulk TILs. PD1-selected TILs and bulk TILs from melanoma (n=2), breast (n=2), and lung (n=2) were analyzed by RNA sequencing. (A) Unique CDR3 (uCDR3) peptide sequences were numbered, and box plots were generated using Python 3.6.3 and the pandas and matplotlib libraries from Anaconda, Inc. (B) The Shannon diversity index was calculated for each sample using iRepertoire, and box plots were generated using Python 3.6.3 and the pandas and matplotlib libraries from Anaconda, Inc. Bars represent the mean percentage for each subset, and vertical lines represent the standard error. Statistical significance was assessed by paired Student's t-tests ***P<0.001, **p<0.01. (C) uCDR3 frequencies were ranked in descending order and reported or summed for each sequenced sample at the indicated intervals (top-ranked uCDR3, 2nd-10th ranked, 11th-20th ranked, etc.). The frequencies were then averaged for each group and plotted using Excel v.1803. (D) Shared uCDR3 clones were identified in complementary bulk TIL and PD1+ derived samples. The sum of the frequencies of each shared unique CDR3 clone is reported in the “Shared%” column. [Figure 51]The proliferated PD1+ TILs function as determined by IFNγ secretion and CD107a recruitment in response to nonspecific stimulation. A) PD1+-derived TILs, PD1-derived TILs, and bulk TILs from melanoma (n=5), lung (n=6), and breast (n=6) were stimulated with plate-bound anti-CD3 for 18 hours. The supernatant was evaluated for IFNγ secretion by ELISA. Results are plotted for individual samples. (B) PD1+-derived TILs, PD1-derived TILs, and bulk TILs from melanoma (n=5), lung (n=7), breast (n=6), and sarcoma (n=1) were evaluated for CD107a cell surface expression in response to 4 hours of PMA stimulation of CD4+ and CD8+ cells by flow cytometry. Results are plotted for individual samples. Horizontal lines represent the mean percentage for each subset, and vertical lines represent the standard error. [Figure 52]Proliferated PD1+ TILs show enhanced autologous melanoma cell lethality and tumor responsiveness compared to PD1- TILs. Tumor responsiveness was evaluated using PD1-selected TIL products from a single melanoma tumor. (A) The entire tumor digest was cleaned up using a dead cell removal kit (Miltenyi). 1e5 live cells were plated into each well of a 96-well plate and adhered in an xCELLigence instrument (ACEA Biosciences, Inc.) at 37°C for 18 hours. Autologous TILs derived from 1e5 PD1+ and PD1- were added to the respective wells to achieve a cell ratio of 1:1 (TIL:target) and incubated for 48 hours. Autologous target cell lethality was recorded as an increase in impedance due to cell detachment. Cell lethality (cell lysis %) (graph on the far left) was calculated using the formula: cell lysis % = [1 - (NCIst) / (AvgNCIRt)] × 100. NCIst is the normalized cell index of the sample, and NCIRt is the mean of the normalized cell index of the matched reference well (digestion only). The graph on the right shows the normalized cell index of the sample. (B) 1e5 cells from whole tumor digests were co-cultured with 1e5 TILs (or digests and TILs only) for 18 hours. The supernatant was evaluated for IFNγ release by ELISA (R&D systems). The bars represent the mean values ​​of the overlapping wells, and the vertical lines represent the standard error. [Figure 53] Selection of PD1+ cells from tumor digests using fluorescence-activated cell sorting. [Figure 54] Identifying methods for digesting tumor tissue. [Figure 55] Identification of a method for digesting tumor tissue using GMP-compliant reagents. [Figure 56] Identification of a method for digesting tumor tissue using GMP-compliant reagents. [Figure 57] Identification of a method for digesting tumor tissue using GMP-compliant reagents. [Figure 58] The sorting yield was higher with fresh tumor digests than with frozen tumor digests. [Figure 59] Similar expression of PD1 in fresh and frozen TILs. [Figure 60] PD1 antibody titration: Variable expression of PD1 using commercially available clones. [Figure 61] Nivolumab inhibits the binding of five commercially available PD1 staining antibodies. [Figure 62] Pembrolizumab differentially inhibits the binding of five commercially available PD1 staining antibodies. [Figure 63] PD-1 MFI was significantly reduced when cells were pre-incubated with pembrolizumab. [Figure 64] TILs co-incubated with pembrolizumab and nivolumab and stained with IgG4 secondary show similar PD-1 expression compared to EH12.2H7 clones. [Figure 65] Incubation of TILs with pembrolizumab and nivolumab did not alter their ability to detect surface PD1 expression. [Figure 66] Selection and propagation results for PD1. [Figure 67] Selection and propagation results for PD1. [Figure 68] Selection and propagation results for PD1. [Figure 69] The optimal seeding density for PD1+-derived TILs is greater than 10,000 cells. [Figure 70] PD1+TIL exhibits a different phenotypic profile compared to PD1-TIL. [Figure 71] PD1+TIL exhibits a different phenotypic profile compared to PD1-TIL. [Figure 72] The frequency of PD1+TILs varied among tumor samples and required two cycles of regeneration to overcome low initial growth rates. [Figure 73] The frequency of PD1+TILs varied among tumor samples and required two cycles of regeneration to overcome the initial proliferative deficit. [Figure 74] PD1+ TILs grown in vitro were phenotypically similar to bulk TILs. [Figure 75]PD1 expression was reduced during in vitro proliferation of PD1+TILs. [Figure 76] The TILs selected in PD1+ are oligoclonal, and the clonal fraction present in bulk TILs is lost. [Figure 77] The TILs selected in PD1+ are oligoclonal, and the clonal fraction present in bulk TILs is lost. [Figure 78] The TILs selected in PD1+ are oligoclonal, and the clonal fraction present in bulk TILs is lost. [Figure 79] The TILs selected in PD1+ are oligoclonal, and the clonal fraction present in bulk TILs is lost. [Figure 80] PD1+-derived TILs function in such a way that they are determined by IFNγ secretion and CD107a recruitment in response to nonspecific stimuli. [Figure 81] PD1+-derived TILs show enhanced lethality in melanoma compared to PD1--derived TILs and bulk TILs. [Figure 82] PD1+-derived TILs showed enhanced lethality in melanoma compared to PD--derived TILs and bulk-derived TILs. [Figure 83] An exemplary embodiment of a method for growing TILs from hematopoietic malignancies using the Gen3 growth platform. [Figure 84] Ex vivo-grown PD1+ TILs showed effector activity in several in vitro assays. The data suggest that PD1+-selected TILs are antigen-specific and possess greater effector function. [Figure 85] A schematic diagram of an exemplary embodiment of the steps of tumor digestion and PD-1+ selection, including PD-1 high selectivity. [Figure 86] TIL data and information selected by PD-1, including uCDR numbers and proliferation data. [Figure 87]Selection strategies and data for PD-1-selected TILs using EH12.2H7 anti-PD-1 antibody rather than M1H4 anti-PD-1 antibody. [Figure 88] Screening data for PD-1-selected TILs, showing the population in a PD-1 high-gating strategy using the EH12.2H7 anti-PD-1 antibody. [Figure 89] PD1+ screening strategy data showing the evaluation of anti-PD1 antibodies for screening M1H4 anti-PD-1 antibodies and EH12.2H7 anti-PD-1 antibodies. [Figure 90] PD-1 staining for TIL selection. The data show that EH12.2H7 and M1H4 exhibit different PD1 profiles in PBMCs and TILs. [Figure 91] Comparative analysis of TILs originating from M1H4 and TILs originating from EH12.2H7. Increased frequency of PD1+ in TILs screened for EH12.2H7. [Figure 92] Reduced magnification of PD1+-derived TILs in REP1 using M1H4 clones. [Figure 93] A comparative analysis of TILs derived from M1H4 and EH12.2H7. Greater oligoclonality (reduced diversity) was observed in TILs selected from M1H4. (Shannon entropy is a standard measure reflecting the number of different species present.) [Figure 94] Compared to EH12.2H7 clones and bulk TILs containing M1H4 clones, PD1+-derived TILs exhibited greater oligoclonality (reduced diversity). (Shannon entropy is a standard measure that reflects the number of different species present.) [Figure 95] Exemplary data demonstrating PD1+ selection: Gating at high PD1+ (high PD-1). [Figure 96] A schematic diagram of an exemplary embodiment of a modified Gen2 process developed for the TIL selected in PD1. [Figure 97]Exemplary data demonstrating PD1+ selection: Gating of different tumor samples with high PD1+ (high PD-1) in small (top) and large (bottom) sizes. [Figure 98] A schematic diagram of an exemplary embodiment of a modified proliferation process developed for TIL selected in PD1. [Figure 99] Data showing initial REP collection at day 17 in PD1+ conditions resulting in 55e9 and 37e9 TILs. [Figure 100] As shown in Figures 96 and 98, IFNγ secretion in two tumor samples under multiple growth process conditions is demonstrated. [Figure 101] As shown in Figures 96 and 98, granzyme B secretion in two tumor samples under multiple growth process conditions is demonstrated. [Figure 102] As shown in Figures 96 and 98, the CD3+CD45+ population in a single tumor sample under multiple growth process conditions is shown. Under the PD1+Gen2 condition, >90% CD3+CD45+ was observed. [Figure 103] As shown in Figures 96 and 98, the CD3+CD45+ population in a single tumor sample under multiple growth process conditions is shown. Under the PD1+Gen2 condition, >90% CD3+CD45+ was observed. [Figure 104] As shown in Figures 96 and 98, the TIL profile characteristics of a single tumor sample under multiple growth process conditions are shown. Purity: TCR a / b+ above 90%, and no detectable NK cells, monocytes, or B cells are present. [Figure 105] As shown in Figures 96 and 98, the TIL profile characteristics of a single tumor sample under multiple growth process conditions are shown. Purity: TCR a / b+ above 90%, and no detectable NK cells, monocytes, or B cells are present. [Figure 106A-B] As shown in Figures 96 and 98, the TIL profile characteristics of two tumor samples under multiple growth process conditions are shown. Differentiation: The PD1+Gen2 differentiation state was equivalent. [Figure 107A-B]As shown in Figures 96 and 98, the TIL profile characteristics of two tumor samples under multiple growth process conditions are shown. Memory: PD1+Gen2 was primarily effector memory TIL. [Figure 108A-B] As shown in Figures 96 and 98, the TIL profile characteristics of two tumor samples under multiple growth process conditions are presented. The activation and exhaustion states in CD4+ were similar. [Figure 109] As shown in Figures 96 and 98, the TIL profile characteristics of two tumor samples under multiple growth process conditions are presented. The activation and exhaustion states in CD8+ were similar. [Figure 110] Exemplary data demonstrating PD1+ selection: Gating of different tumor samples with high PD1+ (high PD-1) levels, compared before and after selection. [Figure 111] Exemplary data demonstrating PD1+ selection: Gating of L4097 tumor samples with high PD1+ (high PD-1). [Figure 112] Exemplary data demonstrating PD1+ selection: Gating of L4089 tumor samples with high PD1+ (high PD-1). [Figure 113] Exemplary data demonstrating PD1+ selection: Gating of H3035 tumor samples with high PD1+ (high PD-1). [Figure 114] Exemplary data demonstrating PD1+ selection: Gating of M1139 tumor samples with high PD1+ (high PD-1). [Figure 115] Exemplary data demonstrating PD1+ selection: Gating of L4100 tumor samples with high PD1+ (high PD-1). [Figure 116] Exemplary data demonstrating PD1+ selection: Gating of OV8030 tumor samples with high PD1+ (high PD-1). [Figure 117] Exemplary data demonstrating PD1+ selection: Gating of L4104 tumor samples with high PD1+ (high PD-1). [Figure 118] Exemplary data demonstrating PD1+ selection: Gating of M1132 tumor samples with high PD1+ (high PD-1). [Figure 119]Exemplary data demonstrating PD1+ selection: Gating of M1136 tumor samples with high PD1+ (high PD-1). [Figure 120] Exemplary data demonstrating PD1+ selection: Gating of H3037 tumor samples with high PD1+ (high PD-1). [Figure 121] Exemplary data demonstrating PD1+ selection: Gating of L4106 tumor samples with high PD1+ (high PD-1). [Figure 122] Exemplary data demonstrating PD1+ selection: Gating of L1141 tumor samples with high PD1+ (high PD-1). [Figure 123] Exemplary data demonstrating PD1+ selection: Gating of L4096 tumor samples with high PD1+ (high PD-1). [Figure 124] Exemplary data demonstrating PD1+ selection: Gating of H3038 tumor samples with high PD1+ (high PD-1). [Figure 125] Exemplary data demonstrating PD1+ selection: Gating at high PD1+ (high PD-1) in L4101 tumor samples. (Note: Potential gating issue at CD8 in the third panel.) [Figure 126] Exemplary data demonstrating PD1+ selection: Gating of L4097 tumor samples with high PD1+ (high PD-1). [Figure 127] Data showing proliferation in selected populations with various PD-1 levels. Cells that proliferated with high PD-1 levels may show reduced proliferation with REP1. [Figure 128] Summary of PD-1 selection, sorting, and proliferation results. Sorting of PD1-enhanced cells using EH12.2H7 anti-PD-1 antibody. [Figure 129] Summary of PD-1 selection, sorting, and proliferation results. Sorting of PD1-enhanced cells using EH12.2H7 anti-PD-1 antibody. [Figure 130] Graphs showing summary data of PD-1 selection, sorting, and proliferation results from Figures 128 and 129. Sorting of PD1-enhanced cells using EH12.2H7 anti-PD-1 antibody. [Figure 131]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, fold these to form a trivalent protein, then bind this to a second trivalent protein by IgG1-Fc (containing CH3 and CH2 domains), and then use this 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 connected by a linker which may contain, for example, hydrophilic residues, as well as Gly and Ser sequences for flexibility, and Glu and Lys for solubility. [Figure 132] The data above shows the collection of 100,000 cells selected in both dropdown menus. We confirmed that the cell populations were correctly gated. The gates were set to high, medium, and low by distinguishing three populations using PBMCs, FMO controls, and the sample itself. [Figure 133] Top left: This is the FMO control. Ensure that medium and high gates are less than 0.5%. Top right: Representative plot where the separation between high and medium is not clear. High background on this day led to a high negative gate. Bottom: Clear display of high and medium. The data suggests that adjustments to the BSC or FSC settings may be necessary. No other channel voltages were adjusted. The PD1 gate was adjusted as needed. [Figure 134]Intrinsic CDR3vβ composition of PD1-selected and unselected TILs. Unselected and PD1-selected TILs grown from two HNSCCs and five NSCLCs were analyzed for their CDR3vβ repertoire. The number of intrinsic CDR3βs, the count of recorded uCDR3s (A.), and the diversity index expressed as Shannon entropy (B.) are plotted for each individual sample. Paired samples are joined by colored lines. P-values ​​calculated by paired t-tests are plotted on each graph. [Figure 135] Graph showing clonal overlap between TILs selected and unselected in PD1. TILs grown from two HNSCCs and five NSCLCs were analyzed for the CDR3vβ repertoire. A. The number of unique CDR3vβ shared between PD1-selected (blue) and unselected (red) TIL samples is shown in the intersection of the Venn diagram for each individual tumor sample. B. and C. The percentage and portion of shared TILs in unselected and PD1-selected TILs are plotted for each individual sample. Paired samples are joined by colored lines. P-values ​​calculated by paired t-tests are indicated in each graph. [Figure 136] Frequencies of the top 10 PD1-selected TIL clones in unselected TIL products. PD1-selected and unselected TILs grown from two HNSCCs and five NSCLCs were analyzed for the CDR3vβ repertoire. Unique CDR3vβ sequences identified in PD1-selected TIL products were ranked from highest to lowest frequency. The individual top 10 frequencies of PD1-selected TIL clones in each paired product are plotted. Paired samples are joined by uncolored lines. P-values ​​calculated by paired t-tests are plotted on each graph. [Figure 137] Description of tumor digestive tissue used in these studies. [Figure 138]Detection of PD1+ cells in tumor digests from various histologies. Legend: PD1 expression in multiple histologies. The percentage of PD1+ TILs in the CD3+ TIL population is plotted for individual samples within each histology. Horizontal lines represent the mean percentage for each subset, and vertical lines represent the standard error. [Figure 139] Description of TILs selected and not selected in PD1 used in this study. [Figure 140] Factor-fold growth in TILs selected by PD1 that decreased during REP1 but not during REP2. Legend: (A) Melanoma, (B) NSCLC, and (C) HNSCC, selected and unselected by PD1, were grown by two 11-day REP cycles. Factor-fold growth for all assayed tumors is shown in (D). The growth factor in the TIL population was calculated using the total cell count at the completion of REP1 and REP2. Results are plotted for individual samples, with black dots representing TILs selected by PD1 and gray triangles representing unselected TILs. Horizontal lines represent the mean percentage for each subset, and vertical lines represent the standard error. Statistical significance was assessed by paired Student's t-tests. * indicates p-value < 0.05. [Figure 141] Growth results from various tumor samples. [Figure 142] Description of PD1-selected and unselected TILs used in this study. PD1-selected and unselected TIL products were obtained from four melanomas, seven NSCLCs, and two HNSCCs according to procedure TMP-18-015. Briefly, whole tumor biopsies were digested using a cocktail of DNAse, hyaluronidase, and collagenase IV. A portion of the resulting single-cell suspension was stained with PD1 and sorted on an FX500 instrument (Sony, HQ, New York). PD1-sorted cells and unselected whole tumor digests were subjected to two 11-day rapid growth phases (REPs) to obtain PD1-selected and unselected TILs, respectively. [Figure 143]PD1-selected and unselected TILs produce IFNγ and granzyme B in response to stimulation with activating beads. Legend: PD1-selected and unselected TILs from 4 melanomas, 7 NSCLCs, and 2 HNSCCs were evaluated for (A) IFNγ and (B) granzyme secretion. Results are plotted for individual samples, with black dots representing the unstimulated state and gray triangles representing the stimulated state with αCD3 / αCD28 / α41BB. Horizontal lines represent the mean percentage for each subset, and vertical lines represent the standard error. Statistical significance was assessed by paired Student's t-tests. ** indicates a p-value < 0.01. [Figure 144] PD1-selected and unselected TILs recruit CD107a in response to PMA / ionomycin stimulation. Legend: PD1-selected and unselected TILs from four melanomas, five NSCLCs, and one HNSCC were evaluated by flow cytometry for CD107a cell surface expression in response to PMA and ionomycin (BioLegend, CA) stimulation. Results are plotted for individual samples, with black dots representing the unstimulated state and gray triangles representing the PMA / ionomycin-stimulated state. Horizontal lines represent the mean percentage for each subset, and vertical lines represent the standard error. [Figure 145] Description of TILs selected and not selected in PD1 used in this study. [Figure 146] TILs selected and unselected by PD1 exhibit autotumor reactivity in vitro. Tumor lethality and reactivity were evaluated in PD1-selected and unselected TILs. (A) Cell index and (B) Tumor cell lethality (% cell lysis) are shown for melanoma samples. Supernatants from two NSCLCs and three melanomas were evaluated by ELISA for (C) IFNγ release. Mean values ​​are plotted as bars, and standard errors are shown as vertical lines. Statistical significance was assessed by paired Student's t-tests. ** indicates a p-value < 0.01. [Figure 147] Description of PD1-selected and unselected TILs used in Example 16. PD1-selected and unselected TIL products were obtained from four melanomas, seven NSCLCs, and two HNSCCs according to procedure TMP-18-015. Briefly, whole tumor biopsies were digested using a cocktail of DNAse, hyaluronidase, and collagenase IV. A portion of the resulting single-cell suspension was stained with PD1 and sorted on an FX500 instrument (Sony, HQ, New York). PD1-selected and unselected TILs were subjected to two 11-day REP cycles. [Figure 148] Figure 1: Comparison of CD4+ and CD8+ T cell levels in PD1-selected and unselected TILs. Legend: PD1-selected and unselected TILs from four melanomas, seven NSCLCs, and two HNSCCs were evaluated for T cell lineage (CD4 and CD8) using flow cytometry. Results are presented as percentages of CD3+ cells. Mean values ​​are plotted as bars, and standard errors are shown as vertical lines. [Figure 149] Comparison of the differentiation status of PD1-selected TILs with that of unselected TILs. Legend: PD1-selected and unselected TILs from 4 melanomas, 7 NSCLCs, and 2 HNSCCs were evaluated for CD27, CD28, CD56, CD57, and KLRG1 expression using flow cytometry. Results are presented as a percentage of CD3+ cells. Mean values ​​are plotted as bars, and standard errors are shown as vertical lines. Statistical significance was assessed by paired Student's t-tests. * indicates p-value < 0.05. [Figure 150]Comparison of the distribution of memory T cell subsets in PD1-selected and unselected TILs. Legend: PD1-selected and unselected TILs from four melanomas, seven NSCLCs, and two HNSCCs were evaluated for the expression of memory markers CD45RA and CCR7 by flow cytometry. T cell memory subsets were determined as shown, and the mean percentage of each subset is plotted as black bars for PD1-selected TILs and gray bars for unselected TILs. Standard error is shown as a vertical line. [Figure 151] Comparison of activation status between PD1-selected and unselected TILs. Legend: PD1-selected and unselected TILs from 4 melanomas, 7 NSCLCs, and 2 HNSCCs were evaluated for CD25, CD69, CD134, and CD137 expression. The mean percentage of CD3+ T cells is plotted as black bars for PD1-selected TILs and gray bars for unselected TILs. Standard errors are shown as vertical lines. Statistical significance was assessed by paired Student's t-tests. * indicates p-value < 0.05. [Figure 152] Comparison of exhaustion / suppression marker expression in PD1-selected and unselected TILs. Legend: PD1-selected and unselected TILs from 4 melanomas, 7 NSCLCs, and 2 HNSCCs were evaluated for LAG3, PD1, TIM3, and CD101 expression by flow cytometry. Mean values ​​are plotted as bars, and standard errors are shown as vertical lines. Statistical significance was assessed by paired Student's t-tests. *** indicates p-value < 0.001. [Figure 153]Comparison of resident memory T cell marker expression in PD1-selected and unselected TILs. PD1-selected and unselected TILs from four melanomas, seven NSCLCs, and two HNSCCs were evaluated for CD39, CD49a, and CD103 expression by flow cytometry. Mean values ​​are plotted as bars, and standard errors are shown as vertical lines. Statistical significance was assessed by paired Student's t-tests. ** indicates a p-value < 0.01. [Figure 154] An embodiment of a full-scale process for PD1 TIL culture. [Figure 155] Small-scale process overview: 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 156] The post-selection purity (%PD-1+) of all three tumors met the criterion of over 80%. The slightly lower purity observed in the melanoma tumors compared to the Hea and Neck tumors is most likely due to lower expression of PD-1+ cells during selection. [Figure 157] Figure 1. Detection of PD-1+ cells in tumor digests from various histologies. PD-1 expression in multiple histologies. The percentage of PD-1+ TILs in the CD3+ TIL population is plotted for individual samples within each histology. Horizontal lines represent the mean percentage for each subset, and vertical lines represent the standard error. [Figure 158] Plot of FACS data. [Figure 159] PD-1+ TILs selected from one ovary, one melanoma, and one HNSCC were screened using either nivolumab or EH12.2H7 to identify PD-1+ TILs. These TILs were evaluated for T cell lineage (CD4 and CD8) using flow cytometry. Results are presented as a percentage of CD3+ cells. Mean values ​​are plotted as bars, and standard errors are shown as vertical lines. [Figure 160]PD-1-selected TILs from one ovarian, one melanoma, and one HNSCC tumor sample, screened using either nivolumab or EH12.2H7 to identify PD-1+ TILs, were evaluated for expression of memory markers CD45RA and CCR7 by flow cytometry. T cell memory subsets (TN / TSCM) were determined as shown, and the mean percentage of each subset was plotted as black bars for nivolumab PD-1-selected TILs and gray bars for EH12.2H7 PD-1-selected TILs. Standard error is shown as a vertical line. [Figure 161A] PD-1-screened TILs from one ovary, one melanoma, and one HNSCC, screened using either nivolumab or EH12.2H7 to identify PD-1+ TILs, were evaluated for PD-1 expression before and after proliferation. The pre-proliferation PD-1+ percentage was determined using the post-screening purity of the PD-1-screened products. Mean values ​​are plotted as bars, and standard errors are shown as vertical lines. Statistical significance was assessed by paired Student's t-tests. ** indicates a p-value < 0.01. [Figure 161B] PD-1-selected TILs from one ovary, one melanoma, and one HNSCC, screened using either nivolumab or EH12.2H7 to identify PD-1+ TILs, were evaluated for (A) IFNγ and (B) granzyme B secretion. Results were plotted for individual samples, with black dots representing the unstimulated state and gray triangles representing the stimulated state with αCD3 / αCD28 / α41BB. Horizontal lines represent the mean percentage for each subset, and vertical lines represent the standard error. [Figure 162]Pre-sorting PD-1 levels in TILs stained with nivolumab and EH12.2H7. Whole tumor digests were divided, stained with either nivolumab or EH12.2H7, and evaluated by flow cytometry. Subsequently, PD-1+ cells identified using each antibody, from one ovary, one melanoma, and one HNSCC, were sorted using an FX500 cell sorter (SONY, NY). [Figure 163] PD-1 levels after sorting in TILs stained with nivolumab and EH12.2H7. [Figure 164] The entire tumor digest was divided, stained with either nivolumab or EH12.2H7, and evaluated by flow cytometry. Subsequently, PD-1+ cells identified using each antibody, from one ovary, one melanoma, and one HNSCC, were sorted using an FX500 cell sorter (SONY, NY). [Figure 165] Detection of PD-1+ cells in tumor digests from various histologies. PD-1 expression in multiple histologies. The percentage of PD-1+ TILs in the CD3+ TIL population is plotted for individual samples within each histology. Horizontal lines represent the mean percentage for each subset, and vertical lines represent the standard error. [Figure 166] In PD-1-selected TILs during the activation phase, digital proliferation decreased, but REP did not. PD-1-selected TILs and whole tumor digests from four melanoma, seven NSCLC, and two HNSCC tumor samples were grown using a two-step process consisting of an 11-day activation step followed by an 11-day REP step. Diagrammatic proliferation for all assayed tumors is shown. The proliferation rate in the TIL population was calculated using the total cell count at the completion of the activation and REP steps. Results are plotted for individual samples, with black dots representing PD-1-selected TILs and gray triangles representing unselected TILs. Horizontal lines represent the mean percentage for each subset, and vertical lines represent the standard error. [Figure 167]Levels of CD4+ and CD8+ T cells in PD-1-selected and unselected TILs. PD-1-selected and unselected TILs from four melanoma, seven NSCLC, and two HNSCC tumor samples were evaluated for T cell lineage (CD4 and CD8) using flow cytometry. Results are presented as percentages of CD3+ cells. Mean values ​​are plotted as bars, and standard errors are shown as vertical lines. [Figure 168] Comparison of memory T cell subset distributions in PD-1-selected and unselected TILs. PD-1-selected and unselected TILs from four melanoma, six NSCLC, and two HNSCC tumor samples were evaluated for expression of memory markers CD45RA and CCR7 by flow cytometry. T cell memory subsets were determined as shown, and the mean percentage of each subset is plotted as black bars for PD-1-selected TILs and gray bars for unselected TILs. Standard error is shown as a vertical line. [Figure 169] PD-1 expression in PD-1+-sorted and unsorted TILs before and after growth. PD-1-sorted TILs and whole tumor digests from 3 melanoma, 7 NSCLC, and 2 HNSCC tumor samples were evaluated for PD-1 expression before and after growth. The percentage of PD-1+ TILs before growth was determined using the purity of the sorted PD-1+ product. Mean values ​​are plotted as bars, and standard errors are shown as vertical lines. Statistical significance was assessed by paired Student's t-tests. *** and **** indicate p-values ​​<0.001 and <0.0001, respectively. [Figure 170]Frequencies of the top 10 PD-1 selected TCRvβ clones in unselected TILs. Legend: PD-1 selected and unselected TILs grown from two HNSCC and five NSCLC tumor samples were analyzed for the CDR3vβ repertoire. Unique CDR3vβ sequences identified in PD-1 selected TIL products were ranked from highest to lowest frequency. The frequencies of the "top 10" (i.e., the 10 most frequent) PD-1 selected TIL clones in each paired product are plotted. Paired samples are joined by an uncolored line. P-values ​​calculated by paired t-tests are indicated in each graph. [Figure 171] TILs selected with PD-1 exhibit superior autotumor reactivity compared to matched, unselected TILs. PD-1 selected TILs and matched, unselected TILs obtained from three melanoma, two NSCLC, one PC, and one TNBC sample were tested for IFN□ secretion by ELISA in response to 18–24 hour incubation with autotumor digests. The difference in IFN□ concentrations measured with and without HLA class I blocking antibodies is shown for each individual sample. Positive values ​​reflect an HLA-specific antitumor response, while zero or negative values ​​reflect a non-specific response. [Figure 172A-B] TILs selected and unselected by PD-1 exhibit autologous tumor lethality. Tumor lethality and responsiveness were evaluated in PD-1 selected and unselected TILs using the xCELLigence real-time cell analysis system. (A) Cell index and (B) Tumor cell lethality (% cell lysis) are shown for melanoma samples. [Figure 172C]PD-1 levels in TILs stained with nivolumab and EH12.2H7. Whole tumor digests were divided, stained with either nivolumab or EH12.2H7, and evaluated by flow cytometry. PD-1+ cells identified using each antibody, from one ovary, one melanoma, and one HNSCC, were then sorted using an FX500 cell sorter (SONY, NY). [Figure 173] Final product yield of PD-1 sorted TILs stained with nivolumab and EH12.2H7. PD-1 sorted TILs derived from nivolumab and EH12.2H7 stained TILs from one ovarian, one melanoma, and one HNSCC were grown using an 11-day activation step followed by 11 days of REP. Number of seeded CD3+ cells, digital proliferation, and extrapolated / actual cell counts are shown. Ovarian and melanoma tumors designated with * were small-scale experiments, while HNSCC designated with ** was performed at full scale. [Figure 174] Expression of CD4+ and CD8+ TILs in PD-1-selected TILs using EH12.2H7 and nivolumab. PD-1-selected TILs, sorted using either nivolumab or EH12.2H7 to identify PD-1+ TILs from one ovary, one melanoma, and one HNSCC, were evaluated for T cell lineage (CD4 and CD8) using flow cytometry. Results are presented as a percentage of CD3+ cells. Mean values ​​are plotted as bars, and standard errors are shown as vertical lines. [Figure 175]Memory populations of PD-1+ TILs sorted with EH12.2H7 and nivolumab. PD-1 selected TILs from one ovarian, one melanoma, and one HNSCC tumor sample, sorted using either nivolumab or EH12.2H7 to identify PD-1+ TILs, were evaluated for expression of memory markers CD45RA and CCR7 by flow cytometry. T cell memory subsets were determined as shown, and the mean percentage of each subset was plotted as black bars for nivolumab PD-1 selected TILs and gray bars for EH12.2H7 PD-1 selected TILs. Standard error is shown as a vertical line. [Figure 176] TIL expression of PD-1 in TILs screened with PD-1 generated using EH12.2H7 and nivolumab, before and after proliferation. PD-1 screened TILs from one ovary, one melanoma, and one HNSCC, screened with either nivolumab or EH12.2H7 to identify PD-1+ TILs, were evaluated for PD-1 expression before and after proliferation. The percentage of PD-1+ before proliferation was determined using the post-screening purity of the PD-1 screened products. Mean values ​​are plotted as bars, and standard errors are shown as vertical lines. Statistical significance was assessed by paired Student's t-tests. ** indicates a p-value < 0.01. [Figure 177] TILs selected with PD-1 using IFNγ produced by PD-1+ TILs screened with EH12.2H7 and nivolumab, and granzyme B, are responsive to nonspecific stimulation. PD-1 selected TILs from one ovary, one melanoma, and one HNSCC, screened with either nivolumab or EH12.2H7 to identify PD-1+ TILs, were evaluated for (A) IFNγ and (B) granzyme B secretion. Results are plotted for individual samples, with black dots representing the unstimulated state and gray triangles representing the stimulated state with αCD3 / αCD28 / α41BB. Horizontal lines represent the mean percentage for each subset, and vertical lines represent the standard error. [Figure 178] Overview of an embodiment of the PD-1+ high Gen-2 process. [Figure 179] FACS plot data.

[0214] A brief explanation of sequence listings Sequence ID 1 is the amino acid sequence of the heavy chain of muromonab.

[0215] Sequence ID 2 is the amino acid sequence of the light chain of muromonab.

[0216] Sequence ID 3 is the amino acid sequence of recombinant human IL-2 protein.

[0217] Sequence ID 4 is the amino acid sequence of aldethleukin.

[0218] Sequence ID 5 is the amino acid sequence of recombinant human IL-4 protein.

[0219] Sequence ID 6 is the amino acid sequence of recombinant human IL-7 protein.

[0220] Sequence ID 7 is the amino acid sequence of recombinant human IL-15 protein.

[0221] Sequence ID 8 is the amino acid sequence of recombinant human IL-21 protein.

[0222] Sequence ID 9 is the amino acid sequence of human 4-1BB.

[0223] Sequence ID 10 is the amino acid sequence of mouse 4-1BB.

[0224] Sequence ID 11 is the heavy chain of the 4-1BB agonist monoclonal antibody utomirumab (PF-05082566).

[0225] Sequence ID 12 is the light chain of the 4-1BB agonist monoclonal antibody utomirumab (PF-05082566).

[0226] Sequence ID 13 is the heavy chain variable region (VH) of the 4-1BB agonist monoclonal antibody utomirumab (PF-05082566).

[0227] Sequence ID 14 is the light chain variable region (VL) of the 4-1BB agonist monoclonal antibody utomirumab (PF-05082566).

[0228] Sequence ID 15 is the heavy chain CDR1 of the 4-1BB agonist monoclonal antibody utomirumab (PF-05082566).

[0229] Sequence ID 16 is the heavy chain CDR2 of the 4-1BB agonist monoclonal antibody utomirumab (PF-05082566).

[0230] Sequence ID No. 17 is the heavy chain CDR3 of the 4-1BB agonist monoclonal antibody utomirumab (PF-05082566).

[0231] Sequence ID No. 18 is the light chain CDR1 of the 4-1BB agonist monoclonal antibody utomirumab (PF-05082566).

[0232] Sequence ID 19 is the light chain CDR2 of the 4-1BB agonist monoclonal antibody utomirumab (PF-05082566).

[0233] Sequence ID No. 20 is the light chain CDR3 of the 4-1BB agonist monoclonal antibody utomirumab (PF-05082566).

[0234] Sequence ID 21 is the heavy chain of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0235] Sequence ID 22 is the light chain of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0236] Sequence ID 23 is the heavy chain variable region (VH) of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0237] Sequence ID No. 24 is the light chain variable region (VL) of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0238] Sequence ID No. 25 is the heavy chain CDR1 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0239] Sequence ID No. 26 is the heavy chain CDR2 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0240] Sequence ID No. 27 is the heavy chain CDR3 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0241] Sequence ID No. 28 is the light chain CDR1 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0242] Sequence ID No. 29 is the light chain CDR2 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0243] Sequence ID No. 30 is the light chain CDR3 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0244] Sequence ID 31 is the Fc domain of the TNFRSF agonist fusion protein.

[0245] Sequence ID 32 is the linker for the TNFRSF agonist fusion protein.

[0246] Sequence ID 33 is the linker for the TNFRSF agonist fusion protein.

[0247] Sequence ID 34 is the linker for the TNFRSF agonist fusion protein.

[0248] Sequence ID 35 is the linker for the TNFRSF agonist fusion protein.

[0249] Sequence ID 36 is the linker for the TNFRSF agonist fusion protein.

[0250] Sequence ID 37 is the linker for the TNFRSF agonist fusion protein.

[0251] Sequence ID 38 is the linker for the TNFRSF agonist fusion protein.

[0252] Sequence ID 39 is the linker for the TNFRSF agonist fusion protein.

[0253] Sequence ID 40 is the linker for the TNFRSF agonist fusion protein.

[0254] Sequence ID 41 is the linker for the TNFRSF agonist fusion protein.

[0255] Sequence ID 42 is the Fc domain of the TNFRSF agonist fusion protein.

[0256] Sequence ID 43 is the linker for the TNFRSF agonist fusion protein.

[0257] Sequence ID 44 is the linker for the TNFRSF agonist fusion protein.

[0258] Sequence ID 45 is the linker for the TNFRSF agonist fusion protein.

[0259] Sequence ID 46 is the amino acid sequence of the 4-1BB ligand (4-1BBL).

[0260] Sequence ID No. 47 is the soluble portion of the 4-1BBL polypeptide.

[0261] Sequence ID 48 is the heavy chain variable region (VH) of the 4-1BB agonist antibody 4B4-1-1 version 1.

[0262] Sequence ID 49 is the light chain variable region (VL) of the 4-1BB agonist antibody 4B4-1-1 version 1.

[0263] Sequence ID 50 is the heavy chain variable region (VH) of the 4-1BB agonist antibody 4B4-1-1 version 2.

[0264] Sequence ID 51 is the light chain variable region (VL) of the 4-1BB agonist antibody 4B4-1-1 version 2.

[0265] Sequence ID 52 is the heavy chain variable region (VH) of the 4-1BB agonist antibody H39E3-2.

[0266] Sequence ID 53 is the light chain variable region (VL) of the 4-1BB agonist antibody H39E3-2.

[0267] Sequence ID 54 is the amino acid sequence of human OX40.

[0268] Sequence ID 55 is the amino acid sequence of mouse OX40.

[0269] Sequence ID 56 is the heavy chain of the OX40 agonist monoclonal antibody tavorixizumab (MEDI-0562).

[0270] Sequence ID 57 is the light chain of the OX40 agonist monoclonal antibody tavorixizumab (MEDI-0562).

[0271] Sequence ID 58 is the heavy chain variable region (VH) of the OX40 agonist monoclonal antibody tavorixizumab (MEDI-0562).

[0272] Sequence ID 59 is the light chain variable region (VL) of the OX40 agonist monoclonal antibody tavorixizumab (MEDI-0562).

[0273] Sequence ID 60 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody tavorixizumab (MEDI-0562).

[0274] Sequence ID 61 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody tavorixizumab (MEDI-0562).

[0275] Sequence ID 62 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody tavorixizumab (MEDI-0562).

[0276] Sequence ID 63 is the light chain CDR1 of the OX40 agonist monoclonal antibody tavorixizumab (MEDI-0562).

[0277] Sequence ID 64 is the light chain CDR2 of the OX40 agonist monoclonal antibody tavorixizumab (MEDI-0562).

[0278] Sequence ID 65 is the light chain CDR3 of the OX40 agonist monoclonal antibody tavorixizumab (MEDI-0562).

[0279] Sequence ID 66 is the heavy chain of the OX40 agonist monoclonal antibody 11D4.

[0280] Sequence ID 67 is the light chain of the OX40 agonist monoclonal antibody 11D4.

[0281] Sequence ID 68 is the heavy chain variable region (VH) of the OX40 agonist monoclonal antibody 11D4.

[0282] Sequence ID 69 is the light chain variable region (VL) of the OX40 agonist monoclonal antibody 11D4.

[0283] Sequence ID 70 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody 11D4.

[0284] Sequence ID 71 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody 11D4.

[0285] Sequence ID 72 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody 11D4.

[0286] Sequence ID 73 is the light chain CDR1 of the OX40 agonist monoclonal antibody 11D4.

[0287] Sequence ID 74 is the light chain CDR2 of the OX40 agonist monoclonal antibody 11D4.

[0288] Sequence ID 75 is the light chain CDR3 of the OX40 agonist monoclonal antibody 11D4.

[0289] Sequence ID 76 is the heavy chain of the OX40 agonist monoclonal antibody 18D8.

[0290] Sequence ID 77 is the light chain of the OX40 agonist monoclonal antibody 18D8.

[0291] Sequence ID 78 is the heavy chain variable region (VH) of the OX40 agonist monoclonal antibody 18D8.

[0292] Sequence ID 79 is the light chain variable region (VL) of the OX40 agonist monoclonal antibody 18D8.

[0293] Sequence ID 80 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody 18D8.

[0294] Sequence ID No. 81 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody 18D8.

[0295] Sequence ID No. 82 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody 18D8.

[0296] Sequence ID No. 83 is the light chain CDR1 of the OX40 agonist monoclonal antibody 18D8.

[0297] Sequence ID 84 is the light chain CDR2 of the OX40 agonist monoclonal antibody 18D8.

[0298] Sequence ID No. 85 is the light chain CDR3 of the OX40 agonist monoclonal antibody 18D8.

[0299] Sequence ID 86 is the heavy chain variable region (VH) of the OX40 agonist monoclonal antibody Hu119-122.

[0300] Sequence ID 87 is the light chain variable region (VL) of the OX40 agonist monoclonal antibody Hu119-122.

[0301] Sequence ID 88 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody Hu119-122.

[0302] Sequence ID 89 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody Hu119-122.

[0303] Sequence ID 90 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody Hu119-122.

[0304] Sequence ID 91 is the light chain CDR1 of the OX40 agonist monoclonal antibody Hu119-122.

[0305] Sequence ID 92 is the light chain CDR2 of the OX40 agonist monoclonal antibody Hu119-122.

[0306] Sequence ID 93 is the light chain CDR3 of the OX40 agonist monoclonal antibody Hu119-122.

[0307] Sequence ID 94 is the heavy chain variable region (VH) of the OX40 agonist monoclonal antibody Hu106-222.

[0308] Sequence ID 95 is the light chain variable region (VL) of the OX40 agonist monoclonal antibody Hu106-222.

[0309] Sequence ID 96 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody Hu106-222.

[0310] Sequence ID 97 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody Hu106-222.

[0311] Sequence ID 98 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody Hu106-222.

[0312] Sequence ID 99 is the light chain CDR1 of the OX40 agonist monoclonal antibody Hu106-222.

[0313] Sequence ID No. 100 is the light chain CDR2 of the OX40 agonist monoclonal antibody Hu106-222.

[0314] Sequence ID 101 is the light chain CDR3 of the OX40 agonist monoclonal antibody Hu106-222.

[0315] Sequence ID 102 is the amino acid sequence of the OX40 ligand (OX40L).

[0316] Sequence ID No. 103 is the soluble portion of the OX40L polypeptide.

[0317] Sequence ID No. 104 is the alternative soluble moiety of the OX40L polypeptide.

[0318] Sequence ID 105 is the heavy chain variable region (VH) of the OX40 agonist monoclonal antibody 008.

[0319] Sequence ID 106 is the light chain variable region (VL) of the OX40 agonist monoclonal antibody 008.

[0320] Sequence ID 107 is the heavy chain variable region (VH) of the OX40 agonist monoclonal antibody 011.

[0321] Sequence ID 108 is the light chain variable region (VL) of the OX40 agonist monoclonal antibody 011.

[0322] Sequence ID 109 is the heavy chain variable region (VH) of the OX40 agonist monoclonal antibody 021.

[0323] Sequence ID No. 110 is the light chain variable region (VL) of the OX40 agonist monoclonal antibody 021.

[0324] Sequence ID 111 is the heavy chain variable region (VH) of the OX40 agonist monoclonal antibody 023.

[0325] Sequence ID 112 is the light chain variable region (VL) of the OX40 agonist monoclonal antibody 023.

[0326] Sequence ID 113 is the heavy chain variable region (VH) of an OX40 agonist monoclonal antibody.

[0327] Sequence ID No. 114 is the light chain variable region (VL) of an OX40 agonist monoclonal antibody.

[0328] Sequence ID No. 115 is the heavy chain variable region (VH) of an OX40 agonist monoclonal antibody.

[0329] Sequence ID 116 is the light chain variable region (VL) of an OX40 agonist monoclonal antibody.

[0330] Sequence ID No. 117 is the heavy chain variable region (VH) of a humanized OX40 agonist monoclonal antibody.

[0331] Sequence ID 118 is the heavy chain variable region (VH) of a humanized OX40 agonist monoclonal antibody.

[0332] Sequence ID No. 119 is the light chain variable region (VL) of a humanized OX40 agonist monoclonal antibody.

[0333] Sequence ID No. 120 is the light chain variable region (VL) of a humanized OX40 agonist monoclonal antibody.

[0334] Sequence ID 121 is the heavy chain variable region (VH) of a humanized OX40 agonist monoclonal antibody.

[0335] Sequence ID No. 122 is the heavy chain variable region (VH) of a humanized OX40 agonist monoclonal antibody.

[0336] Sequence ID 123 is the light chain variable region (VL) of a humanized OX40 agonist monoclonal antibody.

[0337] Sequence ID No. 124 is the light chain variable region (VL) of a humanized OX40 agonist monoclonal antibody.

[0338] Sequence ID No. 125 is the heavy chain variable region (VH) of an OX40 agonist monoclonal antibody.

[0339] Sequence ID No. 126 is the light chain variable region (VL) of an OX40 agonist monoclonal antibody.

[0340] 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 to which the present invention pertains. All patents and publications referenced herein are incorporated in their entirety by reference.

[0341] The term "in vivo" refers to events that occur within the subject's body.

[0342] 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.

[0343] The term "ex vivo" refers to an event involving the treatment or procedure of cells, tissues, and / or organs that have been removed from the subject's body. Appropriately, the cells, tissues, and / or organs may be returned to the subject's body through surgical or therapeutic means.

[0344] The term "rapid growth" 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 growth protocols are outlined below.

[0345] In this specification, “tumor-infiltrating lymphocytes” or “TIL” refers to the initial cell population obtained as leukocytes that have left the bloodstream and migrated into 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 obtained" or "newly isolated"), and "secondary TILs" are any TIL cell populations that are expanded or proliferated as considered herein, and include, but are not limited to, bulk TILs and proliferated TILs ("REP TILs" or "post-REP TILs"). TIL cell populations may include genetically modified TILs.

[0346] In this specification, “cell population” (including TIL) means several cells that share common traits. Generally, a population is approximately 1 × 10⁻⁶ 6 ~1 × 10 10 This is a range of numbers, and different TIL populations contain different numbers. 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 of individual cells. REP proliferation is generally performed with 1.5 × 10⁶ cells for injection. 9 ~1.5×10 10 This is done to provide a collection of individual cells.

[0347] In this specification, “cryopreserved TIL” means primary, bulk, or regenerated (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 of primary TIL.

[0348] In this specification, “thawed cryopreserved TILs” means a population of TILs that have been previously cryopreserved and then treated to return to room temperature or above, including but not limited to cell culture temperature or a temperature at which the TILs may be administered to a patient.

[0349] TILs can generally be defined biochemically using cell surface markers or functionally by their ability to invade tumors and perform therapeutic functions. TILs can generally be classified by expressing one or more of the following biomarkers: 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.

[0350] 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 the cryopreservation medium 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.

[0351] The term "central memory T cell" refers to CD45R0+ cells in humans, and CCR7(CCR7) hi ) and CD62L (CD62 hi This refers to a subset of T cells that constitutively express IL-2, CD3, CD127 (IL-7R), and IL-15R. 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 secrete IL-2 and CD40L primarily as effector molecules. Central memory T cells are dominant in the CD4 compartment of the blood and are proportionally enriched in lymph nodes and tonsils in humans.

[0352] 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 lo ), CD62L expression is heterogeneous or low (CD62L loCentral 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 is BLIMP1. Effector memory T cells rapidly secrete high levels of inflammatory cytokines (including interferon-γ, IL-4, and IL-5) after antigen stimulation. Effector memory T cells are dominant in the CD8 compartment 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.

[0353] 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.

[0354] 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.

[0355] 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.

[0356] The terms “peripheral blood lymphocytes” and “PBL” refer to T cells proliferated from peripheral blood. In some embodiments, PBLs are isolated from whole blood or apheresis products from a donor. In some embodiments, PBLs are isolated from whole blood or apheresis products from a donor by positive or negative selection of T cell phenotypes, such as CD3+CD45+ T cell phenotypes.

[0357] The term "anti-CD3 antibody" refers to an antibody or its variant, such as a monoclonal antibody, and includes human, humanized, chimeric, or mouse antibodies directed against the CD3 receptor in the T cell antigen receptor of mature T cells. Anti-CD3 antibodies include OKT-3, also known as muromonab. Anti-CD3 antibodies also include UHCT1 clones, also known as T3 and CD3ε. Other anti-CD3 antibodies include, for example, otelixizumab, teprizumab, and bicilizumab.

[0358] 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 directed against the CD3 receptor in the T cell antigen receptor of mature T cells, and includes OKT-3 (30 ng / mL, MACS GMP CD3 pure, Miltenyi Biotech, Inc., San Diego, CA, USA) and muromonab, or their variants, conservative amino acid substitutions, glycoforms, or biosimilars, as commercially available forms. The heavy and light chain amino acid sequences of muromonab are shown in Table 1 (SEQ ID NOs: 1 and 2). Hybridomas capable of producing OKT-3 are deposited in the American Type Culture Collection and assigned ATCC accession number CRL8001. The hybridoma capable of producing OKT-3 has also been deposited in the European Collection of Authenticated Cell Cultures (ECACC) and has been assigned catalog number 86022706. Table 1

[0359] 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. The amino acid sequence of recombinant human IL-2 suitable for use in the present invention is shown in Table 2 (SEQ ID NO: 3). For example, the term IL-2 encompasses human recombinant forms of IL-2, such as aldesleukin (PROLEUKIN, commercially available from multiple suppliers at 22 million IU per single-use vial), as well as recombinant IL-2 forms (catalog number CYT-209-b) commercially available from CellGenix, Inc., Portsmouth, NH, USA (CELLGRO GMP) or ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA, and other commercially available equivalents from other vendors. Aldesleukin (des-alanil-1, serine-125 human IL-2) is a non-glycosylated human recombinant form of IL-2 with a molecular weight of approximately 15 kDa. The amino acid sequence of aldesleukin suitable for use in this invention is shown in Table 2 (SEQ ID NO: 4). The term IL-2 also encompasses the pegylated forms of IL-2 described herein, including the pegylated IL-2 prodrug NKTR-214, available from Nektar Therapeutics (South San Francisco, CA, USA). NKTR-214 and pegylated IL-2 suitable for use in the present invention are described in U.S. Patent Application Publication US2014 / 0328791(A1) and International Patent Application Publication WO2012 / 065086(A1), the disclosures of which are incorporated herein by reference.Alternative forms of conjugate IL-2 suitable 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. Formulations of IL-2 suitable 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. [Table 2]

[0360] The term "IL-4" (also referred to herein as "IL4") refers to the cytokine known as interleukin 4, which is produced by Th2 T cells as well as eosinophils, basophils, and mast cells. IL-4 regulates the differentiation of naive helper T cells (Th0 cells) into Th2 T cells. Steinke and Borish, Respir. Res. 2001, 2, 66-70. Upon activation by IL-4, Th2 T cells subsequently produce additional IL-4 in a positive feedback loop. IL-4 also stimulates B cell proliferation and class II MHC expression, inducing class switching from B cells to IgE and IgG1 expression. Recombinant human IL-4 suitable for use in 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).

[0361] 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 γ-chain receptor, which is a set of signals crucial 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).

[0362] 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 their 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). The amino acid sequence of recombinant human IL-15 suitable for use in this invention is shown in Table 2 (SEQ ID NO: 7).

[0363] The term "IL-21" (also referred to herein as "IL21") refers to the pleomorphic cytokine protein known as interleukin-21, and includes all forms of IL-21, including their human and mammalian forms, conserved amino acid substitutions, glycoforms, biosimilars, and variants. IL-21 is described, for example, in Spolski and Leonard, Nat. Rev. Drug. Disc. 2014, 13, 379-95, the disclosure of which is incorporated herein by reference. IL-21 is primarily used in natural killer T cells and activated human CD4 +It is produced by T cells. Recombinant human IL-21 is a single non-glycosylated polypeptide chain containing 132 amino acids with a molecular weight of 15.4 kDa. Recombinant human IL-21 is commercially available from several suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (catalog no. CYT-408-b) and ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-21 recombinant protein, catalog no. 14-8219-80). The amino acid sequence of recombinant human IL-21 suitable for use in this invention is shown in Table 2 (SEQ ID NO: 8).

[0364] Where an "antitumor effective dose," "tumor inhibitory effective dose," or "therapeutic dose" is indicated, the exact amount of the composition of the present invention to be administered can be determined by a physician, taking into account individual differences in the patient's (subject's) age, weight, tumor size, degree of infection or metastasis, and condition. Generally, the pharmaceutical compositions containing tumor-infiltrating lymphocytes (e.g., secondary TILs or genetically modified cytotoxic lymphocytes) described herein are administered at a dose of 10 per kg of body weight. 4 ~10 11 individual cells (for example, 10 per kg of body weight) 5 ~10 6 , 10 5 ~10 10 , 10 5 ~10 11 , 10 6 ~10 10 , 10 6 ~10 11 , 10 7 ~10 11 , 10 7 ~10 10 , 10 8 ~10 11 , 10 8 ~10 10 , 10 9 ~10 11 , or 10 9 ~10 10The cells may be administered in doses of (including all integer values ​​within their range). Tumor-infiltrating lymphocytes (including, in some cases, genetically modified cytotoxic lymphocytes) compositions may also be administered multiple times in these doses. Tumor-infiltrating lymphocytes (including, in some cases, genetically modified ones) may be administered by using infusion techniques commonly known in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). The optimal dose and treatment regimen for a particular patient can be readily determined by those skilled in the medical field by monitoring the patient for signs of the disease and adjusting the treatment accordingly.

[0365] 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 referred to as “liquid tumors.” Hematological malignancies may include, but are not limited to, acute lymphoblastic leukemia (ALL), chronic lymphocytic lymphoma (CLL), small lymphocytic lymphoma (SLL), acute myeloid leukemia (AML), chronic 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.

[0366] 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 cancers of the lung, breast, prostate, colon, rectum, and bladder). The histological structure of a solid tumor includes interdependent tissue compartments containing parenchyma (cancer cells) and supporting stromal cells that may disperse the cancer cells and provide a supporting microenvironment.

[0367] The term “liquid tumor” refers to an abnormal mass of cells that is essentially 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.

[0368] 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.

[0369] In embodiments, the present invention includes a method for treating cancer with 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 embodiments, the non-myeloablative chemotherapy consists of cyclophosphamide 60 mg / kg / day for 2 days (27 and 26 days before TIL infusing) and fludarabine 25 mg / m² 2 This is for 5 days at a rate of / day (27 to 23 days before TIL infusion). In the embodiment, after non-myeloablative chemotherapy according to the present invention and TIL infusion (day 0), the patient receives intravenous infusion of IL-2 at a rate of 720,000 IU / kg every 8 hours until physiological tolerance is reached.

[0370] Experimental results indicate that lymphocyte depletion prior to adoptive transfer of tumor-specific T lymphocytes plays a crucial role in enhancing therapeutic efficacy by eliminating regulatory T cells and competing elements of the immune system ("cytokine sinks"). Therefore, some embodiments of the present invention utilize a lymphocyte depletion step (sometimes referred to as "immunosuppressive conditioning") in the patient before introducing the rTIL of the present invention.

[0371] As used herein, the terms “co-administration,” “administering simultaneously,” “administered in combination,” “administered in combination,” “simultaneous,” and “concurrent” encompass the administration of two or more pharmacoactive ingredients (in preferred embodiments of the present invention, for example, at least one potassium channel agonist in combination with multiple TILs) to a subject so that both the pharmacoactive ingredients and / or their metabolites are present in the subject at the same time. Co-administration includes co-administration in separate compositions, administration in separate compositions at different times, or administration in a composition containing two or more pharmacoactive ingredients. Co-administration in separate compositions and administration in a composition containing both drugs are preferred.

[0372] 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 will induce 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.

[0373] 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 that it completely or partially prevents a disease or its symptoms, and / or therapeutic in that it partially or completely cures the disease and / or any side effects caused by the disease. As used herein, “treatment” encompasses all treatments of diseases in mammals, particularly humans, and includes (a) preventing the development of a disease in a subject that is susceptible to the disease but has not yet been diagnosed with the disease, (b) suppressing the disease, i.e., inhibiting its development or progression, and (c) alleviating the disease, i.e., causing a regression of the disease and / or reducing one or more symptoms of the disease. “Treatment” also means the delivery of a drug to provide a pharmacological effect even when there is no disease or condition. For example, “treatment” includes the delivery of a composition that can induce an immune response or provide immunity, for example, in the case of a vaccine, when there is no disease condition.

[0374] When used in reference to a nucleic acid or a portion of a protein, the term "heterogeneous" indicates that the nucleic acid or protein contains two or more subsequences that are not essentially related to each other. For example, a nucleic acid is typically produced recombinantly and has two or more sequences from unrelated genes arranged to create a new functional nucleic acid, e.g., a promoter from one source and a coding region from another, or a coding region from different sources. Similarly, a heterogeneous protein indicates that the protein contains two or more subsequences that are not essentially related to each other (e.g., a fusion protein).

[0375] 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 correspondence (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 alignment of amino acid or nucleotide sequences are known in the art. A suitable program for determining the sequence identity percentage includes, for example, the BLAST program collection available from the BLAST website of the U.S. National Center for Biotechnology Information. Comparison between two sequences can be performed using either the BLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acid sequences, and BLASTP is used to compare amino acid sequences. ALIGN, ALIGN-2 (Genentech, South San Francisco, California), or MegAlign, available from DNASTAR, are additional publicly available software programs that can be used to align sequences. Those skilled in the art can determine appropriate parameters for maximum alignment using specific alignment software. In certain embodiments, the default parameters of the alignment software are used.

[0376] As used herein, the term “variant” includes, but is not limited to, antibodies or fusion proteins that have 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, for example, involve the substitution 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.

[0377] In this specification, “tumor-infiltrating lymphocytes” or “TIL” refers to the initial cell population obtained as leukocytes that have left the bloodstream and migrated into 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 obtained” 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 proliferated TIL or a second additional proliferated TIL (e.g., one of which includes a TIL referred to as a reREP TIL, as described in step D of Figure 27).

[0378] TILs can generally be defined biochemically using cell surface markers or functionally by their ability to invade tumors and provide therapeutic effects. TILs can generally be classified by expressing one or more of the following biomarkers: 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 the release of interferon (IFN) exceeds approximately 50 pg / mL, approximately 100 pg / mL, approximately 150 pg / mL, or approximately 200 pg / mL.

[0379] The terms “pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” are intended to include all solvents, dispersion media, coatings, antimicrobial and antifungal agents, isotonic and absorption retardants, and inactive components. The use of such pharmaceutically acceptable carriers or pharmaceutically acceptable excipients for pharmaceutically active ingredients is well known in the art. Unless any conventional pharmaceutically acceptable carrier or pharmaceutically acceptable 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.

[0380] The terms “about” and “approximately” mean within a statistically meaningful 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%, more preferably within 10%, and even more preferably within 5%. The permissible variation encompassed by the terms “about” or “approximately” depends on the particular 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 may not be, and do not need to be, exact, but approximate and / or greater or less, reflecting tolerances, conversion factors, rounding, measurement errors, and other factors known to those skilled in the art, as they may be. In general, dimensions, sizes, formulations, parameters, shapes, or other quantities or features are “about” or “approximately,” whether so explicitly stated. It should be noted that very different embodiments of size, shape, and dimensions may use the arrangements described.

[0381] When used in the attached claims, the transitional terms “contains,” “essentially consists of,” and “consist of,” in their original and amended forms, define the claims with respect to additional claim elements or steps not described and, where applicable, are excluded from the claims. The term “contains” is intended to be inclusive or free form and does not exclude any additional, undescribed elements, methods, steps, or materials. The term “consist of” excludes any elements, steps, or materials other than those specified in the claims, and in the latter case, any impurities typically associated with the specified material(s). The term “essentially consists of” limits the claims to any particular element, step, or material(s) and the essential and novel features(s) of the claimed invention, without substantially affecting them. All compositions, methods, and kits described herein that embody the present invention may, in alternative embodiments, be more specifically defined by any of the transitional terms “contains,” “essentially consists of,” and “consist of.”

[0382] "PD-1 high" or "PD-1high" or "PD-1 high The term "PD-1 high TIL" 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.

[0383] "PD-1 Intermediate" or "PD-1 Int" or "PD-1int The term "PD-1-positive TIL" 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-1int 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-1int 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-1int 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-1int TILs.

[0384] "PD-1 negative" or "PD-1neg" or "PD-1 neg The term "PD-1neg" 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-1neg T cells do not express PD-1 protein. In some cases, PD-1neg 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-1neg lymphocytes may express PD-1 at the same levels or range as the majority of lymphocytes in a control population.

[0385] PD-1 high, PD-1 int, and PD-1 neg TILs are distinct and represent different subsets of TILs grown ex vivo according to the methods described herein. In some embodiments, the ex vivo grown TIL population includes PD-1 high TILs, PD-1 int TILs, and PD-1 neg TILs.

[0386] II. TIL Manufacturing Process (Embodiment of the GEN3 process, optionally including synthetic culture medium) Without being limited to any particular theory, it is believed that the first proliferation by priming that primes T cell activation, and the subsequent rapid second proliferation that promotes T cell activation, as described in the method of the present invention, will enable the preparation of proliferated T cells that retain a “younger” phenotype, and therefore the proliferated T cells of the present invention are expected to exhibit greater cytotoxicity against cancer cells than T cells proliferated by other methods. In particular, T cell activation, as taught by the method of the present invention, which is primed by exposure to an anti-CD3 antibody (e.g., OKT-3), IL-2, and optionally antigen-presenting cells (APCs), and then promoted by subsequent additional exposure to anti-CD-3 antibodies (e.g., OKT-3), IL-2, and APCs, is thought to limit or avoid T cell maturation in culture, resulting in a T cell population with a less mature phenotype, which are less exhausted by proliferation in culture and exhibit greater cytotoxicity against cancer cells. In some embodiments, the rapid second proliferation step is divided into multiple 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 a period of 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 from the small-scale culture are cultured in the second container in a larger-scale culture for a period of about 4-7 days to achieve a scale-up of the culture. In some embodiments, the rapid proliferation step is divided into multiple steps: (a) rapidly second proliferation is performed by culturing T cells for a period of 3–4 days in a first small culture in a first container, e.g., a G-REX 100MCS container; and then (b) scaling out of the culture is achieved by 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 such second container are cultured in the second small culture for a period of approximately 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 culture in a first container, e.g., a G-REX 100MCS container, for a period of about 3–4 days, and then (b) transferring and distributing the T cells from the 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, 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 from the small culture transferred to such second containers are cultured in a larger culture for a period of about 4–7 days. In some embodiments, the rapid proliferation step is divided into multiple steps: (a) rapidly second proliferation is performed by culturing T cells in a small culture in a first container, e.g., a G-REX 100MCS container, for a period of about 4 days; and then (b) the T cells from the small culture are transferred and distributed to two, three, or four second containers larger than the first container, e.g., G-REX 500MCS containers, thereby achieving scaling out and scaling up of the culture, in which a portion of the T cells from the small culture transferred to the second container are cultured in a larger culture for a period of about 5 days.

[0387] 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.

[0388] In some embodiments, rapid second proliferation occurs when the activation of T cells brought about by the first proliferation by priming is 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, 45 , 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or after a 100% reduction.

[0389] In some embodiments, rapid second proliferation occurs after the T cell activation resulting from the first proliferation by priming has decreased by a percentage ranging from approximately 1% to 100%.

[0390] In some embodiments, the rapid second proliferation occurs after the T cell activation resulting from the first proliferation by priming has decreased by a percentage in the range of approximately 1%–10%, 10%–20%, 20%–30%, 30%–40%, 40%–50%, 50%–60%, 60%–70%, 70%–80%, 80%–90%, or 90%–100%.

[0391] In some embodiments, rapid second proliferation occurs when the activation of T cells brought about by the first proliferation by priming occurs at least 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 , performed after a reduction 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 or 99%.

[0392] In some embodiments, rapid second proliferation is caused by T cell activation resulting from the first proliferation by priming, at a maximum of 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%.

[0393] In some embodiments, the reduction in T cell activation resulting from the first proliferation by priming is determined by a reduction in the amount of interferon-γ released by T cells in response to antigen stimulation.

[0394] In some embodiments, the first proliferation of T cells due to priming takes place over a period of up to or about 7 or 8 days.

[0395] In some embodiments, the first proliferation of T cells due to priming occurs over a period of up to or about 1, 2, 3, 4, 5, 6, 7, or 8 days.

[0396] 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.

[0397] In some embodiments, the rapid second proliferation of T cells occurs over a period of up to or about 11 days.

[0398] In some embodiments, the rapid second proliferation of T cells occurs over a period of up to or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 days.

[0399] 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.

[0400] In some embodiments, the first proliferation of T cells due to priming occurs over a period of approximately 1 to 7 days, and the rapid second proliferation of T cells occurs over a period of approximately 1 to 11 days.

[0401] In some embodiments, the first proliferation of T cells due to priming occurs over a period of up to 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 or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 days.

[0402] In some embodiments, the first proliferation of T cells due to priming occurs over a period of approximately 1 to 8 days, and the rapid second proliferation of T cells occurs over a period of approximately 1 to 9 days.

[0403] 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.

[0404] In some embodiments, the first proliferation of T cells due to priming occurs over a period of approximately 1 to 7 days, and the rapid second proliferation of T cells occurs over a period of approximately 1 to 9 days.

[0405] 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.

[0406] In some embodiments, T cells are tumor-infiltrating lymphocytes (TILs).

[0407] In some embodiments, T cells are bone marrow-infiltrating lymphocytes (MILs).

[0408] In some embodiments, T cells are peripheral blood lymphocytes (PBLs).

[0409] In some embodiments, T cells are obtained from donors who have cancer.

[0410] In some embodiments, the T cells are TILs obtained from tumors excised from patients with cancer.

[0411] In some embodiments, the T cells are MILs obtained from the bone marrow of patients with hematological malignancies.

[0412] In some embodiments, the T cells are PBLs obtained from peripheral blood mononuclear cells (PBMCs) from a donor. In some embodiments, the donor has cancer. In some embodiments, the donor has hematological malignancies.

[0413] In certain embodiments of this disclosure, immunoeffector cells, such as T cells, may be obtained from units of blood collected from a subject using any of the techniques known to those skilled in the art, such as FICOLL separation. In one preferred embodiment, cells from the circulating blood of an individual are obtained by apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, and B cells, other nucleated leukocytes, erythrocytes, and platelets. In one embodiment, the cells collected by apheresis are washed to remove the plasma fraction, and optionally, the cells can be placed in a suitable buffer or medium for subsequent processing steps. In one embodiment, the cells are washed with phosphate-buffered saline (PBS). In an alternative embodiment, the washing solution may be calcium-deficient, magnesium-deficient, or many, if not all, divalent cations. In one embodiment, T cells are isolated from peripheral blood lymphocytes by lysing erythrocytes and depleting monocytes, for example, by centrifugation with a PERCOLL gradient or by countercurrent centrifugation.

[0414] In some embodiments, the T cells are PBLs isolated from the donor's lymphocyte-enriched whole blood or apheresis product. 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, 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.

[0415] In certain embodiments of this disclosure, immunoeffector cells, such as T cells, may be obtained from units of blood collected from a subject using any of the techniques known to those skilled in the art, such as FICOLL separation. In one preferred embodiment, cells from the circulating blood of an individual are obtained by apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, and B cells, other nucleated leukocytes, erythrocytes, and platelets. In one embodiment, the cells collected by apheresis are washed to remove the plasma fraction, and optionally, the cells can be placed in a suitable buffer or medium for subsequent processing steps. In one embodiment, the cells are washed with phosphate-buffered saline (PBS). In an alternative embodiment, the washing solution may be calcium-deficient, magnesium-deficient, or many, if not all, divalent cations. In one embodiment, T cells are isolated from peripheral blood lymphocytes by lysing erythrocytes and depleting monocytes, for example, by centrifugation with a PERCOLL gradient or by countercurrent centrifugation.

[0416] In some embodiments, T cells are PBLs isolated from lymphocyte-enriched whole blood or apheresis products from a donor. 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, 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. 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 phenotype cells and leaving PBLs. In other embodiments, PBLs are isolated by gradient centrifugation. Once PBLs are isolated from donor tissue, a first proliferation by priming of PBLs is performed according to a first proliferation by priming step of one of the methods described herein, resulting in a suitable number of isolated PBLs (in some embodiments, approximately 1 × 10⁶) for the first proliferation by priming culture. 7 This can be initiated by sowing PBL (Project-Based Learning).

[0417] 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.

[0418] As discussed and generally outlined herein, TILs are collected from patient samples and manipulated to increase in 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. Once thawed, they may also be restimulated to increase their metabolism before injection into the patient.

[0419] In some embodiments, as will be discussed in detail below and in the examples and drawings, the first priming growth (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)) is shortened to 1 to 8 days, and the rapid second growth (a process referred herein as 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)) is shortened to 1 to 9 days. In some embodiments, as will be discussed in detail below and in the examples and drawings, the first priming growth (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)) is shortened to 1 to 8 days, and the rapid second growth (a process referred herein as 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)) is shortened to 1 to 8 days. In some embodiments, as will be discussed in detail below and in the examples and drawings, 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)) is shortened to 1 to 7 days, and the rapid second growth (a process referred herein as 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)) is shortened to 1 to 9 days.In some embodiments, as will be discussed in detail below and in the examples and drawings, 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)) is 1 to 7 days, and the rapid second growth (a process referred herein as 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)) is 1 to 10 days. In some embodiments, the first growth by priming (e.g., the growth described as step B in Figure 1 (in particular, e.g., Figure 1B and / or Figure 1C)) is shortened to 8 days, and the rapid second growth (e.g., the growth described as step D in Figure 1 (in particular, e.g., Figure 1B and / or Figure 1C)) 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)) 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)) is 8-9 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)) 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)) is 7-8 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)) 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)) is 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)) 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)) 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)) 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)) 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)) 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)) 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)) 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)) 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)) 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)) is 7 to 9 days.In some embodiments, 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, e.g., Figure 1B and / or Figure 1C)) is 14 to 16 days, as will be discussed in detail below and in the examples and drawings. In particular, certain embodiments of the present invention are thought to include a first growth step by priming in which the TILs are activated by exposure to OKT-3 in the presence of an anti-CD3 antibody, e.g., 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 step by priming as described above are a first TIL population, i.e., a primary cell population.

[0420] The following “Step” designations A, B, C, etc., refer to non-limiting examples in Figure 1 (especially, for example, Figure 1B and / or Figure 1C) and refer to specific non-limiting embodiments described herein. The order of steps below and in Figure 1 (especially, for example, Figure 1B and / or Figure 1C) is illustrative, and any combination or order of steps, as well as additional steps, repetition of steps, and / or omission of steps, are contemplated in the manner disclosed herein and herein.

[0421] 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 are then grown into larger populations for further processing as described herein, optionally cryopreserved, and optionally evaluated for phenotypic and metabolic parameters as indicators of TIL health.

[0422] 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 can originate from any solid tumor, including primary tumors, invasive tumors, or metastatic tumors. Tumor samples can 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, it has been reported that malignant melanoma tumors have particularly high levels of TILs; therefore, useful TILs are obtained from malignant melanoma tumors.

[0423] Once obtained, tumor specimens are generally prepared using sharp dissection, typically measuring 1 to approximately 8 mm. 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, and then repeating the cycle of mechanical dissociation and incubation 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 / 0244133(A1) (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 growing TILs or treating cancer.

[0424] 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 to 2 hours at 37°C and 5% CO2. In some embodiments, the tumor is digested in an enzyme mixture containing collagenase, DNase, and hyaluronidase for 1 to 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 enzymes to form a tumor digestion reaction mixture.

[0425] In some embodiments, the tumor is reconstituted with lyophilized enzymes in a sterile buffer. In some embodiments, the buffer is sterile HBSS.

[0426] In some embodiments, the enzyme mixture contains collagenase. In some embodiments, the collagenase is collagenase IV. In some embodiments, the action stock of collagenase is a 10-fold action stock at 100 mg / mL.

[0427] In some embodiments, the enzyme mixture contains DNAse. In some embodiments, the action stock of DNAse is a 10-fold action stock of 10,000 IU / mL.

[0428] In some embodiments, the enzyme mixture contains hyaluronidase. In some embodiments, the action stock of hyaluronidase is a 10x action stock of 10 mg / mL.

[0429] In some embodiments, the enzyme mixture comprises 10 mg / mL collagenase, 1000 IU / mL DNAse, and 1 mg / mL hyaluronidase.

[0430] In some embodiments, the enzyme mixture comprises 10 mg / mL collagenase, 500 IU / mL DNAse, and 1 mg / mL hyaluronidase.

[0431] In some embodiments, the enzyme mixture comprises about 10 mg / mL of collagenase, about 1000 IU / mL of DNAse, and about 1 mg / mL of hyaluronidase.

[0432] 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.

[0433] In some embodiments, fragmentation includes physical fragmentation, such as detachment, as well as digestion. In some embodiments, fragmentation is physical fragmentation. In some embodiments, fragmentation is detachment. In some embodiments, fragmentation is by digestion. In some embodiments, TILs may be initially cultured from enzymatic tumor digests and tumor fragments obtained from a patient. In some embodiments, TILs may be initially cultured from enzymatic tumor digests and tumor fragments obtained from a patient.

[0434] In some embodiments where the tumor is a solid tumor, after the tumor sample is obtained, for example, as provided in step A (Figure 1 (particularly, for example, as shown in Figure 1B and / or Figure 1C)), the tumor undergoes physical fragmentation. In some embodiments, the fragmentation occurs before cryopreservation. In some embodiments, the fragmentation occurs after cryopreservation. In some embodiments, the fragmentation occurs after the tumor is obtained and no cryopreservation is performed. In some embodiments, the fragmentation step is an in vitro or ex vivo process. In some embodiments, the tumor is fragmented and 10, 20, 30, 40, or more fragments or pieces are placed in each container for the first proliferation by priming. In some embodiments, the tumor is fragmented and 30 or 40 fragments or pieces are placed in each container for the first proliferation by priming. In some embodiments, the tumor is fragmented and 40 fragments or pieces are placed in each container for the first proliferation by priming. In some embodiments, the plurality of fragments includes from about 4 to about 50 fragments, and each fragment has a volume of about 27 mm 3 In some embodiments, the plurality of fragments includes from about 1300 mm 3 to about 1500 mm 3 and includes from about 30 to about 60 fragments having a total volume. In some embodiments, the plurality of fragments includes about 50 fragments having a total volume of about 1350 mm 3 . In some embodiments, the plurality of fragments includes about 50 fragments having a total mass of from about 1 gram to about 1.5 grams. In some embodiments, the plurality of fragments includes about 4 fragments.

[0435] In some embodiments, TIL is obtained from tumor fragments. In some embodiments, the tumor fragments are obtained by sharp dissection. In some embodiments, the tumor fragments are about 1 mm 3 to 10 mm 3 . In some embodiments, the tumor fragments are about 1 mm 3 to 8 mm 3 . In some embodiments, the tumor fragments are about 1 mm 3It is. In some embodiments, the tumor fragment is about 2 mm 3 It is. In some embodiments, the tumor fragment is about 3 mm 3 It is. In some embodiments, the tumor fragment is about 4 mm 3 It is. In some embodiments, the tumor fragment is about 5 mm 3 It is. In some embodiments, the tumor fragment is about 6 mm 3 It is. In some embodiments, the tumor fragment is about 7 mm 3 It is. In some embodiments, the tumor fragment is about 8 mm 3 It is. In some embodiments, the tumor fragment is about 9 mm 3 It is. In some embodiments, the tumor fragment is about 10 mm 3 It is. 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.

[0436] In some embodiments, the tumor is fragmented to minimize the amount of hemorrhagic, necrotic, and / or fatty tissue on each small piece. In some embodiments, the tumor is fragmented to minimize the amount of hemorrhagic tissue on each small piece. In some embodiments, the tumor is fragmented to minimize the amount of necrotic tissue on each small piece. In some embodiments, the tumor is fragmented to minimize the amount of fatty tissue on each small piece. In certain embodiments, the step of fragmenting the tumor is an in vitro or ex vivo method.

[0437] In some embodiments, tumor fragmentation is performed to preserve the internal structure of the tumor. In some embodiments, tumor fragmentation is performed without using a scalpel or sawing motion. In some embodiments, TIL is obtained from tumor digestate. In some embodiments, tumor digestate is produced by incubation in an enzyme medium, e.g., 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 be mechanically dissociated a third time 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, with or without further incubation at 37°C in 5% CO2 for 30 minutes. In some embodiments, if the cell suspension contained a large number of erythrocytes or dead cells, these cells could be removed at the end of the final incubation by density gradient separation using Ficoll.

[0438] 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 a tumor sample) and may be cryopreserved before proceeding to proliferation as described in step B, as further described below and illustrated in Figure 1 (in particular, e.g., Figure 1B and / or Figure 1C).

[0439] 1. TILs originating from core / small biopsy In some embodiments, TILs are initially obtained from a patient tumor sample ("primary TIL") obtained by core biopsy or a similar procedure, then grown into a larger population for further processing as described herein, optionally cryopreserved, and optionally evaluated for phenotypic and metabolic parameters.

[0440] In some embodiments, a patient's tumor sample may be obtained using methods known in the art, generally via small biopsies, core biopsies, needle biopsies, 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 also 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 comprise multiple tumor samples from a single tumor from the same patient. In some embodiments, a sample may comprise multiple tumor samples from one, two, three, or four tumors from the same patient. In some embodiments, a sample may comprise 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.

[0441] 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, newly acquired TIL cell populations are exposed to cell culture media containing antigen-presenting cells, IL-2, and OKT-3.

[0442] 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 lymph nodes in the neck or axilla region, if available. In some embodiments, a skin lesion or a biopsy thereof is removed. In some embodiments, a lymph node or a biopsy thereof is removed. In some embodiments, metastatic lesions of the lung or liver, or intraperitoneal or thoracic lymph nodes or biopsies thereof may be used.

[0443] In some embodiments, the tumor is a melanoma. In some embodiments, a small biopsy of the melanoma includes a mole or a portion thereof.

[0444] In some embodiments, the small biopsy is a punch biopsy. In some embodiments, the punch biopsy is obtained by pressing a circular blade into the skin. In some embodiments, the punch biopsy is obtained by pressing a circular blade into the skin around the suspected mole. In some embodiments, the punch biopsy is obtained by pressing a circular blade into the skin and a round piece of skin is removed. In some embodiments, the small biopsy is a punch biopsy and a round tumor portion is removed.

[0445] In some embodiments, the small biopsy is an excisional biopsy. In some embodiments, the small biopsy is an excisional biopsy in which the entire mole or growing portion is removed. In some embodiments, the small biopsy is an excisional biopsy in which the entire mole or growing portion is removed along with a small border of normal-looking skin.

[0446] In some embodiments, the microbiome is an incisional biopsy. In some embodiments, the microbiome is an incisional biopsy in which only the most irregular or growing portion of the mole is taken. In some embodiments, the microbiome is an incisional biopsy, which is used when other techniques cannot be completed, such as when the suspected mole is very large.

[0447] 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 placed under anesthesia, 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 remove some tissue. In some embodiments, if the tumor or growing portion cannot be reached by bronchoscopy, a transthoracic needle biopsy may be used. Generally, in the case of a transthoracic needle biopsy, the patient is also placed under anesthesia, and a needle is inserted directly through the skin to the suspected site to remove a small tissue sample. In some embodiments, a transthoracic needle biopsy may require interventional imaging (e.g., the use of X-ray or CT scans 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 illuminated endoscope placed through the mouth into the esophagus). In some embodiments, the small biopsy is obtained surgically.

[0448] 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 removed from the area that appears abnormal. In some embodiments, if the abnormal area is easily accessible, the sample can be taken without hospitalization. In some embodiments, if the tumor is 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 removed using punch forceps.

[0449] In some embodiments, the small biopsy is a cervical biopsy. In some embodiments, the small biopsy is obtained via a vaginal speculum. Generally, the vaginal speculum method involves using 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 remove a larger tissue sample from the cervix. In some embodiments, in addition to helping confirm the diagnosis, a cone biopsy may serve as an initial treatment.

[0450] 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 disperse the cancer cells and provide a supporting microenvironment.

[0451] In some embodiments, samples from the tumor are obtained as fine-needle aspiration (FNA), core biopsies, or microbiops (including, for example, punch biopsies). In some embodiments, the samples are first placed in the G-Rex 10. In some embodiments, if there are one or two core biopsy and / or microbiopsy samples, the samples are first placed in the G-Rex 10. In some embodiments, if there are three, four, five, six, eight, nine, or ten or more core biopsy and / or microbiopsy samples, the samples are first placed in the G-Rex 100. In some embodiments, if there are three, four, five, six, eight, nine, or ten or more core biopsy and / or microbiopsy samples, the samples are first placed in the G-Rex 500.

[0452] 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.

[0453] The TILs described herein may be obtained from FNA samples. In some cases, FNA samples are obtained or isolated from patients using fine-gauge needles in the range of 18-gauge to 25-gauge needles. 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, FNA samples from patients 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.

[0454] In some cases, the TILs described herein are obtained from core biopsy specimens. In some cases, core biopsy specimens are obtained or isolated from patients using surgical or medical needles ranging from 11-gauge to 16-gauge needles. The needles may be 11-gauge, 12-gauge, 13-gauge, 14-gauge, 15-gauge, or 16-gauge. In some embodiments, core biopsy specimens from patients 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.

[0455] Generally, a collected cell suspension is called a "primary cell population" or a "freshly collected" cell population.

[0456] In some embodiments, TILs are not obtained from tumor digests. In some embodiments, the solid tumor core is not fragmented.

[0457] In some embodiments, TIL is obtained from tumor digestate. In some embodiments, tumor digestate is produced by incubation in an enzyme medium, e.g., 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 be mechanically dissociated a third time 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, with or without further incubation at 37°C in 5% CO2 for 30 minutes. In some embodiments, if the cell suspension contained a large number of erythrocytes or dead cells, these cells could be removed at the end of the final incubation by density gradient separation using Ficoll.

[0458] 2. Method for increasing peripheral blood lymphocytes (PBLs) from peripheral blood PBL Method 1. In embodiments of the present invention, PBL is grown using the process described herein. In embodiments of the present invention, this method comprises obtaining a PBMC sample from whole blood. In embodiments, this method comprises enriching T cells by isolating pure T cells from PBMCs using negative selection of the non-CD19+ fraction. In embodiments, this method comprises enriching T cells by isolating pure T cells from PBMCs using magnetic bead-based negative selection of the non-CD19+ fraction.

[0459] In 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).

[0460] PBL Method 2. In embodiments of the present invention, PBL is grown using PBL Method 2, which includes obtaining a PBMC sample from whole blood. T cells from the PBMCs are enriched by incubating the PBMCs at 37°C for at least 3 hours, and then isolating non-adherent cells.

[0461] In 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 per 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.

[0462] PBL Method 3. In embodiments of the present invention, PBL is grown 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.

[0463] In embodiments of the present invention, PBL method 3 is performed as follows: On day 0, cryopreserved PBMCs derived 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).

[0464] In one embodiment, PBMCs are isolated from a whole blood sample. In another embodiment, the PBMC sample is used as a starting material for growing PBLs. In yet another embodiment, the sample is cryopreserved before the growth process. In yet another embodiment, a fresh sample is used as a starting material for growing PBLs. In one embodiment of the present invention, T cells are isolated from PBMCs using methods known in the art. In one embodiment, T cells are isolated using a human Pan T cell isolation kit and an LS column. In one embodiment of the present invention, T cells are isolated from PBMCs using antibody selection methods known in the art, such as CD19 negative selection.

[0465] In embodiments of the present invention, the PBMC sample is incubated for a certain period of time at a desired temperature effective for identifying non-adherent cells. In embodiments of the present invention, the incubation time is approximately 3 hours. In embodiments of the present invention, the temperature is approximately 37°C. The non-adherent cells are then grown using the process described above.

[0466] In some embodiments, PBMC samples are from subjects or patients who have been optionally previously treated with a regimen containing a kinase inhibitor or ITK inhibitor. In some embodiments, tumor samples are from subjects or patients who have been previously treated with a regimen containing a kinase inhibitor or ITK inhibitor. In some embodiments, PBMC samples are from subjects or patients who have been previously treated with a regimen containing a kinase inhibitor or ITK inhibitor and have been receiving 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 more than one year. In another embodiment, PBMCs are derived from patients currently receiving an ITK inhibitor regimen such as ibrutinib.

[0467] In some embodiments, the PBMC sample is from a subject or patient who has been previously treated with a regimen containing a kinase inhibitor or an ITK inhibitor and is refractory to treatment with the kinase inhibitor or an ITK inhibitor such as ibrutinib.

[0468] In some embodiments, PBMC samples are 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 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. In another embodiment, PBMCs are 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.

[0469] In embodiments of the present invention, on day 0, cells are selected for CD19+ and sorted accordingly. In embodiments of the present invention, selection is performed using antibody-conjugated beads. In embodiments of the present invention, pure T cells are isolated from PBMCs on day 0.

[0470] In embodiments of the present invention, for patients who have not been previously treated with ibrutinib or other ITK inhibitors, 10-15 mL of buffy coat is used for approximately 5 × 10 9 This yields PBMCs, which then result in approximately 5.5 × 10⁻⁶ units. 7 This brings about individual PBL (Project-Based Learning).

[0471] In embodiments of the present invention, in patients previously treated with ibrutinib or other ITK inhibitors, the proliferation process is approximately 20 × 10 9 This results in a PBL of 40.3 × 10 6 Each PBMC is approximately 4.7 × 10 5 This brings about individual PBL (Project-Based Learning).

[0472] In any of the embodiments described above, PBMCs may be derived from a whole blood sample, by apheresis, from a buffy coat, or from any other method known in the art for obtaining PBMCs.

[0473] 3. Method for proliferating bone marrow-infiltrating lymphocytes (MILs) from bone marrow-derived PBMCs. MIL Method 3. In embodiments of the present invention, this method includes obtaining PBMCs from bone marrow. On day 0, PBMCs are selected and sorted for CD3+ / CD33+ / CD20+ / CD14+ cells, the non-CD3+ / CD33+ / CD20+ / CD14+ cell fraction is sonicated, and a portion of the sonicated cell fraction is returned to the selected cell fraction.

[0474] In embodiments of the present invention, 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+).

[0475] In embodiments of the present invention, PBMCs are obtained from bone marrow. In embodiments, PBMCs are obtained from bone marrow by apheresis, aspiration, needle biopsy, or other similar means known in the art. In embodiments, PBMCs are fresh. In other embodiments, PBMCs are cryopreserved.

[0476] In embodiments of the present invention, MIL is grown from 10 to 50 ml of bone marrow aspirate. In embodiments of the present invention, 10 ml of bone marrow aspirate is obtained from the patient. In another embodiment, 20 ml of bone marrow aspirate is obtained from the patient. In another embodiment, 30 ml of bone marrow aspirate is obtained from the patient. In another embodiment, 40 ml of bone marrow aspirate is obtained from the patient. In another embodiment, 50 ml of bone marrow aspirate is obtained from the patient.

[0477] In embodiments of the present invention, the number of PBMCs obtained from approximately 10-50 ml of bone marrow aspirate is approximately 5 × 10 7 ~About 10×10 7 This is a PBBC ​​of 10¹⁶. In another embodiment, the number of resulting PMBCs is approximately 7 × 10¹⁶. 7 This is a private-use mobile phone manufacturer (PBMC).

[0478] In embodiments of the present invention, approximately 5 × 10 7 ~About 10×10 7 Each PBMC is approximately 0.5 × 10 6 ~Approx. 1.5×10 6 This yields a number of MILs. In embodiments of the present invention, approximately 1 × 10 6 This number of MILs is produced.

[0479] In embodiments of the present invention, 12 × 10 6 Each PBMC is approximately 1.4 × 10 5 It brings about individual MILs.

[0480] In any of the embodiments described above, PBMCs may be derived from a whole blood sample, from bone marrow, by apheresis, from a buffy coat, or from any other method known in the art for obtaining PBMCs.

[0481] 4. Pre-selection of 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.

[0482] 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 proliferated to a density of 200,000. In some embodiments, the cells are grown or proliferated 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 proliferated to a density of 150,000. In some embodiments, the cells are grown or proliferated 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 proliferated to a density of 250,000.In some embodiments, cells are grown or proliferated 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.

[0483] 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).

[0484] 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., a mouse anti-human PD-1 polyclonal antibody, a 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, M1H4, 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 CT-011, Medivation), anti-PD-1 monoclonal antibody BGB-A317 (BeiGene), and / or anti-PD-1 antibody SHR-1210 (ShangHai Examples include, but are not limited to, the human monoclonal antibody REGN2810 (Regeneron), the human monoclonal antibody MDX-1106 (Bristol-Myers Squibb), and / or the 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. As described herein, as illustrated by steps A-F, other suitable antibodies for use in pre-selection of PD-1 positive TILs for use in the proliferation of TILs by the method of the present invention are the anti-PD-1 antibodies disclosed in U.S. Patent No. 8,008,449, which are incorporated herein by reference. In some embodiments, the anti-PD-1 antibody for use in pre-selection binds to an epitope different from 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 pizilizumab (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 thereof 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.

[0485] 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.

[0486] 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.

[0487] 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.

[0488] 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.

[0489] 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.

[0490] 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.

[0491] 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.

[0492] 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 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) Adding an excess of anti-IgG4 antibody conjugated to a 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).

[0493] In some embodiments, PD-1-positive TILs are PD-1-high TILs.

[0494] 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.

[0495] 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).

[0496] 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 conjugated to PD-1 by the N-terminal loop outside the IgV domain of PD-1; (ii) adding an excess of anti-IgG4 antibody conjugated to a fluorophore; and (iii) performing flow-based 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 conjugate 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) is EH12.2H7 or conjugates to the same epitope as nivolumab.

[0497] In some embodiments, the PD-1 gating method of WO2019 / 156568 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 immunostaining intensity of PD-1 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.

[0498] 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.

[0499] 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.

[0500] B. Step B: First propagation by priming In some embodiments, the method provides younger TILs that may offer additional therapeutic benefits compared to older TILs (i.e., TILs that have undergone more replication rounds before administration to the subject / patient). The characteristics of young TILs have been described in the literature, for example, Donia, at al., Scandinavian Journal of Immunology, 75:157-167 (2012), Dudley et al., Clin Cancer Res, 16:6122-6131 (2010), Huang et al., J Immunother, 28(3):258-267 (2005), Besser et al. al., Clin Cancer Res,19(17):OF1-OF9(2013), Besser et al.,J Immunother 32:415-423(2009), Robbins,et al.,J Immunol 2004;173:7125-7130, Shen et al.,J Immunother,30:123-129(2007), Zhou,et al.,J This is described in Immunother, 28:53-62 (2005), and Tran, et al., J Immunother, 31:742-751 (2008), all of which are incorporated herein by reference in their entirety.

[0501] For example, after detachment or digestion of tumor fragments and / or tumor fragments (e.g., to obtain whole tumor digest and / or whole tumor cell suspension), as described in step A of Figure 1 (especially, e.g., Figure 1B and / or Figure 1C), the resulting cells are cultured in serum containing IL-2, OKT-3, and feeder cells (e.g., antigen-presenting feeder cells or irradiated allogeneic PBMCs) under conditions more favorable to TIL growth than tumor and other cells. In some embodiments, IL-2, OKT-3, and feeder cells are added at the start of culture, along with the tumor digest and / or tumor fragments (e.g., on day 0). In some embodiments, the tumor digest and / or tumor fragments are incubated in a container containing up to 60 fragments per container (in embodiments where fragments are used) and 6000 IU / mL of IL-2. In some embodiments, this primary cell population is cultured for several days, generally 1 to 8 days, resulting in a bulk TIL population, generally about 1 × 10⁶ 8 This generates a bulk TIL population of approximately 1 × 10¹⁶ cells. In some embodiments, this primary cell population is cultured for several days, generally 1 to 7 days, resulting in a bulk TIL population, generally about 1 × 10¹⁶ cells. 8 This generates a bulk TIL population of approximately 1 × 10⁶ cells. In some embodiments, the first proliferation by priming occurs over a period of 1 to 8 days, resulting in a bulk TIL population, generally about 1 × 10⁶ cells. 8 This generates a bulk TIL population of approximately 1 × 10⁶ cells. In some embodiments, the first proliferation by priming occurs over a period of 1 to 7 days, resulting in a bulk TIL population, generally about 1 × 10⁶ cells. 8 This generates a bulk TIL population of approximately 1 × 10⁶ cells. In some embodiments, this priming-induced first proliferation occurs over a period of 5–8 days, resulting in a bulk TIL population, generally about 1 × 10⁶ cells. 8 This generates a bulk TIL population of approximately 1 × 10⁶ cells. In some embodiments, this priming-induced first proliferation occurs over a period of 5–7 days, resulting in a bulk TIL population, generally about 1 × 10⁶ cells. 8 This generates a bulk TIL population of approximately 1 × 10⁶ cells. In some embodiments, this priming-induced first proliferation occurs over a period of about 6–8 days, resulting in a bulk TIL population, generally about 1 × 10⁶ cells. 8This generates a bulk TIL population of approximately 1 × 10⁶ cells. In some embodiments, this priming-induced first proliferation occurs over a period of about 6–7 days, resulting in a bulk TIL population, generally about 1 × 10⁶ cells. 8 This generates a bulk TIL population of approximately 1 × 10⁶ cells. In some embodiments, this priming-induced first proliferation occurs over a period of about 7-8 days, resulting in a bulk TIL population, generally about 1 × 10⁶ cells. 8 This generates a bulk TIL population of approximately 1 × 10⁶ cells. In some embodiments, this priming-induced first proliferation occurs over a period of about 7 days, resulting in a bulk TIL population, generally about 1 × 10⁶ cells. 8 This generates a bulk TIL population of approximately 1 × 10⁶ cells. In some embodiments, this priming-induced first proliferation occurs over a period of about 8 days, resulting in a bulk TIL population, generally about 1 × 10⁶ cells. 8 This generates individual bulk TIL cells.

[0502] In some embodiments, any preferred dose of TIL may be administered. In some embodiments, particularly when the cancer is melanoma, approximately 2.3 × 10 10 ~Approx. 13.7×10 10 TILs were administered, with an average of approximately 7.8 × 10⁶ 10 TIL. In the embodiment, approximately 1.2 × 10 10 ~Approx. 4.3×10 10 TIL is administered. In some embodiments, approximately 3 × 10 10 ~Approx. 12×10 10 TIL is administered. In some embodiments, approximately 4 × 10 10 ~About 10×10 10 TIL is administered. In some embodiments, approximately 5 × 10 10 ~Approx. 8×10 10 TIL is administered. In some embodiments, approximately 6 × 10 10 ~Approx. 8×10 10 TIL is administered. In some embodiments, approximately 7 × 10 10 ~Approx. 8×10 10 TIL is administered. In some embodiments, the therapeutically effective dose is approximately 2.3 × 10⁻⁶ 10 ~Approx. 13.7×10 10In some embodiments, particularly when the cancer is melanoma, the therapeutically effective dose is approximately 7.8 × 10⁻⁶. 10 This is a TIL. In some embodiments, the therapeutically effective dose is approximately 1.2 × 10⁻⁶. 10 ~Approx. 4.3×10 10 This is the TIL. In some embodiments, the therapeutically effective dose is approximately 3 × 10⁻⁶ 10 ~Approx. 12×10 10 This is TIL. In some embodiments, the therapeutically effective dose is approximately 4 × 10⁻⁶ 10 ~About 10×10 10 This is a TIL. In some embodiments, the therapeutically effective dose is approximately 5 × 10⁻⁶ 10 ~Approx. 8×10 10 This is a TIL. In some embodiments, the therapeutically effective dose is approximately 6 × 10⁻⁶ 10 ~Approx. 8×10 10 This is TIL. In some embodiments, the therapeutically effective dose is approximately 7 × 10⁻⁶. 10 ~Approx. 8×10 10 It is TIL.

[0503] In some embodiments, the number of TILs provided in the pharmaceutical composition of the present invention is about 1 × 10 6 , 2×10 6 , 3 x 10 6 , 4×10 6 , 5×10 6 , 6×10 6 , 7×10 6 , 8×10 6 , 9×10 6 , 1 x 10 7 , 2×10 7 , 3 x 10 7 , 4×10 7 , 5×10 7 , 6×10 7 , 7×10 7 , 8×10 7 , 9×10 7 , 1 x 10 8 , 2×10 8 , 3 x 10 8 , 4×10 8 , 5×10 8 , 6×10 8 , 7×10 8, 8×10 8 , 9×10 8 , 1 x 10 9 , 2×10 9 , 3 x 10 9 , 4×10 9 , 5×10 9 , 6×10 9 , 7×10 9 , 8×10 9 , 9×10 9 , 1 x 10 10 , 2×10 10 , 3 x 10 10 , 4×10 10 , 5×10 10 , 6×10 10 , 7×10 10 , 8×10 10 , 9×10 10 , 1 x 10 11 , 2×10 11 , 3 x 10 11 , 4×10 11 , 5×10 11 , 6×10 11 , 7×10 11 , 8×10 11 , 9×10 11 , 1 x 10 12 , 2×10 12 , 3 x 10 12 , 4×10 12 , 5×10 12 , 6×10 12 , 7×10 12 , 8×10 12 , 9×10 12 , 1 x 10 13 , 2×10 13 , 3 x 10 13 , 4×10 13 , 5×10 13 , 6×10 13 , 7×10 13 , 8×10 13 , and 9×10 13 In this embodiment, the number of TILs provided in the pharmaceutical composition of the present invention is 1 × 10⁻⁶. 6 ~5×10 6 , 5×10 6 ~1 × 10 7 , 1 x 10 7 ~5×10 7 , 5×107 ~1 × 10 8 , 1 x 10 8 ~5×10 8 , 5×10 8 ~1 × 10 9 , 1 x 10 9 ~5×10 9 , 5×10 9 ~1 × 10 10 , 1 x 10 10 ~5×10 10 , 5×10 10 ~1 × 10 11 , 5×10 11 ~1 × 10 12 , 1 x 10 12 ~5×10 12 , and 5×10 12 ~1 × 10 13 It is within the range.

[0504] In preferred embodiments, TIL growth may include a process referred to as pre-REP or priming-based REP, as described below and herein, and containing feeder cells from day 0 and / or from the start of culture, a first priming-based growth step (e.g., as described in step B of Figure 1 (e.g., Figure 1B and / or Figure 1C)), followed by rapid second growth (including a process referred to as step D, the rapid growth protocol (REP) step), as described below and herein, followed optionally by cryopreservation, followed by a second step D (including a process referred to as the restimulation REP step), as described below and herein. TILs obtained from this process may be optionally characterized with respect to the phenotypic features and metabolic parameters described herein. In some embodiments, tumor fragments are approximately 1 mm 3 ~10mm 3 That is the case.

[0505] In some embodiments, the first growth culture medium is referred to as "CM," an abbreviation for culture medium. In some embodiments, the CM in step B consists of RPMI 1640 with GlutaMAX, supplemented with 10% human AB serum, 25 mM Hepes, and 10 mg / mL gentamicin.

[0506] In some embodiments, there are 240 or fewer tumor fragments. In some embodiments, there are 240 or fewer tumor fragments placed in 4 or fewer containers. In some embodiments, the containers are GREX100 MCS flasks. In some embodiments, 60 or fewer tumor fragments are placed in one container. In some embodiments, each container contains 500 mL or less of culture medium per container. In some embodiments, the culture medium contains IL-2. In some embodiments, the culture medium contains 6000 IU / mL of IL-2. In some embodiments, the culture medium contains antigen-presenting feeder cells (also referred to herein as "antigen-presenting cells"). In some embodiments, the culture medium contains 2.5 × 10⁶ per container. 8 It contains antigen-presenting feeder cells. In some embodiments, the medium contains OKT-3. In some embodiments, the medium contains 30 ng / mL of OKT-3 per container. In some embodiments, the container is a GREX100 MCS flask. In some embodiments, the medium contains 6000 IU / mL of IL-2, 30 ng of OKT-3, and 2.5 × 10¹⁶ antigen-presenting feeder cells. 8 The medium contains antigen-presenting feeder cells. In some embodiments, the medium contains 6000 IU / mL of IL-2, 30 ng / mL of OKT-3, and 2.5 × 10⁶ cells per container. 8 It contains individual antigen-presenting feeder cells.

[0507] Following the preparation of tumor fragments, whole tumor digests, and / or whole tumor cell suspensions, the resulting cells (i.e., the primary cell population from the fragments and / or digests) are cultured in a medium containing IL-2, antigen-presenting feeder cells, and OKT-3 under conditions more favorable to TIL growth than tumor and other cells, enabling TIL priming and accelerated growth from the start of culture on day 0. In some embodiments, the tumor digest and / or tumor fragments are incubated with 6000 IU / mL of IL-2, as well as antigen-presenting feeder cells and OKT-3. This primary cell population is cultured for several days, generally 1 to 8 days, resulting in a bulk TIL population, generally about 1 × 10⁶. 8 This generates a bulk TIL population of approximately 1 × 10¹⁶ cells. In some embodiments, the growth medium during the first proliferation by priming contains IL-2 or its variants, as well as antigen-presenting feeder cells and OKT-3. In some embodiments, this primary cell population is cultured for several days, generally 1 to 7 days, resulting in a bulk TIL population, generally about 1 × 10¹⁶ cells. 8 This generates a bulk TIL cell population. In some embodiments, the growth medium during the first proliferation by priming contains IL-2 or its variant, as well as antigen-presenting feeder cells and OKT-3. In some embodiments, IL-2 is recombinant human IL-2 (rhIL-2). In some embodiments, the IL-2 stock solution contains 20-30 × 10¹⁶ cells per 1 mg vial. 6 It has a specific activity of IU / mg. In some embodiments, the IL-2 stock solution is 20 × 10⁶ per 1 mg vial. 6 It has a specific activity of IU / mg. In some embodiments, the IL-2 stock solution is 25 × 10⁶ per 1 mg vial. 6 It has a specific activity of IU / mg. In some embodiments, the IL-2 stock solution is 30 × 10⁶ per 1 mg vial. 6 It has a specific activity of IU / mg. In some embodiments, the IL-2 stock solution is 4-8 × 10⁻⁶ 6 The final concentration of IL-2 is IU / mg. In some embodiments, the IL-2 stock solution is 5-7 × 10⁻⁶ 6The final concentration of IL-2 is IU / mg. In some embodiments, the IL-2 stock solution is 6 × 10⁻⁶ 6The final concentration of IL-2 is IU / mg. In some embodiments, the IL-2 stock solution is prepared as described in Example C. In some embodiments, the first growth culture medium by priming contains about 10,000 IU / mL of IL-2, about 9,000 IU / mL of IL-2, about 8,000 IU / mL of IL-2, about 7,000 IU / mL of IL-2, about 6,000 IU / mL of IL-2, or about 5,000 IU / mL of IL-2. In some embodiments, the first growth culture medium by priming contains about 9,000 IU / mL of IL-2 to about 5,000 IU / mL of IL-2. In some embodiments, the first growth culture medium by priming contains about 8,000 IU / mL of IL-2 to about 6,000 IU / mL of IL-2. In some embodiments, the first growth culture medium with priming contains about 7,000 IU / mL of IL-2 to about 6,000 IU / mL of IL-2. In some embodiments, the first growth culture medium with priming contains about 6,000 IU / mL of IL-2. In embodiments, the cell culture medium further contains IL-2. In some embodiments, the first growth cell culture medium with priming contains about 3,000 IU / mL of IL-2. In embodiments, the first growth cell culture medium with priming further contains IL-2. In preferred embodiments, the first growth cell culture medium with priming contains about 3,000 IU / mL of IL-2. In the embodiment, the first cell culture medium for priming contains IL-2 at approximately 1000 IU / mL, 1500 IU / mL, 2000 IU / mL, 2500 IU / mL, 3000 IU / mL, 3500 IU / mL, 4000 IU / mL, 4500 IU / mL, 5000 IU / mL, 5500 IU / mL, 6000 IU / mL, 6500 IU / mL, 7000 IU / mL, 7500 IU / mL, or 8000 IU / mL. In the embodiment, the first cell culture medium for priming contains IL-2 in concentrations of 1000-2000 IU / mL, 2000-3000 IU / mL, 3000-4000 IU / mL, 4000-5000 IU / mL, 5000-6000 IU / mL, 6000-7000 IU / mL, 7000-8000 IU / mL, or approximately 8000 IU / mL.

[0508] In some embodiments, the first growth culture medium by priming includes approximately 500 IU / mL of IL-15, approximately 400 IU / mL of IL-15, approximately 300 IU / mL of IL-15, approximately 200 IU / mL of IL-15, approximately 180 IU / mL of IL-15, approximately 160 IU / mL of IL-15, approximately 140 IU / mL of IL-15, approximately 120 IU / mL of IL-15, or approximately 100 IU / mL of IL-15. In some embodiments, the first growth culture medium by priming includes approximately 500 IU / mL of IL-15 to approximately 100 IU / mL of IL-15. In some embodiments, the first growth culture medium by priming includes approximately 400 IU / mL of IL-15 to approximately 100 IU / mL of IL-15. In some embodiments, the first growth culture medium with priming contains about 300 IU / mL of IL-15 to about 100 IU / mL of IL-15. In some embodiments, the first growth culture medium with priming contains about 200 IU / mL of IL-15. In some embodiments, the first growth culture medium with priming contains about 180 IU / mL of IL-15. In embodiments, the first growth cell culture medium with priming further contains IL-15. In preferred embodiments, the first growth cell culture medium with priming contains about 180 IU / mL of IL-15.

[0509] In some embodiments, the first growth culture medium by priming includes approximately 20 IU / mL of IL-21, approximately 15 IU / mL of IL-21, approximately 12 IU / mL of IL-21, approximately 10 IU / mL of IL-21, approximately 5 IU / mL of IL-21, approximately 4 IU / mL of IL-21, approximately 3 IU / mL of IL-21, approximately 2 IU / mL of IL-21, approximately 1 IU / mL of IL-21, or approximately 0.5 IU / mL of IL-21. In some embodiments, the first growth culture medium by priming includes approximately 20 IU / mL of IL-21 to approximately 0.5 IU / mL of IL-21. In some embodiments, the first growth culture medium by priming includes approximately 15 IU / mL of IL-21 to approximately 0.5 IU / mL of IL-21. In some embodiments, the first growth culture medium with priming contains about 12 IU / mL of IL-21 to about 0.5 IU / mL of IL-21. In some embodiments, the first growth culture medium with priming contains about 10 IU / mL of IL-21 to about 0.5 IU / mL of IL-21. In some embodiments, the first growth culture medium with priming contains about 5 IU / mL of IL-21 to about 1 IU / mL of IL-21. In some embodiments, the first growth culture medium with priming contains about 2 IU / mL of IL-21. In some embodiments, the first growth cell culture medium with priming contains about 1 IU / mL of IL-21. In some embodiments, the first growth cell culture medium with priming contains about 0.5 IU / mL of IL-21. In embodiments, the cell culture medium further contains IL-21. In a preferred embodiment, the first cell culture medium for priming contains approximately 1 IU / mL of IL-21.

[0510] In some embodiments, the first cell culture medium for priming contains OKT-3 antibody. In some embodiments, the first cell culture medium for priming contains about 30 ng / mL of OKT-3 antibody. In some embodiments, the first cell culture medium for priming contains about 0.1 ng / mL, about 0.5 ng / mL, about 1 ng / mL, about 2.5 ng / mL, about 5 ng / mL, about 7.5 ng / mL, about 10 ng / mL, about 15 ng / mL, about 20 ng / mL, about 25 ng / mL, about 30 ng / mL, about 35 ng / mL, about 40 ng / mL, about 50 ng / mL, about 60 ng / mL, about 70 ng / mL, about 80 ng / mL, about 90 ng / mL, about 100 ng / mL, about 200 ng / mL, about 500 ng / mL, and about 1 μg / mL of OKT-3 antibody. In some embodiments, the cell culture medium contains OKT-3 antibody in concentrations of 0.1 ng / mL to 1 ng / mL, 1 ng / mL to 5 ng / mL, 5 ng / mL to 10 ng / mL, 10 ng / mL to 20 ng, 20 ng / mL to 30 ng / mL, 30 ng / mL to 40 ng / mL, 40 ng / mL to 50 ng / mL, and 50 ng / mL to 100 ng / mL. In some embodiments, the cell culture medium contains OKT-3 antibody in concentrations of 15 ng / mL to 30 ng / mL. In some embodiments, the cell culture medium contains OKT-3 antibody in concentrations of 30 ng / mL. In some embodiments, the OKT-3 antibody is muromonab. [Table 3]

[0511] In some embodiments, the first growth cell culture medium primed contains one or more TNFRSF agonists in the cell culture medium. In some embodiments, the TNFRSF agonist contains a 4-1BB agonist. In some embodiments, the TNFRSF agonist is a 4-1BB agonist, which is selected from the group consisting of urelumab, utomirumab, EU-101, fusion proteins, and their fragments, derivatives, variants, biosimilars, and combinations. In some embodiments, the TNFRSF agonist is added at a concentration sufficient to achieve a concentration in the cell culture medium of 0.1 μg / mL to 100 μg / mL. In some embodiments, the TNFRSF agonist is added at a concentration sufficient to achieve a concentration in the cell culture medium of 20 μg / mL to 40 μg / mL.

[0512] In some embodiments, in addition to one or more TNFRSF agonists, the first growth cell culture medium by priming further contains IL-2 at an initial concentration of about 3000 IU / mL and OKT-3 at an initial concentration of about 30 ng / mL, and one or more TNFRSF agonists include a 4-1BB agonist. In some embodiments, in addition to one or more TNFRSF agonists, the first growth cell culture medium by priming further contains IL-2 at an initial concentration of about 6000 IU / mL and OKT-3 at an initial concentration of about 30 ng / mL, and one or more TNFRSF agonists include a 4-1BB agonist.

[0513] In some embodiments, the first growth medium by priming is referred to as "CM," an abbreviation for culture medium. In some embodiments, it is referred to as CM1 (Culture Medium 1). In some embodiments, CM consists of RPMI 1640 with GlutaMAX supplemented with 10% human AB serum, 25 mM Hepes, and 10 mg / mL gentamicin. In some embodiments, CM is CM1 as described in the examples; see Example A. In some embodiments, the first growth by priming occurs in an initial cell culture medium or a first cell culture medium. In some embodiments, the first growth medium by priming or initial cell culture medium or first cell culture medium contains IL-2, OKT-3, and antigen-presenting feeder cells (also referred to herein as feeder cells).

[0514] In some embodiments, the culture medium used in the growth process disclosed herein is serum-free or synthetic. In some embodiments, the serum-free or synthetic medium comprises a basal cell medium and serum supplements and / or serum substitutes. In some embodiments, the serum-free or synthetic medium is used to prevent and / or reduce experimental variability that is partly due to lot-to-lot variability in serum-containing media.

[0515] In some embodiments, serum-free or synthetic media include basal cell media and serum supplements and / or serum substitutes. In some embodiments, basal cell media include, but are not limited to, CTS® OpTmizer® T cell proliferation basal medium, CTS® OpTmizer® T cell proliferation SFM, CTS® AIM-V medium, CTS® AIM-V SFM, LymphoONE® T cell proliferation xeno-free medium, Dulbecco's Modified Eagle Medium (DMEM), Minimum Essential Medium (MEM), Eagle Basal Medium (BME), RPMI 1640, F-10, F-12, Minimum Essential Medium (αMEM), Glasgow Minimum Essential Medium (G-MEM), RPMI Growth Medium, and Iskov Modified Dulbecco's Medium.

[0516] In some embodiments, the serum supplement or serum substitute includes, but is not limited to, one or more of the following: CTS® OpTmizer T cell proliferation serum supplement, CTS® immune cell serum substitute, one or more albumin or albumin substitutes, one or more amino acids, one or more vitamins, one or more transferrin or transferrin substitutes, one or more antioxidants, one or more insulin or insulin substitutes, one or more collagen precursors, one or more antibiotics, and one or more trace elements. In some embodiments, the synthetic medium includes albumin and one or more of the following: glycine, L-histidine, L-isoleucine, L-methionine, L-phenylalanine, L-proline, L-hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, thiamine, reduced glutathione, L-ascorbic acid-2-phosphate, iron-saturated transferrin, insulin, and trace element partial Ag + , Al 3+ Ba 2+ , Cd 2+ Co 2+ , Cr 3+ , Ge 4+ , Se 4+ , Br, T, Mn 2+ , P, Si 4+ , V 5+ Mo 6+ Ni 2+ , Rb + Sn 2+ , and Zr 4+ It comprises one or more components selected from the group consisting of compounds containing the above. In some embodiments, the synthetic medium further comprises L-glutamine, sodium bicarbonate, and / or 2-mercaptoethanol.

[0517] In some embodiments, CTS® OpTmizer® T-cell immune cell serum substitute is used with conventional growth media, including but not limited to CTS® OpTmizer® T-cell proliferation basal medium, CTS® OpTmizer® T-cell proliferation SFM, CTS® AIM-V medium, CST® AIM-V SFM, LymphoONE® T-cell proliferation xeno-free medium, Dulbecco's modified Eagle medium (DMEM), minimal essential medium (MEM), Eagle basal medium (BME), RPMI 1640, F-10, F-12, minimal essential medium (αMEM), Glasgow minimal essential medium (G-MEM), RPMI growth medium, and Iskov modified Dulbecco's medium.

[0518] In some embodiments, the total serum substitute concentration (vol%) in the serum-free or synthetic medium is about 1 vol%, 2 vol%, 3 vol%, 4 vol%, 5 vol%, 6 vol%, 7 vol%, 8 vol%, 9 vol%, 10 vol%, 11 vol%, 12 vol%, 13 vol%, 14 vol%, 15 vol%, 16 vol%, 17 vol%, 18 vol%, 19 vol%, or 20 vol% of the total volume of the serum-free or synthetic medium. In some embodiments, the total serum substitute concentration is about 3% of the total volume of the serum-free or synthetic medium. In some embodiments, the total serum substitute concentration is about 5% of the total volume of the serum-free or synthetic medium. In some embodiments, the total serum substitute concentration is about 10% of the total volume of the serum-free or synthetic medium.

[0519] In some embodiments, the serum-free or synthetic medium is CTS® OpTmizer® T-cell proliferation SFM (ThermoFisher Scientific). Any formulation of CTS® OpTmizer® is useful in the present invention. CTS® OpTmizer® T-cell proliferation SFM is a combination of 1 L of CTS® OpTmizer® T-cell proliferation basal medium and 26 mL of CTS® OpTmizer® T-cell proliferation supplement, which are mixed together before use. In some embodiments, CTS® OpTmizer® T-cell proliferation SFM is supplemented with approximately 3% CTS® Immune Cell Serum Substitute (SR) (ThermoFisher Scientific). In some embodiments, CTS® OpTmizer® T-cell proliferation SFM is supplemented with approximately 3% CTS® Immune Cell Serum Substitute (SR) (ThermoFisher Scientific) along with 55 mM 2-mercaptoethanol. In some embodiments, CTS® OpTmizer® T-cell proliferation SFM is supplemented with approximately 3% CTS® Immune Cell Serum Substitute (SR) (ThermoFisher Scientific), and the final concentration of 2-mercaptoethanol in the culture medium is 55 μM.

[0520] In some embodiments, the synthetic medium is CTS® OpTmizer® T-cell proliferation SFM (ThermoFisher Scientific). Any formulation of CTS® OpTmizer® is useful in the present invention. CTS® OpTmizer® T-cell proliferation SFM is a combination of 1 L of CTS® OpTmizer® T-cell proliferation basal medium and 26 mL of CTS® OpTmizer® T-cell proliferation supplement, which are mixed together before use. In some embodiments, CTS® OpTmizer® T-cell proliferation SFM is supplemented with approximately 3% CTS® Immune Cell Serum Substitute (SR) (ThermoFisher Scientific) along with 55 mM 2-mercaptoethanol. In some embodiments, CTS® OpTmizer® T-cell proliferation SFM is supplemented with approximately 3% CTS® Immune Cell Serum Substitute (SR) (ThermoFisher Scientific), 55 mM 2-mercaptoethanol, and 2 mM L-glutamine. In some embodiments, CTS® OpTmizer® T-cell proliferation SFM is supplemented with approximately 3% CTS® Immune Cell Serum Substitute (SR) (ThermoFisher Scientific), 55 mM 2-mercaptoethanol, and 2 mM L-glutamine, further containing approximately 1000 IU / mL to approximately 8000 IU / mL of IL-2. In some embodiments, CTS® OpTmizer® T-cell proliferation SFM is supplemented with approximately 3% CTS® Immune Cell Serum Substitute (SR) (ThermoFisher Scientific), 55 mM 2-mercaptoethanol, and 2 mM L-glutamine, further containing approximately 3000 IU / mL of IL-2. In some embodiments, CTS® OpTmizer® T-cell proliferation SFM is supplemented with approximately 3% CTS® Immune Cell Serum Substitute (SR) (ThermoFisher Scientific), 55 mM 2-mercaptoethanol, and 2 mM L-glutamine, further containing approximately 6000 IU / mL of IL-2.In some embodiments, CTS® OpTmizer® T-cell proliferation SFM is supplemented with approximately 3% CTS® Immune Cell Serum Substitute (SR) (ThermoFisher Scientific) and 55 mM 2-mercaptoethanol, further containing approximately 1000 IU / mL to approximately 8000 IU / mL of IL-2. In some embodiments, CTS® OpTmizer® T-cell proliferation SFM is supplemented with approximately 3% CTS® Immune Cell Serum Substitute (SR) (ThermoFisher Scientific) and 55 mM 2-mercaptoethanol, further containing approximately 3000 IU / mL of IL-2. In some embodiments, CTS® OpTmizer® T-cell proliferation SFM is supplemented with approximately 3% CTS® Immune Cell Serum Substitute (SR) (ThermoFisher Scientific) and 55 mM 2-mercaptoethanol, further containing approximately 1000 IU / mL to approximately 6000 IU / mL of IL-2. In some embodiments, CTS® OpTmizer® T-cell proliferation SFM is supplemented with approximately 3% CTS® Immune Cell Serum Substitute (SR) (ThermoFisher Scientific) and approximately 2 mM glutamine, further containing approximately 1000 IU / mL to approximately 8000 IU / mL of IL-2. In some embodiments, the CTS® OpTmizer® T-cell proliferation SFM is supplemented with approximately 3% CTS® Immune Cell Serum Substitute (SR) (ThermoFisher Scientific) and approximately 2 mM glutamine, further containing approximately 3000 IU / mL of IL-2. In some embodiments, the CTS® OpTmizer® T-cell proliferation SFM is supplemented with approximately 3% CTS® Immune Cell Serum Substitute (SR) (ThermoFisher Scientific) and approximately 2 mM glutamine, further containing approximately 6000 IU / mL of IL-2. In some embodiments, the CTS® OpTmizer® T-cell proliferation SFM is supplemented with approximately 3% CTS® Immune Cell Serum Substitute (SR) (ThermoFisher Scientific), and the final concentration of 2-mercaptoethanol in the culture medium is 55 μM.

[0521] In some embodiments, serum-free or synthetic media are supplemented with glutamine (i.e., GlutaMAX®) at concentrations of approximately 0.1 mM to approximately 10 mM, 0.5 mM to approximately 9 mM, 1 mM to approximately 8 mM, 2 mM to approximately 7 mM, 3 mM to approximately 6 mM, or 4 mM to approximately 5 mM. In some embodiments, serum-free or synthetic media are supplemented with glutamine (i.e., GlutaMAX®) at a concentration of approximately 2 mM.

[0522] In some embodiments, serum-free or synthetic media are supplemented with 2-mercaptoethanol at concentrations of approximately 5 mM to 150 mM, 10 mM to 140 mM, 15 mM to 130 mM, 20 mM to 120 mM, 25 mM to 110 mM, 30 mM to 100 mM, 35 mM to 95 mM, 40 mM to 90 mM, 45 mM to 85 mM, 50 mM to 80 mM, 55 mM to 75 mM, 60 mM to 70 mM, or approximately 65 mM. In some embodiments, serum-free or synthetic media are supplemented with 2-mercaptoethanol at a concentration of approximately 55 mM. In some embodiments, the final concentration of 2-mercaptoethanol in the medium is 55 μM.

[0523] In some embodiments, the synthetic media described in International PCT Publication WO / 1998 / 030679, incorporated herein by reference, are useful in the present invention. The publication describes serum-free eukaryotic cell culture media. The serum-free eukaryotic cell culture media comprises a basal cell culture medium supplemented with serum-free supplements that can support cell growth in serum-free culture. The serum-free eukaryotic cell culture medium supplement is obtained by comprising, or in combination thereof, one or more components selected from the group consisting of one or more albumin or albumin substitutes, one or more amino acids, one or more vitamins, one or more transferrin or transferrin substitutes, one or more antioxidants, one or more insulin or insulin substitutes, one or more collagen precursors, one or more trace elements, and one or more antibiotics. In some embodiments, the synthetic medium further comprises L-glutamine, sodium bicarbonate, and / or β-mercaptoethanol. In some embodiments, the synthetic medium comprises albumin or an albumin substitute and one or more components selected from the group consisting of one or more amino acids, one or more vitamins, one or more transferrins or transferrin substitutes, one or more antioxidants, one or more insulins or insulin substitutes, one or more collagen precursors, and one or more trace elements. In some embodiments, the synthetic medium comprises albumin and glycine, L-histidine, L-isoleucine, L-methionine, L-phenylalanine, L-proline, L-hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, thiamine, reduced glutathione, L-ascorbic acid-2-phosphate, iron-saturated transferrin, insulin, and trace element partial Ag + , Al 3+ Ba 2+ , Cd 2+ Co 2+ , Cr 3+ , Ge 4+ , Se 4+ , Br, T, Mn 2+ , P, Si 4+ , V 5+ Mo 6+ Ni2+ , Rb + Sn 2+ , and Zr 4+ It comprises one or more components selected from the group consisting of compounds containing [the specified compound]. In some embodiments, the basal cell medium is selected from the group consisting of Dulbecco's modified Eagle medium (DMEM), minimal essential medium (MEM), Eagle basal medium (BME), RPMI 1640, F-10, F-12, minimal essential medium (αMEM), Glasgow minimal essential medium (G-MEM), RPMI growth medium, and Iskov's modified Dulbecco medium.

[0524] In some embodiments, the concentration of glycine in the synthetic medium is in the range of approximately 5 to 200 mg / L, the concentration of L-histidine is approximately 5 to 250 mg / L, the concentration of L-isoleucine is approximately 5 to 300 mg / L, the concentration of L-methionine is approximately 5 to 200 mg / L, the concentration of L-phenylalanine is approximately 5 to 400 mg / L, the concentration of L-proline is approximately 1 to 1000 mg / L, the concentration of L-hydroxyproline is approximately 1 to 45 mg / L, the concentration of L-serine is approximately 1 to 250 mg / L, the concentration of L-threonine is approximately 10 to 500 mg / L, and the concentration of L-tryptophan is approximately 2 to 110 mg / L. The concentrations are approximately 3-175 mg / L for L-tyrosine, 5-500 mg / L for L-valine, 1-20 mg / L for thiamine, 1-20 mg / L for reduced glutathione, 1-20 mg / L for L-ascorbic acid-2-phosphate, 1-200 mg / L for iron-saturated transferrin, 1-50 mg / L for insulin, 1-100 mg / L for sodium selenite, 0.000001-0.0001 mg / L for albumin (e.g., AlbumX® I), and 5,000-50,000 mg / L for albumin.

[0525] In some embodiments, the non-trace element components in the synthetic medium are present in the concentration ranges listed in the column under the heading "Concentration Range in 1x Medium" in Table A below. In other embodiments, the non-trace element components in the synthetic medium are present in the final concentrations listed in the column under the heading "Preferred Embodiment of 1x Medium" in Table A below. In other embodiments, the synthetic medium is a basal cell medium containing a serum-free supplement. In some of these embodiments, the serum-free supplement contains non-trace elements in the types and concentrations listed in the column under the heading "Preferred Embodiment of Supplement" in Table A below. [Table 4]

[0526] In some embodiments, the osmotic pressure of the synthetic medium is approximately 260–350 mOsmol. In some embodiments, the osmotic pressure is approximately 280–310 mOsmol. In some embodiments, the synthetic medium is supplemented with sodium bicarbonate at a maximum concentration of approximately 3.7 g / L, or approximately 2.2 g / L. The synthetic medium may be further supplemented with L-glutamine (final concentration approximately 2 mM), one or more antibiotics, non-essential amino acids (NEAAs, final concentration approximately 100 μM), and 2-mercaptoethanol (final concentration approximately 100 μM).

[0527] In some embodiments, the synthetic media described in Smith, et al., “Ex vivo expansion of human T cells for adoptive immunotherapy using the novel Xeno-free CTS Immune Cell Serum Replacement,” Clin Transl Immunology, 4(1)2015 (doi:10.1038 / cti.2014.31) are useful in the present invention. Briefly, RPMI or CTS® OpTmizer was used as the basal cell medium and supplemented with 0%, 2%, 5%, or 10% of CTS® Immune Cell Serum Replacement.

[0528] In the embodiment, the cell medium in the first and / or second gas-permeable vessel is unfiltered. The use of unfiltered cell medium may simplify the procedure required to grow the number of cells. In the embodiment, the cell medium in the first and / or second gas-permeable vessel is devoid of β-mercaptoethanol (also known as BME or βME, 2-mercaptoethanol, CAS60-24-2).

[0529] In some embodiments, the first priming growth process (including, for example, the process described in step B of Figure 1 (especially, e.g., Figure 1B and / or Figure 1C), which may also be referred to as pre-REP or priming-based REP) is 1 to 8 days, as considered in the examples and drawings. In some embodiments, the first priming growth process (including, for example, the process described in step B of Figure 1 (especially, e.g., Figure 1B and / or Figure 1C), which may also be referred to as pre-REP or priming-based REP) is 2 to 8 days. In some embodiments, the first priming growth process (including, for example, the process described in step B of Figure 1 (especially, e.g., Figure 1B and / or Figure 1C), which may also be referred to as pre-REP or priming-based REP) is 3 to 8 days. In some embodiments, the first priming growth process (including, for example, the process described in step B of Figure 1 (especially, e.g., Figure 1B and / or Figure 1C), which may also be referred to as pre-REP or priming-based REP) is 4 to 8 days, as considered in the examples and drawings. In some embodiments, the first priming growth process (including, for example, the process described in step B of Figure 1 (especially, e.g., Figure 1B and / or Figure 1C), which may also be referred to as pre-REP or priming-based REP) is 1 to 7 days, as considered in the examples and drawings. In some embodiments, the first priming growth process (including, for example, the process described in step B of Figure 1 (especially, e.g., Figure 1B and / or Figure 1C), which may also be referred to as pre-REP or priming-based REP) is 2 to 8 days.In some embodiments, the first priming propagation process (including, for example, the process described in step B of Figure 1 (especially, e.g., Figure 1B and / or Figure 1C), which may also be referred to as pre-REP or priming-based REP) is 2 to 7 days long. In some embodiments, the first priming propagation process (including, for example, the process described in step B of Figure 1 (especially, e.g., Figure 1B and / or Figure 1C), which may also be referred to as pre-REP or priming-based REP) is 3 to 8 days long. In some embodiments, the first priming propagation process (including, for example, the process described in step B of Figure 1 (especially, e.g., Figure 1B and / or Figure 1C), which may also be referred to as pre-REP or priming-based REP) is 3 to 7 days long. In some embodiments, the first priming-based proliferation process (including, for example, the process described in step B of Figure 1 (especially, e.g., Figure 1B and / or Figure 1C), which may also be referred to as pre-REP or priming-based REP) is 4 to 8 days long. In some embodiments, the first priming-based proliferation process (including, for example, the process described in step B of Figure 1 (especially, e.g., Figure 1B and / or Figure 1C), which may also be referred to as pre-REP or priming-based REP) is 4 to 7 days long. In some embodiments, the first priming-based proliferation process (including, for example, the process described in step B of Figure 1 (especially, e.g., Figure 1B and / or Figure 1C), which may also be referred to as pre-REP or priming-based REP) is 5 to 8 days long. In some embodiments, the first proliferation by priming (which may include a process such as that described in step B of Figure 1 (especially, e.g., Figure 1B and / or Figure 1C), and which may also be referred to as pre-REP or REP by priming) lasts for 5 to 7 days.In some embodiments, the first priming-based proliferation process (including, for example, the process described in step B of Figure 1 (especially, e.g., Figure 1B and / or Figure 1C), which may also be referred to as pre-REP or priming-based REP) is 6 to 8 days long. In some embodiments, the first priming-based proliferation process (including, for example, the process described in step B of Figure 1 (especially, e.g., Figure 1B and / or Figure 1C), which may also be referred to as pre-REP or priming-based REP) is 6 to 7 days long. In some embodiments, the first priming-based proliferation process (including, for example, the process provided in step B of Figure 1 (especially, e.g., Figure 1B and / or Figure 1C), which may also be referred to as pre-REP or priming-based REP) is 7 to 8 days long. In some embodiments, the first priming-based proliferation process (including, for example, the process provided in step B of Figure 1 (especially, e.g., Figure 1B and / or Figure 1C), which may also be referred to as pre-REP or priming-based REP) is 8 days long. In some embodiments, the first priming-based proliferation process (including, for example, the process provided in step B of Figure 1 (especially, e.g., Figure 1B and / or Figure 1C), which may also be referred to as pre-REP or priming-based REP) is 7 days long.

[0530] In some embodiments, the first TIL growth by priming can proceed for 1 to 8 days from the time fragmentation occurs and / or the first growth step by priming is initiated. In some embodiments, the first TIL growth by priming can proceed for 1 to 7 days from the time fragmentation occurs and / or the first growth step by priming is initiated. In some embodiments, the first TIL growth by priming can proceed for 2 to 8 days from the time fragmentation occurs and / or the first growth step by priming is initiated. In some embodiments, the first TIL growth by priming can proceed for 2 to 7 days from the time fragmentation occurs and / or the first growth step by priming is initiated. In some embodiments, the first TIL growth by priming can proceed for 3 to 8 days from the time fragmentation occurs and / or the first growth step by priming is initiated. In some embodiments, the first TIL growth by priming can proceed for 3 to 7 days from the time fragmentation occurs and / or the first growth step by priming is initiated. In some embodiments, the first TIL growth by priming can proceed for 4 to 8 days from the time fragmentation occurs and / or the first growth step by priming is initiated. In some embodiments, the first TIL growth by priming can proceed for 4 to 7 days from the time fragmentation occurs and / or the first growth step by priming is initiated. In some embodiments, the first TIL growth by priming can proceed for 5 to 8 days from the time fragmentation occurs and / or the first growth step by priming is initiated. In some embodiments, the first TIL growth by priming can proceed for 5 to 7 days from the time fragmentation occurs and / or the first growth step by priming is initiated.In some embodiments, the first TIL growth by priming can proceed for 6 to 8 days from the time fragmentation occurs and / or the first growth step by priming is initiated. In some embodiments, the first TIL growth by priming can proceed for 6 to 7 days from the time fragmentation occurs and / or the first growth step by priming is initiated. In some embodiments, the first TIL growth by priming can proceed for 7 to 8 days from the time fragmentation occurs and / or the first growth step by priming is initiated. In some embodiments, the first TIL growth by priming can proceed for 8 days from the time fragmentation occurs and / or the first growth step by priming is initiated. In some embodiments, the first TIL growth by priming can proceed for 7 days from the time fragmentation occurs and / or the first growth step by priming is initiated.

[0531] In some embodiments, the first growth of TILs by priming can proceed for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 days. In some embodiments, the first TIL growth can proceed for 1 to 8 days. In some embodiments, the first TIL growth can proceed for 1 to 7 days. In some embodiments, the first TIL growth can proceed for 2 to 7 days. In some embodiments, the first TIL growth can proceed for 3 to 7 days. In some embodiments, the first TIL growth can proceed for 4 to 7 days. In some embodiments, the first TIL growth can proceed for 5 to 7 days. In some embodiments, the first TIL growth can proceed for 6 to 7 days. In some embodiments, the first TIL growth can proceed for 2 to 8 days. In some embodiments, the first TIL growth can proceed for 3 to 8 days. In some embodiments, the first TIL growth can proceed for 4 to 8 days. In some embodiments, the first TIL growth can proceed for 5 to 8 days. In some embodiments, the first TIL growth can proceed for 6 to 8 days. In some embodiments, the first TIL growth can proceed for 2 to 9 days. In some embodiments, the first TIL growth can proceed for 3 to 9 days. In some embodiments, the first TIL growth can proceed for 4 to 9 days. In some embodiments, the first TIL growth can proceed for 5 to 9 days. In some embodiments, the first TIL growth can proceed for 6 to 9 days. In some embodiments, the first TIL growth can proceed for 2 to 10 days. In some embodiments, the first TIL growth can proceed for 3 to 10 days. In some embodiments, the first TIL growth can proceed for 4 to 10 days. In some embodiments, the first TIL growth can proceed for 5 to 10 days. In some embodiments, the first TIL growth can proceed for 6 to 10 days. In some embodiments, the first TIL proliferation can proceed for 2 to 11 days.In some embodiments, the first TIL growth can proceed for 3 to 11 days. In some embodiments, the first TIL growth can proceed for 4 to 11 days. In some embodiments, the first TIL growth can proceed for 5 to 11 days. In some embodiments, the first TIL growth can proceed for 6 to 11 days. In some embodiments, the first TIL growth can proceed for 7 days. In some embodiments, the first TIL growth can proceed for 8 days. In some embodiments, the first TIL growth can proceed for 9 days. In some embodiments, the first TIL growth can proceed for 10 days. In some embodiments, the first TIL growth can proceed for 11 days.

[0532] In some embodiments, combinations of IL-2, IL-7, IL-15, and / or IL-21 are used as a combination during the first propagation by priming. In some embodiments, IL-2, IL-7, IL-15, and / or IL-21, and any combination thereof, may be included during the first propagation by priming, including, for example, the process shown in Figure 1 (especially, for example, Figure 1B) and the process of step B as described herein. In some embodiments, combinations of IL-2, IL-15, and IL-21 are used as a combination during the first propagation by priming. In some embodiments, IL-2, IL-15, and IL-21, and any combination thereof, may be included during the process shown in Figure 1 (especially, for example, Figure 1B and / or Figure 1C) and the process of step B as described herein.

[0533] In some embodiments, the first growth by priming, for example, step B according to Figure 1 (in particular, for example, Figure 1B and / or Figure 1C), is carried out in a closed-system bioreactor. In some embodiments, a closed system is used for TIL growth as described herein. In some embodiments, a bioreactor is used. In some embodiments, the bioreactor is used as a container. In some embodiments, the bioreactor used is, for example, G-REX-10 or G-REX-100. In some embodiments, the bioreactor used is G-REX-100. In some embodiments, the bioreactor used is G-REX-10.

[0534] 1. Feeder cells and antigen-presenting cells In embodiments, the first proliferation procedure by priming described herein (including, for example, the one described in step B from Figure 1 (especially, for example, Figure 1B and / or Figure 1C), and proliferation referred to as pre-REP or priming-based REP) does not require feeder cells (also referred to herein as "antigen-presenting cells") at the start of TIL proliferation, but rather is added during the first proliferation by priming (priming-based REP). In embodiments, the first proliferation procedure by priming described herein (including, for example, the one described in step B from Figure 1 (especially, for example, Figure 1B and / or Figure 1C), and proliferation referred to as pre-REP or priming-based REP) does not require feeder cells (also referred to herein as "antigen-presenting cells") at the start of TIL proliferation, but rather is added during the first proliferation by priming at any point between days 4 and 8. In embodiments, the first proliferation procedure by priming described herein (including, for example, the one described in step B from Figure 1 (especially, for example, Figure 1B and / or Figure 1C), and proliferation referred to as pre-REP or priming-based REP) does not require feeder cells (also referred to herein as "antigen-presenting cells") at the start of TIL proliferation, but rather is added during the first proliferation by priming at any point between days 4 and 7.In embodiments, the first proliferation procedure by priming described herein (including, for example, the one described in step B from Figure 1 (especially, for example, Figure 1B and / or Figure 1C), and proliferation referred to as pre-REP or priming-based REP) does not require feeder cells (also referred to herein as "antigen-presenting cells") at the start of TIL proliferation, but rather is added during the first proliferation by priming at any point between days 5 and 7. In embodiments, the first proliferation procedure by priming described herein (including, for example, the one described in step B from Figure 1 (in particular, e.g., Figure 1B and / or Figure 1C), and proliferation referred to as pre-REP or priming-based REP) does not require feeder cells (also referred to herein as "antigen-presenting cells") at the start of TIL proliferation, but rather is added during the first proliferation by priming at any point on day 6 or 7.In embodiments, the first proliferation procedure by priming described herein (including, for example, the one described in step B from Figure 1 (especially, for example, Figure 1B and / or Figure 1C), and proliferation referred to as pre-REP or priming-based REP) does not require feeder cells (also referred to herein as "antigen-presenting cells") at the start of TIL proliferation, but rather is added during the first proliferation by priming at any point on day 7. In embodiments, the first proliferation procedure by priming described herein (including, for example, the one described in step B from Figure 1 (especially, for example, Figure 1B and / or Figure 1C), and proliferation referred to as pre-REP or priming-based REP) does not require feeder cells (also referred to herein as "antigen-presenting cells") at the start of TIL proliferation, but rather is added during the first proliferation by priming at any point on day 8.

[0535] In embodiments, the first proliferation procedure by priming described herein (including, for example, the one described in step B from Figure 1 (in particular, for example, Figure 1B and / or Figure 1C), and proliferation referred to as pre-REP or REP by priming) requires feeder cells (also referred to herein as “antigen-presenting cells”) at the initiation of TIL proliferation and during the first proliferation by priming. In many embodiments, the feeder cells are peripheral blood mononuclear cells (PBMCs) obtained from a standard whole blood unit from a healthy allogeneic donor. PBMCs are obtained using standard methods such as Ficoll-Paque gradient separation. In some embodiments, 2.5 × 10⁻⁶ cells are used. 8 A number of feeder cells are used during the first proliferation by priming. In some embodiments, 2.5 × 10⁶ cells are used per container. 8 A number of feeder cells are used during the first proliferation by priming. In some embodiments, 2.5 × 10⁶ cells are used per GREX-10. 8 A number of feeder cells are used during the first proliferation by priming. In some embodiments, 2.5 × 10⁶ cells are used per GREX-100. 8A single feeder cell is used during the first proliferation phase priming.

[0536] Generally, homogeneous PBMCs are inactivated by either irradiation or heat treatment and used in the REP procedure, as described in the examples, which provides an exemplary protocol for evaluating the lack of replication ability of irradiated homogeneous PBMCs.

[0537] In some embodiments, if the total number of viable cells on day 14 is less than the initial number of viable cells cultured on day 0 of the first proliferation by priming, the PBMCs are considered to lack replication ability and are acceptable for use in the TIL proliferation procedure described herein.

[0538] In some embodiments, if the total number of viable cells cultured in the presence of OKT3 and IL-2 on day 7 has not increased from the initial number of viable cells cultured on day 0 of the first proliferation by priming, the PBMCs are considered to lack replication ability and are acceptable for use in the TIL proliferation procedure described herein. In some embodiments, the PBMCs are cultured in the presence of 30 ng / mL of OKT3 antibody and 3000 IU / mL of IL-2. In some embodiments, the PBMCs are cultured in the presence of 30 ng / mL of OKT3 antibody and 6000 IU / mL of IL-2.

[0539] In some embodiments, if the total number of viable cells cultured in the presence of OKT3 and IL-2 on day 7 has not increased from the initial number of viable cells cultured on day 0 of the first proliferation by priming, the PBMCs are considered to lack replication ability and are acceptable for use in the TIL proliferation procedure described herein. In some embodiments, the PBMCs are cultured in the presence of 5-60 ng / mL of OKT3 antibody and 1000-6000 IU / mL of IL-2. In some embodiments, the PBMCs are cultured in the presence of 10-50 ng / mL of OKT3 antibody and 2000-5000 IU / mL of IL-2. In some embodiments, the PBMCs are cultured in the presence of 20-40 ng / mL of OKT3 antibody and 2000-4000 IU / mL of IL-2. In some embodiments, the PBMCs are cultured in the presence of 25-35 ng / mL of OKT3 antibody and 2500-3500 IU / mL of IL-2. In some embodiments, PBMCs are cultured in the presence of 30 ng / mL of OKT3 antibody and 6000 IU / mL of IL-2. In some embodiments, PBMCs are cultured in the presence of 15 ng / mL of OKT3 antibody and 3000 IU / mL of IL-2. In some embodiments, PBMCs are cultured in the presence of 15 ng / mL of OKT3 antibody and 6000 IU / mL of IL-2.

[0540] In some embodiments, the antigen-presenting feeder cells are PBMCs. In some embodiments, the antigen-presenting feeder cells are artificial antigen-presenting feeder cells. In embodiments, the ratio of TILs to antigen-presenting feeder cells in the second proliferation is approximately 1:25, 1:50, 1:100, 1:125, 1:150, 1:175, 1:200, 1:225, 1:250, 1:275, 1:300, 1:325, 1:350, 1:375, 1:400, or 1:500. In embodiments, the ratio of TILs to antigen-presenting feeder cells in the second proliferation is 1:50 to 1:300. In embodiments, the ratio of TILs to antigen-presenting feeder cells in the second proliferation is 1:100 to 1:200.

[0541] In embodiments, the first propagation procedure by priming described herein is approximately 2.5 × 10 8 Feeder cells: approximately 100 x 10 6 A ratio of TIL is required. In another embodiment, the first propagation procedure by priming described herein is approximately 2.5 × 10 8 Feeder cell pairs: approximately 50 x 10 6 A ratio of TIL is required. In yet another embodiment, the first growth by priming described herein is about 2.5 × 10 8 Feeder cell pairs: approximately 25 × 10 6 TIL is required. In yet another embodiment, the first growth by priming described herein is about 2.5 × 10 8 Feeder cells are required. In yet another embodiment, the first proliferation by priming requires one-quarter, one-third, five-twelfth, or half the number of feeder cells used in the rapid second proliferation.

[0542] In some embodiments, the culture medium in the first growth by priming contains IL-2. In some embodiments, the culture medium in the first growth by priming contains 6000 IU / mL of IL-2. In some embodiments, the culture medium in the first growth by priming contains antigen-presenting feeder cells. In some embodiments, the culture medium in the first growth by priming contains 2.5 × 10⁶ cells per container. 8 It contains antigen-presenting feeder cells. In some embodiments, the medium in the first proliferation by priming contains OKT-3. In some embodiments, the medium contains 30 ng of OKT-3 per container. In some embodiments, the container is a GREX100 MCS flask. In some embodiments, the medium contains 6000 IU / mL of IL-2, 30 ng / mL of OKT-3, and 2.5 × 10⁶ 8 The medium contains antigen-presenting feeder cells. In some embodiments, the medium contains 6000 IU / mL of IL-2, 30 ng / mL of OKT-3, and 2.5 × 10⁶ cells per container. 8It contains antigen-presenting feeder cells. In some embodiments, the culture medium consists of 500 mL of culture medium and 2.5 × 10⁶ cells per container. 8 Each antigen-presenting feeder cell contains 15 μg of OKT-3. In some embodiments, the medium contains 500 mL of culture medium and 15 μg of OKT-3 per container. In some embodiments, the container is a GREX100 MCS flask. In some embodiments, the medium contains 500 mL of culture medium, as well as 6000 IU / mL of IL-2, 30 ng / mL of OKT-3, and 2.5 × 10 8 The medium contains antigen-presenting feeder cells. In some embodiments, the medium consists of 500 mL of culture medium per container, as well as 6000 IU / mL of IL-2, 15 μg of OKT-3, and 2.5 × 10¹⁶ 8 It contains antigen-presenting feeder cells. In some embodiments, the culture medium consists of 500 mL of culture medium and 2.5 × 10⁶ cells per container. 8 Each antigen-presenting feeder cell contains 15 μg of OKT-3.

[0543] In embodiments, the first proliferation procedure by priming described herein requires an excess of feeder cells exceeding the TIL during the second proliferation. In many embodiments, the feeder cells are peripheral blood mononuclear cells (PBMCs) obtained from a standard whole blood unit from an allogeneic healthy donor. PBMCs are obtained using standard methods such as Ficoll-Paque gradient separation. In embodiments, artificial antigen-presenting (aAPC) cells are used instead of PBMCs.

[0544] Generally, homogeneous PBMCs are inactivated by either irradiation or heat treatment and used in the TIL proliferation procedures described herein, including the exemplary procedures shown in the drawings and examples.

[0545] In the embodiment, artificial antigen-presenting cells are used as a substitute for PBMCs or in combination with PBMCs in the first proliferation by priming.

[0546] 2. Cytokines The growth methods described herein generally use culture media containing high doses of cytokines, particularly IL-2, as is known in the art.

[0547] Alternatively, as generally outlined in International Publications WO2015 / 189356 and WO2015 / 189357 (their entirety expressly incorporated herein by reference), it is also possible to use combinations of cytokines for the first proliferation by priming of TILs, in combinations of two or more of IL-2, IL-15, and IL-21. Thus, possible combinations include IL-2 and IL-15, IL-2 and IL-21, IL-15 and IL-21, and IL-2, IL-15, and IL-21, the latter finding specific applications in many embodiments. The use of cytokine combinations is particularly advantageous for the generation of lymphocytes, and especially T cells as described therein. [Table 5]

[0548] C. Step C: Transition from the first growth phase via priming to the rapid second growth phase. In some cases, a bulk TIL population obtained from a first proliferation by priming (which may include proliferation sometimes referred to as pre-REP), including the TIL population obtained from step B as shown in Figure 1 (especially, for example, Figure 1B and / or Figure 1C), may be subjected to a rapid second proliferation (which may include proliferation sometimes referred to as rapid proliferation protocol (REP)) and then cryopreserved as discussed below. Similarly, if the genetically modified TILs are to be used for therapeutic purposes, the TIL populations grown from the first proliferation by priming, or the TIL populations grown from the rapid second proliferation, may be subjected to genetic modification for suitable therapeutic purposes before the proliferation step, or after the first proliferation by priming and before the rapid second proliferation.

[0549] In some embodiments, TILs obtained from the first growth by priming (e.g., from step B as shown in Figure 1 (especially, e.g., Figure 1B and / or Figure 1C)) are stored until phenotypic determination for selection. In some embodiments, TILs obtained from the first growth by priming (e.g., from step B as shown in Figure 1 (especially, e.g., Figure 1B and / or Figure 1C)) are not stored and proceed directly to rapid second growth. In some embodiments, TILs obtained from the first growth by priming are not cryopreserved after the first growth by priming and before rapid second growth. In some embodiments, the transition from the first growth by priming to the second growth occurs approximately 2, 3, 4, 5, 6, 7, or 8 days after tumor fragmentation occurs and / or the first growth step by priming is initiated. In some embodiments, the transition from priming-induced first growth to rapid second growth occurs approximately 3 to 7 days after fragmentation occurs and / or the first growth step by priming is initiated. In some embodiments, the transition from priming-induced first growth to rapid second growth occurs approximately 3 to 8 days after fragmentation occurs and / or the first growth step by priming is initiated. In some embodiments, the transition from priming-induced first growth to second growth occurs approximately 4 to 7 days after fragmentation occurs and / or the first growth step by priming is initiated. In some embodiments, the transition from priming-induced first growth to second growth occurs approximately 4 to 8 days after fragmentation occurs and / or the first growth step by priming is initiated. In some embodiments, the transition from priming-induced first growth to second growth occurs approximately 5 to 7 days after fragmentation occurs and / or the first growth step by priming is initiated. In some embodiments, the transition from the first growth by priming to the second growth occurs approximately 5 to 8 days after fragmentation occurs and / or the first growth step by priming is initiated.In some embodiments, the transition from the first growth by priming to the second growth occurs approximately 6 to 7 days after fragmentation occurs and / or the first growth step by priming is initiated. In some embodiments, the transition from the first growth by priming to the second growth occurs approximately 6 to 8 days after fragmentation occurs and / or the first growth step by priming is initiated. In some embodiments, the transition from the first growth by priming to the second growth occurs approximately 7 to 8 days after fragmentation occurs and / or the first growth step by priming is initiated. In some embodiments, the transition from the first growth by priming to the second growth occurs approximately 7 days after fragmentation occurs and / or the first growth step by priming is initiated. In some embodiments, the transition from the first growth by priming to the second growth occurs approximately 8 days after fragmentation occurs and / or the first growth step by priming is initiated.

[0550] In some embodiments, the transition from priming-induced first growth to rapid second growth occurs 1, 2, 3, 4, 5, 6, 7, or 8 days after fragmentation occurs and / or the first growth step by priming is initiated. In some embodiments, the transition from priming-induced first growth to rapid second growth occurs 1 to 7 days after fragmentation occurs and / or the first growth step by priming is initiated. In some embodiments, the transition from priming-induced first growth to rapid second growth occurs 1 to 8 days after fragmentation occurs and / or the first growth step by priming is initiated. In some embodiments, the transition from priming-induced first growth to second growth occurs 2 to 7 days after fragmentation occurs and / or the first growth step by priming is initiated. In some embodiments, the transition from the first growth by priming to the second growth occurs 2 to 8 days after fragmentation occurs and / or the first growth step by priming is initiated. In some embodiments, the transition from the first growth by priming to the second growth occurs 3 to 7 days after fragmentation occurs and / or the first growth step by priming is initiated. In some embodiments, the transition from the first growth by priming to the second growth occurs 3 to 8 days after fragmentation occurs and / or the first growth step by priming is initiated. In some embodiments, the rapid transition from the first growth by priming to the second growth occurs 4 to 7 days after fragmentation occurs and / or the first growth step by priming is initiated. In some embodiments, the rapid transition from the first growth by priming to the second growth occurs 4 to 8 days after fragmentation occurs and / or the first growth step by priming is initiated. In some embodiments, the transition from the first growth by priming to the rapid second growth occurs 5 to 7 days after fragmentation occurs and / or the first growth step by priming is initiated.In some embodiments, the transition from priming-induced first growth to rapid second growth occurs 5 to 8 days after fragmentation occurs and / or the first growth step by priming is initiated. In some embodiments, the transition from priming-induced first growth to rapid second growth occurs 6 to 7 days after fragmentation occurs and / or the first growth step by priming is initiated. In some embodiments, the transition from priming-induced first growth to rapid second growth occurs 6 to 8 days after fragmentation occurs and / or the first growth step by priming is initiated. In some embodiments, the transition from priming-induced first growth to rapid second growth occurs 7 to 8 days after fragmentation occurs and / or the first growth step by priming is initiated. In some embodiments, the transition from priming-induced first growth to rapid second growth occurs 7 days after fragmentation occurs and / or the first growth step by priming is initiated. In some embodiments, the transition from the first growth by priming to the rapid second growth occurs 8 days after fragmentation occurs and / or the first growth step by priming is initiated.

[0551] In some embodiments, TILs are not preserved after the primary first growth and before the rapid second growth, and the TILs proceed directly to the rapid second growth (for example, in some embodiments, they are not preserved during the transition from step B to step D, as shown in Figure 1 (in particular, e.g., Figure 1B and / or Figure 1C)). In some embodiments, the transition occurs in a closed system as described herein. In some embodiments, the TILs from the first growth by priming, the second TIL population, proceed directly to the rapid second growth without a transition period.

[0552] In some embodiments, the transition from priming-based first growth to rapid second growth, for example, step C shown in Figure 1 (in particular, for example, Figure 1B), is carried out in a closed-system bioreactor. In some embodiments, a closed system is used for TIL growth as described herein. In some embodiments, a single bioreactor is used. In some embodiments, the single bioreactor used is, for example, GREX-10 or GREX-100. In some embodiments, the closed-system bioreactor is a single bioreactor. In some embodiments, the transition from priming-based first growth to rapid second growth involves scaling up the container size. In some embodiments, priming-based first growth is carried out in a smaller container than rapid second growth. In some embodiments, priming-based first growth is carried out in a GREX-100, and rapid second growth is carried out in a GREX-500.

[0553] In some embodiments, up to 1 × 10 6 The TIL of the cells is obtained at the end of the first proliferation by priming. In some embodiments, 0.1 × 10 6 , 0.2 × 10 6 , 0.3 × 10 6 , 0.4 × 10 6 , 0.5 × 10 6 , 0.6 × 10 6 , 0.7 × 10 6 , 0.8 × 10 6 , 0.9 × 10 6 , 1.0 × 10 6 , 1.1 × 10 6 , 1.2 × 10 6 , 1.3 × 10 6 , 1.4×10 6 , or 0.5 × 10 6A TIL is obtained at the end of the first proliferation by priming. In some embodiments, the TIL at the end of the first proliferation by priming is about 9% to about 40% PD-1+. In some embodiments, the TIL at the end of the first proliferation by priming is about 10% to about 40% PD-1+. In some embodiments, the TIL at the end of the first proliferation by priming is about 15% to about 30% PD-1+. In some embodiments, the TIL at the end of the first proliferation by priming is about 20% to about 40% PD-1+. In some embodiments, the TIL at the end of the first proliferation by priming is about 20% to about 30% PD-1+. In some embodiments, the TIL at the end of the first proliferation by priming is about 10% to about 20% PD-1+. In some embodiments, the TIL at the end of the first proliferation by priming is about 9%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, or about 40% PD-1+. In some embodiments, the TIL at the end of the first proliferation by priming is about 9% to about 40% PD-1 high. In some embodiments, the TIL at the end of the first proliferation by priming is about 15% to about 30% PD-1 high. In some embodiments, the TIL at the end of the first proliferation by priming is about 20% to about 40% PD-1 high. In some embodiments, the TIL at the end of the first proliferation by priming is about 20% to about 30% PD-1 high. In some embodiments, the TIL at the end of the first proliferation by priming is about 10% to about 20% PD-1 high. In some embodiments, the TIL at the end of the first proliferation by priming is PD-1 high of about 9%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, or about 40%.

[0554] D. Step D: Rapid Second Growth In some embodiments, the TIL cell population is further multiplied after a first growth by harvesting and priming, i.e., after steps A and B, and after a transition referred to as step C, as shown in Figure 1 (especially, e.g., Figure 1B and / or Figure 1C). This further growth is referred herein to as rapid second growth, which may include a growth process commonly referred in the art to a rapid growth process (rapid growth protocol or REP), and the process shown in step D of Figure 1 (especially, e.g., Figure 1B and / or Figure 1C). Rapid second growth is generally achieved in a gas-permeable vessel using a culture medium containing several components, including feeder cells, a cytokine source, and an anti-CD3 antibody. In some embodiments, on day 1, 2, 3, or 4 after the start of rapid second growth (i.e., day 8, 9, 10, or 11 of the entire Gen3 process), the TILs are transferred to a large-capacity container.

[0555] In some embodiments, up to 1 × 10 6 TIL is added to the cells at the start of rapid second proliferation. In some embodiments, 0.1 × 10 6 , 0.2 × 10 6 , 0.3 × 10 6 , 0.4 × 10 6 , 0.5 × 10 6 , 0.6 × 10 6 , 0.7 × 10 6 , 0.8 × 10 6 , 0.9 × 10 6 , 1.0 × 10 6 , 1.1 × 10 6 , 1.2 × 10 6 , 1.3 × 10 6 , 1.4×10 6 , or 0.5 × 10 6 TIL is added at the start of rapid second proliferation. In some embodiments, the maximum cell density from the first proliferation by priming is 1e6 cells, providing 1e9 cells to initiate rapid second proliferation.

[0556] In some embodiments, rapid second growth of TIL (which may also be referred to as REP growth, and may include processes such as those shown in step D of Figure 1 (in particular, e.g., Figure 1B and / or Figure 1C)) may be carried out using any TIL flask or container known to those skilled in the art. In some embodiments, second TIL growth may proceed for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days after the start of rapid second growth. In some embodiments, second TIL growth may proceed for about 1 to about 9 days after the start of rapid second growth. In some embodiments, second TIL growth may proceed for about 1 to about 10 days after the start of rapid second growth. In some embodiments, second TIL growth may proceed for about 2 to about 9 days after the start of rapid second growth. In some embodiments, second TIL growth may proceed for about 2 to about 10 days after the start of rapid second growth. In some embodiments, the second TIL growth can proceed for about 3 to 9 days after the onset of rapid second growth. In some embodiments, the second TIL growth can proceed for about 3 to 10 days after the onset of rapid second growth. In some embodiments, the second TIL growth can proceed for about 4 to 9 days after the onset of rapid second growth. In some embodiments, the second TIL growth can proceed for about 4 to 10 days after the onset of rapid second growth. In some embodiments, the second TIL growth can proceed for about 5 to 9 days after the onset of rapid second growth. In some embodiments, the second TIL growth can proceed for about 5 to 10 days after the onset of rapid second growth. In some embodiments, the second TIL growth can proceed for about 6 to 9 days after the onset of rapid second growth. In some embodiments, the second TIL growth can proceed for about 6 to 10 days after the onset of rapid second growth. In some embodiments, the second TIL growth can proceed for about 7 to 9 days after the onset of rapid second growth. In some embodiments, the second TIL growth can proceed for about 7 to 10 days after the onset of rapid second growth.In some embodiments, the second TIL growth can proceed for about 8 to 9 days after the onset of rapid second growth. In some embodiments, the second TIL growth can proceed for about 8 to 10 days after the onset of rapid second growth. In some embodiments, the second TIL growth can proceed for about 9 to 10 days after the onset of rapid second growth. In some embodiments, the second TIL growth can proceed for about 1 day after the onset of rapid second growth. In some embodiments, the second TIL growth can proceed for about 2 days after the onset of rapid second growth. In some embodiments, the second TIL growth can proceed for about 3 days after the onset of rapid second growth. In some embodiments, the second TIL growth can proceed for about 4 days after the onset of rapid second growth. In some embodiments, the second TIL growth can proceed for about 5 days after the onset of rapid second growth. In some embodiments, the second TIL growth can proceed for about 6 days after the onset of rapid second growth. In some embodiments, second TIL growth can proceed for about 7 days after the onset of rapid second growth. In some embodiments, second TIL growth can proceed for about 8 days after the onset of rapid second growth. In some embodiments, second TIL growth can proceed for about 9 days after the onset of rapid second growth. In some embodiments, second TIL growth can proceed for about 10 days after the onset of rapid second growth.

[0557] In some embodiments, rapid second proliferation may be carried out in a gas-permeable vessel using the methods of the present disclosure (e.g., proliferation referred to as REP, and processes such as those shown in step D of Figure 1 (e.g., Figure 1B and / or Figure 1C)). In some embodiments, TILs are grown in rapid second proliferation in the presence of IL-2, OKT-3, and feeder cells (also referred herein as “antigen-presenting cells”). In some embodiments, TILs are grown in rapid second proliferation in the presence of IL-2, OKT-3, and feeder cells, and the feeder cells are added to a final concentration that is 2, 2.4, 2.5, 3, 3.5, or 4 times the concentration of feeder cells present in the first proliferation by priming. For example, TILs can be rapidly grown using nonspecific T cell receptor stimulation in the presence of interleukin-2 (IL-2) or interleukin-15 (IL-15). Nonspecific T cell receptor stimulation may include, for example, anti-CD3 antibodies, such as OKT3 at approximately 30 ng / mL, mouse monoclonal anti-CD3 antibodies (commercially available from Ortho-McNeil, Raritan, NJ or Miltenyi Biotech, Auburn, CA) or UHCT-1 (commercially available from BioLegend, San Diego, CA, USA). TILs may be grown to induce further stimulation of the TIL in vitro by including one or more antigens (including their antigenic moieties), such as cancer epitopes, during a second growth cycle, which can be optionally expressed from a vector such as human leukocyte antigen A2 (HLA-A2) binding peptide, for example, 0.3 μM MART-1:26-35 (27L) or gpl00:209-217 (210M), optionally in the presence of T cell growth factors such as IL-2 or IL-15, optionally at 300 IU / mL. Other suitable antigens may include, for example, NY-ESO-1, TRP-1, TRP-2, tyrosinase cancer antigen, MAGE-A3, SSX-2, and VEGFR2, or their antigenic moieties. TILs can also grow rapidly by restoring antigen-presenting cells expressing HLA-A2 with the same antigen(s) of the cancer pulsed.Alternatively, TILs may be further restimulated, for example, with irradiated autologous lymphocytes, or with irradiated HLA-A2+ allogeneic lymphocytes and IL-2. In some embodiments, restimulation occurs as part of a second proliferation. In some embodiments, the second proliferation occurs in the presence of irradiated autologous lymphocytes, or using irradiated HLA-A2+ allogeneic lymphocytes and IL-2.

[0558] In some embodiments, the cell culture medium further comprises IL-2. In some embodiments, the cell culture medium comprises about 3000 IU / mL of IL-2. In some embodiments, the cell culture medium comprises about 1000 IU / mL, about 1500 IU / mL, about 2000 IU / mL, about 2500 IU / mL, about 3000 IU / mL, about 3500 IU / mL, about 4000 IU / mL, about 4500 IU / mL, about 5000 IU / mL, about 5500 IU / mL, about 6000 IU / mL, about 6500 IU / mL, about 7000 IU / mL, about 7500 IU / mL, or about 8000 IU / mL of IL-2. In the embodiment, the cell culture medium contains IL-2 in concentrations of 1000-2000 IU / mL, 2000-3000 IU / mL, 3000-4000 IU / mL, 4000-5000 IU / mL, 5000-6000 IU / mL, 60...

Claims

1. A method for proliferating 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) Select PD-1-positive TILs from the first TIL population in step (a) to obtain a TIL population rich in PD-1, (c) Producing a second TIL population by performing a first proliferation by priming, wherein the PD-1-rich TIL population is cultured in a cell culture medium containing IL-2, OKT-3, and antigen-presenting cells (APCs), and the first proliferation by priming is performed in a container having a first gas-permeable surface area, and the first proliferation by priming is performed for a first period of 1 to 11 days, thereby obtaining and producing the second TIL population. (d) Producing a third TIL population by rapidly growing 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 1 to 11 days, and the third TIL population is a therapeutic TIL population, and the rapid second growth is carried out in a container having a second gas-permeable surface area. (e) Collecting the therapeutic TIL population obtained from step (d), (f) A method comprising transferring the TIL population collected from step (e) to an injection bag.

2. A method for proliferating 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) Select PD-1-positive TILs from the first TIL population in step (a) to obtain a TIL population rich in PD-1, c) (i) Producing a second TIL population by performing a first proliferation by priming, wherein the PD-1-rich TIL population is cultured in a cell culture medium containing IL-2 and OKT-3, and the first proliferation by priming is performed for a first period of 1 to 11 days, thereby obtaining or producing the second TIL population, or c) (ii) Producing a second TIL population by performing a first proliferation by priming, wherein the PD-1-rich TIL population is cultured in a cell culture medium containing IL-2, OKT-3, and antigen-presenting cells (APCs), and the first proliferation by priming is performed in a container having a first gas-permeable surface area, and the first proliferation by priming is performed for a first period of 1 to 11 days, thereby obtaining 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) A 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 (c)(ii), 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 (c)(ii).

4. A method for proliferating tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, (a) A first TIL population selected to be PD-1 positive, obtained by processing a tumor sample from a subject by tumor digestion and selecting the PD-1 positive TILs, and a second TIL population produced by culturing the first TIL population in a cell culture medium containing IL-2, OKT-3, and antigen-presenting cells (APCs) to perform a first proliferation by priming, wherein the first proliferation by priming is carried out in a container having a first gas-permeable surface area, and the first proliferation by priming is carried out for a first period of 1 to 11 days to obtain and produce the second TIL population. (b) Producing a third TIL population by rapidly growing the second TIL population in contact with a cell culture medium of the second TIL population 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 1 to 11 days, and the third TIL population is a therapeutic TIL population, and the rapid second growth is carried out in a container having a second gas-permeable surface area. (c) A method comprising taking the therapeutic TIL population obtained from step (b).

5. A method for proliferating tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, (a) (i) A first TIL population is cultured in a cell culture medium containing IL-2 and OKT-3 to produce a second TIL population by performing a first proliferation by priming of TILs selected to be PD-1 positive, wherein the first proliferation by priming is performed for a first period of 1 to 11 days to obtain or produce the second TIL population, or (a) (ii) A first TIL population 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 TILs selected to be PD-1 positive, wherein the first proliferation by priming is performed for a first period of 1 to 11 days to obtain and produce the second TIL population. (b) 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. (c) A method comprising taking the therapeutic TIL population obtained from step (b).

6. 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)(ii), or The method according to claim 5, wherein 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)(ii).

7. (i) The selection of PD-1-positive TILs includes (a) exposing the 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; (b) adding an excess of anti-IgG4 antibody conjugated to a fluorophore; and (c) performing flow-based cell sorting based on the fluorophore to obtain a PD-1-rich TIL population, or (ii) 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, 1.5:1 to 10:1, 2:1 to 5:1, 2:1 to 3:1, or (iii) The number of APCs in the first proliferation by priming is 1 × 10 8 Individual APC ~ 3.5 × 10 8 Selected from a range of APCs, the number of APCs in the rapid second growth is 3.5 × 10 8 APCs of 1 x 10 9 Selected from the range of individual APCs, or The number of APCs in the first proliferation by the priming is 1.5 × 10 8 APCs of 3 x 10 8 A selection is made from a range of APCs, and the number of APCs in the rapid second growth is 4 × 10 8 Individual APCs ~ 7.5 × 10 8 Selected from the range of individual APCs, or The number of the APCs in the first proliferation by the priming is selected from the range of 2×10 8 APCs to 2.5×10 8 APCs, and the number of the APCs in the rapid second proliferation is selected from the range of 4.5×10 8 APCs to 5.5×10 8 APCs. The method according to any one of claims 1 to 6.

8. The method, after the step of collecting the therapeutic TIL population, The method according to any one of claims 2 to 7, further comprising the additional step of transferring the collected therapeutic TIL population to an infusion bag.

9. (i) The plurality of tumor fragments are distributed 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, the therapeutic TIL population obtained from the third TIL population is collected from each of the plurality of containers and combined to give rise to the collected TIL population, and / or (ii) The method according to any one of claims 1 to 8, wherein the ratio of the number of TILs in the second TIL group to the number of TILs in the first TIL group is 1.5:1 to 100:

1.

10. (i) 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 / or in the rapid second growth step, the rapid second growth is carried out in a second container having a second gas permeable surface area, and / or (ii) The method according to any one of claims 2 to 9, wherein in the rapid second proliferation step, the APC is layered on the second gas permeable surface with an average thickness of 3 to 5 cell layers, 3.5 to 4.5 cell layers, or 4 cell layers.

11. The method according to any one of claims 2 to 9, wherein, for each container in which the first growth by priming is performed on the first TIL population, the rapid second growth is performed in the same container on the second TIL population produced from the first TIL population, and / or, in the step of the rapid second growth, the APC is layered on the first gas-permeable surface with an average thickness of 3 to 5 cell layers, 3.5 to 4.5 cell layers, or 4 cell layers.

12. The method according to any one of claims 2 to 11, wherein, for each container in which the first proliferation by priming is performed on a first TIL population, in the step of the first proliferation by priming, the first container comprises a first surface area, the cell culture medium comprises antigen-presenting cells (APCs), the APCs are layered on a first gas-permeable surface, 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, 1:1.2 to 1:8, 1:1.3 to 1:7, 1:1.4 to 1:6, 1:1.5 to 1:5, 1:1.6 to 1:4, 1:1.7 to 1:3.5, 1:1.8 to 1:3, or 1:1.9 to 1:2.

5.

13. (i) Two to three days after the rapid second growth step, the cell culture medium is supplemented with additional IL-2 and / or (ii) The method according to any one of claims 1 to 12, further comprising cryopreserving the TIL population collected in the step of collecting the therapeutic TIL population using a cryopreservation process.

14. (i) The antigen-presenting cells are peripheral blood mononuclear cells (PBMCs), 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 The number of cells is 5 × 10¹⁴, and / or, 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 × 10¹⁴. ８ Individual and / or (iii) The step of collecting the therapeutic TIL population is performed using a membrane-based cell processing system, and / or (iv) The cell culture medium is provided in a container selected from the group consisting of G containers and Xuri cell bags, and / or (v) Two to three days after the rapid second growth, the cell culture medium is supplemented with additional IL-2 and / or (vi) The plurality of tumor fragments (a) In the first growth step by priming, each container contains 60 fragments, each fragment measuring 27 mm 3 (b) comprising 50 fragments having a volume of 1 to 1.5 grams in total mass, and / or (b) 1300 to 1500 mm 3 The method according to any one of claims 1 to 13, comprising 30 to 60 fragments having a total volume.

15. The method according to any one of claims 1 to 14, wherein the first growth by priming and the rapid second growth are each carried out individually within a period of 5, 6, 7, 8, 9, 10, or 11 days.

16. (i) The first growth step by priming the therapeutic TIL population by the collection is performed within a period of 14 to 22 days or 15 to 22 days, and / or (ii) The method according to any one of claims 1 to 15, further comprising the step of cryopreserving the collected therapeutic TIL population using a cryopreservation process, wherein the first growth step by priming of the therapeutic TIL population by collection and cryopreservation is performed within 16 days and / or the therapeutic TIL population collected in the step of collecting the therapeutic TIL population contains enough TILs to constitute a therapeutically effective dose.

17. The method according to any one of claims 1 to 16, wherein the antigen-presenting cells are peripheral blood mononuclear cells (PBMCs).

18. (i) The collection of the therapeutic TIL population is performed using a membrane-based cell processing system, or (ii) The plurality of tumor fragments (a) comprising 60 fragments per first gas permeable surface area in step (c), each fragment being 27 mm 3 Having a volume of, or (b) comprising 50 pieces having a total mass of 1 gram to 1.5 grams, or (iii) The method according to any one of claims 1 to 17, wherein the cell culture medium is provided in a container selected from the group consisting of G containers and Xuri cell bags.

19. The method according to any one of claims 1 to 18, wherein the IL-2 concentration is 10,000 IU / mL to 5,000 IU / mL.

20. (i) The first growth by priming and the rapid second growth are each carried out individually within a period of 5, 6, 7, 8, 9, 10, or 11 days, or (ii) The method according to any one of claims 1 to 19, wherein the step of performing the first propagation by priming by transferring the collected TIL population is performed within a period of 14 to 22 days or 15 to 22 days.

21. (i) The collected therapeutic TIL population contains a sufficient amount of TILs to constitute a therapeutically effective dose, and / or (ii) The method according to any one of claims 1 to 20, wherein the vessel in the first proliferation by priming is larger than the vessel in the rapid second proliferation, and / or 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.

22. A population of tumor-infiltrating lymphocytes (TILs) for use in a method for treating a subject having cancer, wherein the method comprises providing proliferating TILs for administration, (a) The step of providing a first TIL population obtained and / or received from tumors excised from the subject by processing a tumor sample obtained from the subject into a plurality of tumor fragments, (b) A step of selecting PD-1-positive TILs from the first TIL population of step (a) to obtain a TIL population rich in PD-1, (c) A step of producing a second TIL population by performing a first proliferation by priming, wherein the PD-1-rich TIL population is cultured in a cell culture medium containing IL-2, OKT-3, and antigen-presenting cells (APCs), the first proliferation by priming being performed in a container having a first gas-permeable surface area, the first proliferation by priming being performed for 1 to 11 days, and the second TIL population being obtained and produced. (d) A step of producing a third TIL population by rapidly growing 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 1 to 11 days to obtain the third TIL population, the third TIL population is a therapeutic TIL population, and the rapid second growth is carried out in a container having a second gas permeable surface area. (e) A step of collecting the therapeutic TIL population obtained from step (d), (f) A step of transferring the TIL sample collected from step (e) to an injection bag, (g) A population of tumor-infiltrating lymphocytes (TILs) for use, comprising the step of providing the TILs from step (f) in a therapeutically effective dose for administration to the subject.

23. (I) The number of TILs sufficient to administer a therapeutically effective dose in step (g) is 1 × 10 ９ ~10 x 10 10 Individual and / or (II) The selection of step (b) includes (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 conjugated to a fluorophore; and (iii) performing flow-based cell sorting based on the fluorophore to obtain a PD-1-rich TIL population, and / or (III) Before administering a therapeutically effective dose of TIL cells in step (g), a non-myeloablative lymphocyte depletion regimen has been administered to the subject, and / or (IV) A tumor-infiltrating lymphocyte (TIL) population for use according to claim 22, further comprising the step of treating the subject with a high-dose IL-2 regimen, which is initiated the day following the administration of the TIL cells to the subject in step (g).

24. (i) The subject has a cancer 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, and / or (i) The container is GREX-10, GREX-100, or GREX-500, and / or (ii) The subject has previously been treated with an anti-PD-1 antibody, The subjects mentioned above have not been previously treated with an anti-PD-1 antibody, and / or (iii) The method according to any one of claims 1 to 21, wherein the PD-1 positive TILs are selected from the first TIL population by the steps of contacting the first TIL population with an anti-PD-1 antibody to form a first complex between the anti-PD-1 antibody and TIL cells in the first TIL population, and then isolating the first complex to obtain the PD-1-rich TIL population.

25. The aforementioned subject previously (i) A first anti-PD1 antibody wherein the PD-1 positive TILs are selected by contacting the first TIL population with the second anti-PD-1 antibody, and 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 on the first TIL population, or (ii) The subject is treated with a first anti-PD1 antibody, wherein the PD-1 positive TILs are selected by bringing the first TIL population into contact with a second anti-PD-1 antibody to form a first complex of the second anti-PD-1 antibody and the first TIL population, and then isolating the first complex to obtain the PD-1-rich TIL population, and / or the second anti-PD-1 antibody is blocked from binding to the first TIL population by the first anti-PD-1 antibody which is insoluble on the first TIL population, and the subject is Previously, the patient was treated with a first anti-PD1 antibody, which is selected by performing a step (b) in which the PD-1 positive TILs are contacted 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 the first TIL population by the first anti-PD-1 antibody which has been insolubilized on the first TIL population, and then the first complex is isolated to obtain the PD-1-rich 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 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, wherein the step of isolating the first complex is performed by isolating the second complex, and / or the subject is previously a first anti-PD-1 antibody, and in step (b), the PD-1 positive TILs undergo the step of contacting the first TIL population with the second anti-PD-1 antibody to form a first complex of the second anti-PD-1 antibody and the first TIL population, and the second anti-PD-1 antibody binds to the PD-1 positive TILs by the first anti-PD-1 antibody insolubilized on the first TIL population. The method according to any one of claims 1 to 21 or 24, wherein the first anti-PD1 antibody is selected by being blocked from and then isolating the first complex to obtain the PD-1-rich TIL population, and the first anti-PD-1 antibody and the second anti-PD-1 antibody include an Fc region, and the method further comprises the steps of contacting the first complex with an anti-Fc antibody bound 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, and contacting the first anti-PD-1 antibody insolubilized on 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 insolubilized on the first TIL population, and isolating the second and third complexes to obtain the PD-1-rich TIL population.

26. (i) Selecting PD-1-positive TILs from the first TIL population to obtain a PD-1-rich TIL population includes selecting a TIL population from the first TIL population in which at least 11.27% to 74.4% are PD-1-positive TILs, and / or (II) The step of selecting the PD-1 positive TIL is, (i) 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, (ii) Adding an excess of anti-IgG4 antibody conjugated to a fluorophore, (iii) a step of obtaining a PD-1-rich TIL population based on the intensity of the fluorophores of the PD-1-positive TILs in the first TIL population compared to the intensity of the PBMC population when performed by fluorescence-activated cell sorting (FACS), and / or (III) The method according to any one of claims 1 to 21 or 24 to 25, wherein the intensity of the fluorophores in both the first population and the PBMC population is used to set up FACS gates to establish low, medium, and high levels of intensity corresponding to PD-1 negative TILs, PD-1 intermediate TILs, and PD-1 positive TILs, respectively.

27. A method for proliferating 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-positive TILs from the first TIL population of step (a) to obtain a PD-1-rich TIL population, wherein at least 10% to 80% of the first TIL population are PD-1-positive TILs. (c) Producing a second TIL population by performing a first proliferation by priming, wherein the PD-1-rich TIL population is cultured in a cell culture medium containing IL-2, OKT-3, and antigen-presenting cells (APCs), and the first proliferation by priming is performed in a container having a first gas-permeable surface area, and the first proliferation by priming is performed for a first period of 1 to 11 days, thereby obtaining and producing the second TIL population. (d) Producing a third TIL population by rapidly growing 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 1 to 11 days, and the third TIL population is a therapeutic TIL population, and the rapid second growth is carried out in a container having a second gas-permeable surface area. (e) Collecting the therapeutic TIL population obtained from step (d), (f) A method comprising transferring the TIL population collected from step (e) to an injection bag.

28. The selection in step (b) is (i) 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, (ii) Adding an excess of anti-IgG4 antibody conjugated to a fluorophore, (iii) The method according to claim 27, comprising the step of obtaining a PD-1-rich TIL population based on the intensity of the fluorophores of the PD-1-positive TILs in the first TIL population compared to the intensity of the PBMC population when performed by fluorescence-activated cell sorting (FACS).

29. (i) Using the intensity of the fluorophores in both the first population and the PBMC population, 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, or (ii) The third TIL group contains at least 1 × 10 units in the container. 8 The container contains at least 1 × 10 TILs, or the third TIL group contains at least 1 × 10 9 The method according to claim 27 or 28, comprising TILs.

30. (i) The subject has a cancer 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, and / or (i) The container is GREX-10, GREX-100, or GREX-500, and / or (ii) The subject has previously been treated with an anti-PD-1 antibody, The subjects mentioned above have not been previously treated with an anti-PD-1 antibody, and / or (iii) A tumor-infiltrating lymphocyte (TIL) population for use according to claim 22 or 23, wherein the PD-1-positive TILs are selected from the first TIL population by the step of 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 PD-1-rich TIL population.

31. The subject is previously (i) A first anti-PD1 antibody wherein the PD-1 positive TILs are selected by contacting the first TIL population with the second anti-PD-1 antibody, and 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 on the first TIL population, or (ii) The subject is treated with a first anti-PD1 antibody, wherein the PD-1 positive TILs are selected by bringing the first TIL population into contact with a second anti-PD-1 antibody to form a first complex of the second anti-PD-1 antibody and the first TIL population, and then isolating the first complex to obtain the PD-1-rich TIL population, and / or the second anti-PD-1 antibody is blocked from binding to the first TIL population by the first anti-PD-1 antibody which is insoluble on the first TIL population, and the subject is Previously, the patient was treated with a first anti-PD1 antibody, which is selected by performing a step (b) in which the PD-1 positive TILs are contacted 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 the first TIL population by the first anti-PD-1 antibody which has been insolubilized on the first TIL population, and then the first complex is isolated to obtain the PD-1-rich 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 further includes the step of contacting the first complex with an anti-Fc antibody that binds 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, wherein the step of isolating the first complex is performed by isolating the second complex, and / or the subject is previously a first anti-PD-1 antibody, and in step (b), the PD-1 positive TILs are contacted with the second anti-PD-1 antibody to form a first complex of the second anti-PD-1 antibody and the first TIL population, and the second anti-PD-1 antibody is blocked from binding to the PD-1 positive TILs by the first anti-PD-1 antibody which has been insolubilized on the first TIL population. A tumor-infiltrating lymphocyte (TIL) population for use according to claim 22 or 23, further comprising the steps of: being treated with a first anti-PD1 antibody, selected by then isolating the first complex to obtain the PD-1-rich TIL population, wherein 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 obtaining the PD-1-rich TIL population, the method further comprises the steps of: contacting the first complex with an anti-Fc antibody bound 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; and contacting the first anti-PD-1 antibody insolubilized on 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 insolubilized on the first TIL population; and isolating the second and third complexes to obtain the PD-1-rich TIL population.

32. (i) Selecting PD-1-positive TILs from the first TIL population to obtain a PD-1-rich TIL population comprises selecting a TIL population from the first TIL population in which at least 11.27% to 74.4% are PD-1-positive TILs, and / or (II) The step of selecting the PD-1 positive TIL is, (i) 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, (ii) Adding an excess of anti-IgG4 antibody conjugated to a fluorophore, (iii) a step of obtaining a PD-1-rich TIL population based on the intensity of the fluorophores of the PD-1-positive TILs in the first TIL population compared to the intensity of the PBMC population when performed by fluorescence-activated cell sorting (FACS), and / or (III) A tumor-infiltrating lymphocyte (TIL) population for use according to claim 22 or 23, wherein the intensity of the fluorophores in both the first population and the PBMC population is used to set up FACS gates to establish low, medium, and high levels of intensity corresponding to PD-1-negative TILs, PD-1 intermediate TILs, and PD-1-positive TILs, respectively.

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