Method for producing tumor-infiltrating lymphocytes and their use in immunotherapy

Abstract

The present invention provides improved and / or shortened methods for expanding TILs and producing therapeutic TIL populations, including novel methods for expanding TIL populations in a closed system that not only reduces microbial contamination but also reduces costs while providing improved efficacy, improved phenotype, and enhanced metabolic health of TILs in a short period of time. Such TILs find use in therapeutic treatment regimens.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 019,917 filed 4 May 2020, U.S. Provisional Patent Application No. 63 / 023,666 filed 12 May 2020, U.S. Provisional Patent Application No. 63 / 146,405 filed 5 February 2021, and U.S. Provisional Patent Application No. 63 / 162,441 filed 17 March 2021, the disclosures thereof incorporated herein by reference in their entirety for all purposes. [Background technology]

[0002] The treatment of large, refractory cancers using adoptive transfer of tumor-infiltrating lymphocytes (TILs) is a powerful treatment option for patients with a poor prognosis. (Gattinoni, et al., Nat. Rev. Immunol. 2006, 6, 383-393). A large number of TILs are required for successful immunotherapy, and commercialization necessitates a robust and reliable process. This has been difficult to achieve due to technical, logistical, and regulatory issues related to cell proliferation. IL-2-based TIL proliferation and subsequent "rapid proliferation processes" (REP) have become the recommended 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 can increase TILs 1,000-fold in 14 days, but often requires a large excess (e.g., 200-fold) of irradiated allogeneic peripheral blood mononuclear cells (PBMCs, also known as mononuclear cells (MNCs)) and anti-CD3 antibody (OKT3) from multiple donors as feeder cells, as well as high doses of IL-2. Dudley, et al., J. Immunother. 2003, 26, 332-42. TILs subjected to the REP procedure produced effective adoptive cell therapy after host immunosuppression in melanoma patients. Current fluid tolerance parameters depend on the compositional readings of the TILs (e.g., CD28, CD8, or CD4 positive) and the proliferation rate and viability of the REP product.

[0003] Current TIL manufacturing processes are limited by length, cost, sterility issues, and other factors described herein. There is an urgent need to provide TIL manufacturing processes and therapies based on such processes that improve cost-effectiveness and scalability in manufacturing and feature more potent anti-cancer phenotypes in TIL preparations produced for the treatment of human patients in multiple clinical centers. The present invention satisfies this need by providing a novel TIL proliferation process that includes antigen-presenting feeder cells from the initiation of proliferation to prime TILs for proliferation, rather than the conventional pre-REP proliferation process, thereby enabling a reduction in the overall time of the proliferation process. [Overview of the project]

[0004] The present invention provides an improved and / or shortened method for growing TILs and producing a therapeutic TIL population.

[0005] This invention provides a method for proliferating tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, and this method (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) Performing a first proliferation priming by culturing a first TIL population in a first TIL cell culture, wherein the first TIL cell culture comprises a first cell culture medium, IL-2, i) A first culture supernatant obtained from a first culture of antigen-presenting feeder cells (APCs), which contains OKT-3, or ii) comprising either APC or OKT-3, The first proliferation priming is performed by culturing the first TIL cell culture in a first container having a first gas-permeable surface area for a first period of approximately 1 to 7 or 8 days in order to obtain a second TIL population, wherein the second TIL population is larger in number than the first TIL population, and priming is performed. (c) To form a second TIL cell culture, to carry out rapid second proliferation by supplementing the first TIL cell culture, the supplementation comprising additional first cell culture medium and IL-2, i) A second culture supernatant obtained from a culture of a second APC, the second culture supernatant containing OKT-3, or ii) By using either APC or OKT-3, Rapid second proliferation is performed by culturing the second TIL cell culture for a second period of approximately 1 to 11 days to obtain a third TIL population, the third TIL population being a therapeutic population for TIL, the first TIL cell culture containing neither the first culture supernatant nor APC, the second TIL cell culture containing neither the second culture supernatant nor supplemental APC, the first cell culture containing no APC and / or the first cell culture containing no supplemental APC, and so on, when rapid second proliferation is performed. (d) Collect the therapeutic TIL population obtained from step (c), (e) Transferring the TIL population collected from step (d) to an infusion bag, etc.

[0006] In some embodiments of this method, in step (b) the first proliferation priming, the first TIL cell culture contains the first culture supernatant, and in step (c) the rapid second proliferation, the first TIL cell culture is supplemented with OKT-3 and APC to form a second TIL cell culture.

[0007] In some embodiments of this method, in step (b) the first proliferation priming, the first TIL cell culture contains OKT-3 and APC, and in step (c) the rapid second proliferation, the first TIL cell culture is supplemented with a second culture supernatant to form a second TIL cell culture.

[0008] In some embodiments of this method, in step (b) the first proliferation priming, the first TIL cell culture contains the first culture supernatant, and in step (c) the rapid second proliferation, the first TIL cell culture is supplemented with the second culture supernatant to form a second TIL cell culture.

[0009] In some embodiments of this method, obtaining a first culture supernatant for use in step (b) is 1) To provide an APC cell culture medium containing IL-2 and OKT-3, 2)1) In the APC cell culture medium, at least about 5 × 10 8 The APCs are cultured for approximately 3-4 days to produce the first culture supernatant, 3) The method includes collecting a first culture supernatant from the cell culture described in 2).

[0010] In some embodiments of this method, obtaining a second culture supernatant for use in step (c) is 1) To provide an APC cell culture medium containing IL-2 and OKT-3, 2)1) In the APC cell culture medium, at least about 1 × 10 7 The individual APCs are cultured for approximately 3-4 days to produce a second culture supernatant, 3) The method includes collecting a second culture supernatant from the cell culture of 2).

[0011] In some embodiments of this method, the rapid second growth in step (c) is performed as follows: i) The step further includes supplementing the second TIL cell culture with additional IL-2 approximately 3 or 4 days after the start of the second period in step (c).

[0012] In some embodiments of this method, APC is exogenous to the subject.

[0013] In some embodiments of this method, APCs are peripheral blood mononuclear cells (PBMCs).

[0014] In some embodiments of this method, the rapid second growth in step (c) is performed as follows: i) After the start of the second period, or after approximately 3 or 4 days, the second TIL cell culture is transferred from the first container to multiple second containers, and a subculture of the second TIL cell culture is formed in each of the multiple second containers. ii) further comprising the step of subculturing the second TIL cell culture in each of a plurality of second vessels for the remainder of the second period.

[0015] In some embodiments of this method, in step i), an equal volume of a second TIL cell culture is transferred to a plurality of second containers.

[0016] In some embodiments of this method, each of the second containers is equal in size to the first container.

[0017] In some embodiments of this method, each of the second containers is larger than the first container.

[0018] In some embodiments of this method, the sizes of the second containers are equal.

[0019] In some embodiments of this method, the second container is larger than the first container.

[0020] In some embodiments of this method, the second container is smaller than the first container.

[0021] In some embodiments of this method, the first container is a G-Rex100 flask.

[0022] In some embodiments of this method, the first container is a G-Rex100 flask, and each of the plurality of second containers is a G-Rex100 flask.

[0023] In some embodiments of this method, the plurality of second containers are selected from the group consisting of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 second containers.

[0024] In some embodiments of this method, the plurality of second containers are five second containers.

[0025] In some embodiments of this method, prior to step ii), the method further includes the step of supplementing each passage of the second TIL cell culture with additional IL-2.

[0026] In some embodiments of this method, prior to step ii), the method further includes the step of supplementing each passage of the second TIL cell culture with the second cell culture medium and IL-2.

[0027] In some embodiments of this method, the first cell culture medium and the second cell culture medium are the same.

[0028] In some embodiments of this method, the first cell culture medium and the second cell culture medium are different.

[0029] In some embodiments of this method, the first cell culture medium is DM1, and the second cell culture medium is DM2.

[0030] This invention provides a method for proliferating tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, and this method (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) To produce a second TIL population, a first proliferation priming is performed by culturing the first TIL population in a cell culture medium containing IL-2, optionally OKT-3, and optionally antigen-presenting cells (APCs) and / or the culture supernatant from a first culture of APCs containing OKT-3, wherein the first proliferation priming is performed for a first period of approximately 1 to 7 / 8 days to obtain a second TIL population, and the number of the second TIL population is greater than the number of the first TIL population. (c) To produce a third TIL population, a rapid second growth is carried out by supplementing the cell culture medium of the second TIL population with additional IL-2, optionally OKT-3, and either antigen-presenting cells (APCs) and / or a second culture of APCs containing OKT-3, wherein the number of APCs added in 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 about 1 to 11 days to obtain a 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. (d) Collect the therapeutic TIL population obtained from step (c), (e) Transferring the TIL population collected from step (d) to an infusion bag, etc.

[0031] This invention provides a method for proliferating tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, and this method (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) Performing a first proliferation priming by culturing the first TIL population in a cell culture medium containing IL-2, optionally OKT-3, and optionally antigen-presenting cells (APCs) and / or the culture supernatant from a first culture of APCs containing OKT-3, wherein the first proliferation priming is performed for a first period of approximately 1 to 7 / 8 days to obtain the second TIL population, and the number of the second TIL population is greater than the number of the first TIL population. (c) To produce a third TIL population, a rapid second growth is carried out by contacting the second TIL population with a cell culture medium containing IL-2, optionally OKT-3, and optionally antigen-presenting cells (APCs) and / or the culture supernatant from a second culture of APCs containing OKT-3, wherein the rapid second growth is carried out for a second period of approximately 1 to 11 days to obtain a third TIL population, the third TIL population being a therapeutic TIL population. (d) Collecting a therapeutic TIL population obtained from step (c), which includes:

[0032] In some embodiments, “obtaining” means that the TIL used in the method and / or process may be directly derived from the sample (including samples from surgical excision, needle biopsy, core biopsy, microbiopsy, or other) as part of the method and / or process steps. In some embodiments, “receiving” means that the TIL used in the method and / or process may be indirectly derived from the sample (including samples from surgical excision, needle biopsy, core biopsy, microbiopsy, or other) as part of the method and / or process steps, and then used in the method and / or process (for example, if step (a) is initiated with TIL derived from the sample by a separate process not included in step (a), such TIL is referred to as “receiving”).

[0033] In some embodiments of this method, 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).

[0034] This invention provides a method for proliferating tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, and this method (a) Performing a first proliferation priming by culturing a first TIL population in order to produce a second TIL population, the first TIL population can be obtained by processing a tumor sample excised from the target tumor into multiple tumor fragments in a cell culture medium containing either IL-2, optionally OKT-3, and optionally antigen-presenting cells (APCs) and / or a culture supernatant from a first culture of APCs containing OKT-3, the first proliferation priming being performed in a container having a first gas-permeable surface area, the first proliferation priming being performed for a first period of approximately 1 to 7 / 8 days to obtain a second TIL population, and the number of second TIL populations being greater than the number of first TIL populations, (b) To produce a third TIL population, a rapid second growth is carried out by contacting the second TIL population with a cell culture medium of the second TIL population containing additional IL-2, optionally OKT-3, and optionally antigen-presenting cells (APCs) and / or the culture supernatant from a second culture of APCs containing OKT-3, wherein the number of APCs during 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 about 1 to 11 days to obtain a 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. (c) Collecting a therapeutic TIL population obtained from step (b), which includes:

[0035] The present invention also provides a method for growing tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, and this method (a) Performing a first proliferation priming by culturing the first TIL population in a cell culture medium containing IL-2, optionally OKT-3, and optionally antigen-presenting cells (APCs) and / or the culture supernatant from a first culture of APCs containing OKT-3, wherein the first proliferation priming is performed for a first period of approximately 1 to 7 / 8 days to obtain the second TIL population, and the number of the second TIL population is greater than the number of the first TIL population. (b) To produce a third TIL population, a rapid second growth is carried out by contacting the second TIL population with a cell culture medium containing IL-2, optionally OKT-3, and optionally antigen-presenting cells (APCs) and / or the culture supernatant from a second culture of APCs containing OKT-3, wherein the rapid second growth is carried out for a second period of approximately 1 to 11 days to obtain a third TIL population, the third TIL population being a therapeutic TIL population. (c) Collecting a therapeutic TIL population obtained from step (b), which includes:

[0036] In some embodiments of this method, in step (a), 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).

[0037] In some embodiments, the ratio of the number of APCs during rapid second growth to the number of APCs during priming of the first growth is selected from a range of about 1.5:1 to about 20:1.

[0038] In some embodiments, the ratio of the number of APCs during rapid second growth to the number of APCs during priming of the first growth is in the range of about 1.5:1 to about 10:1.

[0039] In some embodiments, the ratio of the number of APCs primed for the first proliferation to the number of APCs in the rapidly growing second proliferation is in the range of about 2:1 to about 5:1.

[0040] In some embodiments, the ratio of the number of APCs primed for the first proliferation to the number of APCs in the rapidly growing second proliferation is in the range of about 2:1 to about 3:1.

[0041] In some embodiments, the ratio of the number of APCs primed for the first proliferation to the number of APCs in the rapidly growing second proliferation is about 2:1.

[0042] In some embodiments, the number of APCs in the priming of the first proliferation is about 1.0×10 6 APC / cm 2 ~ about 4.5×10 6 APC / cm 2 is selected from the range of, and the number of APCs in the rapidly growing second proliferation is about 2.5×10 6 APC / cm 2 ~ about 7.5×10 6 APC / cm 2 is selected from the range of.

[0043] In some embodiments, the number of APCs in the priming of the first proliferation is about 1.5×10 6 APC / cm 2 ~ about 3.5×10 6 APC / cm 2 is selected from the range of, and the number of APCs in the rapidly growing second proliferation is about 3.5×10 6 APC / cm 2 ~ about 6.0×10 6 APC / cm 2 is selected from the range of.

[0044] In some embodiments, the number of APCs in the priming of the first proliferation is about 2.0×10 6 APC / cm 2 ~ about 3.0×10 6 APC / cm 2Selected from the range, the number of APCs in rapid second proliferation is approximately 4.0 × 10⁻⁶. 6 APC / cm 2 ~Approx. 5.5×10 6 APC / cm 2 It is selected from the range.

[0045] In some embodiments, the number of APCs in the priming of the first growth is approximately 1 × 10⁶ 8 APC ~ approx. 3.5 × 10 8 Selected from the range of APCs, the number of APCs in rapid second proliferation is approximately 3.5 × 10⁻⁶. 8 APC ~ approx. 1 x 10 9 Selected from the APC range.

[0046] In some embodiments, the number of APCs in the priming of the first growth is approximately 1.5 × 10⁻⁶ 8 APC ~ approx. 3 x 10 8 Selected from the range of APCs, the number of APCs in rapid second proliferation is approximately 4 × 10⁻⁶. 8 APC ~ approx. 7.5 x 10 8 Selected from the APC range.

[0047] In some embodiments, the number of APCs in the priming of the first growth is approximately 2 × 10⁶ 8 APC ~ approx. 2.5 x 10 8 Selected from the range of APCs, the number of APCs in rapid second proliferation is approximately 4.5 × 10⁻⁶. 8 APC ~ approx. 5.5 x 10 8 Selected from the APC range.

[0048] In some embodiments, approximately 2.5 × 10 8 APC was added to the priming of the first growth stage, 5 × 10 8 APC is added to the second rapid growth stage.

[0049] In some embodiments, the ratio of the number of TILs in the second TIL group to the number of TILs in the first TIL group is approximately 1.5:1 to approximately 100:1.

[0050] In some embodiments, the ratio of the number of TILs in the second TIL group to the number of TILs in the first TIL group is approximately 50:1.

[0051] In some embodiments, the ratio of the number of TILs in the second TIL group to the number of TILs in the first TIL group is approximately 25:1.

[0052] In some embodiments, the ratio of the number of TILs in the second TIL group to the number of TILs in the first TIL group is approximately 20:1.

[0053] 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.

[0054] In some embodiments, the number of the second TIL group is at least 50 times greater than the number of the first TIL group.

[0055] In some embodiments, the method, after the step of collecting a therapeutic TIL population, This includes an additional step of transferring the collected therapeutic TIL population to an infusion bag.

[0056] In some embodiments, multiple tumor fragments are distributed into multiple separate containers, in each separate container, a second TIL population is obtained from the first TIL population from the first growth priming step, a third TIL population is obtained from the second TIL population in the 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 obtain the collected TIL populations.

[0057] In some embodiments, the plurality of separate containers include at least two separate containers.

[0058] In some embodiments, the plurality of separate containers includes at least 2 to 20 separate containers.

[0059] In some embodiments, the plurality of separate containers includes at least 2 to 10 separate containers.

[0060] In some embodiments, the plurality of separate containers includes at least two to five separate containers.

[0061] In some embodiments, each of the separate containers includes a first gas-permeable surface area.

[0062] In some embodiments, multiple tumor fragments are distributed into a single container.

[0063] In some embodiments, a single container includes a first gas-permeable surface area.

[0064] In some embodiments, the first proliferation priming step involves the cell culture medium comprising antigen-presenting cells (APCs), which are layered on a first gas-permeable surface region with an average thickness of about 1 to 3 cell layers.

[0065] In some embodiments, during the first proliferation priming step, the APCs are layered on a first gas-permeable surface region with an average thickness of about 1.5 to 2.5 cell layers.

[0066] In some embodiments, during the first proliferation priming step, the APCs are layered on a first gas-permeable surface region with an average thickness of about two cell layers.

[0067] In some embodiments, during a rapid second proliferation step, the APCs are layered on a first gas-permeable surface region with an average thickness of about 3 to 5 cell layers.

[0068] In some embodiments, during rapid second proliferation, the APCs are layered on the first gas-permeable surface region with an average thickness of approximately 3.5 to 4.5 cell layers.

[0069] In some embodiments, during rapid second proliferation, the APCs are layered on the first gas-permeable surface region with an average thickness of approximately 4 cell layers.

[0070] In some embodiments, a first growth priming step is performed in a first vessel having a first gas-permeable surface area, and a rapid second growth step is performed in a second vessel having a second gas-permeable surface area.

[0071] In some embodiments, the second container is larger than the first container.

[0072] In some embodiments, the first proliferation priming step involves the cell culture medium comprising antigen-presenting cells (APCs), which are layered on a first gas-permeable surface region with an average thickness of about 1 to 3 cell layers.

[0073] In some embodiments, during the first proliferation priming step, the APCs are layered on a first gas-permeable surface region with an average thickness of about 1.5 to 2.5 cell layers.

[0074] In some embodiments, during the first proliferation priming step, the APCs are layered on a first gas-permeable surface region with an average thickness of about two cell layers.

[0075] In some embodiments, during rapid second proliferation, the APCs are layered on the second gas-permeable surface region with an average thickness of about 3 to 5 cell layers.

[0076] In some embodiments, during rapid second proliferation, the APCs are layered on the second gas-permeable surface region with an average thickness of approximately 3.5 to 4.5 cell layers.

[0077] In some embodiments, during a rapid second proliferation step, the APCs are layered on a second gas-permeable surface region with an average thickness of approximately four cell layers.

[0078] In some embodiments, for each container in which a first growth priming is performed on a first TIL population, a rapid second growth is performed on a second TIL population generated from the first TIL population within the same container.

[0079] In some embodiments, each container includes a first gas-permeable surface area.

[0080] In some embodiments, the first proliferation priming step involves the cell culture medium comprising antigen-presenting cells (APCs), which are layered on a first gas-permeable surface region with an average thickness of about 1 to 3 cell layers.

[0081] In some embodiments, during the first proliferation priming step, the APCs are layered on a first gas-permeable surface region with an average thickness of about 1.5 to 2.5 cell layers.

[0082] In some embodiments, during the first proliferation priming step, the APCs are layered on a first gas-permeable surface region with an average thickness of about two cell layers.

[0083] In some embodiments, during a rapid second proliferation step, the APCs are layered on a first gas-permeable surface region with an average thickness of about 3 to 5 cell layers.

[0084] In some embodiments, during a rapid second proliferation step, the APCs are layered on a first gas-permeable surface region with an average thickness of approximately 3.5 to 4.5 cell layers.

[0085] In some embodiments, during a rapid second proliferation step, the APCs are layered on a first gas-permeable surface region with an average thickness of approximately 4 cell layers.

[0086] In some embodiments, for each container in which the first growth priming step 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 stacked on a first gas-permeable surface area, and the ratio of the average number of APCs stacked in the first growth priming step to the average number of APCs stacked in the rapid second growth step is selected from the range of about 1:1.1 to about 1:10.

[0087] In some embodiments, the ratio of the average number of APC layers stacked in the first growth priming step to the average number of APC layers stacked in the rapid second growth step is selected from a range of about 1:1.2 to about 1:8.

[0088] In some embodiments, the ratio of the average number of APC layers stacked in the first growth priming step to the average number of APC layers stacked in the rapid second growth step is selected from a range of about 1:1.3 to about 1:7.

[0089] In some embodiments, the ratio of the average number of APC layers stacked in the first growth priming step to the average number of APC layers stacked in the rapid second growth step is selected from a range of about 1:1.4 to about 1:6.

[0090] In some embodiments, the ratio of the average number of APC layers stacked in the first growth priming step to the average number of APC layers stacked in the rapid second growth step is selected from a range of approximately 1:1.5 to approximately 1:5.

[0091] In some embodiments, the ratio of the average number of APC layers stacked in the first growth priming step to the average number of APC layers stacked in the rapid second growth step is selected from a range of about 1:1.6 to about 1:4.

[0092] In some embodiments, the ratio of the average number of APC layers stacked in the first growth priming step to the average number of APC layers stacked in the rapid second growth step is selected from a range of about 1:1.7 to about 1:3.5.

[0093] In some embodiments, the ratio of the average number of APC layers stacked in the first growth priming step to the average number of APC layers stacked in the rapid second growth step is selected from a range of approximately 1:1.8 to approximately 1:3.

[0094] In some embodiments, the ratio of the average number of APC layers stacked in the first growth priming step to the average number of APC layers stacked in the rapid second growth step is selected from a range of about 1:1.9 to about 1:2.5.

[0095] In some embodiments, the ratio of the average number of APC layers stacked in the first growth priming step to the average number of APC layers stacked in the rapid second growth step is approximately 1:2.

[0096] In some embodiments, additional IL-2 is added to the cell culture medium 2-3 days after the rapid second growth step.

[0097] In some embodiments, the method further includes, in the step of collecting a therapeutic TIL population, cryopreserving the collected TIL population using a cryopreservation process.

[0098] In some embodiments, the method further includes the step of freezing and storing the infusion bag.

[0099] In some embodiments, the cryopreservation process is carried out using a 1:1 ratio of TIL population to cryopreservation medium in cryopreservation media.

[0100] In some embodiments, the antigen-presenting cells are peripheral blood mononuclear cells (PBMCs).

[0101] In some embodiments, the PBMCs are irradiated and are homogeneous.

[0102] In some embodiments, the first proliferation priming step involves the cell culture medium containing peripheral blood mononuclear cells (PBMCs), and the total number of PBMCs added to the cell culture medium in the first proliferation priming step is approximately 2.5 × 10⁶ 8 That is the case.

[0103] In some embodiments, the rapid second growth step involves antigen-presenting cells (APCs) in the cell culture medium being peripheral blood mononuclear cells (PBMCs), and the total number of PBMCs added to the cell culture medium in the rapid second growth step is approximately 5 × 10⁶. 8 That is the case.

[0104] In some embodiments, the antigen-presenting cells are artificial antigen-presenting cells.

[0105] In some embodiments, the sampling in the step of collecting therapeutic TIL populations is carried out using a membrane-based cell processing system.

[0106] In some embodiments, the sampling in the step of collecting therapeutic TIL populations is performed using a LOVO cell processing system.

[0107] In some embodiments, the multiple fragments include approximately 60 fragments per container during the priming step of the first growth, with each fragment being approximately 27 mm in size. 3 It has the volume of .

[0108] In some embodiments, the multiple fragments have a total volume of approximately 1300 mm³ 3 ~approx. 1500mm 3 It contains approximately 30 to 60 fragments.

[0109] In some embodiments, the multiple fragments have a total volume of approximately 1350 mm³ 3 It contains approximately 50 fragments.

[0110] In some embodiments, the multiple fragments include about 50 fragments with a total mass of about 1 gram to about 1.5 grams.

[0111] In some embodiments, the cell culture medium is provided in a container selected from the group consisting of G containers and Xuri cell bags.

[0112] In some embodiments, the IL-2 concentration is approximately 10,000 IU / mL to approximately 5,000 IU / mL.

[0113] In some embodiments, the IL-2 concentration is approximately 6,000 IU / mL.

[0114] In some embodiments, the infusion bag used in the step of transferring the collected therapeutic TIL population to an infusion bag is a HypoThermosol-containing infusion bag.

[0115] In some embodiments, the cryopreservation medium contains dimethyl sulfoxide (DMSO).

[0116] In some embodiments, the cryopreservation medium contains 7% to 10% DMSO.

[0117] In some embodiments, the first period in the priming step of the first growth and the second period in the rapid second growth step are carried out separately within periods of 5, 6, or 7 days, respectively.

[0118] In some embodiments, the first period in the first growth priming step is carried out within a period of 5, 6, or 7 days.

[0119] In some embodiments, the second period in the rapid second growth step is carried out within a period of 7, 8, or 9 days.

[0120] In some embodiments, the first period in the priming step of the first growth and the second period in the rapid second growth step are each carried out separately within a period of 7 days.

[0121] In some embodiments, the process from the first proliferation priming step to the collection of therapeutic TILs takes place within a period of approximately 14 to 16 days.

[0122] In some embodiments, the process from the first proliferation priming step to the collection of therapeutic TILs takes place within a period of approximately 15 to 16 days.

[0123] In some embodiments, the process from the first proliferation priming step to the collection of therapeutic TILs is carried out within a period of approximately 14 days.

[0124] In some embodiments, the process from the first proliferation priming step to the collection of therapeutic TILs is carried out within a period of approximately 15 days.

[0125] In some embodiments, the process from the first proliferation priming step to the collection of therapeutic TILs is carried out within a period of approximately 16 days.

[0126] In some embodiments, the method further includes the step of cryopreserving the collected population of therapeutic TILs using a cryopreservation process, wherein the collection and cryopreservation of the therapeutic TIL population from the first proliferation priming step is carried out within 16 days.

[0127] In some embodiments, the therapeutic TIL population collected in the step of collecting a therapeutic TIL population contains enough TILs to constitute a therapeutically effective dose of TILs.

[0128] In some embodiments, the number of TILs sufficient for a therapeutically effective dose is approximately 2.3 × 10⁶ 10 ~Approx. 13.7×10 10 That is the case.

[0129] In some embodiments, the third TIL population in a rapid second growth step provides increased potency, increased interferon-γ production, and / or increased polyclonality.

[0130] In some embodiments, the third TIL population in a rapid second growth step provides at least 1 to 5 times greater interferon-gamma production compared to TILs prepared by a process longer than 18 days.

[0131] In some embodiments, effector T cells and / or central memory T cells obtained from a third TIL population in a rapid second growth step exhibit increased CD8 and CD28 expression compared to effector T cells and / or central memory T cells obtained from a second TIL population in a priming step of the first proliferation.

[0132] In some embodiments, the therapeutic TIL population, obtained from the step of collecting the therapeutic TIL population, is injected into the patient.

[0133] The present invention also provides a method for treating a subject having cancer, the method comprising administering proliferated tumor-infiltrating lymphocytes (TILs), the administration of (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) Priming the first growth in order to produce a second TIL population by culturing the first TIL population in a cell culture medium containing IL-2, optionally OKT-3, and optionally antigen-presenting cells (APCs) and / or the culture supernatant from a first culture of APCs containing OKT-3, wherein the priming of the first growth is performed in a container having a first gas-permeable surface area, and the priming of the first growth is performed for a period of approximately 1 to 7 / 8 days to obtain a second TIL population. (c) To produce a third TIL population, a rapid second growth is carried out by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, and either a second culture of antigen-presenting cells (APCs) and / or OKT-3 containing APCs into the culture supernatant, wherein the number of APCs added in 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 to obtain a third TIL population, 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. (d) Collect the therapeutic TIL population obtained from step (c), (e) Transfer the TIL sample collected from step (d) to an infusion bag, (f) administering a therapeutically effective dose of TIL from step (e), including the above.

[0134] In some embodiments, the number of TILs sufficient to administer a therapeutically effective dose is approximately 2.3 × 10⁶ 10 ~Approx. 13.7×10 10 That is the case.

[0135] In some embodiments, antigen-presenting cells (APCs) are PBMCs.

[0136] 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 (f).

[0137] In some embodiments, the non-myeloablative lymphocyte depletion regimen involves cyclophosphamide at 60 mg / m². 2 / day for 2 days, followed by fludarabine 25 mg / m². 2 The step includes administering the drug daily for 5 days.

[0138] In some embodiments, the method further includes a 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 (f).

[0139] In some embodiments, the high-dose IL-2 regimen is administered as an intravenous bolus infusion of 600,000 or 720,000 IU / kg every 8 hours for 15 minutes until tolerance is achieved.

[0140] In some embodiments, the third TIL population in step (b) provides increased potency, increased interferon-γ production, and / or increased polyclonality.

[0141] In some embodiments, the third TIL population in step (c) provides at least 1 to 5 times greater interferon-γ production compared to TILs prepared by a process longer than 16 days.

[0142] In some embodiments, effector T cells and / or central memory T cells obtained from the third TIL population in step (c) exhibit increased CD8 and CD28 expression compared to effector T cells and / or central memory T cells obtained from the second population in step (b).

[0143] In some embodiments, cancer is a solid tumor.

[0144] In some embodiments, cancer is selected from the group consisting of melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, triple-negative breast cancer, cancer derived from 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.

[0145] In some embodiments, the cancer is selected from the group consisting of melanoma, HNSCC, cervical cancer, NSCLC, glioblastoma (including GBM), and gastrointestinal cancer.

[0146] In some embodiments, the cancer is melanoma.

[0147] In some embodiments, the cancer is HNSCC.

[0148] In some embodiments, the cancer is cervical cancer.

[0149] In some embodiments, the cancer is NSCLC.

[0150] In some embodiments, the cancer is glioblastoma (including GBM).

[0151] In some embodiments, the cancer is gastrointestinal cancer.

[0152] In some embodiments, the cancer is a hypermutant cancer.

[0153] In some embodiments, the cancer is a pediatric hypermutant cancer.

[0154] In some embodiments, the container is a sealed container.

[0155] In some embodiments, the container is a G-container.

[0156] In some embodiments, the container is GREX-10.

[0157] In some embodiments, the sealed container includes GREX-100.

[0158] In some embodiments, the sealed container includes GREX-500.

[0159] The present invention also provides a population of therapeutic tumor-infiltrating lymphocytes (TILs) produced by the methods disclosed herein.

[0160] The present invention also provides a population of therapeutic tumor-infiltrating lymphocytes (TILs) prepared from a patient's tumor tissue, the therapeutic TIL population resulting in increased potency, increased interferon-gamma production, and / or increased polyclonality.

[0161] In some embodiments, the therapeutic TIL population disclosed herein results in increased interferon-gamma production.

[0162] In some embodiments, the therapeutic TIL population disclosed herein results in increased polyclonality.

[0163] In some embodiments, the therapeutic TIL population disclosed herein results in increased efficacy.

[0164] In some embodiments, the therapeutic TIL population described herein is capable of producing at least 1 times more interferon-gamma compared to TILs prepared by processes longer than 16 days.

[0165] In some embodiments, the therapeutic TIL population described herein is capable of producing at least twice as much interferon-gamma compared to TILs prepared by processes longer than 16 days.

[0166] In some embodiments, the therapeutic TIL population described herein is capable of producing at least three times more interferon-gamma compared to TILs prepared by processes longer than 16 days.

[0167] In some embodiments, the present invention provides a population of therapeutic tumor-infiltrating lymphocytes (TILs) capable of producing at least 1 times more interferon-gamma compared to TILs prepared by a process that carries out a first proliferation of TILs without the addition of antigen-presenting cells (APCs).

[0168] In some embodiments, the therapeutic TIL population described herein is capable of producing at least twice as much interferon-gamma as TILs prepared by a process that carries out a first proliferation of TILs without the addition of APC.

[0169] In some embodiments, the therapeutic TIL population described herein is capable of producing at least three times more interferon-gamma compared to TILs prepared by a process that carries out a first proliferation of TILs without the addition of APC.

[0170] In some embodiments, the present invention provides a population of therapeutic tumor-infiltrating lymphocytes (TILs) capable of producing at least 1 times more interferon-gamma compared to TILs prepared by a process that carries out a first proliferation of TILs without the addition of antigen-presenting cells OKT3.

[0171] In some embodiments, the therapeutic TIL population described herein is capable of producing at least twice as much interferon-gamma compared to TILs prepared by a process that carries out a first proliferation of TILs without the addition of OKT3.

[0172] In some embodiments, the therapeutic TIL population described herein is capable of producing at least three times more interferon-gamma compared to TILs prepared by a process that carries out a first proliferation of TILs without the addition of OKT3.

[0173] In some embodiments, the present invention provides a population of therapeutic tumor-infiltrating lymphocytes (TILs) capable of producing at least 1 times more interferon-gamma than TILs prepared by a process that carries out a first proliferation of TILs without the addition of antigen-presenting cells (APCs) and OKT3.

[0174] In some embodiments, the therapeutic TIL population described herein is capable of producing at least twice as much interferon-gamma compared to TILs prepared by a process that carries out a first proliferation of TILs without the addition of antigen-presenting cells (APCs) and OKT3.

[0175] In some embodiments, the therapeutic TIL population described herein is capable of producing at least three times more interferon-gamma compared to TILs prepared by a process that carries out a first proliferation of TILs without the addition of antigen-presenting cells (APCs) and OKT3.

[0176] The present invention also provides tumor-infiltrating lymphocyte (TIL) compositions comprising the therapeutic TIL population and pharmaceutically acceptable carriers described herein.

[0177] The present invention also provides a sterile infusion bag comprising the TIL composition described herein.

[0178] The present invention also provides cryopreserved preparations of therapeutic TIL populations as described herein.

[0179] The present invention also provides tumor-infiltrating lymphocyte (TIL) compositions comprising the therapeutic TIL population and cryopreservation medium described herein.

[0180] In some embodiments, the cryopreservation medium contains DMSO.

[0181] In some embodiments, the cryopreservation medium contains 7% to 10% DMSO.

[0182] The present invention also provides cryopreservation preparations of the TIL composition described herein.

[0183] In some embodiments, the tumor-infiltrating lymphocyte (TIL) compositions described herein are intended for use as pharmaceuticals.

[0184] In some embodiments, the tumor-infiltrating lymphocyte (TIL) compositions described herein are intended for use in the treatment of cancer.

[0185] In some embodiments, the tumor-infiltrating lymphocyte (TIL) compositions described herein are intended for use in the treatment of solid tumor cancers.

[0186] In some embodiments, the tumor-infiltrating lymphocyte (TIL) compositions described herein are intended for use in the treatment of cancers selected from the group consisting of melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, triple-negative breast cancer, cancers derived from human papillomavirus, head and neck cancers (including head and neck squamous cell carcinoma (HNSCC)), glioblastoma (including GBM), gastrointestinal cancer, kidney cancer, and renal cell carcinoma.

[0187] In some embodiments, the tumor-infiltrating lymphocyte (TIL) compositions described herein are intended for use in the treatment of cancers selected from the group consisting of melanoma, HNSCC, cervical cancer, NSCLC, glioblastoma (including GBM), and gastrointestinal cancer.

[0188] In some embodiments, the TIL compositions described herein are for use in the treatment of cancer, where the cancer is melanoma.

[0189] In some embodiments, the TIL compositions described herein are for use in the treatment of cancer, wherein the cancer is HNSCC.

[0190] In some embodiments, the TIL compositions described herein are for use in the treatment of cancer, where the cancer is cervical cancer.

[0191] In some embodiments, the TIL compositions described herein are for use in the treatment of cancer, wherein the cancer is NSCLC.

[0192] In some embodiments, the TIL compositions described herein are for use in the treatment of cancer, where the cancer is glioblastoma (including GBM).

[0193] In some embodiments, the TIL compositions described herein are for use in the treatment of cancer, where the cancer is gastrointestinal cancer.

[0194] In some embodiments, the TIL compositions described herein are for use in the treatment of cancer, which is hypermutant cancer.

[0195] In some embodiments, the TIL compositions described herein are for use in the treatment of cancer, which is a pediatric hypermutant cancer.

[0196] In some embodiments, the present invention provides the use of a tumor-infiltrating lymphocyte (TIL) composition described herein in a method of treating cancer in a subject, comprising administering a therapeutically effective amount of the TIL composition to the subject. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is selected from the group consisting of melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, triple-negative breast cancer, cancer derived from 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, HNSCC, cervical cancer, NSCLC, glioblastoma (including GBM), and gastrointestinal cancer. In some embodiments, the cancer is melanoma. In some embodiments, the cancer is HNSCC. In some embodiments, the cancer is cervical cancer. In some embodiments, the cancer is NSCLC. In some embodiments, the cancer is glioblastoma (including GBM). In some embodiments, the cancer is gastrointestinal cancer. In some embodiments, the cancer is hypermutant cancer. In some embodiments, the cancer is pediatric hypermutant cancer.

[0197] In some embodiments, the tumor-infiltrating lymphocyte (TIL) compositions described herein are for use in a method of treating cancer in a subject, comprising administering a therapeutically effective amount of the TIL composition to the subject. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is selected from the group consisting of melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, triple-negative breast cancer, cancer derived from 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, HNSCC, cervical cancer, NSCLC, glioblastoma (including GBM), and gastrointestinal cancer.

[0198] The present invention also provides a method for treating cancer in a subject, comprising administering a therapeutically effective dose of the tumor-infiltrating lymphocyte (TIL) composition described herein to the subject.

[0199] In some embodiments, cancer is a solid tumor.

[0200] In some embodiments, cancer is selected from the group consisting of melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, triple-negative breast cancer, cancer derived from 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.

[0201] In some embodiments, the cancer is selected from the group consisting of melanoma, HNSCC, cervical cancer, NSCLC, glioblastoma (including GBM), and gastrointestinal cancer. In some embodiments, the cancer is melanoma. In some embodiments, the cancer is HNSCC. In some embodiments, the cancer is cervical cancer. In some embodiments, the cancer is NSCLC. In some embodiments, the cancer is glioblastoma (including GBM). In some embodiments, the cancer is gastrointestinal cancer. In some embodiments, the cancer is hypermutant cancer. In some embodiments, the cancer is pediatric hypermutant cancer.

[0202] The present invention also provides a method for proliferating T cells, and this method is (a) Priming the first proliferation of the first T cell population obtained from the donor by culturing the first T cell population in order to induce proliferation of the first T cell population and to prime its activity, (b) After the activation of the first T cell population primed in step (a) begins to break down, in order to obtain a second T cell population, the first T cell population is cultured to induce proliferation and increase the activation of the first T cell population, thereby rapidly promoting the second proliferation of the first T cell population. (c) including collecting a second T cell population.

[0203] In some embodiments, the first propagation priming in step (a) is carried out over a period of up to 7 days.

[0204] In some embodiments, the rapid second growth in step (b) is carried out over a period of up to 11 days.

[0205] In some embodiments, the rapid second growth in step (b) is carried out over a period of up to 9 days.

[0206] In some embodiments, the first growth priming in step (a) is carried out for 7 days, and the rapid second growth in step (b) is carried out for 9 days.

[0207] In some embodiments, the first propagation priming in step (a) is carried out over a period of up to 8 days.

[0208] In some embodiments, the rapid second growth in step (b) is carried out over a period of up to 8 days.

[0209] In some embodiments, the first growth priming in step (a) is carried out for 8 days, and the rapid second growth in step (b) is carried out for 8 days.

[0210] In some embodiments of this method, in step (a), a first T cell population is cultured in a first culture medium containing OKT-3 and IL-2.

[0211] In some embodiments, the first culture medium comprises OKT-3, IL-2, and antigen-presenting cells (APCs).

[0212] In some embodiments of this method, in step (b), the first population of T cells is cultured in a second culture medium containing OKT-3, IL-2, and antigen-presenting cells (APCs).

[0213] In some embodiments of this method, in step (a), a first population of T cells is cultured in a first culture medium in a container comprising a first gas-permeable surface, the first culture medium optionally comprising OKT-3, IL-2 and optionally a first antigen-presenting cell (APC) population or a culture supernatant from a first APC culture comprising OKT-3, the first APC population being exogenous to the first T cell population, and the first APC population being comprising a first gas-permeable surface The cells are stacked on a surface, and in step (b), the first T cell population is cultured in a second culture medium in a container, the second culture medium containing OKT-3, IL-2 and a second APC population, or a culture supernatant from a second APC culture containing OKT-3, the second APC population being exogenous to the donor of the first T cell population, the second APC population being stacked on the first gas-permeable surface, and the second APC population being larger than the first APC population.

[0214] In some embodiments, the ratio of the number of APCs in the second APC group to the number of APCs in the first APC group is approximately 2:1.

[0215] In some embodiments, the number of APCs in the first APC population is approximately 2.5 × 10 8 Therefore, the number of APCs in the second APC population is approximately 5 × 10⁻⁶. 8 That is the case.

[0216] In some embodiments of this method, in step (a), a first APC group is laminated on a first gas-permeable surface with an average thickness of two layers of APC.

[0217] In some embodiments of this method, in step (b), a second APC ensemble is laminated on the first gas-permeable surface with an average thickness selected from a range of 4 to 8 layers of APC.

[0218] In some embodiments, the ratio of the average number of APC layers laminated on the first gas permeable surface in step (b) to the average number of APC layers laminated on the first gas permeable surface in step (a) is 2:1.

[0219] In some embodiments, APCs are peripheral blood mononuclear cells (PBMCs).

[0220] In some embodiments, the APC comprises PBMCs, which are irradiated and exogenous to the donor of the first T cell population.

[0221] In some embodiments, T cells are tumor-infiltrating lymphocytes (TILs).

[0222] In some embodiments, T cells are medullary lymphocytes (MILs).

[0223] In some embodiments, T cells are peripheral blood lymphocytes (PBLs).

[0224] In some embodiments, the cell culture medium is a limited medium and / or a serum-free medium.

[0225] In some embodiments, the limiting medium comprises (optionally recombinant) transferrin, (optionally recombinant) insulin, and (optionally recombinant) albumin.

[0226] In some embodiments, the serum-free or serum-limited medium comprises a basal cell medium, as well as serum supplements and / or serum substitutes.

[0227] In some embodiments, the basal cell medium includes CTS® OpTmizer® T-cell Expansion Basal Medium, CTS® OpTmizer® T-Cell Expansion SFM, CTS® AIM-V Medium, CTS® AIM-V SFM, LymphoONE® T-Cell Expansion Xeno-Free Medium, Dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), Basal Medium Eagle (BME), RPMI 1640, F-10, F-12, Minimal Essential Medium (αMEM), Glasgow's Minimal Essential Medium (G-MEM), RPMI growth medium, and Iscove's Modified Dulbecco's Medium.

[0228] In some embodiments, the serum supplement or serum replacement is selected from the group consisting of CTS® OpTmizer T-Cell Expansion Serum Supplement and CTS® Immune Cell Serum Replacement.

[0229] In some embodiments, the cell culture medium comprises one or more albumins or albumin substitutes.

[0230] In some embodiments, the cell culture medium contains one or more amino acids.

[0231] In some embodiments, the cell culture medium comprises one or more vitamins, one or more transferrins, or transferrin substitutes.

[0232] In some embodiments, the cell culture medium comprises one or more antioxidants, one or more insulins, or insulin substitutes.

[0233] In some embodiments, the cell culture medium comprises one or more collagen precursors, one or more antibiotics, and one or more trace elements.

[0234] In some embodiments, the cell culture medium contains albumin.

[0235] In some embodiments, the cell culture medium contains albumin, 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

[0236] In some embodiments, the cell culture medium further comprises L-glutamine, sodium bicarbonate, and / or 2-mercaptoethanol.

[0237] In some embodiments, the cell culture medium contains a total serum substitution concentration (vol%) of approximately 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 cell culture medium.

[0238] In some embodiments, the cell culture medium contains a total serum substitution concentration of about 3%, about 5%, or about 10% of the total cell culture medium volume.

[0239] In some embodiments, the cell culture medium further comprises glutamine (i.e., GlutaMAX®) at concentrations of approximately 0.1 mM to 10 mM, 0.5 mM to 9 mM, 1 mM to 8 mM, 2 mM to 7 mM, 3 mM to 6 mM, or 4 mM to 5 mM.

[0240] In some embodiments, the cell culture medium further comprises glutamine (i.e., GlutaMAX®) at a concentration of approximately 2 mM.

[0241] In some embodiments, the cell culture medium further comprises 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.

[0242] In some embodiments, the cell culture medium further comprises 2-mercaptoethanol at a concentration of about 55 mM.

[0243] In some embodiments, the cell culture medium includes a limited medium as described in International PCT Publication No. WO / 1998 / 030679.

[0244] In some embodiments, the cell culture medium contains glycine in the range of approximately 5-200 mg / L, L-histidine in the range of approximately 5-250 mg / L, L-isoleucine in the range of approximately 5-300 mg / L, L-methionine in the range of approximately 5-200 mg / L, L-phenylalanine in the range of approximately 5-400 mg / L, L-proline in the range of approximately 1-1000 mg / L, L-hydroxyproline in the range of approximately 1-45 mg / L, L-serine in the range of approximately 1-250 mg / L, L-threonine in the range of approximately 10-500 mg / L, and L-tryptophan in the range of approximately 2-110 mg / L. It contains L-tyrosine in the range of approximately 3 to 175 mg / L, L-valine in the range of approximately 5 to 500 mg / L, thiamine in the range of approximately 1 to 20 mg / L, reduced glutathione in the range of approximately 1 to 20 mg / L, L-ascorbic acid-2-phosphate in the range of approximately 1 to 200 mg / L, iron-saturated transferrin in the range of approximately 1 to 50 mg / L, insulin in the range of approximately 1 to 100 mg / L, sodium selenite in the range of approximately 0.000001 to 0.0001 mg / L, and / or albumin (e.g., AlbumX® I) in the range of approximately 5,000 to 50,000 mg / L.

[0245] In some embodiments, cell culture media containing one or more non-trace element partials in a limited medium exist in the concentration ranges listed in the column under the heading “Concentration Ranges in 1X Medium” in Table 4 provided herein.

[0246] In some embodiments, the volumetric osmolality of the cell culture medium is approximately 260–350 mOsmol.

[0247] In some embodiments, the cell culture medium further contains about 3.7 g / L or about 2.2 g / L of sodium bicarbonate.

[0248] In some embodiments, the cell culture medium further comprises L-glutamine (final concentration about 2 mM), one or more antibiotics, non-essential amino acids (NEAAs; final concentration about 100 μM), and / or 2-mercaptoethanol (final concentration about 100 μM).

[0249] In some embodiments, the cell culture medium in the first and / or second gas-permeable vessel does not contain beta-mercaptoethanol (BME or βME; also known as 2-mercaptoethanol, CAS60-24-2).

[0250] In some embodiments, the cell culture medium comprises CTSOPTmizerT-Cell Expansion SFM, 3% CTS Immune Cell Serum Replacement, 55 mM BME, and optionally glutamine.

[0251] In some embodiments, the cell culture medium comprises CTS® OpTmizer® T-cell Expansion Basal Medium (26 mL / L) supplemented with CTS® OpTmizer® T-cell Expansion Supplement, 3% CTS® Immune Cell SR, and 2 mM Glutamax, and optionally further comprising 6,000 IU / mL of IL-2.

[0252] In some embodiments, the cell culture medium comprises CTS® OpTmizer® T-cell Expansion Basal Medium (26 mL / L) supplemented with CTS® OpTmizer® T-cell Expansion Supplement, 3% CTS® Immune Cell SR, and 2 mM Glutamax, and optionally further comprising 3,000 IU / mL of IL-2.

[0253] In some embodiments, the tumor sample is one or more small biopsies, core biopsies, or needle biopsies of a tumor in the subject.

[0254] The present invention also provides a method for growing tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, and this method (i) Obtaining and / or receiving a first TIL population from tumor samples obtained from one or more small biopsies, core biopsies, or needle biopsies of a tumor in a subject by culturing tumor samples in a first cell culture medium containing IL-2 for approximately 3 days, (ii) To produce a second TIL population, a first proliferation priming is performed by culturing the first TIL population in a second cell culture medium containing IL-2, OKT-3, and antigen-presenting cells (APCs), wherein the first proliferation priming is performed in a container containing a first gas-permeable surface, the first proliferation priming is performed for a first period of about 7 or 8 days to obtain a second TIL population, and the number of the second TIL population is greater than the number of the first TIL population. (iii) To produce a third TIL population, a rapid second growth is carried out by supplementing the second cell culture medium of the second TIL population with additional IL-2, OKT-3, and APC, wherein the number of APCs added in the rapid second growth is at least twice the number of APCs added in step (ii), the rapid second growth is carried out for a second period of about 11 days to obtain a 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. (iv) Collect the therapeutic TIL population obtained from step (iii), (v) Transferring the TIL population collected from step (iv) to an infusion bag, etc.

[0255] The present invention also provides a method for growing tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, and this method (i) Obtaining and / or receiving a first TIL population from tumor samples obtained from one or more small biopsies, core biopsies, or needle biopsies of a tumor in a subject by culturing tumor samples in a first cell culture medium containing IL-2 for approximately 3 days, (ii) Performing a first proliferation priming by culturing the first TIL population in a second cell culture medium containing IL-2, OKT-3, and antigen-presenting cells (APCs) in order to produce a second TIL population, wherein the first proliferation priming is performed for a first period of about 7 or 8 days to obtain a second TIL population, and the number of the second TIL population is greater than the number of the first TIL population. (iii) To produce a third TIL population, a rapid second growth is carried out by contacting the second TIL population with a third cell culture medium containing IL-2, OKT-3, and APC, wherein the rapid second growth is carried out for a second period of approximately 11 days to obtain the third TIL population, and the third TIL population is the therapeutic TIL population. (iv) Collecting a therapeutic TIL population obtained from step (iii), which includes:

[0256] In some embodiments, after the fifth day of the second period, the culture is divided into two or more subcultures, an additional amount of the third culture medium is added to each subculture, and the cultures are incubated for approximately six days.

[0257] In some embodiments, after the fifth day of the second period, the culture is divided into up to five subcultures.

[0258] In some embodiments, all steps of the method are completed in approximately 22 days.

[0259] The present invention also provides a method for proliferating T cells, and this method is (i) In order to induce proliferation of the first T cell population and prime its activity, the first T cell population is primed from tumor samples obtained from one or more small biopsies, core biopsies, or needle biopsies of the donor tumor, (ii) After the activation of the first T cell population primed in step (a) begins to break down, in order to obtain a second T cell population, the first T cell population is cultured to induce proliferation and increase the activity of the first T cell population, thereby rapidly promoting the second proliferation of the first T cell population. (iv) including collecting a second T cell population.

[0260] In some embodiments, tumor samples are obtained from multiple core biopsies.

[0261] In some embodiments, the multiple core biopsies are selected from the group consisting of 2, 3, 4, 5, 6, 7, 8, 9, and 10 core biopsies.

[0262] The present invention also provides a proliferated tumor-infiltrating lymphocyte (TIL) composition, which is i) Therapeutic tumor-infiltrating lymphocyte (TIL) population, and ii) A limited medium or serum-free medium comprising optionally (optionally recombinant) transferrin, optionally recombinant insulin, and optionally recombinant albumin.

[0263] In some embodiments, the restricted medium or serum-free medium comprises (optionally recombinant) transferrin, (optionally recombinant) insulin, and (optionally recombinant) albumin.

[0264] In some embodiments, the limited medium or serum-free medium comprises a basal cell medium, as well as serum supplements and / or serum substitutes.

[0265] In some embodiments, the basal cell medium includes, but is not limited to, CTS® OpTmizer® T-cell Expansion Basal Medium, CTS® OpTmizer® T-Cell Expansion SFM, CTS® AIM-V Medium, CTS® AIM-V SFM, LymphoONE® T-Cell Expansion Xeno-Free Medium, Dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), Basal Medium Eagle (BME), RPMI 1640, F-10, F-12, Minimal Essential Medium (αMEM), Glasgow's Minimal Essential Medium (G-MEM), RPMI Growth Medium, and Iscove's Modified Dulbecco's Medium.

[0266] In some embodiments, the serum supplement or serum replacement is selected from the group consisting of CTS® OpTmizerT-Cell Expansion Serum Supplement and CTS® Immune Cell Serum Replacement.

[0267] In some embodiments, the limited medium or serum-free medium comprises one or more albumins or albumin substitutes.

[0268] In some embodiments, the limited medium or serum-free medium contains one or more amino acids.

[0269] In some embodiments, the limited medium or serum-free medium comprises one or more vitamins, one or more transferrins, or transferrin substitutes.

[0270] In some embodiments, the limited medium or serum-free medium comprises one or more antioxidants, one or more insulins, or insulin substitutes.

[0271] In some embodiments, the limited medium or serum-free medium comprises one or more collagen precursors, one or more antibiotics, and one or more trace elements.

[0272] In some embodiments, the limited medium or serum-free medium contains albumin.

[0273] In some embodiments, the limited medium or serum-free medium contains 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+ Ni 2+ , Rb + Sn 2+ and Zr 4+ It comprises one or more components selected from the group consisting of compounds containing

[0274] In some embodiments, the limited medium or serum-free medium further comprises L-glutamine, sodium bicarbonate, and / or 2-mercaptoethanol.

[0275] In some embodiments, the limited medium or serum-free medium contains a total serum substitution concentration (vol%) of approximately 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 cell culture medium.

[0276] In some embodiments, the restricted medium or serum-free medium contains a total serum substitute concentration of about 3%, about 5%, or about 10% of the total cell culture medium volume.

[0277] In some embodiments, the limited medium or serum-free medium further comprises glutamine (i.e., GlutaMAX®) at concentrations of about 0.1 mM to about 10 mM, 0.5 mM to about 9 mM, 1 mM to about 8 mM, 2 mM to about 7 mM, 3 mM to about 6 mM, or 4 mM to about 5 mM.

[0278] In some embodiments, the limited medium or serum-free medium further comprises glutamine (i.e., GlutaMAX®) at a concentration of about 2 mM.

[0279] In some embodiments, the limited medium or serum-free medium further comprises 2-mercaptoethanol at concentrations of approximately 5 mM to approximately 150 mM, 10 mM to approximately 140 mM, 15 mM to approximately 130 mM, 20 mM to approximately 120 mM, 25 mM to approximately 110 mM, 30 mM to approximately 100 mM, 35 mM to approximately 95 mM, 40 mM to approximately 90 mM, 45 mM to approximately 85 mM, 50 mM to approximately 80 mM, 55 mM to approximately 75 mM, 60 mM to approximately 70 mM, or approximately 65 mM.

[0280] In some embodiments, the limited medium or serum-free medium further comprises 2-mercaptoethanol at a concentration of about 55 mM.

[0281] In some embodiments, the limited medium or serum-free medium includes the limited medium described in International PCT Publication No. WO / 1998 / 030679.

[0282] In some embodiments, the limited medium or serum-free medium contains glycine in the range of approximately 5–200 mg / L, L-histidine in the range of approximately 5–250 mg / L, L-isoleucine in the range of approximately 5–300 mg / L, L-methionine in the range of approximately 5–200 mg / L, L-phenylalanine in the range of approximately 5–400 mg / L, L-proline in the range of approximately 1–1000 mg / L, L-hydroxyproline in the range of approximately 1–45 mg / L, L-serine in the range of approximately 1–250 mg / L, L-threonine in the range of approximately 10–500 mg / L, and L-tryptopha in the range of approximately 2–110 mg / L. It contains L-tyrosine in the range of approximately 3 to 175 mg / L, L-valine in the range of approximately 5 to 500 mg / L, thiamine in the range of approximately 1 to 20 mg / L, reduced glutathione in the range of approximately 1 to 20 mg / L, L-ascorbic acid-2-phosphate in the range of approximately 1 to 200 mg / L, iron-saturated transferrin in the range of approximately 1 to 50 mg / L, insulin in the range of approximately 1 to 100 mg / L, sodium selenite in the range of approximately 0.000001 to 0.0001 mg / L, and / or albumin (e.g., AlbumX® I) in the range of approximately 5,000 to 50,000 mg / L.

[0283] In some embodiments, the limited medium or serum-free medium containing one or more non-trace element components is available in the concentration ranges listed in the column under the heading "Concentration Ranges in 1X Medium" in Table 4 provided herein.

[0284] In some embodiments, the volumetric osmolality of the limited medium or serum-free medium is approximately 260–350 mOsmol.

[0285] In some embodiments, the limited medium or serum-free medium further contains about 3.7 g / L or about 2.2 g / L of sodium bicarbonate.

[0286] In some embodiments, the limited medium or serum-free medium further comprises L-glutamine (final concentration about 2 mM), one or more antibiotics, non-essential amino acids (NEAAs; final concentration about 100 μM), and / or 2-mercaptoethanol (final concentration about 100 μM).

[0287] In some embodiments, the limited medium or serum-free medium in the first and / or second gas-permeable vessel does not contain beta-mercaptoethanol (BME or βME; also known as 2-mercaptoethanol, CAS 60-24-2).

[0288] In some embodiments, the cell culture medium comprises CTS OpTmizer T-Cell Expansion SFM, 3% CTS Immune Cell Serum Replacement, 55 mM BME, and optionally glutamine.

[0289] In some embodiments, the cell culture medium comprises CTS® OpTmizer® T-cell Expansion Basal Medium (26 mL / L) supplemented with CTS® OpTmizer® T-cell Expansion Supplement, 3% CTS® Immune Cell SR, and 2 mM Glutamax, and optionally further comprising 6,000 IU / mL of IL-2.

[0290] In some embodiments, the cell culture medium comprises CTS® OpTmizer® T-cell Expansion Basal Medium (26 mL / L) supplemented with CTS® OpTmizer® T-cell Expansion Supplement, 3% CTS® Immune Cell SR, and 2 mM Glutamax, and optionally further comprising 3,000 IU / mL of IL-2.

[0291] In some embodiments, the TIL population is a therapeutic TIL population.

[0292] In some embodiments, the therapeutic TIL population exhibits elevated serum IFN-γ levels, where the elevation of IFN-γ is greater than 200 pg / ml, greater than 250 pg / ml, greater than 300 pg / ml, greater than 350 pg / ml, greater than 400 pg / ml, greater than 450 pg / ml, greater than 500 pg / ml, greater than 550 pg / ml, greater than 600 pg / ml, greater than 650 pg / ml, greater than 700 pg / ml, greater than 750 pg / ml, greater than 800 pg / ml, greater than 850 pg / ml, greater than 900 pg / ml, greater than 950 pg / ml, or greater than 1000 pg / ml.

[0293] In some embodiments, the present invention provides a method for growing tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, the method being (a) Obtain a first TIL population from tumors resected from patients by processing tumor samples obtained from patients into multiple tumor fragments, or By processing tumor samples obtained from patients into tumor digests, a first TIL population is obtained from tumors resected from patients. (b) Optionally, adding tumor fragments or tumor digests to the closed system, (c) Performing a first proliferation or priming of the first proliferation by culturing the first TIL population in a cell culture medium containing IL-2, antigen-presenting cells (APCs), and optionally OKT-3, in order to produce a second TIL population, wherein the priming of the first proliferation is performed for approximately 5 to 9 days to obtain a second TIL population, and the first proliferation or priming of the first proliferation is optionally performed in a sealed container providing a first gas-permeable surface area, and the transition from step (b) to step (c) is optionally performed in a closed system without opening the system, (d) To produce a third TIL population, a rapid second growth is carried out by supplementing the cell culture medium of the second TIL population with additional IL-2, optionally OKT-3, and APC, wherein the second growth is carried out for approximately 1 to 5 days to obtain a third TIL population, and the second growth is optionally carried out in a sealed container providing a second gas-permeable surface area, and the transition from step (c) to step (d) is optionally carried out in a closed system without opening the system, thereby carrying out a rapid second growth. (e) Performing a third growth by dividing a third TIL population into a first group of TIL subpopulations, wherein each subpopulation of the first group of TIL subpopulations is seeded in a separate container, and a cell culture medium supplemented with IL-2, optionally OKT-3, and culture material is added to produce a second group of TIL subpopulations, the third growth is performed for approximately 4 to 8 days, and optionally each separate container is a closed container providing a third gas-permeable surface area, and the transition from step (d) to step (e) is performed without opening the system if optionally carried out in a closed system, the third growth is performed. (f) Collecting a second set of TIL subpopulations obtained from step (e), wherein the transition from step (e) to step (f) is performed without opening the system if it is performed arbitrarily in a closed system, and the collection of the second set of TIL subpopulations (g) Transferring the TIL subgroup collected from step (g) to one or more infusion bags, the transfer from step (f) to (g) being performed without opening the system if optionally performed in a closed system, is included in the transfer to the infusion bags.

[0294] In some embodiments, the present invention provides tumor-infiltrating lymphocyte (TIL) compositions comprising a population of therapeutic infiltrating lymphocytes (TILs), the TIL compositions being (a) Obtain a first TIL population from tumors resected from patients by processing tumor samples obtained from patients into multiple tumor fragments, or By processing tumor samples obtained from patients into tumor digests, a first TIL population is obtained from tumors resected from patients. (b) Optionally, adding tumor fragments or tumor digests to the closed system, (c) Performing a first proliferation or priming of the first proliferation by culturing the first TIL population in a cell culture medium containing IL-2, antigen-presenting cells (APCs), and optionally OKT-3, in order to produce a second TIL population, wherein the priming of the first proliferation is performed for approximately 5 to 9 days to obtain a second TIL population, and the first proliferation or priming of the first proliferation is optionally performed in a sealed container providing a first gas-permeable surface area, and the transition from step (b) to step (c) is optionally performed in a closed system without opening the system, (d) To produce a third TIL population, a rapid second growth is carried out by supplementing the cell culture medium of the second TIL population with additional IL-2, optionally OKT-3, and APC, wherein the second growth is carried out for approximately 1 to 5 days to obtain a third TIL population, and the second growth is optionally carried out in a sealed container providing a second gas-permeable surface area, and the transition from step (c) to step (d) is optionally carried out in a closed system without opening the system, thereby carrying out a rapid second growth. (e) Performing a third growth by dividing a third TIL population into a first group of TIL subpopulations, wherein each subpopulation of the first group of TIL subpopulations is seeded in a separate container, and a cell culture medium supplemented with IL-2, optionally OKT-3, and culture material is added to produce a second group of TIL subpopulations, the third growth is performed for approximately 4 to 8 days, and optionally each separate container is a closed container providing a third gas-permeable surface area, and the transition from step (d) to step (e) is performed without opening the system if optionally carried out in a closed system, the third growth is performed. (f) Collecting a second set of TIL subpopulations obtained from step (e), wherein the transition from step (e) to step (f) is performed without opening the system if it is performed arbitrarily in a closed system, and the collection of the second set of TIL subpopulations (g) The TIL subpopulation collected from step (g) is produced by a method comprising transferring to one or more infusion bags, wherein the transfer from step (f) to (g) is performed without opening the system if it is optionally performed in a closed system.

[0295] In some embodiments, the TIL composition is a cryopreserved composition, and the method further comprises (h) using a cryopreservation process to cryopreserve an infusion bag containing the TIL population taken from step (g).

[0296] In some embodiments, the present invention provides a method for treating a subject with cancer, the method comprising administering proliferated tumor-infiltrating lymphocytes (TILs), the administration of (a) Obtain a first TIL population from tumors resected from patients by processing tumor samples obtained from patients into multiple tumor fragments, or By processing tumor samples obtained from patients into tumor digests, a first TIL population is obtained from tumors resected from patients. (b) Optionally, adding tumor fragments or tumor digests to the closed system, (c) Performing a first proliferation or priming of the first proliferation by culturing the first TIL population in a cell culture medium containing IL-2, antigen-presenting cells (APCs), and optionally OKT-3, in order to produce a second TIL population, wherein the priming of the first proliferation is performed for approximately 5 to 9 days to obtain a second TIL population, and the first proliferation or priming of the first proliferation is optionally performed in a sealed container providing a first gas-permeable surface area, and the transition from step (b) to step (c) is optionally performed in a closed system without opening the system, (d) To produce a third TIL population, a rapid second growth is carried out by supplementing the cell culture medium of the second TIL population with additional IL-2, optionally OKT-3, and APC, wherein the second growth is carried out for approximately 1 to 5 days to obtain a third TIL population, and the second growth is optionally carried out in a sealed container providing a second gas-permeable surface area, and the transition from step (c) to step (d) is optionally carried out in a closed system without opening the system, thereby carrying out a rapid second growth. (e) Performing a third growth by dividing a third TIL population into a first group of TIL subpopulations, wherein each subpopulation of the first group of TIL subpopulations is seeded in a separate container, and a cell culture medium supplemented with IL-2, optionally OKT-3, and culture material is added to produce a second group of TIL subpopulations, the third growth is performed for approximately 4 to 8 days, and optionally each separate container is a closed container providing a third gas-permeable surface area, and the transition from step (d) to step (e) is performed without opening the system if optionally carried out in a closed system, the third growth is performed. (f) Collecting a second set of TIL subpopulations obtained from step (e), wherein the transition from step (e) to step (f) is performed without opening the system if it is performed arbitrarily in a closed system, and the collection of the second set of TIL subpopulations (g) Transferring the TIL subgroup collected from step (g) to one or more infusion bags, wherein the transition from step (f) to (g) is performed without opening the system if it is optionally performed in a closed system, and transferring to infusion bags. (h) administering a therapeutically effective dose to the third TIL population from the infusion bag of step (g), which includes the following:

[0297] In some embodiments, the first growth or priming of the first growth is carried out for about 6 to 8 days.

[0298] In some embodiments, rapid second growth is carried out for about 2 to 4 days.

[0299] In some embodiments, the third growth cycle is carried out for approximately 5 to 7 days.

[0300] In some embodiments, the first growth or priming of the first growth is carried out for about 7 days, the rapid second growth is carried out for about 3 days, and the third growth is carried out for about 6 days.

[0301] In some embodiments, steps (c) to (e) are carried out over approximately 14 to 18 days.

[0302] In some embodiments, steps (c) to (e) are carried out over approximately 16 days.

[0303] In some embodiments, steps (c) to (e) are carried out in approximately 18 days or less.

[0304] In some embodiments, steps (c) to (e) are carried out in approximately 16 days or less.

[0305] In some embodiments, step (e) is to divide each subgroup of the first plurality of TIL subgroups into approximately 2 × 10⁻¹⁶ subgroups. 6 cells / cm 2 This includes sowing at a seeding density in a separate container that provides a third gas-permeable surface area.

[0306] In some embodiments, cancer is selected from the group consisting of melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, triple-negative breast cancer, cancer derived from 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.

[0307] In some embodiments, the cancer is selected from the group consisting of melanoma, HNSCC, cervical cancer, NSCLC, glioblastoma (including GBM), and gastrointestinal cancer.

[0308] In some embodiments, the cancer is melanoma.

[0309] In some embodiments, the cancer is HNSCC.

[0310] In some embodiments, the cancer is cervical cancer.

[0311] In some embodiments, the cancer is NSCLC.

[0312] In some embodiments, the cancer is glioblastoma (including GBM).

[0313] In some embodiments, the cancer is gastrointestinal cancer.

[0314] In some embodiments, the cancer is a hypermutant cancer.

[0315] In some embodiments, the cancer is a pediatric hypermutant cancer.

[0316] 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 a first TIL population from a tumor resected from a patient by processing a tumor sample obtained from the patient into multiple tumor fragments, or obtaining a first TIL population from a tumor resected from a patient by processing a tumor sample into tumor digests, (b) Optionally, adding tumor fragments or tumor digests to the closed system, (c) Performing a first proliferation or priming of the first proliferation by culturing the first TIL population in a cell culture medium containing IL-2, antigen-presenting cells (APCs), and optionally OKT-3, in order to produce a second TIL population, wherein the priming of the first proliferation is performed for approximately 5 to 9 days to obtain a second TIL population, and the first proliferation or priming of the first proliferation is optionally performed in a sealed container providing a first gas-permeable surface area, and the transition from step (b) to step (c) is optionally performed in a closed system without opening the system, (d) To produce a third TIL population, a rapid second growth is carried out by supplementing the cell culture medium of the second TIL population with additional IL-2, optionally OKT-3, and APC, wherein the second growth is carried out for approximately 5 to 9 days to obtain a third TIL population, and the second growth is optionally carried out in a sealed container providing a second gas-permeable surface area, and the transition from step (c) to step (d) is optionally carried out in a closed system without opening the system, thereby carrying out a rapid second growth. (e) Performing a third growth by dividing a third TIL population into a first group of TIL subpopulations, wherein each subpopulation of the first group of TIL subpopulations is seeded in a separate container, and a cell culture medium supplemented with IL-2, optionally OKT-3, and culture material is added to produce a second group of TIL subpopulations, the third growth is performed for approximately 5-9 days, and optionally each separate container is a closed container providing a third gas-permeable surface area, and the transition from step (d) to step (e) is performed without opening the system if optionally carried out in a closed system, the third growth is performed. (f) Collecting a second set of TIL subpopulations obtained from step (f), wherein the transition from step (e) to step (f) is performed without opening the system if it is performed arbitrarily in a closed system, and the collection of the second set of TIL subpopulations (g) Transferring the TIL subgroup collected from step (f) to one or more infusion bags, the transfer from step (f) to (g) being performed without opening the system if optionally performed in a closed system, is included in the transfer to the infusion bags.

[0317] In some embodiments, the present invention provides tumor-infiltrating lymphocyte (TIL) compositions comprising a population of therapeutic infiltrating lymphocytes (TILs), the TIL compositions being (a) Obtaining a first TIL population from a tumor resected from a patient by processing a tumor sample obtained from the patient into multiple tumor fragments, or obtaining a first TIL population from a tumor resected from a patient by processing a tumor sample into tumor digests, (b) Optionally, adding tumor fragments or tumor digests to the closed system, (c) Performing a first proliferation or priming of the first proliferation by culturing the first TIL population in a cell culture medium containing IL-2, antigen-presenting cells (APCs), and optionally OKT-3, in order to produce a second TIL population, wherein the priming of the first proliferation is performed for approximately 5 to 9 days to obtain a second TIL population, and the first proliferation or priming of the first proliferation is optionally performed in a sealed container providing a first gas-permeable surface area, and the transition from step (b) to step (c) is optionally performed in a closed system without opening the system, (d) To produce a third TIL population, a rapid second growth is carried out by supplementing the cell culture medium of the second TIL population with additional IL-2, optionally OKT-3, and APC, wherein the second growth is carried out for approximately 5 to 9 days to obtain a third TIL population, and the second growth is optionally carried out in a sealed container providing a second gas-permeable surface area, and the transition from step (c) to step (d) is optionally carried out in a closed system without opening the system, thereby carrying out a rapid second growth. (e) Performing a third growth by dividing a third TIL population into a first group of TIL subpopulations, wherein each subpopulation of the first group of TIL subpopulations is seeded in a separate container, and a cell culture medium supplemented with IL-2, optionally OKT-3, and culture material is added to produce a second group of TIL subpopulations, the third growth is performed for approximately 5-9 days, and optionally each separate container is a closed container providing a third gas-permeable surface area, and the transition from step (d) to step (e) is performed without opening the system if optionally carried out in a closed system, the third growth is performed. (f) Collecting a second set of TIL subpopulations obtained from step (f), wherein the transition from step (e) to step (f) is performed without opening the system if it is performed arbitrarily in a closed system, and the collection of the second set of TIL subpopulations (g) The TIL subpopulation collected from step (f) is produced by transferring it to one or more infusion bags, the transfer from step (f) to (g) being carried out without opening the system if optionally performed in a closed system.

[0318] In some embodiments, the TIL composition is a cryopreserved composition, and the method further comprises (h) using a cryopreservation process to cryopreserve an infusion bag containing the TIL population taken from step (g).

[0319] In some embodiments, the present invention provides a method for treating a subject with cancer, the method comprising administering proliferated tumor-infiltrating lymphocytes (TILs), the administration of (a) Obtaining a first TIL population from a tumor resected from a patient by processing a tumor sample obtained from the patient into multiple tumor fragments, or obtaining a first TIL population from a tumor resected from a patient by processing a tumor sample into tumor digests, (b) Optionally, adding tumor fragments or tumor digests to the closed system, (c) Performing a first proliferation or priming of the first proliferation by culturing the first TIL population in a cell culture medium containing IL-2, antigen-presenting cells (APCs), and optionally OKT-3, in order to produce a second TIL population, wherein the priming of the first proliferation is performed for approximately 5 to 9 days to obtain a second TIL population, and the first proliferation or priming of the first proliferation is optionally performed in a sealed container providing a first gas-permeable surface area, and the transition from step (b) to step (c) is optionally performed in a closed system without opening the system, (d) To produce a third TIL population, a rapid second growth is carried out by supplementing the cell culture medium of the second TIL population with additional IL-2, optionally OKT-3, and APC, wherein the second growth is carried out for approximately 5 to 9 days to obtain a third TIL population, and the second growth is optionally carried out in a sealed container providing a second gas-permeable surface area, and the transition from step (c) to step (d) is optionally carried out in a closed system without opening the system, thereby carrying out a rapid second growth. (e) Performing a third growth by dividing a third TIL population into a first group of TIL subpopulations, wherein each subpopulation of the first group of TIL subpopulations is seeded in a separate container, and a cell culture medium supplemented with IL-2, optionally OKT-3, and culture material is added to produce a second group of TIL subpopulations, the third growth is performed for approximately 5-9 days, and optionally each separate container is a closed container providing a third gas-permeable surface area, and the transition from step (d) to step (e) is performed without opening the system if optionally carried out in a closed system, the third growth is performed. (f) Collecting a second set of TIL subpopulations obtained from step (f), wherein the transition from step (e) to step (f) is performed without opening the system if it is performed arbitrarily in a closed system, and the collection of the second set of TIL subpopulations (g) Transferring the TIL subgroup collected from step (f) to one or more infusion bags, the transfer from step (f) to (g) being performed without opening the system if the system is optionally closed, to an infusion bag. (h) administering a therapeutically effective dose to the third TIL population from the infusion bag of step (g), which includes the following:

[0320] In some embodiments, in step (a), a tumor sample obtained from a patient is processed into multiple tumor fragments by (i) freezing the tumor sample to produce a frozen tumor sample, (ii) thawing the frozen tumor sample to produce a thawed tumor sample, and (iii) fragmenting the thawed tumor sample into multiple tumor fragments.

[0321] In some embodiments, in step (a), the tumor sample obtained from the patient is processed into a tumor digest by (i) freezing the tumor sample to produce a frozen tumor sample, (ii) thawing the frozen tumor sample to produce a thawed tumor sample, and (iii) digesting the thawed tumor sample to produce a tumor digest.

[0322] In some embodiments, in step (a), the tumor sample obtained from the patient is processed into a tumor digest by: (i) cryopreserving the tumor sample to generate a cryopreserved tumor sample; (ii) thawing the cryopreserved tumor sample to produce a thawed tumor sample; (iii) fragmenting the thawed tumor sample into a plurality of tumor fragments; and (iv) digesting the plurality of tumor fragments to produce a tumor digest.

[0323] In some embodiments, step (e) includes seeding each subpopulation of the first plurality of TIL subpopulations at a seeding density of about 2×10 6 cells / cm 2 into a separate container providing a third gas-permeable surface area.

[0324] In some embodiments, the cancer is selected from the group consisting of melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, triple-negative breast cancer, human papillomavirus-derived cancer, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), glioblastoma (including GBM), gastrointestinal cancer, renal cancer, and renal cell carcinoma.

[0325] In some embodiments, the cancer is selected from the group consisting of melanoma, HNSCC, cervical cancer, NSCLC, glioblastoma (including GBM), and gastrointestinal cancer.

[0326] In some embodiments, the cancer is melanoma.

[0327] In some embodiments, the cancer is HNSCC.

[0328] In some embodiments, the cancer is cervical cancer.

[0329] In some embodiments, the cancer is NSCLC.

[0330] In some embodiments, the cancer is glioblastoma (including GBM).

[0331] In some embodiments, the cancer is gastrointestinal cancer.

[0332] In some embodiments, the cancer is a hypermutant cancer.

[0333] In some embodiments, the cancer is a pediatric hypermutant cancer.

[0334] In some embodiments, the present invention provides a method for growing tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, the method being (a)(i) Thawing a cryopreserved tumor digest containing a first TIL population from a tumor that has been cut from a subject, digested after cutting, and cryopreserved after digestion; and (ii) Performing a first proliferation or priming of a first proliferation by culturing the first TIL population in a cell culture medium containing IL-2, antigen-presenting cells (APCs), and optionally OKT-3, thereby producing a second TIL population, wherein the first proliferation or priming of a first proliferation is performed for approximately 5 to 9 days to obtain a second TIL population, and the first proliferation or priming of a first proliferation is optionally performed in a sealed container providing a first gas-permeable surface area. (b) To produce a third TIL population, a rapid second growth is carried out by supplementing the cell culture medium of the second TIL population with additional IL-2, APC, and optionally OKT-3, wherein the second growth is carried out for approximately 5 to 9 days to obtain a third TIL population, and the second growth is optionally carried out in a sealed container providing a second gas-permeable surface area, and the transition from step (a) to step (b) is optionally carried out in a closed system without opening the system, thereby carrying out a rapid second growth. (c) Performing a third growth by dividing a third TIL population into a first plurality of TIL subpopulations, wherein each subpopulation of the first plurality of TIL subpopulations is seeded in a separate container, and a cell culture medium supplemented with IL-2, optionally OKT-3, and culture material is added to produce a second plurality of TIL subpopulations, the third growth is performed for approximately 5 to 9 days, and optionally each separate container is a closed container providing a third gas-permeable surface area, and the transition from step (b) to step (c) is performed without opening the system if optionally carried out in a closed system, the third growth is performed. (d) Collecting a second group of TIL subpopulations obtained from step (c), wherein the transition from step (d) to step (e) is performed without opening the system if it is performed arbitrarily in a closed system, and the collection of the second group of TIL subpopulations (e) Transferring the TIL subgroup collected from step (d) to one or more infusion bags, the transfer from step (d) to (e) being performed without opening the system if optionally performed in a closed system, is included in the transfer to the infusion bags.

[0335] In some embodiments, the present invention provides tumor-infiltrating lymphocyte (TIL) compositions comprising a population of therapeutic infiltrating lymphocytes (TILs), the TIL compositions being (a)(i) Thawing a cryopreserved tumor containing a first TIL population from a tumor that has been cut from a subject and cryopreserved after cutting, and (ii) Culturing the first TIL population in a cell culture medium containing IL-2, antigen-presenting cells (APCs), and optionally OKT-3 to produce a second TIL population, wherein the first growth or priming of the first growth is carried out for approximately 5 to 9 days to obtain a second TIL population, and the first growth or priming of the first growth is optionally carried out in a sealed container providing a first gas-permeable surface area, (b) To produce a third TIL population, a rapid second growth is carried out by supplementing the cell culture medium of the second TIL population with additional IL-2, APC, and optionally OKT-3, wherein the second growth is carried out for approximately 5-9 days to obtain a third TIL population, and the second growth is optionally carried out in a sealed container providing a second gas-permeable surface area, and the transition from step (a) to step (b) is optionally carried out in a closed system without opening the system, thereby carrying out a rapid second growth. (c) Performing a third growth by dividing a third TIL population into a first plurality of TIL subpopulations, wherein each subpopulation of the first plurality of TIL subpopulations is seeded in a separate container, and a cell culture medium supplemented with IL-2, optionally OKT-3, and culture material is added to produce a second plurality of TIL subpopulations, the third growth is performed for approximately 5 to 9 days, and optionally each separate container is a closed container providing a third gas-permeable surface area, and the transition from step (b) to step (c) is performed without opening the system if optionally carried out in a closed system, the third growth is performed. (d) Collecting a second set of TIL subpopulations obtained from step (c), wherein the transition from step (c) to step (d) is performed without opening the system if it is performed arbitrarily in a closed system, and the collection of the second set of TIL subpopulations (e) Transferring the TIL subpopulation collected from step (d) to one or more infusion bags, the transfer from step (d) to (e) being carried out without opening the system if optionally performed in a closed system, is produced by a method including the transfer to infusion bags.

[0336] In some embodiments, the TIL composition is a cryopreserved composition, and the method further comprises (f) using a cryopreservation process to cryopreserve an infusion bag containing the TIL population taken from step (e).

[0337] In some embodiments, the present invention provides a method for treating a subject with cancer, the method comprising administering proliferated tumor-infiltrating lymphocytes (TILs), the administration of (a)(i) Thawing a cryopreserved tumor digest containing a first TIL population from a tumor that has been cut from a subject, digested after cutting, and cryopreserved after digestion; and (ii) Performing a first proliferation or priming of a first proliferation by culturing the first TIL population in a cell culture medium containing IL-2, and optionally OKT-3, and antigen-presenting cells (APCs) to produce a second TIL population, wherein the first proliferation or priming of a first proliferation is performed for approximately 5 to 9 days to obtain a second TIL population, and the first proliferation or priming of a first proliferation is optionally performed in a sealed container providing a first gas-permeable surface area. (b) To produce a third TIL population, a rapid second growth is carried out by supplementing the cell culture medium of the second TIL population with additional IL-2, optionally OKT-3, and APC, wherein the second growth is carried out for approximately 5 to 9 days to obtain a third TIL population, and the second growth is optionally carried out in a sealed container providing a second gas-permeable surface area, and the transition from step (a) to step (b) is optionally carried out in a closed system without opening the system, thereby carrying out a rapid second growth. (c) Performing a third growth by dividing a third TIL population into a first plurality of TIL subpopulations, wherein each subpopulation of the first plurality of TIL subpopulations is seeded in a separate container, and a cell culture medium supplemented with IL-2, optionally OKT-3, and culture material is added to produce a second plurality of TIL subpopulations, the third growth is performed for approximately 5 to 9 days, and optionally each separate container is a closed container providing a third gas-permeable surface area, and the transition from step (b) to step (c) is performed without opening the system if optionally carried out in a closed system, the third growth is performed. (d) Collecting a second group of TIL subpopulations obtained from step (c), wherein the transition from step (d) to step (e) is performed without opening the system if it is performed arbitrarily in a closed system, and the collection of the second group of TIL subpopulations (e) Transferring the TIL subgroup collected from step (d) to one or more infusion bags, the transfer from step (d) to (e) being performed without opening the system if the system is optionally closed, to an infusion bag. (f) administering a therapeutically effective dose to a third TIL population from the infusion bag of step (g), which includes:

[0338] In some embodiments, step (a)(i) comprises thawing a cryopreserved tumor containing a first TIL population from a tumor excised from a subject and cryopreserved after excision in order to produce a thawed tumor, and fragmenting the thawed tumor into a plurality of tumors, and (a)(ii) comprises culturing the plurality of tumor fragments containing the first TIL population.

[0339] In some embodiments, step (c) divides each subgroup of the first plurality of TIL subgroups into approximately 2 × 10 6 cells / cm 2 This includes sowing at a seeding density in a separate container that provides a third gas-permeable surface area.

[0340] In some embodiments, the first growth or priming of the first growth is carried out for about 6 to 8 days.

[0341] In some embodiments, rapid second growth is carried out for approximately 6–8 days.

[0342] In some embodiments, the third growth cycle is carried out for approximately 6 to 8 days.

[0343] In some embodiments, the first growth or priming first growth, rapid second growth, and third growth are carried out over approximately 18 to 24 days.

[0344] In some embodiments, the first growth or priming first growth, rapid second growth, and third growth are carried out over approximately 20-22 days.

[0345] In some embodiments, the first growth or priming first growth, rapid second growth, and third growth are carried out over approximately 21 days.

[0346] In some embodiments, the first growth or priming first growth, rapid second growth, and third growth are carried out within approximately 24 days or less.

[0347] In some embodiments, the first growth or priming first growth, rapid second growth, and third growth are carried out within approximately 22 days or less.

[0348] In some embodiments, the first growth or priming first growth, rapid second growth, and third growth are carried out within approximately 21 days or less.

[0349] In some embodiments, cancer is selected from the group consisting of melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, triple-negative breast cancer, cancer derived from 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.

[0350] In some embodiments, the cancer is selected from the group consisting of melanoma, HNSCC, cervical cancer, NSCLC, glioblastoma (including GBM), and gastrointestinal cancer.

[0351] In some embodiments, the cancer is melanoma.

[0352] In some embodiments, the cancer is HNSCC.

[0353] In some embodiments, the cancer is cervical cancer.

[0354] In some embodiments, the cancer is NSCLC.

[0355] In some embodiments, the cancer is glioblastoma (including GBM).

[0356] In some embodiments, the cancer is gastrointestinal cancer.

[0357] In some embodiments, the cancer is a hypermutant cancer.

[0358] In some embodiments, the cancer is a pediatric hypermutant cancer.

[0359] In some embodiments, the present invention provides a method for growing tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, the method being (a) Performing a first proliferation or priming of a first proliferation by culturing a tumor sample containing a first TIL population cleaved from a patient in a cell culture medium containing IL-2, antigen-presenting cells (APCs), and optionally OKT-3, wherein the first proliferation priming is performed for approximately 5 to 9 days to obtain a second TIL population, and the first proliferation or priming of a first proliferation is optionally performed in a sealed container providing a first gas-permeable surface area. (b) To produce a third TIL population, a rapid second growth is carried out by supplementing the cell culture medium of the second TIL population with additional IL-2, optionally OKT-3, and APC, wherein the second growth is carried out for approximately 1 to 5 days to obtain a third TIL population, and the second growth is optionally carried out in a sealed container providing a second gas-permeable surface area, and the transition from step (a) to step (b) is optionally carried out in a closed system without opening the system, thereby carrying out a rapid second growth. (c) Performing a third growth by dividing a third TIL population into a first plurality of TIL subpopulations, wherein each subpopulation of the first plurality of TIL subpopulations is seeded in a separate container, and a cell culture medium supplemented with IL-2, optionally OKT-3, and culture material is added to produce a second plurality of TIL subpopulations, the third growth is performed for approximately 4 to 8 days, and optionally each separate container is a closed container providing a third gas-permeable surface area, and the transition from step (b) to step (c) is performed without opening the system if optionally carried out in a closed system, the third growth is performed. (d) Collecting a second set of TIL subpopulations obtained from step (c), wherein the transition from step (c) to step (d) is performed without opening the system if it is performed arbitrarily in a closed system, and the collection of the second set of TIL subpopulations (e) Transferring the TIL subgroup collected from step (d) to one or more infusion bags, the transfer from step (d) to (e) being performed without opening the system if optionally performed in a closed system, is included in the transfer to the infusion bags.

[0360] In some embodiments, the present invention provides tumor-infiltrating lymphocyte (TIL) compositions comprising a therapeutic population of TILs, the TIL compositions being (a) Performing a first proliferation or priming of a first proliferation by culturing a tumor sample containing a first TIL population cleaved from a patient in a cell culture medium containing IL-2, antigen-presenting cells (APCs), and optionally OKT-3, wherein the first proliferation priming is performed for approximately 5 to 9 days to obtain a second TIL population, and the first proliferation or priming of a first proliferation is optionally performed in a sealed container providing a first gas-permeable surface area. (b) To produce a third TIL population, a rapid second growth is carried out by supplementing the cell culture medium of the second TIL population with additional IL-2, optionally OKT-3, and APC, wherein the second growth is carried out for approximately 1 to 5 days to obtain a third TIL population, and the second growth is optionally carried out in a sealed container providing a second gas-permeable surface area, and the transition from step (a) to step (b) is optionally carried out in a closed system without opening the system, thereby carrying out a rapid second growth. (c) Performing a third growth by dividing a third TIL population into a first plurality of TIL subpopulations, wherein each subpopulation of the first plurality of TIL subpopulations is seeded in a separate container, and a cell culture medium supplemented with IL-2, optionally OKT-3, and culture material is added to produce a second plurality of TIL subpopulations, the third growth is performed for approximately 4 to 8 days, and optionally each separate container is a closed container providing a third gas-permeable surface area, and the transition from step (b) to step (c) is performed without opening the system if optionally carried out in a closed system, the third growth is performed. (d) Collecting a second set of TIL subpopulations obtained from step (c), wherein the transition from step (c) to step (d) is performed without opening the system if it is performed arbitrarily in a closed system, and the collection of the second set of TIL subpopulations (e) Transferring the TIL subpopulation collected from step (d) to one or more infusion bags, the transfer from step (d) to (e) being carried out without opening the system if optionally performed in a closed system, is produced by a method including the transfer to infusion bags.

[0361] In some embodiments, the TIL composition is a cryopreserved composition, and the method further comprises (f) using a cryopreservation process to cryopreserve an infusion bag containing the TIL population taken from step (e).

[0362] In some embodiments, the present invention provides a method for treating a subject with cancer, the method comprising administering proliferated tumor-infiltrating lymphocytes (TILs), the administration of (a) Performing a first proliferation or priming of a first proliferation by culturing a tumor sample containing a first TIL population cleaved from a patient in a cell culture medium containing IL-2, antigen-presenting cells (APCs), and optionally OKT-3, wherein the first proliferation priming is performed for approximately 5 to 9 days to obtain a second TIL population, and the first proliferation or priming of a first proliferation is optionally performed in a sealed container providing a first gas-permeable surface area. (b) To produce a third TIL population, a rapid second growth is carried out by supplementing the cell culture medium of the second TIL population with additional IL-2, optionally OKT-3, and APC, wherein the second growth is carried out for approximately 1 to 5 days to obtain a third TIL population, and the second growth is optionally carried out in a sealed container providing a second gas-permeable surface area, and the transition from step (a) to step (b) is optionally carried out in a closed system without opening the system, thereby carrying out a rapid second growth. (c) Performing a third growth by dividing a third TIL population into a first plurality of TIL subpopulations, wherein each subpopulation of the first plurality of TIL subpopulations is seeded in a separate container, and a cell culture medium supplemented with IL-2, optionally OKT-3, and culture material is added to produce a second plurality of TIL subpopulations, the third growth is performed for approximately 4 to 8 days, and optionally each separate container is a closed container providing a third gas-permeable surface area, and the transition from step (b) to step (c) is performed without opening the system if optionally carried out in a closed system, the third growth is performed. (d) Collecting a second set of TIL subpopulations obtained from step (c), wherein the transition from step (c) to step (d) is performed without opening the system if it is performed arbitrarily in a closed system, and the collection of the second set of TIL subpopulations (e) Transferring the TIL subgroup collected from step (d) to one or more infusion bags, the transfer from step (d) to (e) being performed without opening the system if the system is optionally closed, to an infusion bag. (f) administering a therapeutically effective dose to a third TIL population from the infusion bag of step (e), which includes:

[0363] In some embodiments, prior to culturing in step (a), the tumor sample is fragmented into multiple tumor fragments containing a first TIL population.

[0364] In some embodiments, the tumor sample is digested before culturing in step (a) to produce a tumor digest containing a first TIL population.

[0365] In some embodiments, the first growth or priming of the first growth is carried out for about 6 to 8 days.

[0366] In some embodiments, rapid second growth is carried out for about 2 to 4 days.

[0367] In some embodiments, the third growth cycle is carried out for approximately 5 to 7 days.

[0368] In some embodiments, the first growth or priming of the first growth is carried out for about 7 days, the rapid second growth is carried out for about 3 days, and the third growth is carried out for about 6 days.

[0369] In some embodiments, steps (a) to (c) are carried out over approximately 14 to 18 days.

[0370] In some embodiments, steps (a) to (c) are carried out over approximately 16 days.

[0371] In some embodiments, steps (a) to (c) are carried out in approximately 18 days or less.

[0372] In some embodiments, steps (a) to (c) are carried out in approximately 16 days or less.

[0373] In some embodiments, step (c) divides each subgroup of the first plurality of TIL subgroups into approximately 2 × 10 6 cells / cm 2 This includes sowing at a seeding density in a separate container that provides a third gas-permeable surface area.

[0374] In some embodiments, cancer is selected from the group consisting of melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, triple-negative breast cancer, cancer derived from 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.

[0375] In some embodiments, the cancer is selected from the group consisting of melanoma, HNSCC, cervical cancer, NSCLC, glioblastoma (including GBM), and gastrointestinal cancer.

[0376] In some embodiments, the cancer is melanoma.

[0377] In some embodiments, the cancer is HNSCC.

[0378] In some embodiments, the cancer is cervical cancer.

[0379] In some embodiments, the cancer is NSCLC.

[0380] In some embodiments, the cancer is glioblastoma (including GBM).

[0381] In some embodiments, the cancer is gastrointestinal cancer.

[0382] In some embodiments, the cancer is a hypermutant cancer.

[0383] In some embodiments, the cancer is a pediatric hypermutant cancer.

[0384] In some embodiments, the present invention provides a method for growing tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, the method being (a) Performing a first proliferation or priming of a first proliferation by culturing a tumor sample containing a first TIL population cleaved from a patient in a cell culture medium containing IL-2, antigen-presenting cells (APCs), and optionally OKT-3, wherein the first proliferation priming is performed for approximately 5 to 9 days to obtain a second TIL population, and the first proliferation or priming of a first proliferation is optionally performed in a sealed container providing a first gas-permeable surface area. (b) To produce a third TIL population, a rapid second growth is carried out by supplementing the cell culture medium of the second TIL population with additional IL-2, APC, and optionally OKT-3, wherein the second growth is carried out for approximately 5 to 9 days to obtain a third TIL population, and the second growth is optionally carried out in a sealed container providing a second gas-permeable surface area, and the transition from step (a) to step (b) is optionally carried out in a closed system without opening the system, thereby carrying out a rapid second growth. (c) Performing a third growth by dividing a third TIL population into a first plurality of TIL subpopulations, wherein each subpopulation of the first plurality of TIL subpopulations is seeded in a separate container, and a cell culture medium supplemented with IL-2, optionally OKT-3, and culture material is added to produce a second plurality of TIL subpopulations, the third growth is performed for approximately 5 to 9 days, and optionally each separate container is a closed container providing a third gas-permeable surface area, and the transition from step (b) to step (c) is performed without opening the system if optionally carried out in a closed system, the third growth is performed. (d) Collecting a second set of TIL subpopulations obtained from step (f), wherein the transition from step (c) to step (d) is performed without opening the system if it is performed arbitrarily in a closed system, and the collection of the second set of TIL subpopulations (e) Transferring the TIL subgroup collected from step (d) to one or more infusion bags, the transfer from step (d) to (e) being performed without opening the system if optionally performed in a closed system, is included in the transfer to the infusion bags.

[0385] In some embodiments, the present invention provides tumor-infiltrating lymphocyte (TIL) compositions comprising a population of therapeutic infiltrating lymphocytes (TILs), the TIL compositions being (a) Performing a first proliferation or priming of a first proliferation by culturing a tumor sample containing a first TIL population cleaved from a patient in a cell culture medium containing IL-2, antigen-presenting cells (APCs), and optionally OKT-3, wherein the first proliferation priming is performed for approximately 5 to 9 days to obtain a second TIL population, and the first proliferation or priming of a first proliferation is optionally performed in a sealed container providing a first gas-permeable surface area. (b) To produce a third TIL population, a rapid second growth is carried out by supplementing the cell culture medium of the second TIL population with additional IL-2, APC, and optionally OKT-3, wherein the second growth is carried out for approximately 5 to 9 days to obtain a third TIL population, and the second growth is optionally carried out in a sealed container providing a second gas-permeable surface area, and the transition from step (a) to step (b) is optionally carried out in a closed system without opening the system, thereby carrying out a rapid second growth. (c) Performing a third growth by dividing a third TIL population into a first plurality of TIL subpopulations, wherein each subpopulation of the first plurality of TIL subpopulations is seeded in a separate container, and a cell culture medium supplemented with IL-2, optionally OKT-3, and culture material is added to produce a second plurality of TIL subpopulations, the third growth is performed for approximately 5 to 9 days, and optionally each separate container is a closed container providing a third gas-permeable surface area, and the transition from step (b) to step (c) is performed without opening the system if optionally carried out in a closed system, the third growth is performed. (d) Collecting a second set of TIL subpopulations obtained from step (f), wherein the transition from step (c) to step (d) is performed without opening the system if it is performed arbitrarily in a closed system, and the collection of the second set of TIL subpopulations (e) Transferring the TIL subpopulation collected from step (d) to one or more infusion bags, the transfer from step (d) to (e) being carried out without opening the system if optionally performed in a closed system, is produced by a method including the transfer to infusion bags.

[0386] In some embodiments, the TIL composition is a cryopreserved composition, and the method further comprises (f) using a cryopreservation process to cryopreserve an infusion bag containing the TIL population taken from step (e).

[0387] In some embodiments, the present invention provides a method for treating a subject with cancer, the method comprising administering proliferated tumor-infiltrating lymphocytes (TILs), the administration of (a) Performing a first proliferation or priming of a first proliferation by culturing a tumor sample containing a first TIL population cleaved from a patient in a cell culture medium containing IL-2, antigen-presenting cells (APCs), and optionally OKT-3, wherein the priming of the first proliferation is performed for about 5 to 9 days to obtain a second TIL population, and the priming of the first proliferation or priming of a first proliferation is optionally performed in a sealed container providing a first gas-permeable surface area. (b) To produce a third TIL population, a rapid second growth is carried out by supplementing the cell culture medium of the second TIL population with additional IL-2, APC, and optionally OKT-3, wherein the second growth is carried out for approximately 5 to 9 days to obtain a third TIL population, and the second growth is optionally carried out in a sealed container providing a second gas-permeable surface area, and the transition from step (a) to step (b) is optionally carried out in a closed system without opening the system, thereby carrying out a rapid second growth. (c) Performing a third growth by dividing a third TIL population into a first plurality of TIL subpopulations, wherein each subpopulation of the first plurality of TIL subpopulations is seeded in a separate container, and a cell culture medium supplemented with IL-2, optionally OKT-3, and culture material is added to produce a second plurality of TIL subpopulations, the third growth is performed for approximately 5 to 9 days, and optionally each separate container is a closed container providing a third gas-permeable surface area, and the transition from step (b) to step (c) is performed without opening the system if optionally carried out in a closed system, the third growth is performed. (d) Collecting a second set of TIL subpopulations obtained from step (f), wherein the transition from step (c) to step (d) is performed without opening the system if it is performed arbitrarily in a closed system, and the collection of the second set of TIL subpopulations (e) Transferring the TIL subgroup collected from step (d) to one or more infusion bags, the transfer from step (d) to (e) being performed without opening the system if the system is optionally closed, to an infusion bag. (f) administering a therapeutically effective dose to a third TIL population from the infusion bag of step (e), which includes:

[0388] In some embodiments, prior to culturing in step (a), the tumor sample is fragmented into multiple tumor fragments containing a first TIL population.

[0389] In some embodiments, the tumor sample is digested before culturing in step (a) to produce a tumor digest containing a first TIL population.

[0390] In some embodiments, the first growth or priming of the first growth is carried out for about 6 to 8 days.

[0391] In some embodiments, rapid second growth is carried out for approximately 6–8 days.

[0392] In some embodiments, the third growth cycle is carried out for approximately 6 to 8 days.

[0393] In some embodiments, the first growth or priming first growth, rapid second growth, and third growth are carried out over approximately 18 to 24 days.

[0394] In some embodiments, the first growth or priming first growth, rapid second growth, and third growth are carried out over approximately 20-22 days.

[0395] In some embodiments, the first growth or priming first growth, rapid second growth, and third growth are carried out over approximately 21 days.

[0396] In some embodiments, the first growth or priming first growth, rapid second growth, and third growth are carried out within approximately 24 days or less.

[0397] In some embodiments, the first growth or priming first growth, rapid second growth, and third growth are carried out within approximately 22 days or less.

[0398] In some embodiments, the first growth or priming first growth, rapid second growth, and third growth are carried out within approximately 21 days or less.

[0399] In some embodiments, step (c) divides each subgroup of the first plurality of TIL subgroups into approximately 2 × 10 6 cells / cm 2 This includes sowing at a seeding density in a separate container that provides a third gas-permeable surface area.

[0400] In some embodiments, cancer is selected from the group consisting of melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, triple-negative breast cancer, cancer derived from 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.

[0401] In some embodiments, the cancer is selected from the group consisting of melanoma, HNSCC, cervical cancer, NSCLC, glioblastoma (including GBM), and gastrointestinal cancer.

[0402] In some embodiments, the cancer is melanoma.

[0403] In some embodiments, the cancer is HNSCC.

[0404] In some embodiments, the cancer is cervical cancer.

[0405] In some embodiments, the cancer is NSCLC.

[0406] In some embodiments, the cancer is glioblastoma (including GBM).

[0407] In some embodiments, the cancer is gastrointestinal cancer.

[0408] In some embodiments, the cancer is a hypermutant cancer.

[0409] In some embodiments, the cancer is a pediatric hypermutant cancer. [Brief explanation of the drawing]

[0410] [Figure 1A] This shows a comparison between the 2A process (a process of approximately 22 days) and an embodiment of the Gen3 process for TIL manufacturing (a process of approximately 14 to 18 days). [Figure 1B] This is an exemplary Gen3 process chart showing an overview of steps A through F (a process lasting approximately 14 to 18 days). [Figure 1C] This chart provides three exemplary Gen3 processes, each of the three process variations, including an overview of steps A through F (a process lasting approximately 14 to 18 days). [Figure 1D] This is an example of a modified Gen2-like process that provides an overview of steps A through F (a process lasting approximately 22 days). [Figure 1E] This is an illustrative Gen3 second-generation process chart that provides an overview of steps A through F (a process lasting approximately 14 to 18 days). [Figure 1F] This is an illustrative Gen3 second-generation process chart that provides an overview of steps A through F (a process lasting approximately 14 to 18 days). [Figure 1G] This is an illustrative Gen3 second-generation process chart that provides an overview of steps A through F (a process lasting approximately 14 to 18 days). [Figure 2] This provides an experimental flowchart for comparing Gen2 (Process 2A) and Gen3. [Figure 3]This document shows a comparison of various Gen2 (2A process) and Gen3.1 process embodiments. [Figure 4] This table describes the various characteristics of the Gen2, Gen2.1, and Gen3.0 process embodiments. [Figure 5] This is an overview of the culture medium conditions for an embodiment of the Gen3 process called Gen3.1. [Figure 6] This is a schematic diagram of an exemplary embodiment of the Gen3 process (a 16-day process). [Figure 7] This is a schematic diagram of an exemplary embodiment for growing TILs from hematopoietic malignancies using the Gen3 process. On day 0, the T cell fraction (CD3+, CD45+) is separated from the apheresis product enriched with lymphocytes, whole blood, or tumor digests (fresh or thawed) by positive or negative selection methods, i.e., removing T cells using T cell markers (CD2, CD3, etc., or other cells that leave T cells), or by gradient centrifugation. [Figure 8] This is a schematic diagram of an exemplary embodiment of the Gen3 process (a 16-day process). [Figure 9] This is a schematic diagram of an exemplary embodiment of the Gen3.1 testing (Gen3.1 optimization) process (a 16-17 day process). [Figure 10A] This is a schematic diagram of an exemplary embodiment of the Gen3 process (a 16-day process). [Figure 10B] This is a schematic diagram of an exemplary embodiment of the Gen3 process (a 16-day process). [Figure 11] This is a schematic diagram of an exemplary embodiment of the preparation timeline for the Gen3 process (16 / 17 day process). [Figure 12] This is a schematic diagram of an exemplary embodiment of the Gen3 process (a 14-16 day process). [Figure 13A] This is a schematic diagram of an exemplary embodiment of the Gen3 process (a 16-day process). [Figure 13B] This is a schematic diagram of an exemplary embodiment of the Gen3 process (a 16-day process). [Figure 14] This is a comparison of the Gen2, Gen2.1, and Gen3 process (16-day process) embodiments. [Figure 15] This is a flowchart comparison of the Gen3 embodiment (Gen3.0, Gen3.1 control, Gen3.1 test). [Figure 16] The components of an exemplary embodiment of the Gen3 process (Gen3 optimization, a 16-17 day process) are shown. [Figure 17] Structures IA and IB are provided, where the cylinders refer to individual polypeptide-binding domains. Structures IA and IB include, for example, three linearly linked TNFRSF-binding domains derived from an antibody binding to 4-1BBL or 4-1BB, which fold to form a trivalent protein, then linked to a second trivalent protein via IgG1-Fc (containing CH3 and CH2 domains), and then linked to two of the trivalent proteins via disulfide bonds (small elongated ovals), stabilizing the structure and providing an agonist capable of binding to the intracellular signaling domains and signaling proteins of six receptors to form a signaling complex. The TNFRSF-binding domains shown as cylinders may be scFv domains containing VH and VL chains linked by a linker that may contain, for example, hydrophilic residues and Gly and Ser sequences for flexibility, as well as Glu and Lys for solubility. [Figure 18] This is an overview of the Gen2 and Gen3 processes using biopsy samples. [Figure 19] This is an exemplary embodiment of the Gen3 process. [Figure 20] This is an exemplary embodiment of the current Gen3 process. [Figure 21] These are feeder proposed conditions for an exemplary Gen3 process and three exemplary second-generation Gen3 processes. [Figure 22] These are exemplary embodiments of Gen2 and Gen3 processes using various starting materials. [Figure 23]Figure 18 shows a comparison of the CD3+CD45+% of core and excised samples for each process, as illustrated. [Figure 24] Figure 18 shows a comparison of IFNγ data from core and excised samples for each process, as illustrated. [Figure 25] Figure 18 shows an overview of the total number of viable cells and product attributes for each process, as illustrated. [Figure 26] Figure 18 illustrates the augmented phenotypic characteristics related to purity, identification, and memory for each process. Note: Less than 3% of B cells, monocytes, or NK cells were detected. [Figure 27] Figure 18 shows a comparison of the phenotypes of the processes illustrated. [Figure 28A] Figure 18 illustrates the characteristics of the proliferated phenotype associated with differentiation, activation, and depletion through the processes shown. [Figure 28B] Figure 18 illustrates the characteristics of the proliferated phenotype associated with differentiation, activation, and depletion through the processes shown. [Modes for carrying out the invention]

[0411] A brief explanation of sequence listings Sequence ID 1 is the amino acid sequence of the heavy chain of muromonab.

[0412] Sequence ID 2 is the amino acid sequence of the light chain of muromonab.

[0413] Sequence ID 3 is the amino acid sequence of recombinant human IL-2 protein.

[0414] Sequence ID 4 is the amino acid sequence of aldethleukin.

[0415] Sequence ID 5 is the amino acid sequence of recombinant human IL-4 protein.

[0416] Sequence ID 6 is the amino acid sequence of recombinant human IL-7 protein.

[0417] Sequence ID 7 is the amino acid sequence of recombinant human IL-15 protein.

[0418] Sequence ID 8 is the amino acid sequence of recombinant human IL-21 protein.

[0419] Sequence ID 9 is the amino acid sequence of human 4-1BB.

[0420] Sequence ID 10 is the amino acid sequence of mouse 4-1BB.

[0421] Sequence ID 11 is the heavy chain of the 4-1BB agonist monoclonal antibody utomilbam (PF-05082566).

[0422] Sequence ID 12 is the light chain of the 4-1BB agonist monoclonal antibody utomilbam (PF-05082566).

[0423] Sequence ID 13 is the heavy chain variable region (VH) of the 4-1BB agonist monoclonal antibody utomilbam (PF-05082566).

[0424] Sequence ID No. 14 is the light chain variable region (VL) of the 4-1BB agonist monoclonal antibody utomilbam (PF-05082566).

[0425] Sequence ID 15 is the heavy chain CDR1 of the 4-1BB agonist monoclonal antibody utomilbam (PF-05082566).

[0426] Sequence ID 16 is the heavy chain CDR2 of the 4-1BB agonist monoclonal antibody utomilbam (PF-05082566).

[0427] Sequence ID 17 is the heavy chain CDR3 of the 4-1BB agonist monoclonal antibody utomilbam (PF-05082566).

[0428] Sequence number 18 is the light chain CDR1 of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0429] Sequence number 19 is the light chain CDR2 of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0430] Sequence number 20 is the light chain CDR3 of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0431] Sequence number 21 is the heavy chain of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0432] Sequence number 22 is the light chain of the 4-1BB agonist monoclonal antibody urelumab (BMS-66�513).

[0433] Sequence number 23 is the heavy chain variable region (V H ) of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0434] Sequence number 24 is the light chain variable region (V L ) of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0435] Sequence number 25 is the heavy chain CDR1 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0436] Sequence number 26 is the heavy chain CDR2 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0437] Sequence number 27 is the heavy chain CDR3 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0438] Sequence ID No. 28 is the light chain CDR1 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0439] Sequence ID No. 29 is the light chain CDR2 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0440] Sequence ID No. 30 is the light chain CDR3 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0441] Sequence ID 31 is the Fc domain of the TNFRSF agonist fusion protein.

[0442] Sequence ID 32 is the linker for the TNFRSF agonist fusion protein.

[0443] Sequence ID 33 is the linker for the TNFRSF agonist fusion protein.

[0444] Sequence ID 34 is the linker for the TNFRSF agonist fusion protein.

[0445] Sequence ID 35 is the linker for the TNFRSF agonist fusion protein.

[0446] Sequence ID 36 is the linker for the TNFRSF agonist fusion protein.

[0447] Sequence ID 37 is the linker for the TNFRSF agonist fusion protein.

[0448] Sequence ID 38 is the linker for the TNFRSF agonist fusion protein.

[0449] Sequence ID 39 is the linker for the TNFRSF agonist fusion protein.

[0450] Sequence ID 40 is the linker for the TNFRSF agonist fusion protein.

[0451] Sequence ID 41 is the linker for the TNFRSF agonist fusion protein.

[0452] Sequence ID 42 is the Fc domain of the TNFRSF agonist fusion protein.

[0453] Sequence ID 43 is the linker for the TNFRSF agonist fusion protein.

[0454] Sequence ID 44 is the linker for the TNFRSF agonist fusion protein.

[0455] Sequence ID 45 is the linker for the TNFRSF agonist fusion protein.

[0456] Sequence ID 46 is the amino acid sequence of the 4-1BB ligand (4-1BBL).

[0457] Sequence ID 47 is the water-soluble portion of the 4-1BBL polypeptide.

[0458] SEQ ID NO: 48 is the heavy chain variable region (V) of the 4-1BB agonist antibody 4B4-1-1 version 1. H )

[0459] SEQ ID NO: 49 is the light chain variable region (V) of the 4-1BB agonist antibody 4B4-1-1 version 1. L )

[0460] SEQ ID NO: 50 is the heavy chain variable region (V) of the 4-1BB agonist antibody 4B4-1-1 version 2. H )

[0461] Sequence ID 51 is the light chain variable region (V) of the 4-1BB agonist antibody 4B4-1-1 version 2. L )

[0462] Sequence ID 52 is the heavy chain variable region (V) of the 4-1BB agonist antibody H39E3-2. H )

[0463] Sequence ID 53 is the light chain variable region (V) of the 4-1BB agonist antibody H39E3-2. L )

[0464] Sequence ID 54 is the amino acid sequence of human OX40.

[0465] Sequence ID 55 is the amino acid sequence of mouse OX40.

[0466] Sequence ID 56 is the heavy chain of the OX40 agonist monoclonal antibody tavorixizumab (MEDI-0562).

[0467] Sequence ID 57 is the light chain of the OX40 agonist monoclonal antibody tavorixizumab (MEDI-0562).

[0468] Sequence ID 58 is the heavy chain variable region (V) of the OX40 agonist monoclonal antibody tavorixizumab (MEDI-0562). H )

[0469] Sequence ID 59 is the light chain variable region (V) of the OX40 agonist monoclonal antibody tavorixizumab (MEDI-0562). L )

[0470] Sequence ID 60 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody tavorixizumab (MEDI-0562).

[0471] Sequence ID 61 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody tavorixizumab (MEDI-0562).

[0472] Sequence ID 62 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody tavorixizumab (MEDI-0562).

[0473] Sequence ID 63 is the light chain CDR1 of the OX40 agonist monoclonal antibody tavorixizumab (MEDI-0562).

[0474] Sequence ID 64 is the light chain CDR2 of the OX40 agonist monoclonal antibody tavorixizumab (MEDI-0562).

[0475] Sequence ID 65 is the light chain CDR3 of the OX40 agonist monoclonal antibody tavorixizumab (MEDI-0562).

[0476] Sequence ID 66 is the heavy chain of the OX40 agonist monoclonal antibody 11D4.

[0477] Sequence ID 67 is the light chain of the OX40 agonist monoclonal antibody 11D4.

[0478] Sequence ID 68 is the heavy chain variable region (V) of the OX40 agonist monoclonal antibody 11D4. H )

[0479] Sequence ID 69 is the light chain variable region (V) of the OX40 agonist monoclonal antibody 11D4. L )

[0480] SEQ ID NO: 70 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody 11D4.

[0481] Sequence ID 71 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody 11D4.

[0482] Sequence ID 72 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody 11D4.

[0483] Sequence ID 73 is the light chain CDR1 of the OX40 agonist monoclonal antibody 11D4.

[0484] Sequence ID 74 is the light chain CDR2 of the OX40 agonist monoclonal antibody 11D4.

[0485] Sequence ID 75 is the light chain CDR3 of the OX40 agonist monoclonal antibody 11D4.

[0486] Sequence ID 76 is the heavy chain of the OX40 agonist monoclonal antibody 18D8.

[0487] Sequence ID 77 is the light chain of the OX40 agonist monoclonal antibody 18D8.

[0488] SEQ ID NO: 78 is the heavy chain variable region (V) of the OX40 agonist monoclonal antibody 18D8. H )

[0489] SEQ ID NO: 79 is the light chain variable region (V) of the OX40 agonist monoclonal antibody 18D8. L )

[0490] SEQ ID NO: 80 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody 18D8.

[0491] Sequence ID No. 81 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody 18D8.

[0492] Sequence ID No. 82 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody 18D8.

[0493] Sequence ID No. 83 is the light chain CDR1 of the OX40 agonist monoclonal antibody 18D8.

[0494] Sequence ID 84 is the light chain CDR2 of the OX40 agonist monoclonal antibody 18D8.

[0495] Sequence ID No. 85 is the light chain CDR3 of the OX40 agonist monoclonal antibody 18D8.

[0496] Sequence ID 86 is the heavy chain variable region (V) of the OX40 agonist monoclonal antibody Hu119-122. H )

[0497] Sequence ID 87 is the light chain variable region (V) of the OX40 agonist monoclonal antibody Hu119-122. L )

[0498] Sequence ID 88 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody Hu119-122.

[0499] Sequence ID 89 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody Hu119-122.

[0500] Sequence ID 90 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody Hu119-122.

[0501] Sequence ID 91 is the light chain CDR1 of the OX40 agonist monoclonal antibody Hu119-122.

[0502] Sequence ID 92 is the light chain CDR2 of the OX40 agonist monoclonal antibody Hu119-122.

[0503] Sequence ID 93 is the light chain CDR3 of the OX40 agonist monoclonal antibody Hu119-122.

[0504] Sequence ID 94 is the heavy chain variable region (V) of the OX40 agonist monoclonal antibody Hu106-222. H )

[0505] Sequence ID 95 is the light chain variable region (V) of the OX40 agonist monoclonal antibody Hu106-222. L )

[0506] Sequence ID 96 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody Hu106-222.

[0507] Sequence ID 97 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody Hu106-222.

[0508] Sequence ID 98 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody Hu106-222.

[0509] Sequence ID 99 is the light chain CDR1 of the OX40 agonist monoclonal antibody Hu106-222.

[0510] Sequence ID No. 100 is the light chain CDR2 of the OX40 agonist monoclonal antibody Hu106-222.

[0511] Sequence ID 101 is the light chain CDR3 of the OX40 agonist monoclonal antibody Hu106-222.

[0512] Sequence ID 102 is the amino acid sequence of the OX40 ligand (OX40L).

[0513] Sequence ID 103 is the water-soluble portion of the OX40L polypeptide.

[0514] Sequence ID No. 104 is the alternative water-soluble portion of the OX40L polypeptide.

[0515] Sequence ID 105 is the heavy chain variable region (V) of the OX40 agonist monoclonal antibody 008. H )

[0516] Sequence ID 106 is the light chain variable region (V) of the OX40 agonist monoclonal antibody 008. L )

[0517] Sequence ID 107 is the heavy chain variable region (V) of OX40 agonist monoclonal antibody 011. H )

[0518] Sequence ID 108 is the light chain variable region (V) of OX40 agonist monoclonal antibody 011. L )

[0519] Sequence ID 109 is the heavy chain variable region (V) of the OX40 agonist monoclonal antibody 021. H )

[0520] Sequence ID 110 is the light chain variable region (V) of the OX40 agonist monoclonal antibody 021. L )

[0521] Sequence ID 111 is the heavy chain variable region (V) of the OX40 agonist monoclonal antibody 023. H )

[0522] Sequence ID 112 is the light chain variable region (V) of the OX40 agonist monoclonal antibody 023. L )

[0523] Sequence ID 113 is the heavy chain variable region (V) of the OX40 agonist monoclonal antibody. H )

[0524] Sequence ID 114 is the light chain variable region (V) of the OX40 agonist monoclonal antibody. L )

[0525] Sequence ID 115 is the heavy chain variable region (V) of the OX40 agonist monoclonal antibody. H )

[0526] Sequence ID 116 is the light chain variable region (V) of the OX40 agonist monoclonal antibody. L )

[0527] Sequence ID 117 is the heavy chain variable region (V) of a humanized OX40 agonist monoclonal antibody. H )

[0528] Sequence ID 118 is the heavy chain variable region (V) of a humanized OX40 agonist monoclonal antibody. H )

[0529] Sequence ID 119 is the light chain variable region (V) of a humanized OX40 agonist monoclonal antibody. L )

[0530] Sequence ID 120 is the light chain variable region (V) of a humanized OX40 agonist monoclonal antibody. L )

[0531] Sequence ID 121 is the heavy chain variable region (V) of a humanized OX40 agonist monoclonal antibody. H )

[0532] Sequence ID 122 is the heavy chain variable region (V) of a humanized OX40 agonist monoclonal antibody. H )

[0533] Sequence ID 123 is the light chain variable region (V) of a humanized OX40 agonist monoclonal antibody. L )

[0534] Sequence ID 124 is the light chain variable region (V) of a humanized OX40 agonist monoclonal antibody. L )

[0535] Sequence ID 125 is the heavy chain variable region (V) of the OX40 agonist monoclonal antibody. H )

[0536] Sequence ID 126 is the light chain variable region (V) of the OX40 agonist monoclonal antibody. L )

[0537] Sequence numbers 127-462 are currently unassigned.

[0538] Sequence ID 463 is the heavy chain amino acid sequence of the PD-1 inhibitor nivolumab.

[0539] Sequence ID 464 is the light chain amino acid sequence of the PD-1 inhibitor nivolumab.

[0540] Sequence ID 465 is the heavy chain variable region (V) of the PD-1 inhibitor nivolumab. H ) This is the amino acid sequence.

[0541] Sequence ID 466 is the light chain variable region (V) of the PD-1 inhibitor nivolumab.L ) This is the amino acid sequence.

[0542] Sequence ID 467 is the heavy chain CDR1 amino acid sequence of the PD-1 inhibitor nivolumab.

[0543] Sequence ID 468 is the heavy chain CDR2 amino acid sequence of the PD-1 inhibitor nivolumab.

[0544] Sequence ID 469 is the heavy chain CDR3 amino acid sequence of the PD-1 inhibitor nivolumab.

[0545] Sequence ID 470 is the CDR1 amino acid sequence of the light chain of the PD-1 inhibitor nivolumab.

[0546] Sequence ID 471 is the CDR2 amino acid sequence of the light chain of the PD-1 inhibitor nivolumab.

[0547] Sequence ID 472 is the CDR3 amino acid sequence of the light chain of the PD-1 inhibitor nivolumab.

[0548] Sequence ID 473 is the heavy chain amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0549] Sequence ID 474 is the light chain amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0550] Sequence ID 475 is the heavy chain variable region (V) of the PD-1 inhibitor pembrolizumab. H ) This is the amino acid sequence.

[0551] Sequence ID 476 is the light chain variable region (V) of the PD-1 inhibitor pembrolizumab. L ) This is the amino acid sequence.

[0552] Sequence ID 477 is the heavy chain CDR1 amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0553] Sequence ID 478 is the heavy chain CDR2 amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0554] Sequence ID 479 is the heavy chain CDR3 amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0555] Sequence ID 480 is the CDR1 amino acid sequence of the light chain of the PD-1 inhibitor pembrolizumab.

[0556] Sequence ID 481 is the CDR2 amino acid sequence of the light chain of the PD-1 inhibitor pembrolizumab.

[0557] Sequence ID 482 is the CDR3 amino acid sequence of the light chain of the PD-1 inhibitor pembrolizumab.

[0558] Sequence ID 483 is the heavy chain amino acid sequence of the PD-L1 inhibitor durvalumab.

[0559] Sequence ID 484 is the light chain amino acid sequence of the PD-L1 inhibitor durvalumab.

[0560] Sequence ID 485 is the heavy chain variable region (V) of the PD-L1 inhibitor durvalumab. H ) This is the amino acid sequence.

[0561] Sequence ID 486 is the light chain variable region (V) of the PD-L1 inhibitor durvalumab. L ) This is the amino acid sequence.

[0562] Sequence ID 487 is the heavy chain CDR1 amino acid sequence of the PD-L1 inhibitor durvalumab.

[0563] Sequence ID 488 is the heavy chain CDR2 amino acid sequence of the PD-L1 inhibitor durvalumab.

[0564] Sequence ID 489 is the heavy chain CDR3 amino acid sequence of the PD-L1 inhibitor durvalumab.

[0565] Sequence ID 490 is the CDR1 amino acid sequence of the light chain of the PD-L1 inhibitor durvalumab.

[0566] Sequence ID 491 is the CDR2 amino acid sequence of the light chain of the PD-L1 inhibitor durvalumab.

[0567] Sequence ID 492 is the CDR3 amino acid sequence of the light chain of the PD-L1 inhibitor durvalumab.

[0568] Sequence ID 493 is the heavy chain amino acid sequence of the PD-L1 inhibitor avelumab.

[0569] Sequence ID 494 is the light chain amino acid sequence of the PD-L1 inhibitor avelumab.

[0570] Sequence ID 495 is the heavy chain variable region (V) of the PD-L1 inhibitor avelumab. H ) This is the amino acid sequence.

[0571] Sequence ID 496 is the light chain variable region (V) of the PD-L1 inhibitor avelumab. L ) This is the amino acid sequence.

[0572] Sequence ID 497 is the heavy chain CDR1 amino acid sequence of the PD-L1 inhibitor avelumab.

[0573] Sequence ID 498 is the heavy chain CDR2 amino acid sequence of the PD-L1 inhibitor avelumab.

[0574] Sequence ID 499 is the heavy chain CDR3 amino acid sequence of the PD-L1 inhibitor avelumab.

[0575] Sequence ID 500 is the CDR1 amino acid sequence of the light chain of the PD-L1 inhibitor avelumab.

[0576] Sequence ID 501 is the CDR2 amino acid sequence of the light chain of the PD-L1 inhibitor avelumab.

[0577] Sequence ID 502 is the CDR3 amino acid sequence of the light chain of the PD-L1 inhibitor avelumab.

[0578] Sequence ID 503 is the heavy chain amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0579] Sequence ID 504 is the light chain amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0580] Sequence ID 505 is the heavy chain variable region (V) of the PD-L1 inhibitor atezolizumab. H ) This is the amino acid sequence.

[0581] Sequence ID 506 is the light chain variable region (V) of the PD-L1 inhibitor atezolizumab. L ) This is the amino acid sequence.

[0582] Sequence ID 507 is the heavy chain CDR1 amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0583] Sequence ID 508 is the heavy chain CDR2 amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0584] Sequence ID 509 is the heavy chain CDR3 amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0585] Sequence ID 510 is the CDR1 amino acid sequence of the light chain of the PD-L1 inhibitor atezolizumab.

[0586] Sequence ID 511 is the CDR2 amino acid sequence of the light chain of the PD-L1 inhibitor atezolizumab.

[0587] Sequence ID 512 is the CDR3 amino acid sequence of the light chain of the PD-L1 inhibitor atezolizumab.

[0588] Sequence ID 513 is the heavy chain amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0589] Sequence ID 514 is the light chain amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0590] Sequence ID 515 is the heavy chain variable region (V) of the CTLA-4 inhibitor ipilimumab. H ) This is the amino acid sequence.

[0591] Sequence ID 516 is the light chain variable region (V) of the CTLA-4 inhibitor ipilimumab. L ) This is the amino acid sequence.

[0592] Sequence ID 517 is the heavy chain CDR1 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0593] Sequence ID 518 is the heavy chain CDR2 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0594] Sequence ID 519 is the heavy chain CDR3 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0595] Sequence ID 520 is the CDR1 amino acid sequence of the light chain of the CTLA-4 inhibitor ipilimumab.

[0596] Sequence ID 521 is the CDR2 amino acid sequence of the light chain of the CTLA-4 inhibitor ipilimumab.

[0597] Sequence ID 522 is the CDR3 amino acid sequence of the light chain of the CTLA-4 inhibitor ipilimumab.

[0598] Sequence ID 523 is the heavy chain amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0599] Sequence ID 524 is the light chain amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0600] Sequence ID 525 is the heavy chain variable region (V) of the CTLA-4 inhibitor tremelimumab. H ) This is the amino acid sequence.

[0601] Sequence ID 526 is the light chain variable region (V) of the CTLA-4 inhibitor tremelimumab. L ) This is the amino acid sequence.

[0602] Sequence ID 527 is the heavy chain CDR1 amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0603] Sequence ID 528 is the heavy chain CDR2 amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0604] Sequence ID 529 is the heavy chain CDR3 amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0605] Sequence ID 530 is the CDR1 amino acid sequence of the light chain of the CTLA-4 inhibitor tremelimumab.

[0606] Sequence ID 531 is the CDR2 amino acid sequence of the light chain of the CTLA-4 inhibitor tremelimumab.

[0607] Sequence ID 532 is the CDR3 amino acid sequence of the light chain of the CTLA-4 inhibitor tremelimumab.

[0608] Sequence ID 533 is the heavy chain amino acid sequence of the CTLA-4 inhibitor zarifremab.

[0609] Sequence ID 534 is the light chain amino acid sequence of the CTLA-4 inhibitor zarifremab.

[0610] Sequence ID 535 is the heavy chain variable region (V) of the CTLA-4 inhibitor zarifremab. H ) This is the amino acid sequence.

[0611] Sequence ID 536 is the light chain variable region (V) of the CTLA-4 inhibitor zarifremab. L ) This is the amino acid sequence.

[0612] Sequence ID 537 is the heavy chain CDR1 amino acid sequence of the CTLA-4 inhibitor zarifremab.

[0613] Sequence ID 538 is the heavy chain CDR2 amino acid sequence of the CTLA-4 inhibitor zarifremab.

[0614] Sequence ID 539 is the heavy chain CDR3 amino acid sequence of the CTLA-4 inhibitor zarifremab.

[0615] Sequence ID 540 is the CDR1 amino acid sequence of the light chain of the CTLA-4 inhibitor zarifremab.

[0616] Sequence ID 541 is the CDR2 amino acid sequence of the light chain of the CTLA-4 inhibitor zarifremab.

[0617] Sequence ID 542 is the CDR3 amino acid sequence of the light chain of the CTLA-4 inhibitor zarifremab.

[0618] Sequence ID 543 is the IL-2 sequence.

[0619] Sequence ID 544 is the IL-2 mutain sequence.

[0620] Sequence ID 545 is the IL-2 mutain sequence.

[0621] Sequence ID 546 is IgG.IL2R67A.H1, which is HCDR1_IL-2.

[0622] Sequence ID 547 is HClR2 of IgG.IL2R67A.H1.

[0623] Sequence ID 548 is IgG.IL2R67A.H1 HCDR3.

[0624] Sequence ID 549 is IgG.IL2R67A.H1 HCDR1_IL-2kabat.

[0625] Sequence ID 550 is HCl2kabat of IgG.IL2R67A.H1.

[0626] Sequence ID 551 is HCl3kabat of IgG.IL2R67A.H1.

[0627] Sequence ID 552 is IgG.IL2R67A.H1 HCDR1_IL-2clothia.

[0628] Sequence ID 553 is IgG.IL2R67A.H1 HCDR2clothia.

[0629] Sequence ID 554 is IgG.IL2R67A.H1 HCDR3clothia.

[0630] Sequence ID 555 is IgG.IL2R67A.H1 HCDR1_IL-2IMGT.

[0631] Sequence ID 556 is IgG.IL2R67A.H1 HCDR2IMGT.

[0632] Sequence ID 557 is IgG.IL2R67A.H1 HCDR3IMGT.

[0633] Sequence ID 558 is the VH chain of IgG.IL2R67A.H1.

[0634] Sequence ID 559 is the heavy chain of IgG.IL2R67A.H1.

[0635] Sequence ID 560 is LCDR1kabat of IgG.IL2R67A.H1.

[0636] Sequence ID 561 is LCDR2kabat for IgG.IL2R67A.H1.

[0637] Sequence ID 562 is LCDR3kabat for IgG.IL2R67A.H1.

[0638] Sequence ID 563 is LCDR1chothia of IgG.IL2R67A.H1.

[0639] Sequence ID 564 is LCDR2chothia of IgG.IL2R67A.H1.

[0640] Sequence ID 565 is IgG.IL2R67A.H1 LCDR3chothia.

[0641] Sequence ID 566 is a VL chain.

[0642] Sequence ID 567 is a light chain.

[0643] Sequence ID 568 is a light chain.

[0644] Sequence ID 569 is a light chain.

[0645] Sequence ID 570 is in the IL-2 form.

[0646] Sequence ID 571 is in the IL-2 form.

[0647] Sequence ID 572 is in the IL-2 form.

[0648] Sequence ID 573 is a mucin domain polypeptide.

[0649] I. Definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as they would ordinarily be understood by those skilled in the art. All patents and publications referenced herein are incorporated herein in their entirety by reference.

[0650] The term "in vivo" refers to events that occur within the subject's body.

[0651] The term "in vitro" refers to events that occur outside the body of the subject. In vitro assays encompass cell-based assays that use living or dead cells, and may also include cell-free assays that do not use intact cells.

[0652] The term "ex vivo" refers to events involving the performance of treatment or procedures on cells, tissues, and / or organs removed from a subject's body. Where appropriate, cells, tissues, and / or organs may be returned to the subject's body by surgical or therapeutic means.

[0653] The term "rapid growth" means an increase in the number of antigen-specific TILs of at least approximately 3 times (or 4 times, 5 times, 6 times, 7 times, 8 times, or 9 times) over one week, more preferably at least approximately 10 times (or 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, or 90 times) over one week, or most preferably at least approximately 100 times over one week. An overview of some rapid growth protocols is provided below.

[0654] In this specification, "tumor-infiltrating lymphocytes" or "TILs" refers to the initial population of cells obtained as leukocytes that have left the bloodstream and migrated into the tumor. TILs include CD8 + Cytotoxic T cells (lymphocytes), Th1 and Th17CD4 + 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 a patient's tissue sample as outlined herein (and may be referred to as "freshly obtained" or "freshly isolated"), and "secondary TILs" are any proliferation or augmented TIL cell populations considered herein, including, but not limited to, bulk TILs and augmented TILs ("REP TILs" or "post-REP TILs"). TIL cell populations may include genetically modified TILs.

[0655] In this specification, "cell population" (including TILs) means a large number of cells that share common traits. Generally, a population is typically 1 × 10⁶ in number. 6 ~1 × 10 10 This ranges, and different TIL populations consist of different numbers. For example, the initial growth of primary TILs in the presence of IL-2 is approximately 1 × 10⁻⁶. 8This results in a bulk TIL population of cells. REP proliferation is generally 1.5 × 10⁶ for injection. 9 ~1.5×10 10 This is done to provide a population of cells. In some embodiments, 2.3 × 10 10 ~13.7×10 10 REP proliferation is performed to provide a population.

[0656] In this specification, “cryopreserved TIL” means primary, bulk, or regenerated (REP TIL) tissue that has been processed and stored at a temperature in the range of approximately -150°C to -60°C. General methods of cryopreservation are also described elsewhere in this specification, including in the examples. For clarity, “cryopreserved TIL” can be distinguished from frozen tissue samples that can be used as a source for primary TIL.

[0657] 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.

[0658] TILs can generally be defined biochemically using cell surface markers or functionally by their ability to infiltrate tumors and influence treatment. 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. In addition, and alternatively, TILs can be functionally defined by their ability to infiltrate solid tumors upon reintroduction into a patient. TILs can be further characterized by potency; for example, a TIL may be considered potent if the release of interferon (IFN) is greater than approximately 50 pg / mL, 100 pg / mL, 150 pg / mL, or 200 pg / mL. For example, a TIL may be considered effective if the interferon (IFNγ) release is greater than approximately 50 pg / mL, 100 pg / mL, 150 pg / mL, or 200 pg / mL, 300 pg / mL, 400 pg / mL, 500 pg / mL, 600 pg / mL, 700 pg / mL, 800 pg / mL, 900 pg / mL, or 1000 pg / mL.

[0659] The term “cryopreservation medium” or “cryopreservation medium” refers to any medium that can be used for cryopreserving cells. Such media contain 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 is sometimes referred to by the trade name “CryoStor® CS10”. CS10 medium is a serum-free, animal-component-free medium containing DMSO.

[0660] The term "central memory T cell" refers to a CD45R0+ cell in humans, and CCR7(CCR7) hi ) and CD62L (CD62 hiThis refers to a subset of T cells that constitutively express IL-2. The surface phenotype of central memory T cells also includes TCR, CD3, CD127 (IL-7R), and IL-15R. The transcription factors of central memory T cells include BCL-6, BCL-6B, MBD2, and BMI1. Central memory T cells secrete mainly IL-2 and CD40L as effector molecules after TCR triggering. Central memory T cells are dominant in the CD4 compartment of the blood and are proportionally concentrated in the lymph nodes and tonsils in humans.

[0661] The term "effector memory T cells" refers to cells that, like central memory T cells, are CD45R0+ but have lost constitutive expression of CCR7 (CCR7 lo ), CD62L expression is heterogeneous or low (CD62L lo Central memory T cells (CD8+) refer to a subset of human or mammalian T cells. The surface phenotype of central memory T cells also includes TCR, CD3, CD127 (IL-7R), and IL-15R. The transcription factor of central memory T cells includes BLIMP1. Effector memory T cells rapidly secrete high levels of inflammatory cytokines, including interferon-γ, IL-4, and IL-5, following antigen stimulation. Effector memory T cells are dominant in the CD8 compartment of the blood and are proportionally concentrated in the lungs, liver, and intestines in humans. CD8+ effector memory T cells possess large amounts of perforin.

[0662] A "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, closed G-containers. Once tumor segments are added to the closed system, the system is not opened to the external environment until the TIL is ready to be administered to the patient.

[0663] The terms “fragmentation,” “fragment,” and “fragmented” as used herein to describe a process for destroying a tumor 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.

[0664] The term "fine-needle aspiration" or FNA refers to a type of biopsy procedure that can be used in sampling or diagnostic procedures, including tumor sampling, which involves taking a sample but not removing or excising the tumor. In fine-needle aspiration, as described herein, a hollow needle, e.g., 25-18 gauge, is inserted into the tumor or an area containing a tumor to obtain fluid and cells (including tissue) for further analysis or growth. With FNA, cells are extracted without preserving the histological structure of the tissue cells. FNA may include tissue-infiltrated lesions (TILs). In some cases, fine-needle aspiration cytology is performed using an ultrasound-guided fine-needle aspiration biopsy needle. FNA needles are commercially available from Becton Dickinson, Covidien, and others.

[0665] The term “core biopsy” or “core needle biopsy” refers to a type of biopsy procedure that can be used in sampling or diagnostic procedures, including tumor sampling, which involves taking a sample but not removing or excising the tumor. In fine-needle aspiration, as described herein, a hollow needle, e.g., 16-11 gauge, is inserted into the tumor or an area containing a tumor to obtain fluid and cells (including tissue) for further analysis or growth. In core biopsy, the larger needle size compared to FNA allows for the extraction of cells with some degree of histological structure preserved. Core biopsy needles are generally of a gauge size that can preserve at least some of the tumor's tissue structure. Core biopsies may include TILs. In some cases, core needle biopsies are performed using biopsy instruments, vacuum-assisted core needle biopsy instruments, stereotactic core needle biopsy instruments, ultrasound-guided core needle biopsy instruments, and MRI-guided core needle biopsy instruments, which are commercially available from companies such as Bard Medical and Becton Dickinson.

[0666] 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 irradiated allogeneic peripheral blood mononuclear cells.

[0667] 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.

[0668] The term "anti-CD3 antibody" refers to an antibody or its variant, such as a monoclonal antibody, including human, humanized, chimeric, or mouse antibodies against the CD3 receptor in the T cell antigen receptor of mature T cells. Anti-CD3 antibodies include OKT-3, also known as muromonab. Anti-CD3 antibodies also include the UHCT1 clone, also known as T3 and CD3ε. Other anti-CD3 antibodies include, for example, otelixizumab, teprizumab, and bicilizumab.

[0669] The term "OKT-3" (also referred to herein as "OKT3") refers to monoclonal antibodies or biosimilars or variants thereof, including human, humanized, chimeric, or mouse antibodies against the CD3 receptor, the T cell antigen receptor of mature T cells, and includes commercially available forms such as OKT-3 (30 ng / mL, MACS GMP CD3pure, Miltenyi Biotech, Inc., San Diego, CA, USA) and muromonab or its variants, conservative amino acid substitutions, glycoforms, or biosimilars thereof. The amino acid sequences of the heavy and light chains of muromonab are shown in Table 1 (SEQ ID NO: 1 and SEQ ID NO: 2). Hybridomas capable of producing OKT-3 are deposited in the American Type Culture Collection and assigned ATCC accession number CRL8001. Hybridomas capable of producing OKT-3 are also deposited in the European Collection of Authenticated Cell Cultures (ECACC) and assigned catalog number 86022706. [Table 1]

[0670] 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 thereof. IL-2 is described, for example, 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 forms of recombinant IL-2 commercially supplied by CellGenix, Inc., Portsmouth, NH, USA (CELLGROGMP) or ProSpec-TanyTechnoGene Ltd., East Brunswick, NJ, USA (Cat. No. CYT-209-b), and other commercial equivalents from other vendors. Aldesleukin (des-alanil-1, serine-125 human IL-2) is a non-glycosylated human recombinant IL-2 with a molecular weight of approximately 15 kDa. The amino acid sequence of aldesleukin suitable for use in this invention is shown in Table 2 (SEQ ID NO: 4). The term IL-2 also refers to the pegylated IL-2 prodrug benpegaldesleukin (NKTR-214), available from Nektar Therapeutics, South San Francisco, CA, USA, as described herein, in which an average of 6 lysine residues are substituted with [(2,7-bis{[methylpoly(oxyethylene)]carbamoyl}-9H-fluoren-9-yl)methoxy]carbonyl. 6This includes pegylated human recombinant IL-2 (SEQ ID NO: 4), or pegylated forms of IL-2 that can be prepared by methods known in the art, as described in International Patent Application Publication WO2018 / 132496A1 of Example 19, or in U.S. Patent Application Publication US2019 / 0275133A1 of Example 1, which are incorporated herein by reference. Benpegaldesleukine (NKTR-214) and other pegylated IL-2 molecules suitable for use in the present invention are described in U.S. Patent Application Publication US2014 / 0328791A1 and International Patent Application Publication WO2012 / 065086, which are incorporated herein by reference. Alternative forms of conjugated IL-2 suitable for use in the present invention are described in U.S. Patents 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.

[0671] In some embodiments, the IL-2 form suitable for use in the present invention is THOR-707, available from Synthorx, Inc. Preparations and characterizations of THOR-707 and further alternative forms of IL-2 suitable for use in the present invention are described in U.S. Patent Application Publications US2020 / 0181220A1 and US 2020 / 0330601, the disclosures of which are incorporated herein by reference. In some embodiments, the IL-2 form suitable for use in the present invention is an interleukin-2 (IL-2) conjugate comprising isolated and purified IL-2 polypeptide; and a conjugate moiety (amino acid residue numbering corresponding to Sequence ID No. 5) that binds to the isolated and purified IL-2 polypeptide at an amino acid position selected from K35, T37, R38, T41, F42, K43, F44, Y45, E61, E62, E68, K64, P65, V69L72, and Y107. In some embodiments, the amino acid position is selected from T37, R38, T41, F42, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107. In some embodiments, the amino acid position is selected from T37, R38, T41, F42, F44, Y45, E61, E62, E68, P65, V69, L72, and Y107. In some embodiments, the amino acid position is selected from T37, T41, F42, F44, Y45, P65, V69, L72, and Y107. In some embodiments, the amino acid position is selected from R38 and K64. In some embodiments, the amino acid position is selected from E61, E62, and E68. In some embodiments, the amino acid position is E62. In some embodiments, an amino acid residue selected from K35, T37, R38, T41, F42, K43, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107 is further mutated to lysine, cysteine, or histidine. In some embodiments, the amino acid residue is mutated to cysteine. In some embodiments, the amino acid residue is mutated to lysine. In some embodiments, K35, T37, R38, T41, F42, K43, F44, Y45, E61, E62, E68, K64, P65, V69, L72,The amino acid residues selected from and Y107 are further mutated into non-natural amino acids. In some embodiments, the non-natural amino acids are N6-azidoethoxy-L-lysine (AzK), N6-propargylethoxy-L-lysine (PraK), BCN-L-lysine, norbornenyllysine, TCO-lysine, methyltetrazinyllysine, allyloxycarbonyllysine, 2-amino-8-oxononanoic acid, 2-amino-8-oxooctanoic acid, p-acetyl-L-phenylalanine, p-azidomethyl-L-phenylalanine (pAMF), p-iodo-L-phenylalanine, m-acetylphenylalanine, 2-amino-8-oxononanoic acid, p-propargyloxyphenylalanine, p-propargyl-phenylalanine, 3-methylphenylalanine, L-DOPA, fluorinated phenylalanine, isopropyl Contains -L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, O-allyl-tyrosine, O-methyl-L-tyrosine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, phosphonotyrosine, tri-O-acetyl-GlcNAcp-serine, L-phosphoserine, phosphonoserine, L-3-(2-naphthyl)alanine, 2-amino-3-((2-((3-(benzyloxy)-3-oxopropyl)amino)ethyl)ceranyl)propanoic acid, 2-amino-3-(phenylceranyl)propaneoic, or selenocysteine. In some embodiments, the IL-2 conjugate exhibits reduced affinity for the IL-2 receptor α (IL-2Rα) subunit compared to the wild-type IL-2 polypeptide. In some embodiments, the reduced affinity represents a decrease in binding affinity to IL-2Rα of approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more than 99% compared to the wild-type IL-2 polypeptide. In some embodiments, the reduced affinity represents a decrease of approximately 1x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 30x, 50x, 100x, 200x, 300x, 500x, 1000x compared to the wild-type IL-2 polypeptide.or more. In some embodiments, the conjugate moiety impairs or blocks the bond between IL-2 and IL-2Rα. In some embodiments, the conjugate moiety comprises a water-soluble polymer. In some embodiments, an additional conjugate moiety comprises a water-soluble polymer. In some embodiments, each of the water-soluble polymers independently comprises polyethylene glycol (PEG), poly(propylene glycol) (PPG), copolymer of ethylene glycol and propylene glycol, poly(oxyethylated polyol), poly(olefin alcohol), poly(vinylpyrrolidone), poly(hydroxyalkyl methacrylamide), poly(hydroxyalkyl methacrylate), poly(sugar), poly(α-hydroxy acid), poly(vinyl alcohol), polyphosphazene, polyoxazoline (POZ), poly(N-acryloylmorpholine), or a combination thereof. In some embodiments, each of the water-soluble polymers independently comprises PEG. In some embodiments, PEG is linear PEG or branched PEG. In some embodiments, each of the water-soluble polymers independently comprises a polysaccharide. In some embodiments, the polysaccharide comprises dextran, polysialic acid (PSA), hyaluronic acid (HA), amylose, heparin, heparan sulfate (HS), dextrin, or hydroxyethyl starch (HES). In some embodiments, each of the water-soluble polymers independently comprises a glycan. In some embodiments, each of the water-soluble polymers independently comprises a polyamine. In some embodiments, the conjugate moiety comprises a protein. In some embodiments, the additional conjugate moiety comprises a protein. In some embodiments, each of the proteins independently comprises albumin, transferrin, or transthyretin. In some embodiments, each of the proteins independently comprises an Fc moiety. In some embodiments, each of the proteins independently comprises an IgG Fc moiety. In some embodiments, the conjugate moiety comprises a polypeptide. In some embodiments, the additional conjugate moiety comprises a polypeptide. In some embodiments, each polypeptide independently comprises an XTEN peptide.The material comprises a glycine-rich homoamino acid polymer (HAP), a PAS polypeptide, an elastin-like polypeptide (ELP), a CTP peptide, or a gelatin-like protein (GLK) polymer. In some embodiments, the isolated and purified IL-2 polypeptide is modified by glutamylation. In some embodiments, the conjugate moiety is directly conjugated to the isolated and purified IL-2 polypeptide. In some embodiments, the conjugate moiety is indirectly conjugated to the isolated and purified IL-2 polypeptide. In some embodiments, the linker comprises a homohomo-bifunctional linker. In some embodiments, the homobifunctional linker is Romant reagent dithiobis(succinimidyl propionate) DSP, 3'3'-dithiobis(sulfosuccinimidyl propionate) (DTSSP), disuccinimidyl suberate (DSS), bis(BS) suberate, disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfoDST), ethylene glycobis(succinimidyl succinate) (EGS), disuccinimidyl glutarate (DSG), N,N'-disuccinimidyl carbonate (DSC), dimethyl adipimidine (DMA), dimethyl pimerimidine (DMP), dimethyl suberidine (DMS), dimethyl-3,3'-dithiobispropionimidate (DTBP), 1,4-di-(3'-(2'-pyridyldithio) Propionamide)butane (DPDPB), bismaleimide hexane (BMH), aryl halide-containing compounds (DFDNB), e.g., 1,5-difluoro-2,4-dinitrobenzene or 1,3-difluoro-4,6-dinitrobenzene, 4,4'-difluoro-3,3'-dinitrophenyl sulfone (DFDNPS), bis-[β-(4-azidosalicylamido)ethyl]disulfide (BASED), formaldehyde, glutaraldehyde, 1,4-butanediol diglycidyl ether, adipic acid dihydrazide, carbohydrazide, o-toluidine, 3,3'-dimethylbenzidine, benzidine, α,α'-p-diaminodiphenyl, diiodo-p-xylenesulfonic acid, N,N'-ethylene-bis(iodoacetamide),or comprising N,N'-hexamethylene-bis(iodoacetamide). In some embodiments, the linker comprises a heterobifunctional linker. In some embodiments, the heterobifunctional linker comprises N-succinimidyl 3-(2-pyridyldithio)propionate (sPDP), long-chain N-succinimidyl 3-(2-pyridyldithio)propionate (LC-sPDP), water-soluble long-chain N-succinimidyl 3-(2-pyridyldithio)propionate (sulfo-LC-sPDP), succinimidyloxycarbonyl-α-methyl-α-(2-pyridyldithio)toluene (sMPT), sulfosuccinimidyl-6-[α-methyl-α-(2-pyridyldithio]toluene [Dyldithio)toluamide]hexanoate (sulfo-LC-sMPT), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sMCC), sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-sMCC), m-maleimidobenzoyl-N-hydroxysuccinimid ester (MB), m-maleimidobenzoyl-N-hydroxysulfosuccinimid ester (sulfo-MB), N-succinimidyl (4- Iodoacetyl)aminobenzoate (sIAB), sulfosuccinimidyl (4-iodoacetyl)aminobenzoate (sulfo-sIAB), succinimidyl-4-(p-maleimidophenyl)butyrate (sMPB), sulfosuccinimidyl-4-(p-maleimidophenyl)butyrate (sulfo-sMPB), N-(γ-maleimidobutyryloxy)succinimidide ester (GMB), N-(γ-maleimidobutyryloxy)sulfosuccinimidide ester (sulfo-GMB), succinimidyl Dyl 6-((iodoacetyl)amino)hexanoate (sIAX), succinimidyl 6-[6-(((iodoacetyl)amino)hexanoyl)ami]hexanoate (slAXX), succinimidyl 4-(((iodoacetyl)amino)methyl)cyclohexane-1-carboxylate (sIAC), succinimidyl 6-(((((4-iodoacetyl)amino)methyl)cyclohexane-1-carbonyl)amino)hexanoate (sIACX), p-nitrophenyl iodoacetate (NPIA),Carbonyl-reactive and sulfhydryl-reactive crosslinking agents such as 4-(4-N-maleimidophenyl)butyrate hydrazide (MPBH), 4-(N-maleimidomethyl)cyclohexane-1-carboxyl-hydrazide-8 (M2C2H), 3-(2-pyridyldithio)propionyl hydrazide (PDPH), N-hydroxysuccinimidyl-4-azidosalicylic acid (NHs-AsA), N-hydroxysulfosuccinimidyl-4-azidosalicylic acid (, Sulfo-NHs-AsA), sulfosuccinimidyl-(4-azidosalicylamide)hexanoate (sulfo-NHs-LC-AsA), sulfosuccinimidyl-2-(p-azidosalicylamide)ethyl-1,3'-dithiopropionate (sAsD), N-hydroxysuccinimidyl-4-azidobenzoate (HsAB), N-hydroxysulfosuccinimidyl-4-azidobenzoic acid (sulfo-HsAB), N-succinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate Noate (sANPAH), sulfosuccinimidyl-6-(4'-azido-2)'-nitrophenylamino)hexanoate (sulfo-sANPAH), N-5-azido-2-nitrobenzoyloxysuccinimide (AN)B-NOs), sulfosuccinimidyl-2-(m-azido-o-nitrobenzamide)-ethyl-1,3'-dithiopropionate (sAND), N-succinimidyl-4(4-azidophenyl)1,3'-dithiopropionate (sADP), N-sulfosuccinimidyl ( 4-azidophenyl)-1,3'-dithiopropionate (sulfo-sADP), sulfosuccinimidyl 4-(ρ-azidophenyl)butyrate (sulfo-sAPB), sulfosuccinimidyl 2-(7-azido-4-methylcoumarin-3)-acetamido)ethyl-1,3'-dithiopropionate (sAED), sulfosuccinimidyl 7-azido-4-methylcoumarin-3-acetate (sulfo-sAMCA), p-nitrophenyldiazopirubate (pNPDP), p-nitrophenyl-2-dia The linker comprises zo-3,3,3-trifluoropropionic acid (PNP-DTP), 1-(ρ-azidosalicylamide)-4-(iodoacetamide)butane (AsIB), N-[4-(ρ-azidosalicylamide)butyl]-3'-(2'-pyridyldithio)propionamide (APDP), benzophenone-4-iodoacetamide, p-azidobenzoylhydrazide (ABH), 4-(p-azidosalicylamide)butylamine (AsBA), or p-azidophenylglyoxal (APG). In some embodiments, the linker comprises a cleavable linker, optionally comprising a dipeptide linker.In some embodiments, the dipeptide linker comprises Val-Cit, Phe-Lys, Val-Ala, or Val-Lys. In some embodiments, the linker comprises an uncleaved linker. In some embodiments, the linker comprises a maleimide group optionally comprising maleimidocaproyl (mc), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sMCC), or sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-sMCC). In some embodiments, the linker comprises a spacer. In some embodiments, the spacer comprises p-aminobenzyl alcohol (PAB), p-aminobenzyloxycarbonyl (PABC), its derivatives, or analogs. In some embodiments, the conjugate moiety can extend the serum half-life of the IL-2 conjugate. In some embodiments, an additional conjugate moiety can extend the serum half-life of the IL-2 conjugate. In some embodiments, the IL-2 form suitable for use in the present invention is a fragment of any of the IL-2 forms described herein. In some embodiments, the IL-2 form suitable for use in the present invention is pegged as disclosed in U.S. Patent Application Publication US2020 / 0181220A1 and U.S. Patent Application Publication US2020 / 0330601A1. In some embodiments, the IL-2 form suitable for use in the present invention is an IL-2 conjugate comprising an IL-2 polypeptide containing N6-azidoethoxy-L-lysine (AzK) covalently bonded to a conjugate portion containing polyethylene glycol (PEG), wherein the IL-2 polypeptide has amino acids having at least 80% identity with SEQ ID NO: 5, and AzK is substituted with amino acids at the positions K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69, or L72, which refer to the amino acid positions of SEQ ID NO: 5. In some embodiments, the IL-2 polypeptide contains a one-residue N-terminal deletion relative to SEQ ID NO: 5.In some embodiments, the IL-2 form suitable for use in the present invention lacks the involvement of the IL-2R alpha chain but retains normal binding to the intermediate affinity IL-2R beta-gamma signaling complex. In some embodiments, the IL-2 form suitable for use in the present invention is an IL-2 conjugate comprising an IL-2 polypeptide containing N6-azidoethoxy-L-lysine (AzK) covalently bonded to a polyethylene glycol (PEG) conjugate portion, wherein the IL-2 polypeptide has amino acids having at least 90% identity with SEQ ID NO: 5, and AzK is substituted with an amino acid at the position K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69, or L72, which reference the amino acid position of SEQ ID NO: 5. In some embodiments, an IL-2 form suitable for use in the present invention is an IL-2 conjugate comprising an IL-2 polypeptide containing N6-azidoethoxy-L-lysine (AzK) covalently bonded to a conjugate portion containing polyethylene glycol (PEG), wherein the IL-2 polypeptide has amino acids having at least 95% identity with SEQ ID NO: 5, and AzK is substituted with an amino acid at the position K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69, or L72, which reference the amino acid position of SEQ ID NO: 5. In some embodiments, an IL-2 form suitable for use in the present invention is an IL-2 conjugate comprising an IL-2 polypeptide containing N6-azidoethoxy-L-lysine (AzK) covalently bonded to a polyethylene glycol (PEG) conjugate portion, wherein the IL-2 polypeptide has amino acids having at least 98% identity with SEQ ID NO: 5, and AzK is substituted with an amino acid at the position K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69, or L72, which reference the amino acid position of SEQ ID NO: 570.

[0672] In some embodiments, the IL-2 form suitable for use in the present invention is Nemvaleukin alpha, also known as ALKS-4230 (SEQ ID NO: 571), available from Alkermes, Inc. Nemvaleukin alpha is produced in glycosylated Chinese hamster ovary (CHO) cells and contains peptidyl linkers. 60 GG 61 ) fuses to human interleukin-2 fragment (62-132) via peptidyl linker ( 133 GSGGGS 138 Human interleukin-2 fragments (1-59) and variants (Cys) fused to human interleukin-2 receptor α chain fragments (139-303) via ) 125 >Ser 51 ); Human interleukin-2 ((IL-2)(75-133)-peptide [Cys] produced in Chinese hamster ovary (CHO) cells, glycoform alpha, fused to human interleukin-2 (IL-2)(4-74)-peptide (62-132) via the G2 peptide linker (60-61), and fused to human interleukin-2 receptor α chain (IL2R subunit alpha, IL2Rα, IL2RA)(1-165)-peptide (139-303) via the GSG3S peptide linker (133-138). 125Also known as (51)>Ser]-mutant(1-59). The amino acid sequence of nemvaleukin alpha is shown in SEQ ID NO: 571. In some embodiments, nemvaleukin alpha exhibits the following post-translational modifications: disulfide crosslinking at positions 31-116, 141-285, 184-242, 269-301, 166-197 or 166-199, 168-199 or 168-197 (using the numbering in SEQ ID NO: 571), and glycosylation at positions N187, N206, T212 (using the numbering in SEQ ID NO: 571). Preparation and characterization of nemvaleukin alpha, as well as further alternative forms of IL-2 suitable for use in the present invention, are described in U.S. Patent Application Publication US2021 / 0038684A1 and U.S. Patent No. 10,183,979, which are incorporated herein by reference. In some embodiments, an IL-2 form suitable for use in the present invention is a protein having at least 80%, at least 90%, at least 95%, or at least 90% sequence identity with respect to SEQ ID NO: 571. In some embodiments, an IL-2 form suitable for use in the present invention has the amino acid sequence shown in SEQ ID NO: 571 or its conserved amino acid substitutions. In some embodiments, an IL-2 form suitable for use in the present invention is a fusion protein, or a variant, fragment, or derivative thereof, containing amino acids 24-452 of SEQ ID NO: 572. In some embodiments, an IL-2 form suitable for use in the present invention is a fusion protein, or a variant, fragment, or derivative thereof, containing an amino acid sequence having at least 80%, at least 90%, at least 95%, or at least 90% sequence identity with respect to amino acids 24-452 of SEQ ID NO: 572. Other IL-2 forms suitable for use in the present invention are described in U.S. Patent No. 10,183,979, the disclosure of which is incorporated herein by reference.In some embodiments, the IL-2 form suitable for use in the present invention is a fusion protein comprising a first fusion partner linked to a second fusion partner by a mucin domain polypeptide linker, wherein the first fusion partner is IL-1Rα or a protein having at least 98% amino acid sequence identity with IL-1Rα and possessing receptor antagonist activity of IL-Rα, wherein the second fusion partner comprises all or part of an immunoglobulin including an Fc region, the mucin domain polypeptide linker comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 573, and the half-life of the fusion protein is improved when comparing the first fusion partner to the second fusion partner in the absence of the mucin domain polypeptide linker. [Table 2]

[0673] In some embodiments, an IL-2 configuration suitable for use in the present invention includes a heavy chain variable region (V) which comprises complementarity determination regions HCDR1, HCDR2, and HCDR3. H ) an antibody cytokine transplant protein containing and a light chain variable region (V) containing LCDR1, LCDR2, LCDR3 L ) and V H or V L The antibody cytokine transplantation protein comprises an IL-2 molecule or fragment thereof transplanted into the CDR, and the antibody cytokine transplantation protein preferentially promotes the proliferation of T effector cells over regulatory T cells. In some embodiments, the antibody cytokine transplantation protein includes complementarity-determining regions HCDR1, HCDR2, HCDR3, and a heavy chain variable region (V H ) an antibody cytokine transplant protein containing and a light chain variable region (V) containing LCDR1, LCDR2, LCDR3 L ) and V H or V LThe regime comprises an IL-2 molecule or fragment thereof transplanted into a CDR, the IL-2 molecule being a mutain, and the antibody cytokine transplant protein preferentially proliferates T effector cells over regulatory T cells. In some embodiments, the IL-2 regime comprises administration of an antibody described in U.S. Patent Application Publication US2020 / 0270334, the disclosure of which is incorporated herein by reference. In some embodiments, the antibody cytokine transplant protein comprises a heavy chain variable region (V) including complementarity-determining regions HCDR1, HCDR2, HCDR3. H ) and the light chain variable region (V) including LCDR1, LCDR2, and LCDR3 L ) and V H or V L The antibody cytokine transplantation protein comprises an IL-2 molecule or fragment thereof transplanted into a CDR, wherein the IL-2 molecule is mutein, and the antibody cytokine transplantation protein preferentially proliferates T effector cells over regulatory T cells, and the antibody further comprises an IgG class heavy chain and an IgG class light chain selected from the group consisting of an IgG class light chain containing SEQ ID NO: 569 and an IgG class heavy chain containing SEQ ID NO: 568, an IgG class light chain containing SEQ ID NO: 567 and an IgG class heavy chain containing SEQ ID NO: 559, an IgG class light chain containing SEQ ID NO: 569 and an IgG class heavy chain containing SEQ ID NO: 559, and an IgG class light chain containing SEQ ID NO: 37 and an IgG class heavy chain containing SEQ ID NO: 568.

[0674] In some embodiments, the IL-2 molecule or a fragment thereof is V H The IL-2 molecule is implanted in HCDR1 and is mutain. In some embodiments, the IL-2 molecule or a fragment thereof is V H The IL-2 molecule is implanted in HCDR2 and is mutain. In some embodiments, the IL-2 molecule or a fragment thereof is V H The IL-2 molecule is implanted in HCDR3 and is mutain. In some embodiments, the IL-2 molecule or a fragment thereof is V L The IL-2 molecule is implanted in LCDR1 and is mutain. In some embodiments, the IL-2 molecule or a fragment thereof is V LThe IL-2 molecule is implanted in LCDR2 and is mutain. In some embodiments, the IL-2 molecule or a fragment thereof is V L It is transplanted into LCDR3, and the IL-2 molecule is mutain.

[0675] The insertion of the IL-2 molecule may occur in or near the N-terminal region of the CDR, in the central region of the CDR, or in or near the C-terminal region of the CDR. In some embodiments, the antibody-cytokine transplant protein contains an IL-2 molecule incorporated into the CDR, and the IL2 sequence does not frameshift the CDR sequence. In some embodiments, the antibody-cytokine transplant protein contains an IL-2 molecule incorporated into the CDR, and the IL2 sequence replaces all or part of the CDR sequence. The substitution by the IL-2 molecule may occur in or near the N-terminal region of the CDR, in the central region of the CDR, or in or near the C-terminal region of the CDR. The substitution by the IL-2 molecule may be just one or two amino acids in the CDR sequence, or it may be the entire CDR sequence.

[0676] In some embodiments, the IL-2 molecule is directly transplanted into the CDR without a peptide linker and without additional amino acids between the CDR sequence and the IL-2 sequence. In some embodiments, the IL-2 molecule is indirectly transplanted into the CDR using a peptide linker, with additional amino acids between the CDR sequence and the IL-2 sequence.

[0677] In some embodiments, the IL-2 molecule described herein is IL-2 mutein. In some cases, IL-2 mutein includes the R67A substitution. In some embodiments, IL-2 mutein includes amino acid sequence number 544 or 545. In some embodiments, IL-2 mutein includes the amino acid sequence in Table 1 of U.S. Patent Application Publication No. US2020 / 0270334, the disclosure of which is incorporated herein by reference.

[0678] In some embodiments, the antibody cytokine transplant protein includes HCDR1 selected from the group consisting of SEQ ID NOs: 546, 549, 552, and 555. In some embodiments, the antibody cytokine transplant protein includes HCDR1 selected from the group consisting of SEQ ID NOs: 7, 10, 543, and 546. In some embodiments, the antibody cytokine transplant protein includes HCDR1 selected from the group consisting of HCDR2 selected from the group consisting of SEQ ID NOs: 547, 550, 553, and 556. In some embodiments, the antibody cytokine transplant protein includes HCDR3 selected from the group consisting of SEQ ID NOs: 548, 551, 554, and 557. In some embodiments, the antibody cytokine transplant protein includes V containing the amino acid sequence of SEQ ID NOs: 558. H Includes a region. In some embodiments, the antibody cytokine transplant protein includes a heavy chain containing the amino acid sequence of SEQ ID NO: 559. In some embodiments, the antibody cytokine transplant protein includes a V containing the amino acid sequence of SEQ ID NO: 566. L Includes a region. In some embodiments, the antibody cytokine transplant protein includes a light chain containing the amino acid sequence of SEQ ID NO: 567. In some embodiments, the antibody cytokine transplant protein includes a V containing the amino acid sequence of SEQ ID NO: 28. H The region and V containing the amino acid sequence of SEQ ID NO: 566 LThe antibody cytokine transplant protein includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 559 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 567. In some embodiments, the antibody cytokine transplant protein includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 559 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 569. In some embodiments, the antibody cytokine transplant protein includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 568 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 567. In some embodiments, the antibody cytokine transplant protein includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 568 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 569. In some embodiments, the antibody cytokine transplant protein comprises IgG.IL2F71A.H1 or IgG.IL2R67A.H1 or its variants, derivatives, or fragments, or their conserved amino acid substitutions, or proteins having at least 80%, at least 90%, at least 95%, or at least 98% sequence identity thereto. In some embodiments, the antibody component of the antibody cytokine transplant protein described herein comprises an immunoglobulin sequence, a framework sequence, or a CDR sequence of palivizumab. In some embodiments, the antibody cytokine transplant protein described herein has a longer serum half-life than a wild-type IL-2 molecule, such as but not limited to aldesleukin or an equivalent molecule. [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4]

[0679] 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 by 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. Res2001, 2, 66-70. When activated by IL-4, Th2 T cells subsequently produce additional IL-4 in a positive feedback loop. IL-4 also stimulates B cell proliferation and the expression of class IIMHC, and induces class switching from B cells to the expression of IgE and IgG1. 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 (Cat. No. CYT-211) and ThermoFisher Scientific, Inc., Waltham, MA, USA (Human IL-15 recombinant protein, Cat. No. 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).

[0680] The term "IL-7" (also referred to herein as "IL7") refers to a glycosylated tissue-derived cytokine known as interleukin-7, which can be obtained from stromal and epithelial cells, as well as dendritic cells. Fry and Mackall, Blood 2002, 99, 3892-904. IL-7 can stimulate T cell development. IL-7 binds to the IL-7 receptor, a heterodimer consisting of the IL-7 receptor alpha and the common gamma chain receptor, which is a set of signals 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 (Cat. No. CYT-254) and ThermoFisher Scientific, Inc., Waltham, MA, USA (Human IL-15 recombinant protein, Cat. No. GibcoPHC0071). Table 2 shows the amino acid sequence of recombinant human IL-7 suitable for use in the present invention (SEQ ID NO: 6).

[0681] The term “IL-15” (also referred to herein as “IL15”) refers to the T cell growth factor known as interleukin-15 and includes all forms of IL-2, including human and mammalian forms, conserved amino acid substitutions, glycoforms, biosimilars, and variants thereof. 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 with a molecular weight of 12.8 kDa, containing 114 amino acids (and N-terminal methionine). Recombinant human IL-15 is commercially available from several suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (Cat. No. Recombinant Human IL-15) and ThermoFisher Scientific, Inc., Waltham, MA, USA (Human IL-15 Recombinant Protein, Cat. No. 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).

[0682] The term "IL-21" (also referred to herein as "IL21") refers to the pleiotropic cytokine protein known as interleukin-21, and includes all forms of IL-21, including human and mammalian forms, conserved amino acid substitutions, glycoforms, biosimilars, and variants thereof. 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 expressed on 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 with a molecular weight of 15.4 kDa and containing 132 amino acids. Recombinant human IL-21 is commercially available from several suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (Cat. No. CYT-408-b) and ThermoFisher Scientific, Inc., Waltham, MA, USA (Human IL-21 Recombinant Protein, Cat. 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).

[0683] Where “antitumor effective dose,” “tumor suppression effective dose,” or “therapeutic dose” is indicated, the exact amount of the composition of the present invention administered can be determined by a physician taking into account differences in the individual’s age, weight, tumor size, degree of infection or metastasis, and the patient’s (subject’s) condition. Generally, the pharmaceutical compositions comprising tumor-infiltrating lymphocytes (e.g., secondary TILs or genetically modified cytotoxic lymphocytes) described herein are 10 4 ~10 11 cells / kg body weight (e.g., 10 5 ~10 6 , 10 5 ~10 10 , 10 5 ~10 11 , 10 6 ~10 10 , 10 6 ~10 11 , 10 7 ~10 11 , 10 7 ~10 10 , 10 8 ~10 11 , 10 8 ~10 10 , 10 9 ~10 11 , or 10 9 ~10 10It may be administered in doses of cells / kg body weight, and it can be said that this includes all integer values ​​within these ranges. Tumor-infiltrating lymphocytes (including, in some cases, genetically modified cytotoxic lymphocytes) compositions may also be administered multiple times in these doses. Tumor-infiltrating lymphocytes (including, in some cases, genetically modified cytotoxic lymphocytes) may be administered 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 easily determined by a person skilled in the art of medicine by monitoring the patient for signs of the disease and adjusting the treatment as appropriate.

[0684] The terms "hematological malignancy" and "hematologic malignancy," or related terms, refer to cancers of mammals and tumors of hematopoietic and lymphoid tissues, including but not limited to the blood, bone marrow, lymph nodes, and lymphoid tissues. Hematological malignancies are also called "humoral malignancies." Hematological malignancies 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 malignancy" refers to hematological malignancies that affect B cells.

[0685] The term "solid tumor" refers to an abnormal mass of tissue that does not typically 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 tissue structure of a solid tumor includes interdependent tissue compartments, which consist of parenchyma (cancer cells) and supporting stromal cells that disperse the cancer cells and provide a supporting microenvironment.

[0686] The term “humoral tumor” refers to an abnormal mass of cells that is essentially fluid. Humoral tumor cancers include, but are not limited to, leukemia, myeloma, and lymphoma, as well as other hematological malignancies. TILs obtained from humoral tumors may also be referred to herein as bone marrow-infiltrating lymphocytes (MILs). TILs obtained from humoral tumors, including humoral 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.

[0687] As used herein, the term “microenvironment” may refer to the microenvironment of a solid or hematological tumor as a whole, or to individual subsets of cells within that microenvironment. As used herein, the tumor microenvironment refers to a complex mixture of “cells, water-soluble factors, signaling molecules, extracellular matrix, and mechanical cues” that promote neoplastic transformation, support tumor growth and invasion, protect tumors from host immunity, and promote treatment resistance. 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.

[0688] In some embodiments, the present invention includes a method for treating cancer with a TIL population, wherein the patient is pre-treated with non-myeloablative chemotherapy prior to the infusion of the TIL according to the present invention. In some embodiments, a TIL population may be provided, and the patient is pre-treated with non-myeloablative chemotherapy prior to the infusion of the TIL according to the present invention. In some embodiments, the non-myeloablative chemotherapy consists of cyclophosphamide 60 mg / kg / day for 2 days (days 27 and 26 prior to TIL infusion) and fludarabine 25 mg / m² 2 The dose is 60 mg / kg / day for 5 days (days 27-23 of TIL infusion). In some embodiments, non-myeloablative chemotherapy is cyclophosphamide 60 mg / kg / day for 2 days (days 27 and 26 prior to TIL infusion) and fludarabine 25 mg / m². 2The dose is 60 mg / kg / day for 3 days (days 27-25 of TIL infusion). In some embodiments, non-myeloablative chemotherapy is cyclophosphamide 60 mg / kg / day for 2 days (days 27 and 26 prior to TIL infusion) and fludarabine 25 mg / m². 2 This is for 3 days (days 25-23 of TIL infusion). In some embodiments, after non-myeloablative chemotherapy and TIL infusion according to the present invention (day 0), the patient receives intravenous infusion of IL-2 at 720,000 IU / kg every 8 hours until it reaches a physiologically acceptable level.

[0689] Experimental results indicate that lymphatic 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 lymphatic depletion step (also referred to as "immunosuppressive conditioning") in the patient before introducing the rTIL of the present invention.

[0690] As used herein, the terms “simultaneous administration,” “administered simultaneously,” “administered in combination,” “administered in combination,” “simultaneous,” and “concurren” encompass the administration of two or more active pharmaceutical ingredients (in preferred embodiments of the present invention, for example, at least one potassium channel agonist combined with multiple TILs) to a subject, so that the active pharmaceutical ingredients and / or their metabolites are present in the subject simultaneously. Simultaneous administration includes simultaneous administration in separate compositions, administration of separate compositions at different time points, or administration in a single composition containing two or more active pharmaceutical ingredients. Simultaneous administration in separate compositions and administration in a composition containing both drugs are preferred.

[0691] 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 an intended application, including but not limited to the treatment of a disease. The therapeutic effective dose may vary depending on the intended application (in vitro or in vivo), the subject and disease state being treated (e.g., the subject’s weight, age, and sex), the severity of the disease state, or the method of administration. The term also applies to doses that elicit a specific response in target cells (e.g., reduced platelet adhesion and / or cell migration). A specific dose may vary depending on the specific compound selected, the dosing plan 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.

[0692] As used herein, terms such as “treatment,” “to treat,” and “to cure” refer to obtaining a desired pharmacological and / or physiological effect. This effect may be prophylactic in that it completely or partially prevents the disease or symptoms thereof, and / or therapeutic in that it partially or completely cures the disease and / or any side effects resulting from it. As used herein, “treatment” encompasses any treatment of a disease in mammals, particularly humans, and includes (a) preventing the onset of the disease in subjects who are susceptible to or at risk of developing the disease but have not yet been diagnosed with it; (b) inhibiting the disease, i.e., preventing its manifestation or progression; and (c) mitigating the disease, i.e., causing its regression and / or reduction. “Treatment” also means the delivery of a drug to provide a pharmacological effect even in the absence of a disease or condition. For example, “treatment” includes the delivery of a composition that can induce an immune response or provide immunity in the absence of a disease condition, for example, in the case of a vaccine.

[0693] When used in part with nucleic acids or proteins, the term “heterogeneous” indicates that the nucleic acid or protein contains two or more sequences or subsequences that are not found in the same relationship to one another. For example, typically, nucleic acids are recombinantly produced to have 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 the other source, or coding regions from different sources. Similarly, heterogeneous proteins indicate that the protein contains two or more subsequences that are not found in the same relationship to one another in nature (e.g., fusion proteins).

[0694] In the context of two or more nucleic acids or polypeptides, the terms “sequence identity,” “percent identity,” and “sequence percent identity” (or their synonyms, e.g., “99% identity”) refer to two or more sequences or subsequences that, when compared and aligned (with gaps introduced as necessary) to obtain the greatest possible match, are identical or have an explicit percentage of the same nucleotide or amino acid residues, without considering conserved amino acid substitutions as part of sequence identity. Percent identity can be measured using sequence comparison software or algorithms, or by visual inspection. Various algorithms and software that can be used to obtain alignment of amino acid or nucleotide sequences are known in the art. Suitable programs for determining percent sequence identity include, for example, the BLAST suite of programs available from the BLAST website of the U.S. government's National Center for Biotechnology Information. Comparison between two sequences can be performed using either the BLASTN or BLASTP algorithm. BLASTN is used for comparing nucleic acid sequences, and BLASTP is used for comparing 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.

[0695] As used herein, the term “variant” includes, but is not limited to, a protein, antibody, or fusion protein having an amino acid sequence different from that of a reference antibody, antibody, or fusion protein due to one or more substitutions, deletions, and / or additions within or adjacent to the amino acid sequence of the reference antibody, antibody, or fusion protein. A variant may have one or more conservative substitutions in its amino acid sequence compared to that of the reference antibody. Conservative substitutions may include, for example, substitutions of similarly charged or uncharged amino acids. A variant retains the ability to specifically bind to the antigen of the reference antibody, protein, or fusion protein. The term “variant” also includes pegylated antibodies or proteins.

[0696] In this specification, "tumor-infiltrating lymphocytes" or "TILs" refers to the initial population of cells obtained as leukocytes that have left the bloodstream and migrated into the tumor. TILs include CD8 + Cytotoxic T cells (lymphocytes), Th1 and Th17CD4 + 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 obtained from a patient's tissue sample as outlined herein (and may be referred to as "freshly obtained" or "freshly isolated"), and "secondary TILs" are any proliferation or proliferationd TIL cell populations as considered herein, and include, but are not limited to, bulk TILs and proliferationd TILs ("REP TILs"), as well as "reREP TILs," as considered herein. reREP TILs may include, for example, a second proliferation TIL or a second additional proliferation TIL (e.g., a TIL referred to as a reREP TIL, as described in step D of Figure 1).

[0697] TILs can generally be defined biochemically using cell surface markers or functionally by their ability to infiltrate tumors and influence treatment. 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. In addition, or alternatively, TILs can be functionally defined by their ability to infiltrate solid tumors upon reintroduction into a patient. TILs can be further characterized by potency; for example, a TIL may be considered potent if the release of interferon (IFN) is greater than approximately 50 pg / mL, 100 pg / mL, 150 pg / mL, or 200 pg / mL. For example, a TIL may be considered effective if the interferon (IFNγ) release is approximately 50 pg / mL, greater than approximately 100 pg / mL, greater than approximately 150 pg / mL, or greater than approximately 200 pg / mL, greater than approximately 300 pg / mL, greater than approximately 400 pg / mL, greater than approximately 500 pg / mL, greater than approximately 600 pg / mL, greater than approximately 700 pg / mL, greater than approximately 800 pg / mL, greater than approximately 900 pg / mL, or greater than approximately 1000 pg / mL.

[0698] The terms “pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” are intended to include any and all solvents, dispersions, coatings, antibacterial and antifungal agents, isotonic and absorption retardants, and inactive components. The use of such pharmaceutically acceptable carriers or pharmaceutically acceptable excipients with active pharmaceutical ingredients is well known in the art. Unless a conventional pharmaceutically acceptable carrier or pharmaceutically acceptable excipient is incompatible with the active pharmaceutical ingredient, its use in the therapeutic compositions of the present invention is intended. Additional active pharmaceutical ingredients, such as other drugs, may also be incorporated into the compositions and methods described.

[0699] The terms “about” and “approximately” mean values ​​within a statistically meaningful range. Such a range may be within one order of magnitude 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 test and is readily understandable to 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 characteristics are not, and do not need to be, exact, and may be approximate and / or greater or less, reflecting tolerances, conversion factors, rounding, measurement errors, and other factors known to those skilled in the art, as necessary. In general, dimensions, sizes, formulations, parameters, shapes, or other quantities or characteristics are “about” or “approximately,” whether so explicitly stated. It should be noted that very different embodiments of size, shape, and dimensions may adopt the configurations described.

[0700] The transitional phrases “contains,” “essentially consists of,” and “consist of,” when used in the original and modified forms in the appended claims, define the claims in terms of whether any additional undescribed claim elements or steps are excluded from the claims. The term “contains” is intended to be inclusive or non-exclusive and does not exclude any additional undescribed elements, methods, steps, or materials. The term “consist of” excludes elements, steps, or materials other than those specified in the claims, and in the latter case, excludes impurities that would ordinarily be associated with the specified materials. The term “essentially consists of” limits the claims to such an extent that it does not substantially affect the specified elements, steps, or materials or the essential and novel features of the claimed invention. All compositions, methods, and kits described herein that embody the present invention may be more specifically defined in alternative embodiments by any of the transitional phrases “contains,” “essentially consists of,” and “consist of.”

[0701] II. TIL Manufacturing Process (Gen3 Process Embodiment, optionally including limited culture medium) In addition to the methods described herein, International Application No. PCT / US2019 / 059718 is incorporated herein by reference in its entirety for all purposes. Not limited to any particular theory, it is believed that the priming of a first proliferation that primes T cell activation, followed by a rapid second proliferation that promotes T cell activation, as described in the methods of the present invention, enables 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, which is primed by exposure to a first culture supernatant obtained from a first culture of APCs supplemented with an anti-CD3 antibody (e.g., OKT-3), IL-2, and optionally antigen-presenting cells (APCs), or IL-2 and anti-CD3 antibody (e.g., OKT-3), and then boosted by subsequent exposure to a second culture supernatant obtained from a second culture of APCs supplemented with additional anti-CD3 antibody (e.g., OKT-3), IL-2, and APCs, or additional IL-2 and anti-CD3 antibody (e.g., OKT-3), is thought to limit or avoid T cell maturation in culture, as taught by the method of the present invention, resulting in a population of T cells with a less mature phenotype, which are thought to be less depleted by proliferation in culture and exhibit greater cytotoxicity against cancer cells. In some embodiments, the rapid second proliferation step is divided into multiple steps to achieve scaling up the culture by: (a) performing rapid second proliferation by culturing T cells in a small culture in a first container, e.g., a G-REX100MCS container, for about 3-4 days; and then (b) resulting in the transfer of T cells in the small culture to a larger second container, e.g., a G-REX500MCS container, and culturing the T cells from the small culture in the larger culture in the second container for about 4-7 days.In some embodiments, the rapid growth step is divided into multiple steps to achieve scaling out of the culture by: (a) carrying out rapid second growth by culturing the T cells of a first small culture in a first container, e.g., a G-REX100MCS container, for about 3-4 days; and then (b) resulting in the transfer and allocation of 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, in each second container a portion of the T cells from the first small culture that have been transferred to the second container are cultured in the second small culture for about 4-7 days. In some embodiments, the rapid growth step is divided into multiple steps to achieve scaling out of the culture by: (a) performing a rapid second growth by culturing a small culture of T cells in a first container, e.g., a G-REX100MCS container, for about 3-4 days; and then (b) resulting in the transfer and assignment of the T cell small culture from the first container 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., a G-REX500MCS container, which are larger in size than the first container, and in each second container, a portion of the T cells from the small culture transferred to the second container are cultured in the larger culture for about 4-7 days. In some embodiments, the rapid growth step is divided into multiple steps to achieve scaling out of the culture by: (a) performing a rapid second growth by culturing a small culture of T cells in a first container, e.g., a G-REX100MCS container, for 4 days; and then (b) resulting in the transfer and allocation of the T cell culture from the first small culture to two, three, or four second containers larger than the first container, e.g., G-REX500MCS containers, in each of the second containers, a portion of the T cells from the small culture transferred to the second container are cultured in the larger culture for about 5 days.

[0702] In some embodiments, rapid second proliferation occurs after the T cell activation brought about by the priming of the first proliferation has begun to decrease, decline, attenuate, or sedate.

[0703] In some embodiments, rapid second proliferation occurs when the T cell activity brought about by priming of the first proliferation is reduced to 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, It is implemented after a decrease of 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%, or approximately that percentage.

[0704] In some embodiments, rapid second proliferation is carried out after the T cell activity brought about by the priming of the first proliferation has decreased by about 1% to 100%, or approximately that percentage.

[0705] In some embodiments, rapid second proliferation is performed after the T cell activity brought about by the priming of the first proliferation has decreased by approximately 1%–10%, 10%–20%, 20%–30%, 30%–40%, 40%–50%, 50%–60%, 60%–70%, 70%–80%, 80%–90%, or 90%–100%, or approximately that percentage.

[0706] In some embodiments, rapid second proliferation occurs when the T cell activity brought about by priming of the first proliferation is 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, 44, 45, It is implemented after a decrease of 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%, or approximately that percentage.

[0707] In some embodiments, rapid second proliferation increases the activity of T cells affected by priming during the first proliferation to 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, It is implemented after a decrease of 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%, or up to approximately that percentage.

[0708] In some embodiments, the decrease in T cell activity resulting from the priming of the first proliferation is determined by a decrease in the amount of interferon-gamma released by T cells in response to antigen stimulation.

[0709] In some embodiments, the priming of the first proliferation of T cells is carried out over a period of up to 7 or 8 days, or approximately that number of days.

[0710] In some embodiments, the priming of the first proliferation of T cells is carried out over a period of up to 1, 2, 3, 4, 5, 6, 7, or 8 days or up to approximately that number of days.

[0711] In some embodiments, the priming of the first proliferation of T cells is carried out over a period of 1, 2, 3, 4, 5, 6, 7, or 8 days.

[0712] In some embodiments, the rapid second proliferation of T cells takes place over a period of up to 11 days or approximately that duration.

[0713] In some embodiments, the rapid second proliferation of T cells takes place over a maximum of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 days, or approximately that duration.

[0714] In some embodiments, the rapid second proliferation of T cells takes place over a period of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 days.

[0715] In some embodiments, the priming of the first proliferation of T cells takes place over a period of 1 to 7 days or approximately that period, and the rapid second proliferation of T cells takes place over a period of 1 to 11 days or approximately that period.

[0716] In some embodiments, the priming of the first proliferation of T cells takes place over a period of up to 1, 2, 3, 4, 5, 6, 7, or 8 days or approximately that many days, and the rapid second proliferation of T cells takes place over a period of up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 days or approximately that many days.

[0717] In some embodiments, the priming of the first proliferation of T cells takes place over a period of 1 to 8 days or approximately that period, and the rapid second proliferation of T cells takes place over a period of 1 to 9 days or approximately that period.

[0718] In some embodiments, the priming of the first T cell proliferation is carried out for 8 days, and the rapid second T cell proliferation is carried out for 9 days.

[0719] In some embodiments, the priming of the first proliferation of T cells takes place over a period of 1 to approximately 7 days or roughly that duration, and the rapid second proliferation of T cells takes place over a period of 1 to approximately 9 days or roughly that duration.

[0720] In some embodiments, the first priming of T cell proliferation is carried out for 7 days, and the rapid second proliferation of T cells is carried out for 9 days.

[0721] In some embodiments, T cells are tumor-infiltrating lymphocytes (TILs).

[0722] In some embodiments, T cells are medullary lymphocytes (MILs).

[0723] In some embodiments, T cells are peripheral blood lymphocytes (PBLs).

[0724] In some embodiments, T cells are obtained from a donor who has cancer.

[0725] In some embodiments, the T cells are TILs obtained from tumors excised from patients with cancer.

[0726] In some embodiments, the T cells are MILs obtained from the bone marrow of patients suffering from hematological malignancies.

[0727] 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 cancer is selected from the group consisting of melanoma, ovarian cancer, endometrial cancer, thyroid cancer, colorectal cancer, cervical cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, cancer derived from 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, triple-negative breast cancer, cancer derived from 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 humoral tumor. In some embodiments, the tumor is a solid tumor. In some embodiments, the donor has a hematological malignancy.

[0728] In certain embodiments of this disclosure, immunoeffector cells, such as T cells, may be obtained from blood units collected from a subject using any number of techniques known to those skilled in the art, such as Ficoll isolation. In one preferred embodiment, cells derived from the circulating blood of an individual are obtained by apheresis. The apheresis product typically contains lymphocytes, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes, and platelets, including T cells. In one embodiment, the cells collected by apheresis may be washed to remove the plasma fraction, and optionally, the cells may be placed in a buffer or medium suitable for subsequent processing steps. In one embodiment, the cells are washed with phosphate-buffered saline (PBS). In alternative embodiments, the washing solution may be calcium-deficient, magnesium-deficient, or lack many but 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 countercurrent centrifugation.

[0729] In some embodiments, the T cells are PBLs isolated from whole blood or lymphocyte-enriched 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, endometrial cancer, thyroid cancer, colorectal cancer, cervical cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, cancer derived from 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, triple-negative breast cancer, cancer derived from 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 humoral tumor. In some embodiments, the tumor is a solid tumor. In some embodiments, the donor has a hematological malignancy. In some embodiments, in the case of a T cell phenotype, PBLs are isolated from whole blood or lymphocyte-enriched apheresis products by using a positive or negative selection method, i.e., using a marker, e.g., CD3+CD45+, or by removing non-T cell phenotype cells to leave the PBLs. In other embodiments, PBLs are isolated by gradient centrifugation. Once PBLs are isolated from the donor tissue, a first proliferation priming of PBLs is performed according to the first proliferation priming step of any of the methods described herein, in a first proliferation priming culture containing a suitable number of isolated PBLs (in some embodiments, approximately 1 × 10⁶). 7 It can be started by sowing seeds (PBL).

[0730] An exemplary TIL process known as Process 3 (also referred to herein as GEN3 or Gen3), which includes some of these features, is shown in Figure 1 (especially, for example, Figures 1B and / or 1C and / or 1E and / or 1F and / or 1G), and some of the advantages of this embodiment of the present invention over Process 2A are described in Figures 1 and 2 (especially, for example, Figures 1B and / or 1C and / or 1E and / or 1F and / or 1G). Two embodiments of Process 3 are shown in Figures 1 and 30 (especially, for example, Figures 1B and / or 1C and / or 1E and / or 1F and / or 1G). Process 2A or Gen2 is described in U.S. Patent Publications 2018 / 0280436 and 2019 / 0231820, which are incorporated herein by reference in their entirety.

[0731] As discussed and outlined herein, TILs are taken from patient samples and manipulated to increase their number before implantation in the patient using a TIL proliferation process described herein and referred to as Gen3. In some embodiments, TILs are manipulated as described below as optional. 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.

[0732] In some embodiments, the first growth priming (a process referred herein as pre-rapid growth (Pre-REP), and including the process shown in Figure 1 (especially, for example, Figures 1B and / or 1C and / or 1E and / or 1F and / or 1G) as step B) 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 in Figure 1 (especially, for example, Figures 1B and / or 1C and / or 1E and / or 1F and / or 1G) as step D) is shortened to 1 to 9 days, which are described in detail below and in the examples and figures. In some embodiments, the first growth priming (a process referred herein as Pre-Rapid Growth (Pre-REP), and including the process shown in Figure 1 (especially, for example, Figures 1B and / or 1C and / or 1E and / or 1F and / or 1G) as step B) 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 in Figure 1 (especially, for example, Figures 1B and / or 1C and / or 1E and / or 1F and / or 1G) as step D) is shortened to 1 to 8 days, which are described below and in the examples and figures. As described below, in some embodiments, the first growth priming (a process referred herein to as pre-rapid growth (Pre-REP), and including as step B the process shown in Figure 1 (especially, for example, Figure 1B and / or Figure 1C and / or Figure 1E and / or Figure 1F and / or Figure 1G)) is shortened to 1 to 7 days, and the rapid second growth (a process referred herein to as rapid growth protocol (REP), and including as step D the process shown in Figure 1 (especially, for example, Figure 1B and / or Figure 1C and / or Figure 1E and / or Figure 1F and / or Figure 1G)) is shortened to 1 to 9 days, which are described in detail below and in the examples and figures.In some embodiments, the first growth priming (a process referred herein as pre-rapid growth (Pre-REP), and including the process shown in Figure 1 (especially, for example, Figures 1B and / or 1C and / or 1E and / or 1F and / or 1G) as step B) is 1 to 7 days, and the rapid second growth (a process referred herein as rapid growth protocol (REP), and including the process shown in Figure 1 (especially, for example, Figures 1B and / or 1C and / or 1E and / or 1F and / or 1G) as step D) is 1 to 10 days, which are described in detail below and in the examples and figures. In some embodiments, the priming of the first growth (e.g., the growth described in step B of Figure 1 (e.g., in particular, e.g., Figures 1B and / or 1C and / or 1E and / or 1F and / or 1G)) is shortened to 8 days, and the rapid second growth (e.g., the growth described in step D of Figure 1 (e.g., in particular, e.g., Figures 1B and / or 1C and / or 1E and / or 1F and / or 1G)) is 7 to 9 days. In some embodiments, the first growth priming (e.g., the growth described in step B of Figure 1 (especially, e.g., Figures 1B and / or 1C and / or 1E and / or 1F and / or 1G)) is shortened to 7 days, and the rapid second growth (e.g., the growth described in step D of Figure 1 (especially, e.g., Figures 1B and / or 1C and / or 1E and / or 1F and / or 1G)) is 7 to 8 days.In some embodiments, the priming of the first growth (e.g., the growth described in step B of Figure 1 (e.g., in particular, e.g., Figures 1B and / or 1C and / or 1E and / or 1F and / or 1G)) is shortened to 8 days, and the rapid second growth (e.g., the growth described in step D of Figure 1 (e.g., in particular, e.g., Figures 1B and / or 1C and / or 1E and / or 1F and / or 1G)) is 8 days. In some embodiments, the priming of the first growth (e.g., the growth described in step B of Figure 1 (e.g., in particular, e.g., Figures 1B and / or 1C and / or 1E and / or 1F and / or 1G)) is 8 days, and the rapid second growth (e.g., the growth described in step D of Figure 1 (e.g., in particular, e.g., Figures 1B and / or 1C and / or 1E and / or 1F and / or 1G)) is 9 days. In some embodiments, the priming of the first growth (e.g., the growth described in step B of Figure 1 (e.g., in particular, e.g., in Figure 1B and / or in Figure 1C and / or in Figure 1E and / or in Figure 1F and / or in Figure 1G)) is 8 days, and the rapid second growth (e.g., the growth described in step D of Figure 1 (e.g., in particular, e.g., in Figure 1B and / or in Figure 1C and / or in Figure 1E and / or in Figure 1F and / or in Figure 1G)) is 10 days. In some embodiments, the priming of the first growth (e.g., the growth described in step B of Figure 1 (e.g., in particular, e.g., in Figure 1B and / or in Figure 1C and / or in Figure 1E and / or in Figure 1F and / or in Figure 1G)) is 7 days, and the rapid second growth (e.g., the growth described in step D of Figure 1 (e.g., in particular, e.g., in Figure 1B and / or in Figure 1C and / or in Figure 1E and / or in Figure 1F and / or in Figure 1G)) is 7 to 10 days. In some embodiments, the first growth priming (e.g., the growth described in step B of Figure 1 (in particular, e.g., Figures 1B and / or 1C and / or 1E and / or 1F and / or 1G)) is 7 days, and the rapid second growth (e.g., the growth described in step D of Figure 1 (in particular, e.g., Figures 1B and / or 1C and / or 1E and / or 1F and / or 1G)) is 8 to 10 days.In some embodiments, the priming of the first growth (e.g., the growth described in step B of Figure 1 (e.g., in particular, e.g., in Figure 1B and / or in Figure 1C and / or in Figure 1E and / or in Figure 1F and / or in Figure 1G)) is 7 days, and the rapid second growth (e.g., the growth described in step D of Figure 1 (e.g., in particular, e.g., in Figure 1B and / or in Figure 1C and / or in Figure 1E and / or in Figure 1F and / or in Figure 1G)) is 9 to 10 days. In some embodiments, the priming of the first growth (e.g., the growth described in step B of Figure 1 (e.g., in particular, e.g., in Figure 1B and / or in Figure 1C and / or in Figure 1E and / or in Figure 1F and / or in Figure 1G)) is shortened to 7 days, and the rapid second growth (e.g., the growth described in step D of Figure 1 (e.g., in particular, e.g., in Figure 1B and / or in Figure 1C and / or in Figure 1E and / or in Figure 1F and / or in Figure 1G)) is 7 to 9 days. In some embodiments, the combination of a first growth priming and a rapid second growth (e.g., the growth described in steps B and D of Figure 1 (especially, e.g., Figures 1B and / or 1C and / or 1E and / or 1F and / or 1G)) lasts for 14 to 16 days, which are described below and in the examples and figures. In particular, certain embodiments of the present invention include a first growth priming step in which TILs are activated by exposure to an anti-CD3 antibody, e.g., OKT-3, in the presence of IL-2, or by exposure to an antigen in the presence of at least IL-2 and an anti-CD3 antibody, e.g., OKT3. In certain embodiments, the TILs activated in the above first growth priming step are a population of first TILs, i.e., a primary cell population.

[0733] The following “Steps” designations A, B, C, etc., refer to non-limiting examples in Figure 1 (e.g., Figure 1B and / or Figure 1C and / or Figure 1E and / or Figure 1F and / or Figure 1G) and to certain non-limiting embodiments described herein. The following and the order of steps in Figure 1 (e.g., Figure 1B and / or Figure 1C and / or Figure 1E and / or Figure 1F and / or Figure 1G) are 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.

[0734] A. Step A: Obtain a tumor sample from the patient. Generally, TILs are initially obtained from patient tumor samples ("primary TILs") or from circulating lymphocytes such as peripheral blood lymphocytes containing TIL-like features, and are subsequently grown into larger populations for further manipulation as described herein, optionally cryopreserved, and optionally evaluated for phenotypic and metabolic parameters as indicators of the health status of the TILs.

[0735] Patient tumor samples can be obtained through methods well known in the art, generally surgical excision, needle biopsy, or other means for obtaining a sample containing a mixture of tumor and TIL cells. Generally, tumor samples may originate from any solid tumor, including primary tumors, invasive tumors, or metastatic tumors. Tumor samples may also be humoral tumors, such as tumors obtained from hematological malignancies. Solid tumors may be any cancer type, including but not limited to breast, pancreatic, prostate, colorectal, lung, brain, kidney, stomach, and skin (including but not limited to squamous cell carcinoma, basal cell carcinoma, and melanoma). In some embodiments, cancers are selected from cervical cancer, head and neck cancer (including 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. Useful TILs are obtained from malignant melanoma tumors, as they have been reported to have particularly high levels of TILs in some embodiments.

[0736] Once obtained, tumor samples are generally cut into 1-8 mm strips using a sharp tool. 3 It is fragmented into small pieces, about 2-3 mm in size. 3 This 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 dissociation instrument). Tumor digests can be produced by placing the tumor in an enzymatic medium, mechanically dissociating the tumor for approximately 1 minute, then incubating in 5% CO2 at 37°C for 30 minutes, followed by 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 performing density gradient separation using FICOLL branched hydrophilic polysaccharides. Other methods known in the art, such as the method described in U.S. Patent Application Publication No. 2012 / 0244133 A1, can be used, and their disclosures are incorporated herein by reference. Any of the aforementioned methods can be used in any of the embodiments described herein for methods of growing TILs or treating cancer.

[0737] The tumor dissociation enzyme mixture may contain one or more dissociation (digestion) enzymes, including but not limited to collagenase (including any blend or type of collagenase), Accutase®, Accumax®, hyaluronidase, neutral protease (dispase), chymotrypsin, chymopapain, trypsin, caseinase, elastase, papain, type XIV protease (pronase), deoxyribonuclease I (DNase), trypsin inhibitors, other dissociation enzymes or proteolytic enzymes, and any combination thereof.

[0738] In some embodiments, the dissociated enzyme is reconstituted from a lyophilized enzyme. In some embodiments, the lyophilized enzyme is reconstituted with a certain amount of sterile buffer such as HBSS.

[0739] In some cases, collagenase (such as animal-free type 1 collagenase) is reconstituted in 10 ml of sterile HBSS or other buffer. The lyophilized stock enzyme may be at a concentration of 2892 PZ U / vial. In some embodiments, collagenase is reconstituted in 5 ml to 15 ml of buffer. In some embodiments, after reconstitution, the collagenase stock is approximately 100 PZ U / ml to approximately 400 PZ U / ml, for example, approximately 100 PZ U / ml to approximately 400 PZ U / ml, approximately 100 PZ U / ml to approximately 350 PZ U / ml, approximately 100 PZ U / ml to approximately 300 PZ U / ml, approximately 150 PZ U / ml to approximately 400 PZ U / ml, approximately 100 PZ U / ml, approximately 150 PZ U / ml, approximately 200 PZ U / ml, approximately 210 PZ U / ml, approximately 220 PZ U / ml, approximately 230 PZ U / ml, approximately 240 PZ U / ml, approximately 250 PZ U / ml, approximately 260 PZ U / ml, approximately 270 PZ U / ml, approximately 280 PZ U / ml, approximately 289.2 PZ U / ml, and approximately 300 PZ U / ml. The range is U / ml, approximately 350 PZ U / ml, or approximately 400 PZ U / ml.

[0740] In some embodiments, the neutral protease is reconstituted in 1 ml of sterile HBSS or other buffer. The lyophilized stock enzyme may be at a concentration of 175 DMCU / vial. The concentration of the lyophilized stock enzyme may be 175 DMC / mL. In some embodiments, after reconstitution, the neutral protease stock may have concentrations of approximately 100 DMC / mL to approximately 400 DMC / mL, for example, approximately 100 DMC / mL to approximately 400 DMC / mL, approximately 100 DMC / mL to approximately 350 DMC / mL, approximately 100 DMC / mL to approximately 300 DMC / mL, approximately 150 DMC / mL to approximately 400 DMC / mL, approximately 100 DMC / mL, approximately 110 DMC / mL The range is approximately 120 DMC / ml, 130 DMC / ml, 140 DMC / ml, 150 DMC / ml, 160 DMC / ml, 170 DMC / ml, 175 DMC / ml, 180 DMC / ml, 190 DMC / ml, 200 DMC / ml, 250 DMC / ml, 300 DMC / ml, 350 DMC / ml, or 400 DMC / ml.

[0741] In some embodiments, DNaseI is reconstituted in 1 ml of sterile HBSS or other buffer. The concentration of the lyophilized stock enzyme was 4 KU / vial. In some embodiments, after reconstitution, the DNaseI stock is in the range of approximately 1 KU / ml to 10 KU / ml, for example, approximately 1 KU / ml, approximately 2 KU / ml, approximately 3 KU / ml, approximately 4 KU / ml, approximately 5 KU / ml, approximately 6 KU / ml, approximately 7 KU / ml, approximately 8 KU / ml, approximately 9 KU / ml, or approximately 10 KU / ml.

[0742] In some embodiments, the enzyme stock may vary, so check the concentration of the freeze-dried stock and adjust the final amount of enzyme to be added to the digestion cocktail accordingly.

[0743] In some embodiments, the enzyme mixture includes a neutral protease, a DNase, and a collagenase.

[0744] In some embodiments, the enzyme mixture contains approximately 10.2 µl of neutral protease (0.36 DMCU / ml), 21.3 µl of collagenase (1.2 PZ / ml), and 250 µl of DNAseI (200 U / ml) in approximately 4.7 ml of sterile HBSS.

[0745] As described 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 undergoes 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. In some embodiments, the tumor is digested in an enzyme mixture containing collagenase, DNase, and hyaluronidase at 37°C in 5% CO2 for 1 to 2 hours by rotation. In some embodiments, the tumor is digested overnight at a constant rotation. In some embodiments, the tumor is digested overnight at 37°C in 5% CO2. In some embodiments, the whole tumor is combined with the enzymes to form a tumor digestion reaction mixture.

[0746] In some embodiments, the tumor is reconstituted with lyophilized enzymes in a sterile buffer. In some embodiments, the buffer is sterile HBSS.

[0747] In some embodiments, the enzyme mixture contains collagenase. In some embodiments, the collagenase is collagenase IV. In some embodiments, the working stock of collagenase is a 100 mg / ml 10× working stock.

[0748] In some embodiments, the enzyme mixture contains DNAse. In some embodiments, the working stock of DNAse is a 10,000 IU / ml 10× working stock.

[0749] In some embodiments, the enzyme mixture contains hyaluronidase. In some embodiments, the working stock of hyaluronidase is a 10 mg / ml 10× working stock.

[0750] In some embodiments, the enzyme mixture comprises 10 mg / ml collagenase, 1000 IU / ml DNAse, and 1 mg / ml hyaluronidase.

[0751] In some embodiments, the enzyme mixture comprises 10 mg / ml collagenase, 500 IU / ml DNAse, and 1 mg / ml hyaluronidase.

[0752] Generally, cell suspensions obtained from tumors are referred to as “primary cell populations” or “newly obtained” or “newly isolated” cell populations. In certain embodiments, a newly obtained TIL cell population is exposed to a cell culture medium containing antigen-presenting cells, IL-12, and OKT-3.

[0753] In some embodiments, fragmentation includes physical fragmentation, such as slicing and digestion. In some embodiments, fragmentation is physical fragmentation. In some embodiments, fragmentation is slicing. In some embodiments, fragmentation is digestion. In some embodiments, TILs can be initially cultured from enzymatic tumor digests and tumor fragments obtained from a patient. In some embodiments, TILs can be initially cultured from enzymatic tumor digests and tumor fragments obtained from a patient.

[0754] In some embodiments, if the tumor is a solid tumor, the tumor undergoes physical fragmentation after obtaining a tumor sample in step A (provided in Figure 1 (particularly, for example, Figures 1B and / or 1C and / or 1E and / or 1F and / or 1G)). In some embodiments, fragmentation occurs before cryopreservation. In some embodiments, fragmentation occurs after cryopreservation. In some embodiments, fragmentation occurs after obtaining the tumor but without cryopreservation. In some embodiments, the fragmentation step is performed in vitro or ex vivo. In some embodiments, the tumor is fragmented, and 10, 20, 30, 40, or more fragments or pieces are placed in each container for priming of the first growth. In some embodiments, the tumor is fragmented, and 30 or 40 fragments or pieces are placed in each container for priming of the first growth. In some embodiments, the tumor is fragmented, and 40 fragments or pieces are placed in each container for priming of the first growth. In some embodiments, the fragments include about 4 to about 50 pieces, and each fragment is about 27 mm 3 It has a volume of . In some embodiments, the multiple fragments have a total volume of about 1300 mm³. 3 ~approx. 1500mm 3 It contains approximately 30 to 60 fragments. In some embodiments, the fragments have a total volume of approximately 1350 mm³. 3 It contains about 50 fragments. In some embodiments, the multiple fragments consist of about 50 fragments with a total mass of about 1 gram to about 1.5 grams. In some embodiments, the multiple fragments consist of about 4 fragments.

[0755] In some embodiments, TILs are obtained from tumor fragments. In some embodiments, tumor fragments are obtained by sharp incision. In some embodiments, tumor fragments are approximately 1 mm in size. 3 ~10mm 3 In some embodiments, the tumor fragment is approximately 1 mm 3 ~8mm 3 In some embodiments, the tumor fragment is approximately 1 mm 3 In some embodiments, the tumor fragment is approximately 2 mm in size.3 In some embodiments, the tumor fragment is approximately 3 mm in size. 3 In some embodiments, the tumor fragment is approximately 4 mm. 3 In some embodiments, the tumor fragment is approximately 5 mm in size. 3 In some embodiments, the tumor fragment is approximately 6 mm 3 In some embodiments, the tumor fragment is approximately 7 mm 3 In some embodiments, the tumor fragment is approximately 8 mm in size. 3 In some embodiments, the tumor fragment is approximately 9 mm 3 In some embodiments, the tumor fragment is approximately 10 mm. 3 In some embodiments, the tumor fragment is 1-4 mm × 1-4 mm × 1-4 mm. In some embodiments, the tumor fragment is 1 mm × 1 mm × 1 mm. In some embodiments, the tumor fragment is 2 mm × 2 mm × 2 mm. In some embodiments, the tumor fragment is 3 mm × 3 mm × 3 mm. In some embodiments, the tumor fragment is 4 mm × 4 mm × 4 mm.

[0756] In some embodiments, the tumor is fragmented to minimize the amount of bleeding, necrosis, and / or adipose tissue in each portion. In some embodiments, the tumor is fragmented to minimize the amount of bleeding tissue in each portion. In some embodiments, the tumor is fragmented to minimize the amount of necrotic tissue in each portion. In some embodiments, the tumor is fragmented to minimize the amount of adipose tissue in each portion. In certain embodiments, the tumor fragmentation step is performed in vitro or ex vivo.

[0757] In some embodiments, tumor fragmentation is performed while preserving the internal structure of the tumor. In some embodiments, tumor fragmentation is performed without performing a cutting operation with a surgical scalpel. In some embodiments, TIL is obtained from the tumor digest. In some embodiments, the tumor digest 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 can be mechanically dissociated for approximately 1 minute. The solution can then be incubated in 5% CO2 at 37°C for 30 minutes, and then mechanically dissociated again for approximately 1 minute. After incubation again in 5% CO2 at 37°C for 30 minutes, the tumor can be mechanically dissociated a third time for approximately 1 minute. In some embodiments, if large tissue fragments are present after the third mechanical disruption, one or two further mechanical separations may be applied to the sample, with or without a further 30-minute incubation in 5% CO2 at 37°C. In some embodiments, if the cell suspension contains a large number of erythrocytes or dead cells at the end of the final incubation, density gradient separation using Ficoll may be performed to remove these cells.

[0758] In some embodiments, the cell suspension prior to the priming step of the first proliferation is referred to as the “primary cell population” or the “freshly obtained” or “freshly isolated” cell population.

[0759] In some embodiments, the cells may be optionally frozen after sample isolation (e.g., after obtaining a tumor sample and / or after obtaining a cell suspension from the tumor sample) and stored by freezing before proceeding to the proliferation described in step B, which is described in detail below and illustrated in Figure 1 (in particular, e.g., Figure B and / or Figure 1C and / or Figure 1E and / or Figure 1F and / or Figure 1G).

[0760] 1. TILs originating from core / small biopsy In some embodiments, TILs are initially obtained from a patient's tumor sample ("primary TIL") by core biopsy or a similar procedure, then grown into larger populations for further operations described herein, optionally cryopreserved, and optionally evaluated for phenotypic and metabolic parameters.

[0761] In some embodiments, a patient's tumor sample can be obtained using methods known in the Art, generally via small biopsy, core biopsy, needle biopsy, or other means for obtaining a sample containing a mixture of tumor 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 humoral tumor, such as a tumor obtained from a hematological malignancy. In some embodiments, a sample may originate from multiple small tumor samples or biopsies. In some embodiments, a sample may include multiple tumor samples originating from a single tumor from the same patient. In some embodiments, a sample may include multiple tumor samples originating from one, two, three, or four tumors from the same patient. In some embodiments, a sample may include multiple tumor samples originating from multiple tumors from the same patient. Solid tumors may be any type of cancer, including, but not limited to, breast, pancreatic, prostate, colorectal, lung, brain, kidney, stomach, and skin (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 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 they have been reported to have particularly high levels of TILs.

[0762] Generally, cell suspensions obtained from tumor cores or fragments are referred to as “primary cell populations” or “newly obtained” or “newly isolated” cell populations. In certain embodiments, the newly obtained TIL cell population is exposed to a cell culture medium containing antigen-presenting cells, IL-12, and OKT-3.

[0763] In some embodiments, if the tumor is metastatic and the primary lesion has been effectively treated / removed in the past, it may be necessary to remove one of the metastatic lesions. In some embodiments, the least invasive approach, if possible, is to remove a skin lesion or lymph nodes in the cervical or axillary region. In some embodiments, a skin lesion is removed or a biopsy thereof is removed. In some embodiments, a lymph node or a biopsy thereof is removed. In some embodiments, metastatic lesions of the lung or liver, or intraperitoneal or thoracic lymph nodes and their biopsies may be used.

[0764] In some embodiments, the tumor is a melanoma. In some embodiments, the small biopsy of the melanoma includes a mole or a portion thereof.

[0765] In some embodiments, the small biopsy is a punch biopsy. In some embodiments, the punch biopsy is obtained by pressing a circular blade against the skin. In some embodiments, the punch biopsy is obtained by pressing a circular blade against the skin, the suspected mole. In some embodiments, the punch biopsy is obtained by pressing a circular blade against the skin, and a round piece of skin is removed. In some embodiments, the small biopsy is a punch biopsy, and a round portion of the tumor is removed.

[0766] 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 growth is removed. In some embodiments, the small biopsy is an excisional biopsy in which the entire mole or growth is removed with a small border of normal-looking skin.

[0767] In some embodiments, the small biopsy is an incisional biopsy. In some embodiments, the small biopsy is an incisional biopsy, and only the most anomalous portion of the mole or growth is taken. In some embodiments, the small biopsy is an incisional biopsy, and the incisional biopsy is used when it cannot be completed by other techniques, such as when the suspected mole is very large.

[0768] In some embodiments, the small biopsy is a lung biopsy. In some embodiments, the small biopsy is obtained by bronchoscopy. Generally, with bronchoscopy, the patient is anesthetized, and a small instrument is inserted through the nose or mouth, down the throat, and into the bronchial passages, where the small instrument is used to remove tissue. In some embodiments, if the tumor or growth cannot be reached by bronchoscopy, transthoracotomy can be used. Generally, with transthoracotomy, the patient is also anesthetized, and a needle is inserted directly through the skin to the suspected site to remove a small tissue sample. In some embodiments, transthoracotomy may require interventional radiology (e.g., the use of X-rays 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 with endoscopic ultrasound (e.g., an endoscope with a light is inserted through the mouth into the esophagus). In some embodiments, the small biopsy is obtained surgically.

[0769] 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, a sample can be taken without hospitalization. In some embodiments, if the tumor is deep 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 lump 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 portion of the suspicious area is removed using punch forceps.

[0770] In some embodiments, the small biopsy is a cervical biopsy. In some embodiments, the small biopsy is obtained by colposcopy. Generally, colposcopy involves using an illuminated proliferative instrument attached to a magnifying binocular (colposcope) to biopsy a small portion 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, and outpatient surgery may be required to remove a larger tissue fragment from the cervix. In some embodiments, in addition to helping confirm the diagnosis, a cone biopsy may serve as an initial treatment.

[0771] The term “solid tumor” refers to an abnormal mass of tissue that does not typically 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, triple-negative breast cancer, prostate, colon, rectum, and bladder cancers. 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, which include parenchyma (cancer cells) and supporting stromal cells that disperse the cancer cells and provide a supporting microenvironment.

[0772] 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 microbiopsies, 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 microbiopsies, 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 microbiopsies, the samples are first placed in the G-Rex 500.

[0773] FNA can be obtained from tumors selected from the group consisting of lung, melanoma, head and neck, cervical, ovarian, pancreatic, 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, NSCLC patients have previously undergone surgical treatment.

[0774] The TILs described herein can be obtained from FNA samples. In some cases, FNA samples are obtained or isolated from patients using fine-gauge needles ranging from 18-gauge to 25-gauge. Fine-gauge needles may be 18-gauge, 19-gauge, 20-gauge, 21-gauge, 22-gauge, 23-gauge, 24-gauge, or 25-gauge. In some embodiments, the FNA sample from the patient 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, 900,000 TILs, 950,000 TILs, or more.

[0775] In some cases, the TILs described herein are obtained from a core biopsy sample. In some cases, the core biopsy sample is obtained or isolated from a patient using a surgical or medical needle ranging from 11 gauge to 16 gauge. The needle may be 11 gauge, 12 gauge, 13 gauge, 14 gauge, 15 gauge, or 16 gauge. In some embodiments, the core biopsy sample from a patient 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, 900,000 TILs, 950,000 TILs, or more.

[0776] Generally, a collected cell suspension is called a "primary cell population" or a "freshly collected" cell population.

[0777] In some embodiments, TILs are not obtained from tumor digests. In some embodiments, solid tumors are not fragmented.

[0778] In some embodiments, TILs are obtained from tumor digests. In some embodiments, tumor digests are 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 (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 in 5% 2CO at 37°C for 30 minutes, and then mechanically disrupted again for approximately 1 minute. After incubation again in 5% CO2 at 37°C for 30 minutes, the tumor can be mechanically disrupted for a third time for approximately 1 minute. In some embodiments, if large tissue fragments are present after the third mechanical disruption, one or two further mechanical dissociations may be applied to the sample, with or without a further 30-minute incubation in 5% CO2 at 37°C. In some embodiments, if the cell suspension contains a large number of red blood cells or dead cells at the end of the final incubation, density gradient separation using Ficoll can be performed to remove these cells.

[0779] In some embodiments, obtaining a first TIL population involves a multi-lesion sampling method.

[0780] The tumor dissociation enzyme mixture may contain one or more dissociation (digestion) enzymes, including but not limited to collagenase (including any blend or type of collagenase), Accutase®, Accumax®, hyaluronidase, neutral protease (dispase), chymotrypsin, chymopapain, trypsin, caseinase, elastase, papain, type XIV protease (pronase), deoxyribonuclease I (DNase), trypsin inhibitors, other dissociation enzymes or proteolytic enzymes, and any combination thereof.

[0781] In some embodiments, the dissociated enzyme is reconstituted from a lyophilized enzyme. In some embodiments, the lyophilized enzyme is reconstituted with a certain amount of a sterile buffer such as HBSS.

[0782] In some cases, collagenase (such as animal-free type 1 collagenase) is reconstituted in 10 ml of sterile HBSS or other buffer. The lyophilized stock enzyme may be at a concentration of 2892 PZ U / vial. In some embodiments, collagenase is reconstituted in 5 ml to 15 ml of buffer. In some embodiments, after reconstitution, the collagenase stock is approximately 100 PZ U / ml to approximately 400 PZ U / ml, for example, approximately 100 PZ U / ml to approximately 400 PZ U / ml, approximately 100 PZ U / ml to approximately 350 PZ U / ml, approximately 100 PZ U / ml to approximately 300 PZ U / ml, approximately 150 PZ U / ml to approximately 400 PZ U / ml, approximately 100 PZ U / ml, approximately 150 PZ U / ml, approximately 200 PZ U / ml, approximately 210 PZ U / ml, approximately 220 PZ U / ml, approximately 230 PZ U / ml, approximately 240 PZ U / ml, approximately 250 PZ U / ml, approximately 260 PZ U / ml, approximately 270 PZ U / ml, approximately 280 PZ U / ml, approximately 289.2 PZ U / ml, and approximately 300 PZ U / ml. The range is U / ml, approximately 350 PZ U / ml, or approximately 400 PZ U / ml.

[0783] In some embodiments, the neutral protease is reconstituted in 1 ml of sterile HBSS or other buffer. The lyophilized stock enzyme may be at a concentration of 175 DMCU / vial. The concentration of the lyophilized stock enzyme may be 175 DMC / mL. In some embodiments, after reconstitution, the neutral protease stock may have concentrations of approximately 100 DMC / mL to approximately 400 DMC / mL, for example, approximately 100 DMC / mL to approximately 400 DMC / mL, approximately 100 DMC / mL to approximately 350 DMC / mL, approximately 100 DMC / mL to approximately 300 DMC / mL, approximately 150 DMC / mL to approximately 400 DMC / mL, approximately 100 DMC / mL, approximately 110 DMC / mL The range is approximately 120 DMC / ml, 130 DMC / ml, 140 DMC / ml, 150 DMC / ml, 160 DMC / ml, 170 DMC / ml, 175 DMC / ml, 180 DMC / ml, 190 DMC / ml, 200 DMC / ml, 250 DMC / ml, 300 DMC / ml, 350 DMC / ml, or 400 DMC / ml.

[0784] In some embodiments, DNaseI is reconstituted in 1 ml of sterile HBSS or other buffer. The concentration of the lyophilized stock enzyme was 4 KU / vial. In some embodiments, after reconstitution, the DNaseI stock is in the range of approximately 1 KU / ml to 10 KU / ml, for example, approximately 1 KU / ml, approximately 2 KU / ml, approximately 3 KU / ml, approximately 4 KU / ml, approximately 5 KU / ml, approximately 6 KU / ml, approximately 7 KU / ml, approximately 8 KU / ml, approximately 9 KU / ml, or approximately 10 KU / ml.

[0785] In some embodiments, the enzyme stock may vary, so check the concentration of the freeze-dried stock and adjust the final amount of enzyme to be added to the digestion cocktail accordingly.

[0786] In some embodiments, the enzyme mixture comprises a neutral protease, a collagenase, and a DNase.

[0787] In some embodiments, the enzyme mixture contains approximately 10.2 µl of neutral protease (0.36 DMCU / ml), 21.3 µl of collagenase (1.2 PZ / ml), and 250 µl of DNAseI (200 U / ml) in approximately 4.7 ml of sterile HBSS.

[0788] 2. Pleural effusion TIL In some embodiments, the sample is a pleural fluid sample. In some embodiments, the source of TILs for growth by the process described herein is a pleural fluid sample. In some embodiments, the sample is a pleural fluid-derived sample. In some embodiments, the source of TILs for growth by the process described herein is a pleural fluid-derived sample. See, for example, the method described in U.S. Patent No. US2014 / 0295426, which is incorporated herein by reference in its entirety for all purposes.

[0789] In some embodiments, any pleural fluid or pleural effusion containing suspected and / or TILs may be used. Such samples may originate from primary or metastatic lung cancer such as NSCLC or SCLC. In some embodiments, the sample may be secondary metastatic cancer cells originating from another organ, e.g., the breast, ovary, colon, or prostate. In some embodiments, the sample for use in the growth method described herein is pleural exudate. In some embodiments, the sample for use in the growth method described herein is pleural effusion. Other biological samples may include other serous fluids containing TILs, e.g., ascites fluid from the abdomen or pancreatic cystic fluid. Ascites fluid and pleural fluid contain very similar chemical systems, and both the abdomen and lung, like malignant tumors, have mesothelial glands and fluid forms in the pleural and abdominal cavities, and in some embodiments, such fluids contain TILs. In some embodiments in which this disclosure exemplified pleural fluid, ascites fluid or other cystic fluid containing TILs may be used and carried out in the same manner having similar results.

[0790] In some embodiments, the pleural fluid is in its untreated form, as taken directly from the patient. In some embodiments, the untreated pleural fluid is placed in a standard blood collection tube, such as an EDTA or heparin tube, before the contact step. In some embodiments, the untreated pleural fluid is placed in a standard CellSave® tube (Veridex) before the contact step. In some embodiments, the sample is placed in a CellSave tube immediately after collection from the patient to avoid a reduction in the number of viable TILs. The number of viable TILs decreases significantly within 24 hours, even at 4°C, if left in untreated pleural fluid. In some embodiments, the sample is placed in a suitable collection tube within 1 hour, 5 hours, 10 hours, 15 hours, or up to 24 hours after excision from the patient. In some embodiments, the sample is placed in a suitable collection tube within 1 hour, 5 hours, 10 hours, 15 hours, or up to 24 hours after excision from the patient, at 4°C.

[0791] In some embodiments, a pleural fluid sample from a selected subject may be diluted. In one embodiment, the dilution is 1:10 pleural fluid to diluent. In another embodiment, the dilution is 1:9 pleural fluid to diluent. In another embodiment, the dilution is 1:8 pleural fluid to diluent. In another embodiment, the dilution is 1:5 pleural fluid to diluent. In another embodiment, the dilution is 1:2 pleural fluid to diluent. In another embodiment, the dilution is 1:1 pleural fluid to diluent. In some embodiments, the diluent includes saline, phosphate-buffered saline, other buffers, or physiologically acceptable diluents. In some embodiments, the sample is placed in a CellSave tube immediately after collection and dilution from the patient to avoid a decrease in viable TILs. This decrease can occur to a considerable extent within 24-48 hours, even at 4°C, if left in untreated pleural fluid. In some embodiments, the pleural fluid sample is collected from the patient and diluted, then placed in a suitable collection tube within 1 hour, 5 hours, 10 hours, 15 hours, 24 hours, 36 hours, and up to 48 hours. In some embodiments, the pleural fluid sample is collected from the patient and diluted, then placed in a suitable collection tube at 4°C within 1 hour, 5 hours, 10 hours, 15 hours, 24 hours, 36 hours, and up to 48 hours.

[0792] In some embodiments, the pleural fluid sample is concentrated by conventional means before further processing steps. In some embodiments, this pretreatment of the pleural fluid is preferred in situations where the pleural fluid must be cryopreserved for transport to the laboratory where the method is performed or for subsequent analysis (e.g., 24-48 hours after collection). In some embodiments, the pleural fluid sample is prepared by centrifugation after the pleural fluid sample is collected from the subject and the centrifugated product or pellet is resuspended in a buffer. In some embodiments, the pleural fluid sample is subjected to multiple centrifugations and resuspensions before transport or cryopreservation for subsequent analysis and / or processing.

[0793] In some embodiments, the pleural fluid sample is concentrated before further processing steps by using a filtration method. In some embodiments, the pleural fluid sample to be used in the contact step is prepared by filtering the fluid through a filter having a known and essentially uniform pore size that allows the pleural fluid to pass through a membrane but retains tumor cells. In some embodiments, the pore diameter of the membrane may be at least 4 μM. In other embodiments, the pore diameter may be 5 μM or greater, and in other embodiments, it may be 6, 7, 8, 9, or 10 μM. After filtration, the cells containing the TILs retained by the membrane can be washed from the membrane and placed in a suitable physiologically acceptable buffer. The cells containing the TILs thus concentrated can then be used in the contact step of the method.

[0794] In some embodiments, a pleural fluid sample (e.g., including untreated pleural fluid), diluted pleural fluid, or resuspended cell pellet is brought into contact with a lysis reagent that selectively lyses anucleated red blood cells present in the sample. In some embodiments, this step is performed before further processing steps, in situations where the pleural fluid contains a significant number of RBCs. Suitable lysis reagents include single lysis reagents or lysis reagents and quench reagents, or lysis reagents, quench reagents, and fixation reagents. Suitable lysis systems are commercially available and include the BD Pharm Lyse® system (Becton Dickenson). Other lysis systems include the Versalyse® system, FACSlyse® system (Becton Dickenson), Immunoprep® system or Erythrolyse II system (Beckman Coulter, Inc.), or ammonium chloride systems. In some embodiments, the lysis reagent may differ depending on the primary requirements, which are efficient lysis of red blood cells and preservation of the phenotypic properties of TILs in the pleural fluid. In addition to using a single reagent for dissolution, a dissolution system useful for the method described herein may include a second reagent, for example, one that quenches or delays the effect of the dissolving reagent during the remaining steps of the method, such as Stabilyse® reagent (Beckman Coulter). Depending on the choice of dissolving reagent or the preferred method, conventional fixation reagents may also be used.

[0795] In some embodiments, untreated, diluted, or multi-centrifugation or treated pleural fluid samples are frozen and stored at a temperature of about -140°C before further processing and / or development as provided herein.

[0796] 3. Methods for increasing peripheral blood lymphocytes (PBLs) from peripheral blood PBL Method 1. In some embodiments of the present invention, PBLs are grown using the processes described herein. In some embodiments of the present invention, the method comprises obtaining a PBMC sample from whole blood. In some embodiments, the method comprises enriching T cells by isolating pure T cells from PBMCs using negative selection of the non-CD19+ fraction. In some embodiments, the method comprises enriching T cells by isolating pure T cells from PBMCs using magnetic bead-based negative selection of the non-CD19+ fraction.

[0797] In some embodiments of the present invention, PBL method 1 is carried out 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).

[0798] PBL Method 2. In some 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 isolating non-adherent cells.

[0799] In some embodiments of the present invention, PBL method 2 is carried out as follows: On day 0, the cryopreserved PMBC sample is thawed, and 6 million PBMC cells per well are seeded in CM-2 medium in a 6-well plate and incubated at 37 degrees Celsius for 3 hours. After 3 hours, non-adherent cells, which are PBL, are removed and counted.

[0800] PBL Method 3. In some 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.

[0801] In some embodiments of the present invention, PBL method 3 is carried out 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). T cells from the non-CD19+ cell fraction are purified using the Human Pan T-cell Isolation Kit and LS Columns (Miltenyi Biotec).

[0802] In some embodiments, PBMCs are isolated from a whole blood sample. In some embodiments, the PBMC sample is used as a starting material for growing PBLs. In some embodiments, the sample is cryopreserved before the growth process. In other embodiments, a fresh sample is used as a starting material to grow PBLs. In some embodiments of the present invention, T cells are isolated from PBMCs using methods known in the art. In some embodiments, T cells are isolated using a Human Pan T-Cell Isolation Kit and LS columns. In some embodiments of the present invention, T cells are isolated from PBMCs using antibody selection methods known in the art, for example, negative selection of CD19.

[0803] In some embodiments of the present invention, a PBMC sample is incubated for a certain period of time at a desired temperature effective for identifying non-adherent cells. In some embodiments of the present invention, the incubation time is about 3 hours. In some embodiments of the present invention, the temperature is about 37°C. The non-adherent cells are then grown using the process described above.

[0804] In some embodiments, PBMC samples are from subjects or patients optionally previously treated with a regimen containing a kinase inhibitor or ITK inhibitor. In some embodiments, tumor samples are from subjects or patients previously treated with a regimen containing a kinase inhibitor or ITK inhibitor. In some embodiments, PBMC samples are from subjects or patients previously treated with a regimen containing a kinase inhibitor or ITK inhibitor for at least one month, at least two months, at least three months, at least four months, at least five months, at least six months, or one year or longer. In other embodiments, PBMCs are derived from patients currently receiving an ITK inhibitor regimen such as ibrutinib.

[0805] In some embodiments, the PBMC samples are from subjects or patients who have been pre-treated with a regimen containing a kinase inhibitor or an ITK inhibitor and are resistant to treatment with the kinase inhibitor or an ITK inhibitor such as ibrutinib.

[0806] 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 more than one year. In other embodiments, PBMCs are derived from patients who have been previously exposed to an ITK inhibitor but have not received treatment for at least three months, at least six months, at least nine months, or at least one year.

[0807] In one embodiment of the present invention, cells are selected for CD19+ on day 0 and sorted accordingly. In some embodiments of the present invention, the selection is performed using antibody-conjugated beads. In some embodiments of the present invention, pure T cells are isolated from PBMCs on day 0.

[0808] In some embodiments of the present invention, in patients who have not been previously treated with ibrutinib or other ITK inhibitors, 10-15 ml of buffy coat is used to approximately 5 × 10 9 It produces PBMCs, which then grow to approximately 5.5 × 10⁻⁶ 7 It produces PBL (Problem-Based Learning).

[0809] In some embodiments of the present invention, in patients previously treated with ibrutinib or other ITK inhibitors, the proliferation process is approximately 20 × 10 9 This leads to PBL. In some embodiments of the present invention, 40.3 × 10 6 The PBMC is approximately 4.7 x 10 5 This brings about PBL (Project-Based Learning).

[0810] In any of the embodiments described above, the PBMC may be derived from a whole blood sample by apheresis, a buffy coat, or any other method known in the art for obtaining a PBMC.

[0811] 4. Method for increasing bone marrow-infiltrating lymphocytes (MILs) from bone marrow-derived PBMCs MIL Method 3. In some embodiments of the present invention, the method includes obtaining PBMCs from bone marrow. On day 0, PBMCs are selected and sorted for CD3+ / CD33+ / CD20+ / CD14+, the non-CD3+ / CD33+ / CD20+ / CD14+ cell fraction is sonicated, and a portion of the sonicated cell fraction is added back to the selected cell fraction.

[0812] In some embodiments of the present invention, MIL method 3 is carried out 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 S3e cell sorting (Bio-Rad). The cells are sorted into two fractions: an immunocell fraction (or MIL fraction) (CD3+CD33+CD20+CD14+) and an AML blast cell fraction (non-CD3+CD33+CD20+CD14+).

[0813] In some embodiments of the present invention, PBMCs are obtained from bone marrow. In some embodiments, PBMCs are obtained from bone marrow by apheresis, aspiration, needle biopsy, or other similar means known in the art. In some embodiments, the PBMCs are fresh. In other embodiments, the PBMCs are cryopreserved.

[0814] In some embodiments of the present invention, MIL is grown from 10 to 50 ml of bone marrow aspirate. In some embodiments of the present invention, 10 ml of bone marrow aspirate is obtained from the patient. In other embodiments, 20 ml of bone marrow aspirate is obtained from the patient. In other embodiments, 30 ml of bone marrow aspirate is obtained from the patient. In other embodiments, 40 ml of bone marrow aspirate is obtained from the patient. In other embodiments, 50 ml of bone marrow aspirate is obtained from the patient.

[0815] In some 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 It is PBBC. In other embodiments, the number of resulting PMBCs is approximately 7 × 10 7 This is a private-use mobile phone manufacturer (PBMC).

[0816] In some embodiments of the present invention, about 5 × 10 7 ~About 10×10 7 The PBMC is approximately 0.5 x 10 6 ~Approx. 1.5×10 6 This yields MIL. In some embodiments of the present invention, about 1 × 10 6 Brings MIL.

[0817] In some embodiments of the present invention, 12 × 10 6 The PBMC is approximately 1.4 x 10 5 Brings MIL.

[0818] In any of the embodiments described above, the PBMC 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.

[0819] B. Step B: Priming for the first proliferation In some embodiments, the method provides younger TILs that may offer additional therapeutic effects than older TILs (i.e., TILs that have undergone more replication rounds before being administered to the subject / patient). The characteristics of young TILs are described in the literature, for example, Donia, et al., Scandinavian Journal of Immunology, 75:157~167 (2012), Dudley et al., ClinCancerRes, 16:6122-6131 (2010), Huang et al., JImmunother, 28(3):258~267 (2005), Besser et al. al., ClinCancerRes,19(17):OF1-OF9(2013), Besser et al., JImmunother32:415~423(2009), Robbins, et al., JImmunol2004;173:7125-7130, Shen et al. al., J Immunother, 30:123~129 (2007), Zhou, et al. This is described in al., Jimmunother, 28:53-62 (2005), and Tran, et al., Jimmunother, 31:742-751 (2008), all of which are incorporated herein by reference in their entirety.

[0820] After slicing or digesting tumor fragments and / or tumor fragments, for example, as described in step A of Figure 1 (in particular, for example, Figures 1B and / or 1C and / or 1E and / or 1F and / or 1G), the resulting cells are cultured in serum containing IL-2, OKT-3, and feeder cells (e.g., antigen-presenting feeder cells) and / or OKT-3, 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., day 0). In some embodiments, the tumor digest and / or tumor fragments are incubated in containers containing up to 60 fragments per container. In some embodiments, the tumor digest and / or tumor fragments are incubated in containers containing up to 80 fragments per container. In some embodiments, the tumor digest and / or tumor fragments are incubated in containers containing up to 100 fragments per container. In some embodiments, tumor digests and / or tumor fragments are incubated in a container containing up to 60 fragments and 6000 IU / mL of IL-2 per container. In some embodiments, tumor digests and / or tumor fragments are incubated in a container containing up to 80 fragments and 6000 IU / mL of IL-2 per container. In some embodiments, tumor digests and / or tumor fragments are incubated in a container containing up to 100 fragments and 6000 IU / mL of IL-2 per container. In some embodiments, this primary cell population is cultured for several days, generally 1 to 8 days, to obtain a bulk TIL population, generally about 1 × 10⁶ 8 A bulk TIL population is obtained. In some embodiments, this period is referred to as activation I. In some embodiments, this primary cell population is cultured for a certain period, generally 1 to 7 days, to obtain a bulk TIL population, generally about 1 × 10⁶. 8 A bulk TIL population of approximately 1 × 10¹⁶ cells is obtained. In some embodiments, a first proliferation priming is performed for 1 to 8 days to obtain a bulk TIL population, generally about 1 × 10¹⁶ cells. 8A bulk TIL population of approximately 1 × 10¹⁶ cells is obtained. In some embodiments, a first proliferation priming is performed for 1 to 7 days to obtain a bulk TIL population, generally about 1 × 10¹⁶ cells. 8 A bulk TIL population of approximately 1 × 10¹⁶ cells is obtained. In some embodiments, a first proliferation priming is performed for 1 to 3 days to obtain a bulk TIL population, generally about 1 × 10¹⁶ cells. 8 A bulk TIL population of approximately 1 × 10¹⁶ cells is obtained. In some embodiments, a first proliferation priming is performed for 1 to 4 days ...

Claims

1. A method for increasing tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, wherein the method is: (a) Obtaining and / or receiving a first TIL population from tumors excised from a subject by processing a tumor sample obtained from the subject into multiple tumor fragments, (b) Performing a first proliferation priming by culturing the first TIL population in a first TIL cell culture, wherein the first TIL cell culture comprises a first cell culture medium, IL-2, i) A first culture supernatant obtained from a culture of first antigen-presenting feeder cells (APCs), the first culture supernatant containing OKT-3, or ii) Including either APC or OKT-3, The first proliferation priming is performed by culturing the first TIL cell culture in a first container having a first gas-permeable surface area for a first period of 1 to 7 or 1 to 8 days to obtain a second TIL population, wherein the second TIL population is larger in number than the first TIL population, and the first proliferation priming is performed. (c) To form a second TIL cell culture, to carry out rapid second proliferation by supplementing the first TIL cell culture, wherein the supplementation comprises additional first cell culture medium, IL-2, i) A second culture supernatant obtained from a culture of the second APC, which contains OKT-3, or ii) Including either APC or OKT-3, The rapid second proliferation is carried out by culturing the second TIL cell culture for a second period of 1 to 11 days to obtain a third TIL population, the third TIL population being a therapeutic population for TILs, the first TIL cell culture not containing either the first culture supernatant or APC, the second TIL cell culture not containing either the second culture supernatant or supplemental APC, the first cell culture not containing APC and / or the second cell culture not containing supplemental APC, and the rapid second proliferation is carried out as described above. (d) Collecting the therapeutic TIL population obtained from step (c), (e) The method comprising transferring the TIL population collected from step (d) to an infusion bag.

2. A method for growing TILs according to claim 1, wherein in step (b) the priming of the first proliferation, the first TIL cell culture comprises the first culture supernatant, and in step (c) the rapid second proliferation, the first TIL cell culture is supplemented with OKT-3 and APC to form the second TIL cell culture, or in step (b) the priming of the first proliferation, the first TIL cell culture comprises OKT-3 and APC, and in step (c) the rapid second proliferation, the first TIL cell culture is supplemented with the second culture supernatant to form the second TIL cell culture, or in step (b) the priming of the first proliferation, the first TIL cell culture comprises the first culture supernatant, and in step (c) the rapid second proliferation, the first TIL cell culture is supplemented with the second culture supernatant to form the second TIL cell culture.

3. To obtain the first culture supernatant for use in step (b), 1) To provide an APC cell culture medium containing IL-2 and OKT-3, 2) At least 5 × 10 in the APC cell culture medium from 1) ８ The first culture supernatant is produced by culturing the individual APCs for 3 to 4 days, 3) Collecting the first culture supernatant from the cell culture in 2), and / or, To obtain the second culture supernatant for use in step (c), 1) To provide an APC cell culture medium containing IL-2 and OKT-3, 2) At least 1 × 10 in the APC cell culture medium from 1). ７ The APCs are cultured for 3 to 4 days to produce the second culture supernatant, 3) Collecting the second culture supernatant from the cell culture in 2), and / or, The rapid second growth in step (c) i) A method for growing TILs according to claim 1, further comprising the step of supplementing the second TIL cell culture with additional IL-2 three or four days after the start of the second period in step (c).

4. The method according to claim 1, wherein the APC is exogenous to the subject and the APC is a peripheral blood mononuclear cell (PBMC).

5. The rapid second growth in step (c) i) After the start of the second period, or after 3 or 4 days, transfer the second TIL cell culture from the first container to a plurality of second containers to form a subculture of the second TIL cell culture in each of the plurality of second containers; ii) The method according to claim 1, further comprising the step of subculturing the second TIL cell culture in each of the plurality of second containers for the remainder of the second period.

6. The method according to claim 5, wherein in step i), an equal amount of the second TIL cell culture is transferred to the plurality of second containers.

7. The method according to claim 6, wherein the plurality of second containers are selected from the group consisting of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 second containers.

8. Prior to step ii), the method further includes the step of supplementing each passage of the second TIL cell culture with additional IL-2, The method according to claim 5, further comprising the step of supplementing each passage of the second TIL cell culture with the second cell culture medium and IL-2 before step ii).

9. A method for increasing tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, wherein the method is: (a) Obtaining and / or receiving a first TIL population from tumors excised from a subject by processing a tumor sample obtained from the subject into multiple tumor fragments, (b) Performing a first growth priming by culturing a first TIL population in a cell culture medium containing IL-2 to produce a second TIL population, wherein the first growth priming is performed in a container having a first gas-permeable surface area, the first growth priming is performed for a first period of 1 to 7 to 1 to 8 days to obtain the second TIL population, and the number of the second TIL population is greater than the number of the first TIL population, (c) To produce a third TIL population, the rapid second growth is carried out by supplementing the cell culture medium of the second TIL population with additional IL-2 and either the culture supernatant from a second APC culture containing APC and OKT-3 or the culture supernatant from a second APC culture containing OKT-3, wherein the number of APCs added in 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 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 the second gas permeable surface area. (d) Collecting the therapeutic TIL population obtained from step (c), (e) Transferring the TIL population collected from step (d) to an infusion bag, or The aforementioned method, (a) Obtaining and / or receiving a first TIL population from tumors excised from a subject by processing tumor samples obtained from the subject into multiple tumor fragments, (b) Performing a first proliferation priming by culturing the first TIL population in a cell culture medium containing IL-2 to produce a second TIL population, wherein the first proliferation priming is performed for a first period of 1 to 7 days or 1 to 8 days to obtain the second TIL population, and the number of the second TIL population is greater than the number of the first TIL population. (c) To produce a third TIL population, a rapid second growth is carried out by contacting the second TIL population with a cell culture medium containing either IL-2, APC, and the culture supernatant from a second APC culture containing OKT-3, or the culture supernatant from a first APC culture containing OKT-3, wherein the rapid second growth is carried out for a second period of 1 to 11 days to obtain the third TIL population, and the third TIL population is a therapeutic TIL population. (d) The method comprising collecting a therapeutic TIL population obtained from step (c).

10. The method according to claim 9, wherein the culture of step (b) further comprises antigen-presenting cells (APCs) and the culture supernatant from a culture of a second APC containing OKT-3, or the culture supernatant from a culture of a first APC containing OKT-3, 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).

11. A method for proliferating tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, wherein the method is: (a) Performing a first growth priming by culturing a first TIL population in order to produce a second TIL population, wherein the first TIL population can be obtained by processing a tumor sample excised from a target tumor into multiple tumor fragments in a cell culture medium containing IL-2, the first growth priming is performed in a container having a first gas-permeable surface area, the first growth priming is performed for a first period of 1 to 7 days or 1 to 8 days in order to obtain the second TIL population, and the number of the second TIL population is greater than the number of the first TIL population, (b) To produce a third TIL population, a rapid second growth is carried out by contacting the second TIL population with a cell culture medium of the second TIL population containing additional IL-2, and either the culture supernatant from a second culture of APC containing OKT-3 or the culture supernatant from a first culture of APC containing OKT-3, wherein the number of APCs during 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 to obtain a 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. (c) The method comprising taking the therapeutic TIL population obtained from step (b).

12. A method for proliferating tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, wherein the method is: (a) Performing a first proliferation priming by culturing a first TIL population in a cell culture medium containing IL-2 in order to produce a second TIL population, wherein the first proliferation priming is performed for a first period of 1 to 7 days or 1 to 8 days to obtain a second TIL population, and the number of the second TIL population is greater than the number of the first TIL population. (b) To produce a third TIL population, a rapid second growth is carried out by contacting the second TIL population with a cell culture medium containing IL-2, APC, and either the culture supernatant from a second culture of APC containing OKT-3 or the culture supernatant from a first culture of APC containing OKT-3, wherein the rapid second growth is carried out for a second period of 1 to 11 days to obtain the third TIL population, and the third TIL population is a therapeutic TIL population. (c) The method comprising taking the therapeutic TIL population obtained from step (b).

13. In step (a), the cell culture medium further comprises antigen-presenting cells (APCs), 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), and (i) The ratio of the number of APCs in the rapid second growth to the number of APCs in the priming of the first growth is in the range of 1.5:1 to 20:1, or (ii) The number of APCs in the priming of the first proliferation is 1.0×10 ６ APC / cm ２ to 4.5×10 ６ APC / cm ２ within the range, and the number of APCs in the rapid second proliferation is 2.5×10 ６ APC / cm ２ to 7.5×10 ６ APC / cm ２ within the range, or (iii) The number of APCs in the first proliferation priming is 1 × 10 ８ APC ~ 3.5 x 10 ８ This is within the range of APCs, and the number of APCs in the rapid second growth is 3.5 × 10 ８ APC ~ 1 x 10 ９ It is within the scope of APC, or (iv) The ratio of the number of TILs in the second TIL group to the number of TILs in the first TIL group is between 1.5:1 and 100:

1. The method according to claim 12.

14. The method, after the step of collecting the therapeutic TIL population, The method according to any one of claims 9 to 13, further comprising the additional step of transferring the collected therapeutic TIL population to an infusion bag.

15. The method according to any one of claims 9 to 14, wherein the plurality of tumor fragments are distributed into a plurality of separate containers, in each separate container the second TIL population is obtained from the first TIL population from the first growth priming step, the third TIL population is obtained from the second TIL population in the rapid second growth step, and the therapeutic TIL population obtained from the third TIL population is collected from each of the plurality of containers and combined to obtain the collected TIL population.

16. The method according to claim 15, wherein the plurality of separate containers comprises at least two separate containers, and each of the separate containers comprises a first gas permeable surface area.

17. The method according to claim 16, wherein in the first proliferation priming step, the cell culture medium comprises antigen-presenting cells (APCs), the APCs are stacked on the first gas-permeable surface region with an average thickness of 1 to 3 cell layers, and / or in the rapid second proliferation step, the APCs are stacked on the first gas-permeable surface region with a thickness of 3 to 5 cell layers.

18. The method according to any one of claims 9 to 14, wherein in the first growth priming step, the first growth priming is performed in a first container having a first gas-permeable surface area, and in the rapid second growth step, the rapid second growth step is performed in a second container having a second gas-permeable surface area, and in the first growth priming step, the cell culture medium comprises antigen-presenting cells (APCs), the APCs being stacked on the first gas-permeable surface area with an average thickness of 1 to 3 cell layers, or in the first growth priming step, the APCs being stacked on the first gas-permeable surface area with an average thickness of 1.5 to 2.5 cell layers, and / or in the rapid second growth step, the APCs being stacked on the second gas-permeable surface area with a thickness of 3 to 5 cell layers.

19. For each container in which the first growth priming is performed on the first TIL population, the rapid second growth is performed on the second TIL population generated from the first TIL population within the same container, and each container includes the first gas permeable surface area. In the first proliferation priming step, the cell culture medium contains antigen-presenting cells (APCs), and the APCs are stacked on the first gas-permeable surface region with an average thickness of 1 to 3 cell layers. The method according to any one of claims 9 to 17, wherein in the rapid second proliferation step, the APCs are stacked on the first gas-permeable surface region to a thickness of 3 to 5 cell layers.

20. The method according to any one of claims 9 to 18, wherein, in the first proliferation priming step, for each container in which the first proliferation 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 stacked on a first gas-permeable surface area, and the ratio of the average number of APCs stacked in the first proliferation priming step to the average number of APCs stacked in the rapid second proliferation step is in the range of 1:1.1 to 1:

10.

21. The method according to any one of claims 1 to 20, wherein, two to three days after the rapid second growth step, additional IL-2 is added to the cell culture medium, and / or, in the step of collecting the therapeutic TIL population, the collected TIL population is cryopreserved using a cryopreservation process.

22. The method according to any one of claims 1 to 21, wherein the first period in the first growth priming step and the second period in the rapid second growth step are each carried out individually within a period of 5 days, 6 days, or 7 days.

23. The method according to any one of claims 9 to 21, wherein the steps from the first proliferation priming step to the collection of the therapeutic TIL population are carried out within a period of 14 to 16 days.

24. The method according to any one of claims 9 to 23, wherein 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.

25. A method for proliferating tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, wherein the method is: (i) Obtaining and / or receiving a first TIL population from tumor samples obtained from one or more micro-biops, core biopsies, or needle biopsies of a tumor in a subject by culturing tumor samples in a first cell culture medium containing IL-2 for three days, (ii) To produce a second TIL population, a first proliferation priming is performed by culturing the first TIL population in a second cell culture medium containing IL-2, OKT-3, antigen-presenting cells (APCs), and either the culture supernatant from a culture of a first APC containing OKT-3 or the culture supernatant from a culture of a first APC containing OKT-3, wherein the first proliferation priming is performed in a container having a first gas-permeable surface area, the first proliferation priming is performed for a first period of 7 to 8 days to obtain the second TIL population, and the number of the second TIL population is greater than the number of the first TIL population, (iii) To produce a third TIL population, a rapid second growth is carried out by supplementing the second cell culture medium of the second TIL population with additional IL-2, OKT-3, or the culture supernatant from a second APC culture containing antigen-presenting cells (APCs) and / or OKT-3, wherein the number of APCs added in the rapid second growth is at least twice the number of APCs added in step (ii), the rapid second growth is carried out for a second period of 11 days to obtain a 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. (iv) Collecting the therapeutic TIL population obtained from step (iii), (v) Transferring the TIL population collected from step (iv) to an infusion bag, or the method thereof, (i) Obtaining and / or receiving a first TIL population from tumor samples obtained from one or more micro-biops, core biopsies, or needle biopsies of a tumor in a subject by culturing tumor samples in a first cell culture medium containing IL-2 for three days, (ii) To produce a second TIL population, a first proliferation priming is performed by culturing the first TIL population in a cell culture medium containing IL-2, OKT-3, antigen-presenting cells (APCs), and either the culture supernatant from a first culture of APCs containing OKT-3 or the culture supernatant from a first culture of APCs containing OKT-3, wherein the first proliferation priming is performed for a first period of 7 or 8 days to obtain a second TIL population, and the number of the second TIL population is greater than the number of the first TIL population. (iii) To produce a third TIL population, a rapid second growth is carried out by contacting the second TIL population with a third cell culture medium containing IL-2, OKT-3, antigen-presenting cells (APCs), and either the culture supernatant from a second culture of APCs containing OKT-3 or the culture supernatant from a first culture of APCs containing OKT-3, wherein the rapid second growth is carried out for a second period of 11 days to obtain the third TIL population, and the third TIL population is a therapeutic TIL population. (iv) The method comprising collecting the therapeutic TIL population obtained from step (iii).

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