Method for Enriching Tumor-Infiltrating Lymphocytes

Abstract

The present disclosure provides improved methods for expanding tumor infiltrating lymphocytes (TILs) and for producing therapeutic populations of TILs in less time than traditional methods. TIL cell therapy products can be produced that easily meet target cell doses while maintaining the desired T cell phenotype.

Description

[Technical field]

[0001] The present invention relates to a method for enriching tumor-infiltrating lymphocytes.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Applications Nos. 63 / 304,288 (filed January 28, 2022), 63 / 411,317 (filed September 29, 2022), and 63 / 423,676 (filed November 8, 2022), each of which is incorporated by reference in its entirety. [Background technology]

[0003] background Adoptive cell therapy using tumor infiltrating lymphocytes (TILs) offers a potential treatment option for cancer. TILs are enriched in polyclonal T cells with diverse antigen specificities. Extraction of tumor fragments and subsequent ex vivo expansion removes TILs from the inhospitable tumor microenvironment and reduces the immunosuppressive effect of intratumoral regulatory T cells. Ex vivo expansion of TILs rejuvenates the cells and generates billions of such cells to be reinfused into the patient. Cell therapy products that can address the broad nature of tumor neo-antigens and the unique array from each patient would lead to the possibility of individualized, targeted responses. Sarnaik et al., J. Clinical Oncology, 2021, 39:2656-2666.

[0004] Effective treatment of cancer using TILs requires ex vivo production of billions of TILs. For production of TILs, a patient's resected tumor is first fragmented and cultured in vitro under conditions that support migration of TILs from the tumor tissue. In a typical protocol, TIL numbers are then expanded with a rapid expansion protocol (REP) using irradiated allogeneic donor peripheral blood mononuclear cells (PBMCs) and anti-CD3 monoclonal antibodies. REP is used to generate sufficient TILs for infusion. Several factors, including the availability and validation of donor PBMCs, make the typical REP protocol lengthy, labor intensive, and expensive. In addition to these negative factors, TILs expanded with the standard protocol are primarily characterized as terminally differentiated phenotypes (TILs) associated with poorer clinical responses. EM , T EMRA ) in TIL-infused products. SCM , T CM ) have been associated with improved anti-tumor responses in patients. Thus, there is a need to replace the usual REP protocol with a more convenient yet GMP-compliant process that can generate TILs with a preferred phenotype using readily available commercial reagents. Summary of the Invention

[0005] General The present disclosure provides a method of expanding tumor infiltrating lymphocytes (TILs), the method comprising (a) culturing a first cell population in a first cell culture medium to generate a second cell population, and (b) contacting the second cell population with a polymer matrix comprising an anti-CD3 antibody and an anti-CD28 antibody or fragments thereof in a second cell culture medium to generate a third cell population. In certain embodiments, the first cell population is obtained from a tumor sample from a patient.

[0006] The third cell population can be TILs or cells to be included in the pharmaceutical composition of the present invention. The third cell population can be TILs or cells to be used in the method of the present invention to treat a subject (e.g., a patient) with cancer.

[0007] In certain embodiments, the second cell culture medium comprises about 100 IU / mL to about 8,000 IU / mL interleukin-2 (IL-2), about 500 IU / mL to about 4,000 IU / mL IL-2, about 1,000 IU / mL, about 2,000 IU / mL, about 3,000 IU / mL, about 4,000 IU / mL, or about 5,000 IU / mL IL-2.

[0008] In certain embodiments, the first cell culture medium comprises about 2,000 IU / mL to about 8,000 IU / mL of IL-2, about 5,000 IU / mL, about 6,000 IU / mL, about 7,000 IU / mL, or about 8,000 IU / mL of IL-2.

[0009] In certain embodiments, in step (a), the first cell population is cultured for about 10 days to about 40 days, about 10 days to about 14 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, or about 17 days.

[0010] The method may further comprise cryopreserving the second population of cells after step (a).

[0011] In certain embodiments, in step (b), the contacting is performed for about 3 days to about 17 days, about 5 days to about 15 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, or about 17 days.

[0012] In certain embodiments, the third cell population is at least 100 times greater in number than the second cell population, or from about 100 to about 2000 times greater in number than the second cell population.

[0013] The tumor sample can be from a solid tumor.Solid tumors include, but are not limited to, sarcoma, hepatocellular carcinoma, glioma, head and neck cancer, bone cancer, brain cancer, breast cancer, anal cancer, anal canal cancer, anorectal cancer, eye cancer, intrahepatic bile duct cancer, joint cancer, cervical cancer, gallbladder cancer, pleural cancer, nasal cancer, nasal cavity cancer, middle ear cancer, oral cancer, vulva cancer, colon cancer, esophageal cancer, cervical cancer, gastrointestinal cancer, hypopharyngeal cancer, laryngeal cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma, nasopharyngeal cancer, ovarian cancer, pancreatic cancer, peritoneal cancer, omental cancer, mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, kidney cancer, small intestine cancer, soft tissue cancer, stomach cancer, testicular cancer, thyroid cancer, ureter cancer, bladder cancer, and combinations thereof.

[0014] The present disclosure provides tumor infiltrating lymphocytes obtained by the methods of the present invention.

[0015] The present disclosure also relates to the following: (i) CD3 + CD3 cells range in percentage from about 3% to about 88% + CD8 + T cells, (ii) CD3 + CD3 cells range in percentage from about 10% to about 96% + CD4 + T cells, (iii) CD4 + CD4 cells range from approximately 50% to approximately 88% + T CM T cells, (iv) CD8 + The percentage of CD8 cells ranges from about 28% to about 82%. + T CM T cells, (v) CD4 + The percentage of CD4 cells ranges from about 11% to about 49%. + T EM T cells, (vi) CD8 + CD8 cells range in percentage from about 11% to about 61% + T EM T cells The present invention also includes a cell population enriched for or expanded from tumor infiltrating lymphocytes, comprising one or more of:

[0016] The cell population was determined to have >80% CD3 + It may comprise T cells.

[0017] The cell population is CD4 + The percentage of CD4 cells ranges from about 10% to about 51%. + CD27 + It may comprise T cells.

[0018] The cell population is CD8 + CD8 cells range in percentage from about 12% to about 72% + CD27 + It may comprise T cells.

[0019] The cell population is CD8 + The percentage of CD8 cells ranges from about 34% to about 95%. + CD28 + It may comprise T cells.

[0020] The cell population is CD4 + CD4 cells range from approximately 82% to approximately 100% + CD28 + It may comprise T cells.

[0021] The cell population is CD4 + CD4 cells range in percentage from about 0.2% to about 5.8% + 4-1BB + It may comprise T cells.

[0022] The cell population is CD8 + CD8 cells range in percentage from about 0.2% to about 11.6% + 4-1BB + It may comprise T cells.

[0023] The cell population is CD4 +CD4 cells range in percentage from about 0.2% to about 19.5% + LAG3 + It may comprise T cells.

[0024] The cell population is CD8 + CD8 cells ranged from approximately 6% to approximately 51.2% + LAG3 + It may comprise T cells.

[0025] The cell population is CD4 + The percentage of CD4 cells ranges from about 0.9% to about 31%. + PD1 + It may comprise T cells.

[0026] The cell population is CD8 + CD8 cells range in percentage from about 1% to about 18% + PD1 + It may comprise T cells.

[0027] The cell population contained less than 10% CD56 + It may comprise NK cells.

[0028] The present disclosure provides cell populations obtainable by the methods of the invention.

[0029] The method includes (a) culturing a first cell population in a first cell culture medium to generate a second cell population, and (b) contacting the second cell population with a polymer matrix comprising an anti-CD3 antibody and an anti-CD28 antibody or fragment thereof in a second cell culture medium to generate a third cell population. In certain embodiments, the first cell population is obtained from a tumor sample from a patient. The third cell population can be a cell population generated by the method.

[0030] The present disclosure provides a method for expanding a cell population enriched for tumor-infiltrating lymphocytes, the method comprising (a) culturing cells obtained from a tumor sample from a patient, and (b) treating the cultured cells to generate a cell population enriched for tumor-infiltrating lymphocytes, wherein the cell population is enriched for tumor-infiltrating lymphocytes by: (i) CD3 + CD3 cells range in percentage from about 3% to about 88% + CD8 + T cells, (ii) CD3 + CD3 cells range in percentage from about 10% to about 96% + CD4 + T cells, (iii) CD4 + CD4 cells range from approximately 50% to approximately 88% + CD45RA - CD62L + Central memory T cells, (iv) CD8 + The percentage of CD8 cells ranges from about 28% to about 82%. + CD45RA - CD62L + Central memory T cells, (v) CD4 + The percentage of CD4 cells ranges from about 11% to about 49%. + CD45RA - CD62L - Effector memory T cells, (vi) CD8 + CD8 cells range in percentage from about 11% to about 61% + T CD45RA - CD62L - Effector memory T cells Contains one or more of the following:

[0031] The present disclosure also includes a method of treating a patient with cancer comprising administering to the patient a tumor-infiltrating lymphocyte of the invention, a cell population of the invention, a cell of the invention (e.g., a third cell population), or a composition of the invention.

[0032] The present disclosure provides pharmaceutical compositions comprising the tumor infiltrating lymphocytes of the invention, the cell populations of the invention, or the cells of the invention (eg, a third cell population).

[0033] In certain embodiments, about 1×10 9 ~Approx. 1×10 11 cells, or approximately 5 x 10 9 ~Approx. 9×10 10 The cells are administered to the patient.

[0034] The cancer may be melanoma, cervical cancer, lung cancer, colorectal cancer, breast cancer or head and neck cancer. The cancer may include sarcoma, hepatocellular carcinoma, glioma, head and neck cancer, bone cancer, brain cancer, breast cancer, anal cancer, anal canal cancer, anorectal cancer, eye cancer, intrahepatic bile duct cancer, joint cancer, cervical cancer, gallbladder cancer, pleural cancer, nasal cancer, nasal cavity cancer, middle ear cancer, oral cancer, vulva cancer, colon cancer, esophageal cancer, cervical cancer, gastrointestinal cancer, hypopharyngeal cancer, laryngeal cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma, nasopharyngeal cancer, ovarian cancer, pancreatic cancer, peritoneal cancer, omental cancer, mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, kidney cancer, small intestine cancer, soft tissue cancer, stomach cancer, testicular cancer, thyroid cancer, ureter cancer, bladder cancer or combinations thereof. [Brief description of the drawings]

[0035] [Figure 1] TIL proliferation and glucose / lactate levels in pre-REP TIL cultures following standardized protocol. In the standardized pre-REP protocol, tumor fragments were placed in a G-Rex® 100M device containing 250 mL of pre-REP complete medium, fed with 1 volume of medium on day 10, and harvested on day 14. Supernatants were collected on days 0, 6, 10, and 14. [Diagram 2] Lineage characterization of pre-REP TILs by standardized protocols: NK, CD45+CD3-CD56+ (within CD45+ cells); B, CD45+CD3-CD19+ (within CD45+ cells); myeloid, CD45+CD3-CD14+ (within CD45+ cells); epithelial, CD45- EPCAM+ (within live cells); CD3+ T cells (within CD45+ cells); CD4+ T and CD8+ T (within CD3+ T cells). [Diagram 3] Expression of activating / inhibitory surface markers and subsets of CD4 + pre-REP TILs generated according to a standardized protocol. [Figure 4] Expression of activating / inhibitory surface markers and subsets of CD8+ pre-REP TILs generated according to standardized protocols. [Diagram 5] Percentage of CD3+ T cells and CD4+ and CD8+ T cell subsets in TILs expanded by traditional REP or TransAct REP. Statistical significance was determined by paired t test: **P<0.01; ns, not significant (n=12). [Figure 6] Memory phenotypes of CD4+ and CD8+ TILs. Top panel: CD4, bottom panel: CD8. Each dot represents one donor. Horizontal bars represent the mean and standard deviation. TN, naive T cells; TSCM, stem cell memory; TCM, central memory; TEM, effector memory; TEFF, effector T cells. P values ​​were obtained by Student's paired t-test. Ns, not significant; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 (n=12). [Figure 7] Analysis of inhibitory and activating markers in CD4+ and CD8+ TIL subsets. Top panel: CD4, bottom panel: CD8. Each dot represents one donor. Horizontal bars represent the mean and standard deviation. P values ​​were obtained by Student's paired t-test. ns, not significant; *P<0.05, **P<0.01, ***P<0.001 (n=12). [Figure 8] Production of IFN-γ and granzyme B in response to αCD3 stimulation. Stimulation was performed in triplicate for every donor, and each dot represents the mean. Horizontal bars represent the total mean of different donors in each condition. P values ​​were obtained by paired t test. ns, not significant; *P<0.05, **P<0.01. [Figure 9]Tumor killing efficacy of C-TIL051 (TransAct REP TIL) and traditional REP TIL during incubation in the presence of autologous tumor cells. TILs from donor T5101016 were labeled with Cytolight Rapid Red and then co-cultured with cancer MOS for 68 h at E:T ratios of 2:1, 3:1, and 4:1 in culture medium containing Caspase 3 / 7 Green. MOS alone was used as a negative control. Higher Caspase 3 / 7 signal indicates higher killing efficacy. [Figure 10] Dynamic changes in IL-2 protein concentration in C-TIL051 REP PD run cultures using TransAct and G-Rex® devices. IL-2 protein concentration was measured by ELISA in REP CM (day 0) or culture supernatant samples collected at different time points (left). When samples from seven REP runs were analyzed, TILs from different donors showed different levels of proliferation (right; data at day 14 represent expected numbers after taking into account the respective split factors at day 9). [Figure 11] Dynamic changes in glucose and lactate levels in C-TIL051 REP PD run cultures using TransAct and G-Rex® devices. Glucose and lactate concentrations in REP CM (day 0) or culture supernatant samples collected at different time points were measured by Glucose-Glo and Lactate-Glo assays, respectively. Samples from seven REP runs were analyzed and TILs from different donors showed different levels of proliferation. [Figure 12] FACS analysis of surface marker expression and memory phenotype of CD4+ and CD8+ TILs after REP by TransAct using a G-Rex® instrument. Data are shown as mean with 95% CI. TN (Tnaive), naive T cells; TSCM (Tscm), stem memory T cells; TCM (Tcm), central memory T cells; TEM (Tem), effector memory T cells; TEFF, effector T cells. [Figure 13] Dynamic changes in IL-2 protein concentration in C-TIL051 REP PD performing cultures using the G-Rex® device with Xuri™ W25 bioreactor sequentially and TransAct. Supernatants from G-Rex® cultures were sampled on day 7, after which cultures were inoculated into Xuri™ Cellbags on the same day and then sampled daily from day 8 until harvest. [Figure 14] Dynamic changes in glucose and lactate concentrations in C-TIL051 REP PD performing cultures with continuous use of the G-Rex® device and Xuri™ W25 bioreactor and TransAct. Supernatant from the G-Rex® culture was sampled on day 7, after which the culture was inoculated into a Xuri™ Cellbag on the same day and then sampled daily from day 8 until harvest. [Figure 15] Cell proliferation, viability and cumulative fold expansion of REP TILs from C-TIL051 PD run with sequential use of G-Rex® device and Xuri™ W25 bioreactor and TransAct. Five pre-REP samples were first activated with TransAct and expanded in a G-Rex® 100M device, then transferred to a Xuri™ W25 bioreactor on day 7. Cells were expanded in the Xuri™ for an additional 8-12 days where they were sampled daily for cell proliferation and viability. [Figure 16] FACS analysis of surface marker expression and memory phenotype of CD4+ and CD8+ TILs after sequential use of the G-Rex® device with a Xuri™ W25 bioreactor and REP with TransAct. Data are shown as mean with 95% CI. TN, naive T cells; TSCM, stem memory T cells; TCM, central memory T cells; TEM, effector memory T cells; TEFF, effector T cells. [Figure 17] IFN-γ production by C-TIL051 from mock runs in response to αCD3 / CD28 stimulation. [Figure 18]Upregulation of T cell activation markers (4-1BB and OX40) by the C-TIL051 Engineer Run in response to αCD3 stimulation. Horizontal lines indicate grand means. [Figure 19] Assessment of C-TIL051-mediated MOS killing using a T cell potency assay. Quantification of relative T5101034 (left) and T5101035 (right) autologous TIL-induced MOS killing observed at E:T ratios of 1:1, 5:1, and 10:1. Co-cultures were initiated on day 3 after MOS establishment and images were acquired up to 96 hours. Mean integrated fluorescence intensity (NIRCU×μm2) in the NIR channel was from viable tumor cells labeled with NIR680 dye. Data were normalized to the MOS-only group. [Figure 20] The requirement for HLA engagement demonstrated the specificity of C-TIL051-mediated MOS killing. Co-cultures of C-TIL051 REP TILs with autologous MOS were performed at an effector:target ratio of 5:1 in the presence or absence of the indicated HLA-blocking antibodies and analyzed for up to 96 hours. The mean integrated fluorescence intensity (NIRCU × μm2) in the NIR channel was from viable tumor cells labeled with NIR680 dye. Data shown are normalized to the MOS-only group. [Figure 21-1] Blockade of HLA class I and II reduced cytokine production in co-cultures of C-TIL051 with autologous MOS. C-TIL051: TIL generated by an embodiment of the method of the present invention. [Figure 21-2] Blockade of HLA class I and II reduced cytokine production in co-cultures of C-TIL051 with autologous MOS. C-TIL051: TIL generated by an embodiment of the method of the present invention.

[0036] Detailed Description The present invention provides a method for expanding tumor infiltrating lymphocytes (TILs). The present method does not require the use of feeder cells, such as PBMCs. Compared to TILs generated / expanded using irradiated PBMCs, TILs generated / expanded using the present method are accompanied by preferential expansion of CD8 TILs and maintain low surface levels of exhaustion markers, such as PD-1, on TILs. In particular, TILs generated / expanded using the present method (1) maintain a relatively high CD8 / CD4 ratio, (2) have a higher proportion of Tnaive / scm and Tcm on CD4 and CD8 T cells, and (3) show a reduced positivity of PD-1 on CD4 and CD8 T cells. The present method can rapidly expand TILs with a high CD8 / CD4 ratio and generate TILs with a higher proportion of CD8 cytotoxic T cells, reduced exhaustion and enhanced memory. Thus, TILs generated by the present method are more effective for cancer immunotherapy.

[0037] The present disclosure also includes autologous TIL therapy using tumor-tissue T cells that can be expanded ex vivo and recognize tumor antigens while maintaining a heterogeneous repertoire of T cells using a centralized manufacturing process.

[0038] In a particular embodiment, the cells of the present invention contain autologous TILs taken from a patient's tumor sample. The tumor is first fragmented and cultured in high-dose IL-2 to promote the release and proliferation of TILs. This initial stage of proliferation of TILs from tumor tissue is called pre-rapid proliferation protocol (pre-REP). After this initial ex vivo culture, TILs can be further propagated in rapid proliferation protocol (REP) until therapeutic dose is reached.

[0039] The present disclosure provides a method for expanding tumor infiltrating lymphocytes (TILs), which may include (a) culturing a first cell population in a first cell culture medium to generate a second cell population, where the first cell population is obtained from a tumor sample from a patient, and (b) contacting the second cell population with a polymer matrix comprising an anti-CD3 antibody and an anti-CD28 antibody or fragments thereof in a second cell culture medium to generate a third cell population, where the second cell culture medium comprises about 100 IU / mL to about 8,000 IU / mL of interleukin-2 (IL-2).

[0040] The method may further comprise cryopreserving the second cell population after the first expansion (pre-REP, or step (a) of the method). The cryopreserved second cell population may then be thawed and subjected to a second expansion (REP, or step (b) of the method, e.g., contacting with a polymer matrix comprising anti-CD3 and anti-CD28 antibodies or fragments thereof in a second cell culture medium). The second cell population may be subjected to a second expansion (REP, or step (b) of the method) immediately after thawing. The second population of cells may be recovered for less than 2 days, less than 48 hours, less than 40 hours, less than 36 hours, less than 30 hours, less than 24 hours, less than 20 hours, less than 15 hours, less than 12 hours, less than 10 hours, less than 9 hours, less than 8 hours, less than 7 hours, less than 6 hours, less than 5 hours, less than 4 hours, less than 3 hours, less than 2 hours or less than 1 hour and then subjected to a second expansion [REP, or step (b) of the method)].

[0041] The second cell population, which contains the pre-REP TILs, can be cryopreserved during the patient's disease progression. After the patient's disease progression, the pre-REP TILs are thawed and expanded according to the Rapid Expansion Protocol (REP).

[0042] In the case of frozen and thawed cell products, the thawed cells can be contacted with a polymer matrix comprising anti-CD3 and anti-CD28 antibodies or fragments thereof, or can be activated after standing overnight. Alternatively, the thawed cells can be contacted with a polymer matrix comprising anti-CD3 and anti-CD28 antibodies or fragments thereof, or can be activated immediately without standing overnight. In one embodiment, the frozen cells are rapidly thawed at 37° C. in a water bath.

[0043] The volume ratio of TransAct to the second cell culture medium (or cell culture medium for second expansion, or REP cell culture medium) can range from about 1:200 to about 1:2, about 1:180 to about 1:2, about 1:150 to about 1:5, about 1:120 to about 1:5, about 1:100 to about 1:8, about 1:80 to about 1:8, about 1:50 to about 1:10, about 1:40 to about 1:10, about 1:30 to about 1:10, about 1:20 to about 1:10, about 1:20 to about 1:15, or about 1:17.5. In certain embodiments, the rapid expansion protocol (REP) uses MACS® GMP T Cell TransAct™, a colloidal polymer nanomatrix covalently linked to humanized recombinant CD3 and CD28 agonists (e.g., antibodies). In one embodiment, 1 mL of TransAct may be used for a final volume of 17.5 mL of REP cell culture medium. The REP TILs are then washed, formulated, and / or cryopreserved.

[0044] "Tumor infiltrating lymphocytes" or "TILs" may refer to a population of cells originally derived as leukocytes that have left the bloodstream of a subject and migrated into a tumor. TILs include T cells (e.g., CD8 + Cytotoxic T cells, Th1 and Th17 CD4 +T cells), B cells, natural killer cells, dendritic cells and macrophages. TILs can include (or be) tumor-infiltrating T cells. TILs include both primary and secondary TILs. Primary TILs are those obtained from patient tissue samples as described herein. Secondary TILs are any TIL cell population that has been expanded or expanded as described herein, including but not limited to bulk TILs and expanded TILs ("REP TILs" or "post-REP TILs"). TIL cell populations can include genetically modified TILs. TILs can generally be defined biochemically using cell surface markers or functionally by their ability to infiltrate tumors and provide therapy. TILs may generally be classified by expression of one or more of the following biomarkers: CD3, CD4, CD8, TCRα / β, CD27, CD28, CCR7, CD62L, CD45RA, CD45RO, CD95, PD-1, TIM-3, LAG-3, 4-1BB, and CD25. Additionally or alternatively, TILs may be functionally defined by their ability to infiltrate solid tumors upon reintroduction into the patient. Fisher et al., Tumor localization of adoptively transferred indium-111 labeled tumor infiltrating lymphocytes in patients with metastatic melanoma, J. Clin. Oncol., 1989, 7(2):250-61.

[0045] The term "IL-2" (or "IL2") refers to the T cell growth factor known as interleukin-2 and includes all forms of IL-2, including human and mammalian forms, conservative amino acid substitutions, glycoforms, biosimilars, and variants thereof. IL-2 is described, for example, in Nelson, J. Immunol. 2004, 172, 3983-88 and Malek, Annu. Rev. Immunol. 2008, 26, 453-79, the disclosures of which are incorporated herein by reference. IL-2 can include human recombinant forms of IL-2, such as aldesleukin (Proleukin®), and forms of recombinant IL-2. Aldesleukin (des-alanyl-1, serine-125 human IL-2) is the non-glycosylated human recombinant form of IL-2. IL-2 may also include pegylated forms of IL-2, including the pegylated IL2 prodrug NKTR-214. Alternative forms of conjugated IL-2 suitable for use in the present invention are described in U.S. Patent Nos. 4,766,106, 5,206,344, 5,089,261 and 4,902,502, the disclosures of which are incorporated herein by reference. Formulations of IL-2 suitable for use in the present invention are described in U.S. Patent No. 6,706,289, the disclosures of which are incorporated herein by reference.

[0046] The first expansion (pre-REP) may take place in a closed vessel having a first gas permeable surface area. The second expansion (REP) may take place in a closed vessel having a second gas permeable surface area. In some embodiments, the closed vessel comprises (or is) a single bioreactor. In some embodiments, the closed vessel may comprise (or is) a G-Rex® vessel and / or a Xuri™ cell bag. In some embodiments, the closed vessel is a G-Rex® device and / or a Xuri™ W25 bioreactor. In some embodiments, the closed vessel comprises (or is) a G-Rex® 10M and / or a G-Rex® 100M. In some embodiments, the closed vessel comprises (or is) a G-Rex® 10M, a G-Rex® 100M and / or a Xuri™ W25 bioreactor. In certain embodiments, the methods (e.g., in the pre-REP and / or REP TIL culture process) include the use of a gas permeable rapid growth (G-Rex®) device as the TIL culture vessel. In one embodiment, the TILs are expanded in a gas permeable bag. In one embodiment, the TILs are expanded using a cell expansion system that expands the TILs in a gas permeable bag, e.g., the Xuri™ W25 bioreactor. In one embodiment, the TILs are expanded using a cell expansion system that expands the TILs in a gas permeable bag, e.g., the WAVE bioreactor system, also known as the Xuri™ Cell Expansion System W5. In one embodiment, the cell expansion system includes a gas permeable cell bag. In one embodiment, the TILs can be expanded in a G-Rex® flask.

[0047] In some embodiments, the closed system uses one container from the time the tumor fragments are obtained until the TILs are ready for administration to the patient or cryopreservation. In some embodiments that use two containers, the first container is a closed G-Rex® container and the population of TILs is centrifuged and transferred to an infusion bag without opening the first closed G-Rex® container.

[0048] The present disclosure also provides tumor-infiltrating lymphocytes obtained by the methods of the present invention.

[0049] The present disclosure also relates to the following: (i) CD3 + CD3 cells range in percentage from about 3% to about 88% + CD8 + T cells, (ii) CD3 + CD3 cells range in percentage from about 10% to about 96% + CD4 + T cells, (iii) CD4 + CD4 cells range from approximately 50% to approximately 88% + T CM T cells, (iv) CD8 + The percentage of CD8 cells ranges from about 28% to about 82%. + T CM T cells, (v) CD4 + The percentage of CD4 cells ranges from about 11% to about 49%. + T EM T cells, (vi) CD8 + CD8 cells range in percentage from about 11% to about 61% + T EM T cells The present invention also includes a cell population enriched for or expanded from tumor infiltrating lymphocytes, comprising one or more of:

[0050] In certain embodiments, the cell population comprises 80% or more CD3 + It may comprise T cells.

[0051] In certain embodiments, the cell population is CD4 + The percentage of CD4 cells ranges from about 10% to about 51%. + CD27 + It may comprise T cells.

[0052] In certain embodiments, the cell population is CD8 + CD8 cells range in percentage from about 12% to about 72% + CD27 + It may comprise T cells.

[0053] In certain embodiments, the cell population is CD8 + The percentage of CD8 cells ranges from about 34% to about 95%. + CD28 + It may comprise T cells.

[0054] In certain embodiments, the cell population is CD4 + CD4 cells range from approximately 82% to approximately 100% + CD28 + It may comprise T cells.

[0055] In certain embodiments, the cell population is CD4 + CD4 cells range in percentage from about 0.2% to about 5.8% + 4-1BB + It may comprise T cells.

[0056] In certain embodiments, the cell population is CD8 + CD8 cells range in percentage from about 0.2% to about 11.6% + 4-1BB + It may comprise T cells.

[0057] In certain embodiments, the cell population is CD4 + CD4 cells range in percentage from about 0.2% to about 19.5% + LAG3 +It may comprise T cells.

[0058] In certain embodiments, the cell population is CD8 + CD8 cells ranged from approximately 6% to approximately 51.2% + LAG3 + It may comprise T cells.

[0059] In certain embodiments, the cell population is CD4 + The percentage of CD4 cells ranges from about 0.9% to about 31%. + PD1 + It may comprise T cells.

[0060] In certain embodiments, the cell population is CD8 + CD8 cells range in percentage from about 1% to about 18% + PD1 + It may comprise T cells.

[0061] In certain embodiments, the cell population has less than 10% CD56 + It may comprise NK cells.

[0062] The present disclosure provides a cell population produced by a method of expanding tumor infiltrating lymphocytes (TILs), which may include (a) culturing a first cell population in a first cell culture medium to produce a second cell population, where the first cell population is obtained from a tumor sample from a patient, and (b) contacting the second cell population with a polymer matrix comprising an anti-CD3 antibody and an anti-CD28 antibody or fragments thereof in a second cell culture medium, where the second cell culture medium comprises about 100 IU / mL to about 8,000 IU / mL of interleukin-2 (IL-2).

[0063] The present disclosure provides a method for expanding a cell population enriched for tumor-infiltrating lymphocytes, the method comprising (a) culturing cells obtained from a tumor sample from a patient, and (b) treating the cultured cells to generate a cell population enriched for tumor-infiltrating lymphocytes, wherein the cell population is enriched for tumor-infiltrating lymphocytes by: (i) CD3 + CD3 cells range in percentage from about 3% to about 88% + CD8 + T cells, (ii) CD3 + CD3 cells range in percentage from about 10% to about 96% + CD4 + T cells, (iii) CD4 + CD4 cells range from approximately 50% to approximately 88% + CD45RA - CD62L + Central memory T cells, (iv) CD8 + The percentage of CD8 cells ranges from about 28% to about 82%. + CD45RA - CD62L + Central memory T cells, (v) CD4 + The percentage of CD4 cells ranges from about 11% to about 49%. + CD45RA - CD62L - Effector memory T cells, and (vi) CD8 + CD8 cells range in percentage from about 11% to about 61% + T CD45RA - CD62L - Effector memory T cells Contains one or more of the following:

[0064] The present disclosure also provides cell populations enriched for tumor infiltrating lymphocytes obtained by the methods of the present invention.

[0065] The method may use any suitable cell culture medium. In certain embodiments, the method uses one or more types of cell culture medium. Cell culture media that may be used include, but are not limited to, AIM-V medium (L-glutamine, 50 μg / ml streptomycin sulfate and 10 μg / ml gentamicin sulfate) (Thermo Fisher) and RPMI 1640 medium.

[0066] The concentration of IL-2 (in the first or second culture medium) may be about 1000 IU / mL to about 10000 IU / mL, about 2000 IU / mL to about 10000 IU / mL, about 3000 IU / mL to about 10000 IU / mL, about 4000 IU / mL to about 10000 IU / mL, about 5000 IU / mL to about 10000 IU / mL, about 2000 IU / mL to about 8000 IU / mL, about 3000 IU / mL to about 7000 IU / mL, or about 4000 IU / mL to about 6000 IU / mL. The concentration of IL-2 (in the first or second culture medium) may be about 6000 IU / mL. The concentration of IL-2 (in the first culture medium or the second culture medium) can also be about 2000 IU / mL, 3000 IU / mL, 4000 IU / mL, 5000 IU / mL, 6000 IU / mL, 7000 IU / mL, 8000 IU / mL, 9000 IU / mL or up to about 10000 IU / mL.

[0067] Once TILs are expanded, they can be subjected to in vitro assays to determine their characteristics and / or functions, such as tumor reactivity.For example, TILs can be evaluated by FACS for expression of CD3, CD4, CD8 and CD58.TILs can also be subjected to co-culture, cytotoxicity, ELISA or ELISPOT assays.

[0068] Tumor samples may be taken from any mammal. The cells or pharmaceutical compositions of the present invention may be used to treat any mammal. The mammal may be a human or a non-human primate. Mammals also include, but are not limited to, mammals of the order Lagomorpha, such as rabbits; the order Carnivora, including Felidae (cats) and Canidae (dogs); the order Artiodactyla, including Bovidae (cattle) and Porcine (pigs); or the order Perissodactyla, including Equidae (horses). The mammal is preferably a non-human primate, such as a non-human primate of the order Primates, Ceboid or Simoid (monkeys), or a non-human primate of the order Anthropoids (humans and apes). In some embodiments, the mammal may be a rodent mammal, such as mice and hamsters.

[0069] Obtaining tumor samples Generally, TILs (a first cell population) are first obtained from a patient tumor sample and then expanded into larger populations for further manipulation as described herein and, optionally, cryopreserved.

[0070] TILs can be grown from tumor samples, or tissues or organs affected by cancer. In some embodiments, TILs are obtained from tumor fragments. In some embodiments, tumors (samples) can be excised from non-cancerous tissues or necrotic areas. A patient's tumor sample can be obtained using methods known in the art, such as surgical resection, needle biopsy, or other means to obtain a sample containing a mixture of tumor and TIL cells. Once a tumor sample is obtained, it can be fragmented (e.g., using sharp dissection) into small pieces. The first cell population can be one or more tumor fragments from a patient (or multiple patients). The first cell population can be obtained from one or more tumor fragments from a tumor excised from a patient. Methods for destroying tumors can include mechanical fragmentation methods, such as crushing, slicing, splitting, and mincing tumor tissue, as well as any other methods for destroying the physical structure of tumor tissue.

[0071] In some embodiments, the tumor fragment is about 1 mm3 ~50mm 3 , about 1mm 3 ~45mm 3 , about 1mm 3 ~40mm 3 , about 1mm 3 ~35mm 3 , about 1mm 3 ~30mm 3 , about 1mm 3 ~25mm 3 , about 1mm 3 ~20mm 3 , about 1mm 3 ~15mm 3 , about 1mm 3 ~10mm 3 , about 1mm 3 ~8mm 3 , about 2 mm 3 ~3mm 3 , about 1mm 3 , about 2 mm 3 , about 3mm 3 , about 4mm 3 , about 5mm 3 , about 6mm 3 , about 7mm 3 , about 8mm 3 , approx. 9mm 3 , about 10mm 3 , approx. 12 mm 3 , about 15mm 3 , approx. 18mm 3 , about 20mm 3 , about 25mm 3 , or about 30 mm 3 In some embodiments, the plurality of fragments includes about 4 to about 50 fragments, each fragment having a volume of about 8 mm. 3 ~approx. 27mm 3 In some embodiments, the pieces have a volume of about 1300 mm 3 ~about 1500mm 3 In some embodiments, the plurality of pieces comprises about 30 to about 60 pieces having a total volume of about 1350 mm 3 In some embodiments, the plurality of pieces comprises about 50 pieces having a total mass of about 1 gram to about 1.5 grams.

[0072] In some embodiments, the tumor may be fragmented to about 2-3 mm in each dimension. In some cases, the tumor may be fragmented to about 0.5 mm to about 5 mm, about 1 mm to about 2 mm, about 2 mm to about 3 mm, about 3 mm to about 4 mm, or about 4 mm to about 5 mm in each dimension.

[0073] TILs can be cultured from these tumor fragments using enzymatic tumor digests. Such tumor digests can be generated by incubation in enzyme (e.g., DNase and collagenase)-containing medium followed by mechanical dissociation (e.g., using a tissue dissociator). In some embodiments, fragmentation includes physical fragmentation (e.g., dissection) and / or digestion.

[0074] The method may include obtaining a bulk population of T cells from tumor sample by any suitable method known in the art.For example, the bulk population of T cells may be obtained from tumor sample by separating tumor sample into cell suspension and selecting specific cell population therefrom.Suitable methods for obtaining a bulk population of T cells may include, but are not limited to, any one or more of mechanically dissociating (e.g., mincing) tumor, enzymatically dissociating (e.g., digesting) tumor, and aspirating (e.g., using a needle).

[0075] In certain embodiments, the tumor sample is from a solid tumor, including a primary tumor, an invasive tumor, or a metastatic tumor. A solid tumor can be benign or malignant. The term "solid tumor cancer" refers to a malignant, neoplastic, or cancerous solid tumor. Solid tumors may include sarcoma, hepatocellular carcinoma, glioma, head and neck cancer, bone cancer, brain cancer, breast cancer, anal cancer, anal canal cancer, anorectal cancer, eye cancer, intrahepatic bile duct cancer, joint cancer, cervical cancer, gallbladder cancer, pleural cancer, nasal cancer, nasal cavity cancer, middle ear cancer, oral cancer, vulva cancer, colon cancer, esophageal cancer, cervical cancer, gastrointestinal cancer, hypopharyngeal cancer, laryngeal cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma, nasopharyngeal cancer, ovarian cancer, pancreatic cancer, peritoneal cancer, omental cancer, mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, kidney cancer, small intestine cancer, soft tissue cancer, stomach cancer, testicular cancer, thyroid cancer, ureter cancer, bladder cancer, or a combination thereof. In some embodiments, the cancer is selected from cervical cancer, head and neck cancer (including, for example, head and neck squamous cell carcinoma (HNSCC)), glioblastoma, ovarian cancer, sarcoma, pancreatic cancer, bladder cancer, breast cancer, and non-small cell lung cancer.

[0076] The tumor sample may also be a liquid tumor, such as a tumor obtained from a hematological malignancy.

[0077] TILs can be expanded from tumor samples from donors at any stage of development, including but not limited to fetal, neonatal, juvenile and adult.

[0078] The tumor fragments can then be cultured in vitro using media and cell stimulants, such as cytokines, In some cases, IL-2 can be used to grow TILs from the tumor fragments.

[0079] First proliferation (pre-REP) Pre-REP TILs expanded by this method can generate REP TILs without a significantly reduced CD8 / CD4 ratio, and the pre-REP TILs can be expanded while maintaining low levels of expression of the exhaustion marker PD-1 in the REP TILs.

[0080] In the first expansion (pre-REP, step (a) of the method), the first cell culture medium contains from about 1,000 IU / mL to about 10,000 IU / mL of IL-2, from about 1,000 IU / mL to about 9,000 IU / mL of IL-2, from about 1,000 IU / mL to about 8,000 IU / mL of IL-2, from about 2,000 IU / mL to about 8,000 IU / mL of IL-2, from about 2,000 IU / mL to about 7,000 IU / mL of IL-2, from about 2,000 IU / mL to about 6,000 IU / mL of IL-2, from about 2,000 IU / mL to about 5,000 IU / mL of IL-2, from about 2,000 IU / mL to about 4,000 IU / mL of IL-2, from about 2,000 IU / mL to about 5,000 IU / mL of IL-2, from about 2,000 IU / mL to about 6,000 IU / mL of IL-2, from about 2,000 IU / mL to about 7,000 IU / mL of IL-2, from about 2,000 IU / mL to about 8,000 IU / mL of IL-2, from about 2,000 IU / mL to about 9,000 IU / mL of IL-2, from about 2,000 IU / mL to about 10,000 IU / mL of IL-2, from about 2,000 IU / mL to about 12,000 IU / mL to about 14,000 IU / mL of IL-2, from about 2,000 IU / mL to about 16,000 IU / mL to about 3,000 IU / mL IL-2, about 3,000 IU / mL to about 8,000 IU / mL IL-2, about 3,000 IU / mL to about 7,000 IU / mL IL-2, about 3,000 IU / mL to about 6,000 IU / mL IL-2, about 3,000 IU / mL to about 5,000 IU / mL IL-2, about 4,000 IU / mL to about 8,000 IU / mL IL-2, about 2,000 IU / mL IL-2, about 3,000 IU / mL IL-2, about 4,000 IU / mL IL-2, about 5,000 IU / mL IL-2, about 6,000 IU / mL IL-2, about 7,000 IU / mL IL-2, about 8,000 In certain embodiments, the first cell culture medium may comprise about 1,000 IU / mL of IL-2, about 9,000 IU / mL of IL-2, or about 10,000 IU / mL of IL-2. In certain embodiments, the first cell culture medium may comprise about 3,000 IU / mL of IL-2. In certain embodiments, the first cell culture medium may comprise about 5,000 IU / mL of IL-2. In certain embodiments, the first cell culture medium may comprise about 6,000 IU / mL of IL-2. In certain embodiments, the first cell culture medium may comprise about 7,000 IU / mL of IL-2. In certain embodiments, the first cell culture medium may comprise about 8,000 IU / mL of IL-2.

[0081] In the first expansion (pre-REP, step (a) of the method), the first cell population may be cultured for about 3 to about 60 days, about 5 to about 50 days, about 7 to about 40 days, about 10 to about 40 days, about 10 to about 30 days, about 10 to about 20 days, about 10 to about 18 days, about 10 to about 17 days, about 10 to about 16 days, about 12 to about 15 days, about 12 to about 14 days, about 13 to about 14 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, or about 20 days (the first expansion may continue for such periods). In certain embodiments, the first cell population is cultured for about 14 days. In certain embodiments, the first expansion continues for about 14 days.

[0082] In the first expansion (pre-REP, step (a) of the method), once the first cell population has been cultured for about 2 or 3 days (on or about day 3, if day 0 is the day when the culture of the first cell population is initiated), about 3 or 4 days (on or about day 4), about 4 or 5 days (on or about day 5), about 5 or 6 days (on or about day 6), about 6 or 7 days (on or about day 7), about 7 or 8 days (on or about day 8), about 8 or 9 days (on or about day 9), about 9 or 10 days (on or about day 10), about 10 or 11 days (on or about day 11), about 11 or 12 days (on or about day 12), about 12 or 13 days (on or about day 13), or about 13 or 14 days (on or about day 14). In certain embodiments, in the first expansion (pre-REP, step (a) of the method), the cell culture medium is added once on or about day 9 (when the first cell population has been cultured for about 8 or 9 days). In certain embodiments, in the first expansion (pre-REP, step (a) of the method), the cell culture medium is added once on or about day 10 (when the first cell population has been cultured for about 9 or 10 days). In certain embodiments, in the first expansion (pre-REP, step (a) of the method), the cell culture medium is added once on or about day 11 (when the first cell population has been cultured for about 10 or 11 days).

[0083] In certain embodiments, in the first expansion (pre-REP, step (a) of the method), the volume of cell culture medium added is about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 1.1x, about 1.2x, about 1.3x, about 1.4x, about 1.5x, about 1.6x, about 1.7x, about 1.8x, about 1.9x, about 2x, about 2.2x, about 2.5x, about 2.7x or about 3x the volume of cell culture (medium) in the cell culture vessel.

[0084] The initial culture volume of the first expansion (pre-REP, step (a) of the method) may be from about 5% to about 100%, from about 10% to about 80%, from about 10% to about 60%, from about 10% to about 50%, from about 10% to about 40%, from about 10% to about 30%, from about 20% to about 80%, from about 20% to about 60%, from about 20% to about 50%, The initial culture volume for the first expansion (pre-REP, step (a) of the method) is about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, or about 40% of the volume of the cell culture vessel / system. In certain embodiments, the initial culture volume for the first expansion (pre-REP, step (a) of the method) is about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, or about 40% of the volume of the cell culture vessel / system. In certain embodiments, the initial culture volume for the first expansion (pre-REP, step (a) of the method) is about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, or about 40% of the volume of the cell culture vessel / system. In certain embodiments, the initial culture volume for the first expansion (pre-REP, step (a) of the method) is about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, or about 40% of the volume of the cell culture vessel / system. 2 For a G-Rex® 100M device (100 cm2 bottom surface area), an initial culture volume of 10 mL, 15 mL, 20 mL, 25 mL, 30 mL, 35 mL or 40 mL is used. 2 For 100 mL, 150 mL, 200 mL, 250 mL, 300 mL, 350 mL or 400 mL initial culture volumes are used.

[0085] The first expansion (pre-REP) consisted of approximately 1 × 10 TILs or cells. 7 ~Approx. 1×10 9 Approximately 2 x 10 TILs or cells 7 ~Approx. 9×10 8 Approximately 3 x 10 TILs or cells 7 ~Approx. 8×10 8 Approximately 4 x 10 TILs or cells 7 ~Approx. 6×10 8 Approximately 5 x 10 TILs or cells 7 ~Approx. 5×10 8 Approximately 6 x 10 TILs or cells 7 ~Approx. 4×10 8 Approximately 8 x 10 TILs or cells 7 ~Approx. 3×10 8 Approximately 9 x 10 TILs or cells7 ~Approx. 2×10 8 Approximately 9 x 10 TILs or cells 7 ~Approx. 1×10 8 or approximately 1 x 10 TILs or cells 8 A range of cell or TIL populations may be generated.

[0086] Secondary proliferation (REP) The second propagation is generally referred to as the rapid propagation process (REP). The second propagation of the method may or may not use feeder cells. In one embodiment, the method or the second propagation of the method is a process that does not use feeder cells.

[0087] In the second expansion (REP, step (b) of the method), the second cell culture medium contains from about 50 IU / mL to about 10,000 IU / mL of IL-2, from about 100 IU / mL to about 8,000 IU / mL of IL-2, from about 200 IU / mL to about 6,000 IU / mL of IL-2, from about 500 IU / mL to about 6,000 IU / mL of IL-2, from about 500 IU / mL to about 4,000 IU / mL of IL-2, from about 500 IU / mL to about 3,000 IU / mL of IL-2, from about 800 IU / mL to about 5,000 IU / mL of IL-2, from about 800 IU / mL to about 4,000 IU / mL of IL-2, from about 1,000 IU / mL to about 6,000 IU / mL of IL-2. IU / mL of IL-2, or from about 1,000 IU / mL to about 4,000 IU / mL of IL-2. In one embodiment, the second cell culture medium comprises about 3,000 IU / mL of IL-2.

[0088] In the second propagation (REP, step (b) of the method), the contact may be for about 3 days to about 17 days, about 3 days to about 60 days, about 5 days to about 50 days, about 7 days to about 40 days, about 10 days to about 40 days, about 10 days to about 30 days, about 10 days to about 20 days, or about 10 days to about 14 days.

[0089] In some embodiments, the second expansion (REP, step (b) of the method) may continue for 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days. In some embodiments, the second TIL expansion may continue for about 7 days to about 14 days. In some embodiments, the second TIL expansion may continue for about 8 days to about 14 days. In some embodiments, the second TIL expansion may continue for about 9 days to about 14 days. In some embodiments, the second TIL expansion may continue for about 10 days to about 14 days. In some embodiments, the second TIL expansion may continue for about 11 days to about 14 days. In some embodiments, the second TIL expansion may continue for about 12 days to about 14 days. In some embodiments, the second TIL expansion may continue for about 13 days to about 14 days. In some embodiments, the second TIL expansion may continue for about 14 days.

[0090] In the second propagation (REP, step (b) of the method), the contacting can be performed in vitro, for example in any container that can accommodate the cells, preferably in a sterile environment. Such containers can be, for example, culture flasks, culture bags, bioreactors, or any device that can be used to grow cells, including in a closed cell culture system or closed container. The closed system or container can provide a gas permeable surface area. In one embodiment, the second propagation (REP, step (b) of the method) can be performed using T-175 flasks, gas permeable bags, and / or gas permeable culture systems (e.g., G-Rex®). In some embodiments, the second propagation (REP, step (b) of the method) is performed in a closed system or closed bioreactor. In some embodiments, the closed bioreactor is a single bioreactor. In one embodiment, the second propagation can be performed in G-Rex® 10M and / or G-Rex® 100M.

[0091] In certain embodiments, the third cell population is at least 100 times more in number than the second cell population, e.g., about 100 to about 2000 times more in number, about 100 to about 1800 times more in number, about 100 to about 1500 times more in number, about 100 to about 1200 times more in number, about 100 to about 1000 times more in number, about 100 to about 800 times more in number, about 100 to about 600 times more in number, or about 100 to about 500 times more in number.

[0092] The third cell population can be a therapeutic population of TILs. In some embodiments, the therapeutic population of TILs comprises sufficient TILs for a therapeutically effective dose of TILs.

[0093] In some embodiments, the third cell population comprises an expanded subpopulation of effector T cells and / or central memory T cells compared to the second TIL population, hi some embodiments, the third TIL population preserves to a greater extent the TCR repertoire of the second TIL population.

[0094] Polymer matrix containing anti-CD3 and anti-CD28 antibodies or fragments thereof - Patents.com In certain embodiments, the methods use a polymer matrix comprising anti-CD3 and anti-CD28 antibodies or fragments thereof to generate, expand, activate and / or enrich TILs.

[0095] The flexible matrix can comprise (or consist essentially of, or consist of) collagen, proteins, peptides, polysaccharides, glycosaminoglycans and / or extracellular matrix compositions. Polysaccharides include, for example, cellulose, agarose, dextran, chitosan, hyaluronic acid or alginate. Other polymers can include polyesters, polyethers, polyanhydrides, polyalkylcyanoacrylates, polyacrylamides, polyorthoesters, polyphosphazenes, polyvinyl acetates, block copolymers, polypropylenes, polytetrafluoroethylene (PTFE) or polyurethanes. The polymer can be lactic acid or a copolymer. The copolymer can include lactic and / or glycolic acid (PLGA). The polymer matrix can comprise (or consist essentially of, or consist of) a polymeric dextran material (or a polymer of dextran).

[0096] The polymer matrix may have an average molecular weight of 40,000 Daltons.

[0097] The polymer matrix may or may not contain magnetic, paramagnetic, superparamagnetic nanocrystals or fluorescent dyes (eg, embedded within the polymer matrix).

[0098] The polymer matrix may have a size of less than 1 μm, less than 500 nm, or less than 200 nm. The polymer matrix may have a size of about 1 nm to about 500 nm, or about 10 nm to about 200 nm.

[0099] The anti-CD3 antibody or fragment thereof and the anti-CD28 antibody or fragment thereof can be bound to the same polymer matrix or can be bound to separate polymer matrices. The anti-CD3 antibody or fragment thereof and the anti-CD28 antibody or fragment thereof can be bound or coupled to the polymer matrix by various methods known in the art. The binding can be covalent or non-covalent, electrostatic or hydrophobic. The binding can be achieved by various binding means including chemical, mechanical, enzymatic or other suitable means. The antibody or fragment thereof can be first directly or indirectly bound to the matrix. For example, the antibody or fragment thereof can be first bound to the matrix via an avidin (or streptavidin) and biotin system. The antibody or fragment thereof can be indirectly bound to the matrix, for example via an anti-isotype antibody. Another example includes using protein A or protein G or other non-specific antibody binding molecules bound to the matrix to bind the antibody or fragment thereof. Alternatively, the antibody or fragment thereof can be bound to the matrix by chemical means such as cross-linking to the matrix.

[0100] Anti-CD3 and / or anti-CD28 antibodies can be polyclonal and monoclonal antibodies, chimeric antibodies, haptens and antibody fragments, and molecules that are equivalent to antibodies in that they specifically bind to an epitope on an antigen. The term "antibody" includes polyclonal and monoclonal antibodies of any isotype (IgA, IgG, IgE, IgD, IgM), or antigen-binding portions thereof, such as, but not limited to, F(ab) and Fv fragments, such as scFv, single-chain antibodies, chimeric antibodies, humanized antibodies, and Fab expression libraries. In certain embodiments, anti-CD3 and / or anti-CD28 antibodies can be monoclonal antibodies. Anti-CD3 antibodies include OKT-3, also known as muromonab. Anti-CD3 antibodies also include UHCT1 clones, also known as CD3ε and T3. Other anti-CD3 antibodies include, for example, otelixizumab, teplizumab, and visilizumab.

[0101] The ratio of anti-CD3 antibody or fragment thereof to anti-CD28 antibody or fragment thereof (bound to the same polymer matrix or to separate polymer matrices) can range from about 100:1 to about 1:100, about 10:1 to about 1:10, or about 2:1 to about 1:2. The anti-CD3 antibody or fragment thereof and / or the anti-CD28 antibody or fragment thereof can be bound to the same or separate matrices at a high density of greater than 25 μg per mg of matrix, or greater than 50 μg per mg of matrix.

[0102] In one embodiment, the polymer matrix is ​​TransAct™. U.S. Patent No. 10,513,687.

[0103] In certain embodiments, the ratio of polymer matrix to cells can be greater than 100:1, greater than 500:1, or greater than 1000:1.

[0104] Pharmaceutical Compositions The present disclosure provides pharmaceutical compositions comprising the cells / TILs or cell populations of the present invention. The pharmaceutical compositions may comprise the cells or cell populations of the present invention and a pharma- ceutically acceptable carrier or excipient. The pharma- ceutically acceptable carrier or excipient may include suitable solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonicity and absorption delaying agents, and inert ingredients. Additional active pharmaceutical ingredients, such as other drugs, may also be incorporated into the compositions and methods described. Suitable pharma- ceutical acceptable carriers for cells for injection may include, for example, any isotonic carrier, such as saline (about 0.9% w / v NaCl in water, about 300 mOsm / L NaCl in water, or about 9.0 g NaCl per liter of water), or about 5% dextrose in water. In one embodiment, the pharma- ceutical acceptable carrier is supplemented with human serum albumin. The compositions of the present disclosure may be formulated for intravenous administration. In one embodiment, the pharmaceutical composition is a suspension of TILs or cells in a sterile buffer.

[0105] The cells or pharmaceutical compositions of the present invention may be used in methods of treating diseases, such as hyperproliferative disorders. They may also be used in the treatment of other disorders. In some embodiments, the hyperproliferative disorder is cancer.

[0106] The cell or cell population of the present invention can be used in a method for treating or preventing cancer.In this regard, the present disclosure provides a method for treating or preventing cancer in a mammal, comprising administering to a mammal an amount of the pharmaceutical composition, cell or cell population of the present invention that is effective for treating or preventing cancer in the mammal.Another embodiment of the present invention provides a method for treating or preventing cancer in a mammal, comprising administering to a mammal an amount of the cell or cell population of the present invention that is effective for treating or preventing cancer in the mammal.

[0107] The present disclosure also includes methods of treating a subject (e.g., a mammal) or patient with cancer. The methods can include administering the TILs of the invention and / or the cell populations of the invention to the subject or patient. The cells / TILs can be administered to the patient as a pharmaceutical composition.

[0108] The cells or pharmaceutical compositions of the present invention may be administered by any suitable route, including intranasal and transdermal routes, intraarterial routes, intravenous, intraperitoneal, parenteral, intramuscular, subcutaneous, topical, implantation or inhalation. The cells or pharmaceutical compositions of the present invention may be administered by injection or infusion. In some embodiments, the cells or pharmaceutical compositions of the present invention are administered by intraarterial or intravenous administration (e.g., infusion). Other suitable routes of administration include intraperitoneal, intrathecal and intralymphatic.

[0109] Any suitable dose of TILs can be administered. In some embodiments, about 2×10 10 ~Approx. 15×10 10 TILs or cells, approximately 1 x 10 10 ~Approx. 5×10 10 TILs or cells, approximately 3 x 10 10 ~Approx. 12×10 10 TILs or cells, approximately 4 x 10 10 ~About 10×10 10 TILs or cells, approximately 5 x 10 10 ~Approx. 8×10 10 TILs or cells, approximately 6 x 10 10 ~Approx. 8×10 10 TILs or cells, approximately 7 x 10 10 ~Approx. 8×10 10 TILs or cells, approximately 8 x 10 10 TILs or cells, or approximately 7 x 10 10 In some embodiments, a therapeutically effective dose is about 2×10 TILs or cells. 10 ~Approx. 15×10 10 In some embodiments, the therapeutically effective dose is about 1 x 10 TILs or cells. 10 ~Approx. 5×10 10In some embodiments, the therapeutically effective dose is about 3×10 TILs or cells. 10 ~Approx. 12×10 10 In some embodiments, the therapeutically effective dose is about 4×10 TILs or cells. 10 ~About 10×10 10 In some embodiments, the therapeutically effective dose is about 5×10 TILs or cells. 10 ~Approx. 8×10 10 In some embodiments, the therapeutically effective dose is about 6×10 TILs or cells. 10 ~Approx. 8×10 10 In some embodiments, the therapeutically effective dose is about 7×10 TILs or cells. 10 ~Approx. 8×10 10 In some embodiments, the effective dose of TILs is about 1×10 6 , 2×10 6 , 3×10 6 , 4×10 6 , 5×10 6 , 6×10 6 , 7×10 6 , 8×10 6 , 9×10 6 , 1×10 7 , 2×10 7 , 3×10 7 , 4×10 7 , 5×10 7 , 6×10 7 , 7×10 7 , 8×10 7 , 9×10 7 , 1×10 8 , 2×10 8 , 3×10 8 , 4×10 8 , 5×10 8 , 6×10 8 , 7×10 8 , 8×10 8 , 9×10 8 , 1×10 9 , 2×10 9 , 3×10 9 , 4×10 9 , 5×10 9 , 6×10 9 , 7×109 , 8×10 9 , 9×10 9 , 1×10 10 , 2×10 10 , 3×10 10 , 4×10 10 , 5×10 10 , 6×10 10 , 7×10 10 , 8×10 10 , 9×10 10 , 1×10 11 , 2×10 11 , 3×10 11 , 4×10 11 , 5×10 11 , 6×10 11 , 7×10 11 , 8×10 11 , 9×10 11 , 1×10 12 , 2×10 12 , 3×10 12 , 4×10 12 , 5×10 12 , 6×10 12 , 7×10 12 , 8×10 12 , 1×10 13 , 2×10 13 , 3×10 13 , 4×10 13 , 5×10 13 , 6×10 13 , 7×10 13 , 8×10 13 and 9×10 13 In some embodiments, the effective dose of TILs is 1×10 6 ~5×10 6 , 5×10 6 ~1×10 7 , 1×10 7 ~5×10 7 , 5×10 7 ~1×10 8 , 1×10 8 ~5×10 8 , 1×10 9 ~5×10 9 , 5×10 9 ~1×10 10 , 1×10 10 ~5×10 10, 5×10 10 ~1×10 11 , 5×10 11 ~1×10 12 , 1×10 12 ~5×10 12 , and 5 × 10 12 ~1×10 13 In certain embodiments, the range is about 1×10 cells / TILs. 9 ~Approx. 1×10 11 cells, or approximately 5 x 10 9 ~Approx. 9×10 10 cells are administered to the patient. The number of cells / TILs is approximately 10 x 10 cells per administration (e.g., infusion). 6 ~About 10×10 11 Approximately 10 x 10 cells per administration (e.g., injection) 9 ~About 10×10 11 or 10 x 10 cells per administration (e.g., injection) 7 ~About 10×10 9 The pharmaceutical composition comprising TILs may be 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 10 , or 10 9 ~10 10 The therapeutic agent may be administered at a dose of 100 to 200 cells / kg body weight (including all integer values ​​within these ranges).

[0110] The TILs provided in the pharmaceutical composition of the present invention can be effective over a wide range of dosages.The exact dosage can depend on the route of administration, the rate of administration, the severity of the disorder or condition, the sex and age of the subject to be treated, and the body weight of the subject to be treated.In appropriate cases, the clinically established dosage of TILs can be used.

[0111] The cells or pharmaceutical compositions of the present invention may be administered in a single dose (single dose) or multiple doses (multiple doses). Such administration may be by injection, for example, intravenous injection. In some embodiments, the cells or pharmaceutical compositions of the present invention are administered as a single intra-arterial or intravenous infusion. Administration may be once, twice, three times, four times, five times, six times, or more than six times per year. Administration may be once per month, once every two weeks, once per week, or once every two days. Administration of TILs may be continued as long as necessary.

[0112] For purposes of the methods of the invention, when a cell population is administered, the cells can be allogeneic or autologous cells to the mammal. In one embodiment, the cells are autologous to the mammal.

[0113] The cancer treated by the cells or pharmaceutical compositions of the present invention may be melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, cancer caused by human papillomavirus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), kidney cancer, renal cell carcinoma, colorectal cancer or other types of cancer. The cells or pharmaceutical compositions of the present invention may be used to treat recurrent or refractory non-small cell lung cancer (NSCLC). Cancers include sarcomas (e.g., synovial sarcoma, osteosarcoma, uterine leiomyosarcoma, and alveolar rhabdomyosarcoma), lymphomas (e.g., Hodgkin's lymphoma and non-Hodgkin's lymphoma), hepatocellular carcinoma, glioma, head and neck cancer, acute lymphoid cancer, acute myeloid leukemia, bone cancer, brain cancer, breast cancer, anal cancer, anal canal cancer, anorectal cancer, eye cancer, intrahepatic bile duct cancer, joint cancer, cervical cancer, gallbladder cancer, pleural cancer, nasal cancer, nasal cavity cancer, middle ear cancer, oral cancer, vulvar cancer, chronic lymphocytic leukemia, The cancer may be any cancer, including any of the following: ovarian cancer, chronic myeloid cancer, colon cancer (e.g., colon carcinoma), esophageal cancer, cervical cancer, gastrointestinal cancer (e.g., gastrointestinal carcinoid tumors), hypopharyngeal cancer, laryngeal cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma, multiple myeloma, nasopharyngeal cancer, ovarian cancer, pancreatic cancer, peritoneal, omental and mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, kidney cancer, small intestine cancer, soft tissue cancer, stomach cancer, testicular cancer, thyroid cancer, ureteral cancer, and bladder cancer.

[0114] In some embodiments, the hyperproliferative disorder is a solid tumor cancer. In some embodiments, the solid tumor cancer is selected from the group consisting of melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, cancer caused by human papillomavirus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), kidney cancer and renal cell carcinoma. In some embodiments, the hyperproliferative disorder is a hematological malignancy. In some embodiments, the solid tumor cancer is chronic lymphocytic leukemia, acute lymphoblastic leukemia, diffuse large B-cell lymphoma, non-Hodgkin's lymphoma, Hodgkin's lymphoma, follicular lymphoma and mantle cell lymphoma.

[0115] In one embodiment, the method of treating cancer in a patient further comprises pretreating the patient with non-myeloablative chemotherapy prior to administration of the cells or pharmaceutical compositions of the invention. In one embodiment, the non-myeloablative chemotherapy comprises administering cyclophosphamide and / or fludarabine prior to TIL infusion. In one embodiment, following the non-myeloablative chemotherapy and TIL infusion, the patient receives an intravenous infusion of IL-2.

[0116] The term "about" in reference to a numerical value refers to ±10% of the numerical value stated. In other words, the numerical value can range from 90% of the stated value to 110% of the stated value.

[0117] Without further elaboration, it is believed that a person skilled in the art can utilize the present disclosure to its fullest extent based on the above description. Accordingly, the following specific embodiments should be construed as merely illustrative, and not limiting in any way to the remainder of the disclosure. All publications cited herein are incorporated by reference for the purposes or subject matter referred to herein.

[0118] Example 1: Development of the TIL pre-REP process and characterization of pre-REP TILs In the pre-rapid expansion protocol (pre-REP), autologous tumor infiltrating lymphocytes (TILs) were harvested from patient NSCLC tumor samples. The tumors were first fragmented and cultured in high dose IL-2 to promote the release and proliferation of TILs. After this initial ex vivo culture, TILs can be further expanded in the rapid expansion protocol (REP) until a therapeutic dose is reached. We have optimized a pre-REP TIL culture process that can be scaled for GMP production.

[0119] The initial T cell material (pre-REP TILs) can be cryopreserved while awaiting patient disease progression following standard treatment. After patient disease progression, the pre-REP TILs can be thawed and expanded by the Rapid Expansion Protocol (REP).

[0120] Our optimized pre-REP TIL culture process involves the use of a gas permeable rapid growth (G-Rex®) device as the TIL culture vessel. The process is performed with shorter culture times and medium change frequencies to maintain a robust GMP manufacturing process that produces pre-REP TILs with high cell yields and favorable T cell phenotypes suitable for further manufacturing. Medium can be added on or about days 8, 9, 10 and / or 11 [day 0 is the day cell culture (first growth or pre-REP) is initiated] and pre-REP TILs can be harvested on or about days 12, 13, 14, 15, 16 or 17. With the optimized protocol, IL-2 and glucose levels remain adequate while lactate, a growth inhibitory metabolite, is at low levels that do not adversely affect TIL proliferation. We demonstrated that after optimization, the average number of TILs generated per tumor fragment was more than four-fold higher than the number of TILs generated before optimization (16.29 × 10 6 ±10.58×10 6 vs. 3.10×10 6 ±3.52×10 6 ), which demonstrated an overall superior yield of pre-REP TILs. Pre-REP TILs exhibited an earlier differentiation state and a higher proportion of CD27 + Cells, the majority of which are T central memory (CD4 + In the compartment, 66.28% ± 12.87%, CD8 + in the IL-1 compartment (59.04% ± 13.47%; n = 8), and a very low percentage of effector cells (CD4 + Cells and CD8 + In the cells, the T N / T SCM The proportion of subsets was also higher, indicating more favorable TIL characteristics. 1 .

[0121] In one embodiment, the Rapid Expansion Protocol (REP) uses MACS® GMP T Cell TransAct™, a colloidal polymer nanomatrix covalently linked to humanized recombinant CD3 and CD28 agonists (e.g., antibodies). The REP TILs are then washed, formulated, and / or cryopreserved.

[0122] Fragmented melanoma tumors grown in the G-Rex® device produced an average of 7.51 × 10 tumors per tumor fragment during pre-REP, with an average of 14-18 days in culture. 6 It has previously been shown to generate TILs 2 Our target cell number for pre-REP production is a minimum of 1 x 10 based on previous publications. 9 Based on a TIL target dose and an estimated REP expansion of 400-fold, 6 x 10 7 CD3 + T cells were 3 Appropriate conditions favoring the expansion and preservation of the early differentiation phenotype of pre-REP TILs will generate high-quality intermediates, which are expected to facilitate the production of a final REP TIL product with high therapeutic potential. 4,5 This study aimed to optimize primary culture conditions to generate high-yield production of pre-REP TILs with an early differentiation phenotype and shortened culture times.

[0123] method Tumor collection Non-small cell lung cancer tumor samples were collected. NSCLC samples were de-identified and each donor was assigned a unique identification number. Briefly, resected tumor samples were placed in tissue preservation solution at 4°C and shipped within 24 hours after surgery. Tumor samples were transferred to the preparation of TILs immediately after receipt.

[0124] Tumor fragmentation and dissemination Only one tumor was treated at a time, and a new set of materials and equipment was used for each tumor.

[0125] Tumor wash medium was added to the wells of a 6-well plate for washing. Tumor wash medium was added to a Petri dish for tumor fragmentation. Using tweezers, the tumor was transferred to the Petri dish. The tumor was cut into fragments of approximately 2-3 mm in each dimension. During fragmentation, areas of necrosis, hemorrhage, and fatty tissue were removed. Each tumor fragment was washed to remove red blood cells. This process was repeated until the entire tumor section was dissected. The number of tumor fragments was recorded, and fragments from the same donor were combined for culture in the same culture device.

[0126] When using the G-Rex® 10M device, 1, 2, 3, 4, 5, 6, 7 or 8 tumor fragments from the same donor were seeded into one G-Rex® 10M device. When using the G-Rex® 100M device, up to 100 tumor fragments from the same donor were seeded into one G-Rex® 100M device.

[0127] Expansion of pre-REP TILs Pre-REP TILs were cultured in pre-REP basal medium supplemented with 300 IU / mL, 1000 IU / mL, 3000 IU / mL or 6000 IU / mL IL-2, containing RPMI-1640, human AB serum, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), β-ME (2-mercaptoethanol), GlutaMax and gentamicin.

[0128] On day 0 after tumor fragmentation, tumor fragments were seeded into G-Rex® 10M culture devices. For small-scale pre-REP, 1-8 fragments were added into the G-Rex® 10M device in an initial medium volume of 10 mL, 20 mL, 25 mL, 30 mL, 50 mL or 100 mL.

[0129] number of cells Cell concentration, viability and size were measured using a NucleoCounter® NC-200™ system.

[0130] Collection of TIL culture medium samples If spent medium was removed before cell counting, 500 μL of spent medium was stored in a −20° C. freezer for later analysis. Alternatively, if cell counting was not performed, small amounts (approximately 500 μL each time) of pre-REP culture medium were collected from near the medium surface. Medium was collected from the G-Rex® 10M or G-Rex® 100M devices at the indicated time points during the pre-REP stage (e.g., days 0, 3, 6, 7, 10, and 14).

[0131] Quantification of glucose and lactate concentrations in cultures Glucose and lactate concentrations in culture supernatants were measured by Glucose-Glo Assay and Lactate-Glo Assay (Promega).

[0132] Quantification of IL-2 concentrations by ELISA To quantify IL-2 protein concentration, an IL-2 ELISA kit from Thermo Fisher Scientific, Inc. was used. Samples were measured in duplicate or triplicate wells. Data are presented as mean ± standard deviation (SD). Protein concentrations (pg / mL) were converted to IU / mL (for IL-2, 1 ng / mL = 16.36 IU / mL).

[0133] Flow cytometry The cells were first stained with LIVE / DEAD™ Fixable Aqua (Thermo Fisher Scientific, Inc.) and then stained with antibodies against various surface markers. Samples were then acquired using a CytoFLEX LX flow cytometer. All data were analyzed using FlowJo V10 software.

[0134] Results and Analysis As an alternative to flasks and bags for immature TIL processes, G-Rex® (Gas Permeable Rapid Proliferation) is a manufacturing platform specifically tailored for immune cell production that is easy to scale up, scale out, and meets the cGMP conditions required for clinical trials. Early TIL expansion (pre-REP) was compared using metastatic melanoma tumors cultured in the G-Rex® 10 device versus 24-well plates. 2 In our study, we used the G-Rex® device as the pre-REP TIL culture vessel.

[0135] We first used four matched tumor sample sets (T5101001, T5101002, T5101004 and T5101005) for pre-REP using G-Rex® 10M. The final pre-REP cell yield was (15.60±25.09)×10 cells for the four donors. 6 From (27.54±22.60)×10 6 The range was 100%.

[0136] Phenotyping of pre-REP TILs + Cells and CD8 + The majority of cells are of effector phenotype T EM (effector memory T cells), but central memory phenotype T CM (central memory T cells).

[0137] To optimize feeding, glucose and lactate levels as well as IL-2 consumption in pre-REP cultures were measured. Next, G-Rex® 100M devices (bottom surface area 100 cm) were used to measure the glucose and lactate levels in pre-REP cultures. 2The process was scaled up to use 1000 mL of 1000-mL PBS (volume 1000 mL). Tumor fragments were used to initiate pre-REP cultures of three donors (T5101009, T5101010 and T5101013). Pre-REP runs for T5101009 and T5101010 were performed prior to the determination of culture time and stopped on days 17 and 20, respectively, without addition or replacement of medium. For T5101013, cell counts were performed and fresh medium was added on or around days 8, 9, 10 or 1. Glucose levels began to fall in T5101010 and T5101013 after day 6, where significant cell proliferation was observed compared to T5101009. Greater lactate production by T5101010 was detected by day 3 and continued to increase throughout the culture. For T5101013, the addition of fresh medium appeared to prevent further increases in lactate levels, and such medium addition is particularly important for donors with rapidly proliferating TILs to maintain lactate at low levels that are tolerated by T cells.

[0138] Furthermore, IL-2 consumption in pre-REP cultures was evaluated. IL-2 protein concentration was measured by ELISA kit, and the concentration (pg / mL) was converted to IU / mL (IL-2: 1 ng / mL = 16.36 IU / mL). During the first 6 days of the two pre-REP runs analyzed, the culture medium was not changed or added because the total number of T cells was low at the initial stage. However, at day 6, the yield of pre-REP TILs for both T5101001 and T5101002 was 2 × 10 6 Although the increase in IL-2 levels was less than 50%, a reduction of approximately 50% in IL-2 levels was still observed.

[0139] TILs derived from single tumor fragments in 24-well plates were seeded in G-Rex® 10M devices and cultured for an additional 8 days without medium replacement / addition during the first 6 days. We found a consistent ~50% decrease in IL-2 levels after 6 days in both T5101001 and T5101002 culture medium samples, but their proliferation was significantly different (T5101001 - 5×10 6 and T5101001 - 10×10 6 For T5101002 - 5×10 6 and T5101002 - 10×10 6 (3.18-fold and 3.57-fold, respectively, for pre-REP TILs). Thus, T cell proliferation did not appear to be a determining factor for the reduced IL-2 concentrations in pre-REP TIL cultures.

[0140] Additionally, IL-2 consumption was examined in the G-Rex® 100M device during the pre-REP phase (same sample set used for glucose and lactate measurements). In this analysis, donors T5101009 and T5101010 showed significantly different pre-REP TIL yields (2.2×10 6 vs 93×10 6 ), whereas the pre-REP run showed comparable IL-2 decline curves. It is noteworthy that for these two donors, no medium change / supplement was performed throughout the pre-REP phase. As a result, no matter how strongly the T cells proliferated, at day 14, approximately 20% of the original IL-2 concentration remained.

[0141] Pre-REP Procedures Day 0 - Tumor Fragmentation and Primary TIL Culture. Briefly, tumors are cut into fragments of approximately 2-3 mm in each dimension and necrosis, hemorrhage and fat deposits are removed. 1, 2, 3, 4 or 5 fragments are cultured per G-Rex® 10M culture device in an initial culture volume of 10 mL, 20 mL, 25 mL, 30 mL, 50 mL or 100 mL. For a total number of fragments up to 100, 1-2 G-Rex® 100M culture devices may be used with an initial culture volume of 100 mL, 200 mL, 250 mL, 500 mL or 1000 mL. All pre-REP culture medium for the entire process may be prepared on day 0 using 500 or 1000 mL of pre-REP basal medium supplemented with 300 IU / mL, 1000 IU / mL, 3000 IU / mL or 6000 IU / mL of IL-2. In one embodiment, the IL-2 concentration is 300 IU / mL. In another embodiment, the IL-2 concentration is 1000 IU / mL. In yet another embodiment, the IL-2 concentration is 3000 IU / mL. In yet another embodiment, the IL-2 concentration is 6000 IU / mL.

[0142] Days 8, 9, 10 or 11 - Addition or replacement of medium. Pre-REP CM (culture medium) prepared on day 0 is warmed to room temperature or 37°C prior to use. If using G-Rex® 10M or G-Rex® 100M devices, a total volume of 1x, 2x or 3x the initial pre-REP CM volume is added or replaced into each culture device. Return culture devices to incubator for continuation of TIL culture.

[0143] Day 13, 14, 15, 16 or 17 - Harvesting of pre-REP TILs. Prepare 100 or 200 mL of harvesting solution using Plasma-LyteA (polyelectrolyte for injection) and human serum albumin (HSA) to obtain a final concentration of 0.5%, 0.7%, 1%, 1.5%, 2% or 5% HSA solution. Harvest the TILs manually. Collect the cell suspension and filter through a cell strainer. Wash the G-Rex® device twice to ensure that all TILs are harvested.

[0144] The cells are centrifuged at 200, 300 or 400 g for 15 or 30 min at 4° C. The supernatant is discarded and the cell pellet is resuspended and combined using recovery solution containing HSA in Plasma-Lyte A. A cell count is performed using an NC200 automated cell counter and the cells are stored frozen.

[0145] The following culture conditions are used:

[0146] Culture conditions (Ai): Day 0 - Initial culture volume is 10 mL for G-Rex® 10M [or 100 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 10% of the volume of the cell culture vessel / system. Day 8 - Add or replace cell culture medium with a volume of 1x the initial culture volume. Day 13 - Pre-REP TIL recovery.

[0147] Culture conditions (A-ii): Day 0 - Initial culture volume is 20 mL for G-Rex® 10M [or 200 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 20% of the volume of the cell culture vessel / system. Day 8 - Add or replace cell culture medium with a volume of 1x the initial culture volume. Day 13 - Pre-REP TIL recovery.

[0148] Culture conditions (A-iii): Day 0 - Initial culture volume is 25 mL for G-Rex® 10M [or 250 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 25% of the volume of the cell culture vessel / system. Day 8 - Add or replace cell culture medium with a volume of 1x the initial culture volume. Day 13 - Pre-REP TIL recovery.

[0149] Culture conditions (A-iv): Day 0 - Initial culture volume is 30 mL for G-Rex® 10M [or 300 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 30% of the volume of the cell culture vessel / system. Day 8 - Add or replace cell culture medium with a volume of 1x the initial culture volume. Day 13 - Pre-REP TIL recovery.

[0150] Culture conditions (Av): Day 0 - Initial culture volume is 50 mL for G-Rex® 10M [or 500 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 50% of the volume of the cell culture vessel / system. Day 8 - Add or replace cell culture medium with a volume of 1x the initial culture volume. Day 13 - Pre-REP TIL recovery.

[0151] Culture conditions (A-vi): Day 0 - Initial culture volume is 100 mL for G-Rex® 10M (1000 mL for G-Rex® 100M). In other words, the initial culture volume for the first expansion is 100% of the volume of the cell culture vessel / system. Day 8 - Replace the cell culture medium with a volume of 1x the initial culture volume. Day 13 - Pre-REP TIL recovery.

[0152] Culture conditions (A-vii): Day 0 - Initial culture volume is 10 mL for G-Rex® 10M [or 100 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 10% of the volume of the cell culture vessel / system. Day 8 - Add or replace cell culture medium in an amount twice the initial culture volume. Day 13 - Pre-REP TIL recovery.

[0153] Culture conditions (A-viii): Day 0 - Initial culture volume is 20 mL for G-Rex® 10M [or 200 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 20% of the volume of the cell culture vessel / system. Day 8 - Add or replace cell culture medium in an amount twice the initial culture volume. Day 13 - Pre-REP TIL recovery.

[0154] Culture conditions (A-ix): Day 0 - Initial culture volume is 25 mL for G-Rex® 10M [or 250 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 25% of the volume of the cell culture vessel / system. Day 8 - Add or replace cell culture medium in an amount twice the initial culture volume. Day 13 - Pre-REP TIL recovery.

[0155] Culture conditions (Ax): Day 0 - Initial culture volume is 30 mL for G-Rex® 10M [or 300 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 30% of the volume of the cell culture vessel / system. Day 8 - Add or replace cell culture medium in an amount twice the initial culture volume. Day 13 - Pre-REP TIL recovery.

[0156] Culture conditions (A-xi): Day 0 - Initial culture volume is 50 mL for G-Rex® 10M [or 500 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 50% of the volume of the cell culture vessel / system. Day 8 - Replace the cell culture medium with twice the amount of the initial culture volume. Day 13 - Pre-REP TIL recovery.

[0157] Culture conditions (A-xii): Day 0 - Initial culture volume is 10 mL for G-Rex® 10M [or 100 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 10% of the volume of the cell culture vessel / system. Day 8 - Add or replace cell culture medium in an amount three times the initial culture volume. Day 13 - Pre-REP TIL recovery.

[0158] Culture conditions (A-xiii): Day 0 - Initial culture volume is 20 mL for G-Rex® 10M [or 200 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 20% of the volume of the cell culture vessel / system. Day 8 - Add or replace cell culture medium in an amount three times the initial culture volume. Day 13 - Pre-REP TIL recovery.

[0159] Culture conditions (A-xiv): Day 0 - Initial culture volume is 25 mL for G-Rex® 10M [or 250 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 25% of the volume of the cell culture vessel / system. Day 8 - Add or replace cell culture medium in an amount three times the initial culture volume. Day 13 - Pre-REP TIL recovery.

[0160] Culture conditions (A-xv): Day 0 - Initial culture volume is 30 mL for G-Rex® 10M [or 300 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 30% of the volume of the cell culture vessel / system. Day 8 - Replace the cell culture medium with an amount three times the initial culture volume. Day 13 - Pre-REP TIL recovery.

[0161] Culture conditions (Bi): Day 0 - Initial culture volume is 10 mL for G-Rex® 10M [or 100 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 10% of the volume of the cell culture vessel / system. Day 9 - Add or replace cell culture medium with a volume of 1x the initial culture volume. Day 13 - Pre-REP TIL recovery.

[0162] Culture conditions (B-ii): Day 0 - Initial culture volume is 20 mL for G-Rex® 10M [or 200 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 20% of the volume of the cell culture vessel / system. Day 9 - Add or replace cell culture medium with a volume of 1x the initial culture volume. Day 13 - Pre-REP TIL recovery.

[0163] Culture conditions (B-iii): Day 0 - Initial culture volume is 25 mL for G-Rex® 10M [or 250 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 25% of the volume of the cell culture vessel / system. Day 9 - Add or replace cell culture medium with a volume of 1x the initial culture volume. Day 13 - Pre-REP TIL recovery.

[0164] Culture conditions (B-iv): Day 0 - Initial culture volume is 30 mL for G-Rex® 10M [or 300 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 30% of the volume of the cell culture vessel / system. Day 9 - Add or replace cell culture medium with a volume of 1x the initial culture volume. Day 13 - Pre-REP TIL recovery.

[0165] Culture conditions (Bv): Day 0 - Initial culture volume is 50 mL for G-Rex® 10M [or 500 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 50% of the volume of the cell culture vessel / system. Day 9 - Add or replace cell culture medium with a volume of 1x the initial culture volume. Day 13 - Pre-REP TIL recovery.

[0166] Culture conditions (B-vi): Day 0 - Initial culture volume is 100 mL for G-Rex® 10M (1000 mL for G-Rex® 100M). In other words, the initial culture volume for the first expansion is 100% of the volume of the cell culture vessel / system. Day 9 - Replace the cell culture medium with a volume of 1x the initial culture volume. Day 13 - Pre-REP TIL recovery.

[0167] Culture conditions (B-vii): Day 0 - Initial culture volume is 10 mL for G-Rex® 10M [or 100 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 10% of the volume of the cell culture vessel / system. Day 9 - Add or replace cell culture medium in an amount twice the initial culture volume. Day 13 - Pre-REP TIL recovery.

[0168] Culture conditions (B-viii): Day 0 - Initial culture volume is 20 mL for G-Rex® 10M [or 200 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 20% of the volume of the cell culture vessel / system. Day 9 - Add or replace cell culture medium in an amount twice the initial culture volume. Day 13 - Pre-REP TIL recovery.

[0169] Culture conditions (B-ix): Day 0 - Initial culture volume is 25 mL for G-Rex® 10M [or 250 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 25% of the volume of the cell culture vessel / system. Day 9 - Add or replace cell culture medium in an amount twice the initial culture volume. Day 13 - Pre-REP TIL recovery.

[0170] Culture conditions (Bx): Day 0 - Initial culture volume is 30 mL for G-Rex® 10M [or 300 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 30% of the volume of the cell culture vessel / system. Day 9 - Add or replace cell culture medium in an amount twice the initial culture volume. Day 13 - Pre-REP TIL recovery.

[0171] Culture conditions (B-xi): Day 0 - Initial culture volume is 50 mL for G-Rex® 10M [or 500 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 50% of the volume of the cell culture vessel / system. Day 9 - Replace the cell culture medium with twice the amount of the initial culture volume. Day 13 - Pre-REP TIL recovery.

[0172] Culture conditions (B-xii): Day 0 - Initial culture volume is 10 mL for G-Rex® 10M [or 100 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 10% of the volume of the cell culture vessel / system. Day 9 - Add or replace cell culture medium in an amount three times the initial culture volume. Day 13 - Pre-REP TIL recovery.

[0173] Culture conditions (B-xiii): Day 0 - Initial culture volume is 20 mL for G-Rex® 10M [or 200 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 20% of the volume of the cell culture vessel / system. Day 9 - Add or replace cell culture medium in an amount three times the initial culture volume. Day 13 - Pre-REP TIL recovery.

[0174] Culture conditions (B-xiv): Day 0 - Initial culture volume is 25 mL for G-Rex® 10M [or 250 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 25% of the volume of the cell culture vessel / system. Day 9 - Add or replace cell culture medium in an amount three times the initial culture volume. Day 13 - Pre-REP TIL recovery.

[0175] Culture conditions (B-xv): Day 0 - Initial culture volume is 30 mL for G-Rex® 10M [or 300 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 30% of the volume of the cell culture vessel / system. Day 9 - Replace the cell culture medium with an amount three times the initial culture volume. Day 13 - Pre-REP TIL recovery.

[0176] Culture conditions (Ci): Day 0 - Initial culture volume is 10 mL for G-Rex® 10M [or 100 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 10% of the volume of the cell culture vessel / system. Day 10 - Add or replace cell culture medium with a volume of 1x the initial culture volume. Day 13 - Pre-REP TIL recovery.

[0177] Culture conditions (C-ii): Day 0 - Initial culture volume is 20 mL for G-Rex® 10M [or 200 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 20% of the volume of the cell culture vessel / system. Day 10 - Add or replace cell culture medium with a volume of 1x the initial culture volume. Day 13 - Pre-REP TIL recovery.

[0178] Culture conditions (C-iii): Day 0 - Initial culture volume is 25 mL for G-Rex® 10M [or 250 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 25% of the volume of the cell culture vessel / system. Day 10 - Add or replace cell culture medium with a volume of 1x the initial culture volume. Day 13 - Pre-REP TIL recovery.

[0179] Culture conditions (C-iv): Day 0 - Initial culture volume is 30 mL for G-Rex® 10M [or 300 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 30% of the volume of the cell culture vessel / system. Day 10 - Add or replace cell culture medium with a volume of 1x the initial culture volume. Day 13 - Pre-REP TIL recovery.

[0180] Culture conditions (Cv): Day 0 - Initial culture volume is 50 mL for G-Rex® 10M [or 500 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 50% of the volume of the cell culture vessel / system. Day 10 - Add or replace cell culture medium with a volume of 1x the initial culture volume. Day 13 - Pre-REP TIL recovery.

[0181] Culture conditions (C-vi): Day 0 - Initial culture volume is 100 mL for G-Rex® 10M [or 1000 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 100% of the volume of the cell culture vessel / system. Day 10 - Replace the cell culture medium with a volume of 1x the initial culture volume. Day 13 - Pre-REP TIL recovery.

[0182] Culture conditions (C-vii): Day 0 - Initial culture volume is 10 mL for G-Rex® 10M [or 100 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 10% of the volume of the cell culture vessel / system. Day 10 - Add or replace cell culture medium with a volume twice the initial culture volume. Day 13 - Pre-REP TIL recovery.

[0183] Culture conditions (C-viii): Day 0 - Initial culture volume is 20 mL for G-Rex® 10M [or 200 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 20% of the volume of the cell culture vessel / system. Day 10 - Add or replace cell culture medium with a volume twice the initial culture volume. Day 13 - Pre-REP TIL recovery.

[0184] Culture conditions (C-ix): Day 0 - Initial culture volume is 25 mL for G-Rex® 10M [or 250 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 25% of the volume of the cell culture vessel / system. Day 10 - Add or replace cell culture medium with a volume twice the initial culture volume. Day 13 - Pre-REP TIL recovery.

[0185] Culture conditions (Cx): Day 0 - Initial culture volume is 30 mL for G-Rex® 10M [or 300 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 30% of the volume of the cell culture vessel / system. Day 10 - Add or replace cell culture medium with a volume twice the initial culture volume. Day 13 - Pre-REP TIL recovery.

[0186] Culture conditions (C-xi): Day 0 - Initial culture volume is 50 mL for G-Rex® 10M [or 500 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 50% of the volume of the cell culture vessel / system. Day 10 - Replace the cell culture medium with a volume twice the initial culture volume. Day 13 - Pre-REP TIL recovery.

[0187] Culture conditions (C-xii): Day 0 - Initial culture volume is 10 mL for G-Rex® 10M [or 100 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 10% of the volume of the cell culture vessel / system. Day 10 - Add or replace cell culture medium with a volume three times the initial culture volume. Day 13 - Pre-REP TIL recovery.

[0188] Culture conditions (C-xiii): Day 0 - Initial culture volume is 20 mL for G-Rex® 10M [or 200 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 20% of the volume of the cell culture vessel / system. Day 10 - Add or replace cell culture medium with a volume three times the initial culture volume. Day 13 - Pre-REP TIL recovery.

[0189] Culture conditions (C-xiv): Day 0 - Initial culture volume is 25 mL for G-Rex® 10M [or 250 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 25% of the volume of the cell culture vessel / system. Day 10 - Add or replace cell culture medium with a volume three times the initial culture volume. Day 13 - Pre-REP TIL recovery.

[0190] Culture conditions (C-xv): Day 0 - Initial culture volume is 30 mL for G-Rex® 10M [or 300 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 30% of the volume of the cell culture vessel / system. Day 10 - Replace the cell culture medium with a volume three times the initial culture volume. Day 13 - Pre-REP TIL recovery.

[0191] Culture conditions (Di): Day 0 - Initial culture volume is 10 mL for G-Rex® 10M [or 100 mL for G-Rex® 100M]. In other words, the initial culture volume in the first expansion is 10% of the volume of the cell culture vessel / system. Day 11 - Add or replace cell culture medium with a volume of 1x the initial culture volume. Day 13 - Pre-REP TIL recovery.

[0192] Culture conditions (D-ii): Day 0 - Initial culture volume is 20 mL for G-Rex® 10M [or 200 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 20% of the volume of the cell culture vessel / system. Day 11 - Add or replace cell culture medium with a volume of 1x the initial culture volume. Day 13 - Pre-REP TIL recovery.

[0193] Culture conditions (D-iii): Day 0 - Initial culture volume is 25 mL for G-Rex® 10M [or 250 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 25% of the volume of the cell culture vessel / system. Day 11 - Add or replace cell culture medium with a volume of 1x the initial culture volume. Day 13 - Pre-REP TIL recovery.

[0194] Culture conditions (D-iv): Day 0 - Initial culture volume is 30 mL for G-Rex® 10M [or 300 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 30% of the volume of the cell culture vessel / system. Day 11 - Add or replace cell culture medium with a volume of 1x the initial culture volume. Day 13 - Pre-REP TIL recovery.

[0195] Culture conditions (Dv): Day 0 - Initial culture volume is 50 mL for G-Rex® 10M [or 500 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 50% of the volume of the cell culture vessel / system. Day 11 - Add or replace cell culture medium with a volume of 1x the initial culture volume. Day 13 - Pre-REP TIL recovery.

[0196] Culture conditions (D-vi): Day 0 - Initial culture volume is 100 mL for G-Rex® 10M [or 1000 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 100% of the volume of the cell culture vessel / system. Day 11 - Replace the cell culture medium with a volume of 1x the initial culture volume. Day 13 - Pre-REP TIL recovery.

[0197] Culture conditions (D-vii): Day 0 - Initial culture volume is 10 mL for G-Rex® 10M [or 100 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 10% of the volume of the cell culture vessel / system. Day 11 - Add or replace cell culture medium with a volume twice the initial culture volume. Day 13 - Pre-REP TIL recovery.

[0198] Culture conditions (D-viii): Day 0 - Initial culture volume is 20 mL for G-Rex® 10M [or 200 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 20% of the volume of the cell culture vessel / system. Day 11 - Add or replace cell culture medium with a volume twice the initial culture volume. Day 13 - Pre-REP TIL recovery.

[0199] Culture conditions (D-ix): Day 0 - Initial culture volume is 25 mL for G-Rex® 10M [or 250 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 25% of the volume of the cell culture vessel / system. Day 11 - Add or replace cell culture medium with a volume twice the initial culture volume. Day 13 - Pre-REP TIL recovery.

[0200] Culture conditions (Dx): Day 0 - Initial culture volume is 30 mL for G-Rex® 10M [or 300 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 30% of the volume of the cell culture vessel / system. Day 11 - Add or replace cell culture medium with a volume twice the initial culture volume. Day 13 - Pre-REP TIL recovery.

[0201] Culture conditions (D-xi): Day 0 - Initial culture volume is 50 mL for G-Rex® 10M [or 500 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 50% of the volume of the cell culture vessel / system. Day 11 - Replace the cell culture medium with a volume twice the initial culture volume. Day 13 - Pre-REP TIL recovery.

[0202] Culture conditions (D-xii): Day 0 - Initial culture volume is 10 mL for G-Rex® 10M [or 100 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 10% of the volume of the cell culture vessel / system. Day 11 - Add or replace cell culture medium with a volume three times the initial culture volume. Day 13 - Pre-REP TIL recovery.

[0203] Culture conditions (D-xiii): Day 0 - Initial culture volume is 20 mL for G-Rex® 10M [or 200 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 20% of the volume of the cell culture vessel / system. Day 11 - Add or replace cell culture medium with a volume three times the initial culture volume. Day 13 - Pre-REP TIL recovery.

[0204] Culture conditions (D-xiv): Day 0 - Initial culture volume is 25 mL for G-Rex® 10M [or 250 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 25% of the volume of the cell culture vessel / system. Day 11 - Add or replace cell culture medium with a volume three times the initial culture volume. Day 13 - Pre-REP TIL recovery.

[0205] Culture conditions (D-xv): Day 0 - Initial culture volume is 30 mL for G-Rex® 10M [or 300 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 30% of the volume of the cell culture vessel / system. Day 11 - Replace the cell culture medium with a volume three times the initial culture volume. Day 13 - Pre-REP TIL recovery.

[0206] Culture conditions (Ei): Day 0 - Initial culture volume is 10 mL for G-Rex® 10M [or 100 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 10% of the volume of the cell culture vessel / system. Day 8 - Add or replace cell culture medium with a volume of 1x the initial culture volume. Day 14-Pre-REP TIL collection.

[0207] Culture conditions (E-ii): Day 0 - Initial culture volume is 20 mL for G-Rex® 10M [or 200 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 20% of the volume of the cell culture vessel / system. Day 8 - Add or replace cell culture medium with a volume of 1x the initial culture volume. Day 14 - Pre-REP TIL recovery.

[0208] Culture conditions (E-iii): Day 0 - Initial culture volume is 25 mL for G-Rex® 10M [or 250 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 25% of the volume of the cell culture vessel / system. Day 8 - Add or replace cell culture medium with a volume of 1x the initial culture volume. Day 14 - Pre-REP TIL recovery.

[0209] Culture conditions (E-iv): Day 0 - Initial culture volume is 30 mL for G-Rex® 10M [or 300 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 30% of the volume of the cell culture vessel / system. Day 8 - Add or replace cell culture medium with a volume of 1x the initial culture volume. Day 14 - Pre-REP TIL recovery.

[0210] Culture conditions (Ev): Day 0 - Initial culture volume is 50 mL for G-Rex® 10M [or 500 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 50% of the volume of the cell culture vessel / system. Day 8 - Add or replace cell culture medium with a volume of 1x the initial culture volume. Day 14 - Pre-REP TIL recovery.

[0211] Culture conditions (E-vi): Day 0 - Initial culture volume is 100 mL for G-Rex® 10M [or 1000 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 100% of the volume of the cell culture vessel / system. Day 8 - Replace the cell culture medium with a volume of 1x the initial culture volume. Day 14 - Pre-REP TIL recovery.

[0212] Culture conditions (E-vii): Day 0 - Initial culture volume is 10 mL for G-Rex® 10M [or 100 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 10% of the volume of the cell culture vessel / system. Day 8 - Add or replace cell culture medium with a volume twice the initial culture volume. Day 14 - Pre-REP TIL recovery.

[0213] Culture conditions (E-viii): Day 0 - Initial culture volume is 20 mL for G-Rex® 10M [or 200 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 20% of the volume of the cell culture vessel / system. Day 8 - Add or replace cell culture medium with a volume twice the initial culture volume. Day 14 - Pre-REP TIL recovery.

[0214] Culture conditions (E-ix): Day 0 - Initial culture volume is 25 mL for G-Rex® 10M [or 250 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 25% of the volume of the cell culture vessel / system. Day 8 - Add or replace cell culture medium with a volume twice the initial culture volume. Day 14 - Pre-REP TIL recovery.

[0215] Culture conditions (Ex): Day 0 - Initial culture volume is 30 mL for G-Rex® 10M [or 300 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 30% of the volume of the cell culture vessel / system. Day 8 - Add or replace cell culture medium with a volume twice the initial culture volume. Day 14 - Pre-REP TIL recovery.

[0216] Culture conditions (E-xi): Day 0 - Initial culture volume is 50 mL for G-Rex® 10M [or 500 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 50% of the volume of the cell culture vessel / system. Day 8 - Replace the cell culture medium with a volume twice the initial culture volume. Day 14 - Pre-REP TIL recovery.

[0217] Culture conditions (E-xii): Day 0 - Initial culture volume is 10 mL for G-Rex® 10M [or 100 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 10% of the volume of the cell culture vessel / system. Day 8 - Add or replace cell culture medium with a volume three times the initial culture volume. Day 14 - Pre-REP TIL recovery.

[0218] Culture conditions (E-xiii): Day 0 - Initial culture volume is 20 mL for G-Rex® 10M [or 200 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 20% of the volume of the cell culture vessel / system. Day 8 - Add or replace cell culture medium with a volume three times the initial culture volume. Day 14 - Pre-REP TIL recovery.

[0219] Culture conditions (E-xiv): Day 0 - Initial culture volume is 25 mL for G-Rex® 10M [or 250 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 25% of the volume of the cell culture vessel / system. Day 8 - Add or replace cell culture medium with a volume three times the initial culture volume. Day 14 - Pre-REP TIL recovery.

[0220] Culture conditions (E-xv): Day 0 - Initial culture volume is 30 mL for G-Rex® 10M [or 300 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 30% of the volume of the cell culture vessel / system. Day 8 - Replace the cell culture medium with a volume three times the initial culture volume. Day 14 - Pre-REP TIL recovery.

[0221] Culture conditions (Fi): Day 0 - Initial culture volume is 10 mL for G-Rex® 10M [or 100 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 10% of the volume of the cell culture vessel / system. Day 9 - Add or replace cell culture medium with a volume of 1x the initial culture volume. Day 14 - Pre-REP TIL recovery.

[0222] Culture conditions (F-ii): Day 0 - Initial culture volume is 20 mL for G-Rex® 10M [or 200 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 20% of the volume of the cell culture vessel / system. Day 9 - Add or replace cell culture medium with a volume of 1x the initial culture volume. Day 14 - Pre-REP TIL recovery.

[0223] Culture conditions (F-iii): Day 0 - Initial culture volume is 25 mL for G-Rex® 10M [or 250 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 25% of the volume of the cell culture vessel / system. Day 9 - Add or replace cell culture medium with a volume of 1x the initial culture volume. Day 14 - Pre-REP TIL recovery.

[0224] Culture conditions (F-iv): Day 0 - Initial culture volume is 30 mL for G-Rex® 10M [or 300 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 30% of the volume of the cell culture vessel / system. Day 9 - Add or replace cell culture medium with a volume of 1x the initial culture volume. Day 14 - Pre-REP TIL recovery.

[0225] Culture conditions (Fv): Day 0 - Initial culture volume is 50 mL for G-Rex® 10M [or 500 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 50% of the volume of the cell culture vessel / system. Day 9 - Add or replace cell culture medium with a volume of 1x the initial culture volume. Day 14 - Pre-REP TIL recovery.

[0226] Culture conditions (F-vi): Day 0 - Initial culture volume is 100 mL for G-Rex® 10M [or 1000 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 100% of the volume of the cell culture vessel / system. Day 9 - Replace the cell culture medium with a volume of 1x the initial culture volume. Day 14 - Pre-REP TIL recovery.

[0227] Culture conditions (F-vii): Day 0 - Initial culture volume is 10 mL for G-Rex® 10M [or 100 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 10% of the volume of the cell culture vessel / system. Day 9 - Add or replace cell culture medium with a volume twice the initial culture volume. Day 14 - Pre-REP TIL recovery.

[0228] Culture conditions (F-viii): Day 0 - Initial culture volume is 20 mL for G-Rex® 10M [or 200 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 20% of the volume of the cell culture vessel / system. Day 9 - Add or replace cell culture medium with a volume twice the initial culture volume. Day 14 - Pre-REP TIL recovery.

[0229] Culture conditions (F-ix): Day 0 - Initial culture volume is 25 mL for G-Rex® 10M [or 250 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 25% of the volume of the cell culture vessel / system. Day 9 - Add or replace cell culture medium with a volume twice the initial culture volume. Day 14 - Pre-REP TIL recovery.

[0230] Culture conditions (Fx): Day 0 - Initial culture volume is 30 mL for G-Rex® 10M [or 300 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 30% of the volume of the cell culture vessel / system. Day 9 - Add or replace cell culture medium with a volume twice the initial culture volume. Day 14 - Pre-REP TIL recovery.

[0231] Culture conditions (F-xi): Day 0 - Initial culture volume is 50 mL for G-Rex® 10M [or 500 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 50% of the volume of the cell culture vessel / system. Day 9 - Replace the cell culture medium with a volume twice the initial culture volume. Day 14 - Pre-REP TIL recovery.

[0232] Culture conditions (F-xii): Day 0 - Initial culture volume is 10 mL for G-Rex® 10M [or 100 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 10% of the volume of the cell culture vessel / system. Day 9 - Add or replace cell culture medium with a volume three times the initial culture volume. Day 14 - Pre-REP TIL recovery.

[0233] Culture conditions (F-xiii): Day 0 - Initial culture volume is 20 mL for G-Rex® 10M [or 200 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 20% of the volume of the cell culture vessel / system. Day 9 - Add or replace cell culture medium with a volume three times the initial culture volume. Day 14 - Pre-REP TIL recovery.

[0234] Culture conditions (F-xiv): Day 0 - Initial culture volume is 25 mL for G-Rex® 10M [or 250 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 25% of the volume of the cell culture vessel / system. Day 9 - Add or replace cell culture medium with a volume three times the initial culture volume. Day 14 - Pre-REP TIL recovery.

[0235] Culture conditions (F-xv): Day 0 - Initial culture volume is 30 mL for G-Rex® 10M [or 300 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 30% of the volume of the cell culture vessel / system. Day 9 - Replace the cell culture medium with a volume three times the initial culture volume. Day 14 - Pre-REP TIL recovery.

[0236] Culture conditions (Gi): Day 0 - Initial culture volume is 10 mL for G-Rex® 10M [or 100 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 10% of the volume of the cell culture vessel / system. Day 10 - Add or replace cell culture medium with a volume of 1x the initial culture volume. Day 14 - Pre-REP TIL recovery.

[0237] Culture conditions (G-ii): Day 0 - Initial culture volume is 20 mL for G-Rex® 10M [or 200 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 20% of the volume of the cell culture vessel / system. Day 10 - Add or replace cell culture medium with a volume of 1x the initial culture volume. Day 14 - Pre-REP TIL recovery.

[0238] Culture conditions (G-iii): Day 0 - Initial culture volume is 25 mL for G-Rex® 10M [or 250 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 25% of the volume of the cell culture vessel / system. Day 10 - Add or replace cell culture medium with a volume of 1x the initial culture volume. Day 14 - Pre-REP TIL recovery.

[0239] Culture conditions (G-iv): Day 0 - Initial culture volume is 30 mL for G-Rex® 10M [or 300 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 30% of the volume of the cell culture vessel / system. Day 10 - Add or replace cell culture medium with a volume of 1x the initial culture volume. Day 14 - Pre-REP TIL recovery.

[0240] Culture conditions (Gv): Day 0 - Initial culture volume is 50 mL for G-Rex® 10M [or 500 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 50% of the volume of the cell culture vessel / system. Day 10 - Add or replace cell culture medium with a volume of 1x the initial culture volume. Day 14 - Pre-REP TIL recovery.

[0241] Culture conditions (G-vi): Day 0 - Initial culture volume is 100 mL for G-Rex® 10M [or 1000 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 100% of the volume of the cell culture vessel / system. Day 10 - Replace the cell culture medium with a volume of 1x the initial culture volume. Day 14 - Pre-REP TIL recovery.

[0242] Culture conditions (G-vii): Day 0 - Initial culture volume is 10 mL for G-Rex® 10M [or 100 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 10% of the volume of the cell culture vessel / system. Day 10 - Add or replace cell culture medium with a volume twice the initial culture volume. Day 14 - Pre-REP TIL recovery.

[0243] Culture conditions (G-viii): Day 0 - Initial culture volume is 20 mL for G-Rex® 10M [or 200 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 20% of the volume of the cell culture vessel / system. Day 10 - Add or replace cell culture medium with a volume twice the initial culture volume. Day 14 - Pre-REP TIL recovery.

[0244] Culture conditions (G-ix): Day 0 - Initial culture volume is 25 mL for G-Rex® 10M [or 250 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 25% of the volume of the cell culture vessel / system. Day 10 - Add or replace cell culture medium with a volume twice the initial culture volume. Day 14 - Pre-REP TIL recovery.

[0245] Culture conditions (Gx): Day 0 - Initial culture volume is 30 mL for G-Rex® 10M [or 300 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 30% of the volume of the cell culture vessel / system. Day 10 - Add or replace cell culture medium with a volume twice the initial culture volume. Day 14 - Pre-REP TIL recovery.

[0246] Culture conditions (G-xi): Day 0 - Initial culture volume is 50 mL for G-Rex® 10M [or 500 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 50% of the volume of the cell culture vessel / system. Day 10 - Replace the cell culture medium with a volume twice the initial culture volume. Day 14 - Pre-REP TIL recovery.

[0247] Culture conditions (G-xii): Day 0 - Initial culture volume is 10 mL for G-Rex® 10M [or 100 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 10% of the volume of the cell culture vessel / system. Day 10 - Add or replace cell culture medium with a volume three times the initial culture volume. Day 14 - Pre-REP TIL recovery.

[0248] Culture conditions (G-xiii): Day 0 - Initial culture volume is 20 mL for G-Rex® 10M [or 200 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 20% of the volume of the cell culture vessel / system. Day 10 - Add or replace cell culture medium with a volume three times the initial culture volume. Day 14 - Pre-REP TIL recovery.

[0249] Culture conditions (G-xiv): Day 0 - Initial culture volume is 25 mL for G-Rex® 10M [or 250 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 25% of the volume of the cell culture vessel / system. Day 10 - Add or replace cell culture medium with a volume three times the initial culture volume. Day 14 - Pre-REP TIL recovery.

[0250] Culture conditions (G-xv): Day 0 - Initial culture volume is 30 mL for G-Rex® 10M [or 300 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 30% of the volume of the cell culture vessel / system. Day 10 - Replace the cell culture medium with a volume three times the initial culture volume. Day 14 - Pre-REP TIL recovery.

[0251] Culture conditions (Hi): Day 0 - Initial culture volume is 10 mL for G-Rex® 10M [or 100 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 10% of the volume of the cell culture vessel / system. Day 11 - Add or replace cell culture medium with a volume of 1x the initial culture volume. Day 14 - Pre-REP TIL recovery.

[0252] Culture conditions (H-ii): Day 0 - Initial culture volume is 20 mL for G-Rex® 10M [or 200 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 20% of the volume of the cell culture vessel / system. Day 11 - Add or replace cell culture medium with a volume of 1x the initial culture volume. Day 14 - Pre-REP TIL recovery.

[0253] Culture conditions (H-iii): Day 0 - Initial culture volume is 25 mL for G-Rex® 10M [or 250 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 25% of the volume of the cell culture vessel / system. Day 11 - Add or replace cell culture medium with a volume of 1x the initial culture volume. Day 14 - Pre-REP TIL recovery.

[0254] Culture conditions (H-iv): Day 0 - Initial culture volume is 30 mL for G-Rex® 10M [or 300 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 30% of the volume of the cell culture vessel / system. Day 11 - Add or replace cell culture medium with a volume of 1x the initial culture volume. Day 14 - Pre-REP TIL recovery.

[0255] Culture conditions (Hv): Day 0 - Initial culture volume is 50 mL for G-Rex® 10M [or 500 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 50% of the volume of the cell culture vessel / system. Day 11 - Add or replace cell culture medium with a volume of 1x the initial culture volume. Day 14 - Pre-REP TIL recovery.

[0256] Culture conditions (H-vi): Day 0 - Initial culture volume is 100 mL for G-Rex® 10M [or 1000 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 100% of the volume of the cell culture vessel / system. Day 11 - Replace the cell culture medium with a volume of 1x the initial culture volume. Day 14 - Pre-REP TIL recovery.

[0257] Culture conditions (H-vii): Day 0 - Initial culture volume is 10 mL for G-Rex® 10M [or 100 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 10% of the volume of the cell culture vessel / system. Day 11 - Add or replace cell culture medium with a volume twice the initial culture volume. Day 14 - Pre-REP TIL recovery.

[0258] Culture conditions (H-viii): Day 0 - Initial culture volume is 20 mL for G-Rex® 10M [or 200 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 20% of the volume of the cell culture vessel / system. Day 11 - Add or replace cell culture medium with a volume twice the initial culture volume. Day 14 - Pre-REP TIL recovery.

[0259] Culture conditions (H-ix): Day 0 - Initial culture volume is 25 mL for G-Rex® 10M [or 250 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 25% of the volume of the cell culture vessel / system. Day 11 - Add or replace cell culture medium with a volume twice the initial culture volume. Day 14 - Pre-REP TIL recovery.

[0260] Culture conditions (Hx): Day 0 - Initial culture volume is 30 mL for G-Rex® 10M [or 300 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 30% of the volume of the cell culture vessel / system. Day 11 - Add or replace cell culture medium with a volume twice the initial culture volume. Day 14 - Pre-REP TIL recovery.

[0261] Culture conditions (H-xi): Day 0 - Initial culture volume is 50 mL for G-Rex® 10M [or 500 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 50% of the volume of the cell culture vessel / system. Day 11 - Replace the cell culture medium with a volume twice the initial culture volume. Day 14 - Pre-REP TIL recovery.

[0262] Culture conditions (H-xii): Day 0 - Initial culture volume is 10 mL for G-Rex® 10M [or 100 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 10% of the volume of the cell culture vessel / system. Day 11 - Add or replace cell culture medium with a volume three times the initial culture volume. Day 14 - Pre-REP TIL recovery.

[0263] Culture conditions (H-xiii): Day 0 - Initial culture volume is 20 mL for G-Rex® 10M [or 200 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 20% of the volume of the cell culture vessel / system. Day 11 - Add or replace cell culture medium with a volume three times the initial culture volume. Day 14 - Pre-REP TIL recovery.

[0264] Culture conditions (H-xiv): Day 0 - Initial culture volume is 25 mL for G-Rex® 10M [or 250 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 25% of the volume of the cell culture vessel / system. Day 11 - Add or replace cell culture medium with a volume three times the initial culture volume. Day 14 - Pre-REP TIL recovery.

[0265] Culture conditions (H-xv): Day 0 - Initial culture volume is 30 mL for G-Rex® 10M [or 300 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 30% of the volume of the cell culture vessel / system. Day 11 - Replace the cell culture medium with a volume three times the initial culture volume. Day 14 - Pre-REP TIL recovery.

[0266] Culture conditions (Ii): Day 0 - Initial culture volume is 10 mL for G-Rex® 10M [or 100 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 10% of the volume of the cell culture vessel / system. Day 8 - Add or replace cell culture medium with a volume of 1x the initial culture volume. Day 15-Pre-REP TIL collection.

[0267] Culture conditions (I-ii): Day 0 - Initial culture volume is 20 mL for G-Rex® 10M [or 200 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 20% of the volume of the cell culture vessel / system. Day 8 - Add or replace cell culture medium with a volume of 1x the initial culture volume. Day 15-Pre-REP TIL collection.

[0268] Culture conditions (I-iii): Day 0 - Initial culture volume is 25 mL for G-Rex® 10M [or 250 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 25% of the volume of the cell culture vessel / system. Day 8 - Add or replace cell culture medium with a volume of 1x the initial culture volume. Day 15-Pre-REP TIL collection.

[0269] Culture conditions (I-iv): Day 0 - Initial culture volume is 30 mL for G-Rex® 10M [or 300 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 30% of the volume of the cell culture vessel / system. Day 8 - Add or replace cell culture medium with a volume of 1x the initial culture volume. Day 15-Pre-REP TIL collection.

[0270] Culture conditions (IV): Day 0 - Initial culture volume is 50 mL for G-Rex® 10M [or 500 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 50% of the volume of the cell culture vessel / system. Day 8 - Add or replace cell culture medium with a volume of 1x the initial culture volume. Day 15-Pre-REP TIL collection.

[0271] Culture conditions (I-vi): Day 0 - Initial culture volume is 100 mL for G-Rex® 10M [or 1000 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 100% of the volume of the cell culture vessel / system. Day 8 - Replace the cell culture medium with a volume of 1x the initial culture volume. Day 15-Pre-REP TIL collection.

[0272] Culture conditions (I-vii): Day 0 - Initial culture volume is 10 mL for G-Rex® 10M [or 100 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 10% of the volume of the cell culture vessel / system. Day 8 - Add or replace cell culture medium with a volume twice the initial culture volume. Day 15-Pre-REP TIL collection.

[0273] Culture conditions (I-viii): Day 0 - Initial culture volume is 20 mL for G-Rex® 10M [or 200 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 20% of the volume of the cell culture vessel / system. Day 8 - Add or replace cell culture medium with a volume twice the initial culture volume. Day 15-Pre-REP TIL collection.

[0274] Culture conditions (I-ix): Day 0 - Initial culture volume is 25 mL for G-Rex® 10M [or 250 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 25% of the volume of the cell culture vessel / system. Day 8 - Add or replace cell culture medium with a volume twice the initial culture volume. Day 15-Pre-REP TIL collection.

[0275] Culture conditions (Ix): Day 0 - Initial culture volume is 30 mL for G-Rex® 10M [or 300 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 30% of the volume of the cell culture vessel / system. Day 8 - Add or replace cell culture medium with a volume twice the initial culture volume. Day 15 - Pre-REP TIL recovery.

[0276] Culture conditions (I-xi): Day 0 - Initial culture volume is 50 mL for G-Rex® 10M [or 500 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 50% of the volume of the cell culture vessel / system. Day 8 - Replace the cell culture medium with a volume twice the initial culture volume. Day 15 - Pre-REP TIL recovery.

[0277] Culture conditions (I-xii): Day 0 - Initial culture volume is 10 mL for G-Rex® 10M [or 100 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 10% of the volume of the cell culture vessel / system. Day 8 - Add or replace cell culture medium with a volume three times the initial culture volume. Day 15 - Pre-REP TIL recovery.

[0278] Culture conditions (I-xiii): Day 0 - Initial culture volume is 20 mL for G-Rex® 10M [or 200 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 20% of the volume of the cell culture vessel / system. Day 8 - Add or replace cell culture medium with a volume three times the initial culture volume. Day 15 - Pre-REP TIL recovery.

[0279] Culture conditions (I-xiv): Day 0 - Initial culture volume is 25 mL for G-Rex® 10M [or 250 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 25% of the volume of the cell culture vessel / system. Day 8 - Add or replace cell culture medium with a volume three times the initial culture volume. Day 15 - Pre-REP TIL recovery.

[0280] Culture conditions (I-xv): Day 0 - Initial culture volume is 30 mL for G-Rex® 10M [or 300 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 30% of the volume of the cell culture vessel / system. Day 8 - Replace the cell culture medium with a volume three times the initial culture volume. Day 15 - Pre-REP TIL recovery.

[0281] Culture conditions (Ji): Day 0 - Initial culture volume is 10 mL for G-Rex® 10M [or 100 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 10% of the volume of the cell culture vessel / system. Day 9 - Add or replace cell culture medium with a volume of 1x the initial culture volume. Day 15 - Pre-REP TIL recovery.

[0282] Culture conditions (J-ii): Day 0 - Initial culture volume is 20 mL for G-Rex® 10M [or 200 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 20% of the volume of the cell culture vessel / system. Day 9 - Add or replace cell culture medium with a volume of 1x the initial culture volume. Day 15 - Pre-REP TIL recovery.

[0283] Culture conditions (J-iii): Day 0 - Initial culture volume is 25 mL for G-Rex® 10M [or 250 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 25% of the volume of the cell culture vessel / system. Day 9 - Add or replace cell culture medium with a volume of 1x the initial culture volume. Day 15 - Pre-REP TIL recovery.

[0284] Culture conditions (J-iv): Day 0 - Initial culture volume is 30 mL for G-Rex® 10M [or 300 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 30% of the volume of the cell culture vessel / system. Day 9 - Add or replace cell culture medium with a volume of 1x the initial culture volume. Day 15 - Pre-REP TIL recovery.

[0285] Culture conditions (Jv): Day 0 - Initial culture volume is 50 mL for G-Rex® 10M [or 500 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 50% of the volume of the cell culture vessel / system. Day 9 - Add or replace cell culture medium with a volume of 1x the initial culture volume. Day 15 - Pre-REP TIL recovery.

[0286] Culture conditions (J-vi): Day 0 - Initial culture volume is 100 mL for G-Rex® 10M [or 1000 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 100% of the volume of the cell culture vessel / system. Day 9 - Replace the cell culture medium with a volume of 1x the initial culture volume. Day 15 - Pre-REP TIL recovery.

[0287] Culture conditions (J-vii): Day 0 - Initial culture volume is 10 mL for G-Rex® 10M [or 100 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 10% of the volume of the cell culture vessel / system. Day 9 - Add or replace cell culture medium with a volume twice the initial culture volume. Day 15 - Pre-REP TIL recovery.

[0288] Culture conditions (J-viii): Day 0 - Initial culture volume is 20 mL for G-Rex® 10M [or 200 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 20% of the volume of the cell culture vessel / system. Day 9 - Add or replace cell culture medium with a volume twice the initial culture volume. Day 15 - Pre-REP TIL recovery.

[0289] Culture conditions (J-ix): Day 0 - Initial culture volume is 25 mL for G-Rex® 10M [or 250 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 25% of the volume of the cell culture vessel / system. Day 9 - Add or replace cell culture medium with a volume twice the initial culture volume. Day 15 - Pre-REP TIL recovery.

[0290] Culture conditions (Jx): Day 0 - Initial culture volume is 30 mL for G-Rex® 10M [or 300 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 30% of the volume of the cell culture vessel / system. Day 9 - Add or replace cell culture medium with a volume twice the initial culture volume. Day 15 - Pre-REP TIL recovery.

[0291] Culture conditions (J-xi): Day 0 - Initial culture volume is 50 mL for G-Rex® 10M [or 500 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 50% of the volume of the cell culture vessel / system. Day 9 - Replace the cell culture medium with a volume twice the initial culture volume. Day 15 - Pre-REP TIL recovery.

[0292] Culture conditions (J-xii): Day 0 - Initial culture volume is 10 mL for G-Rex® 10M [or 100 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 10% of the volume of the cell culture vessel / system. Day 9 - Add or replace cell culture medium with a volume three times the initial culture volume. Day 15 - Pre-REP TIL recovery.

[0293] Culture conditions (J-xiii): Day 0 - Initial culture volume is 20 mL for G-Rex® 10M [or 200 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 20% of the volume of the cell culture vessel / system. Day 9 - Add or replace cell culture medium with a volume three times the initial culture volume. Day 15 - Pre-REP TIL recovery.

[0294] Culture conditions (J-xiv): Day 0 - Initial culture volume is 25 mL for G-Rex® 10M [or 250 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 25% of the volume of the cell culture vessel / system. Day 9 - Add or replace cell culture medium with a volume three times the initial culture volume. Day 15 - Pre-REP TIL recovery.

[0295] Culture conditions (J-xv): Day 0 - Initial culture volume is 30 mL for G-Rex® 10M [or 300 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 30% of the volume of the cell culture vessel / system. Day 9 - Replace the cell culture medium with a volume three times the initial culture volume. Day 15 - Pre-REP TIL recovery.

[0296] Culture conditions (Ki): Day 0 - Initial culture volume is 10 mL for G-Rex® 10M [or 100 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 10% of the volume of the cell culture vessel / system. Day 10 - Add or replace cell culture medium with a volume of 1x the initial culture volume. Day 15 - Pre-REP TIL recovery.

[0297] Culture conditions (K-ii): Day 0 - Initial culture volume is 20 mL for G-Rex® 10M [or 200 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 20% of the volume of the cell culture vessel / system. Day 10 - Add or replace cell culture medium with a volume of 1x the initial culture volume. Day 15 - Pre-REP TIL recovery.

[0298] Culture conditions (K-iii): Day 0 - Initial culture volume is 25 mL for G-Rex® 10M [or 250 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 25% of the volume of the cell culture vessel / system. Day 10 - Add or replace cell culture medium with a volume of 1x the initial culture volume. Day 15 - Pre-REP TIL recovery.

[0299] Culture conditions (K-iv): Day 0 - Initial culture volume is 30 mL for G-Rex® 10M [or 300 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 30% of the volume of the cell culture vessel / system. Day 10 - Add or replace cell culture medium with a volume of 1x the initial culture volume. Day 15 - Pre-REP TIL recovery.

[0300] Culture conditions (Kv): Day 0 - Initial culture volume is 50 mL for G-Rex® 10M [or 500 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 50% of the volume of the cell culture vessel / system. Day 10 - Add or replace cell culture medium with a volume of 1x the initial culture volume. Day 15 - Pre-REP TIL recovery.

[0301] Culture conditions (K-vi): Day 0 - Initial culture volume is 100 mL for G-Rex® 10M [or 1000 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 100% of the volume of the cell culture vessel / system. Day 10 - Replace the cell culture medium with a volume of 1x the initial culture volume. Day 15 - Pre-REP TIL recovery.

[0302] Culture conditions (K-vii): Day 0 - Initial culture volume is 10 mL for G-Rex® 10M [or 100 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 10% of the volume of the cell culture vessel / system. Day 10 - Add or replace cell culture medium with a volume twice the initial culture volume. Day 15 - Pre-REP TIL recovery.

[0303] Culture conditions (K-viii): Day 0 - Initial culture volume is 20 mL for G-Rex® 10M [or 200 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 20% of the volume of the cell culture vessel / system. Day 10 - Add or replace cell culture medium with a volume twice the initial culture volume. Day 15 - Pre-REP TIL recovery.

[0304] Culture conditions (K-ix): Day 0 - Initial culture volume is 25 mL for G-Rex® 10M [or 250 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 25% of the volume of the cell culture vessel / system. Day 10 - Add or replace cell culture medium with a volume twice the initial culture volume. Day 15 - Pre-REP TIL recovery.

[0305] Culture conditions (Kx): Day 0 - Initial culture volume is 30 mL for G-Rex® 10M [or 300 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 30% of the volume of the cell culture vessel / system. Day 10 - Add or replace cell culture medium with a volume twice the initial culture volume. Day 15 - Pre-REP TIL recovery.

[0306] Culture conditions (K-xi): Day 0 - Initial culture volume is 50 mL for G-Rex® 10M [or 500 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 50% of the volume of the cell culture vessel / system. Day 10 - Replace the cell culture medium with a volume twice the initial culture volume. Day 15 - Pre-REP TIL recovery.

[0307] Culture conditions (K-xii): Day 0 - Initial culture volume is 10 mL for G-Rex® 10M [or 100 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 10% of the volume of the cell culture vessel / system. Day 10 - Add or replace cell culture medium with a volume three times the initial culture volume. Day 15 - Pre-REP TIL recovery.

[0308] Culture conditions (K-xiii): Day 0 - Initial culture volume is 20 mL for G-Rex® 10M [or 200 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 20% of the volume of the cell culture vessel / system. Day 10 - Add or replace cell culture medium with a volume three times the initial culture volume. Day 15 - Pre-REP TIL recovery.

[0309] Culture conditions (K-xiv): Day 0 - Initial culture volume is 25 mL for G-Rex® 10M [or 250 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 25% of the volume of the cell culture vessel / system. Day 10 - Add or replace cell culture medium with a volume three times the initial culture volume. Day 15 - Pre-REP TIL recovery.

[0310] Culture conditions (K-xv): Day 0 - Initial culture volume is 30 mL for G-Rex® 10M [or 300 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 30% of the volume of the cell culture vessel / system. Day 10 - Replace the cell culture medium with a volume three times the initial culture volume. Day 15 - Pre-REP TIL recovery.

[0311] Culture conditions (Li): Day 0 - Initial culture volume is 10 mL for G-Rex® 10M [or 100 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 10% of the volume of the cell culture vessel / system. Day 11 - Add or replace cell culture medium with a volume of 1x the initial culture volume. Day 15 - Pre-REP TIL recovery.

[0312] Culture conditions (L-ii): Day 0 - Initial culture volume is 20 mL for G-Rex® 10M [or 200 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 20% of the volume of the cell culture vessel / system. Day 11 - Add or replace cell culture medium with a volume of 1x the initial culture volume. Day 15 - Pre-REP TIL recovery.

[0313] Culture conditions (L-iii): Day 0 - Initial culture volume is 25 mL for G-Rex® 10M [or 250 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 25% of the volume of the cell culture vessel / system. Day 11 - Add or replace cell culture medium with a volume of 1x the initial culture volume. Day 15 - Pre-REP TIL recovery.

[0314] Culture conditions (L-iv): Day 0 - Initial culture volume is 30 mL for G-Rex® 10M [or 300 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 30% of the volume of the cell culture vessel / system. Day 11 - Add or replace cell culture medium with a volume of 1x the initial culture volume. Day 15 - Pre-REP TIL recovery.

[0315] Culture conditions (Lv): Day 0 - Initial culture volume is 50 mL for G-Rex® 10M [or 500 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 50% of the volume of the cell culture vessel / system. Day 11 - Add or replace cell culture medium with a volume of 1x the initial culture volume. Day 15 - Pre-REP TIL recovery.

[0316] Culture conditions (L-vi): Day 0 - Initial culture volume is 100 mL for G-Rex® 10M [or 1000 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 100% of the volume of the cell culture vessel / system. Day 11 - Replace the cell culture medium with a volume of 1x the initial culture volume. Day 15 - Pre-REP TIL recovery.

[0317] Culture conditions (L-vii): Day 0 - Initial culture volume is 10 mL for G-Rex® 10M [or 100 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 10% of the volume of the cell culture vessel / system. Day 11 - Add or replace cell culture medium with a volume twice the initial culture volume. Day 15 - Pre-REP TIL recovery.

[0318] Culture conditions (L-viii): Day 0 - Initial culture volume is 20 mL for G-Rex® 10M [or 200 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 20% of the volume of the cell culture vessel / system. Day 11 - Add or replace cell culture medium with a volume twice the initial culture volume. Day 15 - Pre-REP TIL recovery.

[0319] Culture conditions (L-ix): Day 0 - Initial culture volume is 25 mL for G-Rex® 10M [or 250 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 25% of the volume of the cell culture vessel / system. Day 11 - Add or replace cell culture medium with a volume twice the initial culture volume. Day 15 - Pre-REP TIL recovery.

[0320] Culture conditions (Lx): Day 0 - Initial culture volume is 30 mL for G-Rex® 10M [or 300 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 30% of the volume of the cell culture vessel / system. Day 11 - Add or replace cell culture medium with a volume twice the initial culture volume. Day 15 - Pre-REP TIL recovery.

[0321] Culture conditions (L-xi): Day 0 - Initial culture volume is 50 mL for G-Rex® 10M [or 500 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 50% of the volume of the cell culture vessel / system. Day 11 - Replace the cell culture medium with a volume twice the initial culture volume. Day 15 - Pre-REP TIL recovery.

[0322] Culture conditions (L-xii): Day 0 - Initial culture volume is 10 mL for G-Rex® 10M [or 100 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 10% of the volume of the cell culture vessel / system. Day 11 - Add or replace cell culture medium with a volume three times the initial culture volume. Day 15 - Pre-REP TIL recovery.

[0323] Culture conditions (L-xiii): Day 0 - Initial culture volume is 20 mL for G-Rex® 10M [or 200 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 20% of the volume of the cell culture vessel / system. Day 11 - Add or replace cell culture medium with a volume three times the initial culture volume. Day 15 - Pre-REP TIL recovery.

[0324] Culture conditions (L-xiv): Day 0 - Initial culture volume is 25 mL for G-Rex® 10M [or 250 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 25% of the volume of the cell culture vessel / system. Day 11 - Add or replace cell culture medium with a volume three times the initial culture volume. Day 15 - Pre-REP TIL recovery.

[0325] Culture conditions (L-xv): Day 0 - Initial culture volume is 30 mL for G-Rex® 10M [or 300 mL for G-Rex® 100M]. In other words, the initial culture volume for the first expansion is 30% of the volume of the cell culture vessel / system. Day 11 - Replace the cell culture medium with a volume three times the initial culture volume. Day 15 - Pre-REP TIL recovery.

[0326] Other suitable culture conditions may also be used.

[0327] Success rate before and after optimization Before optimization, the success rate of pre-REP TIL production was low, with an average yield of 3.10 × 10 6 After optimizing the pre-REP procedure, full-scale cultures were performed in a G-Rex® 100M instrument, which yielded 16.29 × 10 cells / fragment. 6 The average yield of cells / fragments was 100% (Table 2). Thus, our optimized procedure resulted in a significantly higher yield of recovered pre-REP TILs than pre-optimization (P=0.00125; pre-optimization run n=12, post-optimization run n=8) (Table 2).

[0328] [Table 1]

[0329] Assessment of glucose and lactate concentrations during a standardized pre-REP protocol To verify that the medium additions in the standardized pre-REP protocol were sufficient for the process, supernatants were collected on days 0, 6, 10, and 14 from seven runs (T5101014-T5101018, T5101021, and T5101022) and final TIL yield, glucose, and lactate concentrations were assessed. Up to day 6 and thereafter, glucose and lactate levels varied in proportion to the degree of TIL proliferation (Figure 1). However, glucose levels at the time of harvest remained high enough to sustain T cell proliferation (7.69 ± 2.86 mM; 95% CI, 5.05-10.33 mM; n = 7) (Table 3). Importantly, lactate levels (6.30 ± 3.92 mM; 95% CI, 2.67-9.93 mM; n = 7) were well below 20 mM. Above this level, CD8 + Killer T cells have been shown to exhibit reduced proliferation, survival and function 14Taken together, our results demonstrated that an optimized pre-REP process could maintain glucose and lactate levels within a range that supports TIL migration and proliferation.

[0330] [Table 2]

[0331] Characterization of pre-REP TILs As expected, pre-REP TILs grown under optimized conditions had high survival rates and high CD3 + The mean viability of pre-REP TILs was 93.68% ± 2.48% (n = 8). + The average percentage of cells was 89.43% ± 5.31% (n = 8).

[0332] CD8 in the pre-REP TIL product + The percentage of cells varied between donors, with the average percentage (37.35%) being similar to other NSCLC pre-REP TIL products 3 The pre-REP material contained only low percentages of NK cells, B cells, myeloid cells, and epithelial cells: NK cell, B cell, myeloid cell, and epithelial cell percentages were 7.74% ± 4.87%, 0.118% ± 0.117%, 0.034% ± 0.032%, and 0.074% ± 0.137%, respectively (n = 8; Figure 2 and Table 4).

[0333] [Table 3]

[0334] We also extensively analyzed the phenotype of our pre-REP TILs by examining the expression of various activating / inhibitory markers and the distribution of T cell subsets. The data showed that these pre-REP TILs showed high expression of CD27 and CD28 [here, CD4 +The positive frequencies in pre-REP TILs were 37.94% ± 7.17% and 94.51% ± 4.72%, respectively (n = 8; Fig. 3, Table 5), and CD8 + The positive frequencies in pre-REP TILs were 34.45% ± 9.62% and 57.66% ± 19.19%, respectively (n = 8; Figure 4, Table 7). + and CD8 + Pre-REP T cells express various inhibitory markers and are CD4 + LAG-3 was least expressed on pre-REP T cells, with a positive frequency of 10.07% ± 3.23% (n = 8; Figure 3, Table 5). + and CD8 + PD-1 was expressed moderately on pre-REP T cells of both types of T cells. + Pre-REP T cells and CD8 + In pre-REP T cells, the percentages were 57.43% ± 19.58% and 34.31% ± 21.67%, respectively. TIM-3 was the most abundant CD8 + TIM-3 was highly expressed on pre-REP TILs. + In pre-REP T cells, it was 53.50% ± 16.12% (n = 8; Fig. 3, Table 5), whereas in CD8 + In pre-REP T cells it was 71.93% ± 17.85% (n = 8; Fig. 4, Table 7).

[0335] One favorable feature of our pre-REP TILs is that they express CD4 + and CD8 + In both pre-rep TILs, T CM A high proportion of, and T EM The proportion of patients with PTSD was low. CM The percentage of CD4 + In pre-REP TILs, it was 66.28% ± 12.87%, whereas in CD8 + In pre-REP TILs, it was 59.04% ± 13.47%. EM The percentage of CD4 +In pre-REP TILs, it was 30.98% ± 13.25% (n = 8; Fig. 3, Table 6), whereas in CD8 + In pre-REP TILs, the rate was 27.66% ± 11.59% (n = 8; Fig. 4, Table 8). EFF Minimum percentage of subset (CD4 + Cells and CD8 + In the cells, the T N / T SCM The proportion of subsets was also high [CD4 + In pre-REP TILs, it was 2.49% ± 2.28% (n = 8), and in CD8 + In pre-REP TILs, the percentage of TILs was 9.04%±8.65% (n=8) (Figure 3, Table 6; and Figure 4, Table 8)]. This indicates a prominent early memory TIL phenotype. This is very important because the clinical benefit of TIL therapy depends heavily on the specific quality of the TIL product generated from pre-REP TILs. Overall, we have now demonstrated a method to generate pre-REP TILs in an efficient and timely manner. The method resulted in high yields and favorable TIL phenotypes, suggesting the success of REP in relation to clinical efficacy.

[0336] [Table 4]

[0337] TIFF2025503959000005.tif200160

[0338] conclusion We optimized the pre-REP TIL culture process to obtain a high percentage of T CM and healthy TIL phenotypes were obtained. Overall, these studies can be utilized to support pre-REP clinical manufacturing and subsequent TIL experiments.

[0339] References 1. Jansen, C. S. et al., An intra-tumoral niche maintains and differentiates stem-like CD8 T cells. Nature 576, 465 - 470 (2019). 2. Jin, J. et al., Simplified method of the growth of human tumor infiltrating lymphocytes in gas-permeable flasks to numbers needed for patient treatment. J Immunother 35, 283 - 292 (2012). 3. Creelan, B. C. et al., Tumor-infiltrating lymphocyte treatment for anti-PD-1-resistant metastatic lung cancer: a phase 1 trial. Nat Med 27, 1410 - 1418 (2021). 4. Rosenberg, S. A. et al., Durable complete responses in heavily pretreated patients with metastatic melanoma using T-cell transfer immunotherapy. Clin Cancer Res 17, 4550 - 4557 (2011). 5. Ando, M., Ito, M., Srirat, T., Kondo, T. & Yoshimura, A. Memory T cell, exhaustion, and tumor immunity. Immunol Med 43, 1 - 9 (2020). 6. Rosenberg, S. A., Spiess, P. & Lafreniere, R. A new approach to the adoptive immunotherapy of cancer with tumor-infiltrating lymphocytes. Science 233, 1318 - 1321 (1986). 7.Huang,J.ら,Survival,persistence,and progressive differentiation of adoptively transferred tumor-reactive T cells associated with tumor regression.J Immunother 28,258-267(2005). 8.Powell,D.J.,Dudley,M.E.,Robbins,P.F.& Rosenberg,S.A.Transition of late-stage effector T cells to CD27+ CD28+ tumor-reactive effector memory T cells in humans after adoptive cell transfer therapy.Blood 105,241-250(2005). 9.Dudley,M.E.,Wunderlich,J.R.,Shelton,T.E.,Even,J.& Rosenberg,S.A.Generation of tumor-infiltrating lymphocyte cultures for use in adoptive transfer therapy for melanoma patients.J Immunother 26,332-342(2003). 10.Wu,R.ら,Adoptive T-cell therapy using autologous tumor-infiltrating lymphocytes for metastatic melanoma: current status and future outlook.Cancer J 18,160-175(2012). 11. Zhou, J., Dudley, ME, Rosenberg, SA & Robbins, PFPersistence of multiple tumor-specific T-cell clones is associated with complete tumor regression in a melanoma patient receiving adoptive cell transfer therapy.J Immunother 28,53-62(2005). 12. Tran, KQ et al., Minimally cultured tumor-infiltrating lymphocytes display optimal characteristics for adoptive cell therapy. J Immunother 31, 742-751 (2008). 13. Pilling, D. et al., High cell density provides potent survival signals for resting T-cells. Cell Mol Biol(Noisy-le-grand) 46, 163-174 (2000). 14. Fischer, K. et al., Inhibitory effect of tumor cell-derived lactic acid on human T cells. Blood 109, 3812-3819 (2007). 15. Ghaffari, S. et al., Optimizing interleukin-2 concentration, seeding density and bead-to-cell ratio of T-cell expansion for adoptive immunotherapy. BMC Immunol 22,43(2021).

[0340] Example 2: Comparison of TILs generated at small scale from traditional REP or feeder cell-free TransAct REP protocols Traditionally, TILs have been grown from single tumor fragments in wells of 24-well plates in the presence of high doses of IL-2, selected for tumor reactivity, and then rapidly expanded. 6 Even if the output from this method is clinically effective, the generation of TILs is challenging because traditional methods of TIL generation, assayed for specific tumor recognition, typically require 6–8 weeks, which leads to T cell exhaustion in vitro and short survival in vivo. 7,8 Additionally, selection of tumor responders results in a lower success rate of TIL production, leading to a dropout rate of over 50% in patients referred for TIL therapy. 9 , which greatly limits its clinical application. 10 An alternative "immature" TIL process has been developed that uses whole tumor tissue in tissue culture flasks and gas-permeable bags, which favors the generation of TILs with properties associated with improved persistence in vivo, such as long telomeres and enhanced expression of CD27 and CD28. 11,12 .

[0341] The REP protocol used here was a feeder cell-free process, which differs from the traditional TIL REP protocol in which irradiated PBMCs and anti-CD3 (OKT3) antibodies are used for expansion. The feeder cell-free REP process may increase the potency and safety of the product and reduce the manufacturing cost. We performed a comparison of small-scale TILs using the traditional REP protocol with those from the TransAct REP process, which does not use feeder cells.

[0342] Comparison of TIL proliferation in TransAct REP and traditional REP Using pre-REP TILs from 12 donors, we found that the mean fold expansion of TILs expanded using the TransAct REP protocol was 389.3±138.8 (Table 9), which was comparable to the traditional REP protocol, suggesting that the TransAct REP protocol could generate sufficient TILs to meet clinical target dose requirements.

[0343] [Table 5]

[0344] Comparison of survival rates and T cell subtypes between TransAct REP and traditional REP TransAct REP TILs showed consistent CD8 + There was a significant increase in the proportion of T cells (Figure 5 and Table 10).

[0345] Comparison of T cell phenotype and inhibitory / activating cell surface markers from TransAct REP or traditional REP For TransAct REP TILs, CD4 + Populations and CD8 + Both populations showed a significant increase in the proportion of cells with an early memory phenotype (Figure 6, Figure 7 and Table 10), while the more differentiated effector T cells (T EM , T EFF ) was decreased (Figure 6 and Table 10). These TIL characteristics have been shown to be associated with favorable outcomes in TIL adoptive cell therapy. 4 .

[0346] REP TILs were also analyzed for expression of immune checkpoint (inhibitory) and activation cell surface markers (Figure 7 and Table 10). Compared to traditional REP TILs, TransAct REP TILs were significantly more potent than CD4 + Compartment and CD8 + Increased proportion of CD27 in both compartments + cells, as well as a reduced proportion of CD4 + and CD8 + T cells, further supporting the early memory phenotype of TransAct REP TILs.

[0347] [Table 6]

[0348] Table 11 shows T cell subsets from another experiment. [Table 7]

[0349] Percentage of REP TIL T cell subtypes determined by FACS analysis from nine donors expanded with irradiated PBMC (irPBMC) + OKT3 mAb or the present method (e.g., using MACS® GMP T cell TransAct™). The average percentage of each population is shown with the range in brackets. P values ​​were determined using a paired t-test in Excel (n=9).

[0350] Comparison of T cell activation in response to αCD3 stimulation with TransAct REP and traditional REP To compare the functionality of TILs expanded by the two REP methods, changes in cell surface activation markers and cytokine secretion of cells recovered from REP TILs in response to αCD3 stimulation were examined. TransAct REP TILs produced much higher levels of IFN-γ and granzyme B upon αCD3 stimulation in the presence or absence of exogenous IL-2 (Figure 8, Table 12). The mean percentage of TILs expressing surface 4-1BB in the presence or absence of exogenous IL-2 was significantly higher than that of CD4 + Populations and CD8 + The mean TIL function was greater in TransAct REP TILs in both the control and control populations (Table 12). These data indicate that compared with traditional REP, TransAct REP supports better functionality of proliferating TILs in response to T cell activation.

[0351] [Table 8]

[0352] In vitro MOS tumor killing of TILs from TransAct REP or traditional REP To compare the tumor cell killing effects of traditional REP and TransAct REP TILs, we performed co-cultures of TILs with autologous tumor-derived micro-organospheres (MOS). REP TILs from donor T5101016 were co-cultured with autologous lung cancer MOS at various E:T ratios of 2:1, 3:1, or 4:1 (20,000-40,000 TILs:10,000 cancer cells in MOS). The co-cultures were imaged every 2 hours for up to 68 hours, and we observed an increase in caspase 3 / 7 signal in the TIL group (Figure 9).

[0353] The assay demonstrated the cytotoxicity of TIL against autologous NSCLC MOS. TransAct REP TIL (C-TIL051) showed significantly enhanced tumor killing activity, as indicated by caspase 3 / 7 signal, at all E:T ratios tested, compared with traditional TIL (Figure 9). These data indicate that TransAct REP TIL has better functionality in response to autologous tumor cells, compared with traditional REP TIL.

[0354] Comparison of TCR repertoires between TransAct REP TILs and traditional REP TILs TCR beta chain sequencing data of TILs from six donors were compared for repertoire diversity before (pre-REP) and after REP expansion with either traditional REP or TransAct REP. The Morishita Index, which is a correlation analysis that considers both the number and relative abundance of shared clones between two samples, is shown for each sample pair with pre-REP and REP. Values ​​range from 0 to 1, with higher values ​​indicating higher similarity between the overall repertoires of those two samples. The Morishita Index of TransAct REP was higher than that of traditional REP, suggesting that TransAct REP may have a higher degree of overall conservation of the pre-REP repertoire (Table 13).

[0355] [Table 9]

[0356] Moreover, combined analyses on various matrices from multiple aspects (including Simpson clonality, TCR overlap between pre-REP TILs and TILs, and preservation of the top 100 pre-REP clones after REP) indicate that the TransAct REP process appears to be better, or at least not inferior, to the traditional REP process in preserving the pre-REP TCR repertoire, further supporting the use of TransAct in the REP process.

[0357] Example 3: Development of the TIL REP process and characterization of the TILs Our small-scale experiments using pre-REP TILs derived from tumors of 11 NSCLC patients showed that the commercially available αCD3 / αCD28 agonist MACS® GMP T-cell TransAct™ (abbreviated as TransAct herein) can meet clinical dose requirements and can be used in place of irradiated feeder cells and αCD3 antibodies to sustain rapid proliferation of TILs. The aim of this study was to develop a REP process using TransAct to expand pre-REP TILs at manufacturing scale. We also aimed to characterize TILs generated in process development (PD) and mock runs from a GMP facility in terms of T-cell phenotype, repertoire diversity and / or tumor killing function.

[0358] Our experimental data indicates that successful REP of TILs can be achieved at manufacturing scale after activation with MACS® GMP T cell TransAct™ in a G-Rex® cell culture device or in sequential use of G-Rex® and Xuri™ W25 bioreactors. Characterization of TILs from process development runs showed that expanded cells exhibited favorable phenotypes and well-conserved T cell receptor (TCR) repertoires representative of cognate pre-REP TILs. TILs also retained the ability to upregulate expression of surface T cell activation markers and produce various cytokines in response to T cell activation signals. Furthermore, characterization of TIL products from mock runs from our GMP facility demonstrated that the autologous cell therapy products we developed possess an early differentiation phenotype and have potent tumor killing capabilities.

[0359] material Pre-REP TILs from 11 donors were used for PD. Pre-REP cells from five donors (T5101001, T5101002, T5101005, T5101008, T5101010) were collected prior to optimization of the pre-REP process and their CD3 + The percentage of T cells ranged from 38.7% to 98.0%. Pre-REP cells from six other donors (T5101014, T5101015, T5101016, T5101017, T5101018, T5101024) were recovered after optimization of the pre-REP process, and their CD3 + The percentage of T cells ranged from 81.8% to 97.0%.

[0360] method G-Rex® 10M and G-Rex® 100M devices in series and REP with TransAct On day 0, pre-REP cells were thawed in a water bath and washed twice with REP complete medium (CM; REP basal medium supplemented with 300 or 1000, 3000 or 6000 IU / mL IL-2). In one embodiment, the IL-2 concentration is 300 IU / mL. In another embodiment, the IL-2 concentration is 1000 IU / mL. In yet another embodiment, the IL-2 concentration is 3000 IU / mL. In yet another embodiment, the IL-2 concentration is 6000 IU / mL. The cells were then resuspended in REP CM and counted with an automated cell counter (NC-200). To initiate culture in the G-Rex® 10M device, approximately 1×10 6 , 2×10 6 , 5×10 6 , 10×10 6 or 20×10 6 Aliquots of cell suspension containing viable cells and TransAct were added into the device at volumetric ratios of TransAct:medium of approximately 1:150, 1:5, 1:10, or 1:17.5, and the final culture volume was adjusted to 10 mL, 15 mL, or 20 mL with REP CM. G-Rex® 10M was then incubated at 37° C., 5% CO 2 The mice were placed in an incubator.

[0361] On days 3, 4 or 5, the initial culture was diluted 10, 20 or 30 fold by adding pre-warmed REP CM to the G-Rex® 10M. 2 Returned to the incubator.

[0362] On days 6, 7, 8, 9 or 10, the cells were resuspended and counted in G-Rex® 10M, and the cultures were then transferred to new G-Rex® 100M devices and diluted 10x, 20x, 30x by adding pre-warmed REP CM. The G-Rex® 100M was then cooled to 4°C for 24 hours. 2 Placed in an incubator.

[0363] Cells were harvested from the G-Rex® 100M on days 12, 13, 14, 15, 16, 17, 18, 19 or 20. An aliquot of the remaining cells was pelleted and stored frozen.

[0364] G-Rex® Device with Xuri™ W25 and TransAct REP On day 0, pre-REP cells were thawed in a water bath and washed twice with REP CM. The cells were then resuspended in REP CM and counted on an NC-200. To initiate cultures in the G-Rex® 100M device, approximately 20×10 6 , 50×10 6 , 1000×10 6 or 200 x 10 6 Aliquots of cell suspension containing viable cells and TransAct were added into the device at a volumetric ratio of TransAct:medium of approximately 1:150, 1:5, 1:10, or 1:17.5, and the final culture volume was adjusted to 50-100 mL with REP CM. Alternatively, when using the closed G-Rex® 100M-CS device, the barrel of a 50 ml syringe was connected to the Luer port of the medium addition tube, and the cell suspension and TransAct were then added to the G-Rex® via the syringe barrel. The medium addition tube was then heat sealed and the syringe barrel removed. The G-Rex® 100M or G-Rex® 100M-CS was then inoculated with CO. 2 Placed in an incubator.

[0365] On days 3, 4 or 5, the initial culture was diluted 10, 15 or 20 fold by adding pre-warmed REP CM to the G-Rex® 100M. When using the G-Rex® 100M-CS, medium was added via a medium transfer bag containing REP CM attached to the medium addition tubing on the G-Rex® 100M-CS. After medium addition, the tubing was heat sealed and the medium transfer bag was removed. The G-Rex® device was placed in a CO 2 Placed back in incubator.

[0366] On or about days 6-10, approximately 25% or 50% of the spent medium was removed from the top of the G-Rex® 100M and stored in a new medium bottle. If using the G-Rex® 100M-CS, spent medium was removed from the medium addition / removal tube and stored in a medium transfer bag. The cells were then resuspended in the G-Rex® device and samples were taken and counted on the NC-200. The G-Rex® device was kept in a CO 2 Placed back in incubator.

[0367] To initiate the Xuri™ REP, a 2L or 10L Xuri™ Cellbag was placed onto the rocking tray of a Xuri™ W25 cell growth system. Fresh CM was then added to the Cellbag and the system was allowed to equilibrate for 2 hours.

[0368] Depending on the total cell number, an aliquot of the cell suspension in the G-Rex® device was collected in a transfer bag (in the case of G-Rex® 100M-CS, a GatheRex cell collection pump was used) so that the cell number met the requirements for Xuri™ Cellbag inoculation. The transfer bag was then connected to the feed line of the Xuri™ Cellbag to transfer the cells. Additional spent medium from the G-Rex® (saved before cell counting) was added to the Xuri™ Cellbag so that the volume of fresh CM in the Cellbag was half of the final total culture volume and the final cell concentration was approximately 0.5×10 6 / mL or 1 × 10 6 / mL or 2 × 10 6 / mL. Xuri™ growth was then initiated with slow rocking speed and angle.

[0369] On days 6, 7 or 8 and beyond, samples were taken from the Xuri™ Cellbag daily to count and monitor cell growth and viability. Fresh REP CM was added after each cell count to adjust the culture to the desired cell concentration until the total culture volume reached 1 liter or 5 liters. Perfusion was then started the following day at 0.5 liters / day, 1 liter / day or 2 liters / day. Depending on the cell concentration, the perfusion rate was gradually increased each day along with the rocking speed and angle.

[0370] On the final day of Xuri™ culture, samples were taken for cell count and FACS analysis, after which REP cells were harvested, washed and cryopreserved.

[0371] αCD3 stimulation REP products from three donors (T5101015, T5101018, and T5101024) were thawed and placed in REP CM for 1-2 days. Cells were then washed and resuspended in REP basal medium in the absence of IL-2 or in the presence of 300 IU / mL IL-2 (final concentration) and plated at 2 × 10 cells / well in a 96-well plate. 5 Cells were seeded. MACS® GMP CD3 Purification (αCD3 antibody, OKT3) was diluted in the same culture medium and added to the wells at a final concentration of 1 μg / mL. Cells were stimulated overnight, then supernatants were harvested for cytokine assays and cells were stained for FACS analysis. Experiments were performed in triplicate for each condition.

[0372] Cytokine assays For analysis of cytokine concentrations in cell culture supernatants, the LEGENDplex™ Human CD8 / NK Panel Kit was used for simultaneous quantification of multiple soluble analytes according to the manufacturer's protocol. Assays were read on a CytoFLEX LX FACS instrument and data were analyzed with the LEGENDplex™ software.

[0373] FACS Cells were first stained with LIVE / DEAD™ Fixable Aqua dye and then with antibodies against various surface markers. Reagents. Samples were then acquired on a CytoFLEX LX FACS instrument. All data was analyzed with FlowJo V10 software.

[0374] Collection of TIL culture media samples For REP with the G-Rex® device, where cells were present at the bottom, approximately 500 μL of the supernatant was sampled at the indicated time points. For REP with the Xuri™ W25 bioreactor, culture samples were taken from the sampling port of the Xuri™ Cellbag and centrifuged at 400 g / min for 5 min. REP culture medium samples (200 μL to 500 μL) were obtained by transferring the supernatant to a new 1.5 mL sterile tube. The culture medium samples were immediately stored in a -20°C freezer and thawed on ice at the time of assay.

[0375] IL-2 enzyme-linked immunosorbent assay (ELISA) Samples were diluted 15-300 times in REP basal medium depending on IL-2 protein concentration. Samples were measured in duplicate or triplicate wells. Data are shown as mean ± standard deviation (SD). The mean IL-2 concentration of three batches of fresh REP CM samples was expressed as the concentration on day 0. Mass concentrations were converted to unit concentrations assuming that 1 ng / mL of IL-2 corresponds to 16.36 IU / mL.

[0376] Quantification of glucose and lactate concentrations in culture supernatants Samples were aliquoted and analyzed using Glucose-Glo and Lactate-Glo Assay Kits following the manufacturer's recommended protocol. Culture supernatants were diluted 1:200-1:300 in PBS. Plates were read for luminescence on a SpectraMax iD3. Standard curves were generated in GraphPad Prism and sample values ​​were calculated in Excel.

[0377] TCR repertoire analysis To compare TCR repertoire diversity before (pre-REP) and after full-scale REP expansion of PD performed using the G-Rex® device and the Xuri™ W25 bioreactor sequentially, TCR beta chain sequencing of TILs from two donors (T5101015-3 and T5101024a) was performed.

[0378] Mock run of TIL from a GMP facility Pre-REP TILs from two donors (T5101034 and T5101035) were expanded in a GMP facility and cryopreserved. + The percentage of T cells was 85.72% and 90.82% for T5101034 and T5101035, respectively. Pre-REP TILs were cryopreserved for 10 and 25 days, respectively, before starting REP manufacturing in the same GMP facility. Standard operating procedures for REP were developed based on the PD run and applied to the engineering run and mock run.

[0379] Characterization of TIL simulated run products The yield of TIL mock production was measured by automated cell counting on an NC-200 cell counter. The T cell phenotype of the product was analyzed by surface marker staining and FACS on a BD FACSLyric™ clinical flow cytometry system. To measure non-specific activation, cryopreserved products were thawed, allowed to recover overnight, and then 1×10 5 Cells were stimulated with Dynabeads™ Human T-Activator CD3 / CD28 (Thermo Fisher) at a 1:1 ratio in a final volume of 200 μL for 24 hours. Cell supernatants were then harvested for IFN-γ ELISA using a commercial kit (Thermo Fisher). Alternatively, after overnight recovery, 2×10 5The cells were stimulated with αCD3 antibody (Miltenyi) at a final concentration of 1 μg / mL for 24 hours in a final volume of 200 μL. The cells were then harvested for FACS analysis of T cell activation markers including 4-1BB and OX40.

[0380] We also characterized TIL simulated production from two donors (T5101034 and T5101035) by direct functional assays involving co-culture of the TIL production with autologous tumor-derived micro-organospheres (MOS) (Ding et al., Patient-derived micro-organospheres enable clinical precision oncology, Cell Stem Cell, 2022, 29, 905-917, e906).

[0381] Results and Analysis C-TIL051 PD Implementation with Sequential Use of G-Rex® 10M and G-Rex® 100M Devices and TransAct We first performed sequential use of the G-Rex® 10M and G-Rex® 100M devices and TransAct to obtain 10×10 6 REP was performed using pre-REP TILs.

[0382] REP Process Conditions REP of TILs requires sufficient nutrients and high IL-2 concentrations, while accumulation of metabolic products such as lactate can reduce T cell proliferation. Glucose, lactate and IL-2 levels were measured from culture supernatants taken before medium exchange / addition and at harvest. IL-2 concentrations were found to be at high levels (>2000 IU / mL) at all time points for all samples tested, regardless of cell proliferation rate (Figure 10, left). During the first 6 days of culture, glucose levels decreased as TILs began to proliferate. Medium was then added or replaced, which maintained glucose levels at approximately 10 mM in most samples until the end of culture (Figure 11), and exponential proliferation was observed from day 6 (Figure 10, right). Consistent with glucose consumption, lactate began to accumulate in the cultures during the first 6 days, after which it was diluted or maintained at similar low levels by medium addition / exchange. Overall, throughout the cultivation process, lactate levels remained well below 20 mM for all samples tested (Figure 11). This is consistent with previous studies 14 According to the results of the present study, the feeding schedule was well tolerated by T cells. These data demonstrated that the feeding schedule developed by the inventors meets the requirements for TIL expansion in the G-Rex® device.

[0383] Fold expansion, viability and T cell composition of TILs from sequential use of G-Rex® 10M and G-Rex® 100M devices and PD performed with TransAct After culture, TILs from eight donors expanded at least 200-fold (Table 14), demonstrating the success of REP. For these samples, we observed a mean expansion fold of 712.4 ± 292.2 [95% confidence interval (CI): 468.1 to 956.7], as well as high cell viability (97.95% ± 1.51%) and T cell purity (97.43% ± 1.50%; Table 14) (CD3+ / -1.01% of total viable cells). + The pre-REP yields of these samples were in a wide range (30 × 10 6 ~783×10 6Thus, the yield of pre-REP TILs does not appear to be related to the REP yield, and two samples (T5101002 and T5101008) with pre-REP yields below 50 million cells showed robust growth with our protocol (Table 14). Considering the high average fold expansion, the REP process developed here produces only 10 × 10 of the intermediate product of pre-REP TILs. 6 10 to produce a target dose of final REP product, i.e., 1 x 10 9 ~100×10 9 Of note, these eight samples contained more than 80% CD3+ TILs in the starting material. + T cells (Table 14). In contrast, a low percentage of CD3 + Two other pre-REP samples containing T cells (T5101005 and T5101010, CD3 + T cell %: 64.0% and 38.7%, respectively, showed poor proliferation at the REP stage (17-fold and 167-fold, respectively). Thus, CD3 + The proportion of T cells may be important for TransAct-mediated REP. Overall, these data demonstrated that TransAct can indeed support REP of NSCLC TILs with a high success rate when using the G-Rex® device at manufacturing scale. The successful expansion of these eight samples also demonstrated that the REP process by TransAct can be initiated immediately after thawing of pre-REP cells without the 2+ day recovery period typically required for traditional REP using irradiated feeder cells.

[0384] [Table 10]

[0385] T cell phenotype of TILs from sequential use of G-Rex® 10M and G-Rex® 100M devices and PD performed with TransAct FACS analysis demonstrated that REP TILs from PD runs using sequential G-Rex® 10M and G-Rex® 100M instruments expressed a relatively high percentage of CD8 + The results showed that CD4 + and CD8 + Both TILs expressed high levels of the major costimulatory molecule CD28 (mean 83.10% ± 30.07% and 66.03% ± 16.81%, respectively; Figure 12 and Table 15), and to a lesser extent CD27 (mean 26.46% ± 20.68% and 40.12% ± 21.15%, respectively; Figure 12 and Table 15). Concerning inhibitory markers, both populations showed low expression of PD-1 (mean 10.98% ± 12.60% and 9.26% ± 14.03%, respectively; Figure 12 and Table 15) and LAG-3 (mean 7.46% ± 6.99% and 28.41% ± 19.40%, respectively; Figure 12 and Table 15), but high expression of TIM-3 (mean 48.96% ± 20.70% and 61.04% ± 18.97%, respectively; Figure 12 and Table 15). Finally, REP TILs mainly expressed central memory (T CM ) cells and effector memory (T EM ) cells (Figure 12). + and CD8 + T in TIL CM The mean percentages of T were 51.88% ± 26.19% and 54.96% ± 13.82%, respectively. EM The mean percentages of were 37.81% ± 23.11% and 29.31% ± 15.44%, respectively (Table 16).

[0386] [Table 11]

[0387] Taken together, the TransAct REP process developed by the inventors using the sequential use of the G-Rex® 10M and G-Rex® 100M instruments is applicable to the manufacture of TIL cell therapy products, which meets cell dose requirements while maintaining the desired T cell phenotype.

[0388] C-TIL051 PD Implementation with G-Rex® Device and Xuri™ W25 Bioreactor and TransAct The TransAct REP process using the G-Rex® 10M and G-Rex® 100M devices in series works well for donors with low pre-REP yields. We have also developed the TransAct REP process using the G-Rex® 100M and Xuri™ W25 bioreactors in series for high yields of pre-REP TILs.

[0389] We performed five REP runs using pre-REP TILs from three donors. After seeding pre-REP TILs into one G-Rex® 100M or its closed system equivalent, G-Rex® 100M-CS (range of seeded cell numbers: 87 × 10 6 ~200×10 6 ), cells were activated with TransAct and grown in a G-Rex® device for 7 days, which resulted in an average of 12-fold cell expansion. Cultures were then transferred to 10L Xuri™ Cellbags and further grown in the Xuri™ W25 system, except for T5101024, for which the culture was split evenly between two 10L Xuri™ Cellbags to compare different feeding schedules.

[0390] Culture conditions TIL expansion in the G-Rex® device and subsequent Xuri™ W25 bioreactor require different feeding schedules. To justify the feeding schedule we developed, we measured glucose, lactate and IL-2 levels from harvested supernatants of G-Rex® (day 7) and Xuri™ (daily) cultures. IL-2 concentrations were found to be high (>1000 IU / mL) at all time points for all samples tested, regardless of cell growth rate (Figure 13).

[0391] Glucose levels were also high (Figure 14), but lactate was at low levels that were largely tolerated by the T cells, except for T5101024a REP, which showed transient high levels of lactate at day 7 at the end of G-Rex® culture (Figure 14). In this REP, lactate levels decreased and remained at low levels during Xuri™ culture as a result of the addition of fresh REP CM, and robust cell growth was observed (Figure 15). These data demonstrated that the feeding schedule we developed met the requirements for TIL growth in both the G-Rex® device and the Xuri™ bioreactor.

[0392] TIL yield, viability and T cell composition from sequential use of the G-Rex® device with a Xuri™ W25 bioreactor and PD performed with TransAct After 8-12 days of Xuri™ growth, the PD run was terminated, with a minimum REP yield of 32.4 × 10 9 cells, which is 1 × 10 9 ~100×10 9 The target dose range of 55.38 ± 21.91 × 10 was reached (mean yield 55.38 ± 21.91 × 10 9 ; Figure 15 and Table 17). The final REP product had high viability (average 98.42% ± 0.08%) and CD3 +(average 97.64%±0.95%), with an average cell expansion fold of 514.6±206.2 for this REP process (Table 17). Notably, as the growth plateau was not reached when the process was terminated, these cells retained the potential to expand further (FIG. 15), and the average cell expansion fold could be further increased by extending the culture time in the Xuri™ bioreactor. Similarly, for samples that grow rapidly and reach the target cell number early, the REP process could be stopped accordingly for harvesting. Thus, the REP process provides flexibility in the future production time of TILs.

[0393] [Table 12]

[0394] T cell phenotypes using sequential G-Rex® devices with Xuri™ W25 bioreactors and performing TIL PD with TransAct T cell phenotype was analyzed by FACS, which indicates that the REP product from the novel process is relatively high in CD8 + The CD4 cell ratio was maintained (mean 51.70% ± 25.40%; Table 17). + and CD8 + Both TILs expressed high levels of CD28 (mean 77.14%±19.77% and 49.26%±26.18%, respectively), and moderate levels of TIM-3 (mean 31.72%±18.70% and 34.68%±31.40%, respectively), but expressed low levels of 4-1BB, CD27, PD-1, and LAG-3 (mean positivity for these markers was approximately 10% or less; Figure 16 and Table 18). REP products were also predominantly expressed in T CM and T EM The CD4 TILs were composed of CD4 T cells (FIG. 16) and had a favorable phenotype similar to the TILs generated from the G-Rex® device process alone. + and CD8 + T in TIL CM The mean percentages of T were 40.16% ± 19.32% and 31.34% ± 19.88%, respectively. EMThe mean percentages of were 53.50% ± 21.66% and 48.86% ± 23.98%, respectively (Table 19).

[0395] [Table 13]

[0396] Taken together, we have determined the culture parameters for optimal performance of Xuri™ REP. The TransAct REP process we developed by sequential use of the G-Rex® device and the Xuri™ bioreactor can be applied to the manufacturing of TIL cell therapy products that can easily meet the target cell dose while maintaining the desired T cell phenotype.

[0397] Characterization of TILs from PD runs by TCR sequencing To compare the repertoire diversity before (pre-REP) and after performing manufacturing-scale REP PD, we sequenced the TCR beta chains of TILs from two donors. REP TILs showed large TCR overlap with their cognate pre-REP TILs (92% for T5101015-3 and 77% for T5101024a, respectively), suggesting that the TCR clones presented by pre-REP TILs mostly survived after the REP process. We also calculated the Morishita Index for each pre-REP and REP sample pair, which is a correlation analysis that takes into account both the number and relative abundance of shared clones between the two samples. Values ​​range from 0 to 1, with higher values ​​indicating higher similarity between the overall repertoires of the two samples. Also, the Morishita index was relatively high (0.80 for T5101015 and 0.54 for T5101024, respectively), again suggesting that REP TILs from PD runs may well preserve the repertoire diversity of pre-REP TILs, which is further supported by the preservation of the top 100 pre-REP clones after REP and additional analysis of Simpson clonality.

[0398] Functional characterization of TILs from PD runs REP from TIL PD To obtain evidence for the functionality of TILs, we investigated the cell activation and cytokine secretion of TILs from three donors after Xuri™ REP in response to αCD3 stimulation. All three PD products were polyfunctional, producing large amounts of IFN-γ, granzyme B and TNF-α upon αCD3 stimulation alone, although the absolute levels varied between donors. For some donors, cytokine production could potentially be enhanced by the addition of IL-2. Consistently, CD4 + Populations and CD8 + In both populations, surface expression of 4-1BB and OX40 was upregulated by αCD3 stimulation alone, and expression of the latter was substantially enhanced by the addition of IL-2. These data demonstrate that C-TIL051 PD-enhanced REP TILs retain functionality to respond to T cell activation signals.

[0399] Characterization of TIL from simulated runs Based on the above PD runs, standard operating procedures were established. Full-scale mock runs of T5101034 and T5101035 were performed in a GMP facility. REP products were tested. Characterization of the TIL final products is shown below.

[0400] Yield, viability and T cell composition of TILs from mock runs Upon harvest, REP yields from both mock runs were greater than our target dose of 1 × 10 with high viability (96.5% and 96.8% for T5101034 and T5101035, respectively). 9 ~100×10 9 cells (1.72 × 10 for T5101034 and T5101035, respectively). 9 and 5.95×10 9 ; Table 20). REP TIL CD3 +The proportions were 86.98% and 97.42% for T5101034 and T5101035, respectively, of which 30.96% and 9.81% were CD8 + T cells (Table 20).

[0401] [Table 14]

[0402] T cell phenotype of C-TIL051 from mock runs T cell activation and exhaustion markers and memory phenotype of the REP TIL products from the mock run were examined. CD4 + In the subset, surface expression of 4-1BB was minimal for both samples (0.20% and 0.87% for T5101034 and T5101035, respectively), whereas surface expression of PD-1 was repeatedly detected (14.56% and 26.90% for T5101034 and T5101035, respectively) (Table 21). CD8 + In a subset, CD4 + Compared to the 100 and 150 subsets, surface expression of 4-1BB was slightly higher (2.36% and 6.13% for T5101034 and T5101035, respectively), whereas surface expression of PD-1 was lower (approximately 5%) in both samples (Table 21). Phenotypic analysis also demonstrated that both TIL products consisted primarily of memory cells, with a high proportion of naïve or stem cell memory (T5101034 and T5101035) for T5101034 and T5101035. N / T SCM ) stage, 19.93% and 22.35%, respectively, and central memory (T CM ) stage, 29.15% and 19.72%, respectively, and effector memory (T EM ) stage, the percentages were 37.56% and 34.37%, respectively (Table 22). EFF ) were 13.36% and 23.56%, respectively, for the two products (Table 22).

[0403] [Table 15]

[0404] Functional characterization of TILs from simulated runs First, to characterize the function of the REP TIL products, an IFN-γ release assay was performed. After overnight stimulation with anti-CD3 / CD28-conjugated Dynabeads, both products released large amounts of IFN-γ (10.46 fg / cell and 16.15 fg / cell for T5101034 and T5101035, respectively; FIG. 17).

[0405] We also examined the surface expression of T cell activation markers by the REP TIL products after overnight stimulation with αCD3 antibody. + or CD8 + In both subsets, surface expression of both 4-1BB and OX40 was strongly induced (FIG. 18).

[0406] Furthermore, the in vitro tumor killing effect of REP TILs from two full-scale mock runs against MOS generated from syngeneic tumor samples was evaluated. Morphological characterization, immunohistochemical staining for EpCAM and immunofluorescent staining for EpCAM and pan-cytokeratin demonstrated that both T5101034 and T5101035 MOS were tumor-derived. In subsequent potency assays, both products were able to eradicate MOS when co-cultured at higher effector-to-target ratios (5:1 and 10:1; Figure 19). This was quantified by measuring the fluorescence intensity from remaining NIR680 dye-positive live tumor cells. In both cases, the effect was detectable after only 8 hours of co-culture and became increasingly pronounced over time (Figure 19). Importantly, the addition of HLA class I blocking antibodies in the co-culture completely abolished the killing effect of the T5101034 TIL product, whereas HLA class II blockade had no effect (Fig. 20, left). This suggests that the killing function is primarily mediated by CD8 +In the case of T5101035, the addition of either HLA class I or class II blocking antibodies was able to abolish the killing effect (Figure 20, right). This suggests that the killing function is mediated by CD4 T cells. + and CD8 + These results suggest that both T cells from T5101034 and T5101035 TILs were required for tumor cell-specific killing. The dependence on HLA recognition in both cases, regardless of donor differences, demonstrated that the tumor-killing function of T5101034 and T5101035 TILs was tumor cell-specific.

[0407] We also collected culture supernatants from cocultures of TIL and MOS (E:T=5:1) on day 3 and performed cytokine assays. Compared with the TIL-only control, the TIL and MOS coculture group showed a significant increase in the levels of IFN-γ and granzyme A in the culture supernatants (Figure 21). The levels of granzyme B in the T5101034 TIL and MOS coculture supernatants were also increased, and the levels of TNF-α in the T5101035 TIL and MOS coculture supernatants were also increased (Figure 21). This result was consistent with the results of TIL tumor killing efficacy already described. Furthermore, the addition of HLA class I blocking antibodies dramatically reduced the cytokine levels in the coculture supernatants of TIL and MOS from both donors, whereas the addition of HLA class II blocking antibodies mainly reduced the cytokine levels in the coculture supernatants of T5101035 TIL and MOS (Figure 21). Regardless of donor differences, the dependence on HLA recognition in both cases demonstrated that the tumor-killing effect of TILs was TCR-mediated. Taken together, these data demonstrated that TILs from mock runs were highly effective in killing autologous tumor cells.

[0408] conclusion We have successfully developed a feeder-free TIL REP process using αCD3 / αCD28 nanoparticles. The TIL REP process can be initiated immediately after thawing of pre-REP cells. Depending on the viable cell number of pre-REP TILs, different cell expansion devices or systems (i.e., G-Rex® 10M, G-Rex® 100M-CS and Xuri™ W25 bioreactor) can be used for the REP process to achieve the target therapeutic dose. T cell repertoire diversity is preserved in TILs. TILs also exhibit the desired T cell phenotype, the ability to upregulate T cell activation markers, and the ability to produce various cytokines in response to T cell activation signals. Furthermore, functional characterization of TILs from mock runs demonstrated that the product is effective in killing autologous tumor cells.

[0409] The scope of the present invention is not limited by what has been specifically shown and described above. Those skilled in the art will recognize that there are suitable alternatives to the described examples of materials, configurations, structures and dimensions. In the description of the present invention, numerous references, including patents and various publications, have been cited and discussed. The citation and discussion of such references is made solely to clarify the description of the present invention, and no reference is admitted to be prior art to the invention described herein. All references cited and described herein are incorporated herein by reference in their entirety. Variations, modifications and other implementations of what is described herein will occur to those skilled in the art without departing from the spirit and scope of the present invention. While specific embodiments of the present invention have been shown and described, it will be apparent to those skilled in the art that changes and modifications may be made without departing from the spirit and scope of the present invention. The matters set forth in the foregoing description are set forth solely by way of example and not by way of limitation.

Claims

1. (a) culturing a first cell population in a first cell culture medium to generate a second cell population, wherein the first cell population is obtained from a tumor sample from a patient; and (b) contacting the second cell population with a polymer matrix comprising anti-CD3 and anti-CD28 antibodies or fragments thereof in a second cell culture medium to generate a third cell population, wherein the second cell culture medium comprises 100 IU / mL to 8,000 IU / mL, 500 IU / mL to 4,000 IU / mL, or 3,000 IU / mL interleukin-2 (IL-2); A method for expanding tumor infiltrating lymphocytes (TILs), comprising:

2. 10. The method of claim 1, wherein the first cell culture medium comprises 2,000 IU / mL to 8,000 IU / mL of IL-2.

3. 2. The method of claim 1, wherein in step (a), the first cell population is cultured for 10 to 40 days, or 10 to 14 days.

4. 2. The method of claim 1, wherein in step (b), the contacting is for 3 days to 17 days.

5. 2. The method of claim 1, wherein the third cell population is at least 100 times greater in number than the second cell population, or 100 to 2000 times greater in number.

6. 2. The method of claim 1, wherein the tumor sample is from a solid tumor, and the solid tumor comprises sarcoma, hepatocellular carcinoma, glioma, head and neck cancer, bone cancer, brain cancer, breast cancer, anal cancer, anal canal cancer, anorectal cancer, eye cancer, intrahepatic bile duct cancer, joint cancer, cervical cancer, gallbladder cancer, pleural cancer, nasal cancer, nasal cavity cancer, middle ear cancer, oral cancer, vulva cancer, colon cancer, esophageal cancer, cervical cancer, gastrointestinal cancer, hypopharyngeal cancer, laryngeal cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma, nasopharyngeal cancer, ovarian cancer, pancreatic cancer, peritoneal cancer, omental cancer, mesentery cancer, pharyngeal cancer, prostate cancer, rectal cancer, kidney cancer, small intestine cancer, soft tissue cancer, stomach cancer, testicular cancer, thyroid cancer, ureter cancer, bladder cancer, or a combination thereof.

7. Tumor-infiltrating lymphocytes obtained by the method of claim 1.

8. A cell population enriched for or expanded from tumor-infiltrating lymphocytes, comprising: (i) CD3 ＋ CD3 percentage ranges from 3% to 88% of cells ＋ CD8 ＋ T cells, (ii) CD3 ＋ CD3 percentages range from 10% to 96% of cells ＋ CD4 ＋ T cells, (iii) CD4 ＋ CD4 percentage ranges from 50% to 88% of cells ＋ T ＣＭ T cells, (iv) CD8 ＋ CD8 percentage ranges from 28% to 82% of cells ＋ T ＣＭ T cells, (v) CD4 ＋ CD4 percentage ranges from 11% to 49% of cells ＋ T ＥＭ T cells, and (vi) CD8 ＋ CD8 percentages ranged from 11% to 61% of cells ＋ T ＥＭ T cell and The cell population comprises 70% or more viable cells, and the cell population is generated from a tumor sample from a patient.

9. 80% or more CD3 in live cells ＋ The cell population of claim 8 , comprising T cells.

10. CD4 ＋ CD4 percentage ranges from 10% to 51% of cells ＋ CD27 ＋ The cell population of claim 8 , comprising T cells.

11. CD8 ＋ CD8 percentages ranged from 12% to 72% of cells ＋ CD27 ＋ The cell population of claim 8 , comprising T cells.

12. CD8 ＋ CD8 percentage ranges from 34% to 95% of cells ＋ CD28 ＋ The cell population of claim 8 , comprising T cells.

13. CD4 ＋ CD4 percentage ranges from 82% to 100% of cells ＋ CD28 ＋ The cell population of claim 8 , comprising T cells.

14. CD4 ＋ CD4 percentage ranges from 0.2% to 5.8% of cells ＋ 4-1BB ＋ The cell population of claim 8 , comprising T cells.

15. CD8 ＋ CD8 percentages ranging from 0.2% to 11.6% of cells ＋ 4-1BB ＋ The cell population of claim 8 , comprising T cells.

16. CD4 ＋ CD4 percentage ranges from 0.2% to 19.5% of cells ＋ LAG3 ＋ The cell population of claim 8 , comprising T cells.

17. CD8 ＋ CD8 percentage ranged from 6% to 51.2% of cells ＋ LAG3 ＋ The cell population of claim 8 , comprising T cells.

18. CD4 ＋ CD4 percentage ranges from 0.9% to 31% of cells ＋ PD1 ＋ The cell population of claim 8 , comprising T cells.

19. CD8 ＋ CD8 percentages range from 1% to 18% of cells ＋ PD1 ＋ The cell population of claim 8 , comprising T cells.

20. CD56 below 10% ＋ The cell population of claim 8 , comprising NK cells.

21. (a) culturing a first cell population in a first cell culture medium to generate a second cell population, wherein the first cell population is obtained from a tumor sample from a patient; and 10. The cell population of claim 8, produced by a method of expanding tumor-infiltrating lymphocytes (TILs), comprising: (b) contacting the second cell population with a polymer matrix comprising an anti-CD3 antibody and an anti-CD28 antibody, or fragments thereof, in a second cell culture medium, wherein the second cell culture medium comprises 100 IU / mL to 8,000 IU / mL of interleukin-2 (IL-2).

22. (a) culturing cells obtained from a tumor sample from a patient; (b) treating the cultured cells to generate a cell population enriched for tumor-infiltrating lymphocytes; 1. A method for expanding a cell population enriched for tumor-infiltrating lymphocytes, comprising: (i) CD3 ＋ CD3 percentage ranges from 3% to 88% of cells ＋ CD8 ＋ T cells, (ii) CD3 ＋ CD3 percentages range from 10% to 96% of cells ＋ CD4 ＋ T cells, (iii) CD4 ＋ CD4 percentage ranges from 50% to 88% of cells ＋ CD45RA － CD62L ＋ central memory T cells, (iv) CD8 ＋ CD8 percentage ranges from 28% to 82% of cells ＋ CD45RA － CD62L ＋ central memory T cells, (v) CD4 ＋ CD4 percentage ranges from 11% to 49% of cells ＋ CD45RA － CD62L － effector memory T cells, and (vi) CD8 ＋ CD8 percentages ranged from 11% to 61% of cells ＋ T CD45RA － CD62L － Effector memory T cells The method includes one or more of:

23. A cell population enriched for tumor-infiltrating lymphocytes obtained by the method of claim 22.

24. The tumor-infiltrating lymphocytes of claim 7, or the cell population of claim 8 or claim 23, for treating cancer in a patient, comprising: the cancer comprises sarcoma, hepatocellular carcinoma, glioma, head and neck cancer, bone cancer, brain cancer, breast cancer, anal cancer, anal canal cancer, anorectal cancer, eye cancer, intrahepatic bile duct cancer, joint cancer, cervical cancer, gallbladder cancer, pleural cancer, nasal cancer, nasal cavity cancer, middle ear cancer, oral cancer, vulva cancer, colon cancer, esophageal cancer, cervical cancer, gastrointestinal cancer, hypopharyngeal cancer, laryngeal cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma, nasopharyngeal cancer, ovarian cancer, pancreatic cancer, peritoneal cancer, omental cancer, mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, kidney cancer, small intestine cancer, soft tissue cancer, stomach cancer, testicular cancer, thyroid cancer, ureter cancer, bladder cancer, or a combination thereof; Tumor-infiltrating lymphocytes or cell populations.

25. The treatment, ９ ~1 x 10 １１ The tumor-infiltrating lymphocyte or cell population of claim 24, comprising administering to a patient 5×10 9 to 9×10 10 cells.

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