Improvement process for culturing therapeutic-use tumor infiltrating lymphocyte

Abstract

To reactivate exhausted tumor infiltrating lymphocytes (TIL) in vitro.SOLUTION: Reactivation includes coculturing an excised TIL including a tumor fragment together with a checkpoint inhibitor, stimulating the TIL by another interleukin which is known to recover exhaustion of T-cells, and / or inhibiting an effect of a regulatory T-cell secretion factor (such as IL-10), and forming a preferable tumor microenvironment (TME) in which T-cells exhausted by the inhibition can proliferate faster than a currently constructed TIL proliferation procedure and to a larger number.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention aims to reactivate exhausted tumor infiltrating lymphocytes (TILs) ex vivo, which involves co-culturing excised TILs containing tumor fragments with tumor microenvironment (TME) stimulators such as immune checkpoint inhibitors (ICIs), stimulating the TILs with other interleukins known to reverse T cell exhaustion, and / or inhibiting the effects of regulatory T cell secreted factors (e.g., inhibiting IL-10), thereby creating a favorable tumor microenvironment in which exhausted T cells can expand faster and to greater numbers than currently established TIL expansion procedures. [Background technology]

[0002] Tumor-infiltrating lymphocytes are associated with improved prognosis and progression-free survival in cancer patients receiving immunotherapy, including the use of immune checkpoint inhibitors (ICIs) against CTLA-4 and PD-1 / PD-L1.

[0003] However, only a portion of patients still have a durable long-term response to such treatment, since many other factors seem to be involved in the tumor microenvironment in reducing immune responsiveness. One of the important factors seems to be T cell exhaustion, which leads to physical elimination and / or dysfunction of antigen-specific T cells. Factors involved in this exhaustion phenomenon include surface markers expressed on tumor cells, lymphocytes, and monocytes, and soluble molecules secreted by regulatory T cells and NK cells in the tumor microenvironment (TME). However, the lack of stimulatory factors such as interferon-gamma and IL-2 in the TME is also evident.

[0004] Reversal of T cell exhaustion represents an important target for the development of a new class of ICIs, either as monotherapy or in combination with established therapies. However, these targets are often responsible for inducing immune tolerance to avoid autoimmune responses, and systemic administration of inhibitors can cause serious side effects. Furthermore, administration of T cell stimulatory molecules such as IL-2 can cause serious and sometimes fatal side effects that must be managed by a skilled clinician. Several approaches have been taken to administer drug candidates locally within the tumor, thereby potentially avoiding systemic side effects. However, the distribution of cancer cells throughout the body in many metastatic patients can call into question the success of this approach in this setting.

[0005] The use of tumor-infiltrating lymphocyte (TIL) therapy has shown remarkable clinical benefit in patients with a variety of malignancies, including metastatic melanoma, head and neck cancer, and cervical cancer, with objective response rates of up to 55% and complete responses in up to 20% of patients. Briefly, this type of therapy utilizes the ex vivo expansion of autologous T lymphocytes by first stimulating fragments from resected tumors with IL-2, anti-CD3 antibodies, and feeder cells, thereby growing these cells to billions before readministering the T cells to patients undergoing lymphodepleting therapy, thereby promoting subsequent tumor regression.

[0006] TIL therapy is costly and time consuming, therefore it would be beneficial to optimize current methods and identify ways to shorten the TIL expansion period, increase the expansion rate, and achieve a more favorable phenotype. Summary of the Invention

[0007] The present invention relates to a method of expanding tumor infiltrating lymphocytes (TILs) into a therapeutic population of TILs to promote regression of cancer in a mammal, the method comprising the steps of: (a) obtaining a first population of TILs from a tumor excised from the mammal and culturing the autologous T cells; (b) performing a first expansion by culturing the first population of TILs in a cell culture medium comprising IL-2 and one or more TME stimulants to generate a second population of TILs; (c) performing a second expansion by supplementing the cell culture medium of the second population of TILs with additional IL-2, anti-CD3 antibody, and antigen presenting cells (APCs) to generate a third population of TILs, which is a therapeutic population; and (d) administering the therapeutic population of T cells to the mammal following administration of non-myeloablative lymphodepleting chemotherapy, wherein administration of the T cells to the mammal promotes regression of cancer in the mammal.

[0008] A further aspect of the invention relates to a method of treating a subject suffering from cancer comprising administering expanded tumor infiltrating lymphocytes (TILs), the method comprising the steps of: (a) harvesting a first population of TILs from a tumor resected from the mammal and culturing the autologous T cells; (b) performing a first expansion by culturing the first population of TILs in cell culture medium comprising IL-2 and one or more TME stimulants to generate a second population of TILs; (c) performing a second expansion by supplementing the cell culture medium of the second population of TILs with additional IL-2, anti-CD3 antibody, and antigen presenting cells (APCs) to generate a third population of TILs, which is a therapeutic population; and (d) administering the therapeutic population of T cells to the mammal following administration of non-myeloablative lymphodepleting chemotherapy, wherein administration of the T cells to the mammal promotes regression of the cancer in the mammal.

[0009] Another aspect of the invention relates to a method of expanding tumor infiltrating lymphocytes (TILs) into a therapeutic population of TILs, comprising the steps of: (a) harvesting a first population of TILs from a tumor excised from a mammal and culturing the autologous T cells; (b) performing a first expansion by culturing the first population of TILs in a cell culture medium comprising IL-2 and one or more TME stimulants to generate a second population of TILs; and (c) performing a second expansion by supplementing the cell culture medium of the second population of TILs with additional IL-2, anti-CD3 antibody, and antigen presenting cells (APCs) to generate a third population of TILs, which is a therapeutic population.

[0010] In one or more embodiments, the one or more TME stimulants are selected from the group consisting of substances capable of antagonizing and / or inhibiting receptors expressed on the surface of T cells (or their ligands) known to cause downregulation, deactivation, and / or exhaustion of T cells; substances capable of stimulating and / or promoting receptors expressed on the surface of T cells known to cause upregulation, activation, and / or reactivation of T cells; substances capable of antagonizing and / or inhibiting soluble molecules and cytokines and their receptors known to cause downregulation, deactivation, and / or exhaustion of T cells; and substances capable of downregulating and / or exhausting regulatory T cells, thereby promoting T cell proliferation in vitro.

[0011] In one or more embodiments, the one or more TME stimuli are one or more check point inhibitors or inhibitors of their ligands, such as anti-PD1, anti-PD-L1, anti-PD-L2, anti-CTLA-4, anti-LAG3, anti-A2AR, anti-B7-H3, anti-B7-H4, anti-BTLA, anti-IDO, anti-HVEM, anti-IDO, anti-TDO, anti-KIR, anti-NOX2, anti-TIM3, anti-galectin 9, anti-VISTA, anti-SIGLEC7 / 9, optionally wherein one or more check point inhibitors or inhibitors of their ligands are also added to the second expansion.

[0012] In one or more embodiments, the agent capable of antagonizing and / or inhibiting receptors expressed on the surface of T cells (or their ligands) known to cause T cell downregulation, inactivation, and / or exhaustion is selected from the group consisting of: A: an agent that acts through a PD-1 receptor on the surface of T cells; B: an agent that acts through a CTLA-4 receptor on the surface of T cells; C: an agent that acts through a LAG-3 receptor on the surface of T cells; D: an agent that acts through a TIGIT / CD226 receptor on the surface of T cells; E: an agent that acts through a KIR receptor on the surface of T cells; F: an agent that acts through a TIM-3 receptor on the surface of T cells; G: an agent that acts through a BTLA receptor on the surface of T cells; and H: an agent that acts through an A2aR receptor on the surface of T cells.

[0013] In one or more embodiments, the Group A agents are selected from one or more of the group consisting of pembrolizumab, nivolumab, cemiplimab, sym021, atezolizumab, avelumab, and durvalumab.

[0014] In one or more embodiments, the group B agent is selected from one or more of the group consisting of ipilimumab and tremelimumab. In one or more embodiments, the group C agent is selected from one or more of the group consisting of leratolimab, eftiragimob alfa, and sym022. In one or more embodiments, the group D agent is tiragolumab. In one or more embodiments, the group E agent is lirilumab. In one or more embodiments, the group F agent is sym023. In one or more embodiments, the group G agent is 40E4 and PJ196.

[0015] In one or more embodiments, the substance capable of stimulating and / or promoting a receptor expressed on the surface of a T cell that is known to cause upregulation, activation, and / or reactivation of T cells is selected from the group consisting of: I: a substance that acts through the OX40 / CD134 receptor on the surface of a T cell; J: a substance that acts through the 4-1BB / CD137 receptor on the surface of a T cell; K: a substance that acts through the CD28 receptor on the surface of a T cell; L: a substance that acts through the ICOS receptor on the surface of a T cell; M: a substance that acts through the GITR receptor on the surface of a T cell; N: a substance that acts through the CD40L receptor on the surface of a T cell; and O: a substance that acts through the CD27 receptor on the surface of a T cell.

[0016] In one or more embodiments, the group J agent is selected from one or more of the group consisting of urelumab and utomilumab. In one or more embodiments, the group K agent is telalzimab. In one or more embodiments, the group O agent is valilumab.

[0017] In one or more embodiments, the substances capable of antagonizing and / or inhibiting soluble molecules and cytokines and their receptors known to cause downregulation, inactivation, and / or exhaustion of T cells are selected from the group consisting of P: substances acting through IDO1 / 2 receptors on the surface of T cells, Q: substances acting through TGFβ receptors on the surface of T cells, R: substances acting through IL-10 receptors on the surface of T cells, and S: substances acting through IL-35 receptors on the surface of T cells.

[0018] In one or more embodiments, the P group agent is epacedostat. In one or more embodiments, the Q group agent is linrodostat. In one or more embodiments, the R group agent is galunisertib.

[0019] In one or more embodiments, the agent capable of downregulating and / or depleting regulatory T cells and thereby promoting T cell proliferation in vitro is selected from the group consisting of: T: cyclophosphamide; U: TKI; V: agents acting through αCD25; and X: IL2 / diphtheria toxin fusion.

[0020] In one or more embodiments, the Group U agent is sunitinib. In one or more embodiments, the Group V agent is selected from one or more of the group consisting of sorafenib, imatinib, and daclizumab. In one or more embodiments, the Group X agent is denileukin diftitox.

[0021] In one or more embodiments, the concentration of the substance is from 0.1 μg / mL to 300 μg / mL, such as from 1 μg / mL to 100 μg / mL, such as from 10 μg / mL to 100 μg / mL, such as from 1 μg / mL to 10 μg / mL.

[0022] In one or more embodiments, the therapeutic population of T cells is used to treat a cancer type selected from the group consisting of: 1: solid tumors, 2: ICI naive tumors, 3: MSI-H tumors, 4: hematological tumors, virally induced tumors, and 5: hypermutated tumors (such as POL-E and POL-D mutant tumors).

[0023] In one or more embodiments, the therapeutic population of T cells is used to treat a cancer type selected from the group consisting of breast cancer, renal cell carcinoma, bladder cancer, melanoma, cervical cancer, gastric cancer, colon cancer, lung cancer, head and neck cancer, ovarian cancer, Hodgkin's lymphoma, pancreatic cancer, liver cancer, and sarcoma.

[0024] In one or more embodiments, the therapeutic population of T cells is used to treat breast cancer. In one or more embodiments, the therapeutic population of T cells is used to treat renal cell carcinoma. In one or more embodiments, the therapeutic population of T cells is used to treat bladder cancer. In one or more embodiments, the therapeutic population of T cells is used to treat melanoma. In one or more embodiments, the therapeutic population of T cells is used to treat cervical cancer. In one or more embodiments, the therapeutic population of T cells is used to treat gastric cancer. In one or more embodiments, the therapeutic population of T cells is used to treat colorectal cancer. In one or more embodiments, the therapeutic population of T cells is used to treat lung cancer. In one or more embodiments, the therapeutic population of T cells is used to treat head and neck cancer. In one or more embodiments, the therapeutic population of T cells is used to treat ovarian cancer. In one or more embodiments, the therapeutic population of T cells is used to treat Hodgkin's lymphoma. In one or more embodiments, the therapeutic population of T cells is used to treat pancreatic cancer. In one or more embodiments, the therapeutic population of T cells is used to treat liver cancer. In one or more embodiments, the therapeutic population of T cells is used to treat sarcoma.

[0025] In one or more embodiments, steps (a)-(c) or (d) are performed within a period of about 20 to about 45 days. In one or more embodiments, steps (a)-(c) or (d) are performed within a period of about 20 to about 40 days. In one or more embodiments, steps (a)-(c) or (d) are performed within a period of about 25 to about 40 days. In one or more embodiments, steps (a)-(c) or (d) are performed within a period of about 30 to about 40 days. In one or more embodiments, steps (a)-(b) are performed within a period of about 10 to about 28 days. In one or more embodiments, steps (a)-(b) are performed within a period of about 10 to about 20 days.

[0026] In one or more embodiments, step (c) is performed within a period of about 12 to about 18 days. In one or more embodiments, step (c) is performed within a period of about 10 to about 28 days. In one or more embodiments, step (c) is performed within a period of about 10 to about 20 days. In one or more embodiments, step (c) is performed within a period of about 12 to about 18 days.

[0027] In one or more embodiments, step (b) comprises administering 1×10 6 ~1×10 7 cells, e.g. 2 x 10 6 ~5×10 6 In one or more embodiments, step (b) produces 5×10 cells. 6 ~1×10 7 In one or more embodiments, step (b) produces 1×10 cells. 6 ~5×10 7 In one or more embodiments, step (b) produces 1×10 cells. 7 ~5×10 7 In one or more embodiments, step (c) generates 1×10 cells. 7 ~1×10 12 cells, e.g. 1 x 10 8 ~5×10 9 cells, e.g. 1 x 10 9 ~5×10 9 cells, e.g. 1 x 10 8 ~5×10 10 cells, e.g. 1 x 10 9 ~5×10 11 In one or more embodiments, step (c) generates 1×10 cells. 7 ~1×10 10 In one or more embodiments, step (c) generates 1×10 cells. 7 ~1×10 9 In one or more embodiments, step (c) generates 1×10 cells. 7 ~1×10 8 Generate cells. In one or more embodiments, the APCs are artificial APCs (aAPCs) or allogeneic feeder cells. In one or more embodiments, a therapeutic population of TILs is infused into a patient.

[0028] In one or more embodiments, the cells are removed from the cell culture and cryopreserved in a storage medium prior to performing step (c).

[0029] In one or more embodiments, the method further comprises transducing the first population of TILs with an expression vector comprising a nucleic acid encoding a chimeric antigen receptor (CAR) comprising a single chain variable fragment antibody fused to at least one endodomain of a T cell signaling molecule.

[0030] In one or more embodiments, step (c) further comprises removing the cells from the cell culture medium.

[0031] In one or more embodiments, step (a) further comprises processing the resected tumor into a plurality of tumor fragments, eg, between 4-50 fragments, between 20-30 fragments, etc.

[0032] In one or more embodiments, the size of the fragments is about 5-50 mm. 3 In one or more embodiments, the size of the fragments is about 50 mm 3 In one or more embodiments, the size of the fragments is about 0.1 to 10 mm. 3 In one or more embodiments, the size of the fragments is about 0.1 to 1 mm. 3 In one or more embodiments, the size of the fragments is about 0.5 to 5 mm. 3 In one or more embodiments, the size of the fragments is about 1-10 mm. 3 In one or more embodiments, the size of the fragments is about 1-3 mm. 3 It is. In one or more embodiments, the mammal is a human.

[0033] In one or more embodiments, the cell culture medium is provided in a container selected from the group consisting of a G-Rex container and a Xuri culture bag.

[0034] One aspect relates to a population of tumor infiltrating lymphocytes (TILs) obtained by the method of any of the preceding embodiments.

[0035] A further aspect relates to expanded tumor infiltrating lymphocytes (TILs) for use in treating a subject suffering from cancer, the treatment comprising the steps of: harvesting a first population of TILs from a tumor resected from the mammal and culturing the autologous T cells; performing a first expansion by culturing the first population of TILs in cell culture medium comprising IL-2 and one or more TME stimulants to generate a second population of TILs; performing a second expansion by supplementing the cell culture medium of the second population of TILs with additional IL-2, anti-CD3, and antigen presenting cells (APCs) to generate a third population of TILs, which is a therapeutic population; and administering the therapeutic population of T cells to the mammal after administering non-myeloablative lymphodepleting chemotherapy, wherein administration of the T cells to the mammal promotes regression of the cancer in the mammal.

[0036] A further aspect relates to a population of tumor infiltrating lymphocytes (TILs) obtained by a method comprising the steps of: harvesting a first population of TILs from a tumor excised from a mammal and culturing the autologous T cells; performing a first expansion by culturing the first population of TILs in a cell culture medium comprising IL-2 and one or more TME stimulants to generate a second population of TILs; and performing a second expansion by supplementing the cell culture medium of the second population of TILs with additional IL-2, anti-CD3, and antigen presenting cells (APCs) to generate a third population of TILs, which is a therapeutic population.

[0037] A further aspect relates to a therapeutic population of TILs comprising IL-2 and one or more TME stimulatory agents.

[0038] A further aspect relates to a therapeutic population of TILs comprising IL-2, one or more TME stimulants, additional IL-2, anti-CD3, and antigen presenting cells (APCs). [Brief description of the drawings]

[0039] [Figure 1]FIG. 1 shows that a tumor containing tumor infiltrating lymphocytes (TILs) excised from a mammal is cut into smaller fragments and placed into one or more multi-well cell culture dishes. These fragments are then cultured in cell culture medium containing interleukin 2 (IL-2) and tumor microenvironment (TME) stimulants during a first expansion to generate a second TIL population. The second TIL population is then further expanded by a second expansion (often referred to as rapid expansion) in cell culture medium containing feeder cells, anti-CD3 antibodies, and IL-2. The second expansion may be up to the procedure, and cell culture may be performed in one or more steps, in one or more vessels in further substeps, but is shown in the figure as only one step for simplicity. Finally, the TILs are harvested to generate a third therapeutic TIL population and resuspended into a final TIL product that is infused back into the mammal to promote cancer regression.

[0040] [Diagram 2] FIG. 2 shows the success rate (>50,000 cells / fragment) of cell expansion using IL-2, Durvalumab (Durva), Avelu, Relatli, Tiragoloumab (Tira), Pembrolizumab (Pembro), Ipilimumab (Ipi), Seralizumab (Thera), Nivolumab (Nivo), or Urelumab / OKT3 (Ure).

[0041] [Diagram 3] FIG. 3 shows the success (black) and failure (white) rates of cell expansion (>50,000 cells / fragment) with IL-2, Durvalumab (Durva), Avelu, Relatli, Tiragomab (Tira), Pembrolizumab (Pembro), Ipilimumab (Ipi), Seralizumab (Thera), Nivolumab (Nivo), or Urelumab / OKT3 (Ure).

[0042] [Figure 4]Figure 4 shows the success rate of cell expansion (>50,000 cells / fragment) using IL-2+ / - TME-S. See Table 43 for specific stimuli for each group.

[0043] [Diagram 5] Figure 5 shows the success rate (black) and failure rate (white) of cell expansion (>50,000 cells / fragment) using IL-2+ / - TME-S. See Table 43 for specific stimuli for each group.

[0044] [Figure 6] Figure 6 shows the total cell number and expansion time of cell cultures in G-Rex flasks with IL-2+ / -TME-S. See Table 43 for specific stimuli for each group. Visible growth indicator lines were added manually for simplicity.

[0045] [Figure 7] FIG. 7 shows total cell numbers in cells grown from head and neck cancer (HN), metastatic melanoma (MM), and ovarian cancer (OC) using low (1 μg / mL), medium (5 μg / mL), and high (25 μg / mL) concentrations of anti-CTLA-4 (ipilimumab).

[0046] [Figure 8] FIG. 8 shows total cell counts in cells grown from head and neck cancer (HN), metastatic melanoma (MM), and ovarian cancer (OC) using low (1 μg / mL), medium (5 μg / mL), and high (25 μg / mL) concentrations of anti-PD1 (pembrolizumab).

[0047] [Figure 9] FIG. 9 shows total cell counts in cells grown from head and neck cancer (HN), metastatic melanoma (MM), and ovarian cancer (OC) using low (2 μg / mL) and medium (10 μg / mL) concentrations of anti-4-1BB (urelumab) with OKT3 (30 ng / mL).

[0048] [Figure 10] FIG. 10 shows total cell numbers in cells grown from head and neck cancer (HN), metastatic melanoma (MM), and ovarian cancer (OC) using low (0.02 μg / mL), medium (0.1 μg / mL), high (2 μg / mL), and ultra-high (2 μg / mL) concentrations of anti-CD28 (celalizumab).

[0049] [Figure 11] Figure 11 shows total cell counts in cells grown from head and neck cancer (HN), metastatic melanoma (MM), and ovarian cancer (OC) using low (2 μg / mL), medium (10 μg / mL), and high (50 μg / mL) concentrations of anti-PD-L1 (avelumab).

[0050] [Figure 12] FIG. 12 shows total cell counts in cells grown from head and neck cancer (HN), metastatic melanoma (MM), and ovarian cancer (OC) using low (2 μg / mL), medium (10 μg / mL), and high (50 μg / mL) concentrations of anti-PD-1 (nivolumab).

[0051] [Figure 13] Figure 13 shows the number of viable cells per fragment after culture of tumor tissue from any cancer type in G-Rex flasks with IL-2+ / -TME-S. See Table 43 for specific stimuli for each group. Statistics performed by two-tailed Mann-Whitney U test comparing each group to control (IL-2), p>0.05 was considered not significant, and significance was classified as *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0052] [Figure 14]Figure 14 shows the number of viable cells per fragment after culture of tumor tissue from cervical cancer in G-Rex flasks with IL-2+ / -TME-S. See Table 43 for specific stimulants for each group. Statistics performed by two-tailed Mann-Whitney U test comparing each group with control (IL-2), p>0.05 was considered not significant, and significance was classified as *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0053] [Figure 15] Figure 15 shows the number of viable cells per fragment after culture of tumor tissue from melanoma in G-Rex flasks with IL-2+ / -TME-S. See Table 43 for specific stimulants for each group. Statistics performed by two-tailed Mann-Whitney U test comparing each group with control (IL-2), p>0.05 was considered not significant, and significance was classified as *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0054] [Figure 16] Figure 16 shows the number of viable cells per fragment after culture of tumor tissue from ovarian cancer in G-Rex flasks with IL-2+ / -TME-S. See Table 43 for specific stimulants for each group. Statistics performed by two-tailed Mann-Whitney U test comparing each group with control (IL-2), p>0.05 was considered not significant, and significance was classified as *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0055] [Figure 17]Figure 17 shows the number of viable cells per fragment after culture of tumor tissue from head and neck cancer in G-Rex flasks with IL-2+ / -TME-S. See Table 43 for specific stimulants for each group. Statistics performed by two-tailed Mann-Whitney U test comparing each group with control (IL-2), p>0.05 was considered not significant, and significance was classified as *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0056] [Figure 18] Figure 18 shows the number of viable cells per fragment after culture of tumor tissue from lung cancer in G-Rex flasks with IL-2+ / -TME-S. See Table 43 for specific stimuli for each group. Statistics performed by two-tailed Mann-Whitney U test comparing each group with control (IL-2), p>0.05 was considered not significant, and significance was classified as *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0057] [Figure 19] Figure 19 shows the number of viable cells per fragment after culture of tumor tissue from colon cancer in G-Rex flasks with IL-2+ / -TME-S. See Table 43 for specific stimuli for each group. Statistics performed by two-tailed Mann-Whitney U test comparing each group with control (IL-2), p>0.05 was considered not significant, and significance was classified as *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0058] [Figure 20]Figure 20 shows the number of viable cells per fragment after culture of tumor tissue from any cancer type in G-Rex flasks with IL-2 + / - antagonists, agonists, CD28 family, reactivation treatments, or depletion treatments. See Table 44 for specific stimuli for each group. Statistics performed by two-tailed Mann-Whitney U test comparing each group to control (IL-2), p>0.05 was considered not significant, and significance was classified as * p <0.05, ** p <0.01, *** p <0.001, ****p<0.0001.

[0059] [Figure 21] Figure 21 shows the number of viable cells per fragment after culture of tumor tissue from melanoma in G-Rex flasks with IL-2 + / - antagonists, agonists, CD28 family, reactivation treatments, or depletion treatments. See Table 44 for specific stimuli for each group. Statistics performed by two-tailed Mann-Whitney U test comparing each group to control (IL-2), p>0.05 was considered not significant, and significance was classified as * p <0.05, ** p <0.01, *** p <0.001, ****p<0.0001.

[0060] [Figure 22] Figure 22 shows the number of viable cells per fragment after culture of tumor tissue from ovarian cancer in G-Rex flasks with IL-2 + / - antagonists, agonists, CD28 family, reactivating treatments, or depleting treatments. See Table 44 for specific stimuli for each group. Statistics performed by two-tailed Mann-Whitney U test comparing each group to control (IL-2), p>0.05 was considered not significant, and significance was classified as * p <0.05, ** p <0.01, *** p <0.001, ****p<0.0001.

[0061] [Diagram 23]Figure 23 shows the number of viable cells per fragment after culture of tumor tissue from lung cancer in G-Rex flasks with IL-2+ / -antagonists, agonists, CD28 family, reactivation treatments, and depletion treatments. See Table 44 for specific stimuli for each group. Statistics performed by two-tailed Mann-Whitney U test comparing each group with control (IL-2), p>0.05 was considered not significant, and significance was classified as *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0062] [Figure 24] Figure 24 shows the number of viable cells per fragment after culture of tumor tissue from cervical cancer in G-Rex flasks with IL-2 + / - antagonists, agonists, CD28 family, reactivation treatments, or depletion treatments. See Table 44 for specific stimuli for each group. Statistics performed by two-tailed Mann-Whitney U test comparing each group with control (IL-2), p>0.05 was considered not significant, and significance was classified as * p <0.05, ** p <0.01, *** p <0.001, ****p<0.0001.

[0063] [Diagram 25] Figure 25 shows the number of viable cells per fragment after culture of tumor tissue from colon cancer in G-Rex flasks with IL-2 + / - antagonists, agonists, CD28 family, reactivation treatments, or depletion treatments. See Table 44 for specific stimuli for each group. Statistics performed by two-tailed Mann-Whitney U test comparing each group to control (IL-2), p>0.05 was considered not significant, and significance was classified as * p <0.05, ** p <0.01, *** p <0.001, ****p<0.0001.

[0064] [Figure 26]Figure 26 shows the number of viable cells per fragment after culture of tumor tissue from head and neck cancer in G-Rex flasks with IL-2 + / - antagonists, agonists, CD28 family, reactivation treatments, or depletion treatments. See Table 44 for specific stimuli for each group. Statistics performed by two-tailed Mann-Whitney U test comparing each group to control (IL-2), p>0.05 was considered not significant, and significance was classified as * p <0.05, ** p <0.01, *** p <0.001, ****p<0.0001.

[0065] [Figure 27] Figure 27 shows the number of viable cells per fragment after culture of tumor tissue from any cancer type in G-Rex flasks with IL-2 + / - PD-1, PD-L1. See Table 43 for specific stimuli for each group. Statistics performed by two-tailed Mann-Whitney U test comparing each group to control (IL-2), p>0.05 was considered not significant, and significance was classified as * p <0.05, ** p <0.01, *** p <0.001, ****p<0.0001.

[0066] [Figure 28] Figure 28 shows the number of viable cells per fragment after culture of tumor tissue from ovarian cancer in G-Rex flasks with IL-2 + / - PD-1, PD-L1. See Table 43 for specific stimuli for each group. Statistics performed by two-tailed Mann-Whitney U test comparing each group to control (IL-2), p>0.05 was considered not significant, and significance was classified as * p <0.05, ** p <0.01, *** p <0.001, ****p<0.0001.

[0067] [Figure 29]Figure 29 shows the number of viable cells per fragment after culture of tumor tissue from melanoma in G-Rex flasks with IL-2 + / - PD-1, PD-L1. See Table 43 for specific stimuli for each group. Statistics performed by two-tailed Mann-Whitney U test comparing each group to control (IL-2), p>0.05 was considered not significant, and significance was classified as * p <0.05, ** p <0.01, *** p <0.001, ****p<0.0001.

[0068] [Diagram 30] Figure 30 shows the number of viable cells per fragment after culture of tumor tissue from lung cancer in G-Rex flasks with IL-2 + / - PD-1, PD-L1. See Table 43 for specific stimuli for each group. Statistics performed by two-tailed Mann-Whitney U test comparing each group with control (IL-2), p>0.05 was considered not significant, and significance was classified as * p <0.05, ** p <0.01, *** p <0.001, ****p<0.0001.

[0069] [Diagram 31] Figure 31 shows the number of viable cells per fragment after culture of tumor tissue from head and neck cancer in G-Rex flasks with IL-2 + / - PD-1, PD-L1. See Table 43 for specific stimuli for each group. Statistics performed by two-tailed Mann-Whitney U test comparing each group to control (IL-2), p>0.05 was considered not significant, and significance was classified as * p <0.05, ** p <0.01, *** p <0.001, ****p<0.0001.

[0070] [Diagram 32]Figure 32 shows the number of viable cells per fragment after culture of tumor tissue from colon cancer in G-Rex flasks with IL-2 + / - PD-1, PD-L1. See Table 43 for specific stimuli for each group. Statistics performed by two-tailed Mann-Whitney U test comparing each group with control (IL-2), p>0.05 was considered not significant, and significance was classified as * p <0.05, ** p <0.01, *** p <0.001, ****p<0.0001.

[0071] [Diagram 33] Figure 33 shows the number of viable cells per fragment after culture of tumor tissue from cervical cancer in G-Rex flasks with IL-2 + / - PD-1, PD-L1. See Table 43 for specific stimulants for each group. Statistics performed by two-tailed Mann-Whitney U test comparing each group with control (IL-2), p>0.05 was considered not significant, and significance was classified as * p <0.05, ** p <0.01, *** p <0.001, ****p<0.0001.

[0072] [Diagram 34] Figure 34 shows the number of viable cells per fragment after culture of tumor tissue from any cancer type in G-Rex flasks with IL-2+ / - group A: pembrolizumab, nivolumab, durvalumab, and avelumab. See Table 43 for specific stimulants for each group. Statistics performed by two-tailed Mann-Whitney U test comparing each group to control (IL-2), p>0.05 was considered not significant, and significance was classified as *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0073] [Diagram 35]Figure 35 shows the number of viable cells per fragment after culturing tumor tissue from ovarian cancer in G-Rex flasks with IL-2+ / - group A, pembrolizumab, nivolumab, durvalumab, and avelumab. See Table 43 for specific stimulants for each group. Statistics performed by two-tailed Mann-Whitney U test comparing each group to control (IL-2), p>0.05 was considered not significant, and significance was classified as *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0074] [Diagram 36] Figure 36 shows the number of viable cells per fragment after culture of tumor tissue from melanoma in G-Rex flasks with IL-2+ / - group A, pembrolizumab, nivolumab, durvalumab, and avelumab. See Table 43 for specific stimulants for each group. Statistics performed by two-tailed Mann-Whitney U test comparing each group to control (IL-2), p>0.05 was considered not significant, and significance was classified as *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0075] [Figure 37] Figure 37 shows the number of viable cells per fragment after culture of tumor tissue from head and neck cancer in G-Rex flasks with IL-2+ / - group A, pembrolizumab, nivolumab, durvalumab, and avelumab. See Table 43 for specific stimulants for each group. Statistics performed by two-tailed Mann-Whitney U test comparing each group to control (IL-2), p>0.05 was considered not significant, and significance was classified as *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0076] [Figure 38]Figure 38 shows the number of viable cells per fragment after culturing tumor tissue from cervical cancer in G-Rex flasks with IL-2+ / - group A: pembrolizumab, nivolumab, durvalumab, and avelumab. For the specific stimulants of each group, see Table 43. Statistics performed by two-tailed Mann-Whitney U test comparing each group with control (IL-2), p>0.05 was considered not significant, and significance was classified as *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0077] [Figure 39] Figure 39 shows the number of viable cells per fragment after culture of tumor tissue from any carcinoma in G-Rex flasks with IL-2+ / - Group A, Group B, or Group A+B. See Table 43 for specific stimulants for each group. Statistics performed by two-tailed Mann-Whitney U test comparing each group to control (IL-2), p>0.05 was considered not significant, and significance was classified as *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0078] [Diagram 40] Figure 40 shows the number of viable cells per fragment after culture of tumor tissue from any cancer type in G-Rex flasks with IL-2+ / -Urelumab / OKT3, ipilimumab (ipi), and pembrolizumab (pembro). Statistics performed by two-tailed Mann-Whitney U test comparing each group to the control (Urelumab / OKT3), p>0.05 was considered not significant, and significance was classified as *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0079] [Diagram 41]Figure 41 shows the number of viable cells per fragment after culture of tumor tissue from any cancer type in G-Rex flasks with IL-2+ / - urelumab / OKT3, ipilimumab (ipi), and pembrolizumab (pembro) with or without a time delay. Statistics performed by two-tailed Mann-Whitney U test, p>0.05 was considered not significant, and significance was classified as *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0080] [Diagram 42] Figure 42 shows the number of viable cells per fragment after culture of tumor tissue from any cancer type in G-Rex flasks with IL-2 + seralizumab, or IL-2 + seralizumab + ipilimumab (ipi) + pembrolizumab (pembro). Statistics performed by two-tailed Mann-Whitney U test, p>0.05 was considered not significant, and significance was classified as *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0081] [Diagram 43] Figure 43 shows the success rate of cell expansion (>50,000 cells / fragment) in all cancer types using IL-2+ / -TME-S. See Table 43 for the specific stimuli for each group.

[0082] [Diagram 44] Figure 44 shows the frequency of T cells (CD3+) in all cancer types using IL-2+ / -TME-S. For stimuli used, see Table 43. Statistics performed by two-tailed Mann-Whitney U test, p>0.05 was considered not significant.

[0083] [Diagram 45]Figure 45 shows the frequency of T cells (CD3+) in all cancer types using IL-2+ / -TME-S. See Table 43 for specific stimuli for each group. Statistics performed by two-tailed Mann-Whitney U test, p>0.05 was considered not significant, and significance was classified as *p<0.05, **p<0.01.

[0084] [Diagram 46] Figure 46 shows the number of viable T cells (CD3+) per tumor fragment in all cancer types using IL-2+ / -TME-S. See Table 43 for stimulants used. Statistics performed by two-tailed Mann-Whitney U test, p>0.05 was considered not significant, significance was classified as *p<0.05, **p<0.01, ***p<0.001.

[0085] [Figure 47] Figure 47 shows the number of viable T cells (CD3+) per tumor fragment in all cancer types using IL-2+ / -TME-S. See Table 43 for specific stimuli for each group. Statistics performed by two-tailed Mann-Whitney U test, p>0.05 was considered not significant, and significance was classified as *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0086] [Figure 48] Figure 48 shows the frequency of effector memory T cells (TEMs) in CD4+ (left) and CD8+ (right) for all cancer types using IL-2+ / -TME-S. See Table 43 for specific stimuli for each group. Statistics performed by two-tailed Mann-Whitney U test, p>0.05 was considered not significant, and significance was classified as *p<0.05.

[0087] [Figure 49]Figure 49 shows the frequency of CD8+ T cells in all cancer types using IL-2+ / -TME-S. For stimulants used, see Table 43. Statistics performed by two-tailed Mann-Whitney U test, p>0.05 was considered not significant, and significance was classified as *p<0.05, **p<0.01.

[0088] [Figure 50] Figure 50 shows the number of viable CD8+ T cells per tumor fragment in all cancer types using IL-2+ / -TME-S. See Table 43 for specific stimuli for each group. Statistics performed by two-tailed Mann-Whitney U test, p>0.05 was considered not significant, and significance was classified as *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0089] [Figure 51] Figure 51 shows the frequency of CD4+ T cells in all cancer types using IL-2+ / -TME-S. See Table 43 for specific stimuli for each group. Statistics performed by two-tailed Mann-Whitney U test, p>0.05 was considered not significant, and significance was classified as *p<0.05, **p<0.01.

[0090] [Figure 52] Figure 52 shows the frequency of NK cells in all cancer types using IL-2+ / -TME-S. See Table 43 for specific stimuli for each group. Statistics performed by two-tailed Mann-Whitney U test, p>0.05 was considered not significant, and significance was classified as *p<0.05, **p<0.01.

[0091] [Figure 53]FIG. 53 shows the frequency of T cells (CD3+) and NK (CD3-CD56+) cells in head and neck cancer using IL-2+ / - low concentration (2 μg / mL) urelumab (Ure) / OKT3 (30 ng / mL) or medium concentration (10 μg / mL) urelumab / OKT3 (30 ng / mL), as well as pembrolizumab (Pembro), ipilimumab (Ipi).

[0092] [Figure 54] FIG. 54 shows the frequency of CD8+ T cells in head and neck cancer using IL-2+ / - low concentration (2 μg / mL) urelumab (Ure) / OKT3 (30 ng / mL) or medium concentration (10 μg / mL) urelumab / OKT3 (30 ng / mL), as well as pembrolizumab (Pembro) and ipilimumab (Ipi).

[0093] [Figure 55] Figure 55 shows the frequency of CD3+, NK, CD8+, and CD4+ cells in all cancer types with IL-2+ / -TME-S without (white bars) and with (black bars) a time delay. See Table 43 for specific stimuli for each group. Statistics performed by two-tailed Mann-Whitney U test, p>0.05 is considered not significant.

[0094] [Figure 56] Figure 56 shows the frequency of CD4+ surface LAG-3 cells (white bars) or CD8+ cells (black bars) in all cancer types using IL-2+ / -TME-S. See Table 43 for specific stimuli for each group. Statistics performed by two-tailed Mann-Whitney U test, p>0.05 was considered not significant, and significance was classified as *p<0.05.

[0095] [Figure 57]Figure 57 shows the frequency of CD4+ surface LAG-3 cells or CD8+ cells in all cancer types with IL-2+ / -TME-S without (white bars) and with (black bars) a time delay. For specific stimuli for each group, see Table 43. Statistics performed by two-tailed Mann-Whitney U test, p>0.05 was considered not significant.

[0096] [Figure 58] Figure 58 shows the frequency of CD8+ surface CD28 cells in all carcinomas using IL-2+ / - TME-S. See Table 43 for specific stimuli for each group. Statistics performed by two-tailed Mann-Whitney U test, p>0.05 was considered not significant. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0097] The present invention aims to reactivate exhausted tumor infiltrating lymphocytes (TILs) ex vivo, for example by co-culturing excised TILs with tumor fragments with checkpoint inhibitors, stimulating TILs with other interleukins known to reverse T cell exhaustion, and / or inhibiting the effects of regulatory T cell secreted factors (such as IL-10), thereby creating a favorable TME capable of expanding exhausted T cells faster and to greater numbers than currently established TIL expansion techniques.

[0098] This approach may be advantageous over systemic therapy because in vitro stimulation can be performed using much higher dose levels than are tolerated in vivo. As an example, the TIL procedure can utilize IL-2 at concentrations of 3-6,000 IU per mL, which is 5-10 times higher than the dose recommended systemically.

[0099] Furthermore, because T cells with higher affinity for tumor antigens may have an increased tendency to become exhausted, targeted ex vivo reactivation may help expand high-affinity T cell clones that can more aggressively target the tumors in which they reside, ultimately improving the clinical outcome of this new approach to TIL therapy.

[0100] Accordingly, the present invention relates to a method of expanding tumor infiltrating lymphocytes (TILs) into a therapeutic population of TILs to promote regression of cancer in a mammal, the method comprising the steps of: (a) obtaining a first population of TILs from a tumor excised from the mammal and culturing the autologous T cells; (b) performing a first expansion by culturing the first population of TILs in a cell culture medium comprising IL-2 and one or more TME stimulants to generate a second population of TILs; (c) performing a second expansion by supplementing the cell culture medium of the second population of TILs with additional IL-2, anti-CD3 antibody, and antigen presenting cells (APCs) to generate a third population of TILs, which is a therapeutic population; and (d) administering the therapeutic population of T cells to the mammal following administration of non-myeloablative lymphodepleting chemotherapy, wherein administration of the T cells to the mammal promotes regression of cancer in the mammal.

[0101] "Tumor infiltrating lymphocytes" or "TILs" as used herein refer to a population of cells originally obtained as white blood cells that have left the subject's bloodstream and migrated into a tumor. TILs include, but are not limited to, CD8+ cytotoxic T cells (lymphocytes), Th1 and Th17 CD4+ T cells (CD4+ helper cells), natural killer cells, dendritic cells, and M1 macrophages. TILs include both primary and secondary TILs. "Primary TILs" are those obtained from a patient tissue sample as outlined herein (sometimes referred to as "freshly harvested"), and "secondary TILs" are any TIL cell population that has been expanded or propagated as described herein. TILs may generally be defined biochemically using cell surface markers or functionally by their ability to infiltrate tumors and effect therapy. TILs may generally be classified by expression of one or more of the following biomarkers: CD4, CD8, TCR ab, CD27, CD28, CD56, CCR7, CD45Ra, CD95, PD-1, and CD25. Additionally and alternatively, TILs may be functionally defined by their ability to infiltrate solid tumors upon reintroduction into a patient. TILs may be further characterized by potency, e.g., TILs may be considered potent if their release of interferon (IFN) is greater than about 50 pg / mL, greater than about 100 pg / mL, greater than about 150 pg / mL, or greater than about 200 pg / mL.

[0102] A further aspect of the invention relates to a method of treating a subject suffering from cancer comprising administering expanded tumor infiltrating lymphocytes (TILs), the method comprising the steps of: (a) culturing autologous T cells by harvesting a first population of TILs from a tumor resected from the mammal; (b) performing a first expansion by culturing the first population of TILs in a cell culture medium comprising IL-2 and one or more TME stimulants to generate a second population of TILs; (c) performing a second expansion by supplementing the cell culture medium of the second population of TILs with additional IL-2, anti-CD3 antibody, and antigen presenting cells (APCs) to generate a third population of TILs, which is a therapeutic population; and (d) administering the therapeutic population of T cells to the mammal after administration of non-myeloablative lymphodepleting chemotherapy, wherein administration of the T cells to the mammal promotes regression of the cancer in the mammal.

[0103] The terms "treatment", "treating", "treat" and the like refer to obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic, in that it completely or partially prevents the disease or its symptoms, and / or it may be therapeutic, in that it partially or completely cures the disease and / or the deleterious effects caused by the disease. As used herein, "treatment" includes any treatment of disease in a mammal, particularly a human, including (a) preventing the disease from occurring in a subject who may be predisposed to the disease but has not yet been diagnosed; (b) inhibiting the disease, i.e., arresting its onset or progression; and (c) relieving the disease, i.e., causing regression of the disease and / or relieving one or more symptoms of the disease. "Treatment" is also meant to encompass the delivery of an agent to provide a pharmacological effect even in the absence of disease or pathology. For example, "treatment" encompasses the delivery of a composition capable of eliciting an immune response or conferring immunity in the absence of disease symptoms, e.g., in the case of a vaccine.

[0104] The term "anti-CD3 antibody" refers to an antibody or variant thereof, e.g., a monoclonal antibody, including a human, humanized, chimeric, or murine antibody, directed against the CD3 receptor in the T cell antigen receptor of mature T cells. Anti-CD3 antibodies include OKT3, also known as muromonab. Anti-CD3 antibodies also include UHCT1 clones, also known as T3 and CD3e. Other anti-CD3 antibodies include, for example, otelixizumab, teplizumab, and vicilizumab. In one embodiment, the cell culture medium includes an OKT3 antibody. In some embodiments, the cell culture medium includes about 30 ng / mL of an OKT3 antibody. In one embodiment, the cell culture medium contains about 0.1 ng / mL, about 0.5 ng / mL, about 1 ng / mL, about 2.5 ng / mL, about 5 ng / mL, about 7.5 ng / mL, about 10 ng / mL, about 15 ng / mL, about 20 ng / mL, about 25 ng / mL, about 30 ng / mL, about 35 ng / mL, about 40 ng / mL, about 50 ng / mL, about 60 ng / mL, about 70 ng / mL, about 80 ng / mL, about 90 ng / mL, about 100 ng / mL, about 200 ng / mL, about 500 ng / mL, and about 1 pg / mL of OKT3 antibody. In one embodiment, the cell culture medium comprises 0.1 ng / mL to 1 ng / mL, 1 ng / mL to 5 ng / mL, 5 ng / mL to 10 ng / mL, 10 ng / mL to 20 ng / mL, 20 ng / mL to 30 ng / mL, 30 ng / mL to 40 ng / mL, 40 ng / mL to 50 ng / mL, and 50 ng / mL to 100 ng / mL of OKT3 antibody. In some embodiments, the cell culture medium does not comprise OKT3 antibody.

[0105] The term "IL-2" (also referred to herein as "IL2") refers to the T-cell growth factor known as interleukin-2, and includes all forms of IL-2, including human and mammalian forms, conservative amino acid substitutions, glycoforms, epigenetic preparations, and variants thereof.

[0106] After preparation of the tumor fragments, the resulting cells (i.e., fragments) are cultured in medium containing IL-2 under conditions that favor the growth of TILs over tumor and other cells. In some embodiments, tumor digests are cultured in 2 mL wells in medium containing inactivated human AB serum containing 6000 IU / mL IL-2 (or optionally in the presence of an aAPC cell population as outlined herein). This primary cell population is cultured over several days, usually 6-14 days, to produce a large TIL population, usually about 1×10 6 ~1×10 8 In some embodiments, the growth medium during the first expansion contains IL-2 or a variant thereof. In some embodiments, the IL is recombinant human IL-2 (rhIL-2). In some embodiments, the IL-2 stock solution contains 20-30×10 for a 1 mg vial. 6 IU / mg specific activity. In some embodiments, the IL-2 stock solution contains 20×10 6 IU / mg specific activity. In some embodiments, the IL-2 stock solution contains 25×10 6 IU / mg specific activity. In some embodiments, the IL-2 stock solution contains 30×10 6 IU / mg specific activity. In some embodiments, the IL-2 stock solution has a specific activity of 4-8×10 6 IU / mg of IL-2. In some embodiments, the IL-2 stock solution contains 5-7×10 6 IU / mg of IL-2. In some embodiments, the IL-2 stock solution contains 6×10 6IU / mg of IL-2. In some embodiments, the IL-2 stock solution is prepared as described in the Examples. In some embodiments, the first expansion culture medium comprises about 10,000 IU / mL of IL-2, about 9,000 IU / mL of IL-2, about 8,000 IU / mL of IL-2, about 7,000 IU / mL of IL-2, about 6,000 IU / mL of IL-2, or about 5,000 IU / mL of IL-2. In some embodiments, the first expansion culture medium comprises about 9,000 IU / mL to about 5,000 IU / mL of IL-2. In some embodiments, the first expansion culture medium comprises about 8,000 IU / mL to about 6,000 IU / mL of IL-2. In some embodiments, the first expansion culture medium comprises about 7,000 IU / mL to about 6,000 IU / mL of IL-2. In some embodiments, the first expansion culture medium comprises about 6,000 IU / mL of IL-2. In one embodiment, the cell culture medium further comprises IL-2. In some embodiments, the cell culture medium comprises about 3,000 IU / mL of IL-2. In one embodiment, the cell culture medium further comprises IL-2. In a preferred embodiment, the cell culture medium comprises about 3,000 IU / mL of IL-2. In one embodiment, the cell culture medium comprises about 1,000 IU / mL, about 1,500 IU / mL, about 2,000 IU / mL, about 2,500 IU / mL, about 3,000 IU / mL, about 3,500 IU / mL, about 4,000 IU / mL, about 4,500 IU / mL, about 5,000 IU / mL, about 5,500 IU / mL, about 6,000 IU / mL, about 6,500 IU / mL, about 7,000 IU / mL, about 7,500 IU / mL, or about 8,000 IU / mL of IL-2. In one embodiment, the cell culture medium comprises 1,000-2,000 IU / mL, 2,000-3,000 IU / mL, 3,000-4,000 IU / mL, 4,000-5,000 IU / mL, 5,000-6,000 IU / mL, 6,000-7,000 IU / mL, 7,000-8,000 IU / mL, or about 8,000 IU / mL of IL-2.

[0107] IL-4, IL-7, IL-15 and / or IL-21 may also be added to steps (b) and / or (c) of the method. The term "IL-4" (also referred to herein as "IL4") refers to the cytokine known as interleukin-4, which is produced by Th2 T cells, as well as by eosinophils, basophils, and mast cells. IL-4 regulates the differentiation of naive helper T cells (ThO cells) to Th2 T cells. The term "IL-7" (also referred to herein as "IL7") refers to the glycosylated tissue-derived cytokine known as interleukin-7, which may be obtained from stromal and epithelial cells and dendritic cells. The term "IL-15" (also referred to herein as "IL15") refers to the T cell growth factor known as interleukin-15, and includes all forms of IL-2, including human and mammalian forms, conservative amino acid substitutions, glycoforms, post-development preparations, and variants thereof. The term "IL-21" (also referred to herein as "IL21") refers to the pleiotropic cytokine protein known as interleukin-21, and includes all forms of IL-21, including human and mammalian forms, conservative amino acid substitutions, glycoforms, epigenetic formulations, and variants thereof.

[0108] Another aspect of the invention relates to a method of expanding tumor infiltrating lymphocytes (TILs) into a therapeutic population of TILs, comprising the steps of: (a) culturing autologous T cells by harvesting a first population of TILs from a tumor excised from a mammal; (b) performing a first expansion by culturing the first population of TILs in a cell culture medium comprising IL-2 and one or more TME stimulants to generate a second population of TILs; and (c) performing a second expansion by supplementing the cell culture medium of the second population of TILs with additional IL-2, anti-CD3 antibody, and antigen presenting cells (APCs) to generate a third population of TILs, which is a therapeutic population.

[0109] Experiments have found that lymphodepletion prior to adoptive transfer of tumor-specific T lymphocytes plays an important role in enhancing therapeutic efficacy by eliminating regulatory T cells and competing elements of the immune system ("cytokine sinks"). Thus, some embodiments of the present invention utilize a lymphodepletion step (also referred to as "immunosuppressive conditioning") in patients prior to introducing the TILs of the present invention.

[0110] The method of the present invention from step (a) to step (c) may be carried out in a closed system. The term "closed system" refers to a system that is closed to the outside environment. Any closed system suitable for cell culture methods may be used in the method of the present invention. Closed systems include, but are not limited to, closed G containers. Once tumor parts are added to the closed system, the system is not opened to the outside environment until the TILs are ready to be administered to the patient.

[0111] The term "TME stimulant" refers to a substance (or agent) that has the ability to create a favorable microenvironment within a tumor, in which exhausted T cells can be reactivated to multiply and restore their anti-tumor function. Thus, in one or more embodiments, the one or more TME stimulants are selected from the group consisting of: (x) one or more substances capable of antagonizing and / or inhibiting receptors expressed on the surface of T cells (or their ligands) known to cause downregulation, deactivation, and / or exhaustion of T cells; (y) one or more substances capable of stimulating and / or promoting receptors expressed on the surface of T cells known to cause upregulation, activation, and / or reactivation of T cells; (z) one or more substances capable of antagonizing and / or inhibiting soluble molecules and cytokines and their receptors known to cause downregulation, deactivation, and / or exhaustion of T cells; (v) one or more substances capable of downregulating and / or exhausting regulatory T cells, thereby promoting the proliferation of ex vivo effector T cells; and (w) one or more substances from group (x), (y), (z) and / or (v). Group (w) may be one, two, or three of the substances from (x), (y), (z), and / or (v). In one or more embodiments, (w) is one or two of the materials from (x). In one or more embodiments, (w) is one or two of the materials from (y). In one or more embodiments, (w) is one or two of the materials from (z). In one or more embodiments, (w) is one or two of the materials from (v). (w) may be any combination of the materials in Table 1 listed in Tables 2-41 and 43-44.

[0112] They may be added at step (b) and / or step (c) of the method, and if they are only required for initial growth, may be removed after 2, 4, 6 or more days during growth. They may be removed by washing the cell culture. Individual TME stimulants may be added together or over time, i.e., at 1 day intervals, or at 2, 3, 4, 5, 6 or 7 day intervals.

[0113] In one or more embodiments, the one or more TME stimulants are one or more check point inhibitors or inhibitors of their ligands, such as, for example, anti-PD1, anti-PD-L1, anti-PD-L2, anti-CTLA-4, anti-LAG3, anti-A2AR, anti-B7-H3, anti-B7-H4, anti-BTLA, anti-IDO, anti-HVEM, anti-IDO, anti-TDO, anti-KIR, anti-NOX2, anti-TIM3, anti-galectin 9, anti-VISTA, anti-SIGLEC7 / 9, and optionally one or more check point inhibitors or inhibitors of their ligands are also added to the second expansion.

[0114] In one or more embodiments, substances capable of antagonizing and / or inhibiting receptors expressed on the surface of T cells (or their ligands) that are known to cause T cell downregulation, inactivation, and / or exhaustion are selected from the group consisting of: A: substances acting through the PD-1 receptor on the surface of T cells; B: substances acting through the CTLA-4 receptor on the surface of T cells; C: substances acting through the LAG-3 receptor on the surface of T cells; D: substances acting through the TIGIT / CD226 receptor on the surface of T cells; E: substances acting through the KIR receptor on T cells; F: substances acting through the TIM-3 receptor on T cells; G: substances acting through the BTLA receptor on the surface of T cells; and H: substances acting through the A2aR receptor on the surface of T cells. Of course, the definition of a substance acting through a given receptor may also encompass the ligand of the same receptor. This means that, for example, in the case of the PD-1 receptor, substances targeting PD-L1 or PD-L2 may also be encompassed. Group A may therefore include agents that act through the PD-1 receptor on the surface of T cells as well as its ligands.

[0115] In one or more embodiments, the group A agent is selected from one or more of the group consisting of pembrolizumab, nivolumab, cemiplimab, sym021, atezolizumab, avelumab, and durvalumab. In one or more embodiments, the group A agent is pembrolizumab. In one or more embodiments, the group A agent is nivolumab. In one or more embodiments, the group A agent is cemiplimab. In one or more embodiments, the group A agent is sym021. In one or more embodiments, the group A agent is atezolizumab. In one or more embodiments, the group A agent is avelumab. In one or more embodiments, the group A agent is durvalumab.

[0116] In one or more embodiments, the group B agent is selected from one or more of the group consisting of ipilimumab and tremelimumab. In one or more embodiments, the group B agent is ipilimumab. In one or more embodiments, the group B agent is tremelimumab. In one or more embodiments, the group C agent is selected from one or more of the group consisting of leratolimab, eftiragimob alfa, and sym022. In one or more embodiments, the group D agent is tiragolumab. In one or more embodiments, the group E agent is lirilumab. In one or more embodiments, the group F agent is sym023. In one or more embodiments, the group G agent is 40E4 and PJ196.

[0117] In one or more embodiments, the substance capable of stimulating and / or promoting a receptor expressed on the surface of a T cell that is known to cause upregulation, activation, and / or reactivation of T cells is selected from the group consisting of: I: a substance that acts through the OX40 / CD137 receptor on the surface of a T cell; J: a substance that acts through the 4-1BB / CD137 receptor on the surface of a T cell; K: a substance that acts through the CD28 receptor on the surface of a T cell; L: a substance that acts through the ICOS receptor on the surface of a T cell; M: a substance that acts through the GITR receptor on the surface of a T cell; N: a substance that acts through the CD40L receptor on the surface of a T cell; and O: a substance that acts through the CD27 receptor on the surface of a T cell.

[0118] In one or more embodiments, the group J agent is selected from one or more of the group consisting of urelumab and utomilumab. In one or more embodiments, the group J agent is urelumab. In one or more embodiments, the group J agent is utomilumab. The group J agent may be used in combination with an anti-CD3 agent such as OKT-3. Thus, one combination may be urelumab and OKT-3 (urelumab / OKT-3). Another combination may be utomilumab and OKT-3 (utomilumab / OKT-3). In one or more embodiments, the group K agent is celalizumab. In one or more embodiments, the group O agent is valilumab.

[0119] In one or more embodiments, one or more agents of group A may be combined with one or more agents of group B. In one or more embodiments, one or more agents of group A may be combined with one or more agents of group B and one or more agents of group J. These combinations have been shown to be effective in the examples of the present disclosure. This means that one or more agents of group A selected from one or more agents of the group consisting of pembrolizumab, nivolumab, cemiplimab, sym021, atezolizumab, avelumab may be combined with one or more agents of group B selected from one or more agents of the group consisting of ipilimumab and tremelimumab. These agents may then be combined with one or more agents of group J selected from one or more agents of the group consisting of urelumab and utomirumab. Agents of group J may be used in combination with anti-CD3 agents such as OKT-3. Thus, one combination may be one or more agents of group A selected from one or more from the group consisting of pembrolizumab, nivolumab, cemiplimab, sym021, atezolizumab, avelumab, in combination with ipilimumab from group B and urelumab from group J. A particular choice may be pembrolizumab in combination with ipilimumab from group B and urelumab from group J, with or without an anti-CD3 agent such as OKT-3.

[0120] In one or more embodiments, the substances capable of antagonizing and / or inhibiting soluble molecules and cytokines and their receptors known to cause downregulation, inactivation, and / or exhaustion of T cells are selected from the group consisting of P: substances acting through IDO1 / 2 receptors on the surface of T cells, Q: substances acting through TGFβ receptors on the surface of T cells, R: substances acting through IL-10 receptors on the surface of T cells, and S: substances acting through IL-35 receptors on the surface of T cells.

[0121] In one or more embodiments, the group P agent is epacedostat. In one or more embodiments, the group Q agent is linrodostat. In one or more embodiments, the group R agent is galunisertib.

[0122] In one or more embodiments, the agent capable of downregulating and / or depleting regulatory T cells and thereby promoting ex vivo effector T cell proliferation is selected from the group consisting of: T: cyclophosphamide, U: TKI, V: agents acting through αCD25, and X: IL2 / diphtheria toxin fusion.

[0123] Groups A to X listed in Table 1 may be used in combination with multiple substances as shown in Tables 2 to 44. Thus, in one or more embodiments, in the first expansion, IL2 is used in any combination with any substance (see Table 1), i.e. in step (b) of the method of the invention, in any combination listed in Tables 2 to 44.

[0124] In one or more embodiments, the group U agent is sunitinib. In one or more embodiments, the group V agent is selected from one or more of the group consisting of sorafenib, imatinib, and daclizumab. In one or more embodiments, the group X agent is denileukin diftitox.

[0125] Example 4 shows that the success of ex vivo TIL expansion was increased when TIL cultures were initiated when TME stimulants were added to the culture medium as described in Example 2. Example 5 shows that the culture time of TILs was reduced when TIL cultures were initiated when TME stimulants were added to the culture medium as performed in Example 2. Example 6, performed as described in Example 2, shows that TIL yields were increased when TIL cultures from various tumor types were initiated when TME stimulants were added to the culture medium at various concentrations.

[0126] Example 9 shown in Figure 27 shows that the addition of TME stimulants to the standard juvenile TIL production procedure as carried out in Example 2 significantly enhanced TIL proliferation, thereby generating a higher number of viable cells per tumor fragment by antagonizing the receptor expressed on the surface of T cells or its ligand (PD-L1) expressed on the surface of tumor cells and other cells in the tumor microenvironment. In Figures 28-33, the effect of adding TME stimulants to primary TIL cultures from the PD-1 or PD-L1 groups versus the standard TIL production procedure was observed in ovarian cancer, melanoma, lung cancer, head and neck cancer, colon cancer, and cervical cancer, respectively. Although not significant in all conditions, this effect showed a similar trend in various cancers as shown by the example of PD-1 / PD-L1 in all tumors in Figure 27. This example showed that targeting either the receptor PD-1 or its ligand PD-L1 is interchangeable and may produce similar effects.

[0127] In Figures 35-38, the effect of adding inhibitors from group A by various manufacturers to primary TIL cultures was observed in ovarian cancer, melanoma, head and neck cancer, and cervical cancer, respectively. As an example of this effect in Figure 34, similar trends were observed in various cancers as an example in all tumors, indicating that similar effects may be obtained using TME stimulants from various manufacturers.

[0128] Figure 39 shows that the addition of TME stimulants from group A (PD-1 inhibitors or their ligands), group B (CTLA-4 inhibitors or their ligands), or both groups A and B significantly increased TIL proliferation compared to standard juvenile TIL production procedures. The combined use of TME stimulants from group A and group B tended to further improve TIL proliferation rates. Figure 40 shows that 4-1BB stimulants and anti-CD3, i.e., urelumab / OKT3 (group J), alone or in combination with the CTLA-4 inhibitor ipilimumab (group B), or in combination with the PD-1 inhibitor pembrolizumab (group A), or in combination with both ipilimumab and pembrolizumab, all three combinations showed very strong TIL proliferation per tumor fragment from various cancers in viable cells. Thus, one embodiment of the present invention relates to the use of one or more TME stimulants from group A for the methods of the present invention. One embodiment of the invention relates to the use of one or more TME stimulants from group B for the methods of the invention. One embodiment of the invention relates to the use of one or more TME stimulants from group J for the methods of the invention. A further embodiment of the invention relates to a combination in which one or more substances from groups A and B are used together as TME stimulants. A further embodiment of the invention relates to a combination in which one or more substances from groups A, B, and J are used together as TME stimulants.

[0129] In Figure 41, we investigated the effect of a time delay in the addition of urelumab / OKT3 to the process of producing young TIL cultures. Here, we compared the triple combination of urelumab / OKT3, ipilimumab, and pembrolizumab added to early young TIL cultures on day 0 (left side of figure) with a time delay condition where ipilimumab and pembrolizumab were added and urelumab / OKT3 was added after waiting 2 days (right side of figure). There was no significant difference between the two conditions, and very strong TIL proliferation was observed in both conditions. In Figure 42, we investigated the effect of adding ceralizumab alone (left side of figure) or in combination with both ipilimumab and pembrolizumab to early young TIL cultures. Although not significant, the triple combination tended to induce a faster proliferation rate in young TILs compared to ceralizumab alone.

[0130] Example 12 shows that the successful rate of in vitro TIL expansion was increased when TIL culture was initiated when TME stimulating agents were added to the culture medium, either alone or in combination. Thus, one embodiment of the present invention relates to the use of a TME stimulating agent in a method according to the present invention, which results in increased in vitro expansion compared to the absence of one or more TME stimulating agents. The methods of the present invention are performed in vitro and not on or in vivo. They refer to the expansion of patient cells in a laboratory and therefore do not require a physician to produce this.

[0131] Example 13 shows that the addition of a TME stimulatory agent to the processing of young TILs provides a novel improvement to existing standard TIL manufacturing procedures that allows for the production of a TIL product that comprises an increased frequency of T cells and an increased number of viable T cells. One embodiment of the invention relates to the use of a TME stimulatory agent in a method according to the invention to produce a TIL product that comprises an increased frequency of T cells and an increased number of viable T cells compared to the absence of one or more TME stimulatory agents.

[0132] Example 14 shows that the addition of a TME stimulatory agent to the manufacturing process of young TILs provides a novel improvement to existing standard TIL procedures that allows for the generation of a TIL product that contains a comparable frequency of effector memory T cells. One embodiment of the invention relates to the use of a TME stimulatory agent in a method according to the invention to generate a TIL product that contains a comparable frequency of effector memory T cells compared to when one or more TME stimulatory agents are not used.

[0133] Example 15 shows that the addition of TME stimulatory agents, alone and in combination, to the manufacturing process of young TILs provides a novel improvement to existing standard TIL procedures that allows for the generation of a TIL product that includes an increased frequency of CD8+ T cells. Thus, one embodiment of the invention relates to the use of a TME stimulatory agent in a method according to the invention, thereby generating a TIL product that has an increased frequency of CD8+ T cells compared to when one or more TME stimulatory agents are not used.

[0134] Example 16 shows that the addition of a TME stimulatory agent to the manufacturing process of young TILs provides a novel improvement to existing standard TIL procedures that allows for the generation of a TIL product that comprises a reduced frequency of CD4+ T cells. One embodiment of the invention relates to the use of a TME stimulatory agent in a method according to the invention to generate a TIL product that comprises a reduced frequency of CD4+ T cells compared to when one or more TME stimulatory agents are not used.

[0135] Example 17 shows that the addition of a TME stimulatory agent to the manufacturing process of young TILs provides a novel improvement to existing standard TIL procedures that allows for the generation of a TIL product that comprises a reduced frequency of NK cells. Thus, one embodiment of the invention relates to the use of a TME stimulatory agent in a method according to the invention to generate a TIL product that comprises a reduced frequency of NK cells compared to when one or more TME stimulatory agents are not used.

[0136] Example 18 shows that the addition of a TME stimulatory agent to the processing of young TILs provides a novel improvement to existing standard TIL procedures that allows for the generation of a TIL product that comprises a reduced frequency of NK cells and an increased frequency of CD8+ T cells. Thus, one embodiment of the invention relates to the use of a TME stimulatory agent in a method according to the invention to generate a TIL product that comprises a reduced frequency of NK cells and an increased frequency of CD8+ T cells compared to the absence of one or more TME stimulatory agents.

[0137] Example 19 shows that the addition of a TME stimulatory agent with a time delay to the processing of young TILs provides a novel improvement to existing standard TIL procedures that allows for the generation of a TIL product that includes increased total frequencies of T cells and CD8+ cells and decreased frequencies of NK cells and CD4+ T cells. One embodiment of the invention relates to the use of a TME stimulatory agent in a method according to the invention to generate a TIL product that includes increased total frequencies of T cells and CD8+ cells and decreased frequencies of NK cells and CD4+ T cells compared to the absence of one or more TME stimulatory agents.

[0138] Example 20 shows that the addition of a TME stimulator to the process of producing young TILs provides a novel improvement to the existing standard TIL procedure that allows the generation of a TIL product that includes an increased frequency of tumor-specific LAG-3+ T cells. Since LAG-3 is a marker of T cell exhaustion and T cells with higher affinity for tumor antigens are known to generally have an increased tendency to be exhausted, clonal expansion of CD8+LAG-3+ T cells may result in a higher proportion of tumor-reactive T cells, which may improve the clinical outcome of this novel approach to TIL therapy. Thus, one embodiment of the present invention relates to the use of a TME stimulator in the method according to the present invention to increase the frequency of tumor-specific LAG-3+ T cells compared to the absence of one or more TME stimulators.

[0139] Example 21 shows that the addition of a TME stimulatory agent to the process for producing young TILs provides a novel improvement to existing standard TIL procedures that allows for the generation of a TIL product that includes an increased frequency of CD8+ T cells with a more immature phenotype that expresses CD28. One embodiment of the invention relates to the use of a TME stimulatory agent in a method according to the invention to increase the frequency of CD8+ T cells with a more immature phenotype that expresses CD28 compared to the absence of one or more TME stimulatory agents.

[0140] Using the techniques presented herein allows for much higher dosage concentrations than are tolerated in vivo. This concentration may therefore be at least 2 times higher than the maximum tolerated dose tolerated in vivo. This concentration may even be higher, for example 5 to 10 times higher than the maximum tolerated dose tolerated in vivo. Thus, in one or more embodiments, the concentration of the substance is 0.1 μg / mL to 300 μg / mL. This concentration may also be 1 μg / mL to 100 μg / mL. This concentration may also be 10 μg / mL to 100 μg / mL. This concentration may also be 1 μg / mL to 10 μg / mL.

[0141] In one or more embodiments, the therapeutic population of T cells is used to treat a cancer type selected from the group consisting of: 1: solid tumors, 2: ICI naive tumors, 3: MSI-H tumors, 4: hematological tumors, 5: hypermutated tumors (e.g., POL-E and POL-D mutated tumors), and 6: virally induced tumors.

[0142] In one or more embodiments, the therapeutic population of T cells is used to treat a cancer type selected from the group consisting of breast cancer, renal cell carcinoma, bladder cancer, melanoma, cervical cancer, gastric cancer, colon cancer, lung cancer, head and neck cancer, ovarian cancer, Hodgkin's lymphoma, pancreatic cancer, liver cancer, and sarcoma.

[0143] In one or more embodiments, the therapeutic population of T cells is used to treat breast cancer. In one or more embodiments, the therapeutic population of T cells is used to treat renal cell carcinoma. In one or more embodiments, the therapeutic population of T cells is used to treat bladder cancer. In one or more embodiments, the therapeutic population of T cells is used to treat melanoma. In one or more embodiments, the therapeutic population of T cells is used to treat cervical cancer. In one or more embodiments, the therapeutic population of T cells is used to treat gastric cancer. In one or more embodiments, the therapeutic population of T cells is used to treat colorectal cancer. In one or more embodiments, the therapeutic population of T cells is used to treat lung cancer. In one or more embodiments, the therapeutic population of T cells is used to treat head and neck cancer. In one or more embodiments, the therapeutic population of T cells is used to treat ovarian cancer. In one or more embodiments, the therapeutic population of T cells is used to treat Hodgkin's lymphoma. In one or more embodiments, the therapeutic population of T cells is used to treat pancreatic cancer. In one or more embodiments, the therapeutic population of T cells is used to treat liver cancer. In one or more embodiments, the therapeutic population of T cells is used to treat sarcoma.

[0144] In one or more embodiments, steps (a)-(c) or (d) are performed within a period of about 20 to about 45 days. In one or more embodiments, steps (a)-(c) or (d) are performed within a period of about 20 to about 40 days. In one or more embodiments, steps (a)-(c) or (d) are performed within a period of about 25 to about 40 days. In one or more embodiments, steps (a)-(c) or (d) are performed within a period of about 30 to about 40 days. In one or more embodiments, steps (a)-(b) are performed within a period of about 10 to about 28 days. In one or more embodiments, steps (a)-(b) are performed within a period of about 10 to about 20 days.

[0145] In some embodiments, the first TIL expansion (step (a)) may be allowed to continue for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days. In some embodiments, the first TIL expansion may be allowed to continue for 1-14 days. In some embodiments, the first TIL expansion may be allowed to continue for 2-14 days. In some embodiments, the first TIL expansion may be allowed to continue for 3-14 days. In some embodiments, the first TIL expansion may be allowed to continue for 4-14 days. In some embodiments, the first TIL expansion may be allowed to continue for 5-14 days. In some embodiments, the first TIL expansion may be allowed to continue for 6-14 days. In some embodiments, the first TIL expansion may be allowed to continue for 7-14 days. In some embodiments, the first TIL expansion may be allowed to continue for 8-14 days. In some embodiments, the first TIL expansion may be allowed to continue for 9-14 days. In some embodiments, the first TIL expansion may be allowed to continue for 10-14 days. In some embodiments, the first TIL expansion may be allowed to continue for 11-14 days. In some embodiments, the first TIL expansion may be allowed to continue for 12-14 days. In some embodiments, the first TIL expansion may be allowed to continue for 13-14 days. In some embodiments, the first TIL expansion may be allowed to continue for 14 days. In some embodiments, the first TIL expansion may be allowed to continue for 1-11 days. In some embodiments, the first TIL expansion may be allowed to continue for 2-11 days. In some embodiments, the first TIL expansion may be allowed to continue for 3-11 days. In some embodiments, the first TIL expansion may be allowed to continue for 4-11 days. In some embodiments, the first TIL expansion may be allowed to continue for 5-11 days. In some embodiments, the first TIL expansion may be allowed to continue for 6-11 days. In some embodiments, the first TIL expansion may be allowed to continue for 7-11 days. In some embodiments, the first TIL expansion may be allowed to continue for 8-11 days. In some embodiments, the first TIL expansion may be allowed to continue for 9-11 days. In some embodiments, the first TIL expansion may be allowed to continue for 10-11 days. In some embodiments, the first TIL expansion may be allowed to continue for 11 days.

[0146] In one or more embodiments, step (b) is performed within a period of about 6 days to about 18 days. In one or more embodiments, step (b) is performed within a period of about 7 days to about 14 days. In one or more embodiments, step (b) is performed within a period of about 7 days to about 10 days. In one or more embodiments, step (b) is performed within a period of about 6 days to about 12 days.

[0147] In one or more embodiments, step (c) is performed within a period of about 12 to about 18 days. In one or more embodiments, step (c) is performed within a period of about 10 to about 28 days. In one or more embodiments, step (c) is performed within a period of about 10 to about 20 days. In one or more embodiments, step (c) is performed within a period of about 12 to about 18 days.

[0148] In some embodiments, the transition from the first expansion to the second expansion occurs 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days from the time fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 1 day to 14 days from the time fragmentation occurs. In some embodiments, the first TIL expansion may be allowed to continue for 2 days to 14 days. In some embodiments, the transition from the first expansion to the second expansion occurs 3 days to 14 days from the time fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 4 days to 14 days from the time fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 5 days to 14 days from the time fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 6 days to 14 days from the time fragmentation occurs. In some embodiments, the transition from the first growth to the second growth occurs 7 to 14 days after the fragmentation occurs. In some embodiments, the transition from the first growth to the second growth occurs 8 to 14 days after the fragmentation occurs. In some embodiments, the transition from the first growth to the second growth occurs 9 to 14 days after the fragmentation occurs. In some embodiments, the transition from the first growth to the second growth occurs 10 to 14 days after the fragmentation occurs. In some embodiments, the transition from the first growth to the second growth occurs 11 to 14 days after the fragmentation occurs. In some embodiments, the transition from the first growth to the second growth occurs 12 to 14 days after the fragmentation occurs. In some embodiments, the transition from the first growth to the second growth occurs 13 to 14 days after the fragmentation occurs. In some embodiments, the transition from the first growth to the second growth occurs 14 days after the fragmentation occurs. In some embodiments, the transition from the first growth to the second growth occurs 1-11 days after fragmentation occurs. In some embodiments, the transition from the first growth to the second growth occurs 2-11 days after fragmentation occurs. In some embodiments, the transition from the first growth to the second growth occurs 3-11 days after fragmentation occurs.In some embodiments, the transition from the first growth to the second growth occurs 4 to 11 days after the fragmentation occurs. In some embodiments, the transition from the first growth to the second growth occurs 5 to 11 days after the fragmentation occurs. In some embodiments, the transition from the first growth to the second growth occurs 6 to 11 days after the fragmentation occurs. In some embodiments, the transition from the first growth to the second growth occurs 7 to 11 days after the fragmentation occurs. In some embodiments, the transition from the first growth to the second growth occurs 8 to 11 days after the fragmentation occurs. In some embodiments, the transition from the first growth to the second growth occurs 9 to 11 days after the fragmentation occurs. In some embodiments, the transition from the first growth to the second growth occurs 10 to 11 days after the fragmentation occurs. In some embodiments, the transition from the first growth to the second growth occurs 11 days after the fragmentation occurs.

[0149] One of the key findings is that more TILs can be delivered faster. This is valuable because it ensures that there is a constant supply of cells needed for therapeutic applications. Getting more cells faster reduces the cost of production and also provides treatment to patients faster. In one or more embodiments, step (b) comprises 1×10 6 ~1×10 7 cells, e.g. 2 x 10 6 ~5×10 6 In one or more embodiments, step (b) produces 5×10 cells. 6 ~1×10 7 In one or more embodiments, step (b) produces 1×10 cells. 6 ~5×10 7 In one or more embodiments, step (b) produces 1×10 cells. 7 ~5×10 7 In one or more embodiments, step (c) generates 1×10 cells. 7 ~1×10 12 cells, e.g. 1 x 10 8 ~5×10 9 cells, e.g. 1 x 109 ~5×10 9 cells, e.g. 1 x 10 8 ~5×10 10 cells, e.g. 1 x 10 9 ~5×10 11 In one or more embodiments, step (c) produces at least 10 cells compared to the number of cells after expansion in step (b). 4 A fold increase, e.g., at least 10 3 A fold increase, e.g., at least 10 2 In one or more embodiments, step (c) results in a 1×10 7 ~1×10 10 In one or more embodiments, step (c) generates 1×10 cells. 7 ~1×10 9 In one or more embodiments, step (c) generates 1×10 cells. 7 ~1×10 8 Generate cells.

[0150] Example 7 shown in FIG. 13 shows that the addition of TME stimulants to the standard blast TIL production procedure carried out as described in Example 2 significantly enhanced TIL proliferation, thereby generating a higher number of viable cells per tumor fragment. Example 8 shown in FIG. 20 shows that the addition of TME stimulants that antagonize receptors expressed on the surface of T cells (or their ligands), stimulate receptors expressed on the surface of T cells (or their ligands), reactivate exhausted T cells (or their ligands), deplete regulatory T cells, and / or target receptors expressed on the surface of T cells from the CD28 family (or their ligands from B7 family proteins) to the processing steps of blast TILs provides a new improvement to the existing standard TIL procedure that allows for a faster production procedure of TIL therapy. In some embodiments, the antigen-presenting feeder cells (APCs) are PBMCs. In some embodiments, the antigen-presenting feeder cells (APCs) are allogeneic feeder cells. In some embodiments, the antigen-presenting feeder cells are artificial antigen-presenting feeder cells. In one embodiment, the ratio of TILs to antigen-presenting feeder cells in the second expansion is about 1:25, about 1:50, about 1:100, about 1:125, about 1:150, about 1:175, about 1:200, about 1:225, about 1:250, about 1:275, about 1:300, about 1:325, about 1:350, about 1:375, about 1:400, or about 1:500. In one embodiment, the ratio of TILs to antigen-presenting feeder cells in the second expansion is 1:50 to 1:300. In one embodiment, the ratio of TILs to antigen-presenting feeder cells in the second expansion is 1:100 to 1:200. In one or more embodiments, the APC is an artificial APC (aAPC).

[0151] In one embodiment, the TILs expanded using the APCs of the present disclosure are administered to the patient as a pharmaceutical composition. In one embodiment, the pharmaceutical composition is a suspension of TILs in a sterile buffer. The TILs expanded using the PBMCs of the present disclosure may be administered by any suitable route known in the art. In some embodiments, the T cells are administered as a single intra-arterial or intravenous infusion, preferably lasting about 30-60 minutes. Other suitable routes of administration include intraperitoneal, intrathecal, and intralymphatic. In one or more embodiments, the therapeutic population of TILs is infused into the patient.

[0152] In one or more embodiments, the cells are removed from the cell culture and cryopreserved in a storage medium prior to performing step (c).

[0153] In one or more embodiments, the method further comprises transducing the first population of TILs with an expression vector comprising a nucleic acid encoding a chimeric antigen receptor (CAR) comprising a single chain variable fragment antibody fused to an endodomain of at least one T cell signaling molecule.

[0154] In one or more embodiments, step (c) further comprises removing the cells from the cell culture medium.

[0155] In one or more embodiments, step (a) further comprises processing the resected tumor into multiple tumor fragments, e.g., 4-50 fragments, e.g., 20-30 fragments. In one or more embodiments, the fragments are about 1-50 mm 3 In one or more embodiments, the fragments have a size of about 5 to 50 mm. 3 In one or more embodiments, the fragments have a size of about 0.1 to 10 mm 3 In one or more embodiments, the fragments have a size of about 0.1 to 1 mm 3 In one or more embodiments, the fragments have a size of about 0.5 to 5 mm. 3 In one or more embodiments, the fragments have a size of about 1-10 mm 3 In one or more embodiments, the fragments have a size of about 1-3 mm3 The terms "fragmenting," "fragment," and "fragmented" as used herein to describe the process of destroying tumors include mechanical methods of fragmenting tumor tissue, such as grinding, cutting, splitting, mincing, and any other method for disrupting the physical structure of tumor tissue.

[0156] In one or more embodiments, the mammal is a human. In some embodiments, the TILs are obtained from tumor fragments. In some embodiments, the tumor fragments are obtained by sharp dissection. In some embodiments, the tumor fragments are about 0.1 mm 3 ~10 mm 3 In some embodiments, the tumor fragment is about 1 mm 3 ~10 mm 3 In some embodiments, the tumor fragment is about 1 mm 3 ~8 mm 3 In some embodiments, the tumor fragment is about 1 mm 3 In some embodiments, the tumor fragment is about 2 mm 3 In some embodiments, the tumor fragment is about 3 mm 3 In some embodiments, the tumor fragment is about 4 mm 3 In some embodiments, the tumor fragment is about 5 mm 3 In some embodiments, the tumor fragment is about 6 mm 3 In some embodiments, the tumor fragment is about 7 mm 3 In some embodiments, the tumor fragment is about 8 mm 3 In some embodiments, the tumor fragment is about 9 mm 3 In some embodiments, the tumor fragment is about 10 mm 3In some embodiments, the tumor is 1-4 mm x 1-4 mm x 1-4 mm. In some embodiments, the tumor is 1 mm x 1 mm x 1 mm. In some embodiments, the tumor is 2 mm x 2 mm x 2 mm. In some embodiments, the tumor is 3 mm x 3 mm x 3 mm. In some embodiments, the tumor is 4 mm x 4 mm x 4 mm. Currently, much larger fragment sizes are required (5 mm 3 (End). The present invention allows the use of smaller fragments, since the cells grow in a more optimized manner and reach the number of cells required for treatment more quickly. The use of smaller fragments means that patients who could not be treated before, for example because their tumors were too small or only small tumor samples could be taken, can now be treated. The size of the fragments used in the method of the present invention is therefore important.

[0157] In some embodiments, tumor fragmentation is performed to maintain the internal structure of the tumor. In some embodiments, tumor fragmentation is performed without performing sawing motion with a scalpel. In some embodiments, TILs are obtained from tumor digests. In some embodiments, tumor digests are generated by culturing in enzyme medium, such as, but not limited to, RPMI 1640, 2 mM GlutaMAX, 10 mg / mL gentamicin, 30 U / mL DNase, and 1.0 mg / mL collagenase, followed by mechanical dissociation (GentleMACS, Miltenyi Biotec, Auburn, CA). After placing the tumor in the enzyme medium, the tumor may be mechanically dissociated for about 1 minute. The solution is then incubated in 5% CO2 at 37°C for 30 minutes, followed by mechanical disruption again for about 1 minute. After again culturing in 5% CO2 at 37°C for 30 minutes, the tumor may be mechanically disrupted again for about 1 minute. In some embodiments, after a third mechanical disruption if large tissue debris was present, one or two additional mechanical separations were performed on the samples with or without an additional incubation for 30 minutes at 37° C. in 5% CO2. In some embodiments, at the end of the final incubation, if the cell suspension contains a large number of red blood cells or dead cells, a density gradient separation using Ficoll can be performed to remove these cells.

[0158] In one or more embodiments, the cell culture medium is provided in a container selected from the group consisting of a G-Rex container and a Xuri culture bag.

[0159] One aspect relates to a tumor infiltrating lymphocyte (TIL) population obtained by the method of any of the preceding embodiments.

[0160] A further aspect relates to expanded tumor infiltrating lymphocytes (TILs) for use in treating a subject suffering from cancer, the treatment comprising culturing autologous T cells by harvesting a first population of TILs from a tumor resected from the mammal; performing a first expansion by culturing the first population of TILs in cell culture medium comprising IL-2 and one or more TME stimulants to generate a second population of TILs; performing a second expansion by supplementing the cell culture medium of the second population of TILs with additional IL-2, anti-CD3, and antigen presenting cells (APCs) to generate a third population of TILs, which is a therapeutic population; and administering the therapeutic population of T cells to the mammal after administration of non-myeloablative lymphodepleting chemotherapy, wherein administration of the T cells to the mammal promotes regression of the cancer in the mammal.

[0161] In one embodiment, the invention includes a method of treating cancer with a population of TILs, or the use of TILs to treat cancer, where the patient has been pre-treated with a non-myeloablative chemotherapy prior to infusion of the TILs according to the present disclosure. In one embodiment, the non-myeloablative chemotherapy comprises administration of cyclophosphamide at 60 mg / kg / day for 2 days (days 7 and 2 prior to TIL infusion) and 25 mg / m 2 In one embodiment, after non-myeloablative chemotherapy and TIL infusion according to the present disclosure (day 0), the patient receives an intravenous infusion of IL-2 at 720,000 IU / kg every 8 hours to physiological tolerance.

[0162] In some embodiments, the disclosure provides a method of treating cancer with a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of: (a) obtaining a first population of TILs from a tumor resected from a patient; (b) performing a first expansion of the first population of TILs in a first cell culture medium comprising IL-2 and one or more TME stimulants to obtain a second population of TILs at least 5-fold greater in number than the first population of TILs; (c) performing rapid expansion of the second population of TILs using a population of myeloid artificial antigen presenting cells (myeloid aAPCs) in a second cell culture medium comprising IL-2 and anti-CD3 to obtain a third population of TILs at least 50-fold greater in number 7 days after initiation of rapid expansion than the second population of TILs; and (d) administering a therapeutically effective amount of the third population of TILs to a patient suffering from cancer. In some embodiments, the disclosure provides a tumor infiltrating lymphocyte (TIL) population for use in treating cancer, the TIL population being obtained by a method comprising the steps of: (b) performing a first expansion of a first TIL population obtained from a tumor resected from a patient in a first cell culture medium comprising IL-2 to obtain a second TIL population at least 5 times greater in number than the first TIL population; (c) performing rapid expansion of the second TIL population using a population of myeloid artificial antigen presenting cells (myeloid aAPCs) in a second cell culture medium comprising IL-2 and anti-CD3 to obtain a third TIL population at least 50 times greater in number 7 days after the initiation of rapid expansion than the second TIL population; and (d) administering a therapeutically effective amount of the third TIL population to a patient suffering from cancer. In some embodiments, the method comprises a first step (a) of obtaining a first TIL population from a tumor resected from a patient. In some embodiments, IL-2 is present in the second cell culture medium at an initial concentration of about 3,000 IU / mL and anti-CD3 antibody is present at an initial concentration of about 30 ng / mL. In some embodiments, the first expansion is performed for a period of 14 days or less. In some embodiments, the first expansion is performed using a gas-permeable container. In some embodiments, the second expansion is performed using a gas-permeable container. In some embodiments, the ratio of the second TIL population to the aAPC population in the rapid expansion is 1:80 to 1:400. In some embodiments, the ratio of the second TIL population to the aAPC population in the rapid expansion is about 1:300.

[0163] A further aspect relates to a tumor infiltrating lymphocyte (TIL) population obtained by a method comprising culturing autologous T cells by harvesting a first TIL population from a tumor excised from a mammal; performing a first expansion by culturing the first TIL population in a cell culture medium comprising IL-2 and one or more TME stimulants to generate a second TIL population; and performing a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, anti-CD3, and antigen presenting cells (APCs) to generate a third TIL population, which is a therapeutic population.

[0164] A further aspect relates to a therapeutic population of TILs comprising IL-2 and one or more TME stimulatory agents.

[0165] A further aspect relates to a therapeutic population of TILs comprising IL-2, one or more TME stimulatory agents, IL-2, anti-CD3, and antigen presenting cells (APCs).

[0166] The following diagrams and examples are provided to illustrate the present invention, they are intended to be illustrative and should not be construed as limiting in any way. table [Table 1] [Table 2] [Table 3] [Table 4] [Table 5] [Table 6] [Table 7]

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[0167] Example 1: One or more immunomodulatory agents reactivate exhausted T cells in vitro

[0168] Step 1.1: Effect of a single immunomodulator a) Resected TIL-containing tumor tissue from various patients was cultured at 1 cm 3 Cut the tumor into 30-50 pieces per tissue and transfer them to a 24-well cell culture dish in 2 mL of cell culture medium containing 6,000 IU / mL IL-2 and either no immunomodulatory agent (control), low, medium, or high concentration of each immunomodulator listed in Table 1. b) Cell culture dishes are incubated at 37°C and 5% CO2 with frequent changes of cell culture medium. Cell cultures are grown at 1.5 x 10 cells per well. 6 The cells should not be expanded to 100 cells and should be split into new wells. c) Cells are harvested several days later, counted to determine cell mass, and analyzed for viability and phenotype using flow cytometry.

[0169] Step 1.2: Effects of co-blockade and / or blockade / stimulation of PD1 a) Because blockade of PD1 has been clearly identified as a key pathway to reactivate exhausted T cells, new studies will be set up involving IL-2, optimal concentrations of PD1 and other immunomodulators listed in Table 1, and specific combinations with IL-2 listed in Tables 2-21, and performed as described above.

[0170] Step 1.3: Effects of co-blockade and / or blockade / stimulation of CTLA4 a) Because blockade of CTLA4 has been clearly identified as a key pathway to reactivate exhausted T cells, new studies will be set up involving IL-2, optimal concentrations of PD1 and other immunomodulators listed in Table 1, and specific combinations with IL-2 listed in Tables 22-41, and performed as described above.

[0171] Step 2: Possible further fine-tuning of the immunomodulator concentration

[0172] Step 3: Elucidation of combination effects a) A new test is set up similarly using the best performing immunomodulator at optimal concentration from the first experiment in a combination approach, adding several immunomodulators at the same time using the same readouts as above to measure possible synergistic effects. b) The above tests are repeated several times to ultimately identify combinations that result in reduced time, improved growth rate, and / or improved phenotype.

[0173] Step 4: Validation of optimal combinations in patients against standard TIL manufacturing procedures a) First TIL culture expansions will be performed in parallel in a large number of patients eligible for TIL therapy to validate the impact in a real patient environment. Example 2: "Young" Tumor Infiltrating Lymphocytes (TILs) Using TME Stimulators

[0174] This example illustrates a manufacturing process for generating "young" tumor infiltrating lymphocytes (TILs) using TME stimulators.

[0175] Tumor material of various histologies was obtained from commercial sources. Fourteen tumors or tumor digests were obtained from separate patients (3 ovarian, 3 metastatic melanoma, 3 head and neck, 2 lung, 2 colon, and 1 cervical cancer; Table 42). Cryopreserved or fresh tumor material was sent to Cbio A / S in sterile freezing or transport medium. Tumor material was handled in a laminar flow hood to maintain sterility.

[0176] TILs were prepared by standard TIL production procedures as detailed previously (Friese, C. et al., CTLA-4 blockade boosts the expansion of tumor-reactive CD8+ tumor-infiltrating lymphocytes in ovarian cancer. Sci Rep 10, 3914 (2020); and Jin, J. et al., Simplified Method of the Growth of Human Tumor Infiltrating Lymphocytes in Gas-permeable Flasks to Numbers Needed for Patient Treatment, Journal of Immunotherapy, 35 - Issue 3 (2012)). Briefly, TIL cultures were established using tumor fragments or tumor digests. Tumors were cultured at 1–3 mm 3 Tumors were divided into 1000 IU / mL fragments and placed in G-Rex 6-well plates (WilsonWolf; 5 fragments per well) with 10 mL of complete medium (CM) containing 6,000 IU / mL IL-2 (6,000 IU / mL, Clinigen) alone (reference) or in combination with low, medium, high, or very high concentrations of each of the TME stimuli listed in Table 43: PD-1 / PD-L1 antagonist (Group A), CTLA-4 antagonist (Group B), LAG-3 antagonist (Group C), TIGIT antagonist (Group D), 4-1BB agonist / anti-CD3 (Group J), and CD28 agonist (Group K) in a humidified incubator at 37°C with 5% CO2. Every 4-5 days, CM was added until a total volume of 40 mL was reached. Every 4-5 days, half of the medium was subsequently removed and replaced with CM and IL-2. TIL cultures from tumor digests were cultured in flat-bottom 96-well plates in 250 μL of CM and IL-2 (6000 IU / mL, Clinigen) overnight in a single-cell suspension (5 × 10 5The cells were initiated by culturing at 100 cells / mL in a humidified incubator containing 5% CO at 37°C. Every 2-3 days, half of the medium was removed and replaced with CM.

[0177] CM consisted of RPMI1640 containing GlutaMAX, 25 mM HEPES pH 7.2 (Gibco), 10% heat-inactivated human AB serum (Sigma-Aldrich), 100 U / mL penicillin, 100 μg / mL streptomycin (Gibco), and 1.25 μg / mL fungizone (Bristol-Myers Squibb).

[0178] This example demonstrates the generation of 10-28 day old "young" tumor infiltrating lymphocytes (TILs) using TME stimulants. Example 3: Phenotypic analysis of "young" TIL cultures using TME stimulants

[0179] This example shows the phenotypic analysis of "young" TIL cultures using TME stimuli performed as described in Example 2.

[0180] When cultures designated as juvenile TIL generation were harvested, their phenotype was assessed by flow cytometry.

[0181] TIL phenotype was determined by assessing viability and both frequency and absolute cell numbers in the CD3+, CD3+CD8+, CD3+CD4+, and NK fractions. In addition, the differentiation state, activation state, expression of exhaustion markers, and senescence of TILs were assessed. Flow cytometry was performed using the following markers: TIL study 1: CD3, CD4, CD8, CD45RA, CD56, CCR7, FVS780, BTLA, LAG-3, PD-1, TIM-3 TIL consideration 2: CD3, CD4, CD8, CD45RA, CD56, CCR7, FVS780, CD27, CD28, CD57, CD69

[0182] Briefly, about 0.5 × 10 per study 6 Young TILs were washed and then incubated with titrated antibodies (BD Biosciences, Table 45) and Brilliant Stain Buffer (BD Biosciences) for 30 min at 4° C. Cells were washed twice with PBS and directly analyzed by flow cytometry (CytoFLEX, Beckman Coulter).

[0183] This example demonstrates the phenotypic analysis of "young" TIL cultures using TME stimulants. Example 4: TME stimulators increased the success rate of TIL expansion in vitro

[0184] This example shows that when TIL cultures were initiated as described in Example 2, the addition of TME stimulants to the culture medium increased the success of TIL expansion in vitro.

[0185] The success rate of TIL expansion was investigated by measuring the number of cells per tumor fragment at the time of harvesting the TIL cultures. 4 TILs / fragment was set as the threshold for judging successful TIL culture.

[0186] Determination of the success rate of TIL expansion, as shown in Figures 2 and 3, indicates that when TME stimulants were added to "juvenile" TIL cultures, the success rate of TIL cultures was higher compared to 61.5% of reference cultures (avelumab: 68%, leratolimab: 70%, tiragolumab: 76.5%, pembrolizumab: 82.1%, ipilimumab: 88.5%, ceralizumab: 90.9%, nivolumab: 92.3%, and urelumab / OKT3: 100%).

[0187] When TME stimulants were grouped according to their targets, the present example also shows that the success rate of TIL cultures was higher in each group compared to 61.5% in the reference culture: Group C (70%, LAG-3 inhibitor), Group A (76.3%, including inhibitors of PD1 and its ligand PD-L1), Group D (76.5%, TIGIT inhibitor), Group B (88.5%, inhibitors of CTLA-4 and its ligand), Group K (90.9%, CD28 agent), and Group J (96.3%, 4-1BB agent with anti-CD3), as shown in Figures 4 and 5, respectively.

[0188] This example demonstrates that in vitro TIL expansion was more successful when TIL cultures were initiated with the addition of TME stimulants to the culture medium compared to standard TIL production procedures. Example 5: Checkpoint blockade or costimulation increased TIL yield and shortened TIL culture time

[0189] This example shows that when TIL cultures were initiated and performed as described in Example 2, the addition of a TME stimulant to the culture medium increased the TIL yield and shortened the culture time of the TILs.

[0190] The TIL yield and the culture time of TILs when harvesting TIL cultures were investigated. This analysis shows that compared to TILs cultured with IL-2 alone, TIL yields increased and culture times were shortened when TIL cultures were initiated when TME stimulants were added to the culture medium (Figure 6). Here, it is shown that not only the TME stimulants of group K and group J shortened the juvenile TIL culture time, but the stimulants of group A, group B, and group C also induced a higher growth rate as well compared to the standard juvenile TIL procedure. Notably, the stimulant of group J was combined in a double or triple fashion with either the stimulants of group A and / or group B, which further accelerated the growth rate.

[0191] This example demonstrates that when TIL culture is initiated, TIL yields are increased and TIL culture times are shortened when TME stimulants are added to the culture medium compared to standard TIL production procedures. Example 6: Different concentrations of TME stimulants induced TIL proliferation in vitro

[0192] This example, performed as described in Example 2, shows that when TIL cultures were initiated from different tumor types, TIL yields increased when TME stimulants were added to the culture medium at different concentrations.

[0193] The yield of TIL proliferation when TIL cultures were harvested was investigated. The first analysis in Figure 7 shows that when TIL cultures were initiated, low, medium, and high concentrations of the CTLA-4 antagonist ipilimumab (group B) were added to the culture medium, which enhanced TIL proliferation by 1.15-fold, 1.85-fold, and 1.53-fold, respectively, compared to IL-2 alone. This example shows that when TIL cultures were initiated in a dose-dependent manner, the TIL yield increased when the CTLA-4 antagonist (group B) was added to the culture medium. The CTLA-4 antagonist used (ipilimumab) is also known to deplete T cells expressing CTLA-4 by antibody-dependent cellular cytotoxicity (ADCC), but this new finding shows how depleting a specific subset of T cells can accelerate the proliferation rate of other T cells, thereby increasing the overall TIL yield.

[0194] Figure 8 shows that when TIL cultures were initiated, the addition of low, medium, or high concentrations of the PD-1 antagonist pembrolizumab (group A) to the culture medium tended to increase TIL yields in a dose-dependent manner compared to the standard young TIL procedure.

[0195] Figures 9-12 show dose-dependent effects of similar TIL yields compared to standard blast TIL procedures for the 4-1BB agonist urelumab with anti-CD3 (OKT3) (group J), the CD28 agonist celalizumab (group K), the PD-L1 inhibitor avelumab (group A: a ligand for PD-1), and another PD-1 inhibitor nivolumab (group A).

[0196] This Example 6 demonstrates how different concentrations of TME stimulants affected TIL proliferation in a dose-dependent manner. Example 7: TME stimulants collectively and from individual groups promote TIL proliferation

[0197] Example 7, shown in Figure 13, demonstrates that the addition of TME stimulatory agents to a standard blast TIL procedure performed as described in Example 2 significantly enhanced TIL proliferation to generate more viable cells per tumor fragment. This was illustrated using several representative tumor fragments from various solid cancers, including ovarian, head and neck, colon, melanoma, cervical, colon, and lung cancers.

[0198] Dividing the TME stimuli into basic subgroups, this example also shows that the addition of inhibitors from group A (such as inhibitors of PD1 and its ligand PD-L1), group B (inhibitors of CTLA-4), group J (4-1BB agents with anti-CD3), and group K (CD28 agents) also significantly increased TIL proliferation. Although not as pronounced in this example, the addition of TME stimuli from group C (LAG-3 inhibitors) and group D (TIGIT inhibitors) also tended to improve TIL proliferation.

[0199] In Figures 14-19, the effect of adding TME stimulants to primary TIL cultures from the same groups as above is illustrated in cervical, melanoma, ovarian, head and neck, lung, and colon cancers, respectively. Although not prominent in all conditions, the effect shows a similar pattern among cancers as in all tumor examples in Figure 13, although the effect and magnitude of individual stimuli in different cancers varied.

[0200] In summary, the addition of TME stimulants to the young TIL processing step provided a novel improvement over the existing standard TIL procedure, allowing for a more rapid production procedure for TIL therapy. Example 8: TIL stimulatory agonists, antagonists, T cell depletors, T cell reactivators, and stimulators of CD28 family origin significantly improved TIL growth rates

[0201] Example 8, shown in Figure 20, demonstrates that the addition of TME stimulatory agents to a standard blast TIL procedure performed as described in Example 2 significantly enhanced TIL proliferation to generate more viable cells per tumor fragment. This was illustrated using several representative tumor fragments from various solid cancers, including ovarian, head and neck, colon, melanoma, cervical, colon, and lung cancers.

[0202] Dividing the TME stimulants into subgroups according to their function, this example also shows that T cell antagonists, agonists, reactivators, depletors, and members of the CD28 family of receptors all had a significant effect on TIL proliferation. Representative amounts of various TME antagonists exemplified herein, including two different PD-1 inhibitors (pembrolizumab and nivolumab), two different PD-L1 inhibitors (avelumab and durvalumab), a CTLA-4 inhibitor (ipilimumab), and a TIGIT inhibitor (tiragolumab), showed a 3-5 fold increase over standard juvenile TIL processes, while TME agonists exemplified herein by 4-1BB (urelumab with anti-CD3 (OKT3)) and stimulants targeting CD28 (celalizumab) appeared to promote proliferation at a much faster rate.

[0203] Further categorizing TME antagonists according to whether they allow depletion of regulatory T cells by antibody-dependent cellular cytotoxicity (ADCC) such as ipilimumab and tiragolumab, or whether they only allow reactivation of T cells by checkpoint blockade, both demonstrated significant improvements in TIL proliferation rates over standard juvenile TIL procedural conditions, as shown in Figure 20.

[0204] Looking specifically to TME stimulants from the CD28 family of proteins, exemplified herein by inhibitors of PD-1, CTLA-4, and CD28, or their ligands from the B7 family of proteins, exemplified herein by two different inhibitors of PD-L1, have also been demonstrated to significantly enhance TIL proliferation compared to standard young TIL procedures. Although not shown here, other receptors expressed on the T cell surface from the CD28 protein family, such as BTLA and ICOS, may have a similar proliferation stimulatory effect on young TIL cultures.

[0205] In Figures 21-26, the effect of adding TME stimulants to primary TIL cultures from the same groups as described above in this example is illustrated for melanoma, ovarian, lung, cervical, colon, and head and neck cancer, respectively. Although not significant in all conditions, the effect shows a similar pattern among cancers for all tumor examples in Figure 20, although the effect and magnitude of individual stimulants in different cancers varied.

[0206] In summary of this example, the addition of TME stimulants that either antagonize receptors expressed on the surface of T cells (or their ligands), act on receptors expressed on the surface of T cells, reactivate exhausted T cells (or their ligands), deplete regulatory T cells, and / or target receptors expressed on the surface of T cells derived from the CD28 family (or their ligands derived from proteins of the B7 family) to the juvenile TIL processing step provides a novel improvement over existing standard TIL procedures that allows for a faster production procedure of TIL therapy. Example 9: Antagonists, TME stimulators that target receptors expressed on the surface of T cells or their ligands, showed similar TIL proliferation stimulatory effects

[0207] Example 9, shown in Figure 27, shows that the addition of TME stimulants to a standard young TIL procedure as performed in Example 2 significantly improved TIL proliferation, resulting in more viable cells per tumor fragment by stimulating either the receptor expressed on the surface of T cells (PD-1 in this case) or its ligand (PD-L1) expressed on the surface of tumor cells and other cells in the tumor microenvironment. This was illustrated using several representative tumor fragments from various solid cancers, including ovarian, head and neck, colon, melanoma, cervical, and lung cancer. This was an example of how inhibition of receptors expressed on the surface of T cells that are known to downregulate T cell activity can have a similar effect to inhibition of their ligands, and how both may produce a similar effect on TIL proliferation that reactivates exhausted T cells and improves young TIL proliferation. The PD1 / PD-L1 example therefore illustrates a more general trend towards receptor / ligand inhibition at other receptors expressed on the T cell surface, such as CTLA-4, LAG-3, TIGIT, KIR, TIM-3, BTLA, and their ligands.

[0208] In Figures 28-33, the effect of adding TME stimulants to primary TIL cultures from either the standard TIL production procedure, the PD-1 group, or the PD-L1 group was illustrated in ovarian, melanoma, lung, head and neck, colon, and cervical cancer, respectively. Although not prominent in all conditions, the effect showed a similar pattern between each cancer as in Figure 27 for PD-1 / PD-L1 in all tumors. Example 10: TME stimulants from different manufacturers showed similar TIL proliferation stimulating effects

[0209] Example 10, shown in Figure 34, shows that the addition of TME stimulants from various manufacturers had a similar effect when added to a standard blast TIL procedure performed as described in Example 2, significantly enhancing TIL proliferation and generating more viable cells per tumor fragment, regardless of manufacturer. This was illustrated using several representative tumor fragments from various solid tumors, including ovarian, melanoma, head and neck, and cervical cancer.

[0210] Two PD-1 inhibitors (pembrolizumab; Merck Sharp & Dome, and nivolumab; Bristol Myers Squibb) and two PD-L1 inhibitors (avelumab; Merck KgaA, and durvalumab; AstraZeneca) were tested in this example. All the different TME stimulants showed a significant improvement over the standard juvenile TIL procedure in their ability to accelerate TIL proliferation. The four different antibodies tended to show similar effects compared to group A and among the individual inhibitors.

[0211] This is an example of how interchangeable TME stimulants from different manufacturers are in general and how their use can optimize the young TIL manufacturing process.

[0212] In Figures 35-38, the effect of adding Group A inhibitors from various manufacturers to primary TIL cultures is demonstrated for ovarian, melanoma, head and neck, and cervical cancer, respectively. While not significant in all conditions, this effect showed a similar pattern between each cancer as exemplified in Figure 34 for all tumors. Example 11: Combination of TME stimulating agents further enhanced proliferation of immature TILs

[0213] This example, performed as described in Example 2, shows that when TIL cultures from various tumor types were initiated, TIL yields were increased when various combinations of TME stimuli were added to the culture medium compared to standard TIL production procedures.

[0214] TIL yield was investigated when harvesting TIL cultures. The first analysis shown in Figure 39 shows that the addition of TME stimuli of group A (PD-1 inhibitor or its ligand) or group B (CTLA-4 inhibitor), or both group A and group B, significantly enhanced TIL proliferation compared to standard juvenile TIL procedure. Although not significant, the simultaneous addition of group A and group B TME stimuli tended to further enhance TIL proliferation rate.

[0215] In another analysis depicted in FIG. 40, urelumab with 4-1BB agonist anti-CD3 (OKT3) (Group J) either alone or in combination with the CTLA-4 inhibitor ipilimumab (Group B), or in combination with the PD-1 inhibitor pembrolizumab (Group A), or in the triple combination with both ipilimumab and pembrolizumab all demonstrated very robust TIL expansion in viable cell counts per tumor fragment from a variety of cancers.

[0216] In Figure 41, the effect of a time delay in the addition of urelumab / OKT3 to young TIL cultures was investigated. Here, the triple combination of urelumab / OKT3, ipilimumab, and pembrolizumab added to early young TIL cultures on day 0 (left side of figure) was compared to a time delay of adding ipilimumab and pembrolizumab and waiting 2 days before adding urelumab / OKT3 (right side of figure). There was no significant difference between the two conditions, and both conditions showed very strong TIL proliferation.

[0217] In Figure 42, the effect of adding celalizumab alone (left side of figure) or in combination with both ipilimumab and pembrolizumab to early juvenile TIL cultures was investigated. Although not significant, the triple combination tended to induce a faster proliferation rate in juvenile TILs compared to celalizumab alone. Example 12: TME stimulators used alone or in combination increased the success of TIL expansion in vitro

[0218] This example shows that the success rate of in vitro TIL expansion was increased when TME stimulants were added to the culture medium during the initiation of TIL culture, which was performed as described in Example 2.

[0219] The success rate of TIL expansion was investigated by measuring the number of cells per tumor fragment at the time of harvesting the TIL cultures. 4 TILs / fragment was set as the threshold for successful TIL culture.

[0220] Measurement of the success rate of TIL proliferation, as shown in Figure 43, indicates that when TME stimulants from the various groups were added either alone or in combination to "young" TIL cultures, the success rate of the TIL cultures increased compared to 61.5% for the reference cultures (Group A: 76.3%, Group B: 88.5%, Group J: 100.0%, Group A+B: 83.3%, Group B+J: 100.0%, Group A+J: 100.0%, and the triple combination of Group A+B+J: 96.0%).

[0221] In this example, it was demonstrated that the successful rate of in vitro TIL expansion was increased when TIL culture was initiated by adding TME stimuli alone or in combination to the culture medium compared to standard TIL production procedures. Example 13: TME stimulators from different groups, together and in combination, improve T cell frequency and number

[0222] Example 13, shown in Figures 44-47, demonstrates that the addition of TME stimulants to a standard blastocyst TIL procedure performed as described in Example 2, and staining of T cells with anti-CD3 flow cytometry antibody as described in Example 3, significantly enhanced TIL proliferation, resulting in higher T cell frequency compared to IL-2 alone (Figure 44), and generated higher numbers of viable T cells per tumor fragment (Figure 46). This was illustrated using several representative tumor fragments from various solid tumors, including ovarian, head and neck, colon, melanoma, cervical, colon, and lung cancers.

[0223] Breaking down the TME stimuli into basic subgroups, this example also showed that the addition of inhibitors from group A (such as inhibitors of PD1 and its ligand PD-L1) or group B (inhibitors of CTLA-4 and its ligand) also significantly increased the frequency of T cells by T cell reactivation compared to IL-2 alone (Figure 45). This may lead to the expansion of more tumor-reactive T cells, as T cells with higher affinity for tumor antigens may have an increased tendency to be exhausted. Furthermore, this example showed that the addition of TME stimuli from group B also significantly increased the frequency of T cells compared to group J (4-1BB agents with anti-CD3) or the combination of groups J, A, and B (Figure 45).

[0224] Breaking down the TME stimuli into basic subgroups, this example also shows that the addition of inhibitors from group A (such as inhibitors of PD1 and its ligand PD-L1), group B (inhibitors of CTLA-4 and its ligand), group K (CD28 agonists), and group J (4-1BB agonists with anti-CD3) significantly increased the number of viable T cells per tumor fragment compared to IL-2 alone (Figure 47). Furthermore, this example also shows that the addition of a combination of TME stimuli from groups J, A, and B also significantly increased the number of viable T cells per tumor fragment compared to groups A, B, or a combination of groups A and B (Figure 47). Furthermore, there was a trend towards a higher number of cells per fragment when the combination of TME stimuli from groups J, A, and B was added compared to the addition of a TME stimuli from group J alone (Figure 47).

[0225] In summary of this example, the addition of TME stimulatory agents to the immature TIL manufacturing process provided a novel improvement over existing standard TIL procedures, allowing for the generation of a TIL product that contains increased T cell frequencies and increased numbers of viable T cells. Example 14: TME stimulants from different groups, collectively and in combination, maintain the frequency of effector memory T cells

[0226] Example 14 shown in Figure 48 shows that the addition of TME stimulants to a standard young TIL production procedure performed as described in Example 2, and staining of T cells with anti-CD3, anti-CD45RA, and anti-CCR7 flow cytometry antibodies as described in Example 3, significantly increased the frequency of effector memory T cells among CD4+ T cells and slightly increased the frequency of effector memory T cells among CD8+ T cells compared to IL-2 alone. This was illustrated using several representative tumor fragments from various solid cancers including ovarian cancer, head and neck cancer, colon cancer, melanoma, cervical cancer, colon cancer, and lung cancer.

[0227] In summary, the addition of TME stimulatory agents to the juvenile TIL manufacturing process provided a novel improvement over existing standard TIL procedures, allowing the generation of a TIL product containing a comparable frequency of effector memory T cells. Example 15: Combination of TME stimulators increases the frequency and number of CD8+ T cells

[0228] Example 15 shown in Figures 49-50 demonstrates that the addition of a combination of TME stimulants from Group J (4-1BB agents with anti-CD3), Group A (including inhibitors of PD1 and its ligand PD-L1), and Group B (inhibitors of CTLA-4 and its ligand) to a standard blast TIL production procedure performed as described in Example 2, and staining of T cells with anti-CD3 and anti-CD-8 flow cytometry antibodies as described in Example 3, significantly enhanced the proliferation of CD8+ T cells, resulting in a significant increase in the frequency (Figure 49) and number (Figure 50) of CD8+ T cells compared to IL-2 alone. This was illustrated using several representative tumor fragments from various solid cancers including ovarian cancer, head and neck cancer, colon cancer, melanoma, cervical cancer, colon cancer, and lung cancer.

[0229] This example also demonstrates that the addition of a combination of TME stimuli from group J (4-1BB agents with anti-CD3), group A (such as inhibitors of PD1 and its ligand PD-L1), and group B (inhibitors of CTLA-4) to normal young TILs showed a trend towards enhancing the proliferation of CD8+ T cells compared to the addition of group A, group B, or group J TME stimuli alone (Figure 49). This example further demonstrates that the addition of a combination of TME stimuli from group J, group A, and group B significantly increased the number of viable CD8+ T cells compared to group A or group B alone, and showed a trend towards increasing the number of viable CD8+ T cells compared to group J alone (Figure 50).

[0230] An increased frequency of CD8+ T cells in TIL infusion products has previously been associated with beneficial clinical outcomes of TIL therapy in patients with metastatic melanoma (Radvanyi, LG et al., Specific lymphocyte subsets predict response to adoptive cell therapy using expanded autologous tumor-infiltrating lymphocytes in metastatic melanoma patients. Clin. Cancer Res. 18, 6758-6770 (2012)). Thus, methods to increase the frequency of CD8+ T cells may be able to induce clinical responses in cancer patients who do not respond to TILs produced using standard TIL procedures.

[0231] In summary, the addition of TME stimulatory agents, both alone and in combination, to the young TIL processing step provided a novel improvement over existing standard TIL manufacturing procedures, allowing the generation of a TIL product containing an increased frequency of CD8+ T cells. Example 16: Combination of TME stimulators reduces the frequency of CD4+ T cells

[0232] Example 16 shown in Figure 51 shows that the addition of a combination of TME stimuli from group J (4-1BB agents with anti-CD3), group A (including inhibitors of PD1 and its ligand PD-L1), and group B (including inhibitors of CTLA-4) to a standard blast TIL procedure performed as described in Example 2, and staining of T cells with anti-CD3 and anti-CD4 flow cytometry antibodies as described in Example 3, significantly reduced the proliferation of CD4+ T cells, resulting in a significantly lower frequency of CD4+ T cells compared to IL-2 alone (Figure 51). This was illustrated using several representative tumor fragments from various solid cancers including ovarian cancer, head and neck cancer, colon cancer, melanoma, cervical cancer, colon cancer, and lung cancer.

[0233] In summary, the addition of TME stimulatory agents to the young TIL processing step provided a novel improvement to existing standard TIL manufacturing procedures, allowing the generation of a TIL product containing a reduced frequency of CD4+ T cells. Example 17: TME stimulants from various groups reduce the frequency of NK cells

[0234] Example 17 shown in Figure 52 demonstrates that the addition of TME stimuli from group A (such as inhibitors of PD1 and its ligand PD-L1) or group B (inhibitors of CTLA-4) to a standard blast TIL procedure performed as described in Example 2, and staining of NK cells with anti-CD3 and anti-CD56 flow cytometry antibodies as described in Example 3, significantly reactivated T cells and resulted in a reduced frequency of NK cells compared to IL-2 alone. This was illustrated using several representative tumor fragments from various solid cancers including ovarian cancer, head and neck cancer, colon cancer, melanoma, cervical cancer, colon cancer, and lung cancer.

[0235] Furthermore, this example demonstrates that the addition of TME stimuli from group B tended to reduce the frequency of NK cells compared to group J (4-1BB agonist with anti-CD3) and group K (CD28 agonist).

[0236] In summary of this example, the addition of TME stimulants to the young TIL processing step provided a novel improvement to existing standard TIL manufacturing procedures that allowed the generation of a TIL product containing reduced frequencies of NK cells. Example 18: TME stimulants from different groups or their combinations positively affect the frequency of NK cells, total T cells and CD8+ T cells

[0237] Example 18 shown in Figure 53 and Figure 54 demonstrates that the addition of urelumab / OKT3 (Group J) alone or in combination with pembrolizumab (Group A) or ipilimumab and pembrolizumab (Group B + Group A) to a standard blast TIL procedure performed as described in Example 2, and staining of NK and T cells with anti-CD3, anti-CD56, and anti-CD8 flow cytometry antibodies as described in Example 3, reactivated NK cells, resulting in increased frequency of NK cells and decreased frequency of T cells compared to IL-2 alone in one head and neck cancer sample (Figure 53). This NK cell reactivation was inhibited by adding ipilimumab together with urelumab / OKT3 to T cell reactivating TIL cultures, resulting in a decreased NK cell frequency and an increased T cell frequency compared to the addition of urelumab / OKT3 alone (group J) or in combination with pembrolizumab (group A) or ipilimumab and pembrolizumab (group B+group A) to the standard blastocytic TIL production protocol. This was illustrated using representative samples of head and neck cancer.

[0238] Furthermore, this example demonstrated that the addition of urelumab / OKT3 (group J) and ipilimumab (group B) reduced the frequency of CD8+ T cells compared to urelumab / OKT3 alone, urelumab / OKT3 and pembrolizumab (group A), and urelumab / OKT3 with ipilimumab and pembrolizumab (Figure 54).

[0239] Thus, this example demonstrates that the addition of urelumab / OKT3 (group J) plus ipilimumab (group B) and pembrolizumab (group A) may be preferable compared to urelumab / OKT3 and ipilimumab alone.

[0240] In summary, the addition of TME stimulatory agents to the young TIL processing step provided a novel improvement over existing standard TIL manufacturing procedures, allowing the generation of a TIL product that contained a reduced frequency of NK cells and an increased frequency of CD8+ T cells. Example 19: Combination of TME stimuli added with a time delay increases the frequency of CD3+ and CD8+ T cells and decreases the frequency of NK cells and CD4+ T cells

[0241] In Example 19 shown in Figure 55, the addition of a combination of TME stimuli from group A (such as inhibitors of PD1 and its ligand PD-L1) and group B (inhibitors of CTLA-4) on day 0 and group J (4-1BB agents with anti-CD3) on day 2 to a standard blast TIL procedure performed as described in Example 2, and staining of T cells with anti-CD3, anti-CD56, anti-CD8, and anti-CD4 flow cytometry antibodies as described in Example 3 demonstrated enhanced proliferation of T cells and CD8+ T cells, resulting in increased frequency of total T cells (CD3+) and CD8+ T cells (Figure 55) and decreased frequency of NK cells and CD4+ T cells, compared to the addition of TME stimuli from the same groups (A, B, and J) on day 0. This was illustrated using several representative tumor fragments from various solid cancers including ovarian cancer, head and neck cancer, colon cancer, melanoma, cervical cancer, colon cancer, and lung cancer. The time delay appears to allow for a stronger effect of the Group A and Group B antagonists in depleting regulatory CD-4+ T cells and reactivating CD-8+ T cells before adding the Group J agents.

[0242] In summary of this example, the addition of a TME stimulant with a time delay to the young TIL processing step provided a novel improvement to existing standard TIL manufacturing procedures that allowed the generation of a TIL product containing increased frequencies of total T cells and CD8+ T cells and decreased frequencies of NK cells and CD4+ T cells. Example 20: TME stimulators alone or in combination increase the frequency of LAG3+ T cells

[0243] Example 20 shown in Figure 56 and Figure 57 demonstrates that the addition of TME stimuli from group A (such as inhibitors of PD1 and its ligand PD-L1), group B (inhibitors of CTLA-4), group K (CD28 agents), or group J (4-1BB agents with anti-CD3) alone or in combination to a standard blast TIL procedure performed as described in Example 2, and staining of T cells with anti-CD3, anti-CD8, anti-CD4, and anti-LAG-3 flow cytometry antibodies as described in Example 3, enhanced reactivation of tumor-specific T cells, resulting in an increased frequency of LAG-3+ T cells in both CD4+ and CD8+ T cells compared to IL-2 alone (Figure 56). In particular, the frequency of CD4+LAG-3+ T cells was significantly higher when TME stimuli from groups A and B were added. This was illustrated using several representative tumor fragments from a variety of solid cancers, including ovarian, head and neck, colon, melanoma, cervical, colorectal, and lung cancer.

[0244] Furthermore, this example showed that the addition of a combination of TME stimuli from Group A (such as inhibitors of PD1 and its ligand PD-L1), Group B (inhibitors of CTLA-4), or Group J (4-1BB agents with anti-CD3) with a time delay as described in Example 19 enhanced the reactivation of tumor-specific CD8+ T cells, resulting in a trend towards increased frequency of CD8+LAG-3+ T cells, compared to the addition of a combination of TME stimuli from Groups A, B, and J (Figure 57).

[0245] In summary, the addition of TME stimulants to the young TIL processing step provided a novel improvement to the existing standard TIL manufacturing procedure, allowing the generation of a TIL product containing an increased frequency of tumor-specific LAG-3+ T cells. Since LAG-3 is a T cell exhaustion marker and T cells with higher affinity for tumor antigens are generally more prone to exhaustion, clonal expansion of CD8+LAG-3+ T cells may result in a higher proportion of tumor-reactive T cells, improving the clinical outcome of this novel approach to TIL therapy. Example 21: TME stimulants increased the frequency of CD8 T cells with a more immature phenotype that was CD28+

[0246] Example 21 shown in Figure 58 shows that the addition of TME stimuli from group A (such as inhibitors of PD1 and its ligand PD-L1), group B (inhibitors of CTLA-4), group K (CD28 agonists), or group J (4-1BB agonists with anti-CD3) alone or in combination to a standard blastic TIL procedure performed as described in Example 2, and staining of T cells with anti-CD3, anti-CD8, and anti-CD28 flow cytometry antibodies as described in Example 3, enhanced the proliferation of T cells with a more blastic phenotype, resulting in an increased frequency of CD8+CD28+ T cells compared to IL-2 alone (Figure 58). This was illustrated using several representative tumor fragments from various solid cancers including ovarian cancer, head and neck cancer, colon cancer, melanoma, cervical cancer, colon cancer, and lung cancer.

[0247] Furthermore, this example showed that the addition of a combination of TME stimuli from Group A (such as inhibitors of PD1 and its ligand PD-L1), Group B (inhibitors of CTLA-4), or Group J (4-1BB agents with anti-CD3) enhanced the expansion of T cells with a more juvenile phenotype, resulting in a trend towards increased frequency of CD8+CD28+ T cells, compared to the addition of a TME stimuli from Group A or Group B alone (Figure 58).

[0248] This example further shows that the addition of a combination of TME stimuli from Group A (such as inhibitors of PD1 and its ligand PD-L1), Group B (inhibitors of CTLA-4), and Group J (4-1BB agents with anti-CD3) with a time delay as described in Example 19 tended to enhance the expansion of T cells with a more juvenile phenotype, resulting in a higher frequency of generation of CD8+CD28+ T cells, compared to the addition of a TME stimuli from Group A or Group B alone or the addition of a combination of TME stimuli from Group A, Group B, and Group J without a time delay (Figure 58).

[0249] In summary, the addition of TME stimulatory agents to the young TIL processing step provided a novel improvement over existing standard TIL manufacturing procedures, allowing the generation of a TIL product that includes an increased frequency of CD8+ T cells with a more young phenotype that expresses CD28. [item]

[0250] Item 1: A method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic population of TILs to promote regression of cancer in a mammal, the method comprising: (a) obtaining a first population of TILs from a tumor excised from a mammal and culturing the autologous T cells; (b) performing a first expansion by culturing the first TIL population in a cell culture medium comprising IL-2 and one or more TME stimulants to generate a second TIL population; (c) performing a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, anti-CD3 antibody, and antigen-presenting cells (APCs) to generate a third TIL population, which is a therapeutic population; and (d) administering to the mammal the therapeutic population of T cells following administration of non-myeloablative lymphodepleting chemotherapy, wherein administration of the T cells to the mammal promotes regression of the cancer in the mammal.

[0251] Item 2: A method of treating a subject suffering from cancer, comprising administering expanded tumor infiltrating lymphocytes (TILs), the method comprising: (a) obtaining a first population of TILs from a tumor excised from a mammal and culturing the autologous T cells; (b) performing a first expansion by culturing the first TIL population in a cell culture medium comprising IL-2 and one or more TME stimulants to generate a second TIL population; (c) performing a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, anti-CD3 antibody, and antigen-presenting cells (APCs) to generate a third TIL population, which is a therapeutic population; and (d) administering to the mammal the therapeutic population of T cells following administration of non-myeloablative lymphodepleting chemotherapy, wherein administration of the T cells to the mammal promotes regression of the cancer in the mammal.

[0252] Item 3: A method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic population of TILs, the method comprising: (a) obtaining a first population of TILs from a tumor excised from a mammal and culturing the autologous T cells; (b) performing a first expansion by culturing the first TIL population in a cell culture medium comprising IL-2 and one or more TME stimulants to generate a second TIL population; and (c) performing a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, anti-CD3 antibody, and antigen presenting cells (APCs) to generate a third TIL population, which is a therapeutic population.

[0253] Item 4: One or more TME stimulants are (x) one or more substances capable of antagonizing and / or inhibiting receptors expressed on the surface of T cells (or their ligands) that are known to cause downregulation, deactivation, and / or exhaustion of T cells; (y) one or more substances capable of stimulating and / or enhancing receptors expressed on the surface of T cells that are known to cause upregulation, activation, and / or reactivation of T cells; (z) one or more substances capable of antagonizing and / or inhibiting soluble molecules and cytokines and their receptors that are known to cause T cell downregulation, deactivation, and / or exhaustion; (v) one or more substances capable of downregulating and / or depleting regulatory T cells, thereby promoting the expansion of ex vivo effector T cells; and (w) The method according to any one of items 1 to 3, wherein the compound is selected from the group consisting of a specific combination of one or more substances from groups (x), (y), (z) and / or (v) listed in Tables 2 to 41.

[0254] Item 5: The method according to any of items 1 to 4, wherein the one or more TME stimulants are one or more check point inhibitors or inhibitors of their ligands, such as, for example, anti-PD1, anti-PD-L1, anti-PD-L2, anti-CTLA-4, anti-LAG3, anti-A2AR, anti-B7-H3, anti-B7-H4, anti-BTLA, anti-IDO, anti-HVEM, anti-IDO, anti-TDO, anti-KIR, anti-NOX2, anti-TIM3, anti-galectin 9, anti-VISTA, anti-SIGLEC7 / 9, and optionally the one or more check point inhibitors or inhibitors of their ligands are also added to the second expansion.

[0255] Item 6: Substances capable of antagonizing and / or inhibiting receptors expressed on the surface of T cells (or their ligands) that are known to cause downregulation, inactivation, and / or exhaustion of T cells are A: Substances that act via the PD-1 receptor on the surface of T cells; B: Substances that act through the CTLA-4 receptor on the surface of T cells; C: Substances acting through the LAG-3 receptor on the surface of T cells; D: Substances acting through the TIGIT / CD226 receptor on the surface of T cells; E: Substances that act through KIR receptors on the surface of T cells; F: Substances that act through the TIM-3 receptor on the surface of T cells; G: Substances that act through the BTLA receptor on the surface of T cells; and H: The method according to any one of items 1 to 5, wherein the substance is selected from the group consisting of substances that act via the A2aR receptor on the surface of T cells.

[0256] Item 7: The method of item 6, wherein the group A agents are selected from one or more of the group consisting of pembrolizumab, nivolumab, cemiplimab, sym021, atezolizumab, avelumab, and durvalumab.

[0257] Item 8: The method of item 6 or 7, wherein the group B agents are selected from one or more of the group consisting of ipilimumab and tremelimumab.

[0258] Item 9: The method according to any one of Items 6 to 8, wherein the group C substance is selected from one or more of the group consisting of leratolimab, eftiragmo alpha, and sym022.

[0259] Item 10: The method of any of items 6 to 9, wherein the Group D agent is tiragolumab.

[0260] Item 11: The method according to any of items 6 to 10, wherein the Group E substance is lirilumab.

[0261] Item 12: The method according to any one of Items 6 to 11, wherein the substance of group F is sym023.

[0262] Item 13: The method according to any one of Items 6 to 12, wherein the substances of group G are 40E4 and PJ196.

[0263] Item 14: Substances capable of stimulating and / or promoting receptors expressed on the surface of T cells that are known to cause upregulation, activation and / or reactivation of T cells include: I: Substances that act via the OX40 / CD134 receptor on the surface of T cells; J: Substances that act via the 4-1BB / CD137 receptor on the surface of T cells; K: Substances that act through the CD28 receptor on the surface of T cells; L: Substances that act via the ICOS receptor on the surface of T cells; M: Substances that act through the GITR receptor on the surface of T cells; N: Substances that act through the CD40L receptor on the surface of T cells; and O: A method according to any one of items 1 to 13, wherein the substance is selected from the group consisting of substances that act via the CD27 receptor on the surface of T cells.

[0264] Item 15: The method of item 14, wherein the group J agent is selected from one or more of the group consisting of urelumab and utomirumab.

[0265] Item 16: The method of item 14, wherein the Group K agent is celalizumab.

[0266] Item 17: The method of item 14, wherein the group O agent is valilumab.

[0267] Item 18: Substances capable of antagonizing and / or inhibiting soluble molecules and cytokines and their receptors known to cause downregulation, inactivation, and / or exhaustion of T cells are P: Substances that act via the IDO1 / 2 receptors on the surface of T cells; Q: Substances that act through the TGFβ receptor on the surface of T cells; R: Substances that act through the IL-10 receptor on the surface of T cells; and S: A method according to any one of items 1 to 17, wherein the substance is selected from the group consisting of substances that act via the IL-35 receptor on the surface of T cells.

[0268] Item 19: The method according to item 14, wherein the group P substance is epacedostat.

[0269] Item 20: The method according to item 14, wherein the group Q substance is linrodostat.

[0270] Item 21: The method of item 14, wherein the group R agent is galunisertib.

[0271] Item 22: Substances capable of downregulating and / or depleting regulatory T cells and thereby promoting in vitro effector T cell proliferation include: T: cyclophosphamide; U: TKI; V: Substances acting through αCD25; and X: IL2 / diphtheria toxin fusion.

[0272] Item 23: The method of item 20, wherein the Group U agent is sunitinib.

[0273] Item 24: The method of item 20, wherein the Group V agent is selected from one or more of the group consisting of sorafenib, imatinib, and daclizumab.

[0274] Item 25: The method of item 20, wherein the group X agent is denileukin diftitox.

[0275] Item 26: The method according to any of Items 1 to 25, wherein the concentration of the substance is 0.1 μg / mL to 300 μg / mL, for example, 1 μg / mL to 100 μg / mL, for example, 10 μg / mL to 100 μg / mL, for example, 1 μg / mL to 10 μg / mL.

[0276] Item 27: Therapeutic populations of T cells are used to 1: solid tumor; 2: ICI-naive tumor; 3: MSI-H tumor; 4: Hematological neoplasms; and 5: The method according to any one of items 1 to 26, for treating a cancer type selected from the group consisting of hypermutated tumors.

[0277] Item 28: The method of any of items 1 to 27, wherein the therapeutic population of T cells is used to treat a cancer type selected from the group consisting of breast cancer, renal cell carcinoma, bladder cancer, melanoma, cervical cancer, gastric cancer, colorectal cancer, lung cancer, head and neck cancer, ovarian cancer, Hodgkin's lymphoma, pancreatic cancer, liver cancer, and sarcoma.

[0278] Item 29: The method of any of items 1 to 28, wherein the therapeutic population of T cells is used to treat breast cancer.

[0279] Item 30: The method of any of items 1 to 29, wherein the therapeutic population of T cells is used to treat renal cell carcinoma.

[0280] Item 31: The method of any of items 1 to 30, wherein a therapeutic population of T cells is used to treat bladder cancer.

[0281] Item 32: The method of any of items 1 to 31, wherein the therapeutic population of T cells is used to treat melanoma.

[0282] Item 33: The method of any of items 1 to 32, wherein the therapeutic population of T cells is used to treat cervical cancer.

[0283] Item 34: The method of any of items 1 to 33, wherein a therapeutic population of T cells is used to treat gastric cancer.

[0284] Item 35: The method of any of items 1 to 34, wherein the therapeutic population of T cells is used to treat colorectal cancer.

[0285] Item 36: The method of any of items 1 to 35, wherein the therapeutic population of T cells is used to treat lung cancer.

[0286] Item 37: The method of any of items 1 to 36, wherein the therapeutic population of T cells is used to treat head and neck cancer.

[0287] Item 38: The method of any of items 1 to 37, wherein the therapeutic population of T cells is used to treat ovarian cancer.

[0288] Item 39: The method of any of items 1 to 38, wherein the therapeutic population of T cells is used to treat Hodgkin's lymphoma.

[0289] Item 40: The method of any of items 1 to 39, wherein a therapeutic population of T cells is used to treat pancreatic cancer.

[0290] Item 41: The method according to any of items 1 to 40, wherein a therapeutic population of T cells is used to treat liver cancer.

[0291] Item 42: The method of any of items 1 to 41, wherein the therapeutic population of T cells is used to treat sarcoma.

[0292] Item 43: The method according to any one of Items 1 to 42, wherein steps (a) to (c) or (d) are carried out within a period of about 20 days to about 45 days.

[0293] Item 44: The method according to any one of Items 1 to 43, wherein steps (a) to (c) or (d) are carried out within a period of about 20 days to about 40 days.

[0294] Item 45: The method according to any one of Items 1 to 44, wherein steps (a) to (c) or (d) are carried out within a period of about 25 days to about 40 days.

[0295] Item 46: The method according to any one of Items 1 to 45, wherein steps (a) to (c) or (d) are carried out within a period of about 30 days to about 40 days.

[0296] Item 47: The method according to any one of Items 1 to 46, wherein steps (a) and (b) are carried out within a period of about 10 days to about 28 days.

[0297] Item 48: The method according to any one of Items 1 to 47, wherein steps (a) and (b) are carried out within a period of about 10 days to about 20 days.

[0298] Item 49: The method according to any one of Items 1 to 48, wherein step (c) is carried out within a period of about 12 days to about 18 days.

[0299] Item 50: The method according to any one of Items 1 to 49, wherein step (c) is carried out within a period of about 10 days to about 28 days.

[0300] Item 51: The method according to any one of Items 1 to 50, wherein step (c) is carried out within a period of about 10 days to about 20 days.

[0301] Item 52: The method according to any one of Items 1 to 51, wherein step (c) is carried out within a period of about 12 days to about 18 days.

[0302] Item 53: Step (b) is 1×10 6 ~1×10 7 cells, e.g. 2 x 106 ~5×10 6 53. The method according to any one of items 1 to 52, wherein the cells are generated.

[0303] Item 54: Step (c) is 1 x 10 7 ~1×10 12 cells, e.g. 1 x 10 8 ~5×10 9 cells, e.g. 1 x 10 9 ~5×10 9 cells, e.g. 1 x 10 8 ~5×10 10 cells, e.g. 1 x 10 9 ~5×10 11 54. The method according to any one of items 1 to 53, wherein the cells are generated.

[0304] Item 55: The method according to any of items 1 to 54, wherein the APC is an artificial APC (aAPC) or an allogeneic feeder cell.

[0305] Item 56: The method of any of items 1 to 55, wherein a therapeutic population of TILs is infused into the patient.

[0306] Item 57: The method according to any of items 1 to 56, wherein the cells are removed from the cell culture and cryopreserved in a storage medium before carrying out step (c).

[0307] Item 58: The method of any of items 1 to 57, further comprising transducing the first TIL population with an expression vector comprising a nucleic acid encoding a chimeric antigen receptor (CAR) comprising a single-chain variable fragment antibody fused to at least one endodomain of a T cell signaling molecule.

[0308] Item 59: The method according to any of Items 1 to 58, wherein step (c) further comprises removing the cells from the cell culture medium.

[0309] Item 60: The method according to any of items 1 to 59, wherein step (a) further comprises processing the resected tumor into a plurality of tumor fragments, for example into 4 to 50 fragments, for example into 20 to 30 fragments.

[0310] Item 61: The size of the fragments is approximately 5-50 mm. 3 , e.g. 20-50 mm 3 Item 61. The method according to Item 60, wherein

[0311] Item 62: The method according to any one of Items 1 to 61, wherein the mammal is a human.

[0312] Item 63: The method of any of items 1 to 62, wherein the cell culture medium is provided in a container selected from the group consisting of a G-Rex container and a Xuri cell bag.

[0313] Item 64: The method of any of items 1 to 63, wherein the anti-CD3 antibody is OKT3.

[0314] Item 65: A tumor-infiltrating lymphocyte (TIL) population obtained by the method according to any one of items 1 to 64.

[0315] Item 66: Expanded tumor infiltrating lymphocytes (TILs) for use in the treatment of a subject suffering from cancer, the treatment comprising: obtaining a first population of TILs from a tumor resected from the mammal and culturing the autologous T cells; performing a first expansion by culturing the first TIL population in a cell culture medium comprising IL-2 and one or more TME stimulating agents to generate a second TIL population; performing a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, anti-CD3, and antigen presenting cells (APCs) to generate a third TIL population, which is a therapeutic population; and The method includes administering to the mammal a therapeutic population of T cells following administration of non-myeloablative lymphodepleting chemotherapy, wherein administration of the T cells to the mammal promotes regression of the cancer in the mammal.

[0316] Item 67: A tumor infiltrating lymphocyte (TIL) population obtained by a method, the method comprising: obtaining a first population of TILs from a tumor resected from the mammal and culturing the autologous T cells; performing a first expansion by culturing the first TIL population in a cell culture medium comprising IL-2 and one or more TME stimulants to generate a second TIL population; and A second expansion is performed by supplementing the cell culture medium of the second TIL population with additional IL-2, anti-CD3, and antigen presenting cells (APCs) to generate a third TIL population that serves as a therapeutic population.

[0317] Item 68: A therapeutic TIL population comprising IL-2 and one or more TME stimulatory agents.

[0318] Item 69: A therapeutic TIL population comprising IL-2, one or more TME stimulatory agents, IL-2, an anti-CD3 antibody, and an antigen presenting cell (APC).

Claims

1. 1. A pharmaceutical composition comprising expanded tumor infiltrating lymphocytes (TILs) for use in the treatment of a subject suffering from cancer, said treatment comprising: obtaining a first population of TILs from a tumor excised from the mammal and culturing the autologous T cells; performing a first expansion by culturing said first TIL population in a cell culture medium comprising IL-2 and one or more TME stimulatory agents to generate a second TIL population, wherein said one or more TME stimulatory agents comprise at least one or more of the Group B agents selected from the group consisting of ipilimumab and tremelimumab; performing a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, anti-CD3 antibody, and antigen presenting cells (APCs) to generate a third TIL population, which is a therapeutic population; and Administering said therapeutic population of T cells to said mammal following administration of non-myeloablative lymphodepleting chemotherapy, thereby promoting regression of cancer in said mammal.

2. 1. A pharmaceutical composition comprising expanded tumor infiltrating lymphocytes (TILs) for use in promoting regression of cancer in a subject suffering from cancer, said regression being characterized by: obtaining a first population of TILs from a tumor excised from the mammal and culturing the autologous T cells; performing a first expansion by culturing said first TIL population in a cell culture medium comprising IL-2 and one or more TME stimulatory agents to generate a second TIL population, wherein said one or more TME stimulatory agents comprise at least one or more of the Group B agents selected from the group consisting of ipilimumab and tremelimumab; performing a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, anti-CD3 antibody, and antigen presenting cells (APCs) to generate a third TIL population, which is a therapeutic population; and 20. The method of claim 1, further comprising administering to said mammal a therapeutic population of T cells following administration of non-myeloablative lymphodepleting chemotherapy, thereby promoting regression of cancer in said mammal.

3. 1. A method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic population of TILs, comprising: (a) obtaining a first population of TILs from a tumor excised from a mammal and culturing the autologous T cells; (b) performing a first expansion by culturing said first TIL population in a cell culture medium comprising IL-2 and one or more TME stimulatory agents to generate a second TIL population, wherein said one or more TME stimulatory agents comprise at least one or more of the Group B agents selected from the group consisting of ipilimumab and tremelimumab; and (c) performing a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, anti-CD3 antibody, and antigen presenting cells (APCs) to generate a third TIL population, which is a therapeutic population.

4. The one or more TME stimulants include at least: One or more of the Group A agents selected from the group consisting of pembrolizumab, nivolumab, cemiplimab, sym021, atezolizumab, avelumab, and durvalumab; and One or more of the Group B agents selected from the group consisting of ipilimumab and tremelimumab The pharmaceutical composition according to claim 1 or 2, comprising:

5. 5. The pharmaceutical composition of claim 4, further comprising one or more Group J agents selected from the group consisting of urelumab and utomirumab.

6. 6. The pharmaceutical composition of claim 5, wherein Group J is used in combination with an anti-CD3 agent.

7. The pharmaceutical composition of claim 6, wherein the anti-CD3 agent is OKT3.

8. 5. The pharmaceutical composition of claim 4, wherein the Group A agent is pembrolizumab.

9. The pharmaceutical composition of any one of claims 1, 2, and 4-8, wherein the group B agent is ipilimumab.

10. 6. The pharmaceutical composition of claim 5, wherein the Group J agent is urelumab.

11. The pharmaceutical composition according to any one of claims 1, 2 and 4 to 10, wherein the concentration of the substance is from 0.1 μg / mL to 300 μg / mL.

12. The pharmaceutical composition of any one of claims 1, 2, and 4-11, wherein steps (a) and (b) are carried out within a period of about 7 days to about 28 days.

13. The pharmaceutical composition of any one of claims 1, 2, and 4-12, wherein step (c) is carried out within a period of about 7 days to about 21 days.

14. 14. The pharmaceutical composition of any one of claims 1, 2, and 4-13, wherein the therapeutic population of T cells is used to treat a cancer type selected from the group consisting of breast cancer, renal cell carcinoma, bladder cancer, melanoma, cervical cancer, gastric cancer, colon cancer, lung cancer, head and neck cancer, ovarian cancer, Hodgkin's lymphoma, pancreatic cancer, liver cancer, and sarcoma.

15. The step (c) is 1×10 ７ ~1×10 １２ The pharmaceutical composition according to any one of claims 1, 2, and 4 to 14, for producing a cell.

16. The pharmaceutical composition of any one of claims 1, 2, and 4 to 15, wherein the anti-CD3 antibody is OKT3.

17. 17. The pharmaceutical composition of any one of claims 1, 2, and 4-16, wherein the multiple TME stimulants are added together or over time, i.e., one day apart or 2, 3, 4, 5, 6, or 7 days apart.

18. The pharmaceutical composition of any one of claims 1, 2, and 4 to 17, wherein the antigen-presenting cells (APCs) are selected from the group consisting of allogeneic feeder cells, PBMCs, and artificial antigen-presenting feeder cells.

19. The pharmaceutical composition of any one of claims 1, 2, and 4-18, wherein the step of culturing the autologous T cells further comprises processing the excised tumor into a plurality of tumor fragments.

20. The size of the fragments is about 1 to 10 mm ３ 20. The pharmaceutical composition of claim 19,

21. A tumor infiltrating lymphocyte (TIL) population obtained by the method of claim 3, comprising: ８ ~5×10 １１ The population being cells.

22. 1. A pharmaceutical composition comprising a population of TILs comprising IL-2 and one or more TME stimulatory agents, said one or more TME stimulants comprising at least one or more of the Group B agents selected from the group consisting of ipilimumab and tremelimumab; The population is 1×10 ８ ~5×10 １１ The pharmaceutical composition is a cell.

23. 1. A pharmaceutical composition comprising a population of TILs comprising IL-2 and one or more TME stimulatory agents, The one or more TME stimulants include at least: One or more of the Group A agents selected from the group consisting of pembrolizumab, nivolumab, cemiplimab, sym021, atezolizumab, avelumab, and durvalumab; and One or more of the Group B agents selected from the group consisting of ipilimumab and tremelimumab Including, The population is 1×10 ８ ~5×10 １１The pharmaceutical composition is a cell.

24. 24. The pharmaceutical composition of claim 23, further comprising at least one or more of Group J agents selected from the group consisting of urelumab and utomirumab.

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