Remnant tumor infiltrating lymphocytes and methods for preparing and using same - Patents.com

Abstract

Remnant tumor-infiltrating lymphocytes and methods for preparing and using same are provided. [Solution] In some embodiments, a method is disclosed for delivering a therapeutically effective amount of an expanded number of tumor-infiltrating lymphocytes obtained from tumor remnants to a patient in need thereof for the treatment of cancer.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This international application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 423,750, filed November 17, 2016, and U.S. Provisional Patent Application No. 62 / 460,441, filed February 17, 2017, the entireties of which are incorporated by reference herein.

[0002] FIELD OF THEINVENTION SUMMARY OF THE DISCLOSURE

[0002] Methods and compositions for expanding tumor-infiltrating lymphocytes from tumor remnants are disclosed in some embodiments. [Background technology]

[0003] 2. Background of the Invention

[0003] The treatment of bulky and refractory cancers with adoptive transfer of tumor infiltrating lymphocytes (TILs) represents a powerful therapeutic approach for patients with poor prognosis. Gattinoni, et al, Nat. Rev. Immunol. 2006, 6, 383-393. Adoptive T cell therapy with autologous TILs achieves objective response rates of up to 55% and durable regressions of >25% in metastatic melanoma patients. Large numbers of TILs are required for successful immunotherapy, and a robust and reliable process is necessary for commercialization. This has been difficult to achieve due to technical, theoretical, and regulatory issues related to cell expansion. IL-2-based TIL expansion followed by the "rapid expansion process" (REP) has become the preferred method of TIL expansion due to its speed and efficiency. Dudley, et al, Science 2002, 298, 850-54;Dudley, et al, J. Clin. Oncol. 2005, 23, 2346-57; Dudley, et al, J. Clin. Oncol. 2008, 26, 5233-39; Riddell, et al, Science 1992, 257, 238-41;Dudley, et al, J. Immunother. 2003, 26, 332-42. The process of generating TILs from resected tumors is similar to that of tumors grown from 1 to 3 mm in size. 3 The method includes dissecting the tumor into small fragments and expanding the TILs in the presence of interleukin 2 (IL-2) in a pre-rapid proliferation protocol (pre-REP or initiation) step. During the pre-REP step, tumor-resident immune cells migrate and proliferate, and these TILs are subjected to a second REP process in which irradiated peripheral blood mononuclear cell (PBMC) feeders, anti-CD3 antibodies (OKT-3, muromonab), and IL-2 greatly expand the number of TILs. To date, all TIL expansion processes discard the residual tumor fragments after the pre-REP process.

[0004]

[0004] Direct enzymatic digestion of resected tumors has previously been explored as an alternative to pre-REP, but has been reported to yield fewer TIL cultures and a reduced ability to obtain TILs than the IL-2-initiated pre-REP process. Dudley, et al, J. Immunother. 2003, 26, 332-42. For this reason, digestion has not been further explored in the development of TILs as a treatment for cancer. Summary of the Invention [Problem to be solved by the invention]

[0005]

[0005] TILs derived from the pre-REP and REP processes have dominated clinical studies of TILs to date, which have provided modest clinical responses. The field remains challenging, particularly in the extension of TIL therapy from melanoma to other tumor types. Goff, et al, J. Clin. Oncol. 2016, 34, 2389-97; Dudley, et al, J. Clin. Oncol. 2008, 26, 5233-39; Rosenberg, et al, Clin. Cancer Res. 2011, 17, 4550-57. Much of the focus has been on specific subsets (e.g., CD8 + It has been defined to select TILs (T cells) or to select proliferating TILs to target driver mutations, such as mutated ERBB2IP epitopes or driver mutations in KRAS oncogenes. Tran, et al., N. Engl. J. Med. 2016, 375, 2255-62; Tran, et al, Science 2014, 344, 641-45. However, such selection approaches, even if developed and can show efficacy in larger clinical trials, significantly increase the time, complexity and cost of implementing TIL therapy, limiting the possibility of widespread use of TIL therapy in various types of cancer. Therefore, there is an urgent need to develop a process that can provide TILs with improved properties for use in cancer therapy. [Means for solving the problem]

[0006]

[0006] The present invention provides the discovery that TILs with improved properties can be obtained from a tumor remnant cell-based process, and unexpectedly, such remnant TILs (rTILs) are phenotypically and functionally distinct from normal emigrant TILs (eTILs). The use of rTILs and the combination of rTILs and eTILs in cancer immunotherapy offers significant advantages over previous eTIL-based therapies.

[0007] Summary of the Invention In one embodiment, the present invention includes a method for preparing remnant tumor infiltrating lymphocytes (rTILs) for adoptive T cell therapy, the method comprising: (a) obtaining tumor tissue from a patient, the tumor tissue comprising tumor infiltrating lymphocytes (TILs); (b) fragmenting the tumor tissue; and (c) treating the tumor tissue in a gas permeable container with the first cell culture medium and interleukin 2 (IL-2) to provide tumor remnants and nascent TILs (eTILs); (d) excluding at least some eTILs; and (e) enzymatically digesting the tumor remnant into tumor remnant cells using a digestion mixture; (f) expanding the tumor remnant cells in a second cell culture medium comprising cell culture medium, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas permeable container to provide an expanded number of remnant tumor infiltrating lymphocytes (rTILs); Including, The rTILs express reduced levels of T cell exhaustion markers relative to eTILs, the T cell exhaustion markers being selected from the group consisting of TIM3, LAG3, TIGIT, PD-1, CTLA-4, and combinations thereof.

[0008] In one embodiment, the present invention includes a method for preparing remnant tumor infiltrating lymphocytes (rTILs) for adoptive T cell therapy, the method comprising: (a) obtaining tumor tissue from a patient, the tumor tissue comprising tumor infiltrating lymphocytes (TILs); (b) fragmenting the tumor tissue; and (c) treating the tumor tissue in a gas permeable container with the first cell culture medium and interleukin 2 (IL-2) to provide tumor remnants and nascent TILs (eTILs); (d) excluding at least some eTILs; and (e) enzymatically digesting the tumor remnant into tumor remnant cells using a digestion mixture; (f) expanding the tumor remnant cells in a second cell culture medium comprising cell culture medium, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas permeable container to provide an expanded number of remnant tumor infiltrating lymphocytes (rTILs); Including, rTILs expressed reduced levels of T cell exhaustion markers relative to eTILs, the T cell exhaustion marker is selected from the group consisting of TIM3, LAG3, TIGIT, PD-1, CTLA-4, and combinations thereof; The tumor tissue is selected from the group consisting of melanoma tumor tissue, head and neck tumor tissue, breast tumor tissue, kidney tumor tissue, pancreatic tumor tissue, glioblastoma tumor tissue, lung tumor tissue, colorectal tumor tissue, sarcoma tumor tissue, triple negative breast tumor tissue, cervical tumor tissue, ovarian tumor tissue, and acute myeloid leukemia bone marrow or tumor tissue.

[0009] In one embodiment, the present invention includes a method for preparing remnant tumor infiltrating lymphocytes (rTILs) for adoptive T cell therapy, the method comprising: (a) obtaining tumor tissue from a patient, the tumor tissue comprising tumor infiltrating lymphocytes (TILs); (b) fragmenting the tumor tissue; and (c) treating the tumor tissue in a gas permeable container with the first cell culture medium and interleukin 2 (IL-2) to provide tumor remnants and nascent TILs (eTILs); (d) excluding at least some eTILs; and (e) enzymatically digesting the tumor remnant into tumor remnant cells using a digestion mixture; (f) expanding the tumor remnant cells in a second cell culture medium comprising cell culture medium, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas permeable container to provide an expanded number of remnant tumor infiltrating lymphocytes (rTILs); Including, rTILs expressed reduced levels of T cell exhaustion markers relative to eTILs, the T cell exhaustion marker is selected from the group consisting of TIM3, LAG3, TIGIT, PD-1, CTLA-4, and combinations thereof; The irradiated feeder cells include irradiated allogeneic peripheral blood mononuclear cells.

[0010] In one embodiment, the present invention includes a method for preparing remnant tumor infiltrating lymphocytes (rTILs) for adoptive T cell therapy, the method comprising: (a) obtaining tumor tissue from a patient, wherein the tumor tissue contains tumor infiltrating lymphocytes (TILs); (b) fragmenting the tumor tissue; and (c) treating the tumor tissue in a gas permeable container with the first cell culture medium and interleukin 2 (IL-2) to provide tumor remnants and nascent TILs (eTILs); (d) excluding at least some eTILs; and (e) enzymatically digesting the tumor remnant into tumor remnant cells using a digestion mixture; (f) expanding the tumor remnant cells in a second cell culture medium comprising cell culture medium, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas permeable container to provide an expanded number of remnant tumor infiltrating lymphocytes (rTILs); Including, rTILs expressed reduced levels of T cell exhaustion markers relative to eTILs, the T cell exhaustion marker is selected from the group consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof; IL-2 is present in the second cell culture medium at an initial concentration of about 3000 IU / mL, and OKT-3 antibody is present in the second cell culture medium at an initial concentration of about 30 ng / mL.

[0011] In one embodiment, the present invention includes a method for preparing remnant tumor infiltrating lymphocytes (rTILs) for adoptive T cell therapy, the method comprising: (a) obtaining tumor tissue from a patient, the tumor tissue comprising tumor infiltrating lymphocytes (TILs); (b) fragmenting the tumor tissue; and (c) treating the tumor tissue in a gas permeable container with the first cell culture medium and interleukin 2 (IL-2) to provide tumor remnants and nascent TILs (eTILs); (d) excluding at least some eTILs; and (e) enzymatically digesting the tumor remnant into tumor remnant cells using a digestion mixture; (f) expanding the tumor remnant cells in a second cell culture medium comprising cell culture medium, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas permeable container to provide an expanded number of remnant tumor infiltrating lymphocytes (rTILs); Including, rTILs expressed reduced levels of T cell exhaustion markers relative to eTILs, the T cell exhaustion marker is selected from the group consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof; CD8 in rTILs + T cells and CD4 + At least one T cell exhaustion marker in T cells is reduced by at least 10% relative to eTILs.

[0012] In one embodiment, the present invention includes a method for preparing remnant tumor infiltrating lymphocytes (rTILs) for adoptive T cell therapy, the method comprising: (a) obtaining tumor tissue from a patient, the tumor tissue comprising tumor infiltrating lymphocytes (TILs); (b) fragmenting the tumor tissue; and (c) treating the tumor tissue in a gas permeable container with the first cell culture medium and interleukin 2 (IL-2) to provide tumor remnants and nascent TILs (eTILs); (d) excluding at least some eTILs; and (e) enzymatically digesting the tumor remnant into tumor remnant cells using a digestion mixture; (f) expanding the tumor remnant cells in a second cell culture medium comprising cell culture medium, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas permeable container to provide an expanded number of remnant tumor infiltrating lymphocytes (rTILs); Including, rTILs expressed reduced levels of T cell exhaustion markers relative to eTILs, the T cell exhaustion marker is selected from the group consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof; T cell exhaustion markers are CD8 + The LAG3 marker in T cells and in rTILs is reduced by at least 2-fold relative to eTILs.

[0013] In one embodiment, the present invention includes a method for preparing remnant tumor infiltrating lymphocytes (rTILs) for adoptive T cell therapy, the method comprising: (a) obtaining tumor tissue from a patient, the tumor tissue comprising tumor infiltrating lymphocytes (TILs); (b) fragmenting the tumor tissue; and (c) treating the tumor tissue in a gas permeable container with the first cell culture medium and interleukin 2 (IL-2) to provide tumor remnants and nascent TILs (eTILs); (d) excluding at least some eTILs; and (e) enzymatically digesting the tumor remnant into tumor remnant cells using a digestion mixture; (f) expanding the tumor remnant cells in a second cell culture medium comprising cell culture medium, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas permeable container to provide an expanded number of remnant tumor infiltrating lymphocytes (rTILs); Including, rTILs expressed reduced levels of T cell exhaustion markers relative to eTILs, the T cell exhaustion marker is selected from the group consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof; T cell exhaustion markers are CD8 + The TIM3 marker in T cells and the LAG3 marker in rTILs are reduced by at least 3-fold relative to eTILs.

[0014] In one embodiment, the present invention includes a method for preparing remnant tumor infiltrating lymphocytes (rTILs) for adoptive T cell therapy, the method comprising: (a) obtaining tumor tissue from a patient, the tumor tissue comprising tumor infiltrating lymphocytes (TILs); (b) fragmenting the tumor tissue; and (c) treating the tumor tissue in a gas permeable container with the first cell culture medium and interleukin 2 (IL-2) to provide tumor remnants and nascent TILs (eTILs); (d) excluding at least some eTILs; and (e) enzymatically digesting the tumor remnant into tumor remnant cells using a digestion mixture; (f) expanding the tumor remnant cells in a second cell culture medium comprising cell culture medium, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas permeable container to provide an expanded number of remnant tumor infiltrating lymphocytes (rTILs); Including, rTILs expressed reduced levels of T cell exhaustion markers relative to eTILs, the T cell exhaustion marker is selected from the group consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof; T cell exhaustion markers are CD4 + The TIM3 marker in T cells and the LAG3 marker in rTILs are reduced by at least 2-fold relative to eTILs.

[0015] In one embodiment, the present invention includes a method for preparing remnant tumor infiltrating lymphocytes (rTILs) for adoptive T cell therapy, the method comprising: (a) obtaining tumor tissue from a patient, the tumor tissue comprising tumor infiltrating lymphocytes (TILs); (b) fragmenting the tumor tissue; and (c) treating the tumor tissue in a gas permeable container with the first cell culture medium and interleukin 2 (IL-2) to provide tumor remnants and nascent TILs (eTILs); (d) excluding at least some eTILs; and (e) enzymatically digesting the tumor remnant into tumor remnant cells using a digestion mixture; (f) expanding the tumor remnant cells in a second cell culture medium comprising cell culture medium, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas permeable container to provide an expanded number of remnant tumor infiltrating lymphocytes (rTILs); Including, rTILs expressed reduced levels of T cell exhaustion markers relative to eTILs, the T cell exhaustion marker is selected from the group consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof; TIM3 and LAG3 markers in rTILs are undetectable by flow cytometry.

[0016] In one embodiment, the present invention includes a method for preparing remnant tumor infiltrating lymphocytes (rTILs) for adoptive T cell therapy, the method comprising: (a) obtaining tumor tissue from a patient, the tumor tissue comprising tumor infiltrating lymphocytes (TILs); (b) fragmenting the tumor tissue; and (c) treating the tumor tissue in a gas permeable container with the first cell culture medium and interleukin 2 (IL-2) to provide tumor remnants and nascent TILs (eTILs); (d) excluding at least some eTILs; and (e) enzymatically digesting the tumor remnant into tumor remnant cells using a digestion mixture; (f) expanding the tumor remnant cells in a second cell culture medium comprising cell culture medium, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas permeable container to provide an expanded number of remnant tumor infiltrating lymphocytes (rTILs); Including, rTILs expressed reduced levels of T cell exhaustion markers relative to eTILs, the T cell exhaustion marker is selected from the group consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof; CD56 in rTILs + Expression of CD56 in eTILs + Expression is reduced by at least 3-fold.

[0017] In one embodiment, the present invention includes a method for preparing remnant tumor infiltrating lymphocytes (rTILs) for adoptive T cell therapy, the method comprising: (a) obtaining tumor tissue from a patient, the tumor tissue comprising tumor infiltrating lymphocytes (TILs); (b) fragmenting the tumor tissue; and (c) treating the tumor tissue in a gas permeable container with the first cell culture medium and interleukin 2 (IL-2) to provide tumor remnants and nascent TILs (eTILs); (d) excluding at least some eTILs; and (e) enzymatically digesting the tumor remnant into tumor remnant cells using a digestion mixture; (f) expanding the tumor remnant cells in a second cell culture medium comprising cell culture medium, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas permeable container to provide an expanded number of remnant tumor infiltrating lymphocytes (rTILs); Including, rTILs expressed reduced levels of T cell exhaustion markers relative to eTILs, the T cell exhaustion marker is selected from the group consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof; CD69 in rTILs + Expression of CD69 in eTILs + Expression is increased by at least 2-fold.

[0018] In one embodiment, the present invention includes a method for preparing remnant tumor infiltrating lymphocytes (rTILs) for adoptive T cell therapy, the method comprising: (a) obtaining tumor tissue from a patient, the tumor tissue comprising tumor infiltrating lymphocytes (TILs); (b) fragmenting the tumor tissue; and (c) treating the tumor tissue in a gas permeable container with the first cell culture medium and interleukin 2 (IL-2) to provide tumor remnants and nascent TILs (eTILs); (d) excluding at least some eTILs; and (e) enzymatically digesting the tumor remnant into tumor remnant cells using a digestion mixture; (f) expanding the tumor remnant cells in a second cell culture medium comprising cell culture medium, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas permeable container to provide an expanded number of remnant tumor infiltrating lymphocytes (rTILs); Including, rTILs expressed reduced levels of T cell exhaustion markers relative to eTILs, the T cell exhaustion marker is selected from the group consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof; The digestion mixture contains deoxyribonuclease, collagenase and hyaluronidase.

[0019] In one embodiment, the present invention includes a method for preparing remnant tumor infiltrating lymphocytes (rTILs) for adoptive T cell therapy, the method comprising: (a) obtaining tumor tissue from a patient, the tumor tissue comprising tumor infiltrating lymphocytes (TILs); (b) fragmenting the tumor tissue; and (c) treating the tumor tissue in a gas permeable container with the first cell culture medium and interleukin 2 (IL-2) to provide tumor remnants and nascent TILs (eTILs); (d) excluding at least some eTILs; and (e) enzymatically digesting the tumor remnant into tumor remnant cells using a digestion mixture; (f) expanding the tumor remnant cells in a second cell culture medium comprising cell culture medium, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas permeable container to provide an expanded number of remnant tumor infiltrating lymphocytes (rTILs); Including, rTILs expressed reduced levels of T cell exhaustion markers relative to eTILs, the T cell exhaustion marker is selected from the group consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof; The first cell culture medium further comprises a cytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21, and combinations thereof.

[0020] In one embodiment, the present invention includes a method for preparing remnant tumor infiltrating lymphocytes (rTILs) for adoptive T cell therapy, the method comprising: (a) obtaining tumor tissue from a patient, the tumor tissue comprising tumor infiltrating lymphocytes (TILs); (b) fragmenting the tumor tissue; and (c) treating the tumor tissue in a gas permeable container with the first cell culture medium and interleukin 2 (IL-2) to provide tumor remnants and nascent TILs (eTILs); (d) excluding at least some eTILs; and (e) enzymatically digesting the tumor remnant into tumor remnant cells using a digestion mixture; (f) expanding the tumor remnant cells in a second cell culture medium comprising cell culture medium, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas permeable container to provide an expanded number of remnant tumor infiltrating lymphocytes (rTILs); Including, rTILs expressed reduced levels of T cell exhaustion markers relative to eTILs, the T cell exhaustion marker is selected from the group consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof; The second cell culture medium further comprises a cytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21, and combinations thereof.

[0021] In one embodiment, the invention includes a method of treating cancer in a patient in need of such treatment, the treatment comprising delivering a therapeutically effective amount of rTILs to the patient, the rTILs comprising: (a) obtaining tumor tissue from a patient, the tumor tissue comprising tumor infiltrating lymphocytes (TILs); (b) fragmenting the tumor tissue; (c) treating the tumor tissue in a gas permeable container with the first cell culture medium and interleukin 2 (IL-2) to provide tumor remnants and nascent TILs (eTILs); (d) removing at least some of the eTILs; (e) enzymatically digesting the tumor remnant into tumor remnant cells using the digestion mixture; (f) growing the tumor remnant cells in a second cell culture medium containing cell culture medium, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas permeable container to expand the number of remnant tumor infiltrating lymphocytes (rTILs); and preparing the product according to a method comprising: rTILs expressed reduced levels of T cell exhaustion markers relative to eTILs, the T cell exhaustion marker is selected from the group consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof; The second cell culture medium further comprises a cytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21, and combinations thereof.

[0022] In one embodiment, the invention includes a method of treating cancer in a patient in need of such treatment, the method comprising: (a) obtaining tumor tissue from a patient, the tumor tissue comprising tumor infiltrating lymphocytes (TILs); (b) fragmenting the tumor tissue; and (c) treating the tumor tissue in a gas permeable container with the first cell culture medium and interleukin 2 (IL-2) to provide tumor remnants and nascent TILs (eTILs); (d) excluding at least some eTILs; and (e) enzymatically digesting the tumor remnant into tumor remnant cells using a digestion mixture; (f) expanding the tumor remnant cells in a second cell culture medium comprising cell culture medium, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas permeable container to provide an expanded number of remnant tumor infiltrating lymphocytes (rTILs); rTILs expressed reduced levels of T cell exhaustion markers relative to eTILs, the T cell exhaustion marker is selected from the group consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof; providing a second cell culture medium further comprising a cytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21, and combinations thereof; (g) treating the patient with a non-myeloablative lymphodepleting regimen prior to administering the rTILs to the patient; (h) administering to the patient a therapeutically effective amount of rTILs; (i) treating the patient with a high-dose IL-2 regimen starting the day after administration of the rTILs to the patient; Includes.

[0023] In one embodiment, the invention includes a method of treating cancer in a patient in need of such treatment, the method comprising: (a) obtaining tumor tissue from a patient, the tumor tissue comprising tumor infiltrating lymphocytes (TILs); (b) fragmenting the tumor tissue; and (c) treating the tumor tissue in a gas permeable container with the first cell culture medium and interleukin 2 (IL-2) to provide tumor remnants and nascent TILs (eTILs); (d) excluding at least some eTILs; and (e) enzymatically digesting the tumor remnant into tumor remnant cells using a digestion mixture; (f) expanding the tumor remnant cells in a second cell culture medium comprising cell culture medium, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas permeable container to provide an expanded number of remnant tumor infiltrating lymphocytes (rTILs); rTILs expressed reduced levels of T cell exhaustion markers relative to eTILs, the T cell exhaustion marker is selected from the group consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof; providing a second cell culture medium further comprising a cytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21, and combinations thereof; (g) treating the patient with a non-myeloablative lymphodepleting regimen prior to administering the rTILs to the patient; (h) administering to the patient a therapeutically effective amount of rTILs, wherein a therapeutically effective amount of eTILs is co-administered to the patient in a mixture with the rTILs; (i) treating the patient with a high-dose IL-2 regimen starting the day after administration of the rTILs to the patient; Includes.

[0024] In one embodiment, the invention includes a method of treating cancer in a patient in need of such treatment, the method comprising: (a) obtaining tumor tissue from a patient, the tumor tissue comprising tumor infiltrating lymphocytes (TILs); (b) fragmenting the tumor tissue; and (c) treating the tumor tissue in a gas permeable container with the first cell culture medium and interleukin 2 (IL-2) to provide tumor remnants and nascent TILs (eTILs); (d) excluding at least some eTILs; and (e) enzymatically digesting the tumor remnant into tumor remnant cells using a digestion mixture; (f) expanding the tumor remnant cells in a second cell culture medium comprising cell culture medium, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas permeable container to provide an expanded number of remnant tumor infiltrating lymphocytes (rTILs); rTILs expressed reduced levels of T cell exhaustion markers relative to eTILs, the T cell exhaustion marker is selected from the group consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof; providing a second cell culture medium further comprising a cytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21, and combinations thereof; (g) treating the patient with a non-myeloablative lymphodepleting regimen prior to administering the rTILs to the patient; (h) administering to the patient a therapeutically effective amount of rTILs, wherein a therapeutically effective amount of eTILs is co-administered to the patient in a mixture with the rTILs; (i) treating the patient with a high-dose IL-2 regimen starting the day after administration of the rTILs to the patient; Including, The cancer is selected from the group consisting of melanoma, double refractory melanoma, ovarian cancer, cervical cancer, lung cancer, bladder cancer, breast cancer, head and neck cancer, renal cell carcinoma, acute myeloid leukemia, colorectal cancer, sarcoma, non-small cell lung cancer (NSCLC) and triple negative breast cancer.

[0025] In one embodiment, the invention includes a method of treating cancer in a patient in need of such treatment, the method comprising: (a) obtaining tumor tissue from a patient, the tumor tissue comprising tumor infiltrating lymphocytes (TILs); (b) fragmenting the tumor tissue; and (c) treating the tumor tissue in a gas permeable container with the first cell culture medium and interleukin 2 (IL-2) to provide tumor remnants and nascent TILs (eTILs); (d) excluding at least some eTILs; and (e) enzymatically digesting the tumor remnant into tumor remnant cells using a digestion mixture; (f) expanding the tumor remnant cells in a second cell culture medium comprising cell culture medium, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas permeable container to provide an expanded number of remnant tumor infiltrating lymphocytes (rTILs); rTILs expressed reduced levels of T cell exhaustion markers relative to eTILs, the T cell exhaustion marker is selected from the group consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof; providing a second cell culture medium further comprising a cytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21, and combinations thereof; (g) treating the patient with a non-myeloablative lymphodepleting regimen prior to administering the rTILs to the patient, the non-myeloablative lymphodepleting regimen being 60 mg / m 2 / day followed by cyclophosphamide at 25 mg / m for 5 days. 2 administering fludarabine at a dose of 100 mg / kg / day to (h) administering to the patient a therapeutically effective amount of rTILs, wherein a therapeutically effective amount of eTILs is co-administered to the patient in a mixture with the rTILs; (i) treating the patient with a high-dose IL-2 regimen starting the day after administration of the rTILs to the patient; Includes.

[0026] In one embodiment, the invention includes a method of treating cancer in a patient in need of such treatment, the method comprising: (a) obtaining tumor tissue from a patient, the tumor tissue comprising tumor infiltrating lymphocytes (TILs); (b) fragmenting the tumor tissue; and (c) treating the tumor tissue in a gas permeable container with the first cell culture medium and interleukin 2 (IL-2) to provide tumor remnants and nascent TILs (eTILs); (d) excluding at least some eTILs; and (e) enzymatically digesting the tumor remnant into tumor remnant cells using a digestion mixture; (f) expanding the tumor remnant cells in a second cell culture medium comprising cell culture medium, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas permeable container to provide an expanded number of remnant tumor infiltrating lymphocytes (rTILs); rTILs expressed reduced levels of T cell exhaustion markers relative to eTILs, the T cell exhaustion marker is selected from the group consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof; providing a second cell culture medium further comprising a cytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21, and combinations thereof; (g) treating the patient with a non-myeloablative lymphodepleting regimen prior to administering the rTILs to the patient; (h) administering to the patient a therapeutically effective amount of rTILs, wherein a therapeutically effective amount of eTILs is co-administered to the patient in a mixture with the rTILs; (i) treating the patient with a high-dose IL-2 regimen starting the day after administration of the rTILs to the patient; Including, High-dose IL-2 regimens include 600,000 or 720,000 IU / kg aldesleukin or its biosimilar or variant administered as a 15-minute bolus intravenously every 8 hours until tolerated.

[0027] In one embodiment, the present invention includes a method for preparing remnant tumor infiltrating lymphocytes (rTILs) for adoptive T cell therapy, the method comprising: (a) obtaining tumor tissue from a patient, the tumor tissue comprising tumor infiltrating lymphocytes (TILs); (b) fragmenting the tumor tissue; and (c) treating the tumor tissue in a gas permeable container with the first cell culture medium and interleukin 2 (IL-2) to provide tumor remnants and nascent TILs (eTILs); (d) excluding eTIL; (e) enzymatically digesting the tumor remnant into tumor remnant cells using a digestion mixture; (f) expanding the tumor remnant cells in a second cell culture medium comprising cell culture medium, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas permeable container to provide an expanded number of remnant tumor infiltrating lymphocytes (rTILs); Including, The rTILs express reduced levels of T cell exhaustion markers relative to eTILs, the T cell exhaustion markers being selected from the group consisting of TIM3, LAG3, TIGIT, PD-1, CTLA-4, and combinations thereof.

[0028] In one embodiment, the present invention includes a method for preparing remnant tumor infiltrating lymphocytes (rTILs) for adoptive T cell therapy, the method comprising: (a) obtaining tumor tissue from a patient, the tumor tissue comprising tumor infiltrating lymphocytes (TILs); (b) fragmenting the tumor tissue; and (c) treating the tumor tissue in a gas permeable container with the first cell culture medium and interleukin 2 (IL-2) to provide tumor remnants and nascent TILs (eTILs); (d) excluding eTIL; (e) enzymatically digesting the tumor remnant into tumor remnant cells using a digestion mixture; (f) expanding the tumor remnant cells in a second cell culture medium comprising cell culture medium, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas permeable container to provide an expanded number of remnant tumor infiltrating lymphocytes (rTILs); Including, rTILs expressed reduced levels of T cell exhaustion markers relative to eTILs, the T cell exhaustion marker is selected from the group consisting of TIM3, LAG3, TIGIT, PD-1, CTLA-4, and combinations thereof; The tumor tissue is selected from the group consisting of melanoma tumor tissue, head and neck tumor tissue, breast tumor tissue, kidney tumor tissue, pancreatic tumor tissue, glioblastoma tumor tissue, lung tumor tissue, colorectal tumor tissue, sarcoma tumor tissue, triple negative breast tumor tissue, cervical tumor tissue, ovarian tumor tissue, and acute myeloid leukemia bone marrow or tumor tissue.

[0029] In one embodiment, the present invention includes a method for preparing remnant tumor infiltrating lymphocytes (rTILs) for adoptive T cell therapy, the method comprising: (a) obtaining tumor tissue from a patient, the tumor tissue comprising tumor infiltrating lymphocytes (TILs); (b) fragmenting the tumor tissue; and (c) treating the tumor tissue in a gas permeable container with the first cell culture medium and interleukin 2 (IL-2) to provide tumor remnants and nascent TILs (eTILs); (d) excluding eTIL; (e) enzymatically digesting the tumor remnant into tumor remnant cells using a digestion mixture; (f) expanding the tumor remnant cells in a second cell culture medium comprising cell culture medium, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas permeable container to provide an expanded number of remnant tumor infiltrating lymphocytes (rTILs); Including, rTILs expressed reduced levels of T cell exhaustion markers relative to eTILs, the T cell exhaustion marker is selected from the group consisting of TIM3, LAG3, TIGIT, PD-1, CTLA-4, and combinations thereof; The irradiated feeder cells include irradiated allogeneic peripheral blood mononuclear cells.

[0030] In one embodiment, the present invention includes a method for preparing remnant tumor infiltrating lymphocytes (rTILs) for adoptive T cell therapy, the method comprising: (a) obtaining tumor tissue from a patient, the tumor tissue comprising tumor infiltrating lymphocytes (TILs); (b) fragmenting the tumor tissue; and (c) treating the tumor tissue in a gas permeable container with the first cell culture medium and interleukin 2 (IL-2) to provide tumor remnants and nascent TILs (eTILs); (d) excluding eTIL; (e) enzymatically digesting the tumor remnant into tumor remnant cells using a digestion mixture; (f) expanding the tumor remnant cells in a second cell culture medium comprising cell culture medium, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas permeable container to provide an expanded number of remnant tumor infiltrating lymphocytes (rTILs); Including, rTILs expressed reduced levels of T cell exhaustion markers relative to eTILs, the T cell exhaustion marker is selected from the group consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof; IL-2 is present in the second cell culture medium at an initial concentration of about 3000 IU / mL, and OKT-3 antibody is present in the second cell culture medium at an initial concentration of about 30 ng / mL.

[0031] In one embodiment, the invention includes a method for preparing remnant tumor infiltrating lymphocytes (rTILs) for adoptive T cell therapy, the method comprising: (a) obtaining tumor tissue from a patient, the tumor tissue comprising tumor infiltrating lymphocytes (TILs); (b) fragmenting the tumor tissue; and (c) treating the tumor tissue in a gas permeable container with the first cell culture medium and interleukin 2 (IL-2) to provide tumor remnants and nascent TILs (eTILs); (d) excluding eTIL; (e) enzymatically digesting the tumor remnant into tumor remnant cells using a digestion mixture; (f) expanding the tumor remnant cells in a second cell culture medium comprising cell culture medium, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas permeable container to provide an expanded number of remnant tumor infiltrating lymphocytes (rTILs); Including, rTILs expressed reduced levels of T cell exhaustion markers relative to eTILs, the T cell exhaustion marker is selected from the group consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof; CD8 in rTILs +T cells and CD4 + At least one T cell exhaustion marker in T cells is reduced by at least 10% relative to eTILs.

[0032] In one embodiment, the present invention includes a method for preparing remnant tumor infiltrating lymphocytes (rTILs) for adoptive T cell therapy, the method comprising: (a) obtaining tumor tissue from a patient, the tumor tissue comprising tumor infiltrating lymphocytes (TILs); (b) fragmenting the tumor tissue; and (c) treating the tumor tissue in a gas permeable container with the first cell culture medium and interleukin 2 (IL-2) to provide tumor remnants and nascent TILs (eTILs); (d) excluding eTIL; (e) enzymatically digesting the tumor remnant into tumor remnant cells using a digestion mixture; (f) expanding the tumor remnant cells in a second cell culture medium comprising cell culture medium, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas permeable container to provide an expanded number of remnant tumor infiltrating lymphocytes (rTILs); Including, rTILs expressed reduced levels of T cell exhaustion markers relative to eTILs, the T cell exhaustion marker is selected from the group consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof; T cell exhaustion markers are CD8 + The LAG3 marker in T cells and in rTILs is reduced by at least 2-fold relative to eTILs.

[0033] In one embodiment, the present invention includes a method for preparing remnant tumor infiltrating lymphocytes (rTILs) for adoptive T cell therapy, the method comprising: (a) obtaining tumor tissue from a patient, the tumor tissue comprising tumor infiltrating lymphocytes (TILs); (b) fragmenting the tumor tissue; and (c) treating the tumor tissue in a gas permeable container with the first cell culture medium and interleukin 2 (IL-2) to provide tumor remnants and nascent TILs (eTILs); (d) excluding eTIL; (e) enzymatically digesting the tumor remnant into tumor remnant cells using a digestion mixture; (f) expanding the tumor remnant cells in a second cell culture medium comprising cell culture medium, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas permeable container to provide an expanded number of remnant tumor infiltrating lymphocytes (rTILs); Including, rTILs expressed reduced levels of T cell exhaustion markers relative to eTILs, the T cell exhaustion marker is selected from the group consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof; T cell exhaustion markers are CD8 + The TIM3 marker in T cells and the LAG3 marker in rTILs are reduced by at least 3-fold relative to eTILs.

[0034] In one embodiment, the present invention includes a method for preparing remnant tumor infiltrating lymphocytes (rTILs) for adoptive T cell therapy, the method comprising: (a) obtaining tumor tissue from a patient, the tumor tissue comprising tumor infiltrating lymphocytes (TILs); (b) fragmenting the tumor tissue; and (c) treating the tumor tissue in a gas permeable container with the first cell culture medium and interleukin 2 (IL-2) to provide tumor remnants and nascent TILs (eTILs); (d) excluding eTIL; (e) enzymatically digesting the tumor remnant into tumor remnant cells using a digestion mixture; (f) expanding the tumor remnant cells in a second cell culture medium comprising cell culture medium, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas permeable container to provide an expanded number of remnant tumor infiltrating lymphocytes (rTILs); Including, rTILs expressed reduced levels of T cell exhaustion markers relative to eTILs, the T cell exhaustion marker is selected from the group consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof; T cell exhaustion markers are CD4 + The TIM3 marker in T cells and the LAG3 marker in rTILs are reduced by at least 2-fold relative to eTILs.

[0035] In one embodiment, the present invention includes a method for preparing remnant tumor infiltrating lymphocytes (rTILs) for adoptive T cell therapy, the method comprising: (a) obtaining tumor tissue from a patient, the tumor tissue comprising tumor infiltrating lymphocytes (TILs); (b) fragmenting the tumor tissue; and (c) treating the tumor tissue in a gas permeable container with the first cell culture medium and interleukin 2 (IL-2) to provide tumor remnants and nascent TILs (eTILs); (d) excluding eTIL; (e) enzymatically digesting the tumor remnant into tumor remnant cells using a digestion mixture; (f) expanding the tumor remnant cells in a second cell culture medium comprising cell culture medium, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas permeable container to provide an expanded number of remnant tumor infiltrating lymphocytes (rTILs); Including, rTILs expressed reduced levels of T cell exhaustion markers relative to eTILs, the T cell exhaustion marker is selected from the group consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof; TIM3 and LAG3 markers in rTILs are undetectable by flow cytometry.

[0036] In one embodiment, the present invention includes a method for preparing remnant tumor infiltrating lymphocytes (rTILs) for adoptive T cell therapy, the method comprising: (a) obtaining tumor tissue from a patient, the tumor tissue comprising tumor infiltrating lymphocytes (TILs); (b) fragmenting the tumor tissue; and (c) treating the tumor tissue in a gas permeable container with the first cell culture medium and interleukin 2 (IL-2) to provide tumor remnants and nascent TILs (eTILs); (d) excluding eTIL; (e) enzymatically digesting the tumor remnant into tumor remnant cells using a digestion mixture; (f) expanding the tumor remnant cells in a second cell culture medium comprising cell culture medium, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas permeable container to provide an expanded number of remnant tumor infiltrating lymphocytes (rTILs); Including, rTILs expressed reduced levels of T cell exhaustion markers relative to eTILs, the T cell exhaustion marker is selected from the group consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof; CD56 in rTILs + Expression of CD56 in eTILs + Expression is reduced by at least 3-fold.

[0037] In one embodiment, the present invention includes a method for preparing remnant tumor infiltrating lymphocytes (rTILs) for adoptive T cell therapy, the method comprising: (a) obtaining tumor tissue from a patient, the tumor tissue comprising tumor infiltrating lymphocytes (TILs); (b) fragmenting the tumor tissue; and (c) treating the tumor tissue in a gas permeable container with the first cell culture medium and interleukin 2 (IL-2) to provide tumor remnants and nascent TILs (eTILs); (d) excluding eTIL; (e) enzymatically digesting the tumor remnant into tumor remnant cells using a digestion mixture; (f) expanding the tumor remnant cells in a second cell culture medium comprising cell culture medium, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas permeable container to provide an expanded number of remnant tumor infiltrating lymphocytes (rTILs); Including, rTILs expressed reduced levels of T cell exhaustion markers relative to eTILs, the T cell exhaustion marker is selected from the group consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof; CD69 in rTILs + Expression of CD69 in eTILs + Expression is increased by at least 2-fold.

[0038] In one embodiment, the present invention includes a method for preparing remnant tumor infiltrating lymphocytes (rTILs) for adoptive T cell therapy, the method comprising: (a) obtaining tumor tissue from a patient, the tumor tissue comprising tumor infiltrating lymphocytes (TILs); (b) fragmenting the tumor tissue; and (c) treating the tumor tissue in a gas permeable container with the first cell culture medium and interleukin 2 (IL-2) to provide tumor remnants and nascent TILs (eTILs); (d) excluding eTIL; (e) enzymatically digesting the tumor remnant into tumor remnant cells using a digestion mixture; (f) expanding the tumor remnant cells in a second cell culture medium comprising cell culture medium, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas permeable container to provide an expanded number of remnant tumor infiltrating lymphocytes (rTILs); Including, rTILs expressed reduced levels of T cell exhaustion markers relative to eTILs, the T cell exhaustion marker is selected from the group consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof; The digestion mixture contains deoxyribonuclease, collagenase and hyaluronidase.

[0039] In one embodiment, the present invention includes a method for preparing remnant tumor infiltrating lymphocytes (rTILs) for adoptive T cell therapy, the method comprising: (a) obtaining tumor tissue from a patient, the tumor tissue comprising tumor infiltrating lymphocytes (TILs); (b) fragmenting the tumor tissue; and (c) treating the tumor tissue in a gas permeable container with the first cell culture medium and interleukin 2 (IL-2) to provide tumor remnants and nascent TILs (eTILs); (d) excluding eTIL; (e) enzymatically digesting the tumor remnant into tumor remnant cells using a digestion mixture; (f) expanding the tumor remnant cells in a second cell culture medium comprising cell culture medium, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas permeable container to provide an expanded number of remnant tumor infiltrating lymphocytes (rTILs); Including, rTILs expressed reduced levels of T cell exhaustion markers relative to eTILs, the T cell exhaustion marker is selected from the group consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof; The first cell culture medium further comprises a cytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21, and combinations thereof.

[0040] In one embodiment, the present invention includes a method for preparing remnant tumor infiltrating lymphocytes (rTILs) for adoptive T cell therapy, the method comprising: (a) obtaining tumor tissue from a patient, the tumor tissue comprising tumor infiltrating lymphocytes (TILs); (b) fragmenting the tumor tissue; and (c) treating the tumor tissue in a gas permeable container with the first cell culture medium and interleukin 2 (IL-2) to provide tumor remnants and nascent TILs (eTILs); (d) excluding eTIL; (e) enzymatically digesting the tumor remnant into tumor remnant cells using a digestion mixture; (f) expanding the tumor remnant cells in a second cell culture medium comprising cell culture medium, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas permeable container to provide an expanded number of remnant tumor infiltrating lymphocytes (rTILs); Including, rTILs expressed reduced levels of T cell exhaustion markers relative to eTILs, the T cell exhaustion marker is selected from the group consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof; The second cell culture medium further comprises a cytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21, and combinations thereof.

[0041] In one embodiment, the invention includes a method of treating cancer in a patient in need of such treatment, the treatment comprising delivering a therapeutically effective amount of rTILs to the patient, the rTILs comprising: (a) obtaining tumor tissue from a patient, the tumor tissue comprising tumor infiltrating lymphocytes (TILs); (b) fragmenting the tumor tissue; (c) treating the tumor tissue in a gas permeable container with the first cell culture medium and interleukin 2 (IL-2) to provide tumor remnants and nascent TILs (eTILs); (d) removing the eTIL; (e) enzymatically digesting the tumor remnant into tumor remnant cells using the digestion mixture; (f) growing the tumor remnant cells in a second cell culture medium containing cell culture medium, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas permeable container to expand the number of remnant tumor infiltrating lymphocytes (rTILs); and preparing the product according to a method comprising: rTILs expressed reduced levels of T cell exhaustion markers relative to eTILs, the T cell exhaustion marker is selected from the group consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof; The second cell culture medium further comprises a cytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21, and combinations thereof.

[0042] In one embodiment, the invention includes a method of treating cancer in a patient in need of such treatment, the method comprising: (a) obtaining tumor tissue from a patient, the tumor tissue comprising tumor infiltrating lymphocytes (TILs); (b) fragmenting the tumor tissue; and (c) treating the tumor tissue in a gas permeable container with the first cell culture medium and interleukin 2 (IL-2) to provide tumor remnants and nascent TILs (eTILs); (d) excluding eTIL; (e) enzymatically digesting the tumor remnant into tumor remnant cells using a digestion mixture; (f) expanding the tumor remnant cells in a second cell culture medium comprising cell culture medium, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas permeable container to provide an expanded number of remnant tumor infiltrating lymphocytes (rTILs); rTILs expressed reduced levels of T cell exhaustion markers relative to eTILs, the T cell exhaustion marker is selected from the group consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof; providing a second cell culture medium further comprising a cytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21, and combinations thereof; (g) treating the patient with a non-myeloablative lymphodepleting regimen prior to administering the rTILs to the patient; (h) administering to the patient a therapeutically effective amount of rTILs; (i) treating the patient with a high-dose IL-2 regimen starting the day after administration of the rTILs to the patient; Includes.

[0043] In one embodiment, the invention includes a method of treating cancer in a patient in need of such treatment, the method comprising: (a) obtaining tumor tissue from a patient, the tumor tissue comprising tumor infiltrating lymphocytes (TILs); (b) fragmenting the tumor tissue; and (c) treating the tumor tissue in a gas permeable container with the first cell culture medium and interleukin 2 (IL-2) to provide tumor remnants and nascent TILs (eTILs); (d) excluding eTIL; (e) enzymatically digesting the tumor remnant into tumor remnant cells using a digestion mixture; (f) expanding the tumor remnant cells in a second cell culture medium comprising cell culture medium, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas permeable container to provide an expanded number of remnant tumor infiltrating lymphocytes (rTILs); rTILs expressed reduced levels of T cell exhaustion markers relative to eTILs, the T cell exhaustion marker is selected from the group consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof; providing a second cell culture medium further comprising a cytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21, and combinations thereof; (g) treating the patient with a non-myeloablative lymphodepleting regimen prior to administering the rTILs to the patient; (h) administering to the patient a therapeutically effective amount of rTILs, wherein a therapeutically effective amount of eTILs is co-administered to the patient in a mixture with the rTILs; (i) treating the patient with a high-dose IL-2 regimen starting the day after administration of the rTILs to the patient; Includes.

[0044] In one embodiment, the invention includes a method of treating cancer in a patient in need of such treatment, the method comprising: (a) obtaining tumor tissue from a patient, the tumor tissue comprising tumor infiltrating lymphocytes (TILs); (b) fragmenting the tumor tissue; and (c) treating the tumor tissue in a gas permeable container with the first cell culture medium and interleukin 2 (IL-2) to provide tumor remnants and nascent TILs (eTILs); (d) excluding eTIL; (e) enzymatically digesting the tumor remnant into tumor remnant cells using a digestion mixture; (f) expanding the tumor remnant cells in a second cell culture medium comprising cell culture medium, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas permeable container to provide an expanded number of remnant tumor infiltrating lymphocytes (rTILs); rTILs expressed reduced levels of T cell exhaustion markers relative to eTILs, the T cell exhaustion marker is selected from the group consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof; providing a second cell culture medium further comprising a cytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21, and combinations thereof; (g) treating the patient with a non-myeloablative lymphodepleting regimen prior to administering the rTILs to the patient; (h) administering to the patient a therapeutically effective amount of rTILs, wherein a therapeutically effective amount of eTILs is co-administered to the patient in a mixture with the rTILs; (i) treating the patient with a high-dose IL-2 regimen starting the day after administration of the rTILs to the patient; Including, The cancer is selected from the group consisting of melanoma, double refractory melanoma, ovarian cancer, cervical cancer, lung cancer, bladder cancer, breast cancer, head and neck cancer, renal cell carcinoma, acute myeloid leukemia, colorectal cancer, sarcoma, non-small cell lung cancer (NSCLC) and triple negative breast cancer.

[0045] In one embodiment, the invention includes a method of treating cancer in a patient in need of such treatment, the method comprising: (a) obtaining tumor tissue from a patient, the tumor tissue comprising tumor infiltrating lymphocytes (TILs); (b) fragmenting the tumor tissue; and (c) treating the tumor tissue in a gas permeable container with the first cell culture medium and interleukin 2 (IL-2) to provide tumor remnants and nascent TILs (eTILs); (d) excluding eTIL; (e) enzymatically digesting the tumor remnant into tumor remnant cells using a digestion mixture; (f) expanding the tumor remnant cells in a second cell culture medium comprising cell culture medium, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas permeable container to provide an expanded number of remnant tumor infiltrating lymphocytes (rTILs); rTILs expressed reduced levels of T cell exhaustion markers relative to eTILs, the T cell exhaustion marker is selected from the group consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof; providing a second cell culture medium further comprising a cytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21, and combinations thereof; (g) treating the patient with a non-myeloablative lymphodepleting regimen prior to administering the rTILs to the patient, the non-myeloablative lymphodepleting regimen being 60 mg / m 2 / day followed by cyclophosphamide at 25 mg / m for 5 days. 2 administering fludarabine at a dose of 100 mg / kg / day to (h) administering to the patient a therapeutically effective amount of rTILs, wherein a therapeutically effective amount of eTILs is co-administered to the patient in a mixture with the rTILs; (i) treating the patient with a high-dose IL-2 regimen starting the day after administration of the rTILs to the patient; Includes.

[0046] In one embodiment, the invention includes a method of treating cancer in a patient in need of such treatment, the method comprising: (a) obtaining tumor tissue from a patient, the tumor tissue comprising tumor infiltrating lymphocytes (TILs); (b) fragmenting the tumor tissue; and (c) treating the tumor tissue in a gas permeable container with the first cell culture medium and interleukin 2 (IL-2) to provide tumor remnants and nascent TILs (eTILs); (d) excluding eTIL; (e) enzymatically digesting the tumor remnant into tumor remnant cells using a digestion mixture; (f) expanding the tumor remnant cells in a second cell culture medium comprising cell culture medium, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas permeable container to provide an expanded number of remnant tumor infiltrating lymphocytes (rTILs); rTILs expressed reduced levels of T cell exhaustion markers relative to eTILs, the T cell exhaustion marker is selected from the group consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof; providing a second cell culture medium further comprising a cytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21, and combinations thereof; (g) treating the patient with a non-myeloablative lymphodepleting regimen prior to administering the rTILs to the patient; (h) administering to the patient a therapeutically effective amount of rTILs, wherein a therapeutically effective amount of eTILs is co-administered to the patient in a mixture with the rTILs; (i) treating the patient with a high-dose IL-2 regimen starting the day after administration of the rTILs to the patient; Including, High-dose IL-2 regimens include 600,000 or 720,000 IU / kg aldesleukin or its biosimilar or variant administered as a 15-minute bolus intravenously every 8 hours until tolerated.

[0047]

[0047] In one embodiment, the present invention includes a process for generating an expanded number of tumor remnant cells including tumor infiltrating lymphocytes (TILs) from a patient for adoptive T cell therapy. In some embodiments, the process of the present invention may include obtaining tumor tissue from a patient, the tumor tissue including TILs. In some embodiments, the process of the present invention may include fragmenting the tumor tissue. In some embodiments, the process of the present invention may include processing the tumor tissue in a gas permeable container with cell culture medium and interleukin 2 (IL-2) and other T cell growth factors or agonist antibodies to provide tumor remnants and an expanded number of TILs. In some embodiments, the process of the present invention may include removing the expanded number of TILs. In some embodiments, the process of the present invention may include enzymatically digesting the tumor remnant into tumor remnant cells. In some embodiments, the process of the present invention may include processing the tumor remnant cells with cell culture medium, irradiated feeder cells, anti-CD3 monoclonal antibody (muromonab or OKT-3), and IL-2 to provide an expanded number of tumor remnant cells. In some embodiments, the tumor remnant cells prepared according to the process of the present invention may comprise TILs that express low levels of at least one marker selected from the group consisting of TIM3, LAG3, PD-1, and combinations thereof. In some embodiments, the tumor tissue may be selected from the group consisting of melanoma tumor tissue, head and neck tumor tissue, breast tumor tissue, kidney tumor tissue, pancreatic tumor tissue, lung tumor tissue, and colorectal tumor tissue.

[0048]

[0048] In one embodiment, the present invention may include a method of treating cancer in a patient in need of such treatment. In some embodiments, the treatment may include delivering a therapeutically effective amount of an expanded number of tumor remnant cells to the patient, and the expanded number of tumor remnant cells may be prepared according to any process described herein.

[0049] BRIEF DESCRIPTION OF THE DRAWINGS

[0049] The foregoing summary, as well as the following detailed description of the present invention, will be better understood when read in conjunction with the appended drawings. [Brief description of the drawings]

[0050] [Figure 1]

[0050] An exemplary diagram of the preparation of tumor digestion solution is shown. [Diagram 2]

[0051] An exemplary flow-through diagram of the tumor digestion procedure is shown. In this example, two digestion methods are run simultaneously and seeded separately as part of two different pre-REPs designed to compare the efficacy of each digestion method. [Diagram 3]

[0052] Differential phenotypic expression of key markers in eTILs and rTILs is shown. [Figure 4]

[0053] 2A and 2B show the study of eTILs and rTILs with 2-(N-(7-nitrobenzo-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG) (A) and Mitotracker (B) to assess metabolic capacity prior to rapid proliferation. [Diagram 5]

[0054] Results are shown for an experiment in which eTILs and rTILs were stimulated overnight with CD3 / CD28 / 4-1BB beads with brefeldin A for CD4+ and CD8+ T cells. PMA and ionomycin were added for 4-5 hours. Interferon-γ was assessed by intracellular flow cytometry analysis (n=3). [Figure 6]

[0055] Results are shown demonstrating that (A) rTILs expand during rapid proliferation and (B) remain phenotypically distinct from eTILs. [Figure 7]

[0056] 1 shows an exemplary process for treating a patient with the rTILs of the present invention. [Figure 8]

[0057] 1 shows an exemplary timeline of the process of treating a patient with the rTILs of the present invention. [Figure 9]

[0058] The diversity of the TCR-vβ repertoire (i.e., diversity score) in eTILs and rTILs is shown. [Figure 10]

[0059] The percentage of shared CDR3 in eTILs and rTILs is shown. [Figure 11]

[0060] Cell proliferation analysis in triple-negative breast, colon, lung, renal and melanoma is shown. eTILs from CD4+ or CD8+ populations in all five tumors demonstrated enhanced proliferative capacity as demonstrated by Cell Trace dye shift (or dye dilution) when compared to eTILs alone immediately after co-culture with rTILs with anti-CD3 antibody. Red represents eTILs and blue represents eTILs when co-cultured with rTILs. [Figure 12]

[0061] A heatmap generated from Nanostring analysis is shown, which shows that the gene expression profiles for eTILs and rTILs are significantly different. [Figure 13]

[0062] Graphs generated from Nanostring analysis are shown, which show that several genes are significantly up- or down-regulated in rTILs compared to eTILs. [Figure 14]

[0063] A clonotype graph showing the top 50 shared CDR3s between eTILs and rTILs (for three eTIL / rTIL pairs) derived from ovarian cancer is shown. [Figure 15]

[0064] A clonotype graph showing the top 50 shared CDR3s between renal cancer-derived eTILs and rTILs (for three eTIL / rTIL pairs) is shown. [Figure 16]

[0065] A clonotype graph showing the top 50 shared CDR3s between eTILs and rTILs (for three eTIL / rTIL pairs) derived from triple-negative breast cancer is shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0051] A brief explanation of sequence listings

[0066] SEQ ID NO:1 is the amino acid sequence of the heavy chain of muromonab.

[0067] SEQ ID NO:2 is the amino acid sequence of the light chain of muromonab.

[0068] SEQ ID NO:3 is the amino acid sequence of recombinant human IL-2 protein.

[0069] SEQ ID NO:4 is the amino acid sequence of aldesleukin.

[0070] SEQ ID NO:5 is the amino acid sequence of recombinant human IL-4 protein.

[0071] SEQ ID NO:6 is the amino acid sequence of recombinant human IL-7 protein.

[0072] SEQ ID NO:7 is the amino acid sequence of recombinant human IL-15 protein.

[0073] SEQ ID NO:8 is the amino acid sequence of recombinant human IL-21 protein.

[0052] Detailed Description of the Invention

[0074] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All patents and publications mentioned herein are incorporated by reference in their entirety.

[0053] definition

[0075] The terms "exhaustion phenotype" and "exhaustion marker" refer to cell surface markers characteristic of T cell exhaustion in response to prolonged stimulation of the T cell receptor (TCR) by an antigen. T cells exhibiting an exhausted phenotype express inhibitory receptors such as T cell immunoglobulin and mucin domain containing 3 (TIM3 or TIM-3), lymphocyte activation gene 3 (LAG3 or LAG-3), T cell immunoreceptor with immunoglobulin domain and ITIM domain (TIGIT) and programmed cell death protein 1 (PD-1), and lack the ability to increase the production of effector cytokines and mount an effective immune response. T cell exhaustion is described in Yi, et al., Immunology 2010, 129, 474-81, the disclosure of which is incorporated herein by reference.

[0054]

[0076] As used herein, the terms "co-administration," "co-administering," "administered in combination," "administering in combination," "simultaneous," and "concurrently" include administration of two or more active pharmaceutical ingredients to a human subject such that both active pharmaceutical ingredients and / or their metabolites are present in the human subject at the same time. Co-administration includes simultaneous administration of separate compositions, administration of separate compositions at different times, or administration of a composition in which two or more active pharmaceutical ingredients are present. Simultaneous administration of separate compositions and administration of a composition in which both agents are present are also encompassed by the methods of the invention.

[0055]

[0077] The term "in vivo" refers to events that take place inside a subject's body.

[0056]

[0078] The term "in vitro" refers to events that take place outside a subject's body. In vitro assays include cell-based assays, in which live or dead cells are used, and can also include cell-free assays, in which no intact cells are used.

[0057]

[0079] The term "antigen" refers to a substance that induces an immune response. In some embodiments, an antigen is a molecule that can be bound by an antibody or TCR (when presented by a major histocompatibility complex (MHC) molecule). The term "antigen" as used herein also encompasses T cell epitopes. Antigens can additionally be recognized by the immune system. In some embodiments, an antigen can induce a humoral or cellular immune response, causing activation of B and / or T lymphocytes. In some cases, this may require that the antigen contains or is associated with a Th cell epitope. An antigen may also have one or more epitopes (e.g., B- and T-epitopes). In some embodiments, an antigen will preferably react with its corresponding antibody or TCR, typically with high specificity and selectivity, and not with the multitude of other antibodies or TCRs that may be induced by other antigens.

[0058]

[0080] The term "effective amount" or "therapeutically effective amount" as described herein refers to an amount of a compound or a combination of compounds sufficient to achieve the intended use, including but not limited to the treatment of a disease. The therapeutically effective amount may vary depending on the intended use (in vitro or in vivo) or the human subject and disease state to be treated (e.g., subject's weight, age and sex), the severity of disease symptoms, the mode of administration, etc., and can be easily determined by one skilled in the art. The term also applies to a dose that will induce a specific response in target cells (e.g., reduced platelet adhesion and / or cell migration). The specific dose will vary depending on the particular compound selected, the dosing regimen followed, whether the compound is administered in combination with other compounds, the timing of administration, the tissue to which it is administered, and the bodily delivery system to which the compound is delivered. The therapeutically effective amount may be an "effective antitumor amount" and / or an "effective tumor-inhibiting amount", which may be the exact amount of the composition of the present invention to be administered, and can be determined by a physician taking into account individual differences in the age, weight, tumor size, degree of infection or metastasis, and condition of the patient (subject). Generally, the pharmaceutical compositions comprising the cytotoxic lymphocytes or rTILs described herein are administered in a dose of 10 4 ~10 11 Cells / kg body weight (e.g., 10 5 ~10 6 , 10 5 ~10 10 , 10 5 ~10 11 , 10 6 ~10 10 , 10 6 ~10 11 , 10 7 ~10 11 , 10 7 ~10 10 , 10 8 ~10 11 , 10 8 ~10 10 , 10 9 ~10 11 or 10 9 ~10 10It can be stated that the cytotoxic lymphocyte or rTIL compositions may be administered at a dosage of 100 mg / kg of cells / kg of body weight (including all integer values ​​within these ranges). The cytotoxic lymphocyte or rTIL compositions may also be administered multiple times at these dosages. The cytotoxic lymphocyte or rTIL may be administered using injection techniques commonly known in immunotherapy (see, e.g., Rosenberg et al., N. Eng. J. Med. 319: 1676, 1988). Optimal dosages and treatment regimes for a particular patient can be readily determined by one of ordinary skill in the art of medicine by monitoring the patient for signs of disease and adjusting the treatment accordingly.

[0059]

[0081] The term "therapeutic benefit" as used herein encompasses therapeutic benefit and / or prophylactic benefit in a human subject. A prophylactic benefit includes delaying or eliminating the onset of a disease or condition, delaying or eliminating the onset of symptoms of a disease or condition, slowing, halting or reversing the progression of a disease or condition, or any combination thereof.

[0060]

[0082] A "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and inactive ingredients. The use of such pharmaceutically acceptable carriers or pharmaceutically acceptable excipients for active pharmaceutical ingredients is well known in the art. Except where any conventional pharmaceutically acceptable carrier or pharmaceutically acceptable excipient is incompatible with the active pharmaceutical ingredient, its use in the therapeutic compositions of the present invention is contemplated. Additional active pharmaceutical ingredients, such as other drugs, may also be incorporated into the compositions and methods described.

[0061]

[0083] The terms "treatment", "treating", "treating" and the like refer to obtaining a desired pharmacological and / or physiological effect. The effect may be preventative in terms of completely or partially preventing the disease or its symptoms, and / or therapeutic in terms of partially or completely treating the disease and / or side effects caused by the disease. As used herein, "treatment" encompasses any treatment of disease in a mammal, particularly a human, including: (a) preventing the disease from developing in a subject who may be prone to the disease but has not yet been diagnosed as suffering from the disease; (b) inhibiting the disease, i.e., suppressing the onset or progression of the disease; and (c) relieving the disease, i.e., causing regression of the disease and / or relieving one or more symptoms. "Treatment" is also meant to encompass the delivery of an agent to produce a pharmacological effect in the absence of disease or even symptoms. 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.

[0062]

[0084] The term "heterologous" when used in reference to portions of a nucleic acid or protein indicates that the nucleic acid or protein comprises two or more subsequences that are not found in the same relationship to each other in nature. For example, nucleic acids are typically generated by recombinant techniques where two or more sequences from unrelated genes, such as a promoter from one source and a coding region from another source, or coding regions from different sources, are assembled to form a new functional nucleic acid. Similarly, a protein of heterologous origin indicates that the protein comprises two or more subsequences that are not found in the same relationship to each other in nature (e.g., a fusion protein).

[0063]

[0085] The term "rapid expansion" refers to an increase in the number of antigen-specific TILs of at least about 3-fold (or 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, or 9-fold) over a period of one week, more preferably at least about 10-fold (or 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, or 90-fold) over a period of one week, and most preferably at least about 100-fold over a period of one week. Several rapid expansion protocols are outlined herein.

[0064]

[0086] As used herein, "tumor infiltrating lymphocytes" or "TILs" refers to a population of cells originally derived as leukocytes that have left the bloodstream of a subject and migrated into a tumor. TILs include, but are not limited to, CD8 + Cytotoxic T cells (lymphocytes), Th1 and Th17 CD4 + These include T cells, natural killer cells, dendritic cells, and M1 macrophages. TILs include both primary and secondary TILs. "Primary TILs" are those obtained from patient tissue samples as outlined herein (sometimes referred to as "freshly harvested"), and "secondary TILs" are any TIL cell populations expanded as discussed herein, including but not limited to bulk TILs and expanded TILs ("REP TILs" or "post-REP TILs"). In certain embodiments, the term "primary TILs" may include rTILs and a mixture of eTILs and rTILs.

[0065]

[0087] A "population of cells" (including TILs) herein refers to a number of cells that share a common trait. Generally, a population is generally between 1×10 6 ~1×10 10 , with different TIL populations containing different numbers. For example, initial expansion of primary TILs in the presence of IL-2 yielded approximately 1 × 10 8 REP expansion typically results in a bulk TIL population of cells of 1.5 x 10 for infusion. 9 ~1.5×10 10 This is done to provide a population of cells.

[0066]

[0088] The terms "peripheral blood mononuclear cells" and "PBMCs" refer to peripheral blood cells with round nuclei, including lymphocytes (e.g., T cells, B cells, and NK cells) and monocytes. Preferably, the peripheral blood mononuclear cells are irradiated allogeneic peripheral blood mononuclear cells. PBMCs are a type of antigen-presenting cell.

[0067]

[0089] By "cryopreserved TILs" herein is meant primary TILs, bulk TILs or expanded TILs (REP TILs) that have been processed and stored in the range of about -150°C to -60°C. General methods for cryopreservation are also described elsewhere herein. For clarity, "cryopreserved TILs" are distinguishable from frozen tissue samples that may be used as a source of primary TILs, including rTILs.

[0068]

[0090] "Thawed cryopreserved TILs" herein refers to a population of TILs (e.g., rTILs) that have previously been cryopreserved and then processed to return to room temperature or above, including, but not limited to, cell culture temperature or a temperature at which the TILs can be administered to a patient.

[0069]

[0091] The terms "sequence identity", "percent identity" and "percent sequence identity" in the context of two or more nucleic acids or polypeptides refer to two or more sequences or subsequences that are the same or have a certain percentage of the same nucleotides or amino acid residues when compared and aligned (with gaps introduced as necessary) for maximum correspondence, without considering any conservative amino acid substitutions as part of the sequence identity. Percent identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software that can be used to obtain alignment of amino acid or nucleotide sequences are known in the art. Suitable programs for determining percent sequence identity include, for example, the BLAST collection of programs available from the U.S. Government's National Center for Biotechnology Information BLAST website. Comparison between two sequences can be performed using the BLASTN or BLASTP algorithms. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. ALIGN, ALIGN-2 (Genentech, South San Francisco, California) or MegAlign (available from DNASTAR) are further publicly available software programs that can be used to align sequences. Those skilled in the art can determine suitable parameters for maximal alignment depending on the particular alignment software. In certain embodiments, the default parameters of the alignment software are used.

[0070]

[0092] The term "conservative amino acid substitution" refers to a modification of an amino acid sequence that does not eliminate the binding of an antibody to an antigen. Conservative amino acid substitutions include the replacement of an amino acid within a class with an amino acid from the same class, where the classes are defined by common physicochemical amino acid side chain properties and high substitution frequency in homologous proteins found in nature, as determined, for example, by standard Dayhoff frequency exchange matrices or BLOSUM matrices. Six general classes of amino acid side chains have been categorized, including: Class I (Cys); Class II (Ser, Thr, Pro, Ala, Gly); Class III (Asn, Asp, Gln, Glu); Class IV (His, Arg, Lys); Class V (Ile, Leu, Val, Met); and Class VI (Phe, Tyr, Trp). For example, the replacement of Asp with another residue from class III, such as Asn, Gln, or Glu, is a conservative substitution. Thus, a predicted non-essential amino acid residue in a protein is preferably replaced with another amino acid residue from the same class. Methods for identifying conservative amino acid substitutions that do not abolish antigen binding are well known in the art (see, e.g., Brummell, et al, Biochemistry 1993, 32, 1180-1187; Kobayashi, et al., Protein Eng. 1999, 12, 879-884 (1999); and Burks, et al, Proc. Natl. Acad. Sci. USA 1997, 94, 412-417).

[0071]

[0093] "PEGylation" refers to the modification of an antibody or fusion protein or fragment thereof that typically reacts with polyethylene glycol (PEG), e.g., a reactive ester or aldehyde derivative of PEG, under conditions in which one or more PEG groups are attached to the antibody or antibody fragment. PEGylation can, for example, increase the biological half-life of the antibody (e.g., serum). Preferably, PEGylation is carried out via an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer). As used herein, the term "polyethylene glycol" refers to the modification of an antibody or fusion protein or fragment thereof that typically reacts with polyethylene glycol, e.g., a reactive ester or aldehyde derivative of PEG, under conditions in which one or more PEG groups are attached to the antibody or antibody fragment. PEGylation can, for example, increase the biological half-life of the antibody (e.g., serum). Preferably, PEGylation is carried out via an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer). As used herein, the term "polyethylene glycol" refers to the modification of an antibody or fusion protein that is typically reacted with other proteins, e.g., mono(C1-C 10 ) is intended to encompass any form of PEG used to derivatize the alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. The protein or antibody to be pegylated may be a non-glycosylated protein or a non-glycosylated antibody. Methods of pegylation are known in the art and may be used with the antibodies of the invention, for example, as described in EP 0154316 and EP 0401384, and U.S. Pat. No. 5,824,778, the disclosures of each of which are incorporated herein by reference.

[0072]

[0094] The term "OKT-3" (also referred to herein as "OKT3") refers to a monoclonal antibody or a biosimilar or variant thereof (including a human, humanized, chimeric or murine antibody) directed against the CD3 receptor in the T cell antigen receptor of mature T cells, including commercially available forms such as OKT-3 (30 ng / mL, MACS GMP CD3 pure, Miltenyi Biotech, Inc., San Diego, CA, USA) and muromonab or variants, conservative amino acid substitutions, glycoforms or biosimilars thereof. The amino acid sequences of the heavy and light chains of muromonab are given in Table 1 (SEQ ID NO: 1 and SEQ ID NO: 2). A hybridoma capable of producing OKT-3 has been deposited at the American Type Culture Collection and assigned ATCC accession number CRL 8001. A hybridoma capable of producing OKT-3 has also been deposited at the European Collection of Authenticated Cell Cultures (ECACC) and assigned catalog number 86022706. Anti-CD3 antibodies also include the UHCT1 clone (commercially available from BioLegend, San Diego, Calif., USA) (also known as T3 and CD3ε).

[0073] [Table 1]

[0074]

[0095] 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, biosimilars, and variants thereof. IL-2 is described, for example, in Nelson, J. Immunol. 2004, 172, 3983-88 and Malek, Annu. Rev. Immunol. 2008, 26, 453-79, the disclosures of which are incorporated herein by reference. The amino acid sequence of recombinant human IL-2 suitable for use in the present invention is provided in Table 2 (SEQ ID NO:3). For example, the term IL-2 encompasses human recombinant forms of IL-2, such as aldesleukin (PROLEUKIN, commercially available from multiple suppliers at 22 million IU per single-use vial) as well as forms of recombinant IL-2 commercially supplied by CellGenix, Inc., Portsmouth, NH, USA (CELLGRO GMP) or ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (catalog number CYT-209-b) and other commercially available equivalents from other vendors. Aldesleukin (des-alanyl-1, serine-125 human IL-2) is a non-glycosylated human recombinant form of IL-2 with a molecular weight of approximately 15 kDa. The amino acid sequence of aldesleukin suitable for use in the present invention is given in Table 2 (SEQ ID NO: 4). The term IL-2 also encompasses pegylated forms of IL-2 as described herein, including the pegylated IL2 prodrug NKTR-214 (available from Nektar Therapeutics, South San Francisco, Calif., USA). NKTR-214 and pegylated IL-2 suitable for use in the present invention are described in U.S. Patent Application Publication No. 2014 / 0328791A1 and WO 2012 / 065086A1, the disclosures of which are incorporated herein by reference.Alternative forms of conjugated IL-2 suitable for use in the present invention are described in U.S. Patent Nos. 4,766,106, 5,206,344, 5,089,261 and 4,902,502, the disclosures of which are incorporated herein by reference. Formulations of IL-2 suitable for use in the present invention are described in U.S. Patent No. 6,706,289, the disclosure of which is incorporated herein by reference.

[0075] [Table 2]

[0076]

[0096] The term "IL-4" (also referred to herein as "IL4") refers to a cytokine known as interleukin 4, which is produced by Th2 T cells and by eosinophils, basophils and mast cells. IL-4 regulates the differentiation of naive helper T cells (Th0 cells) into Th2 T cells. Steinke and Borish, Respir. Res. 2001, 2, 66-70. Upon activation by IL-4, Th2 T cells then produce additional IL-4 in a positive feedback loop. IL-4 also induces B cell proliferation and expression of class II MHC, inducing class switch to IgE and IgG1 expression from B cells. Recombinant human IL-4 suitable for use in the present invention is commercially available from several sources, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (cat. no. CYT-211) and ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-4 recombinant protein, cat. no. Gibco CTP0043). The amino acid sequence of recombinant human IL-4 suitable for use in the present invention is provided in Table 2 (SEQ ID NO:5).

[0077]

[0097] The term "IL-7" (also referred to herein as "IL7") refers to a glycosylated tissue-derived cytokine known as interleukin 7, which can be obtained from stromal and epithelial cells as well as dendritic cells. Fry and Mackall, Blood 2002, 99, 3892-904. IL-7 can stimulate T cell development. IL-7 binds to the IL-7 receptor (a heterodimer consisting of an alpha chain and a common gamma chain receptor, the IL-7 receptor), which in a series of signals is important for the development in the thymus and survival of T cells in the periphery. Recombinant human IL-4 suitable for use in the present invention is commercially available from several sources, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (catalog number CYT-254) and ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-7 recombinant protein, catalog number Gibco PHC0071). The amino acid sequence of a recombinant human IL-7 suitable for use in the present invention is provided in Table 2 (SEQ ID NO:6).

[0078]

[0098] 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, their conservative amino acid substitutions, glycoforms, biosimilars, and variants. IL-15 is described, for example, in Fehniger and Caligiuri, Blood 2001, 97, 14-32, the disclosure of which is incorporated herein by reference. IL-15 shares β and γ signaling receptor subunits with IL-2. Recombinant human IL-15 is a single non-glycosylated polypeptide chain containing 114 amino acids (and an N-terminal methionine) with a molecular weight of 12.8 kDa. Recombinant human IL-15 is commercially available from several sources, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (cat. no. CYT-230-b) and ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-15 recombinant protein, cat. no. 34-8159-82). The amino acid sequence of recombinant human IL-15 suitable for use in the present invention is provided in Table 2 (SEQ ID NO: 7).

[0079]

[0099] 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, biosimilars, and variants thereof. IL-21 is described, for example, in Spolski and Leonard, Nat. Rev. Drug. Disc. 2014, 13, 379-95, the disclosure of which is incorporated herein by reference. IL-21 is primarily involved in the regulation of natural killer T cells and activated human CD4 T cells. +It is produced by T cells. Recombinant human IL-21 is a single non-glycosylated polypeptide chain containing 132 amino acids with a molecular weight of 15.4 kDa. Recombinant human IL-21 is commercially available from several sources, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (catalog number CYT-408-b) and ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-21 recombinant protein, catalog number 14-8219-80). The amino acid sequence of recombinant human IL-21 suitable for use in the present invention is provided in Table 2 (SEQ ID NO: 8).

[0080]

[0100] The term "biosimilar" refers to a biological product, including a monoclonal antibody or a fusion protein, that is highly similar to a reference biological product approved in the United States, despite minor differences in clinically inactive components, for which there are no clinically meaningful differences between the biological product and the reference product in terms of product safety, purity, and potency. Furthermore, a similar biological product or "biosimilar" drug is a biological product that is similar to another biological product already approved for use by the European Medicines Agency. The term "biosimilar" is also used synonymously by regulatory agencies in other countries and regions. A biological product or biological product is a drug product produced by or derived from a biological source, such as bacteria or yeast. They may consist of relatively small molecules, such as human insulin or erythropoietin, or complex molecules, such as monoclonal antibodies. For example, if the reference IL-2 protein is aldesleukin (PROLEUKIN), the protein approved by the drug regulatory authorities for aldesleukin is aldesleukin and a "biosimilar" or "biosimilar" of aldesleukin. In Europe, a similar biopharmaceutical or "biosimilar" medicinal product is a biopharmaceutical product similar to another biopharmaceutical product already approved for use by the European Medicines Agency (EMA). The relevant legal basis for similar biological uses in Europe is Article 6 of Regulation (EC) No 726 / 2004 and Article 10(4) of Directive 2001 / 83 / EC (as amended), so in Europe, a biosimilar may be authorized under Article 6 of Regulation (EC) No 726 / 2004 and Article 10(4) of Directive 2001 / 83 / EC and may be the subject of an application for authorization or authorization. The original biopharmaceutical product already authorized may be called the "reference medicinal product" in Europe. Some of the requirements for a product to be considered a biosimilar are outlined in the CHMP guideline on Similar Biological Medicinal Products.In addition, product specific guidelines, including guidelines for monoclonal antibody biosimilars, are provided by the EMA on a product-by-product basis and are published on its website. The biosimilars described herein may be similar in quality characteristics, biological activity, mechanism of action, safety profile and / or efficacy to the reference pharmaceutical product. In addition, the biosimilars may be used or intended to treat the same symptoms as the reference pharmaceutical product. Thus, the biosimilars described herein may be considered to have similar or highly similar quality characteristics to the reference pharmaceutical product. Alternatively or in addition, the biosimilars described herein may be considered to have similar or highly similar biological activity to the reference pharmaceutical product. Alternatively or in addition, the biosimilars described herein may be considered to have similar or highly similar safety profile to the reference pharmaceutical product. Alternatively or in addition, the biosimilars described herein may be considered to have similar or highly similar efficacy to the reference pharmaceutical product. The biosimilars in Europe described herein are compared to the reference pharmaceutical product approved by the EMA. However, in some cases, a biosimilar may be compared in certain studies to a biopharmaceutical product that has been approved outside the European Economic Area (a non-EEA approved "comparator"). Such studies include, for example, certain clinical studies and in vivo non-clinical studies. The term "biosimilar" as used herein also relates to a biopharmaceutical product that has become or can be compared to a non-EEA approved comparator. Particular biosimilars are proteins, such as antibodies, antibody fragments (e.g., antigen-binding portions) and fusion proteins. Protein biosimilars may have amino acid sequences with minor modifications in the amino acid structure (including, for example, deletions, additions and / or substitutions of amino acids) that do not significantly affect the function of the polypeptide. A biosimilar may include an amino acid sequence that has 97% or more, e.g., 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of its reference pharmaceutical product.A biosimilar may include one or more post-translational modifications, such as, but not limited to, glycosylation, oxidation, deamidation, and / or cleavage, which differ from the post-translational modifications of the reference pharmaceutical product (provided that the difference does not result in a change in the safety and / or efficacy of the pharmaceutical product). A biosimilar may have the same or different glycosylation pattern as the reference pharmaceutical product. In particular, but not exclusively, a biosimilar may have a different glycosylation pattern (provided that the difference addresses or is intended to address a safety issue associated with the reference pharmaceutical product). In addition, a biosimilar may deviate from the reference pharmaceutical product, for example, in its strength, pharmaceutical form, formulation, excipients, and / or presentation (provided that the safety and efficacy of the pharmaceutical product is not compromised). A biosimilar may include differences, for example, in its pharmacokinetic (PK) profile and / or pharmacodynamic (PD) profile compared to the reference pharmaceutical product, but still be considered sufficiently similar to the reference pharmaceutical product to be approved or to be considered suitable for approval. In certain circumstances, biosimilars exhibit different binding properties compared to the reference pharmaceutical product, which are not considered by regulatory authorities, such as the EMA, to be a barrier to approval as a similar biological product. The term "biosimilar" is also used interchangeably by regulatory agencies in other countries and regions.

[0081]

[0101] The term "variant" as used herein includes, but is not limited to, an antibody or fusion protein that comprises an amino acid sequence that differs from the amino acid sequence of a reference antibody by one or more substitutions, deletions and / or additions at specific positions within or adjacent to the amino acid sequence of the reference antibody. A variant may contain one or more conservative substitutions in its amino acid sequence compared to the amino acid sequence of the reference antibody. Conservative substitutions may include, for example, substitutions of similarly charged or uncharged amino acids. A variant retains the ability of the reference antibody to specifically bind to an antigen. The term variant also includes pegylated antibodies or proteins.

[0082]

[0102] "PEGylation" refers to a modified antibody or fragment thereof that is typically reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions in which one or more PEG groups are attached to the antibody, antibody fragment, or protein. PEGylation can, for example, increase the biological half-life of the antibody or protein (e.g., serum). Preferably, PEGylation is carried out via an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer). As used herein, the term "polyethylene glycol" refers to a modified antibody or fragment thereof that is typically reacted with polyethylene glycol, such as a reactive ester or aldehyde derivative of PEG, under conditions in which one or more PEG groups are attached to the antibody, antibody fragment, or protein. PEGylation can, for example, increase the biological half-life of the antibody or protein (e.g., serum). Preferably, PEGylation is carried out via an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer). As used herein, the term "polyethylene glycol" refers to a modified antibody or fragment thereof that is typically reacted with other proteins, such as mono(C1-C 10 ) is intended to encompass any form of PEG used to derivatize the alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. The antibody or protein to be pegylated may be a non-glycosylated antibody. Methods of pegylation are known in the art and may be used with the antibodies of the invention, for example as described in EP 0154316 and EP 0401384.

[0083]

[0103] The term "hematological malignancies" refers to mammalian cancers and tumors of hematopoietic and lymphatic tissues, including but not limited to blood, bone marrow, lymph nodes, and lymphatic tissues. Hematological malignancies are also called "liquid tumors." Hematological malignancies include but are not limited to acute lymphoblastic leukemia (ALL), chronic lymphocytic lymphoma (CLL), small lymphocytic lymphoma (SLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute monocytic leukemia (AMoL), Hodgkin's lymphoma, and non-Hodgkin's lymphoma. The term "B-cell hematological malignancies" refers to hematological malignancies affecting B cells.

[0084]

[0104] The term "solid tumor" refers to an abnormal mass of tissue that does not usually contain cysts or liquid areas. Solid tumors can be benign or malignant. The term "solid tumor cancer" refers to a malignant, neoplastic, or cancerous solid tumor. Solid tumor cancers include, but are not limited to, sarcomas, carcinomas, and lymphomas, such as lung, breast, prostate, colon, rectum, and bladder cancers. The tissue structure of a solid tumor includes interdependent tissue compartments, including parenchyma (cancer cells) and supporting stromal cells, in which the cancer cells are dispersed and which may provide a supportive microenvironment.

[0085]

[0105] The term "liquid tumor" refers to an abnormal mass of cells that is fluid in nature.Liquid tumor cancers include, but are not limited to, leukemia, myeloma and lymphoma and other hematological malignancies.TILs obtained from liquid tumors may also be referred to herein as bone marrow infiltrating lymphocytes (MILs).

[0086]

[0106] The term "microenvironment" as used herein may refer to the microenvironment of a solid or hematological tumor as a whole or to an individual subset of cells within the microenvironment. As used herein, tumor microenvironment refers to a complex mixture of "cells, soluble factors, signaling molecules, extracellular matrix, and mechanical cues that promote malignant transformation, support tumor growth and invasion, protect tumors from host immunity, promote therapeutic resistance, and provide a niche for favorable metastasis to develop," as described in Swartz, et al., Cancer Res., 2012, 72, 2473. Tumors express antigens that should be recognized by T cells, but clearance of tumors by the immune system is rare due to immunosuppression by the microenvironment.

[0087]

[0107] As used herein, the terms "fragmenting," "fragment," and "fragmented" to describe the process of disrupting tumors include mechanical fragmentation methods, such as crushing, slicing, dividing, and mortising tumor tissue, as well as any other method that disrupts the physical structure of tumor tissue.

[0088]

[0108] The terms "about" and "approximately" mean within a statistically significant range of values. Such ranges may be within an order of magnitude, preferably within 50%, more preferably within 20%, even more preferably within 10%, and even more preferably within 5% of a given value or range. The acceptable variation encompassed by the term "about" or "approximately" depends on the particular system under study and can be readily understood by one of ordinary skill in the art. Furthermore, the terms "about" and "approximately" as used herein mean that dimensions, sizes, formulas, parameters, shapes, and other quantities and characteristics are not and need not be exact, but may be approximate and / or greater or smaller reflecting tolerances, conversion factors, rounding, measurement errors, and the like, as well as other factors known to those of ordinary skill in the art, as desired. In general, a dimension, size, formula, parameter, shape, or other quantity or characteristic is "about" or "approximately" whether or not it is expressly stated to be so. It is noted that embodiments with very different sizes, shapes, and dimensions may use the configurations described.

[0089]

[0109] The transitional terms "comprising," "consisting essentially of," and "consisting of," when used in the appended claims in their original and amended forms, define the claim in terms of the exclusion from the claim of additional unrecited claim elements or steps, if any. The term "comprising" is intended to be inclusive or open ended and does not exclude any additional unrecited elements, methods, steps, or materials. The term "consisting of" excludes any element, step, or material other than those specified in the claim, and in later instances, other than impurities normally associated with the specified material. The term "consisting essentially of" limits the claim to those elements, steps, or materials specified and that do not materially affect the basic and novel characteristics of the claimed invention. All compositions, methods, and kits described herein that embody the present invention may be more specifically defined in alternative embodiments by any of the transitional terms "comprising," "consisting essentially of," and "consisting of."

[0090]

[0110] For the avoidance of doubt, it is intended that certain features (e.g., integers, properties, values, uses, diseases, formulas, compounds, or groups) described herein in connection with a particular aspect, embodiment, or example of the invention are to be understood as applicable to any other aspect, embodiment, or example described herein, unless incompatible therewith. Such features may therefore be used where appropriate in connection with any of the definitions, claims, or embodiments defined herein. All of the features disclosed herein (including the accompanying claims, abstract, and drawings) and / or all of the steps of any method or process so disclosed may be combined in any combination, except combinations in which at least some of the features and / or steps are mutually exclusive. The invention is not limited to any of the details of any of the disclosed embodiments. The invention extends to any novel one or novel combination of the features disclosed herein (including the accompanying claims, abstract, and drawings) or any novel one or novel combination of the steps of any method or process so disclosed.

[0091] Method for Proliferating Remnant Tumor-Infiltrating Lymphocytes

[0111] In one embodiment, the invention includes a method of expanding remnant tumor infiltrating lymphocytes (rTILs) following digestion of a tumor, as described herein.

[0092]

[0112] In one embodiment, the invention includes a method of expanding rTILs, the method comprising expanding the rTILs by contacting a population of rTILs comprising at least one rTIL with IL-2.

[0093]

[0113] In one embodiment, the present invention provides a method for expanding a population of rTILs, the method comprising the steps described in Jin, et al., J. Immunotherapy 2012, 35, 283-292, the disclosure of which is incorporated herein by reference. For example, the tumor can be placed in an enzyme medium and mechanically fragmented for approximately 1 minute. The mixture can then be incubated at 37° C. in 5% CO2 for 30 minutes, and then mechanically fragmented again for approximately 1 minute. After the 30-minute incubation at 37° C. in 5% CO2, the tumor can be mechanically fragmented a third time for approximately 1 minute. After the third mechanical disruption, if large pieces of tissue are present, the sample can be subjected to one or two additional mechanical disaggregations, with or without an additional incubation at 37° C. in 5% CO2 for 30 minutes. After 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. TIL cultures were initiated in 24-well plates (Costar 24-well cell culture cluster, flat bottom; Corning Incorporated, Corning, NY) with 1 × 10 cells in 2 mL of complete medium (CM) with IL-2 (6000 IU / mL; Chiron Corp., Emeryville, CA) per well. 6 Tumor digestive cells or tumors with a size of approximately 1-8 mm 3 One tumor fragment per 1000 cells / well can be seeded. CM consists of RPMI 1640 supplemented with GlutaMAX, 10% human Ab serum, 25 mM Hepes, and 10 mg / ml gentamicin. Cultures are grown in 10 cm wells in a 40 mL volume. 2 Cells can be initiated in gas permeable flasks (G-Rex 10; Wilson Wolf Manufacturing, New Brighton) with gas permeable silicone bottoms, and 10–40 × 10 cells / ml in 10–40 mL of CM with IL-2 were cultured in each flask. 6Viable tumor digested cells or 5-30 tumor fragments can be loaded. G-Rex 10 and 24 well plates can be incubated in a humidified incubator at 37°C with 5% CO2, and half the medium can be removed and replaced with fresh CM and IL-2 5 days after initiation of culture, and half the medium can be changed every 2-3 days after day 5. Rapid expansion protocols (REP) for TILs can be performed using T-175 flasks and gas permeable bags or gas permeable G-Rex flasks as described elsewhere herein. For REP in T-175 flasks, 1 x 10 6 The rTILs may be suspended in 150 mL of medium in each flask. The rTILs may be cultured in a 1:1 mixture of CM and AIM-V medium (50 / 50 medium) supplemented with 3000 IU / mL IL-2 and 30 ng / mL anti-CD3 antibody (OKT-3). The T-175 flasks may be incubated at 37°C in 5% CO2. Half the medium may be changed on day 5 with 50 / 50 medium with 3000 IU / mL IL-2. On day 7, the cells from the two T-175 flasks may be combined in a 3L bag, and 300 mL of AIM-V with 5% human AB serum and 3000 IU / mL IL-2 may be added to the 300 mL TIL suspension. The number of cells in each bag may be counted daily or every two days, and fresh medium may be added at a concentration of 0.5-2.0 x 10 6 Additional medium may be added to maintain cell numbers at 100 cm 2 For REP in a 500 mL volumetric flask with a gas permeable silicone bottom (e.g., G-Rex 100, Wilson Wolf Manufacturing, described elsewhere herein), 5×10 6 Or 10 x 10 6The TILs may be cultured in 400 mL of 50 / 50 medium supplemented with 3000 IU / mL IL-2 and 30 ng / mL anti-CD3 antibody (OKT-3). The G-Rex 100 flasks may be incubated at 37° C. in 5% CO2. On day 5, 250 mL of the supernatant may be removed and placed into a centrifuge bottle and centrifuged at 1500 rpm (491 g) for 10 minutes. The resulting TIL pellet may be resuspended in 150 mL of fresh 50 / 50 medium with 3000 IU / mL IL-2 and returned to the G-Rex 100 flask. If the TILs were expanded sequentially in the G-Rex 100 flasks, on day 7, the TILs in each G-Rex 100 may be suspended in the 300 mL of medium present in each flask and the cell suspension may be divided into three 100 mL aliquots that may be used to seed three G-Rex 100 flasks. Approximately 150 mL of AIM-V with 5% human AB serum and 3000 IU / mL IL-2 can then be added to each flask. The G-Rex 100 flasks can then be incubated at 37° C. in 5% CO2, and after day 4, 150 mL of AIM-V with 3000 IU / mL IL-2 can be added to each G-Rex 100 flask. REP can then be completed by harvesting the cells on day 14 of culture.

[0094]

[0114] In one embodiment, the method of growing or treating cancer includes obtaining TILs from a patient tumor sample. Patient tumor samples can be obtained using methods known in the art. For example, TILs can be obtained from enzymatic tumor digests and tumor fragments (about 1 to about 8 mm) from sharp dissection. 3Tumor digests can be cultured from tumor tissue (sizes of 100-200 mm). Such tumor digests can be produced by incubation in enzyme medium (e.g., Roswell Park Memorial Institute (RPMI) 1640 buffer, 2 mM glutamate, 10 mcg / mL gentamicin, 30 units / mL DNase, and 1.0 mg / mL collagenase) followed by mechanical dissociation (e.g., using a tissue dissociator or fragmentator). Tumor digests can be produced by placing the tumor in enzyme medium, mechanically fragmenting the tumor for approximately 1 minute, then incubating at 37° C. in 5% CO2 for 30 minutes, followed by repeated cycles of mechanical dissociation and incubation under the conditions described above until only small tissue fragments are present. After completion of this process, if the cell suspension contains a large number of red blood cells or dead cells, density gradient separation using FICOLL branched hydrophilic polysaccharide can be performed to remove these cells. Alternative methods known in the art may be used, such as those described in U.S. Patent Application Publication No. 2012 / 0244133A1, the disclosure of which is incorporated herein by reference. Any of the above-mentioned methods may be used in the methods of expanding TILs or treating cancer in any of the embodiments described herein.

[0095]

[0115] In one embodiment, REP of rTILs can be carried out using any suitable method in a gas permeable container. For example, rTILs can be rapidly expanded using non-specific T cell receptor stimulation in the presence of interleukin-2 (IL-2), interleukin-15 (IL-15) and / or interleukin-21 (IL-21), as described, for example, in WO 2015 / 189356 A1 and WO 2015 / 189356 A1, the disclosures of each of which are incorporated herein by reference. Non-specific T cell receptor stimulation can include, for example, about 30 ng / mL OKT-3 (monoclonal anti-CD3 antibody) (commercially available from Ortho-McNeil, Raritan, NJ or Miltenyi Biotech, Inc., San Diego, CA, USA). TILs can be rapidly expanded by further in vitro stimulation of the TILs with one or more antigens of the cancer, including antigenic portions thereof, e.g., epitopes, optionally expressed from a vector, optionally in the presence of a T cell growth factor, e.g., 300 IU / mL IL-2 or IL-15 (e.g., human leukocyte antigen A2 (HLA-A2) binding peptide, e.g., 0.3 μM MART-L26-35 (27L) or gpl 00:209-217 (210M)). Other suitable antigens can include, e.g., NY-ESO-1, TRP-1, TRP-2, tyrosinase cancer antigen, MAGE-A3, SSX-2, and VEGFR2, or antigenic portions thereof. TILs can also be rapidly expanded by restimulation with the same antigens of the cancer pulsed on HLA-A2-expressing antigen-presenting cells. Alternatively, TILs can be further restimulated with, for example, irradiated autologous lymphocytes or irradiated HLA-A2+ allogeneic lymphocytes and IL-2.

[0096]

[0116] In one embodiment, the method of expanding TILs may include using about 5000 mL to about 25000 mL of cell culture medium, about 5000 mL to about 10000 mL of cell culture medium, or about 5800 mL to about 8700 mL of cell culture medium. In one embodiment, the method for expanding TILs may include using about 1000 mL to about 2000 mL of cell culture medium, about 2000 mL to about 3000 mL of cell culture medium, about 3000 mL to about 4000 mL of cell culture medium, about 4000 mL to about 5000 mL of cell culture medium, about 5000 mL to about 6000 mL of cell culture medium, about 6000 mL to about 7000 mL of cell culture medium, about 7000 mL to about 8000 mL of cell culture medium, about 8000 mL to about 9000 mL of cell culture medium, about 9000 mL to about 10000 mL of cell culture medium, about 10000 mL to about 15000 mL of cell culture medium, about 15000 mL to about 20000 mL of cell culture medium, or about 20000 mL to about 25000 mL of cell culture medium. In one embodiment, one or less types of cell culture medium are used for expanding the number of TILs. Any suitable cell culture medium may be used, such as AIM-V cell culture medium (L-glutamine, 50 μM streptomycin sulfate and 10 μM gentamicin sulfate) cell culture medium (Invitrogen, Carlsbad Calif.). In this regard, the methods of the invention advantageously reduce the amount of medium and number of medium types required to expand the number of TILs. In one embodiment, expanding the number of TILs may include feeding the cells less frequently than every 3 or 4 days. Expanding the number of cells in a gas permeable container simplifies the procedures required to expand the number of cells by reducing the frequency of feeding required to expand the cells.

[0097]

[0117] In one embodiment, rapid expansion is performed using a gas permeable container. Such an embodiment involves expanding a cell population to approximately 5×10 5 cells / cm 2 From 10×10 6 ~30×10 6 cells / cm 2In one embodiment, the expansion occurs without feeding. In one embodiment, the expansion occurs without feeding as long as the medium is present in the gas permeable flask at a height of about 10 cm. In one embodiment, this is without feeding, but with the addition of one or more cytokines. In one embodiment, the cytokines can be added as a bolus, and there is no need to mix the cytokines with the medium at all. Such containers, devices and methods are known in the art and have been used to expand TILs and are described in U.S. Patent Application Publication Nos. 2014 / 0377739A1, WO 2014 / 210036A1, U.S. Patent Application Publication Nos. 2013 / 0115617A1, WO 2013 / 188427A1, U.S. Patent Application Publication No. 2011 / 0136228A1, U.S. Patent No. 8,809,050, WO 2011 / 07208 8A2, US Patent Application Publication No. 2016 / 0208216A1, US Patent Application Publication No. 2012 / 0244133A1, WO 2012 / 129201A1, US Patent Application Publication No. 2013 / 0102075A1, US Patent No. 8,956,860, WO 2013 / 173835A1 and US Patent Application Publication No. 2015 / 0175966A1, the disclosures of which are incorporated herein by reference. Such a process is also described in Jin, et al., J. Immunotherapy 2012, 35, 283-292, the disclosures of which are incorporated herein by reference.

[0098]

[0118] In one embodiment, the gas permeable container is a G-Rex 10 flask (Wilson Wolf Manufacturing Corporation, New Brighton, MN, USA). 2 In one embodiment, the gas permeable container comprises a 40 mL cell medium capacity. In one embodiment, the gas permeable container provides 100-300 million TILs after two medium changes.

[0099]

[0119] In one embodiment, the gas permeable container is a G-Rex 100 flask (Wilson Wolf Manufacturing Corporation, New Brighton, MN, USA). 2 In one embodiment, the gas permeable container comprises a gas permeable culture surface of 450 mL. In one embodiment, the gas permeable container comprises a cell medium capacity of 450 mL. In one embodiment, the gas permeable container provides 1-3 billion TILs after two medium changes.

[0100]

[0120] In one embodiment, the gas permeable container is a G-Rex 100M flask (Wilson Wolf Manufacturing Corporation, New Brighton, MN, USA). 2 In one embodiment, the gas permeable container comprises a 1000 mL cell medium capacity. In one embodiment, the gas permeable container provides 1-3 billion TILs without medium exchange.

[0101]

[0121] In one embodiment, the gas permeable container is a G-Rex 100 L flask (Wilson Wolf Manufacturing Corporation, New Brighton, MN, USA). 2 In one embodiment, the gas permeable container comprises a gas permeable culture surface of 2000 mL. In one embodiment, the gas permeable container comprises a cell medium capacity of 2000 mL. In one embodiment, the gas permeable container provides 1-3 billion TILs without medium exchange.

[0102]

[0122] In one embodiment, the gas permeable container is a G-Rex 24-well plate (Wilson Wolf Manufacturing Corporation, New Brighton, MN, USA). In one embodiment, the gas permeable container comprises a plate having wells, each well having a diameter of 2 cm.2 In one embodiment, the gas permeable container comprises a plate having wells, each well comprising 8 mL of cell medium volume. In one embodiment, the gas permeable container provides 20-60 million cells per well after two medium changes.

[0103]

[0123] In one embodiment, the gas permeable container is a G-Rex 6-well plate (Wilson Wolf Manufacturing Corporation, New Brighton, MN, USA). In one embodiment, the gas permeable container comprises a plate with wells, each well having a diameter of 10 cm. 2 In one embodiment, the gas permeable container comprises a plate having wells, each well comprising 40 mL of cell medium volume. In one embodiment, the gas permeable container provides 100-300 million cells per well after two medium changes.

[0104]

[0124] In one embodiment, the cell culture medium in the first gas permeable container and / or the second gas permeable container is unfiltered. The use of unfiltered cell culture medium can simplify the steps required to expand cell numbers. In one embodiment, the cell culture medium in the first gas permeable container and / or the second gas permeable container lacks beta-mercaptoethanol (BME).

[0105]

[0125] In one embodiment, the duration of the method includes obtaining a tumor tissue sample from a mammal, culturing the tumor tissue sample in a first gas permeable container containing cell culture medium, obtaining TILs from the tumor tissue sample, and expanding the TIL population in a second gas permeable container containing cell culture medium for a period of about 14 to about 42 days, e.g., about 28 days.

[0106]

[0126] In one embodiment, the ratio of rTIL to PBMC in rapid proliferation is about 1 to 25, about 1 to 50, about 1 to 100, about 1 to 125, about 1 to 150, about 1 to 175, about 1 to 200, about 1 to 225, about 1 to 250, about 1 to 275, about 1 to 300, about 1 to 325, about 1 to 350, about 1 to 375, about 1 to 400, or about 1 to 500. In one embodiment, the ratio of rTIL to PBMC in rapid proliferation is 1 to 50 to 1 to 300. In one embodiment, the ratio of rTIL to PBMC in rapid proliferation is 1 to 100 to 1 to 200.

[0107]

[0127] In one embodiment, the ratio of rTIL to PBMC (rTIL:PBMC) is 1:5, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:85, 1:90, 1:95, 1:100, 1:105, 1:110, 1:115, 1:1 The ratio of rTIL to PBMCs is selected from the group consisting of 1:20, 1:125, 1:130, 1:135, 1:140, 1:145, 1:150, 1:155, 1:160, 1:165, 1:170, 1:175, 1:180, 1:185, 1:190, 1:195, 1:200, 1:225, 1:250, 1:275, 1:300, 1:350, 1:400, 1:450 and 1:500. In a preferred embodiment, the ratio of rTIL to PBMCs (rTIL:PBMC) is about 1:90. In a preferred embodiment, the ratio of TIL to PBMCs (rTIL:PBMC) is about 1:95. In a preferred embodiment, the ratio of rTIL to PBMCs (TIL:PBMC) is about 1:100. In a preferred embodiment, the ratio of rTILs to PBMCs (TILs:PBMCs) is about 1: 105. In a preferred embodiment, the ratio of rTILs to PBMCs (TILs:PBMCs) is about 1: 110.

[0108]

[0128] In one embodiment, the cell culture medium further comprises IL-2. In a preferred embodiment, the cell culture medium comprises about 3000 IU / mL of IL-2. In one embodiment, the cell culture medium comprises about 1000 IU / mL, about 1500 IU / mL, about 2000 IU / mL, about 2500 IU / mL, about 3000 IU / mL, about 3500 IU / mL, about 4000 IU / mL, about 4500 IU / mL, about 5000 IU / mL, about 5500 IU / mL, about 6000 IU / mL, about 6500 IU / mL, about 7000 IU / mL, about 7500 IU / mL, or about 8000 IU / mL of IL-2. In one embodiment, the cell culture medium contains 1000-2000 IU / mL, 2000-3000 IU / mL, 3000-4000 IU / mL, 4000-5000 IU / mL, 5000-6000 IU / mL, 6000-7000 IU / mL, 7000-8000 IU / mL, or 8000 IU / mL of IL-2.

[0109]

[0129] In one embodiment, the cell culture medium comprises an OKT-3 antibody. In a preferred embodiment, the cell culture medium comprises about 30 ng / mL of an OKT-3 antibody. In one embodiment, the cell culture medium comprises 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, or about 1 μg / mL of an OKT-3 antibody. In one embodiment, the cell culture medium contains 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, or 50 ng / mL to 100 ng / mL of OKT-3 antibody.

[0110]

[0130] In one embodiment, the rapid expansion process for TILs can be carried out using T-175 flasks and gas permeable bags (Tran, et al., J. Immunother. 2008, 31, 742-51; Dudley, et al, J. Immunother. 2003, 26, 332-42) or gas permeable incubators (G-Rex flasks available from Wilson Wolf Manufacturing Corporation, New Brighton, MN, USA) as previously described. For TIL rapid expansion in T-175 flasks, 1×10 cells suspended in 150 mL of medium were used. 6 TILs can be added to each T-175 flask. TILs can be cultured in a 1:1 mixture of CM and AIM-V medium, supplemented with 3000 IU (injection units) of IL-2 per mL and 30 ng of anti-CD3 antibody (e.g., OKT-3) per mL. T-175 flasks can be incubated at 37°C in 5% CO2. Half the medium can be replaced on day 5 with 50 / 50 medium with 3000 IU of IL-2 per mL. On day 7, cells from two T-175 flasks can be combined in a 3 liter bag, and 300 mL of AIM-V with 5% human AB serum and 3000 IU of IL-2 per mL was added to the 300 ml TIL suspension. The number of cells in each bag can be counted daily or every two days, and fresh medium can be added at 0.5-2.0 x 10 6 Added to maintain cell numbers in cells / mL.

[0111]

[0131] In one embodiment, for rapid TIL expansion in a 500 mL gas permeable flask with a 100 cm gas permeable silicone bottom (G-Rex 100, commercially available from Wilson Wolf Manufacturing Corporation, New Brighton, MN, USA), 5×10 6 Or 10 x 10 6TILs may be cultured in 50 / 50 medium supplemented with 5% human AB serum, 3000 IU IL-2 per mL, and 30 ng anti-CD3 (OKT-3) per mL. The G-Rex 100 flask may be incubated at 37° C. in 5% CO2. On day 5, 250 mL of supernatant may be removed and placed into a centrifuge bottle and centrifuged at 1500 rpm (revolutions per minute; 491×g) for 10 minutes. The TIL pellet may be resuspended in 150 mL of fresh medium with 5% human AB serum, 3000 IU IL-2 per mL, and returned to the original G-Rex 100 flask. If TILs are expanded sequentially in G-Rex 100 flasks, on day 7, the TILs in each G-Rex 100 can be suspended in the 300 mL of medium present in each flask, and the cell suspension can be divided into three 100 mL aliquots that can be used to seed three G-Rex 100 flasks. Then, 150 mL of AIM-V with 5% human AB serum and 3000 IU of IL-2 per mL can be added to each flask. The G-Rex 100 flasks can be incubated at 37° C. in 5% CO2, and after day 4, 150 mL of AIM-V with 3000 IU of IL-2 per mL can be added to each G-Rex 100 flask. The cells can be harvested on day 14 of culture.

[0112]

[0132] In one embodiment, the TILs can be prepared as follows: 3Tumor fragments were cultured in complete medium (CM) consisting of AIM-V medium (Invitrogen Life Technologies, Carlsbad, CA) supplemented with 2 mM glutamine (Mediatech, Inc. Manassas, VA), 100 U / mL penicillin (Invitrogen Life Technologies), 100 μg / mL streptomycin (Invitrogen Life Technologies), 5% heat-inactivated human AB serum (Valley Biomedical, Inc. Winchester, VA), and 600 IU / mL rhIL-2 (Chiron, Emeryville, CA). For enzymatic digestion of solid tumors, tumor specimens were diced into RPMI-1640, washed, centrifuged at 800 rpm for 5 min at 15-22°C, resuspended in enzymatic digestion buffer (0.2 mg / mL collagenase and 30 units / ml DNase in RPMI-1640) and rotated overnight at room temperature. TILs established from fragments are expanded in CM for 3–4 weeks and can be either freshly grown in heat-inactivated HAB serum with 10% dimethyl sulfoxide (DMSO) or cryopreserved and stored at −180°C until time of study. Tumor-associated lymphocytes (TAL) from ascites collections are cultured at 3 × 10 in CM in 24-well plates. 6 Cells / well were seeded. TIL proliferation was examined approximately every other day using a low magnification inverted microscope.

[0113]

[0133] In one embodiment, the TILs are grown in a gas permeable container. Gas permeable containers have been used to grow TILs with PBMCs using methods, compositions and devices known in the art, including those described in U.S. Patent Application Publication No. 2005 / 0106717A1, the disclosure of which is incorporated herein by reference. In one embodiment, the TILs are grown in a gas permeable bag. In one embodiment, the TILs are grown by a cell expansion system that grows TILs in a gas permeable bag, such as the Xuri Cell Expansion System W25 (GE Healthcare). In one embodiment, the TILs are grown by a cell expansion system that grows TILs in a gas permeable bag, such as the WAVE Bioreactor System, also known as the Xuri Cell Expansion System W5 (GE Healthcare). In one embodiment, the cell expansion system comprises a gas permeable cell bag having a volume selected from the group consisting of about 100 mL, about 200 mL, about 300 mL, about 400 mL, about 500 mL, about 600 mL, about 700 mL, about 800 mL, about 900 mL, about 1 L, about 2 L, about 3 L, about 4 L, about 5 L, about 6 L, about 7 L, about 8 L, about 9 L, about 10 L, about 11 L, about 12 L, about 13 L, about 14 L, about 15 L, about 16 L, about 17 L, about 18 L, about 19 L, about 20 L, about 25 L, and about 30 L. In one embodiment, the cell growth system comprises a gas permeable cell bag having a volume range selected from the group consisting of 50-150 mL, 150-250 mL, 250-350 mL, 350-450 mL, 450-550 mL, 550-650 mL, 650-750 mL, 750-850 mL, 850-950 mL, and 950-1050 mL. In one embodiment, the cell expansion system comprises a gas permeable cell bag having a volume range selected from the group consisting of 1L-2L, 2L-3L, 3L-4L, 4L-5L, 5L-6L, 6L-7L, 7L-8L, 8L-9L, 9L-10L, 10L-11L, 11L-12L, 12L-13L, 13L-14L, 14L-15L, 15L-16L, 16L-17L, 17L-18L, 18L-19L, and 19L-20L.In one embodiment, the cell expansion system comprises a gas permeable cell bag having a volume range selected from the group consisting of 0.5L-5L, 5L-10L, 10L-15L, 15L-20L, 20L-25L, and 25L-30L. In one embodiment, the cell expansion system utilizes a rocking time of about 30 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 24 hours, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, about 21 days, about 22 days, about 23 days, about 24 days, about 25 days, about 26 days, about 27 days, and about 28 days. In one embodiment, the cell growth system utilizes rocking times of 30 minutes to 1 hour, 1 hour to 12 hours, 12 hours to 1 day, 1 day to 7 days, 7 days to 14 days, 14 days to 21 days, and 21 days to 28 days. In one embodiment, the cell growth system utilizes rocking speeds of about 2 locks / min, about 5 locks / min, about 10 locks / min, about 20 locks / min, about 30 locks / min, and about 40 locks / min. In one embodiment, the cell growth system utilizes rocking speeds of 2 locks / min to 5 locks / min, 5 locks / min to 10 locks / min, 10 locks / min to 20 locks / min, 20 locks / min to 30 locks / min, and 30 locks / min to 40 locks / min. In one embodiment, the cell growth system utilizes rocking angles of about 2°, about 3°, about 4°, about 5°, about 6°, about 7°, about 8°, about 9°, about 10°, about 11°, and about 12°. In one embodiment, the cell growth system utilizes rocking angles of 2°-3°, 3°-4°, 4°-5°, 5°-6°, 6°-7°, 7°-8°, 8°-9°, 9°-10°, 10°-11°, and 11°-12°.

[0114]

[0134] In one embodiment, the method for expanding rTIL further comprises selecting rTIL for good tumor reactivity.Any selection method known in the art can be used.For example, the method described in US Patent Application Publication No. 2016 / 0010058A1 (the disclosure of which is incorporated herein by reference) can be used to select TIL for good tumor reactivity.

[0115] Characteristics of rTILs

[0135] In one embodiment, the rTILs of the present invention exhibit an exhausted T cell phenotype characterized by one or more T cell exhaustion markers. In one embodiment, the rTILs of the present invention exhibit an exhausted T cell phenotype characterized by one or more T cell exhaustion markers using flow cytometry analysis. In one embodiment, the T cell exhaustion marker is PD-1. In one embodiment, the T cell exhaustion marker is LAG3. In one embodiment, the T cell exhaustion marker is TIM3.

[0116]

[0136] In one embodiment, PD-1 expression in rTILs is reduced by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, or at least 150% relative to eTILs. In one embodiment, PD-1 expression in rTILs is reduced by at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold relative to eTILs.

[0117]

[0137] In one embodiment, LAG3 expression in rTILs is reduced by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, or at least 150% relative to eTILs. In one embodiment, LAG3 expression in rTILs is reduced by at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold relative to eTILs. In one embodiment, LAG3 expression in rTILs is undetectable by flow cytometry.

[0118]

[0138] In one embodiment, TIM3 expression in rTILs is reduced by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, or at least 150% relative to eTILs. In one embodiment, TIM3 expression in rTILs is reduced by at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold relative to eTILs. In one embodiment, TIM3 expression in rTILs is undetectable by flow cytometry.

[0119]

[0139] In one embodiment, TIGIT expression in rTILs is reduced by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, or at least 150% relative to eTILs. In one embodiment, TIGIT expression in rTILs is reduced by at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold relative to eTILs. In one embodiment, TIGIT expression in rTILs is undetectable by flow cytometry.

[0120]

[0140] In one embodiment, CTLA-4 expression in rTILs is reduced by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, or at least 150% relative to eTILs. In one embodiment, CTLA-4 expression in rTILs is reduced by at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold relative to eTILs. In one embodiment, CTLA-4 expression in rTILs is undetectable by flow cytometry.

[0121]

[0141] In one embodiment, CD69 expression in rTILs is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, or at least 150% relative to eTILs. In one embodiment, CD69 expression in rTILs is increased by at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold relative to eTILs.

[0122]

[0142] In one embodiment, S1PR1 (sphingosine-1-phosphate receptor 1) expression in rTILs is reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, or at least 150% relative to eTILs. In one embodiment, S1PR1 expression in rTILs is reduced by at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold relative to eTILs.

[0123]

[0143] In one embodiment, telomere length in rTILs is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, or at least 150% relative to eTILs. In one embodiment, telomere length in rTILs is increased by at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold relative to eTILs.

[0124]

[0144] In one embodiment, CD28 expression in rTILs is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, or at least 150% relative to eTILs. In one embodiment, CD28 expression in rTILs is increased by at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold relative to eTILs.

[0125]

[0145] In one embodiment, CD27 expression in rTILs is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, or at least 150% relative to eTILs. In one embodiment, CD27 expression in rTILs is increased by at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold relative to eTILs.

[0126]

[0146] In some embodiments, the methods described herein may include optional cryopreservation of eTILs and / or rTILs in storage medium (e.g., medium containing 5% DMSO) before performing further steps described herein or after completion of the REP steps described herein before transporting, thawing, and / or administering to a patient. In some embodiments, the methods described herein may include thawing the cryopreserved TILs (e.g., cryopreserved eTILs, cryopreserved rTILs, or combinations or mixtures thereof) before performing further steps described herein. In some embodiments, the further step may be additional or repeated expansion of eTILs and / or rTILs (e.g., reREP), which may be performed on the thawed cells using supplemented cell medium including, for example, IL-2, OKT-3, and / or feeder cells (e.g., antigen presenting cells), typically including peripheral blood mononuclear cells (PBMCs; or alternatively using antigen presenting cells), and the further expansion step may be performed for at least 14 days. In some embodiments, such media may contain a combination of IL-2, IL-15 and / or IL-23 rather than IL-2 alone.

[0127]

[0147] Cryopreservation as discussed herein can occur at multiple points throughout the TIL expansion process. In some embodiments, the bulk TIL population after expansion (e.g., eTILs, rTILs, or a combination or mixture thereof) can be cryopreserved. Cryopreservation can typically be accomplished by placing the TIL population in a freezing solution, such as 85% supplemented inactivated AB serum and 15% dimethyl sulfoxide (DMSO). The cells in the solution are placed in a cryovial and stored at -80°C for 24 hours, and optionally transferred to a gas nitrogen freezer for cryopreservation. See Sadeghi, et al, Acta Oncologica 2013, 52, 978-986. In some embodiments, the TILs described herein can be cryopreserved in 5% DMSO. In some embodiments, the TILs described herein can be cryopreserved in cell culture medium plus 5% DMSO.

[0128]

[0148] Where appropriate, cryopreserved cells as described herein, such as cryopreserved rTILs, are removed from the freezer and thawed in a 37°C water bath until approximately 4 / 5 of the solution has thawed. The cells are typically resuspended in complete medium and optionally washed one or more times. In some embodiments, the thawed TILs can be counted and assessed for viability as known in the art.

[0129] How to digest tumors to obtain rTILs

[0149] In one embodiment, the method for obtaining rTILs comprises digesting the tumor with one or more enzymes. Enzymes suitable for tumor digestion are described in Volvitz, et al., BMC Neuroscience 2016, 17, 30, the disclosure of which is incorporated herein by reference.

[0130]

[0150] In some embodiments, the present invention may include a method of obtaining rTILs, which comprises digesting a tumor, which may include tumor tissue or a portion thereof, with deoxyribonuclease, collagenase, hyaluronidase, or a combination thereof.

[0131]

[0151] In one embodiment, the method of obtaining rTIL comprises digesting the tumor with any enzyme that catalyzes the hydrolytic cleavage of phosphodiester bonds in the DNA backbone, thus degrading DNA. In one embodiment, the method of obtaining rTIL comprises digesting the tumor with deoxyribonuclease (DNase). In one embodiment, the method of obtaining rTIL comprises digesting the tumor with deoxyribonuclease and at least one other enzyme. In one embodiment, the deoxyribonuclease is deoxyribonuclease I. In one embodiment, the deoxyribonuclease is deoxyribonuclease II. In one embodiment, the deoxyribonuclease is deoxyribonuclease I from bovine pancreas (Sigma D5025 or equivalent). In one embodiment, the deoxyribonuclease is recombinant deoxyribonuclease I from bovine expressed in Pichia pastoris (Sigma D2821 or equivalent). In one embodiment, the deoxyribonuclease is recombinant human deoxyribonuclease I (rhDNAase I, also known as dornase alfa, commercially available as PULMOZYME from Genentech, Inc.). In one embodiment, the deoxyribonuclease is deoxyribonuclease II from bovine spleen (Sigma D8764 or equivalent). In one embodiment, the deoxyribonuclease is deoxyribonuclease II from porcine spleen (Sigma D4138 or equivalent). In one embodiment, any of the above deoxyribonucleases are present in the tumor digest. The preparation and properties of deoxyribonucleases suitable for use in the present invention are described in U.S. Pat. Nos. 5,783,433; 6,391,607; 7,407,785; and 7,297,526, as well as WO 2016 / 108244 A1, the disclosures of each of which are incorporated herein by reference.

[0132]

[0152] In one embodiment, the method of obtaining rTILs comprises digesting the tumor with any enzyme that catalyzes the cleavage of peptide bonds in collagen, thus degrading collagen. In one embodiment, the method of obtaining rTILs comprises digesting the tumor with collagenase. In one embodiment, the method of obtaining rTILs comprises digesting the tumor with collagenase and at least one other enzyme. In one embodiment, the collagenase is collagenase from Clostridium histolyticum. In one embodiment, the collagenase is clostridiopeptidase A. In one embodiment, the collagenase is collagenase I. In one embodiment, the collagenase is collagenase II. In one embodiment, the collagenase is collagenase from Clostridium histolyticum (Sigma C5138 or equivalent). The preparation and properties of collagenases suitable for use in the present invention are described in U.S. Pat. Nos. 3,201,325; 3,705,083; 3,821,364; 5,177,017; 5,422,261; 5,989,888; and 9,211,316, the disclosures of each of which are incorporated herein by reference.

[0133]

[0153] In one embodiment, the method for obtaining rTILs comprises digesting the tumor with any enzyme that catalyzes the degradation of hyaluronic acid. In one embodiment, the method for obtaining rTILs comprises digesting the tumor with hyaluronidase. In one embodiment, the method for obtaining rTILs comprises digesting the tumor with hyaluronoglucosidase. In one embodiment, the hyaluronidase is hyaluronidase type I from bovine testis (Sigma H3506 or equivalent). In one embodiment, the hyaluronidase is hyaluronidase type II from ovine testis (Sigma H2126 or equivalent). In one embodiment, the hyaluronidase is hyaluronidase type III. In one embodiment, the hyaluronidase is hyaluronidase type IV (type IV-S) from bovine testis (Sigma H3884 or equivalent). In one embodiment, the hyaluronidase is hyaluronidase type V from ovine testis (Sigma H6254 or equivalent). In one embodiment, the hyaluronidase is hyaluronidase type VIII from bovine testis (Sigma H3757 or equivalent). In one embodiment, the hyaluronidase is recombinant human hyaluronidase (commercially available as HYLENEX from Halozyme, Inc.). The preparation and properties of hyaluronidase suitable for use in the present invention are described in U.S. Pat. Nos. 4,820,516; 5,593,877; 6,057,110; 6,123,938; 7,767,429; 8,202,517; 8,431,124; and 8,431,380, the disclosures of each of which are incorporated herein by reference.

[0134]

[0154] In one embodiment, the method for obtaining rTILs comprises digesting the tumor with deoxyribonuclease and hyaluronidase. In one embodiment, the method for obtaining rTILs comprises digesting the tumor with deoxyribonuclease and collagenase. In one embodiment, the method for obtaining rTILs comprises digesting the tumor with hyaluronidase and collagenase. In one embodiment, the method for obtaining rTILs comprises digesting the tumor with deoxyribonuclease, hyaluronidase and collagenase.

[0135]

[0155] In one embodiment, the method for obtaining rTIL comprises digesting the tumor with DNase and hyaluronidase and at least one further enzyme. In one embodiment, the method for obtaining rTIL comprises digesting the tumor with DNase and collagenase and at least one further enzyme. In one embodiment, the method for obtaining rTIL comprises digesting the tumor with hyaluronidase and collagenase and at least one further enzyme. In one embodiment, the method for obtaining rTIL comprises digesting the tumor with DNase, hyaluronidase and collagenase and at least one further enzyme. In any of the above embodiments, the further enzyme is selected from the group consisting of caseinase, clostripain, trypsin, and combinations thereof.

[0136]

[0156] In one embodiment, the method of obtaining rTILs comprises digesting the tumor with any of the enzymes described above, and further comprises mechanically disrupting or fragmenting the tumor before, during, or after digestion.

[0137]

[0157] In one embodiment, the method for obtaining rTILs comprises digesting the tumor with any of the enzymes described above, wherein the digestion is carried out for a period selected from the group consisting of 15 minutes, 30 minutes, 45 minutes, 1 hour, 90 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 18 hours, 24 hours, 36 hours and 48 hours.

[0138]

[0158] In one embodiment, the method for obtaining rTILs comprises digesting the tumor with any of the enzymes described above, wherein the digestion is carried out for a period selected from the group consisting of about 15 minutes, about 30 minutes, about 45 minutes, about 1 hour, about 90 minutes, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 18 hours, about 24 hours, about 36 hours and about 48 hours.

[0139]

[0159] In one embodiment, the method for obtaining rTILs comprises digesting the tumor with any of the enzymes described above, wherein the digestion is carried out for a period selected from the group consisting of less than 15 minutes, less than 30 minutes, less than 45 minutes, less than 1 hour, less than 90 minutes, less than 2 hours, less than 3 hours, less than 4 hours, less than 5 hours, less than 6 hours, less than 7 hours, less than 8 hours, less than 9 hours, less than 10 hours, less than 11 hours, less than 12 hours, less than 18 hours, less than 24 hours, less than 36 hours and less than 48 hours.

[0140]

[0160] In one embodiment, the method for obtaining rTILs comprises digesting the tumor with any of the enzymes described above, wherein the digestion is carried out for a period selected from the group consisting of more than 15 minutes, more than 30 minutes, more than 45 minutes, more than 1 hour, more than 90 minutes, more than 2 hours, more than 3 hours, more than 4 hours, more than 5 hours, more than 6 hours, more than 7 hours, more than 8 hours, more than 9 hours, more than 10 hours, more than 11 hours, more than 12 hours, more than 18 hours, more than 24 hours, more than 36 hours and more than 48 hours.

[0141]

[0161] In one embodiment, the method for obtaining rTILs comprises digesting the tumor with any of the enzymes described above, wherein the digestion is carried out for a period selected from the group consisting of 30 minutes to 1 hour, 1 hour to 2 hours, 2 hours to 3 hours, 3 hours to 4 hours, 4 hours to 5 hours, 5 hours to 6 hours, 6 hours to 12 hours, 12 hours to 18 hours, 18 hours to 24 hours and 24 hours to 48 hours.

[0142]

[0162] In one embodiment, the method for obtaining rTILs comprises digesting the tumor with any of the enzymes described above, wherein the digestion is carried out at a temperature selected from the group consisting of about 20°C, about 25°C, about 30°C, about 35°C, about 40°C, about 45°C, about 50°C, about 55°C, about 60°C, about 65°C, about 70°C, about 75°C and about 80°C.

[0143]

[0163] In one embodiment, the method for obtaining rTIL comprises digesting the tumor with any of the enzymes described above, wherein the digestion is carried out at a temperature selected from the group consisting of 20°C to 25°C, 25°C to 30°C, 30°C to 35°C, 35°C to 40°C, 40°C to 45°C, 45°C to 50°C, 50°C to about 55°C, 55°C to 60°C, 60°C to 65°C, 65°C to 70°C, 70°C to 75°C, and 75°C to 80°C.

[0144]

[0164] In one embodiment, the method for obtaining rTILs comprises digesting the tumor with any of the enzymes described above, where if the tumor remnants (after pre-REP) are digested, the time and temperature of digestion are respectively decreased. In one embodiment, the method for obtaining rTILs comprises digesting the tumor with any of the enzymes described above, where if the entire tumor fragment (without pre-REP) is digested, the time and temperature of digestion are respectively increased.

[0145] Methods for modulating the ratio of rTILs to eTILs

[0165] In one embodiment, the concentration of rTILs relative to eTILs can be adjusted or controlled by use of any of the expansion and digestion steps described herein (including pre-REP) such that the therapeutic TIL product used to treat cancers described herein can contain a desired ratio of rTILs to eTILs. In one embodiment, the invention provides a method for removing eTILs from a mixture of eTILs and rTILs. In one embodiment, the invention provides a method for removing rTILs from a mixture of eTILs and rTILs.

[0146]

[0166] In some embodiments of the methods of the invention, eTILs and / or rTILs may be added to the culture prior to the first expansion step (e.g., pre-REP) and / or in a second expansion step (e.g., REP). In some embodiments of the methods of the invention, eTILs may be cultured separately through one, two, three or more expansions according to the culture or expansion steps described herein and added to the population of rTILs and eTILs at a selected rTIL to eTIL ratio. In some embodiments of the methods of the invention, rTILs may be cultured separately through one, two, three or more expansions according to the culture or expansion steps described herein and added to the population of eTILs to provide a mixture of rTILs and eTILs at a selected rTIL to eTIL ratio.

[0147]

[0167] In one embodiment, eTILs prepared according to the methods described herein can be added to a population of rTILs to provide a selected rTIL to eTIL ratio in the resulting rTIL / eTIL mixture. In one embodiment, rTILs prepared according to the methods described herein can be added to a population of eTILs to provide a selected rTIL to eTIL ratio in the resulting rTIL / eTIL mixture.

[0148]

[0168] In one embodiment, the invention provides a method of treating cancer, the treatment comprising delivering a therapeutically effective amount of TILs to a patient, wherein the ratio of rTILs to eTILs in the TILs (e.g., the selected rTIL to eTIL ratio) is selected from the group consisting of about 0:100, about 1:99, about 5:95, about 10:90, about 15:85, about 20:80, about 25:75, about 30:70, about 35:65, about 40:60, about 45:55, about 50:50, about 55:45, about 60:40, about 65:35, about 70:30, about 75:25, about 80:20, about 85:15, about 90:10, about 95:5, about 99:1 and about 100:0 rTIL to eTIL.

[0149]

[0169] In one embodiment, the rTIL to eTIL ratio is adjusted using a selection method, which can be used by one of skill in the art to increase or decrease rTIL to eTIL as desired. In one embodiment, the selection method is based on a deficiency in exhaustion markers, including TIM3, LAG3, TIGIT, PD-1, and CTLA-4. In one embodiment, the selection method is based on improved CD69 expression. In one embodiment, the selection method is based on higher mitochondrial mass. In one embodiment, the selection method is based on a subset of cell surface proteins. In one embodiment, the selection method is based on phenotype. In one embodiment, the selection method is based on function.

[0150]

[0170] In one embodiment, the rTIL to eTIL ratio is adjusted by co-culturing rTIL and eTIL in the same cell culture medium until the desired ratio is achieved. In one embodiment, the rTIL to eTIL ratio is adjusted by co-culturing rTIL and eTIL in the same cell culture medium until the desired ratio is achieved, including the addition of rTIL or eTIL to the cell culture medium at various times during expansion. In one embodiment, rTIL expansion is preferentially expanded in cell culture medium with the addition of cytokines other than IL-2, including IL-4, IL-7, IL-15 and / or IL-21.

[0151]

[0171] In one embodiment, the ratio of rTILs to eTILs (e.g., a selected rTIL to eTIL ratio) provided by the methods described herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45% , 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% rTILs versus eTILs.

[0152]

[0172] In one embodiment, the ratio of rTILs to eTILs (e.g., a selected rTIL to eTIL ratio) provided by the methods described herein is at most 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, The rTILs to eTILs may be 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9%.

[0153]

[0173] In one embodiment, the ratio of rTILs to eTILs provided by the methods described herein (e.g., a selected rTIL to eTIL ratio) is about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 109%, 109%, 104%, 105%, 106%, 107%, 6%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% rTILs versus eTILs.

[0154] Methods of Treating Cancer and Other Diseases

[0174] The rTILs and combinations of rTILs and eTILs described herein can be used in methods of treating disease in humans. In one embodiment, they are used to treat hyperproliferative disorders. In some embodiments, the hyperproliferative disorder is cancer. In some embodiments, the hyperproliferative disorder is a solid tumor cancer. In some embodiments, the solid tumor cancer is selected from the group consisting of melanoma, double refractory melanoma (i.e., melanoma refractory to at least two prior treatments including chemotherapy and checkpoint inhibitors), ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, cancer caused by human papillomavirus, head and neck cancer, kidney cancer, renal cell carcinoma, and sarcoma. In some embodiments, the hyperproliferative disorder is a hematological malignancy (or liquid tumor cancer). In some embodiments, the hematological malignancy is selected from the group consisting of acute myeloid leukemia, chronic lymphocytic leukemia, acute lymphoblastic leukemia, diffuse large B-cell lymphoma, non-Hodgkin's lymphoma, Hodgkin's lymphoma, follicular lymphoma, and mantle cell lymphoma. The rTILs and combinations of rTILs and eTILs described herein may also be used to treat other disorders described herein and in the following paragraphs.

[0155]

[0175] In one embodiment, the invention includes a method of treating cancer in a patient in need of such treatment, the treatment comprising delivering to the patient a therapeutically effective amount of rTILs, the rTILs comprising: (a) obtaining tumor tissue from a patient, the tumor tissue comprising tumor infiltrating lymphocytes (TILs); (b) fragmenting the tumor tissue; (c) treating the tumor tissue in a gas permeable container with the first cell culture medium and interleukin 2 (IL-2) to provide tumor remnants and nascent TILs (eTILs); (d) removing at least some of the eTILs; (e) enzymatically digesting the tumor remnant into tumor remnant cells using the digestion mixture; (f) growing the tumor remnant cells in a second cell culture medium containing cell culture medium, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas permeable container to expand the number of remnant tumor infiltrating lymphocytes (rTILs); and preparing the product according to a method comprising: rTILs expressed reduced levels of T cell exhaustion markers relative to eTILs, the T cell exhaustion marker is selected from the group consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof; The second cell culture medium further comprises a cytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21, and combinations thereof.

[0156]

[0176] In one embodiment, the invention includes a method of treating cancer in a patient in need of such treatment, the method comprising: (a) obtaining tumor tissue from a patient, the tumor tissue comprising tumor infiltrating lymphocytes (TILs); (b) fragmenting the tumor tissue; and (c) treating the tumor tissue in a gas permeable container with the first cell culture medium and interleukin 2 (IL-2) to provide tumor remnants and nascent TILs (eTILs); (d) excluding at least some eTILs; and (e) enzymatically digesting the tumor remnant into tumor remnant cells using a digestion mixture; (f) expanding the tumor remnant cells in a second cell culture medium comprising cell culture medium, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas permeable container to provide an expanded number of remnant tumor infiltrating lymphocytes (rTILs); rTILs expressed reduced levels of T cell exhaustion markers relative to eTILs, the T cell exhaustion marker is selected from the group consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof; providing a second cell culture medium further comprising a cytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21, and combinations thereof; (g) treating the patient with a non-myeloablative lymphodepleting regimen prior to administering the rTILs to the patient; (h) administering to the patient a therapeutically effective amount of rTILs; (i) treating the patient with a high-dose IL-2 regimen starting the day after administration of the rTILs to the patient; Includes.

[0157]

[0177] In one embodiment, the invention includes a method of treating cancer in a patient in need of such treatment, the method comprising: (a) obtaining tumor tissue from a patient, the tumor tissue comprising tumor infiltrating lymphocytes (TILs); (b) fragmenting the tumor tissue; and (c) treating the tumor tissue in a gas permeable container with the first cell culture medium and interleukin 2 (IL-2) to provide tumor remnants and nascent TILs (eTILs); (d) excluding at least some eTILs; and (e) enzymatically digesting the tumor remnant into tumor remnant cells using a digestion mixture; (f) expanding the tumor remnant cells in a second cell culture medium comprising cell culture medium, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas permeable container to provide an expanded number of remnant tumor infiltrating lymphocytes (rTILs); rTILs expressed reduced levels of T cell exhaustion markers relative to eTILs, the T cell exhaustion marker is selected from the group consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof; providing a second cell culture medium further comprising a cytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21, and combinations thereof; (g) treating the patient with a non-myeloablative lymphodepleting regimen prior to administering the rTILs to the patient; (h) administering to the patient a therapeutically effective amount of rTILs, wherein a therapeutically effective amount of eTILs is co-administered to the patient in a mixture with the rTILs; (i) treating the patient with a high-dose IL-2 regimen starting the day after administration of the rTILs to the patient; Includes.

[0158]

[0178] In one embodiment, the invention includes a method of treating cancer in a patient in need of such treatment, the method comprising: (a) obtaining tumor tissue from a patient, the tumor tissue comprising tumor infiltrating lymphocytes (TILs); (b) fragmenting the tumor tissue; and (c) treating the tumor tissue in a gas permeable container with the first cell culture medium and interleukin 2 (IL-2) to provide tumor remnants and nascent TILs (eTILs); (d) excluding at least some eTILs; and (e) enzymatically digesting the tumor remnant into tumor remnant cells using a digestion mixture; (f) expanding the tumor remnant cells in a second cell culture medium comprising cell culture medium, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas permeable container to provide an expanded number of remnant tumor infiltrating lymphocytes (rTILs); rTILs expressed reduced levels of T cell exhaustion markers relative to eTILs, the T cell exhaustion marker is selected from the group consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof; providing a second cell culture medium further comprising a cytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21, and combinations thereof; (g) treating the patient with a non-myeloablative lymphodepleting regimen prior to administering the rTILs to the patient; (h) administering to the patient a therapeutically effective amount of rTILs, wherein a therapeutically effective amount of eTILs is co-administered to the patient in a mixture with the rTILs; (i) treating the patient with a high-dose IL-2 regimen starting the day after administration of the rTILs to the patient; Including, The cancer is selected from the group consisting of melanoma, double refractory melanoma, ovarian cancer, cervical cancer, lung cancer, bladder cancer, breast cancer, head and neck cancer, renal cell carcinoma, acute myeloid leukemia, colorectal cancer, sarcoma, non-small cell lung cancer (NSCLC) and triple negative breast cancer.

[0159]

[0179] In one embodiment, the invention includes a method of treating cancer in a patient in need of such treatment, the method comprising: (a) obtaining tumor tissue from a patient, the tumor tissue comprising tumor infiltrating lymphocytes (TILs); (b) fragmenting the tumor tissue; and (c) treating the tumor tissue in a gas permeable container with the first cell culture medium and interleukin 2 (IL-2) to provide tumor remnants and nascent TILs (eTILs); (d) excluding at least some eTILs; and (e) enzymatically digesting the tumor remnant into tumor remnant cells using a digestion mixture; (f) expanding the tumor remnant cells in a second cell culture medium comprising cell culture medium, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas permeable container to provide an expanded number of remnant tumor infiltrating lymphocytes (rTILs); rTILs expressed reduced levels of T cell exhaustion markers relative to eTILs, the T cell exhaustion marker is selected from the group consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof; providing a second cell culture medium further comprising a cytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21, and combinations thereof; (g) treating the patient with a non-myeloablative lymphodepleting regimen prior to administering the rTILs to the patient, the non-myeloablative lymphodepleting regimen being 60 mg / m 2 / day followed by cyclophosphamide at 25 mg / m for 5 days. 2 administering fludarabine at a dose of 100 mg / kg / day to (h) administering to the patient a therapeutically effective amount of rTILs, wherein a therapeutically effective amount of eTILs is co-administered to the patient in a mixture with the rTILs; (i) treating the patient with a high-dose IL-2 regimen starting the day after administration of the rTILs to the patient; Includes.

[0160]

[0180] In some embodiments of the methods described herein, the step of removing at least a plurality of eTILs is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 109%, 109%, 104%, 105%, 106%, 107%, 108%, 109%, 109%, 109 This includes excluding 9%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or 100% of the eTIL.

[0161]

[0181] The efficacy of the compounds and combinations of compounds described herein in treating, preventing and / or managing the indicated diseases or disorders can be tested using various models known in the art, which provide guidance for the treatment of human diseases.For example, a model for determining the efficacy of treatment of ovarian cancer is described, for example, in Mullany, et al., Endocrinology 2012, 153, 1585-92; and Fong, et al., J. Ovarian Res. 2009, 2, 12. A model for determining the efficacy of treatment of pancreatic cancer is described, for example, in Herreros-Villanueva, et al, World J. Gastroenterol. 2012, 18, 1286-1294. A model for determining the efficacy of treatment of breast cancer is described, for example, in Fantozzi, Breast Cancer Res. 2006, 8, 212. Models for determining the effectiveness of melanoma treatments are described, for example, in Damsky, et al, Pigment Cell & Melanoma Res. 2010, 23, 853-859. Models for determining the effectiveness of lung cancer treatments are described, for example, in Meuwissen, et al, Genes & Development, 2005, 19, 643-664. Models for determining the effectiveness of lung cancer treatments are described, for example, in Kim, Clin. Exp. Otorhinolaryngol. 2009, 2, 55-60; and Sano, Head Neck Oncol. 2009, 1, 32.

[0162] Coadministration of IL-2

[0182] In one embodiment, the present invention provides a method of treating cancer in a patient in need of such treatment, comprising: (a) obtaining rTILs from a tumor resected from a patient according to the methods described herein; (b) treating the patient with a non-myeloablative lymphodepleting regimen prior to administering the rTILs to the patient; (c) administering to the patient a therapeutically effective amount of rTILs; (d) treating the patient with an IL-2 regimen starting the day after administration of the rTILs to the patient; The present invention provides a method comprising:

[0163]

[0183] In one embodiment, the IL-2 regimen comprises a high dose IL-2 regimen comprising aldesleukin or a biosimilar or variant thereof administered intravenously beginning the day after administration of a therapeutically effective portion of the third population of TILs, where aldesleukin or a biosimilar or variant thereof is administered using a 15 minute bolus intravenous infusion every 8 hours until tolerated for a maximum of 14 doses at a dose of 600,000 or 720,000 IU / kg of patient weight. After 9 rest days, this schedule can be repeated for an additional 14 doses, for a total of up to 28 doses.

[0164]

[0184] In one embodiment, the IL-2 regimen comprises a high dose IL-2 regimen comprising aldesleukin or a biosimilar or variant thereof administered intravenously beginning the day after administration of a therapeutically effective portion of the third population of TILs, where aldesleukin or a biosimilar or variant thereof is administered using a 15 minute bolus intravenously every 8 hours until tolerated for a maximum of 14 doses at a dose of 0.037 mg / kg or 0.044 mg / kg IU / kg of patient weight. After 9 rest days, this schedule can be repeated for an additional 14 doses, for a total of up to 28 doses.

[0165]

[0185] In one embodiment, the invention includes a method of treating cancer in a patient in need of such treatment, the method comprising: (a) obtaining tumor tissue from a patient, the tumor tissue comprising tumor infiltrating lymphocytes (TILs); (b) fragmenting the tumor tissue; and (c) treating the tumor tissue in a gas permeable container with the first cell culture medium and interleukin 2 (IL-2) to provide tumor remnants and nascent TILs (eTILs); (d) excluding at least some eTILs; and (e) enzymatically digesting the tumor remnant into tumor remnant cells using a digestion mixture; (f) expanding the tumor remnant cells in a second cell culture medium comprising cell culture medium, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas permeable container to provide an expanded number of remnant tumor infiltrating lymphocytes (rTILs); rTILs expressed reduced levels of T cell exhaustion markers relative to eTILs, the T cell exhaustion marker is selected from the group consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof; providing a second cell culture medium further comprising a cytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21, and combinations thereof; (g) treating the patient with a non-myeloablative lymphodepleting regimen prior to administering the rTILs to the patient; (h) administering to the patient a therapeutically effective amount of rTILs, wherein a therapeutically effective amount of eTILs is co-administered to the patient in a mixture with the rTILs; (i) treating the patient with a high-dose IL-2 regimen starting the day after administration of the rTILs to the patient; Including, High-dose IL-2 regimens include 600,000 or 720,000 IU / kg aldesleukin or its biosimilar or variant administered as a 15-minute bolus intravenously every 8 hours until tolerated.

[0166]

[0186] In one embodiment, the IL-2 regimen comprises a decrescendo IL-2 regimen, as described in O'Day, et al., J. Clin. Oncol. 1999, 17, 2752-61 and Eton, et al., Cancer 2000, 88, 1703-9, the disclosures of which are incorporated herein by reference. In one embodiment, the decrescendo IL-2 regimen comprises 18×10 IL-2 administered intravenously over 6 hours. 6 IU / m 2 , followed by 18 × 10 6 IU / m 2 , followed by 18 × 10 6 IU / m 2 , followed by 4.5 × 10 administered intravenously over the subsequent 72 hours. 6 IU / m 2 This treatment cycle may be repeated every 28 days for up to four cycles. In one embodiment, the tapered IL-2 regimen includes 18,000,000 IU / m on day 1. 2 , 9,000,000 IU / m on the second day 2 and 4,500,000 IU / m on days 3 and 4. 2 Includes.

[0167]

[0187] In one embodiment, the invention includes a method of treating cancer in a patient in need of such treatment, the method comprising: (a) obtaining tumor tissue from a patient, the tumor tissue comprising tumor infiltrating lymphocytes (TILs); (b) fragmenting the tumor tissue; and (c) treating the tumor tissue in a gas permeable container with the first cell culture medium and interleukin 2 (IL-2) to provide tumor remnants and nascent TILs (eTILs); (d) excluding at least some eTILs; and (e) enzymatically digesting the tumor remnant into tumor remnant cells using a digestion mixture; (f) expanding the tumor remnant cells in a second cell culture medium comprising cell culture medium, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas permeable container to provide an expanded number of remnant tumor infiltrating lymphocytes (rTILs); rTILs expressed reduced levels of T cell exhaustion markers relative to eTILs, the T cell exhaustion marker is selected from the group consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof; providing a second cell culture medium further comprising a cytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21, and combinations thereof; (g) treating the patient with a non-myeloablative lymphodepleting regimen prior to administering the rTILs to the patient; (h) administering to the patient a therapeutically effective amount of rTILs, wherein a therapeutically effective amount of eTILs is co-administered to the patient in a mixture with the rTILs; (i) treating the patient with a tapered IL-2 regimen starting the day after administration of the rTILs to the patient; Including, The tapered IL-2 regimen consisted of 18 × 10 6 IU / m 2 , followed by 18 × 10 6 IU / m 2 , followed by 18 × 10 6 IU / m 2 , followed by 4.5 × 10 administered intravenously over the subsequent 72 hours. 6 IU / m 2 Repeated every 28 days for up to four cycles.

[0168]

[0188] In one embodiment, the IL-2 regimen includes administration of pegylated IL-2, including pegylated aldesleukin, in one embodiment, the IL-2 regimen includes administration of pegylated IL-2 at a dose of 0.10 mg / day to 50 mg / day every 1, 2, 4, 6, 7, 14, or 21 days.

[0169]

[0189] In one embodiment, the invention includes a method of treating cancer in a patient in need of such treatment, the method comprising: (a) obtaining tumor tissue from a patient, the tumor tissue comprising tumor infiltrating lymphocytes (TILs); (b) fragmenting the tumor tissue; and (c) treating the tumor tissue in a gas permeable container with the first cell culture medium and interleukin 2 (IL-2) to provide tumor remnants and nascent TILs (eTILs); (d) excluding at least some eTILs; and (e) enzymatically digesting the tumor remnant into tumor remnant cells using a digestion mixture; (f) expanding the tumor remnant cells in a second cell culture medium comprising cell culture medium, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas permeable container to provide an expanded number of remnant tumor infiltrating lymphocytes (rTILs); rTILs expressed reduced levels of T cell exhaustion markers relative to eTILs, the T cell exhaustion marker is selected from the group consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof; providing a second cell culture medium further comprising a cytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21, and combinations thereof; (g) treating the patient with a non-myeloablative lymphodepleting regimen prior to administering the rTILs to the patient; (h) administering to the patient a therapeutically effective amount of rTILs, wherein a therapeutically effective amount of eTILs is co-administered to the patient in a mixture with the rTILs; (i) treating the patient with a pegylated IL-2 regimen starting the day after administration of the rTILs to the patient; Including, The pegylated IL-2 regimen involves administration of pegylated IL-2 at a dose of 0.10 mg / day to 50 mg / day every 1, 2, 4, 6, 7, 14 or 21 days.

[0170] Nonmyeloablative lymphodepletion by chemotherapy

[0190] In one embodiment, the invention includes a method of treating cancer with a population of rTILs, where the patient is pretreated with a non-myeloablative chemotherapy prior to infusion of the rTILs according to the invention. In some embodiments, a population of rTILs may be provided along with a population of eTILs, where the patient is pretreated with a non-myeloablative chemotherapy prior to infusion of the rTILs and eTILs according to the invention. In one embodiment, the non-myeloablative chemotherapy comprises cyclophosphamide 60 mg / kg / d for 2 days (days 27 and 26 prior to rTIL infusion) and fludarabine 25 mg / m for 5 days (days 27-23 prior to rTIL infusion). 2 In one embodiment, after non-myeloablative chemotherapy and rTIL infusion according to the invention (day 0), patients receive IL-2 intravenously at 720,000 IU / kg every 8 hours to physiological tolerance.

[0171]

[0191] Experimental findings indicate that lymphodepletion prior to adoptive transfer of tumor-specific T lymphocytes plays a key role in enhancing treatment 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 (sometimes also referred to as "immunosuppressive conditioning") in patients prior to the introduction of the rTILs of the present invention.

[0172]

[0192] Generally, lymphodepletion is achieved using administration of fludarabine or cyclophosphamide (the active form is called mafosfamide) and combinations thereof. Such methods are described in Gassner, et al, Cancer Immunol. Immunother. 2011, 60, 75-85; Muranski, et al, Nat. Clin. Pract. Oncol, 2006, 3, 668-681; Dudley, et al, J. Clin. Oncol. 2008, 26, 5233-5239 and Dudley, et al, J. Clin. Oncol. 2005, 23, 2346-2357, all of which are incorporated herein by reference in their entirety.

[0173]

[0193] In some embodiments, fludarabine is administered at a fludarabine concentration of 0.5 μg / mL to 10 μg / mL. In some embodiments, fludarabine is administered at a fludarabine concentration of 1 μg / mL. In some embodiments, fludarabine treatment is administered for 1, 2, 3, 4, 5, 6, or 7 days or more. In some embodiments, fludarabine is administered at a dosage of 10 mg / kg / day, 15 mg / kg / day, 20 mg / kg / day, 25 mg / kg / day, 30 mg / kg / day, 35 mg / kg / day, 40 mg / kg / day, or 45 mg / kg / day. In some embodiments, fludarabine treatment is administered at 35 mg / kg / day for 2 to 7 days. In some embodiments, fludarabine treatment is administered at 35 mg / kg / day for 4 to 5 days. In some embodiments, fludarabine treatment is administered at 25 mg / kg / day for 4-5 days.

[0174]

[0194] In some embodiments, mafosfamide (the active form of cyclophosphamide) is provided by administration of cyclophosphamide at a concentration of 0.5 μg / mL to 10 μg / mL. In some embodiments, mafosfamide (the active form of cyclophosphamide) is provided by administration of cyclophosphamide at a concentration of 1 μg / mL. In some embodiments, cyclophosphamide treatment is administered for 1, 2, 3, 4, 5, 6, or 7 days or more. In some embodiments, cyclophosphamide is administered at a concentration of 100 mg / m 2 / day, 150mg / m 2 / day, 175mg / m 2 / day, 200mg / m 2 / day, 225mg / m 2 / day, 250mg / m 2 / day, 275mg / m 2 / day or 300 mg / m 2 In some embodiments, cyclophosphamide is administered at a dosage of 35 mg / kg / day for 2-7 days. In some embodiments, cyclophosphamide treatment is administered at a dosage of 250 mg / kg / day. In some embodiments, cyclophosphamide is administered intravenously (iv). In some embodiments, cyclophosphamide treatment is administered at a dosage of 35 mg / kg / day for 2-7 days. In some embodiments, cyclophosphamide treatment is administered at a dosage of 250 mg / kg / day. 2 / day iv for 4-5 days. In some embodiments, cyclophosphamide treatment is administered at 250 mg / m 2 / day iv for 4 days.

[0175]

[0195] In some embodiments, lymphodepletion is performed by administering fludarabine, and cyclophosphamide is administered to the patient concomitantly. In some embodiments, fludarabine is administered at a dose of 25 mg / m 2 / day iv, and cyclophosphamide was administered at 250 mg / m 2 / day iv for 4 days.

[0176]

[0196] In one embodiment, lymphodepletion is with 60 mg / m 2 / day followed by cyclophosphamide at 25 mg / m for 5 days. 2The procedure is performed by administering fludarabine at a dose of 100 mg / day.

[0177] Pharmaceutical Compositions, Dosages and Dosing Regimens

[0197] In one embodiment, the rTILs expanded using the method of the invention are administered to a patient as a pharmaceutical composition. In one embodiment, the pharmaceutical composition is a suspension of rTILs in a sterile buffer. The rTILs expanded using the method of the invention may be administered by any suitable route known in the art. Preferably, the rTILs are administered as a single intra-arterial or intravenous infusion, preferably lasting approximately 30-60 minutes. Other suitable routes of administration include intraperitoneal, intrathecal and intralymphatic administration.

[0178]

[0198] In one embodiment, the rTILs and eTILs expanded using the methods of the invention are administered to a patient as a pharmaceutical composition. In one embodiment, the pharmaceutical composition is a suspension of rTILs and eTILs in a sterile buffer. The rTILs and eTILs expanded using the methods of the invention may be administered by any suitable route known in the art. Preferably, the rTILs and eTILs are administered as a single intra-arterial or intravenous infusion, preferably lasting approximately 30-60 minutes. Other suitable routes of administration include intraperitoneal, intrathecal and intralymphatic administration.

[0179]

[0199] Any suitable dose of rTILs may be administered. Preferably, the dose is about 2.3×10 10 ~Approx. 13.7×10 10 rTILs were administered, with an average of approximately 7.8 × 10 10 In one embodiment, about 1.2×10 rTILs are administered. 10 ~Approx. 4.3×10 10 rTILs are administered.

[0180]

[0200] Any suitable dose of rTILs and eTILs may be administered. Preferably, the dose is about 2.3×10 10~Approx. 13.7×10 10 rTILs and eTILs were administered, with an average of approximately 7.8×10 10 In one embodiment, about 1.2×10 rTILs and eTILs are administered. 10 ~Approx. 4.3×10 10 rTILs and eTILs will be administered.

[0181]

[0201] In some embodiments, the number of rTILs provided in the pharmaceutical composition of the present invention is about 1×10 6 , 2×10 6 , 3×10 6 , 4×10 6 , 5×10 6 , 6×10 6 , 7×10 6 , 8×10 6 , 9×10 6 , 1×10 7 , 2×10 7 , 3×10 7 , 4×10 7 , 5×10 7 , 6×10 7 , 7×10 7 , 8×10 7 , 9×10 7 , 1×10 8 , 2×10 8 , 3×10 8 , 4×10 8 , 5×10 8 , 6×10 8 , 7×10 8 , 8×10 8 , 9×10 8 , 1×10 9 , 2×10 9 , 3×10 9 , 4×10 9 , 5×10 9 , 6×10 9 , 7×10 9 , 8×10 9 , 9×10 9 , 1×10 10 , 2×10 10 , 3×10 10 , 4×10 10 , 5×10 10 , 6×1010 , 7×10 10 , 8×10 10 , 9×10 10 , 1×10 11 , 2×10 11 , 3×10 11 , 4×10 11 , 5×10 11 , 6×10 11 , 7×10 11 , 8×10 11 , 9×10 11 , 1×10 12 , 2×10 12 , 3×10 12 , 4×10 12 , 5×10 12 , 6×10 12 , 7×10 12 , 8×10 12 , 9×10 12 , 1×10 13 , 2×10 13 , 3×10 13 , 4×10 13 , 5×10 13 , 6×10 13 , 7×10 13 and 8 x 10 13 , 9×10 13 In one embodiment, the number of rTILs provided in the pharmaceutical composition of the present invention is 1×10 6 ~5×10 6 , 5×10 6 ~1×10 7 , 1×10 7 ~5×10 7 , 5×10 7 ~1×10 8 , 1×10 8 ~5×10 8 , 5×10 8 ~1×10 9 , 1×10 9 ~5×10 9 , 5×10 9 ~1×10 10 , 1×10 10 ~5×10 10 , 5×10 10 ~1×10 11 , 5×10 11 ~1×10 12 , 1×1012 ~5×10 12 and 5 x 10 12 ~1×10 13 It is within the range of individuals.

[0182]

[0202] In some embodiments, the number of rTILs and eTILs provided in the pharmaceutical composition of the present invention is about 1×10 6 , 2×10 6 , 3×10 6 , 4×10 6 , 5×10 6 , 6×10 6 , 7×10 6 , 8×10 6 , 9×10 6 , 1×10 7 , 2×10 7 , 3×10 7 , 4×10 7 , 5×10 7 , 6×10 7 , 7×10 7 , 8×10 7 , 9×10 7 , 1×10 8 , 2×10 8 , 3×10 8 , 4×10 8 , 5×10 8 , 6×10 8 , 7×10 8 , 8×10 8 , 9×10 8 , 1×10 9 , 2×10 9 , 3×10 9 , 4×10 9 , 5×10 9 , 6×10 9 , 7×10 9 , 8×10 9 , 9×10 9 , 1×10 10 , 2×10 10 , 3×10 10 , 4×10 10 , 5×10 10 , 6×10 10 , 7×10 10 , 8×10 10 , 9×10 10 , 1×10 11 , 2×1011 , 3×10 11 , 4×10 11 , 5×10 11 , 6×10 11 , 7×10 11 , 8×10 11 , 9×10 11 , 1×10 12 , 2×10 12 , 3×10 12 , 4×10 12 , 5×10 12 , 6×10 12 , 7×10 12 , 8×10 12 , 9×10 12 , 1×10 13 , 2×10 13 , 3×10 13 , 4×10 13 , 5×10 13 , 6×10 13 , 7×10 13 , 8×10 13 and 9 x 10 13 In one embodiment, the number of rTILs and eTILs provided in the pharmaceutical composition of the present invention is 1×10 6 ~5×10 6 , 5×10 6 ~1×10 7 , 1×10 7 ~5×10 7 , 5×10 7 ~1×10 8 , 1×10 8 ~5×10 8 , 5×10 8 ~1×10 9 , 1×10 9 ~5×10 9 , 5×10 9 ~1×10 10 , 1×10 10 ~5×10 10 , 5×10 10 ~1×10 11 , 5×10 11 ~1×10 12 , 1×10 12 ~5×10 12 and 5 x 10 12 ~1×10 13 It is within the range of individuals.

[0183]

[0203] In some embodiments, the concentration of rTIL provided in the pharmaceutical composition of the present invention may be, for example, 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.0% or more of the pharmaceutical composition. 7%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002% or 0.0001% w / w, w / v or v / v.

[0184]

[0204] In some embodiments, the concentration of rTIL and eTIL provided in the pharmaceutical composition of the present invention may be, for example, 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, ... 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002% or 0.0001% w / w, w / v or v / v.

[0185]

[0205] In some embodiments, the concentration of rTIL provided in the pharmaceutical composition of the present invention is 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19.75%, 19.50%, 19.25%, 19%, 18.75%, 18.50%, 18.25%, 18%, 17.75%, 17.50%, 17.25%, 17%, 16.75%, 16.50%, 16.25%, 16%, 15.75%, 15.5%, 16 ... 0%, 15.25%, 15%, 14.75%, 14.50%, 14.25%, 14%, 13.75%, 13.50%, 13.25%, 13%, 12.75%, 12.50%, 12.25%, 12%, 11.75%, 11.50%, 11.25%, 11%, 10.75%, 10.50%, 10.25%, 10%, 9.75%, 9.50%, 9.25%, 9%, 8.75%, 8.50%, 8.25%, 8%, 7.75% ,7.50%,7.25%,7%,6.75%,6.50%,6.25%,6%,5.75%,5.50%,5.25%,5%,4.75%,4.50%,4.25%,4%,3.75%,3.50%,3.25%,3%,2.75%,2.50%,2.25%,2%,1.75%,1.50%,125%,1%,0.5%,0.4%,0.3%,0.2%,0.1%,0.09%,0.08%,0.07%,0. 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002% or greater than 0.0001% w / w, w / v or v / v.

[0186]

[0206] In some embodiments, the concentration of rTIL and eTIL provided in the pharmaceutical composition of the present invention is 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19.75%, 19.50%, 19.25%, 19%, 18.75%, 18.50%, 18.25%, 18%, 17.75%, 17.50%, 17.25%, 17%, 16.75%, 16.50%, 16.25%, 16%, 15.75% of the pharmaceutical composition. ,15.50%,15.25%,15%,14.75%,14.50%,14.25%,14%,13.75%,13.50%,13.25%,13%,12.75%,12.50%,12.25%,12%,11.75%,11.50%,11.25%,11%,10.75%,10.50%,10.25%,10%,9.75%,9.50%,9.25%,9%,8.75%,8.50%,8.25%,8%,7. 75%,7.50%,7.25%,7%,6.75%,6.50%,6.25%,6%,5.75%,5.50%,5.25%,5%,4.75%,4.50%,4.25%,4%,3.75%,3.50%,3.25%,3%,2.75%,2.50%,2.25%,2%,1.75%,1.50%,125%,1%,0.5%,0.4%,0.3%,0.2%,0.1%,0.09%,0.08%,0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002% or greater than 0.0001% w / w, w / v or v / v.

[0187]

[0207] In some embodiments, the concentration of rTIL provided in the pharmaceutical composition of the present invention is from about 0.0001% to about 50%, from about 0.001% to about 40%, from about 0.01% to about 30%, from about 0.02% to about 29%, from about 0.03% to about 28%, from about 0.04% to about 27%, from about 0.05% to about 26%, from about 0.06% to about 25%, from about 0.07% to about 24% of the pharmaceutical composition. , about 0.08% to about 23%, about 0.09% to about 22%, about 0.1% to about 21%, about 0.2% to about 20%, about 0.3% to about 19%, about 0.4% to about 18%, about 0.5% to about 17%, about 0.6% to about 16%, about 0.7% to about 15%, about 0.8% to about 14%, about 0.9% to about 12%, or about 1% to about 10% w / w, w / v or v / v.

[0188]

[0208] In some embodiments, the concentration of rTIL and eTIL provided in the pharmaceutical composition of the present invention is from about 0.0001% to about 50%, from about 0.001% to about 40%, from about 0.01% to about 30%, from about 0.02% to about 29%, from about 0.03% to about 28%, from about 0.04% to about 27%, from about 0.05% to about 26%, from about 0.06% to about 25%, from about 0.07% to about 24%, about 0.08% to about 23%, about 0.09% to about 22%, about 0.1% to about 21%, about 0.2% to about 20%, about 0.3% to about 19%, about 0.4% to about 18%, about 0.5% to about 17%, about 0.6% to about 16%, about 0.7% to about 15%, about 0.8% to about 14%, about 0.9% to about 12%, or about 1% to about 10% w / w, w / v or v / v.

[0189]

[0209] In some embodiments, the concentration of rTIL provided in the pharmaceutical composition of the present invention is within the range of about 0.001% to about 10%, about 0.01% to about 5%, about 0.02% to about 4.5%, about 0.03% to about 4%, about 0.04% to about 3.5%, about 0.05% to about 3%, about 0.06% to about 2.5%, about 0.07% to about 2%, about 0.08% to about 1.5%, about 0.09% to about 1%, about 0.1% to about 0.9% w / w, w / v or v / v of the pharmaceutical composition.

[0190]

[0210] In some embodiments, the concentration of rTIL and eTIL provided in the pharmaceutical composition of the present invention is within the range of about 0.001% to about 10%, about 0.01% to about 5%, about 0.02% to about 4.5%, about 0.03% to about 4%, about 0.04% to about 3.5%, about 0.05% to about 3%, about 0.06% to about 2.5%, about 0.07% to about 2%, about 0.08% to about 1.5%, about 0.09% to about 1%, about 0.1% to about 0.9% w / w, w / v or v / v of the pharmaceutical composition.

[0191]

[0211] In some embodiments, the amount of rTIL provided in the pharmaceutical composition of the present invention is 10 g, 9.5 g, 9.0 g, 8.5 g, 8.0 g, 7.5 g, 7.0 g, 6.5 g, 6.0 g, 5.5 g, 5.0 g, 4.5 g, 4.0 g, 3.5 g, 3.0 g, 2.5 g, 2.0 g, 1.5 g, 1.0 g, 0.95 g, 0.9 g, 0.85 g, 0.8 g, 0.75 g, 0.7 g, 0.65 g, 0.6 g, 0.55 g, 0.5 g, 0.45 g, 0.4 g, 0.35 g, 0.3 g, 0.25 g, , 0.2g, 0.15g, 0.1g, 0.09g, 0.08g, 0.07g, 0.06g, 0.05g, 0.04g, 0.03g, 0.02g, 0.01g, 0.009g, 0.008g, 0.007g, 0.006g, 0.005g, 0.004g, 0.003g, 0.002g, 0.001g, 0.0009g, 0.0008g, 0.0007g, 0.0006g, 0.0005g, 0.0004g, 0.0003g, 0.0002g or 0.0001g.

[0192]

[0212] In some embodiments, the amount of rTIL and eTIL provided in the pharmaceutical composition of the present invention is 10g, 9.5g, 9.0g, 8.5g, 8.0g, 7.5g, 7.0g, 6.5g, 6.0g, 5.5g, 5.0g, 4.5g, 4.0g, 3.5g, 3.0g, 2.5g, 2.0g, 1.5g, 1.0g, 0.95g, 0.9g, 0.85g, 0.8g, 0.75g, 0.7g, 0.65g, 0.6g, 0.55g, 0.5g, 0.45g, 0.4g, 0.35g, 0.3g, 0.5g, 0.6g, 0.8g, 0.9 ...g, 0.8g, 0.9g, 0.8g, 0.8g, 0.9g, 0.8g, 0.8g, 0.9g, 0.8g, 0.8g, 0.9g, 0.8g, 0.8g, 0.8g, 0.8g, 0.8g, 0.8g, 0.8g, 0.8g, 0.8g, 0.8g, 25g, 0.2g, 0.15g, 0.1g, 0.09g, 0.08g, 0.07g, 0.06g, 0.05g, 0.04g, 0.03g, 0.02g, 0.01g, 0.009g, 0.008g, 0.007g, 0.006g, 0.005g, 0.004g, 0.003g, 0.002g, 0.001g, 0.0009g, 0.0008g, 0.0007g, 0.0006g, 0.0005g, 0.0004g, 0.0003g, 0.0002g, or 0.0001g.

[0193]

[0213] In some embodiments, the amount of rTIL provided in the pharmaceutical composition of the present invention is 0.0001g, 0.0002g, 0.0003g, 0.0004g, 0.0005g, 0.0006g, 0.0007g, 0.0008g, 0.0009g, 0.001g, 0.0015g, 0.002 ... 5g, 0.003g, 0.0035g, 0.004g, 0.0045g, 0.005g, 0.0055g, 0.006g, 0.0065g, 0.00 7g, 0.0075g, 0.008g, 0.0085g, 0.009g, 0.0095g, 0.01g, 0.015g, 0.02g, 0.025g, 0 .03g, 0.035g, 0.04g, 0.045g, 0.05g, 0.055g, 0.06g, 0.065g, 0.07g, 0.075g, 0.0 8g, 0.085g, 0.09g, 0.095g, 0.1g, 0.15g, 0.2g, 0.25g, 0.3g, 0.35g, 0.4g, 0.45g, 0 .5g, 0.55g, 0.6g, 0.65g, 0.7g, 0.75g, 0.8g, 0.85g, 0.9g, 0.95g, 1g, 1.5g, 2g, 2. 5g, 3g, 3.5g, 4g, 4.5g, 5g, 5.5g, 6g, 6.5g, 7g, 7.5g, 8g, 8.5g, 9g, 9.5g or more than 10g.

[0194]

[0214] In some embodiments, the amount of rTIL and eTIL provided in the pharmaceutical composition of the present invention is 0.0001g, 0.0002g, 0.0003g, 0.0004g, 0.0005g, 0.0006g, 0.0007g, 0.0008g, 0.0009g, 0.001g, 0.0015g, 0.002g, 0.0025g, 0.003g, 0.0035g, 0.004g, 0.0045g, 0.005g, 0.0055g, 0.006g, 0.0065g, 0.007g, 0.0075g, 0.008g, 0.0085g, 0.009g, 0.0095g, 0.01g, 0.015g, 0.02g, 0.025 g, 0.03g, 0.035g, 0.04g, 0.045g, 0.05g, 0.055g, 0.06g, 0.065g, 0.07g, 0.075g, 0 .08g, 0.085g, 0.09g, 0.095g, 0.1g, 0.15g, 0.2g, 0.25g, 0.3g, 0.35g, 0.4g, 0.45g , 0.5g, 0.55g, 0.6g, 0.65g, 0.7g, 0.75g, 0.8g, 0.85g, 0.9g, 0.95g, 1g, 1.5g, 2g, 2 .5g, 3g, 3.5g, 4g, 4.5g, 5g, 5.5g, 6g, 6.5g, 7g, 7.5g, 8g, 8.5g, 9g, 9.5g or more than 10g.

[0195]

[0215] The rTIL provided in the pharmaceutical composition of the present invention is effective over a wide dosage range. The exact dosage will depend on the route of administration, the form in which the compound is administered, the sex and age of the subject to be treated, the weight of the subject to be treated, and the preference and experience of the attending physician. Where appropriate, clinically established dosages of rTIL may be used. The amount of pharmaceutical composition, e.g., dosage of rTIL, administered using the methods herein will depend on the human or mammal to be treated, the severity of the disorder or condition, the rate of administration, the pharmacokinetics of the active pharmaceutical ingredient, and the discretion of the treating physician.

[0196]

[0216] The rTIL and eTIL provided in the pharmaceutical composition of the present invention are effective over a wide dosage range. The exact dosage will depend on the route of administration, the form in which the compound is administered, the sex and age of the subject to be treated, the weight of the subject to be treated, and the preference and experience of the attending physician. Where appropriate, clinically established dosages of rTIL and eTIL may be used. The amount of pharmaceutical composition administered using the methods herein, e.g., dosage of rTIL and eTIL, will depend on the human or mammal to be treated, the severity of the disorder or condition, the rate of administration, the pharmacokinetics of the active pharmaceutical ingredient, and the discretion of the treating physician.

[0197]

[0217] In some embodiments, rTILs can be administered in a single dose. Such administration can be by injection, for example, intravenous injection. In some embodiments, rTILs can be administered in multiple doses. Administration can be once, twice, three times, four times, five times, six times or more than six times per year. Administration can be once a month, once every two weeks, once a week or once every other day. Administration of rTILs can continue for as long as necessary.

[0198]

[0218] In some embodiments, rTIL and eTIL can be administered in a single dose. Such administration can be by injection, for example, intravenous injection. In some embodiments, rTIL can be administered in multiple doses. Dosing can be once, twice, three times, four times, five times, six times, or more than six times per year. Dosing can be once a month, once every two weeks, once a week, or once every other day. Administration of rTIL and eTIL can continue for as long as necessary.

[0199]

[0219] In some embodiments, an effective dosage of rTILs is about 1×10 6 , 2×10 6 , 3×10 6 , 4×10 6 , 5×10 6 , 6×10 6 , 7×10 6 , 8×10 6 , 9×10 6 , 1×107 、2×10 7 、3×10 7 、4×10 7 、5×10 7 、6×10 7 、7×10 7 、8×10 7 、9×10 7 、1×10 8 、2×10 8 、3×10 8 、4×10 8 、5×10 8 、6×10 8 、7×10 8 、8×10 8 、9×10 8 、1×10 9 、2×10 9 、3×10 9 、4×10 9 、5×10 9 、6×10 9 、7×10 9 、8×10 9 、9×10 9 、1×10 10 、2×10 10 、3×10 10 、4×10 10 、5×10 10 、6×10 10 、7×10 10 、8×10 10 、9×10 10 、1×10 11 、2×10 11 、3×10 11 、4×10 11 、5×10 11 、6×10 11 、7×10 11 、8×10 11 、9×10 11 、1×10 12 、2×10 12 、3×10 12 、4×10 12 、5×10 12 、6×10 12 、7×10 12 、8×10 12 、9×10 12 、1×10 13 、2×10 13, 3×10 13 , 4×10 13 , 5×10 13 , 6×10 13 , 7×10 13 and 8 x 10 13 , 9×10 13 In some embodiments, the effective dosage of rTILs is 1×10 6 ~5×10 6 , 5×10 6 ~1×10 7 , 1×10 7 ~5×10 7 , 5×10 7 ~1×10 8 , 1×10 8 ~5×10 8 , 5×10 8 ~1×10 9 , 1×10 9 ~5×10 9 , 5×10 9 ~1×10 10 , 1×10 10 ~5×10 10 , 5×10 10 ~1×10 11 , 5×10 11 ~1×10 12 , 1×10 12 ~5×10 12 and 5 x 10 12 ~1×10 13 It is within the range of individuals.

[0200]

[0220] In some embodiments, the effective dosage of rTILs and eTILs is about 1×10 6 , 2×10 6 , 3×10 6 , 4×10 6 , 5×10 6 , 6×10 6 , 7×10 6 , 8×10 6 , 9×10 6 , 1×10 7 , 2×10 7 , 3×10 7 , 4×10 7 , 5×10 7 , 6×10 7 , 7×10 7、8×10 7 、9×10 7 、1×10 8 、2×10 8 、3×10 8 、4×10 8 、5×10 8 、6×10 8 、7×10 8 、8×10 8 、9×10 8 、1×10 9 、2×10 9 、3×10 9 、4×10 9 、5×10 9 、6×10 9 、7×10 9 、8×10 9 、9×10 9 、1×10 10 、2×10 10 、3×10 10 、4×10 10 、5×10 10 、6×10 10 、7×10 10 、8×10 10 、9×10 10 、1×10 11 、2×10 11 、3×10 11 、4×10 11 、5×10 11 、6×10 11 、7×10 11 、8×10 11 、9×10 11 、1×10 12 、2×10 12 、3×10 12 、4×10 12 、5×10 12 、6×10 12 、7×10 12 、8×10 12 、9×10 12 、1×10 13 、2×10 13 、3×10 13 、4×10 13 、5×10 13 、6×10 13 、7×10 13 、8×10 13 、9×1013 In some embodiments, the effective dosage of rTILs and eTILs is 1×10 6 ~5×10 6 , 5×10 6 ~1×10 7 , 1×10 7 ~5×10 7 , 5×10 7 ~1×10 8 , 1×10 8 ~5×10 8 , 5×10 8 ~1×10 9 , 1×l0 9 ~5×10 9 , 5×10 9 ~1×10 10 , 1×10 10 ~5×10 10 , 5×10 10 ~1×10 11 , 5×10 11 ~1×10 12 , 1×10 12 ~5×10 12 , 5×10 12 ~1×10 13 It is within the range of individuals.

[0201]

[0221] In some embodiments, an effective dosage of rTIL is about 0.01 mg / kg to about 4.3 mg / kg, about 0.15 mg / kg to about 3.6 mg / kg, about 0.3 mg / kg to about 3.2 mg / kg, about 0.35 mg / kg to about 2.85 mg / kg, about 0.15 mg / kg to about 2.85 mg / kg, about 0.3 mg / kg to about 2.15 mg / kg, about 0.45 mg / kg to about 1.7 mg / kg, about 0.15 mg / kg to about 1.3 mg / kg, about 0.3 mg / kg to about 1.15 mg / kg, about 0.45 mg / kg to about 1 mg / kg, about 0.55 mg / kg to about 0.85 mg / kg, about 0.65 mg / kg to about 0.8 mg / kg, about 0.7 mg / kg g to about 0.75 mg / kg, about 0.7 mg / kg to about 2.15 mg / kg, about 0.85 mg / kg to about 2 mg / kg, about 1 mg / kg to about 1.85 mg / kg, about 1.15 mg / kg to about 1.7 mg / kg, about 1.3 mg / kg to about 1.6 mg / kg, about 1.35 mg / kg to about 1.5 mg / kg, about 2.15 mg / kg to about 3.6 mg / kg, about 2.3 mg / kg to about 3.4 mg / kg, about 2.4 mg / kg to about 3.3 mg / kg, about 2.6 mg / kg to about 3.15 mg / kg, about 2.7 mg / kg to about 3 mg / kg, about 2.8 mg / kg to about 3 mg / kg, or about 2.85 mg / kg to about 2.95 mg / kg.

[0202]

[0222] In some embodiments, effective dosages of rTILs and eTILs include about 0.01 mg / kg to about 4.3 mg / kg, about 0.15 mg / kg to about 3.6 mg / kg, about 0.3 mg / kg to about 3.2 mg / kg, about 0.35 mg / kg to about 2.85 mg / kg, about 0.15 mg / kg to about 2.85 mg / kg, about 0.3 mg / kg to about 2.15 mg / kg, about 0.45 mg / kg to about 1.7 mg / kg, about 0.15 mg / kg to about 1.3 mg / kg, about 0.3 mg / kg to about 1.15 mg / kg, about 0.45 mg / kg to about 1 mg / kg, about 0.55 mg / kg to about 0.85 mg / kg, about 0.65 mg / kg to about 0.8 mg / kg, about 0.7 mg / kg to about 1.8 mg / kg, about 0.8 mg / kg to about 1.9 mg / kg, about 0.9 mg / kg to about 2.2 mg / kg, about 0.9 mg / kg to about 2.3 mg / kg, about 0.9 mg / kg to about 2.4 mg / kg, about 0.9 mg / kg to about 2.5 mg / kg, about 0.9 mg / kg to about 3.2 mg / kg, about 0.9 mg / kg to about 3.2 mg / kg, about 0.9 mg / kg to about 3.2 mg / kg, about 0.9 mg / kg to about 2.3 ...2.4 mg / kg, about 0.9 mg / kg to about 3 g / kg to about 0.75 mg / kg, about 0.7 mg / kg to about 2.15 mg / kg, about 0.85 mg / kg to about 2 mg / kg, about 1 mg / kg to about 1.85 mg / kg, about 1.15 mg / kg to about 1.7 mg / kg, about 1.3 mg / kg to about 1.6 mg / kg, about 1.35 mg / kg to about 1.5 mg / kg, about 2.15 mg / kg to about 3.6 mg / kg, about 2.3 mg / kg to about 3.4 mg / kg, about 2.4 mg / kg to about 3.3 mg / kg, about 2.6 mg / kg to about 3.15 mg / kg, about 2.7 mg / kg to about 3 mg / kg, about 2.8 mg / kg to about 3 mg / kg, or about 2.85 mg / kg to about 2.95 mg / kg.

[0203]

[0223] In some embodiments, an effective dosage of rTIL is about 1 mg to about 500 mg, about 10 mg to about 300 mg, about 20 mg to about 250 mg, about 25 mg to about 200 mg, about 1 mg to about 50 mg, about 5 mg to about 45 mg, about 10 mg to about 40 mg, about 15 mg to about 35 mg, about 20 mg to about 30 mg, about 23 mg to about 28 mg, about 50 mg to about 150 mg, about 60 mg to about 140 mg, about 25 mg to about 200 mg, about 25 mg to about 300 mg, about 25 mg to about 200 mg, about 25 mg to about 200 mg, about 25 mg to about 300 mg, about 25 mg to about 300 mg, about 30 mg to about 40 mg, about 40 mg to about 50 mg, about 50 mg to about 150 mg, about 60 mg to about 14 ... g, about 70 mg to about 130 mg, about 80 mg to about 120 mg, about 90 mg to about 110 mg, about 95 mg to about 105 mg, about 98 mg to about 102 mg, about 150 mg to about 250 mg, about 160 mg to about 240 mg, about 170 mg to about 230 mg, about 180 mg to about 220 mg, about 190 mg to about 210 mg, about 195 mg to about 205 mg, or about 198 mg to about 207 mg.

[0204]

[0224] In some embodiments, effective dosages of rTIL and eTIL are from about 1 mg to about 500 mg, from about 10 mg to about 300 mg, from about 20 mg to about 250 mg, from about 25 mg to about 200 mg, from about 1 mg to about 50 mg, from about 5 mg to about 45 mg, from about 10 mg to about 40 mg, from about 15 mg to about 35 mg, from about 20 mg to about 30 mg, from about 23 mg to about 28 mg, from about 50 mg to about 150 mg, from about 60 mg to about 140 mg, from about 150 mg to about 25 ... 0 mg, about 70 mg to about 130 mg, about 80 mg to about 120 mg, about 90 mg to about 110 mg, about 95 mg to about 105 mg, about 98 mg to about 102 mg, about 150 mg to about 250 mg, about 160 mg to about 240 mg, about 170 mg to about 230 mg, about 180 mg to about 220 mg, about 190 mg to about 210 mg, about 195 mg to about 205 mg, or about 198 mg to about 207 mg.

[0205]

[0225] Effective amounts of rTILs and / or eTILs can be administered in single or multiple doses by any accepted mode of administration of agents of similar utility, including injection into the bloodstream, injection into a tumor, intra-arterially, intravenously, intraperitoneally, parenterally, intramuscularly, subcutaneously, local injection, intranasally, by implantation or by inhalation. EXAMPLES

[0206] Working Example

[0226] The embodiments encompassed herein are described with reference to the following examples, which are set forth for illustrative purposes only, and the disclosure encompassed herein should in no way be construed as being limited to these examples, but rather as encompassing any and all variations that become evident as a result of the teachings set forth herein.

[0207] Example 1 – Expansion of rTILs from tumor digests

[0227] Tumor remnants were digested according to the following exemplary procedure, which describes the viable digestion of fresh human tumor samples into single cell suspensions to obtain and isolate tumor-infiltrating lymphocytes; the DNase-collagenase-hyaluronidase (DCH) method (described herein) can be used to isolate human tumors, or the MACS Human Tumor Isolation Kit (TDK) (Miltenyi Biotech, Inc., San Diego, Calif., USA) digestion protocol can be used.

[0208]

[0228] CM1+IL-2 working medium is prepared as follows: 500 mL RPMI 1640, 200 mM L-glutamine and 100 mL human AB serum are placed in a 37° C. water bath and equilibrated for at least 30 minutes. The contents of this mixture are transferred from the water bath to a biosafety cabinet along with 1000×β-ML stock solution and 50 mg / mL gentamicin stock solution from the refrigerator. Remove 50 mL from the RPMI 1640 and add 50 mL human AB serum, 5 mL 200 mM L-glutamine, 500 μL 1000×β-ML and 500 μL 50 mg / mL gentamicin. Add 500 μL of 6000 U / mL reconstituted human rhIL-2 (CellGenix, Inc., Portsmouth, ML USA) to complete medium 1.

[0209]

[0229] A 10x DCH stock solution is prepared using the following procedure, which is shown in Figure 1. First, calculate the volume needed to reconstitute each enzyme to obtain the desired working solution concentration. For example, reconstitute 150,000 U (International Units) of deoxyribonuclease in 15 mL to obtain a working solution of 10,000 U / mL. Aliquot the remaining working solution. Reconstitute the lyophilized enzyme in the amount of sterile Hank's Balanced Salt Solution (HBSS, Sigma H6648, Sigma-Aldrich Co., St. Louis, MO, USA or equivalent) precalculated above at room temperature. Remove any residual powder from the sides of the bottle and the protective foil. Pipette up and down several times to swirl to ensure complete reconstitution. Add 100,000 U of DNase (deoxyribonuclease I from bovine pancreas, Sigma D5025 or equivalent), 1 g of collagenase (Sigma C5138 from Clostridium histolyticum or equivalent) and 100 mg of hyaluronidase (type V from sheep testis, Sigma H6254 or equivalent) to a final volume of 100 mL sterile HBSS to obtain a 10x triple enzyme digestion stock solution for human tumors. Aliquot the remaining enzyme working solution into 10,000 U / mL DNase, 10 mg / mL collagenase and 1 mg / mL hyaluronidase. In a final volume of 100 mL, the 10x DCH stock solution has the following concentrations: DNase I 1000 U / mL, collagenase 10 mg / mL and hyaluronidase 1 mg / mL. Dilute the 10x DCH stock solution to 1x DCH in HBSS for tumor digestion.

[0210]

[0230] At the same time, for comparison of DCH digestion with TDK in MACS, prepare the reagents contained in TDK in MACS to the manufacturer's specifications as necessary. Thaw the aliquots stored at -20°C at room temperature.

[0211]

[0231] Tumors can be prepared for digestion as follows: Remove the tumor from its primary and secondary packaging, weigh the vial to record the mass, and transfer to a biosafety cabinet. Cut the tumor into fragments or morselize the tumor. Select some fragments to use in the digestion protocol and retain additional fragments for histology and DNA extraction as needed.

[0212]

[0232] An exemplary DCH-based tumor digestion procedure is shown in Figure 2 and includes the following steps: 10x DCH stock solution must be diluted to 1x working concentration for digestion. Calculate the total volume required for tumor digestion. This is the volume required for 1cm of tumor. 2 Approximately 5 mL of solution per tube. Dilute the DCH working solution to 1x by adding 1 part DCH to 9 parts HBSS. Transfer the tumor fragments to a 50 mL Falcon conical tube with the volume of HBSS calculated above. Add the amount of 10x DCH calculated above and cap and optionally seal the tube. Transfer to a MACS tube rotator (Miltenyi Biotech, Inc., San Diego, CA, USA) in a humidified incubator at 37°C and 5% CO2 with constant rotation for 1-2 hours. Alternatively, the tumor fragments can be digested overnight at room temperature, again with constant rotation. Attach a 0.70 μm strainer to a sterile Falcon conical tube. Remove the digest from the incubator with a pipette and add the entire contents of the digest to the strainer. Use the butt of a sterile syringe plunger to push any solids through the strainer. Cap the tube containing the DCH-digested rTILs. Cells can be washed, free of the digest cocktail, counted, and resuspended in medium for REP propagation as described elsewhere herein.

[0213]

[0233] If a pre-REP step is desired to provide eTILs for comparison with rTILs (as in the example below), seed G-REX flasks with DCH digested rTILs for pre-REP. Label the required number of G-REX 10 flasks and add digests. Add CM1+IL-2 to obtain a final volume of 40 mL. Place flasks in a humidified incubator at 37° C., 5% CO2. Cell counts and viability can be performed using a Nexcelom Cellometer K2 using 40 μL samples to 40 μL of Acridine Orange and Propidium Iodide Dual Staining Solution (AOPI), diluting as necessary and counting in duplicate for each digest or condition. Mix samples thoroughly to avoid clumping and pipette AOPI quickly before running each sample to ensure that viability is not obscured by the cytotoxic effects of propidium iodide.

[0214]

[0234] The DCH procedure described above was surprisingly found to be superior to the MACS TDK enzymatic digestion mixture and procedure for several reasons. Three independent experiments with the MACS TDK mixture were performed. The first experiment was performed with melanoma tumors, where CD4 + / CD8 + A large downregulation of the population was observed by flow cytometry using the MACS system. No such effect was observed in DCH-digested rTILs, indicating that the MACS digestion procedure may adversely affect the expression of surface markers. In a second experiment, estrogen receptor-positive (ER+) / progesterone receptor-positive (PR+) breast tumors were used, and the MACS digestion revealed debris in 24-well G-REX plates, whereas the DCH digestion yielded clear material, indicating that the digestion was insufficient for the MACS enzyme cocktail. Finally, a second digestion of a different ER+ / PR+ breast tumor using the MACS TDK enzyme digestion mixture and procedure resulted in poor both rTIL yield and viability.

[0215] Example 2 –Phenotypic characterization of rTILs derived from tumor digests

[0235] During pre-REP, tumor-resident TILs migrate and proliferate as eTILs. The length of pre-REP used to prepare eTILs for comparison with rTILs can vary from 11 to 21 days depending on cell proliferation. Residual tumor fragments (remnants) are usually discarded and the expanded eTILs are subjected to REP with irradiated PBMC feeders, anti-CD3 and IL-2. Viable TILs remaining in the tumor remnants (rTILs) after pre-REP were examined after digestion according to Example 1 described above to evaluate their function and phenotype in comparison with eTILs.

[0216]

[0236] Cell populations (i.e. expanded cell populations) (n=17) from tumor remnant and pre-REP suspensions in melanoma, head and neck, breast, kidney, pancreas, lung and colorectal tumors were evaluated and compared. Interestingly, rTILs are consistently phenotypically distinct from eTILs, as determined and shown herein by differential expression of various markers, including LAG3, TIM-3, PD-1, CD69, CD45RO, CD27, CD56, CD57 and HLA-DR. REPs from tumor remnant and pre-REP populations resulted in comparable proliferation, but similar to the pre-REP results, the phenotypic signature varied between the two populations with respect to LAG3, TIM-3, HLA-DR and CD28.

[0217]

[0237] rTILs and eTILs (n=9) obtained from melanoma, breast, renal, pancreatic, lung and colorectal tumors were evaluated and compared. Tumor rTILs were consistently phenotypically distinct from eTILs as determined by differential expression of various markers (Table 3 and Figure 3).

[0218] [Table 3]

[0219]

[0238] The fundamental difference between rTILs and eTILs is the increased expression of CD69 (CD4+ 7-fold higher median fluorescence intensity (MFI) in CD8+ mice (p<.0001), and reduced LAG3 expression (CD8+ + T cells) (p<0.05) and attenuated TIM3 expression (CD8 + and CD4 + 3-fold and 2-fold MFI in T cells, respectively (p<0.05 / 0.01), and attenuated CD154 expression (CD4 + There was a 3-fold increase in MFI in T cells (p<0.01) and attenuated CD56 expression (5%) (p<0.05). Surprisingly, REP of rTILs and eTILs resulted in comparable proliferation similar to the pre-REP results described in Example 4. The phenotypic signature of rTILs persisted after fidelity REP and was expressed to the individual levels of LAG3, TIM3 and CD28 as described in Example 4. Furthermore, because CD57 is a receptor associated with terminal differentiation, the results suggest that rTILs are less terminally differentiated (i.e., less likely to die) than eTILs.

[0220]

[0239] The results reported in Figure 3 and Table 3 show that eTILs and rTILs show clear but consistent differences in phenotypic expression in various tumor histologies. Most notably, there was a decrease in the expression of so-called "exhaustion markers" (LAG3 and TIM3) in rTILs. Interestingly, PD-1 was similarly expressed in eTILs and rTILs. In addition, there was an enhancement of CD69 expression in rTILs compared to eTILs, yet KLRG1 was similar between the two populations (data not shown). This provides further evidence that rTILs do not appear to be terminally differentiated, but are phenotypically similar to tissue-resident effector memory T cells.

[0221]

[0240] Collectively, these results identified significant differences in the biology of cell populations that remain within or proliferate and progress out of tumors, as well as signals associated with migration and retention.

[0222] Example 3 –Functional characterization of rTILs derived from tumor digests

[0241] T cell dysfunction is directly associated with loss of mitochondrial function. Sharping, et al., Immunity 2016, 45, 374-88. Furthermore, reprogramming T cells to favor mitochondrial biogenesis may increase the persistence and function of intratumoral T cells. Thus, metabolically more active T cells are important for mounting an effective immune response against tumors. In an effort to functionally evaluate eTILs and rTILs, eTILs and rTILs were compared for metabolic capacity via Mitotracker and 2-(N-(7-nitrobenzo-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG). 2-NBDG can be used to measure glucose uptake but does not identify the primary metabolic process, i.e., complete oxidation or glycolysis only within mitochondria and the production of lactate. Mitotracker dye (ThermoFisher Scientific, Inc., Waltham, MA, USA) can be used to measure mitochondrial mass. Comparison of eTILs and rTILs by this approach demonstrated enhanced glucose updating in rTILs, as shown in Figure 4. This result is surprising because rTILs released directly from the tumor would be expected to be more glycolytic. However, when mitochondrial mass in rTILs was assessed, rTILs showed slightly enhanced Mitotracker levels compared to eTILs. These results demonstrate that rTILs were more metabolically active than eTILs, when expected to be less active, and suggest that rTILs may have a greater ability to mount an immune response against tumors than eTILs.

[0223]

[0242] To further evaluate functional capacity, rTILs were stimulated overnight with Brefeldin A and anti-CD3, anti-CD28 and anti-CD137 antibody-coated beads (DYNABEADS, Cat. No. 11162D, commercially available from ThermoFisher Scientific, Inc., Waltham, MA, USA) to measure IFN-γ. Cells were harvested and intracellularly stained for IFN-γ after permeabilization and assessed by flow cytometry. Results are shown in FIG. 5.

[0224]

[0243] The ability of TILs to produce cytokines was also assessed by stimulating cells with phorbol 12-myristate 13-acetate (PMA) and ionomycin. The results are also shown in FIG. 5. Granzyme B, TNF-α and IL-17A levels were also assessed. There was only little IL-17A and no difference in TNFα or granzyme B between rTILs and eTILs (data not shown). Surprisingly, CD4 + In subset (CD8 + Slightly elevated IFN-γ levels in rTILs (but not in T cells) were observed in anti-CD3 / anti-CD28 / anti-CD137 bead and PMA / ionomycin conditions (n=3). This data suggests that rTILs are functionally competent cells and show evidence of greater functional competence than eTILs.

[0225] Example 4 – Comparison of REPs of rTILs and eTILs derived from tumor digests

[0244] eTILs and rTILs were subjected to a rapid proliferation protocol (REP) with irradiated PBMC feeders, anti-CD3 antibody (OKT3) and IL-2 for 14 days. Viability and cell number were assessed in duplicate in three independent tumors (n=3). Phenotypic expression was assessed by flow cytometry. Successful initiation of mini-REP experiments was observed for both rTILs and eTILs. The REP performance of TILs with anti-CD3 antibody and feeders was similar, but rTILs surprisingly showed slightly enhanced cell number compared to eTILs (p<0.08) (Figure 6A). As observed before REP (see Example 2), eTILs and rTILs obtained after REP were phenotypically different. Many phenotypic differences observed in pre-REP were preserved during REP, such as the reduced expression of LAG3 and TIM3 in rTILs (Figure 6B).

[0226]

[0245] Additional characteristics of eTILs and rTILs can be compared based on the results of (1) deep TCR sequencing, (2) co-culture growth (rTIL / eTIL co-culture with cytokine mixture) and further functional assays, and (3) transcriptional profiling assays (e.g., using NanoString Technologies' NCOUNTER system). TCR sequencing can assess clonality and / or diversity of TCR repertoire, including Vb repertoire. Telomere length can also be assessed to compare rTILs with eTILs.

[0227] Example 5 –Treatment of human diseases with rTILs and combinations of rTILs and eTILs

[0246] As described herein, the rTILs of the present invention can be used to treat cancer. An overall process flow diagram for the expansion of rTILs from a patient's tumor and treatment of the patient is shown in FIG. 7. The process allows for tailoring of the rTIL to eTIL ratio in the TIL product infused into the patient, as shown. The ratio of rTIL to eTIL can be selected by affinity assays or other cell sorting assays known by those skilled in the art based on differential expression of CD69 and / or T-cell-encompassing markers in rTILs and eTILs.

[0228]

[0247] FIG. 8 shows a timeline illustrating an exemplary process of obtaining rTILs from a patient's tumor, expanding rTILs from tumor remnants using the pre-REP and then REP stages, performing lymphodepletion, and injecting the rTILs into the patient, along with a parallel eTIL process.

[0229] Example 6 –Studies evaluating the Vβ repertoire in eTILs and rTILs

[0248] eTILs and rTILs were assessed for differences in the Vβ T cell receptor repertoire with respect to diversity and frequency.

[0230]

[0249] In this study, six pre-REP eTIL / rTIL pairs were harvested from the following tissue structures: ovarian cancer; renal cancer (n=2); and breast cancer (TNBC n=2, ER+PR+ n=1). Cell pellets on dry ice were shipped to iRepertoire (Huntsville, AL, USA) for RNA extraction and Vβ sequencing.

[0231]

[0250] The results of this study are shown in Figures 9, 10 and 14-16. In particular, Figures 9 and 10 show the diversity scores and % of shared CDR3, respectively. Additionally, three clonotype graphs showing the top 50 shared CDR3 are shown in Figures 14, 15 and 16 for ovarian cancer, renal cancer and triple-negative breast cancer, respectively.

[0232]

[0251] Surprisingly, the diversity of the TCRvβ repertoire is greater in rTILs than in eTILs (Figure 9). Approximately 30-50% of the total CDR3s in eTILs and rTILs were shared (Figure 10), demonstrating that a high percentage of the total CDR3s are differentially expressed in the two populations. However, among the shared CDR3s, the top 50 clones were shared between the two populations in most cases. This suggests that eTILs and rTILs have clones with similar antigen specificity (see Figures 14-16). Furthermore, the frequency of the top 50 clones varied. This again suggests that eTILs and rTILs are surprisingly distinct T cell populations.

[0233] Example 7 – Co-culture proliferation assay studies

[0252] Immediately after co-culture, eTILs and rTILs were assessed to determine whether rTILs could alter the proliferation status of eTILs (or vice versa).

[0234]

[0253] In this study, five pre-REP eTIL / rTIL pairs were harvested from the following tissue structures: renal carcinoma, triple-negative breast cancer (TNBC), melanoma, lung cancer, and colorectal cancer. rTILs were isolated from tumor remnants by enzymatic digestion for 60 min at 37°C. eTILs were stained with Cell Trace Yellow and rTILs with Cell Trace Red to track the two distinct populations independently. 1e6 of eTILs, 5e5 eTILs + 5e5 rTILs, and 1e6 rTILs were cultured with IL-2+ / - and OKT3 (anti-CD3 antibody) for 4 days at 37°C and assessed for proliferation by flow cytometry.

[0235]

[0254] The results of this study are shown in Figure 11. In Figure 11, eTILs from either the CD4+ or CD8+ populations in all five tumors demonstrated enhanced proliferative capacity as demonstrated by Cell Trace dye shift (or dye dilution) when compared to eTILs alone immediately after co-culture with rTILs with anti-CD3 antibody. Red represents eTILs and blue represents eTILs when co-cultured with rTILs.

[0236] Example 8 – Co-culture proliferation assay studies

[0255] eTILs and rTILs were evaluated to identify similarities and / or differences in the gene expression profiles of rTILs and eTILs.

[0237]

[0256] In this study, Nanostring's nCounter technology was utilized, which provides a digital readout of gene expression using color-coded barcodes multiplexed to mRNA. Purified RNA (RNeasy, Qiagen) from six matched eTIL and rTIL samples was hybridized with the nCounter Immunology V2 panel code set for 16 hours on a thermocycler. The code set consists of a mixture of capture and reporter probes that are multiplexed with the target RNA through a 22 bp interaction during thermocycling. Samples were loaded into 12-well SPRINT cartridges and run on the nCounter SPRINT instrument. Count data was exported in a custom RCC format to match RLF files, which match gene names to probe IDs. Normalization and analysis were performed with nSolver 3.0 (NanoString Technologies, Inc.).

[0238]

[0257] The results of this study are shown in Figures 12 and 13. As shown in Figures 12 and 13, gene expression profiles are significantly different when comparing eTILs and rTILs (see heatmap in Figure 12). Several genes are significantly up- or down-regulated in rTILs compared to eTILs (Figure 13).

Claims

1. A therapeutically effective amount of remnant tumor infiltrating lymphocytes (rTILs) for treating cancer in a patient in need of said treatment, said rTILs being obtained by enzymatically digesting tumor remnants separated from nascent tumor infiltrating lymphocytes (eTILs) into tumor remnant cells using a digestion mixture comprising deoxyribonuclease, collagenase and hyaluronidase, and expanding said tumor remnant cells in a second cell culture medium comprising cell culture medium, irradiated feeder cells, OKT-3 antibody and interleukin 2 (IL-2) to provide an expanded number of rTILs; The tumor remnants and eTILs are derived from tumor tissue obtained from the patient, the tumor tissue being fragmented and treated with a first cell culture medium containing IL-2.

2. The rTIL of claim 1, wherein the tumor tissue is selected from the group consisting of melanoma tumor tissue, head and neck tumor tissue, breast tumor tissue, kidney tumor tissue, pancreatic tumor tissue, glioblastoma tumor tissue, lung tumor tissue, colorectal tumor tissue, sarcoma tumor tissue, triple negative breast tumor tissue, cervical tumor tissue, ovarian tumor tissue, and acute myeloid leukemia bone marrow or tumor tissue.

3. The rTIL of claim 1 or 2, wherein the irradiated feeder cells comprise irradiated allogeneic peripheral blood mononuclear cells.

4. The rTIL of any one of claims 1 to 3, wherein IL-2 is present in the second cell culture medium at an initial concentration of 3000 IU / mL and OKT-3 antibody is present in the second cell culture medium at an initial concentration of 30 ng / mL.

5. CD56 in the rTILs ＋ Expression of CD56 in the eTILs ＋ The rTIL of any one of claims 1 to 4, wherein expression is reduced by at least 3-fold.

6. CD69 in the rTILs ＋ Expression of CD69 in the eTILs ＋ The rTIL of any one of claims 1 to 5, which increases expression by at least 2-fold.

7. The rTIL of any one of claims 1 to 6, wherein the first cell culture medium further comprises a cytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21, and combinations thereof.

8. The rTIL of any one of claims 1 to 7, wherein the second cell culture medium further comprises a cytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21, and combinations thereof.

9. The rTIL of any one of claims 1 to 8, wherein the rTIL is cryopreserved prior to obtaining the rTIL.

10. The rTIL of claim 9, wherein the cryopreserved rTIL population is thawed prior to obtaining the rTIL.

11. The rTIL of any one of claims 1 to 10, wherein the patient is administered a non-myeloablative lymphodepleting regimen prior to administering the rTIL to the patient, and the patient is administered a high-dose IL-2 regimen starting the day after administering a therapeutically effective amount of the rTIL to the patient.

12. The non-myeloablative lymphodepleting regimen consisted of 60 mg / m ２ The patient was administered cyclophosphamide at a dose of 25 mg / m2 / day followed by 5 days of cyclophosphamide. ２ The method of claim 11, further comprising administering fludarabine to the patient at a dose of 100 mg / day.

13. The rTIL of claim 11 or 12, wherein the high dose IL-2 regimen comprises 600,000 or 720,000 IU / kg aldesleukin or a biosimilar or variant thereof administered to the patient as a 15 minute bolus intravenous infusion every 8 hours until tolerated.

14. The rTIL of any one of claims 1 to 13, wherein the cancer is selected from the group consisting of melanoma, double refractory melanoma, uveal melanoma, ovarian cancer, cervical cancer, lung cancer, bladder cancer, breast cancer, head and neck cancer, renal cell carcinoma, acute myeloid leukemia, colorectal cancer and sarcoma.

15. The rTIL of claim 14, wherein the cancer is breast cancer, and the breast cancer is selected from the group consisting of triple negative breast cancer, estrogen receptor positive breast cancer, progesterone receptor positive breast cancer, and estrogen receptor positive / progesterone receptor positive breast cancer.

16. The rTIL of claim 14, wherein the cancer is lung cancer, and the lung cancer is selected from the group consisting of non-small cell lung cancer and small cell lung cancer.

17. The rTIL of any one of claims 1 to 16, wherein the therapeutically effective amount of rTIL is provided in a pharmaceutical composition, and the concentration of the rTIL in the pharmaceutical composition is greater than 50%.

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