Methods for cryopreservation of solid tumor fragments

Abstract

The present invention provides improved methods for expanding TILs and producing therapeutic TIL populations, including novel methods for cryopreserving tumor tissue, which result in improved efficacy, improved phenotype, and increased metabolic health of TILs in shorter periods of time while allowing for reduced microbial contamination and reduced costs. Such TILs have uses in therapeutic treatment regimens.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 224,766, filed July 22, 2021, the disclosure of which is incorporated herein in its entirety. [Background technology]

[0002] The treatment of bulky, refractory cancers using adoptive autologous transfer of tumor-infiltrating lymphocytes (TILs) offers a powerful approach to treating patients with poor prognosis. Gattinoni, et al., Nat. Rev. Immunol. 2006, 6, 383-393. TILs are dominated by T cells, and 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. Many approaches to improve response to TIL therapy in melanoma and to extend TIL therapy to other tumor types have met with limited success, and the field remains challenging. 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. Combination studies with single immune checkpoint inhibitors have also been described, but further research is ongoing and additional treatment options are needed (Kverneland et al., Oncotarget, 2020, 11(22), 2092-2105).

[0003] Furthermore, current TIL production and treatment processes are limited by length, cost, sterility concerns, and other factors described herein, severely limiting the ability to treat patients who are refractory to other checkpoint inhibitor therapies. There is an urgent need to provide TIL production processes and therapies based on such processes that are suitable for use in treating patients who have few or no remaining viable treatment options. The present invention fills this need by providing an abbreviated production process for use in generating TILs.

[0004] The present invention provides improved and / or shortened processes and methods for preparing TILs, including novel methods for cryopreserving tumor tissue, to prepare therapeutic TIL populations with increased therapeutic efficacy for the treatment of cancer with TILs. Summary of the Invention

[0005] Provided herein are methods for cryopreserving tumor tissue using slow freezing techniques.

[0006] In some embodiments, the present invention provides a method for cryopreserving tumor tissue, comprising: (i) adding a cryopreservation medium to a closable container; (ii) pre-cooling the closeable container in a controlled rate freezing device; (iii) fragmenting the tumor tissue to obtain tumor fragments; (iv) placing the tumor fragments in a closable container containing a cryopreservation medium and closing the container; (v) incubating the sealed container containing the tumor fragments and cryopreservation medium for about 30-60 minutes at a temperature of about 2-8°C; (vi) slow-freezing the container in a controlled rate freezer; (vii) transferring the container to a liquid nitrogen freezer.

[0007] In some embodiments, the present invention provides a method for cryopreserving tumor tissue, comprising: (i) placing tumor fragments obtained from fragmenting tumor tissue into a pre-chilled, closable container containing a cryopreservation medium and closing the container; (ii) incubating the sealed container containing the tumor fragments and cryopreservation medium for about 30-60 minutes at a temperature of about 2-8°C; (iii) slow-freezing the container in a controlled rate freezer; (iv) transferring the container to a liquid nitrogen freezer.

[0008] In some embodiments, the present invention provides a method for cryopreserving tumor tissue, comprising: (i) placing tumor digests obtained from digesting tumor tissue in an enzymatic medium or tumor fragments produced from fragmenting tumor tissue into a pre-chilled closable container containing a cryopreservation medium and closing the container; (ii) incubating the sealed container containing the tumor digest and cryopreservation medium for about 30-60 minutes at a temperature of about 2-8°C; (iii) slow-freezing the container in a controlled rate freezer; (iv) transferring the container to a liquid nitrogen freezer.

[0009] In some embodiments, the present invention provides a method for cryopreserving tumor tissue, comprising: (i) adding a cryopreservation medium to a closable container; (ii) pre-cooling the closeable container in a controlled rate freezing device; (iii) digesting the tumor tissue in an enzymatic medium to obtain a tumor digest; (iv) placing the tumor digest in a cryopreservation medium in a closable container and closing the container; (v) incubating the sealed container containing the tumor digest and cryopreservation medium for about 30-60 minutes at a temperature of about 2-8°C; (vi) slow-freezing the container in a controlled rate freezer; (vii) transferring the container to a liquid nitrogen freezer.

[0010] In some embodiments, the enzyme medium comprises DNase.

[0011] In some embodiments, the enzyme medium comprises collagenase.

[0012] In some embodiments, the enzyme medium comprises a neutral protease.

[0013] In some embodiments, the enzyme medium comprises hyaluronidase.

[0014] In some embodiments, the present invention provides a cryopreserved tumor tissue, comprising the steps of: (i) adding a cryopreservation medium to a closable container; (ii) transferring the closeable container to a controlled rate freezing device; (iii) fragmenting the tumor tissue to obtain tumor fragments; (iv) placing the tumor fragments in a closable container containing a cryopreservation medium and closing the container; (v) slow-freezing the container containing the tumor fragments and cryopreservation medium; (vi) transferring the container to liquid nitrogen.

[0015] In some embodiments, the present invention provides a cryopreserved tumor tissue, comprising the steps of: (i) placing tumor fragments obtained from fragmenting tumor tissue into a pre-chilled closable container containing a cryopreservation medium and closing the container; (ii) incubating the sealed container containing the tumor fragments and cryopreservation medium for about 30-60 minutes at a temperature of about 2-8°C; (iii) slow-freezing the container in a controlled rate freezer; (iv) transferring the container to a liquid nitrogen freezer.

[0016] In some embodiments, the present invention provides a cryopreserved tumor digest comprising the steps of: (i) placing a tumor digest obtained from digesting tumor tissue in an enzymatic medium or tumor fragments produced from fragmenting tumor tissue into a pre-chilled closable container containing a cryopreservation medium and closing the container; (ii) incubating the sealed container containing the tumor digest and cryopreservation medium for about 30-60 minutes at a temperature of about 2-8°C; (iii) slow-freezing the container in a controlled rate freezer; (iv) transferring the container to a liquid nitrogen freezer.

[0017] In some embodiments, the present invention provides a cryopreserved tumor digest comprising the steps of: (i) adding a cryopreservation medium to a closable container; (ii) pre-cooling the closeable container in a controlled rate freezer; (iii) digesting the tumor tissue in an enzymatic medium to obtain a tumor digest; (iv) placing the tumor digest in a cryopreservation medium in a closable container and closing the container; (v) incubating the sealed container containing the tumor digest and cryopreservation medium for about 30-60 minutes at a temperature of about 2-8°C; (vi) slow-freezing the container in a controlled rate freezer; (vii) transferring the container to a liquid nitrogen freezer.

[0018] In some embodiments, the enzyme medium comprises DNase.

[0019] In some embodiments, the enzyme medium comprises collagenase.

[0020] In some embodiments, the enzyme medium comprises a neutral protease.

[0021] In some embodiments, the enzyme medium comprises hyaluronidase.

[0022] In some embodiments, the present invention provides a method for expanding tumor infiltrating lymphocytes (TILs), comprising: (a) obtaining and / or receiving a first population of TILs from tumor tissue resected from a subject or patient and storing the tumor tissue in a frozen state, the method comprising: (i) adding a cryopreservation medium to a closable container; (ii) pre-cooling the closeable container in a controlled rate freezing device; (iii) fragmenting the tumor tissue to obtain tumor fragments; (iv) placing the tumor fragments in a closable container containing a cryopreservation medium and sealing the container; (v) slow-freezing the container containing the tumor fragments and cryopreservation medium; (vi) storing, including transferring the container to liquid nitrogen; (b) culturing the first TIL population in a culture medium to expand the first TIL population.

[0023] In some embodiments, the present invention provides a method for expanding tumor infiltrating lymphocytes (TILs), comprising: (a) obtaining and / or receiving a first population of TILs from tumor tissue resected from a subject or patient and storing the tumor tissue in a frozen state, the method comprising: (i) placing tumor fragments obtained from fragmenting tumor tissue into a pre-chilled, closable container containing a cryopreservation medium and closing the container; (ii) incubating the sealed container containing the tumor fragments and cryopreservation medium for about 30-60 minutes at a temperature of about 2-8°C; (iii) slow-freezing the container in a controlled rate freezer; (iv) storing the container, including transferring the container to a liquid nitrogen freezer; (b) culturing the first TIL population in a culture medium to expand the first TIL population.

[0024] In some embodiments, the present invention provides a method for expanding tumor infiltrating lymphocytes (TILs), comprising: (a) obtaining and / or receiving a first population of TILs from tumor tissue resected from a subject or patient and storing the tumor tissue in a frozen state, the method comprising: (i) placing tumor digests obtained from digesting tumor tissue in an enzymatic medium or tumor fragments produced from fragmenting tumor tissue into a pre-chilled, closable container containing a cryopreservation medium and closing the container; (ii) incubating the sealed container containing the tumor digest and cryopreservation medium for about 30-60 minutes at a temperature of about 2-8°C; (iii) slow-freezing the container in a controlled rate freezer; (iv) storing the container, including transferring the container to a liquid nitrogen freezer; (b) culturing the first TIL population in a culture medium to expand the first TIL population.

[0025] In some embodiments, the present invention provides a method for expanding tumor infiltrating lymphocytes (TILs), comprising: (a) obtaining and / or receiving a first population of TILs from tumor tissue resected from a subject or patient and storing the tumor tissue in a frozen state, the method comprising: (i) placing the tumor tissue produced from fragmenting the tumor tissue into a pre-chilled, closable container containing a cryopreservation medium and closing the container; (ii) incubating the sealed container containing the tumor fragments and cryopreservation medium for about 30-60 minutes at a temperature of about 2-8°C; (iii) slow-freezing the container in a controlled rate freezer; (iv) storing the container, including transferring the container to a liquid nitrogen freezer; (b) digesting the tumor fragments in an enzymatic medium to produce a tumor digest; (c) culturing the first TIL population in a culture medium to expand the first TIL population.

[0026] In some embodiments, the present invention provides a method for expanding tumor infiltrating lymphocytes (TILs), comprising: (a) obtaining and / or receiving a first population of TILs from tumor tissue resected from a subject or patient and storing the tumor tissue in a frozen state, the method comprising: (i) adding a cryopreservation medium to a closable container; (ii) pre-cooling the closeable container in a controlled rate freezing device; (iii) digesting the tumor tissue produced from fragmenting the tumor tissue or tumor fragments in an enzymatic medium to obtain a tumor digest; (iv) placing the tumor digest into a pre-chilled closable container containing cryopreservation medium and closing the container; (v) incubating the sealed container containing the tumor digest and cryopreservation medium for about 30-60 minutes at a temperature of about 2-8°C; (vi) slow-freezing the container in a controlled rate freezer; (vii) storing the container, including transferring the container to a liquid nitrogen freezer; (b) culturing the first TIL population in a culture medium to expand the first TIL population.

[0027] In some embodiments, the present invention provides a method for expanding tumor infiltrating lymphocytes (TILs), comprising: (a) obtaining and / or receiving a first population of TILs from tumor tissue resected from a subject or patient and storing the tumor tissue in a frozen state, the method comprising: (i) adding a cryopreservation medium to a closable container; (ii) pre-cooling the closeable container in a controlled rate freezing device; (iii) fragmenting the tumor tissue to obtain tumor fragments; (iv) placing the tumor fragments in a closable container containing a cryopreservation medium and closing the container; (v) slow-freezing the container containing the tumor fragments and cryopreservation medium; (vi) storing, including transferring the container to liquid nitrogen; (b) digesting the tumor fragments in an enzymatic medium to produce a tumor digest; (c) culturing the first TIL population in a culture medium to expand the first TIL population.

[0028] In some embodiments, the enzyme medium comprises DNase.

[0029] In some embodiments, the enzyme medium comprises collagenase.

[0030] In some embodiments, the enzyme medium comprises a neutral protease.

[0031] In some embodiments, the enzyme medium comprises hyaluronidase.

[0032] In some embodiments, the present invention provides a method for rapid expansion of tumor infiltrating lymphocytes (TILs), comprising: (a) obtaining and / or receiving a first population of TILs from tumor tissue resected from a subject or patient and storing the tumor tissue in a frozen state, the method comprising: (i) adding a cryopreservation medium to a closable container; (ii) pre-cooling the closable container containing the cryopreservation medium in a controlled rate freezing device; (iii) fragmenting the tumor tissue to obtain tumor fragments; (iv) placing the tumor fragments in a closable container containing a cryopreservation medium and closing the container; (v) incubating the sealed container containing the tumor fragments and cryopreservation medium for about 30-60 minutes at a temperature of about 2-8°C; (vi) slow-freezing the container in a controlled rate freezer; (vii) storing the container, including transferring the container to a liquid nitrogen freezer; (b) culturing the first TIL population in a culture medium containing IL-2, OKT-3 (anti-CD3 antibody), and antigen-presenting cells (APCs), resulting in rapid expansion of the first TIL population to produce a second TIL population.

[0033] In some embodiments, the present invention provides a method for rapid expansion of tumor infiltrating lymphocytes (TILs), comprising: (a) obtaining and / or receiving a first population of TILs from tumor tissue resected from a subject or patient and storing the tumor tissue in a frozen state, the method comprising: (i) placing tumor fragments obtained from fragmenting tumor tissue into a pre-chilled, closable container containing a cryopreservation medium and closing the container; (ii) incubating the sealed container containing the tumor fragments and cryopreservation medium for about 30-60 minutes at a temperature of about 2-8°C; (iii) slow-freezing the container in a controlled rate freezer; (iv) storing the container, including transferring the container to a liquid nitrogen freezer; (b) culturing the first TIL population in a culture medium containing IL-2, OKT-3 (anti-CD3 antibody), and antigen-presenting cells (APCs), resulting in rapid expansion of the first TIL population to produce a second TIL population.

[0034] In some embodiments, the present invention provides a method for rapid expansion of tumor infiltrating lymphocytes (TILs), comprising: (a) obtaining and / or receiving a first population of TILs from tumor tissue resected from a subject or patient and storing the tumor tissue in a frozen state, the method comprising: (i) placing tumor digests obtained from digesting tumor tissue in an enzymatic medium or tumor fragments produced from fragmenting tumor tissue into a pre-chilled, closable container containing a cryopreservation medium and closing the container; (ii) incubating the sealed container containing the tumor digest and cryopreservation medium for about 30-60 minutes at a temperature of about 2-8°C; (iii) slow-freezing the container in a controlled rate freezer; (iv) storing the container, including transferring the container to a liquid nitrogen freezer; (b) culturing the first TIL population in a culture medium containing IL-2, OKT-3 (anti-CD3 antibody), and antigen-presenting cells (APCs), resulting in rapid expansion of the first TIL population to produce a second TIL population.

[0035] In some embodiments, the present invention provides a method for rapid expansion of tumor infiltrating lymphocytes (TILs), comprising: (a) obtaining and / or receiving a first population of TILs from tumor tissue resected from a subject or patient and storing the tumor tissue in a frozen state, the method comprising: (i) placing tumor fragments obtained from fragmenting tumor tissue into a pre-chilled, closable container containing a cryopreservation medium and closing the container; (ii) incubating the sealed container containing the tumor fragments and cryopreservation medium for about 30-60 minutes at a temperature of about 2-8°C; (iii) slow-freezing the container in a controlled rate freezer; (iv) storing the container, including transferring the container to a liquid nitrogen freezer; (b) digesting the tumor fragments in an enzymatic medium to produce a tumor digest; (c) culturing the first TIL population in a culture medium containing IL-2, OKT-3 (anti-CD3 antibody), and antigen-presenting cells (APCs), resulting in rapid expansion of the first TIL population to produce a second TIL population.

[0036] In some embodiments, the present invention provides a method for rapid expansion of tumor infiltrating lymphocytes (TILs), comprising: (a) obtaining and / or receiving a first population of TILs from tumor tissue resected from a subject or patient and storing the tumor tissue in a frozen state, the method comprising: (i) adding a cryopreservation medium to a closable container; (ii) pre-cooling the closable container containing the cryopreservation medium in a controlled rate freezing device; (iii) fragmenting the tumor tissue to obtain tumor fragments; (iv) placing the tumor fragments in a closable container containing a cryopreservation medium and closing the container; (v) incubating the sealed container containing the tumor fragments and cryopreservation medium for about 30-60 minutes at a temperature of about 2-8°C; (vi) slow-freezing the container in a controlled rate freezer; (vii) storing the container, including transferring the container to a liquid nitrogen freezer; (b) digesting the tumor fragments in an enzymatic medium to produce a tumor digest; (c) culturing the first TIL population in a culture medium containing IL-2, OKT-3 (anti-CD3 antibody), and antigen-presenting cells (APCs), resulting in rapid expansion of the first TIL population to produce a second TIL population.

[0037] In some embodiments, the present invention provides a method for rapid expansion of tumor infiltrating lymphocytes (TILs), comprising: (a) obtaining and / or receiving a first population of TILs from tumor tissue resected from a subject or patient and storing the tumor tissue in a frozen state, the method comprising: (i) adding a cryopreservation medium to a closable container; (ii) pre-cooling the closeable container in a controlled rate freezing device; (iii) digesting the tumor tissue produced from fragmenting the tumor tissue or tumor fragments in an enzymatic medium to obtain a tumor digest; (iv) placing the tumor digest into a pre-chilled closable container containing cryopreservation medium and closing the container; (v) incubating the sealed container containing the tumor digest and cryopreservation medium for about 30-60 minutes at a temperature of about 2-8°C; (vi) slow-freezing the container in a controlled rate freezer; (vii) storing the container, including transferring the container to a liquid nitrogen freezer; (b) culturing the first TIL population in a culture medium containing IL-2, OKT-3 (anti-CD3 antibody), and antigen-presenting cells (APCs), resulting in rapid expansion of the first TIL population to produce a second TIL population.

[0038] In some embodiments, the enzyme medium comprises DNase.

[0039] In some embodiments, the enzyme medium comprises collagenase.

[0040] In some embodiments, the enzyme medium comprises a neutral protease.

[0041] In some embodiments, the enzyme medium comprises hyaluronidase.

[0042] In some embodiments, the present invention provides a method for preparing expanded tumor infiltrating lymphocytes (TILs), comprising: (a) obtaining and / or receiving a first population of TILs from tumor tissue resected from a subject or patient and storing the tumor tissue in a frozen state, the method comprising: (i) adding a cryopreservation medium to a closable container; (ii) pre-cooling the closeable container in a controlled rate freezing device; (iii) fragmenting the tumor tissue to obtain tumor fragments; (iv) placing the tumor fragments in a closable container containing a cryopreservation medium and closing the container; (v) incubating the sealed container containing the tumor fragments and cryopreservation medium for about 30-60 minutes at a temperature of about 2-8°C; (vi) slow-freezing the container in a controlled rate freezer; (vii) storing the container, including transferring the container to a liquid nitrogen freezer; (b) culturing the first TIL population in a first cell culture medium containing IL-2 for about 3 to 14 days to produce a second TIL population; (c) culturing the second TIL population in a second cell culture medium comprising antigen-presenting cells (APCs), OKT-3, and IL-2 for about 7 to 14 days to provide an expanded number of TILs.

[0043] In some embodiments, the present invention provides a method for preparing expanded tumor infiltrating lymphocytes (TILs), comprising: (a) obtaining and / or receiving a first population of TILs from tumor tissue resected from a subject or patient and storing the tumor tissue in a frozen state, the method comprising: (i) placing tumor fragments obtained from fragmenting tumor tissue into a pre-chilled, closable container containing a cryopreservation medium and closing the container; (ii) incubating the sealed container containing the tumor fragments and cryopreservation medium for about 30-60 minutes at a temperature of about 2-8°C; (iii) slow-freezing the container in a controlled rate freezer; (iv) storing the container, including transferring the container to a liquid nitrogen freezer; (b) culturing the first TIL population in a first cell culture medium containing IL-2 for about 3 to 14 days to produce a second TIL population; (c) culturing the second TIL population in a second cell culture medium comprising antigen-presenting cells (APCs), OKT-3, and IL-2 for about 7 to 14 days to provide an expanded number of TILs.

[0044] In some embodiments, the present invention provides a method for preparing expanded tumor infiltrating lymphocytes (TILs), comprising: (a) obtaining and / or receiving a first population of TILs from tumor tissue resected from a subject or patient and storing the tumor tissue in a frozen state, the method comprising: (i) placing tumor digests obtained from digesting tumor tissue in an enzymatic medium or tumor fragments produced from fragmenting tumor tissue into a pre-chilled, closable container containing a cryopreservation medium and closing the container; (ii) incubating the sealed container containing the tumor digest and cryopreservation medium for about 30-60 minutes at a temperature of about 2-8°C; (iii) slow-freezing the container in a controlled rate freezer; (iv) storing the container, including transferring the container to a liquid nitrogen freezer; (b) culturing the first TIL population in a first cell culture medium containing IL-2 for about 3 to 14 days to produce a second TIL population; (c) culturing the second TIL population in a second cell culture medium comprising antigen-presenting cells (APCs), OKT-3, and IL-2 for about 7 to 14 days to provide an expanded number of TILs.

[0045] In some embodiments, the present invention provides a method for preparing expanded tumor infiltrating lymphocytes (TILs), comprising: (a) obtaining and / or receiving a first population of TILs from tumor tissue resected from a subject or patient and storing the tumor tissue in a frozen state, the method comprising: (i) placing tumor fragments obtained from fragmenting tumor tissue into a pre-chilled, closable container containing a cryopreservation medium and closing the container; (ii) incubating the sealed container containing the tumor fragments and cryopreservation medium for about 30-60 minutes at a temperature of about 2-8°C; (iii) slow-freezing the container in a controlled rate freezer; (iv) storing the container, including transferring the container to a liquid nitrogen freezer; (b) digesting the tumor fragments in an enzymatic medium to produce a tumor digest; (c) culturing the first TIL population in a first cell culture medium containing IL-2 for about 3 to 14 days to produce a second TIL population; (d) culturing the second TIL population in a second cell culture medium comprising antigen-presenting cells (APCs), OKT-3, and IL-2 for about 7 to 14 days to provide an expanded number of TILs.

[0046] In some embodiments, the present invention provides a method for preparing expanded tumor infiltrating lymphocytes (TILs), comprising: (a) obtaining and / or receiving a first population of TILs from tumor tissue resected from a subject or patient and storing the tumor tissue in a frozen state, the method comprising: (i) adding a cryopreservation medium to a closable container; (ii) pre-cooling the closeable container in a controlled rate freezing device; (iii) fragmenting the tumor tissue to obtain tumor fragments; (iv) placing the tumor fragments in a closable container containing a cryopreservation medium and closing the container; (v) incubating the sealed container containing the tumor fragments and cryopreservation medium for about 30-60 minutes at a temperature of about 2-8°C; (vi) slow-freezing the container in a controlled rate freezer; (vii) storing the container, including transferring the container to a liquid nitrogen freezer; (b) digesting the tumor fragments in an enzymatic medium to produce a tumor digest; (c) culturing the first TIL population in a first cell culture medium containing IL-2 for about 3 to 14 days to produce a second TIL population; (d) culturing the second TIL population in a second cell culture medium comprising antigen-presenting cells (APCs), OKT-3, and IL-2 for about 7 to 14 days to provide an expanded number of TILs.

[0047] In some embodiments, the present invention provides a method for preparing expanded tumor infiltrating lymphocytes (TILs), comprising: (a) obtaining and / or receiving a first population of TILs from tumor tissue resected from a subject or patient and storing the tumor tissue in a frozen state, the method comprising: (i) adding a cryopreservation medium to a closable container; (ii) pre-cooling the closeable container in a controlled rate freezing device; (iii) digesting the tumor tissue produced from fragmenting the tumor tissue or tumor fragments in an enzymatic medium to obtain a tumor digest; (iv) placing the tumor digest into a pre-chilled closable container containing cryopreservation medium and closing the container; (v) incubating the sealed container containing the tumor digest and cryopreservation medium for about 30-60 minutes at a temperature of about 2-8°C; (vi) slow-freezing the container in a controlled rate freezer; (vii) storing the container, including transferring the container to a liquid nitrogen freezer; (b) culturing the first TIL population in a first cell culture medium containing IL-2 for about 3 to 14 days to produce a second TIL population; (c) culturing the second TIL population in a second cell culture medium comprising antigen-presenting cells (APCs), OKT-3, and IL-2 for about 7 to 14 days to provide an expanded number of TILs.

[0048] In some embodiments, the enzyme medium comprises DNase.

[0049] In some embodiments, the enzyme medium comprises collagenase.

[0050] In some embodiments, the enzyme medium comprises a neutral protease.

[0051] In some embodiments, the enzyme medium comprises hyaluronidase.

[0052] In some embodiments, the step of culturing the first TIL population is carried out for about 1 to 11 days.

[0053] In some embodiments, the step of culturing the second TIL population is carried out for about 7 to 11 days.

[0054] In some embodiments, the steps of culturing the first TIL population and culturing the second TIL population are completed within a period of about 22 days.

[0055] In some embodiments, the step of culturing the second TIL population is carried out by culturing the second TIL population in a second cell culture medium for a first period of about 5 days, at the end of the first period, dividing the culture into multiple subcultures, and culturing each of the multiple subcultures in a third culture medium containing IL-2 for a second period of about 6 days, and at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0056] In some embodiments, the step of culturing the first population of TILs is carried out for about 7 days.

[0057] In some embodiments, the step of culturing the second population of TILs is carried out for about 14 days.

[0058] In some embodiments, the step of culturing the second TIL population is carried out by culturing the second TIL population in a second cell culture medium for a first period of about 7 days, at the end of the first period, dividing the culture into multiple subcultures, each of the multiple subcultures being cultured in a third culture medium containing IL-2 for a second period of about 7 days, and at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0059] In some embodiments, the present invention provides a method for preparing expanded tumor infiltrating lymphocytes (TILs), comprising: (a) obtaining and / or receiving a first population of TILs from tumor tissue resected from a subject or patient and storing the tumor tissue in a frozen state, the method comprising: (i) adding a cryopreservation medium to a closable container; (ii) pre-cooling the closeable container in a controlled rate freezing device; (iii) fragmenting the tumor tissue to obtain tumor fragments; (iv) placing the tumor fragments in a closable container containing a cryopreservation medium and closing the container; (v) incubating the sealed container containing the tumor fragments and cryopreservation medium for about 30-60 minutes at a temperature of about 2-8°C; (vi) slow-freezing the container in a controlled rate freezer; (vii) storing the container, including transferring the container to a liquid nitrogen freezer; (b) performing an initial expansion (or first expansion by priming) of the first TIL population in a first cell culture medium to obtain a second TIL population, wherein the first cell culture medium comprises IL-2, optionally OKT-3 (anti-CD3 antibody), and optionally antigen-presenting cells (APCs), and the first expansion by priming occurs over a period of about 1 to 8 days; and (c) performing a rapid second expansion of the second TIL population in a second cell culture medium to obtain an expanded number of TILs, wherein the second cell culture medium comprises IL-2, OKT-3 (an anti-CD3 antibody), and APCs, and the rapid expansion is performed over a period of 14 days or less, and optionally, the rapid second expansion can proceed for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days after the initiation of the rapid second expansion to obtain an expanded number of TILs.

[0060] In some embodiments, the present invention provides a method for preparing expanded tumor infiltrating lymphocytes (TILs), comprising: (a) obtaining and / or receiving a first population of TILs from tumor tissue resected from a subject or patient and storing the tumor tissue in a frozen state, the method comprising: (i) placing tumor fragments obtained from fragmenting tumor tissue into a pre-chilled, closable container containing a cryopreservation medium and closing the container; (ii) incubating the sealed container containing the tumor fragments and cryopreservation medium for about 30-60 minutes at a temperature of about 2-8°C; (iii) slow-freezing the container in a controlled rate freezer; (iv) storing the container, including transferring the container to a liquid nitrogen freezer; (b) performing an initial expansion (or first expansion by priming) of the first TIL population in a first cell culture medium to obtain a second TIL population, wherein the first cell culture medium comprises IL-2, optionally OKT-3 (anti-CD3 antibody), and optionally antigen-presenting cells (APCs), and the first expansion by priming occurs over a period of about 1 to 8 days; and (c) performing a rapid second expansion of the second TIL population in a second cell culture medium to obtain an expanded number of TILs, wherein the second cell culture medium comprises IL-2, OKT-3 (an anti-CD3 antibody), and APCs, and the rapid expansion is performed over a period of 14 days or less, and optionally, the rapid second expansion can proceed for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days after the initiation of the rapid second expansion to obtain an expanded number of TILs.

[0061] In some embodiments, the present invention provides a method for preparing expanded tumor infiltrating lymphocytes (TILs), comprising: (a) obtaining and / or receiving a first population of TILs from tumor tissue resected from a subject or patient and storing the tumor tissue in a frozen state, the method comprising: (i) placing tumor digests obtained from digesting tumor tissue in an enzymatic medium or tumor fragments produced from fragmenting tumor tissue into a pre-chilled, closable container containing a cryopreservation medium and closing the container; (ii) incubating the sealed container containing the tumor digest and cryopreservation medium for about 30-60 minutes at a temperature of about 2-8°C; (iii) slow-freezing the container in a controlled rate freezer; (iv) storing the container, including transferring the container to a liquid nitrogen freezer; (b) performing an initial expansion (or first expansion by priming) of the first TIL population in a first cell culture medium to obtain a second TIL population, wherein the first cell culture medium comprises IL-2, optionally OKT-3 (anti-CD3 antibody), and optionally antigen-presenting cells (APCs), and the first expansion by priming occurs over a period of about 1 to 8 days; and (c) performing a rapid second expansion of the second TIL population in a second cell culture medium to obtain an expanded number of TILs, wherein the second cell culture medium comprises IL-2, OKT-3 (an anti-CD3 antibody), and APCs, and the rapid expansion is performed over a period of 14 days or less, and optionally, the rapid second expansion can proceed for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days after the initiation of the rapid second expansion to obtain an expanded number of TILs.

[0062] In some embodiments, the present invention provides a method for preparing expanded tumor infiltrating lymphocytes (TILs), comprising: (a) obtaining and / or receiving a first population of TILs from tumor tissue resected from a subject or patient and storing the tumor tissue in a frozen state, the method comprising: (i) placing tumor fragments obtained from fragmenting tumor tissue into a pre-chilled, closable container containing a cryopreservation medium and closing the container; (ii) incubating the sealed container containing the tumor fragments and cryopreservation medium for about 30-60 minutes at a temperature of about 2-8°C; (iii) slow-freezing the container in a controlled rate freezer; (iv) storing the container, including transferring the container to a liquid nitrogen freezer; (b) digesting the tumor fragments in an enzymatic medium to produce a tumor digest; (c) performing an initial expansion (or first expansion by priming) of the first TIL population in a first cell culture medium to obtain a second TIL population, wherein the first cell culture medium comprises IL-2, optionally OKT-3 (anti-CD3 antibody), and optionally antigen-presenting cells (APCs), and the first expansion by priming occurs over a period of about 1 to 8 days; and (d) performing a rapid second expansion of the second TIL population in a second cell culture medium to obtain an expanded number of TILs, wherein the second cell culture medium comprises IL-2, OKT-3 (an anti-CD3 antibody), and APCs, and the rapid expansion is performed over a period of 14 days or less, and optionally, the rapid second expansion can proceed for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days after the initiation of the rapid second expansion to obtain an expanded number of TILs.

[0063] In some embodiments, the present invention provides a method for preparing expanded tumor infiltrating lymphocytes (TILs), comprising: (a) obtaining and / or receiving a first population of TILs from tumor tissue resected from a subject or patient and storing the tumor tissue in a frozen state, the method comprising: (i) adding a cryopreservation medium to a closable container; (ii) pre-cooling the closeable container in a controlled rate freezing device; (iii) fragmenting the tumor tissue to obtain tumor fragments; (iv) placing the tumor fragments in a closable container containing a cryopreservation medium and closing the container; (v) incubating the sealed container containing the tumor fragments and cryopreservation medium for about 30-60 minutes at a temperature of about 2-8°C; (vi) slow-freezing the container in a controlled rate freezer; (vii) storing the container, including transferring the container to a liquid nitrogen freezer; (b) digesting the tumor fragments in an enzymatic medium to produce a tumor digest; (c) performing an initial expansion (or first expansion by priming) of the first TIL population in a first cell culture medium to obtain a second TIL population, wherein the first cell culture medium comprises IL-2, optionally OKT-3 (anti-CD3 antibody), and optionally antigen-presenting cells (APCs), and the first expansion by priming occurs over a period of about 1 to 8 days; and (d) performing a rapid second expansion of the second TIL population in a second cell culture medium to obtain an expanded number of TILs, wherein the second cell culture medium comprises IL-2, OKT-3 (an anti-CD3 antibody), and APCs, and the rapid expansion is performed over a period of 14 days or less, and optionally, the rapid second expansion can proceed for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days after the initiation of the rapid second expansion to obtain an expanded number of TILs.

[0064] In some embodiments, the present invention provides a method for preparing expanded tumor infiltrating lymphocytes (TILs), comprising: (a) obtaining and / or receiving a first population of TILs from tumor tissue resected from a subject or patient and storing the tumor tissue in a frozen state, the method comprising: (i) adding a cryopreservation medium to a closable container; (ii) pre-cooling the closeable container in a controlled rate freezing device; (iii) digesting the tumor tissue produced from fragmenting the tumor tissue or tumor fragments in an enzymatic medium to obtain a tumor digest; (iv) placing the tumor digest into a pre-chilled closable container containing cryopreservation medium and closing the container; (v) incubating the sealed container containing the tumor digest and cryopreservation medium for about 30-60 minutes at a temperature of about 2-8°C; (vi) slow-freezing the container in a controlled rate freezer; (vii) storing the container, including transferring the container to a liquid nitrogen freezer; (b) performing an initial expansion (or first expansion by priming) of the first TIL population in a first cell culture medium to obtain a second TIL population, wherein the first cell culture medium comprises IL-2, optionally OKT-3 (anti-CD3 antibody), and optionally antigen-presenting cells (APCs), and the first expansion by priming occurs over a period of about 1 to 8 days; and (c) performing a rapid second expansion of the second TIL population in a second cell culture medium to obtain an expanded number of TILs, wherein the second cell culture medium comprises IL-2, OKT-3 (an anti-CD3 antibody), and APCs, and the rapid expansion is performed over a period of 14 days or less, and optionally, the rapid second expansion can proceed for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days after the initiation of the rapid second expansion to obtain an expanded number of TILs.

[0065] In some embodiments, the enzyme medium comprises DNase.

[0066] In some embodiments, the enzyme medium comprises collagenase.

[0067] In some embodiments, the enzyme medium comprises a neutral protease.

[0068] In some embodiments, the enzyme medium comprises hyaluronidase.

[0069] In some embodiments, the first culture medium comprises APCs.

[0070] In some embodiments, the number of APCs in the second culture medium is greater than the number of APCs in the first culture medium.

[0071] In some embodiments, the first expansion step by priming is carried out for a period of about 7 or 8 days.

[0072] In some embodiments, the rapid second expansion step is carried out for about 7-10 days.

[0073] In some embodiments, the rapid expansion step is carried out by culturing the second TIL population in a second cell culture medium for a first period of about 3-4 days, at the end of the first period, dividing the culture into multiple subcultures, and culturing each of the multiple subcultures in a third culture medium containing IL-2 for a second period of about 4-6 days, and at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0074] In some embodiments, the present invention provides a method for preparing expanded tumor infiltrating lymphocytes (TILs), comprising: (a) obtaining and / or receiving a first population of TILs from tumor tissue resected from a subject or patient and storing the tumor tissue in a frozen state, the method comprising: (i) adding a cryopreservation medium to a closable container; (ii) pre-cooling the closeable container in a controlled rate freezing device; (iii) fragmenting the tumor tissue to obtain tumor fragments; (iv) placing the tumor fragments in a closable container containing a cryopreservation medium and closing the container; (v) incubating the sealed container containing the tumor fragments and cryopreservation medium for about 30-60 minutes at a temperature of about 2-8°C; (vi) slow-freezing the container in a controlled rate freezer; (vi) storing the container, including transferring the container to a liquid nitrogen freezer; (b) performing a first expansion by culturing the first TIL population in a first cell culture medium comprising interleukin 2 (IL-2), and optionally OKT-3, and optionally antigen-presenting cells (APCs) to provide a second TIL population; (c) performing a second expansion by culturing the second TIL population in a second cell culture medium comprising APCs, OKT-3, and IL-2 to provide an expanded number of TILs.

[0075] In some embodiments, the present invention provides a method for preparing expanded tumor infiltrating lymphocytes (TILs), comprising: (a) obtaining and / or receiving a first population of TILs from tumor tissue resected from a subject or patient and storing the tumor tissue in a frozen state, the method comprising: (i) placing tumor fragments obtained from fragmenting tumor tissue into a pre-chilled, closable container containing a cryopreservation medium and closing the container; (ii) incubating the sealed container containing the tumor fragments and cryopreservation medium for about 30-60 minutes at a temperature of about 2-8°C; (iii) slow-freezing the container in a controlled rate freezer; (iv) storing the container, including transferring the container to a liquid nitrogen freezer; (b) performing a first expansion by culturing the first TIL population in a first cell culture medium comprising interleukin 2 (IL-2), and optionally OKT-3, and optionally antigen-presenting cells (APCs) to provide a second TIL population; (c) performing a second expansion by culturing the second TIL population in a second cell culture medium comprising APCs, OKT-3, and IL-2 to provide an expanded number of TILs.

[0076] In some embodiments, the present invention provides a method for preparing expanded tumor infiltrating lymphocytes (TILs), comprising: (a) obtaining and / or receiving a first population of TILs from tumor tissue resected from a subject or patient and storing the tumor tissue in a frozen state, the method comprising: (i) placing tumor digests obtained from digesting tumor tissue in an enzymatic medium or tumor fragments produced from fragmenting tumor tissue into a pre-chilled, closable container containing a cryopreservation medium and closing the container; (ii) incubating the sealed container containing the tumor digest and cryopreservation medium for about 30-60 minutes at a temperature of about 2-8°C; (iii) slow-freezing the container in a controlled rate freezer; (iv) storing the container, including transferring the container to a liquid nitrogen freezer; (b) performing a first expansion by culturing the first TIL population in a first cell culture medium comprising interleukin 2 (IL-2), and optionally OKT-3, and optionally antigen-presenting cells (APCs) to provide a second TIL population; (c) performing a second expansion by culturing the second TIL population in a second cell culture medium comprising APCs, OKT-3, and IL-2 to provide an expanded number of TILs.

[0077] In some embodiments, the present invention provides a method for preparing expanded tumor infiltrating lymphocytes (TILs), comprising: (a) obtaining and / or receiving a first population of TILs from tumor tissue resected from a subject or patient and storing the tumor tissue in a frozen state, the method comprising: (i) placing tumor fragments obtained from fragmenting tumor tissue into a pre-chilled, closable container containing a cryopreservation medium and closing the container; (ii) incubating the sealed container containing the tumor fragments and cryopreservation medium for about 30-60 minutes at a temperature of about 2-8°C; (iii) slow-freezing the container in a controlled rate freezer; (iv) storing the container, including transferring the container to a liquid nitrogen freezer; (b) digesting the tumor fragments in an enzymatic medium to produce a tumor digest; (c) performing a first expansion by culturing the first TIL population in a first cell culture medium comprising interleukin 2 (IL-2), and optionally OKT-3, and optionally antigen-presenting cells (APCs) to provide a second TIL population; (d) performing a second expansion by culturing the second TIL population in a second cell culture medium comprising APCs, OKT-3, and IL-2 to provide an expanded number of TILs.

[0078] In some embodiments, the present invention provides a method for preparing expanded tumor infiltrating lymphocytes (TILs), comprising: (a) obtaining and / or receiving a first population of TILs from tumor tissue resected from a subject or patient and storing the tumor tissue in a frozen state, the method comprising: (i) adding a cryopreservation medium to a closable container; (ii) pre-cooling the closeable container in a controlled rate freezing device; (iii) fragmenting the tumor tissue to obtain tumor fragments; (iv) placing the tumor fragments in a closable container containing a cryopreservation medium and closing the container; (v) incubating the sealed container containing the tumor fragments and cryopreservation medium for about 30-60 minutes at a temperature of about 2-8°C; (vi) slow-freezing the container in a controlled rate freezer; (vii) storing the container, including transferring the container to a liquid nitrogen freezer; (b) digesting the tumor fragments in an enzymatic medium to produce a tumor digest; (c) performing a first expansion by culturing the first TIL population in a first cell culture medium comprising interleukin 2 (IL-2), and optionally OKT-3, and optionally antigen-presenting cells (APCs) to provide a second TIL population; (d) performing a second expansion by culturing the second TIL population in a second cell culture medium comprising APCs, OKT-3, and IL-2 to provide an expanded number of TILs.

[0079] In some embodiments, the present invention provides a method for preparing expanded tumor infiltrating lymphocytes (TILs), comprising: (a) obtaining and / or receiving a first population of TILs from tumor tissue resected from a subject or patient and storing the tumor tissue in a frozen state, the method comprising: (i) adding a cryopreservation medium to a closable container; (ii) pre-cooling the closeable container in a controlled rate freezing device; (iii) digesting the tumor tissue produced from fragmenting the tumor tissue or tumor fragments in an enzymatic medium to obtain a tumor digest; (iv) placing the tumor digest into a pre-chilled closable container containing cryopreservation medium and closing the container; (v) incubating the sealed container containing the tumor digest and cryopreservation medium for about 30-60 minutes at a temperature of about 2-8°C; (vi) slow-freezing the container in a controlled rate freezer; (vii) storing the container, including transferring the container to a liquid nitrogen freezer; (b) performing a first expansion by culturing the first TIL population in a first cell culture medium comprising interleukin 2 (IL-2), and optionally OKT-3, and optionally antigen-presenting cells (APCs) to provide a second TIL population; (c) performing a second expansion by culturing the second TIL population in a second cell culture medium comprising APCs, OKT-3, and IL-2 to provide an expanded number of TILs.

[0080] In some embodiments, the enzyme medium comprises DNase.

[0081] In some embodiments, the enzyme medium comprises collagenase.

[0082] In some embodiments, the enzyme medium comprises a neutral protease.

[0083] In some embodiments, the enzyme medium comprises hyaluronidase.

[0084] In some embodiments, the first culture medium comprises APC and OKT-3.

[0085] In some embodiments, the number of APCs in the second culture medium is greater than the number of APCs in the first culture medium.

[0086] In some embodiments, the present invention provides a method for expanding tumor-infiltrating lymphocytes into a therapeutic TIL population, the method comprising: (a) obtaining and / or receiving a first population of TILs from a sample of tumor tissue produced by surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining tumor tissue from a patient or subject, and storing the sample of tumor tissue in a frozen state, the method comprising: (i) adding a cryopreservation medium to a closable container; (ii) pre-cooling the closeable container in a controlled rate freezing device; (iii) optionally fragmenting the sample of tumor tissue to obtain tumor fragments; (iv) placing a sample of tumor tissue or a tumor fragment into the closable container containing the cryopreservation medium and closing the container; (v) incubating the sealed container containing the sample and cryopreservation medium for about 30 to 60 minutes at a temperature of about 2 to 8°C; (vi) slow-freezing the container in a controlled rate freezer; (vii) a storing step, comprising transferring the container to a liquid nitrogen freezer; (b) adding a tumor tissue sample or tumor fragment to a closed system and performing a first expansion by culturing the first TIL population in a first cell culture medium containing IL-2 to produce a second TIL population, wherein the first expansion is performed in a closed container providing a first gas permeable surface area, and the first expansion is performed for about 3 to 14 days to obtain the second TIL population; (c) performing a second expansion by culturing the second TIL population in a second cell culture medium comprising IL-2, OKT-3, and antigen-presenting cells (APCs) to produce a third TIL population, wherein the second expansion is performed for about 7-14 days to obtain the third TIL population, wherein the second expansion is performed in a sealed container that provides a second gas-permeable surface area, wherein the third TIL population is a therapeutic TIL population, and wherein the transition from step (b) to step (c) occurs without opening the system. (d) harvesting the therapeutic TIL population obtained from step (c), wherein the transition from step (c) to step (d) occurs without opening the system.

[0087] In some embodiments, the present invention provides a method for expanding tumor-infiltrating lymphocytes into a therapeutic TIL population, the method comprising: (a) obtaining and / or receiving a first population of TILs from a sample of tumor tissue produced by surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining tumor tissue from a patient or subject, and storing the sample of tumor tissue in a frozen state, wherein the method of storing the sample comprises: (i) placing a sample of tumor tissue or tumor fragments produced by fragmenting a sample of tumor tissue into a pre-chilled, closable container containing a cryopreservation medium and closing the container; (ii) incubating the sealed container containing the sample and cryopreservation medium for about 30 to 60 minutes at a temperature of about 2 to 8°C; (iii) slow-freezing the container in a controlled rate freezer; (iv) a storing step, comprising transferring the container to a liquid nitrogen freezer; (b) adding the sample or tumor tissue or tumor fragment to a closed system and performing a first expansion by culturing the first TIL population in a first cell culture medium containing IL-2 to produce a second TIL population, wherein the first expansion is performed in a closed container that provides a first gas permeable surface area, and the first expansion is performed for about 3 to 14 days to obtain the second TIL population; (c) performing a second expansion by culturing the second TIL population in a second cell culture medium comprising IL-2, OKT-3, and antigen-presenting cells (APCs) to produce a third TIL population, wherein the second expansion is performed for about 7 to 14 days to obtain the third TIL population, wherein the second expansion is performed in a sealed container that provides a second gas-permeable surface area, wherein the third TIL population is a therapeutic TIL population, and wherein the transition from step (b) to step (c) occurs without opening the system. (d) harvesting the therapeutic TIL population obtained from step (c), wherein the transition from step (c) to step (d) occurs without opening the system.

[0088] In some embodiments, the present invention provides a method for expanding tumor-infiltrating lymphocytes into a therapeutic TIL population, the method comprising: (a) obtaining and / or receiving a first population of TILs from a sample of tumor tissue produced by surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining tumor tissue from a patient or subject, and storing the sample of tumor tissue in a frozen state, the method comprising: (i) placing a sample of tumor tissue or a tumor digest obtained from digesting tumor fragments produced from fragmenting a sample of tumor tissue in an enzymatic medium into a pre-chilled closable container containing a cryopreservation medium and closing the container; (ii) incubating the sealed container containing the tumor digest and cryopreservation medium for about 30-60 minutes at a temperature of about 2-8°C; (iii) slow-freezing the container in a controlled rate freezer; (iv) a storing step, comprising transferring the container to a liquid nitrogen freezer; (b) adding tumor digest to a closed system and culturing the first TIL population in a first cell culture medium containing IL-2 to perform a first expansion to produce a second TIL population, wherein the first expansion is performed in a closed container that provides a first gas permeable surface area, and the first expansion is performed for about 3 to 14 days to obtain the second TIL population; (c) performing a second expansion by culturing the second TIL population in a second cell culture medium comprising IL-2, OKT-3, and antigen-presenting cells (APCs) to produce a third TIL population, wherein the second expansion is performed for about 7-14 days to obtain the third TIL population, wherein the second expansion is performed in a sealed container that provides a second gas-permeable surface area, wherein the third TIL population is a therapeutic TIL population, and wherein the transition from step (b) to step (c) occurs without opening the system. (d) harvesting the therapeutic TIL population obtained from step (c), wherein the transition from step (c) to step (d) occurs without opening the system.

[0089] In some embodiments, the present invention provides a method for expanding tumor-infiltrating lymphocytes into a therapeutic TIL population, the method comprising: (a) obtaining and / or receiving a first population of TILs from a sample of tumor tissue produced by surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining tumor tissue from a patient or subject, and storing the sample of tumor tissue in a frozen state, the method comprising: (i) placing a sample of tumor tissue or tumor fragments produced by fragmenting a sample of tumor tissue into a pre-chilled, closable container containing a cryopreservation medium and closing the container; (ii) incubating the sealed container containing the sample and cryopreservation medium for about 30 to 60 minutes at a temperature of about 2 to 8°C; (iii) slow-freezing the container in a controlled rate freezer; (iv) a storing step, comprising transferring the container to a liquid nitrogen freezer; (b) digesting the tumor tissue sample or tumor fragment in an enzymatic medium to produce a tumor digest; (c) adding tumor digest to a closed system and culturing the first TIL population in a first cell culture medium containing IL-2 to perform a first expansion to produce a second TIL population, wherein the first expansion is performed in a closed container that provides a first gas permeable surface area, and the first expansion is performed for about 3 to 14 days to obtain the second TIL population; (d) performing a second expansion by culturing the second TIL population in a second cell culture medium comprising IL-2, OKT-3, and antigen presenting cells (APCs) to produce a third TIL population, wherein the second expansion is performed for about 7 to 14 days to obtain the third TIL population, wherein the second expansion is performed in a sealed container that provides a second gas permeable surface area, wherein the third TIL population is a therapeutic TIL population, and wherein the transition from step (c) to step (d) occurs without opening the system. (e) harvesting the therapeutic TIL population obtained from step (d), wherein the transition from step (d) to step (e) occurs without opening the system.

[0090] In some embodiments, the present invention provides a method for expanding tumor-infiltrating lymphocytes into a therapeutic TIL population, the method comprising: (a) obtaining and / or receiving a first population of TILs from a sample of tumor tissue produced by surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining tumor tissue from a patient or subject, and storing the sample of tumor tissue in a frozen state, the method comprising: (i) adding a cryopreservation medium to a closable container; (ii) pre-cooling the closeable container in a controlled rate freezing device; (iii) optionally fragmenting the sample of tumor tissue to obtain tumor fragments; (iv) placing the tumor tissue sample or tumor fragment in a closable container containing a cryopreservation medium and closing the container; (v) incubating the sealed container containing the sample and cryopreservation medium for about 30 to 60 minutes at a temperature of about 2 to 8°C; (vi) slow-freezing the container in a controlled rate freezer; (vi) a storing step, comprising transferring the container to a liquid nitrogen freezer; (b) digesting the tumor tissue sample or tumor fragment in an enzymatic medium to produce a tumor digest; (c) adding tumor digest to a closed system and culturing the first TIL population in a first cell culture medium containing IL-2 to perform a first expansion to produce a second TIL population, wherein the first expansion is performed in a closed container that provides a first gas permeable surface area, and the first expansion is performed for about 3 to 14 days to obtain the second TIL population; (d) performing a second expansion by culturing the second TIL population in a second cell culture medium comprising IL-2, OKT-3, and antigen presenting cells (APCs) to produce a third TIL population, wherein the second expansion is performed for about 7 to 14 days to obtain the third TIL population, wherein the second expansion is performed in a sealed container that provides a second gas permeable surface area, wherein the third TIL population is a therapeutic TIL population, and wherein the transition from step (c) to step (d) occurs without opening the system. (e) harvesting the therapeutic TIL population obtained from step (c), wherein the transition from step (d) to step (e) occurs without opening the system.

[0091] In some embodiments, the present invention provides a method for expanding tumor-infiltrating lymphocytes into a therapeutic TIL population, the method comprising: (a) obtaining and / or receiving a first population of TILs from a sample of tumor tissue produced by surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining tumor tissue from a patient or subject, and storing the sample of tumor tissue in a frozen state, the method comprising: (i) adding a cryopreservation medium to a closable container; (ii) pre-cooling the closeable container in a controlled rate freezing device; (iii) digesting the tumor tissue sample or tumor fragments produced by fragmenting the tumor tissue sample in an enzymatic medium to obtain a tumor digest; (iv) placing the tumor digest into a pre-chilled closable container containing cryopreservation medium and closing the container; (v) incubating the sealed container containing the tumor digest and cryopreservation medium for about 30-60 minutes at a temperature of about 2-8°C; (vi) slow-freezing the container in a controlled rate freezer; (vii) a storing step, comprising transferring the container to a liquid nitrogen freezer; (b) adding tumor digest to a closed system and culturing the first TIL population in a first cell culture medium containing IL-2 to perform a first expansion to produce a second TIL population, wherein the first expansion is performed in a closed container that provides a first gas permeable surface area, and the first expansion is performed for about 3 to 14 days to obtain the second TIL population; (c) performing a second expansion by culturing the second TIL population in a second cell culture medium comprising IL-2, OKT-3, and antigen-presenting cells (APCs) to produce a third TIL population, wherein the second expansion is performed for about 7-14 days to obtain the third TIL population, wherein the second expansion is performed in a sealed container that provides a second gas-permeable surface area, wherein the third TIL population is a therapeutic TIL population, and wherein the transition from step (b) to step (c) occurs without opening the system. (d) harvesting the therapeutic TIL population obtained from step (c), wherein the transition from step (c) to step (d) occurs without opening the system.

[0092] In some embodiments, the enzyme medium comprises DNase.

[0093] In some embodiments, the enzyme medium comprises collagenase.

[0094] In some embodiments, the enzyme medium comprises a neutral protease.

[0095] In some embodiments, the enzyme medium comprises hyaluronidase.

[0096] In some embodiments, the first expansion is carried out for about 1 to 11 days.

[0097] In some embodiments, the second expansion is carried out for about 7 to 11 days.

[0098] In some embodiments, the first expansion and the second expansion are completed within a period of about 22 days.

[0099] In some embodiments, the second expansion comprises the following steps: (i) culturing the second population of TILs in a second culture medium for a first period of about 5 days; (ii) subdividing the culture of step (i) into a plurality of subcultures, wherein each of the plurality of subcultures is transferred to a separate sealed container providing a third gas permeable surface and cultured in a third culture medium comprising IL-2 for a second period of about 6 days, the transition from step (i) to step (ii) being performed without opening the system; (iii) combining the multiple subcultures to produce a third TIL population, wherein the transition from step (ii) to step (iii) is performed without opening the system.

[0100] In some embodiments, the first expansion is carried out for about 7 days.

[0101] In some embodiments, the second expansion is carried out for about 14 days.

[0102] In some embodiments, the second expansion comprises the following steps: (i) culturing the second population of TILs in a second culture medium for a first period of about 7 days; (ii) subdividing the culture of step (i) into a plurality of subcultures, wherein each of the plurality of subcultures is transferred to a separate sealed container providing a third gas permeable surface and cultured in a third culture medium comprising IL-2 for a second period of about 7 days, wherein the transition from step (i) to step (ii) is performed without opening the system; (iii) combining the multiple subcultures to produce a third TIL population, wherein the transition from step (ii) to step (iii) is performed without opening the system.

[0103] In some embodiments, the present invention provides a method for expanding tumor-infiltrating lymphocytes into a therapeutic TIL population, the method comprising: (a) obtaining and / or receiving a first population of TILs from a sample of tumor tissue produced by surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining tumor tissue from a patient or subject, and storing the sample of tumor tissue in a frozen state, the method comprising: (i) adding a cryopreservation medium to a closable container; (ii) pre-cooling the closeable container in a controlled rate freezing device; (iii) optionally fragmenting the sample of tumor tissue to obtain tumor fragments; (iv) placing the tumor tissue sample or tumor fragment in a closable container containing a cryopreservation medium and closing the container; (v) incubating the sealed container containing the tumor tissue sample or tumor fragment and the cryopreservation medium for about 30 to 60 minutes at a temperature of about 2 to 8°C; (vi) slow-freezing the container in a controlled rate freezer; (vii) a storing step, comprising transferring the container to a liquid nitrogen freezer; (b) culturing the first TIL population in a first cell culture medium containing IL-2 for about 1-3 days; (c) performing an initial expansion (or first expansion by priming) of the first TIL population in a second cell culture medium to obtain a second TIL population, wherein the second cell culture medium comprises IL-2, OKT-3 (anti-CD3 antibody), and antigen-presenting cells (APCs), and the first expansion by priming occurs for about 1 to 11 days; (d) performing a second rapid expansion of the second TIL population in a third cell culture medium to obtain a third TIL population, wherein the third TIL population is a therapeutic TIL population, the third cell culture medium comprising IL-2, OKT-3 (an anti-CD3 antibody), and APCs, and the rapid expansion is performed for 1 to 11 days after the initiation of the second rapid expansion; (e) harvesting the therapeutic TIL population.

[0104] In some embodiments, the present invention provides a method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population, the method comprising: (a) obtaining and / or receiving a first population of TILs from a sample of tumor tissue produced by surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining tumor tissue from a patient or subject, and storing the sample of tumor tissue in a frozen state, the method comprising: (i) placing a sample of tumor tissue or tumor fragments produced by fragmenting a sample of tumor tissue into a pre-cooled closable container and closing the container; (ii) incubating the sealed container containing the sample and cryopreservation medium for about 30 to 60 minutes at a temperature of about 2 to 8°C; (iii) slow-freezing the container in a controlled rate freezer; (iv) a storing step, comprising transferring the container to a liquid nitrogen freezer; (b) culturing the first TIL population in a first cell culture medium containing IL-2 for about 1-3 days; (c) performing an initial expansion (or first expansion by priming) of the first TIL population in a second cell culture medium to obtain a second TIL population, wherein the second cell culture medium comprises IL-2, OKT-3 (anti-CD3 antibody), and antigen-presenting cells (APCs), and the first expansion by priming occurs for about 1 to 11 days; (d) performing a second rapid expansion of the second TIL population in a third cell culture medium to obtain a third TIL population, wherein the third TIL population is a therapeutic TIL population, the third cell culture medium comprising IL-2, OKT-3 (an anti-CD3 antibody), and APCs, and the rapid expansion is performed for 1 to 11 days after the initiation of the second rapid expansion; (e) harvesting the therapeutic TIL population.

[0105] In some embodiments, the present invention provides a method for expanding tumor-infiltrating lymphocytes into a therapeutic TIL population, the method comprising: (a) obtaining and / or receiving a first population of TILs from a sample of tumor tissue produced by surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining tumor tissue from a patient or subject, and storing the sample of tumor tissue in a frozen state, the method comprising: (i) placing a sample of tumor tissue or a tumor digest obtained from digesting tumor fragments produced from fragmenting a sample of tumor tissue in an enzymatic medium into a pre-chilled closable container containing a cryopreservation medium and closing the container; (ii) incubating the sealed container containing the tumor digest and cryopreservation medium for about 30-60 minutes at a temperature of about 2-8°C; (iii) slow-freezing the container in a controlled rate freezer; (iv) a storing step, comprising transferring the container to a liquid nitrogen freezer; (b) culturing the first TIL population in a first cell culture medium containing IL-2 for about 1-3 days; (c) performing an initial expansion (or first expansion by priming) of the first TIL population in a second cell culture medium to obtain a second TIL population, wherein the second cell culture medium comprises IL-2, OKT-3 (anti-CD3 antibody), and antigen-presenting cells (APCs), and the first expansion by priming occurs for about 1 to 11 days; (d) performing a second rapid expansion of the second TIL population in a third cell culture medium to obtain a third TIL population, wherein the third TIL population is a therapeutic TIL population, the third cell culture medium comprising IL-2, OKT-3 (an anti-CD3 antibody), and APCs, and the rapid expansion is performed for 1 to 11 days after the initiation of the second rapid expansion; (e) harvesting the therapeutic TIL population.

[0106] In some embodiments, the present invention provides a method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population, the method comprising: (a) obtaining and / or receiving a first population of TILs from a sample of tumor tissue produced by surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining tumor tissue from a patient or subject, and storing the sample of tumor tissue in a frozen state, the method comprising: (i) placing a sample of tumor tissue or tumor fragments produced by fragmenting a sample of tumor tissue into a pre-cooled closable container and closing the container; (ii) incubating the sealed container containing the sample and cryopreservation medium for about 30 to 60 minutes at a temperature of about 2 to 8°C; (iii) slow-freezing the container in a controlled rate freezer; (iv) a storing step, comprising transferring the container to a liquid nitrogen freezer; (b) digesting the tumor fragments in an enzymatic medium to produce a tumor digest; (c) culturing the first TIL population in a first cell culture medium containing IL-2 for about 1-3 days; (d) performing an initial expansion (or first expansion by priming) of the first TIL population in a second cell culture medium to obtain a second TIL population, wherein the second cell culture medium comprises IL-2, OKT-3 (anti-CD3 antibody), and antigen-presenting cells (APCs), and the first expansion by priming occurs for about 1 to 11 days; (e) performing a second rapid expansion of the second TIL population in a third cell culture medium to obtain a third TIL population, wherein the third TIL population is a therapeutic TIL population, the third cell culture medium comprising IL-2, OKT-3 (anti-CD3 antibody), and APC, and the rapid expansion is performed for 1 to 11 days after the initiation of the second rapid expansion; (f) harvesting the therapeutic TIL population.

[0107] In some embodiments, the present invention provides a method for expanding tumor-infiltrating lymphocytes into a therapeutic TIL population, the method comprising: (a) obtaining and / or receiving a first population of TILs from a sample of tumor tissue produced by surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining tumor tissue from a patient or subject, and storing the sample of tumor tissue in a frozen state, the method comprising: (i) adding a cryopreservation medium to a closable container; (ii) pre-cooling the closeable container in a controlled rate freezing device; (iii) optionally fragmenting the sample of tumor tissue to obtain tumor fragments; (iv) placing the tumor tissue sample or tumor fragment in a closable container containing a cryopreservation medium and closing the container; (v) incubating the sealed container containing the tumor fragments and cryopreservation medium for about 30-60 minutes at a temperature of about 2-8°C; (vi) slow-freezing the container in a controlled rate freezer; (vii) a storing step, comprising transferring the container to a liquid nitrogen freezer; (b) digesting the tumor tissue sample or tumor fragment in an enzymatic medium; (c) culturing the first TIL population in a first cell culture medium containing IL-2 for about 1-3 days; (d) performing an initial expansion (or first expansion by priming) of the first TIL population in a second cell culture medium to obtain a second TIL population, wherein the second cell culture medium comprises IL-2, OKT-3 (anti-CD3 antibody), and antigen-presenting cells (APCs), and the first expansion by priming occurs for about 1 to 11 days; (e) performing a second rapid expansion of the second TIL population in a third cell culture medium to obtain a third TIL population, wherein the third TIL population is a therapeutic TIL population, the third cell culture medium comprising IL-2, OKT-3 (anti-CD3 antibody), and APC, and the rapid expansion is performed for 1 to 11 days after the initiation of the second rapid expansion; (f) harvesting the therapeutic TIL population.

[0108] In some embodiments, the present invention provides a method for expanding tumor-infiltrating lymphocytes into a therapeutic TIL population, the method comprising: (a) obtaining and / or receiving a first population of TILs from a sample of tumor tissue produced by surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining tumor tissue from a patient or subject, and storing the sample of tumor tissue in a frozen state, the method comprising: (i) adding a cryopreservation medium to a closable container; (ii) pre-cooling the closeable container in a controlled rate freezing device; (iii) digesting the tumor tissue sample or tumor fragments produced by fragmenting the tumor tissue sample in an enzymatic medium to obtain a tumor digest; (iv) placing the tumor digest into a pre-chilled closable container containing cryopreservation medium and closing the container; (v) incubating the sealed container containing the tumor digest and cryopreservation medium for about 30-60 minutes at a temperature of about 2-8°C; (vi) slow-freezing the container in a controlled rate freezer; (vii) a storing step, comprising transferring the container to a liquid nitrogen freezer; (b) culturing the first TIL population in a first cell culture medium containing IL-2 for about 1-3 days; (c) performing an initial expansion (or first expansion by priming) of the first TIL population in a second cell culture medium to obtain a second TIL population, wherein the second cell culture medium comprises IL-2, OKT-3 (anti-CD3 antibody), and antigen-presenting cells (APCs), and the first expansion by priming occurs for about 1 to 11 days; (d) performing a second rapid expansion of the second TIL population in a third cell culture medium to obtain a third TIL population, wherein the third TIL population is a therapeutic TIL population, the third cell culture medium comprising IL-2, OKT-3 (an anti-CD3 antibody), and APCs, and the rapid expansion is performed for 1 to 11 days after the initiation of the second rapid expansion; (e) harvesting the therapeutic TIL population.

[0109] In some embodiments, the enzyme medium comprises DNase.

[0110] In some embodiments, the enzyme medium comprises collagenase.

[0111] In some embodiments, the enzyme medium comprises a neutral protease.

[0112] In some embodiments, the enzyme medium comprises hyaluronidase.

[0113] In some embodiments, the number of APCs in the third culture medium is greater than the number of APCs in the second culture medium.

[0114] In some embodiments, the first expansion by priming is carried out for about 3 to 11 days.

[0115] In some embodiments, the rapid second expansion is carried out for about 7 to 11 days.

[0116] In some embodiments, the priming primary expansion and rapid secondary expansion are completed within a period of about 22 days.

[0117] In some embodiments, the rapid second expansion is carried out by culturing the second TIL population in a third cell culture medium for a first period of about 5 days, at the end of the first period, dividing the culture into multiple subcultures, and culturing each of the multiple subcultures in a fourth culture medium containing IL-2 for a second period of about 6 days, and at the end of the second period, combining the multiple subcultures to provide the therapeutic TIL population.

[0118] In some embodiments, the present invention provides a method for expanding tumor-infiltrating lymphocytes into a therapeutic TIL population, the method comprising: (a) obtaining and / or receiving a first population of TILs from a sample of tumor tissue produced by surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining tumor tissue from a patient or subject, and storing the sample of tumor tissue in a frozen state, the method comprising: (i) pre-cooling the closeable container in a controlled rate freezing device; (ii) optionally fragmenting the sample of tumor tissue to obtain tumor fragments; (iii) placing the tumor tissue sample or tumor fragment in a closable container containing a cryopreservation medium and closing the container; (iv) incubating the closed container containing the tumor tissue sample or tumor fragment and the cryopreservation medium for about 30-60 minutes at a temperature of about 2-8°C; (v) slow freezing the container in a controlled rate freezer; (vi) a storing step, comprising transferring the container to a liquid nitrogen freezer; (b) adding the sample or tumor tissue or tumor fragment to a closed system and culturing a first TIL population in a first cell culture medium to perform initial expansion (or first expansion by priming) to produce a second TIL population, wherein the first expansion is performed in a sealed container providing a first gas permeable surface area, the first cell culture medium comprising IL-2, optionally OKT-3 (anti-CD3 antibody), and optionally antigen presenting cells (APCs), and the first expansion by priming occurs over a period of about 1 to 8 days to produce a second TIL population; (c) performing a second rapid expansion of the second TILs in a second cell culture medium comprising IL-2, OKT-3, and antigen presenting cells (APCs) to produce a third TIL population, wherein the second rapid expansion is performed in a sealed container providing a second gas permeable surface area, wherein the third TIL population is a therapeutic TIL population, and wherein the rapid expansion is performed over a period of no more than 14 days, and optionally, the rapid second expansion can proceed over about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days after initiation of the rapid second expansion, and wherein the transition from step (b) to step (c) occurs without opening the system; (d) harvesting the therapeutic TIL population obtained from step (c), wherein the transition from step (c) to step (d) occurs without opening the system.

[0119] In some embodiments, the present invention provides a method for expanding tumor-infiltrating lymphocytes into a therapeutic TIL population, the method comprising: (a) obtaining and / or receiving a first population of TILs from a sample of tumor tissue produced by surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining tumor tissue from a patient or subject, and storing the sample of tumor tissue in a frozen state, the method comprising: (i) placing a sample of tumor tissue, or tumor fragments obtained from fragmenting tumor tissue, into a pre-chilled, closable container containing a cryopreservation medium and closing the container; (ii) incubating the sealed container containing the tumor tissue sample or tumor fragment and the cryopreservation medium for about 30 to 60 minutes at a temperature of about 2 to 8°C; (iii) slow-freezing the container in a controlled rate freezer; (iv) a storing step, comprising transferring the container to a liquid nitrogen freezer; (b) adding the sample or tumor tissue or tumor fragment to a closed system and culturing a first TIL population in a first cell culture medium to perform initial expansion (or first expansion by priming) to produce a second TIL population, wherein the first expansion is performed in a sealed container providing a first gas permeable surface area, the first cell culture medium comprising IL-2, optionally OKT-3 (anti-CD3 antibody), and optionally antigen presenting cells (APCs), and the first expansion by priming occurs over a period of about 1 to 8 days to produce a second TIL population; (c) performing a second rapid expansion of the second TILs in a second cell culture medium comprising IL-2, OKT-3, and antigen presenting cells (APCs) to produce a third TIL population, wherein the second rapid expansion is performed in a sealed container providing a second gas permeable surface area, wherein the third TIL population is a therapeutic TIL population, and wherein the rapid expansion is performed over a period of no more than 14 days, and optionally, the rapid second expansion can proceed over about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days after initiation of the rapid second expansion, and wherein the transition from step (b) to step (c) occurs without opening the system; (d) harvesting the therapeutic TIL population obtained from step (c), wherein the transition from step (c) to step (d) occurs without opening the system.

[0120] In some embodiments, the present invention provides a method for expanding tumor-infiltrating lymphocytes into a therapeutic TIL population, the method comprising: (a) obtaining and / or receiving a first population of TILs from a sample of tumor tissue produced by surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining tumor tissue from a patient or subject, and storing the sample of tumor tissue in a frozen state, the method comprising: (i) placing a sample of tumor tissue or a tumor digest obtained from digesting tumor fragments produced from fragmenting a sample of tumor tissue in an enzymatic medium into a pre-chilled closable container containing a cryopreservation medium and closing the container; (ii) incubating the closed container containing the tumor digest and cryopreservation medium for about 30-60 minutes at a temperature of about 2-8°C; (iii) slow-freezing the container in a controlled rate freezer; (iv) a storing step, comprising transferring the container to a liquid nitrogen freezer; (b) adding tumor digest to a closed system and culturing a first TIL population in a first cell culture medium to perform an initial expansion (or first expansion by priming) to produce a second TIL population, wherein the first expansion is performed in a sealed container providing a first gas permeable surface area, the first cell culture medium comprising IL-2, optionally OKT-3 (an anti-CD3 antibody), and optionally antigen presenting cells (APCs), and the first expansion by priming occurs over a period of about 1 to 8 days; (c) performing a second rapid expansion of the second TILs in a second cell culture medium comprising IL-2, OKT-3, and antigen presenting cells (APCs) to produce a third TIL population, wherein the second rapid expansion is performed in a sealed container providing a second gas permeable surface area, wherein the third TIL population is a therapeutic TIL population, and wherein the rapid expansion is performed over a period of no more than 14 days, and optionally, the rapid second expansion can proceed over about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days after initiation of the rapid second expansion, and wherein the transition from step (b) to step (c) occurs without opening the system; (d) harvesting the therapeutic TIL population obtained from step (c), wherein the transition from step (c) to step (d) occurs without opening the system.

[0121] In some embodiments, the present invention provides a method for expanding tumor-infiltrating lymphocytes into a therapeutic TIL population, the method comprising: (a) obtaining and / or receiving a first population of TILs from a sample of tumor tissue produced by surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining tumor tissue from a patient or subject, and storing the sample of tumor tissue in a frozen state, the method comprising: (i) pre-cooling the closeable container in a controlled rate freezing device; (ii) optionally fragmenting the sample of tumor tissue to obtain tumor fragments; (iii) placing the tumor tissue sample or tumor fragment in a closable container containing a cryopreservation medium and closing the container; (iv) incubating the closed container containing the tumor tissue sample or tumor fragment and the cryopreservation medium for about 30-60 minutes at a temperature of about 2-8°C; (v) slow freezing the container in a controlled rate freezer; (vi) a storing step, comprising transferring the container to a liquid nitrogen freezer; (b) digesting the tumor tissue sample or tumor fragment in an enzymatic medium to produce a tumor digest; (c) adding tumor digest to a closed system and culturing a first TIL population in a first cell culture medium for initial expansion (or first expansion by priming) to produce a second TIL population, wherein the first expansion is performed in a sealed container providing a first gas permeable surface area, the first cell culture medium comprising IL-2, optionally OKT-3 (an anti-CD3 antibody), and optionally antigen presenting cells (APCs), and the first expansion by priming occurs over a period of about 1 to 8 days; (d) performing a second rapid expansion of the second TILs in a second cell culture medium comprising IL-2, OKT-3, and antigen presenting cells (APCs) to produce a third TIL population, wherein the second rapid expansion is performed in a sealed container providing a second gas permeable surface area, wherein the third TIL population is a therapeutic TIL population, and wherein the rapid expansion is performed over a period of no more than 14 days, and optionally, the rapid second expansion can proceed over about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days after initiation of the rapid second expansion, and wherein the transition from step (c) to step (d) occurs without opening the system; (e) harvesting the therapeutic TIL population obtained from step (d), wherein the transition from step (d) to step (e) occurs without opening the system.

[0122] In some embodiments, the present invention provides a method for expanding tumor-infiltrating lymphocytes into a therapeutic TIL population, the method comprising: (a) obtaining and / or receiving a first population of TILs from a sample of tumor tissue produced by surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining tumor tissue from a patient or subject, and storing the sample of tumor tissue in a frozen state, the method comprising: (i) placing a sample of tumor tissue, or tumor fragments obtained from fragmenting tumor tissue, into a pre-chilled, closable container containing a cryopreservation medium and closing the container; (ii) incubating the sealed container containing the tumor tissue sample or tumor fragment and the cryopreservation medium for about 30 to 60 minutes at a temperature of about 2 to 8°C; (iii) slow-freezing the container in a controlled rate freezer; (iv) a storing step, comprising transferring the container to a liquid nitrogen freezer; (b) digesting the tumor tissue sample or tumor fragment in an enzymatic medium to produce a digest; (c) adding tumor digest to a closed system and culturing a first TIL population in a first cell culture medium for initial expansion (or first expansion by priming) to produce a second TIL population, wherein the first expansion is performed in a sealed container providing a first gas permeable surface area, the first cell culture medium comprising IL-2, optionally OKT-3 (an anti-CD3 antibody), and optionally antigen presenting cells (APCs), and the first expansion by priming occurs over a period of about 1 to 8 days; (d) performing a second rapid expansion of the second TILs in a second cell culture medium comprising IL-2, OKT-3, and antigen presenting cells (APCs) to produce a third TIL population, wherein the second rapid expansion is performed in a sealed container providing a second gas permeable surface area, wherein the third TIL population is a therapeutic TIL population, and wherein the rapid expansion is performed over a period of no more than 14 days, and optionally, the rapid second expansion can proceed over about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days after initiation of the rapid second expansion, and wherein the transition from step (c) to step (d) occurs without opening the system; (e) harvesting the therapeutic TIL population obtained from step (c), wherein the transition from step (d) to step (e) occurs without opening the system.

[0123] In some embodiments, the present invention provides a method for expanding tumor-infiltrating lymphocytes into a therapeutic TIL population, the method comprising: (a) obtaining and / or receiving a first population of TILs from a sample of tumor tissue produced by surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining tumor tissue from a patient or subject, and storing the sample of tumor tissue in a frozen state, the method comprising: (i) pre-cooling the closeable container in a controlled rate freezing device; (ii) optionally fragmenting the sample of tumor tissue to obtain tumor fragments; (iii) digesting the tumor tissue sample or tumor fragment in an enzymatic medium to produce a tumor digest; (iv) placing the tumor digest into a closable container containing a cryopreservation medium and closing the container; (v) incubating the sealed container containing the tumor tissue sample or tumor fragment and the cryopreservation medium for about 30 to 60 minutes at a temperature of about 2 to 8°C; (vi) slow-freezing the container in a controlled rate freezer; (vii) a storing step, comprising transferring the container to a liquid nitrogen freezer; (b) adding tumor digest to a closed system and culturing a first TIL population in a first cell culture medium to perform an initial expansion (or first expansion by priming) to produce a second TIL population, wherein the first expansion is performed in a sealed container providing a first gas permeable surface area, the first cell culture medium comprising IL-2, optionally OKT-3 (an anti-CD3 antibody), and optionally antigen presenting cells (APCs), and the first expansion by priming occurs over a period of about 1 to 8 days; (c) performing a second rapid expansion of the second TILs in a second cell culture medium comprising IL-2, OKT-3, and antigen presenting cells (APCs) to produce a third TIL population, wherein the second rapid expansion is performed in a closed container providing a second gas permeable surface area, wherein the third TIL population is a therapeutic TIL population, and wherein the rapid expansion is performed over a period of no more than 14 days, and optionally, the rapid second expansion can proceed over about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days after initiation of the rapid second expansion, and wherein the transition from step (b) to step (c) occurs without opening the system; (d) harvesting the therapeutic TIL population obtained from step (c), wherein the transition from step (c) to step (d) occurs without opening the system.

[0124] In some embodiments, the enzyme medium comprises DNase.

[0125] In some embodiments, the enzyme medium comprises collagenase.

[0126] In some embodiments, the enzyme medium comprises a neutral protease.

[0127] In some embodiments, the enzyme medium comprises hyaluronidase.

[0128] In some embodiments, the first culture medium comprises OKT-3.

[0129] In some embodiments, the first culture medium comprises APCs.

[0130] In some embodiments, the number of APCs in the second culture medium is greater than the number of APCs in the first culture medium.

[0131] In some embodiments, the first expansion step by priming is carried out for a period of about 7 or 8 days.

[0132] In some embodiments, the rapid second expansion is carried out for about 7-10 days.

[0133] In some embodiments, the rapid expansion step is carried out by culturing the second TIL population in a second cell culture medium for a first period of about 3-4 days, at the end of the first period, dividing the culture into multiple subcultures, and culturing each of the multiple subcultures in a third culture medium containing IL-2 for a second period of about 4-6 days, and at the end of the second period, combining the multiple subcultures to provide the therapeutic TIL population.

[0134] In some embodiments, the present invention provides a method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population, comprising: (a) obtaining and / or receiving a first population of TILs from a sample of tumor tissue produced by surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining tumor tissue from a patient or subject, and storing the sample of tumor tissue in a frozen state, the method comprising: (i) pre-cooling the closeable container in a controlled rate freezing device; (ii) optionally fragmenting the sample of tumor tissue to obtain tumor fragments; (iii) digesting the tumor tissue sample or tumor fragment in an enzymatic medium to produce a tumor digest; (iv) placing the tumor digest into a closable container containing a cryopreservation medium and closing the container; (v) incubating the sealed container containing the tumor tissue sample or tumor fragment and the cryopreservation medium for about 30 to 60 minutes at a temperature of about 2 to 8°C; (vi) slow-freezing the container in a controlled rate freezer; (vii) storing the container, including transferring the container to a liquid nitrogen freezer; (b) selecting PD-1 positive TILs from the first TIL population in the tumor digest of step (a) to obtain a PD-1 enriched TIL population; (c) performing a first priming expansion by culturing the PD-1-enriched TIL population in a first cell culture medium comprising IL-2, OKT-3, and antigen-presenting cells (APCs) to produce a second TIL population, wherein the first priming expansion is performed in a container comprising a first gas permeable surface area, and the first priming expansion is performed for a first period of about 1 to 7, 8, 9, 10, or 11 days to obtain a second TIL population; (d) performing a second rapid expansion by culturing the second TIL population in a second culture medium comprising IL-2, OKT-3, and APCs, wherein the number of APCs added to the rapid second expansion is at least twice the number of APCs added in step (c), and the rapid second expansion is performed for a second period of time of about 1 to 11 days to obtain a therapeutic TIL population, and the rapid second expansion is performed in a container comprising a second gas permeable surface area; (e) harvesting the therapeutic TIL population obtained from step (d).

[0135] In some embodiments, the present invention provides a method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population, comprising: (a) obtaining and / or receiving a first population of TILs from a sample of tumor tissue produced by surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining tumor tissue from a patient or subject, and storing the sample of tumor tissue in a frozen state, the method comprising: (i) placing a sample of tumor tissue or a tumor digest obtained from digesting tumor fragments produced from fragmenting a sample of tumor tissue in an enzymatic medium into a pre-chilled closable container containing a cryopreservation medium and closing the container; (ii) incubating the sealed container containing the tumor digest and cryopreservation medium for about 30-60 minutes at a temperature of about 2-8°C; (iii) slow-freezing the container in a controlled rate freezer; (iv) a storing step, comprising transferring the container to a liquid nitrogen freezer; (b) selecting PD-1 positive TILs from the first TIL population in the tumor digest of step (a) to obtain a PD-1 enriched TIL population; (c) performing a first priming expansion of the PD-1-enriched TIL population by culturing it in a first cell culture medium comprising IL-2, OKT-3, and antigen-presenting cells (APCs) to produce a second TIL population, wherein the first priming expansion is performed in a container comprising a first gas permeable surface area, and the first priming expansion is performed for a first period of about 1 to 7, 8, 9, 10, or 11 days to obtain a second TIL population; (d) performing a second rapid expansion by culturing the second TIL population in a second culture medium comprising IL-2, OKT-3, and APCs, wherein the number of APCs added to the rapid second expansion is at least twice the number of APCs added in step (c), and the rapid second expansion is performed for a second period of time of about 1 to 11 days to obtain a therapeutic TIL population, and the rapid second expansion is performed in a container comprising a second gas permeable surface area; (e) harvesting the therapeutic TIL population obtained from step (d).

[0136] In some embodiments, the present invention provides a method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population, comprising: (a) obtaining and / or receiving a first population of TILs from a sample of tumor tissue produced by surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining tumor tissue from a patient or subject, and storing the sample of tumor tissue in a frozen state, the method comprising: (i) pre-cooling the closeable container in a controlled rate freezing device; (ii) optionally fragmenting the sample of tumor tissue to obtain tumor fragments; (iii) placing the tumor tissue sample or tumor fragment in a closable container containing a cryopreservation medium and closing the container; (iv) incubating the sealed container containing the tumor tissue sample or tumor fragment and the cryopreservation medium for about 30 to 60 minutes at a temperature of about 2 to 8°C; (v) slow freezing the container in a controlled rate freezer; (vi) a storing step, comprising transferring the container to a liquid nitrogen freezer; (b) digesting the tumor tissue sample or tumor fragment in an enzymatic medium to produce a tumor digest; (c) selecting PD-1 positive TILs from the first TIL population in the tumor digest of step (b) to obtain a PD-1 enriched TIL population; (d) performing a first priming expansion by culturing the PD-1-enriched TIL population in a first cell culture medium comprising IL-2, OKT-3, and antigen-presenting cells (APCs) to produce a second TIL population, wherein the first priming expansion is performed in a container comprising a first gas permeable surface area, and the first priming expansion is performed for a first period of about 1 to 7, 8, 9, 10, or 11 days to obtain a second TIL population; (e) performing a second rapid expansion by culturing the second TIL population in a second culture medium comprising IL-2, OKT-3, and APCs, wherein the number of APCs added to the rapid second expansion is at least twice the number of APCs added in step (c), and the rapid second expansion is performed for a second period of time of about 1 to 11 days to obtain a therapeutic TIL population, and the rapid second expansion is performed in a container comprising a second gas permeable surface area; (f) harvesting the therapeutic TIL population obtained from step (e).

[0137] In some embodiments, the PD-1 selection step comprises: (i) exposing a first population of TILs and a population of PBMCs to an excess of a monoclonal anti-PD-1 IgG4 antibody that binds to PD-1 by the N-terminal loop outside the IgV domain of PD-1; (ii) adding an excess of anti-IgG4 antibody conjugated to a fluorophore; (iii) obtaining a PD-1-enriched TIL population based on the intensity of the fluorophore of PD-1-positive TILs in the first TIL population compared to the intensity of the PBMC population as determined by fluorescence-activated cell sorting (FACS).

[0138] In some embodiments, the enzyme medium comprises DNase.

[0139] In some embodiments, the enzyme medium comprises collagenase.

[0140] In some embodiments, the enzyme medium comprises a neutral protease.

[0141] In some embodiments, the enzyme medium comprises hyaluronidase.

[0142] In some embodiments, the number of APCs in the second culture medium is greater than the number of APCs in the first culture medium.

[0143] In some embodiments, the first expansion step by priming is carried out for about 11 days.

[0144] In some embodiments, the rapid second expansion step is carried out for about 11 days.

[0145] In some embodiments, the rapid expansion step is carried out by culturing the second TIL population in a second cell culture medium for a first period of about 5 days, at the end of the first period, dividing the culture into multiple sub-cultures, and culturing each of the multiple sub-cultures in a third culture medium containing IL-2 for a second period of about 6 days, and at the end of the second period, combining the multiple sub-cultures to provide the therapeutic TIL population.

[0146] In some embodiments, the tumor tissue is derived from a dissected tumor.

[0147] In some embodiments, the dissected tumor is less than 8 hours old.

[0148] In some embodiments, 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 HPV-positive tumor tissue.

[0149] In some embodiments, the tumor tissue is fragmented into roughly spherical pieces having a diameter of about 1.5 mm to 6 mm.

[0150] In some embodiments, the tumor tissue is fragmented into approximately spherical fragments having a diameter of about 3 mm or about 6 mm.

[0151] In some embodiments, the tumor tissue is fragmented into generally rectangular pieces having a shortest edge length of at least 1.5 mm and a longest edge length of about 6 mm.

[0152] In some embodiments, the tumor tissue is fragmented into generally cubic pieces having edge lengths of about 3 mm or about 6 mm.

[0153] In some embodiments, the tumor fragments are washed with physiologically buffered isotonic saline prior to incubation.

[0154] In some embodiments, the washing comprises three successive washes of at least three minutes each, with physiologically buffered isotonic saline being replaced after each successive wash.

[0155] In some embodiments, the present invention provides a method for expanding peripheral blood lymphocytes (PBLs) from peripheral blood, comprising: (a) obtaining a sample of peripheral blood mononuclear cells (PBMCs) from the peripheral blood of a patient, the patient optionally being pre-treated with an ITK inhibitor, and storing the sample of PBMCs in a frozen state; (i) pre-cooling the closeable container in a controlled rate freezing device; (ii) placing the sample of PBMCs in a closable container containing a cryopreservation medium and closing the container; (iii) incubating the closable container containing the sample of PBMCs and the cryopreservation medium for about 30-60 minutes at a temperature of about 2-8°C; (iv) slow freezing the container in a controlled rate freezer; (v) storing the container, including transferring the container to a liquid nitrogen freezer; (b) optionally washing the PBMCs by centrifugation; (c) mixing magnetic beads selective for CD3 and CD28 with PBMCs; (d) culturing the mixture in a cell culture medium containing IL-2; (e) harvesting the PBL product from the cell culture medium.

[0156] In some embodiments, the present invention provides a method for expanding peripheral blood lymphocytes (PBLs) from peripheral blood, comprising: (a) obtaining a sample of peripheral blood mononuclear cells (PBMCs) from the peripheral blood of a patient, the patient optionally being pre-treated with an ITK inhibitor, and storing the sample of PBMCs in a frozen state; (i) pre-cooling the closeable container in a controlled rate freezing device; (ii) placing said sample of PBMCs in a closable container containing a cryopreservation medium and closing the container; (iii) incubating the closable container containing the sample of PBMCs and the cryopreservation medium for about 30-60 minutes at a temperature of about 2-8°C; (iv) slow freezing the container in a controlled rate freezer; (v) storing the container, including transferring the container to a liquid nitrogen freezer; (b) optionally washing the PBMCs by centrifugation; (c) mixing magnetic beads selective for CD3 and CD28 with PBMCs; (d) culturing the mixture in a cell culture medium containing IL-2; (e) removing the magnetic beads using a magnet; (f) harvesting the PBL product from the cell culture medium.

[0157] In some embodiments, the present invention provides a method for expanding peripheral blood lymphocytes (PBLs) from peripheral blood, comprising: (a) obtaining a sample of peripheral blood mononuclear cells (PBMCs) from the peripheral blood of a patient, the patient optionally being pre-treated with an ITK inhibitor, and storing the sample of PBMCs in a frozen state; (i) pre-cooling the closeable container in a controlled rate freezing device; (ii) placing the sample of PBMCs in a closable container containing a cryopreservation medium and closing the container; (iii) incubating the closable container containing the sample of PBMCs and the cryopreservation medium for about 30-60 minutes at a temperature of about 2-8°C; (iv) slow freezing the container in a controlled rate freezer; (v) storing the container, including transferring the container to a liquid nitrogen freezer; (b) optionally washing the PBMCs by centrifugation; (c) mixing magnetic beads selective for CD3 and CD28 with PBMCs to form a mixture; (d) seeding the PBMCs in the mixture into a container that provides a gas permeable surface and culturing them in a cell culture medium containing about 3000 IU / mL of IL-2 for about 4 to about 6 days; (e) feeding the PBMCs using a medium containing about 3000 IU / mL of IL-2 and culturing the PBMCs for about 5 days, such that the total culture period of steps (d) and (e) is about 9 to about 11 days; (f) removing the magnetic beads using a magnet; (g) harvesting PBMCs from the cell culture medium; (h) removing residual B cells using magnetic activated cell sorting and CD19+ beads to produce a PBL product.

[0158] In some embodiments, the PBL product is formulated and, optionally, cryopreserved.

[0159] In some embodiments, no more than about 50 mL of the patient's peripheral blood is obtained in step (a).

[0160] In some embodiments, the seeding density of the PBMCs in step (d) is about 2×10 to the surface area of ​​the gas permeable surface. 5 / cm 2 ~Approx. 1.6×10 3 / cm 2 is.

[0161] In some embodiments, the seeding density of the PBMCs in step (d) is about 25,000 cells / cm of surface area of ​​the gas permeable surface. 2 ~about 50,000 cells / cm 2 is.

[0162] In some embodiments, a sample of PBMCs is obtained from the patient's peripheral blood by density gradient centrifugation.

[0163] In some embodiments, the density gradient centrifugation is Ficoll density gradient centrifugation.

[0164] In some embodiments, the present invention provides therapeutic populations of tumor infiltrating lymphocyte (TIL) products produced by the methods described herein.

[0165] In some embodiments, the present invention provides methods for treating cancer in a patient, comprising administering to the patient an effective amount of a therapeutic TIL population produced by the methods described herein.

[0166] In some embodiments, the cancer is selected from the group consisting of glioblastoma (GBM), gastrointestinal cancer, melanoma, ovarian cancer, endometrial cancer, thyroid cancer, colorectal cancer, cervical cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, endometrial cancer, cholangiocarcinoma, cancer caused by human papillomavirus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), renal cancer, renal cell carcinoma, multiple myeloma, chronic lymphocytic leukemia, acute lymphocytic leukemia, diffuse large B-cell lymphoma, non-Hodgin's lymphoma, Hodgin's lymphoma, follicular lymphoma, and mantle cell lymphoma.

[0167] In some embodiments, the cancer is selected from the group consisting of cutaneous melanoma, ocular melanoma, uveal melanoma, conjunctival malignant melanoma, pleomorphic xanthoastrocytoma, dysembryoplastic neuroepithelial tumor, ganglioglioma, and pilocytic astrocytoma, endometrial adenocarcinoma with marked mucinous differentiation (ECMD), papillary thyroid carcinoma, low-grade serous or borderline ovarian carcinoma, hairy cell leukemia, and Langerhans cell histiocytosis.

[0168] In some embodiments, the invention provides a PBL product produced by the methods described herein.

[0169] In some embodiments, the present invention provides methods for treating cancer in a patient, comprising administering to the patient an effective amount of a PBL product described herein.

[0170] In some embodiments, the cancer is selected from the group consisting of acute myeloid leukemia (AML), mantle cell lymphoma (MCL), follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), activated B-cell (ABC) DLBCL, germinal center B-cell (GCB) DLBCL, chronic lymphocytic leukemia (CLL), CLL with Richter's transformation (or Richter's syndrome), small lymphocytic leukemia (SLL), non-Hodgkin's lymphoma (NHL), Hodgkin's lymphoma, relapsed and recurrent leukemia (RELATED). and / or a hematological malignancy selected from the group consisting of refractory Hodgkin's lymphoma, B-cell acute lymphoblastic leukemia (B-ALL), mature B-ALL, Burkitt's lymphoma, Waldenstrom's macroglobulinemia (WM), multiple myeloma, myelodysplastic syndrome, myelofibrosis, chronic myeloid leukemia, follicle center lymphoma, indolent NHL, human immunodeficiency (HIV)-associated B-cell lymphoma, and Epstein-Barr virus (EBV)-associated B-cell lymphoma.

[0171] In some embodiments, the cryopreservation medium comprises about 2% v / v DMSO to about 15% v / v DMSO.

[0172] In some embodiments, the cryopreservation medium comprises about 10% v / v DMSO.

[0173] In some embodiments, the cryopreservation medium comprises at least one antimicrobial agent.

[0174] In some embodiments, the cryopreservation medium comprises gentamicin at a concentration of at least 50 μg / mL.

[0175] In some embodiments, the closable container is a cryogenic vial.

[0176] In some embodiments, the closable container is filled with cryopreservation medium to about 50% to about 85% volume.

[0177] In some embodiments, the controlled rate freezer is an IPA-free controlled rate freezer that cools at a rate of about -0.1°C / min to about -10°C / min.

[0178] In some embodiments, the controlled rate freezer is an IPA-free controlled rate freezer that cools at a rate of about -1°C / min.

[0179] In some embodiments, all of the positions of the controlled rate freezing device are filled with a closable container containing a cryopreservation medium.

[0180] In some embodiments, slow freezing comprises incubating a controlled rate freezer at a temperature of about -70°C to about -90°C.

[0181] In some embodiments, slow freezing comprises incubating a controlled rate freezer at a temperature of about -80°C for about 3-5 hours.

[0182] In some embodiments, the slow freezing comprises incubating a controlled rate freezer at a temperature of about -80°C for about 4 hours.

[0183] In some embodiments, the slow freezing comprises incubating a controlled rate freezing device containing dry ice.

[0184] In some embodiments, the slow freezing comprises incubating a controlled rate freezing device in a -80°C freezer.

[0185] In some embodiments, the slow freezing occurs at a cooling rate of about -0.1°C / min to about -10°C / min.

[0186] In some embodiments, the slow freezing occurs at a cooling rate of about -1°C / min.

[0187] In some embodiments, after recovery from freezing, the cells have a post-thaw viability of at least about 80%.

[0188] In some embodiments, IL-2 is present in the cell culture medium at an initial concentration of 1000 IU / mL to 6000 IU / mL in the first expansion.

[0189] In some embodiments, in the rapid second expansion step, IL-2 is present at an initial concentration of 1000 IU / mL to 6000 IU / mL and the OKT-3 antibody is present at an initial concentration of about 30 ng / mL.

[0190] In some embodiments, the first expansion is performed using a gas-permeable container.

[0191] In some embodiments, the second expansion is performed using a gas-permeable container.

[0192] In some embodiments, 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.

[0193] In some embodiments, the second cell culture medium and / or the third culture medium further comprises a cytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21, and combinations thereof.

[0194] In some embodiments, the method further comprises treating the patient with a non-myeloablative lymphodepletion regimen prior to administering the TIL or PBL product to the patient.

[0195] In some embodiments, the method further comprises treating the patient with an IL-2 regimen starting the day after administering the TIL or PBL product to the patient.

[0196] In some embodiments, the method further comprises treating the patient with an IL-2 regimen starting on the same day that the TIL or PBL product is administered to the patient.

[0197] In some embodiments, the IL-2 regimen includes aldesleukin, nemvaleukin, or a biosimilar or variant thereof.

[0198] In some embodiments, a therapeutically effective amount of TIL product is about 2.3 x 10 10 ~Approx. 13.7×10 10 Including TIL.

[0199] In some embodiments, the second TIL population is at least 50-fold more numerous than the first TIL population.

[0200] 10. Use of an effective amount of a therapeutic TIL population or PBL product produced by the method of any of the preceding claims for the treatment of cancer.

[0201] 10. The TIL of any of the preceding claims, wherein the TIL is gene edited according to any of the methods described herein. [Brief explanation of the drawings]

[0202] [Figure 1] An exemplary Gen2 (Process 2A) chart providing an overview of steps A-F. [Figure 2A] 1 is a process flow diagram of an embodiment of Gen2 (Process 2A) for TIL fabrication. [Figure 2B] 1 is a process flow diagram of an embodiment of Gen2 (Process 2A) for TIL fabrication. [Figure 2C] 1 is a process flow diagram of an embodiment of Gen2 (Process 2A) for TIL fabrication. [Figure 3] FIG. 1 shows a diagram of an embodiment of an exemplary manufacturing process (approximately 22 days) for cryopreserved TILs. [Figure 4] FIG. 1 shows a diagram of an embodiment of Gen2 (Process 2A), a 22-day process for TIL fabrication. [Figure 5] 1 is a comparison table of steps A-F from exemplary embodiments of Process 1C and Gen2 (Process 2A) for TIL fabrication. [Figure 6] Detailed comparison of Process 1C and Gen2 (Process 2A) embodiments for TIL fabrication. [Figure 7] 1. Exemplary Gen3 TIL fabrication process. [Figure 8A] A comparison of embodiments of the 2A process (an approximately 22 day process) and the Gen3 process (an approximately 14-16 day process) for TIL fabrication is shown. [Figure 8B] An exemplary Process Gen3 chart providing an overview of steps A-F (approximately a 14-16 day process). [Figure 8C] A chart providing three exemplary Gen3 processes along with an overview of steps A-F (approximately 14- to 16-day processes) for each of the three process variations. [Figure 8D] An exemplary modified Gen2-like process (approximately a 22-day process) providing an overview of steps A-F. [Figure 9] 1 provides an experimental flow chart for the comparison between Gen2 (Process 2A) and Gen3 processes. [Figure 10] 1 shows a comparison of various Gen2 (Process 2A) and Gen3.1 process embodiments. [Figure 11] 1 is a table illustrating various features of embodiments of the Gen2, Gen2.1, and Gen3.0 processes. [Figure 12] Summary of media conditions for an embodiment of the Gen3 process, designated Gen3.1. [Figure 13] 1 is a table illustrating various features of embodiments of the Gen2, Gen2.1, and Gen3.0 processes. [Figure 14] 1 is a table comparing various features of embodiments of the Gen2 and Gen3.0 processes. [Figure 15] 1 is a table providing media use in various embodiments of the described expansion process. [Figure 16] Schematic of an exemplary embodiment of the Gen3 process (16-day process). [Figure 17]Schematic of an exemplary embodiment of a method for expanding T cells from hematopoietic malignancies using the Gen3 expansion platform. [Figure 18] Structures IA and IB are provided. The cylinders refer to individual polypeptide binding domains. Structures IA and IB comprise three linearly linked TNFRSF-binding domains, e.g., derived from antibodies that bind to 4-1BBL or 4-1BB, that fold to form a trivalent protein, which is then linked to a second trivalent protein via IgG1-Fc (comprising the CH3 and CH2 domains), which is then used to link two of the trivalent proteins together via disulfide bonds (small oblong ellipses), stabilizing the structure and providing an agonist that can bring together the six receptor and intracellular signaling domains of the signaling protein to form a signaling complex. The TNFRSF-binding domains shown as cylinders can be, for example, scFv domains comprising VH and VL chains connected by a linker that may contain hydrophilic residues and Gly and Ser sequences for flexibility, and Glu and Lys for solubility. [Figure 19] Schematic of an exemplary embodiment of the Gen3 process (16-day process). [Figure 20] 1 provides a process overview of an exemplary embodiment of the Gen3.1 process (16-day process). [Figure 21] Schematic of an exemplary embodiment of the Gen3.1 testing process (16-17 day process). [Figure 22] Schematic of an exemplary embodiment of the Gen3 process (16-day process). [Figure 23] 1 is a comparison table of an exemplary Gen2 process and an exemplary Gen3 process. [Figure 24] Schematic of an exemplary embodiment of the preparation timeline for the Gen3 process (16-17 day process). [Figure 25] Schematic of an exemplary embodiment of the Gen3 process (14-16 day process). [Figure 26A] Schematic of an exemplary embodiment of the Gen3 process (16-day process). [Figure 26B] Schematic of an exemplary embodiment of the Gen3 process (16-day process). [Figure 27] Schematic of an exemplary embodiment of the Gen3 process (16-day process). [Figure 28] Comparison of Gen2, Gen2.1, and Gen3 process (16-day process) embodiments. [Figure 29] Comparison of Gen2, Gen2.1, and Gen3 process (16-day process) embodiments. [Figure 30] Components of a Gen3 embodiment. [Figure 31] Flowchart comparison of Gen3 embodiments (Gen3.0, Gen3.1 control, Gen3.1 test). [Figure 32] Components of an exemplary embodiment of the Gen3 process (16-17 day process) are shown. [Figure 33] Approval criteria table. [Figure 34] Comparison of slow and fast freezing methods for cryopreservation of tumor tissue on day 11 of TIL culture. [Figure 35] Comparison of slow and fast freezing methods for cryopreservation of tumor tissue at day 22 of TIL culture.

[0203] Brief Description of Sequence Listing SEQ ID NO: 1 is the amino acid sequence of the heavy chain of muromonab.

[0204] SEQ ID NO: 2 is the amino acid sequence of the light chain of muromonab.

[0205] SEQ ID NO: 3 is the amino acid sequence of recombinant human IL-2 protein.

[0206] SEQ ID NO: 4 is the amino acid sequence of aldesleukin.

[0207] SEQ ID NO: 5 is an IL-2 form.

[0208] SEQ ID NO: 6 is the amino acid sequence of nemvaleukin alpha.

[0209] SEQ ID NO: 7 is an IL-2 form.

[0210] SEQ ID NO: 8 is a mucin domain polypeptide.

[0211] SEQ ID NO: 9 is the amino acid sequence of the human IL-4 protein.

[0212] SEQ ID NO: 10 is the amino acid sequence of recombinant human IL-7 protein.

[0213] SEQ ID NO: 11 is the amino acid sequence of recombinant human IL-15 protein.

[0214] SEQ ID NO: 12 is the amino acid sequence of recombinant human IL-21 protein.

[0215] SEQ ID NO: 13 is the IL-2 sequence.

[0216] SEQ ID NO: 14 is the IL-2 mutein sequence.

[0217] SEQ ID NO: 15 is the sequence of an IL-2 mutein.

[0218] SEQ ID NO: 16 is HCDR1_IL-2 of IgG.IL2R67A.H1.

[0219] SEQ ID NO: 17 is HCDR2 of IgG.IL2R67A.H1.

[0220] SEQ ID NO: 18 is the HCDR3 of IgG.IL2R67A.H1.

[0221] SEQ ID NO: 19 is HCDR1_IL-2 Kabat of IgG.IL2R67A.H1.

[0222] SEQ ID NO: 20 is the HCDR2 Kabat of IgG.IL2R67A.H1.

[0223] SEQ ID NO: 21 is the HCDR3 Kabat of IgG.IL2R67A.H1.

[0224] SEQ ID NO: 22 is the HCDR1_IL-2 clotia of IgG.IL2R67A.H1.

[0225] SEQ ID NO: 23 is the HCDR2 clone of IgG.IL2R67A.H1.

[0226] SEQ ID NO: 24 is the HCDR3 clone of IgG.IL2R67A.H1.

[0227] SEQ ID NO: 25 is HCDR1_IL-2 IMGT of IgG.IL2R67A.H1.

[0228] SEQ ID NO: 26 is HCDR2 IMGT of IgG.IL2R67A.H1.

[0229] SEQ ID NO: 27 is the HCDR3 IMGT of IgG.IL2R67A.H1.

[0230] SEQ ID NO: 28 is the V H It's a chain.

[0231] SEQ ID NO: 29 is the heavy chain of IgG.IL2R67A.H1.

[0232] SEQ ID NO: 30 is the LCDR1 Kabat of IgG.IL2R67A.H1.

[0233] SEQ ID NO: 31 is the LCDR2 Kabat of IgG.IL2R67A.H1.

[0234] SEQ ID NO: 32 is the LCDR3 Kabat of IgG.IL2R67A.H1.

[0235] SEQ ID NO: 33 is the LCDR1 Clothiase of IgG.IL2R67A.H1.

[0236] SEQ ID NO: 34 is the LCDR2 clone of IgG.IL2R67A.H1.

[0237] SEQ ID NO: 35 is the LCDR3 clone of IgG.IL2R67A.H1.

[0238] SEQ ID NO: 36 is V L It's a chain.

[0239] SEQ ID NO: 37 is the light chain.

[0240] SEQ ID NO: 38 is the light chain.

[0241] SEQ ID NO: 39 is the light chain.

[0242] SEQ ID NO: 40 is the amino acid sequence of human 4-1BB.

[0243] SEQ ID NO: 41 is the amino acid sequence of mouse 4-1BB.

[0244] SEQ ID NO: 42 is the heavy chain of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0245] SEQ ID NO: 43 is the light chain of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0246] SEQ ID NO: 44 represents the heavy chain variable region (V) of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566). H )

[0247] SEQ ID NO: 45 is the light chain variable region (V) of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566). L )

[0248] SEQ ID NO: 46 is the heavy chain CDR1 of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0249] SEQ ID NO: 47 is the heavy chain CDR2 of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0250] SEQ ID NO: 48 is the heavy chain CDR3 of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0251] SEQ ID NO: 49 is the light chain CDR1 of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0252] SEQ ID NO: 50 is the light chain CDR2 of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0253] SEQ ID NO: 51 is the light chain CDR3 of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0254] SEQ ID NO: 52 is the heavy chain of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0255] SEQ ID NO: 53 is the light chain of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0256] SEQ ID NO: 54 is the heavy chain variable region (VH) of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0257] SEQ ID NO: 55 is the light chain variable region (VL) of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0258] SEQ ID NO: 56 is the heavy chain CDR1 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0259] SEQ ID NO: 57 is the heavy chain CDR2 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0260] SEQ ID NO: 58 is the heavy chain CDR3 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0261] SEQ ID NO: 59 is the light chain CDR1 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0262] SEQ ID NO: 60 is the light chain CDR2 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0263] SEQ ID NO: 61 is the light chain CDR3 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0264] SEQ ID NO: 62 is the Fc domain of the TNFRSF agonist fusion protein.

[0265] SEQ ID NO: 63 is the linker of the TNFRSF agonist fusion protein.

[0266] SEQ ID NO: 64 is the linker of the TNFRSF agonist fusion protein.

[0267] SEQ ID NO: 65 is the linker of the TNFRSF agonist fusion protein.

[0268] SEQ ID NO: 66 is the linker of the TNFRSF agonist fusion protein.

[0269] SEQ ID NO: 67 is the linker for the TNFRSF agonist fusion protein.

[0270] SEQ ID NO: 68 is the linker for the TNFRSF agonist fusion protein.

[0271] SEQ ID NO: 69 is the linker for the TNFRSF agonist fusion protein.

[0272] SEQ ID NO: 70 is the linker of the TNFRSF agonist fusion protein.

[0273] SEQ ID NO: 71 is the linker for the TNFRSF agonist fusion protein.

[0274] SEQ ID NO: 72 is the linker for the TNFRSF agonist fusion protein.

[0275] SEQ ID NO: 73 is the Fc domain of the TNFRSF agonist fusion protein.

[0276] SEQ ID NO: 74 is the linker for the TNFRSF agonist fusion protein.

[0277] SEQ ID NO: 75 is the linker for the TNFRSF agonist fusion protein.

[0278] SEQ ID NO: 76 is the linker for the TNFRSF agonist fusion protein.

[0279] SEQ ID NO: 77 is the 4-1BB ligand (4-1BBL) amino acid sequence.

[0280] SEQ ID NO: 78 is the soluble portion of the 4-1BBL polypeptide.

[0281] SEQ ID NO: 79 is the heavy chain variable region (V) of 4-1BB agonist antibody 4B4-1-1 version 1 H )

[0282] SEQ ID NO: 80 represents the light chain variable region (V) of 4-1BB agonist antibody 4B4-1-1 version 1 L )

[0283] SEQ ID NO: 81 is the heavy chain variable region (V H )

[0284] SEQ ID NO: 82 is the light chain variable region (V) of 4-1BB agonist antibody 4B4-1-1 version 2 L )

[0285] SEQ ID NO: 83 identifies the heavy chain variable region (V H )

[0286] SEQ ID NO: 84 identifies the light chain variable region (V L )

[0287] SEQ ID NO: 85 is the amino acid sequence of human OX40.

[0288] SEQ ID NO: 86 is the amino acid sequence of mouse OX40.

[0289] SEQ ID NO: 87 is the heavy chain of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0290] SEQ ID NO: 88 is the light chain of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0291] SEQ ID NO: 89 is the heavy chain variable region (V) of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562). H )

[0292] SEQ ID NO: 90 is the light chain variable region (V) of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562). L )

[0293] SEQ ID NO: 91 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0294] SEQ ID NO: 92 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0295] SEQ ID NO: 93 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0296] SEQ ID NO: 94 is the light chain CDR1 of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0297] SEQ ID NO: 95 is the light chain CDR2 of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0298] SEQ ID NO: 96 is the light chain CDR3 of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0299] SEQ ID NO: 97 is the heavy chain of the OX40 agonist monoclonal antibody 11D4.

[0300] SEQ ID NO: 98 is the light chain of the OX40 agonist monoclonal antibody 11D4.

[0301] SEQ ID NO: 99 is the heavy chain variable region (V) of the OX40 agonist monoclonal antibody 11D4. H )

[0302] SEQ ID NO: 100 is the light chain variable region (V) of the OX40 agonist monoclonal antibody 11D4. L )

[0303] SEQ ID NO: 101 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody 11D4.

[0304] SEQ ID NO: 102 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody 11D4.

[0305] SEQ ID NO: 103 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody 11D4.

[0306] SEQ ID NO: 104 is the light chain CDR1 of the OX40 agonist monoclonal antibody 11D4.

[0307] SEQ ID NO: 105 is the light chain CDR2 of the OX40 agonist monoclonal antibody 11D4.

[0308] SEQ ID NO: 106 is the light chain CDR3 of the OX40 agonist monoclonal antibody 11D4.

[0309] SEQ ID NO: 107 is the heavy chain of the OX40 agonist monoclonal antibody 18D8.

[0310] SEQ ID NO: 108 is the light chain of the OX40 agonist monoclonal antibody 18D8.

[0311] SEQ ID NO: 109 is the heavy chain variable region (V) of the OX40 agonist monoclonal antibody 18D8. H )

[0312] SEQ ID NO: 110 is the light chain variable region (V) of the OX40 agonist monoclonal antibody 18D8. L )

[0313] SEQ ID NO: 111 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody 18D8.

[0314] SEQ ID NO: 112 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody 18D8.

[0315] SEQ ID NO: 113 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody 18D8.

[0316] SEQ ID NO: 114 is the light chain CDR1 of the OX40 agonist monoclonal antibody 18D8.

[0317] SEQ ID NO: 115 is the light chain CDR2 of the OX40 agonist monoclonal antibody 18D8.

[0318] SEQ ID NO: 116 is the light chain CDR3 of the OX40 agonist monoclonal antibody 18D8.

[0319] SEQ ID NO: 117 is the heavy chain variable region (V) of the OX40 agonist monoclonal antibody Hu119-122. H )

[0320] SEQ ID NO: 118 is the light chain variable region (V) of the OX40 agonist monoclonal antibody Hu119-122. L )

[0321] SEQ ID NO: 119 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody Hu119-122.

[0322] SEQ ID NO: 120 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody Hu119-122.

[0323] SEQ ID NO: 121 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody Hu119-122.

[0324] SEQ ID NO: 122 is the light chain CDR1 of the OX40 agonist monoclonal antibody Hu119-122.

[0325] SEQ ID NO: 123 is the light chain CDR2 of the OX40 agonist monoclonal antibody Hu119-122.

[0326] SEQ ID NO: 124 is the light chain CDR3 of the OX40 agonist monoclonal antibody Hu119-122.

[0327] SEQ ID NO: 125 is the heavy chain variable region (V) of the OX40 agonist monoclonal antibody Hu106-222. H )

[0328] SEQ ID NO: 126 is the light chain variable region (V) of the OX40 agonist monoclonal antibody Hu106-222. L )

[0329] SEQ ID NO: 127 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody Hu106-222.

[0330] SEQ ID NO: 128 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody Hu106-222.

[0331] SEQ ID NO: 129 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody Hu106-222.

[0332] SEQ ID NO: 130 is the light chain CDR1 of the OX40 agonist monoclonal antibody Hu106-222.

[0333] SEQ ID NO: 131 is the light chain CDR2 of the OX40 agonist monoclonal antibody Hu106-222.

[0334] SEQ ID NO: 132 is the light chain CDR3 of the OX40 agonist monoclonal antibody Hu106-222.

[0335] SEQ ID NO: 133 is the OX40 ligand (OX40L) amino acid sequence.

[0336] SEQ ID NO: 134 is the soluble portion of the OX40L polypeptide.

[0337] SEQ ID NO: 135 is an alternative soluble portion of the OX40L polypeptide.

[0338] SEQ ID NO: 136 is the heavy chain variable region (V H )

[0339] SEQ ID NO: 137 is the light chain variable region (V) of OX40 agonist monoclonal antibody 008. L )

[0340] SEQ ID NO: 138 is the heavy chain variable region (V) of OX40 agonist monoclonal antibody 011. H )

[0341] SEQ ID NO: 139 is the light chain variable region (V) of the OX40 agonist monoclonal antibody 011. L )

[0342] SEQ ID NO: 140 is the heavy chain variable region (V) of the OX40 agonist monoclonal antibody 021. H )

[0343] SEQ ID NO: 141 is the light chain variable region (V) of the OX40 agonist monoclonal antibody 021. L )

[0344] SEQ ID NO: 142 is the heavy chain variable region (V) of the OX40 agonist monoclonal antibody 023. H )

[0345] SEQ ID NO: 143 is the light chain variable region (V) of the OX40 agonist monoclonal antibody 023. L )

[0346] SEQ ID NO: 144 is the heavy chain variable region (V H )

[0347] SEQ ID NO: 145 is the light chain variable region (V) of the OX40 agonist monoclonal antibody L )

[0348] SEQ ID NO: 146 is the heavy chain variable region (V H )

[0349] SEQ ID NO: 147 is the light chain variable region (V) of the OX40 agonist monoclonal antibody L )

[0350] SEQ ID NO: 148 is the heavy chain variable region (V H )

[0351] SEQ ID NO: 149 is the heavy chain variable region (V) of a humanized OX40 agonist monoclonal antibody. H )

[0352] SEQ ID NO: 150 is the light chain variable region (V) of a humanized OX40 agonist monoclonal antibody. L )

[0353] SEQ ID NO: 151 is the light chain variable region (V) of a humanized OX40 agonist monoclonal antibody. L )

[0354] SEQ ID NO: 152 is the heavy chain variable region (V H )

[0355] SEQ ID NO: 153 is the heavy chain variable region (V) of a humanized OX40 agonist monoclonal antibody. H )

[0356] SEQ ID NO: 154 is the light chain variable region (V) of a humanized OX40 agonist monoclonal antibody. L )

[0357] SEQ ID NO: 155 is the light chain variable region (V) of a humanized OX40 agonist monoclonal antibody. L )

[0358] SEQ ID NO: 156 is the heavy chain variable region (V H )

[0359] SEQ ID NO: 157 is the light chain variable region (V) of the OX40 agonist monoclonal antibody L )

[0360] SEQ ID NO: 158 is the heavy chain amino acid sequence of the PD-1 inhibitor nivolumab.

[0361] SEQ ID NO: 159 is the light chain amino acid sequence of the PD-1 inhibitor nivolumab.

[0362] SEQ ID NO: 160 represents the heavy chain variable region (V) of the PD-1 inhibitor nivolumab H ) amino acid sequence.

[0363] SEQ ID NO: 161 represents the light chain variable region (V) of the PD-1 inhibitor nivolumab L ) amino acid sequence.

[0364] SEQ ID NO: 162 is the heavy chain CDR1 amino acid sequence of the PD-1 inhibitor nivolumab.

[0365] SEQ ID NO: 163 is the heavy chain CDR2 amino acid sequence of the PD-1 inhibitor nivolumab.

[0366] SEQ ID NO: 164 is the heavy chain CDR3 amino acid sequence of the PD-1 inhibitor nivolumab.

[0367] SEQ ID NO: 165 is the light chain CDR1 amino acid sequence of the PD-1 inhibitor nivolumab.

[0368] SEQ ID NO: 166 is the light chain CDR2 amino acid sequence of the PD-1 inhibitor nivolumab.

[0369] SEQ ID NO: 167 is the light chain CDR3 amino acid sequence of the PD-1 inhibitor nivolumab.

[0370] SEQ ID NO: 168 is the heavy chain amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0371] SEQ ID NO: 169 is the light chain amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0372] SEQ ID NO: 170 represents the heavy chain variable region (V) of the PD-1 inhibitor pembrolizumab H ) amino acid sequence.

[0373] SEQ ID NO: 171 is the light chain variable region (V) of the PD-1 inhibitor pembrolizumab L ) amino acid sequence.

[0374] SEQ ID NO: 172 is the heavy chain CDR1 amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0375] SEQ ID NO: 173 is the heavy chain CDR2 amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0376] SEQ ID NO: 174 is the heavy chain CDR3 amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0377] SEQ ID NO: 175 is the light chain CDR1 amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0378] SEQ ID NO: 176 is the light chain CDR2 amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0379] SEQ ID NO: 177 is the light chain CDR3 amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0380] SEQ ID NO: 178 is the heavy chain amino acid sequence of the PD-L1 inhibitor durvalumab.

[0381] SEQ ID NO: 179 is the light chain amino acid sequence of the PD-L1 inhibitor durvalumab.

[0382] SEQ ID NO: 180 represents the heavy chain variable region (V) of the PD-L1 inhibitor durvalumab H ) amino acid sequence.

[0383] SEQ ID NO: 181 represents the light chain variable region (V) of the PD-L1 inhibitor durvalumab L ) amino acid sequence.

[0384] SEQ ID NO: 182 is the heavy chain CDR1 amino acid sequence of the PD-L1 inhibitor durvalumab.

[0385] SEQ ID NO: 183 is the heavy chain CDR2 amino acid sequence of the PD-L1 inhibitor durvalumab.

[0386] SEQ ID NO: 184 is the heavy chain CDR3 amino acid sequence of the PD-L1 inhibitor durvalumab.

[0387] SEQ ID NO: 185 is the light chain CDR1 amino acid sequence of the PD-L1 inhibitor durvalumab.

[0388] SEQ ID NO: 186 is the light chain CDR2 amino acid sequence of the PD-L1 inhibitor durvalumab.

[0389] SEQ ID NO: 187 is the light chain CDR3 amino acid sequence of the PD-L1 inhibitor durvalumab.

[0390] SEQ ID NO: 188 is the heavy chain amino acid sequence of the PD-L1 inhibitor avelumab.

[0391] SEQ ID NO: 189 is the light chain amino acid sequence of the PD-L1 inhibitor avelumab.

[0392] SEQ ID NO: 190 represents the heavy chain variable region (V) of the PD-L1 inhibitor avelumab H ) amino acid sequence.

[0393] SEQ ID NO: 191 is the light chain variable region (V) of the PD-L1 inhibitor avelumab L ) amino acid sequence.

[0394] SEQ ID NO: 192 is the heavy chain CDR1 amino acid sequence of the PD-L1 inhibitor avelumab.

[0395] SEQ ID NO: 193 is the heavy chain CDR2 amino acid sequence of the PD-L1 inhibitor avelumab.

[0396] SEQ ID NO: 194 is the heavy chain CDR3 amino acid sequence of the PD-L1 inhibitor avelumab.

[0397] SEQ ID NO: 195 is the light chain CDR1 amino acid sequence of the PD-L1 inhibitor avelumab.

[0398] SEQ ID NO: 196 is the light chain CDR2 amino acid sequence of the PD-L1 inhibitor avelumab.

[0399] SEQ ID NO: 197 is the light chain CDR3 amino acid sequence of the PD-L1 inhibitor avelumab.

[0400] SEQ ID NO: 198 is the heavy chain amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0401] SEQ ID NO: 199 is the light chain amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0402] SEQ ID NO: 200 represents the heavy chain variable region (V) of the PD-L1 inhibitor atezolizumab H ) amino acid sequence.

[0403] SEQ ID NO: 201 is the light chain variable region (V) of the PD-L1 inhibitor atezolizumab L ) amino acid sequence.

[0404] SEQ ID NO: 202 is the heavy chain CDR1 amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0405] SEQ ID NO: 203 is the heavy chain CDR2 amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0406] SEQ ID NO: 204 is the heavy chain CDR3 amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0407] SEQ ID NO: 205 is the light chain CDR1 amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0408] SEQ ID NO: 206 is the light chain CDR2 amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0409] SEQ ID NO: 207 is the light chain CDR3 amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0410] SEQ ID NO: 208 is the heavy chain amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0411] SEQ ID NO: 209 is the light chain amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0412] SEQ ID NO: 210 represents the heavy chain variable region (V) of the CTLA-4 inhibitor ipilimumab H ) amino acid sequence.

[0413] SEQ ID NO: 211 represents the light chain variable region (V) of the CTLA-4 inhibitor ipilimumab L ) amino acid sequence.

[0414] SEQ ID NO: 212 is the heavy chain CDR1 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0415] SEQ ID NO: 213 is the heavy chain CDR2 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0416] SEQ ID NO: 214 is the heavy chain CDR3 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0417] SEQ ID NO: 215 is the light chain CDR1 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0418] SEQ ID NO: 216 is the light chain CDR2 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0419] SEQ ID NO: 217 is the light chain CDR3 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0420] SEQ ID NO: 218 is the heavy chain amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0421] SEQ ID NO: 219 is the light chain amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0422] SEQ ID NO: 220 represents the heavy chain variable region (V) of the CTLA-4 inhibitor tremelimumab H ) amino acid sequence.

[0423] SEQ ID NO: 221 represents the light chain variable region (V) of the CTLA-4 inhibitor tremelimumab L ) amino acid sequence.

[0424] SEQ ID NO: 222 is the heavy chain CDR1 amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0425] SEQ ID NO: 223 is the heavy chain CDR2 amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0426] SEQ ID NO: 224 is the heavy chain CDR3 amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0427] SEQ ID NO: 225 is the light chain CDR1 amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0428] SEQ ID NO: 226 is the light chain CDR2 amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0429] SEQ ID NO: 227 is the light chain CDR3 amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0430] SEQ ID NO: 228 is the heavy chain amino acid sequence of the CTLA-4 inhibitor zalifrelimab.

[0431] SEQ ID NO: 229 is the light chain amino acid sequence of the CTLA-4 inhibitor zalifrelimab.

[0432] SEQ ID NO: 230 is the heavy chain variable region (V) of the CTLA-4 inhibitor zalifrelimab H ) amino acid sequence.

[0433] SEQ ID NO: 231 is the light chain variable region (V) of the CTLA-4 inhibitor zalifrelimab L ) amino acid sequence.

[0434] SEQ ID NO: 232 is the heavy chain CDR1 amino acid sequence of the CTLA-4 inhibitor zalifrelimab.

[0435] SEQ ID NO: 233 is the heavy chain CDR2 amino acid sequence of the CTLA-4 inhibitor zalifrelimab.

[0436] SEQ ID NO: 234 is the heavy chain CDR3 amino acid sequence of the CTLA-4 inhibitor zalifrelimab.

[0437] SEQ ID NO: 235 is the light chain CDR1 amino acid sequence of the CTLA-4 inhibitor zalifrelimab.

[0438] SEQ ID NO: 236 is the light chain CDR2 amino acid sequence of the CTLA-4 inhibitor zalifrelimab.

[0439] SEQ ID NO: 237 is the light chain CDR3 amino acid sequence of the CTLA-4 inhibitor zalifrelimab.

[0440] I. Definition 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 referenced herein are incorporated by reference in their entirety.

[0441] As used herein, the terms "co-administration," "co-administering," "administered in combination," "administering in combination," "simultaneous," and "concurrent" encompass administration of two or more active pharmaceutical ingredients (e.g., multiple TILs in preferred embodiments of the present invention) to a subject such that both active pharmaceutical ingredients and / or their metabolites are present in the subject at the same time. Simultaneous administration includes simultaneous administration in separate compositions, administration at different times in separate compositions, or administration in a composition in which two or more active pharmaceutical ingredients are present. Concurrent administration in separate compositions and administration in a composition in which both agents are present are preferred.

[0442] The term "in vivo" refers to events that take place inside a subject's body.

[0443] The term "in vitro" refers to events that occur outside a subject's body. In vitro assays include cell-based assays, in which living or dead cells are used, and can also include cell-free assays, in which no intact cells are used.

[0444] The term "ex vivo" refers to events involving the administration of a therapy or treatment to cells, tissues, and / or organs that have been removed from a subject's body. Suitably, the cells, tissues, and / or organs may be returned to the subject's body in a surgical or therapeutic manner.

[0445] The term "rapid expansion" refers to an increase in the number of antigen-specific TILs by at least about 3-fold (or 4, 5, 6, 7, 8, or 9-fold) over a one-week period, more preferably by at least about 10-fold (or 20, 30, 40, 50, 60, 70, 80, or 90-fold) over a one-week period, or most preferably by at least about 100-fold over a one-week period. Several rapid expansion protocols are described herein.

[0446] As used herein, "tumor infiltrating lymphocytes" or "TILs" refers to a population of cells that are initially acquired as leukocytes that have left the bloodstream of a subject and migrated to a tumor. TILs include CD8 + Cytotoxic T cells (lymphocytes), Th1 and Th17 CD4 + These include, but are not limited to, T cells, natural killer cells, dendritic cells, and M1 macrophages. TILs include both primary and secondary TILs. "Primary TILs" are those obtained from a patient tissue sample as outlined herein (sometimes referred to as "freshly harvested"), and "secondary TILs" are any expanded or propagated TIL cell populations discussed herein, including, but not limited to, bulk TILs and expanded TILs ("REP TILs" or "post-REP TILs"). TIL cell populations may include genetically modified TILs.

[0447] As used herein, a "population of cells" (including TILs) refers to several cells that share a common trait. Generally, a population is generally on the order of 1 x 10 6 ~1×10 10 The number of TILs ranges from approximately 1 x 10 to 1 x 10, with different TIL populations containing different numbers. For example, the initial growth of primary TILs in the presence of IL-2 is approximately 1 x 10 8 REP expansion typically yields a bulk TIL population of 1.5 x 10 cells for injection. 9 ~1.5×10 10 This is done to provide a population of cells.

[0448] As used herein, "cryopreserved TILs" refers to TILs, either primary, bulk, or expanded (REP TILs), that are processed and stored at temperatures ranging from approximately -150°C to -60°C. General methods for cryopreservation are described elsewhere herein, including in the Examples. For clarity, "cryopreserved TILs" can be distinguished from frozen tissue samples that may be used as a source of primary TILs.

[0449] As used herein, "thawed cryopreserved TILs" refers to a population of TILs that have been previously 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.

[0450] TILs can generally be defined either biochemically using cell surface markers or functionally by their ability to infiltrate tumors and achieve therapy. TILs can generally be classified by expression of one or more of the following biomarkers: CD4, CD8, TCRαβ, CD27, CD28, CD56, CCR7, CD45Ra, CD95, PD-1, and CD25. Additionally and alternatively, TILs can be functionally defined by their ability to infiltrate solid tumors upon reintroduction into patients.

[0451] The term "cryopreservation media" or "cryopreservation medium" refers to any medium that can be used for cryopreserving cells. Such media can include media containing 7% to 10% DMSO. Exemplary media include CryoStor CS10, Hyperthermasol, and combinations thereof. The term "CS10" refers to cryopreservation media obtained from Stemcell Technologies or Biolife Solutions. CS10 medium may be referred to by the trade name "CryoStor® CS10." CS10 medium is a serum-free, animal-component-free medium that contains DMSO. In some embodiments, CS10 medium contains 10% DMSO.

[0452] The term "central memory T cells" refers to cells that are CD45R0+ and CCR7 (CCR7 高 ) and CD62L (CD62 高) is a subset of T cells that constitutively express the CD4 receptor. The surface phenotype of central memory T cells also includes TCR, CD3, CD127 (IL-7R), and IL-15R. Transcription factors of central memory T cells include BCL-6, BCL-6B, MBD2, and BMI1. After TCR triggering, central memory T cells primarily secrete IL-2 and CD40L as effector molecules. Central memory T cells predominate in the CD4 compartment in the blood and are proportionally enriched in lymph nodes and tonsils in humans.

[0453] The term "effector memory T cells" refers to T cells that are CD45R0+ like central memory T cells, but have lost constitutive expression of CCR7 (CCR7 低 ), heterogeneous or low CD62L expression (CD62L 低 ), refers to a subset of human or mammalian T cells. The surface phenotype of central memory T cells also includes TCR, CD3, CD127 (IL-7R), and IL-15R. Transcription factors of central memory T cells include BLIMP1. Effector memory T cells rapidly secrete high levels of inflammatory cytokines after antigen stimulation, including interferon gamma, IL-4, and IL-5. Effector memory T cells predominate in the CD8 compartment in the blood and are proportionally enriched in the lung, liver, and intestine in humans. CD8+ effector memory T cells carry large amounts of perforin.

[0454] The term "closed system" refers to a system that is closed to the external environment. Any closed system suitable for cell culture methods can be used in the methods of the present invention. Closed systems include, but are not limited to, sealed G containers. Once tumor segments are added to the closed system, the system is not opened to the external environment until the TILs are ready to be administered to a patient.

[0455] The terms "fragmenting," "fragments," and "fragmented" as used herein to describe processes for destroying tumors include mechanical fragmentation methods such as crushing, slicing, dividing, and mincing tumor tissue, as well as any other method for disrupting the physical structure of tumor tissue.

[0456] The terms "peripheral blood mononuclear cells" and "PBMCs" refer to peripheral blood cells with round nuclei, including lymphocytes (T cells, B cells, NK cells) and monocytes. When used as antigen-presenting cells (PBMCs are a type of antigen-presenting cell), the peripheral blood mononuclear cells are preferably irradiated allogeneic peripheral blood mononuclear cells.

[0457] The terms "peripheral blood lymphocytes" and "PBLs" refer to T cells expanded from peripheral blood. In some embodiments, PBLs are isolated from whole blood or apheresis products from a donor. In some embodiments, PBLs are isolated from whole blood or apheresis products from a donor by positive or negative selection of a T cell phenotype, such as a CD3+CD45+ T cell phenotype.

[0458] The term "anti-CD3 antibody" refers to an antibody or variant thereof, e.g., a monoclonal antibody, including a human, humanized, chimeric, or murine antibody, directed against the CD3 receptor in the T cell antigen receptor of mature T cells. Anti-CD3 antibodies include OKT-3, also known as muromonab. Anti-CD3 antibodies also include the UHCT1 clone, also known as T3 and CD3ε. Other anti-CD3 antibodies include, for example, otelixizumab, teplizumab, and visilizumab.

[0459] The term "OKT-3" (also referred to herein as "OKT3") refers to a monoclonal antibody, including a human, humanized, chimeric, or murine antibody against the CD3 receptor in the T cell antigen receptor of mature T cells, or a biosimilar or variant thereof, 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 shown in Table 1 (SEQ ID NO: 1 and SEQ ID NO: 2). A hybridoma capable of producing OKT-3 has been deposited with the American Type Culture Collection and assigned ATCC accession number CRL8001. A hybridoma capable of producing OKT-3 has also been deposited with the European Collection of Authenticated Cell Cultures (ECACC) and assigned catalog number 86022706. [Table 1]

[0460] 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. IL-2 is described, for example, in Nelson, J. Immunol. 2004, 172, 3983-88 and Malek, Annu. Rev. Immunol. 2008, 26, 453-79, the disclosures of which are incorporated herein by reference. The amino acid sequence of recombinant human IL-2 suitable for use in the present invention is shown in Table 2 (SEQ ID NO: 3). For example, the term IL-2 encompasses human recombinant IL-2 forms such as aldesleukin (PROLEUKIN, commercially available from multiple suppliers at 22 million IU per single-use vial), as well as the recombinant IL-2 form (catalog number CYT-209-b) marketed by CellGenix, Inc., Portsmouth, NH, USA (CELLGRO GMP) or ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA, and other commercial equivalents from other vendors. Aldesleukin (des-alanyl-1, serine-125 human IL-2) is a non-glycosylated human recombinant IL-2 form with a molecular weight of approximately 15 kDa. The amino acid sequence of aldesleukin suitable for use in the present invention is shown in Table 2 (SEQ ID NO: 4). The term IL-2 also refers to the pegylated IL-2 prodrug bempegaldesleukin (NKTR-214, an IL-2 prodrug in which an average of six lysine residues are replaced with [(2,7-bis{[methylpoly(oxyethylene)]carbamoyl}-9H-fluoren-9-yl)methoxy]carbonyl). 6The present invention also encompasses pegylated forms of IL-2 described herein, including pegylated human recombinant IL-2 such as SEQ ID NO: 4, which is available from Nektar Therapeutics (South San Francisco, CA, USA) or can be prepared by methods known in the art, such as the method described in Example 19 of International Patent Application Publication No. WO2018 / 132496 A1 or Example 1 of U.S. Patent Application Publication No. US2019 / 0275133 A1, the disclosures of which are incorporated herein by reference. Benpegaldesleukin (NKTR-214) and other pegylated IL-2 molecules suitable for use in the present invention are described in U.S. Patent Application Publication No. US2014 / 0328791 A1 and International Patent Application Publication No. WO2012 / 065086 A1, 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.

[0461] In some embodiments, a suitable IL-2 form for use in the present invention is THOR-707, available from Synthorx, Inc. The preparation and properties of THOR-707 and additional alternative forms of IL-2 suitable for use in the present invention are described in U.S. Patent Application Publication Nos. US2020 / 0181220 A1 and US2020 / 0330601 A1, the disclosures of which are incorporated herein by reference. In some embodiments, a suitable IL-2 form for use in the present invention is an interleukin-2 (IL-2) complex comprising an isolated and purified IL-2 polypeptide and a conjugation moiety that binds to the isolated and purified IL-2 polypeptide at an amino acid position selected from K35, T37, R38, T41, F42, K43, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107, wherein the numbering of the amino acid residues corresponds to SEQ ID NO:5. In some embodiments, the amino acid position is selected from T37, R38, T41, F42, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107. In some embodiments, the amino acid position is selected from T37, R38, T41, F42, F44, Y45, E61, E62, E68, P65, V69, L72, and Y107. In some embodiments, the amino acid position is selected from T37, T41, F42, F44, Y45, P65, V69, L72, and Y107. In some embodiments, the amino acid position is selected from R38 and K64. In some embodiments, the amino acid position is selected from E61, E62, and E68. In some embodiments, the amino acid position is E62. In some embodiments, an amino acid residue selected from K35, T37, R38, T41, F42, K43, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107 is further mutated to lysine, cysteine, or histidine. In some embodiments, the amino acid residue is mutated to cysteine. In some embodiments, the amino acid residue is mutated to lysine. In some embodiments, K35, T37, R38, T41, F42, K43, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107 are ...and Y107 are further mutated to an unnatural amino acid. In some embodiments, the unnatural amino acid is selected from N6-azidoethoxy-L-lysine (AzK), N6-propargylethoxy-L-lysine (PraK), BCN-L-lysine, norbornene lysine, TCO-lysine, methyltetrazine lysine, allyloxycarbonyl lysine, 2-amino-8-oxononanoic acid, 2-amino-8-oxooctanoic acid, p-acetyl-L-phenylalanine, p-azidomethyl-L-phenylalanine (pAMF), p-iodo-L-phenylalanine, m-acetylphenylalanine, 2-amino-8-oxononanoic acid, p-propargyloxyphenylalanine, p-propargyl-phenylalanine, 3-methyl-phenylalanine, L-dopa, fluorinated phenylalanine, isopropyl and selenocysteine, or selenocysteine. In some embodiments, the IL-2 complex has a reduced affinity for the IL-2 receptor alpha (IL-2Rα) subunit compared to a wild-type IL-2 polypeptide. In some embodiments, the reduced affinity is about a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or greater than a 99% reduction in binding affinity for IL-2Rα compared to a wild-type IL-2 polypeptide. In some embodiments, the reduced affinity is about a 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 30-fold, 50-fold, 100-fold, 200-fold, 300-fold, 500-fold, or greater reduction in binding affinity for IL-2Rα compared to a wild-type IL-2 polypeptide.1000-fold or more. In some embodiments, the conjugated moiety impairs or blocks the binding of IL-2 to IL-2Rα. In some embodiments, the conjugated moiety comprises a water-soluble polymer. In some embodiments, the additional conjugated moiety comprises a water-soluble polymer. In some embodiments, each of the water-soluble polymers independently comprises polyethylene glycol (PEG), poly(propylene glycol) (PPG), copolymers of ethylene glycol and propylene glycol, poly(oxyethylated polyol), poly(olefinic alcohol), poly(vinylpyrrolidone), poly(hydroxyalkyl methacrylamide), poly(hydroxyalkyl methacrylate), poly(saccharide), poly(α-hydroxy acid), poly(vinyl alcohol), polyphosphazene, polyoxazoline (POZ), poly(N-acryloylmorpholine), or a combination thereof. In some embodiments, each of the water-soluble polymers independently comprises PEG. In some embodiments, the PEG is linear PEG or branched PEG. In some embodiments, each of the water-soluble polymers independently comprises a polysaccharide. In some embodiments, the polysaccharide comprises dextran, polysialic acid (PSA), hyaluronic acid (HA), amylose, heparin, heparan sulfate (HS), dextrin, or hydroxyethyl starch (HES). In some embodiments, each of the water soluble polymers independently comprises a glycan. In some embodiments, each of the water soluble polymers independently comprises a polyamine. In some embodiments, the conjugation moiety comprises a protein. In some embodiments, the additional conjugation moieties comprise a protein. In some embodiments, each of the proteins independently comprises albumin, transferrin, or transthyretin. In some embodiments, each of the proteins independently comprises an Fc portion. In some embodiments, each of the proteins independently comprises an Fc portion of an IgG. In some embodiments, the conjugation moiety comprises a polypeptide. In some embodiments, the additional conjugation moieties comprise a polypeptide. In some embodiments, each of the proteins independently comprises an XTEN peptide, a glycine-rich homoamino acid polymer (HAP),The conjugated moiety may comprise a PAS polypeptide, an elastin-like polypeptide (ELP), a CTP peptide, or a gelatin-like protein (GLK) polymer. In some embodiments, the isolated and purified IL-2 polypeptide is modified by glutamylation. In some embodiments, the conjugated moiety is directly attached to the isolated and purified IL-2 polypeptide. In some embodiments, the conjugated moiety is indirectly attached to the isolated and purified IL-2 polypeptide via a linker. In some embodiments, the linker comprises a homobifunctional linker. In some embodiments, the homobifunctional linker is selected from the group consisting of the Romant reagents dithiobis(succinimidyl propionate) DSP, 3'3'-dithiobis(sulfosuccinimidyl propionate) (DTSSP), disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl) suberate (BS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfoDS T), ethylene glycobis(succinimidyl succinate) (EGS), disuccinimidyl glutarate (DSG), N,N'-disuccinimidyl carbonate (DSC), dimethyl adipimidate (DMA), dimethyl pimelimidate (DMP), dimethyl suberimidate (DMS), dimethyl-3,3'-dithiobispropionimidate (DTBP), 1,4-di-(3'-(2'-pyridyldithio) (e) propionamido) butane (DPDPB), bismaleimidohexane (BMH), halogenated aryl-containing compounds (DFDNB), such as 1,5-difluoro-2,4-dinitrobenzene or 1,3-difluoro-4,6-dinitrobenzene, 4,4'-difluoro-3,3'-dinitrophenyl sulfone (DFDNPS), bis-[β-(4-azidosalicylamido)ethyl] disulfide (BASED), formaldehyde, glutaraldehyde, 1,4-butanediol diglycidyl ether, adipic acid dihydrazide, carbohydrazide, o-toluidine, 3,3'-dimethylbenzidine, benzidine, α,α'-p-diaminodiphenyl, diiodo-p-xylene sulfonic acid, N,N'-ethylene-bis(iodoacetamide), or N,N'-hexamethylenebis(iodoacetamide). In some embodiments, the linker isHeterobifunctional linkers include, in some embodiments, N-succinimidyl 3-(2-pyridyldithio)propionate (sPDP), long-chain N-succinimidyl 3-(2-pyridyldithio)propionate (LC-sPDP), water-soluble long-chain N-succinimidyl 3-(2-pyridyldithio)propionate (sulfo-LC-sPDP), succinimidyloxycarbonyl-α-methyl-α-(2-pyridyldithio)toluene (sMPT), sulfosuccinimidyl-6-[α-methyl-α-(2 -pyridyldithio)toluamide]hexanoate (sulfo-LC-sMPT), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sMCC), sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-sMCC), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MB), m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (sulfo-MB), N-succinimide succinimidyl (4-iodoacetyl) aminobenzoate (sIAB), sulfosuccinimidyl (4-iodoacetyl) aminobenzoate (sulfo-sIAB), succinimidyl-4-(p-maleimidophenyl) butyrate (sMPB), sulfosuccinimidyl-4-(p-maleimidophenyl) butyrate (sulfo-sMPB), N-(γ-maleimidobutyryloxy) succinimide ester (GMB), N-(γ-maleimidobutyryloxy) sulfosuccinimide ester (sulfo-GM B), succinimidyl 6-((iodoacetyl)amino)hexanoate (sIAX), succinimidyl 6-[6-(((iodoacetyl)amino)hexanoyl)amino]hexanoate (slAXX), succinimidyl 4-(((iodoacetyl)amino)methyl)cyclohexane-1-carboxylate (sIAC), succinimidyl 6-(((((4-iodoacetyl)amino)methyl)cyclohexane-1-carbonyl)amino)hexanoate (sIACX), p-nitrophenyl iodoacetate (NPIA), carbonyl-reactive and sulfhydryl-reactive crosslinkers, such as 4-(4-N-maleimidophenyl)butyric acid hydrazide (MPBH),4-(N-maleimidomethyl)cyclohexane-1-carboxyl-hydrazide-8 (M2C2H), 3-(2-pyridyldithio)propionylhydrazide (PDPH), N-hydroxysuccinimidyl-4-azidosalicylate (NHs-AsA), N-hydroxysulfosuccinimidyl-4-azidosalicylate (sulfo-NHs-AsA), sulfosuccinimidyl, -(4-Azidosalicylamido)hexanoate (sulfo-NHs-LC-AsA), sulfosuccinimidyl-2-(p-azidosalicylamido)ethyl-1,3′-dithiopropionate (sAsD), N-hydroxysuccinimidyl-4-azidobenzoate (HsAB), N-hydroxysulfosuccinimidyl-4-azidobenzoate (sulfo-HsAB), N-succinimidyl-6-(4′-azido-2′-nitrophenylamino)hexanoate (sANPAH), sulfosuccinimidyl-6-(4′-azido-2′-nitrophenylamino)hexanoate (sulfo-sANPAH), N-5-azido-2-nitrobenzoyloxysuccinimide (ANB-NO), sulfosuccinimidyl-6-(4′-azido-2′-nitrophenylamino)hexanoate (sulfo- ... sulfosuccinimidyl-6-(4′-azido-2′-nitrophenylamino)hexanoate (sulfo-sANPAH), sulfosuccinimidyl-6-(4′-azido-2′-nitrophenylamino)hexanoate (sulfo-sANPAH), sulfosuccinimidyl-6-(4′-azido-2′-nitrophenylamino)hexanoate (sulfo-sANPAH), sulfosuccinimidyl-6-(4′-azido-2′-nitrophenylamino)hexanoate (sulfo Fosuccinimidyl-2-(m-azido-o-nitrobenzamido)-ethyl-1,3′-dithiopropionate (sAND), N-succinimidyl-4(4-azidophenyl)-1,3′-dithiopropionate (sADP), N-sulfosuccinimidyl (4-azidophenyl)-1,3′-dithiopropionate (sulfo-sADP), sulfosuccinimidyl 4-(ρ-azidophenyl)butyrate (sulfo-sAPB), sulfosuccinimidyl 2-(7-azido-4-methylcoumarin-3-acetamido)ethyl-1,3′-dithiopropionate (sAED), sulfosuccinimidyl 7-azido-4-methylcoumarin-3-acetate (sulfo-sAMCA), p-nitrophenyl Examples of suitable linkers include diazopyruvate (pNPDP), p-nitrophenyl-2-diazo-3,3,3-trifluoropropionate (PNP-DTP), 1-(ρ-azidosalicylamido)-4-(iodoacetamido)butane (AsIB), N-[4-(ρ-azidosalicylamido)butyl]-3′-(2′-pyridyldithio)propionamide (APDP), benzophenone-4-iodoacetamide, p-azidobenzoyl hydrazide (ABH), 4-(ρ-azidosalicylamido)butylamine (AsBA), or p-azidophenylglyoxal (APG). In some embodiments, the linker optionally comprises a non-cleavable linker, including a dipeptide linker. In some embodiments, the dipeptide linker comprises Val-Cit, Phe-Lys, Val-Ala, or Val-Lys.In some embodiments, the linker comprises a non-cleavable linker. In some embodiments, the linker optionally comprises a maleimide group, including maleimidocaproyl (mc), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sMCC), or sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-sMCC). In some embodiments, the linker further comprises a spacer. In some embodiments, the spacer comprises p-aminobenzyl alcohol (PAB), p-aminobenzyloxycarbonyl (PABC), a derivative, or an analog thereof. In some embodiments, the conjugated moiety is capable of extending the serum half-life of the IL-2 conjugate. In some embodiments, the additional conjugated moiety is capable of extending the serum half-life of the IL-2 conjugate. In some embodiments, the IL-2 form suitable for use in the present invention is a fragment of any of the IL-2 forms described herein. In some embodiments, IL-2 forms suitable for use in the present invention are pegylated as disclosed in U.S. Patent Application Publication Nos. US2020 / 0181220 A1 and US2020 / 0330601 A1. In some embodiments, IL-2 forms suitable for use in the present invention are IL-2 conjugates comprising an IL-2 polypeptide comprising N6-azidoethoxy-L-lysine (AzK) covalently attached to a conjugation moiety comprising polyethylene glycol (PEG), wherein the IL-2 polypeptide comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO:5, and AzK substitutes an amino acid at position K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69, or L72 relative to amino acid position within SEQ ID NO:5. In some embodiments, the IL-2 polypeptide comprises a one-residue N-terminal deletion relative to SEQ ID NO:5. In some embodiments, forms of IL-2 suitable for use in the present invention lack IL-2R alpha chain association but retain normal binding to the intermediate affinity IL-2R beta-gamma signaling complex.In some embodiments, a suitable IL-2 form for use in the present invention is an IL-2 conjugate comprising an IL-2 polypeptide comprising N6-azidoethoxy-L-lysine (AzK) covalently attached to a conjugation moiety comprising polyethylene glycol (PEG), wherein the IL-2 polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:5, and wherein AzK substitutes an amino acid at position K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69, or L72 relative to amino acid position within SEQ ID NO:5. In some embodiments, a suitable IL-2 form for use in the present invention is an IL-2 conjugate comprising an IL-2 polypeptide comprising N6-azidoethoxy-L-lysine (AzK) covalently attached to a conjugation moiety comprising polyethylene glycol (PEG), wherein the IL-2 polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:5, and wherein AzK substitutes an amino acid at position K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69, or L72 relative to amino acid position within SEQ ID NO:5. In some embodiments, a suitable IL-2 form for use in the present invention is an IL-2 conjugate comprising an IL-2 polypeptide comprising N6-azidoethoxy-L-lysine (AzK) covalently attached to a conjugation moiety comprising polyethylene glycol (PEG), wherein the IL-2 polypeptide comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO:5, and wherein AzK substitutes an amino acid at position K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69, or L72 relative to amino acid position in SEQ ID NO:5.

[0462] In some embodiments, a form of IL-2 suitable for use in the present invention is nembareukin alfa, also known as ALKS-4230 (SEQ ID NO: 6), available from Alkermes, Inc. Nembareukin alfa is a nucleotide analogue of IL-2 that is linked via a peptidyl linker ( 60 GG 61 ) and fused to human interleukin-2 fragment (62-132) via a peptidyl linker ( 133 GSGGGS 138Human interleukin-2 receptor α-chain fragment (139-303) fused via a nucleotide sequence (Cys), produced in Chinese hamster ovary (CHO) cells, and glycosylated. 125 >Ser 51 ; human interleukin-2 (IL-2) (4-74)-peptide (62-132) fused via a G2 peptide linker (60-61) and human interleukin-2 receptor alpha chain (IL2R subunit alpha, IL2Rα, IL2RA) (1-165)-peptide (139-303) fused via a GSG3S peptide linker (133-138), produced in Chinese hamster ovary (CHO) cells, and alpha-glycosylated human interleukin-2 (IL-2) (75-133)-peptide [Cys 125(51)>Ser]-mutant (1-59). The amino acid sequence of nemvaleukin alfa is set forth in SEQ ID NO: 6. In some embodiments, nemvaleukin alfa exhibits the following post-translational modifications: disulfide bridges at the following positions: 31-116, 141-285, 184-242, 269-301, 166-197, or 166-199, 168-199, or 168-197 (using the numbering of SEQ ID NO: 6), and glycosylation sites at the following positions: N187, N206, T212, using the numbering of SEQ ID NO: 6. The preparation and properties of nemvaleukin alfa, as well as additional alternative forms of IL-2 suitable for use in the present invention, are described in U.S. Patent Application Publication No. US2021 / 0038684 A1 and U.S. Patent No. 10,183,979, the disclosures of which are incorporated herein by reference. In some embodiments, an IL-2 form suitable for use in the present invention is a protein having at least 80%, at least 90%, at least 95%, or at least 90% sequence identity to SEQ ID NO:6. In some embodiments, an IL-2 form suitable for use in the present invention has the amino acid sequence set forth in SEQ ID NO:6 or conservative amino acid substitutions thereof. In some embodiments, an IL-2 form suitable for use in the present invention is a fusion protein comprising amino acids 24-452 of SEQ ID NO:7, or a variant, fragment, or derivative thereof. In some embodiments, an IL-2 form suitable for use in the present invention is a fusion protein comprising an amino acid sequence having at least 80%, at least 90%, at least 95%, or at least 90% sequence identity to amino acids 24-452 of SEQ ID NO:7, or a variant, fragment, or derivative thereof. Other IL-2 forms suitable for use in the present invention are described in U.S. Pat. No. 10,183,979, the disclosure of which is incorporated herein by reference.Optionally, in some embodiments, a form of IL-2 suitable for use in the present invention is a fusion protein comprising a first fusion partner linked to a second fusion partner by a mucin domain polypeptide linker, wherein the first fusion partner is IL-1Rα or a protein having at least 98% amino acid sequence identity to IL-1Rα and having receptor antagonist activity for IL-Rα, the second fusion partner comprises all or a portion of an immunoglobulin comprising an Fc region, and the mucin domain polypeptide linker comprises SEQ ID NO:8 or an amino acid sequence having at least 90% sequence identity to SEQ ID NO:8, and wherein the half-life of the fusion protein is improved compared to the fusion of the first fusion partner with the second fusion partner in the absence of the mucin domain polypeptide linker. [Table 2-1] [Table 2-2]

[0463] In some embodiments, IL-2 forms suitable for use in the present invention comprise a heavy chain variable region (V) comprising complementarity determining regions HCDR1, HCDR2, and HCDR3. H ) and a light chain variable region (V L ) and V H or V L and an IL-2 molecule or a fragment thereof grafted onto the CDR of a heavy chain variable region (V) comprising complementarity determining regions HCDR1, HCDR2, and HCDR3. H ) and a light chain variable region (V L ) and V H or V Land an IL-2 molecule or fragment thereof grafted onto the CDRs of: H ) and a light chain variable region (V L ) and an IL-2 molecule or a fragment thereof grafted into the CDR of VH or VL, wherein the IL-2 molecule is a mutein, and the antibody cytokine graft protein preferentially expands T effector cells over regulatory T cells, and the antibody further comprises an IgG class heavy chain and an IgG class light chain selected from the group consisting of an IgG class light chain comprising SEQ ID NO: 39 and an IgG class heavy chain comprising SEQ ID NO: 38, an IgG class light chain comprising SEQ ID NO: 37 and an IgG class heavy chain comprising SEQ ID NO: 29, an IgG class light chain comprising SEQ ID NO: 39 and an IgG class heavy chain comprising SEQ ID NO: 29, an IgG class light chain comprising SEQ ID NO: 37 and an IgG class heavy chain comprising SEQ ID NO: 38.

[0464] In some embodiments, the IL-2 molecule or fragment thereof is V H In some embodiments, the IL-2 molecule or fragment thereof is a mutein. H In some embodiments, the IL-2 molecule or fragment thereof is a mutein. H In some embodiments, the IL-2 molecule or fragment thereof is a mutein. L In some embodiments, the IL-2 molecule or a fragment thereof is grafted onto LCDR1 of V LIn some embodiments, the IL-2 molecule or fragment thereof is grafted onto LCDR2 of V L The LCDR3 of the IL-2 molecule is grafted onto the LCDR3 of the IL-2 molecule, and the IL-2 molecule is a mutein.

[0465] The insertion of the IL-2 molecule can be at or near the N-terminal region of the CDR, the middle region of the CDR, or at or near the C-terminal region of the CDR. In some embodiments, the antibody cytokine graft protein comprises an IL-2 molecule incorporated into the CDR, and the IL2 sequence does not frameshift the CDR sequence. In some embodiments, the antibody cytokine graft protein comprises an IL-2 molecule incorporated into the CDR, and the IL-2 sequence replaces all or part of the CDR sequence. The replacement with the IL-2 molecule can be at or near the N-terminal region of the CDR, the middle region of the CDR, or the C-terminal region of the CDR. The replacement with the IL-2 molecule can be as little as one or two amino acids of the CDR sequence, or the entire CDR sequence.

[0466] In some embodiments, the IL-2 molecule is directly grafted onto the CDR without a peptide linker and without additional amino acids between the CDR sequence and the IL-2 sequence. In some embodiments, the IL-2 molecule is indirectly grafted onto the CDR using a peptide linker with one or more additional amino acids between the CDR sequence and the IL-2 sequence.

[0467] In some embodiments, the IL-2 molecule described herein is an IL-2 mutein. In some cases, the IL-2 mutein comprises an R67A substitution. In some embodiments, the IL-2 mutein comprises the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 15. In some embodiments, the IL-2 mutein comprises the amino acid sequence of Table 1 of U.S. Patent Application Publication No. US2020 / 0270334 A1, the disclosure of which is incorporated herein by reference.

[0468] In some embodiments, the antibody cytokine transplant protein comprises an HCDR1 selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22, and SEQ ID NO: 25. In some embodiments, the antibody cytokine transplant protein comprises an HCDR1 selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 13, and SEQ ID NO: 16. In some embodiments, the antibody cytokine transplant protein comprises an HCDR1 selected from the group consisting of an HCDR2 selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 20, SEQ ID NO: 23, and SEQ ID NO: 26. In some embodiments, the antibody cytokine transplant protein comprises an HCDR3 selected from the group consisting of SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO: 24, and SEQ ID NO: 27. In some embodiments, the antibody cytokine transplant protein comprises a V comprising the amino acid sequence of SEQ ID NO: 28. H In some embodiments, the antibody cytokine transplant protein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 29. In some embodiments, the antibody cytokine transplant protein comprises a V region comprising the amino acid sequence of SEQ ID NO: 36. L In some embodiments, the antibody cytokine transplant protein comprises a light chain comprising the amino acid sequence of SEQ ID NO: 37. In some embodiments, the antibody cytokine transplant protein comprises a V region comprising the amino acid sequence of SEQ ID NO: 28. H V comprising the region and the amino acid sequence of SEQ ID NO: 36 LIn some embodiments, the antibody cytokine transplant protein comprises a heavy chain region comprising the amino acid sequence of SEQ ID NO:29 and a light chain region comprising the amino acid sequence of SEQ ID NO:37. In some embodiments, the antibody cytokine transplant protein comprises a heavy chain region comprising the amino acid sequence of SEQ ID NO:29 and a light chain region comprising the amino acid sequence of SEQ ID NO:39. In some embodiments, the antibody cytokine transplant protein comprises a heavy chain region comprising the amino acid sequence of SEQ ID NO:38 and a light chain region comprising the amino acid sequence of SEQ ID NO:37. In some embodiments, the antibody cytokine transplant protein comprises a heavy chain region comprising the amino acid sequence of SEQ ID NO:38 and a light chain region comprising the amino acid sequence of SEQ ID NO:39. In some embodiments, the antibody cytokine transplant protein comprises IgG.IL2F71A.H1 or IgG.IL2R67A.H1 of U.S. Patent Application Publication No. 2020 / 0270334 A1, or a variant, derivative, or fragment thereof, or conservative amino acid substitutions thereof, or a protein having at least 80%, at least 90%, at least 95%, or at least 98% sequence identity thereto. In some embodiments, the antibody component of the antibody cytokine transplant proteins described herein comprises immunoglobulin sequences, framework sequences, or CDR sequences of palivizumab. In some embodiments, the antibody cytokine transplant proteins described herein have a longer serum half-life than a wild-type IL-2 molecule, such as, but not limited to, aldesleukin or an equivalent molecule. In some embodiments, the antibody cytokine transplant proteins described herein have a sequence set forth in Table 3. [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4]

[0469] The term "IL-4" (also referred to herein as "IL4") refers to the cytokine known as interleukin 4, which is produced by Th2 T cells, as well as eosinophils, basophils, and mast cells. IL-4 regulates the differentiation of naive helper T cells (Th0 cells) into Th2 T cells. Steinke and Borish, Respir. Res. 2001, 2, 66-70. Upon activation by IL-4, Th2 T cells subsequently produce additional IL-4 in a positive feedback loop. IL-4 also stimulates B cell proliferation and class II MHC expression, and induces class switching from B cells to IgE and IgG1 expression. Recombinant human IL-4 suitable for use in the present invention is commercially available from several suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (catalog number CYT-211) and ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-15 recombinant protein, catalog number Gibco CTP0043). The amino acid sequence of recombinant human IL-4 suitable for use in the present invention is shown in Table 2 (SEQ ID NO: 9).

[0470] The term "IL-7" (also referred to herein as "IL7") refers to a glycosylated tissue-derived cytokine known as interleukin-7, which can be obtained from stromal and epithelial cells, as well as dendritic cells. Fry and Mackall, Blood 2002, 99, 3892-904. IL-7 can stimulate T cell development. IL-7 binds to the IL-7 receptor, a heterodimer consisting of the IL-7 receptor alpha and the common gamma chain receptor, which is a series of signals important for T cell development in the thymus and survival in the periphery. Recombinant human IL-7 suitable for use in the present invention is commercially available from several suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (catalog number CYT-254) and ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-15 recombinant protein, catalog number Gibco PHC0071). The amino acid sequence of a recombinant human IL-7 suitable for use in the present invention is shown in Table 2 (SEQ ID NO: 10).

[0471] The term "IL-15" (also referred to herein as "IL15") refers to the T-cell growth factor known as interleukin-15 and includes all forms of IL-2, including human and mammalian forms, conservative amino acid substitutions, glycoforms, 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 suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (catalog number CYT-230-b) and ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-15 recombinant protein, catalog number 34-8159-82). The amino acid sequence of recombinant human IL-15 suitable for use in the present invention is shown in Table 2 (SEQ ID NO: 11).

[0472] 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. 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 primarily stimulates natural killer T cells and activated human CD4 +It is produced by T cells. Recombinant human IL-21 is a single, non-glycosylated polypeptide chain containing 132 amino acids with a molecular weight of 15.4 kDa. Recombinant human IL-21 is commercially available from several suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (catalog 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 shown in Table 2 (SEQ ID NO: 21).

[0473] When an "antitumor effective amount," "tumor inhibiting effective amount," or "therapeutic amount" is indicated, the exact amount of the composition of the present invention to be administered can be determined by a physician, taking into account individual differences in the age, weight, tumor size, extent of infection or metastasis, and health status of the patient (subject). Generally, tumor-infiltrating lymphocytes (e.g., secondary TILs or genetically modified cytotoxic lymphocytes) described herein are administered at a dose of 10 per kg of body weight. 4 ~10 11 cells (e.g., 10 per kg of body weight) 5 ~10 6 , 10 5 ~10 10 , 10 5 ~10 11 , 10 6 ~10 10 , 10 6 ~10 11 , 10 7 ~10 11 , 10 7 ~10 10 , 10 8 ~10 11 , 10 8 ~10 10 , 10 9 ~10 11 , or 10 9 ~10 10It may be stated that the TIL (optionally including genetically modified cytotoxic lymphocytes) compositions may be administered at doses of 1000-20 ...

[0474] The terms "hematological malignancies," "blood system malignancies," or terms of related meaning, refer to cancers and tumors of mammalian hematopoietic and lymphatic tissues, including, but not limited to, blood, bone marrow, lymph nodes, and lymphatic tissues. Hematological malignancies are also referred to as "liquid tumors." Hematological malignancies may include, but are not limited to, acute lymphoblastic leukemia (ALL), chronic lymphocytic lymphoma (CLL), small lymphocytic lymphoma (SLL), acute myeloid leukemia (AML), chronic myelogenous leukemia (CML), multiple myeloma, 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.

[0475] 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, as well as other hematological malignancies. TILs obtained from liquid tumors may also be referred to herein as bone marrow-infiltrating lymphocytes (MILs). TILs obtained from liquid tumors, including those circulating in peripheral blood, may also be referred to herein as PBLs. The terms MILs, TILs, and PBLs are used interchangeably herein and differ only based on the tissue type from which the cells are derived.

[0476] The term "microenvironment" as used herein may refer to the solid or hematological tumor microenvironment as a whole, or to individual subsets of cells within the microenvironment. As used herein, the tumor microenvironment refers to a complex mixture of "cells, soluble factors, signaling molecules, extracellular matrix, and mechanical cues that promote neoplastic transformation, support tumor growth and invasion, protect tumors from host immunity, foster therapeutic resistance, and provide a niche for successful and dominant metastasis," as described in Swartz, et al., Cancer Res., 2012, 72, 2473. Tumors express antigens that are recognized by T cells, but tumor clearance by the immune system is rare due to immunosuppression by the microenvironment.

[0477] In some embodiments, the invention includes methods of treating cancer with a TIL population, wherein the patient is pretreated with non-myeloablative chemotherapy prior to infusion of TILs according to the invention. In some embodiments, a TIL population may be provided, wherein the patient is pretreated with non-myeloablative chemotherapy prior to infusion of TILs according to the invention. In some embodiments, the non-myeloablative chemotherapy is cyclophosphamide 60 mg / kg / day for 2 days (27 and 26 days before TIL infusion) and fludarabine 25 mg / m2 / day for 5 days (27-23 days before TIL infusion). In some embodiments, after non-myeloablative chemotherapy and TIL infusion according to the invention (day 0), the patient receives an intravenous infusion of IL-2 at 720,000 IU / kg every 8 hours to physiological tolerance.

[0478] Experimental findings indicate that lymphodepletion prior to adoptive transfer of tumor-specific T lymphocytes plays an important role in enhancing therapeutic efficacy by eliminating regulatory T cells and competing elements of the immune system ("cytokine sinks"). Accordingly, some embodiments of the present invention utilize a lymphodepletion step (sometimes referred to as "immunosuppressive conditioning") on patients prior to introducing the TILs of the present invention.

[0479] The term "effective amount" or "therapeutically effective amount" refers to an amount of a compound or combination of compounds described herein sufficient to achieve the intended use, including, but not limited to, disease treatment. A therapeutically effective amount may vary depending on the intended use (in vitro or in vivo), the subject and condition being treated (e.g., the subject's weight, age, and sex), the severity of the condition, or the method of administration. The term also applies to a dose that induces 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 to be followed, whether the compound is administered in combination with other compounds, the timing of administration, the tissue to which it is administered, and the physical delivery system by which the compound is delivered.

[0480] The terms "treatment," "treating," "treating," and the like refer to obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic, in terms of completely or partially preventing the disease or condition, and / or therapeutic, in terms of partially or completely curing 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, and includes (a) preventing the disease from occurring in a subject who may be susceptible to the disease but has not yet been diagnosed with it; (b) suppressing the disease, i.e., arresting its onset or progression; and (c) palliating the disease, i.e., causing regression of the disease and / or alleviating one or more disease symptoms. "Treatment" is also intended to encompass the delivery of an agent to provide a pharmacological effect even in the absence of a disease or condition. For example, "treatment" encompasses the delivery of a composition capable of eliciting an immune response or conferring immunity in the absence of a pathology, e.g., in the case of a vaccine.

[0481] The term "heterologous," when used with 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 produced recombinantly, with two or more sequences from unrelated genes arranged to create a new functional nucleic acid, e.g., a promoter from one source and a coding region from another source, or coding regions from different sources. Similarly, a heterologous protein 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).

[0482] The terms "sequence identity," "percent identity," and "percent sequence identity" (or their synonyms, e.g., "99% identical") 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 specified percentage of the same nucleotide or amino acid residues when compared and aligned for maximum correspondence (introducing gaps, if necessary), 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 suite of programs available from the BLAST website of the U.S. government's National Center for Biotechnology Information. Comparison between two sequences can be performed using either the BLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acid sequences, and BLASTP is used to compare amino acid sequences. ALIGN, ALIGN-2 (Genentech, South San Francisco, California), or MegAlign, available from DNASTAR, are additional publicly available software programs that can be used to align sequences. Those skilled in the art can determine the appropriate parameters for maximum alignment depending on the particular alignment software. In certain embodiments, the default parameters of the alignment software are used.

[0483] As used herein, the term "variant" includes, but is not limited to, an antibody or fusion protein comprising 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.

[0484] As used herein, "tumor infiltrating lymphocytes" or "TILs" refers to a population of cells that are initially acquired as leukocytes that have left the bloodstream of a subject and migrated to a tumor. TILs include CD8 + Cytotoxic T cells (lymphocytes), Th1 and Th17 CD4 + These include, but are not limited to, T cells, natural killer cells, dendritic cells, and M1 macrophages. TILs include both primary and secondary TILs. "Primary TILs" are those obtained from a patient tissue sample as outlined herein (sometimes referred to as "freshly harvested"). "Secondary TILs" are any TIL cell populations that have been expanded or propagated as discussed herein, including, but not limited to, bulk TILs, expanded TILs ("REP TILs"), and "reREP TILs" as discussed herein. reREP TILs can include, for example, second expanded TILs or second additional expanded TILs (e.g., those described in step D of FIG. 8, including TILs designated as reREP TILs).

[0485] TILs can generally be defined either biochemically using cell surface markers or functionally by their ability to infiltrate tumors and achieve therapy. TILs can generally be classified by the expression of one or more of the following biomarkers: CD4, CD8, TCRαβ, CD27, CD28, CD56, CCR7, CD45Ra, CD95, PD-1, and CD25. Additionally or alternatively, TILs can be functionally defined by their ability to infiltrate solid tumors upon reintroduction into patients. TILs can be further characterized by efficacy; for example, TILs can be considered potent if their interferon (IFN) release is greater than about 50 pg / mL, greater than about 100 pg / mL, greater than about 150 pg / mL, or greater than about 200 pg / mL. For example, TILs can be considered potent if interferon (IFNγ) release is greater than about 50 pg / mL, greater than about 100 pg / mL, greater than about 150 pg / mL, or greater than about 200 pg / mL, greater than about 300 pg / mL, greater than about 400 pg / mL, greater than about 500 pg / mL, greater than about 600 pg / mL, greater than about 700 pg / mL, greater than about 800 pg / mL, greater than about 900 pg / mL, or greater than about 1000 pg / mL.

[0486] The term "deoxyribonucleotide" encompasses natural and synthetic, unmodified and modified deoxyribonucleotides. Modifications include changes to the sugar moiety, the base moiety, and / or the linkages between deoxyribonucleotides in an oligonucleotide.

[0487] The term "RNA" defines a molecule containing at least one ribonucleotide residue. The term "ribonucleotide" defines a nucleotide having a hydroxyl group at the 2' position of a bD-ribofuranose moiety. The term RNA includes double-stranded RNA, single-stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, and altered RNA that differs from naturally occurring RNA by the addition, deletion, substitution, and / or alteration of one or more nucleotides. The nucleotides of the RNA molecules described herein can also include non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs may be referred to as analogs or analogs of naturally occurring RNA.

[0488] The term "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 insofar as any conventional pharmaceutically acceptable carrier or pharmaceutically acceptable excipient is incompatible with the active pharmaceutical ingredient, its use in the therapeutic compositions of the invention is contemplated. Additional active pharmaceutical ingredients, such as other drugs, can also be incorporated into the compositions and methods described.

[0489] The terms "about" and "approximately" mean within a statistically significant range of values. Such a range may be within one order of magnitude of a given value or range, preferably within 50%, more preferably within 20%, even more preferably within 10%, and even more preferably within 5%. The acceptable deviation 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, as used herein, the terms "about" and "approximately" mean that dimensions, sizes, formulations, parameters, shapes, and other quantities and characteristics are not, and need not be, exact, but may be approximate and / or larger or smaller, as appropriate, reflecting tolerances, conversion factors, rounding, measurement errors, etc., and other factors known to those of ordinary skill in the art. In general, a dimension, size, formulation, parameter, shape, or other quantity or characteristic is "about" or "approximately" whether or not expressly stated as such. It should be noted that embodiments of widely different sizes, shapes, and dimensions may employ the described configurations.

[0490] When used in the appended claims, the transitional terms "comprising," "consisting essentially of," and "consisting of" define the claim in its original and amended form, in terms of whether additional unrecited claim elements or steps, if any, are excluded from the claim. 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 the latter case, also excludes impurities normally associated with the specified material(s). The term "consisting essentially of" limits the claim to the specified element, step, or material(s) and does not materially affect the basic and novel feature(s) of the claimed invention. All compositions, methods, and kits described herein embodying the present invention may, in alternative embodiments, be more specifically defined by any of the transitional terms "comprising," "consisting essentially of," and "consisting of."

[0491] The terms "antibody" and its plural "antibodies" refer to whole immunoglobulins and any antigen-binding fragment ("antigen-binding portion") or single chains thereof. "Antibody" also refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or antigen-binding portions thereof. Each heavy chain contains a heavy chain variable region (referred to herein as V H Each light chain is composed of a light chain variable region (abbreviated herein as V) and a heavy chain constant region. The heavy chain constant region is composed of three domains: CH1, CH2, and CH3. L The light chain constant region consists of one domain, C L The V of the antibody H and V L The regions can be further subdivided into regions of hypervariability, termed complementarity-determining regions (CDRs) or hypervariable regions (HVRs), which may be interspersed with more conserved regions called framework regions (FRs). H and V Lis composed of three CDRs and four FRs arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with one or more antigen epitopes. The constant region of the antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0492] 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. As used herein, the term "antigen" also encompasses T cell epitopes. An antigen can additionally be recognized by the immune system. In some embodiments, an antigen can induce a humoral or cellular immune response, leading to the activation of B and / or T lymphocytes. In some cases, this may require that the antigen contain or be bound by a Th cell epitope. An antigen may also have one or more epitopes (e.g., B- and T-epitopes). In some embodiments, an antigen preferably reacts with a corresponding antibody or TCR, typically in a highly specific and selective manner, and not with many other antibodies or TCRs that may be induced by other antigens.

[0493] The terms "monoclonal antibody," "mAb," "monoclonal antibody composition," or their plurals, refer to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition exhibits a single binding specificity and affinity for a particular epitope. Monoclonal antibodies specific for a particular receptor can be produced using knowledge and techniques in the art by injecting a test subject with an appropriate antigen and then isolating hybridomas expressing antibodies with the desired sequence or functional characteristics. DNA encoding the monoclonal antibody is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to genes encoding the heavy and light chains of the monoclonal antibody). Hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA can be placed into an expression vector and then transfected into host cells such as E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not produce immunoglobulin protein to obtain the synthesis of the monoclonal antibody in the recombinant host cells. Recombinant production of antibodies is described in more detail below.

[0494] As used herein, the term "antigen-binding portion" or "antigen-binding fragment" of an antibody (or simply "antibody portion" or "fragment") refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed by the term "antigen-binding portion" of an antibody include: (i) V L , V H , C L (ii) a Fab fragment, which is a monovalent fragment consisting of two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a V H and an Fd fragment consisting of the CH1 domain, (iv) a V of a single arm of an antibody L and V H Fv fragment consisting of domains, (v) V H or VL and (v) isolated complementarity-determining regions (CDRs). L and V H are encoded by separate genes, they can be synthesized using recombinant methods L and V H The domains may be joined by a synthetic linker that allows them to pair as a single protein chain to form a monovalent molecule known as a single-chain Fv (scFv; see, e.g., Bird, et al., Science 1988, 242, 423-426, and Huston, et al., Proc. Natl. Acad. Sci. USA 1988, 85, 5879-5883). Such scFv antibodies are also intended to be encompassed by the term "antigen-binding portion" or "antigen-binding fragment" of an antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as intact antibodies. In some embodiments, the scFv protein domain comprises a V H Part and V L The scFv molecule contains a V L If the domain is the N-terminal portion of the scFv molecule, V L -LV H , or V H If the domain is the N-terminal portion of the scFv molecule, V H -LV L Methods for producing scFv molecules and designing suitable peptide linkers are described in U.S. Pat. Nos. 4,704,692, 4,946,778, R. Raag and M. Whitlow, "Single Chain Fvs," FASEB Vol. 9:73-80 (1995), and RE Bird and BW Walker, Single Chain Antibody Variable Regions, TIBTECH, Vol. 9:132-137 (1991), the disclosures of which are incorporated herein by reference.

[0495] As used herein, the term "human antibody" is intended to include antibodies having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region also is derived from human germline immunoglobulin sequences. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). As used herein, the term "human antibody" is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0496] The term "human monoclonal antibody" refers to antibodies displaying a single binding specificity which have variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. In some embodiments, human monoclonal antibodies are produced by hybridomas comprising B cells obtained from a transgenic non-human animal, e.g., a transgenic mouse, whose genome comprises human heavy chain and light chain transgenes fused to an immortalized cell.

[0497] As used herein, the term "recombinant human antibody" includes all human antibodies prepared, expressed, created, or isolated by recombinant means, such as (a) antibodies isolated from animals (such as mice) that are transgenic or transchromosomal for human immunoglobulin genes or hybridomas prepared therefrom (described further below); (b) antibodies isolated from host cells transformed to express human antibodies, e.g., from transfectomas; (c) antibodies isolated from recombinant combinatorial human antibody libraries; and (d) antibodies prepared, expressed, created, or isolated by any other means, including splicing human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable regions in which the framework and CDR regions are derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies may be subjected to in vitro mutagenesis (or, when animals transgenic for human Ig sequences are used, in vivo somatic mutagenesis) to thereby modify the V sequences of the recombinant antibodies. H and V L The amino acid sequence of the region is human germline V H and V L These are sequences that are derived from and related to sequences, but may not naturally occur within the human antibody germline repertoire in vivo.

[0498] As used herein, "isotype" refers to the antibody class (e.g., IgM or IgG1) that is encoded by heavy chain constant region genes.

[0499] The phrases "an antibody that recognizes an antigen" and "an antibody that is specific for an antigen" are used interchangeably herein with the term "an antibody that specifically binds to an antigen."

[0500] The term "human antibody derivative" refers to any modified form of a human antibody, including a conjugate of the antibody with another active pharmaceutical ingredient or antibody. The terms "conjugate," "antibody drug conjugate," "ADC," or "immunoconjugate" refer to an antibody or fragment thereof conjugated to another therapeutic moiety, which can be conjugated to the antibodies described herein using methods available in the art.

[0501] The terms "humanized antibody," "humanized antibodies," and "humanization" are intended to refer to antibodies in which CDR sequences derived from the germline of another mammalian species, such as mouse, have been grafted onto human framework sequences. Additional framework region modifications can be made within the human framework sequences. Humanized forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. In most cases, humanized antibodies are human immunoglobulins (recipient antibodies) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate having the desired specificity, affinity, and capacity. In some cases, Fv framework (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may contain residues that are not found in the recipient or donor antibody. These modifications are made to further refine antibody performance. Generally, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence. The humanized antibody will also optionally comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones, et al., Nature 1986, 321, 522-525; Riechmann, et al., Nature 1988, 332, 323-329; and Presta, Curr. Op. Struct. Biol. 1992, 2, 593-596. The antibodies described herein can also be modified to use any Fc variant known to confer improved (e.g., reduced) effector function and / or FcR binding.Fc variants are described in, for example, International Patent Application Publication Nos. WO1988 / 07089A1, WO1996 / 14339A1, WO1998 / 05787A1, WO1998 / 23289A1, WO1999 / 51642A1, WO99 / 58572A1, WO2000 / 09560A2, WO2000 / 32767A1, WO2000 / 42072A2, and WO2002 / 4 4215A2, WO2002 / 060919A2, WO2003 / 074569A2, WO2004 / 016750A2, WO2004 / 029207A2, WO2004 / 03 5752A2, WO2004 / 063351A2, WO2004 / 074455A2, WO2004 / 099249A2, WO2005 / 040217A2, WO2005 / 07 0963A1, WO2005 / 077981A2, WO2005 / 092925A2, WO2005 / 123780A2, WO2006 / 019447A1, WO2006 / 047350A2, and WO2006 / 085967A2, as well as U.S. Pat. Nos. 5,648,260, 5,739,277, 5,834,250, 5,869,046, 6,096 ,871, 6,121,022, 6,194,551, 6,242,195, 6,277,375, 6,528,624, 6,538,124, 6,737,056, 6,821,505, 6,998,253, and 7,083,784 (the disclosures of which are incorporated herein by reference).

[0502] The term "chimeric antibody" is intended to refer to an antibody in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, e.g., an antibody in which the variable region sequences are derived from a murine antibody and the constant region sequences are derived from a human antibody.

[0503] A "diabody" is a small antibody fragment that has two antigen-binding sites. The fragments bind to the same polypeptide chain (VH -V L or V L -V H ) in the light chain variable domain (V L ) connected to the heavy chain variable domain (V H ). When a linker that is too short to pair the two domains on the same chain is used, the domains are forced to pair with complementary domains on another chain and create two antigen-binding sites. Bispecific antibodies are more fully described, for example, in European Patent No. EP 404,097, International Patent Publication No. WO 93 / 11161, and Bolliger, et al., Proc. Natl. Acad. Sci. USA 1993, 90, 6444-6448.

[0504] The term "glycosylation" refers to modified derivatives of antibodies. An aglycosylated antibody lacks glycosylation. Glycosylation can be altered to, for example, increase the affinity of an antibody for an antigen. Such carbohydrate modifications can be achieved, for example, by altering one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions can be made that result in the elimination of one or more variable region framework glycosylation sites, thereby eliminating glycosylation at those sites. As described in U.S. Pat. Nos. 5,714,350 and 6,350,861, aglycosylation may increase the affinity of an antibody for an antigen. Additionally or alternatively, antibodies can be generated with altered types of glycosylation, such as hypofucosylated antibodies with reduced amounts of fucosyl residues or antibodies with increased bisecting GlcNac structures. Such altered glycosylation patterns have been demonstrated to increase antibody potency. Such carbohydrate modifications can be achieved, for example, by expressing the antibody in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells to express the recombinant antibodies of the present invention, thereby producing antibodies with altered glycosylation. For example, the cell lines Ms704, Ms705, and Ms709 lack the fucosyltransferase gene FUT8 (alpha(1,6) fucosyltransferase), such that antibodies expressed in these cell lines lack fucose on their carbohydrates. The Ms704, Ms705, and Ms709 FUT8- / - cell lines were generated by targeted disruption of the FUT8 gene in CHO / DG44 cells using two replacement vectors (see, e.g., U.S. Patent Publication No. 2004 / 0110704 or Yamane-Ohnuki, et al., Biotechnol. Bioeng., 2004, 87, 614-622).As another example, European Patent No. EP 1,176,195 describes cell lines with a functionally disrupted FUT8 gene encoding a fucosyltransferase, thereby resulting in antibodies expressed in such cell lines exhibiting hypofucosylation by reducing or eliminating alpha-1,6 bond-related enzymes. It also describes cell lines with reduced or no enzymatic activity for adding fucose to N-acetylglucosamine attached to the Fc region of antibodies, such as the rat myeloma cell line YB2 / 0 (ATCC CRL 1662). International Patent Publication No. WO 03 / 035835 describes a variant CHO cell line, Lec 13 cells, that has a reduced ability to attach fucose to Asn(297)-linked carbohydrates, further resulting in hypofucosylation of antibodies expressed in the host cells (see also Shields, et al., J. Biol. Chem. 2002, 277, 26733-26740). International Patent Publication No. WO 99 / 54342 describes cell lines engineered to express glycoprotein-modifying glycosyltransferases (e.g., beta(1,4)-N-acetylglucosaminyltransferase III (GnTIII)), such that antibodies expressed in the engineered cell lines exhibit increased bisecting GlcNac structures, resulting in increased ADCC activity of the antibodies (see also Umana, et al., Nat. Biotech. 1999, 17, 176-180). Alternatively, fucose residues of antibodies can be cleaved using a fucosidase enzyme. For example, the fucosidase alpha-L-fucosidase removes fucosyl residues from antibodies, as described in Tarentino, et al., Biochem. 1975, 14, 5516-5523.

[0505] "PEGylation" refers to a modified antibody or fragment thereof that has been reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions that result in one or more PEG groups being attached to the antibody or antibody fragment. PEGylation can, for example, increase the biological (e.g., serum) half-life of the antibody. 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 mono(C1-C2)-poly(ethylene glycol ... 10 PEG is intended to encompass any of the forms of PEG that have been used to derivatize other proteins, such as alkoxy- or aryloxy-polyethylene glycol, or polyethylene glycol-maleimide. The antibody to be pegylated may be an aglycosylated antibody. Methods for pegylation are known in the art and can be applied to the antibodies of the invention, for example, as described in European Patent Nos. EP 0 154 316 and EP 0 401 384, and U.S. Pat. No. 5,824,778 (the disclosures of each of which are incorporated herein by reference).

[0506] The term "biosimilar" refers to a biological product that is highly similar to a reference biological product approved in the United States, despite minor differences in clinically inactive components, including monoclonal antibodies or proteins, and that has no clinically meaningful differences between the biological product and the reference product in terms of product safety, purity, and potency. In Europe, a similar biological or "biosimilar" drug is a biological product 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. Biological products or biological products are medicines made by or derived from biological sources, such as bacteria or yeast. They can 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), a protein approved by drug regulatory authorities for aldesleukin is a "biosimilar" of aldesleukin or a "biosimilar of" aldesleukin. In Europe, a similar biological or "biosimilar" medicinal product is a biological product similar to another biological product already authorized for use by the European Medicines Agency (EMA). The legal basis for similar biological uses in Europe is Article 6 of Regulation (EC) No. 726 / 2004, as amended, and Article 10(4) of Directive 2001 / 83 / EC. Therefore, in Europe, biosimilars may be authorized or approved for authorization or licensing purposes under Article 6 of Regulation (EC) No. 726 / 2004 and Article 10(4) of Directive 2001 / 83 / EC. The original biological product already authorized is sometimes referred to as 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 biosimilar medicinal products. Additionally, product-specific guidelines, including those related to monoclonal antibody biosimilars, are provided by the EMA on a product-by-product basis and are available on its website.Biosimilars described herein may be similar to the reference medicinal product in terms of quality characteristics, biological activity, mechanism of action, safety profile, and / or efficacy. Furthermore, biosimilars may be used or intended for use to treat the same condition as the reference medicinal product. Thus, biosimilars described herein may be considered to have similar or very similar quality characteristics to the reference medicinal product. Alternatively, or in addition, biosimilars described herein may be considered to have similar or very similar biological activity to the reference medicinal product. Alternatively, or in addition, biosimilars described herein may be considered to have a similar or very similar safety profile to the reference medicinal product. Alternatively, or in addition, biosimilars described herein may be considered to have similar or very similar efficacy to the reference medicinal product. As described herein, biosimilars in Europe are compared to reference medicinal products authorized by the EMA. However, in some cases, biosimilars may be compared in specific studies to biopharmaceuticals authorized outside the European Economic Area (non-EEA-authorized "comparators"). Such studies include, for example, specific clinical studies and in vivo nonclinical studies. As used herein, the term "biosimilar" also refers to a biopharmaceutical that has been 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 to the amino acid structure (e.g., including amino acid deletions, additions, and / or substitutions) that do not significantly affect the function of the polypeptide. A biosimilar may have an amino acid sequence that has 97% or more sequence identity, e.g., 97%, 98%, 99%, or 100%, to the amino acid sequence of its reference pharmaceutical. A biosimilar may include one or more post-translational modifications, such as, but not limited to, glycosylation, oxidation, deamidation, and / or cleavage, that differ from the post-translational modifications of the reference pharmaceutical, provided that the differences do not result in a change in the safety and / or efficacy of the pharmaceutical. A biosimilar may have the same or a different glycosylation pattern as the reference pharmaceutical, provided that the differences do not result in a change in the safety and / or efficacy of the pharmaceutical.In particular, but not exclusively, biosimilars may have different glycosylation patterns if the differences address or are intended to address safety concerns associated with the reference drug. Additionally, biosimilars may deviate from the reference drug, for example, in its strength, dosage form, formulation, excipients, and / or presentation, provided that the drug's safety and efficacy are not compromised. Biosimilars may contain differences, for example, in their pharmacokinetic (PK) and / or pharmacodynamic (PD) profiles, compared to the reference drug, but are still considered sufficiently similar to the reference drug to be approved or deemed suitable for approval. In certain circumstances, biosimilars exhibit different binding characteristics compared to the reference drug, and these different binding characteristics 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.

[0507] II. Cryopreservation method Provided herein are methods for cryopreserving tumor tissue using slow freezing techniques.

[0508] In some embodiments, the present invention provides a method for cryopreserving tumor tissue, as well as cryopreserved tumor tissue, comprising the steps of: (i) adding a cryopreservation medium to a closable container; (ii) pre-cooling the closeable container in a controlled rate freezer; (iii) fragmenting the tumor tissue to obtain tumor fragments; (iv) placing the tumor fragments in a closable container containing a cryopreservation medium and closing the container; (v) optionally, incubating the sealed container containing the tumor fragments and cryopreservation medium; (vi) slow-freezing the container in a controlled rate freezer; (vii) transferring the container to a liquid nitrogen freezer.

[0509] In some embodiments, the present invention provides a method for cryopreserving tumor tissue, as well as cryopreserved tumor tissue, comprising the steps of: (i) placing tumor fragments obtained from fragmenting tumor tissue into a pre-chilled closable container containing a cryopreservation medium and closing the container; (ii) optionally incubating the sealed container containing the tumor fragments and cryopreservation medium; (iii) slow-freezing the container in a controlled rate freezer; (iv) transferring the container to a liquid nitrogen freezer.

[0510] In some embodiments, the present invention provides a method for cryopreserving tumor tissue, as well as cryopreserved tumor tissue, comprising the steps of: (i) placing a tumor digest obtained from digesting tumor tissue in an enzymatic medium or tumor fragments produced from fragmenting tumor tissue into a pre-chilled closable container containing a cryopreservation medium and closing the container; (ii) optionally incubating the sealed container containing the tumor digest and cryopreservation medium; (iii) slow-freezing the container in a controlled rate freezer; (iv) transferring the container to a liquid nitrogen freezer.

[0511] In some embodiments, the present invention provides a method for cryopreserving tumor tissue, as well as cryopreserved tumor tissue, comprising the steps of: (i) adding a cryopreservation medium to a closable container; (ii) pre-cooling the closeable container in a controlled rate freezer; (iii) digesting the tumor tissue in an enzymatic medium to obtain a tumor digest; (iv) placing the tumor digest in a cryopreservation medium in a closable container and closing the container; (v) optionally, incubating the sealed container containing the tumor digest and cryopreservation medium; (vi) slow-freezing the container in a controlled rate freezer; (vii) transferring the container to a liquid nitrogen freezer.

[0512] In view of the present disclosure, any suitable cryopreservation medium known to one of skill in the art can be used in the methods described herein. Examples of suitable cryopreservation media include, but are not limited to, CryoStor® CS10, HypoThermosol®, or combinations thereof. In some embodiments, the cryopreservation medium comprises about 2% v / v DMSO to about 15% v / v DMSO. In some embodiments, the cryopreservation medium comprises about 2% v / v DMSO. In some embodiments, the cryopreservation medium comprises about 2% v / v DMSO. In some embodiments, the cryopreservation medium comprises about 3% v / v DMSO. In some embodiments, the cryopreservation medium comprises about 4% v / v DMSO. In some embodiments, the cryopreservation medium comprises about 5% v / v DMSO. In some embodiments, the cryopreservation medium comprises about 6% v / v DMSO. In some embodiments, the cryopreservation medium comprises about 7% v / v DMSO. In some embodiments, the cryopreservation medium comprises about 8% v / v DMSO. In some embodiments, the cryopreservation medium comprises about 9% v / v DMSO. In some embodiments, the cryopreservation medium comprises about 10% v / v DMSO. In some embodiments, the cryopreservation medium comprises about 11% v / v DMSO. In some embodiments, the cryopreservation medium comprises about 12% v / v DMSO. In some embodiments, the cryopreservation medium comprises about 13% v / v DMSO. In some embodiments, the cryopreservation medium comprises about 14% v / v DMSO. In some embodiments, the cryopreservation medium comprises about 15% v / v DMSO. In some embodiments, the cryopreservation medium comprises at least one antimicrobial agent. Any suitable antimicrobial agent known to those of skill in the art in light of the present disclosure can be used in the methods described herein. In some embodiments, the cryopreservation medium comprises gentamicin. In some embodiments, the cryopreservation medium comprises gentamicin at a concentration of at least 50 μg / mL. In some embodiments, the cryopreservation medium comprises gentamicin at a concentration of at least 40 μg / mL. In some embodiments, the cryopreservation medium comprises gentamicin at a concentration of at least 30 μg / mL.In some embodiments, the cryopreservation medium comprises gentamicin at a concentration of at least 20 μg / mL.

[0513] In view of the present disclosure, any suitable closable container known to one of skill in the art can be used in the methods described herein. Examples of suitable closable containers include, but are not limited to, capped microcentrifuge tubes, lidded microcentrifuge tubes, and cryogenic specimen storage vials. The term "cryogenic sample storage vial" is intended to encompass terms such as cryogenic sample storage vials, cryocontainers, and cryotubes, including closed, sealed, or reclosable containers (e.g., with screw caps or friction-sealing snap caps) that allow the container to be safely and securely stored at low temperatures (meaning temperatures below -80°C, and optionally immersed in liquid nitrogen or suspended in the gas phase above liquid nitrogen at temperatures of approximately -196°C). Capped or lidded microcentrifuge tubes and cryovials, typically made from polyethylene or polypropylene, are often used as cryogenic sample storage vials.

[0514] In some embodiments, the closable container is filled with cryopreservation medium at about 50% to about 85% volume. In some embodiments, the closable container is filled with cryopreservation medium at about 50% to about 85% volume. In some embodiments, the closable container is filled with cryopreservation medium at about 50% to about 75% volume. In some embodiments, the closable container is filled with cryopreservation medium at about 50% to about 65% volume. In some embodiments, the closable container is filled with cryopreservation medium at about 50% to about 55% volume. In some embodiments, the closable container is filled with cryopreservation medium at about 60% to about 85% volume. In some embodiments, the closable container is filled with cryopreservation medium at about 60% to about 75% volume. In some embodiments, the closable container is filled with cryopreservation medium at about 60% to about 65% volume. In some embodiments, the closable container is filled with cryopreservation medium at about 70% to about 85% volume. In some embodiments, the closable container is filled with cryopreservation medium to about 70% to about 75% of its volume, hi some embodiments, the closable container is filled with cryopreservation medium to about 80% to about 85% of its volume.

[0515] In some embodiments, the pre-chilling step comprises placing the closable container in a controlled rate freezer for a period of at least about 5 minutes to about 8 hours at a temperature of about −80° C. to about 8° C. In some embodiments, the pre-chilling step comprises freezing the closable container at about −80° C., about −79° C., about −78° C., about −77° C., about −76° C., about −75° C., about −70° C., about −65° C., about −60° C., about −55° C., about −50° C., about −45° C., about −40° C., about −35° C., about −30° C., about −25° C., about −20° C., about −15° C., about −10° C., about −5° C., about 0° C., about 1° C., about 2° C., about 3° C., about 4° C., about 5° C., about 6° C., about 7° C., about 8° C., or any temperature in between, for at least about 5 minutes to at least about 10 minutes. , at least about 15 minutes, at least about 20 minutes, at least about 25 minutes, at least about 30 minutes, at least about 35 minutes, at least about 40 minutes, at least about 45 minutes, at least about 50 minutes, at least about 55 minutes, at least about 1 hour, at least about 1.5 hours, at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 6 hours, at least about 7 hours, at least about 8 hours, or more.

[0516] In some embodiments, the sealed container containing the tumor fragments and cryopreservation medium is incubated at a temperature of about 2-8° C. for about 30-60 minutes before slow-freezing the container in a controlled rate freezer. In some embodiments, the container containing the tumor fragments and cryopreservation medium is incubated at about 2° C., about 3° C., about 4° C., about 5° C., about 6° C., about 7° C., about 8° C., or any temperature therebetween, for about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 60 minutes, or longer, before slow-freezing the container in a controlled rate freezer.

[0517] In view of the present disclosure, any suitable controlled rate freezer known to one of skill in the art can be used in the methods described herein. Examples of suitable controlled rate freezers include, but are not limited to, a Corning CoolCell™ device or a Nalgene Mr. Frosty™ device. In some embodiments, the controlled rate freezer is an IPA-free controlled rate freezer that cools at a rate of about -0.1°C / min to about -10°C / min. In some embodiments, the controlled rate freezer is an IPA-free controlled rate freezer that cools at a rate of about -0.1°C / min to about -10°C / min, about -0.2°C / min to about -5°C / min, about -0.5°C / min to about -2.5°C / min, or about -1°C / min to about -2°C / min. In some embodiments, the controlled rate freezer is an IPA-free controlled rate freezer that cools at a rate of about -1°C / min.

[0518] In some embodiments, all of the locations of the controlled rate freezing device are filled with closable containers containing cryopreservation medium. In some embodiments, 90% or more of the locations of the controlled rate freezing device are filled with closable containers containing cryopreservation medium. In some embodiments, 80% or more of the locations of the controlled rate freezing device are filled with closable containers containing cryopreservation medium. In some embodiments, 70% or more of the locations of the controlled rate freezing device are filled with closable containers containing cryopreservation medium. In some embodiments, 60% or more of the locations of the controlled rate freezing device are filled with closable containers containing cryopreservation medium. In some embodiments, 50% or more of the locations of the controlled rate freezing device are filled with closable containers containing cryopreservation medium. In some embodiments, 40% or more of the locations of the controlled rate freezing device are filled with closable containers containing cryopreservation medium.

[0519] As used herein, the term "slow freezing" refers to a process in which a sample is cooled at a controlled rate in a refrigerated environment prior to final cryopreservation, such as in liquid nitrogen. In some embodiments, the cooling rate is from about -0.1°C / min to about -10°C / min, from about -0.2°C / min to about -5°C / min, from about -0.5°C / min to about -2.5°C / min, or from about -1°C / min to about -2°C / min. In some embodiments, the cooling rate is about -1°C / min. In some embodiments, the cooling environment is a -80°C freezer or dry ice set to about -90°C to about -70°C, e.g., about -90°C, about -89°C, about -88°C, about -87°C, about -86°C, about -85°C, about -84°C, about -83°C, about -82°C, about -81°C, about -80°C, about -79°C, about -78°C, about -77°C, about -76°C, about -75°C, about -74°C, about -73°C, about -72°C, about -72°C, about -71°C, or any temperature in between.

[0520] In some embodiments, slow freezing comprises incubating the controlled rate freezer at a temperature of about -70°C to about -90°C. In some embodiments, slow freezing comprises incubating the controlled rate freezer at a temperature of about -75°C to about -85°C. In some embodiments, slow freezing comprises incubating the controlled rate freezer at a temperature of about -78°C to about -80°C. In some embodiments, slow freezing comprises incubating the controlled rate freezer with dry ice. In some embodiments, slow freezing comprises incubating the controlled rate freezer in a -80°C freezer. In some embodiments, slow freezing comprises incubating the controlled rate freezer in dry ice.

[0521] In some embodiments, slow freezing comprises incubating a controlled rate freezer at a temperature of about -80°C for about 3-5 hours. In some embodiments, slow freezing comprises incubating a controlled rate freezer at a temperature of about -80°C for about 3 hours. In some embodiments, slow freezing comprises incubating a controlled rate freezer at a temperature of about -80°C for about 4 hours. In some embodiments, slow freezing comprises incubating a controlled rate freezer at a temperature of about -80°C for about 5 hours.

[0522] In some embodiments, after recovery from freezing, the cells have a post-thaw viability of at least about 80%. In some embodiments, after recovery from freezing, the cells have a post-thaw viability of at least about 75%. In some embodiments, after recovery from freezing, the cells have a post-thaw viability of at least about 70%. In some embodiments, after recovery from freezing, the cells have a post-thaw viability of at least about 65%. In some embodiments, after recovery from freezing, the cells have a post-thaw viability of at least about 60%. In some embodiments, after recovery from freezing, the cells have a post-thaw viability of at least about 55%. In some embodiments, after recovery from freezing, the cells have a post-thaw viability of at least about 50%. In some embodiments, after recovery from freezing, the cells have a post-thaw viability of at least about 45%. In some embodiments, after recovery from freezing, the cells have a post-thaw viability of at least about 40%. In some embodiments, after recovery from freezing, the cells have a post-thaw viability of at least about 35%. In some embodiments, after recovery from freezing, the cells have a post-thaw viability of at least about 30%. In some embodiments, after recovery from freezing, the cells have a post-thaw viability of at least about 25%. In some embodiments, after recovery from freezing, the cells have a post-thaw viability of at least about 20%. In light of the present disclosure, any suitable method for measuring or determining post-thaw viability known in the art can be used in the methods described herein.

[0523] In some embodiments, tumor digests are generated by incubating tumors in an enzyme medium, such as, but not limited to, RPMI 1640, 2 mM GlutaMAX, 10 mg / mL gentamicin, 30 U / mL DNase, and 1.0 mg / mL collagenase, followed by mechanical dissociation (GentleMACS, Miltenyi Biotec, Auburn, CA). In some embodiments, tumors are placed in a tumor dissociation enzyme mixture, including one or more dissociation (digestion) enzymes, such as, but not limited to, collagenase (including any blend or type of collagenase), Accutase™, Accumax™, hyaluronidase, neutral protease (dispase), chymotrypsin, chymopapain, trypsin, caseinase, elastase, papain, protease type XIV (pronase), deoxyribonuclease I (DNase), trypsin inhibitor, any other dissociation enzyme or proteolytic enzyme, and any combination thereof. In other embodiments, the tumor is placed in a tumor dissociation enzyme cocktail comprising collagenase (including any blend or type of collagenase), neutral protease (dispase), and deoxyribonuclease I (DNase).

[0524] In some embodiments, the present invention provides a method for expanding tumor infiltrating lymphocytes (TILs), comprising: (a) obtaining and / or receiving a first population of TILs from tumor tissue excised from a subject or patient and storing the tumor tissue in a frozen state, wherein methods of storing tumor tissue include methods of cryopreserving tumor tissue described herein; (b) culturing the first TIL population in a culture medium to expand the first TIL population.

[0525] In some embodiments, the present invention provides a method for expanding tumor infiltrating lymphocytes (TILs), comprising: (a) obtaining and / or receiving a first population of TILs from tumor tissue excised from a subject or patient and storing the tumor tissue in a frozen state, wherein methods of storing tumor tissue include methods of cryopreserving tumor tissue described herein; (b) digesting the tumor fragments in an enzymatic medium to produce a tumor digest; (c) culturing the first TIL population in a culture medium to expand the first TIL population.

[0526] In some embodiments, the present invention provides a method for rapid expansion of tumor infiltrating lymphocytes (TILs), comprising: (a) obtaining and / or receiving a first population of TILs from tumor tissue excised from a subject or patient and storing the tumor tissue in a frozen state, wherein methods of storing tumor tissue include methods of cryopreserving tumor tissue described herein; (b) culturing the first TIL population in a culture medium containing IL-2, OKT-3 (anti-CD3 antibody), and antigen-presenting cells (APCs), resulting in rapid expansion of the first TIL population to produce a second TIL population.

[0527] In some embodiments, the present invention provides a method for rapid expansion of tumor infiltrating lymphocytes (TILs), comprising: (a) obtaining and / or receiving a first population of TILs from tumor tissue excised from a subject or patient and storing the tumor tissue in a frozen state, wherein methods of storing tumor tissue include methods of cryopreserving tumor tissue described herein; (b) digesting the tumor fragments in an enzymatic medium to produce a tumor digest; (c) culturing the first TIL population in a culture medium containing IL-2, OKT-3 (anti-CD3 antibody), and antigen-presenting cells (APCs), resulting in rapid expansion of the first TIL population to produce a second TIL population.

[0528] In some embodiments, the present invention provides a method for preparing expanded tumor infiltrating lymphocytes (TILs), comprising: (a) obtaining and / or receiving a first population of TILs from tumor tissue excised from a subject or patient and storing the tumor tissue in a frozen state, wherein methods of storing tumor tissue include methods of cryopreserving tumor tissue described herein; (b) culturing the first TIL population in a first cell culture medium containing IL-2 for about 3 to 14 days to produce a second TIL population; (c) culturing the second TIL population in a second cell culture medium comprising antigen-presenting cells (APCs), OKT-3, and IL-2 for about 7 to 14 days to provide an expanded number of TILs.

[0529] In some embodiments, the present invention provides a method for preparing expanded tumor infiltrating lymphocytes (TILs), comprising: (a) obtaining and / or receiving a first population of TILs from tumor tissue excised from a subject or patient and storing the tumor tissue in a frozen state, wherein methods of storing tumor tissue include methods of cryopreserving tumor tissue described herein; (b) digesting the tumor fragments in an enzymatic medium to produce a tumor digest; (c) culturing the first TIL population in a first cell culture medium containing IL-2 for about 3 to 14 days to produce a second TIL population; (d) culturing the second TIL population in a second cell culture medium comprising antigen-presenting cells (APCs), OKT-3, and IL-2 for about 7 to 14 days to provide an expanded number of TILs.

[0530] In some embodiments, the step of culturing the first TIL population is carried out for about 1 to 11 days. In some embodiments, the step of culturing the first TIL population is carried out for about 3 to 11 days, about 4 to 11 days, about 5 to 11 days, about 6 to 11 days, about 7 to 11 days, about 8 to 11 days, about 9 to 11 days, about 10 to 11 days, about 3 to 10 days, about 4 to 10 days, about 5 to 10 days, about 6 to 10 days, about 7 to 10 days, about 8 to 10 days, about 9 to 10 days, or about The step of culturing the first TIL population is carried out for 3 to 9 days, about 4 to 9 days, about 5 to 9 days, about 6 to 9 days, about 7 to 9 days, about 8 to 9 days, about 3 to 8 days, about 4 to 8 days, about 5 to 8 days, about 6 to 8 days, about 7 to 8 days, about 3 to 7 days, about 4 to 7 days, about 5 to 7 days, about 6 to 7 days, about 3 to 6 days, about 4 to 6 days, about 5 to 6 days, about 3 to 5 days, about 4 to 5 days, or about 3 to 4 days. In some embodiments, the step of culturing the first TIL population is carried out for about 1 day. In some embodiments, the step of culturing the first TIL population is carried out for about 2 days. In some embodiments, the step of culturing the first TIL population is carried out for about 3 days. In some embodiments, the step of culturing the first TIL population is carried out for about 4 days. In some embodiments, the step of culturing the first TIL population is carried out for about 5 days. In some embodiments, culturing the first TIL population is performed for about 6 days. In some embodiments, culturing the first TIL population is performed for about 7 days. In some embodiments, culturing the first TIL population is performed for about 8 days. In some embodiments, culturing the first TIL population is performed for about 9 days. In some embodiments, culturing the first TIL population is performed for about 10 days. In some embodiments, culturing the first TIL population is performed for about 11 days.

[0531] In some embodiments, the rapid second expansion is carried out for about 7-11 days. In some embodiments, the rapid second expansion is carried out for about 7-11 days, about 8-11 days, about 9-11 days, about 10-11 days, about 7-10 days, about 8-10 days, about 9-10 days, about 7-9 days, about 8-9 days, or about 7-8 days. In some embodiments, the rapid second expansion is carried out for about 7 days. In some embodiments, the rapid second expansion is carried out for about 8 days. In some embodiments, the rapid second expansion is carried out for about 9 days. In some embodiments, the rapid second expansion is carried out for about 10 days. In some embodiments, the rapid second expansion is carried out for about 11 days. In some embodiments, the rapid second expansion is carried out for about 7-12 days, about 8-12 days, about 9-12 days, about 10-12 days, or about 11-12 days. In some embodiments, the rapid second expansion is performed for about 7-13 days, about 8-13 days, about 9-13 days, about 10-13 days, about 11-13 days, or about 12-13 days. In some embodiments, the rapid second expansion is performed for about 7-14 days, about 8-14 days, about 9-14 days, about 10-14 days, about 11-14 days, about 12-14 days, or about 13-14 days. In some embodiments, the rapid second expansion is performed for about 12 days. In some embodiments, the rapid second expansion is performed for about 13 days. In some embodiments, the rapid second expansion is performed for about 14 days.

[0532] In some embodiments, the steps of culturing the first TIL population and culturing the second TIL population are completed within a period of about 22 days. In some embodiments, the steps of culturing the first TIL population and culturing the second TIL population are completed within a period of about 8 days. In some embodiments, the steps of culturing the first TIL population and culturing the second TIL population are completed within a period of about 9 days. In some embodiments, the steps of culturing the first TIL population and culturing the second TIL population are completed within a period of about 10 days. In some embodiments, the steps of culturing the first TIL population and culturing the second TIL population are completed within a period of about 11 days. In some embodiments, the steps of culturing the first TIL population and culturing the second TIL population are completed within a period of about 12 days. In some embodiments, the steps of culturing the first TIL population and culturing the second TIL population are completed within a period of about 13 days. In some embodiments, the steps of culturing the first TIL population and culturing the second TIL population are completed within a time period of about 14 days. In some embodiments, the steps of culturing the first TIL population and culturing the second TIL population are completed within a time period of about 15 days. In some embodiments, the steps of culturing the first TIL population and culturing the second TIL population are completed within a time period of about 16 days. In some embodiments, the steps of culturing the first TIL population and culturing the second TIL population are completed within a time period of about 17 days. In some embodiments, the steps of culturing the first TIL population and culturing the second TIL population are completed within a time period of about 18 days. In some embodiments, the steps of culturing the first TIL population and culturing the second TIL population are completed within a time period of about 19 days. In some embodiments, the steps of culturing the first TIL population and culturing the second TIL population are completed within a time period of about 20 days. In some embodiments, the steps of culturing the first TIL population and culturing the second TIL population are completed within a period of about 21 days.

[0533] In some embodiments, the step of culturing the second TIL population is carried out by culturing the second TIL population in a second cell culture medium for a first period of about 5 days, at the end of the first period, dividing the culture into multiple subcultures, and culturing each of the multiple subcultures in a third culture medium containing IL-2 for a second period of about 6 days, and at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0534] In some embodiments, the step of culturing the second TIL population is carried out by culturing the second TIL population in a second cell culture medium for a first period of about 7 days, at the end of the first period, dividing the culture into multiple subcultures, each of the multiple subcultures being cultured in a third culture medium containing IL-2 for a second period of about 7 days, and at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0535] In some embodiments, the present invention provides a method for preparing expanded tumor infiltrating lymphocytes (TILs), comprising: (a) obtaining and / or receiving a first population of TILs from tumor tissue excised from a subject or patient and storing the tumor tissue in a frozen state, wherein methods of storing tumor tissue include methods of cryopreserving tumor tissue described herein; (b) performing an initial expansion (or first expansion by priming) of the first TIL population in a first cell culture medium to obtain a second TIL population, wherein the first cell culture medium comprises IL-2, optionally OKT-3 (anti-CD3 antibody), and optionally antigen-presenting cells (APCs), and the first expansion by priming occurs over a period of about 1 to 8 days; and (c) performing a rapid second expansion of the second TIL population in a second cell culture medium to obtain an expanded number of TILs, wherein the second cell culture medium comprises IL-2, OKT-3 (an anti-CD3 antibody), and APCs, and the rapid expansion is performed over a period of 14 days or less, and optionally, the rapid second expansion can proceed for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days after the initiation of the rapid second expansion to obtain an expanded number of TILs.

[0536] In some embodiments, the present invention provides a method for preparing expanded tumor infiltrating lymphocytes (TILs), comprising: (a) obtaining and / or receiving a first population of TILs from tumor tissue excised from a subject or patient and storing the tumor tissue in a frozen state, wherein methods of storing tumor tissue include methods of cryopreserving tumor tissue described herein; (b) digesting the tumor tissue in an enzymatic medium to produce a tumor digest; (c) performing an initial expansion (or first expansion by priming) of the first TIL population in a first cell culture medium to obtain a second TIL population, wherein the first cell culture medium comprises IL-2, optionally OKT-3 (anti-CD3 antibody), and optionally antigen-presenting cells (APCs), and the first expansion by priming occurs over a period of about 1 to 8 days; and (d) performing a rapid second expansion of the second TIL population in a second cell culture medium to obtain an expanded number of TILs, wherein the second cell culture medium comprises IL-2, OKT-3 (an anti-CD3 antibody), and APCs, and the rapid expansion is performed over a period of 14 days or less, and optionally, the rapid second expansion can proceed for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days after the initiation of the rapid second expansion to obtain an expanded number of TILs.

[0537] In some embodiments, the first culture medium comprises APCs. In some embodiments, the number of APCs in the second culture medium is greater than the number of APCs in the first culture medium.

[0538] In some embodiments, the first expansion step by priming is carried out for a period of about 7 or 8 days. In some embodiments, the first expansion step by priming is carried out for about 7 days. In some embodiments, the first expansion step by priming is carried out for about 8 days.

[0539] In some embodiments, the rapid second expansion step is performed for about 7-10 days. In some embodiments, the rapid second expansion step is performed for about 8-10 days. In some embodiments, the rapid second expansion step is performed for about 9-10 days. In some embodiments, the rapid second expansion step is performed for about 7-9 days. In some embodiments, the rapid second expansion step is performed for about 8-9 days. In some embodiments, the rapid second expansion step is performed for about 7-8 days. In some embodiments, the rapid second expansion step is performed for about 7 days. In some embodiments, the rapid second expansion step is performed for about 8 days. In some embodiments, the rapid second expansion step is performed for about 9 days. In some embodiments, the rapid second expansion step is performed for about 10 days.

[0540] In some embodiments, the rapid expansion step is carried out by culturing the second TIL population in a second cell culture medium for a first period of about 3-4 days, at the end of the first period, dividing the culture into multiple subcultures, and culturing each of the multiple subcultures in a third culture medium containing IL-2 for a second period of about 4-6 days, and at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0541] In some embodiments, the present invention provides a method for preparing expanded tumor infiltrating lymphocytes (TILs), comprising: (a) obtaining and / or receiving a first population of TILs from tumor tissue excised from a subject or patient and storing the tumor tissue in a frozen state, wherein methods of storing tumor tissue include methods of cryopreserving tumor tissue described herein; (b) performing a first expansion by culturing the first TIL population in a first cell culture medium comprising interleukin 2 (IL-2), and optionally OKT-3, and optionally antigen-presenting cells (APCs) to provide a second TIL population; (c) performing a second expansion by culturing the second TIL population in a second cell culture medium comprising APCs, OKT-3, and IL-2 to provide an expanded number of TILs.

[0542] In some embodiments, the present invention provides a method for preparing expanded tumor infiltrating lymphocytes (TILs), comprising: (a) obtaining and / or receiving a first population of TILs from tumor tissue excised from a subject or patient and storing the tumor tissue in a frozen state, wherein methods of storing tumor tissue include methods of cryopreserving tumor tissue described herein; (b) digesting the tumor tissue in an enzymatic medium to produce a tumor digest; (c) performing a first expansion by culturing the first TIL population in a first cell culture medium comprising interleukin 2 (IL-2), and optionally OKT-3, and optionally antigen-presenting cells (APCs) to provide a second TIL population; (d) performing a second expansion by culturing the second TIL population in a second cell culture medium comprising APCs, OKT-3, and IL-2 to provide an expanded number of TILs.

[0543] In some embodiments, the first culture medium comprises APCs and / or OKT-3. In some embodiments, the first culture medium comprises APCs. In some embodiments, the first culture medium comprises OKT-3. In some embodiments, the first culture medium comprises APCs and OKT-3. In some embodiments, the number of APCs in the second culture medium is greater than the number of APCs in the first culture medium.

[0544] In some embodiments, the present invention provides a method for expanding tumor-infiltrating lymphocytes into a therapeutic TIL population, the method comprising: (a) obtaining and / or receiving a first population of TILs from a sample of tumor tissue produced by surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining tumor tissue from a patient or subject, and storing the sample of tumor tissue in a frozen state, wherein the method for storing the sample of tumor tissue includes the method for cryopreserving tumor tissue described herein; (b) adding a sample of tumor tissue, or tumor fragments obtained from fragmenting a sample of tumor tissue, to a closed system and performing a first expansion by culturing the first TIL population in a first cell culture medium containing IL-2 to produce a second TIL population, wherein the first expansion is performed in a closed container providing a first gas permeable surface area, and the first expansion is performed for about 3 to 14 days to obtain the second TIL population; (c) performing a second expansion by culturing the second TIL population in a second cell culture medium comprising IL-2, OKT-3, and antigen-presenting cells (APCs) to produce a third TIL population, wherein the second expansion is performed for about 7-14 days to obtain the third TIL population, wherein the second expansion is performed in a sealed container that provides a second gas-permeable surface area, wherein the third TIL population is a therapeutic TIL population, and wherein the transition from step (b) to step (c) occurs without opening the system. (d) harvesting the therapeutic TIL population obtained from step (c), wherein the transition from step (c) to step (d) occurs without opening the system.

[0545] In some embodiments, the present invention provides a method for expanding tumor-infiltrating lymphocytes into a therapeutic TIL population, the method comprising: (a) obtaining and / or receiving a first population of TILs from a sample of tumor tissue produced by surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining tumor tissue from a patient or subject, and storing the sample of tumor tissue in a frozen state, wherein the method for storing the sample of tumor tissue includes the method for cryopreserving tumor tissue described herein; (b) digesting a sample of tumor tissue, or tumor fragments obtained from fragmenting a sample of tumor tissue, in an enzymatic medium to produce a tumor digest; (c) adding tumor digest to a closed system and culturing the first TIL population in a first cell culture medium containing IL-2 to perform a first expansion to produce a second TIL population, wherein the first expansion is performed in a closed container that provides a first gas permeable surface area, and the first expansion is performed for about 3 to 14 days to obtain the second TIL population; (d) performing a second expansion by culturing the second TIL population in a second cell culture medium comprising IL-2, OKT-3, and antigen presenting cells (APCs) to produce a third TIL population, wherein the second expansion is performed for about 7 to 14 days to obtain the third TIL population, wherein the second expansion is performed in a sealed container that provides a second gas permeable surface area, wherein the third TIL population is a therapeutic TIL population, and wherein the transition from step (c) to step (d) occurs without opening the system. (e) harvesting the therapeutic TIL population obtained from step (d), wherein the transition from step (d) to step (e) occurs without opening the system.

[0546] In some embodiments, the first expansion is performed for about 1 to 11 days. In some embodiments, the first expansion is performed for about 2 to 11 days, about 3 to 11 days, about 4 to 11 days, about 5 to 11 days, about 6 to 11 days, about 7 to 11 days, about 8 to 11 days, about 9 to 11 days, about 10 to 11 days, about 2 to 10 days, about 3 to 10 days, about 4 to 10 days, about 5 to 10 days, about 6 to 10 days, about 7 to 10 days, about 8 to 10 days, about 9 to 10 days, about 2 to 9 days, about 3 to 9 days, about 4 to 9 days, about 5 to 10 days, about 6 to 10 days, about 7 to 10 days, about 8 to 10 days, about 9 to 10 days, about 2 to 9 days, about 3 to 9 days, about 4 to 9 days, about 5 to 10 days, about 9 to 10 days, about 9 to 10 days, about 10 to 1 ... The first expansion is performed for about 1-9 days, about 6-9 days, about 7-9 days, about 8-9 days, about 2-8 days, about 3-8 days, about 4-8 days, about 5-8 days, about 6-8 days, about 7-8 days, about 2-7 days, about 3-7 days, about 4-7 days, about 5-7 days, about 6-7 days, about 2-6 days, about 3-6 days, about 4-6 days, about 5-6 days, about 2-5 days, about 3-5 days, about 4-5 days, about 2-4 days, about 3-4 days, or about 2-3 days. In some embodiments, the first expansion is performed for about 1 day. In some embodiments, the first expansion is performed for about 2 days. In some embodiments, the first expansion is performed for about 3 days. In some embodiments, the first expansion is performed for about 4 days. In some embodiments, the first expansion is performed for about 5 days. In some embodiments, the first expansion is performed for about 6 days. In some embodiments, the first expansion is performed for about 7 days. In some embodiments, the first expansion is performed for about 8 days. In some embodiments, the first expansion is performed for about 9 days. In some embodiments, the first expansion is performed for about 10 days. In some embodiments, the first expansion is performed for about 11 days.

[0547] In some embodiments, the second expansion is performed for about 7-11 days. In some embodiments, the second expansion is performed for about 7-11 days, about 8-11 days, about 9-11 days, about 10-11 days, about 7-10 days, about 8-10 days, about 9-10 days, about 7-9 days, about 8-9 days, or about 7-8 days. In some embodiments, the second expansion is performed for about 7 days. In some embodiments, the second expansion is performed for about 8 days. In some embodiments, the second expansion is performed for about 9 days. In some embodiments, the second expansion is performed for about 10 days. In some embodiments, the second expansion is performed for about 11 days. In some embodiments, the second expansion is performed for about 7-12 days, about 8-12 days, about 9-12 days, about 10-12 days, or about 11-12 days. In some embodiments, the second expansion is carried out for about 7-13 days, about 8-13 days, about 9-13 days, about 10-13 days, about 11-13 days, or about 12-13 days. In some embodiments, the second expansion is carried out for about 7-14 days, about 8-14 days, about 9-14 days, about 10-14 days, about 11-14 days, about 12-14 days, or about 13-14 days. In some embodiments, the second expansion is carried out for about 12 days. In some embodiments, the second expansion is carried out for about 13 days. In some embodiments, the second expansion is carried out for about 14 days.

[0548] In some embodiments, the first expansion and the second expansion are completed within a period of about 22 days. In some embodiments, the first expansion and the second expansion are completed within a period of about 8 days. In some embodiments, the first expansion and the second expansion are completed within a period of about 9 days. In some embodiments, the first expansion and the second expansion are completed within a period of about 10 days. In some embodiments, the first expansion and the second expansion are completed within a period of about 11 days. In some embodiments, the first expansion and the second expansion are completed within a period of about 12 days. In some embodiments, the first expansion and the second expansion are completed within a period of about 13 days. In some embodiments, the first expansion and the second expansion are completed within a period of about 14 days. In some embodiments, the first expansion and the second expansion are completed within a period of about 15 days. In some embodiments, the first expansion and the second expansion are completed within a period of about 16 days. In some embodiments, the first expansion and the second expansion are completed within a period of about 17 days. In some embodiments, the first expansion and second expansion are completed within a period of about 18 days. In some embodiments, the first expansion and second expansion are completed within a period of about 19 days. In some embodiments, the first expansion and second expansion are completed within a period of about 20 days. In some embodiments, the first expansion and second expansion are completed within a period of about 21 days.

[0549] In some embodiments, the second expansion comprises the following steps: (i) culturing the second population of TILs in a second culture medium for a first period of about 5 days; (ii) subdividing the culture of step (i) into a plurality of subcultures, wherein each of the plurality of subcultures is transferred to a separate sealed container providing a third gas permeable surface and cultured in a third culture medium comprising IL-2 for a second period of about 6 days, the transition from step (i) to step (ii) being performed without opening the system; (iii) combining the multiple subcultures to produce a third TIL population, wherein the transition from step (ii) to step (iii) is performed without opening the system.

[0550] In some embodiments, the second expansion comprises the following steps: (i) culturing the second population of TILs in a second culture medium for a first period of about 7 days; (ii) subdividing the culture of step (i) into a plurality of subcultures, wherein each of the plurality of subcultures is transferred to a separate sealed container providing a third gas permeable surface and cultured in a third culture medium comprising IL-2 for a second period of about 7 days, wherein the transition from step (i) to step (ii) is performed without opening the system; (iii) combining the multiple subcultures to produce a third TIL population, wherein the transition from step (ii) to step (iii) is performed without opening the system.

[0551] In some embodiments, the present invention provides a method for expanding tumor-infiltrating lymphocytes into a therapeutic TIL population, the method comprising: (a) obtaining and / or receiving a first population of TILs from a sample of tumor tissue produced by surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining tumor tissue from a patient or subject, and storing the sample of tumor tissue in a frozen state, wherein the method for storing the sample of tumor tissue includes the method for cryopreserving tumor tissue described herein; (b) culturing the first TIL population in a first cell culture medium containing IL-2 for about 1-3 days; (c) performing an initial expansion (or first expansion by priming) of the first TIL population in a second cell culture medium to obtain a second TIL population, wherein the second cell culture medium comprises IL-2, OKT-3 (anti-CD3 antibody), and antigen-presenting cells (APCs), and the first expansion by priming occurs for about 1 to 11 days; (d) performing a second rapid expansion of the second TIL population in a third cell culture medium to obtain a third TIL population, wherein the third TIL population is a therapeutic TIL population, the second cell culture medium comprising IL-2, OKT-3 (an anti-CD3 antibody), and APCs, and the rapid expansion is performed for 1 to 11 days after the initiation of the second rapid expansion; (e) harvesting the therapeutic TIL population.

[0552] In some embodiments, the present invention provides a method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population, the method comprising: (a) obtaining and / or receiving a first population of TILs from a sample of tumor tissue produced by surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining tumor tissue from a patient or subject, and storing the sample of tumor tissue in a frozen state, wherein the method for storing the sample of tumor tissue includes the method for cryopreserving tumor tissue described herein; (b) digesting a sample of tumor tissue, or tumor fragments obtained from fragmenting a sample of tumor tissue, in an enzymatic medium to produce a tumor digest; (c) culturing the first TIL population in a first cell culture medium containing IL-2 for about 1-3 days; (d) performing an initial expansion (or first expansion by priming) of the first TIL population in a second cell culture medium to obtain a second TIL population, wherein the second cell culture medium comprises IL-2, OKT-3 (anti-CD3 antibody), and antigen-presenting cells (APCs), and the first expansion by priming occurs for about 1 to 11 days; (e) performing a second rapid expansion of the second TIL population in a third cell culture medium to obtain a third TIL population, wherein the third TIL population is a therapeutic TIL population, the second cell culture medium comprising IL-2, OKT-3 (anti-CD3 antibody), and APCs, and the rapid expansion is performed for 1 to 11 days after the initiation of the second rapid expansion; (f) harvesting the therapeutic TIL population.

[0553] In some embodiments, the number of APCs in the third culture medium is greater than the number of APCs in the second culture medium.

[0554] In some embodiments, the first expansion by priming is carried out for about 3 to 11 days. In some embodiments, the first expansion by priming is carried out for about 3-11 days, about 4-11 days, about 5-11 days, about 6-11 days, about 7-11 days, about 8-11 days, about 9-11 days, about 10-11 days, about 3-10 days, about 4-10 days, about 5-10 days, about 6-10 days, about 7-10 days, about 8-10 days, about 9-10 days, about 3-9 days, about 4-9 days, about 5-9 days, about 6-9 days, about 7-9 days, about 8-9 days, about 3-8 days, about 4-8 days, about 5-8 days, about 6-8 days, about 7-8 days, about 3-7 days, about 4-7 days, about 5-7 days, about 6-7 days, about 3-6 days, about 4-6 days, about 5-6 days, about 3-5 days, about 4-5 days, or about 3-4 days. In some embodiments, the first priming expansion is carried out for about 3 days. In some embodiments, the first priming expansion is carried out for about 4 days. In some embodiments, the first priming expansion is carried out for about 5 days. In some embodiments, the first priming expansion is carried out for about 6 days. In some embodiments, the first priming expansion is carried out for about 7 days. In some embodiments, the first priming expansion is carried out for about 8 days. In some embodiments, the first priming expansion is carried out for about 9 days. In some embodiments, the first priming expansion is carried out for about 10 days. In some embodiments, the first priming expansion is carried out for about 11 days.

[0555] In some embodiments, the rapid second expansion is carried out for about 7-11 days. In some embodiments, the rapid second expansion is carried out for about 7-11 days, about 8-11 days, about 9-11 days, about 10-11 days, about 7-10 days, about 8-10 days, about 9-10 days, about 7-9 days, about 8-9 days, or about 7-8 days. In some embodiments, the rapid second expansion is carried out for about 7 days. In some embodiments, the rapid second expansion is carried out for about 8 days. In some embodiments, the rapid second expansion is carried out for about 9 days. In some embodiments, the rapid second expansion is carried out for about 10 days. In some embodiments, the rapid second expansion is carried out for about 11 days. In some embodiments, the rapid second expansion is carried out for about 7-12 days, about 8-12 days, about 9-12 days, about 10-12 days, or about 11-12 days. In some embodiments, the rapid second expansion is performed for about 7-13 days, about 8-13 days, about 9-13 days, about 10-13 days, about 11-13 days, or about 12-13 days. In some embodiments, the rapid second expansion is performed for about 7-14 days, about 8-14 days, about 9-14 days, about 10-14 days, about 11-14 days, about 12-14 days, or about 13-14 days. In some embodiments, the rapid second expansion is performed for about 12 days. In some embodiments, the rapid second expansion is performed for about 13 days. In some embodiments, the rapid second expansion is performed for about 14 days.

[0556] In some embodiments, the first expansion by priming and the rapid second expansion are completed within a period of about 22 days. In some embodiments, the first expansion by priming and the rapid second expansion are completed within a period of about 10 days. In some embodiments, the first expansion by priming and the rapid second expansion are completed within a period of about 11 days. In some embodiments, the first expansion by priming and the rapid second expansion are completed within a period of about 12 days. In some embodiments, the first expansion by priming and the rapid second expansion are completed within a period of about 13 days. In some embodiments, the first expansion by priming and the rapid second expansion are completed within a period of about 14 days. In some embodiments, the first expansion by priming and the rapid second expansion are completed within a period of about 15 days. In some embodiments, the first expansion by priming and the rapid second expansion are completed within a period of about 16 days. In some embodiments, the first expansion by priming and the rapid second expansion are completed within a period of about 17 days. In some embodiments, the first expansion by priming and the rapid second expansion are completed within a period of about 18 days. In some embodiments, the first expansion by priming and the rapid second expansion are completed within a period of about 19 days. In some embodiments, the first expansion by priming and the rapid second expansion are completed within a period of about 20 days. In some embodiments, the first expansion by priming and the rapid second expansion are completed within a period of about 21 days.

[0557] In some embodiments, the rapid second expansion is carried out by culturing the second TIL population in a third cell culture medium for a first period of about 5 days, at the end of the first period, dividing the culture into multiple subcultures, and culturing each of the multiple subcultures in a fourth culture medium containing IL-2 for a second period of about 6 days, and at the end of the second period, combining the multiple subcultures to provide the therapeutic TIL population.

[0558] In some embodiments, the present invention provides a method for expanding tumor-infiltrating lymphocytes into a therapeutic TIL population, the method comprising: (a) obtaining and / or receiving a first population of TILs from a sample of tumor tissue produced by surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining tumor tissue from a patient or subject, and storing the sample of tumor tissue in a frozen state, wherein the method for storing the sample of tumor tissue includes the method for cryopreserving tumor tissue described herein; (b) adding a sample of tumor tissue, or tumor fragments obtained from fragmenting a sample of tumor tissue, to a closed system and performing an initial expansion (or first expansion by priming) by culturing a first population of TILs in a first cell culture medium to produce a second population of TILs, wherein the first expansion is performed in a closed container providing a first gas permeable surface area, the first cell culture medium comprising IL-2, optionally OKT-3 (an anti-CD3 antibody), and optionally antigen presenting cells (APCs), and the first expansion by priming occurs over a period of about 1 to 8 days to produce a second population of TILs; (c) performing a second rapid expansion of the second TILs in a second cell culture medium comprising IL-2, OKT-3, and antigen presenting cells (APCs) to produce a third TIL population, wherein the second rapid expansion is performed in a sealed container providing a second gas permeable surface area, wherein the third TIL population is a therapeutic TIL population, and wherein the rapid expansion is performed over a period of no more than 14 days, and optionally, the rapid second expansion can proceed over about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days after initiation of the rapid second expansion, and wherein the transition from step (b) to step (c) occurs without opening the system; (d) harvesting the therapeutic TIL population obtained from step (c), wherein the transition from step (c) to step (d) occurs without opening the system.

[0559] In some embodiments, the present invention provides a method for expanding tumor-infiltrating lymphocytes into a therapeutic TIL population, the method comprising: (a) obtaining and / or receiving a first population of TILs from a sample of tumor tissue produced by surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining tumor tissue from a patient or subject, and storing the sample of tumor tissue in a frozen state, wherein the method for storing the sample of tumor tissue includes the method for cryopreserving tumor tissue described herein; (b) digesting a sample of tumor tissue, or tumor fragments obtained by fragmenting a sample of tumor tissue, in an enzymatic medium to produce a digest; (c) adding tumor digest to a closed system and culturing a first TIL population in a first cell culture medium for initial expansion (or first expansion by priming) to produce a second TIL population, wherein the first expansion is performed in a sealed container providing a first gas permeable surface area, the first cell culture medium comprising IL-2, optionally OKT-3 (an anti-CD3 antibody), and optionally antigen presenting cells (APCs), and the first expansion by priming occurs over a period of about 1 to 8 days; (d) performing a second rapid expansion of the second TILs in a second cell culture medium comprising IL-2, OKT-3, and antigen presenting cells (APCs) to produce a third TIL population, wherein the second rapid expansion is performed in a sealed container providing a second gas permeable surface area, wherein the third TIL population is a therapeutic TIL population, and wherein the rapid expansion is performed over a period of no more than 14 days, and optionally, the rapid second expansion can proceed over about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days after initiation of the rapid second expansion, and wherein the transition from step (c) to step (d) occurs without opening the system; (e) harvesting the therapeutic TIL population obtained from step (c), wherein the transition from step (d) to step (e) occurs without opening the system.

[0560] In some embodiments, the first culture medium comprises APCs and / or OKT-3. In some embodiments, the first culture medium comprises APCs. In some embodiments, the first culture medium comprises OKT-3. In some embodiments, the first culture medium comprises APCs and OKT-3. In some embodiments, the number of APCs in the second culture medium is greater than the number of APCs in the first culture medium.

[0561] In some embodiments, the rapid expansion step is carried out by culturing the second TIL population in a second cell culture medium for a first period of about 3-4 days, at the end of the first period, dividing the culture into multiple subcultures, and culturing each of the multiple subcultures in a third culture medium containing IL-2 for a second period of about 4-6 days, and at the end of the second period, combining the multiple subcultures to provide the therapeutic TIL population.

[0562] In some embodiments, the present invention provides a method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population, comprising: (a) obtaining and / or receiving a first population of TILs from a sample of tumor tissue produced by surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining tumor tissue from a patient or subject, and storing the sample of tumor tissue in a frozen state, wherein the method for storing the sample of tumor tissue includes the method for cryopreserving tumor tissue described herein; (b) selecting PD-1 positive TILs from a first population of TILs in a tumor digest produced from digesting a sample of tumor tissue in an enzymatic medium to obtain a PD-1 enriched population of TILs; (c) performing a first priming expansion by culturing the PD-1-enriched TIL population in a first cell culture medium comprising IL-2, OKT-3, and antigen-presenting cells (APCs) to produce a second TIL population, wherein the first priming expansion is performed in a container comprising a first gas permeable surface area, and the first priming expansion is performed for a first period of about 1 to 7, 8, 9, 10, or 11 days to obtain a second TIL population; (d) performing a second rapid expansion by culturing the second TIL population in a second culture medium comprising IL-2, OKT-3, and APCs, wherein the number of APCs added to the rapid second expansion is at least twice the number of APCs added in step (c), and the rapid second expansion is performed for a second period of time of about 1 to 11 days to obtain a therapeutic TIL population, and the rapid second expansion is performed in a container comprising a second gas permeable surface area; (e) harvesting the therapeutic TIL population obtained from step (d).

[0563] In some embodiments, the present invention provides a method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population, comprising: (a) obtaining and / or receiving a first population of TILs from a sample of tumor tissue produced by surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining tumor tissue from a patient or subject, and storing the sample of tumor tissue in a frozen state, wherein the method for storing the sample of tumor tissue includes the method for cryopreserving tumor tissue described herein; (b) digesting the tumor tissue sample or tumor fragment in an enzymatic medium to produce a tumor digest; (c) selecting PD-1 positive TILs from the first TIL population in the tumor digest of step (b) to obtain a PD-1 enriched TIL population; (d) performing a first priming expansion by culturing the PD-1-enriched TIL population in a first cell culture medium comprising IL-2, OKT-3, and antigen-presenting cells (APCs) to produce a second TIL population, wherein the first priming expansion is performed in a container comprising a first gas permeable surface area, and the first priming expansion is performed for a first period of about 1 to 7, 8, 9, 10, or 11 days to obtain a second TIL population; (e) performing a second rapid expansion by culturing the second TIL population in a second culture medium comprising IL-2, OKT-3, and APCs, wherein the number of APCs added to the rapid second expansion is at least twice the number of APCs added in step (c), and the rapid second expansion is performed for a second period of time of about 1 to 11 days to obtain a therapeutic TIL population, and the rapid second expansion is performed in a container comprising a second gas permeable surface area; (f) harvesting the therapeutic TIL population obtained from step (e).

[0564] In some embodiments, the PD-1 selection step comprises: (i) exposing a first population of TILs and a population of PBMCs to an excess of a monoclonal anti-PD-1 IgG4 antibody that binds to PD-1 by the N-terminal loop outside the IgV domain of PD-1; (ii) adding an excess of anti-IgG4 antibody conjugated to a fluorophore; (iii) obtaining a PD-1-enriched TIL population based on the intensity of the fluorophore of PD-1-positive TILs in the first TIL population compared to the intensity of the PBMC population as determined by fluorescence-activated cell sorting (FACS).

[0565] In some embodiments, the number of APCs in the second culture medium is greater than the number of APCs in the first culture medium.

[0566] In some embodiments, the first expansion step by priming is carried out for about 11 days.

[0567] In some embodiments, the rapid second expansion step is carried out for about 11 days.

[0568] In some embodiments, the rapid expansion step is carried out by culturing the second TIL population in a second cell culture medium for a first period of about 5 days, at the end of the first period, dividing the culture into multiple sub-cultures, and culturing each of the multiple sub-cultures in a third culture medium containing IL-2 for a second period of about 6 days, and at the end of the second period, combining the multiple sub-cultures to provide the therapeutic TIL population.

[0569] In some embodiments, the tumor tissue is derived from a dissected tumor.

[0570] In some embodiments, the dissected tumor is less than 8 hours old.

[0571] In some embodiments, 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 HPV-positive tumor tissue.

[0572] In some embodiments, the tumor tissue is fragmented into approximately spherical fragments having a diameter of about 1.5 mm to 6 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments having a diameter of about 2 mm to 6 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments having a diameter of about 2.5 mm to 6 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments having a diameter of about 3 mm to 6 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments having a diameter of about 3.5 mm to 6 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments having a diameter of about 4 mm to 6 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments having a diameter of about 4.5 mm to 6 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments having a diameter of about 5 mm to 6 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments having a diameter of about 5.5 mm to 6 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments having a diameter of about 1.5 mm to 5 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments having a diameter of about 2 mm to 5 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments having a diameter of about 2.5 mm to 5 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments having a diameter of about 3 mm to 5 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments having a diameter of about 3.5 mm to 5 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments having a diameter of about 4 mm to 5 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments having a diameter of about 4.5 mm to 5 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments having a diameter of about 1.5 mm to 4 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments having a diameter of about 2 mm to 4 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments having a diameter of about 2.5 mm to 4 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments having a diameter of about 3 mm to 4 mm.In some embodiments, the tumor tissue is fragmented into approximately spherical fragments having a diameter of about 3.5 mm to 4 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments having a diameter of about 1.5 mm to 3 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments having a diameter of about 2 mm to 3 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments having a diameter of about 2.5 mm to 3 mm. In some embodiments, the tumor tissue is fragmented into approximately spherical fragments having a diameter of about 1.5 mm to 2 mm.

[0573] In some embodiments, the tumor tissue is fragmented into generally rectangular fragments having a shortest edge length of at least 1.5 mm and a longest edge length of about 6 mm. In some embodiments, the tumor tissue is fragmented into generally rectangular fragments having a shortest edge length of at least 2 mm and a longest edge length of about 6 mm. In some embodiments, the tumor tissue is fragmented into generally rectangular fragments having a shortest edge length of at least 2.5 mm and a longest edge length of about 6 mm. In some embodiments, the tumor tissue is fragmented into generally rectangular fragments having a shortest edge length of at least 3 mm and a longest edge length of about 6 mm. In some embodiments, the tumor tissue is fragmented into generally rectangular fragments having a shortest edge length of at least 3.5 mm and a longest edge length of about 6 mm. In some embodiments, the tumor tissue is fragmented into generally rectangular fragments having a shortest edge length of at least 4 mm and a longest edge length of about 6 mm. In some embodiments, the tumor tissue is fragmented into generally rectangular fragments having a shortest edge length of at least 4.5 mm and a longest edge length of about 6 mm. In some embodiments, the tumor tissue is fragmented into generally rectangular pieces having a shortest edge length of at least 5 mm and a longest edge length of about 6 mm. In some embodiments, the tumor tissue is fragmented into generally rectangular pieces having a shortest edge length of at least 5.5 mm and a longest edge length of about 6 mm.

[0574] In some embodiments, the tumor tissue is fragmented into generally cubic fragme...

Claims

1. A method for cryopreserving tumor tissue, comprising: (i) Freezing placing tumor fragments obtained by fragmenting the tumor tissue in a pre-cooled closable container containing a cryopreservation medium, and closing the container; (ii) incubating the sealed container containing the tumor fragments and the cryopreservation medium at a temperature of about 2 to 8 °C for about 30 to 60 minutes; (iii) slowly freezing the container in a controlled-rate freezer; (iv) transferring the container to a liquid nitrogen freezer.

2. The method according to claim 1, wherein the tumor tissue is fragmented into substantially spherical fragments having a diameter of about 1.5 mm to 6 mm.

3. The method according to claim 2, wherein the tumor tissue is fragmented into substantially spherical fragments having a diameter of about 3 mm or about 6 mm.

4. The method according to claim 1, wherein the tumor tissue is fragmented into generally rectangular fragments having a shortest edge length of at least 1.5 mm and a longest edge length of about 6 mm.

5. The method according to claim 4, wherein the tumor tissue is fragmented into generally cubic fragments having an edge length of about 3 mm or about 6 mm.

6. The method or product according to any one of claims 1 to 5, wherein the cryopreservation medium contains about 2% v / v DMSO to about 15% v / v DMSO.

7. The method or product according to claim 6, wherein the cryopreservation medium contains about 10% v / v DMSO.

8. The method or product according to any one of claims 1 to 5, wherein the cryopreservation medium contains at least one antibacterial agent.

9. The method or product according to claim 8, wherein the cryopreservation medium contains gentamicin at a concentration of at least 50 μg / mL.

10. The method or product according to any one of claims 1 to 5, wherein the closable container is a cryovial.

11. The method or product according to any one of claims 1 to 5, wherein the closable container is filled with the cryopreservation medium at a volume of about 50% to about 85%.

12. The method or product according to any one of claims 1 to 5, wherein the controlled-rate freezer is an IPA-free controlled-rate freezer that cools at a rate of about -0.1 °C / min to about -10 °C / min.

13. The method or product according to claim 12, wherein the controlled-rate freezer is an IPA-free controlled-rate freezer that cools at a rate of about -1 °C / min.

14. The method or product according to any one of claims 1 to 5, wherein all of the positions of the controlled-rate freezer are filled with a closable container containing the cryopreservation medium.

15. The method or product according to any one of claims 1 to 5, wherein said slow freezing comprises incubating the controlled-rate freezer at a temperature of from about -70 °C to about -90 °C.

16. The method or product according to any one of claims 1 to 5, wherein said slow freezing comprises incubating the controlled-rate freezer at a temperature of about -80 °C for about 3 to 5 hours.

17. The method or product according to claim 16, wherein said slow freezing comprises incubating the controlled-rate freezer at a temperature of about -80 °C for about 4 hours.

18. The method or product according to any one of claims 1 to 5, wherein said slow freezing comprises incubating the controlled-rate freezer containing dry ice.

19. The method or product according to any one of claims 1 to 5, wherein said slow freezing comprises incubating the controlled-rate freezer in a -80 °C freezer.

20. The method or product according to any one of claims 1 to 5, wherein after recovery from freezing, the cells have a post-thaw viability of at least about 80%.

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