Proliferation of tumor-infiltrating lymphocytes by potassium channel agonists and their therapeutic use

Abstract

Proliferation of tumor-infiltrating lymphocytes by potassium channel agonists and therapeutic uses thereof are provided. [Solution] K Ca Disclosed herein are methods for expanding tumor infiltrating lymphocytes (TILs) using potassium channel agonists, such as 3.1 (IK channel) agonists, and the use of such expanded TILs in the treatment of diseases such as cancer.

Description

Technical Field

[0001] Cross - reference to Related Applications

[0001] This international application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 504,385, filed May 10, 2017; U.S. Provisional Patent Application No. 62 / 466,921, filed March 3, 2017; and U.S. Provisional Patent Application No. 62 / 443,519, filed January 6, 2017, the entire contents of which are incorporated herein by reference.

[0002] Field of the Invention

[0002] K Ca A method of growing tumor - infiltrating lymphocytes (TIL) using a potassium channel agonist comprising 3.1 and a composition of a population of TIL obtained therefrom are disclosed herein. Further, the therapeutic use of TIL grown using a potassium channel agonist, including the treatment of diseases such as cancer, is disclosed herein.

Background Art

[0003] Background of the Invention

[0003] The treatment of bulky and refractory cancers using adoptive autologous transfer of tumor-infiltrating lymphocytes (TILs) represents a powerful approach to therapy for patients with poor prognoses. Gattinoni, et al, Nat.Rev. Immunol.2006, 6, 383-393. TILs are T cell-dominant, and IL-2-based TIL expansion followed by the "rapid expansion process" (REP) has become the preferred method for 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. Several approaches have been explored to improve the response to TIL therapy in melanoma and to expand TIL therapy to other types of tumors, but with limited success, and this 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.

[0004]

[0004] Potassium channels (K + channels) are a type of transmembrane ion-conducting channels commonly found in cells. K + channels are divided into four major classes: (1) voltage-gated potassium channels (K v ) that open or respond to fluctuations in the membrane potential across the membrane; (2) calcium-activated potassium channels (K Ca ) that open in response to the presence of calcium ions or other signaling molecules; (3) inwardly rectifying potassium channels (K IR); and (4) tandem pore-type potassium channels that are constitutively open or have high basal activation (K 2 p). A very large number of K + channel subtypes are recognized within these four classes. However, only two K + channel subtypes are known to be expressed by T cells. Activated effector T cells express high levels of K v 1.3 (encoded by voltage-dependent potassium channel, shaker-related subfamily member 3, KCNA3), and activated naive and central memory T cell subsets express high levels of K Ca 3.1 (intermediate conductance Ca 2+ activated K + channel, also known as the IK channel or SK4 channel, and encoded by KCNN4). Feske, et al., Annu.Rev. Immunol.2015, 33, 291-353; Di, et al, Proc.Nat’l Acad.Sci.USA 2010, 107, 1541-46. Inhibition of K Ca 3.1 suppresses mouse T cell proliferation and cytokine production. Di, et al, Proc.Nat’l Acad.Sci.USA 2010, 107, 1541-46. Necrotic tissue releases high intracellular stores of K + into the extracellular space, and high intracellular K + concentrations suppress interferon-γ (IFN-γ) production in T cells, and overexpression of K v 1.3 in mouse T cells improves antitumor immunity and host survival. Eil, et al., Nature 2016, 537, 539-543. However, the effect of K + channel manipulation on the proliferation and performance of TILs as a treatment for diseases such as cancer has not been investigated. SUMMARY OF THE INVENTION MEANS FOR SOLVING THE PROBLEM

[0005]

[0005] The present invention relates to KCa 3.1 K channels agonists, openers or activators + Provided is the surprising finding that potassium channel agonists, openers or activators, when used in the TIL expansion process, result in improved TIL phenotypic characteristics and less TIL differentiation.

[0006] SUMMARY OF THE INVENTION

[0006] In one embodiment, the present invention is a method for treating cancer using a population of tumor infiltrating lymphocytes (TIL), comprising: (a) excising a tumor from a patient, the tumor comprising a first population of TIL; (b) fragmenting the tumor into tumor fragments; (c) contacting the tumor fragments with a first cell culture medium; (d) initially expanding the first population of TIL in the first cell culture medium to obtain a second population of TIL, wherein the number of the second population of TIL is at least five times greater than the first population of TIL, and the first cell culture medium comprises IL-2; (e) rapidly expanding the second population of TIL in a second cell culture medium to obtain a third population of TIL, wherein the number of the third population of TIL is at least 50 times greater than the second population of TIL 7 days after the start of the rapid expansion, the second cell culture medium comprises IL-2, OKT-3 (anti-CD3 antibody) and irradiated allogeneic peripheral blood mononuclear cells (PBMC), and the rapid expansion is carried out over a period of 14 days or less; (f) recovering the third population of TIL; and (g) administering a therapeutically effective portion of the third population of TIL to a patient having cancer wherein either the first cell culture medium or the second cell culture medium or both the first cell culture medium and the second cell culture medium further comprise a potassium channel agonist.

[0007]

[0007] In one embodiment, the present invention is a method for treating cancer using a population of tumor infiltrating lymphocytes (TIL), comprising: (a) A step of removing a tumor from a patient, wherein the tumor comprises a first population of tumor-infiltrating lymphocytes (TIL); (b) A step of fragmenting the tumor into tumor fragments; (c) A step of contacting the tumor fragments with a first cell culture medium; (d) A step of performing initial expansion of the first population of TIL in the first cell culture medium to obtain a second population of TIL, wherein the number of the second population of TIL is at least five times more than that of the first population of TIL, and the first cell culture medium contains IL-2; (e) A step of performing rapid expansion of the second population of TIL in a second cell culture medium to obtain a third population of TIL, wherein the number of the third population of TIL is at least 50 times more than that of the second population of TIL 7 days after the start of rapid expansion, the second cell culture medium contains IL-2, OKT-3 (anti-CD3 antibody) and irradiated allogeneic peripheral blood mononuclear cells (PBMC), and the rapid expansion is performed over a period of 14 days or less; (f) A step of recovering the third population of TIL; and (g) A step of administering a therapeutically effective portion of the third population of TIL to a patient having cancer comprising, either the first cell culture medium or the second cell culture medium or both the first cell culture medium and the second cell culture medium further contain a potassium channel agonist, and the potassium channel agonist is a K Ca 3.1 (IK channel) agonist, and provides a method.

[0008]

[0008] In one embodiment, the present invention is a method for treating cancer using a population of tumor-infiltrating lymphocytes (TIL), (a) A step of removing a tumor from a patient, wherein the tumor comprises a first population of TIL; (b) A step of fragmenting the tumor into tumor fragments; (c) A step of contacting the tumor fragments with a first cell culture medium; (d) A step of initially expanding a first population of TILs in a first cell culture medium to obtain a second population of TILs, wherein the number of the second population of TILs is at least five times more than that of the first population of TILs, and the first cell culture medium contains IL-2; (e) A step of rapidly expanding the second population of TILs in a second cell culture medium to obtain a third population of TILs, wherein the number of the third population of TILs is at least 50 times more than that of the second population of TILs 7 days after the start of the rapid expansion, the second cell culture medium contains IL-2, OKT-3 (anti-CD3 antibody) and irradiated allogeneic peripheral blood mononuclear cells (PBMCs), and the rapid expansion is carried out over a period of 14 days or less; (f) A step of recovering the third population of TILs; and (g) A step of administering a therapeutically effective portion of the third population of TILs to a patient having cancer comprising, either the first cell culture medium or the second cell culture medium or both the first cell culture medium and the second cell culture medium further contain a potassium channel agonist, and the potassium channel agonist is a K Ca 3.1 (IK channel) agonist, and the second population of TILs contains a population of T cells having a phenotype selected from the group consisting of increased CD8 + CD28 + , CD8 + CD27 + , CD8 + CD27 + CD28 + , CCR7 + and combinations thereof, and the phenotype in the second population of TILs is increased by at least 5% compared to the reference population of TILs obtained without the potassium channel agonist. In some examples, the increase is about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 200%, about 300%, about 400%, about 500%, about 600%, about 700%, about 800%, about 900% or about 1000%.

[0009]

[0009] In one embodiment, the present invention is a method for treating cancer using a population of tumor infiltrating lymphocytes (TILs), comprising: (a) excising a tumor from a patient, the tumor comprising a first population of TILs; (b) fragmenting the tumor into tumor fragments; (c) contacting the tumor fragments with a first cell culture medium; (d) performing an initial expansion of the first population of TILs in the first cell culture medium to obtain a second population of TILs, wherein the number of the second population of TILs is at least five-fold greater than the first population of TILs, and the first cell culture medium comprises IL-2; (e) performing a rapid expansion of the second population of TILs in a second cell culture medium to obtain a third population of TILs, wherein the number of the third population of TILs is at least 50-fold greater than the second population of TILs seven days after the start of the rapid expansion, the second cell culture medium comprises IL-2, OKT-3 (anti-CD3 antibody) and irradiated allogeneic peripheral blood mononuclear cells (PBMCs), and the rapid expansion is performed over a period of 14 days or less; (f) recovering the third population of TILs; and (g) administering a therapeutically effective portion of the third population of TILs to a patient having cancer wherein either the first cell culture medium or the second cell culture medium or both the first cell culture medium and the second cell culture medium further comprise a potassium channel agonist, the potassium channel agonist is a K Ca 3.1 (IK channel) agonist, and the third population of TILs has an increase in CD8 + CD28 + 、CD8 + CD27 + 、CD8 + CD27 + CD28 + 、CCR7 +A method is provided that includes a population of T cells having a phenotype selected from the group consisting of and combinations thereof, wherein the phenotype in the third population of TILs is at least 5% increased relative to a reference population of TILs. In some examples, the increase is about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 200%, about 300%, about 400%, about 500%, about 600%, about 700%, about 800%, about 900% or about 1000%.

[0010]

[0010] In one embodiment, the present invention is a method of treating cancer using a population of tumor infiltrating lymphocytes (TILs), comprising: (a) excising a tumor from a patient, the tumor comprising a first population of TILs; (b) fragmenting the tumor into tumor fragments; (c) contacting the tumor fragments with a first cell culture medium; (d) performing an initial expansion of the first population of TILs in the first cell culture medium to obtain a second population of TILs, wherein the number of the second population of TILs is at least five-fold greater than the first population of TILs, and the first cell culture medium comprises IL-2; (e) performing a rapid expansion of the second population of TILs in a second cell culture medium to obtain a third population of TILs, wherein the number of the third population of TILs is at least 50-fold greater than the second population of TILs 7 days after the start of the rapid expansion, the second cell culture medium comprises IL-2, OKT-3 (anti-CD3 antibody) and irradiated allogeneic peripheral blood mononuclear cells (PBMCs), and the rapid expansion is performed over a period of 14 days or less; (f) recovering the third population of TILs; and (g) administering a therapeutically effective portion of the third population of TILs to a patient having cancer wherein the potassium channel agonist is K Ca 3.1 (IK channel) agonist, and both the first cell culture media are K Ca3.1 Further comprising an agonist, the second cell culture medium being K Ca 3.1 Provide a method further comprising an agonist.

[0011]

[0011] In one embodiment, the present invention is a method for treating cancer using a population of tumor-infiltrating lymphocytes (TILs), (a) Removing a tumor from a patient, the tumor comprising a first population of TILs; (b) Fragmenting the tumor into tumor fragments; (c) Contacting the tumor fragments with a first cell culture medium; (d) Performing initial expansion of the first population of TILs in the first cell culture medium to obtain a second population of TILs, the number of the second population of TILs being at least five times more than that of the first population of TILs, the first cell culture medium comprising IL-2; (e) Performing rapid expansion of the second population of TILs in a second cell culture medium to obtain a third population of TILs, the number of the third population of TILs being at least 50 times more than that of the second population of TILs 7 days after the start of rapid expansion, the second cell culture medium comprising IL-2, OKT-3 (anti-CD3 antibody) and irradiated allogeneic peripheral blood mononuclear cells (PBMCs), and the rapid expansion being performed over a period of 14 days or less; (f) Recovering the third population of TILs; and (g) Administering a therapeutically effective portion of the third population of TILs to a patient having cancer wherein either the first cell culture medium or the second cell culture medium or both the first cell culture medium and the second cell culture medium further comprise a potassium channel agonist, and the potassium channel agonist is K Ca 3.1 (IK channel) agonist, and the concentration of the K Ca 3.1 agonist in the first cell culture medium is 1 - 1000 nM, to provide a method.

[0012]

[0012] In one embodiment, the present invention is a method for treating cancer using a population of tumor-infiltrating lymphocytes (TILs), (a) A step of removing a tumor from a patient, wherein the tumor comprises a first population of tumor-infiltrating lymphocytes (TIL); (b) A step of fragmenting the tumor into tumor fragments; (c) A step of contacting the tumor fragments with a first cell culture medium; (d) A step of performing initial expansion of the first population of TIL in the first cell culture medium to obtain a second population of TIL, wherein the number of the second population of TIL is at least five times more than that of the first population of TIL, and the first cell culture medium comprises IL-2; (e) A step of performing rapid expansion of the second population of TIL in a second cell culture medium to obtain a third population of TIL, wherein the number of the third population of TIL is at least 50 times more than that of the second population of TIL 7 days after the start of the rapid expansion, the second cell culture medium comprises IL-2, OKT-3 (anti-CD3 antibody) and irradiated allogeneic peripheral blood mononuclear cells (PBMC), and the rapid expansion is performed over a period of 14 days or less; (f) A step of recovering the third population of TIL; and (g) A step of administering a therapeutically effective portion of the third population of TIL to a patient having cancer comprising, either the first cell culture medium or the second cell culture medium or both the first cell culture medium and the second cell culture medium further comprises a potassium channel agonist, and the potassium channel agonist is a K Ca 3.1 (IK channel) agonist, and the concentration of the K Ca 3.1 agonist in the first cell culture medium is about 100 nM, providing a method.

[0013]

[0013] In one embodiment, the present invention is a method for treating cancer using a population of tumor-infiltrating lymphocytes (TIL), (a) A step of removing a tumor from a patient, wherein the tumor comprises a first population of TIL; (b) A step of fragmenting the tumor into tumor fragments; (c) A step of contacting the tumor fragments with a first cell culture medium; (d) A step of initially expanding a first population of tumor-infiltrating lymphocytes (TILs) in a first cell culture medium to obtain a second population of TILs, wherein the number of the second population of TILs is at least five times more than that of the first population of TILs, and the first cell culture medium contains IL-2; (e) A step of rapidly expanding the second population of TILs in a second cell culture medium to obtain a third population of TILs, wherein the number of the third population of TILs is at least 50 times more than that of the second population of TILs 7 days after the start of the rapid expansion, the second cell culture medium contains IL-2, OKT-3 (anti-CD3 antibody) and irradiated allogeneic peripheral blood mononuclear cells (PBMCs), and the rapid expansion is carried out over a period of 14 days or less; (f) A step of recovering the third population of TILs; and (g) A step of administering a therapeutically effective portion of the third population of TILs to a patient having cancer comprising, either the first cell culture medium or the second cell culture medium or both the first cell culture medium and the second cell culture medium further contain a potassium channel agonist, and the potassium channel agonist is a K Ca 3.1 (IK channel) agonist, and the concentration of the K Ca 3.1 agonist in the second cell culture medium is 0.1 - 100 mM, providing a method.

[0014]

[0014] In one embodiment, the present invention is a method for treating cancer using a population of tumor-infiltrating lymphocytes (TILs), (a) A step of excising a tumor from a patient, wherein the tumor contains a first population of TILs; (b) A step of fragmenting the tumor into tumor fragments; (c) A step of contacting the tumor fragments with a first cell culture medium; (d) A step of initially expanding a first population of TILs in a first cell culture medium to obtain a second population of TILs, wherein the number of the second population of TILs is at least five times more than that of the first population of TILs, and the first cell culture medium contains IL-2; (e) A step of rapidly expanding a second population of tumor infiltrating lymphocytes (TIL) in a second cell culture medium to obtain a third population of TIL, wherein the number of the third population of TIL is at least 50 times more than that of the second population of TIL 7 days after the start of rapid expansion, the second cell culture medium contains IL-2, OKT-3 (anti-CD3 antibody) and irradiated allogeneic peripheral blood mononuclear cells (PBMC), and the rapid expansion is carried out over a period of 14 days or less; (f) A step of collecting the third population of TIL; and (g) A step of administering a therapeutically effective portion of the third population of TIL to a patient having cancer comprising, either the first cell culture medium or the second cell culture medium or both the first cell culture medium and the second cell culture medium further contain a potassium channel agonist, and the potassium channel agonist is K Ca 3.1 (IK channel) agonist, and the concentration of the K Ca 3.1 agonist in the second cell culture medium is about 50 mM, providing a method.

[0015]

[0015] In one embodiment, the present invention is a method for treating cancer using a population of tumor infiltrating lymphocytes (TIL), comprising (a) A step of excising a tumor from a patient, wherein the tumor contains a first population of TIL; (b) A step of fragmenting the tumor into tumor fragments; (c) A step of contacting the tumor fragments with a first cell culture medium; (d) A step of initially expanding a first population of TIL in the first cell culture medium to obtain a second population of TIL, wherein the number of the second population of TIL is at least 5 times more than that of the first population of TIL, and the first cell culture medium contains IL-2; (e) A step of rapidly growing a second population of TILs in a second cell culture medium to obtain a third population of TILs, wherein the number of the third population of TILs is at least 50 times more than that of the second population of TILs 7 days after the start of the rapid growth, the second cell culture medium contains IL-2, OKT-3 (anti-CD3 antibody) and irradiated allogeneic peripheral blood mononuclear cells (PBMCs), and the rapid growth is carried out over a period of 14 days or less; (f) A step of collecting the third population of TILs; and (g) A step of administering a therapeutically effective portion of the third population of TILs to a patient having cancer comprising, either the first cell culture medium or the second cell culture medium or both the first cell culture medium and the second cell culture medium further contain a potassium channel agonist, and the potassium channel agonist is a K Ca 3.1 (IK channel) agonist, and the initial growth is carried out over a period of 21 days or less, providing a method.

[0016]

[0016] In one embodiment, the present invention is a method for treating cancer using a population of tumor infiltrating lymphocytes (TILs), (a) A step of excising a tumor from a patient, wherein the tumor contains a first population of TILs; (b) A step of fragmenting the tumor into tumor fragments; (c) A step of contacting the tumor fragments with a first cell culture medium; (d) A step of initially growing a first population of TILs in the first cell culture medium to obtain a second population of TILs, wherein the number of the second population of TILs is at least 5 times more than that of the first population of TILs, and the first cell culture medium contains IL-2; (e) A step of rapidly growing a second population of TILs in a second cell culture medium to obtain a third population of TILs, wherein the number of the third population of TILs is at least 50 times more than that of the second population of TILs 7 days after the start of the rapid growth, the second cell culture medium contains IL-2, OKT-3 (anti-CD3 antibody) and irradiated allogeneic peripheral blood mononuclear cells (PBMCs), and the rapid growth is carried out over a period of 14 days or less; (f) Step of collecting the third population of TILs; and (g) Step of administering a therapeutically effective portion of the third population of TILs to a patient having cancer comprising, wherein either the first cell culture medium or the second cell culture medium or both the first cell culture medium and the second cell culture medium further comprises a potassium channel agonist, and the potassium channel agonist is a K Ca 3.1 (IK channel) agonist, and the initial proliferation is carried out over a period of 11 days or less, to provide a method.

[0017]

[0017] In one embodiment, the present invention is a method of treating cancer using a population of tumor infiltrating lymphocytes (TILs), comprising: (a) Step of excising a tumor from a patient, wherein the tumor comprises a first population of TILs; (b) Step of fragmenting the tumor into tumor fragments; (c) Step of contacting the tumor fragments with a first cell culture medium; (d) Step of performing initial proliferation of the first population of TILs in the first cell culture medium to obtain a second population of TILs, wherein the number of the second population of TILs is at least 5 times more than that of the first population of TILs, and the first cell culture medium comprises IL-2; (e) Step of performing rapid proliferation of the second population of TILs in the second cell culture medium to obtain a third population of TILs, wherein the number of the third population of TILs is at least 50 times more than that of the second population of TILs 7 days after the start of the rapid proliferation, and the second cell culture medium comprises IL-2, OKT-3 (anti-CD3 antibody) and irradiated allogeneic peripheral blood mononuclear cells (PBMCs), and the rapid proliferation is carried out over a period of 14 days or less; (f) Step of collecting the third population of TILs; and (g) Step of administering a therapeutically effective portion of the third population of TILs to a patient having cancer comprising, wherein either the first cell culture medium or the second cell culture medium or both the first cell culture medium and the second cell culture medium further comprises a potassium channel agonist, and the potassium channel agonist is a KCa A method that is a 3.1 (IK channel) agonist and in which rapid expansion is carried out over a period of 7 days or less is provided.

[0018]

[0018] In one embodiment, the present invention is a method for treating cancer using a population of tumor infiltrating lymphocytes (TIL), comprising: (a) excising a tumor from a patient, wherein the tumor comprises a first population of TIL; (b) fragmenting the tumor into tumor fragments; (c) contacting the tumor fragments with a first cell culture medium; (d) performing initial expansion of the first population of TIL in the first cell culture medium to obtain a second population of TIL, wherein the number of the second population of TIL is at least 5-fold more than that of the first population of TIL, and the first cell culture medium contains IL-2; (e) performing rapid expansion of the second population of TIL in a second cell culture medium to obtain a third population of TIL, wherein the number of the third population of TIL is at least 50-fold more than that of the second population of TIL 7 days after the start of rapid expansion, the second cell culture medium contains IL-2, OKT-3 (anti-CD3 antibody) and irradiated allogeneic peripheral blood mononuclear cells (PBMC), and rapid expansion is carried out over a period of 14 days or less; (f) recovering the third population of TIL; and (g) administering a therapeutically effective portion of the third population of TIL to a patient having cancer wherein either the first cell culture medium or the second cell culture medium or both the first cell culture medium and the second cell culture medium further contain a potassium channel agonist, and the potassium channel agonist is a K Ca 3.1 (IK channel) agonist, and the K Ca 3.1 agonist has the formula (1):

Chemical formula

[0019]

[0019] In one embodiment, the present invention is a method for treating cancer using a population of tumor infiltrating lymphocytes (TIL), comprising: (a) removing a tumor from a patient, the tumor comprising a first population of TIL; (b) fragmenting the tumor into tumor fragments; (c) contacting the tumor fragments with a first cell culture medium; (d) performing initial expansion of the first population of TIL in the first cell culture medium to obtain a second population of TIL, wherein the number of the second population of TIL is at least five times more than that of the first population of TIL, and the first cell culture medium contains IL-2; (e) performing rapid expansion of the second population of TIL in a second cell culture medium to obtain a third population of TIL, wherein the number of the third population of TIL is at least 50 times more than that of the second population of TIL 7 days after the start of rapid expansion, the second cell culture medium contains IL-2, OKT-3 (anti-CD3 antibody) and irradiated allogeneic peripheral blood mononuclear cells (PBMC), and the rapid expansion is performed over a period of 14 days or less; (f) recovering the third population of TIL; and (g) administering a therapeutically effective portion of the third population of TIL to a patient having cancer wherein either the first cell culture medium or the second cell culture medium or both the first cell culture medium and the second cell culture medium further contain a potassium channel agonist, and the potassium channel agonist is a K Ca 3.1 (IK channel) agonist, and the K Ca 3.1 agonist is naphtho[1,2-d]thiazol-2-ylamine (SKA-31):

Chemical formula

[0020] In one embodiment, the present invention is a method of treating cancer using a population of tumor-infiltrating lymphocytes (TIL), comprising: (a) removing a tumor from a patient, the tumor comprising a first population of TIL; (b) fragmenting the tumor into tumor fragments; (c) contacting the tumor fragments with a first cell culture medium; (d) performing initial expansion of the first population of TIL in the first cell culture medium to obtain a second population of TIL, wherein the number of the second population of TIL is at least five-fold greater than the first population of TIL, and the first cell culture medium comprises IL-2; (e) performing rapid expansion of the second population of TIL in a second cell culture medium to obtain a third population of TIL, wherein the number of the third population of TIL is at least 50-fold greater than the second population of TIL 7 days after the start of rapid expansion, the second cell culture medium comprises IL-2, OKT-3 (anti-CD3 antibody) and irradiated allogeneic peripheral blood mononuclear cells (PBMC), and the rapid expansion is performed over a period of 14 days or less; (f) recovering the third population of TIL; and (g) administering a therapeutically effective portion of the third population of TIL to a patient having cancer wherein either the first cell culture medium or the second cell culture medium or both the first cell culture medium and the second cell culture medium further comprise a potassium channel agonist, and the potassium channel agonist is a K Ca 3.1 (IK channel) agonist, and the K Ca 3.1 agonist is anthra[2,1-d]thiazol-2-ylamine (SKA-20): [Chemical formula] or a pharmaceutically acceptable salt, co-crystal, solvate or prodrug thereof. Provided is a method.

[0021] In one embodiment, the present invention is a method of treating cancer using a population of tumor-infiltrating lymphocytes (TIL), comprising: (a) A step of removing a tumor from a patient, wherein the tumor comprises a first population of tumor-infiltrating lymphocytes (TIL); (b) A step of fragmenting the tumor into tumor fragments; (c) A step of contacting the tumor fragments with a first cell culture medium; (d) A step of performing initial expansion of the first population of TIL in the first cell culture medium to obtain a second population of TIL, wherein the number of the second population of TIL is at least five times more than that of the first population of TIL, and the first cell culture medium contains IL-2; (e) A step of performing rapid expansion of the second population of TIL in a second cell culture medium to obtain a third population of TIL, wherein the number of the third population of TIL is at least 50 times more than that of the second population of TIL 7 days after the start of rapid expansion, the second cell culture medium contains IL-2, OKT-3 (anti-CD3 antibody) and irradiated allogeneic peripheral blood mononuclear cells (PBMC), and the rapid expansion is performed over a period of 14 days or less; (f) A step of recovering the third population of TIL; and (g) A step of administering a therapeutically effective portion of the third population of TIL to a patient having cancer comprising, either the first cell culture medium or the second cell culture medium or both the first cell culture medium and the second cell culture medium further contain a potassium channel agonist, and the potassium channel agonist is a K Ca 3.1 (IK channel) agonist, and the K Ca 3.1 agonist is 6,7-dichloro-1H-indole-2,3-dione 3-oxime (NS309): [Chemical formula] or a pharmaceutically acceptable salt, co-crystal, solvate or prodrug thereof, provides a method.

[0022] In one embodiment, the present invention is a method for treating cancer using a population of tumor-infiltrating lymphocytes (TIL), (a) A step of removing a tumor from a patient, wherein the tumor comprises a first population of tumor-infiltrating lymphocytes (TIL); (b) A step of fragmenting the tumor into tumor fragments; (c) A step of contacting the tumor fragments with a first cell culture medium; (d) A step of performing initial expansion of the first population of TIL in the first cell culture medium to obtain a second population of TIL, wherein the number of the second population of TIL is at least five times greater than that of the first population of TIL, and the first cell culture medium contains IL-2; (e) A step of performing rapid expansion of the second population of TIL in a second cell culture medium to obtain a third population of TIL, wherein the number of the third population of TIL is at least 50 times greater than that of the second population of TIL 7 days after the start of rapid expansion, the second cell culture medium contains IL-2, OKT-3 (anti-CD3 antibody) and irradiated allogeneic peripheral blood mononuclear cells (PBMC), and the rapid expansion is performed over a period of 14 days or less; (f) A step of harvesting the third population of TIL; and (g) A step of administering a therapeutically effective portion of the third population of TIL to a patient having cancer comprising, either the first cell culture medium or the second cell culture medium or both the first cell culture medium and the second cell culture medium further contain a potassium channel agonist, and the potassium channel agonist is a K Ca 3.1 (IK channel) agonist, and the K Ca 3.1 agonist is riluzole: [Chemical formula] or a pharmaceutically acceptable salt, co-crystal, solvate or prodrug thereof, to provide a method.

[0023]

[0023] In one embodiment, the present invention is a method for treating cancer using a population of tumor-infiltrating lymphocytes (TIL), (a) A step of removing a tumor from a patient, wherein the tumor comprises a first population of TIL; (b) A step of fragmenting the tumor into tumor fragments; (c) Step of contacting the tumor fragment with a first cell culture medium; (d) Step of performing initial proliferation of a first population of TILs in the first cell culture medium to obtain a second population of TILs, wherein the number of the second population of TILs is at least five times more than that of the first population of TILs, and the first cell culture medium contains IL-2; (e) Step of performing rapid proliferation of the second population of TILs in a second cell culture medium to obtain a third population of TILs, wherein the number of the third population of TILs is at least 50 times more than that of the second population of TILs 7 days after the start of the rapid proliferation, the second cell culture medium contains IL-2, OKT-3 (anti-CD3 antibody) and irradiated allogeneic peripheral blood mononuclear cells (PBMCs), and the rapid proliferation is performed over a period of 14 days or less; (f) Step of recovering the third population of TILs; and (g) Step of administering a therapeutically effective portion of the third population of TILs to a patient having cancer comprising, either the first cell culture medium or the second cell culture medium or both the first cell culture medium and the second cell culture medium further contain a potassium channel agonist, and the potassium channel agonist is K Ca 3.1 (IK channel) agonist, and the K Ca 3.1 agonist is 5-methylnaphtho[1,2-d]oxazol-2-amine; 5-ethylnaphtho[1,2-d]oxazol-2-amine; 5-propylnaphtho[1,2-d]oxazol-2-amine; 5-cyclopropylnaphtho[1,2-d]oxazol-2-amine; 5-(tert-butyl)naphtho[1,2-d]oxazol-2-amine; 5-fluoronaphtho[1,2-d]oxazol-2-amine; 5-chloronaphtho[1,2-d]oxazol-2-amine; 5-bromonaphtho[1,2-d]oxazol-2-amine; 5-iodonaphtho[1,2-d]oxazol-2-amine; 2-Aminonaphtho[1,2-d]oxazole-5-carbonitrile; Naphtho[1,2-d]oxazole-2,5-diamine; N 5 -Methylnaphtho[1,2-d]oxazole-2,5-diamine; N 5 ,N 5 -Dimethylnaphtho[1,2-d]oxazole-2,5-diamine; N 5 -Ethylnaphtho[1,2-d]oxazole-2,5-diamine; 5-(Pyrrolidin-1-yl)naphtho[1,2-d]oxazol-2-amine; 5-Methoxynaphtho[1,2-d]oxazol-2-amine; 5-Trifluoromethylnaphtho[1,2-d]oxazol-2-amine; 5-Methylnaphtho[2,1-d]oxazol-2-amine; 5-Ethylnaphtho[2,1-d]oxazol-2-amine; 5-Propylnaphtho[2,1-d]oxazol-2-amine; 5-Cyclopropylnaphtho[2,1-d]oxazol-2-amine; 5-(tert-Butyl)naphtho[2,1-d]oxazol-2-amine; 5-Fluoronaphtho[2,1-d]oxazol-2-amine; 5-Chloronaphtho[2,1-d]oxazol-2-amine; 5-Bromonaphtho[2,1-d]oxazol-2-amine; 5-Iodonaphtho[2,1-d]oxazol-2-amine; 2-Aminonaphtho[2,1-d]oxazole-5-carbonitrile; Naphtho[2,1-d]oxazole-2,5-diamine; N5-Methylnaphtho[2,1-d]oxazole-2,5-diamine; N5,N5-Dimethylnaphtho[2,1-d]oxazole-2,5-diamine; N5-Ethylnaphtho[2,1-d]oxazole-2,5-diamine; 5-(Pyrrolidin-1-yl)naphtho[2,1-d]oxazol-2-amine; 5-Methoxynaphtho[2,1-d]oxazol-2-amine; 5-Trifluoromethylnaphtho[2,1-d]oxazol-2-amine; and its pharmaceutically acceptable salts, co-crystals, solvates or prodrugs A method is provided that is selected from the group consisting of.

[0024]

[0024] In one embodiment, the present invention is a method of treating cancer using a population of tumor infiltrating lymphocytes (TIL), comprising: (a) removing a tumor from a patient, the tumor comprising a first population of TIL; (b) fragmenting the tumor into tumor fragments; (c) contacting the tumor fragments with a first cell culture medium; (d) performing an initial expansion of the first population of TIL in the first cell culture medium to obtain a second population of TIL, wherein the number of the second population of TIL is at least five times greater than the first population of TIL, and the first cell culture medium comprises IL-2; (e) performing a rapid expansion of the second population of TIL in a second cell culture medium to obtain a third population of TIL, wherein the number of the third population of TIL is at least 50 times greater than the second population of TIL 7 days after the start of the rapid expansion, the second cell culture medium comprises IL-2, OKT-3 (anti-CD3 antibody) and irradiated allogeneic peripheral blood mononuclear cells (PBMC), and the rapid expansion is performed over a period of 14 days or less; (f) recovering the third population of TIL; and (g) administering a therapeutically effective portion of the third population of TIL to a patient having cancer comprising, wherein either the first cell culture medium or the second cell culture medium or both the first cell culture medium and the second cell culture medium further comprise a potassium channel agonist, and IL-2 is present at an initial concentration of 1000 IU / mL to 6000 IU / mL in the first cell culture medium, a method is provided.

[0025]

[0025] In one embodiment, the present invention is a method for treating cancer using a population of tumor infiltrating lymphocytes (TIL), (a) a step of excising a tumor from a patient, wherein the tumor comprises a first population of TIL; (b) a step of fragmenting the tumor into tumor fragments; (c) a step of contacting the tumor fragments with a first cell culture medium; (d) a step of performing initial expansion of the first population of TIL in the first cell culture medium to obtain a second population of TIL, wherein the number of the second population of TIL is at least 5 times more than that of the first population of TIL, and the first cell culture medium comprises IL-2; (e) a step of performing rapid expansion of the second population of TIL in a second cell culture medium to obtain a third population of TIL, wherein the number of the third population of TIL is at least 50 times more than that of the second population of TIL 7 days after the start of rapid expansion, the second cell culture medium comprises IL-2, OKT-3 (anti-CD3 antibody) and irradiated allogeneic peripheral blood mononuclear cells (PBMC), and the rapid expansion is performed over a period of 14 days or less; (f) a step of recovering the third population of TIL; and (g) a step of administering a therapeutically effective portion of the third population of TIL to a patient having cancer comprising, wherein either the first cell culture medium or the second cell culture medium or both the first cell culture medium and the second cell culture medium further comprise a potassium channel agonist, in the second cell culture medium, 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, a method is provided.

[0026]

[0026] In one embodiment, the present invention is a method of treating cancer using a population of tumor-infiltrating lymphocytes (TILs), comprising: (a) removing a tumor from a patient, the tumor comprising a first population of TILs; (b) fragmenting the tumor into tumor fragments; (c) contacting the tumor fragments with a first cell culture medium; (d) performing an initial expansion of the first population of TILs in the first cell culture medium to obtain a second population of TILs, wherein the number of the second population of TILs is at least five-fold greater than the first population of TILs, and the first cell culture medium comprises IL-2; (e) performing a rapid expansion of the second population of TILs in a second cell culture medium to obtain a third population of TILs, wherein the number of the third population of TILs is at least 50-fold greater than the second population of TILs 7 days after the start of the rapid expansion, the second cell culture medium comprises IL-2, OKT-3 (anti-CD3 antibody) and irradiated allogeneic peripheral blood mononuclear cells (PBMCs), and the rapid expansion is performed over a period of 14 days or less; (f) recovering the third population of TILs; and (g) administering a therapeutically effective portion of the third population of TILs to a patient having cancer wherein either the first cell culture medium or the second cell culture medium or both the first cell culture medium and the second cell culture medium further comprise a potassium channel agonist, and the initial expansion is performed using a gas-permeable container.

[0027]

[0027] In one embodiment, the present invention is a method of treating cancer using a population of tumor-infiltrating lymphocytes (TILs), comprising: (a) removing a tumor from a patient, the tumor comprising a first population of TILs; (b) fragmenting the tumor into tumor fragments; (c) contacting the tumor fragments with a first cell culture medium; (d) A step of initially expanding a first population of tumor-infiltrating lymphocytes (TILs) in a first cell culture medium to obtain a second population of TILs, wherein the number of the second population of TILs is at least five times more than that of the first population of TILs, and the first cell culture medium contains IL-2; (e) A step of rapidly expanding the second population of TILs in a second cell culture medium to obtain a third population of TILs, wherein the number of the third population of TILs is at least 50 times more than that of the second population of TILs 7 days after the start of the rapid expansion, the second cell culture medium contains IL-2, OKT-3 (anti-CD3 antibody) and irradiated allogeneic peripheral blood mononuclear cells (PBMCs), and the rapid expansion is carried out over a period of 14 days or less; (f) A step of recovering the third population of TILs; and (g) A step of administering a therapeutically effective portion of the third population of TILs to a patient having cancer comprising, either the first cell culture medium or the second cell culture medium or both the first cell culture medium and the second cell culture medium further contain a potassium channel agonist, and the rapid expansion is carried out using a gas-permeable container, and provides a method.

[0028]

[0028] In one embodiment, the present invention is a method for treating cancer using a population of tumor-infiltrating lymphocytes (TILs), (a) A step of excising a tumor from a patient, wherein the tumor contains a first population of TILs; (b) A step of fragmenting the tumor into tumor fragments; (c) A step of contacting the tumor fragments with a first cell culture medium; (d) A step of initially expanding a first population of TILs in a first cell culture medium to obtain a second population of TILs, wherein the number of the second population of TILs is at least five times more than that of the first population of TILs, and the first cell culture medium contains IL-2; (e) A step of rapidly expanding a second population of tumor infiltrating lymphocytes (TIL) in a second cell culture medium to obtain a third population of TIL, wherein the number of the third population of TIL is at least 50 times more than that of the second population of TIL 7 days after the start of the rapid expansion, the second cell culture medium contains IL-2, OKT-3 (anti-CD3 antibody) and irradiated allogeneic peripheral blood mononuclear cells (PBMC), and the rapid expansion is carried out over a period of 14 days or less; (f) A step of collecting the third population of TIL; and (g) A step of administering a therapeutically effective portion of the third population of TIL to a patient having cancer comprising, either the first cell culture medium or the second cell culture medium or both the first cell culture medium and the second cell culture medium further contain a potassium channel agonist, and the first cell culture medium further contains a cytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21 and combinations thereof, a method is provided.

[0029]

[0029] In one embodiment, the present invention is a method for treating cancer using a population of tumor infiltrating lymphocytes (TIL), (a) A step of excising a tumor from a patient, wherein the tumor contains a first population of TIL; (b) A step of fragmenting the tumor into tumor fragments; (c) A step of contacting the tumor fragments with a first cell culture medium; (d) A step of initially expanding a first population of TIL in a first cell culture medium to obtain a second population of TIL, wherein the number of the second population of TIL is at least 5 times more than that of the first population of TIL, and the first cell culture medium contains IL-2; (e) A step of rapidly expanding a second population of TIL in a second cell culture medium to obtain a third population of TIL, wherein the number of the third population of TIL is at least 50 times more than that of the second population of TIL 7 days after the start of the rapid expansion, the second cell culture medium contains IL-2, OKT-3 (anti-CD3 antibody) and irradiated allogeneic peripheral blood mononuclear cells (PBMC), and the rapid expansion is carried out over a period of 14 days or less; (f) Step of recovering a third population of TILs; and (g) Step of administering a therapeutically effective portion of the third population of TILs to a patient having cancer comprising, wherein either the first cell culture medium or the second cell culture medium or both the first cell culture medium and the second cell culture medium further comprises a potassium channel agonist, and the second cell culture medium further comprises a cytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21, and combinations thereof, a method is provided.

[0030]

[0030] In one embodiment, the present invention is a method for treating cancer using a population of tumor infiltrating lymphocytes (TILs), (a) Step of excising a tumor from a patient, wherein the tumor comprises a first population of TILs, the step; (b) Step of fragmenting the tumor into tumor fragments; (c) Step of contacting the tumor fragments with a first cell culture medium; (d) Step of performing initial expansion of the first population of TILs in the first cell culture medium to obtain a second population of TILs, wherein the number of the second population of TILs is at least five times more than the first population of TILs, and the first cell culture medium comprises IL-2, the step; (e) Step of performing rapid expansion of the second population of TILs in the second cell culture medium to obtain a third population of TILs, wherein the number of the third population of TILs is at least 50 times more than the second population of TILs 7 days after the start of rapid expansion, and the second cell culture medium comprises IL-2, OKT-3 (anti-CD3 antibody), and irradiated allogeneic peripheral blood mononuclear cells (PBMCs), and the rapid expansion is performed over a period of 14 days or less, the step; (f) Step of recovering a third population of TILs; and (g) Step of administering a therapeutically effective portion of the third population of TILs to a patient having cancer comprising, wherein either the first cell culture medium or the second cell culture medium or both the first cell culture medium and the second cell culture medium further comprise a potassium channel agonist, the method further comprising the step of treating the patient with a potassium channel agonist starting on the day after the administration of the third population of TILs to the patient, provides a method.

[0031]

[0031] In one embodiment, the present invention is a method for treating cancer using a population of tumor infiltrating lymphocytes (TILs), (a) the step of excising a tumor from a patient, wherein the tumor comprises a first population of TILs; (b) the step of fragmenting the tumor into tumor fragments; (c) the step of contacting the tumor fragments with a first cell culture medium; (d) the step of performing an initial expansion of the first population of TILs in the first cell culture medium to obtain a second population of TILs, wherein the number of the second population of TILs is at least five times more than the first population of TILs, and the first cell culture medium comprises IL-2; (e) the step of performing a rapid expansion of the second population of TILs in a second cell culture medium to obtain a third population of TILs, wherein the number of the third population of TILs is at least 50 times more than the second population of TILs 7 days after the start of the rapid expansion, the second cell culture medium comprises IL-2, OKT-3 (anti-CD3 antibody) and irradiated allogeneic peripheral blood mononuclear cells (PBMCs), and the rapid expansion is performed over a period of 14 days or less; (f) the step of harvesting the third population of TILs; and (g) the step of administering a therapeutically effective portion of the third population of TILs to a patient having cancer comprising, wherein either the first cell culture medium or the second cell culture medium or both the first cell culture medium and the second cell culture medium further comprise a potassium channel agonist, the method further comprising the step of treating the patient with a potassium channel agonist before the step of excising the tumor from the patient, provides a method.

[0032]

[0032] In one embodiment, the present invention is a method of treating cancer using a population of tumor-infiltrating lymphocytes (TIL), comprising: (a) removing a tumor from a patient, wherein the tumor comprises a first population of TIL; (b) fragmenting the tumor into tumor fragments; (c) contacting the tumor fragments with a first cell culture medium; (d) initially expanding the first population of TIL in the first cell culture medium to obtain a second population of TIL, wherein the number of the second population of TIL is at least five-fold greater than the first population of TIL, and the first cell culture medium comprises IL-2; (e) rapidly expanding the second population of TIL in a second cell culture medium to obtain a third population of TIL, wherein the number of the third population of TIL is at least 50-fold greater than the second population of TIL 7 days after the start of rapid expansion, the second cell culture medium comprises IL-2, OKT-3 (anti-CD3 antibody) and irradiated allogeneic peripheral blood mononuclear cells (PBMC), and the rapid expansion is carried out over a period of 14 days or less; (f) recovering the third population of TIL; and (g) administering a therapeutically effective portion of the third population of TIL to a patient having cancer wherein either the first cell culture medium or the second cell culture medium or both the first cell culture medium and the second cell culture medium further comprise a potassium channel agonist, and the method further comprises treating the patient with a non-myeloablative lymphocyte depletion regimen prior to administering the third population of TIL to the patient.

[0033]

[0033] In one embodiment, the present invention is a method of treating cancer using a population of tumor-infiltrating lymphocytes (TIL), comprising: (a) removing a tumor from a patient, wherein the tumor comprises a first population of TIL; (b) fragmenting the tumor into tumor fragments; (c) contacting the tumor fragments with a first cell culture medium; (d) A step of initially expanding a first population of tumor-infiltrating lymphocytes (TIL) in a first cell culture medium to obtain a second population of TIL, wherein the number of the second population of TIL is at least five times more than that of the first population of TIL, and the first cell culture medium contains IL-2; (e) A step of rapidly expanding the second population of TIL in a second cell culture medium to obtain a third population of TIL, wherein the number of the third population of TIL is at least 50 times more than that of the second population of TIL 7 days after the start of the rapid expansion, the second cell culture medium contains IL-2, OKT-3 (anti-CD3 antibody) and irradiated allogeneic peripheral blood mononuclear cells (PBMC), and the rapid expansion is carried out over a period of 14 days or less; (f) A step of recovering the third population of TIL; and (g) A step of administering a therapeutically effective portion of the third population of TIL to a patient having cancer comprising, either the first cell culture medium or the second cell culture medium or both the first cell culture medium and the second cell culture medium further contain a potassium channel agonist, the method further comprises a step of treating the patient with a non-myeloablative lymphocyte depletion regimen before administering the third population of TIL to the patient, and the non-myeloablative lymphocyte depletion regimen comprises administering cyclophosphamide at a dose of 60 mg / m 2 / day for 2 days, followed by administering fludarabine at a dose of 25 mg / m 2 / day for 5 days, to provide a method.

[0034]

[0034] In one embodiment, the present invention is a method for treating cancer using a population of tumor-infiltrating lymphocytes (TIL), (a) A step of excising a tumor from a patient, wherein the tumor contains a first population of TIL; (b) A step of fragmenting the tumor into tumor fragments; (c) A step of contacting the tumor fragments with a first cell culture medium; (d) A step of initially expanding a first population of tumor infiltrating lymphocytes (TILs) in a first cell culture medium to obtain a second population of TILs, wherein the number of the second population of TILs is at least five times more than that of the first population of TILs, and the first cell culture medium contains IL-2; (e) A step of rapidly expanding the second population of TILs in a second cell culture medium to obtain a third population of TILs, wherein the number of the third population of TILs is at least 50 times more than that of the second population of TILs 7 days after the start of the rapid expansion, the second cell culture medium contains IL-2, OKT-3 (anti-CD3 antibody) and irradiated allogeneic peripheral blood mononuclear cells (PBMCs), and the rapid expansion is carried out over a period of 14 days or less; (f) A step of collecting the third population of TILs; and (g) A step of administering a therapeutically effective portion of the third population of TILs to a patient having cancer comprising, either the first cell culture medium or the second cell culture medium or both the first cell culture medium and the second cell culture medium further contain a potassium channel agonist, and the method further comprises a step of treating the patient with a high-dose IL-2 regimen that starts on the day after the administration of the third population of TILs to the patient.

[0035]

[0035] In one embodiment, the present invention is a method for treating cancer using a population of tumor infiltrating lymphocytes (TILs), (a) A step of excising a tumor from a patient, wherein the tumor contains a first population of TILs; (b) A step of fragmenting the tumor into tumor fragments; (c) A step of contacting the tumor fragments with a first cell culture medium; (d) A step of initially expanding a first population of TILs in a first cell culture medium to obtain a second population of TILs, wherein the number of the second population of TILs is at least five times more than that of the first population of TILs, and the first cell culture medium contains IL-2; (e) A step of rapidly expanding a second population of TILs in a second cell culture medium to obtain a third population of TILs, wherein the number of the third population of TILs is at least 50 times more than that of the second population of TILs 7 days after the start of rapid expansion, the second cell culture medium contains IL-2, OKT-3 (anti-CD3 antibody) and irradiated allogeneic peripheral blood mononuclear cells (PBMCs), and the rapid expansion is carried out over a period of 14 days or less; (f) A step of recovering the third population of TILs; and (g) A step of administering a therapeutically effective portion of the third population of TILs to a patient having cancer comprising, either the first cell culture medium or the second cell culture medium or both the first cell culture medium and the second cell culture medium further contain a potassium channel agonist, and the potassium channel agonist is a K Ca 3.1 (IK channel) agonist, and the method further comprises a step of treating the patient with a high-dose IL-2 regimen that starts on the day following the administration of the third population of TILs to the patient, and the high-dose IL-2 regimen comprises 600,000 or 720,000 IU / kg of aldesleukin or its biosimilar or variant administered as a 15-minute bolus intravenous injection every 8 hours up to the tolerated volume, to provide a method.

[0036]

[0036] In one embodiment, the present invention is a method for treating cancer using a population of tumor-infiltrating lymphocytes (TILs), (a) A step of excising a tumor from a patient, wherein the tumor contains a first population of TILs; (b) A step of fragmenting the tumor into tumor fragments; (c) A step of contacting the tumor fragments with a first cell culture medium; (d) A step of initially expanding a first population of TILs in the first cell culture medium to obtain a second population of TILs, wherein the number of the second population of TILs is at least 5 times more than that of the first population of TILs, and the first cell culture medium contains IL-2; (e) A step of rapidly expanding a second population of TILs in a second cell culture medium to obtain a third population of TILs, wherein the number of the third population of TILs is at least 50 times more than that of the second population of TILs 7 days after the start of the rapid expansion, the second cell culture medium contains IL-2, OKT-3 (anti-CD3 antibody) and irradiated allogeneic peripheral blood mononuclear cells (PBMCs), and the rapid expansion is carried out over a period of 14 days or less; (f) A step of recovering the third population of TILs; and (g) A step of administering a therapeutically effective portion of the third population of TILs to a patient having cancer comprising, either the first cell culture medium or the second cell culture medium or both the first cell culture medium and the second cell culture medium further contain a potassium channel agonist, and the potassium channel agonist is a K Ca 3.1 (IK channel) agonist, and the cancer is selected from the group consisting of melanoma, ovarian cancer, cervical cancer, lung cancer, bladder cancer, breast cancer, head and neck cancer, renal cell carcinoma, acute myeloid leukemia, colorectal cancer and sarcoma, provides a method.

[0037]

[0037] In one embodiment, the present invention is a method for treating cancer using a population of tumor infiltrating lymphocytes (TILs), (a) A step of excising a tumor from a patient, wherein the tumor contains a first population of TILs; (b) A step of fragmenting the tumor into tumor fragments; (c) A step of contacting the tumor fragments with a first cell culture medium; (d) A step of initially expanding the first population of TILs in the first cell culture medium to obtain a second population of TILs, wherein the number of the second population of TILs is at least 5 times more than that of the first population of TILs, and the first cell culture medium contains IL-2; (e) A step of rapidly growing a second population of tumor infiltrating lymphocytes (TIL) in a second cell culture medium to obtain a third population of TIL, wherein the number of the third population of TIL is at least 50 times more than that of the second population of TIL 7 days after the start of the rapid growth, the second cell culture medium contains IL-2, OKT-3 (anti-CD3 antibody) and irradiated allogeneic peripheral blood mononuclear cells (PBMC), and the rapid growth is carried out over a period of 14 days or less; (f) A step of recovering the third population of TIL; and (g) A step of administering a therapeutically effective portion of the third population of TIL to a patient having cancer comprising, either the first cell culture medium or the second cell culture medium or both the first cell culture medium and the second cell culture medium further contain a potassium channel agonist, and the cancer is non-small cell lung cancer (NSCLC), estrogen receptor positive (ER + ) breast cancer, progesterone receptor positive (PR + ) breast cancer, human epidermal growth factor receptor 2 (HER2 + ) breast cancer, triple positive breast cancer (ER + / PR + / HER2 + ), triple negative breast cancer (ER - / PR - / HER2 - ), and is selected from the group consisting of double resistant melanoma and uveal (intraocular) melanoma, a method is provided.

[0038]

[0038] In one embodiment, the present invention is a process for preparing a population of tumor infiltrating lymphocytes (TIL) from a tumor, (a) A step of fragmenting the tumor; (b) A step of initially growing a first population of TIL in a first cell culture medium to obtain a second population of TIL, wherein the number of the second population of TIL is at least 5 times more than that of the first population of TIL, and the first cell culture medium contains IL-2; (b) A step of rapidly expanding a second population of TILs in a second cell culture medium to obtain a third population of TILs, wherein the number of the third population of TILs is at least 50 times more than that of the second population of TILs 7 days after the start of rapid expansion, the second cell culture medium contains IL-2, OKT-3 (anti-CD3 antibody), irradiated allogeneic peripheral blood mononuclear cells (PBMCs), and the rapid expansion is carried out over a period of 14 days or less; (c) A step of recovering the third population of TILs including, either the first cell culture medium or the second cell culture medium or both the first cell culture medium and the second cell culture medium further contain a potassium channel agonist, and provides a process.

[0039]

[0039] In one embodiment, the present invention is a process for preparing a population of tumor infiltrating lymphocytes (TILs) from a tumor, (a) A step of fragmenting the tumor; (b) A step of initially expanding a first population of TILs in a first cell culture medium to obtain a second population of TILs, wherein the number of the second population of TILs is at least 5 times more than that of the first population of TILs, and the first cell culture medium contains IL-2; (b) A step of rapidly expanding a second population of TILs in a second cell culture medium to obtain a third population of TILs, wherein the number of the third population of TILs is at least 50 times more than that of the second population of TILs 7 days after the start of rapid expansion, the second cell culture medium contains IL-2, OKT-3 (anti-CD3 antibody), irradiated allogeneic peripheral blood mononuclear cells (PBMCs), and the rapid expansion is carried out over a period of 14 days or less; (c) A step of recovering the third population of TILs including, either the first cell culture medium or the second cell culture medium or both the first cell culture medium and the second cell culture medium further contain a potassium channel agonist, and the first population of TILs is obtained from a tumor or a part thereof, and provides a process.

[0040]

[0040] In one embodiment, the present invention is a process for preparing a population of tumor-infiltrating lymphocytes (TILs) from a tumor, comprising: (a) fragmenting the tumor; (b) performing an initial expansion of a first population of TILs in a first cell culture medium to obtain a second population of TILs, wherein the number of the second population of TILs is at least five times greater than that of the first population of TILs, and the first cell culture medium contains IL-2; (b) performing a rapid expansion of the second population of TILs in a second cell culture medium to obtain a third population of TILs, wherein the number of the third population of TILs is at least 50 times greater than that of the second population of TILs 7 days after the start of the rapid expansion, the second cell culture medium contains IL-2, OKT-3 (anti-CD3 antibody), irradiated allogeneic peripheral blood mononuclear cells (PBMCs), and the rapid expansion is carried out over a period of 14 days or less; (c) recovering the third population of TILs and either the first cell culture medium or the second cell culture medium or both the first cell culture medium and the second cell culture medium further contain a potassium channel agonist, and the tumor or a part thereof is excised from a patient.

[0041]

[0041] In one embodiment, the present invention is a process for preparing a population of tumor-infiltrating lymphocytes (TILs) from a tumor, comprising: (a) fragmenting the tumor; (b) performing an initial expansion of a first population of TILs in a first cell culture medium to obtain a second population of TILs, wherein the number of the second population of TILs is at least five times greater than that of the first population of TILs, and the first cell culture medium contains IL-2; (b) A step of rapidly growing a second population of TILs in a second cell culture medium to obtain a third population of TILs, wherein the number of the third population of TILs is at least 50 times more than that of the second population of TILs 7 days after the start of the rapid growth, the second cell culture medium contains IL-2, OKT-3 (anti-CD3 antibody), irradiated allogeneic peripheral blood mononuclear cells (PBMCs), and the rapid growth is carried out over a period of 14 days or less; (c) A step of recovering the third population of TILs comprising, either the first cell culture medium or the second cell culture medium or both the first cell culture medium and the second cell culture medium further contain a potassium channel agonist, and the potassium channel agonist is K Ca 3.1 (IK channel) agonist, provides a process.

[0042]

[0042] In one embodiment, the present invention is a process for preparing a population of tumor infiltrating lymphocytes (TILs) from a tumor, comprising (a) A step of fragmenting the tumor; (b) A step of initially growing a first population of TILs in a first cell culture medium to obtain a second population of TILs, wherein the number of the second population of TILs is at least 5 times more than that of the first population of TILs, and the first cell culture medium contains IL-2; (b) A step of rapidly growing a second population of TILs in a second cell culture medium to obtain a third population of TILs, wherein the number of the third population of TILs is at least 50 times more than that of the second population of TILs 7 days after the start of the rapid growth, the second cell culture medium contains IL-2, OKT-3 (anti-CD3 antibody), irradiated allogeneic peripheral blood mononuclear cells (PBMCs), and the rapid growth is carried out over a period of 14 days or less; (c) A step of recovering the third population of TILs comprising, either the first cell culture medium or the second cell culture medium or both the first cell culture medium and the second cell culture medium further contain a potassium channel agonist, and the second population of TILs is increased relative to a reference population of TILs obtained without a potassium channel agonist, CD8+ CD28 + 、CD8 + CD27 + 、CD8 + CD27 + CD28 + 、CCR7 + and a population of T cells having a phenotype selected from the group consisting of and combinations thereof, wherein the phenotype in the second population of TILs is increased by at least 5% relative to the reference population of TILs, provides a process. In some examples, the increase is about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 200%, about 300%, about 400%, about 500%, about 600%, about 700%, about 800%, about 900% or about 1000%.

[0043]

[0043] In one embodiment, the present invention is a process for preparing a population of tumor infiltrating lymphocytes (TILs) from a tumor, comprising (a) fragmenting the tumor; (b) initially expanding a first population of TILs in a first cell culture medium to obtain a second population of TILs, wherein the number of the second population of TILs is at least five-fold greater than the first population of TILs, and the first cell culture medium contains IL-2; (b) rapidly expanding the second population of TILs in a second cell culture medium to obtain a third population of TILs, wherein the number of the third population of TILs is at least 50-fold greater than the second population of TILs 7 days after the start of the rapid expansion, and the second cell culture medium contains IL-2, OKT-3 (anti-CD3 antibody), irradiated allogeneic peripheral blood mononuclear cells (PBMCs), and the rapid expansion is carried out over a period of 14 days or less; (c) recovering the third population of TILs comprising, and either the first cell culture medium or the second cell culture medium or both the first cell culture medium and the second cell culture medium further comprise a potassium channel agonist, and a third population of TILs is increased relative to a reference population of TILs obtained without a potassium channel agonist, CD8 + CD28 + CD8 + CD27 + CD8 + CD27 + CD28 + CCR7 + and comprising a population of T cells having a phenotype selected from the group consisting of and combinations thereof, wherein the phenotype in the third population of TILs is increased by at least 5% relative to the reference population of TILs. In some examples, the increase is about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 200%, about 300%, about 400%, about 500%, about 600%, about 700%, about 800%, about 900% or about 1000%.

[0044]

[0044] In one embodiment, the present invention is a process for preparing a population of tumor infiltrating lymphocytes (TILs) from a tumor, (a) fragmenting the tumor; (b) initially expanding a first population of TILs in a first cell culture medium to obtain a second population of TILs, wherein the number of the second population of TILs is at least five times greater than the first population of TILs, and the first cell culture medium comprises IL-2; (b) rapidly expanding the second population of TILs in a second cell culture medium to obtain a third population of TILs, wherein the number of the third population of TILs is at least 50 times greater than the second population of TILs 7 days after the start of rapid expansion, and the second cell culture medium comprises IL-2, OKT-3 (anti-CD3 antibody), irradiated allogeneic peripheral blood mononuclear cells (PBMCs), and the rapid expansion is carried out over a period of 14 days or less; (c) Step of recovering the third population of TILs comprising, either the first cell culture medium or the second cell culture medium or both the first cell culture medium and the second cell culture medium further comprises a potassium channel agonist, and the potassium channel agonist is K Ca 3.1 (IK channel) agonist, and both of the first cell culture media are K Ca 3.1 agonist, and the second cell culture medium further comprises a K Ca 3.1 agonist, providing a process.

[0045]

[0045] In one embodiment, the present invention is a process for preparing a population of tumor infiltrating lymphocytes (TILs) from a tumor, comprising (a) Step of fragmenting the tumor; (b) Step of initially expanding the first population of TILs in the first cell culture medium to obtain a second population of TILs, wherein the number of the second population of TILs is at least 5 times more than that of the first population of TILs, and the first cell culture medium comprises IL-2; (b) Step of rapidly expanding the second population of TILs in the second cell culture medium to obtain a third population of TILs, wherein the number of the third population of TILs is at least 50 times more than that of the second population of TILs 7 days after the start of the rapid expansion, and the second cell culture medium comprises IL-2, OKT-3 (anti-CD3 antibody), irradiated allogeneic peripheral blood mononuclear cells (PBMC), and the rapid expansion is carried out over a period of 14 days or less; (c) Step of recovering the third population of TILs comprising, either the first cell culture medium or the second cell culture medium or both the first cell culture medium and the second cell culture medium further comprises a potassium channel agonist, and the potassium channel agonist is K Ca 3.1 (IK channel) agonist, and the K Ca 3.1 agonist concentration in the first cell culture medium is 1 - 1000 nM, providing a process.

[0046]

[0046] In one embodiment, the present invention is a process for preparing a population of tumor-infiltrating lymphocytes (TIL) from a tumor, comprising: (a) fragmenting the tumor; (b) performing an initial expansion of a first population of TIL in a first cell culture medium to obtain a second population of TIL, wherein the number of the second population of TIL is at least five times greater than the first population of TIL, and the first cell culture medium contains IL-2; (b) performing a rapid expansion of the second population of TIL in a second cell culture medium to obtain a third population of TIL, wherein the number of the third population of TIL is at least 50 times greater than the second population of TIL 7 days after the start of the rapid expansion, the second cell culture medium contains IL-2, OKT-3 (anti-CD3 antibody), irradiated allogeneic peripheral blood mononuclear cells (PBMC), and the rapid expansion is carried out over a period of 14 days or less; (c) recovering the third population of TIL and either the first cell culture medium or the second cell culture medium or both the first cell culture medium and the second cell culture medium further contain a potassium channel agonist, and the potassium channel agonist is a K Ca 3.1 (IK channel) agonist, and the concentration of the K Ca 3.1 agonist in the first cell culture medium is about 100 nM, providing a process.

[0047]

[0047] In one embodiment, the present invention is a process for preparing a population of tumor-infiltrating lymphocytes (TIL) from a tumor, comprising: (a) fragmenting the tumor; (b) performing an initial expansion of a first population of TIL in a first cell culture medium to obtain a second population of TIL, wherein the number of the second population of TIL is at least five times greater than the first population of TIL, and the first cell culture medium contains IL-2; (b) A step of rapidly expanding a second population of TILs in a second cell culture medium to obtain a third population of TILs, wherein the number of the third population of TILs is at least 50 times more than that of the second population of TILs 7 days after the start of the rapid expansion, the second cell culture medium contains IL-2, OKT-3 (anti-CD3 antibody), irradiated allogeneic peripheral blood mononuclear cells (PBMCs), and the rapid expansion is carried out over a period of 14 days or less; (c) A step of recovering the third population of TILs comprising, either the first cell culture medium or the second cell culture medium or both the first cell culture medium and the second cell culture medium further contain a potassium channel agonist, and the potassium channel agonist is K Ca 3.1 (IK channel) agonist, and the concentration of the K Ca 3.1 agonist in the second cell culture medium is 0.1 to 100 mM, providing a process.

[0048]

[0048] In one embodiment, the present invention is a process for preparing a population of tumor infiltrating lymphocytes (TILs) from a tumor, (a) A step of fragmenting the tumor; (b) A step of initially expanding a first population of TILs in a first cell culture medium to obtain a second population of TILs, wherein the number of the second population of TILs is at least 5 times more than that of the first population of TILs, and the first cell culture medium contains IL-2; (b) A step of rapidly expanding a second population of TILs in a second cell culture medium to obtain a third population of TILs, wherein the number of the third population of TILs is at least 50 times more than that of the second population of TILs 7 days after the start of the rapid expansion, the second cell culture medium contains IL-2, OKT-3 (anti-CD3 antibody), irradiated allogeneic peripheral blood mononuclear cells (PBMCs), and the rapid expansion is carried out over a period of 14 days or less; (c) A step of recovering the third population of TILs comprising, either the first cell culture medium or the second cell culture medium or both the first cell culture medium and the second cell culture medium further comprising a potassium channel agonist, the potassium channel agonist being a K Ca 3.1 (IK channel) agonist, and the concentration of the K Ca 3.1 agonist in the first cell culture medium is about 50 mM, provides a process.

[0049]

[0049] In one embodiment, the present invention is a process for preparing a population of tumor infiltrating lymphocytes (TIL) from a tumor, (a) fragmenting the tumor; (b) initially expanding a first population of TIL in a first cell culture medium to obtain a second population of TIL, wherein the number of the second population of TIL is at least five times greater than the first population of TIL, and the first cell culture medium comprises IL-2; (b) rapidly expanding the second population of TIL in a second cell culture medium to obtain a third population of TIL, wherein the number of the third population of TIL is at least 50 times greater than the second population of TIL 7 days after the start of the rapid expansion, and the second cell culture medium comprises IL-2, OKT-3 (anti-CD3 antibody), irradiated allogeneic peripheral blood mononuclear cells (PBMC), and the rapid expansion is carried out over a period of 14 days or less; (c) recovering the third population of TIL comprising, either the first cell culture medium or the second cell culture medium or both the first cell culture medium and the second cell culture medium further comprising a potassium channel agonist, and the initial expansion is carried out over a period of 21 days or less, provides a process.

[0050]

[0050] In one embodiment, the present invention is a process for preparing a population of tumor infiltrating lymphocytes (TIL) from a tumor, (a) fragmenting the tumor; (b) A step of initially expanding a first population of tumor infiltrating lymphocytes (TIL) in a first cell culture medium to obtain a second population of TIL, wherein the number of the second population of TIL is at least five times more than that of the first population of TIL, and the first cell culture medium contains IL-2; (b) A step of rapidly expanding a second population of TIL in a second cell culture medium to obtain a third population of TIL, wherein the number of the third population of TIL is at least 50 times more than that of the second population of TIL 7 days after the start of the rapid expansion, the second cell culture medium contains IL-2, OKT-3 (anti-CD3 antibody), irradiated allogeneic peripheral blood mononuclear cells (PBMC), and the rapid expansion is carried out over a period of 14 days or less; (c) A step of collecting the third population of TIL including, either the first cell culture medium or the second cell culture medium or both the first cell culture medium and the second cell culture medium further contain a potassium channel agonist, and the initial expansion is carried out over a period of 11 days or less, providing a process.

[0051]

[0051] In one embodiment, the present invention is a process for preparing a population of tumor infiltrating lymphocytes (TIL) from a tumor, (a) A step of fragmenting the tumor; (b) A step of initially expanding a first population of TIL in a first cell culture medium to obtain a second population of TIL, wherein the number of the second population of TIL is at least five times more than that of the first population of TIL, and the first cell culture medium contains IL-2; (b) A step of rapidly expanding a second population of TIL in a second cell culture medium to obtain a third population of TIL, wherein the number of the third population of TIL is at least 50 times more than that of the second population of TIL 7 days after the start of the rapid expansion, the second cell culture medium contains IL-2, OKT-3 (anti-CD3 antibody), irradiated allogeneic peripheral blood mononuclear cells (PBMC), and the rapid expansion is carried out over a period of 14 days or less; (c) A step of collecting the third population of TIL A process is provided that includes and in which either the first cell culture medium or the second cell culture medium or both the first cell culture medium and the second cell culture medium further include a potassium channel agonist, and rapid proliferation is carried out over a period of 7 days or less.

[0052]

[0052] In one embodiment, the present invention is a process for preparing a population of tumor infiltrating lymphocytes (TIL) from a tumor, comprising: (a) fragmenting the tumor; (b) initially expanding a first population of TIL in a first cell culture medium to obtain a second population of TIL, wherein the number of the second population of TIL is at least 5-fold greater than the first population of TIL, and the first cell culture medium includes IL-2; (b) rapidly expanding the second population of TIL in a second cell culture medium to obtain a third population of TIL, wherein the number of the third population of TIL is at least 50-fold greater than the second population of TIL 7 days after the start of rapid proliferation, and the second cell culture medium includes IL-2, OKT-3 (anti-CD3 antibody), irradiated allogeneic peripheral blood mononuclear cells (PBMC), and rapid proliferation is carried out over a period of 14 days or less; (c) recovering the third population of TIL including, and in which either the first cell culture medium or the second cell culture medium or both the first cell culture medium and the second cell culture medium further include a potassium channel agonist, and the potassium channel agonist is a K Ca 3.1 (IK channel) agonist, and the K Ca 3.1 agonist has the formula (1):

Chemical formula

[0053]

[0053] In one embodiment, the present invention is a process for preparing a population of tumor-infiltrating lymphocytes (TIL) from a tumor, comprising (a) Step of fragmenting the tumor; (b) Step of initially growing a first population of tumor-infiltrating lymphocytes (TILs) in a first cell culture medium to obtain a second population of TILs, wherein the number of the second population of TILs is at least five times more than that of the first population of TILs, and the first cell culture medium contains IL-2; (b) Step of rapidly growing the second population of TILs in a second cell culture medium to obtain a third population of TILs, wherein the number of the third population of TILs is at least 50 times more than that of the second population of TILs 7 days after the start of the rapid growth, the second cell culture medium contains IL-2, OKT-3 (anti-CD3 antibody), and irradiated allogeneic peripheral blood mononuclear cells (PBMCs), and the rapid growth is carried out over a period of 14 days or less; (c) Step of collecting the third population of TILs comprising, either the first cell culture medium or the second cell culture medium or both the first cell culture medium and the second cell culture medium further contain a potassium channel agonist, and the potassium channel agonist is a K Ca 3.1 (IK channel) agonist, and the K Ca 3.1 agonist is naphtho[1,2-d]thiazol-2-ylamine (SKA-31): [Chemical formula] or a pharmaceutically acceptable salt, co-crystal, solvate or prodrug thereof, provides a process.

[0054] In one embodiment, the present invention is a process for preparing a population of tumor-infiltrating lymphocytes (TILs) from a tumor, comprising (a) Step of fragmenting the tumor; (b) Step of initially growing a first population of TILs in a first cell culture medium to obtain a second population of TILs, wherein the number of the second population of TILs is at least five times more than that of the first population of TILs, and the first cell culture medium contains IL-2; (b) A step of rapidly expanding a second population of TILs in a second cell culture medium to obtain a third population of TILs, wherein the number of the third population of TILs is at least 50 times more than that of the second population of TILs 7 days after the start of rapid expansion, the second cell culture medium contains IL-2, OKT-3 (anti-CD3 antibody), irradiated allogeneic peripheral blood mononuclear cells (PBMCs), and the rapid expansion is carried out over a period of 14 days or less; (c) A step of collecting the third population of TILs including, either the first cell culture medium or the second cell culture medium or both the first cell culture medium and the second cell culture medium further contain a potassium channel agonist, and the potassium channel agonist is K Ca 3.1 (IK channel) agonist, and the K Ca 3.1 agonist is anthra[2,1-d]thiazol-2-ylamine (SKA-20):

Chemical formula

[0055]

[0055] In one embodiment, the present invention is a process for preparing a population of tumor-infiltrating lymphocytes (TILs) from a tumor, (a) A step of fragmenting the tumor; (b) A step of initially expanding a first population of TILs in a first cell culture medium to obtain a second population of TILs, wherein the number of the second population of TILs is at least 5 times more than that of the first population of TILs, and the first cell culture medium contains IL-2; (b) A step of rapidly expanding a second population of TILs in a second cell culture medium to obtain a third population of TILs, wherein the number of the third population of TILs is at least 50 times more than that of the second population of TILs 7 days after the start of rapid expansion, the second cell culture medium contains IL-2, OKT-3 (anti-CD3 antibody), irradiated allogeneic peripheral blood mononuclear cells (PBMCs), and the rapid expansion is carried out over a period of 14 days or less; (c) Step of recovering the third population of TILs comprising, wherein either the first cell culture medium or the second cell culture medium or both the first cell culture medium and the second cell culture medium further comprises a potassium channel agonist, and the potassium channel agonist is K Ca 3.1 (IK channel) agonist, and K Ca 3.1 agonist is 6,7-dichloro-1H-indole-2,3-dione 3-oxime (NS309): [Chemical formula] or a pharmaceutically acceptable salt, co-crystal, solvate or prodrug thereof, providing a process.

[0056]

[0056] In one embodiment, the present invention is a process for preparing a population of tumor infiltrating lymphocytes (TILs) from a tumor, comprising (a) a step of fragmenting the tumor; (b) a step of initially expanding a first population of TILs in a first cell culture medium to obtain a second population of TILs, wherein the number of the second population of TILs is at least five times more than that of the first population of TILs, and the first cell culture medium comprises IL-2; (b) a step of rapidly expanding the second population of TILs in a second cell culture medium to obtain a third population of TILs, wherein the number of the third population of TILs is at least 50 times more than that of the second population of TILs 7 days after the start of the rapid expansion, and the second cell culture medium comprises IL-2, OKT-3 (anti-CD3 antibody), irradiated allogeneic peripheral blood mononuclear cells (PBMC), and the rapid expansion is carried out over a period of 14 days or less; (c) a step of recovering the third population of TILs comprising, wherein either the first cell culture medium or the second cell culture medium or both the first cell culture medium and the second cell culture medium further comprises a potassium channel agonist, and the potassium channel agonist is K Ca 3.1 (IK channel) agonist, and K Ca 3.1 agonist is riluzole: [Chemical formula] or a pharmaceutically acceptable salt, co-crystal, solvate or prodrug thereof, provides a process.

[0057]

[0057] In one embodiment, the present invention is a process for preparing a population of tumor-infiltrating lymphocytes (TIL) from a tumor, (a) fragmenting the tumor; (b) initially expanding a first population of TIL in a first cell culture medium to obtain a second population of TIL, wherein the number of the second population of TIL is at least five times more than that of the first population of TIL, and the first cell culture medium contains IL-2; (b) rapidly expanding the second population of TIL in a second cell culture medium to obtain a third population of TIL, wherein the number of the third population of TIL is at least 50 times more than that of the second population of TIL 7 days after the start of the rapid expansion, and the second cell culture medium contains IL-2, OKT-3 (anti-CD3 antibody), irradiated allogeneic peripheral blood mononuclear cells (PBMC), and the rapid expansion is carried out over a period of 14 days or less; (c) recovering the third population of TIL comprising, either the first cell culture medium or the second cell culture medium or both the first cell culture medium and the second cell culture medium further contain a potassium channel agonist, and the potassium channel agonist is K Ca 3.1 (IK channel) agonist, and the K Ca 3.1 agonist is 5-methylnaphtho[1,2-d]oxazol-2-amine; 5-ethylnaphtho[1,2-d]oxazol-2-amine; 5-propylnaphtho[1,2-d]oxazol-2-amine; 5-cyclopropylnaphtho[1,2-d]oxazol-2-amine; 5-(tert-butyl)naphtho[1,2-d]oxazol-2-amine; 5-Fluoronaphtho[1,2-d]oxazol-2-amine; 5-Chloronaphtho[1,2-d]oxazol-2-amine; 5-Bromonaphtho[1,2-d]oxazol-2-amine; 5-Iodonaphtho[1,2-d]oxazol-2-amine; 2-Aminonaphtho[1,2-d]oxazole-5-carbonitrile; Naphtho[1,2-d]oxazole-2,5-diamine; N 5 -Methylnaphtho[1,2-d]oxazole-2,5-diamine; N 5 ,N 5 -Dimethylnaphtho[1,2-d]oxazole-2,5-diamine; N 5 -Ethylnaphtho[1,2-d]oxazole-2,5-diamine; 5-(Pyrrolidin-1-yl)naphtho[1,2-d]oxazol-2-amine; 5-Methoxynaphtho[1,2-d]oxazol-2-amine; 5-Trifluoromethylnaphtho[1,2-d]oxazol-2-amine; 5-Methylnaphtho[2,1-d]oxazol-2-amine; 5-Ethylnaphtho[2,1-d]oxazol-2-amine; 5-Propylnaphtho[2,1-d]oxazol-2-amine; 5-Cyclopropylnaphtho[2,1-d]oxazol-2-amine; 5-(tert-Butyl)naphtho[2,1-d]oxazol-2-amine; 5-Fluoronaphtho[2,1-d]oxazol-2-amine; 5-Chloronaphtho[2,1-d]oxazol-2-amine; 5-Bromonaphtho[2,1-d]oxazol-2-amine; 5-Iodonaphtho[2,1-d]oxazol-2-amine; 2-Aminonaphtho[2,1-d]oxazole-5-carbonitrile; Naphtho[2,1-d]oxazole-2,5-diamine; N5-Methylnaphtho[2,1-d]oxazole-2,5-diamine; N5,N5-Dimethylnaphtho[2,1-d]oxazole-2,5-diamine; N5-Ethylnaphtho[2,1-d]oxazole-2,5-diamine; 5-(Pyrrolidin-1-yl)naphtho[2,1-d]oxazol-2-amine; 5-Methoxynaphtho[2,1-d]oxazol-2-amine; 5-Trifluoromethylnaphtho[2,1-d]oxazol-2-amine; and its pharmaceutically acceptable salts, co-crystals, solvates or prodrugs A process is provided that is selected from the group consisting of.

[0058]

[0058] In one embodiment, the present invention is a process for preparing a population of tumor-infiltrating lymphocytes (TIL) from a tumor, comprising (a) Fragmenting the tumor; (b) Initiating the initial expansion of a first population of TIL in a first cell culture medium to obtain a second population of TIL, wherein the number of the second population of TIL is at least five times greater than the first population of TIL, and the first cell culture medium contains IL-2; (b) Rapidly expanding the second population of TIL in a second cell culture medium to obtain a third population of TIL, wherein the number of the third population of TIL is at least 50 times greater than the second population of TIL 7 days after the start of the rapid expansion, and the second cell culture medium contains IL-2, OKT-3 (anti-CD3 antibody), irradiated allogeneic peripheral blood mononuclear cells (PBMC), and the rapid expansion is carried out over a period of 14 days or less; (c) Recovering the third population of TIL comprising, wherein either the first cell culture medium or the second cell culture medium or both the first cell culture medium and the second cell culture medium further comprise a potassium channel agonist, and IL-2 is present in the first cell culture medium at an initial concentration of 1000 IU / mL to 6000 IU / mL, provides a process.

[0059]

[0059] In one embodiment, the present invention is a process for preparing a population of tumor infiltrating lymphocytes (TIL) from a tumor, (a) fragmenting the tumor; (b) initially expanding a first population of TIL in a first cell culture medium to obtain a second population of TIL, wherein the number of the second population of TIL is at least five times greater than the first population of TIL, and the first cell culture medium comprises IL-2; (b) rapidly expanding the second population of TIL in a second cell culture medium to obtain a third population of TIL, wherein the number of the third population of TIL is at least 50 times greater than the second population of TIL 7 days after the start of rapid expansion, the second cell culture medium comprises IL-2, OKT-3 (anti-CD3 antibody), irradiated allogeneic peripheral blood mononuclear cells (PBMC), and the rapid expansion is carried out over a period of 14 days or less; (c) recovering the third population of TIL comprising, wherein either the first cell culture medium or the second cell culture medium or both the first cell culture medium and the second cell culture medium further comprise a potassium channel agonist, in the second cell culture medium, 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, provides a process.

[0060]

[0060] In one embodiment, the present invention is a process for preparing a population of tumor infiltrating lymphocytes (TIL) from a tumor, (a) fragmenting the tumor; (b) A step of initially expanding a first population of TILs in a first cell culture medium to obtain a second population of TILs, wherein the number of the second population of TILs is at least five times more than that of the first population of TILs, and the first cell culture medium contains IL-2; (b) A step of rapidly expanding a second population of TILs in a second cell culture medium to obtain a third population of TILs, wherein the number of the third population of TILs is at least 50 times more than that of the second population of TILs 7 days after the start of the rapid expansion, the second cell culture medium contains IL-2, OKT-3 (anti-CD3 antibody), irradiated allogeneic peripheral blood mononuclear cells (PBMCs), and the rapid expansion is carried out over a period of 14 days or less; (c) A step of recovering the third population of TILs including, either the first cell culture medium or the second cell culture medium or both the first cell culture medium and the second cell culture medium further contain a potassium channel agonist, and the initial expansion is carried out using a gas-permeable container, provides a process.

[0061]

[0061] In one embodiment, the present invention is a process for preparing a population of tumor-infiltrating lymphocytes (TILs) from a tumor, (a) A step of fragmenting the tumor; (b) A step of initially expanding a first population of TILs in a first cell culture medium to obtain a second population of TILs, wherein the number of the second population of TILs is at least five times more than that of the first population of TILs, and the first cell culture medium contains IL-2; (b) A step of rapidly expanding a second population of TILs in a second cell culture medium to obtain a third population of TILs, wherein the number of the third population of TILs is at least 50 times more than that of the second population of TILs 7 days after the start of the rapid expansion, the second cell culture medium contains IL-2, OKT-3 (anti-CD3 antibody), irradiated allogeneic peripheral blood mononuclear cells (PBMCs), and the rapid expansion is carried out over a period of 14 days or less; (c) A step of recovering the third population of TILs comprising, either the first cell culture medium or the second cell culture medium or both the first cell culture medium and the second cell culture medium further comprising a potassium channel agonist, and rapid expansion being carried out using a gas permeable container, provides a process.

[0062]

[0062] In one embodiment, the present invention is a process for preparing a population of tumor infiltrating lymphocytes (TIL) from a tumor, comprising: (a) fragmenting the tumor; (b) initially expanding a first population of TIL in a first cell culture medium to obtain a second population of TIL, wherein the number of the second population of TIL is at least five times more than the first population of TIL, and the first cell culture medium comprises IL-2; (b) rapidly expanding the second population of TIL in a second cell culture medium to obtain a third population of TIL, wherein the number of the third population of TIL is at least 50 times more than the second population of TIL 7 days after the start of rapid expansion, and the second cell culture medium comprises IL-2, OKT-3 (anti-CD3 antibody), irradiated allogeneic peripheral blood mononuclear cells (PBMC), and rapid expansion is carried out over a period of 14 days or less; (c) recovering the third population of TIL comprising, either the first cell culture medium or the second cell culture medium or both the first cell culture medium and the second cell culture medium further comprising a potassium channel agonist, and the first cell culture medium further comprising a cytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21, and combinations thereof, provides a process.

[0063]

[0063] In one embodiment, the present invention is a process for preparing a population of tumor infiltrating lymphocytes (TIL) from a tumor, comprising: (a) fragmenting the tumor; (b) A step of initially expanding a first population of tumor infiltrating lymphocytes (TILs) in a first cell culture medium to obtain a second population of TILs, wherein the number of the second population of TILs is at least five times more than that of the first population of TILs, and the first cell culture medium contains IL-2; (b) A step of rapidly expanding a second population of TILs in a second cell culture medium to obtain a third population of TILs, wherein the number of the third population of TILs is at least 50 times more than that of the second population of TILs 7 days after the start of the rapid expansion, the second cell culture medium contains IL-2, OKT-3 (anti-CD3 antibody), irradiated allogeneic peripheral blood mononuclear cells (PBMCs), and the rapid expansion is carried out over a period of 14 days or less; (c) A step of recovering the third population of TILs including, either the first cell culture medium or the second cell culture medium or both the first cell culture medium and the second cell culture medium further contain a potassium channel agonist, and the second cell culture medium further contains a cytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21 and combinations thereof, provides a process.

[0064]

[0064] In one embodiment, the present invention is a process for preparing a population of tumor infiltrating lymphocytes (TILs) from a tumor, (a) A step of fragmenting the tumor; (b) A step of initially expanding a first population of TILs in a first cell culture medium to obtain a second population of TILs, wherein the number of the second population of TILs is at least five times more than that of the first population of TILs, and the first cell culture medium contains IL-2; (b) A step of rapidly expanding a second population of TILs in a second cell culture medium to obtain a third population of TILs, wherein the number of the third population of TILs is at least 50 times more than that of the second population of TILs 7 days after the start of the rapid expansion, the second cell culture medium contains IL-2, OKT-3 (anti-CD3 antibody), irradiated allogeneic peripheral blood mononuclear cells (PBMCs), and the rapid expansion is carried out over a period of 14 days or less; (c) A step of recovering the third population of TILs comprising, wherein either the first cell culture medium or the second cell culture medium or both the first cell culture medium and the second cell culture medium further comprise a potassium channel agonist, and the potassium channel agonist is a K Ca 3.1 (IK channel) agonist, and the process is an ex vivo method, provides a process.

[0065]

[0065] In one embodiment, the present invention provides a population of TILs for the treatment of cancer in a patient, the population of TILs obtainable by any one of the aforementioned processes. In one embodiment, the present invention provides a population of TILs for the treatment of cancer in a patient, the population of TILs obtainable by any one of the aforementioned processes, wherein the cancer is selected from melanoma, ovarian cancer, cervical cancer, lung cancer, bladder cancer, breast cancer, head and neck cancer, renal cell carcinoma, acute myeloid leukemia, colorectal cancer, sarcoma, non-small cell lung cancer (NSCLC) or triple-negative breast cancer, double-resistant melanoma and uveal (intraocular) melanoma. In one embodiment, the present invention provides a population of TILs for the treatment of cancer by intratumoral injection or intravenous infusion in a patient, the population of TILs obtainable by any one of the aforementioned processes, wherein the cancer is selected from melanoma, ovarian cancer, cervical cancer, lung cancer, bladder cancer, breast cancer, head and neck cancer, renal cell carcinoma, acute myeloid leukemia, colorectal cancer, sarcoma, non-small cell lung cancer (NSCLC) or triple-negative breast cancer, double-resistant melanoma and uveal (intraocular) melanoma. In any of the aforementioned embodiments, the population of TILs can be administered in combination with a chemotherapeutic agent.

[0066]

[0066] In one embodiment, the present invention provides a cell culture medium comprising IL-2 and a potassium channel agonist.

[0067]

[0067] In one embodiment, the present invention provides a cell culture medium comprising IL-2, an anti-CD3 antibody or a fragment, variant or biosimilar thereof, peripheral blood mononuclear cells (PBMCs) and a potassium channel agonist.

[0068]

[0068] In one embodiment, the present invention provides a cell culture medium comprising IL-2 and a potassium channel agonist, wherein the potassium channel agonist is a K Ca 3.1 agonist.

[0069]

[0069] In one embodiment, the present invention provides a cell culture medium comprising IL-2, an anti-CD3 antibody or a fragment, variant or biosimilar thereof, peripheral blood mononuclear cells (PBMCs), and a potassium channel agonist, wherein the potassium channel agonist is a K Ca 3.1.

[0070]

[0070] In one embodiment, the present invention provides a cell culture medium comprising IL-2 and a potassium channel agonist, wherein the potassium channel agonist is a K Ca 3.1 agonist, and the K Ca 3.1 agonist has the formula (1):

Chemical formula

[0071]

[0071] In one embodiment, the present invention provides a cell culture medium comprising IL-2 and a potassium channel agonist, wherein the potassium channel agonist is a K Ca 3.1 agonist, and the K Ca 3.1 agonist is naphtho[1,2-d]thiazol-2-ylamine (SKA-31):

Chemical formula

[0072]

[0072] In one embodiment, the present invention provides a cell culture medium comprising IL-2 and a potassium channel agonist, wherein the potassium channel agonist is a K Ca 3.1 agonist, and the K Ca 3.1 agonist is anthra[2,1-d]thiazol-2-ylamine (SKA-20):

Chemical formula

[0073]

[0073] In one embodiment, the present invention provides a cell culture medium comprising IL-2 and a potassium channel agonist, wherein the potassium channel agonist is a K Ca 3.1 agonist, and the K Ca 3.1 agonist is 6,7-dichloro-1H-indole-2,3-dione 3-oxime (NS309):

Chemical formula

[0074]

[0074] In one embodiment, the present invention provides a cell culture medium comprising IL-2 and a potassium channel agonist, wherein the potassium channel agonist is a K Ca 3.1 agonist, and the K Ca 3.1 agonist is riluzole:

Chemical formula

[0075]

[0075] In one embodiment, the present invention provides a cell culture medium comprising IL-2 and a potassium channel agonist, wherein the potassium channel agonist is a K Ca 3.1 agonist, and the KCa 3.1 The agonists are 5-methylnaphtho[1,2-d]oxazol-2-amine; 5-ethylnaphtho[1,2-d]oxazol-2-amine; 5-propylnaphtho[1,2-d]oxazol-2-amine; 5-cyclopropylnaphtho[1,2-d]oxazol-2-amine; 5-(tert-butyl)naphtho[1,2-d]oxazol-2-amine; 5-fluoronaphtho[1,2-d]oxazol-2-amine; 5-chloronaphtho[1,2-d]oxazol-2-amine; 5-bromonaphtho[1,2-d]oxazol-2-amine; 5-iodonaphtho[1,2-d]oxazol-2-amine; 2-aminonaphtho[1,2-d]oxazole-5-carbonitrile; naphtho[1,2-d]oxazole-2,5-diamine; N 5 -methylnaphtho[1,2-d]oxazole-2,5-diamine; N 5 ,N 5 -dimethylnaphtho[1,2-d]oxazole-2,5-diamine; N 5 -ethylnaphtho[1,2-d]oxazole-2,5-diamine; 5-(pyrrolidin-1-yl)naphtho[1,2-d]oxazol-2-amine; 5-methoxynaphtho[1,2-d]oxazol-2-amine; 5-trifluoromethylnaphtho[1,2-d]oxazol-2-amine; 5-methylnaphtho[2,1-d]oxazol-2-amine; 5-ethylnaphtho[2,1-d]oxazol-2-amine; 5-propylnaphtho[2,1-d]oxazol-2-amine; 5-cyclopropylnaphtho[2,1-d]oxazol-2-amine; 5-(tert-Butyl)naphtho[2,1-d]oxazol-2-amine; 5-Fluoronaphtho[2,1-d]oxazol-2-amine; 5-Chloronaphtho[2,1-d]oxazol-2-amine; 5-Bromonaphtho[2,1-d]oxazol-2-amine; 5-Iodonaphtho[2,1-d]oxazol-2-amine; 2-Aminonaphtho[2,1-d]oxazole-5-carbonitrile; Naphtho[2,1-d]oxazole-2,5-diamine; N5-Methylnaphtho[2,1-d]oxazole-2,5-diamine; N5,N5-Dimethylnaphtho[2,1-d]oxazole-2,5-diamine; N5-Ethylnaphtho[2,1-d]oxazole-2,5-diamine; 5-(Pyrrolidin-1-yl)naphtho[2,1-d]oxazol-2-amine; 5-Methoxynaphtho[2,1-d]oxazol-2-amine; 5-Trifluoromethylnaphtho[2,1-d]oxazol-2-amine; and its pharmaceutically acceptable salts, co-crystals, solvates or prodrugs A cell culture medium is provided, which is selected from the group consisting of.

[0076]

[0076] In one embodiment, the present invention provides a cell culture medium comprising IL-2 and a potassium channel agonist, wherein the potassium channel agonist is a K Ca 3.1 agonist.

[0077]

[0077] In one embodiment, the present invention provides a kit comprising a cell culture medium comprising IL-2 and a potassium channel agonist, wherein the potassium channel agonist is a K Ca 3.1 agonist.

[0078]

[0078] In one embodiment, the present invention provides a kit comprising a cell culture medium containing IL-2, a potassium channel agonist, and a tumor, wherein the potassium channel agonist is K Ca 3.1 agonist.

[0079]

[0079] In one embodiment, the present invention provides the use of a potassium channel agonist in the manufacture of a population of TILs for the treatment of cancer. Suitably, the potassium channel agonist is K Ca 3.1 (IK channel) agonist.

[0080]

[0080] In one embodiment, the present invention provides the use of a potassium channel agonist in the manufacture of a population of TILs for the treatment of cancer, wherein the potassium channel agonist is K Ca 3.1 agonist. In one embodiment, the present invention provides the use of a potassium channel agonist in the manufacture of a population of TILs for the treatment of cancer, wherein the potassium channel agonist is the K Ca 3.1 agonist described herein. Suitably, the potassium channel agonist is SKA-31, SKA-20, NS309, riluzole or a pharmaceutically acceptable salt, co-crystal, solvate or prodrug thereof.

[0081] Brief Description of the Drawings

[0081] The foregoing summary and the following detailed description of the invention will be better understood when read in conjunction with the accompanying drawings.

Brief Description of the Drawings

[0082]

Figure 1

[0082] Illustrates the sorting strategy used in a flow cytometry experiment for the analysis of KCa3.1 expression in T cell subsets.

Figure 2

[0083] Shows the results of the flow cytometry experiment conducted using the strategy shown in Fig. 1. The upper panel shows the CD3+CD4+ subset, and the lower panel shows the CD3+CD8+ subset. Both the KCa3.1+ and KCa3.1- subsets are shown.

Figure 3

[0084] Shows the expression of four T cell subsets: naive, central memory (TCM), effector memory (TEF), and effector memory RA+ (TEMRA) cells. The upper panel shows the CD3+CD4+ subset, and the lower panel shows the CD3+CD8+ subset. Both the KCa3.1+ and KCa3.1- subsets are shown.

Figure 4

[0085] Shows the dynamic expression of KCa3.1 in two healthy donor peripheral blood mononuclear cell (PBMC) lines and three melanoma TIL lines. Cells were harvested and stained with anti-CD3, anti-CD8, anti-KCa3.1, and Amcyan on days 1, 3, and 7 after activation to determine the dynamic expression of KCa3.1.

Figure 5

[0086] Shows flow cytometry data indicating the percentage of KCa3.1 in the CD3+CD4+ subset (upper) and CD3+CD8+ subset (lower) at day 0 and day 3 after TCR activation.

Figure 6

[0087] Shows the dynamic expression of KCa3.1 within 3 days in the CD3+CD4+ (upper) and CD3+CD8+ (lower) subsets.

Figure 7

[0088] Shows flow cytometry data indicating KCa3.1 expression in the CD3+CD4+ (upper) and CD3+CD8+ (lower) subsets of healthy donor PBMCs compared to TIL.

Figure 8

[0089] Shows the percentage of KCa3.1 expression observed in the CD3+CD4+ (upper) and CD3+CD8+ (lower) subsets of healthy donor PBMCs compared to TIL (p-values represent the difference between normal PBMCs and pre-REP-TIL using Student's one-sided T-test, and p-values less than 0.05 are considered statistically significant).

Figure 9

[0090] Shows the fold expansion of TILs from different strains (''SKA-31'') in the presence of the KCa3.1 agonist SKA-31 compared to a control experiment performed without SKA-31 (''no treatment'').

Figure 10

[0091] Shows the percentage of CD8+CD27+CD28+ T cell subset observed in TILs treated with the KCa3.1 agonist SKA-31 (''K+ agonist'') compared to TILs treated without SKA-31 during REP (''no treatment'').

Figure 11

[0092] Shows the percentage of CD8+CD28+ T cell subset observed in TILs treated with the KCa3.1 agonist SKA-31 (''K+ agonist'') compared to TILs treated without SKA-31 during REP (''no treatment'').

Figure 12

[0093] Shows the percentage of CD8+CD27+ T cell subset observed in TILs treated with the KCa3.1 agonist SKA-31 (''K+ agonist'') compared to TILs treated without SKA-31 during REP (''no treatment'').

Figure 13

[0094] Shows the increase in CCR7+ expression in CD4+ and CD8+ TILs (''K+ agonist'') obtained from three tumor fragments (kidney, estrogen receptor positive (ER+) breast, and melanoma) treated with the KCa3.1 agonist SKA-31 compared to TILs treated without SKA-31 during pre-REP (''no treatment'').

Figure 14

[0095] Shows the results of a representative flow cytometry experiment performed to measure CCR7+ expression.

Figure 15

[0096] Shows a statistically significant increase in CCR7+ expression in CD4+ and CD8+ TILs obtained from 14 tumor fragments.

Figure 16

[0097] Shows the results of a representative flow cytometry experiment performed to measure CCR7+ expression.

Figure 17

[0098] It shows a statistically significant increase in CD25+ expression in CD4+ and CD8+ TILs obtained from 16 tumor fragments.

Figure 18

[0099] It shows the results of a representative flow cytometry experiment conducted to measure CD25+ expression.

Figure 19

[0100] It shows the absolute cell numbers obtained for ovarian tumors in pre-REP treated with the KCa3.1 agonist SKA-31 (「SKA-31」) compared to TILs treated without SKA-31 (「untreated」).

Figure 20

[0101] It shows the absolute cell numbers obtained for breast tumors in pre-REP treated with the KCa3.1 agonist SKA-31 (「SKA-31」) compared to TILs treated without SKA-31 (「untreated」).

Figure 21

[0102] It shows the results of IFN-γ secretion from TILs after co-culturing melanoma tumor cells and TILs for 24 hours at an effector:target (E:T) ratio of 3:1 using TILs prepared with or without SKA-31. NT refers to untreated (i.e., TILs prepared without SKA-31).

Figure 22

[0103] It shows the killing titers (measured by caspase-3) of the M1032 TIL cell line at different E:T ratios with MHC-I blockade (using an antibody) and without MHC-I blockade (「non-blockade」).

Figure 23

[0104] It shows the killing titers (measured by caspase-3) of the M1041 TIL cell line at different E:T ratios with MHC-I blockade (using an antibody) and without MHC-I blockade (「non-blockade」).

Figure 24

[0105] Illustrates the TIL expansion and treatment process. The potassium channel agonists of the present disclosure, including one or more KCa3.1 agonists of the present disclosure, can be used in both the pre-REP stage (upper half of the figure) and / or the REP stage (lower half of the figure), and can be added when adding IL-2 to each cell culture. Step 1 refers to the addition of 4 tumor fragments to 10 G-Rex 10 flasks. In step 2, approximately 40×106 or more TIL are obtained. In step 3, splitting occurs into 36 G-Rex 100 flasks for REP. In step 4, the TIL are harvested by centrifugation. Fresh TIL product is obtained in step 5 after a total process time of approximately 43 days, at which point the TIL can be infused into the patient.

Figure 25

[0106] Shows a treatment protocol for use with TIL expanded with the potassium channel agonists of the present disclosure. Surgery (and tumor resection) is performed at the start, and lymphodepleting chemotherapy refers to myeloablative lymphodepletion with chemotherapy as described elsewhere herein. The potassium channel agonists of the present disclosure can also be used during treatment as described herein after administration of the TIL.

Mode for Carrying Out the Invention

[0083] Brief Description of the Sequence Listing

[0107] SEQ ID NO: 1 is the amino acid sequence of the heavy chain of muromonab.

[0084]

[0108] SEQ ID NO: 2 is the amino acid sequence of the light chain of muromonab.

[0085]

[0109] SEQ ID NO: 3 is the amino acid sequence of the recombinant human IL-2 protein.

[0086]

[0110] SEQ ID NO: 4 is the amino acid sequence of aldesleukin.

[0087]

[0111] SEQ ID NO: 5 is the amino acid sequence of the recombinant human IL-4 protein.

[0088]

[0112] SEQ ID NO: 6 is the amino acid sequence of recombinant human IL-7 protein.

[0089]

[0113] SEQ ID NO: 7 is the amino acid sequence of recombinant human IL-15 protein.

[0090]

[0114] SEQ ID NO: 8 is the amino acid sequence of recombinant human IL-21 protein.

[0091] DETAILED DESCRIPTION OF THE INVENTION

[0115] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All patents and publications mentioned herein are incorporated herein by reference in their entirety.

[0092] DEFINITIONS

[0116] As used herein, the terms “co-administered,” “co-administering,” “administered in combination with,” “administering in combination with,” “simultaneous” and “concurrent” include the administration to a subject of two or more active pharmaceutical ingredients (in a preferred embodiment of the invention, for example, at least one potassium channel agonist in combination with a plurality of TILs) such that both active pharmaceutical ingredients and / or their metabolites are present in the subject at the same time. Co-administration includes administration in separate compositions at the same time, administration at different times in separate compositions, or administration in a composition in which two or more active pharmaceutical ingredients are present. Administration in separate compositions at the same time and administration in a composition in which both agents are present are preferred.

[0093]

[0117] The term “in vivo” refers to events occurring within the body of a mammalian subject.

[0094]

[0118] The term “ex vivo” refers to events occurring outside the body of a mammalian subject in an artificial environment.

[0095]

[0119] The term "in vitro" refers to events occurring in a test system. In vitro assays include cell-based assays in which viable or dead cells can be used, and may also include cell-free assays in which intact cells are not used.

[0096]

[0120] The term "rapid proliferation" means an increase in the number of antigen-specific TILs of at least about 3-fold (or 4-fold, 5-fold, 6-fold, 7-fold, 8-fold or 9-fold), more preferably at least about 10-fold (or 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold or 90-fold), or most preferably at least about 100-fold over a one-week period. Several rapid proliferation protocols are described herein.

[0097]

[0121] As used herein, the terms "fragmentation", "fragment" and "fragmented" used to describe the process of breaking up a tumor include mechanical fragmentation methods such as the crushing, slicing, dividing and mincing of tumor tissue and other methods of disrupting the physical structure of tumor tissue.

[0098]

[0122] As used herein, "tumor-infiltrating lymphocytes" or "TIL" means a population of cells originally obtained as white blood cells that have migrated from the bloodstream of a subject into a tumor. TILs include, but are not limited to, CD8 + cytotoxic T cells (lymphocytes), Th1 and Th17 CD4 + T cells, natural killer cells, dendritic cells and M1 macrophages. TILs include both primary and secondary TILs. "Primary TIL" is obtained from a tissue sample of a patient (sometimes referred to as "freshly harvested") as outlined herein, and "secondary TIL" is any population of TIL cells that has been expanded or proliferated (as discussed herein) including, but not limited to, bulk TIL and proliferated TIL ("REP TIL" or "TIL post-REP"). A TIL cell population may include genetically modified TILs.

[0099]

[0123] As used herein, "population of cells" (including TIL) means several cells sharing a common trait. Generally, the population is generally in the range of 1×10 6 to 1×10 10 , and different TIL populations contain different numbers. For example, the initial expansion of primary TIL in the presence of IL-2 results in a population of bulk TIL of approximately 1×10 8 cells. REP expansion is generally performed to provide a population of 1.5×10 9 to 1.5×10 10 cells for injection.

[0100]

[0124] As used herein, "cryopreserved TIL" means that any TIL of primary, bulk, or expanded (REP TIL) is treated and stored in the range of about -150°C to -60°C. General methods for cryopreservation are also described elsewhere in this specification, including in the examples. For clarity, "cryopreserved TIL" is distinguishable from cryopreserved tissue samples that can be used as a source of primary TIL.

[0101]

[0125] As used herein, "thawed cryopreserved TIL" means a population of TIL that has been previously cryopreserved and then treated to return to room temperature or above, including but not limited to cell culture temperature or a temperature at which the TIL can be administered to a patient.

[0102]

[0126] TIL can generally be defined biochemically using cell surface markers or functionally by their ability to infiltrate tumors and affect treatment. TIL can generally be classified by expressing one or more of the following biomarkers: CD4, CD8, TCRαβ, CD27, CD28, CD56, CCR7, CD45Ra, CD95, PD-1, and CD25. Additionally and alternatively, TIL can be defined functionally by their ability to infiltrate solid tumors upon reintroduction into a patient.

[0103]

[0127] The term "central memory T cell" refers to a subset of T cells in humans that are CD45R0+ and constitutively express CCR7 (CCR7 hi ) and CD62L (CD62 hi ). The surface phenotype of central memory T cells also includes TCR, CD3, CD127 (IL-7R), and IL-15R. The transcription factors of central memory T cells include BCL-6, BCL-6B, MBD2, and BMI1. Central memory T cells secrete mainly IL-2 and CD40L as effector molecules after TCR triggering. Central memory T cells are predominant in the CD4 compartment of the blood and are proportionally concentrated in lymph nodes and tonsils in humans.

[0104]

[0128] The term "effector memory T cell" is, like central memory T cells, CD45R0+, but has lost constitutive expression of CCR7 (CCR7 lo ) and is heterogeneous or low in CD62L expression (CD62L lo ) and 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. The transcription factors of central memory T cells include BLIMP1. Effector memory T cells rapidly secrete high levels of inflammatory cytokines, including interferon-γ, IL-4, and IL-5, after antigen stimulation. Effector memory T cells are predominant in the CD8 compartment of the blood and are proportionally concentrated in the lung, liver, and intestine in humans. CD8+ effector memory T cells carry a large amount of perforin.

[0105]

[0129] 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 with the method of the present invention. Examples of closed systems include, but are not limited to, sealed G containers (G-containers). When 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 the patient.

[0106]

[0130] The terms "peripheral blood mononuclear cells" and "PBMC" refer to peripheral blood cells with round nuclei, including lymphocytes (T cells, B cells, NK cells) and monocytes. Preferably, the peripheral blood mononuclear cells are irradiated allogeneic peripheral blood mononuclear cells.

[0107]

[0131] The term "anti-CD3 antibody" refers to an antibody or a variant thereof, such as a monoclonal antibody, and includes human, humanized, chimeric or mouse antibodies 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. Other anti-CD3 antibodies include, for example, otrexup, teprotumumab and visilizumab.

[0108]

[0132] The term "OKT-3" (also referred to herein as "OKT3") refers to a monoclonal antibody or biosimilar or a variant thereof, including human, humanized, chimeric or mouse antibodies directed against the CD3 receptor in the T cell antigen receptor of mature T cells, and includes commercially available forms such as OKT-3 (30 ng / mL, MACS GMP CD3 pure, Miltenyi Biotech, Inc., San Diego, CA, USA) and muromonab or a variant thereof, conservative amino acid substitutions, glycoforms or biosimilars. The amino acid sequences of the heavy and light chains of muromonab are shown in Table 1 (SEQ ID NO: 1 and SEQ ID NO: 2). Hybridomas capable of producing OKT-3 have been deposited with the American Type Culture Collection and are assigned the ATCC accession number CRL 8001. Hybridomas capable of producing OKT-3 have also been deposited with the European Collection of Authenticated Cell Cultures (ECACC) and are assigned the catalogue number 86022706.

[0109]

Table 1

[0110]

[0133] The term "IL-2" (also referred to herein as "IL2") refers to the T cell growth factor known as interleukin-2 and includes all forms of IL-2, including human and mammalian forms, conservative amino acid substitutions, glycoforms, biosimilars, and variants thereof. IL-2 is described, for example, in Nelson, J. Immunol. 2004, 172, 3983-88 and Malek, Annu. Rev. Immunol. 2008, 26, 453-79, the disclosures of which are incorporated herein by reference. The amino acid sequence of recombinant human IL-2 suitable for use in the present invention is shown in Table 2 (SEQ ID NO: 3). For example, the term IL-2 includes human recombinant forms of IL-2 such as aldesleukin (PROLEUKIN, commercially available from multiple suppliers at 22 million IU per single-use vial) and recombinant IL-2 forms commercially supplied from CellGenix, Inc., Portsmouth, NH, USA (CELLGRO GMP) or ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (catalog number CYT-209-b), and other commercially available equivalents from other vendors. Aldesleukin (des-alanyl-1, serine-125 human IL-2) is a non-glycosylated human recombinant form of IL-2 with a molecular weight of approximately 15 kDa. The amino acid sequence of aldesleukin suitable for use in the present invention is shown in Table 2 (SEQ ID NO: 4). The term IL-2 also includes pegylated forms of IL-2, including the pegylated IL2 prodrug NKTR-214 available from Nektar Therapeutics, South San Francisco, CA, USA, as described herein. NKTR-214 and pegylated IL-2 suitable for use in the present invention are described in US Patent Application Publication No. 2014 / 0328791A1 and International Publication No. 2012 / 065086A1, the disclosures of which are incorporated herein by reference. Alternative forms of conjugated IL-2 suitable for use in the present invention are described in US 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.

[0111]

Table 2

[0112]

[0134] 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. When activated by IL-4, Th2 T cells subsequently produce additional IL-4 in a positive feedback loop. IL-4 stimulates B cell proliferation and class II MHC expression and also induces class switching to IgE and IgG 1 expression from B cells. Recombinant human IL-4 suitable for use in the present invention is commercially available from a plurality of suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (catalog number CYT-211) and ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-4 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: 5).

[0113]

[0135] 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 cells, epithelial cells, and dendritic cells. Fry and Mackall, Blood 2002, 99, 3892-904. IL-7 can stimulate the expression of T cells. IL-7 binds to the IL-7 receptor, a heterodimer consisting of the IL-7 receptor alpha and the common gamma chain receptor, in 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 multiple suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (catalog number CYT-254) and ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-7 recombinant protein, catalog number Gibco PHC0071). The amino acid sequence of recombinant human IL-7 suitable for use in the present invention is shown in Table 2 (SEQ ID NO: 6).

[0114]

[0136] 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-15, including human and mammalian forms, conservative amino acid substitutions, glycoforms, biosimilars, and variants thereof. IL-15 is described, for example, in Fehniger and Caligiuri, Blood 2001, 97, 14-32, the disclosure of which is incorporated herein by reference. IL-15 shares the 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 multiple 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: 7).

[0115]

[0137] The term "IL-21" (also referred to herein as "IL21") refers to the pleiotropic cytokine protein known as interleukin-21 and includes all forms of IL-21, including human and mammalian forms, conservative amino acid substitutions, glycoforms, biosimilars, and variants thereof. IL-21 is described, for example, in Spolski and Leonard, Nat.Rev. Drug.Disc.2014, 13, 379-95, the disclosure of which is incorporated herein by reference. IL-21 is mainly expressed in 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 multiple 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: 8).

[0116]

[0138] When an "anti-tumor effective amount", "tumor-suppressing 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 age, body weight, tumor size, degree of infection or metastasis, and the condition of the patient (subject). Generally, a pharmaceutical composition containing tumor-infiltrating lymphocytes (e.g., secondary TIL or genetically engineered cytotoxic lymphocytes) described herein contains all integer values within these ranges, 10 4 ~10 11 cells / kg body weight (e.g., 10 5 ~10 6 、10 5 ~10 10 、10 5 ~10 11 、10 6 ~10 10 、10 6 ~10 11 ,10 7 ~10 11 、10 7 ~10 10 、10 8 ~10 11 、10 8 ~10 10 、10 9 ~10 11 or 10 9 ~10 10It can be stated that it can be administered at a dose of cells / kg body weight. Tumor-infiltrating lymphocyte (in some cases, including genetically engineered cytotoxic lymphocytes) compositions can also be administered multiple times at these doses. Tumor-infiltrating lymphocytes (in some cases, including genetically engineered ones) can be administered by using infusion techniques commonly known in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). The optimal dose and treatment regimen for a particular patient can be readily determined by those skilled in the art by monitoring the patient for signs of the disease and adjusting the treatment accordingly.

[0117]

[0139] The term "liquid tumor" refers to an abnormal mass of cells that is inherently fluid. Liquid tumor cancers include, but are not limited to, leukemia, myeloma, and lymphoma, as well as other blood malignancies. TILs obtained from liquid tumors are also referred to herein as marrow-infiltrating lymphocytes (MILs).

[0118]

[0140] The term "blood malignancy" refers to cancers of mammals and tumors of the hematopoietic and lymphatic tissues that include, but are not limited to, blood, bone marrow, lymph nodes, and lymphatic tissues. Blood malignancies are also referred to as "liquid tumors." Blood malignancies include, but are not limited to, acute lymphoblastic leukemia (ALL), chronic lymphocytic lymphoma (CLL), small lymphocytic lymphoma (SLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute monocytic leukemia (AMoL), Hodgkin lymphoma, and non-Hodgkin lymphoma. The term "B-cell blood malignancy" refers to a blood malignancy that affects B cells.

[0119]

[0141] The term "solid tumor" refers to an abnormal mass of tissue that typically does not contain cysts or liquid areas. Solid tumors can be either benign or malignant. The term "solid tumor cancer" refers to a malignant, neoplastic, or cancerous solid tumor. Solid tumor cancers include, but are not limited to, sarcomas, carcinomas, and lymphomas such as lung cancer, breast cancer, prostate cancer, colon cancer, rectal cancer, and bladder cancer. The tissue structure of a solid tumor includes interdependent tissue compartments containing soft tissue (cancer cells) and supporting stromal cells in which cancer cells can be dispersed and which provide a supportive microenvironment.

[0120]

[0142] As used herein, the term "microenvironment" can refer to the solid or blood tumor microenvironment as a whole or to individual subsets of cells within the microenvironment. As described in Swartz, et al., Cancer Res., 2012, 72, 2473, the tumor microenvironment as used herein 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 the tumor from the host immune system, foster treatment resistance, and provide a niche for successful metastatic seeding." Tumors express antigens that should be recognized by T cells, but elimination of tumors by the immune system is rare due to immune suppression by the microenvironment.

[0121]

[0143] The term "pharmaceutically acceptable salt" refers to salts derived from a variety of organic and inorganic counterions known in the art. Pharmaceutically acceptable acid addition salts can be formed using inorganic and organic acids. Preferred inorganic acids from which salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, and phosphoric acid. Preferred organic acids from which salts can be derived include, for example, acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, and salicylic acid. Pharmaceutically acceptable base addition salts can be formed using inorganic and organic bases. Inorganic bases from which salts can be derived include, for example, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, and aluminum. Organic bases from which salts can be derived include, for example, primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins. Specific examples include isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine. In some embodiments, the pharmaceutically acceptable base addition salts are selected from ammonium salts, potassium salts, sodium salts, calcium salts, and magnesium salts. The term "cocrystal" refers to a molecular complex derived from several cocrystal formers known in the art. Unlike salts, cocrystals typically do not involve hydrogen transfer between the cocrystal and the drug, but instead involve intermolecular interactions such as hydrogen bonding, aromatic ring stacking, or dispersion forces between the cocrystal former and the drug in the crystal structure.

[0122]

[0144] The terms "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" are intended to include any solvent, dispersion medium, coating, antibacterial and antifungal agents, isotonic and absorption delaying agents, and inert ingredients. The use of such pharmaceutically acceptable carriers or pharmaceutically acceptable excipients for active pharmaceutical ingredients is well known in the art. The use thereof in the therapeutic compositions of the present invention is contemplated, except where any conventional pharmaceutically acceptable carrier or pharmaceutically acceptable excipient is incompatible with the active pharmaceutical ingredient. Additional active pharmaceutical ingredients, such as other drugs, can also be incorporated into the described compositions and methods.

[0123]

[0145] The term "solvate" refers to a compound that is physically associated with one or more molecules of a pharmaceutically acceptable solvent. The term solvate includes hydrates in which water is physically associated with the compound in the solid state and organic solvates.

[0124]

[0146] The term "prodrug" is intended to describe a compound that can be converted to the biologically active compounds described herein under physiological conditions or by solvolysis. Thus, the term "prodrug" refers to a precursor of a pharmaceutically acceptable biologically active compound. A prodrug can be inactive when administered to a subject but is converted to the active compound in vivo, for example, by hydrolysis. Prodrug compounds often provide advantages in solubility, tissue compatibility, or delayed release in mammals (see, e.g., Bundgaard, H., Design of Prodrugs (1985) (Elsevier, Amsterdam)). The term "prodrug" is also intended to include any covalently attached carrier that releases the active compound in vivo when administered to a subject. Prodrugs of the active compounds described herein can be prepared by modifying the functional groups present in the active compounds in such a way that the modification is cleaved, either in a routine procedure or in vivo, to yield the active parent compound. Prodrugs include, for example, compounds in which a hydroxy, amino, or mercapto group is bonded to any group that cleaves to form a free hydroxy, free amino, or free mercapto group, respectively, when the prodrug of the active compound is administered to a mammalian subject. Examples of prodrugs include, but are not limited to, acetate, formate, and benzoate derivatives of alcohols, various ester derivatives of carboxylic acids, or acetamide, formamide, and benzamide derivatives of amine functional groups in the active compound.

[0125]

[0147] "Alkyl" refers to a straight or branched hydrocarbon chain radical consisting only of carbon and hydrogen atoms, having 1 to 10 carbon atoms and containing no unsaturation (e.g., (C 1~10 )alkyl or C 1~10refers to "(alkyl)". Whenever shown in this specification, a numerical range such as "1 to 10" always refers to each integer within the given range. For example, "1 to 10 carbon atoms" means that the alkyl group can consist of a maximum of 10 carbon atoms such as 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc. However, this definition also intends to cover the occurrence of the term "alkyl" when the numerical range is not specifically specified. Typical alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, n - butyl, isobutyl, sec - butyl isobutyl, tert - butyl, pentyl, isopentyl, neopentyl, hexyl, heptyl, octyl, nonyl, and decyl. The alkyl moiety can be bonded to the rest of the molecule by a single bond, such as methyl (Me), ethyl (Et), n - propyl (Pr), 1 - methylethyl (isopropyl), n - butyl, n - pentyl, 1,1 - dimethylethyl (t - butyl), and 3 - methylhexyl, etc. Unless otherwise specifically stated in this specification, the alkyl group can independently be heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, hydroxy, halo, cyano, trifluoromethyl, trifluoromethoxy, nitro, trimethylsilyl, -OR a , -SR a , -OC(O)-R a , -N(R a ) 2 , -C(O)R a , -C(O)OR a , -OC(O)N(R a ) 2 , -C(O)N(R a ) 2 , -N(R a )C(O)OR a , -N(R a )C(O)R a , -N(R a )C(O)N(R a ) 2 , N(R a )C(NR a )N(R a ) 2 , -N(Ra )S(O) t R a (where t is 1 or 2), -S(O) t OR a (where t is 1 or 2), -S(O) t N(R a ) 2 (where t is 1 or 2) or PO 3 (R a ) 2 (each R a is independently hydrogen, fluoroalkyl, carbocyclic, carbocyclic alkyl, aryl, aralkyl, heterocycloalkyl, heterocycloalkyl alkyl, heteroaryl or heteroaryl alkyl) and is optionally substituted by one or more substituents.

[0126]

[0148] "Alkoxy" refers to an -O-alkyl group containing 1 to 8 carbon atoms in a straight chain, branched, cyclic arrangement and combinations thereof, bonded to the parent structure through oxygen. Examples include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, cyclopropyloxy and cyclohexyloxy. "Lower alkoxy" refers to an alkoxy group containing 1 to 6 carbons. The term "substituted alkoxy" refers to an alkoxy in which the alkyl component is substituted (i.e., -O-(substituted alkyl)). Unless otherwise specified herein, the alkyl portion of an alkoxy group is independently alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, hydroxy, halo, cyano, trifluoromethyl, trifluoromethoxy, nitro, trimethylsilyl, -OR a , -SR a , -OC(O)-R a , -N(R a ) 2 , -C(O)R a , -C(O)OR a , -OC(O)N(R a ) 2 , -C(O)N(R a ) 2 , -N(Ra )C(O)OR a 、 -N(R a )C(O)R a 、 -N(R a )C(O)N(R a ) 2 、 N(R a )C(NR a )N(R a ) 2 、 -N(R a )S(O) t R a (t is 1 or 2), -S(O) t OR a (t is 1 or 2), -S(O) t N(R a ) 2 (t is 1 or 2) or PO 3 (R a ) 2 (each R a is independently hydrogen, alkyl, fluoroalkyl, carbocyclic, carbocyclic alkyl, aryl, aralkyl, heterocycloalkyl, heterocycloalkyl alkyl, heteroaryl or heteroaryl alkyl) and is optionally substituted by one or more substituents.

[0127]

[0149] "Amino" or "amine", unless otherwise specified, refers to the -N(R a ) 2 radical group, and each R a is independently hydrogen, alkyl, fluoroalkyl, carbocyclic, carbocyclic alkyl, aryl, aralkyl, heterocycloalkyl, heterocycloalkyl alkyl, heteroaryl or heteroaryl alkyl. When the -N(R a ) 2 group has two R a substituents other than hydrogen, they can together with the nitrogen atom form a 4-, 5-, 6- or 7-membered ring. For example, -N(R a ) 2is intended to include, but is not limited to, 1-pyrrolidinyl and 4-morpholinyl. Unless otherwise specified herein, an amino group is independently alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, hydroxy, halo, cyano, trifluoromethyl, trifluoromethoxy, nitro, trimethylsilyl, -OR a , -SR a , -OC(O)-R a , -N(R a ) 2 , -C(O)R a , -C(O)OR a , -OC(O)N(R a ) 2 , -C(O)N(R a ) 2 , -N(R a )C(O)OR a , -N(R a )C(O)R a , -N(R a )C(O)N(R a ) 2 , N(R a )C(NR a )N(R a ) 2 , -N(R a )S(O) t R a (t is 1 or 2), -S(O) t OR a (t is 1 or 2), -S(O) t N(R a ) 2 (t is 1 or 2) or PO 3 (R a ) 2 (each R a is independently hydrogen, alkyl, fluoroalkyl, carbocyclic, carbocyclic alkyl, aryl, aralkyl, heterocycloalkyl, heterocycloalkyl alkyl, heteroaryl or heteroaryl alkyl) and is optionally substituted by one or more substituents. The term "substituted amino" means, as described above, the group -NHR dand NR d R d also refers to the N-oxide thereof. The N-oxide can be prepared by treating the corresponding amino group with, for example, hydrogen peroxide or m-chloroperbenzoic acid.

[0128]

[0150] "Aromatic" or "aryl" or "Ar" refers to an aromatic radical having 6 to 14 ring atoms (e.g., phenyl, fluorenyl, and naphthyl) having at least one ring (e.g., phenyl, fluorenyl, and naphthyl) having a carbocyclic conjugated π electron system (e.g., C 6 ~C 14 aromatic or C 6 ~C 14 aryl). A divalent radical formed from a substituted benzene derivative and having a free valence on a ring atom is called a substituted phenylene radical. A divalent radical derived from a monovalent polycyclic hydrocarbon radical ending with "-yl" by removing one hydrogen atom from a carbon atom having a free valence is named by adding "-idene" to the name of the corresponding monovalent radical. For example, a naphthyl group having two bonding points is naphthylidene. As always shown in this specification, a numerical range such as "6 to 10" refers to each integer within a given range. For example, "6 to 10 ring atoms" means that an aryl group can consist of a maximum of 10 ring atoms such as 6 ring atoms and 7 ring atoms. This term includes monocyclic or fused-ring polycyclic (i.e., rings sharing adjacent pairs of ring atoms) groups. Unless otherwise specified herein, the aryl moiety is independently alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, hydroxy, halo, cyano, trifluoromethyl, trifluoromethoxy, nitro, trimethylsilyl, -OR a 、-SR a 、-OC(O)-R a 、-N(R a ) 2 、-C(O)R a 、-C(O)OR a 、-OC(O)N(R a ) 2 、-C(O)N(Ra ) 2 ,-N(R a )C(O)OR a ,-N(R a )C(O)R a ,-N(R a )C(O)N(R a ) 2 、N(R a )C(NR a )N(R a ) 2 ,-N(R a )S(O) t R a (t is 1 or 2), -S(O) t OR a (t is 1 or 2), -S(O) t N(R a ) 2 (t is 1 or 2) or PO 3 (R a ) 2 (each R a is independently hydrogen, alkyl, fluoroalkyl, carbocyclic, carbocyclic alkyl, aryl, aralkyl, heterocycloalkyl, heterocycloalkyl alkyl, heteroaryl or heteroaryl alkyl) and is optionally substituted by one or more substituents.

[0129]

[0151] "Cycloalkyl" refers to a monocyclic or polycyclic radical that contains only carbon and hydrogen and may be saturated or partially unsaturated. A cycloalkyl group is a group having 3 to 10 ring atoms (i.e., (C 3~10 )cycloalkyl or C 3~10It includes (cycloalkyl). Whenever shown in this specification, a numerical range such as "3 to 10" refers to each integer within the given range. For example, "3 to 10 carbon atoms" means that the cycloalkyl group can consist of a maximum of 10 carbon atoms such as 3 carbon atoms. Examples of cycloalkyl groups include, but are not limited to, moieties such as cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, norbornyl, etc. Unless otherwise specifically stated in this specification, the cycloalkyl group is independently alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, hydroxy, halo, cyano, trifluoromethyl, trifluoromethoxy, nitro, trimethylsilyl, -OR a , -SR a , -OC(O)-R a , -N(R a ) 2 , -C(O)R a , -C(O)OR a , -OC(O)N(R a ) 2 , -C(O)N(R a ) 2 , -N(R a )C(O)OR a , -N(R a )C(O)R a , -N(R a )C(O)N(R a ) 2 , N(R a )C(NR a )N(R a ) 2 , -N(R a )S(O) t R a (t is 1 or 2), -S(O) t OR a (t is 1 or 2), -S(O) t N(R a ) 2 (t is 1 or 2) or PO 3 (R a )2 (Each R a is independently optionally substituted by one or more substituents which are hydrogen, alkyl, fluoroalkyl, carbocyclyl, carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl or heteroarylalkyl).

[0130]

[0152] "Cycloalkoxy" refers to a cycloalkyl group bonded to the parent structure through oxygen. Examples include, but are not limited to, cyclopropyloxyl and cyclohexyloxyl.

[0131]

[0153] "Cyano" refers to the -CN radical.

[0132]

[0154] "Fluoroalkyl" refers to an alkyl radical as defined above substituted by one or more fluoro radicals as defined above, such as trifluoromethyl, difluoromethyl, 2,2,2-trifluoroethyl, 1-fluoromethyl-2-fluoroethyl, etc. The alkyl portion of the fluoroalkyl radical may be optionally substituted as defined above for alkyl groups.

[0133]

[0155] "Halo", "halide" or alternatively "halogen" is intended to mean fluoro, chloro, bromo or iodo. The term "haloC n~m alkyl" includes a C n~m alkyl structure substituted by one or more halo groups or combinations thereof. For example, the terms "fluoroalkyl" and "trifluoroalkyl" include haloalkyl where halo is fluorine.

[0134]

[0156] "Heteroaryl", or "heteroaromatic", or "HetAr" is a 5- to 18-membered aromatic radical which may be monocyclic, bicyclic, tricyclic or tetracyclic and which contains one or more ring heteroatoms selected from nitrogen, oxygen and sulfur (e.g., C 5 ~C13refers to (heteroaryl). Whenever shown in this specification, a numerical range such as "5 to 18" always refers to each integer within the given range. For example, "5 to 18 ring atoms" means that a heteroaryl group can consist of a maximum of 18 ring atoms such as 5 ring atoms, 6 ring atoms, etc. A divalent radical derived from a monovalent heteroaryl radical whose name ends with "-yl" by removing one hydrogen atom from an atom with a free valence is named by adding "-iden" to the name of the corresponding monovalent radical. For example, a pyridyl group having two bonding points is pyridinylene. An N-containing "heteroaromatic" or "heteroaryl" moiety refers to an aromatic group in which at least one of the ring backbone atoms is a nitrogen atom. A polycyclic heteroaryl group can be either a fused type or a non-fused type. The heteroatoms in a heteroaryl radical are optionally oxidized. When one or more nitrogen atoms are present, it is optionally quaternized. A heteroaryl can be bonded to the rest of the molecule via any atom of the ring. Examples of heteroaryl include, but are not limited to, azepinyl, acridinyl, benzimidazolyl, benzindolyl, 1,3-benzodioxolyl, benzofuranyl, benzoxazolyl, benzo[d]thiazolyl, benzothiadiazolyl, benzo[b][1,4]dioxepinyl, benzo[b][1,4]oxazinyl, 1,4-benzodioxanyl, benzonaphthofuranyl, benzoxazolyl, benzodioxolyl, benzodioxinyl, benzoxazolyl, benzopyranyl, benzopyranonyl, benzofuranyl, benzofuranonyl, benzofurazanyl, benzothiazolyl, benzothienyl (benzothiophenyl), benzothieno[3,2-d]pyrimidinyl, benzotriazolyl, benzo[4,6]imidazo[1,2-a]pyridinyl, carbazolyl, cinnolinyl, cyclopenta[d]pyrimidinyl, 6,7-dihydro-5H-cyclopenta[4,5]thieno[2,3-d]pyrimidinyl, 5,6-dihydrobenzo[h]quinazolinyl, 5,6-dihydrobenzo[h]cinnolinyl, 6,7-dihydro-5H-benzo[6,7]cyclohepta[1,2-c]pyridazinyl, dibenzofuranyl, dibenzothiophenyl, furanyl, furazanyl, furanonyl, furo[3,2-c]pyridinyl, 5,6,7,8,9,10 - hexahydrocycloocta[d]pyrimidinyl, 5,6,7,8,9,10 - hexahydrocycloocta[d]pyridazinyl, 5,6,7,8,9,10 - hexahydrocycloocta[d]pyridinyl, isothiazolyl, imidazolyl, indazolyl, indolyl, indazolyl, isoindolyl, indolinyl, isoindolinyl, isoquinolyl, indolizinyl, isoxazolyl, 5,8 - methano - 5,6,7,8 - tetrahydroquinazolinyl, naphthyridinyl, 1,6 - naphthyridinonyl, oxadiazolyl, 2 - oxoazepinyl, oxazolyl, oxiranyl, 5,6,6a,7,8,9,10,10a - octahydrobenzo[h]quinazolinyl, 1 - phenyl - 1H - pyrrolyl, phenazinyl, phenothiazinyl, phenoxazinyl, phthalazinyl, pteridinyl, purinyl, pyranyl, pyrrolyl, pyrazolyl, pyrazolo[3,4 - d]pyrimidinyl, pyridinyl, pyrido[3,2 - d]pyrimidinyl, pyrido[3,4 - d]pyrimidinyl, pyrazinyl, pyrimidinyl, pyridazinyl, pyrrolyl, quinazolinyl, quinoxalinyl, quinolinyl, isoquinolinyl, tetrahydroquinolinyl, 5,6,7,8 - tetrahydroquinazolinyl, 5,6,7,8 - tetrahydrobenzo[4,5]thieno[2,3 - d]pyrimidinyl, 6,7,8,9 - tetrahydro - 5H - cyclohepta[4,5]thieno[2,3 - d]pyrimidinyl, 5,6,7,8 - tetrahydropyrido[4,5 - c]pyridazinyl, thiazolyl, thiadiazolyl, thiapyryl, triazolyl, tetrazolyl, triazinyl, thieno[2,3 - d]pyrimidinyl, thieno[3,2 - d]pyrimidinyl, thieno[2,3 - c]pyridinyl and thiophenyl (i.e., thienyl) are included. Unless otherwise specified herein, the heteroaryl moiety is independently alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, hydroxy, halo, cyano, nitro, oxo, thioxo, trimethylsilyl, - OR, a , - SR a , - OC(O) - R a , - N(R a ) 2, -C(O)R a , -C(O)OR a , -OC(O)N(R a ) 2 , -C(O)N(R a ) 2 , -N(R a )C(O)OR a , -N(R a )C(O)R a , -N(R a )C(O)N(R a ) 2 , N(R a )C(NR a )N(R a ) 2 , -N(R a )S(O) t R a (t is 1 or 2), -S(O) t OR a (t is 1 or 2), -S(O) t N(R a ) 2 (t is 1 or 2) or PO 3 (R a ) 2 (each R a is independently hydrogen, alkyl, fluoroalkyl, carbocyclic, carbocyclic alkyl, aryl, aralkyl, heterocycloalkyl, heterocycloalkyl alkyl, heteroaryl or heteroaryl alkyl) and is optionally substituted by one or more substituents. Substituted heteroaryl also includes ring systems substituted with one or more oxide (-O-) substituents such as pyridinyl N-oxide. Other non-limiting examples of heterocycles include azetidine, pyrrolidine, imidazolidine, piperidine and piperazine.

[0135]

[0157] "Hydroxy" refers to the -OH radical.

[0136]

[0158] "Nitro" refers to the -NO 2 radical.

[0137]

[0159] The term "antibody" and its plural form "antibodies" refer to immunoglobulins in their entirety and any antigen-binding fragments ("antigen-binding portions") or single chains thereof. "Antibody" further refers to a glycoprotein containing at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen-binding portion thereof. Each heavy chain is composed of a heavy-chain variable region (abbreviated herein as V H for short) and a heavy-chain constant region. The heavy-chain constant region is composed of three domains, CH1, CH2, and CH3. Each light chain is composed of a light-chain variable region (abbreviated herein as V L for short) and a light-chain constant region. The light-chain constant region is composed of one domain, C L . The V H and V L regions of the antibody are referred to as complementarity-determining regions (CDRs) or hypervariable regions (HVRs), and can be further subdivided into regions having hypervariability that can be dispersed in more conserved regions (called framework regions (FRs)). Each V H and V L is composed of three CDRs and four FRs arranged in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus towards the carboxy terminus. 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 can 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 of the classical complement system (Clq).

[0138]

[0160] 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 a TCR when presented by a major histocompatibility complex (MHC) molecule. As used herein, the term "antigen" also encompasses T cell epitopes. An antigen can be further recognized by the immune system. In some embodiments, an antigen can induce a humoral or cellular immune response that results in the activation of B lymphocytes and / or T lymphocytes. In some cases, this may require that the antigen contains or binds to a Th cell epitope. An antigen can also have one or more epitopes (e.g., B epitopes and T epitopes). In some embodiments, an antigen preferably reacts with its corresponding antibody or TCR in a highly specific and selective manner, typically, and does not react with a number of other antibodies or TCRs that can be induced by other antigens.

[0139]

[0161] The terms "monoclonal antibody", "mAb", "monoclonal antibody composition" or plural forms thereof refer to a preparation of antibody molecules of a single molecular composition. A monoclonal antibody composition exhibits a single binding specificity and affinity for a particular epitope. A monoclonal antibody specific for a particular receptor can be made using knowledge and techniques in the art of injecting an appropriate antigen into a test subject and then isolating a hybridoma that expresses an antibody having the desired sequence or functional characteristics. DNA encoding a monoclonal antibody can be 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. The DNA can be placed into an expression vector once isolated and then transfected into host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells or myeloma cells that do not otherwise produce immunoglobulin proteins in other ways to obtain the synthesis of monoclonal antibodies in recombinant host cells. The recombinant production of antibodies is described in more detail below.

[0140]

[0162] 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 F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; and (iii) a V(ab')2 fragment, which is a monovalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region. H and an Fd fragment consisting of the CH1 domain; (iv) a V of a single arm of an antibody. L and V H (v) an Fv fragment consisting of a V H Or V L and (vi) isolated complementarity determining regions (CDRs). In addition, the two domains of the Fv fragment, V L and V H are encoded by separate genes, which can be synthesized by recombinant methods. L and V H The domain pairs can be linked by a synthetic linker, allowing them to be produced as a single protein chain forming 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 within the term "antigen-binding portion" or "antigen-binding fragment" of an antibody. These antibody fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as intact antibodies.

[0141]

[0163] As used herein, the term "human antibody" is intended to include antibodies having a variable region in which both the framework region and the CDR regions are derived from human germline immunoglobulin sequences. Further, when the antibody contains a constant region, the constant region is also 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-directed mutagenesis in vitro or somatic mutations in vivo). The term "human antibody" as used herein 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.

[0142]

[0164] The term "human monoclonal antibody" refers to an antibody that exhibits a single binding specificity and has a variable region in which both the framework region and the CDR regions are derived from human germline immunoglobulin sequences. In one embodiment, the human monoclonal antibody is produced by a hybridoma comprising B cells having a genome that includes a human heavy chain transgene and a light chain transgene that have been fused to immortalized cells and obtained from a transgenic non-human animal, e.g., a transgenic mouse.

[0143]

[0165] As used herein, the term "recombinant human antibody" includes all human antibodies prepared, expressed, produced or isolated by recombinant means, such as (a) antibodies isolated from transgenic or transchromosomal animals (such as mice) carrying human immunoglobulin genes or hybridomas prepared therefrom (further described below), (b) antibodies isolated from host cells transformed to express human antibodies, such as transfectomas, (c) antibodies isolated from recombinant combinatorial human antibody libraries, and (d) antibodies prepared, expressed, produced or isolated by any other means including splicing of 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 can be subjected to in vitro mutagenesis (or in vivo somatic mutagenesis when using transgenic animals for human Ig sequences), and thus, the amino acid sequences of the V H and V L regions are derived from and related to the human germline V H and V L sequences, while being sequences that may not naturally occur within the human antibody germline repertoire in vivo.

[0144]

[0166] As used herein, "isotype" refers to the antibody class (e.g., IgM or IgG1) encoded by the heavy chain constant region gene.

[0145]

[0167] The phrases "antibody that recognizes an antigen" and "antibody specific for an antigen" are used interchangeably herein with the term "antibody that specifically binds to an antigen".

[0146]

[0168] The term "human antibody derivative" refers to any variant form of a human antibody that includes an antibody and another active pharmaceutical ingredient or a conjugate with an antibody. The terms "conjugate", "antibody-drug conjugate", "ADC" or "immunoconjugate" refer to an antibody or a fragment thereof conjugated to another therapeutic moiety, which can be conjugated to the antibodies described herein using methods available in the art.

[0147]

[0169] The terms "humanized antibody" and "humanized" are intended to refer to antibodies in which the CDR sequences derived from the germline of another mammalian species, such as a mouse, have been transplanted into a human framework sequence. Further modifications of framework regions within the human framework sequence may be made. A humanized form of a non-human (e.g., mouse) antibody is a chimeric antibody that contains minimal sequences derived from non-human immunoglobulins. For the most part, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from the hypervariable regions of the recipient have been replaced by residues from the 15 hypervariable regions (donor antibody) of a non-human species such as a mouse, rat, rabbit, or non-human primate that have the desired specificity, affinity, and capacity. In some examples, the Fv framework region (FR) residues of the human immunoglobulin are replaced by the corresponding non-human residues. Additionally, a humanized antibody may contain residues not found in either the recipient antibody or the donor antibody. These modifications are made to further improve antibody performance. Generally, a humanized antibody contains substantially all of at least one, typically two, variable domains, where all or substantially all of the hypervariable loops correspond to those of the non-human immunoglobulin and all or substantially all of the FR regions are those of the human immunoglobulin sequence. A humanized antibody may optionally also include at least a portion of the 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 may also be modified to use any Fc variant known to confer an improvement (e.g., a decrease) in effector function and / or FcR binding.Fc variants can include any one of the amino acid substitutions disclosed in, for example, WO 1988 / 07089 A1, WO 1996 / 14339 A1, WO 1998 / 05787 A1, WO 1998 / 23289 A1, WO 1999 / 51642 A1, WO 99 / 58572 A1, WO 2000 / 09560 A2, WO 2000 / 32767 A1, WO 2000 / 42072 A2, WO 2002 / 44215 A2, WO 2002 / 060919 A2, WO 2003 / 074569 A2, WO 2004 / 016750 A2, WO 2004 / 029207 A2, WO 2004 / 035752 A2, WO 2004 / 063351 A2, WO 2004 / 074455 A2, WO 2004 / 099249 A2, WO 2005 / 040217 A2, WO 2005 / 070963 A1, WO 2005 / 077981 A2, WO 2005 / 092925 A2, WO 2005 / 123780 A2, WO 2006 / 019447 A1, WO 2006 / 047350 A2 and WO 2006 / 085967 A2; and US 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.

[0148]

[0170] The term "chimeric antibody" is intended to refer to an antibody in which the variable region sequence is derived from one species and the constant region sequence is derived from another species, such as an antibody in which the variable region sequence is derived from a mouse antibody and the constant region sequence is derived from a human antibody.

[0149]

[0171] A "diabody" is a small antibody fragment having two antigen-binding sites. The fragment is the same polypeptide chain (V H -V L or V L -V H) and a heavy chain variable domain (V L ) connected to the light chain variable domain within H ). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains on the other chain, generating two antigen-binding sites. Diabodies are described in more detail, for example, in European Patent No. 404,097, International Publication No. 93 / 11161; and Bolliger, et al, Proc. Natl. Acad. Sci. USA 1993, 90, 6444-6448.

[0150]

[0172] The term "glycosylation" refers to modified derivatives of antibodies. Aglycosylated antibodies lack glycosylation. Glycosylation can be modified, for example, to increase the affinity of the antibody for an antigen. Such carbohydrate modifications can be achieved, for example, by changing one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions can be made that result in the removal of one or more variable region framework glycosylation sites, thereby removing glycosylation at that site. As described in U.S. Patent Nos. 5,714,350 and 6,350,861, aglycosylation can increase the affinity of the antibody for an antigen. Additionally or alternatively, antibodies can be made in which the type of glycosylation is altered, such as hypofucosylated antibodies having a reduced amount of fucosyl residues or antibodies having an increased bisecting GlcNac structure. Such modified glycosylation patterns have been demonstrated to increase the ability of the antibody. Such carbohydrate modifications can be achieved, for example, by altering the glycosylation machinery to express the antibody in a host cell. Cells with modified glycosylation machinery have been described in the art and can be used as host cells for expressing the recombinant antibodies of the present invention, thereby producing antibodies having modified glycosylation. For example, the cell lines Ms704, Ms705, and Ms709 lack the fucosyltransferase gene, FUT8 (alpha(1,6)fucosyltransferase), and thus the antibodies expressed in the Ms704, Ms705, and Ms709 cell lines lack fucose on their carbohydrates. The Ms704, Ms705, and Ms709 FUT8- / - cell lines were generated by targeting the disruption of the FUT8 gene in CHO / DG44 cells using two replacement vectors (see, for example, U.S. Patent Application Publication No. 2004 / 0110704 or Yamane-Ohnuki, et al, Biotechnol. Bioeng., 2004, 87, 614-622).As another example, European Patent No. 1,176,195 describes a cell line having a functionally disrupted FUT8 gene encoding a fucosyltransferase such that the antibody expressed in such a cell line exhibits hypofucosylation by reducing or eliminating an alpha1,6-linkage related enzyme, and also describes a cell line having low or no enzyme activity for adding fucose to N-acetylglucosamine that binds to the Fc region of the antibody, such as the rat myeloma cell line YB2 / 0 (ATCC CRL 1662). International Publication No. 03 / 035835 describes a mutant CHO cell line, Lec13 cell, which has a reduced ability to bind fucose to the carbohydrate linked to Asn(297) and results in hypofucosylation of the antibody expressed in its host cell (see also Shields, et al, J. Biol Chem.2002, 277, 26733-26740). International Publication No. 99 / 54342 describes a cell line (e.g., beta(1,4)-N-acetylglucosaminyltransferase III (GnTIII)) that has been engineered to express a glycoprotein-modifying glycosyltransferase such that the antibody expressed in the engineered cell line exhibits an increased bisecting GlcNac structure that results in an increase in the ADCC activity of the antibody (see also Umana, et al., Nat.Biotech.1999, 77, 176-180). Alternatively, the fucose residue of the antibody can be cleaved using a fucosidase enzyme. For example, alpha-L-fucosidase, a fucosidase, removes fucosyl residues derived from the antibody as described in Tarentino, et al., Biochem.1975, 14, 5516-5523.

[0151]

[0173] "Pegylation" refers to a modified antibody or a fragment thereof that reacts with PEG, such as a reactive ester or aldehyde derivative of polyethylene glycol (PEG), under conditions such that one or more PEG groups become 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 effected via an acylation or alkylation reaction with a reactive PEG molecule (or similar reactive water-soluble polymer). As used herein, the term "polyethylene glycol" is intended to encompass any form of PEG that has been used to derivatize other proteins, such as mono(C 1 ~C 10 )alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. The antibody to be pegylated can be an aglycosylated antibody. Methods of pegylation are known in the art and can be applied to the antibodies of the present invention as described, for example, in European Patent Nos. 0154316 and 0401384 and U.S. Patent No. 5,824,778, the disclosures of each of which are incorporated herein by reference.

[0152]

[0174] The term "fusion protein" or "fusion polypeptide" refers to a protein that combines the properties of two or more individual proteins. Such a protein has at least two heterologous polypeptides covalently linked either directly or via an amino acid linker. The polypeptides forming the fusion protein are typically linked from the C-terminus to the N-terminus, but can also be linked from C-terminus to C-terminus, N-terminus to N-terminus, or N-terminus to C-terminus. The polypeptides of the fusion protein can be in any order and can include any two or more or both of the constituent polypeptides. This term includes conservatively modified variants, polymorphic variants, alleles, mutants, subsequences, interspecies homologs, and immunogenic fragments of the antigens that make up the fusion protein. The fusion proteins of the present disclosure can also include additional copies of the component antigens or their immunogenic fragments. The fusion protein can include one or more binding domains that bind to each other and further bind to an Fc domain such as an IgG Fc domain. The fusion proteins can be further linked together to mimic a monoclonal antibody and provide six or more binding domains. The fusion proteins can be produced by recombinant methods as known in the art. The preparation of fusion proteins is known in the art and is described, for example, in International Publication Nos. WO 1995 / 027735 A1, WO 2005 / 103077 A1, WO 2008 / 025516 A1, WO 2009 / 007120 A1, WO 2010 / 003766 A1, WO 2010 / 010051 A1, WO 2010 / 078966 A1, US Patent Application Publication Nos. 2015 / 0125419 A1 and 2016 / 0272695 A1, and US Patent No. 8,921,519, the disclosures of each of which are incorporated herein by reference.

[0153]

[0175] The term "heterologous" when used with respect to a nucleic acid or protein moiety indicates that the nucleic acid or protein contains two or more sub-sequences that are not found in nature in the same relationship to each other. For example, a nucleic acid typically is recombinantly produced and has 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 contains two or more sub-sequences that are not found in nature in the same relationship to each other (e.g., a fusion protein).

[0154]

[0176] The term "conservative amino acid substitution" means an amino acid sequence modification that does not abrogate binding of an antibody or fusion protein to its antigen. Conservative amino acid substitutions include substitutions of one amino acid of a certain class with another amino acid of the same class, where the classes are defined by common physicochemical amino acid side chain properties and high substitution frequencies in naturally occurring homologous proteins, as determined, for example, by standard Dayhoff frequency exchange matrices or BLOSUM matrices. Six general classes of amino acid side chains have been classified and include class I (Cys); class II (Ser, Thr, Pro, Ala, Gly); class III (Asn, Asp, Gln, Glu); class IV (His, Arg, Lys); class V (Ile, Leu, Val, Met); and class VI (Phe, Tyr, Trp). For example, substitution of Asp with another class III residue such as Asn, Gln or Glu is a conservative substitution. Thus, predicted non-essential amino acid residues in an antibody are preferably substituted with another amino acid residue from the same class. Methods for identifying amino acid conservative substitutions that do not eliminate antigen binding are well known in the art (see, e.g., Brummell, et al., Biochemistry 1993, 32, 1180-1187; Kobayashi, et al., Protein Eng. 1999, 12, 879-884 (1999); and Burks, et al, Proc. Natl. Acad. Sci. USA 1997, 94, 412-417).

[0155]

[0177] In connection with two or more nucleic acids or polypeptides, the terms "sequence identity", "percent identity" and "sequence percent identity" (or synonyms thereof, e.g., "99% identical") 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 for maximum correspondence without considering conservative amino acid substitutions as part of the sequence identity and aligned (introducing gaps if necessary). The percent identity can be measured using sequence comparison software or algorithms or by visual inspection. A variety of algorithms and software are known in the art for obtaining an alignment of amino acid or nucleotide sequences. Suitable programs for determining percent sequence identity include, for example, the BLAST programs available from the BLAST website of the National Center for Biotechnology Information of the United States government. The comparison between two sequences can be carried out using either the BLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acid sequences while BLASTP is used to compare amino acid sequences. ALIGN, ALIGN-2 (Genentech, South San Francisco, California) or MegAlign available from DNASTAR are additional publicly available software programs that can be used to align sequences. One of ordinary skill in the art can determine appropriate parameters for maximum alignment with a particular alignment software. In certain embodiments, the default parameters of the alignment software are used.

[0156]

[0178] 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 include one or more conservative substitutions in its amino acid sequence as compared to the amino acid sequence of the reference antibody. Conservative substitutions can include, for example, substitutions of amino acids that are similarly charged or uncharged. A variant retains the ability to specifically bind to the antigen of the reference antibody. The term "variant" also includes pegylated antibodies or proteins.

[0157]

[0179] A nucleic acid sequence implicitly includes its conservatively modified variants (e.g., degenerate codon substitutions) and complementary sequences, as well as the explicitly shown sequence. Specifically, degenerate codon substitutions can be achieved by generating a sequence in which the third position of one or more selected (or all) codons is substituted with a mixed base and / or a deoxyinosine residue. Batzer, et al, Nucleic Acid Res. 1991, 79, 5081; Ohtsuka, et al, J. Biol. Chem. 1985, 260, 2605-2608; Rossolini, et al., Mol. Cell. Probes 1994, 8, 91-98. The term "nucleic acid" is used interchangeably with cDNA, mRNA, oligonucleotide and polynucleotide.

[0158]

[0180] The term "biosimilar" means a biological product, including a monoclonal antibody or protein, which is highly similar to an approved reference biological product in the United States, notwithstanding minor differences in clinically inactive components, and there are no clinically meaningful differences between the biological product and the reference product in terms of product safety, purity, and potency. Further, a similar biological or "biosimilar" medicine is a biological medicine similar to another biological medicine that has already been approved for use by the European Medicines Agency. The term "biosimilar" is also used synonymously by regulatory authorities in other countries and regions. A biological preparation or biopharmaceutical is a medicine made or derived from a biological source 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 the pharmaceutical regulatory authority with respect to Aldesleukin is a "biosimilar to" Aldesleukin or "its biosimilar" of Aldesleukin. In Europe, a similar biological or "biosimilar" medicine is a biological medicine similar to another biological medicine that has already been approved for use by the European Medicines Agency (EMA). The relevant legal basis for similar biological uses in Europe is Article 6 of the amended Regulation (EC) No 726 / 2004 and Article 10(4) of Directive 2001 / 83 / EC. Thus, in Europe, a biosimilar can be approved, have its approval approved, or be the subject of an approval application under Article 6 of Regulation (EC) No 726 / 2004 and Article 10(4) of Directive 2001 / 83 / EC. In Europe, the original biological medicine that has already been approved is sometimes called the "reference medicine". Some of the requirements for a product to be considered a biosimilar are outlined in the CHMP guidelines for similar biological medicines. Further, product-specific guidelines, including guidelines for monoclonal antibody biosimilars, are provided by the EMA for each product and are publicly available on its website.The 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, the biosimilar may be used or intended to be used for treating the same condition as the reference medicinal product. Thus, the biosimilars described herein may be considered to have quality characteristics similar or very similar to those of the reference medicinal product. Alternatively or additionally, the biosimilars described herein may be considered to have biological activity similar or very similar to that of the reference medicinal product. Alternatively or additionally, the biosimilars described herein may be considered to have a safety profile similar or very similar to that of the reference medicinal product. Alternatively or additionally, the biosimilars described herein may be considered to have efficacy similar or very similar to that of the reference medicinal product. As described herein, biosimilars in Europe are compared with the reference medicinal product authorized by the EMA. However, in some cases, the biosimilar may be compared with a biological medicinal product authorized outside the European Economic Area (a non-EEA authorized "comparator") in a particular study. Such tests include, for example, certain clinical trials and in vivo non-clinical trials. As used herein, the term "biosimilar" also relates to biological medicinal products that have been or may be compared with non-EEA authorized comparators. Certain biosimilars are proteins such as antibodies, antibody fragments (e.g., antigen-binding portions) and fusion proteins. Protein biosimilars may have an amino acid sequence with minor modifications to the amino acid structure (including, for example, amino acid deletions, additions and / or substitutions) that do not significantly affect the function of the polypeptide. The biosimilar may include an amino acid sequence having at least 97% sequence identity, such as 97%, 98%, 99% or 100%, to the amino acid sequence of its reference medicinal product. The biosimilar may include one or more post-translational modifications different from those of the reference medicinal product, such as, but not limited to, glycosylation, oxidation, deamidation and / or cleavage, provided that the differences do not result in a change in the safety and / or efficacy of the medicinal product. The biosimilar may have a glycosylation pattern identical or different from that of the reference medicinal product.Although not exclusive, biosimilars may have different glycosylation patterns, particularly when the differences are intended to address or are intended to address concerns regarding the safety of the reference medicinal product. Furthermore, biosimilars may deviate from the reference medicinal product in terms of, for example, its strength, pharmaceutical form, formulation, excipients and / or presentation, provided that the safety and efficacy of the medicinal product are not impaired. Biosimilars may include, for example, differences in the pharmacokinetic (PK) and / or pharmacodynamic (PD) profiles when compared to the reference medicinal product, but are still considered to be sufficiently similar to the reference medicinal product to be approved or considered suitable for approval. In certain circumstances, biosimilars exhibit different binding properties compared to the reference medicinal product, where the different binding properties 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 synonymously by regulatory agencies in other countries and regions.

[0159]

[0181] The terms "effective amount" or "therapeutically effective amount" refer to the amount of a compound or combination of compounds described herein that is sufficient to achieve the intended use, including but not limited to the treatment of a disease. The therapeutically effective amount can vary depending on the intended use (in vitro or in vivo) or the subject being treated and the disease state (e.g., the weight, age and gender of the subject), the severity of the disease state or the method of administration. This term also applies to the dose that induces a specific response (e.g., a decrease in platelet adhesion and / or cell migration) in the target cells. The specific dose varies depending on the specific 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 carried.

[0160]

[0182] "Therapeutic effect", as the term is used herein, encompasses therapeutic and / or prophylactic benefits. A prophylactic effect includes delaying or precluding the occurrence of a disease or condition, delaying or precluding the onset of symptoms of a disease or condition, delaying, arresting, or reversing the progression of a disease or condition, or any combination thereof.

[0161]

[0183] The terms "QD", "qd", or "q.d." mean once a day, once daily, or once every day. The terms "BID", "bid", or "b.i.d." mean twice a day, twice daily, or twice every day. The terms "TID", "tid", or "t.i.d." mean three times a day, three times daily, or three times every day. The terms "QID", "qid", or "q.i.d." mean four times a day, four times daily, or four times every day.

[0162]

[0184] To avoid ambiguity, it is intended that in this specification, any particular feature (e.g., an integer, property, value, use, disease, formula, compound, or group) described in connection with a particular aspect, embodiment, or example of the invention is to be understood as applicable to any other aspect, embodiment, or example described herein, unless incompatible. Thus, such features can be used, as appropriate, in conjunction with any definition, claim, or embodiment defined herein. All features (including the appended claims, abstract, and drawings) disclosed in this specification and / or all steps of any method or process so disclosed may be combined in any combination, except combinations where at least some of the features and / or steps are mutually exclusive. The invention is not limited to any details of the disclosed embodiments. The invention extends to any novel or novel combination of the features (including the appended claims, abstract, and drawings) disclosed herein or to any novel or novel combination of the steps of any method or process so disclosed.

[0163]

[0185] The terms "about" and "approximately" mean within a statistically significant range of values. Such range can be within one digit, preferably within 50%, more preferably within 20%, even more preferably within 10%, and even more preferably within 5% of a given value or range. The allowable variations encompassed by the terms "about" or "approximately" depend on the particular system under study and can be readily understood by one of ordinary skill in the art. Further, 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 can be approximate and / or larger or smaller, as appropriate, reflecting tolerances, conversion factors, rounding, measurement errors, etc., and other factors known to one of ordinary skill in the art. Generally, dimensions, sizes, formulations, parameters, shapes, or other quantities or features are "about" or "approximately", whether or not expressly stated to be so. It should be noted that embodiments of very different sizes, shapes, and dimensions may employ the described mechanisms.

[0164]

[0186] In the original and modified forms, when used in the appended claims, the transitional terms "comprising," "consisting essentially of," and "consisting of" define the subject matter of the claims with respect to what is excluded from the subject matter of the claims if there are additional elements or steps of the claims not recited. The term "comprising" is intended to be inclusive or open-ended and does not exclude any additional unrecited elements, method, steps, or materials. The term "consisting of" excludes any element, step, or material other than those recited in the claims and, in the case of materials, ordinary impurities associated with the recited materials. The term "consisting essentially of" limits the claims to the specified elements, steps, or materials and those that do not substantially affect the basic and novel characteristics of the claimed invention. All compositions, methods, and kits described herein embodying the invention can be more specifically defined in alternative embodiments by any of the transitional terms "comprising," "consisting essentially of," and "consisting of."

[0165] Potassium channel agonist

[0187] In one embodiment, the potassium channel agonist is an IK channel agonist, activator or opener and a K Ca 3.1 channel agonist, activator or opener, also known as a K Ca 3.1 agonist, activator or opener. K Ca 3.1 is also known as hIKCa, hKCa4, hSK4, intermediate conductance calcium-activated potassium channel, and small conductance calcium-activated potassium channel 4.

[0166]

[0188] In one embodiment, the K Ca 3.1 agonist is a benzothiazole derivative or a pharmaceutically acceptable salt, co-crystal, solvate or prodrug thereof. In one embodiment, the K Ca3.1 The agonist is a benzimidazole derivative or a pharmaceutically acceptable salt, cocrystal, solvate or prodrug thereof. Suitable benzothiazole and benzimidazole derivatives are described in Sankaranarayanan, et al, Mol.Pharmacol.2009, 75, 281-95, the disclosure of which is incorporated herein by reference.

[0167]

[0189] In one embodiment, K Ca 3.1 The agonist is of formula (1):

Chemical formula

[0168]

[0190] In one embodiment, the K Ca 3.1 agonist is a compound according to formula (1) or a pharmaceutically acceptable salt, co-crystal, solvate or prodrug thereof, R a is selected from thiol and -NH 2 ; R b and R c are independently selected from hydrogen, (C 1~3 ) alkyl, (C 1~3 ) alkoxyl, halo, nitro, unsubstituted aryl, (C 1~3 ) alkyl, and / or R b and R c together with the carbon atom to which they are attached form a 5- or 6-membered ring selected from the group consisting of aryl, heteroaryl, cycloalkyl and heterocycloalkyl rings; and R d and R e are independently selected from hydrogen, (C 1~3 ) alkyl, (C 1~3 ) alkoxyl, halo, nitro, unsubstituted aryl, (C 1~3 ) alkyl, and / or R d and R eTogether with the carbon atoms to which they are both attached, form a 5- or 6-membered ring selected from the group consisting of aryl, heteroaryl, cycloalkyl and heterocycloalkyl rings, provided that R b and R c form a ring, R d and R e do not form a ring, and when R d and R e form a ring, R b and R c do not form a ring.

[0169]

[0191] In one embodiment, the K Ca 3.1 agonist is naphtho[1,2-d]thiazol-2-ylamine (Formula (2)):

Chemical Formula

[0170]

[0192] In one embodiment, the K Ca 3.1 agonist is anthra[2,1-d]thiazol-2-ylamine (Formula (3)):

Chemical Formula

[0171]

[0193] In one embodiment, K Ca The 3.1 agonist is 6,7-dichloro-1H-indole-2,3-dione 3-oxime (Formula (4)):

Chemical Formula

[0172]

[0194] In one embodiment, K Ca The 3.1 agonist is Formula (5):

Chemical Formula

[0172] or a pharmaceutically acceptable salt, co-crystal, solvate or prodrug thereof. The properties and synthesis of the compound according to formula (5) are described in US Patent No. 6,969,729, the disclosure of which is incorporated herein by reference.

[0173]

[0195] In one embodiment, the K Ca 3.1 agonist is 1-ethyl-1,3-dihydro-2H-benzimidazol-2-one and 1-ethyl-2-benzimidazolinone (formula (6)):

Chemical formula

[0174]

[0196] In one embodiment, the K Ca 3.1 agonist is 5,6-dichloro-1-ethyl-1,3-dihydro-2H-benzimidazol-2-one and DC-EBIO (formula (7)):

Chemical formula

[0175]

[0197] In one embodiment, K Ca 3.1 agonist is 2 - amino - 6 - trifluoromethoxybenzothiazole or 6 - (trifluoromethoxy)benzo[d]thiazol - 2 - amine (Formula (8)):

Chemical formula

[0176]

[0198] In one embodiment, K Ca 3.1 agonist is a compound according to formula (9a) or a compound according to formula (9b):

Chemical formula

[0177]

[0199] In one embodiment, the K Ca 3.1 agonist is 5-methylnaphtho[1,2-d]oxazol-2-amine (formula (10a)) or 5-methylnaphtho[2,1-d]oxazol-2-amine (formula (10b), also known as SKA-121):

Chemical formula

[0178]

[0200] In one embodiment, the K Ca 3.1 agonist is 6-(trifluoromethoxy)-1H-benzo[d]imidazol-2-amine, also known as SKA-12 (formula (11)):

Chemical formula

[0179]

[0201] In one embodiment, the K Ca 3.1 agonist is 6-methoxybenzothiazole-2-thiol, also known as SKA-5 (Formula (12)):

Chemical formula

[0180]

[0202] In one embodiment, the K Ca 3.1 agonist is 6-nitrobenzothiazole-2-thiol, also known as SKA-6 (Formula (13)):

Chemical formula

[0181]

[0203] In one embodiment, the K Ca 3.1 agonist is 6-(trifluoromethoxy)-1H-benzimidazole-2-thiol, also known as SKA-46 (Formula (14)):

Chemical formula

[0182]

[0204] In one embodiment, the K Ca 3.1 agonist is 6-(difluoromethoxy)-1H-benzo[d]imidazole-2-thiol, also known as SKA-47 (Formula (15)):

Chemical formula

[0183]

[0205] In one embodiment, the K Ca 3.1 agonist is 4-(4-(trifluoromethoxy)phenyl)thiazol-2-amine, also known as SKA-41 (Formula (17)):

Chemical formula

[0184]

[0206] In one embodiment, the K Ca 3.1 agonist is benzothiazol-2-amine, also known as SKA-1 (Formula (18)):

Chemical formula

[0185]

[0207] In one embodiment, the K Ca 3.1 agonist is benzothiazol-2-amine, also known as SKA-36 (Formula (19)):

Chemical formula

[0186]

[0208] In one embodiment, the K Ca 3.1 agonist is 6-nitrobenzothiazol-2-amine, also known as SKA-4 (Formula (20)):

Chemical formula

[0187]

[0209] In one embodiment, the K Ca 3.1 agonist is 6-(methylsulfonyl)benzothiazol-2-amine, also known as SKA-16 (Formula (21)):

Chemical formula

[0188]

[0210] In one embodiment, K Ca 3.1 agonist is 1-(2-aminobenzothiazol-6-yl)ethan-1-one, also known as SKA-24 (Formula (22)):

Chemical formula

[0189]

[0211] In one embodiment, K Ca 3.1 agonist is 6-methoxybenzothiazol-2-amine, also known as SKA-2 (Formula (23)):

Chemical formula

[0190]

[0212] In one embodiment, K Ca 3.1 agonist is 6-methoxybenzothiazol-2-amine, also known as SKA-17 (Formula (24)):

Chemical formula

[0191]

[0213] In one embodiment, K Ca 3.1 agonist is 5-chloro-6-methoxybenzothiazol-2-amine, also known as SKA-13 (Formula (25)):

Chemical formula

[0192]

[0214] In one embodiment, K Ca 3.1 agonist is 6-benzylbenzothiazol-2-amine, also known as SKA-7 (Formula (26)):

Chemical formula

[0193]

[0215] In one embodiment, K Ca 3.1 agonist is 6-phenoxybenzothiazol-2-amine, also known as SKA-32 (Formula (27)):

Chemical formula

[0194]

[0216] In one embodiment, the K Ca 3.1 agonist is also known as SKA-22 (Formula (28)) (2-aminobenzothiazol-6-yl)(phenyl)methanone:

Chemical formula

[0195]

[0217] In one embodiment, the K Ca 3.1 agonist is also known as SKA-48 (Formula (29)) (2-aminobenzothiazol-6-yl)(phenyl)methanone:

Chemical formula

[0196]

[0218] In one embodiment, the K Ca 3.1 agonist is also known as SKA-18 (Formula (29)) 6-fluorobenzothiazol-2-amine:

Chemical formula

[0197]

[0219] In one embodiment, the K Ca 3.1 agonist is 5,6-difluorobenzothiazol-2-amine, also known as SKA-42 (Formula (30)):

Chemical formula

[0198]

[0220] In one embodiment, the K Ca 3.1 agonist is 6-chlorobenzothiazol-2-amine, also known as SKA-3 (Formula (31)):

Chemical formula

[0199]

[0221] In one embodiment, the K Ca 3.1 agonist is 5-(trifluoromethoxy)benzothiazol-2-amine, also known as SKA-8 (Formula (32)):

Chemical formula

[0200]

[0222] In one embodiment, the K Ca 3.1 agonist is 4-(trifluoromethoxy)benzo[d]thiazol-2-amine, also known as SKA-35 (Formula (33)):

Chemical Structure

[0201]

[0223] In one embodiment, the K Ca 3.1 agonist is 6-(trifluoromethyl)benzo[d]thiazol-2-amine, also known as SKA-51 (Formula (34)):

Chemical Structure

[0202]

[0224] In one embodiment, the K Ca 3.1 agonist is 5-(trifluoromethyl)benzo[d]thiazol-2-amine, also known as SKA-34 (Formula (35)):

Chemical Structure

[0203]

[0225] In one embodiment, the K Ca 3.1 agonist is 6-((trifluoromethyl)thio)benzo[d]thiazol-2-amine, also known as SKA-19 (Formula (36)):

Chemical formula

[0204]

[0226] In one embodiment, the K Ca 3.1 agonist is 6-(chlorodifluoromethoxy)benzo[d]thiazol-2-amine, also known as SKA-11 (Formula (37)):

Chemical formula

[0205]

[0227] In one embodiment, the K Ca 3.1 agonist is [2,4’bibenzo[d]thiazol]-2’-amine, also known as SKA-53 (Formula (38)):

Chemical formula

[0206]

[0228] In one embodiment, the K Ca 3.1 agonist is 6,7-dihydro-5H-inden[5,6-d]thiazol-2-amine, also known as SKA-29 (Formula (39)):

Chemical formula

[0207]

[0229] In one embodiment, the K Ca 3.1 agonist is 6,7,8,9-tetrahydronaphtho[2,1-d]thiazol-2-amine, also known as SKA-44 (Formula (40)):

Chemical formula

[0208]

[0230] In one embodiment, the K Ca 3.1 agonist is 6,7,8,9-tetrahydronaphtho[1,2-d]thiazol-2-amine, also known as SKA-49 (Formula (41)):

Chemical formula

[0209]

[0231] In one embodiment, the K Ca 3.1 agonist is naphtho[2,1-d]thiazol-2-amine, also known as SKA-45 (Formula (42)):

Chemical formula

[0210]

[0232] In one embodiment, the K Ca 3.1 agonist is 2-amino-6a,10a-dihydroanthra[2,1-d]thiazole-6,11-dione, also known as SKA-21 (Formula (43)):

Chemical formula

[0211]

[0233] In one embodiment, the K Ca 3.1 agonist is 2-amino-7H-chromeno[6,5-d]thiazol-7-one, also known as SKA-26 (Formula (44)):

Chemical formula

[0212]

[0234] In one embodiment, K Ca 3.1 agonist is benzo[1,2-d:4,5-d']bis(thiazole)-2,6-diamine, also known as SKA-50 (Formula (45)):

Chemical formula

[0213]

[0235] In one embodiment, K Ca 3.1 agonist is benzo[1,2-d:4,3-d']bis(thiazole)-2-amine, also known as SKA-25 (Formula (46)):

Chemical formula

[0214]

[0236] In one embodiment, K Ca 3.1 agonist is thiazolo[4,5-c]quinolin-2-amine, also known as SKA-56 (Formula (47)):

Chemical formula

[0215]

[0237] In one embodiment, K Ca The 3.1 agonist is 6,7-dihydro-[1,4]dioxino[2’,3’:4,5]benzo[1,2-d]thiazol-2-amine, also known as SKA-30 (Formula (48)):

Chemical Formula

[0216]

[0238] In one embodiment, K Ca The 3.1 agonist is a benzothiazole derivative selected from any one of the aforementioned benzothiazole compounds or a pharmaceutically acceptable salt, co-crystal, solvate or prodrug thereof. In one embodiment, K Ca The 3.1 agonist is a benzimidazole derivative selected from any one of the aforementioned benzimidazole compounds or a pharmaceutically acceptable salt, co-crystal, solvate or prodrug thereof.

[0217]

[0239] In one embodiment, K Ca The 3.1 agonist is 5-ethylnaphtho[1,2-d]oxazol-2-amine; 5-propylnaphtho[1,2-d]oxazol-2-amine; 5-cyclopropylnaphtho[1,2-d]oxazol-2-amine; 5-(tert-Butyl)naphtho[1,2-d]oxazol-2-amine; 5-Fluoronaphtho[1,2-d]oxazol-2-amine; 5-Chloronaphtho[1,2-d]oxazol-2-amine; 5-Bromonaphtho[1,2-d]oxazol-2-amine; 5-Iodonaphtho[1,2-d]oxazol-2-amine; 2-Aminonaphtho[1,2-d]oxazole-5-carbonitrile; Naphtho[1,2-d]oxazole-2,5-diamine; N 5 -Methylnaphtho[1,2-d]oxazole-2,5-diamine; N 5 ,N 5 ,N-Dimethylnaphtho[1,2-d]oxazole-2,5-diamine; N 5 -Ethylnaphtho[1,2-d]oxazole-2,5-diamine; 5-(Pyrrolidin-1-yl)naphtho[1,2-d]oxazol-2-amine; 5-Methoxynaphtho[1,2-d]oxazol-2-amine; 5-Trifluoromethylnaphtho[1,2-d]oxazol-2-amine; and its pharmaceutically acceptable salts, co-crystals, solvates or prodrugs selected from the group consisting of. The properties and synthesis of these compounds are described in International Publication No. WO 2015 / 164816 A2, the disclosure of which is incorporated herein by reference.

[0218]

[0240] In one embodiment, the K Ca 3.1 agonist is 5-Ethylnaphtho[2,1-d]oxazol-2-amine; 5-Propylnaphtho[2,1-d]oxazol-2-amine; 5-Cyclopropylnaphtho[2,1-d]oxazol-2-amine; 5-(tert-Butyl)naphtho[2,1-d]oxazol-2-amine; 5-Fluoronaphtho[2,1-d]oxazol-2-amine; 5-Chloronaphtho[2,1-d]oxazol-2-amine; 5-Bromonaphtho[2,1-d]oxazol-2-amine; 5-Iodonaphtho[2,1-d]oxazol-2-amine; 2-Aminonaphtho[2,1-d]oxazole-5-carbonitrile; Naphtho[2,1-d]oxazole-2,5-diamine; N 5 -Methylnaphtho[2,1-d]oxazole-2,5-diamine; N 5 ,N 5 -Dimethylnaphtho[2,1-d]oxazole-2,5-diamine; N 5 -Ethylnaphtho[2,1-d]oxazole-2,5-diamine; 5-(Pyrrolidin-1-yl)naphtho[2,1-d]oxazol-2-amine; 5-Methoxynaphtho[2,1-d]oxazol-2-amine; 5-Trifluoromethylnaphtho[2,1-d]oxazol-2-amine; and its pharmaceutically acceptable salts, co-crystals, solvates or prodrugs selected from the group consisting of. The properties and synthesis of these compounds are described in WO 2015 / 164816 A2, the disclosure of which is incorporated herein by reference.

[0219]

[0241] In one embodiment, the K Ca 3.1 agonist is 2,3,3-Trimethyl-3H-benzo[g]indole (also known as SKA-92 and CAS number 74470-85-2); 2-Methylnaphtho[2,3-d]oxazole (also known as SKA-104 and CAS number 20686-66-2); 2-Methylnaphtho[1,2-d]oxazole (also known as SKA-103 and CAS number 85-15-4); Naphtho[1,2-d]oxazol-2-amine (also known as SKA-102 and CAS number 858432-45-8); 2-Methylnaphtho[1,2-d]thiazole (also known as SKA-74 and CAS number 2682-45-3); 2-Amino-4-(1-naphthyl)thiazole (also known as SKA-75, CAS number 56503-96-9); 2-Amino-4-(2-naphthyl)thiazole (also known as SKA-76, CAS number 21331-43-1); and its pharmaceutically acceptable salts, co-crystals, solvates or prodrugs selected from the group consisting of. The properties and synthesis of these compounds are described in International Publication No. 2015 / 164816A2, the disclosure of which is incorporated herein by reference.

[0220]

[0242] In one embodiment, the K Ca 3.1 agonist is 5-Methylnaphtho[1,2-d]thiazol-2-amine (also known as SKA-111) and its pharmaceutically acceptable salts, co-crystals, solvates or prodrugs. The properties and synthesis of the SKA-111 compound are described in International Publication No. 2015 / 164816A2 and Coleman, et al., Mol.Pharmacol.2014, 86, 342-57, the disclosures of which are incorporated herein by reference.

[0221] Method for proliferating tumor infiltrating lymphocytes

[0243] In one embodiment, the present invention provides a method for proliferating TILs, comprising contacting a population of TILs comprising at least one TIL with a potassium channel agonist described herein. In one embodiment, the present invention provides a method for proliferating TILs, comprising the step of contacting a population of TILs with one or more potassium channel agonists in a cell culture medium.

[0222]

[0244] In one embodiment, the present invention is a process for preparing a population of tumor infiltrating lymphocytes (TIL) from a tumor, comprising: (a) contacting the fragmented tumor with a first cell culture medium; (b) performing an initial expansion (pre-REP) of a first population of TIL in the first cell culture medium to obtain a second population of TIL, wherein the number of the second population of TIL is at least five times greater than that of the first population of TIL, and the first cell culture medium contains IL-2; (b) performing a rapid expansion (REP) of the second population of TIL in a second cell culture medium to obtain a third population of TIL, wherein the number of the third population of TIL is at least 50 times greater than that of the second population of TIL 7 days after the start of the rapid expansion, the second cell culture medium contains IL-2, OKT-3 (anti-CD3 antibody), irradiated allogeneic peripheral blood mononuclear cells (PBMC), and the rapid expansion is performed over a period of 14 days or less; (c) recovering the third population of TIL wherein either the first cell culture medium or the second cell culture medium or both the first cell culture medium and the second cell culture medium further contain a potassium channel agonist.

[0223]

[0245] In one embodiment, the potassium channel agonist may be different in the pre-REP step and the REP step.

[0224]

[0246] In one embodiment, the potassium channel agonist is a K Ca 3.1 (IK channel) agonist.

[0225]

[0247] In one embodiment, the present invention is a process for expanding a population of TILs, including a pre-rapid expansion (pre-REP) process and a rapid expansion process (REP), wherein the cell culture medium used for expansion contains IL-2 at a concentration selected from the group consisting of 100 IU / mL to 10,000 IU / mL, 200 IU / mL to 5,000 IU / mL, 300 IU / mL to 4,800 IU / mL, 400 IU / mL to 4,600 IU / mL, 500 IU / mL to 4,400 IU / mL, 600 IU / mL to 4,200 IU / mL, 700 IU / mL to 4,000 IU / mL, 800 IU / mL to 3,800 IU / mL, 900 IU / mL to 3,600 IU / mL, 1,000 IU / mL to 3,400 IU / mL, 1,100 IU / mL to 3,200 IU / mL, 1,200 IU / mL to 3,000 IU / mL, 1,300 IU / mL to 2,800 IU / mL, 1,400 IU / mL to 2,600 IU / mL, 1,500 IU / mL to 2,400 IU / mL, 1,600 IU / mL to 2,200 IU / mL, 1,700 IU / mL to 2,000 IU / mL, 5,500 IU / mL to 9,500 IU / mL, 6,000 IU / mL to 9,000 IU / mL, 6,500 IU / mL to 8,500 IU / mL, 7,000 IU / mL to 8,000 IU / mL, and 7,500 IU / mL to 8,000 IU / mL.

[0226]

[0248] In one embodiment, the present invention provides a process for expanding a population of TILs, including a pre-rapid expansion (pre-REP) process and a rapid expansion process (REP), wherein the cell culture medium used for the expansion contains IL-2 at a concentration selected from the group consisting of about 100 IU / mL, about 200 IU / mL, about 300 IU / mL, about 400 IU / mL, about 100 IU / mL, about 100 IU / mL, about 100 IU / mL, about 100 IU / mL, about 100 IU / mL, about 500 IU / mL, about 600 IU / mL, about 700 IU / mL, about 800 IU / mL, about 900 IU / mL, about 1,000 IU / mL, about 1,100 IU / mL, about 1,200 IU / mL, about 1,300 IU / mL, about 1,400 IU / mL, about 1,500 IU / mL, about 1,600 IU / mL, about 1,700 IU / mL, about 1,800 IU / mL, about 1,900 IU / mL, about 2,000 IU / mL, about 2,100 IU / mL, about 2,200 IU / mL, about 2,300 IU / mL, about 2,400 IU / mL, about 2,500 IU / mL, about 2,600 IU / mL, about 2,700 IU / mL, about 2,800 IU / mL, about 2,900 IU / mL, about 3,000 IU / mL, about 3,100 IU / mL, about 3,200 IU / mL, about 3,300 IU / mL, about 3,400 IU / mL, about 3,500 IU / mL, about 3,600 IU / mL, about 3,700 IU / mL, about 3,800 IU / mL, about 3,900 IU / mL, about 4,000 IU / mL, about 4,100 IU / mL, about 4,200 IU / mL, about 4,300 IU / mL, about 4,400 IU / mL, about 4,500 IU / mL, about 4,600 IU / mL, about 4,700 IU / mL, about 4,800 IU / mL, about 4,900 IU / mL, about 5,000 IU / mL, about 5,100 IU / mL, about 5,200 IU / mL, about 5,300 IU / mL, about 5,400 IU / mL, about 5,500 IU / mL, about 5,600 IU / mL, about 5,700 IU / mL, about 5,800 IU / mL, about 5,900 IU / mL, about 6,000 IU / mL, about 6,500 IU / mL, about 7,000 IU / mL, about 7,500 IU / mL, about 8,000 IU / mL, about 8,500 IU / mL, about 9,000 IU / mL, about 9,500 IU / mL and about 10,000 IU / mL.

[0227]

[0249] In one embodiment, the present invention provides a process for expanding a population of TILs, including a pre-rapid expansion (pre-REP) process. In one embodiment, the present invention is a pre-REP process for expanding a population of TILs, comprising the step of contacting the population of TILs with one or more potassium channel agonists in a cell culture medium, wherein the cell culture medium further comprises IL-2 at an initial concentration of 1000 IU / mL to 6000 IU / mL.

[0228]

[0250] In one embodiment, the present invention is a pre-REP process for expanding a population of TILs, comprising the step of contacting the population of TILs with one or more potassium channel agonists in a cell culture medium, wherein the cell culture medium further comprises IL-2 at an initial concentration of about 6000 IU / mL.

[0229]

[0251] In one embodiment, the present invention is a pre-REP process for expanding a population of TILs, comprising the step of contacting the population of TILs with one or more potassium channel agonists in a cell culture medium, wherein the cell culture medium further comprises IL-2 at an initial concentration of 1000 IU / mL to 6000 IU / mL, and the one or more potassium channel agonists comprise K Ca 3.1 (IK channel) agonist.

[0230]

[0252] In one embodiment, the present invention is a pre-REP process for expanding a population of TILs, comprising the step of contacting the population of TILs with one or more potassium channel agonists in a cell culture medium, the cell culture medium further comprising IL-2 at an initial concentration of 1000 IU / mL to 6000 IU / mL, and the one or more potassium channel agonists being at 1 picomolar (pM) to 1000 pM, 1 pM to 500 pM, 50 pM to 450 pM, 100 pM to 400 pM, 150 pM to 350 pM, 200 pM to 300 pM, 550 pM to 950 pM, 600 pM to 900 pM, 650 pM to 850 pM, 700 pM to 800 pM, 1 nanomolar (nM) to 1000 nM, 1 nM to 500 nM, 50 nM to 450 nM, 100 nM to 400 nM, 150 nM to 350 nM, 200 nM to 300 nM, 550 nM to 950 nM, 600 nM to 900 nM, 650 nM to 850 nM, 700 nM to 800 nM, 100 nM to 500 nM, 200 nM to 500 nM, 300 nM to 500 nM, 400 nM to 500 nM, 500 nM to 600 nM, 600 nM to 700 nM, 700 nM to 800 nM, 250 nM to 500 nM, 10 nM to 200 nM, 50 nM to 200 nM, 1 micromolar (μM) to 1000 μM, 1 μM to 500 μM, 50 μM to 450 μM, 100 μM to 400 μM, 150 μM to 350 μM, 200 μM to 300 μM, 550 μM to 950 μM, 600 μM to 900 μM, 650 μM to 850 μM, 700 μM to 800 μM, 100 μM to 500 μM, 200 μM to 500 μM, 300 μM to 500 μM, 400 μM to 500 μM, 500 μM to 600 μM, 600 μM to 700 μM, 700 μM to 800 μM, 250 μM to 500 μM, 10 μM to 200 μM, 50 μM to 200 μM, 1 millimolar (mM) to 1000 mM, 1 mM to 500 mM, 50 mM to 450 mM, 100 mM to 400 mM, 150 mM to 350 mM, 200 mM to 300 mM, 550 mM to 950 mM, 600 mM to 900 mM, 650 mM to 850 mM, 700 mM to 800 mM, 100 mM to 500 mM, 200 mM to 500 mM, 300 mM to 500 mM, 400 mM to 500 mM, 500 mM to 600 mM, 600 mM to 700 mM, 700 mM to 800 mM, 250 mM to 500 mM,K at a concentration selected from the group consisting of 10 mM to 200 mM and 50 mM to 200 mM Ca Provide a process comprising a 3.1 (IK channel) agonist.

[0231]

[0253] In one embodiment, the present invention is a pre-REP process for expanding a population of TILs, comprising the step of contacting the population of TILs with one or more potassium channel agonists in a cell culture medium, the cell culture medium further comprising IL-2 at an initial concentration of 1000 IU / mL to 6000 IU / mL, and the one or more potassium channel agonists are at a concentration of K selected from the group consisting of 1 nanomolar (nM) to 100 nM, 100 nM to 200 nM, 200 nM to 300 nM, 300 nM to 400 nM, 400 nM to 500 nM, 500 nM to 600 nM, 600 nM to 700 nM, 700 nM to 800 nM, 800 nM to 900 nM, 900 nM to 1 micromolar (μM), 1 μM to 2 μM, 2 μM to 5 μM, 5 μM to 10 μM, and 10 μM to 100 μM Ca Provide a process that may comprise a 3.1 (IK channel) agonist.

[0232]

[0254] In one embodiment, the present invention is a pre-REP process for growing a population of TILs, comprising the step of contacting the population of TILs with one or more potassium channel agonists in a cell culture medium, the cell culture medium further comprising IL-2 at an initial concentration of 1000 IU / mL to 6000 IU / mL, and the one or more potassium channel agonists being at a concentration of about 1 pM, about 50 pM, about 100 pM, about 150 pM, about 200 pM, about 250 pM, about 300 pM, about 350 pM, about 400 pM, about 450 pM, about 500 pM, about 550 pM, about 600 pM, about 650 pM, about 700 pM, about 750 pM, about 800 pM, about 850 pM, about 900 pM, about 950 pM, about 1 nM, about 25 nM, about 50 nM, about 75 nM, about 100 nM, about 125 nM, about 150 nM, about 175 nM, about 200 nM, about 225 nM, about 250 nM, about 275 nM, about 300 nM, about 325 nM, about 350 nM, about 375 nM, about 400 nM, about 425 nM, about 450 nM, about 475 nM, about 500 nM, about 525 nM, about 550 nM, about 575 nM, about 600 nM, about 625 nM, about 650 nM, about 675 nM, about 700 nM, about 750 nM, about 800 nM, about 850 nM, about 900 nM, about 950 nM, about 1 μM, about 2 μM, about 5 μM, about 10 μM, about 20 μM, about 30 μM, about 40 μM, about 50 μM, about 60 μM, about 70 μM, about 80 μM, about 90 μM, about 100 μM, about 125 μM, about 150 μM, about 175 μM, about 200 μM, about 225 μM, about 250 μM, about 275 μM, about 300 μM, about 325 μM, about 350 μM, about 375 μM, about 400 μM, about 425 μM, about 450 μM, about 500 μM, about 550 μM, about 600 μM, about 650 μM, about 700 μM, about 750 μM, about 800 μM, about 850 μM, about 900 μM, about 950 μM, about 1 millimolar (mM), about 2 mM, about 5 mM, about 10 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM, about 95 mM and about 100 mM, selected from the group consisting of K Ca Provide a process comprising a 3.1 (IK channel) agonist.

[0233]

[0255] In one embodiment, the present invention is a pre-REP process for growing a population of TILs, comprising the step of contacting the population of TILs with one or more potassium channel agonists in a cell culture medium, the cell culture medium further comprising IL-2 at an initial concentration of 1000 IU / mL to 6000 IU / mL, and the one or more potassium channel agonists having a concentration selected from the group consisting of greater than 1 pM, greater than 50 pM, greater than 100 pM, greater than 150 pM, greater than 200 pM, greater than 250 pM, greater than 300 pM, greater than 350 pM, greater than 400 pM, greater than 450 pM, greater than 500 pM, greater than 550 pM, greater than 600 pM, greater than 650 pM, greater than 700 pM, greater than 750 pM, greater than 800 pM, greater than 850 pM, greater than 900 pM, greater than 950 pM, greater than 1 nM, greater than 25 nM, greater than 50 nM, greater than 75 nM, greater than 100 nM, greater than 125 nM, greater than 150 nM, greater than 175 nM, greater than 200 nM, greater than 225 nM, greater than 250 nM, greater than 275 nM, greater than 300 nM, greater than 325 nM, greater than 350 nM, greater than 375 nM, greater than 400 nM, greater than 425 nM, greater than 450 nM, greater than 475 nM, greater than 500 nM, greater than 525 nM, greater than 550 nM, greater than 575 nM, greater than 600 nM, greater than 625 nM, greater than 650 nM, greater than 675 nM, greater than 700 nM, greater than 750 nM, greater than 800 nM, greater than 850 nM, greater than 900 nM, greater than 950 nM, greater than 1 μM, greater than 2 μM, greater than 5 μM, greater than 10 μM, greater than 20 μM, greater than 30 μM, greater than 40 μM, greater than 50 μM, greater than 60 μM, greater than 70 μM, greater than 80 μM, greater than 90 μM, greater than 100 μM, greater than 125 μM, greater than 150 μM, greater than 175 μM, greater than 200 μM, greater than 225 μM, greater than 250 μM, greater than 275 μM, greater than 300 μM, greater than 325 μM, greater than 350 μM, greater than 375 μM, greater than 400 μM, greater than 425 μM, greater than 450 μM, greater than 500 μM, greater than 550 μM, greater than 600 μM, greater than 650 μM, greater than 700 μM, greater than 750 μM, greater than 800 μM, greater than 850 μM, greater than 900 μM, greater than 950 μM, greater than 1 millimolar (mM), greater than 2 mM, greater than 5 mM, greater than 10 mM, greater than 25 mM, greater than 30 mM, greater than 35 mM, greater than 40 mM, greater than 45 mM, greater than 50 mM, greater than 55 mM, greater than 60 mM, greater than 65 mM, greater than 70 mM, greater than 75 mM, greater than 80 mM, greater than 85 mM, greater than 90 mM, greater than 95 mM, and greater than 100 mM of K Ca Provide a process comprising a 3.1 (IK channel) agonist.

[0234]

[0256] In one embodiment, the present invention is a pre-REP process for growing a population of TILs, comprising the step of contacting the population of TILs with one or more potassium channel agonists in a cell culture medium, the cell culture medium further comprising IL-2 at an initial concentration of 1000 IU / mL to 6000 IU / mL, and the one or more potassium channel agonists being less than 1 pM, less than 50 pM, less than 100 pM, less than 150 pM, less than 200 pM, less than 250 pM, less than 300 pM, less than 350 pM, less than 400 pM, less than 450 pM, less than 500 pM, less than 550 pM, less than 600 pM, less than 650 pM, less than 700 pM, less than 750 pM, less than 800 pM, less than 850 pM, less than 900 pM, less than 950 pM, less than 1 nM, less than 25 nM, less than 50 nM, less than 75 nM, less than 100 nM, less than 125 nM, less than 150 nM, less than 175 nM, less than 200 nM, less than 225 nM, less than 250 nM, less than 275 nM, less than 300 nM, less than 325 nM, less than 350 nM, less than 375 nM, less than 400 nM, less than 425 nM, less than 450 nM, less than 475 nM, less than 500 nM, less than 525 nM, less than 550 nM, less than 575 nM, less than 600 nM, less than 625 nM, less than 650 nM, less than 675 nM, less than 700 nM, less than 750 nM, less than 800 nM, less than 850 nM, less than 900 nM, less than 950 nM, less than 1 μM, less than 2 μM, less than 5 μM, less than 10 μM, less than 20 μM, less than 30 μM, less than 40 μM, less than 50 μM, less than 60 μM, less than 70 μM, less than 80 μM, less than 90 μM, less than 100 μM, less than 125 μM, less than 150 μM, less than 175 μM, less than 200 μM, less than 225 μM, less than 250 μM, less than 275 μM, less than 300 μM, less than 325 μM, less than 350 μM, less than 375 μM, less than 400 μM, less than 425 μM, less than 450 μM, less than 500 μM, less than 550 μM, less than 600 μM, less than 650 μM, less than 700 μM, less than 750 μM, less than 800 μM, less than 850 μM, less than 900 μM, less than 950 μM, less than 1 millimolar (mM), less than 2 mM, less than 5 mM, less than 10 mM, less than 25 mM, less than 30 mM, less than 35 mM, less than 40 mM, less than 45 mM, less than 50 mM, less than 55 mM, less than 60 mM, less than 65 mM, less than 70 mM, less than 75 mM, less than 80 mM, less than 85 mM, less than 90 mM,K at a concentration selected from the group consisting of less than 95 mM and less than 100 mM Ca Provided is a process comprising a 3.1 (IK channel) agonist.

[0235]

[0257] In one embodiment, the present invention is a pre-REP process for expanding a population of TILs, comprising contacting the population of TILs with one or more potassium channel agonists in a cell culture medium, the cell culture medium further comprising IL-2 at an initial concentration of 1000 IU / mL to 6000 IU / mL, and the one or more potassium channel agonists being K Ca 3.1 (IK channel) agonist, and CD8 + CD28 + , CD8 + CD27 + , CD8 + CD27 + CD28 + , CCR7 + A population of TILs comprising T cells having a phenotype selected from the group consisting of CD8

[0236]

[0258] In one embodiment, the present invention is a pre-REP process for growing a population of TILs, comprising the step of contacting the population of TILs with one or more potassium channel agonists in a cell culture medium, the cell culture medium further comprising IL-2 at an initial concentration of 1000 IU / mL to 6000 IU / mL, and the one or more potassium channel agonists being K Ca 3.1 (IK channel) agonist, and the population of TILs is grown over a period selected from the group consisting of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 25 days, 30 days, 35 days, and 40 days.

[0237]

[0259] In one embodiment, the present invention is a pre-REP process for growing a population of TILs, comprising the step of contacting the population of TILs with one or more potassium channel agonists in a cell culture medium, the cell culture medium further comprising IL-2 at an initial concentration of 1000 IU / mL to 6000 IU / mL, and the one or more potassium channel agonists being K Ca 3.1 (IK channel) agonist, and the population of TILs is grown over a period selected from the group consisting of less than 1 day, less than 2 days, less than 3 days, less than 4 days, less than 5 days, less than 6 days, less than 7 days, less than 8 days, less than 9 days, less than 10 days, less than 11 days, less than 12 days, less than 13 days, less than 14 days, less than 15 days, less than 16 days, less than 17 days, less than 18 days, less than 19 days, less than 20 days, less than 21 days, less than 25 days, less than 30 days, less than 35 days, and less than 40 days.

[0238]

[0260] In one embodiment, the present invention is a method for growing a population of TILs, comprising the step of contacting the population of TILs with one or more potassium channel agonists in a cell culture medium, the cell culture medium further comprising IL-2 at an initial concentration of 1000 IU / mL to 6000 IU / mL and OKT-3 antibody at an initial concentration of about 30 ng / mL.

[0239]

[0261] In one embodiment, the present invention provides a REP process for expanding a population of TILs, the process comprising contacting the population of TILs with one or more potassium channel agonists in a cell culture medium, the cell culture medium further comprising IL-2 at an initial concentration of about 6000 IU / mL and OKT-3 antibody at an initial concentration of about 30 ng / mL.

[0240]

[0262] In one embodiment, the present invention provides a REP process for expanding a population of TILs, the process comprising contacting the population of TILs with one or more potassium channel agonists in a cell culture medium, the cell culture medium further comprising IL-2 at an initial concentration of about 6000 IU / mL and OKT-3 antibody at an initial concentration of about 30 ng / mL, and the one or more potassium channel agonists comprising a K Ca 3.1 (IK channel) agonist.

[0241]

[0263] In one embodiment, the present invention is a REP process for growing a population of TILs, comprising the step of contacting the population of TILs with one or more potassium channel agonists in a cell culture medium, the cell culture medium further comprising IL-2 at an initial concentration of 1000 IU / mL to 6000 IU / mL and OKT-3 antibody at an initial concentration of about 30 ng / mL, and the one or more potassium channel agonists being at 1 picomolar (pM) to 1000 pM, 1 pM to 500 pM, 50 pM to 450 pM, 100 pM to 400 pM, 150 pM to 350 pM, 200 pM to 300 pM, 550 pM to 950 pM, 600 pM to 900 pM, 650 pM to 850 pM, 700 pM to 800 pM, 1 nanomolar (nM) to 1000 nM, 1 nM to 500 nM, 50 nM to 450 nM, 100 nM to 400 nM, 150 nM to 350 nM, 200 nM to 300 nM, 550 nM to 950 nM, 600 nM to 900 nM, 650 nM to 850 nM, 700 nM to 800 nM, 100 nM to 500 nM, 200 nM to 500 nM, 300 nM to 500 nM, 400 nM to 500 nM, 500 nM to 600 nM, 600 nM to 700 nM, 700 nM to 800 nM, 250 nM to 500 nM, 10 nM to 200 nM, 50 nM to 200 nM, 1 micromolar (μM) to 1000 μM, 1 μM to 500 μM, 50 μM to 450 μM, 100 μM to 400 μM, 150 μM to 350 μM, 200 μM to 300 μM, 550 μM to 950 μM, 600 μM to 900 μM, 650 μM to 850 μM, 700 μM to 800 μM, 100 μM to 500 μM, 200 μM to 500 μM, 300 μM to 500 μM, 400 μM to 500 μM, 500 μM to 600 μM, 600 μM to 700 μM, 700 μM to 800 μM, 250 μM to 500 μM, 10 μM to 200 μM, 50 μM to 200 μM, 1 millimolar (mM) to 1000 mM, 1 mM to 500 mM, 50 mM to 450 mM, 100 mM to 400 mM, 150 mM to 350 mM, 200 mM to 300 mM, 550 mM to 950 mM, 600 mM to 900 mM, 650 mM to 850 mM, 700 mM to 800 mM, 100 mM to 500 mM, 200 mM to 500 mM, 300 mM to 500 mM, 400 mM to 500 mM, 500 mM to 600 mM, 600 mM to 700 mM, 700 mM to 800 mM, 250 mM to 500 mM,K at a concentration selected from the group consisting of 10 mM to 200 mM and 50 mM to 200 mM Ca Providing a process comprising a 3.1 (IK channel) agonist.

[0242]

[0264] In one embodiment, the present invention is a REP process for expanding a population of TILs, comprising contacting the population of TILs with one or more potassium channel agonists in a cell culture medium, the cell culture medium further comprising IL-2 at an initial concentration of 1000 IU / mL to 6000 IU / mL and OKT-3 antibody at an initial concentration of about 30 ng / mL, and the one or more potassium channel agonists being at a concentration of K selected from the group consisting of 1 nanomolar (nM) to 100 nM, 100 nM to 200 nM, 200 nM to 300 nM, 300 nM to 400 nM, 400 nM to 500 nM, 500 nM to 600 nM, 600 nM to 700 nM, 700 nM to 800 nM, 800 nM to 900 nM, 900 nM to 1 micromolar (μM), 1 μM to 2 μM, 2 μM to 5 μM, 5 μM to 10 μM, 10 μM to 100 μM, 100 μM to 200 μM, 200 μM to 300 μM, 300 μM to 400 μM, 400 μM to 500 μM, 500 μM to 600 μM, 600 μM to 700 μM, 700 μM to 800 μM, 800 μM to 900 μM, 900 μM to 1 mM, 1 mM to 10 mM, 10 mM to 100 mM, 100 mM to 200 mM, 200 mM to 300 mM, 300 mM to 400 mM, and 400 mM to 500 mM Ca Providing a process comprising a 3.1 (IK channel) agonist.

[0243]

[0265] In one embodiment, the present invention is a REP process for growing a population of TILs, comprising the step of contacting the population of TILs with one or more potassium channel agonists in a cell culture medium, the cell culture medium further comprising IL-2 at an initial concentration of 1000 IU / mL to 6000 IU / mL and OKT-3 antibody at an initial concentration of about 30 ng / mL, and the one or more potassium channel agonists are at a concentration of about 1 pM, about 50 pM, about 100 pM, about 150 pM, about 200 pM, about 250 pM, about 300 pM, about 350 pM, about 400 pM, about 450 pM, about 500 pM, about 550 pM, about 600 pM, about 650 pM, about 700 pM, about 750 pM, about 800 pM, about 850 pM, about 900 pM, about 950 pM, about 1 nM, about 25 nM, about 50 nM, about 75 nM, about 100 nM, about 125 nM, about 150 nM, about 175 nM, about 200 nM, about 225 nM, about 250 nM, about 275 nM, about 300 nM, about 325 nM, about 350 nM, about 375 nM, about 400 nM, about 425 nM, about 450 nM, about 475 nM, about 500 nM, about 525 nM, about 550 nM, about 575 nM, about 600 nM, about 625 nM, about 650 nM, about 675 nM, about 700 nM, about 750 nM, about 800 nM, about 850 nM, about 900 nM, about 950 nM, about 1 μM, about 2 μM, about 5 μM, about 10 μM, about 20 μM, about 30 μM, about 40 μM, about 50 μM, about 60 μM, about 70 μM, about 80 μM, about 90 μM, about 100 μM, about 125 μM, about 150 μM, about 175 μM, about 200 μM, about 225 μM, about 250 μM, about 275 μM, about 300 μM, about 325 μM, about 350 μM, about 375 μM, about 400 μM, about 425 μM, about 450 μM, about 500 μM, about 550 μM, about 600 μM, about 650 μM, about 700 μM, about 750 μM, about 800 μM, about 850 μM, about 900 μM, about 950 μM, about 1 millimolar (mM), about 2 mM, about 5 mM, about 10 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM, about 95 mM, about 100 mM, about 125 mM, about 150 mM, about 175 mM, about 200 mM, about 300 mM, about 400 mM, and about 500 mM, and selected from the group consisting of K CaProvided is a process comprising a (IK channel) agonist.

[0244]

[0266] In one embodiment, the present invention is a REP process for growing a population of TILs, comprising the step of contacting the population of TILs with one or more potassium channel agonists in a cell culture medium, the cell culture medium further comprising IL-2 at an initial concentration of 1000 IU / mL to 6000 IU / mL and OKT-3 antibody at an initial concentration of about 30 ng / mL, and the one or more potassium channel agonists having a concentration selected from the group consisting of greater than 1 pM, greater than 50 pM, greater than 100 pM, greater than 150 pM, greater than 200 pM, greater than 250 pM, greater than 300 pM, greater than 350 pM, greater than 400 pM, greater than 450 pM, greater than 500 pM, greater than 550 pM, greater than 600 pM, greater than 650 pM, greater than 700 pM, greater than 750 pM, greater than 800 pM, greater than 850 pM, greater than 900 pM, greater than 950 pM, greater than 1 nM, greater than 25 nM, greater than 50 nM, greater than 75 nM, greater than 100 nM, greater than 125 nM, greater than 150 nM, greater than 175 nM, greater than 200 nM, greater than 225 nM, greater than 250 nM, greater than 275 nM, greater than 300 nM, greater than 325 nM, greater than 350 nM, greater than 375 nM, greater than 400 nM, greater than 425 nM, greater than 450 nM, greater than 475 nM, greater than 500 nM, greater than 525 nM, greater than 550 nM, greater than 575 nM, greater than 600 nM, greater than 625 nM, greater than 650 nM, greater than 675 nM, greater than 700 nM, greater than 750 nM, greater than 800 nM, greater than 850 nM, greater than 900 nM, greater than 950 nM, greater than 1 μM, greater than 2 μM, greater than 5 μM, greater than 10 μM, greater than 20 μM, greater than 30 μM, greater than 40 μM, greater than 50 μM, greater than 60 μM, greater than 70 μM, greater than 80 μM, greater than 90 μM, greater than 100 μM, greater than 125 μM, greater than 150 μM, greater than 175 μM, greater than 200 μM, greater than 225 μM, greater than 250 μM, greater than 275 μM, greater than 300 μM, greater than 325 μM, greater than 350 μM, greater than 375 μM, greater than 400 μM, greater than 425 μM, greater than 450 μM, greater than 500 μM, greater than 550 μM, greater than 600 μM, greater than 650 μM, greater than 700 μM, greater than 750 μM, greater than 800 μM, greater than 850 μM, greater than 900 μM, greater than 950 μM, greater than 1 millimolar (mM), greater than 2 mM, greater than 5 mM, greater than 10 mM, greater than 25 mM, greater than 30 mM, greater than 35 mM, greater than 40 mM, greater than 45 mM, greater than 50 mM, greater than 55 mM, greater than 60 mM, greater than 65 mM, greater than 70 mM, greater than 75 mM, greater than 80 mM, greater than 85 mM, greater than 90 mM, greater than 95 mM, greater than 100 mM, greater than 125 mM, greater than 150 mM, greater than 175 mM, greater than 200 mM, greater than 300 mM, greater than 400 mM, and greater than 500 mM of K CaProvide a process comprising a (IK channel) agonist.

[0245]

[0267] In one embodiment, the present invention is a REP process for growing a population of TILs, comprising the step of contacting the population of TILs with one or more potassium channel agonists in a cell culture medium, the cell culture medium further comprising IL-2 at an initial concentration of 1000 IU / mL to 6000 IU / mL and OKT-3 antibody at an initial concentration of about 30 ng / mL, and the one or more potassium channel agonists being less than 1 pM, less than 50 pM, less than 100 pM, less than 150 pM, less than 200 pM, less than 250 pM, less than 300 pM, less than 350 pM, less than 400 pM, less than 450 pM, less than 500 pM, less than 550 pM, less than 600 pM, less than 650 pM, less than 700 pM, less than 750 pM, less than 800 pM, less than 850 pM, less than 900 pM, less than 950 pM, less than 1 nM, less than 25 nM, less than 50 nM, less than 75 nM, less than 100 nM, less than 125 nM, less than 150 nM, less than 175 nM, less than 200 nM, less than 225 nM, less than 250 nM, less than 275 nM, less than 300 nM, less than 325 nM, less than 350 nM, less than 375 nM, less than 400 nM, less than 425 nM, less than 450 nM, less than 475 nM, less than 500 nM, less than 525 nM, less than 550 nM, less than 575 nM, less than 600 nM, less than 625 nM, less than 650 nM, less than 675 nM, less than 700 nM, less than 750 nM, less than 800 nM, less than 850 nM, less than 900 nM, less than 950 nM, less than 1 μM, less than 2 μM, less than 5 μM, less than 10 μM, less than 20 μM, less than 30 μM, less than 40 μM, less than 50 μM, less than 60 μM, less than 70 μM, less than 80 μM, less than 90 μM, less than 100 μM, less than 125 μM, less than 150 μM, less than 175 μM, less than 200 μM, less than 225 μM, less than 250 μM, less than 275 μM, less than 300 μM, less than 325 μM, less than 350 μM, less than 375 μM, less than 400 μM, less than 425 μM, less than 450 μM, less than 500 μM, less than 550 μM, less than 600 μM, less than 650 μM, less than 700 μM, less than 750 μM, less than 800 μM, less than 850 μM, less than 900 μM, less than 950 μM, less than 1 millimolar (mM), less than 2 mM, less than 5 mM, less than 10 mM, less than 25 mM, less than 30 mM, less than 35 mM, less than 40 mM, less than 45 mM, less than 50 mM, less than 55 mM, less than 60 mM, less than 65 mM, less than 70 mM, less than 75 mM, less than 80 mM, less than 85 mM,K at a concentration selected from the group consisting of less than 90 mM, less than 95 mM, less than 100 mM, less than 125 mM, less than 150 mM, less than 175 mM, less than 200 mM, less than 300 mM, less than 400 mM, and less than 500 mM Ca Provided is a process comprising a 3.1 (IK channel) agonist.

[0246]

[0268] In one embodiment, the present invention provides a REP process for expanding a population of tumor infiltrating lymphocytes (TILs), the process comprising contacting the population of TILs with one or more potassium channel agonists in a cell culture medium, wherein the population of TILs is expanded at least 50-fold over 7 days in the cell culture medium.

[0247]

[0269] In one embodiment, the present invention provides a REP process for expanding a population of tumor infiltrating lymphocytes (TILs), the process comprising contacting the population of TILs with one or more potassium channel agonists in a cell culture medium, wherein the population of TILs is expanded at least 50-fold over 7 days in the cell culture medium and the expansion is carried out using a gas permeable container.

[0248]

[0270] In one embodiment, the present invention provides a REP process for expanding a population of tumor infiltrating lymphocytes (TILs), the process comprising contacting the population of TILs with one or more potassium channel agonists in a cell culture medium, wherein the population of TILs is expanded at least 50-fold over 7 days in the cell culture medium and the expansion is carried out using a gas permeable container, and the gas permeable container is a gas permeable bag or a gas permeable flask.

[0249]

[0271] In one embodiment, the present invention provides a REP process for expanding a population of TILs, the process comprising contacting the population of TILs with one or more potassium channel agonists in a cell culture medium, wherein the cell culture medium further comprises IL-2 at an initial concentration of 1000 IU / mL to 6000 IU / mL and OKT-3 antibody at an initial concentration of about 30 ng / mL, and the one or more potassium channel agonists are KCa 3.1 (IK channel) agonist, and the population of TILs is rapidly expanded over a period selected from the group consisting of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 25 days, 30 days, 35 days, and 40 days, to provide a process.

[0250]

[0272] In one embodiment, the present invention is a REP process for expanding a population of TILs, comprising the step of contacting the population of TILs with one or more potassium channel agonists in a cell culture medium, the cell culture medium further comprising IL-2 at an initial concentration of 1000 IU / mL to 6000 IU / mL and OKT-3 antibody at an initial concentration of about 30 ng / mL, and the one or more potassium channel agonists being K Ca 3.1 (IK channel) agonist, and the population of TILs is rapidly expanded over a period selected from the group consisting of less than 1 day, less than 2 days, less than 3 days, less than 4 days, less than 5 days, less than 6 days, less than 7 days, less than 8 days, less than 9 days, less than 10 days, less than 11 days, less than 12 days, less than 13 days, less than 14 days, less than 15 days, less than 16 days, less than 17 days, less than 18 days, less than 19 days, less than 20 days, less than 21 days, less than 25 days, less than 30 days, less than 35 days, and less than 40 days, to provide a process.

[0251]

[0273] In one embodiment, the REP can be performed in a gas-permeable container using the potassium channel agonists of the present disclosure by any suitable method. For example, TILs can be rapidly expanded using non-specific T cell receptor stimulation in the presence of interleukin-2 (IL-2) or interleukin-15 (IL-15). Non-specific T cell receptor stimulation can include, for example, about 30 ng / mL of OKT-3, a monoclonal anti-CD3 antibody (commercially available from Ortho-McNeil, Raritan, NJ or Miltenyi Biotech, Auburn, CA). TILs can optionally be further expanded in vitro by stimulating them with one or more antigens, including the antigenic portion thereof, such as cancer epitopes, that can be expressed from vectors such as 0.3 μM MART-1:26-35 (27L) or gpl00:209-217 (210M), optionally in the presence of a T cell growth factor such as 300 IU / mL of IL-2 or IL-15. Other suitable antigens can include, for example, NY-ESO-1, TRP-1, TRP-2, tyrosinase cancer antigen, MAGE-A3, SSX-2, and VEGFR2 or antigenic portions thereof. TILs can also be rapidly expanded by restimulation with the same cancer antigen pulsed on ITLA-A2-expressing antigen-presenting cells. Alternatively, TILs can be further restimulated, for example, with irradiated autologous lymphocytes or irradiated ITLA-A2+ allogeneic lymphocytes and IL-2.

[0252]

[0274] In one embodiment, a method for expanding TILs may include using from about 5000 mL to about 25000 mL of cell culture medium, from about 5000 mL to about 10000 mL of cell culture medium, or from about 5800 mL to about 8700 mL of cell culture medium. In one embodiment, a method for expanding TILs may include using from about 1000 mL to about 2000 mL of cell culture medium, from about 2000 mL to about 3000 mL of cell culture medium, from about 3000 mL to about 4000 mL of cell culture medium, from about 4000 mL to about 5000 mL of cell culture medium, from about 5000 mL to about 6000 mL of cell culture medium, from about 6000 mL to about 7000 mL of cell culture medium, from about 7000 mL to about 8000 mL of cell culture medium, from about 8000 mL to about 9000 mL of cell culture medium, from about 9000 mL to about 10000 mL of cell culture medium, from about 10000 mL to about 15000 mL of cell culture medium, from about 15000 mL to about 20000 mL of cell culture medium, or from about 20000 mL to about 25000 mL of cell culture medium. In one embodiment, one or fewer types of cell culture media are used to increase the number of TILs. Any suitable cell culture medium, such as AIM-V cell culture medium (L-glutamine, 50 μM streptomycin sulfate and 10 μM gentamicin sulfate) cell culture medium (Invitrogen, Carlsbad CA) can be used. In this regard, the method of the present invention advantageously reduces the amount of medium and the number of types of medium required to increase the number of TILs. In one embodiment, increasing the number of TILs may include feeding the cells at a frequency of once every 3 or 4 days or less. Increasing the number of cells in a gas-permeable container simplifies the procedure required to increase the number of cells by reducing the feeding frequency required to grow the cells.

[0253]

[0275] In one embodiment, rapid expansion is performed using a gas-permeable container. Such embodiments are where the cell population is from about 5×10 5 cells / cm 2 to 10×10 6 ~30×10 6 cells / cm 2to enable growth. In one embodiment, this growth occurs without feeding. In one embodiment, this growth occurs without feeding as long as the medium is at a height of about 10 cm within a gas-permeable flask. In one embodiment, this is without feeding but involves the addition of one or more cytokines. In one embodiment, the cytokine can be added as a bolus without the need to mix it with the medium. Such containers, devices, and methods are known in the art and have been used for the expansion of TILs and are described in U.S. Patent Application Publication No. 2014 / 0377739A1, International Publication No. 2014 / 210036A1, U.S. Patent Application Publication No. 2013 / 0115617A1, International Publication No. 2013 / 188427A1, U.S. Patent Application Publication No. 2011 / 0136228A1, U.S. Patent No. 8,809,050, International Publication No. 2011 / 072088A2, U.S. Patent Application Publication No. 2016 / 0208216A1, U.S. Patent Application Publication No. 2012 / 0244133A1, International Publication No. 2012 / 129201A1, U.S. Patent Application Publication No. 2013 / 0102075A1, U.S. Patent No. 8,956,860, International Publication No. 2013 / 173835A1, and U.S. Patent Application Publication No. 2015 / 0175966A1, the disclosures of which are incorporated herein by reference. Such a process is also described in Jin, et al., J. Immunotherapy 2012, 35, 283-292, the disclosure of which is incorporated herein by reference.

[0254]

[0276] In one embodiment, the gas-permeable container is a G-Rex 10 flask (Wilson Wolf Manufacturing Corporation, New Brighton, MN, USA). In one embodiment, the gas-permeable container is 10 cm 2 in gas-permeable culture surface. In one embodiment, the gas-permeable container contains a 40 mL cell culture medium volume. In one embodiment, the gas-permeable container provides 100 million to 300 million TILs after two medium exchanges.

[0255]

[0277] In one embodiment, the gas-permeable container is a G-Rex 100 flask (Wilson Wolf Manufacturing Corporation, New Brighton, MN, USA). In one embodiment, the gas-permeable container has a gas-permeable culture surface of 100 cm 2 and includes a cell culture medium volume of 450 mL. In one embodiment, the gas-permeable container provides 1 billion to 3 billion TILs after two medium exchanges.

[0256]

[0278] In one embodiment, the gas-permeable container is a G-Rex 100M flask (Wilson Wolf Manufacturing Corporation, New Brighton, MN, USA). In one embodiment, the gas-permeable container has a gas-permeable culture surface of 100 cm 2 and includes a cell culture medium volume of 1000 mL. In one embodiment, the gas-permeable container provides 1 billion to 3 billion TILs without medium exchange.

[0257]

[0279] In one embodiment, the gas-permeable container is a G-Rex 100L flask (Wilson Wolf Manufacturing Corporation, New Brighton, MN, USA). In one embodiment, the gas-permeable container has a gas-permeable culture surface of 100 cm 2 and includes a cell culture medium volume of 2000 mL. In one embodiment, the gas-permeable container provides 1 billion to 3 billion TILs without medium exchange.

[0258]

[0280] In one embodiment, the gas-permeable container is a G-Rex 24-well plate (Wilson Wolf Manufacturing Corporation, New Brighton, MN, USA). In one embodiment, the gas-permeable container includes a plate having wells, and each well has a gas-permeable culture surface of 2 cm 2It includes a gas-permeable culture surface. In one embodiment, the gas-permeable container includes a plate having wells, and each well contains a cell culture medium volume of 8 mL. In one embodiment, the gas-permeable container provides 20 million to 60 million cells per well after two medium exchanges.

[0259]

[0281] In one embodiment, the gas-permeable container is a G-Rex 6-well plate (Wilson Wolf Manufacturing Corporation, New Brighton, MN, USA). In one embodiment, the gas-permeable container includes a plate having wells, and each well has a gas-permeable culture surface of 10 cm 2 It includes. In one embodiment, the gas-permeable container includes a plate having wells, and each well contains a cell culture medium volume of 40 mL. In one embodiment, the gas-permeable container provides 100 million to 300 million cells per well after two medium exchanges.

[0260]

[0282] In one embodiment, the cell culture medium in the first and / or second gas-permeable containers is not filtered. The use of unfiltered cell culture medium can simplify the procedures necessary to expand the cell number. In one embodiment, the cell culture medium in the first and / or second gas-permeable containers lacks beta-mercaptoethanol (BME).

[0261]

[0283] In one embodiment, the period of the method includes obtaining a tumor tissue sample from a mammal; culturing the tumor tissue sample in a first gas-permeable container containing cell culture medium therein; obtaining TIL from the tumor tissue sample; using a potassium channel agonist to increase the number of TIL in a second gas-permeable container containing cell culture medium therein over a period of about 14 to about 42 days, for example, over a period of about 28 days.

[0262]

[0284] In one embodiment, the ratio of TIL to potassium channel agonist (cells to moles) during rapid proliferation is about 1:0.00000001, about 1:0.0000001, about 1:0.00001, about 1:0.0001, about 1:0.001, about 1:0.01, about 1:0.1, about 1:0.1, about 1:1 or about 1:1. In one embodiment, the molar ratio of TIL to potassium channel agonist during rapid proliferation is from 1:0.00000001 to 1:0.0001. In one embodiment, the ratio of TIL to potassium channel agonist during rapid proliferation is from 1:0.00000001 to 1:0.000001.

[0263]

[0285] In one embodiment, the ratio of TIL to potassium channel agonist (TIL: potassium channel agonist, cells to molecules) is from about 1:1 to about 1:10, from about 1:10 to about 1:100, from about 1:100 to about 1:1000, from about 1:1000 to about 1:10 4 , about 1:10 4 to about 1:10 5 , about 1:10 5 to about 1:10 6 , about 1:10 6 to about 1:10 7 , about 1:10 7 to about 1:10 8 and about 1:10 8 to about 1:10 9 and is selected from the group consisting of.

[0264]

[0286] In one embodiment, the cell culture medium contains IL-2. In a preferred embodiment, the cell culture medium contains about 3000 IU / mL of IL-2. In one embodiment, the cell culture medium contains about 1000 IU / mL, about 1500 IU / mL, about 2000 IU / mL, about 2500 IU / mL, about 3000 IU / mL, about 3500 IU / mL, about 4000 IU / mL, about 4500 IU / mL, about 5000 IU / mL, about 5500 IU / mL, about 6000 IU / mL, about 6500 IU / mL, about 7000 IU / mL, about 7500 IU / mL or about 8000 IU / mL of IL-2. In one embodiment, the cell culture medium contains IL-2 at a concentration of 1000 - 2000 IU / mL, 2000 - 3000 IU / mL, 3000 - 4000 IU / mL, 4000 - 5000 IU / mL, 5000 - 6000 IU / mL, 6000 - 7000 IU / mL, 7000 - 8000 IU / mL or 8000 IU / mL.

[0265]

[0287] In one embodiment, the cell culture medium contains the OKT-3 antibody. In a preferred embodiment, the cell culture medium contains about 30 ng / mL of the OKT-3 antibody. In one embodiment, the cell culture medium contains about 0.1 ng / mL, about 0.5 ng / mL, about 1 ng / mL, about 2.5 ng / mL, about 5 ng / mL, about 7.5 ng / mL, about 10 ng / mL, about 15 ng / mL, about 20 ng / mL, about 25 ng / mL, about 30 ng / mL, about 35 ng / mL, about 40 ng / mL, about 50 ng / mL, about 60 ng / mL, about 70 ng / mL, about 80 ng / mL, about 90 ng / mL, about 100 ng / mL, about 200 ng / mL, about 500 ng / mL and about 1 μg / mL of the OKT-3 antibody. In one embodiment, the cell culture medium contains the OKT-3 antibody at a concentration of 0.1 ng / mL - 1 ng / mL, 1 ng / mL - 5 ng / mL, 5 ng / mL - 10 ng / mL, 10 ng / mL - 20 ng / mL, 20 ng / mL - 30 ng / mL, 30 ng / mL - 40 ng / mL, 40 ng / mL - 50 ng / mL and 50 ng / mL - 100 ng / mL.

[0266]

[0288] In one embodiment, the TILs are grown in a gas-permeable container. Gas-permeable containers are used to grow TILs using PBMCs, using methods, compositions, and devices known in the art, including those described in U.S. Patent Application Publication No. 2005 / 0106717A1, the disclosure of which is incorporated herein by reference. In one embodiment, the TILs are grown in a gas-permeable bag. In one embodiment, the TILs are grown using a cell expansion system that grows TILs in a gas-permeable bag, such as the Xuri Cell Expansion System W25 (GE Healthcare). In one embodiment, the TILs are grown using a cell expansion system that grows TILs in a gas-permeable bag, such as the WAVE Bioreactor System, also known as the Xuri Cell Expansion System W5 (GE Healthcare). In one embodiment, the cell expansion system includes a gas-permeable cell bag having a volume selected from the group consisting of about 100 mL, about 200 mL, about 300 mL, about 400 mL, about 500 mL, about 600 mL, about 700 mL, about 800 mL, about 900 mL, about 1 L, about 2 L, about 3 L, about 4 L, about 5 L, about 6 L, about 7 L, about 8 L, about 9 L, about 10 L, about 11 L, about 12 L, about 13 L, about 14 L, about 15 L, about 16 L, about 17 L, about 18 L, about 19 L, about 20 L, about 25 L, and about 30 L. In one embodiment, the cell expansion system includes a gas-permeable cell bag having a volume in the range selected from the group consisting of 50 - 150 mL, 150 - 250 mL, 250 - 350 mL, 350 - 450 mL, 450 - 550 mL, 550 - 650 mL, 650 - 750 mL, 750 - 850 mL, 850 - 950 mL, and 950 - 1050 mL. In one embodiment, the cell expansion system includes a gas-permeable cell bag having a volume in the range selected from the group consisting of 1 L - 2 L, 2 L - 3 L, 3 L - 4 L, 4 L - 5 L, 5 L - 6 L, 6 L - 7 L, 7 L - 8 L, 8 L - 9 L, 9 L - 10 L, 10 L - 11 L, 11 L - 12 L, 12 L - 13 L, 13 L - 14 L, 14 L - 15 L, 15 L - 16 L, 16 L - 17 L, 17 L - 18 L, 18 L - 19 L, and 19 L - 20 L.In one embodiment, the cell growth system includes a gas-permeable cell bag having a volume in the range selected from the group consisting of 0.5 L to 5 L, 5 L to 10 L, 10 L to 15 L, 15 L to 20 L, 20 L to 25 L, and 25 L to 30 L. In one embodiment, the cell growth system uses rocking times of about 30 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 24 hours, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, about 21 days, about 22 days, about 23 days, about 24 days, about 25 days, about 26 days, about 27 days, and about 28 days. In one embodiment, the cell growth system uses rocking times of 30 minutes to 1 hour, 1 hour to 12 hours, 12 hours to 1 day, 1 day to 7 days, 7 days to 14 days, 14 days to 21 days, and 21 days to 28 days. In one embodiment, the cell growth system uses rocking speeds of about 2 rockings per minute, about 5 rockings per minute, about 10 rockings per minute, about 20 rockings per minute, about 30 rockings per minute, and about 40 rockings per minute. In one embodiment, the cell growth system uses rocking speeds of 2 rockings per minute to 5 rockings per minute, 5 rockings per minute to 10 rockings per minute, 10 rockings per minute to 20 rockings per minute, 20 rockings per minute to 30 rockings per minute, and 30 rockings per minute to 40 rockings per minute. In one embodiment, the cell growth system uses rocking angles of about 2°, about 3°, about 4°, about 5°, about 6°, about 7°, about 8°, about 9°, about 10°, about 11°, and about 12°. In one embodiment, the cell growth system uses rocking angles of 2° to 3°, 3° to 4°, 4° to 5°, 5° to 6°, 6° to 7°, 7° to 8°, 8° to 9°, 9° to 10°, 10° to 11°, and 11° to 12°.

[0267]

[0289] In one embodiment, a method of growing TIL using a potassium channel agonist further includes a step of selecting TIL for excellent tumor reactivity. Any selection method known in the art can be used. For example, the method described in US Patent Application Publication No. 2016 / 0010058A1, the disclosure of which is incorporated herein by reference, can be used for the selection of TIL for excellent tumor reactivity.

[0268]

[0290] In one embodiment, the present invention provides a method of expanding a population of TILs using any of the potassium channel agonists of the present disclosure, the method including the steps as described in Jin, et al., J. Immunotherapy 2012, 35, 283-292, the disclosure of which is incorporated herein by reference. For example, a tumor or a part thereof can be placed in an enzyme medium and mechanically separated for approximately 1 minute. Then, the mixture can be incubated at 37° C. in 5% CO 2 for 30 minutes and then mechanically disrupted again for approximately 1 minute. After incubation at 37° C. in 5% CO 2 for 30 minutes, the tumor or a part thereof can be mechanically disrupted a third time for approximately 1 minute. After the third mechanical disruption, if large tissue pieces are present, one or two additional mechanical separations can be applied to the sample, with or without an additional 30-minute incubation at 37° C. in 5% CO 2 . At the end of the final incubation, if the cell suspension contains a large number of red blood cells or dead cells, density gradient separation using Ficoll can be performed to remove these cells. TIL culture is initiated in 24-well plates (Costar 24-well cell culture clusters, flat bottom; Corning Incorporated, Corning, NY), and each well is seeded with 1×10 6 tumor-digested cells or one tumor fragment approximately 1-8 mm 3 in size in 2 mL of complete medium (CM) containing IL-2 (6000 IU / mL; Chiron Corp., Emeryville, CA). CM contains Roswell Park Memorial Institute (RPMI) 1640 buffer containing GlutaMAX supplemented with 10% human AB serum, 25 mM Hepes, and 10 mg / mL gentamicin. The culture can be initiated in gas-permeable flasks (G-Rex 10; Wilson Wolf Manufacturing, New Brighton) having a gas-permeable silicon bottom with a volume of 40 mL and a diameter of 10 cm, and each flask is seeded with 10-40×10 2 in 10-40 mL of CM containing IL-2.6 It can be filled with individual viable tumor digest cells or 5 to 30 tumor fragments. G-Rex 10 and 24-well plates can be incubated in a humidified incubator at 37°C in 5% CO 2 and can be incubated in a humidified incubator at 37°C. Five days after the start of culture, half of the medium can be removed and replaced with fresh CM and IL-2. After day 5, half of the medium can be replaced every 2 to 3 days. Using the potassium channel agonists of the present disclosure, as described elsewhere herein, a rapid expansion protocol (REP) of TIL can be performed using a T-175 flask and a gas-permeable bag or a gas-permeable G-Rex flask. In the REP in a T-175 flask, 1×10 6 TIL can be suspended in 150 mL of medium in each flask. TIL can be cultured with the potassium channel agonists of the present disclosure at the ratios described herein in a 1:1 mixture (50 / 50 medium) of CM and AIM-V medium supplemented with 3000 IU / mL of IL-2 and 30 ng / mL of anti-CD3 antibody (OKT-3). The T-175 flask can be incubated at 37°C in 5% CO 2 and can be incubated at 37°C. Half of the medium can be replaced on day 5 with 50 / 50 medium containing 3000 IU / mL of IL-2. On day 7, the cells from two T-175 flasks can be mixed in a 3 L bag, and 300 mL of AIM-V containing 5% human AB serum and 3000 IU / mL of IL-2 can be added to 300 mL of the TIL suspension. The cell number in each bag can be counted daily or every other day, and fresh medium can be added to maintain the cell number at 0.5 to 2.0×10 6 cells / mL. In the REP in a 500 mL capacity flask (e.g., G-Rex 100, Wilson Wolf Manufacturing) with a 100 cm 2 gas-permeable silicon bottom as described elsewhere herein, 5×10 6 or 10×10 6Individual TILs can be cultured with a potassium channel agonist at the ratios described herein (e.g., 1:100) in 400 mL of 50 / 50 medium supplemented with 3000 IU / mL of IL-2 and 30 ng / mL of anti-CD3 antibody (OKT-3). G-Rex 100 flasks can be incubated at 37°C in 5% CO 2 2. They can be centrifuged at 1500 rpm (491 g) for 10 minutes in centrifuge bottles after removing 250 mL of the supernatant on day 5. The resulting TIL pellet can be resuspended in 150 mL of fresh 50 / 50 medium containing 3000 IU / mL of IL-2 and returned to and added to the G-Rex 100 flask. When continuously growing TILs in the G-Rex 100 flask, on day 7, the TILs in each G-Rex 100 are suspended in the 300 mL of medium present in each flask, and the cell suspension can be divided into three 100 mL aliquots that can be used to inoculate three G-Rex 100 flasks. Then, approximately 150 mL of AIM-V containing 5% human AB serum and 3000 UI of IL-2 per mL can be added to each flask. Then, the G-Rex 100 flask can be incubated at 37°C in 5% CO 2 2. After 4 days, 150 mL of AIM-V with 3000 IU / mL of IL-2 can be added to each G-Rex 100 flask. Thereafter, REP can be completed by harvesting the cells on day 14 of the culture.

[0269]

[0291] In one embodiment, a method of growing or treating cancer comprises the step of obtaining TILs from a patient's tumor sample. The patient's tumor sample can be obtained using methods known in the art. For example, TILs can be cultured from enzymatic tumor digests and tumor fragments (sized approximately 1 to about 8 mm) by sharp dissection. Such tumor digests can be produced by incubation in an enzyme medium (e.g., Roswell Park Memorial Institute (RPMI) 1640 buffer, 2 mM glutamate, 10 mcg / mL gentamicin, 30 Units / mL of DNase, and 1.0 mg / mL of collagenase), followed by mechanical separation (e.g., using a tissue dissociator). The tumor is placed in the enzyme medium, the tumor is mechanically separated for approximately 1 minute, followed by incubation at 37 °C in 5% CO 2 2 for 30 minutes, and then the cycle of mechanical separation and incubation is repeated under the aforementioned conditions until only small tissue pieces are present, whereby a tumor digest can be produced. At the end of this process, if the cell suspension contains a large number of red blood cells or dead cells, density gradient separation using FICOLL branched hydrophilic polysaccharide can be performed to remove these cells. Alternative methods known in the art, such as those described in U.S. Patent Application Publication No. 2012 / 0244133A1, can be used, the disclosure of which is incorporated herein by reference. Any of the aforementioned methods can be used in any of the embodiments described herein for a method of growing TILs or treating cancer.

[0270]

[0292] In one embodiment, as described above, the rapid expansion process of TILs can be carried out using a T-175 flask and a gas-permeable bag (Tran, et al, J. Immunother. 2008, 31, 742-51; Dudley, et al, J. Immunother. 2003, 26, 332-42), or using a gas-permeable culture vessel (G-Rex flask, commercially available from Wilson Wolf Manufacturing Corporation, New Brighton, MN, USA). For the rapid expansion of TILs in a T-175 flask, 1×10 6 TILs suspended in 150 mL of medium can be added to each T-175 flask. The TILs can be cultured with the potassium channel agonist of the present disclosure at a ratio of 1 TIL to 100 potassium channel agonist molecules, and the cells can be cultured in a 1:1 mixture of CM and AIM-V medium supplemented with 3000 IU (International Units) / mL of IL-2 and 30 ng / mL of anti-CD3 antibody (e.g., OKT-3). The T-175 flasks can be incubated at 37 °C in 5% CO 2 . Half of the medium can be replaced on day 5 with a 50 / 50 medium containing 3000 IU of IL-2 per mL. On day 7, the cells from two T-175 flasks are mixed in a 3 L bag, and 300 mL of AIM-V containing 5% human AB serum and 3000 IU of IL-2 per mL are added to the 300 mL TIL suspension. The cell count in each bag is counted daily or every other day, and fresh medium is added to maintain the cell count at 0.5 - 2.0×10 6 cells / mL.

[0271]

[0293] In one embodiment, for the rapid expansion of TILs in a 500 mL capacity gas-permeable flask (G-Rex 100, commercially available from Wilson Wolf Manufacturing Corporation, New Brighton, MN, USA) having a 100 cm 2 gas-permeable silicon bottom, 5×10 6 or 10×10 6Individual TILs can be cultured with a potassium channel agonist in 400 mL of 50 / 50 medium supplemented with 5% human AB serum, 3000 IU / mL of IL-2, and 30 ng / mL of anti-CD3 (OKT-3). G-Rex 100 flasks can be incubated at 37°C in 5% CO 2 2. On day 5, 250 mL of supernatant can be removed and placed into a centrifuge bottle and centrifuged at 1500 rpm (revolutions per minute; 491×g) for 10 minutes. The TIL pellet can be resuspended in 150 mL of fresh medium containing 5% human AB serum and 3000 IU of IL-2 per mL and returned to and added to the original G-Rex 100 flask. When continuously growing TILs in a G-Rex 100 flask, on day 7, the TILs in each G-Rex 100 flask can be suspended in 300 mL of medium present in each flask and the cell suspension can be divided into three 100 mL aliquots that can be used to inoculate three G-Rex 100 flasks. Then, 150 mL of AIM-V containing 5% human AB serum and 3000 UI of IL-2 per mL can be added to each flask. The G-Rex 100 flasks can be incubated at 37°C in 5% CO 2 2 and incubated at 37°C. Four days later, 150 mL of AIM-V with 3000 IU of IL-2 per mL can be added to each G-Rex 100 flask. The cells can be harvested on day 14 of culture.

[0272]

[0294] In one embodiment, TILs can be prepared as follows. 2 mm 3Tumor fragments are cultured in complete medium (CM) containing AIM-V medium (Invitrogen Life Technologies, Carlsbad, CA) supplemented with 2 mM glutamine (Mediatech, Inc. Manassas, VA), 100 U / mL penicillin (Invitrogen Life Technologies), 100 μg / mL streptomycin (Invitrogen Life Technologies), 5% heat-inactivated human AB serum (Valley Biomedical, Inc. Winchester, VA), and 600 IU / mL rhIL-2 (Chiron, Emeryville, CA). For enzymatic digestion of solid tumors, tumor specimens are diced into RPMI-1640, washed, centrifuged at 800 rpm for 5 min at 15-22°C, and resuspended in enzymatic digestion buffer (0.2 mg / mL collagenase and 30 Units / ml DNase in RPMI-1640), followed by overnight rotation at room temperature. TILs generated from the fragments can be expanded in CM for 3-4 weeks and either grown fresh or cryopreserved in heat-inactivated HAB serum containing 10% dimethyl sulfoxide (DMSO) and stored at -180 °C until time of study. Tumor associated lymphocytes (TAL) obtained from ascites collections were cultured at 3 × 10 in 24-well plates in CM. 6 Cells were seeded at 1000 x g / well. TIL proliferation was examined every other day using a low magnification inverted microscope.

[0273]

[0295] In one embodiment, the potassium channel agonist of the present invention can be used to expand T cells. Any of the above-mentioned embodiments of the present invention described for the expansion of TILs can also be applied to the expansion of T cells. In one embodiment, the potassium channel agonist of the present invention can be used to expand CD8 T cells. In one embodiment, the potassium channel agonist of the present invention can be used to expand CD4 T cells. +It can be used to proliferate T cells. In one embodiment, the potassium channel agonist of the present invention can be used to proliferate T cells transduced with a chimeric antigen receptor (CAR-T). In one embodiment, the potassium channel agonist of the present invention can be used to proliferate T cells comprising a modified T cell receptor (TCR). CAR-T cells can be targeted to any suitable antigen, including CD19, as described in, for example, U.S. Patent Nos. 7,070,995; 7,446,190; 8,399,645; 8,916,381; and 9,328,156, the disclosures of which are incorporated herein by reference. Modified TCR cells can be targeted to any suitable antigen, including NY-ESO-1, TRP-1, TRP-2, tyrosinase cancer antigen, MAGE-A3, SSX-2, and VEGFR2 or antigenic portions thereof, as described in, for example, U.S. Patent Nos. 8,367,804 and 7,569,664, the disclosures of which are incorporated herein by reference.

[0274] Optional cryopreservation of TIL

[0001] In some embodiments, either a bulk TIL population or an expanded TIL population can be optionally cryopreserved. In some embodiments, cryopreservation is performed on a therapeutic TIL population. In some embodiments, cryopreservation is performed on TILs recovered after expansion. In some embodiments, the TILs are cryopreserved within an infusion bag. In some embodiments, the TILs are cryopreserved before being placed in the infusion bag. In some embodiments, the TILs are cryopreserved and not in an infusion bag. In some embodiments, cryopreservation is performed using a cryopreservation medium. In some embodiments, the cryopreservation medium contains dimethyl sulfoxide (DMSO). This is generally accomplished by placing the TIL population in a cryopreservation solution, such as 85% complement-inactivated AB serum and 15% dimethyl sulfoxide (DMSO). The cells in the solution are placed in a cryovial and optionally transferred to a gaseous nitrogen freezer for cryopreservation and stored at -80°C for 24 hours. See Sadeghi, et al, Acta Oncologica 2013, 52, 978-986.

[0275]

[0002] When appropriate, the cells are removed from the freezer and thawed in a 37°C water bath until approximately 4 / 5 of the solution is thawed. The cells are generally resuspended in complete medium and optionally washed one or more times. In some embodiments, as is known in the art, the thawed TILs can be counted and evaluated for viability.

[0276]

[0003] In a preferred embodiment, the population of TILs is cryopreserved using CS10 cryopreservation medium (CryoStor 10, BioLife Solutions). In a preferred embodiment, the population of TILs is cryopreserved using a cryopreservation medium containing dimethyl sulfoxide (DMSO). In a preferred embodiment, the population of TILs is cryopreserved using a 1:1 (vol:vol) ratio of CS10 and cell culture medium. In a preferred embodiment, the population of TILs is cryopreserved using a 1:1 (vol:vol) ratio of CS10 and cell culture medium that further contains additional IL-2.

[0277]

[0004] As described herein, cryopreservation can be performed at multiple time points throughout the TIL expansion process. In some embodiments, the bulk TIL population after the first expansion or the expanded population of TILs after one or more second expansions can be cryopreserved. Cryopreservation can generally be achieved by placing the TIL population in a cryopreservation solution, such as 85% complement-inactivated AB serum and 15% dimethyl sulfoxide (DMSO). The cells in the solution are placed in cryovials and optionally transferred to a gaseous nitrogen freezer for cryopreservation and stored at -80°C for 24 hours. See Sadeghi, et al, Acta Oncologica 2013, 52, 978-986.

[0278]

[0005] When appropriate, the cells are removed from the freezer and thawed in a 37°C water bath until approximately 4 / 5 of the solution is thawed. The cells are generally resuspended in complete medium and optionally washed one or more times. In some embodiments, as is known in the art, the thawed TILs can be counted and evaluated for viability.

[0279]

[0006] In some cases, the TIL population can be cryopreserved immediately using the protocols described herein.

[0280] TIL Pharmaceutical Compositions, Dosages, and Administration Regimens

[0296] In one embodiment, TILs expanded using the methods of the present disclosure are administered to a patient as a pharmaceutical composition. In one embodiment, the pharmaceutical composition is a suspension of TILs in a sterile buffer. TILs expanded using the methods of the present disclosure can be administered by any suitable route known in the art. Preferably, the TILs are administered as a single intraarterial or intravenous infusion, which preferably lasts approximately 30 - 60 minutes. Other suitable routes of administration include intraperitoneal administration, intrathecal administration, and intralymphatic administration.

[0281]

[0297] Any suitable dose of TIL can be administered. Particularly when the cancer is melanoma, preferably, the average is about 7.8×10 10 TIL is about 2.3×10 10 ~about 13.7×10 10 TIL is administered. In one embodiment, about 1.2×10 10 ~about 4.3×10 10 of TIL is administered.

[0282]

[0298] In some embodiments, the number of TIL provided in the pharmaceutical composition of the present invention is about 1×10 6 , 2×10 6 , 3×10 6 , 4×10 6 , 5×10 6 , 6×10 6 , 7×10 6 , 8×10 6 , 9×10 6 , 1×10 7 , 2×10 7 , 3×10 7 , 4×10 7 , 5×10 7 , 6×10 7 , 7×10 7 , 8×10 7 , 9×10 7 , 1×10 8 , 2×10 8 , 3×10 8 , 4×10 8 , 5×10 8 , 6×10 8 , 7×10 8 , 8×10 8 , 9×10 8 , 1×10 9 , 2×10 9 , 3×10 9 , 4×10 9 , 5×10 9 , 6×10 9 , 7×10 9 , 8×10 9 , 9×10 9 , 1×10 10 , 2×10 10 , 3×10 10 , 4×10 10 , 5×1010 and 6×10 10 and 7×10 10 and 8×10 10 and 9×10 10 and 1×10 11 and 2×10 11 and 3×10 11 and 4×10 11 and 5×10 11 and 6×10 11 and 7×10 11 and 8×10 11 and 9×10 11 and 1×10 12 and 2×10 12 and 3×10 12 and 4×10 12 and 5×10 12 and 6×10 12 and 7×10 12 and 8×10 12 and 9×10 12 and 1×10 13 and 2×10 13 and 3×10 13 and 4×10 13 and 5×10 13 and 6×10 13 and 7×10 13 and 8×10 13 and 9×10 13 and are in number. In one embodiment, the number of TILs provided in the pharmaceutical composition of the present invention is 1×10 6 to 5×10 6 and 5×10 6 to 1×10 7 and 1×10 7 to 5×10 7 and 5×10 7 to 1×10 8 and 1×10 8 to 5×10 8 and 5×10 8 to 1×10 9 and 1×10 9 to 5×10 9 and 5×10 9 to 1×10 10 and 1×10 10 to 5×10 10 and 5×10 10 to 1×10 11 and 5×10 11 to 1×10 12, 1×10 12 ~5×10 12 and 5×10 12 ~1×10 13 is within the range of.

[0283]

[0299] In some embodiments, the concentration of TILs provided in the pharmaceutical composition of the present invention is, for example, 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002% or 0.0001% w / w, w / v or v / v lower than that of the pharmaceutical composition.

[0284]

[0300] In some embodiments, the concentration of TILs provided in the pharmaceutical composition of the present invention is higher than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19.75%, 19.50%, 19.25%, 19%, 18.75%, 18.50%, 18.25%, 18%, 17.75%, 17.50%, 17.25%, 17%, 16.75%, 16.50%, 16.25%, 16%, 15.75%, 15.50%, 15.25%, 15%, 14.75%, 14.50%, 14.25%, 14%, 13.75%, 13.50%, 13.25%, 13%, 12.75%, 12.50%, 12.25%, 12%, 11.75%, 11.50%, 11.25%, 11%, 10.75%, 10.50%, 10.25%, 10%, 9.75%, 9.50%, 9.25%, 9%, 8.75%, 8.50%, 8.25%, 8%, 7.75%, 7.50%, 7.25%, 7%, 6.75%, 6.50%, 6.25%, 6%, 5.75%, 5.50%, 5.25%, 5%, 4.75%, 4.50%, 4.25%, 4%, 3.75%, 3.50%, 3.25%, 3%, 2.75%, 2.50%, 2.25%, 2%, 1.75%, 1.50%, 125%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002% or 0.0001% w / w, w / v or v / v of the pharmaceutical composition.

[0285]

[0301] In some embodiments, the concentration of TILs provided in the pharmaceutical composition of the present invention is within the range of about 0.0001% to about 50%, about 0.001% to about 40%, about 0.01% to about 30%, about 0.02% to about 29%, about 0.03% to about 28%, about 0.04% to about 27%, about 0.05% to about 26%, about 0.06% to about 25%, about 0.07% to about 24%, about 0.08% to about 23%, about 0.09% to about 22%, about 0.1% to about 21%, about 0.2% to about 20%, about 0.3% to about 19%, about 0.4% to about 18%, about 0.5% to about 17%, about 0.6% to about 16%, about 0.7% to about 15%, about 0.8% to about 14%, about 0.9% to about 12% or about 1% to about 10% w / w, w / v or v / v of the pharmaceutical composition.

[0286]

[0302] In some embodiments, the concentration of TILs provided in the pharmaceutical composition of the present invention is within the range of about 0.001% to about 10%, about 0.01% to about 5%, about 0.02% to about 4.5%, about 0.03% to about 4%, about 0.04% to about 3.5%, about 0.05% to about 3%, about 0.06% to about 2.5%, about 0.07% to about 2%, about 0.08% to about 1.5%, about 0.09% to about 1%, about 0.1% to about 0.9% w / w, w / v or v / v of the pharmaceutical composition.

[0287]

[0303] In some embodiments, the amount of TILs provided in the pharmaceutical composition of the present invention is 10 g, 9.5 g, 9.0 g, 8.5 g, 8.0 g, 7.5 g, 7.0 g, 6.5 g, 6.0 g, 5.5 g, 5.0 g, 4.5 g, 4.0 g, 3.5 g, 3.0 g, 2.5 g, 2.0 g, 1.5 g, 1.0 g, 0.95 g, 0.9 g, 0.85 g, 0.8 g, 0.75 g, 0.7 g, 0.65 g, 0.6 g, 0.55 g, 0.5 g, 0.45 g, 0.4 g, 0.35 g, 0.3 g, 0.25 g, 0.2 g, 0.15 g, 0.1 g, 0.09 g, 0.08 g, 0.07 g, 0.06 g, 0.05 g, 0.04 g, 0.03 g, 0.02 g, 0.01 g, 0.009 g, 0.008 g, 0.007 g, 0.006 g, 0.005 g, 0.004 g, 0.003 g, 0.002 g, 0.001 g, 0.0009 g, 0.0008 g, 0.0007 g, 0.0006 g, 0.0005 g, 0.0004 g, 0.0003 g, 0.0002 g or 0.0001 g or less.

[0288]

[0304] In some embodiments, the amount of TILs provided in the pharmaceutical composition of the present invention is greater than 0.0001 g, 0.0002 g, 0.0003 g, 0.0004 g, 0.0005 g, 0.0006 g, 0.0007 g, 0.0008 g, 0.0009 g, 0.001 g, 0.0015 g, 0.002 g, 0.0025 g, 0.003 g, 0.0035 g, 0.004 g, 0.0045 g, 0.005 g, 0.0055 g, 0.006 g, 0.0065 g, 0.007 g, 0.0075 g, 0.008 g, 0.0085 g, 0.009 g, 0.0095 g, 0.01 g, 0.015 g, 0.02 g, 0.025 g, 0.03 g, 0.035 g, 0.04 g, 0.045 g, 0.05 g, 0.055 g, 0.06 g, 0.065 g, 0.07 g, 0.075 g, 0.08 g, 0.085 g, 0.09 g, 0.095 g, 0.1 g, 0.15 g, 0.2 g, 0.25 g, 0.3 g, 0.35 g, 0.4 g, 0.45 g, 0.5 g, 0.55 g, 0.6 g, 0.65 g, 0.7 g, 0.75 g, 0.8 g, 0.85 g, 0.9 g, 0.95 g, 1 g, 1.5 g, 2 g, 2.5, 3 g, 3.5, 4 g, 4.5 g, 5 g, 5.5 g, 6 g, 6.5 g, 7 g, 7.5 g, 8 g, 8.5 g, 9 g, 9.5 g or 10 g.

[0289]

[0305] The TILs provided in the pharmaceutical composition of the present invention are effective over a wide dosage range. The exact dosage will depend on the route of administration, the form in which the compound is administered, the gender and age of the subject to be treated, the weight of the subject to be treated, as well as the preference and experience of the attending physician. Clinically established dosages of TILs may also be used where appropriate. The amount of the pharmaceutical composition administered using the methods herein, such as the dosage of TILs, will depend on the human or mammal to be treated, the severity of the disease or condition, the rate of administration, the nature of the active pharmaceutical ingredient, and the discretion of the prescribing physician.

[0290]

[0306] In some embodiments, the TILs can be administered as a single dose. Such administration can be by injection, for example, intravenous injection. In some embodiments, the TILs can be administered in multiple doses. The administration can be once a year, twice a year, three times a year, four times a year, five times a year, six times a year or more than six times. The administration can be once a month, once every two weeks, once a week or every other day. The administration of TILs can be continued as long a...

Claims

A pharmaceutical composition comprising a population of tumor-infiltrating lymphocytes (TILs) for adoptive T cell therapy for the treatment of cancer in a patient, wherein the TILs are: (a) a step of initially expanding a first population of TILs in a first cell culture medium to obtain a second population of TILs, wherein the first cell culture medium contains IL-2 and the first population of TILs is obtained from a tumor excised from the patient; (b) a step of rapidly expanding the second population of TILs in a second cell culture medium to obtain a third population of TILs, wherein the second cell culture medium contains IL-2, OKT-3 and irradiated allogeneic peripheral blood mononuclear cells (PBMCs), and the rapid expansion is carried out over a period of 14 days or less; and (c) a step of recovering the third population of TILs Obtained by, one or both of the first cell culture medium and the second cell culture medium contain a K Ｃａ 3.1 (IK channel) agonist further, Said K Ｃａ 3.1 The agonist is naphtho[1,2-d]thiazol-2-ylamine (SKA-31) 【Chemical 1】 A pharmaceutical composition comprising the above steps.

2. An ex vivo method for preparing a therapeutic population of tumor-infiltrating lymphocytes (TILs), comprising: (a) a step of initially expanding a first population of TILs in a first cell culture medium to obtain a second population of TILs, wherein the first cell culture medium contains IL-2 and the first population of TILs is obtained from a tumor; (b) a step of rapidly expanding the second population of TILs in a second cell culture medium to obtain a third population of TILs, wherein the second cell culture medium contains IL-2, OKT-3 (anti-CD3 antibody), irradiated allogeneic peripheral blood mononuclear cells (PBMCs), and the rapid expansion is carried out over a period of 14 days or less; (c) a step of recovering the third population of TILs, wherein at least a part of the third population of TILs is a therapeutic population of TILs comprising, wherein one or both of the first cell culture medium and the second cell culture medium contain K Ｃａ 3.1 (IK channel) agonist, and further comprising Said K Ｃａ 3.1 The agonist is naphtho[1,2-d]thiazol-2-ylamine (SKA-31) 【Chemical Formula 2】 A method comprising the above steps.

3. Both the first cell culture medium and the second cell culture medium are the K Ｃａ The pharmaceutical composition according to claim 1, further comprising a 3.1 agonist.

4. (i) The concentration of the K in the first cell culture medium Ｃａ The concentration of the 3.1 agonist is 1 to 1000 nM, or (ii) The concentration of the K Ｃａ 3.1 agonist is 0.1 to 100 mM, or (iii) the initial expansion is carried out over a period of 21 days or less and / or (iv) the rapid expansion is carried out over a period of 7 days or less, The pharmaceutical composition according to claim 1 or 3.

5. (i) the IL-2 is present in the first cell culture medium at an initial concentration of 1000 IU / mL to 6000 IU / mL, or (ii) in the second cell culture medium, the 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, or (iii) the initial expansion is carried out using a gas-permeable container, (iv) Is the rapid expansion carried out using a gas-permeable container, or (v) Does the first cell culture medium further contain a cytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21, and combinations thereof, and / or (vi) Does the second cell culture medium further contain a cytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21, and combinations thereof, the pharmaceutical composition according to claim 1, 3, or 4.

6. (i) the second population of TILs is increased relative to a reference population of TILs obtained without the potassium channel agonist, ＋ CD28 ＋ , CD8 ＋ CD27 ＋ , CD8 ＋ CD27 ＋ CD28 ＋ , CCR7 ＋ and combinations thereof, wherein the phenotype in the second population of TILs is increased by at least 5% relative to the reference population of TILs; (ii) The third population of the TILs includes a population of T cells having a phenotype selected from the group consisting of CD8 ＋ CD28 ＋ , CD8 ＋ CD27 ＋ , CD8 ＋ CD27 ＋ CD28 ＋ , CCR7 ＋ , and combinations thereof, and the phenotype in the third population of the TILs is increased by at least 5% relative to the reference population of the TILs obtained without the potassium channel agonist. ＋ CD28 ＋ CD8 ＋ CD27 ＋ CD8 ＋ CD27 ＋ CD28 ＋ CCR7 ＋ or (iii) Does the second population of the TILs include a population of T cells having a phenotype with a lower degree of differentiation compared to a reference population of TILs obtained without the potassium channel agonist, and / or (iv) Does the third population of the TILs include a population of T cells having a phenotype with a lower degree of differentiation compared to a reference population of TILs obtained without the potassium channel agonist, the pharmaceutical composition according to any one of claims 1, 3 to 5.

7. (i) Is the cancer selected from melanoma, ovarian cancer, cervical cancer, lung cancer, bladder cancer, breast cancer, head and neck cancer, renal cell cancer, acute myeloid leukemia, colorectal cancer, sarcoma, non-small cell lung cancer (NSCLC), or triple-negative breast cancer, double-resistant melanoma, and uveal (intraocular) melanoma, or (ii) The cancer is selected from the group consisting of non-small cell lung cancer (NSCLC), estrogen receptor-positive (ER ＋ ) breast cancer, progesterone receptor-positive (PR ＋ ) breast cancer, human epidermal growth factor receptor 2 (HER2 ＋ ) breast cancer, triple-positive breast cancer (ER ＋ / PR ＋ / HER2 ＋ ), triple-negative breast cancer (ER － / PR － / HER2 － ), double-resistant melanoma and uveal (intraocular) melanoma The pharmaceutical composition according to any one of claims 1, 3 to 6.

8. The adoptive T cell therapy further comprises: (i)Starting on the day after administration of the third population of the TILs to the patient, the K Ｃａ Step of treating the patient with a 3.1 (IK channel) agonist; (ii) prior to said step of removing the tumor from said patient, said K Ｃａ 3.1 (IK channel) treating said patient with a channel agonist; (iii) treating the patient with a non-myeloablative lymphocyte depletion regimen prior to administering the third population of TILs to the patient, optionally, the non-myeloablative lymphocyte depletion regimen comprises administering cyclophosphamide at a dose of 60 mg / m ２ / day for 2 days, followed by administering fludarabine at a dose of 25 mg / m ２ / day for 5 days; and / or (iv) Treating the patient with a high-dose IL-2 regimen that starts on the day after the administration of the third population of the TILs to the patient, optionally, the high-dose IL-2 regimen is administered as a 15-minute bolus intravenous injection every 8 hours up to the tolerated volume, and includes 600,000 or 720,000 IU / kg of aldesleukin or its biosimilar or variant, the pharmaceutical composition according to any one of claims 1, 3 to 5.

9. IL-2 and K Ｃａ 3.1 A cell culture medium for culturing tumor-infiltrating lymphocytes (TIL) comprising an agonist, an anti-CD3 antibody or a fragment, variant or biosimilar thereof, and peripheral blood mononuclear cells (PBMC), wherein Said K Ｃａ 3.1 The agonist is Naphtho[1,2-d]thiazol-2-ylamine (SKA-31) 【Chemical 3】 A cell culture medium containing.

10. A kit containing the cell culture medium according to claim 9 for preparing tumor-infiltrating lymphocytes (TILs) from a tumor.

11. Both the first cell culture medium and the second cell culture medium are the K Ｃａ 3.1 agonist further comprises the method according to claim 2.

12. (i) The concentration of the K in the first cell culture medium Ｃａ 3.1 The concentration of the agonist is 1 to 1000 nM, or (ii) The concentration of the K in the first cell culture medium Ｃａ 3.1 The concentration of the agonist is 0.1 to 100 mM, or (iii) Is the initial expansion carried out over a period of 21 days or less, and / or (iv) Is the rapid expansion carried out over a period of 7 days or less, The method according to claim 2 or 11.

13. (i) the IL-2 is present in the first cell culture medium at an initial concentration of 1000 IU / mL to 6000 IU / mL, or (ii) in the second cell culture medium, the 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, or (iii) the initial proliferation is performed using a gas-permeable container, or (iv) the rapid proliferation is performed using a gas-permeable container, or (v) 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, and / or (vi) the second cell culture medium further comprises a cytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21, and combinations thereof, the method according to claim 2, 11 or 12.

14. (i) The second population of the TILs includes a population of T cells having a phenotype selected from the group consisting of CD8, ＋ CD28, ＋ , CD8, ＋ CD27, ＋ , CD8, ＋ CD27, ＋ CD28, ＋ , CCR7, ＋ , and combinations thereof, and the phenotype in the second population of the TILs is increased by at least 5% relative to the reference population of the TILs, or ＋ CD28 ＋ CD8 ＋ CD27 ＋ CD8 ＋ CD27 ＋ CD28 ＋ CCR7 ＋ and the phenotype in the second population of the TILs is increased by at least 5% relative to the reference population of the TILs, or (ii) The third population of the TILs includes a population of T cells having a phenotype selected from the group consisting of CD8 ＋ CD28 ＋ , CD8 ＋ CD27 ＋ , CD8 ＋ CD27 ＋ CD28 ＋ , CCR7 ＋ , and combinations thereof, and the phenotype in the third population of the TILs is increased by at least 5% relative to the reference population of the TILs, or ＋ CD28 ＋ CD8 ＋ CD27 ＋ CD8 ＋ CD27 ＋ CD28 ＋ CCR7 ＋ and the phenotype in the third population of the TILs is increased by at least 5% relative to the reference population of the TILs, or (iii) the second population of the TILs comprises a population of T cells having a phenotype with a lower degree of differentiation compared to a reference population of TILs obtained without the potassium channel agonist, and / or (iv) the third population of the TILs comprises a population of T cells having a phenotype with a lower degree of differentiation compared to a reference population of TILs obtained without the potassium channel agonist, the method according to any one of claims 2, 11 to 13.

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