Patent Application
20230117388
This document relates to methods and materials for expanding tumor infiltrating gamma-delta (γδ) T cells (e.g., tumor infiltrating γδ T cells) in culture. For example, this document provides methods and materials for expanding large numbers of tumor infiltrating γδ T cells (e.g., tumor infiltrating γδ T cells that are predominantly Vδ1+) from tissue obtained from a mammal having cancer (e.g., a tumor sample). This document also provides populations of such tumor infiltrating γδ T cells and methods and materials for using such tumor infiltrating γδ T cells and/or such populations to treat cancer within a mammal (e.g., a human).
Cancer immunotherapies including adoptive cell therapy (ACT) with tumor infiltrating lymphocytes (TIL) depend on T cell effector functions. These αβ T cell receptor (TCR) expressing cells target cancer cells through recognition of peptide or lipid antigens presented by major histocompatibility complex (MHC) Class I and II and MHC-like CD1 molecules. TIL therapies that include lymphodepletion, adoptive transfer of ex vivo expanded autologous TIL, and post infusion administration of high dose interleukin-2 (IL-2) has provided durable complete responses in patients with treatment refractory metastatic melanoma, cervical cancer, and other epithelial cancers. With current TIL therapy protocols providing objective clinical response and in particular, complete responses, in many treated patients, improvements in the understanding of the mechanisms of treatment response can help broaden the application of these treatments (Dafni et al., Ann. Oncol., 30:1902-1913 (2019)).
Clinical manifestation of cancer often occurs following years of cancer immune editing with the emergence of poorly immunogenic tumor cell variants, many of which have lost Class I MHC molecules (Schreiber et al., Science, 331:1565-1570 (2011)). Despite efforts to reinvigorate immune responses with ACT, genomic instability of cancer cells promotes Darwinian selection processes associated with mutational downregulation or complete loss of immune reactive tumor associated peptide antigens that provide a means of immune escape (Dudley et al., J Clin. Oncol., 23:2346-2357 (2005); Khong et al., Nat. Immunol., 3:999-1005 (2002); Zitvogel et al., Nat. Rev. Immunol., 6:715-727 (2006); and Orlando et al., Nat. Med., 24:1504-1506 (2018)).
As noted, immune evasion is also mediated by reduced expression or lack of MHC-Class 1 antigen presentation that is pervasive across several solid tumors and limits the efficacy of αβ T cell immunotherapy (Dhatchinamoorthy et al., Front. Immunol., 12:636568 (2021); Tran et al., N. Engl. J. Med., 375:2255-2262 (2016); and Chowell et al., Science, 359:582-587 (2018)). More recently, T cell intrinsic factors, including functional exhaustion associated with lack of effective co-stimulation, inhibitory receptor expression and abrogation of stem cell like memory differentiation dictate persistence and response to immunotherapy (Ahmadzadeh et al., Blood, 114:1537-1544 (2009); Baitsch et al., J Clin. Invest., 121:2350-2360 (2011); Miller et al., Nat. Immunol., 20:326-336 (2019); Sade-Feldman et al., Cell, 175:998-1013 e1020 (2018); Jansen et al., Nature, 576:465-470 (2019); and Krishna et al., Science, 370:1328-1334 (2020)). Therapeutic interventions that can overcome challenges inherent to tumor cell immune escape and suppression paradigms can further improve immunotherapy treatment outcomes.
γδ TCR expressing cells are an evolutionarily conserved lymphocytic subset whose MHC-unrestricted recognition of pathogen derived or host cell non-peptide metabolites and stress antigens provide compelling opportunities to discern their utility in immunosurveillance and cancer immunotherapy (Vantourout et al., Nat. Rev. Immunol., 13:88-100 (2013); Silva-Santos et al., Nat. Rev. Immunol., 15:683-691 (2015); Silva-Santos et al., Nat. Rev. Cancer, 19:392-404 (2019); Sebestyen et al., Nat. Rev. Drug Discov., 19:169-184 (2020); and Ribot et al., Nat. Rev. Immunol., 21:221-232 (2021)). γδ T cells, especially Vδ1+ cells, are predominantly tissue resident immune effectors that display diverse roles in mediating TCR- and natural cytotoxicity receptor (NCR)-dependent tumor surveillance. As such, they coordinate and mediate both innate and adaptive immune responses (Vantourout et al., Nat. Rev. Immunol., 13:88-100 (2013); Silva-Santos et al., Nat. Rev. Immunol., 15:683-691 (2015); Silva-Santos et al., Nat. Rev. Cancer, 19:392-404 (2019); Sebestyen et al., Nat. Rev. Drug Discov., 19:169-184 (2020); Ribot et al., Nat. Rev. Immunol., 21:221-232 (2021); and Davey et al., Trends Immunol., 39:446-459 (2018)). The presence of these cells is associated with better outcomes in patients with many types of cancer. For example, patients with leukemia recovering an increased number of γδ T cells following bone marrow transplantation experienced greater long-term survival (Godder et al., Bone Marrow Transplant., 39:751-757 (2007)). Furthermore, a meta-analysis of infiltrating immune cell gene expression signatures of 25 solid tumor types from the cancer genome atlas (TCGA) identified γδ T cells to be the most significant cell type associated with favorable prognosis (Gentles et al., Nat. Med., 21:938-945 (2015)). Early and ongoing efforts targeting phosphoantigen reactive, blood resident Vγ9Vδ2 cells have established the clinical feasibility and safety of γδ cancer cell therapy (Sebestyen et al., Nat. Rev. Drug Discov., 19:169-184 (2020)).
This document provides methods and materials for expanding tumor infiltrating γδ T cells (e.g., tumor infiltrating γδ T cells) in culture. For example, this document provides methods and materials for expanding tumor infiltrating γδ T cells obtained from tissue (e.g., a tumor sample) to obtain large numbers (e.g., greater than 1×107, greater than 1×108, greater than 5×108, or greater than 1×109) of tumor infiltrating γδ T cells (e.g., tumor infiltrating γδ T cells that are predominantly Vδ1+) within, for example, 25 to 30 days.
As described herein, γδ T cells obtained from tumor tissue (and/or healthy tissue that is within 30 mm of a tumor) can be expanded in vitro using a combination of cytokines (e.g., IL-2 plus IL-4 plus IL-15 (IL-2/IL-4/IL-15)) to produce populations of tumor infiltrating γδ T cells having desired percentages of cells having desired phenotypes. For example, this document provides methods and materials for expanding tumor infiltrating γδ T cells by culturing a first population containing tumor infiltrating γδ T cells in the presence of IL-2 for 5 to 15 days (e.g., 6 to 15 days, 7 to 15 days, 8 to 15 days, 9 to 15 days, 9 to 13 days, 10 to 12 days, or 7 to 10 days) to produce a second population of cells, and subsequently culturing the second population of cells in the presence of IL-2, IL-4, and IL-15 (and optionally PBMCs such as irradiated allogeneic PBMCs and optionally an anti-CD3 agonistic antibody) for 8 to 21 days (e.g., 10 to 21 days, 12 to 21 days, 14 to 21 days, 8 to 18 days, 8 to 16 days, 8 to 14 days, 10 to 20 days, 10 to 18 days, 12 to 18 days, 10 to 16 days, 12 to 16 days, or 13 to 15 days) to produce an expanded population of tumor infiltrating γδ T cells. In some cases, a population of expanded tumor infiltrating γδ T cells can be obtained by (a) obtaining a tissue sample containing a tumor and/or healthy tissue that was within 30 mm of a tumor, (b) obtaining a first cell population containing tumor infiltrating γδ T cells from that tissue, (c) optionally enriching that first cell population so that the resulting enriched population contains a higher ratio of tumor infiltrating γδ T cells to total CD3+ cells, and (d) culturing the first cell population (or the optional enriched population) in the presence of IL-2, IL-4, IL-15, PBMCs (e.g., irradiated PBMCs), and an anti-CD3 antibody for 8 to 21 days (e.g., 10 to 21 days, 12 to 21 days, 14 to 21 days, 8 to 18 days, 8 to 16 days, 8 to 14 days, 10 to 20 days, 10 to 18 days, 12 to 18 days, 10 to 16 days, 12 to 16 days, or 13 to 15 days) to obtain a population of expanded tumor infiltrating γδ T cells.
In some cases, greater than 85 percent of the CD3+ cells of an expanded population provided herein can be γδ TCR+ cells, less than 10 percent of the CD3+ cells of that population can be αβ TCR+ cells, less than 10 percent of the CD45+ cells of that population can be NK cells, greater than 30 percent of the γδ TCR+ cells of that population can be Vδ1+ cells, less than 60 percent of the γδ TCR+ cells of that population can be Vδ1−Vδ2− cells, less than 25 percent of the γδ TCR+ cells of that population can be Vδ2+ cells, greater than 70 percent of the γδ TCR+ cells of that population can be TEM cells, less than 25 percent of the γδ TCR+ cells of that population can be TEMRA cells, as high as 10 percent of the γδ TCR+ cells of that population can be CD69+ CD103+ Tissue resident memory (TRM) cells, as high as 50 percent of the γδ TCR+ cells of that population can be CD56+ cells, from 1 to 40 percent of the γδ TCR+ cells of that population can be CD137+ cells, less than 25 percent of the γδ TCR+ cells of that population can be PD-1+ cells, from 5 to 40 percent of the γδ TCR+ cells of that population can be BTLA+ cells, greater than 60 percent of the γδ TCR+ cells of that population can be NKG2D+ cells, and greater than 20 percent of the γδ TCR+ cells of that population can be NKp46+ cells.
As also described herein, the populations of tumor infiltrating γδ T cells provided herein can be administered to a mammal (e.g., human) having cancer to treat cancer within that mammal. For example, a population of tumor infiltrating γδ T cells provided herein can be administered (e.g., intravenously administered) to a mammal (e.g., a human) having cancer as an adoptive cellular therapy to treat that cancer either alone or in combination with (a) tumor infiltrating αβ T cells and/or (b) one or more therapeutic agents such as one or more checkpoint inhibitors (e.g., anti-PD-1 antibodies and/or anti-PD-L1 antibodies), IL-2, one or more lymphodepleting chemotherapy agents (e.g., cyclophosphamide and/or fludarabine), one or more tumor infiltrating lymphocyte enhancement agents (e.g., CpG and/or oncolytic viruses such as vaccinia viruses), brachytherapy, or combinations thereof. In such cases, the administered tumor infiltrating γδ T cells can provide effective immune responses against cancer cells within the mammal, thereby reducing the number of cancer cells within the mammal.
In general, one aspect of this document features a method for producing a cell population comprising γδ T cells. The method comprises (or consists essentially of or consists of) culturing a first cell population comprising γδ T cells in the presence of IL-2, IL-4, and IL-15 for 8 to 21 days to obtain a second cell population, wherein the second cell population comprises at least 10 times more γδ T cells than the first cell population. The γδ T cells can be human cells. The γδ T cells can be tumor infiltrating γδ T cells. The first cell population can be (i) a population of tumor infiltrating γδ T cells obtained from (a) tissue comprising a tumor or (b) healthy tissue that was within 30 mm of a tumor, (ii) a population of γδ T cells obtained from healthy tissue, (iii) a population of γδ T cells obtained from infected tissue, or (iv) a population of γδ T cells obtained from tissue harboring autoimmune T cells. The method can comprise obtaining the first cell population from the tissue comprising the tumor. The method can comprise obtaining the first cell population from the healthy tissue that was within 30 mm of the tumor. The first cell population can be a cell population that was cultured in the presence of 50 international units/mL to 6000 international units/mL of IL-2 and in the absence of IL-4 and IL-15 for 3 to 15 days prior to the culturing in the presence of IL-2, IL-4, and IL-15. The first cell population can be a cell population that was cultured in the presence of 100 international units/mL to 4000 international units/mL of IL-2 and in the absence of IL-4 and IL-15 for 8 to 15 days prior to the culturing in the presence of IL-2, IL-4, and IL-15. The first cell population can be a cell population that was enriched for tumor infiltrating γδ T cells. The first cell population can be a cell population that was enriched for tumor infiltrating γδ T cells via (a) the removal of at least some αβ T cells or (b) the isolation of at least some γδ T cells. The method can comprise removing at least some αβ T cells from a cell population to obtain the first cell population. The removing can comprise positively selecting αβ T cells and removing the positively selected αβ T cells. The method can comprise isolating at least some γδ T cells from a cell population to obtain the first cell population. The isolating can comprise positively selecting γδ T cells and isolating the positively selected γδ T cells. The culturing the first cell population comprising γδ T cells in the presence of IL-2, IL-4, and IL-15 for the 8 to 21 days can comprise culturing the first cell population comprising γδ T cells in the presence of IL-2, IL-4, IL-15, irradiated PBMCs, and an anti-CD3 antibody for the 8 to 21 days. The culturing the first cell population comprising γδ T cells in the presence of IL-2, IL-4, and IL-15 can be for 12 to 16 days. The culturing the first cell population comprising γδ T cells in the presence of IL-2, IL-4, and IL-15 can be for 13 to 15 days. The second cell population can comprise at least 50 times more γδ T cells than the first cell population, at least 100 times more γδ T cells than the first cell population, at least 200 times more γδ T cells than the first cell population, at least 300 times more γδ T cells than the first cell population, or at least 400 times more γδ T cells than the first cell population. The second cell population can comprise greater than 1×108 γδ T cells. The IL-2 can be a human IL-2. The IL-4 can be a human IL-4. The IL-15 can be a human IL-15. Greater than 85 percent of the CD3+ cells the second cell population can be γδ TCR+ cells. Less than 10 percent of the CD3+ cells of the second cell population can be αβ TCR+ cells. Less than 10 percent of the CD45+ cells of the second cell population can be NK cells. Greater than 30 percent of the γδ TCR+ cells of the second cell population can be Vδ1+ cells. Less than 60 percent of the γδ TCR+ cells of the second cell population can be Vδ1−Vδ2− cells. Less than 25 percent of the γδ TCR+ cells of the second cell population can be Vδ2+ cells. Greater than 70 percent of the γδ TCR+ cells of the second cell population can be TEM cells. Less than 25 percent of the γδ TCR+ cells of the second cell population can be TEMRA cells. Less than 10 percent of the γδ TCR+ cells of the second cell population can be CD69+ CD103+ TRM cells. From 1 to 10 percent of the γδ TCR+ cells of the second cell population can be CD69+ CD103+ TRM cells. Less than 50 percent of the γδ TCR+ cells of the second cell population can be CD56+ cells. From 1 to 50 percent of the γδ TCR+ cells of the second cell population can be CD56+ cells. From 1 to 40 percent of the γδ TCR+ cells of the second cell population can be CD137+ cells. Less than 25 percent of the γδ TCR+ cells of the second cell population can be PD-1+ cells. From 5 to 40 percent of the γδ TCR+ cells of the second cell population can be BTLA+ cells. Greater than 60 percent of the γδ TCR+ cells of the second cell population can be NKG2D+ cells. Greater than 20 percent of the γδ TCR+ cells of the second cell population can be NKp46+ cells.
In another aspect, this document features an isolated cell population comprising (or consisting essentially of or consisting of) polyclonal γδ T cells, wherein the population comprises greater than 1×108 γδ T cells. Greater than 85 percent of the CD3+ cells the cell population can be γδ TCR+ cells. Less than 10 percent of the CD3+ cells of the cell population can be αβ TCR+ cells. Less than 10 percent of the CD45+ cells of the cell population can be NK cells. Greater than 30 percent of the γδ TCR+ cells of the cell population can be Vδ1+ cells. Less than 60 percent of the γδ TCR+ cells of the cell population can be Vδ1−Vδ2− cells. Less than 25 percent of the γδ TCR+ cells of the cell population can be Vδ2+ cells. Greater than 70 percent of the γδ TCR+ cells of the cell population can be TEM cells. Less than 25 percent of the γδ TCR+ cells of the cell population can be TEMRA cells. Less than 10 percent of the γδ TCR+ cells of the cell population can be CD69+ CD103+ TRM cells. From 1 to 10 percent of the γδ TCR+ cells of the cell population can be CD69+ CD103+ TRM cells. Less than 50 percent of the γδ TCR+ cells of the cell population can be CD56+ cells. From 1 to 50 percent of the γδ TCR+ cells of the cell population can be CD56+ cells. From 1 to 40 percent of the γδ TCR+ cells of the cell population can be CD137+ cells. Less than 25 percent of the γδ TCR+ cells of the cell population can be PD-1+ cells. From 5 to 40 percent of the γδ TCR+ cells of the cell population can be BTLA+ cells. Greater than 60 percent of the γδ TCR+ cells of the cell population can be NKG2D+ cells. Greater than 20 percent of the γδ TCR+ cells of the cell population can be NKp46+ cells. The cells of the cell population can be human cells. The γδ T cells can be tumor infiltrating γδ T cells. The cell population can be a cell population that was produced using a method for producing a cell population comprising γδ T cells as described in any statement or combination of statements from the following paragraph.
The method can comprise (or can consist essentially of or can consist of) culturing a first cell population comprising γδ T cells in the presence of IL-2, IL-4, and IL-15 for 8 to 21 days to obtain a second cell population, wherein the second cell population comprises at least 10 times more γδ T cells than the first cell population. The γδ T cells can be human cells. The γδ T cells can be tumor infiltrating γδ T cells. The first cell population can be (i) a population of tumor infiltrating γδ T cells obtained from (a) tissue comprising a tumor or (b) healthy tissue that was within 30 mm of a tumor, (ii) a population of γδ T cells obtained from healthy tissue, (iii) a population of γδ T cells obtained from infected tissue, or (iv) a population of γδ T cells obtained from tissue harboring autoimmune T cells. The method can comprise obtaining the first cell population from the tissue comprising the tumor. The method can comprise obtaining the first cell population from the healthy tissue that was within 30 mm of the tumor. The first cell population can be a cell population that was cultured in the presence of 50 international units/mL to 6000 international units/mL of IL-2 and in the absence of IL-4 and IL-15 for 3 to 15 days prior to the culturing in the presence of IL-2, IL-4, and IL-15. The first cell population can be a cell population that was cultured in the presence of 100 international units/mL to 4000 international units/mL of IL-2 and in the absence of IL-4 and IL-15 for 8 to 15 days prior to the culturing in the presence of IL-2, IL-4, and IL-15. The first cell population can be a cell population that was enriched for tumor infiltrating γδ T cells. The first cell population can be a cell population that was enriched for tumor infiltrating γδ T cells via (a) the removal of at least some αβ T cells or (b) the isolation of at least some γδ T cells. The method can comprise removing at least some αβ T cells from a cell population to obtain the first cell population. The removing can comprise positively selecting αβ T cells and removing the positively selected αβ T cells. The method can comprise isolating at least some γδ T cells from a cell population to obtain the first cell population. The isolating can comprise positively selecting γδ T cells and isolating the positively selected γδ T cells. The culturing the first cell population comprising γδ T cells in the presence of IL-2, IL-4, and IL-15 for the 8 to 21 days can comprise culturing the first cell population comprising γδ T cells in the presence of IL-2, IL-4, IL-15, irradiated PBMCs, and an anti-CD3 antibody for the 8 to 21 days. The culturing the first cell population comprising γδ T cells in the presence of IL-2, IL-4, and IL-15 can be for 12 to 16 days. The culturing the first cell population comprising γδ T cells in the presence of IL-2, IL-4, and IL-15 can be for 13 to 15 days. The second cell population can comprise at least 50 times more γδ T cells than the first cell population, at least 100 times more γδ T cells than the first cell population, at least 200 times more γδ T cells than the first cell population, at least 300 times more γδ T cells than the first cell population, or at least 400 times more γδ T cells than the first cell population. The second cell population can comprise greater than 1×108 γδ T cells. The IL-2 can be a human IL-2. The IL-4 can be a human IL-4. The IL-15 can be a human IL-15. Greater than 85 percent of the CD3+ cells the second cell population can be γδ TCR+ cells. Less than 10 percent of the CD3+ cells of the second cell population can be αβ TCR+ cells. Less than 10 percent of the CD45+ cells of the second cell population can be NK cells. Greater than 30 percent of the γδ TCR+ cells of the second cell population can be Vδ1+ cells. Less than 60 percent of the γδ TCR+ cells of the second cell population can be Vδ1−Vδ2− cells. Less than 25 percent of the γδ TCR+ cells of the second cell population can be Vδ2+ cells. Greater than 70 percent of the γδ TCR+ cells of the second cell population can be TEM cells. Less than 25 percent of the γδ TCR+ cells of the second cell population can be TEMRA cells. Less than 10 percent of the γδ TCR+ cells of the second cell population can be CD69+ CD103+ TRM cells. From 1 to 10 percent of the γδ TCR+ cells of the second cell population can be CD69+ CD103+ TRM cells. Less than 50 percent of the γδ TCR+ cells of the second cell population can be CD56+ cells. From 1 to 50 percent of the γδ TCR+ cells of the second cell population can be CD56+ cells. From 1 to 40 percent of the γδ TCR+ cells of the second cell population can be CD137+ cells. Less than 25 percent of the γδ TCR+ cells of the second cell population can be PD-1+ cells. From 5 to 40 percent of the γδ TCR+ cells of the second cell population can be BTLA+ cells. Greater than 60 percent of the γδ TCR+ cells of the second cell population can be NKG2D+ cells. Greater than 20 percent of the γδ TCR+ cells of the second cell population can be NKp46+ cells.
In another aspect, this document features a method for providing a mammal with γδ T cells. The method comprises (or consists essentially of or consists of) administering, to a mammal, a cell population produced as described in any statement or combination of statements from the preceding paragraph. The mammal can be a human. The mammal can be a mammal having cancer. The cells of the first cell population can be allogenic or autologous to the mammal administered the cell population. The method can comprise administering αβ T cells to the mammal.
In another aspect, this document features a method for providing a mammal with γδ T cells. The method comprises (or consists essentially of or consists of) administering a cell population (e.g., an isolated cell population) to a mammal. The mammal can be a human. The mammal can be a mammal having cancer, an autoimmune condition, or an infection. The cells of the cell population can be allogenic or autologous to the mammal. The method can comprise administering αβ T cells to the mammal. The cell population (e.g., isolated cell population) can comprise (or can consist essentially of or can consist of) polyclonal γδ T cells, wherein the population comprises greater than 1×108 γδ T cells. Greater than 85 percent of the CD3+ cells the cell population can be γδ TCR+ cells. Less than 10 percent of the CD3+ cells of the cell population can be αβ TCR+ cells. Less than 10 percent of the CD45+ cells of the cell population can be NK cells. Greater than 30 percent of the γδ TCR+ cells of the cell population can be Vδ1+ cells. Less than 60 percent of the γδ TCR+ cells of the cell population can be Vδ1−Vδ2− cells. Less than 25 percent of the γδ TCR+ cells of the cell population can be Vδ2+ cells. Greater than 70 percent of the γδ TCR+ cells of the cell population can be TEM cells. Less than 25 percent of the γδ TCR+ cells of the cell population can be TEMRA cells. Less than 10 percent of the γδ TCR+ cells of the cell population can be CD69+ CD103+ TRM cells. From 1 to 10 percent of the γδ TCR+ cells of the cell population can be CD69+ CD103+ TRM cells. Less than 50 percent of the γδ TCR+ cells of the cell population can be CD56+ cells. From 1 to 50 percent of the γδ TCR+ cells of the cell population can be CD56+ cells. From 1 to 40 percent of the γδ TCR+ cells of the cell population can be CD137+ cells. Less than 25 percent of the γδ TCR+ cells of the cell population can be PD-1+ cells. From 5 to 40 percent of the γδ TCR+ cells of the cell population can be BTLA+ cells. Greater than 60 percent of the γδ TCR+ cells of the cell population can be NKG2D+ cells. Greater than 20 percent of the γδ TCR+ cells of the cell population can be NKp46+ cells. The cells of the cell population can be human cells. The γδ T cells can be tumor infiltrating γδ T cells. The cell population can be a cell population that was produced using a method for producing a cell population comprising γδ T cells as described in any statement or combination of statements from the following paragraph.
The method can comprise (or can consist essentially of or can consist of) culturing a first cell population comprising γδ T cells in the presence of IL-2, IL-4, and IL-15 for 8 to 21 days to obtain a second cell population, wherein the second cell population comprises at least 10 times more γδ T cells than the first cell population. The γδ T cells can be human cells. The γδ T cells can be tumor infiltrating γδ T cells. The first cell population can be (i) a population of tumor infiltrating γδ T cells obtained from (a) tissue comprising a tumor or (b) healthy tissue that was within 30 mm of a tumor, (ii) a population of γδ T cells obtained from healthy tissue, (iii) a population of γδ T cells obtained from infected tissue, or (iv) a population of γδ T cells obtained from tissue harboring autoimmune T cells. The method can comprise obtaining the first cell population from the tissue comprising the tumor. The method can comprise obtaining the first cell population from the healthy tissue that was within 30 mm of the tumor. The first cell population can be a cell population that was cultured in the presence of 50 international units/mL to 6000 international units/mL of IL-2 and in the absence of IL-4 and IL-15 for 3 to 15 days prior to the culturing in the presence of IL-2, IL-4, and IL-15. The first cell population can be a cell population that was cultured in the presence of 100 international units/mL to 4000 international units/mL of IL-2 and in the absence of IL-4 and IL-15 for 8 to 15 days prior to the culturing in the presence of IL-2, IL-4, and IL-15. The first cell population can be a cell population that was enriched for tumor infiltrating γδ T cells. The first cell population can be a cell population that was enriched for tumor infiltrating γδ T cells via (a) the removal of at least some αβ T cells or (b) the isolation of at least some γδ T cells. The method can comprise removing at least some αβ T cells from a cell population to obtain the first cell population. The removing can comprise positively selecting αβ T cells and removing the positively selected αβ T cells. The method can comprise isolating at least some γδ T cells from a cell population to obtain the first cell population. The isolating can comprise positively selecting γδ T cells and isolating the positively selected γδ T cells. The culturing the first cell population comprising γδ T cells in the presence of IL-2, IL-4, and IL-15 for the 8 to 21 days can comprise culturing the first cell population comprising γδ T cells in the presence of IL-2, IL-4, IL-15, irradiated PBMCs, and an anti-CD3 antibody for the 8 to 21 days. The culturing the first cell population comprising γδ T cells in the presence of IL-2, IL-4, and IL-15 can be for 12 to 16 days. The culturing the first cell population comprising γδ T cells in the presence of IL-2, IL-4, and IL-15 can be for 13 to 15 days. The second cell population can comprise at least 50 times more γδ T cells than the first cell population, at least 100 times more γδ T cells than the first cell population, at least 200 times more γδ T cells than the first cell population, at least 300 times more γδ T cells than the first cell population, or at least 400 times more γδ T cells than the first cell population. The second cell population can comprise greater than 1×108 γδ T cells. The IL-2 can be a human IL-2. The IL-4 can be a human IL-4. The IL-15 can be a human IL-15. Greater than 85 percent of the CD3+ cells the second cell population can be γδ TCR+ cells. Less than 10 percent of the CD3+ cells of the second cell population can be αβ TCR+ cells. Less than 10 percent of the CD45+ cells of the second cell population can be NK cells. Greater than 30 percent of the γδ TCR+ cells of the second cell population can be Vδ1+ cells. Less than 60 percent of the γδ TCR+ cells of the second cell population can be Vδ1−Vδ2− cells. Less than 25 percent of the γδ TCR+ cells of the second cell population can be Vδ2+ cells. Greater than 70 percent of the γδ TCR+ cells of the second cell population can be TEM cells. Less than 25 percent of the γδ TCR+ cells of the second cell population can be TEMRA cells. Less than 10 percent of the γδ TCR+ cells of the second cell population can be CD69+CD103+ TRM cells. From 1 to 10 percent of the γδ TCR+ cells of the second cell population can be CD69+ CD103+ TRM cells. Less than 50 percent of the γδ TCR+ cells of the second cell population can be CD56+ cells. From 1 to 50 percent of the γδ TCR+ cells of the second cell population can be CD56+ cells. From 1 to 40 percent of the γδ TCR+ cells of the second cell population can be CD137+ cells. Less than 25 percent of the γδ TCR+ cells of the second cell population can be PD-1+ cells. From 5 to 40 percent of the γδ TCR+ cells of the second cell population can be BTLA+ cells. Greater than 60 percent of the γδ TCR+ cells of the second cell population can be NKG2D+ cells. Greater than 20 percent of the γδ TCR+ cells of the second cell population can be NKp46+ cells.
In another aspect, this document features a method for treating cancer. The method comprises (consists essentially of or consists of) administering, to a mammal having cancer, a cell population produced as described in any statement or combination of statements from the preceding paragraph. The mammal can be a human. The cells of the first cell population can be allogenic or autologous to the mammal having cancer. The method can comprise administering αβ T cells to the mammal.
In another aspect, this document features a method for treating cancer. The method comprises (consists essentially of or consists of) administering a cell population (e.g., an isolated cell population) to a mammal having cancer. The mammal can be a human. The cells of the cell population can be allogenic or autologous to the mammal having cancer. The method can comprise administering αβ T cells to the mammal. The cell population (e.g., isolated cell population) can comprise (or can consist essentially of or can consist of) polyclonal γδ T cells, wherein the population comprises greater than 1×108 γδ T cells. Greater than 85 percent of the CD3+ cells the cell population can be γδ TCR+ cells. Less than 10 percent of the CD3+ cells of the cell population can be αβ TCR+ cells. Less than 10 percent of the CD45+ cells of the cell population can be NK cells. Greater than 30 percent of the γδ TCR+ cells of the cell population can be Vδ1+ cells. Less than 60 percent of the γδ TCR+ cells of the cell population can be Vδ1−Vδ2− cells. Less than 25 percent of the γδ TCR+ cells of the cell population can be Vδ2+ cells. Greater than 70 percent of the γδ TCR+ cells of the cell population can be TEM cells. Less than 25 percent of the γδ TCR+ cells of the cell population can be TEMRA cells. Less than 10 percent of the γδ TCR+ cells of the cell population can be CD69+ CD103+ TRM cells. From 1 to 10 percent of the γδ TCR+ cells of the cell population can be CD69+ CD103+ TRM cells. Less than 50 percent of the γδ TCR+ cells of the cell population can be CD56+ cells. From 1 to 50 percent of the γδ TCR+ cells of the cell population can be CD56+ cells. From 1 to 40 percent of the γδ TCR+ cells of the cell population can be CD137+ cells. Less than 25 percent of the γδ TCR+ cells of the cell population can be PD-1+ cells. From 5 to 40 percent of the γδ TCR+ cells of the cell population can be BTLA+ cells. Greater than 60 percent of the γδ TCR+ cells of the cell population can be NKG2D+ cells. Greater than 20 percent of the γδ TCR+ cells of the cell population can be NKp46+ cells. The cells of the cell population can be human cells. The γδ T cells can be tumor infiltrating γδ T cells. The cell population can be a cell population that was produced using a method for producing a cell population comprising γδ T cells as described in any statement or combination of statements from the following paragraph.
The method can comprise (or can consist essentially of or can consist of) culturing a first cell population comprising γδ T cells in the presence of IL-2, IL-4, and IL-15 for 8 to 21 days to obtain a second cell population, wherein the second cell population comprises at least 10 times more γδ T cells than the first cell population. The γδ T cells can be human cells. The γδ T cells can be tumor infiltrating γδ T cells. The first cell population can be (i) a population of tumor infiltrating γδ T cells obtained from (a) tissue comprising a tumor or (b) healthy tissue that was within 30 mm of a tumor, (ii) a population of γδ T cells obtained from healthy tissue, (iii) a population of γδ T cells obtained from infected tissue, or (iv) a population of γδ T cells obtained from tissue harboring autoimmune T cells. The method can comprise obtaining the first cell population from the tissue comprising the tumor. The method can comprise obtaining the first cell population from the healthy tissue that was within 30 mm of the tumor. The first cell population can be a cell population that was cultured in the presence of 50 international units/mL to 6000 international units/mL of IL-2 and in the absence of IL-4 and IL-15 for 3 to 15 days prior to the culturing in the presence of IL-2, IL-4, and IL-15. The first cell population can be a cell population that was cultured in the presence of 100 international units/mL to 4000 international units/mL of IL-2 and in the absence of IL-4 and IL-15 for 8 to 15 days prior to the culturing in the presence of IL-2, IL-4, and IL-15. The first cell population can be a cell population that was enriched for tumor infiltrating γδ T cells. The first cell population can be a cell population that was enriched for tumor infiltrating γδ T cells via (a) the removal of at least some αβ T cells or (b) the isolation of at least some γδ T cells. The method can comprise removing at least some αβ T cells from a cell population to obtain the first cell population. The removing can comprise positively selecting αβ T cells and removing the positively selected αβ T cells. The method can comprise isolating at least some γδ T cells from a cell population to obtain the first cell population. The isolating can comprise positively selecting γδ T cells and isolating the positively selected γδ T cells. The culturing the first cell population comprising γδ T cells in the presence of IL-2, IL-4, and IL-15 for the 8 to 21 days can comprise culturing the first cell population comprising γδ T cells in the presence of IL-2, IL-4, IL-15, irradiated PBMCs, and an anti-CD3 antibody for the 8 to 21 days. The culturing the first cell population comprising γδ T cells in the presence of IL-2, IL-4, and IL-15 can be for 12 to 16 days. The culturing the first cell population comprising γδ T cells in the presence of IL-2, IL-4, and IL-15 can be for 13 to 15 days. The second cell population can comprise at least 50 times more γδ T cells than the first cell population, at least 100 times more γδ T cells than the first cell population, at least 200 times more γδ T cells than the first cell population, at least 300 times more γδ T cells than the first cell population, or at least 400 times more γδ T cells than the first cell population. The second cell population can comprise greater than 1×108 γδ T cells. The IL-2 can be a human IL-2. The IL-4 can be a human IL-4. The IL-15 can be a human IL-15. Greater than 85 percent of the CD3+ cells the second cell population can be γδ TCR+ cells. Less than 10 percent of the CD3+ cells of the second cell population can be αβ TCR+ cells. Less than 10 percent of the CD45+ cells of the second cell population can be NK cells. Greater than 30 percent of the γδ TCR+ cells of the second cell population can be Vδ1+ cells. Less than 60 percent of the γδ TCR+ cells of the second cell population can be Vδ1−Vδ2− cells. Less than 25 percent of the γδ TCR+ cells of the second cell population can be Vδ2+ cells. Greater than 70 percent of the γδ TCR+ cells of the second cell population can be TEM cells. Less than 25 percent of the γδ TCR+ cells of the second cell population can be TEMRA cells. Less than 10 percent of the γδ TCR+ cells of the second cell population can be CD69+CD103+ TRM cells. From 1 to 10 percent of the γδ TCR+ cells of the second cell population can be CD69+ CD103+ TRM cells. Less than 50 percent of the γδ TCR+ cells of the second cell population can be CD56+ cells. From 1 to 50 percent of the γδ TCR+ cells of the second cell population can be CD56+ cells. From 1 to 40 percent of the γδ TCR+ cells of the second cell population can be CD137+ cells. Less than 25 percent of the γδ TCR+ cells of the second cell population can be PD-1+ cells. From 5 to 40 percent of the γδ TCR+ cells of the second cell population can be BTLA+ cells. Greater than 60 percent of the γδ TCR+ cells of the second cell population can be NKG2D+ cells. Greater than 20 percent of the γδ TCR+ cells of the second cell population can be NKp46+ cells.
In another aspect, this document features a method for treating an autoimmune condition. The method comprises (consists essentially of or consists of) administering a cell population (e.g., an isolated cell population) to a mammal having an autoimmune condition. The mammal can be a human. The cells of the cell population can be allogenic or autologous to the mammal having the autoimmune condition. The method can comprise administering αβ T cells to the mammal. The cell population (e.g., isolated cell population) can comprise (or can consist essentially of or can consist of) polyclonal γδ T cells, wherein the population comprises greater than 1×108 γδ T cells. Greater than 85 percent of the CD3+ cells the cell population can be γδ TCR+ cells. Less than 10 percent of the CD3+ cells of the cell population can be αβ TCR+ cells. Less than 10 percent of the CD45+ cells of the cell population can be NK cells. Greater than 30 percent of the γδ TCR+ cells of the cell population can be Vδ1+ cells. Less than 60 percent of the γδ TCR+ cells of the cell population can be Vδ1−Vδ2− cells. Less than 25 percent of the γδ TCR+ cells of the cell population can be Vδ2+ cells. Greater than 70 percent of the γδ TCR+ cells of the cell population can be TEM cells. Less than 25 percent of the γδ TCR+ cells of the cell population can be TEMRA cells. Less than 10 percent of the γδ TCR+ cells of the cell population can be CD69+ CD103+ TRM cells. From 1 to 10 percent of the γδ TCR+ cells of the cell population can be CD69+ CD103+ TRM cells. Less than 50 percent of the γδ TCR+ cells of the cell population can be CD56+ cells. From 1 to 50 percent of the γδ TCR+ cells of the cell population can be CD56+ cells. From 1 to 40 percent of the γδ TCR+ cells of the cell population can be CD137+ cells. Less than 25 percent of the γδ TCR+ cells of the cell population can be PD-1+ cells. From 5 to 40 percent of the γδ TCR+ cells of the cell population can be BTLA+ cells. Greater than 60 percent of the γδ TCR+ cells of the cell population can be NKG2D+ cells. Greater than 20 percent of the γδ TCR+ cells of the cell population can be NKp46+ cells. The cells of the cell population can be human cells. The γδ T cells can be tumor infiltrating γδ T cells. The cell population can be a cell population that was produced using a method for producing a cell population comprising γδ T cells as described in any statement or combination of statements from the following paragraph.
The method can comprise (or can consist essentially of or can consist of) culturing a first cell population comprising γδ T cells in the presence of IL-2, IL-4, and IL-15 for 8 to 21 days to obtain a second cell population, wherein the second cell population comprises at least 10 times more γδ T cells than the first cell population. The γδ T cells can be human cells. The γδ T cells can be tumor infiltrating γδ T cells. The first cell population can be (i) a population of tumor infiltrating γδ T cells obtained from (a) tissue comprising a tumor or (b) healthy tissue that was within 30 mm of a tumor, (ii) a population of γδ T cells obtained from healthy tissue, (iii) a population of γδ T cells obtained from infected tissue, or (iv) a population of γδ T cells obtained from tissue harboring autoimmune T cells. The method can comprise obtaining the first cell population from the tissue comprising the tumor. The method can comprise obtaining the first cell population from the healthy tissue that was within 30 mm of the tumor. The first cell population can be a cell population that was cultured in the presence of 50 international units/mL to 6000 international units/mL of IL-2 and in the absence of IL-4 and IL-15 for 3 to 15 days prior to the culturing in the presence of IL-2, IL-4, and IL-15. The first cell population can be a cell population that was cultured in the presence of 100 international units/mL to 4000 international units/mL of IL-2 and in the absence of IL-4 and IL-15 for 8 to 15 days prior to the culturing in the presence of IL-2, IL-4, and IL-15. The first cell population can be a cell population that was enriched for tumor infiltrating γδ T cells. The first cell population can be a cell population that was enriched for tumor infiltrating γδ T cells via (a) the removal of at least some αβ T cells or (b) the isolation of at least some γδ T cells. The method can comprise removing at least some αβ T cells from a cell population to obtain the first cell population. The removing can comprise positively selecting αβ T cells and removing the positively selected αβ T cells. The method can comprise isolating at least some γδ T cells from a cell population to obtain the first cell population. The isolating can comprise positively selecting γδ T cells and isolating the positively selected γδ T cells. The culturing the first cell population comprising γδ T cells in the presence of IL-2, IL-4, and IL-15 for the 8 to 21 days can comprise culturing the first cell population comprising γδ T cells in the presence of IL-2, IL-4, IL-15, irradiated PBMCs, and an anti-CD3 antibody for the 8 to 21 days. The culturing the first cell population comprising γδ T cells in the presence of IL-2, IL-4, and IL-15 can be for 12 to 16 days. The culturing the first cell population comprising γδ T cells in the presence of IL-2, IL-4, and IL-15 can be for 13 to 15 days. The second cell population can comprise at least 50 times more γδ T cells than the first cell population, at least 100 times more γδ T cells than the first cell population, at least 200 times more γδ T cells than the first cell population, at least 300 times more γδ T cells than the first cell population, or at least 400 times more γδ T cells than the first cell population. The second cell population can comprise greater than 1×108 γδ T cells. The IL-2 can be a human IL-2. The IL-4 can be a human IL-4. The IL-15 can be a human IL-15. Greater than 85 percent of the CD3+ cells the second cell population can be γδ TCR+ cells. Less than 10 percent of the CD3+ cells of the second cell population can be αβ TCR+ cells. Less than 10 percent of the CD45+ cells of the second cell population can be NK cells. Greater than 30 percent of the γδ TCR+ cells of the second cell population can be Vδ1+ cells. Less than 60 percent of the γδ TCR+ cells of the second cell population can be Vδ1−Vδ2− cells. Less than 25 percent of the γδ TCR+ cells of the second cell population can be Vδ2+ cells. Greater than 70 percent of the γδ TCR+ cells of the second cell population can be TEM cells. Less than 25 percent of the γδ TCR+ cells of the second cell population can be TEMRA cells. Less than 10 percent of the γδ TCR+ cells of the second cell population can be CD69+CD103+ TRM cells. From 1 to 10 percent of the γδ TCR+ cells of the second cell population can be CD69+ CD103+ TRM cells. Less than 50 percent of the γδ TCR+ cells of the second cell population can be CD56+ cells. From 1 to 50 percent of the γδ TCR+ cells of the second cell population can be CD56+ cells. From 1 to 40 percent of the γδ TCR+ cells of the second cell population can be CD137+ cells. Less than 25 percent of the γδ TCR+ cells of the second cell population can be PD-1+ cells. From 5 to 40 percent of the γδ TCR+ cells of the second cell population can be BTLA+ cells. Greater than 60 percent of the γδ TCR+ cells of the second cell population can be NKG2D+ cells. Greater than 20 percent of the γδ TCR+ cells of the second cell population can be NKp46+ cells.
In another aspect, this document features a method for treating an infection. The method comprises (consists essentially of or consists of) administering a cell population (e.g., an isolated cell population) to a mammal having an infection. The mammal can be a human. The cells of the cell population can be allogenic or autologous to the mammal having the infection. The method can comprise administering αβ T cells to the mammal. The cell population (e.g., isolated cell population) can comprise (or can consist essentially of or can consist of) polyclonal γδ T cells, wherein the population comprises greater than 1×108 γδ T cells. Greater than 85 percent of the CD3+ cells the cell population can be γδ TCR+ cells. Less than 10 percent of the CD3+ cells of the cell population can be αβ TCR+ cells. Less than 10 percent of the CD45+ cells of the cell population can be NK cells. Greater than 30 percent of the γδ TCR+ cells of the cell population can be Vδ1+ cells. Less than 60 percent of the γδ TCR+ cells of the cell population can be Vδ1−Vδ2− cells. Less than 25 percent of the γδ TCR+ cells of the cell population can be Vδ2+ cells. Greater than 70 percent of the γδ TCR+ cells of the cell population can be TEM cells. Less than 25 percent of the γδ TCR+ cells of the cell population can be TEMRA cells. Less than 10 percent of the γδ TCR+ cells of the cell population can be CD69+ CD103+ TRM cells. From 1 to 10 percent of the γδ TCR+ cells of the cell population can be CD69+ CD103+ TRM cells. Less than 50 percent of the γδ TCR+ cells of the cell population can be CD56+ cells. From 1 to 50 percent of the γδ TCR+ cells of the cell population can be CD56+ cells. From 1 to 40 percent of the γδ TCR+ cells of the cell population can be CD137+ cells. Less than 25 percent of the γδ TCR+ cells of the cell population can be PD-1+ cells. From 5 to 40 percent of the γδ TCR+ cells of the cell population can be BTLA+ cells. Greater than 60 percent of the γδ TCR+ cells of the cell population can be NKG2D+ cells. Greater than 20 percent of the γδ TCR+ cells of the cell population can be NKp46+ cells. The cells of the cell population can be human cells. The γδ T cells can be tumor infiltrating γδ T cells. The cell population can be a cell population that was produced using a method for producing a cell population comprising γδ T cells as described in any statement or combination of statements from the following paragraph.
The method can comprise (or can consist essentially of or can consist of) culturing a first cell population comprising γδ T cells in the presence of IL-2, IL-4, and IL-15 for 8 to 21 days to obtain a second cell population, wherein the second cell population comprises at least 10 times more γδ T cells than the first cell population. The γδ T cells can be human cells. The γδ T cells can be tumor infiltrating γδ T cells. The first cell population can be (i) a population of tumor infiltrating γδ T cells obtained from (a) tissue comprising a tumor or (b) healthy tissue that was within 30 mm of a tumor, (ii) a population of γδ T cells obtained from healthy tissue, (iii) a population of γδ T cells obtained from infected tissue, or (iv) a population of γδ T cells obtained from tissue harboring autoimmune T cells. The method can comprise obtaining the first cell population from the tissue comprising the tumor. The method can comprise obtaining the first cell population from the healthy tissue that was within 30 mm of the tumor. The first cell population can be a cell population that was cultured in the presence of 50 international units/mL to 6000 international units/mL of IL-2 and in the absence of IL-4 and IL-15 for 3 to 15 days prior to the culturing in the presence of IL-2, IL-4, and IL-15. The first cell population can be a cell population that was cultured in the presence of 100 international units/mL to 4000 international units/mL of IL-2 and in the absence of IL-4 and IL-15 for 8 to 15 days prior to the culturing in the presence of IL-2, IL-4, and IL-15. The first cell population can be a cell population that was enriched for tumor infiltrating γδ T cells. The first cell population can be a cell population that was enriched for tumor infiltrating γδ T cells via (a) the removal of at least some αβ T cells or (b) the isolation of at least some γδ T cells. The method can comprise removing at least some αβ T cells from a cell population to obtain the first cell population. The removing can comprise positively selecting αβ T cells and removing the positively selected αβ T cells. The method can comprise isolating at least some γδ T cells from a cell population to obtain the first cell population. The isolating can comprise positively selecting γδ T cells and isolating the positively selected γδ T cells. The culturing the first cell population comprising γδ T cells in the presence of IL-2, IL-4, and IL-15 for the 8 to 21 days can comprise culturing the first cell population comprising γδ T cells in the presence of IL-2, IL-4, IL-15, irradiated PBMCs, and an anti-CD3 antibody for the 8 to 21 days. The culturing the first cell population comprising γδ T cells in the presence of IL-2, IL-4, and IL-15 can be for 12 to 16 days. The culturing the first cell population comprising γδ T cells in the presence of IL-2, IL-4, and IL-15 can be for 13 to 15 days. The second cell population can comprise at least 50 times more γδ T cells than the first cell population, at least 100 times more γδ T cells than the first cell population, at least 200 times more γδ T cells than the first cell population, at least 300 times more γδ T cells than the first cell population, or at least 400 times more γδ T cells than the first cell population. The second cell population can comprise greater than 1×108 γδ T cells. The IL-2 can be a human IL-2. The IL-4 can be a human IL-4. The IL-15 can be a human IL-15. Greater than 85 percent of the CD3+ cells the second cell population can be γδ TCR+ cells. Less than 10 percent of the CD3+ cells of the second cell population can be αβ TCR+ cells. Less than 10 percent of the CD45+ cells of the second cell population can be NK cells. Greater than 30 percent of the γδ TCR+ cells of the second cell population can be Vδ1+ cells. Less than 60 percent of the γδ TCR+ cells of the second cell population can be Vδ1−Vδ2− cells. Less than 25 percent of the γδ TCR+ cells of the second cell population can be Vδ2+ cells. Greater than 70 percent of the γδ TCR+ cells of the second cell population can be TEM cells. Less than 25 percent of the γδ TCR+ cells of the second cell population can be TEMRA cells. Less than 10 percent of the γδ TCR+ cells of the second cell population can be CD69+CD103+ TRM cells. From 1 to 10 percent of the γδ TCR+ cells of the second cell population can be CD69+ CD103+ TRM cells. Less than 50 percent of the γδ TCR+ cells of the second cell population can be CD56+ cells. From 1 to 50 percent of the γδ TCR+ cells of the second cell population can be CD56+ cells. From 1 to 40 percent of the γδ TCR+ cells of the second cell population can be CD137+ cells. Less than 25 percent of the γδ TCR+ cells of the second cell population can be PD-1+ cells. From 5 to 40 percent of the γδ TCR+ cells of the second cell population can be BTLA+ cells. Greater than 60 percent of the γδ TCR+ cells of the second cell population can be NKG2D+ cells. Greater than 20 percent of the γδ TCR+ cells of the second cell population can be NKp46+ cells.
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 pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
Other features and advantages of the invention will be apparent from the following detailed description and from the claims.
This document provides methods and materials for expanding tumor infiltrating γδ T cells (e.g., tumor infiltrating γδ T cells) in culture. For example, this document provides methods and materials for expanding tumor infiltrating γδ T cells obtained from tissue (e.g., a tumor sample) to obtain large numbers (e.g., greater than 1×107, greater than 1×108, greater than 5×108, or greater than 1×109) of tumor infiltrating γδ T cells (e.g., tumor infiltrating γδ T cells that are predominantly Vδ1+) than can be permissible for therapeutic use.
As described herein, tissue containing a tumor (or healthy tissue that is within 30 mm of a tumor, or healthy tissue that is within 20 mm of a tumor, or healthy tissue that is within 10 mm of a tumor) can contain tumor infiltrating γδ T cells and can be obtained from a mammal (e.g., a human cancer patient). In some cases, one or more lymph nodes adjacent to a tumor and/or one or more tumor draining lymph nodes can contain tumor infiltrating γδ T cells and can be obtained from a mammal (e.g., a human cancer patient). For example, lung tissue containing a lung tumor (or healthy lung tissue that is within 30 mm (e.g., within 20 mm or within 10 mm) of a lung tumor or a tumor draining lymph node of a lung tumor) can be obtained from a mammal (e.g., a human lung cancer patient) and used as a source of tumor infiltrating γδ T cells. In another example, skin tissue containing a skin tumor (or healthy skin tissue that is within 30 mm (e.g., within 20 mm or within 10 mm) of a skin tumor or a tumor draining lymph node of a skin tumor) can be obtained from a mammal (e.g., a human skin cancer patient) and used as a source of tumor infiltrating γδ T cells. Other examples of tissues that can be obtained and used as described herein include, without limitation, tissue containing a glioblastoma (or healthy tissue within 30 mm (e.g., within 20 mm or within 10 mm) of a glioblastoma), tissue containing a head & neck squamous cell carcinoma (or healthy tissue within 30 mm (e.g., within 20 mm or within 10 mm) of a head & neck squamous cell carcinoma), tissue containing a cutaneous melanoma (or healthy tissue within 30 mm (e.g., within 20 mm or within 10 mm) of a cutaneous melanoma), tissue containing a lung adenocarcinoma (or healthy tissue within 30 mm (e.g., within 20 mm or within 10 mm) of a lung adenocarcinoma), tissue containing a lung squamous cell carcinoma (or healthy tissue within 30 mm (e.g., within 20 mm or within 10 mm) of a lung squamous cell carcinoma), tissue containing a breast carcinoma (or healthy tissue within 30 mm (e.g., within 20 mm or within 10 mm) of a breast carcinoma), tissue containing a mesothelioma (or healthy tissue within 30 mm (e.g., within 20 mm or within 10 mm) of a mesothelioma), tissue containing a liver hepatocellular carcinoma (or healthy tissue within 30 mm (e.g., within 20 mm or within 10 mm) of a liver hepatocellular carcinoma), tissue containing a pancreatic ductal adenocarcinoma (or healthy tissue within 30 mm (e.g., within 20 mm or within 10 mm) of a pancreatic ductal adenocarcinoma), tissue containing a kidney renal cell carcinoma (or healthy tissue within 30 mm (e.g., within 20 mm or within 10 mm) of a kidney renal cell carcinoma), tissue containing a bladder urothelial carcinoma (or healthy tissue within 30 mm (e.g., within 20 mm or within 10 mm) of a bladder urothelial carcinoma), tissue containing a cervical squamous cell carcinoma and endocervical adenocarcinoma (or healthy tissue within 30 mm (e.g., within 20 mm or within 10 mm) of a cervical squamous cell carcinoma and endocervical adenocarcinoma), tissue containing a lymph node metastases, tissue containing a peritoneum tumor (or healthy tissue within 30 mm (e.g., within 20 mm or within 10 mm) of a peritoneum tumor), tissue containing a bone tumor (or healthy tissue within 30 mm (e.g., within 20 mm or within 10 mm) of a bone tumor), tissue containing an endocrine gland tumor (or healthy tissue within 30 mm (e.g., within 20 mm or within 10 mm) of an endocrine gland tumor), tissue containing a reproductive organ tumor (or healthy tissue within 30 mm (e.g., within 20 mm or within 10 mm) of a reproductive organ tumor), or tissue containing a brain (or healthy tissue within 30 mm (e.g., within 20 mm or within 10 mm) of a brain tumor).
Once a tissue is obtained, the tissue can be cultured in a manner that promotes the isolation of tumor infiltrating γδ T cells from the tissue. For example, one or more pieces (e.g., 2-3 mm3 pieces) of the tissue can be cultured in the presence of IL-2 for 5 to 15 days (e.g., 6 to 15 days, 7 to 15 days, 8 to 15 days, 9 to 15 days, 9 to 13 days, 10 to 12 days, 7 to 10 days, or 8 to 14 days). In some cases, the one or more pieces (e.g., 2-3 mm3 pieces) of the tissue can be cultured in a gas permeable rapid expansion flask. Any appropriate concentration of IL-2 can be used to promote the isolation of tumor infiltrating γδ T cells from the tissue. For example, from about 50 international units (IU) to about 6000 IU (e.g., from about 100 IU to about 6000 IU, from about 500 IU to about 6000 IU, from about 1000 IU to about 6000 IU, from about 1500 IU to about 6000 IU, from about 2000 IU to about 6000 IU, from about 2500 IU to about 6000 IU, from about 3000 IU to about 6000 IU, from about 3500 IU to about 6000 IU, from about 2500 IU to about 4000 IU, or from about 2500 IU to about 3500 IU) of IL-2 per mL of culture medium can be used.
In some cases, tissue (e.g., tumor tissue) can be mechanically and/or enzymatically digested, and a single cell tumor digest suspension can be cultured for a period of time or γδ T cells can be directly isolated at this time.
After culturing tissue containing tumor infiltrating γδ T cells with IL-2 for 5 to 15 days (e.g., 6 to 15 days, 7 to 15 days, 8 to 15 days, 9 to 15 days, 9 to 13 days, 10 to 12 days, 7 to 10 days, or 8 to 14 days), a cell population that exited the tissue can be harvested. In some cases, the harvested cell population can include tumor infiltrating αβ T cells and tumor infiltrating γδ T cells. In some cases, the harvested cell population can include a greater number of tumor infiltrating up T cells than the number of tumor infiltrating γδ T cells. In some cases, an anti-αβ TCR antibody, an anti-CD28 antibody, an anti-4-1BBL antibody, an anti-GITR antibody, an anti-CD27 antibody, or a combination thereof can be used to promote a cell population that is enriched for γδ T cells from the harvested cell population.
Once the harvested cell population is obtained, an optional enrichment for γδ T cells can be performed. For example, magnetic beads containing an anti-αβ TCR antibody can be used in a negative selection process to remove αβ T cells from the harvested cell population to obtain a cell population enriched for γδ T cells. In some cases, an anti-TCR γδ antibody, an anti-Vδ1 antibody, an anti-NKG2D antibody, or a combination thereof can be used in a positive selection process to isolate γδ T cells from the harvested cell population to obtain a cell population enriched for γδ T cells.
Briefly, when using antibodies to remove non-γδ T cells (e.g., αβ T cells) from or to isolate γδ T cells from the harvested cell population to obtain a cell population enriched for γδ T cells, the antibodies can be biotinylated and can be attached to a magnetic substrate (e.g., a magnetic bead) via streptavidin. In some cases, flow activated cell sorting (FACS) can be used to remove non-γδ T cells (e.g., αβ T cells) from or to isolate γδ T cells from the harvested cell population to obtain a cell population enriched for γδ T cells.
In some cases, the harvested cell population (or a portion thereof) can be used for expanding the number of γδ T cells without the optional enrichment step.
Once the harvested cell population (with or without the optional enrichment for γδ T cells) is obtained, the harvested cell population (or a portion thereof) can be used to perform an expansion step that increases the number of γδ T cells present. In some cases, this expansion step can increase the starting number of γδ T cells present in the starting cell population to a number of γδ T cells present in the resulting cell population that is from 10 to 1000 fold greater (e.g., 10 to 600 fold, 20 to 600 fold, 30 to 600 fold, 40 to 600 fold, 50 to 600 fold, 75 to 600 fold, 100 to 600 fold, 200 to 1000 fold, 250 to 1000 fold, 300 to 1000 fold, 350 to 1000 fold, 400 to 1000 fold, 450 to 1000 fold, 500 to 1000 fold, 200 to 1000 fold, 250 to 1000 fold, 300 to 51000 00 fold, 350 to 1000 fold, 400 to 1000 fold, or 450 to 1000 fold) greater than that starting number. In some cases, this expansion step can increase the starting number of γδ T cells present in the starting cell population to a number of γδ T cells present in the resulting cell population that is more than 200 fold greater (e.g., more than 250 fold greater, more than 300 fold greater, more than 350 fold greater, more than 400 fold greater, or more than 450 fold greater) than that starting number. In some cases, this expansion step can expand the starting number of γδ T cells present in the starting cell population to a number of γδ T cells present in the resulting cell population that is 200 to 600 fold (e.g., 200 to 600 fold, 250 to 600 fold, 300 to 600 fold, 350 to 600 fold, 400 to 600 fold, 450 to 600 fold, 500 to 600 fold, 200 to 550 fold, 250 to 550 fold, 300 to 550 fold, 350 to 550 fold, 400 to 550 fold, 450 to 550 fold, 500 to 550 fold, 200 to 500 fold, 250 to 500 fold, 300 to 500 fold, 350 to 500 fold, 400 to 500 fold, or 450 to 500 fold) greater than that starting number. In some cases, this expansion step can increase the starting number of γδ T cells present in the starting cell population to a number of γδ T cells present in the resulting cell population that is greater than 25 percent (e.g., greater than 50 percent, greater than 75 percent, or greater than 95 percent) enriched in γδ T cells.
Any appropriate method can be used to promote the expansion of γδ T cells of a harvested cell population (or a portion thereof) or a harvested, γδ T cell-enriched cell population (or a portion thereof). For example, a harvested cell population (with or without the optional enrichment for γδ T cells) or a portion thereof can be cultured in the presence of IL-2, IL-4, and IL-15 to promote the expansion of γδ T cells. The amount of IL-2 can be from about 50 IU to about 6000 IU (e.g., from about 100 IU to about 6000 IU, from about 500 IU to about 6000 IU, from about 1000 IU to about 6000 IU, from about 1500 IU to about 6000 IU, from about 2000 IU to about 6000 IU, from about 2500 IU to about 6000 IU, from about 3000 IU to about 6000 IU, from about 3500 IU to about 6000 IU, from about 2500 IU to about 4000 IU, or from about 2500 IU to about 3500 IU) of IL-2/mL of culture medium. The amount of IL-4 can be from about 10 ng to about 200 ng (e.g., from about 20 ng to about 200 ng, from about 50 ng to about 200 ng, from about 75 ng to about 200 ng, from about 10 ng to about 150 ng, from about 10 ng to about 100 ng, from about 50 ng to about 150 ng, or from about 90 ng to about 110 ng) of IL-4/mL of culture medium. The amount of IL-15 can be from about 10 ng to about 200 ng (e.g., from about 20 ng to about 200 ng, from about 50 ng to about 200 ng, from about 75 ng to about 200 ng, from about 10 ng to about 150 ng, from about 10 ng to about 100 ng, from about 50 ng to about 150 ng, from about 50 ng to about 90 ng, or from about 60 ng to about 90 ng) of IL-15/mL of culture medium.
In some cases, a harvested cell population (with or without the optional enrichment for γδ T cells) or a portion thereof can be cultured in the presence of IL-2, IL-4, and IL-15 with the optional inclusion of IL-7 and/or IL-21. When optionally including IL-7, amount of IL-7 can be from about 10 ng to about 200 ng (e.g., from about 20 ng to about 200 ng, from about 50 ng to about 200 ng, from about 75 ng to about 200 ng, from about 10 ng to about 150 ng, from about 10 ng to about 100 ng, from about 50 ng to about 150 ng, or from about 90 ng to about 110 ng) of IL-7/mL of culture medium. When optionally including IL-21, amount of IL-21 can be from about 10 ng to about 200 ng (e.g., from about 20 ng to about 200 ng, from about 50 ng to about 200 ng, from about 75 ng to about 200 ng, from about 10 ng to about 150 ng, from about 10 ng to about 100 ng, from about 50 ng to about 150 ng, or from about 90 ng to about 110 ng) of IL-21/mL of culture medium.
Any appropriate IL-2, IL-4, and IL-15 (and optionally included IL-7 and/or IL-21) can be used to expand γδ T cells as described herein. For example, when expanding human γδ T cells, then human IL-2, human IL-4, and human IL-15 can be used to expand the human γδ T cells. In another example, when expanding horse γδ T cells, then horse IL-2, horse IL-4, and horse IL-15 can be used to expand the horse γδ T cells. In another example, when expanding monkey γδ T cells, then monkey IL-2, monkey IL-4, and monkey IL-15 can be used to expand the monkey γδ T cells. In another example, when expanding dog γδ T cells, then dog IL-2, dog IL-4, and dog IL-15 can be used to expand the dog γδ T cells.
The harvested cell population (with or without the optional enrichment for γδ T cells) or a portion thereof can be cultured in the presence of IL-2, IL-4, and IL-15 for any appropriate length of time to promote the expansion of γδ T cells. For example, a harvested cell population (with or without the optional enrichment for γδ T cells) or a portion thereof can be culture in the presence of IL-2, IL-4, and IL-15 for 8 to 21 days (e.g., 10 to 21 days, 12 to 21 days, 14 to 21 days, 8 to 18 days, 8 to 16 days, 8 to 14 days, 10 to 20 days, 10 to 18 days, 12 to 18 days, 10 to 16 days, 12 to 16 days, or 13 to 15 days). In some cases, the IL-2, IL-4, and IL-15 in the culture can be replenished every 3 days, every 4-6 days, or every 2-3 days.
In some cases, the culture containing IL-2, IL-4, and IL-15 and being used to expand the number of γδ T cells can contain one or more additional agents. For example, in addition to IL-2, IL-4, and IL-15, the culture can contain anti-CD3 antibodies (e.g., soluble and/or immobilized anti-CD3 antibodies), anti-CD28 antibodies (e.g., soluble and/or immobilized anti-CD28 antibodies), irradiated PBMCs (e.g., irradiated PBMCs that are autologous to the mammal to be treated with the expanded γδ T cells), agonistic anti-γδ TCR antibodies (e.g., soluble and/or immobilized anti-γδ TCR antibodies such as Vδ1 antibodies; about 1 μg/mL; see, e.g., Zhou et al., Cell Mol. Immunol., 9(1):34-44 (2012)), anti-4-1BB antibodies (e.g., soluble and/or immobilized anti-4-1BB antibodies such as Urelumab; about 10 μg/mL; see, e.g., Sakellariou-Thompson et al., Clin. Cancer Res., 23(23):7263-7275 (2017)), anti-TIGIT antibodies (e.g., soluble and/or immobilized anti-TIGIT antibodies; 1 μg/mL; see, e.g., Chauvin et al., J Clin. Invest., 125(5):2046-58 (2015)), high glucose (e.g., from 5 mM to 25 mM, from 8 mM to 20 mM, from 8 mM to 12 mM, or from 9 mM to 11 mM of glucose; see, e.g., Lopes et al., Nat. Immunol., 22:179-192 (2021)), irradiated artificial antigen presenting cells (e.g., cloned K562 cells transfected with 4-1BBL, CD86, IL-15/membrane bound IL-15; 1:100 ratio; see, e.g., Deniger et al., Clin. Cancer Res., 20(22):5708-5719 (2014)), PHA (about 1 μg/mL), irradiated EBV transfected B cell lines (1:100 ratio; see, e.g., Ma et al., J Exp. Med., 208(3):491-503 (2011)), anti-OX40 antibodies (e.g., soluble and/or immobilized anti-OX40 antibodies), phosphoinositide 3-kinase (PI 3-kinase) inhibitors (e.g., Idelalisib, Copanlisib, Duvelisib, Alpelisib, or Umbralisib), CDK4/6 inhibitors (e.g., palbociclib, ribociclib, or abemaciclib; see, e.g., Lelliott et al., Cancer Discov., 11(10):2582-2601 (2021)), CBL-B inhibitors (e.g., NX-0255 or NX-1607; see, e.g., Rountree et al., Cancer Res., Jul. 1, 2021 (81) (13 Supplement):1595), STS1 inhibitors (see, e.g., Hwang et al., Exp. Mole. Med., 52:750-761 (2020)), CISH (see, e.g., Palmer et al., J. Exp. Med., 212(12):2095-2113 (2015)), TET2 (see, e.g., Fraietta et al., Nature, 558(7709):307-312 (2018)), or combinations thereof. For example, in addition to IL-2, IL-4, and IL-15, the culture can contain anti-CD3 antibodies (e.g., soluble anti-CD3 antibodies) and irradiated PBMCs. The amount of anti-CD3 antibodies can be from about 0.1 μg to about 1 μg of anti-CD3 antibodies per mL of culture medium. The amount of anti-CD28 antibodies can be from about 500 ng to about 5 μg of anti-CD28 antibodies per mL of culture medium. The amount of irradiated PBMCs can be based on the number of input γδ T cells such that the ratio of γδ T cells:PBMCs is from about 1:25 to about 1:200 (e.g., 1:100).
After expanding the number of γδ T cells in the presence of IL-2, IL-4, and IL-15, the cells can be washed to remove any particular components of the culture medium. For example, after the expansion step is completed, the resulting cell population can be washed to remove any remaining IL-2, IL-4, IL-15, anti-CD3 antibodies, and/or anti-CD28 antibodies, and/or the expanded γδ T cells can be concentrated. In some cases, after the expansion step, the expanded γδ T cells can be cultured in the absence of IL-2, IL-4, and/or IL-15 for any appropriate length of time. For example, after the rapid expansion step, the population of expanded γδ T cells can be cultured in the absence of IL-2, IL-4, and/or IL-15 for 10 to 75 days (e.g., 10 to 60 days, 10 to 50 days, or 10 to 25 days). In some cases, expanded γδ T cells can be obtained from multiple donors (e.g., multiple humans) and pooled to provide a population of γδ T cells for treating one or more patients (e.g., one or more humans).
As described herein, a cell population containing expanded γδ T cells that results from a γδ T cell expansion in the presence of IL-2, IL-4, and IL-15 as described herein can have a particularly desired make up of cells. For example, in some cases, greater than 85 percent (e.g., greater than 90 percent, greater than 91 percent, greater than 92 percent, greater than 93 percent, greater than 94 percent, greater than 95 percent, greater than 96 percent, greater than 97 percent, greater than 98 percent, or greater than 99 percent) of the CD3+ cells of a population provided herein can be γδ TCR+ cells. In some cases, less than 10 percent (e.g., less than 9 percent, less than 8 percent, less than 7 percent, less than 6 percent, less than 5 percent, less than 4 percent, less than 3 percent, less than 2 percent, or less than 1 percent) of the CD3+ cells of a population provided herein can be αβ TCR+ cells. In some cases, less than 10 percent (e.g., less than 9 percent, less than 8 percent, less than 7 percent, less than 6 percent, less than 5 percent, less than 4 percent, less than 3 percent, less than 2 percent, or less than 1 percent) of the CD45+ cells of a population provided herein can be NK cells.
In some cases, a cell population containing expanded γδ T cells that results from a γδ T cell expansion in the presence of IL-2, IL-4, and IL-15 as described herein can vary and can include not only αβ T cells, but also phenotypic NKT, NK and B cells in various proportions.
In some cases, greater than 85 percent (e.g., greater than 90 percent, greater than 91 percent, greater than 92 percent, greater than 93 percent, greater than 94 percent, greater than 95 percent, greater than 96 percent, greater than 97 percent, greater than 98 percent, or greater than 99 percent) of the CD3+ cells of a population provided herein can be γδ TCR+ cells and less than 10 percent (e.g., less than 9 percent, less than 8 percent, less than 7 percent, less than 6 percent, less than 5 percent, less than 4 percent, less than 3 percent, less than 2 percent, or less than 1 percent) of the CD3+ cells of that population can be αβ TCR+ cells.
In some cases, greater than 85 percent (e.g., greater than 90 percent, greater than 91 percent, greater than 92 percent, greater than 93 percent, greater than 94 percent, greater than 95 percent, greater than 96 percent, greater than 97 percent, greater than 98 percent, or greater than 99 percent) of the CD3+ cells of a population provided herein can be γδ TCR+ cells and less than 10 percent (e.g., less than 9 percent, less than 8 percent, less than 7 percent, less than 6 percent, less than 5 percent, less than 4 percent, less than 3 percent, less than 2 percent, or less than 1 percent) of the CD45+ cells of that population can be NK cells.
In some cases, greater than 85 percent (e.g., greater than 90 percent, greater than 91 percent, greater than 92 percent, greater than 93 percent, greater than 94 percent, greater than 95 percent, greater than 96 percent, greater than 97 percent, greater than 98 percent, or greater than 99 percent) of the CD3+ cells of a population provided herein can be γδ TCR+ cells, less than 10 percent (e.g., less than 9 percent, less than 8 percent, less than 7 percent, less than 6 percent, less than 5 percent, less than 4 percent, less than 3 percent, less than 2 percent, or less than 1 percent) of the CD3+ cells of that population can be αβ TCR+ cells, and less than 10 percent (e.g., less than 9 percent, less than 8 percent, less than 7 percent, less than 6 percent, less than 5 percent, less than 4 percent, less than 3 percent, less than 2 percent, or less than 1 percent) of the CD45+ cells of that population can be NK cells.
In some cases, greater than 30 percent (e.g., greater than 35 percent, greater than 40 percent, greater than 45 percent, greater than 50 percent, greater than 55 percent, greater than 60 percent, greater than 65 percent, greater than 70 percent, greater than 75 percent, greater than 80 percent, greater than 85 percent, greater than 90 percent, or greater than 95 percent) of the γδ TCR+ cells of a population provided herein can be Vδ1+ cells.
In some cases, less than 60 percent (e.g., less than 55 percent, less than 50 percent, less than 45 percent, less than 40 percent, less than 35 percent, less than 30 percent, less than 25 percent, less than 20 percent, less than 15 percent, less than 10 percent, less than 5 percent, or less than 2 percent) of the γδ TCR+ cells of a population provided herein can be VDδ1− Vδ2− cells.
In some cases, less than 25 percent (e.g., less than 20 percent, less than 15 percent, less than 10 percent, less than 5 percent, less than 4 percent, less than 3 percent, less than 2 percent, or less than 1 percent) of the γδ TCR+ cells of a population provided herein can be Vδ2+ cells.
In some cases, greater than 30 percent (e.g., greater than 35 percent, greater than 40 percent, greater than 45 percent, greater than 50 percent, greater than 55 percent, greater than 60 percent, greater than 65 percent, greater than 70 percent, greater than 75 percent, greater than 80 percent, greater than 85 percent, greater than 90 percent, or greater than 95 percent) of the γδ TCR+ cells of a population provided herein can be Vδ1+ cells and less than 60 percent (e.g., less than 55 percent, less than 50 percent, less than 45 percent, less than 40 percent, less than 35 percent, less than 30 percent, less than 25 percent, less than 20 percent, less than 15 percent, less than 10 percent, less than 5 percent, or less than 2 percent) of the γδ TCR+ cells of that population can be VDδ1−Vδ2− cells.
In some cases, greater than 30 percent (e.g., greater than 35 percent, greater than 40 percent, greater than 45 percent, greater than 50 percent, greater than 55 percent, greater than 60 percent, greater than 65 percent, greater than 70 percent, greater than 75 percent, greater than 80 percent, greater than 85 percent, greater than 90 percent, or greater than 95 percent) of the γδ TCR+ cells of a population provided herein can be Vδ1+ cells and less than 25 percent (e.g., less than 20 percent, less than 15 percent, less than 10 percent, less than 5 percent, less than 4 percent, less than 3 percent, less than 2 percent, or less than 1 percent) of the γδ TCR+ cells of that population can be Vδ2+ cells.
In some cases, greater than 30 percent (e.g., greater than 35 percent, greater than 40 percent, greater than 45 percent, greater than 50 percent, greater than 55 percent, greater than 60 percent, greater than 65 percent, greater than 70 percent, greater than 75 percent, greater than 80 percent, greater than 85 percent, greater than 90 percent, or greater than 95 percent) of the γδ TCR+ cells of a population provided herein can be Vδ1+ cells, less than 60 percent (e.g., less than 55 percent, less than 50 percent, less than 45 percent, less than 40 percent, less than 35 percent, less than 30 percent, less than 25 percent, less than 20 percent, less than 15 percent, less than 10 percent, less than 5 percent, or less than 2 percent) of the γδ TCR+ cells of that population can be VDδ1−Vδ2− cells, and less than 25 percent (e.g., less than 20 percent, less than 15 percent, less than 10 percent, less than 5 percent, less than 4 percent, less than 3 percent, less than 2 percent, or less than 1 percent) of the γδ TCR+ cells of that population can be Vδ2+ cells.
In some cases, greater than 70 percent (e.g., greater than 75 percent, greater than 80 percent, greater than 85 percent, greater than 90 percent, or greater than 95 percent) of the γδ TCR+ cells of a population provided herein can be TEM cells.
In some cases, less than 25 percent (e.g., less than 20 percent, less than 15 percent, less than 10 percent, less than 5 percent, less than 4 percent, less than 3 percent, less than 2 percent, or less than 1 percent) of the γδ TCR+ cells of a population provided herein can be TEMRA cells.
In some cases, greater than 70 percent (e.g., greater than 75 percent, greater than 80 percent, greater than 85 percent, greater than 90 percent, or greater than 95 percent) of the γδ TCR+ cells of a population provided herein can be TEM cells and less than 25 percent (e.g., less than 20 percent, less than 15 percent, less than 10 percent, less than 5 percent, less than 4 percent, less than 3 percent, less than 2 percent, or less than 1 percent) of the γδ TCR+ cells of that population can be TEMRA cells.
In some cases, a population provided herein can have a higher percentage (e.g., a percentage that is 2 to 40 percentage points higher, 5 to 40 percentage points higher, 10 to 40 percentage points higher, 15 to 40 percentage points higher, 20 to 40 percentage points higher, 5 to 35 percentage points higher, 5 to 30 percentage points higher, 5 to 25 percentage points higher, 5 to 20 percentage points higher, 5 to 15 percentage points higher, or 5 to 10 percentage points higher) of γδ TCR+ TEM cells following cell expansion in the presence of IL-2, IL-4, and IL-15 than the starting population had before cell expansion in the presence of IL-2, IL-4, and IL-15.
In some cases, a population provided herein can have a lower percentage (e.g., a percentage that is 2 to 30 percentage points lower, 5 to 30 percentage points lower, 10 to 30 percentage points lower, 15 to 30 percentage points lower, 20 to 30 percentage points lower, 5 to 25 percentage points lower, 5 to 20 percentage points lower, 5 to 15 percentage points lower, or 5 to 10 percentage points lower) of γδ TCR+ TEMRA cells following cell expansion in the presence of IL-2, IL-4, and IL-15 than the starting population had before cell expansion in the presence of IL-2, IL-4, and IL-15.
In some cases, a population provided herein can have a higher percentage (e.g., a percentage that is 2 to 40 percentage points higher, 5 to 40 percentage points higher, 10 to 40 percentage points higher, 15 to 40 percentage points higher, 20 to 40 percentage points higher, 5 to 35 percentage points higher, 5 to 30 percentage points higher, 5 to 25 percentage points higher, 5 to 20 percentage points higher, 5 to 15 percentage points higher, or 5 to 10 percentage points higher) of γδ TCR+ TEM cells and a lower percentage (e.g., a percentage that is 2 to 30 percentage points lower, 5 to 30 percentage points lower, 10 to 30 percentage points lower, 15 to 30 percentage points lower, 20 to 30 percentage points lower, 5 to 25 percentage points lower, 5 to 20 percentage points lower, 5 to 15 percentage points lower, or 5 to 10 percentage points lower) of γδ TCR+ TEMRA cells following cell expansion in the presence of IL-2, IL-4, and IL-15 than the starting population had before cell expansion in the presence of IL-2, IL-4, and IL-15.
In some cases, less than 10 percent (e.g., less than 9 percent, less than 8 percent, less than 7 percent, less than 6 percent, less than 5 percent, less than 4 percent, less than 3 percent, less than 2 percent, or less than 1 percent) of the γδ TCR+ cells of a population provided herein can be CD69+ CD103+ TRM. In some cases, from 1 to 10 percent (e.g., from 1 to 9 percent, from 1 to 8 percent, from 1 to 7 percent, from 1 to 6 percent, from 1 to 5 percent, from 1 to 4 percent, from 2 to 10 percent, from 3 to 10 percent, from 4 to 10 percent, from 5 to 10 percent, from 6 to 10 percent, from 2 to 8 percent, from 2 to 6 percent, from 4 to 8 percent, or from 4 to 6 percent) of the γδ TCR+ cells of a population provided herein can be CD69+ CD103+ TRM cells.
In some cases, less than 50 percent (e.g., less than 45 percent, less than 40 percent, less than 35 percent, less than 30 percent, less than 25 percent, less than 20 percent, less than 15 percent, less than 10 percent, less than 5 percent, or less than 2 percent) of the γδ TCR+ cells of a population provided herein can be CD56+ cells. In some cases, from 1 to 50 percent (e.g., from 1 to 45 percent, from 1 to 40 percent, from 1 to 35 percent, from 1 to 30 percent, from 1 to 25 percent, from 1 to 20 percent, from 5 to 50 percent, from 10 to 50 percent, from 15 to 50 percent, from 20 to 50 percent, from 10 to 40 percent, from 15 to 35 percent, or from 20 to 30 percent) of the γδ TCR+ cells of a population provided herein can be CD56+ cells.
In some cases, from 1 to 40 percent (e.g., from 1 to 35 percent, from 1 to 30 percent, from 1 to 25 percent, from 1 to 20 percent, from 1 to 15 percent, from 1 to 10 percent, from 5 to 40 percent, from 10 to 40 percent, from 15 to 40 percent, from 20 to 40 percent, from 5 to 35 percent, from 10 to 30 percent, or from 15 to 25 percent) of the γδ TCR+ cells of a population provided herein can be CD137+ cells.
In some cases, a population provided herein can have a higher percentage (e.g., a percentage that is 2 to 50 percentage points higher, 5 to 50 percentage points higher, 2 to 40 percentage points higher, 5 to 40 percentage points higher, 10 to 40 percentage points higher, 15 to 40 percentage points higher, 20 to 40 percentage points higher, 5 to 35 percentage points higher, 5 to 30 percentage points higher, 5 to 25 percentage points higher, 5 to 20 percentage points higher, 5 to 15 percentage points higher, or 5 to 10 percentage points higher) of CD137+γδ TCR+ cells following cell expansion in the presence of IL-2, IL-4, and IL-15 than the starting population had before cell expansion in the presence of IL-2, IL-4, and IL-15.
In some cases, less than 25 percent (e.g., less than 20 percent, less than 15 percent, less than 10 percent, less than 5 percent, less than 4 percent, less than 3 percent, less than 2 percent, or less than 1 percent) of the γδ TCR+ cells of a population provided herein can be PD-1+ cells.
In some cases, a population provided herein can have a lower percentage (e.g., a percentage that is 5 to 90 percentage points lower, 5 to 80 percentage points lower, 5 to 75 percentage points lower, 5 to 70 percentage points lower, 5 to 75 percentage points lower, 5 to 70 percentage points lower, 5 to 65 percentage points lower, 5 to 60 percentage points lower, 5 to 55 percentage points lower, 5 to 50 percentage points lower, 5 to 45 percentage points lower, 5 to 40 percentage points lower, 5 to 35 percentage points lower, 5 to 30 percentage points lower, 5 to 25 percentage points lower, 5 to 20 percentage points lower, 5 to 15 percentage points lower, 5 to 10 percentage points lower, 10 to 90 percentage points lower, 10 to 80 percentage points lower, 10 to 75 percentage points lower, 10 to 70 percentage points lower, 10 to 75 percentage points lower, 10 to 70 percentage points lower, 10 to 65 percentage points lower, 10 to 60 percentage points lower, 10 to 55 percentage points lower, 10 to 50 percentage points lower, 10 to 45 percentage points lower, 10 to 40 percentage points lower, 10 to 35 percentage points lower, 10 to 30 percentage points lower, 10 to 25 percentage points lower, 10 to 20 percentage points lower, 10 to 15 percentage points lower, 25 to 90 percentage points lower, 25 to 80 percentage points lower, 25 to 75 percentage points lower, 25 to 70 percentage points lower, 25 to 75 percentage points lower, 25 to 70 percentage points lower, 25 to 65 percentage points lower, 25 to 60 percentage points lower, 25 to 55 percentage points lower, 25 to 50 percentage points lower, 25 to 45 percentage points lower, 25 to 40 percentage points lower, 25 to 35 percentage points lower, or 25 to 30 percentage points lower) of PD-1+γδ TCR+ cells following cell expansion in the presence of IL-2, IL-4, and IL-15 than the starting population had before cell expansion in the presence of IL-2, IL-4, and IL-15.
In some cases, from 5 to 40 percent (e.g., from 5 to 35 percent, from 5 to 30 percent, from 5 to 25 percent, from 5 to 20 percent, from 5 to 15 percent, from 10 to 40 percent, from 10 to 35 percent, from 10 to 30 percent, from 10 to 25 percent, from 10 to 20 percent, or from 15 to 25 percent) of the γδ TCR+ cells of a population provided herein can be BTLA+ cells.
In some cases, from 5 to 40 percent (e.g., from 5 to 35 percent, from 5 to 30 percent, from 5 to 25 percent, from 5 to 20 percent, from 5 to 15 percent, from 10 to 40 percent, from 10 to 35 percent, from 10 to 30 percent, from 10 to 25 percent, from 10 to 20 percent, or from 15 to 25 percent) of the αβ TCR+ cells of a population provided herein can be BTLA+ cells.
In some cases, greater than 60 percent (e.g., greater than 65 percent, greater than 70 percent, greater than 75 percent, greater than 80 percent, greater than 85 percent, greater than 90 percent, or greater than 95 percent) of the γδ TCR+ cells of a population provided herein can be NKG2D+ cells.
In some cases, greater than 20 percent (e.g., greater than 25 percent, greater than 30 percent, greater than 35 percent, greater than 40 percent, greater than 45 percent, greater than 50 percent, greater than 55 percent, greater than 60 percent, greater than 65 percent, greater than 70 percent, greater than 75 percent, greater than 80 percent, greater than 85 percent, greater than 90 percent, or greater than 95 percent) of the γδ TCR+ cells of a population provided herein can be NKp46+ cells.
In some cases, a population provided herein can have a higher percentage (e.g., a percentage that is 5 to 90 percentage points higher, 5 to 85 percentage points higher, 5 to 80 percentage points higher, 5 to 75 percentage points higher, 5 to 70 percentage points higher, 5 to 65 percentage points higher, 5 to 60 percentage points higher, 5 to 55 percentage points higher, 5 to 50 percentage points higher, 5 to 45 percentage points higher, 5 to 40 percentage points higher, 5 to 35 percentage points higher, 5 to 30 percentage points higher, 5 to 25 percentage points higher, 10 to 90 percentage points higher, 10 to 85 percentage points higher, 10 to 80 percentage points higher, 10 to 75 percentage points higher, 10 to 70 percentage points higher, 10 to 65 percentage points higher, 10 to 60 percentage points higher, 10 to 55 percentage points higher, 10 to 50 percentage points higher, 10 to 45 percentage points higher, 10 to 40 percentage points higher, 10 to 35 percentage points higher, 10 to 30 percentage points higher, 10 to 25 percentage points higher, 15 to 90 percentage points higher, 15 to 85 percentage points higher, 15 to 80 percentage points higher, 15 to 75 percentage points higher, 15 to 70 percentage points higher, 15 to 65 percentage points higher, 15 to 60 percentage points higher, 15 to 55 percentage points higher, 15 to 50 percentage points higher, 15 to 45 percentage points higher, 15 to 40 percentage points higher, 15 to 45 percentage points higher, 15 to 30 percentage points higher, 15 to 25 percentage points higher, or 20 to 40 percentage points higher) of NKp46+ cells following cell expansion in the presence of IL-2, IL-4, and IL-15 than the starting population had before cell expansion in the presence of IL-2, IL-4, and IL-15.
In some cases, (a) greater than 85 percent (e.g., greater than 90 percent, greater than 91 percent, greater than 92 percent, greater than 93 percent, greater than 94 percent, greater than 95 percent, greater than 96 percent, greater than 97 percent, greater than 98 percent, or greater than 99 percent) of the CD3+ cells of a population provided herein can be γδ TCR+ cells, (b) less than 10 percent (e.g., less than 9 percent, less than 8 percent, less than 7 percent, less than 6 percent, less than 5 percent, less than 4 percent, less than 3 percent, less than 2 percent, or less than 1 percent) of the CD3+ cells of that population can be αβ TCR+ cells, (c) less than 10 percent (e.g., less than 9 percent, less than 8 percent, less than 7 percent, less than 6 percent, less than 5 percent, less than 4 percent, less than 3 percent, less than 2 percent, or less than 1 percent) of the CD45+ cells of that population can be NK cells, (d) greater than 30 percent (e.g., greater than 35 percent, greater than 40 percent, greater than 45 percent, greater than 50 percent, greater than 55 percent, greater than 60 percent, greater than 65 percent, greater than 70 percent, greater than 75 percent, greater than 80 percent, greater than 85 percent, greater than 90 percent, or greater than 95 percent) of the γδ TCR+ cells of a population provided herein can be Vδ1+ cells, (e) less than 60 percent (e.g., less than 55 percent, less than 50 percent, less than 45 percent, less than 40 percent, less than 35 percent, less than 30 percent, less than 25 percent, less than 20 percent, less than 15 percent, less than 10 percent, less than 5 percent, or less than 2 percent) of the γδ TCR+ cells of that population can be VDδ1−Vδ2− cells, (f) less than 25 percent (e.g., less than 20 percent, less than 15 percent, less than 10 percent, less than 5 percent, less than 4 percent, less than 3 percent, less than 2 percent, or less than 1 percent) of the γδ TCR+ cells of that population can be Vδ2+ cells, (g) greater than 70 percent (e.g., greater than 75 percent, greater than 80 percent, greater than 85 percent, greater than 90 percent, or greater than 95 percent) of the γδ TCR+ cells of a population provided herein can be TEM cells, (h) less than 25 percent (e.g., less than 20 percent, less than 15 percent, less than 10 percent, less than 5 percent, less than 4 percent, less than 3 percent, less than 2 percent, or less than 1 percent) of the γδ TCR+ cells of that population can be TEMRA cells, (i) less than 10 percent (e.g., less than 9 percent, less than 8 percent, less than 7 percent, less than 6 percent, less than 5 percent, less than 4 percent, less than 3 percent, less than 2 percent, or less than 1 percent) of the γδ TCR+ cells of that population can be CD69+ CD103+ TRM or from 1 to 10 percent (e.g., from 1 to 9 percent, from 1 to 8 percent, from 1 to 7 percent, from 1 to 6 percent, from 1 to 5 percent, from 1 to 4 percent, from 2 to 10 percent, from 3 to 10 percent, from 4 to 10 percent, from 5 to 10 percent, from 6 to 10 percent, from 2 to 8 percent, from 2 to 6 percent, from 4 to 8 percent, or from 4 to 6 percent) of the γδ TCR+ cells of that population can be CD69+ CD103+ TRM cells, (j) less than 50 percent (e.g., less than 45 percent, less than 40 percent, less than 35 percent, less than 30 percent, less than 25 percent, less than 20 percent, less than 15 percent, less than 10 percent, less than 5 percent, or less than 2 percent) of the γδ TCR+ cells of that population can be CD56+ cells or from 1 to 50 percent (e.g., from 1 to 45 percent, from 1 to 40 percent, from 1 to 35 percent, from 1 to 30 percent, from 1 to 25 percent, from 1 to 20 percent, from 5 to 50 percent, from 10 to 50 percent, from 15 to 50 percent, from 20 to 50 percent, from 10 to 40 percent, from 15 to 35 percent, or from 20 to 30 percent) of the γδ TCR+ cells of that population can be CD56+ cells, (k) from 1 to 40 percent (e.g., from 1 to 35 percent, from 1 to 30 percent, from 1 to 25 percent, from 1 to 20 percent, from 1 to 15 percent, from 1 to 10 percent, from 5 to 40 percent, from 10 to 40 percent, from 15 to 40 percent, from 20 to 40 percent, from 5 to 35 percent, from 10 to 30 percent, or from 15 to 25 percent) of the γδ TCR+ cells of that population can be CD137+ cells, (1) less than 25 percent (e.g., less than 20 percent, less than 15 percent, less than 10 percent, less than 5 percent, less than 4 percent, less than 3 percent, less than 2 percent, or less than 1 percent) of the γδ TCR+ cells of that population can be PD-1+ cells, (m) from 5 to 40 percent (e.g., from 5 to 35 percent, from 5 to 30 percent, from 5 to 25 percent, from 5 to 20 percent, from 5 to 15 percent, from 10 to 40 percent, from 10 to 35 percent, from 10 to 30 percent, from 10 to 25 percent, from 10 to 20 percent, or from 15 to 25 percent) of the γδ TCR+ cells of that population can be BTLA+ cells, (n) greater than 60 percent (e.g., greater than 65 percent, greater than 70 percent, greater than 75 percent, greater than 80 percent, greater than 85 percent, greater than 90 percent, or greater than 95 percent) of the γδ TCR+ cells of that population can be NKG2D+ cells, and (o) greater than 20 percent (e.g., greater than 25 percent, greater than 30 percent, greater than 35 percent, greater than 40 percent, greater than 45 percent, greater than 50 percent, greater than 55 percent, greater than 60 percent, greater than 65 percent, greater than 70 percent, greater than 75 percent, greater than 80 percent, greater than 85 percent, greater than 90 percent, or greater than 95 percent) of the γδ TCR+ cells of that population can be NKp46+ cells.
In addition to providing the cell populations described herein and the methods for producing those cell populations as described herein, this document provides methods for using the cell populations described herein to treat any appropriate disease, disorder, or condition. For example, the cell populations described herein can be used to treat autoimmune conditions such as rheumatoid arthritis, systemic lupus erythematosus, and scleroderma, infections such as HIV infections, malaria, tuberculosis, hepatitis B, and SARS-CoV-2 infections, and/or cancer. For example, a cell population described herein can be administered to a mammal for use in, for example, adoptive cellular therapies to treat cancer. Any appropriate mammal can be treated with a cell population described herein. For example, humans, horses, cattle, pigs, dogs, cats, mice, and rats can be treated with a population of expanded tumor infiltrating γδ T cells described herein. Any appropriate number of cells can be within the cell population described herein that is administered to a mammal (e.g., a human) to treat cancer. For example, a cell population described herein can have from about 1×107 to about 1×1012 cells (e.g., from 5×107 to 1×1011 cells, from 1×108 to 1×1011 cells, from 5×108 to 1×1011 cells, from 1×109 to 1×1011 cells, or from 1×1010 to 1×1012 cells) and can be administered to a mammal (e.g., a human) to treat cancer. In some cases, a cell population described herein can be administered to a mammal (e.g., a human) to treat cancer such that from about 1×107 to about 1×1012 (e.g., from 5×107 to 1×1011, from 1×108 to 1×1011, from 5×108 to 1×1011, from 1×109 to 1×1011, or from 1×1010 to 1×1012) of γδ T cells are delivered to the mammal.
Any appropriate cancer can be treated using a cell population described herein. For example, glioblastomas, head & neck squamous cell carcinomas, cutaneous melanomas, lung adenocarcinomas, lung squamous cell carcinomas, breast carcinomas, mesotheliomas, liver hepatocellular carcinomas, pancreatic ductal adenocarcinomas, kidney renal cell carcinomas, bladder urothelial carcinomas, cervical squamous cell carcinomas and endocervical adenocarcinomas, esophageal carcinomas, stomach adenocarcinomas, colorectal adenocarcinomas, rectal adenocarcinomas, ovarian serous cystadenocarcinomas, uterine corpus endometrial carcinomas, prostate adenocarcinomas, and sarcomas can be treated using a cell population described herein.
Any appropriate route of administration can be used to administer a cell population described herein to a mammal. For example, a cell population described herein can be administered intravenously, intraperitoneally, or intratumorally.
When treating a mammal having a condition (e.g., an autoimmune condition), a disease, or an infection (e.g., an HIV infection, malaria, tuberculosis, hepatitis B, and SARS-CoV-2 infection) other than cancer, any appropriate tissue source can be used to obtained γδ T cells. For example, when expanding γδ T cells to treat an autoimmune condition, tissue involved in the autoimmune condition containing γδ T cells or uninvolved tissue containing γδ T cells (e.g., involved or uninvolved skin, liver, kidney, esophagus, small bowel, and/or colon tissue) can be used as a tissue source to obtain γδ T cells as described herein. When expanding γδ T cells to treat an infection, tissue involved in the infection containing γδ T cells or uninvolved tissue containing γδ T cells (e.g., involved or uninvolved skin, liver, kidney, esophagus, small bowel, and/or colon tissue) can be used as a tissue source to obtain γδ T cells as described herein.
The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.
A retrospective series of n=10 low grade (AJCC 8th edition Grade 1) PMP tumors specimens were identified from the University of Pittsburgh Medical Center Digestive Diseases Tissue Repository for immune repertoire sequencing following pathologic review for evidence of lymphocytic infiltrate (
Prospective TIL expansion was completed on n=26 consenting patients with peritoneal surface malignancies (PMP or colon cancer) undergoing standard of care CRS-HIPEC at the University of Pittsburgh Medical Center as part of a non-interventional tumor registry and tissue procurement clinical protocol (
The pre-rapid expansion protocol (pre-REP) modulation of γδ TIL was assessed on n=15 tumor digests from patients undergoing resection for melanoma as part of a non-interventional tumor banking clinical protocol (
Total RNA was extracted from FFPE tissue scrolls using the Covaris M220 focused ultasonicator and truXTRAC FFPE total NA magnetic bead Ultra Kit according to the manufacturer's protocol. The iR-RepSeq-plus 7-Chain DAM-PCR amplification sequence kit (iRepertoire Inc) was used to generate next generation sequencing libraries covering the human TCR-Vα, -Vβ, -Vγ, and -Vδ, and BCR IgH, Igκ, and Igλ chains. 1000 ng of extracted RNA was amplified in a single assay incorporating unique molecular identifiers (UMIs) during the reverse transcription (RT) step by the Biomek-i5 workstation (Beckman Coulter). Amplified libraries were multiplexed and pooled for sequencing on the Illumina NovaSeq platform with a 500-cycle kit. Each sample was allotted 5 million total sequencing reads. Raw data was demultiplexed and UMI guided assembly was performed using migec v1.2.9, and the resulting consensus fastqs were aligned and assembled into clonotypes using mixcr v3.0.14. The output T cell receptor sequence covers FR2 to FR4, as well as the beginning of the constant region.
Raw data were analyzed using the iRmap program (iRepertoire Inc.). Total reads were normalized to generate UMIs, and unique CDR3s (uCDR3s), mean CDR3 length, and sample Shannon true entropy scores were compared across all seven chains. The IgH chain immunoglobulin fraction was assembled with the TRUST algorithm and correlated with TCR and BCR repertoire metrics. Corollary immune repertoire analysis was completed on n=68 pancreatic tumor specimens from patients receiving neoadjuvant chemotherapy and curative resection and n=238 healthy donor PBMCs (iRepertoire). The publicity of PMP TCRs and BCRs was determined by the percent sharing with the n=238 health donor PBMCs. The generational probability of shared PMP specific BCR IgH clonotypes was calculated with the OLGA algorithm.
Mucinous peritoneal tumors were dissected to remove necrotic or fatty tissue and n=40 spatially distinct 2-3 mm3 tumor fragments (
Following randomization and selection of n=40 spatially distinct 2-3 mm3 tumor fragments, the remaining tumor fragments (if available) were enzymatically and mechanically digested into a single cell suspension with the human tumor dissociation kit (Miltenyi Biotec) and OctoMACS with Heaters Disassociater (Miltenyi Biotec) according to the manufacturer's protocol. Digested single cell suspensions were filtered with a 70 μm strainer, treated with 10 mL ACK lysis buffer (Gibco) for 5 minutes, washed twice with PBS, counted, and cryopreserved as described above.
Whole blood was collected in BD Vacutainer® EDTA tubes, diluted 1:1 with PBS and centrifuged atop 15 mL Lymphoprep™ density gradient media (Stemcell Technologies) in a SepMate™ 50 Tube at 1200 G, 20 minutes. Plasma and mononuclear cells were removed, washed with PBS, treated with 10 mL ACK lysis buffer (Gibco) for 5 minutes, washed twice with PBS, counted, and cryopreserved as described above.
Day 11 bulk TIL cultures, αβ TCR+ depleted cells, and post-REP Day 25 TIL cultures were utilized for spectral cytometry to assess the phenotypic expression of T cell memory, activation, exhaustion, and NCRs. Cells were strained with a 30 μm filter, washed with cytometry buffer (2% FBS in 4° C. PBS), incubated 5 minutes with Human TruStain FcX™ block (Biolegend), washed, and stained with a master mix of fluorescent conjugated antibodies and Brilliant Stain Buffer (BD) for 25 minutes at 4° C. protected from light. Samples were washed and resuspended in 200 μL of cytometry buffer and analyzed on the 5 laser Cytek® Aurora Spectral Cytometer. Single color spectral signatures were measured with UltraComp eBeadsrm (Invitrogen) and spectrally resolved along with TIL autofluorescence spectral signature using the SpectroFlo® software. Following gating of single cell, live, CD45+ immune cell, CD56+ NK cells, CD3+ cells, αβ TCR+ CD4+ and CD8+ T cells, and γδ TCR+Vδ1+, Vδ2+, and Vδ1−Vδ2− T cells (
To assess the autologous tumor reactivity of expanded αβ (IL-2 only) and γδ (IL-2, IL-4, and IL-15) TIL, cryopreserved TIL were thawed and rested overnight in IL-2 (3,000 IU/mL) media, washed twice with PBS, counted and plated (1×105 cells) in a 96 well round bottom plate in IL-2 free complete media alone, with CD3-CD28 stimulation (Dynabeads, 2.5 μL/well, Invitrogen), 1×105 autologous PBMC, or 1×105 autologous tumor digest single cell suspension with culture volume normalized to 200 μL for 24 hours in a humidified incubator at 37° C. in 5% CO2 as described elsewhere (Dudley et al., J. Immunother., 26:332-342 (2003)). 50 μL of supernatant was harvested from duplicate co-cultures, diluted 1:2, and assessed for production of IFNγ with the Human IFNγ ELISA Kit (Invitrogen) according to the manufacturer's instructions. TIL-autologous tumor digest reactivity was compared with co-culture with autologous PBMC and between paired γδ and αβ TIL. In certain cases, γδ TIL or autologous tumor digest were also incubated with blocking antibodies (TIL: isotype control mouse IgG (Invitrogen, 10 μg/mL), anti-γδ TCR (Novus Biologicals, clone 7A5, 3 μg/mL), or anti-NKG2D (BD, clone 1D11, 10 μg/mL) or tumor digest: isotype control mouse IgG (10 μg/mL) or anti-MHC-1 (Invitrogen, W6/32 10 μg/mL) for 2 hours prior to co-culture.
To assess the MHC unrestricted recognition of TIL, γδ and αβ TIL were similarly co-cultured with 1×105 cancer cell lines (K562, HCT116, RKO, SW480, or SW48; all from ATCC, authenticated and Mycoplasma negative (eMyco™ plus PCR kit)) following a minimum of two passages of culture in complete media in a humidified incubator at 37° C. in 5% CO2.
To evaluate the expression of NKG2D ligands in the tested cancer cell lines, mRNA Z-scores of MICA, MICA, ULBP1, ULBP2, and ULBP3 were queried for the K562, HCT116, RKO, SW480, and SW48 cells from the Cancer Cell Line Encyclopedia using the cBioPortal for cancer genomics.
The tumor specific Vδ1 infiltration and prognostic ability was assessed in the 20 most common primary solid tumors (NCI) of bulk RNA sequencing data in The Cancer Genome Atlas (TCGA) with the Gene Expression Profiling Interactive Analysis Server 2 (GEPIA2). Mean expression (log transcripts per million) of γδ TIL subsets (TRDV1, TRDV2, and TRDV3) and αβ TIL (TRBC2 Beta Chain 2 Constant Region) were calculated. Kaplan Meier survival analysis by normalized (ACTB beta actin) TRDV1 expression above (high) or below (low) the median for selected tumor types was completed with calculation of Log rank p value and 95% confidence interval of survival estimates. TRDV1 expression was directly correlated with TRBC2 expression across selected tumors, and corresponding Pearson correlation coefficient and P values were calculated.
Data were expressed as mean±standard deviation. Graphical visualization and statistical analysis were performed using Microsoft Excel and GraphPad Prism 9. Descriptive statistics, Two-tailed non-parametric test, Mann-Whitney U tests (unpaired), and Wilcoxon signed-rank (paired, for all comparisons of αβ and γδ TIL) tests were used. Correlations were calculated with the Pearson correlation coefficient and plotted with nonlinear regression and 95% confidence bands. P values<0.05 were considered statistically significant, and significance levels were set to * P<0.05, ** P<0.01, *** P<0.001, and **** P<0.0001.
Low Grade Pseudomyxoma Peritonei (PMP) Display Elevated B Cell Receptor (BCR) IgE Fraction Associated with TCR Vδ
Following pathologic analysis of previously resected peritoneal tumor specimens (
With limited prior understanding of the adaptive immune response to this understudied tumor type, full TCR and BCR sequencing of the resected FFPE tumor specimen from the first operative resection (
Comparison with a cohort of healthy donor PBMC repertoires (n=238) revealed that this low grade PMP cohort displayed a highly private repertoire with only Vu (0.06% of chain), Igκ (3.7%), and Igλ (1.9%), demonstrating shared public CDR3s (
Given the unexpected abundance of BCR transcripts, the immunoglobin fraction of the total low grade PMP IgH repertoire was further analyzed, which revealed an expected distribution dominated by IgG (53.4±12.0%) and IgA (21.7±9.9%) (
With the understanding that peritoneal γδ TIL display a diverse polyclonal and private repertoire, the following was performed to prospectively assess γδ TIL. Tumor specimens were collected from consenting patients with peritoneal surface malignancies undergoing CRS-HIPEC (n=26) (
Mucinous peritoneal tumors were dissected into spatially distinct 2-3 mm3 fragments (
Multispectral flow cytometry was utilized to define the composition and phenotype of the bulk proliferating peritoneal TIL populations. IL-2 reactive CD56+ CD3− Natural Killer (NK) cells and CD3+ T cells were the major constituents of the CD45+ TIL population (
γδ TIL Display a Tissue Resident Effector Memory Phenotype with Reduced PD-1, but Greater NKG2D and CD137 Expression Compared to αβ TIL
To better understand the phenotype of the γδ and up TIL populations, markers of T cell memory, differentiation, and activation, inhibitory receptors associated with T cell exhaustion, and expression of natural cytotoxicity receptors (NCRs) were assessed (
The majority of γδ TIL displayed an effector memory phenotype (TEM: CD45RO+ CD62L−, 75.5±15.8%) that was comparable to that observed in αβ TIL (71.2±20.8%,
Given that the composition of ex vivo expanded TIL populations is highly dependent on spatial heterogeneity and culture conditions promoting the proliferation of tumor dominant and minority populations associated with differential tumor reactivity, expression of activation and exhaustion molecules (Poschke et al., Clin. Cancer Res., 26:4289-4301 (2020)) were compared. Expanded γδ (92.2%) and αβ (97.4%) TIL displayed high levels of CD2 (
Inhibitory immune receptor expression are simultaneous markers of tumor reactivity, immune exhaustion, and potential for suppression (Ahmadzadeh et al., Blood, 114:1537-1544 (2009); Baitsch et al., J Clin. Invest., 121:2350-2360 (2011); Miller et al., Nat. Immunol., 20:326-336 (2019); and Gros et al., J Clin. Invest., 124:2246-2259 (2014)). With the exception of PD-L1, γδ TIL displayed more variable expression of PD-1, LAG-3, TIGIT, and BTLA compared to αβ TIL (
The innate-like NK cell properties of γδ T cells, including expression of the NCRs NKG2D and NKp46 confer additional reactivity to stress antigens and antitumor potential (Silva-Santos et al., Nat. Rev. Cancer, 19:392-404 (2019); Wu et al., Sci. Transl. Med., 11(513):aax9364 (2019); Mikulak et al., JCI Insight, 4(24):e125884 (2019); and Foord et al., Sci. Transl. Med., 13(577):abb0192 (2021)). While expression of NKG2D was uniformly high on γδ TIL (72.8±7.9%) and higher than αβ TIL (38.0±19.8%, p=0.007), NKp46 expression was more heterogenous (17.4±22.4%) and did not differ from αβ TIL (23.6±30.1%) (
To consider the adoptive transfer of γδ TIL displaying a favorable tissue resident effector memory phenotype with limited exhaustion and enhanced expression of CD137 and NKG2D, an expansion protocol was designed to generate a clinically feasible number of γδ TIL. γδ TIL were negatively selected with depletion of αβ TCR+ cells. Then, 1×106 γδ TIL (or bulk αβ TIL for comparison) were expanded for 14 days with mitogenic CD3 stimulation (OKT-3, 30 ng/mL), high concentrations of IL-2 (3,000 IU/mL), and irradiated allogenic healthy donor PBMCs (
Different combinations of cytokines were evaluated (in combination with anti-CD3 and irradiated PBMCs) to determine if a population of γδ TIL having a desired phenotype can be obtained in appropriate numbers and percentages. While addition of IL-15 (25.6 fold expansion) or IL-7 (164.3 fold expansion) increased expansion of selected γδ TIL, a combination of IL-2, IL-4, and IL-15 (453.8±100.8 fold expansion) demonstrated considerably enhanced γδ TIL expansion (p=0.0008) that was largely comparable to that observed for the IL-2 only expansion of native αβ TIL (725.5±153 fold expansion) (
Spectral cytometric phenotyping of the negatively selected γδ TIL that were IL-2/IL-4/IL-15 expanded (
The IL-2/IL-4/IL-15 expansion of the negatively selected γδ TIL resulted in increased proliferation of TEM γδ TIL (87.1±7.2% vs 75.5±15.8%, p=0.034), with reduced TEMRA (7.0±6.3% vs 14.9±12.9%, p=0.031) compared to the negatively selected γδ TIL preparation before IL-2/IL-4/IL-15 expansion (
Following IL-2/IL-4/IL-15 expansion of negatively selected γδ TIL (5.3±2.7% vs 20.8±16.2%, p<0.0001) and IL-2 only expansion of native αβ TIL (1.7±1.5% vs 12.3 13.0%, p=0.004), the number of CD69+ CD103+ TRM cells were reduced compared to the pre-expansion TIL, but higher in the γδ TIL population (p=0.004,
Completed and ongoing trials of TIL therapy in patients with metastatic epithelial cancer have identified in vitro TIL reactivity to autologous patient tumor as a key determinant of objective clinical response (Tran et al., Science, 344:641-645 (2014); Stevanovic et al., J. Clin. Oncol. 33:1543-1550 (2015); Stevanovic et al., Clin. Cancer Res., 25:1486-1493 (2019); Chandran et al., Lancet Oncol., 18:792-802 (2017); and Zacharakis et al., Nat. Med., 24:724-730 (2018)). To measure the tumor reactivity of the expanded peritoneal TIL, in patients with available specimens (n=11), IFNγ production was assessed following 24-hour co-culture of a 1:1 ratio of autologous tumor digest cryopreserved at the time of resection and either IL-2/IL-4/IL-15 expanded, negatively selected γδ TIL or IL-2 only expanded, native αβ TIL (
Given that γδ TIL possess MHC unrestricted TCRs, their reactivity against a series of HLA unmatched cancer cell lines also was evaluated (
Given the established role of γδ T cell NCR mediated recognition of target cells and uniformly high expression of NKG2D within this cohort of expanded peritoneal γδ TIL, the following was performed to identify its role, along with the γδ TCR, in mediating autologous tumor reactivity (Silva-Santos et al., Nat. Rev. Immunol., 15:683-691 (2015); and Silva-Santos et al., Nat. Rev. Cancer, 19:392-404 (2019)). Following co-culture of IL-2/IL-4/IL-15 expanded, negatively selected γδ TIL with autologous tumor digests (n=7), combinations of anti-MHC-1 (W6/32), anti-NKG2D (1D11), anti-γδ TCR (7A5), or isotype control (mouse IgG) mAb were utilized to block the corresponding receptor binding and signaling (
To identify additional factors associated with γδ TIL autologous tumor reactivity, the production of IFNγ following autologous tumor digest co-culture with IL-2/IL-4/IL-15 expanded, negatively selected γδ TIL phenotypic characteristics were compared. The percent composition of Vδ1 positively correlated (r=+0.719, p=0.012) with IFNγ production, supporting earlier reports of the enhanced anti-tumor potential of Vδ1 cells over that observed with other γδ subsets (
Pre-Rapid Expansion Protocol Modulation of γ Chain Cytokines and CD137 Engagement does not Improve γδ TIL Expansion
Given the enhanced autologous tumor reactivity of γδ TIL in comparison to αβ TIL, methods for the specific expansion of γδ TIL during the pre-REP culture period, which determines the input number of γδ TIL available for REP, were investigated. With the increased number of γδ TIL following isolation and culture with IL-2, IL-4, and IL-15 during the REP, this γ chain combination was evaluated in a retrospective cohort of cryopreserved tumor digests (n=15,
While the γ chain combination increased the total number of viable expanded TIL following 11 days of culture, due to increased CD3+αβ TCR+ CD4 and CD8 TIL, no differences in the number of γδ TCR+ or Vδ1+ cells were observed with or without CD137 stimulation compared to IL-2 alone (
With multiple clinical studies of TIL therapy identifying infusion of increased number of tumor reactive T cells associated with objective clinical response, and the aforementioned sparse infiltration of γδ TIL, the following was performed to identify target indications with increased γδ TIL and determine their impact on long term survival (Radvanyi et al., Clin. Cancer Res., 18:6758-6770 (2012); Goff et al., J. Clin. Oncol., 34:2389-2397 (2016) and Chandran et al., Lancet Oncol., 18:792-802 (2017)). Using bulk RNA sequencing data of the 20 most prevalent solid tumors from The Cancer Genome Atlas (TCGA), the expression of γδ and αβ TIL was identified with the Gene Expression Profiling Interactive Analysis 2 (GEPIA 2) tool (
Given the predominant infiltration of the Vδ1 subset across tumors compared to the other γδ T cell subsets and association with autologous tumor reactivity, the prognostic impact of TRDV1 expression on overall survival in the selected tumors was evaluated. Following normalization of TRDV1 expression with beta actin (ACTB), the cohorts were split into high and low expression groups based on the median expression level of TRDV1 of individual tumor types. When including all TCGA tumors available for analysis on the GEPIA 2 server, high expression of TRDV1 was associated with considerable survival benefit (p<0.00001,
Taken together, the results provided herein demonstrate that tumor infiltrating γδ T cells, displaying diverse, patient specific repertoires, tissue resident effector memory phenotypes, and superior autologous tumor reactivity, can be successfully expanded by themselves or in parallel with αβ TIL to unleash the full TCR repertoire against cancer.
Embodiment 1. A method for producing a cell population comprising γδ T cells, wherein said method comprises culturing a first cell population comprising γδ T cells in the presence of IL-2, IL-4, and IL-15 for 8 to 21 days to obtain a second cell population, wherein said second cell population comprises at least 10 times more γδ T cells than said first cell population.
Embodiment 2. The method of Embodiment 1, wherein said γδ T cells are human cells.
Embodiment 3. The method of any one of Embodiments 1-2, wherein said γδ T cells are tumor infiltrating γδ T cells.
Embodiment 4. The method of any one of Embodiments 1-3, wherein said first cell population is:
(i) a population of tumor infiltrating γδ T cells obtained from (a) tissue comprising a tumor or (b) healthy tissue that was within 30 mm of a tumor,
(ii) a population of γδ T cells obtained from healthy tissue,
(iii) a population of γδ T cells obtained from infected tissue, or
(iv) a population of γδ T cells obtained from tissue harboring autoimmune T cells.
Embodiment 5. The method of Embodiment 4, wherein said method comprises obtaining said first cell population from said tissue comprising said tumor.
Embodiment 6. The method of Embodiment 4, wherein said method comprises obtaining said first cell population from said healthy tissue that was within 30 mm of said tumor.
Embodiment 7. The method of any one of Embodiments 1-6, wherein said first cell population is a cell population that was cultured in the presence of 50 international units/mL to 6000 international units/mL of IL-2 and in the absence of IL-4 and IL-15 for 3 to 15 days prior to said culturing in the presence of IL-2, IL-4, and IL-15.
Embodiment 8. The method of any one of Embodiments 1-6, wherein said first cell population is a cell population that was cultured in the presence of 100 international units/mL to 4000 international units/mL of IL-2 and in the absence of IL-4 and IL-15 for 8 to 15 days prior to said culturing in the presence of IL-2, IL-4, and IL-15.
Embodiment 9. The method of any one of Embodiments 1-8, wherein said first cell population is a cell population that was enriched for tumor infiltrating γδ T cells.
Embodiment 10. The method of any one of Embodiments 1-8, wherein said first cell population is a cell population that was enriched for tumor infiltrating γδ T cells via (a) the removal of at least some αβ T cells or (b) the isolation of at least some γδ T cells.
Embodiment 11. The method of any one of Embodiments 9-10, wherein said method comprises removing at least some αβ T cells from a cell population to obtain said first cell population.
Embodiment 12. The method of Embodiment 11, wherein said removing comprises positively selecting αβ T cells and removing the positively selected αβ T cells.
Embodiment 13. The method of any one of Embodiments 9-10, wherein said method comprises isolating at least some γδ T cells from a cell population to obtain said first cell population.
Embodiment 14. The method of Embodiment 13, wherein said isolating comprises positively selecting γδ T cells and isolating the positively selected γδ T cells.
Embodiment 15. The method of any one of Embodiments 1-14, wherein said culturing said first cell population comprising γδ T cells in the presence of IL-2, IL-4, and IL-15 for said 8 to 21 days comprises culturing said first cell population comprising γδ T cells in the presence of IL-2, IL-4, IL-15, irradiated PBMCs, and an anti-CD3 antibody for said 8 to 21 days.
Embodiment 16. The method of any one of Embodiments 1-15, wherein said culturing said first cell population comprising γδ T cells in the presence of IL-2, IL-4, and IL-15 is for 12 to 16 days.
Embodiment 17. The method of any one of Embodiments 1-15, wherein said culturing said first cell population comprising γδ T cells in the presence of IL-2, IL-4, and IL-15 is for 13 to 15 days.
Embodiment 18. The method of any one of Embodiments 1-17, wherein said second cell population comprises:
at least 50 times more γδ T cells than said first cell population,
at least 100 times more γδ T cells than said first cell population,
at least 200 times more γδ T cells than said first cell population,
at least 300 times more γδ T cells than said first cell population, or
at least 400 times more γδ T cells than said first cell population.
Embodiment 19. The method of any one of Embodiments 1-18, wherein said second cell population comprises greater than 1×108 γδ T cells.
Embodiment 20. The method of any one of Embodiments 1-19, wherein said IL-2 is a human IL-2, wherein said IL-4 is a human IL-4, and wherein said IL-15 is a human IL-15.
Embodiment 21. The method of any one of Embodiments 1-20, wherein greater than 85 percent of the CD3+ cells of said second cell population are γδ TCR+ cells.
Embodiment 22. The method of any one of Embodiments 1-21, wherein less than 10 percent of the CD3+ cells of said second cell population are αβ TCR+ cells.
Embodiment 23. The method of any one of Embodiments 1-22, wherein less than 10 percent of the CD45+ cells of said second cell population are NK cells.
Embodiment 24. The method of any one of Embodiments 1-23, wherein greater than 30 percent of the γδ TCR+ cells of said second cell population are Vδ1+ cells.
Embodiment 25. The method of any one of Embodiments 1-24, wherein less than 60 percent of the γδ TCR+ cells of said second cell population are Vδ1−Vδ2− cells.
Embodiment 26. The method of any one of Embodiments 1-25, wherein less than 25 percent of the γδ TCR+ cells of said second cell population are Vδ2+ cells.
Embodiment 27. The method of any one of Embodiments 1-26, wherein greater than 70 percent of the γδ TCR+ cells of said second cell population are TEM cells.
Embodiment 28. The method of any one of Embodiments 1-27, wherein less than 25 percent of the γδ TCR+ cells of said second cell population are TEMRA cells.
Embodiment 29. The method of any one of Embodiments 1-28, wherein less than 10 percent of the γδ TCR+ cells of said second cell population are CD69+ CD103+ TRM cells.
Embodiment 30. The method of any one of Embodiments 1-29, wherein from 1 to 10 percent of the γδ TCR+ cells of said second cell population are CD69+ CD103+ TRM cells.
Embodiment 31. The method of any one of Embodiments 1-30, wherein less than 50 percent of the γδ TCR+ cells of said second cell population are CD56+ cells.
Embodiment 32. The method of any one of Embodiments 1-31, wherein from 1 to 50 percent of the γδ TCR+ cells of said second cell population are CD56+ cells.
Embodiment 33. The method of any one of Embodiments 1-32, wherein from 1 to 40 percent of the γδ TCR+ cells of said second cell population are CD137+ cells.
Embodiment 34. The method of any one of Embodiments 1-33, wherein less than 25 percent of the γδ TCR+ cells of said second cell population are PD-1+ cells.
Embodiment 35. The method of any one of Embodiments 1-34, wherein from 5 to 40 percent of the γδ TCR+ cells of said second cell population are BTLA+ cells.
Embodiment 36. The method of any one of Embodiments 1-35, wherein greater than 60 percent of the γδ TCR+ cells of said second cell population are NKG2D+ cells.
Embodiment 37. The method of any one of Embodiments 1-36, wherein greater than 20 percent of the γδ TCR+ cells of said second cell population are NKp46+ cells.
Embodiment 38. An isolated cell population comprising polyclonal γδ T cells, wherein said population comprises greater than 1×108 γδ T cells.
Embodiment 39. The cell population of Embodiment 38, wherein greater than 85 percent of the CD3+ cells said cell population are γδ TCR+ cells.
Embodiment 40. The cell population of any one of Embodiments 38-39, wherein less than 10 percent of the CD3+ cells of said cell population are αβ TCR+ cells.
Embodiment 41. The cell population of any one of Embodiments 39-40, wherein less than 10 percent of the CD45+ cells of said cell population are NK cells.
Embodiment 42. The cell population of any one of Embodiments 39-41, wherein greater than 30 percent of the γδ TCR+ cells of said cell population are Vδ1+ cells.
Embodiment 43. The cell population of any one of Embodiments 39-42, wherein less than 60 percent of the γδ TCR+ cells of said cell population are Vδ1−Vδ2− cells.
Embodiment 44. The cell population of any one of Embodiments 39-43, wherein less than 25 percent of the γδ TCR+ cells of said cell population are Vδ2+ cells.
Embodiment 45. The cell population of any one of Embodiments 39-44, wherein greater than 70 percent of the γδ TCR+ cells of said cell population are TEM cells.
Embodiment 46. The cell population of any one of Embodiments 39-45, wherein less than 25 percent of the γδ TCR+ cells of said cell population are TEMRA cells.
Embodiment 47. The cell population of any one of Embodiments 39-46, wherein less than 10 percent of the γδ TCR+ cells of said cell population are CD69+ CD103+ TRM cells.
Embodiment 48. The cell population of any one of Embodiments 39-47, wherein from 1 to 10 percent of the γδ TCR+ cells of said cell population are CD69+ CD103+ TRM cells.
Embodiment 49. The cell population of any one of Embodiments 39-48, wherein less than 50 percent of the γδ TCR+ cells of said cell population are CD56+ cells.
Embodiment 50. The cell population of any one of Embodiments 39-49, wherein from 1 to 50 percent of the γδ TCR+ cells of said cell population are CD56+ cells.
Embodiment 51. The cell population of any one of Embodiments 39-50, wherein from 1 to 40 percent of the γδ TCR+ cells of said cell population are CD137+ cells.
Embodiment 52. The cell population of any one of Embodiments 39-51, wherein less than 25 percent of the γδ TCR+ cells of said cell population are PD-1+ cells.
Embodiment 53. The cell population of any one of Embodiments 39-52, wherein from 5 to 40 percent of the γδ TCR+ cells of said cell population are BTLA+ cells.
Embodiment 54. The cell population of any one of Embodiments 39-53, wherein greater than 60 percent of the γδ TCR+ cells of said cell population are NKG2D+ cells.
Embodiment 55. The cell population of any one of Embodiments 39-54, wherein greater than 20 percent of the γδ TCR+ cells of said cell population are NKp46+ cells.
Embodiment 56. The cell population of any one of Embodiments 39-55, wherein the cells of said cell population are human cells.
Embodiment 57. The cell population of any one of Embodiments 39-56, wherein said γδ T cells are tumor infiltrating γδ T cells.
Embodiment 58. The cell population of any one of Embodiments 39-57, wherein cell population was produced using the method of any one of Embodiments 1-37.
Embodiment 59. A method for providing a mammal with γδ T cells, wherein said method comprises administering a cell population produced as set forth in any one of Embodiments 1-37 to a mammal.
Embodiment 60. The method of Embodiment 59, wherein said mammal is a human.
Embodiment 61. The method of any one of Embodiments 59-60, wherein said mammal has cancer.
Embodiment 62. The method of any one of Embodiments 59-61, wherein the cells of said first cell population are allogenic or autologous to said mammal administered said cell population.
Embodiment 63. A method for providing a mammal with γδ T cells, wherein said method comprises administering said cell population of any one of Embodiments 38-58 to a mammal.
Embodiment 64. The method of Embodiment 63, wherein said mammal is a human.
Embodiment 65. The method of any one of Embodiments 63-64, wherein said mammal has cancer, an autoimmune condition, or an infection.
Embodiment 66. The method of any one of Embodiments 59-65, wherein the cells of said cell population are allogenic or autologous to said mammal.
Embodiment 67. A method for treating cancer, wherein said method comprises administering a cell population produced as set forth in any one of Embodiments 1-37 to a mammal having cancer.
Embodiment 68. The method of Embodiment 67, wherein said mammal is a human.
Embodiment 69. The method of any one of Embodiments 67-68, wherein the cells of said first cell population are allogenic or autologous to said mammal having cancer.
Embodiment 70. A method for treating cancer, wherein said method comprises administering said cell population of any one of Embodiments 38-58 to a mammal having cancer.
Embodiment 71. The method of Embodiment 70, wherein said mammal is a human.
Embodiment 72. The method of any one of Embodiments 70-71, wherein the cells of said cell population are allogenic or autologous to said mammal having cancer.
Embodiment 73. The method of any one of Embodiments 59-72, wherein said method comprises administering αβ T cells to said mammal.
Embodiment 74. A method for treating an autoimmune condition, wherein said method comprises administering said cell population of any one of Embodiments 38-58 to a mammal having an autoimmune condition.
Embodiment 75. The method of Embodiment 74, wherein said mammal is a human.
Embodiment 76. The method of any one of Embodiments 74-75, wherein the cells of said cell population are allogenic or autologous to said mammal having said autoimmune condition.
Embodiment 77. The method of any one of Embodiments 74-76, wherein said method comprises administering αβ T cells to said mammal.
Embodiment 78. A method for treating an infection, wherein said method comprises administering said cell population of any one of Embodiments 38-58 to a mammal having an infection.
Embodiment 79. The method of Embodiment 78, wherein said mammal is a human.
Embodiment 80. The method of any one of Embodiments 78-79, wherein the cells of said cell population are allogenic or autologous to said mammal having said infection.
Embodiment 81. The method of any one of Embodiments 78-80, wherein said method comprises administering αβ T cells to said mammal.
It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.
This application claims the benefit of U.S. Provisional Application Ser. No. 63/257,805, filed Oct. 20, 2021. The disclosure of the prior application is considered part of (and is incorporated by reference in) the disclosure of this application.
| Number | Date | Country | |
|---|---|---|---|
| 63257805 | Oct 2021 | US |
