Methods for generating tissue-resident memory T cells and their use

Abstract

Provided herein are methods for generating tissue-resident memory-like T cells through a combination of hypoxia and TGFβ. Additionally, provided herein are methods for using tissue-resident memory T cells as adoptive cell therapy.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 62 / 747,523, filed Oct. 18, 2018, and U.S. Provisional Patent Application No. 62 / 846,270, filed May 10, 2019, the entire contents of both of which are incorporated herein by reference.

[0002] Incorporation of Sequence Listing The sequence listing contained in the file named "UTFCP1408WO_ST25.txt", which is 8 KB (measured in Microsoft Windows®) and created on Oct. 17, 2019, is filed with the present application by electronic application and is incorporated herein by reference together with this specification.

Background Art

[0003] 1. Field The present invention generally relates to the fields of pharmacy and immunology. More particularly, it relates to methods for generating tissue - resident memory - like T cells and their use.

Summary of the Invention

Problems to be Solved by the Invention

[0004] 2. Description of Related Art Tissue - resident memory cells (T RM ) are a recently identified subset of memory T cells that are important in the local front - line defense against viral diseases. Recent reports also suggest that cells with this phenotype play an important role in anti - tumor immunity. Little is relatively known about T RM differentiation, and the fact that endogenous tissue - resident memory cells are difficult to isolate hinders their study in basic research and their application in adoptive cell therapy. Therefore, the need for methods for generating tissue - resident memory cells has not been met.

Means for Solving the Problems

[0005] In one embodiment, the present disclosure provides an in vitro method for generating tissue-resident memory-like T cells (T RM -like T cells), comprising: (a) obtaining a starting population of T cells; (b) culturing the starting population of T cells under hypoxic conditions or in the presence of a hypoxia inducer to generate early effector cells; and (c) further culturing the early effector cells in the presence of transforming growth factor β1 (TGF-β1) to generate T RM -like T cells.

[0006] In another embodiment, the present disclosure provides an in vitro method for generating tissue-resident memory-like T cells (T RM -like T cells), comprising: (a) obtaining a starting population of T cells; (b) culturing the starting population of T cells under hypoxic conditions or in the presence of a hypoxia inducer to generate early effector cells; and (c) further culturing the early effector cells in the presence of transforming growth factor β1 (TGF-β1), transforming growth factor β2 (TGF-β2), transforming growth factor β3 (TGF-β3), or transforming growth factor β4 (TGF-β4) to generate T RM -like T cells. In some embodiments, the culturing comprises activating the starting population of T cells to generate early effector cells.

[0007] In yet another embodiment, the present disclosure provides an in vitro method for generating T RM -like T cells, comprising: (a) obtaining a starting population of T cells; (b) culturing the starting population of T cells under hypoxic conditions or in the presence of a hypoxia inducer; and (c) further culturing the starting population of T cells in the presence of TGF-β1 to generate T RM -like T cells.

[0008] In some aspects, the starting population of T cells is CD8 +It is a peripheral blood T cell. In a specific embodiment, CD8 + The peripheral blood T cell is a human CD8 + It is a peripheral blood T cell. In a specific embodiment, it is a human CD8 + Obtaining the peripheral blood T cell involves CD45RA from a peripheral blood sample + CCR7 + CD8 +Including selecting naive T cells. In some embodiments, the peripheral blood sample is obtained from a healthy subject. In some embodiments, the peripheral blood sample is obtained from a subject diagnosed with cancer or suspected of having cancer. In some embodiments, the peripheral blood sample is obtained from a subject diagnosed with a viral disease or suspected of having a viral disease. In certain embodiments, the starting population of T cells is generated by stimulation of naive T cells with antigen-presenting cells pulsed with peptides, full-length antigens, or cell lysates. In certain embodiments, the T cells are obtained from the tumor site or are tumor-infiltrating lymphocytes. In some embodiments, the T cells are naive T cells. For example, the cell lysate is a tumor lysate. In a specific embodiment, the antigen is a cancer antigen. In some embodiments, the peptide is a peptide derived from a protein that is differentially expressed or highly expressed by cancer cells. In some embodiments, the peptide is a peptide derived from a neoantigen or a protein containing a mutation. In certain embodiments, the starting population of T cells is enriched for T cells specific for the antigen of interest. In certain embodiments, the starting population of T cells is purified to enrich for CD8-positive peptide-MHC tetramer-positive cells. In some embodiments, the starting population of T cells is purified by fluorescence-activated cell sorting. In certain embodiments, the starting population of T cells is a modified T cell. In some embodiments, the modified T cell is generated by introduction of a cloned T cell receptor (TCR) into a population of host cells. In certain embodiments, the population of host cells is peripheral blood mononuclear cells. In some embodiments, the cloned TCR is introduced into the population of host cells by a non-viral method, such as an episomal vector or a transposon-transposase system. In certain embodiments, the cloned TCR is introduced into the population of host cells by transduction. In some embodiments, the population of host cells is transduced with a viral vector containing the TCRα chain and the TCRβ chain. In certain embodiments, the viral vector is a lentiviral vector. In some embodiments, the transduced population of host cells is purified to enrich for CD8-positive peptide-MHC tetramer-positive cells.In certain embodiments, the modified T cells expressed a chimeric antigen receptor. In a specific embodiment, the chimeric antigen receptor comprises a cloned TCR. In some embodiments, the starting population of T cells is tumor infiltrating lymphocytes obtained from a subject.

[0009] In certain embodiments, the hypoxic condition is further defined as less than 5% oxygen, such as 4%, 3%, 2%, 1%, or less oxygen. In some embodiments, the hypoxia inducer is a hypoxia mimetic. In certain embodiments, the hypoxia inducer or hypoxia mimetic is cobalt chloride (CoCl2), deferoxamine mesylate (DFOM), dimethyloxalyglycine (DMOG), or a prolyl hydroxylase inhibitor, such as a 2-OG analog. In some embodiments, the prolyl hydroxylase inhibitor is roxadustat (FG-4592).

[0010] In some embodiments, the culture of step (b) is performed in the presence of TCR stimulation and co-stimulation. In certain embodiments, the TCR stimulation and co-stimulation include anti-CD3 and anti-CD28 antibodies, anti-CD3 and anti-CD28 beads, feeder cells, antigen presenting cells, artificial antigen presenting cells, peptide and / or protein antigens, or combinations thereof. In some embodiments, the TCR stimulation and co-stimulation include anti-CD3 and anti-CD28 beads. In certain embodiments, the culture of step (b) is performed for 3 to 5 days, such as 4 days. In certain embodiments, the culture of step (b) is performed under normoxic conditions, such as 20% oxygen. In certain embodiments, the step of the culture of step (b) is performed in the presence of, for example, 25 to 100 IU / mL, such as 25, 50, or 75 IU / mL of IL-2. In some embodiments, the culture of step (b) is performed under hypoxic conditions, such as 2% oxygen. In certain embodiments, the culture is performed in the presence of IL-15. In some embodiments, IL-15 is present at a concentration of 5 to 20 ng / mL, such as 7 to 12 ng / mL, specifically 7, 8, 9, 10, 11, or 12 ng / mL.

[0011] In certain embodiments, TGF-β1 is further defined as recombinant human TGF-β1 (rhTGF-β1). In some embodiments, rhTGF-β1 is present at a concentration of 0.1 to 5 ng / mL, such as 1 to 1.5 ng / mL, specifically 1.1, 1.15, 1.2, 1.25, 1.3, 1.35, 1.4, 1.45, or 1.5 ng / mL. In some embodiments, rhTGF-β1 is present at a concentration of about 2, 3, 4, 5, 6, 7, 8, 9, 10 ng / mL. In yet other embodiments, rhTGF-β1 is present at a concentration of about 15, 20, 25, 30, 35, 40, 45 or 50 ng / mL. In some embodiments, the culture in step (c) is carried out under hypoxic conditions or in the presence of a hypoxia inducer. In certain embodiments, the culture in step (c) is carried out for 1 to 3 days, such as 2 days.

[0012] In some embodiments, T RM -like T cells are CD69 + CD103 + positive. In certain embodiments, at least 30%, such as 40%, 45%, 50%, 55%, 60% or more, of the cells generated in step (c) are CD69 + CD103positive cells. In some embodiments, T + RM -like T cells express PD-1, CD101, and / or CD49a. In certain embodiments, PD-1, CD101, and / or CD49a expressed on T RM -like T cells are measured as cell surface expression (e.g., via flow cytometry). In certain embodiments, T RMT-like cells have a higher expression of CD69, ITGAE, PDCD1, and / or CD101 compared to cells cultured under atmospheric oxygen conditions. In certain embodiments, the higher expression of CD69, ITGAE, PDCD1, and / or CD101 is a higher expression of the CD69, ITGAE, PDCD1, and / or CD101 proteins compared to cells cultured under atmospheric oxygen conditions. In other embodiments, the higher expression of CD69, ITGAE, PDCD1, and / or CD101 is a higher expression of the CD69, ITGAE, PDCD1, and / or CD101 mRNA transcripts compared to cells cultured under atmospheric oxygen conditions.

[0013] In certain embodiments, T RM T-like cells have a higher expression of TNFA, GZMB, SLC2A1, and / or VEGF compared to cells cultured under atmospheric oxygen conditions. In certain embodiments, the higher expression of TNFA, GZMB, SLC2A1, and / or VEGF is a higher expression of the TNFα, GZMB, GLUT1, and / or VEGF proteins compared to cells cultured under atmospheric oxygen conditions. In other embodiments, the higher expression of TNFA, GZMB, SLC2A1, and / or VEGF is a higher expression of the TNFA, GZMB, SLC2A1, and / or VEGF mRNA transcripts compared to cells cultured under atmospheric oxygen conditions.

[0014] In some embodiments, T RM T-like cells have a decreased expression of S1PR1, KLF2, and / or SELL compared to cells cultured under atmospheric oxygen conditions. In some embodiments, T RM T-like cells have a decreased expression of S1PR1, KLF2, and / or CD62L protein compared to cells cultured under atmospheric oxygen conditions. In some embodiments, T RM T-like cells have a decreased expression of S1PR1, KLF2, and / or SELL mRNA transcripts compared to cells cultured under atmospheric oxygen conditions.

[0015] In specific embodiments, T RMThe T-like cells essentially do not have the expression of CXCR6 protein. In certain embodiments, the T RM -like cells have essentially undetectable cell surface expression of CXCR6 protein.

[0016] In some embodiments, the T RMLike T cells have higher expression of GNLY, MYO7A, ITGAE, EGR2, CCL20, ATP1B1, NR4A3, PERP, RASGEF1B, NR4A1, BMF, EGR1, CXCL13, PDCD1, ITGA1, CCL22, CA10, RGS1, ITGA1, CD101, TNFRSF9 (4-1BB), CCL4, CCL5, NOTCH1, RBPJ, STRIP2, ARHGEF40, DBH, SRGAP3, CSGALNACT1, GPR25, RGS16, DAPK2, NCS1, COL6A3, GDPD4, SLC1A4, CDK14, LMCD1, ILDR2, and / or ADCY3 compared to cells cultured under atmospheric oxygen conditions. In certain embodiments, higher expression of GNLY, MYO7A, ITGAE, EGR2, CCL20, ATP1B1, NR4A3, PERP, RASGEF1B, NR4A1, BMF, EGR1, CXCL13, PDCD1, ITGA1, CCL22, CA10, RGS1, ITGA1, CD101, TNFRSF9 (4-1BB), CCL4, CCL5, NOTCH1, RBPJ, STRIP2, ARHGEF40, DBH, SRGAP3, CSGALNACT1, GPR25, RGS16, DAPK2, NCS1, COL6A3, GDPD4, SLC1A4, CDK14, LMCD1, ILDR2, and / or ADCY3 protein. In certain embodiments, higher expression of GNLY, MYO7A, ITGAE, EGR2, CCL20, ATP1B1, NR4A3, PERP, RASGEF1B, NR4A1, BMF, EGR1, CXCL13, PDCD1, ITGA1, CCL22, CA10, RGS1, ITGA1, CD101, TNFRSF9 (4-1BB), CCL4, CCL5, NOTCH1, RBPJ, STRIP2, ARHGEF40, DBH, SRGAP3, CSGALNACT1, GPR25, RGS16, DAPK2, NCS1, COL6A3, GDPD4, SLC1A4, CDK14, LMCD1, ILDR2, and / or ADCY3 mRNA transcripts.

[0017] In some embodiments, T RMLike T cells have higher expression of GNLY, MYO7A, ITGAE, EGR2, CCL20, ATP1B1, NR4A3, PERP, RASGEF1B, NR4A1, BMF, EGR1, CXCL13, PDCD1, ITGA1, CCL22, CA10, and / or RGS1 compared to cells cultured under atmospheric oxygen conditions. In certain embodiments, the higher expression of GNLY, MYO7A, ITGAE, EGR2, CCL20, ATP1B1, NR4A3, PERP, RASGEF1B, NR4A1, BMF, EGR1, CXCL13, PDCD1, ITGA1, CCL22, CA10, and / or RGS1 is higher expression of the GNLY, MYO7A, ITGAE, EGR2, CCL20, ATP1B1, NR4A3, PERP, RASGEF1B, NR4A1, BMF, EGR1, CXCL13, PDCD1, ITGA1, CCL22, CA10, and / or RGS1 protein. In certain embodiments, the higher expression of GNLY, MYO7A, ITGAE, EGR2, CCL20, ATP1B1, NR4A3, PERP, RASGEF1B, NR4A1, BMF, EGR1, CXCL13, PDCD1, ITGA1, CCL22, CA10, and / or RGS1 is higher expression of the GNLY, MYO7A, ITGAE, EGR2, CCL20, ATP1B1, NR4A3, PERP, RASGEF1B, NR4A1, BMF, EGR1, CXCL13, PDCD1, ITGA1, CCL22, CA10, and / or RGS1 mRNA transcript.

[0018] In some embodiments, T RMThe like T cells have higher expression of ITGAE, ITGA1, PDCD1, CD101, TNFRSF9 (4-1BB), CXCL13, CCL20, NOTCH1, RBPJ, NR4A1, EGR2, and / or RGS1 as compared to cells cultured under atmospheric oxygen conditions. In certain embodiments, the higher expression of ITGAE, ITGA1, PDCD1, CD101, TNFRSF9 (4-1BB), CXCL13, CCL20, NOTCH1, RBPJ, NR4A1, EGR2, and / or RGS1 is higher expression of the ITGAE, ITGA1, PDCD1, CD101, TNFRSF9 (4-1BB), CXCL13, CCL20, NOTCH1, RBPJ, NR4A1, EGR2, and / or RGS1 protein. In certain embodiments, the higher expression of ITGAE, ITGA1, PDCD1, CD101, TNFRSF9 (4-1BB), CXCL13, CCL20, NOTCH1, RBPJ, NR4A1, EGR2, and / or RGS1 is higher expression of the ITGAE, ITGA1, PDCD1, CD101, TNFRSF9 (4-1BB), CXCL13, CCL20, NOTCH1, RBPJ, NR4A1, EGR2, and / or RGS1 mRNA transcript.

[0019] In some embodiments, T RMThe T-like cells have higher expression of MYO7A, STRIP2, ARHGEF40, ITGAE, DBH, SRGAP3, CSGALNACT1, GPR25, RGS16, DAPK2, NCS1, COL6A3, GDPD4, SLC1A4, CXCL13, CDK14, LMCD1, ILDR2, and / or ADCY3 compared to cells cultured under atmospheric oxygen conditions. In certain embodiments, the higher expression of MYO7A, STRIP2, ARHGEF40, ITGAE, DBH, SRGAP3, CSGALNACT1, GPR25, RGS16, DAPK2, NCS1, COL6A3, GDPD4, SLC1A4, CXCL13, CDK14, LMCD1, ILDR2, and / or ADCY3 is higher expression of the MYO7A, STRIP2, ARHGEF40, ITGAE, DBH, SRGAP3, CSGALNACT1, GPR25, RGS16, DAPK2, NCS1, COL6A3, GDPD4, SLC1A4, CXCL13, CDK14, LMCD1, ILDR2, and / or ADCY3 protein. In certain embodiments, the higher expression of MYO7A, STRIP2, ARHGEF40, ITGAE, DBH, SRGAP3, CSGALNACT1, GPR25, RGS16, DAPK2, NCS1, COL6A3, GDPD4, SLC1A4, CXCL13, CDK14, LMCD1, ILDR2, and / or ADCY3 is higher expression of the MYO7A, STRIP2, ARHGEF40, ITGAE, DBH, SRGAP3, CSGALNACT1, GPR25, RGS16, DAPK2, NCS1, COL6A3, GDPD4, SLC1A4, CXCL13, CDK14, LMCD1, ILDR2, and / or ADCY3 mRNA transcript.

[0020] In some embodiments, T RMThe γδ T cells have lower expression of CD58, NR3C1, RAP1GAP2, SELP, CXCR2, TBX21, ITGAL, SELL, KLF3, KLF2, RASGRP2, FAM65B, SERPINE2, ITGAM, KLRB1, TGFBR3, SMAD3, TNFSF8, DUSP2, PLEK, GOLGA2P7, FOSB, PLCG2, SLAMF7, SLC6A8, SOCS3, and / or PTGER2 compared to cells cultured under atmospheric oxygen conditions. In certain embodiments, the lower expression of CD58, NR3C1, RAP1GAP2, SELP, CXCR2, TBX21, ITGAL, SELL, KLF3, KLF2, RASGRP2, FAM65B, SERPINE2, ITGAM, KLRB1, TGFBR3, SMAD3, TNFSF8, DUSP2, PLEK, GOLGA2P7, FOSB, PLCG2, SLAMF7, SLC6A8, SOCS3, and / or PTGER2 is lower expression of the CD58, NR3C1, RAP1GAP2, SELP, CXCR2, TBX21, ITGAL, SELL, KLF3, KLF2, RASGRP2, FAM65B, SERPINE2, ITGAM, KLRB1, TGFBR3, SMAD3, TNFSF8, DUSP2, PLEK, GOLGA2P7, FOSB, PLCG2, SLAMF7, SLC6A8, SOCS3, and / or PTGER2 protein. In certain embodiments, the lower expression of CD58, NR3C1, RAP1GAP2, SELP, CXCR2, TBX21, ITGAL, SELL, KLF3, KLF2, RASGRP2, FAM65B, SERPINE2, ITGAM, KLRB1, TGFBR3, SMAD3, TNFSF8, DUSP2, PLEK, GOLGA2P7, FOSB, PLCG2, SLAMF7, SLC6A8, SOCS3, and / or PTGER2 is lower expression of the CD58, NR3C1, RAP1GAP2, SELP, CXCR2, TBX21, ITGAL, SELL, KLF3, KLF2, RASGRP2, FAM65B, SERPINE2, ITGAM, KLRB1, TGFBR3, SMAD3, TNFSF8, DUSP2, PLEK, GOLGA2P7, FOSB, PLCG2, SLAMF7, SLC6A8, SOCS3, and / or PTGER2 mRNA transcript.

[0021] In some embodiments, T RM regulatory T cells have lower expression of CD58, NR3C1, RAP1GAP2, SELP, CXCR2, TBX21, ITGAL, SELL, KLF3, RASGRP2, ITGAM, KLRB1, TGFBR3, SMAD3, and / or TNFSF8 compared to cells cultured under atmospheric oxygen conditions. In certain embodiments, the lower expression of CD58, NR3C1, RAP1GAP2, SELP, CXCR2, TBX21, ITGAL, SELL, KLF3, RASGRP2, ITGAM, KLRB1, TGFBR3, SMAD3, and / or TNFSF8 is lower expression of the CD58, NR3C1, RAP1GAP2, SELP, CXCR2, TBX21, ITGAL, SELL, KLF3, RASGRP2, ITGAM, KLRB1, TGFBR3, SMAD3, and / or TNFSF8 protein. In certain embodiments, the lower expression of CD58, NR3C1, RAP1GAP2, SELP, CXCR2, TBX21, ITGAL, SELL, KLF3, RASGRP2, ITGAM, KLRB1, TGFBR3, SMAD3, and / or TNFSF8 is lower expression of the CD58, NR3C1, RAP1GAP2, SELP, CXCR2, TBX21, ITGAL, SELL, KLF3, RASGRP2, ITGAM, KLRB1, TGFBR3, SMAD3, and / or TNFSF8 mRNA transcript.

[0022] In some embodiments, T RMLike T cells have higher expression of GNLY, MYO7A, ITGAE, EGR2, CCL20, ATP1B1, NR4A3, PERP, RASGEF1B, NR4A1, BMF, EGR1, CXCL13, PDCD1, ITGA1, CCL22, CA10, and / or RGS1 compared to cells cultured under atmospheric oxygen conditions. In certain embodiments, the higher expression of GNLY, MYO7A, ITGAE, EGR2, CCL20, ATP1B1, NR4A3, PERP, RASGEF1B, NR4A1, BMF, EGR1, CXCL13, PDCD1, ITGA1, CCL22, CA10, and / or RGS1 is higher expression of the GNLY, MYO7A, ITGAE, EGR2, CCL20, ATP1B1, NR4A3, PERP, RASGEF1B, NR4A1, BMF, EGR1, CXCL13, PDCD1, ITGA1, CCL22, CA10, and / or RGS1 protein. In certain embodiments, the higher expression of GNLY, MYO7A, ITGAE, EGR2, CCL20, ATP1B1, NR4A3, PERP, RASGEF1B, NR4A1, BMF, EGR1, CXCL13, PDCD1, ITGA1, CCL22, CA10, and / or RGS1 is higher expression of the GNLY, MYO7A, ITGAE, EGR2, CCL20, ATP1B1, NR4A3, PERP, RASGEF1B, NR4A1, BMF, EGR1, CXCL13, PDCD1, ITGA1, CCL22, CA10, and / or RGS1 mRNA transcript.

[0023] In some embodiments, T RMThe γδ T cells have higher expression of MYO7A, STRIP2, ARHGEF40, ITGAE, DBH, SRGAP3, CSGALNACT1, GPR25, RGS16, DAPK2, NCS1, COL6A3, GDPD4, SLC1A4, CXCL13, CDK14, LMCD1, ILDR2, and / or ADCY3 compared to cells cultured under atmospheric oxygen conditions. In certain embodiments, the higher expression of MYO7A, STRIP2, ARHGEF40, ITGAE, DBH, SRGAP3, CSGALNACT1, GPR25, RGS16, DAPK2, NCS1, COL6A3, GDPD4, SLC1A4, CXCL13, CDK14, LMCD1, ILDR2, and / or ADCY3 is higher expression of the MYO7A, STRIP2, ARHGEF40, ITGAE, DBH, SRGAP3, CSGALNACT1, GPR25, RGS16, DAPK2, NCS1, COL6A3, GDPD4, SLC1A4, CXCL13, CDK14, LMCD1, ILDR2, and / or ADCY3 protein. In certain embodiments, the higher expression of MYO7A, STRIP2, ARHGEF40, ITGAE, DBH, SRGAP3, CSGALNACT1, GPR25, RGS16, DAPK2, NCS1, COL6A3, GDPD4, SLC1A4, CXCL13, CDK14, LMCD1, ILDR2, and / or ADCY3 is higher expression of the MYO7A, STRIP2, ARHGEF40, ITGAE, DBH, SRGAP3, CSGALNACT1, GPR25, RGS16, DAPK2, NCS1, COL6A3, GDPD4, SLC1A4, CXCL13, CDK14, LMCD1, ILDR2, and / or ADCY3 mRNA transcript.

[0024] In a further embodiment, the method further comprises generating γδ T cells having specificity for an antigen of interest. In some embodiments, the γδ T cells having specificity for an antigen of interest RM are modified by transducing the γδ T cells with a T cell receptor (TCR) specific for the antigen of interest. In other embodiments, the γδ T cells having specificity for an antigen of interest RM RM RMThe T-like cells are generated by using a starting population of T cells having specificity for the antigen of interest. In some embodiments, the T RM -like cells are activated by culturing the starting population of T cells with peptide-pulsed antigen-presenting cells (APCs), such as artificial APCs (aAPCs), during step (b). In some embodiments, the APC is a mature dendritic cell. In a specific embodiment, steps (b) and (c) are repeated at least once. In some embodiments, the T RM -like cells are cultured in the presence of a histone deacetylase (HDAC) inhibitor during step (b) and / or step (c). In certain embodiments, the HDAC inhibitor is selected from the group consisting of trichostatin A, trapoxin B, phenylbutyric acid, valproic acid, vorinostat (suberanilohydroxamic acid or SAHA, commercially available as Zolinza®), belinostat (PXD101, commercially available as Beleodaq®), panobinostat (commercially available as Farydaq®), dacinostat (LAQ824), entinostat (SNDX-275 or MS-275), tacedinaline (CI994), and mocetinostat (MGCD0103).

[0025] In some embodiments, the antigen of interest is intended to target or treat lung cancer (including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), peritoneal cancer, gastric or stomach cancer (including gastrointestinal cancer and gastrointestinal stromal cancer), pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, various types of head and neck cancer, or melanoma.

[0026] Furthermore, T RM -like cells that do not express, substantially do not express, or essentially do not express the CXCR6 protein are provided herein. In some embodiments, the non-expression of the CXCR6 protein is non-cell surface expression of the CXCR6 protein. In other embodiments, the TRM γδ T cells express CXCR6 mRNA transcripts, but do not express CXCR6 protein or express CXCR6 protein on the cell surface. In some embodiments, the γδ RM T cells are specific for the antigen of interest. In another embodiment, a pharmaceutical composition comprising a population of γδ RM T cells as presented above is provided. In another embodiment, a pharmaceutical composition comprising a population of γδ T cells substantially without the expression of CXCR6 protein and a pharmaceutically acceptable carrier is provided. In some embodiments, the γδ RM T cells are generated by the methods of the present embodiment. In some embodiments, the γδ RM T cells express PD-1, CD101, and / or CD49a. In certain embodiments, at least 40%, at least 45%, at least 50%, at least 55%, at least 60% or more of the cells are CD69 RM CD103 + CD103 + cells. In certain embodiments, the γδ RM T cells are CD69 + CD103 + cells. In some embodiments, the γδ RM T cells have higher expression of GNLY, MYO7A, ITGAE, EGR2, CCL20, ATP1B1, NR4A3, PERP, RASGEF1B, NR4A1, BMF, EGR1, CXCL13, PDCD1, ITGA1, CCL22, CA10, RGS1, ITGA1, CD101, TNFRSF9 (4-1BB), CCL4, CCL5, NOTCH1, RBPJ, STRIP2, ARHGEF40, DBH, SRGAP3, CSGALNACT1, GPR25, RGS16, DAPK2, NCS1, COL6A3, GDPD4, SLC1A4, CDK14, LMCD1, ILDR2, and / or ADCY3 compared to cells cultured under atmospheric oxygen conditions. In some embodiments, the γδ RMThe γδ T cells have higher expression of GNLY, MYO7A, ITGAE, EGR2, CCL20, ATP1B1, NR4A3, PERP, RASGEF1B, NR4A1, BMF, EGR1, CXCL13, PDCD1, ITGA1, CCL22, CA10, and / or RGS1 compared to cells cultured under atmospheric oxygen conditions. In some embodiments, the γδ RM T cells have higher expression of ITGAE, ITGA1, PDC1, CD101, TNFRSF9 (4-1BB), CXCL13, CCL20, NOTCH1, RBPJ, NR4A1, EGR2, and / or RGS1 compared to cells cultured under atmospheric oxygen conditions. In some embodiments, the γδ RM T cells have higher expression of MYO7A, STRIP2, ARHGEF40, ITGAE, DBH, SRGAP3, CSGALNACT1, GPR25, RGS16, DAPK2, NCS1, COL6A3, GDPD4, SLC1A4, CXCL13, CDK14, LMCD1, ILDR2, and / or ADCY3 compared to cells cultured under atmospheric oxygen conditions. In some embodiments, the γδ RM T cells have lower expression of CD58, NR3C1, RAP1GAP2, SELP, CXCR2, TBX21, ITGAL, SELL, KLF3, KLF2, RASGRP2, FAM65B, SERPINE2, ITGAM, KLRB1, TGFBR3, SMAD3, and / or TNFSF8, DUSP2, PLEK, GOLGA2P7, FOSB, PLCG2, SLAMF7, SLC6A8, SOCS3, and / or PTGER2 compared to cells cultured under atmospheric oxygen conditions. In some embodiments, the γδ RM T cells have lower expression of CD58, NR3C1, RAP1GAP2, SELP, CXCR2, TBX21, ITGAL, SELL, KLF3, RASGRP2, ITGAM, KLRB1, TGFBR3, SMAD3, and / or TNFSF8 compared to cells cultured under atmospheric oxygen conditions. In some embodiments, the γδ RM T cells have lower expression of KLF2, KLF3, SELL, FAM65B, and / or SERPINE2 compared to cells cultured under atmospheric oxygen conditions. In some embodiments, the γδRM The γδ T cells have lower expression of DUSP2, PLEK, GOLGA2P7, FOSB, PLCG2, ITGAM, FOS, KLF3, SLAMF7, TNFSF8, SLC6A8, KLF2, SOCS3, and / or PTGER2 compared to cells cultured under atmospheric oxygen conditions.

[0027] In another embodiment, a composition comprising, in an effective amount, γδ T cells that are essentially free of CXCR6 protein expression, for example, γδ T cells generated by the methods of this embodiment, is provided for treating an immune-related disorder in a subject. RM γδ T cells, such as γδ T cells generated by the methods of this embodiment, RM are provided. In certain embodiments, the γδ T cells have specificity for an antigen of interest. RM The γδ T cells have specificity for an antigen of interest.

[0028] Further provided herein is the use of, in an effective amount, γδ T cells that are essentially free of CXCR6 protein expression, for example, γδ T cells generated by the methods of this embodiment, for treating an immune-related disorder in a subject. In certain embodiments, the γδ T cells have specificity for an antigen of interest. RM γδ T cells, such as γδ T cells generated by the methods of this embodiment, RM are provided. In certain embodiments, the γδ T cells have specificity for an antigen of interest. RM The γδ T cells have specificity for an antigen of interest.

[0029] In some embodiments, the antigen of interest is intended to target or treat lung cancer (including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), peritoneal cancer, gastric or stomach cancer (including gastrointestinal cancer and gastrointestinal stromal tumor), pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, various types of head and neck cancer, or melanoma.

[0030] In a further embodiment, a method for treating an immune-related disorder in a subject, the method comprising administering, in an effective amount, γδ T cells that are essentially free of CXCR6 protein expression, for example, γδ T cells generated by the methods of this embodiment, RM γδ T cells, such as γδ T cells generated by the methods of this embodiment, RMA method is provided that includes administering to a subject an effective amount of γδ T cells. In some embodiments, the subject is human.

[0031] In some embodiments, the immune-related disorder is cancer, autoimmune deficiency, graft-versus-host disease, allograft rejection, or an inflammatory condition. In certain embodiments, the subject has received a tissue or organ transplant.

[0032] In further embodiments, the method further includes administering at least one therapeutic agent. In some embodiments, the at least one second therapeutic agent includes chemotherapy, immunotherapy, surgery, radiation therapy, or biological therapy. In some embodiments, the γδ RM T cells and / or the at least one second therapeutic agent are administered intravenously, intraperitoneally, intratracheally, intratumorally, intramuscularly, endoscopically, intralesionally, percutaneously, subcutaneously, locally, or by direct injection or perfusion. In certain embodiments, the γδ RM T cells are administered prior to the second therapeutic agent. In some embodiments, the γδ RM T cells are administered after the second therapeutic agent. In certain embodiments, the γδ RM T cells are administered concurrently with the second therapeutic agent. In a specific embodiment, the immunotherapy is a 4-1BB agonist. In certain embodiments, the 4-1BB agonist is a 4-1BB antibody. In other embodiments, the second therapeutic agent is an immune checkpoint inhibitor. In certain embodiments, the immune checkpoint inhibitor is an anti-CTLA-4, anti-PD1, or anti-PD-L1 inhibitor.

[0033] In another embodiment, a method of treating a viral infection in a subject, the method comprising administering to the subject an effective amount of γδ T cells that are essentially devoid of CXCR6 expression, such as γδ T cells generated by the method, such as γδ T cells having specificity for one or more viral antigens. RM such as γδ T cells generated by the method RM such as γδ T cells having specificity for one or more viral antigens RM A method is provided that includes administering to a subject an effective amount of γδ T cells.

[0034] Other objects, features, and advantages of the present invention will become apparent from the following detailed description. However, while the detailed description and specific examples represent preferred embodiments of the present invention, it is to be understood that various changes and modifications within the spirit and scope of the present invention will become apparent to those skilled in the art from this detailed description, which is presented by way of illustration only.

[0035] The following drawings form a part of this specification and are included to further illustrate specific aspects of the present disclosure. The present disclosure may be more fully understood by referring to one or more of these drawings in combination with the detailed description of the specific embodiments presented herein.

Brief Description of the Drawings

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Figure 1

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DETAILED DESCRIPTION OF THE INVENTION

[0046] Tissue-resident memory cells (T RMare non-recirculating memory T cells present within tissues, lacking the molecules that enable their release from tissues and migration to lymph nodes, and act as frontline responders (Mami-Chouaib and Tartour, 2019). T RM Regarding differentiation, relatively little is known. Effector T cells that invade tissues can become T RM by upregulating or downregulating genes that enable tissue retention. In this study, it was found that hypoxia and TGF-β1 can induce a T + -like phenotype in human peripheral blood CD8 RM T cells. In this study, when human peripheral blood T cells, such as CD8 + T cells or CD4 + T cells, are differentiated in vitro under hypoxia and TGF-β1, they express a T RM phenotype and are shown to express protein markers and genes generally associated with tissue-resident memory cells (Table 1). These findings identify previously unreported factors regarding T RM differentiation and enable an easy means of generating T RM -phenotype cells for basic research and translational applications such as adoptive cell therapy.

[0047] Accordingly, certain embodiments of the present disclosure provide a method for generating T RM -phenotype cells. The terms "T RM -phenotype cells" and "T RMThe term "like cells" is used interchangeably herein to refer to the cells provided by the present method. The method can include culturing peripheral blood T cells under hypoxic conditions or in the presence of an agent that induces or mimics hypoxia. Exemplary hypoxia mimics include, but are not limited to, cobalt chloride (CoCl2), deferoxamine mesylate (DFOM), dimethyloxalylglycine (DMOG), or prolyl hydroxylase inhibitors such as roxadustat. During this period, the cells can be polyclonally activated, for example, by anti-CD3 and anti-CD28 beads, to generate early effector cells. The term "early effector cells" refers to cells within one week of activation from the naive state. Activation can include culturing in the presence of TCR stimulation and costimulation, including, but not limited to, anti-CD3 / anti-CD28 antibodies, anti-CD3 / anti-CD28 beads, feeder cells, antigen-presenting cells, artificial antigen-presenting cells, peptide and / or protein antigens, or combinations thereof. After activation to generate early effector cells, the cells are further cultured in the presence of TGF-β1 to generate T RM phenotype cells. Thus, hypoxia and TGF-β1 can be used to induce a CD8 RM cell population expressing human T + -associated markers CD69 + and CD103 + . Human CD8 T cells differentiated under hypoxia and TGF-β1 have T + -like transcriptional characteristics. RM

[0048] T RM -like cells can be antigen-specific. One method can include polyclonal activation of naive T cells under the conditions described herein for generating T RM -like cells, followed by transduction to express an antigen-specific TCR. In a modified version of the ETC stimulation method, naive T cells can be activated via peptide-pulsed antigen-presenting cells (or artificial antigen-presenting cells) under hypoxia and then further cultured in the presence of rh TGF-β1. This activation can be performed twice to generate antigen-specific cells. In another method, antigen-specific TRM Like cells may be generated by a combination of hypoxia and epigenetic modifiers such as TGF-β1 and HDAC inhibitors to differentiate the already expanded antigen-specific cells into a T RM phenotype.

[0049] The present T cells, for example, the starting population of T cells, may be modified T cells. In certain embodiments, the modified T cells include T cells expressing a chimeric antigen receptor (CAR T cells). In certain embodiments, the modified T cells include T cells expressing a recombinant T cell receptor capable of binding to a tumor-specific epitope or neoepitope. In some embodiments, the modified T cells are constructed using any of a number of well-established gene transfer methods known to those skilled in the art. In certain embodiments, the modified cells are constructed using a gene transfer method based on a viral vector for introducing a nucleic acid encoding a chimeric antigen receptor specific for a desired target tumor antigen or a nucleic acid encoding a recombinant TCR specific for a desired tumor-specific epitope or neoepitope. In certain embodiments, the modified cells are constructed using a gene transfer method based on a non-viral vector for introducing a nucleic acid encoding a chimeric antigen receptor specific for a desired target tumor antigen or a nucleic acid encoding a recombinant TCR specific for a desired tumor-specific epitope or neoepitope. In certain embodiments, the gene transfer method based on a viral vector includes a lentiviral vector. In certain embodiments, the gene transfer method based on a viral vector includes a retroviral vector. In certain embodiments, the gene transfer method based on a viral vector includes an adenovirus or an adeno-associated viral vector. The gene transfer method based on a non-viral vector may include an episomal vector or a transposon-transposase system. For example, the transposon-transposase system can be the well-known Sleeping Beauty, Frog Prince transposon-transposase system, or the PiggyBac transposon system specific for TTAA. In certain embodiments, the gene transfer method based on a non-viral vector includes a gene editing method selected from the group consisting of zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated protein 9 (Cas9) nucleases.In certain embodiments, gene editing methods based on non-viral vectors include transfection or transformation methods selected from the group consisting of lipofection, nucleofection, particle bombardment, virosomes, liposomes, polycations or lipid: nucleic acid conjugates, naked DNA, artificial virions, and agent-enhanced uptake of DNA.

[0050] In certain embodiments, CAR T cells express a CAR construct comprising an extracellular antigen-binding domain, an optional spacer sequence, a transmembrane domain, one or more intracellular signaling domains, and one or more optional control sequences for activating or inactivating the CAR T cells.

[0051] In certain embodiments, the extracellular antigen-binding domain comprises a portion capable of specifically binding to a desired target. In certain embodiments, the portion capable of specifically binding to a desired target comprises a monoclonal antibody or an antigen-binding fragment thereof. In certain embodiments, the antigen-binding fragment comprises a single-chain variable fragment (scFv) of a monoclonal antibody capable of specifically binding to a desired target. In certain embodiments, the desired target is a tumor-specific antigen. In certain embodiments, the tumor-specific antigen is selected from the group consisting of CD19, CD20, CD22, carcinoembryonic antigen, alpha-fetoprotein, CA-125, MUC-1, epithelial tumor antigen, melanoma-associated antigen (MAGE) (e.g., MAGE-1, MAGE-11, or MAGE-A), mutant p53, mutant ras, HER2 / Neu, ERBB2, folate-binding protein, HIV-1 envelope glycoprotein gp120, HIV-1 envelope glycoprotein gp41, GD2, CD123, CD23, CD30, CD56, c-Met, mesothelin, GD3, HERV-K, IL-11Rα, kappa chain, lambda chain, CSPG4, ERBB2, EGFRvIII, VEGFR2, and human papillomavirus (HPV). In certain embodiments, the desired target is a tumor-specific neoepitope. In certain embodiments, the tumor-specific neoepitope is identified by in silico analysis. In certain embodiments, the tumor-specific neoepitope is identified and purified from a population of autologous TILs derived from a human subject.

[0052] In certain embodiments, the transmembrane domain comprises any synthetic or natural amino acid sequence capable of forming a structure that can crosslink cell membranes. In certain embodiments, the structure capable of crosslinking cell membranes comprises an α helix. In certain embodiments, the transmembrane region is derived from a naturally occurring transmembrane protein selected from the group consisting of CD3ζ, CD3ε, CD4, CD5, CD8, CD9, CD16, CD22, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, 4-1BB / CD137, CD154, inducible T cell co-stimulatory molecule (ICOS) / CD278, glucocorticoid-induced TNFR-related protein (GITR) / CD357, NKG2D, TCRα, and TCRβ. In certain embodiments, the transmembrane region derived from a naturally occurring transmembrane protein comprises one or more amino acid substitutions within a sequence known to be involved in the interaction with other signaling proteins.

[0053] In certain embodiments, one or more intracellular signaling domains comprise one or more intracellular tyrosine-based activation motifs (「ITAM」). In certain embodiments, one or more ITAMs are present on the CD3 zeta (CD3ζ) molecule. In certain embodiments, one or more intracellular signaling domains further comprise a co-stimulatory signaling domain selected from the group consisting of CD28, 4-1BB / CD137, ICOS, OX40, CD2, CD40L, CD27, Light-R, GITR, or combinations thereof.

[0054] In certain embodiments, the T cells comprise a recombinant T cell receptor capable of binding to a tumor-specific epitope or neoepitope. In certain embodiments, the recombinant T cell receptor comprises a naturally occurring TCR cloned from T cells isolated from a subject. In certain embodiments, the recombinant TCR comprises a heterodimer (i.e., TCRαβ) comprising a TCR alpha (TCRα) polypeptide and a TCR beta (TCRβ) polypeptide. In certain embodiments, the recombinant TCR comprises a heterodimer (i.e., TCRγδ) comprising a TCR gamma (TCRγ) polypeptide and a TCR delta (TCRδ) polypeptide.

[0055] In certain embodiments, the recombinant TCRαβ comprises a cloned TCRαβ that is isolated from a subject and specific for a peptide antigen derived from a desired target. In certain embodiments, the subject is a mammal. In certain embodiments, the mammal is a human. In certain embodiments, the desired target is a tumor-specific antigen selected from the group consisting of CD19, CD20, CD22, carcinoembryonic antigen, alpha-fetoprotein, CA-125, MUC-1, epithelial tumor antigen, melanoma-associated antigen, mutant p53, mutant ras, HER2 / Neu, ERBB2, folate-binding protein, HIV-1 envelope glycoprotein gp120, HIV-1 envelope glycoprotein gp41, GD2, CD123, CD23, CD30, CD56, c-Met, mesothelin, GD3, HERV-K, IL-11Rα, kappa chain, lambda chain, CSPG4, ERBB2, EGFRvIII, and VEGFR2. In certain embodiments, the recombinant TCRγδ comprises a cloned TCRγδ that is isolated from a subject and specific for a peptide antigen derived from a desired target. In certain embodiments, the subject is a mammal. In certain embodiments, the mammal is a human. In certain embodiments, the desired target is a tumor-specific antigen selected from the group consisting of CD19, CD20, CD22, carcinoembryonic antigen, alpha-fetoprotein, CA-125, MUC-1, epithelial tumor antigen, melanoma-associated antigen, mutant p53, mutant ras, HER2 / Neu, ERBB2, folate-binding protein, HIV-1 envelope glycoprotein gp120, HIV-1 envelope glycoprotein gp41, GD2, CD123, CD23, CD30, CD56, c-Met, mesothelin, GD3, HERV-K, IL-11Rα, kappa chain, lambda chain, CSPG4, ERBB2, EGFRvIII, and VEGFR2.

[0056] Furthermore, provided herein are methods contemplated for the use of the T RM cells provided herein, for example, in adoptive cell therapy for treating cancer or viral diseases. The cells may be used for immunosuppression, for example, in subjects having graft-versus-host disease (GVHD), tissue or organ rejection, or an autoimmune condition.

[0057]

Table 1

[0058] I. Definitions As used herein, "essentially free of" means, with respect to a particular component, that none of the particular components are intentionally formulated in the composition and / or are present only as contaminants or in trace amounts. Thus, the total amount of the particular component due to any unintentional contamination of the composition is well less than 0.05%, preferably less than 0.01%. Most preferably, the composition is such that the amount of the particular component cannot be detected by standard analytical methods.

[0059] As used herein, "a" or "an" may mean one or more. When used in the claims herein and in conjunction with the term "comprising", the term "a" or "an" may mean one or more than one.

[0060] The use of the term "or" in the claims is used to mean "and / or" unless explicitly stated otherwise to refer only to alternatives or that the alternatives are mutually exclusive, although the disclosure supports definitions that refer only to alternatives and "and / or". As used herein, "another" may mean at least a second or more. The terms "about", "substantially" and "approximately" generally mean plus or minus 5% of a specified value.

[0061] "Autoimmune disease" refers to a disease in which the immune system generates an immune response (e.g., a B cell or T cell response) against an antigen that is part of the normal host (i.e., a self-antigen), resulting in tissue damage. The self-antigen may be derived from host cells or from symbiotic organisms such as microorganisms that normally colonize mucosal surfaces (known as symbionts).

[0062] The term "graft-versus-host disease (GVHD)" refers to a serious complication common to bone marrow or other tissue transplants, where a reaction of immunologically competent lymphocytes provided against the recipient's own tissues is observed. GVHD is a complication that can occur with any graft that uses or contains stem cells derived from either a related or unrelated donor. In some embodiments, GVHD is chronic GVHD (cGVHD).

[0063] As used herein, the terms "chimeric antigen receptor", "CAR", "chimeric T cell receptor", "artificial T cell receptor", or "chimeric immune receptor" refer to a modified chimeric receptor construct that confers a desired non-MHC-restricted antigen-binding specificity onto an immune effector cell, such as an effector T cell. A CAR may include, for example, an extracellular antigen-binding domain (e.g., an antibody or antibody fragment, such as a single-chain variable fragment (scFv) having a desired antigen specificity), a spacer sequence, a transmembrane domain, and one or more intracellular signaling domains. Exemplary intracellular signaling domains may include one or more intracellular tyrosine-based activation motifs ("ITAM"), such as CD3 zeta (CD3ζ), and / or one or more co-stimulatory signaling domains, such as CD28, 4-1BB / CD137, ICOS, OX40, or combinations thereof.

[0064] As used herein, the terms "treat", "treatment", "treating", etc. refer to the process of alleviating, reducing, or mitigating the symptoms of a disease or condition in a subject, for example, by administering a therapeutic agent to the subject or by performing a surgical, clinical, or other medical procedure on the subject.

[0065] As used herein, the terms "subject" or "patient" are used interchangeably herein to refer to an individual, such as a human or non-human organism, such as a primate, mammal, or vertebrate.

[0066] As used throughout this application, the terms "therapeutic benefit" or "therapeutically effective" refer to those that promote or enhance the health of a subject with respect to the medical treatment of this condition. This includes, but is not limited to, a decrease in the frequency or severity of the signs or symptoms of the disease. For example, the treatment of cancer may include, for example, a decrease in tumor size, a reduction in tumor invasiveness, a decrease in the cancer growth rate, or prevention of metastasis. The treatment of cancer may also refer to prolonging the survival of a subject having cancer.

[0067] As generally used herein, "pharmaceutically acceptable" refers to compounds, materials, compositions, and / or dosage forms that are within the scope of sound medical judgment, suitable for use in contact with human and animal tissues, organs, and / or body fluids, without undue toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable risk-benefit ratio.

[0068] "Pharmaceutically acceptable salts", as defined above, mean salts of the compounds disclosed herein that are pharmaceutically acceptable and have the desired pharmacological activity. Such salts include acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid; or 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, 2-naphthalenesulfonic acid, 3-phenylpropionic acid, 4,4'-methylenebis(3-hydroxy-2-ene-1-carboxylic acid), 4-methylbicyclo[2.2.2]oct-2-ene-1-carboxylic acid, acetic acid, aliphatic mono- and dicarboxylic acids, aliphatic sulfuric acids, aromatic sulfuric acids, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, carbonic acid, cinnamic acid, citric acid, cyclopentanepropionic acid, ethanesulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, heptanoic acid, hexanoic acid, hydroxynaphthoic acid, lactic acid, lauryl sulfuric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, muconic acid, o-(4-hydroxybenzoyl)benzoic acid, oxalic acid, p-chlorobenzenesulfonic acid, phenyl-substituted alkanoic acids, propionic acid, p-toluenesulfonic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, tartaric acid, tertiary butylacetic acid, trimethylacetic acid and other organic acids. Pharmaceutically acceptable salts also include base addition salts that may be formed when the acidic proton present is capable of reacting with an inorganic or organic salt. Acceptable inorganic salts include sodium hydroxide, sodium carbonate, potassium hydroxide, aluminum hydroxide and calcium hydroxide. Acceptable organic salts include ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine and the like. It should be understood that the specific anions or cations that form part of any salt of the present invention are not critical as long as the salt as a whole is pharmaceutically acceptable. Further examples of pharmaceutically acceptable salts and their preparation and use are presented in Handbook of Pharmaceutical Salts: Properties, and Use (P.H. Stahl & C.G. Wermuth eds., Verlag Helvetica Chimica Acta, 2002).

[0069] "Pharmaceutically acceptable carrier", "drug carrier", or simply "carrier" is a pharmaceutically acceptable substance formulated with an active ingredient formulation that is involved in transporting, delivering, and / or transporting a chemical. For example, a drug carrier for improving drug delivery and effectiveness, including sustained release techniques for modulating drug bioavailability, reducing drug metabolism, and / or reducing drug toxicity, may be used. Some drug carriers may enhance the effectiveness of drug delivery to a specific target site. Examples of carriers include liposomes, microparticles (e.g., made of lactic acid-glycolic acid copolymer), albumin microspheres, synthetic polymers, nanofibers, protein-DNA complexes, protein conjugates, red blood cells, virosomes, and dendrimers.

[0070] The term "culturing" refers to the in vitro maintenance, differentiation, and / or proliferation of cells in a suitable medium. "Enriched" means a composition containing cells that are present in a higher percentage of total cells than is found in the tissue when present in an organism.

[0071] An "isolated" biological component (a part of a blood material, e.g., a blood component) refers to a component that is substantially separated or purified from other biological components of an organism in which it naturally occurs. An isolated cell is one that is substantially separated or purified from other biological components of an organism in which it naturally occurs.

[0072] II. Method of Use In some embodiments, the present disclosure provides a method for adoptive cell therapy, the method comprising administering an effective amount of the T RM cells of the present disclosure. In certain embodiments of the present disclosure, a cancer or viral disease is treated by adoptive transfer of a T RM cell population that induces an immune response. In some embodiments, the T RM cell population itself will mediate an immune response. The T RMOnce activated in vivo, cells may produce various pro-inflammatory factors, such as chemokines and cytokines, which will induce an immune response. A method for treating or delaying the progression of cancer in an individual, the method comprising administering to the individual an effective amount of a T RM cell population is provided herein. The method may be applied for treating immune disorders, solid tumors, blood cancers, and viral infections. For example, the viral infection to be treated according to an embodiment may be an HIV, HBV, or herpes virus infection.

[0073] The tumors in cases where the present treatment method is useful include those found in any malignant cell type, such as solid tumors or blood tumors. Exemplary solid tumors include, but are not limited to, tumors of organs selected from the group consisting of pancreas, colon, cecum, stomach, brain, head, neck, ovary, kidney, larynx, sarcoma, lung, bladder, melanoma, prostate, and chest. Exemplary blood tumors include tumors of the bone marrow, T cell or B cell malignancies, leukemia, lymphoma, blastoma, myeloma, and the like. Further examples of cancers that may be treated using the methods provided herein include, but are not limited to, lung cancer (including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous cell carcinoma of the lung), peritoneal cancer, gastric or stomach cancer (including gastrointestinal cancer and gastrointestinal stromal cancer), pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, various types of head and neck cancer, and melanoma.

[0074] Cancer may specifically be of the following histological types, which are neoplasm, malignant; cancer; cancer, undifferentiated; giant cell and spindle cell cancer; small cell cancer; papillary cancer; squamous cell cancer; lymphoepithelial cancer; basal cell cancer; trichoblastoma; transitional cell cancer; papillary transitional cell cancer; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular and cholangiocarcinoma; columnar adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial adenomatous polyposis; solid cancer; carcinoid tumor, malignant; bronchioloalveolar adenocarcinoma; papillary adenocarcinoma; chromophobic cancer; eosinophilic cancer; eosinophilic adenocarcinoma; basophilic cancer; clear cell adenocarcinoma; granular cell cancer; follicular adenocarcinoma; papillary-follicular adenocarcinoma; unencapsulated sclerosing carcinoma; adrenocortical carcinoma; endometrioid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous gland carcinoma; ceruminous gland carcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating ductal carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; Paget's disease, breast; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma associated with squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; capsular cell tumor, malignant; granulosa cell tumor, malignant; androblastoma, malignant; sertoli cell carcinoma; leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extra-mammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficially spreading melanoma; malignant lentigo melanoma; acral lentiginous melanoma; nodular melanoma; malignant melanoma in giant congenital nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; fetal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; müllerian duct mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymal tumor, malignant; Brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; undifferentiated embryonal cell tumor; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; angiosarcoma; vascular endothelioma, malignant; Kaposi sarcoma; hemangioendothelioma, malignant; lymphangiosarcoma; osteosarcoma; parosteal osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; Ewing sarcoma; odontogenic tumor, malignant; ameloblastic odontogenic sarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; epithelioma;Astrocytoma; Protoplasmic astrocytoma; Fibrous astrocytoma; Astroblastoma; Glioblastoma; Anaplastic glioma; Anaplastic glioblastoma; Undifferentiated neuroectodermal; Cerebellar sarcoma; Ganglioblastoma; Neuroblastoma; Retinoblastoma; Olfactory neurogenic tumor; Meningioma, malignant; Neurofibrosarcoma; Schwannoma, malignant; Granular cell tumor, malignant; Malignant lymphoma; Hodgkin's disease; Hodgkin's lymphoma (hodgkin’s); Lateral granuloma; Malignant lymphoma, small lymphocytic; Malignant lymphoma, large cell, diffuse; Malignant lymphoma, follicular; Mycosis fungoides; Other specified non-Hodgkin lymphoma; B-cell lymphoma; Low-grade / follicular non-Hodgkin lymphoma (NHL); Small lymphocytic (SL) NHL; Intermediate-grade / follicular NHL; Intermediate-grade diffuse NHL; High-grade immunoblastic NHL; High-grade lymphoblastic NHL; High-grade small non-cleaved cell NHL; Large tumor lesion NHL; Mantle cell lymphoma; AIDS-related lymphoma; Waldenström macroglobulinemia; Malignant histiocytosis; Multiple myeloma; Mast cell sarcoma; Immunoproliferative small intestinal disease; Leukemia; Lymphocytic leukemia; Plasma cell leukemia; Erythroleukemia; Lymphosarcoma cell leukemia; Myelogenous leukemia; Basophilic leukemia; Eosinophilic leukemia; Monocytic leukemia; Mast cell leukemia; Megakaryoblastoid leukemia; Myelosarcoma; Hairy cell leukemia; Chronic lymphocytic leukemia (CLL); Acute lymphoblastic leukemia (ALL); Acute myelogenous leukemia (AML); And is not limited to chronic myelogenous leukemia.

[0075] Certain embodiments of the present disclosure relate to methods of treating leukemia. Leukemia is a cancer of the blood or bone marrow, characterized by the abnormal proliferation (production by proliferation) of blood cells, usually white blood cells (leukocytes). It is part of a broad group of diseases known as blood tumors. Leukemia is a broad term encompassing the spectrum of the disease. Leukemia is clinically and pathologically classified as acute and chronic diseases.

[0076] In certain embodiments of the present disclosure, T RMThe cells are delivered to an individual in need thereof, such as an individual having cancer or an infection, such as a bacterial or viral infection. Next, the cells attack each of the cancerous or pathogenic cells by enhancing the individual's immune system. In some cases, the individual is provided with one or more doses of the cells. When the individual is provided with two or more doses of immune cells, the period between administrations needs to be sufficient to allow for a period of growth in the individual. In a specific embodiment, the period between administrations is 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or more.

[0077] Certain embodiments of the present disclosure provide methods for treating or preventing immune-mediated disorders. In one embodiment, the subject has an autoimmune disease. Non-limiting examples of autoimmune diseases include alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune adrenal diseases, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune oophoritis and orchitis, autoimmune thrombocytopenia, Behçet's disease, bullous pemphigoid, cardiomyopathy, celiac spate-dermatitis, chronic fatigue immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, cicatricial pemphigoid, CREST syndrome, cold agglutinin disease, Crohn's disease, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, glomerulonephritis, Graves' disease, Guillain-Barré, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (ITP), IgA neuropathy, juvenile arthritis, lichen planus, lupus erythematosus, Ménière's disease, mixed connective tissue disease, multiple sclerosis, type 1 or immune-mediated diabetes, myasthenia gravis, nephrotic syndrome (such as minimal change group, focal segmental glomerulosclerosis, or membranous nephropathy), pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndrome, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynaud's phenomenon, Reiter's syndrome, rheumatoid arthritis, sarcoidosis, scleroderma, Sjögren's syndrome, stiff-man syndrome, systemic lupus erythematosus, ulcerative colitis, uveitis, vasculitis (such as polyarteritis nodosa, Takayasu arteritis, temporal arteritis / giant cell arteritis, or herpes zoster vasculitis), vitiligo, and Wegener's granulomatosis. Thus, some examples of autoimmune diseases treatable using the methods disclosed herein include, but are not limited to, multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, type I diabetes, Crohn's disease; ulcerative colitis, myasthenia gravis, glomerulonephritis, ankylosing spondylitis, vasculitis, or psoriasis. The subject may also have an allergic disorder such as asthma.

[0078] In yet another embodiment, the subject is a recipient of a transplanted organ, and stem cells and T RM cells are used to prevent and / or treat rejection. In certain embodiments, the subject has or is at risk of developing graft-versus-host disease. GVHD is a complication that can occur in any transplant that uses or contains stem cells from either a related or unrelated donor. There are two types of GVHD, acute and chronic. Acute GVHD appears within three months of transplantation. Symptoms of acute GVHD include a reddish rash on the hands and feet that may be accompanied by peeling or blistering skin, which spreads and can become more severe. Acute GVHD can also affect the gastrointestinal tract, in which case it presents with cramps, nausea, and diarrhea. Jaundice of the skin and eyes indicates that acute GVHD is affecting the liver. Chronic GVHD is ranked based on its severity: stage / grade 1 is mild; stage / grade 4 is severe. Chronic GVHD develops three months or more after transplantation. The symptoms of chronic GVHD are similar to those of acute GVHD, but chronic GVHD can also affect the mucous membranes of the eyes, the salivary glands of the mouth, and the glands that lubricate the stomach wall and intestines. Any of the populations of immune cells disclosed herein can be utilized. Examples of transplanted organs include solid organ grafts such as kidneys, livers, skin, pancreas, lungs, and / or hearts, or cell grafts such as islets, hepatocytes, myoblasts, bone marrow, or hematopoietic or other stem cells. The graft can be a composite graft such as facial tissue. The immune cells can be administered before, simultaneously with, or after transplantation. In some embodiments, the immune cells are administered before transplantation, for example, at least 1 hour, at least 12 hours, at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, or at least 1 month before transplantation. In one specific non-limiting example, administration of a therapeutically effective amount of immune cells is made 3 to 5 days before transplantation.

[0079] In some embodiments, the subject is a T RMPrior to the cell population, non-myeloablative lymphocyte depletion chemotherapy can be administered. The non-myeloablative lymphocyte depletion chemotherapy can be any suitable such treatment method and can be administered by any suitable route. The non-myeloablative lymphocyte depletion chemotherapy can include, for example, the administration of cyclophosphamide and fludarabine, particularly when the cancer is melanoma and it can be metastatic. An exemplary route for administering cyclophosphamide and fludarabine is intravenously. Similarly, cyclophosphamide and fludarabine can be administered at any suitable dose. In certain embodiments, about 60 mg / kg of cyclophosphamide is administered for 2 days, followed by 2 about 25 mg / m

[0080] of fludarabine administered for 5 days. RM In certain embodiments, a growth factor that promotes the proliferation and activation of the T RM cell population is administered to the subject either simultaneously with the T RM cell population or subsequent to the immune cells. The growth factor can be any suitable growth factor that promotes the proliferation and activation of the T

[0081] cell population. Examples of suitable immune cell growth factors include interleukin (IL)-2, IL-7, IL-15, and IL-12, which can be used alone or in various combinations such as IL-2 and IL-7, IL-2 and IL-15, IL-7 and IL-15, IL-2, IL-7 and IL-15, IL-12 and IL-7, IL-12 and IL-15, or IL-12 and IL2. RM A therapeutically effective amount of T

[0082] cells can be administered by several routes including parenteral administration, for example, intravenous, intraperitoneal, intramuscular, intrasternal, or intra-articular injection, or infusion. RM The therapeutically effective amount of T RMIt may be the amount of cells. It may be the amount necessary to reduce symptoms associated with inflammation, such as pain, edema, and elevated body temperature. It may also be the amount necessary to reduce or prevent rejection of a transplanted organ.

[0083] T RM The cells can be administered according to a treatment plan that conforms to standard treatments for treating a disease, such as a single or multiple administrations over 1 day to several days to remit the condition, or long-term periodic administrations to inhibit disease progression and prevent disease recurrence. The exact dosage to be utilized in the formulation will also depend on the route of administration and the severity of the disease or disorder and will need to be determined according to the judgment of the practitioner and the circumstances of each patient. The therapeutically effective amount of immune cells will depend on the subject being treated, the severity and type of affliction, and the mode of administration. In some embodiments, the dosage that can be used in the treatment of a human subject is at least 3.8×10 4 cells, at least 3.8×10 5 cells, at least 3.8×10 6 cells, at least 3.8×10 7 cells, at least 3.8×10 8 cells, at least 3.8×10 9 cells, or at least 3.8×10 10 cells / m 2 of the body. In certain embodiments, the dosage used in the treatment of a human subject is in the range of about 3.8×10 9 cells to about 3.8×10 10 cells / m 2 of the body. In further embodiments, the therapeutically effective amount of immune cells is about 5×10 6 cells / kg body weight to about 7.5×10 8 cells / kg body weight, such as about 2×10 7 cells / kg body weight to about 5×10 8 cells / kg body weight, or about 5×10 7 cells / kg body weight to about 2×10 8It can vary by the number of cells / kg body weight. The exact amount of immune cells can be readily determined by those skilled in the art based on the age, weight, gender, and physiological state of the subject. The effective dose can be extrapolated from the dose-response curve derived from in vitro or animal model test systems.

[0084] T RM The cells may be administered in combination with one or more other therapeutic agents for the treatment of immune-mediated disorders. Examples of combination therapy agents include, but are not limited to, one or more antimicrobial agents (such as antibiotics, antiviral drugs, and antifungal drugs), antitumor agents (such as fluorouracil, methotrexate, paclitaxel, fludarabine, etoposide, doxorubicin, or vincristine), immune-depleting agents (such as fludarabine, etoposide, doxorubicin, or vincristine), immunosuppressive agents (such as azathioprine, or glucocorticoids such as dexamethasone or prednisone), anti-inflammatory agents (such as glucocorticoids such as hydrocortisone, dexamethasone or prednisone, or non-steroidal anti-inflammatory agents such as acetylsalicylic acid, ibuprofen or naproxen sodium), cytokines (such as interleukin-10 or transforming growth factor-β), hormones (such as estrogen), or vaccines. Furthermore, but not limited to, calcineurin inhibitors (such as cyclosporine and tacrolimus); mTOR inhibitors (such as rapamycin); mycophenolate mofetil, antibodies (such as those recognizing CD3, CD4, CD40, CD154, CD45, IVIG, or B cells); chemotherapeutic agents (such as methotrexate, treosulfan, busulfan); irradiation; or immunosuppressive or immunotolerogenic agents including chemokines, interleukins or their inhibitors (such as BAFF, IL-2, anti-IL-2R, IL-4, JAK kinase inhibitors) can be administered. Such additional pharmaceuticals can be administered before, during, or after the administration of the immune cells, depending on the desired effect. This administration of the cells and the agents can be by the same route or by different routes, and at the same site or different sites.

[0085] A. Combination Therapy In certain embodiments, the methods provided herein further comprise administering to the subject at least one additional therapeutic agent. All additional therapeutic agents disclosed herein will be administered to the subject in accordance with good clinical practice standards for each specific composition or treatment, taking into account any potential toxicity, possible side effects, and any other relevant factors.

[0086] In certain embodiments, the additional therapy may be immunotherapy, radiation therapy, surgery (e.g., surgical resection of a tumor), chemotherapy, bone marrow transplantation, or a combination of the foregoing. The additional therapy may be a targeted therapy. In certain embodiments, the additional therapy is administered prior to primary treatment (i.e., in the case of adjuvant therapy). In certain embodiments, the additional therapy is administered after primary treatment (i.e., in the case of neoadjuvant therapy).

[0087] In certain embodiments, the additional therapy includes immunotherapy. In certain embodiments, the immunotherapy includes immune checkpoint inhibitors. In certain embodiments, the immune checkpoint inhibitors inhibit immune checkpoint proteins selected from the group consisting of programmed cell death pathway 1 (PD-1 / CD279) and its ligands (PD-L1 / CD274 and PD-L2 / CD273), cytotoxic T lymphocyte-associated antigen 4 (CTLA-4 / CD152), lymphocyte activation gene 3 (LAG-3 / CD223), B and T lymphocyte attenuator (BTLA), T cell immunoreceptor with Ig and immunoreceptor tyrosine-based inhibitory motif (ITIM) domain (TIGIT), T cell immunoglobulin domain and mucin domain 3 (TIM-3 / HAVcr2), killer immunoglobulin-like receptor (KIR / CD158), V domain immunoglobulin suppressor of T cell activation (VISTA), and adenosine A2a receptor (A2aR). In some aspects, the immunotherapeutic agent is a 4-1BB agonist. Exemplary 4-1BB agonists include, but are not limited to, 4-1BB agonist antibodies (e.g., utomilumab), recombinant 4-1BB (including, but not limited to, soluble, matrix-bound scaffold-bound forms), and 4-1BB aptamers.

[0088] In certain embodiments, the immune checkpoint inhibitor is a PD-1 binding antagonist. In certain embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody. In certain embodiments, the anti-PD-1 antibody is selected from the group consisting of nivolumab, pembrolizumab, and CT-011. In certain embodiments, the PD-1 binding antagonist is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular or PD-1 binding portion of PDL1 or PDL2 fused to an immunoglobulin constant region (e.g., the Fc region of an immunoglobulin sequence)).

[0089] In certain embodiments, the immune checkpoint inhibitor is a CTLA-4 binding antagonist. In certain embodiments, the CTLA-4 binding antagonist is an anti-CTLA-4 antibody. In certain embodiments, the anti-CTLA-4 antibody is selected from the group consisting of ipilimumab and tremelimumab.

[0090] In certain embodiments, the additional therapeutic agent includes treatment by radiotherapy. In certain embodiments, the radiotherapy is selected from the group consisting of gamma ray (γ-ray), X-ray, microwave, proton beam irradiation, ultraviolet irradiation, and direct delivery of a radioisotope to the tumor. In certain embodiments, the radiotherapy includes treatment by X-ray. In certain embodiments, the X-ray is administered at a daily dose of 50 to 200 roentgens over 3 to 4 weeks. In certain embodiments, the X-ray is administered at a single dose of 2000 to 6000 roentgens. In certain embodiments, the radiotherapy includes direct delivery of a radioisotope to the tumor. The dose range for the radioisotope varies widely depending on the half-life of the isotope, the intensity and type of the emitted radiation, and the degree of uptake by the tumor cells, but the determination of an appropriate therapeutically effective amount is within the level of those skilled in the art.

[0091] In certain embodiments, the additional therapeutic agent includes administration of an agent for treating side effects (such as nausea, cachexia, etc.) associated with the primary treatment. In certain embodiments, the additional treatment method includes immunotherapy. In certain embodiments, the additional treatment method includes radiotherapy. In some embodiments, the radiotherapy includes gamma ray irradiation. In certain embodiments, the additional treatment method includes surgery. In certain embodiments, the additional treatment method includes a combination of radiotherapy and surgery. In certain embodiments, the additional treatment method includes treatment by a class of chemotherapeutic agents selected from the group consisting of alkylating agents, anthracyclines, cytoskeleton disrupting agents, epothilones, histone deacetylase inhibitors, topoisomerase I inhibitors, topoisomerase II inhibitors, kinase inhibitors, nucleotide analogs and nucleotide precursor analogs, peptide antibiotics, platinum-based compounds, retinoids, vinca alkaloids, and derivatives thereof.

[0092] Additional therapies contemplated herein may be administered before, after, or simultaneously with the administration of the compositions provided herein. In certain embodiments, the additional therapy is administered before the composition provided herein. In certain embodiments, the additional therapy is administered after the composition provided herein. In certain embodiments, the additional therapy is administered at one or more intervals before or after the administration of the composition provided herein. Determining the appropriate intervals for administering the additional therapy so that the subject being treated benefits from the combination therapy is within the level of skill of one of ordinary skill in the art.

[0093] B. Pharmaceutical Compositions In another aspect, T RM Pharmaceutical compositions and formulations comprising the cells and a pharmaceutically acceptable carrier are provided herein.

[0094] Pharmaceutical compositions and formulations as described herein comprise an active ingredient (such as an antibody or polypeptide) having the desired purity in one or more optional pharmaceutically acceptable carriers (Remington’s Pharmaceutical Sciences 22 ndIt can be prepared by mixing with an aqueous solution, for example, normal saline (e.g., 0.9%) and human serum albumin (e.g., 10%) in the form of a 2012 edition. Pharmaceutically acceptable carriers are generally non-toxic to the recipient at the dosages and concentrations utilized, and include, but are not limited to, buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (octadecyl dimethyl benzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol, etc.); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other sugars such as glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., zinc-protein complexes); and / or nonionic surfactants such as polyethylene glycol (PEG).

[0095] III. Kit In some embodiments, for example, T RM A kit may be provided that includes one or more media and components for generating cells. Such formulations are T RMThe factor cocktail may be included in a form suitable for combination with cells. The reagent system may be packaged, if desired, either in an aqueous medium or in lyophilized form. The container means of the kit will generally include at least one vial, test tube, flask, bottle, syringe or other container means into which the components are placed and preferably may be suitably aliquoted. If two or more components are present in the kit, the kit will also generally include a second, third or other additional container into which the additional components may be separately placed. However, various combinations of components may be included in the vial. The components of the kit may be provided as dry powders. When the reagent and / or components are provided as dry powders, the powders may be reconstituted by the addition of a suitable solvent. It is contemplated that the solvent may also be provided in a separate container means. The kit will also typically include means for containing the kit components, which are sealed for commercial use. Such containers may include injection or blow molded plastic containers in which the desired vials are held. The kit may also include instructions for use, for example in printed or electronic format, such as digital format.

Examples

[0096] IV. Examples The following examples are included to demonstrate preferred embodiments of the invention. It should be understood by those skilled in the art that the techniques disclosed in the following examples represent techniques discovered by the inventors to function well in the practice of the invention and, thus, can be considered to constitute preferred modes for its practice. However, those skilled in the art will understand, in light of the present disclosure, that many changes can be made in the specific embodiments disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

[0097] Example 1 - Generation of Tissue Resident Memory T Cells Peripheral blood samples were obtained from healthy human subjects. Blood was sorted using FACS to naive (CD45RA + CCR7+ ) CD8 + T cells were isolated. Next, the T cells were activated polyclonally under atmospheric oxygen (about 20%) or hypoxia (2% O2) for 4 days to generate "naive effectors". Next, the naive effectors were cultured for an additional 2 days in the presence of 1.25 ng / mL of rhTGF-β1. Next, the cells were collected and analyzed for the expression of T RM -related genes and surface markers.

[0098] The combination of hypoxia and TGF-β1 induced a CD69 + CD103 + population, and it was found that human T-related markers including CD69 and CD103 were expressed (Figure 3). T RM -like T cells were further analyzed for changes in the expression of additional genes. It was found that cells cultured under hypoxic versus atmospheric oxygen conditions had changes in gene expression related to the T RM -like phenotype (Figure 2). RM

[0099] Naive CD8 + T cells were activated by hypoxia in the presence or absence of the addition of rhTGF-β1. The frequency of the CD69 + CD103 + population was evaluated by flow cytometry. It was found that hypoxia and TGF-β1 cooperate to induce CD69 + CD103 + cells (Figure 4).

[0100] Furthermore, it was found that the T RM -like phenotype cells induced by hypoxia and TGF-β1 showed transcriptional differences similar to those reported for endogenous T RM . It was found that CD69 - CD103 - , CD69 + CD103 - , and CD69 + CD103 +T cells were generated and selected, followed by RNA isolation and transcriptome analysis via RNA sequencing (n=3). RM Figure 9B shows a heat map depicting the expression of selected genes reported in the transcriptome analysis of CD8 from human lung. + CD69 + T cells vs. CD8 + CD69 - T cells and CD69 + CD103 + T induced in vitro with hypoxia and TGF-β1 RM (iT RM ) versus normal cell culture conditions (20% O2 without TGF-β1) - CD103 - Heatmaps comparing transcriptional differences (log2FC) in cells are shown. Differential expression was determined by |log2FC|≧1 and FDR<0.05.

[0101] This study demonstrated that hypoxia and TGF-β1 mediate the expression of CD8 + CD69 + CD103 + Induce cell populations and generate human T RM Expressing related markers and endogenous human T RM This study demonstrated that the in vivo T RM The antigen-specific T cells express another possible driver of differentiation and may allow for the development of adoptive cell therapy utilizing this unique cell type. RM This method provides the basis for an in vitro method for generating T-like cells. RM The phenotype can be used to generate cells having the phenotype.

[0102] Example 2 - Materials and Methods Cell Isolation and In Vitro Cell Culture: Peripheral blood mononuclear cells (PBMCs) from healthy donors were collected by leukapheresis and stored in liquid nitrogen until use. All human sample collections were performed with informed consent and approved by the Institutional Review Board (IRB) of UT MD Anderson Cancer Center. CD8 + T cells were enriched from healthy donor PBMCs using the StemCell EasySep™ kit. Next, cells were stained with fluorescent dye-conjugated antibodies against CD8, CD45RA, and CCR7. Naïve CD8 + CD45RA + CCR7 + cells were sorted using a FACSAria™ III or Fusion cell sorter (BD Biosciences). The sorted naïve cells were resuspended in cell culture medium (RPMI with 10% FBS, L-glutamine, and penicillin-streptomycin) containing 10 IU / ml of IL-2 (Prometheus) and equilibrated overnight in 2% oxygen in a hypoxic chamber (Coy Laboratory Products) or atmospheric oxygen (approximately 20%) in a standard cell culture incubator (Thermo Fisher). After equilibration, cells were activated with anti-CD3 / anti-CD28 beads (Dynabeads®, Gibco) for 4 days. On day 4, 1.25 ng / ml of recombinant human TGF-β1 (Biolegend) was added and cells were cultured for an additional 2 days. Antigen-specific CD8 + T cells generated via stimulation with autologous peptide-pulsed dendritic cells or TCR transduction followed by tetramer-based sorting were expanded using either a normal rapid expansion protocol (REP) or a TRM-modified REP. Anti-CD3 (OKT3) at 30 ng / mL was used to expand tetramer-positive cells for 10 - 14 days, either i) with the addition of IL-2 (50 IU / ml) in 20% oxygen and normal REP, or ii) with the addition of IL-15 (10 ng / ml) and rhTGF-β1 (1.25 ng / ml, starting on day 4) in 2% oxygen and modified REP, with 200 irradiated allogeneic PBMCs and LCLs as feeder cells to induce the T RM phenotype.

[0103] Flow cytometry: human T RM For the analysis of related markers, beads were removed from the cells, and the cells were washed once with staining buffer, and stained with Live / Dead Fixable Aqua (Life Technologies) and fluorescent dye-conjugated antibodies against CD8, CD69, CD103, PD-1, CD101, CXCR6, and CD49a (all from Biolegend). After staining, the cells were fixed with 4% paraformaldehyde fixation buffer (Biolegend), washed, and stored in staining buffer until analysis. The stained cells were analyzed using an ACEA Novocyte® 3000 flow cytometer. Compensation beads (UltraComp™, eBioscience) stained with a single fluorescent dye and fluorescence minus one (FMO) samples were used as controls. FlowJo software (BD Biosciences) was used to analyze the data.

[0104] Quantitative real-time PCR (qRT-PCR or qPCR): human T RM For the analysis of related gene expression, beads were removed from the cells, and the cells were washed once with PBS. The Qiagen RNeasy® Plus Mini Kit was used to isolate RNA according to the manufacturer's instructions. If necessary, the Qiagen RNeasy® MinElute Cleanup Kit was used to further purify and / or concentrate the RNA according to the manufacturer's instructions. First-strand cDNA was synthesized using M-MLV reverse transcriptase (Thermo Fisher). Quantitative real-time PCR was performed using a QuantStudio® 5 real-time PCR system and PowerUp™ SYBR® Green Master Mix (Applied Biosystems, ThermoFisher Scientific). Relative mRNA gene expression was normalized to the housekeeping gene RPL13A. The primers used are listed in the table below.

[0105]

Table 2

[0106] RNA-Sequence Determination Transcriptome Analysis: Cells were sorted using a FACSAria™ IIIu cell sorter (BD Biosciences), and then RNA was isolated using the Qiagen RNeasy® Plus Mini Kit, followed by the Qiagen RNeasy® MinElute Cleanup Kit. Libraries were constructed using the Illumina TruSeq Stranded mRNA Kit. RNA sequencing was performed using the Illumina NextSeq® 500 platform. Raw reads were mapped to the human (Homo sapiens) reference genome and transcriptome (GRCh38, GENCODE V23) using HISAT2 (version: 2.1.0). Counts for genes were obtained using Htseq-count (version: 2.1.0). Differentially expressed genes were identified using the R and Bioconductor package DESeq2 (version 1.14.1). Genes with FDR < 0.05 and |fold change| > 2 (mRNA only, obtaining protein-coding genes for P-value correction) were considered differentially expressed. The R and Bioconductor package fgsea (version 1.10.0) was used to determine whether a predefined gene set showed statistically significant concordant differences between two biological states (e.g., phenotypes). Gene sets were derived from several previously published T cell signatures. T RM Signatures were constructed from several experiments. Lung T RM and breast cancer TIL signatures were downloaded from the Gene Expression Omnibus (GEO), GSE61397 and GSE110938, respectively. False discovery rate (FDR) - corrected P-values less than 0.05 were considered significant or "true".

[0107] Functional analysis of genes differentially expressed (FDR < 0.05 and |fold change| > 2) was performed using Ingenuity® Pathway Analysis (IPA) software (version 48207413, Qiagen) with all genes in the Ingenuity Knowledge Base as a reference set, and by right-sided Fisher's exact test in a core analysis to determine whether a pathway was significantly altered between conditions (-log10[P-value] > 1.3).

[0108] Statistical analysis: GraphPad Prism (version 7, GraphPad software, San Diego, CA) was used to perform graphical representation and statistical analysis of the data. Data are presented as mean ± standard error of the mean. Results between experimental groups were compared using the statistical tests described in the figure legends (analysis of variance followed always by Tukey's multiple comparison test). p < 0.05 was considered statistically significant.

[0109] Example 3 - Hypoxia acts as an environmental factor in the differentiation of human tissue-resident memory T cells Human CD8 + T cells differentiated under hypoxia and TGF-β1 acquire a T RM -like phenotype: Under conditions of relative hypoxia within inflamed tissues, it was hypothesized that hypoxia could provide an additional factor for T RM differentiation. To determine whether hypoxia could contribute to the induction of the T RM phenotype, naive (CD45RA + CCR7 + ) CD8 + T cells were isolated from human peripheral blood and activated for 4 days under hypoxia (2% O2) or normal cell culture conditions (atmospheric oxygen, ~20% O2) to generate "early effectors", and then cultured for an additional 2 days in the presence of rhTGF-β1.

[0110] Quantitative real-time PCR (qPCR) was used to evaluate a large panel of genes related to T RM transcriptional profiles. Cells differentiated under 2% O2 + TGF-β1 were human T RMshowed upregulation of most of the genes identified by Kumar et al. 2017 (incorporated herein by reference) as core transcriptional signatures (i.e., CD69, ITGAE (CD103), PDCD1 (PD-1), CD101, and CXCR6) (Figure 2A). In particular, no difference was observed in the transcript levels of ITGA1 (CD49a). Furthermore, transcripts of genes important in T cell recirculation (S1PR1, KLF2, SELL (CD62L)) were downregulated, further suggesting a resident memory phenotype. (Figure 2B). Previous reports in mouse models have shown that downregulation of S1PR1 and KLF2 is important for T RM differentiation, and reduced levels of these genes have been shown to be observed in endogenous human T RM as well. Transcripts in the transcription factor Eomes were dramatically reduced (Figure 2C). Tests in mice have shown that ablation of Eomes is required for TGF-β1 responsiveness and T RM establishment. IRF4 and RUNX3 were upregulated. The specific role of IRF4 in T RM is still unclear, but its upregulation has been reported in human T RM . The transcription factor RUNX3 has recently been identified as a major regulator of T RM differentiation. Finally, increased levels of transcripts encoding the effector molecules TNF-α and granzyme b were observed, similar to findings reported in human lung T RM (Figure 2D). Increased expression of the classical hypoxia-responsive genes SLC2A1 (Glut-1) and VEGF confirmed that the cells were responding to hypoxic conditions (Figure 1E). Overall, these results indicate that human CD8 + T cells acquire tissue-resident memory-like transcriptional characteristics when differentiated under hypoxia in combination with TGF-β1.

[0111] Human T RMFlow cytometry analysis to evaluate the protein-level expression of markers that are thought to be the core signature. In all healthy donors tested, under 2% O2 + TGF-β1 conditions, CD69 showed an increase in cells compared to 20% O2 + TGF-β1 conditions (Figure 2A). Cell viability was equivalent or superior in 2% O2 + TGF-β1 cells compared to 20% O2 + TGF-β1 cells (Figure 7B). These CD69 + CD103 + cells expressed PD-1, CD101, and CD49a (Figure 3B). In particular, CXCR6 surface protein expression was not observed despite transcriptional upregulation. The expression of both CD69 and CD103 is currently commonly used to clarify T + CD103 + but is partly due to the heterogeneous expression of CD69 and CD103 in endogenous resident memory cells, and there is still debate about which of these surface markers is most suitable for use in identifying T RM Therefore, from the 2% O2 + TGF-β1 condition, the expression levels of the related markers PD-1, CD101, and CD49a were compared among the CD69 RM CD103 - CD103 + CD69 + CD103 + CD69 + CD103 - populations. As expected, the CD69 RM CD103 + CD103 + population had the highest levels of PD-1 and CD101 surface expression (Figure 3C). CD49a expression was also high but equal to the expression levels observed in the CD69 + CD103 - population. In the comparison of different oxygen conditions, the most significant increase in the fold change of the population was found in the CD69 + CD103 + population (Figures 7C, 7D). Based on these results, CD69 as an in vitro-induced T RM cells + CD103 +It was chosen to focus on further analysis of the population.

[0112] Hypoxia and TGF-β1 exposure are T RM Synergistic factors for obtaining the phenotype: T cells are exposed to non-hypoxic oxygen levels in circulation because the atmospheric oxygen level under normal tissue culture conditions is higher than that experienced by T cells in vivo where the effect of 10% O2 (which is physiologically important) was evaluated. CD69 in 10% O2 + TGF-β1 + CD103 + There was a slight increase in T cells in 20% O2 + TGF-β1 compared to that in 20% O2 + TGF-β1, but after correction in multiple comparisons, it was revealed that there was no significant difference between the two conditions in the expression of signature genes (Figure 8A-E). Furthermore, CD69 RM CD103 + CD103 + The fold change of the population (O2 + TGF-β1 above 20%) was significantly greater in the 2% O2 + TGF-β1 condition versus the 10% O2 + TGF-β1 condition (Figure 8F).

[0113] Hypoxia and TGF-β1 of T RM To evaluate the respective contributions of hypoxia and TGF-β1 to the induction of phenotypic cells, in vitro differentiation experiments were performed under 2% O2 or 20% O2, in the presence or absence of the addition of rhTGF-β1. Consistent with published reports that hypoxia and TGF-β can each drive the expression of these markers, hypoxia mainly induced CD69 + cells while TGF-β1 induced CD103 + cells. Hypoxia or TGF-β1 alone did indeed induce a medium-sized population of CD69 + CD103 + cells, but the combination of hypoxia and TGF-β1 was seen to cooperate in the induction of the resident phenotype as the combined effect was significantly greater than the additive effect of either condition alone (Figure 4A). CD69 + CD103 + cells induced by hypoxia and TGF-β1 are T RMMarkers PD-1, CD101, and CD49a were expressed at high levels compared to the majority of the population under 20% O2 and 2% O2 conditions (each, CD69 - CD103 - and CD69 + CD103 - ) (Figure 4B).

[0114] In vitro-induced T RM showed enrichment in the endogenous human T RM gene signature: CD69 - CD103 - (20% O2) cells, CD69 + CD103 - (2% O2) cells, and CD69 + CD103 + (2% O2 + TGF-β1) cells, suggesting that differences in T RM marker expression represented distinct populations. Therefore, each phenotype was sorted and their transcriptional characteristics were analyzed by RNA sequencing. According to principal component analysis (PCA), these three populations were confirmed to be different from each other (Figure 5A). According to unsupervised hierarchical clustering, CD69 - CD103 - cells and CD69 + CD103 + cells had different gene signatures, while CD69 + CD103 - cells were shown to have somewhat intermediate transcriptional characteristics (Figures 5B, 5C). Comparison of the top differentially expressed genes between CD69 + CD103 + cells and CD69 - CD103 - cells showed gene expression patterns consistent with those reported for endogenous human T RM , such as increased expression of ITGAE, EGR2, GNLY, BMF, RASGEF1B, and NR4A1, and decreased expression of SELL, KLF2, and KLF3, indicating a non-recirculating transcriptional program (Figures 5B, 5C). The KLF2 target gene S1PR1 was also downregulated but did not meet the fold change threshold. CD69+ CD103 + The cells showed increased expression of ITGA1, PDCD1, CD101, and TNFRSF9, all of which are RM constantly reported to be upregulated in endogenous human T RM in vivo. An increase in the levels of the transcription factor NOTCH1, which is known to contribute to the

[0115] maintenance of endogenous T RM cells, and RBPJ, which plays a central role in Notch signaling, was observed (Figure 5C). RM Endogenous T + CD103 + cells often express various chemokines, presumably as part of their "alarm function" when mobilizing other immune cells to local tissues. Consistent with the chemokine properties of endogenous T RM cells, in vitro-induced CD69

[0116] CD103 - effector memory cells in the present study or CD103 - T cells within the same tissue site upregulated CXCL13 and CCL20, as well as CCL4, CCL5, and CCL22 (Figure 5C). Changes in the expression of genes that have often been reported in endogenous human T + T RM cells, such as upregulation of MYO7A and RGS1 and downregulation of SERPINE2, RAP1GAP2, RASGRP2, and FAM65B, were also observed, although their roles are unclear. - CD103 - To gain insights into the physiological relevance of these findings, gene set enrichment analysis (GSEA) was performed using gene signatures from published analyses of endogenous human CD103 + CD103 + compared to effector memory cells from peripheral blood or CD103 EM T cells within the same tissue site. According to the results, the transcriptional profiles of CD69 - CD103 RM cells were similar to the signatures of T+ CD103 - CD69 Compared to Cells + CD103 + Cells are exclusively T within the same tissue RM vs CD103 - was shown to be similar to the signature of T cells (Figure 5D). These results reflect the degree of difference in oxygen pressure during circulation relative to local tissues. Even among TILs within the same tumor, T cells within the tissue will be exposed to more similar oxygen levels compared to T cells in circulation, and multiple recent characteristics of TILs in various solid tumor types are related to CD103 + Resident memory-like TIL (TIL RM ) have been reported. Therefore, CD69 induced under hypoxia + TGF-β1 + CD103 + cells compared to CD69 + CD103 - cells induced under hypoxia alone, CD8 + CD69 + CD103 + vs CD8 + CD69 + CD103 - enrichment of the gene signature reported for breast cancer TIL was observed (Figure 5E, 5F). Since hypoxia and TGF-β are common features of the tumor microenvironment, as a result, these conditions may contribute to the generation of CD103 + TIL in vivo.

[0117] CD69 + CD103 + cells compared to CD69 - CD103 - cells according to Ingenuity Pathway Analysis (IPA), many of the differentially expressed genes were shown to be components of the glycolysis, gluconeogenesis, and TGF-β signaling pathways (Figure 6A). Given that the cells were exposed to hypoxia and TGF-β and assuming that hypoxia is a major regulator of cell metabolism, these results were expected. Enrichment of genes in the Notch signaling pathway was also observed, which is in endogenous human lung T RMreported within (Figure 6A). These findings raise questions about the role of metabolism in T RM differentiation. Hombrink et al. suggested that the major role of Notch signaling in lung T RM is the regulation of metabolic programs, as chemical inhibition of Notch signaling affected genes involved in glycolysis, oxidative phosphorylation, and fatty acid metabolic pathways. Similar to hypoxia, deletion of the purinergic receptor P2RX7, a known modifier of aerobic glycolysis, suggested that impaired metabolism via dysregulation of T RM formation, as P2RX7-deficient cells exhibited reduced mitochondrial mass and function, defects in aerobic glycolysis, and impaired glucose uptake.

[0118] Enrichment of differentially expressed genes involved in leukocyte extravasation signaling. Epithelial adhesion junction signaling, integrin signaling, and paxillin signaling were observed, all of which were involved in focal adhesion signaling and suggested changes in the programming of migration (Figure 6B). Consistent with previous reports that PI(3)K (phosphatidylinositol-3-OH kinase) signaling is involved in cytokine-induced downregulation of KLF2 and may play a role in the generation of T RM in vivo, multiple pathways involved in inositol phosphate signaling, namely, 3-phosphoinositide biosynthesis, the superpathway of inositol phosphate compounds, D-myo-inositol (1,4,5,6)-tetraphosphate biosynthesis, and D-myo-inositol (3,4,5,6)-tetraphosphate biosynthesis, were also enriched.

[0119] To more fully understand the functional relevance of the transcriptional characteristics induced by hypoxia + TGF-β, pathway analysis was also performed on the transcriptional data published by Kumar et al., and changes in Th1 and Th2 activation and granulocyte adhesion and extravasation pathways were observed in their endogenous lung T RM and in vitro-induced T RM and in vitro-induced T RMwere found to be common. In particular, across all datasets, axon guidance pathways were also highly significantly differentially regulated. Axon guidance was initially seemingly unrelated to resident memory T cells on the surface and current T RM has not been reported in the literature, but it is a process when environmental factors affect the migration pattern of cells. Many of the same elements that govern axon guidance are also known to regulate immune cell trafficking and can be regulated by hypoxia and / or TGF-β.

[0120] To evaluate the effect of the HIF prolyl hydroxylase inhibitor FG-4592 (roxadustat) in combination with TGFB1, further tests were conducted. Naïve CD8 + T cells isolated from peripheral blood were activated for 4 days under 20% O2 (AtmosO2) in the presence of the HIF prolyl hydroxylase inhibitor FG-4592 (roxadustat), and then further activated for 2 days with rhTGF-β1. On day 4, cells activated under 2% O2 (hypoxia) with the addition of rhTGF-β1 are shown in Figure 10A for comparison. The combination was found to induce + CD103 + CD69 cells (Figure 10A).

[0121] Next, naïve CD8 + T cells were stimulated with autologous monocyte-derived dendritic cells pulsed with the MART-1 (M27) peptide for 7 days under 20% O2 (AtmosO2) or 2% O2 on day 4 with the addition of rhTGF-β1, and + CD103 + antigen-specific (tetramer + ) T cells were generated (detected by flow cytometry) (Figure 10B). Used in an improved rapid expansion protocol with hypoxia and rhTGF-β1, within antigen-specific T cells RMThe phenotype was induced. Antigen-specific T cells were generated via stimulation with autologous MART-1 peptide-pulsed dendritic cells (ETC) or transduction of the TCR specific for gp100 (TCRT), labeled with a fluorescent dye-conjugated tetramer, and sorted. Next, the sorted antigen-specific T cells were stimulated with anti-CD3 (OKT3) and irradiated feeder cells under 20% O2, and IL-2 was added (normal REP) or IL-15 was added under 2% O2, and rhTGF-β1 was added after day 4. Thus, with the improved rapid expansion protocol, T RM phenotype was also shown to be induced (Figure 10C).

[0122] Therefore, in this study, in vitro T RM phenotype was reproduced from human peripheral blood-derived T cells, and hypoxia was identified as a potential factor in T RM differentiation. While there are obvious limitations to experiments feasible in humans, the described studies, supported by the observation that hypoxia + TGF-β-induced T RM mimics the transcriptional and proteomic landscapes of endogenous T RM as well as the pathways related to migration and metabolism, provide compelling evidence that hypoxia is an environmental factor that can contribute to the acquisition of the T RM phenotype.

[0123] All of the methods disclosed and claimed in this specification can be constructed and implemented without undue experimentation in light of the present disclosure. While the compositions and methods of the invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that changes may be applied to the methods described herein and to the steps or series of steps of the methods without departing from the concept, spirit, and scope of the invention. More specifically, it will be apparent that the same or similar results may be obtained if certain chemically and physiologically related agents are substituted for the agents described herein. All such similar substitutions and modifications apparent to those skilled in the art are deemed to be within the scope of the spirit, scope, and concept of the invention as defined by the appended claims. References The following references are specifically incorporated herein by reference to the extent that they provide exemplary procedures or other details that supplement the presentation herein. 1) Atkuri, K.R., et al. (2005). Culturing at atmospheric oxygen levels impacts lymphocyte function. Proc National Acad Sci 102, 3756 - 3759. 2) Caldwell, C.C., et al. (2001). Differential effects of physiologically relevant Hypoxic conditions on T lymphocyte development and effector functions. Journal of immunology (Baltimore, Md: 1950) 167, 6140 - 6149. 3) Kumar, B.V., et al. (2017). Human Tissue-Resident Memory T Cells Are Defined by Core Transcriptional and Functional Signatures in Lymphoid and Mucosal Sites. Cell Rep 20, 2921 - 2934. 4) Mackay, L.K., et al. (2013). The developmental pathway for CD103(+)CD8 + tissue-resident memory T cells of skin. Nat Immunol 14, 1294 - 1301. 5) Hombrink, P., et al. (2016). Programs for the persistence, vigilance and control of Human CD8(+) lung-resident memory T cells. Nat Immunol 17, 1467 - 1478. 6) Skon, C.N., et al. (2013). Transcriptional downregulation of S1pr1 is required for the establishment of resident memory CD8 + T cells. Nat Immunol 14, 1285 - 1293. 7) Mackay, L.K., and Kallies, A. (2017). Transcriptional Regulation of Tissue-Resident Lymphocytes. Trends Immunol 38, 94 - 103. 8) Mackay, L.K., et al. (2015). T-box Transcription Factors Combine with the Cytokines TGF-beta and IL-15 to Control Tissue-Resident Memory T Cell Fate. Immunity 43, 1101 - 1111. 9) Mackay, L.K., et al. (2016). Hobit and Blimp1 instruct a universal transcriptional program of tissue residency in lymphocytes. Science 352, 459 - 463. 10) Mami0Chouaib and Tartour. (2019) Editorial: Tissue Resident Memory T Cells. Front. Immunol 10, 1 - 3. 11) Milner, J.J., et al. (2017). Runx3 programsCD8(+)T cell residency in non-lymphoid tissues and tumours. Nature 552, 253 - 257.

Claims

**Claim 1** An in vitro method for generating tissue-resident memory-like T cells (T ＲＭ -like T cells), comprising: (a) Culturing a starting population of T cells under hypoxic conditions or in the presence of a hypoxia-inducing agent to generate initial effector cells, wherein the starting population of T cells comprises CD45RA+CCR7+CD8+ naive T cells in a peripheral blood sample or antigen-specific (tetramer+) T cells; (b) T ＲＭ To generate the T-like T cells, further culturing the initial effector cells in the presence of transforming growth factor β1 (TGF-β1); A method comprising the steps of: **Claim 2** The method according to claim 1, wherein the peripheral blood sample is a sample obtained from a healthy subject, a subject diagnosed with cancer, or a subject suspected of having cancer. **Claim 3** The method according to claim 1, wherein the T cells of the starting population are stimulated by antigen-presenting cells pulsed with a peptide, full-length antigen, or cell lysate prior to culturing. **Claim 4** The method according to claim 3, wherein the T cells are T cells obtained from a tumor site or tumor-infiltrating lymphocytes. **Claim 5** The method according to claim 4, wherein the T cells are naive T cells. **Claim 6** The method according to claim 4, wherein the starting population of T cells is purified to enrich for cells that are CD8 positive and peptide-MHC tetramer positive. **Claim 7** The method according to claim 1, wherein the hypoxic conditions are further defined as less than 5% oxygen. **Claim 8** The method according to claim 1, wherein the hypoxia-inducing agent is a hypoxia mimetic. **Claim 9** The hypoxia inducer or hypoxia mimetic is cobalt chloride (CoCl ２ ), deferoxamine mesylate (DFOM), dimethyloxaly glycine (DMOG), or a prolyl hydroxylase inhibitor, the method according to claim 8. **Claim 10** The method according to claim 9, wherein the prolyl hydroxylase inhibitor is a 2-OG analog or roxadustat (FG-4592). **Claim 11** The method according to claim 1, wherein the culturing of step (b) is performed in the presence of TCR stimulation and costimulation, comprising anti-CD3 and anti-CD28 antibodies, anti-CD3 and anti-CD28 beads, feeder cells, antigen-presenting cells, artificial antigen-presenting cells, peptides and / or protein antigens, or combinations thereof. **Claim 12** The method according to claim 11, wherein the culturing of step (b) is performed in the presence of IL-15. **Claim 13** The method according to claim 12, wherein the IL-15 is present at a concentration of 5-20 ng / mL or 10 ng / mL. **Claim 14** The method according to claim 1, wherein TGF-β1 is further defined as recombinant human TGF-β1 (rhTGF-β1). **Claim 15** The method according to claim 14, wherein the rhTGF-β1 is present at a concentration of 0.1-5 ng / mL, 1-1.5 ng / mL, or 1.25 ng / mL. **Claim 16** The method according to claim 1, wherein the culture in step (b) is carried out under hypoxic conditions or in the presence of a hypoxia inducer.

17. Said T ＲＭ such T cells are CD69 ＋ CD103 ＋ The method according to claim 1, wherein it is

18. At least 30% or 40% of the cells generated in step (b) are CD69 ＋ CD103 ＋ The method according to claim 17, wherein the cells are

19. Said T ＲＭ The method according to claim 1, wherein the T cells express PD-1, CD101, and / or CD49a.

20. Said T ＲＭ The method according to claim 1, wherein the T cells have a higher expression of CD69, ITGAE, PDCD1 and / or CD101 compared to cells cultured under atmospheric oxygen conditions.

21. Said T ＲＭ The method according to claim 1, wherein the like T cells have higher expression of TNFα, GZMB, SLC2A1, and / or VEGF as compared to cells cultured under atmospheric oxygen conditions.

22. Said T ＲＭ The method according to claim 1, wherein the T cells essentially do not have the expression of CXCR6 protein.

23. Antigen-specific T ＲＭ The method according to claim 1, further comprising generating γδ T cells.

24. Antigen-specific T ＲＭ generating the T-like T cells includes culturing the cells in the presence of a histone deacetylase (HDAC) inhibitor during step (a) and / or step (b), wherein the HDAC inhibitor is selected from the group consisting of trichostatin A, trapoxin B, phenylbutyric acid, valproic acid, vorinostat (suberanilohydroxamic acid or SAHA, commercially available as Zolinza (registered trademark)), belinostat (PXD101, commercially available as Belinodaq (registered trademark)), panobinostat (commercially available as Farydaq (registered trademark)), dacinostat (LAQ824), entinostat (SNDX-275 or MS-275), tacedinaline (CI994), and mocetinostat (MGCD0103), the method according to claim 23.

25. In vitro cultured and isolated T ＲＭ helper T cells that essentially do not have the expression of CXCR6 protein, ＲＭ wherein the T ＲＭ helper T cells are T helper T cells produced by the method according to any one of claims 1 to 24.

26. T ＲＭ such T cells are (a) expressing PD-1, CD101, and / or CD49a (b) CD69 ＋ CD103 ＋ is (c) expressing GNLY, MYO7A, ITGAE, EGR2, CCL20, ATP1B1, NR4A3, PERP, RASGEF1B, NR4A1, BMF, EGR1, CXCL13, PCD1, ITGA1, CCL22, CA10, RGS1, ITGA1, CD101, TNFRSF9 (4-1BB), CCL4, CCL5, NOTCH1, RBPJ, STRIP2, ARHGEF40, DBH, SRGAP3, CSGALNACT1, GPR25, RGS16, DAPK2, NCS1, COL6A3, GDPD4, SLC1A4, CDK14, LMCD1, ILDR2, and / or ADCY3 (d) expressing GNLY, MYO7A, ITGAE, EGR2, CCL20, ATP1B1, NR4A3, PERP, RASGEF1B, NR4A1, BMF, EGR1, CXCL13, PCD1, ITGA1, CCL22, CA10, and / or RGS1 (e) expressing ITGAE, ITGA1, PCD1, CD101, TNFRSF9 (4-1BB), CXCL13, CCL20, NOTCH1, RBPJ, NR4A1, EGR2, and / or RGS1 (f) expressing MYO7A, STRIP2, ARHGEF40, ITGAE, DBH, SRGAP3, CSGALNACT1, GPR25, RGS16, DAPK2, NCS1, COL6A3, GDPD4, SLC1A4, CXCL13, CDK14, LMCD1, ILDR2, and / or ADCY3 (g) not expressing or essentially not having the expression of CD58, NR3C1, RAP1GAP2, SELP, CXCR2, TBX21, ITGAL, SELL, KLF3, KLF2, RASGRP2, FAM65B, SERPINE2, ITGAM, KLRB1, TGFBR3, SMAD3, TNFRSF8, DUSP2, PLEK, GOLGA2P7, FOSB, PLCG2, SLAMF7, SLC6A8, SOCS3, and / or PTGER2 (h) Does not express or essentially does not have the expression of CD58, NR3C1, RAP1GAP2, SELP, CXCR2, TBX21, ITGAL, SELL, KLF3, RASGRP2, ITGAM, KLRB1, TGFBR3, SMAD3, and / or TNFSF8, (i) Does not express or essentially does not have the expression of KLF2, KLF3, SELL, FAM65B, and / or SERPINE2, and / or (j) Does not express or essentially does not have the expression of DUSP2, PLEK, GOLGA2P7, FOSB, PLCG2, ITGAM, FOS, KLF3, SLAMF7, TNFSF8, SLC6A8, KLF2, SOCS3, and / or PTGER2, The T according to claim 25 ＲＭ type T cells. [

27. ] An effective amount of T-like T cells substantially free of the expression of CXCR6 protein, wherein the T-like T cells are generated by the method according to any one of claims 1 to 24, for treating an immune-related disorder in a subject. ＲＭ ＲＭ ​​ [

28. ] The agent according to claim 27, wherein the immune-related disorder is cancer, autoimmune deficiency, graft-versus-host disease, allograft rejection, or an inflammatory condition.

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