Method for producing cytotoxic effector memory T cells for cancer T cell therapy

Abstract

As used herein, CD161 + A method for expanding T cells is provided. A modified CD161 containing chimeric antigen receptor (CAR) + Methods and compositions for generating T cells are also provided. In certain embodiments, CAR-expressing T cells are produced, expanded, and / or used in the treatment of disease (e.g., cancer).

Description

[Technical Field]

[0001] Reference to related applications This application claims priority to U.S. Provisional Application No. 62 / 931,670, filed on November 6, 2019, the entirety of which is incorporated herein by reference.

[0002] Description of research funded by the federal government. This invention was made with government support under grant number AI127387, awarded by the National Institutes of Health. The U.S. Government reserves certain rights in this invention.

[0003] 1. Field This disclosure generally relates to the fields of medicine, immunology, cell biology, and molecular biology. In certain aspects, the field of this disclosure relates to immunotherapy. More specifically, this disclosure relates to the generation of improved chimeric antigen receptor (CAR) T cells and therapeutic methods using such cells. [Background technology]

[0004] 2. Explanation of related technologies Pancreatic ductal adenocarcinoma (PDAC) is a highly invasive tumor with a 5-year survival rate of less than 9% despite aggressive surgery, radiation, and high-dose chemotherapy (Ansari et al., 2015). In recent years, adoptive chimeric antigen receptor (CAR) T-cell therapy has been used to treat cancer, specifically selected CD19 +CARs have shown great potential as a treatment for malignant tumors (Maude et al., 2018, Neelapu et al., 2017). CAR constructs consist of a single-chain variable region fragment (scFv) that targets cell surface tumor antigens, a transmembrane domain, a hinge region, and an intracellular signaling domain of CD3ζ typically fused to either a 4-1BB or CD28 costimulatory molecule (van der Stegen et al., 2015). In a pilot Phase I clinical trial, adoptive cell therapy with autologous mesothelin-specific CAR-T cells showed safe and mild efficacy in a small number of patients with chemotherapy-refractory metastatic human PDACs (Beatty et al., 2018), but CAR T cell therapy for pancreatic tumors remains underdeveloped. In fact, at present, CAR-based therapies have not shown any significant efficacy in solid tumors.

[0005] A key feature of cell-mediated immunity against viral infection is the establishment of a population of long-lived memory T cells that provide resilient immunity to subsequent challenges through accelerated growth and cytotoxic dynamics (Seaman et al., 2004). Several groups have previously identified interesting subsets of such memory T cells, which can be identified by the expression of the innate cytotoxic receptor NK1.1 in mice or CD161 in humans (Martin et al., 2009, Turtle et al., 2009, Northfield et al., 2008, Takahashi et al., 2006, Assarsson et al., 2000, Billerbeck et al., 2010, Fergusson et al., 2011, Fergusson et al., 2016, Fergusson et al., 2014). TCR invariant or CD8αα + CD161 +In contrast to cells, polyclonal αβ cell populations have a distinct transcriptional profile with stem cell-like capabilities for self-renewal and differentiation, significant upregulation of genes from the granzyme superfamily (Fergusson et al., 2011, Fergusson et al., 2014), characteristic antiviral specificities (Fergusson et al., 2008, Billerbeck et al., 2010, Havenith et al., 2012, Neelapu et al., 2005), as well as tissue homing properties (Billerbeck et al., 2010). Typically, CD161 is known as a congenital NK cell receptor but can also be expressed on CD4, CD8, and NKT cells (Fergusson et al., 2016). Although also found in the circulation, CD8 + CD161 + cells contribute to tissue pathogenesis due to tissue residency properties and / or a tendency for extravasation during chronic viral infections and autoimmune conditions (Assarsson et al., 2000, Billerbeck et al., 2010, Annibali et al., 2011). Furthermore, high expression levels of CD161 in tumor-resident immune infiltrates are associated with substantially improved clinical outcomes and survival in NSCLC (Braud et al., 2018).

Summary of the Invention

[0006] In a first embodiment, (a) obtaining a sample of cells, wherein the sample comprises CD161 + T cells, and (b) culturing the T cells in the presence of IL-7, IL-15, and IL-21, thereby providing a population of T cells in which the number of CD161 + cells is increased compared to non-CD161 + cells. An in vitro or ex vivo method is provided that includes the above. In certain embodiments, the T cells comprise CD8 + CD161 + T cells. In further embodiments, the T cells comprise CD4 + CD161 + T cells.

[0007] IL-7 may be present at approximately 5–20 ng / ml, IL-15 at approximately 2.5–10 ng / ml, and / or IL-21 at approximately 20–40 ng / ml, for example, 10 ng / ml of IL-7, 5 ng / ml of IL-15, and / or 30 ng / ml of IL-21. This method involves, before step (b), the CD8 in the sample. + CD161 + The method may further include purifying or enriching T cells to determine the presence of cells. After step (b), the CD8 in the sample + CD161 + The presence of cells may further include purifying or enriching T cells. Enriching T cells in a sample may include fluorescent cell sorting, magnetic or paramagnetic bead separation. Culturing may be continued for up to 7, 14, 21, 28, 35, or 42 days.

[0008] In some embodiments, cells are further cultured in a medium containing a CD3 and / or CD28 stimulant. In some embodiments, the CD3 and / or CD28 stimulant comprises a CD3 and / or CD28-conjugated antibody. In some embodiments, cells are further cultured in a medium containing a CD3, CD28, and / or CD161 stimulant. In some embodiments, the CD3, CD28, and / or CD161 stimulant comprises a CD3, CD28, and / or CD161-conjugated antibody. In some embodiments, cells are further cultured in a medium containing a CD3-conjugated antibody, a CD28-conjugated antibody, Clec2d, and / or a CD161-stimulating antibody. In some embodiments, cells are further cultured in a medium containing approximately 0.1–5.0, 0.3–3.0, or 0.5–2.0 μg / ml of CD3-conjugated antibody, CD28-conjugated antibody, Clec2d, and / or a CD161-stimulating antibody.

[0009] In one embodiment, CD8 + CD161 + cells, CD8 + CD161 negCells and bulk PBMCs are stimulated with plate-bound anti-CD3 / CD28 and enlarged in a cytokine cocktail consisting of 10 ng / ml IL-7, 5 ng / ml IL-15, and 30 ng / ml IL-21 (all from Peprotech, Rocky Hill, NJ). In one embodiment, CD8 + CD161 + Cells were isolated and cultured in response to 1 ug / mL of anti-CD / CD28 / CD161, and then augmented in RPMI-1640, 10% FBS, and 2 mmol / l GlutaMAX in a cytokine cocktail consisting of 10 ng / ml IL-7, 5 ng / ml IL-15, and 30 ng / ml IL-21. The cells were placed in a humidified chamber at 37°C for 48 hours. After 48 hours, the cells were augmented with the IL7 / 15 / 21 cytokine cocktail without antibody stimulation.

[0010] This method may further include obtaining cells from the subject, for example, by apheresis or venipuncture. The sample may be a cryopreserved sample. The sample may be derived from umbilical cord blood. The sample may be a peripheral blood sample from the subject. The sample may have an increased percentage of CD8 compared to an equivalent sample obtained from the subject. + CD161 + It may include subpopulations of T cells, including cellular cells. The sample may be obtained from a third-party institution.

[0011] The method may further include introducing nucleic acids encoding CARs into T cells in the sample, for example, using a viral vector or through a method that does not require viral transduction into T cells. The introduction of nucleic acids encoding CARs or transgenic TCRs into T cells may be performed before or after step (b). The T cells may be inactivated in terms of the expression of endogenous T cell receptors and / or endogenous HLA.

[0012] This method may further include introducing a nucleic acid encoding a membrane-bound Cγ cytokine into T cells, such that the membrane-bound Cγ cytokine is membrane-bound IL-15. The membrane-bound Cγ cytokine may be an IL-15-IL-15Rα fusion protein.

[0013] Culturing may involve culturing T cells in the presence of dendritic cells or artificial antigen-presenting cells (aAPCs). aAPCs may include CAR-binding or transgenic TCR-binding antibodies or fragments thereof expressed on the surface of the aAPC. aAPCs may include additional molecules that activate or co-stimulate T cells. These additional molecules may include membrane-bound Cγ cytokines. Culturing T cells in the presence of aAPCs may involve culturing cells in a ratio of approximately 10:1 to approximately 1:10 (CAR cells to aAPCs).

[0014] The method may further include cryopreserving samples of a population of transgenic CAR or transgenic TCR cells. The CAR or transgenic TCR can target cancer cell antigens, such as CD19, CD20, ROR1, CD22 carcinoembryonic antigens, alpha-fetoprotein, CA-125, 5T4, MUC-1, epithelial tumor antigens, prostate-specific antigens, melanoma-associated antigens, mutant p53, mutant ras, HER2 / Neu, folate-binding proteins, HIV-1 envelope glycoprotein gp120, HIV-1 envelope glycoprotein gp41, GD2, CD123, CD33, CD138, CD23, CD30, CD56, c-Met, meoterin, GD3, HERV-K, IL-11Rα, κ chain, λ chain, CSPG4, ERBB2, EGFRvIII, VEGFR2, HER2-HER3 combinations, or HER1-HER2 combinations. CARs or transgenic TCRs may target pathogenic antigens such as fungal, viral, or bacterial pathogens. The pathogens may be Plasmodium, Trypanosoma, Aspergillus, Candida, HSV, HIV, RSV, EBV, CMV, JC virus, BK virus, or Ebola pathogen.

[0015] This method involves determining the CD8 of the sample before step (b), after step (b), or both before and after step (b), for example, by cell counting / flow cytometry. + CD161 + This may further include evaluating the cell content.

[0016] T cell compositions prepared by the method described herein are also provided.

[0017] Further embodiments involve a method for providing a T-cell response in a human subject having a disease, comprising administering an effective amount of T cells as described herein. The disease may be cancer, and the CAR or transgenic TCR targets cancer cell antigens. The subject may have previously received anticancer therapy. The subject may be in remission or not have symptoms of cancer, but may contain detectable cancer cells.

[0018] [Invention 1001] (a) Obtain a sample of cells, wherein the sample is CD161 + The fact that it includes T cells, (b) Culture the T cells in the presence of IL-7, IL-15, and IL-21, Therefore, CD161 + The number of non-CD161 cells + To provide a population of T cells that are increasing in size compared to other cells, In vitro or ex vivo methods, including those mentioned above. [Invention 1002] (a) Obtaining a sample of cells, wherein the sample is CD8 + CD161 + The fact that it includes T cells, (b) Culture the T cells in the presence of IL-7, IL-15, and IL-21, Therefore, CD8 + CD161 + The number of T cells is non-CD8 + CD161 + To provide a population of T cells that are increasing in size compared to other cells, A method of the present invention 1001, including the method of the present invention 1001. [Invention 1003] (a) Obtaining a cell sample, wherein the sample is CD4+ CD161 + The fact that it includes T cells, (b) Culture the T cells in the presence of IL-7, IL-15, and IL-21, Therefore, CD4 + CD161 + The number of T cells is non-CD4 + CD161 + To provide a population of T cells that are increasing in size compared to other cells, A method of the present invention 1001, including the method of the present invention 1001. [Invention 1004] The method according to any one of the present invention 1001 to 1003, wherein the cells are further cultured in a medium containing a CD3 and / or CD28 stimulant. [Invention 1005] The method of the present invention 1004, wherein the CD3 and / or CD28 stimulant comprises a CD3 and / or CD28-conjugated antibody. [Invention 1006] The method of the present invention 1004, wherein the cells are further cultured in a medium containing a CD3-conjugated antibody, a CD28-conjugated antibody, Clec2d, and / or a CD161-conjugated antibody. [Invention 1007] The method of the present invention 1006, wherein the cells are further cultured in a medium containing approximately 0.1–5.0, 0.3–3.0, or 0.5–2.0 μg / ml of CD3-conjugated antibody, CD28-conjugated antibody, Clec2d, and / or CD161-conjugated antibody. [Invention 1008] (a1) Obtain a sample of cells, wherein the sample is CD161 + The fact that it includes T cells, (a2) Culturing the T cells in the presence of a CD3 and / or CD28 stimulant, and in the presence of IL-7, IL-15, and IL-21, (b) Culturing the T cells in the presence of IL-7, IL-15, and IL-21. A method of the present invention, including any of the methods described in 1001 to 1003. [Invention 1009] (a1) Obtain a sample of cells, wherein the sample is CD161 + The fact that it includes T cells, (a2) Culturing the T cells in the presence of a CD3-conjugated antibody, a CD28-conjugated antibody, a CD161-conjugated antibody, and / or Clec2d, and in the presence of IL-7, IL-15, and IL-21, (b) Culturing the T cells in the presence of IL-7, IL-15, and IL-21. A method of the present invention, including any of the methods described in 1001 to 1003. [Invention 1010] The method of the present invention 1009, wherein the culture in step (b) essentially does not contain a CD3-conjugated antibody, a CD28-conjugated antibody, a Clec2d antibody, and / or a CD161-conjugated antibody. [Invention 1011] The method of the present invention 1010, wherein the culture in step (a2) lasts for approximately 12 to 72 hours, 24 to 58 hours, or 24 to 36 hours. [Invention 1012] The method of the present invention 1010, wherein the culture in step (b) lasts for at least about 12 hours. [Invention 1013] The method of the present invention 1012, wherein the culture in step (b) lasts for at least about 1, 2, 3, 4, 5, 6, or 7 days. [Invention 1014] The method of the present invention 1009, wherein the culture in step (b) essentially does not contain CD3-conjugated antibody and CD28-conjugated antibody. [Invention 1015] A method according to any one of the present invention 1001 to 1003, wherein IL-7 is present at approximately 5 to 20 ng / ml, IL-15 is present at approximately 2.5 to 10 ng / ml, and / or IL-21 is present at approximately 20 to 40 ng / ml, for example, 10 ng / ml of IL-7, 5 ng / ml of IL-15, and / or 30 ng / ml of IL-21. [Invention 1016] Before step (b), the CD8 in the sample + CD161 + The method of the present invention 1001 or 1015, further comprising purifying or enriching T cells for the presence of cells. [Invention 1017] After step (b), the CD8 in the sample + CD161 + The method of the present invention 1001 or 1015, further comprising purifying or enriching T cells for the presence of cells. [Invention 1018] The method of the present invention 1016 or 1017, wherein enriching the T cells in the sample includes fluorescent cell sorting, magnetic bead separation, or paramagnetic bead separation. [Invention 1019] A method according to any of the invention 1001-1018, wherein the culture is sustained for a maximum of 7, 14, 21, 28, 35, or 42 days. [Invention 1020] The method of the present invention described above, wherein the culture is performed in a serum-containing culture medium, any one of the methods described in 1001 to 1019. [Invention 1021] The method of the present invention described above, wherein the culture is performed in a serum-free medium, as described in any of the methods described in 1001 to 1019. [Invention 1022] Any method 1001 to 1003 of the present invention, further comprising obtaining the cells from a target. [Invention 1023] The method of the present invention 1022, wherein the sample is obtained by apheresis. [Invention 1024] The method according to any one of the present invention 1001 to 1003, wherein the sample is a frozen and stored sample. [Invention 1025] The method according to any one of the present invention 1001 to 1003, wherein the sample is derived from umbilical cord blood. [Invention 1026] The method according to any one of the present invention 1001 to 1003, wherein the sample is a peripheral blood sample from the subject. [Invention 1027] The aforementioned sample is obtained by apheresis, according to any one of the methods 1001 to 1003 of the present invention. [Invention 1028] The aforementioned sample is obtained by any of the methods 1001 to 1003 of the present invention, wherein the sample is obtained by venipuncture. [Invention 1029] The aforementioned sample has a CD8 increase of the percentage compared to an equivalent sample obtained from the subject. + CD161 + A method according to any one of the present invention 1001 to 1003, comprising a subpopulation of T cells including cellular cells. [Invention 1030] The method according to any of the present invention 1001 to 1003, wherein obtaining the aforementioned sample includes obtaining the aforementioned sample from a third-party organization. [Invention 1031] Any method of the present invention 1001 to 1030 further comprises introducing nucleic acids encoding a chimeric antigen receptor (CAR) or a transgenic T cell receptor (TCR) into T cells in the aforementioned sample. [Invention 1032] The method of the present invention 1031, wherein the introduction does not require infecting the T cells with a virus or transduction. [Invention 1033] The method of the present invention 1031 or 1032, wherein the introduction of nucleic acids encoding a CAR or transgenic TCR into the T cells is performed prior to step (b). [Invention 1034] The method of the present invention 1031 or 1032, wherein the introduction of nucleic acids encoding a CAR or transgenic TCR into the T cells is performed after step (b). [Invention 1035] The method of the present invention 1031, wherein the T cells are inactivated with respect to the expression of endogenous T cell receptors and / or endogenous HLA. [Invention 1036] The method of the present invention 1031, further comprising introducing a nucleic acid encoding a membrane-bound Cγ cytokine into the T cells. [Invention 1037] The method of the present invention 1036, wherein the membrane-bound Cγ cytokine is membrane-bound IL-15. [Invention 1038] The method of the present invention 1036, wherein the membrane-bound Cγ cytokine is an IL-15-IL-15Rα fusion protein. [Invention 1039] The method of the present invention 1031, wherein culturing comprises culturing the T cells in the presence of dendritic cells or artificial antigen-presenting cells (aAPCs). [Invention 1040] The method of the present invention 1039, wherein the aAPC comprises a CAR-binding antibody or a fragment thereof expressed on the surface of the aAPC. [Invention 1041] The method of the present invention 1039, wherein the aAPC comprises additional molecules that activate or co-stimulate T cells. [Invention 1042] The method of the present invention 1040, wherein the additional molecule comprises a membrane-bound Cγ cytokine. [Invention 1043] The method of the present invention 1039, wherein culturing the T cells in the presence of aAPCs comprises culturing the cells in a ratio of approximately 10:1 to approximately 1:10 (CAR cells to aAPCs). [Invention 1044] The method of the present invention 1031 further comprises cryopreserving a sample of the population of transgenic CAR or transgenic TCR cells. [Invention 1045] The method of the present invention 1031, wherein the CAR or the transgenic TCR targets a cancer cell antigen. [Invention 1046] The method of the present invention 1045, wherein the cancer cell antigen is CD19, CD20, ROR1, CD22 carcinoembryonic antigen, alpha-fetoprotein, CA-125, 5T4, MUC-1, epithelial tumor antigen, prostate-specific antigen, melanoma-associated antigen, mutant p53, mutant ras, HER2 / Neu, folate-binding protein, HIV-1 envelope glycoprotein gp120, HIV-1 envelope glycoprotein gp41, GD2, CD123, CD33, CD138, CD23, CD30, CD56, c-Met, meoterin, GD3, HERV-K, IL-11Rα, κ chain, λ chain, CSPG4, ERBB2, EGFRvIII, VEGFR2, a combination of HER2-HER3, or a combination of HER1-HER2. [Invention 1047] The method of the present invention 1031, wherein the CAR or the transgenic TCR targets a pathogen antigen. [Invention 1048] The method of the present invention 1047, wherein the pathogen is a fungal, viral, or bacterial pathogen. [Invention 1049] The method of the present invention 1047, wherein the pathogen is Plasmodium, Trypanosoma, Aspergillus, Candida, HSV, HIV, RSV, EBV, CMV, JC virus, BK virus, or the Ebola pathogen. [Invention 1050] Before step (b), after step (b), or both before and after step (b), for example, by cell counting / flow cytometry, the CD161 of the sample is determined. + Any method of the present invention 1001 to 1003, further comprising evaluating the T cell content. [Invention 1051] A T cell composition prepared by any of the methods described in Invention 1001 to 1050. [Invention 1052] A method for providing a T cell response in a human subject having a disease, comprising administering an effective amount of T cells of the present invention 1031 or 1032 to the subject. [Invention 1053] The method of the present invention 1052, wherein the disease is cancer, and the CAR or the transgenic TCR targets cancer cell antigens. [Invention 1054] The method of the present invention 1053, wherein the subject has previously undergone anti-cancer therapy. [Invention 1055] The method of the present invention 1054, wherein the subject is in a state of remission or does not have the symptoms of the cancer, but includes detectable cancer cells. Other purposes, features, and advantages of this disclosure will become apparent from the following detailed description. However, please understand that the detailed description and specific examples are provided for illustrative purposes only, while illustrating preferred embodiments of this disclosure, as various changes and modifications within the intent and scope of this disclosure will become apparent to those skilled in the art from this detailed description. [Brief explanation of the drawing]

[0019] The following drawings form part of this specification and are included to further illustrate certain aspects of the disclosure. This disclosure may be better understood by referring to one or more of these drawings in conjunction with the detailed description of the specific embodiments presented herein.

[0020] [Figure 1]Gene expression analysis of antigen-stimulated T cells using microarrays showed significant upregulation of cytotoxicity and congenital-like properties of CD8+NK1.1+ cells. A cohort of 15 mice received dendritic cell-based vaccination in combination with chemotherapy for mouse pancreatic ductal adenocarcinoma. Sixty days after tumor inoculation, spleens were harvested and pooled into three groups of five each, and activated overnight with dendritic cells loaded with tumor antigens. Antigen-stimulated cells were then sorted into NK1.1neg and NK1.1+ subsets by flow cytometry, gating over the CD8+CD69+ population. The volcanic plot shows 1642 genes that are significantly regulated between CD8+NK1.1neg and CD8+NK1.1+ cells at a univariate significance level of 0.1. The top 15 genes that are differentially regulated at an FDR of 0.05 are labeled on the plot. [Figure 2]Figures 2A–F define a memory population in which CD8+NK1.1+ cells provide durable protection against influenza infection and melanoma tumors, improving survival rates. In the influenza model, splenocytes were collected from mice upon recovery after influenza infection, sorted into CD8+NK1.1neg and CD8+NK1.1+ cells, and then adopted into naive mice challenged with influenza. In the melanoma model, tumor-bearing mice were vaccinated with dendritic cells loaded with tumor antigens. After 3 weeks, splenocytes were collected, sorted into CD8+NK1.1neg and CD8+NK1.1+ cells, and adopted into mice with palpable tumors. Adoptive transfer of antigen-experienced CD8+NK1.1+ cells provided durable protection against influenza infection (Figures 2A–C) and melanoma tumors (Figures 2D–F). (Figure 2A) Mice that received CD8+NK1.1+ cells recovered their body weight during influenza infection compared to mice that received CD8+NK1.1neg and naive CD8+ cells. (Figure 2B) Mice that received CD8+NK1.1+ cells had a 100% survival rate compared to the groups that received CD8+NK1.1neg and naive CD8+ cells. (Figure 2C) Analysis of PBMCs two weeks after infection showed a 40% increase in circulating CD3+CD8+IFN-γ+ cells among mice that received CD8+NK1.1+ cells compared to adoptive cohorts of naive and CD8+NK1.1neg (p<0.003). (Figure 2D~E) In a melanoma model, mice that received CD8+NK1.1+ cells showed delayed tumor growth and improved survival. (Figure 2F) Analysis of peripheral blood lymphocytes 3 weeks after tumor transplantation showed significantly elevated levels of the memory markers CD62L and CCR7 between GP100 tetramer-specific CD8+ cells in the cohort adopted with CD8+NK1.1neg or naive splenocytes compared to the cohort adopted with CD8+NK1.1neg or naive splenocytes. Each experiment consisted of n=10 mice per group. Error bars = + / - SEM, *p<0.05, one-way ANOVA. [Figure 3]The mouse CD3+CD8+NK1.1+ cell population is phenotypically conserved among its human CD3+CD8+CD161+ counterparts. Human equivalents of CD3+CD8+CD161+ and CD3+CD8+CD161neg cells, mouse CD3+CD8+NK1.1+ cells, and CD3+CD8+NK1.1neg cells were magnetically sorted from the peripheral blood of six human donors, and gene expression profiles were analyzed by microarray. The volcanic plot, showing differential gene regulation between CD8+CD161+ and CD8+CD161neg cells, highlights CD161 receptor upregulation with an ellipse. [Figure 4] CD8+CD161+, CD8+CD161neg, and unmodified bulk PBMCs were freshly isolated from human peripheral blood products. Isolated cells were immediately tested for cytotoxicity in a 4-hour killing assay using a 51Cr-labeled allogeneic 293-HEK target. As shown, CD8+CD161+ cells were able to induce 100% target lysis at an E:T ratio of 25:1, while bulk PBMCs and CD8+CD161neg cells exhibited 22% and 15% lysis at a maximum E:T ratio of 50:1, respectively (p<0.002 at 50:1, p<0.0007 at 25:1, and p<0.00002 at 5:1, by one-way ANOVA). X-axis - E:T ratio. Y-axis - killing rate. Error bars = + / -SD. [Figure 5] Ex vivo augmentation of CD8+CD161+ cells with IL7 / 15 / 21, combined with plate-bound stimulation with anti-CD3 / CD28 / Clec2d, enhanced the central memory phenotype (CD45RA-CCR7+). CD8+CD161+ cells were selected from normal donors, and ex vivo stimulation conditions were optimized. Cells were not CAR transduced. Compared to stimulation with IL2, IL-2 / 7 / 15, and IL2 / 7 / 15 / 21, the combination of IL7 / 15 / 21 and plate-bound stimulation with anti-CD3 / CD28 / Clec2d resulted in significant upregulation of central memory (CD45RA-CCR7+). [Figure 6]Ex vivo augmentation of CD8+CD161+ cells with IL7 / 15 / 21, combined with plate-bound stimulation with anti-CD3 / CD28 / Clec2d, enhanced cytotoxic granzyme production. CD8+CD161+ cells were selected from normal donors, and ex vivo stimulation conditions were optimized. Cells were not CAR transduced. Compared to stimulation with IL2, IL-2 / 7 / 15, and IL2 / 7 / 15 / 21, the combination of IL7 / 15 / 21 and plate-bound stimulation with anti-CD3 / CD28 / Clec2d resulted in significant upregulation of cytotoxic molecules, granzymes, and perforins. [Figure 7A] CD8+NK1.1+ cells have been identified as important circulating memory cells in multiple mouse models of disease. To verify that the protective effect of CD8+NK1.1+ cells is model-independent, adoptive transfer experiments of CD8+NK1.1+ cells were performed in influenza infection and melanoma tumor models. (Figure 7A) Naive mice were exposed to a sublethal dose of influenza, allowed to recover from infection, and splenocytes were collected 3 weeks after infection and magnetically sorted into CD8+NK1.1neg and CD8+NK1.1+ cells. 5 × 10⁵ cells / mouse from each NK1.1 group were adopted into a naive cohort lethally challenged with the same influenza virus strain 24 hours after adoptive transfer. Mice receiving naive CD8+ splenocytes served as controls. (Figure 7B) Naive mice were subcutaneously inoculated with 2 × 10⁵ B16 melanoma cells and vaccinated with a cell-based vaccine loaded with B16 antigen on days 7 and 14 post-inoculation. On day 21, the mice were sacrificed, splenocytes were collected, and sorted into CD8+NK1.1neg and CD8+NK1.1+ cell populations. Subsequently, the naive cohort inoculated with palpable B16 tumors was adopted by intraperitoneal injection with 1.5 × 10⁶ CD8+NK1.1neg and CD8+NK1.1+ cells, respectively. Mice receiving naive CD8+ splenocytes served as controls. [Figure 7B] Please refer to the explanation in Figure 7A. [Figure 8-1]TCR-Vβ spectratyping revealed CD3+CD8+CD161+ cells to be essentially polyclonal. To confirm the clonality of CD8+CD161+ cells, TCR-Vβ spectratyping was performed on donor-derived cells. Histograms from amplifications of 30 TCR Vβ family cells showed an unskewed Gaussian distribution of CDR3 size, suggesting the polyclonal nature of these cells. [Figure 8-2] Please refer to the explanation in Figure 8-1. [Figure 9] Interspecies comparative gene analysis has revealed conserved gene signatures of 206 genes that are differentially regulated between two populations. Based on nomenclature, 206 common genes were identified between mouse (15 pooled samples) and human (6 corresponding samples) microarray analysis. The expression patterns of these genes were similar between activated CD8+NK1.1+ cells and quiescent CD8+CD161+ cells, demonstrating the highly conserved nature of the gene signatures. [Modes for carrying out the invention]

[0021] Description of Exemplary Embodiments As mentioned above, CAR-T therapy has shown promise in the treatment of cancers such as metastatic mouse tubular adenocarcinoma (PDAC). In our previous study, we introduced adoptively transferred antigen-experienced CD8 + NK1.1 + We demonstrated that the cells can mediate durable protection in a PDAC model. Interestingly, these cells were present 9 months after initial exposure to the antigen and were subsequently highly protected when adopted into naive mice challenged with the parental PDAC cell line (Konduri et al., 2016). Building on these results, we attempted to characterize the further biological and functional properties of these cells in various in vivo model systems, including a SCID xenograft model for CAR T cell therapy for the treatment of PDAC. The results showed that CD8 + CD161+ We have demonstrated that T cells contain an excellent platform for CAR T cell therapy, provided that they are adapted to prevent differentiation of the starting cell population during transduction and growth. Furthermore, we have developed an improved method that now allows for the ex vivo growth of such cells, thereby making it easier to deliver CAR-T therapy to subjects who need it. These and other features of this disclosure are described in more detail below.

[0022] I. Definition As used herein, "a" or "an" may mean one or more. As used in the claims herein, when used in conjunction with the word "comprising," "a" or "an" may mean one or more.

[0023] The use of the term “or” in the claims is used to mean “and / or” unless it is expressly indicated that it refers only to substitutes, or if the substitutes are mutually exclusive; however, this disclosure supports the definition that refers only to substitutes and “and / or.” Where used herein, “another” may mean at least two or more.

[0024] Throughout this application, the term "approximately" is used to indicate that the value includes an inherent variation in the error relating to the device, a method used to determine the value, a variation present between the subjects of study, or a value within 10 percent of the stated value.

[0025] As used herein, the term “chimeric antigen receptor (CAR)” may refer to, for example, an artificial T cell receptor, a chimeric T cell receptor, a transgenic T cell receptor, or a chimeric immune receptor, and encompasses engineered receptors that impart artificial specificity to specific immune effector cells. CARs may also be used to confer specificity to monoclonal antibodies to T cells, thereby enabling the generation of a large number of specific T cells, for example, for use in adoptive cell therapy. In certain embodiments, CARs direct, for example, the specificity of cells to tumor-associated antigens. In some embodiments, a CAR comprises an intracellular activation domain, a transmembrane domain, and an extracellular domain including a tumor-associated antigen binding region. In certain embodiments, a CAR comprises a fusion of a single-chain variable fragment (scFv) derived from a monoclonal antibody fused to a CD3-zeta transmembrane domain and an endodomain. Specificity in other CAR designs may derive from the receptor ligand (e.g., a peptide) or from a pattern recognition receptor such as dectin. In certain cases, the spacing of the antigen recognition domains may be modified to reduce activation-induced cell death. In certain cases, CARs may contain domains for additional co-stimulatory signaling, such as CD3-zeta, FcR, CD27, CD28, CD137, DAP10, and / or OX40. In some cases, molecules including co-stimulatory molecules, reporter genes for imaging (e.g., positron emission tomography), gene products that conditionally remove T cells upon prodrug addition, homing receptors, chemokines, chemokine receptors, cytokines, and cytokine receptors can be co-expressed with CARs.

[0026] As used herein, the term “T cell receptor (TCR)” refers to a protein receptor on a T cell that is composed of an alpha (α) and beta (β) chain heterodimer, although in some cells, the TCR consists of gamma and delta (γ / δ) chains. In embodiments of this disclosure, the TCR may be modified on any cell containing a TCR that includes, for example, helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells, and gamma-delta T cells.

[0027] The terms “tumor-associated antigen” and “cancer cell antigen” are used synonymously herein. In each case, the terms refer to proteins, glycoproteins, or carbohydrates that are specifically or preferentially expressed by cancer cells.

[0028] II. Chimeric Antigen Receptors As used herein, the term “antigen” refers to a molecule that can be bound by an antibody or a T cell receptor. Antigens can further induce humoral and / or cellular immune responses that result in the production of B lymphocytes and / or T lymphocytes.

[0029] Embodiments of this disclosure include nucleic acids encoding antigen-specific chimeric antigen receptor (CAR) polypeptides, including CARs that have been humanized to reduce immunogenicity (hCAR), comprising an intracellular signaling domain, a transmembrane domain, and an extracellular domain comprising one or more signaling motifs. In certain embodiments, the CAR may recognize an epitope consisting of a shared space between one or more antigens. Pattern recognition receptors such as Dectin-1 can be used to derive specificity for carbohydrate antigens. In certain embodiments, the binding region may include a complementarity-determining region of a monoclonal antibody, a variable region of a monoclonal antibody, and / or its antigen-binding fragment. In another embodiment, the specificity is derived from a peptide (e.g., cytokine) that binds to the receptor. A complementarity-determining region (CDR) is a short amino acid sequence found in the variable domain of an antigen receptor (e.g., immunoglobulin and T cell receptor) protein that complements the antigen and thus provides the specificity of its receptor for that particular antigen. Each polypeptide chain of an antigen receptor contains three CDRs (CDR1, CDR2, and CDR3). Antigen receptors typically consist of two polypeptide chains; therefore, there are six CDRs for each antigen receptor that can come into contact with an antigen, with each heavy and light chain containing three CDRs. Since most sequence mutations associated with immunoglobulin and T cell receptors are found within the CDRs, these regions are sometimes called hypervariable domains. Of these, CDR3 exhibits the greatest variability because it is encoded by recombination of the VJ (VDJ in the case of the heavy chain and TCR αβ chain) region.

[0030] Human CAR nucleic acids are envisioned to be human genes for augmenting cellular immunotherapy for human patients. In certain embodiments, this disclosure includes full-length CAR cDNA or coding regions. The antigen-binding region or domain is a single-chain variable fragment (scFv) derived from a specific human monoclonal antibody, such as that described in U.S. Patent No. 7,109,304, which is incorporated herein by reference. H and V LThe chain may contain fragments. The fragments may also be any number of different antigen-binding domains of a human antigen-specific antibody. In a more specific embodiment, the fragment is an antigen-specific scFv encoded by a sequence optimized for the use of human codons for expression in human cells.

[0031] The structure may be a multimer, such as a diabody or a multimer. The multimer is most likely formed by the cross-pairing of the variable portions of the light and heavy chains into what Winters calls a diabody. The hinge portion of the construct can have several alternatives, from being completely deleted, maintaining the first cysteine, maintaining proline instead of serine substitution, and cleaving down to the first cysteine. The Fc portion may be deleted. Any protein that is stable and / or dimerizes can serve this purpose. Only one of the Fc domains, e.g., either the CH2 or CH3 domain from human immunoglobulin, can be used. Alternatively, the hinge, CH2 and CH3 regions of human immunoglobulin modified to improve dimerization can also be used. Only the hinge portion of immunoglobulin can be used. The CD8α portion can also be used.

[0032] The intracellular signaling domain of the chimeric receptor described herein is responsible for the activation of at least one of the normal effector functions of the immune cell in which the chimeric receptor is located. The term “effector function” refers to a specific function of a differentiated cell. The effector function of a T cell may be, for example, cytolytic activity or helper activity including cytokine secretion. Effector functions in naive, memory, or memory T cells include antigen-dependent proliferation. Thus, the term “intracellular signaling domain” refers to the portion of a protein that transduces the effector function signal, causing the cell to perform the specific function. Usually, the entire intracellular signaling domain is used, but often it is not necessary to use the entire intracellular polypeptide. To the extent that a truncated portion of the intracellular signaling domain can be used, such truncated portion may be used in place of the intact chain, as long as it still transduces the effector function signal. Thus, the term intracellular signaling domain means that it includes any truncated portion of the intracellular signaling domain that is sufficient to transduce the effector function signal. Examples include the zeta chain of the T cell receptor or any of its homologs (e.g., eta, delta, gamma, or epsilon), MB1 chain, B29, Fc RIII, Fc RI, and combinations of signaling molecules such as CD3ζ and CD28, CD27, 4-1BB, DAP-10, OX40, as well as other similar molecules and fragments. Intracellular signaling portions of other members of the activating protein family, such as FcγRIII and FcεRI, can be used. In preferred embodiments, the human CD3ζ intracellular domain is taken up for activation.

[0033] The antigen-specific extracellular domain and intracellular signaling domain may be linked by transmembrane domains such as the human IgG4Fc hinge and Fc region. Alternatives include the human CD4 transmembrane domain, the human CD28 transmembrane domain, the human transmembrane CD3ζ domain, or the cysteine-mutated human CD3ζ domain, or other transmembrane domains derived from other human transmembrane signaling proteins such as CD16 and CD8 and the erythropoietin receptor.

[0034] In some embodiments, the CAR nucleic acid includes a transmembrane domain and sequences encoding other costimulatory receptors, such as a modified CD28 intracellular signaling domain. Other costimulatory receptors include, but are not limited to, one or more of CD28, CD27, OX-40 (CD134), DAP10, and 4-1BB (CD137). In addition to the primary signal initiated by CD3ζ, the additional signaling provided by human costimulatory receptors inserted into human CARs is crucial for the full activation of T cells and can contribute to improving the in vivo persistence and therapeutic success of adoptive immunotherapy.

[0035] In certain embodiments, the disclosure relates to isolated nucleic acid segments and expression cassettes incorporating DNA sequences encoding a CAR. The vectors of the disclosure are primarily designed to deliver a desired gene to immune cells, preferably T cells, under the control of a controlled eukaryotic promoter, such as the MNDU3 promoter, CMV promoter, EF1α promoter, or ubiquitin promoter. The vectors may also contain selectable markers to facilitate in vitro manipulation, where there is no other reason. In other embodiments, the CAR may be expressed from mRNA transcribed in vitro from a DNA template.

[0036] Chimeric antigen receptor molecules are recombinant and distinguished by their ability to both bind to antigens and transduce activation signals via an immunoreceptor activation motif (ITAM) present in their cytoplasmic tail. Receptor constructs that utilize the antigen-binding moiety (e.g., generated from single-chain antibodies (scFv)) offer the additional advantage of being "universal" in that they bind to native antigens on the target cell surface in an HLA-independent manner. For example, several laboratories have reported scFv constructs fused to sequences encoding the zeta chain (ζ) of the CD3 complex, the Fc receptor gamma chain, and the intracellular portion of the sky tyrosine kinase (Eshhar et al., 1993; Fitzer-Attas et al., 1998). Redirected T-cell effector mechanisms, including tumor recognition and lysis by CTLs, have been documented in several mouse and human antigen-scFv:ζ systems (Eshhar, 1997; Altenschmidt et al., 1997; Brocker et al., 1998).

[0037] To date, non-human antigen-binding regions have typically been used in the construction of chimeric antigen receptors. Potential problems associated with the use of non-human antigen-binding regions, such as mouse monoclonal antibodies, include the lack of human effector function and the inability to penetrate tumor masses. In other words, such antibodies may not be able to destroy cells expressing CARs by mediating complement-dependent lysis or lysing human target cells via antibody-dependent cytotoxicity or Fc receptor-mediated phagocytosis. Furthermore, non-human monoclonal antibodies can be recognized by the human host as foreign proteins, and therefore, repeated injection of such foreign antibodies may lead to the induction of an immune response resulting in adverse hypersensitivity reactions. In the case of mouse-based monoclonal antibodies, this is often called a human anti-mouse antibody (HAMA) response. Therefore, the use of human antibodies is preferable because it does not induce as potent a HAMA response as mouse antibodies. Similarly, the use of human sequences in CARs can avoid immunomediated recognition and thus avoid elimination by endogenous T cells that recognize antigens present in the recipient and processed in relation to HLA.

[0038] In some embodiments, the chimeric antigen receptor comprises an extracellular domain including a) an intracellular signaling domain, b) a transmembrane domain, and c) an antigen-binding domain.

[0039] In certain embodiments, the intracellular receptor signaling domain within the CAR may be, for example, CD3, or a T cell antigen receptor complex such as the Fcγ RIII costimulatory signaling domain, CD28, CD27, DAP10, CD137, OX40, CD2, either alone or in conjunction with the CD3 zeta chain. In specific embodiments, the intracellular domain (which may also be called the cytoplasmic domain) may include one or more portions or all of the following: TCR zeta chain, CD28, CD27, OX40 / CD134, 4-1BB / CD137, FcεRIγ, ICOS / CD278, IL-2R beta / CD122, IL-2Rα / CD132, DAP10, DAP12, and CD40. In some embodiments, any portion of the endogenous T cell receptor complex in the intracellular domain is used. A so-called third-generation CAR may use one or more cytoplasmic domains, for example, having at least two or three signaling domains fused together for additive or synergistic effects.

[0040] In certain embodiments of the chimeric antigen receptor, the antigen-specific portion of the receptor (which may be called the extracellular domain, including the antigen-binding region) includes a pathogen-specific antigen-binding domain containing a tumor-associated antigen or a carbohydrate antigen recognized by a pattern recognition receptor such as Dectin-1. The tumor-associated antigen may be any type, as long as it is expressed on the cell surface of tumor cells. Exemplary embodiments of tumor-associated antigens include CD19, CD20, carcinoembryonic antigen, α-fetoprotein, CA-125, MUC-1, CD56, EGFR, c-Met, AKT, Her2, Her3, epithelial tumor antigen, melanoma-associated antigen, mutant p53, mutant ras, and the like. In certain embodiments, CAR may be co-expressed with membrane-bound cytokines to improve persistence when small amounts of tumor-associated antigen are present. For example, CAR may be co-expressed with membrane-bound IL-15.

[0041] In certain embodiments, intracellular tumor-associated antigens such as HA-1, survivin, WT1, and p53 may be targeted. This can be achieved by CARs expressed on universal T cells that recognize processed peptides described from intracellular tumor-associated antigens in relation to HLA. Furthermore, universal T cells may be genetically modified to express T cell receptor pairings that recognize intracellularly processed tumor-associated antigens in relation to HLA.

[0042] The pathogen may be of any kind, but in certain embodiments, the pathogen may be, for example, a fungus, a bacterium, or a virus. Exemplary viral pathogens include the adenoviridae family, Epstein-Barr virus (EBV), cytomegalovirus (CMV), respiratory syncytial virus (RSV), JC virus, BK virus, HSV, the HHV family of viruses, the picornaviridae, herpesviridae, hepadnaviridae, flaviviridae, retroviridae, orthomyxoviridae, paramyxoviridae, papovaviridae, poliomaviridae, rhabdoviridae, and togaviridae. Exemplary pathogenic viruses cause smallpox, influenza, mumps, measles, varicella, Ebola, and rubella. Exemplary pathogenic fungi include Candida, Aspergillus, Cryptococcus, Histoplasma, Pneumocystis, and Stachybotrys. Exemplary pathogenic bacteria include Streptococcus, Pseudomonas, Shigella, Campylobacter, Staphylococcus, Helicobacter, E. coli, Rickettsia, Bacillus, Bordetella, Chlamydia, Spirochetes, and Salmonella. In one embodiment, the pathogen receptor Dectin-1 may be used to generate CARs that recognize carbohydrate structures on the cell wall of fungi. T cells genetically modified to express CARs based on the specificity of Dectin-1 can recognize Aspergillus and target hyphal growth. In another embodiment, CARs may be produced based on antibodies that recognize viral determinants (e.g., glycoproteins derived from CMV and Ebola) to interfere with viral infection and pathology.

[0043] In some embodiments, the pathogenic antigen is an Aspergillus carbohydrate antigen in which the extracellular domain of the CAR recognizes the carbohydrate pattern of the fungal cell wall via Dectin-1 or the like.

[0044] The chimeric immunoreceptors described herein may be produced by any means known in the art, but are preferably produced using DNA recombination techniques. Nucleic acid sequences encoding several regions of the chimeric receptor can be prepared and assembled into a complete coding sequence by standard molecular cloning techniques (e.g., genome library screening, PCR, primer-assisted ligation, yeast and bacterial scFv libraries, site-directed mutagenesis). The resulting coding regions can be inserted into an expression vector and used to transform a suitable expression host allogeneic T cell line.

[0045] As used herein, nucleic acid constructs, nucleic acid sequences, or polynucleotides are intended to mean DNA molecules that can be transformed or introduced into T cells, transcribed, and translated to produce products (e.g., chimeric antigen receptors).

[0046] In the exemplary nucleic acid constructs (polynucleotides) used in this disclosure, promoters are ligated to the nucleic acid sequences encoding the chimeric receptors of this disclosure, i.e., they are positioned to promote the transcription of messenger RNA from the DNA encoding the chimeric receptors. Promoters can be of genomic origin or synthetically generated. Various promoters for use in T cells are well known in the art (e.g., the CD4 promoter disclosed by Marodon et al., 2003). Promoters may be constitutive or inductive, for example, if induction is associated with a particular cell type or a particular level of maturity. Alternatively, many well known viral promoters are also suitable. Promoters of interest include the β-actin promoter, the SV40 early and late promoters, the immunoglobulin promoter, the human cytomegalovirus promoter, the retroviral promoter, and the Friend spleen-limited foci virus promoter. Promoters may or may not associate with enhancers, and enhancers may spontaneously associate with certain promoters or with different promoters.

[0047] The sequences of open reading frames encoding chimeric receptors can be obtained from genomic DNA sources, cDNA sources, or synthesized (e.g., via PCR), or a combination thereof. Depending on the size of the genomic DNA and the number of introns, it may be preferable to use cDNA or a combination thereof, as introns have been found to stabilize mRNA or lead to T cell-specific expression (Barthel and Goldfeld, 2003). Furthermore, it may be even more advantageous to use endogenous or exogenous non-coding regions to stabilize mRNA.

[0048] For the expression of the chimeric antigen receptor of this disclosure, the chimeric receptor can be generated in a target host using a native or endogenous transcription initiation region of a nucleic acid sequence encoding the N-terminal component of the chimeric receptor. Alternatively, an exogenous transcription initiation region can be used to enable constitutive or inductive expression, where the expression can be controlled depending on the target host, the desired expression level, the properties of the target host, and so on.

[0049] Similarly, the signal sequence that guides the chimeric receptor to the surface membrane may be the endogenous signal sequence of the N-terminal component of the chimeric receptor. Selectively, in some cases, it may be desirable to replace this sequence with a different signal sequence. However, the selected signal sequence should be compatible with the T cell secretory pathway so that the chimeric receptor is presented on the T cell surface.

[0050] Similarly, the termination region may be provided by a native or endogenous transcriptional termination region of the nucleic acid sequence encoding the C-terminal component of the chimeric receptor. Alternatively, the termination region may be derived from a different source. In most cases, the source of the termination region is generally considered irrelevant to recombinant protein expression, and a wide variety of termination regions may be used without adversely affecting expression.

[0051] As those skilled in the art will understand, in some cases, several amino acids at the terminal end of the antigen-binding domain within a CAR may be deleted, for example, typically 10 or fewer residues, and more typically 5 or fewer. It may also be desirable to introduce a small number of amino acids into the boundary, typically 10 or fewer, and more typically 5 or fewer residues. Deletion or insertion of amino acids can result from the needs of the construct, such as providing convenient restriction sites, ease of manipulation, and improved expression levels. In addition, substitution of one or more amino acids with different amino acids may occur for similar reasons, but typically, no more than approximately 5 amino acids are substituted within any single domain.

[0052] Chimeric constructs encoding the chimeric receptor according to this disclosure can be prepared by conventional methods. In most cases, native sequences may be used, and the native genes may be isolated and manipulated as necessary to enable proper binding of various components. Thus, the nucleic acid sequences encoding the N-terminal and C-terminal proteins of the chimeric receptor can be isolated using polymerase chain reaction (PCR) with appropriate primers that result in deletion of undesirable parts of the gene. Alternatively, the chimeric construct can be generated using restriction digestion of the cloned gene. In either case, sequences can be selected to provide restriction sites that are blunt-ended or have complementary duplication.

[0053] Various operations for preparing chimeric constructs can be performed in vitro, and in certain embodiments, the chimeric construct is introduced into a vector for cloning and expression in a suitable host using standard transformation or transfection methods. Thus, after each operation, the construct obtained from the binding of DNA sequences is cloned, the vector is isolated, and the sequence is screened to ensure that the sequence encodes the desired chimeric receptor. Sequences can be screened by restriction analysis, sequencing, etc.

[0054] The chimeric constructs of this disclosure find applications in subjects with or suspected of having cancer by reducing tumor size or preventing tumor growth or regrowth in these subjects. Accordingly, this disclosure relates to a method of reducing growth or preventing tumor formation in a subject by introducing the chimeric constructs of this disclosure into isolated T cells of a subject and reintroducing the transformed T cells into the subject, thereby eliciting an antitumor response to reduce or eliminate tumors in the subject. Suitable T cells that may be used include cytotoxic lymphocytes (CTLs) or any cells having T cell receptors that require destruction. As is well known to those skilled in the art, various methods for isolating these cells from a subject are readily available. For example, cell surface marker expression or commercially available kits (e.g., ISOCELL® from Pierce, Rockford, Ill.) are used.

[0055] The chimeric construct is expected to be introduced into the subject's own T cells as naked DNA or in a suitable vector. Methods for stably transfecting T cells by electroporation using naked DNA are known in the art. See, for example, U.S. Patent No. 6,410,319. Naked DNA generally refers to the DNA encoding the chimeric receptor of this disclosure contained in a plasmid expression vector in an appropriate orientation for expression. Advantageously, the use of naked DNA reduces the time required to produce T cells expressing the chimeric receptor of this disclosure.

[0056] Alternatively, the chimeric construct can be introduced into T cells using a viral vector (e.g., a retroviral vector, adenovirus vector, adeno-associated virus vector, or lentiviral vector). Vectors suitable for use according to the methods disclosed herein do not replicate in the target T cells. Numerous virus-based vectors are known, and the number of viral copies maintained within the cell is low enough to maintain cell viability. Exemplary vectors include the pFB-neo-vector (STRATAGENE®) disclosed herein, as well as vectors based on HIV, SV40, EBV, HSV, or BPV.

[0057] Once it is established that transfected or transduced T cells can express chimeric receptors as surface membrane proteins at desired levels and regulatory levels, it can be determined whether the chimeric receptors function in host cells and result in desired signal induction. The transduced T cells are then reintroduced or administered to a target to activate an antitumor response in the target. To facilitate administration, the transduced T cells according to this disclosure can be made into a pharmaceutical composition or, with appropriate carriers or diluents, into implants suitable for in vivo administration, which may further be pharmaceutically acceptable. Means for preparing such compositions or implants have been described in the art (see, for example, Remington's Pharmaceutical Sciences, 16th Ed., Mack, ed., 1980). If necessary, the transduced T cells can be formulated in the usual manner for each route of administration into semi-solid or liquid preparations such as capsules, solutions, injections, inhalants, or aerosols. By means known in the art, the release and absorption of the composition can be prevented or minimized, or sustained release of the composition can be ensured, until the composition reaches the target tissue or organ. However, preferably, a pharmaceutically acceptable form is used that does not inactivate cells expressing the chimeric receptor. Therefore, transduced T cells can preferably be prepared in a pharmaceutically acceptable composition containing an equilibrium salt solution, preferably a Hanks equilibrium salt solution, or ordinary physiological saline.

[0058] III. Methods and compositions related to embodiments In certain aspects, this disclosure relates to antigen-specific CD8 + CD161 +The present invention includes a method for producing and / or amplifying T cells, which comprises transfecting T cells with an expression vector containing a DNA construct encoding an hCAR, and then optionally stimulating the cells with an antigen-positive cell, recombinant antigen, or antibody against a receptor to cause cell proliferation. As described in the examples, specific combinations of interleukins-IL-7, IL-15, and IL-21 are used in CD8 + CD161 + It provides a substantially improved increase in T cells.

[0059] In another embodiment, methods are provided for stably transfecting and redirecting T cells by electroporation or other non-viral gene transfer (such as sonoporation, but not limited to) using naked DNA. Most researchers have used viral vectors to deliver heterologous genes to T cells. By using naked DNA, the time required to produce redirected T cells can be reduced. "Naked DNA" means DNA encoding a chimeric T cell receptor (cTCR) contained in an expression cassette or vector in an appropriate orientation for expression. The electroporation method of this disclosure expresses a chimeric TCR (cTCR) and produces a stable transfectant to be supported on its surface.

[0060] A "chimeric TCR" refers to a receptor expressed by T cells that includes an intracellular signaling domain, a transmembrane domain, and an extracellular domain, the extracellular domain being able to specifically bind to antigens that are not normally bound by T cell receptors in a manner unrestricted by MHC. Stimulation of T cells with an antigen under appropriate conditions results in cell proliferation (increase) and / or IL-2 production. The exemplary chimeric receptor in this application is an example of a chimeric TCR. However, this method is applicable to transfection using chimeric TCRs that are specific to other target antigens, such as chimeric TCRs that are specific to HER2 / Neu (Stancovski et al., 1993), ERBB2 (Moritz et al., 1994), folate-binding protein (Hwu et al., 1995), renal cell carcinoma (Weitjens et al., 1996), and HIV-1 envelope glycoproteins gp120 and gp41 (Roberts et al., 1994). Other cell surface target antigens include, but are not limited to, CD20, carcinoembryonic antigen, mesothelin, ROR1, c-Met, CD56, GD2, GD3, α-fetoprotein, CD23, CD30, CD123, IL-11Rα, κ chain, λ chain, CD70, CA-125, MUC-1, EGFR and its variants, and epithelial tumor antigens.

[0061] In certain embodiments, the T cells are primary human T cells, e.g., T cells derived from human peripheral blood mononuclear cells (PBMCs), PBMCs collected after stimulation with G-CSF, bone marrow, or umbilical cord blood. Conditions include the use of mRNA and DNA, as well as the use of electroporation. After transfection, the cells may be injected immediately or stored. In certain embodiments, after transfection, the cells may be propagated ex vivo as a bulk population for several days, weeks, or months, for approximately 1, 2, 3, 4, or 5 days or longer after gene transfer to the cells. In further embodiments, after transfection, the transfectant is cloned to produce a clone showing the presence of a single integrated or episome-maintained expression cassette or plasmid, and the expression of the chimeric receptor is increased ex vivo. The clone selected for increase exhibits the ability to specifically recognize target cells. Recombinant T cells may be enlarged by stimulation with IL-2 or other cytokines that bind to a common gamma chain (e.g., IL-7, IL-12, IL-15, IL-21, and others). Recombinant T cells may be enlarged by stimulation with artificial antigen-presenting cells. Recombinant T cells may be enlarged on artificial antigen-presenting cells or with antibodies such as OKT3 that crosslink CD3 on the T cell surface. A subset of recombinant T cells may be deleted on artificial antigen-presenting cells or with antibodies such as Campath that bind to CD52 on the T cell surface. In a further embodiment, genetically modified cells may be cryopreserved.

[0062] Post-injection T cell proliferation (survival) can be evaluated by (i) q-PCR using CAR-specific primers, (ii) flow cytometry using CAR-specific antibodies, and / or (iii) soluble TAA.

[0063] In certain embodiments of this disclosure, CAR cells are delivered to an individual in need, for example, an individual with cancer or an infection. The cells then enhance the individual's immune system to attack the respective cancer or pathogenic cells. In some cases, the individual is provided with one or more doses of antigen-specific CAR T cells. If the individual is provided with two or more doses of antigen-specific CAR T cells, the time interval between doses should be sufficient to allow time for reproduction in the individual, and in certain embodiments, the time interval between doses is 1, 2, 3, 4, 5, 6, 7 days, or longer.

[0064] The source of allogeneic T cells, modified to contain both chimeric antigen receptors and lacking a functional TCR, may be of any type, but in certain embodiments, the cells are obtained, for example, from umbilical cord blood, peripheral blood, human embryonic stem cells, or a bank of induced pluripotent stem cells. A dose suitable for therapeutic effect is, for example, preferably, at least 10 per dose in a series of administration cycles. 5 1 or about 10 5 ~about 10 10 These would be individual cells. An exemplary dosing regimen involves at least approximately 10 on day 0. 5 Starting with a single cell, for example, within a few weeks of initiating an intra-patient dose escalation scheme, approximately 10 10 The treatment consists of four dose-escalation, one-week administration cycles, gradually increasing the dose to the target dose per cell. Preferred administration methods include intravenous, subcutaneous, intracavitary (e.g., via a reservoir access device), intraperitoneal, and direct injection into the tumor mass.

[0065] The pharmaceutical compositions of this disclosure can be used alone or in combination with other established agents useful for the treatment of cancer. Whether delivered alone or in combination with other agents, the pharmaceutical compositions of this disclosure can be delivered via various routes and to various sites within the mammalian body, particularly the human body, to achieve a particular effect. Those skilled in the art will recognize that multiple routes can be used for administration, but that certain routes may provide a more immediate and effective response than others. For example, intradermal delivery may be more advantageous than inhalation for the treatment of melanoma. Local or systemic delivery can be achieved by administration including application or dropping of the formulation into body cavities, inhalation or insufflation of aerosols, or parenteral delivery including intramuscular, intravenous, intraportal, intrahepatic, intraperitoneal, subcutaneous, or intradermal administration.

[0066] The compositions of this disclosure may be provided in unit dosage forms, and each dosage unit, for example, an injectable, contains a predetermined amount of the composition, either alone or in appropriate combination with other activators. As used herein, the term unit dosage form refers to a physically distinct unit suitable as a single dosage form for human and animal subjects, each unit containing a predetermined amount of the composition of this disclosure, calculated, where appropriate, in relation to a pharmaceutically acceptable diluent, carrier, or vehicle, in an amount sufficient to produce the desired effect, either alone or in combination with other activators. The specifications of the novel unit dosage forms of this disclosure depend on the specific pharmacodynamics relating to the pharmaceutical composition in a particular subject.

[0067] Preferably, an effective amount or sufficient number of isolated and transduced T cells are present in the composition and introduced into the subject, thereby establishing a long-term specific antitumor response that reduces tumor size or eliminates tumor growth or regrowth more effectively than would be achieved by other means in the absence of such treatment. Preferably, the amount of transduced T cells reintroduced into the subject results in a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 100% reduction in tumor size compared to other identical conditions in which transduced T cells are absent.

[0068] Therefore, the amount of transduced T cells administered should take into account the route of administration and ensure that a sufficient number of transduced T cells are introduced to achieve the desired therapeutic response. Furthermore, the amount of each activator contained in the compositions described herein (e.g., the amount per cell in contact or the amount per specific body weight) may vary for different applications. Generally, the concentration of transduced T cells is preferably at least about 1 × 10⁻⁶. 6 ~Approx. 1×10 9 More preferably, approximately 1 × 10⁶ transduced T cells 7 ~Approx. 5×10 8 The transduced T cells should be sufficient to provide the target being treated, but any preferred quantity exceeding the above, for example, 5 × 10⁶ 8 A quantity exceeding or falling below the above, for example, 1 × 10 7 It can be used in any number of doses less than 100. The administration schedule can be based on well-established cell-based therapies (see, for example, Topalian and Rosenberg, 1987, U.S. Patent No. 4,690,915), or an alternative sequential infusion strategy can be used.

[0069] These values ​​provide general guidance on the range of transduced T cells that practitioners may utilize when optimizing the methods of this disclosure for carrying out the disclosure. Such enumeration of ranges herein does not in any way preclude the use of higher or lower amounts of components, as can be ensured in specific applications. For example, actual doses and schedules may vary depending on whether the composition is administered in combination with other pharmaceutical compositions, or on individual differences in pharmacokinetics, drug dynamics, and metabolism. Those skilled in the art can easily make any necessary adjustments depending on the urgency of the particular situation.

[0070] IV. Exemplary Human Antigen Receptor T Cells As stated above, this disclosure is CD8 + CD161 +Relates to the culture and use of T cells.

[0071] CD8 (cluster of differentiation 8) is a transmembrane glycoprotein that functions as a coreceptor for the T cell receptor (TCR). Similar to the TCR, CD8 binds to major histocompatibility complex (MHC) molecules, but is specific for class I MHC proteins. There are two isoforms of this protein, α and β, which are encoded by different genes. In humans, both genes are located on chromosome 2 at position 2p12.

[0072] The CD8 coreceptor is mainly expressed on the surface of cytotoxic T cells, but can also be found on natural killer cells, cortical thymocytes, and dendritic cells. The CD8 molecule is a marker for the cytotoxic T cell population. It is expressed in T cell lymphoblastic lymphoma and mycosis fungoides.

[0073] To function, CD8 forms a dimer consisting of a pair of CD8 chains. The most common form of CD8 is composed of CD8-α and CD8-β chains, both of which are members of the immunoglobulin superfamily with an immunoglobulin variable (IgV)-like extracellular domain connected to the membrane by a thin stalk, and an intracellular tail. Non-covalent homodimers of the CD-8α chain are also expressed on some cells. The molecular weight of each CD8 chain is approximately 34 kDa. The structure of the CD8 molecule was determined by X-ray diffraction at a resolution of 2.6 Å by Leahy, D.J., Axel, R., and Hendrickson, W.A. The structure was determined to have an immunoglobulin-like β-sandwich fold and 114 amino acid residues. 2% of the protein is coiled in an α-helix, 46% is coiled in a β-sheet, and the remaining 52% of the molecule remains in the loop portion.

[0074] The extracellular IgV-like domain of CD8-α interacts with the α3 portion of class I MHC molecules. This affinity maintains a close binding between the T cell receptor of cytotoxic T cells and the target cell during antigen-specific activation. Cytotoxic T cells possessing the CD8 surface protein are called CD8+ cells. The primary recognition site is the flexible loop in the α3 domain of the MHC molecule. This was discovered through mutational analysis. The flexible α3 domain is located between residues 223 and 229 in the genome. In addition to assisting cytotoxic T cell antigen interaction, the CD8 coreceptor also plays a role in T cell signaling. The cytoplasmic tail of the CD8 coreceptor interacts with Lck (lymphocyte-specific protein tyrosine kinase). When the T cell receptor binds to its specific antigen Lck, the cytoplasmic CD3 and ζ chain of the TCR complex are phosphorylated, initiating a phosphorylation cascade that ultimately leads to the activation of transcription factors such as NFAT, NF-κB, and AP-1, which affect the expression of specific genes.

[0075] CD161, also known as KLRB1 or NKR-P1A, is classified as a type II membrane protein because it has an external C-terminus. CD161 recognizes lectin-like transcript-1 (LLT1) as a functional ligand. Natural killer (NK) cells are lymphocytes that mediate cytotoxicity and secrete cytokines after immune stimulation. Several genes in the C-type lectin superfamily, including the rodent NKRP1 family of glycoproteins, are expressed by NK cells and may be involved in regulating NK cell function. CD161 contains an extracellular domain, a transmembrane domain, and a cytoplasmic domain, each possessing several motifs characteristic of C-type lectins.

[0076] In one embodiment, the composition and method of this embodiment are used to express human CD8 (a chimeric antigen receptor (or CAR) polypeptide). + CD161 +The CAR may have arbitrary antigen-binding specificity, but it includes typical intracellular signaling domains, transmembrane domains, and extracellular domains present in CAR constructs. The extracellular domain will include a given binding region depending on the intended use for which the CAR-T is designed. The binding region is F(ab')2, Fab', Fab, Fv, or scFv. The binding region may include an amino acid sequence that is at least, at most, or about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the wild-type amino acid sequence. The intracellular domain may include the intracellular signaling domain of human CD3ζ and may further include a human CD28 intracellular segment. In certain embodiments, the transmembrane domain is the CD28 transmembrane domain.

[0077] In further embodiments, the composition may comprise a nucleic acid encoding the polypeptide described above. In certain embodiments, the nucleic acid sequence is optimized for human codon use.

[0078] In further embodiments, the composition may include cells expressing the polypeptide described herein. The T cells may include an expression cassette encoding the CAR polypeptide. The expression cassette may be contained in a non-viral vector such as a transposon, a human transposon, or a recombinant variant thereof. The expression cassette may be contained in a viral vector or a recombinant variant thereof. The expression cassette may be genomically integrated, episomal maintained, or expressed from mRNA.

[0079] In further embodiments, the disclosure includes a method for producing T cells expressing a human CAR, comprising introducing an expression cassette into cells, wherein the expression cassette encodes a polypeptide comprising an extracellular binding domain, a transmembrane domain, and one or more intracellular signaling domains. The method may further include stimulating cells with an antibody against a target antigen or receptor to cause the cells to proliferate, kill, and / or produce cytokines, for example, cells may be stimulated to proliferate or grow in target antigen-carrying artificial antigen-presenting cells.

[0080] In certain embodiments, the disclosure includes a method for treating a human disease condition, comprising injecting a patient with recombinant cells expressing a sufficient amount of human CAR to treat the human disease condition, wherein the human CAR comprises an extracellular target-binding domain, a transmembrane domain, and an intracellular signaling domain. The condition may be, for example, cancer, an autoimmune disease, or an infection.

[0081] The hCAR may be a chimeric receptor containing one or more activating endodomains, such as a CD3-ζ-derived activating domain. Additional T cell activation motifs include, but are not limited to, CD28, CD27, OX-40, DAP10, and 4-1BB. In certain embodiments, the activating domain may also include a CD28 transmembrane and / or activating domain. In further embodiments, an hCAR coding region and / or expression cassette codon optimized for expression in human cells and subjects, for example, in one embodiment, an scFv region obtained from the VH and VL sequences of a target-specific human antibody, is incorporated into the binding segment of the hCAR. In another embodiment, the hCAR expression cassette is maintained episomalally or incorporated into the genome of recombinant cells. In certain embodiments, the expression cassette is contained in a nucleic acid that can be incorporated by using an integrase mechanism, a viral vector such as a retroviral vector, or a non-viral vector such as a transposon mechanism. In further embodiments, the expression cassette is contained in a transposon-based nucleic acid. In certain embodiments, the expression cassette is part of a two-component Sleeping Beauty (SB) or piggyBac system that utilizes transposons and transposases for enhanced nonviral gene transfer.

[0082] Recombinant hCAR-expressing cells can be quantitatively increased to clinically significant numbers. One example of such increase is the use of artificial antigen-presenting cells (aAPCs). Recombinant hCAR-expressing cells can be verified and identified by flow cytometry and Western blot analysis. Recombinant hCARs expressing T cells can recognize and kill target cells by expressing the CAR. In a further embodiment, hCARs can be expressed in universal cells that can be injected across the transplantation barrier to help prevent immunogenicity. In cases of cytotoxicity, hCARs can be used with human genes for imaging (by positron emission tomography, PET, etc.) and conditional ablation of T cells. The recombinant cells of this disclosure may be used in certain cell therapies.

[0083] V. Exemplary membrane-bound IL-15 co-expressing chimeric antigen receptors or transgenic TCR T cells for targeting minimal residual disease Chemotherapy for lineage B acute lymphoblastic leukemia (B-ALL) in adults and children has disease relapse rates of 65% and 20%, respectively, due to drug-resistant residual lesions. The high incidence of B-ALL relapses, particularly in the poor-prognosis group, has led to calls for the use of immunotherapy with allogeneic hematopoietic stem cell transplantation (HSCT). This therapy relies on allogeneic reactive cells present in the donor graft to eradicate residual leukemia cells or minimal residual disease to improve disease-free survival. Donor lymphocyte infusion has been used to enhance the ability of engrafted T cells to target residual B-ALL after allogeneic HSCT; however, this therapeutic approach for such patients achieves remission rates of less than 10% and is associated with high morbidity and mortality due to the frequency and severity of graft-versus-host disease (GVHD). In these refractory malignancies, where recurrence is a common and often fatal problem, adoptive therapy using peripheral blood mononuclear cell (PBMC)-derived T cells after HSCT can enhance the antitumor effect or graft-versus-leukemia (GVL) effect by retargeting the specificity of donor T cells against tumor-associated antigens (TAAs).

[0084] Currently, CAR-modified T cells rely on obtaining CAR-mediated survival signaling that occurs only upon encounter with tumor antigens. In the clinical context in which these CAR-modified T cells are injected into patients with large lesions, sufficient tumor antigens are present to provide adequate activation and survival signaling via CAR. However, patients with relapsed B-ALL often undergo myelo-depleting chemotherapy, followed by HSCT, resulting in minimal residual disease (MRD). In this case, patients have a low tumor cell load, and minute levels of TAA severely limit the CAR-mediated signaling necessary to support the injected T cells, consequently impairing their therapeutic potential. Alternative CAR-independent means to improve T cell persistence are expected to improve the engraftment of CAR-modified T cells.

[0085] Cytokines within the common gamma-chain receptor family (γC) are important costimulatory molecules for T cells, which are crucial for lymphoid function, survival, and proliferation. IL-15 possesses several desirable attributes for adoptive therapy. IL-15 is a homeostatic cytokine that supports the survival of long-lived memory cytotoxic T cells, promotes tumor eradication by mitigating the functional suppression of tumor resident cells, and inhibits AICD.

[0086] IL-15 is tissue-limited and is observed only under pathological conditions, at any level in serum, or systemically. Unlike other γC cytokines secreted into the surrounding environment, IL-15 is trans-presented to T cells in association with the IL-15 receptor alpha (IL-15Rα) by producing cells. The unique delivery mechanism of this cytokine to T cells and other responding cells is: (i) highly targeted and localized, (ii) increases the stability and half-life of IL-15, and (iii) results in a qualitatively different signaling pathway than that achieved by soluble IL-15.

[0087] In one embodiment, the disclosure provides a method for generating chimeric antigen receptor (CAR) modified T cells or transgenic TCR T cells with extended lifespan in vivo potential, for example, for the treatment of leukemia patients exhibiting minimal residual disease (MRD). Overall, the method describes a method for increasing therapeutic potential by fusing soluble molecules such as cytokines to the cell surface. At the core of the method is the co-modification of CAR T cells or transgenic TCR T cells with a human cytokine mutant protein (mutein) of interleukin-15 (IL-15) (hereinafter referred to as mIL15). The mIL15 fusion protein consists of a codon-optimized cDNA sequence of IL-15 fused to the full-length IL15 receptor α via a flexible serine-glycine linker. This IL-15 mutant protein is designed as follows: (i) mIL15 expression is CAR + or transgenic TCR +Restricted to the surface of T cells, it restricts the diffusion of cytokines into the non-target in vivo environment, thereby potentially improving its safety profile if exogenous soluble cytokine administration results in toxicity, and (ii) in the context of IL-15Rα, it presents IL-15 to mimic physiologically appropriate and qualitative signaling and perform the stabilization and recycling of the IL-15 / IL-15Ra complex over a longer cytokine half-life. T cells expressing mIL15 are capable of continuous supportive cytokine signaling, which is important for survival after injection. mIL15 generated by non-viral Sleeping Beauty system gene modification on a clinically applicable platform and subsequent ex vivo expansion + CAR + T cell or mIL15 + Transgenic TCR + T cells obtained a T cell injection product with improved persistence after injection in mouse models with high tumor cell amounts, low tumor cell amounts, or no tumor cell amounts. Furthermore, mIL15 + CAR + T cells also showed improved anti-tumor efficacy in both models with high or low tumor cell amounts.

[0088] CAR in the model with high tumor cell amounts + mIL15 for T cells + CAR + The improved persistence and anti-tumor activity of T cells is such that mIL15 + CAR + T cells are, CAR + T cells may be more effective than shown. Thus, mIL15 + CAR + T cells can, in the most extensive applications, replace CAR + T cells in adoptive therapy. mIL15 + CAR +The ability of T cells to survive independently of survival signaling via CAR enables these modified T cells to persist after injection despite the lack of tumor antigen. Thus, this is expected to have the greatest impact on treatment efficacy in the context of MRD treatment, particularly in patients who have received myeloablative chemotherapy and hematopoietic stem cell transplantation. These patients receive adoptive T cell transfer together with mIL15 + CAR + to treat MRD and prevent relapse.

[0089] Membrane-bound cytokines such as mIL15 have a wide-ranging impact. In addition to membrane-bound IL-15, other membrane-bound cytokines are envisioned. Membrane-bound cytokines can also extend to the cell surface expression of other molecules associated with the activation and proliferation of cells used for human applications. These include, but are not limited to, cytokines, chemokines, and other molecules that contribute to the activation and growth of cells used for human applications.

[0090] Membrane-bound cytokines such as mIL15 can be used ex vivo to prepare cells for human applications and can be used on injected cells (e.g., T cells) used for human applications. For example, membrane-bound IL-15 can be expressed on artificial antigen-presenting cells (aAPCs) such as those derived from K562 to stimulate T cells and NK cells (and other cells) for activation and / or proliferation. The population of T cells activated / proliferated by mIL15 on aAPCs includes genetically modified lymphocytes but also tumor infiltrating lymphocytes and other immune cells. These aAPCs are not injected. In contrast, mIL15 (and other membrane-bound molecules) can be expressed on injected T cells and other cells.

[0091] The treatment efficacy of MRD treatment with CAR-modified T cells is hampered by the lack of persistence after adoptive transfer of T cells. mIL15 + CAR + T cells or mIL15 + transgenic TCR +The ability of T cells to survive in vivo for extended periods, independently of tumor antigens, presents great potential for treating patients with MRD. In this case, since current approaches to MRD patients are inadequate, mIL15 and the persistent T cells that support it address the need. The persistence of T cells and other lymphocytes injected into patients with MRD is a key factor in CAR + This applies beyond T cells. Any immune cells used to treat and prevent malignant tumors, infections, or autoimmune diseases must be able to persist over long periods in order to achieve a sustained therapeutic effect. Therefore, activating T cells for persistence beyond signals derived from endogenous T cell receptors or introduced immune receptors is important in many aspects of adoptive immunotherapy. Thus, the expression of membrane-bound cytokines can be used to enhance the therapeutic potential and persistence of injected T cells and other immune cells for various pathological conditions.

[0092] The inventors of this invention have found that CAR + T cells or transgenic TCRs + We generated an IL-15 mutant protein expressed on T cells as a membrane-bound fusion protein of IL-15 and IL-15Rα (mIL15). The mIL15 construct was co-electro-transferred (on day 0) into primary human T cells as two Sleeping Beauty DNA transposon plasmids, along with a CD19-specific CAR. + Co-culturing artificial antigen-presenting cells on supplemented IL-21 revealed a clinically relevant number of mIL-15 + CAR + T cells were generated. Signaling through the IL-15 receptor complex in genetically modified T cells was verified by phosphorylation of STAT5 (pSTAT5), and these T cells were CAR + CD19 equivalent to T cells +The tumor target showed redirected, specific lysis. Furthermore, after antigen depletion, signaling generated by mIL15 increased the prevalence of T cells with a less differentiated / younger phenotype, possessing memory-related attributes including specific cell surface markers, transcription factors, and the ability to secrete IL-2. These features are desirable traits in T cells used for adoptive transfer, and they correlate with T cell subsets that have demonstrated the ability to persist over long periods in vivo. (Disseminated CD19) + In immunodeficient NSG mice with leukemia, mIL15 + CAR + T cells showed both sustained and antitumor effects, but CAR + The T cell counterpart failed to maintain significant persistence despite the presence of TAA. mIL15 + / - CAR + T cells initially engraft for 6 days, followed by disseminated CD19 + In the leukemia-induced prophylactic mouse (NSG) model, mIL15 + CAR + It was found that only T cells persisted and tumor engraftment was prevented. mIL15 + CAR + To test whether T cells were persistent regardless of stimulation from TAA, mRNA was used. + / - CAR + T cells were adopted into tumor-free NSG mice. mIL15 + CAR + Only T cells were able to persist in this in vivo environment without the support of exogenous cytokines or the presence of CD19 TAAs. These data suggest that mIL15 is CAR + T cells or transgenic TCRs +Demonstrate that it can be co-expressed on T cells and improve in vivo persistence without the support of TAAs or exogenous cytokines. Briefly, this cytokine fusion molecule provides a stimulatory signal via pSTAT5 that enhances in vivo T cell persistence while maintaining tumor-specific functionality, (ii) maintains a subset of T cells that promote a memory-like phenotype, (iii) eliminates the need and cost of clinical-grade IL-2 for in vitro and in vivo T cell expansion and persistence, and (iv) reduces the need for clinical-grade soluble IL-15.

[0093] VI. Pancreatic adenocarcinoma Pancreatic cancer occurs when cells in the pancreas, an organ behind the stomach that produces digestive enzymes, begin to grow out of control and form tumors. These cancer cells have the ability to invade other parts of the body. There are many types of pancreatic cancer. The most common type of pancreatic adenocarcinoma accounts for about 85% of cases, and the term "pancreatic cancer" may be used to refer only to this type. These adenocarcinomas begin in the part of the pancreas that makes digestive enzymes. Several other types of cancer, which collectively account for most non-adenocarcinomas, can also arise from these cells. 1-2 percent of cases of pancreatic cancer are neuroendocrine tumors, which arise from hormone-producing cells in the pancreas. These are generally less invasive than pancreatic adenocarcinoma.

[0094] The most common signs and symptoms of pancreatic cancer can include yellowing of the skin, abdominal or back pain, unexplained weight loss, pale stools, dark urine, and loss of appetite. Usually, there are no symptoms in the early stages of the disease, and symptoms specific enough to suggest pancreatic cancer do not develop until the disease has reached an advanced stage. By the time of diagnosis, pancreatic cancer has often spread to other parts of the body.

[0095] Pancreatic cancer rarely develops before the age of 40, with more than half of pancreatic adenocarcinoma cases occurring after the age of 70. Risk factors for pancreatic cancer include smoking, obesity, diabetes, and certain rare genetic conditions. Approximately 25% of cases are associated with smoking, and 5-10% are associated with inherited genes. Pancreatic cancer is usually diagnosed through a combination of medical imaging techniques such as ultrasound or computed tomography, blood tests, and tissue biopsy. The disease is divided into stages from early (stage I) to late (stage IV). Screening of the general population has not been proven effective.

[0096] The risk of developing pancreatic cancer is low among non-smokers and those who maintain a healthy weight and limit their intake of red or processed meats. If smokers quit smoking, their chances of developing the disease decrease and, after 20 years, are almost the same as the chances of other people. Pancreatic cancer can be treated with surgery, radiation therapy, chemotherapy, palliative care, or a combination of these. Treatment options are based in part on the stage of the cancer. Surgery is the only treatment that can cure pancreatic adenocarcinoma and may also be performed to improve quality of life even without the possibility of a cure. Medications to manage pain and improve digestion may be necessary. Even for those undergoing treatment aimed at a cure, early palliative care is recommended.

[0097] In 2015, all types of pancreatic cancer caused 411,600 deaths worldwide. Pancreatic cancer is the fifth leading cause of cancer death in the UK and the third leading cause in the US. The disease is very common in developed countries, where it accounted for approximately 70% of new cases in 2012. Pancreatic adenocarcinoma typically has a very poor prognosis, with only 25% surviving one year and 5% surviving five years after diagnosis. For cancers diagnosed early, the five-year survival rate can rise to around 20%. Neuroendocrine cancers have a better outcome, with 65% of those diagnosed surviving five years after diagnosis, although survival rates vary significantly depending on the type of tumor.

[0098] VII. The Immune System and Immunotherapy In some embodiments, medical disorders are treated by the transfer of redirected T cells that induce a specific immune response. In one embodiment of this disclosure, a B-cell lineage malignancy or disorder is treated by the transfer of redirected T cells that induce a specific immune response. Therefore, a basic understanding of the immune response is necessary.

[0099] The cells of the adaptive immune system are a type of white blood cell called lymphocytes. B cells and T cells are the main types of lymphocytes. B cells and T cells originate from the same pluripotent hematopoietic stem cells and are indistinguishable from each other until activated. B cells play a major role in humoral immune responses, while T cells are closely involved in cell-mediated immune responses. They can be distinguished from other lymphocyte types, such as B cells and NK cells, by the presence of special receptors on their cell surface called T cell receptors (TCRs). In almost all other vertebrates, B cells and T cells are produced by stem cells in the bone marrow. T cells migrate to the thymus and develop there, from which their name is derived. In humans, about 1% to 2% of the lymphocyte pool is constantly recirculated to optimize the chances of antigen-specific lymphocytes finding their specific antigens in secondary lymphoid tissues.

[0100] T lymphocytes originate from hematopoietic stem cells in the bone marrow and typically migrate to the thymus to mature. T cells express their own antigen-binding receptors (T cell receptors) on their membranes, which can recognize antigens only when associated with major histocompatibility complex (MHC) molecules on the surface of other cells. There are at least two populations of T cells, known as T helper cells and T cytotoxic cells. T helper cells and T cytotoxic cells are primarily distinguished by the display of membrane-bound glycoproteins CD4 and CD8, respectively. T helper cells harbor a variety of lymphokines essential for the activation of B cells, T cytotoxic cells, macrophages, and other cells of the immune system. In contrast, T cytotoxic cells that recognize antigen-MHC complexes proliferate and differentiate into effector cells called cytotoxic T lymphocytes (CTLs). CTLs eliminate antigen-showing cells of the body, such as virus-infected cells and tumor cells, by producing substances that lead to cytolysis. Natural killer cells (or NK cells) are a type of cytotoxic lymphocyte that constitutes a major component of the innate immune system. NK cells play a major role in rejecting tumor and virus-infected cells. The cells kill target cells by apoptosis by releasing small cytoplasmic granules of proteins called perforin and granzymes.

[0101] Antigen-presenting cells, including macrophages, B lymphocytes, and dendritic cells, are distinguished by the expression of specific MHC molecules. APCs internalize antigens and re-express a portion of those antigens along with MHC molecules on their outer cell membranes. The major histocompatibility complex (MHC) is a large gene complex with multiple loci. MHC loci encode two major classes of MHC membrane molecules called class I and class II MHC. Helper T lymphocytes generally recognize antigens associated with MHC class II molecules, while T-cytotoxic lymphocytes recognize antigens associated with MHC class I molecules. In humans, MHC is called the HLA complex, and in mice, it is called the H-2 complex.

[0102] The T cell receptor, or TCR, is a molecule found on the surface of T lymphocytes (or T cells) and is generally responsible for recognizing antigens bound to the major histocompatibility complex (MHC) molecule. While 95% of T cells have a heterodimer TCR consisting of α and β chains, 5% of T cells have a TCR consisting of gamma and delta chains. Engagement of the TCR with the antigen and MHC leads to the activation of the T lymphocyte through a series of biochemical events mediated by relevant enzymes, co-receptors, and specialized accessory molecules. In immunology, the CD3 antigen (CD stands for differential antigens) is a protein complex in mammals consisting of four distinct chains (CD3γ, CD3δ, and 2xCD3ε) that associates with molecules known as the T cell receptor (TCR) and ζ chain to generate an activation signal in T lymphocytes. The TCR, ζ chain, and CD3 molecule all contain the TCR complex. CD3γ, CD3δ, and CD3ε chains are highly related cell surface proteins of the immunoglobulin superfamily, each containing a single extracellular immunoglobulin domain. The transmembrane region of the CD3 chain is negatively charged, a feature that allows these chains to associate with positively charged TCR chains (TCRα and TCRβ). The intracellular tail of the CD3 molecule contains a single conserved motif known as the immunoreceptor tyrosine system activating motif, or ITAM for short, which is essential for TCR signaling.

[0103] CD28 is one of the molecules expressed on T cells that provides the co-stimulatory signals required for T cell activation. CD28 is a receptor for B7.1 (CD80) and B7.2 (CD86). When activated by Toll-like receptor ligands, B7.1 expression is upregulated in antigen-presenting cells (APCs). B7.2 expression on antigen-presenting cells is constitutive. CD28 is the only B7 receptor that is constitutively expressed on naive T cells. Stimulation via CD28, in addition to the TCR, can provide potent co-stimulatory signals to T cells for the production of various interleukins (particularly IL-2 and IL-6).

[0104] Strategies for isolating and amplifying antigen-specific T cells as therapeutic interventions for human diseases have been validated in clinical trials (Riddell et al., 1992; Walter et al., 1995; Heslop et al., 1996).

[0105] Autoimmune diseases, or autoimmunity, are conditions in which an organism fails to recognize its own components (down to the submolecular level) as "self," resulting in an immune response against its own cells and tissues. Any disease resulting from such an abnormal immune response is called an autoimmune disease. Notable examples include celiac disease, type 1 diabetes mellitus (IDDM), systemic lupus erythematosus (SLE), Sjögren's syndrome, multiple sclerosis (MS), Hashimoto's thyroiditis, Graves' disease, idiopathic thrombocytopenic purpura, and rheumatoid arthritis (RA).

[0106] Inflammatory diseases, including autoimmune diseases, are also a class of diseases associated with B-cell damage. Examples of autoimmune diseases include acute idiopathic thrombocytopenic purpura, chronic idiopathic thrombocytopenic purpura, dermatomyositis, Sydenham's chorea, myasthenia gravis, systemic lupus erythematosus, lupus nephritis, rheumatic fever, polyglandular syndrome, bullous pemphigus, diabetes mellitus, Henoch-Schönlein purpura, post-streptococcal nephritis, erythema nodosum, Takayasu's arteritis, Addison's disease, rheumatoid arthritis, multiple sclerosis, sarcoidosis, ulcerative colitis, erythema multiforme, IgA nephritis, polyarteritis nodosa, ankylosing spondylitis, Goodpasture syndrome, thromboangiitis ubiterans, Sjögren's syndrome, primary biliary cirrhosis, Hashimoto's thyroiditis, hyperthyroidism, scleroderma, chronic active hepatitis, polymyositis / dermatomyositis, polychondritis, pemphigus vulgaris, Wegener's granulomatosis, membranous nephropathy, amyotrophic lateral sclerosis, tabes dorsalis, giant cell arteritis / polymyalgia, pernicious anemia, rapidly progressive glomerulonephritis, psoriasis, and fibrotic alveolitis are among the most common treatments, but are not limited to these. The most common treatments are corticosteroids and cytotoxic drugs, which can be highly toxic. These drugs also suppress the entire immune system, can lead to serious infections, and adversely affect the bone marrow, liver, and kidneys. Other therapeutic agents used to treat class III autoimmune diseases have been directed at T cells and macrophages. More effective methods are needed to treat autoimmune diseases, especially class III autoimmune diseases.

[0107] VIII.Artificial antigen presenting cells In some embodiments, aAPC is useful in the preparation of therapeutic compositions and cell therapy products of the embodiments. For general guidance on the preparation and use of antigen-presenting systems, see, for example, U.S. Patents 6,225,042, 6,355,479, 6,362,001 and 6,790,662, U.S. Patent Application Publications 2009 / 0017000 and 2009 / 0004142, and International Publication 2007 / 103009.

[0108] aAPCs are typically incubated with a peptide of optimal length that allows for direct binding of the peptide to an MHC molecule without additional processing. Alternatively, cells can express the antigen of interest (i.e., in the case of MHC-independent antigen recognition). In addition to the peptide-MHC molecule or antigen of interest, the aAPC system may also include at least one exogenous co-molecule. Any preferred number and combination of co-molecules may be used. Co-molecules may be selected from co-stimulatory molecules and adhesion molecules. Exemplary co-stimulatory molecules include CD70 and B7.1 (B7.1 was previously known as B7 and also known as CD80), which, among other things, bind to CD28 and / or CTLA-4 molecules on the surface of T cells, thereby influencing, for example, T cell growth, Th1 differentiation, short-term T cell survival, and cytokine secretion such as interleukin (IL)-2 (see Kim et al., 2004). Examples of adhesion molecules include carbohydrate-binding glycoproteins such as selectins, transmembrane glycoproteins such as integrins, calcium-dependent proteins such as cadherins, and single-pass transmembrane immunoglobulin (Ig) superfamily proteins such as intercellular adhesion molecules (ICAMs), which promote intercellular or cell-pair matrix contact. Exemplary adhesion molecules include ICAMs such as LFA-3 and ICAM-1. Techniques, methods, and reagents useful for the selection, cloning, preparation, and expression of exemplary accessory molecules, including co-stimulatory and adhesion molecules, are exemplified, for example, in U.S. Patents 6,225,042, 6,355,479, and 6,362,001.

[0109] Cells selected to become aAPCs preferably have defects in intracellular antigen processing, intracellular peptide transport, and / or intracellular MHC class I or class II molecular peptide loading, or are poikilothermic (i.e., less sensitive to temperature challenges than mammalian cell lines), or possess both defects and poikilothermic properties. Preferably, cells selected to become aAPCs also express at least one endogenous counterpart to an exogenous MHC class I or class II molecule (e.g., the endogenous MHC class I or class II molecule and / or endogenous auxiliary molecule described above) and lack the ability to assist auxiliary molecular components introduced into the cell. Furthermore, aAPCs preferably retain the defects and poikilothermic properties that the cell possessed before their modification to generate aAPCs. Exemplary aAPCs constitute or are derived from transporters associated with antigen processing (TAP) deficient cell lines, such as insect cell lines. Exemplary poikilothermic insect cell lines include Drosophila cell lines such as the Schneider II cell line (see, for example, Schneider, 1972). Exemplary methods for the preparation, proliferation, and culture of Schneider II cells are provided in U.S. Patents 6,225,042, 6,355,479, and 6,362,001.

[0110] In one embodiment, aAPC is also subjected to a freeze-thaw cycle. In an exemplary freeze-thaw cycle, aAPC may be frozen by bringing a suitable container containing aAPC into contact with an appropriate amount of liquid nitrogen, solid carbon dioxide (i.e., dry ice), or a similar cryogenic material so that freezing occurs rapidly. The frozen aAPC is then thawed by either removing the aAPC from the cryogenic material and exposing it to ambient room temperature conditions, or by an easy thawing process that facilitates a short thawing time using a lukewarm water bath or warm hands. Furthermore, aAPC may be frozen before thawing and stored for a long period of time. The frozen aAPC may be thawed before further use and then freeze-dried. Preferably, preservatives that may have adverse effects on the freeze-thaw procedure, such as dimethyl sulfoxide (DMSO), polyethylene glycol (PEG), and other preservatives, are not present in the medium containing aAPC undergoing the freeze-thaw cycle, or are essentially removed, for example, by transferring the aAPC to a medium that is essentially free of such preservatives.

[0111] In other preferred embodiments, heterologous nucleic acids and nucleic acids endogenous to aAPCs may be inactivated by crosslinking, resulting in the absence of essential cellular proliferation, replication, or expression of nucleic acids after inactivation. In one embodiment, aAPCs are inactivated at a point after the expression of exogenous MHC and auxiliary molecules, the presentation of such molecules on the surface of the aAPC, and the loading of the presented MHC molecules with selected peptides. Thus, such inactivated and selected peptide-loaded aAPCs retain their selected peptide-presenting function while essentially becoming unable to proliferate or replicate. Preferably, crosslinking also results in aAPCs that are essentially free of contaminating microorganisms such as bacteria and viruses without substantially reducing the antigen-presenting cellular function of the aAPCs. Therefore, crosslinking helps to maintain the important APC function of aAPCs while simultaneously mitigating safety concerns for cell therapy products developed using aAPCs. For methods relating to crosslinking and aAPCs, see, for example, U.S. Patent Application Publication 2009 / 0017000, incorporated herein by reference.

[0112] IX. Kits of this Disclosure Any of the compositions described herein may be included in the kit. In some embodiments, allogeneic CAR T cells are provided in the kit, which may also include a culture medium, aAPC, growth factors, antibodies (e.g., for sorting or characterizing CAR T cells), and / or reagents suitable for cell growth, such as plasmids encoding CAR or transposases.

[0113] In non-limiting examples, a chimeric receptor expression construct, one or more reagents for generating the chimeric receptor expression construct, cells for transfection of the expression construct, and / or one or more instruments for obtaining allogeneic cells for transfection of the expression construct (such instruments may be syringes, pipettes, forceps, and / or any such medically approved device).

[0114] In some embodiments, an expression construct for eliminating endogenous TCR α / β expression, one or more reagents for generating the construct, and / or CAR + T cells are provided in the kit. In some embodiments, an expression construct encoding a zinc finger nuclease is included.

[0115] In some embodiments, the kit includes reagents or apparatus for the electroporation of cells.

[0116] A kit may comprise one or more suitably divided compositions or reagents of the Disclosure for the purpose of producing the compositions of the Disclosure. The components of the kit may be packaged in either an aqueous medium or a lyophilized form. The container means of the kit may comprise at least one vial, test tube, flask, bottle, syringe, or other container means in which the components may be arranged, preferably suitably divided. If there are multiple components in the kit, the kit generally also comprises a second, third, or other additional container in which additional components may be arranged separately. However, various combinations of components may be contained in vials. The kits of the Disclosure also typically comprise means for sealing and housing the chimeric receptor constructs and any other reagent containers for commercial sale. Such containers may comprise, for example, injection or blow-molded plastic containers in which the desired vials are held. [Examples]

[0117] The following embodiments are included to demonstrate preferred embodiments of the Disclosure. Those skilled in the art will understand that the techniques disclosed in the following embodiments represent techniques discovered by the inventors to function well in the implementation of the Disclosure and can therefore be considered to constitute a preferred mode for its implementation. However, those skilled in the art will understand that many modifications can be made in light of the Disclosure to obtain similar or similar results without departing from the spirit and scope of the Disclosure.

[0118] Example 1 - Materials and Method Mouse microarray analysis. CD8 + NK1.1 + Cells and CD8 + NK1.1 neg Cells were isolated from mice previously receiving pancreatic tumors and therapeutically treated with a cell-based vaccine in conjunction with gemcitabine chemotherapy (Konduri et al., 2016). The isolated cells were activated with PDAC antigen-loaded autoDCs, and total RNA was isolated using the RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions. CD8 + NK1.1 + and CD8 + NK1.1 neg Cellular gene expression profiling was performed using the Affymetrix mouse transcriptome array 1.0 chip (Affymetrix, Santa Clara, CA, USA) at the sequencing and microarray facility of the University of Texas MD Anderson Cancer Center (Houston, TX).

[0119] Influenza model. To generate T cells for adoptive transfer, C57 / BL6 mice were challenged with influenza A / HongKong / 8 / 68(H3N2), a Swiss mouse lung-adapted strain of H3N2 influenza A virus, courtesy of Dr. Brian Gilbert, as described (Liang et al., 2017). Infection was carried out by 20 minutes of sprayed aerosol exposure of influenza virus diluted in MEM medium + 0.05% gelatin using an Aerotech II nebulizer supplied with 10 L / min of room air produced from an Aridyne 2000 air compressor. All mice infected in any given experiment were infected simultaneously in a single exposure chamber. Two weeks after infection, the mice were sacrificed, their spleens were harvested, and CD8 + Cells were negatively selected (Miltenyi Biotec). Isolated CD8 +The cells are further processed by NK1.1 + and NK1.1 neg The group was magnetically separated (Miltenyi Biotec). Subsequently, 500,000 CD8s were processed. + NK1.1 + and CD8 + NK1.1 neg Cells were transferred to naive mice that had been challenged with the influenza virus.

[0120] Melanoma model. To generate T cells for adoptive transfer, C57 / BL6 mice were subcutaneously inoculated with 250,000 B16F10 melanoma tumor cells (American Type Culture Collection, Manassas, VA) suspended in 100 μl of PBS. As described, DCs were loaded with melanoma tumor antigen (Konduri et al., 2016). One week after tumor inoculation, 200,000 antigen-loaded DCs suspended in 50 μl of PBS were injected into the soles of the feet with a booster vaccine administered 7 days later. Ten days after booster, the vaccinated mice were sacrificed and CD8 + Splenocytes were isolated by negative selection (Miltenyi Biotec). Isolated CD8 + The cells are further processed by NK1.1 + and NK1.1 neg The population was magnetically sorted (Miltenyi Biotec). Three groups of eight naive mice were subcutaneously injected with 250,000 B16F10 tumor cells. Tumor size was recorded and the animals were randomized so that each group had similar mean tumor size and standard error. Seven days after tumor inoculation, the treated mice were transferred to CD8 by intraperitoneal adoptive transfer. + NK1.1 + Cells or CD8 + NK1.1 - Each mouse received 1.5 million cells. Naive mice served as untreated controls. Tumor size was determined by external caliper measurement using the formula (length × width). 2The calculation was performed using the formula ) × π / 6. If the tumor cell count in the control group exceeded the acceptable limit set by the Center for Comparative Medicine (CCM), the mice were euthanized 22 days after tumor inoculation.

[0121] Analysis of mouse PBMCs. PBMCs were collected from adoptive mice by blood collection from the posterior orbital venous plexus two weeks after influenza infection or three weeks after tumor transplantation. Red blood cells were lysed by treatment with ammonium chloride (Sigma-Aldrich) according to the manufacturer's instructions. The leukocyte pellet was washed once with PBS and resuspended in AIM-V medium containing 10% mouse serum. Cells were stained with anti-CD3, CD4, CD8, CD25, and IFN-γ for flow cytometry analysis. All flow cytometry analyses were performed using an LSR II flow cytometer (BD Biosciences) and analyzed for OS-X using FlowJo version 10.0.00003 (Tree Star Inc., Ashland, OR).

[0122] Human microarray analysis. CD8 + CD161 + and CD8 + CD161 neg Cells were magnetically isolated from peripheral blood derived from three healthy donors and three PDAC patients. The isolated cells were not activated. Total RNA from the cells was isolated using the RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions. CD8 + CD161 + and CD8 + CD161 negCellular gene expression profiling was performed using the Affymetrix Human Transcriptome Array 1.0 chip (Affymetrix, Santa Clara, CA, USA) at the sequencing and microarray facility of the University of Texas MD Anderson Cancer Center (Houston, TX). Detailed descriptions of sample requirements and pre-analysis of data are available on the facility's website (world-wide-web at mdanderson.org / research / research-resources / core-facilities / sequencing-and-microarray-facility-smf / services-and-fees / microarray-services-overview.html). The data were analyzed and visualized using Transcriptome Analysis Console v3.0 (Affymetrix).

[0123] TCR Vβ Spectratyping. CD8 isolated from peripheral blood of a normal donor. + CD161 + Cells were spectrally typed using the Mayo Clinic method. The resulting images consist of clusters of fluorescence peaks with single-nucleotide separations and varying fluorescence intensities, roughly corresponding to the number of fragments of their size represented in the donor's original RNA. Peak patterns were reviewed for tissue (number of peaks), relative intensity between peaks, and size distribution.

[0124] Cytotoxic assay. CD8 + CD161 + CD8 + CD161 neg To evaluate the cytotoxicity of cells and bulk PBMCs, a chromium-based short-term cytotoxicity assay was performed in vitro. CD8 + CD161 + CD8 + CD161 neg, and unprocessed bulk PBMCs were newly isolated from human peripheral blood products. The isolated cells were... 51 In a 4-hour killing assay using a Cr-labeled allogeneic 293-HEK target, cytotoxicity was immediately tested at T cell:target cell ratios of 5:1, 25:1, and 50:1. Cell lysis was determined by the amount of chromium released into the culture medium, read using a Wizard2 gamma counter (Perkin Elmer).

[0125] CD8 + CD161 + Ex vivo culture conditions for cells. CD8 isolated from apheresis products of normal donors. + CD161 + cells, CD8 + CD161 neg Cells and bulk PBMCs were stimulated with plate-bound anti-CD3 / CD28 and enlarged in a cytokine cocktail consisting of 10 ng / ml IL-7, 5 ng / ml IL-15, and 30 ng / ml IL-21 (all from Peprotech, Rocky Hill, NJ). CD8 + CD161 + Cells were isolated from healthy donor apheresis products and cultured in 1 ug / mL of anti-CD3 / CD28 / Clec2d (anti-human CD3-eBioscience catalog no. 16-0037-8, anti-human CD28 from BD Biosciences catalog no. 555725, recombinant human Clec2d, Novus Biologicals catalog no. NBP2-22966), and augmented in RPMI-1640, 10% FBS, and 2 mmol / L GlutaMAX (Invitrogen) in a cytokine cocktail consisting of 10 ng / mL IL-7, 5 ng / mL IL-15, and 30 ng / mL IL-21 (all from Peprotech, Rocky Hill, NJ). Cells were placed in a humidified chamber at 37°C for 48 hours. After 48 hours, cells were augmented with the IL7 / 15 / 21 cytokine cocktail without antibody stimulation.

[0126] Statistical Analysis. Unless otherwise specified, the significance of differences was determined by two-way or one-way analysis of variance (ANOVA) using Bonferroni's post-hoc test for multiple comparisons. Kaplan-Meier survival significance was determined by the log-rank (Mantel-Cox) test. All data are presented as mean ± SEM unless otherwise specified, and all analyses were performed using Prism software (GraphPad software). Statistical significance was defined as p ≤ 0.05.

[0127] Example 2 - Results T cell expression profiling after chemoimmunotherapy for PDAC is CD3 + CD8 + NK1.1 + Identify the innate-like cytotoxic properties of cells. Previous studies have shown that spleen CD8 isolated 9 months after chemoimmunotherapy for the treatment of orthotopic PDACs. + NK1.1 + We demonstrated that a very small number of cells (less than 1,500 per mouse) can still provide rapid and robust antitumor protection against the parental PDAC cell line in a model of metastatic disease (Konduri et al., 2016). The inventors of this NK1.1 + CD3 + CD8 + To gain insights into the key functional characteristics of T cells, Kras G12D / p53 - / - A cohort of mice with PDAC tumors were orthotopically transplanted and subsequently cured using a previously published (Konduri et al., 2016) immunotherapy protocol. Two months after treatment and cure, CD8 + Spleen cells were negatively selected, and NK1.1 + and NK1.1 neg The cells were subdivided into fractions. These fractions were then co-cultured separately overnight with mature DCs loaded with PDAC to identify PDAC antigen-specific cells, which were then isolated by upregulated CD69 expression. Microarrays showed that upon antigen stimulation, 1642 genes were identified as CD8 + NK1.1 +and CD8 + NK1.1 neg The results showed differential regulation between cells and the receptors at a univariate significance level of 0.1 (Figure 1). While many different pathways may have been affected (Table 1), the most significant differences were observed in cytolytic granzyme serine proteases, particularly non-classical granzyme isoforms F, D, G, and C, as well as innate-like cytotoxic receptors (Table 2). These results were found in CD8 + NK1.1 + The cells possess CD8, which has significantly enhanced cytolytic ability. + This suggested that it represents a population of T cells.

[0128] (Table 1) Upregulated and downregulated genes at the top level. TIFF0007856563000001.tif204160TIFF0007856563000002.tif235160TIFF0007856563000003.tif41160

[0129] (Table 2) Changes in magnification and P-values ​​for genes grouped into the granzyme pathway and killer cell receptor subfamily pathways. TIFF0007856563000004.tif180160TIFF0007856563000005.tif238160TIFF0007856563000006.tif16160

[0130] NK1.1 identifies important circulating memory T cell populations in multiple mouse models of disease. To verify that NK1.1 can identify similar important populations of cytolytic memory cells in a model-independent manner, we performed adoptive transfer experiments in a second tumor model and an infectious disease model. First, a donor cohort of 6-8 week old mice was inoculated with a sublethal dose of H2N3 mouse-adapted influenza virus. Three weeks after inoculation and recovery from weight loss, splenocytes were collected and negative selection was performed to identify CD8 + Non-adherent cells were isolated. NK1.1 was selected by positive selection. + and NK1.1 negAfter separation into fractions, 5 × 10⁻¹⁰ of each NK1.1 group 5 Individual cells / mice were adopted into a naive cohort that was lethally challenged with the same influenza virus strain 24 hours after adoption (Figure 7A). Body weight was recorded as an indicator of recovery, and survival rates were determined by Kaplan-Meier analysis. Donor CD8 + NK1.1 + The cohort adopted via cell transfer regained full body weight and survived the infection, but CD8 + NK1.1 neg The cohorts that were adopted and transplanted via cells all experienced weight loss and naive CD8 + They died at the same rate as the controls adopted via splenocytes (Figure 2A-B). Analysis of PBMCs 7 days after infection showed naive and CD8 + NK1.1 neg Compared to the adopted cohort, CD8 + NK1.1 + Circulating CD3 between mice that received cells + CD8 + IFN-γ + A 40% increase was observed in cells (p<0.003) (Figure 2C).

[0131] In the second model system, the donor mouse cohort was divided into 2 × 10⁶ 5 Individual B16 melanoma cells were subcutaneously inoculated, and on days 7 and 14 post-inoculation, the mice were vaccinated with a cell-based vaccine loaded with B16. On day 21, the mice were sacrificed, and splenocytes were collected again and tested for CD8 + NK1.1 + and CD8 + NK1.1 neg The cells were sorted into populations. Next, naive cohorts inoculated with palpable B16 tumors were divided into 1.5 × 10⁶ groups. 6 CD8 + NK1.1 + or CD8 + NK1.1 neg Cellular adoptive transfer was performed (Figure 7B). CD8 + NK1.1 + Mice that received the cells showed a significant delay in tumor growth, accompanied by a survival benefit, but CD8 +NK1.1 neg The cohort that received the cells survived identically to the control cohort that was adopted with naive splenocytes (Figure 2D-E). Peripheral blood lymphocyte analysis was performed using CD8 + NK1.1 neg Or, compared to a cohort adopted with naive splenocytes, CD8 + NK1.1 + GP100 tetramer-specific CD8 in cell-adoptive-transfer cohorts + The levels of the intercellular memory markers CD62L and CCR7 were significantly elevated (Figure 2F). Combining these results, the CD161 homolog NK1.1 was found to be the major CD8 in a simple disease model among adolescent mice with a single pathogenic injury. + This suggests the demarcation of memory cell populations.

[0132] Mouse CD3 + CD8 + NK1.1 + The cell population is human CD3 + CD8 + CD161 + It is phenotypically preserved between its counterparts. CD8 in various systems + NK1.1 + Prompted by a protective memory response provided by cells, the inventors then identified memory CD8 defined by NK1.1 expression. + A subset of T cells has similar CD3 in the peripheral circulation. + CD8 + CD161 + We investigated whether the phenotypic and transcriptional conservation occurred in the human population of the cell population. For this analysis, we used CD8 + CD161 + and CD8 + CD161 neg Cells were differentially isolated from six different human donors. CD161 was identified by TCR-Vβ spectrtyping. +After confirming that the cells were polyclonal (Figure 8), transcriptional profiling of each population was performed by microarray analysis. These cells, despite being inactive and in a homeostatic state prior to analysis, reproduced the upregulated granzyme and intrinsic cytotoxic receptor profiles in these cells with a single-variable significance level of 0.1 (Figure 3, Table 3). Interspecies comparative gene analysis between activated mouse cells and inactivated human cells identified conserved signatures of 206 genes using a common nomenclature differentially regulated between the two populations (Figure 9). Reactome pathway analysis of upregulated human genes included HDAC deacetylation, DNA and histone methylation, nucleosome assembly, RNA polymerase I promoter escape, transcriptional regulation by small RNAs, and RNA-mediated gene silencing, encompassing <5 × 10⁶ -4 We identified signatures related to differentiation and regulation in FDR.

[0133] (Table 3) Mouse CD8 + NK1.1 + The phenotypic characteristics of the cells include increased expression of granzyme and killer lectin-like receptor genes in CD8 + CD161 + This is reproduced during the quiescent phase of a cell. TIFF0007856563000007.tif149160

[0134] Development of a PDAC model system for CAR T cell therapy. Based on its novel biological potential, the inventors have developed a human CD8 + CD161 + We hypothesized that a subset of these cells might offer more functional and sustained antitumor efficacy than conventional bulk PBMCs in association with solid tumor CAR T-cell therapy.

[0135] CD8 with IL7 / 15 / 21 combined with plate-bound stimulation using anti-CD3 / CD28 / Clec2d + CD161 + Ex vivo enlargement of cells leads to a central memory phenotype (CD45RA- CCR7 + ) was enhanced. CD8 + CD161 + Cells were selected from normal donors, and ex vivo stimulation conditions were optimized. Compared to IL2, IL-2 / 7 / 15, and IL2 / 7 / 15 / 21 stimulation, the combination of IL7 / 15 / 21 and plate-bound stimulation with anti-CD3 / CD28 / Clec2d showed improved central memory (CD45RA). - CCR7 + This resulted in significant upward regulation (Figure 5).

[0136] CD8 with IL7 / 15 / 21 combined with plate-bound stimulation using anti-CD3 / CD28 / Clec2d + CD161 + Ex vivo enlargement of cells enhanced cytotoxic granzyme production. CD8 + CD161 + Cells were selected from normal donors, and ex vivo stimulation conditions were optimized. Compared to IL2, IL-2 / 7 / 15, and IL2 / 7 / 15 / 21 stimulation, the combination of IL7 / 15 / 21 and plate-bound stimulation with anti-CD3 / CD28 / Clec2d resulted in significant upregulation of cytotoxic molecules, granzymes, and perforins (Figure 6).

[0137] CD8 + CD161 + The cells exhibit an inherent killing advantage in vitro. CD8 + CD161 + CD8 + CD161 neg To evaluate the cytotoxicity of cells and bulk PBMCs, a chromium-based short-term cytotoxicity assay was performed in vitro. CD8 + CD161 + CD8 + CD161 neg , and unprocessed bulk PBMCs were newly isolated from human peripheral blood products. The isolated cells were... 51Cytotoxicity was immediately tested in a 4-hour killing assay using a Cr-labeled allogeneic 293-HEK target. As shown in Figure 4, CD8 + CD161 + The cells induced 100% targeted lysis with an E:T ratio of 25:1, while bulk PBMCs and CD8 + CD161 neg The cells exhibited lysogenic activity of 22% and 15% at a maximum E:T ratio of 50:1, respectively (p<0.002 at 50:1, p<0.0007 at 25:1, and p<0.00002 at 5:1, one-way ANOVA). These data were obtained using CD8 + CD161 + T cells, CD8 + CD161 neg Alternatively, it demonstrated enhanced cytotoxicity that is not reproducible in bulk PBMC counterparts.

[0138] Example 3 - Discussion Based on the expression of surface molecules and secreted cytokines, lymphocytes are classified into different subsets and lineages. However, this classification is constantly changing with the identification of new cell subsets that occasionally express markers from previously identified cell subsets and lineages. One such surface molecule is CD161, which is known to be expressed on NK cells, NKT cells, and other T cell lineages (Fergusson et al., 2011). CD161 shares 47% homology with its mouse counterpart NK1.1 and is expressed by up to one-quarter of peripheral T cells (Neelapu et al., 1994). Since NK-T cells constitute less than 1% of peripheral T cells, CD3 + CD161 + The cells represent a distinct lineage of T cells, considering that they constitute more than 5% of circulating T cells (Takahashi et al., 2006). CD8 + In T cells, CD161 expression is defined as either intermediate or high, but CD4 expressing CD161 + Such distinctions are not clear among T cells (Takahashi et al., 2006). CD8 +CD161 high The cells have been previously defined as MAIT cells (Martin et al., 2009, Goldfinch et al., 2010), Tc17 cells (Northfield et al., 2008, Billerbeck et al., 2010), or memory stem cells (Turtle et al., 2009). Transcriptional profile analysis of different CD161-expressing cells is performed using CD8 + CD161 + Conserved CD161 enriched in T cells ++ / MAIT identifies the cell transcription signature, which is CD4 + CD161 + and TCRγδ + CD161 + This can be extended to T cells (Fergusson et al., 2014). Furthermore, CD161 T cell expression populations share a congenital-like TCR-independent response to interleukin (IL)-12 + IL-18. This response is independent of regulation by CD161, which acts as a co-stimulator in relation to T cell receptor stimulation. Therefore, CD161 expression identifies transcriptional and functional phenotypes shared among human T lymphocytes that are independent of both T cell receptor (TCR) expression and cell lineage. + CD161 + and CD4 + CD161 + The role of cells has been defined in viral infections (Northfield et al., 2008, Billerbeck et al., 2010, Rowan et al., 2008) and autoimmune diseases (Annibali et al., 2011, Cosmi et al., 2008, Kleinschek et al., 2009), but to date, the role of CD8 in cancer biology has been defined. + CD161 + No role of the cell has been clearly defined. In this study, the inventors investigated CD8 + CD161 + We started with the goal of understanding the biological and functional properties of cells.

[0139] The inventors previously used CD161 + CD8 in the mouse counterpart of cells + NK1.1 + We have reported on the functional significance of these cells and have observed an increase in their number under conditions that mimic viral infection (Konduri et al., 2016).

[0140] Mouse CD8 + NK1.1 + Microarray analysis of cells is used for CD8 + NK1.1 neg Compared to its counterpart, these cells demonstrated significant upregulation of granzyme production upon antigen stimulation. Differences were observed in the expression of innate genes and pathways that play a role in cytotoxicity. CD161 + Human equivalents have also been previously reported to constitutively express cytotoxic mediators granzyme B and perforin. In contrast, a quarter of cells lacking CD161 expression express naive CD8 + These are T cells that express lower levels of granzyme B and perforin, even within the memory population (Neelapu et al., 2018). CD8 + CD161 + The expression of CCR4 and CCR6 on cells indicates their ability to maintain tissue residentity and reach different organs. A similar expression pattern is observed in the circulating blood of CD161 in MS patients. high It has been observed in cells, enhancing their entry into the CNS and contributing to pathogenesis (Annibali et al., 2011). The inventors of this invention have found that resting CD8 + CD161 neg We also found that cells express higher levels of CXCR3, and that CXCR3 is CD8 + It is an effector memory marker that leads T cell differentiation into short-lived effectors with limited potential for memory (Kurachi et al., 2011).

[0141] CAR T cell therapy uses CD19 +While effective against hematological malignancies, it was ineffective in targeting solid tumors (Neelapu et al., 2016, Abken, 2015). One major challenge is overcoming inhibitory signaling by tregs and enhancing effector and memory functions (Klebanoff et al., 2012). Enhancing the persistence of effector and memory T cells can lead to more efficient CAR-T cell therapy. In preclinical models, CD8 + and CD4 + Both subsets expressed synergistic antitumor CAR-T activity (Sommermeyer et al., 2016). Similar results were obtained with manipulated CD4 + and CD8 + Preclinical mouse experiments have shown that combinations of T cells induce potent tumor rejection (Moeller et al., 2005; Shedlock and Shen, 2003). Recent clinical trial data in patients with non-Hodgkin lymphoma and chronic lymphocytic leukemia have shown that CD8 cells, which were separately augmented in vitro and injected in a 1:1 ratio, induced potent tumor rejection. + and CD4 + The high anticancer activity of CD19-CAR-T cells generated from a T cell subset composition was demonstrated (Turtle et al., 2016a). Similar results were obtained in a clinical trial involving patients with B-cell acute lymphoblastic leukemia (Turtle et al., 2016b). Isolated CD8 + T CM Have you been treated with a first-generation CD19-CAR-T using a subset, or with CD8? + and CD4 + T CM Another clinical study involving patients with either high-risk intermediate-grade B lineage non-Hodgkin lymphoma treated with second-generation CD19-CAR-T using both subsets demonstrated the feasibility and safety of both approaches (Turtle et al., 2016c), but CD4 + and CD8 + T CMThe CAR-T cell group and the second-generation CAR-T cell group exhibited better persistence. These studies highlight the need to evaluate different subsets of T cells and lymphocytes in CAR-T cell therapy. Lymphocyte subsets with intrinsic killing potential, such as NK, NKT, and γδT cells, have been evaluated for CAR potential (Ngai et al., 2018, Liu et al., 2018, Zoon et al., 2015). CD8 + CD161 + The cells contained less than 1% of the central memory marker CD62L. + CCR7 + CD161 expresses high CD8 + CD45RA neg It remains defined as an effector memory phenotype with cells (Takahashi et al., 2006). Previous reports indicated that CD161-negative cells did not alter CD161 expression upon anti-CD2, CD3, or CD28 stimulation, and that influenza-specific cells did not express CD161 after restimulation, even in the presence of cytokines (Northfield et al., 2008), suggesting that CD161 is not simply a marker of activation but defines a distinct lineage.

[0142] Bulk PBMC preparations typically used in CAR T cell production represent a heterogeneous population of cells, including highly differentiated antigen-experienced subsets. n ), stem cell memory (T scm ), and central memory (T cmIt has been previously reported that a subset of IL-7 and IL-15 resulted in a stronger antitumor response (Wang et al., 2011; Berger et al., 2008; Gattinoni et al., 2011; Gattinoni et al., 2005). While poorly differentiated cells may be more beneficial, ex vivo culture methods (cytokine composition and culture duration) can promote T cell differentiation (Alizadeh et al., 2019). Inclusion of IL-7 and IL-15 is beneficial for lymphocyte development, differentiation, and homeostasis during ex vivo T cell augmentation and has been shown to result in higher in vivo viability compared to IL-2 augmented CAR-T cells (Xu et al., 2014; Rochman et al., 2009). Several studies have shown that using IL-7 and IL-15 together can promote T cell differentiation. scm This has shown that phenotype preservation and enhancement of CAR-T cell efficacy can be achieved (Rochman et al., 2009, Cieri et al., 2013). Ex vivo augmentation of CD3 / CD28-CAR-T cells in the presence of IL-7 and IL-15 enhanced effector activity while preserving stem / memory potential against the GD2 tumor antigen (Gargett et al., 2015). CAR-T cells with increased IL-15 exhibited a stem cell memory phenotype (CD62L + CD45RA + CCR7 + It has also been demonstrated that IL-15 reduces the expression of exhaustion markers and increases proliferation during antigen challenge (Alizadeh et al., 2019). Other studies have shown that IL-21 also reduces CD2 + CD28 + CD8 +It has been shown that IL-7 promotes T cell growth (Santegoets et al., 2013) and enhances the potency of CD19-CAR-T cells (Rosenberg, 2014). The addition of IL-15 and IL-21 has been previously reported to help improve and maintain the memory potential of NKT cells (Ngai et al., 2018). Ex vivo growth of lymphocytes based on a combination of IL-7, IL-15, and IL-21 has been reported to enhance memory cells, reduce metastasis, and improve survival against mouse melanoma (Zoon et al., 2015). In this study, we found that ex vivo culture and bulk T cell growth using a cocktail of IL-7, IL-15, and IL-21 primarily enhances bulk PBMCs or CD8 cells grown without IL-21 or with IL-2 alone. + CD161 neg Without significantly altering the cellular phenotype, CD8 + CD161 + We found that it brings benefits to the cell population.

[0143] In summary, the inventors of the present invention have found that CD8 + CD161 + Cells and their mouse equivalents CD8 + NK1.1 + We report that the cells exhibit extremely high potential for cytotoxicity. Microarray-based gene expression profile analysis revealed NK1.1 neg and CD161 neg Compared to their counterparts, these cells showed increased expression levels of granzymes, perforins, and innate-like receptors when activated. In vitro, CD8 + CD161 + T cells are bulk PBMCs or CD8 + CD161 neg They were killed with higher efficiency than the population. The use of this subset for T-cell-based therapies offers exciting new opportunities for the effective treatment of solid tumors, including PDACs.

[0144] All methods disclosed and claimed herein can be prepared and performed without excessive experimentation in light of this disclosure. While the compositions and methods of this disclosure have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that modifications can be applied to the methods and steps, or the order of the steps of the methods, herein without departing from the concepts, spirit, and scope of this disclosure. More specifically, it will be apparent that certain chemically and physiologically relevant agents can be substituted for the agents described herein, while achieving the same or similar results. Such similar substitutes and modifications, which will be apparent to those skilled in the art, will be considered within the spirit, scope, and concepts of this disclosure as defined by the appended claims.

[0145] XI. References The following references are incorporated herein by reference insofar as they provide exemplary procedural or other details that supplement what is described herein. U.S. Patent No. 4,690,915 U.S. Patent No. 6,225,042 U.S. Patent No. 6,225,042 U.S. Patent No. 6,355,479 U.S. Patent No. 6,355,479 U.S. Patent No. 6,362,001 U.S. Patent No. 6,362,001 U.S. Patent No. 6,410,319 U.S. Patent No. 6,790,662 U.S. Patent No. 7,109,304 U.S. Patent Application Publication No. 2009 / 0004142 U.S. Patent Application Publication No. 2009 / 0017000 International Publication No. 2007 / 103009 Abken, Immunotherapy 7,535-544,2015. Ahmed et al., Cancer Res 67,5957-5964,2007. Ahmed et al., Clin Cancer Res 16,474-485,2010. Ahmed et al., J Clin Oncol 33, 1688-1696, 2015. Alizadeh et al., Cancer Immunol Res 7,759-772,2019. Altenschmidt et al., 1997. Annibali et al., Brain 134, 542-554, 2011. Ansari et al., World J Gastroenterol 21, 3157-3165, 2015. Assarsson et al., J Immunol 165, 3673-3679, 2000. Barthel and Goldfeld, 2003. Beatty et al.,Gastroenterology 155,29-32,2018. Berger et al., J Clin Invest 118, 294-305, 2008. Billerbeck et al., Proc Natl Acad Sci USA 107, 3006-3011, 2010. Braud et al., Oncoimmunology 7, e1423184, 2018. Brocker et al., 1998. Cieri et al., Blood 121,573-584, 2013. Cosmi et al., J Exp Med 205, 1903-1916, 2008. Eshhar et al., 1993. Eshhar, 1997. Fergusson et al., Cell Rep 9, 1075-1088, 2014. Fergusson et al., Front Immunol 2,36,2011. Fergusson et al., Mucosal Immunol 9,401-413, 2016. Fitzer-Attas et al., 1998. Gargett et al., Cytotherapy 17,487-495, 2015. Gattinoni et al., J Clin Invest 115, 1616-1626, 2005. Gattinoni et al., Nat Med 17, 1290-1297, 2011. Goldfinch et al., Vet Res 41,62,2010. Grada et al., Mol Ther Nucleic Acids 2, e105, 2013. Havenith et al., Int Immunol 24,625-636,2012. Hegde et al.,Mol Ther 21,2087-2101,2013. Heslop et al., 1996. Hwu et al., 1995. Kim et al., Nature, Vol.22(4), pp.403-410, 2004. Klebanoff et al., J Immunother 35,651-660,2012. Kleinschek et al., J Exp Med 206, 525-534, 2009. Konduri et al., Oncoimmunology 5, e1213933, 2016. Kurachi et al., J Exp Med 208, 1605-1620, 2011. Liang et al., Front Immunol 8, 1801, 2017. Liu et al., Leukemia 32,520-531, 2018. Marodon et al., 2003. Martin et al.,PLoS Biol 7,e54,2009. Maude et al.,N Engl J Med 378,439-448,2018. Moeller et al.,Blood 106,2995-3003,2005. Moritz et al.,1994. Neelapu et al.,Cancers(Basel)8,,2016. Neelapu et al.,Immunology,,2018. Neelapu et al.,J Immunol 153,2417-2428,1994. Neelapu et al.,Mod Pathol 28,428-436,2015. Neelapu et al.,N Engl J Med 377,2531-2544,2017. Neelapu et al.,Viral Immunol 18,513-522,2005. Ngai et al.,J Immunol 201,2141-2153,2018. Northfield et al.,Hepatology 47,396-406,2008. Remington's Pharmaceutical Sciences,16th Ed.,Mack,ed.,1980. Riddell et al.,1992. Roberts et al.,1994. Rochman et al.,Nat Rev Immunol 9,480-490,2009. Rosenberg,J Immunol 192,5451-5458,2014. Rowan et al.,J Immunol 181,4485-4494,2008. Santegoets et al.,J Transl Med 11,37,2013. Schneider,J.Embryol.Exp.Morph,Vol 27,pp.353-365,1972. Seaman et al.,J Virol 78,206-215,2004. Shedlock and Shen,Science 300,337-339,2003. Sommermeyer et al.,Leukemia 30,492-500,2016. Stancovski et al.,1993. Takahashi et al.,J Immunol 176,211-216,2006. Topalian and Rosenberg,1987. Turtle et al.,Clin Pharmacol Ther 100,252-258,2016b. Turtle et al.,Immunity 31,834-844,2009. Turtle et al.,J Clin Invest 126,2123-2138,2016a. Turtle et al.,Sci Transl Med 8,355ra116,2016c. van der Stegen et al.,Nat Rev Drug Discov 14,499-509,2015. Vera et al.,Blood 108,3890-3897,2006. Walter et al.,1995. Wang et al.,Blood 118,1255-1263,2011. Weitjens et al.,1996. Xu et al.,Blood 123,3750-3759,2014. Zoon et al.,Int J Mol Sci 16,8744-8760,2015.

Claims

1. CD8 with upregulated expression of granzyme and perforin ＋ CD161 ＋ An in vitro or ex vivo method for providing a population of T cells, (a) CD8 ＋ CD161 ＋ Prepare a sample that has already been collected and contains T cells, (b) CD8 in the presence of IL-7, IL-15, IL-21, CD3-conjugated antibody, CD28-conjugated antibody, and Clec2d ＋ CD161 ＋ T cells are cultured, Thereby, CD8 T cells not cultured in the presence of IL-7, IL-15, IL-21, CD3-binding antibody, CD28-binding antibody, and Clec2d ＋ CD161 ＋ Compared to T cells, CD8 T cells with upregulated expression of granzyme and perforin ＋ CD161 ＋ To provide a population of T cells The method, including the method described above.

2. (c) CD8 in the presence of IL-7, IL-15, and IL-21 without antibody stimulation ＋ CD161 ＋ Culturing T cells The method according to claim 1, further comprising:

3. The method according to claim 2, wherein the culture in step (c) does not include a CD3-conjugated antibody, a CD28-conjugated antibody, and Clec2d.

4. The method according to claim 3, wherein the culture in step (b) is carried out for 12 to 72 hours, 24 to 58 hours, or 24 to 36 hours.

5. The method according to claim 3, wherein the culture in step (c) lasts for at least 12 hours.

6. The method according to claim 1, wherein IL-7 is present at 5 to 20 ng / ml, IL-15 is present at 2.5 to 10 ng / ml, and / or IL-21 is present at 20 to 40 ng / ml.

7. The method according to claim 6, wherein IL-7 is present at 10 ng / ml, IL-15 is present at 5 ng / ml, and / or IL-21 is present at 30 ng / ml.

8. Before step (b), the CD8 in the sample ＋ CD161 ＋ The method according to claim 1, further comprising purifying or concentrating T cells for the presence of cells.

9. After step (b), the CD8 in the sample ＋ CD161 ＋ The method according to claim 1, further comprising purifying or concentrating T cells for the presence of cells.

10. The method according to claim 8 or 9, wherein enriching the T cells in the sample includes fluorescent cell sorting, magnetic bead separation, or paramagnetic bead separation.

11. The method according to any one of claims 1 to 10, wherein the culture is performed in a serum-containing culture medium.

12. The method according to any one of claims 1 to 10, wherein the culture is performed in a serum-free medium.

13. The method according to claim 1, wherein the cell sample is derived from the subject.

14. The method according to claim 13, wherein the sample is obtained by apheresis.

15. The method according to claim 1, wherein the sample is a frozen and stored sample.

16. The method according to claim 1, wherein the sample is derived from umbilical cord blood.

17. The method according to claim 13, wherein the sample is a peripheral blood sample from the subject.

18. The method according to claim 1, wherein the sample is obtained by apheresis.

19. The method according to claim 1, wherein the sample is obtained by venipuncture.

20. CD8 in the aforementioned sample ＋ CD161 ＋ The method according to any one of claims 1 to 19, further comprising introducing a nucleic acid encoding a chimeric antigen receptor (CAR) or a transgenic T cell receptor (TCR) into a T cell.

21. The aforementioned CD8 ＋ CD161 ＋ The method according to claim 20, wherein introducing nucleic acids encoding CAR or transgenic TCR into T cells is performed prior to step (b).

22. The aforementioned CD8 ＋ CD161 ＋ The method according to claim 20, wherein introducing nucleic acids encoding CAR or transgenic TCR into T cells is performed after step (b).

23. The aforementioned CD8 ＋ CD161 ＋ The method according to claim 20, wherein T cells are inactivated with respect to the expression of endogenous T cell receptors and / or endogenous HLA.

24. The nucleic acid encoding the membrane-bound Cγ cytokine is the CD8 ＋ CD161 ＋ The method according to claim 20, further comprising introducing it into T cells.

25. The method according to claim 24, wherein the membrane-bound Cγ cytokine is membrane-bound IL-15.

26. The method according to claim 24, wherein the membrane-bound Cγ cytokine is an IL-15-IL-15Rα fusion protein.

27. The above culturing is the CD8 ＋ CD161 ＋ The method according to claim 20, comprising culturing T cells in the presence of dendritic cells or artificial antigen-presenting cells (aAPCs).

28. The method according to claim 27, wherein the aAPC comprises a CAR-binding antibody or an antigen-binding fragment thereof expressed on the surface of the aAPC.

29. The method according to claim 27, wherein the aAPC comprises additional molecules that activate or co-stimulate T cells.

30. The method according to claim 29, wherein the additional molecule comprises a membrane-bound Cγ cytokine.

31. The aforementioned CD8 ＋ CD161 ＋ The method according to claim 27, wherein culturing T cells in the presence of aAPC includes culturing the cells in a ratio of 10:1 to 1:10 (CAR cells to aAPC).

32. The method according to claim 20, further comprising cryopreserving a sample of the population of CAR or transgenic TCR cells.

33. The method according to claim 20, wherein the CAR or the transgenic TCR targets a cancer cell antigen.

34. The method according to claim 33, wherein the cancer cell antigen is CD19, CD20, ROR1, CD22 carcinoembryonic antigen, alpha-fetoprotein, CA-125, 5T4, MUC-1, epithelial tumor antigen, prostate-specific antigen, melanoma-associated antigen, variant p53, variant ras, HER2 / Neu, folate-binding protein, HIV-1 envelope glycoprotein gp120, HIV-1 envelope glycoprotein gp41, GD2, CD123, CD33, CD138, CD23, CD30, CD56, c-Met, meoterin, GD3, HERV-K, IL-11Rα, κ light chain, λ light chain, CSPG4, ERBB2, EGFRvIII, VEGFR2, a combination of HER2-HER3, or a combination of HER1-HER2.

35. The method according to claim 20, wherein the CAR or the transgenic TCR targets a pathogen antigen.

36. The method according to claim 35, wherein the pathogen is a fungus, a virus, or a bacterial pathogen.

37. The method according to claim 35, wherein the pathogen is Plasmodium, Trypanosoma, Aspergillus, Candida, HSV, HIV, RSV, EBV, CMV, JC virus, BK virus, or the Ebola pathogen.

38. Before step (b), after step (b), or both before and after step (b), the CD8 of the sample ＋ CD161 ＋ The method according to claim 1, further comprising evaluating the T cell content.

39. The method according to claim 38, wherein the evaluation is performed by cell counting / flow cytometry.

40. Effective amounts of CD8 to provide a T cell response in human subjects with disease ＋ CD161 ＋ A pharmaceutical composition containing T cells, The aforementioned CD8 ＋ CD161 ＋ T cells are those into which nucleic acids encoding chimeric antigen receptors (CARs) or transgenic T cell receptors (TCRs) have been introduced. The aforementioned CD8 ＋ CD161 ＋ The T cells were cultured in the presence of IL-7, IL-15, IL-21, CD3-conjugated antibody, CD28-conjugated antibody, and Clec2d, and The aforementioned CD8 ＋ CD161 ＋ T cells that have not been cultured in the presence of IL-7, IL-15, IL-21, CD3-conjugated antibody, CD28-conjugated antibody, and Clec2d, and CD8 ＋ CD161 ＋ Compared to T cells, the expression of granzymes and perforins is upregulated. The aforementioned pharmaceutical composition.

41. The pharmaceutical composition according to claim 40, wherein the disease is cancer, and the CAR or the transgenic TCR targets cancer cell antigens.

42. The pharmaceutical composition according to claim 41, wherein the subject has previously received anticancer therapy.

43. The pharmaceutical composition according to claim 42, wherein the subject is in a state of remission or does not have the symptoms of cancer but contains detectable cancer cells.

Images