Methods for generating cytotoxic effector memory T cells for T cell therapy of cancer

Abstract

Provided herein are methods of expanding CD161 + T cells. Also provided are methods and compositions for producing the modified CD161 + T cells comprising a chimeric antigen receptor (CAR). In particular aspects, CAR-expressing T cells are generated, expanded, and / or used in the treatment of diseases (e.g., cancer).

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to U.S. Provisional Application No. 62 / 931,670, filed on November 6, 2019, the entire contents of which are incorporated herein by reference.

[0003] Statement Regarding Federally Funded Research

[0004] This invention was made with government support under Grant No. AI 127387 awarded by the National Institutes of Health. The government has certain rights in this invention. Technical Field

[0005] The present disclosure generally relates to the fields of medicine, immunology, cell biology and molecular biology. In some aspects, the field of the present disclosure relates to immunotherapy. More specifically, the field relates to generating improved chimeric antigen receptor (CAR) T cells and methods of treating using such cells. Background Art

[0006] Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive tumor with a poor five-year survival rate of <9% despite aggressive surgery, radiotherapy, and high-dose chemotherapy (Ansari et al., 2015). In recent years, adoptive chimeric antigen receptor (CAR) T cell therapy has shown strong potential as a treatment modality for cancer, particularly for selected CD19 T cells. + malignant tumors (Maude et al., 2018; Neelapu et al., 2017). CAR constructs consist of a single-chain fragment variable region (scFv) targeting a cell surface tumor antigen, a transmembrane domain, a hinge region, and an intracellular signaling domain of CD3ζ, typically fused to those of 4-1BB or CD28 co-stimulatory molecules (van der Stegen et al., 2015). In a Phase I clinical trial, adoptive cell therapy with autologous mesothelin-specific CAR-T cells was shown to be safe and moderately effective for chemotherapy-refractory metastatic human PDAC in a small number of patients (Beatty et al., 2018); however, progress in CAR T cell therapy for pancreatic tumors remains slow. In fact, few CAR-based therapies have shown any significant efficacy in the solid tumor setting.

[0007] The key feature of cell-mediated immunity for viral infection is the establishment of a long-lived population of memory T cells that provides persistent immunity to subsequent challenges by accelerating expansion and cytotoxicity kinetics (Seaman et al., 2004). Several groups have previously identified interesting subpopulations of such memory T cells (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), which can be identified by the expression of the natural cytotoxic receptor NK1.1 in mice or CD161 in humans. With TCR invariants or CD8αα + CD161 + Compared to NK cells, polyclonal αβ cell populations exhibit stem cell-like self-renewal and differentiation capabilities, distinct transcriptional profiles with significantly upregulated genes from the granzyme superfamily (Fergusson et al., 2011; Fergusson et al., 2014); unique antiviral specificity (Fergusson et al., 2008; Billerbeck et al., 2010; Havenith et al., 2012; Neelapu et al., 2005); and tissue homing properties (Billerbeck et al., 2010). Typically, CD161 is known as the innate 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 + Due to their tissue-resident properties and / or extravasation propensity, cells contribute to tissue pathogenesis during chronic viral infections as well as during autonomous immune 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 significantly improved clinical outcomes and survival in NSCLC (Braud et al., 2018). Summary of the Invention

[0008] In a first embodiment, an in vitro or ex vivo method is provided, comprising: (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 CD161 + The number of cells compared to non-CD161 +In some aspects, the T cells include CD8 + CD161 + In another aspect, the T cells include CD4 + CD161 + T cells.

[0009] IL-7 may be present at about 5-20 ng / ml, IL-15 may be present at about 2.5-10 ng / ml and / or IL-21 may be present at about 20-40 ng / ml, such as 10 ng / ml IL-7, 5 ng / ml IL-15 and / or 30 ng / ml IL-21. The method may further comprise, before step (b), purifying or enriching the sample for CD8 + CD161 + The method may further comprise, after step (b), purifying or enriching the sample for CD8 T cells. + CD161 + The enrichment of T cells in the sample may include fluorescent cell sorting, magnetic bead separation, or paramagnetic bead separation. The culture may last for up to 7 days, 14 days, 21 days, 28 days, 35 days, or 42 days.

[0010] In some aspects, the cells are further cultivated in a culture medium comprising CD3 and / or CD28 stimulants. In some aspects, the CD3 and / or CD28 stimulants include CD3 and / or CD28 binding antibodies. In some aspects, the cells are further cultivated in a culture medium comprising CD3, CD28 and / or CD161 stimulants. In some aspects, the CD3, CD28 and / or CD161 stimulants include CD3, CD28 and / or CD161 binding antibodies. In some aspects, the cells are further cultivated in a culture medium comprising CD3 binding antibodies, CD28 binding antibodies, Clec2d and / or CD161 stimulating antibodies. In some aspects, the cells are further cultivated in a culture medium comprising approximately 0.1 to 5.0, 0.3 to 3.0 or 0.5 to 2.0 μg / ml of CD3 binding antibodies, CD28 binding antibodies, Clec2d and / or CD161 stimulating antibodies.

[0011] On the one hand, CD8 + CD161 + cells, CD8 + CD161 negCells and bulk PBMCs were stimulated with plate-bound anti-CD3 / CD28 and expanded in a cytokine cocktail containing 10 ng / ml IL-7, 5 ng / ml IL-15, and 30 ng / ml IL-21 (all from Peprotech, Rocky Hill, NJ). In one aspect, CD8 + CD161 + Cells were isolated, cultured, and expanded in a cytokine cocktail containing 10 ng / ml IL-7, 5 ng / ml IL-15, and 30 ng / ml IL-21 in RPMI-1640, 10% FBS, and 2 mmol / l GlutaMAX. The cells were placed in a humidified chamber at 37°C for 48 hours. After 48 hours, the cells were expanded with the IL7 / 15 / 21 cytokine cocktail without antibody stimulation.

[0012] The method may further comprise obtaining the cells from the subject, such as by apheresis or venipuncture. The sample may be a cryopreserved sample. The sample may be from umbilical cord blood. The sample may be a peripheral blood sample from the subject. The sample may comprise CD8 T cells compared to a comparable sample obtained from the subject. + CD161 + The sample may be obtained from a third party.

[0013] The method may further include introducing the nucleic acid encoding CAR into the T cells in the sample, such as with a viral vector or by a method not involving transducing the T cells with a virus. Introducing the nucleic acid encoding CAR or transgenic TCR into the T cells may occur before step (b) or after step (b). The T cells expressing endogenous T cell receptors and / or endogenous HLA may be inactivated.

[0014] The method may further comprise introducing a nucleic acid encoding a membrane-bound Cγ cytokine into the T cell, such as wherein the membrane-bound Cγ cytokine is membrane-bound IL-15. The membrane-bound Cγ cytokine may be an IL-15-IL-15Rα fusion protein.

[0015] The culture may include culturing the T cells in the presence of dendritic cells or artificial antigen presenting cells (aAPC). The aAPC may include CAR binding antibodies or transgenic TCR binding antibodies or fragments thereof expressed on the surface of the aAPC. The aAPC may include other molecules that activate or costimulate T cells. The other molecules may include membrane-bound Cγ cytokines. The culture of T cells in the presence of aAPC may include culturing the cells with a (CAR cell to aAPC) ratio of about 10: 1 to about 1: 10.

[0016] The method may further include freezing and preserving a sample of a transgenic CAR cell group or a transgenic TCR cell group. The CAR or transgenic TCR can target cancer cell antigens such as CD19, CD20, ROR1, CD22 carcinoembryonic antigen, α-fetoprotein, CA-125, 5T4, MUC-1, epithelial tumor antigen, prostate specific antigen, melanoma associated antigen, mutated p53, mutated 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, mesothelin, GD3, HERV-K, IL-11Rα, kappa chain, lambda chain, CSPG4, ERBB2, EGFRvIII, VEGFR2, HER2-HER3 combination or HER1-HER2 combination. The CAR or transgenic TCR can target pathogen antigens, such as fungi, viruses or bacterial pathogens. The pathogen can be Plasmodium, Trypanosoma, Aspergillus, Candida, HSV, HIV, RSV, EBV, CMV, JC virus, BK virus or Ebola pathogen.

[0017] The method may further comprise assessing the CD8 + CD161 + Cell content, such as by cell counting / flow cytometry.

[0018] Also provided is a T cell composition produced by the method as described herein.

[0019] Additional embodiments relate to a method of providing a T cell response in a human subject having a disease, the method comprising administering an effective amount of T cells as described herein. The disease can be cancer, and wherein the CAR or transgenic TCR targets a cancer cell antigen. The subject may have undergone prior anti-cancer therapy. The subject may be in remission or have no symptoms of the cancer, but may include detectable cancer cells.

[0020] Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. However, it should be understood that although the detailed description and specific examples represent preferred embodiments of the present disclosure, they are given by way of illustration only, as various changes and modifications within the spirit and scope of the present disclosure will become apparent to those skilled in the art based on this detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The following drawings form part of the specification of the present invention and are included to further illustrate certain aspects of the present disclosure. The present disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0022] Figure 1 Gene expression analysis of antigenically stimulated T cells by microarray revealed that CD8 + NK1.1 + A cohort of 15 mice received a combination of chemotherapy and dendritic cell-based vaccination against murine pancreatic ductal adenocarcinoma. 60 days after tumor inoculation, spleens were harvested, pooled into three groups of five, and activated overnight with tumor antigen-loaded dendritic cells. + CD69 + Populations were gated and antigen-stimulated T cells were sorted into NK1.1 by flow cytometry. neg and NK1.1 + The volcano plot shows the difference between CD8 + NK1.1 neg cells and CD8 + NK1.1 + 1642 genes significantly regulated between cells. The top 15 genes differentially regulated at an FDR of 0.05 are marked on the graph.

[0023] Figure 2A-F .CD8 + NK1.1 +The cells defined a memory population that provided long-lasting protection and improved survival against influenza infection and melanoma tumors. In the influenza model, splenocytes were collected from mice that recovered from influenza infection and sorted into CD8 + NK1.1 neg and CD8 + NK1.1 + cells and adoptively transferred into naive mice that were subsequently challenged with influenza. In a melanoma model, tumor-bearing mice were vaccinated with dendritic cells loaded with tumor antigens. Splenocytes were harvested three weeks later and sorted into CD8 + NK1.1 neg and CD8 + NK1.1 + cells and adoptively transferred into mice bearing palpable tumors. + NK1.1 + Adoptive transfer of cells provided durable protection against influenza infection (Figure 2A-C) and melanoma (Figure 2D-F). (Figure 2A) + NK1.1 neg and naive CD8 + Compared to mice receiving CD8 + NK1.1 + Mice receiving CD8 + NK1.1 neg and naive CD8 + Compared with the group receiving CD8 + NK1.1 + One hundred percent survival was observed in mice infected with CD8 T cells. (Figure 2C) Analysis of PBMCs two weeks after infection showed that the PBMCs were significantly different from those of naive and CD8 T cells. + NK1.1 neg Compared with the adoptive transfer cohort, the + NK1.1 + In mice with circulating CD3 + CD8 + IFN-γ + In the melanoma model, CD8 + NK1.1 + Mice treated with CD8 T cells showed delayed tumor growth and improved survival. (Figure 2F) Analysis of peripheral blood lymphocytes three weeks after tumor transplantation showed that the + NK1.1 neg Compared with the cohorts adoptively transferred with CD8 + NK1.1+ GP100 tetramer-specific CD8 + The levels of memory markers CD62L and CCR7 were significantly increased in the cells. For each experiment, n = 10 mice per group. Error bars = + / - SEM, * p<0.05, one-way ANOVA.

[0024] Figure 3 . Mouse CD3 + CD8 + NK1.1 + Human CD3 + CD8 + CD161 + Phenotypically conserved among counterparts. CD3 + CD8 + CD161 + and CD3 + CD8 + CD161 neg cells, mouse CD3 + CD8 + NK1.1 + cells and CD3 + CD8 + NK1.1 neg Human equivalents of cells were magnetically sorted from the peripheral blood of six human donors and subjected to gene expression profiling by microarray. + CD161 + cells and CD8 + CD161 neg Volcano plot of differentially regulated genes between cells highlights upregulation of the CD161 receptor in the oval.

[0025] Figure 4 CD8+CD161+, CD8+CD161neg and unmanipulated native PBMCs were freshly isolated from human peripheral blood products. 51 The isolated cells were immediately tested for their cytotoxic capacity in a four-hour killing assay using Cr-labeled allogeneic 293-HEK targets. As indicated, CD8 + CD161 + cells could induce 100% target lysis at an E:T ratio of 25:1, while native PBMC and CD8 + CD161 negCells exhibited 22% and 15% lytic capacity 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 - percent killing. Error bars = + / - SD.

[0026] Figure 5 .Use IL7 / 15 / 21 to CD8 + CD161 + The combination of ex vivo expansion of cells and stimulation with plate-bound anti-CD3 / CD28 / Clec2d enhances the central memory phenotype (CD45RA - CCR7 + ). CD8 + CD161 + Cells were sorted from normal donors and ex vivo stimulation conditions were optimized. Cells were not CAR-transduced. The combination of IL7 / 15 / 21 and plate-bound anti-CD3 / CD28 / Clec2d stimulation resulted in a decrease in central memory (CD45RA) compared to IL2, IL-2 / 7 / 15, and IL2 / 7 / 15 / 21 stimulation. - CCR7 + ) were significantly upregulated.

[0027] Figure 6 .Use IL7 / 15 / 21 to CD8 + CD161 + The combination of ex vivo expansion of cells and stimulation with plate-bound anti-CD3 / CD28 / Clec2d enhances cytotoxic granzyme production. + CD161 + Cells were sorted from normal donors, and ex vivo stimulation conditions were optimized. Cells were not CAR-transduced. The combination of IL7 / 15 / 21 and plate-bound anti-CD3 / CD28 / Clec2d stimulation resulted in significant upregulation of cytotoxic molecules, granzymes, and perforin compared to IL2, IL-2 / 7 / 15, or IL2 / 7 / 15 / 21 stimulation.

[0028] Figure 7A -B.CD8 + NK1.1 + Cells have been identified as key circulating memory cells in multiple mouse disease models. + NK1.1 + The protective effect of the cells was model-independent and was demonstrated in influenza infection models and melanoma tumor models. + NK1.1 + Adoptive cell transfer experiment. Figure 7A) naive mice were exposed to a sublethal dose of influenza, mice were allowed to recover from infection, and splenocytes were harvested three weeks post-infection and magnetically sorted for CD8 + NK1.1 neg and CD8 + NK1.1 + 5x10 cells per NK1.1 group 5 cells / mouse were adoptively transferred into a naive cohort that was lethally challenged with the same influenza virus strain 24 hours after adoptive transfer. + Mice with splenocytes served as controls. Figure 7B )Use 2x10 5 Naive mice were inoculated subcutaneously with B16 melanoma cells and vaccinated with a cell-based vaccine loaded with B16 antigen on days 7 and 14 post-inoculation. On day 21, mice were sacrificed and splenocytes were harvested and sorted into CD8 + NK1.1 neg Cell populations and CD8 + NK1.1 + Then 1.5x10 6 CD8 + NK1.1 neg cells and CD8 + NK1.1 + Cells were adoptively transferred into naive cohorts inoculated with palpable B16 tumors. + Splenocyte-deficient mice served as controls.

[0029] Figure 8 .TCR-Vβ spectral typing showed that CD3 + CD8 + CD161 + The cells are polyclonal in nature. + CD161 + To determine the clonal nature of the cells, TCR-Vβ spectral typing was performed on the donor-derived cells. Histograms of the 30 amplified TCR Vβ families showed an unbiased Gaussian distribution of CDR3 sizes, indicating the polyclonal nature of these cells.

[0030] Figure 9 Cross-species comparative gene analysis revealed a conserved gene signature of 206 genes that were differentially regulated between the two populations. 206 common genes were identified between mouse (15 pooled samples) and human (6 paired samples) microarray analyses based on nomenclature. The expression patterns of these genes were significantly different in activated CD8 + NK1.1+ cells and resting CD8 + CD161 + The patterns were similar between cells, indicating the highly conserved nature of the genetic signature. DETAILED DESCRIPTION

[0031] As discussed above, CAR-T therapy has shown great promise in the treatment of cancers such as metastatic murine ductal adenocarcinoma (PDAC). In previous work, the inventors demonstrated that adoptively transferred, antigen-treated CD8 + NK1.1 + Cells can regulate lasting protection in PDAC models. Interestingly, these cells exist for nine months after initial exposure to the antigen, and when adoptively transferred to naive mice and subsequently attacked with parental PDAC cell lines, the cells are highly protective (Konduri et al., 2016). By expanding these results, the inventors attempted to characterize the additional biological and functional properties of these cells in various in vivo model systems, including SCID xenograft models of CAR T cell therapy for the treatment of PDAC. The results show that if adjustments are made to prevent differentiation of the starting cell population during transduction and amplification, CD8 + CD161 + T cells comprise an excellent platform for CAR T cell therapy. In addition, improved methods have now been developed by which such cells can be expanded ex vivo, thereby making it easier to provide CAR-T therapy to subjects in need. These and other features of the present disclosure are described in more detail below.

[0032] I. Definition

[0033] As used in the specification herein, "a" or "an" may mean one or more. As used in the claims herein, when used in conjunction with the word "comprising", the word "a" or "an" may mean one or more than one.

[0034] The use of the term "or" in the claims is intended to mean "and / or" unless explicitly stated to refer only to alternatives or the alternatives are mutually exclusive, but this disclosure supports a definition referring only to alternatives and "and / or." As used herein, "another" can mean at least a second or more.

[0035] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the device, method being employed to determine the value, or the variation that exists among study subjects, or is within 10% of the stated value.

[0036] As used herein, the term "chimeric antigen receptor (CAR)" may refer to, for example, artificial T cell receptors, chimeric T cell receptors, genetically modified T cell receptors, or chimeric immune receptors, and encompasses engineered receptors that are artificially specific and transplanted onto specific immune effector cells. CAR can be used to impart the specificity of monoclonal antibodies to T cells, thereby allowing the production of a large number of specific T cells, for example, for adoptive cell therapy. In a particular embodiment, for example, CAR guides the specificity of cells to tumor-associated antigens. In certain embodiments, CAR includes an intracellular activation domain, a transmembrane domain, and an extracellular domain including a tumor-associated antigen binding region. In certain aspects, CAR includes a fusion of a single-chain variable fragment (scFv) derived from a monoclonal antibody with a CD3-ζ transmembrane domain and an intracellular domain. The specificity of other CAR designs can be derived from a ligand (e.g., peptide) of a receptor or from a pattern recognition receptor, such as Dectin. In some cases, the interval of the antigen recognition domain can be modified to reduce activation-induced cell death. In some cases, CAR includes a domain for additional costimulatory signal conduction, such as CD3-ζ, FcR, CD27, CD28, CD137, DAP10 and / or OX40. In some cases, the molecule can be co-expressed with CAR, and the molecule includes a costimulatory molecule, a reporter gene for imaging (for example, for positron emission tomography), a gene product, homing receptor, chemokine, chemokine receptor, cytokine and cytokine receptor for conditionally ablating T cells when a prodrug is added.

[0037] As used herein, the term "T cell receptor (TCR)" refers to a protein receptor on T cells composed of a heterodimer of α (alpha) and β (beta) chains, although in some cells, the TCR is composed of γ and δ (γ / δ) chains. In embodiments of the present disclosure, the TCR can be modified on any cell that includes a TCR, including, for example, helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells, and γδ T cells.

[0038] The terms "tumor-associated antigen" and "cancer cell antigen" are used interchangeably herein. In each case, the terms refer to a protein, glycoprotein, or carbohydrate that is specifically or preferentially expressed by cancer cells.

[0039] II. Chimeric Antigen Receptors

[0040] As used herein, the term "antigen" is a molecule that can be bound by an antibody or T cell receptor. Antigens are further capable of inducing a humoral immune response and / or a cellular immune response, thereby leading to the production of B lymphocytes and / or T lymphocytes.

[0041] Embodiments of the present disclosure relate to nucleic acids comprising nucleic acids encoding antigen-specific chimeric antigen receptor (CAR) polypeptides, comprising CAR (hCAR) that has been humanized to reduce immunogenicity, including an intracellular signaling domain, a transmembrane domain, and an extracellular domain comprising one or more signaling motifs. In certain embodiments, CAR can recognize an epitope comprising a shared space between one or more antigens. Pattern recognition receptors, such as Dectin-1, can be used to obtain 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 an antigen-binding fragment thereof. In another embodiment, the specificity is derived from a peptide (e.g., a cytokine) that binds to the receptor. A complementarity determining region (CDR) is a short amino acid sequence present in the variable domain of an antigen receptor (e.g., immunoglobulin and T cell receptor) protein that complements the antigen and thus provides the receptor with its specificity for the specific antigen. Each polypeptide chain of an antigen receptor contains three CDRs (CDR1, CDR2, and CDR3). Since antigen receptors are typically composed of two polypeptide chains, there are six CDRs for each antigen receptor that can contact the antigen—three CDRs for each heavy chain and three for each light chain. Since most of the sequence variability associated with immunoglobulins and T cell receptors is found in the CDRs, these regions are sometimes referred to as hypervariable domains. Of these, CDR3 shows the greatest variability because it is encoded by recombination of the VJ (VDJ in the case of heavy chains and TCR αβ chains) regions.

[0042] It is envisioned that human CAR nucleic acid is a human gene to enhance cellular immunotherapy for human patients. In a specific embodiment, the present disclosure comprises a full-length CAR cDNA or coding region. The antigen binding region or domain may include a V domain of a single chain variable fragment (scFv) derived from a specific human monoclonal antibody. H and V L The fragments of the antibody chain can be fragments of the antibody chain, such as those described in U.S. Patent No. 7,109,304, which is incorporated herein by reference. The fragments can also be any number of different antigen-binding domains of human antigen-specific antibodies. In more specific embodiments, the fragments are antigen-specific scFvs encoded by sequences optimized for human codons expressed in human cells.

[0043] The arrangement can be multimeric, such as a diabody or a multimer. Multimers are most likely formed by cross-pairing the variable portions of the light and heavy chains into what Winters has called a diabody. The hinge portion of the construct can have a variety of alternatives, from complete deletion to maintaining the first cysteine, to proline substitution rather than serine substitution, to truncation until the first cysteine. The Fc portion can be deleted. Any stable and / or dimerizing protein can achieve this purpose. Only one of the Fc domains can be used, for example, the CH2 or CH3 domain from a human immunoglobulin. The hinge region, CH2 region, and CH3 region of a human immunoglobulin that has been modified to improve dimerization can also be used. Only the hinge portion of an immunoglobulin can also be used. Parts of CD8α can also be used.

[0044] The intracellular signaling domain of the chimeric receptor of the present disclosure is responsible for activating at least one of the normal effector functions of the immune cell into which the chimeric receptor has been placed. The term "effector function" refers to a specialized function of a differentiated cell. For example, the effector function of a T cell can be cytolytic activity or helper activity, including the secretion of cytokines. Effector function in naive, memory, or anamnestic T cells includes antigen-dependent proliferation. Thus, the term "intracellular signaling domain" refers to a portion of a protein that transduces an effector function signal and directs the cell to perform a specialized function. Although the entire intracellular signaling domain will generally be employed, in many cases, it is not necessary to use the entire intracellular polypeptide. To the extent that a truncated portion of an intracellular signaling domain can be used, such a truncated portion can be used in place of the complete chain, as long as it still transduces an effector function signal. Thus, the term intracellular signaling domain is intended to encompass any truncated portion of an intracellular signaling domain that is sufficient to transduce an effector function signal. Examples include the ζ chain of the T cell receptor or any homolog thereof (e.g., η, δ, γ or ε), MB1 chain, B29, Fc RIII, Fc RI and a combination of signaling molecules such as CD3ζ and CD28, CD27, 4-1BB, DAP-10, OX40 and combinations thereof, 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 a preferred embodiment, the human CD3ζ intracellular domain is employed for activation.

[0045] The antigen-specific extracellular domain and the intracellular signaling domain can be connected by a transmembrane domain, such as the human IgG4 Fc hinge region and Fc region. Alternatives include the human CD4 transmembrane domain, the human CD28 transmembrane domain, the transmembrane human CD3 zeta domain, or a cysteine-mutated human CD3 zeta domain or other transmembrane domains from other human transmembrane signaling proteins, such as CD16 and CD8 and the erythropoietin receptor.

[0046] In certain embodiments, CAR nucleic acid includes sequences encoding other costimulatory receptors, such as transmembrane domains and modified CD28 intracellular signaling domains.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 CD3 ζ, the other signals provided by the human costimulatory receptors inserted in human CAR are important for the complete activation of T cells and can help improve the therapeutic success of persistence and adoptive immunotherapy in vivo.

[0047] In a particular embodiment, the present disclosure relates to an isolated nucleic acid fragment and an expression cassette incorporating a DNA sequence encoding a CAR. The vector of the present disclosure is designed to be primarily used to deliver the desired gene to immune cells, preferably T cells under the control of a regulated eukaryotic promoter, for example, MNDU3 promoter, CMV promoter, EF1α promoter or ubiquitin promoter. Moreover, the vector may include a selectable marker, if for no other reason, to facilitate its in vitro manipulation. In other embodiments, CAR can be expressed from mRNA transcribed in vitro from a DNA template.

[0048] Chimeric antigen receptor molecules are recombinant and differ in their ability to bind antigens and transduce activation signals through the immunoreceptor activation motifs (ITAM's) present in their cytoplasmic tails. Receptor constructs utilizing antigen binding moieties (e.g., produced from single-chain antibodies (scFv)) provide a "universal" additional advantage because the construct binds to the original antigen on the target cell surface in an HLA-independent manner. For example, several laboratories have reported scFv constructs fused to sequences encoding the intracellular portion of the ζ chain (ζ), Fc receptor γ chain, and sky tyrosine kinase for the CD3 complex (Eshhar et al., 1993; Fitzer-Attas et al., 1998). Redirected T cell effector mechanisms and CTL lysis involving tumor recognition have been documented in several mouse and human antigen scFv:ζ systems (Eshhar, 1997; Altenschmidt et al., 1997; Brocker et al., 1998).

[0049] So far, non-human antigen binding regions are generally used to construct chimeric antigen receptors. The potential problem with non-human antigen binding regions such as mouse monoclonal antibodies is the lack of human effector function and the inability to penetrate into the tumor mass. In other words, such antibodies may not be able to regulate complement-dependent lysis or phagocytosis regulated by antibody-dependent cellular toxicity or Fc receptors to cause human target cells to lyse to destroy cells expressing CAR. In addition, non-human monoclonal antibodies can be recognized as foreign proteins by the human host, and therefore, repeated injections of such foreign antibodies may cause induction of immune responses, thereby causing harmful hypersensitivity reactions. For mouse-based monoclonal antibodies, this is generally referred to as human anti-mouse antibody (HAMA) responses. Therefore, it is more preferred to use human antibodies because they do not cause strong HAMA responses like mouse antibodies. Similarly, using human sequences in CAR can avoid the recognition of immune regulation, and therefore avoid being eliminated by endogenous T cells residing in the recipient and recognize processed antigens in the context of HLA.

[0050] In some embodiments, the chimeric antigen receptor comprises: a) an intracellular signaling domain; b) a transmembrane domain; and c) an extracellular domain comprising an antigen binding region.

[0051] In a specific embodiment, the intracellular receptor signaling domain in the CAR includes those of the T cell antigen receptor complex, such as the ζ chain of CD3, and the FcγRIII costimulatory signaling domain, CD28, CD27, DAP10, CD137, OX40, CD2, for example, alone or in series with CD3ζ. In the specific embodiment attached, the intracellular domain (which may be referred to as the cytoplasmic domain) includes part or all of one or more of the TCRζ chain, CD28, CD27, OX40 / CD134, 4-1BB / CD137, FcεRIγ, ICOS / CD278, IL-2Rβ / CD122, IL-2Rα / CD132, DAP10, DAP12, and CD40. In some embodiments, any part of the endogenous T cell receptor complex in the intracellular domain is used. For example, one or more cytoplasmic domains can be used because the so-called third generation CAR has at least two or three signaling domains fused together to produce an additive effect or a synergistic effect.

[0052] In certain embodiments of the chimeric antigen receptor, the antigen-specific portion of the receptor (which may be referred to as an extracellular domain including an antigen binding region) includes a tumor-associated antigen or a pathogen-specific antigen binding domain, comprising a sugar antigen recognized by a pattern recognition receptor, such as Dectin-1. Tumor-associated antigens can be of any kind, as long as they are 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 antigens, melanoma-associated antigens, mutated p53, mutated ras, etc. In certain embodiments, CAR can be co-expressed with membrane-bound cytokines to improve persistence when there is a small amount of tumor-associated antigen. For example, CAR can be co-expressed with membrane-bound IL-15.

[0053] In certain embodiments, intracellular tumor-associated antigens can be targeted, such as HA-1, survivin, WT1, and p53. This can be achieved by expressing a CAR on universal T cells that recognizes a processed peptide described according to an intracellular tumor-associated antigen in the context of HLA. In addition, universal T cells can be genetically modified to express a T cell receptor pairing that recognizes an intracellular processed tumor-associated antigen in the context of HLA.

[0054] Pathogen can be any kind, but in a specific embodiment, pathogen is for example fungus, bacterium or virus.Exemplary viral pathogens include adenoviridae, Epstein-Barr virus (Epstein-Barr virus, EBV), cytomegalovirus (CMV), respiratory syncytial virus (RSV), JC virus, BK virus, HSV, HHV virus family, Picornaviridae, Herpesviridae, Hepadnaviridae, Flaviviridae, Retroviridae, Orthomyxoviridae, Paramyxoviridae, Papovaviridae, Polyomavirus, Rhabdoviridae and Togaviridae.Exemplary pathogenic viruses cause smallpox, influenza, mumps, measles, varicella, Ebola virus and rubella.Exemplary pathogenic fungi include Candida, Aspergillus, Cryptococcus, Histoplasma, Pneumocystis and Stachybotrys. Exemplary pathogenic bacteria include Streptococcus, Pseudomonas, Shigella, Campylobacter, Staphylococcus, Helicobacter, Escherichia coli, Rickettsia, Bacillus, Bordetella, Chlamydia, Spirochete and Salmonella. In one embodiment, the pathogen receptor Dectin-1 can be used to produce a CAR that identifies the carbohydrate structure on the cell wall of the fungus. T cells genetically modified to express CAR based on Dectin-1 specificity can identify Aspergillus and target hyphae growth. In another embodiment, CAR can be manufactured based on antibodies (e.g., glycoproteins from CMV and Ebola viruses) that identify viral determinants to interfere with viral infection and pathology.

[0055] In some embodiments, the pathogenic antigen is an Aspergillus carbohydrate antigen, against which the extracellular domain in the CAR recognizes carbohydrate patterns of the fungal cell wall, such as by Dectin-1.

[0056] Chimeric immunoreceptors according to the present disclosure can be produced by any means known in the art, although preferably produced using recombinant DNA technology. Nucleic acid sequences encoding several regions of the chimeric receptor can be prepared by standard techniques of molecular cloning (genomic library screening, PCR, primer-assisted ligation, scFv libraries from yeast and bacteria, site-directed mutagenesis, etc.) and assembled into a complete coding sequence. The resulting coding regions can be inserted into expression vectors and used to transform appropriate expression host allogeneic T cell lines.

[0057] As used herein, nucleic acid construct or nucleic acid sequence or polynucleotide is intended to mean a DNA molecule that can be transformed or introduced into a T cell and transcribed and translated to produce a product (eg, a chimeric antigen receptor).

[0058] In the exemplary nucleic acid constructs (polynucleotides) employed in the present disclosure, a promoter is operably linked to a nucleic acid sequence encoding a chimeric receptor of the present disclosure, i.e., the two are positioned so as to promote transcription of messenger RNA from the DNA encoding the chimeric receptor. The promoter may be of genomic origin or synthetically produced. Various promoters for T cells are well known in the art (e.g., the CD4 promoter disclosed by Marodon et al., 2003). For example, the promoter may be constitutive or inducible, wherein induction is associated with a specific cell type or a specific level of maturation. Alternatively, a variety of well-known viral promoters are also suitable. Promoters of interest include the β-actin promoter, the SV40 early and late promoters, immunoglobulin promoters, the human cytomegalovirus promoter, retroviral promoters, and the Friend spleen focus-forming virus promoter. The promoter may be associated with an enhancer or may not be associated with the enhancer, wherein the enhancer may be naturally associated with a specific promoter or with a different promoter.

[0059] The sequence encoding the open reading frame of the chimeric receptor can be obtained from a genomic DNA source, a cDNA source, or can be synthesized (e.g., by PCR), or a combination thereof. Depending on the size of the genomic DNA and the number of introns, the use of cDNA or a combination thereof may be desirable, as introns have been found to stabilize mRNA or provide T cell-specific expression (Barthel and Goldfeld, 2003). Furthermore, the use of endogenous or exogenous non-coding regions to stabilize mRNA may be further advantageous.

[0060] For expression of the chimeric antigen receptors of the present disclosure, naturally occurring or endogenous transcriptional initiation regions of the nucleic acid sequence encoding the N-terminal component of the chimeric receptor can be used to produce the chimeric receptor in the target host. Alternatively, exogenous transcriptional initiation regions that allow constitutive or inducible expression can be used, wherein expression can be controlled depending on the target host, the desired expression level, the properties of the target host, etc.

[0061] Likewise, the signal sequence that directs the chimeric receptor to the surface membrane can be the endogenous signal sequence of the N-terminal component of the chimeric receptor. Optionally, in some cases, it may be desirable to exchange this sequence for a different signal sequence. However, the selected signal sequence should be compatible with the secretory pathway of the T cell so that the chimeric receptor is presented on the surface of the T cell.

[0062] Similarly, the termination region can be provided by a naturally occurring or endogenous transcriptional termination region of the nucleic acid sequence encoding the C-terminal component of the chimeric receptor. Alternatively, the termination region can be derived from a different source. In most cases, the source of the termination region is not generally considered to be critical for recombinant protein expression, and a variety of termination regions can be used without adversely affecting expression.

[0063] As will be appreciated by those skilled in the art, in some cases, several amino acids at the end of the antigen binding domain in the CAR may be deleted, for example, typically no more than 10 residues, more typically no more than 5 residues. Moreover, it may be desirable to introduce a small number of amino acids at the border, typically no more than 10 residues, more typically no more than 5 residues. The deletion or insertion of amino acids may be a result of construction needs, thereby providing convenient restriction sites, ease of manipulation, increased expression levels, and the like. Additionally, for similar reasons, substitution of one or more amino acids with different amino acids may occur, typically without substitution of more than about five amino acids in any one domain.

[0064] Chimeric constructs according to the coding chimeric receptors of the present disclosure can be prepared in a conventional manner. Because in most cases, natural sequences can be adopted, natural genes can be appropriately separated and manipulated to allow the appropriate connection of various components. Therefore, using appropriate primers that cause the undesirable partial deletion of genes, the nucleic acid sequences of the N-terminal protein and the C-terminal protein of the coding chimeric receptor can be separated by adopting polymerase chain reaction (PCR). Alternatively, restriction digestion of cloned genes can be used to produce chimeric constructs. In either case, the sequence can be selected to provide blunt ends or have complementary overlapping restriction sites.

[0065] The various manipulations used to prepare chimeric constructs can be performed in vitro, and in certain embodiments, the chimeric constructs are introduced into vectors for cloning and expression in appropriate hosts using standard transformation or transfection methods. Thus, after each manipulation, the resulting constructs from the DNA sequence ligation are cloned, the vectors are isolated, and the sequences are screened to ensure that the sequences encode the desired chimeric receptor. The sequences can be screened by restriction analysis, sequencing, etc.

[0066] The chimeric constructs of the present disclosure are applied to subjects suffering from or suspected of having cancer by reducing the size of the tumor or preventing the growth or regrowth of the tumor in these subjects. Therefore, the present disclosure further relates to a method for reducing growth or preventing tumor formation by introducing the chimeric constructs of the present disclosure into the isolated T cells of the subject and reintroducing the transformed T cells into the subject, thereby affecting the anti-tumor response to reduce or eliminate the tumor in the subject. Suitable T cells that can be used include cytotoxic lymphocytes (CTLs) or any cells with T cell receptors that need to be destroyed. As is well known to those skilled in the art, various methods are easily able to isolate these cells from the subject. For example, using cell surface marker expression or using commercially available kits (e.g., ISOCELL from Pierce, Rockford, Ill.) TM ).

[0067] It is envisioned that the chimeric construct can be introduced into the subject's own T cells as naked DNA or a suitable vector. Methods for stably transfecting T cells using naked DNA by electroporation 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 the present invention contained in a plasmid expression vector that is appropriately oriented for expression. Advantageously, the use of naked DNA reduces the time required to generate T cells expressing the chimeric receptor of the present invention.

[0068] Alternatively, viral vectors (e.g., retroviral vectors, adenoviral vectors, adeno-associated viral vectors, or lentiviral vectors) can be used to introduce the chimeric construct into T cells. Suitable vectors for use according to the methods of the present disclosure are not replicable in the T cells of the subject. A large number of viral-based vectors are known in which the copy number of the virus maintained in the cell is low enough to maintain the viability of the cell. Illustrative vectors include the pFB-neo vector disclosed herein. and vectors based on HIV, SV40, EBV, HSV, or BPV.

[0069] Once it is confirmed that the transfected or transduced T cells are able to express the chimeric receptor as a surface membrane protein with desired regulation and expressed at a desired level, it is possible to determine whether the chimeric receptor works in the host cell to provide the desired signal induction. Subsequently, the transduced T cells are reintroduced into the subject or applied to the subject to activate the anti-tumor response in the subject. In order to promote administration, the transduced T cells according to the present disclosure can be made into a pharmaceutical composition or an implant suitable for in vivo administration with an appropriate carrier or diluent, and the carrier or diluent can further be pharmaceutically acceptable. The manner of making such compositions or implants has been described in the art (see, for example, Remington's Pharmaceutical Sciences, 16th edition, Mack edited, 1980). Where appropriate, the transduced T cells can be formulated into a semisolid or liquid preparation, such as a capsule, solution, injection, inhalant or aerosol, for their corresponding route of administration. Methods known in the art can be used to prevent or minimize the release and absorption of the composition until it reaches the target tissue or target organ, or to ensure the timed release of the composition. However, it is desirable to use a pharmaceutically acceptable form that allows cells expressing the chimeric receptor to be produced. Thus, it is desirable to prepare the transduced T cells into a pharmaceutical composition containing a balanced salt solution, preferably Hanks' balanced salt solution or normal saline.

[0070] III. Methods and Compositions Related to the Examples

[0071] In certain aspects, the present disclosure comprises a method for producing and / or expanding antigen-specific CD8 + CD161 + A method for expressing T cells, comprising: transfecting T cells with an expression vector containing a DNA construct encoding hCAR; and then optionally stimulating the cells with antigen-positive cells, recombinant antigens, or antibodies against the receptor to cause cell proliferation. As described in the examples, a specific combination of interleukins, namely IL-7, IL-15, and IL-21, provides significantly improved CD8 + CD161 + Expansion of T cells.

[0072] In another aspect, a method for stably transfecting and redirecting T cells by electroporation or other non-viral gene transfer using naked DNA (such as, but not limited to, sonoporation) is provided. Most researchers have used viral vectors to carry heterologous genes into T cells. By using naked DNA, the time required to generate 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 the present disclosure produces a stable transfectant that expresses and carries a chimeric TCR (cTCR) on its surface.

[0073] "Chimeric TCR" means a receptor expressed by a T cell and including an intracellular signaling domain, a transmembrane domain, and an extracellular domain, wherein the extracellular domain can specifically bind to an antigen in an MHC-unrestricted manner, and the antigen is generally not bound by a T cell receptor in the manner described. Antigens stimulate T cells under appropriate conditions to cause cell proliferation (amplification) and / or IL-2 production. The exemplary chimeric receptors of the present application are examples of chimeric TCRs. However, the method is suitable for transfection with chimeric TCRs that are specific for other target antigens, such as 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, epithelial tumor antigens, etc.

[0074] In some aspects, T cells are primary human T cells collected after stimulation with G-CSF, bone marrow or umbilical cord blood, such as T cells derived from human peripheral blood mononuclear cells (PBMC) PBMC. Conditions include the use of mRNA and DNA and electroporation. After transfection, cells can be infused immediately or can be stored. In some aspects, after transfection, cells can be propagated in vitro as a body colony for several days, weeks or months in about 1 day, 2 days, 3 days, 4 days, 5 days or longer after gene transfer to cells. In other aspects, after transfection, transfectants are cloned and clones show the presence of a single integrated or somatically maintained expression cassette or plasmid, and the expression of chimeric receptors is amplified in vitro. The clones selected for amplification demonstrate the ability to specifically recognize target cells. Recombinant T cells can be amplified by stimulating with IL-2 or other cytokines (e.g., IL-7, IL-12, IL-15, IL-21, etc.) combined with common gamma chains. Recombinant T cells can be amplified by stimulating with artificial antigen presenting cells. Recombinant T cells can be expanded on artificial antigen-presenting cells or with antibodies such as OKT3 that crosslink CD3 on the surface of T cells. Subpopulations of recombinant T cells can be deleted on artificial antigen-presenting cells or with antibodies such as Campath that bind to CD52 on the surface of T cells. In another aspect, the genetically modified cells can be cryopreserved.

[0075] T cell proliferation (survival) after infusion can be assessed by: (i) q-PCR using primers specific for the CAR; (ii) flow cytometry using antibodies specific for the CAR; and / or (iii) soluble TAA.

[0076] In certain embodiments of the present disclosure, CAR cells are delivered to individuals in need, such as individuals with cancer or infection. The cells then enhance the individual's immune system to attack the corresponding cancer cells or pathogenic cells. In some cases, one or more doses of antigen-specific CAR T cells are provided to an individual. In the case of providing two or more doses of antigen-specific CAR T cells to an individual, the duration between administrations should be sufficient to allow for the time of reproduction in the individual, and in a specific embodiment, the duration between doses is 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days or more days.

[0077] The source of allogeneic T cells modified to contain a chimeric antigen receptor and lack a functional TCR can be of any kind, but in specific embodiments, the cells are obtained from, for example, umbilical cord blood, peripheral blood, human embryonic stem cells, or induced pluripotent stem cell banks. A suitable dose for therapeutic effect will be at least 10 5 cells per dose or approximately 10 5 cells per dose with approximately 1010 Cells per dose, for example, preferably in a series of dosing cycles. An exemplary dosing regimen consists of four one-week ascending dosing cycles, starting with at least about 10 cells on day 0. 5 Starting with approximately 10 cells, for example, gradually increasing to approximately 10 cells within several weeks after initiation of the intra-patient dose escalation regimen. 10 Suitable routes of administration include intravenous, subcutaneous, intracavitary (eg, via a reservoir access device), intraperitoneal, and direct injection into the tumor mass.

[0078] The pharmaceutical composition of the present disclosure can be used alone or in combination with other generally recognized medicaments that can be used for treating cancer. No matter whether it is delivered alone or delivered in combination with other medicaments, the pharmaceutical composition of the present disclosure can be delivered by various routes and delivered to mammals, particularly various parts of the human body, to achieve a specific effect. It will be understood by those skilled in the art that although more than one approach can be used for administration, a specific approach can provide a more direct and more effective reaction than another approach. For example, intradermal delivery rather than by inhalation can be advantageously used to treat melanoma. Local or systemic delivery can be completed by administration, and the administration includes applying or instilling the formulation into the body cavity, inhaling or blowing into an aerosol or introducing by parenteral administration, including intramuscular administration, intravenous administration, administration in the portal vein, intrahepatic administration, peritoneal administration, subcutaneous administration or intradermal administration.

[0079] The compositions of the present disclosure can be provided in unit dosage form, wherein each dosage unit, such as an injection, contains a predetermined amount of the composition, alone or in appropriate combination with other active agents. As used herein, the term unit dosage form refers to a physically discrete unit suitable as a unit dose for human and animal subjects, where appropriate, each unit containing a predetermined amount of the compositions of the present disclosure, alone or in combination with other active agents, and a pharmaceutically acceptable diluent, carrier or vehicle, the predetermined amount being calculated in an amount sufficient to produce the desired effect. The specifications of the novel unit dosage form of the present disclosure depend on the specific pharmacodynamics associated with the pharmaceutical composition in a particular subject.

[0080] It is desirable that an effective amount or sufficient number of isolated transduced T cells be present in the composition and introduced into a subject such that a long-term specific anti-tumor response is established to reduce the size of the tumor or eliminate tumor growth or regrowth that would otherwise result in the absence of such treatment. It is desirable that reintroduction of the transduced T cells into the subject results in a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 100% reduction in tumor size when compared to otherwise identical conditions in the absence of the transduced T cells.

[0081] Therefore, the amount of transduced T cells administered should take into account the route of administration and should be such that a sufficient number of transduced T cells will be introduced to achieve the desired therapeutic response. In addition, the amount of each active agent included in the compositions described herein (e.g., the active dose per cell to be contacted or the active dose per a certain body weight) can vary in different applications. Generally, it is desired that the concentration of transduced T cells should be sufficient to provide at least about 1x10 6 to about 1x10 9 transduced T cells, and even more desirably, about 1x10 7 to about 5x10 8 transduced T cells, although any suitable amount may be used, e.g., greater than 5 x 10 8 cells, or less than, for example, less than 1x10 7 Dosing regimens can be based on established cell-based therapies (see, eg, Topalian and Rosenberg, 1987; US Patent 4,690,915), or alternative continuous infusion strategies can be employed.

[0082] These values ​​provide general guidance for practitioners regarding the range of transduced T cells to be utilized when optimizing the methods of the present disclosure for practicing the present disclosure. Such ranges described herein in no way preclude the use of higher or lower amounts of components, which may be desirable in specific applications. For example, actual dosages and regimens may vary depending on whether the composition is administered in combination with other pharmaceutical compositions, or according to individual differences in pharmacokinetics, drug disposition, and metabolism. Those skilled in the art can readily make any necessary adjustments at any time according to the exigencies of a particular situation.

[0083] IV. Exemplary Human Antigen Receptor T Cells

[0084] As discussed above, the present disclosure relates to CD8 + CD161 + T cell culture and use.

[0085] CD8 (cluster of differentiation 8) is a transmembrane glycoprotein that acts as a co-receptor 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 protein subtypes, α and β, each encoded by a different gene. In humans, both genes are located on chromosome 2 at position 2p12.

[0086] The CD8 co-receptor is primarily 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 of cytotoxic T cell populations. It is expressed in T-cell lymphoblastic lymphoma and hypopigmented mycosis fungoides.

[0087] In order 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 immunoglobulin variable (IgV)-like extracellular domains, which are connected to the membrane by a thin stem and an intracellular tail. Less common homodimers of the CD8-α 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 Leahy, DJ, Axel, R. and Hendrickson, WA by X-ray diffraction at a resolution of 2.6 Å. The structure was determined to have an immunoglobulin-like β-sandwich fold and 114 amino acid residues. 2% of the protein is wound into α helices and 46% is wound into β sheets, with the remaining 52% of the molecule retained in the loop portion.

[0088] The extracellular IgV-like domain of CD8-α interacts with the α3 portion of the class I MHC molecule. This affinity brings the T cell receptor of cytotoxic T cells and target cells into close contact during antigen-specific activation. Cytotoxic T cells that possess the CD8 surface protein are called CD8+ T cells. The primary recognition site is a 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 facilitating cytotoxic T cell antigen interactions, the CD8 co-receptor also plays a role in T cell signaling. The cytoplasmic tail of the CD8 co-receptor interacts with Lck (lymphocyte-specific protein tyrosine kinase). Once the T cell receptor binds to its specific antigen, Lck phosphorylates the cytoplasmic CD3 and ζ chain of the TCR complex. This initiates a phosphorylation cascade that ultimately leads to the activation of transcription factors such as NFAT, NF-κB, and AP-1, thereby influencing the expression of certain genes.

[0089] 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 regulate cytotoxicity and secrete cytokines after immune stimulation. Several genes of the C-type lectin superfamily, including the rodent NKRP1 family of glycoproteins, are expressed by NK cells and may participate in the regulation of NK cell function. CD161 contains an extracellular domain with several motifs characteristic of C-type lectins, a transmembrane domain, and a cytoplasmic domain.

[0090] In one aspect, the compositions and methods of the embodiments relate to human CD8 expressing chimeric antigen receptor (or CAR) polypeptides. + CD161 + T cells. CAR can have any antigen binding specificity, but will include a typical intracellular signaling domain, a transmembrane domain, and an extracellular domain present in the CAR construct. The extracellular domain will include a given binding region, which depends on the purpose of the CAR-T design. 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 some aspects, the transmembrane domain is a CD28 transmembrane domain.

[0091] In other aspects, the composition can comprise a nucleic acid encoding the above-mentioned polypeptide.In certain aspects, the nucleic acid sequence is optimized for human codon usage.

[0092] In still other aspects, the composition may comprise cells expressing the polypeptides described herein. The T cells may comprise an expression cassette encoding a CAR polypeptide. The expression cassette may be included in a non-viral vector, such as a transposon or human transposon or a recombinant variant thereof. The expression cassette may be included in a viral vector or a recombinant variant thereof. The expression cassette may be genomic-integrated or maintained episomally or expressed from mRNA.

[0093] In yet another aspect, the present disclosure includes a method for manufacturing a T cell expressing a human CAR, the method comprising introducing an expression cassette into a cell, 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 comprise stimulating the cell with a target antigen or an antibody against the receptor to proliferate the cell, kill the cell, and / or cause the cell to produce cytokines; for example, the cell may be stimulated to proliferate or amplify with an artificial antigen-presenting cell carrying the target antigen.

[0094] In certain aspects, the present disclosure includes a method for treating a human disease condition, the method comprising infusing to the patient a recombinant cell expressing a human CAR in an amount sufficient to treat the condition, wherein the human CAR includes an extracellular target binding domain, a transmembrane domain, and an intracellular signaling domain. For example, the condition can be cancer, an autoimmune disease, or an infectious disease.

[0095] hCAR can be a chimeric receptor including one or more activation intracellular domains, such as an activation domain derived from CD3-ζ. Additional T cell activation motifs include but are not limited to CD28, CD27, OX-40, DAP10, and 4-1BB. In some aspects, the activation domain may also include CD28 transmembrane and / or activation domains. In other aspects, the hCAR coding region and / or expression cassette codons are optimized for expression in human cells and subjects. For example, in one embodiment, the scFv region obtained from the VH sequence and VL sequence of the target-specific human antibody is incorporated into the binding segment of hCAR. In another embodiment, the hCAR expression cassette is maintained episomically or integrated into the genome of the recombinant cell. In some aspects, the expression cassette is included in a nucleic acid that can be integrated using an integrase mechanism, a viral vector such as a retroviral vector, or a non-viral vector such as a transposon mechanism. In other embodiments, the expression cassette is included in a nucleic acid based on a transposon. In certain embodiments, the expression cassette is part of the two-component Sleeping Beauty (SB) or piggyBac system, which utilizes transposons and transposases to enhance non-viral gene transfer.

[0096] The number of recombinant hCAR expressing cells can be expanded to a clinically significant number. An example of this amplification uses artificial antigen presenting cells (aAPC). Recombinant hCAR expressing cells can be verified and identified by flow cytometry and Western blot analysis. Expressing T cells, i.e., recombinant hCAR expressing CAR can recognize and kill target cells. In another aspect, hCAR can be expressed as universal cells, which can be infused across the transplant barrier to help prevent immunogenicity. When cytotoxicity occurs, hCAR can be imaged together with human genes (such as by positron emission tomography, PET) and conditional ablation of T cells. The recombinant cells disclosed herein can be used for specific cell therapies.

[0097] V. Exemplary Membrane-Bound IL-15 Co-Expressing Chimeric Antigen Receptor or Transgenic TCRT Cells for Targeting Minimal Residual Disease

[0098] The disease relapse rate of chemotherapy-treated B-lineage acute lymphoblastic leukemia (B-ALL) in adults and children is 65% and 20%, respectively, due to drug-resistant residual disease. The high incidence of B-ALL relapse, especially in the poor prognosis group, has promoted the use of immune-based therapies using allogeneic hematopoietic stem cell transplantation (HSCT). This therapy relies on the presence of alloreactive cells in the donor graft to eradicate the remaining leukemic cells or minimal residual disease to improve disease-free survival. Donor lymphocyte infusion has been used to enhance the ability of transplanted T cells to target residual B-ALL after allogeneic HSCT, but this treatment approach for such patients achieves a remission rate of less than 10% and is associated with high morbidity and mortality due to the frequency and severity of graft-versus-host disease (GVHD). Because relapse is a common and lethal problem in these refractory malignancies, adoptive therapy using peripheral blood mononuclear cell (PBMC)-derived T cells after HSCT can be used to enhance the antitumor or graft-versus-leukemia (GVL) effect by retargeting the specificity of donor T cells to tumor-associated antigens (TAAs).

[0099] At present, CAR-modified T cells rely on obtaining survival signal conduction by CAR, which only occurs when encountering tumor antigens. In the clinical situation where these CAR-modified T cells are infused into patients with large-volume diseases, there are sufficient tumor antigens, which are manifested as providing sufficient activation and survival signal conduction by CAR. However, patients with relapsed B-ALL usually receive myeloablative chemotherapy and then undergo HSCT, and show minimal residual disease (MRD). In this case, the patient's tumor burden is low, and the tiny TAA level severely limits the signal conduction of the CAR regulation required for supporting the infused T cells, thereby compromising therapeutic potential. It is expected that the alternative CAR-independent way for improving T cell persistence will improve the transplantation of CAR-modified T cells.

[0100] Cytokines in the common gamma chain receptor family (γC) are important co-stimulatory molecules of T cells that are crucial for lymphoid function, survival, and proliferation. IL-15 possesses several properties that are desirable for adoptive therapy. IL-15 is a homeostatic cytokine that supports the survival of long-lived memory cytotoxic T cells, promotes the eradication of established tumors by alleviating functional suppression of tumor-resident cells, and inhibits AICD.

[0101] IL-15 is tissue restricted and can only be observed at any level in serum or systemically under pathological conditions. Unlike other γC cytokines that are secreted into the surrounding environment, IL-15 is trans-presented by producing cells to T cells in the context of IL-15 receptor α (IL-15Rα). This cytokine has a unique delivery mechanism for T cells and other responder cells: (i) it is highly targeted and localized, (ii) it increases the stability and half-life of IL-15, and (iii) it produces a signaling pathway that is qualitatively different from that achieved by soluble IL-15.

[0102] In one embodiment, the present disclosure provides a method for producing chimeric antigen receptor (CAR) modified T cells or transgenic TCR T cells with long-term in vivo potential for treating leukemia patients who exhibit minimal residual disease (MRD), for example. In general, this method describes how soluble molecules such as cytokines can be fused to the cell surface to increase therapeutic potential. The core of this method relies on co-modification of CAR T cells or transgenic TCR T cells and human cytokine mutant protein of interleukin-15 (IL-15) (hereinafter referred to as mIL15). The mIL15 fusion protein comprises a codon-optimized IL-15 cDNA sequence fused to the full-length IL15 receptor α via a flexible serine-glycine linker. This IL-15 mutant protein is designed in this way to: (i) limit mIL15 expression to the CAR + or transgenic TCR + mIL15 is expressed on the surface of T cells to limit the spread of cytokines to non-target in vivo environments, thereby potentially improving its safety profile, as exogenous soluble cytokine administration has led to toxicity; and (ii) IL-15 is present in the context of IL-15Rα to mimic physiologically relevant and qualitative signaling and stabilization and recycling of the IL-15 / IL-15Ra complex to achieve a longer cytokine half-life. T cells expressing mIL15 are able to continue to support cytokine signaling, which is critical for their survival after infusion. mIL15 is generated by non-viral Sleeping Beauty system genetic modification and subsequent ex vivo expansion on a clinically applicable platform + CAR + T cells or mIL15 + Genetically modified TCR + T cells produced enhanced persistence of T cell infusion products after infusion in murine models with high, low, or no tumor burden. + CAR + T cells also showed enhanced anti-tumor efficacy in both high or low tumor burden models.

[0103] In high tumor burden models, mIL15+ CAR + T cells vs CAR + T cells have higher persistence and anti-tumor activity, suggesting that mIL15 + CAR + T cells may be more effective than CAR T cells in treating leukemia patients with active disease with a prevalent tumor burden. + T cells are more effective. Therefore, in the broadest application, mIL15 + CAR + T cells could replace CARs in adoptive therapy + T cells. mIL15 + CAR + The ability of T cells to survive independently of survival signaling via the CAR allows these modified T cells to persist after infusion in the absence of tumor antigens. Therefore, this is expected to have the greatest impact on treatment efficacy in the MRD setting, particularly in patients who have received myeloablative chemotherapy and hematopoietic stem cell transplantation. These patients will be treated with mIL15 + CAR + T cells were adopted by adoptive T cell transfer to treat their MRD and prevent relapse.

[0104] Membrane-bound cytokines, such as mIL-15, are of broad interest. In addition to membrane-bound IL-15, other membrane-bound cytokines are contemplated. Membrane-bound cytokines can also be extended to cell surface expression of other molecules associated with activation and proliferation of cells for human applications. These include, but are not limited to, cytokines, chemokines, and other molecules that contribute to activation and proliferation of cells for human applications.

[0105] Membrane-bound cytokines, such as mIL15, can be used in vitro to prepare cells for human applications and can be used on infused cells (e.g., T cells) for human applications. For example, membrane-bound IL-15 can be expressed on artificial antigen-presenting cells (aAPCs), such as cells derived from K562, to stimulate T cells and NK cells (and other cells) to activate and / or proliferate. The T cell population activated / propagated on aAPCs by mIL15 includes genetically modified lymphocytes, but also tumor-infiltrating lymphocytes and other immune cells. These aAPCs are not infused. In contrast, mIL15 (and other membrane-bound molecules) can be expressed on infused T cells and other cells.

[0106] The therapeutic efficacy of MRD therapy using CAR-modified T cells is hampered by the lack of persistence of T cells after adoptive transfer. + CAR + T cells or mIL15 + Genetically modified TCR+ The ability of T cells to persist in vivo for extended periods independent of tumor antigens suggests significant potential for treating patients with MRD. In this setting, mIL15 and the persistent T cells it supports will fill a need, as current approaches for patients with MRD are insufficient. The persistence of infused T cells and other lymphocytes in patients with MRD exceeds that of CARs. + T cell persistence. Any immune cell used to treat or prevent malignancies, infections, or autoimmune diseases must be able to persist over the long term if a sustained therapeutic effect is to be achieved. Therefore, the ability to activate T cells and allow them to persist beyond signals from endogenous T cell receptors or introduced immune receptors is important for many aspects of adoptive immunotherapy. Therefore, the expression of membrane-bound cytokines can be used to enhance the therapeutic potential and persistence of T cells and other immune cells infused for various pathological conditions.

[0107] The inventors have generated a mutant protein of IL-15 that is expressed as a CAR + T cells or genetically modified TCRs + Membrane-bound fusion protein of IL-15 and IL-15Rα (mIL15) on T cells. The mIL15 construct was co-electrotransferred into primary human T cells with a CD19-specific CAR (day 0) as two Sleeping Beauty DNA transposon plasmids. Clinically relevant amounts of mIL15 + CAR + T cells express CD19 + Artificial antigen presenting cells co-cultured with IL-21 produced and supplemented. Signaling through the IL-15 receptor complex in genetically modified T cells was confirmed by phosphorylation of STAT5 (pSTAT5), and these T cells showed equivalent CAR + CD19 on T cells + Redirected specific lysis of tumor targets. In addition, after antigen withdrawal, signaling generated by mIL15 increases the prevalence of T cells with a less differentiated / younger phenotype that has memory-related properties, including specific cell surface markers, transcription factors, and the ability to secrete IL-2. These properties are ideal in T cells for adoptive transfer because they are associated with T cell subsets in which the ability to persist in vivo for a long time has been demonstrated. + In immunocompromised NSG mice with leukemia, mIL15 + CAR + T cells showed both persistence and anti-tumor effects, and their CAR + T cell counterparts were unable to maintain significant persistence despite the presence of TAAs. In a preventive mouse (NSG) model, mIL15+ / - CAR + T cells were maintained for six days, followed by the introduction of disseminated CD19 + Leukemia, only mIL15 was found + CAR + T cells persist and prevent tumor transplantation. + CAR + Whether T cells can persist independently of TAA stimulation, mIL15 + / - CAR + T cells were adoptively transferred into tumor-free NSG mice. Only mIL15 + CAR + T cells were able to persist in this in vivo environment without exogenous cytokine support or the presence of CD19 TAA. These data suggest that mIL15 can be a key player in CAR + T cells or genetically modified TCRs + In summary, this cytokine fusion molecule: (i) provides a stimulatory signal through pSTAT5, leading to enhanced T cell persistence in vivo while maintaining tumor-specific functions, (ii) maintains T cell subsets that promote a memory-like phenotype, (iii) eliminates the requirement and cost of clinical-grade IL-2 for T cell expansion and persistence in vitro and in vivo, and (iv) alleviates the need for clinical-grade soluble IL-15.

[0108] VI. Pancreatic cancer

[0109] Pancreatic cancer develops when cells in the pancreas, a glandular organ behind the stomach, begin to multiply out of control and form a mass. These cancer cells have the ability to invade other parts of the body. There are many types of pancreatic cancer. The most common is pancreatic adenocarcinoma, which accounts for about 85% of cases, and the term "pancreatic cancer" is sometimes used to refer to just this type. These adenocarcinomas begin in the part of the pancreas that produces digestive enzymes. Several other types of cancer, which together represent the majority of non-adenocarcinomas, can also arise from these cells. One to two percent of pancreatic cancer cases are neuroendocrine tumors, which arise from the hormone-producing cells of the pancreas. These are generally less aggressive than pancreatic adenocarcinomas.

[0110] Signs and symptoms of the most common form of pancreatic cancer can include yellowing of the skin, abdominal or back pain, unexplained weight loss, light-colored stools, dark urine, and loss of appetite. There are often no symptoms in the early stages of the disease, and specific symptoms sufficient to indicate pancreatic cancer usually don't develop until the disease is advanced. By the time it's diagnosed, pancreatic cancer has usually spread to other parts of the body.

[0111] Pancreatic cancer rarely occurs before the age of 40, and more than half of pancreatic cancer cases occur in people over the age of 70. Risk factors for pancreatic cancer include smoking, obesity, diabetes and certain rare genetic conditions. About 25% of cases are related to smoking, and 5-10% of cases are related to genetic genes. Pancreatic cancer is usually diagnosed by a combination of medical imaging techniques such as ultrasound or computer tomography, blood tests and tissue sample examination (biopsy). The disease is divided into several stages, from early stage (Phase I) to late stage (Phase IV). Effective screening of the general population has not yet been found.

[0112] Non-smokers and those who maintain a healthy weight and limit their consumption of red or processed meats have a lower risk of pancreatic cancer. If smokers quit, their chances of developing the disease decrease, returning almost to the level of the rest of the population after 20 years. 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 curative treatment for pancreatic cancer and can improve quality of life even when a cure is not possible. Medications for pain management and improved digestion are sometimes needed. Even for those receiving treatment aimed at curing the disease, early palliative care is recommended.

[0113] In 2015, all types of pancreatic cancer caused 411,600 deaths worldwide. Pancreatic cancer is the fifth most common cause of cancer death in the UK and the third most common cause in the US. The disease is most common in developed countries, where approximately 70% of new cases originated in 2012. Pancreatic cancer generally has a poor prognosis: 25% of people survive one year after diagnosis, and 5% survive five years. For cancers diagnosed early, the five-year survival rate rises to approximately 20%. Neuroendocrine cancers have a better outcome; within five years of diagnosis, 65% of those diagnosed are alive, although survival rates vary considerably depending on the type of tumor.

[0114] VII. Immune System and Immunotherapy

[0115] In some embodiments, medical conditions are treated by transferring redirected T cells that elicit a specific immune response. In one embodiment of the present disclosure, a B-cell lineage malignancy or condition is treated by transferring redirected T cells that elicit a specific immune response. Therefore, a basic understanding of immune responses is essential.

[0116] 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 are derived from the same multipotent hematopoietic stem cells and cannot distinguish between each other until they are activated. B cells play an important 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 called T cell receptors (TCRs) on their cell surfaces. In almost all other vertebrates, B cells and T cells are produced by stem cells in the bone marrow. T cells enter and develop in the thymus, from which they get their name. In humans, approximately 1%-2% of the lymphocyte pool recirculates every hour to optimize the chances of antigen-specific lymphocytes finding their specific antigens within secondary lymphoid tissues.

[0117] T lymphocytes are produced by hematopoietic stem cells in the bone marrow and usually migrate to the thymus until maturity. T cells express unique antigen-binding receptors (T cell receptors) on their membranes, which can only recognize antigens associated with major histocompatibility complex (MHC) molecules on the surface of other cells. There are at least two T cell populations, known as T helper cells and T cytotoxic cells. T helper cells and T cytotoxic cells are mainly distinguished by their display of membrane-bound glycoproteins CD4 and CD8, respectively. T helper cells secrete various lymphokines, which are crucial 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 body cells that display antigens, such as virus-infected cells and tumor cells, by producing substances that cause cell lysis. Natural killer cells (or NK cells) are a type of cytotoxic lymphocytes that constitute the main components of the innate immune system. NK cells play a major role in rejecting tumors and virus-infected cells. Cells kill by releasing small cytoplasmic granules of proteins called perforins and granzymes that cause the target cell to die by apoptosis.

[0118] Antigen presenting cells, including macrophages, B lymphocytes and dendritic cells, are distinguished by their expression of specific MHC molecules. APCs internalize antigens and re-express a portion of the antigen, as well as the MHC molecules on their outer cell membranes. The major histocompatibility complex (MHC) is a large genetic complex with multiple loci. The MHC locus encodes two major classes of MHC membrane molecules, referred to as class I and class II MHC. T helper lymphocytes typically recognize antigens associated with MHC class II molecules, and T cytotoxic lymphocytes recognize antigens associated with MHC class I molecules. In humans, MHC is referred to as the HLA complex and in mice as the H-2 complex.

[0119] T cell receptor or TCR is a molecule that is present on the surface of T lymphocytes (or T cells) and is generally responsible for identifying the antigen bound to the major histocompatibility complex (MHC) molecule. It is a heterodimer composed of the α chain and β chain in 95% of T cells, while 5% of T cells have a TCR composed of γ chain and δ chain. The combination of TCR with antigen and MHC causes its T lymphocyte to activate through a series of biochemical events regulated by related enzymes, co-receptors and specialized auxiliary molecules. In immunology, the CD3 antigen (CD stands for cluster of differentiation) is a protein complex composed of four different chains (CD3γ, CD3δ and two CD3ε) in mammals, which is associated with the molecule and ζ chain known as the T cell receptor (TCR) to produce activation signals in T lymphocytes. TCR, ζ chain and CD3 molecules constitute the TCR complex together. CD3γ chain, CD3δ chain and CD3ε chain are highly correlated cell surface proteins of the immunoglobulin superfamily containing a single extracellular immunoglobulin domain. The transmembrane regions of the CD3 chains are negatively charged, a property that allows these chains to associate with the positively charged TCR chains (TCRα and TCRβ). The intracellular tail of the CD3 molecule contains a single conserved motif known as the immunoreceptor tyrosine-based activation motif, or ITAM, which is crucial for the signaling ability of the TCR.

[0120] 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 constitutively expressed on naive T cells. In addition to the TCR, stimulation by CD28 can provide T cells with potent co-stimulatory signals to produce various interleukins (specifically IL-2 and IL-6).

[0121] Isolation and expansion of antigen-specific T cells has been validated in clinical trials as a strategy for therapeutic intervention in human disease (Riddell et al., 1992; Walter et al., 1995; Heslop et al., 1996).

[0122] An autoimmune disease or autoimmunity is the inability of an organism to recognize its own components (down to the submolecular level) as "self," which results in an immune response against its own cells and tissues. Any disease caused by this abnormal immune response is known as an autoimmune disease. Prominent 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).

[0123] Inflammatory diseases, including autoimmune diseases, are also a class of diseases associated with B cell disorders. Examples of autoimmune diseases include, but are not limited to, acute idiopathic thrombocytopenic purpura, chronic idiopathic thrombocytopenic purpura, dermatomyositis, Sydenham's chorea, myasthenia gravis, systemic lupus erythematosus, lupus nephritis, rheumatic fever, polyglandular syndrome, bullous pemphigoid, diabetes mellitus, Henoch-Schonlein purpura, poststreptococcal nephritis, erythema nodosum, Takayasu's arteritis, Addison's disease, and leukemia. Disease, rheumatoid arthritis, multiple sclerosis, sarcoidosis, ulcerative colitis, erythema multiforme, IgA nephropathy, polyarteritis nodosa, ankylosing spondylitis, Goodpasture's syndrome, thromboangiitis obliterans, Sjögren's syndrome, primary biliary cirrhosis, Hashimoto's thyroiditis, thyrotoxicosis, 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 fibrosing alveolitis. The most common treatments are corticosteroids and cytotoxic drugs, which can be very toxic. These drugs also suppress the entire immune system, can lead to serious infections, and have adverse effects on the bone marrow, liver, and kidneys. To date, other therapeutic approaches for treating Class III autoimmune diseases have been directed against T cells and macrophages.There is a need for more effective methods to treat autoimmune diseases, particularly Class III autoimmune diseases.

[0124] VIII. Artificial Antigen Presenting Cells

[0125] In some cases, aAPCs can be used to prepare the therapeutic compositions and cell therapy products of the embodiments. For general guidance on the preparation and use of antigen presentation systems, see, for example, U.S. Patent Nos. 6,225,042, 6,355,479, 6,362,001, and 6,790,662; U.S. Patent Application Publication Nos. 2009 / 0017000 and 2009 / 0004142; and International Publication No. WO2007 / 103009.

[0126] aAPC is usually incubated with a peptide of optimal length, which allows the peptide to bind directly to the MHC molecule without the need for additional processing. Alternatively, the cell can express the antigen of interest (i.e., in the case of MHC-independent antigen recognition). In addition to the peptide-MHC molecule or the antigen of interest, the aAPC system can also include at least one exogenous helper molecule. Any suitable number of helper molecules and helper molecule combinations can be used. Helper molecules can be selected from helper molecules such as costimulatory molecules and adhesion molecules. Exemplary costimulatory molecules include, among others, CD70 and B7.1 (B7.1 was previously referred to as B7 and also referred to as CD80), which bind to CD28 and / or CTLA-4 molecules on the T cell surface, thereby affecting, for example, T cell expansion, Th1 differentiation, short-term T cell survival rate and cytokine secretion, such as interleukin (IL) -2 (see Kim et al., 2004). Adhesion molecules can include carbohydrate-binding glycoproteins such as selectins, transmembrane binding 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, for example, cell-to-cell or cell-to-matrix contact. Exemplary adhesion molecules include LFA-3 and ICAMs, such as ICAM-1. Techniques, methods, and reagents for selecting, cloning, preparing, and expressing exemplary helper molecules, including costimulatory molecules and adhesion molecules, are exemplified in, for example, U.S. Patent Nos. 6,225,042, 6,355,479, and 6,362,001.

[0127] Cells selected to become aAPCs are preferably defective in intracellular antigen processing, intracellular peptide transport and / or intracellular MHC class I or class II peptide loading, or are poikilothermic (i.e., less sensitive to temperature attack than mammalian cell lines) or have both defects and poikilothermic properties. Preferably, cells selected to become aAPCs also lack the ability to express at least one endogenous counterpart (e.g., endogenous MHC class I or class II molecules and / or endogenous accessory molecules as described above) to exogenous MHC class I or class II molecules and the accessory molecule components introduced into the cell. In addition, aAPCs preferably retain the defects and poikilothermic properties that the cells had before they were modified to produce aAPCs. Exemplary aAPCs are composed of or derived from transporters associated with antigen processing (TAP)-deficient cell lines such as insect cell lines. Exemplary poikilothermic insect cell lines are Drosophila cell lines, such as the Schneider 2 cell line (see, e.g., Schneider, 1972). Illustrative methods for the preparation, growth, and culture of Schneider 2 cells are provided in US Patent Nos. 6,225,042, 6,355,479, and 6,362,001.

[0128] In one embodiment, aAPCs are also subjected to freeze-thaw cycles. In an exemplary freeze-thaw cycle, aAPCs can be frozen by contact with a suitable receptacle containing aAPCs 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 aAPCs are then thawed by removing the aAPCs from the cryogenic material and exposing them to ambient room temperature conditions, or by promoting a thawing process that shortens the thawing time using a warm water bath or warm hands. In addition, aAPCs can be frozen and stored for an extended period of time before thawing. Frozen aAPCs can also be thawed and then lyophilized before further use. Preferably, preservatives that may adversely affect the freeze-thaw procedure, such as dimethyl sulfoxide (DMSO), polyethylene glycol (PEG), and other preservatives, are not present in the culture medium containing aAPCs undergoing freeze-thaw cycles, or are substantially removed, such as by transferring the aAPCs to a culture medium that is substantially free of such preservatives.

[0129] In other preferred embodiments, heterologous nucleic acids and nucleic acids endogenous to aAPCs can be inactivated by crosslinking, such that substantially no cell growth, replication, or nucleic acid expression occurs following inactivation. In one embodiment, aAPCs are inactivated at a point after expression of exogenous MHC and helper molecules, presentation of such molecules on the surface of the aAPC, and presentation of the loaded MHC molecule with the selected peptide or peptides. Thus, such inactivated and selected peptide-loaded aAPCs, while substantially unable to proliferate or replicate, retain the selected peptide-presenting function. Preferably, crosslinking also produces aAPCs that are substantially free of contaminating microorganisms, such as bacteria and viruses, without substantially reducing the antigen-presenting cell function of the aAPCs. Thus, crosslinking maintains the important APC functions of aAPCs while helping to alleviate safety concerns regarding cell therapy products developed using aAPCs. For methods related to crosslinking and aAPCs, see, for example, U.S. Patent Application Publication No. 20090017000, which is incorporated herein by reference.

[0130] IX. Kits of the Disclosure

[0131] Any composition described herein can be included in a kit. In certain embodiments, allogeneic CART cells are provided in a kit, which may also include reagents suitable for amplifying cells, such as culture medium, aAPC, growth factors, antibodies (e.g., for sorting or characterizing CAR T cells) and / or plasmids encoding CAR or transposase.

[0132] In non-limiting examples, a chimeric receptor expression construct, one or more reagents for producing a chimeric receptor expression construct, cells for transfecting the expression construct, and / or one or more instruments for obtaining allogeneic cells for transfecting the expression construct (such instruments can be syringes, pipettes, forceps, and / or any such medically approved devices).

[0133] In some embodiments, an expression construct for eliminating endogenous TCRαβ expression, one or more reagents for producing the construct, and / or a CAR are provided in a kit. + In some embodiments, this comprises an expression construct encoding a zinc finger nuclease.

[0134] In some aspects, the kit includes reagents or equipment for electroporation of cells.

[0135] The test kit may include one or more suitable aliquots of the compositions or reagents of the present disclosure to produce the compositions of the present disclosure. The components of the test kit may be packaged in aqueous media or lyophilized form. The container means of the test kit may include at least one vial, test tube, flask, bottle, syringe or other container means in which the components may be placed and preferably in suitable aliquots. When there is more than one component in the test kit, the test kit will generally also contain a second, third or other container in which other components may be placed separately. However, various combinations of components may be included in the vial. The test kit of the present disclosure generally also includes a device for accommodating the chimeric receptor construct and any other reagent containers for commercial sale in a tightly sealed manner. Such containers may include, for example, injection or blow-molded plastic containers in which the desired vial is retained.

[0136] X. Examples

[0137] The following examples are included to illustrate preferred embodiments of the present disclosure. It will be appreciated by those skilled in the art that the techniques disclosed in the following examples represent techniques discovered by the inventors to function well in the practice of the present disclosure and, therefore, can be considered to constitute preferred modes for its practice. However, in light of the present disclosure, it will be appreciated by those skilled in the art that many changes may be made to the specific embodiments disclosed and still obtain the same or similar results without departing from the spirit and scope of the present disclosure.

[0138] Example 1 - Materials and Methods

[0139] Mouse microarray analysis of CD8 isolated from mice previously harboring pancreatic tumors + NK1.1 + cells and CD8 + NK1.1 neg Cells were treated with a combination of cell-based vaccines and gemcitabine chemotherapy (Konduri et al., 2016). The isolated cells were activated with autologous DC loaded with PDAC antigens and total RNA was isolated using RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions. CD8 + NK1.1 + cells and CD8 +NK1.1 neg Cells were subjected to gene expression profiling analysis.

[0140] Influenza model. To generate T cells for adoptive transfer, C57 / BL6 mice were challenged with a Swiss mouse lung-adapted strain of H3N2 influenza A virus generously provided by Dr. Brian Gilbert as described (Liang et al., 2017). Infection was performed by exposure to aerosols of influenza virus diluted in MEM medium + 0.05% gelatin using an Aerotech II nebulizer using an Aridyne 2000 compressor generating room air at 10 liters / minute. All mice infected in any given experiment were infected simultaneously in a single exposure chamber. Two weeks after infection, mice were sacrificed, spleens were harvested, and CD8 + The cells were negatively selected (Miltenyi Biotec). + Cells were further magnetically sorted for NK1.1 + Groups and NK1.1 neg Group (Miltenyi Biotec). 500,000 CD8 + NK1.1 + cells and CD8 + NK1.1 neg The cells were adoptively transferred into naive mice, which were then challenged with influenza virus.

[0141] 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 PBS. DCs were loaded with melanoma tumor antigens as described (Konduri et al., 2016). One week after tumor inoculation, 200,000 antigen-loaded DCs suspended in 50 μl PBS were injected into the footpad and boosted seven days later. Ten days after the boost, the vaccinated mice were sacrificed and CD8 + Splenocytes (Miltenyi Biotec). + Cells were further magnetically sorted for NK1.1 + Groups and NK1.1 neg(Miltenyi Biotec). Three groups of eight naive mice were injected subcutaneously with 250,000 B16F10 tumor cells. Tumor size was recorded and animals were randomly divided into groups so that each group had a similar mean tumor size and standard error. Seven days after tumor inoculation, mice in the treatment group each received 1.5 million CD8 + NK1.1 + cells or CD8 + NK1.1 - cells. Naive mice served as untreated controls. Tumor size was determined by external caliper measurements and calculated using the formula (length × width 2 ) × π / 6. Once the tumor burden in the control group exceeded the permissible limit set by the Center for Comparative Medicine (CCM), mice were euthanized 22 days after tumor inoculation.

[0142] Murine PBMC analysis. PBMCs were collected from adoptively transferred mice by retroorbital bleeding two weeks after influenza infection or three weeks after tumor implantation. Red blood cells were lysed by treatment with ammonium chloride (Sigma-Aldrich) according to the manufacturer's instructions. The white blood cell pellet was washed once with PBS and resuspended in AIM-V medium with 10% mouse serum. Cells were stained with anti-CD3, CD4, CD8, CD25, and IFN-γ for flow cytometric analysis. All flow cytometric analyses were performed using an LSRII flow cytometer (BD Biosciences) and OS-X was analyzed using FlowJo version 10.0.00003 (Tree Star Inc., Ashland, OR).

[0143] Human microarray analysis. + CD161 + cells and CD8 + CD161 neg Cells were magnetically separated from peripheral blood derived from 3 healthy donors and 3 PDAC patients. The isolated cells were not activated. Total RNA was isolated from the cells by RNeasy mini kit (Qiagen) according to the manufacturer's instructions. CD8 + CD161 + cells and CD8 + CD161 negGene expression profiling of cells was performed. Detailed instructions for sample requirements and data pre-analysis 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). Data were analyzed and visualized using the Transcriptome Analysis Console v3.0 (Affymetrix).

[0144] TCR Vβ spectral typing. CD8 isolated from peripheral blood of normal donors + CD161 + Cells were spectrally typed by the Mayo Clinic. The resulting image is a cluster of fluorescent peaks separated by single base pairs and of varying fluorescence intensity, roughly corresponding to the number of fragments of that size represented in the original donor RNA. The organization of the peak pattern (number of peaks), relative intensity across peaks, and size distribution are reviewed.

[0145] Cytotoxicity assay. To assess CD8 + CD161 + cells relative to CD8 + CD161 neg The cytotoxic capacity of cells and bulk PBMCs was assessed in vitro using a short-term chromium-based cytotoxicity assay. + CD161 + 、CD8 + CD161 neg Unmanipulated native PBMCs were freshly isolated from human peripheral blood products. 51 The isolated cells were immediately tested for their cytotoxic capacity in a four-hour killing assay using Cr-labeled allogeneic 293-HEK targets at T cell:target cell ratios of 5:1, 25:1, and 50:1. Cell lysis was determined by chromium release into the culture medium and read using a Wizard2 gamma counter (Perkin Elmer).

[0146] CD8 + CD161 + Cell culture conditions in vitro. CD8 + CD161 + cells, CD8 + CD161 negCells and native PBMCs were stimulated with plate-bound anti-CD3 / CD28 and expanded in a cytokine cocktail containing 10 ng / ml IL-7, 5 ng / ml IL-15, and 30 ng / ml IL-21 (all from Peptek, Rocky Hill, NJ). + CD161 + Cells are separated from healthy donors' apheresis products and are cultured for stimulation of each of the anti-CD3 / CD28 / Clec2d (anti-human CD3-eBioscience cat #16-0037-8, anti-human CD28 from BD Biosciences cat #555725, recombinant human Clec2d, Novus Biologicals cat #NBP2-22966) of 1 ug / mL, and amplified (all from the Pipetech company in Rocky Hill, New Jersey) in a cytokine mixture comprising 10 ng / ml IL-7, 5 ng / ml IL-15, and 30 ng / ml IL-21. The cells are placed in a humidified chamber at 37°C for 48 hours. After 48 hours, in the absence of antibody stimulation, the cells are amplified with IL7 / 15 / 21 cytokine mixture.

[0147] Statistical analysis. Unless otherwise stated, significant differences were determined by two-way analysis of variance (ANOVA) or one-way ANOVA with Bonferroni post hoc test for multiple comparisons. Kaplan–Meier survival significance was determined by the log-rank (Mantel-Cox) test. Unless otherwise stated, all data are shown as mean ± SEM, and all analyses were performed using Prism software (GraphPad Software). Statistical significance was defined as p ≤ 0.05.

[0148] Example 2 - Results

[0149] T cell profiling after chemoimmunotherapy for PDAC identified CD3 + CD8 + NK1.1 + Previous work has shown that very small numbers (<1,500 per mouse) of splenic CD8 + NK1.1 +The cells can still provide rapid and robust antitumor protection against the parental PDAC cell line in a metastatic disease model (Konduri et al., 2016). + CD3 + CD8 + Key functional characteristics of T cell subsets, the inventors implanted orthotopically G12D / p53 - / - Two months after treatment and cure, the CD8 + Splenocytes were negatively selected and subdivided into NK1.1 + fractions and NK1.1 neg These fractions were then co-cultured with mature DCs loaded with PDAC overnight, and PDAC antigen-specific cells were identified and isolated by upregulating CD69 expression. Microarray showed that at a univariate significance level of 0.1, PDAC antigen-specific cells were significantly upregulated in CD8 + NK1.1 + cells and CD8 + NK1.1 neg 1642 genes differentially regulated between cells ( Figure 1 Although many different pathways may be affected (Table 1), the most striking differences were found in the cytolytic granzyme serine proteases, particularly the atypical granzyme isoforms F, D, G, and C, and innate-like cytotoxicity receptors (Table 2). These results suggest that CD8 + NK1.1 + cells expressed CD8 with significantly enhanced cytolytic capacity + T cell population.

[0150] Table 1: Top up-regulated and down-regulated genes.

[0151]

[0152]

[0153]

[0154] Table 2: Fold changes and P values ​​for genes grouped into the granzyme pathway and killer cell-like receptor subfamily pathway

[0155]

[0156]

[0157] NK1.1 identifies key circulating memory T cell populations in multiple mouse disease models. To verify that NK1.1 can identify similar key populations of cytolytic memory cells in a model-independent manner, the inventors 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 harvested and CD8 + Non-adherent cells were isolated into NK1.1 cells by positive selection. + fractions and NK1.1 neg After fractionation, 5x10 5 cells / mouse were adoptively transferred into a naive cohort that was lethally challenged with the same influenza virus strain 24 hours after adoptive transfer ( Figure 7A ). Body weight was recorded as an indicator of recovery and survival was determined by Kaplan-Meier assay. + NK1.1 + The adoptive cell transfer cohort fully recovered body weight and survived infection, whereas those using CD8 + NK1.1 neg The adoptive cell transfer cohort lost weight and showed a similar response to that of naive CD8 + The control group of adoptively transferred splenocytes died at the same rate (Figure 2A-B). Analysis of PBMCs 7 days after infection showed that the primary and CD8 + NK1.1 neg Compared with the adoptive transfer cohort, the + NK1.1 + In mice with circulating CD3 + CD8 + IFN-γ + cells (p<0.003) increased by 40% (Fig. 2C).

[0158] In the second model system, 2x10 5 Cohorts of donor mice were inoculated subcutaneously with B16 melanoma cells and vaccinated with B16-loaded cell-based vaccines on days 7 and 14 post-inoculation. On day 21, mice were sacrificed and splenocytes were harvested and sorted for CD8 + NK1.1 + Cell populations and CD8 + NK1.1 neg Then use 1.5x10 6 CD8 + NK1.1 + cells or CD8+ NK1.1 neg Cells were adoptively transferred into naive cohorts inoculated with palpable B16 tumors ( Figure 7B ). Accept CD8 + NK1.1 + Mice receiving CD8 + NK1.1 neg The survival of the cohort of cells adoptively transferred with naive splenocytes was the same as that of the control cohort (Figure 2D-E). Analysis of peripheral blood lymphocytes showed that the survival of the cohort of cells adoptively transferred with CD8 + NK1.1 neg Compared with the cohorts adoptively transferred with CD8 + NK1.1 + GP100 tetramer-specific CD8 + In the NK1.1 cell line, the levels of memory markers CD62L and CCR7 were significantly increased (Figure 2F). These results indicate that the CD161 homolog NK1.1 defines the major CD8 + Memory cell population.

[0159] Mouse CD3 + CD8 + NK1.1 + Human CD3 + CD8 + CD161 + Phenotypic conservation among counterparts. + NK1.1 + To stimulate protective memory responses provided by cells in various systems, the inventors next interrogated the role of memory CD8 + Do T cell subsets in the peripheral circulation have similar CD3 + CD8 + CD161 + The cell population is phenotypically and transcriptionally conserved across human populations. For this analysis, CD8 + CD161 + and CD8 + CD161 neg Cells were differentially isolated from six different human donors. CD161 was validated by TCR-Vβ spectral typing. + Cells are polyclonal ( Figure 8) were followed by transcriptional profiling of each population by microarray analysis. Despite the fact that these cells had not been activated and were in a steady-state resting state before analysis, the profiles of upregulated granzymes and natural cytotoxicity receptors were recapitulated in these cells at a univariate significance level of 0.1 ( Figure 3 , Table 3). Cross-species gene comparison analysis between activated mouse cells and non-activated human cells identified a conserved signature of 206 genes with a common nomenclature that were differentially regulated in the two populations ( Figure 9 Reactome pathway analysis of upregulated human genes identified genes with expression of <5x10 -4 The FDRs were used to characterize differentiation and regulation-related features, including HDAC deacetylation, DNA and histone methylation, nucleosome assembly, RNA polymerase I promoter escape, transcriptional regulation by small RNAs, and RNA gene silencing.

[0160] Table 3: Mouse CD8 + NK1.1 + The phenotypic characteristics of CD8 + CD161 + The quiescent phase of cells is recapitulated, with elevated expression of granzyme and killer lectin-like receptor genes.

[0161]

[0162]

[0163] Development of a model system for CAR T cell therapy of PDAC. Based on its potential for novel biology, the inventors hypothesized that human CD8 + CD161 + Subpopulations may provide more functional and durable antitumor efficacy than conventional bulk PBMCs in the context of solid tumor CAR T cell therapy.

[0164] Using IL7 / 15 / 21 to detect CD8 + CD161 + The combination of ex vivo expansion of cells and stimulation with plate-bound anti-CD3 / CD28 / Clec2d enhances the central memory phenotype (CD45RA - CCR7 + ). CD8 + CD161 + Cells were sorted from normal donors and ex vivo stimulation conditions were optimized. The combination of IL7 / 15 / 21 and plate-bound anti-CD3 / CD28 / Clec2d stimulation resulted in central memory (CD45RA) activation compared to IL2, IL-2 / 7 / 15, and IL2 / 7 / 15 / 21 stimulation. - CCR7+ ) was significantly upregulated ( Figure 5 ).

[0165] The combination of ex vivo expansion of CD8+CD161+ cells with IL7 / 15 / 21 and stimulation with plate-bound anti-CD3 / CD28 / Clec2d enhances cytotoxic granzyme production. + CD161 + Cells were sorted from normal donors and ex vivo stimulation conditions were optimized. The combination of IL7 / 15 / 21 and plate-bound anti-CD3 / CD28 / Clec2d stimulation resulted in a significant upregulation of cytotoxic molecules, granzymes, and perforin compared to IL2, IL-2 / 7 / 15, and IL2 / 7 / 15 / 21 stimulation ( Figure 6 ).

[0166] CD8 + CD161 + To assess the potential of CD8 + CD161 + cells relative to CD8 + CD161 neg The cytotoxic capacity of cells and bulk PBMCs was assessed in vitro using a short-term chromium-based cytotoxicity assay. + CD161 + 、CD8 + CD161 neg Unmanipulated native PBMCs were freshly isolated from human peripheral blood products. 51 The isolated cells were immediately tested for their cytotoxicity in a four-hour killing assay using Cr-labeled allogeneic 293-HEK targets. Figure 4 As shown, CD8 + CD161 + cells could induce 100% target lysis at an E:T ratio of 25:1, while native PBMC and CD8 + CD161 neg The cells showed 22% and 15% lytic capacity 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). These data indicate that CD8 + CD161 + T cells have enhanced cytotoxicity, and in CD8 + CD161 neg or absent in bulk PBMC counterparts.

[0167] Example 3 - Discussion

[0168] Lymphocytes are classified into different subsets and lineages based on the expression of surface molecules and secreted cytokines. However, classification is dynamic, with new cell subsets occasionally being identified that express markers from previously identified cell subsets and lineages. One such surface molecule is CD161, known to be expressed on NK cells, NKT cells, and other T cell lineages (Fergusson et al., 2011). CD161 shares 47% homology with its murine counterpart NK1.1 and is expressed by up to a quarter of peripheral T cells (Neelapu et al., 1994). Since NK-T cells account for less than 1% of peripheral T cells, CD3 + CD161 + The cells represent a different lineage of T cells, as they account for more than 5% of circulating T cells (Takahashi et al., 2006). + On T cells, CD161 expression was defined as moderate or high, whereas on CD4 T cells expressing CD161 + There is no such distinction between T cells (Takahashi et al., 2006). + CD161 high cells have been previously defined as MAIT cells (Martin et al., 2009; Goldfinch et al., 2010), Tc 17 cells (Northfield et al., 2008; Billerbeck et al., 2010), or memory stem cells (Turtle et al., 2009). Transcriptional profiling of cells with different CD161 expression profiles identified cells enriched for CD8 + CD161 + Conserved CD161 on T cells ++ / MAIT cell transcriptional signature, which can be extended to CD4 + CD161 + and TCRγδ + CD161 + T cells (Fergusson et al., 2014). In addition, populations expressing CD161 T cells share an innate-like TCR-independent response to interleukin (IL)-12 plus IL-18. This response is independent of the regulation of CD161, which acts as a co-stimulatory molecule in the context of T cell receptor stimulation. Therefore, the expression of CD161 identifies transcriptional and functional phenotypes that are shared across human T lymphocytes and are independent of both T cell receptor (TCR) expression and cell lineage. CD8 + CD161 + cells and CD4 + CD161 +The role of CD8 T cells during viral infection (Northfield et al., 2008; Billerbeck et al., 2010; Rowan et al., 2008) and in autoimmune diseases (Annibali et al., 2011; Cosmi et al., 2008; Kleinschek et al., 2009) has been defined, but to date, CD8 + CD161 + The role of CD8 in cancer biology is not yet clearly defined. In the current study, the inventors set out to understand + CD161 + Biological and functional properties of cells.

[0169] The inventors have previously reported that CD161 + cells, CD8 + NK1.1 + The functional significance of these cells is confirmed by their mouse counterparts, and the numbers of these cells have been found to increase under conditions that simulate viral infection (Konduri et al., 2016).

[0170] Mouse CD8 + NK1.1 + Microarray analysis of cells revealed that CD8 + NK1.1 neg Compared with their counterparts, these cells showed a significant upregulation of granzyme production after antigen stimulation. Innate genes and pathways that play a role in cytotoxic function were differentially expressed. + The human equivalents also constitutively express the cytotoxic mediators granzyme B and perforin. In contrast, a quarter of the cells lacking CD161 expression were naive CD8 + T cells, and even in the memory population express lower levels of granzyme B and perforin (Neelapu et al., 2018). CCR4 and CCR6 are expressed in CD8 + CD161 + Expression on cells indicates their ability to maintain tissue residency and homing to different organs. 高 Similar expression patterns were observed in CD8 T cells, thereby enhancing their entry into the CNS and contributing to pathogenesis (Annibali et al., 2011). The inventors found that resting CD8 + CD161 neg The cells also expressed higher levels of CXCR3, which led to CD8 + Effector memory markers are where T cells differentiate into short-lived effectors with limited memory potential (Kurachi et al., 2011).

[0171] Although targeting CD19 + CAR-T cell therapy is effective in hematological malignancies, but it is ineffective in targeting solid tumors (Neelapu et al., 2016; Abken, 2015). A major challenge is to overcome the inhibitory signaling of Tregs and enhance effector and memory functions (Klebanoff et al., 2012). Enhancing the persistence of effector and memory T cells could lead to highly effective CAR-T cell therapy. In preclinical models, CD8 + Subpopulations and CD4 + Both subpopulations expressed synergistic anti-tumor CAR-T activity (Sommermeyer et al., 2016). Similar results were observed in preclinical mouse experiments, where engineered CD4 + and CD8 + Combinations of T cells induce potent tumor rejection (Moeller et al., 2005; Shedlock and Shen, 2003). Recent clinical trial data on patients with non-Hodgkin lymphoma and chronic lymphocytic leukemia suggest that CD8 T cells are involved in the + and CD4 + High anticancer activity of CD19-CAR-T cells generated by a combination of T cell subsets that were expanded separately in vitro and infused at a 1:1 ratio (Turtle et al., 2016a). Similar results were obtained in a clinical trial of patients with B-cell acute lymphoblastic leukemia (Turtle et al., 2016b). Another clinical study of patients with high-risk intermediate-stage B-lineage non-Hodgkin's lymphoma treated with the following demonstrated the feasibility and safety of these two approaches: using isolated CD8 + T CM The first generation of CD19-CAR-T or CD8 + and CD4 + T CM Second-generation CD19-CAR-T therapy for both subpopulations (Turtle et al., 2016c) despite CD4 + and CD8 + T CM The CAR-T group and the second-generation CAR-T cells showed better persistence. These studies highlight the need to evaluate different subsets of T cells and lymphocytes in CAR-T cell therapy. Lymphocyte subsets with inherent 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 +Cells were previously defined as effector memory phenotypes, with less than 1% expressing CD161 高 CD8 + CD45RA neg Cells express the central memory marker CD62L + CCR7 + (Takahashi et al., 2006). According to previous reports, CD161-negative cells did not change CD161 expression by anti-CD2, anti-CD3, or anti-CD28 stimulation, and influenza-specific cells did not express CD161 after restimulation, even in the presence of cytokines (Northfield et al., 2008), suggesting that CD161 is not just a marker of activation but defines different lineages.

[0172] The bulk PBMC preparations commonly used for CAR T cell generation represent a heterogeneous group of cells that also contain highly differentiated antigen-responsive subpopulations. n ) subpopulations, stem cell memory (T scm ) subpopulations and central memory (T cm ) subsets lead to more potent anti-tumor responses (Wang et al., 2011; Berger et al., 2008; Gattinoni et al., 2011; Gattinoni et al., 2005). Less differentiated cells may be more beneficial; however, 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 has been shown to benefit lymphocyte development, differentiation, and homeostasis during ex vivo expansion of T cells and to improve in vivo survival compared to IL-2-expanded CAR-T cells (Xu et al., 2014; Rochman et al., 2009). Some studies have shown that the use of IL-7 and IL-15 together can preserve T scm phenotype and enhance the efficacy of CAR-T cells (Rochman et al., 2009; Cieri et al., 2013). Ex vivo expansion of CD3 / CD28-CAR-T in the presence of IL-7 and IL-15 enhances effector activity while retaining stem / memory potential against GD2 tumor antigens (Gargett et al., 2015). It has also been shown that CAR-T cells expanded with IL-15 retain the stem cell memory phenotype (CD62L + CD45RA + CCR7 + ). IL-15 also reduces the expression of exhaustion markers and increases proliferation after antigen challenge (Alizadeh et al., 2019). Others have shown that IL-21 promotes CD2 +CD28 + CD8 + T cell amplification (Santegoets et al., 2013) and enhance the efficacy of CD19-CAR-T (Rosenberg, 2014). It has been previously reported that the addition of IL-15 and IL-21 helps to enhance and maintain the memory potential of NKT cells (Ngai et al., 2018). It has been reported that a combination of lymphocyte ex vivo expansion based on IL-7, IL-15 and IL-21 enhances memory cells, reduces metastasis and improves survival against murine melanoma (Zoon et al., 2015). In the present study, the inventors found that ex vivo culture and expansion of bulk T cells with a mixture of IL-7, IL-15 and IL-21 mixtures is mainly beneficial to CD8 + CD161 + The cell population did not change significantly in the presence of either native PBMC or CD8 + CD161 neg Cell phenotype.

[0173] In summary, the inventors report that CD8 + CD161 + cells and their mouse equivalents CD8 + NK1.1 + Cells exhibited unusually high cytotoxic potential. Gene expression profiling by microarray revealed that NK1.1 neg counterparts and CD161 neg Compared to their counterparts, these cells expressed enhanced levels of granzymes, perforins, and innate-like receptors upon activation. + CD161 + The killing efficiency of T cells is higher than that of native PBMC or CD8 + CD161 neg Exploiting this subpopulation for T cell-based therapies offers exciting new opportunities for the effective treatment of solid tumors encompassing PDAC.

[0174] ***

[0175] According to the present disclosure, all methods disclosed and claimed herein can be prepared and performed without undue experimentation. Although the compositions and methods of the present disclosure have been described in accordance with preferred embodiments, it will be apparent to those skilled in the art that the steps or sequence of steps of the methods and methods described herein can be changed without departing from the concept, spirit and scope of the present disclosure. More specifically, it will be apparent that certain agents related to chemistry and physiology can replace the agents described herein while achieving the same or similar results. It will be apparent to those skilled in the art that all such similar substitutions and modifications are considered to be within the spirit, scope and concept of the present disclosure as defined by the appended claims.

[0176] XI. References

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Claims

1. A CD8 + CD161 + Use of a composition of T cells for the preparation of a medicament for providing a T cell response in a human subject suffering from a disease, wherein the CD8 + CD161 + T cell populations have upregulated granzyme and perforin expression, and wherein the CD8 + CD161 + T cells are cultured using an ex vivo method comprising: (a) obtaining a sample of cells, said sample comprising CD8 + CD161 + T cells; and (b) culturing the T cells in the presence of IL-7, IL-15, IL-21, a CD3 binding antibody, a CD28 binding antibody, and Clec2d, This provides a comparison of CD8 T cells cultured in the absence of IL-7, IL-15, IL-21, CD3 binding antibodies, CD28 binding antibodies, and Clec2d. + CD161 + CD8 cells with upregulated expression of granzymes and perforin + CD161 + T cell population.

2. The use according to claim 1, wherein the disease is cancer.

3. The use according to claim 2, wherein the subject has undergone previous anti-cancer therapy.

4. The use according to claim 3, wherein the subject is in remission or has no symptoms of the cancer but comprises detectable cancer cells. The use according to claim 1 , wherein Clec2d is present in the culture medium at 0.1 to 5.0 μg / ml.

6. The use according to claim 1, wherein the ex vivo method further comprises: (c) culturing the T cells in the presence of IL-7, IL-15 and IL-21 without antibody stimulation.

7. The method according to claim 5, wherein the culturing step (c) does not contain CD3 binding antibodies, CD28 binding antibodies, Clec2d and / or CD161 binding antibodies.

8. The use according to claim 1, wherein the culturing of step (b) lasts for 12 to 72 hours.

9. The use according to claim 6, wherein the culturing of step (b) lasts for at least 12 hours.

10. The use according to claim 6, wherein the culturing step (c) does not contain CD3 binding antibodies and CD28 binding antibodies.

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

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

13. The use according to claim 1, 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.

14. The use according to claim 1, wherein IL-7 is present at 10 ng / ml, IL-15 is present at 5 ng / ml, and IL-21 is present at 30 ng / ml.

15. The use according to claim 1, further comprising, before step (b), purifying or enriching the CD8 + CD161 + cells of T cells.

16. The use according to claim 1, further comprising, after step (b), purifying or enriching the CD8 + CD161 + cells of T cells. The use according to claim 15 , wherein enriching T cells in the sample comprises fluorescent cell sorting or magnetic bead separation. The use according to claim 16 , wherein enriching T cells in the sample comprises fluorescent cell sorting or magnetic bead separation.

19. The use according to claim 1, wherein the culturing is continued for at most 7, 14, 21, 28, 35 or 42 days.

20. The use according to claim 1, wherein the culturing is performed in a medium containing serum.

21. The use according to claim 1, wherein the culturing is performed in a serum-free medium.

22. The use according to claim 1, wherein the sample is a frozen sample.

23. The use according to claim 1, wherein the sample is from umbilical cord blood.

24. The use according to claim 1, wherein the sample is a peripheral blood sample from a subject.

25. The use according to claim 1, wherein the sample is obtained by apheresis.

26. The use according to claim 1, wherein the sample is obtained by venipuncture.

27. The use according to claim 1, wherein obtaining the sample comprises obtaining the sample from a third party.

28. The use according to any one of claims 1 to 26, wherein the CD8 + CD161 + T cells expressing chimeric antibody receptors (CARs).

29. The use according to claim 27, wherein the CD8 + CD161 + T cell expression of endogenous T cell receptors and / or endogenous HLA is inactivated.

30. The use according to claim 1, wherein the sample comprises CD8 + CD161 + The percentage of cells is increased in T cell subsets.

31. The method of claim 1, further comprising evaluating the CD8 + CD161 + T cell content.

32. The use according to claim 28, wherein said assessment is by cell counting or flow cytometry.

33. A CD8 + CD161 + A T cell composition prepared by the following method: CD8 cells were cultured in the presence of IL-7, IL-15, IL-21, CD3-binding antibody, CD28-binding antibody, and Clec2d. + CD161 + T cells, This provides a comparison of CD8 T cells cultured in the absence of IL-7, IL-15, IL-21, CD3 binding antibodies, CD28 binding antibodies, and Clec2d. + CD161 + CD8 cells with upregulated expression of granzymes and perforin + CD161 + T cell population.

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