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

Abstract

The present disclosure provides methods for expanding CD161+ T cells. Also provided are methods and compositions for generating modified CD161+ T cells comprising a chimeric antigen receptor (CAR). In certain aspects, CAR-expressing T cells are generated, expanded, and / or used for treating diseases (e.g., cancer).

Description

[0001] Cross - Reference to Related Applications

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

[0003] Statement Regarding Federally Sponsored Research

[0004] This invention was made with government support under Grant No. AI127387 awarded by the National Institutes of Health. The government has certain rights in the invention. FIELD OF THE INVENTION

[0005] The present disclosure generally relates to the fields of medicine, immunology, cell biology, and molecular biology. In certain 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 treatment using such cells. BACKGROUND OF THE INVENTION

[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 great potential as a form of cancer treatment, particularly for the selection of CD19 + malignancies (Maude et al., 2018; Neelapu et al., 2017). The CAR construct consists of a single-chain variable fragment (scFv) targeting a cell surface tumor antigen, a transmembrane domain, a hinge region, and an intracellular signaling domain of CD3ζ that is typically fused to those 4-1BB or CD28 costimulatory molecules (van der Stegen et al., 2015). In a phase I clinical trial, adoptive cell therapy with autologous mesothelin-specific CAR-T cells showed safety and moderate efficacy in a small subset of patients with chemotherapy-refractory metastatic human PDAC (Beatty et al., 2018); however, CART cell therapy for pancreatic tumors remains slow to progress. In fact, currently, few CAR-based therapies have shown any significant efficacy in the solid tumor setting.

[0007] The key property of cellular regulatory immunity against viral infection lies in the establishment of a population of memory T cells with long-term survival, which provides persistent immunity to subsequent challenges through accelerated expansion and cytotoxic kinetics (Seaman et al., 2004). Several groups have previously identified interesting subsets 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. Compared with TCR invariant or CD8αα + CD161 + 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 specificities (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 referred to as an 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 + cells contribute to tissue pathogenesis during chronic viral infections and autoimmune conditions due to tissue residency properties and / or extravasation tendencies (Assarsson et al., 2000; Billerbeck et al., 2010; Annibali et al., 2011). Further, high expression levels of CD161 in tumor-resident immune infiltrates are associated with significantly improved clinical outcomes and survival rates in NSCLC (Braud et al., 2018). Summary of the Invention

[0008] In a first embodiment, there is provided an in vitro or ex vivo method, the method comprising: (a) obtaining a sample of cells, the sample comprising CD161 + T cells; and (b) culturing the T cells in the presence of IL-7, IL-15, and IL-21, thereby providing a number of CD161 + cells that is compared to non-CD161 +A population of T cells with an expanded number of cells. In some aspects, the T cells include CD8 + CD161 + T cells. In other aspects, the T cells include CD4 + CD161 + T cells.

[0009] IL-7 can be present at about 5 - 20 ng / ml, IL-15 can be present at about 2.5 - 10 ng / ml and / or IL-21 can 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 can further include, prior to step (b), purifying or enriching the T cells present in the sample that are CD8 + CD161 + cells. The method can further include, after step (b), purifying or enriching the T cells present in the sample that are CD8 + CD161 + cells. Enriching the T cells in the sample can include fluorescence-activated cell sorting, magnetic bead separation or paramagnetic bead separation. The culture can be continued for up to 7 days, 14 days, 21 days, 28 days, 35 days or 42 days.

[0010] In some aspects, the cells are further cultured in a medium comprising a CD3 and / or CD28 stimulator. In some aspects, the CD3 and / or CD28 stimulator includes a CD3 and / or CD28 binding antibody. In some aspects, the cells are further cultured in a medium comprising a CD3, CD28 and / or CD161 stimulator. In some aspects, the CD3, CD28 and / or CD161 stimulator includes a CD3, CD28 and / or CD161 binding antibody. In some aspects, the cells are further cultured in a medium comprising a CD3 binding antibody, a CD28 binding antibody, Clec2d and / or a CD161 stimulatory antibody. In some aspects, the cells are further cultured in a medium comprising about 0.1 to 5.0, 0.3 to 3.0 or 0.5 to 2.0 μg / ml of a CD3 binding antibody, a CD28 binding antibody, Clec2d and / or a CD161 stimulatory antibody.

[0011] In one aspect, CD8 + CD161 + cells, CD8 + CD161 negCells and bulk PBMCs were stimulated with plate-bound anti-CD3 / CD28 and expanded in a cytokine mixture 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 and cultured and expanded in RPMI-1640, 10% FBS, and 2 mmol / l GlutaMAX in a cytokine mixture containing 10 ng / ml IL-7, 5 ng / ml IL-15, and 30 ng / ml IL-21. The cells were placed in a humidified chamber at 37°C for 48 hours. After 48 hours, the cells were expanded with the IL7 / 15 / 21 cytokine mixture in the absence of antibody stimulation.

[0012] The method can further include obtaining the cells from a subject, such as by apheresis or venipuncture. The sample can be a cryopreserved sample. The sample can be from umbilical cord blood. The sample can be a peripheral blood sample from the subject. The sample can include a T cell subset with an increased percentage of CD8 + CD161 + cells compared to a comparable sample obtained from the subject. The sample can be obtained from a third party.

[0013] The method can further include introducing a nucleic acid encoding a CAR into the T cells in the sample, such as with a viral vector or by a method that does not involve viral transduction of the T cells. Introduction of the nucleic acid encoding a CAR or transgenic TCR into the T cells can occur before step (b) or after step (b). The T cells expressing an endogenous T cell receptor and / or endogenous HLA can be inactivated.

[0014] The method can further include introducing a nucleic acid encoding a membrane-bound Cγ cytokine into the T cells, such as where the membrane-bound Cγ cytokine is membrane-bound IL-15. The membrane-bound Cγ cytokine can be an IL-15-IL-15Rα fusion protein.

[0015] The culturing can include culturing the T cells in the presence of dendritic cells or artificial antigen-presenting cells (aAPCs). The aAPCs can include a CAR-binding antibody or a transgenic TCR-binding antibody or a fragment thereof expressed on the surface of the aAPC. The aAPCs can include additional molecules that activate or co-stimulate the T cells. The additional molecules can include membrane-bound Cγ cytokines. The culturing of the T cells in the presence of aAPCs can include culturing the cells at a (CAR cells to aAPCs) ratio of from about 10:1 to about 1:10.

[0016] The method can further include cryopreserving a sample of the transgenic CAR cell population or the transgenic TCR cell population. The CAR or transgenic TCR can target a cancer cell antigen, such as CD19, CD20, ROR1, CD22 carcinoembryonic antigen, α-fetoprotein, CA-125, 5T4, MUC-1, epithelial tumor antigen, prostate-specific antigen, melanoma-associated antigen, mutant p53, mutant ras, HER2 / Neu, folate-binding protein, HIV-1 envelope glycoprotein gp120, HIV-1 envelope glycoprotein gp41, GD2, CD123, CD33, CD138, CD23, CD30, CD56, c-Met, 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 a pathogen antigen, such as a fungal, viral, or bacterial pathogen. The pathogen can be Plasmodium, Trypanosoma, Aspergillus, Candida, HSV, HIV, RSV, EBV, CMV, JC virus, BK virus, or an Ebola pathogen.

[0017] The method can further include assessing the CD8 + CD161 + cell content of the sample, such as by cell counting / flow cytometry, before step (b), after step (b), or before and after step (b).

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

[0019] Another embodiment relates 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 a prior anti-cancer therapy. The subject may be in remission or have no symptoms of the cancer, but includes detectable cancer cells.

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

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

[0022] Figure 1 . Gene expression analysis of a microarray of T cells stimulated by antigenic stimulation showed a significant upregulation of the cytotoxicity and innate-like properties of CD8 + NK1.1 + cells. A cohort of 15 mice received a combination of chemotherapy for murine pancreatic ductal adenocarcinoma with dendritic cell-based vaccination. Sixty days after tumor inoculation, spleens were harvested, pooled into three groups of five each, and activated overnight with dendritic cells loaded with tumor antigen. Then, antigenically stimulated T cells were sorted into NK1.1 + CD69 + populations by gating on the CD8 neg and NK1.1 + subpopulations by flow cytometry. The volcano plot shows 1642 genes significantly regulated between CD8 + NK1.1 neg cells and CD8 + NK1.1 + cells at a univariate significance level of 0.1. The top 15 genes differentially regulated at an FDR of 0.05 are marked on the graph.

[0023] Figures 2A - F . CD8 + NK1.1 +Cells defined a memory population that provided durable protection and improved survival against influenza infection and melanoma tumors. In the influenza model, splenocytes were harvested from mice after recovery from influenza infection and sorted into CD8 + NK1.1 neg and CD8 + NK1.1 + cells, and adoptively transferred into naïve mice that were subsequently challenged with influenza. In the melanoma model, tumor-bearing mice were vaccinated with dendritic cells loaded with tumor antigen. After three weeks, splenocytes were harvested and sorted into CD8 + NK1.1 neg and CD8 + NK1.1 + cells, and adoptively transferred into mice with palpable tumors. Adoptive transfer of CD8 + NK1.1 + cells that had experienced antigen provided durable protection against influenza infection (Figure 2A-C) and melanoma (Figure 2D-F). (Figure 2A) Mice receiving CD8 + NK1.1 neg and naïve CD8 + cells regained their body weight after influenza infection compared to mice receiving CD8 + NK1.1 + cells. (Figure 2B) A hundred percent survival rate was observed in mice receiving CD8 + NK1.1 neg cells compared to the group receiving CD8 + cells and naïve CD8 + NK1.1 + cells. (Figure 2C) Analysis of PBMCs two weeks after infection showed that circulating CD3 + NK1.1 neg cells increased by 40% (p<0.003) in mice receiving CD8 + NK1.1 + cells compared to the naïve and CD8 + CD8 + IFN-γ + adoptive transfer cohorts. (Figure 2D-E) In the melanoma model, mice receiving CD8 + NK1.1 + cells showed delayed tumor growth and improved survival. (Figure 2F) Analysis of peripheral blood lymphocytes three weeks after tumor transplantation showed that, compared to the cohorts adoptively transferred with CD8 + NK1.1 neg or naïve splenocytes, in the cohort adoptively transferred with CD8 + NK1.1+ GP100 tetramer - specific CD8 cells in the adoptive transfer cohort of cells + showed significantly elevated levels of the memory markers CD62L and CCR7. For each experiment, n = 10 mice per group. Error bars = + / - SEM, * p < 0.05, one - way ANOVA.

[0024] Figure 3 . Murine CD3 + CD8 + NK1.1 + cell populations are phenotypically conserved in the human CD3 + CD8 + CD161 + counterparts. CD3 + CD8 + CD161 + and CD3 + CD8 + CD161 neg cells, murine CD3 + CD8 + NK1.1 + cells and the human equivalents of CD3 + CD8 + NK1.1 neg cells were magnetically sorted from the peripheral blood of six human donors and analyzed for gene expression profiles by microarray. The volcano plot showing differential regulation of genes between CD8 + CD161 + cells and CD8 + CD161 neg cells highlights up - regulation of the CD161 receptor in the oval.

[0025] Figure 4 . CD8+CD161 +, CD8+CD161neg, and unmanipulated naïve PBMCs were freshly isolated from human peripheral blood products. The cytotoxic capabilities of the isolated cells were tested immediately using 51 Cr - labeled allogeneic 293 - HEK targets in a four - hour killing assay. As shown, CD8 + CD161 + cells could induce 100% target lysis at an E:T ratio of 25:1, while naïve PBMCs and CD8 + CD161 negCells exhibited lysis capabilities of 22% and 15% respectively at a maximum E:T ratio of 50:1 (by one-way ANOVA, p < 0.002 at 50:1, p < 0.0007 at 25:1, and p < 0.00002 at 5:1). X-axis - E:T ratio. Y-axis - percentage of killing. Error bars = + / - SD.

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

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

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

[0029] Figure 8 . TCR-Vβ spectratyping indicated that CD3 + CD8 + CD161 + cells were essentially polyclonal. To confirm the clonal nature of CD8 + CD161 + cells, TCR-Vβ spectratyping was performed on donor-derived cells. Histograms from 30 TCR Vβ families amplified 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 differentially regulated between the two populations. 206 common genes were identified between nomenclature-based mouse (15 pooled samples) and human (6 paired samples) microarray analyses. The expression patterns of these genes in activated CD8 + NK1.1+ Cells are similar to resting CD8 + CD161 + cells, indicating a highly conserved nature of the gene signature. DETAILED DESCRIPTION

[0031] As discussed above, CAR-T therapy shows great promise in the treatment of cancers such as metastatic murine ductal adenocarcinoma (PDAC). In past work, the inventors demonstrated that adoptive transfer of antigen-experienced CD8 + NK1.1 + cells can mediate durable protection in a PDAC model. Interestingly, these cells persisted for nine months after initial exposure to antigen and were highly protective when adoptively transferred into naive mice and subsequently challenged with the parental PDAC cell line (Konduri et al., 2016). Building on these results, the inventors sought to characterize 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 demonstrated that CD8 + CD161 + T cells represent an excellent platform for CAR T cell therapy if engineered to prevent differentiation of the starting cell population during transduction and expansion. 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 disclosure are shown in more detail below.

[0032] I. DEFINITIONS

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

[0034] The use of the term "or" in the claims is used to mean "and / or" unless explicitly indicated to refer to only alternatives or the alternatives are mutually exclusive, but the disclosure supports both only alternatives and the definition of "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 encompasses the inherent variation of error of the device, method for measuring the value, or variation that exists among subjects being studied or values within 10% of the stated value.

[0036] As used herein, the term "chimeric antigen receptor (CAR)" can refer to, for example, an artificial T cell receptor, chimeric T cell receptor, transgenic T cell receptor, or chimeric immune receptor, and encompasses engineered receptors that transplant artificial specificity onto specific immune effector cells. A CAR can be used to confer the specificity of a monoclonal antibody onto T cells, thereby allowing for the generation of large numbers of specific T cells, for example, for adoptive cell therapy. In a specific embodiment, for example, the CAR directs the specificity of the cell to a tumor-associated antigen. In some embodiments, the CAR includes an intracellular activation domain, a transmembrane domain, and an extracellular domain that includes a tumor-associated antigen binding region. In a particular aspect, the CAR includes a fusion of a single-chain variable fragment (scFv) derived from a monoclonal antibody fused to a CD3-ζ transmembrane domain and intracellular domain. The specificity of other CAR designs can be derived from a ligand of the receptor (e.g., a peptide) or from a pattern recognition receptor, such as Dectin. In some cases, the spacing of the antigen recognition domain can be modified to reduce activation-induced cell death. In some cases, the CAR includes domains for additional co-stimulatory signaling, such as CD3-ζ, FcR, CD27, CD28, CD137, DAP10, and / or OX40. In some cases, molecules can be co-expressed with the CAR, the molecules comprising co-stimulatory molecules, a reporter gene for imaging (e.g., for positron emission tomography), a gene product that conditionally ablates T cells upon addition of a prodrug, homing receptors, chemokines, chemokine receptors, cytokines, and cytokine receptors.

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

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

[0039] II. Chimeric Antigen Receptor

[0040] As used herein, the term "antigen" is a molecule capable of being bound by an antibody or a T cell receptor. An antigen is additionally capable of inducing a humoral immune response and / or a cellular immune response, resulting in the production of B lymphocytes and / or T lymphocytes.

[0041] Embodiments of the present disclosure relate to nucleic acids that comprise a nucleic acid encoding an antigen - specific chimeric antigen receptor (CAR) polypeptide, comprising a CAR that has been humanized to reduce immunogenicity (hCAR), including an intracellular signaling domain, a transmembrane domain, and an extracellular domain comprising one or more signaling motifs. In certain embodiments, the CAR can recognize an epitope that comprises a shared space between one or more antigens. Pattern recognition receptors, such as Dectin - 1, can be used to achieve specificity for glyco - antigens. In certain embodiments, the binding region can include complementarity - determining regions of a monoclonal antibody, variable regions of a monoclonal antibody, and / or antigen - binding fragments thereof. In another embodiment, the specificity is derived from a peptide (e.g., a cytokine) that binds to a receptor. Complementarity - determining regions (CDRs) are short amino acid sequences present in the variable domains of antigen receptors (e.g., immunoglobulins and T - cell receptors) that complement the antigen and thus provide the receptor with its specificity for the particular 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 in each heavy chain and three in each light chain. Since most of the sequence variability associated with immunoglobulins and T - cell receptors resides in the CDRs, these regions are sometimes referred to as hypervariable domains. Among these, CDR3 shows the greatest variability because it is encoded by the recombination of VJ (VDJ in the case of heavy chains and TCRαβ chains) regions.

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

[0043] The arrangement can be multimeric, such as diabodies or multimers. Multimers are most likely formed by cross - pairing the variable parts of the light and heavy chains into what has been called diabodies by Winters. The hinge part of the construct can have various alternatives, from complete deletion to maintaining the first cysteine, to proline substitution instead of serine substitution, to truncation until the first cysteine. The Fc part can be absent. Any stable and / or dimerizing protein can serve this purpose. Only one of the Fc domains can be used, for example, the CH2 or CH3 domain from human immunoglobulin. The hinge region, CH2 region, and CH3 region of human immunoglobulin that have been modified to improve dimerization can also be used. Only the hinge part of the 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 cells in which the chimeric receptor has been placed. The term "effector function" refers to the specialized functions of differentiated cells. For example, the effector functions of T cells can be cytolytic activity or helper activity, including the secretion of cytokines. The effector functions in naive, memory, or memory - like T cells include antigen - dependent proliferation. Thus, the term "intracellular signaling domain" refers to the part of a protein that transduces the effector function signal and directs the cell to perform specialized functions. Although the entire intracellular signaling domain will usually be employed, in many cases, it is not necessary to use the entire intracellular polypeptide. To the extent that truncated portions of the intracellular signaling domain can be found to be used, such truncated portions can be used in place of the full - length chain as long as they still transduce the effector function signal. Thus, the term intracellular signaling domain is intended to include any truncated portion of the intracellular signaling domain that is sufficient to transduce the effector function signal. Examples include the ζ - chain of the T - cell receptor or any of its homologs (e.g., η, δ, γ, or ε), the MB1 chain, B29, Fc RIII, Fc RI, and combinations of signaling molecules such as CD3ζ and CD28, CD27, 4 - 1BB, DAP - 10, OX40, and their combinations, and other similar molecules and fragments. The intracellular signaling parts of other members of the activation 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 linked by a transmembrane domain, such as the hinge region and Fc region of human IgG4Fc. Alternatives include the human CD4 transmembrane domain, the human CD28 transmembrane domain, the transmembrane human CD3ζ domain, or the cysteine - mutated human CD3ζ domain, or other transmembrane domains from other human transmembrane signaling proteins, such as CD16, CD8, and the erythropoietin receptor.

[0046] In some embodiments, the CAR nucleic acid includes sequences encoding other co-stimulatory receptors, such as transmembrane domains and modified CD28 intracellular signaling domains. Other co-stimulatory receptors include, but are not limited to, one or more of CD28, CD27, OX-40 (CD134), DAP10, and 4-1BB (CD137). In addition to the primary signal initiated by CD3ζ, the additional signals provided by human co-stimulatory receptors inserted in the human CAR are important for the full activation of T cells and can help improve in vivo persistence and the therapeutic success of adoptive immunotherapy.

[0047] In certain embodiments, the present disclosure relates to isolated nucleic acid fragments and expression cassettes incorporating DNA sequences encoding CARs. The vectors of the present disclosure are designed primarily for delivery of the desired gene to immune cells, preferably T cells under the control of a regulated eukaryotic promoter, e.g., the MNDU3 promoter, CMV promoter, EF1α promoter, or ubiquitin promoter. Moreover, the vector can contain selectable markers, if for no other reason, to facilitate its manipulation in vitro. In other embodiments, the CAR can be expressed from mRNA transcribed in vitro from a DNA template.

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

[0049] To date, non-human antigen-binding regions have generally been used to construct chimeric antigen receptors. A potential problem with non-human antigen-binding regions such as murine monoclonal antibodies is the lack of human effector functions and the inability to penetrate tumor masses. In other words, such antibodies may not be able to mediate complement-dependent lysis or cause lysis of human target cells by antibody-dependent cell cytotoxicity or Fc receptor-mediated phagocytosis to destroy cells expressing the CAR. In addition, non-human monoclonal antibodies can be recognized by the human host as foreign proteins, and thus, repeated injection of such foreign antibodies may lead to induction of an immune response, resulting in harmful hypersensitivity reactions. For murine-based monoclonal antibodies, this is generally referred to as the human anti-mouse antibody (HAMA) response. Thus, the use of human antibodies is more preferred because they do not elicit a strong HAMA response like murine antibodies. Similarly, the use of human sequences in CARs can avoid immunomodulatory recognition and thus avoid elimination by endogenous T cells resident in the recipient and recognition of 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 specific embodiments, the intracellular receptor signaling domain in the CAR comprises those of the T cell antigen receptor complex, such as the ζ chain of CD3, as well as the FcγRIII costimulatory signaling domain, CD28, CD27, DAP10, CD137, OX40, CD2, alone or in tandem with CD3ζ, for example. In specific embodiments, the intracellular domain (which may be referred to as the cytoplasmic domain) comprises a portion or all of one or more of 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 portion of the endogenous T cell receptor complex in the intracellular domain is employed. For example, one or more cytoplasmic domains may be employed because so-called third-generation CARs have at least two or three signaling domains fused together to produce additional or synergistic effects.

[0052] In certain embodiments of the chimeric antigen receptor, the antigen - specific portion of the receptor (which may be referred to as the extracellular domain comprising the antigen - binding region) comprises a tumor - associated antigen or a pathogen - specific antigen - binding domain, including a glycan antigen recognized by a pattern - recognition receptor, such as Dectin - 1. The tumor - associated antigen can be of any type as long as it is expressed on the cell surface of tumor cells. Exemplary embodiments of tumor - associated antigens include CD19, CD20, carcinoembryonic antigen, alpha - fetoprotein, CA - 125, MUC - 1, CD56, EGFR, c - Met, AKT, Her2, Her3, epithelial tumor antigen, melanoma - associated antigen, mutant p53, mutant ras, etc. In certain embodiments, the CAR can be co - expressed with a membrane - bound cytokine to enhance persistence when there is a low amount of tumor - associated antigen. For example, the 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 a CAR expressed on a universal T - cell that recognizes the processed peptide described according to the intracellular tumor - associated antigen in the context of HLA. Additionally, the universal T - cell can be genetically modified to express a T - cell receptor pair that recognizes the intracellularly processed tumor - associated antigen in the context of HLA.

[0054] The pathogen can be of any kind, but in a specific embodiment, the pathogen is, for example, a fungus, a bacterium, or a virus. Exemplary viral pathogens include Adenoviridae, 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, chickenpox, 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 (E. coli), Rickettsia, Bacillus, Bordetella, Chlamydia, Spirochete, and Salmonella. In one embodiment, the pathogen receptor Dectin-1 can be used to generate a CAR that recognizes the carbohydrate structure on the cell wall of fungi. T cells genetically modified to express a CAR based on Dectin-1 specificity can recognize Aspergillus and target hyphal growth. In another embodiment, the CAR can be manufactured based on antibodies that recognize viral determinants (e.g., glycoproteins from CMV and Ebola virus) to interfere with viral infection and pathology.

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

[0056] Chimeric immune receptors according to the present disclosure can be generated by any means known in the art, although preferably they are generated 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 region can be inserted into an expression vector and used to transform a suitable expressing host allogeneic T cell line.

[0057] As used herein, a 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 (e.g., a chimeric antigen receptor).

[0058] In an exemplary nucleic acid construct (polynucleotide) employed in the present disclosure, a promoter is operably linked to the nucleic acid sequence encoding the chimeric receptor of the present disclosure, i.e., the two are positioned such that it facilitates transcription of messenger RNA from the DNA encoding the chimeric receptor. The promoter can 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 can be constitutive or inducible, where induction is related to 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, SV40 early and late promoters, immunoglobulin promoters, human cytomegalovirus promoters, retroviral promoters, and Friend spleen focus-forming virus promoters. The promoter can be associated with an enhancer or can be unassociated with an enhancer, where the enhancer can be naturally associated with a particular promoter or associated with a different promoter.

[0059] The sequence of the open reading frame encoding 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). Moreover, it can be further advantageous to use endogenous or exogenous non-coding regions to stabilize the mRNA.

[0060] For the expression of the chimeric antigen receptors of the present disclosure, the naturally occurring or endogenous transcriptional start region of the nucleic acid sequence encoding the N-terminal component of the chimeric receptor can be used to produce the chimeric receptor in a target host. Alternatively, an exogenous transcriptional start region allowing constitutive or inducible expression can be used, where the expression can be controlled according to the target host, desired expression level, nature of the target host, etc.

[0061] Similarly, 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 the 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 generally not considered critical for recombinant protein expression, and a variety of termination regions can be employed without adversely affecting expression.

[0063] As will be understood by those skilled in the art, in some cases, several amino acids at the end of the antigen-binding domain in the CAR can be deleted, e.g., generally no more than 10 residues, more typically no more than 5 residues. Also, it may be desirable to introduce a small number of amino acids at the boundary, generally no more than 10 residues, more typically no more than 5 residues. The deletion or insertion of amino acids can occur as a result of the construction requirements, so as to provide convenient restriction sites, be easy to manipulate, improve the expression level, etc. Additionally, for similar reasons, the substitution of one or more amino acids with different amino acids can occur, generally substituting no more than about five amino acids in any one domain.

[0064] The chimeric construct encoding the chimeric receptor according to the present disclosure can be prepared in a conventional manner. Since in most cases natural sequences can be employed, the natural genes can be appropriately isolated and manipulated to allow for the proper ligation of the various components. Thus, using appropriate primers that result in the deletion of unwanted portions of the gene, the nucleic acid sequences encoding the N-terminal and C-terminal proteins of the chimeric receptor can be isolated by polymerase chain reaction (PCR). Alternatively, restriction digestion of the cloned gene can be used to generate the chimeric construct. In either case, the sequences can be selected to provide blunt ends or restriction sites with complementary overlaps.

[0065] The various manipulations for preparing chimeric constructs can be performed in vitro, and in certain embodiments, the chimeric constructs are introduced into a vector for cloning and expression in a suitable host using standard transformation or transfection methods. Thus, after each manipulation, the resulting construct from the DNA sequence ligation is cloned, the vector is isolated, and the sequence is screened to ensure that the sequence encodes the desired chimeric receptor. The sequence can be screened by restriction analysis, sequencing, etc.

[0066] The chimeric constructs of the present disclosure are applied to subjects having or suspected of having cancer by reducing the size of tumors or preventing the growth or regrowth of tumors in these subjects. Thus, 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 isolated T cells of a subject and reintroducing the transformed T cells into the subject, thereby affecting an anti-tumor response to reduce or eliminate tumors in the subject. Suitable T cells that can be used include cytotoxic lymphocytes (CTLs) or any cells having a T cell receptor that needs to be disrupted. As is well known to those skilled in the art, these cells can be readily isolated from a subject using various methods. For example, using cell surface marker expression or using a commercially available kit (e.g., ISOCELL from Pierce, Rockford, Ill.). TM )

[0067] It is contemplated that the chimeric constructs 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 DNA encoding the chimeric receptors of the present disclosure contained in a plasmid expression vector in a suitable orientation for expression. Advantageously, using naked DNA reduces the time required to generate T cells expressing the chimeric receptors of the present disclosure.

[0068] Alternatively, viral vectors (e.g., retroviral vectors, adenoviral vectors, adeno-associated viral vectors, or lentiviral vectors) can be used to introduce the chimeric constructs into T cells. Suitable vectors for use in the methods of the present disclosure are non-replicable in the subject's T cells. A large number of virus-based vectors are known, where the copy number of the virus maintained in the cell is low enough to maintain cell viability. Exemplary 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 can express the chimeric receptor as a surface membrane protein with desired regulation and at a desired level, it can be determined whether the chimeric receptor functions in the host cell to provide the desired signal induction. Subsequently, the transduced T cells are reintroduced into or administered to the subject to activate the anti-tumor response in the subject. To facilitate administration, the transduced T cells according to the present disclosure can be formulated into a pharmaceutical composition or an implant suitable for in vivo administration with a suitable carrier or diluent, which can further be pharmaceutically acceptable. The ways of formulating such compositions or implants have been described in the art (see, for example, Remington's Pharmaceutical Sciences, 16th edition, edited by Mack, 1980). In appropriate cases, the transduced T cells can be formulated into preparations in semi-solid or liquid form, such as capsules, solutions, injections, inhalants or aerosols, in a conventional manner for their corresponding routes of administration. Ways 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 organ, or to ensure the timed release of the composition. However, it is desired that a pharmaceutically acceptable form of the cells expressing the chimeric receptor is employed. Thus, it is desired that the transduced T cells can be formulated into a pharmaceutical composition containing a balanced salt solution, preferably Hanks' balanced salt solution or physiological saline.

[0070] III. Methods and Compositions Related to Examples

[0071] In certain aspects, the present disclosure encompasses a method of manufacturing and / or expanding antigen-specific CD8 + CD161 + T cells, the method 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 antigen or an antibody 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 expansion of CD8 + CD161 + T cells.

[0072] In another aspect, a method is provided 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. 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 a proper orientation for expression. The electroporation method of the present disclosure produces stable transfectants that express and carry the chimeric TCR (cTCR) on their surface.

[0073] "Chimeric TCR" means a receptor expressed by a T cell and comprising an intracellular signaling domain, a transmembrane domain, and an extracellular domain, wherein the extracellular domain is capable of specifically binding an antigen in an MHC-unrestricted manner, the antigen not normally being bound by the T cell receptor in such a manner. Stimulation of the T cell by the antigen under appropriate conditions results in cell proliferation (expansion) and / or production of IL-2. Exemplary chimeric receptors of the present application are examples of chimeric TCRs. However, the method is suitable for transfection with chimeric TCRs specific for other target antigens, such as chimeric TCRs specific for 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, alpha-fetoprotein, CD23, CD30, CD123, IL-11Rα, kappa chain, lambda chain, CD70, CA-125, MUC-1, EGFR and its variants, epithelial tumor antigen, and the like.

[0074] In some aspects, the T cells are primary human T cells collected after stimulation with G-CSF, bone marrow, or cord blood, such as T cells derived from human peripheral blood mononuclear cells (PBMCs). The conditions include the use of mRNA and DNA and electroporation. After transfection, the cells can be immediately infused or the cells can be stored. In some aspects, after transfection, the cells can be propagated ex vivo as a bulk population for days, weeks, or months within about 1 day, 2 days, 3 days, 4 days, 5 days, or longer after gene transfer into the cells. In additional aspects, after transfection, the transfectants are cloned and the clones are shown to have a single integrated or episomally maintained expression cassette or plasmid, and the expression of the chimeric receptor is amplified ex vivo. The clones selected for amplification are shown to have the ability to specifically recognize target cells. The recombinant T cells can be amplified by stimulation with IL-2 or other cytokines that bind to the common gamma chain (e.g., IL-7, IL-12, IL-15, IL-21, etc.). The recombinant T cells can be amplified by stimulation with artificial antigen-presenting cells. The recombinant T cells can be amplified on artificial antigen-presenting cells or in the presence of an antibody such as OKT3 that crosslinks CD3 on the T cell surface. A subset of the recombinant T cells can be depleted on artificial antigen-presenting cells or in the presence of an antibody such as Campath that binds to CD52 on the T cell surface. In additional aspects, the genetically modified cells can be cryopreserved.

[0075] The proliferation (survival) of the T cells after infusion can be evaluated by: (i) q-PCR using primers specific for the CAR; (ii) flow cytometry using an antibody specific for the CAR; and / or (iii) soluble TAA.

[0076] In certain embodiments of the present disclosure, CAR cells are delivered to an individual in need, such as an individual suffering from cancer or an infection. The cells then enhance the individual's immune system to attack the corresponding cancer cells or pathogenic cells. In some cases, the individual is provided with one or more doses of antigen-specific CAR T cells. In the case where the individual is provided with two or more doses of antigen-specific CAR T cells, the duration between administrations should be sufficient to allow time for proliferation 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 lacking a functional TCR can be of any kind, but in a specific embodiment, the cells are obtained from, for example, cord blood, peripheral blood, human embryonic stem cells, or induced pluripotent stem cell banks. A suitable dose for a therapeutic effect will be at least 10 5 cells per dose or about 10 5 cells per dose and about 1010 between per dose of cells, e.g., preferably in a series of dosing cycles. An exemplary dosing regimen consists of four one-week escalating dose dosing cycles starting at at least about 10 5 cells on day 0 and gradually increasing to a target dose of about 10 10 cells, for example, within several weeks after initiation of the patient's internal dose escalation protocol. Suitable modes of administration include intravenous, subcutaneous, intracavitary (e.g., via a reservoir access device), intraperitoneal, and direct injection into the tumor mass.

[0078] The pharmaceutical compositions of the present disclosure can be used alone or in combination with other well-recognized agents useful for treating cancer. Whether delivered alone or in combination with other agents, the pharmaceutical compositions of the present disclosure can be delivered by various routes and to various parts of a mammalian, particularly a human, body to achieve a specific effect. Those skilled in the art will understand that although more than one route can be used for administration, a particular route can provide a more direct and effective response than another route. For example, intradermal delivery rather than by inhalation can be advantageously used to treat melanoma. Local or systemic delivery can be accomplished by administration, which includes applying or instilling the formulation into a body cavity, inhaling or insufflating an aerosol, or by parenteral introduction, including intramuscular administration, intravenous administration, intraportal administration, intrahepatic administration, peritoneal administration, subcutaneous administration, or intradermal administration.

[0079] The compositions of the present disclosure can be provided in unit dosage forms, where each dosage unit, e.g., an injection, contains a predetermined amount of the composition, alone or in suitable combination with other active agents. As used herein, the term unit dosage form refers to, where appropriate, a physically discrete unit suitable as a unit dose for human and animal subjects, each unit containing a predetermined amount of the composition 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 forms of the present disclosure depend on the specific pharmacodynamics associated with the pharmaceutical composition in a particular subject.

[0080] It is desired that an effective amount or a 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 desired that re-introducing the amount of transduced T cells into a subject results in a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 100% reduction in tumor size compared to other identical conditions in the absence of transduced T cells.

[0081] Accordingly, the dosage of the transduced T cells administered should take into account the route of administration and should be such that a sufficient number of transduced T cells are 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 certain body weight) may vary in different applications. Generally, it is desirable that the concentration of the 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 as described above may be used, e.g., greater than 5x10 8 cells, or lower, e.g., less than 1x10 7 cells. The dosing regimen may be based on established cell-based therapies (see, e.g., Topalian and Rosenberg, 1987; U.S. Patent 4,690,915), or an alternative continuous infusion strategy may be employed.

[0082] These values provide general guidance to the practitioner regarding the range of transduced T cells to be utilized when optimizing the methods of the present disclosure for the practice of the present disclosure. Such ranges recited herein in no way exclude the use of higher or lower amounts of the components, which may be required in specific applications. For example, the actual dose and regimen 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 in light of the exigencies of the particular situation.

[0083] IV. Exemplary Human Antigen Receptor T Cells

[0084] As discussed above, the present disclosure relates to the culturing and use of CD8 + CD161 + T cells.

[0085] CD8 (cluster of differentiation 8) is a transmembrane glycoprotein that serves 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 coreceptor is mainly expressed on the surface of cytotoxic T cells, but can also be present on natural killer cells, cortical thymocytes, and dendritic cells. The CD8 molecule is a marker of the cytotoxic T cell population. It is expressed in T cell lymphoblastic lymphoma and hypopigmented mycosis fungoides.

[0087] 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 that are connected to the membrane by a thin stalk and an intracellular tail. A less common homodimer of the CD8-α chain is also expressed on some cells. The molecular weight of each CD8 chain is approximately 34 kDa. The structure of the CD8 molecule was determined by X-ray diffraction at 2.6 Å resolution by Leahy, D.J., Axel, R., and Hendrickson, W.A. The structure was determined to have an immunoglobulin-like β-sandwich fold and 114 amino acid residues. 2% of the protein is coiled into α-helices, and 46% is coiled into β-sheets, with the remaining 52% of the molecule remaining in loop regions.

[0088] The extracellular IgV-like domain of CD8-α interacts with the α3 portion of class I MHC molecules. This affinity brings the T cell receptor of cytotoxic T cells and the target cell into close proximity during antigen-specific activation. Cytotoxic T cells with the CD8 surface protein are called CD8+ T cells. The major recognition site is a flexible loop at the α3 domain of the MHC molecule. This was discovered by performing mutagenesis 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 coreceptor also plays a role in T cell signaling. The cytoplasmic tail of the CD8 coreceptor 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 chains of the TCR complex, which initiates a phosphorylation cascade that ultimately leads to the activation of transcription factors such as NFAT, NF-κB, and AP-1, thereby affecting 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 extracellular 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 can participate in the regulation of NK cell function. CD161 contains an extracellular domain with several motifs with C-type lectin properties, a transmembrane domain, and a cytoplasmic domain.

[0090] In one aspect, the compositions and methods of the embodiments relate to human CD8 + CD161 + T cells that express a chimeric antigen receptor (or CAR) polypeptide. The CAR can have any antigen-binding specificity but will include the typical intracellular signaling domain, transmembrane domain, and extracellular domain present in the CAR construct. The extracellular domain will include a given binding region, depending on the use for which the CAR-T is designed. The binding region is F(ab')2, Fab', Fab, Fv, or scFv. The binding region can 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 can include the intracellular signaling domain of human CD3ζ and can further include the intracellular segment of human CD28. In certain aspects, the transmembrane domain is the CD28 transmembrane domain.

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

[0092] In still additional aspects, the composition can contain cells that express the polypeptides described herein. The T cells can include an expression cassette encoding the CAR polypeptide. The expression cassette can be included in a non-viral vector, such as a transposon or a human transposon or a recombinant variant thereof. The expression cassette can be included in a viral vector or a recombinant variant thereof. The expression cassette can be genomically integrated or episomally maintained or expressed from mRNA.

[0093] In yet additional aspects, the present disclosure encompasses a method of making T cells that express 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 can further include stimulating the cells with a target antigen or an antibody against the receptor to proliferate the cells, kill the cells, and / or cause the cells to produce cytokines; for example, the cells can be stimulated to proliferate or expand with an artificial antigen-presenting cell carrying the target antigen.

[0094] In some aspects, the present disclosure includes methods of treating a human disease condition, the methods comprising infusing into a patient an amount of recombinant cells expressing a human CAR sufficient to treat the condition, wherein the human CAR comprises 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] The hCAR can be a chimeric receptor comprising one or more activating intracellular domains, such as a CD3-ζ-derived activating domain. Additional T cell activation motifs include, but are not limited to, CD28, CD27, OX-40, DAP10, and 4-1BB. In some aspects, the activating domain can also comprise the CD28 transmembrane and / or activating domain. In additional aspects, the hCAR coding region and / or expression cassette codons are optimized for expression in human cells and subjects. For example, in one embodiment, an scFv region obtained from the VH and VL sequences of a target-specific human antibody is incorporated into the binding segment of the hCAR. In another embodiment, the hCAR expression cassette is maintained episomally or integrated into the genome of the recombinant cell. In some aspects, the expression cassette comprises nucleic acid capable of being integrated by using an integrase mechanism, such as a viral vector like a retroviral vector, or a non-viral vector like a transposon mechanism. In additional embodiments, the expression cassette is contained in transposon-based nucleic acid. In a particular embodiment, the expression cassette is part of a two-component Sleeping Beauty (SB) or piggyBac system that utilizes a transposon and a transposase to enhance non-viral gene transfer.

[0096] The number of recombinant hCAR-expressing cells can be expanded to a clinically meaningful number. An example of such expansion uses artificial antigen-presenting cells (aAPCs). The recombinant hCAR-expressing cells can be verified and identified by flow cytometry and Western blot analysis. The expressed T cells, i.e., the recombinant hCAR-expressing CAR, can recognize and kill target cells. In additional aspects, the hCAR can be expressed as a universal cell that can be infused across transplantation barriers to help prevent immunogenicity. Upon cytotoxicity, the hCAR can be imaged (e.g., by positron emission tomography, PET) together with the human gene and conditionally ablated in T cells. The recombinant cells of the present disclosure can be used in specific cell therapies.

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

[0098] Due to drug-resistant residual disease, the disease recurrence rates of chemotherapy treatment for adult and pediatric B-lineage acute lymphoblastic leukemia (B-ALL) are 65% and 20%, respectively. The high incidence of B-ALL recurrence, especially in groups with poor prognosis, has promoted the use of immunotherapy-based approaches using allogeneic hematopoietic stem cell transplantation (HSCT). This therapy relies on alloreactive cells present in the donor graft to eradicate residual leukemia cells or minimal residual disease to improve disease-free survival. Donor lymphocyte infusion has been used to enhance the ability of transplanted T cells to target residual B-ALL after allogeneic HSCT, but this treatment approach for such patients achieves remission rates of less than 10% and is associated with high morbidity and mortality in terms of the frequency and severity of graft-versus-host disease (GVHD). Since recurrence is a common and fatal problem in these refractory malignancies, adoptive therapy using peripheral blood mononuclear cell (PBMC)-derived T cells after HSCT can be used to increase the anti-tumor effect or graft-versus-leukemia (GVL) effect by re-targeting the specificity of donor T cells to tumor-associated antigens (TAAs).

[0099] Currently, CAR-modified T cells rely on obtaining survival signaling through the CAR, which occurs only upon encounter with tumor antigens. In the clinical situation of infusing these CAR-modified T cells into patients with bulky disease, there are sufficient tumor antigens, manifested as providing sufficient activation and survival signaling through the CAR. However, patients with relapsed B-ALL typically receive myeloablative chemotherapy followed by HSCT and present with minimal residual disease (MRD). In this situation, the patient's tumor burden is low, and the low levels of minor TAAs severely limit the CAR-mediated signaling required to support the infused T cells, thereby compromising the therapeutic potential. Alternative CAR-independent ways expected to enhance T cell persistence will improve the engraftment of CAR-modified T cells.

[0100] Cytokines in the common γ-chain receptor family (γC) are important co-stimulatory molecules crucial for T cell lymphoid function, survival, and proliferation. IL-15 has several properties 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 inhibition of tumor-resident cells, and inhibits AICD.

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

[0102] In one embodiment, the present disclosure provides a method for generating chimeric antigen receptor (CAR)-modified T cells or transgenic TCR T cells with long-term in vivo potential for treating, for example, leukemia patients presenting with minimal residual disease (MRD). Generally speaking, 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-modifying CAR T cells or transgenic TCR T cells and a human cytokine mutant protein of interleukin-15 (IL-15) (hereinafter referred to as mIL15). The mIL15 fusion protein contains the cDNA sequence of codon-optimized IL-15, which is fused to the full-length IL15 receptor α through a flexible serine-glycine linker. This IL-15 mutant protein is designed in this way to: (i) restrict the expression of mIL15 to the surface of CAR + or transgenic TCR + T cells to limit the diffusion of the cytokine to the non-target in vivo environment, thereby potentially improving its safety profile, as the administration of exogenous soluble cytokines has led to toxicity; and (ii) the presence of IL-15 in the context of IL-15Rα to mimic physiologically relevant and qualitative signaling as well as the stabilization and recycling of the IL-15 / IL-15Ra complex to obtain a longer cytokine half-life. T cells expressing mIL15 are able to continue to support cytokine signaling, which is crucial for their survival rate after infusion. Generated by non-viral sleeping beauty system gene modification and subsequent ex vivo expansion on a clinically applicable platform, mIL15 + CAR + T cells or mIL15 + transgenic TCR + T cells produce a persistently enhanced T cell infusion product after infusion in murine models with high, low, or no tumor burden. In addition, mIL15 + CAR + T cells also show improved anti-tumor efficacy in both high-tumor burden models and low-tumor burden models.

[0103] In high-tumor burden models, mIL15+ CAR + T cells are more persistent and have higher anti-tumor activity than CAR + T cells, indicating that mIL15 + CAR + T cells may be more effective than CAR when treating leukemia patients with active disease with widespread tumor burden. Therefore, in the broadest applications, mIL15 + CAR + T cells can replace CAR + T cells in adoptive therapy. mIL15 + T cells. mIL15 + CAR + The ability of T cells to survive independently of survival signaling through CAR allows these modified T cells to persist after infusion in the absence of tumor antigen. Therefore, this is expected to have the greatest impact on therapeutic efficacy in the MRD treatment setting, especially in patients who have received myeloablative chemotherapy and hematopoietic stem cell transplantation. These patients will receive adoptive T cell transfer with mIL15 + CAR + T cells to treat their MRD and prevent relapse.

[0104] Membrane-bound cytokines, such as mIL15, have broad implications. In addition to membrane-bound IL-15, other membrane-bound cytokines are envisioned. Membrane-bound cytokines can also extend to the cell surface expression of other molecules related to the activation and propagation of cells for human applications. These include but are not limited to cytokines, chemokines, and other molecules that contribute to the activation and proliferation of cells for human applications.

[0105] Membrane-bound cytokines, such as mIL15, can be used ex vivo to prepare cells for human applications and can be 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) for activation and / or proliferation. The population of T cells activated / propagated by mIL15 on aAPCs contains genetically modified lymphocytes but also contains 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 treatment with CAR-modified T cells is hampered by the lack of persistence after adoptive T cell transfer. mIL15 + CAR + T cells or mIL15 + transgenic TCR+ The ability of T cells to survive long-term in vivo independently of tumor antigens indicates great potential for treating patients with MRD. In this context, mIL15 and the persistent T cells it supports would meet the need, as current approaches for patients with MRD are inadequate. The persistence of T cells and other lymphocytes infused in patients with MRD exceeds that of CAR + T cells. For a sustained therapeutic effect to be achieved, any immune cells used to treat and prevent malignancies, infections, or autoimmune diseases must be able to persist long-term. Thus, activating T cells to persist beyond signals from the endogenous T cell receptor or the introduced immune receptor 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 mutant proteins of IL-15 that are expressed as CAR + T cells or transgenic TCR + membrane-bound fusion proteins of IL-15 and IL-15Rα (mIL15) on T cells. The mIL15 construct was co-electroporated 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 were generated and supplemented with IL-21 by co-culture on CD19 + artificial antigen-presenting cells. Signaling through the IL-15 receptor complex in the gene-modified T cells was verified by phosphorylation of STAT5 (pSTAT5), and these T cells showed redirected specific lysis of CD19 + tumor targets equivalent to CAR + T cells. In addition, after antigen withdrawal, signaling by mIL15 increased the prevalence of T cells with a less differentiated / younger phenotype that had 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, as they are associated with T cell subsets in which the ability to persist long-term in vivo has been demonstrated. In immunocompromised NSG mice bearing disseminated CD19 + leukemia, mIL15 + CAR + T cells showed both persistence and anti-tumor effects, while their CAR + T cell counterparts could not maintain significant persistence despite the presence of TAA. In a prophylactic mouse (NSG) model, mIL15 was first transplanted+ / - CAR + T cells persisted for six days and then disseminated CD19 was introduced + leukemia, and only mIL15 was found + CAR + T cells persisted and prevented tumor engraftment. To test mIL15 + CAR + whether T cells could persist independently of TAA stimulation, mIL15 + / - CAR + T cells were adoptively transferred into NSG mice without tumors. Only mIL15 + CAR + T cells were able to persist in this in vivo environment without exogenous cytokine support or in the presence of CD19 TAA. These data indicate that mIL15 can be co-expressed on CAR + T cells or transgenic TCR + T cells, thereby enhancing in vivo persistence without TAA or exogenous cytokine support. In summary, this cytokine fusion molecule: (i) provides a stimulatory signal through pSTAT5, resulting in enhanced in vivo T cell persistence while maintaining tumor-specific function, (ii) maintains a subset of T cells that promote a memory-like phenotype, (iii) eliminates the need and cost of clinical-grade IL-2 for in vitro and in vivo T cell expansion and persistence, and (iv) reduces the need for clinical-grade soluble IL-15.

[0108] VI. Pancreatic cancer

[0109] Pancreatic cancer occurs 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 only to refer to this type. These adenocarcinomas start 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 be caused by these cells. One to two percent of pancreatic cancer cases are neuroendocrine tumors, which are caused by hormone-producing cells of the pancreas. These are generally less aggressive than pancreatic adenocarcinoma.

[0110] The 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 usually no symptoms in the early stages of the disease, and specific symptoms sufficient to indicate pancreatic cancer generally do not occur until the disease has reached an advanced stage. By the time of diagnosis, 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 70. Risk factors for pancreatic cancer include smoking, obesity, diabetes, and certain rare genetic conditions. Approximately 25% of cases are related to smoking, and 5 - 10% of cases are related to genetic factors. Pancreatic cancer is usually diagnosed through a combination of medical imaging techniques such as ultrasound or computed tomography scans, blood tests, and examination of tissue samples (biopsies). The disease is divided into several stages, from early stage (stage I) to advanced stage (stage IV). There is no effective screening for the general population yet.

[0112] Non - smokers and those who maintain a healthy weight and limit their consumption of red or processed meat have a lower risk of developing pancreatic cancer. If smokers quit smoking, their chance of developing the disease decreases, and after 20 years, it almost returns to the level of other groups. Pancreatic cancer can be treated by surgery, radiotherapy, chemotherapy, palliative care, or a combination of these. The treatment plan is based in part on the cancer stage. Surgery is the only treatment that can cure pancreatic cancer and can also improve the quality of life when there is no potential for cure. Sometimes medications for pain management and to improve digestion are needed. Even for those who receive treatment aimed at cure, early palliative care is recommended.

[0113] In 2015, all types of pancreatic cancer caused 411,600 deaths globally. 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 developed countries in 2012. Pancreatic cancer usually has a poor prognosis: after diagnosis, 25% of people survive for one year, and 5% of people survive for five years. For cancers diagnosed at an early stage, the five - year survival rate rises to approximately 20%. Neuroendocrine cancers have better outcomes; 65% of those diagnosed are alive within five years after diagnosis, although the survival rate varies quite a bit depending on the type of tumor.

[0114] VII. Immune System and Immunotherapy

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

[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 pluripotent hematopoietic stem cells and cannot be distinguished from each other until they are activated. B cells play an important role in the humoral immune response, while T cells are closely involved in cell-mediated immune responses. It can be distinguished from other lymphocyte types such as B cells and NK cells by a special receptor called the T cell receptor (TCR) present on its cell surface. In almost all other vertebrates, B cells and T cells are produced by stem cells in the bone marrow. T cells enter the thymus and develop there, and their name is derived from the thymus. In humans, approximately 1%-2% of the lymphocyte pool recycles every hour to optimize the chance for antigen-specific lymphocytes to find their specific antigen within secondary lymphoid tissues.

[0117] T lymphocytes are produced from hematopoietic stem cells in the bone marrow and typically migrate to the thymus until they mature. 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 surfaces of other cells. There are at least two populations of T cells, called T helper cells and cytotoxic T cells. T helper cells and cytotoxic T cells are mainly distinguished by their respective display of the membrane-bound glycoproteins CD4 and CD8. T helper cells secrete various lymphokines that are crucial for the activation of B cells, cytotoxic T cells, macrophages, and other cells of the immune system. In contrast, cytotoxic T cells that recognize antigen-MHC complexes proliferate and differentiate into effector cells called cytotoxic T lymphocytes (CTLs). CTLs eliminate body cells displaying the antigen, 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 lymphocyte that constitutes a major component 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 perforin and granzyme, which cause the target cells to die by apoptosis.

[0118] Antigen-presenting cells, which include 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, along with MHC molecules on their outer cell membranes. The major histocompatibility complex (MHC) is a large genetic complex with multiple loci. MHC loci encode two major classes of MHC membrane molecules, called class I and class II MHC. T helper lymphocytes typically recognize antigens associated with MHC class II molecules, and cytotoxic T lymphocytes recognize antigens associated with MHC class I molecules. In humans, the MHC is called the HLA complex, and in mice it is called the H-2 complex.

[0119] The T cell receptor or TCR is a molecule present on the surface of T lymphocytes (or T cells) that is typically responsible for recognizing antigens bound to major histocompatibility complex (MHC) molecules. It is a heterodimer composed of an alpha chain and a beta chain in 95% of T cells, while 5% of T cells have a TCR composed of a gamma chain and a delta chain. The binding of the TCR to an antigen and MHC leads to the activation of its T lymphocytes through a series of biochemical events regulated by associated enzymes, co-receptors, and specialized accessory 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 are associated with a molecule called the T cell receptor (TCR) and the ζ chain to generate an activation signal in T lymphocytes. The TCR, ζ chain, and CD3 molecules together constitute the TCR complex. The CD3γ chain, CD3δ chain, and CD3ε chain are highly related cell surface proteins of the immunoglobulin superfamily that contain a single extracellular immunoglobulin domain. The transmembrane region of the CD3 chains is 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 called the immunoreceptor tyrosine-based activation motif or simply ITAM, which is crucial for the signaling capacity of the TCR.

[0120] CD28 is one of the molecules expressed on T cells that provides a co-stimulatory signal 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). The expression of B7.2 on antigen-presenting cells is constitutive. CD28 is the only B7 receptor that is constitutively expressed on naive T cells. In addition to the TCR, stimulation through CD28 can provide a potent co-stimulatory signal for T cells to produce various interleukins (specifically IL-2 and IL-6).

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

[0122] Autoimmune diseases or autoimmunity is a failure of an organism to recognize its own components (down to the submolecular level) as "self", which leads to an immune response against its own cells and tissues. Any disease caused by such an abnormal immune response is called an autoimmune disease. Prominent examples include celiac disease, type 1 diabetes (IDDM), systemic lupus erythematosus (SLE), Sjogren'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, Henoch-Schonlein purpura, post-streptococcal glomerulonephritis, erythema nodosum, Takayasu's arteritis, Addison's disease, rheumatoid arthritis, multiple sclerosis, sarcoidosis, ulcerative colitis, erythema multiforme, IgA nephropathy, polyarteritis nodosa, ankylosing spondylitis, Goodpasture's syndrome, thromboangiitis obliterans, Sjogren'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 rheumatica, 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 severe infections, and have adverse effects on the bone marrow, liver, and kidneys. To date, other treatments for class III autoimmune diseases have targeted T cells and macrophages. More effective methods are needed to treat autoimmune diseases, specifically 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 Examples. For general guidance on the preparation and use of antigen presenting 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] aAPCs are typically incubated with peptides of optimal length, which allows the peptides to bind directly to MHC molecules without additional processing. Alternatively, cells can express the antigen of interest (i.e., in the case of MHC-independent antigen recognition). In addition to the peptide-MHC molecule or the antigen of interest, the aAPC system can also include at least one exogenous accessory molecule. Any suitable number and combination of accessory molecules can be employed. Accessory molecules can be selected from accessory molecules such as costimulatory molecules and adhesion molecules. Exemplary costimulatory molecules include, among others, CD70 and B7.1 (B7.1 was previously known as B7 and is also known as CD80), which bind to CD28 and / or CTLA-4 molecules on the surface of T cells, thereby influencing, for example, T cell expansion, Th1 differentiation, short-term T cell survival, 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 molecule (ICAM), which promote, for example, cell-cell or cell-matrix contact. Exemplary adhesion molecules include LFA-3 and ICAM, such as ICAM-1. Techniques, methods, and reagents for the selection, cloning, preparation, and expression of exemplary accessory molecules, including costimulatory molecules and adhesion molecules, are illustrated, for example, in U.S. Patent Nos. 6,225,042, 6,355,479, and 6,362,001.

[0127] Cells selected to be aAPCs are preferably defective in intracellular antigen processing, intracellular peptide transport, and / or intracellular peptide loading of MHC class I or II molecules, or are poikilothermic (i.e., less sensitive to temperature shock than mammalian cell lines), or have both defects and poikilothermic properties. Preferably, cells selected to be aAPCs also lack the ability to express at least one endogenous counterpart (e.g., endogenous MHC class I or II molecules and / or endogenous co-stimulatory molecules as described above) onto exogenous MHC class I or II molecules and co-stimulatory molecule components introduced into the cell. In addition, aAPCs preferably retain the defects and poikilothermic properties that the cells had prior to their modification to produce aAPCs. Exemplary aAPCs are constituted or derived from transporters associated with antigen processing (TAP)-deficient cell lines such as insect cell lines. An exemplary poikilothermic insect cell line is the Drosophila cell line, 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 U.S. Patent Nos. 6,225,042, 6,355,479, and 6,362,001.

[0128] In one embodiment, the aAPCs are also subjected to freeze-thaw cycles. In an exemplary freeze-thaw cycle, the aAPCs can be frozen by contacting them with a suitable receptacle containing aAPC and an appropriate amount of liquid nitrogen, solid carbon dioxide (i.e., dry ice), or similar cryogenic material such that freezing occurs rapidly. The frozen aAPCs are then thawed by removing the aAPC from the cryogenic material and exposing it to ambient room temperature conditions, or by facilitating a thawing process that uses a warm water bath or a warm hand to promote a shortened thawing time. Additionally, the aAPCs can be frozen and stored for an extended period of time prior to thawing. The frozen aAPCs can also be thawed and then lyophilized prior to further use. Preferably, preservatives that may have a detrimental effect on the freeze-thaw procedure, such as dimethyl sulfoxide (DMSO), polyethylene glycol (PEG), and other preservatives, are absent from the medium containing aAPCs that have undergone freeze-thaw cycles, or are substantially removed, such as by transferring the aAPCs to a medium that is substantially free of such preservatives.

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

[0130] IX. Kits of the Present Disclosure

[0131] Any of the compositions described herein can be included in a kit. In some embodiments, allogeneic CART cells are provided in the kit, which can also contain reagents suitable for expanding the cells, such as media, 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, chimeric receptor expression constructs, one or more reagents for generating chimeric receptor expression constructs, cells for transfecting the expression constructs, and / or one or more instruments for obtaining allogeneic cells for transfecting the expression constructs (such instruments can be syringes, pipettes, forceps, and / or any such medically approved device).

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

[0134] In some aspects, the kit includes reagents or devices for cell electroporation.

[0135] The 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 kit may be packaged in an aqueous medium or in a lyophilized form. The container device of the kit may comprise at least one vial, test tube, flask, bottle, syringe or other container device in which the components may be placed and preferably are suitably aliquoted. When there is more than one component in the kit, the kit will generally also contain a second, third or other additional container in which the additional components may be placed separately. However, various combinations of components may be included in the vial. The kits of the present disclosure generally also contain a device for containing the chimeric receptor construct and any other reagent containers in a tightly sealed manner for commercial sale. Such containers may comprise, for example, an injection or blow-molded plastic container that retains the desired vials therein.

[0136] X. Examples

[0137] The following examples are included to illustrate the preferred embodiments of the present disclosure. Those skilled in the art should understand that the techniques disclosed in the following examples represent techniques that have been found by the inventors to work well in the practice of the present disclosure and thus may be considered to constitute preferred modes of its practice. However, according to the present disclosure, those skilled in the art should understand that many changes may be made to the specific embodiments disclosed without departing from the spirit and scope of the present disclosure and still obtain the same or similar results.

[0138] Example 1 - Materials and Methods

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

[0140] Influenza model. As described, to generate T cells for adoptive transfer, C57 / BL6 mice were challenged with the influenza A / Hong Kong / 8 / 68 (H3N2) Swiss mouse lung adapted virus strain of influenza A virus H3N2, generously provided by Dr. Brian Gilbert (Liang et al., 2017). Since the Aridyne 2000 compressor generates room air at 10 liters per minute, infection was carried out by exposure to an aerosol of influenza virus atomized in MEM medium + 0.05% gelatin for 20 minutes using an Aerotech II nebulizer. All mice infected in any given experiment were infected simultaneously in a single exposure chamber. Two weeks after infection, the mice were sacrificed, spleens were harvested and CD8 + cells were negatively selected (Miltenyi Biotec). The isolated CD8 + cells were further magnetically sorted into NK1.1 + populations and NK1.1 neg populations (Miltenyi Biotec). Subsequently, 500,000 CD8 + NK1.1 + cells and CD8 + NK1.1 neg cells were adoptively transferred into naïve mice and 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 footpads and a booster vaccination was given seven days later. Ten days after the boost, the vaccinated mice were sacrificed and CD8 + spleen cells were isolated by negative selection (Miltenyi Biotec). The isolated CD8 + cells were further magnetically sorted into NK1.1 + populations and NK1.1 negPopulation (Miltenyi Biotec). Three groups of eight naïve mice were each subcutaneously injected with 250,000 B16F10 tumor cells. Tumor size was recorded and the animals were randomly grouped such that each group had a similar mean tumor size and standard error. Seven days after tumor inoculation, the treated mice each received 1.5 million CD8 + NK1.1 + cells or CD8 + NK1.1 - cells by intraperitoneal adoptive transfer. The naïve mice served as untreated controls. Tumor size was determined by external caliper measurement and calculated by the formula (length × width 2 ) × π / 6. Once the tumor burden in the control group exceeded the allowable limit set by the Comparative Medicine Center (CCM), the mice were euthanized 22 days after tumor inoculation.

[0142] Mouse PBMC analysis. Two weeks after influenza infection or three weeks after tumor implantation, PBMC were collected from the mice that received adoptive transfer by retro-orbital bleeding. Erythrocytes were lysed by treatment with ammonium chloride (Sigma-Aldrich) according to the manufacturer's instructions. The leukocyte pellet was washed once with PBS and resuspended in AIM-V medium with 10% mouse serum. Cells were stained with anti-CD3, CD4, CD8, CD25, IFN-γ for flow cytometry analysis. All flow cytometry analyses were performed using an LSR II flow cytometer (BD Biosciences) and analyzed with FlowJo version 10.0.00003 (Tree Star Inc., Ashland, OR).

[0143] Human microarray analysis. CD8 + CD161 + cells and CD8 + CD161 neg cells were magnetically isolated from the peripheral blood of three healthy donors and three PDAC patients. The isolated cells were not activated. Total RNA was isolated from the cells by the RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions. CD8 + CD161 + cells and CD8 + CD161 negCells were subjected to gene expression profiling. Detailed instructions for sample requirements and data pre - analysis can be found on the facility's website (world - wide web at mdanderson.org / research / research - resources / core - facilities / sequencing - and - microarray - facility - smf / services - and - fees / microarray - services - overview.html). The data was analyzed and visualized using Transcriptome Analysis Console v3.0 (Affymetrix).

[0144] TCR Vβ spectratyping. CD8 + CD161 + cells isolated from the peripheral blood of normal donors were spectratyped by Mayo Clinic. The resulting images are clusters of fluorescent peaks with single - base - pair resolution and different fluorescence intensities, approximately corresponding to the number of fragments of the indicated size represented in the donor's native RNA. The organization of the peak pattern (number of peaks), relative intensity across peaks, and size distribution were reviewed.

[0145] Cytotoxicity assays. To evaluate the cytotoxicity of CD8 + CD161 + cells relative to CD8 + CD161 neg cells and autologous PBMCs, a chromium - based short - term cytotoxicity assay was performed in vitro. CD8 + CD161 + 、CD8 + CD161 neg and unmanipulated autologous PBMCs were freshly isolated from human peripheral blood products. The cytotoxicity of the isolated cells was immediately tested in a four - hour killing assay using 51 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 the chromium released into the culture medium and read using a Wizard2γ counter (Perkin Elmer).

[0146] CD8 + CD161 + Ex vivo culture conditions. CD8 + CD161 + cells isolated from apheresis blood component products of normal donors, CD8 + CD161 negCells and bulk PBMC were stimulated with plate-bound anti-CD3 / CD28 and expanded in a cytokine mixture containing 10 ng / ml IL-7, 5 ng / ml IL-15, and 30 ng / ml IL-21 (all from PepTcell, Inc., Rocky Hill, NJ). CD8 + CD161 + cells were isolated from healthy donor apheresis blood components and cultured with stimulation with anti-CD3 / CD28 / Clec2d at 1 μg / mL each (anti-human CD3 - eBioscience cat #16 - 0037 - 8, anti-human CD28 from BD Biosciences cat #555725, recombinant human Clec2d, Novus Biologicals cat #NBP2 - 22966), and expanded in RPMI - 1640, 10% FBS, and 2 mmol / l GlutaMAX (Invitrogen) in a cytokine mixture containing 10 ng / ml IL-7, 5 ng / ml IL-15, and 30 ng / ml IL-21 (all from PepTcell, Inc., Rocky Hill, NJ). 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 mixture without antibody stimulation.

[0147] Statistical analysis. Unless otherwise stated, significant differences were determined by two-way ANOVA or one-way ANOVA with multiple comparisons using the Bonferroni post hoc test. 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 expression profiles after PDAC chemoimmunotherapy identified CD3 + CD8 + NK1.1 + innate-like and cytotoxic properties of cells. Previous work showed that very small numbers (<1,500 per mouse) of splenic CD8 + NK1.1 +Cells can still provide rapid and robust anti-tumor protection against parental PDAC cell lines in metastatic disease models (Konduri et al., 2016). To gain insight into the key functional properties of this NK1.1 + CD3 + CD8 + T cell subset, the inventors orthotopically implanted a cohort of mice with Kras G12D / p53 - / - PDAC tumors and subsequently cured them with a previously disclosed (Konduri et al., 2016) immune-based treatment protocol. Two months after treatment and cure, CD8 + splenocytes were negatively selected and subdivided into NK1.1 + fractions and NK1.1 neg fractions. These fractions were then co-cultured overnight with PDAC-loaded mature DCs, and PDAC antigen-specific cells were identified and isolated by upregulation of CD69 expression. Microarray indicated 1642 genes ([[]] Figure 1 ) differentially regulated between CD8 + NK1.1 + cells and CD8 + NK1.1 neg cells at a univariate significant level of 0.1. Although many different pathways may be affected (Table 1), the most significant differences were found in the lytic granzyme serine proteases, particularly the atypical granzyme isoforms F, D, G, and C, and the innate-like cytotoxicity receptors (Table 2). These results indicate that CD8 + NK1.1 + cells represent a CD8 + T cell population with significantly enhanced lytic capacity.

[0150] Table 1: Top upregulated and downregulated genes.

[0151]

[0152]

[0153] Table 2: Fold change and P-value of genes grouped into granzyme pathways and killer cell-like receptor subfamily pathways

[0154]

[0155]

[0156] NK1.1 Identifies Key Circulating Memory T Cell Populations in Multiple Mouse Disease Models. To verify that NK1.1 can identify a similar key population of lytic memory cells in a model-independent manner, the inventors conducted 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 sub-lethal dose of H2N3 mouse-adapted influenza virus. Three weeks after inoculation and after recovery from weight loss, splenocytes were harvested and CD8 + non-adherent cells were isolated by negative selection. After isolation into NK1.1 + fractions and NK1.1 neg fractions by positive selection, 5 x 10 5 cells / mouse of each NK1.1 group were adoptively transferred into a naïve 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. Cohorts adoptively transferred with donor CD8 + NK1.1 + cells fully recovered body weight and survived the infection, while those adoptively transferred with CD8 + NK1.1 neg cells all lost weight and died at the same rate as the control group adoptively transferred with naïve CD8 + splenocytes (Figure 2A-B). Analysis of PBMCs 7 days after infection showed that circulating CD3 + NK1.1 neg cells increased by 40% (p < 0.003) in mice receiving CD8 + NK1.1 + cells compared to naïve and CD8 + CD8 + IFN-γ + adoptive transfer cohorts (Figure 2C).

[0157] In a second model system, a donor mouse cohort was subcutaneously inoculated with 2 x 10 5 B16 melanoma cells and vaccinated on days 7 and 14 after inoculation with a cell-based vaccine loaded with B16. On day 21, the mice were sacrificed and splenocytes were harvested again and sorted into CD8 + NK1.1 + cell populations and CD8 + NK1.1 neg cell populations. Then 1.5 x 10 6 CD8 + NK1.1 + cells or CD8+ NK1.1 neg Cells were each subjected to adoptive transfer of a primary cohort inoculated with palpable B16 tumors ( Figure 7B ). Mice receiving CD8 + NK1.1 + cells exhibited significantly delayed tumor growth and a survival benefit, while the cohort receiving CD8 + NK1.1 neg cells had the same survival as the control cohort adoptively transferred with primary splenocytes (Figure 2D - E). Analysis of peripheral blood lymphocytes showed that levels of the memory markers CD62L and CCR7 were significantly elevated in GP100 tetramer - specific CD8 + NK1.1 neg cells in the cohort adoptively transferred with CD8 + NK1.1 + cells compared to the cohorts adoptively transferred with CD8 + NK1.1 + cells or primary splenocytes (Figure 2F). These results indicate that the CD161 homolog NK1.1 defines the major CD8

[0158] mouse CD3 + CD8 + NK1.1 + cell population is phenotypically conserved in the human CD3 + CD8 + CD161 + counterpart. Inspired by the protective memory responses provided by CD8 + NK1.1 + cells in various systems, the inventors next asked whether the memory CD8 + T - cell subset defined by NK1.1 expression is phenotypically and transcriptionally conserved in the analogous CD3 + CD8 + CD161 + cell population in the human population. For this analysis, CD8 + CD161 + and CD8 + CD161 neg cells were differentially isolated from six different human donors. After verifying that CD161 + cells are polyclonal by TCR - Vβ spectratyping ( Figure 8Subsequently, each population was subjected to transcriptional profiling by microarray analysis. Despite the fact that these cells were not activated prior to analysis and were in a steady-state resting condition, the profiles of upregulated granzyme and natural cytotoxicity receptors were recapitulated in these cells at a univariate significant level of 0.1 ( Figure 3 , Table 3). Cross-species gene comparison analysis between activated murine cells and non-activated human cells identified a conserved signature of 206 genes with common nomenclature that were differentially regulated in the two populations ( Figure 9 ). Reactome pathway analysis of the upregulated human genes identified signatures related to differentiation and regulation at an FDR of <5x10 -4 , which included HDAC deacetylation, DNA and histone methylation, nucleosome assembly, RNA polymerase I promoter escape, transcriptional regulation of small RNAs, and gene silencing of RNAs.

[0159] Table 3: Phenotypic characteristics of murine CD8 + NK1.1 + cells were recapitulated during the resting phase of CD8 + CD161 + cells, in which the expression of granzyme and killer lectin-like receptor genes was elevated

[0160]

[0161]

[0162] Development of a model system for CAR T cell therapy for PDAC. Based on the potential of its novel biology, the inventors hypothesized that the human CD8 + CD161 + subset could provide more functional and durable anti-tumor efficacy than conventional autologous PBMCs in the context of solid tumor CAR T cell therapy.

[0163] Ex vivo expansion of CD8 + CD161 + cells in combination with plate-bound anti-CD3 / CD28 / Clec2d stimulation enhanced 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 with plate-bound anti-CD3 / CD28 / Clec2d stimulation led to a central memory (CD45RA - CCR7+ ) with significant upregulation Figure 5 ).

[0164] Ex vivo expansion of CD8+CD161+ cells with IL7 / 15 / 21 in combination with plate-bound anti-CD3 / CD28 / Clec2d stimulation enhanced cytotoxic granzyme production. CD8 + CD161 + cells were sorted from normal donors and ex vivo stimulation conditions were optimized. The combination of IL7 / 15 / 21 with plate-bound anti-CD3 / CD28 / Clec2d stimulation led to significant upregulation of cytotoxic molecules, granzyme, and perforin compared to IL2, IL-2 / 7 / 15, IL2 / 7 / 15 / 21 stimulation( Figure 6 ).

[0165] CD8 + CD161 + cells showed an inherent killing advantage in vitro. To evaluate the cytotoxic ability of CD8 + CD161 + cells relative to CD8 + CD161 neg cells and autologous PBMCs, a chromium-based short-term cytotoxicity assay was performed in vitro. CD8 + CD161 + -, CD8 + CD161 neg and unmanipulated autologous PBMCs were freshly isolated from human peripheral blood products. Using 51 Cr-labeled allogeneic 293-HEK targets, the cytotoxic ability of the isolated cells was immediately tested in a four-hour killing assay. As Figure 4 shown, CD8 + CD161 + cells could induce 100% target lysis at an E:T ratio of 25:1, while autologous PBMCs and CD8 + CD161 neg cells showed lysis capabilities of 22% and 15% respectively at a maximum E:T ratio of 50:1 (by one-way ANOVA, p < 0.002 at 50:1, p < 0.0007 at 25:1 and p < 0.00002 at 5:1). These data indicate that CD8 + CD161 + T cells have enhanced cytotoxicity, which is absent in CD8 + CD161 neg or autologous PBMC counterparts.

[0166] Example 3 - Discussion

[0167] Based on the expression of surface molecules and secreted cytokines, lymphocytes are classified into different subsets and lineages. However, the classification is dynamic, with the identification of new cell subsets that occasionally express markers from previously identified cell subsets and lineages. One such surface molecule is CD161, which is known to be expressed on NK cells, NKT cells, and other T cell lineages (Fergusson et al., 2011). CD161 shares 47% homology with the murine counterpart NK1.1 and is expressed by up to one-quarter of peripheral T cells (Neelapu et al., 1994). Since NK-T cells account for less than 1% of peripheral T cells, CD3 + CD161 + cells represent a distinct lineage of T cells as they account for more than 5% of circulating T cells (Takahashi et al., 2006). On CD8 + T cells, CD161 expression is defined as medium or high, while there is no such distinction among CD4 + T cells that express CD161 (Takahashi et al., 2006). CD8 + CD161 高 cells have previously been defined as MAIT cells (Martin et al., 2009; Goldfinch et al., 2010), Tc17 cells (Northfield et al., 2008; Billerbeck et al., 2010), or memory stem cells (Turtle et al., 2009). Transcriptional profiling of different CD161-expressing cells identified a conserved CD161 + CD161 + / MAIT cell transcriptional signature in CD8 ++ T cells, which could be extended to CD4 + CD161 + and TCRγδ + CD161 + T cells (Fergusson et al., 2014). In addition, populations of CD161-expressing 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. Thus, CD161 expression 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 cells during viral infections (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, any role of CD8 + CD161 + cells in cancer biology has not been clearly defined. In the present study, the inventors set out to understand the biology and functional properties of CD8 + CD161 + cells.

[0168] The inventors had previously reported the functional significance of CD161 + cells, the murine counterpart of CD8 + NK1.1 + and had found an increase in the number of these cells under conditions mimicking viral infection (Konduri et al., 2016).

[0169] Microarray analysis of murine CD8 + NK1.1 + cells revealed that, compared to the CD8 + NK1.1 neg counterpart, granzyme production by these cells was significantly upregulated following antigen stimulation. Innate genes and pathways involved in cytotoxic function were differentially expressed. It has previously been reported that the human equivalent of CD161 + also constitutively expresses the cytotoxic mediators granzyme B and perforin. In contrast, a quarter of the cells lacking CD161 expression were naive CD8 + T cells and expressed low levels of granzyme B and perforin even in the memory population (Neelapu et al., 2018). CCR4 and CCR6 expression on CD8 + CD161 + cells suggests their ability to maintain tissue residency and home to different organs. A similar expression pattern was observed in CD161 高 cells in the circulating blood of MS patients, enhancing their entry into the CNS and contributing to pathogenesis (Annibali et al., 2011). The inventors found that resting CD8 + CD161 neg cells also expressed higher levels of CXCR3, an effector memory marker that drives CD8 + T cells to differentiate into short-lived effectors with limited memory potential (Kurachi et al., 2011).

[0170] Although effective against CD19 + hematological malignancies, CAR-T cell therapy 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 can lead to efficient CAR-T cell therapy. In preclinical models, both CD8 + subsets and CD4 + subsets expressed co-antitumor CAR-T activity (Sommermeyer et al., 2016). Similar results were observed in preclinical mouse experiments, in which engineered CD4 + and CD8 + T cell combinations induced 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 showed high anti-cancer activity of CD19-CAR-T cells produced from a composition of CD8 + and CD4 + T cell subsets that were separately expanded in vitro and infused at a 1:1 ratio (Turtle et al., 2016a). The same results were also obtained in a clinical trial of patients with B cell acute lymphoblastic leukemia (Turtle et al., 2016b). Another clinical study on patients with high-risk intermediate B-lineage non-Hodgkin lymphoma treated with the following demonstrated the feasibility and safety of both methods: first-generation CD19-CAR-T using isolated CD8 + T CM subsets or second-generation CD19-CAR-T treatment using both CD8 + and CD4 + T CM subsets (Turtle et al., 2016c), although the CAR-T group with CD4 + and CD8 + T CM 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 intrinsic killing potential, such as NK, NKT, and γδ T 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 phenotype, in which less than 1% of 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 alter CD161 expression upon stimulation with anti-CD2, anti-CD3 or anti-CD28, and influenza-specific cells also 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 and defines a distinct lineage.

[0171] The autologous PBMC preparations commonly used for CAR T cell generation represent a heterogeneous group of cells, which also contain highly differentiated subsets that have experienced antigen. It has been previously reported that the naive (T n ) subset, stem cell memory (T scm ) subset and central memory (T cm ) subset used for CAR engineering result in 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, in vitro culture methods (cytokine composition and culture duration) can promote T cell differentiation (Alizadeh et al., 2019). The inclusion of IL-7 and IL-15 has been shown to be beneficial for lymphocyte development, differentiation and homeostasis during in vitro expansion of T cells, and has higher in vivo survival rates 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 the 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 enhanced effector activity while preserving the stem / memory potential against the GD2 tumor antigen (Gargett et al., 2015). It has also been demonstrated 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 + Expansion of T cells (Santegoets et al., 2013) and enhancement of the potency of CD19-CAR-T (Rosenberg, 2014). It has been previously reported that the addition of IL-15 and IL-21 contributes to enhancing and maintaining 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 studies of the present invention, the inventors found that ex vivo culture and expansion of autologous T cells with a mixture of IL-7, IL-15, and IL-21 mixtures is mainly beneficial for CD8 + CD161 + cell populations without significantly altering the phenotype of autologous PBMCs or CD8 + CD161 neg cells grown without IL-21 alone or even IL-2 alone.

[0172] In summary, the inventors report that CD8 + CD161 + cells and their murine equivalent CD8 + NK1.1 + cells exhibit an abnormally high cytotoxic potential. Gene expression profiling by microarray revealed that, compared to NK1.1 neg counterparts and CD161 neg counterparts, the expression levels of granzymes, perforin, and innate-like receptors are enhanced in these cells upon activation. In vitro, the killing efficiency of CD8 + CD161 + T cells is higher than that of autologous PBMCs or CD8 + CD161 neg populations. Using this subset for T cell-based therapies provides exciting new opportunities for the effective treatment of solid tumors including PDAC.

[0173] ***

[0174] According to the present disclosure, all of the methods disclosed and claimed herein can be prepared and executed without undue experimentation. While the compositions and methods of the present disclosure have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that the methods, as well as the steps or the sequence of steps of the methods described herein, can be varied without departing from the concepts, spirit, and scope of the present disclosure. More specifically, it will be apparent that certain chemically and physiologically related agents can replace the agents described herein while achieving the same or similar results. It is apparent to those skilled in the art that all such similar substitutions and modifications are considered to be within the spirit, scope, and concepts of the present disclosure as defined by the appended claims.

[0175] XI. References

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Claims

1. A method for providing CD8 + CD161 + T cell populations in vitro or ex vivo, wherein the CD8 + CD161 + T cell populations have upregulated granzyme and perforin expression, and the method comprises: (a) Obtain a sample of cells, the 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, Thus provided is a CD8 + CD161 + cell population in which the expression of granzyme and perforin is upregulated compared to CD8 + CD161 + T cells cultured in the absence of IL-7, IL-15, IL-21, CD3-binding antibody, CD28-binding antibody, and Clec2d.

2. The method according to claim 1, wherein Clec2d is present in the culture medium at 0.1 to 5.0 μg / ml.

3. The method according to claim 2, wherein Clec2d is present in the culture medium at 0.3 to 3.0 μg / ml.

4. The method according to claim 2, wherein Clec2d is present in the culture medium at 0.5 to 2.0 μg / ml.

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

6. The method according to claim 5, wherein the culturing step (c) is free of a CD3-binding antibody, a CD28-binding antibody, Clec2d, and / or a CD161-binding antibody.

7. The method according to claim 1, wherein the culturing in step (b) is carried out for 12 to 72 hours.

8. The method according to claim 7, wherein the culturing in step (b) is carried out for 24 to 58 hours.

9. The method according to claim 7, wherein the culturing in step (b) is carried out for 24 to 36 hours.

10. The method according to claim 5, wherein the culturing in step (b) is carried out for at least 12 hours.

11. The method according to claim 5, wherein the culturing in step (b) is carried out for at least 1 day.

12. The method according to claim 5, wherein the culturing step (c) is free of a CD3-binding antibody and a CD28-binding antibody.

13. The method 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.

14. The method 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.

15. The method according to claim 1, further comprising purifying or enriching T cells present in the sample that are CD8 + CD161 + prior to step (b).

16. The method according to claim 1, further comprising, after step (b), purifying or enriching the T cells present in the sample that are CD8 + CD161 + cells.

17. The method according to claim 15, wherein enriching the T cells in the sample comprises fluorescence-activated cell sorting, magnetic bead separation.

18. The method according to claim 17, wherein the magnetic beads are paramagnetic beads.

19. The method according to claim 16, wherein enriching the T cells in the sample comprises fluorescence-activated cell sorting, magnetic bead separation.

20. The method according to claim 19, wherein the magnetic beads are paramagnetic beads.

21. The method according to claim 1, wherein the culturing is carried out for at most 7 days.

22. The method according to claim 1, wherein the culturing is carried out in a serum-containing culture medium.

23. The method according to claim 1, wherein the culturing is carried out in a serum-free culture medium.

24. The method according to claim 1, further comprising obtaining the cells from a subject.

25. The method according to claim 24, wherein the sample is obtained by apheresis.

26. The method according to claim 1, wherein the sample is a cryopreserved sample.

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

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

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

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

31. The method according to claim 1, wherein the sample comprises a T cell subset having an increased percentage of cells comprising CD8 + CD161 + compared to a comparable sample obtained from a subject.

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

33. The method according to claim 1, further comprising assessing the CD161 + T cell content of the sample before step (b), after step (b), or before and after step (b).

34. The method according to claim 33, wherein the assessment is by cell counting or flow cytometry.

35. A T cell composition prepared by the method according to any one of claims 1 to 32.

36. Use of the T cell composition according to claim 35 for the preparation of a medicament that provides a T cell response in a human subject suffering from a disease.

37. The use according to claim 36, wherein the disease is cancer and wherein the CAR or transgenic TCR targets a cancer cell antigen.

38. The use according to claim 37, wherein the subject has undergone a prior anti-cancer therapy.

39. The use according to claim 38, wherein the subject is in remission or has no symptoms of the cancer but includes detectable cancer cells.

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