Immune response cells expressing dominant negative FAS and uses thereof

Abstract

The present disclosure provides methods and compositions for enhancing immune responses against cancer and pathogens. The present disclosure relates to cells comprising an antigen recognition receptor (e.g., a chimeric antigen receptor (CAR) or a T cell receptor (TCR)) and a dominant negative Fas polypeptide. In certain embodiments, the cells are antigen-directed and exhibit enhanced cell persistence and enhanced anti-target therapeutic efficacy.

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 62 / 738,317, filed on September 28, 2018, the entire contents of which are incorporated herein by reference and for which priority is claimed.

[0003] Funding Information

[0004] This invention was made with government support under grants numbered ZIA BC011586 and ZIA BC010763, awarded by the NCI's Intramural Research Program of the NIH Cancer Research Center. The government has certain rights in this invention.

[0005] Sequence Listing

[0006] This application contains a sequence listing submitted electronically in ASCII format, the entire contents of which are incorporated herein by reference. The ASCII copy was created on October 16, 2019, has a name of 072734_0934_SL.txt, and is 42,831 bytes in size. Field of the Invention

[0007] The subject matter of the present disclosure provides methods and compositions for enhancing immune responses against cancer and pathogens. The present disclosure relates to immune response cells comprising dominant - negative Fas polypeptides. The immune response cells can further comprise antigen - recognition receptors (e.g., chimeric antigen receptors (CARs) or T - cell receptors (TCRs)). Background Art

[0008] Adoptive cell immunotherapy with genetically engineered autologous or allogeneic T cells has shown evidence of therapeutic efficacy against a variety of human cancers, including but not limited to melanoma and various B - cell malignancies. By introducing genes encoding artificial T - cell receptors, called chimeric antigen receptors (CARs) or T - cell receptors (TCRs), T cells can be modified to target tumor - associated antigens, thereby conferring specificity to antigens expressed by cancer or virus - infected cells. Immunotherapy is a targeted therapy with the potential to provide cancer treatment.

[0009] Adoptive cell transfer (ACT) using genetically engineered T cells has entered the standard of care for patients with refractory B-cell malignancies, including pediatric acute lymphoblastic leukemia (1) and adult aggressive B-cell lymphoma (2). Regardless of institution, gene vector, or cell composition, the superior efficacy of ACT in hematologic lymphoid malignancies has been consistently observed in multiple clinical trials (3-8). In contrast, patients with solid malignancies, which are the leading cause of cancer-related death in adults overall (9), have relatively little response to adoptive immunotherapy (10-13). Thus, there remains a need for new strategies to enhance the potency of transferred T cells. SUMMARY OF THE INVENTION

[0010] The subject matter of the present disclosure provides cells (e.g., T cells, tumor-infiltrating lymphocytes, or natural killer (NK) cells) comprising a dominant-negative Fas polypeptide. In certain embodiments, the cell comprises: (a) an antigen recognition receptor that binds to an antigen (e.g., a CAR or a TCR), and (b) an exogenous dominant-negative Fas polypeptide. In certain embodiments, the dominant-negative Fas polypeptide comprises at least one modification in the cytoplasmic death domain. In certain embodiments, the at least one modification is selected from a mutation, a deletion, or an insertion. In certain embodiments, the at least one modification is in the cytoplasmic death domain of human Fas. In certain embodiments, the at least one modification in the cytoplasmic death domain prevents the binding between the dominant-negative Fas polypeptide and the FADD polypeptide. In certain embodiments, the dominant-negative Fas polypeptide comprises a deletion of amino acids 230-314 of human Fas having the amino acid sequence shown in SEQ ID NO: 10. In certain embodiments, the dominant-negative Fas polypeptide comprises an amino acid sequence having at least about 80% identity to the amino acid sequence shown in SEQ ID NO: 12. In certain embodiments, the dominant-negative Fas polypeptide has the amino acid sequence shown in SEQ ID NO: 12.

[0011] In certain embodiments, the dominant-negative Fas polypeptide comprises a point mutation at position 260 of human Fas having the amino acid sequence shown in SEQ ID NO: 10. In certain embodiments, the point mutation of human Fas is D260V. In certain embodiments, the dominant-negative Fas polypeptide comprises an amino acid sequence having at least about 80% identity to the amino acid sequence shown in SEQ ID NO: 14. In certain embodiments, the dominant-negative Fas polypeptide has the amino acid sequence shown in SEQ ID NO: 14.

[0012] In certain embodiments, the exogenous dominant-negative Fas polypeptide enhances the cellular persistence of immune-responsive cells. In certain embodiments, the exogenous dominant-negative Fas polypeptide reduces apoptosis or anergy of immune-responsive cells.

[0013] In certain embodiments, the antigen recognition receptor is exogenous or endogenous (e.g., the natural antigen specificity of T cells from peripheral blood after in vitro sensitization and / or selection, or tumor infiltrating lymphocytes). In certain embodiments, the antigen recognition receptor is recombinantly expressed. In certain embodiments, the antigen recognition receptor is expressed from a vector.

[0014] In certain embodiments, the exogenous dominant negative Fas polypeptide is expressed from a vector.

[0015] In certain embodiments, the cell is an immune response cell. In certain embodiments, the cell is a cell of the lymphoid lineage or a cell of the myeloid lineage. In certain embodiments, the cell is selected from T cells, natural killer (NK) cells, B cells, monocytes, and macrophages. In certain embodiments, the cell is a T cell. In certain embodiments, the T cell is a cytotoxic T lymphocyte (CTL), a regulatory T cell, or a natural killer T (NKT) cell. In certain embodiments, the immune response cell is autologous or allogeneic to the intended recipient.

[0016] In certain embodiments, the antigen is a tumor antigen or a pathogen antigen. In certain embodiments, the antigen is a tumor antigen. In certain embodiments, the tumor antigen is selected from CD19, MUC16, MUC1, CA1X, CEA, CD8, CD7, CD10, CD20, CD22, CD30, CLL1, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD133, CD138, EGP-2, EGP-40, EpCAM, erb-B2,3,4, FBP, fetal acetylcholine receptor, folate receptor-a, GD2, GD3, HER-2, hTERT, IL-13R-a2, K-light chain, KDR, mutant KRAS, mutant PIK3CA, mutant IDH, mutant p53, mutant NRAS, LeY, L1 cell adhesion molecule, MAGE-A1, mesothelin, ERBB2, MAGEA3, CT83 (also known as KK-LC-1), p53, MART1, GP100, proteinase 3 (PR1), tyrosinase, survivin, hTERT, EphA2, NKG2D ligand, NY-ES0-1, carcinoembryonic antigen (h5T4), PSCA, PSMA, ROR1, TAG-72, VEGF-R2, WT-1, BCMA, CD123, CD44V6, NKCS1, EGF1R, EGFR-VIII, and CD99, CD70, ADGRE2, CCR1, LILRB2, PRAME, HPV E6 oncoprotein, HPV E7 oncoprotein, and ERBB. In certain embodiments, the tumor antigen is CD19.

[0017] In certain embodiments, the antigen is a pathogen-associated antigen. In certain embodiments, the pathogen-associated antigen is a viral antigen present in cytomegalovirus (CMV), Epstein-Barr virus (EBV), human immunodeficiency virus (HIV), or influenza virus.

[0018] In certain embodiments, the antigen recognition receptor is a T cell receptor (TCR) or a chimeric antigen receptor (CAR). In certain embodiments, the antigen recognition receptor is an endogenous TCR that recognizes a pathogen-associated antigen, and the cell is a pathogen-specific T cell. In certain embodiments, the antigen recognition receptor is an endogenous TCR that recognizes a tumor antigen, and the cell is a tumor-specific T cell. In certain embodiments, the antigen recognition receptor is a CAR. In certain embodiments, the CAR includes an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. In certain embodiments, the CAR further includes a co-stimulatory signaling domain. In certain embodiments, at least one co-stimulatory signaling domain includes a CD28 polypeptide.

[0019] In certain embodiments, the cell further includes a suicide gene. In certain embodiments, the suicide gene is herpes simplex virus thymidine kinase (hsv-tk), inducible caspase 9 suicide gene (iCasp-9), or truncated human epidermal growth factor receptor (EGFRt) polypeptide.

[0020] The subject matter of the present disclosure provides a composition (e.g., a pharmaceutical composition) comprising an effective amount of the cells disclosed herein. In certain embodiments, the composition is a pharmaceutical composition that further comprises a pharmaceutically acceptable carrier. In certain embodiments, the composition is for treating and / or preventing neoplasia and / or pathogen infection.

[0021] The subject matter of the present disclosure provides a method of inducing and / or enhancing an immune response to a target antigen. In certain embodiments, the method comprises administering to a subject an effective amount of the cells disclosed herein or a pharmaceutical composition comprising the same.

[0022] The subject matter of the present disclosure provides a method of reducing the tumor burden in a subject. In certain embodiments, the method comprises administering to a subject an effective amount of the cells disclosed herein or a pharmaceutical composition comprising the same. In certain embodiments, the method reduces the number of tumor cells. In certain embodiments, the method reduces the size of the tumor. In certain embodiments, the method eradicates the tumor in the subject.

[0023] The subject matter of the present disclosure provides methods for treating and / or preventing tumorigenesis or prolonging the survival of a subject having tumorigenesis. In certain embodiments, the method comprises administering to the subject an effective amount of a cell or a pharmaceutical composition comprising the same.

[0024] In certain embodiments, the tumor or tumorigenesis is selected from blood cancers, B-cell leukemia, multiple myeloma, lymphocytic leukemia (ALL), chronic lymphocytic leukemia, non-Hodgkin lymphoma, myeloid leukemia, and myelodysplastic syndrome (MDS). In certain embodiments, the tumorigenesis is B-cell leukemia, multiple myeloma, lymphocytic leukemia (ALL), chronic lymphocytic leukemia, or non-Hodgkin lymphoma, and the antigen is CD19. In certain embodiments, the tumorigenesis is selected from solid cancers. Selected solid malignancies can include cancers derived from the brain, breast, lung, gastrointestinal tract (including the esophagus, stomach, small intestine, large intestine, and rectum), pancreas, prostate, soft tissue / bone, uterus, cervix, ovary, kidney, skin, thymus, testis, head and neck, or liver.

[0025] The subject matter of the present disclosure provides methods for treating blood cancers in a subject. In certain embodiments, the method comprises administering to the subject an effective amount of T cells, wherein the T cells comprise an antigen recognition receptor that binds to an antigen and an exogenous dominant-negative Fas polypeptide. In certain embodiments, the blood cancer is selected from B-cell leukemia, multiple myeloma, acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia, non-Hodgkin lymphoma, myeloid leukemia, and myelodysplastic syndrome (MDS).

[0026] The subject matter of the present disclosure provides methods for treating solid tumors in a subject. In certain embodiments, the method comprises administering to the subject an effective amount of T cells, wherein the T cells comprise an antigen recognition receptor that binds to an antigen and an exogenous dominant-negative Fas polypeptide. In certain embodiments, the solid tumor is selected from tumors derived from the brain, breast, lung, gastrointestinal tract (including the esophagus, stomach, small intestine, large intestine, and rectum), pancreas, prostate, soft tissue / bone, uterus, cervix, ovary, kidney, skin, thymus, testis, head and neck, or liver.

[0027] The subject matter of the present disclosure provides methods for preventing and / or treating pathogen infections in a subject. In certain embodiments, the method comprises administering to the subject an effective amount of the cells disclosed herein or a pharmaceutical composition comprising the same. In certain embodiments, the pathogen is selected from viruses, bacteria, fungi, parasites, and protozoa that can cause disease.

[0028] The subject matter of the present disclosure provides methods of making antigen - specific cells. In certain embodiments, the method comprises introducing into a cell (a) a first nucleic acid sequence encoding an antigen - recognition receptor that binds to an antigen; and (b) a second nucleic acid sequence encoding an exogenous dominant - negative Fas polypeptide. In certain embodiments, one or both of the first nucleic acid sequence and the second nucleic acid sequence are operably linked to a promoter element. In certain embodiments, one or both of the first nucleic acid sequence and the second nucleic acid sequence are included in a vector. In certain embodiments, the vector is a retroviral vector.

[0029] The subject matter of the present disclosure provides a nucleic acid composition comprising (a) a first nucleic acid sequence encoding an antigen - recognition receptor and (b) a second nucleic acid sequence encoding an exogenous dominant - negative Fas polypeptide. In certain embodiments, one or both of (a) and (b) are operably linked to a promoter element. In certain embodiments, one or both of the first nucleic acid sequence and the second nucleic acid sequence are included in a vector. In certain embodiments, the vector is a retroviral vector.

[0030] The subject matter of the present disclosure also provides a vector comprising the nucleic acid composition disclosed herein.

[0031] The subject matter of the present disclosure provides a kit comprising the cells disclosed herein, the nucleic acid composition disclosed herein, or the vector disclosed herein. In certain embodiments, the kit further comprises written instructions for treating and / or preventing tumor formation and / or pathogen infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The following detailed description, given by way of example, can be understood in conjunction with the accompanying drawings, which are not intended to limit the invention to the specific embodiments described.

[0033] Figure 1A-1FDepicts overexpression of death-inducing ligand FASLG in the human tumor microenvironment. (A) Pan-cancer analysis of FASLG expression in the microenvironment of 26 different tumor types relative to matched normal primary tissues. RNA sequencing (RNA-seq) data of human cancers and matched normal tissues extracted from The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression datasets were analyzed using UCSC Xena and shown as normalized RNA-Seq by RSEM values. Statistical comparison of expression between tumor and normal tissues was performed using Mann-Whitney t-test and Bonferroni correction; ***P < 0.001, **P < 0.01, *P < 0.05. (B) Selected, pre-ranked gene set enrichment analysis (GSEA) of all KEGG pathways for genes positively correlated with the averaged FASLG expression in 26 TCGA histologies. The diameter of the circle reflects the number of genes identified in the GSEA signal set. The nominal P values and FDR q values for all shown GSEAs were < 0.001. (C) Pearson correlation coefficients of the top 200 related genes with FASLG gene expression in 26 human cancers in the TCGA database. Selected immune-related genes associated with the GSEA signal sets listed in panel (B) were identified. (D, E) Representative histogram (D) and summary plot (E) of Fas MFI on phenotypically defined CD8a + T cell subsets. The data shown are from peripheral blood T cells of 47 patients and HD. The CD8 + T cell subsets in panels (D) and (E) are defined as follows: TN cells, CD8a + CD45RA + CD45RO - CCR7 + CD62L + CD27 + CD28 + Fas - ; TCM, CD8a + CD45RO + CD45RA - CCR7 + CD62L + ; TEM, CD8a + CD45RO + CD45RA - CCR7 - CD62L - ; TEMRA, CD8a +CD45RA + CCR7 - CD62L - 。(F) At the time of enrollment in the adoptive immunotherapy clinical trial, among age-matched healthy donors (HD; n = 39; left), melanoma patients (MEL; n = 20; middle), and diffuse large B-cell lymphoma patients (DLBCL; n = 17; right), the proportion of TN among all CD8a + T cells. ***P < 0.001, ns = not significant (two-way ANOVA).

[0034] Figure 2A-2D Depicts the prevention of FasL-mediated apoptosis by murine T cells engineered with Fas DNR. (A) Schematic of physiological Fas signaling and the design of two murine Fas dominant-negative receptors (DNR). The retrovirally encoded FasDNR was designed to prevent the recruitment of Fas-associated death domain protein (FADD) by: (i) substituting asparagine for the isoleucine residue at position 246 of the death domain (DD; Fas I246N ), or (ii) truncating most of the intracellular death domain (Fas ΔDD ). Wild-type Fas (Fas WT ) and empty vector were used as controls. The receptors were cloned into a bicistronic vector containing the Thy1.1 reporter gene. EC, extracellular domain; TM, transmembrane domain; T2A, Thosea asigna virus 2A self-cleaving peptide. (B) Experimental schedule for the stimulation, retroviral transduction, expansion, and detection of lz-FasL-mediated apoptosis of WT CD8α I246N T cells modified with Fas ΔDD , Fas WT , Fas + or empty vector control. (C) Representative FACS plots and (D) summary bar graphs showing the frequency of apoptotic Annexin V -1 / PI + -transduced T cells at rest and 6 h after exposure to lz-FasL (50 ng mL + ). Results are shown after gating on transduced Thy1.1 + cells. The data shown represent 6 independently conducted experiments and are shown as mean ± SEM, n = 3 for each condition. ***P < 0.001, ns = not significant (two-way ANOVA).

[0035] Figure 3A-3H Depicts the enhanced survival of Fas DNR-engineered T cells in the tumor microenvironment. (A) T cells engineered with Fas ΔDD DNR (Ly5.1+ Thy1.1 + ) or empty vector control (Ly5.1 - Thy1.1 + ) engineered to be inherently distinguishable WT pmel-1 CD8α + Experimental protocol for the generation and co - infusion of T cells. Prior to reconstitution, transduced T cells were enriched to a ~1:1 mixture with anti - Thy1.1 microbeads, and then a total of 8e 6 T cells were intravenously injected into Thy1.1 - Ly5.1 - mice bearing established B16 melanoma tumors (10d) that had been sublethally irradiated (6Gy). Recipient mice received IL - 2 daily by intraperitoneal injection for 3d, and spleens and tumors were harvested on d7 for analysis. (B) Relative persistence of Fas ΔDD DNR - modified T cells relative to empty vector - modified T cells. Gated live CD8α + Thy1.1 + lymphocytes are shown. Results are representative of two independent experiments, with n = 5 - 8 mice per experiment. ***P < 0.001 (unpaired two - tailed Student’s t test). (C) Representative FACS plots and (D) summary bar graphs of T - cell viability after overnight culture in cytokine - free medium alone, in the presence of B16 melanoma, or with lz - FasL (50 ng mL -1 ) together. Prior to starting the overnight culture, T cells were transduced with Fas ΔDD DNR or empty vector control without bead enrichment. Data are shown after gating on Thy1.1 + and Thy1.1 - lymphocytes. Bar graphs are shown as mean ± SEM and represent four independent experiments, with n = 3 replicates per condition. (E) Relative persistence of Fas ΔDD DNR - modified T cells relative to empty vector - modified T cells in the spleens and tumors of recipient mice. Gated live CD8α + Thy1.1 + lymphocytes. Results are representative of two independent experiments, with n = 5 - 8 mice per experiment. ****P < 0.0001, **P < 0.01, paired two - tailed Student’s t test. (F) Viable Ly5.1 ΔDD transduced with empty or Fas + CD8α + Vβ13 +Total number of cells. (G) Relative fold expansion of empty construct Fas found in the spleen on the designated day, normalized. ΔDD (H) Percentage of live Ly5.1 + CD8α + Vβ13 + cells. Representative plots from 2 independent experiments. Data are shown as mean ± SEM, n = 3 for each condition. *P < 0.05, Wilcoxon rank sum test.

[0036] Figure 4A-4E Depicts that adoptive transfer of Fas DNR-modified T cells does not result in acquired autoimmune lymphoproliferative syndrome (ALPS). Recipients received a sublethal dose of 6 Gy irradiation and then adoptive transfer of 5e 5 purified with beads modified with Fas ΔDD DNR or empty vector control of Thy1.1 + CD3 in the spleens of WT mice of pmel-1T cells + B220 + CD4 - CD8α - (A) Representative FACS plots and (B) summary bar graphs of the frequency of double-negative T cells. Recipient mice also received IL-2 by intraperitoneal injection daily for 3 d. Age-matched wild-type mice and Fas-deficient lpr / lpr mice served as negative and positive controls, respectively. Shown are the persistence and surface phenotypes of transferred Fas ΔDD DNR- or empty vector control-modified pmel-1T cells >6 months later, (C) representative FACS plots (D) summary scatter plots. All data shown represent 5 independent experiments, n = 5 - 8 mice per cohort per experiment. ***P < 0.001, *P < 0.05 (one-way ANOVA). (E) Analysis of Fas ΔDD or empty vector control-modified WT pmel-1CD8α + T cells in B6 mice for long-term persistence experimental design.

[0037] Figure 5A-5H Depicts that adoptive transfer of Fas DNR-modified T cells enhances anti-tumor efficacy, independent of T cell differentiation status. (A) Experimental design for generating WT pmel-1CD8 ΔDD Fas I246N or empty vector control-modified T cells. No treatment as control or received Fas + T cells. ΔDD Fas I246Nor 5×10 modified with empty vector control 5 Thy1.1 purified by beads + (B) Tumor regression and (C) survival of mice with 10d-established B16 melanoma tumors that received pmel-1 cells. Before cell infusion, all treated mice received a sublethal dose of radiation (6 Gy), followed by 3d of intraperitoneal injection of IL-2. (D) Shows CD62L modified with Fas DNR or empty vector control sorted before infusion + CD44 + Thy1.1 + Representative FACS plots of the purity of TCM-like pmel-1 T cells. Untreated or received 5×10 5 Sorted and purified TCM-like Thy1.1 + (E) Tumor regression and (F) survival of mice with 10d-established B16 melanoma tumors that received cells modified with sorted and purified TCM-like Thy1.1. Untreated or received 5×10 5 Sorted and purified TCM-like Thy1.1 + (G) Tumor regression and (H) survival of mice with 10d-established B16 melanoma tumors that received cells modified with sorted and purified TCM-like Thy1.1. All tumor measurements were performed blindly by independent investigators. Representative results from two independent experiments are shown as mean ± SEM, using n = 5-8 mice / cohort. Statistical comparisons were made using the Wilcoxon rank sum test (B, E, G) or the Log-rank Mantel Cox test (C, F, H). **P < 0.01; *P < 0.05.

[0038] Figure 6A-6D Depicts that genetic modification with Fas DNR protects human T cells from FasL-induced apoptosis. (A) Schematic of physiological Fas signaling and the design of two human Fas dominant negative receptors (DNR). The retrovirally encoded human Fas DNR was designed to prevent the recruitment of Fas-associated death domain protein (FADD) by: (i) replacing the aspartic acid residue at position 260 of the death domain with valine (DD; hFas D260V ); or (ii) truncating most of the human intracellular death domain (hFas ΔDD ; ΔDD = deletion of human Fas amino acids 230-314). The empty vector was used as a negative control. The receptor was cloned into a bicistronic vector containing the Thy1.1 reporter gene. EC, extracellular domain; TM, transmembrane domain; T2A, 2A self-cleaving peptide derived from Thosea asigna virus 2A. (B) Stimulation, retroviral transduction, amplification, and detection with Fas D260V 、Fas ΔDDHuman CD8+ T cells derived from peripheral blood mononuclear cells (PBMC) modified with or without empty vector control + Experimental schedule of lz-FasL-mediated apoptosis of T cells. (C) Representative FACS plots and (D) summary plots showing Annexin V apoptosis after 6 h of rest and exposure to titrated concentrations of lz-FasL + Frequency of T cells. Gated transduced (Thy1.1 + ) or untransduced (Thy1.1 - ) T cells are shown. Data are shown as mean ± SEM, n = 3 for each condition, representing 3 independent experiments. *P < 0.05, ns = not significant (Wilcoxon rank sum test).

[0039] Figure 7A-7D Depicts the design and expression of retrovirally encoded murine Fas DNR constructs and controls in murine CD8 + T cells. (A) Schematic of the design of retroviral constructs encoding murine wild-type (WT) Fas or Fas mutants with impaired ability to bind Fas-associated intracellular adaptor molecules via the death domain. WT Fas, Fas with asparagine substituted for isoleucine at position 246 (Fas I246N ) or Fas with truncated intracellular death domain (Fas ΔDD ) were cloned into the MSGV1 expression vector and placed in front of the T2A cleavage site and the Thy1.1 reporter gene. An empty vector containing only the Thy1.1 reporter gene (empty) was used as a negative control. (B) Representative FACS plots and summary bar graphs showing Fas expression 4 d after (C) Thy1.1 and (D) retroviral transduction of Fas-deficient lpr / lpr or WT CD8α + T cells. In the flow plots, the percentage of gated Thy1.1 + or Fas + cells is shown in black, and the MFI of Thy1.1 + or Fas + cells is shown in red. Data in (C) and (D) are shown as mean ± SEM, n = 3 for each condition, representing 12 independent experiments.

[0040] Figure 8A-8D Depicts that Fas DNR blocks lz-FasL-induced AKT activation and T cell differentiation. (A, B) Iz-FasL exposure vs. (A) phospho-AKT S473 and (B) phospho-S6 S235 / 236 in CD8α I246N -, Fas ΔDD - or empty vector control-transduced+ Representative FACS histograms (upper panels) and summary plots (lower panels) of the dose-response relationship in T cells. Results are shown 6 days after activation, retroviral transduction, and expansion in the continuous presence of lz-FasL at the indicated concentrations. (C) In the absence of exogenous FasL, 11 days after transducing CD8α + T cells with Fas I246N , Fas ΔDD or empty vector control, representative FACS plots of T cell differentiation (upper panels) and intracellular IFNγ / IL-2 production (lower panels). Intracellular cytokine staining was measured after incubation with PMA / ionomycin in brefeldin A and monensin for approximately 5 hours. (D) Memory T cell subset composition of CD8α + T cells 11 days after activation, transduction, and expansion in culture. Plots show mean ± SEM for n = 3 for each condition, representing 3 (A, B) and 5 (C, D) independent experiments. *P < 0.05, (Wilcoxon rank sum test).

[0041] Figure 9A-9E Depicts the role of Fas DNR and anti-CD19 CAR-modified T cell therapy in a leukemia mouse model. (A) Experimental design of treatment with syngeneic T cells co-transduced with anti-CD19 CAR and Fas ΔDD or empty vector control in a mouse leukemia model. Before cell infusion, all treated mice received a sublethal dose of radiation (5 Gy), followed by 3 days of intraperitoneal injection of IL-2. Representative FACS plots of (B) co-transduction efficiency and (C) purity of sorted Thy1.1 ΔDD T cells modified with anti-CD19 CAR and Fas + or empty vector control. Survival of mice with 10-day established E2a:PBX pre-B ALL tumors receiving a high CAR T cell dose (5.5 × 10 ΔDD ) of sorted and purified Thy1.1 + T cells modified with anti-CD19 CAR and Fas 5 or empty vector control (D). (E) Survival of mice with 10-day established E2a:PBX pre-B ALL tumors receiving a low CAR T cell dose (1.8 × 10 ΔDD ) of sorted and purified Thy1.1 + T cells modified with anti-CD19 CAR and Fas 5 or empty vector control. All tumor measurements were performed blindly by independent investigators.

[0042] Figure 10A-10G Shown is that the expression of Fas DNR enhanced the anti-apoptotic function and in vivo persistence of the anti-CD19 CAR model. Representative flow plots (A) and summary data (B) of dual transduction of B6 CD8α + T cells with a retroviral construct encoding anti-CD19 CAR and empty or Fas DNR. Analysis was performed on day 11 after Thy1.1 bead enrichment on day 6. (C) Summary bar graphs of relative T cell viability (relative to Fas -1 ) after overnight culture alone in cytokine-free medium, together with lz-FasL (100 ng / ml -1 ), each of anti-CD3 and anti-CD28 at 2 μg / ml ΔDD ) or E2a-PBX in cytokine-free medium. Data shown after gating on Thy1.1 + lymphocytes represent 3 independently conducted experiments and are shown as mean ± SEM, n = 3 per condition. *P < 0.05, ****P < 0.0001, two-way ANOVA. (D) Experimental protocol for the generation and infusion of WT CD8α ΔDD T cells engineered to express anti-CD19 CAR as well as Fas + or empty vector control. Transduced T cells were enriched with Thy1.1 beads before infusion and then the T cells were intravenously infused into sub-lethally irradiated (5 Gy) mice with 4-day established E2a-PBX leukemia. Spleens and BM were harvested on day 14 for analysis. Co-Td, co-transduction. (E) Summary data of viable CD8α + Thy1.1 + lymphocytes in recipient mice spleens and BM. (F) Summary data of E2a-PBX leukemia frequency in the BM of recipient mice. Results in E and F represent 2 independent experiments, n = 3 - 5 mice each. *P < 0.05, **P < 0.01, ****P < 0.0001, one-way ANOVA, corrected with Tukey multiple comparisons. (G) Survival of mice with 4-day established E2a-PBX leukemia untreated or receiving 3 × 10 5 (left) or 2 × 10 5 (right) anti-CD19 CAR + Thy1.1 + -modified cells. Representative results from 4 independent experiments are shown as mean ± SEM using n = 5 mice / cohort. Statistical comparisons were made using the log-rank Mantel-Cox test; *P < 0.05 **P < 0.01.

[0043] Figure 11Depicts that Fas DNR can protect untransduced cells from FasL-mediated apoptosis. The summary bar graph shows the relative frequencies of cell viability of untransduced and transduced T cells 20 hours after exposure to lz-FasL (100 ng mL -1 ). Results are shown after gating on live CD8α + lymphocytes, and viability relative to medium for each transduction condition. Data shown are representative of 3 independent experiments and are shown as mean ± SEM, n = 3 for each condition. ****P < 0.0001, ns = not significant (one-way ANOVA, corrected with Tukey's multiple comparisons).

[0044] Figure 12A-12D Shows that Fas I246N expression in T cells does not cause reversion to WT Fas. (A) Experimental schedule for stimulation, retroviral transduction, and analysis of WT CD8α WT T cells modified with Fas I246N or Fas + . (B) Representative FACS plots of Thy1.1 expression in Fas WT - or Fas I246N -transduced cells on days 6 and 12. (C) Experimental schedule for stimulation, transduction, Thy1.1 enrichment, and sequencing of WT CD8α WT T cells modified with Fas I246N or Fas + . (D) Representative sequencing data showing that WT Fas maintains the A-T-C sequence encoding isoleucine at amino acid position 246, while the Fas I246N sequence is A-A-C, which encodes asparagine at amino acid position 246 in the introduced Fas DNR construct.

[0045] Figure 13 Depicts that IFNγ upregulates FasL on the surface of B16 tumor cells. B16 cells were treated with vehicle (PBS) or IFNγ (100 ng mL -1 ) for 24 hours, and then surface expression of MHC class I (H-2Db; left panel) or FasL (right panel) was analyzed by flow cytometry.

[0046] Figure 14 Illustrates that T cells engineered with Fas DNR are protected from apoptosis induced by various stimuli. The summary bar graph shows the relative frequencies of cell viability of transduced T cells 20 hours after exposure to lz-FasL (100 ng mL -1 ). Results are shown after gating on Thy1.1 + cells, and show viability relative to Fas ΔDDThe data shown are representative of 10 independently performed experiments and are shown as mean ± SEM, n = 3 for each condition. *P < 0.05 **P < 0.01 ****P < 0.0001, ns = not significant (one-way ANOVA, corrected for Tukey's multiple comparisons).

[0047] Figure 15A-15H Fas DNR expression does not induce lymphoproliferation in the ALPS-susceptible MRL strain. (A) Schematic diagram comparing the onset of lymphoproliferation in C57BL / 6B6-lpr mice at 6-9 months (top) and the MRL-lpr strain at 3-4 months. (B) Analysis of CD8α expressing WT anti-CD19 CAR modified with FasΔDD or empty vector control in WT MRL-Mp mice + Experimental design for long-term persistence of T cells. A total of 3 × 10 6 Anti-CD19 CAR + CD8α + T cells were infused intravenously into mice that had received sublethally irradiated (6 Gy XRT). Recipient mice received daily intraperitoneal injections of IL-2 for 3 days, and spleens were harvested 93 days later for analysis. (C) Total spleen weights in recipient mice compared to age-matched wild-type mice and Fas-deficient B6-lpr mice (negative and positive controls, respectively). (D, E) CD3 + B220 + Representative FACS plots (D) and (E) summary bar graphs of the frequency of double negative lymphocytes. (F) Summary bar graphs of the levels of antinuclear antibody (ANA) Ig (top) and anti-dsDNA Ig (bottom) measured by ELISA. (G, H) Summary bar graphs showing the expression of Fas ΔDD Transferred Thy1.1 modified by DNR or empty vector control + T cell persistence (G) and surface phenotype (H). n = 27 mice per cohort. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05, ns = not significant (one-way ANOVA with Tukey's multiple comparison correction).

[0048] Figure 16A-16BDepicts that adoptively transferred T cells modified with Fas DNR do not induce inflammatory infiltration in the lungs of ALPS-susceptible MRL host mice. (A) Representative H&E-stained micrographs, and (B) summary graphs, showing the intensity of inflammatory mononuclear cell infiltration in the lungs of treated mice. Arrows and asterisks point to areas of dense perivascular and peribronchial mononuclear inflammatory infiltration, respectively. Scale bar = 300 μm. All images were scored blindly by an interpretive pathologist. ***P<0.001, ns = not significant (one-way ANOVA, corrected with Tukey's multiple comparisons).

[0049] Figure 17A-17E Shows genetic co-modification of primary human T cells with a Fas dominant-negative receptor (ADD), an antigen-specific TCR (NY-ESO-1, 1G4), and a traceable suicide switch (truncated EGFR). (A) Design of the human retroviral constructs used in these experiments. (B) Schematic of primary human T cells co-modified with TCR and Fas DNR co-modification. (C) Co-expression of human Fas DNR and the tEGFR suicide switch. (D) Production of antigen-specific cytokines and (E) response to lz-FasL.

[0050] Figure 18A-18D Depicts genetic co-modification of primary human T cells with a Fas dominant-negative receptor (ADD), an antigen-specific CAR (anti-CD19, 28z), and a traceable suicide switch (truncated EGFR). (A) Design of the human retroviral constructs used in these experiments. (B) Schematic of primary human T cells co-modified with CAR and Fas DNR co-modification. (C) Time-dependent induction of apoptosis in human T cells modified with tEGFR alone or in combination with hFas DNR after exposure to lz-FasL. (D) Release of antigen-specific cytokines and degranulation in human T cells modified with anti-CD19 CAR alone or in combination with hFas DNR co-modification. Detailed implementation

[0051] The subject matter of the present disclosure provides cells that include genetically modified immune response cells (e.g., T cells or NK cells) that include a dominant negative Fas polypeptide. In certain embodiments, the immune response cells further include an antigen recognition receptor (e.g., a TCR or a CAR). The subject matter of the present disclosure also provides methods of using such cells to induce and / or enhance an immune response against a target antigen, and / or to treat and / or prevent tumor formation, pathogen infection, or other diseases / conditions (e.g., diseases / conditions that require an increased antigen-specific immune response). The subject matter of the present disclosure is at least partially based on the discovery that dominant negative Fas polypeptides enhance cell persistence, prevent activation-induced cell death, prevent FasL-induced cell death, and / or improve the anti-tumor effect of immune response cells.

[0052] 1. Definitions

[0053] Unless otherwise defined, all technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. The following references provide one of ordinary skill in the art with a general definition of many of the terms used in the subject matter of the present disclosure: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, unless otherwise indicated, the following terms have the meanings ascribed to them below.

[0054] As used herein, the terms “about” or “approximately” refer to an acceptable error range around a particular value, as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, in accordance with the practice in the art, “about” can mean within 3 or more standard deviations. Alternatively, “about” can mean within a range of at most 20%, preferably at most 10%, more preferably at most 5%, and still more preferably at most 1% of a given value. Alternatively, particularly with respect to biological systems or methods, the term can mean within an order of magnitude of the numerical value, preferably within 5-fold, more preferably within 2-fold.

[0055] As used herein, the term "immune response cell" refers to a cell that functions in an immune response, or its progenitor or progeny cells.

[0056] "Activating an immune response cell" refers to the induction of signal transduction or a change in protein expression in a cell, resulting in the initiation of an immune response. For example, when the CD3 chains aggregate in response to ligand binding and immunoreceptor tyrosine-based inhibitory motifs (ITAMs), a signal transduction cascade is generated. In certain embodiments, when an endogenous TCR or an exogenous CAR binds to an antigen, the formation of an immune synapse occurs, which includes the aggregation of many molecules near the bound receptor (e.g., CD4 or CD8, CD3γ / δ / ε / ζ, etc.). This aggregation of membrane-bound signaling molecules causes phosphorylation of the ITAM motifs included in the CD3 chains. This phosphorylation, in turn, initiates the T cell activation pathway, ultimately activating transcription factors such as NF-κB and AP-1. These transcription factors induce the overall gene expression of T cells to increase the production of IL-2 for the proliferation and expression of master regulatory T cell proteins, in order to initiate a T cell-mediated immune response.

[0057] "Stimulating an immune response cell" refers to a signal that results in a strong and sustained immune response. In various embodiments, this occurs after activation of immune cells (e.g., T cells) or is mediated simultaneously through receptors including, but not limited to, CD28, CD137 (4-1BB), OX40, CD40, and ICOS. Receiving multiple stimulatory signals is important for establishing a strong and long-term T cell-mediated immune response. T cells can rapidly become suppressed and unresponsive to antigens. Although the effects of these co-stimulatory signals may vary, they generally result in increased gene expression to generate long-lived, proliferative, and anti-apoptotic T cells that respond strongly to antigens for thorough and persistent eradication.

[0058] As used herein, the term "antigen recognition receptor" refers to a receptor that is capable of activating an immune or immune response cell (e.g., a T cell) in response to its binding to an antigen. Non-limiting examples of antigen recognition receptors include natural or endogenous T cell receptors ("TCRs") and chimeric antigen receptors ("CARs").

[0059] As used herein, the term "antibody" refers not only to intact antibody molecules but also to fragments of antibody molecules that retain the ability to bind immunogen. Such fragments are also well known in the art and are frequently used both in vitro and in vivo. Thus, as used herein, the term "antibody" refers not only to intact immunoglobulin molecules but also to the well-known active fragments F(ab')2 and Fab. F(ab')2 and Fab fragments that lack the Fc fragment of the intact antibody are cleared from the circulation more rapidly and may have less non-specific tissue binding of the intact antibody (Wahl et al., J. Nucl. Med. 24:316-325 (1983)). As used herein, antibodies include intact native antibodies, bispecific antibodies; chimeric antibodies; Fab, Fab', single-chain variable region fragments (scFv), fusion polypeptides, and unconventional antibodies. In certain embodiments, an antibody is a glycoprotein that comprises at least two heavy (H) chains and two light (L) chains that are interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as V H ) and a heavy chain constant (C H ) region. The heavy chain constant region consists of three domains, CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated herein as V L ) and a light chain constant C L region. The light chain constant region consists of one domain C L . The V H region and the V L region can be further subdivided into hypervariable regions, called complementarity-determining regions (CDRs), which are interspersed with more conserved regions, called framework regions (FRs). Each V H and V L consists of three CDRs and four FRs, which are arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with an antigen. The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1q).

[0060] As used herein, "CDR" is defined as the amino acid sequence of the complementarity determining regions of an antibody, which are the hypervariable regions of the immunoglobulin heavy and light chains. See, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 4th Ed., Department of Health and Human Services, National Institutes of Health (1987). Generally, an antibody includes three heavy chain and three light chain CDRs or CDR regions in the variable region. The CDRs provide most of the contact residues for binding of the antibody to an antigen or epitope. In certain embodiments, the CDR regions are delineated using the Kabat system (Kabat, E.A., et al. (1991) Sequences of Proteins of Immunological Interest, 5th Ed., Department of Health and Human Services, NIH Publication No. 91-3242).

[0061] As used herein, the term "single-chain variable fragment" or "scFv" is a fusion protein of the variable regions of an immunoglobulin heavy chain (V H ::V L ) and light chain (V H ) that are covalently linked to form a V L ::V H ::V L heterodimer. V H and V L are directly linked, or linked by a peptide-encoding linker (e.g., 10, 15, 20, 25 amino acids) that connects the N-terminus of V H to the C-terminus of V L , or the C-terminus of V H to the N-terminus of V L . The linker is generally rich in glycine to increase flexibility and rich in serine or threonine to increase solubility. Although the constant regions are removed and a linker is introduced, the scFv protein retains the specificity of the original immunoglobulin. Single-chain Fv polypeptide antibodies can be prepared as described by Huston et al. (Proc. Nat. Acad. Sci. USA, 85:5879-5883, 1988) including V H and V LNucleic acid expression of the coding sequence. Additionally, see U.S. Pat. Nos. 5,091,513, 5,132,405, and 4,956,778; and U.S. Patent Publications Nos. 20050196754 and 20050196754. Antagonistic scFvs with inhibitory activity have been described (see, e.g., Zhao et al., Hybridoma (Larchmt) 2008 27(6):455-51; Peter et al., J Cachexia Sarcopenia Muscle Aug. 12, 2012; Shieh et al., J Immunol 2009 183(4):2277-85; Giomarelli et al., Thromb Haemost 2007 97(6):955-63; Fife et al., J Clin Invest 2006 116(8):2252-61; Brocks et al., Immunotechnology 1997 3(3):173-84; Moosmayer et al., Ther Immunol 1995 2(1):31-40). Agonistic scFvs with stimulatory activity have been described (see, e.g., Peter et al., J Biol Chem 2003 25278(38):36740-7; Xie et al., Nat Biotech 1997 15(8):768-71; Ledbetter et al., Crit Rev Immunol 1997 17(5-6):427-55; Ho et al., Biochim Biophys Acta 2003 1638(3):257-66).

[0062] As used herein, the term "affinity" refers to a measure of the strength of binding. Affinity can depend on the tightness of the stereochemical fit between the antibody binding site and the antigenic determinant, the size of the contact area between them, and / or the distribution of charged and hydrophobic groups. As used herein, the term "affinity" also includes "antibody avidity," which refers to the strength of the antigen-antibody bond after formation of a reversible complex. Methods for calculating the affinity of an antibody for an antigen are known in the art and include, but are not limited to, various antigen-binding assays, such as functional assays (e.g., flow cytometry assays).

[0063] As used herein, the term "chimeric antigen receptor" or "CAR" refers to a molecule comprising an extracellular antigen-binding domain and a transmembrane domain fused to an intracellular signaling domain capable of activating or stimulating an immune response cell. In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a scFv. The scFv can be derived from the variable heavy and light regions of a fused antibody. Additionally or alternatively, the scFv can be derived from Fab’s (as opposed to antibodies, e.g., obtained from a Fab library). In certain embodiments, the scFv is fused to a transmembrane domain and then to an intracellular signaling domain. In certain embodiments, the CAR is selected to have a high binding affinity or antibody avidity for the antigen.

[0064] As used herein, the term "nucleic acid molecule" includes any nucleic acid molecule encoding a polypeptide of interest (e.g., a dominant negative Fas polypeptide) or a fragment thereof. Such nucleic acid molecules need not have 100% homology or identity with an endogenous nucleic acid sequence, but can exhibit substantial identity. Polynucleotides having "substantial identity" or "substantial homology" with an endogenous sequence are generally capable of hybridizing to at least one strand of a double-stranded nucleic acid molecule. "Hybridization" refers to the pairing of complementary polynucleotide sequences (e.g., the genes described herein) or portions thereof to form a double-stranded molecule under various stringent conditions. (See, e.g., Wahl, G.M. and S.L.Berger (1987) Methods Enzymol. 152:399; Kimmel, A.R. (1987) Methods Enzymol. 152:507).

[0065] As used herein, the term "conservative sequence modification" refers to an amino acid modification that does not significantly affect or alter the binding characteristics of a CAR of the present disclosure that includes an amino acid sequence (e.g., the extracellular antigen-binding domain of a CAR). Conservative modifications can include amino acid substitutions, additions, and deletions. Modifications can be introduced into the human scFv of a CAR of the present disclosure by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Amino acids can be grouped according to their physicochemical properties (e.g., charge and polarity). A conservative amino acid substitution is an amino acid substitution in which an amino acid residue is replaced with an amino acid within the same group. For example, amino acids can be classified by charge: positively charged amino acids include lysine, arginine, and histidine; negatively charged amino acids include aspartic acid and glutamic acid; and neutral charge amino acids include alanine, asparagine, cysteine, glutamine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. Additionally, amino acids can be classified by polarity: polar amino acids include arginine (basic polar), asparagine, aspartic acid (acidic polar), glutamic acid (acidic polar), glutamine, histidine (basic polar), lysine (basic polar), serine, threonine, and tyrosine; nonpolar amino acids include alanine, cysteine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, and valine. In certain embodiments, conservative substitutions include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In certain embodiments, one or more amino acid residues within or outside of the CDR regions can be replaced with other amino acid residues from the same group, and the retained function of the altered antibody (i.e., the functions described in (c) to (l) above) can be tested using the functional assays described herein. In certain embodiments, no more than one, no more than two, no more than three, no more than four, or no more than five residues are altered in the specified sequences outside of the CDR regions or within the CDR regions.

[0066] As used herein, the percent homology between two amino acid sequences is equivalent to the percent identity between the two sequences. The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology = number of identical positions # / total number of positions # × 100), where the number of gaps and the length of each gap are taken into account and the gaps are introduced to achieve the best alignment of the two sequences. Comparison of sequences and determination of the percent identity between two sequences can be accomplished using mathematical algorithms.

[0067] The percent homology between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4: 11-17 (1988)), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Additionally, the percent homology between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol. Biol. 48: 444-453 (1970)) algorithm, which has been incorporated into the GAP program in the GCG software package (available from www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4, and a length weight of 1, 2, 3, 4, 5, or 6.

[0068] Additionally or alternatively, the amino acid sequences of the subject matter of the present disclosure can further be used as a "query sequence" to search public databases, e.g., to identify related sequences. Such a search can be performed using the XBLAST program (version 2.0) of Altschul et al. (1990) J. Mol. Biol. 215: 403-10. The BLAST protein search can be performed using the XBLAST program with a score = 50 and wordlength = 3 to obtain amino acid sequences homologous to the designated sequences disclosed herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be used as described in Altschul et al., (1997) Nucleic Acids Res. 25(17): 3389-3402. When using the BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

[0069] Moreover, sequence identity can be measured using sequence analysis software (e.g., the sequence analysis software package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX programs). These software programs match identical or similar sequences by assigning a degree of homology to various substitutions, deletions, and / or other modifications.

[0070] "Substantial identity" or "substantial homology" means that a polypeptide or nucleic acid molecule exhibits at least about 50% homology or identity with a reference amino acid sequence (e.g., any amino acid sequence described herein) or nucleic acid sequence (e.g., any nucleic acid sequence described herein). In certain embodiments, such a sequence has at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99% or at least about 100% homology or identity with the amino acid or nucleic acid sequence used for comparison.

[0071] In an exemplary method for determining the degree of identity, the BLAST program can be used, and its probability scores are between e -3 and e -100 , indicating closely related sequences.

[0072] "Analogue" means a structurally related polypeptide or nucleic acid molecule having the function of a reference polypeptide or nucleic acid molecule.

[0073] As used herein, the term "ligand" refers to a molecule that binds to a receptor. In certain embodiments, the ligand binds to a receptor on another cell, thereby allowing cell-cell recognition and / or interaction.

[0074] As used herein, the term "constitutive expression" or "constitutively expressed" means expressed or being expressed under all physiological conditions.

[0075] "Disease" means any condition, disorder or illness that impairs or interferes with the normal function of a cell, tissue or organ, such as tumor formation and pathogen infection of cells.

[0076] "Effective amount" (or "therapeutically effective amount") is an amount sufficient to produce a beneficial or desired clinical result after treatment. The effective amount can be administered to a subject in one or more doses. For treatment, the effective amount is an amount sufficient to relieve, improve, stabilize, reverse or slow the progression of the disease or otherwise reduce the pathological consequences of the disease. The effective amount is usually determined by a physician according to the specific situation and is within the ability of those skilled in the art. When determining the appropriate dose to achieve the effective amount, several factors are usually considered. These factors include the age, sex and weight of the subject, the disease being treated, the severity of the disease, and the form and effective concentration of the cells being administered.

[0077] "Enhanced tolerance" means blocking the activity of autoreactive cells or immunoresponsive cells targeting the transplanted organ or tissue.

[0078] "Endogenous" means that a nucleic acid molecule or polypeptide is normally expressed in a cell or tissue.

[0079] "Exogenous" means that a nucleic acid molecule or polypeptide is not endogenously present in a cell. Thus, the term "exogenous" will encompass any recombinant nucleic acid molecule or polypeptide expressed in a cell, such as foreign, heterologous, and overexpressed nucleic acid molecules and polypeptides. An "exogenous" nucleic acid refers to a nucleic acid that is not present in a native wild-type cell. For example, an exogenous nucleic acid can differ from its endogenous counterpart by sequence, location / orientation, or both. For clarity, an exogenous nucleic acid can have the same or a different sequence relative to its native endogenous counterpart; it can be introduced into the cell itself or its progenitor cells by genetic modification and can optionally be operably linked to additional optional control sequences, such as a non-native promoter or a secretion sequence.

[0080] "Heterologous nucleic acid molecule or polypeptide" means a nucleic acid molecule (e.g., a cDNA, DNA, or RNA molecule) or polypeptide that is not normally present in a cell or a sample obtained from a cell. The nucleic acid can be from another organism or can be, for example, an mRNA molecule that is not normally expressed in the cell or sample.

[0081] "Modulate" means a positive or negative change. Exemplary modulations include a change of about 1%, about 2%, about 5%, about 10%, about 25%, about 50%, about 75%, or about 100%.

[0082] "Increase" means a positive change of at least about 5%. The change can be about 5%, about 10%, about 25%, about 30%, about 50%, about 75%, about 100%, or more.

[0083] "Decrease" means a negative change of at least about 5%. The change can be about 5%, about 10%, about 25%, about 30%, about 50%, about 75%, or even about 100%.

[0084] "Isolated cell" means a cell that is separated from the molecules and / or cellular components that are naturally associated with the cell.

[0085] The terms "isolated", "purified", or "biologically pure" refer to a substance that is, to varying degrees, free from the components that are normally associated with it in its native state. "Isolated" indicates the degree of separation from its original source or environment. "Purified" indicates a higher degree of isolation than "isolated". A "purified" or "biologically pure" protein is sufficiently free of other substances such that any impurities do not substantially affect the biological properties of the protein or cause other adverse consequences. That is, a nucleic acid or peptide is purified if it is produced by recombinant DNA techniques and is substantially free of cellular material, viral material, or culture medium, or if it is chemically synthesized and is substantially free of chemical precursors or other chemicals. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. The term "purified" can indicate that a nucleic acid or protein gives rise to substantially one band on an electrophoretic gel. For proteins that can be modified (e.g., phosphorylated or glycosylated), different modifications can result in different isolated proteins, which can be purified separately.

[0086] As used herein, the term "antigen-binding domain" refers to a domain that is capable of specifically binding to a particular antigenic determinant or group of antigenic determinants present on a cell.

[0087] As used herein, "linker" shall refer to a functional group (e.g., chemical or polypeptide) that covalently attaches two or more polypeptides or nucleic acids so as to link them to each other. As used herein, "peptide linker" refers to one or more amino acids used to couple two proteins together (e.g., to couple V H and V L domains). In certain embodiments, the linker comprises the sequence shown in GGGGSGGGGSGGGGS [SEQ ID NO:1].

[0088] "Neoplasm" refers to a disease characterized by the pathological proliferation of cells or tissues and their subsequent migration or invasion into other tissues or organs. The growth of a neoplasm is typically uncontrolled and progressive and occurs under conditions that do not cause or result in the cessation of normal cell proliferation. Neoplasms can affect a variety of cell types, tissues, or organs, including but not limited to the following organs: bladder, bone, brain, breast, cartilage, glia, esophagus, fallopian tube, gallbladder, heart, intestine, kidney, liver, lung, lymph node, nervous tissue, ovary, pancreas, prostate, skeletal muscle, skin, spinal cord, spleen, stomach, testis, thymus, thyroid, trachea, urogenital tract, ureter, urethra, uterus, and vagina, or their tissue or cell types. Neoplasms include cancers such as sarcomas, carcinomas, or plasmacytomas (malignant tumors of plasma cells).

[0089] "Receptor" refers to a polypeptide or a portion thereof that is present on the cell membrane and selectively binds one or more ligands.

[0090] "Recognition" refers to the selective binding to a target. T cells that recognize tumors can express receptors (such as TCR or CAR) that bind to tumor antigens.

[0091] "Reference" or "control" refers to a standard for comparison. For example, the level of scFv-antigen binding of cells expressing CAR and scFv can be compared with the level of scFv-antigen binding of corresponding cells expressing only CAR.

[0092] "Secreted" means that a polypeptide is released from a cell through the secretory pathway via the endoplasmic reticulum, Golgi apparatus, and vesicles that transiently fuse at the cytoplasmic membrane to release the protein extracellularly.

[0093] "Signal sequence" or "leader sequence" refers to a peptide sequence (such as 5, 10, 15, 20, 25, or 30 amino acids) present at the N-terminus of a newly synthesized protein that directs them into the secretory pathway. Exemplary leader sequences include, but are not limited to, the IL-2 signal sequence: MYRMQLLSCIALSLALVTNS [SEQ ID NO: 2] (human), MYSMQLASCVTLTLVLLVNS [SEQ ID NO: 3] (mouse), the κ leader sequence: METPAQLLFLLLLWLPDTTG [SEQ ID NO: 4] (human), METDTLLLWVLLLWVPGSTG [SEQ ID NO: 5] (mouse); the CD8 leader sequence: MALPVTALLLPLALLLHAARP [SEQ ID NO: 6] (human); the truncated human CD8 signal peptide: MALPVTALLLPLALLLHA [SEQ ID NO: 7] (human); the albumin signal sequence: MKWVTFISLLFSSAYS [SEQ ID NO: 8] (human); and the prolactin signal sequence: MDSKGSSQKGSRLLLLLVVSNLLLCQGVVS [SEQ ID NO: 9] (human). "Soluble" means that a polypeptide can freely diffuse in an aqueous environment (e.g., not membrane-bound).

[0094] "Specifically binds" means that a polypeptide or a fragment thereof recognizes and binds to a biomolecule of interest (such as a polypeptide) but substantially does not recognize and bind to other molecules in a sample (such as a biological sample, which naturally includes the polypeptides disclosed herein).

[0095] As used herein, the term "tumor antigen" refers to an antigen (e.g., polypeptide) that is uniquely or differentially expressed on tumor cells as compared to normal or non-IS neoplastic cells. In certain embodiments, tumor antigens include any polypeptide expressed by a tumor that is capable of activating or inducing an immune response through an antigen recognition receptor (e.g., CD19, MUC-16), or that is capable of inhibiting an immune response through receptor-ligand binding (e.g., CD47, PD-L1 / L2, B7.1 / 2).

[0096] The terms "comprising," "containing" are intended to have the broad meaning given to them in U.S. patent law and can mean "including," "encompassing," etc.

[0097] As used herein, "treatment" refers to a clinical intervention that attempts to alter the course of a disease in an individual or cell being treated and can be used to prevent or during the clinical pathological process. Therapeutic effects of treatment include, but are not limited to, preventing the occurrence or recurrence of a disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease state, and remission or improvement of prognosis. By preventing the progression of a disease or disorder, treatment can prevent deterioration due to the disorder in an affected or diagnosed subject or a subject suspected of having the disorder, and treatment can prevent the onset of the disorder or the symptoms of the disorder in a subject at risk of having or suspected of having the disorder.

[0098] "Individual" or "subject" herein refers to a vertebrate, such as a human or non-human animal, such as a mammal. Mammals include, but are not limited to, humans, primates, farm animals, sport animals, rodents, and pets. Non-limiting examples of non-human animal subjects include rodents such as mice, rats, hamsters, and guinea pigs; rabbits; dogs; cats; sheep; pigs; goats; cows; horses; and non-human primates such as apes and monkeys. As used herein, the term "immunocompromised" refers to a subject having an immunodeficiency. Such a subject is highly susceptible to opportunistic infections, which are caused by organisms that typically do not cause disease in people with a healthy immune system but that can affect people with a poor or suppressed immune system function.

[0099] Other aspects of the subject matter of the present disclosure are described in the following disclosures, all of which are within the scope of the present disclosure.

[0100] 2. Dominant negative Fas polypeptide

[0101] The Fas cell surface death receptor (Fas) is also known as APT1; CD95; FAS1; APO-1; FASTM; ALPS1A; TNFRSF6. GenBank ID: 355 (human), 14102 (mouse), 246097 (rat), 282488 (cow), 486469 (dog). The protein products of Fas include, but are not limited to, NCBI Reference Sequences NP_000034.1, NP_001307548.1, NP_690610.1 and NP_690611.1.

[0102] Fas is a member of the TNF receptor superfamily and contains a death domain. It is involved in the regulation of programmed cell death and is associated with the pathogenesis of various malignancies and immune system diseases. The interaction of Fas with its ligand allows the formation of a death-inducing signaling complex with other components such as Fas-associated death domain protein (FADD), which can induce programmed cell death.

[0103] In certain embodiments, the Fas polypeptide is a human Fas polypeptide. In certain embodiments, the human Fas polypeptide comprises or has the amino acid sequence of NCBI Reference No.: NP_000034.1 (SEQ ID NO: 10), which is provided hereinbelow. In certain embodiments, the human Fas polypeptide comprises or has an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99% or at least about 100% homology or identity to the sequence shown in SEQ ID NO: 10.

[0104]

[0105] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 10 is shown as SEQ ID NO: 11, which is provided below.

[0106]

[0107] In certain embodiments, the term "dominant-negative Fas polypeptide" refers to a dominant-negative form of the Fas polypeptide, which is the gene product of a dominant-negative mutation of the Fas gene. In certain embodiments, the dominant-negative mutation (also known as "antimorphic mutation") has an altered gene product that antagonizes the wild-type allele. In certain embodiments, the dominant-negative Fas polypeptide adversely affects the normal wild-type Fas polypeptide within the same cell. In certain embodiments, the dominant-negative Fas polypeptide interacts with the wild-type Fas polypeptide but blocks its signal transduction to downstream molecules such as FADD.

[0108] In certain non-limiting embodiments, the dominant negative Fas polypeptide comprises a heterologous signal peptide, such as an IL-2 signal peptide, a κ leader sequence, a CD8 leader sequence, or a peptide having substantially equivalent activity.

[0109] In certain embodiments, the dominant negative Fas polypeptide comprises at least one modification in the intracellular domain. In certain embodiments, the at least one modification prevents Fas from binding to the FADD polypeptide. In certain embodiments, the at least one modification is within the death domain. In certain embodiments, the at least one modification is within amino acids from about 200 to about 320 of SEQ ID NO: 10. In certain embodiments, the at least one modification is within amino acids from about 200 to about 319 of SEQ ID NO: 10. In certain embodiments, the at least one modification is within amino acids from about 202 to about 319 of SEQ ID NO: 10. In certain embodiments, the at least one modification is within amino acids from about 226 to about 319 of SEQ ID NO: 10. The death domain of the Fas protein is disclosed in Tartaglia LA et al. Cell. (1993); 74(5):845-53; Itoh and Nagata. J Biol Chem. (1993); 268(15):10932; Boldin MP et al. J Biol Chem. (1995); 270(14); 7795-8; and Huang B et al. Nature (1996); 384(6610):638-41, which are hereby incorporated by reference in their entirety.

[0110] In certain embodiments, the modification is selected from a mutation, a deletion, and an insertion. In certain embodiments, the mutation is a point mutation.

[0111] In certain embodiments, the modification is a deletion. In certain embodiments, the dominant negative Fas polypeptide comprises a partial or complete deletion of the death domain. In certain embodiments, the dominant negative Fas polypeptide comprises or has a deletion of amino acid residues 230-314 of a human wild-type Fas polypeptide (e.g., a polypeptide having the amino acid sequence shown in SEQ ID NO: 10). In certain embodiments, the dominant negative Fas polypeptide having a deletion of amino acid residues 230-314 of a human wild-type Fas polypeptide having an amino acid sequence as shown in SEQ ID NO: 10 is designated "hFas" ΔDD ". hFas ΔDD has the amino acid sequence shown in SEQ ID NO: 12. SEQ ID NO: 12 is provided below.

[0112]

[0113] An exemplary nucleotide sequence encoding the amino acid sequence SEQ ID NO: 12 is as set forth in SEQ ID NO: 13, which is provided below.

[0114]

[0115] In certain embodiments, the dominant negative Fas polypeptide comprises or has an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99% or at least about 100% homology or identity to the amino acid sequence shown in SEQ ID NO: 12. In certain embodiments, the dominant negative Fas polypeptide having an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99% or at least about 100% homology or identity to the amino acid sequence shown in SEQ ID NO: 12 comprises or has a deletion of amino acid residues 230 - 314 of the human Fas polypeptide (e.g., the polypeptide having the amino acid sequence shown in SEQ ID NO: 10).

[0116] In certain embodiments, the modification is a point mutation. In certain embodiments, the dominant negative Fas polypeptide comprises or has a point mutation at position 260 of the human Fas polypeptide (e.g., the polypeptide having the amino acid sequence shown in SEQ ID NO: 10). In certain embodiments, the point mutation is D260V. In certain embodiments, the dominant negative Fas polypeptide having the point mutation D260V of the human wild - type Fas polypeptide is designated "hFas" D260V ". hFas D260V has the amino acid sequence shown in SEQ ID NO: 14. SEQ ID NO: 14 is provided below.

[0117]

[0118] An exemplary nucleotide sequence encoding the amino acid sequence SEQ ID NO: 14 is as set forth in SEQ ID NO: 15, which is provided below.

[0119]

[0120] In certain embodiments, the dominant negative Fas polypeptide comprises or has an amino acid sequence that has at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or at least about 100% homology or identity to the amino acid sequence shown in SEQ ID NO: 14. In certain embodiments, the dominant negative Fas polypeptide having an amino acid sequence that has at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or at least about 100% homology or identity to the amino acid sequence shown in SEQ ID NO: 14 comprises or has a point mutation D260V of a human Fas polypeptide (e.g., a polypeptide having the amino acid sequence shown in SEQ ID NO: 10).

[0121] In certain non-limiting embodiments, the dominant negative Fas polypeptide comprises a heterologous signal peptide, such as an IL-2 signal peptide, a κ leader sequence, a CD8 leader sequence, or a peptide having substantially equivalent activity.

[0122] 3. Antigen recognition receptor

[0123] The present disclosure provides an antigen recognition receptor that binds to an antigen. In certain embodiments, the antigen recognition receptor is a chimeric antigen receptor (CAR). In certain embodiments, the antigen recognition receptor is a T cell receptor (TCR). The antigen recognition receptor can bind to a tumor antigen or a pathogen antigen.

[0124] 3.1. Antigen

[0125] In certain embodiments, the antigen recognition receptor binds to a tumor antigen. Any tumor antigen (antigenic peptide) can be used in the tumor-related embodiments described herein. Antigen sources include, but are not limited to, cancer proteins. The antigen can be expressed as a peptide, or as a full-length protein or a portion thereof. The full-length protein or a portion thereof can be natural or mutagenized. Non-limiting examples of tumor antigens include CD19, MUC16, MUC1, CA1X, CEA, CD8, CD7, CD10, CD20, CD22, CD30, CLL1, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD133, CD138, EGP-2, EGP-40, EpCAM, erb-B2,3,4, FBP, fetal acetylcholine receptor, folate receptor-a, GD2, GD3, HER-2, hTERT, IL-13R-a2, K-light chain, KDR, mutant KRAS (including but not limited to G12V, G12D, G12C), mutant PIK3CA (including but not limited to E52K, E545K, H1047R, H1047L), mutant IDH (including but not limited to R132H), mutant p53 (including but not limited to R175H, Y220C, G245D, G245S, R248L, R248Q, R248W, R249S, R273C, R273L, R273H and R282W), mutant NRAS (including but not limited to Q61K)), LeY, L1 cell adhesion molecule, MAGE-A1, mesothelin, ERBB2, MAGEA3, CT83 (also known as KK-LC-1), p53, MART1, GP100, proteinase 3 (PR1), tyrosinase, survivin, hTERT, EphA2, NKG2D ligand, NY-ES0-1, carcinoembryonic antigen (h5T4), PSCA, PSMA, ROR1, TAG-72, VEGF-R2, WT-1, BCMA, CD123, CD44V6, NKCS1, EGF1R, EGFR-VIII and CD99, CD70, ADGRE2, CCR1, LILRB2, PRAME, HPV E6 oncoprotein, HPV E7 oncoprotein and ERBB. In certain embodiments, the tumor antigen is CD19.

[0126] In certain embodiments, the antigen recognition receptor binds to a human CD19 polypeptide. In certain embodiments, the human CD19 polypeptide comprises the amino acid sequence shown in SEQ ID NO: 16, which is provided below.

[0127]

[0128] In certain embodiments, the antigen recognition receptor binds to the extracellular domain of the human CD19 protein.

[0129] In certain embodiments, the antigen recognition receptor binds to a pathogen antigen, e.g., for treating and / or preventing pathogen infection or other infectious diseases, e.g., in an immunocompromised subject. Non-limiting examples of pathogens include viruses, bacteria, fungi, parasites, and protozoa capable of causing disease.

[0130] Non-limiting examples of viruses include the Retroviridae (e.g., human immunodeficiency virus, such as HIV-1 (also known as HDTV-III, LAVE, or HTLV-III / LAV or HIV-III; and other isolates, such as HIV-LP)); Picornaviridae (e.g., poliovirus, hepatitis A virus, enteroviruses, human coxsackieviruses, rhinoviruses, echoviruses); Calciviridae (e.g., strains that cause gastroenteritis); Togaviridae (e.g., equine encephalitis virus, rubella virus); Flaviridae (e.g., dengue virus, encephalitis virus, yellow fever virus); Coronoviridae (e.g., coronavirus); Rhabdoviridae (e.g., vesicular stomatitis virus, rabies virus); Filoviridae (e.g., Ebola virus); Paramyxoviridae (e.g., parainfluenza virus, mumps virus, measles virus, respiratory syncytial virus); Orthomyxoviridae (e.g., influenza virus); Bunyaviridae (e.g., hantavirus, bunga virus, phlebovirus, and nairovirus); Arenaviridae (hemorrhagic fever viruses); Reoviridae (e.g., reovirus, orbiviruses, and rotaviruses); Birnaviridae; Hepadnaviridae (hepatitis B virus); Parvoviridae (parvovirus); Papovaviridae (papillomavirus, polyomavirus); Adenoviridae (most adenoviruses); Herpesviridae (herpes simplex virus (HSV) 1 and 2, varicella-zoster virus, cytomegalovirus (CMV), herpesvirus); Poxviridae (variola virus, vaccinia virus, poxvirus); and Iridoviridae (e.g., African swine fever virus); and unclassified viruses (e.g., the agent of hepatitis D (thought to be a defective satellite of hepatitis B virus), the agents of non-A, non-B hepatitis (group 1 = enterically transmitted; group 2 = parenterally transmitted (i.e., hepatitis C); Norwalk and related viruses, and astroviruses); human papillomavirus (i.e., HPV), JC virus, Epstein-Barr virus, Merkel cell polyomavirus).

[0131] Non-limiting examples of bacteria include Staphylococci, Streptococcus, Escherichia coli, Pseudomonas species, and Salmonella species. Specific examples of infectious bacteria include, but are not limited to, Helicobacter pyloris, Borelia burgdorferi, Legionella pneumophilia, Mycobacteria sps (e.g., Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium kansaii, Mycobacterium gordonae), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes (Group A Streptococcus), Streptococcus agalactiae (Group B Streptococcus), Streptococcus (viridans group), Streptococcus faecalis, Streptococcus bovis, Streptococcus (anaerobic genus), Streptococcus pneumoniae, pathogenic Campylobacter sp., Enterococcus sp., Haemophilus influenzae, Bacillus antracis, corynebacterium diphtheriae, corynebacterium sp.) Erysipelothrix rhusiopathiae, Clostridium perfringers, Clostridium tetani, Enterobacter aerogenes, Klebsiella pneumoniae, Pasturella multocida, Bacteroides sp., Fusobacterium nucleatum, Streptobacillus moniliformis, Treponema pallidium, Treponema pertenue, Leptospira, Rickettsia, Clostridium difficile, and Actinomyces israelii.

[0132] In certain embodiments, the pathogen antigen is a viral antigen present in cytomegalovirus (CMV), a viral antigen present in Epstein Barr Virus (EBV), a viral antigen present in human immunodeficiency virus (HIV), or a viral antigen present in influenza virus.

[0133] 3.2. T cell receptor (TCR)

[0134] In certain embodiments, the antigen recognition receptor is a TCR. A TCR is a disulfide-linked heterodimeric protein composed of two variable chains that are expressed as part of a complex with invariant CD3 chain molecules. TCRs are found on the surface of T cells and are responsible for recognizing antigen as a peptide bound to a major histocompatibility complex (MHC) molecule. In certain embodiments, the TCR comprises an α chain and a β chain (encoded by TRA and TRB, respectively). In certain embodiments, the TCR comprises a γ chain and a δ chain (encoded by TRG and TRD, respectively).

[0135] Each chain of the TCR consists of two extracellular domains: a variable (V) region and a constant (C) region. The constant region is close to the cell membrane, followed by a transmembrane region and a short cytoplasmic tail region. The variable region binds to the peptide / MHC complex. The variable domains of both chains each have three complementarity-determining regions (CDRs).

[0136] In certain embodiments, the TCR can form a receptor complex with three dimer signal transduction modules CD3δ / ε, CD3γ / ε, and CD247ζ / ζ or ζ / η. When the TCR complex binds to its antigen and MHC (peptide / MHC), the T cell expressing the TCR complex is activated.

[0137] In certain embodiments, the TCR is an endogenous TCR. In certain embodiments, the TCR recognizes viral antigens. In certain embodiments, the TCR is expressed in virus-specific T cells. In certain embodiments, the virus-specific T cells are derived from an individual immune to viral infection, such as BK virus, human herpesvirus 6, Epstein-Barr virus (EBV), cytomegalovirus, or adenovirus. In certain embodiments, the virus-specific T cells are the T cells disclosed in Leen et al., Blood, Vol. 121, No. 26, 2013; Barker et al., Blood, Vol. 116, No. 23, 2010; Tzannou et al., Journal of Clinical Oncology, Vol. 35, No. 31, 2017; or Bollard et al., Blood, Vol. 32, No. 8, 2014, the entire contents of which are incorporated herein by reference in their entirety. In certain embodiments, the TCR recognizes tumor antigens. In certain embodiments, the TCR is expressed in tumor-specific T cells. In certain embodiments, the tumor-specific T cells are tumor-infiltrating T cells generated by culturing T cells with explants of tumors such as melanoma or epithelial carcinoma. In certain embodiments, the tumor-specific T cells are the T cells disclosed in Stevanovic et al., Science, 356, 200-205, 2017; Dudley et al., Journal of Immunotherapy, 26(4): 332-342, 2003; or Goff et al., Journal of Clinical Oncology, Vol. 34, No. 20, 2016, the entire contents of which are incorporated herein by reference in their entirety.

[0138] In certain embodiments, the antigen recognition receptor is a recombinant TCR. In certain embodiments, the antigen recognition receptor is a non-naturally occurring TCR. In certain embodiments, the non-naturally occurring TCR differs from any naturally occurring TCR by at least one amino acid residue. In certain embodiments, the non-naturally occurring TCR differs from any naturally occurring TCR by at least about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, 12, about 13, about 14, about 15, about 20, about 25, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100 or more amino acid residues. In certain embodiments, the non-naturally occurring TCR is obtained by modification of a naturally occurring TCR by at least one amino acid residue. In certain embodiments, the non-naturally occurring TCR is obtained by modification of a naturally occurring TCR by at least about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 20, about 25, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100 or more amino acid residues.

[0139] 3.3. Chimeric Antigen Receptor (CAR)

[0140] In certain embodiments, the antigen recognition receptor is a CAR. A CAR is an engineered receptor that confers or transfers specificity for a target to an immune effector cell or an immune response cell. CARs can be used to transfer the specificity of a monoclonal antibody onto T cells; the transfer of their coding sequences is facilitated by retroviral vectors.

[0141] There are three generations of CARs in total. "First-generation" CARs typically consist of an extracellular antigen-binding domain (e.g., scFv) that is fused to a transmembrane domain, which in turn is fused to a cytoplasmic / intracellular signaling domain. "First-generation" CARs can provide de novo antigen recognition and activate CD4 + and CD8 + T cells via the CD3ζ chain signaling domain in a single fusion molecule, independent of HLA-mediated antigen presentation. "Second-generation" CARs add intracellular signaling domains from various costimulatory molecules (e.g., CD28, 4-1BB, ICOS, OX40) to the cytoplasmic tail region of the CAR to provide additional signals to T cells. "Second-generation" CARs include CARs that provide both costimulation (e.g., CD28 or 4-1BB) and activation (CD3ζ). "Third-generation" CARs include CARs that provide multiple costimulations (e.g., CD28 or 4-1BB) and activation (CD3ζ). In certain embodiments, the antigen recognition receptor is a first-generation CAR.

[0142] In certain non-limiting embodiments, the dissociation constant (K d ) of the extracellular antigen-binding domain of the CAR (embodied as, for example, an scFv or an analogue thereof) binding to the antigen is about 2×10 -7 M or less. In certain embodiments, K d is about 2×10 -7 M or less, about 1×10 -7 M or less, about 9×10 -8 M or less, about 1×10 -8 M or less, about 9×10 -9 M or less, about 5×10 -9 M or less, about 4×10 -9 M or less, about 3×10 -9 M or less, about 2×10 -9 M or less, or about 1×10 -9 M or less. In certain non-limiting embodiments, K d is about 3×10 -9 M or less. In certain non-limiting embodiments, K d is about 1×10 -9 M to about 3×10 -7 M. In certain non-limiting embodiments, K d is about 1.5×10 -9 M to about 3×10 -7 M. In certain non-limiting embodiments, K d is about 1.5×10 -9 M to about 2.7×10 -7 M.

[0143] Binding of the extracellular antigen-binding domain (e.g., in an scFv or analog thereof) can be assayed by, for example, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS analysis, biological assay (e.g., growth inhibition), or Western Blot assay. Each of these assays generally detects the presence of a specific target protein-antibody complex by employing a labeled reagent (e.g., an antibody or scFv) specific for the target complex. For example, an scFv can be radiolabeled and used in a radioimmunoassay (RIA) (see, e.g., Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, Endocrine Society, March 1986, incorporated herein by reference). The radioisotope can be detected by methods such as using a gamma counter or scintillation counter or by autoradiography. In certain embodiments, the extracellular antigen-binding domain of the CAR is labeled with a fluorescent label. Non-limiting examples of fluorescent labels include green fluorescent protein (GFP), blue fluorescent proteins (e.g., EBFP, EBFP2, Azurite, and mKalamal), cyan fluorescent proteins (e.g., ECFP, Cerulean, and CyPet), and yellow fluorescent proteins (e.g., YFP, Citrine, Venus, and YPet).

[0144] According to the subject matter of the present disclosure, a CAR comprises an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen-binding domain specifically binds an antigen, which can be a tumor antigen or a pathogen antigen.

[0145] In certain embodiments, the CAR comprises an extracellular antigen-binding domain that binds to CD19. In certain embodiments, the CAR is one described in Kochenderder, IN et al., Blood. 2010 Nov 11;116(19):3875-86, which is incorporated herein by reference in its entirety.

[0146] 3.3.1. Extracellular antigen-binding domain of CAR

[0147] In certain embodiments, the extracellular antigen-binding domain specifically binds an antigen. In certain embodiments, the antigen is a tumor antigen. In certain embodiments, the tumor antigen is CD19. In certain embodiments, the extracellular antigen-binding domain is a scFv. In certain embodiments, the scFv is a human scFv. In certain embodiments, the scFv is a humanized scFv. In certain embodiments, the scFv is a murine scFv. In certain embodiments, the extracellular antigen-binding domain is a Fab, which is optionally cross-linked. In certain embodiments, the extracellular antigen-binding domain is an F(ab)2. In certain embodiments, any of the foregoing molecules may be included in a fusion protein having a heterologous sequence to form the extracellular antigen-binding domain. In certain embodiments, the scFv is identified by screening a scFv phage library with an antigen-Fc fusion protein. In certain embodiments, the antigen is a tumor antigen. In certain embodiments, the antigen is a pathogen antigen.

[0148] 3.3.2. Transmembrane domain of CAR

[0149] In certain non-limiting embodiments, the transmembrane domain of the CAR comprises a hydrophobic α-helix spanning at least a portion of the membrane. Different transmembrane domains result in different receptor stabilities. Upon antigen recognition, the receptors aggregate and signals are transmitted to the cell. According to the subject matter of the present disclosure, the transmembrane domain of the CAR can comprise a CD8 polypeptide, a CD28 polypeptide, a CD3ζ polypeptide, a CD4 polypeptide, a 4-1BB polypeptide, an OX40 polypeptide, an ICOS polypeptide, a synthetic peptide (not based on a protein associated with an immune response), or a combination thereof.

[0150] In certain embodiments, the transmembrane domain comprises a CD8 polypeptide. In certain embodiments, the CD8 polypeptide comprises or has an amino acid sequence or a fragment thereof having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% homology or identity (homology herein can be determined using standard software such as BLAST or FASTA) with the sequence having NCBI reference number NP_001139345.1 (SEQ ID NO: 17), and / or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions. In certain embodiments, the CD8 polypeptide comprises or has an amino acid sequence that is a contiguous portion of SEQ ID NO: 17, having a length of at least 20, or at least 30, or at least 40, or at least 50 and at most 235 amino acids. Additionally or alternatively, and in various non-limiting embodiments, the CD8 polypeptide comprises or has the amino acid sequence of amino acids 1 to 235, 1 to 50, 50 to 100, 100 to 150, 137 to 209, 150 to 200 or 200 to 235 of SEQ ID NO: 17. In certain embodiments, the CAR comprises the transmembrane domain of CD8 (e.g., human CD8) or a portion thereof. In certain embodiments, the CAR of the present disclosure comprises a transmembrane domain that comprises a CD8 polypeptide, which CD8 polypeptide comprises or has the amino acid sequence of amino acids 137 to 209 of SEQ ID NO: 17. SEQ ID NO: 17 is provided below.

[0151]

[0152] In certain embodiments, the CD8 polypeptide comprises or has an amino acid sequence or a fragment thereof having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% homology or identity (homology herein can be determined using standard software such as BLAST or FASTA) to the sequence with NCBI reference number AAA92533.1 (SEQ ID NO: 18), and / or may optionally include up to one or up to two or up to three conservative amino acid substitutions. In certain embodiments, the CD8 polypeptide comprises or has an amino acid sequence that is a contiguous portion of SEQ ID NO: 18, which is at least about 20, or at least about 30, or at least about 40, or at least about 50, or at least about 60, or at least about 70, or at least about 100, or at least about 200 and at most 247 amino acids in length. Additionally or alternatively, and in various non-limiting embodiments, the CD8 polypeptide comprises or has the amino acid sequence of amino acids 1 to 247, 1 to 50, 50 to 100, 100 to 150, 150 to 200, 151 to 219, or 200 to 247 of SEQ ID NO: 18. In certain embodiments, the CAR comprises the transmembrane domain of CD8 (such as murine CD8) or a portion thereof. In certain embodiments, the CAR of the present disclosure comprises a transmembrane domain that comprises a CD8 polypeptide that comprises or has the amino acid sequence of amino acids 151 to 219 of SEQ ID NO: 18. SEQ ID NO: 18 is provided below.

[0153]

[0154] According to the subject matter of the present disclosure, a "CD8 nucleic acid molecule" refers to a polynucleotide encoding a CD8 polypeptide.

[0155] In some embodiments, the transmembrane domain of the CAR of the present disclosure comprises a CD28 polypeptide. The CD28 polypeptide may comprise or have an amino acid sequence or a fragment thereof having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% homology or identity with the sequence having NCBI reference number NP_006130 (SEQ ID NO: 19), and / or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions. In some non-limiting embodiments, the CD28 polypeptide comprises or has an amino acid sequence that is a continuous portion of SEQ ID NO: 19, which is at least 20, or at least 30, or at least 40, or at least 50 and at most 220 amino acids in length. Additionally or alternatively, in various non-limiting embodiments, the CD28 polypeptide comprises or has the amino acid sequence of amino acids 1 to 220, 1 to 50, 50 to 100, 100 to 150, 114 to 220, 150 to 200, 153 to 179, or 200 to 220 of SEQ ID NO: 19. In some embodiments, the CD28 polypeptide comprises or has the amino acid sequence of amino acids 114 to 220 of SEQ ID NO: 19. In some embodiments, the CAR comprises the transmembrane domain of CD28 (e.g., human CD28) or a portion thereof. In some embodiments, the CAR comprises a CD28 polypeptide that comprises or has the amino acid sequence of amino acids 153 to 179 of SEQ ID NO: 19. SEQ ID NO: 19 is provided below:

[0156]

[0157] An exemplary nucleic acid sequence encoding amino acids 153 to 179 of SEQ ID NO: 19 is shown as SEQ ID NO: 20, which is provided below.

[0158]

[0159] In certain embodiments, the transmembrane domain of the CAR of the present disclosure comprises a CD28 polypeptide. The CD28 polypeptide can have an amino acid sequence or a fragment thereof having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% homology or identity with the sequence having NCBI reference number NP_031668.3 (SEQ ID NO: 21), and / or can optionally comprise up to one or up to two or up to three conservative amino acid substitutions. In certain non-limiting embodiments, the CD28 polypeptide comprises or has an amino acid sequence that is a contiguous portion of SEQ ID NO: 21, which is at least 20, or at least 30, or at least 40, or at least 50 and at most 218 amino acids in length. Additionally or alternatively, in various non-limiting embodiments, the CD28 polypeptide comprises or has the amino acid sequence of amino acids 1 to 218, 1 to 50, 50 to 100, 100 to 150, 114 to 220, 150 to 200, 151 to 177, or 200 to 220 of SEQ ID NO: 21. In certain embodiments, the CD28 polypeptide comprises or has the amino acid sequence of amino acids 114 to 220 of SEQ ID NO: 21. In certain embodiments, the CAR comprises the transmembrane domain of CD28 (e.g., murine CD28) or a portion thereof. In certain embodiments, the CAR comprises a CD28 polypeptide that comprises or has the amino acid sequence of amino acids 151 to 177 of SEQ ID NO: 21. SEQ ID NO: 21 is provided as follows:

[0160]

[0161] According to the subject matter of the present disclosure, a "CD28 nucleic acid molecule" refers to a polynucleotide encoding a CD28 polypeptide.

[0162] In certain non-limiting embodiments, the CAR can further comprise a spacer region that links the extracellular antigen-binding domain to the transmembrane domain. The spacer region can have sufficient flexibility to allow the antigen-binding domain to be oriented in different directions to facilitate antigen recognition. The spacer region can be the hinge region from IgG1, or a CH2CH3 region of an immunoglobulin and a portion of CD3, a portion of the CD28 polypeptide (e.g., a portion of SEQ ID NO: 19 or SEQ ID NO: 21), a portion of the CD8 polypeptide (e.g., a portion of SEQ ID NO: 17 or a portion of SEQ ID NO: 18), a variant having at least about 80%, at least about 85%, at least about 90% or at least about 95% homology or identity with any of the foregoing, or a synthetic spacer sequence.

[0163] 3.3.3. Intracellular signaling domain of CAR

[0164] In certain non-limiting embodiments, the intracellular signaling domain of the CAR comprises a CD3ζ polypeptide, which can activate or stimulate a cell (e.g., a cell of the lymphoid lineage, such as a T cell). Wild-type (“native”) CD3ζ includes three immunoreceptor tyrosine-based activation motifs (“ITAMs”) (e.g., ITAM1, ITAM2, and ITAM3), and transmits an activation signal to a cell (e.g., a cell of the lymphoid lineage, such as a T cell) upon antigen binding. The intracellular signaling domain of the native CD3ζ-chain is the primary transmitter of signals from the endogenous TCR.

[0165] In certain embodiments, the intracellular signaling domain of the CAR comprises a native CD3ζ polypeptide. In certain embodiments, the CD3ζ polypeptide comprises or has an amino acid sequence or a fragment thereof having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homology or identity to the sequence with NCBI reference number NP_932170 (SEQ ID NO: 22), and / or may optionally include up to one or up to two or up to three conservative amino acid substitutions. In certain non-limiting embodiments, the CD3ζ polypeptide comprises or has an amino acid sequence that is at least 20, or at least 30, or at least 40, or at least 50 and at most 164 amino acids in length as a contiguous portion of SEQ ID NO: 22. Additionally or alternatively, in various non-limiting embodiments, the CD3ζ polypeptide comprises or has the amino acid sequence of amino acids 1 to 164, 1 to 50, 50 to 100, 100 to 150, 52 to 164, or 150 to 164 of SEQ ID NO: 22. In certain non-limiting embodiments, the intracellular signaling domain of the CAR comprises a CD3ζ polypeptide having the amino acid sequence of amino acids 52 to 164 of SEQ ID NO: 22. SEQ ID NO: 22 is provided below:

[0166]

[0167] In certain embodiments, the CD3ζ polypeptide comprises or has an amino acid sequence that has at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homology or identity with the sequence having NCBI Reference No: NP_001106864.2 (SEQ ID No: 23), or a fragment thereof, and / or may optionally include up to one or up to two or up to three conservative amino acid substitutions. In certain non-limiting embodiments, the CD3ζ polypeptide comprises or has an amino acid sequence that is a contiguous portion of SEQ ID NO: 23 and is at least about 20, or at least about 30, or at least about 40, or at least about 50, or at least about 90, or at least about 100, and at most 188 amino acids in length. Additionally or alternatively, in various non-limiting embodiments, the CD3ζ polypeptide comprises or has the amino acid sequence of amino acids 1 to 164, 1 to 50, 50 to 100, 52 to 142, 100 to 150, or 150 to 188 of SEQ ID NO: 23. SEQ ID NO: 23 is provided below:

[0168]

[0169] In certain non-limiting embodiments, the intracellular signaling domain of the CAR comprises a CD3ζ polypeptide that comprises or has the amino acid sequence as set forth in SEQ ID NO: 24. SEQ ID NO: 24 is provided below.

[0170]

[0171] In certain embodiments, the intracellular signaling domain of the CAR comprises a murine CD3ζ polypeptide. In certain embodiments, the intracellular signaling domain of the CAR comprises a human CD3ζ polypeptide.

[0172] In certain non-limiting embodiments, the intracellular signaling domain of the CAR does not include a co-stimulatory signaling region, i.e., the CAR is a first-generation CAR.

[0173] In certain non-limiting embodiments, the intracellular signaling domain of the CAR further comprises at least one co-stimulatory signaling region. In certain embodiments, the co-stimulatory region comprises at least one co-stimulatory molecule, which can provide optimal lymphocyte activation. As used herein, a "co-stimulatory molecule" refers to a cell surface molecule other than an antigen receptor or its ligand that is required for an effective response of lymphocytes to an antigen. The at least one co-stimulatory signaling region can comprise a CD28 polypeptide, a 4-1BB polypeptide, an OX40 polypeptide, an ICOS polypeptide, a DAP-10 polypeptide, or a combination thereof. The co-stimulatory molecule can bind to a co-stimulatory ligand, which is a protein expressed on the cell surface that, upon binding to its receptor, generates a co-stimulatory response, i.e., affects the intracellular response to the stimulation generated when an antigen binds to its CAR molecule. Co-stimulatory ligands include, but are not limited to, CD80, CD86, CD70, OX40L, and 4-1BBL. As an example, the 4-1BB ligand (i.e., 4-1BBL) can bind to 4-1BB (also known as "CD137") to provide an intracellular signal that, in combination with the CAR signal, induces the effector cell function of CAR + T cells. CARs comprising an intracellular signaling domain comprising a co-stimulatory signaling region comprising 4-1BB, ICOS, or DAP-10 are disclosed in U.S. Patent 7,446,190, which is incorporated herein by reference in its entirety.

[0174] In certain embodiments, the intracellular signaling domain of the CAR comprises a co-stimulatory signaling region comprising a CD28 polypeptide (e.g., the intracellular domain of CD28 or a portion thereof). In certain embodiments, the intracellular signaling domain of the CAR comprises a co-stimulatory signaling region comprising a CD28 polypeptide (e.g., the intracellular domain of human CD28 or a portion thereof). In certain embodiments, the CD28 polypeptide comprises or has an amino acid sequence having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% homology or identity to the amino acid sequence shown in SEQ ID NO: 19, or a fragment thereof, and / or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions. In certain non-limiting embodiments, the CD28 polypeptide comprises or has an amino acid sequence that is a contiguous portion of SEQ ID NO: 19, which is at least 20, or at least 30, or at least 40, or at least 50 and at most 220 amino acids in length. Additionally or alternatively, in various non-limiting embodiments, the CD28 polypeptide comprises or has the amino acid sequence of amino acids 1 to 220, 1 to 50, 50 to 100, 100 to 150, 114 to 220, 150 to 200, 181 to 220, or 200 to 220 of SEQ ID NO: 19. In certain embodiments, the CD28 polypeptide comprises or has the amino acid sequence of amino acids 181 to 220 of SEQ ID NO: 19.

[0175] In certain embodiments, the intracellular signaling domain of the CAR comprises a co-stimulatory signaling region comprising a CD28 polypeptide (e.g., the intracellular domain of murine CD28 or a portion thereof). In certain embodiments, the CD28 polypeptide comprises or has an amino acid sequence having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homology or identity to the amino acid sequence shown in SEQ ID NO: 21, or a fragment thereof, and / or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions. In certain non-limiting embodiments, the CD28 polypeptide comprises or has an amino acid sequence that is a continuous portion of SEQ ID NO: 21, which is at least about 20, or at least about 30, or at least about 40, or at least about 50 and at most 218 amino acids in length. Additionally or alternatively, in various non-limiting embodiments, the CD28 polypeptide comprises or has the amino acid sequence of amino acids 1 to 218, 1 to 50, 50 to 100, 100 to 150, 114 to 218, 115 to 218, 150 to 200, 178 to 218, or 200 to 218 of SEQ ID NO: 21. In certain embodiments, the CD28 polypeptide comprises or has the amino acid sequence of amino acids 115 to 218 of SEQ ID NO: 21.

[0176] According to the subject matter of the present disclosure, a "CD28 nucleic acid molecule" refers to a polynucleotide encoding a CD28 polypeptide.

[0177] In certain embodiments, the intracellular signaling domain of the CAR comprises the murine intracellular signaling domain of CD28. In certain embodiments, the intracellular signaling domain of the CAR comprises the human intracellular signaling domain of CD28.

[0178] In certain embodiments, the intracellular signaling domain of the CAR comprises a co-stimulatory signaling region that comprises two co-stimulatory molecules: CD28 and 4-1BB, or CD28 and OX40.

[0179] In certain embodiments, the intracellular signaling domain of the CAR includes a co-stimulatory signaling region that includes a 4-1BB polypeptide. In certain embodiments, the intracellular signaling domain of the CAR includes a co-stimulatory signaling region that includes the intracellular domain of 4-1BB or a portion thereof. In certain embodiments, the intracellular signaling domain of the CAR includes a co-stimulatory signaling region that includes the intracellular domain of human 4-1BB or a portion thereof. 4-1BB can act as a tumor necrosis factor (TNF) ligand and has stimulatory activity. In certain embodiments, the 4-1BB polypeptide comprises or has an amino acid sequence having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homology or identity to the sequence with NCBI Reference No. NP_001552 (SEQ ID NO: 25), or a fragment thereof, and / or can optionally include up to one or up to two or up to three conservative amino acid substitutions. In certain non-limiting embodiments, the 4-1BB polypeptide comprises or has an amino acid sequence that is a contiguous portion of SEQ ID NO: 25 that is at least about 20, or at least about 30, or at least about 40, or at least about 50 and at most 255 amino acids in length. Additionally or alternatively, in various non-limiting embodiments, the 4-1BB polypeptide comprises or has the amino acid sequence of amino acids 1 to 255, 1 to 50, 50 to 100, 100 to 150, 150 to 200, 214 to 255, or 200 to 255 of SEQ ID NO: 25. In certain embodiments, the 4-1BB polypeptide comprises or has the amino acid sequence of amino acids 214 to 255 of SEQ ID NO: 24. SEQ ID NO: 25 is provided below:

[0180]

[0181] According to the subject matter of the present disclosure, a "4-1BB nucleic acid molecule" refers to a polynucleotide encoding a 4-1BB polypeptide.

[0182] In certain embodiments, the intracellular signaling domain of the CAR includes the intracellular signaling domain of human 4-1BB or a portion thereof. In certain embodiments, the intracellular signaling domain of the CAR includes the intracellular signaling domain of murine 4-1BB or a portion thereof.

[0183] In certain embodiments, the intracellular signaling domain of the CAR includes a co-stimulatory signaling region that includes an OX40 polypeptide. In certain embodiments, the intracellular signaling domain of the CAR includes a co-stimulatory signaling region that includes the intracellular domain of OX40 or a portion thereof. In certain embodiments, the intracellular signaling domain of the CAR includes a co-stimulatory signaling region that includes the intracellular domain of human OX40 or a portion thereof. In certain embodiments, the OX40 polypeptide comprises or has an amino acid sequence having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homology or identity to the sequence with NCBI reference number NP_003318 (SEQ ID NO: 26), or a fragment thereof, and / or may optionally include up to one or up to two or up to three conservative amino acid substitutions. In certain non-limiting embodiments, the OX40 polypeptide comprises or has an amino acid sequence that is a contiguous portion of SEQ ID NO: 26 that is at least about 20, or at least about 30, or at least about 40, or at least about 50 and up to 277 amino acids in length. Additionally or alternatively, in various non-limiting embodiments, the OX40 polypeptide comprises or has the amino acid sequence of amino acids 1 to 277, 1 to 50, 50 to 100, 100 to 150, 150 to 200, or 200 to 277 of SEQ ID NO: 26. SEQ ID NO: 26 is provided below:

[0184]

[0185] According to the subject matter of the present disclosure, an "OX40 nucleic acid molecule" refers to a polynucleotide encoding an OX40 polypeptide.

[0186] In certain embodiments, the intracellular signaling domain of the CAR includes a co-stimulatory signaling region that includes an ICOS polypeptide. In certain embodiments, the intracellular signaling domain of the CAR includes a co-stimulatory signaling region that includes the intracellular domain of ICOS or a portion thereof. In certain embodiments, the intracellular signaling domain of the CAR includes a co-stimulatory signaling region that includes the intracellular domain of human ICOS or a portion thereof. In certain embodiments, the ICOS polypeptide includes or has an amino acid sequence that has at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homology or identity with the sequence having NCBI reference number NP_036224 (SEQ ID NO: 27), or a fragment thereof, and / or may optionally include up to one or up to two or up to three conservative amino acid substitutions. In certain non-limiting embodiments, the ICOS polypeptide includes or has an amino acid sequence that is a contiguous portion of SEQ ID NO: 27 and is at least about 20, or at least about 30, or at least about 40, or at least about 50 and up to 199 amino acids in length. Additionally or alternatively, in various non-limiting embodiments, the ICOS polypeptide includes or has the amino acid sequence of amino acids 1 to 277, 1 to 50, 50 to 100, 100 to 150, or 150 to 199 of SEQ ID NO: 27. SEQ ID NO: 27 is provided below:

[0187]

[0188] According to the subject matter of the present disclosure, an "ICOS nucleic acid molecule" refers to a polynucleotide that encodes an ICOS polypeptide.

[0189] 3.3.4. Exemplary CAR

[0190] In certain embodiments, the CARs of the present disclosure include: an extracellular antigen-binding domain that binds to a CD19 polypeptide (such as a human CD19 polypeptide), a transmembrane domain that includes a CD28 polypeptide (such as the transmembrane domain of human CD28 or a portion thereof), an intracellular signaling domain that includes a CD3ζ polypeptide, and a co-stimulatory signaling domain that includes a CD28 polypeptide (such as the intracellular domain of human CD28 or a portion thereof). In certain embodiments, the CAR is designated "CD1928ζ". In certain embodiments, the CAR (e.g., CD1928ζ) includes the amino acid sequence shown in SEQ ID NO: 28. SEQ ID NO: 28 is provided below.

[0191]

[0192] An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 28 is shown as SEQ ID NO: 29. SEQ ID NO: 29 is provided below.

[0193]

[0194] 4. Cells

[0195] The subject matter of the present disclosure provides cells comprising a dominant negative Fas polypeptide disclosed herein. In certain embodiments, the cell further comprises an antigen recognition receptor that binds to an antigen (e.g., a CAR or a TCR). In certain embodiments, the dominant negative Fas polypeptide is an exogenous dominant negative Fas polypeptide. In certain embodiments, the antigen recognition receptor is capable of activating the cell. In certain embodiments, the dominant negative Fas polypeptide (e.g., an exogenous dominant negative Fas polypeptide) is capable of promoting the anti-tumor effect of the cell. The cell can be transduced with an antigen recognition receptor and an exogenous dominant negative Fas polypeptide such that the cell co-expresses the antigen recognition receptor and the exogenous dominant negative Fas polypeptide.

[0196] In certain embodiments, the cell is an immune response cell. In certain embodiments, the cell is a cell of the lymphoid lineage. Cells of the lymphoid lineage can provide antibody production, regulation of the cellular immune system, detection of foreign agents in the blood, detection of host foreign cells, etc. Non-limiting examples of cells of the lymphoid lineage include T cells, natural killer (NK) cells, B cells, dendritic cells, and stem cells from which lymphoid cells can differentiate. In certain embodiments, the stem cell is a pluripotent stem cell (e.g., an embryonic stem cell or an induced pluripotent stem cell).

[0197] In certain embodiments, the cell is a T cell. T cells can be lymphocytes that mature in the thymus and are mainly responsible for cell-mediated immunity. T cells are involved in the adaptive immune system. The T cells of the subject matter of the present disclosure can be any type of T cell, including but not limited to helper T cells, cytotoxic T cells, memory T cells (including central memory T cells, stem cell-like memory T cells (or stem-like memory T cells), and two types of effector memory T cells: e.g., T EM cells and T EMRA cells), regulatory T cells (also known as suppressor T cells), tumor-infiltrating lymphocytes (TILs), natural killer T cells, mucosa-associated invariant T cells, and γδ T cells. Cytotoxic T cells (CTLs or killer T cells) are a subset of T lymphocytes capable of inducing the death of infected somatic cells or tumor cells. A patient's own T cells can be genetically modified to target specific antigens by the introduction of an antigen recognition receptor (e.g., a CAR or a TCR). In certain embodiments, the cell is a T cell. T cells can be CD4 + T cells or CD8+ T cells. In certain embodiments, the T cells are CD4 + T cells. In certain embodiments, the T cells are CD8 + T cells.

[0198] In certain embodiments, the cells are virus-specific T cells. In certain embodiments, the virus-specific T cells comprise an endogenous TCR that recognizes a viral antigen. In certain embodiments, the cells are tumor-specific T cells. In certain embodiments, the tumor-specific T cells comprise an endogenous TCR that recognizes a tumor antigen.

[0199] In certain embodiments, the cells are NK cells. Natural killer (NK) cells can be lymphocytes that are part of cell-mediated immunity and play a role in the innate immune response. NK cells do not require prior activation to perform cytotoxic effects on target cells.

[0200] The types of human lymphocytes of the present disclosure include, but are not limited to, peripheral donor lymphocytes, such as those disclosed in the following references: Sadelaín, M. et al., 2003, Nat Rev Cancer 3:35-45 (disclosing peripheral donor lymphocytes genetically modified to express a CAR); Morgan, R.A. et al., 2006 Science 314:126-129 (disclosing peripheral donor lymphocytes genetically modified to express a full-length tumor antigen recognition T cell receptor complex comprising an α and a β heterodimer); Panelli, M.C., et al. 2000 J Immunol 164:495-504; Panelli, M.C., et al. 2000 J Immunol 164:4382-4392 (disclosing lymphocyte cultures derived from tumor-infiltrating lymphocytes (TIL) in tumor biopsies); and Dupont, J., et al. 2005 Cancer Res 65:5417-5427; Papanicolaou, G.A., et al. 2003 Blood 102:2498-2505 (disclosing antigen-specific peripheral blood leukocytes selectively expanded in vitro using artificial antigen-presenting cells (AAPCs) or pulsed dendritic cells). Immune response cells (e.g., T cells) can be autologous, non-autologous (e.g., allogeneic), or derived in vitro from engineered progenitor or stem cells.

[0201] In certain embodiments, the cell is a cell of the myeloid lineage. Non-limiting examples of cells of the myeloid lineage include monocytes, macrophages, basophils, neutrophils, eosinophils, mast cells, erythrocytes, megakaryocytes, platelet cells, and stem cells from which myeloid cells can differentiate. In certain embodiments, the stem cell is a pluripotent stem cell (e.g., an embryonic stem cell or an induced pluripotent stem cell).

[0202] The immune response cells of the present disclosure are capable of modulating the tumor microenvironment. Tumors have a microenvironment that is hostile to the host immune response, which involves a series of mechanisms by which malignant cells protect themselves from immune recognition and elimination. This "hostile tumor microenvironment" includes a variety of immunosuppressive factors, which include infiltrating regulatory CD4 + T cells (Tregs), myeloid-derived suppressor cells (MDSC), tumor-associated macrophages (TAM), immunosuppressive cytokines including TGF-β, and the expression of ligands that target immunosuppressive receptors (CTLA-4 and PD-1) expressed by activated T cells. These immunosuppressive mechanisms play a role in maintaining tolerance and suppressing inappropriate immune responses, but within the tumor microenvironment, these mechanisms prevent an effective anti-tumor immune response. After encountering target tumor cells, these immunosuppressive factors can jointly induce significant anergy or apoptosis of adoptively transferred CAR-modified T cells.

[0203] In certain embodiments, the cells of the present disclosure have increased cell persistence. In certain embodiments, the cells of the present disclosure have reduced apoptosis and / or anergy.

[0204] 5. Compositions and Vectors

[0205] The subject matter of the present disclosure provides compositions comprising a dominant negative Fas polypeptide disclosed herein (e.g., as disclosed in Section 2) and an antigen recognition receptor disclosed herein (e.g., as disclosed in Section 3). Cells (e.g., immune response cells) comprising such compositions are also provided.

[0206] In certain embodiments, the dominant negative Fas polypeptide is operably linked to a first promoter. In certain embodiments, the antigen recognition receptor is operably linked to a second promoter.

[0207] Furthermore, the subject matter of the present disclosure provides nucleic acid compositions comprising a first polynucleotide encoding a dominant negative Fas polypeptide disclosed herein (e.g., as disclosed in Section 2) and a second polynucleotide encoding an antigen recognition receptor disclosed herein (e.g., as disclosed in Section 3). Cells comprising such nucleic acid compositions are also provided.

[0208] In certain embodiments, the nucleic acid composition further comprises a first promoter operably linked to a dominant negative Fas polypeptide. In certain embodiments, the nucleic acid composition further comprises a second promoter operably linked to an antigen recognition receptor.

[0209] In certain embodiments, one or both of the first promoter and the second promoter are endogenous or exogenous. In certain embodiments, the exogenous promoter is selected from the elongation factor (EF)-1 promoter, CMV promoter, SV40 promoter, PGK promoter, long terminal repeat (LTR) promoter, and metallothionein promoter. In certain embodiments, one or both of the first promoter and the second promoter are inducible promoters. In certain embodiments, the inducible promoter is selected from the NFAT transcriptional response element (TRE) promoter, CD69 promoter, CD25 promoter, IL-2 promoter, IL-12 promoter, P40 promoter, and Bcl-xL promoter.

[0210] The above compositions and nucleic acid compositions can be administered to a subject and / or delivered into cells by methods known in the art or as described herein to a subject. Genetic modification of cells (e.g., T cells) can be accomplished by transducing a substantially homogeneous cell composition with a recombinant DNA construct. In certain embodiments, a retroviral vector (γ-retroviral vector or lentiviral vector) is used to introduce the DNA construct into the cells. For example, a first polynucleotide encoding an antigen recognition receptor and a second polynucleotide encoding a dominant negative Fas polypeptide can be cloned into a retroviral vector, and expression can be driven by its endogenous promoter, by the retroviral long terminal repeat, or by a promoter specific for the target cell type of interest. Non-viral vectors can also be used.

[0211] For the initial genetic modification of cells to include a dominant negative Fas polypeptide and an antigen recognition receptor (e.g., a CAR or a TCR), retroviral vectors are commonly used for transduction. However, any other suitable viral vector or non-viral delivery system can also be used. The antigen recognition receptor and the dominant negative Fas polypeptide can be constructed in a single polycistronic expression cassette, multiple expression cassettes of a single vector, or multiple vectors. Examples of elements that produce polycistronic expression cassettes include, but are not limited to, various viral and non-viral internal ribosome entry sites (IRESs, e.g., FGF-1 IRES, FGF-2 IRES, VEGF IRES, IGF-III IRES, NF-κB IRES, RUNX1 IRES, p53 IRES, hepatitis A IRES, hepatitis C IRES, pestivirus IRES, foot-and-mouth disease virus IRES, picornavirus IRES, poliovirus IRES, and encephalomyocarditis virus IRES) and cleavable linkers (e.g., 2A peptides, e.g., P2A, T2A, E2A, and F2A peptides). Combinations of retroviral vectors and suitable packaging systems are also suitable, where the capsid protein will have the function of infecting human cells. Various amphotropic cell lines are known, which include, but are not limited to, PA12 (Miller et al., (1985) Mol. Cell. Biol. 5:431-437); PA317 (Miller et al., (1986) Mol. Cell. Biol. 6:2895-2902); and CRIP (Danos et al., (1988) Proc. Acad. Sci. USA 85:6460-6464). Non-amphotropic particles are also suitable, e.g., particles pseudotyped with VSVG, RD114, or GALV envelopes and any other pseudotyping known in the art.

[0212] Possible transduction methods also include direct co-culture of cells with producer cells, e.g., by the method of Bregni et al. (1992) Blood 80:1418-1422, or culture alone with viral supernatants or concentrated vector stocks with or without appropriate growth factors and polycations, e.g., by the methods of Xu, et al. (1994) Exp. Hemat. 22:223-230; and Hughes, et al. (1992) J. Clin. Invest. 89:1817.

[0213] Other transduction viral vectors can be used to modify cells. In certain embodiments, the selected vectors exhibit high infection efficiency and stable integration and expression (see, e.g., Cayouette et al., Human Gene Therapy 8:423-430, 1997; Kido et al., Current Eye Research 15:833-844, 1996; Bloomer et al., Journal of Virology 71:6641-6649, 1997; Naldini et al., Science 272:263-267, 1996; and Miyoshi et al., Proc. Natl. Acad. Sci. U.S.A. 94:10319, 1997). Other viral vectors that can be used include, for example, adenovirus, lentivirus, and adeno-associated virus vectors, vaccinia virus, bovine papillomavirus, or herpesvirus, such as Epstein-Barr virus (also see, e.g., Miller, Human Gene Therapy 15-14, 1990; Friedman, Science 244:1275-1281, 1989; Eglitis et al., BioTechniques 6:608-614, 1988; Tolstoshev et al., Current Opinion in Biotechnology 1:55-61, 1990; Sharp, The Lancet 337:1277-1278, 1991; Cornetta et al., Nucleic Acid Research and Molecular Biology 36:311-322, 1987; Anderson, Science 226:401-409, 1984; Moen, Blood Cells 17:407-416, 1991; Miller et al., Biotechnology 7:980-990, 1989; LeGal La Salle et al., Science 259:988-990, 1993; and Johnson, Chest 107:77S-83S, 1995). Retroviral vectors have been particularly well developed and have been used clinically (Rosenberg et al., N. Engl. J. Med 323:370, 1990; Anderson et al., U.S. Pat. No. 5,399,346).

[0214] Non-viral methods can also be used for the genetic modification of immune response cells. For example, nucleic acid molecules can be introduced into immune response cells by the following methods: administration of nucleic acids in the case of lipofection (Feigner et al., Proc. Natl. Acad. Sci. U.S.A. 84:7413, 1987; Ono et al., Neuroscience Letters 17:259, 1990; Brigham et al., Am. J. Med. Sci. 298:278, 1989; Staubinger et al., Methods in Enzymology 101:512, 1983), asialoorosomucoid-polylysine conjugation (Wu et al., Journal of Biological Chemistry 263:14621, 1988; Wu et al., Journal of Biological Chemistry 264:16985, 1989), or microinjection under surgical conditions (Wolff et al., Science 247:1465, 1990). Other non-viral gene transfer methods include in vitro transfection using calcium phosphate, DEAE-dextran, electroporation, and protoplast fusion. Liposomes may also be useful for delivering DNA into cells. The transplantation of a normal gene into an affected tissue of a subject can also be accomplished by transferring normal nucleic acids into cell types that can be cultured ex vivo (e.g., autologous or heterologous primary cells or their progeny), and then injecting the cells (or their progeny) into the target tissue or systemically. Recombinant receptors can also be derived or obtained using transposases or targeted nucleases (e.g., zinc finger nucleases, meganucleases, or TALE nucleases, CRISPR). Transient expression can be obtained by RNA electroporation.

[0215] Any targeted genome editing method can also be used to deliver the dominant negative Fas polypeptides and / or antigen recognition receptors disclosed herein to cells or subjects. In certain embodiments, the CRISPR system is used to deliver the dominant negative Fas polypeptides and / or antigen recognition receptors disclosed herein. In certain embodiments, zinc finger nucleases are used to deliver the dominant negative Fas polypeptides and / or antigen recognition receptors disclosed herein. In certain embodiments, the TALEN system is used to deliver the dominant negative Fas polypeptides and / or antigen recognition receptors disclosed herein.

[0216] The clustered regularly interspaced short palindromic repeats (CRISPR) system is a genome editing tool found in prokaryotic cells. When used for genome editing, the system includes Cas9 (a protein that can use crRNA as its guide to modify DNA), CRISPR RNA (crRNA, which contains the RNA that Cas9 uses to guide it to the correct segment of host DNA, and a region that binds to tracrRNA (usually in the form of a hairpin loop) to form an active complex with Cas9), trans-activating crRNA (tracrRNA, which binds to crRNA and forms an active complex with Cas9), and an optional segment of a DNA repair template (DNA that directs the cell's repair process to allow insertion of a specific DNA sequence). CRISPR / Cas9 typically uses plasmids to transfect target cells. The crRNA needs to be designed for each application because this is the sequence that Cas9 uses to recognize and directly bind to the target DNA in the cell. The repair template carrying the CAR expression cassette also needs to be designed for each application because it must overlap the sequences on either side of the incision and encode the inserted sequence. Multiple crRNAs and tracrRNAs can be packaged together to form a single guide RNA (sgRNA). The sgRNA can be ligated to the Cas9 gene and made into a plasmid for transfection into cells.

[0217] Zinc finger nucleases (ZFNs) are artificial restriction endonucleases generated by fusing zinc finger DNA-binding domains to DNA cleavage domains. The zinc finger domains can be engineered to target specific DNA sequences, which allows ZFNs to target desired sequences within the genome. The DNA-binding domains of individual ZFNs typically consist of multiple independent zinc finger repeats, and each can recognize multiple base pairs. The most common method for generating new zinc finger domains is to combine smaller zinc finger "modules" with known specificities. The most common cleavage domain in ZFNs is the non-specific cleavage domain from the type II restriction endonuclease FokI. Using the endogenous homologous recombination (HR) mechanism and a homologous DNA template carrying the CAR expression cassette, ZFNs can be used to insert the CAR expression cassette into the genome. When the target sequence is cleaved by ZFNs, the HR mechanism searches for homology between the damaged chromosome and the homologous DNA template, and then replicates the sequence of the template between the two broken ends of the chromosome, thereby integrating the homologous DNA template into the genome.

[0218] Transcription activator-like effector nucleases (TALENs) are restriction endonucleases that can be engineered to cut specific DNA sequences. The TALEN system works in much the same way as ZFNs. They are created by fusing a transcription activator-like effector DNA-binding domain to a DNA cleavage domain. Transcription activator-like effectors (TALEs) consist of 33–34 amino acid repeat motifs with two variable positions that have a strong ability to recognize specific nucleotides. By assembling arrays of these TALEs, the TALE DNA-binding domain can be engineered to bind to a desired DNA sequence, thus directing the nuclease to cut a specific location in the genome.

[0219] Polynucleotide therapies can be directed by any suitable promoter (such as the human cytomegalovirus (CMV), simian virus 40 (SV40), or metallothionein promoter) and regulated by any appropriate mammalian regulatory element or intron (such as the elongation factor 1a enhancer / promoter / intron construct). For example, if desired, enhancers known to preferentially direct gene expression in specific cell types can be used to direct the expression of the nucleic acid. The enhancers used can include, but are not limited to, those characterized as tissue- or cell-specific enhancers. Additionally optionally, if genomic clones are used as therapeutic constructs, regulation can be mediated by homologous regulatory sequences or, if desired, by regulatory sequences derived from heterologous sources, including any of the aforementioned promoters or regulatory elements.

[0220] Methods for delivering genome editing agents / systems can vary depending on need. In some embodiments, the components of the selected genome editing method are delivered as a DNA construct in one or more plasmids. In some embodiments, the components are delivered via viral vectors. Common delivery methods include, but are not limited to, electroporation, microinjection, gene gun, impalefection, hydrodynamic pressure, continuous infusion, sonication, magnetofection, adeno-associated virus, pseudotyping of viral vectors with envelope proteins, cis and trans elements of replicating vectors, herpes simplex virus, and chemical mediators (such as oligonucleotides, lipid complexes, polyplexes, polycations, dendrimers, inorganic nanoparticles, and cell-penetrating peptides).

[0221] The resulting cells can be grown under conditions similar to those of unmodified cells, thus expanding the modified cells for a variety of purposes.

[0222] 6. Polypeptides and analogs

[0223] Also included in the subject matter of the present disclosure are CD19, CD28, 4-1BB, CD8, CD3ζ, and Fas polypeptides or fragments thereof that are modified in a manner that enhances their anti-tumor activity when expressed in immune response cells. The subject matter of the present disclosure provides methods for optimizing amino acid sequences or nucleic acid sequences by generating sequence variations. These variations can include certain mutations, deletions, insertions, or post-translational modifications. The subject matter of the present disclosure also includes analogs of any of the naturally occurring polypeptides disclosed herein (including, but not limited to, CD19, CD28, 4-1BB, CD8, CD3ζ, and Fas). The analogs can differ from the naturally occurring polypeptides disclosed herein by amino acid sequence differences, by post-translational modifications, or by both. The analogs can exhibit at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more homology with all or a portion of the naturally occurring amino acid sequences of the subject matter of the present disclosure. The length of the sequence comparison is at least 5, 10, 15, or 20 amino acid residues, such as at least 25, 50, or 75 amino acid residues, or more than 100 amino acid residues. Similarly, in an exemplary method for determining the degree of identity, the BLAST program can be used, and probability scores between e -3 and e -100 indicate that the sequences are closely related. Modifications include in vivo and in vitro chemical derivatization of polypeptides, such as acetylation, carboxylation, phosphorylation, or glycosylation; these modifications can occur during polypeptide synthesis or processing or after treatment with isolated modifying enzymes. The analogs can also differ from the naturally occurring polypeptides by changes in the primary sequence. These include natural and induced genetic variations (e.g., as described in Sambrook, Fritsch, and Maniatis, Molecular Cloning: A Laboratory Manual (2nd ed.), CSH Press, 1989, or Ausubel et al., supra, which are generated by random mutagenesis by irradiation or exposure to ethyl methanesulfonate or by site-specific mutagenesis). Also included are cyclic peptides, molecules, and analogs that contain residues other than L-amino acids, such as D-amino acids or non-naturally occurring or synthetic amino acids, such as β or γ amino acids.

[0224] In addition to full-length polypeptides, the subject matter of the present disclosure also provides fragments of any of the polypeptides or peptide domains disclosed herein. As used herein, the term "fragment" refers to at least 5, 10, 13, or 15 amino acids. In certain embodiments, the fragment comprises at least 20 contiguous amino acids, at least 30 contiguous amino acids, or at least 50 contiguous amino acids. In certain embodiments, the fragment comprises at least 60 to 80, 100, 200, 300, or more contiguous amino acids. Fragments can be generated by methods known to those of skill in the art or can result from normal protein processing (e.g., removal of amino acids not required for biological activity from a nascent polypeptide or removal of amino acids by alternative mRNA splicing or alternative protein processing events).

[0225] Non-protein analogs have chemical structures designed to mimic the functional activities of the proteins disclosed herein (e.g., dominant-negative Fas polypeptides). Such analogs may exceed the physiological activity of the original polypeptide. Methods for analog design are well known in the art, and analogs can be synthesized according to these methods by modifying the chemical structure to increase the anti-tumor activity of the original polypeptide when the resulting analog is expressed in immune-responsive cells. These chemical modifications include, but are not limited to, substituting alternative R groups and altering the degree of saturation at specific carbon atoms of the reference polypeptide. In certain embodiments, the protein analogs are relatively resistant to in vivo degradation, resulting in a longer-lasting therapeutic effect after administration. Assays for measuring functional activity include, but are not limited to, those described in the following examples.

[0226] 7. Administration

[0227] The cells of the present disclosure or compositions comprising them can be administered systemically or directly to a subject to induce and / or enhance an immune response against an antigen and / or to treat and / or prevent tumor formation and / or pathogen infection. In certain embodiments, the cells of the present disclosure or compositions comprising them are directly injected into the organ of interest (e.g., the organ affected by tumor formation). Alternatively, for example, the cells of the present disclosure or compositions comprising them can be administered indirectly to the organ of interest by administering to the circulatory system (e.g., tumor vasculature). Amplification and differentiation agents can be provided before, during, or after administration of the cells or compositions to increase the production of T cells or NK cells in vitro or in vivo.

[0228] The cells of the present disclosure can be administered in any physiologically acceptable carrier, typically by intravascular administration, although they can also be introduced into bone or other convenient sites where the cells can find suitable sites for regeneration and differentiation (e.g., the thymus). Typically, at least about 1×10 5 cells will be administered, ultimately reaching about 1×10 10One or more. The cells of the present disclosure may include a purified cell population. Those skilled in the art can readily determine the percentage of the cells of the present disclosure in the population using various well-known methods, such as fluorescence-activated cell sorting (FACS). In a population comprising the cells of the present disclosure, suitable ranges of purity are from about 50% to about 55%, from about 5% to about 60%, and from about 65% to about 70%. In certain embodiments, the purity is from about 70% to about 75%, from about 75% to about 80%, or from about 80% to about 85%. In certain embodiments, the purity is from about 85% to about 90%, from about 90% to about 95%, and from about 95% to about 100%. The dose can be readily adjusted by those skilled in the art (e.g., a decrease in purity may require an increase in dose). The cells can be introduced by injection, catheter, etc.

[0229] The compositions of the present disclosure can be pharmaceutical compositions comprising the cells of the present disclosure or their progenitor cells and a pharmaceutically acceptable carrier. Administration can be autologous or allogeneic. For example, cells or progenitor cells can be obtained from a subject and administered to the same subject or a different compatible subject. Cells or their progeny derived from peripheral blood (e.g., of in vivo, ex vivo, or in vitro origin) can be administered by local injection, including catheter administration, systemic injection, local injection, intravenous injection, or parenteral administration. When administering a therapeutic composition of the subject matter of the present disclosure, it can be formulated into a unit-dose injectable form (solution, suspension, emulsion).

[0230] 8. Dosage form

[0231] Compositions comprising the cells of the present disclosure can be conveniently provided in the form of a sterile liquid preparation, such as an isotonic aqueous solution, suspension, emulsion, dispersion, or viscous composition, which can be buffered to a selected pH. Liquid preparations are generally easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are somewhat more convenient for administration, especially by injection. On the other hand, viscous compositions can be formulated within an appropriate viscosity range to provide a longer contact time with a particular tissue. The liquid or viscous composition can include a carrier, which can be a solvent or dispersion medium, including, for example, water, saline, phosphate-buffered saline, polyols (such as glycerol, propylene glycol, liquid polyethylene glycol, etc.), and suitable mixtures thereof.

[0232] A sterile injectable solution can be prepared by incorporating the genetically modified immune response cells in a required amount of a suitable solvent and incorporating different amounts of other ingredients as needed. Such compositions can be mixed with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, dextrose, etc. The compositions can also be lyophilized. The compositions can include auxiliary substances such as wetting agents, dispersing agents, or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling agents or thickening agents, preservatives, flavoring agents, coloring agents, etc., depending on the route of administration and the desired formulation. Standard textbooks such as "REMINGTON'S PHARMACEUTICAL SCIENCE", 17th edition, 1985, incorporated herein by reference, can be consulted to prepare suitable formulations without undue experimentation.

[0233] Various additives that enhance the stability and sterility of the compositions can be added, including antimicrobial preservatives, antioxidants, chelating agents, and buffering agents. The action of microorganisms can be ensured to be prevented by various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, etc. The absorption of injectable drug forms can be prolonged by using agents that delay absorption such as aluminum monostearate and gelatin. However, according to the subject matter of the present disclosure, any vehicle, diluent, or additive used will have to be compatible with the genetically modified immune response cells or their progenitor cells.

[0234] The compositions can be isotonic, i.e., they can have the same osmotic pressure as blood and tears. The required isotonicity of the compositions can be achieved using sodium chloride or other pharmaceutically acceptable agents such as glucose, boric acid, sodium tartrate, propylene glycol, or other inorganic or organic solutes. Sodium chloride is particularly suitable for buffers containing sodium ions.

[0235] If desired, pharmaceutically acceptable thickening agents can be used to maintain the viscosity of the compositions at a selected level. For example, methylcellulose is readily available economically and is easy to use. Other suitable thickening agents include, for example, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, carbomer, etc. The concentration of the thickening agent can depend on the reagent selected. It is important to use an amount that can achieve the selected viscosity. Obviously, the choice of suitable carrier and other additives will depend on the exact route of administration and the nature of the particular dosage form, such as a liquid dosage form (e.g., whether the composition is formulated as a solution, suspension, gel, or other liquid form, such as a timed-release form or a liquid-filled form).

[0236] For the subject being treated, the number of cells to be administered will vary. In one embodiment, about 10 4 to about 10 10 、about 10 5 to about 10 9 、or about 106 to about 10 8 cells of the present disclosure are administered to a human subject. More effective cells can be administered in a smaller number. In certain embodiments, at least about 1×10 8 , about 2×10 8 , about 3×10 8 , about 4×10 8 or about 5×10 8 cells of the present disclosure are administered to a human subject. The precise determination of the effective dose can be considered based on the individual factors of each subject, including its size, age, gender, weight, and the condition of the specific subject. A person skilled in the art can readily determine the dose from the present disclosure and general knowledge in the art.

[0237] A person skilled in the art can readily determine the amounts of cells and optional additives, vehicles, and / or carriers administered in the composition and in the method. Generally, any additive (other than the active cells and / or agents) is present in a phosphate buffered saline solution in an amount of 0.001% to 50% (by weight) solution, and the active ingredient is present in the order of micrograms to milligrams, such as about 0.0001 wt% to about 5 wt%, about 0.0001 wt% to about 1 wt%, about 0.0001 wt% to about 0.05 wt% or about 0.001 wt% to about 20 wt%, about 0.01 wt% to about 10 wt% or about 0.05 wt% to about 5 wt%. For any composition to be administered to an animal or a human, the following can be determined: toxicity, such as by determining the lethal dose (LD) and LD50 in a suitable animal model such as a rodent like a mouse; the dose of the composition, the concentration of the components therein, and the timing of administering the composition that elicits a suitable response. Such determination does not require undue experimentation based on the knowledge of the skilled person, the present disclosure, and the literature cited herein. Also, the duration of continuous administration can be determined without excessive experimentation.

[0238] 9. Method of treatment

[0239] The subject matter of the present disclosure provides methods for inducing and / or increasing an immune response in a subject in need thereof. The cells of the present disclosure and compositions comprising the same can be used for treating and / or preventing tumorigenesis in a subject. The cells of the present disclosure and compositions comprising the same can be used for prolonging the survival of a subject suffering from tumorigenesis. The cells of the present disclosure and compositions comprising the same can also be used for treating and / or preventing tumorigenesis in a subject. The cells of the present disclosure and compositions comprising the same can also be used for reducing the tumor burden in a subject. The cells of the present disclosure and compositions comprising the same can also be used for treating and / or preventing pathogen infections or other infectious diseases in a subject such as an immunocompromised human subject. These methods comprise administering an effective amount of the cells of the present disclosure or a composition comprising the same (e.g., a pharmaceutical composition) to achieve a desired effect, whether alleviating an existing condition or preventing recurrence. For treatment, the amount administered is an amount effective to produce the desired effect. The effective amount can be provided in a single or series of administrations. The effective amount can be provided in a large dose or by continuous perfusion.

[0240] For adoptive immunotherapy using antigen-specific T cells, a cell dose in the range of about 10 6 -10 10 (e.g., about 10 9 ) is typically infused. When the cells of the present disclosure are administered to a host and subsequently differentiate, the T cells are induced to be specific for a particular antigen. The modified cells can be administered by any method known in the art, which includes, but is not limited to, intravenous, subcutaneous, intranodal, intratumoral, intrathecal, intrapleural, intraperitoneal, and direct administration to the thymus.

[0241] The subject matter of the present disclosure provides methods for treating and / or preventing tumorigenesis in a subject. The method comprises administering an effective amount of the cells of the present disclosure or a composition comprising the same to a subject suffering from tumorigenesis.

[0242] In certain embodiments, the tumorigenesis or tumor is a cancer having increased FASLG RNA expression relative to a matched normal source tissue. See Yamamoto et al., J Clin Invest. (2019); 129(4):1551-1565, which is incorporated herein by reference.

[0243] Non-limiting examples of tumor formation include blood cancers (such as leukemia, lymphoma, and myeloma), ovarian cancer, breast cancer, bladder cancer, brain cancer, colon cancer, bowel cancer, liver cancer, lung cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer, glioblastoma, laryngeal cancer, melanoma, neuroblastoma, adenocarcinoma, glioma, soft tissue sarcoma, and various carcinomas (including prostate cancer and small cell lung cancer). Suitable cancers also include any known cancer in the field of oncology, including, but not limited to, astrocytoma, fibrosarcoma, myxosarcoma, liposarcoma, oligodendroglioma, ependymoma, medulloblastoma, primitive neuroectodermal tumor (PNET), chondrosarcoma, osteosarcoma, pancreatic ductal adenocarcinoma, small cell and large cell lung adenocarcinoma, chordoma, angiosarcoma, endotheliosarcoma, squamous cell carcinoma, bronchioloalveolar carcinoma, epithelial adenocarcinoma and its liver metastases, lymphangiosarcoma, lymphangioendotheliosarcoma, liver cancer, cholangiocarcinoma, synovioma, mesothelioma, Ewing's sarcoma, rhabdomyosarcoma, colon cancer, basal cell carcinoma, sweat gland cancer, papillary carcinoma, sebaceous gland carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, testicular tumor, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, retinoblastoma, leukemia, multiple myeloma, Waldenstrom's macroglobulinemia, and heavy chain disease, breast tumors such as ductal and lobular adenocarcinoma, squamous and adenocarcinoma of the cervix, uterine and ovarian epithelial cancer, prostatic adenocarcinoma, transitional squamous cell carcinoma of the bladder, B and T cell lymphoma (nodular and diffuse), plasmacytoma, acute and chronic leukemia, malignant melanoma, soft tissue sarcoma, and leiomyosarcoma. In certain embodiments, tumor formation is selected from blood cancers (such as leukemia, lymphoma, and myeloma), ovarian cancer, prostate cancer, breast cancer, bladder cancer, brain cancer, colon cancer, bowel cancer, liver cancer, lung cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer, glioblastoma, and laryngeal cancer. In certain embodiments, the immune response cells and compositions comprising them of the present disclosure can be used to treat and / or prevent blood cancers (such as leukemia, lymphoma, and myeloma) or ovarian cancer that are not suitable for conventional therapeutic interventions.

[0244] In certain embodiments, the tumor is a solid cancer or a solid tumor. In certain embodiments, the solid tumor or solid cancer is selected from glioblastoma, prostatic adenocarcinoma, renal papillary cell carcinoma, sarcoma, ovarian cancer, pancreatic adenocarcinoma, rectal adenocarcinoma, colon adenocarcinoma, esophageal cancer, uterine corpus endometrial carcinoma, breast cancer, cutaneous melanoma, lung adenocarcinoma, gastric adenocarcinoma, cervical and endocervical cancer, renal clear cell carcinoma, testicular germ cell tumor, and aggressive B cell lymphoma.

[0245] The subject may have an advanced form of the disease, in which case the treatment goals may include alleviating or reversing disease progression and / or reducing side effects. The subject may have a history of having been treated, in which case the treatment goals typically include reducing or delaying the risk of recurrence.

[0246] Suitable human subjects for treatment generally include two treatment groups that can be distinguished by clinical criteria. Subjects with "advanced disease" or "high tumor burden" are those with clinically measurable tumors. Clinically measurable tumors are tumors that can be detected based on the tumor mass (e.g., by palpation, CAT scan, ultrasound, mammogram, or x-ray; a positive biochemical or histopathological marker alone is not sufficient to identify this population). The pharmaceutical composition is administered to these subjects to elicit an anti-tumor response, with the aim of alleviating their condition. Ideally, the result is a reduction in the tumor mass, but any clinical improvement can constitute a benefit. Clinical improvement includes reducing the risk or rate of progression or reducing the pathological consequences of the tumor.

[0247] A second group of suitable subjects is referred to in the art as the "adjuvant group". These are individuals with a history of tumor formation but who respond to another form of treatment. Previous therapies may include, but are not limited to, surgical resection, radiotherapy, and conventional chemotherapy. As a result, these individuals do not have clinically measurable tumors. However, they are suspected of having a risk of disease progression near the primary tumor site or of metastasis. This group can be further subdivided into high-risk and low-risk individuals. Subdivision is based on characteristics observed before or after the initial treatment. These characteristics are known in the clinical field and are appropriately defined for each different tumor. Typical characteristics of the high-risk subgroup are that the tumor has invaded adjacent tissues or shows lymph node involvement.

[0248] Another group has a genetic susceptibility to tumors but has not yet demonstrated clinical signs of tumors. For example, women of childbearing age who test positive for a gene mutation associated with breast cancer may wish to receive one or more of the immune response cells described herein for prophylactic treatment to prevent the occurrence of tumors until prophylactic surgery is appropriate.

[0249] As a result of the surface expression of antigen recognition receptors that bind to tumor antigens and dominant negative Fas polypeptides (such as exogenous Fas polypeptides), the anti-tumor effects of cells comprising the antigen recognition receptors and dominant negative Fas polypeptides are enhanced, and adoptively transferred T or NK cells are endowed with enhanced and selective cytolytic activity at the tumor site. In addition, after their localization to and proliferation in tumor or virus-infected sites, T cells convert the tumor or virus-infected sites into highly conductive environments for a wide range of immune cells (tumor-infiltrating lymphocytes, NK cells, NKT cells, dendritic cells, and macrophages) that participate in the physiological anti-tumor or antiviral responses.

[0250] In addition, the subject matter of the present disclosure provides methods for treating and / or preventing pathogen infections (such as viral infections, bacterial infections, fungal infections, parasitic infections, or protozoan infections) in, for example, immunocompromised subjects. The methods can include administering to a subject suffering from a pathogen infection an effective amount of the cells of the present disclosure or a composition comprising the same. Exemplary viral infections that are amenable to treatment include, but are not limited to, cytomegalovirus (CMV), Epstein-Barr virus (EBV), human immunodeficiency virus (HIV), and influenza virus infections.

[0251] The cells of the present disclosure (e.g., T cells) can be further modified to avoid or minimize the risk of immune complications (referred to as "malignant T cell transformation"), such as graft-versus-host disease (GvHD), or when healthy tissues express the same target antigen as tumor cells, which can lead to results similar to GvHD. A potential solution to this problem is to engineer a suicide gene into the cells of the present disclosure. Suitable suicide genes include, but are not limited to, herpes simplex virus thymidine kinase (hsv-tk), inducible Caspase 9 suicide gene (iCasp-9), and truncated human epidermal growth factor receptor (EGFRt) polypeptide. In certain embodiments, the suicide gene is the EGFRt polypeptide. The EGFRt polypeptide can effect T cell elimination by administering an anti-EGFR monoclonal antibody (e.g., cetuximab). The EGFRt can be covalently linked upstream of the antigen recognition receptor. The suicide gene can be included in a vector comprising the nucleic acid encoding the CAR of the present disclosure. In this way, administration of a prodrug designed to activate the suicide gene (e.g., a prodrug (e.g., AP1903, which can activate iCasp-9)) during malignant T cell transformation (e.g., GVHD) will trigger apoptosis of T cells expressing the receptor (e.g., expressing the CAR) in which the suicide gene is activated. Incorporating the suicide gene into the antigen recognition receptor (e.g., CAR) of the present disclosure increases the safety level and enables elimination of most of the receptor-expressing (e.g., CAR-expressing) T cells within a very short time. The cells of the present disclosure (e.g., T cells) incorporating the suicide gene can be preemptively eliminated at a given time point after T cell infusion, or eradicated at the earliest signs of toxicity.

[0252] 10. Kit

[0253] The subject matter of the present disclosure provides a kit for inducing and / or enhancing an immune response and / or treating and / or preventing tumor formation or pathogen infection in a subject. In certain embodiments, the kit comprises an effective amount of the cells of the present disclosure or a pharmaceutical composition comprising the same. In certain embodiments, the kit comprises a sterile container; such a container can be a box, ampoule, bottle, vial, tube, bag, sachet, blister pack, or other suitable container forms known in the art. These containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for containing a drug. In certain non-limiting embodiments, the kit comprises an isolated nucleic acid molecule encoding an antigen recognition receptor (e.g., CAR or TCR) specific for an antigen of interest and an isolated nucleic acid molecule encoding a dominant negative Fas polypeptide in an expression form, and the nucleic acid molecules can optionally be included in the same or different vectors.

[0254] If desired, the cells and / or nucleic acid molecules are provided together with instructions for administering the cells or nucleic acid molecules to a subject having or at risk of developing neoplasia, a pathogen or an immune disorder. The instructions typically include information regarding the use of the composition for treating and / or preventing neoplasia or pathogen infection. In certain embodiments, the instructions include at least one of the following: a description of the therapeutic agent; a dosage schedule and administration for treating or preventing neoplasia, pathogen infection or an immune disorder or symptoms thereof; precautions; warnings; indications; contraindications; overdose information; adverse reactions; animal pharmacology; clinical studies; and / or references. The instructions may be printed directly on the container (if any), or provided as a label affixed to the container, or as a separate sheet, booklet, card or folder inside or with the container.

[0255] Example

[0256] Unless otherwise indicated, the practice of the present disclosure employs conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the capabilities of those skilled in the art. These techniques are explained fully in the following references: for example, "Molecular Cloning: A Laboratory Manual", 2nd edition (Sambrook, 1989); "Oligonucleotide Synthesis" (Gait, 1984); "Animal Cell Culture" (Freshney, 1987); "Methods in Enzymology", "Handbook of Experimental Immunology" (Weir, 1996); "Gene Transfer Vectors for Mammalian Cells" (Miller and Calos, 1987); "Current Protocols in Molecular Biology" (Ausubel, 1987); "PCR: The Polymerase Chain Reaction", (Mullis, 1994); "Current Protocols in Immunology" (Coligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides disclosed herein and are thus contemplated in the preparation and implementation of the subject matter disclosed herein. Specific available techniques for specific embodiments will be discussed in the following sections.

[0257] The following examples are put forward to provide those of ordinary skill in the art with a complete disclosure and description of how to prepare and use the cells and compositions of the present disclosure, and are not intended to limit the scope of what the inventors regard as their invention.

[0258] Example 1 - T Cells Engineered to Overcome Death Signaling within the Tumor Microenvironment Enhance Adoptive Cancer Immunotherapy

[0259] Introduction

[0260] Multiple variables can affect the success or failure of adoptive T cell-mediated cancer regression (15). These can include the T cell differentiation state (16) and local immunosuppressive factors present in the tumor-bearing host (17). Despite these complexities, one of the most consistent response-related single factors observed in both hematological cancers (2-5,7) and solid cancers (10,18,19) is the expansion and / or persistence of adoptively transferred T cells following infusion.

[0261] The following hypothesis was proposed: that disruption of factors that negatively regulate T cell proliferation and survival might represent potential actionable pathways to enhance adoptive immunotherapy. Multiple clinical trials have tested whether extracellular approaches can improve the persistence of adoptively transferred T cells, including co-administration of immune checkpoint inhibitors (20,21). However, these drugs do not always effectively penetrate the solid tumor microenvironment (22) and may cause non-specific immune activation, leading to systemic toxicity without contributing to efficacy (23). Therefore, a cell-intrinsic strategy was sought to specifically enhance functions within tumor-specific T cells, thereby controlling the risk of systemic toxicity and leveraging the ability to reliably genetically engineer human T cells for clinical use.

[0262] Using pan-cancer analysis to identify candidate ligands that can limit the ability of T cells to expand and persist in tumor-bearing hosts, it was found that the canonical apoptosis-inducing ligand FASLG is preferentially expressed in the majority of human tumor microenvironments. Additionally, it was found that most therapeutic T cells used for constitutive adoptive immunotherapy express Fas (the cognate receptor for FasL). Based on these findings, a series of Fas dominant-negative receptors (DNRs) were developed that function in both primary murine T cells and human T cells to block FasL-induced apoptosis. Adoptively transferred Fas DNR-engineered T cells showed enhanced T cell persistence and anti-tumor immunity without causing uncontrolled lymphoproliferation. Collectively, these results provide a potential general strategy to improve the persistence and viability of adoptively transferred T cells across a broad range of human malignancies following ACT.

[0263] Methods and Materials

[0264] Human specimens: In the adoptive immunotherapy clinical protocol, peripheral blood mononuclear cells (PBMCs) were obtained from age- and sex-matched healthy donors or melanoma patients and diffuse large B cell lymphoma (DLBCL) patients. All anonymous NIH blood bank donors and cancer patients providing PBMC samples were enrolled in a clinical trial approved by the NIH Clinical Center and the NCI Institutional Review Board. Each patient signed an informed consent form and received a patient information sheet prior to participation.

[0265] Pan-cancer bioinformatics analysis of The Cancer Genome Atlas (TCGA): RNA sequencing (RNA-seq) data from 26 human cancers in the TCGA dataset and matched normal tissues from the GTEx dataset were collected and analyzed in the form of normalized RNA-seq by RSEM (RNA-Seq by Expectation Maximization) values via UCSC Xena. FASLG gene expression (as normalized RSEM counts) was analyzed separately. Statistical data were corrected by the Mann-Whitney U test. To identify genes positively correlated with FASLG expression, pre-ranked gene set enrichment was performed for all KEGG pathways in the mSigDB database. Pearson correlation was performed on the top 1000 genes that were positively correlated with FASLG expression on average across 26 TCGA histologies.

[0266] Mice: Adult male or female C57BL / 6NCR (B6; Ly5.2 + ) were purchased from Charles River Laboratories of NCI Frederick. B6.SJL-Ptprc a Pepc b / BoyJ (Ly5.l + ), B6.129S7-Rag1Lm / Mom / J (Rag), B6.MRL-Fas l p r / J (lpr), B6.Cg-Thy1 a / Cy Tg(TcraTcrb)8Rest / J (pmel-1(67)), MRL / MpJ (MRL-Mp) and MRL / MpJ-Faslpr / J (MRL-lpr) mice were purchased from Jackson Laboratory. When indicated, pmel-1 mice were crossed with Ly5.1, Rag or Rag x lpr backgrounds. All mice were maintained under specific pathogen-free conditions. Animal experiments were approved by the Institutional Animal Care and Use Committees of the NCI and conducted in accordance with NIH guidelines.

[0267] Retroviral vectors and murine and human CD8 +Transduction of T cells: Synthetic murine and human Fas cDNA sequences were synthesized and cloned (Genscript) into the MSGV retroviral plasmid, upstream of the T2A skipping sequence and the selectable marker Thy1.1. Murine T cell transduction was performed as previously described (68). Briefly, prior to transfection, Platinum-E amphotropic packaging cells (CellBioLabs) were plated in a BioCoat 10 cm dish (Corning) overnight. The next day, 24 μg of retroviral plasmid DNA encoding MSGV-Thy1.1 (empty), MSGV-WT-mFas-Thy1.1 (mWT), MSGV-I246N-mFas-Thy1.1 (Fas I246N ), or MSGV-ΔDD-mFas-Thy1.1 (Fas ΔDD ), or MSGV-1D3-28Z (anti-CD19 CAR) (71) was separately mixed with 6 μg of pCL-Eco plasmid DNA and applied to Platinum-E cells in antibiotic-free 10% medium together with 60 μL of Lipofectamine 2000 (ThermoFisher) in OptiMEM for 7 h.

[0268] Plasmids encoding human Fas mutant genes were subcloned into the murine leukemia virus-based SFG retroviral vector as described by Maher et al., Nat Biotechnol (2002); 20:70-75.

[0269] After 7 h, the medium was changed; after 48 h, the viral supernatant was collected from the cells and centrifuged to remove debris. The retroviral supernatant was centrifuged at 2000 × g at 32 °C for 2 h on a non-tissue culture-treated 24-well plate that had been coated overnight with 20 μg / mL Retronectin (Takara Bio). Activated CD8α + T cells that had been activated for 24 h were added to the plate, but 100 μL of the viral supernatant was removed from the plate, centrifuged at 1500 rpm at 32 °C for 5 min, and then incubated overnight. The next day, transduction was repeated a second time in the same manner. For human T cell transduction, 293T cells (69) and RD114 were used instead of Platinum-E cells, and transfection and virus harvest were performed according to the murine virus production procedure described above.

[0270] T cell culture and Fas death assay: Human PBMCs were obtained from healthy donors or patients by leukapheresis or venipuncture and centrifuged on a Ficoll-Hypaque (Lonza) gradient to remove red blood cells and isolate lymphocytes. Cells were washed twice with PBS containing 1 mM EDTA, stained with a fixable viability dye in PBS (Thermo Fisher), and then washed twice with PBS supplemented with 2% FBS and 1 mM EDTA (FACS buffer). Human CD8α + T cells were isolated using a human CD8 isolation kit (Stem Cell Technologies). Mouse and human T cells, as well as E2a-PBX leukemia cells (72), were maintained in RPMI 1640 (Gibco) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1% penicillin / streptomycin (100 U / mL and 100 μg / mL, respectively; Gibco), gentamicin (10 μg / mL), MEM non-essential amino acids (Gibco), sodium pyruvate (1 nM), GlutaMAX (2 mM), 0.011 mM 2-mercaptoethanol, and amphotericin B (250 ng / mL). B16-mhgp100 tumor cells, Platinum-E cells, and 293T cells were maintained in DMEM (Gibco) supplemented with 10% FBS and the above additives.

[0271] Uninfected mouse CD8α + T cells were isolated from splenocytes using a MACS CD8 + negative selection kit (Miltenyi Biotec) and stimulated in tissue culture-treated 24-well plates with plate-bound anti-CD3 (2 μg / mL, clone 145-2C11, BD Biosciences), soluble anti-CD28 (1 μg / mL, clone 37-51, BD Biosciences), and IL-2 (5 ng / mL). Pmel-1 T cells were stimulated in whole splenocyte cultures with 1 μg / mL human gp100 (25-33) peptide and IL-2 (5 ng / mL, Prometheus). Human PBMCs or CD8α +T cells were stimulated with plate-bound anti-CD3 (1 μg / mL, clone OKT3, BD Biosciences) and soluble anti-CD28 (1 μg / mL, clone CD28.2, BD Biosciences) for 2 days, and then IL-2 (20 ng / mL) was given during the remaining culture period. Cells were stimulated for 24 hours prior to transduction with viral supernatant on days 1 and 2 of culture. On day 3, cells were removed from the Retronectin-coated plates and returned to tissue culture-treated 24-well plates or flasks. Notably, cells were grown in medium or with lz-FasL (recombinant form of oligomeric FasL) at the indicated concentrations (43, 52). Five to six days after stimulation, T cells were washed twice in PBS and seeded at 1 - 2×10 5 cells / well in 24-well plates with the indicated concentration of lz-FasL and incubated at 37 °C, 5% CO2 for 6 or 24 hours. Cells were then washed twice and stained for annexin V and PI positivity, or with a live / dead fixation dye (Thermo Fisher) and for CD8α (clone 53-6.7, BD Biosciences) and Thy1.1 (clone HIS51, eBioscience).

[0272] Flow cytometry, intracellular cytokine staining, and phosphoflow analysis: Cells were stained with a fixable viability dye (Thermo Fisher) in PBS and then washed twice with PBS supplemented with 2% FBS and 1 mM EDTA (FACS buffer). Cells were stained with antibodies conjugated to the following fluorochromes: CD3 (UCHT1), CCR7 (3D12), CD45RA (HI100), CD45RO (UCHL1), CD28 (CD28.2), CD95 (DX2) (BD Biosciences); and CD27 (M-T271), CD62L (DREG-56), CD8α (SK1), CD4 (OKT4) (BioLegend).

[0273] Mouse T cells, BM, and splenocytes were stained with fixable live / dead dye and then stained with the following antibodies: CD3 (145-2C11), CD8α (53-6.7), Vβ13 (MR12-3), Ly5.1 (A20), Ly5.2 (104), CD62L (MEL-14), CD95 (Jo2), B220 (RA3-6B2) (BD Biosciences); CD44 (IM7), CD19 (6D5), CD93 (AA4.1) (BioLegend); Thy1.1 (HIS51, eBioscience). For anti-CD19 CAR detection (67), streptavidin L (Genscript) was used.

[0274] For phospho-flow analysis, BD Phosflow reagents were used and cells were fixed and permeabilized according to the manufacturer's protocol. After permeabilization, cells were stained with pAkt (S473) (D9E) and pS6 (S235 / 236) (D57.2.2E) from Cell Signaling. For intracellular cytokine staining, cells were stained with fixable live / dead dye in PBS and then surface antibody staining was performed in FACS buffer, followed by fixation and permeabilization (BD Biosciences), and staining for IFNγ (XMG1.2, BD Biosciences) and IL-2 (JES6-5H4, BioLegend). For FasL staining, tumor cells were incubated with vehicle (PBS) or murine IFN-γ (100 ng ml -1 , Bio-Legend) for 24 h and then stained with FasL (Kay-10) and H-2Db (KH95) (BD Biosciences). All flow cytometry data were acquired using a BD Fortessa flow cytometer (Becton Dickinson) and analyzed using FlowJo v.9.9 software (TreeStar).

[0275] Sanger sequencing analysis: Genomic DNA was extracted from Thy1.1-enriched empty vector or Fas I246N transduced cells using the AllPrep DNR / RNA Mini kit (QIAGEN). Primers (IDT) were designed such that the forward primer was located upstream of the Fas I246N point mutant Fas and the reverse primer was located in the Thy1.1 reporter gene. After PCR amplification (Invitrogen), Sanger sequencing was performed.

[0276] Adoptive cell transfer, T cell counting, and cancer therapy: For in vivo persistence analysis, male or female B6 mice aged 6 to 12 weeks were subjected to 6 Gy total body irradiation. One day later, they were injected via the tail vein with 5 × 10 5 syngeneic labeled pmel-1 T cells transduced with a reporter gene construct containing Thy1.1. Mice were sacrificed at the indicated days, and the steady-state expansion of pmel-1 T cells in splenocytes was analyzed.

[0277] For cancer therapy experiments, male or female B6 mice aged 6 - 12 weeks were injected with 5 × 10 5 cells of the previously described B16 melanoma line (57) (which overexpresses the chimeric human / mouse gp100 antigen KVPRNQDWL (a.a. 25 - 33)) or 1 × 10 6 CD19 + E2a-PBX leukemia cells. At the indicated days, tumor-bearing mice were subjected to 6 Gy total body irradiation. Mice were left untreated as controls or received the indicated doses of syngeneic labeled pmel-1 or anti-CD19 CAR-transduced T cells (which were modified with a reporter gene construct containing Thy1.1) via the tail vein. To analyze the persistence of anti-CD19 CAR-transduced T cells and the leukemia burden, mice were sacrificed after 14 days and cell analysis of the spleen and BM was performed.

[0278] For MRL-Mp mouse experiments, 8-week-old female mice were subjected to 6 Gy total body irradiation. One day later, the mice were injected with 3 × 10 6 anti-CD19 CAR-transduced CD8α + T cells, which were also transduced with a reporter gene construct containing Thy1.1. Age-matched MRL-lpr female mice were left unmanipulated as an ALPS positive control. Immediately before infusion, all transduced T cells were bead-enriched to >92% purity using anti-Thy1.1 magnetic beads (Miltenyi Biotec). All treated mice received 12 μg IL-2 intraperitoneally once daily for 3 days. All tumor measurements were performed blindly by independent investigators.

[0279] T cell and tumor cell co-culture assay: After culturing for approximately 6 days, pmel-1 T cells were washed twice in PBS and seeded at 5 × 10 4 cells per well in a 96-well round-bottom plate in T cell medium without IL-2. The T cells were incubated alone, together with plate-bound anti-CD3 / CD28 (both at 2 μg ml -1 ), and with 1.5 × 10 5Incubate with Bl6-mhgp100 cells, or with 100 ng / mL lz-FasL. Culture the cells together for 6 or 24 hours, then wash and stain to determine cell viability.

[0280] ELISA assay: Analyze serum anti-nuclear and anti-dsDNA antibodies on 1:5 diluted serum; ELISA was performed according to the manufacturer's instructions (Alpha Diagnostic International).

[0281] Histopathology: Fix lung tissues in buffered 10% formalin and stain with H&E. Score the tissue sections blindly by an interpreting pathologist. The scoring is as follows: 0, no definite finding; 1, mild infiltration; 2, minimal infiltration; 3, moderate infiltration; 4, severe infiltration.

[0282] Statistical analysis: Plot the product of the perpendicular tumor diameters as the mean ± SEM for each data point and compare tumor treatment plots using the Wilcoxon rank sum test, and evaluate animal survival analysis using the Log-rank Mantel Cox test. For all other experiments, use unpaired two-tailed Student's t test to compare data, perform multiple comparisons by Bonferroni correction, or use one-way or two-way ANOVA with repeated measures (as indicated). In all cases, a P value less than 0.05 was considered significant. Statistical calculations were performed using Prism 7 GraphPad software (GraphPad Software Inc.).

[0283] Results

[0284] Human tumor microenvironment overexpresses death-inducing ligand FASLG

[0285] In human ACT clinical trials for blood and solid cancers, the expansion and persistence of in vivo T cells are positively correlated with clinical responses (3 - 5, 10, 19). These observations led to the hypothesis that disrupting pathways that impair T cell proliferation and survival might represent potential actionable targets to improve outcomes after adoptive transfer. To determine whether ligands that negatively regulate T cell proliferation and survival are enriched in the human tumor microenvironment, RNA sequencing data were compared using tumor samples from the TCGA database relative to matched normal tissue sources. Given recent evidence that tissues adjacent to resected tumors have an inflamed transcriptomic signature, reflecting an intermediate state between transformed and untransformed tissues (24), expression data from the Genotype-Tissue Expression (GTEx) database (25) were used as normal controls. A total of 9,330 samples obtained from 26 different cancer types for which appropriate matched tissue sources were available were analyzed (Table 1). Raw data from each dataset were extracted and normalized in the same way using the RNA-Seq by Expectation-Maximization (RSEM) method (26).

[0286] Table 1

[0287]

[0288]

[0289]

[0290] It has been found that, relative to normal tissues, the expression of FASLG (the gene encoding the classical inducer of apoptosis FasL (CD178)) is overexpressed in most of the evaluated cancer types ( Figure 1A ). These include cancers responsive to immunotherapy, such as cutaneous melanoma (SKCM), clear cell renal carcinoma (KIRC), lung adenocarcinoma (LUAD), and gastro-esophageal cancer (STAD / ESCA), as well as cancers relatively refractory to existing immunotherapies, such as breast cancer (BRCA), colorectal adenocarcinoma (READ / COAD), glioblastoma multiforme (GMB), ovarian cancer (OV), pancreatic cancer (PAAD), and prostate cancer (PRAD). Overall, 73% (19 / 26) of the evaluated human tumor types showed significant differential expression of FASLG within the tumor mass relative to normal tissue controls (P < 0.05 to P < 0.001; Mann-Whitney U test, Bonferroni correction). In contrast, only 19% (5 / 26) of cancer types did not show significant differential expression, and only a few (8%; 2 / 26) showed evidence of decreased FASLG expression when tumor samples were compared to normal tissues.

[0291] To gain a deeper understanding of the nature of FASLG expression in the human tumor microenvironment, gene set enrichment analysis (GSEA) was performed using genes positively correlated with FASLG in all 26 evaluated cancer types (27)( Figure 1B ). The expression profiles of many immune-related pathways, including NK cell cytotoxicity, antigen processing and presentation, TCR signaling, primary immunodeficiency, and apoptosis, were significantly enriched (nominal P value < 0.001, FDR q value < 0.001). Consistent with these findings, examination of the top 200 genes positively correlated with FASLG revealed an overrepresentation of markers associated with both lymphocyte activation (such as IFNG, PRF1, 41BB, and ICOS) and immune counterregulation (such as PDCD1, LAG3, and IL10RA)( Figure 1C and Table 2). Collectively, these data indicate that death-inducing ligands that may compromise T cell survival are significantly overexpressed in the majority of human cancer microenvironments and are highly associated with the expression signatures of immune activation and regulation.

[0292] Table 2

[0293]

[0294]

[0295] Next, it was determined whether the FasL homologous receptor Fas (CD95) is expressed on the surface of T cells used for clinical adoptive immunotherapy. Fas was previously found to be expressed on all non-naive human T cell subsets from healthy donors (HD), including central memory T cells (TCM), effector memory T cells (TEM), and effector memory T cells co-expressing CD45RA (TEMRA) (28, 29). The frequencies of CD8α + T cell subsets and Fas expression in each subset were analyzed in melanoma and aggressive B cell lymphoma patients from apheresis products used to generate therapeutic T cells for ACT. In these patients, Fas was found to be highly expressed on the TCM, TEM, and TEMRA subsets( Figure 1D and 1E ). Additionally, the frequencies of naive CD8α + T cells (TN) in these patients were compared to a group of age-matched HD. It was found that the percentage of Fas - TN cells in HD was significantly higher compared to melanoma and lymphoma patients( Figure 1F), this finding may reflect the impact of prior immunostimulatory and lymphodepleting therapies on the cancer patients analyzed (5, 30, 31). Thus, a significant proportion of the human T cells used for ACT express known death receptors, and these cells are transferred into a tumor microenvironment rich in the expression of their cognate ligands.

[0296] T cells engineered with a Fas dominant-negative receptor prevent FasL-mediated apoptosis

[0297] The results showed that patient-derived T cells for adoptive immunotherapy were biased towards a subset expressing Fas, which was subsequently transferred into a tumor microenvironment rich in FASLG. Based on these data, it was next examined whether disrupting Fas signaling in adoptively transferred T cells could prevent their apoptosis and improve in vivo persistence. In addition to triggering T cell apoptosis, FasL is also an essential effector molecule for T cell-mediated tumor killing (32). Moreover, systemic administration of anti-FasL antibody or Fas-Fc fusion protein can both induce toxicity, including the development of lymphoproliferative syndrome and the accumulation of abnormal populations of double-negative (DN) CD3 + B220 + CD4 - CD8 - TCRα / β + lymphocytes (33, 34). For these reasons, a cell-intrinsic genetic modification strategy was sought to abrogate Fas signaling only within tumor-reactive T cells to maintain anti-tumor efficacy and minimize the risk of systemic toxicity.

[0298] Physiologically, FasL initiates apoptotic signaling by first inducing the oligomerization of Fas receptors into trimers or larger oligomers on the cell membrane ( Figure 2A )(35). Fas oligomers recruit intracellular adapter molecules via the Fas-associated death domain (FADD) through the homotypic death domain (DD) present in each molecule (36, 37). The aggregation of FADD recruits the cysteine-aspartic protease procaspase 8 through the homologous death effector domain in each molecule (38), forming a death-inducing signaling complex (DISC) that can initiate the apoptotic signaling cascade (39). Based on this mechanism of action, it was hypothesized that overexpression of a mutant Fas variant genetically altered to block FADD binding would act as a dominant-negative receptor (DNR) when expressed in Fas-sensitive wild-type (WT) T cells for adoptive immunotherapy. Currently, virus-based constructs are the most commonly used method for stably modifying human T cells for clinical applications (40). Therefore, a series of retroviral constructs were created that encoded the murine Fas sequence (where the asparagine residue at position 246 in the DD was replaced with isoleucine (Fas I246N), a natural mutant of murine Fas that cannot bind FADD (41, 42)), or encodes a Fas mutant in which most of the intracellular DD is truncated (del aa222-306; Fas ΔDD ) to prevent FADD binding)( Figure 2A and 7A ). As a control, an empty vector construct and a construct encoding the full WT sequence of Fas (Fas WT ) were generated. To identify transduced cells, all vectors contained a Thy1.1 reporter gene separated from Fas using T2A "self-cleavage".

[0299] T cells were isolated from Fas-competent WT mice, activated in the presence of IL-2, and transduced with the empty construct, the Fas WT construct, the Fas I246N construct, or the Fas ΔDD construct( Figure 2B ). Phenotypic analysis 6 days after activation and transduction showed that all constructs had a high transduction efficiency, as measured by Thy1.1 expression( Figure 7B and 7C ). Notably, for constructs containing WT (6.8-fold higher Fas MFI) or mutant Fas variants (Fas I246N and Fas ΔDD , with 43-fold and 98-fold higher Fas MFI, respectively), ectopic Fas expression was measurably higher than endogenous Fas expression levels( Figure 7B and 7D ). After 6 days of culture, transduced T cells were stimulated with recombinant FasL molecules oligomerized through a leucine zipper domain (lz-FasL) to mimic the function of membrane-bound FasL (43), or left untreated as a control. In the absence of lz-FasL, T cells transduced with each construct remained viable with similar rates( Figure 2C ). However, after exposure to lz-FasL, a significant proportion of Thyl.l WT T cells transduced with the empty vector control or Fas + were converted into apoptotic Annexin V + PI + populations( Figure 2C and 2D ; P < 0.001). Interestingly, overexpression of Fas WT consistently led to higher levels of apoptosis relative to T cells transduced with the empty vector, indicating that Fas expression above physiological levels sensitizes T cells to FasL-mediated cell death. In contrast, transduction with Fas I246N or Fas ΔDDVector-transduced T cells were almost completely protected from lz-FasL-induced apoptosis. Among the T cell repertoire transduced with Fas I246N or Fas ΔDD , protection from apoptosis was restricted solely to the Thy1.1 + population, indicating a cell-intrinsic function of Fas DNR( Figure 11 ). This suggests that Fas I246N and Fas ΔDD may also function to protect neighboring T cells from apoptosis by acting as a "sink" for local FasL. In T cells modified with Fas I246N , no functional or genetic evidence of reversion to the WT sequence was found. After serial in vitro restimulation, selective enrichment of T cells modified with Fas I246N and Fas WT was measured, indicating that DNR remained functionally intact over time( Figure 12A and 12B ). Furthermore, Sanger sequencing of Fas I246N -transduced T cells after serial restimulation showed no evidence of reversion of the I246N point mutation to the WT Fas sequence( Figure 12C and 12D ). Thus, overexpression of Fas variants abrogates their ability to bind FADD function in a dominant-negative manner, preventing FasL-mediated apoptosis of WT T cells.

[0300] Finally, we sought to determine whether Fas DNR protects adoptively transferred T cells from other apoptosis-inducing stimuli that they may encounter in vivo. These include activation-induced cell death (AICD), cytokine withdrawal, and proximity to tumor cells. For these assays, pmel-1 T cells specific for the cancer antigen gp100 and B16 melanoma cells engineered to express human gp100 (B16 cells) were utilized. Although B16 cells do not express FasL at rest, incubation with IFN-γ upregulates FasL expression to a measurable extent( Figure 13 ). Pmel-1 T cells transduced with Fas I246N or Fas WT were equally protected from apoptosis induced by lz-FasL or tumor co-culture( Figure 14 ). In contrast, after AICD induction by anti-CD3 / CD28 restimulation or acute cytokine withdrawal, T cells transduced with Fas I246N resulted in significantly increased cell viability relative to cells modified with Fas ΔDD . These findings may be attributed to Fas I246NThe ability of the variant to bind to FADD with reduced efficiency under certain conditions (73). Thus, given its superior functional properties, for all in vivo experiments, the present disclosure subsequently focused only on Fas ΔDD DNR. This allowed for a clearer determination of the impact of Fas signaling ablation on the in vivo function of adoptively transferred T cells.

[0301] Adoptive transfer of T cells engineered with Fas DNR results in excellent persistence

[0302] Next, it was determined whether the expression of Fas DNR in T cells after adoptive transfer into tumor-bearing hosts led to superior in vivo persistence.

[0303] Allogeneically labeled, genetically modified pmel-1 T cells were adoptively transferred into sub-lethally irradiated Thy1.1 - C57BL / 6 (B6) mice to induce in vivo homeostatic proliferation, and the expansion and persistence of the transferred cells were measured over time. Fas ΔDD or empty vector control-transduced T cells were identified by the expression of the Thy1.1 reporter gene. To measure T cell proliferation, the T cells were co-stained for the cell proliferation marker Ki-67.

[0304] One day after transfer, Fas ΔDD - and empty vector-modified pmel-1 T cells were engrafted at similar levels and expressed Ki-67 almost uniformly ( Figure 3F-3H ). Starting within three days of transfer, the multi-log expansion of the two modified cell populations was measured. However, at the peak of expansion, a ~50-fold increase in the number of Fas ΔDD -modified T cells was observed relative to control-modified cells. This in turn led to a >10-fold higher level of persistence of Fas DNR-modified T cells at day 30 ( Figure 3F and 3G ). Over time, a comparable decline in Ki-67 expression was observed in both modified T cell populations ( Figure 3H ), which was associated with the reconstitution of the host endogenous T cell compartment. These data indicate that in vivo proliferation was comparable between the two modified T cell populations. However, Fas DNR-modified T cells may exhibit superior overall expansion and mid-term persistence through reduced apoptosis.

[0305] Next, it was sought to determine whether genetic modification with Fas DNR led to superior T cell persistence within the TME. To ensure that the modified T cells were exposed to the same microenvironmental factors in any given tumor, a co-infusion experiment was performed.

[0306] From Ly5.1- / Thy1.1 - or Ly5.1 + / Thy1.1 - Background Obtaining congenitally distinguishable pmel-1 CD8+ T cells specific for the cancer antigen gp100 + T cells were transduced separately with Fas ΔDD DNR or an empty vector control expressing Thy1.1. Subsequently, the transduced T cells expressing Thy1.1 were purified using anti-Thy1.1 microbeads, reconstituted at approximately a 1:1 ratio, and then co-infused into sub-lethally irradiated Ly5.1 - / Thy1.1 - mice bearing 10-day established B16 melanoma tumors ( Figure 3A ). As is currently done in many solid tumor ACT clinical trials, the treated mice received a limited course of IL-2 after metastasis (13, 18, 44 - 46). Seven days after infusion, the spleens and tumors of the recipient mice were collected and analyzed for the presence of adoptively transferred, genetically modified Thy1.1 + pmel-1 T cells. Consistently found in the spleens and tumors of the recipient mice, Ly5.1-Thy1.1 + Fas ΔDD -modified T cells were significantly enriched relative to Ly5.1-Thy1.1 + empty vector-modified T cells ( Figure 3B and 3E ; P < 0.01, P < 0.001). To test whether T cells engineered with Fas ΔDD DNR could enhance T cell survival in a microenvironment enriched in tumor cells, an in vitro co-culture assay was performed. Pmel-1 T cells expressing Fas ΔDD or an empty vector control were plated alone overnight without IL-2 or co-cultured with B16 melanoma tumors. As a positive control for cell death, T cells were cultured in the presence of lz-FasL. In this experiment, T cells were not enriched with Thy1.1 beads to achieve an additional internal control. After 24 hours, T cell viability was obtained by FACS analysis. Although substantial cell death was induced in empty vector-transduced pmel-1 T cells by co-culture with B16 or addition of lz-FasL, this was not observed in the Fas ΔDD -transduced counterparts ( Figure 3C ). In addition, untransduced cells in both groups exhibited comparable cell viability in response to B16 co-culture or lz-FasL ( Figure 3D)。In summary, these results indicate that genetic modification with Fas DNR enhances adoptive cell transfer and the engraftment and viability of tumor-reactive T cells following exposure to a tumor-rich microenvironment.

[0307] ACT of Fas DNR-modified T cells does not result in the ALPS phenotype

[0308] Mice and humans with germline defects in components of normal apoptotic signaling, such as Fas, can have profound alterations in normal lymphocyte homeostasis and development. These abnormalities are collectively referred to as autoimmune lymphoproliferative syndrome (ALPS) and include abnormal CD3 + B220 + CD4 - CD8 - lymphocyte population accumulation and autoantibody formation, resulting in impaired survival (47, 48). Given the potential safety concerns associated with disabling normal Fas signaling in mature T cells, detailed, long-term immune monitoring was performed on animals that had received Fas ΔDD DNR-modified T cells for more than 6 months ( Figure 4E ). This time point was chosen because mice with germline defects in Fas typically develop overt clinical manifestations within the first 3.5 - 5 months after birth, depending on the background strain (49, 50). Unmanipulated WT and Fas-deficient lpr / lpr mice were used as negative and positive controls for the ALPS phenotype, respectively, to evaluate the frequency of CD3 ΔDD DNR or empty vector control-modified Vβ3 13 + pmel-1 T cells in the spleens of mice that had received ACT. As expected, the spleens of lpr / lpr mice showed a significant accumulation of abnormal CD3 + B220 + lymphocytes relative to WT controls ( + B220 + ; P < 0.05, P < 0.001). In contrast, mice that received T cells modified with either empty vector control or Fas DNR did not show a significant increase in this population. To rule out transformation of the modified T cell population, the long-term persistence and phenotype of the transferred Vβ3 13 Figure 4A and 4B ; P < 0.05, P < 0.001). In contrast, mice that received T cells modified with either empty vector control or Fas DNR did not show a significant increase in this population. To rule out transformation of the modified T cell population, the long-term persistence and phenotype of the transferred Vβ3 13 + Thy1.1 + -engineered T cells were evaluated. Over 200 days, T cells engineered with Fas ΔDD DNR persisted in higher numbers compared to cells modified with empty vector control ( Figure 4C and 4D; P < 0.05). Long-term persistent Fas DNR-modified T cells maintained a conventional CD3 + B220 - phenotype. These data indicate that adoptively transferred pmel-1 T cells expressing Fas DNR did not undergo abnormal lymphocyte proliferation in B6 hosts.

[0309] Previously, it was shown that the expression of transgenic TCRs crossed with the Fas-deficient lpr background could limit the development of ALPS (74). In addition, compared with other strains, the B6 strain exhibits lymphoproliferative symptoms at a slower rate (49, 50, 75). Therefore, by adoptively transferring an open T cell pool engineered with Fas DNR or an empty control gene into the ALPS-susceptible MRL-Mp strain, additional experiments were conducted to evaluate the safety of Fas ΔDD DNR modification. Fas-deficient mice on an MRL background (MRL-lpr mice) develop autoantibodies, nephritis, and splenomegaly more severely and several months earlier than B6-lpr mice ( Figure 15A )(49, 50, 75). To induce the activation and expansion of adoptively transferred T cells in this model, open pool T cells from MRL-Mp mice were co-transduced with the previously described second-generation anti-CD19 28ζCAR (71) and Fas ΔDD or a control vector. In these experiments, the use of anti-CD19 CAR promoted strong in vivo proliferation of T cells by recognizing host CD19 + B cells. Notably, recently published data indicate that T cells modified with CAR can still be stimulated through their TCR (72, 76).

[0310] Spleens of MRL-Mp mice receiving cell-free (PBS) or anti-CD19 CAR transduced with Fas ΔDD or an empty control were analyzed and compared with the spleens of age-matched Fas-deficient MRL-lpr mice ( + ). The spleens of age-matched MRL-lpr mice were significantly heavier compared with the spleens from all other treatment groups. Importantly, no differences in spleen size were observed between PBS-treated mice and mice receiving cells transduced with anti-CD19 CAR modified with Fas Figure 15C ) or a control. Flow cytometry analysis of splenocytes showed a large expansion of abnormal DN CD3 ΔDD B220 + lymphocytes in the spleens of MLR-lpr mice, accounting for more than 30% of all lymphocytes in total ( + ) and Figure 15D and 15E)。In contrast, the frequencies of CD3 ΔDD and B220 + lymphocytes in mice treated with empty vector and Fas + T cells were similar to the levels observed in PBS control mice.

[0311] To assess the development of autoimmunity, serum analysis was performed on all treated animals using samples from MRL-1pr mice as positive controls. Mice receiving anti-CD19 CAR ΔDD or empty vector-modified Fas + T cells had low antinuclear and anti-dsDNA antibody titers comparable to those of PBS controls ( Figure 15F ). In contrast, sera from MRL-1pr positive control mice showed high titers of both types of autoantibodies. In the absence of uncontrolled lymphocyte proliferation and autoantibody formation, anti-CD19 CAR + T cells co-transduced with Fas DNR persisted at significantly higher levels in the spleens of recipient MRL-Mp mice compared to control-modified anti-CD19 CAR + T cells ( Figure 15G ). In addition, compared to control-modified CAR + cells, persistent Fas DNR-modified CAR + T cells did not acquire a greater proportion of abnormal CD3 + and B220 + cells ( Figure 15H ). These results directly reflect the findings from the transfer of Fas ΔDD -modified pmel-1T cells into B6 hosts ( Figure 4C and 4D ).

[0312] Finally, to assess whether ALPS-susceptible MRL-Mp recipient mice developed pulmonary pathology after adoptive transfer of Fas DNR-modified T cells, blinded pathological evaluations of H&E-stained lung specimens were performed. Consistent with previous reports (77), Fas-deficient MRL-lpr mice developed dense perivascular and peribronchial mononuclear cell inflammatory lung infiltrates ( Figure 16A and 16B ). In contrast, mice treated with Fas ΔDD or control-modified T cells did not show evidence of increased inflammatory infiltrates relative to PBS-treated control injections. In addition, no evidence of pulmonary fibrosis was observed.

[0313] In summary, these data in the B6 and MRL-Mp strains indicate that, although Fas ΔDDThe relative survival period of DNR T cells was increased, but no evidence was detected of uncontrolled lymphatic accumulation, formation of Thy1.l + CD3 + B220 + clinical evidence of a population or autoimmunity. Based on these data, infusion of mature T cells with impaired Fas signaling does not result in an acquired lymphoproliferative phenotype.

[0314] T cell-intrinsic disruption of Fas signaling enhances antitumor efficacy after ACT

[0315] It has been determined that adoptively transferred T cells engineered with Fas DNR result in enhanced persistence without long-term toxicity. Next, the anti-tumor efficacy of these cells was evaluated. Pmel-1 T cells were stimulated and transduced with Fas I246N 、Fas ΔDD or empty vector control pairs and retroviruses. After that, restimulation and further expansion were performed to mimic the more differentiated T cell population present in the circulation of cancer patients (5,31)( Figure 1D and 5A ). Eleven days later, the transduced T cells under each condition were isolated using anti-Thy1.1 microbeads to a purity of >98%, and then injected separately into sub-lethally irradiated mice bearing established B16 melanoma tumors. The treated mice additionally received IL-2 by intraperitoneal injection. Relative to untreated controls, all mice receiving adoptively transferred pmel-1 T cells experienced a significant delay in tumor growth( Figure 5B ). However, relative to control-modified pmel-1 cells, those mice receiving T cells engineered with Fas I246N or Fas ΔDD DNR showed enhanced tumor control ability( Figure 5B ; P<0.001), and the animal survival rate was significantly increased( Figure 5C ; P<0.05 and P<0.01)

[0316] Recently, it has been found that Fas stimulation can induce non-apoptotic Akt / mTOR signaling, leading to enhanced T cell differentiation (51,52). Consistent with previous results, it was found that exposure to lz-FasL led to phosphorylation (p)Akt in T cells transduced with empty vector control S473 and pS6S235,S236 to increase in a dose-dependent manner( Figure 8A and 8B ).

[0317] Expansion of control-modified cells led to the accumulation of TEM-like cells and a reduced ability to produce IL-2( Figure 8C and 8D)。In contrast, T cells transduced with Fas I246N or Fas ΔDD failed to show Akt or S6 phosphorylation and were protected from enhanced Akt-mediated T cell differentiation. These cells retained the major TCM-like phenotype and the ability to produce IL-2. In several different animal models (29, 53, 54) and clinical trials (10, 55), compared with the transfer of TEM-like cells, the transfer of TCM-like cells was associated with superior tumor regression. These findings increase the possibility that the superior tumor regression observed with cells modified with Fas DNR may be attributed to differences in cell differentiation rather than protection from Fas-mediated T cell death. To test this possibility, transduced TCM-like phenotype cells (Thy1.1 + CD44 hi g h CD62L + ) were isolated to >96% purity by FACS sorting, and the T cell differentiation status was normalized at the time of cell infusion ( Figure 5D ). Subsequently, central memory-like sorted T cells were transferred into sub-lethally irradiated B16 tumor-bearing mice as Figure 5A shown. It was found that even when normalized for the TCM-like differentiation status, adoptive transfer of T cells modified with Fas DNR led to superior tumor regression and animal survival ( Figure 5E-5H ; P < 0.05).

[0318] In summary, preventing Fas-mediated cell death in adoptively transferred, tumor-reactive T cells engineered with Fas DNR results in superior tumor regression and animal survival.

[0319] Genetic engineering with Fas DNR protects human T cells from Fas-mediated apoptosis

[0320] To determine the clinical feasibility of engineering human T cells with Fas DNR, retroviral constructs encoding human Fas sequences mutated to prevent FADD binding were designed. This included a human Fas variant (hFas D244V ) containing a point mutation (replacing the aspartic acid residue at position 244 with valine) (56, 57), and a human Fas (del aa230 - 314; hFas ΔDD ) with most of the intracellular death domain truncated ( Figure 6A ) (56, 57).

[0321] CD8 + T cells were isolated from HD PBMC, stimulated with anti-CD3 / CD28 and IL-2, and then transduced with hFas D244V , hFas ΔDDor an empty vector control were transduced ( Figure 6B ). In the absence of additional stimuli, transduced Thy1.1 - and transduced Thy1.1 + T cells both maintained similar viability, as measured by annexin V and PI staining ( Figure 6C ). However, when these cells were cultured in the presence of increasing doses of lz-FasL, T cells transduced with the empty vector showed a significant and dose-dependent increase in both the frequency of annexin V + apoptotic and necrotic cells ( Figure 6C and 6D ). In contrast, T cells modified with hFas D244V or hFas ΔDD were significantly protected from lz-FasL-mediated apoptosis. This protection was mainly T cell-intrinsic, as untransduced Thy1.1 D244V or hFas ΔDD transduced Thy1.1 + T cells showed a significantly higher frequency of annexin V - positive cells compared to Thy1.1 + T cells transduced with hFas. Thus, genetic modification with Fas DNR can protect primary human T cells from FasL-induced cell death, providing a new approach to protect T cells from secondary metastasis in the human tumor microenvironment.

[0322] Discussion

[0323] The results of the pan-cancer analysis reported here strongly suggest that the canonical death-inducing ligand FASLG is overexpressed in the majority of human cancer microenvironments. A significant proportion of human T cells used for adoptive immunotherapy co-express Fas, the cognate receptor of FasL. Based on these findings, a cell-intrinsic strategy was tested that uses a series of Fas DNRs to "insulate" Fas-sensitive murine and human T cells from FasL-induced apoptosis. Functionally, adoptively transferred FasDNR-modified T cells showed excellent persistence both peripherally and in tumors of tumor-bearing animals, resulting in excellent tumor regression and overall survival. Importantly, although FasDNR-modified T cells showed increased survival at 6 months post-transfer compared to control-modified T cells, no evidence of uncontrolled lymphoproliferation or autoimmunity was detected. Thus, these findings provide a novel and potentially general genetic modification strategy to enhance the function of adoptively transferred T cells against a wide range of human malignancies, including advanced solid cancers.

[0324] Previously reported, in addition to its typical function of inducing apoptosis, Fas can also promote the differentiation of murine and human T cells in an AKT-dependent manner (51, 52). Consistent with these findings, T cells transduced with Fas DNR were protected from lz-FasL-mediated induction of pAKT S473 and pS6 S235,S236 . Thus, this blockade in AKT / mTOR signaling minimized T cell differentiation, promoted the accumulation of TCM-like cells, retained the expression of the lymphoid homing marker CD62L, and the ability to produce IL-2. In multiple preclinical models (29, 53, 54) and in a retrospective analysis of human clinical trials (10, 55), compared with TEM-like cells, the infusion of TCM-like cells was associated with better anti-tumor efficacy. These findings increase the possibility that the excellent therapeutic effects obtained using Fas DNR-modified cells may be due to the infusion of less differentiated T cells rather than the blockade of apoptosis. To address this possibility, the anti-tumor efficacy of phenotypically matched, FACS-sorted T CM -like cells modified with Fas DNR or empty vector control was compared. Even after normalizing the surface phenotype, Fas DNR-modified T CM showed excellent therapeutic efficacy compared to control-modified T CM . Mechanistically, the major contribution to enhancing the in vivo anti-tumor efficacy using Fas DNR-modified T cells can be attributed to the disruption of cell death rather than the infusion of less differentiated cells. These findings are also consistent with recent papers by Zhu et al., Horton et al., and Lakins et al., demonstrating that FasL-induced apoptosis of tumor-infiltrating lymphocytes limits the efficacy of immune checkpoint inhibitors (17, 58, 59).

[0325] Although analysis has shown that FASLG expression is enriched in the microenvironment of many human tumors, they have not defined which specific cell types are expressing the ligand. Using immunohistochemical protein staining, previous studies have determined that FasL can be expressed directly on the surface of many solid cancers identified in a pan-cancer analysis. This includes breast cancer, colon cancer, brain cancer, kidney cancer, and cervical cancer (60,61). Additionally, recent studies have determined that FasL is expressed along the luminal surface of newly formed blood vessels surrounding human ovarian and brain cancers, creating a tumor endothelial death barrier that restricts T cell infiltration (60,62). Finally, it is possible that FasL can be expressed by cells of both the innate and adaptive immune systems in the tumor microenvironment. This possibility has been shown previously by others (17), and further shown by our own analysis, indicating a high correlation between FASLG and many immune-related genes. Finally, functional data indicate that Fas DNR modification can also provide protection from other apoptosis-inducing stimuli that T cells may experience following adoptive transfer of cells into tumor or infected hosts. These include activation-induced cell death (AICD), cytokine withdrawal, and proximity to antigen-expressing tumor cells. Collectively, these data suggest that the source of FasL may be histology-dependent. Thus, a cell-intrinsic Fas DNR approach that does not compromise the FasL-mediated tumor killing ability of transferred T cells may have broad applicability in multiple cancer types.

[0326] Fas DNR now joins a list of other candidate DNRs with which T cells can be modified to intrinsically disrupt signaling by immunosuppressive factors present in the tumor microenvironment, including the TGFβ receptor (63) and PD1 (64). The use of short hairpin RNA approaches to disrupt Fas in human T cells in vitro has been reported (65). However, due to relatively poor Fas knockout efficiency, this approach requires lengthy in vitro selection. Additionally, the in vivo anti-tumor ability of these cells was not tested. Although enhanced cell persistence was observed using T cells modified with Fas DNR, no evidence of double-negative T cell formation or uncontrolled lymphocyte proliferation was observed.

[0327] Germline loss-of-function in Fas signaling can lead to autoimmune lymphoproliferative disease in mice and humans, which is a potential safety consideration for the Fas DNR approach. Although the survival of FasΔDD-modified T cells was prolonged, no uncontrolled lymphoid accumulation or abnormal CD3 + B220 +Evidence of lymphocyte formation or autoimmunity using two different mouse strains. This includes adoptive transfer of polyclonal T cell populations into ALPS-susceptible MRL-Mp strains. Based on these data, infusion of mature T cells impaired in Fas signaling is unlikely to result in acquired lymphoproliferative syndrome.

[0328] Although Fas is a key mediator initiating the extrinsic apoptosis signaling cascade, the intrinsic apoptosis pathway remains intact in cells. Thus, competition for homeostatic cytokines ignored due to lack of antigen and T cell exhaustion can both regulate the in vivo homeostasis of Fas DNR cells in vivo. Despite these reassuring safety data in mice, improvements in the clinical application of this approach could also include the introduction of a suicide mechanism, such as truncated EGFR(66) upstream of Fas DNR.

[0329] In summary, in many patients receiving adoptive immunotherapy for the treatment of solid cancers, the FasL / Fas pathway is expected to be activated. Novel dominant-negative receptors have been developed that inherently abrogate the apoptosis-inducing function of this pathway in primary murine and human T cells, resulting in enhanced cell persistence and enhanced anti-tumor efficacy. These data lay the foundation for a potentially general strategy to enhance the potency of adoptive immunotherapy against solid and hematological cancers.

[0330] Example 2 - Role of Fas DNR and anti-CD19 CAR-modified T cell therapy in a leukemia mouse model

[0331] Next, the therapeutic efficacy of adoptively transferred T cells engineered with Fas DNR and CAR was evaluated. An independent tumor model in which CAR targets hematological malignancies was used. A recently developed syngeneic B cell ALL (B-ALL) line (72,78) driven by a physiologically relevant E2a-PBX translocation in a murine second-generation 28ζ anti-CD19 CAR therapeutic model was used. There were two reasons for choosing the syngeneic model over the more commonly used xenogeneic anti-CD19 CAR therapeutic model. First, in addition to tumor cells and adoptively transferred T cells expressing FasL, it was necessary to ensure that the transferred T cells were fully responsive to host-derived FasL. Second, to avoid potential confounding effects of xenoreactivity on AICD induction in the transferred T cells.

[0332] T cells were subjected to stimulation and retrovirally transduced with anti-CD19 CAR and Fas ΔDD or empty vector control. The co-transduction efficiency and purity of the transduced T cells are shown in Figure 9B-9C and Figure 10A-10B . When using Fas ΔDDWhen compared to the empty vector control, protein L was used to identify CAR-transduced T cells (79), and the co-transduction efficiency was equally effective. Next, it was determined how the co-transduced anti-CD19 CAR T cells responded to various apoptosis-inducing stimuli, including exogenous FasL, cytokine withdrawal, AICD, and exposure to antigen-expressing B-ALL tumor cells( Figure 10C ). Similar to the results obtained with pmel-1 T cells expressing TCR, relative to the CAR + T cells transduced with the empty vector control, the expression of Fas ΔDD protected the CAR-modified T cells from each of these death-inducing stimuli.

[0333] Figure 9A and 10D showed the experimental design of treatment with syngeneic T cells co-transduced with anti-CD19 CAR and Fas ΔDD or the empty vector control in a murine leukemia model.

[0334] The treated mice received daily IL-2 injections for 3 days to support the expansion of adoptively transferred T cells. Fourteen days after cell infusion, the persistence of adoptively transferred cells in the spleen and BM (two disease sites of E2a-PBX B-ALL) was analyzed. Higher levels of Thy1.1 + Fas ΔDD cells were observed at the two disease sites compared to mice receiving T cells transduced with the empty vector( Figure 10E ). E2a-PBX leukemia expresses classical B-ALL precursor markers, including CD19, B220, and CD93 (80). As Figure 10F shown, 14 days after T cell treatment, the BM in untreated (PBS) mice and mice treated with the empty vector contained approximately 70% leukemic cells. However, mice receiving Fas ΔDD -modified cells contained less than 1% leukemic cells in the BM. These data indicate that CAR + T cells expressing Fas DNR cells were able to mediate excellent leukemia clearance relative to T cells transduced with the empty vector.

[0335] Eleven days later, the T cells transduced under each condition were separated to a purity >98% using anti-Thy1.1 microbeads and then injected separately into sub-lethally irradiated mice bearing established E2a:PBX pre-B ALL tumors. The treated mice also received IL-2 by intraperitoneal injection. All mice receiving high-dose CAR T cells (5.5×10 5 ) experienced a significant delay in tumor growth relative to the untreated control( Figure 9D ). However, when using low-dose CAR T cells (1.8×105 ) When processed, compared to the control, only those mice that received T cells modified with Fas ΔDD DNR showed significantly improved animal survival rates( Figure 9E ).

[0336] In another experimental setting, the survival of leukemic mice after adoptive transfer of two different doses of second-generation 28ζ anti-CD19 CAR-transduced T cells co-modified with Fas ΔDD or an empty control was analyzed. To provide a therapeutic window, a dose of CAR-modified T cells that had previously been shown to be sub-therapeutic in this model (72) was transferred. Compared to untreated mice, at a higher cell dose (3×10 5 CAR + cells), adoptive transfer of control or Fas ΔDD -modified CAR + T cells led to a significant increase in animal survival rates( Figure 10G , left). However, while all mice that received Fas DNR-modified CAR + T cells survived, the survival of mice that received control-modified CAR + T cells did not exceed 55 days. When the dose of CAR + cells was further reduced (2×10 5 ), Fas DNR-modified T cells continued to provide long-term survival in 100% of the treated mice, while control-modified T cells completely lost their efficacy( Figure 10G , right). Previous reports have shown that second-generation CARs containing 4-1BB express higher levels of anti-apoptotic proteins compared to CARs containing the CD28 domain (80). These data in the solid cancer B16 melanoma and hematological E2a-PBX leukemia models indicate that expression of Fas DNR in adoptively transferred T cells leads to excellent in vivo cell persistence and anti-tumor efficacy, regardless of whether the antigen-targeting construct is a TCR or a 28ζCAR.

[0337] Example 3 - FasDNR protects cells from FasL-induced apoptosis and does not affect T cell tumor targeting function

[0338] Methods

[0339] Cell culture。Platinum-GP retroviral packaging cells (Cell Biolabs) were cultured in RPMI supplemented with 10% fetal bovine serum, 10 mM HEPES (Gibco), and 25 units / ml PenStrep (Gibco). Primary T cells were cultured in RPMI supplemented with 10% heat-inactivated human serum, 25 mM HEPES (Gibco), and 50 units / ml PenStrep (Gibco).

[0340] Isolation and expansion of human T cells 。Buffy coats were obtained from healthy donors at the New York Blood Center. Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation using Lymphocyte Separation Medium (Coming). EasySep human CD8 + T cell isolation kit (Stemcell) was used to isolate CD8 + T cells. CD8 + T cells were activated on plates coated with 5 μg / ml anti-CD3 (MiltenyiBiotec) antibody and 1 μg / ml soluble anti-CD28 (Miltenyi Biotec). For viral transduction, T cells were treated with 50 IU / ml of IL-2 (PeproTech) for 2 days prior to transduction.

[0341] Plasmid design and viral transduction 。All plasmids for virus packaging were designed based on the SFGγ retroviral vector. Feline endogenous retroviral envelope RD114 was used for co-transfection with the SFGγ vector into Platinum-GP cells. Platinum-GP cell co-transfection was performed using Lipofectamine 3000 (ThermoFisher).

[0342] Primary T cells were transduced with the viral supernatant on plates coated with Retronectin (Takara). Briefly, plates were coated with 20 μg / ml Retronectin overnight at 4°C and then blocked with PBS with 2% FBS for 30 minutes at room temperature. The plates were washed with PBS and loaded with the viral supernatant. Centrifugation was performed at 2000 g, 32°C for 2 hours. The supernatant was aspirated and the cells were loaded into each well. The plates were centrifuged again at 1200 rpm, 32°C for 5 minutes and incubated at 37°C for 2 days.

[0343] Flow cytometry and intracellular staining 。Conjugated antibodies for flow cytometry included Brilliant Violet421 TMAnti-human EGFR (AY13, Biolegend), PE / Cy5 anti-human CD95 Fas (DX2, Biolegend), APC / Cyanine7 anti-human CD95 Fas (DX2, Biolegend), PerCP / Cyanine5.5 anti-human TNF-α (Mabl1, Biolegend). For NY-ESO-targeted TCR, PE anti-TCR Vβ13.1 (IMMU 222, Beckman Coulter) was used. For CAR staining, AlexaFluor 647 AffiniPure F(ab')2 fragment goat anti-mouse IgG, F(ab')2 antibody (Jackson ImmunoResearch) was used.

[0344] FasL apoptosis assay . For all apoptosis assays, a soluble form of FasL oligomerized via a leucine zipper motif (FasL-LZ) was used at 100 ng / ml. Cells were treated with FasL-LZ at the indicated time points at 37 °C. Cells were washed and surface antibody staining was performed. Cells were stained with CellEvent TM Caspase-3 / 7 Green Detection Reagent (ThermoFisher) in FACS buffer for 25 minutes at 37 °C and washed twice. Then cells were stained with APC Annexin V (Biolegend) in Annexin V binding buffer (Biolegend) for 25 minutes at room temperature. Cells were washed twice and resuspended in Annexin V binding buffer for flow cytometry.

[0345] Statistical analysis . All statistical analyses were performed using Prism 7 (GraphPad) software. No statistical method was used to determine the sample size. All analyses were performed on samples in triplicate. Statistical comparisons between two groups were calculated by paired Student's t-test on matched samples. P < 0.05 was considered statistically significant.

[0346] Results

[0347] The functions of T cells engineered with Fas DNR and antigen recognition receptors (TCR and CAR) were additionally evaluated. Multiple constructs were designed as Figure 17A shown. The resulting engineered human primary T cells expressed Fas that protected the T cells from FasL-induced apoptosis, DNR a T cell receptor (TCR) targeting the NY-ESO1 antigen, and an EGFRt that could be targeted by monoclonal antibodies to induce antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicityFigure 17B )。The cells express Fas DNR and tEGFR. After antigen stimulation, both control and FasDNR cells showed increased TNFα staining ( Figure 17D ). Moreover, after exposure to Fas ligand leucine zipper (FasL-1z) at different time points, T cells expressing Fas DNR showed reduced staining for apoptosis markers.

[0348] Similarly, after co-engineering primary human T cells with Fas DNR , a traceable truncated EGFR, and an antigen-specific CAR anti-CD19 (CD1928ζ), the T cell function was ([[]] Figure 18A and 18B ). After exposure to Fas ligand leucine zipper (lz-FasL), T cells expressing Fas DNR were protected from apoptosis, independent of the expression of anti-CD19 CAR ( Figure 18C ). Moreover, after co-incubation with K562 cells expressing CD19, T cells expressing only anti-CD19 CAR (1928ζ) or T cells expressing Fas DNR (tEGFR-hFASDNR+CD1928ζ) showed comparable antigen-specific cytokine release and degranulation ( Figure 18D ). Thus, Fas DNR reduced FasL-induced apoptosis without altering T cell function.

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[0430] Embodiments of the subject matter of the present disclosure

[0431] It will be apparent from the foregoing description that changes and modifications can be made to the subject matter of the present disclosure to adapt it to various uses and conditions. Such embodiments are also within the scope of the appended claims.

[0432] The list of elements in any variable definition described herein includes any single element or combination (or sub-combination) of elements that defines the variable as the listed elements. The embodiments described herein include taking the embodiment as any single embodiment or in combination with any other embodiment or part thereof.

[0433] All patents and publications mentioned in this specification are incorporated herein by reference to the same extent as if each individual patent and publication were specifically and individually indicated to be incorporated by reference.

Claims

1. A cell, comprising: (a) an antigen recognition receptor that binds to an antigen, and (b) an exogenous dominant negative Fas polypeptide having an amino acid sequence as set forth in SEQ ID NO: 12 or SEQ ID NO:

14.

2. The cell according to claim 1, wherein the at least one modification in the cytoplasmic death domain prevents the binding between the dominant negative Fas polypeptide and the FADD polypeptide.

3. The cell according to claim 1, wherein the exogenous dominant negative Fas polypeptide enhances the cell persistence of immune response cells.

4. The cell according to claim 1, wherein the exogenous dominant negative Fas polypeptide reduces apoptosis or anergy of immune response cells.

5. The cell according to claim 1, wherein the antigen recognition receptor is exogenous or endogenous.

6. The cell according to claim 1, wherein the antigen recognition receptor is recombinantly expressed.

7. The cell according to claim 1, wherein the antigen recognition receptor is expressed from a vector.

8. The cell according to any one of claims 1-7, wherein the exogenous dominant negative Fas polypeptide is expressed from a vector.

9. The cell according to any one of claims 1-8, wherein the cell is an immune response cell.

10. The cell according to claim 9, wherein the cell is a cell of lymphoid lineage or a cell of myeloid lineage.

11. The cell according to claim 9, wherein the cell is selected from T cells, natural killer (NK) cells, B cells, monocytes, and macrophages.

12. The cell according to claim 9, wherein the cell is a T cell.

13. The cell according to claim 12, wherein the T cell is a cytotoxic T lymphocyte (CTL), a regulatory T cell, or a natural killer T (NKT) cell.

14. The cell according to any one of claims 1-13, wherein the cell is autologous or allogeneic to an intended recipient.

15. The cell according to any one of claims 1-14, wherein the antigen is a tumor antigen or a pathogen antigen.

16. The cell according to claim 15, wherein the antigen is a tumor antigen.

17. The cell according to claim 16, wherein the tumor antigen is selected from CD19, MUC16, MUC1, CA1X, CEA, CD8, CD7, CD10, CD20, CD22, CD30, CLL1, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD133, CD138, EGP-2, EGP-40, EpCAM, erb-B2, erb-B3, erb-B4, FBP, fetal acetylcholine receptor, folate receptor-a, GD2, GD3, HER-2, hTERT, IL-13R-a2, K-light chain, KDR, mutant KRAS, mutant PIK3CA, mutant IDH, mutant p53, mutant NRAS, LeY, L1 cell adhesion molecule, MAGE-A1, mesothelin, ERBB2, MAGEA3, CT83 (also known as KK-LC-1), p53, MART1, GP100, proteinase 3 (PR1), tyrosinase, survivin, hTERT, EphA2, NKG2D ligand, NY-ES0-1, carcinoembryonic antigen (h5T4), PSCA, PSMA, ROR1, TAG-72, VEGF-R2, WT-1, BCMA, CD123, CD44V6, NKCS1, EGF1R, EGFR-VIII and CD99, CD70, ADGRE2, CCR1, LILRB2, PRAME, HPV E6 oncoprotein, HPV E7 oncoprotein and ERBB.

18. The cell according to claim 17, wherein the antigen is CD19.

19. The cell according to claim 15, wherein the antigen is a pathogen-associated antigen.

20. The cell according to claim 19, wherein the pathogen-associated antigen is a viral antigen present in cytomegalovirus (CMV), Epstein-Barr virus (EBV), human immunodeficiency virus (HIV) or influenza virus.

21. The cell according to any one of claims 1-20, wherein the antigen recognition receptor is a T cell receptor (TCR) or a chimeric antigen receptor (CAR).

22. The cell according to any one of claims 1-21, wherein the antigen recognition receptor is a CAR.

23. The cell according to claim 22, wherein the CAR comprises an extracellular antigen-binding domain, a transmembrane domain and an intracellular signaling domain.

24. The cell according to claim 23, wherein the intracellular signaling domain further comprises at least one co-stimulatory signaling domain.

25. The cell according to claim 24, wherein the at least one co-stimulatory signaling domain comprises a CD28 polypeptide.

26. The cell according to any one of claims 1-25, which further comprises a suicide gene.

27. The cell according to claim 26, wherein the suicide gene is herpes simplex virus thymidine kinase (hsv-tk), inducible caspase 9 suicide gene (iCasp-9), or truncated human epidermal growth factor receptor (EGFRt) polypeptide.

28. A composition comprising an effective amount of the cell according to any one of claims 1-27.

29. The composition according to claim 28, wherein the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable excipient.

30. Use of an effective amount of the cell according to any one of claims 1-27 or the composition according to claim 28 or 29 in the preparation of a drug for inducing and / or enhancing an immune response to a target antigen.

31. Use of an effective amount of the cell according to any one of claims 1-27 or the composition according to claim 28 or 29 in the preparation of a drug for reducing the tumor burden in a subject.

32. The use according to claim 31, wherein the drug reduces the number of tumor cells in the subject, reduces the size of the tumor, and / or eradicates the tumor.

33. Use of an effective amount of the cell according to any one of claims 1-27 or the composition according to claim 28 or 29 in the preparation of a drug for treating and / or preventing tumorigenesis.

34. Use of an effective amount of the cell according to any one of claims 1-27 or the composition according to claim 28 or 29 in the preparation of a drug for prolonging the survival period of a subject suffering from tumorigenesis.

35. The use according to any one of claims 30-34, wherein the tumor or tumorigenesis is selected from blood cancer, B-cell leukemia, multiple myeloma, lymphocytic leukemia (ALL), chronic lymphocytic leukemia, non-Hodgkin lymphoma, myeloid leukemia, and myelodysplastic syndrome (MDS).

36. The use according to claim 35, wherein the tumorigenesis is B-cell leukemia, multiple myeloma, lymphocytic leukemia (ALL), chronic lymphocytic leukemia, or non-Hodgkin lymphoma, and the antigen is CD19.

37. Use of an effective amount of T cells in the preparation of a drug for treating blood cancer in a subject in need thereof, wherein the T cells comprise an antigen recognition receptor that binds to an antigen and an exogenous dominant-negative Fas polypeptide, and the amino acid sequence of the exogenous dominant-negative Fas polypeptide is as shown in SEQ ID NO: 12 or SEQ ID NO:

14.

38. The use according to claim 37, wherein the blood cancer is selected from B-cell leukemia, multiple myeloma, acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia, non-Hodgkin lymphoma, myeloid leukemia, and myelodysplastic syndrome (MDS).

39. Use of an effective amount of T cells in the preparation of a medicament for treating solid tumors in a subject in need thereof, wherein the T cells comprise an antigen recognition receptor that binds to an antigen and an exogenous dominant-negative Fas polypeptide, and the amino acid sequence of the exogenous dominant-negative Fas polypeptide is as shown in SEQ ID NO: 12 or SEQ ID NO:

14.

40. The use according to claim 39, wherein the solid tumor is a tumor derived from the brain, breast, lung, gastrointestinal tract, pancreas, prostate, soft tissue / bone, uterus, cervix, ovary, kidney, skin, thymus, testis, head and neck, or liver.

41. The use according to claim 40, wherein the gastrointestinal tract includes the esophagus, stomach, small intestine, large intestine, and rectum.

42. Use of an effective amount of the cell according to any one of claims 1-27 or the composition according to claim 28 or 29 in the preparation of a medicament for preventing and / or treating pathogen infections in a subject.

43. The use according to claim 42, wherein the pathogen is selected from viruses, bacteria, fungi, parasites, and protozoa that can cause diseases.

44. A method for producing antigen-specific cells, the method comprising introducing into the cells (a) a first nucleic acid sequence encoding an antigen recognition receptor that binds to an antigen; and (b) a second nucleic acid sequence encoding an exogenous dominant-negative Fas polypeptide, and the amino acid sequence of the exogenous dominant-negative Fas polypeptide is as shown in SEQ ID NO: 12 or SEQ ID NO:

14.

45. The method according to claim 44, wherein one or both of the first nucleic acid sequence and the second nucleic acid sequence are operably linked to a promoter element.

46. The method according to claim 44 or 45, wherein one or both of the first nucleic acid sequence and the second nucleic acid sequence are comprised in a vector.

47. The method according to claim 46, wherein the vector is a retroviral vector.

48. A nucleic acid composition comprising (a) a first nucleic acid sequence encoding an antigen recognition receptor and (b) a second nucleic acid sequence encoding an exogenous dominant-negative Fas polypeptide, and the amino acid sequence of the exogenous dominant-negative Fas polypeptide is as shown in SEQ ID NO: 12 or SEQ ID NO:

14.

49. The nucleic acid composition according to claim 48, wherein one or both of the first nucleic acid sequence and the second nucleic acid sequence are operably linked to a promoter element.

50. The nucleic acid composition according to claim 48 or 49, wherein one or both of the first nucleic acid sequence and the second nucleic acid sequence are comprised in a vector.

51. The nucleic acid composition according to claim 50, wherein the vector is a retroviral vector.

52. A vector comprising the nucleic acid composition according to any one of claims 48-51.

53. A kit, which comprises the cells according to any one of claims 1-27, the composition according to claim 28 or 29, the nucleic acid composition according to any one of claims 48-51, or the vector according to claim 52.

54. The kit according to claim 53, wherein the kit further comprises written instructions for treating and / or preventing tumor formation or pathogen infection.

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