Death receptor 5-targeted chimeric antigen receptors and uses thereof

Abstract

The present disclosure provides modified immune cells or precursors thereof (e.g., gene edited modified T cells) comprising chimeric antigen receptors (CARs) specific for Death receptor-5 (DR5), and compositions and methods related thereto.

Description

[0001] DEATH RECEPTOR 5-TARGETED CHIMERIC ANTIGEN RECEPTORS

[0002] AND USES THEREOF

[0003] CROSS-REFERENCE TO RELATED APPLICATION

[0004] The present application is entitled to priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 63 / 702,449 filed on October 2, 2024, which is herein incorporated by reference in its entirety.

[0005] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0006] This invention was made with government support under CA174523. CA261608 and CA258113 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0007] REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0008] This application contains a sequence listing as a ST.26 file named “046483- 7476xx.xml," created on September 25, 2025 and having a size of 175,435 bytes, which is herein incorporated by reference.

[0009] BACKGROUND

[0010] The development of the chimeric antigen receptor (CAR) and its successful clinical use to direct T cells against specific types of cancers has been an important advancement in cancer immunotherapy. Despite the efficacy of some of these approaches, CAR constructs must be individually designed and developed for each disease, and target molecules must be selected that minimize unintended toxi cities to normal cell populations, which also express them. The risk of on-target, off-tumor toxicity can be high, as illustrated by toxicity in Her2- CAR T cell and GD2-CAR T cell clinical trials, for example.

[0011] Thus, there is a need for novel and effective compositions and methods for cancer treatment.

[0012] SUMMARY OF THE INVENTION

[0013] As described herein, the present invention relates to modified immune cells or precursors thereof comprising chimeric antigen receptors (CARs) specific for death receptor 5 (DR5). In one aspect, the present invention provides a nucleic acid comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR) comprising an antigen binding domain, a transmembrane domain, and an intracellular domain, wherein the antigen binding domain specifically binds DR-5.

[0014] In some embodiments, the antigen binding domain comprises an antibody or an antigen-binding fragment thereof. In some embodiments, the antigen-binding fragment is selected from the group consisting of a Fab. a single-chain variable fragment (scFv). or a single-domain antibody. In some embodiments, the antigen-binding fragment is an scFv. In some embodiments, the antigen binding domain comprises a heavy7chain variable region comprising an amino acid sequence having at least 95%-99% identity to the amino acid sequence of SEQ ID NO: 1, 4. 7, or 10; and / or a light chain variable region comprising an amino acid sequence having at least 95-99% identity to the amino acid sequence of SEQ ID NO: 2, 5, 8, or 11. In some embodiments, the heavy chain variable region comprises the amino acid sequence of any one of SEQ ID NO: 1, 4, 7, or 10; and / or the light chain variable region comprises the amino acid sequence of any one of SEQ ID NO: 2, 5, 8. or 11. In some embodiments, the antigen binding domain comprises the ammo acid sequence of SEQ ID NO: 3, 6, 9, or 12; or an amino acid sequence having at least 95%-99% identity7to the amino acid sequence of SEQ ID NO: 3, 6, 9, or 12.

[0015] In some embodiments, the transmembrane domain is selected from the group consisting of an artificial hydrophobic sequence, and a transmembrane domain of a type I transmembrane protein, an alpha, beta, or zeta chain of a T cell receptor, CD28, CD3epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, 0X40 (CD134), 4-1BB (CD137), and CD154. In some embodiments, the transmembrane domain comprises a transmembrane domain of CD8. In some embodiments, the transmembrane domain of CD8 comprises the amino acid sequence of SEQ ID NO: 16. In some embodiments, the transmembrane domain of CD8 is encoded by the nucleotide sequence of SEQ ID NO: 77;

[0016] In some embodiments, the CAR further comprises a hinge domain. In some embodiments, the hinge domain is selected from the group consisting of an Fc fragment of an antibody, a hinge region of an antibody, a CH2 region of an antibody, a CH3 region of an antibody, an artificial hinge domain, a hinge comprising an amino acid sequence of a CD8 hinge, or any combination thereof. In some embodiments, the hinge domain is a CD8 hinge. In some embodiments, the CD8 hinge comprises the amino acid sequence of SEQ ID NO: 14. In some embodiments, the CD8 hinge is encoded by the nucleotide sequence of SEQ ID NO: 74.

[0017] In some embodiments, the intracellular domain comprises a costimulatory signaling domain and an intracellular signaling domain. In some embodiments, the costimulatory signaling domain comprises a costimulatory domain of a protein selected from the group consisting of proteins in the TNFR superfamily, CD28, 4-1BB (CD137), 0X40 (CD134), PD-1, CD7, LIGHT. CD83L, DAP10. DAP12, CD27. CD2, CD5, ICAM-1, LFA-1, Lek, TNFR-I, TNFI-II, Fas, CD30, CD40, ICOS, NKG2C, MyD88, and B7-H3 (CD276), or a variant thereof. In some embodiments, the costimulatory signaling domain comprises a costimulatory domain of 4- IBB. In some embodiments, the costimulatory signaling domain comprises the amino acid sequence of SEQ ID NO: 20. In some embodiments, the costimulatory signaling domain is encoded by the nucleotide sequence of SEQ ID NO: 81;

[0018] In some embodiments, the intracellular signaling domain comprises an intracellular domain selected from the group consisting of cytoplasmic signaling domains of a human CD3 zeta chain (CD3Q, FcyRIII, FcsRI, a cytoplasmic tail of an Fc receptor, an immunoreceptor tyrosine-based activation motif (ITAM) bearing cytoplasmic receptor, TCR zeta, FcR gamma, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d, or a variant thereof. In some embodiments, the intracellular signaling domain comprises an intracellular domain of CD3^, In some embodiments, the intracellular domain of CD3^ comprises the amino acid sequence of SEQ ID NO: 24 or 25. In some embodiments, the intracellular domain of CD3^ is encoded by the nucleotide sequence of SEQ ID NO: 85 or 86.

[0019] In some embodiments, the CAR comprises the antigen binding domain, a CD8a hinge region, a CD8a transmembrane domain, a 4-1BB costimulatory domain, and a CD3^ intracellular signaling domain.

[0020] In some embodiments, the CAR comprises the amino acid sequence of any one of SEQ ID NOs 26-29; or an amino acid sequence having at least 95%-99% identity to the amino acid sequence of any one of SEQ ID NOs: 26-29.

[0021] In some embodiments, the CAR is encoded by nucleotide sequence set forth in of any one of SEQ ID NOs: 87-90; or a nucleotide sequence having at least 95%-99% identity to any one of SEQ ID NOs: 87-90.

[0022] In some embodiments, the nucleic acid further comprises a second polynucleotide sequence encoding a second polypeptide. In some embodiments, the nucleic acid encodes a self-cleaving 2A peptide domain between the CAR and the second polypeptide. In some embodiments, the self-cleaving peptide domain is selected from the group consisting of T2A, P2A, E2A and F2A.

[0023] In some embodiments, the nucleic acid comprises an internal ribosome binding site (IRES) between the CAR and the second polypeptide.

[0024] In another aspect, the present invention provides a nucleic acid encoding a CAR as described herein. In some embodiments, the nucleic acid comprises a vector. In some embodiments, the vector is an expression vector. In some embodiments, the vector is a viral vector selected from the group consisting of a retroviral vector, a lentiviral vector, an adenoviral vector, and an adeno-associated viral vector. In some embodiments, the vector is a lentiviral vector. In some embodiments, the lentiviral vector is a self-inactivating HIV vector comprising an LTR comprising a U3 deletion. In some embodiments, the HIV vector comprises a partial gag sequence, optionally wherein the partial gag sequence comprises the nucleotide sequence of SEQ ID NO: 33. In some embodiments, the HIV vector further comprises a central polypurine tract (cPPT). optionally wherein the cPPT comprises the nucleotide sequence of SEQ ID NO: 34. In some embodiments, expression vector comprises a Woodchuck Hepatitis virus post-transcriptional regulator}' element (WPRE), optionally wherein the WPRE comprises the nucleotide sequence of SEQ ID NO: 35. In some embodiments, the nucleic acid is a lentivirus expression construct comprising the polynucleotide sequence of any one of SEQ ID NOs: 37-40.

[0025] In another aspect, the present invention provides a chimeric antigen receptor (CAR) encoded by a nucleic acid or expression vector construct described herein. In some embodiments, the CAR comprises an antigen binding domain, a transmembrane domain, and an intracellular domain, wherein the antigen binding domain specifically binds DR-5. In some embodiments, the antigen binding domain comprises an antibody or an antigen-binding fragment thereof. In some embodiments, the antigen-binding fragment is selected from the group consisting of a Fab, a single-chain variable fragment (scFv), or a single-domain antibody. In some embodiments, the antigen-binding fragment is a scFv.

[0026] In some embodiments, the antigen binding domain comprises a heavy chain variable region comprising an amino acid sequence having at least 95%-99% identity to the amino acid sequence of SEQ ID NO: 1, 4, 7, or 10; and / or a light chain variable region comprising an amino acid sequence having at least 95-99% identity to the amino acid sequence of SEQ ID NO: 2, 5, 8. or 11. In some embodiments, the heavy chain variable region comprises the amino acid sequence of any one of SEQ ID NO: 1, 4, 7, or 10; and / or the light chain variable region comprises the amino acid sequence of any one of SEQ ID NO: 2, 5, 8. or 11.

[0027] In some embodiments, the antigen binding domain comprises an scFv comprising the amino acid sequence of SEQ ID NO: 3, 6, 9, or 12; or an amino acid sequence having at least 95%-99% identity to the amino acid sequence of SEQ ID NO: 3, 6, 9, or 12.

[0028] In some embodiments, the CAR comprises a transmembrane domain selected from the group consisting of an artificial hydrophobic sequence, and a transmembrane domain of a ty pe I transmembrane protein, an alpha, beta, or zeta chain of a T cell receptor, CD28, CD3epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, 0X40 (CD 134), 4- IBB (CD 137), and CD 154. In some embodiments, the transmembrane domain comprises a transmembrane domain of CD8. In some embodiments, the transmembrane domain of CD8 comprises the amino acid sequence of SEQ ID NO: 16. In some embodiments, the transmembrane domain of CD8 is encoded by the nucleotide sequence of SEQ ID NO: 77;

[0029] In some embodiments, the CAR further comprises a hinge domain. In some embodiments, the hinge domain is selected from the group consisting of an Fc fragment of an antibody, a hinge region of an antibody, a CH2 region of an antibody, a CH3 region of an antibody, an artificial hinge domain, a hinge comprising an amino acid sequence of a CD8 hinge, or any combination thereof. In some embodiments, the hinge domain is a CD8 hinge. In some embodiments, the CD8 hinge comprises the amino acid sequence of SEQ ID NO: 14. In some embodiments, the CD8 hinge is encoded by the nucleotide sequence of SEQ ID NO: 74;

[0030] In some embodiments, the intracellular domain comprises a costimulatory signaling domain and an intracellular signaling domain. In some embodiments, the costimulatory signaling domain comprises a costimulatory domain of a protein selected from the group consisting of proteins in the TNFR superfamily, CD28, 4-1BB (CD137), 0X40 (CD134), PD-1, CD7, LIGHT, CD83L, DAP10, DAP12, CD27, CD2, CD5, ICAM-1, LFA-1, Lek, TNFR-I, TNFI-II, Fas, CD30, CD40, ICOS, NKG2C, and B7-H3 (CD276), or a variant thereof. In some embodiments, the costimulatory signaling domain comprises a costimulatory domain of 4-1BB. In some embodiments, the costimulatory signaling domain comprises the amino acid sequence of SEQ ID NO: 20. In some embodiments, the costimulatoiy signaling domain is encoded by the nucleotide sequence of SEQ ID NO: 81;

[0031] In some embodiments, the intracellular signaling domain comprises an intracellular domain selected from the group consisting of cytoplasmic signaling domains of a human CD3 zeta chain (CD3Q, FcyRIII, FcsRI. a cytoplasmic tail of an Fc receptor, an immunoreceptor tyrosine-based activation motif (ITAM) bearing cytoplasmic receptor, TCR zeta, FcR gamma, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d, or a variant thereof. In some embodiments, the intracellular signaling domain comprises an intracellular domain of CD3^, In some embodiments, the intracellular domain of CD3^ comprises the amino acid sequence of SEQ ID NO: 24 or 25. In some embodiments, the intracellular domain of CD3^ is encoded by the nucleotide sequence of SEQ ID NO: 85 or 86.

[0032] In some embodiments, the CAR comprises the antigen binding domain, a CD8a hinge region, a CD8a transmembrane domain, a 4-1BB costimulatory domain, and a CD3^ intracellular signaling domain.

[0033] In some embodiments, the CAR comprises an amino acid sequence set forth in any one of SEQ ID NOs: 26-29; or an amino acid sequence having at least 95%-99% identity to the amino acid sequence of SEQ ID NOs: 26-29. Alternatively, in some embodiments, the CAR is encoded by a lentivirus expression construct comprising the nucleotide sequence of any one of SEQ ID NOs: 87-90; or a nucleotide sequence having at least 95%-99% identity to any one of SEQ ID NOs: 87-90.

[0034] In some embodiments, the CAR further comprising a second polypeptide.

[0035] In some embodiments, the CAR further comprises a self-cleaving 2A peptide domain between the CAR and the second polypeptide. In some embodiments, the self-cleaving peptide domain is selected from the group consisting of T2A, P2A, E2A and F2A.

[0036] In another aspect, the present invention provides a cell comprising a nucleic acid, expression vector construct, or CAR described herein. In some embodiments, the cell is an immune cell or precursor cell thereof. In some embodiments, the cell is a natural killer (NK) cell or a T cell. In some embodiments, the cell is a gamma delta T cell. In some embodiments, the cell is an autologous cell. In some embodiments, the cell is an allogeneic cell. In some embodiments, the cell is from an NK92 cell line.

[0037] In another aspect, the present invention provides a pharmaceutical composition comprising a cell as described herein and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition is administered intratumorally. In some embodiments, the pharmaceutical composition is administered intravenously.

[0038] In another aspect, the present invention provides a method for killing a DR5+ cell expressing a death receptor 5 antigen, comprising: contacting the cell with a cell as described herein. In some embodiments, the DR5+ cell is a melanoma cell, a pancreatic cancer cell, a lung cancer cell, a glioma cell, a breast cancer cell, a liver cancer cell, or an ovarian cancer cell. In some embodiments, the DR5+ cell is a myeloid-derived suppressor cell (MDSC). In some embodiments, the DR5+ cell is a cancer-associated fibroblast (CAF).

[0039] In another aspect, the present invention provides a method for treating cancer in a subject in need thereof, comprising: administering an effective amount of a pharmaceutical composition described herein to the subject, thereby treating the cancer.

[0040] In some embodiments, the cancer is a solid tumor. In some embodiments, the solid tumor is selected from the group consisting of melanoma, glioma, breast cancer, liver cancer, lung cancer, ovarian cancer, cervical cancer, uterus cancer, head and neck cancer, hepatocellular carcinoma, colon cancer, rectal cancer, colorectal carcinoma, kidney cancer, prostate cancer, gastric cancer, bronchus cancer, pancreatic cancer, urinary bladder cancer, and brain cancer. In some embodiments, the solid tumor is melanoma, pancreatic cancer, lung cancer, glioma, breast cancer, liver cancer, or ovarian cancer.

[0041] In some embodiments, the cancer is a hematologic cancer. In some embodiments, the hematologic cancer is a myeloid malignancy selected from the group consisting of acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), myelodysplastic neoplasm, and myeloproliferative neoplasm.

[0042] In another aspect, the present invention provides a method for preparing a transduced cell described herein, comprising: introducing into the cell a nucleic acid or vector construct as described herein.

[0043] BRIEF DESCRIPTION OF THE DRAWINGS

[0044] The foregoing and other features and advantages of the present invention will be more fully understood from the following detailed description of illustrative embodiments taken in conjunction with the accompanying drawings.

[0045] FIGs. 1 A-1B: DR5 mRNA and protein expression correlated with melanoma. (A) Expression of DR5 gene in cancer tissues from the TCGA database. (B) Expression of DR5 mRNA in clinical melanoma tissues using in situ hybridization and protein using immunohistochemistry. The bar indicated 100 pm.

[0046] FIGs. 2A-2H: DR5 CAR-T cells are cytotoxic to DR5+ cancer cells. (A) Schematic of DR5 chimeric antigen receptors construct architecture. (B) Expression of the CAR on transduced T cells after magnetic bead purification. (C) DR5 expression on Luciferase-GFP transfected melanoma cell line A375, ovarian cancer cell line OVCAR5, and hepatocellular carcinoma cell line HepG2. (D) Luciferase-based cytotoxicity assay after 16 hours of coculture of DR5 CAR-T cells with cancer cells at indicated E:T ratio, n=3. (E) Representative images of 3D bicellular spheroids of A375 cells (GFP+Hoechst33342+) and fibroblast cell line BJ (Hoechst33342+) coculture with indicated DR5 CAR-T cells at E:T ratio of 1. Bar=100pm. (F) Cytotoxicity of DR5 CAR-T to A375 / BJ bicellular spheroids, target cell lysis data shown as loss of GFP signal density, n=3. (G) PRRA assay analysis of representative T cell activation gene expression after co-culture with A375 melanoma cells for 4 hours, n=5. (H) Heat map of cell death and apoptosis-associated protein expression using RPPA after co-culture of A375 melanoma cells with CAR-T cells for 4 hours, data shown as average values of 5 samples (n=5). All data shown as Mean±S.E.M., *P<0.05, **P<0.01, ***P<0.001.

[0047] FIGs. 3A-3B: DR5 CAR-T cells are cytotoxic to DR5+ cancer cells. Related to Fig. 2. (A) Schematic of lentiviral vector for lentiviral DR5 CAR expression. (B) Heat map of T cell activation gene expression of RPPA assay analysis, data shown as average values of 5 samples.

[0048] FIGs. 4A-4F: DR5 CAR-T cells are cytotoxic to myeloid-derived suppressor cells (MDSCs). (A) PBMC-Monocyte derived MDSCs are typical CD1 lb+CD33+PDLlhi cells by FACS. They also express HLD-DR and CD14. Representative flow charts of induced MDSCs. (B) Induced MDSCs inhibit T-cell proliferation. CFSE staining data after coculturing T cells and induced MDSCs 3 days. (C) DR5 expression on the surface of MDSCs and A375 melanoma cells. (D) Cytotoxicity of DR5 CAR-T cells against MDSCs. LDH- releasing assays were performed after co-culture of T cells-target cells at E:T ratio 5: 1 for 16 hours, n=3. (E) Representative images of 3D spheroids with A375 cells (GFP+Hoechst33342+) and MDSCs (Hoechst33342+) coculture with indicated DR5 CAR-T cells at E:T ratio of 1: 1. Bar=100pm. (F) Statistical analysis of cytotoxicity of DR5 CAR-T cells against A375-MDSC spheroids, target cell lysis data shown as PI signal density, n=3-5. All data are shown as Mean±S.E.M., *P<0.05, **P<0.01 by Student’s t-test, n.s., no statistically significant difference.

[0049] FIGs. 5A-5H: Membrane DR5-scFvs induce target cell death. (A) DR5-scFv membrane expression on non-cytotoxic SupTl cells. SupTl cells were transfected with CD19 and DR5 CAR constructs. CAR expression on the cell surface was detected by FACS. (B) SupTl cells do not express cytolytic proteins. Cytotoxic T cell functional protein expression in SupTl cells by FACS. (C) Cytotoxicity of DR5 CAR-SupTl cells against melanoma cells. LDH-rel easing assays were performed 16 hours after co-culture of DR5 CAR-SupTl and melanoma cells at indicated E:T ratios, n=3. (D) Representative images of Hoechst33342 / PI double staining assays showing DR5 CAR-SupTl cells kill A375 cells in 2D culture at an E:T ratio of 1 : 1, n=3. CD19 CAR-SupTl and wild-type SupTl cells were used as controls. (E) Statistical analysis of cytotoxicity7of DR5 SupTl to A375 cells. The number of PI+ (Red) cells in different fields was quantified and averaged, n=3. (F) Western blot analysis showing the sEV marker CD63 expression in SupTl-derived sEVs. (G) Size analysis indicated the sEV size at -lOOnm. The EV size was measured by the NanoSight NS300 instrument. (H) Cytotoxicity of DR5 CAR-SupTl sEVs. LDH releasing assay after coculture of sEVs from CAR-SupTl cells and A375 melanoma at the indicated amount for 16 hrs, n=3. All data are shown as Mean±S.E.M., *P<0.05, **P<0.01, ***P<0.001 by Student’s t-test, n.s., no statistically significant difference.

[0050] FIGs. 6A-6E: Effects of DR5-CAR-SupTl cells on K562-CD19 cells. (A) DR5 expression levels in different cell lines. DR5 expression on A375, K562-CD19, and SupTl cells were measured by FACS. (B) DR5 expression in a panel of melanoma cell lines by FACS analysis._,(C-D) Effects of DR5-CAR-SupTl cells on K562-CD19 cells. SupTl or DR5 CAR-SupTl cells were cocultured with K562-CD19 cells at an E:T ratio of 1:1 for 16 hrs. PI and Annexin V were used to measure cell apoptosis. Representative flow chart after coculture of SupTl cells with K562-CD19. (C). Statistical analysis of apoptosis and cell death of K562-CD19 cells after treatment n=3. (D). (E) Cytotoxicity7assay of large EVs (size>200nm) derived from DR5 CAR-T cells to A375 cells. Luciferase-based assays were performed after coculture of large EVs (1 pg / ml or 5 pg / ml) from DR5 CAR-SupTl with different A375 cells for 16 hrs, n=3. All data are shown as Mean±S.E.M., *P<0.05, **P<0.01, ***P<0.001, n.s., no statistically significant difference.

[0051] FIGs. 7A-7B: CAR-T cell viability after culturing. (A) Viability of cultured DR5 CAR-T cells after transduced 4 days, n=5. (B) Viability of cultured DR5 CAR-T cells after transduced 3 days and cocultured with target cells (A375) for 24 hours, n=5. All data are shown as Mean±S.E.M., *P<0.05, **P<0.01, ***P<0.001, n.s., no statistically significant difference.

[0052] FIGs. 8A-8F: Affinity of DR5-scFv to its target. (A) Representative scFv structure of anti-DR5 (1#) with light chain region (pink), heavy chain region (green), and G4S linker (blue). (B) Representative binding of anti-DR5 scFv (Green) and DR5 protein (Deep Red). (C) Schematics of DR5 whole antibodies (up) and DR5 scFv-antibodies (down). (D) Image panels of On-Cell Western (OCW) assay showing DR5 antibody binding intensity at the indicated dilutions. Pre-fixed A375 cells were used in the binding assays. (E) Normalized OCW data of DR5 antibody binding intensity to A375 cells, shown as Mean±S.E.M. of 3 wells. (F) Representative flow cytometry data showing the DR5 antibody binding to A375 cells. Different DR5 whole and scFv antibodies (4pg) were used in the assays.

[0053] FIGs. 9A-9B: Computational analysis of DR5 scFvs binding to their targets. (A) HADDOCK scores of affinity of DR5 scFvs to their targets. (B) Computational models of DR5 scFvs binding to DR5 protein. The binding sites are highlighted with a teal color.

[0054] FIGs. 10A-10G: DR5 CAR-T cells are not cytotoxic to DR5- cells. (A) DR5 expression levels on different cell lines. HaCaT cells expressed a lower level of DR5 than A375 cells. (B) DR5 CAR-T cells with little toxicity’ to HaCaT cells at indicated E:T ratios, n=3. (C) Membrane DR5 expression levels in DR5 knockout (KO) and over-expression (OE) A375 cells. FACS was used to measure DR5 expression in these cell lines. (D) DR5 expression levels in DR5KO and DR5OE A375 cells. Western blot was used to measure DR5 expression in these cell lines. (E) Cytotoxicity assay of DR5 CAR-T cells to A375 cells. Luciferase-based assays were performed after coculture of DR5 CAR-T cells with different A375 cells for 16 hrs at the indicated E:T ratio of 5: 1, n=3. (F) Cytotoxicity' assay of sEVs derived from DR5 CAR-T cells to A375 cells. Luciferase-based assays were performed after coculture of sEVs (1 pg / ml) from DR5 CAR-T cells with different A375 cells for 16 hrs, n=3. (G) Luciferase assays after 4 or 16 hours of DR5 CAR SupTl-A375 coculture at indicated effector to target (E:T) ratios to measure cytotoxicity' by DR5 CAR-SupTl cells against DR5 wild type and knockout A375 cells, n=3. All data shown as Mean±S.E.M.. *P<0.05. **P<0.01, ***P<0.001.

[0055] FIGs. 11 A-l 1G: Effects of DR5 CAR-T cells in vivo. (A) Treatment schema of intratumoral DR5 CAR-T cells in A375 melanoma xenograft model. 5x106 A375-DR5OE melanoma cells were intratumorally injected into the flanks of nude mice, 5 mice per group. (B) Representative tumor bioluminescence images and Day 20 bright field tumor images. (C) Tumor bioluminescence kinetics of A375-DR5-GFP-FFLuc tumor grow th in the xenograft nude mice model shown in (B), n=5. (D) Body weight curves of tumor-burden mice, n=5. (E) Treatment schema of systemic DR5 CAR-T cells in the patient-derived melanoma xenograft models. 10 mice were used in each group. (F) WM3907 PDX tumor growth curves after systemic DR5 CAR-T cell treatment with Two-way' ANOVA analysis. (G) Survival curve of A375 WT xenograft mice, n=5. All data are shown as mean±SEM. *P<0.05, **P<0.01, ***p<0.001, n.s., no statistically significant difference. FIGs. 12A-12N: Effects of DR5 CAR-T cells in MPDOs and MSCs. (A) Representative fluorescence images of MPDOs co-cultured with UTD T cells (Pink) and DR5 CAR-T cells (Green). Arrows indicate the DR5 CAR-T cells, and arrowheads indicate dead tumor cells. Bars indicate 200 pm. Hoechst33342 and PI were used to label nuclei and dead cells, respectively. (B) Confocal images of MPDOs cocultured with DR5 CAR-T cells (CFSE) for 24 hours. Hoechst33342 and PI were used to label nuclei and dead cells, respectively, n=3. (C) DR5 CAR-T cell infiltration and tumor killing in MDPOs. CAR-T cells are labeled with CFSE. Hoechst33342 and PI were used to label nuclei and dead cells, respectively. Fluorescent density was digitally analyzed, n=3. (D) Effects of DR5 CAR-T cells in MSCs. MSCs from five different patients were used in the experiment. One to two slices per patient were included in the study. Each dot represents a tumor slice. The MSCs were incubated with DR5 CAR-T cells, untransduced cells, or vehicles for 48 hours. The tissues were washed and digested into single cells for FACS analysis. All data are shown as Mean ± S.E.M., *P<0.05, **P<0.01. ***P<0.001. (E) DR5 CAR-T cells infiltrating MPDOs. UTD and CAR-T cells were labeled with far-red fluorescent dye. MPDOs from three melanoma patients were incubated with labeled UTD or CAR-T cells for 48 hours. After treatment, the MPDOs were collected and disassociated into single cells for FACS analysis. Far-red+CD3+ T cells are quantified. Each dot represents an independent well. (F-I) Effects of DR5 CAR-T cells on MDSCs. MPDOs from four melanoma patients were incubated with labeled UTD or CAR-T cells for 48 hours. After treatment, the MPDOs were collected and disassociated into single cells for FACS analysis. More cell death in the MPDOs was observed after CAR-T cell treatment (F). CDl lb+CD45+HLA-DR- myeloid cells (G), CD33+CDl lb+ MDSCs (H), CD14+CDlb+CD33+ M-MDSCs (I) were significantly decreased after CAR-T cell treatment, n=17, and each dot represents an independent well. (J- L) Tumor-resident CD8+ T cells were activated after CAR-T cell treatment. UTD and CAR- T cells were labeled with far-red fluorescent dye. MPDOs from three melanoma patients were incubated with labeled UTD or CAR-T cells for 48 hours. After treatment, the MPDOs were collected and dissociated into single cells for FACS analysis. Far-red-CD8+ T cells (J), Gran zy me B+far-red-CD8+ T cells (K), Ki67+far-red-CD8+ T cells (L) were significantly increased after CAR-T cell treatment. n=5, and each dot represents an independent well. (M- N) Effects of DR5 CAR-T cells on MSCs. MSCs from five different patients were used in the experiment. One to two slices per patient were included in the study. Each dot represents a tumor slice. The MSCs were incubated with DR5 CAR-T cells or UTD cells for 48 hours. The tissues were washed and digested into single cells for FACS analysis. Cytotoxicity (M) and CD3+ T cells (N) are significantly increased after CAR-T cell treatment. All data are shown as Mean±S.E.M., *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001

[0056] FIG. 13: DR5 CAR-T cells induced tumor lysis in the MPDOs. Representative bright field images of MPDOs from four melanoma patients cocultured with DR5 CAR-T cells for 48 hours show organoid disintegration.

[0057] FIGs. 14A-14C: Membrane DR5-scFvs induce target cell death. (A) DR5-scFv membrane expression on non-cytotoxic SupTl cells. SupTl cells are transfected with CD19 and DR5 CAR constructs. CAR expression on the cell surface is detected using FACS. (B) SupTl cells do not express cytolytic proteins. Cytotoxic T cell functional protein expression in SupTl cells by FACS. (C) Cytotoxicity of DR5 CAR-SupTl cells against melanoma cells. LDH-releasing assays are performed 16 hours after co-culture of DR5 CAR-SupTl and melanoma cells at indicated E:T ratios, n=3.

[0058] FIG. 15: Cytotoxicity of large EVs derived from DR5 CAR-SupTl cells to A375 cells. Luciferase-based assays were performed after coculture of large EVs (1 pg / ml or 5 pg / ml) from DR5 CAR-SupTl with A375 cells for 16 hrs, n=3. All data are shown as Mean±S.E.M., *P<0.05, **P<0.01, ***P<0.001, n.s., no statistically significant difference.

[0059] FIGs. 16A-16G: DR5 CAR-T cells are not cytotoxic to DR5-negative cells. (A) DR5 expression levels on different cell lines. HaCaT cells expressed a lower level of DR5 than A375 cells. (B) DR5 CAR-T cells with little toxicity to HaCaT cells at indicated E:T ratios, n=3. (C) Membrane DR5 expression levels in DR5 knockout (KO) and over-expression (OE) A375 cells. FACS was used to measure DR5 expression in these cell lines. (D) DR5 expression levels in DR5K0and DR5OEA375 cells. Western blot was used to measure DR5 expression in these cell lines. (E) Cytotoxicity assay of DR5 CAR-T cells to A375 cells. Luciferase-based assays were performed after coculture of DR5 CAR-T cells with different A375 cells for 16 hrs at the indicated E:T ratio of 5: 1 , n=3. (F) Cytotoxicity assay of sEVs derived from DR5 CAR-T cells to A375 cells. Luciferase-based assays are performed after coculture of sEVs (1 pg / ml) from DR5 CAR-T cells with different A375 cells for 16 hrs, n=3. (G) Luciferase assays after 4 or 16 hours of DR5 CAR SupTl-A375 coculture at indicated effector to target (E:T) ratios to measure cytotoxicity by DR5 CAR-SupTl cells against DR5 wild type and knockout A375 cells, n=3. All data shown as Mean±S.E.M., *P<0.05, **P<0.01, ***P<0.0. FIG. 17: Representative flow cytometry data showing the DR5 antibody binding to A375 cells. Four different DR5 whole and scFv antibodies (4pg) were used in the assays.

[0060] FIGs. 18A-18B: CAR-T cell viability after expansion and activation. (A) Viability of cultured DR5 CAR-T cells after expansion for 4 days, n=5. (B) Viability of cultured DR5 CAR-T cells after expansion for 3 days and cocultured with target cells (A375) for 24 hours, n=5. All data are shown as Mean±S.E.M., *P<0.05, **P<0.01, ***P<0.001, n.s., no statistically significant difference.

[0061] FIGs. 19A-19B: MDSCs inhibit T cell proliferation. (A) PBMC-monocyte-derived MDSCs were typically identified as CDl lb+CD33+PDLlhi cells by FACS. They also express HLD-DR and CD14. Representative flow charts of induced MDSCs. (B) Induced MDSCs inhibited T-cell proliferation. CFSE staining data after co-culturing T cells and induced MDSCs for 3 days.

[0062] FIGs. 20A-20H: Effects of DR5 CAR-T cells in vivo. (A) Treatment schema of intratumoral DR5 CAR-T cells in A375 melanoma xenograft model. 5><106A375-DR5OE(B- D) or A375 wildtype (WT) (E-G) melanoma cells are intratumorally injected into the flanks of nude mice, 5xl06CAR+ T cells and same number of UTD cells in lOOul PBS were injected. (B) Representative A375-DR5OEtumor bioluminescence images and Day 20 bright field tumor images. (C) Tumor bioluminescence kinetics of A375-DR5-GFP-FFLuc tumor growth in the xenograft nude mice model shown in (B), n=5. (D) Body weight curves of tumor-burden mice, n=5. (E) Tumor volumes of A375 WT tumor growth in the xenograft nude mice model, n=5. (F) Body weight curves of tumor-burden mice of E, n=5. (G) Survival curve of A375 WT xenograft mice, n=5. All data are shown as mean±SEM. *P<0.05, **P<0.01, ***P<0.001, n.s., no statistically significant difference.

[0063] FIG. 21: Representative flow charts showing murine DR5 CAR-T cells express BFP.

[0064] FIG. 22: Body weight of C57 mice injected with murine DR5 CAR-T cells. Nontumor bearing mice were injected with 5xl06murine DR5 CAR-T cells via the tail veins. Body w eight of the treated mice was measured twice a week, n=5.

[0065] FIGs. 23A-23C: Murine DR5 CAR-T cells are cytotoxic to murine melanoma cells. Luciferase cytotoxicity assay after coculture of murine DR5 CAR-T cells with cancer cells for 24 hours at indicated E:T ratio, n=3. All data are shown as mean±SEM. *P<0.05, **P<0.01, ***P<0.001, n.s., no statistically significant difference.

[0066] FIG. 24: DR5 CAR-T cells inhibit PMN-MDSCs in the MPDOs. MPDOs from four melanoma patients are incubated with labeled UTD or CAR-T cells for 48 hours. After treatment, the MPDOs were collected and dissociated into single cells for FACS analysis. CD15+CDlb+CD33+ PMN-MDSCs were significantly decreased after CAR-T cell treatment. n=17, and each dot represents an independent well.

[0067] FIGs. 25A-25F: DR5 CAR enhances the cytotoxicity of y5 T cells against A375 melanoma cells. A375 cells were co-cultured with unmodified y5 T cells or DR5 CAR- modified y5 T cells at effector- to-target (E: T) ratios of 2.5: 1 or 5: 1 for 12, 24, or 48 hours. Cytotoxicity was measured by flow cytometry. (A-C) E:T ratio of 5: 1 at 12 (A), 24 (B), and 48 hours (C). (D-F) E:T ratio of 2.5: 1 at 12 (D). 24 (E). and 48 hours (F). y5T cells were expanded using an anti-TCR PAN y / 5 antibody. Data presented as mean + SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns=not significant.

[0068] FIGs. 26A-26D: DR5 CAR enhances the killing ability of yoT cells in melanoma patient derived tissue slices. Tumor tissue slices were treated with different cells for 48 hours (n=12 per group, 1 million y5T cells for each 11x8x5 mm3). (A) Cytotoxicity of CAR-y5T cells, (B) Ki-67+CD8+ cells in the tissue slices. (C) Granzyme B+CD8+ T cells in the tissue slides (D) Perforin+CD8+ cells in the tissue slides. Percentage of Ki-67+, Granzyme B+, and Perforin+ cells among CD8+ T cells. Data presented as mean + SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns=not significant.

[0069] FIGs. 27A-27F: DR5 CAR enhances the killing ability ofyST cells in melanoma patient-derived organoids (MPDOs). MPDOs were generated using fresh melanoma tissues and then treated with different cells for 48 hours (n=12 per group). (A) Cytotoxicity of CAR- y5T cells, (B-C) immune cell infiltration in the MPDOs, (D) Ki-67+CD8+ cells in the MPDOs, (E) Granzyme B+CD8+ cells in the MPDOs, and (F) Perforin+CD8+ cells in the MPDOs. Cytotoxicity measured by flow cytometry. Data presented as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns=not significant.

[0070] FIGs; 28A-28D: DR5 CAR enhances the killing ability of y5T cells to myeloid derived suppressor cells (MDSCs) in MPDOs. MPODs were treated with different cells for 48 hours. (A) increasing cytotoxicity of CAR-yST cells, (B) Decreasing DR5+ cells in the MPDOs, (C) Increasing immune cell infiltration in the MPDOs, and (D) Decreasing MDSC levels in MPDOs. Control group, n=19; y5T group, n=17; y5T-94CAR group, n=15. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns=not significant.

[0071] FIGs. 29A-29E: Comparison of treatment response to a|3 T cells, y6 T cells, or DR5- y5 T cells in tumor bearing mice. Quantification of bioluminescence signal from tumors at days 3, 10, 15, and 20 post-treatment for each group. (A, B) Representative images of tumors in mice from each treatment group at days 3, 10, 15, and 20. (C) Kinetics of tumor bioluminescence over the 20-day treatment period. Each line represents the mean total flux for a treatment group. Error bars indicate SEM. (D) Tumor volume measurements over time for mice in each treatment group. Tumor volumes were calculated using caliper measurements and the formula V = (L x WA2) / 2. Data shown as mean ± SEM. *p<0.05, **p<0.01 compared to aP T cell group. (E) Body weights of mice from each treatment group measured over the course of the experiment to assess treatment toxicity. Data presented as mean ± SEM. The results demonstrate that y5 T cells expressing DR5 CAR exhibited the most potent anti-tumor activity, significantly reducing tumor growth and bioluminescence signal compared to untransduced aP T cells and yd T cells, without significant changes in body weight across all groups.

[0072] FIGs. 30A-30B: Effect of DR5 CAR yoT cells on WM4380 patient derived xenograft (PDX). NSG mice bearing PDXs were treated with vehicle, untransduced y5 T cells or DR5 CAR transfected y5 T (gdT) cells. (A) Tumor volume measurements over time for mice in each treatment group. Tumor volumes were calculated using caliper measurements and the formula V = (L x WA2) / 2. (B) Survival curve of NSG mice bearing PDXs, n=5. Data shown as mean ± SEM. *p<0.05, **p<0.01 compared to aP T cell group. Untransduced yd T cells inhibited tumor growth. The effects were significantly increased after treating with DR5 CAR yd T cells.

[0073] FIGs. 31A-31B: Effects ofDR5 CAR-gdT cell on glioma blastoma multiforme (GBM). 20,000 U251(A) or U87(B) GBM cells in each were treated with DR5 CAR-gdT cells or untransduced gdT cells. The E:T ratio =5, 2.5, 1 .25, 0.625. Treatment is 12 to 24 hours. Luciferase assay was performed to measure cytotoxicity. Each cycle represents an independent experiment. * p<0.05, **p<0.01, *** p<0.001.

[0074] DETAILED DESCRIPTION

[0075] The present invention provides compositions and methods of using genetically modified immune cells or precursors thereof (e.g., modified T cells) comprising an engineered chimeric antigen receptor (CAR) specific for Death Receptor 5 (DR5).

[0076] Definitions

[0077] Unless otherwise defined, scientific and technical terms used herein have the meanings that are commonly understood by those of ordinary skill in the art. In the event of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. The use of “or’ means “and / or” unless stated otherwise. The use of the term “including,” as well as other forms, such as “includes” and “included,” is not limiting.

[0078] Generally, nomenclature used in connection with cell and tissue culture, molecular biology , immunology', microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein is well-known and commonly used in the art. The methods and techniques provided herein are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications, as commonly accomplished in the art, or as described herein. The nomenclatures used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those w ell-known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

[0079] That the disclosure may be more readily understood, select terms are defined below.

[0080] The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By w ay of example, “an element” means one element or more than one element.

[0081] “About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

[0082] “Activation,” as used herein, refers to the state of a T cell that has been sufficiently stimulated to induce detectable cellular proliferation. Activation can also be associated with induced cytokine production, and detectable effector functions. The term “activated T cells” refers to, among other things, T cells that are undergoing cell division.

[0083] As used herein, to “alleviate” a disease means reducing the severity of one or more symptoms of the disease.

[0084] As used herein, the term “ambiguous” is used in Table 1 with reference to generic nucleotides wherein N (any nucleotide) is A or C or G or T or U; R (purine) is A or G; Y (pyrimidine) is T or C; K (keto) is G or T: M (amino) is A or C; S (strong interaction, 3 H bonds) is G or C: W (weak interaction, 2 H bonds) is A or T; B is Not A (C or G or T): D is Not C (A or G or T); H is Not G (A or C or T); V is Not T or U (A or C or G) .

[0085] The term "antigen'' as used herein is defined as a molecule that provokes an immune response. This immune response may involve either antibody production, or the activation of specific immunologically-competent cells, or both. The skilled artisan will understand that any macromolecule, including virtually all proteins or peptides, can serve as an antigen.

[0086] Furthermore, antigens can be derived from recombinant or genomic DNA. A skilled artisan will understand that any DNA, which comprises a nucleotide sequence or a partial nucleotide sequence encoding a protein that elicits an immune response therefore encodes an “antigen"’ as that term is used herein. Furthermore, one skilled in the art will understand that an antigen need not be encoded solely by a full-length nucleotide sequence of a gene. It is readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene and that these nucleotide sequences are arranged in various combinations to elicit the desired immune response. Moreover, a skilled artisan will understand that an antigen need not be encoded by a “gene” at all. It is readily apparent that an antigen can be generated synthesized or can be derived from a biological sample. Such a biological sample can include, but is not limited to a tissue sample, a tumor sample, a cell, or a biological fluid.

[0087] As used herein, the term “autologous” is meant to refer to any material derived from the same individual to which it is later to be re-introduced into the individual.

[0088] A “co-stimulatory molecule” refers to the cognate binding partner on a T cell that specifically binds with a co-stimulatory ligand, thereby mediating a co-stimulatory response by the T cell, such as, but not limited to, proliferation. Co-stimulatory molecules include, but are not limited to an MHC class I molecule, BTLA and a Toll ligand receptor.

[0089] A “co-stimulatory signal,” as used herein, refers to a signal, which in combination with a primary signal, such as TCR / CD3 ligation, leads to T cell proliferation and / or upregulation or downregulation of key molecules.

[0090] A “disease” is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal’s health continues to deteriorate. In contrast, a "‘disorder” in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal’s state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal’s state of health. The term “downregulation'’ as used herein refers to the decrease or elimination of gene expression of one or more genes.

[0091] “Effective amount” or “therapeutically effective amount” are used interchangeably herein, and refer to an amount of a compound, formulation, material, or composition, as described herein effective to achieve a particular biological result or provides a therapeutic or prophylactic benefit. Such results may include, but are not limited to an amount that when administered to a mammal, causes a detectable level of immune suppression or tolerance compared to the immune response detected in the absence of the composition of the invention. The immune response can be readily assessed by a plethora of art-recognized methods. The skilled artisan would understand that the amount of the composition administered herein varies and can be readily determined based on a number of factors such as the disease or condition being treated, the age and health and physical condition of the mammal being treated, the severity of the disease, the particular compound being administered, and the like.

[0092] “Encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

[0093] As used herein “endogenous” refers to any material from or produced inside an organism, cell, tissue, or system.

[0094] The term “epitope” as used herein is defined as a small chemical molecule on an antigen that can elicit an immune response, inducing B and / or T cell responses. An antigen can have one or more epitopes. Most antigens have many epitopes; i.e., they are multivalent. In general, an epitope is roughly about 10 amino acids and / or sugars in size. Preferably, the epitope is about 4-18 amino acids, more preferably about 5-16 amino acids, and even more most preferably 6-14 amino acids, more preferably about 7-12, and most preferably about 8- 10 amino acids. One skilled in the art understands that generally the overall three- dimensional structure, rather than the specific linear sequence of the molecule, is the main criterion of antigenic specificity and therefore distinguishes one epitope from another. Based on the present disclosure, a peptide used in the present invention can be an epitope.

[0095] As used herein, the term '‘exogenous” refers to any material introduced from or produced outside an organism, cell, tissue, or system.

[0096] The term “expand” as used herein refers to increasing in number, as in an increase in the number of T cells. In one embodiment, the T cells that are expanded ex vivo increase in number relative to the number originally present in the culture. In another embodiment, the T cells that are expanded ex vivo increase in number relative to other cell types in the culture. The term "ex vivo " as used herein, refers to cells that have been removed from a living organism, (e.g., a human) and propagated outside the organism (e.g., in a culture dish, test tube, or bioreactor).

[0097] The term '‘expression” as used herein is defined as the transcription and / or translation of a particular nucleotide sequence driven by its promoter.

[0098] “Expression vector” refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g, naked or contained in liposomes) and viruses (e.g., Sendai viruses, lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.

[0099] As used herein, “Fab” refers to a fragment of an antibody structure that binds to an antigen but is monovalent and does not have a Fc portion, for example, an antibody digested by the enzyme papain yields two Fab fragments and an Fc fragment (e.g., a heavy (H) chain constant region; Fc region that does not bind to an antigen).

[0100] As used herein, “F(ab')2” refers to an antibody fragment generated by pepsin digestion of whole IgG antibodies, w herein this fragment has tw o antigen binding (ab') (bivalent) regions, wherein each (ab') region comprises two separate amino acid chains, a part of a H chain and a light (L) chain linked by an S — S bond for binding an antigen and where the remaining H chain portions are linked together. A “F(ab')2” fragment can be split into two individual Fab' fragments.

[0101] “Identity” as used herein refers to the subunit sequence identity between two polymeric molecules particularly between two amino acid molecules, such as, between two polypeptide molecules. When two amino acid sequences have the same residues at the same positions; e.g., if a position in each of two polypeptide molecules is occupied by an arginine, then they are identical at that position. The identity or extent to which two amino acid sequences have the same residues at the same positions in an alignment is often expressed as a percentage. The identity between two amino acid sequences is a direct function of the number of matching or identical positions; e.g., if half (e.g., five positions in a polymer ten amino acids in length) of the positions in two sequences are identical, the two sequences are 50% identical; if 90% of the positions (e.g., 9 of 10), are matched or identical, the two amino acids sequences are 90% identical.

[0102] The term “immune response’’ as used herein is defined as a cellular response to an antigen that occurs when lymphocytes identify antigenic molecules as foreign and induce the formation of antibodies and / or activate lymphocytes to remove the antigen.

[0103] The term “immunosuppressive” is used herein to refer to reducing overall immune response.

[0104] “Insertion / deletion,” commonly abbreviated “indel,” is a type of genetic polymorphism in which a specific nucleotide sequence is present (insertion) or absent (deletion) in a genome.

[0105] “Isolated” means altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.” An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.

[0106] The term “knockout” as used herein refers to the ablation of gene expression of one or more genes from a cell.

[0107] A “lentivirus” as used herein refers to a genus of the Retroviridae family. Lentiviruses are unique among the retroviruses in being able to infect non-dividing cells; they can deliver a significant amount of genetic information into the DNA of the host cell, so they are one of the most efficient methods of a gene delivery7vector. HIV, SIV, and FIV are all examples of lentiviruses. Vectors derived from lentiviruses offer the means to achieve significant levels of gene transfer in vivo.

[0108] By the term “modified” as used herein, is meant a changed state or structure of a molecule or cell of the invention. Molecules may be modified in many ways, including chemically, structurally, and functionally. Cells may be modified through the introduction of nucleic acids. By the term ‘‘modulating,’" as used herein, is meant mediating a detectable increase or decrease in the level of a response in a subject compared with the level of a response in the subject in the absence of a treatment or compound, and / or compared with the level of a response in an otherwise identical but untreated subject. The term encompasses perturbing and / or affecting a native signal or response thereby mediating a beneficial therapeutic response in a subject, preferably, a human.

[0109] As used herein, the term “non-ambiguous” nucleotides is used in Table 1 with reference to the following abbreviations for the commonly occurring nucleotides, where “A” refers to adenosine, “C” refers to cytosine, “G” refers to guanosine, “T” refers to thymidine, and “U” refers to uridine.

[0110] The term “oligonucleotide” typically refers to short polynucleotides. It will be understood that when a nucleotide sequence is represented by a DNA sequence (i.e., A, T, C, G), this also includes an RNA sequence (i.e., A, U, C, G) in which “U” replaces “T.”

[0111] Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence that encodes a protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some versions contain an intron(s).

[0112] “Parenteral” administration of an immunogenic composition includes, e.g, subcutaneous (s.c.), intravenous (i.v.). intramuscular (i.m.). or intrastemal injection, or infusion techniques.

[0113] The term “polynucleotide” as used herein is defined as a chain of nucleotides. Furthermore, nucleic acids are polymers of nucleotides. Thus, nucleic acids and polynucleotides as used herein are interchangeable. One skilled in the art has the general knowledge that nucleic acids are polynucleotides, which can be hydrolyzed into the monomeric “nucleotides.” The monomeric nucleotides can be hydrolyzed into nucleosides. As used herein polynucleotides include, but are not limited to, all nucleic acid sequences which are obtained by any means available in the art, including, without limitation, recombinant means, i.e.. the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCR, and the like, and by synthetic means.

[0114] As used herein, the terms “peptide,” “polypeptide.” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein's or peptide's sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides, and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. "Polypeptides" include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.

[0115] By the term "specifically binds,” as used herein with respect to an antibody, is meant an antibody which recognizes a specific antigen, but does not substantially recognize or bind other molecules in a sample. For example, an antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more species. But such crossspecies reactivity does not itself alter the classification of an antibody as specific. In another example, an antibody that specifically binds to an antigen may also bind to different allelic forms of the antigen. However, such cross reactivity does not itself alter the classification of an antibody as specific. In some instances, the terms “specific binding” or “specifically binding,” can be used in reference to the interaction of an antibody, a protein, or a peptide with a second chemical species, to mean that the interaction is dependent upon the presence of a particular structure (e.g, an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a specific protein structure rather than to proteins generally. If an antibody is specific for epitope “A”, the presence of a molecule containing epitope A (or free, unlabeled A), in a reaction containing labeled “A” and the antibody, will reduce the amount of labeled A bound to the antibody.

[0116] By the term “stimulation,” is meant a primary response induced by binding of a stimulatory molecule (e.g., a TCR / CD3 complex) with its cognate ligand thereby mediating a signal transduction event, such as, but not limited to, signal transduction via the TCR / CD3 complex. Stimulation can mediate altered expression of certain molecules, such as upregulation of interferon-gamma, and / or reorganization of cytoskeletal structures, and the like.

[0117] A “stimulatory molecule,” as the term is used herein, means a molecule on a T cell that specifically binds with a cognate stimulatory ligand present on an antigen presenting cell. A “stimulatory ligand,"’ as used herein, means a ligand that when present on an antigen presenting cell (e.g., an aAPC, a dendritic cell, a B-cell, and the like) can specifically bind with a cognate binding partner (referred to herein as a “stimulatory molecule”) on a T cell, thereby mediating a primary response by the T cell, including, but not limited to, activation, initiation of an immune response, proliferation, and the like. Stimulatory ligands are well-known in the art and encompass, inter alia, an MHC Class I molecule loaded with a peptide, an anti-CD3 antibody, a superagonist anti-CD28 antibody, and a superagonist anti- CD2 antibody.

[0118] The term “subject” is intended to include living organisms in which an immune response can be elicited (e g., mammals). A “subject"’ or “patient,” as used therein, may be a human or non-human mammal. Non-human mammals include, for example, livestock and pets, such as ovine, bovine, porcine, canine, feline and murine mammals. Preferably, the subject is human.

[0119] A “target site” or “target sequence” refers to a nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule may specifically bind under conditions sufficient for binding to occur. In some embodiments, a target sequence refers to a genomic nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule may specifically bind under conditions sufficient for binding to occur.

[0120] As used herein, the term “T cell receptor” or “TCR” refers to a complex of membrane proteins that participate in the activation of T cells in response to the presentation of antigen. The TCR is responsible for recognizing antigens bound to major histocompatibility complex molecules. TCR is composed of a heterodimer of an alpha (a) and beta ( ) chain, although in some cells the TCR consists of gamma and delta (y / 5) chains. TCRs may exist in alpha / beta and gamma / delta forms, which are structurally similar but have distinct anatomical locations and functions. Each chain is composed of two extracellular domains, a variable and constant domain. In some embodiments, the TCR may be modified on any cell comprising a TCR, including, for example, a helper T cell, a cytotoxic T cell, a memory T cell, regulatory T cell, natural killer T cell, and gamma delta T cell.

[0121] The term “therapeutic” as used herein means a treatment and / or prophylaxis. A therapeutic effect is obtained by suppression, remission, or eradication of a disease state.

[0122] “Transplant” refers to a biocompatible lattice or a donor tissue, organ, or cell, to be transplanted. An example of a transplant may include but is not limited to skin cells or tissue, bone marrow, and solid organs such as heart, pancreas, kidney, lung, and liver. A transplant can also refer to any material that is to be administered to a host. For example, a transplant can refer to a nucleic acid or a protein.

[0123] The term '‘transfected” or ‘'transformed” or ‘'transduced” as used herein refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell. A “transfected” or “transformed” or “transduced” cell is one which has been transfected, transformed, or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[0124] To “treat” a disease as the term is used herein, means to reduce the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject.

[0125] A “vector” is a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term “vector” includes an autonomously replicating plasmid or a virus. The term should also be constmed to include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as. for example, polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, Sendai viral vectors, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, lentiviral vectors, and the like.

[0126] Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3. 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0127] As further described below; the present invention provides DR5-specific chimeric antigen receptors (CARs; e.g., an “anti-DR5 CAR” or “DR5 CAR”) and modified cells comprising the same. Also provided are compositions and methods for utilizing DR5- specific CARs to kill or eliminate cancer cells and myeloid-derived suppressor cells (MDSCs) and to treat DR5+cancers and other DR5+associated diseases or disorders. Chimeric Antigen Receptor (CAR)

[0128] In some aspects, the present invention provides chimeric antigen receptor (CAR) comprising an antigen binding domain, a transmembrane domain, and an intracellular domain, wherein the antigen binding domain specifically binds DR-5.

[0129] The present invention provides compositions and methods for modified immune cells or precursor cells thereof, e.g., modified T cells, comprising a chimeric antigen receptor (CAR) having affinity for Glycoprotein Nonmetastatic Melanoma Protein B (DR5). In one aspect, an anti-DR5 CAR of the invention comprises an antigen binding domain (e.g., DR5 binding domain), a transmembrane domain, a costimulatory signaling domain, and an intracellular signaling domain. The anti-DR5 CAR may further comprise a hinge domain (or region). In some embodiments, each of the domains of the DR5 CAR is separated by a linker or spacer.

[0130] DR5 Antigen Binding Domain

[0131] The antigen binding domain of the DR5 CAR is an extracellular region of the CAR for binding to a DR5 molecule on a cell surface. In an embodiment, the DR5 CAR comprises specific binding affinity for DR5+cancer cells. In another embodiment, the DR5 CAR comprises specific binding affinity7for DR5 myeloid-derived suppressor cells (MDSCs). In some embodiments, the CAR comprises specific binding affinity for DR5 on a cell coincident with a particular status or state of differentiation of the cell.

[0132] In certain embodiments, the antigen binding domain of the invention comprises an antibody or fragment thereof, that binds to a DR5 molecule. In certain exemplary embodiments, the antigen binding domain is an scFv antibody that binds to DR5. In some embodiments, the DR5 binding domain is a murine DR5 binding domain, e.g., the DR5 binding domain is of murine origin. In some embodiments, the DR5 binding domain is a humanized DR5 binding domain. In some embodiments, the DR5 binding domain is a human DR5 binding domain, e.g, the DR5 binding domain is of human origin. In some embodiments, the antigen binding domain is derived from the same species in which the CAR will ultimately be used. For example. ))for use in humans, the antigen binding domain of the CAR may comprise a human antibody.

[0133] In one embodiment, the DR5 binding domain comprises a heavy chain variable (VH) region comprising an amino acid sequence set forth in SEQ ID NOs: 1, 4, 7, or 10. In another embodiment, the DR5 binding domain binding domain comprises a light chain variable (VL) region comprising an amino acid sequence set forth in SEQ ID NOs: 2, 5, 8, or 11.

[0134] In some embodiments, the antigen binding domain comprises a heavy chain variable (VH) region comprising an amino acid sequence having at least 95%-99% identity to the amino acid sequence of SEQ ID NO: 1, 4, 7, or 10; and / or a light chain variable (VL) region comprising an amino acid sequence having at least 95-99% identity to the amino acid sequence of SEQ ID NO: 2, 5. 8, or 11.

[0135] In another embodiment, the VL region comprises the amino acid sequence of any one of SEQ ID NO: 1, 4, 7, or 10; and / or the VL region comprises the amino acid sequence of any one of SEQ ID NO: 2, 5, 8, or 11.

[0136] In another embodiment, the DR5 binding comprises a VH region comprising the amino acid sequence of SEQ ID NO: 1 and a Vi, region comprising the amino acid sequence of SEQ ID NO: 2. In another embodiment, the DR5 binding comprises a VH region comprising the amino acid sequence of SEQ ID NO: 4 and a VL region comprising the amino acid sequence of SEQ ID NO: 5. In another embodiment, the DR5 binding comprises a VH region comprising the amino acid sequence of SEQ ID NO: 7 and a VL region comprising the amino acid sequence of SEQ ID NO: 8. In another embodiment, the DR5 binding comprises a VH region comprising the amino acid sequence of SEQ ID NO: 10 and a VL region comprising the amino acid sequence of SEQ ID NO: 11.

[0137] In other embodiments, the antigen binding domain comprises the amino acid sequence of SEQ ID NO: 3, 6, 9, or 12; or an amino acid sequence having at least 95%-99% identity to the amino acid sequence of SEQ ID NO: 3, 6, 9, or 12.

[0138] In an embodiment, the antigen binding domain in scFv-Gl comprises a VH region encoded by the nucleotide sequence of any one of SEQ ID NOs: 91, 93, 95, or 97. In some embodiments, the antigen binding domain in scFv-Gl comprises a VH region encoded by the nucleotide sequence of any one of SEQ ID NOs: 92, 94, 96, or 98.

[0139] In another embodiment, the antigen binding domain in scFv-G2 comprises a VH region encoded by the nucleotide sequence of any one of SEQ ID NOs: 99. 101, 103, or 105. In some embodiments, the antigen binding domain in scFv-G2 comprises a VH region encoded by the nucleotide sequence of any one of SEQ ID NOs: 100, 102, 104, or 106.

[0140] In another embodiment, the antigen binding domain in scFv-G3 comprises a VH region encoded by the nucleotide sequence of any one of SEQ ID NOs: 107, 109, 111, or 113. In some embodiments, the antigen binding domain in scFv-G3 comprises a VH region encoded by the nucleotide sequence of any one of SEQ ID NOs: 108, 110, 112, or 114. In another embodiment, the antigen binding domain in scFv-G4 comprises a VH region encoded by the nucleotide sequence of any one of SEQ ID NOs: 115. 117. 119, or 121. In some embodiments, the antigen binding domain in scFv-G4 comprises a VH region encoded by the nucleotide sequence of any one of SEQ ID NOs: 116, 118, 120, or 122.

[0141] The antigen binding domain can include any domain that binds to the antigen and may include, but is not limited to. a monoclonal antibody, a polyclonal antibody, a synthetic antibody, a human antibody, a humanized antibody, a non-human antibody, and any fragment thereof. Thus, in one embodiment, the antigen binding domain portion comprises a mammalian antibody or a fragment thereof. In another embodiment, the antigen binding domain of the CAR is selected from the group consisting of an anti-DR5 antibody or a fragment thereof. In some embodiments, the antigen binding domain is selected from the group consisting of an antibody, an antigen binding fragment (Fab), and a single-chain variable fragment (scFv). In some embodiments, a DR5 binding domain of the present invention is selected from the group consisting of a DR5-specific antibody, a DR5-specific Fab, and a DR5-specific scFv. In one embodiment, a DR5 binding domain is a DR5-specific antibody. In one embodiment, a DR5 binding domain is a DR5-specific Fab. In one embodiment, a DR5 binding domain is a DR5-specific scFv.

[0142] As used herein, the term “single-chain variable fragment” or “scFv” is a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of an immunoglobulin (e.g., mouse or human) covalently linked to form a VH:VL heterodimer. The heavy (VH) and light chains (VL) are either joined directly or joined by a peptide- encoding linker or spacer, which connects the N-terminus of the VH with the C-terminus of the VL, or the C-terminus of the VH with the N-terminus of the VL. The terms “linker” and “spacer” are used interchangeably herein. In some embodiments, the antigen binding domain (e.g., DR5 binding domain) comprises an scFv having the configuration from N-terminus to C-terminus, VH - linker - VL. In some embodiments, the antigen binding domain (e.g., DR5 binding domain) comprises an scFv having the configuration from N-terminus to C-terminus, VL - linker - VH. Those of skill in the art would be able to select the appropriate configuration for use in the present invention.

[0143] The linker is typically rich in glycine for flexibility, as well as serine or threonine for solubility. The linker can link the heavy chain variable region and the light chain variable region of the extracellular antigen-binding domain. Non-limiting examples of linkers are disclosed in Shen et al., Anal. Chem. 80(6): 1910-1917 (2008) and WO 2014 / 087010. the contents of which are hereby incorporated by reference in their entireties. Various linker sequences are known in the art, including, without limitation, glycine serine (GS) linkers such as (GS)n, (GSGGS)n (SEQ ID NO: 41), (GGGS)n (SEQ ID NO: 42), and (GGGGS)n (SEQ ID NO: 43), where n represents an integer of at least 1. Exemplary linker sequences can comprise amino acid sequences including, without limitation, GGSG (SEQ ID NO: 44), GGSGG (SEQ ID NO: 45), GSGSG (SEQ ID NO: 46), GSGGG (SEQ ID NO: 47), GGGSG (SEQ ID NO: 48), GSSSG (SEQ ID NO: 49), GGGGS (SEQ ID NO: 50), (GGGGS)? (SEQ ID NO: 51), (GGGGS)4 (SEQ ID NO: 52), and the like. Those of skill in the art would be able to select the appropriate linker sequence for use in the present invention. In some embodiments, an antigen binding domain (e.g, DR5 binding domain) of the present invention comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL is separated by the linker sequence having the amino acid sequence (GGGGS)? (SEQ ID NO: 51).

[0144] Despite removal of the constant regions and the introduction of a linker, scFv proteins retain the specificity of the original immunoglobulin. Single chain Fv polypeptide antibodies can be expressed from a nucleic acid comprising VH- and VL-encoding sequences as described by Huston, et al. (Proc. Nat. Acad. Sci. USA. 85:5879-5883. 1988). See, also. U.S. Patent Nos. 5,091,513, 5,132,405 and 4,956,778; and U.S. Patent Publication Nos. 20050196754 and 20050196754. Antagonistic scFvs having inhibitory activity7have been described (see, e.g., Zhao et al., Hybridoma (Larchmt) 2008 27(6):455-51; Peter et al., J Cachexia Sarcopenia Muscle 2012 August 12; Shieh et al., J Imunol 2009 183(4):2277-85; Giomarelli et al., Thromb Haemost 2007 97(6):955-63; Fife eta., J Clin Invst 2006 116(8):2252-61; Brocks et al., Immunotechnology 1997 3(3): 173-84; Moosmayer et al., Ther Immunol 1995 2(10:31-40). Agonistic scFvs having stimulatory activity7have 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).

[0145] In one embodiment, the DR5 antigen binding domain is an scFv comprising the amino acid sequence of SEQ ID NO: 3. In another embodiment, the DR5 antigen binding domain is an scFv comprising the amino acid sequence of SEQ ID NO: 6. In another embodiment, the DR5 antigen binding domain is an scFv comprising the amino acid sequence of SEQ ID NO: 9. In another embodiment, the DR5 antigen binding domain is an scFv comprising the amino acid sequence of SEQ ID NO: 12.

[0146] Tolerable variations of the DR5 binding domain will be known to those of skill in the art, while maintaining specific binding to DR5. For example, in some embodiments the DR5 binding domain comprises an amino acid sequence that has at least 90%, at least 91%, at least 92%. at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to any of the corresponding amino acid sequences set forth in Table 1, as exemplified by SEQ ID NOs: 3, 6, 9, and 12.

[0147] The antigen binding domain may be operably linked to another domain of the CAR, such as a transmembrane domain and costimulatory signaling domain, both described elsewhere herein. In one embodiment, a nucleic acid encoding the antigen binding domain is operably linked to a nucleic acid encoding a transmembrane domain and a nucleic acid encoding a costimulatory signaling domain.

[0148] The antigen binding domains described herein, such as the antibody or fragment thereof that binds to DR5, can be combined with any of the transmembrane domains described herein, any of the intracellular domains or cytoplasmic domains described herein, or any of the other domains described herein that may be included in the CAR, unless specifically described to the contrary or clearly evident to one skilled in the art.

[0149] Transmembrane Domain

[0150] With respect to the transmembrane domain, the DR5 CAR of the present invention comprises a transmembrane domain that connects the antigen binding domain of the CAR to the intracellular domain. The transmembrane domain is a region that is capable of spanning the plasma membrane of a cell (e.g. an immune cell or precursor thereof). The transmembrane domain is for insertion into a cell membrane, e.g, a eukaryotic cell membrane. In some embodiments, the transmembrane domain is interposed between the antigen binding domain and the intracellular domain of the DR5 CAR. In some embodiments, the transmembrane domain is interposed between a hinge region and the intracellular domain of the DR5 CAR.

[0151] In an embodiment, the transmembrane domain is naturally associated with one or more of the domains in the CAR. In some instances, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.

[0152] The transmembrane domain may be derived either from a natural or from a synthetic source. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein, e.g., a Type I transmembrane protein. Where the source is synthetic, the transmembrane domain may be any artificial sequence that facilitates insertion of the CAR into a cell membrane, e.g., an artificial hydrophobic sequence. Examples of the transmembrane regions of particular use in this invention include, without limitation, transmembrane domains derived from (i.e., comprise at least the transmembrane region(s) of) the alpha, beta or zeta chain of the T-cell receptor, CD28, CD2, CD3 epsilon, DR5, CD4, CD5, CD7, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134 (OX-40), CD137 (4-1BB), CD154 (CD40L), CD278 (ICOS), CD357 (GITR), Toll-like receptor 1 (TLR1), TLR2, TLR3. TLR4, TLR5, TLR6. TLR7. TLR8, and TLR9. In some embodiments, the transmembrane domain may be synthetic, in which case it will comprise predominantly hydrophobic residues such as leucine and valine. In certain exemplary embodiments, a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain.

[0153] In some embodiments, the transmembrane domain comprises a CD8a transmembrane domain. In one embodiment, the CD8a transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 16.

[0154] In some embodiments, the transmembrane domain comprises a CD28 transmembrane domain. In one embodiment, the CD28 transmembrane domain comprising the amino acid sequence set forth in SEQ ID NO: 17.

[0155] In some embodiments, the transmembrane domain comprises a ICOS transmembrane domain. In one embodiment, the ICOS transmembrane domain comprising the amino acid sequence set forth in SEQ ID NO: 18.

[0156] In some embodiments, the transmembrane domain comprises an 0X40 transmembrane domain. In one embodiment, the 0X40 CD28 transmembrane domain comprising the amino acid sequence set forth in SEQ ID NO: 19.

[0157] The transmembrane domains described herein can be combined with any of the antigen binding domains described herein, any of the costimulatory signaling domains described herein, any of the intracellular signaling domains described herein, or any of the other domains described herein that may be included in the DR5 CAR, unless specifically described to the contrary or clearly evident to one skilled in the art.

[0158] In some embodiments, the DR5 CAR further includes a hinge region. The hinge region of the CAR is a hydrophilic region which is located between the antigen binding domain and the transmembrane domain. In some embodiments, this domain facilitates proper protein folding for the CAR. The hinge region is an optional component for the CAR. The hinge region may include a domain selected from Fc fragments of antibodies, hinge regions of antibodies, CH2 regions of antibodies, CH3 regions of antibodies, artificial hinge sequences or combinations thereof. Examples of hinge regions include, without limitation, a CD8a hinge, artificial hinges made of polypeptides which may be as small as, three glycines (Gly), as well as CHI and CH3 domains of IgGs (such as human IgG4).

[0159] In some embodiments, the DR5 CAR of the present disclosure includes a hinge region that connects the antigen binding domain with the transmembrane domain, which, in turn, connects to the intracellular domain. The hinge region is capable of supporting the antigen binding domain to recognize and bind to the target antigen on the target cells (see, e.g.. Hudecek et al., Cancer Immunol. Res. (2015) 3(2): 125-135). Generally, the hinge region is a flexible domain, thus allowing the antigen binding domain to have a structure to optimally recognize the specific structure and density of the target antigens on a cell such as tumor cell. The flexibility of the hinge region permits the hinge region to adopt many different conformations.

[0160] In some embodiments, the hinge region is an immunoglobulin heavy chain hinge region. In some embodiments, the hinge region is a hinge region polypeptide derived from a receptor (e.g., a CD8-derived hinge region).

[0161] The hinge region can have a length of from about 4 amino acids to about 50 amino acids, e.g., from about 4 amino acids to about 10 amino acids, from about 10 amino acids to about 15 amino acids, from about 15 amino acids to about 20 amino acids, from about 20 amino acids to about 25 amino acids, from about 25 amino acids to about 30 amino acids, from about 30 amino acids to about 40 amino acids, or from about 40 amino acids to about 50 amino acids.

[0162] Suitable hinge regions can be readily selected and can be of any of a number of suitable lengths, such as from 1 amino acid (e.g., Gly) to 20 amino acids, from 2 amino acids to 15 amino acids, from 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and can be 1, 2, 3, 4, 5, 6, or 7 amino acids.

[0163] For example, hinge regions include glycine polymers (G)n, glycine-serine polymers (including, for example, (GS)n, (GSGGS)n (SEQ ID NO: 41), (GGGS)n(SEQ ID NO: 42), and (GGGGS)n, where n is an integer of at least one), glycine-alanine polymers, alanineserine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers can be used; both Gly and Ser are relatively unstructured, and therefore can serve as a neutral tether between components. Glycine polymers can be used; glycine accesses significantly more phi-psi space than even alanine, and is much less restricted than residues with longer side chains (see, e.g, Scheraga, Rev. Computational. Chem. (1992) 2: 73-142). Exemplary hinge regions can comprise amino acid sequences including, but not limited to, GGSG (SEQ ID NO: 44), GGSGG (SEQ ID NO: 45). GSGSG (SEQ ID NO: 46), GSGGG (SEQ ID NO: 47), GGGSG (SEQ ID NO: 48), GSSSG (SEQ ID NO: 49), GGGGS (SEQ ID NO: 50), and the like.

[0164] In some embodiments, the hinge region is an immunoglobulin heavy chain hinge region. Immunoglobulin hinge region amino acid sequences are known in the art; see, e.g. Tan et al., Proc. Natl. Acad. Sci. USA (1990) 87(1): 162-166; and Huck et al.. Nucleic Acids Res. (1986) 14(4): 1779-1789. As non-limiting examples, an immunoglobulin hinge region can include one of the following amino acid sequences: DKTHT (SEQ ID NO: 53); CPPC (SEQ ID NO: 54); CPEPKSCDTPPPCPR (SEQ ID NO: 55) (see, e.g., Glaser et al., J. Biol. Chem. (2005) 280:41494-41503); ELKTPLGDTTHT (SEQ ID NO: 56); KSCDKTHTCP (SEQ ID NO: 57); KCCVDCP (SEQ ID NO: 58); KYGPPCP (SEQ ID NO: 59); EPKSCDKTHTCPPCP (SEQ ID NO: 60) (human IgGl hinge); ERKCCVECPPCP (SEQ ID NO: 61) (human IgG2 hinge); ELKTPLGDTTHTCPRCP (SEQ ID NO: 62) (human IgG3 hinge); SPNMVPHAHHAQ (SEQ ID NO: 63) (human IgG4 hinge); and the like.

[0165] The hinge region can comprise an amino acid sequence of a human IgGl. IgG2, IgG3. or IgG4, hinge region. In one embodiment, the hinge region can include one or more amino acid substitutions and / or insertions and / or deletions compared to a wild-type (naturally- occurring) hinge region. For example, His229 of human IgGl hinge can be substituted with Tyr, so that the hinge region comprises the sequence EPKSCDKTYTCPPCP (SEQ ID NO: 60); see, e.g., Yan et al., J. Biol. Chem. (2012) 287: 5891 -5897. In one embodiment, the CAR comprises a human CD8 hinge domain comprising the amino acid sequence of SEQ ID NO: 14. In one embodiment, the CAR comprises a human CD28 hinge domain comprising the amino acid sequence of SEQ ID NO: 15.

[0166] The transmembrane domain may be combined with any hinge domain and / or may comprise one or more transmembrane domains described herein. For example, in one embodiment, the DR5 CAR comprises a CD8a hinge domain of SEQ ID NO: 14 and a CD8a transmembrane domain of SEQ ID NO: 16.

[0167] The transmembrane domains described herein, such as a transmembrane region of alpha, beta or zeta chain of the T-cell receptor, CD28, CD2, CD3 epsilon, CD45, CD4, CD5, CD7, CD8, CD9, CD 16, CD22, CD33, CD37, CD64, CD80, CD86, CD134 (OX-40), CD137 (4-1BB), CD154 (CD40L), CD278 (ICOS), CD357 (GITR), Toll-like receptor 1 (TLR1), TLR2, TLR3. TLR4, TLR5, TLR6, TLR7. TLR8, and TLR9, can be combined with any of the antigen binding domains described herein, any of the costimulatory signaling domains or intracellular domains or cytoplasmic domains described herein, or any of the other domains described herein that may be included in the CAR.

[0168] In one embodiment, the transmembrane domain may be synthetic, in which case it will comprise predominantly hydrophobic residues such as leucine and valine. In exemplary embodiments, a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain.

[0169] Tolerable variations of the transmembrane and / or hinge domain will be known to those of skill in the art, while maintaining its intended function. For example, in some embodiments a transmembrane domain or hinge domain comprises an amino acid sequence that has at least 90%, at least 91%, at least 92%. at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to their corresponding domains from where they are derived or with respect to the sequences in Table 1 herein.

[0170] Intracellular Domain

[0171] The DR5 CAR of the present invention also includes an intracellular domain. The intracellular domain of the CAR is responsible for activation of at least one of the effector functions of the cell in which the CAR is expressed (e.g, immune cell). The intracellular domain transduces the effector function signal and directs the cell (e.g, immune cell) to perform its specialized function, e.g., harming and / or destroying a target cell.

[0172] The intracellular domain or otherwise the cytoplasmic domain of the CAR is responsible for activation of the cell in which the CAR is expressed. Examples of an intracellular domain for use in the invention include, but are not limited to, the cytoplasmic portion of a surface receptor, co-stimulatory molecule, and any molecule that acts in concert to initiate signal transduction in the T cell, as well as any derivative or variant of these elements and any synthetic sequence that has the same functional capability.

[0173] In certain embodiments, the intracellular domain comprises a costimulatory signaling domain. In certain embodiments, the intracellular domain comprises an intracellular signaling domain. In certain embodiments, the intracellular domain comprises a costimulatory signaling domain and an intracellular signaling domain.

[0174] In certain embodiments, the intracellular domain comprises a costimulatory signaling domain comprising any suitable portion of one or more co-stimulatory molecules, such as at least one signaling domain from CD2, CD3, CD8. CD27, CD28, 0X40, ICOS, 4-1BB, PD-1, any derivative or variant thereof, any synthetic sequence thereof that has the same functional capability, and any combination thereof. In some embodiments, the costimulatory domain is derived from 4- IBB, CD28, ICOS, or 0X40. In an embodiment, the costimulatory domain is derived from 4- IBB and / or comprises an amino acid sequence set forth in SEQ ID NO: 20. In another embodiment, the costimulatory domain is derived from CD28 and / or comprises an amino acid sequence set forth in SEQ ID NO: 21. In another embodiment, the costimulatory domain is derived from ICOS and / or comprises an amino acid sequence set forth in SEQ ID NO: 22. In another embodiment, the costimulatory domain is derived from 0X40 and / or comprises an amino acid sequence set forth in SEQ ID NO: 23.

[0175] Intracellular signaling domains suitable for use in the DR5 CAR of the present invention include any desired signaling domain that provides a distinct and detectable signal (e.g., increased production of one or more cytokines by the cell; change in transcription of a target gene; change in activity of a protein; change in cell behavior, e.g, cell death; cellular proliferation; cellular differentiation; cell survival; modulation of cellular signaling responses; etc.) in response to activation of the CAR (i.e., activated by antigen and dimerizing agent). In some embodiments, the intracellular signaling domain includes at least one (e.g.. one, two. three, four, five. six. etc.) IT AM motifs as described below. In some embodiments, the intracellular signaling domain includes DAP10 / CD28 type signaling chains. In some embodiments, the intracellular signaling domain is not covalently attached to the membrane bound CAR, but is instead diffused in the cytoplasm.

[0176] Intracellular signaling domains suitable for use in the DR5 CAR of the present invention include immunoreceptor tyrosine-based activation motif (ITAM)-containing intracellular signaling polypeptides. In some embodiments, an ITAM motif is repeated twice in an intracellular signaling domain, where the first and second instances of the ITAM motif are separated from one another by 6 to 8 amino acids. In one embodiment, the intracellular signaling domain in the DR5 CAR comprises 3 ITAM motifs. In some embodiments, intracellular signaling domains includes the signaling domains of human immunoglobulin receptors that contain immunoreceptor tyrosine-based activation motifs (IT AMs) such as, but not limited to, Fc gamma RI, Fc gamma RIIA, Fc gamma RIIC, Fc gamma RIIIA, FcRL5 (see, e.g.. Gillis et al., Front. (2014) Immunol. 5:254).

[0177] A suitable intracellular signaling domain can be an ITAM motif-containing portion that is derived from a polypeptide that contains an ITAM motif. For example, a suitable intracellular signaling domain can be an ITAM motif-containing domain from any ITAM motif-containing protein. Thus, a suitable intracellular signaling domain need not contain the entire sequence of the entire protein from which it is derived. Examples of suitable ITAM motif-containing polypeptides include, but are not limited to: DAP12, FCER1G (Fc epsilon receptor I gamma chain), CD3D (CD3 delta), CD3E (CD3 epsilon), CD3G (CD3 gamma). CD3Z (CD3 zeta), and CD79A (antigen receptor complex-associated protein alpha chain).

[0178] Examples of the intracellular signaling domain include, without limitation, the £ chain of the T cell receptor complex or any of its homologs, e.g, q chain, FcsRIy and P chains, MB 1 (Iga) chain, B29 (Ig) chain, etc., human CD3 zeta chain, CD3 polypeptides (A, 5 and e), syk family tyrosine kinases (Syk, ZAP 70. etc.), src family tyrosine kinases (Lek. Fyn, Lyn, etc.), and other molecules involved in T cell transduction, such as CD2, CD5 and CD28. In one embodiment, the intracellular signaling domain may be human CD3 zeta chain, FcyRIII, FcsRI, cytoplasmic tails of Fc receptors, an immunoreceptor tyrosine-based activation motif (IT AM) bearing cytoplasmic receptors, and combinations thereof.

[0179] Other examples of the intracellular domain include a fragment or domain from one or more molecules or receptors including, but are not limited to, TCR, CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, CD86, common FcR gamma, FcR beta (Fc Epsilon Rib), CD79a, CD79b, Fc gamma R1 la, DAP10, DAP12, T cell receptor (TCR), CD8, CD27. CD28, 4-1BB (CD137), 0X9, 0X40. CD30. CD40, PD-1. ICOS, a KIR family protein, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, CD5, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD127, CD160, CD19, CD4. CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6. CD49L ITGAD, CD lid, ITGAE, CD 103, ITGAL, CD1 l a, LFA-1 , ITGAM, CD lib, ITGAX, CD1 1c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD 160 (BY55), PSGL1, CD 100 (SEMA4D). CD69, SLAMF6 (NTB-A. Lyl08), SLAM (SLAMFL CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD 162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, other co-stimulatory molecules described herein, any derivative, variant, or fragment thereof, any synthetic sequence of a costimulatory molecule that has the same functional capability, and any combination thereof.

[0180] Additional examples of intracellular domains include, without limitation, intracellular signaling domains of several types of various other immune signaling receptors, including, but not limited to, first, second, and third generation T cell signaling proteins including CD3, B7 family costimulatory, and Tumor Necrosis Factor Receptor (TNFR) superfamily receptors (see, e.g, Park and Brentjens, J. Clin. Oncol. (2015) 33(6): 651-653). Additionally, intracellular signaling domains may include signaling domains used by NK and NKT cells (see, e.g, Hermanson and Kaufman. Front. Immunol. (2015) 6: 195) such as signaling domains ofNKp30 (B7-H6) (see, e.g., Zhang et al., J. Immunol. (2012) 189(5): 2290-2299), and DAP 12 (see, e.g., Topfer et al., J. Immunol. (2015) 194(7): 3201-3212), NKG2D, NKp44, NKp46, DAP 10, and CD3z.

[0181] While usually the entire intracellular signaling domain can be employed, in manycases it is not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such truncated portion may be used in place of the intact chain as long as it transduces the effector function signal. The intracellular signaling domain includes any truncated portion of the intracellular signaling domain sufficient to transduce the effector function signal.

[0182] In one embodiment, the intracellular signaling domain in the CAR includes the cytoplasmic signaling domain of human CD3 zeta. In an exemplary embodiment, the intracellular signaling domain of human CD3 zeta comprises the amino acid sequence set forth in SEQ ID NO: 24 or 25.

[0183] The intracellular signaling domains described herein can be combined with any of the costimulatory signaling domains described herein, any of the antigen binding domains described herein, any of the transmembrane domains described herein, or any of the other domains described herein that may be included in the CAR. For example, in some embodiments, the CAR comprises a 4- IBB costimulatory domain comprising the amino acid sequence SEQ ID NO: 20 and a CD3 zeta intracellular signaling domain comprising the amino acid sequence SEQ ID NO: 24 or 25.

[0184] Tolerable variations of the intracellular domain will be known to those of skill in the art, while maintaining specific activity. For example, in some embodiments the intracellular domain comprises an amino acid sequence that has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to any of the costimulatory or intracellular signaling domains described in Table 1.

[0185] In some embodiments, the DR5 CAR may further comprise a spacer domain between the extracellular domain and the transmembrane domain of the CAR, and / or between the transmembrane domain and an intracellular domain of the CAR. As used herein, the term “spacer domain’" generally means any oligo- or polypeptide that functions to link the transmembrane domain to. either the extracellular domain or. the intracellular domain in the polypeptide chain. A spacer domain may comprise up to 300 amino acids, e.g., 10 to 100 amino acids, or 25 to 50 amino acids. In some embodiments, the spacer domain may be a short oligo- or polypeptide linker, e.g., between 2 and 10 amino acids in length. For example, a glycine-serine doublet provides a particularly suitable linker between the transmembrane domain and the intracellular signaling domain of the DR5 CAR.

[0186] Various spacer sequences are known in the art, including, without limitation, glycine serine (GS) spacers such as (GS)n. GSG, (GSGGS)n (SEQ ID NO: 41), (GGGS)n (SEQ ID NO: 42). and (GGGGS)n (SEQ ID NO: 43). where n represents an integer of at least I. Exemplary spacer sequences can comprise amino acid sequences including, without limitation, GGSG (SEQ ID NO: 44), GGSGG (SEQ ID NO: 45), GSGSG (SEQ ID NO: 46), GSGGG (SEQ ID NO: 47), GGGSG (SEQ ID NO: 48), GSSSG (SEQ ID NO: 49), GGGGS (SEQ ID NO: 50), (G4S)3 (SEQ ID NO: 51), (G4S)4 (SEQ ID NO: 52), and the like. Those of skill in the art would be able to select the appropriate spacer sequence for use in the present invention.

[0187] The DR5 binding domains described herein may be combined with any of the transmembrane domains, any of the costimulatory signaling domains, any of the intracellular signaling domains, or any of the other domains described herein that may be included in a CAR of the present invention.

[0188] In an embodiment, the anti-DR5 CAR comprises a DR5 antigen binding domain, a hinge region, a transmembrane domain, and an intracellular domain, wherein the DR5 binding domain comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1 , 4, 7 or 10, a light chain variable region comprising the amino acid sequence of SEQ ID NO: 2, 5, 8 or 11, a CD8a transmembrane domain; a CD8a hinge region, a 4-lBB costimulatory signaling domain, and a CD3 zeta intracellular signaling domain, wherein the CAR specifically binds to DR5. In another embodiment, the anti-DR5 CAR comprises an anti-DR5 scFv binding domain comprising the amino acid sequence of SEQ ID NO: 3, 6, 9 or 12.

[0189] In another embodiment, the anti-DR5 CAR comprises a DR5 antigen binding domain, a hinge region, a transmembrane domain, and an intracellular domain, wherein the DR5 binding domain comprises a heavy chain variable region comprising the ammo acid sequence of SEQ ID NO: 1, 4, 7 or 10, a light chain variable region comprising the amino acid sequence of SEQ ID NO: 2, 5, 8 or 11, a CD8a transmembrane domain; a CD8a hinge region, a CD28 costimulatory signaling domain, and a CD3 zeta intracellular signaling domain, wherein the CAR specifically binds to DR5. In another embodiment, the anti-DR5 CAR comprises an anti-DR5 scFv binding domain comprising the amino acid sequence of SEQ ID NO: 3, 6, 9 or 12.

[0190] In another embodiment, the anti-DR5 CAR comprises a DR5 antigen binding domain, a hinge region, a transmembrane domain, and an intracellular domain, wherein the DR5 binding domain comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1, 4, 7 or 10, a light chain variable region comprising the amino acid sequence of SEQ ID NO: 2, 5. 8 or 11, a CD8a transmembrane domain; a CD8a hinge region, a 4-lBB costimulatory signaling domain, a CD28 costimulatory signaling domain, and a CD3 zeta intracellular signaling domain, wherein the CAR specifically binds to DR5. In another embodiment, the anti-DR5 CAR comprises an anti-DR5 scFv binding domain comprising the amino acid sequence of SEQ ID NO: 3, 6. 9 or 12. In another embodiment, the anti-DR5 CAR comprises an anti-DR5 scFv binding domain comprising the amino acid sequence of SEQ ID NO: 3, 6, 9 or 12.

[0191] In some embodiments, the present invention further provides a genetically modified immune cell (e g., T cell, NK cell, NKT cell, gamma delta T cell) or precursor cell thereof comprises a CAR having a specific binding affinity for DR5. which can be used to kill or eliminate cancer cells and myeloid-derived suppressor cells (MDSCs).

[0192] Modified Immune Cells

[0193] The present invention provides a modified immune cell or precursor cell thereof (e.g, a modified T cell, a modified NK cell, a gamma-delta T cell) comprising the DR5 CAR. Accordingly, such modified cells possess the specificity directed by the CAR expressed therefrom. For example, a modified cell of the present invention comprising a DR5 CAR possesses specificity for a target cell expressing DR5.

[0194] Any modified cell comprising a CAR may be envisioned to comprise any antigen binding domain, any hinge, any transmembrane domain, any intracellular costimulatory domain, and any intracellular signaling domain described herein, and can readily be understood and made by a person of skill in the art in view of the disclosure herein.

[0195] In some embodiments, the modified cell is an immune cell or precursor cell thereof. In one embodiment, the modified cell is a T cell. In a particular embodiment, the T cell is a gamma-delta T cell. In another embodiment, the modified immune cell is an NKT cell. In another embodiment, the modified immune cell is an NK cell.

[0196] In some embodiments, the modified immune cell is an autologous cell. In some embodiments, the modified immune cells is an allogeneic cell. In some embodiments, a modified cell of the present invention is genetically edited to disrupt the expression of one or more endogenously expressed genes to reduce the risk of autoreactivity and / or increase the efficacy of the modified cells. In certain embodiments, the gene-edited immune cells (e.g.. T cells, NK cells) have a reduction, deletion, elimination, knockout, or disruption in expression of one or more endogenously expressed receptor. In one embodiment, the modified cell is genetically edited to disrupt the expression of an endogenous TCR gene product (e.g.. gene products of T Cell Receptor Alpha Constant (TRAC) and T Cell Receptor Beta Constant (TRBC)), Beta-2-microglobulin (B2M), and Class II Major Histocompatibility Complex Transactivator (CIITA).

[0197] In certain embodiments, the modified cell of the present disclosure is genetically edited to disrupt the expression of endogenous PD-1 gene products. In certain embodiments, disrupting the expression of endogenous PD-1 may create “checkpoint” resistant modified cells, resulting in increased tumor control. Checkpoint resistant modified cells may also be created by disrupting the expression of, for example, without limitation, the Adenosine A2A receptor (A2AR), B7-H3 (CD276), B7-H4 (VTCN1), the B and T Lymphocyte Attenuator protein (BTLA / CD272). CD96, the Cytotoxic T-Lymphocyte Associated protein 4 (CTLA- 4 / CD152), Indoleamine 2,3-dioxygenase (IDO), the Killer-cell Immunoglobulin-like Receptor (KIR), the Lymphocyte Activation Gene-3 (LAG3), the T cell immunoreceptor with Ig and ITIM domains (TIGIT), T-cell Immunoglobulin domain and Mucin domain 3 (TIM-3), or the V-domain Ig suppressor of T cell activation (VISTA).

[0198] Nucleic Acids

[0199] In another aspect, the present invention provides a nucleic acid encoding any of the CARs described herein, such as a DR5 CAR comprising an DR5 antigen binding domain, a transmembrane domain, an intracellular domain, and optionally a hinge domain. Generally, the nucleic acid is provided for producing or expressing the DR5 CAR as described herein, e.g., in a mammalian cell. In an exemplary7embodiment, the antigen binding domain of the DR5 CAR of the present invention is encoded by a nucleotide sequences set forth in SEQ ID NOs: 26-29.

[0200] In some embodiments, a nucleic acid of the present disclosure further comprises a leader sequence encoding a signal peptide at the N-terminal end of the CAR and / or other encoded proteins, which is cleaved from the antigen binding domain during cellular processing and localization of the CAR and / or marker protein to the cellular membrane. Suitable signal peptide sequences are known to those of skill in the art. In an exemplary embodiment, the nucleic acid encodes a CD8a signal peptide comprising the amino acid sequence of SEQ ID NO: 13.

[0201] In some embodiments, the nucleic acid is operably linked to a transcriptional control element, e.g., a promoter, enhancer, etc. Suitable promoter and enhancer elements are known to those of skill in the art. In certain embodiments, the promoter is an EF-1 alpha promoter. An exemplary EF-1 alpha comprises the nucleotide sequence of SEQ ID NO: 30. In other embodiments, the promoter is a Rous sarcoma virus (RSV) promoter. An exemplary RSV promoter comprises the nucleotide sequence of SEQ ID NO: 31. Other constitutive promoter sequences may also be used, including, but not limited to a cytomegalovirus (CMV) immediate early promoter, a simian virus 40 (SV40) early promoter, a mouse mammary tumor virus (MMTV) or human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, a MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, as well as human gene promoters such as, but not limited to, an actin promoter, a myosin promoter, a hemoglobin promoter, and a creatine kinase promoter.

[0202] Other suitable promoters include, but are not limited to cell-type specific promoters, including light and / or heavy chain immunoglobulin gene promoters, a CD8 cell-specific promoter, a CD4 cell-specific promoter, a neutrophil-specific promoter, an NK. cell-specific promoter, and various art-known tissue specific promoters.

[0203] Inducible promoters are also contemplated as part of the invention. The use of an inducible promoter provides a molecular switch capable of turning on expression of the polynucleotide sequence which it is operatively linked when such expression is desired, or turning off the expression when expression is not desired. Examples of inducible promoters include, but are not limited to tetracycline regulated promoters, (e.g., promoter systems including Tet Activators, TetON, TetOFF, etc.), steroid regulated promoters (e.g., rat glucocorticoid receptor promoter systems, human estrogen receptor promoter systems, retinoid promoter systems, glucocorticoid promoters, progesterone promoters, and thyroid promoter systems, ecdysone promoter systems, mifepristone promoter systems, etc.), metal regulated promoters (e.g., metallothionein promoter systems, etc.), pathogenesis-related regulated promoters (e.g., salicylic acid regulated promoters, ethylene regulated promoters, benzothiadiazole regulated promoters, etc.), temperature regulated promoters (e.g, heat shock inducible promoters (e.g, HSP-70, HSP-90, soybean heat shock promoter, etc.), light regulated promoters, synthetic inducible promoters, and the like.

[0204] In some embodiments, the locus or construct or transgene containing the suitable promoter is irreversibly switched through the induction of an inducible system. Suitable systems for induction of an irreversible switch are well known in the art, e.g., induction of an irreversible switch may make use of a Cre-lox-mediated recombination (see, e.g., Fuhrmann- Benzakein, et al.. Proc. Natl. Acad. Sci. USA (2000) 28:e99, the disclosure of which is incorporated herein by reference). Any suitable combination of recombinase, endonuclease, ligase, recombination sites, etc. know n to the art may be used in generating an irreversibly switchable promoter. Methods, mechanisms, and requirements for performing site-specific recombination, described elsewhere herein, find use in generating irreversibly switched promoters and are well known in the art, see, e.g., Grindley et al. Annual Review of Biochemistry (2006) 567-605; and Tropp, Molecular Biology (2012) (Jones & Bartlett Publishers, Sudbury, Mass.), the disclosures of which are incorporated herein by reference.

[0205] In some embodiments, the nucleic acid comprises a polynucleotide comprising a nucleotide sequence encoding a chimeric receptor inducible expression cassette. In one embodiment, the chimeric receptor inducible expression cassette is for the production of a transgenic polypeptide product that is released upon chimeric receptor signaling. See, e.g., Chmielewski and Abken, Expert Opin. Biol. Ther. (2015) 15(8): 1145-1154; and Abken. Immunotherapy (2015) 7(5): 535-544. In some embodiments, a nucleic acid of the present disclosure comprises one or more transgenes operably linked to a T-cell activation responsive promoter (e.g., NF AT).

[0206] In some embodiments, the nucleic acid comprises polynucleotide comprising a nucleotide sequence encoding one or more additional protein(s) of interest, including but not limited to, a cytokine, a cytokine antagonist, an immune checkpoint inhibitor, a chemokine, a switch receptor, a marker protein, or a combination thereof.

[0207] Exemplary cytokines include, but are not limited to, IL-2, IL-7, IL-9, IL-12, IL-21, IL-22, and IL-23.

[0208] Exemplary cytokine antagonists include, but are not limited to, those inhibiting cytokine release syndrome (CRS), such as anti-IL-6, anti-IL-1, anti-IFN-y, anti-TNFa, anti- IL-8, anti-ILlO, anti-GM-SCM, including scFvs and / or nanobodies thereof.

[0209] Exemplary immune checkpoint inhibitor include, but are not limited to, antagonists of a protein selected from the group consisting of the Programmed Death 1 receptor (PD-1), the Adenosine A2A receptor (A2AR), B7-H3 (CD276), B7-H4 (VTCN1), the B and T Lymphocyte Attenuator protein (BTLA / CD272), CD96, the Cytotoxic T-Lymphocyte Associated protein 4 (CTLA-4 / CD152), Indoleamine 2,3-dioxygenase (IDO), the Killer-cell Immunoglobulin-like Receptor (KIR), the Lymphocyte Activation Gene-3 (LAG3), the T cell immunoreceptor with Ig and ITIM domains (TIGIT), T-cell Immunoglobulin domain and Mucin domain 3 (TIM-3), the V-domain Ig suppressor of T cell activation, and combinations thereof.

[0210] Exemplary immune checkpoint inhibitors include, but are not limited to PD1, PD-L1, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, and TGFR beta.

[0211] Exemplary chemokines include, but are not limited to CXCL9 and CCL19.

[0212] The term “switch receptor” refers to a molecule designed to switch a negative signal transduction signal into a positive signal. In some embodiments, the switch receptor is a chimeric protein comprising a first protein or fragment thereof associated with a negative signal, and a second protein or fragment thereof associated with a positive signal. Examples of proteins associated with a negative signal include, without limitation, CTLA-4, PD-1 , BTLA. TIM-3 and the like. Examples of proteins associated with a positive signal include, without limitation, CD28, ICOS, 4-1BB, IL-12R, an extracellular domain of a TGFPR (e. , TGF0RI or TGFpRII) and the like. Exemplary switch receptors include, but are not limited to, PD1-CD28, TIM3-CD28, PD1-4-1BB, PD1A132L-4-1BB, PDlA132L_cD28, TGFpRI-IL- 12Rpi , TGFPRII-IL-12RP2, and the dominant-negative TGFpRII switch receptor, TGFPRIIDN. Other suitable switch receptors for use in the present invention are described in US Patent No. 10,981,969 B2, the disclosure of which is incorporated herein by reference.

[0213] Exemplary markers (or reporters) include, but are not limited to, minimal low' affinity nerve growth factor (mLNGFR), truncated LNGFR (tLNGFR), truncated EGFR (tEGFR), truncated CD 19 (tCD19), truncated CD34 (tCD34). blue fluorescent protein (BFP), green fluorescent protein (GFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), and combinations thereof.

[0214] In some embodiments, the nucleic acid comprises one or more polynucleotides comprising nucleotide sequence(s) encoding one or more additional CARs for co-expression with the DR5 CAR of the present invention. In certain embodiments, the additional CAR(s) include an antigen binding domain targeting a tumor associated antigen. In certain embodiments, the tumor associated antigen is overexpressed in a solid tumor. Exemplary tumor associated antigens include, but are not limited to, alpha feto-protein (AFP) / HLA-A2, AXL, B7-H3, B7H4. BCMA, CA-IX. CD2. CD3. CD4. CD5. CD7, CD8, CD19. CD20, CD22, CD30, CD33, CD38, CD44v6, CD70, CD79a, CD79b, CD80, CD86, CD117, CD123, CD133, CD147, CD171, CD276, CEA, CEACAM5, claudin 18.2, c-Met, DLL3, DR5, EGFR, EGFRvIII, EpCAM, EphA2, FAP, folate receptor alpha (FRa) / folate binding protein (FBP), GD-2, Glycolipid F77. glypican-2 (GPC2), glypican-3 (GPC3). HER2. HLA-A2, ICAM1, IL3Ra, IL13Ra2, LAGE-1, Lewis Y, LMP1 (EBV), MAGE-A1, MAGE-A3, MAGE-A4, Melan A, mesothelin, MG7 (glycosylated CEA), MMP, MUC1, Nectin4 / FAP, NKG2D-Ligands (MIC-A. MIC-B, and the ULBPs 1 to 6), NY-ESO-1. Pl 6, PD-L1. PSCA, PSMA, ROR1, ROR2, TIM-3, TM4SF1, TnMucl, VEGFR2, and any combination thereof.

[0215] In certain embodiments, the additional CAR(s) include an antigen binding domain targeting a tumor associated antigen B cell antigen expressed in a non-solid tumor. Exemplary B cell antigens include but are not limited to CD5, CD10, CD19, CD20, CD21, CD22, CD23, CD24. CD25. CD27, CD30, CD33 (IL3Ra), CD34, CD37. CD38, CD40, CD52, CD53, CD69, CD72, CD73, CD74, CD75, CD77, CD79a, CD79b, CD80, CD81, CD82, CD83, CD84, CD85, CD86, CD123, CD135, CD138, CD179, CD269. Flt3, ROR1, BCMA, FcRn5, FcRn2, CS-L CXCR4, 5, 7, IL-7 / 3R, IL7 / 4 / 3R, and L4R, and variants thereof.

[0216] In embodiments comprising or expressing an additional CAR, any of the CAR components described herein for use in the DR5 CAR may be used in a similar manner for the additional CAR(s).

[0217] In some embodiments, the additional protein(s) of interest is / are operatively linked to a different promoter than the one directing expression of the DR5 CAR. In other embodiments, the additional protein(s) is / are co-expressed with the DR5 CAR from a common promoter using self-cleaving 2A peptides fused in-frame between two or more polypeptides, using an internal ribosome entry site (IRES) between one or more polypeptides, or a combination thereof.

[0218] As used herein, a “self-cleaving 2A peptide’’ or “2A peptide” refers to an oligopeptide that allow multiple proteins to be encoded as polyproteins, which dissociate into component proteins upon translation. Use of the term “self-cleaving” is not intended to imply a proteolytic cleavage reaction. Various self-cleaving or 2A peptides are known to those of skill in the art, including, without limitation, those found in members of the Picomaviridae virus family, e.g., foot-and-mouth disease virus (FMDV), equine rhinitis A virus (ERAVO, Thosea asigna virus (TaV), and porcine tescho virus-1 (PTV-1); and carioviruses such as Theilovirus and encephalomyocarditis viruses. 2A peptides derived from FMDV, ERAV, PTV-1. and TaV are referred to herein as “F2A.” “E2A.” “P2A,” and “T2A,” respectively.

[0219] In one embodiment, the nucleic acid of the present disclosure encodes the T2A selfcleaving peptide of SEQ ID NO: 64. In another embodiment, the nucleic acid encodes the P2A self-cleaving peptide of SEQ ID NO: 65. In another embodiment, the nucleic acid encodes the E2A self-cleaving peptide of SEQ ID NO: 66. In another embodiment, the nucleic acid encodes the F2A self-cleaving peptide of SEQ ID NO: 67. Those of skill in the art would be able to select the appropriate self-cleaving peptide for use in the present invention.

[0220] In some embodiments, a polynucleotide comprising an internal ribosome entry site (IRES) is interposed between two or more polypeptides. As used herein, “an internal ribosome entry site” or “IRES” refers to an element that promotes direct internal ribosome entry to the initiation codon, such as ATG, of a protein coding region, thereby leading to capindependent translation of the gene. Various internal ribosome entry sites are known to those of skill in the art, including, without limitation, IRES obtainable from viral or cellular mRNA sources, e.g., immunoglobulin heavy-chain binding protein (BiP); vascular endothelial growth factor (VEGF); fibroblast growth factor 2; insulin-like growth factor; translational initiation factor eIF4G; yeast transcription factors TFIID and HAP4; and IRES obtainable from, e.g.. cardiovirus, rhinovirus, aphthovirus, HCV, Friend murine leukemia virus (FrMLV), and Moloney murine leukemia virus (MoMLV). An exemplary IRES, encephalomyocarditis virus (EMCV) IRES, comprises the nucleotide sequence of SEQ ID NO: 68. Those of skill in the art would be able to select the appropriate IRES for use in the present invention.

[0221] A nucleic acid of the present disclosure may be present within an expression vector and / or a cloning vector.

[0222] Expression Vectors and Expression Constructs

[0223] In another aspect, the present invention provides a cloning vector, expression vector or expression construct comprising a nucleic acid encoding a targeting protein (e.g., anti-DR5 targeting protein) described herein. Suitable expression vectors include, e.g, plasmids, viral vectors, and the like. Large numbers of suitable vectors and promoters are known to those of skill in the art; many are commercially available for generating a subject recombinant construct. The following vectors are provided by w ay of example, and should not be construed in any way as limiting: Bacterial: pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene, La Jolla, Calif., USA); pTrc99A, pKK223-3. pKK233-3, pDR540, and pRIT5 (Pharmacia, Uppsala, Sweden). Eukaryotic: pWLneo, pSV2cat, pOG44, PXR1, pSG (Stratagene) pSVK3, pBPV, pMSG and pSVL (Pharmacia).

[0224] Expression vectors generally have convenient restriction sites located near the promoter sequence to provide for the insertion of nucleic acid sequences encoding heterologous proteins. A selectable marker operative in the expression host may be present. Suitable expression vectors include, but are not limited to, viral vectors (e.g, viral vectors based on vaccinia virus; poliovirus; adenovirus (see, e.g., Li et al., Invest. Opthalmol. Vis. Set. (1994) 35: 2543-2549; Borras et al., Gene Ther. (1999) 6: 515-524; Li and Davidson, Proc. Natl. Acad. Sci. USA (1995) 92: 7700-7704; Sakamoto et al., H. Gene Ther. (1999) 5: 1088-1097; WO 94 / 12649, WO 93 / 03769; WO 93 / 19191; WO 94 / 28938; WO 95 / 11984 and WO 95 / 00655); adeno-associated virus (see, e.g, Ali et al., Hum. Gene Ther. (1998) 9: 81- 86. Flannery et al.. Proc. Natl. Acad. Sci. USA (1997) 94: 6916-6921; Bennett et al., Invest. Opthalmol. Vis. Sci. (1997) 38: 2857-2863; Jomary et al., Gene Ther. (1997) 4:683 690, Rolling et al., Hum. Gene Ther. (1999) 10: 641-648; Ali et al., Hum. Mol. Genet. (1996) 5: 591-594; Srivastava in WO 93 / 09239, Samulski et al., J. Vir. (1989) 63: 3822-3828;

[0225] Mendelson et al., Virol. (1988) 166: 154-165; and Flotte et al., Proc. Natl. Acad. Sci. USA (1993) 90: 10613-10617); SV40; herpes simplex virus; human immunodeficiency virus (see, e.g., Miyoshi et al., Proc. Natl. Acad. Sci. USA (1997) 94: 10319-23; Takahashi et al., J.

[0226] Virol. (1999) 73: 7812-7816); a retroviral vector (e.g.. murine leukemia virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous sarcoma virus, Harvey sarcoma virus, avian leukosis virus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus); and the like.

[0227] Additional expression vectors suitable for use are, e.g., without limitation, a lentivirus vector, a gamma retrovirus vector, a foamy virus vector, an adeno-associated virus vector, an adenovirus vector, a pox virus vector, a herpes virus vector, an engineered hybrid virus vector, a transposon mediated vector, and the like. Viral vector technology is well known in the art and is described, for example, in Sambrook et al., 2012, Molecular Cloning: A Laboratory Manual, volumes 1-4, Cold Spring Harbor Press, NY), and in other virology and molecular biology manuals. Viruses, which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno- associated viruses, herpes viruses, and lentiviruses.

[0228] In certain embodiments, the vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers, (e.g., WO 01 / 96584; WO 01 / 29058; and U.S. Pat. No. 6,326,193).

[0229] In some embodiments, an expression vector (e.g., a lentiviral vector) may be used for expressing the DR5 CAR and / or any other additional transgenes. In some embodiments, the expression vector (e.g., lentiviral vector) will comprise additional elements that will aid in the functional expression of the targeting protein or targeting protein encoded therein. In some embodiments, an expression vector comprises a mammalian promoter. In one embodiment, the vector comprises an elongation-factor- 1 -alpha promoter (EF-1 a promoter. Use of an EFla promoter may increase the efficiency in expression of downstream transgenes, such as the nucleic acid encoding the targeting protein. Physiologic promoters (e.g., an EF-la promoter) may be less likely to induce integration mediated genotoxicity, and may abrogate the ability of the retroviral vector to transform stem cells Other physiological promoters suitable for use in a vector (e.g., a lenti viral vector) are known to those of skill in the art and may be incorporated into a vector of the present invention.

[0230] In some embodiments, the vector (e.g., a lentiviral vector) further comprises a non- requisite cA-acting sequence that may improve titers and gene expression. One non-limiting example of a non-requisite cis acting sequence is the central polypurine tract and central termination sequence (cPPT / CTS) which is important for efficient reverse transcription and nuclear import. Other non-requisite cis acting sequences are known to those of skill in the art and may be incorporated into a vector (e.g., lentiviral vector) of the present invention.

[0231] In some embodiments, the vector further comprises a posttranscriptional regulatory- element. Posttranscriptional regulatory elements may improve RNA translation, improve transgene expression, and stabilize RNA transcripts. One example of a posttranscriptional regulatory element is the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE). Accordingly, in some embodiments a vector for the present invention further comprises a WPRE sequence. Various posttranscriptional regulator elements are known to those of skill in the art and may be incorporated into a vector (e.g, a lentiviral vector) of the present invention.

[0232] A vector of the present invention may further comprise additional elements such as a rev response element (RRE) for RNA transport, packaging sequences, and 5' and 3’ long terminal repeats (LTRs). The term "long terminal repeat” or LTR ' refers to domains of base pairs located at the ends of retroviral DNAs which comprise U3, R and U5 regions. LTRs generally provide functions required for the expression of retroviral genes (e.g., promotion, initiation, and polyadenylation of gene transcripts) and to viral replication. In one embodiment, a vector (e.g., lentiviral vector) of the present invention includes a 3‘ U3 deleted LTR. Accordingly, a vector (e.g. lentiviral vector) of the present invention may comprise any combination of the elements described herein to enhance the efficiency of functional expression of transgenes. For example, a vector (e.g., lentiviral vector) of the present invention may comprise a WPRE sequence, cPPT sequence, RRE sequence, 5'LTR, 3‘ U3 deleted LTR' in addition to a nucleic acid encoding for targeting protein (e.g., anti-DR5 targeting protein). Vectors of the present invention may be self-inactivating vectors. As used herein, the term “self-inactivating vector” refers to vectors in which the 3’ LTR enhancer promoter region (U3 region) has been modified (e.g, by deletion or substitution). A self-inactivating vector may prevent viral transcription beyond the first round of viral replication. Consequently, a self-inactivating vector may be capable of infecting and then integrating into a host genome (e.g, a mammalian genome) only once, and cannot be passed further. Accordingly, self-inactivating vectors may greatly reduce the risk of creating a replication- competent virus.

[0233] In one embodiment, the lentiviral vector is a self-inactivating HIV vector comprising a 5’ U3 LTR (e.g., SEQ ID NO: 69). a 3’ U3 deleted LTR (e.g.. SEQ ID NO: 70), a partial HIV gag sequence comprising an RRE sequence and packaging signal (i|i) (e.g., SEQ ID NO: 71), and / or an HIV central polypurine tract (cPPT) sequence (SEQ ID NO: 72). In some embodiments, the expression vector and / or HIV vector comprises a Woodchuck Hepatitis virus post-transcriptional regulatory element (WPRE). In an exemplary embodiment, the WPRE comprises the nucleotide sequence of SEQ ID NO: 73.

[0234] In some embodiments, a nucleic acid encoding an anti-DR5 CAR comprises a lentivirus vector. In one embodiment, the nucleic acid encoding the anti-DR5 CAR comprises a lentivirus expression construct comprising a nucleotide sequence as set forth in e.g., SEQ ID NOs: 37-40.

[0235] In order to assess the expression of a polypeptide or portions thereof, the expression vector or construct to be introduced into a cell may also contain either a selectable marker gene or a reporter gene, or both, to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected through viral vectors. In some embodiments, the selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells. Useful selectable markers include, without limitation, antibiotic-resistance genes.

[0236] Reporter genes may be used e.g., for tracking T and NK cells expressing the DR5 CARs as described herein. In general, a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and that encodes a polypeptide whose expression is manifested by some easily detectable property7, e.g, flow cytometry7or enzymatic activity. Expression of the reporter gene is assessed at a suitable time after the DNA has been introduced into the recipient cells. In some embodiments, a nucleic acid of the present invention may be RNA, e.g., in vitro synthesized RNA. Methods for in vitro synthesis of RNA are known to those of skill in the art; any known method can be used to synthesize RNA comprising a sequence encoding a chimeric receptor of the present disclosure. Methods for introducing RNA into a host cell are known in the art. See, e.g., Zhao et al. Cancer Res. (2010) 15: 9053. Introducing RNA comprising a nucleotide sequence encoding a chimeric receptor of the present disclosure into a host cell can be carried out in vitro, ex vivo or in vivo. For example, a host cell (e.g.. an NK cell, a cytotoxic T lymphocyte, etc.) can be electroporated in vitro or ex vivo with RNA comprising a nucleotide sequence encoding a chimeric receptor of the present disclosure. Nucleotide and amino sequences of exemplary CAR components, vector sequences, and expression constructs can be found in Table 1.

[0237] Table 1: Nucleotide and Amino Acid Sequences

[0238] Methods of Generating Modified Immune Cells

[0239] The present invention provides methods for producing / generating a modified immune cell or precursor cell thereof (e.g. a T cell, NK. NKT. gamma-delta T cell). The cells are engineered by introducing a nucleic acid encoding the anti-DR5 CAR and / or additional proteins of interest. In some embodiments, the CAR may be accompanied by a gene modification system that is capable of modifying the expression or sequence of an endogenous gene.

[0240] Methods of introducing nucleic acids or expression constructs into a cell include physical, biological, and chemical methods. Physical methods for introducing a polynucleotide, such as RNA, into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. RNA can be introduced into target cells using commercially available methods which include electroporation (AmaxaNucleofector-II (Amaxa Biosystems, Cologne, Germany)), (ECM 830 (BTX) (Harvard Instruments, Boston, MA) or the Gene Pulser II (BioRad, Denver, CO), Multiporator (Eppendorf, Hamburg Germany). RNA can also be introduced into cells using cationic liposome mediated transfection using lipofection, using polymer encapsulation, using peptide mediated transfection, or using biolistic particle delivery systems such as “gene guns” (see, for example, Nishikawa, et al. Hum Gene Ther.. 12(8): 861 -70 (2001).

[0241] Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, and especially retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g, human cells. Other viral vectors can be derived from lentivirus, poxviruses, herpesviruses, adenoviruses, and adeno-associated viruses, and the like. See, for example, U.S. Pat. Nos. 5,350,674 and 5,585,362.

[0242] In some embodiments, a nucleic acid encoding the DR5 CAR of the invention is introduced into a cell by an expression vector. Expression vectors comprising a nucleic acid encoding DR5 CAR are provided herein. Suitable expression vectors include lentivirus vectors, gamma retrovirus vectors, foamy virus vectors, adeno associated virus (AAV) vectors, adenovirus vectors, engineered hybrid viruses, naked DNA, including but not limited to transposon mediated vectors, such as Sleeping Beauty, Piggyback, and Integrases such as Phi31 . Other suitable expression vectors include Epstein-Barr virus (EBV), herpes simplex virus (HSV), and retrovirus expression vectors.

[0243] Adenovirus expression vectors are based on adenoviruses, which have a low capacity for integration into genomic DNA but a high efficiency for transfecting host cells. Adenovirus expression vectors contain adenovirus sequences sufficient to: (a) support packaging of the expression vector and (b) to ultimately express the DR5 CAR in the host cell. In some embodiments, the adenovirus genome is a 36 kb, linear, double stranded DNA, where a foreign DNA sequence (e.g, a nucleic acid encoding the DR5 CAR) may be inserted to substitute large pieces of adenoviral DNA in order to make the expression vector of the present invention (see, e.g, Danthinne and Imperiale, Gene Therapy (2000) 7(20): 1707- 1714).

[0244] Another expression vector is based on an adeno associated virus, which takes advantage of the adenovirus coupled systems. This AAV expression vector has a high frequency of integration into the host genome. It can infect non-dividing cells, thus making it useful for delivery of genes into mammalian cells, for example, in tissue cultures or in vivo. The AAV vector has a broad host range for infectivity. Details concerning the generation and use of AAV vectors are described in U.S. Patent Nos. 5,139,941 and 4,797,368.

[0245] Retrovirus expression vectors are capable of integrating into the host genome, delivering a large amount of foreign genetic material, infecting a broad spectrum of species and cell types, and being packaged in special cell lines. The retrovirus vector is constructed by inserting a nucleic acid (e.g.. a nucleic acid encoding the DR5 CAR) into the viral genome at certain locations to produce a virus that is replication defective. Though the retrovirus vectors are able to infect a broad variety of cell ty pes, integration, and stable expression of the DR5 CAR, requires the division of host cells.

[0246] Lentivirus vectors are derived from lentiviruses. which are complex retroviruses that, in addition to the common retroviral genes gag, pol, and env, contain other genes with regulatory or structural function (see, e.g., U.S. Patent Nos. 6,013,516 and 5,994, 136). Some examples of lentiviruses include the human immunodeficiency viruses (HIV-1, HIV-2) and the simian immunodeficiency virus (SIV). Lentivirus vectors have been generated bymultiply attenuating the HIV virulence genes, for example, the genes env, vif, vpr. vpu and nef are deleted making the vector biologicalty safe. Lentivirus vectors are capable of infecting non-dividing cells and can be used for both in vivo and ex vivo gene transfer and expression, e.g, of a nucleic acid encoding the DR5 CAR (see, e.g., U.S. Patent No. 5,994,136).

[0247] Expression vectors including a nucleic acid of the present disclosure can be introduced into a host cell by any means know n to persons skilled in the art. The expression vectors may include viral sequences for transfection, if desired. Alternatively, the expression vectors may be introduced by fusion, electroporation, biolistics, transfection, lipofection. or the like. The host cell may be grown and expanded in culture before introduction of the expression vectors, followed by the appropriate treatment for introduction and integration of the vectors. The host cells are then expanded and may be screened by virtue of a marker present in the vectors. Various markers that may be used are known in the art, and may include hprt, neomycin resistance, thymidine kinase, hygromycin resistance, etc. As used herein, the terms “cell,” “cell line,” and “cell culture” may be used interchangeably. In some embodiments, the host cell is an immune cell or precursor thereof, e.g., a T cell, an NK cell, or an NKT cell.

[0248] The present invention also provides genetically engineered cells which include and stably express the DR5 CAR of the present disclosure. In some embodiments, the genetically engineered cells are genetically engineered T-lymphocytes (T cells), regulatory T cells (Tregs), naive T cells (TN), memory T cells (for example, central memory T cells (TCM), effector memory cells (TEM)), natural killer cells (NK cells), natural killer T cells (NKT cells) and macrophages capable of giving rise to therapeutically relevant progeny. In one embodiment, the genetically engineered cells are autologous cells.

[0249] Modified cells (e.g, comprising the DR5 CAR) may be produced by stably transfecting host cells with an expression vector including a nucleic acid of the present disclosure. Additional methods to generate a modified cell of the present disclosure include, without limitation, chemical transformation methods (e.g. , using calcium phosphate, dendrimers, liposomes and / or cationic polymers), non-chemical transformation methods (e.g, electroporation, optical transformation, gene electrotransfer and / or hydrodynamic delivery) and / or particle-based methods (e.g., impalefection, using a gene gun and / or magnetofection). Transfected cells expressing the DR5 CAR of the present disclosure may be expanded ex vivo.

[0250] Physical methods for introducing an expression vector into host cells include calcium phosphate precipitation, hpofection. particle bombardment, microinjection, electroporation, and the like. Methods for producing cells including vectors and / or exogenous nucleic acids are well-known in the art. See, e.g, Sambrook et al. (2001), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory , New York.

[0251] Chemical means for introducing a polynucleotide into a host cell include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary' colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g, an artificial membrane vesicle).

[0252] Lipids suitable for use can be obtained from commercial sources. For example, dimyristyl phosphatidylcholine (“DMPC”) can be obtained from Sigma, St. Louis, MO; dicetyl phosphate (“DCP”) can be obtained from K & K Laboratories (Plainview, NY); cholesterol (“Choi”) can be obtained from Calbiochem-Behring; dimyristyl phosphatidylglycerol (“DMPG”) and other lipids may be obtained from Avanti Polar Lipids. Inc. (Birmingham, AL). Stock solutions of lipids in chloroform or chloroform / methanol can be stored at about -20°C. Chloroform is used as the only solvent since it is more readily evaporated than methanol. “Liposome” is a generic term encompassing a variety of single and multilamellar lipid vehicles formed by the generation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as having vesicular structures with a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5: 505-10). However, compositions that have different structures in solution than the normal vesicular structure are also encompassed. For example, the lipids may assume a micellar structure or merely exist as nonuniform aggregates of lipid molecules. Also contemplated are lipofectamine-nucleic acid complexes.

[0253] Regardless of the method used to introduce exogenous nucleic acids into a host cell or otherwise expose a cell to the inhibitor of the present invention, in order to confirm the presence of the nucleic acids in the host cell, a variety’ of assays may be performed. Such assays include, for example, “molecular biological’' assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR and PCR; “biochemical” assays, such as detecting the presence or absence of a particular peptide, e.g.. by immunological means (ELISAs and Western blots) or by assays described herein to identify agents falling within the scope of the invention.

[0254] Moreover, the nucleic acids may be introduced by any means, such as transducing the expanded T cells, transfecting the expanded T cells, and electroporating the expanded T cells. One nucleic acid may be introduced by one method and another nucleic acid may be introduced into the T cell by a different method.

[0255] Sources of Immune Cells

[0256] Prior to expansion, a source of immune cells is obtained from a subject for ex vivo manipulation. Sources of target cells for ex vivo manipulation may' also include, e.g., autologous or heterologous donor blood, cord blood, or bone marrow. For example, the source of immune cells may be from the subject to be treated with the modified immune cells of the invention, e.g., the subject's blood, the subject's cord blood, or the subject’s bone marrow. Non-limiting examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof. In certain exemplary’ embodiments, the subject is a human.

[0257] Immune cells can be obtained from a number of sources, including blood, peripheral blood mononuclear cells, bone marrow, lymph node tissue, spleen tissue, umbilical cord, lymph, or lymphoid organs. Immune cells are cells of the immune system, such as cells of the innate or adaptive immunity, e.g, myeloid or lymphoid cells, including lymphocytes, ty pically T cells and / or NK cells and / or NKT cells. Other exemplary cells include stem cells, such as multipotent and pluripotent stem cells, including induced pluripotent stem cells (iPSCs). In certain aspects, the cells are human cells. With reference to the subject to be treated, the cells may be allogeneic and / or autologous. The cells ty pically are primary' cells, such as those isolated directly from a subject and / or isolated from a subject and frozen.

[0258] In certain embodiments, the immune cell is a T cell, e.g., a CD8+ T cell (e.g. a CD8+ naive T cell, central memory T cell, or effector memory7T cell), a CD4+ T cell, a natural killer T cell (NKT cells), a gamma-delta T cell, a regulatory T cell (Treg), a stem cell memory T cell, a lymphoid progenitor cell, a hematopoietic stem cell, a natural killer cell (NK cell), a natural killer T cell (NK cell) or a dendritic cell. In some embodiments, the cells are monocytes or granulocytes, e.g, myeloid cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils, and / or basophils. In an embodiment, the target cell is an induced pluripotent stem (iPS) cell or a cell derived from an iPS cell, e.g, an iPS cell generated from a subject, manipulated to alter (e.g., induce a mutation in) or manipulate the expression of one or more target genes, and differentiated into, e.g.. a T cell, e.g.. a CD8+ T cell (e.g, a CD8+ naive T cell, central memory T cell, or effector memory T cell), a CD4+ T cell, a stem cell memory' T cell, a lymphoid progenitor cell or a hematopoietic stem cell.

[0259] In some embodiments, the cells include one or more subsets of T cells or other cell types, such as whole T cell populations, CD4+ cells, CD8+ cells, and subpopulations thereof, such as those defined by function, activation state, maturity, potential for differentiation, expansion, recirculation, localization, and / or persistence capacities, antigen- specificity7, ty pe of antigen receptor, presence in a particular organ or compartment, marker or cytokine secretion profile, and / or degree of differentiation. Among the sub-types and subpopulations of T cells and / or of CD4+ and / or of CD8+ T cells are naive T (TN) cells, effector T cells (TEFF), memory' T cells and sub-types thereof, such as stem cell memory7T (TSCM), central memory7T (TCM), effector memory T (TEM), or terminally differentiated effector memory7T cells, tumor-infiltrating lymphocytes (TIL), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosa-associated invariant T (MAIT) cells, naturally occurring and adaptive regulatory T (Treg) cells, helper T cells, such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, alpha / beta T cells, and gamma / delta T cells. In certain embodiments, any number of T cell lines available in the art, may be used. In some embodiments, the methods include isolating immune cells from the subject, preparing, processing, culturing, and / or engineering them. In some embodiments, preparation of the engineered cells includes one or more culture and / or preparation steps. The cells for engineering as described may be isolated from a sample, such as a biological sample, e.g, one obtained from or derived from a subject. In some embodiments, the subject from which the cell is isolated is one having the disease or condition or in need of a cell therapy or to which cell therapy will be administered. The subject in some embodiments is a human in need of a particular therapeutic intervention, such as the adoptive cell therapy for which cells are being isolated, processed, and / or engineered. Accordingly, the cells in some embodiments are primary cells, e.g., primary human cells. The samples include tissue, fluid, and other samples taken directly from the subject, as well as samples resulting from one or more processing steps, such as separation, centrifugation, genetic engineering (e.g., transduction with viral vector), washing, and / or incubation. The biological sample can be a sample obtained directly from a biological source or a sample that is processed. Biological samples include, but are not limited to, body fluids, such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat, tissue and organ samples, including processed samples derived therefrom.

[0260] In certain aspects, the sample from which the cells are derived or isolated is blood or a blood-derived sample, or is or is derived from an apheresis or leukapheresis product. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs), leukocytes, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, gut associated lymphoid tissue, mucosa associated lymphoid tissue, spleen, other lymphoid tissues, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testes, ovaries, tonsil, or other organ, and / or cells derived therefrom. Samples include, in the context of cell therapy, e.g, adoptive cell therapy, samples from autologous and allogeneic sources.

[0261] In some embodiments, the transduced cells are gamma-delta T cells.

[0262] Wang et al., JImmunother Cancer. 2021; 9(12): e003339 and U.S. Patent Publication No. 2024 / 197876 Al are incorporated herein by reference for their disclosures of compositions and methods for isolating gamma-delta T cells from a subject, and preparing, processing, culturing, and / or engineering them.

[0263] In some embodiments, the cells are derived from cell lines, e.g. T cell lines. The cells in some embodiments are obtained from a xenogeneic source, for example, from mouse, rat. non-human primate, and pig. In some embodiments, isolation of the cells includes one or more preparation and / or non-affinity -based cell separation steps. In some examples, cells are washed, centrifuged, and / or incubated in the presence of one or more reagents, for example, to remove unwanted components, enrich for desired components, and lyse or remove cells sensitive to particular reagents. In some examples, cells are separated based on one or more property7, such as density, adherent properties, size, sensitivity7and / or resistance to particular components.

[0264] In some examples, cells from the circulating blood of a subject are obtained, e.g.. by apheresis or leukapheresis. The samples, in certain aspects, contain lymphocytes, including T .cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and / or platelets, and in certain aspects contains cells other than red blood cells and platelets. In some embodiments, the blood cells collected from the subject are washed, e.g., to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps. In some embodiments, the cells are washed with phosphate buffered saline (PBS). In some embodiments , a washing step is accomplished by tangential flow filtration (TFF) according to the manufacturer's instructions. In certain embodiments, the cells are resuspended in a variety of biocompatible buffers after washing. In certain embodiments, components of a blood cell sample are removed, and the cells directly resuspended in culture media. In some embodiments, the methods include density -based cell separation methods, such as the preparation of white blood cells from peripheral blood by lysing the red blood cells and centrifugation through a Percoll or Ficoll gradient.

[0265] In one embodiment, immune cells are obtained from the circulating blood of an individual are obtained by apheresis or leukapheresis. The apheresis product ty pically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. The cells collected by apheresis may be washed to remove the plasma fraction and to place the cells in an appropriate buffer or media, such as phosphate buffered saline (PBS) or wash solution lacks calcium and may lack magnesium or may lack many if not all divalent cations, for subsequent processing steps. As those of ordinary skill in the art would readily appreciate a washing step may be accomplished by methods known to those in the art. such as by using a semi-automated “flow-through” centrifuge (for example, the Cobe 2991 cell processor, the Baxter CytoMate, or the Haemonetics Cell Saver 5) according to the manufacturer's instructions. After washing, the cells may be resuspended in a variety7of biocompatible buffers, such as, for example. Ca2+-free, Mg2+-free PBS, PlasmaLyte A, or another saline solution with or without buffer. In some embodiments, the undesirable components of the apheresis sample may be removed, and the cells directly resuspended in culture media.

[0266] In some embodiments, the isolation methods include the separation of different cell types based on the expression or presence in the cell of one or more specific molecules, such as surface markers, e.g., surface proteins, intracellular markers, or nucleic acid. In some embodiments, any known method for separation based on such markers may be used. In some embodiments, the separation is affinity- or immunoaffinity -based separation. For example, the isolation in certain aspects includes separation of cells and cell populations based on the cells' expression or expression level of one or more markers, typically cell surface markers, for example, by incubation with an antibody or binding partner that specifically binds to such markers, followed generally by washing steps and separation of cells having bound the antibody or binding partner, from those cells having not bound to the antibody or binding partner. Such separation steps can be based on positive selection, in which the cells having bound the reagents are retained for further use, and / or negative selection, in which the cells having not bound to the antibody or binding partner are retained. In some examples, both fractions are retained for further use. In certain aspects, negative selection can be particularly useful where no antibody is available that specifically identifies a cell type in a heterogeneous population, such that separation is best carried out based on markers expressed by cells other than the desired population. The separation need not result in 100% enrichment or removal of a particular cell population or cells expressing a particular marker. For example, positive selection of or enrichment for cells of a particular ty pe, such as those expressing a marker, refers to increasing the number or percentage of such cells, but need not result in a complete absence of cells not expressing the marker. Likewise, negative selection, removal, or depletion of cells of a particular type, such as those expressing a marker, refers to decreasing the number or percentage of such cells, but need not result in a complete removal of all such cells.

[0267] In certain exemplary embodiments, multiple rounds of separation steps are carried out, where the positively or negatively selected fraction from one step is subjected to another separation step, such as a subsequent positive or negative selection. In certain exemplary embodiments, a single separation step can deplete cells expressing multiple markers simultaneously, such as by incubating cells with a plurality of antibodies or binding partners, each specific for a marker targeted for negative selection. Likewise, multiple cell types can simultaneously be positively selected by incubating cells with a plurality of antibodies or binding partners expressed on the various cell types. In some embodiments, one or more of the T cell populations is enriched for or depleted of cells that are positive for (marker+) or express high levels (marker111811) of one or more particular markers, such as surface markers, or that are negative for (marker') or express relatively low levels (markerlow) of one or more markers. For example, in certain aspects, specific subpopulations of T cells, such as cells positive or expressing high levels of one or more surface markers, e.g., CD28+, CD62L+, CCR7+, CD27+. CD127+. CD4+, CD8+, CD45RA+, and / or CD45RO+ T cells, are isolated by positive or negative selection techniques. In some cases, such markers are those that are absent or expressed at relatively low levels on certain populations of T cells (such as non-memory cells) but are present or expressed at relatively higher levels on certain other populations of T cells (such as memory cells). In one embodiment, the cells (such as the CD8+ cells or the T cells, e.g., CD3+ cells) are enriched for (i.e., positively selected for) cells that are positive or expressing high surface levels of CD45RO, CCR7, CD28, CD27, CD44, CD127, and / or CD62L and / or depleted of (e.g., negatively selected for) cells that are positive for or express high surface levels of CD45RA. In some embodiments, cells are enriched for or depleted of cells positive or expressing high surface levels of CD122, CD95, CD25. CD27. and / or IL7-Ra (CD127). In certain exemplary embodiments, CD8+ T cells are enriched for cells positive for CD45RO (or negative for CD45RA) and for CD62L. For example, CD3+, CD28+ T cells can be positively selected using CD3 / CD28 conjugated magnetic beads (e.g., DYNABEADS® M- 450 CD3 / CD28 T Cell Expander).

[0268] In some embodiments, T cells are separated from a PBMC sample by negative selection of markers expressed on non-T cells, such as B cells, monocytes, or other white blood cells, such as CD 14. In certain aspects, a CD4+ or CD8+ selection step is used to separate CD4+ helper and CD8+ cytotoxic T cells. Such CD4+ and CD8+ populations can be further sorted into sub-populations by positive or negative selection for markers expressed or expressed to a relatively higher degree on one or more naive, memory, and / or effector T cell subpopulations. In some embodiments, CD8+ cells are further enriched for or depleted of naive, central memory, effector memory, and / or central memory stem cells, such as by positive or negative selection based on surface antigens associated with the respective subpopulation. In some embodiments, enrichment for central memory T (TCM) cells is carried out to increase efficacy, such as to improve long-term survival, expansion, and / or engraftment following administration, which in certain aspects is particularly robust in such sub-populations. In some embodiments, combining TCM-enriched CD8+ T cells and CD4+ T cells further enhances efficacy. In some embodiments, memoiy T cells are present in both CD62L+ and CD62L- subsets of CD8+ peripheral blood lymphocytes. PBMC can be enriched for or depleted of CD62L-CD8+ and / or CD62L+CD8+ fractions, such as using anti-CD8 and anti-CD62L antibodies. In some embodiments, a CD4+ T cell population and / or a CD8+ T population is enriched for central memory (TCM) cells. In some embodiments, the enrichment for central memory T (TCM) cells is based on positive or high surface expression of CD45RO, CD62L, CCR7. CD28, CD3, and / or CD 127; in certain aspects, it is based on negative selection for cells expressing or highly expressing CD45RA and / or granzyme B. In certain aspects, isolation of a CD8+ population enriched for TCM cells is carried out by depletion of cells expressing CD4, CD 14, CD45RA, and positive selection or enrichment for cells expressing CD62L. In one aspect, enrichment for central memory T (TCM) cells is carried out starting with a negative fraction of cells selected based on CD4 expression, which is subjected to a negative selection based on expression of CD 14 and CD45RA, and a positive selection based on CD62L. Such selections in certain aspects are carried out simultaneously and in other aspects are carried out sequentially, in either order. In some embodiments, the same CD4 expression-based selection step used in preparing the CD8+ cell population or subpopulation, also is used to generate the CD4+ cell population or sub-population, such that both the positive and negative fractions from the CD4-based separation are retained and used in subsequent steps of the methods, optionally following one or more further positive or negative selection steps.

[0269] CD4+ T helper cells are sorted into naive, central memory', and effector cells by identifying cell populations that have cell surface antigens. CD4+ lymphocytes can be obtained by standard methods. In some embodiments, naive CD4+ T lymphocytes are CD45RO-, CD45RA+, CD62L+, CD4+ T cells. In some embodiments, central memory CD4+ cells are CD62L+ and CD45RO+. In some embodiments, effector CD4+ cells are CD62L- and CD45RO. In one example, to enrich for CD4+ cells by negative selection, a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CDl lb, CD16, HLA-DR, and CD8. In some embodiments, the antibody or binding partner is bound to a solid support or matrix, such as a magnetic bead or paramagnetic bead, to allow for separation of cells for positive and / or negative selection.

[0270] In some embodiments, the cells are incubated and / or cultured prior to or in connection with genetic engineering. The incubation steps can include culture, cultivation, stimulation, activation, and / or propagation. In some embodiments, the compositions or cells are incubated in the presence of stimulating conditions or a stimulatory agent. Such conditions include those designed to induce proliferation, expansion, activation, and / or survival of cells in the population, to mimic antigen exposure, and / or to prime the cells for genetic engineering, such as for the introduction of a recombinant antigen receptor. The conditions can include one or more of particular media, temperature, oxygen content, carbon dioxide content, time, agents, e.g., nutrients, amino acids, antibiotics, ions, and / or stimulatory factors, such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agents designed to activate the cells. In some embodiments, the stimulating conditions or agents include one or more agent, e.g, ligand, which is capable of activating an intracellular signaling domain of a TCR complex. In certain aspects, the agent turns on or initiates TCR / CD3 intracellular signaling cascade in a T cell. Such agents can include antibodies, such as those specific for a TCR component and / or costimulatory receptor, e.g., anti-CD3, anti-CD28, for example, bound to solid support such as a bead, and / or one or more cytokines. Optionally, the expansion method may further comprise the step of adding anti- CD3 and / or anti CD28 antibody to the culture medium (e.g, at a concentration of at least about 0.5 ng / ml). In some embodiments, the stimulating agents include IL-2 and / or IL-15, for example, an IL-2 concentration of at least about 10 units / mL.

[0271] In another embodiment, T cells are isolated from peripheral blood by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLL™ gradient. Alternatively, T cells can be isolated from an umbilical cord. In any event, a specific subpopulation of T cells can be further isolated by positive or negative selection techniques.

[0272] The cord blood mononuclear cells so isolated can be depleted of cells expressing certain antigens, including, but not limited to, CD34, CD8, CD14, CD19, and CD56. Depletion of these cells can be accomplished using an isolated antibody, a biological sample comprising an antibody, such as ascites, an antibody bound to a physical support, and a cell bound antibody.

[0273] Enrichment of a T cell population by negative selection can be accomplished using a combination of antibodies directed to surface markers unique to the negatively selected cells. An exemplary method is cell sorting and / or selection via negative magnetic immunoadherence or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface markers present on the cells negatively selected. For example, to enrich for CD4+cells by negative selection, a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CDl lb, CD16, HLA-DR. and CD8. For isolation of a desired population of cells by positive or negative selection, the concentration of cells and surface (e g., particles such as beads) can be varied. In certain embodiments, it may be desirable to significantly decrease the volume in which beads and cells are mixed together (i.e., increase the concentration of cells), to ensure maximum contact of cells and beads. For example, in one embodiment, a concentration of 2 billion cells / ml is used. In one embodiment, a concentration of 1 billion cells / ml is used. In a further embodiment, greater than 100 million cells / ml is used. In a further embodiment, a concentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells / ml is used. In yet another embodiment, a concentration of cells from 75, 80, 85, 90, 95, or 100 million cells / ml is used. In further embodiments, concentrations of 125 or 150 million cells / ml can be used. Using high concentrations can result in increased cell yield, cell activation, and cell expansion.

[0274] T cells can also be frozen after the washing step, which does not require the monocyte-removal step. While not wishing to be bound by theory', the freeze and subsequent thaw step provides a more uniform product by removing granulocytes and to some extent monocytes in the cell population. After the washing step that removes plasma and platelets, the cells may be suspended in a freezing solution. While many freezing solutions and parameters are known in the art and will be useful in this context, in a non-limiting example, one method involves using PBS containing 20% DMSO and 8% human serum albumin, or other suitable cell freezing media. The cells are then frozen to -80°C at a rate of 1°C per minute and stored in the vapor phase of a liquid nitrogen storage tank. Other methods of controlled freezing may be used as w ell as uncontrolled freezing immediately at -20°C or in liquid nitrogen.

[0275] In one embodiment, the population of T cells is comprised within cells such as peripheral blood mononuclear cells, cord blood cells, a purified population of T cells, and a T cell line. In another embodiment, peripheral blood mononuclear cells comprise the population of T cells. In yet another embodiment, purified T cells comprise the population of T cells.

[0276] Expansion of Immune Cells

[0277] Whether prior to or after modification of cells to express the DR5 CAR, the cells can be activated and expanded in number using methods as described, for example, in U.S. Patent Nos. 6,352,694; 6,534.055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6.905,681; 7,144,575; 7.067,318; 7.172,869; 7,232.566; 7,175.843; 5,883,223; 6,905,874; 6.797,514; 6,867,041; and U.S. Publication No. 20060121005. For example, the immune cells of the invention may be expanded by contact with a surface having attached thereto an agent that stimulates a CD3 / TCR complex associated signal and a ligand that stimulates a costimulatory molecule on the surface of the immune cells. In particular, immune cell populations may be stimulated by contact with an anti-CD3 antibody, or an antigen-binding fragment thereof, or an anti-CD2 antibody immobilized on a surface, or by contact with a protein kinase C activator (e.g, bryostatin) in conjunction with a calcium ionophore. For costimulation of an accessory molecule on the surface of the immune cells, a ligand that binds the accessory molecule is used. For example, immune cells can be contacted with an anti- CD3 antibody and an anti-CD28 antibody, under conditions appropriate for stimulating proliferation of the immune cells. Examples of an anti-CD28 antibody include 9.3, B-T3, XR-CD28 (Diaclone, Besancon. France) and these can be used in the invention, as can other methods and reagents known in the art (see, e.g., ten Berge et al., Transplant Proc. (1998) 30(8): 3975-3977; Haanen et al., J. Exp. Med. (1999) 190(9): 1319-1328; and Garland et al., J. Immunol. Methods (1999) 227(1-2): 53-63).

[0278] Expanding the immune cells by the methods disclosed herein can be multiplied by about 10-fold, 20-fold. 30-fold, 40-fold. 50-fold, 60-fold. 70-fold, 80-fold. 90-fold. 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700 fold, 800-fold, 900-fold, 1000-fold, 2000-fold, 3000-fold, 4000-fold, 5000-fold, 6000-fold, 7000-fold, 8000-fold, 9000-fold, 10,000-fold, 100,000-fold, 1,000,000-fold, 10,000,000-fold, or greater, and any and all whole or partial integers therebetween. In one embodiment, the immune cells expand in the range of about 20-fold to about 50-fold.

[0279] Following culturing, the immune cells can be incubated in cell medium in a culture apparatus for a period of time or until the cells reach confluency or high cell density for optimal passage before passing the cells to another culture apparatus. The culturing apparatus can be of any culture apparatus commonly used for culturing cells in vitro. In certain exemplary embodiments, the level of confluence is 70% or greater before passing the cells to another culture apparatus. In particularly exemplary' embodiments, the level of confluence is 90% or greater. A period of time can be any time suitable for the culture of cells in vitro. The immune cell medium may be replaced during the culture of the immune cells at any time. In certain exemplary embodiments, the immune cell medium is replaced about every 2 to 3 days. The immune cells are then harvested from the culture apparatus whereupon the immune cells can be used immediately or cryopreserved to be stored for use at a later time. In one embodiment, the invention includes cry opreserving the expanded immune cells. The cryopreserved immune cells are thawed prior to introducing nucleic acids into the immune cell.

[0280] In another embodiment, the method comprises isolating immune cells and expanding the immune cells. In another embodiment, the invention further comprises cry opreserving the immune cells prior to expansion. In yet another embodiment, the cryopreserved immune cells are thawed for electroporation with the RNA encoding the chimeric membrane protein.

[0281] Another procedure for ex vivo expansion cells is described in U.S. Pat. No. 5.199,942 (incorporated herein by reference). Expansion, such as described in U.S. Pat. No. 5,199,942 can be an alternative or in addition to other methods of expansion described herein. Briefly, ex vivo culture and expansion of immune cells comprises the addition to the cellular growth factors, such as those described in U.S. Pat. No. 5,199,942, or other factors, such as Flt3-L, IL-1, IL-3, and c-kit ligand. In one embodiment, expanding the immune cells comprises culturing the immune cells with a factor selected from the group consisting of Flt3-L, IL-1, IL-3, and c-kit ligand.

[0282] The culturing step as described herein (contact with agents as described herein or after electroporation) can be very short, for example less than 24 hours such as 1, 2. 3. 4, 5, 6. 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 hours. The culturing step as described further herein (contact with agents as described herein) can be longer, for example 1, 2, 3, 4. 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more days.

[0283] Various terms are used to describe cells in culture. Cell culture refers generally to cells taken from a living organism and grown under controlled condition. A primary cell culture is a culture of cells, tissues or organs taken directly from an organism and before the first subculture. Cells are expanded in culture when they are placed in a growth medium under conditions that facilitate cell growth and / or division, resulting in a larger population of the cells. When cells are expanded in culture, the rate of cell proliferation is typically measured by the amount of time required for the cells to double in number, otherwise known as the doubling time.

[0284] Each round of subculturing is referred to as a passage. When cells are subcultured, they are referred to as having been passaged. A specific population of cells, or a cell line, is sometimes referred to or characterized by the number of times it has been passaged. For example, a cultured cell population that has been passaged ten times may be referred to as a P10 culture. The primary culture, i.e., the first culture following the isolation of cells from tissue, is designated P0. Following the first subculture, the cells are described as a secondary culture (Pl or passage 1). After the second subculture, the cells become a tertiary' culture (P2 or passage 2), and so on. It will be understood by those of skill in the art that there may be many population doublings during the period of passaging. Therefore, the number of population doublings of a culture is greater than the passage number. The expansion of cells (i.e., the number of population doublings) during the period between passaging depends on many factors, including but is not limited to the seeding density, substrate, medium, and time between passaging.

[0285] In one embodiment, the cells may be cultured for several hours (about 3 hours) to about 14 days or any hourly integer value in between. Conditions appropriate for immune cell culture include an appropriate media (e.g.. Minimal Essential Media or RPMI Media 1640 or, X-vivo 15, (Lonza)) that may contain factors necessary for proliferation and viability , including serum (e.g, fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN- gamma, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGF-beta, and TNF-a or any other additives for the grow th of cells known to the skilled artisan. Other additives for the growth of cells include, but are not limited to, surfactant, plasmanate, and reducing agents such as N- acetyl-cysteine and 2-mercaptoethanol. Media can include RPMI 1640. AIM-V, DMEM, MEM, a-MEM, F-12, X-Vivo 15. and X-Vivo 20. Optimizer, with added amino acids, sodium pyruvate, and vitamins, either serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones, and / or an amount of cytokine(s) sufficient for the growth and expansion of immune cells. Antibiotics, e.g., penicillin and streptomycin, are included only in experimental cultures, not in cultures of cells that are to be infused into a subject. The target cells are maintained under conditions necessary to support growth, for example, an appropriate temperature (e.g., 37° C) and atmosphere (e.g, air plus 5% CO2).

[0286] The medium used to culture the immune cells may include an agent that can costimulate the immune cells. For example, an agent that can stimulate CD3 is an antibody to CD3, and an agent that can stimulate CD28 is an antibody to CD28. This is because, as demonstrated by the data disclosed herein, a cell isolated by the methods disclosed herein can be expanded approximately 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80- fold, 90-fold. 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold. 900-fold, 1000-fold, 2000-fold, 3000-fold, 4000-fold, 5000-fold, 6000-fold, 7000-fold, 8000- fold, 9000-fold, 10,000-fold, 100,000-fold, 1,000,000-fold, 10,000,000-fold, or greater. In one embodiment, the immune cells expand in the range of about 2-fold to about 50-fold, or more by culturing the electroporated population. In one embodiment, human T regulatory cells are expanded via anti-CD3 antibody coated KT64.86 artificial antigen presenting cells (aAPCs). Methods for expanding and activating immune cells can be found in U.S. Patent Numbers 7,754,482, 8.722,400, and 9.555,105, the contents of which are incorporated herein in their entirety.

[0287] In one embodiment, the method of expanding the immune cells can further comprise isolating the expanded immune cells for further applications. In another embodiment, the method of expanding can further comprise a subsequent electroporation of the expanded immune cells followed by culturing. The subsequent electroporation may include introducing a nucleic acid encoding an agent, such as a transducing the expanded immune cells, transfecting the expanded immune cells, or electroporating the expanded immune cells with a nucleic acid, into the expanded population of immune cells, wherein the agent further stimulates the immune cell. The agent may stimulate the immune cells, such as by stimulating further expansion, effector function, or another immune cell function.

[0288] Methods

[0289] In one aspect, the present invention provides a method for killing a DR5+ cell, comprising contacting the cell with an anti-DR5 CAR T cell, an anti-DR5 CAR NK cell, or an anti-DR5 expression construct, as described herein. In one embodiment, the DR5+ cell is a melanoma cell, breast cancer cell, liver cancer cell, or ovarian cancer cell. In another embodiment, the DR5+ cell is a myeloid-derived suppressor cell (MDSC). In another embodiment, the DR5+ cell is a cancer-associated fibroblast (CAF).

[0290] In another aspect, the present invention provides a method for treating a disease or disorder comprising administering to a subject in need thereof an anti-DR5 CAR T cell, an anti-DR5 CAR NK cell, or pharmaceutical composition thereof (as described herein), in an amount effective for treating a disease or disorder. In some embodiments, the disease is cancer. In certain embodiments, the cancer comprises a solid tumor. In other embodiments, the cancer is a hematological malignancy. In some embodiments, the DR5 CAR-T or DR5 CAR-NK cells described herein may be included in a pharmaceutical composition for immunotherapy or adoptive cell therapy. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable earner.

[0291] The DR5 CAR-T / CAR-NK cells of the present invention can be administered to an animal, preferably a mammal, even more preferably a human, to treat a DR5+cancer to produce an apoptotic response and / or cell-mediated immune response against tumor cells, where it is desirable to treat or alleviate the disease. In an embodiment, the cancer to be treated is a solid tumor. In some embodiments, the cancer is melanoma, lung cancer, liver cancer, pancreatic cancer, stomach cancer, colon cancer, kidney cancer, brain cancer, head and neck cancer, breast cancer, skin cancer, rectal cancer, uterine cancer, cervical cancer, ovarian cancer, testicular cancer, skin cancer, gastric cancer, esophageal cancer, and / or the cancer includes a sarcoma cell, a rhabdoid cancer cell, a neuroblastoma cell, retinoblastoma cell, or a medulloblastoma cell, and / or the cancer is uterine carcinosarcoma (UCS), gastrointestinal stromal tumor (GIST), brain lower grade glioma (LGG), thymoma (THYM), testicular germ cell tumors (TGCT), glioblastoma multiforme (GBM),skin cutaneous melanoma (SKCM), mucosal melanoma, acral melanoma, liver hepatocellular carcinoma (LIHC), uveal melanoma (UVM), kidney chromophobe (KICH), thyroid cancer (THCA), kidney renal clear cell carcinoma (KIRC), kidney renal papillary cell carcinoma (KIRP), stomach adenocarcinoma (STAD), cholangiocarcinoma (CHOL), adenoid cystic carcinoma (ACC), prostate adenocarcinoma (PRAD), pheochromocytoma and paraganglioma (PCPG), DLBC, lung adenocarcinoma (LU AD), head-neck squamous cell carcinoma (HNSC), pancreatic adenocarcinoma (PAAD), breast cancer (BRCA), mesothelioma (MESO), colon and rectal adenocarcinoma (COAD), rectum adenocarcinoma (READ), esophageal carcinoma (ESCA), ovarian cancer (OV), lung squamous cell carcinoma (LUSC), bladder urothelial carcinoma (BLCA), sarcoma (SARC), or uterine corpus endometrial carcinoma (UCEC).

[0292] In some embodiments, the solid tumor is selected from the group consisting of melanoma, breast cancer, liver cancer, lung cancer, ovarian cancer, cervical cancer, uterus cancer, head and neck cancer, hepatocellular carcinoma, colon cancer, rectal cancer, colorectal carcinoma, kidney cancer, prostate cancer, gastric cancer, bronchus cancer, pancreatic cancer, urinary bladder cancer, and brain cancer.

[0293] In another embodiment, the cancer to be treated is a hematological cancer. In one embodiment, the cancer is a leukemia. In another embodiment, the cancer is a myeloma. In another embodiment, the cancer is a lymphoma. Exemplar}' hematological cancers be treated with the compositions of the present invention include those selected from the group consisting of chronic lymphocytic leukemia (CLL). mantle cell lymphoma (MCL). multiple myeloma, acute lymphoid leukemia (ALL), Hodgkin lymphoma, B-cell acute lymphoid leukemia (BALL), T-cell acute lymphoid leukemia (TALL), small lymphocytic leukemia (SLL), B cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt lymphoma, diffuse large B cell lymphoma (DLBCL), DLBCL associated with chronic inflammation, follicular lymphoma, pediatric follicular lymphoma, hairy cell leukemia, small cell- or a large cell-follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma (extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue), Marginal zone lymphoma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom macroglobulinemia, splenic marginal zone lymphoma, splenic lymphoma / leukemia, splenic diffuse red pulp small B-cell lymphoma, hairy cell leukemia-variant, lymphoplasmacytic lymphoma, a heavy chain disease, plasma cell myeloma, solitary plasmocytoma of bone, extraosseous plasmocytoma, nodal marginal zone lymphoma, pediatric nodal marginal zone lymphoma, primary cutaneous follicle center lymphoma, lymphomatoid granulomatosis, primary mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, ALK.+ large B-cell lymphoma, large B-cell lymphoma arising in HHV8-associated multicentric Castleman disease, primary effusion lymphoma, B-cell lymphoma, or unclassifiable lymphoma.

[0294] In certain exemplary embodiments, the CAR-T / NK cells of the invention are used to treat a myeloma, or a condition related to myeloma. Examples of myeloma or conditions related thereto include, without limitation, light chain myeloma, non-secretory myeloma, monoclonal gamopathy of undetermined significance (MGUS), plasmacytoma (e.g., solitary, multiple solitary, extramedullary’ plasmacytoma), amyloidosis, and multiple myeloma. In one embodiment, a method of the present disclosure is used to treat multiple myeloma. In one embodiment, a method of the present disclosure is used to treat refractory’ myeloma. In one embodiment, a method of the present disclosure is used to treat relapsed myeloma.

[0295] In some embodiments, the subject has been treated with a therapeutic agent prior to administration of the DR5 CAR-T / NK cells. In some embodiments, the subject is refractory or non-responsive to the other therapeutic agent. In some embodiments, the subject has persistent or relapsed disease, e.g., following treatment with another therapeutic intervention, including chemotherapy, radiation, and / or hematopoietic stem cell transplantation (HSCT), e.g., allogenic HSCT, and / or other CAR-T / NK cell therapy. In some embodiments, the administration effectively treats the subject despite the subject having become resistant to another therapy.

[0296] In some embodiments, the subject is responsive to the other therapeutic agent, and treatment with the DR5 CAR-T / NK cells reduces disease burden. In some embodiments, the subject is initially responsive to the other therapeutic agents, but exhibits a relapse of the disease or condition over time. In some embodiments, the subject has not relapsed. In some such embodiments, the subject is determined to be at risk for relapse, such as at a high risk of relapse, and thus the DR5 CAR-I7NK cells are administered prophylactically, e.g, to reduce the likelihood of or prevent relapse. In some aspects, the subject has not received prior treatment with another therapeutic agent.

[0297] In some embodiments, the subject has persistent or relapsed disease, e.g., following treatment with another therapeutic intervention, including chemotherapy, radiation, hematopoietic stem cell transplantation (HSCT), e.g., allogenic HSCT, and / or other CAR-T cell therapies. In some embodiments, the administration effectively treats the subject despite the subject having become resistant to another therapy.

[0298] Administration of the DR5 CAR-T / NK cells of the invention may be carried out in any convenient manner known to those of skill in the art. In some embodiments, the DR5 CAR-T / NK cells may be administered to a subject by injection or transfusion. In some embodiments, the DR5 CAR-T / NK cells described herein may be administered to a patient intratumorally, transarterially, subcutaneously, intradermally, intranodally, intramedullary, intramuscularly, by intravenous (i.v.) injection, or intraperitoneally. In other instances, the DR5 CAR-T / NK cells of the invention are injected directly into a site of inflammation in the subject, a local disease site in the subject, a lymph node, an organ, a tumor, and the like.

[0299] For the prevention or treatment of disease, the appropriate dosage may depend on the type of disease to be treated, the type of targeting proteins or target proteins, the severity and course of the disease, whether the DR5 CAR-T / NK cells are administered for preventive or therapeutic purposes, previous therapy, the subject's clinical history and response to the DR5 CAR-T / NK cells, and / or the discretion of the attending physician. In some embodiments, the compositions in some embodiments are suitably administered to the subject at one time or over a series of treatments.

[0300] In some embodiments, the cells are administered at a desired dosage, which in some aspects includes a desired dose or number of cells or cell type(s) and / or a desired ratio of cell ty pes. Thus, the dosage of cells in some embodiments is based on a total number of cells (or number per kg body weight) and a desired ratio of the individual populations or sub-types, such as the CD4+ to CD8+ ratio. In some embodiments, the dosage of cells is based on a desired total number (or number per kg of body weight) of cells in the individual populations or of individual cell types. In some embodiments, the dosage is based on a combination of such features, such as a desired number of total cells, desired ratio, and desired total number of cells in the individual populations.

[0301] In certain embodiments, the cells, or individual populations of sub-types of cells, are administered to the subject at a range of about one million to about 100 billion cells, such as. e.g., 1 million to about 50 billion cells (e.g., about 5 million cells, about 25 million cells, about 500 million cells, about 1 billion cells, about 5 billion cells, about 20 billion cells, about 30 billion cells, about 40 billion cells, or a range defined by any two of the foregoing values), such as about 10 million to about 100 billion cells (e.g., about 20 million cells, about 30 million cells, about 40 million cells, about 60 million cells, about 70 million cells, about 80 million cells, about 90 million cells, about 10 billion cells, about 25 billion cells, about 50 billion cells, about 75 billion cells, about 90 billion cells, or a range defined by any two of the foregoing values), and in some cases about 100 million cells to about 50 billion cells (e.g., about 120 million cells, about 250 million cells, about 350 million cells, about 450 million cells, about 650 million cells, about 800 million cells, about 900 million cells, about 3 billion cells, about 30 billion cells, about 45 billion cells) or any value in between these ranges.

[0302] In some embodiments, the dose of total cells and / or dose of individual subpopulations of cells is within a range of between at or about IxlO5cells / kg to about IxlO11cells / kg 104and at or about 1011cells / kilograms (kg) body weight, such as between 105and 106cells / kg body weight, for example, at or about 1 x 105cells / kg, 1.5 x 105cells / kg, 2 x 105cells / kg, or 1 x 106cells / kg body weight. For example, in some embodiments, the cells are administered at, or within a certain range of error of, betw een at or about 104and at or about 109T cells / kilograms (kg) body weight, such as between 105and 106T cells / kg body weight, for example, at or about 1 x 105T cells / kg, 1.5 x 105T cells / kg, 2 x 105T cells / kg, or 1 x 106T cells / kg body weight. In other exemplary embodiments, a suitable dosage range of modified cells for use in a method of the present disclosure includes, without limitation, from about IxlO5cells / kg to about IxlO6cells / kg, from about IxlO6cells / kg to about IxlO7cells / kg, from about IxlO7cells / kg about IxlO8cells / kg, from about IxlO8cells / kg about IxlO9cells / kg, from about IxlO9cells / kg about IxlO10cells / kg, from about IxlO10cells / kg about IxlO11cells / kg. In an exemplary embodiment, a suitable dosage for use in a method of the present disclosure is about IxlO8cells / kg. In an exemplary embodiment, a suitable dosage for use in a method of the present disclosure is about IxlO7cells / kg. In other embodiments, a suitable dosage is from about IxlO7total cells to about 5xl07total cells. In some embodiments, a suitable dosage is from about IxlO8total cells to about 5x108total cells. In some embodiments, a suitable dosage is from about 1.4xl07total cells to about 1. IxlO9total cells. In an exemplary7embodiment, a suitable dosage for use in a method of the present disclosure is about 7xl09total cells.

[0303] In some embodiments, the cells are administered at or within a certain range of error of between at or about 104and at or about 109CD4+and / or CD8+cells / kilograms (kg) body weight, such as between 105and 106CD4+and / or CD8+cells / kg body weight, for example, at or about 1 x 105CD4+and / or CD8+cells / kg, 1.5 x 105CD4+and / or CD8+cells / kg, 2 x 105CD4+and / or CD8+cells / kg, or 1 x 106CD4+and / or CD8+cells / kg body weight. In some embodiments, the cells are administered at or within a certain range of error of, greater than, and / or at least about 1 x 106, about 2.5 x 106, about 5 x 106, about 7.5 x 106, or about 9 x 106CD4+cells, and / or at least about 1 x 106, about 2.5 x 106, about 5 x 106, about 7.5 x 106, or about 9 x 106CD8+ cells, and / or at least about 1 x 106, about 2.5 x 106. about 5 x 106, about 7.5 x 106, or about 9 x 106T cells. In some embodiments, the cells are administered at or within a certain range of error of between about 108and 1012or between about 1010and 1011T cells, between about 108and 1012or between about IO10and 1011CD4+cells, and / or between about 108and 1012or between about IO10and 1011CD8+cells.

[0304] In some embodiments, the cells are administered at or within a tolerated range of a desired output ratio of multiple cell populations or sub-types, such as CD4+ and CD8+ cells or sub-types. In some aspects, the desired ratio can be a specific ratio or can be a range of ratios, for example, in some embodiments, the desired ratio (e.g., ratio of CD4+to CD8+cells) is between at or about 5: 1 and at or about 5: 1 (or greater than about 1 :5 and less than about 5 : 1 ), or between at or about 1 : 3 and at or about 3 : 1 (or greater than about 1 : 3 and less than about 3: 1), such as between at or about 2: 1 and at or about 1 :5 (or greater than about 1 :5 and less than about 2: 1, such as at or about 5: 1, 4.5: 1, 4: 1, 3.5: 1, 3: 1, 2.5: 1, 2: 1, 1.9: 1, 1.8: 1, 1.7: 1. 1.6: 1, 1.5: 1, 1.4: 1. 1.3: 1, 1.2: 1. 1.1: 1, 1 : 1, 1 : 1.1, 1 : 1.2, 1: 1.3. 1: 1.4, 1 : 1 .5, 1 : 1 .6, 1 : 1 .7, 1 : 1 .8, 1 : 1 .9: 1 :2, 1 :2.5, 1 :3, 1 :3.5, 1 :4, 1 :4.5, or 1 :5. In some aspects, the tolerated difference is within about 1%, about 2%, about 3%, about 4% about 5%, about 10%, about 15%. about 20%, about 25%, about 30%, about 35%, about 40%. about 45%, about 50% of the desired ratio, including any value in between these ranges.

[0305] In some embodiments, a dose of modified cells is administered to a subject in need thereof, in a single dose or multiple doses. In some embodiments, a dose of modified cells is administered in multiple doses, e.g, once a week or every 7 days, once every 2 weeks or every 14 days, once every 3 weeks or every 21 days, once every 4 weeks or every 28 days. In an exemplary embodiment, a single dose of modified cells is administered to a subject in need thereof. In an exemplary embodiment, a single dose of modified cells is administered to a subject in need thereof by rapid intravenous infusion.

[0306] For the prevention or treatment of disease, the appropriate dosage may depend on the type of disease to be treated, the type of cells or recombinant receptors, the severity and course of the disease, whether the cells are administered for preventive or therapeutic purposes, previous therapy, the subject's clinical history and response to the cells, and the discretion of the attending physician. The compositions and cells are in some embodiments suitably administered to the subject at one time or over a series of treatments.

[0307] In some embodiments, the cells are administered as part of a combination treatment, such as simultaneously with or sequentially with, in any order, another therapeutic intervention, such as an antibody or engineered cell or receptor or agent, such as a cytotoxic or therapeutic agent. The cells in some embodiments are co-administered with one or more additional therapeutic agents or in connection with another therapeutic intervention, either simultaneously or sequentially in any order. In some contexts, the cells are co-administered with another therapy sufficiently close in time such that the cell populations enhance the effect of one or more additional therapeutic agents, or vice versa. In some embodiments, the cells are administered prior to the one or more additional therapeutic agents. In some embodiments, the cells are administered after the one or more additional therapeutic agents. In some embodiments, the one or more additional agents includes a cytokine, such as IL-2, for example, to enhance persistence. In some embodiments, the methods comprise administration of a chemotherapeutic agent.

[0308] In certain embodiments, the modified cells of the invention (e.g, a modified cell comprising a CAR) may be administered to a subject in combination with an immune checkpoint antibody (e.g, an anti-PDl, anti-CTLA-4, or anti-PDLl antibody). For example, the modified cell may be administered in combination with an antibody or antibody fragment targeting, for example, PD-1 (programmed death 1 protein). Examples of anti-PD-1 antibodies include, but are not limited to, pembrolizumab (KEYTRUDA®, formerly lambrolizumab, also known as MK-3475), and nivolumab (BMS-936558, MDX-1106, ONO- 4538, OPDIVA®) or an antigen-binding fragment thereof. In certain embodiments, the modified cell may be administered in combination with an anti-PD-Ll antibody or antigenbinding fragment thereof. Examples of anti-PD-Ll antibodies include, but are not limited to, BMS-936559, MPDL3280A (TECENTRIQ®, Atezolizumab), and MEDI4736 (Durvalumab, Imfinzi). In certain embodiments, the modified cell may be administered in combination with an anti-CTLA-4 antibody or antigen-binding fragment thereof. An example of an anti- CTLA-4 antibody includes, but is not limited to, Ipilimumab (trade name Yervoy). Other types of immune checkpoint modulators may also be used including, but not limited to, small molecules, siRNA, miRNA, and CRISPR systems. Immune checkpoint modulators may be administered before, after, or concurrently with the modified cell comprising the CAR. In certain embodiments, combination treatment comprising an immune checkpoint modulator may increase the therapeutic efficacy of a therapy comprising a modified cell of the present invention.

[0309] Following administration of the cells, the biological activity of the engineered cell populations in some embodiments is measured, e.g., by any of a number of known methods. Parameters to assess include specific binding of an engineered or natural T cell or other immune cell to antigen, in vivo, e.g.. by imaging, or ex vivo, e.g., by ELISA or flow cytometry. In certain embodiments, the ability of the engineered cells to destroy target cells can be measured using any suitable method known in the art, such as cytotoxicity assays described in, for example, Kochenderfer et al., J. Immunotherapy, 32(7): 689-702 (2009), and Herman et al. J. Immunological Methods, 285(1): 25-40 (2004). In certain embodiments, the biological activity of the cells is measured by assaying expression and / or secretion of one or more cytokines, such as CD 107a, IFNy, IL-2, and TNF. In some aspects the biological activity’ is measured by assessing clinical outcome, such as reduction in tumor burden or load.

[0310] In certain embodiments, the subject is provided a secondary treatment. Secondary' treatments include but are not limited to chemotherapy, radiation, surgery’, and medications.

[0311] In some embodiments, the subject can be administered a conditioning therapy prior to CAR-T or CAR-NK cell therapy. In some embodiments, the conditioning therapy comprises administering an effective amount of cyclophosphamide to the subject. In some embodiments, the conditioning therapy comprises administering an effective amount of fludarabine to the subject. In preferred embodiments, the conditioning therapy comprises administering an effective amount of a combination of cyclophosphamide and fludarabine to the subject. Administration of a conditioning therapy prior to CAR-T or CAR-NK cell therapy may increase the efficacy of the CAR-T or CAR-NK cell therapy. Methods of conditioning patients for T cell therapy are described in U.S. Patent No. 9,855,298. which is incorporated herein by reference in its entirety.

[0312] In some embodiments, a specific dosage regimen of the present disclosure includes a lymphodepletion step prior to the administration of the modified T cells. In an exemplary’ embodiment, the lymphodepletion step includes administration of cyclophosphamide and / or fludarabine.

[0313] In some embodiments, the lymphodepletion step includes administration of cyclophosphamide at a dose of between about 200 mg / m2 / day and about 2000 mg / m2 / day (e.g, 200 mg / m2 / day, 300 mg / m2 / day, or 500 mg / m2 / day). In an exemplary' embodiment, the dose of cyclophosphamide is about 300 mg / m2 / day. In some embodiments, the lymphodepletion step includes administration of fludarabine at a dose of between about 20 mg / m2 / day and about 900 mg / m2 / day (e.g, 20 mg / m2 / day, 25 mg / m2 / day, 30 mg / m2 / day, or 60 mg / m2 / day). In an exemplary embodiment, the dose of fludarabine is about 30 mg / m2 / day.

[0314] In some embodiment, the lymphodepletion step includes administration of cyclophosphamide at a dose of between about 200 mg / m2 / day and about 2000 mg / m2 / day (e.g, 200 mg / m2 / day, 300 mg / m2 / day. or 500 mg / m2 / day), and fludarabine at a dose of between about 20 mg / m2 / day and about 900 mg / m2 / day (e.g.. 20 mg / m2 / day. 25 mg / m2 / day, 30 mg / m2 / day, or 60 mg / m2 / day). In an exemplary embodiment, the lymphodepletion step includes administration of cyclophosphamide at a dose of about 300 mg / m2 / day, and fludarabine at a dose of about 30 mg / m2 / day.

[0315] In an exemplary embodiment, the dosing of cyclophosphamide is 300 mg / m2 / day over three days, and the dosing of fludarabine is 30 mg / m2 / day over three days.

[0316] Dosing of lymphodepletion chemotherapy may be scheduled on Days -6 to -4 (with a -1-day window, i.e., dosing on Days -7 to -5) relative to T cell (e.g., CAR-T, TCR-T, a modified T cell, etc.) infusion on Day 0.

[0317] In an exemplary embodiment, for a subject having cancer, the subject receives lymphodepleting chemotherapy including 300 mg / m2of cyclophosphamide by intravenous infusion 3 days prior to administration of the modified T cells. In an exemplary embodiment, for a subject having cancer, the subject receives lymphodepleting chemotherapy including 300 mg / m2of cyclophosphamide by intravenous infusion for 3 days prior to administration of the modified T cells.

[0318] In an exemplary embodiment, for a subject having cancer, the subject receives lymphodepleting chemotherapy including fludarabine at a dose of between about 20 mg / m2 / day and about 900 mg / m2 / day (e.g, 20 mg / m2 / day, 25 mg / m2 / day, 30 mg / m2 / day, or 60 mg / m2 / day). In an exemplary embodiment, for a subject having cancer, the subject receives lymphodepleting chemotherapy including fludarabine at a dose of 30 mg / m2for 3 days.

[0319] In an exemplary embodiment, for a subject having cancer, the subject receives lymphodepleting chemotherapy including cyclophosphamide at a dose of between about 200 mg / m2 / day and about 2000 mg / m2 / day (e.g, 200 mg / m2 / day, 300 mg / m2 / day, or 500 mg / m2 / day), and fludarabine at a dose of between about 20 mg / m2 / day and about 900 mg / m2 / day (e.g, 20 mg / m2 / day, 25 mg / m2 / day, 30 mg / m2 / day, or 60 mg / m2 / day). In an exemplary embodiment, for a subject having cancer, the subject receives lymphodepleting chemotherapy including cyclophosphamide at a dose of about 300 mg / m2 / day, and fludarabine at a dose of 30 mg / m2for 3 days.

[0320] Cells of the invention can be administered in dosages and routes and at times to be determined in appropriate pre-clinical and clinical experimentation and trials. Cell compositions may be administered multiple times at dosages within these ranges. Administration of the cells of the invention may be combined with other methods useful to treat the desired disease or condition as determined by those of skill in the art.

[0321] It is known in the art that one of the adverse effects following infusion of CAR-T or CAR-NK cells is the onset of immune activation, know n as cytokine release syndrome (CRS). CRS is immune activation resulting in elevated inflammatory cytokines. CRS is a known on-target toxicity, development of which likely correlates with efficacy. Clinical and laboratory measures range from mild CRS (constitutional symptoms and / or grade-2 organ toxicity) to severe CRS (sCRS; grade >3 organ toxicity, aggressive clinical intervention, and / or potentially life threatening). Clinical features include high fever, malaise, fatigue, myalgia, nausea, anorexia, tachycardia / hypotension, capillary leak, cardiac dysfunction, renal impairment, hepatic failure, and disseminated intravascular coagulation. Dramatic elevations of cytokines including interferon-gamma, granulocyte macrophage colony-stimulating factor, IL- 10, and IL-6 have been shown following CAR T-cell infusion. One CRS signature is elevation of cytokines including IL-6 (severe elevation), IFN-gamma, TNF-alpha (moderate), and IL-2 (mild). Elevations in clinically available markers of inflammation including ferritin and C-reactive protein (CRP) have also been observed to correlate with the CRS syndrome. The presence of CRS generally correlates with expansion and progressive immune activation of adoptively transferred cells. It has been demonstrated that the degree of CRS severity is dictated by disease burden at the time of infusion as patients with high tumor burden experience a more sCRS.

[0322] Accordingly, the invention provides for, following the diagnosis of CRS, appropriate CRS management strategies to mitigate the physiological symptoms of uncontrolled inflammation without dampening the antitumor efficacy of the engineered cells (e.g., CAR-T or CAR-NK cells). CRS management strategies are known in the art. For example, systemic corticosteroids may be administered to rapidly reverse symptoms of sCRS (e.g., grade 3 CRS) without compromising initial antitumor response.

[0323] In some embodiments, an anti-IL-6R antibody may be administered. An example of an anti-IL-6R antibody is the Food and Drug Administration-approved monoclonal antibody tocilizumab, also known as atlizumab (marketed as Actemra, or RoActemra). Tocilizumab is a humanized monoclonal antibody against the interleukin-6 receptor (IL-6R). Administration of tocilizumab has demonstrated near-immediate reversal of CRS.

[0324] CRS is generally managed based on the severity of the observed syndrome and interventions are tailored as such. CRS management decisions may be based upon clinical signs and symptoms and response to interventions, not solely on laboratory values alone.

[0325] Mild to moderate cases generally are treated with symptom management with fluid therapy, non-steroidal anti-inflammatory drug (NS AID) and antihistamines as needed for adequate symptom relief. More severe cases include patients with any degree of hemodynamic instability; with any hemodynamic instability, the administration of tocilizumab is recommended. The first-line management of CRS may be tocilizumab, in some embodiments, at the labeled dose of 8 mg / kg IV over 60 minutes (not to exceed 800 mg / dose); tocilizumab can be repeated Q8 hours. If suboptimal response to the first dose of tocilizumab, additional doses of tocilizumab may be considered. Tocilizumab can be administered alone or in combination with corticosteroid therapy. Patients with continued or progressive CRS symptoms, inadequate clinical improvement in 12-18 hours or poor response to tocilizumab. may be treated with high-dose corticosteroid therapy, generally hydrocortisone 100 mg IV or methylprednisolone 1-2 mg / kg. In patients with more severe hemodynamic instability or more severe respiratory' symptoms, patients may be administered high-dose corticosteroid therapy early in the course of the CRS. CRS management guidance may be based on published standards (Lee et al. (2019) Biol Blood Marrow Transplant, doi.org / 10. 1016 / j.bbmt.2018.12.758; Neelapu et al. (2018) Nat Rev Clin Oncology, 15:47; Teachey et al. (2016) Cancer Dis cov, 6(6):664-679).

[0326] Features consistent with Macrophage Activation Syndrome (MAS) or Hemophagocytic lymphohistiocytosis (HLH) have been observed in patients treated with CAR-T therapy (Henter, 2007), coincident with clinical manifestations of the CRS. MAS appears to be a reaction to immune activation that occurs from the CRS, and should therefore be considered a manifestation of CRS. MAS is similar to HLH (also a reaction to immune stimulation). The clinical syndrome of MAS is characterized by high grade non-remitting fever, cytopenias affecting at least two of three lineages, and hepatosplenomegaly. It is associated with high serum ferritin, soluble interleukin-2 receptor, and triglycerides, and a decrease of circulating natural killer (NK) activity.

[0327] Pharmaceutical and Formulations Also provided are populations of immune cells of the invention, compositions containing such cells and / or enriched for such cells, such as in which cells expressing the recombinant receptor make up at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more of the total cells in the composition or cells of a certain ty pe such as T cells or CD8+ or CD4+ cells. Among the compositions are pharmaceutical compositions and formulations for administration, such as for adoptive cell therapy. Also provided are therapeutic methods for administering the cells and compositions to subjects, e.g, patients.

[0328] Also provided are compositions including the cells for administration, including pharmaceutical compositions and formulations, such as unit dose form compositions including the number of cells for administration in a given dose or fraction thereof. The pharmaceutical compositions and formulations generally include one or more optional pharmaceutically acceptable carrier or excipient. In some embodiments, the composition includes at least one additional therapeutic agent.

[0329] The term "pharmaceutical formulation" refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered. A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to. a buffer, excipient, stabilizer, or preservative. In some aspects, the choice of carrier is determined in part by the particular cell and / or by the method of administration. Accordingly, there are a variety' of suitable formulations. For example, the pharmaceutical composition can contain preservatives. Suitable preservatives may include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. In some aspects, a mixture of two or more preservatives is used. The preservative or mixtures thereof are typically present in an amount of about 0.0001% to about 2% by weight of the total composition. Carriers are described, e.g. by Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980). Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3- pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn- protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG).

[0330] Buffering agents in some aspects are included in the compositions. Suitable buffering agents include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. In some aspects, a mixture of two or more buffering agents is used. The buffering agent or mixtures thereof are typically present in an amount of about 0.001% to about 4% by weight of the total composition. Methods for preparing administrable pharmaceutical compositions are known. Exemplary methods are described in more detail in, for example, Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins; 21st ed. (May 1, 2005).

[0331] The formulations can include aqueous solutions. The formulation or composition may also contain more than one active ingredient useful for the particular indication, disease, or condition being treated with the cells, preferably those with activities complementary to the cells, where the respective activities do not adversely affect one another. Such active ingredients are suitably present in combination in amounts that are effective for the purpose intended. Thus, in some embodiments, the pharmaceutical composition further includes other pharmaceutically active agents or drugs, such as chemotherapeutic agents, e.g., asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, and / or vincristine. The pharmaceutical composition in some embodiments contains the cells in amounts effective to treat or prevent the disease or condition, such as a therapeutically effective or prophylactically effective amount. Therapeutic or prophylactic efficacy in some embodiments is monitored by periodic assessment of treated subjects. The desired dosage can be delivered by a single bolus administration of the cells, by multiple bolus administrations of the cells, or by continuous infusion administration of the cells.

[0332] Formulations include those for oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration. In some embodiments, the cell populations are administered parenterally. The term "parenteral," as used herein, includes intravenous, intramuscular, subcutaneous, rectal, vaginal, and intraperitoneal administration. In some embodiments, the cells are administered to the subject using peripheral systemic delivery by intravenous, intraperitoneal, or subcutaneous injection. Compositions in some embodiments are provided as sterile liquid preparations, e.g., isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which may in some aspects be buffered to a selected pH. Liquid preparations are normally easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are somewhat more convenient to administer, especially by injection. Viscous compositions, on the other hand, can be formulated within the appropriate viscosity range to provide longer contact periods with specific tissues. Liquid or viscous compositions can comprise carriers, which can be a solvent or dispersing medium containing, for example, water, saline, phosphate buffered saline, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol) and suitable mixtures thereof.

[0333] Sterile injectable solutions can be prepared by incorporating the cells in a solvent, such as in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, dextrose, or the like. The compositions can contain auxiliary substances such as wetting, dispersing, or emulsifying agents (e.g. methylcellulose). pH buffering agents, gelling or viscosity’ enhancing additives, preservatives, flavoring agents, and / or colors, depending upon the route of administration and the preparation desired. Standard texts may in some aspects be consulted to prepare suitable preparations.

[0334] Various additives which enhance the stability and sterility of the compositions, including antimicrobial preservatives, antioxidants, chelating agents, and buffers, can be added. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, and sorbic acid. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0335] The formulations to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, e.g., by filtration through sterile filtration membranes.

[0336] The contents of the articles, patents, and patent applications, and all other documents and electronically available information mentioned or cited herein, are hereby incorporated by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. Applicants reserve the right to physically incorporate into this application any and all materials and information from any such articles, patents, patent applications, or other physical and electronic documents. While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made, and equivalents may be substituted without departing from the true spirit and scope of the invention. It will be readily apparent to those skilled in the art that other suitable modifications and adaptations of the methods described herein may be made using suitable equivalents without departing from the scope of the embodiments disclosed herein. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit, and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto. Having now described certain embodiments in detail, the same will be more clearly understood by reference to the following examples, which are included for purposes of illustration only and are not intended to be limiting.

[0337] EXPERIMENTAL EXAMPLES

[0338] The invention is now described with reference to the following Examples. These Examples are provided for the purpose of illustration only, and the invention is not limited to these Examples, but rather encompasses all variations that are evident as a result of the teachings provided herein.

[0339] Example 1: DR5 CAR-T cells are highly cytotoxic to DR5+ tumor cells

[0340] To evaluate whether elevated DR5 expression is associated with solid tumors, DR5 expression in the TCGA database was analyzed. Additionally, in situ hybridization and immunohistochemistry of DR5 were performed on clinical melanoma tissues. The results demonstrated that DR5 was highly expressed in different tumor types, including melanoma (FIG. 1 A). Furthermore, DR5 mRNA and protein were highly expressed in clinical melanoma samples (FIG. IB).

[0341] Protein sequences of four human DR5-scFvs w ere reverse-translated into DNA sequences, codon-optimized, and cloned into pTRPE lentiviral vectors (FIGs. 2A, 3A) and confirmed by Sanger sequencing. T cells from freshly collected human peripheral blood mononuclear cells (PBMC) were expanded and transfected with lentiviruses carrying four different DR5-scFv CAR constructs (l#-4#). After magnetic bead sorting, most of the T cells expressed DR5-scFvs (FIG. 2B). Melanoma (A375), ovarian cancer (OVCAR5), and hepatocellular carcinoma (HepG2) expressed similar levels of DR5 (FIG. 2C). DR5 CAR-T cells were highly cytotoxic to these DR5+ tumor cells (FIG. 2D). In addition, DR5 CAR-T cells 1# and 3# showed more cytotoxicity to tumor cells than the other two DR5 CAR-T cell clones in vitro.

[0342] To study the treatment efficacy of DR5 CAR-T cells against melanoma cells in 3D cultures, bicellular spheroids composed of GFP+ A375 cells and BJ fibroblasts were cocultured with DR5 CAR-T cells at an E:T ratio of 1 : 1. Cells in the spheroids were stained with Hoechst33342 and GPF intensity was analyzed (FIGs. 2E-2F). Untransduced (UTD) T cells and CD 19 CAR-T cells were included as controls. The results showed that DR5 CAR-T cells effectively killed DR5+ melanoma cells in 3D spheroids.

[0343] To further evaluate the activation, function, and target cell death of DR5 CAR T cells, reverse-phase protein arrays (RPPA) was performed to analyze CAR T cell activation after coculture with A375 target cells (FIGs. 2H, 3B). Representative proteins are shown in FIG. 2G. The data indicated that DR5 CAR T cells were activated upon incubation with target cells, whereas UTD cells remained in a resting state. Proteins associated with cell death and apoptosis were highly expressed in the target cells cocultured with DR5 CAR T cells, compared to those cocultured with UTD and target cells alone (FIGs. 2H). These findings demonstrate that DR5 CAR T cells are effective in targeting DR5 expressing tumor cells in both 2D and 3D models.

[0344] Example 2: DR5 CAR-T cells are cytotoxic to MDSCs in vitro

[0345] DR5 is highly expressed in myeloid-derived suppressor cells (MDSCs)(Condamine, 2014). To investigate the effects of DR5 CAR-T cells on MDSCs, MDSCs from PBMC- monocytes were generated and their phenotype was confirmed by flow cytometry', which showed a typical CD1 lb+CD33+PDLlhlexpression pattern (FIG. 19A). DR5 CAR-T cells were then cocultured with MDSCs and T cell proliferation was assessed by CFSE staining. MDSCs were found to significantly suppress T cell proliferation compared to T cells alone (FIG. 19B). Next, DR5 expression was evaluated on the surface of MDSCs by flow cytometry. The results of this analysis showed higher expression levels of DR5 in MDSCs than the A375 melanoma cells (FIG. 4C). To evaluate the cytotoxicity of DR5 CAR-T cells against MDSCs, LDH-releasing assays were performed after 16 hours of CAR-T cell-MDSC coculture. The results showed that selected 3# DR5 CAR-T cells were significantly cytotoxic to MDSCs in vitro (FIG. 4D). Next, the treatment efficacy of DR5 CAR-T cells against 3D spheroids composed of GFP+ A375 cells and MDSCs at an E:T ratio of 1: 1 was examined. The spheroids were stained with Hoechst33342 and PI and imaged using a fluorescent microscope (FIG. 4E). The results showed that DR5 CAR-T cells effectively induced lysis of cells in the spheroids based on overall PI staining (FIG. 4F). UTD-T cells or CD 19 CAR-T cells were included as controls. These results demonstrated that DR5 CAR-T cells are cytotoxic to tumor cells in the presence of MDSCs.

[0346] Example 3: Cell and EV membrane-anchored DR5 CAR-scFvs effectively induce target cell lysis

[0347] Since agonistic DR5 antibodies can directly activate the DR5 pathway and induce target cell apoptosis (Y uan, 2019; Snajdauf, 2021), it was of interest to see whether DR5- scFvs can directly induce target cell death without T cell activation. SupTl cell is a T lymphoblast cell line that is not cytotoxic to other cells. Stable DR5 CAR-expressing SupTl cells were generated and expression of DR5 CARs on the cell surface was confirmed (FIG. 5 A). SupTl cells are not cytotoxic and do not express cytotoxic T cell functional molecules, such as granzy me B, perforin and IFN-y (FIG. 5B) or the target antigen DR5 (FIG. 6A). The cytotoxicity of DR5 CAR-SupTl cells expressing varying levels of DR5 on melanoma cells was examined (FIG. 6B). DR5 CAR-SupTl cells effectively killed these melanoma cells in a dose- and DR5 expression level-dependent manner (FIG. 5C). Hoechst33342 / PI double staining assays confirmed the tumor killing by DR5 CAR-SupTl cells (FIGs. 5D-E). The effect of DR5-CAR SupTl cells on non-adherent K562-CD19 lymphoblast cells, which express a lower level of DR5 than A375 melanoma cells was further examined (FIG. 6A). The DR5-CAR SupTl cells were found to induce cell apoptosis and death of the K562-CD19 lymphoblast cells (FIGs. 6C-6D). In addition, SupTl cells were transfected with CD19- CAR. How ever, the CD19-CAR SupTl cells were not cytotoxic to K562-CD19 cells (FIGs. 6C-6D). These data indicate that membrane DR5 scFvs induce DR5 activation in target cells.

[0348] Extracellular vesicles (EVs) are nanosized lipid bilayer particles that are naturally released from virtually all cell types (Weber, 2018). Based on their size and isolation methods, EVs are further divided into small extracellular vesicles (sEVs) and microvesicles (MVs) (Herman, 2021). To see whether sEVs and MVs from DR5-CAR-SupTl are cytotoxic to A375 melanoma cells, CD63 -containing sEVs having a size of -100 nm were enriched (FIGs. 5F-G) and tested for their ability to induce A375 melanoma cell death. The ability of both sEVs and MVs (>200nm) from DR5-CAR-SupTl cells to effectively kill A375 cells was confirmed (FIGs. 5H, 6E). These findings are consistent with membrane DR5 CAR-scFvs inducing target cell death by directly activating DR5 pathways independent of T-cell activation. Example 4: DR5-scFv affinity to DR5 correlates with its target-killing efficacy

[0349] CAR affinity to its target is crucial for CAR-T cell function and CAR-T cell-induced adverse reactions (Flugel, 2023; Olson, 2022; Duan, 2022). It was observed that the viability of DR5 CAR-T cells following transfection with different clones of DR5-scFv CAR lentiviruses was variable in the absence of target cells 72 hours after transduction or incubation with A375 cells for 4 hours, and the 1# clone had the least viable cells (FIGs. 7A- B). To select the best CAR for further development, the affinity of all four DR5 scFvs to DR5 was analyzed using in silico computational methods (FIGs. 8A-8B). The DR5 scFvs appear to bind to slightly different areas of DR5, however, the HADDOCK Scores of DR5- scFvs to DR5 binding affinity were not statistically (FIGs. 9A-9B). and 3# appeared to have lower affinity than other clones (FIGs. 9A-B).

[0350] To study the binding affinity experimentally, four different DR5 whole-antibodies and their corresponding DR5-scFv-antibodies were constructed (FIG. 8C). Then, the binding capacity of these DR5 antibodies to pre-fixed A375 cells was evaluated using On-Cell Western assays (FIG. 8D). The normalized data of DR5 whole-antibody and DR5-scFv- antibody-binding to A375 cells is shown in FIG. 8E. The DR5 antibodies were found to bind to their target with different affinities. Clone 1# DR5-scFv-antibodies showed the highest binding affinity to DR5 on the A375 cells, followed by clone 3#, which was different from the results generated by computational analysis. Flow cytometry data further demonstrated that DR5 whole-antibodies and DR5-scFv-antibodies similarly bound to A375 cells, consistent with the On-Cell Western assay results (FIGs. 8F and 17). Next, the effects of CAR expression on T cell proliferation was tested. The viability of cultured DR5 CAR-T cells was measured, and clone 1# had the least viable cells (FIG. 18 A). Similar results were observed after DR5 CAR-T cells were cocultured with A375 cells for 24 hours (FIG. 18B). Therefore, clone 3# CAR was selected for further development in the in vivo studies.

[0351] Example 5: DR5 CAR-T cells lack off-target toxicity to DR5-cells

[0352] To evaluate the specificity and off-target toxicity of DR5 CAR-T cells, their cytotoxicity was tested against DR5" HaCaT keratinocyte cells (FIG. 10A). None of the four DR5 CAR-T cell clones were found to be cytotoxic to HaCaT cells (FIG. 10B). Next, DR5 overexpressing (OE) or knockout (KO) A375 melanoma cells were generated. Flow cytometry and western blot analyses confirmed the expression of DR5 in these cells, respectively (FIGs. 10C-D). Luciferase-releasing assays showed that neither DR5 CAR-T cells nor EVs from CAR-T cells showed significant toxicity to DR5K0cells (FIGs. 10E-F). Similarly, surface-anchored DR5-scFvs on SupTl cells also demonstrated no significant cytotoxic effects on DR5K0A375 cells (FIG. 10G). These results show that DR5 CAR-T cells are target-specific and do not exhibit off-target toxicity to DR5" cells.

[0353] Example 6: DR5 CAR-T cells inhibit melanoma growth in vivo without significant adverse effects

[0354] To investigate the effect of DR5 CAR-T cells in vivo, an A375 melanoma xenograft model was established by injecting 5xl06A375-DR5OEwith GFP and FFLuc melanoma cells into the flanks of nude mice. The treatment schema is shown in FIG. 11 A. Mice were treated with intratumoral injection of untransduced (UTD) T cells or DR5 CAR-T cells. Tumor bioluminescence images and Day 20 bright field tumor images are shown in FIG. 1 IB. The tumor bioluminescence kinetics of A375 tumor growth in the xenograft model is shown in FIG. 11C. The results indicated a significant reduction of tumor burden after DR5 CAR-T cell treatment compared to the UTD T cell control group. Moreover, no apparent systemic toxicity was observed as evidenced by the treated mice showing no significant body weight loss (FIG. 1 ID). Histological analysis of the tumor samples collected on day 20 showed no signs of off-target toxicity as evidenced by the absence of any pathological changes in the major organs of the mice. To further confirm the effect of DR5 CAR-T cells in vivo. WT A375 melanoma cells were treated with intratumoral UTD T cells or DR5 CAR- T cells. Tumor growth was significantly reduced and mouse survival was prolonged significantly (FIGs. 1 IE-11G). No apparent systemic toxicity w as observed in the major organs in the treated mice by histology. These results indicated that DR5 CAR-T cells can control melanoma tumor growth in vivo. AR-T cells infiltrate and kill tumor cells in -derived and melanoma slice cultures

[0355] To evaluate the DR5 CAR-T cell efficacy against melanoma in patients’ tissues, fresh viable human melanoma tissues were obtained from surgically resected specimens, and the tumor tissues were sectioned into small fragments and cultured to form melanoma patient- derived organoids (MPDOs). MPDOs w ere cultured with a mixture of labeled untransduced T cells (pink. CellTrace Far Red) and DR5 CAR-T cells (green. CFSE). Propidium iodide (PI) staining was used to demonstrate cell death (FIG. 12A). The live cell imaging studies demonstrated that few dead cells (red, PI+) were present in the MPDOs at the beginning of the experiments. Killing of the cells in the organoids was relatively fast, and by 120 minutes, many dead cells in the MPDOs were stained positive for PI (FIG. 12 A). Confocal images of MPDOs cocultured with DR5 CAR-T cells for 24 hours demonstrated that more infiltrating DR5 CAR-T cells and Pl-positive dead cells were present in the MPDOs than MPDOs treated with untransduced (UTD) T-cells (FIG. 12B). MPDOs from three different patients were tested. CFSE-labeled DR5-CAR-T cells penetrated deep into the organoids, while UTD T- cells were mostly attached to the surface of the organoids.

[0356] Imaging analysis was performed to quantify the fluorescence density in MPDOs and confirmed observation (FIG. 12C). After coculturing the MPDOs with DR5 CAR-T cells for 48 hours, the organoids disintegrated as the organoids lost sharp tissue edges; the disintegration was not observed in the untreated MPDOs or MPDOs treated with UTD T- cells (FIG. 13).

[0357] The effect of DR5 CAR-T cells on patient-derived organotypic melanoma slice cultures (MSCs) was further tested. MSCs represent a physiologically relevant culture system that preserves the original TME (Kenerson, 2020, 2021 ; Dimou, 2022; Sivakumar, 2019) and is useful for predicting responses to small molecules (28) and checkpoint inhibitors (Voabil, 2021). 300 pm thick fresh melanoma slices from 5 patients were incubated with vehicle, untransduced T-cells, and DR5 CAR-T cells (2xl06) for 48 hours. Since spontaneous melanoma necrosis is common, some tumor slices contain necrotic areas. DR5 CAR-T cell treatment was found to significantly increase the number of dead cells compared to MSCs treated with untransduced T cells (FIG. 12D). MSCs were washed and lysed to form the single-cell suspension for FACS analysis. MSCs incubated with untransduced T cells had a similar number of CD3+ T cells in the tissue. In contrast, MSCs incubated with DR5 CAR-T cells had significantly more CD3+ T cells in the tissue, which is indicative of more tumor-infiltrating DR5 CAR-T cells in the tissues. Significantly more CD8+Granzyme B+ and CD8+Ki67+ T cells were also present in the tissues after DR5 CAR-T cell treatment (FIG. 12D). These data support the tumor-infiltrating and killing ability7of DR5 CAR-T cells in the patient-derived melanoma organoid models.

[0358] MPDOs embedded in the Matrigel were treated with far-red-labeled DR5-CAR-T cells or UTD T cells for 48 hours. MPDOs were collected and dissociated into a single-cell suspension. FACS analysis showed that 3-fold more far-red+CD3+ CAR-T cells than far red+CD3+ UTD T cells were detected in the MPDOs (FIG. 12E). consistent with the imaging analysis data (FIG. 12C). Significantly more dead cells were present in the MPDOs after DR5 CAR-T cell treatment (FIG. 12F). CD1 lb+ myeloid cells (FIG. 12G), CD1 lb+CD33+ MDSCs (FIG. 12H), CD14+CD1 lb+CD33+ M-MDSCs (FIG. 121), and CD15+CD1 lb+CD33+ PMN-MDSCs (FIG. 25) were significantly decreased after DR5 CAR-T cell treatment compared to UTD T cell treatment. On the contrary, tumor-resident far-red-CD8+ T cells (FIG. 12J) were significantly increased, and these far-red-CD8+ T cells expressed significantly more granzyme B (FIG. 12K) and Ki67 (FIG. 12L), indicating activation of tumor-resident CD8+ T cells after DR5 CAR-T cell treatment. To further confirm the effect of DR5 CAR-T cells, we also tested their effects in MSCs. MSCs represent a physiologically relevant culture system that preserves the original TME (Kenerson, 2020, 2021; Dimou, 2022; Sivakumar 2019), demonstrating utility in predicting responses to small molecules (Kenerson, 2020) and checkpoint inhibitors (Voabil, 2021). 300 pm thick fresh melanoma slices from 5 patients were incubated with UTD T-cells and DR5 CAR-T cells (2xl06) for 48 hours. DR5 CAR-T cells significantly increased the PI+ dead cells compared to MSCs treated with UTD T cells (FIG. 12M). MSCs were washed and lysed to form a single-cell suspension for FACS analysis. MSCs incubated with DR5 CAR-T cells had a significantly higher number of CD3+ T cells in the tissue than tissues treated with UTD T cells, supporting more tumor-infiltrating DR5 CAR-T cells in the tissues (FIG. 12N). These data suggest that DR5 CAR-T cells can infiltrate patient-derived melanoma tissues, inhibit the MDSCs, and activate tumor-resident CD8+ T cells.

[0359] Example 8; DR5 CAR-scFvs on cell and EV surfaces effectively induce target cell lysis

[0360] Since agonistic DR5 antibodies can directly activate the DR5 pathway and induce target cell apoptosis (Y uan, 2018; Snajdauf, 2021), it was of interest to determine whether the DR5-scFvs can directly induce target cell death without the need for T cell activation. SupTl cell is a T lymphoblast cell line that is not cytotoxic to other cells. Stable DR5 CAR- expressing SupTl cells were generated and their expression of DR5 CARs on the cell surface was confirmed (FIG. 14 A). SupTl cells were not cytotoxic and did not express cytotoxic T cell functional molecules, such as granzy me B, perforin and IFN-y (FIG. 14B) or the target antigen DR5 (FIG. 6A). The cytotoxic effect of DR5 CAR-SupTl cells on melanoma cells was evaluated with various levels of DR5 expression (FIG. 6B). DR5 CAR-SupTl cells effectively killed these melanoma cells in a dose- and DR5 expression level-dependent manners (FIG. 14C). Hoechst33342 / PI double staining assays were performed and the results confirmed the tumor-killing by DR5 CAR-SupTl cells (FIGs. 5D-5E). The effect of DRSCAR SupTl cells was also tested on non-adherent lymphoblast cell K562-CD19 cells, which express a lower level of DR5 than A375 (FIG. 6A), DR5-CAR SupTl cells induced K562- CD19 cell apoptosis and death (FIGs. 6C-6D). Additionally, SupTl cells were transfected with CD19-CAR. In contrast, the CD19-CAR SupTl cells did not show similar cytotoxic effects to K562-CD19 cells (FIGs. 6C-D). These data show that membrane DR5 scFvs induce DR5 activation in target cells.

[0361] Extracellular vesicles (EVs) are nanosized lipid bilayer particles that are naturally released from almost all types of cells (Herrmann. 2021). Based on size and isolation methods. EVs are further divided into small extracellular vesicles (sEVs) and microvesicles (MVs)(Nederveen, 2021). The cytotoxicity of sEVs and MVs derived from DR5-CAR-SupTl cells on melanoma cells w as investigated. sEVs were enriched in CD63 and had a size of -100 nm (FIGs. 5F-5G). These sEVs effectively induced A375 melanoma cell death (FIG. 5H). Microvesicles (MV) (>200nm) from DR5-CAR-SupTl cells also had similar effects (FIG. 15). These findings support that membrane DR5 CAR-scFvs can induce target cell death by directly activating DR5 pathways, independent of T-cell activation

[0362] Example 9: DR5 CAR-T cells are not cytotoxic to DR5 cells

[0363] To evaluate the specificity and off-target toxicity of DR5 CAR-T cells, their cytotoxicity to DR5" HaCaT keratinocyte cells was examined (FIG. 16A). The results demonstrated that all four DR5 CAR-T cells w ere not cytotoxic to HaCaT cells (FIG. 16B). Next. DR5 overexpression (OE) or knockout (KO) A375 melanoma cells w ere generated. Flow cytometry and western blot confirmed the expression of DR5 in these cells, respectively (FIGs. 16C-16D). Luciferase-releasing assays demonstrated that neither DR5 CAR-T cells nor EVs derived from CAR-T cells exhibited significant toxicity to DR5KOcells (FIGs. 16E- 16F). Furthermore, surface-anchored DR5-scFvs on SupTl cells demonstrated no significant cytotoxic effect on DR5KOA375 cells (FIG. 16G). These results support that DR5 CAR-T cells are target-specific and do not exhibit off-target toxicity to DR5' cells.

[0364] Example 10: DR5 CAR-T cells are cytotoxic to MDSCs in vitro

[0365] DR5 is highly expressed in MDSCs (Condamine, 2014). To investigate the effects of DR5 CAR-T cells on MDSCs. MDSCs from PBMC-monocytes were generated and their phenotype was confirmed by flow cytometry, which showed typical CDl lb+CD33+PDLlhlexpression (FIG. 19A). DR5 CAR-T cells were then co-cultured with MDSCs and T cell proliferation was assessed by CFSE staining. MDSCs significantly suppressed T cell proliferation compared to T cells alone (FIG. 19B). Next. DR5 expression on the surface of MDSCs was evaluated by flow cytometry. MDSCs expressed a higher level of DR5 than the A375 melanoma cells (FIG. 8G). To evaluate the cytotoxicity of DR5 CAR-T cells against MDSCs, LDH-releasing assays were performed after 16 hours of CAR-T-cells and MDSC coculture. The results showed that selected 3# DR5 CAR-T cells were significantly cytotoxic to MDSCs in vitro (FIG. 15H). Next, the treatment efficacy of DR5 CAR-T cells against 3D spheroids composed of GFP+A375 cells and MDSCs was examined at an E:T ratio of 1:1. The spheroids were stained with Hoechst33342 and PI and imaged using a fluorescent microscope (FIG. 81). The results showed that the DR5 CAR-T cells effectively induced lysis of target A375 cells in the spheroids based on overall GFP and PI staining (FIGs. 8I-8J). UTD-T cells or CD 19 CAR-T cells were included as controls. These results demonstrate that DR5 CAR-T cells are cytotoxic to tumor cells in the presence of MDSCs.

[0366] Example 11: DR5 CAR-T cells are efficacious and safe in the syngeneic melanoma models

[0367] Since DR5 may be expressed in some normal tissues, a CAR was designed against murine DR5. Murine CAR-T cells allowed for an evaluation of the safety and efficacy in an immunocompetent setting. The murine CAR construct was designed based on the M5-1 monoclonal antibody (Takeda, 2004) and was engineered with traceable BFP and nLuc marker expression as previously described (Zhang, 2024). Murine DR5 CAR-T cells were manufactured using mouse splenocytes (FIG. 20A), and 31.4% of the CAR-T cells showed BFP expression (FIG. 21). When these murine DR5 CAR cells were injected into healthy C57 mice via the tail veins, these mice maintained their body weight (FIG. 22) and did not die during treatment.

[0368] Next, DR5 expression was examined in several mouse melanoma cell lines. The results of this analysis showed that over 90% of Yumml.7 and BRAFV600EpTEN' ' cells expressed DR5 and 59% of B16-F10 cells expressed DR5 (FIG. 20B). Murine DR5 CAR-T cells induced significant cytotoxicity in Yumml.7 and BRAFV600EpTEN' / ' cells even at E:T ratio of 0.6:1 after coculturing 12 hours (FIG. 20C) and 24 hours (FIGs. 23A-23C). To further evaluate toxicity and the in vivo efficacy, Yumm 1.7 syngeneic melanomas were established in C57 mice. When the tumors became palpable, murine DR5 CAR cells were injected once through the tail veins. Systemic CAR-T cells led to significant tumor grow th reduction and prolonged survival compared to UTD treated mice (FIGs. 20D-20F). No weight loss (FIG. 20F) or histological changes were detected in the major organs. Flowcytometry confirmed the presence of circulating CAR-T cells after sacrificing the mice at the end of the experiment, indicating persistence of CAR-T cells following infusion (FIGs. 20G- 20H). These findings support the safety and therapeutic potential of DR5 -targeted CAR-T cells in an intact immune system.

[0369] Example 12: DR5 CAR enhances the cytotoxicity of T cells against A375 melanoma cells

[0370] A375 cells were co-cultured with unmodified y5 T cells or DR5 CAR-modified y8 T cells at effector-to-target (E: T) ratios of 2.5: 1 or 5: 1 for 12. 24. or 48 hours. Cytotoxicity was measured by flow cytometry. As shown in FIGs. 25A-25F, yo T cells were cytotoxic to A375 melanoma cells and their effects were significantly increased following transfection with DR5 CAR.

[0371] Example 13: DR5 CAR enhances the killing ability of vST cells in melanoma patient derived tissue slices

[0372] Melanoma patient-derived tumor tissue slices were treated with different cells for 48 hours as indicated in FIGs. 26A-26D (n=12 per group, 1 million y8T cells for each 11x8x5 mm3). As shown in these figures, while untransduced y5 T cells were cytotoxic to cells in the melanoma tissue slices, DR5 CAR y8 T cells (GDT-94car) were significantly more cytotoxic than untransduced y5 T cells (FIG. 26A) (FIG. 26B), Granzyme B (FIG. 26C), and perforin (FIG. 26D), supporting the reinvigoration of the tumor-resident T cells.

[0373] Example 14: DR5 CAR enhances the killing ability of yST cells in melanoma patient- derived organoids (MPDOs)

[0374] Melanoma patient-derived organoids (MPDOs) were generated using fresh melanoma tissues and then treated with different cells for 48 hours (n=12 per group) as indicated in FIGs. 27-27F. As shown in these figures, while untransduced y8 T cells were cytotoxic to cells in MPDOs, DR5 CAR y8 T cells (GDT-94car) were significantly more cytotoxic to these cells than untransduced y8 T cells (FIG. 27A). Moreover, following CAR-T cell treatment, there were more CD45+ immune cells (FIG. 27B) and CD3+ T cells (FIG. 27C), and the tumor-resident CD8+ T cells expressed more Ki-67 (FIG. 27D), Granzyme B (FIG. 27E), and perforin (FIG. 27F), supporting the reinvigoration of the tumor-resident T cells.

[0375] Example 15: DR5 CAR enhances the killing ability of y6T cells to myeloid derived suppressor cells (MDSCs) in MPDOs MPDOs in mice were treated with different cells for 48 hours as indicated in FIGs. 28A-28D. As shown in these figures, while untransduced y5 T cells were cytotoxic to cells in MPDOs, DR5 CAR y5 T cells (GDT-94car) were significantly more cytotoxic to cells in the MPDOs than untransduced y5 T cells (FIG. 28A). After CAR-T cell treatment, there were significantly less DR5+ cells (FIG. 28B) and MDSCs (FIG. 28D) in the MPDOs, and more CD45+ immune cells in the MPDOs, consistent with the killing of MDSCs in the melanoma tissues.

[0376] Example 16; DR5 CAR

[0377] A375 melanoma tumor xenografts in mice were treated for 20 days with untransduced (UTD) aP T cells or DR5 CAR aP T and DR5 CAR y5 T cells. The bioluminescence (FIG. 29A) and tumor volumes (FIG. 29B) associated with the treatment over the course of the 20 day period is shown. As reflected in FIGs. 29A-29D, while UTD aP T cells were cytotoxic to the A375 melanoma cells in the xenografts, DR5 CAR aP T and y5 T cells were significantly more cytotoxic than the UTD aP T cells lin addition, the treatment was safe in mice as these treated mice did not lose their body weight (FIG, 29E).

[0378] Example 17 ; DR5 CAR yS T cells inhibited melanoma PDX growth in vivo

[0379] DR5 CAR y5 T cells were found to significantly inhibit melanoma patient-derived xenografts in mice as compared to untransduced y5 T cells (gdT) or vehicle as quantified by tumor size measurements (FIG. 30A). Further, as shown in FIG. 30B, the treatment prolonged the lifespan of the treated mice. Untransduced y5 T cells (gdT) also had some effects but the effects were less than the CAR y5 T cells.

[0380] Example 18; Effects of DR5 CAR-yS T cells on glioblastoma multiforme (GBM)

[0381] 20,000 cells from each of tw o glioblastoma multiforme (GBM) cell lines, U251(A) and U87(B) GBM cells were separately treated for 12-24 hours with DR5 CAR-gdT cells or untransduced gdT cells at E:T ratios of 5, 2.5, 1.25, 0.625 as indicated in FIGs. 31A-31B. The results of this expenment showed that while y5 T cells were cytotoxic to the GBM cell lines, their cytopathic effects were significantly enhanced by transfection with DR5 CAR (FIGs. 31A-31B).

[0382] Example 13. Materials and methods Human DR5 CAR vector construction

[0383] Four anti-human-DR5 antibody sequences were obtained from the International Immunogenetics Information System (IMGT, mAB-DB ID 183, 224, 234, and 348). The amino acid sequences of humanized DR5 scFvs were converted to cDNAs and custom synthesized by Integrated DNA Technologies, Inc. (CA, USA). The DNA fragments of the variable domain of heavy’ chain (VH) and light chain (VL) were fused and linked with a - (G4S)3- linker and flanked by a human-CD8 leader. These cDNAs were then cloned into an engineered pTRPE CAR encoding lentiviral backbone, which contains the EFla promoter, CD8a leader, CD8a hinge extracellular domain, and transmembrane domain, followed by 4- 1BB and CD3^ endodomains, as previously described (Milone, 2009; Richman, 2018).

[0384] Lentivirus packaging

[0385] CAR lentiviral vector packaging was carried out using HEK293T cells (Richman, 2018). A transfection mixture was prepared by combining 10 pg of the lentiviral vector plasmid, 7.5 pg of pMDLg / pRRE, 2.5 pg of pRSV-Rev, and 2.5 pg of pCMV-VSVG in 500 pL of serum-free RPMI medium. Premixed lipofectamine 2000 was added to the mixture and incubated for 20 minutes at room temperature. The transfection mix was added to the cells and incubated for 6-8 hours at 37°C in a 5% CO2 incubator. Half volume of full RPMI- 1640 medium containing 10% FBS was added after transfection. After 24 and 48 hours, lentivirus-containing supernatant was collected, filtered, and concentrated overnight by centrifugation at 12,000 rpm. The final lentivirus particles were aliquoted and can be stored at -80°C until needed.

[0386] Primary cells and cell lines

[0387] Human peripheral blood mononuclear cells (PBMCs) and monocytes from healthy donors were obtained from the Human Immunology7Core at the Perelman School of Medicine at the University7of Pennsylvania (Wang, 2021). Human melanoma cell lines A375, A2058, WM9, and 903 were obtained from Meenhard Herlyn's laboratory (The Wistar Institute, Philadelphia, Pennsylvania, USA), and they were routinely tested for mycoplasma and DNA fingerprinted (Liu, 2018). Other cells were from ATCC.

[0388] T cell expansion and CAR-T cell transduction

[0389] T-cell expansion was started from PBMCs using the method described (Richman, 2018; Hu, 2017). PBMCs were cultured with CD3 / 28 Dynabeads at a 1: 1 cell-to-bead ratio in RPMI1640 media supplemented with 10% FBS (HyClone; GE Healthcare, Utah, USA), 100 U / mL penicillin-streptomycin, 2 mM L-glutamine. 1 / 1000 2-mercaptoethanol (2-Me) (Gibco; Thermo Fisher Scientific, Massachusetts, USA) and 100 units / mL of recombinant human interleukin (IL)-2 (PeproTech, New Jersey, USA), new media added and supplemented every other day. The cells were incubated at 37°C with 5% CO2 for 24 hours before transduction with CAR lentiviral vectors at MOI=1. The medium and IL2 were replenished even' 24-48 hours as required. After 7 days, the activated T cells were harvested for further purification, analysis, and downstream applications.

[0390] MDSC culture

[0391] Isolated healthy donor PBMCs were used to culture MDSCs, with fresh cells being used every time. The cells were plated at a density of 1-2 x 10A6 cells / mL in RPMI-1640 full medium and supplemented with 50 ng / ml of human GM-CSF and 20 ng / ml of human IL-6 in a T25 flask. Fresh culture medium was added every other day. After 7-10 days of culture, adherent cells were harvested and analyzed by flow cytometry to confirm MDSC phenotype, demonstrating that the cells were CD1 lb+CD33+ with other checkpoint markers (Bronte, 2016). DR5 expression was assessed and compared to that of melanoma A375 cells using flow cytometry'. This method was reliable and efficient for generating MDSCs for further experimentation.

[0392] Melanoma xenografts

[0393] Animal procedures in this study were approved by the Institutional Animal Care and Use Committees at the University of Pennsylvania (804874)(Liu. 2018). All mice were bred and housed in a pathogen-free facility under controlled temperature (22 °C) and lighting (12: 12 h light-dark cycle) and were fed a chow diet. Melanoma xenografts were established by injecting a single-cell suspension of A375 cells (2 million in 200 pL PBS) subcutaneously into the left flank of each nude mouse. Female nude mice from Jackson Laboratories were used in this study, and 5 mice were in each group. Each treatment group, including a negative control group, consisted of five mice. Treatment was initiated once tumors were palpable and had reached a size of approximately 50 mm3. Treatments were administered 5e6 CAR+ T cells and same number of UTD cells in lOOpl PBS as twice a week for 3 weeks. Tumor volumes were calculated by measuring bioluminescence imaging (Liu, 2018). The instrument held 3 mice in one session. Tumor volume was measured using a caliper.

[0394] Patient-derived xenograft (PDX) model

[0395] All animal care and procedures for the establishment and maintenance of PDXs were conducted in accordance with The Wistar Institute's animal ethics approvals (201545). Experimental and control mice were co-housed. To generate PDX models, the WM4380-2 melanoma tumor was cut into small pieces of 1 mm3. These tumor fragments were suspended in 50% Matrigel and then implanted subcutaneously in 4-6-week-old female NOD-SCID- / IL2X-receptor null (NSG) mice under isoflurane anesthesia. After allowing the tumor to grow gradually for 45 days, tumor tissues were collected, cut into small pieces, and frozen in aliquots in FBS with 10% DMSO. Frozen cells were stored at -140 °C until use. PDXs were routinely validated by DNA and RNA sequencing, STR profiling, and pathological assessment. Each animal bearing PDX tumors of 50-100 mm3was distributed into experimental groups following the principles of single-mouse trials. The day of PDX implantation was designated as day 0. To assess in vivo response to CAR-T cells, CAR-T cells or untransfected T cells (5 x 1OA6 cells) were suspended in 100 pl PBS and injected intravenously into mice when tumors became palpable on day 8. CAR-T cells were injected via tail vein once a week for three weeks. Tumor volume and body weight were measured twice weekly.

[0396] Cytotoxicity assays

[0397] The Promega CytoTox 96 Non-Radioactive Cytotoxicity Assay kit (G1780; Promega, Wisconsin, USA) was utilized to measure the cytotoxicity of DR5 CAR T cells against melanoma cells and other cancer cells through lactate dehydrogenase (LDH) release, following previously described protocols (Wang, 2021). Target cells were seeded at a density of 4xl04cells / well in 50 pL of standard grow th medium in 96-well plates, while DR5 CAR-T cells were seeded at the indicated effector: target (E:T) ratio in the same volume simultaneously. After the stated time, the plates were briefly centrifuged, and 50 pL of supernatants were harvested for further analysis. The absorbance at 490 nm was measured using a BioTek Synergy HT reader (BioTek Instruments, Vermont, USA) following LDH activity detection. The percentage of cytotoxicity w as calculated as (experimental-effector spontaneous-target spontaneous) / (target maximum-target spontaneous) x 100. Additionally, a luciferase-releasing assay system was employed to evaluate DR5 CAR-T cell cytotoxicity against target cells expressing luciferase. After incubation of target cells and DR5 CAR-T cells, the plates w ere briefly spun down, and the supernatant was carefully removed. Then, 25 pL of lysis buffer was added, and the plate was incubated on ice for 10 minutes before adding 100 pL of luciferin substrate of luciferase. The plate was read immediately using a luminometer. The percentage of cytotoxicity was calculated as (target maximum signal - experimental signal) / target maximum signal x 100.

[0398] Flow cytometry T cells and cancer cells were washed with a PBS solution containing 3% FBS. After washing, 0.5- 1x106cells were aliquoted into separate tubes and incubated with an antibody mixture in 100-150 pl of staining buffer for 20 minutes at 4°C in the dark. The cells w ere then washed once with the w ashing buffer and resuspended in 300-500pl of washing buffer. If fixing was necessary , the cells w ere w ashed with PBS and fixed with a fixing buffer containing 1.5% PFA. Data acquisition was carried out using either BD LSR or Fortessa flow cytometers, and FlowJo software was used to analyze the data (Wang. 2021).

[0399] Western blot

[0400] Western blotting was performed as described previously (Liu, 2018; Bronte, 2016). Cells or extracellular vesicles (EVs) were lysed using RIP A buffer, and the protein concentrations were quantified using a BCA Protein Assay Kit (Bio-Rad, CA, USA). Subsequently, 20-50 pg of protein was separated by SDS-PAGE and e-transferred onto PVDF membranes. The membranes were then blocked with 5% non-fat milk at room temperature for 1 hour, followed by overnight incubation at 4 °C with primary antibodies. Afterward, the membranes w ere incubated with HRP-conjugated secondary antibodies (Cell Signaling Technology) at room temperature for 1 hour before being visualized using an ECL detection reagent.

[0401] Analysis ofTCGA data

[0402] DR5 (TNFRSF10B) expression was analyzed using The Human Protein Atlas and The Cancer Genome Atlas Program (TCGA). Human DR5 mRNA expression in tumor tissues was obtained from TCGA (https: / / ww w .cancer.gov / tcga).

[0403] Reverse phase protein array assays (RPPA)

[0404] RPPA sample preparation and protocol, as previously described (Wang, 2021; Liu, 2018). Briefly, protein lysates were prepared using a lysis buffer and 4x SDS sample buffer, with added protease and phosphatase inhibitors. The lysates were extracted from frozen tumors and analyzed using the RPPA platform at the MD Anderson Functional Proteomics Core facility. Detailed information on the RPPA method, data normalization, and the antibodies used is available on the core facility's website. Heatmaps were generated using Cluster and then analyzed using R and The Database for Annotation, Visualization, and Integrated Discovery' (DAVID)(Sherman, 2022; Huang, 2009).

[0405] Computational analysis ofscFv binding affinity to DR5 To model the single-chain variable fragment (scFv), the variable light and heavychains of each antibody were obtained from the International Immunogenetics Information System (Kaas, 2004; Ehrenmann, 2010). These, along with the linker sequence, were then entered into the Antibody Structure Prediction Panel of Maestro suite (2023-2) by Schrodinger [Schrodinger, L., and Warren DeLano. "PyMOL." (2023)]. The Antibody Structure Prediction Panel employed a homology search for the framework region to model the scFvs, utilizing the Enhanced Clothia numbering scheme. The highest-scoring homolog framework, defined by the average of the heavy and light chain similarity scores of the framework region to that of the inputted sequence, was chosen for each scFv. After the framework region selection, the scFv models were submitted for the complementarity determining region (CDR) loop modeling, where antibody databases were searched for loops of the same length as those in the query sequence. The loops are then clustered structurally. The modeled scFv is inspected in the workspace where the hydrogen bonding network was optimized within the structure, followed by a minimization step and water removal.

[0406] Once homology modeling was done, the scFvs were further prepared for docking studies using PDB-Tools (v2.3.1) webserver (Rodrigues, 2018). The linker was extracted, leaving only the variable domain of the scFvs, followed by sequential renumbering of the residue sequence. A final clean-up was conducted to remove any irrelevant lines that could disturb the residue renumbering. Once the .pdb file for each scFv was prepared, the high- resolution 2.2 A crystal structure of DR5 (PDB ID: lD4V)(Mongkolsapaya, 1999) was obtained from the online RCSB Protein Data Bank as .pdb files. It was then opened on PyMOL (2.5.5), where water molecules and any non-applicable bound ligands were removed. The final structures were then exported as a .pdb file to be used for molecular docking studies on the web server, High Ambiguity Driven protein-protein Docking (HADDOCK) (v2.4)(Honorato, 2021; Van Zundert, 2016). A loose definition of the epitope and paratope residues were specified to guide the docking.

[0407] Expression of mouse-anti-human DR5 recombinant antibodies

[0408] To generate mouse-anti-human DR5 recombinant antibodies, the DR5 CAR scFv cDNAs were cloned into pFUSEss-CHIg-mG2a and pFUSE2ss-CLIg-mK vectors from Invivogene, while the anti-hDR5 VL or VH cDNAs were cloned into pFUSE2ss-CLIg-mK or pFUSEss-CHIg-mG2a vectors, respectively (Richman, 2018). FreeStyle 293-F cells were cultured in FreeSty le 293 Expression Medium under shaking conditions at 90 rpm and in an incubator at 37°C and 5% CO2 until they reached a density of 2-3 x 10A6 cells / mL. The paired DR5 pFUSEss-CHIg / CLIg plasmids were transfected into the cells using 293fectin Transfection Reagent. After 24-48 hours of growth, the supernatant was collected, and cell debris was removed by centrifugation at 10,000 x g for 20 minutes. Recombinant antibody concentrations were determined using a BCA Protein Assay Kit and SDS-PAGE / Silver Stain, and the purified antibodies were stored at -20°C or 4°C until further use.

[0409] On-Cell-Western (OCW) assay

[0410] OCW assays were performed at the Wistar Institute. A375 melanoma cells with overexpressed GFP and DR5 were utilized as target cells for the OCW assay. For the live cell assay, 8,000 cells were seeded and allowed to attach for 24 hours. Antibodies (Abs) were dissolved in culture medium at dilutions of 1 :2, 1 :4, and 1:8. A total of 30 pL of the Ab solution was added to each well and incubated at 37°C for 30 minutes. The cell media was removed, and 50 pL of 4% paraformaldehyde was added to fix the cells. The plate was incubated at room temperature for 20 minutes without agitation. After washing the plate twice with TBS, 50 pL of Intercept Blocking Buffer was added to each well and incubated for 1 hour at room temperature with moderate shaking to allow7for blocking. A fluorescently labeled secondary antibody, IRDye 800 CW, was diluted in 0.2% Tween-20 Intercept Blocking Buffer at a ratio of 1 :800, and LI-COR CellTag 700 Stain was diluted at 1:500. 15 pL of the diluted secondary antibody solution with CellTag 700 Stain was added to testing wells, while 15 pL of the secondary antibody solution without CellTag 700 Stain was added to background wells. The plate was incubated on a plate shaker for 45 minutes, protecting it from light. The plate was washed 5 times with IX TBS with 0.1% Tween-20 washing solution, and the final wash solution was removed by tapping or blotting the plate. The plate was then scanned on the LI-COR Odyssey CLx with the 700 and 800 nm channels using an Odyssey® Imager. The data was analyzed with Image Studio under the On-Cell Western Software protocol and graphed using Prism GraphPad 8. The experiments were performed in triplicate.

[0411] Extracellular vesicle preparation and characterization

[0412] To isolate exosomes from cell culture, the cell culture supernatant is collected and centrifuged at 300 x g for 10 minutes to remove any cells or debris. The resulting supernatant is then transferred to ultracentrifuge tubes and centrifuged at 2,000 x g for 10 minutes to remove any remaining cell debris. After filtering the supernatant through a 0.22 pm filter, it is centrifuged at 100,000 x g for 2 hours to pellet the exosomes. The supernatant is then discarded, and the exosome pellet is washed and resuspended in a small volume of PBS or another appropriate buffer, followed by another round of ultracentrifugation at 100,000 x g for 2 hours. Finally, the supernatant is discarded, and the exosome pellet is resuspended in a small volume of PBS or another appropriate buffer for downstream applications. The NanoSight NS300 (Malvern Instruments), which comes with particletracking software and fast video capture capabilities, was used to measure the size and concentration of sEVs obtained from Sup-T cell culture supernatants (Guo. 2025; Chen, 2018).

[0413] Bicellular spheroid culturing

[0414] The method for generating bicellular spheroids and co-culture was previously described (Ou, 2022; Ou, 2023; Wang, 2021; Ou, 2021)(. In summary, 50 pl of 1.5% agarose gel (A9539, Sigma- Aldrich, St. Louis, Missouri) was used to prime the culturing well of a flat bottom 96-well tissue culture multiplate to form an unattached support matrix. Bicellular spheroids were created by seeding 3 x 103A375 melanoma cells and 6 x 103BJ fibroblasts (tumor cells:fibroblasts = 1:2) or 3 x 103A375 melanoma cells and the same number of MDSCs (tumor cells:MDSCs = 1: 1) into the agarose-primed wells. After 48 hours of coculturing, the spheroids were checked under a microscope and then co-cultured with 3 x 103CAR-T cells (tumor cells: effectors = 1 : 1). Prior to fluorescence microscope scanning after 24 hours of co-culturing, Hoechst33342 (Ipg / ml) and PI (I pg / ml) were stained. FUJI imaging was used to analyze imaging data and all experiments were set up in triplicate.

[0415] Murine DR5 CAR construct and murine CAR-T cell manufacture

[0416] The murine DR5 CAR is designed based on scFv sequence of the DR5 M5-1 monoclonal antibody (Takeda, 2004) and the DNA construct has traceable BFP and nLuc as we previously described (Zhang, 2024). Mouse CAR T cells were generated using a retroviral transduction protocol. First, Plat-E cells were transfected with a pMMLV-based vector encoding the CAR construct along with fluorescent and bioluminescent reporters. The resulting retroviral supernatant was harvested and used to transduce mouse CD3+ T cells isolated from splenocytes and activated with plate-bound anti-CD3 / CD28 antibodies. RetroNectin-coated plates and centrifugation were used to facilitate viral transduction. After transduction. CAR T cells were expanded in IL-2-containing media for up to six days. Transduction efficiency was confirmed by flow cytometry using both reporter fluorescence and CAR-specific staining.

[0417] Melanoma patient-derived organoids (MPDOs) Fresh melanoma tissues were obtained from the University' of Pennsylvania Hospital Department of Pathology’ and Laboratory Medicine. All procedures were approved by the Institutional Review Board at the University7of Pennsylvania. The fresh tissues were maintained in RPMI-1640 medium until processing. The tissue specimen blocks and organoids used in this study were prepared as previously described (Ou, 2023; Jacob, 2020). To optimize the growth of melanoma organoids, an FBS-free medium specifically designed for melanoma cells independently was developed, which comprised of 50% M594 media, 50% Advanced DMEM media, and 1% each of B27, NEAA, and P / S.

[0418] Patient-derived organotypic melanoma slice culture (MSC)

[0419] The fresh tumor tissues were kept on ice and sliced using a vibrating microtome. Tissue preparation was performed according to a previously described protocol (Kenerson, 2021). Briefly, the tissues were collected and embedded in 2% agarose for slicing. After the agarose solidified, it was fixed to a specimen disc and sliced using Compresstome VF-310-0Z at a thickness of 300 pm. The tissue slices were placed into a Millicell insert (PIHP01250, Millipore, MO, USA) in a 24-well plate and cultured in RPMI 1640 medium with 10% FBS, 2 mM L-glutamine (A2916801, Gibco), 10 mM HEPES (15630080, Gibco), 1 mM sodium pyruvate (11360070. Gibco), 5.5 pM 2-mercaptoethanol, 1% penicillin-streptomycin, and 200 lU / mL IL-2.

[0420] Immunofluorescence and live imaging

[0421] Immunofluorescence and live image pictures were captured and analyzed using a Zeiss microscope with lOx and 20x objective lenses. To visualize the nuclei and cell viability, cells were stained with Hoechst33342 and PI, respectively. Depending on the specific experiments, T-cells were stained with CFSE or CellTrace Far Red. Zeiss ZEN software was employed to analyze the fluorescent images, which were synthesized into video files. Representative images were used to present the data, while statistical analyses were based on at least three independent datasets.

[0422] In Situ Hybridization (ISH)

[0423] Formalin-Fixed-Paraffm-Embedded (FFPE) tissues were sectioned in 5 pm thickness. Sectioned tissues on slides were used for in situ hybridization to probe specific mRNA following the protocol of RNAscope 2.5 HD Reagent Kit- Red (ACDscope, 322350). Probes specific to DR5 w7ere purchased from ACDscope (Hs-TNFRSF10B-Cl,1048531-Cl). Fast- Red was used to develop the positive probe signal, also included in the kit. Representative bright field images are captured under a microscope. Immunohistochemistry (IHC)

[0424] Formalin-Fixed-Paraffin-Embedded (FFPE) tissues were sectioned in 5 pm thickness. Slides were boiled for 10 min in 1 mM citrate buffer (w / v) with pH 6.0 for antigen retrieval. ImmPRESS Excel Amplified HRP Polymer Staining kit (Vector Laboratories; MP-7601) was used for IHC staining. Primary antibody against DR5 (Invitrogen, MA5-32693) was diluted 1:200 in PBST buffer and incubated with slides overnight at 40 C. 3.3 '-Diaminobenzi dine (DAB) as used as chromogen (Vector Laboratories; MP-7601). Images were captured under a microscope.

[0425] Statistical analysis

[0426] Statistical analysis was performed using GraphPad (Prism software package version) and Microsoft Excel software. Data are presented as mean±SEM. and significant differences were examined with Student’s t-test. A P value of <0.05 was considered statistically significant.

[0427] Enumerated Embodiments

[0428] The following enumerated embodiments are provided, the numbering of which is not to be construed as designating levels of importance.

[0429] Embodiment 1 provides a nucleic acid comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR) comprising an antigen binding domain, a transmembrane domain, and an intracellular domain, wherein the antigen binding domain specifically binds DR-5.

[0430] Embodiment 2 provides the nucleic acid of embodiment 1, wherein the antigen binding domain comprises an antibody or an antigen-binding fragment thereof.

[0431] Embodiment 3 provides the nucleic acid of embodiment 1 or 2, wherein the antigenbinding fragment is selected from the group consisting of a Fab, a single-chain variable fragment (scFv). or a single-domain antibody.

[0432] Embodiment 4 provides the nucleic acid of any one of embodiments 1-3, wherein the antigen-binding fragment is a scFv.

[0433] Embodiment 5 provides the nucleic acid of any one of embodiments 1-4, wherein the antigen binding domain comprises a heavy chain variable region comprising an amino acid sequence having at least 95%-99% identity to the amino acid sequence of SEQ ID NO: 1, 4, 7, or 10; and / or a light chain variable region comprising an amino acid sequence having at least 95-99% identity to the amino acid sequence of SEQ ID NO: 2, 5, 8, or 11. Embodiment 6 provides the nucleic acid of embodiment 5, wherein the heavy chain variable region comprises the amino acid sequence of any one of SEQ ID NO: 1. 4, 7, or 10; and / or the light chain variable region comprises the amino acid sequence of any one of SEQ ID NO: 2, 5, 8, or 11.

[0434] Embodiment 7 provides the nucleic acid of embodiment 5 or 6, wherein the antigen binding domain comprises the amino acid sequence of SEQ ID NO: 3, 6, 9, or 12; or an amino acid sequence having at least 95%-99% identity to the amino acid sequence of SEQ ID NO: 3, 6, 9, or 12.

[0435] Embodiment 8 provides the nucleic acid of any one of embodiments 1-7, wherein the transmembrane domain is selected from the group consisting of an artificial hydrophobic sequence, and a transmembrane domain of a type I transmembrane protein, an alpha, beta, or zeta chain of a T cell receptor, CD28, CD3epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, 0X40 (CD134), 4-1BB (CD137), and CD154.

[0436] Embodiment 9 provides the nucleic acid of embodiment 7, wherein the transmembrane domain comprises a transmembrane domain of CD8.

[0437] Embodiment 10 provides the nucleic acid of embodiment 9. wherein the transmembrane domain of CD8 comprises the amino acid sequence of SEQ ID NO: 16.

[0438] Embodiment 11 provides the nucleic acid of embodiment 6, wherein the transmembrane domain of CD8 is encoded by the nucleotide sequence of SEQ ID NO: 77.

[0439] Embodiment 12 provides the nucleic acid of any one of embodiments 1-11. wherein the CAR further comprises a hinge domain.

[0440] Embodiment 13 provides the nucleic acid of embodiment 12, wherein the hinge domain is selected from the group consisting of an Fc fragment of an antibody, a hinge region of an antibody, a CH2 region of an antibody, a CH3 region of an antibody, an artificial hinge domain, a hinge comprising an amino acid sequence of a CD8 hinge, or any combination thereof.

[0441] Embodiment 14 provides the nucleic acid of embodiment 12, wherein the hinge domain is a CD8 hinge.

[0442] Embodiment 15 provides the nucleic acid of embodiment 14, wherein the CD8 hinge comprises the amino acid sequence of SEQ ID NO: 14.

[0443] Embodiment 16 provides the nucleic acid of embodiment 15, wherein the CD8 hinge is encoded by the nucleotide sequence of SEQ ID NO: 74; Embodiment 17 provides the nucleic acid of any one of embodiments 1-16, wherein the intracellular domain comprises a costimulatory signaling domain and an intracellular signaling domain.

[0444] Embodiment 18 provides the nucleic acid of embodiment 17, wherein the costimulatory signaling domain comprises a costimulatory domain of a protein selected from the group consisting of proteins in the TNFR superfamily, CD28, 4-1BB (CD137), 0X40 (CD134), PD-1, CD7, LIGHT. CD83L, DAP10. DAP12, CD27, CD2, CD5, ICAM-1, LFA- 1, Lek, TNFR-I, TNFI-II, Fas, CD30, CD40, ICOS, NKG2C, and B7-H3 (CD276), or a variant thereof.

[0445] Embodiment 19 provides the nucleic acid of embodiment 17 or 18, wherein the costimulatory signaling domain comprises a costimulatory domain of 4- IBB.

[0446] Embodiment 20 provides the nucleic acid of embodiment 19, wherein the costimulatory signaling domain comprises the amino acid sequence of SEQ ID NO: 20.

[0447] Embodiment 21 provides the nucleic acid of embodiment 20, wherein the costimulatory signaling domain is encoded by the nucleotide sequence of SEQ ID NO: 81;

[0448] Embodiment 22 provides the nucleic acid of any one of embodiments 17-21, wherein the intracellular signaling domain comprises an intracellular domain selected from the group consisting of cytoplasmic signaling domains of a human CD3 zeta chain (CD3Q, FcyRIII, FcsRI, a cytoplasmic tail of an Fc receptor, an immunoreceptor tyrosine-based activation motif (ITAM) bearing cytoplasmic receptor, TCR zeta, FcR gamma, CD3 gamma. CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d, or a variant thereof.

[0449] Embodiment 23 provides the nucleic acid of embodiment 15, wherein the intracellular signaling domain comprises an intracellular domain of CD3q.

[0450] Embodiment 24 provides the nucleic acid of embodiment 23, wherein the intracellular domain of CD3£ comprises the amino acid sequence of SEQ ID NO: 24 or 25.

[0451] Embodiment 25 provides the nucleic acid of embodiment 24, wherein the intracellular domain of CD3^ is encoded by the nucleotide sequence of SEQ ID NO: 85 or 86.

[0452] Embodiment 26 provides the nucleic acid of embodiment 1. wherein the CAR comprises the antigen binding domain, a CD8a hinge region, a CD8a transmembrane domain, a 4-1BB costimulatory domain, and a CD3^ intracellular signaling domain.

[0453] Embodiment 27 provides the nucleic acid of any one of embodiments 1-26, wherein the CAR comprises the amino acid sequence of any one of SEQ ID NOs: 1-12; or an amino acid sequence having at least 95%-99% identity to any one of SEQ ID NOs: 1-12. Embodiment 28 provides the nucleic acid of any one of embodiments 1-27, wherein the CAR comprises the amino acid sequence of any one of SEQ ID NOs: 26-29; or an amino acid sequence having at least 95%-99% identity to any one of SEQ ID NOs: 26-29.

[0454] Embodiment 29 provides the nucleic acid of any one of embodiments 1-28, wherein the CAR is encoded by the nucleotide sequence of any one of SEQ ID NOs: 87-90; or a nucleotide sequence having at least 95%-99% identity to any one of SEQ ID NOs: 87-90.

[0455] Embodiment 30 provides the nucleic acid of any one of embodiments 1-28 further comprising a second polynucleotide sequence encoding a second polypeptide.

[0456] Embodiment 31 provides the nucleic acid of any one of embodiments 1-30, wherein the nucleic acid encodes a self-cleaving 2A peptide domain between the CAR and the second polypeptide.

[0457] Embodiment 32 provides the nucleic acid of embodiment 31, wherein the selfcleaving peptide domain is selected from the group consisting of T2A, P2A, E2A and F2A.

[0458] Embodiment 33 provides the nucleic acid of any one of embodiments 29-32, wherein the nucleic acid comprises an internal ribosome binding site (IRES) between the CAR and the second polypeptide.

[0459] Embodiment 34 provides a vector comprising the nucleic acid of any one of embodiments 1-33.

[0460] Embodiment 35 provides the vector of embodiment 34, wherein the vector is an expression vector.

[0461] Embodiment 36 provides the vector of embodiment 34 or 35, wherein the vector is a viral vector selected from the group consisting of a retroviral vector, a lentiviral vector, an adenoviral vector, and an adeno-associated viral vector.

[0462] Embodiment 37 provides the vector of embodiment 36, wherein the vector is a lentiviral vector.

[0463] Embodiment 38 provides the vector of embodiment 37, wherein the lentiviral vector is a self-inactivating HIV vector comprising an LTR comprising a U3 deletion.

[0464] Embodiment 39 provides the vector of embodiment 38, wherein the HIV vector comprises a partial gag sequence, optionally wherein the partial gag sequence comprises the nucleotide sequence of SEQ ID NO: 33.

[0465] Embodiment 40 provides the vector of embodiment 38 or 39, wherein the HIV vector further comprises a central polypurine tract (cPPT), optionally wherein the cPPT comprises the nucleotide sequence of SEQ ID NO: 34. Embodiment 41 provides the vector of any one of embodiments 38-40 further comprising a Woodchuck Hepatitis virus post-transcriptional regulatory element (WPRE), optionally wherein the WPRE comprises the nucleotide sequence of SEQ ID NO: 35.

[0466] Embodiment 42 provides the vector of embodiment 38, comprising the polynucleotide sequence of any one of SEQ ID NOs: 37-40.

[0467] Embodiment 43 provides a chimeric antigen receptor (CAR) encoded by the nucleic acid of any one of embodiments 1-33; or the vector of any one of embodiments 34-42.

[0468] Embodiment 44 provides a chimeric antigen receptor (CAR) comprising an antigen binding domain, a transmembrane domain, and an intracellular domain, wherein the antigen binding domain specifically binds DR-5.

[0469] Embodiment 45 provides the CAR of embodiment 44, wherein the antigen binding domain comprises an antibody or an antigen-binding fragment thereof.

[0470] Embodiment 46 provides the CAR of embodiment 44 or 45, wherein the antigenbinding fragment is selected from the group consisting of a Fab, a single-chain variable fragment (scFv), or a single-domain antibody.

[0471] Embodiment 47 provides the CAR of any one of embodiments 44-46. wherein the antigen-binding fragment is a scFv.

[0472] Embodiment 48 provides the CAR of any one of embodiments 44-47, wherein the antigen binding domain comprises a heavy chain variable region comprising an amino acid sequence having at least 95%-99% identity to the amino acid sequence of SEQ ID NO: 1, 4, 7, or 10; and / or a light chain variable region comprising an amino acid sequence having at least 95-99% identify to the amino acid sequence of SEQ ID NO: 2, 5, 8, or 11.

[0473] Embodiment 49 provides the CAR of embodiment 48. wherein the heavy chain variable region comprises the amino acid sequence of any one of SEQ ID NO: 1. 4, 7, or 10; and / or the light chain variable region comprises the amino acid sequence of any one of SEQ ID NO: 2, 5, 8, or 11.

[0474] Embodiment 50 provides the CAR of embodiment 48 or 49, wherein the antigen binding domain comprises the amino acid sequence of SEQ ID NO: 3, 6, 9, or 12; or an amino acid sequence having at least 95%-99% identify to the amino acid sequence of SEQ ID NO: 3, 6, 9, or 12.

[0475] Embodiment 51 provides the CAR of any one of embodiments 44-50, wherein the transmembrane domain is selected from the group consisting of an artificial hydrophobic sequence, and a transmembrane domain of a type I transmembrane protein, an alpha, beta, or zeta chain of a T cell receptor, CD28, CD3epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37. CD64, CD80, CD86, 0X40 (CD134). 4-1BB (CD137). and CD154.

[0476] Embodiment 52 provides the CAR of embodiment 51, wherein the transmembrane domain comprises a transmembrane domain of CD8.

[0477] Embodiment 53 provides the CAR of embodiment 52, wherein the transmembrane domain of CD8 comprises the amino acid sequence of SEQ ID NO: 16.

[0478] Embodiment 54 provides the CAR of embodiment 53. wherein the transmembrane domain of CD8 is encoded by the nucleotide sequence of SEQ ID NO: 77;

[0479] Embodiment 55 provides the CAR of any one of embodiments 44-54, wherein the CAR further comprises a hinge domain.

[0480] Embodiment 56 provides the CAR of embodiment 55, wherein the hinge domain is selected from the group consisting of an Fc fragment of an antibody, a hinge region of an antibody, a CH2 region of an antibody, a CH3 region of an antibody, an artificial hinge domain, a hinge comprising an amino acid sequence of a CD8 hinge, or any combination thereof.

[0481] Embodiment 57 provides the CAR of embodiment 55, wherein the hinge domain is a CD8 hinge.

[0482] Embodiment 58 provides the CAR of embodiment 57, wherein the CD8 hinge comprises the amino acid sequence of SEQ ID NO: 14.

[0483] Embodiment 59 provides the CAR of embodiment 58, wherein the CD8 hinge is encoded by the nucleotide sequence of SEQ ID NO: 74;

[0484] Embodiment 60 provides the CAR of any one of embodiments 44-59, wherein the intracellular domain comprises a costimulatory signaling domain and an intracellular signaling domain.

[0485] Embodiment 61 provides the CAR of embodiment 60, wherein the costimulatory signaling domain comprises a costimulatory domain of a protein selected from the group consisting of proteins in the TNFR superfamily, CD28, 4-1BB (CD137), 0X40 (CD134), PD-1, CD7, LIGHT, CD83L, DAP10, DAP12, CD27, CD2, CD5, ICAM-1, LFA-1, Lek, TNFR-I, TNFI-II, Fas. CD30, CD40, ICOS. NKG2C. and B7-H3 (CD276), or a variant thereof.

[0486] Embodiment 62 provides the CAR of embodiment 60 or 61, wherein the costimulatory signaling domain comprises a costimulatory domain of 4-1BB.

[0487] Embodiment 63 provides the CAR of embodiment 62, wherein the costimulatory signaling domain comprises the amino acid sequence of SEQ ID NO: 20. Embodiment 64 provides the CAR of embodiment 63. wherein the costimulatory signaling domain is encoded by the nucleotide sequence of SEQ ID NO: 81;

[0488] Embodiment 65 provides the CAR of any one of embodiments 60-64. wherein the intracellular signaling domain comprises an intracellular domain selected from the group consisting of cytoplasmic signaling domains of a human CD3 zeta chain (CD3Q, FcyRIII, FcsRI, a cytoplasmic tail of an Fc receptor, an immunoreceptor tyrosine-based activation motif (ITAM) bearing cytoplasmic receptor, TCR zeta, FcR gamma, CD3 gamma. CD3 delta. CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d, or a variant thereof.

[0489] Embodiment 66 provides the CAR of embodiment 65, wherein the intracellular signaling domain comprises an intracellular domain of CD3^,

[0490] Embodiment 67 provides the CAR of embodiment 66, wherein the intracellular domain of CD3^ comprises the amino acid sequence of SEQ ID NO: 24 or 25.

[0491] Embodiment 68 provides the CAR of embodiment 67, wherein the intracellular domain of CD3^ is encoded by the nucleotide sequence of SEQ ID NO: 85 or 86.

[0492] Embodiment 69 provides the CAR of embodiment 44, wherein the CAR comprises the antigen binding domain, a CD8a hinge region, a CD8a transmembrane domain, a 4-1BB costimulatory domain, and a CD3^ intracellular signaling domain.

[0493] Embodiment 70 provides the CAR of any one of embodiments 44-69, wherein the CAR comprises the amino acid sequence of any one of SEQ ID NOs: 1-12 or 26-29, or comprises an amino acid sequence having at least 95%-99% identity to the amino acid sequence of SEQ ID NOs: 1 -12 or 26-29.

[0494] Embodiment 71 provides the CAR of any one of embodiments 44-70, wherein the CAR is encoded by a nucleic acid sequence comprising the sequence of any one of SEQ ID NOs: 87-90; or a nucleotide sequence having at least 95%-99% identity to any one of SEQ ID NOs: 87-90.

[0495] Embodiment 72 provides the CAR of any one of embodiments 44-69 further comprising a second polypeptide.

[0496] Embodiment 73 provides the CAR of embodiment 72, further comprising a selfcleaving 2A peptide domain between the CAR and the second polypeptide.

[0497] Embodiment 74 provides the CAR of embodiment 73, wherein the self-cleaving peptide domain is selected from the group consisting of T2A, P2A, E2A and F2A.

[0498] Embodiment 75 provides a cell comprising the nucleic acid of any one of embodiments 1-33, the vector of any one of embodiments 34-42, or the CAR of any one of embodiments 43-74. Embodiment 76 provides the cell of embodiment 75, wherein the cell is an immune cell or precursor cell thereof.

[0499] Embodiment 77 provides the cell of embodiment 75 or 76, wherein the cell is a natural killer (NK) cell or a T cell.

[0500] Embodiment 78 provides the cell of any one of embodiments 75-78, wherein the cell is a gamma delta T cell.

[0501] Embodiment 79 provides the cell of any one of embodiments 75-78, wherein the cell is an autologous cell.

[0502] Embodiment 80 provides the cell of any one of embodiments 75-78, wherein the cell is an allogeneic cell.

[0503] Embodiment 81 provides the cell of any one of embodiments 75-77, wherein the cell is from an NK92 cell line.

[0504] Embodiment 82 provides a pharmaceutical composition comprising the cell of any one of embodiments 75-81 and a pharmaceutically acceptable carrier.

[0505] Embodiment 83 provides a method for killing a DR+ 5 cell expressing a death receptor 5 antigen, comprising: contacting the DR5+ cell with the cell of any one of embodiments 75-81.

[0506] Embodiment 84 provides the method of embodiment 83, wherein the DR5+ cell is a melanoma cell, a pancreatic cancer cell, a lung cancer cell, a glioma cell, a breast cancer cell, a liver cancer cell, or an ovarian cancer cell.

[0507] Embodiment 85 provides the method of embodiment 83, wherein the DR5+ cell is a myeloid-derived suppressor cell (MDSC).

[0508] Embodiment 86 provides the method of embodiment 83, wherein the DR5+ cell is a cancer-associated fibroblast (CAF).

[0509] Embodiment 87 provides a method for treating cancer in a subject in need thereof, comprising: administering an effective amount of the pharmaceutical composition of embodiment 82 to the subject, thereby treating the cancer.

[0510] Embodiment 88 provides the method of embodiment 87, wherein the cancer is a solid tumor.

[0511] Embodiment 89 provides the method of embodiment 43, wherein the solid tumor is selected from the group consisting of melanoma, glioma, breast cancer, liver cancer, lung cancer, ovarian cancer, cervical cancer, uterus cancer, head and neck cancer, hepatocellular carcinoma, colon cancer, rectal cancer, colorectal carcinoma, kidney cancer, prostate cancer, gastric cancer, bronchus cancer, pancreatic cancer, urinary bladder cancer, and brain cancer. Embodiment 90 provides the method of embodiment 89, wherein the solid tumor is melanoma, pancreatic cancer, lung cancer, glioma, breast cancer, liver cancer, or ovarian cancer.

[0512] Embodiment 91 provides the method of any one of embodiments 87-90, wherein the pharmaceutical composition is administered intratumorally.

[0513] Embodiment 92 provides the method of embodiment 87, wherein the cancer is a hematologic cancer.

[0514] Embodiment 93 provides the method of embodiment 92, wherein the hematologic cancer is a myeloid malignancy selected from the group consisting of acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML). myelodysplastic neoplasm, and myeloproliferative neoplasm.

[0515] Embodiment 94 provides a method for preparing the cell of any one of embodiments 75-81, comprising: introducing into the cell the nucleic acid of any one of embodiments 1-33, or the vector of any one of embodiments 34-42.

[0516] Other Embodiments

[0517] The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

[0518] The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety. While this disclosure has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this disclosure may be devised by others skilled in the art without departing from the true spirit and scope of the disclosure. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

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Claims

CLAIMSWhat is claimed:

1. A nucleic acid comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR) comprising an antigen binding domain, a transmembrane domain, and an intracellular domain, wherein the antigen binding domain specifically binds DR-5.

2. The nucleic acid of claim 1, wherein the antigen binding domain comprises an antibody or an antigen-binding fragment thereof.

3. The nucleic acid of claim 1 or 2, wherein the antigen-binding fragment is selected from the group consisting of a Fab, a single-chain variable fragment (scFv), or a single-domain antibody.

4. The nucleic acid of any one of claims 1-3, wherein the antigen-binding fragment is a scFv.

5. The nucleic acid of any one of claims 1-4, wherein the antigen binding domain comprises a heavy chain variable region comprising an amino acid sequence having at least 95%-99% identity to the amino acid sequence of SEQ ID NO: 1, 4, 7, or 10; and / or a light chain variable region comprising an amino acid sequence having at least 95-99% identity to the amino acid sequence of SEQ ID NO: 2, 5, 8, or 11.

6. The nucleic acid of claim 5, wherein the heavy chain variable region comprises the amino acid sequence of any one of SEQ ID NO: 1, 4, 7, or 10; and / or the light chain variable region comprises the amino acid sequence of any one of SEQ ID NO: 2, 5, 8, or 11.

7. The nucleic acid of claim 5 or 6, wherein the antigen binding domain comprises the amino acid sequence of SEQ ID NO: 3, 6, 9, or 12; or an amino acid sequence having at least 95%-99% identity to the amino acid sequence of SEQ ID NO: 3, 6, 9, or 12.

8. The nucleic acid of any one of claims 1-7, wherein the transmembrane domain is selected from the group consisting of an artificial hydrophobic sequence, and a transmembrane domain ofa type T transmembrane protein, an alpha, beta, or zeta chain of a T cell receptor, CD28, CD3epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, 0X40 (CD134), 4-1BB (CD137), and CD154.

9. The nucleic acid of claim 7, wherein the transmembrane domain comprises a transmembrane domain of CD8.

10. The nucleic acid of claim 9, wherein the transmembrane domain of CD8 comprises the amino acid sequence of SEQ ID NO: 16.

11. The nucleic acid of claim 6, wherein the transmembrane domain of CD8 is encoded by the nucleotide sequence of SEQ ID NO: 77.

12. The nucleic acid of any one of claims 1-11, wherein the CAR further comprises a hinge domain.

13. The nucleic acid of claim 12, wherein the hinge domain is selected from the group consisting of an Fc fragment of an antibody, a hinge region of an antibody, a CH2 region of an antibody, a CH3 region of an antibody, an artificial hinge domain, a hinge comprising an amino acid sequence of a CD8 hinge, or any combination thereof.

14. The nucleic acid of claim 12, wherein the hinge domain is a CD8 hinge.

15. The nucleic acid of claim 14, wherein the CD8 hinge comprises the amino acid sequence of SEQ ID NO: 14.

16. The nucleic acid of claim 15, wherein the CD8 hinge is encoded by the nucleotide sequence of SEQ ID NO: 74;17. The nucleic acid of any one of claims 1-16, wherein the intracellular domain comprises a costimulatory signaling domain and an intracellular signaling domain.

18. The nucleic acid of claim 17, wherein the costimulatory signaling domain comprises a costimulatory domain of a protein selected from the group consisting of proteins in the TNFR superfamily, CD28, 4-1BB (CD137), 0X40 (CD134), PD-1, CD7, LIGHT, CD83L, DAP10, DAP12, CD27, CD2, CD5, ICAM-1, LFA-1, Lek, TNFR-I, TNFI-II, Fas, CD30, CD40, ICOS, NKG2C, and B7-H3 (CD276), or a variant thereof.

19. The nucleic acid of claim 17 or 18, wherein the costimulatory signaling domain comprises a costimulatory domain of 4- IBB.

20. The nucleic acid of claim 19, wherein the costimulatory signaling domain comprises the amino acid sequence of SEQ ID NO: 20.

21. The nucleic acid of claim 20, wherein the costimulatory signaling domain is encoded by the nucleotide sequence of SEQ ID NO: 81.

22. The nucleic acid of any one of claims 17-21, wherein the intracellular signaling domain comprises an intracellular domain selected from the group consisting of cytoplasmic signaling domains of a human CD3 zeta chain (CD3Q, FcyRIII, FcsRI, a cytoplasmic tail of an Fc receptor, an immunoreceptor tyrosine-based activation motif (IT AM) bearing cytoplasmic receptor, TCR zeta, FcR gamma, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d, or a variant thereof.

23. The nucleic acid of claim 15, wherein the intracellular signaling domain comprises an intracellular domain of CD3(^.

24. The nucleic acid of claim 23, wherein the intracellular domain of CD3(^ comprises the amino acid sequence of SEQ ID NO: 24 or 25.

25. The nucleic acid of claim 24, wherein the intracellular domain of CD3^ is encoded by the nucleotide sequence of SEQ ID NO: 85 or 86.

26. The nucleic acid of claim 1, wherein the CAR comprises the antigen binding domain, a CD8a hinge region, a CD8a transmembrane domain, a 4- IBB costimulatory domain, and a CD3(^ intracellular signaling domain.

27. The nucleic acid of any one of claims 1-26, wherein the CAR comprises the amino acid sequence of any one of SEQ ID NOs: 1-12; or an amino acid sequence having at least 95%-99% identity to any one of SEQ ID NOs: 1-12.

28. The nucleic acid of any one of claims 1-27, wherein the CAR comprises the amino acid sequence of any one of SEQ ID NOs: 26-29; or an amino acid sequence having at least 95%- 99% identity to any one of SEQ ID NOs: 26-29.

29. The nucleic acid of any one of claims 1-28, wherein the CAR is encoded by the nucleotide sequence of any one of SEQ ID NOs: 87-90; or a nucleotide sequence having at least 95%-99% identity to any one of SEQ ID NOs: 87-90.

30. The nucleic acid of any one of claims 1-28 further comprising a second polynucleotide sequence encoding a second polypeptide.

31. The nucleic acid of any one of claims 1-30, wherein the nucleic acid encodes a self-cleaving 2A peptide domain between the CAR and the second polypeptide.

32. The nucleic acid of claim 31, wherein the self-cleaving peptide domain is selected from the group consisting of T2A, P2A, E2A and F2A.

33. The nucleic acid of any one of claims 29-32, wherein the nucleic acid comprises an internal ribosome binding site (IRES) between the CAR and the second polypeptide.

34. A vector comprising the nucleic acid of any one of claims 1-33.

35. The vector of claim 34, wherein the vector is an expression vector.

36. The vector of claim 34 or 35, wherein the vector is a viral vector selected from the group consisting of a retroviral vector, a lentiviral vector, an adenoviral vector, and an adeno-associated viral vector.

37. The vector of claim 36, wherein the vector is a lentiviral vector.

38. The vector of claim 37, wherein the lentiviral vector is a self-inactivating HIV vector comprising an LTR comprising a U3 deletion.

39. The vector of claim 38, wherein the HIV vector comprises a partial gag sequence, optionally wherein the partial gag sequence comprises the nucleotide sequence of SEQ ID NO: 33.

40. The vector of claim 38 or 39, wherein the HIV vector further comprises a central polypurine tract (cPPT), optionally wherein the cPPT comprises the nucleotide sequence of SEQ ID NO: 34.

41. The vector of any one of claims 38-40 further comprising a Woodchuck Hepatitis virus post- transcriptional regulatory element (WPRE), optionally wherein the WPRE comprises the nucleotide sequence of SEQ ID NO: 35.

42. The vector of claim 38, comprising the polynucleotide sequence of any one of SEQ ID NOs: 37-40.

43. A chimeric antigen receptor (CAR) encoded by the nucleic acid of any one of claims 1-33; or the vector of any one of claims 34-42.

44. A chimeric antigen receptor (CAR) comprising an antigen binding domain, a transmembrane domain, and an intracellular domain, wherein the antigen binding domain specifically binds DR- 5.

45. The CAR of claim 44, wherein the antigen binding domain comprises an antibody or an antigen-binding fragment thereof.

46. The CAR of claim 44 or 45, wherein the antigen-binding fragment is selected from the group consisting of a Fab, a single-chain variable fragment (scFv), or a single-domain antibody.

47. The CAR of any one of claims 44-46, wherein the antigen-binding fragment is a scFv.

48. The CAR of any one of claims 44-47, wherein the antigen binding domain comprises a heavy chain variable region comprising an amino acid sequence having at least 95%-99% identity to the amino acid sequence of SEQ ID NO: 1, 4, 7, or 10; and / or a light chain variable region comprising an amino acid sequence having at least 95-99% identity to the amino acid sequence of SEQ ID NO: 2, 5, 8, or 11.

49. The CAR of claim 48, wherein the heavy chain variable region comprises the amino acid sequence of any one of SEQ ID NO: 1, 4, 7, or 10; and / or the light chain variable region comprises the amino acid sequence of any one of SEQ ID NO: 2, 5, 8, or 11.

50. The CAR of claim 48 or 49, wherein the antigen binding domain comprises the amino acid sequence of SEQ ID NO: 3, 6, 9, or 12; or an amino acid sequence having at least 95%-99% identity to the amino acid sequence of SEQ ID NO: 3, 6, 9, or 12.

51. The CAR of any one of claims 44-50, wherein the transmembrane domain is selected from the group consisting of an artificial hydrophobic sequence, and a transmembrane domain of a type I transmembrane protein, an alpha, beta, or zeta chain of a T cell receptor, CD28, CD3epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, 0X40 (CD134), 4-1BB (CD137), and CD154.

52. The CAR of claim 51, wherein the transmembrane domain comprises a transmembrane domain of CD8.

53. The CAR of claim 52, wherein the transmembrane domain of CD8 comprises the amino acid sequence of SEQ ID NO: 16.

54. The CAR of claim 53, wherein the transmembrane domain of CD8 is encoded by the nucleotide sequence of SEQ ID NO: 77;55. The CAR of any one of claims 44-54, wherein the CAR further comprises a hinge domain.

56. The CAR of claim 55, wherein the hinge domain is selected from the group consisting of an Fc fragment of an antibody, a hinge region of an antibody, a CH2 region of an antibody, a CH3 region of an antibody, an artificial hinge domain, a hinge comprising an amino acid sequence of a CD8 hinge, or any combination thereof.

57. The CAR of claim 55, wherein the hinge domain is a CD8 hinge.

58. The CAR of claim 57, wherein the CD8 hinge comprises the amino acid sequence of SEQ ID NO: 14.

59. The CAR of claim 58, wherein the CD8 hinge is encoded by the nucleotide sequence of SEQ ID NO: 74;60. The CAR of any one of claims 44-59, wherein the intracellular domain comprises a costimulatory signaling domain and an intracellular signaling domain.

61. The CAR of claim 60, wherein the costimulatory signaling domain comprises a costimulatory domain of a protein selected from the group consisting of proteins in the TNFR superfamily, CD28, 4-1BB (CD137), 0X40 (CD134), PD-1, CD7, LIGHT, CD83L, DAP10, DAP12, CD27, CD2, CD5, ICAM-1, LFA-1, Lek, TNFR-I, TNFI-II, Fas, CD30, CD40, ICOS, NKG2C, and B7-H3 (CD276), or a variant thereof.

62. The CAR of claim 60 or 61, wherein the costimulatory signaling domain comprises acostimulatory domain of 4-1BB.

63. The CAR of claim 62, wherein the costimulatory signaling domain comprises the amino acid sequence of SEQ ID NO: 20.

64. The CAR of claim 63, wherein the costimulatory signaling domain is encoded by the nucleotide sequence of SEQ ID NO: 81;65. The CAR of any one of claims 60-64, wherein the intracellular signaling domain comprises an intracellular domain selected from the group consisting of cytoplasmic signaling domains of a human CD3 zeta chain (CD3Q, FcyRIII, FcsRI, a cytoplasmic tail of an Fc receptor, an immunoreceptor tyrosine-based activation motif (IT AM) bearing cytoplasmic receptor, TCR zeta, FcR gamma, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d, or a variant thereof.

66. The CAR of claim 65, wherein the intracellular signaling domain comprises an intracellular domain of CD3^,67. The CAR of claim 66, wherein the intracellular domain of CD3^ comprises the amino acid sequence of SEQ ID NO: 24 or 25.

68. The CAR of claim 67, wherein the intracellular domain of CD3^ is encoded by the nucleotide sequence of SEQ ID NO: 85 or 86.

69. The CAR of claim 44, wherein the CAR comprises the antigen binding domain, a CD8a hinge region, a CD8a transmembrane domain, a 4- IBB costimulatory domain, and a CD3(^ intracellular signaling domain.

70. The CAR of any one of claims 44-69, wherein the CAR comprises the amino acid sequence of any one of SEQ ID NOs: 1-12 or 26-29, or comprises an amino acid sequence having at least 95%-99% identity to the amino acid sequence of SEQ ID NOs: 1-12 or 26-29.

71. The CAR of any one of claims 44-70, wherein the CAR is encoded by a nucleic acid sequence comprising the sequence of any one of SEQ ID NOs: 87-90; or a nucleotide sequence having at least 95%-99% identity to any one of SEQ ID NOs: 87-90.

72. The CAR of any one of claims 44-69 further comprising a second polypeptide.

73. The CAR of claim 72, further comprising a self-cleaving 2A peptide domain between the CAR and the second polypeptide.

74. The CAR of claim 73, wherein the self-cleaving peptide domain is selected from the group consisting of T2A, P2A, E2A and F2A.

75. A cell comprising the nucleic acid of any one of claims 1-33, the vector of any one of claims 34-42, or the CAR of any one of claims 43-74.

76. The cell of claim 75, wherein the cell is an immune cell or precursor cell thereof.

77. The cell of claim 75 or 76, wherein the cell is a natural killer (NK) cell or a T cell.

78. The cell of any one of claims 75-78, wherein the cell is a gamma delta T cell.

79. The cell of any one of claims 75-78, wherein the cell is an autologous cell.

80. The cell of any one of claims 75-78, wherein the cell is an allogeneic cell.

81. The cell of any one of claims 75-77, wherein the cell is from an NK92 cell line.

82. A pharmaceutical composition comprising the cell of any one of claims 75-81 and a pharmaceutically acceptable carrier.

83. A method for killing a DR5+ cell expressing a death receptor 5 antigen, comprising: contacting the DR5+ cell with the cell of any one of claims 75-81.

84. The method of claim 83, wherein the DR5+ cell is a melanoma cell, a pancreatic cancer cell, a lung cancer cell, a glioma cell, a breast cancer cell, a liver cancer cell, or an ovarian cancer cell.

85. The method of claim 83, wherein the DR5+ cell is a myeloid-derived suppressor cell (MDSC).

86. The method of claim 83, wherein the DR5+ cell is a cancer-associated fibroblast (CAF).

87. A method for treating cancer in a subject in need thereof, comprising: administering an effective amount of the pharmaceutical composition of claim 82 to the subject, thereby treating the cancer.

88. The method of claim 87, wherein the cancer is a solid tumor.

89. The method of claim 43, wherein the solid tumor is selected from the group consisting of melanoma, glioma, breast cancer, liver cancer, lung cancer, ovarian cancer, cervical cancer, uterus cancer, head and neck cancer, hepatocellular carcinoma, colon cancer, rectal cancer, colorectal carcinoma, kidney cancer, prostate cancer, gastric cancer, bronchus cancer, pancreatic cancer, urinary bladder cancer, and brain cancer.

90. The method of claim 89, wherein the solid tumor is melanoma, pancreatic cancer, lung cancer, glioma, breast cancer, liver cancer, or ovarian cancer.

91. The method of any one of claims 87-90, wherein the pharmaceutical composition is administered intratumorally.

92. The method of claim 87, wherein the cancer is a hematologic cancer.

93. The method of claim 92, wherein the hematologic cancer is a myeloid malignancy selected from the group consisting of acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), myelodysplastic neoplasm, and myeloproliferative neoplasm.

94. A method for preparing the cell of any one of claims 75-81, comprising: introducing into the cell the nucleic acid of any one of claims 1-33, or the vector of any one of claims 34-42.

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