Fig 2: DSG2-directed CAR-T cells are safe in a human DSG2 transgenic mouse model.(A) Safety study design. Mice were randomized (n=6/group; 3 male, 3 female) and twice pre-conditioned with cyclophosphamide (CTX; 100mg/kg i.p.). Blood was collected prior to control or αDSG2 CAR-T cell treatment (day 0) for baseline circulating cytokine quantification and comparison to peak cytokine levels 3 days after CAR-T cell administration. At two weeks post-treatment, blood and tissues were collected for organ-toxicity biomarker and histopathology evaluation, respectively. (B) Percent body weight change normalized to day 0 starting body weight prior to CAR-T cell treatment. (C) Serum cytokine levels in peripheral blood pre- (day 0) and post- (day 3) CAR-T cell treatment. (D-F) Serum biomarkers associated with liver (D), heart (E), and kidney (F) pathology in peripheral blood at study end (day 14). ALP, alkaline phosphatase; AST, aspartate aminotransferase; ALT, alanine aminotransferase; BUN, blood urine nitrogen. (G) Treatment-blinded histopathology evaluation of organs collected at study end (day 14). Color gradient denotes inflammation score: 1–4. Rows indicate individual animals. All data in B-F are presented as means ± SD. Body weight significance (B) determined by two-way ANOVA. Organ-damage biomarker analysis (D-F) was performed by unpaired t test with Welch’s correction. P > 0.05 was considered not statistically significant, ns. See also Supplementary Fig. S8-S9.

Fig 3: DSG2-directed CAR-T cells recognize and eliminate DSG2-expressing cancer cells in vitro.(A) Schematic diagram of the third-generation αDSG2 CAR, T2A, GFP reporter construct. (B) Representative αDSG2 CAR lentiviral transduction efficiency (GFP fluorescence) in human T cells. (C) Effector cytokine secretion in supernatants of αCD19 (control) or αDSG2 CAR-T cells co-cultured with wildtype (DSG2+) or DSG2-deficient (DSG2-) DLD-1 colorectal cancer cells in three donors after 24 hours. Data represented as mean ± SD; n=3 technical replicates/donor. (D) Cytolysis of wildtype (top) and DSG2-deficient (bottom) DLD-1 cells by αDSG2 CAR-T cells at 5:1 E:T (Effector:Target) ratio compared to donor-matched untransduced and control CAR-T cells. (E) Comparison of DLD-1 target cell cytolysis between αDSG2 (top) and control CAR-T cells (bottom) at a decreasing E:T ratio. (F) Composite cytolysis of DLD-1 target cells at decreasing E:T ratios of untransduced, control, or αDSG2CAR-T cells from E at 36 hours. (G) Cytolysis of DLD-1 target cells by αDSG2 CAR-T cells compared to control CAR-T cells in three separate donors (5:1 E:T). (H) Composite heat map of αDSG2CAR-T cell cytolysis across various solid tumor cell lines [colorectal (dark blue), pancreatic (purple), lung (grey), prostate (cyan), breast (pink), and liver (green)] relative to control CAR-T cells. Individual cytolysis curves for H are shown in Supplementary Figure S6B. All cytolysis graphs (D-H) depict group mean cytolysis ± SD; n≥3 technical replicates/CAR or T cell type. Cytolysis curve significance determined by area under curve (AUC) calculation and compared using an unpaired t test with Welch’s correction. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. See also Supplementary Fig. S3-S6.

Fig 4: DSG2-directed CAR-T cells eliminate DSG2-expressing solid tumor cell xenografts in vivo.(A) Tumor growth curves (top) and mouse overall survival (bottom) of subcutaneous DLD-1 colorectal tumors after control (CD19) or DSG2-directed CAR-T cell treatment (vertical dotted line denotes treatment on day 15). (B-D) αDSG2 CAR-T cell dose response in mice bearing peritoneal DLD-1 metastases. (B) Schematic of luciferase-expressing DLD-1 i.p. implantation and treatment with control or decreasing quantities of αDSG2 CAR-T cells. (C) Bioluminescence images of mice with i.p. luciferase-expressing DLD-1 tumors; arrow denotes treatment on day 14. (D) Tumor burden (BLI) over time (left) and associated overall survival (right). Individual mouse BLI are shown with a bolded overlay indicating the median BLI of all mice per group. Vertical dotted line denotes treatment on day 14. (E-I) Metastatic tumor models were established in NSG mice. Tumor burden (via BLI) and overall survival were monitored after control or αDSG2 CAR-T cell treatment. Vertical dotted lines denote treatment day. (E) BxPC-3 metastatic pancreatic tumor model (i.p.). Treatment on day 22. (F) A549 lung adenocarcinoma model (i.v.). Treatment on day 18. (G) DU145 metastatic prostate cancer model (i.v.). Treatment on day 39. (H) MDA-MB-231 metastatic breast cancer model (i.v.). Treatment on day 34. (I) HepG2 metastatic liver cancer model (i.p.). Treatment on day 11. All survival was analyzed using the log-rank (Mantel-Cox) test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant. All panels employed 5×106 CAR+ T cells, except B-D as indicated. See also Supplementary Fig. S7.

Fig 5: DSG2-directed CAR-T cells eliminate orthotopic and patient-derived pancreatic tumors.(A-C) Tumor burden quantified over time by BLI (A), circulating serum CEA levels (B), and overall survival (C) in mice surgically implanted with luciferase-expressing AsPC-1 pancreatic cancer cells in the pancreas following control or αDSG2 CAR-T cell treatment; vertical dotted line denotes treatment on day 7. (D-F) Tumor burden quantified over time via caliper measurement (D), circulating serum CEA levels (E), and overall survival (F) in mice challenged subcutaneously with patient-derived pancreatic cancer xenograft tissue. Mice received either control or αDSG2 CAR-T cells when tumors reached ~150 mm3. Longitudinal dotted line indicates euthanasia criterion. Individual mouse BLI (A) and serum CEA concentrations (B,E) are shown with a bolded overlay indicating the median of all mice per group. Tumor growth and serum CEA were analyzed by two-way ANOVA. All survival was analyzed using the log-rank (Mantel-Cox) test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.