Fig 2: HAdV-D49K interacts with CAR but is not dependent upon it as an entry receptor. (A) SPR was used to detect potential interactions between HAdV-D49K or HAdV-D49.KO1.K and CAR, CD46, and DSG2. (B) Antibody binding inhibition assays were used to assess the ability of recombinant HAdV-D49K to inhibit antibody binding to CHO-CAR or CHO-BC1 cells. (C and D) Blocking of HAdV-C5-mediated transduction was studied by preincubation of CHO-CAR cells with HAdV-C5K (C) or HAdV-D49K (D). (E and F) Blocking of HAdV-C5/D49K-mediated transduction was studied by preincubation of CHO-CAR cells with HAdV-C5K (E) or HAdV-D49K (F). (G and H) Blocking of HAdV-C5/D49K-mediated transduction was studied by preincubation of CHO-K1 cells with HAdV-C5K (G) or HAdV-D49K (H). Cells were infected with 5,000 viral particles per cell of replication-deficient HAdV-C5 or HAdV-C5/D49K expressing a luciferase transgene, with or without blockade by 20 μg of recombinant HAdV-C5 or HAdV-D49 fiber knob protein. n = 3; error is expressed ± the SD. *, P < 0.05; **, P < 0.01 (based on a nonparametric Mann-Whitney test).

Fig 3: Desmoglein 2 is unlikely to be a receptor for HAdV-D26K or HAdV-D48K. The dissociation constant was calculated for HAdV-B3K binding to DSG2, but kinetics were too fast to determine KOn or KOff (a), the KD curve is shown for HAdV-B3K while HAdV-D26K and HAdV-D48K are seen to form no interaction with DSG2 (b). nm (not measured) indicates that the kinetics were too fast to measure, nb denotes no binding

Fig 4: 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 5: DSG2 is abundantly and universally expressed across epithelia-derived solid tumors.(A) DSG2mRNA expression in TCGA-cataloged tumors. (B) Percentage of patient tumors scoring medium/high for DSG2 protein via Human Protein Atlas (HPA) IHC. Percentage based on number of medium/high IHC-scored tissues divided by total number of available patient specimens per tumor type. CAB025122 dataset. (C) 2023 indexed Surveillance, Epidemiology, and End Results (SEER) cancer statistics highlighting the six tumor types producing the most U.S. cancer-related deaths. (D) Representative DSG2 protein IHC in HPA-cataloged solid tumors representing the six largest cancer mortality contributors. CAB025122 dataset. Scale bar, 200 μm. (E) Total DSG2 protein in various cancer cell line lysates measured by immunoblot and cataloged by tumor type: colorectal (dark blue), pancreatic (purple), lung (grey), prostate (cyan), breast (pink), liver (green), and pancreatic patient-derived xenograft tumor tissue (purple). (F) DSG2 cell-surface expression measured by flow cytometry relative to isotype-matched controls (left; dotted light grey). Inset values (right) indicate average DSG2 molecules per cell line. n=2 replicates/cell line. See also Supplementary Fig. S1-S3.