Figure 4 from Preclinical Evaluation of AZD6422, an Armored Chimeric Antigen Receptor T Cell Targeting CLDN18.2 in Gastric, Pancreatic, and Esophageal Cancers

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Rationale for selection of TGFβ armoring and proof of mechanism in vitro and in vivo. A, Quantitative analysis of intensity and prevalence and representative images of TGFβ staining in a subset of patient tumor samples [gastric cancer (GC), n = 130; esophageal adenocarcinoma (EAc), n = 15; PDAC, n = 71]. Data represent the pooled scores of tumor and stromal compartments. B, Schematic representation of second-generation CAR-T lentivirus design, including an IgG4P hinge, CD28 transmembrane, CD28 costimulatory domain, and CD3z (unarmored CAR-T cells) or additional T2A self-cleaving peptide and dnTGFβRII (armored CAR-T cells). Average fold expansion across multiple healthy donors is shown. There was no significant difference in expansion across groups (one-way ANOVA). Representative FC plots show CAR and TGFβRII surface expression at day 10 after lentivirus transduction compared with UT control. C, FACS-purified, serum-starved CAR-T cells were stimulated with 1 ng/mL rhTGFβ for various time periods. Western blotting was used to determine protein levels of p-SMAD2/3 and total SMAD2/3; β-actin was used as the loading control. D, Percent cytolysis of BxPC3 + CLDN18.2 cells as determined by xCELLigence RTCA assay after 72 hours of co-culture at a 1:1 ratio with CLDN18.2 CAR-T cells in the presence or absence of 10 ng/mL rhTGFβ. Results were analyzed by using paired t tests. E, NSG mice bearing pancreatic PDX (PANC22, H-score = 225, TGFβ intermediate) were dosed by tail vein with 3e6 CAR+ unarmored or dnTGFβRII CLDN18.2 CAR-T cells; the total T-cell infusion number was matched across groups. Tumor volume and body weight were measured biweekly (n = 5). Serum levels of IFNγ were measured at 7 and 14 days after infusion (n = 3). Representative images of CLDN18.2 and TGFβ staining IHC expression (20× scan) are shown. All data represent mean ± SEM of replicate experiments or animals.

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