Evaluation of CLDN18.2-targeting CAR-T cells in vivo. A, NSG mice bearing PaTu8988s HS xenografts (CLDN18.2 H-score = 268) were dosed by tail vein with 9e6 CAR+ CLDN18.2 Bz CAR-T cells; total T-cell infusion number was matched across groups. Tumor volume and body weight were measured biweekly (n = 9). Serum levels of IFNγ were measured at 4, 7, and 14 days after infusion (n = 3). B, Schematic representation of a second-generation CAR-T design modified to replace 4-1BB with a CD28 costimulatory domain (28z). The average transduction efficiency (CAR+, day 9) of multiple healthy donors for clone 9 28z is shown. Representative FC plots of CAR surface expression at day 9 after lentivirus transduction were compared with UT control for a single donor. C, NSG mice bearing PaTu8988s HS xenografts were dosed as described in A with clone 9 CD28z or Bz CAR-T (n = 6) at indicated doses. Serum levels of IFNγ were measured at 4, 7, and 14 days after infusion (n = 3). D, Representative images of CLDN18.2 (top row) and CD3 (bottom row) staining in the stomachs of mice dosed with clone 9 CAR-T cells from C at indicated time points. All data represent mean ± SEM of replicate experiments or animals.
ARTICLE ABSTRACT
Claudin 18.2 (CLDN18.2) is a surface membrane protein that is crucial for maintaining tight junctions in gastric mucosal cells and is highly expressed in gastric, esophageal, and pancreatic cancers. Thus, CLDN18.2 is suited for exploration as a clinical target for chimeric antigen receptor T-cell (CAR-T) therapy in these indications. Although CAR-T therapies show promise, a challenge faced in their development for solid tumors is the immunosuppressive tumor microenvironment, which is often characterized by the presence of immune and stromal cells secreting high levels of TGFβ. The addition of TGFβ armoring can potentially expand CAR-T activity in solid tumors. We report on the preclinical development of a CLDN18.2-targeting CAR-T therapy showing effectiveness in patient models with CLDN18.2-positive gastric, esophageal, and pancreatic tumors.
The lead lentivirus product contains a unique single-chain variable fragment; CD28 and CD3z costimulatory and signaling domains; and dominant-negative TGF-β receptor armoring, enhancing targeting and safety and counteracting suppression. We developed a shortened cell manufacturing process to enhance the potency of the final product AZD6422.
AZD6422 exhibited significant antitumor activity and tolerability in multiple patient-derived tumor xenograft models with various CLDN18.2 and TGF-β levels, as determined by IHC. The efficacy of armored CAR-T cells in tumor models with elevated TGFβ was increased in vitro and in vivo. In vitro restimulation assays established greater persistence and cytolytic function of AZD6422 compared with a traditionally manufactured CAR-T.
AZD6422 was safe and efficacious in patient-derived, CLDN18.2-positive murine models of gastrointestinal cancers. Our data support further clinical development of AZD6422 for patients with these cancers.
