Development and evaluation of CLDN18.2-targeting CAR-T cells in vitro. A, Schematic representation of second-generation CAR-T lentivirus design, which includes a 4-1BB costimulatory domain (Bz). The table shows for each CLDN18.2-reactive clone the relative binding affinity (human and mouse), reactivity to mutant CLDN18.2 (M149L), and average transduction efficiency (CAR+, day 9) of multiple healthy donors. Representative FC plots of CAR surface expression at day 9 after lentivirus transduction were compared with UT control for a single donor. B, CLDN18.2 cell surface expression of various cell lines as determined by FC with 5 μg/mL CLDN18.2-reactive clones compared with nonspecific isotype antibody (R347). C, Epitope characterization of CLDN18.2-reactive clones. The AlphaFold structure on the far left (red) represents all sites of point mutation in HEK293 cells that vary between CLDN18.1 and CLDN18.2 in the first extracellular loop; the color-coded diagrams represent sites that influence respective clone binding. D, Percent cytolysis of HEK293 + huCLDN18.1, HEK293 + huCLDN18.2, HEK293 + muCLDN18.1, HEK293 + muCLDN18.2, and PaTu8988s HS cells determined by xCELLigence RTCA assay after 48 hours of co-culture with CLDN18.2 CAR-T cells at a 1:1 E:T ratio. The supernatants from the xCELLigence assay were collected at 24 hours for cytokine assessment (Meso Scale Discovery) assay. All data represent mean ± SEM of replicate experiments.
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.
