Decoding the response and resistant features to the Claudin18.2-specific CAR-T cell CT041 in gastric cancer: A multi-omics exploratory biomarker analysis of the phase 1 clinical trial.

4015 Background: CT041, a Claudin18.2-specific CAR-T cell therapy against gastric cancer (GC), has demonstrated encouraging efficacy and a manageable safety performance. However, the characteristics of response and resistance to CT041 therapy remain unclear. Methods: An exploratory biomarker analysis of the phase 1 CT041-CG4006 clinical trial (NCT03874897) was conducted. Baseline samples were collected from primary tumors (PT), and dynamic samples were collected from peripheral blood and malignant ascites at day 0, 3, and 7 of 35 patients. These samples underwent single-cell RNA sequencing (n = 41), spatial transcriptomics (ST, n = 8 for Visium V2 and n = 5 for Xenium 5K), and multiplex immunofluorescence detection (n = 28). Results: In the responders, significantly higher GZMK+CD8+ T-lymphocyte infiltrates, which were in a progenitor-like exhausted (Tpex) state, were identified within PT. More GZMK+ Tpex infiltration also correlated with better prognosis (high vs. low: progression-free survival 9.98 vs. 5.45 months, P = 0.020; overall survival 18.03 vs. 8.03 months, P = 0.038), manifesting superior potential as a predictor of CT041 response (AUC 0.87, 95%CI 0.72-1.00). Moreover, strong and positive associations between GZMK+ Tpex and IRF8+ B-lymphocytes were discovered. ST detection further uncovered that they tended to co-localize within tertiary lymphoid structures and intimately interacted via the MHC-I and CCL5-CXCR4 pathways. In cases resistant to CT041, significantly higher infiltrates of IQGAP3+ cancer cells, a subset characterized by augmented proliferative activity, were discovered. While IQGAP3+ cancer cells hindered the infiltration of GZMK+ Tpex, they exhibited a co-infiltrated pattern with IL6+ fibroblasts, wherein enhanced epithelial-to-mesenchymal transition pathway activity was noted. Through dynamically parsing the blood and ascites samples, an initial activated and subsequent exhausted state of GZMK+ cytotoxic T-lymphocyte was observed after the infusion of CT041. Additionally, significant up-regulation of NR4A1, a crucial regulator of Tpex, and CCR9, a key homing molecule to the gastrointestinal mucosa, of peripheral T-lymphocytes was detected in responsive cases. Conclusions: This exploratory analysis first unveiled the biological underpinnings and clinical implications of GZMK+ Tpex in determining response and resistance to the Claudin18.2-specific CAR-T therapy.