Abstract 6113: Cytotoxic and antitumoral effect of an anti-CD44v6 CAR-T against gastric cancer

Abstract Background: Gastric cancer ranks among the top 5 cancer types in terms of both incidence and mortality, with a 5-year overall survival rate of only 29%. CD44 variant 6 (CD44v6) is an isoform of CD44 and is identified in 70% of gastric cancer cases. CD44v6 is considered a marker of poor prognosis, and its expression varies among different types of gastric cancer. CD44v6 has a role in apoptosis resistance, metastasis, and tumor progression, and is shown to be enriched in recurrent tumors. CD44v6 expression in normal tissue is very low and is restricted to certain cell types. Therefore, CD44v6 is a promising target for targeted therapies against gastric cancer. In this study, we aim to develop a chimeric antigen receptor (CAR) T cell therapy targeting CD44v6 and evaluate its functionality, cytotoxic activity and antitumoral effect. The scFv was derived from the monoclonal antibody (mAb) known as bivatuzumab (BIWA-4), a humanized mAb that has been used in clinical studies as radiotherapy and as antibody drug conjugates. Methods: Plasmid containing the CAR sequence with the BIWA-4 scFv was synthesized, and Jurkat T cell line and primary human T cells were transduced with lentivirus containing the CAR sequence. Jurkat cell line was used to measure binding to recombinant protein and assess CD69 expression after antigen exposure. Primary T cells transduced with CAR were co-cultured at different E:T ratios with gastric cancer cell lines (AGS, SNU 638, and MKN45) with different levels of expression of CD44v6. Target cell killing was measured by flow cytometry, bioluminescence, and fluorescence microscopy. T cell exhaustion and exhaustion phenotype were evaluated prior killing assays. For in vivo experiments, NSG mice will be engrafted with gastric cancer cell lines and tumor progression will be evaluated by IVIS. Results: While high affinity variants of bivatuzumab have been used to construct CAR (BIWA-8), ours is the first study that used medium affinity variant known as BIWA-4. Jurkat cells transduced with the car construct demonstrated specific binding to the recombinant monomeric CD44v6, which triggered surface expression of early T cell activation marker CD69. With varying concentrations of monomeric CD44v6, the affinity of CAR to CD44v6 was determined to be ∼100 nM KD. Transduction efficiency of primary T cells remained stable, reaching ∼60% after 10 days of expansion. Most CAR T cells exhibited effector memory phenotype, with low levels of PD1. CD44v6 CAR T killing in vitro was correlated with the E: T ratio and the level of antigen expression, demonstrating specificity of CAR and avoiding off-target effects with CD44v6 negative cells. Conclusion: We have successfully developed CAR T cells possessing medium affinity against CD44v6 and confirmed CAR-specific killing of CD44v6 positive gastric cancer cells. Citation Format: gonzalo gonzalez, Moonsoo Jin, Yanping Yang, Elizabeth Ortiz, Yogindra Vedvyas. Cytotoxic and antitumoral effect of an anti-CD44v6 CAR-T against gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6113.