Abstract 4807: Claudin-3-targeting CAR-T using C-terminal domain of Clostridium perfringens enterotoxin for TKI-resistant EGFR-mutated NSCLC
Authors: Oh Min Kwon, Yunjoo Joo, You Won Lee, Seunghyun Jeon, Jungwon Kim, Hwa Jin Lee, Mi Ran Yun, Byoung Chul Cho
Journal: Cancer Research (2025)

Abstract Introduction: Epidermal growth factor receptor (EGFR) mutations (MT) are oncogenic drivers in non-small cell lung cancer (NSCLC). However, acquired resistance to EGFR tyrosine kinase inhibitors (TKIs) remains a significant challenge for the treatment of NSCLC. Claudin-3 (CLDN3), a tight junction protein involved in maintaining epithelial integrity, has been reported to be overexpressed via EGFR downstream pathways or mislocalized in NSCLC, contributing to its malignant potential and making it a promising therapeutic target. The Clostridium perfringens enterotoxin (CPE), with its receptor-binding C-terminal domain (cCPE), offers a capability to specifically target CLDN3. This study aims to develop a novel CAR-T therapy utilizing cCPE to target CLDN3, focusing on overcoming resistance in post-TKI NSCLC. Methods: CLDN3 expression was evaluated through scRNA sequencing (scRNA-seq) of primary lung tissues from 95 patients: EGFR-WT (n=35), treatment-naïve EGFR-MT (n=55), post-EGFR-TKI (n=5), and normal tissues (n=22). Protein expression was validated using ATCC and patient-derived cell lines (PDCs), including NCI-H1703 (EGFR-WT, CLDN3-negative) and EGFR-MT cell lines: HCC827 (E19del), HCC827_AR (E19del/MET amplification), YU1092 (L861Q), YU1157 (L858R), YU1162 (E19del), YU1150 (L858R/T790M), YU1182 (L858R/C797S), and YU1097 (E19del/T790M/C797S). CLDN3-overexpressing cell line (H1703Cld3) was engineered via viral transduction. CAR constructs were designed with various cCPE lengths (amino acids 116-319 [C3L], 184-319 [C3M], 194-319 [C3S]) and linked to 4-1BB and CD3ζ intracellular signaling domains via CD8α hinge and transmembrane regions. CAR expression was analyzed using flow cytometry. Antitumor activity was assessed through RTCA, LDH assays, and ELISA. In vivo efficacy was evaluated in xenograft models using H1703, H1703Cld3, and CLDN3-positive PDCs. Results: scRNA-seq analysis revealed significantly elevated CLDN3 expression in post-EGFR-TKI patients compared to EGFR-WT and -MT. H1703 was selected as a CLDN3-, and HCC827, HCC827_AR, YU1092, YU1157, YU1162, YU1150, YU1182 and YU1097 as CLDN3+ target cells. Among the CARs, C3M showed optimal transduction efficiency (46.2%) and balanced activity, producing high IFN-γ (70,000pg/mL) against H1703Cld3 without auto-reactivity observed in C3S. C3M-CAR-T cells showed specifically higher secretion of IL-2, IFN-γ, and TNF-α against CLDN3+ target cells compared to CLDN3-. LDH assay and RTCA confirmed robust tumor lysis of CLDN3+ tumor cells than mock T cells. In vivo, C3M-CAR-T treatment led to significant tumor regression in xenografts of H1703Cld3 and PDCs. Conclusions: This study highlights CLDN3 as a promising therapeutic target using cCPE-based CAR-T in NSCLC for overcoming resistance to EGFR-TKI. The C3M-CAR-T demonstrated robust anti-tumor efficacy across in vitro and in vivo models. Citation Format: Oh Min Kwon, Yunjoo Joo, You Won Lee, Seunghyun Jeon, Jungwon Kim, Hwa Jin Lee, Mi Ran Yun, Byoung Chul Cho. Claudin-3-targeting CAR-T using C-terminal domain of Clostridium perfringens enterotoxin for TKI-resistant EGFR-mutated NSCLC [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 4807.