Overcoming target heterogeneity in gastric cancer with an eKiH-engineered CLDN18.2×HER2 bispecific ADC

Background: Target heterogeneity, mosaic and cluster expression of CLDN18.2 and HER2 is common in gastric cancer (GC) and undermines the efficacy of single-antigen therapies. We previously developed DA3501 (AT-211), a CLDN18.2-targeting ADC currently advancing toward Phase 1 evaluation. The DA3501 antibody clone demonstrates enhanced affinity and internalization compared with zolbetuximab. To more comprehensively address heterogeneous tumors, we engineered a CLDN18.2×HER2 bispecific ADC built on Dong-A ST BsAb platform incorporating an enhanced knob-into-hole (eKiH) interface to improve heavy-chain pairing fidelity and bispecific assembly. Methods: Antibody discovery and optimization yielded lead clones for CLDN18.2 and HER2 with improved binding and internalization. Bispecifics were assembled using Dong-A ST BsAb platform. Conjugation to monomethyl auristatin E (MMAE), exatecan (a topoisomerase-I inhibitor), or dual-payload configurations was performed using site-selective chemistry. Target prevalence and co-expression were evaluated in human GC using single-cell RNA sequencing (scRNA-seq) of tumor specimens and multiplex immunohistochemistry (mIHC) on tissue microarrays (TMAs). Immune correlates were analyzed to assess combinatorial potential with immune checkpoint inhibitors (ICIs). Results: scRNA-seq revealed CLDN18.2 and HER2 expression in mutually exclusive as well as co-expressing tumor cell subsets, corroborated by mIHC showing intra-tumoral mosaic and clustered patterns. The DA3501 anti-CLDN18.2 clone exhibited superior binding kinetics and internalization versus zolbetuximab. ATS1002 retained high-affinity binding to both targets and demonstrated efficient internalization in dual-positive and single-positive (CLDN18.2 or HER2) models, supporting a “catch-all” approach for heterogeneous lesions. ADCs conjugated with MMAE, exatecan, or dual payloads showed potent in vitro cytotoxicity across CLDN18.2-high, HER2-high, and co-expressing GC lines. Immune-correlative analyses indicated potential synergy with ICIs in tumors exhibiting activated T-cell and myeloid signatures. Conclusions: This BsAb is a rationally engineered CLDN18.2×HER2 bispecific ADC designed to overcome target heterogeneity in GC. Together with the DA3501 program, it broadens therapeutic coverage of CLDN18.2/HER2-expressing tumors and combination-strategy studies, including ICI co-therapy.