Dual targeting of HER2 and AXL by an eKiH-engineered bispecific ADC to overcome resistance in solid tumors

Background: AXL signaling is a key mediator of drug resistance, metastasis, and immune modulation in solid tumors. Its activation drives epithelial-mesenchymal transition (EMT) and promotes tumor aggressiveness, underscoring its role in therapeutic resistance. HER2, a clinically validated ADC target, is similarly associated with oncogenic signaling and metastatic progression. Dual targeting of HER2 and AXL offers a rational approach to suppress complementary resistance pathways and overcome tumor heterogeneity, particularly in patients refractory to standard therapies. We therefore developed a HER2×AXL bispecific ADC using Dong-A ST BsAb platform with an enhanced knob-into-hole (eKiH) interface to improve heavy-chain pairing fidelity and bispecific assembly. Methods: Lead HER2 and AXL antibodies were selected for high affinity and improved internalization; the AXL lead outperformed benchmark anti-AXL antibodies in binding and internalization assays. Bispecifics were assembled using Dong-A ST BsAb platform and site-selectively conjugated to monomethyl auristatin E (MMAE), exatecan (a topoisomerase-I inhibitor), or dual-payload configurations. Single-cell RNA-seq (scRNA-seq) datasets from lung, breast, and gastric cancers were analyzed to profile HER2 heterogeneity and AXL-associated epithelial-mesenchymal transition (EMT) signatures. Multiplex immunohistochemistry (mIHC) on tissue microarrays quantified spatial co-expression and heterogeneity. Combination potential with immune checkpoint inhibitors (ICIs) was explored via immune-correlative analyses and ongoing co-treatment studies. Results: scRNA-seq revealed pronounced intra- and inter-tumoral HER2 heterogeneity across indications, while AXL-high tumors exhibited elevated EMT-related markers. mIHC confirmed mosaic/cluster-type co-expression and heterogeneous distribution of HER2 and AXL across multiple tumor types. HER2xAXL BsAb retained high-affinity binding to both targets and efficient internalization, with preliminary in-vitro cytotoxicity observed in HER2-high, AXL-high, and co-expressing models using MMAE, exatecan, and dual-payload ADCs. Immune-correlative data support the mechanistic rationale for ICI combinations, and formal co-treatment evaluations are in progress. Conclusions: HER2xAXL is a rationally engineered HER2×AXL bispecific ADC designed to mitigate target heterogeneity and therapy resistance. These preclinical data support investigation in HER2-expressing solid tumors-including lung, breast, and gastric cancers-particularly in patients who have failed standard first-/second-line regimens.