Abstract 5451: JSKN021, an innovative site-specific dual-payload bispecific antibody drug conjugate targeting EGFR and HER3 exhibits potent preclinical activities

Abstract Background: EGFR and HER3, both belong to the EGFR family, are widely overexpressed in human malignancies, rendering them promising targets for cancer treatment. Cancer is a heterogeneous disease. Inter- and intratumor heterogeneity is believed to be a major factor contributing to recurrence, metastasis and resistance to current socs. To address these therapeutic challenges, we generated JSKN021, a dual payload ADC targeting EGFR/HER3. Both payloads, a novel DNA topoisomerase I inhibitor (T01, Alphatecan) and MMAE, are conjugated to glycan on Fc via cleavable linkers. The conjugation processes were carried out via combination of glycan transferase and click reaction. The streamlined process generated site specific and homogeneous conjugation with DAR4(T01)and DAR2 (MMAE).In this study, the pharmacological activity of JSKN021 was evaluated using relevant in vitro cell killing assays and in vivo models. Methods: A novel Topo 1 inhibitor T01 and MMAE were stably conjugated to an EGFR/HER3 mAb using a glycan based site-specific manner. The conjugated products were characterized by LC-MS. Binding specificity of JSKN021 was confirmed by ELISA. In addition, release of payload were evaluated by LC-HRMS. At last, in vitro cell growth inhibitory and in vivo antitumor activities of JSKN021 were evaluated using different cancer cell lines and CDX models in comparison to single payload ADCs. Results: After conjugation, LC-MS results showed that JSKN021 had a homogeneous DAR4 for T01 payload and DAR2 for MMAE. JSKN021 was demonstrated to bind EGFR and HER3 simultaneously. In addition, both T01 and MMAE were detected in cell culture supernatant and suspension of HCC827. Yet, no other metabolites were found detectable. JSKN021 demonstrated excellent stability in rats, mice, monkeys, and human serum, with minimal payload release. The stability was derived from the advantage of glycan based conjugation. JSKN021 inhibited the growth of cancer cells with either Her3 or EGFR or both expression, such as HCC827, MDA-MB-468, A431 and NCI-H1975. Furthermore, JSKN021 showed stronger tumor inhibition efficacy than mono payload ADCs in multiple CDX models. Conclusion: The preclinical studies suggest JSKN021, as a dual-payload EGFR/HER3 bispecific ADC, might be a promising novel antitumor therapeutic agent. Citation Format: Ting Xu, Fan Li, Zhilong Lai, Rupeng Qu. JSKN021, an innovative site-specific dual-payload bispecific antibody drug conjugate targeting EGFR and HER3 exhibits potent preclinical activities [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 5451.