TSY-310, a novel bispecific EGFR×ROR1 ADC: Assessment of antitumor activity in heterogeneous breast tumors through enhanced internalization and bystander cytotoxicity.

Abstract

3086 Background: Epidermal growth factor receptor (EGFR) is a clinically validated target and is highly expressed in various solid tumors, including triple-negative breast cancer (TNBC). Clinical efficacy of EGFR-directed therapies in TNBC, however, is often limited by intratumoral heterogeneity, acquired resistance, and mutations conferring insensitivity to tyrosine kinase inhibitors. TSY-310 is a novel bispecific EGFR×ROR1 nanobody-Fc fusion antibody–drug conjugate (BsADC) designed to address these challenges by simultaneously targeting EGFR and receptor tyrosine kinase-like orphan receptor 1 (ROR1). TSY-310 carries an average of 3.6 molecules of monomethyl auristatin E (MMAE) via a protease-cleavable valine–citrulline linker, enabling potent and tumor-selective cytotoxicity, and induces strong tumor regression across multiple NSCLC PDX models representing a range of EGFR and ROR1 expression levels. Methods: Cytotoxicity was evaluated in cell lines with distinct EGFR/ROR1 expression profiles. Bystander killing was assessed in co-culture systems of antigen-positive and antigen-negative cells. Cellular internalization and trafficking were analyzed by immunofluorescence microscopy. In vivo efficacy and bystander activity are investigated in xenograft models with heterogeneous antigen expression. Results: TSY-310 exhibited sub-nanomolar cytotoxicity (IC 50 = 0.45 nM) against MDA-MB-468 (ROR1⁺/EGFR⁺) TNBC cells in correlation with dual receptor expression levels, and demonstrated robust bystander killing, eliminating neighboring antigen-negative A427-Luc and MDA-MB-453-Luc cells through MMAE diffusion in co-culture systems. Rapid internalization of TSY-310 was observed in dual-positive MDA-MB-468 cells within 6 hours, consistent with enhanced cytotoxic activity. In a EGFR⁺/ROR1⁺ HBCx-28 TNBC PDX model, it achieved complete and sustained tumor regressions in all treated mice (5/5) and demonstrated superior efficacy compared with the monospecific ROR1-directed ADC on a payload-equivalent basis. In vivo studies to assess bystander activity and efficacy in heterogeneous tumor models are ongoing. Conclusions: These findings highlight TSY-310 as a first-in-class bispecific ADC capable of overcoming tumor heterogeneity through dual binding and bystander killing, supporting its potential as an effective therapeutic agent for heterogeneous solid tumors such as TNBC, where existing EGFR-targeted therapies have shown limited benefit.