Title: HER2-mediated internalization of cytotoxic agents in ERBB2 amplified or mutant lung cancers Running title: Antitumoral activity of anti-HER2 ADCs in lung cancer

Amplification and oncogenic mutations of ERBB2 , the gene encoding the HER2 receptor tyrosine kinase, promote receptor hyperactivation and tumor growth. Here we demonstrate that HER2 ubiquitination and internalization, rather than its overexpression, are key mechanisms underlying endocytosis and consequent efficacy of the anti-HER2 antibody-drug conjugates (ADCs) ado-trastuzumab emtansine (T-DM1) and trastuzumab deruxtecan (T-DXd) in lung cancer cell lines and patient-derived xenograft models. These data translated into a 51% response rate in a clinical trial of T-DM1 in 49 patients with ERBB2 /HER2-amplified or mutant lung cancers. We show that co-treatment with irreversible pan-HER inhibitors enhances receptor ubiquitination and consequent ADC internalization and efficacy. We also demonstrate that ADC switching to T-DXd, which harbors a different cytotoxic payload, achieves durable responses in a patient with lung cancer and corresponding xenograft model developing resistance to T-DM1. Our findings may help guide future clinical trials and expand the field of ADC as cancer therapy. inhibitors or ADC therapy switching can improve the efficacy of these agents. Our findings can potentially be extended to other HER2-activated tumor types, for which pharmacological HER2 signaling blockade is insufficient to elicit strong and durable responses and may help guide future clinical trials and expand the field of ADC as cancer therapy. M.G. K. , J.T. P., R.S. , J.T. , G.A. U. , E. de S. , C.M. R. , M.S. designed and analyzed the experiments. R.A. F. and N.U. L. provided the clinical data of the breast cancer patient treated with neratinib in combination with T-DM1. B.T. L. , R.S. , H-Y.T. and P.R. assisted with statistical analysis. B.T. L. , H-Y.T. , M.L. M. , C.X. , M.O. , R.S. , M.E. A. , G.A. U. assisted with prospective genomic and clinical data collection and sample annotation. S.T. , F.C. , W-L.L. and A.B performed the mass spectrometry and analyzed the proteomic data. J.T. P., N.R. and C.M. R. contributed with intellectual insights for the study design. B.T. L. , F.M. , S.M. , E.C. , M.S. wrote the manuscript with help from all the authors. **=<0.01 at the indicated time point, ns: non-significant. (n = 2 independent experiments, ≥80 cells analyzed in total per condition, per time point). E) Western blot analysis of MCF10A isogenic models incubated with T-DM1 (10 μg/mL) or vehicle as control for 24 hours. Total and phospho-HER2, as well as and total phospho-tyrosine were assayed. Cleaved PARP was used to determine the apoptosis induction. Actin was included as loading control. F) Western blot analysis of NCI-H2030 isogenic models incubated with T-DM1 (10 μg/mL) or vehicle as control for 24 hours. Total and phospho-HER2, as well as and total phospho-tyrosine were assayed. Cleaved PARP was used to determine the apoptosis induction. Actin was included as loading control. G, H) 89 Zr-Trastuzumab PET/CT scan representative images of ERBB2 amplified (G) and ERBB2 mutant (H) NSCLC patients (from a total of n=4 ERBB2 amplified and n=4 ERBB2 mutant NSCLC patients evaluated by 89 Zr-Trastuzumab PET/CT scan). I) In vivo efficacy study of an ERBB2 YVMA mutant lung PDX treated with T-DM1 (15 mg/kg, i.v. once a week). Measurements show average tumor volumes +/- SEM, n=7 animals per vivo study of a ERBB2 S310F mutant and amplified lung PDX treated with T-DM1 Comparisons between the two groups for each time point were performed using 2-way ANOVA test, at time