Zongertinib (BI 1810631), an Irreversible HER2 TKI, Spares EGFR Signaling and Improves Therapeutic Response in Preclinical Models and Patients with HER2-Driven Cancers

Abstract

Mutations in ERBB2 (encoding HER2) occur in 2% to 4% of non-small cell lung cancer (NSCLC) and confer poor prognosis. ERBB-targeting tyrosine kinase inhibitors, approved for treating other HER2-dependent cancers, are ineffective in HER2-mutant NSCLC due to dose-limiting toxicities or suboptimal potency. We report the discovery of zongertinib (BI 1810631), a covalent HER2 inhibitor. Zongertinib potently and selectively blocks HER2, while sparing EGFR, and inhibits the growth of cells dependent on HER2 oncogenic driver events, including HER2-dependent human cancer cells resistant to trastuzumab deruxtecan. Zongertinib displays potent antitumor activity in HER2-dependent human NSCLC xenograft models and enhances the activities of antibody-drug conjugates and KRASG12C inhibitors without causing obvious toxicities. The preclinical efficacy of zongertinib translates in objective responses in patients with HER2-dependent tumors, including cholangiocarcinoma (SDC4-NRG1 fusion) and breast cancer (V777L HER2 mutation), thus supporting the ongoing clinical development of zongertinib. Significance: HER2-mutant NSCLC poses a challenge in the clinic due to limited options for targeted therapies. Pan-ERBB blockers are limited by wild-type EGFR-mediated toxicity. Zongertinib is a highly potent and wild-type EGFR-sparing HER2 inhibitor that is active in HER2-driven tumors in the preclinical and clinical settings.

Figure 1.

Zongertinib is a highly selective covalent HER2 inhibitor. A, Potency of zongertinib and the noncovalent matched pair binder BI-3999 in a cellular phosphorylation assay using HEK cells and antiproliferative assays using Ba/F3 and human cancer cell lines. B, Physicochemical parameters and in vitro DMPK of zongertinib. CL_int, intrinsic clearance; d, dog; f_u, fraction unbound; FaSSIF, fasted state–simulated intestinal fluid; FeSSIF, fed state–simulated intestinal fluid; h, human; m, mouse; PPB, plasma–protein binding; r, rat. PPB data were obtained using the methods reported in the Methods section. C, Chemical structures of zongertinib and the noncovalent matched pair binder BI-3999. D, Kinase selectivity profile of zongertinib: Phylogenetic tree for protein kinases with the size and color of nodes indicating inhibition at 1 μmol/L in in vitro kinase assays. The kinase assays reported in the figure were performed as single measurements. E, IC₅₀ values of zongertinib in a set of selected kinases in a biochemical kinase assay, n = 1. F, Summary of the prediction of zongertinib PK and therapeutic dose in human. AUC_{ss, tau 24 hours}, AUC at steady state at a dosing interval of 24 hours; DMPK, x; F, oral bioavailability; k_a, absorption rate constant; ; t_max, time of maximum plasma concentration; V_ss, volume of distribution at steady state.

Figure 2.

Zongertinib is active on HER2 mutation or overexpression–dependent cell lines and spares EGFR^WT. A, Selectivity index plot for proliferation assay in Ba/F3 cells dependent on HER2^YVMA in reference to EGFR^WT. Formula for the calculation of the slectivity index is provided in (49). The screen of TKIs was conducted in a single experiment without repetition except for zongertinib (n = 3). Each dot represents one (except zongertinib, where the dot represents the mean) indicated compound measurement. B, Dose–response curves of zongertinib and poziotinib in Ba/F3 cells dependent on HER2 and EGFR variants. C, IC₅₀ values for zongertinib in Ba/F3 cells dependent on EGFR^WT, HER2^WT, or indicated HER2 variants. Each dot represents the result from an independent dose–response experiment using the indicated cell line. Y-axis: IC₅₀ in nmol/L. D, Antiproliferative activity of zongertinib in a panel of cancer cells. X-axis: cell lines arranged according to oncogenic mechanism. Y-axis: IC₅₀ in nmol/L. EGFR and HER2 status is indicated in the inset. Individual points indicate individual measurements in fully independent experiments. E, Modulation of HER2 signaling at the level of HER2 and downstream mediators in NCI-N87 cells upon zongertinib treatment at different doses and time points. Plots show total protein for HER2, ERK, AKT, cyclin D1, p27, cleaved caspase 3, and cleaved PARP as well as phosphorylated HER2 (Y1196), ERK, and AKT (S473). Actin used as the loading control is also shown. F, Modulation of HER2 and EGFR as well as their downstream mediators in H2170 HER2^YVMA, MDA-MB-175-VII, NCI-N87 (all HER2-driven), and A-431 (EGFR^WT-driven) cells upon zongertinib or poziotinib treatment. Plots show total protein for HER2, EGFR, ERK, AKT, S6, and DUSP6 as well as phosphorylated HER2, EGFR, ERK, S6, and AKT. GAPDH used as the loading control is also shown. G, Downregulation of MPAS genes (subset of MAP kinase pathway downstream genes) in PC-9 EGFR^KO,HER2^YVMA cells upon zongertinib treatment in vitro. H, Modulation of MAPK downstream genes in PC-9 EGFR^KO,HER2^YVMA cells upon zongertinib treatment. I, Downregulation of MPAS genes in NCI-N87 cells upon zongertinib treatment in vitro. NOS, x; TNBC, x.

Figure 3.

Zongertinib activity correlates with the HER2 status in cell lines. A, Sensitivity to zongertinib in 846 cancer cell lines color-coded by HER2 expression levels using a cutoff of ≥ TPM 250 (250 ; HER2-high). Cell lines are arranged from left to right in order of decreasing sensitivity to zongertinib. B, Correlation analysis of HER2 protein and mRNA levels in cancer cell lines (left). Proteome- and transcriptome-wide correlation analysis of protein and mRNA levels across cell lines. X-axis: Pearson correlation coefficients binned (right). C, Validation of PRISM screen in selected cancer cell lines spanning a spectrum of HER2 expression levels. X-axis: HER2 mRNA expression in TPM; Y-axis: IC₅₀ in nmol/L (CellTiter-Glo assay). D, Western blot for the total and phosphorylated ERBB members and downstream mediators in indicated cell lines from C in untreated setting (baseline). Note: p-AKT (S473). E, Prevalence of HER2 amplifications and overexpression in indicated patient cohorts across a total of >60,000 tumor samples from Tempus AI pan-cancer gene expression records. A HER2 overexpression cutoff was computed using logistic regression on all HER2-amplified (CN ≥6) samples. Samples were then grouped by indication, and the prevalence of ERRB2-amplified, HER2-overexpressed/ERBB2 low-amplified, and HER2-overexpressed/ERBB2 nonamplified cases per cohort is visualized. Indications are ranked by the overall prevalence per cohort, and the number of cases in each cohort is indicated. CN, copy number; Dox, doxycycline; rel. expr., relative expression.

Figure 4.

Zongertinib reduces tumor growth in preclinical models with different HER2 oncogenic mechanisms. A, Growth curves of tumors derived from HER2^WT-amplified NCI-N87 cells in mice treated with indicated doses and dosing schedules of zongertinib or the control natrosol. The mean tumor volume ± SEM is plotted for all in vivo experiments. B, Modulation of HER2 phosphorylation (ELISA), DUSP6 expression (QuantSeq), and AKT phosphorylation (S473; ELISA) in mice engrafted with NCI-N87 tumors upon zongertinib treatment. Animals were dosed twice daily, 6 hours apart, for 3 days; timepoints are relative to the first dose on day 3. C, Schematic outline of the cell line engineering strategy. Endogenous ERBB2 and EGFR were knocked out in PC-9 cells while overexpressing a HER2^YVMA construct containing an ARTi-RNAi target sequence. Knockdown is achieved by doxycycline-induced expression of an ARTi–shRNA coupled to GFP. D, In vivo experiment comparing doxycyclin-induced PC-9 EGFR^KO,HER2^YVMA-ARTi knockdown to control treatment. E, Doxycycline-induced knockdown of HER2 in PC-9 EGFR^KO,HER2^YVMA-ARTi cells and concurrent downmodulation of pERK and pAKT. F, Growth curves of tumors derived from PC-9 EGFR^KO, HER2^YVMA cells in mice treated with indicated doses and dosing schedules of zongertinib or the control natrosol. G, Growth curves of tumors derived from a PDX model (CTG-2543) carrying a HER2 exon 20 insertion (HER2^YVMA) treated with indicated doses of zongertinib, the EGFR inhibitor erlotinib, the pan-ERBB inhibitor poziotinib, or the control natrosol. H, Downregulation of MPAS gene signature in the PC-9 EGFR^KO, HER2^YVMA xenograft model upon zongertinib treatment. Each group contained up to five animals. Some groups show a lower number of replicates in the case the respective samples did not pass quality control. Dosing schemata (every day and twice a day) are highlighted on the figure. I, Modulation of HER2 phosphorylation (ELISA), DUSP6 expression (hybridization-based), and ERK phosphorylation (ELISA) in mice engrafted with PC-9 EGFR^KO, HER2^YVMA tumors upon zongertinib treatment. Animals were treated once per day with the indicated doses for 3 days, and timepoints are relative to the last dose. shRNA, short hairpin RNA.

Figure 5.

Zongertinib is a rational combination partner for HER2- and KRAS-targeted therapies. A, Schematic of the experiment for data in B and C. T-DXd–resistant tumors are generated in vivo, harvested, and cultured in vitro and then tested for sensitivities to T-DXd, the T-DXd payload deruxtecan, and zongertinib. B, Reduction of phosphorylated HER2 (Y1196) upon zongertinib but not T-DXd treatment in parental and T-DXd–resistant NCI-N87 cells. C, Dose–response curves (each line represents an independent experiment) of parental and T-DXd–resistant NCI-N87 cells to indicated treatments in vitro. D, Growth curves of tumors derived from NCI-N87 cells in mice treated with zongertinib or T-DXd individually or in combination. E, Growth curves of tumors derived from NCI-N87 cells in mice treated with zongertinib or T-DM1 individually or in combination. F, Growth curves of tumors derived from PC-9 EGFR^KO,HER2^YVMA cells in mice treated with zongertinib or T-DM1 individually or in combination. G, Growth curves of tumors derived from NCI-H2122 cells in mice treated with zongertinib or adagrasib individually or in combination. H, Growth curves of tumors derived from ST-524, a KRAS^G12C-dependent but HER2-independent colorectal PDX, in mice treated with zongertinib or adagrasib individually or in combination.

Figure 6.

Zongertinib reduces tumor burden in patients. A, Pre- and on-treatment CT scans of a patient having stage IV cholangiocarcinoma (baseline panel) with a SDC4–NRG1 fusion treated with zongertinib and showing a response (day 129 panel). Quantification of the tumor marker CA19-9 in the blood at indicated dates (x-axis; right). B, Pre- and on-treatment CT scans of a patient having stage IV breast cancer (baseline panel) with a HER2^V777L exon 20 mutation treated with zongertinib and showing a response at the first evaluation (day 38 panel). Quantification of the tumor marker CA15.3 in the blood at indicated dates (x-axis; right). C, Pre- and on-treatment CT scans of a patient having stage IV breast cancer (baseline panel) with ERBB2 amplification treated with zongertinib and showing a tumor response of the locoregional recurrence (right axilla) at the first evaluation (day 37 panel). No biomarker data are available for this patient.