EGFR inhibition enhances the cellular uptake and antitumor-activity of the HER3 antibody drug conjugate HER3-DXd

Abstract

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI) are the standard-of-care treatment for EGFR-mutant non-small cell lung cancers (NSCLC). However, most patients develop acquired drug resistance to EGFR TKIs. HER3 is a unique pseudokinase member of the ERBB family that functions by dimerizing with other ERBB family members (EGFR and HER2) and is frequently overexpressed in EGFR-mutant NSCLC. Although EGFR TKI resistance mechanisms do not lead to alterations in HER3, we hypothesized that targeting HER3 might improve efficacy of EGFR TKI. HER3-DXd is an antibody-drug conjugate (ADC) comprised of HER3-targeting antibody linked to a topoisomerase I inhibitor currently in clinical development. In this study, we evaluated the efficacy of HER3-DXd across a series of EGFR inhibitor-resistant, patient-derived xenografts and observed it to be broadly effective in HER3-expressing cancers. We further developed a preclinical strategy to enhance the efficacy of HER3-DXd through osimertinib pre-treatment, which increased membrane expression of HER3 and led to enhanced internalization and efficacy of HER3-DXd. The combination of osimertinib and HER3-DXd may be an effective treatment approach and should be evaluated in future clinical trials in EGFR-mutant NSCLC patients.

INTRODUCTION

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) are the standard of care for advanced EGFR mutant NSCLC (1,2). For patients with the advanced EGFR mutant cancer harboring the common EGFR exon 19 deletion and L858R mutations, osimertinib is the preferred first line EGFR inhibitor of choice given superior progression free and overall survivals compared to prior generation EGFR TKIs (3). However, resistance to osimertinib invariably develops and the mechanisms of resistance are quite diverse (4,5). Although targeting specific osimertinib resistance mechanisms is feasible, it may not be practical and frequently no targetable resistance mechanism is identified (4). HER3 is overexpressed in various cancer types, including lung cancer (6,7), making it an attractive therapeutic target. Both EGFR and HER3 are ubiquitously expressed in primary NSCLC tumors, and EGFR/HER3 expression was previously detected in 67 % of circulating tumor cells of NSCLC patients (7). Furthermore, alterations in HER3 have not been described as resistance mechanisms to EGFR TKIs and as such targeting HER3 maybe a novel approach to treat drug resistant forms of EGFR mutant cancers

Patritumab deruxtecan (HER3-DXd; U3–1402) is an antibody drug conjugate (ADC) comprised of a human HER3-targeting antibody (patritumab) linked to a topoisomerase I inhibitor (DX-8951 derivative, or DXd) (8). Patritumab (also known as U3–1287) has been tested as a single agent in 57 patients (20 were NSCLC patients and the majority had been treated with prior EGFR inhibitors) and demonstrated no single agent activity (9). HER3-DXd is currently being evaluated as a single agent in EGFR inhibitor resistant EGFR mutant non-small cell lung cancer (NSCLC), HER3 positive metastatic breast cancer, and metastatic colorectal cancer (NCT03260491, NCT02980341, NCT04479436). The determinants of the HER3-DXd efficacy are presently not well understood.

In the current study we queried the single agent efficacy of HER3-DXd in preclinical models of EGFR tyrosine kinase inhibitor (TKI) resistant NSCLCs harboring different drug resistance mechanisms. While the efficacy of HER3-DXd as a single agent was variable across the models, we sought to develop a strategy to enhance the efficacy of HER3-DXd through pre-treatment with osimertinib.

MATERIALS AND METHODS

Antibody internalization assay

Cells were seeded into 96-well plate the previous day of the assay start (6000 cells / well) to obtain 30–40 % confluency. Next day the cells were treated with either DMSO or osimertinib and pre-imaged for 6–8 hours before addition of the conjugated antibodies. HER3-DXd (or control IgG) was conjugated with fab-pHrodo fragments (Essen Biosciences) using 1:3 molar ratio. Antibodies were conjugated 20 min in the dark, +37 °C, after which the conjugated ADCs were administrated to the cells, and the imaging was continued immediately. Imaging and analysis were performed using Incucyte Zoom / S3 live cell imagers (Essen Biosciences) and quantified using the Incucyte softwares.

Cell lines

Cell line name	Source	Fingerprinting information
PC-9; human EGFR-mutant	Dr. Kazuto Nishio (Kindai University, Osaka, Japan)	Fingerprinted; RRID: CVCL_B260
HCC4006; human EGFR-mutant NSCLC, male	ATTC	(CRL-2871); RRID: CVCL_1269
HCC827; human EGFR-mutant NSCLC, female	ATCC	N/A
H1975	ATCC	(CRL-5908); RRID: CVCL_1511
DFCI-284	Established in the Jänne laboratory	N/A
DFCI-243	Established in the Jänne laboratory	N/A

Cell culture & Reagents

Cell lines were obtained from ATCC and cultured in RPMI medium (Gibco) with 10 % FBS (Sigma) and 1 % penicillin / streptomycin (Life Technologies). DFCI-284 and DFCI-243 were established by using previously published protocol (10). Cells were tested for mycoplasma routinely by PCR testing. HER3-DXd was a kind gift of Daiihi Sankyo. Osimertinib and gefitinib were purchased from Selleck (#S7297, #S1025). Antibodies used in the study are described in Supplementary Table S1.

Cell confluency and apoptosis assays

1000 cells / well were seeded on the first day in 96-well plates. Next day cells were treated, and CellEvent Caspase 3/7 green apoptosis detection reagent (Thermo Fisher Scientific) was added to the cells. Cells were imaged every 2 hours, and medium and drugs were changed every week.

Cell titer glo assay

Cell titer glo (Promega) was conducted according to manufacturer’s instructions.

Cycloheximide chase experiment

Cells were pre-treated for 24 hrs with gefitinib or osimertinib and then continuously treated together with 30 ug/ml cycloheximide. Protein lysates were harvested at the indicated timepoints.

Data analysis

When two groups were compared, two-tailed unpaired t-test was used to calculate significance. One-way ANOVA was used when comparing 3 or more groups, Tukey’s multiple comparisons test was used for post-hoc analysis. Fisher’s exact test was used to analyze the regrowth of the PDX tumors after treatment (Supplementary Figure S5G, S5J). The used statistical tests are indicated in the figure legends. Data are presented as mean −/+ stdev or sem, indicated in the figure legends. p ≤ 0.05 was considered significant, and all assays were repeated using 3–6 biological replicates. GraphPad Prism 7.04 software was used for statistical analyses. Graphical abstract was created with BioRender.com (Publication agreement number: QA22SQP9UO).

Flow cytometry for cell lines

Cells were detached with Accutase (Thermo Fisher Scientific), and both cells and culture medium were collected. Cells were counted with Countess cell counter (Invitrogen), suspension confluency was adjusted to 1×10⁶ cells / ml, after which the cells were stained with 1:100 zombie viability dye for 20 min in dark, RT. Cells were spin down and washed with PBS, followed by 30 min staining with conjugated antibodies in flow buffer (10 % FBS-PBS), on ice and dark. Samples were spin washed 3 times, resuspended into flow buffer and acquired using Fortessa analyzer (BD). The data were analyzed with FlowJo version 10.6 (TreeStar).

Flow cytometry for patient-derived xenograft tumors

Patient-derived xenograft tumors were collected and minced with a scalpel, after which they were shook 140 RPM in 0.2 % collagenase A and cell culture medium for 3–4 hrs, +37 °C. Cell suspension was treated with TrypLe Express solution (Thermo Fisher Scientific) for 20 minutes +37 °C to obtain a single cell suspension. Cell suspension was adjusted to 1×10⁶ cells / ml and stained with zombie viability dye for 20 min RT dark. Cells were spin down and washed with PBS, followed by 30 min staining with conjugated antibodies in flow buffer (10 % FBS-PBS), on ice and dark. Samples were spin washed 3 times, resuspended into flow buffer and acquired using Fortessa analyzer (BD). The data were analyzed with FlowJo version 10.6 (TreeStar).

Immunofluorescence

Cells grown and treated on coverslips were fixed with 4 % PFA and permeabilized with 0.1 % Triton X-100. 2D immunofluorescence stainings were performed using standard protocols. 3D cultured samples were fixed with 2% PFA, permeabilized with 0.25 % Triton X-100, and blocked with 10% goat serum in PBS. Primary antibodies were diluted in blocking buffer and 3D cultures were stained overnight. After three washes with IF buffer (0.1 % BSA, 0.2 % Triton X-100, 0.05 % Tween-20 in PBS), the cells were incubated with secondary antibodies in blocking buffer and washed again. Nuclei were counterstained with 1 μg/ml DAPI (Cell Signaling Technology) and mounted with Immu-Mount reagent (Fisher Scientific). Imaging was performed using Nikon Eclipse 80i and Leica SP5 confocal microscopes (DFCI Confocal and Light Microscopy Core Facility). Quantifications for nuclear staining, Ki67, and pH2aX were performed using ImageJ software.

Immunohistochemistry

Four-micron thick formalin-fixed, paraffin embedded (FFPE) tissue sections were stained at the Brigham & Women’s Hospital Pathology Core. After deparaffinization, sections were treated with the EDTA based (pH 9.0) Epitope Retrieval Solution at 90^oC. Endogenous peroxidase was blocked for 15 min. Antibodies were diluted 1:100 and incubated for 30 min. Envision + polymer system (DAKO, Cat# 4011) was used to detect rabbit antibody for 30 minutes. Sections were detected with DAB and counterstained with Hematoxylin. HER3 was visually scored by a pathologist for the generation of H-score according to the formula: [3 × (% cells 3+) + 2 × (% cells 2+) +1 × (% cells 1+)].

Incucyte cell confluency assay

Cells were seeded in a 96-well plate, 1000 cells / well, and the following days the cells were treated using HP D300e digital dispenser. Imaging and analysis were performed using Incucyte Zoom / S3 imagers (Essen Biosciences).

Luciferase assay for promoter activity

Cells were transiently transfected with 1ug of the Firefly luciferase-expressing Her3 promoter reporter plasmid (ErbB-3-pGL3, gift from Frederick Domann, Addgene #60899) and 100ng of the Renilla luciferase-expressing pRL-CMV control plasmid (Promega, Cat# E2261) for 24hrs using Fugene HD transfection reagent (Promega Cat#E2311). Cells were then drug treated and luciferase activities were measured using the Dual-Glo® Luciferase Assay system (Promega Cat# E2920).

Cell line-derived xenograft and patient-derived xenografts

Patient derived xenografts were generated from tumor biopsies or pleural effusions from EGFR mutant patients undergoing clinical biopsies and propagated in mice. All patients provided written informed consent. The study was conducted in accordance with the Declaration of Helsinki and was approved by the Dana Farber Cancer Institute. All animal studies were conducted at Dana-Farber Cancer Institute with the approval of the Institutional Animal Care and Use Committee in an AAALAC accredited vivarium. PDX tumors for DFCI-161, DFCI-284, DFCI-295, DFCI-306, DFCI-359 and DFCI-429 were derived from pleural effusions collected from patients as part of routine clinical care. Effusions were immune depleted and enriched for cancer cells, and these cancer cells were cultured on plastic for three days in RPMI-1640 media supplemented with 10% fetal bovine serum and 1% antibiotic prior to subcutaneous implantation. The PDX tumor for DFCI-315 was derived from a surgical specimen and implanted subcutaneously. The PDX tumor for DFCI-243 was derived from a surgical biopsy and implanted into sub-renal capsule for expansion. Following initial implantation, all PDX models were expanded and passaged continually in mice as subcutaneous tumors. All tumors used in efficacy studies were implanted subcutaneously. For the HCC4006 xenograft model, cells were grown in RPMI-1640 media supplemented with 10% fetal bovine serum and mice were implanted subcutaneously with 5 × 10⁶ cells/mouse in 50% Matrigel (Corning, 356231). All PDX tumors and HCC4006 cells were implanted in 8 to 10-week-old female NSG (NOD.Cg-Prkdc^scid Il2rg^tm1WjI/SzJ) mice purchased from Jackson Labs (005557-NSG; RRID: IMSR_JAX:005557). Following implantation, tumor establishment and growth were monitored by caliper measurements twice per week. Typically, when tumors reached 150–250 mm³, mice were randomized by tumor volume into various treatment groups. Mice harboring tumors were treated with either human IgG control (Bethyl Laboratories, Montgomery, TX) diluted with PBS, HER3-DXd diluted with 10 mM acetate buffer with 5% sorbitol, pH 5.5 or osimertinib formulated as a fine suspension in 0.5% hydroxypropyl methylcellulose (HPMC) in water. IgG control or HER3-DXd were dosed intravenously either once weekly or once every three weeks, while osimertinib was administered orally once daily. During treatment, tumor measurements were taken using calipers and body weights monitored twice a week. Animals were euthanized if the tumor volume exceeded 2,000 mm³ or if the tumors became ulcerated/necrotic, and tumor samples were harvested and snap frozen for subsequent analysis.

Osimertinib resistant cell lines

Cas9 targeting crRNAs to target EGFR exon 20 were designed using the MIT CRISPR design tool. Homologous repair template encoding T790M/C797S EGFR was supplied to parental PC9 cells as a short single stranded linear DNA. Silent mutations were incorporated into the donor strand where possible to protect the template from re-cleavage by Cas9 following integration, and to assess editing efficiency through sequencing. To minimize the effects of codon bias on protein translation, silent mutations incorporated within the donor template were either natural variants or corresponded to codons encoding tRNAs found at comparable frequency in the cell as those encoded by the endogenous codon. Targeting constructs and donor constructs were electroporated into cells alongside nuclear-targeted recombinant active Cas9 protein. Following gene editing, successfully recombined cells were selected with osimertinib, beginning 72 hours after nucleofection. Colonies, including the PC9 T790M/C797S clone used in the studies, were picked by hand under a light microscope and expanded in separate wells under continual osimertinib selection. Allele frequencies were determined through deep amplicon sequencing through the MGH CCIB DNA core CRISPR Sequencing platform. Osimertinib was washed out from PC9 T790M/C797S cells for at least one week prior to initiation of in vitro and in vivo studies.

Osimertinib resistant DFCI-243 cells were created by treating the cells with increasing doses of osimertinib for multiple weeks, ending up with cells resistant to up to 100 nM osimertinib.

For in vivo experiment The PC9 T790M C797S cells were harvested, and 5 × 10⁶ cells with 50% Matrigel (Fisher Scientific) were implanted subcutaneously in the right flank of the 9-week-old female NCr nude mice. Tumors were allowed to establish with an average tumor size of 164 mm³ before randomization into various treatment groups. Mice were treated as above.

Patient derived xenograft 3D culture

The 3D culture sample was carefully minced with a blade and incubated 3 hours with gentle shaking (140 rpm) +37 °C in RPMI medium containing 0.2% of Collagenase A (Sigma), 10% serum, and 1% penicillin/streptomycin (Life Technologies). The samples were centrifuged at 1400 rpm for 5 min and the pellet was washed with 5 ml PBS. Fragments were recentrifuged, resuspended in Matrigel (BD) and seeded to 8-well chamber slides (Nunc) for 3D culture or 96-well plates for cell titer glo assay. Samples were cultured in RPMI medium with 10% serum and 1% penicillin/streptomycin.

Proximity ligation assay

Cells grown and treated on glass coverslips were fixed with 4 % PFA and permeabilized with 0.1 % Triton X-100. Proximity ligation assay was performed using manufacturer’s protocol (Sigma Aldrich). For formalin-fixed paraffin-embedded tissues, paraffin was removed from the slides with xylene and ethanol series and the samples were permeabilized using 0.25 % Triton-X-100 for 25 min. Antigens were recovered in microwave for 20 minutes using citrate buffer (Abcam), and the slides were mouse-on-mouse blocked 30 min RT using Background Buster (Innovex). After this proximity ligation assay was performed using manufacturer’s protocol. Nuclei were stained with 1 μg/ml DAPI (Cell Signaling Technology) for 10 minutes and the slides were mounted using Immu-Mount (Fisher Scientific). PLA quantification was performed with ImageJ software.

Western blot

Cell lines were lysed with RIPA lysis buffer. SDS-page and western blot were done using standard protocols.

RESULTS

Single agent efficacy of HER3-DXd in EGFR inhibitor resistant models of non-small cell lung cancer

To study the relevance of HER2 and HER3 in EGFR-inhibitor resistant cancers, we evaluated the protein expression levels of these two receptors using immunohistochemistry in EGFR mutant patient-derived xenograft models (PDX) resistant to either first- or third-generation EGFR inhibitors, harboring a broad range of acquired resistance mechanisms. (Fig 1A, Supplementary Table S2). Although most models expressed both HER2 and HER3, high HER3 expression was more prevalent, and unlike HER2, HER3 expression was detected in all of the models. We also noted HER3 expression in HER2 mutant and amplified models, although in those models HER2 expression was more prevalent. The widespread HER3 expression across all tumor models suggested, that HER3 could serve as a therapeutic target independent of the specific mechanism of resistance to EGFR inhibitors.

Figure 1: Single agent activity of HER3-DXd in EGFR inhibitor resistant patient-derived xenografts.

(A) HER2 and HER3 expression across EGFR inhibitor resistant patient-derived xenograft models. HER2 and HER3 were stained with immunohistochemistry and quantified using H score. The models used for HER3-DXd efficacy studies are bolded and marked with an asterisk. (B)-(E) Immunohistochemistry stainings for HER3 and single agent efficacy of HER3-DXd in PDX models with variable baseline HER3 expression. 10 mg/kg HER3-DXd or IgG control, qw. Scale bar is 50 μm. Student’s t test (unpaired), SEM. (F) Waterfall plot for all PDX models treated with HER3-DXd. The graph shows maximal tumor volume change induced by HER3-DXd treatment. (G) In vivo- grafted PC-9 T790M/C797S cells are resistant to osimertinib but are still sensitive for HER3-DXd. Mice were treated with either 25 mg/kg osimertinib qd., or 10 mg/kg HER3-DXd, qw. ANOVA, SEM.

**** p<0.0001, *** p < 0.001, ** p < 0.01, ns = not significant.

Prior preclinical studies suggest, that the efficacy of HER3-DXd is linked to the baseline HER3 expression in breast cancer (8). To evaluate the effects of HER3-DXd in NSCLC, we determined the efficacy of single-agent HER3-DXd in eight PDX models covering a wide range of HER3 expression (Fig 1B–E, Supplementary Fig. S1A–B, Supplementary Table S2). Single treatment of HER3-DXd (10 mg/kg, qw.), compared to IgG control, led to a significant tumor growth delay in the majority of the models, except in DFCI-306 which has negligible HER3 expression. The drug was well tolerated (Supplementary Fig. S1C–E). HER3-DXd efficacy was dose-dependent (Supplementary Fig. S1F). In two models, DFCI-161 (EGFR L858R/MET amplified) and DFCI-315 (HER2 exon20 V777_G778insGSP) HER3-DXd treatment was associated with substantial tumor regressions while in the other models it led to tumor growth delay (Figure 1F). Control IgG antibody didn’t affect the growth of the tumors (Supplementary Figure S1G).

We further evaluated the efficacy of HER3-DXd in a model containing the EGFR C797S (11). As we were unable to generate a PDX model from an osimertinib resistant patient with this mutation, we engineered a model using CRISPR-Cas9. Using the EGFR mutant PC-9 cell line, we introduced EGFR T790M and C797S in cis as commonly detected in NSCLC patients. The resulting cells were resistant to EGFR inhibitors compared to the parental population (Supplementary Fig. S1H–I, Supplementary Table S3). We next evaluated the efficacy of both osimertinib and HER3-DXd in this model. The PC-9 T790M/C797S tumors also expressed HER3 and were resistant to single agent osimertinib while single agent HER3-DXd treatment led to a significant tumor growth delay (Figure 1G, Supplementary Fig. S1J–K).

EGFR inhibitors increase the membrane and total expression of HER3 in cell lines and patient-derived xenografts

Our initial studies demonstrated variable efficacy of HER3-DXd as a single agent in EGFR mutant PDX models and no efficacy in the PDX model that lacked HER3 expression. We previously demonstrated that EGFR inhibitor treatment leads to an increase in HER3 levels in vitro (12) and as such we aimed to determine if EGFR inhibition could potentially enhance the efficacy of HER3-DXd.

To systematically evaluate the impact of EGFR inhibition on HER2 and HER3 expression, six EGFR-mutant NSCLC cell lines were treated with gefitinib or osimertinib after which HER2 and HER3 membrane expression were evaluated using flow cytometry (Figure 2A). To make the analysis more comprehensive, the membrane expression of HER2 and HER3 in viable single cells was further divided into negative, low, and high expression groups (Supplementary Fig. S2A). Twenty-four-hour pre-treatment with EGFR inhibitors increased the HER3 membrane levels in all six cell lines in a dose-dependent manner, and the increase was observed in both the amount of HER3 positive cells and the intensity of HER3 expression (Figure 2B, Supplementary Fig. S2B). In half of the models HER2 membrane expression levels also increased, but in the remaining models HER2 was already highly expressed at baseline, and further increase could not be detected (Supplementary Fig. S2C). In the cell lines carrying the T790M resistance mutation making the cells resistant to gefitinib, osimertinib treatment more effectively increased HER3 membrane expression, suggesting that the effect was dependent on on-target EGFR inhibition. In a time-course assay we observed that the increase in HER3 expression peaked between 8 and 24 hours following the start of EGFR inhibitor treatment (Figure 2C–D). There was no further increase in HER3 expression when treatment was extended beyond 24 hours (Supplementary Fig. S2D). Treatment with the anti-EGFR monoclonal antibody cetuximab did not lead to an increase in HER3 surface expression (Supplementary Fig. 2E).

Figure 2: EGFR inhibitors increase the membrane and total expression of HER3 in cell lines and patient-derived xenografts.

(A) Schematic for the experimental design. EGFR mutant non-small cell lung cancer lines were treated with gefitinib or osimertinib for 24 hr after which they were stained for the viability marker, HER2, and HER3. (B) HER3 membrane expression in cell lines after EGFR inhibitor treatment. HER3 positive cells were categorized into low and high expression groups. N >3 biological replicates. Student’s t test (unpaired), SD. (C-D) Time series for the HER3 membrane expression after EGFR inhibitor treatment in HCC4006 and DFCI243 cell lines. N = 3 biological replicates. Student’s t test (unpaired), SD. (E) EGFR inhibitors increase HER3:EGFR interaction measured by proximity-ligation assay (PLA). The cells were treated with 100 nM gefitinib or osimertinib for 24 hr before the assay. Scale bar is 20 μm. (F) Quantification of the HER3:EGFR PLA. N = 4 biological replicates. Student’s t test (unpaired), SD. (G-H) EGFR inhibitors stabilize HER3 in protein level. Cells were treated with 100 nM gefitnib or osimertinib for 24 hr before adding 30 μg/ml cycloheximide (CHX). Graph shows the average of quantifications of western blots from 3 biological replicates. ANOVA (one way), SD. (I) Treatment scheme for PDX tumors. Tumor-bearing mice were treated once with vehicle or 25 mg/kg osimertinib for 24 hrs, after which the tumors were collected for analysis. (J) Osimertinib pre-treatment increases total HER3 protein levels. Quantification of the bands is shown below the blot. Long exposure = longer blot exposure time. Hsp90 is the loading control. (K) Osimertinib pre-treatment increases the amount of viable HER3+ EpCam+ cells in PDX tumors. EpCam is an epithelial cell marker. N >3 / group. Student’s t test (unpaired), SD. (L) 48 hr treatment does not further increase HER3 membrane expression. Tumor-bearing mice were treated either for 24 hr or 48 hr with vehicle or 25 mg/kg osimertinib before HER3:EGFR interaction in vivo. (N) Quantification of the PLA assay. N >3 mice / group, 6–8 fields of views / tumor. Student’s t test (unpaired). Scale bar is 200 μm.

**** p<0.0001, *** p < 0.001, ** p < 0.01, ns = not significant.

Proximity ligation assay (PLA) is a useful tool for measuring protein-protein interactions in situ and has been used to study and quantify the dimerization interactions of EGFR family (13,14). A positive PLA signal is detected when the distance between two proteins is less than 40 nm. We used PLA to test if the EGFR TKI induced increase in membrane HER3 could also lead to formation of HER3:EGFR dimers, suggesting active HER3 signaling. In all three studied cell lines, we noticed a significant increase in membrane HER3:EGFR proximity following EGFR inhibitor treatment (Figure 2E–F), suggesting that the increase in HER3 expression was also associated with the formation of EGFR:HER3 heterodimers.

Next, we examined whether the increase in membrane expression of HER3 was regulated at the level of mRNA transcription or through protein stability. In a cycloheximide chase assay, HER3 protein levels increased after EGFR inhibitor treatment, suggesting that HER3 increase was regulated at the level of protein stability (Figure 2G–H, Supplementary Fig. S2F–G). However, we also observed minor increases in HER3 promoter activity measured using a luciferase promoter reporter assay (Supplementary Fig. S2H–I), and mRNA expression (Supplementary Fig. S2J), suggesting that the upregulation was partly also occurring at the level of transcription. Inhibition of EGFR downstream signaling by either an AKT inhibitor (MK-2206) or a MEK inhibitor (trametinib) partially phenocopied the effect of EGFR inhibition in PC-9 cells (Supplementary Fig. S2K), suggesting that inhibition of both signaling pathways contributed to the observed HER3 feedback upregulation.

To study if EGFR inhibition alters HER3 expression also in vivo, four PDX models with variable baseline HER3 expression levels were treated with vehicle or osimertinib for 24 hours followed by analysis of HER3 expression (Figure 2I). We observed that in all four models osimertinib pre-treatment increased HER3 total protein expression (Figure 2J). In addition, the number of HER3 positive viable epithelial cells (marked by EpCam) also increased following osimertinib treatment (Figure 2K). We noted a substantial increase in the protein levels of HER3 even in two additional models that were originally low or negative for HER3, including the DFCI-306 model (Supplementary Fig. S2L–M). HER3 expression did not increase further by extending the treatment time to 48 hrs (Figure 2L). PLA analysis from the osimertinib treated PDX tumors also revealed an increase in HER3:EGFR proximity analogous to the in vitro observations (Figure 2M–N).

EGFR inhibitor pre-treatment enhances the internalization and DNA damaging effects of HER3-DXd

Since membrane expression of the target is a key factor for the binding and internalization of antibody drug conjugates (15), we further evaluated the biological significance of the increased HER3 membrane expression on HER3-DXd. We tracked the internalization of HER3-DXd over time, using a pH-sensitive pHrodo Red conjugate, which is non-fluorescent outside the cell in neutral pH, but fluoresces once it is internalized and processed in the endosomes and lysosomes which have a more acidic environment (16). The cells were pretreated for 8 hours either with vehicle, gefitinib, or osimertinib, after which the pHrodo labelled control IgG-ADC or HER3-DXd were added to the cells, and the internalization was measured every two hours using live cell imaging (Figure 3A). The 8-hour time point was selected to avoid possible non-specific internalization caused by EGFR inhibitor induced cell death. Cell lines with different baseline HER3 levels were tested (Figure 3B).

Figure 3: EGFR inhibitor- induced HER3 expression increases the internalization and efficacy of HER3-DXd in vitro.

(A) Schematic for the ADC internalization assay. HER3-DXd (or control IgG-ADC) was conjugated with pH-sensitive pHrodo Red fluorescent probes. After conjugation the labeled antibodies were added to the cells pre-treated either with vehicle or 100 nM osimertinib for 8 hr, and fluorescence was measured over time using Incucyte live cell imager. (B) Western blot showing the baseline expression of (p)HER3 and (p)EGFR in the used cell lines. Hsp90 is a loading control. Lanes are from the same blot; dashed line indicates where the blot was cut. (C) Representative images of HER3-DXd intake in all three cell lines. Scale bar is 60 μm. (D) Area under curve for HER3-DXd internalization in 3 different cell lines. Each cell line was normalized to the parental control cell line. ANOVA, N= 4 biological replicates, SD. (E) Quantification of the HER3-DXd internalization in DFCI-243 cells. N= 3 biological replicates, SD. (F) Representative pictures from the ADC internalization assay in DFCI-243 cells over time. HER3-DXd internalization is shown in red, green is cleaved caspase 3. Scale bar is 200 μm. (G)-(H) Western blots showing the DNA damage and apoptosis response followed by single agent or combination treatment with 50 nM osimertinib and 10 μg/ml HER3-DXd. Hsp90 is the loading control. (I) Immunofluorescence staining for pH2aX and Ki67 after 24 hr osimertinib (50 nM for HCC4006 and H1975, 10nM for DFCI-243) pre-treatment followed by 24 hrs of 10 μg/ml HER3-DXd. Scale bar is 25 μm. (J)-(M) Quantification for pH2aX and Ki67 immunofluorescence. N = 3 biological replicates. **** p<0.0001, *** p < 0.001, ** p < 0.01, ns = not significant.

In all three cell lines HER3-DXd was internalized to a greater extent than the control IgG-ADC, suggesting that the internalization was dependent on antibody binding (Supplementary Fig. S3A). In both DFCI-243 and H1975 cell lines there was a 3- to 5-fold higher intake of HER3-DXd over time in cells pre-treated with osimertinib, compared to vehicle (Figure 3D–E, Supplementary Fig. S3A). In HCC4006 cell line, with very high baseline HER3 expression, there was an increase in the ADC intake, but the fold increase in the internalization was less compared to the two lower HER3 expressing lines (Figure 3C–E, Supplementary Fig. S3B–C). In DFCI-243 cell line expressing low baseline HER3, osimertinib pre-treatment significantly increased the uptake of HER3-DXd over time (Figure 3F–G). DFCI-243 harbors EGFR T790M gefitinib resistance mutation, and gefitinib treatment did not lead to enhanced HER3-DXd internalization, suggesting that the increased uptake was dependent on target engagement with EGFR. Importantly, EGFR TKI pre-treatment did not increase IgG-ADC internalization in any of the cell lines (Supplementary Fig. S3D), suggesting that the increase in internalization was specific to the EGFR-inhibition induced HER3 and not due an increase in non-specific internalization.

HER3-DXd is linked to a topoisomerase I inhibitor (exatecan derivative (DXd), which induces DNA damage following internalization and lysosomal cleavage. To further study the potential therapeutic effect of combining osimertinib with HER3-DXd combination treatment, we evaluated the impact of the single agents and the combination on DNA damage. The combination treatment increased the levels of activating phosphorylation in ATM kinase in all three cell lines, followed by increased phosphorylation of its downstream target H2aX, indicating higher amount of DNA double-strand breaks (DSB) more compared to the single agents (Figure 3G–H, Supplementary Fig. S3E). In addition, the combination led to a greater increased in both cleaved caspase-3 and PARP. We further measured the amount of DSB (pH2aX) and monitored cell proliferation (Ki-67 positivity) using immunofluorescence. Indeed, we observed that the osimertinib/HER3-DXd combination induced more DSB damage than the single agents alone and led to decrease in Ki-67 positive cells (Figure 3I–M). Collectively, these findings demonstrate that EGFR inhibitor pre-treatment enhances the cellular uptake of HER3-DXd resulting in a greater degree of DNA damage.

Osimertinib enhances the HER3-DXd efficacy in vitro and ex vivo

To study the therapeutic potential of the osimertinib / HER3-DXd combination, we tested the efficacy of HER3-DXd in EGFR mutant cell lines with and without osimertinib pre-treatment. The cells were first pre-treated with osimertinib for 24 hours, followed by continuous treatment with both osimertinib and HER3-DXd. In both the HCC406 and H1975 cells, the combination was more effective than each of the single agents (Figure 4A–B). The DFCI-243 was quite sensitive to single agent osimertinib and as such we compared the regrowth of the cells following drug withdrawal and noted a significant difference in the regrowth for the combination compared to osimertinib alone (Figure 4C). Consistent with the effects on growth, we observed an increase in apoptosis in the combination groups compared to the single agents (Figure 4D–F).

Figure 4: Osimertinib pre-treatment increases the HER3-DXd efficacy in cell lines and in ex vivo patient-derived xenografts.

(A)-(B) Growth of HCC4006 and H1975 cells treated with DMSO, 50 nM osimertinib, 10 μg/ml HER3-DXd, or combination of osimertinib and HER3-DXd. Arrows indicate when medium and drugs were changed to the cells. ANOVA, SD. (C) Regrowth of DFCI-243 cells treated with 10 nM osimertinib, 10 μg/ml HER3-DXd, or combination of osimertinib and HER3-DXd. Cells were treated for 1 week after which the drug was withdrawn. ANOVA, SD. (D)-(F) Apoptosis quantification in cells treated like A-C. Values were normalized to cell confluency. ANOVA, SD. (G) Increased HER3 membrane expression by EGFR inhibition is target-dependent in PC-9 cells. HER3 membrane expression in PC-9, PC-9 gefitinib resistant cells (GR) and PC-9 osimertinib resistant (T/C: T790M/C797S) cells. Cells were treated with DMSO, 100 nM gefitnib, 100 nM osimertinib, or 10 ng/ml cetuximab for 24 hours. Students t test, SD. (H) Western blot showing the total HER3 levels and inhibition of EGFR/pERK signaling by on-target EGFR inhibition. GAPDH is a loading control. Cells were treated like in (G). (I) Example images from patient-derived xenograft fragment cultures stained for cleaved caspase-3 and epithelial marker E-Cadherin. Scale bar is 50 μm (J)-(K) Viability of PDX fragments on day 7 and day 12 (cell titer glo assay), normalized to baseline viability. ANOVA, SD.

Our studies suggest that on-target EGFR inhibition is necessary for HER3 upregulation and as such for the efficacy of the osimertinib / HER3-Dxd combination. We tested PC-9 cells resistant to gefitinib (PC-9 GR) or osimertinib (PC-9 T790M/C797S) for their HER3 expression and for the efficacy of the osimertinib / HER3-Dxd combination. We noted that HER3 membrane and total expression was dependent on EGFR target engagement, since gefitinib did not lead to increased membrane/total HER3 expression in PC-9 GR cells, whereas osimertinib did not increase HER3 in PC-9 T790M/C797S cells (Figure 4G–H). Same pattern was observed in the DFCI-243 cells that were made resistant to osimertinib by treating the cells with increasing doses of osimertinib (Supplementary Figure 4A). PC-9 cells were very sensitive to single-agent osimertinib and HER3-Dxd and as such, there was only a slight increase in efficacy following treatment with the combination of the two drugs in both the PC-9 and PC-9 GR cells. In contrast there was no increase in efficacy of the combination in the PC9 T790M/C797S cells, suggesting that on-target EGFR inhibition is important for the combination efficacy (Supplementary Figure S4B–D).

We next studied the efficacy of the osimertinib/HER3-DXd combination using ex vivo 3D cultured PDX tumor fragments. PDX tumors were isolated from mice and tumor-derived fragments were treated with either single agents or the combination analogous to the cell lines. Viability was measured on days 7 and 12. We observed significant effect of the combination at day 12 in both the DFCI-282 and DFCI-243 models compared to the single agent (Figures 4I–K), In contrast, in the DFCI-249 model, HER3-DXd was effective as a single agent, and there was no significant added benefit from osimertinib pre-treatment.

Osimertinib enhances the HER3-DXd efficacy in vivo

The in vitro findings suggested that the osimertinib/HER3-DXd combination should be further evaluated in an in vivo setting. To first evaluate the potential toxicities of the combination, non-tumor bearing mice were treated with osimertinib (25 mg/kg qd) and HER3-DXd (10 mg/kg, qw) for 21 days. We observed no significant toxicities of the combination as measured by mouse body weights and by examining complete blood counts (Supplementary Fig. S5A–C). To evaluate the combination efficacy, we chose DFCI-243 PDX model, since in the DFCI-243 derived cell line we detected low HER3 levels (Figure 3B), modest single agent HER3-DXd activity (Figure 4C) and added benefit from the osimertinib combination in both 2D cell line and 3D cultured PDX-derived tumor fragments (Figure 4C, Figure 4G–I). Like in the in vitro models, the mice were first treated with osimertinib and 24 hours following which they were treated with continuous daily osimertinib (10 mg/kg) in combination with weekly HER3-DXd (3mg/kg or 10mg/kg) for 28 days (Figure 5A). We saw tumor growth delays with both single agent osimertinib and HER3-DXd (Figure 5B–D), however all the osimertinib treated tumors regrew (Figure 5C) after the drug withdrawal, as did the majority of the HER3-DXd treated tumors (Figure 5D). The combination was associated with a greater maximal tumor reduction (Supplemental Figure 5D) and the fewest number of mice exhibiting tumor re-growth following drug withdrawal (Figure 5E, Supplementary Figure 4E–G). Interestingly, in the combination group using the lower HER3-DXd dose (3 mg/kg) 2/8 tumors were able to regrow, while in the higher dose (10mg/kg HER3-DXd) group only 1/8 tumor regrew. The difference in regrowth between single agent HER3-DXd and Osimertinib+HER3-DXd groups was statistically significant.

Figure 5: Osimertinib pre-treatment increases the HER3-DXd efficacy ex vivo and in vivo patient-derived xenografts.

(A) Treatment scheme for DFCI-243 PDX. Tumor-bearing mice were first treated with vehicle or osimertinib (10 mg/kg) for 24 hr, after which HER3-DXd (3/10 mg/kg) was added. Mice were treated for 28 days,and followed up for tumor regrowth. (B)-(E) Individual tumor growths for all treated mice (DFCI-243 PDX). (F)-(I) Individual tumor growths for all treated mice (HCC4006 xenograft). Osimertinib vs U3–1402 p<0.0001 ****, Osimertinib vs U3–1402 + Osimertinib p<0.0002 ***, U3–1402 vs U3–1402 Osimertinib p<0.0001 ****, 2-way ANOVA.

**** p<0.0001, *** p < 0.001, ** p < 0.01, ns = not significant.

To test the efficacy of the combination in a second model, we chose HCC4006 which has moderate single-agent HER3-DXd sensitivity in vitro (Figure 4A). HCC4006 model was more sensitive to single-agent osimertinib than DFCI-243 model (Figure 5F–G) but had variable responses to the single agent HER3-DXd (Figure 5H). Greatest maximal tumor reduction and slowest tumor regrowth after withdrawal was again associated with the combination group (Figure 5I, Supplementary Figure S5H). The tumor growth difference between the single agent treatment groups and the combination groups was statistically significant (Supplementary Figure 54I). The difference in regrowth between single agent HER3-DXd and Osimertinib+HER3-DXd groups was statistically significant (Supplementary Figure S5J).

DISCUSSION

Targeting HER3 represents an attractive strategy to treat EGFR inhibitor resistant EGFR mutant NSCLC. HER3 is expressed in the majority of EGFR mutant cancers, and HER3 is not a known resistance mechanism to EGFR kinase inhibitors. In addition, as resistance mechanisms can be diverse, or sometimes no known or targetable mechanism is identified, targeting HER3 represents a practical and uniform approach to treat drug resistant cancers (4,17). HER3-DXd is undergoing clinical development as a single agent in EGFR inhibitor resistant EGFR mutant cancers (NCT03260491) and efficacy (response rate: 39 % and progression free survival 8.2 months) has thus far been observed (18). However, as efficacy of HER3-DXd is not uniform, we aimed to develop a strategy that could potentially enhance the efficacy of single agent therapy.

Our studies demonstrate, that EGFR inhibition not only leads to increased HER3 expression but that this is associated with an increase in HER3-DXd internalization, DNA damaging effects and efficacy in vitro and in vivo. Previous studies of HER2 TKI lapatinib and EGFR/HER2 targeting numberTKI poziotinib have demonstrated that TKIs can induce increased cell-surface accumulation of HER2 by reduced receptor ubiquitination, and pozitionib-induced HER2 was found to sensitize cells to HER2 antibody-drug conjugate T-DM1 in various cancer types (19,20). Similarly, co-treatment with irreversible pan-HER inhibitors neratinib or afatinib enhanced HER2 ubiquitination and receptor internalization resulting in increased T-DM1 efficacy in preclinical models of ERBB2-amplified lung cancer (21), suggesting that TKI-induced receptor internalization could be a generalized strategy to enhance efficacy of ADCs. However, the mechanistic basis, following TKI treatment, differs for HER2 and HER3.

There are several potential clinical scenarios where a combination of osimertinib and HER3-Dxd could be evaluated including in patients that have developed osimertinib resistance. Our findings however suggest that EGFR inhibition is necessary for both an increase in HER3 membrane expression and for the combination efficacy of the two drugs. As such, patients that have developed an on-target EGFR mutation as their resistance mechanism to osimertinib, such as EGFR C797S, would not be expected to benefit from this combination. The optimal clinical scenario for the combination is where EGFR mutant cancers are sensitive to the single agents.

Although both osimertinib and HER3-DXd can independently, and through different mechanisms, be effective in EGFR mutant cancers, our findings suggest that the combination maybe even more effective given this unique interaction. At the moment, single agent osimertinib is the standard of care for advanced EGFR mutant NSCLC. However, the efficacy of osimertinib is limited by the development of acquired drug resistance. The combination of osimertinib and HER3-DXd may thus be one way to limit or delay resistance. These preclinical studies have led to the initiation of a phase I clinical trial combining osimertinib with HER3-DXd (NCT04676477). The study will include evaluation of the combination of osimertinib and HER3-Dxd in patients previously treated with osimertinib and those that are EGFR inhibitor naïve.

SIGNIFICANCE.

EGFR inhibition leads to increased HER3 membrane expression and promotes HER3-DXd antibody drug conjugate internalization and efficacy, supporting the clinical development of the EGFR inhibitor/HER3-DXd combination in EGFR mutant lung cancer.