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Table S1 contains IC50 values for cetuximab inhibition, EGFR mutation and amplification status of tested cell lines. Figure S1 shows cetuximab binding to surface EGFR on HN5 and cetuximab resistant HN5 cell lines. Figure S2 shows EGFR levels in parental HN5 cells and cetuximab resistant clones. Figure S3 Overexposed image of immunoblot presented in Figure 2B. Figure S4 shows EGFR degradation upon treatment with anti-EGFR 2mixture for 48h. Figure S5 shows LNA-mediated knockdown of EGFR inhibit growth and proliferation of HN5, HN5 CR2, and HN5 CR14. Figure S6 EGFR levels in FaDu versus cetuximab resistant FaDu cell lines. Figure S7 HER3 and IGF1R plasticity as a mechanism of acquired cetuximab resistance in HN5 CR14. Figure S8 Full dose-response curves of cetuximab and EGFR+HER3+IGF1R 5 mixture in a panel of SCCUAT cell lines.

Short description of methods and materials used for supplemmentary figures. BioLayer Interferometry, ADCC and CDC assays.

Supplementary Tables and Figures. Table S1 contains affinities for antibodies binding to HER2. Table S2 contains results from epitope bin analysis of the antibodies. Figure S1 shows a comparison of Perjeta and the pertuzumab analogue used in this paper. Figure S2 shows sensograms for the anti-HER2 antibodies. Figure S3 contains dose-response curves for anti-HER2 lead mixtures in the cell lines BT474, SK-BR3, HCC202 and NCI-N87. Figure S4 shows immunoblot data on the levels of HER2, pHER2, EGFR, pEGFR, HER3 and pHER3 in the eight cell lines OE19, N87, MCF7, MDA-MB-175, BT474, HCC202 SK-BR3 and ZE-75-30. Figure S5 shows activity of anti-HER2 mAbs and mixtures in ADCC and CDC assays. Figure S6 demonstrates synergy of the tripartite mixture in OE19 cell line. Figure S7 shows quantification of HER2 and pHER2 levels in OE19 and HCC202 cell lines upon treatment with anti-HER2 mAbs and mixtures. Figure S8 shows activity of tripartite mixtures of trastuzumab, pertuzumab and lead anti-HER2 antibodies in a cell viability assay

Supplementary Figures S1-8. Figure S1 - Analysis of nonlinear blending synergy for the pairs of antibodies that constitute Pan-HER. Figure S2 - In vitro comparison of Pan-HER and a combination of cetuximab, trastuzumab and MM-121. Figure S3 - In vivo assessment of nonlinear blending synergy for the target specificities in the Pan-HER mixture. Figure S4 - Dose titration of Pan-HER in the BxPC3 xenograft model. Figure S5 - IHC analysis of Calu-3 tumors. Figure S6 - Assessment of cell death and cell cycle arrest. Figure S7 - Assessment of ADCC in a panel of cell lines. Figure S8 - Analysis of the effect of receptor internalization on effector functions.

Supplementary Methods. Description of methods used for assessment of synergy, immunohistochemistry, cell death, cell cycle arrest, ADCC and CDC.

Supplementary Tables S1-2. Table S1 - Source, origin, subtype and growth medium for each cell line. Table S2 - Characteristics of tested patient-derived xenograft models.

Purpose: Accumulating evidence indicates a high degree of plasticity and compensatory signaling within the human epidermal growth factor receptor (HER) family, leading to resistance upon therapeutic intervention with HER family members.Experimental Design/Results: We have generated Pan-HER, a mixture of six antibodies targeting each of the HER family members EGFR, HER2, and HER3 with synergistic pairs of antibodies, which simultaneously remove all three targets, thereby preventing compensatory tumor promoting mechanisms within the HER family. Pan-HER induces potent growth inhibition in a range of cancer cell lines and xenograft models, including cell lines with acquired resistance to therapeutic antibodies. Pan-HER is also highly efficacious in the presence of HER family ligands, indicating that it is capable of overcoming acquired resistance due to increased ligand production. All three target specificities contribute to the enhanced efficacy, demonstrating a distinct benefit of combined HER family targeting when compared with single-receptor targeting.Conclusions: Our data show that simultaneous targeting of three receptors provides broader efficacy than targeting a single receptor or any combination of two receptors in the HER family, especially in the presence of HER family ligands. Pan-HER represents a novel strategy to deal with primary and acquired resistance due to tumor heterogeneity and plasticity in terms of HER family dependency and as such may be a viable alternative in the clinic. Clin Cancer Res; 21(18); 4110–22. ©2015 AACR.See related commentary by Yarden and Sela, p. 4030

It is well known that the development of drug resistance in cancer cells can lead to changes in cell morphology. Here, we describe the use of deep neural networks to analyze this relationship, demonstrating that complex cell morphologies can encode states of signaling networks and unravel cellular mechanisms hidden to conventional approaches. We perform high-content screening of 17 cancer cell lines, generating more than 500 billion data points from similar to 850 million cells. We analyze these data using a deep learning model, resulting in the identification of a continuous 27-dimension space describing all of the observed cell morphologies. From its morphology alone, we could thus predict whether a cell was resistant to ErbB-family drugs, with an accuracy of 74%, and predict the potential mechanism of resistance, subsequently validating the role of MET and insulin-like growth factor 1 receptor (IGF1R) as drivers of cetuximab resistance in in vitro models of lung and head/neck cancer.

Squamous cell carcinomas (SCC) arising in upper parts of the aerodigestive tract are among the leading causes of death worldwide. EGFR has been found to play an essential role in driving the malignancy of SCC of the upper aerodigestive tract (SCCUAT), but, despite this, clinical results using a range of different EGFR-targeted agents have been disappointing. Cetuximab is currently the only EGFR-targeted agent approved by the FDA for treatment of SCCUAT. However, intrinsic and acquired cetuximab resistance is a major problem for effective therapy. Thus, a better understanding of the mechanisms responsible for cetuximab resistance is valuable for development of the next generation of antibody therapeutics. In order to better understand the underlying mechanisms of cetuximab resistance in SCCUAT, we established from cetuximab-sensitive models cell lines with acquired resistance to cetuximab by continuous selective pressure in vitro and in vivo. Our results show that resistant clones maintain partial dependency on EGFR and that receptor tyrosine kinase plasticity mediated by HER3 and IGF1R plays an essential role. A multitarget mAb mixture against EGFR, HER3, and IGF1R was able to overcome cetuximab resistance in vitro. To our surprise, these findings could be extended to include SCCUAT cell lines with intrinsic resistance to cetuximab, suggesting that the triad consisting of EGFR, HER3, and IGF1R plays a key role in SCCUAT. Our results thus provide a rationale for simultaneous targeting of EGFR, HER3, and IGF1R in SCCUAT. Mol Cancer Ther; 15(7); 1614–26. ©2016 AACR.

HER2 plays an important role in the development and maintenance of the malignant phenotype of several human cancers. As such, it is a frequently pursued therapeutic target and two antibodies targeting HER2 have been clinically approved, trastuzumab and pertuzumab. It has been suggested that optimal inhibition of HER2 is achieved when utilizing two or more antibodies targeting nonoverlapping epitopes. Superior clinical activity of the trastuzumab plus pertuzumab combination in metastatic breast cancer supports this hypothesis. Because trastuzumab and pertuzumab were not codeveloped, there may be potential for further optimizing HER2 targeting. The study herein evaluated functional activity of anti-HER2 antibody combinations identifying optimal epitope combinations that provide efficacious HER2 inhibition. High-affinity antibodies to all four extracellular domains on HER2 were identified and tested for ability to inhibit growth of different HER2-dependent tumor cell lines. An antibody mixture targeting three HER2 subdomains proved to be superior to trastuzumab, pertuzumab, or a combination in vitro and to trastuzumab in two in vivo models. Specifically, the tripartite antibody mixture induced efficient HER2 internalization and degradation demonstrating increased sensitivity in cell lines with HER2 amplification and high EGFR levels. When compared with individual and clinically approved mAbs, the synergistic tripartite antibody targeting HER2 subdomains I, II, and IV demonstrates superior anticancer activity. Mol Cancer Ther; 14(3); 669–80. ©2015 AACR.

It is well known that the development of drug resistance in cancer cells can lead to a change in cell morphology. We reasoned that machine-learning techniques could thus be used to elucidate far greater insight into the relationship between cell shape and signaling. To test this hypothesis we performed a large high content screen on drug sensitive and drug resistance cancer cells, and analysed the shape of these cells using a deep neural network. Our model identified a continuous 27-dimension space describing all of the observed cell morphologies from which we were able to predict drug resistance with an accuracy of 74%. In addition, analyzing changes in cell morphology identified signaling networks that, when perturbed, caused the death of drug resistant cells. These findings suggests that complex morphologies can decode states of signaling networks seemingly unrelated to cell shape, and that analysis of this information can unravel cellular mechanisms hidden to conventional measurements.

Head and neck squamous cell carcinoma (HNSCC) is among the leading causes of death worldwide. HNSCC originates from the squamous epithelium of the upper aerodigestive tract, including the lip, oral cavity, pharynx, larynx and paranasal sinuses. The epidermal growth factor receptor (EGFR) has been found to play an essential role in driving the malignancy of HNSCC. The monoclonal antibody targeting EGFR, cetuximab, has been approved for clinical use in HNSCC patients, in combination with either radiation or chemotherapy. However, intrinsic and acquired cetuximab resistance is a major problem for effective therapy. Thus, a better understanding of the mechanisms responsible for cetuximab resistance is valuable for development of the next generation of antibody therapeutics. In order to investigate the molecular mechanisms behind cetuximab resistance in HNSCC, we screened a panel of cell lines for sensitivity to cetuximab. As expected, the cell lines exhibited a varied response to cetuximab. The most sensitive cell line, HN5, was used to establish a number of cetuximab-resistant clones. These resistant clones had lower EGFR levels but remained partially dependent on EGFR, as demonstrated by EGFR gene knockdown and treatment with a mixture of two anti-EGFR antibodies. HER3 and IGF1R are known to be major drivers of resistance to EGFR targeting therapies, and therefore we investigated the role of the two receptors in resistant clones with a mixture of antibodies against EGFR, HER3 and IGF1R. The antibody mixture, which simultaneously blocked EGFR, HER3 and IGF1R signaling, resulted in near total growth inhibition of cetuximab resistant clones. These results were extended to HNSCC cell lines with intrinsic resistance to cetuximab and also in these cell lines the three target mAb mixture led to effective growth inhibition. In conclusion, our results demonstrate that HNSCC cell lines have a heterogeneous response to cetuximab and that HER3 and IGF1R effectively compensate for EGFR inhibition in both the acquired and intrinsic cetuximab resistant setting. Our results thus provide a rationale for simultaneous targeting of EGFR, HER3, and IGF1R in HNSCC. Citation Format: Ida Kjaer, Trine Lindsted, Camilla Fröhlich, Ivan D. Horak, Michael Kragh, Jesper V. Olsen, Mikkel W. Pedersen. HER3 and IGF1R are major mediators of both acquired and intrinsic cetuximab resistance in head and neck squamous cell carcinomas. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3585. doi:10.1158/1538-7445.AM2015-3585