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Integrin associated protein (CD47) is an important target in immunotherapy, as it is expressed as a “don't eat me” signal on many tumor cells. Interference with its counter molecule signal regulatory protein alpha (SIRPα), expressed on myeloid cells, can be achieved with blocking Abs, but also by inhibiting the enzyme glutaminyl cyclase (QC) with small molecules. Glutaminyl cyclase inhibition reduces N‐terminal pyro‐glutamate formation of CD47 at the SIRPα binding site. Here, we investigated the impact of QC inhibition on myeloid effector cell‐mediated tumor cell killing by epidermal growth factor receptor (EGFR) Abs and the influence of Ab isotypes. SEN177 is a QC inhibitor and did not interfere with EGFR Ab‐mediated direct growth inhibition, complement‐dependent cytotoxicity, or Ab‐dependent cell‐mediated cytotoxicity (ADCC) by mononuclear cells. However, binding of a human soluble SIRPα‐Fc fusion protein to SEN177 treated cancer cells was significantly reduced in a dose‐dependent manner, suggesting that pyro‐glutamate formation of CD47 was affected. Glutaminyl cyclase inhibition in tumor cells translated into enhanced Ab‐dependent cellular phagocytosis by macrophages and enhanced ADCC by polymorphonuclear neutrophilic granulocytes. Polymorphonuclear neutrophilic granulocyte‐mediated ADCC was significantly more effective with EGFR Abs of human IgG2 or IgA2 isotypes than with IgG1 Abs, proposing that the selection of Ab isotypes could critically affect the efficacy of Ab therapy in the presence of QC inhibition. Importantly, QC inhibition also enhanced the therapeutic efficacy of EGFR Abs in vivo. Together, these results suggest a novel approach to specifically enhance myeloid effector cell‐mediated efficacy of EGFR Abs by orally applicable small molecule QC inhibitors., Inhibition of CD47/signal regulatory protein alpha (SIRPα) interactions is of great interest in cancer immunotherapy. In this manuscript, we investigated the impact of glutaminyl cyclase inhibition by small molecules to interfere with CD47/SIRPa interactions in epidermal growth factor receptor Ab‐mediated tumor immunotherapy in vitro and in vivo.

To examine the influence of epidermal growth factor (EGF) and its receptor (EGFR) on axial ocular elongation, we intraocularly injected an EGF antibody and an EGFR antibody into young guinea pigs with lens-induced axial elongation (myopization). Mean axial elongation was reduced in the eyes injected with the EGF/EGFR-antibody compared with the contralateral control eyes injected with PBS (phosphate-buffered solution) (0.43 ± 0.13 mm vs 0.53 ± 0.13 mm; P < .001). The intereye difference in axial length increased (P = .005) as the doses of the EGF antibody and EGFR antibody increased. As a corollary, the thickness of the retina at the posterior pole was dose-dependently increased in the injected eyes compared to the contralateral control eyes. Immunohistochemical staining for EGF and the relative mRNA expression of EGF and EGFR were the highest in eyes not injected with the EGF antibody or EGFR antibody and decreased (P < .05) as the dose of EGF antibody or EGFR antibody increased. In an in vitro study, EGF had a stimulating effect and the EGF antibody had an inhibitory effect on the proliferation and migration of RPE cells. The findings showed that the intravitreal application of an EGF antibody and EGFR antibody is associated with a dose-dependent reduction in lens-induced axial elongation in young guinea pigs. The EGFR family may play a role in axial elongation of the eye and in the development of myopia.

BackgroundAs receptor-ligand based strategies emerge for surgical imaging, the relative importance of receptor expression in different tumor types is unknown. Near-infrared (NIR) labeled epidermal growth factor receptor (EGFR) antibody, panitumumab-IRDye800, was evaluated across three cancers to demonstrate its clinical utilities and a holistic analysis framework.MethodsThirty-one patients diagnosed with high-grade glioma (HGG, n=5, NCT03510208), head and neck squamous cell carcinoma (HNSCC, n=23, NCT02415881) or lung adenocarcinoma (LAC, n=3, NCT03582124) received systemic administration of 50 mg panitumumab-IRDye800 days prior to surgery. Intraoperative NIR laparoscopic or open-field images of the surgical field were acquired and tissue mimicking phantoms were constructed to identify optimal imaging conditions. Margin distance was correlated to fluorescence on resected specimen surface. Panitumumab-IRDye800 distribution was registered to histology in fixed tissue sections. Immunohistochemistry characterized EGFR expression.ResultsIntraoperative NIR imaging enhanced tumor contrast against surrounding healthy tissue by 5.2-fold, 3.4-fold and 1.4-fold in HGG, HNSCC and LAC, respectively. Imaging quality was optimal at the lowest gain possible under ambient light. Ex vivo NIR fluorescence identified 78 – 97% of at-risk resection margins, with 72 – 92% sensitivity and 67 – 96% specificity for tumor in fixed tissue sections. Intratumoral panitumumab-IRDye800 concentration correlated with total tumoral EGFR expression (HGG > HNSCC > LAC) and delivery barrier. Cellular EGFR expression (80%) and tumor cell density (3000 cells/mm2) was highest in HGG.ConclusionsIn multiple tumor types, EGFR-targeting in fluorescence-guided surgery translated to enhanced macroscopic tumor contrast and successful margin assessment despite disparate tumor cell density and heterogeneous delivery of pantimumab-IRDye800.Translational relevanceAs receptor-ligand based strategies emerge for surgical imaging, the relative importance of receptor expression in different tumor types remains unexamined. Near-infrared labeled epidermal growth factor receptor (EGFR) antibody was evaluated across three cancers to demonstrate its clinical utilities and a holistic analysis framework. Targeted fluorescence imaging was performed in patients infused with a consistent dose of panitumumab-IRDye800 using the same fluorescence imaging devices. In high-grade glioma, head and neck squamous cell carcinoma and lung adenocarcinoma, EGFR-targeting in fluorescence-guided surgery translated to enhanced macroscopic tumor contrast and successful margin assessment. While total EGFR expression and delivery barrier corresponded to overall panitumumab-IRDye800 distribution in tissue, tumor depth and operating room lighting can have greater influence over the intraoperative fluorescence contrast at specific tissue surface locations of a certain patient. These factors can facilitate selecting appropriate molecular target and tumor qualities to streamline translation across institutions and regulatory approval of new imaging probes.

Purpose: While mutations in BRAF in metastatic colorectal cancer (mCRC) most commonly occur at the V600 amino acid, with the advent of next-generation sequencing, non-V600 BRAF mutations are increasingly identified in clinical practice. It is unclear whether these mutants, like BRAF V600E, confer resistance to anti-EGFR therapy. Experimental Design: We conducted a multicenter pooled analysis of consecutive patients with non-V600 BRAF-mutated mCRCs identified between 2010 and 2017. Non-V600 BRAF mutations were divided into functional classes based on signaling mechanism and kinase activity: activating and RAS-independent (class 2) or kinase-impaired and RAS-dependent (class 3). Results: Forty patients with oncogenic non-V600 BRAF–mutant mCRC received anti-EGFR antibody treatment [n = 12 (30%) class 2 and n = 28 (70%) class 3]. No significant differences in clinical characteristics were observed by mutation class. In contrast, while only 1 of 12 patients with class 2 BRAF mCRC responded, 14 of 28 patients with class 3 BRAF responded to anti-EGFR therapy (response rate, 8% and 50%, respectively, P = 0.02). Specifically, in first- or second-line, 1 of 6 (17%) patients with class 2 and 7 of 9 (78%) patients with class 3 BRAF mutants responded (P = 0.04). In third- or later-line, none of 6 patients with class 2 and 7 of 19 (37%) patients with class 3 BRAF mutants responded (P = 0.14). Conclusions: Response to EGFR antibody treatment in mCRCs with class 2 BRAF mutants is rare, while a large portion of CRCs with class 3 BRAF mutants respond. Patients with colorectal cancer with class 3 BRAF mutations should be considered for anti-EGFR antibody treatment. See related commentary by Fontana and Valeri, p. 6896

The discovery of epidermal growth factor (EGF) and its receptor (EGFR) revealed the connection between EGF-like ligands, signaling from the EGFR family members and cancer. Over the next fifty years, analysis of EGFR expression and mutation led to the use of monoclonal antibodies to target EGFR in the treatment of metastatic colorectal cancer (mCRC) and this treatment has improved outcomes for patients. The use of the RAS oncogene mutational status has helped to refine patient selection for EGFR antibody therapy, but an effective molecular predictor of likely responders is lacking. This review analyzes the potential utility of measuring the expression, levels and activation of EGF-like ligands and associated processes as prognostic or predictive markers for the identification of patient risk and more effective mCRC therapies.

Recent clinical data have linked KRAS/TP53 co-mutation (mut) to resistance to radiotherapy (RT), but supporting laboratory in vivo evidence is lacking. In addition, the ability of different radiation doses, with/without epidermal growth factor receptor (EGFR)-directed treatment, to achieve local tumour control as a function of KRAS status is unknown. Here, we assessed clonogenic radiation survival of a panel of annotated lung cancer cell lines. KRASmut/TP53mut was associated with the highest radioresistance in non-isogenic and isogenic comparisons. To validate these findings, isogenic TP53mut NCI-H1703 models, KRASmut or wild-type (wt), were grown as heterotopic xenografts in nude mice. A clinical RT schedule of 30 fractions over 6 weeks was employed. The dose that controlled 50% of tumours (TCD(50)) was calculated. The TCD(50) for KRASwt/TP53mut xenografts was 43.1 Gy whereas KRASmut/TP53mut tumours required a 1.9-fold higher TCD(50) of 81.4 Gy. The EGFR inhibitor erlotinib radiosensitized KRASmut but not KRASwt cells and xenografts. The TCD(50) associated with adding erlotinib to RT was 58.8 Gy for KRASmut, i.e., a ~1.4-fold dose enhancement. However, the EGFR antibody cetuximab did not have a radiosensitizing effect. In conclusion, we demonstrate for the first time that KRASmut in a TP53mut background confers radioresistance when studying a clinical RT schedule and local control rather than tumour growth delay. Despite the known unresponsiveness of KRASmut tumours to EGFR inhibitors, erlotinib radiosensitized KRASmut tumours. Our data highlight KRAS/TP53 co-mutation as a candidate biomarker of radioresistance that can be at least partially reversed by dose escalation or the addition of a targeted agent.

Background:The incidence and mortality associated with lung cancers are increasing at an alarming rate. Studies have shown that activation of Epidermal Growth Factor Receptor (EGFR) triggers the tumorigenesis in these cancers. The potential role of analyzing EGFR expression in these carcinomas could go a long way in devising screening tools for early detection of lung carcinoma. This study was carried out to evaluate the prevalence and factors associated with EGFR expression. Methods: This cross sectional study was carried out among 75 paraffin block specimens of lung carcinoma received in our tertiary care center for a period of five years. Three micron thick paraffin sections were cut and Hemotoxylin & Eosin staining was done. All the adenocarcinoma cases were immunostained with EGFR antibody and the results were analyzed. Results: Majority of the tumors were moderately differentiated (46%) and were negative for lymph node metastasis (69%). With regards to EGFR positivity, majority of the tumors showed EGFR expression 3+ (57.3%) flowed by 2+ (16%). Among the EGFR negative cases, ALK expression was positive in 5% of the cases. Conclusion: The present study has reinforced the fact that Indian patients have high expression of EGFR and therefore can be benefitted by targeted therapy.IHC coupled with molecular analysis would be of maximum benefit to patients with EGFR & ALK mutations.

Objectives To establish a circulating tumor cell (CTC) enrichment system for non-small cell lung cancer (NSCLC) patients who received first-line treatment with epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (EGFR-TKI), using EGFR magnetic liposomes (EGFR-ML). Materials and methods An inverted evaporation method was used to develop antibody modified EGFR-ML. Peripheral blood was collected from NSCLC patients who underwent first-line EGFR-TKI treatment for CTC enumeration. Results Protein electrophoresis, magnetic saturation curve, and ultraviolet absorption spectrum showed successful incorporation of the EGFR antibody on the surface of the magnetic microspheres, and the development of EGFR-ML was ascertained based on cell morphology and particle size. Using EGFR-ML, CTC were successfully enriched from blood samples and were identified in 77.3% (99/128) of the cohort. When compared to the 21L858R variant, EGFR-19del showed lower CTC counts by EGFR-ML (CTCEGFR). At one month after EGFR-TKI, a lower CTCEGFR was associated with partial response (PR) during treatment (CTCEGFR Conclusion We showed that establishing a CTC enrichment system by antibody modified EGFR-ML in NSCLC is feasible. CTC enumeration by EGFR-ML may have the potential to supplement RECIST in dynamically monitoring the response of NSCLC patients’ to first-line EGFR-TKI.

Epidermal growth factor receptor (EGFR) ectodomain variants mediating primary resistance or secondary treatment failure in cancer patients treated with cetuximab or panitumumab support the need for more resistance-preventive or personalized ways of targeting this essential pathway. Here, we tested the hypothesis that the EGFR nanobody 7D12 fused to an IgG1 Fc portion (7D12-hcAb) would overcome EGFR ectodomain–mediated resistance because it targets a very small binding epitope within domain III of EGFR. Indeed, we found that 7D12-hcAb bound and inhibited all tested cell lines expressing common resistance-mediating EGFR ectodomain variants. Moreover, we assessed receptor functionality and binding properties in synthetic mutants of the 7D12-hcAb epitope to model resistance to 7D12-hcAb. Because the 7D12-hcAb epitope almost completely overlaps with the EGF-binding site, only position R377 could be mutated without simultaneous loss of receptor functionality, suggesting a low risk of developing secondary resistance toward 7D12-hcAb. Our binding data indicated that if 7D12-hcAb resistance mutations occurred in position R377, which is located within the cetuximab and panitumumab epitope, cells expressing these receptor variants would retain sensitivity to these antibodies. However, 7D12-hcAb was equally ineffective as cetuximab in killing cells expressing the cetuximab/panitumumab-resistant aberrantly N-glycosylated EGFR R521K variant. Yet, this resistance could be overcome by introducing mutations into the Fc portion of 7D12-hcAb, which enhanced immune effector functions and thereby allowed killing of cells expressing this variant. Taken together, our data demonstrate a broad range of activity of 7D12-hcAb across cells expressing different EGFR variants involved in primary and secondary EGFR antibody resistance.

Background RAS short variant (SV) mutations in colorectal cancer (CRC) are associated with lack of benefit from epidermal growth factor receptor (EGFR) monoclonal antibody (EGFRmAb). However, the clinical implications for RAS amplification (RASa) as a biomarker for anti-EGFR therapy in CRC remain ill defined. Methods Genomic analysis was performed using the Foundation Medicine (FM) comprehensive genomic profiling database of 37,233 CRC cases. Clinical outcomes were assessed using two independent cohorts: the City of Hope (COH) cohort of 338 patients with metastatic CRC (mCRC) and the Flatiron Health–FM real-world clinicogenomic database (CGDB) of 3,904 patients with mCRC. Results RASa was detected in 1.6% (614/37,233) of primarily mCRC. RASa 6–9 (n = 241, 39%), 10–19 (n = 165, 27%), and ≥ 20 (n = 209, 34%) copy number subsets had co-RAS SV/BRAF V600E in 63%/3%, 31%/0.6%, and 4.8%/0% of cases, respectively. In the COH cohort, six patients with RASa (13–54 copies) received EGFRmAb, four of six had progressive disease, two had stable disease, and median time to treatment discontinuation (TTD) was 2.5 months. Of the CGDB EGFRmAb-treated patients, those with RASa (n = 9) had median TTD of 4.7 months and overall survival (OS) of 11.4 months, those with RAS SV (n = 101) had median TTD and OS of 5.3 and 9.4 months, and those with RAS/BRAF wild-type (n = 608) had median TTD and OS of 7.6 and 13.7 months. Conclusion Patients with RASa without RAS mutations (1.1% of mCRC) may have poor outcomes on EGFRmAb, although numbers herein were small, and interpretation is confounded by combination chemotherapy. Larger independent studies are warranted to determine if RASa, including degree of amplification, may act similarly to RAS mutation as a resistance mechanism to EGFRmAb therapies. Implications for Practice Genomic data suggest that RAS amplification occurs as the sole RAS/RAF alteration in >1% of colorectal cancer cases and that degree of amplification inversely correlates with co-occurring MAPK pathway alterations. Preliminary clinical evidence suggests that RAS amplification may function similarly to RAS mutation as a negative predictor of benefit from anti-epidermal growth factor receptor therapies in colorectal cancer. More clinical data are needed, and comprehensive genomic profiling, including detection of RAS amplification, should be used in trial design to inform therapy selection.

Epidermal growth factor receptor (EGFR) is a theranostic biomarker for a variety of cancer types. The aim of the present study was to develop an 18F radiolabeled EGFR targeting RNA aptamer, and to investigate its ability to visualize and quantify EGFR in xenograft models. Biolayer interferometry binding assay was used to detect the binding affinity of the alkyne-modified EGFR aptamer MinE07 (denoted as ME07) with recombinant human wild-type EGFR protein and the mutant EGFRvIII protein. Cy5-conjugated ME07 was used for flow cytometry and immunofluorescence staining, and an Alexa Fluor 488-labeled EGFR antibody (ab193244) was used as a control. 18F-Fluorobenzoyl (FB) azide was employed as a synthon to produce 18F-FB-ME07 via click chemistry, and the cellular uptake and internalization characteristics of 18F-FB-ME07 were investigated. Static PET scans, 60-min dynamic scans, and biodistribution study of 18F-FB-ME07 were performed in three types of tumor models. The Kd values of ME07 to wtEGFR and EGFRvIII proteins were 0.3 nM and 271 nM respectively. The A431, U87MG, and HCT-116 cells showed strong, weak, and negative binding with Cy5-ME07, which is consistent with EGFR expression level in these cells. Peak cell uptake values of 18F-FB-ME07 in A431, U87MG and HCT-116 cells were 2.86%, 2.19% and 0.88% of the added dose respectively. The mean internalization of 18F-FB-ME07 in these cells were 60.02%, 53.1%, and 52.8% of the total accumulated radioactivity. In static PET imaging, despite high uptake in the liver and kidneys, 18F-FB-ME07 showed reasonable accumulation in A431 tumors (1.02 ± 0.13 %ID/g at 30 min after injection). Of note, the uptake of 18F-FB-ME07 in A431 xenografts was significantly higher than that in U87MG and HCT-116 xenografts. In A431 xenografted mice, the tumor/blood ratio was 3.89 and the tumor/muscle ratio reached 8.65. We for the first time generated an aptamer-derived EGFR targeting PET tracer 18F-FB-ME07, which showed highly selective targeting ability in mouse tumor models expressing different levels of EGFR. Our results suggest that 18F-FB-ME07 is a potential EGFR targeting molecular imaging probe for future clinical translation.

Acquired resistance to cetuximab, an antibody that targets the EGFR, impacts clinical benefit in head and neck, and colorectal cancers. One of the mechanisms of resistance to cetuximab is the acquisition of mutations that map to the cetuximab epitope on EGFR and prevent drug binding. We find that necitumumab, another FDA-approved EGFR antibody, can bind to EGFR that harbors the most common cetuximab-resistant substitution, S468R (or S492R, depending on the amino acid numbering system). We determined an X-ray crystal structure to 2.8 Å resolution of the necitumumab Fab bound to an S468R variant of EGFR domain III. The arginine is accommodated in a large, preexisting cavity in the necitumumab paratope. We predict that this paratope shape will be permissive to other epitope substitutions, and show that necitumumab binds to most cetuximab- and panitumumab-resistant EGFR variants. We find that a simple computational approach can predict with high success which EGFR epitope substitutions abrogate antibody binding. This computational method will be valuable to determine whether necitumumab will bind to EGFR as new epitope resistance variants are identified. This method could also be useful for rapid evaluation of the effect on binding of alterations in other antibody/antigen interfaces. Together, these data suggest that necitumumab may be active in patients who are resistant to cetuximab or panitumumab through EGFR epitope mutation. Furthermore, our analysis leads us to speculate that antibodies with large paratope cavities may be less susceptible to resistance due to mutations mapping to the antigen epitope. Mol Cancer Ther; 17(2); 521–31. ©2017 AACR.

Epidermal growth factor receptor (EGFR) is overexpressed in many cancers and therefore serves as an excellent target for prodrug activation. Functionalised trans-cyclooctenes (TCO) were conjugated to an EGFR antibody (cetuximab), providing a reagent for pre-targeting and localisation of the bioorthogonal reagent. The TCOs react with a 4-azidobenzyl carbamate doxorubicin prodrug via a [3 + 2]-cycloaddition and subsequent self-immolation leads to release of doxorubicin (click-and-release). In vitro cell-based assays demonstrated proof-of-concept, that cetuximab conjugated to highly strained TCO (AB-d-TCO) could bind to the EGFR in a melanoma cell line, and selectively activate the doxorubicin prodrug. In a non-EGFR expressing melanoma cell line, no significant prodrug activation was observed. In vivo experiments using this combination of AB-d-TCO and the azido-doxorubicin prodrug in a murine melanoma model revealed no significant anti-tumour activity or increased survival, suggesting there was insufficient prodrug activation and drug release at the tumour site.

Intraoperative definition of tumor free resection margins in head and neck cancer is challenging. In the current proof-of-principle study we evaluated a novel silica nanoparticle-based agent for its potential use as contrast enhancer. We synthesized silica nanoparticles with an average size of 45 nm and modified these particles with the fluorescence stain fluorescein isocyanate (FITC) for particle detection and with epidermal growth factor receptor (EGFR)-targeting antibodies for enhanced tumor specificity. The nanoparticles exhibited good biocompatibility and could be detected in vitro and in vivo by confocal laser scanning microscopy. Additionally, we show in an ex vivo setting that these modified nanoparticles specifically bind to tumor samples and could be detected using a handheld confocal fluorescence endomicroscope. From a clinical point of view, we believe that this method could be used for tumor border contrast enhancement and for better intraoperative definition of R-0 tumor resection.

Although antibodies are routinely used to label and isolate a desired cell type from a more complex mixture of cells, via either fluorescence-activated cell sorting (FACS) or magnetic-activated cell sorting (MACS), such antibody labeling is not easily reversible. We describe an FACS and MACS compatible method to reversibly label and purify cells using aptamers. Magnetic beads loaded with the epidermal growth factor receptor (EGFR)-binding antagonistic aptamer E07 specifically isolated EGFR-expressing cells, and pure, label-free cells were recovered via treatment with an "antidote" oligonucleotide complementary to the aptamer. Additionally, while FACS sorting cells with E07 or EGFR antibody yielded EGFR(+) cells with impeded EGFR signaling, stripping off the aptamer via antidote treatment restored receptor function, returning cells to their native state, which was not possible with the antibody. The ability to reversibly label or isolate cells without compromising their function is a valuable, versatile tool with important implications for both the laboratory and clinic.

Introduction: Glioblastoma (GBM) is the most common and devastating primary brain tumor. The recurrence rate remains high with a median survival of 15 months. GBM’s infiltrative nature results in ill-defined margins that makes maximal tumor resection with minimal morbidity a challenge. Epidermal growth factor receptor (EGFR) is the most frequently amplified gene in GBM (35-45% of tumors) and is associated with overexpression in about 40-98% of cases, a characteristic of more aggressive phenotypes. We hypothesize that fluorescence labeled anti-EGFR monoclonal antibodies (mAb), panitumumab-IRDye800 (pan800) and cetuximab-IRDye800 (cet800), could be leveraged to enhance tumor contrast during surgical resection and improve patient outcome. Methods: 50mg fluorescently labeled corresponding study drugs, pan800 and cet800 respectively, were administered 1-2 days in glioblastoma patients with contrast enhancing (CE) tumors prior to surgery following 100 mg loading dose of unlabeled cetuximab or panitumumab. Near-infrared fluorescence imaging of tumor and histologically negative peri-tumoral tissue was performed intraoperatively and ex vivo. Fluorescence was measured as mean fluorescence intensity (MFI), and tumor-to-background ratios (TBRs) were calculated by comparing MFIs of tumor and histologically uninvolved tissue. Results: Despite heterogeneous drug uptake across all resected brain tissues, mean fluorescence intensity (MFI) correlated strongly (R^2=0.97) with tumor volume among histologically confirmed tumor tissues. The smallest detectable tumor size in a closed-field setting was 4.2 x 2.7 mm^2 (8.2 mg) for pan800 and 8.5 x 6.6 mm^2 (70mg) for cet800. Tumor tissues from pan800 infusion had significantly higher mean TBR (8.1 ± 4.6) than cet800 infused ones in intraoperative imaging (3.3 ± 2.7; P = 0.004). NIR fluorescence from both test drugs provided high contrast to identify as few as a cluster of (5 ± 1) tumor cells in macroscopic imaging of whole sections of paraffin embedded tissues. Sensitivity and specificity of MFI for viable tumor detection was calculated and fluorescence was found to be highly sensitive (64.4% for pan800, 73.0% for cet800) and specific (98.0% for pan800, 66.3% for cet800) for viable tumor tissue while normal peri-tumoral tissue showed minimal fluorescence. No related grade-2 adverse events were observed 30 days beyond the infusion of either study drugs. Conclusion: EGFR antibody based imaging for contrast-enhanced glioblastomas proved safe in human patients and specific intratumoral delivery of NIR fluorescence provided high optical contrast and resolution for intraoperative image-guided resection. Fully humanized panitumumab-IRDye800 demonstrated superior detection sensitivity and tumor specificity over the chimeric cetuximab-IRDye800.

Background Laryngeal cancer is a common malignant tumor of the head and neck. Clinical treatment methods mainly include radiotherapy and chemotherapy, but the toxicity and side effects of these treatments seriously affect the quality of life of patients. Currently, there are no specific anti-laryngeal cancer drugs available. Therefore, it is necessary to develop new targeted drugs for laryngeal cancer. Methods We established a cell model of laryngeal cancer in vitro and a TU686 xenograft model in vivo. We then carried out the related research through a series of experiments [including laser confocal microscopy, enzyme linked immune sorbent assay (ELISA) and Western blot]. Results The results showed that the epidermal growth factor receptor (EGFR) antibody antagonist 6E-C could not only specifically bind to EGFR, but also specifically inhibit the binding of EGF to EGFR. Further analysis indicated that 6E-C could inhibit the EGFR-mediated intracellular signaling pathway. Furthermore, 6E-C inhibited xenograft tumor growth in vivo. Conclusions In summary, we have successfully prepared a new anti-EGFR antibody antagonist, which exhibited anti-laryngeal cancer effects in vitro and in vivo. The current research demonstrates that the EGFR antibody antagonist 6E-C shows potential as an effective anti-laryngeal cancer agent, with potential clinical application value. This study therefore provides a solid foundation for related research in the future.

This study investigated whether antibody-modified immunomagnetic microspheres (IMs) can be used to detect and quantify circulating tumor cells (CTCs) originating from gliomas in liquid biopsies and whether CTC counts in clinical samples are related to the degree of malignancy of gliomas based on clinical data. Epidermal growth factor receptor (EGFR)-antibody-modified and epithelial cell adhesion molecule (EpCAM)-modified IMs were developed, their physical properties, including particle size and zeta potential as well as their biocompatibility, were characterized, and their ability to detect CTCs originating from gliomas was evaluated using a mouse xenograft model and clinical specimens [cerebrospinal fluid (CSF) and peripheral blood collected from 30 patients with gliomas]. The results showed that EGFR-IMs and EpCAM-IMs had low cytotoxicity and that they could capture CTCs in mouse and human biofluids with high capture efficiency. Moreover, CTC counts in the human CSF were positively correlated with glioma grade. Thus, EGFR-IMs and EpCAM-IMs have clinical applicability for the diagnosis of glioma and for monitoring treatment response in patients.

Therapeutic targeting of ASCT2, a glutamine transporter that plays a major role in glutamine uptake in cancer cells, is challenging because ASCT2 also has a biological role in normal tissues. In this study, we report our novel finding that ASCT2 is physically associated in a molecular complex with epidermal growth factor receptor (EGFR), which is often overexpressed in human head and neck squamous cell carcinoma (HNSCC). Furthermore, we found that ASCT2 can be co-targeted by cetuximab, an EGFR antibody approved for treating metastatic HNSCC. We demonstrated that cetuximab downregulated ASCT2 in an EGFR expression-dependent manner via cetuximab-mediated EGFR endocytosis. Downregulation of ASCT2 by cetuximab led to decreased intracellular uptake of glutamine and subsequently a decreased glutathione level. Cetuximab thereby sensitized HNSCC cells to reactive oxygen species (ROS)-induced apoptosis and, importantly, it is independent of effective inhibition of EGFR downstream signaling by cetuximab. In contrast, knockdown of EGFR by siRNA or inhibition of EGFR kinase with gefitinib, an EGFR kinase inhibitor, failed to sensitize HNSCC cells to ROS-induced apoptosis. Our findings support a novel therapeutic strategy for EGFR-overexpressing and cetuximab-resistant cancers by combining cetuximab with an oxidative therapy.

Three FDA-approved epidermal growth factor receptor (EGFR) antibodies (cetuximab, panitumumab, necitumumab) are clinically available to treat patients with different types of cancers. Interestingly, panitumumab is of human IgG2 isotype, which is often considered to have limited immune effector functions. Unexpectedly, our studies unraveled that human IgG2 antibodies against EGFR mediated effective CDC when combined with another noncross-blocking EGFR antibody. This second antibody could be of human IgG1 or IgG2 isotype. Furthermore, EGFR antibodies of human IgG2 isotype were highly potent in recruiting myeloid effector cells such as M1 macrophages and PMN for tumor cell killing by ADCC. Tumor cell killing by PMN was more effective with IgG2 than with IgG1 antibodies if tumor cells expressed lower levels of EGFR. Additionally, lower expression levels of the “don′t eat me” molecule CD47 on tumor cells enabled ADCC also by M2 macrophages, and improved PMN and macrophage-mediated ADCC. A TCGA enquiry revealed broadly varying CD47 expression levels across different solid tumor types. Together, these results demonstrate that human IgG2 antibodies against EGFR can promote significant Fc-mediated effector functions, which may contribute to their clinical efficacy. The future challenge will be to identify clinical situations in which myeloid effector cells can optimally contribute to antibody efficacy.

Increasing the detection specificity and sensitivity of the epidermal growth factor receptor (EGFR) will benefit the diagnosis of a number of cancers. Terahertz metamaterial biosensors are very sensitive and able to detect trace biomolecules. In this work, we fabricated a bow-tie THz metamaterial biosensor and functionalized it with EGFR antibody for specific EGFR detection. The results demonstrate that the proposed strategy can achieve EGFR detection with high specificity and sensitivity. This has the potential to be introduced into clinical use for the fast diagnosis of EGFR related diseases.

Epidermal growth factor receptor (EGFR) is a clinically validated target and often overexpressed in some solid tumors. Both EGFR tyrosine kinase inhibitors and ligand-blocking antibodies have been approved for treatment of NSCLC, head and neck cancers and colorectal cancers. However, clinical response is limited and often accompanied by significant toxicities due to normal tissue expression. To improve the effectiveness of targeting EGFR while minimizing the toxicities on normal tissues, we developed a low-affinity anti-EGFR antibody drug conjugate (ADC), RN765C. Potent in vitro cytotoxicity of RN765C, with nanomolar to subnanomolar EC50, was observed on a panel of cancer cell lines expressing moderate to high level of EGFR. In contrast, RN765C was less effective in killing normal human keratinocytes, presumably due to its lower receptor expression. Mechanistically, RN765C has multiple modes of action: inducing payload mediated mitotic arrest and cell death, blocking EGFR pathway signal and mediating antibody dependent cell cytotoxicity. In preclinical studies, a single dose of RN765C at 1.5-3 mg/kg was generally sufficient to induce tumor regression in multiple cell line and patient-derived xenograft models, including those that are resistant to EGFR-directed tyrosine kinase inhibitors. Our data support further investigation of RN765C in the clinic to treat EGFR expressing solid tumors.

Epidermal growth factor family of receptor tyrosine kinases (ERBB) family cell surface receptors, including epidermal growth factor receptor (EGFR/ERBB1), are phosphorylated upon binding by various EGF family ligands and signal via multiple kinase pathways. EGFR signaling is enhanced because of mutational activation of EGFR in almost half of glioblastomas, the most common malignant primary brain tumor. Therapeutic targeting of EGFR in glioblastoma has remained largely unsuccessful. Here, we profiled nine long-term (LTC) and five glioma-initiating (GIC) cell lines for expression and activation of ERBB family receptors and expression of their ligands. Receptors and ligands were abundantly expressed, with patterns overall similar to glioblastoma expression profiles in vivo as deposited in The Cancer Genome Atlas database. No differences between LTC and GIC emerged. Irrespective of ligand or receptor expression, neither an EGFR antibody, erbitux, nor an EGFR tyrosine kinase inhibitor, gefitinib, were particularly active against LTC or GIC at clinically relevant concentrations. Self-renewal capacity of GIC was severely compromised by epidermal growth factor (EGF) withdrawal, but rescued by transforming growth factor alpha (TGF-α), although not by neuregulin-1 (NRG-1). Subcellular fractionation indicated high levels of nuclear phosphorylated EGFR in all LTC and GIC. In LN-229 cells, pERBB2 and pERBB3 were also detected in the nucleus. Nuclear pERBB2 was less sensitive, whereas pERBB3 was induced, in response to gefitinib. This study provides an extensive characterization of human glioma cell models, including stem-like models, with regard to ERBB receptor/ligand expression and signaling. Redundant signaling involving multiple ERBB family ligands and receptors may contribute to the challenges of developing more effective EGFR-targeted therapies for glioblastoma.

The objective of this study was to investigate the effect of Tamoxifen (TAM) treatment on epidermal growth factor receptor (EGFR) expression in the pubertal mice ovary. In this study, 80 female mice (8 week-old) were used. Animals divided four groups; non-injected (control A), injected with TAM’s vehicle solution (control B). The mice in groups TAM 0.5 and TAM 1.5 were treated with tamoxifen at a dose 0.5 and 1.5 mg/mouse/day respectively. TAM was dissolved with 10% ethanol: 90% corn oil. Mice were given daily subcutaneously injections for 5 days. Ovarian sections were immunostained with EGFR antibody and triple staining for examine the general structure. We observed that follicular atresia was increased, follicular cysts were formed in stroma, and interstitial cells were increased with TAM dose treatment. In the present study, there were no significant differences in the EGFR expression in mouse ovary of all groups. EGFR expression was not determined in the granulosa cells. While strong EGFR immunoreaction was observed in the interstitial cells and oocyte cytoplasma, weak EGFR immunoreactions was observed in theca cells of follicles. As a result, it was observed that the administered doses of TAM have not significant influence on EGFR expression.

// Friederike Braig 1 , Manuela Marz 1 , Aneta Schieferdecker 1 , Alexander Schulte 2 , Mareike Voigt 1 , Alexander Stein 1 , Tobias Grob 3 , Malik Alawi 4 , Daniela Indenbirken 5 , Malte Kriegs 6 , Erik Engel 7 , Udo Vanhoefer 8 , Adam Grundhoff 5 , Sonja Loges 1,9 , Kristoffer Riecken 10 , Boris Fehse 10 , Carsten Bokemeyer 1 and Mascha Binder 1 1 Department of Oncology and Hematology, BMT with section Pneumology, Hubertus Wald Tumorzentrum / UCCH, University Medical Center Hamburg-Eppendorf, Hamburg, Germany 2 Department of Neurosurgery, Laboratory for Brain Tumor Biology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany 3 Department of Pathology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany 4 Bioinformatics Service Facility, University Medical Center Hamburg-Eppendorf, Hamburg, Germany 5 Heinrich-Pette-Institute, Leibniz-Institute for Experimental Virology (HPI), Hamburg, Germany 6 Radiation Biology and Radio-Oncology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany 7 Hamatologisch-onkologische Praxis Altona (HOPA), Hamburg, Germany 8 Marienkrankenhaus, Zentrum fur Innere Medizin, Hamburg, Germany 9 Institute for Tumor Biology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany 10 Research Department Cell and Gene Therapy, Department of Stem Cell Transplantation, University Medical Center Hamburg-Eppendorf, Hamburg, Germany Correspondence to: Mascha Binder, email: // Keywords : panitumumab, cetuximab, EGFR antibody resistance, mutation, circulating tumor DNA Received : January 30, 2015 Accepted : February 20, 2015 Published : March 14, 2015 Abstract Acquired resistance to epidermal growth factor receptor (EGFR) targeted antibodies represents a clinical challenge in the treatment of gastrointestinal tumors such as metastatic colorectal cancer, but its molecular mechanisms are incompletely understood. We scanned KRAS exon 2/3/4, NRAS exon 2/3/4 and the overlapping epitopes of the EGFR antibodies cetuximab and panitumumab for mutations in pre- and post-treatment tumor tissue of 21 patients with gastrointestinal cancer treated with chemotherapy +/- EGFR antibodies by next-generation sequencing (“tumor tissue” cohort). We describe a novel EGFR exon 12 mutation acquired in tumors of 1 out of 3 patients treated with panitumumab. The EGFR G465R mutation introduces a positive charge within the overlap of the panitumumab and cetuximab epitopes. It abrogates antibody binding and mediates cross-resistance to both antibodies in EGFR G465R-transfected Ba/F3 cells. In circulating tumor DNA from an independent “liquid biopsy” cohort of 27 patients, we found this novel mutation in 1 out of 6 panitumumab-treated cases while about one third of patients show acquired RAS mutations. We show that acquired resistance by epitope-changing mutations also emerges during panitumumab treatment, which can be easily detected by a liquid biopsy approach even before clinical resistance occurs and this may help in tailoring EGFR-targeted therapies.

Solid tumors can often be hypoxic in regions, and cancer cells can respond to hypoxia with an increase in proliferation and a decrease in apoptosis, leading to a net increase in viable cell numbers. We have recently found that in an osteosarcoma cell line, hypoxia‐induced proliferation depends on arginase II induction. Epidermal growth factor receptor (EGFR) has been shown to mediate the hypoxia‐induced cellular proliferation in some cancer cell lines. We hypothesized that hypoxia‐induced proliferation of HeLa cells would depend on arginase II induction and that this induction of arginase II would occur through EGFR activation. Exposure of HeLa cells to hypoxia resulted in an upregulation of arginase II mRNA and protein levels, with no effect on arginase I expression. Hypoxia also resulted in significantly greater viable cell numbers than did normoxia. The hypoxia‐induced increase in viable cell numbers was prevented by either a small molecule inhibitor of arginase or siRNA targeting arginase II. Overexpression of arginase II resulted in an increase in viable cell numbers both in normoxia and hypoxia. Hypoxia caused a substantial induction of both epidermal growth factor (EGF) and EGFR. Preventing hypoxia‐induced EGFR expression using siRNA abolished hypoxia‐induced arginase II expression and the increase in viable cell numbers. Treatment with EGF in normoxia not only induced arginase II expression but also resulted in an increase in viable cell numbers. Blocking EGF interactions with EGFR using either an EGF neutralizing antibody or an EGFR antibody prevented the hypoxia‐induced increase in viable cell numbers. These results demonstrate an EGF/EGFR/arginase II pathway that is necessary for hypoxic proliferation in HeLa cells.

Gefitinib (GF) is a US Food and Drug Administration-approved epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor for treating the lung cancers. We fabricated colloidal gold nanoparticle (AuNP) conjugates of the GF anticancer drug by self-assembly to test their potency against A549, NCI-H460, and NCI-H1975 lung cancer cells. GF adsorption on AuNP surfaces was examined by UV–vis absorption spectra and surface-enhanced Raman scattering. Density functional theory calculations were performed to estimate the energetic stabilities of the drug-AuNP composites. The N1 nitrogen atom of the quinazoline ring of GF was calculated to be more stable than the N3 in binding Au cluster atoms. The internalizations of GF-coated AuNPs were examined by transmission electron and dark-field microscopy. A cell viability test of AuNP–GF conjugates with the EGFR antibody exhibited much higher reductions than free GF for A549, NCI-H460, and NCI-H1975 lung cancer cells after treatment for 48 h.

Interaction between the epidermal growth factor receptor (EGFR) and the insulin-like growth factor receptor (IGF-1R) has been well established in many cancer types. We investigated the effects of cetuximab (EGFR antibody) and IMC-A12 (IGF-1R antibody) on the response of head and neck squamous cell carcinoma (HNSCC) to radiation therapy (RT). The effects of cetuximab and IMC-A12 on cell viability and radiosensitivity were determined by clonogenic cell survival assay. Formation of nuclear γ-H2AX and 53BP1 foci was monitored by immunofluorescence. Alterations in target signaling were analyzed by Western blots. In vivo tumor growth delay assay was performed to determine the efficacy of triple therapy with IMC-A12, cetuximab, and RT. In vitro data showed that cetuximab differentially affected the survival and the radiosensitivity of HNSCC cells. Cetuximab suppressed DNA repair that was evident by the prolonged presence of nuclear γ-H2AX and 53BP1 foci. IMC-A12 did not have any effect on the cell survival. However, it increased the radiosensitivity of one of the cell lines. EGFR inhibition increased IGF-1R expression levels and also the association between EGFR and IGF-1R. Addition of IMC-A12 to cetuximab did not increase the radiosensitivity of these cells. Tumor xenografts exhibited enhanced response to RT in the presence of either cetuximab or IMC-A12. Concurrent treatment regimen failed to further enhance the tumor response to cetuximab and/or RT. Taken together our data suggest that concomitant inhibition of both EGFR and IGF-1R pathways did not yield additional therapeutic benefit in overcoming resistance to RT.

Preclinical studies demonstrate that epidermal growth factor receptor (EGFR) signals through both kinase-dependent and independent pathways and that combining a small-molecule EGFR inhibitor, EGFR antibody, and/or anti-angiogenic agent is synergistic. We conducted a dose-escalation, phase I study combining erlotinib, cetuximab, and bevacizumab. The subset of patients with metastatic colorectal cancer was analyzed for safety and antitumor activity. Forty-one patients with heavily pretreated metastatic colorectal cancer received treatment on a range of dose levels. The most common treatment-related grade ≥2 adverse events were rash (68%), hypomagnesemia (37%), and fatigue (15%). Thirty of 34 patients (88%) treated at the full FDA-approved doses of all three drugs tolerated treatment without drug-related dose-limiting effects. Eleven patients (27%) achieved stable disease (SD) ≥6 months and three (7%) achieved a partial response (PR) (total SD>6 months/PR= 14 (34%)). Of the 14 patients with SD≥6 months/PR, eight (57%) had received prior sequential bevacizumab and cetuximab, two (5%) had received bevacizumab and cetuximab concurrently, and four (29%) had received prior bevacizumab but not cetuximab or erlotinib (though three had received prior panitumumab). The combination of bevacizumab, cetuximab, and erlotinib was well tolerated and demonstrated antitumor activity in heavily pretreated patients with metastatic colorectal cancer.

Lessons LearnedGC1118 is a novel fully human anti-epidermal growth factor receptor (EGFR) antibody with unique binding epitopes and different ligand-binding inhibitory activity compared with cetuximab or panitumumab. GC1118 showed promising antitumor activity, especially in patients with colorectal cancer resistant to prior EGFR antibody. Skin toxicities were more common and diarrhea was less frequent compared with other anti-EGFR antibodies.BackgroundGC1118 is a novel monoclonal antibody targeting epidermal growth factor receptor (EGFR) with more potent ligand inhibition than cetuximab or panitumumab. We conducted a first-in-human, phase I study of GC118 in patients with refractory solid tumors.MethodsIn the dose escalation part, GC1118 was administered on days 1, 8, 15, and 22, followed by a 2-week rest, during which dose-limiting toxicities (DLTs) were evaluated. In the expansion part, patients were enrolled into three cohorts (Cohort 1 [C1], patients with colorectal cancer [CRC] without prior anti-EGFR treatment; Cohort 2 [C2], patients with CRC with tumors resistant to anti-EGFR therapy; Cohort 3 [C3], EGFR-overexpressing gastric cancer).ResultsIn the dose escalation part, 24 patients were treated at five dose levels: 0.3, 1.0, 3.0, 4.0, and 5.0 mg/kg. In the 5.0 mg/kg cohort, two patients experienced DLTs (skin toxicities). The maximum-tolerated dose (MTD) was 4.0 mg/kg. Common adverse events were skin toxicities. In the expansion part, 39 patients were enrolled. In Cohort 1, stable disease (SD) was observed in 58%; in Cohort 2, partial response (PR) 17% and SD 8%; in Cohort 3, PR 8% and SD 17%.ConclusionGC1118 showed promising antitumor activity and was well tolerated. Infrequent diarrhea compared with other anti-EGFR antibodies might be advantageous for further development.

To define the molecular basis of secondary resistance to epidermal growth factor receptor (EGFR)-specific antibodies is crucial to increase clinical benefit in patients. The limited access to posttreatment tumor samples constitutes the major barrier to conduct these studies, representing preclinical experimentation as a useful alternative. Anti-EGFR antibody-based therapy has been reported to mediate tumor regression by interrupting oncogenic signals and, more recently, by inducing antitumor immunological responses. However, resistance models have been focused only on tumor escape associated with EGFR blockade, whereas studies describing immune-associated escape mechanisms have not been reported thus far. To address this idea, we modeled resistance induction in D122 metastasis-bearing C57BL/6 mice treated with 7A7 (an anti-murine EGFR antibody). Similarly to patients receiving EGFR-specific antibodies, 7A7 resistance promotion represents an important drawback to successful therapy. Characterization of primary cultures derived from metastasis in 7A7-treated mice revealed a high frequency of tumor variants resistant to in vivo and in vitro antibody treatment. We showed, for the first time, the convergence of alterations in oncogenic and immunological pathways in 7A7-resistant variants. To identify key molecules behind resistance, seven 7A7-resistant variants were screened. HER3 overexpression and PTEN deficiency leading to hyperactivation of protumoral downstream signaling were found in these variants as a consequence of 7A7-mediated EGFR inhibition. Concomitantly, we found a high percentage of resistant variants carrying abnormalities in the constitutive and/or interferon gamma (IFN-γ)-inducible major histocompatibility complex I (MHC-I) expression. A significant decrease in mRNA levels for MHC-I heavy chains, β2-microglogulin and antigen processing machinery genes as well as transcriptional alterations in IFN-γ pathway components were identified as the main mechanisms underlying MHC-I expression defects in 7A7-resistant variants. Notably, these defects have not been previously associated with EGFR-specific antibody resistance, providing novel immunological escape mechanisms. This study has strong implications for the development of new combination strategies to overcome anti-EGFR antibodies refractoriness.

The main challenge for neural progenitor cell (NPC)-mediated repair of spinal cord injury (SCI) is lack of favorable environment to direct its differentiation towards neurons rather than glial cells. The myelin associated inhibitors have been demonstrated to promote NPC differentiation into glial lineage. Herein, to inhibit the downstream signaling activated by myelin associated inhibitors, cetuximab, an epidermal growth factor receptor (EGFR) neutralizing antibody, functionalized collagen scaffold has been developed as a vehicle for NPC implantation. It was found that collagen-cetuximab 1 μg scaffolds enhanced neuronal differentiation and inhibited astrocytic differentiation of NPCs exposed to myelin proteins significantly in vitro. To test the therapeutic effect in vivo, NPCs expressing green fluorescent protein (GFP)-embedded scaffolds have been implanted into the 4 mm-long hemisection lesion of rats. We found that the collagen-cetuximab 5 μg scaffolds induced neuronal differentiation and decreased astrocytic differentiation of NPCs, enhanced axon regeneration, and promoted functional recovery markedly. A well-functionalized scaffold was constructed to improve the recovery of SCI, which could promote the neuronal differentiation of neural progenitor cells in vivo.

Induction of Selective Cell Death of Oral Squamous Carcinoma Ce lls by Integrin α 2 Antibody and EGFR Antibody Yeon-Sik Choi, Gyoo-Cheon Kim 1 , Sik Yoon 2 , Dae-Seok Hwang, Cheol-Hun Kim 3 , Young-Chan Jeon 4 , June-Ho Byun 5 , Sang-Hun Shin, Uk-Kyu Kim Departments of Oral and Maxillofacial Surgery, 1 Oral Anatomy, 4 Prothodontics, School of Dentistry, Pusan National University, 2 Department of Anatomy, School of Medicine, Pusan National Unive rsity, 3 Department of Oral and Maxillofacial Surgery, College of Medicine, Dong-A University, 5 Department of Oral and Maxillofacial Surgery, School of Medicin e, Gyeongsang National UniversityPurpose: This study was to find efficacy of integrin alpha2 ( α 2 ) and epidermal growth factor receptor (EGFR) as tumor marker of oral squamous cell carcinoma (SCC) and clarify the se lective cell death effect of anti-integrin α 2 and anti-EGFR on SCC cells, additionally testify conjugated gold nanoparticle s (GNP) with air plasma for selective cell death of oral SCC.Methods: Expression of integrin α

Tyrosine phosphorylation is an essential posttranslational modification in intracellular signaling molecules. Since tyrosine phosphorylation occurs in less than 0.1 % of all phosphorylated amino acids in mammalian cells, it is difficult to detect the nascent phosphotyrosine at a high signal-to-noise ratio due to high intracellular backgrounds (i.e., unexpected crosstalks among endogenous signaling molecules). In order to address this issue, we reconstituted the mammalian signaling pathway involving an extracellular ligand and a receptor tyrosine kinase (RTK) in Saccharomyces cerevisiae, a lower eukaryote that lacks endogenous tyrosine kinases. In this chapter, we describe a method for high-throughput analysis of ligand-receptor interaction by combining the yeast cell-surface display technique with an automated single-cell analysis and isolation system. Yeast cells coexpressing the cell-wall-anchored form of the human epidermal growth factor (EGF) and the human EGF receptor (EGFR) fused with a signal peptide at the N terminus facilitated the interaction of EGF with EGFR in an autocrine manner, followed by EGFR oligomerization and subsequent autophosphorylation. Furthermore, yeast cells expressing cell-wall-anchored forms of a conformationally constrained random peptide library instead of EGF are treated with a fluorophore-labeled anti-phosphorylated EGFR antibody and then subjected to the automated single-cell analysis and isolation system. The yeast cells with the highest level of fluorescence were shown to display novel and efficient EGFR agonistic peptides. Thus, our yeast display technique serves as a quantitative measurement for RTK activation, which is applicable to high-throughput de novo screening of RTK agonistic peptides.

// Martin Pool 1 , Arjan Kol 1 , Marjolijn N. Lub-de Hooge 2, 3 , Christian A. Gerdes 4 , Steven de Jong 1 , Elisabeth G.E. de Vries 1 , Anton G.T. Terwisscha van Scheltinga 2 1 Department of Medical Oncology, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands 2 Department of Clinical Pharmacy and Pharmacology, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands 3 Department of Nuclear Medicine and Molecular Imaging, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands 4 Department of Roche Pharma Research and Early Development, Roche Innovation Center Zurich, Schlieren, Switzerland Correspondence to: Anton G.T. Terwisscha van Scheltinga, email: a.g.t.terwisscha.van.scheltinga@umcg.nl Keywords: imgatuzumab, EGFR, 89 Zr, immunoPET, shedding Received: January 06, 2016 Accepted: August 27, 2016 Published: September 02, 2016 ABSTRACT Preclinical positron emission tomography (PET) imaging revealed a mismatch between in vivo epidermal growth factor receptor (EGFR) expression and EGFR antibody tracer tumor uptake. Shed EGFR ectodomain (sEGFR), which is present in cancer patient sera, can potentially bind tracer and therefore influence tracer kinetics. To optimize EGFR-PET, we examined the influence of sEGFR levels on tracer kinetics and tumor uptake of EGFR monoclonal antibody 89 Zr-imgatuzumab in varying xenograft models. Human cancer cell lines A431 (EGFR overexpressing, epidermoid), A549 and H441 (both EGFR medium expressing, non-small cell lung cancer) were xenografted in mice. Xenografted mice received 10, 25 or 160 μg 89 Zr-imgatuzumab, co-injected with equal doses 111 In-IgG control. MicroPET scans were made 24, 72 and 144 h post injection, followed by biodistribution analysis. sEGFR levels in liver and plasma samples were determined by ELISA. 89 Zr-imgatuzumab uptake in A431 tumors was highest (29.8 ± 5.4 %ID/g) in the 160 μg dose group. Contrary, highest uptake in A549 and H441 tumors was found at the lowest (10 μg) 89 Zr-imgatuzumab dose. High 89 Zr-imgatuzumab liver accumulation was found in A431 xenografted mice, which decreased with antibody dose increments. 89 Zr-imgatuzumab liver uptake in A549 and H441 xenografted mice was low at all doses. sEGFR levels in liver and plasma of A431 bearing mice were up to 1000-fold higher than levels found in A549, H441 and non-tumor xenografted mice. 89 Zr-imgatuzumab effectively visualizes EGFR-expressing tumors. High sEGFR levels can redirect 89 Zr-imgatuzumab to the liver, in which case tumor visualization can be improved by increasing tracer antibody dose.

Using nanotechnology for optical manipulation of molecular processes in cells with high spatial and temporal precision promises new therapeutic options. Especially tumor therapy may profit as it requires a combination of both selectivity and an effective cell killing mechanism. Here we show a dual targeting approach for selective and efficient light-controlled killing of cells which are positive for epidermal growth factor receptor (EGFR) and Ki-67. Liposomes with the covalently linked EGFR antibody Erbitux enabled selective uptake of FITC-labeled Ki-67 antibody TuBB-9 in EGFR-positive cells pre-loaded with the photoactive dye BPD. After irradiation at 690 nm, BPD disrupted the endosomal membranes and delivered the antibodies to the nucleoli of the cells. The second irradiation at 490 nm activated the FITC-labeled TuBB-9, which caused inactivation of the Ki-67 protein and subsequent cell death via apoptosis. Efficient cell killing was possible at nanomolar concentrations of TuBB-9 due to the effective transport by immune liposomes and the high efficacy of the Ki-67 light-inactivation. Delivery of the liposomal constructs and cell destruction correlated well with the EGFR expression pattern of different cell lines (HeLa, OVCAR-5, MCF-7, and human fibroblasts), demonstrating an excellent selectivity.

The epidermal growth factor receptor (EGFR) and the insulin-like growth factor-1 receptor (IGF-1R) play critical roles in tumor growth, providing a strong rationale for the combined inhibition of IGF-1R and EGFR signaling in cancer therapy. We describe the design, affinity maturation, in vitro and in vivo characterization of the bispecific anti-IGF-1R/EGFR antibody XGFR*. XGFR* is based on the bispecific IgG antibody XGFR, which enabled heterodimerization of an IGF-1R binding scFab heavy chain with an EGFR-binding light and heavy chain by the “knobs-into-holes” technology. XGFR* is optimized for monovalent binding of human EGFR and IGF-1R with increased binding affinity for IGF-1R due to affinity maturation and highly improved protein stability to oxidative and thermal stress. It bears an afucosylated Fc-portion for optimal induction of antibody-dependent cell-mediated cytotoxicity (ADCC). Stable Chinese hamster ovary cell clones with production yields of 2–3 g/L were generated, allowing for large scale production of the bispecific antibody. XGFR* potently inhibits EGFR- and IGF-1R-dependent receptor phosphorylation, reduces tumor cell proliferation in cells with heterogeneous levels of IGF-1R and EGFR receptor expression and induces strong ADCC in vitro. A comparison of pancreatic and colorectal cancer lines demonstrated superior responsiveness to XGFR*-mediated signaling and tumor growth inhibition in pancreatic cancers that frequently show a high degree of IGF-1R/EGFR co-expression. XGFR* showed potent anti-tumoral efficacy in the orthotopic MiaPaCa-2 pancreatic xenograft model, resulting in nearly complete tumor growth inhibition with significant number of tumor remissions. In summary, the bispecific anti-IGF-1R/EGFR antibody XGFR* combines potent signaling and tumor growth inhibition with enhanced ADCC induction and represents a clinical development candidate for the treatment of pancreatic cancer.

Many tumors over-express collagen, which constitutes the physical scaffold of tumor microenvironment. Collagen has been considered to be a target for cancer therapy. The collagen-binding domain (CBD) is a short peptide, which could bind to collagen and achieve the sustained release of CBD-fused proteins in collagen scaffold. Here, a collagen-binding EGFR antibody fragment was designed and expressed for targeting the collagen-rich extracellular matrix in tumors. The antibody fragment (Fab) of cetuximab was fused with CBD (CBD-Fab) and expressed in Pichia pastoris. CBD-Fab maintained antigen binding and anti-tumor activity of cetuximab and obtained a collagen-binding ability in vitro. The results also showed CBD-Fab was mainly enriched in tumors and had longer retention time in tumors in A431 s.c. xenografts. Furthermore, CBD-Fab showed a similar therapeutic efficacy as cetuximab in A431 xenografts. Although CBD-Fab hasn’t showed better therapeutic effects than cetuximab, its smaller molecular and special target may be applicable as antibody–drug conjugates (ADC) or immunotoxins.

In this study we present novel bispecific antibodies that simultaneously target the insulin-like growth factor receptor type I (IGF-1R) and epidermal growth factor receptor (EGFR). For this purpose disulfide stabilized scFv domains of the EGFR/ADCC antibody GA201 were fused via serine-glycine connectors to the C-terminus of the heavy (XGFR2) or light chain (XGFR4), or the N-termini of the light (XGFR5) or heavy chain (XGFR3) of the IGF-1R antibody R1507 as parental IgG1 antibody. The resulting bispecific IGF-1R-EGFR antibodies XGFR2, XGFR3 and XGFR4 were successfully generated with yields and stability comparable to conventional IgG1 antibodies. They effectively inhibited IGF-1R and EGFR phosphorylation and 3D proliferation of H322M and H460M2 tumor cells, induced strong down-modulation of IGF-1R as well as enhanced EGFR down-modulation compared to the parental EGFR antibody GA201 and were ADCC competent. The bispecific XGFR derivatives showed a strong format dependent influence of N- or C-terminal heavy and light chain scFv attachment on ADCC activity and an increase in receptor downregulation over the parental combination in vitro. XGFR2 and XGFR4 were selected for in vivo evaluation and showed potent anti-tumoral efficacy comparable to the combination of monospecific IGF-1R and EGFR antibodies in subcutaneous BxPC3 and H322M xenograft models. In summary, we have managed to overcome issues of stability and productivity of bispecific antibodies, discovered important antibody fusion protein design related differences on ADCC activity and receptor downmodulation and show that IGF-1R-EGFR antibodies represent an attractive therapeutic strategy to simultaneously target two key components de-regulated in multiple cancer types, with the ultimate goal to avoid the formation of resistance to therapy.

Squamous cell carcinoma of head and neck (SCCHN) is the most common neoplasm of the upper aerodigestive tract. In this paper, we attempt to summarize the role and applications of the epidermal growth factor receptor (EGFR) inhibitors monoclonal antibodies (moAbs) and tyrosine kinase inhibitors (TKIs) locally advanced as well as metastatic SCCHN. Targeted therapy in SCCHN is now incorporated in the first-line regimes for advanced disease. Novel targeted agents, including the EGFR antibody, cetuximab, have been approved for use as single agents or in combination with radiation therapy or chemotherapy in treatment of recurrent metastatic or locally advanced SCCHN. Refractory mechanisms that bypass the pathway of EGFR inhibitors activity are identified explaining resistance to targeted therapy. Strategies of cotargeting EGFR and other pathways are under investigation. Examples of targeted therapy being used include mammalian target of rapamycin (mtor) inhibitors, antivascular endothelial growth factor (VEGF) moAb, and other inhibitors. We will be focusing our paper on the preclinical and clinical aspects of EGFR inhibition in SCCHN and touch upon other targeted therapies in application.

Background: Circulating tumor cells (CTCs) are not only a surrogate of distant metastasis, but also useful for material of liquid biopsy. "Universal" CTC-chip is a polymeric microfluidic device that can capture CTCs according to their surface marker from whole blood. We have examined the capture efficiency of lung cancer and mesothelioma cells in the blood with CTC-chip coated with antibodies against various surface marker. Methods: Lung cancer cell lines (PC-9) or mesothelioma cell lines (ACC-MESO-4, MSTO-211H) were used in this study. Cells were CFSE-labeled and added in the blood of healthy donor. A polymeric CTC-chip was coated with two steps: base- and capture-antibody. As capture antibody, we used anti-EpCAM, podoplanin, and EGFR antibody. After flowing these samples, capture efficiency was calculated (number of captured cells/number of flowed cells). Results: Capture efficiency of PC-9, MESO-4, and METO-211H was 100.0%, 9.5% and 9.1% (EpCAM-chip; anti-EpCAM antibody clone HEA125); 5.8%, 85.9% and 9.0 % (podoplanin-chip; anti-podoplanin antibody clone E1); 38.6, 112.5%, 11.7% (podoplanin-chip; anti-podoplanin antibody clone NZ-1); 30.2%, 21.8%, 38.5% (EGFR-chip; anti-EGFR antibody clone 528), respectively. Conclusion: Using "Universal CTC-chip" with various antibodies, it was possible to capture nonepithelial tumor cells such as mesothelioma cells. Citation Format: Kazue Yoneda, Taiji Kuwata, Fumihiro Tanaka. Capture of tumor cells in blood with "Universal CTC-chip" [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1599.

(1) To determine the safety of the epidermal growth factor receptor (EGFR) antibody cetuximab with concurrent gemcitabine and abdominal radiation in the treatment of patients with locally advanced adenocarcinoma of the pancreas. (2) To evaluate the feasibility of pancreatic cancer cell epithelial-mesenchymal transition (EMT) molecular profiling as a potential predictor of response to anti-EGFR treatment.Patients with non-metastatic, locally advanced pancreatic cancer were treated in this dose escalation study with gemcitabine (0-300 mg/m(2)/week) given concurrently with cetuximab (400 mg/m(2) loading dose, 250 mg/m(2) weekly maintenance dose) and abdominal irradiation (50.4 Gy). Expression of E-cadherin and vimentin was assessed by immunohistochemistry in diagnostic endoscopic ultrasound fine-needle aspiration (EUS-FNA) specimens.Sixteen patients were enrolled in 4 treatment cohorts with escalating doses of gemcitabine. Incidence of grade 1-2 adverse events was 96%, and incidence of 3-4 adverse events was 9%. There were no treatment-related mortalities. Two patients who exhibited favorable treatment response underwent surgical exploration and were intraoperatively confirmed to have unresectable tumors. Median overall survival was 10.5 months. Pancreatic cancer cell expression of E-cadherin and vimentin was successfully determined in EUS-FNA specimens from 4 patients.Cetuximab can be safely administered with abdominal radiation and concurrent gemcitabine (up to 300 mg/m(2)/week) in patients with locally advanced adenocarcinoma of the pancreas. This combined therapy modality exhibited limited activity. Diagnostic EUS-FNA specimens could be analyzed for molecular markers of EMT in a minority of patients with pancreatic cancer.

Background: The validation of KRAS mutations as a negative marker of response to anti-epidermal growth factor receptor (EGFR) antibodies has meant a seminal advance towards treatment individualisation of colorectal cancer (CRC) patients. However, as a KRAS wild-type status does not guarantee a response to anti-EGFR antibodies, a current challenge is the identification of other biomarkers of response. On the basis of pre-clinical evidence, we hypothesised that mitogen-activated protein kinase phosphatase-1 (MKP-1), a phosphatase that inactivates MAPKs, could be a mediator of resistance to anti-EGFR antibodies. Methods: Tumour specimens from 48 metastatic CRC patients treated with cetuximab-based chemotherapy were evaluated for KRAS and BRAF mutational status and MKP-1 expression as assessed by immunohistochemistry. Results: As expected, clinical benefit was confined to wild-type KRAS and BRAF patients. Mitogen-activated protein kinase phosphatase-1 was overexpressed in 16 patients (33%) and was not associated with patient baseline clinicopathological characteristics and KRAS mutational status. All patients with BRAF mutations (n=3) had MKP-1 overexpression. Among KRAS wild-type patients, MKP-1 overexpressors had a 7% response rate (RR), whereas patients not overexpressing MKP-1 had a 44% RR (P=0.03). Moreover, median time to progression was significantly longer in MKP-1 non-overexpressing patients (32 vs 13 weeks, P=0.009). Conclusion: These results support the concept of MKP-1 as a promising negative marker of response to cetuximab-based treatment in CRC patients with wild-type KRAS.

Purpose: Epidermal growth factor receptor (EGFR) antibody therapy is established in patients with wild-type KRAS colorectal carcinoma; however, up to 50% of these patients do not respond to this therapy. To identify the possible causes of this therapy failure, we searched for mutations in different EGFR-dependent signaling proteins and analyzed their distribution patterns in primary tumors and corresponding metastases. Experimental Design: Tumor tissues, macrodissected from tumor centers, invasion fronts (n = 100), lymph nodes (n = 55), and distant metastases (n = 20), respectively, were subjected to DNA extraction and mutation analysis of KRAS, BRAF, and PIK3CA. Results: Activating mutations were detected in 41% (KRAS), 7% (BRAF), and 21% (PIK3CA) of the primary tumors. By comparing tumor centers and invasion fronts, the intratumoral heterogeneity of KRAS, BRAF, and PIK3CA mutations was observed in 8%, 1%, and 5% of primary tumors, respectively. Heterogeneity between primary tumors and lymph node metastases was found in 31% (KRAS), 4% (BRAF), and 13% (PIK3CA) of the cases. Heterogeneity between primary tumors and distant metastases was present in two patients (10%) for KRAS and one patient for PIK3CA (5%), but not for BRAF. Discordant results between primary tumors and metastases could markedly be reduced by testing the additional tumor samples. Conclusions: Failure of EGFR antibody therapy in patients with wild-type KRAS colorectal cancer may result from activating BRAF or PIK3CA mutations and false-negative sequencing results caused by intratumoral heterogeneity. Due to the particularly high rates of heterogeneity between primary tumors and lymph node metastases, the latter are least suitable for diagnostic mutation analysis. Clin Cancer Res; 16(3); 790–9

Selective drug delivery is an important approach with great potential for overcoming problems associated with the systemic toxicity and poor bioavailability of antineoplastic drugs. Nanomedicine plays a pivotal role by delivering drugs in a targeted manner to the malignant tumor cells thereby reducing the systemic toxicity of the anticancer drugs. The objective of this study was to prepare and characterize rapamycin loaded polymeric poly(lactide-co-glycolide) (PLGA) nanoparticles (NP) that were surface conjugated with antibodies to epidermal growth factor receptor (EGFR), highly expressed on breast cancer cells, using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) mediated cross linking agents. To potentiate the anticancer efficiency of the formulations, in vitro cytotoxicity of native rapamycin, rapamycin loaded nanoparticles and EGFR antibody conjugated rapamycin loaded nanoparticles (EGFR-Rapa-NPs) were evaluated on malignant MCF 7 breast cancer cell lines. IC(50) doses as determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium (MTT) assay showed the superior antiproliferative activity of EGFR-Rapa-NPs over unconjugated nanoparticles and native rapamycin due to higher cellular uptake on malignant breast cancer cells. Cell cycle arrest and cellular apoptosis induced by the above formulations were confirmed by flow cytometry. Molecular basis of apoptosis studied by western blotting revealed the involvement of a cytoplasmic protein in activating the programmed cell death pathway. Thus it was concluded that EGFR-Rapa-NPs provide an efficient and targeted delivery of anticancer drugs, presenting a promising active targeting carrier for tumor selective therapeutic treatment in near future.

Immunohistochemistry is widely used to assess epidermal growth factor receptor (EGFR) expression on colorectal carcinomas to select patients for treatment with cetuximab, an anti-EGFR antibody. The data comparing different commercial EGFR antibodies is limited, and no cost comparisons have been made. We analyzed 65 advanced colorectal cancers from 36 patients using the EGFR pharmDx kit (DakoCytomation) and Clone 31G7 (Zymed Laboratories, Inc). EGFR expression was seen in 35 (53%) tumors (21 primary, 14 metastatic) with the Dako pharmDx kit. The Zymed antibody showed positive results in 41 (63%) tumors (25 primary, 16 metastatic). The cost per test was $40.00 with the pharmDx kit and $3.52 with the Zymed antibody. The Zymed antibody detects 10% more cases of colorectal cancer as EGFR positive, and is 10 times cheaper than the Dako pharmDx kit. There is little justification for the use of expensive kits for testing EGFR expression, when other available antibodies without the kit can give comparable or superior results.

Bionanocapsules (BNCs) are hollow nanoparticles that are composed of L protein (the hepatitis B virus surface antigen) and show specific affinity for human hepatocytes. The pre-S1 peptide displayed on the surface of BNCs is the specific ligand for binding to the receptor on human hepatocytes. Therefore, BNCs are not delivered to other tissues, such as the brain. The aim of the present study was to develop a novel drug delivery system (DDS) targeting brain tumors using BNCs that selectively targeted brain tumors. Epidermal growth factor receptor (EGFR), especially a constitutively active genomic sequence deletion variant of EGFR (EGFRvIII), is overexpressed in human glioblastoma. In the present study, we replaced the pre-S1 peptide with the antibody affinity motif of protein A and made hybrid BNCs conjugated with anti-human EGFR antibody recognizing EGFRvIII. The hybrid BNCs were efficiently delivered to glioma cells but not normal glial cells. Moreover, we confirmed the specific delivery of the hybrid BNCs to brain tumors in an in vivo brain tumor model. These results suggest that this new approach using BNCs is a promising system for brain tumor-targeted drug delivery.

Effectiveness of epidermal growth factor receptor(EGFR)-targeted, long circulating and temperature-sensitive liposomes(TSLs) is described using sterically stabilized gemcitabine-loaded liposomes in vitro. Development of long-circulating formulation of TSLs with the EGFR antibody attached was designed to expect an increase in binding and drug delivery efficiency to the target cells such as non-small cell lung cancer cells(A549) and human pancreatic carcinoma cells(PANC- 1). New TSLs were prepared using DPPC:DMPC:DSPC(4:1:1 molar ratio) by the REV method. Differential scanning calorimetry of TSLs showed the phase-transition around 402. Release of a self-quenching fluorescent probe, calcein, from TSLs was studied for evaluation of temperaturesensitivity. Anti-proliferation effect of gemcitabine-loaded TSLs and antibody-conjugated TSLs in A549 and PANC-1 were higher than free drug. We conclude that sterically stabilized immunoliposomes exhibited good stability, ability to recognize target cells, and higher potency. Further studies, including in vivo animal study, are under investigation.

Purpose: To optimally integrate epidermal growth factor receptor (EGFR) inhibitors into the clinical treatment of head and neck cancer, two important questions must be answered: (a) does EGFR inhibition add to the effects of radiochemotherapy, and (b) if so, which method of inhibiting EGFR is superior (an EGFR antibody versus a small molecule tyrosine kinase inhibitor)? We designed an in vivo study to address these questions. Experimental Design: Nude mice with UMSCC-1 head and neck cancer xenografts received either single, double, or triple agent therapy with an EGFR inhibitor (either cetuximab or gefitinib), gemcitabine, and/or radiation for 3 weeks. Tumor volumes and animal weights were measured for up to 15 weeks. Immunoblotting and immunofluorescent staining were done on tumors treated with either cetuximab or gefitinib alone. Results: The addition of an EGFR inhibitor significantly delayed the tumor volume doubling time, from a median of 40 days with radiochemotherapy (gemcitabine and radiation) alone, to 106 days with cetuximab and 66 days with gefitinib (both P < 0.005). Cetuximab resulted in significantly less weight loss than gefitinib. Immunoblot analysis and immunofluorescent staining of tumors show that although levels of phosphorylated AKT and extracellular signal–regulated kinase were decreased similarly in response to cetuximab or gefitinib, cetuximab caused prolonged suppression of pEGFR, pSTAT3, and BclXL compared with gefitinib. Conclusions: EGFR inhibition, particularly with cetuximab, improves the effectiveness of radiochemotherapy in this model of head and neck cancer. The correlation of response with prolonged suppression of EGFR, STAT3, and BclXL offers the possibility that these may be candidate biomarkers for response.

We hypothesized that aberrations activating epidermal growth factor receptor (EGFR) via dimerization would be more sensitive to anti-dimerization agents (e.g., cetuximab). EGFR exon 19 abnormalities (L747_A750del; deletes amino acids LREA) respond to reversible EGFR kinase inhibitors (TKIs). Exon 20 in-frame insertions and/or duplications (codons 767 to 774) and T790M mutations are clinically resistant to reversible/some irreversible TKIs. Their impact on protein function/therapeutic actionability are not fully elucidated.In our study, the index patient with non-small cell lung cancer (NSCLC) harbored EGFR D770_P772del_insKG (exon 20). A twenty patient trial (NSCLC cohort) (cetuximab-based regimen) included two participants with EGFR TKI-resistant mutations ((i) exon 20 D770>GY; and (ii) exon 19 LREA plus exon 20 T790M mutations). Structural modeling predicted that EGFR exon 20 anomalies (D770_P772del_insKG and D770>GY), but not T790M mutations, stabilize the active dimer configuration by increasing the interaction between the kinase domains, hence sensitizing to an agent preventing dimerization. Consistent with predictions, the two patients harboring D770_P772del_insKG and D770>GY, respectively, responded to an EGFR antibody (cetuximab)-based regimen; the T790M-bearing patient showed no response to cetuximab combined with erlotinib. In silico modeling merits investigation of its ability to optimize therapeutic selection based on structural/functional implications of different aberrations within the same gene.