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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.

Background MAP2K1 mutations, otherwise known as MEK mutations, are rare oncogenic alterations that have been implicated in MAPK pathway activation. The impact of MAP2K1 mutations in colorectal cancer on EGFR antibody response has not been characterized. Patients and Methods Antitumor activity was assessed in mouse xenograft models with SW48 cell lines harboring MAP2K1 mutation, and protein expression of the RAS signaling pathway was studied by Western blot analysis. We retrospectively identified patients with MAP2K1-mutated metastatic colorectal cancer patients treated at City of Hope Comprehensive Cancer Center between 2015 and 2020 using next-generation sequencing. Patients’ tumor characteristics, treatment response, and outcome are described. Additional patients with the MAP2K1 mutation were identified from The Cancer Genome Atlas and Memorial Sloan Kettering Cancer Center oncogenomic databases. Results Antitumor activity in mouse xenograft models demonstrated efficacy with combination therapy with EGFR and MEK inhibition with either BRAF or ERK inhibitors. Five patients treated at City of Hope between 2015 and 2020 harbored a MAP2K1 mutation at a frequency of 1%. APC and TP53 were common coalterations. All disease was RAS and BRAF wild type, except 1 case that harbored a concurrent KRAS mutation. Four RAS/BRAF wild-type MAP2K1-mutated patients was treated with anti-EGFR, anti-EGFR + MEK and BRAF inhibitors, and anti-EGFR + ERK inhibitors. All 4 patients experienced disease progression. Conclusion MAP2K1 mutation in colorectal cancer is associated with poor response to EGFR inhibition. EGFR inhibition with or without MEK, BRAF, or ERK inhibitors did not result in any clinical benefit in our limited experience.

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.

Urothelial carcinomas represent 90% of all primary bladder cancers. Muscle invasion is a critical prognosticdeterminant in urothelial carcinoma. The overexpression of EMMPRIN and EGFR was found in urothelialcarcinoma. The association between the two markers has not been reported in urothelial carcinoma,therefore we aimed to analyze the expression of EMMPRIN and EGFR and investigate their association withurothelial carcinoma invasiveness. Paraffin-embedded tissues were obtained from 54 urothelial carcinomapatients which then underwent immunohistochemistry staining for EMMPRIN and EGFR antibody. Thecomparison of EMMPRIN and EGFR expression was tested using the Mann Whitney U test. The correlationwas analyzed using the Spearman test. Results showed a significant difference of EMMPRIN expressionbetween non-muscle-invasive and muscle-invasive bladder cancer (p = 0.000), and EMMPRIN expressionwas significantly correlated with the muscle invasion (rs = 0.481, p = 0.000). A significant difference ofEGFR expression between the non-muscle-invasive and muscle-invasive bladder cancer was also found(p = 0.020), and EGFR expression was significantly correlated with the muscle invasion (rs = 0.319, p =0.019). The expression of EMMPRIN was positively correlated with EGFR in urothelial carcinoma (rs =0.322, p = 0.018). The expression of EMMPRIN and EGFR are two potential biomarkers for urothelialcarcinoma invasiveness which may be helpful to differentiate between muscle-invasive and non-muscleinvasive bladder cancer.

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.

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.

The benefit of EGFR–TKI in non–small cell lung cancer has been demonstrated in mutant EGFR tumors as first-line treatment but the benefit in wild-type EGFR tumors is marginal as well as restricted to maintenance therapy in pretreated patients. This work aimed at questioning the effects of cisplatin initial treatment on the EGFR pathway in non–small cell lung cancer and the functional consequences in vitro and in in vivo animal models of patient-derived xenografts (PDX). We establish here that cisplatin pretreatment specifically sensitizes wild-type EGFR-expressing cells to erlotinib, contrary to what happens in mutant EGFR cells and with a blocking EGFR antibody, both in vitro and in vivo. The sensitization entails the activation of the kinase Src upstream of EGFR, thereafter transactivating EGFR through a ligand-independent activation. We propose a combination of markers that enable to discriminate between the tumors sensitized to erlotinib or not in PDX models, which should be worth testing in patients. These markers might be useful for the selection of patients who would benefit from erlotinib as a maintenance therapy. Mol Cancer Ther; 16(8); 1634–44. ©2017 AACR.

ABT-414 is an antibody-drug conjugate (ADC) being developed for the treatment of tumors harboring amplification of the epidermal growth factor receptor (EGFR). This study evaluated the potential of ABT-414 to prolong the QT interval as part of the initial phase 1 study (NCT01741727). Data from patients who received ABT-414 monotherapy at a dose of 1–4 mg/kg once every 3 weeks or 1 or 1.5 mg/kg weekly for 2 out of every 3 weeks (alternate schedule) by intravenous infusion were included in the analysis of triplicate 12-lead ECGs obtained before dosing and through 168 h after dosing. Data from time-matched pharmacokinetic samples and QT interval assessments were evaluated using linear mixed-effects modeling to determine the effects of ABT-414, total ABT-806, and cysteine-maleimidocaproyl monomethyl auristatin F (Cys-mcMMAF) on the QT interval corrected using Fridericia’s formula (QTcF). Fifty-one patients were included in the analyses. ABT-414 had no clinically meaningful effect on QTcF. Using pooled data from doses ≥2 mg/kg, the estimated mean ∆QTcF reached a maximum of 4.30 ms after dosing, with a one-sided 95% upper confidence bound of 8.32 ms. The exposure–response analysis showed no statistically significant relationship between ΔQTcF and the concentration of any analyte (P > 0.05). No patient had a QTcF value >480 ms or a ∆QTcF value >30 ms. ABT-414 had no clinically meaningful effect on the QTcF interval at doses being evaluated for treatment of patients with solid tumors.

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.

Background: Topical chemotherapy of skin cancers is a promising strategy for reduction of side effects and for improvement of patient compliance. The combination of the chemotherapeutic 5-fluouracil (5-FU) and the anti- EGFR antibody cetuximab is a strategy to inhibit tumor growth. Their skin penetration, however, is hampered by their high hydrophilicity, which could be improved by encapsulation in delivery systems. Furthermore, it is a challenge to encapsulate hydrophilic drugs. The conjugation of an antibody to a liposome, maintaining its activity, is also a difficult task. Objective: Thus, we aimed to develop 5-FU liposomes and cetuximab-conjugated liposomes (immunoliposomes) of 5- FU to improve drug cytotoxicity against skin cancer cells. Method: We characterized them by particle size, zeta potential, loading efficiency and antibody integrity. To optimize the loading efficiency of 5-FU, a series of liposomes were prepared, using different methods and drug-to-lipid ratios. Results: Liposomes containing DSPC and Chol at drug-to-lipid ratio 0.1 prepared by the thin lipid hydration method resulted in the best 5-FU encapsulation and were chosen to conjugate with cetuximab. Cetuximab was directly coupled to preformed liposomes using DSPE-mPEG2000-Mal as an anchor. In A431 skin carcinoma cells, at 72 h, 5-FU liposomes showed a 5-fold lower IC50 than 5-FU solution. Additionally, 5-FU immunoliposomes resulted in a 4-fold lower cetuximab IC50 than cetuximab solution, demonstrating synergism with a combination index lower than 1 and potential to improve 5-FU and cetuximab cytotoxicity. Conclusion: Liposomes and immunoliposomes containing 5-FU were developed and cetuximab remained active as demonstrated in cell culture studies.

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.

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.

PURPOSE: Although EGFR exon20 insertion (ex20ins) mutations account for 4~12 % of EGFR mutant NSCLC patients, there is no effective and selectable anticancer drugs targeting ex20ins mutations so far due to various variant mutations in ex20ins mutations. Moreover, most EGFR ex20ins mutants show primary resistance to EGFR TKIs. JNJ-61186372 (JNJ-372), a bispecific antibody that targets the EGFR and cMet receptors, is currently being explored in a first-in-human study in patients with NSCLC. To better understand the mechanism of JNJ-372 activity in this patient population, we conducted preclinical studies exploring the activity of JNJ-372 in different EGFR ex20ins models. METHODS: To elucidate whether JNJ-372 has antitumor effect in EGFR ex20ins mutants via EGFR and c-MET inhibition, cell viability, western blot, cell cycle, colony formation assay, FACS analysis were performed in JNJ-372 treated BaF3 cells, PDCs, PDOs, and PDX expressing EGFR ex20ins mutation. For mouse tumor models, JNJ-372 was administered i.p. twice a week at 10 mg/kg or 30 mg/kg. Antibody dependent cellular cytotoxicity (ADCC) assay was assessed to figure out whether JNJ-372 had ADCC effects. Referenced patients with ex20ins disease were administered 1050 mg JNJ-372 i.v. weekly for the first 4-week cycle, then biweekly for each subsequent cycle RESULTS: JNJ-372 inhibited the growth of BaF3 cells, PDCs, and a PDO harboring a range of ex20ins, which were resistant to osimertinib and gefitinib. Mechanistic assays revealed the reduction of EGFR and cMet receptor levels and decreases in phospho-EGFR and c-Met, as well as inhibition of their downstream signaling pathways. Cleaved caspase-3 and BIMEL were upregulated at anti-proliferative doses, suggesting caspase-mediated cell death. JNJ-372 demonstrated corresponding antitumor activity in PDC and PDX models harboring different ex20ins; inhibition of signaling and engagement of the apoptotic pathway was confirmed in tumors of JNJ-372-treated mice. In PDCs with EGFR ex20ins mutation, we verified that JNJ-372 had a significant ADCC effect compared to EGFR antibody drug cetuximab. In the first-in-human trial, CT scans from two patients treated with JNJ-372 revealed reductions in tumor burden. A 58-year patient harboring H773delinsNPY showed -63% tumor reduction with progression-free survival of > 20 months and a 48-year patient harboring S768_D770dup showed -38.9% tumor reduction. CONCLUSION: JNJ-372 drives antitumor activity in preclinical models of EGFR ex20ins, which have no therapeutic options in clinic, by decreasing EGFR and cMet receptor levels, inhibiting downstream signaling cascades, activating apoptotic signaling as well as ADCC. These results provide a promising therapeutic option to patients with EGFR ex20ins mutations and an understanding of the activity of JNJ-372 being observed in the first-in-human study. Citation Format: Jiyeon Yun, Han Na Kang, Soo-Hwan Lee, Seo-Yoon Jeong, Chae Won Park, Jae-Hwan Kim, Kyoung-Ho Pyo, Ji Min Lee, Seok-Young Kim, Min Hee Hong, Hye Ryun Kim, Meena Thayu, Joshua Curtin, Roland Knoblauch, Matthew Lorenzi, Byoung Chul Cho. JNJ-61186372, an EGFR-cMet bispecific antibody, in EGFR Exon 20 insertion-driven advanced non-small cell lung cancer (NSCLC) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5199.

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.

Anti-epidermal growth factor receptor (EGFR) antibody therapy is used in EGFR expressing cancers including lung, colon, head and neck, and bladder cancers, however results have been modest. Near infrared photoimmunotherapy (NIR-PIT) is a highly selective tumor treatment that employs an antibody-photo-absorber conjugate which is activated by NIR light. NIR-PIT is in clinical trials in patients with recurrent head and neck cancers using cetuximab-IR700 as the conjugate. However, its use has otherwise been restricted to mouse models. This is an effort to explore larger animal models with NIR-PIT. We describe the use of a recombinant canine anti-EGFR monoclonal antibody (mAb), can225IgG, conjugated to the photo-absorber, IR700DX, in three EGFR expressing canine transitional cell carcinoma (TCC) cell lines as a prelude to possible canine clinical studies. Can225-IR700 conjugate showed specific binding and cell-specific killing after NIR-PIT on EGFR expressing cells in vitro. In the in vivo study, can225-IR700 conjugate demonstrated accumulation of the fluorescent conjugate with high tumor-to-background ratio. Tumor-bearing mice were separated into 4 groups: (1) no treatment; (2) 100 µg of can225-IR700 i.v. only; (3) NIR light exposure only; (4) 100 µg of can225-IR700 i.v., NIR light exposure. Tumor growth was significantly inhibited by NIR-PIT treatment compared with the other groups (p < 0.001), and significantly prolonged survival was achieved (p < 0.001 vs. other groups) in the treatment groups. In conclusion, NIR-PIT with can225-IR700 is a promising treatment for canine EGFR-expressing cancers, including invasive transitional cell carcinoma in pet dogs, that could provide a pathway to translation to humans.

A nanodiamonds-based anti-cancer drug targeted delivery system, Epidermal Growth Factor (EGF)–nanodiamonds (NDs)–cisplatin (ENC) bioconjugate was developed. Due to the effect of the specific biorecognition process of EGF (a kind of targeting molecule), human liver hepatocellular carcinoma (HepG2) cells could be selectively killed by the ENC system, even in a lower concentration of the cisplatin (a kind of anti-cancer drug) comparing to the traditional drug therapy. The cytotoxicity of ENC could be blocked by EGFR antibody in cancer cells, revealing the specificity of its targeting ability. Because the morphological change of HepG2 cells treated by ENC was observed, from epithelial to mesenchymal-like shape, the related mechanism was evaluated. The results showed that E-cadherin expression declined after ENC treatment, while vimentin expression did not change. The decrease of E-cadherin expression means the loss of cell–cell adhesion. Loose cells provided more specific surface area for drug absorption. Based on these advantages, ENC delivery system would be a promising targeting drug delivery system. Moreover, we located the spatial distribution of ENC system in cells by using the NDs existing in ENC as Raman probe through 3-dimensional (3D) confocal Raman imaging, i.e., we were able to visualize the process of uptake of ENC into living cancer cells, which is helpful to observe the specific biorecognition process between EGF and its receptor (EGFR) in a physiology condition and the cellular uptake of ENC drug delivery system. The present study demonstrates that drug-loaded NDs have a potential as novel intravascular probes for both diagnostic (e.g., imaging) and therapeutic purposes (e.g., targeted drug delivery).

Background: The human epidermal growth factor receptor (EGFR) is an important target for cancer treatment. Currently, only the EGFR antibodies cetuximab and panitumumab are approved for the treatment of patients with colorectal cancer. However, a major clinical challenge is a short-term response owing to development of acquired resistance during the course of the treatment. Methods: In this study, we investigated the molecular mechanisms underlying development of acquired resistance in DiFi colorectal cancer cells to the anti-EGFR mAb ICR62 (termed DiFi62) and to the small molecule tyrosine kinase inhibitor (TKI) gefitinib (termed DiFiG) using a range of techniques. Results: Compared with the findings from parental DiFi and DiFiG cells, development of acquired resistance to anti-EGFR mAb ICR62 in DiFi62 cells was accompanied by an increase in cell surface EGFR and increased phosphorylation of HER-2 and HER-3. Interestingly, DiFi62 cells also acquired resistance to treatment with anti-EGFR mAbs cetuximab and ICR61, which bind to other distinct epitopes on the extracellular domain of EGFR, but these cells remained equally sensitive as the parental cells to treatment with pan-HER inhibitors such as afatinib. Conclusions: Our results provide a novel mechanistic insight into the development of acquired resistance to EGFR antibody-based therapy in colorectal cancer cells and justify further investigations on the therapeutic benefits of pan-HER family inhibitors in the treatment of colorectal cancer patients once acquired resistance to EGFR antibody-based therapy is developed.

K-ras mutation status has remained elusive in the metastatic liver tumors of colorectal cancer (CRC) in contrast to the primary CRC tumors. In this study, K-ras mutational status of the primary and corresponding liver metastatic tumors was investigated in the 43 CRC patients. Codons 12 and 13 of K-ras were directly sequenced, and a K-ras mutation was evident in 17 cases (39.5%). In 6 cases, the K-ras mutation was evident only in the liver metastasis, but not in the primary CRC, where the mutation was found in codon 13. This discrepancy between primary and metastatic lesions with regard to codon 13 of the K-ras gene may explain the clinical discrepancy of EGFR antibody therapy. In conclusion, the current data may lead to the development of personalized medicine for recurrent CRC, although further validation study is still required.

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.

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.

The EGFR antibody cetuximab is used to treat numerous cancers, but intrinsic and acquired resistance to this agent is a common clinical outcome. In this study, we show that overexpression of the oncogenic receptor tyrosine kinase AXL is sufficient to mediate acquired resistance to cetuximab in models of non–small cell lung cancer (NSCLC) and head and neck squamous cell carcinoma (HNSCC), where AXL was overexpressed, activated, and tightly associated with EGFR expression in cells resistant to cetuximab (CtxR cells). Using RNAi methods and novel AXL-targeting agents, we found that AXL activation stimulated cell proliferation, EGFR activation, and MAPK signaling in CtxR cells. Notably, EGFR directly regulated the expression of AXL mRNA through MAPK signaling and the transcription factor c-Jun in CtxR cells, creating a positive feedback loop that maintained EGFR activation by AXL. Cetuximab-sensitive parental cells were rendered resistant to cetuximab by stable overexpression of AXL or stimulation with EGFR ligands, the latter of which increased AXL activity and association with the EGFR. In tumor xenograft models, the development of resistance following prolonged treatment with cetuximab was associated with AXL hyperactivation and EGFR association. Furthermore, in an examination of patient-derived xenografts established from surgically resected HNSCCs, AXL was overexpressed and activated in tumors that displayed intrinsic resistance to cetuximab. Collectively, our results identify AXL as a key mediator of cetuximab resistance, providing a rationale for clinical evaluation of AXL-targeting drugs to treat cetuximab-resistant cancers. Cancer Res; 74(18); 5152–64. ©2014 AACR.

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.

Colorectal cancer (CRC) is the third most common cancer and the second most common cause of cancer death globally. Significant improvements in survival have been made in patients with metastasis by new therapies. For example, Cetuximab and Panitumumab are monoclonal antibodies that inhibit the epidermal growth receptor (EGFR). KRAS mutations in codon 12 and 13 are the recognized biomarkers that are analyzed in clinics before the administration of anti-EGFR therapy. Genetic analyses have revealed that mutations in KRAS predict a lack of response to Panitumumab and Cetuximab in patients with metastatic CRC (mCRC). Notably, it is estimated that 35–45% of CRC patients harbor KRAS mutations. Therefore, KRAS mutation testing should be performed in all individuals with the advanced CRC in order to identify the patients who will not respond to the monoclonal EGFR antibody inhibitors. New techniques for KRAS testing have arisen rapidly, and each technique has advantages and disadvantages. Herein, we review the latest published literature specific to KRAS mutation testing techniques. Since reliability and feasibility are important issues in clinical analyses. Therefore, this review also summarizes the effectiveness and limitations of numerous KRAS mutation testing techniques.

Mutation status of Kirsten rat sarcoma viral oncogene homolog (KRAS) may serve as a negative predictive marker of Cetuximab in treating colorectal cancer. The present study was to determine the role of KRAS status in EGFR antibody treatment for gastric cancer. KRAS status was clarified in SGC-7901 (wild type) and YCC-2 (G→A mutation) gastric cancer cell lines. Anti-proliferative and pro-apoptotic effects of Cetuximab were tested both in vitro and in vivo, the expression of phosphorylated (p) ERK, a downstream protein in EGFR-RAS-MEK pathway, was also analyzed. No significant in vitro anti-proliferative or pro-apoptotic effects of Cetuximab were observed in both cell lines. The growth of either SGC-7901 or YCC-2 gastric cancer xenograft was significantly inhibited by Cetuximab. Apoptosis was induced in SGC-7901 but not in YCC-2 xenografts after Cetuximab treatment. The expression of pERK was up-regulated in YCC-2 but not SGC-7901 xenografts after Cetuximab treatment. In conclusion, KRAS (G→A) mutation does not affect in vivo anti-cancer efficacy of Cetuximab.

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.

Purpose: Epidermal growth factor receptor (EGFR) represents an important target for treatment of locally advanced breast cancer (LABC), since about 30% of patients with LABC have triple negative breast tumor, and these tumors are often EGFR-positive. Our aim was to synthesize and characterize a novel form of “gold nanobomb” (GNB) for localized targeted radioimmunotherapy of LABC. GNB uses gold nanoparticles (AuNPs) as a carrier to conjugate both EGFR targeting monoclonal antibody (panitumumab) and the β-particle emitting radionuclide 177Lu. Ultimately, these GNB would be administered locally in LABC for tumor treatment while minimizing normal tissue exposure. Methods: The basic idea for construction of this new-targeted agent is to use different length of poly(ethylene glycol) (PEG) spacers to conjugate antibody and DOTA metal chelators for 177Lu. First, OPSS-PEG-NH2 was used to synthesize OPSS-PEG-DOTA for complexing 177Lu and OPSS-PEG-SVA was used to link antibody. Then these PEG spacers were linked to 30 nm AuNPs to form “GNB”. OPSS-PEG-antibody was analyzed by SDS-PAGE and trinitrobenzenesulfonic acid (TNBSA) assay to determine the degree of amine substitution after PEGylation. A radiometric assay was used to determine the number of PEGylated antibody and DOTA-PEG-OPSS per AuNPs. Specificity of GNB for EGFR and internalization into MBA-MD-468 (1×106 EGFR/cell), MBA-MD-231 (1×105 EGFR/cell) and MCF-7 (1×104 EGFR/cell) was confirmed by confocal and darkfield fluorescence microscopy. Binding assay and cell fractionation study were used to determine the binding affinity to EGFR and the total amount of GNB internalized. Results: Reacting panitumumab with increasing ratios of PEG resulted in an increase in molecular weight from 147 to 250 kDa. This result was in accordance with TNBSA assay where increasing ratio of PEG was associated with increasing lysine substitution (1.4 to 50.9 PEGylated lysine per antibody). PEGylation of 1.4 ± 1.7 lysine per antibody resulted in retention of immunoreactivity and when the immunoconjugates were linked to AuNPs, 8.09 ± 0.38 antibodies were attached to one AuNP. The total number of OPSS-PEG-DOTA per AuNP was estimated to be 812 ± 53. The total size of GNB was 67.3 ± 0.6 nm and there was no particle precipitation or aggregation after incubation in PBS. Competitive binding assay revealed that GNB was targeted strongly to EGFR (Kd antibody: 2.7 ± 0.3 nM; Kd GNB: 5.0 ± 4.9 nM) and darkfield microscopy showed that low PEG to antibody ratio increased the targeting efficiency when compared to high PEG to antibody ratio. Confocal microscopy revealed specific GNB uptake in MDA-MB-468 cells and cell fractionation study demonstrated receptor-mediated internalization of GNB, where the total amount of GNB internalized was about 3.1 and 22.2 fold higher in MDA-MB-468 cells than in MDA-MB-231 or MCF-7 cells. Conclusion: This GNB is very effective in targeting and in internalizing into EGFR, thus the GNB labeled with 177Lu emitting β-particles may be a promising new localized treatment for LABC. We plan to now examine the cytotoxicity of GNB both in vitro and in vivo will be explored for treatment of EGFR positive tumors. Supported by a grant from the Canadian Breast Cancer Foundation. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-15-04.

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.

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

// 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.

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.

KRAS mutations are proved to confer dramatic resistance to EGFR target therapy of cancer patients. The aim of this study was to establish a convenient and accurate method to screen plasma KRAS mutations of cancer patients since tumor specimens were not always available in clinical practice. A modified PNA-PCR method was established and evaluated in plasma of 19 pancreatic cancer patients. Our results showed that the modified PNA-PCR assay was a sensitive (87.5%, 14/16) and accurate (92.9%, 13/14) method to screen plasma KRAS mutations of pancreatic cancer patients and there was a high consistency of KRAS mutation status between plasma samples and tumor specimens. The modified protocol could not only screen plasma KRAS mutations rapidly and accurately but also had potential to quantify KRAS mutant DNA to predict treatment response of cancer patients and monitor disease progression. It's should be indicated that this modified assay was only confirmed in the pancreatic cancer patients in this study and need to be verified in other cancer patients.

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.

Necitumumab (EGFR inhibitor) in combination with chemotherapy provides a modest, yet a significant improvement in overall survival over chemotherapy alone in patients with advanced squamous non-small cell lung carcinoma (NSCLC). However, combination therapies targeting EGFR and PD-1 pathway blockers may represent a better way to extend clinical benefit to more cancer patients given that PD-(L)1 antibodies have emerged as a standard of care in NSCLC. Antibodies targeting EGFR have the potential to promote an inflamed tumor microenvironment through engagement of Fc-gamma receptors (FcγR) on innate immune cells resulting in an improved antigen presentation and T cell priming. Therefore, the present study was initiated to understand the combinatorial effect of immune checkpoint (PD-(L)1) inhibitors with necitumumab. Preclinical modeling of EGFR/PD-(L)1 mAb combination in mice is challenging due to the lack of cross-reactivity of necitumumab with mouse EGFR. Syngeneic mouse tumor models widely used to study effects of immunomodulatory agents express low or no EGFR. To overcome this limitation, we used two immunocompetent model systems to study the combination effect of EGFR mAb with PD-1 (RMP1-14) or PD-L1 (178G7) mAbs: 1) genetically engineered mouse model of lung adenocarcinoma (TD model) driven by mutant forms of human EGFR (exon 19 deletion and T790M mutation) and 2) CT26 syngeneic mouse tumor model with ectopic expression of human EGFR (CT26-hEGFR). To engage mouse immune cells more efficiently, a murinized version of necitumumab was generated through antibody engineering, with human EGFR binding Fabs and a mouse Fc backbone. Intratumor immune response was evaluated by immunohistochemistry and a custom-made immune profiling Quantigene Plex (QGP) gene expression panel. In both models, targeting EGFR and PD-1 pathway resulted in the combinatorial antitumor efficacy exemplified by decreased tumor burden compared to the monotherapy groups. QGP analysis of CT26-hEGFR tumor tissue revealed that the combination treatment enhanced intra-tumor immune response exemplified by an upregulation of immune-related genes indicative of T cell infiltration (Cd3e, Cd4, Cd8b1), T cell activation (Ifng, Cd274, Pdcd1lg2, Icos, Tnfrsf4, Tnfrsf18, Cd69, Ido1, Havcr2, Lag3), myeloid cell infiltration (Cd86, Timd4, Vista, Cd68, Mpo, Nos2). Histopathological analysis confirmed an increase in T cell infiltration indicating an improved immune response in the combination therapy group. Taken together, these results provide a rationale for further evaluation of EGFR and PD-(L)1 mAbs in clinical setting. Citation Format: Veena Kandaswamy, Amelie Forest, Marianne Deroose, David A. Schaer, Ting Chen, Shengwu Liu, David Surguladze, Yung-mae Yao, Thompson Doman, Gerald Hall, Kwok-Kin Wong, Michael Kalos, Ruslan D. Novosiadly. Combination of EGFR antibody with PD-1 pathway inhibitors improves anti-tumor efficacy and enhances intra-tumor immune response in preclinical mouse tumor models [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 3632.

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.

3555Background: MET amp is a well described driver of acquired EGFR antibody (Ab) resistance. Blood-based genomic profiling of cell free (cf)DNA is a safe and efficient means to identify pts with a...

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

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.