Overexpression of the epidermal growth factor receptor (EGFR) is observed in a large number of neoplasms. The monoclonal antibody cetuximab/Erbitux is frequently applied to treat EGFR-expressing tumors. However, the application of cetuximab alone or in combination with radio- and/or chemotherapy often yields only little benefit for patients. In the present study, we describe a mechanism that explains resistance of both tumor cell lines and cultured primary human glioma cells to cetuximab. Treatment of these cells with cetuximab promoted DNA synthesis in the absence of increased proliferation, suggesting that DNA repair pathways were activated. Indeed, we observed that cetuximab promoted the activation of the DNA damage response pathway and prevented the degradation of essential meiotic endonuclease 1 homolog 1 (Eme1), a heterodimeric endonuclease involved in DNA repair. The increased levels of Eme1 were necessary for enhanced DNA repair, and the knockdown of Eme1 was sufficient to prevent efficient DNA repair in response to ultraviolet-C light or megavoltage irradiation. These treatments reduced the survival of tumor cells, an effect that was reversed by cetuximab application. Again, this protection was dependent on Eme1. Taken together, these results suggest that cetuximab initiates pathways that result in the stabilization of Eme1, thereby resulting in enhanced DNA repair. Accordingly, cetuximab enhances DNA repair, reducing the effectiveness of DNA-damaging therapies. This aspect should be considered when using cetuximab as an antitumor agent and suggests that Eme1 is a negative predictive marker.

Cetuximab, an anti-EGFR monoclonal antibody (mAb), is approved for advanced head and neck squamous cell carcinoma (HNSCC) but benefits a minority. An established tumor-intrinsic resistance mechanism is cross-talk between the EGFR and hepatocyte growth factor (HGF)/cMet pathways. Dual pathway inhibition may overcome cetuximab resistance. This Phase I study evaluated the combination of cetuximab and ficlatuzumab, an anti-HGF mAb, in patients with recurrent/metastatic HNSCC. The primary objective was to establish the recommended Phase II dose (RP2D). Secondary objectives included overall response rate (ORR), progression-free survival (PFS), and overall survival (OS). Mechanistic tumor-intrinsic and immune biomarkers were explored. Thirteen patients enrolled with no dose-limiting toxicities observed at any dose tier. Three evaluable patients were treated at Tier 1 and nine at Tier 2, which was determined to be the RP2D (cetuximab 500 mg/m2 and ficlatuzumab 20 mg/kg every 2 weeks). Median PFS and OS were 5.4 (90% CI = 1.9-11.4) and 8.9 (90% CI = 2.7-15.2) months, respectively, with a confirmed ORR of 2 of 12 (17%; 90% CI = 6-40%). High circulating soluble cMet levels correlated with poor survival. An increase in peripheral T cells, particularly the CD8+ subset, was associated with treatment response whereas progression was associated with expansion of a distinct myeloid population. This well-tolerated combination demonstrated promising activity in cetuximab-resistant, advanced HNSCC.

Non-melanoma skin cancer is the most common malignancy in US, with an annual incidence of in excess of 1.5 million cases. In the majority of cases, locoregional treatment is curative and systemic therapy is not indicated. Platinum-based chemotherapy regimens have been used most commonly in refractory cases. The use of cetuximab, a monoclonal antibody targeting epidermal growth factor receptor [EGFR], has been reported for skin cancer treatment. This current study evaluated eight cases of locally advanced and refractory basal cell or squamous cell cancers which were treated with cetuximab.

Although the epidermal growth factor receptor (EGFR) is an established target in head-and-neck cancer (HNC), resistance to EGFR-targeted therapy mediated by various mechanisms has been reported. Therefore, a combination strategy to overcome resistance to EGFR mono-targeted therapy is clinically required. We have previously demonstrated that non-thermal atmospheric pressure plasma (NTP) induces death of various cancer cells, including oral squamous cancer (OSCC) cells. In this study, we report for the first time that combining NTP treatment with cetuximab led to inhibition of migration and invasion in cetuximab-resistant OSCC cells, which could be a promising strategy to overcome resistance to anti-EGFR therapy. NTP induced deactivation of NF-κB in SCCQLL1 cells, but not in MSKQLL1 cells. In addition, NTP increased the expression level of E-cadherin, and decreased those of vimentin, Slug, Snail, matrix metalloproteinase (MMP)-2, -9, and activities of MMPs. Moreover, NF-κB upregulation using cDNA diminished the combination effect of NTP on invasion, migration and related signals. Taken together, these results indicate that the combination of NTP with cetuximab can decrease invasiveness in cetuximab-resistant OSCCs through a novel mechanism involving the NF-κB pathway. These findings show the therapeutic potential of treatment that combines NTP and cetuximab in OSCC.

Despite recent advances, the treatment of head and neck squamous cell carcinoma (HNSCC) remains an area of high unmet medical need. HNSCC is frequently associated with either amplification or mutational changes in the PI3K pathway, making PI3K an attractive target particularly in cetuximab-resistant tumors. Here, we explored the antitumor activity of the selective, pan-class I PI3K inhibitor copanlisib with predominant activity towards PI3Kα and δ in monotherapy and in combination with cetuximab using a mouse clinical trial set-up with 33 patient-derived xenograft (PDX) models with known HPV and PI3K mutational status and available data on cetuximab sensitivity. Treatment with copanlisib alone resulted in moderate antitumor activity with 12/33 PDX models showing either tumor stabilization or regression. Combination treatment with copanlisib and cetuximab was superior to either of the monotherapies alone in the majority of the models (21/33), and the effect was particularly pronounced in cetuximab-resistant tumors (14/16). While no correlation was observed between PI3K mutation status and response to either cetuximab or copanlisib, increased PI3K signaling activity evaluated through gene expression profiling showed a positive correlation with response to copanlisib. Together, these data support further investigation of PI3K inhibition in HNSCC and suggests gene expression patterns associated with PI3K signaling as a potential biomarker for predicting treatment responses.

Cetuximab, a chimeric IgG1 monoclonal antibody that targets the ligand-binding domain of the epidermal growth factor receptor (EGFR), is active in metastatic colorectal cancer (mCRC). As an IgG1 antibody, cetuximab may exert its antitumor efficacy through both EGFR antagonism and antibody-dependent cell-mediated cytotoxicity. Clinical trials established the role of cetuximab, particularly with irinotecan, in irinotecan-refractory/heavily pretreated patients. More recent studies show promising activity in second-line treatment after oxaliplatin-based therapy failure, and with first-line chemotherapy, where increased response rates seen with adding cetuximab to first-line therapy for mCRC may increase chances for curative surgery in a population for whom the therapy goal would otherwise be palliative. Cetuximab is generally well tolerated; common toxicities are acne-form rash and hypomagnesemia. Rash intensity is associated with clinical efficacy, and in the future, may be used as a marker for optimal drug exposure. Cetuximab activity in mCRC is not correlated with EGFR expression, and consequently other markers will be needed to identify the most likely responders. Cetuximab has clinically emerged as a core agent, along with 5-fluorouracil, irinotecan, oxaliplatin, and bevacizumab, for overall mCRC management to optimize survival. Ongoing studies are exploring best combinations of cetuximab with these other agents to maximize patient outcome.

There is increasing demand for improved production and purification systems for biosimilar or biobetter humanised monoclonal antibodies and animal production systems offer one such possibile option. Cetuximab, also known as 'Erbitux', is a humanised monoclonal antibody widely used in cancer therapy. We have previously reported on a genetically engineered goat system to produce cetuximab (gCetuximab) in milk. Herein we report that gCetuximab has similar bioactivity and pharamacokinetic properties compared with the commercial product produced in mammalian cell culture. In particular both forms have very similar efficacy in a HT29 colorectal cancer xenograft model alone or when conjugated to the toxin MMAE. This also demonstrates that the gCetuximab will be a viable vehicle for antibody drug conjugate based therapies. Taken together, this shows that the goat milk monoclonal antibody production system is an effective way of producing a biosimilar form of cetuximab.

KRAS mutation is a predictive biomarker for resistance to cetuximab (Erbitux) in metastatic colorectal cancer (mCRC). This study sought to determine if KRAS mutant CRC lines could be sensitized to cetuximab using dasatinib (BMS-354825, Sprycel), a potent, orally bioavailable inhibitor of several tyrosine kinases, including the Src family kinases (SFKs). We analyzed 16 CRC lines for: (1) KRAS mutation status, (2) dependence on mutant KRAS signaling and (3) expression level of epidermal growth factor receptor (EGFR) and SFKs. From these analyses, we selected three KRAS mutant (LS180, LoVo and HCT116) cell lines and two KRAS wild-type cell lines (SW48 and CaCo2). In vitro, using poly-D-lysine/laminin plates, KRAS mutant cell lines were resistant to cetuximab, whereas KRAS wild-type lines showed sensitivity to cetuximab. Treatment with cetuximab and dasatinib showed a greater antiproliferative effect on KRAS mutant lines when compared with either agent alone in vitro and in vivo. To investigate potential mechanisms for this antiproliferative response in the combinatorial therapy, we performed Human Phospho-Kinase Antibody Array analysis, measuring the relative phosphorylation levels of 39 intracellular proteins in untreated, cetuximab, dasatinib or the combinatorial treatment in the KRAS mutant lines LS180, LoVo and HCT116 cells. The results of this experiment showed a decrease in a broad spectrum of kinases centered on the β-catenin pathway, the mitogen-activated protein kinase (MAPK) pathway, AKT/mammalian target of rapamycin (mTOR) pathway and the family of signal transducers and activators of transcription (STATs) when compared with the untreated control or monotherapy treatments. Next, we analyzed tumor growth with cetuximab, dasatinib or their combination in vivo. KRAS mutant xenografts showed resistance to cetuximab therapy, whereas KRAS wild type demonstrated an antitumor response when treated with cetuximab. KRAS mutant tumors exhibited minimal response to dasatinib monotherapy. However, as in vitro, KRAS mutant lines exhibited a response to the combination of cetuximab and dasatinib. Combinatorial treatment of KRAS mutant xenografts resulted in decreased cell proliferation, as measured by Ki67, and higher rates of apoptosis, as measured by TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling). The data presented in this study indicate that dasatinib can sensitize KRAS mutant CRC tumors to cetuximab and may do so by altering the activity of several key signaling pathways. Furthermore, these results suggest that signaling via EGFR and SFKs may be necessary for cell proliferation and survival of KRAS mutant CRC tumors. These data strengthen the rationale for clinical trials combining cetuximab and dasatinib in the KRAS mutant CRC genetic setting.

Cetuximab improves the survival of patients with metastatic colorectal cancer. The main limitation is primary and secondary resistance, the underlying mechanism of which requires extensive investigation. We proved that PRSS expression levels are significantly negatively associated with the sensitivity of cancer cells to cetuximab. Detailed mechanistic analysis indicated that PRSS can cleave cetuximab, leading to resistance. Cetuximab or bevacizumab combined with SPINK1, a PRSS inhibitor, inhibited cell growth more efficiently than cetuximab or bevacizumab alone in xenograft models. PRSS levels in the serum of 156 patients with mCRC were analyzed, and poor efficacy of cetuximab therapy was observed in patients with aberrant PRSS expression. PRSS expression in monoclonal antibody (mAb)-treated patients with cancer from The Cancer Genome Atlas database was also evaluated to determine whether patients with higher PRSS expression have significantly reduced progression-free survival. Our work provides a strong scientific rationale for targeting PRSS in combination with cetuximab therapy.

Cetuximab is a monoclonal antibody blocking the epidermal growth factor receptor (EGFR) in metastatic colorectal cancer (mCRC). However, cetuximab treatment has no clinical benefits in patients affected by mCRC with KRAS mutation or in the presence of constitutive activation of signalling pathways acting downstream of the EGFR. The aim of this study was to improve cetuximab's therapeutic action by conjugating cetuximab with the type 1 ribosome inactivating protein (RIP) quinoin isolated from quinoa seeds. A chemical conjugation strategy based on the use of heterobifunctional reagent succinimidyl 3-(2-pyridyldithio)propionate (SPDP) was applied to obtain the antibody-type 1 RIP chimeric immunoconjugate. The immunotoxin was then purified by chromatographic technique, and its enzymatic action was evaluated compared to quinoin alone. Functional assays were performed to test the cytotoxic action of the quinoin cetuximab immunoconjugate against the cetuximab-resistant GEO-CR cells. The novel quinoin cetuximab immunoconjugate showed a significant dose-dependent cytotoxicity towards GEO-CR cells, achieving IC50 values of 27.7 nM (~5.0 μg/mL) at 72 h compared to cetuximab (IC50 = 176.7 nM) or quinoin (IC50 = 149.3 nM) alone assayed in equimolar amounts. These results support the therapeutic potential of quinoin cetuximab immunoconjugate for the EGFR targeted therapy, providing a promising candidate for further development towards clinical use in the treatment of cetuximab-resistant metastatic colorectal cancer.

The optimal maintenance strategy after induction chemotherapy with anti-epidermal growth factor receptor antibody for patients with RAS wild-type metastatic colorectal cancer (mCRC) remains to be debated.

Squamous cell carcinoma of the head and neck (SCCHN) is the sixth most common cancer worldwide. It has recently been appreciated that human papillomavirus (HPV) status (or p16 status, which is a frequently used surrogate for HPV status) is prognostic for oropharyngeal SCCHN. Here, we review and contextualize existing p16 and HPV data, focusing on the cetuximab registration trials in previously untreated, locoregionally advanced, nonmetastatic SCCHN (LA SCCHN) and in recurrent and/or metastatic SCCHN (R/M SCCHN): the IMCL-9815 and EXTREME clinical trials, respectively. Taken together, the available data suggest that, while p16 and HPV are prognostic biomarkers in patients with LA SCCHN and R/M SCCHN, it could not be shown that they are predictive for the outcomes of the described cetuximab-containing trial regimens. Consequently, although HPV status provides prognostic information, it is not shown to predict therapy response, and so is not helpful for assigning first-line therapy in patients with SCCHN. In addition, we discuss assays currently used to assess p16 and HPV status, as well as the differentiation between these two biomarkers. Ultimately, we believe HPV E6/E7 polymerase chain reaction-based mRNA testing may represent the most informative technique for assessing HPV status in patients with SCCHN. While p16 is a valid surrogate for HPV status in oropharyngeal carcinoma (OPC), there is a higher risk of discordance between p16 and HPV status in non-OPC SCCHN. Collectively, these discussions hold key implications for the clinical management of SCCHN.

It is known that cetuximab (an epidermal growth factor receptor [EGFr] inhibitor) is a radiosensitizer. Also, cetuximab is known to only partially inhibit the signal transducer and activator of transcription - 3 (STAT-3); a mediator of protection from apoptosis. Studies were performed to determine if the radiosensitizing effects of cetuximab could be enhanced with the addition of an inhibitor of STAT-3.

The use of cetuximab anti-epidermal growth factor receptor (anti-EGFR) antibodies has opened the era of targeted and personalized therapy in colorectal cancer (CRC). Poor response rates have been unequivocally shown in mutant KRAS and are even observed in a majority of wild-type KRAS tumors. Therefore, patient selection based on mutational profiling remains problematic. We previously identified methylglyoxal (MGO), a by-product of glycolysis, as a metabolite promoting tumor growth and metastasis. Mutant KRAS cells under MGO stress show AKT-dependent survival when compared with wild-type KRAS isogenic CRC cells. MGO induces AKT activation through phosphatidylinositol 3-kinase (PI3K)/mammalian target of rapamycin 2 (mTORC2) and Hsp27 regulation. Importantly, the sole induction of MGO stress in sensitive wild-type KRAS cells renders them resistant to cetuximab. MGO scavengers inhibit AKT and resensitize KRAS-mutated CRC cells to cetuximab in vivo. This study establishes a link between MGO and AKT activation and pinpoints this oncometabolite as a potential target to tackle EGFR-targeted therapy resistance in CRC.

Cancer progression is a highly complex process that is driven by a constellation of deregulated signaling pathways and key molecular events. In non-small-cell lung cancer (NSCLC), as in several other cancer types, the epidermal growth factor receptor (EGFR) and its downstream signaling components represent a key axis that has been found not only to trigger cancer progression but also to support advanced disease leading to metastasis. Two major therapeutic approaches comprising monoclonal antibodies and small molecule tyrosine kinase inhibitors have so far been used to target this pathway, with a combination of positive, negative, and inconsequential results, as judged by patient survival indices. Since these drugs are expensive and not all patients derive benefits from taking them, it has become both pertinent and paramount to identify biomarkers that can predict not only beneficial response but also resistance. This review focuses on the chimeric monoclonal antibody, cetuximab, its application in the treatment of NSCLC, and the biomarkers that may guide its use in the clinical setting. A special emphasis is placed on the EGFR, including its structural and mechanistic attributes.

To report our experience with a sequential regimen of induction TPF-C followed by radioimmunotherapy with cetuximab in patients with locally advanced head and neck squamous cell carcinoma (HNSCC).

The epidermal growth factor receptor (EGFR) is overexpressed by 80-90% of squamous cell carcinoma of head and neck (HNSCC). In addition to inhibiting EGFR signal transduction, cetuximab, a monoclonal antibody targeting EGFR can also bind to fragment crystallisable domain of immunoglobulins G1 present on natural killer (NK), causing antibody-dependent cellular cytotoxicity (ADCC). However, presence of cetuximab resistance limits effective clinical management of HNSCC.

Therapeutic monoclonal antibodies (mAbs) represent one of the most important classes of pharmaceutical proteins to treat human diseases. Most are produced in cultured mammalian cells which is expensive, limiting their availability. Goats, striking a good balance between a relatively short generation time and copious milk yield, present an alternative platform for the cost-effective, flexible, large-scale production of therapeutic mAbs. Here, we focused on cetuximab, a mAb against epidermal growth factor receptor, that is commercially produced under the brand name Erbitux and approved for anti-cancer treatments. We generated several transgenic goat lines that produce cetuximab in their milk. Two lines were selected for detailed characterization. Both showed stable genotypes and cetuximab production levels of up to 10 g/L. The mAb could be readily purified and showed improved characteristics compared to Erbitux. The goat-produced cetuximab (gCetuximab) lacked a highly immunogenic epitope that is part of Erbitux. Moreover, it showed enhanced binding to CD16 and increased antibody-dependent cell-dependent cytotoxicity compared to Erbitux. This indicates that these goats produce an improved cetuximab version with the potential for enhanced effectiveness and better safety profile compared to treatments with Erbitux. In addition, our study validates transgenic goats as an excellent platform for large-scale production of therapeutic mAbs.

Neurofibromin 1 (NF1) is a tumor suppressor that has been previously reported to regulate RAS‑MAPK signaling. The present study investigated the possible relationship between NF1 expression and anti‑EGFR antibody (cetuximab) sensitivity in colorectal cancer cell lines. In addition, primary or metastatic colorectal cancer samples from patients treated with cetuximab were assessed for the association of cetuximab sensitivity. The quantities of the NF1 transcript, NF1‑related pathway enrichment and NF1 mutation profile were measured and investigated using RNA sequencing and targeted DNA sequencing. Based on growth inhibition and colony formation assay results, cell lines were designated to be cetuximab‑sensitive (NCI‑H508 and Caco2) or cetuximab‑resistant (KM12C and SM480). Western blotting revealed NF1 was highly expressed in cetuximab‑sensitive cell lines whilst there was little expression in their cetuximab‑resistant counterparts. Knocking down NF1 expression using small interfering RNA in the cetuximab‑sensitive cell lines enhanced the phosphorylation of MEK and ERK according to western blotting. NF1 knockdown also reduced apoptosis, as observed by the decreased number of apoptotic bodies by DAPI nuclear staining and reduced cleavage of caspase and poly‑(ADP ribose) polymerase. NF1 overexpression by transfection with GTPase‑activating protein‑related domain subunit rendered the cetuximab‑resistant cell lines, KM12C and SW480, more susceptible to cetuximab‑induced apoptosis. RNA sequencing of 111 RAS and BRAFV600 wild‑type tumor samples collected from cetuximab‑treated patients with metastatic colorectal cancer revealed that the pre‑treatment NF1 expression levels were not associated with the cetuximab response. However, tumor samples obtained after cetuximab treatment displayed slightly lower NF1 transcript levels compared with those in the pre‑treatment samples, suggesting that exposure to the anti‑EGFR antibody may be associated with reduced NF1 expression levels. Next‑generation sequencing revealed that the frequency of inactivating mutations in NF1 were rare (1.8%) in patients with colorectal cancer and were not associated with the protein expression levels of NF1 except for in a small number of cases (0.5%), where the biallelic inactivation of NF1 was observed. To conclude, the present study showed that modification of NF1 expression can affect sensitivity to cetuximab in colorectal cancer cell lines, though a limitation exists in terms of its potential application as a biomarker for RAS and BRAFV600 wild‑type tumors.

We have developed a theranostic nanoparticle, ie, cet-PEG-dexSPIONs, by conjugation of the anti-epidermal growth factor receptor (EGFR) monoclonal antibody, cetuximab, to dextran-coated superparamagnetic iron oxide nanoparticles (SPIONs) via periodate oxidation. Approximately 31 antibody molecules were conjugated to each nanoparticle. Cet-PEG-dexSPIONs specifically bind to EGFR-expressing tumor cells and enhance image contrast on magnetic resonance imaging. Cet-PEG-dexSPION-treated A431 cells showed significant inhibition of epidermal growth factor-induced EGFR phosphorylation and enhancement of EGFR internalization and degradation. In addition, a significant increase in apoptosis was detected in EGFR-overexpressing cell lines, A431 and 32D/EGFR, after 24 hours of incubation at 37°C with cet-PEG-dexSPIONs compared with cetuximab alone. The antibody-dependent cell-mediated cytotoxicity of cetuximab was observed in cet-PEG-dexSPIONs. The results demonstrated that cet-PEG-dexSPIONs retained the therapeutic effect of cetuximab in addition to having the ability to target and image EGFR-expressing tumors. Cet-PEG-dexSPIONs represent a promising targeted magnetic probe for early detection and treatment of EGFR-expressing tumor cells.