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https://doi.org/10.1007/s00262-007-0313-4. did not possess any impact on tumour regression or animal survival. We conclude that 7A7 does not identify mouse EGFR and therefore cannot be used as the mouse equivalent of cetuximab use in humans. As a number of groups possess spent effort and resources with similar issues we feel that publication is definitely a responsible approach. and pre-clinical platform with which to model cetuximab, using the monoclonal antibody 7A7. This would enable investigation of the effect that these therapies have CFM 4 on immune system activation and allow the assessment of underlying immune mechanisms of tumour rejection. Therefore, in order to develop our mouse experiments, we tested the capacity of 7A7 to bind murine EGFR and to induce tumour regression. Like a control, we parallel tested 7A7 Fc Silent, which contains key mutations that abrogate binding of Fc receptors, CFM 4 abolishing the antibody mediated cytotoxicity (ADCC) effector function of 7A7. Theoretically, both 7A7 and 7A7 Fc Silent should identify mouse EGFR and cross-react with human being EGFR. We demonstrate that neither 7A7 nor 7A7 Fc Silent specifically identify either mouse or human being EGFR. 7A7 was unable to effect tumour growth in an EGFR-expressing HPV38 transplantable SCC tumour model. Our study is an exhaustive and characterization of the 7A7 monoclonal antibody. We trust our results can allow researchers to make an informed decision when considering 7A7 for EGFR-targeted preclinical studies in mice. RESULTS EGFR mRNA manifestation in human being and murine cell lines We selected 6 cell lines to analyze the specificity of 7A7 to detect graded levels of mouse and human being EGFR. We quantified EGFR mRNA by quantitative RT-PCR in 2 human being cell lines (A431 and MCF-7) and 4 murine cell lines (3T3-L1, NIH-3T3, HPV38, and TC-1). The human being cell collection A431 indicated more than one thousand-fold EGFR in the mRNA level when compared to MCF-7 cells (Number ?(Figure1A),1A), in line with previously published data [15]. Open in a separate window Number 1 EGFR mRNA manifestation in 2 human being cell lines (A431 and MCF-7) and 4 murine cell lines (3T3-L1, HPV38, NIH-3T3, and TC-1)(A) A431 indicated more than 1 thousand-fold EGFR compared to MCF-7 cells. (B) 3T3-L1 and HPV38 indicated detectable levels of Egfr. NIH-3T3 and TC-1 did not communicate detectable levels of Egfr. The = 3 (* 0.05, **** 0.0001, n.s: not significant). Data demonstrated is definitely representative of one experiment of three with related results. Based on a earlier report describing the presence of Egfr manifestation in 3T3-L1 cells [16], we selected 3T3-L1 cells like a comparator for Egfr manifestation in our cohort of murine cell lines. As demonstrated in Figure ?Number1B,1B, Egfr mRNA levels varied widely among the 4 cell types tested. A SCC cell collection that we developed in-house, HPV38, showed high Egfr mRNA manifestation similar to that of 3T3-L1 cells. In contrast, NIH-3T3 and TC-1 cells showed no significant evidence of Egfr mRNA manifestation, however, TC-1 cells are known to be tumourigenic when injected into mice (of relevance later on) and hence TC-1 CFM 4 cells were selected for further study in subsequent experiments. Characterizations of 7A7 binding to EGFR in human being and murine cell lines Having validated and recognized EGFR positive and negative human being CFM 4 and murine cell lines at an RNA level, we evaluated the capacity of 7A7 mAb to detect EGFR protein by SDS-PAGE Western blot (SDS WB). Immunoblotting of human being A431 and murine 3T3-L1 and HPV38 protein extracts with the polyclonal goat anti-mouse antibody AF1280 showed an 170 KDa band which corresponded with the expected size of EGFR (Number ?(Figure2A)2A) [17]. A similar band was not recognized for CLEC4M TC-1 cells, as expected. However, in our study, we did not observe specific binding of.