Production of Recombinant Human EGFR CR2 Domain and Generation of Monoclonal Antibodies Discriminating The R and K Variants.

Abstract Background: The epidermal growth factor receptor (EGFR) was the first molecular marker to be targeted successfully in monoclonal antibodies-based cancer immunotherapy. The human EGFR displays two common natural genetic variants (R521K) located in the molecule extracellular CR2 domain. This polymorphism affects the outcome of cancer immunotherapy using anti-EGFR mAbs.Results: In this paper, we report the production, purification and characterization of recombinant forms of the EGFR/CR2-R and CR2-K variants as Glutathione S-transferase (GST) fusion protein in E. Coli BL21. We used these two proteins to generate three different murine monoclonal antibodies to the EGFR CR2 domain (anti-CR2R, anti-CR2K and anti-CR2-RK). We carried out the molecular characterization of the anti-CR2-RK mAb. Analysis using Western blot, ELISA and Immunohistochemistry of various tumor tissues samples, showed that anti-CR2RK mAb was specific of the human EGFR CR2 extracellular domain and recognizes equally both the CR2-K and CR2-R natural genetic variants. In addition, the affinity binding of anti-CR2-RK mAb, as determined by Surface Plasmon Resonance, was equal to 27.7KD μM. Conclusions: We produced recombinant forms of the human EGFR CR2 domain R and K variants and generated mAbs that discriminate between them. These mAbs can be engineered into novel cancer therapeutic and or diagnostic tools tailored to the patients EGFR genetic makeup.