Characterization of self-assembled monolayers (SAM) of peptide mimotopes of CD20 antigen and their binding with rituximab (R) using Quartz Crystal Microbalance (QCM) Piezo-Immunosensors: A novel technique to study CD20 antigen interaction.

Abstract

e14004 Background: CD20 antigen is an established target for antibody therapy like R. Understanding interactions of CD20 antigens is important for developing better agents and overcoming resistance. We have developed a new innovative biosensor based quantitative technique to study real time interaction of CD20 antigens with R using QCM Piezo-immunosensor, which can be used to characterize cell surface antigen, study interactions and understand mechanisms of resistance. Methods: Binding of R with SAM of peptide mimotopes of CD20 antigen was systematically characterized. Four peptide mimotopes of CD 20 antigen were selected from literature and redesigned to allow their SAM immobilizations on gold electrodes through a peptide linker with cysteine. The bindings of these peptides with R and control antibodies (Trastuzumab, Bevacizumab) were characterized by Quartz Crystal Microbalance (QCM). Among the four peptide mimotopes initially selected, the peptide designated as CN-14 was the most selective and sensitive for R binding, which was further characterized by ellipsometry, atomic force microscopy and QCM. Results: We have shown a systematic approach for using QCM technique to quantify the apparent binding of CD20 and R. As the GLU residue pKa of CN-14 increased, the affinity of the SAM to Rituximab increased while the selectivity decreased. This was attributed to the weakening of the salt bridge between the CN-14 ARG-10 and GLU-13 at higher pKa values for GLU-13. Conclusions: Interaction of CD20 antigens with antibodies will help physicians to determine the clinical efficacy and resistance mechanisms to antibodies like R. QCM is low cost, highly sensitive, fast, synthetic, assay which could be used as a basis for developing a new generation of affinity-based Immunosensor assays. Our study shows that peptide mimotopes have potential benefit in sensor applications as the peptide-peptide interactions in the SAM could be manipulated by the addition of functional groups to the peptide to influence binding of the target protein as well as for surface immobilization via SAM.