Development of recombinant monoclonal antibodies as tools for functional characterization of structurally–complex transmembrane amino acid carrier

The high affinity and specificity of monoclonal antibodies (mAbs) against a single epitope, combined with the actual possibility to produce them in large quantities through different well–established technologies, make mAbs extremely important research tools for a broad spectrum of biochemistry, molecular and cellular biology applications and sharp devices in medical contexts, both for diagnostic and therapeutic purposes. However, the development of new approaches for the generation, isolation and production of mAbs is critical to address the limited ability of hybridoma technologies to represent the antibody repertoire diversity generated following immunization of wild type or Hu–Ig transgenic mice, or the display technologies propensity to lose the naturally selected heavy and light chains variable regions pairing. Structurally–complex transmembrane protein families such as GPCRs, ion channels and solute carrier (SLC) membrane transporters are functional in many important cellular processes and involved in several pathological conditions. For these reasons they are very attractive targets for the development of monoclonal antibodies either for diagnostic or therapeutic applications. However, the technical difficulties encountered in their generation make these potential therapeutic targets still poorly exploited. The aim of this PhD project is to define an effective method for the production of monoclonal antibodies targeting structurally–complex transmembrane proteins, in order to facilitate the development of potential diagnostics and therapeutics mAbs towards hard–to–target antigens. Combining single antigen–specific Antibody Secreting Cells (ASCs) isolation and cloning of immunoglobulin (Ig) heavy– and light–chain variable regions (VH and VL) it was possible to identify monoclonal antibodies recognizing different epitopes and conformational structures of a complex multi–spanning membrane protein from mice immunized with the protein natively overexpressed on cell surface or with a recombinant fragment of it. One of the newly generated antibodies efficiently recognizes a cell–surface exposed conformational epitope on a cancer cell line. To date, there is no evidence in the literature of existing antibodies capable of specifically recognizing the selected target protein in its conformational form. The results achieved allow the validation of the method described in this work. At the same time, the new developed molecular tool will be very useful for an in–depth functional characterization of a transmembrane amino acid carrier involved in several diseases; a tool that could be eventually further developed for diagnostic and therapeutic applications.