Development and engineering of specific antibodies as potential anti-cancer tools

Today, monoclonal antibosies (mAbs) are the fastest growing group of biotechnology-derived molecules in clinical trials. Thanks to their ability to bind a single epitope, mAb technology has provided a powerful tool for the generation of highly specific antibodies with multiple applications. This work of thesis is part of those studies focused on identifying membrane proteins as potential new targets to be exploited in research, diagnostic and clinical fields. The final goal is the production of full antibodies and antibody fragments against such emerging targets, like the type two anti-Müllerian hormone receptor (AMHRII). Anti-Müllerian hormone (AMH) is a peptide growth factor belonging to the TGF-β family, well known for its role in sexual differentiation. Recently, the scientific community (mainly oncologists) has developed an increasing interest in this hormone for two main reasons: a) “hyper-physiological” AMH dosage inhibits gynecological and breast cancer growth, by interfering with cell cycle progression and inducing apoptosis (Kim et al, 2014; Segev et al, 2000; Anttonen, et al 2012). This ability has been demontrated in vitro and in vivo in rodent ovarian cancer cells (La Marca et al, 2007). These results may be translatable to humans because ovarian cancer cells express AMHRII and their in vitro growth is inhibited using recombinant AMH (Kim et al, 2014). b) AMHRII is selectively expressed in multiple tumor tissues and could therefore be a candidate as a tumor specific antigen (Zugmaier et al, 1990; Masiakos et al, 1999; Segev et al,2001; Barbie et al, 2003). Tumor specific antigens are expressed on tumor cells, but not in the corresponding normal counterpart. Therefore, they are considered potential targets for the development of innovative therapies with a high efficacy and reduced side effects. It has been known that the production of molecules directed towards the receptor rather than the ligand, allows to obtain potential tools able to up-regulate the physiological downstream cascade. Based on these premises, the present work is focused on developing new antibodies able to bind the AMHRII receptor and to up-regulate the signaling cascade physiologically triggered by the hormone AMH. We have produced two hybridomas population able to secrete antibodies (n°7 and n°16) that are capable to bind both conformational and linearized epitopes. Then, we have selected two clones D5 and D7 able to produce monoclonal antibodies (D5/D7 anti-AMHRII) directed against AMHRII, providing evidence that these molecules are able to recognize the antigen, also in a pathological scenario as the one represented by the triple negative breast cancer (TNBC). Moreover, D7 anti-AMHRII mAb has emerged as a possible strongly positive allosteric AMHRII modulator, and preliminary tests demonstrate its efficacy in inhibiting the breast cancer cell growth. Such ability will be better characterized in the future, exploiting its in vivo application. Afterwards, starting from the full antibody, we have developed a single-chain variable fragment (scFv) molecule, scFv-AMHRII-D7 (scFv-AMHRII construct). We have optimized the protocol for its expression in E. Coli, then we performed a small-scale production of the protein. The engineered antibody maintains the ability to up-regulate the AMH/AMHRII-dependent signaling cascade. In conclusion, cD7 anti-AMHRII mAb and scFv anti-AMHRII have been shown to be able to bind the antigen and to functionally affect its signaling cascades. The choice of developing antibodies against the membrane receptor (AMHRII), rather than the ligand (AMH) turned out to be the right strategy to obtain potential therapeutic tools for two main reasons: a) targeting a tumor specific antigen (AMHRII), we can minimize or even avoid the recurrent side effects we have using biological drugs; b) we can avoid the well-known problems related to high costs and difficult large-scale production of recombinant ligands (AMH). In conclusion, we have developed patentable potential diagnostic and therapeutic tools able to mimick AMH function with a comparable efficacy, but with a predictable high specificity thanks to the AMHRII selective targeting.