Selection and Characterization of Synthetic Antibodies Against Human Rad51

Abstract

Chemotherapy is the predominant approach for treating cancer. However, disease relapse frequently occurs because chemotherapy often fails to eliminate all tumor cells due to intrinsic or acquired drug resistance. The failure of conventional chemotherapeutics regimes for cancer highlights the need for novel therapeutic interventions. Recent studies have shown that human Rad51, an evolutionarily conserved DNA recombinase required for homologous recombination, exhibits elevated expression in many cancer cells and is implicated in drug resistance after chemotherapy. Targeted inhibition of human Rad51 has been explored as a way to sensitize cancer cells to chemotherapy. Given the properties of antibodies and their fragments as high-affinity inhibitors, we used antibody phage display to generate antigen-binding fragments (Fabs) against human Rad51. We first isolated human Rad51 specific Fabs by screening a synthetic Fab phage display library against recombinant human Rad51. We isolated a human Rad51 Fab, referred to as Fab F2, which bound human Rad51 with a KD of 8.1 nM. Fab F2 inhibited the DNA binding activity of human Rad51 but did not inhibit human Rad51 ATP hydrolysis activity. We converted Fab F2 into an scFv-Fc fragment (scFv: single-chain variable fragment; Fc: glycosylated crystallizable fragment) for expression in human embryonic kidney 293T cells. Overexpression of scFv-Fc fragment in human embryonic kidney 293T cells increased 4.48-fold more sensitivity to the DNA-damaging agent methyl methanesulfonate in clonogenic survival assays. To enable the delivery of Fab F2 into cells we fused it to a cell membrane import tag (FabItag I2) based on a patent (WO 2014005219 A1) from iProgen Biotech Inc. We labeled FabItag I2 with an 800CW fluorophore and showed that FabItag I2 permeated human embryonic kidney 293T cells using fluorescence microscopy and flow cytometry. FabItag I2 increased the sensitivity of human embryonic kidney 293T cells to methyl methanesulfonate by 2 folds in clonogenic survival assays.