Selection, in depth characterization and site-specific bio-functionalization of nanobodies for the early detection of ovarian cancer

Abstract

Ovarian cancer (OC), of which the incidence increases with age, ranks fifth in cancer deaths among women1. The common symptoms of OC are indistinct and similar to other benign observations2. Most women are therefore diagnosed at an advanced stage III or IV of the disease, at which the 5-year relative survival rate is low (around 39% for stage III and only 17% for stage IV)2,3. OC can be successfully treated (with survival rate 94%) if it is detected early. Serum biomarkers may offer new possibilities to diagnose OC at an early stage. In this study, several nanobodies4 (Nbs) specifically targeting OC biomarkers such as Human epididymis protein 4 (HE4)2, Secretory leukocyte protease inhibitor (SLPI)3 and Progranulin (PGRN)3 were evaluated based on their expression level as well as their target binding affinity using enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR) methods. The binding properties of the selected Nbs were also determined by epitope mapping. The nanobodies with high expression level (~ 8 mg/ mL, 21 mg/ mL and 11 mg/ mL for HE4, SLPI and PGRN, respectively) and high binding affinity (KD value around 10-10– 10-8 M) were selected as best candidates for the development of multi-array biosensors that are capable of detecting multiple targets rapidly and combine high selectivity, high sensitivity and specificity for the detection of OC in early stage. The selected nanobodies were successfully site-specifically alkynated at their C-terminus using the Expressed protein ligation (EPL) technique and were coupled to an azidified PEG counterpart using “click”-chemistry5. The attachment of a click-chemistry functionality at the C-terminus of the nanobodies would pave the way to sensor platforms at which all nanobodies are covalently coupled with a unique and uniform orientation, allowing optimal target binding and resulting in improved sensitivity and selectivity since all nanobodies will have their active region accessible for the target binding.