CHARACTERIZATION OF SPECIFIC CAMELID SINGLE DOMAIN ANTIBODIES, VHH OR NANOBODIES, AGAINST HUMAN IgG4 FOR DIAGNOSTIC APPLICATION IN IgG4 RELATED DISEASE

MARCHESE F; BOZZO J; GRIPPO V

Virtual

Congreso; Reunión Anual de Sociedades de Biociencias; 2021

SAIC-SAI-AAFE-NanoMedAR

The adaptive immune systems of camelids comprise classical antibodies and heavy-chain only antibodies (HCAb), where antigen-binding is mediated by one variable domain, called VHH or nanobody (Nb). Nbs present unique characteristics conferring great potential in the development of more sensitive diagnostic methods, including high solubility, physicochemical stability and low production cost. Moreover, Nbs are able to recognize cryptic epitopes, not targeted by conventional antibodies. On the other hand, IgG4 is increased in the serum of patients with IgG4 related disease (IgG4-RD). Along with other characteristic symptoms, serum IgG4 levels allow the diagnosis of IgG4-RD. For this reason, we aimed to characterize specific VHH against human IgG4 (hIgG4) for the development of new tools in IgG4-RD diagnosis. To achieve our goal, two immune VHH libraries were previously constructed starting from llama blood. Specific Nbs against hIgG4 were selected by Phage Display methodology. The reactivity of 90 selected Nbs was studied against the four human IgGs by ELISA. As a result, 61 out of 90 VHH were specifically reactive against hIgG4. The diversity of these Nbs was analyzed by fingerprinting, showing 20 different digestion patterns. From these VHH, the 10 most reactive Nbs were confirmed as unique by sequencing. Six of these Nbs were expressed as soluble protein in E. coli WK6 strain. Then, reactivity of soluble-expressed Nbs against sera of different species was assessed by Dot Blot. Furthermore, the reactivity against all human IgGs was studied by ELISA, confirming IgG4 specificity. In conclusion, 61 specific Nbs against hIgG4 were successfully selected by Phage Display. Moreover, the 10 most reactive out of these 61 VHH showed to be unique. Six out of these Nbs were expressed as soluble protein and showed high reactivity against only hIgG4 in Dot Blot and ELISA. Further characterization of these Nbs will allow us to develop new tools for diagnostic innovation in IgG4-RD.