Generation and characterization of novel recombinant anti-hERG1 scFv antibodies for cancer molecular imaging

// Claudia Duranti 1 , Laura Carraresi 2 , Angelica Sette 1, 4 , Matteo Stefanini 2 , Tiziano Lottini 1 , Silvia Crescioli 1, 5 , Olivia Crociani 1 , Luisa Iamele 3 , Hugo De Jonge 3 , Ermanno Gherardi 3 and Annarosa Arcangeli 1 1 Department of Experimental and Clinical Medicine, Section of Internal Medicine, University of Florence, Florence, Italy 2 DIVAL Toscana Srl, Sesto Fiorentino, Florence, Italy 3 Department of Molecular Medicine, University of Pavia, Pavia, Italy 4 Present address: Crescendo Biologics Ltd., Babraham Research Campus, Cambridge, UK 5 Present address: Department of Dermatology, King’s College, London, UK Correspondence to: Annarosa Arcangeli, email: annarosa.arcangeli@unifi.it Keywords: cancer diagnostics; molecular imaging; hERG1 potassium channels; antibody engineering Received: August 01, 2018      Accepted: September 21, 2018      Published: October 09, 2018 ABSTRACT Modern molecular imaging techniques have greatly improved tumor detection and post-treatment follow-up of cancer patients. In this context, antibody-based imaging is rapidly becoming the gold standard, since it combines the unique specificity of antibodies with the sensitivity of the different imaging technologies. The aim of this study was to generate and characterize antibodies in single chain Fragment variable (scFv) format directed to an emerging cancer biomarker, the human ether-a-go-go-related gene-1 (hERG1) potassium channel, and to obtain a proof of concept for their potential use for in vivo molecular imaging. The anti-hERG1scFv was generated from a full length monoclonal antibody and then mutagenized, substituting a Phenylalanine residue in the third framework of the V H domain with a Cysteine residue. The resulting scFv-hERG1-Cys showed much higher stability and protein yield, increased affinity and more advantageous binding kinetics, compared to the “native” anti-hERG1scFv. The scFv-hERG1-Cys was hence chosen and characterized: it showed a good binding to the native hERG1 antigen expressed on cells, was stable in serum and displayed a fast pharmacokinetic profile once injected intravenously in nude mice. The calculated half-life was 3.1 hours and no general toxicity or cardiac toxic effects were detected. Finally, the in vivo distribution of an Alexa Fluor 750 conjugated scFv-hERG1-Cys was evaluated both in healthy and tumor-bearing nude mice, showing a good tumor-to-organ ratio, ideal for visualizing hERG1-expressing tumor masses in vivo . In conclusion, the scFv-hERG1-Cys possesses features which make it a suitable tool for application in cancer molecular imaging.