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

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-à-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 VH 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.

The Activity of Kv 11.1 Potassium Channel Modulates F-Actin Organization During Cell Migration of Pancreatic Ductal Adenocarcinoma Cells

“…IF was performed using mAb-hERG1-Alexa488 (final concentration of 5 μg/mg protein), Ts2/16 (Biolegend, San Diego, CA, USA, final concentration of 5 μg/mg protein), anti-paxillin antibody (Biolegend, final concentration 5 μg/mg protein). Incubation and revealing steps were performed as in [39].…”

Ion Channels Orchestrate Pancreatic Ductal Adenocarcinoma Progression and Therapy

“…However, due to the cardiovascular side effects seen for many drugs, targeting the K V 11.1 channel with single-chain function blocking antibodies is also challenging and not preferred. K V 11.1 channel–specific antibodies, such as the single-chain variable fragment (scFv), anti-Kv11.1 ScFv, are more relevant for cancer diagnosis, rather than for cancer treatment ( Duranti et al, 2018 ). The risk of cardiac side effects can be reduced by employing bispecific antibodies.…”

Monoclonal antibody-based molecular imaging strategies and theranostic opportunities

“…Examples of applications of antibody-derived products in molecular imaging include the use of a scFv against the ion channel hERG1 for cancer optical imaging 42, the use of a minibody against PSCA using PET 43 and the use of a PSCA-targeted diabody in a PET/optical imaging hybrid44. In all these studies, the incentive to use antibody fragments was mainly due to the faster clearance from the circulation.…”