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.
Highlights
Molecular imaging techniques are continuously progressing, allowing non-invasive in vivo visualization of biological and pathological processes at the molecular and cellular levels
variable regions of heavy (VH) and VL were sequentially cloned into the pHenIX phagemid, which contains the sequence for the peptide linker necessary to join the carboxyl terminus of VH to the amino terminus of VL, and allows the proper assembly of the single chain Fragment variable (scFv)-human ether-à-go-gorelated gene-1 (hERG1) encoding cassette
The scFv-hERG1 expression cassette was moved from pHenIX into pPIC9K and used to transform the GS115 P. pastoris yeast strain through the spheroplasting technique
Summary
Molecular imaging techniques are continuously progressing, allowing non-invasive in vivo visualization of biological and pathological processes at the molecular and cellular levels. Intact monoclonal antibodies (mAbs) are large (150 kDa) molecules, with generally slow pharmacokinetics, slow blood clearance, sub optimal tumor penetration and accumulation, and are often difficult to produce [6]. These characteristics can delay the time point for imaging, and often results in sub-optimal contrast between the tumor mass and the surrounding normal tissue. It is possible to conjugate the recombinant proteins with quantum dot, fluorescent dyes or other moieties, potentiating their exploitability for in vivo imaging For these reasons, scFv antibodies are becoming the ideal candidates in imaging applications, especially for cancer diagnostics [2,3,4,5]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
