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

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.


Slot blot analysis
Yeast supernatants (200 µl) were applied to a PVDF membrane (Amersham) assembled in a slot blot device between two sheets of 3 MM Whatman paper. The samples were left in incubation for 15 minutes. Blocking was performed with T-PBS 5% BSA for 45 minutes and the membrane was then washed 10 min with T-PBS. The membrane was finally incubated for 1 hour with anti-6xHis-HRP conjugated antibody (Sigma) diluted 1:2000 in T-PBS plus 5% BSA.

Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE)
15 µl of each sample were separated on a 15% acrylamide gel. Electrophoretic runs were performed at 150 V. Gels were either stained with Coomassie Brilliant Blue or transferred to PVDF membranes (see below) for western blot analysis to assess the proper molecular weight (around 30 KDa) of the protein.

Western blot (WB)
After SDS-PAGE, gels were transferred to PVDF membrane (Amersham) in transfer buffer (200 mM glycine, 25 mM Tris, 20% methanol) at 100 V for 1 h. Membranes were washed in T-PBS (PBS 0.1% Tween) and then blocked O/N with T-PBS containing 5% BSA. Membranes were exposed to primary antibody peroxidasecoupled (Sigma) diluted in T-PBS containing 5% BSA for one hour at room temperature. After washing the membranes three times for 10 minutes, each signal was visualized using ECL reagent (Amersham). WB were performed using the following antibodies: anti-myc (Santa Cruz Biotechnology) 1:1000 and anti-6xHis-HRP conjugated antibody (Sigma) 1:2000.

Indirect IF (I-IF) on fixed cells
At the end of incubation, cells were washed once with PBS and fixed with 4% paraformaldehyde (PFA) for 20 min at room temperature. Blocking was performed with 10% BSA for 2 h at room temperature. I-IF was performed using scFv-hERG1-G3, scFv-hERG1-D8Cys, diluted to a final concentration of 20 µg/ml in blocking solution and incubated for 2.5 hours, followed by O/N incubation with the anti-His antibody (1:250) (Abcam) in a blocking solution. The following day, cells were washed three times with PBS and incubated with anti-mouse Alexa 488 (Invitrogen) for 1 hour. Cells were then incubated with Hoechst (1:1000) for 30 minutes and mounted with propyl gallate. Cells were visualized on a confocal microscope (Nikon, C1). IF quantification was performed using ImageJ software: for each image, the measure of three different areas was performed and the mean was calculated.

Direct IF (D-IF)
D-IF was performed using labelled scFv-hERG1 and scFv-hERG1-Cys. To this purpose, scFv-hERG1 and scFv-hERG1Cys were conjugated with Alexa Fluor ® 488 Microscale Protein Labeling Kit (Thermo Fisher Scientific), according to the indications in the protocol. The scFvs were diluted 1:20 in blocking solution and incubated O/N at 4° C. IF on live cells was performed on cells grown O/N on 60 mm Petri dishes (Sarstedt) using an agarose (15 g/l) ring in order to isolate cells and minimize the volumes of reagents needed for incubations. The following day, cells were washed three times with PBS and incubated with Hoechst (1:1000) for 30 min and mounted with propyl gallate. Cells were visualized on a confocal microscope (Nikon, C1). IF quantification was performed as described for I-IF.

IF on spheroids
One thousand PANC-1 cells were seeded on an agarose base layer (1.5 g/l) in a 96 well plate and grown for 72 hours in a humidified incubator at 37° C and 5% CO 2 . For IF experiments, spheroids were incubated with scFv-hERG1-D8Cys (conjugated with Alexa Fluor 488), following the protocol previously described for IF on live cells.
IF intensity (A.U.) calculated using Image J Software (ImageJ 1.38, U.S. National Institutes of Health). For each image, the mean of the fluorescence intensity of three different areas was calculated after the subtraction of the blue channels values (which refers to nuclei staining).
Primers used for the assembly of scFv-hERG1 antibody construct Supplementary