A New Generation of PET/SPECT Tracers

Immuno imaging is a non-invasive and quantitative method for imaging of target distribution and temporal dynamics in vivo without the limitations of error-prone biopsies. In contrast to randomly conjugated antibody PET-tracers, site-specific labeling at the Fc glycan sites using the GlyCLICK technology generates tracers that exhibit increased target-affinity, improved stability and a lower off-target accumulation and for significantly improved performance in vivo.


GlyCLICK Workflow - PET-tracers

A Fully Preserved Immunoreactivity

Conventional radiolabeling of antibodies is limited by the presence of multiple conjugation sites that results in conjugates with undefined stochiometric distribution of radioisotopes across the antibody backbone. The potential label occupancy in the antigen-binding Fab can further impair the immunoreactivity of the intended tracers. To evaluate the antigen-binding capacity after conjugation, trastuzumab was site-specifically labeled (DOL=2) with the desferrioxamine (DFO) chelator using GlyCLICK and analyzed by Surface Plasmon Resonance (SPR). The resulting response curves show that the full immunoreactivity of the antibody is preserved using the GlyCLICK technology (Fig. 1).


SPR analysis - PET-tracers


Figure 1.  SPR analysis. Anti-human IgG (Fc) captured trastuzumab: native or GlyCLICK-conjugated to desferrioxamine (DFO) (DOL=2). HER2 was injected in a range to ensure sufficient curvature. All data were fitted against a 1:1 mathematical model.

Homogenous & Less Hydrophobic Conjugates

Random radiolabeling results in poorly defined and heterogenous antibody tracers due to the insufficient chemoselectivity of the conjugation method. Apart from affecting both targeting properties and stability, random labeling also increases hydrophobicity with excessive labeling of the intended tracer. To demonstrate the performance of site-specific conjugation, trastuzumab was labeled with the DFO chelator using a GlyCLICK kit or by random labeling at lysine residues. The hydrophobicity of each conjugate was studied using Hydrophobic Interaction Chromatography (HIC). The resulting separations show a single peak for site-specifically labeled trastuzumab obtained using GlyCLICK, indication in more homogenous and less hydrophopic batch of tracers compared to the randomly conjugated material (Fig. 2).



GlyCLICK in Predictive PET Imaging


Figure 2. Hydrophobic interaction analysis of GlyCLICK- or randomly conjugated trastuzumab injected on a TSKgel® Butyl-NPR column (Tosoh Bioscience) in 25 mM NaP pH 7, 1.5 M ammonium sulfate and eluted with a salt gradient with 20% isopropanol. Randomly conjugated trastuzumab was unresolved due to the sample heterogeneity.

Enhanced Tumor Uptake & Circulation

Antibody radiolabeled using the site-specific GlyCLICK technology or produced by random conjugation at lysines were analyzed using PET/CT imaging to evaluate their performance in vivo. Once chelated, the tracers were labeled with the standard radioisotope Zirconium-89 (89Zr) before injection into tumor-bearing mice. Analysis of the injected mice was carried out using PET/CT imaging to measure the biodistribution and tumor uptake of the tracers.

The results demonstrate the superior tumor uptake with significantly higher %ID/g and longer circulation time displayed by the site-specific GlyCLICK tracers, as compared to the randomly labeled tracers (Fig. 3). The consistent number of label attached to each antibody (DOL = 2) using the GlyCLICK kit does not only improve performance in vivo, but also allows for better quantitation possibilities in PET-imaging experiments.

GlyCLICK PET-tracers

Figure 3. PET/CT imaging analysis of SK-OV-3 tumor-bearing mice showing left) mean tumor uptake over a time interval of 0-168 hours post injection and right) ex vivo biodistribution of 89ZR-DFO-trastuzumab in major organs.