Biomarkers, capable of identifying the stage and aggressiveness of idiopathic pulmonary fibrosis (IPF) at the time of diagnosis, would improve the accuracy of prognostication and facilitate targeting anti-fibrotic drug therapy to patients who are most likely to benefit. The integrin αvβ6 is highly expressed in injured lung epithelium and is a key activator of transforming growth factor β (TGF-β), which plays a crucial role in the initiation and maintenance of fibrosis. Positron emission tomography (PET) imaging tracers that permit visualisation of lung αvβ6 expression have potential as novel therapeutic and prognostic biomarkers in IPF.
Several αvβ6 integrin-binding cyclic peptides were synthesised using solid phase supported peptide synthesis and functionalised via their N-terminus or side chain amino group with a 6-amino- or a 6-azidohexanoic acid spacer to provide a convenient attachment site for either 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) or an 2-ethynyl-6-[18F]-fluoropyridine prosthetic group for radiolabelling with 68Ga or 18F, respectively. The αvβ6-binding capability of these peptide derivatives was assessed by competitive binding enzyme-linked immunosorbent assay (ELISA) and flow cytometry. Peptide derivatives that displayed strong affinity for αvβ6 were taken forward to “hot” cell surface binding experiments to evaluate their selectivity for the target. Stability of radiolabelled peptides was measured in human serum.
Competitive binding ELISA experiments (Figure 1) and flow cytometry experiments, showed that cRGD1 and cRGD4 were able to inhibit the binding of αvβ6-specific 10D5 mAb to αvβ6 with IC50 values of 6.6 nM and 1.6 nM, respectively. Labelling cRGD1 and cRGD4 with fluorine and gallium resulted in IC50 values of 12.2 nM and 1.2 nM for 19F-Pyr-cRGD1 and 68Ga-DOTA-cRGD4, respectively. Serum stability studies of 68Ga-DOTA-cRGD4 have shown that this tracer is >90% stable after 2 half-lives of 68Ga (136 min).
Radiolabelled cyclic RGD peptides have showed favourable binding and stability characteristics to warrant their investigation by PET imaging in vivo. A xenograft murine model using αvβ6+ and αvβ6- tumours is currently under development to validate tracer uptake and biodistribution in vivo.