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 α_vβ₆ 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 α_vβ₆ expression have potential as novel therapeutic and prognostic biomarkers in IPF.

Several α_vβ₆ 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 α_vβ₆-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 α_vβ₆ 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 α_vβ₆-specific 10D5 mAb to α_vβ₆ 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 α_vβ₆⁺ and α_vβ₆^- tumours is currently under development to validate tracer uptake and biodistribution in vivo.

• Download figure
• Open in new tab
• Download powerpoint

Abstract P40 Figure 1

α_vβ_e competitive binding ELISA between labelled/non-labelled cyclic peptides and the α_vβ_e –specific mAb (clone10D5)