Comparative Study of the Novel RGD Motif-containing αvβ6 Integrin Binding Peptides SFLAP3 and SFITGv6 for Diagnostic Application in HNSCC

Alpha v beta 6 (αvβ6) integrin is overexpressed by several carcinomas and thus considered as target for diagnostic imaging and anti-cancer therapies. Recently, we presented SFITGv6 as a novel potential tracer for imaging and targeted therapy of αvβ6 integin-positive carcinomas. Here, we analyzed affinity and specificity of five native αvβ6 integrin-specific binders in comparison to SFITGv6. Methods: Sunflower trypsin inhibitor (SFTI)-1-based peptides containing arginine-glycine-aspartic acid (RGD) motif-spanning octamers of fibronectin (SFFN1), tenascin C (SFTNC), vitronectin (SFVTN), and latency-associated peptides (LAP) 1 (SFLAP1), and 3 (SFLAP3) were synthesized and their binding potential to αvβ6 integrin-expressing head and neck squamous cell carcinoma (HNSCC) cell lines was evaluated. Subsequently, stability, affinity and specificity were assessed in vitro using radio-high pressure liquid chromatography, surface plasmon resonance (SPR) assay, and binding experiments including competition, kinetics, internalization, and efflux. αvβ6 integrin binding specificity was further evaluated by peptide histochemistry. Finally, in vivo binding properties were assessed using small-animal positron emission tomography (PET) imaging and biodistribution experiments in HNSCC-bearing mice and 68 Ga-DOTA-SFLAP3 was applied for diagnostic positron emission tomography/computed tomography (PET/CT) of a HNSCC patient. Results: Comparing the newly designed peptides significant binding (>20%) to several HNSCC cell lines (HNO97, HNO399, and HNO223) and a fast internalization of up to 60% and 70% was observed for SFLAP3 (G RGD LGRL) and SFITGv6 (F RGD LMQL). In contrast, the other peptides demonstrated binding specificity of SFLAP3 to HNSCC tumor cells. Finally, PET/CT scan of an HNSCC patient showed specific SFLAP3 accumulation in the primary tumor lesion (standardized uptake values (SUV)max = 5.1) and in a corresponding lymph node metastases (SUVmax = 4.1). Conclusion: SFLAP3 represents a promising tracer for prognostic assessment, diagnostic imaging and possibly targeted therapy of αvβ6 integrin-expressing tumors.