Identification of a Novel ITGα_vβ₆-Binding Peptide Using Protein Separation and Phage Display.

Abstract

<b>Purpose:</b> Targeted therapies are regarded as promising approaches to increase 5-year survival rate of head and neck squamous cell carcinoma (HNSCC) patients.<b>Experimental design:</b> For the selection of carcinoma-specific peptides membrane proteome of HNO97 tumor cells fractionated by the ProteomeLab PF2D system and corresponding HNO97 cells were deployed for an alternating biopanning using a sunflower trypsin inhibitor1-based phage display (SFTI8Ph) library. Stability, binding properties and affinity of novel candidates were assessed <i>in vitro</i> using radio-HPLC, binding experiments and surface plasmon resonance assay (SPR), respectively. Subsequently, the affinity of the peptide was verified <i>in situ</i> by using peptide histochemistry, <i>in vitro</i> using flow cytometry, and <i>in vivo</i> by positron emissions tomography (PET/CT).<b>Results:</b> We identified a novel ITGα_vβ₆ binding peptide (SFITGv6) containing the amino acid sequence FRGDLMQL. SFITGv6 provides stability over a period of 24 hours and demonstrates high affinity (<i>K</i>_D = 14.8 nmol/L) for ITGα_vβ₆ In HNO97 cells, a maximal uptake and internalization of up to 37.3% and 37.5%, respectively, was measured. Small-animal PET imaging and biodistribution studies of HNO97 xenografted Balb/c nu/nu mice showed tumor-specific accumulation of ⁶⁸Ga- and ¹⁷⁷Lu-labeled DOTA-SFITGv6, respectively, 30 to 60 minutes after injection. Moreover, peptide histochemistry revealed a strong and homogenous binding of biotin-labeled SFITGv6 to HNSCC tumors and breast- and lung cancer-derived brain metastases. Finally, first PET/CT scans of HNSCC and NSCLC patients displayed SFITGv6 accumulation specifically in tumors, but not in inflammatory lesions.<b>Conclusions:</b> Thus, SFITGv6 represents a novel powerful tracer for imaging and possibly for endoradiotherapy of ITGα_vβ₆-positive carcinoma. <i>Clin Cancer Res; 23(15); 4170-80. ©2017 AACR</i>.