Identification of a Novel ITGαvβ6-Binding Peptide Using Protein Separation and Phage Display

Abstract

Targeted therapies are regarded as promising approaches to increase 5-year survival rate of head and neck squamous cell carcinoma (HNSCC) patients. 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 using radio-HPLC, binding experiments and surface plasmon resonance assay (SPR), respectively. Subsequently, the affinity of the peptide was verified by using peptide histochemistry, using flow cytometry, and by positron emissions tomography (PET/CT). We identified a novel ITGαβ binding peptide (SFITGv6) containing the amino acid sequence FRGDLMQL. SFITGv6 provides stability over a period of 24 hours and demonstrates high affinity ( = 14.8 nmol/L) for ITGαβ 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 Ga- andLu-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. Thus, SFITGv6 represents a novel powerful tracer for imaging and possibly for endoradiotherapy of ITGαβ-positive carcinoma. .

Preclinical Development and First-in-Human Imaging of the Integrin αvβ6 with [18F]αvβ6-Binding Peptide in Metastatic Carcinoma

“…In vivo studies using the paired DX3purob6/DX3puro cell xenograft mouse model confirmed that [ 18 F]a v b 6 -BP met expectations for a v b 6 targeting and overall pharmacokinetics, with good tumor retention and kidney clearance. The tumor uptake values were similar to those noted by Altmann and colleagues for their SFITGv6 peptide which demonstrated good binding in mice bearing HNO97 xenografts (37).…”

“…In vitro affinity and selectivity of [ 18 F]a v b 6 -BP for integrin a v b 6 were excellent and compared favorably with those reported by Altmann and colleagues for their SFTI-1 derivatives; however, it is noted that the experimental methods were somewhat different, and different cells lines with varying expression levels of the integrin a v b 6 were used (37). In vivo studies using the paired DX3purob6/DX3puro cell xenograft mouse model confirmed that [ 18 F]a v b 6 -BP met expectations for a v b 6 targeting and overall pharmacokinetics, with good tumor retention and kidney clearance.…”

Identification of Ligands and Translation to Clinical Applications

“…This approach has been used in various applications, such as in the mapping and mimicking of epitopes, the identification of new receptors and natural ligands, high-affinity antibodies and analogs, the isolation of specific antigens binding to bioactive compounds, the production of novel enzyme inhibitors and DNA-binding proteins, and the probing of cellular and tissue-specific processes. Phage display has been successfully applied for the identification of peptides with a high specificity for target tumor neovasculature or a variety of human tumor cells (16,(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33).…”

“…When libraries based on the trypsin inhibitors sunflower trypsin inhibitor and Min23 were applied, peptides were identified by phage display as well as ribosome display for Dll4 and a v b 6 -integrin (31)(32)(33). The Dll4 peptides showed a high affinity, in the range of 4-40 nM, and a serum stability of up to 10 d. When a membrane proteome of HNO97 tumor cells (head and neck squamous cell carcinoma) fractionated by the ProteomeLab PF2D system (Beckman Coulter) and corresponding HNO97 cells for phage display with a sunflower trypsin inhibitor library were used, a novel a v b 6 -integrin-binding peptide (SFITGv6) with stability over a period of 24 h, a high affinity (diffusion constant, 14.8 nM) for a v b 6 -integrin, and an internalization ratio of 37.5% was identified (33).…”

“…The Dll4 peptides showed a high affinity, in the range of 4-40 nM, and a serum stability of up to 10 d. When a membrane proteome of HNO97 tumor cells (head and neck squamous cell carcinoma) fractionated by the ProteomeLab PF2D system (Beckman Coulter) and corresponding HNO97 cells for phage display with a sunflower trypsin inhibitor library were used, a novel a v b 6 -integrin-binding peptide (SFITGv6) with stability over a period of 24 h, a high affinity (diffusion constant, 14.8 nM) for a v b 6 -integrin, and an internalization ratio of 37.5% was identified (33). Small-animal PET imaging and biodistribution studies of mice with HNO97 xenografts showed tumor-specific accumulation of 68 Ga-and 177 Lu-labeled DOTA-SFITGv6.…”