Comparison of the RGD Motif–Containing α_vβ₆Integrin–Binding Peptides SFLAP3 and SFITGv6 for Diagnostic Application in HNSCC

Abstract

αβ integrin is overexpressed by several carcinomas and thus considered a target for diagnostic imaging and anticancer therapies. Recently, we presented the αβ integrin-binding peptide SFITGv6 as a novel potential tracer for imaging and targeted therapy of αβ integrin-positive carcinomas. Here, we analyzed the affinity and specificity of 5 native αβ integrin-specific binders in comparison to SFITGv6. Sunflower trypsin inhibitor 1 (SFTI1)-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 αβ 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 assay, and binding experiments including competition, kinetics, internalization, and efflux. αβ integrin binding specificity was further evaluated by peptide histochemistry. Finally, in vivo binding properties were assessed using small-animal PET imaging and biodistribution experiments in HNSCC-bearing mice, andGa-DOTA-SFLAP3 was applied for diagnostic PET/CT of an HNSCC patient. When the newly designed peptides were compared, significant binding (>20%) to several HNSCC cell lines (HNO97, HNO399, and HNO223) and a fast internalization of up to 60% and 70% were observed for SFLAP3 (GRGDLGRL) and SFITGv6 (FRGDLMQL). In contrast, the other peptides displayed binding that was moderate (SFLAP1, 4.1%-12.1%) to marginal (SFFN1, SFTNC, and SFVTN, <1%) and were therefore excluded from further analysis. Notably, SFLAP3 exhibited improved affinity for αβ integrin (mean half-maximal inhibitory concentration, 3.5 nM; dissociation constant, 7.4). Moreover, small-animal PET imaging and biodistribution studies of HNSCC xenograft mice revealed an increased tumor-specific accumulation 30-60 min after injection of Ga-labeled orLu-labeled DOTA-SFLAP3. Peptide staining further demonstrated binding specificity for SFLAP3 to HNSCC tumor cells. Finally, PET/CT scanning of an HNSCC patient showed specific SFLAP3 accumulation in the primary tumor lesion (SUV, 5.1) and in corresponding lymph node metastases (SUV, 4.1). SFLAP3 represents a promising tracer for prognostic assessment, diagnostic imaging, and possibly targeted therapy of αβ integrin-expressing tumors.

Preclinical evaluation of peptide-based radiotracers for integrin αvβ6-positive pancreatic carcinoma

“…A strong correlation of the cellular SFLAP3 binding and the integrin αvβ6 expression (flow cytometry) on the different tumor cell lines was detectable. The measured K D of SFLAP3 with 7.4 nM to integrin αvβ6 using SPR spectroscopy analysis is comparable to the measured IC 50 on Capan-2 cells with 6.2 nM [21]. The specificity of the ligand for integrin αvβ6 was also ensured by SPR analysis [21].…”

“…In previous experiments SFITGV6 and its improved variant SFLAP3 showed excellent binding affinities to αvβ6 positive tumors, especially to head and neck cancer [20,21]. From the pancreatic cancer cell lines studied in this work, Capan-2 revealed the highest values in the flow cytometry analysis as well as the highest re-▶Table 1 SUV (max) values of the metastases (patient in ▶ Fig.…”

“…For small animal PET imaging 68 Ga-labeled peptides (approximately 25 GBq/µmol) were injected into the tail vein of an anesthetized (3-4 % isoflurane) animal. Images were acquired on an Inveon small animal PET scanner (Siemens) for 140 min as described [20,21].…”

“…In previous studies, we identified by phage display a peptidic lead structure embedded in the sunflower trypsin inhibitor scaffold [20]. The binding loop of this structure has been modified by exchange of three amino acids to yield the binding sequence of SFLAP3 [21]. Both peptides have been applied in preclinical models of head and neck tumors and in patients with lung and head and neck cancer.…”

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

“…The high contrast in signal gained due to aberrant, but specific expression of a v b 6 suggests that a v b 6targeted agents have significant potential in the treatment and management of a multitude of malignancies. This has led to the development of several preclinical PET and single photon emission computed tomography (SPECT) radiotracers and, most recently, clinical reports in patients with head and neck squamous cell carcinoma and NSCLC (34).…”