^99mTc-labeled cystine knot peptide targeting integrin α_vβ₆ for tumor SPECT imaging

Integrin α_vβ₆ is overexpressed in a variety of cancers, and its expression is often associated with poor prognosis. Therefore, there is a need to develop affinity reagents for noninvasive imaging of integrin α_vβ₆ expression since it may provide early cancer diagnosis, more accurate prognosis, and better treatment planning. We recently engineered and validated highly stable cystine knot peptides that selectively bind integrin α_vβ₆ with no cross-reactivity to integrins α_vβ₅, α₅β₁, or α_vβ₃, also known to be overexpressed in many cancers. Here, we developed a single photon emission computed tomography (SPECT) probe for imaging integrin α_vβ₆ positive tumors. Cystine knot peptide, S₀2, was first conjugated with a single amino acid chelate (SAAC) and labeled with [^99mTc(H₂O)₃(CO) ₃]⁺. The resulting probe, ^99mTc-SAAC-S ₀2, was then evaluated by in vitro cell uptake studies using two α_vβ₆ positive cell lines (human lung adenocarcinoma cell line HCC4006 and pancreatic cancer cell line BxPC-3) and two α_vβ₆ negative cell lines (human lung adenocarcinoma cell line H838 and human embryonic kidney cell line 293T). Next, SPECT/CT and biodistribution studies were performed in nude mice bearing HCC4006 and H838 tumor xenografts to evaluate the in vivo performance of ^99mTc-SAAC-S₀2. Significant differences in the uptake of ^99mTc-SAAC-S₀2 were observed in α_vβ ₆ positive vs negative cells (P < 0.05). Biodistribution and small animal SPECT/CT studies revealed that ^99mTc-SAAC-S₀2 accumulated to moderate levels in antigen positive tumors (∼2% ID/g at 1 and 6 h postinjection, n = 3 or 4/group). Moreover, the probe demonstrated tumor-to-background tissue ratios of 6.81 ± 2.32 (tumor-to-muscle) and 1.63 ± 0.18 (tumor-to-blood) at 6 h postinjection in α _vβ₆ positive tumor xenografts. Co-incubation of the probe with excess amount of unlabeled S₀2 as a blocking agent demonstrated significantly reduced tumor uptake, which is consistent with specific binding to the target. Renal filtration was the main route of clearance. In conclusion, knottin peptides are excellent scaffolds for which to develop highly stable imaging probes for a variety of oncological targets. ^99mTc-SAAC-S₀2 demonstrates promise for use as a SPECT agent to image integrin α_vβ₆ expression in living systems.