99mTc-Labeled cystine-knot peptide for integrin αvβ6 targeted tumor imaging

Objectives It is highly important to develop agents for non-invasive imaging integrin αvβ6 expression since it may provide a technique for cancer early diagnosis, prognosis and treatment planning. We recently discovered and engineered highly stable cystine knot peptides with high integrin αvβ6-binding specificity. In this study, we aimed to develop a single photon emission computed tomography (SPECT) probe for imaging integrin αvβ6.

Methods An engineered cystine knot peptide, S02, was conjugated with a Single Amino Acid Chelate (SAAC) and then labeled with [99mTc(H2O)3(CO)3]+. The resulting probe, 99mTc(CO)3-SAAC-S02, was first evaluated by in vitro cell uptake studies using two αvβ6-positive (HCC4006 and BxPC-3) and negative cell lines (H838 and 293T), and SPECT/CT and biodistribution studies in nude mice bearing HCC4006 and H838 tumor xenografts were then performed to investigate the in vivo performance of the probe.

Results There were significant differences in uptake of 99mTc(CO)3-SAAC-S02 over time between αvβ6 positive and negative cells (P < 0.05). High accumulations were found in both HCC4006 and BxPC-3 cells, and the uptakes were markedly reduced by co-incubation with excess amount of unlabeled S02 as a blocking agent. Biodistribution and small-animal SPECT/CT studies revealed that 99mTc(CO)3-SAAC-S02 was mainly excreted through the kidneys, and it also displayed good tumor uptake and retention (2.02 ± 0.44%ID/g and 2.09 ± 0.55%ID/g at 1 and 6 h post-injection, respectively, n=4/group), high tumor-to-background tissue ratios (for example: tumor-to-muscle and tumor-to-blood ratio are 6.81±2.32 and 1.63±0.18 at 6 h post-injection, respectively, n=4/group) in αvβ6 positive human lung adenocarcinoma HC4006 tumor xeongrafts.

Conclusions Knottin peptide is an excellent platform to develop imaging probes for a variety of targets including integrin αvβ6. 99mTc(CO)3-SAAC-S02 displays promise properties as a SPECT agent for imaging integrin αvβ6 positive tumors.

Research Support This work was partially supported by DOE Stanford Molecular Imaging Research and Training Program (SMIRTP) and NCI CA128908, and the National Natural Science Foundation of China (NSFC) Program (No. 86271600).