I111n-Labeled Cystine-Knot Peptides Based on the Agouti-Related Protein for Targeting Tumor Angiogenesis

Abstract EN

Agouti-related protein (AgRP) is a 4-kDa cystine-knot peptide of human origin with four disulfide bonds and four solvent-exposed loops.

The cell adhesion receptor integrin αvβ3 is an important tumor angiogenesis factor that determines the invasiveness and metastatic ability of many malignant tumors.

AgRP mutants have been engineered to bind to integrin αvβ3 with high affinity and specificity using directed evolution.

Here, AgRP mutants 7C and 6E were radiolabeled with 111In and evaluated for in vivo targeting of tumor integrin αvβ3 receptors.

AgRP peptides were conjugated to the metal chelator 1, 4, 7, 10-tetra-azacyclododecane- N, N′, N″, N‴-tetraacetic acid (DOTA) and radiolabeled with 111In.

The stability of the radiopeptides 111In-DOTA-AgRP-7C and 111In-DOTA-AgRP-6E was tested in phosphate-buffered saline (PBS) and mouse serum, respectively.

Cell uptake assays of the radiolabeled peptides were performed in U87MG cell lines.

Biodistribution studies were performed to evaluate the in vivo performance of the two resulting probes using mice bearing integrin-expressing U87MG xenograft tumors.

Both AgRP peptides were easily labeled with 111In in high yield and radiochemical purity (>99%).

The two probes exhibited high stability in phosphate-buffered saline and mouse serum.

Compared with 111In-DOTA-AgRP-6E, 111In-DOTA-AgRP-7C showed increased U87MG tumor uptake and longer tumor retention (5.74±1.60 and 1.29±0.02%ID/g at 0.5 and 24 h, resp.), which was consistent with measurements of cell uptake.

Moreover, the tumor uptake of 111In-DOTA-AgRP-7C was specifically inhibited by coinjection with an excess of the integrin-binding peptidomimetic c(RGDyK).

Thus, 111In-DOTA-AgRP-7C is a promising probe for targeting integrin αvβ3 positive tumors in living subjects.