Evaluation of a (64)Cu-labeled cystine-knot peptide based on agouti-related protein for PET of tumors expressing alphavbeta3 integrin

Abstract

Recently, a truncated form of the agouti-related protein (AgRP), a 4-kDa cystine-knot peptide of human origin, was used as a scaffold to engineer mutants that bound to alpha(v)beta(3) integrin with high affinity and specificity. In this study, we evaluated the potential of engineered integrin-binding AgRP peptides for use as cancer imaging agents in living subjects.

Methods: Engineered AgRP peptides were prepared by solid-phase peptide synthesis and were folded in vitro and purified by reversed-phase high-performance liquid chromatography. Competition assays were used to measure the relative binding affinities of engineered AgRP peptides for integrin receptors expressed on the surface of U87MG glioblastoma cells. The highest-affinity mutant, AgRP clone 7C, was site-specifically conjugated with 1,4,7,10-tetra-azacyclododecane-N,N',N''N'''-tetraacetic acid (DOTA). The resulting bioconjugate, DOTA-AgRP-7C, was radiolabeled with (64)Cu for biodistribution analysis and small-animal PET studies in mice bearing U87MG tumor xenografts. In addition to serum stability, the in vivo metabolic stability of (64)Cu-DOTA-AgRP-7C was assessed after injection and probe recovery from mouse kidney, liver, tumor, and urine.

Results: AgRP-7C and DOTA-AgRP-7C bound with high affinity to integrin receptors expressed on U87MG cells (half maximal inhibitory concentration values, 20 +/- 4 and 14 +/- 2 nM, respectively). DOTA-AgRP-7C was labeled with (64)Cu with high radiochemical purity (>99%). In biodistribution and small-animal PET studies, (64)Cu-DOTA-AgRP-7C displayed rapid blood clearance, good tumor uptake and retention (2.70 +/- 0.93 percentage injected dose per gram [%ID/g] and 2.37 +/- 1.04 %ID/g at 2 and 24 h, respectively), and high tumor-to-background tissue ratios. The integrin-binding specificity of (64)Cu-DOTA-AgRP-7C was confirmed in vitro and in vivo by showing that a large molar excess of the unlabeled peptidomimetic c(RGDyK) could block probe binding and tumor uptake. Serum stability and in vivo metabolite assays demonstrated that engineered AgRP peptides are sufficiently stable for in vivo molecular imaging applications.

Conclusion: A radiolabeled version of the engineered AgRP peptide 7C showed promise as a PET agent for tumors that express the alpha(v)beta(3) integrin. Collectively, these results validate AgRP-based cystine-knot peptides for use in vivo as molecular imaging agents and provide support for the general use of AgRP as a scaffold to develop targeting peptides, and hence diagnostics, against other tumor receptors.

FIGURE 1

U87MG competition binding assay. Varying concentrations of unlabeled AgRP peptides were incubated with ¹²⁵I-echistatin and allowed to compete for binding to integrin receptors expressed on surface of U87MG glioblastoma cells. Fraction of ¹²⁵I-echistatin bound to cell surface is plotted vs. concentration of unlabeled AgRP-FN (●, 1,400 ± 700 nM), AgRP-3F (▲, 900 ± 300 nM), AgRP-6E (▼, 130 ± 20 nM), AgRP-6F (■, 410 ± 90 nM), AgRP-7C (◆, 20 ± 4 nM), DOTA-AgRP-7C (◇, 14 ± 2 nM), and echistatin (○, 3.3 ± 0.6 nM). IC₅₀ values are reported for each peptide in parentheses. Data represent mean values of binding experiments performed on 3 separate days, and error bars represent SD.

FIGURE 2

In vitro cell binding and uptake assay. U87MG cells were incubated with 111 kBq (3 μCi) of ⁶⁴Cu-DOTA-AgRP-7C for various time points at 4°C or 37°C, with or without addition of 2 μg of c(RGDyK) integrin–blocking peptide. Data represents mean percentage of total radioactivity added, measured in triplicate, and error bars represent SD.

FIGURE 3

Small-animal PET and quantification. (A) Representative coronal and transverse small-animal PET images of U87MG tumor–bearing mice at 0.5, 1, 2, 4, and 24 h after injection of 2.22 MBq (≈60 μCi) of ⁶⁴Cu-DOTAAgRP-7C. Small-animal PET images acquired at 1 and 2 h after coinjection of 330 μg of c(RGDyK) (right). (B) Quantification analysis of radioactivity accumulation in selected organs at different time points after injection of ⁶⁴Cu-DOTA-AgRP-7C, reported as %ID/g. (C) Quantification analysis of radioactivity accumulation of probe in tumor with or without coinjection of c(RGDyK) at 1 and 2 h after injection.

FIGURE 4

Dynamic small-animal PET scans. Time–activity plots of major organs in a U87MG tumor–bearing mouse after intravenous injection of ≈ 3.7 MBq (≈100 μCi) of ⁶⁴Cu-DOTA-AgRP-7C.