⁶⁸Ga-labeling and in vivo evaluation of a uPAR binding DOTA- and NODAGA-conjugated peptide for PET imaging of invasive cancers

Abstract

Introduction

The urokinase-type plasminogen activator receptor (uPAR) is a well-established biomarker for tumor aggressiveness and metastatic potential. DOTA-AE105 and DOTA-AE105-NH₂ labeled with ⁶⁴Cu have previously been demonstrated to be able to noninvasively monitor uPAR expression using positron emission tomography (PET) in human cancer xenograft mice models. Here we introduce ⁶⁸Ga-DOTA-AE105-NH₂ and ⁶⁸Ga-NODAGA-AE105-NH₂ and evaluate their imaging properties using small-animal PET.

Methods

Synthesis of DOTA-AE105-NH₂ and NODAGA-AE105-NH₂ was based on solid-phase peptide synthesis protocols using the Fmoc strategy. ⁶⁸GaCl₃ was eluted from a ⁶⁸Ge/⁶⁸Ga generator. The eluate was either concentrated on a cation-exchange column or fractionated and used directly for labeling. For in vitro characterization of both tracers, partition coefficient, buffer and plasma stability, uPAR binding affinity and cell uptake were determined. To characterize the in vivo properties, dynamic microPET imaging was carried out in nude mice bearing human glioma U87MG tumor xenograft.

Results

In vitro experiments revealed uPAR binding affinities in the lower nM range for both conjugated peptides and identical to AE105. Labeling of DOTA-AE105-NH₂ and NODAGA-AE105-NH₂ with ⁶⁸Ga was done at 95°C and room temperature, respectively. The highest radiochemical yield and purity were obtained using fractionated elution, whereas a negative effect of acetone on labeling efficiency for NODAGA-AE105-NH₂ was observed. Good stability in phosphate-buffered saline and mouse plasma was observed. High cell uptake was found for both tracers in U87MG tumor cells. Dynamic microPET imaging demonstrated good tumor-to-background ratio for both tracers. Tumor uptake was 2.1% ID/g and 1.3% ID/g 30 min postinjection and 2.0% ID/g and 1.1% ID/g 60 min postinjection for ⁶⁸Ga-NODAGA-AE105-NH₂ and ⁶⁸Ga-DOTA-AE105-NH₂, respectively. A significantly higher tumor-to-muscle ratio (P<.05) was found for ⁶⁸Ga-NODAGA-AE105-NH₂ 60 min postinjection.

Conclusions

The use of ⁶⁸Ga-DOTA-AE105-NH₂ and ⁶⁸Ga-NODAGA-AE105-NH₂ as the first gallium-68 labeled uPAR radiotracers for noninvasive PET imaging is reported, which combine versatility with good imaging properties. These new tracers thus constitute an interesting alternative to the ⁶⁴Cu-labeled version (⁶⁴Cu-DOTA-AE105 and 64Cu-DOTA-AE105-NH₂) for detecting uPAR expression in tumor tissue. In our hands, the fractionated elution approach was superior for labeling of peptides, and ⁶⁸Ga-NODAGA-AE105-NH₂ is the favored tracer as it provides the highest tumor-to-background ratio.

Introduction

The urokinase-type plasminogen activator (uPA) and its receptor (uPAR) have been implicated in cancer as a marker for poor prognosis in a variety of human malignancies such as breast, colorectal and gastric cancer [1], [2], [3], [4]. uPAR expression is particularly abundant at the invasive front of tumors or in the surrounding stroma cells. uPAR is therefore generally recognized as a molecular marker for tumor invasion and metastatic disease and is therefore also considered an important target in cancer research [3]. The ability to visualize and quantify uPAR expression noninvasively in vivo is thus attractive from a clinical perspective [5], [6], [7], [8].

Based on an unbiased selection in a naive phage display library by cell lines expressing high levels of uPAR, a family of linear peptide antagonists of the uPA·uPAR interaction was developed after affinity maturation [9]. The resulting 9-mer lead peptide denoted AE105 [9] forms a tight 1:1 complex with purified human uPAR displaying a K_D of 0.4 nM with a k_off of 2×10⁻⁴ s⁻¹ as measured by surface plasmon resonance. AE105 is a potent competitive inhibitor of the uPA·uPAR interaction, displaying an IC₅₀ value of 11 nM in a purified system [9].

We have recently explored the use of this peptide for positron emission tomography (PET) imaging of uPAR expression [10], [11]. In both studies, DOTA was conjugated to the N-terminal of the targeting peptides (DOTA-AE105 and DOTA-AE105-NH₂), which were subsequently labeled with the long-lived PET isotope ⁶⁴Cu (T_1/2=12.7 hr, β⁺=17.8%) (⁶⁴Cu-DOTA-AE105) and investigated in a human cancer xenograft mice model. A quantitative correlation between uPAR expression and the tumor uptake of ⁶⁴Cu-DOTA-AE105-NH₂ in several different human xenograft in mice was recently reported [10], thus illustrating the ability to noninvasively detect uPAR expression in vivo. Because of the limited availability due to the cyclotron-dependent production of ⁶⁴Cu, the use in medical centers worldwide is complicated logistically. With the increased use of the ⁶⁸Ge/⁶⁸Ga generator during the last decade, the advancement of ⁶⁸Ga-based PET imaging agents has begun offering a very cost-effective alternative to the on-site cyclotron [12], [13]. ⁶⁸Ga has some promising physical characteristics (T_1/2=68 min, β⁺=89%) for imaging since the physical half-life more resembles the half-life of peptides in vivo and it has a higher positron abundance than ⁶⁴Cu.

Here we introduce the first ⁶⁸Ga-labeled peptides for PET imaging of uPAR. The amide form of the small linear peptide AE105 was conjugated with the macrocyclic chelators DOTA (DOTA-AE105-NH₂) and NODAGA [14] (NODAGA-AE105-NH₂) in the N-terminal. Both peptides were labeled with ⁶⁸GaCl₃ eluate after either a cation-exchange column purification step or a fractionation of the eluate [15] in order to compare the two different approaches. Finally, the in vitro uPAR binding properties and stability were investigated together with dynamic in vivo PET imaging in nude mice bearing tumor xenograft of the uPAR-positive human glioblastoma cell line U87MG [10].