68Ga-labeling and in vivo evaluation of a uPAR binding DOTA- and NODAGA-conjugated peptide for PET imaging of invasive cancers
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 KD of 0.4 nM with a koff 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 (T1/2=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 (T1/2=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].
Section snippets
Chemical and biological reagents
All commercial chemicals were of analytical grade. They were all used without further purification. 2-(4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclo-dodecan-1-yl)-acetic acid(DOTA-tris(tBu)ester and 4-(4,7-bis(2-(tert-butoxy)-2-oxoethyl)-1,4,7-triazacyclononan-1-yl)-5-(tert-butoxy)-5-oxopentanoic acid [NODAGA-tris(tBu)ester] were purchased from CheMatech (Dijon, France). 68GaCl3 was eluted in 0.1 M HCl obtained from a 68Ge/Ga68 generator (Eckert & Ziegler) at the Department of
Affinity of the DOTA- and NODAGA-conjugated peptides
The interactions of the peptide AE105-NH2, together with the chelator-conjugated analogue NODAGA-AE105-NH2, with immobilized human uPAR in solution were then measured in real time by surface plasmon resonance (Fig. 2). No reduction in the efficacy to compete the uPA·uPAR interaction was found between AE105-NH2 and the conjugated versions (AE105-NH2: IC50=7.6±2.0 nM, DOTA-AE105-NH2: IC50=6.7±0.9 nM [10] and NODAGA-AE105-NH2: IC50=3.4±0.4 nM), indicating that neither DOTA nor NODAGA conjugation
Discussion
In this study, the radiolabeling and feasibility of using 68Ga-labeled peptides as PET tracers for imaging of uPAR expression are reported as an interesting alternative to cyclotron-dependent radionuclides. The use of 68Ga-labeled peptides for cancer imaging has recently attracted considerable interest since 68Ga can be easily obtained from a 68Ge/68Ga generator. 68Ga also possess a medium half-life of 68 min, which is optimal compared to the pharmacokinetics of many synthetic peptides.
The
Acknowledgments
This work was supported by The Danish National Research Foundation (Centre for Proteases and Cancer), Danish Medical Research Council, the Danish National Advanced Technology Foundation, the Novo Nordisk Foundation, the Lundbeck Foundation, Svend Andersen Foundation, Research Foundation of Rigshospitalet, and the A.P. Moeller Foundation.
The authors declare that they have no conflict of interest.
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