Radiolabeled NGR-Based Heterodimers for Angiogenesis Imaging: A Review of Preclinical Studies

Abstract

Since angiogenesis/neoangiogenesis has a major role in tumor development, progression and metastatic spread, the establishment of angiogenesis-targeting imaging and therapeutic vectors is of utmost significance. Aminopeptidase N (APN/CD13) is a pivotal biomarker of angiogenic processes abundantly expressed on the cell surface of active vascular endothelial and various neoplastic cells, constituting a valuable target for cancer diagnostics and therapy. Since the asparagine-glycine-arginine (NGR) sequence has been shown to colocalize with APN/CD13, the research interest in NGR-peptide-mediated vascular targeting is steadily growing. Earlier preclinical experiments have already demonstrated the imaging and therapeutic feasibility of NGR-based probes labeled with different positron emission tomography (PET) and single-photon emission computed tomography (SPECT) radionuclides, including Gallium-68 (⁶⁸Ga), Copper-64 (⁶⁴Cu), Technetium-99m (^99mTc), Lutetium-177 (¹⁷⁷Lu), Rhenium-188 (¹⁸⁸Re) or Bismuth-213 (²¹³Bi). To improve the tumor binding affinity and the retention time of single-receptor targeting peptides, NGR motifs containing heterodimers have been introduced to identify multi-receptor overexpressing malignancies. Preclinical studies with various tumor-bearing experimental animals provide useful tools for the investigation of the in vivo imaging behavior of NGR-based heterobivalent ligands. Herein, we review the reported preclinical achievements on NGR heterodimers that could be highly relevant for the development of further target-specific multivalent compounds in diagnostic and therapeutic settings.

Keywords: Aminopeptidase N (APN/CD13); angiogenesis; asparagine–glycine–arginine (NGR); heterodimer; preclinical; radiolabeled.

Figure 1

Schematic representation of the structure of target-specific peptide-based radiopharmaceuticals. This figure demonstrates the linkage of the targeting moiety and the radioisotope to form a radiopharmaceutical that specifically binds to a cancer-related target molecule. A radiopharmaceutical consists of a target-specific, biologically active binding molecule (e.g., NGR tripeptide, RGD) attached via a chelator/linker to a labeling radioisotope. The radioactive substances—radiotracers—selectively target biomarkers expressed on the surface of the tumor cells (e.g., APN/CD13, α_νβ₃ integrin). In diagnostic settings, a radionuclide with β⁺ or γ emission is bound to the peptide-chelator core, which allows for the in vivo assessment of biological processes at the molecular level using PET or SPCET techniques. On the other hand, radiolabeling with γ- or β-emitting therapeutic isotopes makes selective radiotherapy and tumor killing possible. The use of target-specific peptides as carriers of cytotoxic or proapoptotic drugs (e.g., tTF, VEGI, CLB) and tumor-affine molecules such as HA seems to be successful in the enhancement of their tumor-homing potential and cytotoxic effect.

Figure 2

Schematic overview of NGR-based heterodimers targeting angiogenesis in the tumor microenvironment.

Figure 3

PET/CT imaging and quantitative assessment of [⁶⁸Ga]Ga-NGR-RGD uptake in preclinical mouse models of breast and ovarian tumors. (A) Representative PET/CT images of MCF-7, MDA-MB-231, MDA-MB-468, and MX-1 breast cancer-bearing female BALB/c nude mice 30 min after the intravenous injection of 6.5–9 MBq of [⁶⁸Ga]Ga-NGR-RGD. The white arrows point to the tumors. (B) Quantitative assessment of [⁶⁸Ga]Ga-NGR-RGD uptake in MCF-7, MDA-MB-231, MDA-MB-468, and MX-1 tumors. The uptake values are obtained as mean %ID/g ± SD. (C) Representative PET/CT images of SKOV-3 and ES-2 ovarian tumor-bearing female BALB/c nude mice 1 h after the intravenous injection of 4–5.5 MBq of [⁶⁸Ga]Ga-NGR-RGD. The white arrows demonstrate the localization of the subcutaneous tumors. (D) Quantitative evaluation of [⁶⁸Ga]Ga-NGR-RGD accumulation in SKOV-3 and ES-2 xenografts. The results are expressed as %ID/g ± SD. ** p < 0.01, *** p < 0.001, NS: non significant. (A,B) With permission from [37]. (C,D) With permission from [83].