Radiolabeled probes for imaging of tumor angiogenesis

Introduction

Angiogenesis, the formation of new blood vessels from existing ones, is an essential process if solid tumors are to grow beyond 2 to 3 mm3, since diffusion is no longer sufficient to supply the tissue with oxygen and nutrients. Tumor-induced angiogenesis is a complex multistep process that follows a characteristic cascade of events mediated and controlled by growth factors, cellular receptors and adhesion molecules. Based on a balance between pro-angiogenic and anti-angiogenic factors, a tumor can stay dormant for a very long time period until the so-called “angiogenic switch” occurs. In most tissues tumors can only grow to a life threatening size if the tumor is able to trigger angiogenesis. In tissues with high vessel densities (e.g. liver, brain, among others), tumors may also progress via angiogenesis-independent

co-option of the pre-existent vasculature.

Inhibition of angiogenesis is a new cancer treatment strategy that is now widely investigated clinically. Researchers have begun to search for objective measures that indicate pharmacological responses to anti-angiogenic drugs. Therefore there is a great interest in techniques to visualize angiogenesis in growing tumors noninvasively. During the past decade several markers of angiogenesis have been identified and specific tracers targeting these markers have been developed.

The αvβ3 integrin receptor

The αvβ3 integrin is a transmembrane protein consisting of two non-covalently bound subunits, α and β. Integrin αvβ3 is minimally expressed on normal quiescent endothelial cells, but significantly upregulated on activated endothelial cells during angiogenesis. In addition, αvβ3 is expressed on the cell membrane of various tumor cell types such as: ovarian cancer, neuroblastoma, breast cancer, melanoma, among others.

This integrin interacts with the arginine-glycine-aspartic acid (RGD) amino acid sequence present in extracellular matrix proteins such as vitronectin, fibrinogen, and laminin.

Several research groups have investigated the potential of RGD-containing peptides to target with gamma- or positron-emitting radionuclides αvβ3 expressed in tumors [Table 1]. Over the past decade, many radiolabeled cyclic RGD peptides have been evaluated as potential radiotracers for noninvasive imaging of integrin αvβ3-positive tumors by SPECT or PET. After a systematic search, the group in Munich developed a stable 18F-labeled galactosylated cyclic pentapeptide ([18F]Galacto-RGD), with a high affinity and selectivity for αvβ3 that accumulates specifically in αvβ3-positive tumors and clears rapidly via the kidneys. [18F]Galacto-RGD and [18F]-AH111585 (of which the core peptide sequence was originally discovered from a phage display library as ACDRGDCFCG), are currently under clinical investigation. Recently, a 99mTc-labeled RGD-containing peptide (NC100692) was evaluated in ischemic models and showed high uptake in areas of neovascularization with αvβ3 integrin overexpression. In these models it was shown that NC100692 bund to αvβ3-expressing endothelial cells in the regions of angiogenesis.

Clinical studies

[18F]Galacto-RGD was the first radiotracer applied in patients and could successfully image αvβ3 expression in human tumors with good tumor-to-background ratios. It has been shown that molecular imaging in humans of αvβ3 expression using [18F]Galacto-RGD correlated with αvβ3 expression as determined by immunohistochemistry. In another study, the tracer uptake of [18F]FDG was compared with that of [18F]Galacto-RGD in patients with non-small cell lung cancer (NSCLC, n=10) and various other tumors (n=8). It was found that [18F]FDG uptake in tumor lesions did not correlate with [18F]Galacto-RGD uptake. These results showed that αvβ3 expression and glucose metabolism are not closely correlated in tumor lesions and consequently [18F]FDG cannot provide similar information as [18F]Galacto-RGD [Figure 1].

The second radiotracer which was applied in patients was 99mTc-NC100692. A clinical study was performed to provide an initial indication of the efficacy and safety of imaging malignant breast tumors. Nineteen out of 22 tumors were detected with this radiotracer. In an additional study, integrin scintimammography with 99mTc-NC100692 using a dedicated γ-camera was performed to investigate the ability to detect malignant breast cancer lesions. All patients were known to have lesions highly suspicious of malignancy. Dedicated integrin scintimammography (DISM) detected malignant lesions in seven out of eight patients with focal uptake in all but two tumor lesions. In a subsequent open-label, multicenter, phase 2a study in late-stage cancer patients, 99mTc-NC100692 was able to detect lung and brain metastases from breast and lung cancer with scintigraphy.

Multimeric RGD peptides

To improve the efficiency of tumor targeting and to obtain better in vivo imaging properties, multimeric RGD peptides were synthesized and characterized. The first cyclic RGD multimers that were developed, were E[c(RGDfK)]2-based dimers. Subsequently, the use of E[c(RGDyK)]2-based dimers labeled with 64Cu or 18F for PET imaging was reported.

During the last years, various other RGD dimers, tetramers, and even octamers labeled with different radionuclides have been developed and studied in vitro and in vivo. Generally, the results of these studies have demonstrated that increasing the multiplicity of the peptide can significantly enhance the integrin αvβ3-binding affinity of RGD peptides and improve tumor targeting capability of the radiotracer. In addition, incorporation of the right spacer between the RGD motifs can enhance the affinity for αvβ3 and improve the tumor uptake even further. Among mono-, di-, tetra- and octameric cyclo(RGDfK)-based peptides, the octamer had the highest αvβ3 affinity and usually the highest tumor uptake. From this point of view, further increase of RGD peptide multiplicity may result in formation of oligomeric or polymeric cyclic RGD peptides with improved integrin αvβ3-binding affinity and tumor targeting efficacy. So far, no radiolabeled multimeric RGD peptide have been tested in patients. The studies on multimeric RGD peptides have recently been reviewed [1].

VEGF receptors

Vascular endothelial growth factor (VEGF) is a key regulator of angiogenesis during embryogenesis, skeletal growth and reproductive functions. The expression of  VEGF is upregulated by environmental stress caused by hypoxia, anemia, myocardial ischemia and tumor progression to initiate neovascularization. Via alternative mRNA splicing, the human VEGF-A gene gives rise to four isoforms having 121, 165, 189 and 206 amino acids (VEGF121, VEGF165, VEGF189 and VEGF206, respectively).

VEGF binds two related receptor tyrosine kinases (RTKs), VEGFR-1 and VEGFR-2. Both receptors consist of seven Ig-like domains in the extracellular domain, a single transmembrane region and a consensus tyrosine kinase sequence that is interrupted by a kinase-insert domain. VEGFR-1 binds VEGF with a higher affinity compared to VEGFR-2 (Kd : 25 vs. 75-250 pM).

Bevacizumab is a humanized variant of the anti-VEGF-A monoclonal antibody (mAb) A.4.6.1. Nagengast et al were the first to demonstrate non-invasive VEGF imaging using radiolabeled bevacizumab [2]. In their study, they demonstrated the potential of 89Zr-bevacizumab and 111In-bevacizumab as a specific VEGF tracer in nude mice with human SKOV-3 ovarian tumor xenografts. At the same time, our group showed specific imaging of VEGF-A expression using 111In-bevacizumab in mice with s.c. human colon carcinoma xenografts LS174T [Figure 2]. Recently, the potential of 111In-labeled bevacizumab to image the expression of VEGF-A in tumors was investigated in cancer patients. In a study in colorectal cancer patients with liver metastases, the liver metastases in nine out of 12 patients were visualized with 111In-bevacizumab. In this study, the liver metastases were resected after scintigraphic imaging allowing further immunohistochemical analysis. The VEGF-A expression in these resected liver metastases was determined by in situ hybridization and by ELISA. Surprisingly, no correlation was found between the level of antibody accumulation and expression of VEGF-A.

Cai et al. labeled VEGF121 with 64Cu via DOTA for PET imaging of VEGFR expression [3]. Small-animal PET imaging revealed rapid, specific and prominent uptake of 64Cu-DOTA-VEGF121 in highly vascularized small U87MG human glioblastoma tumors (high VEGFR expression), and significantly lower uptake in large U87MG tumors (low VEGFR expression).

Conclusions

Numerous markers of tumor vasculature have been identified, but only a few radiotracers of angiogenesis have been tested clinically. The most extensively studied marker of angiogenesis is the integrin αvβ3. For this marker the SPECT-tracer, 99mTc-NC100692, and the PET-tracer 18F-galacto-RGD have been successfully tested in cancer patients.

Other targets exclusively expressed on activated endothelial cells may eventually be better targets for imaging angiogenesis.

In conclusion, a few radiotracers for imaging angiogenesis in tumors have been tested in humans. The role of these tracers in assessing the response to anti-angiogenic therapies has yet to be assessed.