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Microbubble-conjugated vascular endothelial growth factor receptor 2 binding peptide


, PhD, , PhD, , PhD, , MD, PhD, and , PhD.

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Created: ; Last Update: July 11, 2012.

Chemical name:Microbubble-conjugated vascular endothelial growth factor receptor
2 binding peptide
Image BR55v1.jpg
Abbreviated name:BR55
Agent Category:Peptide
Target:Vascular endothelial growth factor receptor 2 (VEGFR2)
Target Category:Receptor
Method of detection:Ultrasound
Source of signal / contrast:Microbubbles
  • Checkbox In vitro
  • Checkbox Rodents
Structure of BR55



The vascular endothelial growth factor receptor 2 (VEGFR2; also known as the kinase insert domain receptor) is a primary signal transducer for angiogenesis and the development of pathological conditions such as cancer, diabetic retinopathy, and neovascular age-related macular degeneration (1-3). This receptor is expressed mainly by endothelial cells, and in order to meet the increased nutritional demands of neoplastic lesions its expression is upregulated in the tumor vasculature to promote angiogenesis (2, 4, 5). Therefore, the inhibition of VEGFR2 activity and its downstream signaling pathways are important targets for the treatment of diseases involving angiogenesis (for an illustration of angiogenesis, see Deshpande et al. (6)). The United States Food and Drug Administration (FDA) has approved several drugs and monoclonal antibodies that target the VEGFR2 receptor for the treatment of cancerous tumors and other angiogenic diseases (7). In addition, many antiangiogenic agents are under investigation in ongoing clinical trials. For any therapy to be successful, it is important to select patients who are likely to respond to the treatment and to be able to rapidly evaluate the response after initiation of the therapy. In this respect, the evaluation of anti-VEGFR2 therapies has been performed with various molecular imaging modalities, including ultrasound imaging (8). The main advantage of using ultrasound imaging over other modalities to visualize active tumor angiogenesis by targeting VEGFR2 is that ultrasound generates a purely vascular signal that can be viewed in real time because the contrast agents used with this imaging modality are restricted to the vascular compartment due to their size (9). In addition, the equipment used to perform this technique does not require the use of radioactivity, and it is readily available, inexpensive, and easy to operate (9). With the advancement of ultrasound technology, microbubble (MB)-based contrast agents that target disease-specific receptors or molecules have been developed and used to visualize or monitor angiogenesis and hard-to-detect pathological lesions (6). Previously, biotinylated anti-VEGFR2 antibodies (Abs) coupled to streptavidin-containing MBs through the biotin-strepavidin linkage have been used to assess the expression of VEGFR2 in mouse tumor models, but such contrast agents cannot be used in the clinic because strepavidin and/or the Abs can be immunogenic in humans (10). To circumvent the immunogenicity issues of the Ab-directed biotin-streptavidin–based MB contrast agents, it was necessary to develop contrast agents that would use non-immunogenic molecules to link the VEGFR2-seeking molecules to the MBs.

For this, a novel MB contrast agent (BR55) that targets VEGFR2 and has the potential for translation to the clinic was developed (11). A phage display library was used to identify and create a heterodimeric peptide that showed a selective and high affinity for human VEGFR2 (12). The heterodimeric peptide was then used to generate lipopeptides that were inserted into the phospholipid shells of MBs to produce a targeted ultrasound contrast agent (BR55). This agent was then evaluated for the visualization of xenograft or orthotopic tumors that expressed VEGFR2 (11) and to quantify the receptor in tumors of nude mice undergoing antiangiogenic therapy (10).



The heterodimeric peptide was synthesized on a peptide synthesizer (13) and coupled to the amino group of 1,2-distearoyl-sn-glycerol-3-phosphoethanolamine-N-[amino (polyethylene glycol)-2000 to create a heterodimeric lipopeptide (14). After purification, the heterodimeric lipopeptides were incorporated into the phospholipid membrane of MBs containing perfluorobutane gas to generate BR55 as described elsewhere (11). For storage, the BR55 preparation was freeze-dried in vials containing a mixture of nitrogen and perfluorobutane. Before use, the freeze-dried contents of the vial were reconstituted in 2 mL 5% glucose solution. The reconstituted solution contained 2 × 109 MBs/mL. The BR55 MBs had a mean diameter of 1.5 μm as determined with size distribution analysis, and there were ~4 × 105 lipopeptide molecules specific for VEGF-2 linked to each MB (11).

In Vitro Studies: Testing in Cells and Tissues


It was determined from surface plasmon resonance (SPR) measurements that the heterodimeric peptide (lacking the lipid tail) had a Kd of 0.22 ± 0.02 nM for human VEGR-2/Fc (recombinant VEGFR2 fused to the Fc portion of human IgG1), indicating that the peptide had a high affinity for the receptor (14). Under the same experimental conditions the heterodimer had a Kd of 1.60 ± 0.57 nM for mouse VEGFR2/Fc (approximately eight-fold weaker than for the human receptor), suggesting that the peptide bound preferentially to the human receptor (14). SPR measurements showed that the heterodimer-peptide-phospholipid-PEG2000 conjugate had a Kd of 4.8 nM for the VEGFR2/Fc protein (14).

Using 125I-VEGF165 as the ligand in a receptor binding assay with human umbilical cord vascular endothelial cells (HUVECs; these cells express VEGFR2), the IC50 of BR55 was determined to be 3 nM (12). In another assay, the heterodimeric peptide was reported to have an IC50 of 0.5 nM for the inhibition of VEGF-dependent phosphorylation of VEGFR2 in HUVECs (12). The peptide had an IC50 of 1 nM for the inhibition of VEGF-dependent endothelial cell migration (12). VEGF and an anti-VEGFR2 antibody were reported to have IC50 values of 71 pM and 160 nM, respectively, for the inhibition of BR55 binding to HUVECs (11).

Animal Studies



BR55 was evaluated for the visualization of tumors in female rats bearing orthotopic rat mammary adenocarcinoma cell lesions (MatBIII tumors) (11). Baseline ultrasound imaging was performed on the animals (n = 5 rats/group, under 2% isoflurane in air anesthesia) before the administration of a bolus injection of 2.4 × 107 MBs through the jugular vein. After the injection, imaging was performed on the rats for up to 10 min postinjection (p.i.), during which time any circulating bubbles had disappeared from general circulation (11). With BR55 the tumors were clearly visible at 20 s p.i. and remained visible even at 10 min p.i., indicating that the targeted MBs had accumulated on the VEGFR2-expressing endothelium of the tumor vasculature. Another group of rats that received a dose of untargeted MBs also showed enhanced peak intensity in the tumors at 20 s p.i.; however, at 10 min p.i. the lesions were not clearly visible in these animals (11). In another study, intravital microscopy of rats bearing MatBIII tumors in a dorsal-skin fold chamber (n = 7 animals) showed that the fluorescent BR55 MBs remained bound to the neovasculature of the tumors for >10–15 min (15).

The use of BR55 was evaluated for the visualization of Dunning R-3327 rat prostate adenocarcinoma cell tumors in Copenhagen rats (16). Animals under anesthesia (n = 11 rats) were injected with BR55 (1.3 × 108 MBs/kg body weight) through the tail vein. Ultrasound imaging at 10 s p.i. showed that the tumors were clearly visible in the animals and had a two-fold higher peak intensity (rms2 = 170 ± 71; P < 0.0001) than the healthy prostate tissue (rms2 = 80 ± 32). At this time point the signal from the untargeted bubbles in the tumor was similar to that of BR55. At 10 m p.i., the intensity in the tumor was significantly higher (P < 0.0001) than that observed in the healthy part of the prostate (rms2 = 12 ± 6.8 versus 0.8 ± 1.0, respectively), and the tumor/healthy prostate ratio was 25.7 ± 23.8 at this time point. Almost no signal from the untargeted bubbles in the tumor was observed at this time point. A linear dose-response effect of BR55 was evident between the peak intensity and the late-phase ultrasound signal enhancement in the tumors. Immunofluorescence staining of the tumor and healthy prostate tissues (with a rabbit polyclonal anti-VEGFR2 Ab as the primary Ab) showed there was an increased expression of VEGFR2 in the lesions compared with the normal tissue, and the stain was confined primarily to the tumor neovasculature (16). Using the same animal model, it was shown that BR55 is comparable to magnetic resonance imaging for the detection and determination of the size of prostate cancer tumors in rats (17).

The use of BR55 and BR38 (a non-targeted, MB-based, ultrasound contrast agent) was assessed with ultrasound imaging to distinguish between differentially aggressive xenograft tumors derived from human breast cancer MDA-MB-231 cells (estrogen-independent, highly aggressive; n = 5 animals) and minimally aggressive tumors derived from MCF-7 cells (estrogen-dependent; n = 6 animals) in mice (18). This study showed that both BR38 and BR55 were suitable to characterize and distinguish between breast cancer tumors on the basis of angiogenesis and growth aggressiveness. Ultrasound imaging with BR38 showed that the MDA-MB-231 tumors were highly vascularized compared with the MCF-7 lesions. The images showed that the binding of BR55 was significantly higher (P = 0.049) in the MDA-MB-231 tumors than in the MCF-7 tumors. From this study, the investigators concluded that BR38 and BR55 were suitable for the characterization of breast cancer tumors on the basis of angiogenesis and aggressiveness, but the molecular information alone (the late phase signal of BR55) is better suited to discriminate differently aggressive tumors compared to the functional information (BR55 signal) alone (18).

BR55 was assessed for the visualization of VEGFR2 and its ability to monitor antiangiogenic therapy in mice bearing human colon cancer LS174T cell xenograft tumors (10). Imaging was performed on the rodents using BR55 by measuring the ultrasound signal originating from tumors with or without anti-VEGF Ab treatment (n = 6 mice/group) for up to 6 days after initiation of therapy (the Ab targeted both human and mouse VEGF; the animals were given an intraperitoneal injection of the Ab every 24 h). At 1 d after initiation of treatment, the imaging signal decreased by ~46% (P = 0.02) in the treated tumors compared with the untreated tumors, and the signal remained significantly low (range, 46% to 84% lower; P = 0.38) for the next 5 days. After completion of the imaging protocol, the tumors were removed from the treated and untreated animals for ex vivo immunostaining to determine the microvessel density (the staining is for the CD31 antigen that is found only on the endothelial cells) and the level of VEGFR2 expression in the lesions (10). Immunostaining of tumor sections for CD31 showed that there was a significantly reduced (P = 0.04) number of microvessels in the tumors of animals that received the antiangiogenic treatment (7.3 ± 4.7 microvessels/mm2) compared with the untreated animals (22.0 ± 9.4 microvessels/mm2). In addition, immunostaining for VEGFR2 showed that there was a significantly reduced expression of VEGFR2 (P = 0.03) in the microvessel endothelial cells of tumors obtained from the treated mice compared with similar tissue from the untreated animals.

Other Non-Primate Mammals


No publications are currently available.

Non-Human Primates


No publications are currently available.

Human Studies


No publications are currently available.

Supplemental Information


No information is currently available.

NIH Support

Some of the work reported in this chapter was supported by the National Institutes of Health grants NCI ICMIC CA114747 P50 and NIH R21 CA139279.


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This MICAD chapter is not included in the Open Access Subset, because it was authored / co-authored by one or more investigators who was not a member of the MICAD staff.


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