Microbubble-conjugated vascular endothelial growth factor receptor 2 binding peptide

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).