Ultrasound Molecular Imaging of the Breast Cancer Neovasculature using Engineered Fibronectin Scaffold Ligands: A Novel Class of Targeted Contrast Ultrasound Agent

Abstract

Molecularly-targeted microbubbles (MBs) are increasingly being recognized as promising contrast agents for oncological molecular imaging with ultrasound. With the detection and validation of new molecular imaging targets, novel binding ligands are needed that bind to molecular imaging targets with high affinity and specificity. In this study we assessed a novel class of potentially clinically translatable MBs using an engineered 10th type III domain of human-fibronectin (MB-FN3VEGFR2) scaffold-ligand to image VEGFR2 on the neovasculature of cancer. The in vitro binding of MB-FN3VEGFR2 to a soluble VEGFR2 was assessed by flow-cytometry (FACS) and binding to VEGFR2-expressing cells was assessed by flow-chamber cell attachment studies under flow shear stress conditions. In vivo binding of MB-FN3VEGFR2 was tested in a transgenic mouse model (FVB/N Tg(MMTV/PyMT634Mul) of breast cancer and control litter mates with normal mammary glands. In vitro FACS and flow-chamber cell attachment studies showed significantly (P<0.01) higher binding to VEGFR2 using MB-FN3VEGFR2 than control agents. In vivo ultrasound molecular imaging (USMI) studies using MB-FN3VEGFR2 demonstrated specific binding to VEGFR2 and was significantly higher (P<0.01) in breast cancer compared to normal breast tissue. Ex vivo immunofluorescence-analysis showed significantly (P<0.01) increased VEGFR2-expression in breast cancer compared to normal mammary tissue. Our results suggest that MBs coupled to FN3-scaffolds can be designed and used for USMI of breast cancer neoangiogenesis. Due to their small size, stability, solubility, the lack of glycosylation and disulfide bonds, FN3-scaffolds can be recombinantly produced with the advantage of generating small, high affinity ligands in a cost efficient way for USMI.

In vitro characterization and in vivo ultrasound molecular imaging of nucleolin-targeted microbubbles

“…Potential targeting ligands include antibodies [22, 53–55], peptides [10, 26, 30, 31, 56], or engineered scaffold ligands [57, 58]. Methods to conjugate ligands onto the surface of contrast agents have included biotin-avidin coupling [22, 43, 53–55] and covalent bonds [10, 26, 30, 56, 59].…”

Development and Differentiation in Monobodies Based on the Fibronectin Type 3 Domain

“…In spite of this clinical failure, the Adnectin-anti-VEGFR2 has become a model protein for development of technologies supporting the monobody scaffold, with recent studies aiming to enhance the Adenctin's pharmacokinetic properties through PASylation [50] or improve CMC through bacterial expression [51]. The wider lesson learnt from this first clinical attempt was primarily to select targets and applications that differentiate the FN3 scaffold instead of following established antibody methodologies, such as the re-purposing of the Adnectin-anti-VEGFR2 as an ultrasound imaging agent [52] or in CAR-T (Chimeric Antigen Receptor-T Cell) formats [53].…”

“…[58]. Tumour imaging with monobodies conjugated to (B) radioisotopes [62] and (C) microbubbles [52]. (D) Targeted degradation of endogenous intracellular proteins [63] and (E) targeted intracellular fluorescence reporters for endogenous proteins [64].…”

Interactions Between Tumor Biology and Targeted Nanoplatforms for Imaging Applications

“…The MB‐FN3 VEGFR2 was developed for in vivo ultrasound molecular imaging (USMI) of breast cancer neovasculature with specific binding to VEGFR2, which was significantly higher in breast cancer compared to normal breast tissue. The FN3‐scaffold could be produced via recombinant technology, with small size, solubility, lack of glycosylation, good stability, and disulfide bonds, leading to generation of small, high affinity ligands for USMI 59…”