Targeted Systemic Radiotherapy with scVEGF/¹⁷⁷Lu Leads to Sustained Disruption of the Tumor Vasculature and Intratumoral Apoptosis

Abstract

Tumor vessels abundantly express receptors for vascular endothelial growth factor (VEGF), despite treatment with conventional or antiangiogenic drugs. We wished to determine whether the high levels of VEGF receptor (VEGFR) within the tumor vasculature could be leveraged for intracellular delivery of therapeutically significant doses of scVEGF/ 177 Lu, a novel radiopharmaceutical based on a recombinant single-chain (sc) derivative of VEGF, in orthotopic breast cancer models. Methods: scVEGF-PEG (polyethylene gycol)-DOTA conjugates containing 2.0-, 3.4-, or 5.0-kDa PEG linkers site-specifically conjugated to a cysteinecontaining tag (Cys-tag) in scVEGF were radiolabeled with 177 Lu (scVEGF/ 177 Lu) for in vivo studies. Human MDA231luc and mouse 4T1luc cell lines were injected orthotopically to establish breast carcinoma tumors in immunodeficient and immunocompetent hosts, respectively. The effects of scVEGF/ 177 Lu were defined by analysis of changes in tumor growth and immunohistochemical staining for the endothelial markers CD31 and VEGFR-2 and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining for intratumoral apoptosis. Results: Biodistribution assays and dosimetric calculations established that scVEGF/ 177 Lu with a 3.4-kDa PEG linker delivered the highest dose of radiation to tumors (69.9 cGy/MBq/g of tissue) and the lowest dose to the kidneys (33.3 cGy/MBq/ organ). Total doses below 40 MBq/mouse of scVEGF/ 177 Lu did not affect renal function, and 3 divided doses of 6.3 MBq/mouse or a bolus dose of 18.9 MBq/mouse induced only transient lymphopenia and weight loss (,10% baseline weight). In mice with orthotopic mammary breast carcinoma, intravenous injections of well-tolerated bolus and fractionated doses of scVEGF/ 177 Lu in the range from 6.3 to 18.9 MBq/mouse (25-76 MBq/m 2 ) resulted in dose-dependent tumor growth inhibition. Immunohistochemical analysis of tumors at 4-5 wk after single injections of scVEGF/ 177 Lu indicated dose-dependent regression of tumor vasculature and widespread intratumoral apoptosis. A single dose of 7.4 MBq/mouse of scVEGF/ 177 Lu given before a course of bevacizumab or sunitinib treatment enhanced the antiangiogenic effects of both drugs. Conclusion: Selective targeting of VEGFR in tumor vasculature with well-tolerated doses of scVEGF/ 177 Lu is effective in orthotopic breast cancer models. As high levels of VEGFR expression in the tumor vasculature are a common feature in a variety of cancers, targeting tumor angiogenesis with scVEGF/ 177 Lu warrants further exploration.

Direct measurements of VEGF–VEGFR2 binding affinities reveal the coupling between ligand binding and receptor dimerization

“…scVEGF is a fully functional form of VEGF-A121 as it binds and activates VEGFR2 just like endogenous VEGFs. It is widely used in translational research and has already proven its utility as an imaging and a therapeutic agent (28,30,31).…”

Pharmacokinetics, microscale distribution, and dosimetry of alpha-emitter-labeled anti-PD-L1 antibodies in an immune competent transgenic breast cancer model

“…Cell-specific targeting is possible using targeted radiotherapy. With the exception of efforts to target tumor vasculature [12–14], the focus of such a targeted radiation delivery paradigm is to identify tumor-specific or tumor-associated antigens and use radiolabeled antibodies to specifically target tumor cells [15–19]. The anti-PD-L1 Ab conjugated to a therapeutic radionuclide would therefore target both a PD-L1-expressing tumor and PD-L1-positive cells in its microenvironment.…”

Imaging Key Biomarkers of Tumor Angiogenesis

“…6, Right Panel) retention of delivered fluorescent dyes 80, 85, 86. Furthermore, recent experiments indicated that scVEGF-PEG-DOTA conjugates do not stimulate tumor growth even at cumulative doses that are at least an order of magnitude higher than those needed for imaging 87.…”