PET Imaging of VEGFR-2 Expression in Lung Cancer with ⁶⁴Cu-Labeled Ramucirumab

Abstract

Lung cancer accounts for 17% of cancer-related deaths worldwide, and most patients present with locally advanced or metastatic disease. Novel PET imaging agents for assessing vascular endothelial growth factor receptor-2 (VEGFR-2) expression can be used for detecting VEGFR-2–positive malignancies and subsequent monitoring of therapeutic response to VEGFR-2–targeted therapies. Here, we report the synthesis and characterization of the antibody-based imaging agent for PET imaging of VEGFR-2 expression in vivo. Methods Ramucirumab (named RamAb), a fully humanized IgG1 monoclonal antibody, was conjugated to 2-S-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (p-SCN-Bn-NOTA) and labeled with 64Cu. Flow cytometry analysis and microscopy studies were performed to compare the VEGFR-2 binding affinity of RamAb and NOTA-RamAb. PET imaging and biodistribution studies were performed in nude mice bearing HCC4006 and A549 xenograft tumors. Ex vivo histopathology was performed to elucidate the expression patterns of VEGFR-2 in different tissues and organs to validate in vivo results. Results Flow cytometry examination revealed the specific binding capacity of FITC-RamAb to VEGFR-2, and no difference in VEGFR-2 binding affinity was seen between RamAb and NOTA-RamAb. After being labeled with 64Cu, PET imaging revealed specific and prominent uptake of 64Cu-NOTA-RamAb in VEGFR-2–positive HCC4006 tumors (9.4 ± 0.5 percentage injected dose per gram at 48 h after injection; n = 4) and significantly lower uptake in VEGFR-2–negative A549 tumors (4.3 ± 0.2 percentage injected dose per gram at 48 h after injection; n = 3). Blocking experiments revealed significantly lower uptake in HCC4006 tumors, along with histology analysis, further confirming the VEGFR-2 specificity of 64Cu-NOTA-RamAb. Conclusion This study provides initial evidence that 64Cu-NOTA-RamAb can function as a PET imaging agent for visualizing VEGFR-2 expression in vivo, which may also find potential applications in monitoring the treatment response of VEGFR-2 targeted cancer therapy.

Radionuclide imaging of VEGFR2 in glioma vasculature using biparatopic affibody conjugate: proof-of-principle in a murine model

“…Finding the optimal injected protein dose and the optimal imaging window was the focus of the initial in vivo characterization. The overall biodistribution pattern of the new agent was in good agreement with other VEGFR2-targeting agents ( 40 - 43 , as shown particularly in Figure 8 in 44 ). The in vivo data showed a pronounced influence of injected protein dose on the imaging contrast ( Figure 3 A-B ).…”

“…The blood clearance of activity was fast, and the tumor-to-blood ratios 2 h pi were approximately 10 for MS1 (endothelial) and 3 for PC-3 (epithelial) tumors, reflecting different target density in a tumor composed of endothelial cells compared to an epithelial tumor with VEGFR2 expression confined to the host-derived tumor vasculature. Remarkably, the tumor-to-blood ratios provided by [ 111 In]In-NODAGA-Z VEGFR2 -Bp 2 in both tumor models were superior to 64 Cu-labeled ramucirumab (~1 at 48 h pi), 61/64 Cu-labeled VEGF121 (~1 at 8 h pi for 61 Cu and ~2 at 16 h pi for 64 Cu), 68 Ga -labeled VEGF121 (<1 at 4 h pi), and scVR2 (<2 at 2 h pi) 26 , 40 - 43 . A high tumor-to-blood ratio is important for imaging.…”

ImmunoPET for assessing the differential uptake of a CD146-specific monoclonal antibody in lung cancer

“…Immunofluorescence staining was conducted following a previously described methodology to evaluate the expression of CD146 and CD31 in tumor sections of six lung cancer models [15]. Briefly, tumors extracted after the last PET imaging time point (48 h) and embedded in Tissue-Tek optimal cutting temperature (O.C.T.)…”

PET Imaging of VEGFR with a Novel 64Cu-Labeled Peptide

“…18,23 More recently, several VEGFR-2-specific monoclonal antibodies labeled with copper-64 or zirconium-89 have shown encouraging results for the selective imaging of VEGFR-2. 27,33 Despite these advances, mAb-based imaging agents have shown unfavorable pharmacokinetics (PK) and high nonspecific uptake. Besides, several research efforts have been put toward affibody and peptide-based radiotracers.…”