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

Objectives Lung cancer accounts for 17% of cancer-related deaths worldwide and most patients present with locally advanced or metastatic disease. Novel positron emission tomography (PET) imaging agents for assessing vascular endothelial growth factor receptor-2 (VEGFR-2) expression can be utilized for detecting VEGFR-2+ 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 detection 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. Confocal imaging was utilized to validate the specific binding of RamAb to VEGFR-2 using a cell line known to express high basal levels of VEGFR-2, HUVEC, and a cell line that expresses minimal VEGFR-2, A549. Flow cytometry analysis and microscopy studies were performed to compare 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 were performed to elucidate the expression patterns of VEGFR-2 and VEGFR1 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. In addition, the lack of uptake by A549 cells further confirmed that NOTA conjugation did not compromise the binding affinity or specificity of RamAb for VEGFR-2. After labeled with 64Cu, PET imaging revealed specific and prominent uptake of 64Cu-NOTA-RamAb in VEGFR-2 positive HCC4006 tumors (9.4 ± 0.5 %ID/g at 48 h post-injection; n = 4) and significantly lower uptake in VEGFR-2 negative A549 tumors (4.3 ± 0.2 %ID/g at 48 h post-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. Immunofluorescence results displayed that fluorescence from FITC-RamAb was shown to overlay with the tumor cells, depicted in blue; whereas A549 sections showed minimal expression of VEGFR-2 that did not overlay with tumor vasculature (CD31) or cells (DAPI). Blocking of VEGFR-2 effectively resulted in minimal RamAb binding, providing further evidence of RamAbs selectivity. VEGFR-1 immunofluorescence staining in A549 and HCC4006 tumors showed that A549 tumor sections expressed significantly higher levels of VEGFR-1 in comparison to HCC4006.

Conclusions 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.

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