Molecular Imaging of Melanoma VEGF-expressing Tumors through [99mTc]Tc-HYNIC-Fab(Bevacizumab)


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Abstract

Background:Angiogenesis is a process that many tumors depend on for growth, development, and metastasis. Vascular endothelial growth factor (VEGF) is one of the major players in tumor angiogenesis in several tumor types, including melanoma. VEGF inhibition is achieved by bevacizumab, a humanized monoclonal antibody that binds with high affinity to VEGF and prevents its function. In order to successfully enable in vivo VEGF expression imaging in a murine melanoma model, we previously labeled bevacizumab with [99mTc]Tc. We observed that this was feasible, but it had prolonged blood circulation and delayed tumor uptake.

Objective:The aim of this study was to develop a radiolabeled Fab bevacizumab fragment, [99mTc]Tc-HYNICFab( bevacizumab), for non-invasive in vivo VEGF expression molecular imaging.

Methods:Flow cytometry was used to examine VEGF presence in the murine melanoma cell line (B16-F10). Bevacizumab was digested with papain for six hours at 37°C to produce Fab(bevacizumab), which was then conjugated to NHS-HYNIC-Tfa for radiolabeling with [99mTc]Tc. Stability and binding affinity assays were also evaluated. Biodistribution and single photon emission computed tomography/computed tomography (SPECT/CT) were performed at 1, 3, and 6 h (n = 4) after injection of [99mTc]Tc-HYNIC-Fab(Bevacizumab) in normal and B16-F10 tumor-bearing C57Bl/6J mice.

Results:Using flow cytometry, it was shown that the B16-F10 murine melanoma cell line has intracellular VEGF expression. Papain incubation resulted in the complete digestion of bevacizumab with good purity and homogeneity. The radiolabeling yield of [99mTc]Tc-HYNIC-Fab(bevacizumab) was 85.00 ± 6.06%, with a specific activity of 291.87 ± 18.84 MBq/mg (n=3), showing in vitro stability. Binding assays demonstrated significant intracellular in vitro VEGF expression. Fast blood clearance and high kidney and tumor uptake were observed in biodistribution and SPECT/CT studies.

Conclusions:We present the development and evaluation of [99mTc]Tc-HYNIC-Fab(bevacizumab), a novel molecular VEGF expression imaging agent that may be used for precision medicine in melanoma and potentially in other VEGF-expressing tumors.

About the authors

Marcelo Fernández

Departamento de Radiofarmacia, Centro de Investigaciones Nucleares, Facultad de Ciencias, Universidad de la República

Email: info@benthamscience.net

Ana Laura Reyes

Investigation and Development Department, Centro uruguayo de imagenología Molecular

Email: info@benthamscience.net

Andrea Paolino

Investigation and Development Department, Centro uruguayo de imagenología Molecular

Email: info@benthamscience.net

Eduardo Savio

Investigation and Development Department, Centro uruguayo de imagenología Molecular

Email: info@benthamscience.net

Hugo Cerecetto

Departamento de Radiofarmacia, Centro de Investigaciones Nucleares, Facultad de Ciencias, Universidad de la República

Email: info@benthamscience.net

Pablo Cabral

Departamento de Radiofarmacia, Centro de Investigaciones Nucleares, Facultad de Ciencias, Universidad de la República

Email: info@benthamscience.net

Juan Gambini

Investigation and Development Department, Centro uruguayo de imagenología Molecular

Author for correspondence.
Email: info@benthamscience.net

Ximena Camacho

Departamento de Radiofarmacia, Centro de Investigaciones Nucleares, Facultad de Ciencias, Universidad de la República

Author for correspondence.
Email: info@benthamscience.net

Carolina Perroni

Departamento de Radiofarmacia, Centro de Investigaciones Nucleares, Facultad de Ciencias, Universidad de la República

Email: info@benthamscience.net

Lucía Alfaya

Departamento de Radiofarmacia, Centro de Investigaciones Nucleares, Facultad de Ciencias, Universidad de la República

Email: info@benthamscience.net

Mirel Cabrera

Departamento de Radiofarmacia, Centro de Investigaciones Nucleares, Facultad de Ciencias, Universidad de la República

Email: info@benthamscience.net

Marcos Tassano

Departamento de Radiofarmacia, Centro de Investigaciones Nucleares, Facultad de Ciencias, Universidad de la República

Email: info@benthamscience.net

María García

Departamento de Radiofarmacia, Centro de Investigaciones Nucleares, Facultad de Ciencias, Universidad de la República

Email: info@benthamscience.net

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