Development of a Freeze-Dried Bevacizumab Kit for ^99mTc Radiolabeling and Preclinical Evaluation In Vitro and in...

Bevacizumab, a humanized monoclonal antibody targeting vascular endothelial growth factor (VEGF), was lyophilized and radiolabeled with ^99mTc for tumor-targeted imaging. In this study, a freeze-dried kit was developed, the formulation was optimized, and the radiolabeled bevacizumab was evaluated in vitro and in vivo for preclinical evaluation.

The preparation process was optimized by investigating pH, temperature, time, and stabilizers. The radiochemical purity (RCP) and immunoreactivity of the lyophilized antibody were determined and confirmed by specific binding to VEGF immobilized on Ni-NTA agarose. Preclinical evaluations included binding assays on three human cancer cell lines (A-549, MCF-7, and HT-29), in which binding affinity (K_d) and maximum binding capacity (B_max) were calculated. Biodistribution studies were performed in normal mice.

The optimized kit contained 2.0 mg bevacizumab per vial in phosphate buffer at pH 7.5, ensuring reproducible reconstitution and high labeling efficiency. Quality control demonstrated RCP >97% and in vitro stability of ^99mTc-bevacizumab for at least 6 h post-labeling, with the freeze-dried form stable for 12 months. The ^99mTc-bevacizumab retained >90% immunoreactivity. Binding assays revealed approximately 80% specific binding, with a K_d of 2.8-9.9 nM and a B_max of 1.6-2.0 amol/cell, corresponding to about one million molecules per cell. Biodistribution revealed high initial blood retention, which was moderate in the liver, with a blood clearance half-life of 76.97 min, primarily via renal excretion.

The freeze-dried bevacizumab enables convenient ^99mTc radiolabeling, providing a preclinical proof-of-concept supporting further development as a tumor imaging-agent.