Radiolabeled antibodies to validate blood brain barrier penetration.

Objectives: Detecting and treating diseases of the central nervous system is extremely limited by the capacities of imaging agents and drugs to cross the blood brain barrier (BBB). Our groups have developed high-diversity libraries of human antibodies and protocols to select and evaluate in vitro BBB-crossing antibodies. The aim of this project was to develop a robust and simple in vivo method to validate the antibody penetration through the BBB. Methods: We have conjugated the antibodies with NOTA chelator for ⁶⁴Cu radiolabeling and confirmed their in vitro transport properties at the BBB and in vivo stability. We have compared different modes of delivery (ie. intravenous (IV), intra-arterial (IA)) via the carotid artery to estimate the brain uptake and assessed the biodistribution profiles of the new [⁶⁴Cu]NOTA-antibody conjugates in healthy rats. Results: NOTA was conjugated in high yield to the antibodies (81-89%) at a molar ratio ranging from 1:1 to 2:1 for chelate:antibody. The labeling with ⁶⁴Cu was easily achieved in 35 minutes at room temperature with a radiolabeling yield >95% and a molar activity of 150-160 MBq/nmole. We showed that the injection in the right carotid artery of the new ⁶⁴Cu-NOTA-antibodies resulted in a brief but important radioactivity exposure in the right brain hemisphere, which persisted after exsanguination. Under these conditions, the brain uptake of ⁶⁴Cu-NOTA-conjugates in the right hemisphere was significantly higher as compare to the left hemisphere. IV and IA injection did not show specific uptake for the control ⁶⁴Cu-NOTA-antibody. The right to left hemisphere ratio remained constant across the different concentrations of unlabeled conjugate but exceed that obtained with the control ⁶⁴Cu-NOTA-antibody. Conclusion: Our new ⁶⁴Cu-NOTA-antibodies shows transitory and specific BBB uptake. This successful result is promising for the use of bi-specific radiolabeled antibody for the delivery of drugs in the brain.