Factors influencing transferrin receptor-mediated brain delivery : Evaluating preclinical antibody-based proteins for PET imaging in Alzheimer’s disease

Sammanfattning: Antibody-based proteins targeting amyloid-beta (Aβ) could be used as radioligands in positron emission tomography (PET) to study Alzheimer’s disease (AD) pathology in the living brain. The prospective advantages of antibody-based PET are to detect pathology earlier, with higher sensitivity, and to evaluate treatment effects of emerging immunotherapies against Aβ. However, antibodies and other proteins are too large to cross the blood-brain barrier (BBB). This can be circumvented by fusing antibodies with transferrin-receptor (TfR) binders that penetrate the BBB via receptor-mediated transcytosis. In this thesis, I evaluated different bispecific antibody-based proteins that bind both TfR and Aβ. The overall aim was to determine which factors are important for TfR-mediated brain delivery of these proteins and their use as PET radioligands. In paper I, we studied a large, high TfR-avidity antibody compared with a smaller antibody fragment fusion with lower TfR avidity. The small antibody had fast elimination from blood and was cleared from the brain earlier than the large antibody, thus providing better signal-to-noise ratio for brainPET. In paper II, antibody-like proteins (affibodies), even smaller than the previously studied antibody, had enhanced TfR-mediated brain delivery but had an imbalance in binding to TfR and Aβ. This resulted in poor pathology-related retention of 125I-radiolabeled affibodies. In paper III, we observed that aged mice had poorer brain delivery of the bispecific antibody, mAb3D6-scFv8D3, compared with young mice. Age was also related to increased blood cell binding of the bispecific antibody, and a lower dose resulted in higher relative delivery to the brain parenchyma. In paper IV, we evaluated single domain llama-based antibodies, VHHs, which bound both mouse and human TfR, and were characterized by rapid elimination from blood and brain. The VHHs were fused to an Aβ binding antibody fragment, scFv3D6, which enabled increased brain retention of the 125I-radiobeled antibodies in an AD mouse model, and, thus, provided high contrast to healthy controls.In conclusion, antibody format, size, mouse age, dose, and TfR binding were important factors influencing brain delivery and retention.