Background <p>Transferrin receptor (TfR)-mediated transcytosis is a well-established method for delivering biologic therapeutics and diagnostics to the brain. Although moderate affinity towards TfR is beneficial for TfR-mediated brain delivery at therapeutic doses, emerging evidence has indicated that high TfR affinity may be more beneficial at tracer doses. With the development of antibody-based PET radioligands for neurodegenerative diseases, such as Alzheimer’s disease, understanding the pharmacokinetics of TfR-binders at tracer dose is essential. Thus, this study aimed to evaluate the effect of TfR affinity on brain uptake at a tracer dose in both wild-type (WT) and amyloid-beta (Aβ) pathology presenting mice and to demonstrate the usability of TfR-mediated brain delivery of immunoPET diagnostic radioligands to visualize intrabrain Aβ pathology in vivo.</p> Methods <p>Three different affinity variants of anti-mouse TfR-binding antibody 8D3, engineered by alanine point mutations, were selected. Bispecific antibodies were designed with knob-into-hole technology with one arm targeting TfR (8D3) and the other arm targeting human Aβ (bapineuzumab). Antibody affinities were measured in an in vitro cell assay. In vivo pharmacokinetic analyses of radioiodinated bispecific antibodies and bapineuzumab in brain, blood and peripheral organs were performed over 7 days post-injection in WT mice and a model of Aβ pathology (<i>App</i>^{<i>NL−G−F</i>}). The strongest TfR affinity bispecific antibody was also evaluated as a positron emission tomography (PET) radioligand for detecting Aβ pathology in WT and <i>App</i>^{<i>NL−G−F</i>} mice.</p> Results <p>The three bispecific antibodies bound to TfR with affinities of 10 nM, 20 nM and 240 nM. Independent of genotype, stronger TfR-affinity resulted in higher initial brain uptake. The two higher-affinity bispecific antibodies behaved similarly and differentiated between WT and <i>App</i>^{<i>NL−G−F</i>} mice earlier than the lowest affinity variant. Finally, the 10 nM bispecific antibody was able to clearly differentiate between WT and <i>App</i>^{<i>NL−G−F</i>} mice when used as a PET radioligand.</p> Conclusion <p>This study supports the hypothesis that stronger TfR affinity enhances brain uptake at a tracer dose. With the more effective detection of Aβ pathology, stronger TfR affinity is a crucial design feature for future bispecific immunoPET radioligands for intrabrain targets via TfR-mediated transcytosis.</p>