NGS-Guided Discovery of Cross-Reactive TfR1 VHH Antibodies with Distinct Brain Localization Profiles

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ID: 332073
2026
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Abstract
Antibody delivery across the blood-brain barrier (BBB) remains challenging. We combined phage display with next-generation sequencing (NGS)-guided clone sequence-diverse selection to identify transferrin receptor 1 (TfR1)-binding VHH antibodies. Following alpacas immunization with human and rat TfR1, representative clones from distinct phylogenetic groups were screened for human/mouse cross-reactivity. Three clones (A1, Y11, and Z1) were reformatted as human Fc-mutant fusion proteins and characterized by ELISA, flow cytometry, and fluorescence imaging. All three clones recognized human and mouse TfR1, with ELISA EC₅₀ >100 nM. After intravenous administration in mice, clone Y11 showed sustained brain-associated fluorescence up to 48 h, whereas A1 showed an initially high signal that progressively declined. Fluorescent microscopy revealed preferential localization of Y11 to NeuN-positive neuronal regions, whereas A1 to lectin-positive vascular structures, indicating distinct brain localization profiles among the isolated clones. These findings identify cross-reactive TfR1-targeting VHHs with distinct brain-localization profiles and potential utility as platforms for compartment-selective CNS drug delivery.
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Authors Muhammad Feisal Jatnika, Asaki Nagashima, Wakana Miyahara, Abdur Rafique, Yuji Ito
Journal The Journal of Biochemistry
Year 2026
DOI
10.1093/jb/mvag070
URL
https://doi.org/10.1093/jb/mvag070
Keywords Keywords not found

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