Uncovering temporospatial sensitive TBI targeting strategies via in vivo phage display.

The heterogeneous pathophysiology of traumatic brain injury (TBI) is a barrier to advancing diagnostics and therapeutics, including targeted drug delivery. We used a unique discovery pipeline to identify novel targeting motifs that recognize specific temporal phases of TBI pathology. This pipeline combined in vivo biopanning with domain antibody (dAb) phage display, next-generation sequencing analysis, and peptide synthesis. We identified targeting motifs based on the complementarity-determining region 3 structure of dAbs for acute (1 day post-injury) and subacute (7 days post-injury) post-injury time points in a preclinical TBI model (controlled cortical impact). Bioreactivity and temporal sensitivity of the targeting motifs were validated via immunohistochemistry. Immunoprecipitation-mass spectrometry indicated that the acute TBI targeting motif recognized targets associated with metabolic and mitochondrial dysfunction, whereas the subacute TBI motif was largely associated with neurodegenerative processes. This pipeline successfully discovered temporally specific TBI targeting motif/epitope pairs that will serve as the foundation for the next-generation targeted TBI therapeutics and diagnostics.