Increasing the affinity between a targeted axonal import (TAxI) peptide and its target receptor hLamR

Abstract

An efficient delivery approach for trafficking therapeutics into central nervous system (CNS) is a critical unmet need. Previously, we identified a target axonal import (TAxI) peptide that delivers protein cargo into the central nervous system (CNS), especially the spinal cord and hindbrain, via retrograde axonal transport mechanism after peripheral administration. Mass spectrometry analysis of a TAxI peptide pull-down assay suggested that human laminin receptor (hLamR) is the potential binding target for TAxI peptide. However, the evidence of interaction between TAxI and hLamR is still lacking. Moreover, the binding affinity of TAxI peptide has yet to be optimized. Here, we report that TAxI binds to cells that express hLamR on the cell membrane. Moreover, a fluorescence activated cell sort (FACS) correlation study proved that TAxI binds hLamR directly. Through sequence modification and multivalent polymerization, the affinity of TAxI-based peptides and co-polymer formulations was optimized and demonstrated increased hLamR binding-affinity. In particular, the binding between Poly-TAxI and hLamR was significantly enhanced due to avidity effects in high hLamR expressing cells. Chondroitin sulfate is shown to be an allosteric modulator for facilitating association between TAxI and hLamR. Interestingly, TAxI showed robust binding to multiple cancer cells with relative abundant membrane level of hLamR, compared to the non-cancer cell. In addition, we also present the development and optimization of a workflow to express and purify recombinant hLamR protein expressed in eukaryotic cells. TAxI-based peptide and co-polymer formulations are able to target high hLamR expressing cells, and thus demonstrates the protential potential to target the delivery of promising therapeutics into the CNS.