The binding of programmed death ligand 1 protein (PD-L1) to its receptor programmed death protein 1 (PD-1) mediates immunoevasion in cancer and chronic viral infections, presenting an important target for therapeutic intervention. Several monoclonal antibodies targeting the PD-L1/PD-1 signaling axis are undergoing clinical trials; however, the epitopes of these antibodies have not been described. We have combined orthogonal approaches to localize and characterize the epitope of a monoclonal antibody directed against PD-L1 at good resolution and with high confidence. Limited proteolysis and mass spectrometry were applied to reveal that the epitope resides in the first immunoglobulin domain of PD-L1. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) was used to identify a conformational epitope comprised of discontinuous strands that fold to form a beta sheet in the native structure. This beta sheet presents an epitope surface that significantly overlaps with the PD-1 binding interface, consistent with a desired PD-1 competitive mechanism of action for the antibody. Surface plasmon resonance screening of mutant PD-L1 variants confirmed that the region identified by HDX-MS is critical for the antibody interaction and further defined specific residues contributing to the binding energy. Taken together, the results are consistent with the observed inhibitory activity of the antibody on PD-L1-mediated immune evasion. This is the first report of an epitope for any antibody targeting PD-L1 and demonstrates the power of combining orthogonal epitope mapping techniques.
Adeno-associated virus (AAV) vectors have emerged as the leading delivery platforms for gene therapy. Throughout the life cycle of the virions, the capsid vector carries out diverse functions, ranging from cell surface receptor engagement, cellular entry, endosomal escape, nuclear import to new particle packaging, and assembly. Each of these steps is mediated by exquisite structure features of the viral capsid and its interaction with viral genome, Rep proteins, and cellular organelle and apparatus. In this brief review, we provide an overview of results from over a decade of extensive biophysical studies of the capsid employing various techniques. The remaining unaddressed questions and perspective are also discussed. The detailed understanding of the structure and function interplay would provide insight to the strategy for improving the efficacy and safety of the viral vectors.
An efficient and convenient purifying procedure for recombinant peptide was established. Thereby, the aimed antimicrobial peptide T1 containing the conservative sequences derived from cecropin was successfully expressed and purified. The composition of amino acid of the purified peptide T1 was consistent with that of theoretical design. The significant antimicrobial activity of T1 against gram-positive and gram-negative bacteria was demonstrated, suggesting that the conservative sequences in cecropin play an important role in the antimicrobial mechanism and that antimicrobial peptide T1 has the potential to be used as the food preservative.
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