Abstract
AbstractAntibodies have found applications in several fields, including, medicine, diagnostics, and nanotechnology, yet methods to modulate antibody–antigen binding using an external agent remain limited. Here, we have developed photoactive antibody fragments by genetic site‐specific replacement of single tyrosine residues with photocaged tyrosine, in an antibody fragment, 7D12. A simple and robust assay is adopted to evaluate the light‐mediated binding of 7D12 mutants to its target, epidermal growth factor receptor (EGFR), on the surface of cancer cells. Presence of photocaged tyrosine reduces 7D12‐EGFR binding affinity by over 20‐fold in two out of three 7D12 mutants studied, and binding is restored upon exposure to 365 nm light. Molecular dynamics simulations explain the difference in effect of photocaging on 7D12‐EGFR interaction among the mutants. Finally, we demonstrate the application of photoactive antibodies in delivering fluorophores to EGFR‐positive live cancer cells in a light‐dependent manner.
Highlights
Chemists and biochemists have successfully designed molecular systems that can be controlled in a defined manner in response to external agents, such as pH, light, and small molecules.[1]
Using an on-cell assay, we show that the presence of a photocaging group at specific tyrosine residues in the antigen binding region of 7D12 inhibits its binding to epidermal growth factor receptor (EGFR) on the surface of cancer cells and the binding is restored upon irradiation with 365 nm light
To find an optimal plasmid system and aminoacyl-tRNA synthetase (aaRS)/tRNA pair for incorporation of photocaged tyrosine (pcY) in 7D12, we screened five suppressor plasmids containing either MjCNFRS/MjtRNACUA pair (MjCNFRS is an M. jannaschii Tyrosyl-tRNA synthetase (MjRS) evolved for incorporation of 4-cyanoL-phenylalanine) or the Pyrrolysyl-tRNA synthetase (PylRS)/tRNACUA pair (Supporting information, S3 and S4, and Figure S3 and Figure S4). pULTRA plasmid with MjCNFRS/MjtRNACUA pair, and pCDF plasmid with PylRS/tRNACUA pair show most efficient genetic incorporation of unnatural amino acids
Summary
Chemists and biochemists have successfully designed molecular systems that can be controlled in a defined manner in response to external agents, such as pH, light, and small molecules.[1] Controlling the activity of small molecules and biomolecules has allowed development of molecular machines, novel drugs, and nano-delivery systems, that have found widespread applications.[2] Monoclonal antibodies are arguably one of the most versatile biomolecules that can be adapted to bind to different substrates with high selectively and specificity.
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