Genetic incorporation of unnatural amino acids into single chain variable fragments for generation of stable antibody-imaging probe conjugates for cancer imaging
- Walter and Eliza Hall Institute, 1G Royal Parade, Parkville, VIC 3052.
- University of Melbourne, Parkville, VIC 3010.
- University of Queensland, St Lucia, QLD 4072.
The linker chemistry utilised for the generation of antibody-imaging dye, nanomaterial or drug conjugates plays a crucial role in the development of stable and clinically successful antibody-targeted diagnostics or therapeutics. The incorporation of an unnatural amino acid having orthogonal chemical reactivity into single chain variable fragments (scFv) in vivo provides a chemical handle for the generation of such stable antibody conjugates. An unnatural amino acid having an azide functional group, 4-azido phenylalanine or p-azido phenylalanine (pAzF) was incorporated into anti-EGFR scFv expressed in Escherichia coli. A stable scFv-imaging dye conjugate was generated via a strong triazole linkage formed by strain-promoted copper-free click reaction between the azide functional group of the pAzF anti-EGFR scFv and DBCO functionalized fluorescent dyes, cyanine 5 or cyanine 7. The pAzF anti-EGFR scFv showed significant binding to recombinant EGFR in vitro and also, through flow cytometry, the target specificity of the pAzF anti-EGFR scFv-DBCO cy5 conjugates to native EGFR expressing MDA MB 468 breast cancer cells was validated in vitro. In addition, in vivo optical imaging studies performed with MDA MB 468 tumour xenograft models showed targeted accumulation of pAzF anti-EGFR scFv-DBCO cy7 in MDA MB 468 cancer, thereby establishing both the targeting and imaging potential of the conjugates. This study therefore demonstrates the incorporation of an unnatural amino acid with orthogonal chemical reactivity site-specificially into an scFv as well as the potential of unnatural amino acids to form stable antibody-imaging dye conjugates for use in tumour imaging.