A Versatile Approach for the Site‐Specific Modification of Recombinant Antibodies Using a Combination of Enzyme‐Mediated Bioconjugation and Click Chemistry

Abstract

Au nique two-step modular system for site-specific antibody modification and conjugation is reported. The first step of this approach uses enzymatic bioconjugation with the transpeptidase Sortase Af or incorporation of strained cyclooctyne functional groups.T he second step of this modular approach involves the azide-alkyne cycloaddition click reaction. The versatility of the two-step approach has been exemplified by the selective incorporation of fluorescent dyes and apositron-emitting copper-64 radiotracer for fluorescence and positron-emission tomography imaging of activated platelets,p latelet aggregates,a nd thrombi, respectively.T his flexible and versatile approach could be readily adapted to incorporate al arge arrayo ft ailor-made functional groups using reliable click chemistry whilst preserving the activity of the antibody or other sensitive biological macromolecules.

Sortase A‐mediated site‐specific labeling of camelid single‐domain antibody‐fragments: a versatile strategy for multiple molecular imaging modalities

“…Not surprisingly, SrtA‐mediated conjugation also found its way into the field of in vivo molecular imaging. So far it was employed for the functionalization of scFv's (single‐chain variable fragment) and sdAbs with iron oxide particles for use in magnetic resonance imaging (MRI) , microbubbles for ultrasound imaging and the radiolabels 64 Cu and 18 F for PET . Here we applied SrtA‐mediated conjugation for the site‐specific labeling of sdAbs with 111 In, 68 Ga and Cy5 to produce imaging tracers for SPECT, PET and FRI, respectively.…”

Sortase-click strategy for defined protein conjugation on a heptavalent cyclodextrin scaffold

“…Recently, we exploited the peptide ligation propensity of transpeptidase sortase for installing orthogonal groups compatible with click chemistry for synthesis of chemically defined protein dendrimers [18] . A similar two-step approach using a combination of sortase-mediated labelling and click chemistry was earlier employed for site-specific modification of recombinant antibodies [26] .…”

Site-specific chelator-antibody conjugation for PET and SPECT imaging with radiometals

“…Bacterial enzyme sortase A ( SrtA ) catalyses a transpeptidase reaction between an N-terminal glycine, and a specific amino acid sequence, -Lys-Pro-X-Thr-Gly- (where X is any amino acid) [117] , [118] , [119] . SrtA (i) recognizes and cleaves this peptide sequence between threonine and glycine, and (ii) catalyses the formation of a new amide bond between threonine and an N-terminal glycine-containing species.…”

“…6c) [119]. In the second, the same scFv (in the presence of Srt A) was reacted with an N-terminal glycine attached to a strained cyclooctyne [118]. Once purified, the new scFv-cyclooctyne bioconjugate was reacted chemoselectively with an azide appended to a sarcophagine ligand.…”