A Reagent for Amine‐Directed Conjugation to IgG1 Antibodies

Abstract

Functionalized antibodies are an indispensable resource for diagnosis, therapy and as a research tool for chemical biology. However, simpler and better methodologies are often required to improve the labeling of antibodies in terms of selectivity and scalability. Herein, we report the development of an easily available chemical reagent that allows site‐directed labeling of native human IgG1 antibodies in good yield and mono‐labeling selectivity. The salicylaldehyde moiety of the reagent reacts with surface exposed lysine residues to transiently form an iminium ion, and this positions a semi‐reactive ester in proximity of a second lysine residue that reacts with the ester to form an amide. Interestingly, it appears that the formation of the iminium ion also has a significant activating effect of the ester. We use flow cytometry and bio‐layer interferometry to confirm that the labeled antibodies retain antigen binding.

Reactivity and Selectivity Principles in Native Protein Bioconjugation

“…Ottmann and coworkers argued that the imine generation could act as a molecular glue to hold the protein interactome [40] . In a recent contribution, Gothelf and coworkers used the LDM concept for linchpin‐directed conjugation to IgG1 antibodies [41] . Moreover, it would be interesting to see how the LDM platform enables the development of covalent inhibitors.…”

A Wireframe DNA Cube: Antibody Conjugate for Targeted Delivery of Multiple Copies of Monomethyl Auristatin E

“…The DNA antibody construct is prepared using a lysine directed labeling reagent (LDLR) recently published from our lab. [44] The method allows for easy fabrication of antibodies functionalized with azides, with high control of number of labels pr. antibody.…”

“…Before using the Cube-C MMAE -Ab construct for cell cytotoxicity, we wanted to ensure that the antibody retains antigen binding. As the conjugation approach used is not 100 % site specific, [44] and considering the size of the attachment, we were concerned that the antibody might be sterically to hindered to bind its antigen favorably. To investigate the antigen binding of the cube antibody construct we prepared a Sulfo-Cyanine 3 (Cy3) loaded wireframe DNA cube.…”