Recently, smart chemical modification of native antibodies attracts attention for various purposes, e.g. producing antibody-drug conjugates (ADCs) and fluorescent-labelling, because traditional random reactions between active esters and several unspecific Lys residues sometimes impede antibody performance due to modifications at undesired position. Thus, many site-specific modification strategies are reported (1). Under these circumstances, our group created CCAP (chemical conjugation by affinity peptide) method in 2019 exploiting affinity peptides against Fc moiety of native IgG. This method enabled modification at very specific Lys residues in the hinge moiety of the antibody (2,3). Furthermore, based on this methodology, AJICAP method has been established (4). All these methods adopt specially designed “CCAP reagents” consisting of three moieties: payload, affinity peptide, and active ester. By mixing such reagents and native IgG antibodies, CCAP reagent containing affinity peptides is site-specifically attached to the antibody via a covalent bond formed between active ester and the specific Lys residues of the antibody.
Here we report next generation tCAP reagents containing payload, active acyl donor, and affinity peptide in this order for payload “transfer”. By mixing tCAP reagents and native IgG antibodies, payload such as Ac-Lys(N3)-Gly-Gly was efficiently transferred to antibodies. Moreover, because self-activatable latent acyl donor structure was adopted in tCAP reagents, long shelf-life was also achieved. Eventually, site-specifically di-azidated IgG antibodies thus obtained were further modified with several DBCO compounds by standard strain-promoted azide-alkyne cycloaddition (SPAAC) to afford divalently modified antibody conjugates. We also discuss several characteristics of obtained antibody conjugates including affinities with Fc receptors. This simple but robust technology for modification of native IgG antibodies would be important toward further industrial and pharmaceutical applications.