Traditionally, non-specific chemical conjugation, such as acylation of amines on lysine or alkylation of thiols on cysteines, are widely used; however, they have several shortcomings. First, the lack of site-specificity results in heterogeneous products and irreproducible processes. Second, potential modifications near the complementarity determining region (CDR) may reduce binding affinity and specificity. Conversely, site-specific methods produce well-defined and more homogenous antibody conjugates, ensuring developability and clinical applications. Moreover, several recent side-by-side comparisons of site-specific and stochastic methods have demonstrated that site-specific approaches are more likely to achieve their desired properties and functions, such as increased plasma stability, less variability in dose-dependent studies (particularly at low concentrations), enhanced binding efficiency, as well as increased tumor uptake. Herein we review several standard and practical site-specific bioconjugation methods for native antibodies, i.e., those without recombinant engineering. First, chemo-enzymatic techniques, namely transglutaminase (TGase)-mediated transamidation of a conserved glutamine residue and glycan remodeling of a conserved asparagine N-glycan (GlyCLICK), both in the Fc region. Second, chemical approaches such as selective reduction of disulfides (ThioBridge) and N-terminal amine modifications. Furthermore, we list site-specific antibody-drug conjugates (ADCs) in clinical trials along with the future perspectives of these site-specific methods.
Keywords: antibody-drug conjugate (ADC); bioconjugation; glycan remodeling; hybrid modality; transglutaminase.