Site‐specific Bioconjugation and Convergent Click Chemistry Enhances Antibody–Chromophore Conjugate Binding Efficiency

Photosensitizer (PS)-antibody conjugates (photoimmunoconjugates, PICs) enable cancer-cell targeted photodynamic therapy (PDT). Non-specific chemical bioconjugation is widely used to synthesize PICs but gives rise to several shortcomings. The conjugates are heterogenous, and the process is not easily reproducible. Moreover, modifications at or near the binding sites alter both binding affinity and specificity. To overcome these limitations, we introduce convergent assembly of PICs via a chemo-enzymatic site-specific approach. First, an antibody is conjugated to a clickable handle via site-specific modification of glutamine (Gln) residues catalyzed by transglutaminase (TGase, EC 2.3.2.13). Second, the modified antibody intermediate is conjugated to a compatible chromophore via click chemistry. Utilizing cetuximab, we compared this site-specific conjugation protocol to the non-specific chemical acylation of amines using N-hydroxysuccinimide (NHS) chemistry. Both the heavy and light chains were modified via the chemical route, whereas, only a glutamine 295 in the heavy chain was modified via chemo-enzymatic conjugation. Furthermore, a 2.3-fold increase in the number of bound antibodies per cell was observed for the site-specific compared to non-specific method, suggesting that multiple stochastic sites of modification perturbs the antibody-antigen binding. Altogether, site-specific bioconjugation leads to homogenous, reproducible, and well-defined PICs, conferring higher binding efficiency and probability of clinical success.