A universal approach to the construction of antibody-drug conjugates (ADCs) has been developed. It relies on periodate oxidation of naturally present glycans of immunoglobulin G, followed by oxime ligation and, optionally, copper(I)-catalyzed alkyne-azide cycloaddition for conjugation with a toxic payload. The introduction of highly absorbing cyanine dyes into the linker allows for facile determination of the drug-antibody ratio. We applied this methodology to the synthesis of cytotoxic conjugates of an antibody against the tumor-associated antigen PRAME with doxorubicin and monomethyl auristatin E (MMAE). The resultant conjugates retained their affinity to a large extent, yet their cytotoxicity in vitro varied dramatically: while the doxorubicin-based conjugate did not produce any effect on cells, the MMAE-based one demonstrated specific activity against PRAME-expressing cancer cell lines. Importantly, the latter conjugate constitutes the first reported example of a PRAME-targeting ADC.

Fluorescent antibodies have proved to be an invaluable tool for molecular biology and diagnostics. They are routinely produced by modification of lysine residues, which leads to high heterogeneity. As such, their affinity may be compromised if the antigen-binding site is affected, the probability of which increases along with the degree of labeling. In this work, we propose a methodology for the synthesis of site-specific antibody-dye conjugates with a high degree of labeling. To this end, we synthesized two oxyamine-based branched triazide linkers and coupled them with a periodate-oxidized anti-PRAME antibody 6H8; two oxyamine-based linear monoazide linkers of similar structure were used as controls. The azide-labeled antibodies were subsequently conjugated with fluorescent dyes via SPAAC, a copper-free click reaction. Compared to their counterparts made with linear linkers, the branched conjugates possessed a higher degree of labeling. The utility of the methodology was demonstrated in the detection of the PRAME protein on the surface of the cell by flow cytometry.