High-Yield Site-Specific Conjugation of Fibroblast Growth Factor 1 with Monomethylauristatin E via Cysteine Flanked by Basic Residues

Site-specific conjugation is a leading trend in the development of protein conjugates, including antibody–drug conjugates (ADCs), suitable for targeted cancer therapy. Here, we present a very efficient strategy for specific attachment of a cytotoxic drug to fibroblast growth factor 1 (FGF1), a natural ligand of FGF receptors (FGFRs), which are over-expressed in several types of lung, breast, and gastric cancers and are therefore an attractive molecular target. Recently, we showed that FGF1 fused to monomethylauristatin E (vcMMAE) was highly cytotoxic to cells presenting FGFRs on their surface and could be used as a targeting agent alternative to an antibody. Unfortunately, conjugation via maleimide chemistry to endogenous FGF1 cysteines or a cysteine introduced at the N-terminus proceeded with low yield and led to nonhomogeneous products. To improve the conjugation, we introduced a novel Lys–Cys–Lys motif at either FGF1 terminus, which increased cysteine reactivity and allowed us to obtain an FGF1 conjugate with a defined site of conjugation and a yield exceeding 95%. Using FGFR-expressing cancer lines, we confirmed specific cytotoxity of the obtained C-terminal FGF1–vcMMAE conjugate and its selective endocytososis as compared with FGFR1-negative cells. This simple and powerful approach relying on the introduction of a short sequence containing cysteine and positively charged amino acids could be used universally to improve the efficiency of the site-specific chemical modification of other proteins