5 research outputs found
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 antibodyâdrug 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 LysâCysâLys 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 FGF1âvcMMAE 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