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

Abstract

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.

• Authors: Michal Lobocki, Malgorzata Zakrzewska, Anna Szlachcic, Mateusz A Krzyscik, Aleksandra Sokolowska-Wedzina, Jacek Otlewski
• Journal: Bioconjugate chemistry (Bioconjug Chem) Vol. 28 Issue 7 Pg. 1850-1858 (07 19 2017) ISSN: 1520-4812 [Electronic] United States
• PMID: 28598150 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Antineoplastic Agents
• Oligopeptides
• Fibroblast Growth Factor 1
• Receptor, Fibroblast Growth Factor, Type 1
• Cysteine
• monomethyl auristatin E

Topics

• Amino Acid Motifs
• Animals
• Antineoplastic Agents (chemistry)
• Binding Sites
• Cell Death (drug effects)
• Cell Line, Tumor
• Cysteine (chemistry)
• Drug Delivery Systems (methods)
• Endocytosis (drug effects)
• Fibroblast Growth Factor 1 (chemistry, metabolism)
• Humans
• Oligopeptides (chemistry, pharmacokinetics, pharmacology)
• Receptor, Fibroblast Growth Factor, Type 1 (metabolism)