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

Bioconjug Chem. 2017 Jul 19;28(7):1850-1858. doi: 10.1021/acs.bioconjchem.7b00158. Epub 2017 Jun 23.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 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*
  • Fibroblast Growth Factor 1 / metabolism
  • Humans
  • Oligopeptides / chemistry*
  • Oligopeptides / pharmacokinetics
  • Oligopeptides / pharmacology
  • Receptor, Fibroblast Growth Factor, Type 1 / metabolism

Substances

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