Engineering THIOMABs for site-specific conjugation of thiol-reactive linkers

Methods Mol Biol. 2013:1045:189-203. doi: 10.1007/978-1-62703-541-5_11.

Abstract

Antibody conjugates are used in many therapeutic and research applications and are generated by chemically linking a cysteine or lysine residue to potent chemotherapeutic drugs or other functional groups through a flexible linker. Recently, we have engineered THIOMABs (antibodies with engineered reactive cysteine residues) for site-specific conjugation and showed that these antibody conjugates display homogeneous labeling with optimal in vitro and in vivo characteristics. Here, we describe protocols for engineering, selection, and site-specific conjugation of THIOMABs with thiol-reactive linkers.

MeSH terms

  • Binding Sites
  • Cysteine / chemistry*
  • HEK293 Cells
  • Humans
  • Immunoconjugates / chemistry*
  • Immunoconjugates / genetics*
  • Mutagenesis, Site-Directed
  • Protein Engineering / methods*
  • Substrate Specificity

Substances

  • Immunoconjugates
  • Cysteine