Efficient Preparation of Site-Specific Antibody-Drug Conjugates Using Cysteine Insertion

Mol Pharm. 2017 May 1;14(5):1501-1516. doi: 10.1021/acs.molpharmaceut.6b00995. Epub 2017 Mar 16.


Antibody-drug conjugates (ADCs) are a class of biopharmaceuticals that combine the specificity of antibodies with the high-potency of cytotoxic drugs. Engineering cysteine residues in the antibodies using mutagenesis is a common method to prepare site-specific ADCs. With this approach, solvent accessible amino acids in the antibody have been selected for substitution with cysteine for conjugating maleimide-bearing cytotoxic drugs, resulting in homogeneous and stable site-specific ADCs. Here we describe a cysteine engineering approach based on the insertion of cysteines before and after selected sites in the antibody, which can be used for site-specific preparation of ADCs. Cysteine-inserted antibodies have expression level and monomeric content similar to the native antibodies. Conjugation to a pyrrolobenzodiazepine dimer (SG3249) resulted in comparable efficiency of site-specific conjugation between cysteine-inserted and cysteine-substituted antibodies. Cysteine-inserted ADCs were shown to have biophysical properties, FcRn, and antigen binding affinity similar to the cysteine-substituted ADCs. These ADCs were comparable for serum stability to the ADCs prepared using cysteine-mutagenesis and had selective and potent cytotoxicity against human prostate cancer cells. Two of the cysteine-inserted variants abolish binding of the resulting ADCs to FcγRs in vitro, thereby potentially preventing non-target mediated uptake of the ADCs by cells of the innate immune system that express FcγRs, which may result in mitigating off-target toxicities. A selected cysteine-inserted ADC demonstrated potent dose-dependent anti-tumor activity in a xenograph tumor mouse model of human breast adenocarcinoma expressing the oncofetal antigen 5T4.

Keywords: SG3249; antibody engineering; antibody−drug conjugates; cysteine mutagenesis; cysteine-inserted antibody; oncofetal antigen 5T4; pyrrolobenzodiazepine dimers; site-specific conjugation; tesirine.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antibodies, Monoclonal / chemistry*
  • Antibodies, Monoclonal / therapeutic use*
  • Antibodies, Monoclonal, Humanized / chemistry
  • Antibodies, Monoclonal, Humanized / therapeutic use
  • Antineoplastic Agents / chemistry*
  • Antineoplastic Agents / therapeutic use*
  • Cell Line, Tumor
  • Cysteine / chemistry*
  • Female
  • Humans
  • Immunoconjugates / chemistry
  • Immunoconjugates / therapeutic use
  • Mammary Neoplasms, Experimental / drug therapy
  • Mice
  • Mice, Nude
  • Trastuzumab / chemistry
  • Trastuzumab / therapeutic use
  • Xenograft Model Antitumor Assays


  • Antibodies, Monoclonal
  • Antibodies, Monoclonal, Humanized
  • Antineoplastic Agents
  • Immunoconjugates
  • Cysteine
  • Trastuzumab