Sortase Enzyme-Mediated Generation of Site-Specifically Conjugated Antibody Drug Conjugates with High In Vitro and In Vivo Potency

PLoS One. 2015 Jul 1;10(7):e0131177. doi: 10.1371/journal.pone.0131177. eCollection 2015.


Antibody drug conjugates (ADCs) have recently been proven to be highly potent anti-tumor drugs, typically exceeding the efficacy of conventional monoclonal antibodies (mAbs). ADCs are currently produced by chemical conjugation of a small-molecule toxin to the mAb through lysine or cysteine side chains. This leads to heterogeneous mixtures of ADCs in which variable numbers of drugs are conjugated to individual antibodies and in which the site of conjugation cannot be defined. Consequently, there is currently significant interest in further development of drug conjugation technologies, with a particular focus on site-specific payload conjugation. Here, we present an enzymatic conjugation platform based on the S. aureus sortase A-mediated transpeptidation reaction, allowing the efficient generation of ADCs with toxins conjugated to pre-defined sites at pre-defined drug-to-antibody ratios. For this, two modifications were introduced: first, immunoglobulin heavy (IgH) and light (IgL) chains were modified at their C-termini by addition of the sortase A recognition motif LPETG, and second, the small molecule tubulin polymerization inhibitors monomethylauristatin E (MMAE) and maytansine were modified by addition of a pentaglycine peptide, thus making them suitable substrates for sortase A-mediated transpeptidation. We demonstrate efficient generation and characterization of the anti-CD30 ADC Ac10-vcPAB-MMAE, an enzymatically conjugated counterpart of brentuximab vedotin (Adcetris), as well as several anti-HER-2 ADCs including trastuzumab-maytansine, the counterpart of trastuzumab emtansine (Kadcyla). ADCs generated in this manner were found to display in vitro cell killing activities indistinguishable from the classic conjugates. Further, when tested in vivo in a HER-2-overexpressing ovarian cancer xenograft mouse model, enzymatically generated trastuzumab-maytansine was found to lead to complete regression of established tumors, similar to Kadcyla.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Ado-Trastuzumab Emtansine
  • Aminoacyltransferases / chemistry*
  • Aminoacyltransferases / immunology
  • Animals
  • Antibodies, Monoclonal / chemistry
  • Antibodies, Monoclonal / immunology
  • Antibodies, Monoclonal / pharmacology*
  • Antibodies, Monoclonal, Humanized / chemistry
  • Antibodies, Monoclonal, Humanized / immunology
  • Antibodies, Monoclonal, Humanized / pharmacology*
  • Antineoplastic Agents / chemistry
  • Antineoplastic Agents / immunology
  • Antineoplastic Agents / pharmacology*
  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / immunology
  • Brentuximab Vedotin
  • Cysteine Endopeptidases / chemistry*
  • Cysteine Endopeptidases / immunology
  • Female
  • Humans
  • Immunoconjugates / chemistry
  • Immunoconjugates / immunology
  • Immunoconjugates / pharmacology*
  • Ki-1 Antigen / antagonists & inhibitors
  • Ki-1 Antigen / genetics
  • Ki-1 Antigen / immunology
  • Maytansine / analogs & derivatives*
  • Maytansine / chemistry
  • Maytansine / immunology
  • Maytansine / pharmacology
  • Mice
  • Mice, Nude
  • Oligopeptides / chemistry
  • Oligopeptides / immunology
  • Ovarian Neoplasms / drug therapy*
  • Ovarian Neoplasms / immunology
  • Ovarian Neoplasms / pathology
  • Protein Engineering
  • Receptor, ErbB-2 / antagonists & inhibitors
  • Receptor, ErbB-2 / genetics
  • Receptor, ErbB-2 / immunology
  • Staphylococcus aureus / chemistry
  • Staphylococcus aureus / enzymology
  • Trastuzumab
  • Xenograft Model Antitumor Assays


  • Antibodies, Monoclonal
  • Antibodies, Monoclonal, Humanized
  • Antineoplastic Agents
  • Bacterial Proteins
  • Immunoconjugates
  • Ki-1 Antigen
  • Oligopeptides
  • Maytansine
  • Brentuximab Vedotin
  • Aminoacyltransferases
  • sortase A
  • ERBB2 protein, human
  • Receptor, ErbB-2
  • Cysteine Endopeptidases
  • Trastuzumab
  • Ado-Trastuzumab Emtansine
  • monomethyl auristatin E

Grant support

NBE-Therapeutics AG provided support in the form of salaries for authors R.R.B., T.H., A.S.M. and U.G., but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.