Biotechnol Adv. 2015 Nov 1;33(6 Pt 1):775-84. doi: 10.1016/j.biotechadv.2015.05.001.

Current methods for the synthesis of homogeneous antibody-drug conjugates.

Sochaj AM1, Świderska KW1, Otlewski J2.
  • 1Faculty of Biotechnology, Department of Protein Engineering, University of Wroclaw, Joliot-Curie 14a, 50-383 Wroclaw, Poland.
  • 2Faculty of Biotechnology, Department of Protein Engineering, University of Wroclaw, Joliot-Curie 14a, 50-383 Wroclaw, Poland. Electronic address:



Development of efficient and safe cancer therapy is one of the major challenges of the modern medicine. Over the last few years antibody-drug conjugates (ADCs) have become a powerful tool in cancer treatment with two of them, Adcetris® (brentuximab vedotin) and Kadcyla® (ado-trastuzumab emtansine), having recently been approved by the Food and Drug Administration (FDA). Essentially, an ADC is a bioconjugate that comprises a monoclonal antibody that specifically binds tumor surface antigen and a highly potent drug, which is attached to the antibody via either cleavable or stable linker. This approach ensures specificity and efficacy in fighting cancer cells, while healthy tissues remain largely unaffected. Conventional ADCs, that employ cysteine or lysine residues as conjugation sites, are highly heterogeneous. This means that the species contain various populations of the ADCs with different drug-to-antibody ratios (DARs) and different drug load distributions. DAR and drug-load distribution are essential parameters of ADCs as they determine their stability and efficacy. Therefore, various drug-loaded forms of ADCs (usually from zero to eight conjugated molecules per antibody) may have distinct pharmacokinetics (PK) in vivo and may differ in clinical performance. Recently, a significant progress has been made in the field of site-specific conjugation which resulted in a number of strategies for synthesis of the homogeneous ADCs. This review describes newly-developed methods that ensure homogeneity of the ADCs including use of engineered reactive cysteine residues (THIOMAB), unnatural amino acids, aldehyde tags, enzymatic transglutaminase- and glycotransferase-based approaches and novel chemical methods. Furthermore, we briefly discuss the limitation of these methods emphasizing the need for further improvement in the ADC design and development.

KEYWORDS: ADCs; Cytotoxic agents; Homogeneity; Monoclonal antibodies; Site-specific conjugation; Targeted cancer therapy

PMID: 25981886



Monoclonal antibodies against tumor markers can specifically deliver cytotoxic payloads to cancer cells. This reduces undesired effect of the treatment and enable to use drugs which are a thousand times more potent than conventional chemotherapeutics. An intense work on ADCs (Antibody-Drug Conjugates) began in mid-1980s. Currently, two ADCs, brentuximab vedotin (Seattle Genetics) and ado-trastuzumab emtansine (Genentech) are approved by FDA for marketing and more than 40 are being evaluated in clinical trials. ADCs are very powerful and promising tools for fighting cancer, but they still require further improvement to ensure efficacy and safety of treatment.

Conventional ADCs, including brentuximab vedotin and ado-trastuzumab emtansine are based on  non-specific conjugation of a cytotoxic moiety to the antibody. This result in a mixture of ADCs species which have different drug loading and different drug positions (Figure 1). Heterogeneity of the ADC therapeutics impairs their pharmacokinetics and efficacy (1, 2). Over the last few years researchers have proposed various methods which allowed them to obtain homogeneous ADCs. In this review we present and discuss these new approaches which can potentially result in approval of the first site-specific ADCs.

In our laboratory we are also developing site-specific cytotoxic conjugates. Our construct is based on a small molecule called affibody that recognizes HER2 receptor on cancer cells (3). The affibody does not contain cysteines in its amino acid sequence. To generate a site-specific conjugate we introduced a single cysteine to the affibody and took advantage of maleimide chemistry to conjugate drug molecule. Currently, the affibody-based conjugate is being tested in our laboratory.


Figure 1 Supplement



1) Hamblett, K.J., Senter, P.D., Chace, D.F., et al., 2004. Effects of drug loading on the antitumor activity of a monoclonal antibody drug conjugate. Clin. Cancer Res. 10, 7063–707

2) Shen, B.Q., Xu, K., Liu, L., et al., 2012. Conjugation sitemodulates the in vivo stability and therapeutic activity of antibody–drug conjugates. Nat. Biotechnol. 30, 184–189

3) Feldwisch J., Tolmachev V., Lendel C., et al. 2010. Design of an optimized scaffold for affibody molecules. J Mol Biol. 398, 232-247.



The study was supported by supported by the National Science Centre, Poland (grant number 2013/08/S/NZ1/00845). AMS is a fellow of the National Science Centre, Poland.



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