Abstract
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.
Highlights
Cancer therapies have significantly improved in recent years due to the development of antibody-based therapeutics that confer high selectivity for either direct tumor targeting [1], or for the stimulation of anti-tumor immunity [2]
The CD30-specific chimeric monoclonal antibodies (mAbs) cAc10 [33], principal component of the marketed Antibody Drug Conjugates (ADCs) brentuximab vedotin (Adcetris) [4], was produced with each of its C-termini tagged with a sortase A recognition motif, and a Strep II affinity purification tag [34]
Sortase-enzyme mediated conjugation of small molecular weight toxic payloads to full-length antibodies represents a novel approach for the development of site- conjugated ADCs
Summary
Cancer therapies have significantly improved in recent years due to the development of antibody-based therapeutics that confer high selectivity for either direct tumor targeting [1], or for the stimulation of anti-tumor immunity [2]. Due to the high immunogenicity of the bacterial toxins in humans, immunotoxins are typically associated with a significant immune response, limiting repeated treatment cycles [7]. Since ADCs typically consist largely of human sequences their immunogenicity is manageable [10,11], and numerous treatment cycles are typically possible. Given this specific advantage of ADCs and the commercial success of the two ADC approved for therapy, more than 30 ADCs are currently at various stages of clinical development [12,13]
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