During the last decade monoclonal antibodies (mAbs) have been proven to be the most effective macromolecular therapeutics for the treatment of cancer and inflammatory diseases. Despite all success there are a plethora of attempts to further improve the therapeutic potential of this class of compounds by structural modifications and derivatizations. One such strategy is the covalent conjugation of small cytotoxic molecules to the protein amino acid side-chains, resulting in so-called antibody-drug conjugates (ADCs). ADCs are thought to bind to tumor-associated cell surface antigens, followed by endosmal-lysosomal internalization and intracellular drug release from vesicles into cytoplasm. Thus ADCs combine the extraordinary affinity and specificity of mAbs with the antitumoral potential of highly-toxic small molecules.

Heidelberg Pharma has developed a new ADC technology based on the most potent RNA-polymerase II inhibitor amanitin, a bicyclic octapeptide isolated from the Green Deathcap mushroom Amanita phalloides. Amanitin interferes with the eukaryotic transcription process at very low intracellular concentrations, making it an ideal drug for the use with antibodies binding at low-copy number antigens. Heidelberg Pharma and its cooperation partners have shown outstanding activity of amanitin-based ADCs in therapy-resistant tumor cells, e.g. cells expressing multi-drug resistant transporters, tumor-initiating cells and non-dividing cells at picomolar concentrations. The tolerability and therapeutic window of amanitin-ADCs has been determined in different rodent and non-human primate models. Furthermore, amanitin has a water-soluble structure, resulting in ADCs with low tendency for aggregation, even using higher drug to antibody ratios.

A prerequisite for an effective ADC is the conjugation of the drug moiety to the antibody by a chemical linker group that provides high stability in circulation and substantial drug release after internalization into tumor cells. Heidelberg Pharma has identified sites in amanitin that are suitable for linker integration without diminishing its binding to the target enzyme RNA polymerase II. The linker structures were sucessfully modified with reducible disulfide groups and protease-cleavage sites and thus optimized for the individual antibody structure.