A Chemical Approach Toward the Therapeutic Optimization of Cytotoxic Bioconjugates

The delivery of cytotoxicity to specific cells was recognized to be the key to the effective, chemical treatment of infectious and neoplastic diseases well over a century ago. In the context of oncology, the primary therapeutic challenge is the distinction between healthy and pathological cells despite their pronounced pheno- and genotypic similarity. Bioconjugates, such as antibody-drug conjugates (ADCs), are equipped to solve the issue by virtue of their orthogonal make-up: A protein carrier (antibody) confers tumor selectivity, while a small molecule (“drug”) delivers cytotoxicity. The thesis at hand addresses the analytical challenges which are consequences of an ADC’s orthogonal structure. Paper I critically compares the available methods for the assessment of the stoichiometric ratio between antibody and drug (the drug-antibody ratio, DAR). It concludes with a series of recommendations aimed at entrants new to the field. Paper II introduces a novel method for the removal of small molecule impurities prior to mass-spectrometric analysis. Using the outlined procedure, it is possible to remove almost all potentially interferential buffer components while recovering approximately 90% of the analyte. Synthetic research lies at the thesis’s core. Paper III describes the design and biological evaluation of a series of novel cytotoxins (“azastatins”), whose total synthesis was accomplished in 22-23 steps. The compounds are highly potent inhibitors of tubulin polymerization and appear to only possess cellular permeability while protein-bound, thus eliminating the risk of unintended harm to healthy tissues. Moreover, unpublished research details an ultimately unsuccessful attempt at a solid-phase method for the synthesis of a peptide linker commonly used to attach cytotoxic drugs to antibodies. Satisfactory yields for the detachment of the target compounds from the resin were not achieved, which was rationalized based on pseudo-intramolecular, irreversible solid-phase reattachment. Paper IV introduces a novel methodology for the bioconjugation of small molecules to proteins by means of Wittig chemistry. The procedure afforded nine different conjugates of two different proteins in moderate to excellent yields and may allow for the subsequent functionalization of the newly generated, highly electron deficient alkenes. It is potentially applicable to the challenging generation of protein heterodimers.Prof. Neri has left ETH Zürich in September 2020 and now serves as CEO and CSO of Philogen S.p.A.