A new trastuzumab-ageritin-based immunotoxin that specifically kills breast cancer cells.

Antibody-drug conjugates (ADCs) and other similar drug conjugates, constitute a new class of highly effective therapeutic agents, many of which are clinically approved. A subset of ADCs are immunotoxins (ITs) which contain a protein toxin instead of small cytotoxic agents. Several toxins have been investigated, including small proteins from fungi that inhibit protein synthesis by cleaving a phosphodiester bond within the conserved alpha-sarcin loop (SRL) of 23-28S rRNAs. We prepared a prototypical immunotoxin obtained by linking the ribotoxin-like ageritin to the Fab' free cysteines liberated by pepsin treatment of full antibody. The resulting molecule containing two copies of ageritin linked to 1 copy of Fab' (named ageritin2-Fab) was obtained via a three-step site-specific conjugation approach involving alkylation of the toxin free cysteine, enzyme-mediated introduction of a maleimide group and final alkylation of Fab' two cysteines. Ageritin2-Fab was isolated together with a rough 30% Fab' linked to one copy of ageritin. The resulting mixed product, named Fab-ageritin IT, shows cytotoxicity on a Her2-positive breast cancer cell line that is superior to that of the isolated Fab' and ageritin, due to Fab'-driven toxin internalization. Data suggest that the Fab-ageritin IT and the individual purified components preserve the toxin and Fab' activity but show enhanced cell toxicity, thus resulting in a structurally well-defined, target-specific new type of immunotoxin. The scheme outlined for its preparation is easily extendable to other therapeutic IgG1 thus providing a significant contribution to the ongoing effort to explore new combinations of payloads and targeting platforms for next-generation immunotoxins.