A deimmunised form of the ribotoxin, α-sarcin, lacking CD4⁺T cell epitopes and its use as an immunotoxin warhead

Abstract

Fungal ribotoxins that block protein synthesis can be useful warheads in the context of a targeted immunotoxin. α-Sarcin is a small (17 kDa) fungal ribonuclease produced by Aspergillus giganteus that functions by catalytically cleaving a single phosphodiester bond in the sarcin–ricin loop of the large ribosomal subunit, thus making the ribosome unrecognisable to elongation factors and leading to inhibition of protein synthesis. Peptide mapping using an ex vivo human T cell assay determined that α-sarcin contained two T cell epitopes; one in the N-terminal 20 amino acids and the other in the C-terminal 20 amino acids. Various mutations were tested individually within each epitope and then in combination to isolate deimmunised α-sarcin variants that had the desired properties of silencing T cell epitopes and retention of the ability to inhibit protein synthesis (equivalent to wild-type, WT α-sarcin). A deimmunised variant (D9T/Q142T) demonstrated a complete lack of T cell activation in in vitro whole protein human T cell assays using peripheral blood mononuclear cells from donors with diverse HLA allotypes. Generation of an immunotoxin by fusion of the D9T/Q142T variant to a single-chain Fv targeting Her2 demonstrated potent cell killing equivalent to a fusion protein comprising the WT α-sarcin. These results represent the first fungal ribotoxin to be deimmunised with the potential to construct a new generation of deimmunised immunotoxin therapeutics.

Fungal Ribotoxins: A Review of Potential Biotechnological Applications

“…This result can be considered the proof of concept that immunotoxins based on ribotoxins may be a unique therapeutic tool against different tumor pathologies. Moreover, an immunotoxin containing a deimmunized variant of α-sarcin showing a complete lack of T cell activation in in vitro assays have been recently described [ 76 ]. These results support the design of a new generation of therapeutic deimmunized ribotoxins-based immunotoxins.…”

Inclusion of a Furin Cleavage Site Enhances Antitumor Efficacy against Colorectal Cancer Cells of Ribotoxin α-Sarcin- or RNase T1-Based Immunotoxins

“…Interestingly, ribonucleases (RNases) have acquired a significant importance due to their ideal features for being included as immunotoxin toxic domains [27,28,29,30,31,32]. In particular, ribotoxins stand out within the family of extracellular fungal RNases, as part of the toxic domain of immunotoxins, due to their small size, high thermostability and resistance to proteases, poor immunogenicity, and especially because they are highly effective to inactivate ribosomes [33,34,35,36,37,38]. As proven by the previous results obtained within α-sarcin-based immunotoxins, α-sarcin arises as the most promising ribotoxin to be included in these antitumoral therapeutic designs [36,38,39,40].…”

A novel Carcinoembryonic Antigen (CEA)-Targeted Trimeric Immunotoxin shows significantly enhanced Antitumor Activity in Human Colorectal Cancer Xenografts

“…Recently, the use of α-sarcin as an suitable toxic moiety in recombinant immunotoxins against colorectal cancer has been reported. This immunotoxin showed potent and specific antitumor effectiveness, due to a decrease in cancer cell proliferation and tumor angiogenesis and increased in apoptotic activity, while avoiding unspecific and cytotoxicity against normal cells 25,46 . At the same time, toxin accumulation within targeted cells was considerably increased by the high-affinity binding of the scFv antibody domain 16,25 .…”

“…In this sense, within the family of extracellular fungal ribonucleases, ribotoxins stand out for their potential use as part of immunotoxins because of their small size, high thermostability, poor immunogenicity, resistance to proteases, and their highly efficient ability to inactivate ribosomes 19,25,41–44 . As proved by its functional properties and previous uses within immunotoxins, α-sarcin seems to be one of the most promising candidate to be included in immunotoxin therapeutic designs 16,19,25,45,46 . Its specific ribonucleolytic activity against one single bond, strategically located at the large rRNA, causes ribosome inactivation and, thereby, protein biosynthesis inhibition and apoptosis 47–49 .…”