A deimmunised form of the ribotoxin, ?-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. ?-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 ?-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 ?-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 ?-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 ?-sarcin. These results represent the first fungal ribotoxin to be deimmunised with the potential to construct a new generation of deimmunised immunotoxin therapeutics.

SUBMITTER: Jones TD 

PROVIDER: S-EPMC5081043 | biostudies-literature | 2016 Nov

REPOSITORIES: biostudies-literature