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

作者：Jones Tim D; Hearn Arron R; Holgate Robert G E; Kozub Dorota; Fogg Mark H; Carr Francis J; Baker Matthew P; Lacadena Javier; Gehlsen Kurt R^*

来源：Protein Engineering Design and Selection, 2016, 29(11): 531-540.

DOI：10.1093/protein/gzw045

• alpha-sarcin
• deimmunisation
• immunogenicity
• immunotoxin
• T cell epitope

Fungal ribotoxins that block protein synthesis can be useful warheads in the context of a targeted immunotoxin. alpha-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 alpha-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 alpha-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 alpha-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 alpha-sarcin. These results represent the first fungal ribotoxin to be deimmunised with the potential to construct a new generation of deimmunised immunotoxin therapeutics.

• 出版日期2016-11