title: 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.
authors: Abzena plc., Babraham Research Campus, Babraham, CambridgeCB22 3AT, UK.
authors: Tim D
authors: TD
authors: Jones
authors: Abzena plc., Babraham Research Campus, Babraham, CambridgeCB22 3AT, UK.
authors: Arron R
authors: AR
authors: Hearn
authors: Abzena plc., Babraham Research Campus, Babraham, CambridgeCB22 3AT, UK.
authors: Robert G E
authors: RGE
authors: Holgate
authors: Abzena plc., Babraham Research Campus, Babraham, CambridgeCB22 3AT, UK.
authors: Dorota
authors: D
authors: Kozub
authors: Abzena plc., Babraham Research Campus, Babraham, CambridgeCB22 3AT, UK.
authors: Mark H
authors: MH
authors: Fogg
authors: Abtelum Biomedical, Inc. 175 Briar Lane, Westwood, MA 02090, USA.
authors: Francis J
authors: FJ
authors: Carr
authors: Abzena plc., Babraham Research Campus, Babraham, CambridgeCB22 3AT, UK.
authors: Matthew P
authors: MP
authors: Baker
authors: Departamento de Bioquimica y Biologia Molecular I, Facultad de Ciencias Químicas, Universidad Complutense de Madrid, Avenida Complutense s/n, Madrid 28040, Spain.
authors: Javier
authors: J
authors: Lacadena
authors: Research Corporation Technologies Inc., 5210 E. Williams Circle #240, Tucson, AZ 85711, USA.
authors: Kurt R
authors: KR
authors: Gehlsen
keywords: T cell epitope; deimmunisation; immunogenicity; immunotoxin; α-sarcin