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

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Jones, T.D. and Hearn, A.R. and Holgate, R.G. and Kozub, D. and Fogg, M.H. and Carr, F.J. and Baker, M.P. and Lacadena, Javier and Gehlsen, K.R. (2016) A deimmunised form of the ribotoxin, α-sarcin, lacking CD4+ T cell epitopes and its use as an immunotoxin warhead. Protein Engineering, Design & Selection . pp. 1-10. ISSN 1741-0134 (Online) ; 1741-0126 (Print)

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Official URL: http://peds.oxfordjournals.org/content/early/2016/08/30/protein.gzw045



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.


Item Type:Article
Uncontrolled Keywords:α-sarcin, deimmunisation, immunogenicity, immunotoxin, T cell epitope
Subjects:Medical sciences > Biology > Biochemistry
ID Code:39126
Deposited On:21 Sep 2016 10:43
Last Modified:23 Sep 2016 07:59

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