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

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.Depto. de Bioquímica y Biología MolecularFac. de Ciencias QuímicasTRUEpu

DIFFERENTIAL NEURAL CREST CELL ATTACHMENT AND MIGRATION ON AVIAN LAMININ ISOFORMS

A number of laminin isoforms have recently been identified and proposed to exert different functions during embryonic development. In the present study, we describe the purification and partial characterization of several isoforms isolated from chick heart and gizzard, and provide data on the molecular mechanisms underlying the interaction of avian neural crest cells with these molecules in vitro. Laminins extracted from heart and gizzard tissues were separated by gel filtration and purified to homogeneity by sequential lectin and immunoaffinity chromatography by utilizing monoclonal antibodies directed against the avian alpha 2, beta 2 and gamma 1 laminin chains. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) banding pattern of the polypeptide complexes obtained and immunoblotting with polyclonal antisera allowed the identification of Laminin-2 (alpha 2 beta 1 gamma 1), Laminin-4 (alpha 2 beta 2 gamma 1), and laminins comprising the beta 1, beta 2 and gamma 1 chains associated with a shorter alpha chain which, in SDS-PAGE, co-migrate with the beta/gamma complex in the 200 kDa region. These latter laminins, which are here arbitrarily denoted Laminin-alpha x (heart tissue) and Laminin-G (gizzard tissue), are somewhat distinct in their apparent molecular weight, are differentially associated with nidogen, and appear as "T"-shaped particles similar to Laminin-6 and Laminin-7 when analyzed by transmission electron microscopy following rotary shadowing. In contrast, the avian Laminin-2 and Laminin-4 isoforms exhibit the characteristic cruciform shape described previously for their mammalian counterparts. Isolated neural crest cells differentially attached and migrated on these laminin isoforms, showing a clear preference for Laminin-G. Similarly to the EHS Laminin-1, neural crest cells recognized all avian isoforms through their alpha 1 beta 1 integrin, shown previously to be the primary laminin-binding receptor on these cells. Neural crest cell interaction with the avian laminins was dependent upon maintenance of the secondary and tertiary structure of the molecules, as shown by the marked reduction in cell attachment and migration upon disruption of the alpha-helical coiled-coil structure of their constituent chains. The results demonstrate that different laminin isoforms may be differentially involved in the regulation of neural crest cell migration and suggest that this regulation operates through interaction of the cells with a structurally conserved cell binding site recognized by the alpha 1 beta 1 integrin