The plasmid pO61 that was isolated from an E. coli genomic DNA library and codes for O6-alkylguanine (O6AG) DNA alkyltransferase (ATase) activity (1) has been further characterised. Subclones of the 9 Kb insert of pO61 showed that the ATase activity was encoded in a 2Kb Pst1 fragment but a partial restriction endonuclease map of this was different to that of the E. coli ada gene that codes for O6-AG and alkylphosphotriester dual ATase protein. Fluorographic analyses confirmed that the molecular weight of the pO61-encoded ATase was 19KDa i.e. similar to that of the O6AG ATase function that is cleaved from the 39KDa ada protein but rabbit polyclonal antibodies to the latter reacted only very weakly with the pO61-encoded protein. A different set of hybridisation signals was produced when E. coli DNA, which had been digested with a variety of restriction endonucleases was probed with 2Kb Pst 1 fragment or the ada gene. These results provided evidence for the existence of a second ATase gene in E. coli. The 2Kb Pst-1 fragment of pO61 was therefore sequenced and an open reading frame (ORF) that would give rise to a 19KDa protein was identified. The derived amino acid sequence of this showed a 93 residue region with 49% homology with the O6AG ATase region of the ada protein and had a pentamer and a heptamer of identical sequence separated by 34 amino acids in both proteins. The pentamer included the alkyl accepting cysteine residue of the ada O6AG ATase. The hydrophobic domains were similarly distributed in both proteins. Shine-Dalgarno, -10 and -35 sequences were identified and the origin of transcription was located by primer extension and S1 nuclease mapping. The amino-terminal amino acid sequence of the protein was as predicted from the ORF.

Le(y)-reactive monoclonal antibodies (mAbs) were generated in mice by immunization with synthetic Le(y) neoglycoproteins or with Le(y)-expressing cells. Serological analysis indicated that mAbs raised against synthetic Le(y) (i) reacted strongly with synthetic Le(y) but poorly with natural Le(y), (ii) cross-reacted with Le(x) or H-type 2 structures, and (iii) were IgG1, IgG2a, or IgG2b. mAbs raised against Le(y)-expressing cells (i) reacted with both synthetic Le(y) and natural Le(y), (ii) were of two types: cross-reactive with Le(x) or H-type 2 structures or specific for Le(y), and (iii) were IgM or IgG3. One of the mAbs raised against natural Le(y), mAb 3S193 (IgG3), showed high specificity for Le(y) in ELISA tests with synthetic Le(y) and Le(y) containing glycoproteins and glycolipids; it also reacted strongly in rosetting assays and cytotoxic tests with Le(y)-expressing cells. mAb 3S193 did not lyse O, A, AB, and B human erythrocytes in the presence of human complement. In flow cytometry, there was weak reactivity with granulocytes, a reactivity also observed with two previously described highly specific Le(y) mouse mAbs--BR55-2 (IgG3) and B3 (IgG1). A humanized version of mAb 3S193 has been constructed, and the specificity pattern and reactivity for Le(y) remain very similar to mouse mAb 3S193.

No abstract

Protein therapeutics offer distinct advantages over other classes of drugs largely due to the high level of target specificity and generally low toxicity. Problems have, however, been encountered with some protein therapeutics inducing undesirable immune responses in patients. This immunogenicity can produce pleiotropic effects including the development of a high affinity B cell-mediated humoral response that is often directed against the therapeutic. Opinions are divided as to the principal causes of clinical immunogenicity and, as a result, this area has been the subject of much research. One thing that has emerged as a result of this intense activity is the development of pre-clinical models that can provide a level of prediction of the immunogenic potential of novel protein therapeutics before administration in man.

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.