Site-directed polyethylene glycol modification of trichosanthin: Effects on its biological activities, pharmacokinetics, and antigenicity

Trichosanthin (TCS), a type I ribosome-inactivating protein (RIP), was modi fied with polyethylene glycol (PEG) in order to reduce its antigenicity and prolong its half-life. Computer modeling identified three potential antige nic sites namely Q219, K173 and S7. By site-directed mutagenesis, these sit es were changed into cysteine through which PEG can be covalently attached. The resulting TCS had a PEG coupled directly above one of its potential an tigenic determinants, hence masking the antigenic region and prevent bindin g of antibodies specific to this site. In general, mutation did not bring a bout significant changes in ribosome-inactivating activity, cytotoxicity, a nd abortifacient activity of TCS. However, the in vitro activities of PEG m odified (PEGylated) TCS muteins were 3-20 folds lower and the in vivo activ ity 50% less than that of nTCS. Pharmacokinetics study indicated that all t hree PEGylated TCS muteins showed 6-fold increase in mean residence time as compared to unmodified muteins. The binding affinity of an TgE monoclonal antibody (TE1) to TCS was greatly reduced after PEG modification (PEGylatio n) at position Q219, suggesting that TE1 recognized an epitope very near to residue Q219. PEGylated TCS muteins induced similar IgG response but 4-16 fold lower IgE response in mice compared with nTCS.