Disulfide bridging protein conjugation

Over the last 20 years, protein based medicines have been acquiring an increasingly important role in the treatment of serious medical diseases. They are now the fastest growing sector of new medicines. Their rapid clearance from the body led to them being linked to poly(ethylene glycol) (PEG) as a means by which to prolong the protein's circulation time in the body and reduce its toxicity. This approach has been very successful. However, these current PEG technologies are severely limited and are only available to patients in resource rich countries. There is therefore an urgent need for innovative and cost-effective biotechnology that makes PEGylated proteins more widely available for patients with infectious diseases in resource poor countries. We describe a novel biotechnology that solves this problem, and illustrate its application to IFN-a2 for treating Hepatitis C infection, and to an anti-CD4 antibody fragment that prevents the cell-to-cell transmission of HIV-1.

This proposal seeks to unequivocally prove our hypothesis that we can exploit for PEGylation the chemical selectivity of the thiols in the natural disulfides that exist in therapeutically relevant proteins while retaining the biological function of the protein. We describe preliminary results that demonstrate that it is possible with an immunomodulating protein such as interferon to partially reduce one of its disulfide bonds to site-specifically covalently conjugate both thiols and effectively reanneal the disulfide. Similar preliminary results have been obtained for the Fab for the CD4 antibody.