A COMPARISON OF CRYSTAL STRUCTURES OF ENGINEERED ANTIBODY FRAGMENTS. M J Banfield, D J King* & R L Brady. Dept of Biochemistry, University of Bristol BRISTOL BS8 1TD. UK; *Celltech Therapeutics Ltd., 216 Bath Road, Slough SL1 4EN.

The 3-dimensional structure of the Fab-fragment from the murine monoclonal antibody A5B7 which binds Carinoembryonic antigen (CEA), a protein expressed on carcinoma cell surfaces has been determined to a resolution of 2.1 Å. Forms of this antibody have potential application in the treatment of colerectal cancer (Lane et al., 1995). A5B7 has been the subject of extensive protein engineering studies to produce engineered human constructs that retain high antigen binding. However, although less immunogenic than the murine form, these constructs often have reduced antigen binding, and hence reduced efficacy.

In addition to the murine form of A5B7 we have determined the crystal structure of a engineered human construct that retains ~50% binding affinity. A comparison of the binding site of the two forms shows small changes, some of which might be explained by packing in a different crystal lattice, although others partially explain the observed alterations in antigen affinity. A further engineered human construct of A5B7 that retains 100% antigen binding affinity is also under study.

A second engineered human antibody, derived from the mouse antibody CTM01, which binds to Polymorphic Epithelial Mucin (Muc1), is also being investigated. For this antibody we have a peptide mimic of the antigen and hope to analyse the structure of this Fab-fragment in both native, and peptide-bound forms.

This series of Fab crystal structures provides a unique opportunity to examine the molecular consequences of antibody humanisation, and further our understanding of protein engineering in general.

Lane, D.M., Eagle, K.F., Begent, R.H.J., Hope-Stone, L.D., Green A.J., Casey J.L., Keep, P.A., Kelly, A.M.B., Lederman, J.A., Glaser, M.G. & Hilson A.J.W. (1994) British Journal of Cancer 70, p.521-525.