THERAPEUTIC ANTIBODY STRUCTURES OF RAT AND HUMANIZED (CAMPATH-1) Fab FRAGMENTS Anne C.Bloomer, Graham M. T.Cheetham^*, Geoffrey Hale^*, Herman Waldmann^*, MRC Lab. of Molecular Biology, Hills Rd, Cambridge, CB2 2QH, UK, ^*Sir William Dunn Sch. of Pathology, S. Parks Rd, Oxford, OX1 3RE, UK.

The Campath-1 family of antibodies are able systematically to lyse human lymphocytes with human complement by targeting the small cell-surface glycoprotein CD52, commonly called the Campath-1 antigen. Three of these antibodies have been used clinically for several years providing therapy for patients with an increasing variety of immunologically mediated diseases. We report here the first X-ray crystallographic structures of a Fab fragment from an original rat monoclonal antibody and its humanized counterpart, into which the six complementarity -determining regions of the rat antibody have been introduced.

Crystal structures for this pair of Fab fragments have been refined at 2.6 Å and 3.25 Å, respectively. Translational pseudo-symmetry is observed in the rat Campath-1G crystals (P2₁2₁2₁, Z=4) and also in one crystal form of the humanized Campath-1H antibody (P6₅22, Z=1 and P6₅22, Z=3).

The light chain variable domains of adjacent molecules of Campath-1H form a symmetrical dimer with an extended inter-molecular beta-sheet. Such VL domain dimers have implications for light chain disease and amyloidosis.

Within the antibody-combining sites, which are dominated by the protrusion of side chains Lys54 and Lys56 from hypervariable loop H2, both charge distribution and overall integrity are highly conserved, but large changes in the position of loop H1 are observed with an altered conformation of loop H2. The major determinants of this change are the VH domain framework residues 74 and 24, whose identity differs between these two antibodies.

Sequence data for two different antibodies, having higher and lower affinity for the same antigen, point to essential interactions with the antigen CD52 - a small glycoprotein with a GPI anchor - and its octapeptide mimotope.

These structures provide both a detailed structural insight into the transplantation of an intact antibody-combining site between a rodent and a human framework and also an increased understanding of the specificity and antigen affinity of this pair of Campath-1 antibodies for CD52. This study forms the structural basis for future modification and design of more effective antibodies to this important cell-surface antigen.