NOVEL SELECTION METHOD FOR ENGINEERED ANTIBODIES USING THE MECHANISM OF FV FRAGMENT STABILIZATION IN THE PRESENCE OF ANTIGEN

Although the heavy and light chain domains of some antibody variable r egion fragments (Fvs) readily dissociate under physiological condition s, the Fvs are stable in the presence of antigen, This 'antigen-driven Fv stabilization mechanism' was applied to the selection of clones wi th specificity toward target antigens, The results can be summarized a s follows, (i) Some of the residues in the heavy chain complementarity determining region 2 (HCDR2) of anti-hen egg white lysozyme (HEL) mon oclonal antibody HyHEL10 heavy chain variable region (VH) were randomi zed, (ii) The randomized VH fragments of HyHEL10 were displayed on a f ilamentous bacteriophage and mixed with the target antigen, before bei ng applied to a light chain variable region (VL) which was immobilized on microtiter plates and subjected to selection by panning. (iii) Aft er four rounds of panning, four clones that showed significant binding to human lysozyme (hL), which HyHEL10 recognized poorly, were selecte d from the HCDR2 library, (iv) The soluble Fv fragments selected were expressed in Escherichia coli, purified, and subjected to an inhibitio n assay of lysozyme enzymatic activities and an isothermal titration c alorimetry, These Fv fragments had increased affinity toward hL, and t hermodynamic analysis suggested that the reduced entropy loss due to b inding by the replacement of residues in HCDR2 resulted in the higher hL binding activity.