Molecular Cloning of Antiground-State Proteolytic Antibody Fragments

The variable regions of antibody subunits are the “business ends” of these molecules responsible for interactions with antigens and activation of the biological functions of the constant regions. Rearrangement of variable-, diversity-, and joining-region genes and the hypermutability of the complementarity determining regions (CDRs) permit development of new antigen-binding specificities. With the discovery of natural catalytic activity in autoantibodies (1 –3 ) and the confirmation of this phenomenon (4 ,5 ), it has become clear that hypervariability in antibody genes may permit a natural evolution of enzyme-like sites in antibodies. Additional evidence for this hypothesis consists of observations that monoclonal antibodies (MAb) to a natural antigen, vasoactive intestinal polypeptide (VIP), display proteolytic activity (6 ,7 ). As the first step toward establishing the physicochemical basis of the catalytic activity, we have cloned and expressed the light (L)-chain subunit and single-chain F_V (scF_V ) fragment of one such antibody. The recombinant L chain and the scF_V display interactions with VIP typical of an antibody-combining site. Both molecules are efficient catalysts (8 ). The distinctive feature of catalytic hydrolysis of VIP by these recombinant proteins is high-affinity substrate binding (low K _m ), a property not shared by conventional enzymes. The L chain also catalyzes the hydrolysis of a protease substrate unrelated in sequence to VIP, but this activity appears to be the result of low affinity recognition of basic residues (8 ). By primary structure analyses, molecular modeling, and kinetic examination of the catalytic activities, it appears that the residues in the antibody active site providing important contributions in the binding and hydrolysis steps may be different. Site-directed mutagenesis of the recombinant protein active sites is ongoing to validate this hypothesis.