Antigen-specific Proteolysis by Hybrid Antibodies Containing Promiscuous Proteolytic Light Chains Paired with an Antigen-binding Heavy Chain

The antigen recognition site of antibodies consists of the heavy and light chain variable domains (V_L and V_H domains). V_L domains catalyze peptide bond hydrolysis independent of V_H domains (Mei, S., Mody, B., Eklund, S. H., and Paul, S. (1991) J. Biol. Chem. 266, 15571–15574). V_H domains bind antigens noncovalently independent of V_L domains (Ward, E. S., Güssow, D., Griffiths, A. D., Jones, P. T., and Winter, G. (1989) Nature 341, 544–546). We describe specific hydrolysis of fusion proteins of the hepatitis C virus E2 protein with glutathione S-transferase (GST-E2) or FLAG peptide (FLAG-E2) by antibodies containing the V_H domain of an anti-E2 IgG paired with promiscuously catalytic V_L domains. The hybrid IgG hydrolyzed the E2 fusion proteins more rapidly than the unpaired light chain. An active site-directed inhibitor of serine proteases inhibited the proteolytic activity of the hybrid IgG, indicating a serine protease mechanism. The hybrid IgG displayed noncovalent E2 binding in enzyme-linked immunosorbent assay tests. Immunoblotting studies suggested hydrolysis of FLAG-E2 at a bond within E2 located ∼11 kDa from the N terminus. GST-E2 was hydrolyzed by the hybrid IgG at bonds in the GST tag. The differing cleavage pattern of FLAG-E2 and GST-E2 can be explained by the split-site model of catalysis, in which conformational differences in the E2 fusion protein substrates position alternate peptide bonds in register with the antibody catalytic subsite despite a common noncovalent binding mechanism. These studies provide proof-of-principle that the catalytic activity of a light chain can be rendered antigen-specific by pairing with a noncovalently binding heavy chain subunit.Antibodies (Abs)² are composed of light and heavy chain subunits linked by intra- and inter-chain disulfide bonds. The noncovalent antigen binding site of Abs is formed mainly by amino acids located in the complementarity determining regions of the light and heavy chain variable domains (V_L and V_H domains). Physiological Ab-antigen binding reactions require both Ab subunits. The individual light and heavy chains can bind antigens independent of each other, but the binding affinity of the isolated subunits is often lower than the intact Abs from which they are derived (1–4). From crystallography analyses of Ab-antigen complexes, it appears that antigen contact areas with the V_H domain are somewhat greater than the V_L domain (5, 6). Recombinant IgG Abs composed of the heavy chain drawn from antigen-specific IgGs paired with irrelevant light chains retain antigen binding activity, albeit at reduced levels (1, 3).Following the initial noncovalent antigen binding step, some Abs proceed to catalyze hydrolysis of peptide bonds (7–12). The chemical catalysis step entails nucleophilic attack on the electrophilic carbonyl of peptide bonds by serine protease-like sites present in Ab V domains followed by hydrolysis of the covalent reaction intermediate if a water molecule is available (13–15). Unlike reversible binding, the catalytic function offers a means to permanently inactivate the antigen by its hydrolysis into smaller fragments. Reversibly binding Abs bind the antigen stoichiometrically (e.g. 2 antigen molecules/IgG molecule). As catalysts are reusable, a single catalytic Ab molecule can hydrolyze multiple antigen molecules. This offers the possibility of increased antigen neutralizing potency. Therefore, there is considerable interest in developing catalytic Abs directed to individual polypeptide antigens. The serine protease-like activity is a heritable trait encoded by germline Ab V genes, and Abs in the preimmune repertoire can hydrolyze peptides with diverse sequence promiscuously (13, 14, 16, 17). However, the adaptive immune system has evolved to maximize noncovalent binding affinity of Abs over the course of B cell differentiation. Physiological immune mechanisms do not favor retention and improvement of the catalytic function. B cell clonal proliferation is driven by antigen binding to B cell receptors (Abs associated with signal transducing proteins). Antigen hydrolysis by catalytic B cell receptors is followed by release of the antigen fragments, resulting in reduced B cell receptor occupancy and loss of the proliferative stimulus for the cells. Therefore, unlike the noncovalent antigen binding activity, the catalytic function is poorly selectable. Indeed, other than Abs to autoantigen and B cell superantigen substrates, there are no examples of antigen-specific catalytic Abs generated by physiological adaptive mechanisms (18).Much effort has been devoted to developing antigen-specific catalytic Abs by immune and protein engineering strategies. Based on the premise that binding to the transition state reduces the activation energy of the catalytic reaction, immunization with transition state analogs has been applied to raise Abs that catalyze ester bonds in small haptens (19). Attempts to improve the esterase activity by random mutagenesis followed by isolation of transition state analog-binding Abs have also been described (20). Developing antigen-specific proteolytic Abs, however, has been difficult because peptide bond hydrolysis is an energetically demanding reaction. Moreover, there is no viable engineering strategy available to render catalytic Abs specific for individual polypeptide antigens. We (8, 21, 22) and others (23, 24) have identified Ab light chains that hydrolyze peptide bonds promiscuously without participation from the heavy chain subunit. Disruption of the serine protease-like catalytic triad in an Ab light chain by site-directed mutagenesis was without effect on its ability to bind the polypeptide antigen by noncovalent means (13), and a discrete peptide epitope remote from the bond hydrolyzed by a proteolytic Ab preparation has been identified (25). This lead to a split-site model of proteolysis, in which distinct subsites present within the Ab combining site are responsible for initial noncovalent antigen binding and the ensuing peptide bond hydrolysis reaction (26). If this model is correct, it should be possible to develop hybrid proteolytic Abs specific for individual antigens by pairing light chains containing a promiscuous catalytic subsite with heavy chains that contribute the noncovalent subsite responsible for specific antigen binding. We describe proof-of-principle for this engineering approach using previously described catalytic light chains paired with the heavy chain of a monoclonal IgG that binds the hepatitis C virus (HCV) E2 coat protein. This protein is thought to be important in viral entry into hepatocytes and B cells by virtue of its ability to bind receptors expressed on the host cells (27, 28).