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Catalytic antibodies have exhibited interesting functions against some infectious viruses such as HIV, rabies virus, and influenza virus in vitro as well as in vivo. In some cases, a catalytic antibody light chain takes on several structures from the standpoint of molecular size (monomer, dimer, etc.) and/or isoelectronic point. In this study, we prepared a monomeric 23D4 light chain by mutating the C-terminal Cys to Ala of the wild-type. The mutated 23D4 molecule took a simple monomeric form, which could hydrolyze synthetic 4-methyl-coumaryl-7-amide substrates and a plasmid DNA. Because the monomeric 23D4 light chain suppressed the infection of influenza virus A/Hiroshima/37/2001 in an in vitro assay, the corresponding experiments were conducted in vivo, after the virus strain (which was taken from a human patient) was successfully adapted into BALB/cN Sea mice. In the experiments, a mixture of the monomeric 23D4 and the virus was nasally administered 1) with preincubation and 2) without preincubation. As a result, the monomeric 23D4 clearly exhibited the ability to suppress the influenza virus infection in both cases, indicating a potential drug for preventing infection of the influenza A virus., (© FASEB.)

The ultimate goal of catalytic antibody research is to develop new patient therapies that use the advantages offered by human catalytic antibodies. The establishment of a high-throughput method for obtaining valuable candidate catalytic antibodies must be accelerated to achieve this objective. In this study, based on our concept that we can find antibody light chains with a high probability of success if they include a serine protease-like catalytic triad composed of Ser, His, and Asp on a variable region of the antibody structure, we amplified and cloned DNAs encoding human antibody light chains from germline genes of subgroup II by seminested PCR using two primer sets designed for this purpose. Seven DNA fragments encoding light chains in 17 clones were derived from germline gene A18b, 6 DNA fragments from A3/A19, 2 DNA fragments from A17, and a clone DNA fragment from A5 and O11/O1. All light chains expressed in Escherichia coli and highly purified under nondenaturing conditions exhibited amidolytic activity against synthetic peptides. Some of the light chains exhibited unique features that suppressed the infectious activity of the rabies virus. Furthermore, the survival rate of mice in which a lethal level of the rabies virus was coinoculated directly into the brain with light chain 18 was significantly improved. In the case of humans, these results demonstrate that high-throughput selection of light chains possessing catalytic functions and specificity for a target molecule can be attained from a light-chain DNA library amplified from germline genes belonging to subgroup II.

Antibodies with nucleophilic or catalytic properties often have these characteristics encoded in their germ line genes. Because hydrolytic activity has been reported to be associated with light chain V regions, we have begun an analysis of germ line light chain proteins that could be the basis for affinity maturation into hydrolytic or other reactive antibodies. We produced the germ line A18b light chain and characterized its hydrolytic, nucleophilic, and tertiary structural activities. This light chain was purified to >99% purity and found to hydrolyze aminomethylcoumarin-peptide and larger protein substrates and bind a fluorophosphonate probe. Mutation of putative catalytic residues only resulted in loss of activity of a tetrameric but not dimeric form of the light chain. These biochemical properties provide a framework for understanding the structure-function relationships of germ line antibodies.

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 approximately 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.

We have successfully developed a catalytic antibody capable of degrading the active site of the urease of Helicobacter pylori and eradicating the bacterial infection in a mouse stomach. This monoclonal antibody UA15 was generated using a designed recombinant protein UreB, which contained the crucial region of the H. pylori urease beta-subunit active site, for immunization. The light chain of this antibody (UA15-L) by itself showed a proteolytic activity to substantially degrade both UreB and the intact urease. Oral administration of UA15-L also significantly reduced the number of H. pylori in a mouse stomach. This is the first example of a monoclonal catalytic antibody capable of functioning in vivo, and such an antibody may have a therapeutic utility in the future.

Lc-WT, the wild-type light chain of antibody, and Lc-Triad, its double mutant with E1D and T27aS designing for the construction of catalytic triad within Asp1, Ser27a, and original His93 residues, were displayed on the cell surface of the protease-deficient yeast strain BJ2168. When each cell suspension was reacted with BODIPY FL casein and seven kinds of peptide-MCA substrates, respectively, a remarkable difference in hydrolytic activities toward Suc-GPLGP-MCA (succinyl-Gly-Pro-Leu-Gly-Pro-MCA), a substrate toward collagenase-like peptidase, was observed between the constructs: Lc-Triad-displaying cells showed higher catalytic activity than Lc-WT-displaying cells. The difference disappeared in the presence of the serine protease inhibitor diisopropylfluorophosphate, suggesting that the three amino acid residues, Ser27a, His93, and Asp1, functioned as a catalytic triad responsible for the proteolytic activity in a similar way to the anti-vasoactive intestinal peptide (VIP) antibody light chain. A serine protease-like catalytic triad (Ser, His, and Asp) is considered to be directly involved in the catalytic mechanism of the anti-VIP antibody light chain, which moderately catalyzes the hydrolysis of VIP. These results suggest the possibility of new approach for the creation of tailor-made proteases beyond limitations of the traditional immunization approach.

Generation of antibodies with the ability to destroy targeted viral coat proteins or tumor antigens is an important aim in current research aimed at developing superior catalytic antibodies. To this end, we raised a monoclonal antibody against a discrete sequence of the envelope gp41, RGPDRPEGIEEEGGERDRD, which is a highly conserved sequence in many human immunodeficiency virus (HIV)-1 strains. The light chain subunit of this antibody catalytically decomposed the targeted peptide antigen. The degradation of the immunized peptide antigen by the light chain was initiated by the hydrolytic scission of the peptide bond between Glu12-Gly13, followed by the successive cleavage reactions of the additional peptic bonds into small peptides and amino acids. The decomposition by the light chain obeyed Michaelis-Menten kinetics (k(cat)/K(m) = 2. 8 x 10(5) M(-1) min(-1)). A characteristic feature of the reaction was a slow initial degradation step, followed by an increase in the rate of catalysis. Removal of the light chain by immunoadsorption at either stage of the reaction resulted in recession of catalysis. The light chain also cleaved recombinant gp41 molecule, but did not degrade proteins unrelated in the sequence to the peptide immunogen (bovine and human serum albumins).

A recombinant antibody light chain (L chain) maintained under non-denaturing conditions displayed preferential cleavage of synthetic peptides conjugated to methylcoumarinamide (MCA) on the C-terminal side of Arg and Lys residues. The same L chain renatured from a denaturing solvent (guanidine hydrochloride) acquired the capability of cleaving Tyr-MCA and Leu-MCA bonds, and its ability to cleave MCA linked to basic residues was decreased. The altered cleavage preference was accompanied by a conformational transition in the protein, evident from the fluorescence emission spectra. These observations suggest the feasibility of redirecting the cleavage specificity via alterations in the conformation of proteolytic antibody combining sites.

A complex of an independent L chain from anti-mesotetrakis (4-carboxyphenyl) porphyrin (TCPP) monoclonal antibody 13-1 and TCPP Fe(III) was designated as L-zyme and shown to exhibit high peroxidase activity and high optimal reaction temperature (90 degrees C). Heat denaturation study and circular dichroism (CD) spectra analysis suggested that refolded structure of 13-1 L chain exhibited significantly reduced inactivation rate after heat treatment. The secondary structure of 13-1 L chain changed slightly by the encapsulation of TCPP Fe(III) and the complex was found to be less thermostable than the L chain alone. Furthermore, by characterization of truncated forms of the L chain, it was revealed that the hydrophobic region (115-146) and hydrophilic region (147-189) in CL are important for thermostability and activity, respectively. Tertiary structure of L-zyme was predicted by AbM. Comparison of residues of L-zyme with those in the active centre of known structure of the peroxidase from Arthromyces ramosus (ARP) indicated that His38(CDR1), His94(CDR3), Arg96(CDR3) of L-zyme are important residues for peroxidase activity. Moreover, the steric arrangements of these residues in both L-zyme and ARP are similar, respectively. Distance between proximal His and distal His in L-zyme is 9.09 A, whereas that of ARP is 7.8 A.