Direct conversion of a general antibody to its catalytic antibody and corresponding applications -Importance and role of Pro95 in CDR-3.

Catalytic antibodies possess unique features capable of both recognizing and enzymatically degrading antigens. Therefore, they are more beneficial than monoclonal antibodies (mAbs). Catalytic antibodies exhibit the ability to degrade peptides, antigenic proteins, DNA, and physiologically active molecules. However, they have a significant drawback in terms of their production. The production of a desired catalytic antibody has extensive costs, in terms of time and effort. We herein describe an evolutionary method to produce a desired catalytic antibody via conversion of a general antibody by the deletion of Pro95, which resides in complementarity-determining region-3. As over thousands of mAbs have been produced since 1975, using the novel technology discussed herein, the catalytic feature cleaving the antigen can be conferred to the mAb. In this review article, we discussed in detail not only the role of Pro95 but also the unique features of the converted catalytic antibodies. This technique will accelerate research on therapeutic application of catalytic antibodies.

Obtaining Highly Active Catalytic Antibodies Capable of Enzymatically Cleaving Antigens.

A catalytic antibody has multiple functions compared with a monoclonal antibody because it possesses unique features to digest antigens enzymatically. Therefore, many catalytic antibodies, including their subunits, have been produced since 1989. The catalytic activities often depend on the preparation methods and conditions. In order to elicit the high catalytic activity of the antibodies, the most preferable methods and conditions, which can be generally applicable, must be explored. Based on this view, systematic experiments using two catalytic antibody light chains, #7TR and H34, were performed by varying the purification methods, pH, and chemical reagents. The experimental results obtained by peptidase activity tests and kinetic analysis, revealed that the light chain's high catalytic activity was observed when it was prepared under a basic condition. These data imply that a small structural modulation of the catalytic antibody occurs during the purification process to increase the catalytic activity while the antigen recognition ability is kept constant. The presence of NaCl enhanced the catalytic activity. When the catalytic light chain was prepared with these preferable conditions, #7TR and H34 hugely enhanced the degradation ability of Amyloid-beta and PD-1 peptide, respectively.

A new catalytic site functioning in antigen cleavage by H34 catalytic antibody light chain.

The cleavage reactions of catalytic antibodies are mediated by a serine protease mechanism involving a catalytic triad composed of His, Ser, and Asp residues, which reside in the variable region. Recently, we discovered a catalytic antibody, H34 wild type (H34wt), that is capable of enzymatically cleaving an immune-check point PD-1 peptide and recombinant PD-1; however, H34wt does not contain His residues in the variable region. To clarify the reason behind the catalytic features of H34wt and the amino acid residues involved in the catalytic reaction, we performed site-directed mutagenesis focusing on the amino acid residues involved in the cleavage reaction, followed by catalytic activity tests, immunological reactivity evaluation, and molecular modeling. The results revealed that the cleavage reaction by H34wt proceeds through the action of a new catalytic site composed of Arg, Thr, and Gln. This new scheme differs from that of the serine protease mechanism of catalytic antibodies.

Finding and characterizing a catalytic antibody light chain, H34, capable of degrading the PD-1 molecule.

Programmed cell death 1 (PD-1) is an immune checkpoint molecule regulating T-cell function. Preventing PD-1 binding to its ligand PD-L1 has emerged as an important tool in immunotherapy. Here, we describe a unique human catalytic antibody light chain, H34, which mediates enzymatic degradation of human PD-1 peptides and recombinant human PD-1 protein and thus functions to prevent the binding of PD-1 with PD-L1. H34 degraded one half of the PD-1 molecules within about 6 h under the experimental conditions. Investigating the acquisition of the catalytic function by H34, which belongs to subgroup I and lacks a Pro95 residue in CDR-3, revealed the importance of this sequence, as a Pro95-reconstituted mutant (H34-Pro95(+)) exhibited very little catalytic activity to cleave PD-1. Interestingly, EDTA inhibited the catalytic activity of H34, which could work as a metallo-protease. Zn2+ or Co2+ ions may work as a cofactor. It is meaningfull that H34 was obtained from the human antibody gene taken from a healthy volunteer, suggesting that we potentially have such unique molecules in our body.

A new algorithm to convert a normal antibody into the corresponding catalytic antibody.

Over thousands of monoclonal antibodies (mAbs) have been produced so far, and it would be valuable if these mAbs could be directly converted into catalytic antibodies. We have designed a system to realize the above concept by deleting Pro95, a highly conserved residue in CDR-3 of the antibody light chain. The deletion of Pro95 is a key contributor to catalytic function of the light chain. The S35 and S38 light chains have identical amino acid sequences except for Pro95. The former, with Pro95 did not show any catalytic activity, whereas the latter, without Pro95, exhibited peptidase activity. To verify the generality of this finding, we tested another light chain, T99wt, which had Pro95 and showed little catalytic activity. In contrast, a Pro95-deleted mutant enzymatically degraded the peptide substrate and amyloid-beta molecule. These two cases demonstrate the potential for a new method of creating catalytic antibodies from the corresponding mAbs.