Highly efficient method of preparing human catalytic antibody light chains and their biological characteristics

Abstract

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.

Biochemical Features of a Catalytic Antibody Light Chain, 22F6, Prepared from Human Lymphocytes

“…By joining Asp 1 or Asp 34 , it might be preferable for constructing such a triad. The amino acids Asp 1 , Ser 27a , and His 27d are identical to those of the A18b germ line gene that were found, as previously reported, to all possess amidase activity (16). …”

“…The tendency of the amidase activity of the light chain 22F6 to cleave synthetic substrates differs considerably from that of the other human light chains, such as chains 1, 6, 8, 10, 11, and 18, reported in a previous study (16). The 22F6 light chain seems to possess an enzymatic feature partly of trypsin and partly of factor Xa.…”

“…They also reported the good efficacy of a mouse-type catalytic antibody heavy chain in suppressing infection of influenza virus type A in an in vitro assay (15). In addition, they have recently developed a human type catalytic light chain capable of increasing the survival rate of suckling mice infected with the rabies virus in an in vivo experiment (16). …”

Metal-dependent amyloid β-degrading catalytic antibody construct

“…IgV 2E6 uses the same serine protease-like nucleophilic mechanism for Aβ hydrolysis as catabodies directed to other polypeptide targets (Planque et al 2004; Reshetnyak et al 2007; Hifumi et al 2012). As the nucleophilic reactivity is a germline antibody property developed by Darwinian evolution, the consistent serine protease protease-like mechanism of catabodies is not surprising.…”

Constant Domain-regulated Antibody Catalysis

“…We described antibodies and antibody fragments to HIV gp120 (34), the Staphylococcus aureus virulence factor Efb (20) and the autoantigen amyloid ␤ (47) with neutralizing activity attributable to the catalytic function. The group of Uda and Hifumi has described catalytic antibody fragments to bacterial and viral target antigens that reduce infection in experimental animal models (48,49). Autoimmune disease is associated with increased catalytic antibodies to autoantigens, and numerous examples of pathogenic catalytic autoantibodies have been reported (50 -54; for review see Ref.…”