Highly efficient method of preparing human catalytic antibody light chains and their biological characteristics.
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.
Research Center for Applied Medical Engineering, Faculty of Engineering, Oita University, 700 Dan-noharu, Oita-shi, Oita 870-1192, Japan.
Emi
E
Hifumi
Eijiro
E
Honjo
Naoko
N
Fujimoto
Mitsue
M
Arakawa
Akira
A
Nishizono
Taizo
T
Uda
D000465:Algorithms; D000595:Amino Acid Sequence; D000818:Animals; D000833:Animals, Suckling; D017151:Antibodies, Catalytic / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D004247:DNA / Q000235:genetics / Q000378:metabolism; D005854:Germ Cells; D006801:Humans; D007147:Immunoglobulin Light Chains / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction / Q000379:methods*; D011487:Protein Conformation; D011820:Rabies virus / Q000276:immunology; D016415:Sequence Alignment; D015996:Survival Rate
D017151:Antibodies, Catalytic; D007147:Immunoglobulin Light Chains; D004247:DNA