A novel domain antibody rationally designed against TNF-alpha using variable region of human heavy chain antibody as scaffolds to display antagonistic peptides.
Neutralizing of TNF-alpha has been proved effective in treatment of some autoimmune diseases, e.g. rheumatoid arthritis and Crohn's disease. Low molecular weight synthetic peptides can mimic the binding sites of TNF-alpha receptors and block the activity of TNF-alpha. In order to stabilize the conformation, increase the affinity and bioactivity, in this study, heavy chain variable region of human antibody was used as a scaffold to simultaneously display three peptides, which were designed on the interaction between TNF-alpha and it's neutralizing monoclonal antibody. On the basis of the structural character and physical-chemical property of the families of seven kinds of heavy chain variable regions (VH) in human antibodies, the fifth type of VH was screened as scaffold to display the antagonist peptide. Based on the computer-guided molecular design method, a novel domain antibody against TNF-alpha (named as ATD5) was designed as TNF-alpha antagonist. The theoretical study showed that ATD5 was more stable than displayed antagonist peptide. The binding activity with TNF-alpha was higher than free peptides. After expression and purification in Escherichia coli, ATD5 could bind directly with TNF-alpha and inhibit the binding of TNF-alpha to its two receptors, TNFR1 and TNFR2. ATD5 could also reduce the TNF-alpha-mediated cytotoxicity and inhibit TNF-alpha-mediated caspase activation on L929 cells in a dose dependent manner. The activity of ATD5 was significantly stronger than three peptides displayed by ATD5. This study provides a novel strategy for the development of new TNF-alpha inhibitors. This study demonstrates that it is possible to screen potential antagonists of TNF-alpha using in vitro analysis systems in combination with the computer-aided modeling method.
Qin
Weisong
W
Research Institute of Nephrology, Jinling Hospital, Nanjing, PR China.
Feng
Jiannan
J
Li
Yan
Y
Lin
Zhou
Z
Shen
Beifen
B
D000595:Amino Acid Sequence; D000818:Animals; D019138:Antibodies, Blocking / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000276:immunology* / Q000302:isolation & purification; D000937:Antigen-Antibody Reactions / Q000187:drug effects / Q000276:immunology; D020169:Caspases / Q000378:metabolism; D003602:Cytotoxicity, Immunologic / Q000187:drug effects; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000187:drug effects / Q000276:immunology*; D007135:Immunoglobulin Variable Region / Q000187:drug effects / Q000276:immunology*; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000378:metabolism; D010455:Peptides / Q000494:pharmacology*; D017434:Protein Structure, Tertiary / Q000187:drug effects; D047888:Receptors, Tumor Necrosis Factor, Type I / Q000378:metabolism; D047889:Receptors, Tumor Necrosis Factor, Type II / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000037:antagonists & inhibitors* / Q000276:immunology* / Q000494:pharmacology
D019138:Antibodies, Blocking; D000911:Antibodies, Monoclonal; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D050505:Mutant Proteins; D010455:Peptides; D047888:Receptors, Tumor Necrosis Factor, Type I; D047889:Receptors, Tumor Necrosis Factor, Type II; D014409:Tumor Necrosis Factor-alpha; D020169:Caspases