Humanization and epitope mapping of neutralizing anti-human Fas ligand monoclonal antibodies: structural insights into Fas/Fas ligand interaction.
Fas ligand (L)/CD95L, a proapoptotic member of the TNF family, is a potential target for clinical intervention in various diseases. In the present study, we generated a humanized anti-human FasL mAb and characterized the epitopes of neutralizing mAbs by extensive alanine-scanning mutagenesis of human FasL. The predicted molecular model of FasL trimer revealed that the mAbs recognize largely overlapped conformational epitopes that are composed of two clusters, one around the outer tip-forming D-E loop and another near the top of FasL. Both of these sites on FasL are critically involved in the direct interaction with the corresponding receptor, Fas. These results suggest that the mAbs efficiently neutralize FasL cytotoxicity by masking both of these FasL/Fas contact sites.
Nisihara
T
T
The Chemo-Sero-Therapeutic Research Institute, Kumamoto, Japan.
Ushio
Y
Y
Higuchi
H
H
Kayagaki
N
N
Yamaguchi
N
N
Soejima
K
K
Matsuo
S
S
Maeda
H
H
Eda
Y
Y
Okumura
K
K
Yagita
H
H
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D000918:Antibody Specificity; D017209:Apoptosis; D001666:Binding Sites, Antibody; D016466:CHO Cells; D019556:COS Cells; D002522:Chlorocebus aethiops; D003198:Computer Simulation; D006224:Cricetinae; D003412:Cricetulus; D003602:Cytotoxicity, Immunologic; D018604:Epitope Mapping; D000939:Epitopes / Q000737:chemistry / Q000276:immunology; D053222:Fas Ligand Protein; D015322:Gene Rearrangement, B-Lymphocyte; D005803:Genes, Immunoglobulin; D006801:Humans; D046911:Macromolecular Substances; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000276:immunology*; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009500:Neutralization Tests; D011487:Protein Conformation; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D013329:Structure-Activity Relationship; D019014:fas Receptor / Q000276:immunology
D000911:Antibodies, Monoclonal; D000939:Epitopes; C505661:FASLG protein, human; D053222:Fas Ligand Protein; C505658:Fasl protein, mouse; D046911:Macromolecular Substances; D008562:Membrane Glycoproteins; D019014:fas Receptor