Studies of the interactions between the anticytokeratin 8 monoclonal antibody TS1, its antigen and its anti-idiotypic antibody alphaTS1.
The monoclonal antibody TS1 against cytokeratin 8 and its antiidiotype alphaTS1 have been used for immunotargeting and therapy of carcinomas in experimental tumor model systems. The interaction surfaces between mab TS1, the cytokeratin 8 epitope, and its anti-idiotypic antibody, alphaTS1, were studied in detail in order to make future veneering of the interactions possible. The V-genes of TS1 and alphaTS1 were cloned and sequenced and the CDRs and the framework residues were identified. Amino acids participating in the interactions were identified following chemical modifications of residues in non-protected and protected molecules of cytokeratin 8, alphaTS1 and TS1. From the sequences, the three-dimensional structures were generated using computer modelling of the antibody variable regions. Several charged amino acid, histidine and tyrosine residues were displayed in the antibody surfaces implicated in the interactions and chemical modification confirmed the importance of these amino acids. The cytokeratin 8 epitope has previously been identified by Johansson et al. and it displays negatively charged amino acid residues which could be identified in the chemical modification. It was also revealed that the TS1 binding to cytokeratin 8 and alphaTS1 respectively are partly overlapping; a histidine identified in TS1 is probably involved only in the interaction with alphaTS1. Furthermore, the chemical modification demonstrated that exchanging aspartic-glutamic acids to asparagine-glutamine residues in TS1 increased the binding of TS1 to cytokeratin 8, indicating that there is at least one acidic amino acid that is an obstacle in the TS1-CK8 binding. The detailed assembly of the interaction surfaces will facilitate the future use of site directed mutagenesis to improve the TS1-CK8 association rate and the clearing of TS1 with alphaTS1 in vivo.
Erlandsson
Ann
A
Department of Immunology, Umeå University, S-901 85 Umeå, Sweden.
Holm
Patrik
P
Ullén
Amanda
A
Stigbrand
Torgny
T
Sundström
Birgitta E
BE
D000595:Amino Acid Sequence; D000818:Animals; D000888:Antibodies, Anti-Idiotypic / Q000276:immunology*; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000276:immunology*; D000941:Antigens / Q000276:immunology*; D002460:Cell Line; D003001:Cloning, Molecular; D003198:Computer Simulation; D007633:Keratins / Q000276:immunology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D016415:Sequence Alignment
D000888:Antibodies, Anti-Idiotypic; D000911:Antibodies, Monoclonal; D000941:Antigens; D007633:Keratins