Single-chain antibody construction and functional mapping of the monoclonal antibody TS1

Antibodies are proteins with the ability to bind antigens rapidly and specifically. Antibodies consist of several different parts. The complementary determining regions (CDR) are the parts that recognize the antigen are the focus of this study. The aims of this study were to synthesize and produce a single-chain antibody (scFv) of the anti-cytokeratin 8 monoclonal IgG antibody TS1 and to functionally map amino acid residues important for the interaction with its antigen and the anti-idiotypic antibody αTS1. Cytokeratins are present in significant amount in the necrotic areas of carcinomas and are not released into the circulation since they have low solubility. The TS1 antibody has been shown to be effective in binding cytokeratin 8 (CK8) exposed in tumors in vivo and is proposed to be useful in immunotargeting and/or immunotherapy. The anti-idiotypic antibody αTS1 can be used to regulate the tumor:non-tumor ratio. Mutagenesis of certain amino acid residues can be used to alter the affinity to improve the tumor:nontumor ratio further. In the present study, the TS1 IgG was chemically modified to specify groups of amino acid residues important for the interaction with both CK8 and αTS1. If important residues were found in the CDRs, they were mutated in the TS1 scFv construct and the effect was studied using ELISA. The main conclusions drawn from this study are that the important amino acid residues in TS1 for the interaction with both CK8 and αTS1 are mainly tyrosines, charged residues and a tryptophan. A central interacting interface was identified with the somewhat unusual participation of residues in the CDR 2 of the light chain. Mutations which resulted in increased affinity to both CK8 and αTS1 were also identified.