PLASMID DESIGN FOR PRODUCTION OF CHIMERIC ANTIBODIES WITH DEFINED SPECIFICITY IN EUKARYOTES

Abstract

Aim. In this study we aimed to design the universal genetic construction expressing the light and heavy chains of a chimeric antibody, to develop methodological approaches for the production of chimeric antibodies with defined specificity using the monoclonal antibodies to diphtheria toxin (DT) DT-17 in the CHO cells as an example and to evaluate their immunochemical and effector properties. Materials and methods. Variable region genes of the light and heavy chains of mouse antibodies DT-17 to diphtheria toxin were obtained by PCR method and cloned into pCI-neo plasmid vector. The S V DT -17neo «supervector» containing the genes of a chimeric antibody was constructed by using of genetic engineering techniques. CHO cells were transfected with «supervector» and a highly productive clone secreting chimeric antibodies to DT were collected. Immunochemical methods were used to evaluate antibody activity, and affinity chromatography was used to prepare preparative amounts of the antibodies. Results. U ni versal vectors pLK DT -17 and pHG DT-17 containing light and heavy chain genes of the chimeric antibodies DT -17 to DT were constructed. The variable and constant region genes were flanked by endonuclease restriction sites, which allows to change the specificity of the antibodies. In the future it will make possible to study the modifications of the class and species specificity of the chimeric immunoglobulins. When the CHO cell culture was transfected with the designed vectors, the accumulation of antibodies to DT in the culture medium was detected. The yield of purified DT-17 chimeric antibodies was 4 mg per 1 liter of culture medium. The minimum concentration of chimeric antibodies necessary for DT neutralization in the CHO cells was 30 pg/mL. Conclusion. Universal plasmids encoding the synthesis of light and heavy chains of chimeric DT -17 antibody have been designed. On the basis of these vectors, a «supervector» and a highly productive clone secreting specific antibodies that had neutralizing activity against DT were obtained.

Keywords

• «супервектор»
• immunoglobulin genes
• «supervector»
• chimeric antibodies
• diphtheria toxin
• toxin neutralizing activity