Reconstruction Of Anti-CD20 Monoclonal Antibody With Computer-aided Molecular Modeling Design

The clinical application of murine monoclonal antibodies is associated with a number of drawbacks, including inducement of human anti-mouse antibody (HAMA), short circulating time in vivo, and the inability to fully harness the effectors (ADCC) and complement (CDC) repertoire of the human immune system. Genetic engineering has been used to humanize mouse mAb and can overcome the drawbacks significantly. Our group developed a mouse anti-human CD20 monoclonal antibody 1-28, which possessed potential clinical values. The immunological characteristics of mAb 1-28 were tested. With computer-aided molecular modeling design method and DNA recombination technology, chimeric anti-CD20 antibody and single-chain Fv (scFv) with human lgG1 hinge and Fc regions, designated 5S, were produced. Their binding activity and complement-dependent cytotoxicity (CDC) were analyzed. 1 The immunological characters study on mAb 1-28Specific binding activity of mAb 1-28 was verified by flow cytometry method. The relative binding affinity of mAb 1-28 was only about 1/12th of Rituxan. K value of 1-28 was 1.6×10¹⁰M^-1 measured by Scatchard analysis. The results of competitive binding assay showed that both 2H7 (mouse anti-human CD20 antibody) and Rituxan (chimeric anti-CD20 antibody) could not inhibit the binding of 1-28 to CD20 antigen expressed on the surface of Raji cells. 1-28 could alone inhibit CD20+ cells' proliferation and was sufficient to induce apoptosis of Raji cells and Daudi cells. 1-28 could also fix and activate complement to kill CD20+ cells, such as Raji, while it's ineffective to Jurkat cells.2 Expression and characterization of Chimeric Anti-CD20 AntibodyThe light- and heavy-chain genes were amplified from hybridoma cell line 1-28 secreting anti-human CD20 mAb by RT-PCR method, and then cloned to T vector, sequenced. MAb 1-28 protein was separated by reduced SDS-PAGE, and the light- and heavy-chain bands were excised from preparative gel, digested by trypsin, and subjected to peptide mass fingerprinting. The light- and heavy-chain DNA sequences were verified by their protein sequences. To acquire a positive control in the experiments, V genes of 2H7 were synthesized by overlapping PCR method. Both V genes of 1-28 and 2H7 were cloned into chimeric antibody expression vector (pCMV-VH & pCMV-VL), generating chimeric anti-human CD20 mAb expression vectors. Plasmids were transfected into 293T cells with Lipofectamine? 2000. High level expression of the two anti-human CD20 chimeric mAbs were obtained and their m.w. in agreement with that of human IgG. The binding activity of C2H7 was well reserved, but C1-28 lost its binding activity to Daudi and Raji cells completely. In complement-dependent cytotoxicity assay, C2H7 could fix and activate both human complement and rabbit complement to kill target cells specifically.3 Design of a new kind of anti-CD20 antibodyThe spatial structure of mAb 1-28 variable region was modeled theoretically with homology method carried on http://www.expasy.ch. The stability of mAb 1-28 Fv was analyzed according to inter-molecular hydrogen-bond forming theory and reaction free energy theory. The result showed that the stability of the complex Fv was weak due to the structural non-match and electrostatic non-complementarity. The weak stability of the complex Fv induced the low bioactivity. The residues characters of N and C terminal of antibody 1-28 were studied based on distance geometry and computer graphics technology. The single chain Fv antibody (abbr. scFv) was constructed and the mode was VH-Linker-VL. The result showed that the stability of the antibody 1-28 was increased while the active domain conformation was not changed due to the...