Development Of A Human-Mouse Chimeric Antibody Anti-GPâ…¡b/â…¢a

The objective of this study: To increase anti-GPIIb/IIIa monoclonal antibodies therapeutic usefulness, we chose the best one (PI40) from three kinds of anti-GPIIb/IIIa monoclonal antibodies and used genetic engineering techniques to replace the murine constant regions with human counterparts while retaining the murine antigen binding regions. We successfully developed a chimeric antibody CP140 reconstructed from McAb PI40 which consists of murine antigen binding region (from PI40) and human constant region (from human IgG1). We hope that it can be used for therapy of acute myocardial infarction, unstable angina, and ischemic disease.At first, the best mouse anti-human GPIIb/IIIa monoclonal antibody P140 was selected by the methods of indirect immunofluorescence and inhibition of platelet aggregation. The cDNA fragments of Fd and Light chain encoding 140Fab antibody were amplified by RT-PCR from mRNA extracted from hybridoma cell of P140. The genes were sequenced and consistent with Sequences of Proteins of Immunological Interest. The genes of P140Fab were recombined into p3MH plasmid. After identified by the restriction enzyme digestion, the recombinant clone was transformed into the competent expressive cells of E.coli XLI-Blu. P140Fab was induced by IPTG and further purified by TALON metal affinity resins. We obtained a soluble protein with molecular weigh of 47kD. Identified by ELISA and Western blot and Test of platelet aggragation, a highly specific P140Fab, which can effectively inhibit platelet aggragation, was purified. The results showed that soluble PHOFab was successfully highly expressed.The intact variable genes of P140 were amplified by RT-PCR from mRNA extracted from hybridoma cell of P140 by RACE. The genes were sequenced and consistent with Sequences of Proteins of Immunological Interest. The intact variable genes of P140 were recombined into plasmid VH and VK , and the eukaryotic expression vectors VH/CP140 and V K. /CP140 were constructed. After identified by the restriction enzyme digestion, VH/CP140 and VK /CP140 were transfected into CHO cell line. The RT-PCR analysis showed that the transfected CHO cell has the gene transcription of human-mouse antibody. The result of ELISA showed the human-mouse chimeric antibody CP140 was expressed in the supernatant. In order to improve the product of the antibody, the variable genes of P140 with leader were recombined into plasmid PWS3, and the eukaryotic expression vector PWS3-P140 was constructed. After identified by the restriction enzyme digestion, PWS3-P140 was transfected into CHO cell line. The results of ELISA showed the human-mouse chimeric antibody CP140 was high-expressed in the supernatant.Conclusion: A new anti-human GPIIb/IIIa P140Fab was successfully developed. A new anti-human GPIIb/IIIa human-mouse chimeric antibody CP140 was successfully expressed in eukaryotic cells.