Humanization Of Anti-human CD40 Antibody And Preliminary Study Of Enhanced Expression In Mammalian Cells

This research was based on the human CD40 antagonist mouse monoclonal antibody (5C11) and human muCD40 antagonist mouse monoclonal antibody (5H6) established by us. We constructed and expressed mouse-human chimeric antibody against CD40 in 293T cells, analyze and modify the amino acids sequence of 5C11 in order to enhance the expression amount and characterized its primary biological function.Part I: Construction, Expression and Primary Biological Function of Mouse - Human Chimeric Antibody against CD40Fd and L chain gene were amplified by RT-PCR using degenerate primer from 5C11 murine hybridoma cell line. Design the primers according to heavy and light chain gene sequences and amplify VH and VL gene. Treat the vectors which contain signal peptide sequences and human Fc, CH1 and Cκgene of human IgG1 by Double digestion. Clone the VH and VL gene into chimeric antibody expression vector (pCMV-VH & pCMV-VL ), generating the expression vectors of chimeric anti-human CD40 mAb including light chain expression vector 5C11L-pCMV and heavy chain expression vector 5C11H-pCMV. Recombinant plasmid was transfected into 293T cells using EntransterTM-D. ch5C11 expression was detected by sandwich ELISA and the specificity binding was analyzed by flow cytometry. ch5C11 antibody how to act on proliferation of Daudi cells (conjunction ratio > 90%) was detected by CCK-8. Restriction endonuclease digestion and PCR showed that recombinant genes have been cloned into vector 5C11H-pCMV and 5C11L-pCMV. Chimeric antibody could transiently express in 293T cells。ch5C11 antibody identifies mCD40 on Daudi cells (conjunction ratio 95.5%). ch5C11 effectively inhibits growth.Analyze and modify the amino acids sequence of 5C11 in order to enhance the expression amount. Recombinant plasmid 5C11L-pCMV and 5C11H(M)-pCMV was transfected into 293T cells using EntransterTM-D. ch5C11(M) expression was detected by sandwich ELISA and the specificity binding was analyzed by flow cytometry. ch5C11(M) antibody how to act on proliferation of Daudi cells (conjunction ratio > 90%) was detected by CCK-8. Results showed that ch5C11(M)antibody identifies mCD40 on Daudi cells (conjunction ratio 95.5%). ch5C11 effectively inhibits growth and the expression amount was enhanced effectively.Part II: Construction, Expression and Primary Biological Function of Mouse - Human Chimeric Antibody against muCD40Fd and L chain gene were amplified by RT-PCR using degenerate primer from 5H6 murine hybridoma cell line. Design the primers according to heavy and light chain gene sequences and amplify VH and VL gene. Treat the vectors which contain signal peptide sequences and human Fc, CH1 and Cκgene of human IgG1 by Double digestion. Clone the VH and VL gene into chimeric antibody expression vector (pCMV-VH & pCMV-VL ), generating the expression vectors of chimeric anti-human muCD40 mAb including light chain expression vector 5H6L-pCMV and heavy chain expression vector 5H6H-pCMV. Recombinant plasmid was transfected into 293T cells using EntransterTM-D. ch5H6 expression was detected by sandwich ELISA and the specificity binding was analyzed by flow cytometry.In conclusion, our study has constructed recombinant plasmid 5C11H-pCMV and 5C11L-pCMV, expressed mouse-human chimeric antibody against human CD40 in 293T cells successfully. ch5C11 antibody could depress proliferation of Daudi cells and the expression amount was enhanced effectively by analyze and modify the amino acids sequence. ch5C11 antibody has potency on tumor immunotherapy and graft rejection.And we has constructed recombinant plasmid 5H6H-pCMV and 5H6L-pCMV, expressed mouse-human chimeric antibody against human muCD40 in 293T cells successfully too.