Chimeric Anti-cd20 Genetically Engineered Antibody Construct The Expression And Function

To make monoclonal antibody HI47 enter the clinic therapy of B cell lymphoma, genes of variable region of HI47 were cloned by phage display technology, chimeric antibody was constructed to low HAMA of HI47 and expressed in mammalian cell and Ecoli.1 Cloning of antibody variable region genes and construction of anti-CD20 ScFv phage display libraryExtract total RNA of HI47 hybridoma cell, amplify the genes of antibody variable region of heave chain(VH) and light chain(VL) by RT-PCR, VH and VL were linked by overlap PCR with (G₄S)₃ linker to construct ScFv, ScFv was then cloned into the vector pCANTAB 5E to construct the anti-CD20 ScFv phage display library, through panning, screening by ELISA with Daudi cell as target cell and getting several clones binding specially to CD20, result from the DNA sequencing of clone 6 indicated that this sequence is an antibody gene sequence.2 Construction, expression and function study of chimeric anti-CD20 antibodyAmplified genes of VH and VL of HI47 from the anti-CD20 ScFv expression vector pCANTAB5Ecd20 by PCR, then cloned VH and VL into the vector pYZF containing human antibody CH1 and CL to construct Fab' expression vector pYZFcd20, transformed 16C9 with pYZFcd20 and expressed Fab' in 16C9, The yield of Fab' in 16C9 is about 3mg/ml.Bioactivities of Fab' was studied in vivo and vitro. competitive inhibition of immune fluorescence experiment show that the Fab' inhibits the binding of monoclonal antibody HI47 to CD20 on the surface of Raji cell. Growth inhibition of Daudi and Raji cell by Fab' were observed by MTT method. Fab' inhibited the synthesis of RNA but not DNA of Daudi cell and Raji cell. Experimental therapy of Raji cell lymphoma in dose of 50mg/kg, every four days once, 6 times in a nude mouse xenograft model of Raji cell showed that Fab' has a good anti-tumor effect on Raji cell lymphoma, the tumor growth inhibition rate is about 56%. Induction of tumor cell apoptosis maybe one of anti-tumor mechanisms of chimeric anti-CD20 Fab' antibody fragment .VH and VL genes were cloned into pDR2 containing human antibody CH1,...