Construction And Activity Of The Mutant Hi47f Of Anti-cd20 Chimeric Antibody (ab ') <sub> 2 </ Sub>

In recent years,antibody therapies for tumor were concerned in clinic.Monoclonal antibodies (mAb) directed against CD20,including their radiolabed forms,have been successfully exploited in clinic as effective therapeutic agents in the management of non-Hodgkin's B-cell lymphoma.Three anti-CD20 antibodies (Rituximab,Bexxar,Zevalin) have been approved by Americn Food and Drug Administration(FDA),which indicates that anti-CD20 antibodies are promising in clinic.HI47 was a murine anti-CD20 antibody, developed in 1990 by the institute of Hematology.Using HI47 as parent,we had already constructed its Fab' by random mutation,with the mutant strain named CD20-7.The present study was designed for a constrction of F(ab')₂ and to study its activity in vitro and in vivo and further to optimize its puritification and idonatation condition.By adding a sequence encoding a small peptide, (CPP)3, to the base sequence of pYZFcd20, a new phasmid pYZcpp3 was constructed.Using the pYZcpp3 to transform E.coli. 16c9, the genetically engineered bacteria was obtained. The product of the F(ab')₂ fragment was 3.8 mg/L,and the percentage of F(ab')₂was 15%.Inoculatingv olumn,aerating capacity,germ's age,temperature,pH,fermentation cycle were explored one by one to determine the optimal condition.High density culture was carried out in NLF to produce the F(ab')₂ fragment.The final OD of the fermentation was 140,and the weight of the wet bacterium was 200g/L.The product of the fragment was 241mg/L,and the percentage of F(ab')₂ was 15%, which reached our primely aim.Portein-G was used to prepurified the sample,which obtained the mixture of the F(ab')₂ and Fab'.Gel filtration chromatography,hydrophobic interaction chromatography and ion exchange chromatography were explored to find the best method to purify the F(ab')₂.The purity of these methods was 90.5%,89.3%,92.3%, respectly.And the yield of F(ab')₂ was 65%,67%,70.2%.The ion exchange chromatography was then determined as the best method,because of its large capacity,high yield and purity.The activity in vitro and in vivo of F(ab')2 was evaluated. The F(ab')2 can distinguish and bind to the CD20⁺ B lymphoma cell. Under the same level, F(ab')₂ could compete HI47 binding to the CD20 more efficaciously than Fab'. F(ab')₂ could inhibit the proliferation of CD20⁺ B lymphoma cell more intensely in vitro...