Biological Function Research Of A Mouse Anti Human CD22 Monoclonal Antibody HIB22 And Development Of Single Chain Fv Derived From HIB22 Hybridoma

CD22 is an inhibitory coreceptor of the B-cell receptor(BCR),and is required to modulate the antigen receptor signal in response to cues from the local microenvironment. CD22 selectively expresses on the surface of normal and malignant mature B cells,and can be tested expressing on more than 85%B-cell NHL(non-Hodgkin's lymphoma).The ligation of natural ligand or antibody with CD22 expressed on mature or malignant B cells can trigger a rapid internalization of the CD22-ligand complex.Also,CD22 expresses stably on malignancies,and does not have any change in response to the microenvironment.All these advantages endow CD22 an excellent target for the therapy of NHL and autoimmunity diseases.Research about CD22-targeted therapy is mainly focus on the development of anti-CD22 monoclonal antibodies and drugs derived from these antibodies,while Epratuzumab is one of the most widely used anti-CD22 antibodies in clinical research.HIB22 is a mouse anti human CD22 monoclonal antibody that was developed in Dept.of Immunology, Institute of Hematology,Chinese Academy of Medical Sciences,and has a different recognizing epitope from Epratuzumab.CD22 can regulate B cells through ligand-dependent or-independent mechanisms,so the use of different monoclonal antibodies can result in a series of clinical efficacy.In this research,FACS,MTT testing,RT-PCR and confocal microscopy was used for the in vitro biological function research of HIB22:the effect on the proliferation and apoptosis of NHL relevant cell line Daudi and Raji was investigated;the impact on the clone formation and CD25 expression of PWM stimulated PBMC from health donor was investigated;and the internalization of HIB22-CD22 complex was observed.An anti-CD22 single chain Fv was also developed from HIB22 hybridoma:DNA encoding variable light and heavy chain was synthesized using RT-PCR from HIB22 hybridoma respectively;the fusion gene encoding HIB22-scFv was constructed by overlap-PCR, and then cloned into the pET22b(+) vector,which as a result formed a reconstructive vector pET22b-HIB22-scFv;recombinant vector was then transformed into the E.coli strain BL21(DE3)~9,and the expression was induced by IPTG;The soluble expression product was purified by Nickel Sepharose,and its binding ability and specificity to CD22~+ Raji cells was analyzed by FACS.The in vitro biological function research of HIB22 showed that:HIB22 can efficiently inhibit the proliferation of NHL relevant cell lines Daudi and Raji,and can also impair the over-transcription of proto-oncogene c-myc;HIB22 can significantly inhibit the clone formation and B-cell activation in PWM-activated PBMC from health donor,and HIB22 has certain influence on the number of CD4~+CD25~+T cells and CD8~+CD25~+T cells;also, the internalization of HIB22-CD22 complex was observed clearly.In the genetic reconstruction of HIB22 monoclonal antibody,the fusion protein prokaryotic expression vector pET22b-HIB22-scFv was constructed successfully.Soluble scFv was obtained from E.coli expression system induced by IPTG.Biological activity assay showed that the expressed HIB22-scFv had a specific binding activity with CD22~+ Raji cell line,and the competitive inhibition test indicated that both the HIB22-scFv fusion protein and its parental monoclonal antibody HIB22 has the same binding epitope.