Screening Of CD22 Humanized Genetically Engineering Antibodies

CD19-specific Chimeric Antigen Receptor T cell(CART)has achieved excellent progress in clinical trials of acute lymphoblastic leukemia(ALL),and has significantly improved the clinical treatment of hematological tumors treatment methods.Although CAR-CD19 cell therapy has achieved great success,about 30% of patients have relapsed.Studies have shown that in patients with CAR-CD19 recurrence,the CD19 antigen on the surface of B cells is significantly down-regulated or even turned down negatively.This may be because the use of cells.The root cause of immune escape after treatment.Recently,many studies have extensively elaborated the reasons for the immune escape of B cells.The reasons for recurrence include a series of recurrence mechanisms such as alternative splicing,pedigree switching,biallelic frameshift mutations and intron retention.Therefore,finding new targets or dual-target combination therapy is of great significance for the later clinical treatment of ALL.CD22 is mainly expressed on the surface of normal mature B cells and tumor cells derived from B cells,it mainly inhibits B cell antigen receptor(BCR).CD22 can still be detected on the surface of B cells in patients with recurrence,and it can be used as a very promising target for the treatment of patients with recurrence of CART-CD19.Some CD22-related monoclonal antibody drugs have also been successfully advertised for the treatments of lymphocytic leukemia derived from B cells.Presently,phage antibody library technology has become an important way to obtain humanized high-affinity antibodies,and has been widely used in the fields of medicine and biology.Through repeated rounds of panning,enrichment and elution in vitro or in vivo,and genetically engineered antibodies with high affinity can be quickly screened.In order to further develop cell therapy products or antibody drugs against CART-CD19 recurrence and CD22 as the target disease,this study uses phage antibody library screening technology to screen from commercial humanized phage antibody libraries Domain Antibody Library and Tomlinson I Library Two genetically engineered antibodies with high affinity,one is a single domain antibody(sd Ab)and the other is a single chain variable fragment(sc Fv).The main results of the study are as follows:1.The CD22 extracellular domain expression plasmid was constructed,and a large amount of CD22 extracellular domain protein were expressed by transient transfection of HEK293 FT suspension cells,and high purity and fully glycosylated CD22 extracellular domain were obtained by affinity chromatography,ion exchange chromatography and molecular sieves..At the same time,a 293 cell line capable of stably expressing the full length of CD22 was constructed and screened.2.Using the commercial humanized phage antibody library Domain Antibody Library and Tomlinson I Library,after one round of panning at the level of cells expressing full-length CD22293,and three rounds of streptavidin-biotin magnetic beads solid phase panning,Obtained the single domain antibody secondary library and the single chain antibody secondary library against the CD22 target respectively.Enzyme-linked immunosorbent assay(Enzyme Linked Immune Sorbent Assay,ELISA)was used to screen individual clones for the two antibody libraries enriched by panning,and one antibody fragment with good affinity was obtained respectively.The two clones were sequenced and antibody sequence were also analyzed,and the affinity of the two antibodies was preliminarily determined through ELISA.