Studies On The Production And Antitumor Activities Of Novel Bispecific Proteins Activating Natural Kliller Cells

NK cells are the body’s first line of defense against cancer cell and virus infections,and can directly kill tumor cells non-specifically.This natural killing activity requires neither antigen sensitization nor antibody participation,and is not restricted by MHC.Unlike T cells,NK cells can not cause graft-versus-host disease(GvHD),and are expected to become more widely used and convenient tumor immunotherapy effector cells.ULBP1 is poper macrophage virus glycoprotein UL16 binding protein,and is one of the ligands of the important activating receptor NKG2D on the surface of NK cells.ULBP1 Ligand protein is highly expressed on tumor cells but not in nonmal cells.The binding of ULBP1 and NKG2D can directly activate NK cells,thus exerting the killing effect of NK cells on target cells.Once activated,NK cells infiltrate tissues,secreting perforins and TNF,which attack tumor cells and virus-infected cells.In this study,based on the principle of bispecific T cell engager(BiTE),two new types of bispecific fusion proteins were designed.The results of previous studies were tried to express bispecific fusion proteins.These proteins activate NK At the same time,the cells can target some tumor cells that specifically express antigen protein,which can improve the killing effect on tumor cells.One end of these two fusion proteins is composed of NKG2D ligand ULBP1,and the other end is a single-chain antibody and variable domain of tumor-associated B19 tumor antigen CD 19 antibody or a specific affinity antigen epidermal factor The receptor(Epdermal growth factor receptor,EGFR)affinity body(ZEGFR)is fused to obtain the composition,that is,ULBP1×CD19 scFv or ULBP1×ZEGFR.For the dual-specific fusion protein ULBP1×CD19 scFv,the pPICZα-ULBP1×CD19 scFv recombinant plasmid was established in the amplification laboratory to re-establish the pPICZα-ULBP1×CD19 scFv/X33 expression engineering bacteria.Identify positive transformants,select high-expression clones and perform large-scale expression of ULBP1×CD19 scFv bispecific fusion protein under optimal conditions The target protein was purified by ammonium sulfate fractionation precipitation and histidine tag affinity chromatography to obtain ULBP1×CD19 scFv with high purity.ULBP1×CD19 scFv can increase the killing activity of NK-92MI cells against CD19 overexpressing Raji cells.For the bispecific fusion protein ULBPI×ZEGFR,the gene encoding the fusion protein was obtained by overlapping PCR,and the gene was cloned into the pPICZaA expression vector to obtain the pPICZa-ULBP 1×ZEGFR recombinant plasmid,and the pPICZα-ULBP1×ZEGFR/X33 expression engineering bacteria was established.The Pichia pastoris secretory expression system was used to prepare the target protein to obtain the ULBP1×zEGFR fusion protein with higher purity.Subsequently,the secondary structure of the dual-specific fusion protein was analyzed by circular dichroism spectroscopy,and the specific affinity of the protein for H460,a highly expressed EGFR lung cancer tumor cell,was identified using flow cytometry.The recombinant protein ULBP1×ZEGFR increased NK-The killing activity of 92MI cells on H460 cells with high expression of EGFR.The above results indicate that the ULBP1 ligand protein can be used as an activation element to construct a bispecific fusion protein for NK cell targeting tumor cell killing activity,and the Pichia pastoris secretory expression system can be used as a platform for foreign gene expression technology to express such proteins.