The Expression And Functional Identification Of A HER2-Targeted Immunopyroptotic Molecule

Immunoapoptotic strategy for selective induction of apoptosis in HER2-positive tumor cells is composed of three parts: an anti-HER2 single-chain antibody(sc Fv),translocation domain and human apoptotic effectors.Immunoapoptotic molecules specifically identify and will be internalized into HER2-positive tumor cells,and the C-terminal apoptotic effector molecules were released and translocated to the cytoplasm under the cleavage of specific proteases in the endosomes,causing an activation of cascade reaction which exerts anti-tumor effects consequently.In our previous studies,we have optimized the above three components and constructed humanized immunoapoptotic molecule P1h3-Fdt-t Bid,which has killing activity against a variety of HER2-positive tumor cells in vivo and in vitro.We have also screened out D1 linker peptide which is more stable than Fdt in eukaryotic expression secretion system.However,there still exist the problems of large doses and high treatment frequency in animal experiments,which are a barrier to clinical application.Aiming at this key problem,the present project intends to upgrade the killing strategy.By replacing the apoptotic effector molecule with a pyroptotic effector molecule,the immunopyroptotic molecule was constructed in order to upgrade the apoptosis induction of tumor cells to the more efficient pyroptosis induction strategy,which would effectively enhance the anti-tumor killing capabilities and lay a foundation for further clinical application transformation.Firstly,we constructed eukaryotic expression vectors of the active pyroptotic effector molecule GSDMD-N and its mutant m GSDMD-N,with the active apoptotic effector molecule t Bid as a positive control.At 16 hours after transient transfection of 293 T cells,cell death rates were observed and analyzed.The death rate of GSDMD-N transfected cells was up to 55%,while that of t Bid-transfected cells was only 29%,which showed that the active pyroptotic effector molecule had more efficient killing activities.Mutant GSDMD-N(m GSDMD-N)was designed to inhibit its cellular membrane perforation function,and the death rate of m GSDMD-N transfected cells was significantly reduced,indicating that the killing effects of m GSDMD-N was mediated via pyroptosis.Then we incorporated the active pyroptosis effector molecule GSDMD-N into our previous immunoapoptotic strategy.The P1h3,Fc segment,D1 linker peptide and GSDMD-N(GN)were successively fused to construct the prokaryotic expression vector of immunopyroptotic molecule P1h3-Fc-D1-GN.Meanwhile,the prokaryotic expression vector of immunoapoptotic molecule P1h3-Fc-D1-t Bid was constructed as the control.Despite the success in inducing the expression of the target protein,the purification process encountered obstacles that hindered subsequent functional experiments.Next,we constructed eukaryotic expression vectors of P1h3-Fc-D1-GN and P1h3-Fc-D1-t Bid,respectively.Culture supernatant containing the target proteins was obtained from 293 F transfectant cells,and then incubated with tumor cells with different expression level of HER2 for validation of their cytotoxic function in vitro.PI staining assay showed that the PI positive rate of HER2-expressing tumor cells in the P1h3-Fc-D1-GN group was higher than that in the P1h3-Fc-D1-t Bid group and the control group,and there was no significant difference in the PI positive rate of HER2 negative target cells.It suggested that the immunopyroptotic molecules have a specific killing effect on HER2-positive tumor cells,and that it is feasible to apply the active pyroptotic effector molecule to the targeted anti-tumor strategy.The results of luciferase activity detection showed that the killing of immunopyroptotic molecules to tumor cells with high expression of HER2 was significantly stronger than that of immunoapoptotic molecules,and the same trend was observed in tumor cells with low expression of HER2 although not statistically significant.The results showed that the antitumor effect of the immunopyroptotic strategy was more effective than that of the immunoapoptotic strategy.Finally,in order to solve the difficulty of purification in prokaryotic expression and the problem of poor secretion in eukaryotic expression of immunopyroptotic molecules,the prokaryotic expression vectors of the modified immunopyroptotic molecules GSDMD(D1)-P1h3 were constructed by fusing the active pyroptosis effector molecule GSDMD-N,cathepsin D restriction site D1,pyroptosis self-inhibition domain GSDMD-C,semi-flexible linker peptide and humanized single chain antibody P1h3.GSDMD(D1)-P1h3 was successfully expressed and was optimized for purification.In summary,the strategy of applying active pyroptotic effector molecules to targeted tumor killing is feasible.The immunopyroptotic protein is more effective than the immunoapoptotic protein in killing a variety of HER2-positive tumor cells,which has potential application in enhancing anti-tumor function and reducing drug doses.