Construction And Functional Identification Of T Cells Expressing Long-acting Immunopyroptotic Molecules

Targeted therapy of tumors has made a rapid progress,and the research on targeted drugs is more in-depth.In the case of antibody-mediated fusion proteins,our group has previously established an immunoapoptotic strategy to specifically induce tumor cell apoptosis.A series of immunoapoptotic molecules have been generated by fusing an anti-HER2 single-chain antibody(sc Fv),a membrane translocation domain,and a human apoptotic effector.Our previous studies confirmed specific killing of HER2-positive tumor cells by the immunoapoptotic molecules.However,in order to facilitate practical use of immunoapoptotic proteins,there remain two problems to be solved.The first concern is to further reduce therapeutic doses in vivo.The second concern is to reduce the administration frequency.To solve the first problem,the present study aimed to upgrade a novel tumor killing strategy by transforming apoptosis induction to pyroptosis induction,and thus novel immunopyroptotic molecules were constructed by replacing the apoptotic effector t Bid with the pyroptotic effector GSDMD-N(GN).To solve the second problem,these immunopyroptotic molecules were modified by inserting an albumin-binding peptide(ABD035 or d Ab7h8,vs.Fc fragment as positive control)to prolong lifetime of the fusion proteins.The main data are obtained as follows.1.Three kinds of novel long-acting immunopyroptotic genes were constructed and their expression was verified.The controls included long-acting immunoapoptotic genes,immunoapoptotic genes and immunopyroptotic genes.2.Four fusion molecules were functionally tested.Jurkat cells were infected by recombinant lentiviruses carrying p LVX-p1h3-ABD-GN,p LVX-p1h3-78-GN,p LVX-p1h3-GN and p LVX-p1h3-t Bid,and gene-modified Jurkat cells were examined for their killing activity against tumor cells in vitro.The results showed that the Jurkat cells expressing the long-acting immunopyroptotic protein P1h3-ABD-GN and the immunoapoptotic protein P1h3-t Bid both had a killing effect on HER2-positive tumor cells,with P1h3-ABD-GN showing stronger cytotoxicity in vitro.Such data suggested that the long-acting immunopyroptotic molecules exerted stronger killing activities than the immunoapoptotic molecules,and that a cell-based therapy was feasible via secreting immunopyroptotic proteins.3.Human CD3-positive T cells were obtained by separating,negatively selecting and activating human peripheral blood mononuclear cells,followed by gene transduction of the antibody-conjugated fusion genes.Then the gene-modified T cells were evaluated for their killing of tumor cells in vitro.The results showed that T cells secreting p1h3-ABD-GN,p1h3-Fc-GN,p1h3-FC-t Bid and p1h3-t Bid could target HER2 and kill the HER2 positive tumor cells N87 and SKBR3.Notably,the killing effect of p1h3-ABD-GN-expressing T cells was stronger than that of p1h3-t Bid-expressing T cells,indicating functional advantages of this immunopyroptotic strategy over our previous immunoapoptotic strategy and feasibility of primary T cell-based administration of targeted therapeutic proteins.4.An xenograft Balb/c nude mouse model of HER2-positive gastric cancer cell N87 was established and treated by tail vein injection of Jurkat cells secreting P1h3-ABD-GN,P1h3-GN and P1h3-t Bid.The results showed that in the N87-bearing subcutaneous tumor mouse model,the long-acting immunopyroptotic molecules had a stronger ability to inhibit tumor growth in vivo,similar to the previous in vitro conclusion.Meanwhile,immunohistochemistry and TUNEL staining data confirmed HER2 targeting by showing accumulation of more long-acting immunopyroptotic proteins in tumors.This was presumably due to the albumin-binding capability,which allowed for slower in vivo clearance of the secreted fusion proteins and caused a stronger effect on inducing tumor cell death.Conclusions:The long-acting immune immunopyroptotic molecules can kill HER2 positive tumor cells in vitro and in vivo.This killing effect is stronger than that of immunoapoptotic molecules and immunopyroptotic molecules.It is confirmed that the induction of cell pyrosis is more effective than that of cell apoptosis,and the added albumin binding peptide can make the secreted protein play its role longer.