Structural Modification Of Immunopyroptotins And Antitumor Functional Validation Of The Gene-Engineered T Cells

Cancer has always been a major factor threatening human life and health.HER2,a member of the human epidermal growth factor receptor family,is overexpressed in a variety of tumor cells and is a recognized antitumor target.Important progress has been made in anti-tumor therapy targeting HER2:The monoclonal antibody that recognizes HER2 has been used in the clinic,but the problems of low single-drug response rate and drug resistance have appeared in the process of use.Immunotoxins targeting HER2have entered clinical trials,but have not yet effectively addressed issues such as neutralizing antibody production and cardiotoxicity.Other immunoconjugated proteins are still in the preclinical research phase.We have established an immunoapoptotic strategy for selective induction of apoptosis in HER2-positive tumor cells with decreased immunogenicity.The development of immunomotropy is composed of three parts,and each part is formed by single-chain antibody recognition HER2,and the entire fusion molecule is internalized into tumor cells,and the membrane interposition domain is specifically.The protease cut,releases apoptotic molecules to kill tumor cells.However,the transformation and application of immunoapoptotic molecules faces technical bottlenecks and needs to be improved and optimized.There are three main aspects,The first is to reduce the molecular weight as much as possible to improve the protein expression efficiency and tissue permeability;the second is to strengthen the killing effect to reduce the dosage of purified protein;the third is to prolong the serum half-life to increase the dosing interval.In this paper,based on the original immune-apoptotic strategy,the following design and reconstruction are carried out.In order to reduce the molecular weight,the anti-HER2 single-chain antibody P1h3(239aa)was replaced with a HER2-directed short peptide P12(12aa)to construct a targeted fusion molecule.In order to improve the killing efficiency,the apoptotic effector molecule t Bid was replaced with the pyroptotic effector molecule GSDMD-N to obtain a new type of immunopyroptotic molecule.In order to reduce the in vivo clearance rate,albumin-binding peptides were introduced into the immunopyroptotic molecules,albumin-binding domain ABD035 derived from bacteria,human-derived anti-human albumin single-domain antibody d Ab7h8 were selected,and the Fc segment of Ig G antibody was used as a control.to construct a long-acting immunopyroptotic molecule.The above fusion gene was packaged into a recombinant lentivirus,introduced into Jurkat cells to establish a line,and its effect on killing HER2-positive tumors was verified by in vitro and in vivo experiments.The specific research results are as follows1.Construction of short peptide-directed long-acting immunopyroptosis geneConstruction of long-acting immunopromoting pyroptotic molecules,Using techniques such as PCR amplification and double-enzyme digestion of the target fragment,the short peptide P12 that recognizes HER2,the albumin-binding peptide ABD035 or d Ab7h8,the membrane translocation domain B2-E5C3,and the active pyroptotic molecule GSDMD-N(GN)gene fragments were sequentially recombined to obtain P12-ABD-B2-E5C3-GN and P12-78-B2-E5C3-GN,which were identified by PCR as1278bp and 1368bp respectively.In addition,a long-acting control P12-Fc-B2-E5C3-GN containing the Fc fragment of an Ig G antibody was constructed,with a size of 1695bp.Construction of a long-acting immunoapoptotic molecular control,The P12 short peptide,the Fc segment of Ig G antibody,the membrane translocation domain B2-E5C3,and the active pro-apoptotic molecule t Bid were sequentially recombined by PCR technology,and the size was 1182bp identified by PCR.Construction of short-acting immunoapoptotic molecules as controls,The P12 short peptide,membrane translocation domain B2-E5C3,and pro-apoptotic molecule t Bid was identified by PCR as 618bp.2.Establishment and in vitro functional verification of genetically modified Jurkat cells293T cells were used to transiently transfect the constructed immune pyroptotic molecules and apoptotic molecules,and the fusion gene was verified to be expressed in the cells at the protein level.Through lentiviral packaging,the virus was concentrated to infect Jurkat cells,genetically modified Jurkat cells were constructed,and the expression of the target protein was verified at the protein level.The established cells were co-cultured with tumor cells,and it was observed under different effector-target ratios and different action time conditions that Jurkat cells expressing long-acting immune pyroptotic molecules could specifically kill HER2-positive cells,but PC9~-cells have no effect.The above experiments yielded the following results,the long-acting immuneapoptotic molecules have a stronger killing effect on tumors than the immunoapoptotic molecules,and show obvious killing effects in a low-efficiency target ratio and in a short period of time.3.In vivo anti-tumor function verification of established Jurkat cellsN87 human gastric cancer cells were used to build a Balb/c nude mouse gastric cancer subcutaneous tumor model,and the killing of the tumor by Jurkat cells established by long-acting immunoapoptotic molecules was detected.group and naked Jurkat cell group as negative control.It was observed through in vivo imaging of animals that the effect of long-acting immunoscorch gene-modified Jurkat cells in inhibiting tumor growth was stronger than that of Jurkat cells modified by short-acting apoptotic genes.After the end of treatment,the tumor tissue was stripped away,immunohistochemistry and TUNEL staining were performed,and more long-acting immune focus-dying proteins were observed in the tumor tissue,causing more tumor cell death.the long-acting immuneapoptotic molecules have a stronger killing effect on tumors than the immunoapoptotic molecules,and show obvious killing effects in a low-efficiency target ratio and in a short period of time.