Research Of Inducing T-cell Activation And Produce A Specific Immune Response To Tumor Cells Labeled With HBV-derived Epitopes

Objective:Crispr/Cas9 gene editing technology was used to transform the mutation site of the tumor into specific coding sequence of antigen epitope.This antigen can be presented by MHC-I molecules and be recognized by TCR of T cells,as a result,so as to activate immune cells and induce anti-tumor immune response.Research methods:A specific mutation in the TP53 region of tumor cell K562 through high-throughput genome sequencing was observed.A designed sg RNA for this mutation and directional knockout of the mutation site.Based on this,in this research,we use the sequence encoding a specific HBV surface antigen epitope-HBsAg_335-343 epitope as a Donor,and the genome editing technology was integrated into the mutation site of tumor cells.The edited single cells were obtained by flow sorting,and the stable K562-A2-HBVs E cell line was obtained after expanded culture.The gene modified tumor cells were co-cultured with specifically differentiated T cells in vitro to test the killing efficiency,and K562-HBV humanized tumor model was constructed by NOD-SCID mice in vivo to test the killing efficiency.The main experimental processes are as follows:1.Constructing K562-A2 stable rotation system;2.Constructing K562-A2-HBV stable transformation system;3.Inducing/preparing T cells that recognize the specific epitope;4.Evaluation of antitumor effect targeting this specific antigen epitope.Research results:1.Since the presentation of HBV antigen peptides depends on MHC-I molecules,MHC-I molecules need to be artificially introduced into K562cells.In the early stage of the laboratory,BIMAS and SYFPEITHI were used to predict and verify the affinity between HBsAg_335-343 peptide and HLA-A2 MHC-I molecules,and a lentivirus carrying HLA-A2 MHC-I molecular coding gene was synthesized.The virus also carries puromycin resistance gene.After infecting K562 cells with the virus,we obtained a K562 cell line stably expressing HLA-A2 MHC-I molecules after puromycin screening and culture for one week,and named it K562-A2.2.The third-generation gene editing technology Crispr/Cas9system was used in this research,HBsAg_335-343 antigen peptide was knocked into the mutation site of tumor cell K562.CRISPR website laboratory（http://crispr.mit.edu/）was used in the early stage which provided targeted editing site tool designed the sg RNA sequence and constructed the p X458 targeted editing plasmid.The HBsAg_335-343 antigen epitope was synthesized by molecular cloning technology,and the T-Donor homologous template Donor plasmid was constructed.The two plasmids were co-transfected into K562-A2 cells by electroporation,p X458 plasmid carries EGFP green fluorescence and T-Donor plasmid carries mCherry red fluorescence,we can obtain edited single cells by flow sorting.After expanded culture,K562-A2 cell line stably expressing HBsAg_335-343 antigen peptide was obtained and named K562-A2-HBVs E.3.Induction/preparation of T cells that can specifically recognize HBsAg_335-343 peptide（1）Expressing specific TCR by Jurkat cells:TA1 and TA2,which can specifically be recognized by HBVs E antigen peptides,were TCR genes and obtained from differentiated T cells in the early stage of the laboratory.The plasmid pc DNA3.1（-）-TA1 and pc DNA3.1（-）-TA2 were constructed.The plasmid was transfected into Jurkat cells by electroporation,and Jurkat cells expressing specific TCR were obtained.（2）Induction of T cell specific differentiation:PBMC was isolated from peripheral blood of volunteers,and HBsAg_335-343 peptide specific CTL was induced by stimulation of exogenous peptides HBsAg_335-343 and IL-2.4.Detecting the targeted killing efficiency of effector T cells on modified K562 tumor cells:（1）In vitro co-incubation test:after the target cells K562 and K562-A2-HBVs E were incubated by calcein staining,peptide specific CTL was added for co-incubation.The cytotoxicity test showed that the cleavage rate of K562 cells was 33.16%,and that of K562-A2-HBVs E cells was51.74%.Compared with wild-type K562,the ratio of K562-A2-HBVs E killed by specific CTL after transformation was significantly improved.（2）Construction of animal model in vivo:using the immune deficiency of NOD-SCID mice,we can transplant human cells into animals to build a stable animal model.Tumor cells K562-A2-HBVs E were injected into the axillary part of NOD-SCID mice by subcutaneous injection and tumor block transplantation,and into the tail-by-tail vein injection.The growth size of tumor block at the injection site and the survival cycle of mice were recorded to evaluate the transplantation success rate and transplantation stability of K562-A2-HBVs E humanized tumor model.Finally,the subcutaneous tumor bearing method was selected to construct a stable tumor model with high tumor formation rate.The model mice formed tumors about 2 weeks after tumor bearing,and the survival time of mice was more than 45 days.Conclusion:Although new antigens in tumors may not produce spontaneously,mutations are inevitable.We can use known exogenous epitope peptides with strong ability to induce immune response to specifically mark these mutations.In theory,they will also induce immune response.In this paper,we used Crispr/Cas9 technology to specifically edit the genome of specific mutations in K562 cells,inserted the coding sequence of epitope peptide derived from HBV surface antigen,and prepared immune cells that specifically recognized the epitope peptide.Through in vitro killing experiment,it was preliminarily shown that the inserted epitope peptide can induce specific immune response,it provides a new idea for antitumor research.