| Bioimmunotherapy has been recognized as an effective treatment model in the field of blood tumor treatment.Hematologic tumors differ from solid tumors in their biological characteristics because tiny residual malignant cells can cause disease recurrence.At the same time,blood cells are responsible for important physiological and immune system functions of the body,so the course of blood tumors is more dangerous.Traditional treatments cannot eliminate it completely.For example,chemotherapy,radiotherapy and even bone marrow transplantation often have serious toxic and side effects in the treatment process,which restricts its further extensive application in clinical practice and makes it impossible to achieve complete cure.Therefore,it is urgent to find a new safe and effective treatment.In recent years,targeted immune cell therapy represented by T cells modified by chimeric antigen receptor(CAR)has attracted extensive attention.The technology works by transfection T cells with chimeric antigen receptors that can specifically target tumor surface antigens.Meanwhile,artificial chimeric receptor intracellular domain can provide T cell activation signal,making it a new generation of cell therapy with precise targeting and continuous killing activity,showing good therapeutic effect in the treatment of a variety of tumors,especially blood system tumors.At present,most studies on CAR mainly focus on T cells.However,side effects such as cytokine storm and off-target effect have become important factors limiting the clinical application of CAR-T cells.In this regard,we focused our attention on the artificial chimeric receptor modification of non-specific immune cells represented by natural killer(NK)cells and cytokine induced killer cells.NK cells are an important part of innate immune system and have special advantages in tumor immunotherapy.First,NK cells are not limited by MHC recognition and can activate an autoimmune response without prior sensitization.Secondly,NK cells in adoptive cell transfusion therapy have a short survival cycle in vivo and do not induce anti-graft host reaction.Compared with T cells,NK cells do not secrete cytokines such as IL-6,which can induce cytokine storms.However,in the immune microenvironment,human tumor cells evade the non-specific killing effect of NK cells through various immune escape mechanisms,which limits the role of NK cells in tumor immunotherapy.Based on the above factors and the previous foundation and strategy in CAR-CIK cell research,we introduced chimeric antigen receptor modification to NK cells to guide NK cells to kill blood tumor cells,and at the same time,this study used the expression of chimeric receptor intracellular activation signal to enhance the immune cells' own activity.First,we targeted CD19,a specific target of B lymphoblastic leukemia cells,and carried out the modification of the intracellular segments and transmembrane domain of artificial chimeric receptors for the NK cell lines and the specific activation signals of NK cells from umbilical cord blood.In the process of intracellular section optimization,we will have the function of NK cell activation of IL-15 functional areas,4-1 bb cell activation signal area,CD3? chain intracellular area,CD8 guide chain and intracellular Kozak sequences activation integration in tandem structure domain related molecular sequence.Integrated into the chimeric antigen receptor structure,the artificial chimeric receptor recombinant plasmid for NK cells was successfully constructed.Next,in vitro transduction was carried out to successfully construct artificial chimeric receptor cells with targeted recognition and killing function.At the same time,the NK cell feeding method and cytokines in vitro culture and activation system were optimized and improved,which laid a foundation for a large number of in vitro and in vivo experiments as well as future clinical trials and applications.In order to further verify the targeted killing ability of chimeric receptor modified cells,this study verified and analyzed the killing effect of constructed CAR-CIK,CAR-NK-92 and CAR-NK cells by lactate dehydrogenase,killer factor detection and flow cytometer,etc.Pharmacokinetics was further used to analyze the killing effect and safety of our constructed chimeric receptor modified cells in vivo in animal models.Through analysis,the artificial chimeric receptor modified cells constructed in the study have obvious targeted killing ability and reliable biosafety.In addition,single-cell sequencing technology,gene expression level and bioinformatics analysis were used to study the gene expression profile changes of cord blood NK cells before and after induction differentiation and during proliferation.The results showed that the functionally mature NK cells represented by CX3CR1 had high expression and unique transcriptional characteristics.At the same time,this study explored the proliferation and differentiation of NK cells and the molecular mechanism of cell development biology,and prepared for the clinical application of CD19-CAR-NK cell technology.The innovation of this study is that to try to break through the technical bottleneck of artificial CAR cell method to transform non-specific cells,and optimize and construct a set of innovative CAR-NK preparation system to provide technical support for the expansion of artificial CAR cell therapy.At the same time,the expression of CAR intracellular activation signal can be used to enhance the activity of immune cells themselves,which will be beneficial for CAR related technologies to play a more important role in the immunotherapy process and provide new methods and ideas for further clinical application research.The primary outcome:1.CAR-CIK cells and CAR-NK-92 cell preparation systems targeting CD 19 based on CAR-T technology were designed and constructed.2.A system of CAR-NK cells targeting CD 19,activation and effector based on umbilical cord blood was designed and constructed.3.In vitro experiments verified that CD19-CAR-NK generated after NK cell transduction had significant target-induced cell release function,as well as specific cell killing and showed dose-dependent characteristics.4.The activation system of umbilical cord blood NK cells in vitro culture was optimized,and the optimal NK cell transduction method for CAR-NK cells was found.NK cells can be optimized for transduction using lentivirus vectors or retrovirus vectors.NK cells cultured through optimized culture system can obtain better transduction efficiency.5.The changes of gene expression profile and transcriptional characteristics of umbilical cord blood NK cells before and after differentiation induction and proliferation were studied by using single cell transcriptomic sequencing technology.6.The results of the mouse model of B lymphocytic leukemia showed that the tumor suppression rate of CD19-CAR-NK cells was 61%on the 7th day after infusion,which was much higher than the tumor suppression rate of 12%in the control group.The good biosecurity and effectiveness of CD19-CAR-NK cells were verified without the associated toxicity and tumor-cause teratogenic conditions... |
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