Construction And Functional Identification Of Chimeric Antigen Receptor-engineered Immune Cells Targeting Folate Receptor α

Despite the rapid development of modern medicine,the incidence and mortality of tumors are still increasing year by year,and it has become the “human health killer” second only to cardiovascular and cerebrovascular diseases.Therefore,the situation of tumor prevention and treatment is extremely severe.Conventional treatment methods for tumors mainly include surgical resection,chemotherapy,radiotherapy,and targeted therapy.Although the above-mentioned therapies have made great progress,in actual clinical practice,the inherent shortcomings of these therapies themselves are difficult to overcome.Thus,it is still urgent to explore new tumor treatment methods and strategies.In recent years,the emerging immunotherapy has shown excellent clinical efficacy in a large number of clinical trials.In particular,the chimeric antigen receptor-engineered immune cells and immune checkpoint blockade antibodies have been given priority approval by the Food and Drug Administration for marketing,which has changed people’s traditional understanding of tumor therapy and brought tumor therapy into a new era.Therefore,this research focused on chimeric antigen receptor-engineered immune cells(CAR-engineered immune cells),a research hotspot in the field of tumor immunotherapy.The following studies were carried out from the aspects of tumor antigen selection,construction of CAR-engineered immune cells,interaction mechanism between CAR-engineered immune cells and tumor cells,and novel synergistic strategies.Part Ⅰ Construction and expression identification of αFR-CAR-engineered immune cellsObjective:Folate receptor α(αFR)is a well-recognized tumor-associated antigen and is considered an ideal target for CAR construction.In addition,NK-92 cells are a kind of highly potential CAR-loaded immune cells.However,CAR-engineered NK-92 cells targeting αFR has not been reported yet.Therefore,the research in this chapter aims to constructαFR-CAR-engineered NK-92 cells.Methods:1.According to the structural characteristics of CAR,the first,second and third generation αFR-CARs were designed.2.The gene sequences of each structural molecular fragment of CAR were queried in UniProt,EMBL and NCBI databases to obtain the first,second and third generationαFR-CAR gene sequences.3.The above sequences were synthesized,inserted into lentivirus vector,packed lentivirus and infected NK-92 cells.The NK-92 cell line stably expressing αFR-CAR was selected by puromycin.4.The expression of αFR-CAR in lentivirus-infected NK-92 cells was detected by real-time quantitative polymerase chain reaction(qPCR)and flow cytometry.Results:1.The first,second and third generation αFR-CARs were designed based on the single-chain variable region fragment of the human anti-αFR monoclonal antibody C4.The extracellular segment of these three generations of αFR-CAR is composed of CD8α signal peptide,scFv fragment derived from human anti-αFR antibody C4,human Ig G1 Fc fragment and CD8α hinge region fragment in series,and the transmembrane segment is CD28 transmembrane region fragment.In addition,the first generation of αFR-CAR intracellular segment is the human CD3ζ intracellular signal transduction domain fragment,and the second generation of αFR-CAR intracellular segment consists of the human CD28 intracellular signal transduction domain fragment and the human CD3ζ intracellular signal transduction domain fragment.The intracellular fragment of the third generation αFR-CAR is composed of human CD28 intracellular signal transduction domain fragment,CD137(4-1BB)intracellular signal transduction domain fragment and human CD3ζ intracellular signal transduction domain fragment.2.The NK-92 cell lines stably expressing the first,second and third generationαFR-CAR were selected and named as NK-92-αFR-ζ,NK-92-αFR-28ζ and NK-92-αFR-28BBζ,respectively.3.The results of qPCR and flow cytometry showed that αFR-CAR was highly expressed in NK-92-αFR-ζ,NK-92-αFR-28ζ and NK-92-αFR-28BBζ cells.Conclusion:The first,second and third generation αFR-CAR-engineered NK-92 cells were successfully constructed.Part Ⅱ Evaluation of the specific killing effect of αFR-CAR-engineered immune cells on αFR-positive tumor cellsObjective:In the previous chapter,the first,second and third generation αFR-CAR-engineered NK-92 cells have been successfully constructed,but whether they have strong tumor killing ability.And whether the first,second and third generation αFR-CAR has an effect on the antigen-related proliferation and apoptosis of NK-92 cells needs further study.Therefore,the research in this chapter aimed to evaluate the specific tumoricidal activity ofαFR-CAR-engineered NK-92 cells,and to explore the antigen-specific expansion and antigen-induced apoptosis effects of αFR-CAR-engineered NK-92 cells.Methods:1.SK-OV-3 and A2780 cells with high αFR expression,HCT 116 cells with low αFR expression and A-431 cells without αFR expression were selected as target cells.The in vitro killing effect of αFR-CAR-engineered NK-92 cells was evaluated by lactate dehydrogenase(LDH)detection kit.2.Cell Counting Kit-8(CCK-8),enzyme linked immunosorbent assay(ELISA)and Annexin V-FITC/PI double staining apoptosis detection kit were used to analyze the antigen-specific expansion capacity and antigen-induced apoptotic effects ofαFR-CAR-engineered NK-92 cells.3.Cytokine secretion levels of αFR-CAR-engineered NK-92 cells co-cultured with tumor cells were detected by IFN-γ and TNF-α ELISA kits.4.The killing process of αFR-CAR-engineered NK-92 cells against αFR-positive tumor cells was recorded in real time using a live cell workstation.5.A peritoneal αFR-positive tumor model was constructed in severe combined immunodeficiency mice(B-NDG mice),and the in vivo tumoricidal effect ofαFR-CAR-engineered NK-92 cells was evaluated based on the model and in vivo imaging of animals.Results:1.The results of in vitro cytotoxicity assay showed that αFR-CAR-engineered NK-92 cells could specifically kill αFR-positive tumor cells,and the third generation ofαFR-CAR-engineered NK-92 cells(NK-92-αFR-28BBζ cells)showed the strongest antitumor activity.2.Antigen-specific amplification and antigen-induced apoptosis assays showed that compared with NK-92-αFR-ζ and NK-92-αFR-28ζ cells,NK-92-αFR-28BBζ cells not only had stronger antigen-specific proliferation ability,but also exhibited lower levels of antigen-induced apoptosis.3.The results of the cytokine secretion level detection experiment showed thatαFR-positive tumor cells can make NK-92-αFR-28BBζ cells secrete interferon-γ(IFN-γ)and tumor necrosis factor-α(TNF-α)in large quantities,and the secretion levels of IFN-γ and TNF-α were positively correlated with the expression level of αFR on tumor cells.4.The killing process of NK-92-αFR-28BBζ cells on αFR-positive tumor cells recorded by live cell imaging system showed that SK-OV-3 cells were surrounded by several NK-92-αFR-28BBζ cells,and over time,SK-OV-3 cells decreased in volume,increased in cytoplasmic density,formed vesicles in their membranes,and eventually ruptured.5.The results of in vivo killing experiments showed that NK-92-αFR-28BBζ cells could effectively eliminate αFR-positive tumor cells,inhibit tumor progression,and prolong the survival time of tumor-bearing mice.Conclusion:It was confirmed that αFR-CAR-engineered NK-92 cells had antigen-specific anti-tumor activity in vitro and in vivo,and the third-generation αFR-CAR-engineered NK-92 cells had the strongest killing effect on αFR-positive tumor cells.In addition,the third-generationαFR-CAR-engineered NK-92 cells had stronger antigen-specific proliferation ability and lower levels of antigen-induced apoptosis.Part Ⅲ Molecular mechanism of IFN-γ and TNF-α secreted byαFR-CAR-engineered immune cells to synergistically induce high expression of CXCL10 in tumor cellsObjective:When CAR-engineered immune cells recognize tumor cells,on the one hand,they can directly perforate the cell membrane and rupture the tumor cells by secreting cytotoxic substances such as perforin and granzyme.On the other hand,a large number of cytokines mainly IFN-γ and TNF-α are secreted.These cytokines act as information carriers between different immune cells and between immune cells and tumor cells,and widely regulate the biological behavior of immune cells and tumor cells.However,there is still little research on whether the IFN-γ and TNF-α secreted by CAR-engineered immune cells will have biological effects on tumor cells.Therefore,the research in this chapter aimed to explore the biological effects of IFN-γ and TNF-α secreted by αFR-CAR-engineered NK-92 cells on tumor cells,and to clarify the molecular mechanisms involved.Methods:1.CXC chemokine ligand 10(CXCL10)ELISA kit and qPCR were used to detect the expression level of CXCL10 after tumor cells were co-cultured with αFR-CAR-engineered NK-92 cells(NK-92-αFR-CAR cells,namely NK-92-αFR-28BBζ cells in the previous study,the same below).2.When NK-92-αFR-CAR cells were co-cultured with αFR-positive tumor cells,IFN-γor/and TNF-α-specific neutralizing antibodies were added.The expression levels of CXCL10 in tumor cells were detected by ELISA and qPCR.The same two methods were used to analyze the induction effect of IFN-γ or/and TNF-α stimulation on the expression of CXCL10 in tumor cells.3.Through signal pathway inhibition experiment based on small molecule inhibitors such as Ruxolitinib and BAY 11-7082,si RNA interference experiment,luciferase reporter gene experiment and knockdown and complementation experiment,the signal pathway of IFN-γ or/and TNF-α-induced tumor cells to express CXCL10 was explored.Results:1.When αFR-positive tumor cells were co-cultured with NK-92-αFR-CAR cells,CXCL10 was abundantly secreted by tumor cells.2.The addition of IFN-γ or/and TNF-α specific neutralizing antibody can significantly reduce the expression level of CXCL10 in tumor cells.Either IFN-γ or TNF-α stimulation alone can induce tumor cells to express CXCL10,and simultaneous stimulation of IFN-γ and TNF-α can significantly increase the expression level of CXCL10 in tumor cells.3.The results of signaling pathway inhibition experiments suggested that the JAK1/2-STAT1 signaling pathway may mediate the induction of CXCL10 expression by IFN-γ,and the NF-κB signaling pathway may mediate the induction of CXCL10 expression by TNF-α.The results of si RNA interference experiments and luciferase reporter gene experiments confirmed that IFN-γ or TNF-α induced tumor cells to express CXCL10 by activating the intracellular JAK1/2-STAT1 signaling pathway or NF-κB signaling pathway,respectively.4.The results of cytokine stimulation experiments and si RNA interference experiments showed that combined stimulation of IFN-γ and TNF-α can up-regulate the expression level of interferon-gamma receptor 1(IFNGR1)in tumor cells,which can enhance the activation level of the intracellular JAK1/2-STAT1 signaling pathway,thereby significantly increasing the expression of CXCL10.The results of si RNA interference experiments and luciferase reporter gene experiments showed that when IFN-γ and TNF-α were used in combination to stimulate tumor cells,TNF-α induced the transcriptional expression of IFNGR1 gene by activating the intracellular NF-κB signaling pathway.The results of knockdown and complementation experiments confirmed that when IFN-γ and TNF-α co-stimulated tumor cells,TNF-α upregulated the expression of IFNGR1,which in turn enhanced the activation level of the IFN-γ-IFNGR-JAK1/2-STAT1-CXCL10 signaling axis.Conclusion:It was found that IFN-γ and TNF-α secreted by αFR-CAR-engineered NK-92 cells synergically induced CXCL10 expression in tumor cells,and the specific molecular mechanism was elucidated.When IFN-γ and TNF-α were co-stimulated,on the one hand,IFN-γ and TNF-α induced tumor cells to express CXCL10 through JAK1/2-STAT1 signaling pathway and NF-κB signaling pathway,respectively;On the other hand,TNF-α upregulated the expression of IFNGR1 through the NF-κB signaling pathway,which in turn enhanced the activation level of the IFN-γ-IFNGR-JAK1/2-STAT1-CXCL10 signaling axis.These two aspects of the mechanism jointly mediated the high expression of CXCL10 in tumor cells.Part Ⅳ Construction and functional characterization of self-chemotropicαFR-CAR-engineered immune cells co-expressing CXCR3AObjective:Based on the results of the previous chapter-“IFN-γ and TNF-α secreted byαFR-CAR-engineered NK-92 cells can synergistically induce high expression of chemokine CXCL10 in tumor cells”,this study envisages that the CAR and CXC-chemokine receptor 3A(CXCR3A),the receptor of CXCL10,will be highly expressed on NK-92 cells at the same time.In this way,when αFR-CAR-engineered NK-92 cells co-expressing CXCR3 A to kill tumor cells,the secreted IFN-γ and TNF-α synergistically induced the high expression of CXCL10 in tumor cells.Under the chemotaxis of CXCL10,αFR-CAR-engineered NK-92 cells co-expressing CXCR3 A continuously migrated into the tumor tissue,further enhancing the tumoricidal effect.At the same time,the secretion of IFN-γ and TNF-α was further increased,which in turn could synergistically induce tumor cells to express a greater amount of CXCL10.With the increased expression of CXCL10,more αFR-CAR-engineered NK-92 cells co-expressing CXCR3 A were recruited to the tumor site.Positive feedback ofαFR-CAR-engineered NK-92 cells self-chemotaxis was thus formed.Therefore,the research in this chapter aimed to construct self-chemotropic αFR-CAR-engineered NK-92 cells co-expressing CXCR3 A,and to evaluate their specific antitumor activity.Methods:1.The constructed third-generation αFR-CAR sequence and CXCR3 A sequence were spliced in tandem using the self-cleaving polypeptide 2A sequence.The αFR-CAR-CXCR3 A gene sequence co-expressing αFR-CAR and CXCR3 A was designed.2.The above gene sequence was synthesized,inserted into lentiviral vector,packaged lentivirus and infected NK-92 cells.The NK-92 cell line stably co-expressing αFR-CAR and CXCR3 A was obtained by puromycin selecting(NK-92-αFR-CAR-CXCR3A).3.The expression of αFR-CAR and CXCR3 A in lentivirus-infected NK-92 cells was detected by qPCR and flow cytometry.4.The in vitro killing effect of NK-92-αFR-CAR-CXCR3 A cells was evaluated by LDH detection kit.5.The cytokine secretion levels of NK-92-αFR-CAR-CXCR3 A cells co-cultured with tumor cells were detected by IFN-γ and TNF-α ELISA kits.6.The chemotaxis of NK-92-αFR-CAR-CXCR3 A cells was evaluated by transwell experiments.7.Subcutaneous αFR-positive tumor model was constructed in B-NDG mice.The in vivo antitumor effect of NK-92-αFR-CAR-CXCR3 A cells was evaluated based on this model.In addition,the infiltration of NK-92-αFR-CAR-CXCR3 A cells in tumor tissue was detected by immunohistochemistry.Results:1.The αFR-CAR sequence and the CXCR3 A sequence were linked using the self-cleaving polypeptide 2A sequence.Among them,the aforementioned third-generationαFR-CAR was selected for the study in this chapter.The structure of the CAR is composed of CD8α signal peptide,scFv fragment derived from human anti-αFR antibody C4,human Ig G1 Fc fragment,CD8α hinge region fragment,CD28 transmembrane region fragment,human CD28 intracellular signal transduction domain fragment,CD137(4-1BB)intracellular signal transduction domain fragment and human CD3ζ intracellular signal transduction domain fragment.2.The NK-92 cell lines stably expressing αFR-CAR and stably co-expressing αFR-CAR and CXCR3 A were selected and named NK-92-αFR-CAR and NK-92-αFR-CAR-CXCR3 A,respectively.3.The results of qPCR and flow cytometry showed that αFR-CAR was highly expressed in NK-92-αFR-CAR and NK-92-αFR-CAR-CXCR3 A cells.Furthermore,high expression levels of CXCR3 A were detected only in NK-92-αFR-CAR-CXCR3 A cells.4.The results of in vitro killing experiments showed that both NK-92-αFR-CAR and NK-92-αFR-CAR-CXCR3 A cells could efficiently and specifically kill αFR-positive tumor cells.5.The results of cytokine secretion level detection showed that both NK-92-αFR-CAR and NK-92-αFR-CAR-CXCR3 A cells could secrete IFN-γ and TNF-α in a large amount in an antigen-specific manner.6.The results of transwell experiments showed that CXCL10 could effectively induce the directional migration of NK-92-αFR-CAR-CXCR3 A cells.7.The results of in vivo killing experiments showed that NK-92-αFR-CAR-CXCR3 A cells could effectively kill αFR-positive tumor cells and significantly inhibit tumor growth.In addition,the results of immunohistochemistry showed that NK-92-αFR-CAR-CXCR3 A cells could chemotactically infiltrate into tumor tissue in large numbers.Conclusion:The self-chemotropic αFR-CAR-engineered NK-92 cells co-expressing CXCR3 A were successfully designed and constructed.It is confirmed that it not only has antigen-specific anti-tumor activity,but also migrates into tumor tissue in large quantities under the chemotaxis of CXCL10.In conclusion,this study was the first to construct CAR-engineered NK-92 cells targetingαFR,and used a variety of in vitro and in vivo experiments to verify its antitumor function.Secondly,aim at whether IFN-γ and TNF-α secreted by CAR-engineered immune cells will have biological effects on tumor cells,this study not only found that IFN-γ and TNF-αsecreted by αFR-CAR-engineered NK-92 cells could synergistically induce the high expression of chemokine CXCL10 in tumor cells,but also explored the specific molecular mechanism.Finally,based on the above results and the chemotactic function of CXCL10 itself,this study designed and constructed self-chemotropic αFR-CAR-engineered NK-92 cells co-expressing CXCR3 A,and evaluated its antitumor function and chemotaxis by various in vitro and in vivo experiments.The research results obtained in this subject not only confirm the feasibility of constructing the above two CAR-engineered NK-92 cells targeting αFR,but also provide a new therapeutic weapon with great application potential for patients withαFR-positive tumors.