The Application Of Chimeric Antigen Receptor Macrophages That Promote M1 Polarization In Solid Tumor Immunotherapy

The situation of prevention and control of cancer,which is one of the serious diseases threatening human life and health,is serious.Nowadays,the rapidly developing immunotherapy is considered as the third revolution in the treatment of cancer,among which,adoptive cell therapy has shown great potential in tumor immunotherapy.As one of the major types of adoptive cell therapies,chimeric antigen receptor T-cell(CAR-T)immunotherapy gains critical breakthroughs for treating hematologic malignancies.Chimeric antigen receptors(CARs),which gene sequences are synthesized in vitro by genetic engineering techniques and transferred into cytotoxic immune cells to express on the surface of cell membrane,refer to a class of engineered receptors that specifically track,recognize and mediate tumor-killing effects.Several CAR-T products being approved and used to treat patients with relapsed or refractory leukemia and myeloma.However,similar results are difficult to be obtained when CAR-T therapies are used to treat solid tumors,which represent 90%of all cancers.The underlying reasons for that are the difficulty in getting CAR-T cells inside tumor tissues and the immunosuppressive factors in the tumor microenvironment(TME)that can interfere with the antitumor function of CAR-T cells.Macrophages,an important component of TME,accounting for 30-50%of all immune cells in solid tumors.Because of their high plasticity,macrophages can shift between pro-inflammatory M1 phenotype and anti-inflammatory M2 phenotype to inhibit or promote tumor growth by regulating TME,respectively.Among them,M1 type macrophages can directly phagocytose tumor cells or secrete immunoreactive factors to inhibit the growth of tumor cells.Based on these characteristics,macrophages are considered to have potential as CAR carriers,and their ability to kill and suppress tumor can be enhanced after modification with CARs.Related studies have also confirmed that macrophages loaded with CAR can polarize to a pro-inflammatory M1 phenotype to exert more potent tumor suppressive effects directly or indirectly.Therefore,the aim of this study was to design a CAR that could mediate the polarization of macrophages toward the M1 phenotype,then macrophages expressing this CAR were activated by the target antigen and polarized toward the M1 phenotype to exert positive anti-tumor immune functions in TME.First of all,using Tlr4 and/or Ifngr,the receptor for IFN-y,in the activation pathway of M1 macrophage,as the activation and co-stimulation signal for CAR,and using VEGFR2 as the target antigen for CAR based on its critical role in tumor angiogenesis and its high expression in TME,five kinds of CARs were designed and transferred into RAW264.7 macrophages by lentiviral vector system in this study.After confirming that VEGFR2-CAR was expressed on the surface of cell membranes using flow cytometry,the therapeutic effect of VEGFR2-CAR-Ms was evaluated in 4T1 breast cancer mice model.After three replicate validations,this study found that VEGFR2-CAR-Ms with an intracellular segment of Tlr4,i.e.,mmC,could exert stable and most potent tumor suppressive effects in mice with breast cancer.Next,this research explored the mechanism of VEGFR2-CAR-Ms.Compared to wildtype RAW264.7 cells,the expression of molecules marking the M1 phenotype,MHC-Ⅱ.CD86 and Nos2,was differentially elevated on activated VEGFR2-CAR-Ms,suggesting that VEGFR2-CAR-Ms are able to polarize into M1 macrophages upon activation.Further,the study analyzed the expression and secretion of pro-inflammatory cytokines by VEGFR2-CAR-Ms.It was found that the secretion of TNF-α by VEGFR2-CAR-Ms(mmB、mmC、mmD 和 mmE),except for mmA,was significantly higher compared to RAW264.7 cells transfected with the empty plasmid.There was a 10-fold increase in the secretion of TNF-α in all groups after stimulating by HUVEC cells with high VEGFR2 expression.Moreover,after activation of VEGFR2,mmC followed by the addition of 4T1 cells significantly increased the expression and secretion of TNF-α and IL-6.TNF-α is a critical inducer of apoptosis and it can initiate apoptosis of tumor cells through the TNF-α/TNF-R1 signaling pathway.Further findings indicated that the effect of VEGFR2-CAR-Ms in promoting 4T1 apoptosis was increased 1-fold in the presence of HUVEC,whereas their induction of 4T1 apoptosis was largely abolished after blocking TNF-R1.Meanwhile,this study also examined the phagocytic function of VEGFR2-CAR-Ms,and found no increase in the phagocytic capacity of mmC,which combined with the above results suggested that tumor killing by mmC was dependent on the secretion of TNF-α to induce apoptosis of tumor cells.Finally,safety of VEGFR2-CAR-Ms was evaluated in this study.It was found that they did not phagocytose VEGFR2-expressing non-tumor cells in vitro and did not affect organ and vascular tissues of mice with breast cancer.In order to explore the application of CAR-Ms in different types of tumors,it is important to select effective and favorable targets for macrophage recognition.For this reason,based on the successful application of VEGFR2 targeting tumor angiogenesis,to search for new effective targets,the research initially explored the expression of ganglioside GD2,with a high expression in malignant tumors,in lung cancer.The results showed that the expression of GD2 was upregulated on lung cancer stem cells,and the stemness property of GD2+lung cancer cells was significantly enhanced.GD2 could serve as a marker for lung cancer stem cells and it has the potential to be a new target for CAR-Ms to treat lung cancer.In summary,this study provides a promising CAR design which suitable for macrophages.It can mediate the polarization of macrophages to M1 phenotype and the secretion of TNF-α to induce apoptosis of tumor cells after being activated by VEGFR2 in vitro.And it can effectively inhibit the growth of breast cancer in vivo by targeting VEGFR2.In expanding the application of CAR-Ms,the study explored and identified the tumor-associated antigen GD2 can as a marker for lung cancer stem cells,providing a potential target antigen for CAR-Ms targeting lung cancer stem cells.