Application Of Conjugated Anti-EGFRvⅢ And Calreticulin In The Immunotherapy Of Gliomas

Since the 2016 WHO Classification of central nervous system tumors included the concept of molecular diagnosis,gliomas are gradually changing from histo-morphological classification to molecular biology classification,and molecular targeted therapy has gradually been put on the agenda of clinical pathological diagnosis.Meanwhile,as the understanding of the immune network of the nervous system and the immunology of glioma is growing,immunotherapy has recently entered the limelight as novel strategies for gliomas.Among these new strategies,the most in-depth clinically researched is targeting the mutant form of epidermal growth factor receptor EGFRvⅢ.It has been demonstrated that EGFRvⅢ is expressed in about 30%of newly diagnosed GBM cases.More importantly,EGFRvⅢ is tumor-specific,as it has not been found in normal tissues,suggesting a potential role of EGFRvⅢ as an ideal target for tumor diagnosis and therapy.EGFRvⅢ protein has theoretically the advantages of high specificity and low off-target effect as a target antigen for treatment;however,EGFRv Ⅲ targeted therapy have been unsuccessful to date in previous trials in glioblastoma.The potential reason for these failures may include the ignorance of immune "escape"mechanism of tumor cells,which failed to mobilize immune response to the fullest extent.If anti-EGFRvⅢ therapy acts in synergy with other approaches to enhance tumor immunity,such as improving antigen presentation and phagocytosis effects,the therapeutic efficiency will be improved.As an endoplasmic reticulum retention protein,calreticulin(CRT)belongs to the heat shock protein superfamily.It not only participates in the elimination of apoptotic cells,but more importantly,affects the phagocytic function of antigen-presenting cells,which has become a hot spot for anti-tumor immunotherapy.If used in combination with antibody target therapy,it is expected to exert more efficient anti-tumor capabilities.Based on this,we designed an anti-EGFRvⅢ(mAb806)and CRT couplet-CRT806-for the treatment of EGFRvⅢ highly-expressed gliomas.The anti-EGFRvⅢ part of the couplet inhibits the EGFRvⅢ-STAT3 signaling pathway by targeting the EGFRvⅢ antigen expressed on the surface of tumor cells and aims to exert a tumoricidal effect while CRT acts on the LRP(low-density lipoprotein receptor-related protein)receptor on the surface of tumor cells to form a phagocytic signal,which leads to the activation of phagocytes and enhances their phagocytosis of tumor cells.The two parts acts in synergy to amplificate the anti-tumor effects.The thesis consists of three parts:to explore the mechanism of CRT806’s anti-tumor effect in vitro;to verify its effectiveness in anti-tumor activity in vivo and finally,to briefly evaluate the potential safety concerns of CRT806.Part Ⅰ Effect of CRT806 antibody coupling drugs on the function of antigen-presenting cellsObjective:To test whether CRT806 can enhance anti-tumor activity targeted EGFRvⅢhigh gliomas by enhancing the immune function of anti gen-presenting cellsMethods:① Prepare the antibody coupling drug binding mAb806 with CRT and demonstrate that the coupler retains its targeting effect on EGFRvⅢ;②Transfect EGFRvⅢ into mouse GL261 glioma cells and detect the function of CRT806 on two antigen-presenting cells by low cytometry;③ By transfecting cOVA to GL261EGFRvⅢ cells,we aimed to confirm whether the antigen presentation ability of the phagocytes is enhanced,and whether T cells can be further activated to exert stronger anti-tumor effects.Results:① CRT806 was prepared by coupling the anti-EGFRvⅢ antibody mAb806 and CRT with the streptavidin-biotin system.The molecular weight of mAb806 was about 145kD and the molecular weight of mAb806-streptavidin was about 200kD;the molecular weight of CRT is about 47kD,and the molecular weight of CRT-biotin is about 48kD.The protein molecular weight of the coupling product is about 250kD,which is in line with the sum of the molecular weight of each small molecule drug;② The bone marrow cells were isolated and purified and cultured in the primary culture and it was shown that the CD11b+F4/80+cell ratio was found to be 94.82%±0.42%by flow cytometry on the 7th day after stimulation with the supernatant of L929 cells.Therefore,they can be used as a follow-up experiment of macrophages.The bone marrow cells obtained by the same method added with 10ng/ml GM-CSF+10ng/ml IL-4 produced>70%CD11c+cells,which were used in subsequent experiments as dendritic cells③ CRT806 can enhance the phagocytic ability of macrophages(22.16 ± 3.13%)and dendritic cells(21.05 ± 4.19%)against GL261EGFRvⅢ cells through CRT-LRP phagocytic signals;and enhance their antigen cross presentation ability.The presenting ability of T cells eventually caused the activation of CD8+T cells and exerted anti-tumor effects;④ In the presence of CRT806,phagocytosis experiments with human Thp1(17.66±2.12%)and MUTZ-3(16.37)± 4.69%)cells against vⅢ cells is also enhanced;⑤ Use Western Blot to detect different levels of signals in the cGAS-STING pathway of DC cells suggests that the phosphorylation level of STING,IRF-3,NF-κB and IKKα increased significantly.Conclusion:The various primary cultured cells involved in this section can be cultured smoothly and can meet the needs of subsequent experiments;In vitro experiments confirmed that CRT806 can promote the immune function of myeloid cells and exert stronger an anti-tumor effect.Part Ⅱ To explore the immunotherapy effect of CRT806 on EGFRvⅢhigh glioma in vivoObjective:To further study the in vivo therapeutic effects of CRT806 on EGFRvⅢhigh glioma cellsMethods:① An intracranial GL261EGFRvⅢ glioma model was established to detect the treatment results of CRT806;② Use the small animal live imaging system to detect the changes in the volume of gliomas and analyze the initial time of clinically relevant symptoms of each group;③ Use the ELISA eto detect the ecretion of IFN-γ,IL-2and TNF-α in the periphery blood;④ Immunohistochemistry was used to detect the infiltration of T lymphocytes and macrophage/microglia inside tumor microenvironment,and ⑤ the germinal center of the spleen was analyzed by H&E staining.Results:Compared with the control,the survival time of GL261EGFRvⅢ mice in the CRT806 treatment group was significantly longer;the time of clinical symptoms was delayed,and the secretion of peripheral blood IFN-γ,IL-2 and TNF-α was significantly increased after CRT806 application;the germinal center of the spleen of the mice in the treatment group enlarged significantly.After neutralizing the CD8+T lymphocytes of mice with InVivoMAb anti-mouse CD8α,it was found that the anti-tumor effects of CRT806 were disappeared.To be specific,the ratio of IFNγ+T cells in CRT806 group was 11.77±1.83 while that of aCD8+CRT806 group was 1.40±0.25(n=5).The tumor volume in CRT806 group was 13.53±4.38(n=5)and that of aCD8+CRT806 was 24.28 ± 9.04(n=5,P<0.05),which indicates that the anti-tumor effect of CRT806 depends on CD8+cytotoxic T cells.Conclusion:CRT806 can significantly suppress the development of glioma in situ in GL261EGFRvⅢ mice,prolong the survival time of tumor-bearing mice,and delay the onset of symptoms of nervous system defects;the ultimate anti-tumor effect of CRT806 depends on the acquired immune response mediated by cytotoxic CD8+T cells.Part Ⅲ the safety concern of CRT806 antibody coupling drugsObjective:To briefly detect the safety of CRT806 antibody coupling drugMethods:① CCK-8 experiment was used to detect the effects of CRT806 antibody coupling drugs on the vitality of neurons and astrocytes;②peripheral blood routine analysis was carried to detect the effects of CRT806 on the blood system of experimental animals;③ macrophages phagocytosis test was carried to detect whether CRT806 treatment would affect the phagocytosis of neurons and astrocytes by macrophage;③ H&E staining of the major organs such as the heart,lung,kidney and liver of experimental animals was conducted to observe whether they had obvious pathological changes.Results:Different concentrations of CRT806 had no significant effect on the proliferation of neurons and astrocytes;compared with the IgG control group,CRT806 had a transient effect on the blood routine of experimental animals;the presence of CRT806 did not affect the phagocytosis of normal neurons and astrocytes by macrophages;H&E staining indicated that the main organs of the mice after CRT806 treatment group did not show any obvious pathological changes.Conclusion:CRT806 has no obvious side effects or toxic effects on the animal models,but in-depth biological safety evaluation still needs to be further explored.