PD-L1 Blockage Combined With Demethylation Treatment Enhances The Anti-breast Cancer Effect For MAGE-A11-specific CTL

Breast cancer is the most common malignant tumor in women.The number of patients in the world is more than 1.7 million each year,and about 520,000 people patients die from breast cancer every year.In recent years,the incidence of breast cancer in our country has significantly increased,surpassing that of lung cancer which ranks first in the incidence of malignant tumors among women.Breast cancer accounts for 15% of all female cancer,and the standardized incidence rate of mortality and age is increasing year by year.Although the effect of surgery,radiotherapy,chemotherapy and endocrine therapy is obvious,the injury to the patient itself is great,which causes physical and psychological harm.Tumor immunotherapy has the characteristics of strong specificity and small side effects,which is a hot spot of research in recent years.In tumor immunotherapy,the application of tumor specific T lymphocyte to kill tumor has a good prospect.The key to this treatment is to find target antigens that can be identified by T lymphocytes.Melanoma associated antigen(MAGEs),which belongs to the Cancer/testis antigen(CTA)subfamily,is a tumor associated antigen,which has immunogenicity and restrictive expression.MAGE-A is the most widely studied family,consisting of 12 members of the A1-A12.MAGE-A antigen can be identified by CTL cells and induce specific killing of corresponding tumor cells.Therefore,MAGE-A is a potential target molecule in tumor specific immunotherapy.The study of the application of MAGE-A as a target antigen in the treatment of tumors has been widely reported in recent years.The effect of tumor immunotherapy not only requires the restriction of target antigen expression,but also depends on the expression rate and expression of target antigen.The expression of MAGE-A11 in breast cancer cells is heterogeneous,some cells are highly expressed,some are low expressed,and some are not expressed.In immunotherapy,this heterogeneity will cause some tumor cells to escape and eventually affect the treatment effect.If all breast cancer cells are able to express MAGE-A11,they will overcome these limitations.In recent years,it has been reported that the cytotoxic function of cytotoxic T cells is enhanced by improving the expression of tumor antigen.But this strategy is mainly used in the tumor of the blood system,which has not been reported in solid tumors,especially in breast cancer.The gene expression of MAGE-A subfamily is regulated by promoter methylation.The demethylation reagent can improve the expression of tumor antigen and increase the killing function of tumor specific T cells.Decitabine and 5-azacytidine,a demethylation reagent,can promote the expression of MAGE-A genes.Although these two drugs have shown good remission in the treatment of liquid tumors,the side effects are relatively large and are unstable in the solution.In addition,the growth rate of solid tumors is slow and the half-life of these two drugs is short,and so their effect in solid tumors is not good.Therefore,there is an urgent need for a low toxic new apparent modifier for solid tumors.Zebularine,a new DNA methylation inhibitor,has a spectral antitumor effect with low side effects.It is also found that it can induce the re-expression of certain silent genes such as p21.However,whether zebularine has an impact on the expression of MAGE-A has not been reported.The two important ways of tumor immune-escape are the loss of tumor antigen and immunosuppression.Tumor cells can express a variety of immunosuppressive signal proteins,by mediating immune cell dysfunction and inducing cell apoptosis,to escape the attack of the immune system.The programmed cell death ligand(PD-L1)is one of the important suppressor molecules.Some study found that PD-L1 was highly expressed in a variety of tumors and was associated with poor prognosis in patients with.The combination of PD-L1 expressed by tumor cells and PD-1 on the surface of T cells can lead to abnormal function of T lymphocytes by inhibiting the activation and proliferation of T cells.As an important cell cycle checkpoint,PD-1/PD-L pathway plays a specific negative regulatory role.It is one of the targets of drug intervention and anti-tumor immunotherapy,and has potential application value.By blocking the interaction between PD-1/PD-L to improve the immune response,it is widely used in the treatment of cancer and shows good prospects for application.It provides a theoretical basis for blocking this signal immunotherapy.To sum up,we first screened a series of candidate epitope peptides from MAGE-A11 based on the amino acid sequence of MAGE-A11,and identified a polypeptide that could stimulate CTL immune response.Then we observed the expression of MAGE-A11 in breast cancer cells by DNA methylation inhibitor,and evaluated whether the increased expression of MAGE-A11 could enhance the recognition and killing effect of antigen specific T cells.Finally,combined PD-L1 blocking and epigenetic drugs to improve the anti-tumor effect of CTL,and strive to play the best effect of immunotherapy,and lay the foundation for breast cancer immunotherapy.The main results are as follows: Part one Prediction,synthesis and identification of tumor antigen MAGE-A11 epitope peptideObjective: To predict the HLA-A*0201 restricted epitope of MAGE-A11 by bioinformatics technology,and then to synthesize antigen peptide according to amino acid sequence,and to identify the immune response that can stimulate HLA-A*0201 restricted CTL,and lay the foundation for vaccine design and immunotherapy of breast cancer.Methods:1 The bioinformatics prediction tool was used to predict the HLA-A*0201 restrictive epitopes of MAGE-A11 and to synthesize the candidate epitopes.It was analyzed and purified by high performance liquid chromatography,and its molecular weight was analyzed by mass spectrometry.2 The relative affinity and stability of the epitope of the candidate antigen epitopes and HLA-A*0201 were detected by indirect fluorescence labeling.The morphology of mature dendritic cells cultured in vitro was observed under3 Light microscopy,and the molecular phenotype of mature cells was detected by flow cytometry.4 ELISPOT test detected the specific CTL frequency of secreting and secreting the ability to secrete IFN-gamma.5 CCK-8 test was used to detect the killing function of induced specific CTL on MCF-7,MDA-MB-453,MDA-MB-231 and BT-549 in breast cancer cells.Results:1.In the way of comprehensive comparison,five candidate peptides P221(ILHDKIIDL),P225(KIIDLVHLL),P350(FLFGEPKRL),P384(FLWGPRAHA)and P421(ALREEGEGV)were selected for synthesis.2.The purity of the newly synthesized antigen polypeptides was over 95% through HPLC analysis.The results of mass spectrometry showed that the molecular weight of the synthesized polypeptide was consistent with the theoretical value,indicating that the synthesized polypeptide is the desired polypeptide we need.3.P221 has the highest affinity for HLA-A * 0201,followed by P350 and P384,and P421 has the lowest affinity.In terms of stability,P350 showed the highest stability(DC50 =8h).The followed is P221,P384,the worst of the stability is P225,and the DC50 value is less than 2h.4.The isolated peripheral blood mononuclear cells were adherent to remove the suspended cells.Under microscope,the cells were smaller and grew more adherent and round.They were distributed uniformly,with a few semi-suspension cells.After induction of differentiation into mature dendritic cells,cell bodies increased,irregular shape,rough surface and burr shaped surface.5.Flow cytometry was used to detect the phenotypic changes of CD1 a,CD83,CD80 and CD86 on the surface of immature and mature DC.Before antigen stimulation,DC costimulatory molecules CD1 a and CD83 positive rates were 5.73±3.22%,9.21±4.18%.The positive rate of CD80 and CD86 were 5.94±4.13%,3.37±2.95%.After the stimulation of CD1 a and CD83 positive rates were68.86±9.78%,45.67±10.98%.The positive rate of CD80 and CD86 were 58.81±8.72%,59.75±5.37%.6.In vitro induced CTLs from different healthy individuals showed that P221,P350 and P350 could stimulate the production of a number of skin specific CTL that release IFN-gamma by ELISPOT.The effect of P221 and P384 in donor 1 and 2 was better,and P221 and P350 had better effect in donor 3.7.Cytotoxicity test showed that CTL induced by P221,P350 and P384 could lyse MAGE-A11 positive MCF-7 and MDA-MB-453 cells,but could not lyse MAGE-A11 negative MDA-MB-231 and BT-549 cells.In MCF-7 and MDA-MB-453 cells,the specific killing percentage of the effector cells increased with the ratio of E / T from 10:1 to 40:1.8.With HLA-A2 monoclonal antibody blocking,the killing effect of CTL on MCF-7 and MDA-MB-453 decreased significantly.Brief Conclusions:1.Candidate peptide P350 is an ideal candidate peptide.2.The killing effect of CTL induced by candidate peptides on breast cancer cells is HLA-restricted.3.The CTL induced by candidate peptides can kill the breast cancer cells expressing MAGE-A11.Part two Effect of demethylation on the expression of MAGE-A11 in breast cancer cells and its effect on CTL killing activityObjective: To investigate the effect of Zebularine on the expression of MAGE-A11 in breast cancer cells,and to observe the effect of combination of Zebularine and CTL on the cytotoxicity of breast cancer cells in vitro.Methods:1 PCR and Western Blot were used to detect the effect of zebularine on the expression of MAGE-A11 in MCF-7,MDA-MB-453,MDA-MB-231 and BT-549 of breast cancer cells.2 The changes of proliferation activity of breast cancer cells and antigen specific T cells were observed before and after zebularine treatment.3 The effects of zebularine on the antitumor effect of specific CTL were observed by CCK-8 and IFN-gamma test.4 After transfection of siRNA,the expression of MAGE-A11 in MCF-7 and MDA-MB-453 cells were decreased,and the change of specific CTL on its killing activity were detected.5 The cytotoxic effects of zebularine and MAGE-A11 specific CTL on tumor cells were verified in the primary cells of breast cancer.Results:1.MAGE-A11 mRNA is relatively highly expressed in MDA-MB-453,is low in MCF-7 and is not expressed in MDA-MB-231 and BT-549.50μM zebularine increased MAGE-A11 expression in MDA-MB-453 and MCF-7 and induced the re-expression of MAGE-A11 in MDA-MB-231 and BT-549.100 M zebularine induced MAGE-A11 expression increased significantly(P<0.01).2.MDA-MB-231 and BT-549 cells did not express MAGE-A11 protein.Zebularine(50 mu M)can induce the expression of MAGE-A11.In MCF-7 and MDA-MB-453,the effect of zebularine on the expression of MAGE-A11 was dose-dependent.3.The effect of Zebularine on the proliferation activity of breast cancer cells was time dependent and dose dependent.Zebularine significantly reduced the proliferative activity of MCF-7,MDA-MB-453,MDA-MB-231 and BT-549 cells at 72 h,respectively,at 100μM,50μM,25μM and 50μM.4.MCF-7 and MDA-MB-453 express MAGE-A11,which can also be identified by CTL without zebularine induction.However,zebularine treatment increased cytotoxicity,especially at target ratios of 20: 1 and 40: 1.When MDA-MB-231 and BT-549 are not treated with zebularine,they did not express MAGE-A11 and could not be killed by CTL.After zebularine treatment,the killing phenomenon totumor cells can be observed.5.When MAGE-A11 expression was down regulated in breast cancer cells MCF-7 and MDA-MB-453,the killing rate of CTL to MCF-7 and MDA-MB-453 cells was significantly lower than that in untransfected group and control group(P<0.05)under the same target efficiency ratio.6.Western blot results showed that MAGE-A11 was expressed in primary breast cancer cells,and MAGE-A11 expression increased after 3 days treatment with zebularine(100 μM).In the group of combination of Zebularine and MAGE-A11-specific CTL,the average dissolution rate was 64%,significantly higher than that in the single use group.Brief Conclusions:1.DNA demethylation inhibitor zebularine can induce the increase or re expression of MAGE-A11 expression in breast cancer cells.2.The increase of MAGE-A11 expression in breast cancer cells can improve the identification and killing of antigen specific CTL cells to breast cancer cells.3.The combined use of zebularine and antigen specific CTL cells can improve the killing effect on breast cancer cells and primary cells.Part three Effect of Combined PD-1 / PD-L1 Inhibitors and Demethylated Drugs on CTL Cell KillingObjective: To observe the immune killing effect of PD-L1 monoclonal antibody combined with zebularine on breast cancer cells in vitro and provide a scientific basis for future animal experiments and clinical trials.Methods:1 Flow cytometry(FCM)was used to detect the expression of PD-L1 on the surface of breast cancer cells and the expression of PD-1 on the surface of T lymphocytes.2 ELISPOT method was used to detect the effect of PD-L1 blocking on the activation of T lymphocyte.3 CCK-8 assay was used to determine the changes in the cytotoxicity of CTL cells to breast cancer cells before and after PD-L1 blockage.4 TO observe the effect of zebularine on the PD-1 in CTL cells and PD-L1 expression in tumor cells.5 combination of zebularine and PD-L1 monoclonal antibody to enhance CTL on breast cancer cells MDA-MB-231 and BT-549 killing effect.6 Effect of combined PD-1 / PD-L1 inhibitors and demethylated drugs on CTL cell killing using primary breast cancer cells.Results:1.PD-L1 molecules was not expressed on MCF-7 and MDA-MB-453 cells,but was low-regulated on BT-549 cells(8.1 ± 2.5)% and highly expressed on MDA-MB-231(43.7 + 11.4)%.DC stimulation of the load antigen peptide can increase the expression of PD-1 molecules in T cells.2.ELIDPOT results showed that the number of T lymphocytes after PD-L1 blockade increased compared with that before blocking,but there was no significant difference between before and after blocking(P <0.05).3.Alone PD-L1 blockade,the killing effect of CTL cells on MCF-7,MDA-MB-453,MDA-MB-231 and BT-549 cells did not significantly increase at the same target ratio.4.There was no significant difference in the expression of PD-L1 in MCF-7,MDA-MB-453,MDA-MB-231 and BT-549 cells before and after Zebularine treatment,and in the expression of PD-1 in CTL cells.This Suggestted that PD-L1 expression on breast cancer cells might not be affected by DNA methylation.5.MDA-MB-231 and BT-549 cells expressing PD-L1 were selected as target cells.Under the same target ratio,the cell lysis rate of CTL +Zebularine+PD-L1 mAb group was higher than that of CTL group,CTL +Zebularine group and CTL +PD-L1 mAb group,the difference was statistically significant,and the killing activity increased with the increase of target effect ratio.6.there was a significant difference in the content of IFN-gamma in cell culture supernatant of different treatment groups.The IFN-gamma content in CTL +Zebularine+PD-L1 McAb group was significantly higher than that in other groups,the difference was statistically significant.7.The cultured primary breast cancer cell surface expression of PD-L1 molecules.The expression of MAGE-A11 was positive by Western blot.The cytotoxicity in CTL +Zebularine group and CTL +PD-L1 monoclonal antibody group to tumor cells was significantly higher than that of CTL group.The highest killing effect group in these four groups was CTL +Zebularine+PD-L1 monoclonal antibody group.Brief Conclusions:1.Zebularine treatment did not affect PD-L1 expression in breast cancer cells.2.PD-L1 monoclonal antibody combined with Zebularine can enhance CTL cytotoxicity on breast cancer cells and primary cells.Conclusions:1.HLA restrictive candidate peptide P350,derived from MAGE-A11,can bind to HLA molecules and induce MAGE-A11 specific CTL to kill the breast cancer cells expressing MAGE-A11.2.The use of DNA demethylation therapy can induce the expression of MAGE-A11 in breast cancer cells,thus inducing antigen specific CTL cell recognition,killing breast cancer cell lines and primary breast cancer cells.3.Blocking PD-L1 combined with DNA demethylation drug Zebularine can enhance the killing effect of CTL on breast cancer cell lines and primary breast cancer cells.