Screening And Identification Of HLA-A2 Restricted CTL Epitopes Of Ovarian Cancer-associated Antigen TM4SF1 And Its Antitumor Immune Effects

Biological immunotherapy is an important aid for the treatment of malignant tumors beyond surgery,chemotherapy and radiotherapy.Tumor-specific immunotherapy is based on cytotoxic T lymphocyte(CTL),which can specifically recognize antigenic peptides present on the surface of tumor cells.This recognition is restricted by the major histocompatibility complex(major histocompatibility complex,MHC)-I class molecules.Tumor antigen peptides will be recognized by CTLs when they are presented on the surface of APCs by antigen-presenting cells(APCs)in the form of MHC-peptide molecular complexes,and CTLs are also Activated hyperplasia differentiated into tumor-killing CTLs that specifically kill tumor cells [1].The antigen recognized by T cells is a polypeptide composed of 8 to 12 amino acids presented by MHC class I or class II molecules of target cells or antigen presenting cells,so the CTL epitope peptides of tumor-associated antigens are predicted and prepared accordingly.Peptide vaccines are very significant.CTL epitopes of related antigenic peptides can be presented by human leukocyte antigen(HLA)A2 and A3 molecules,and HLA-A2 is the most common type in Chinese population.The positive rate is as high as 53%[2],so the screening of HLA-A2-restricted CTL epitope peptides are more representative.The identification of HLA-A2-restricted CTL epitopes mainly through the following four steps [3]: 1 using the super-motif,quantification motif and other software and database and other epitope prediction methods to predict the possible T-cell epitope from the target antigen cell epitopes;2 Synthesize the corresponding polypeptides,use TAP-defective T2 cell line for MHC molecular binding force analysis;3 In vitro stimulation of naive T cells with peptides;4 Use of tumor cells expressing the antigen of interest and the corresponding MHC phenotype as Target cells identify the activity of effector CTLs.On the basis of this technical route,dozens of HLA-A2.1-restricted CTL epitopes have been identified from the relevant tumor antigens [4-6].Ovarian cancer is the highest mortality among all gynecologic malignancies and the fifth most common cause of female cancer death [7].Every year,over 230,000 new cases and 151,900 women die from ovarian cancer in the world [8].Mainly due to the lack of typical clinical symptoms,effective screening and early diagnosis methods to detect the disease.More than 75% of patients are diagnosed with advanced disease(stage III or IV)and the 10-year survival rate is only 5%-21% [9].Tumor cytoreductive surgery combined with platinum and paclitaxel-based chemotherapy is the standard first-line therapy for patients with advanced ovarian cancer.This standard treatment makes the complete response rate of advanced ovarian cancer reach 40%-60%,but more than 90% of patients will relapse after 18 months,and chemotherapy resistance,and ultimately died of ovarian cancer [10].Although the current treatment of ovarian cancer has made some progress,it is still not ideal for the treatment and prevention of recurrence and metastasis in advanced patients.Although immunotherapy is currently mainly used as a clinical treatment aid,it is considered to be the most promising treatment.The transmembrane 4 L6 family member 1(TM4SF1),also known as tumor-associated antigen L6(TAL6),is highly expressed in pancreatic cancer [11],liver cancer [12],esophageal cancer [13],breast cancer [14] and other tissues,and with lower expression in the corresponding normal tissues.In our previous study,the related antigen TM4SF1 was screened from a c DNA library constructed from ovarian cancer ascites tumor cells.The expression of gene and protein with TM4SF1 in epithelial ovarian cancer was significantly higher than that of ovarian benign tumor tissue and normal ovarian tissue.With the malignant transformation of the lesions and the increase of clinical stages,the expression of TM4SF1 also gradually increased,suggesting a close correlation between TM4SF1 and the development of ovarian cancer [15-16].Therefore,TM4SF1 has the potential of immunotherapeutic targets and the HLA-A2-restricted polypeptide derived from TM4SF1 may inhibit tumor growth by activating CTLs to kill TM4SF1-expressing ovarian cancer cells.Our group's previous research combined with the current international commonly used quantitative matrix method,artificial neural network prediction method,based on three-dimensional structure and Hidden Markov model prediction methods [17-19],to predict ovarian cancer associated antigen TM4SF1 HLA-A2 restrictive CTL epitopes and 10 predicted epitope peptides of the ovarian cancer associated antigen TM4SF1 HLA-A2 were screened.According to their screening scores,we named those peptides with P1-P10(specific sequences: P1 LLMLLPAFV;P2 MLLPAFVFI;P3 SLFSILLAL;P4 CLIQVINGV;P5 MLSSVLAAL;P6 GLAEGPLCL;P7 AMLSSVLAA;P8 FVWFFSGIV;P9 ALGGIEFIL;P10 TKYASENHL),and four peptides(P1,P2,P8,P10)were selected for immunoreactivity verification.Epitope peptides P1 and P10 all have immunogenicity,and P10 has a higher immunoactivity.In this phase of the experiment,6 other peptides were tested for immunoreactivity,and immunoreactive peptides were screened and immunoreactive.The peptides were further verified in vitro and the results confirmed that the epitope peptide P10:TKYASENHL can induce specific CTLs in peripheral blood mononuclear cells(PBMCs)from healthy HLA-A2-positive volunteers.Those CTLs can specifically kill the HLA-A2 positive HO8910 PM cell line which expressing TM4SF1,providing a theoretical basis for the study of TM4SF1 as an immunotherapeutic target.The full text is divided into the following five parts:Part ? Detection of binding stability between HLA-A2 restricted epitopes and ovarian cancer-associated antigen TM4SF1 HLA-A2.Objective To test the binding stability of the predicted HLA-A2 candidate CTL epitopes of TM4SF1 with HLA-A2 molecules.Methods Six candidate epitope peptides and positive and negative control peptides were synthesized by commercial companies.T2 was cultured in vitro.T2 cells were stimulated with peptides of different concentrations(10 ?g/ml,20 ?g/ml,30 ?g/ml,40 ?g/ml)to detect MIF,and the correlation between MIF and stimulatory concentrations was compared.Results Under a certain range of peptide stimulating concentrations,the MIFs detected by candidate epitope peptides P3,P4,P5,P7,and P9 stimulated T2 cells to increase with increasing peptide concentrations(P<0.05).The MIF of candidate epitope peptide P6 stimulated by T2 cells did not increase with increasing peptide concentration(P>0.05),and the correlation coefficients were P3(0.679),P4(0.817),P5(0.924),P6(-0.107),P7(0.625),and P9(0.939).Conclusion The candidate epitope peptides P3,P4,P5,P7,P9 have good binding stability with HLA-A2 molecular complexes,while peptide P6 hasn't.Part ? The breeding and identification of HLA-A2 transgenic mice,and the detection of ovarian cancer-associated antigen TM4SF1 HLA-A2-restricted CTL epitope in vivo immunoreactivityObjective To breed and identify HLA-A2 transgenic mice and detect the immunoreactivity of the HLA-A2 restricted CTL epitope polypeptide of ovarian cancer-associated antigen TM4SF1 in HLA-A2 transgenic mice.Methods The inbreeding system was used for long-term cohabitation and the HLA-A2 gene was identified in the offspring mice.The transgenic positive female mice were selected and the candidate epitope polypeptides(P3,P4,P5,P6,and P9)were combined.The mice were immunized with HLA-A2 gene mice and spleen lymphocytes were isolated from the mice for pre-incubation ELISPOT experiments.The net T of spot formation number SFC(T = SFC of experimental wells-SFC of negative control wells)and the mean spot size were compared,and the ability of activated CTLs and the level of IFN-? secreted by single cells were analyzed.The ELISPOT results of the epitope peptides were analyzed and compared,and epitope peptides with immunological activity were initially identified.Results Negative control peptide,P4,P6,and P9 did not reach the positive standard SFC.Positive peptide,P3,and P5 all had positive SFC.Conclusion 1.Transgenic mice can be successfully bred using the long-term cohabitation method of inbreds.Mice can be mated again within a few hours after delivery to increase the yield of offspring mice.2.The ovarian cancer-associated antigen TM4SF1 HLA-A2 restricted CTL epitope peptides P3 and P5 are all immunogenic,and the epitope peptides P4,P6 and P9 are not immunogenic,and P5 is more immunogenic than P3.Part ? T-cell Immune Response Assay for HLA-A2 Positive Ovarian CancerObjective To determine whether a meaningful peptide can stimulate specific CTL immune responses in peripheral blood of HLA-A2-positive ovarian cancer patients.Methods Patients with HLA-A2 positive ovarian cancer were selected and PBMCs from peripheral blood mononuclear cells were collected and incubated for 48 h with peptides P1,P5 and P10,respectively.Pre-incubation ELISPOT assays were performed.The net T value of the spot formation number SFC(T = SFC in the experimental well-SFC in the negative control well)and the mean spot size were compared,respectively,and the activated CTL capability and the level of IFN-? secreted by a single cell were analyzed.Results The results of pre-incubation ELISPOT assay indicated that the negative control peptide did not reach the positive standard SFC,positive peptides,P1,P5,and P10 all had positive SFCs,and peptides P1 and P10 stimulated single cells to secrete INF-? factors.The level is higher than P5.Conclusion P1,P5 and P10 can stimulate the immune response of peripheral blood CTL in ovarian cancer patients with HLA-A2 positive.P1 and P10 have stronger immune activity.Part ? In vitro Killing Effects of Specific CTL Induced by Dendritic Cells Loaded with TM4SF1 HLA-A2 Restricted Epitope PeptideObjective To detect the killing ability of dendritic cells derived from human peripheral blood mononuclear cells loaded with epitope peptides on tumor cells.Methods The CCK-8 method was used to detect the killing activity of co-culture of DCs loaded with epitope antigen peptides and CD8+ T cells on human ovarian cancer cells HO8910 PM and anti-HLA-A2 pretreated HO8910 PM.Results The killing effect of CTLs induced by DCs loaded with P10 antigen peptides against human ovarian cancer cell lines HO8910PM(HLA-A2 +,TM4SF1 +)expressing both TM4SF1 antigen and HLA-A2 was:(53.31 +/-2.93)% at 40:1.;(20.44 +/-10.81)% at 20:1;(8.58 +/-7.30)%10?1,significantly higher than the other 3 peptides(P < 0.05)and the killing effect of anti-HLA-A2 pretreated HO8910PM(HLA-A2+,TM4SF1+)was significantly decreased:(28.54±2.04)% at 40:1;(15.47±2.98)% at 20:1,(1.49±1.26)% at 10:1.CD8+ T cells stimulated with DCs that were not loaded with polypeptide had no killing effect on HO8910PM(HLA-A2+,TM4SF1+).Conclusions 1.The HLA-A2-restricted CTL epitope antigen polypeptide TM4SF1 of the ovarian cancer associated antigen P10 loaded DC co-cultured with CD8+ T cells had high killing capacity.2.CD8+ T cells stimulated with DCs not loaded with polypeptide have no killing effect on HO8910PM(HLA-A2+,TM4SF1+).3.Peptide P10 is HLA-A*0201-restricted and expressed on the surface of TM4SF1-positive tumor cells and can be naturally presented and is an HLA-A*0201-restricted CTL epitope of the TM4SF1 antigen.Part 5 Peptide-Induced Killing of Human Ovarian Cancer Cells by Specific CTL in MiceObjective The TM4SF1 epitope peptides were used to immunize HLA-A2 transgenic mice,and spleen cells were collected as effector cells to detect the effect of polypeptide-specific CTL-induced killing of human ovarian cancer cells.Methods Significant peptide immunizations were performed on HLA-A2 miceand spleen cells were obtained.CTLs killing activity induced by CTL epitopeantigen peptides were detected by CCK-8 method on human ovarian cancer celllines HO8910PM(HLA-A2+,TM4SF1+)and HO8910PM(HLA-A2+,TM4SF1-).Results The killing effect of CTL induced by splenocytes of mice immunized with P10 antigen peptide on human ovarian cancer cell line HO8910PM(HLA-A2+,TM4SF1+)at different effect ratios was:(18.98 ± 2.12)% at 12.5:1,(29.63±4.46)% at 25:1,(34.26±2.89)% at 50:1 and(51.38±5.56)% at 100:1,was significantly higher than the other 3 peptides(P < 0.05).However,the killing effect of HO8910PM(HLA-A2+,TM4SF1-)which silenced TM4SF1 gene was significantly decreased:(14.47±3.55)% at 12.5:1,(14.14±2.67)% at 25:1,(17.17±5.25)% at 50:1and(16.50±5.56)% at 100:1.Conclusion The CTL induced by mouse spleen cells immunized with the ovarian cancer-associated antigen TM4SF1 HLA-A2 restricted CTL epitope P10 antigen peptide specifically kills target cells in vitro.