Induction Of Specific CD8~+T Cells Against Hepatocellular Carcinoma-associated Neoantigens

Hepatocellular carcinoma(HCC)is one of the most common malignant carcinomas in China.Hepatitis B virus(HBV)infection is the major cause of HCC in China.Currently,hepatic resection is still the mainstay of curative treatment for HCC which is confined to the liver with satisfactory liver function.While,the patients in the late stage,who received transcatheter arterial chemoembolization or chemotherapy or radiotherapy,had poor progress.The 5-year survival rate of HCC patients in China is about 12.1%.Recently,the FDA of United States had expanded the indications of immune checkpoint inhibitors or regorafenib to include the treatment of patients with HCC progression after tolerating sorafenib therapy.PD-1/PD-L1 immune checkpoint inhibitors have been tested in several types of cancers.The rational of the PD-1/PD-L1 immune checkpoint inhibitor therapy is to reverse the T cell exhaustion and restore their anti-tumor immunity.Generally,the HCC tumor tissues displayed infiltration of immune cells in large amount.A meta-analysis showed that about 17%of the HCC patients showed responses to the therapy with PD-1/PD-L1 immune checkpoint inhibitors.This is might be due to the lack of high quality of tumor antigen-specific T cells in HCC tumor tissues.The technological advances in whole genomic sequencing and the bioinformatic analysis have made it possible to dissect the immune response to personalized neoantigens encoded by tumor somatic cell mutations.Neoantigens,which are derived from tumor-specific somatic mutations of the tumor,are exempt from central tolerance and can function as bona fide antigens that facilitate tumor rejection.Therefore,the personalized vaccines by using the neoantigens could generate robust immune responses.In the patients with melanoma the personalized neoantigen vaccination seem to be very efficient in eradicating established tumors.It is unknown whether this tumor vaccine by using neoantigen is effective in patients with HCC.In this study,we predicted the HCC tumor neoantigen epitopes,and examined their activity in inducing the generation of these antigen-specific CTLs,and analyzed their effects against the tumor cells without injury to normal cells.We analyzed the data of whole-genome sequencing and exome sequencing of HCC tumor and matched non-tumor liver tissues from 49 patients to identify the HCC-associated somatic mutations.Elevated expression in the protein level in the tumor tissues after somatic mutations might be ideal as the neoantigens.We identified that the mutation frequency of tumor protein p53(TP53)gene was 40%,and was 8%in the Laminin Subunit Alpha 3(LAMA3)in these HCC tissues.The mutated TP53 and LAMA3 may be used as the potential mutation-associated neoantigens.We applied GPL 10687 platform to examine the gene expression differences between the tumor and normal tissues of the selected genes in GSE25097,one of the GEO databases.The mRNA expression levels of TP53 and LAMA3 in GSE25097 database were higher in tumors than in matched no-tumor tissue(P<0,001).We obtained the HCC tumor tissues and their matched normal tissues from 10 HCC cases.The differences of expression levels of TP53 and LAMA3 in tumor and no-tumor tissues were validated by using quantitative real-time PCR(qRT-qPCR)and immunohistochemistry.Both the expression levels of TP53 and LAMA 3 were higher in tumors.Therefore,the products from TP53 mutation and LAAMA3 may be used as the mutation-associated neoantigens.We summarized the reports of human leucocyte antigen(HLA)alleles in China.HLA-A is one of the major histocompatibility complex class I(MHC-I)molecules.Among the Chinese population,HLA-A2 and HLA-A11 and HLA-A24 accounted for 70%,representing the major MHC-I molecules.All mutations in exomes of TP53 and LAMAS were translated to amino acid alterations using the Consensus Coding Sequence database from National Center for Biotechnology Information.We set 9-mer or 10-mer or 11-mer epitopes across each mutation site as the antigen epitope.By using NetMHC 4.0 software we predicted the binding strength of each mutant amino acid fragment to each of the HLA-A2 and HLA-A11 and HLA-A24.The predicted binding affinity(Rank%)<2.0 was considered to be a potential epitope.The results showed that mutation at TP 53 R249S had excellent binding affinity with HLA-A2 and HLA-A11.The mutation at LAMA3 R1603S showed binding affinity to HLA-A2 allele.However,in HLA-A24 allele no binding affinity was predicted in the mutated TP53 and LAMA3.TP53 241-255:SCMGGMNRSPILTII(15aa)and LAMA3 1601-1513:VSSEELMTVLSRL(13aa)were selected as the potential mutation-associated neoantigens.Human samples were collected and anonymously coded.Study protocols and usage of human samples were approved by the ethics committee of NCC/CAMS(NCC2015 YZ-22).The single suspension was prepared from lymph nodes that were collected during the surgery on patients who head and neck neoplasm.The individuals with HLA-A2+(n=5)were chosen after being stained with PE anti-human HLA-A2 antibody.T cells were isolated and CTLs were induced by the predicted peptides by using the autologous dendritic cells.Further,we examine their cytotoxicity on different target cells,including the 174 x CEM.T2 cells pulsed with mutated peptide,or pulsed with no-mutated peptide and the peripheral blood mononuclear cells(PBMC)of the same patient.The results showed that the induced CTLs by predicted peptides showed increased cytotoxicity to the 174 x CEM.T2 pulsed with mutated peptide,with no effect on the 174 x CEM.T2 pulsed with no-mutated peptides.At the ratio of 10 effectors to one target,and at 25 effectors to one target cell,the cytotoxicity of induced CTLs by TP53241-255 SCMGGMNRSPILTII was 14.6%±0.5%,19.6%±0.6%to the target pulsed with mutated peptide,and was 10.4%±0.4%,7.7%±0.2%to the target pulsed with no-mutated peptide.At the same ratio,the cytotoxicity of induced CTLs by LAMA3 1601-1613:VSSEELMTVLSRL was 14.6%±0.9%,15.5%±1.4%to the target pulsed with mutated peptide,and 8.6%±0.9%,10.5%±0.4%to the target pulsed with no-mutated peptide.Using the autologous PBMC,we further confirmed CTLs induced by TP53 241-255 SCMGGMNRSPILTII and LAMA31601-1613:VSSEELMTVLSRL showed no obvious cytotoxicity on them with the killing effect of less than 5%.Conclusions1)Higher mutation frequencies of TP53 and LAMA3 were observed in HCC tumor tissues.The protein expression levels of TP53 and LAMA3 showed higher in tumor than in no-tumor tissues.2)The mutation at TP53 R249S showed binding affinity with HLA-A2 and HLA-A11.The mutation at LAMA3 R1603S showed binding affinity to HLA-A2.3)The HLA-A2 restricted TP53241-255:SCMGGMNRSPILTII(15aa)and LAMA31601-1613:VSSEELMTVLSRL(13aa)could induce the generation of antigen-specific CTLs.These induced CTLs could specific kill the target cells with without injury to normal cells.The above predicted peptide epitopes might be potential mutation-associated neoantigens.