Efficacy And Molecular Mechanism Of Neoantigen Peptides Vaccine Combined With PD-1 Blockade Against Hepatocellular Carcinoma

ObjectivesHepatocellular carcinoma(HCC)was associated with worse malignancy and poor prognosis.Lately,immunotherapies such as anti-programmed cell death 1(PD-1)have shown great anti-tumor activity.However,there are still a considerable number of HCC patients who cannot benefit from PD-1 blockade(α-PD1)monotherapy due to restricted tumor infiltrating lymphocytes(TILs).Thus,there is an urgent need to develop novel strategies for increasing tumor-specific T cell infiltration and improving efficacy of immunotherapy in HCC.Tumor neoantigen,a kind of tumor-specific antigen,could induce robust antitumor immune response in multiple cancers,whose efficacy could be further enhanced by combining α-PD1(ranging from 27%-60%)in clinical trials.However,the corresponding immune response and synergistic mechanisms remain largely unclear.Based on these factors,we aimed to assess the antitumor efficacy and immune response of NeoVAC combined with α-PD1 in HCC and further explore the potential antitumor molecular mechanism to provide a new insight for HCC immunotherapy.Methods1.DNA and RNA extracted from Hepa1-6 cells were subjected to whole exome sequencing and RNA sequencing respectively and further analyzed by bioinformatics algorithms to screen the potential neoantigen peptides.Antitumor efficacy of NeoVAC was evaluated by HCC subcutaneous mouse model.2.Synergistic antitumor and tumor prevention efficacy of NeoVAC combined with αPD1 were evaluated by orthotopic HCC mouse model,immune activation was assessed by flow cytometry,neoantigen-specific T cells infiltration and immune memory were assessed by tetramer flow cytometry combined with immunofluorescence and Interferon-γ enzyme-linked immunospot(ELISPOT).3.Immune microenvironment changes and potential antitumor mechanisms of immune cell subsets induced by NeoVAC/α-PD 1 combination therapy were analyzed by single-cell RNA sequencing,single-cell V(D)J analysis,T cell developmental trajectory and cell-cell interaction analysis.The Cancer Genome Atlas(TCGA)database was used to validate the correlation between immune cell subset and overall survival(OS)and recurrence-free survival(RFS).4.The relationship between neoantigen-specific T cell and the vital immune cell subset was validated by flow cytometry.The vitro,vivo and potential clinical antitumor ability of this cell subset was assessed by coincubation,adoptive cell therapy experiments and patient-derived cell(PDC).Results1.Seven out of 20 neoantigen peptides with potential higher immunogenicity to H2Kb assessed by bioinformatics algorithms and ELISPOT were selected for preparing neoantigen peptides vaccine(NeoVAC).NeoVAC showed antitumor efficacy in HCC subcutaneous mouse model.The results of immunofluorescence revealed that the infiltration of both CD8+and CD4+T cells was obviously increased by NeoVAC.2.Most significantly,the tumor growth of NeoVAC plus α-PD1 treated mice showed most dramatic tumor suppress with 80%of durable tumor regression when compared with other 3 treated groups by inducing significantly stronger neoantigen-specific T cell infiltration in tumor.Kaplan-Meier analysis further indicated that mice with NeoVAC plus α-PD1 treatment had significantly longer overall survival than those treated with PBS,NeoVAC or α-PD1 alone.Furthermore,combining NeoVAC with α-PD1 could induce long-term neoantigen-specific immune memory effects for preventing HCC recurrence and metastasis.3.Single cell RNA sequencing analysis showed that CD8+tissue-resident memory T cells(CD8+TRMs)a subset of CD8+T cells mainly from NeoVAC and α-PD1 treated group but was very limited in the other three groups.Pseudotime analysis found that the trajectory began with CD8+naive T cells,followed by CD8+central memory T cells,CD8+effector T cells and CD8+Cd7+memory T cells and then transformed into CD8+TRMs.Ligand-receptor analysis suggested that the recruit of Th2 by CD8+TRMs via CCL5/CCR5 interaction might lead to better antitumor response.In addition,Kaplan-Meier analysis of TCGA HCC dataset also confirmed that the CD8+TRMs was a significant predictor of HCC prognosis,with tumors expressing higher CD69_CD8a signature showed significantly longer OS(p=0.001)and RFS(p=0.002).4.Flow cytometry analysis confirmed the increased infiltration of CD8+TRMs and the rate of neoantigen-specific CD8+T cells in this immune cell subset after combinational therapy.CD8+TRMs showed pivotal tumor-killing role of CD8+TRMs in vitro and vivo experiments and PDC model.Conclusions1.NeoVAC composed of neoantigen peptides and poly(I:C)with high immunogenicity and safety showed antitumor ability in HCC subcutaneous mouse model.2.NeoVAC plus α-PD1 could induce powerful antitumor effect and long-term tumorspecific immune memory in HCC model by inducing massive infiltration of neoantigen-specific T cells.3.CD8+TRMs were found to be significantly increased in HCC tissues after combinational treatment.Recruit of Th2 by CD 8+TPMs via CCL5/CCR5 interaction might lead to better antitumor response.In addition,CD8+TRMs was a significant predictor of HCC prognosis.4.CD8+TRMs containing relative high proportion of neoantigen-specific T cells,as an potential target,was significantly related to the anti-tumor efficacy.