Identification Of Neoantigens For Personalized Cancer Immunotherapy In Advanced Refractory Solid Tumor Patients

Background and Purpose:In recent years,immunotherapy has made great progress and become one of the most promising therapeutic methods for tumor treatment.Accumulating evidence suggests that neoepitope-reactive T cells is a dominant factor that mediate clinical responses in cancer patients received tumor-infiltrating lymphocytes,immune checkpoint inhibitors or TCR-modified T cells therapy.Recent genomic and bioinformatic technological advances have made it possible to dissect the immune response to personalized neoantigens encoded by tumor-specific mutations.However,rapid and efficient identification of neoantigens is still fraught with difficulty,and a systematic evaluation of personalized neoantigens based immunotherapy in advanced refractory solid tumors is lacking.Here,we established two different piplines for rapid and efficient personalized neoantigen identification including the classical pattern of denovo antigen peptides synthesis based on targeted sequencing and bioinformatics analysis,and the customized driver mutation-derived neoantigens peptide library mode.Moreover,neoantigen based personalized immunotherapy was evaluated in advanced refractory solid tumor patients.Methods:A cohort of 26 patients with advanced refractory solid tumors were underwent mutational profiling by cancer-associated genes panel.Genomic nonsynonymous somatic mutations of tumor tissue and ctDNA samples were matihced with normal bood samples.Neoantigens identification were performed by two strategies:(1)As classic mode,somatic mutations were subjected to in silico analysis to predict potential high-affinity epitopes and mutated peptides were denovo synthesized;(2)Customized shared neoantigens peptide library mode:By systematically mining the hot-spot mutations of common solid tumor in TCGA and COSMIC databases,then combined with mutiple epitope prediction programs to optimized and synthesized neoantigen peptide library for human high-frequency HLA class ?molecules(HLA-A*0201,HLA-A*0203,HLA-A*0206,HLA-A*1101,HLA-A*2402)Patients' hotspot mutations were matched to our customized neoantigens peptide library.The expression of IFN-? and CD 137 were detected by ELISPOT and flow cytometry in vitro to identify irnunogenicity neoepitopes of the above candidate peptides.HLA-peptide binding efficiency and the cytotoxicity of neoantigen specific T cells were also evaluated.Peripheral blood mononuclear cells(PBMCs)were collected with COBE SpectraTM MNC program to generate DCs vaccine and neoantigen Specific T cells for personlized immunotherapy following mmunomodulatory chemotherapy or radiotherapy in andvanced refractory solid tumor patients,and the efficacy and safety were evaluatedResults:Among the sequenced patients,1?2 neoantigens recognized by autologous T cells have been successfully identified in 3 of 4 patients who utilized the classic mode and 6 of 12 patients who performed customized neoantigens library,respectively.A median of 35 somatic missense mutations(range g?73)and a median of 55 predicted HLA class I restricted neoantigens(range 8?140)were identified by a 416 genes panel sequencing in 17 advanced refractory solid tumor patients.Subsequently,a total number of 6 patients received approximately 108 DC vaccine and 1010 bulk T cells composed of 109 neoantigen reactive CD8+T cells targeting personalized neoantigen following immunomodulatory chemotherapy or radiotherapy.To date,one patient with metastatic thymoma who failed multi-line treatment is achieving a complete and durable response beyond 10 months.In addition,immune related partial response was observed in another advanced pancreatic cancer patient.The remaining 4 patients achieved prolonged stabilization of disease with median PFS of 8.6 months.No serious adverse events were oberserved except for localized redness and fever,which recovered after symptomatic treatment.Conclusions:Our costomized neoantigens library can provide a novel approach for neoantigens screening in advanced solid tumor patients.Besides,targeted sequencing is sufficient for somatic variant and neoantigen identification.The combination of two strategies can accelerate the neoantigen-based translational immunotherapy research into the paradigm of precision medicine.