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A complete peptidome workflow for neoantigen identification and validation by mass spectrometry-based immunopeptidomics

ABSTRACT: We proposed a complete workflow for neoantigen identification and validation based solely on MS immunopeptidomics.We first developed a new Neo DiscoveryTM immunopeptidome enrichment Kit, which can be used for HLA peptide purification from a small amount of biopsied tissue as low as 18mg. To achieve high sensitivity with such a low amount of sample, we leveraged the power of deep learning and a massive amount of MS-based immunopeptidomics data to build DeepNovo-HLA, a de novo sequencing model specialized for HLA peptides. We also developed DeepImmu, a personalized model for immunogenicity prediction based on the central tolerance of T cells, i.e. the positive and negative selection of T cells in an individual patient.

INSTRUMENT(S): timsTOF Pro 2

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cervical Carcinoma Cell

SUBMITTER: Ngoc Hieu Tran

LAB HEAD: Baozhen Shan

PROVIDER: PXD046370 | Pride | 2024-05-04

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	checksum.txt	Txt
	neo_SH14_HLAI.zip	Other
	patient1.peptideDb.peptides.csv	Csv
	patient2.peptideDb.peptides.csv	Csv
	patient3peptideDb.peptides.csv	Csv

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Project description:Background. Dendritic cell (DC)-based neoantigen vaccination holds potential as a safe and effective adjuvant therapy for patients with early-stage, resectable NSCLC, a tumor type typically characterized by high mutational loads. DCs have the unique ability to elicit robust antitumoral T-cell responses, while neoantigens are ideal targets to elicit high-affinity T cell responses with exquisite tumor specificity. Here, we present the results of a phase I clinical trial in which a novel DC vaccine targeting neoantigens was evaluated in six patients with early stage, resected NSCLC. Methods. Tumor samples were subjected to a comprehensive neoantigen identification approach encompassing genomics, transcriptomics and immunopeptidomics. Using genomics and transcriptopmics data, tumor-specific antigens were identified by a bioinformatics approach. Additionally, immunopeptidomics was performed by immunoprecipitation of human MHC class I molecule followed by immunopeptides enrichment and LC-MS/MS analysis. Two immunopeptidomics screens were performed. In the first immunopeptidomics screen, patient-derived tumors were analyzed to uncover neoepitopes specific for the patient. In the second, screen, patient-derived EBV-immortilized B cell lines overexpressing the selected neoepitopes were analyzed to verify that the predicted neoepitopes can bind to the HLA haplotypes of the patients. For anti-tumor vaccination, patients underwent leukapheresis for the manufacturing of monocyte-derived DCs loaded with neoantigens (Neo-mDCs) according to a four-day protocol. Neo-mDCs were injected intravenously following an intrapatient dose escalation scheme. Primary endpoint of the trial was safety. Secondary endpoints were feasibility, immunogenicity, and relapse-free survival. Results. In the first immunopeptidomics screen, one neoepitopes derived was identified from the tumor of one patient. In the second immunopeptidomics screen, several predicted neoepitopes were confirmed to be presented on the HLA haplotypes of the patients by analyzing the patient-derived EBV-immortilized B cell lines. Additionally, the vaccine was demonstrated to be feasible and safe. T cell responses were observed in 5 of 6 vaccinated patients and were dominated by CD8+ T cells, which could be detected ex vivo at high frequencies >1.5 years after the last dose. Furthermore, single cell analysis indicated that the CD8+ T cell responsive population was polyclonal and exhibited the near entire spectrum of T cell differentiation states, including a naïve-like state associated with long lasting memory but excluding exhausted cell states. Three of six vaccinated patients experienced disease relapse. Conclusion. Neo-mDC vaccination is safe and feasible. Vaccination induces large populations of neoantigen-specific T-cell responses containing long lasting memory and effector cells in early-stage NSCLC patients, suggesting clinical potential.

Project description:Treatment of cancer cells with anti-cancer drugs often fails to achieve complete remission. Yet, such drug treatments may induce alteration in the tumorâs gene expression patterns, including those of Cancer/Testis Antigens (CTA). The degradation products of such antigens can be presented as HLA peptides on the surface of the tumor cells and be developed into anti-cancer immunotherapeutics. For example, the DNA methyl transferase inhibitor, 5-aza-2'-deoxycytidine (Decitabine) has limited anti-tumor efficacy, yet it induces the expression of many genes, including CTAs that are normally silenced in the healthy adult tissues. In this study, the presentation of many new HLA peptides derived from CTAs and induced by Decitabine was demonstrated in three human Glioblastoma cell lines. Such presentation of CTA-derived HLA peptides can be exploited for development of new treatment modalities, combining drug treatment with anti-CTA targeted immunotherapy. The Decitabine-induced HLA peptidomes include many CTAs that are not normally detected in healthy tissues or in cancer cells, unless treated with the drug. In addition, the study included large-scale analyses of the simultaneous effects of Decitabine on the transcriptomes, proteomes and HLA peptidomes of the human Glioblastoma cells. It demonstrates the poor correlations between these three levels of gene expression, both in their total levels and in their response to the drug. The transcriptomes, proteomes and HLA peptidomes of the U-87, T98G and LNT-229 GBM human cell lines were analyzed before and after treatment with Decitabine. Overall, the RNA-Seq transcriptome analyses resulted in the identification of above 26000 transcripts, the proteome analyses identified about 7500 proteins and the HLA class I peptidome analyses resulted in above 25000 identified HLA peptides. Two biological repetitions of the transcriptome, three of the proteome and three of the HLA peptidome were performed with each of the cell lines and treatment, resulting in highly reproducible datasets.

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A complete peptidome workflow for neoantigen identification and validation by mass spectrometry-based immunopeptidomics

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