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Identification of neoantigen-specific T cells and their targets: implications for immunotherapy of head and neck squamous cell carcinoma.

ABSTRACT: To develop a practically applicable method for T-cell receptor (TCR)-engineered T cell immunotherapy targeting neoantigens, we have been attempting to identify neoantigen-specific T cell receptors (TCRs) and establish TCR-engineered T cells in a 3-4-month period. In this study, we report the characterization of T cell repertoires in tumor microenvironment (TME) and identification of neoantigen-specific TCRs after stimulation of patient-derived T cells. We screened 15 potential neoantigen peptides and successfully identified two CD8⁺HLA-dextramer⁺ T cells, which recognized MAGOHB_G17A and ZCCHC14_P368L. All three dominant TCR clonotypes from MAGOHB_G17A-HLA dextramer-sorted CD8⁺ T cells were also found in T cells in TME, while none of dominant TCR clonotypes from ZCCHC14_P368L-HLA dextramer-sorted CD8⁺ T cells was found in the corresponding TME. The most dominant TCRA/TCRB pairs for these two neoantigens were cloned into HLA-matched healthy donors' T lymphocytes to generate TCR-engineered T cells. The functional assay showed MAGOHB_G17A TCR-engineered T cells could be significantly activated in a mutation-specific, HLA-restricted and peptide-dose-dependent manner while ZCCHC14_P368L TCR-engineered T cells could not. Our data showed neoantigen-reactive T cell clonotypes that were identified in the patient's peripheral blood could be present in the corresponding TME and might be good TCRs targeting neoantigens.

SUBMITTER: Ren L

PROVIDER: S-EPMC6422382 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Identification of neoantigen-specific T cells and their targets: implications for immunotherapy of head and neck squamous cell carcinoma.

Ren Lili L Leisegang Matthias M Deng Boya B Matsuda Tatsuo T Kiyotani Kazuma K Kato Taigo T Harada Makiko M Park Jae-Hyun JH Saloura Vassiliki V Seiwert Tanguy T Vokes Everett E Agrawal Nishant N Nakamura Yusuke Y

Oncoimmunology 20190206 4

To develop a practically applicable method for T-cell receptor (TCR)-engineered T cell immunotherapy targeting neoantigens, we have been attempting to identify neoantigen-specific T cell receptors (TCRs) and establish TCR-engineered T cells in a 3-4-month period. In this study, we report the characterization of T cell repertoires in tumor microenvironment (TME) and identification of neoantigen-specific TCRs after stimulation of patient-derived T cells. We screened 15 potential neoantigen peptide ...[more]

PMID: 30906664

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Project description:Adoptive cell therapy using TCR-engineered T cells (TCR-T cells) represents a promising strategy for treating relapsed and metastatic cancers. We previously established methods to identify neoantigen-specific TCRs based on patients' PBMCs. However, in clinical practice isolation of PBMCs from advanced-stage cancer patients proves to be difficult. In this study, we substituted blood-derived T cells for tumor-infiltrating lymphocytes (TILs) and used an HLA-matched cell line of antigen-presenting cells (APCs) to replace autologous dendritic cells. Somatic mutations were determined in head and neck squamous cell carcinoma resected from two patients. HLA-A*02:01-restricted neoantigen libraries were constructed and transferred into HLA-matched APCs for stimulation of patient TILs. TCRs were isolated from reactive TIL cultures and functionality was tested using TCR- T cells in vitro and in vivo. To exemplify the screening approach, we identified the targeted neoantigen leading to recognition of the minigene construct that stimulated the strongest TIL response. Neoantigen peptides were used to load MHC-tetramers for T cell isolation and a TCR was identified targeting the KIAA1429D1358E mutation. TCR-T cells were activated, exhibited cytotoxicity, and secreted cytokines in a dose-dependent manner, and only when stimulated with the mutant peptide. Furthermore, comparable to a neoantigen-specific TCR that was isolated from the patient's PBMCs, KIAA1429D1358E-specific TCR T cells destroyed human tumors in mice. The established protocol provides the required flexibility to methods striving to identify neoantigen-specific TCRs. By using an MHC-matched APC cell line and neoantigen-encoding minigene libraries, autologous TILs can be stimulated and screened when patient PBMCs and/or tumor material are not available anymore. Abbreviations: Head and neck squamous cell carcinoma (HNSCC); adoptive T cell therapy (ACT); T cell receptor (TCR); tumor-infiltrating lymphocytes (TIL); cytotoxic T lymphocyte (CTL); peripheral blood mononuclear cell (PBMC); dendritic cell (DC); antigen-presenting cells (APC).

Project description:To show the relationship between antibody delivery and therapeutic efficacy in head and neck cancers, in this study we evaluated the pharmacokinetics and pharmacodynamics of epidermal growth factor receptor (EGFR)-targeted immunotherapy and radioimmunotherapy by quantitative positron emission tomography (PET) imaging.EGFR expression on UM-SCC-22B and SCC1 human head and neck squamous cell cancer (HNSCC) cells were determined by flow cytometry and immunostaining. Tumor delivery and distribution of cetuximab in tumor-bearing nude mice were evaluated with small animal PET using (64)Cu-DOTA-cetuximab. The in vitro toxicity of cetuximab to HNSCC cells was evaluated by MTT assay. The tumor-bearing mice were then treated with four doses of cetuximab at 10 mg/kg per dose, and tumor growth was evaluated by caliper measurement. FDG PET was done after the third dose of antibody administration to evaluate tumor response. Apoptosis and tumor cell proliferation after cetuximab treatment were analyzed by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling and Ki-67 staining. Radioimmunotherapy was done with (90)Y-DOTA-cetuximab.EGFR expression on UM-SCC-22B cells is lower than that on SCC1 cells. However, the UM-SCC-22B tumors showed much higher (64)Cu-DOTA-cetuximab accumulation than the SCC1 tumors. Cetuximab-induced apoptosis in SCC1 tumors and tumor growth was significantly inhibited, whereas an agonistic effect of cetuximab on UM-SCC-22B tumor growth was observed. After cetuximab treatment, the SCC1 tumors showed decreased FDG uptake, and the UM-SCC-22B tumors had increased FDG uptake. UM-SCC-22B tumors are more responsive to (90)Y-DOTA-cetuximab treatment than SCC1 tumors, partially due to the high tumor accumulation of the injected antibody.Cetuximab has an agonistic effect on the growth of UM-SCC-22B tumors, indicating that tumor response to cetuximab treatment is not necessarily related to EGFR expression and antibody delivery efficiency, as determined by PET imaging. Although PET imaging with antibodies as tracers has limited function in patient screening, it can provide guidance for targeted therapy using antibodies as delivery vehicles.

Project description:BackgroundIn the context of head-and-neck squamous cell carcinoma (HNSCC), dendritic cells (DCs) assume pivotal responsibilities, acting as architects of antigen presentation and conductors of immune checkpoint modulation. In this study, we aimed to identify hub genes associated with DCs in HNSCC and explore their prognostic significance and implications for immunotherapy.MethodsIntegrated clinical datasets from The Cancer Genome Atlas (TCGA)-HNSCC and GSE65858 cohorts underwent meticulous analysis. Employing weighted gene co-expression network analysis (WGCNA), we delineated candidate genes pertinent to DCs. Through the application of random survival forest and least absolute shrinkage and selection operator (LASSO) Cox's regression, we derived key genes of significance. Lisa (epigenetic Landscape In Silico deletion Analysis and the second descendent of MARGE) highlighted transcription factors, with Dual-luciferase assays confirming their regulatory role. Furthermore, immunotherapeutic sensitivity was assessed utilizing the Tumor Immune Dysfunction and Exclusion online tool.ResultsThis study illuminated the functional intricacies of HNSCC DC subsets to tailor innovative therapeutic strategies. We leveraged clinical data from the TCGA-HNSCC and GSE65858 cohorts. We subjected the data to advanced analysis, including WGCNA, which revealed 222 DC-related candidate genes. Following this, a discerning approach utilizing random survival forest analysis and LASSO Cox's regression unveiled seven genes associated with the prognostic impact of DCs, notably ACP2 and CPVL, associated with poor overall survival. Differential gene expression analysis between ACP2 + and ACP2 - DC cells revealed 208 differential expressed genes. Lisa analysis identified the top five significant transcription factors as STAT1, SPI1, SMAD1, CEBPB, and IRF1. The correlation between STAT1 and ACP2 was confirmed through quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Dual-luciferase assays in HEK293T cells. Additionally, TP53 and FAT1 mutations were more common in high-risk DC subgroups. Importantly, the sensitivity to immunotherapy differed among the risk clusters. The low-risk cohorts were anticipated to exhibit favorable responses to immunotherapy, marked by heightened expressions of immune system-related markers. In contrast, the high-risk group displayed augmented proportions of immunosuppressive cells, suggesting a less conducive environment for immunotherapeutic interventions.ConclusionsOur research may yield a robust DC-based prognostic system for HNSCC; this will aid personalized treatment and improve clinical outcomes as the battle against this challenging cancer continues.

Dataset Information

Identification of neoantigen-specific T cells and their targets: implications for immunotherapy of head and neck squamous cell carcinoma.

Publications

Identification of neoantigen-specific T cells and their targets: implications for immunotherapy of head and neck squamous cell carcinoma.

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