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ESPEC-SUIT: a versatile and robust platform to identify and track antigen-specific T cell receptors in patients with cancer

Background Methods to identify and characterize antigen-reactive T cell receptors (TCRs) represent important tools to understand and exploit T cell responses in patients with cancer and beyond. Current methods are hampered by the rarity of individual T cell clones and limited applicability to monito...

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Main Authors: Lindner, Katharina (Author) , Stange, Saskia R. (Author) , Hlawatsch, Julian Benedikt (Author) , Hamberger, Moritz (Author) , Boschert, Tamara (Author) , Tan, Chin Leng (Author) , Hülsmeyer, Ingrid (Author) , Sachse, Samira Moreen (Author) , Jünger, Simone (Author) , Ernst, Alexander (Author) , Głatki, Olga (Author) , Vinogradova, Sofiya (Author) , Fleischhacker, Lena (Author) , Eichmüller, Stefan B. (Author) , Fröhling, Stefan (Author) , Hassel, Jessica C. (Author) , Zörnig, Inka (Author) , Lyu, Yanhong (Author) , Huang, Zhiqin (Author) , Jäger, Dirk (Author) , Momburg, Frank (Author) , Meyer, Marten (Author) , Bunse, Lukas (Author) , Green, Edward W. (Author) , Platten, Michael (Author) , Poschke, Isabel (Author)
Format: Article (Journal)
Language:English
Published: 21 September 2025
In: Journal for ImmunoTherapy of Cancer
Year: 2025, Volume: 13, Pages: 1-16
ISSN:2051-1426
DOI:10.1136/jitc-2025-012216
Online Access:Verlag, kostenfrei, Volltext: https://doi.org/10.1136/jitc-2025-012216
Verlag, kostenfrei, Volltext: https://jitc.bmj.com/content/13/9/e012216
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Author Notes:Katharina Lindner, Saskia R. Stange, Julian Benedikt Hlawatsch, Moritz Hamberger, Tamara Boschert, Chin Leng Tan, Ingrid Hülsmeyer, Samira Moreen Sachse, Simone Jünger, Alexander Ernst, Olga Głatki, Sofiya Vinogradova, Lena Fleischhacker, Stefan B. Eichmüller, Stefan Fröhling, Jessica C. Hassel, Inka Zörnig, Yanhong Lyu, Zhiqin Huang, Dirk Jäger, Frank Momburg, Marten Meyer, Lukas Bunse, Edward William Green, Michael Platten, Isabel Poschke
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Summary:Background Methods to identify and characterize antigen-reactive T cell receptors (TCRs) represent important tools to understand and exploit T cell responses in patients with cancer and beyond. Current methods are hampered by the rarity of individual T cell clones and limited applicability to monitor both major histocompatibility complex (MHC) class I-restricted and MHC class II-restricted responses, hence insufficiently reflecting the entire antigen-reactive repertoire of a patient. To obtain broad and deep insight into polyclonal, antigen-specific TCR repertoires, we developed the ‘epitope-specific expansion culture with subsequent identification of TCRs’ (ESPEC-SUIT) assay to identify and track antigen-specific TCRs.Methods In vitro stimulation of peripheral blood mononuclear cells with (vaccine-targeted) neoantigens was verified in cytokine secretion assays and read-out by TCRβ repertoire sequencing (TCRseq). Candidate antigen-reactive clonotypes were defined by specific expansion in cultures stimulated with relevant antigen, followed by TCR cloning and validation in co-cultures of TCR transgenic effector cells and peptide-presenting targets. Using TCRseq information, candidate and validated clonotypes were traced and characterized in bulk and single-cell repertoire sequencing data of longitudinally collected blood samples and tumor tissue.Results In a cohort of 32 patients with cancer, we demonstrate that ESPEC-SUIT supports strong, robust and reproducible expansion of CD4 + and CD8+ T cells in response to various antigens. TCRseq revealed highly polyclonal neoepitope-specific T cell responses, which can be further characterized with respect to cross-reactivity, affinity or human leukocyte antigen (HLA) restriction. In a subcohort of 10 patients, we selected 341 ESPEC-SUIT-derived TCRs for cloning and in vitro functional validation from >2000 candidates and confirmed antigen-reactivity for >75%. We exemplify the usefulness of this TCR discovery method for downstream analysis in neoepitope vaccinated patients with glioma, where we found longitudinal changes in candidate TCR frequencies in blood mirroring antigen-specific ex vivo Enzyme-Linked ImmunoSpot (ELISpot) responses. Furthermore, up to 67% of candidates could be detected in on-treatment brain tumor tissue and exhibited gene expression signatures overlapping with clonotypes of confirmed specificity to the vaccine antigen.Conclusion ESPEC-SUIT provides unprecedented insight into highly polyclonal, antigen-specific T cell responses and enables discovery of large numbers of TCRs for a given antigen. It represents an efficient, cost-effective and scalable framework for the interrogation of tumor-reactive T cell responses in patients with cancer.
Item Description:Gesehen am 27.10.2025
Physical Description:Online Resource
ISSN:2051-1426
DOI:10.1136/jitc-2025-012216

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