Inhaltszusammenfassung

Engineering tumor antigen-specific αβ T-cell receptor (TCR) genes into autologous T cells for adoptive T cell therapy (ACT) has demonstrated prolonged tumor regression in numerous cancers. Clinical advancement of ACT is hindered by the difficulty in retrieving potent and tumor-specific TCRs. Further, the paucity in murine TCR candidates impedes pre-clinical studies needed to advance the clinical success of TCR-based therapies. Despite advances in single cell TCR sequencing (scTCRseq) methods,...Engineering tumor antigen-specific αβ T-cell receptor (TCR) genes into autologous T cells for adoptive T cell therapy (ACT) has demonstrated prolonged tumor regression in numerous cancers. Clinical advancement of ACT is hindered by the difficulty in retrieving potent and tumor-specific TCRs. Further, the paucity in murine TCR candidates impedes pre-clinical studies needed to advance the clinical success of TCR-based therapies. Despite advances in single cell TCR sequencing (scTCRseq) methods, drawbacks still exist, including low-throughput, high costs and complex protocols. This thesis aimed to develop a high-throughput and easily applicable murine scTCRseq platform to facilitate the discovery of shared tumor antigen and neoepitope-specific murine TCRs. A plate-based scTCRseq platform compatible with next-generation sequencing (NGS) was developed. Sample barcoding permits early pooling of single cell samples and 96-well plate multiplexing allows little hands-on work at a high-throughput. The use of standard laboratory equipment renders the platform easily applicable. Only 25 primers are used and both α and β TCR chains are amplified in the same polymerase chain reaction. An automated data analysis pipeline retrieves αβTCR information from the 2x300 bp paired-end MiSeq data. As an advantage over synthetic gene orders, TCRs of interest can directly be cloned for functional characterization using remaining cDNA. The platform was optimized improving αβTCR detection rates and minimizing platform noise, costs and complexity. Performance evaluation revealed an average paired αβTCR detection rate of 49 %. The scTCRseq platform revealed its superior performance compared to a previously published scTCR cloning platform as well as its ability to provide insight into the TCR repertoire. The scTCRseq platform was applied to establish a library of human and murine HLA-restricted TCRs specific for oncoviral and cancer/testis epitopes, as well as neoantigens. The latter were strictly mutation specific and the majority of the TCRs could recognize the respective tumor cell line. We show that frequent retrieval of the same αβTCR, high read counts and CDR3 homology were indicative of antigen specificity. Moreover, a gene-specific primer panel of commonly used T cell markers was integrated into the platform to allow the additional NGS-based phenotyping of single T cells. This scTCRPhenoSeq platform was applied to determine the phenotype and clonality of chimeric antigen receptor T cells after ACT and subsequent RNA-based in vivo expansion. Together, a powerful tool was developed for the rapid discovery of TA-specific murine TCRs allowing the full exploitation of such TCRs for pre-clinical and clinical advancements of ACT for the benefit of cancer patients.» weiterlesen» einklappen

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Klassifikation

DDC Sachgruppe:
Biowissenschaften, Biologie