<> "The repository administrator has not yet configured an RDF license."^^ . <> . . "Identification and validation of neoepitope-specific T cell receptors for glioma immunotherapy"^^ . "Immunotherapy is a promising tool for cancer treatment and there is great interest to implement T cell-based therapies in patient care. Development of T cell-based therapies has focused on highly immunogenic tumors. Due to their low-mutational load and limited immune infiltration, gliomas are considered difficult targets for immune intervention and thus remained understudied, despite the urgent need to define new therapeutic options for these aggressive brain tumors.\r\nThis thesis aimed at (I) identification and characterization of T cell responses elicited upon vaccination of glioma patients against the recurrent driver mutation R132H in isocitrate dehydrogenase 1 (IDH1), (II) identifying tumor-reactive T cell receptors (TCRs) targeting tumor-private antigens and (III) antigen-agnostic TCR identification in non-vaccinated patients with the overall aim to define a gene expression signature characteristic for tumor-reactive T cells.\r\nThe IDH1R132H oncogenic driver mutation is found in about 70% of WHO grade 2 and 3 gliomas. Its homogenous exclusive expression in tumor tissue makes it an attractive target for immunotherapy, and vaccinations with an IDH1R132H encoding long peptide were shown to efficiently mediate tumor shrinkage in pre-clinical mouse models. The vaccine was proven to be immunogenic in primary WHO grade 3 and 4 IDH1-mutant astrocytomas in the completed NOA16 phase I clinical trial, and also shows immunogenicity in the ongoing NOA21 phase I clinical trial, where combination of the vaccine with the PD-L1 inhibitor avelumab is tested in patients with IDH1 mutant recurrent gliomas.\r\nTo gain a deeper understanding of the T cell response following vaccination, I established “epitope specific expansion cultures” (ESPEC) with “subsequent identification of TCRs” (SUIT) to pre-select TCRs for testing their reactivity. Based on ESPEC, a total of 120 CD4-derived TCRs from seven patients were selected for cloning and functional validation in in vitro co-culture assays, of which 106 were shown to be reactive. IDH1R132H-reactive clones were found to be enriched intratumorally as compared to concurrently sampled blood of vaccinated patients. 49 identified IDH1R132H-reactive TCRs showed in vitro reactivity against wildtype IDH1 if the peptide was used at supraphysiological concentrations, but not at lower peptide concentrations as shown for a selection of TCRs. Analysis of HLA restrictions indicates promiscuous binding of the IDH1R132H peptide to HLA-DR alleles, which is in accordance with the high immunogenicity of the vaccine in an HLA-diverse patient population.\r\nContrary to what was reported so far, I also observed CD8+ T cell responses against IDH1R132H in two of three tested patients. Mass spectrometry confirmed presentation of short IDH1R132H peptides on HLA-B*07:05 and HLA-B*35:01, with these HLA alleles being representative for 12.3% of the German population taking HLA supertype families into account. Using ESPEC-SUIT, one CD8-derived TCR reactive against IDH1R132H was identified. The TCR was found to infiltrate the tumor, making up 0.24% of the T cell repertoire and being positive for granulysin, which could be an indication of cytotoxic function. Extending the screening for reactive CD8-derived TCRs to further patients is planned to gain deeper insights into the vaccine-induced immune response.\r\nESPEC-SUIT was also used to identify TCRs reactive to patient-individual antigens. Private neoepitopes, both from SNVs and fusion events, were predicted based on whole exome and RNA sequencing data of the tumor, and used to stimulate autologous PBMCs. ESPEC cultures were performed with individual peptides or in peptide pools to screen for reactive TCRs against 18-189 peptides per patient. For a colon carcinoma patient (POC-001), 25 of 28 screened TCRs were reactive, covering reactivities against 14 of 18 predicted neoantigens. For patient POC-004 with liposarcoma (12 mutations included for testing) and patient POC-005 with metastatic melanoma (50 mutations included for testing), several hundred expanded TCRs have been identified and await functional validation. While POC-001 and POC-004 were vaccinated with long peptides representative for a selection of mutanome encoded antigens prior to ESPEC, patient POC-005 did not receive such vaccination, but T cells were found to be expanded in post-ESPEC cultures for three short peptides and 32 long peptides, underlining the sensitivity of the ESPEC approach.\r\nTo further understand the role of anti-tumor T cell immunity in brain tumors, tumor infiltrating lymphocytes (TILs) of non-vaccinated patients were used for single cell sequencing with the overall aim to identify a gene signature that can distinguish between tumor-reactive clones and non-reactive bystander clones for future antigen-agnostic identification of reactive TCRs. While gene signatures based on published markers identified no TCRs in primary brain tumors, a total of 46 reactive TCRs were identified when screening the top 83 TIL clonotypes from a melanoma brain metastasis tumor sample. On basis of this data, Chin Leng Tan (DKFZ Heidelberg, Germany) is working on establishing a gene signature, which could be used for antigen-agnostic discovery of reactive TCRs.\r\nCollectively, this thesis presents two strategies on how putatively tumor-reactive TCRs can be selected and validated on a larger scale. Screening for IDH1R132H-reactive TCRs allowed to gain deeper insights into the peripheral and intratumoral immune response elicited upon vaccination with the IDH1R132H peptide vaccine in brain tumor patients. The antigen-targeted approach was shown to be highly efficient for selecting reactive TCRs, which could potentially help to implement TCR-transgenic T cell therapies in patient care."^^ . "2023" . . . . . . . "Katharina"^^ . "Lindner"^^ . "Katharina Lindner"^^ . . . . . . "Identification and validation of neoepitope-specific T cell receptors for glioma immunotherapy (PDF)"^^ . . . "THESIS_KatharinaLindner_FINAL-VERSION_PDF-A.pdf"^^ . . . "Identification and validation of neoepitope-specific T cell receptors for glioma immunotherapy (Other)"^^ . . . . . . "lightbox.jpg"^^ . . . "Identification and validation of neoepitope-specific T cell receptors for glioma immunotherapy (Other)"^^ . . . . . . "preview.jpg"^^ . . . "Identification and validation of neoepitope-specific T cell receptors for glioma immunotherapy (Other)"^^ . . . . . . "medium.jpg"^^ . . . "Identification and validation of neoepitope-specific T cell receptors for glioma immunotherapy (Other)"^^ . . . . . . "small.jpg"^^ . . . "Identification and validation of neoepitope-specific T cell receptors for glioma immunotherapy (Other)"^^ . . . . . . "indexcodes.txt"^^ . . "HTML Summary of #33910 \n\nIdentification and validation of neoepitope-specific T cell receptors for glioma immunotherapy\n\n" . "text/html" . . . "000 Allgemeines, Wissenschaft, Informatik"@de . "000 Generalities, Science"@en . . . "500 Naturwissenschaften und Mathematik"@de . "500 Natural sciences and mathematics"@en . . . "570 Biowissenschaften, Biologie"@de . "570 Life sciences"@en . . . "600 Technik, Medizin, angewandte Wissenschaften"@de . "600 Technology (Applied sciences)"@en . .