Biology subjects

Genzel, N.

Publications and source records attributed to Genzel, N..

3 recordsLinked to original sources

bioRxiv2026

Tumor-versus-non-tumor T cell enrichment supported by single-cell and spatial phenotyping enables antigen-agnostic prioritization of candidate tumor-reactive TCRs

Introduction: Tumor-reactive T cell receptors (TCRs) are promising candidates for the manufacturing of safe and effective adoptive TCR-T cell therapies, but their efficient identification remains a major challenge. We previously established an antigen-agnostic TCR-repertoire analysis workflow that prioritizes candidate TCRs based on intratumoral clonal expansion and high tumor-to-non-tumor (T/N) frequency ratios. Here, we investigated whether T/N-selected clonotypes show independent transcriptional and spatial features consistent with tumor association. Methods: Bulk and single-cell TCR repertoire sequencing of paired tumor and adjacent non-tumor tissues was used to identify clonotypes with high T/N ratios across 15 cancer cases, comprising six non-small cell lung cancers, five pancreatic cancers, two breast cancers, and two colorectal cancers. Candidate clonotypes were further characterized by paired single-cell TCR and gene expression profiling and, in two PDAC cases, by Xenium spatial transcriptomics to assess their cellular phenotype, spatial localization, and proximity to malignant cells. Results: High T/N ratios consistently marked T cell clonotypes with features of tumor reactivity across the studied solid tumor entities. The analysis included Cluster-A, a lead T cell cluster comprising highly similar TCRs shared among 14 HLA-A*02:01-positive patients in the present cohort. Cluster-A clonotypes were consistently enriched in tumor relative to matched non-tumor tissue, and previous functional studies demonstrated tumor recognition by Cluster-A members. In a focused analysis of a pancreatic cancer case, Cluster-A CD8 T cells were selectively detected in tumor tissue, preferentially localized near malignant epithelial cells, and displayed an antigen-experienced effector-memory phenotype with prominent expression of granzymes A and K (GZMA/GZMK). Importantly, in another PDAC case that lacked Cluster-A clonotypes, these features extended to independent clonotypes, revealing a continuous tumor-association axis in which increasing T/N ratios were associated with progressively closer localization to malignant cells and increased GZMA/GZMK expression. Thus, repertoire enrichment, spatial tumor proximity, and effector-associated transcriptional states converged across independent tumor-enriched clonotypes. Outlook: These findings support the T/N ratio as a robust primary criterion for identifying candidate tumor-reactive TCRs. Integration of single-cell and spatial transcriptomics provides supporting biological evidence of tumor association and a framework for prioritizing therapeutic TCR candidates when functional validation is limited.

immunology↗

bioRxiv2025

A new clustering approach identifies tumor-specific common TCRs with pan-cancer reactivity

Tumor-specific T-cells are key in combating cancer as shown in adoptive cell therapy with tumor infiltrating lymphocytes (TILs) and checkpoint inhibitor therapy. Studies in many types of cancer have shown that preexisting tumor reactive T-cells are not only tumor-but typically also patient-specific, requiring personalized treatment options. For viral infections, public T-cell receptors (TCRs) with substantial sequence homologies suggest shared immune-dominant targets in human leucocyte antigen (HLA)-matched individuals. We hypothesized that also in the complex TCR repertoires of tumors subsets of tumor-specific TCRs exist that can be found in different patients with identical or near identical TCRs. This paper presents a TCR-V(D)J-sequence clustering approach identifying clusters of tumor-specific common TCRs mainly from TILs of non-small cell lung cancer (NSCLC) patients. Using two TCR-clusters as examples, we show that T-cells engineered genetically to only express those common TCRs recognized HLA-matched allogeneic tumor cell lines in a cluster typical manner. Recognition of allogeneic tumors was dependent on the HLA allele inferred by the cluster and could be blocked by HLA antibodies. In addition to NSCLC, TCR repertoire analyses in pancreatic ductal adenocarcinoma and a smaller number of breast and colorectal cancer samples revealed TCRs highly homologous or even identical to NSCLC cluster TCRs. TCR-T cells expressing TCRs from these tumors assigned to a specific cluster recognized allogeneic tumor lines in the expected cluster-typical manner. These findings suggest a pan-cancer therapeutic potential of tumor-specific common TCRs.

immunology↗

bioRxiv2024

T-cell receptors identified by a personalized antigen-agnostic screening approach target shared neoantigen KRAS Q61H

Adoptive cell therapy (ACT) with TCR-engineered T-cells represents a promising alternative to TIL- or CAR-T therapies for patients with advanced solid cancers. Currently, selection of therapeutic TCRs critically depends on knowing the target antigens, a condition excluding most patients from treatment. Direct antigen-agnostic identification of tumor-specific T-cell clonotypes and TCR-T manufacturing using their TCRs can advance ACT for patients with aggressive solid cancers. We present a method to identify tumor-specific clonotypes from surgical specimens by comparing TCR{beta}-chain repertoires of TILs and adjacent tissue-resident lymphocytes. In seven NSCLC-patients, tumor-specific clonotypes were selected based on TIL-abundance and high tumor-to-nontumor frequency ratios. In two of the patients, we demonstrate that predicted tumor-specific clonotypes reacted against autologous tumors. In a third patient, we engineered TCR T-cells with four candidate tumor-specific TCRs that showed reactivity against the patients tumor and HLA-matched NSCLC cell lines. The TCR-T cells were then used to screen for candidate neoantigens and aberrantly expressed antigens. Three TCRs recognized recurrent driver-mutation KRAS Q61H-peptide ILDTAGHEEY presented by HLA-A*01:01. The TCRs were also dominant in a tumor relapse, one was found in cell free DNA. The finding of homologous TCRs in independent KRAS Q61H-positive cancers suggests a therapeutic opportunity for HLA-matched patients with KRAS Q61H-expressing tumors.

immunology↗