Discovery, selection, and proof of concept evaluation of novel TCR selective targets in Ewing sarcoma

Thesis

Ewing sarcoma (EwS) is a rare and aggressive malignancy of bone and soft tissue that primarily affects adolescents and young adults. Although multimodal therapy results in a 5-year overall survival rate of about 70%, the outlook for patients with metastatic or relapsed disease remains poor, and no immunologically targeted therapies have been developed in decades. Unlike many cancers caused by the accumulation of various mutations, EwS is driven by a single characteristic fusion event: a chromosomal translocation between EWSR1 and ETS family transcription factors, most often EWSR1– FLI1. While breakpoints can vary, a small set of transcripts typically accounts for most EwS cases. These fusion proteins that define the disease have largely remained “undruggable” by traditional methods.

Here, I outline a systematic pipeline for discovering and functionally validating EwS-specific T cell receptors (TCRs) that target fusion-derived neoantigens. Wholetranscriptome sequencing data from over 140 EwS tumours (both public and in-house) were analysed using machine learning algorithms to predict breakpoint-spanning neoantigens and their restricting HLA alleles. Recurrent candidate peptides were synthesised and used to stimulate T cells from healthy donors with high-affinity HLA subtypes. The antigen-responsive T cells were enriched, single-cell profiled, and their paired TCRs reconstructed.

Using this approach, I identified 19 previously novel TCRs that recognise common EWSR1–ETS breakpoint peptides. Functional testing in Jurkat triple-reporter assays demonstrated 70% reactivity, with representative clones exhibiting EC₅₀ values of approximately 500 nM. Ongoing studies are determining MHC restriction and peptide specificity, paving the way for targeted therapy against EwS fusion neoantigens.

This study is the first large-scale discovery and functional validation of fusionneoantigen–specific TCRs in EwS, establishing oncogenic breakpoint peptides as targetable, disease-defining immunological markers. By demonstrating MHC-restricted antigen presentation and specific TCR recognition, I offer a mechanistic framework for designing personalised mRNA vaccine sequences and evaluating them in preclinical in vivo studies. Overall, these findings lay the foundation for companion diagnostics, mRNA vaccine strategies, and next-generation TCR-based immunotherapies, including adoptive cell therapies and bispecifics such as ImmTACs that can extend the therapeutic reach of these fusion-specific receptors.

Authors: Qian, Q

• DOI: 10.5287/ora-agzpgnnvj
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: T cell receptor; Ewing Sarcoma; neoantigen
• Subjects: Cancer--Immunotherapy