Development of a Neoepitope Prioritization Platform to Identify Immunogenic Neoantigens from Oncogenic FGFR3-TACC3 Fusion in Glioblastoma
type: CreativeWork
description: This study investigates the FGFR3-TACC3 fusion as a tumor-specific neoantigen source and establishes an end-to-end computational and experimental framework for the identification, prioritization, and validation of immunogenic neoantigens derived from chimeric RNAs. The FGFR3-TACC3 fusion, generated through an intrachromosomal rearrangement on chromosome 4, produces a unique junctional sequence that constitutively activates oncogenic signaling while remaining absent in normal tissues, thereby representing a highly specific therapeutic target. To evaluate its neoantigenic potential, 27 peptides (9-11 mers) spanning the fusion junction were assessed for HLA class I binding using NetMHCpan 4.1, MixMHCpred 3.0, and BigMHC 1.0, alongside an in vitro flow cytometry-based binding assay. Eleven peptides, including 10 fusion-derived neoantigens, were validated as strong binders to HLA-A*01:01. NetMHCpan 4.1 demonstrated superior predictive performance, exhibiting a significant inverse correlation with experimental binding and high classification accuracy (AUC = 0.9669). An experimentally derived threshold (EL Rank < 32.7) achieved maximal sensitivity while maintaining high specificity. This was further refined to a more stringent cutoff (EL Rank < 25.7) using an expanded multi-fusion dataset comprising FGFR3-TACC3, CD74-NRG1, and ESR1-PRKN across multiple HLA alleles, improving predictive robustness and specificity. Functional validation using ELISpot assays demonstrated that 9 of 10 fusion derived in vitro validated binders induced significant IFN-γ secretion from CD8 T cells, confirming their immunogenicity. Cross-reactivity assessment using the CrossDome pipeline identified two neoantigens with potential T cell off-target effects, enabling safety-informed prioritization of candidate peptides. Integration of single-cell transcriptomic and TCR repertoire analyses revealed that peptide stimulation induces coordinated activation of immune programs, including T cell activation,…
abstract: This study investigates the FGFR3-TACC3 fusion as a tumor-specific neoantigen source and establishes an end-to-end computational and experimental framework for the identification, prioritization, and validation of immunogenic neoantigens derived from chimeric RNAs. The FGFR3-TACC3 fusion, generated through an intrachromosomal rearrangement on chromosome 4, produces a unique junctional sequence that constitutively activates oncogenic signaling while remaining absent in normal tissues, thereby representing a highly specific therapeutic target. To evaluate its neoantigenic potential, 27 peptides (9-11 mers) spanning the fusion junction were assessed for HLA class I binding using NetMHCpan 4.1, MixMHCpred 3.0, and BigMHC 1.0, alongside an in vitro flow cytometry-based binding assay. Eleven peptides, including 10 fusion-derived neoantigens, were validated as strong binders to HLA-A*01:01. NetMHCpan 4.1 demonstrated superior predictive performance, exhibiting a significant inverse correlation with experimental binding and high classification accuracy (AUC = 0.9669). An experimentally derived threshold (EL Rank < 32.7) achieved maximal sensitivity while maintaining high specificity. This was further refined to a more stringent cutoff (EL Rank < 25.7) using an expanded multi-fusion dataset comprising FGFR3-TACC3, CD74-NRG1, and ESR1-PRKN across multiple HLA alleles, improving predictive robustness and specificity. Functional validation using ELISpot assays demonstrated that 9 of 10 fusion derived in vitro validated binders induced significant IFN-γ secretion from CD8 T cells, confirming their immunogenicity. Cross-reactivity assessment using the CrossDome pipeline identified two neoantigens with potential T cell off-target effects, enabling safety-informed prioritization of candidate peptides. Integration of single-cell transcriptomic and TCR repertoire analyses revealed that peptide stimulation induces coordinated activation of immune programs, including T cell activation,…
author: Thevasagayampillai, Shiyanth 1994-
language: English
keywords: TCR, Cancer, Vaccine, FGFR3-TACC3, Fusion, Neoantigen
date published: 2026-05
date modified: 2026-07-24T02:32:32Z
provider: University of Houston
publisher: Networked Digital Library of Theses and Dissertations
type: WebPage
source organization: University of Houston

PropertyValue
property ID: Handle
value: https://hdl.handle.net/10657/21509