Paper Details

ABSTRACT

Introduction: Novel unannotated open reading frames (nuORFs) are emerging as an untapped source of non-mutated tumor antigens. Their ability to generate immunogenic peptides could be especially valuable in pediatric cancers such as T-cell acute lymphoblastic leukemia (T-ALL), where a low mutational burden limits conventional neoantigens for immune targeting. Although chemotherapy induces remission in many patients, relapsed T-ALL remains difficult to treat, and effective immunotherapies are still lacking. We therefore investigated whether nuORFs could reveal a new class of targetable antigens in T-ALL.

Results:

By integrating HLA class I immunopeptidomics with transcriptomic profiling of 20 T-ALL cell lines and patient-derived primagrafts, we identified 637 presented nuORF-derived peptides. These peptides arose from diverse genomic regions, including introns, noncoding RNAs, intergenic regions, endogenous retroelements, untranslated regions, and alternative reading frames within protein-coding genes. These findings demonstrate that nuORFs expand the antigen repertoire of T-ALL cells.

We hypothesized that nuORF-derived peptides with restricted expression in healthy tissues would be the most promising immunotherapeutic targets. We therefore examined source-region RNA expression in matched T-ALL primagrafts and retained peptides whose source regions were transcriptionally detected. Using the TARGET T-ALL cohort (n=265), we determined the expression threshold associated with a >5% probability of HLA-I peptide presentation. We retained 97 candidates whose expression in normal thymocytes was below this threshold and prioritized 40 significantly enriched in T-ALL primagrafts and the TARGET cohort compared with normal thymocytes. Because expression in other cell populations may limit therapeutic targeting, we excluded candidates whose expression exceeded the presentation threshold in medullary thymic epithelial cells, lymphoid and myeloid populations, or GTEx tissues. This approach identified 5 antigens with limited expression across normal cells and tissues. Each candidate was significantly overexpressed in 3 independent primary T-ALL patient cohorts (n=327) compared with healthy cells and tissues. Across the three cohorts, 100% of patients expressed ≥1 candidate, 99.7% expressed ≥2, 98.5% expressed ≥3, 90.2% expressed ≥4, and 59.0% expressed all 5 candidates. Together, these findings demonstrate broad expression across patients and support their classification as tumor-associated antigens (TAAs).

All 5 candidates were predicted to be immunogenic by PRIME, which integrates HLA class I binding with T-cell receptor recognition propensity. For each candidate, the predicted presenting HLA allele was expressed in the corresponding T-ALL sample.

Finally, we evaluated the immunogenicity of two tumor associated antigens (TAA1 and TAA2). PBMCs from healthy donors expressing HLA-A*03:01 or HLA-B*55:01 were stimulated with TAA1 or TAA2, respectively, and expanded in vitro for 14–27 days. TAA1 elicited peptide-specific responses in 3 donors, with a 3- to 13-fold increase in IFN-γ ELISpot responses relative to irrelevant peptide controls. Flow cytometry further demonstrated at least a 2-fold increase in polyfunctional IFN-γ+TNF-α+ CD8 T cells and degranulating CD107a+IFN-γ+ or CD107a+TNF-α+ CD8 T cells. TAA2 elicited a response in 1 donor, producing a >50-fold increase in IFN-γ ELISpot responses, a 5-fold increase in IFN-γ+TNF-α+ CD8 T cells, an 8-fold increase in CD107a+IFN-γ+ CD8 T cells, and a 5-fold increase in CD107a+TNF-α+ CD8 T cells. Together, these results demonstrate that naturally presented nuORF-derived peptides can elicit functional, antigen-specific CD8 T-cell responses characterized by both cytokine production and degranulation.

Conclusions: Our findings expand the antigenic landscape of T-ALL by revealing nuORFs as an underexplored source of targetable tumor antigens. Importantly, they provide a framework for uncovering non-canonical antigens in low-mutational-burden cancers and developing new immunotherapeutic strategies for pediatric leukemia.