Purpose:

Tumor antigens are central to antitumor immunity. Recent evidence suggests that peptides from noncanonical (nonC) aberrantly translated proteins can be presented on HLA-I by tumor cells. Here, we investigated the immunogenicity of nonC tumor HLA-I ligands (nonC-TL) to better understand their contribution to cancer immunosurveillance and their therapeutic applicability.

Experimental Design:

Peptides presented on HLA-I were identified in 9 patient-derived tumor cell lines from melanoma, gynecologic, and head and neck cancer through proteogenomics. A total of 507 candidate tumor antigens, including nonC-TL, neoantigens, cancer-germline, or melanocyte differentiation antigens, were tested for T-cell recognition of preexisting responses in patients with cancer. Donor peripheral blood lymphocytes (PBL) were in vitro sensitized against 170 selected nonC-TL to isolate antigen-specific T-cell receptors (TCR) and evaluate their therapeutic potential.

Results:

We found no recognition of the 507 nonC-TL tested by autologous ex vivo expanded tumor-reactive T-cell cultures while the same cultures demonstrated reactivity to mutated, cancer-germline, or melanocyte differentiation antigens. However, in vitro sensitization of donor PBL against 170 selected nonC-TL, led to the identification of TCRs specific to three nonC-TL, two of which mapped to the 5′ UTR regions of HOXC13 and ZKSCAN1, and one mapping to a noncoding spliced variant of C5orf22C. T cells targeting these nonC-TL recognized cancer cell lines naturally presenting their corresponding antigens. Expression of the three immunogenic nonC-TL was shared across tumor types and barely or not detected in normal cells.

Conclusions:

Our findings predict a limited contribution of nonC-TL to cancer immunosurveillance but demonstrate they may be attractive novel targets for widely applicable immunotherapies.

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©2023 The Authors; Published by the American Association for Cancer Research
2023
American Association for Cancer Research
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Supplementary data

Supplementary Data S3

Tumor cell line HLA-I typing and tumor type

- xlsx file
Supplementary Data S1

Includes a large list of all the peptides and details of the HLA-I ligands identified at <=0.01 FDR and >=30 ALC score

- xlsx file
Supplementary Data S2

Includes the list of peptides derived from NSM identified at 0.02 FDR and ALC>=30

- xlsx file
Supplementary Table S1

Show patient characteristics and relevant experimental information

- docx file
Supplementary Figures S1-S15

Supplementary Figure 1. Validation of HLA-I ligand tumor antigen candidates with synthetic peptides Supplementary Figure 2. Validation of selected HLA-I ligand tumor antigen candidates identified in Mel-1 and Mel-3 with isotope-labeled peptides Supplementary Figure 3. Frequency of nonC peptides detected in HLA-I. Supplementary Fig. 4. HLA-I allele binding motif of all patients included in the study. Supplementary Figure 5. A fraction of nonC-TL can be detected in publicly available immunopeptidomics datasets from human tumor biopsies Supplementary Figure 6. Evaluation of pre-existing T-cell responses to candidate tumor antigens in cancer patients Supplementary Figure 7. Enrichment of antigen-specific T cells from cancer patients by flow cytometry-based sorting. Supplementary Figure 8. Deconvolution of peptide pools containing recognized CGA or melanoma-associated antigens. Supplementary Figure 9. Functional avidity and specificity of neoantigen-specific T cells isolated from cancer patients. Supplementary Figure 10. HLA restriction element and recognition of autologous tumor cell lines by antigen-specific T cells isolated from cancer patients. SupplementaryFigure 11. Genomic location of the three immunogenic nonC-TL identified through IVS. Supplementary Figure 12. Recognition of the predicted ORFs encoding the immunogenic nonC-TL by TCR transduced cells. Supplementary Figure 13. Loss of recognition of TCL transfected with Cas9-sgRNA targeting the predicted 5’U-HOXC13 ORF. Supplementary Figure 14. Assessment of expression and translation of the immunogenic nonC-TL in human healthy cells. Supplementary Figure 15. NonC-TL are the main source of candidate tumor antigens identified by proteogenomics but tumor-reactive T cells preferentially recognize neoantigens derived from NSM.

- pdf file