Identification of TTLL8, POTEE, and PKMYT1 as immunogenic cancer-associated antigens and potential immunotherapy targets in ovarian cancer

Abstract

Most high-grade serous ovarian cancers (OC) do not respond to current immunotherapies. To identify potential new actionable tumor antigens in OC, we performed immunopeptidomics on a human OC cell line expressing the HLA-A02:01 haplotype, which is commonly expressed across many racial and ethnic groups. From this dataset, we identified TTLL8, POTEE, and PKMYT1 peptides as candidate tumor antigens with low expression in normal tissues and upregulated expression in OC. Using tissue microarrays, we assessed the protein expression of TTLL8 and POTEE and their association with patient outcomes in a large cohort of OC patients. TTLL8 was found to be expressed in 56.7% of OC and was associated with a worse overall prognosis. POTEE was expressed in 97.2% of OC patients and had no significant association with survival. In patient TILs, increases in cytokine production and tetramer-positive populations identified antigen-specific CD8 T cell responses, which were dependent on antigen presentation by HLA class I. Antigen-specific T cells triggered cancer cell killing of antigen-pulsed OC cells. These findings suggest that TTLL8, POTEE, and PKMYT1 are potential targets for the development of antigen-targeted immunotherapy in OC.

Keywords: Cancer immunology; immunotherapy; ovarian cancer; tumor antigen.

Figure 1.

Immunopeptidomics analysis of the HLA-A02:01 OVCAR-5 cell line. (a) Schematic overview of the immunopeptidomics approach. OVCAR-5 cells were collected and lysed, followed by the immunoprecipitation and purification of HLA-peptide complexes. HLA-bound peptides were eluted and identified by LC-MS/MS. Created with BioRender.com. (b) Distribution of identified peptide length in HLA-A02:01-positive OVCAR-5 cell line. (c) Deconvolution of peptide data according to the predicted binding to each HLA allele using MHCMotifDecon. (d) Experimentally derived HLA motifs in OVCAR-5 using MHCMotifDecon. (e) Assessment of peptide binding affinity using NetMHCcons . Peptides were filtered to include only the top 2% HLA binders.

Figure 2.

Immunopeptidomics identifies putative CT antigens in ovarian cancer. (a) The predicted binding affinities of CT peptides were evaluated for their binding affinity to HLA-A02:01 using NetMHCcons. TTLL8, PKMYT1, PKMYT1–2, and POTEE peptides are indicated. The dashed line indicates 5,000 nM. (b) mRNA expression of POTEE, TTLL8, and PKMYT1 across normal tissues in the GTEx Portal (www.Gtexportal.org). (c) Data from the TCGA TARGET GTEx study was used to compare mRNA expression of POTEE, TTLL8, and PKMYT1 in primary tumors, recurrent tumors, and normal ovaries. Data was downloaded using the UCSC Xena Browser (). The rank prediction of all possible (d) POTEE, (e) TTLL8, and (f) PKMYT1 peptides from each whole protein. The rank represents the relative binding strength of peptides to HLA-A02:01 and was analyzed using NetMHCpan for each protein. Peptides identified by immunopeptidomics are indicated.

Figure 3.

TTLL8 and POTEE are frequently expressed in clinical ovarian cancer. (a) Representative images of immunohistochemistry-based evaluation of TTLL8 and POTEE protein expression in indicated tissues. (b, c) Kaplan-Meier curves showing overall survival (b) and progression-free survival (c) comparing patients with no TTLL8 expression (score: 0) versus patients with TTLL8 expression (scores: 1, 2, or 3) in their tumors. (d, e) Kaplan-Meier curves showing overall survival (d) and progression-free survival (e) comparing patients with no or little POTEE expression (score: 0 or 1) versus patients with high POTEE expression (scores: 2 or 3) in their tumors.

Figure 4.

CT antigens activate peptide-specific T cell responses in patient TILs. (a, b) Intracellular cytokine staining (ICS) for IFNγ in CD8 T cells from HLA-A02:01+ patients 1 and 2 and (C, D) ICS for TNFα in CD8 T cells from HLA-A02:01+ patients 1 and 2 with HGSOC. T cells were stimulated with each indicated peptide prior to staining and assessed via flow cytometry. (e, f) Tetramer staining of CD8 T cells from HLA-A02:01+ patient 1. Peptide-expanded T cells were stained with custom pre-labeled tetramers containing TTLL8, PKMYT1, PKMYT1–2, or POTEE peptides.

Figure 5.

CT antigens activate T cell-mediated killing of ovarian cancer cells. (a) Assessment of peptide-specific T cell cytotoxicity at 96-hour incubation with peptide-pulsed OVCAR-5 cells at the indicated E:T ratios. Data is normalized to OVCAR-5 cells alone at 96-hour. ****p < 0.0001. (b) Representative images of the tumor cell killing assay after 96 hours at a 10:1 E:T ratio. The 0:1 ratio represents OVCAR-5 alone without TILs, establishing the baseline confluency. Scale bar: 250 µm. (c) HLA class I blocking assay. ICS for IFNγ and TNFα in CD8 T cells from two HLA-A02:01+ patients with ovarian cancer. CD8 T cells were pre-activated with the TTLL8 peptide and then co-cultured with TTLL8-pulsed OVCAR-5 for 8 hours with W6/32 or IgG control prior to staining. An HLA-A02:01-restricted HIV peptide was used as the negative control.