Open AccessArticle
Immunogenicity of Non-Mutated Ovarian Cancer-Specific Antigens
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Leslie Hesnard, Catherine Thériault, Maxime Cahuzac, Chantal Durette, Krystel Vincent, Marie-Pierre Hardy, Joël Lanoix, Gabriel Ouellet Lavallée, Juliette Humeau, Pierre Thibault and Claude Perreault
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Abstract
Epithelial ovarian cancer (EOC) has not significantly benefited from advances in immunotherapy, mainly because of the lack of well-defined actionable antigen targets. Using proteogenomic analyses of primary EOC tumors, we previously identified 91 aberrantly expressed tumor-specific antigens (TSAs) originating from unmutated genomic sequences.
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Epithelial ovarian cancer (EOC) has not significantly benefited from advances in immunotherapy, mainly because of the lack of well-defined actionable antigen targets. Using proteogenomic analyses of primary EOC tumors, we previously identified 91 aberrantly expressed tumor-specific antigens (TSAs) originating from unmutated genomic sequences. Most of these TSAs derive from non-exonic regions, and their expression results from cancer-specific epigenetic changes. The present study aimed to evaluate the immunogenicity of 48 TSAs selected according to two criteria: presentation by highly prevalent HLA allotypes and expression in a significant fraction of EOC tumors. Using targeted mass spectrometry analyses, we found that pulsing with synthetic TSA peptides leads to a high-level presentation on dendritic cells. TSA abundance correlated with the predicted binding affinity to the HLA allotype. We stimulated naïve CD8 T cells from healthy blood donors with TSA-pulsed dendritic cells and assessed their expansion with two assays: MHC-peptide tetramer staining and TCR Vβ CDR3 sequencing. We report that these TSAs can expand sizeable populations of CD8 T cells and, therefore, represent attractive targets for EOC immunotherapy.
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Tables S1 and S3 for details on modules and donors, respectively). Each module is color-coded and annotated with its respective HLA and the number of TSAs. (
b) Bars depict the peptide copy number per cell detected by MS analyses for nine control peptides known as immunogenic, at t = 0 h after moDCs pulsing with synthetic peptides (module 12;
Table S4). The positive control peptides were analyzed for every donor included in these MS experiments, expressing the proper HLA (eight donors, three to four replicates for each HLA allotype of interest; see
Table S3 for details on donors). (
c) Peptide copy numbers obtained at t = 0 h after pulsing were compared between TSAs and positive control peptides using a non-parametric
t-test (Mann–Whitney), showing no significant difference (ns) between these two groups. Each symbol represents the mean of replicates per peptide. (
d) Correlation between the TSAs abundance detected by MS (in fmol) at t = 0 h after pulsing and the predicted binding affinity obtained from NetMHCpan_4.1 (low value in nM means high affinity). ****
p < 0.0001. (
e) The peptide copy number per cell was obtained from MS analysis for the 48 TSAs at t = 24 h after moDCs pulsing with peptide modules. Panels (
a,
b,
e): ** detected and precisely quantified in at least two replicates, * detected but not precisely quantified in at least two replicates, no star = detected in only one replicate. (
f) The abundance of peptides detected by MS at t = 0 h after pulsing (in fmol) for peptides detected (at least one replicate) or not by MS 24 h after pulsing. The two groups were compared using non-parametric
t-tests (Mann–Whitney), ***;
p-value = 0.0002. (
g) Comparison of peptide copy number/cell detected by MS at t = 0 h and t = 24 h after peptide pulsing for each peptide detected in at least one replicate, 24 h after peptide pulsing. The two groups were compared using a Wilcoxon matched-pairs signed rank test, ***;
p-value = 0.0005. (
h) Correlation between the TSAs abundance detected by MS (in fmol) at t = 24 h and the predicted half-life stability of peptide–HLA complexes obtained from NetMHCstabpan_1.0. **;
p-value = 0.0074. Panels (
d,
h): Spearman correlation coefficients and
p values are indicated below the titles.
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38]. Only clonotypes classified as significantly expanded in one peptide-pulsed condition (compared to the unpulsed condition) are represented. (
a) Frequencies of expanded clonotypes among all clonotypes sequenced in each sample obtained with donors D15, (
b) D16, (
c) D17, (
d) D22, and (
e) D33. Each symbol represents the frequency for one expanded clonotype in the peptide-pulsed condition (circles) and its respective frequency in the unpulsed condition (triangles). (
a–
e) As positive controls, known immunogenic peptides were included according to the HLA genotype of each donor (MelanA binding HLA-A*02:01 or ctl7 binding HLA-B*08:01, highlighted in grey). Each TSA was tested in one donor (panels (
a–
d)), except peptides from module 2 (HLA-A*02:01, TSAs 6 to 10 in orange) tested in 2 different donors (panels (
c,
e)). The number of significantly expanded clonotypes found per TSA or control peptide is represented above each sample. (
f) The sum of all expanded clonotype frequencies was calculated for each peptide condition (
Table S5). Each symbol represents one peptide condition, and results obtained from TSAs were compared to positive control peptides. (
g) Fold change of expansion (frequency in [peptide-pulsed/unpulsed] condition) is represented for each significantly expanded clonotype (each point). Results are grouped by donors and compared between TSAs and positive control conditions. (
f,
g) pink boxes represent TSAs; grey boxes represent positive control peptides. (
h) PRIME score prediction of T-cell recognition (
Table S6): immunogenic control peptides extracted from the literature (14), the 48 TSAs analyzed in this study, and a selection of immunogenic and non-immunogenic peptides used to train PRIME2.0 (48 peptides each). Pink represents the TSAs, grey represents the groups of immunogenic peptides, and white represents the non-immunogenic peptides. (
f–
h) statistical analyses were done using non-parametric
t-tests (Mann–Whitney), where ns stands for not significant; ****
p-value < 0.0001.
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Table S3) and with 2 or 3 different experimental conditions for peptide loading (
a–
c). The unpulsed moDCs condition is used as a negative control for specific expansion. (
d) The positive control condition assessed the proportion of tetramer-positive cells following stimulation with moDCs pulsed with ctl6, ctl7, or MelanA (according to the donor HLA genotype) in each donor from panels (
a–
c). Panels (
a–
d): The frequencies of tetramer-positive CD8 T cells (in %) are represented following a blue intensity gradient. Tetramer-positive CD8 T cells were only considered expanded and described in the figure if expansion folds ≥ 10 compared to the negative control condition. (
e) Frequencies of tetramer-positive CD8 T cells detected after functional expansion for TSAs and positive control peptides. (
f) Compared to the unpulsed condition, the expansion fold of tetramer-positive CD8 T cells is represented for each TSA and positive control peptide. (
g) The expansion fold of tetramer-positive CD8 T cells for all TSAs (in blue) was compared to the ctl7/ctl6 and MelanA groups. Individual points represent replicates of significantly expanded ag-specific T cells. (
h) Frequencies of tetramer-positive CD8 T cells obtained in the unpulsed condition (see
Section 2.6.2 for details). Panel (
e–
h): Only peptides for which expansion was observed in panels (
a–
c) or (
d) are represented. (
i) PRIME score comparison between TSAs groups for which we detected (13) or not (26) T-cell expansion by tetramers, immunogenic epitopes from the literature (14), and a selection of immunogenic or non-immunogenic peptides used to train PRIME2.0 (48 peptides each) (
Table S6). Panels (
g–
i); groups were compared using non-parametric
t-tests (Mann–Whitney), where ns stands for not significant; ****
p-value <0.0001, ***
p-value = 0.0009 (panel (
g)) or
p-value = 0.006 (panel
i), and **
p-value = 0.0095 (panel (
h)).
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