A total of 5,143 cancer cases were analyzed in the High-tech Omics-based Patient Evaluation (HOPE) project, which has been conducted in Shizuoka Cancer Center since 2014 using multiomics analyses, such as whole-exome sequencing (WES) and gene expression profiling (27). For the WES analysis, somatic mutations were identified by comparing data on tumors and corresponding blood samples. Total exonic mutations for each sequenced tumor included single-nucleotide variants and indel/frameshift mutations. All nonsynonymous mutations detected in the cases were collected and screened.

We used the NetMHCpan 4.1 (Immune Epitope Database, http://www.iedb.org/) to assess HLA presentation of 8 to 11-mer peptide epitopes from cancer driver gene mutation to predict affinity (IC₅₀) on the basis of the binding affinity (BA) model or to predict rank scores (%Rank) on the basis of the eluted ligand (EL) model.

TISI human B-lymphoblastoid cell line (B-LCL) (CVCL_E851) with homozygous HLA-class-I loci (26) were supplied by Takara Shuzo Co., Ltd. (Otsu, Shiga, Japan), and we verified the HLA types by Sanger sequencing using SeCore SBT kit (#5300025, One Lambda, Thermo Fisher Scientific, Waltham, Massachusetts, USA). T2 cells were purchased from American Type Culture Collection (#CRL-1992, ATCC, Manassas, VA, USA). These cell lines were maintained in RPMI-1640 medium (#R8758, Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) supplemented with 10% (v/v) FBS (#10437, Gibco, Thermo Fisher Scientific, Waltham, Massachusetts, USA) and 1× penicillin-streptomycin (#151401222, Gibco). Newly constructed TAP2-KO HLA-ABC monoallelic cell clones were maintained in RPMI-1640 medium supplemented with 10% (v/v) FBS, 100 units/ml penicillin, 100 µg/ml streptomycin, 1 mM sodium pyruvate (#11360070, Gibco), 1× MEM nonessential amino acids (#11140050, Gibco), and 55 µg/ml 2-mercaptoethanol (#21985-023, Gibco).

Monoallelic cell cloning was performed using a PE-labeled anti-HLA-ABC monoclonal antibody (clone: G46-2.6, BD Biosciences, Franklin Lakes, New Jersey, USA) to stain HLA-ABC KO or knock-in (KI) cells and a FACSAria cell sorter (BD Biosciences) for sorting single cells to 96-well plates. For the MHC stabilization assay, we used PE-labeled anti-HLA-ABC antibody clone G46-2.6 diluted 1:10 and clone W6/32 (Dako Denmark A/S, Dako North America, Inc., Carpinteria, California, USA) diluted 10 µg/ml with FACSCanto flow cytometer (BD Biosciences) for analysis. PE-labeled anti-mouse Ig polyclonal antibody (#550589, BD Biosciences) was used as the secondary antibody. FlowJo software ver.8.8.7 (Tomy digital biology Co., Ltd. Tokyo, Japan) was used for analysis.

Using the CRISPR/Cas9 system (Invitrogen, Thermo Fisher Scientific), TAP2 and HLA-A, HLA-B, and HLA-C genes were knocked out in the TISI cell line using synthetic gRNAs (#A35510, Invitrogen) (Table SI). Each synthetic HLA-class-I allele cDNA (Genewiz, Tokyo, Japan) corresponding to a TISI-HLA-A*24:02:01:01-deleted site was transcribed to single-stranded DNA and isolated from double strand using agarose gel (#50070, Lonza) electrophoresis with ethidium bromide (#315-90051, Nippongene), and Purified single-stranded DNA was knocked in the TAP2- and HLA-ABC-KO TISI subclone using a paired sgRNA. Each KO and KI site was confirmed by PCR and Sanger sequencing. All sgRNAs and primer pairs are shown in Tables SI and SII.

Fmoc-amino acids were obtained from CEM Corp. (Matthews, NC, USA). Fmoc-amino acid-Wang-resins were obtained from Gyros Protein Technologies Inc. (Tucson, AZ, USA). H-Pro-Barlos resin and reagents for peptide synthesis, 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU), N,N'-diisopropylcarbodiimide (DIC), Oxyma Pure [Ethyl-2-cyano-2-(hydroxyimino)acetate], N,N-diisopropylethylamine (DIPEA), trifluoroacetic acid (TFA), triisopropylsilane (TIPS), and methylene chloride (DCM), were purchased from Watanabe Chem. IND., LTD. (Hiroshima, Japan). Piperidine, pyrrolidine, phenol and ethanedithiol (EDT) were obtained from Fujifilm Wako Pure Chem. Corp. (Tokyo, Japan). N,N'-Dimethylformamide (DMF), N-methyl-2-pyrrolidone (NMP), HPLC grade acetonitrile (ACN) and water were obtained from Kanto Chem. Co. Inc. (Tokyo, Japan).

Neoantigen peptides were synthesized via solid-phase chemistry using an Fmoc protection strategy. Starting from 0.05 mmol of Fmoc-amino acid-Wang-resin, protected peptide resins were constructed using 0.5 mmol of Fmoc-amino acids with tert-butyl-based side-chain-protecting groups on peptide synthesizers with the peptide synthesizer Tribute (Gyros Protein Technologies Inc.) at room temperature or with the microwave peptide synthesizer Liberty PRIME (CEM Corp.) at high temperature (105°C). As Fmoc removal (deprotection) and peptide bond formation (coupling) reagents, 20% piperidine/NMP and HATU/DIPEA for Tribute and 20% pyrrolidine/DMF and DIC/Oxyma for Liberty PRIME, respectively, were used. The synthesis of peptides bearing Pro at the C-terminus was carried out starting with H-Pro-Barlos-resin using a Tribute synthesizer. After final deprotection of the Nα-Fmoc group of the N-terminal amino acid, the partially protected peptide resin obtained was washed with DCM and MeOH and dried in vacuo. Removal of side-chain-protecting groups and cleavage of the resin were performed by treatment with a TFA-containing scavenger cocktail (TFA-phenol-EDT-thioanisole-H2O-TIPS; 90-2-2-2-2-2) at room temperature for 1.5-3 h. The product was precipitated with ether, collected by centrifugation, and dried in vacuo. The resulting crude peptides were purified using a 1525 Binary HPLC Pump equipped with a 2489UV/VIS detector (Waters Corp., Milford, MA, USA) with a YMC-Actus Triart C18 column, 5 µm, 20×150 mm (YMC Corp. Kyoto, Japan). The purity and mass spectrum of the purified peptides were analyzed using an Alliance e2695 HPLC System equipped with a 2489UV/VIS detector and an ACQITY QDa mass detector (Waters Corp.) with an XSelect CSH C18 column, 2.5 µm, 3×75 mm column (Waters Corp.). All synthetic peptides are shown in Table SIII.

The TAP2-KO monoallelic HLA class I-expressing TISI cell lines were precultured with 10,000 U interferon (IFN)-α (Smiferon300, Sumitomo Pharma, Osaka, Japan) to restore baseline HLA expression. 1×10⁵ HLA monoallelic cells were incubated with 25 µM peptide in RPMI 1640 medium with 3 µg/ml beta-2 microglobulin (#21985023, Sigma-Aldrich), 55 µM 2-melcaptoethanol and 0.1% BSA (#A7906, Sigma-Aldrich) at 37°C with 5% CO₂ for 18 h. Cells cultured with 0.5% DMSO without peptide were used as negative controls. Following incubation, the cells were stained with PE-labeled anti-HLA-ABC antibody (clone: G46-2.6) diluted 1:10 at RT (23°C) for 60 min, washed 3 times with PBS supplemented with 0.5% BSA and 2 mM EDTA at 4°C, and fixed with 0.4% paraformaldehyde in PBS with 0.1% BSA and 2 mM EDTA. HLA class I expression increase (ΔHLA) was measured using a FACSCanto flow cytometer (BD Biosciences). The ΔHLA was obtained by calculating the geometric mean of the fluorescence intensity (MFI) using the following formula:

ΔHLA (% control)={(Sample MFI)-(DMSO cont. MFI)}/{(DMSO cont. MFI)-(Unstained background MFI)} ×100

To determine the significance of differences between HLA expression levels, Kruskal-Wallis test followed by Steel's multiple comparison test was performed and P<0.05 was considered to indicate a statistically significant difference. The Kruskal-Wallis test, the Steel's multiple comparison test, and Pearson or Spearman correlation coefficients were calculated using EZR version 1.55 (Saitama Medical Center, Jichi Medical University, Saitama, Japan) (28). All means and standard deviations in the tables are based on three or more independent experiments.

The MHC stabilization assay was improved with the use of HLA-partially-null B-lymphoblastoid cell lines to confirm epitope peptide presentation by HLA molecules (21,22,24). To perform a more reliable MHC stabilization assay, HLA-A-, HLA-B-, HLA-C- and TAP2-KO clones derived from the TISI cell line were generated (Fig. S1, Fig. S2, Fig. S3) and the respective single HLA alleles were knocked in using the CRISPR/Cas9 system (Fig. S4). TISI cells were selected as they constitute a B-lymphocyte line with the ability to cross-present exogenous antigens on HLA molecules and carry homologous HLA-class-I loci. The HLA allele cDNA was knocked in the loci of HLA-A*24:02:01 (Fig. 1A and B). HLA class I expression was not detected in the TAP2, HLA-A, HLA-B and HLA-C-KO TISI clone (Fig. 1C), and the cells subjected to HLA class I cDNA knock-in exhibited HLA class I on their cell surface (Fig. 1D). Each clone was confirmed using genomic DNA sequencing.

The principle of the MHC stabilization assay is that peptides binding to HLA molecules stabilize and increase HLA expression levels on the cell surface. The first study was based on TAP2-deficient and HLA-A*02:01 monoallelic T2 cells and antibody clone W6/32, which can bind HLA-ABC associated with β-2 microglobulin (β2m) and requires 26°C culture conditions to prevent β2m dissociation (21,29). On the other hand, antibody clone G46-2.6 binds to HLA-ABC with or without β2m, which permits evaluation under physiological 37°C culture conditions, preventing low-affinity peptide binding at a low temperature (30) or the effect of bovine β2m in FBS (31). The present study therefore used clone G46-2.6, which enabled clearer observations. The addition of β2m to the assay culture supernatant was necessary for the detection of HLA by antibody clone W6/32, although it exerted a minimal effect on HLA expression (Figs. 2A and S5). In addition, interferons in the culture supernatant can easily alter HLA expression levels. Since HLA expression was extremely low immediately following knock-in cell cloning, the assays were performed after culturing the cells with IFN-α for 1 week to restore the HLA expression levels.

The results revealed increased HLA expression levels (ΔHLA) in MHC stabilization assays using the in-house-generated monoallelic HLA knock-in cell lines, although the predicted peptide/HLA IC₅₀ did not necessarily associate with ΔHLA (Fig. 2B). To assess the usability of the novel monoallelic HLA cells, the MHC stabilization assays were compared between the T2 cells and the monoallelic HLA-A*02:01 knock-in cell line using synthetic peptides (Table SIII). Although the knock-in cell line exhibited low HLA-A expression levels, it exhibited a good correlation with the T2 cells in the original method (Fig. 2C). Furthermore, there was a slight difference between the HLA-A*11:01 homo knock-in and hemi knock-in cell lines (Fig. 2D).

The HOPE cohort comprised 5,521 tumor specimens derived from 5,143 patients treated at the Shizuoka Cancer Center Hospital between January, 2014 and March, 2019. The major cancer types were colon (18.4%), lung (16.5%), rectal (13.3%) and stomach (10.8%) cancers (27). As cancer-driving gene mutations are high-frequency mutations at specific sites in the genome due to evolutionary convergence (32), mutation frequency data were used to select the driver mutations assessed in the present study. This approach is consistent with the objective of screening for mutations that are common in the cancer patient population.

The results revealed 21 AAS mutations with >1% frequency in the HOPE cohort, and these mutations were found in only five cancer driver genes (Fig. 3A). The highly frequent mutations mostly overlapped with those in The Cancer Genome Atlas (TCGA) Cancer HotSpots (33). In the Japanese population, the KRAS G12S and G13D mutants were the most common, whereas KRAS G12R was less common. EGFR mutations were also more common in the Japanese population; however, as the resected primary tumors were the main samples, the EGFR T790M mutation associated with drug resistance was found in only 1 patient in this cohort (Table SIV). The eight most frequent TP53 AAS mutations were prominent at six positions. TP53 is a tumor suppressor gene; however, these dominant-negative mutations appear to be high-frequency targets as the mutants function as oncogenes (Fig. 3B).

The present study first evaluated the frequent mutation-derived AAS epitope presentation on HLA molecules through the NetMHCpan4.1 binding-affinity (BA) model and the eluted-ligand (EL) model to predict binding affinities (IC₅₀) and rank scores (%Rank). In the Japanese dominant HLA alleles (34), a significant number of peptides were predicted to have a high affinity for HLA-A and a smaller number for HLA-B and HLA-C (Fig. 3C).

The present study compared in silico predictions and in vitro assays of the epitopes derived from driver mutations with >0.2% frequency. A total of 138 epitope peptides were synthesized with a predicted affinity (IC₅₀) <500 nM or IC₅₀ <1,000 nM and %Rank <1% for the 10 most frequently appearing HLA-A, -B and -C alleles in the Japanese population, and two peptides derived from cytomegalovirus (Table SIII). For each HLA allele, synthesized peptides with predicted IC₅₀ <10,000 nM were used for the assays, and six to eight peptides with predicted IC₅₀ >10,000 nM were used as negative controls. All assay results are presented in Table SV.

As was expected, ΔHLA correlated with the predicted scores and affinities, particularly for HLA-A*33:03 with the peptide predicted IC₅₀ <1,000 nM (Figs. 4 and 5). The BA model IC₅₀ <1,000 nM or the EL model %Rank <1% are often used as cut-off criteria in the epitope predictions; however, for HLA-A*24:02 and HLA-A*11:01, peptides with predicted IC₅₀ >1,000 nM or with %Rank ranging from 2 to 4% were found to increase HLA expression. Of note, two 9-mer epitopes derived from TP53 driver mutations exhibited extremely high responses to HLA-A*11:01 despite predicted IC₅₀ >5,000 nM, which was more notable than the two 10-mer epitopes with higher predicted affinities (Fig. 5); thus, these mutations are potential new target candidates. Furthermore, the majority of the peptides, including the KRAS G12 or G13 mutation and rich in valine were presented on HLA-A*11:01 regardless of the variant of the mutation.

The epitopes candidates were screened on the basis of the following criteria (Fig. 6): BA_IC₅₀ <1,000 nM, EL_%Rank <1%, no proline in the three forward positions to interfere with peptide processing (35), cysteine content <2 molecules (36,37) and ΔHLA >20% in the MHC stabilization assay with the synthetic peptides. Finally, 27 candidate epitopes were identified in the present study (Table I).