Syro 1

The peptides were synthesized using standard solid-phase fluorenylmethyloxycarbonyl chloride (Fmoc) chemistry on a SyroI (Biotage) instrument. The synthesis was started on 25 µmol of rink amide resin (Novabiochem). Amino acids were coupled in a 4-fold excess using HOBT/HBTU activation. The peptides were cleaved using TFA containing phenol, triisopropylsilane, and 5% water for 3 h. The peptides were then precipitated with tributyl methyl ether and recovered by centrifugation at 2000 × g. The ether washing/centrifugation step was repeated three times.

Peptides were produced on solid phase (Fmoc-Rink Amide MBHA, capacity = 0.67 mmol/g) resin in an automated peptide synthesizer (Syro-I, Biotage, Uppsala, Sweden) using standard Fmoc/tBu strategy with DIC/HOBt coupling reagents. Cf was coupled to the N-terminus of the peptides by using DIC/HOBt coupling method. Peptides were cleaved from the resin with TFA/H₂O/TIS (9.5:2.5:2.5, v/v) mixture (2 h, RT). After filtration, compounds were precipitated in cold diethyl ether, centrifuged (4,000 rpm, 5 min) and freeze-dried from water.
RP-HPLC purification was performed on an UltiMate 3000 Semiprep HPLC (Thermo Fisher Scientific) with a Phenomenex Jupiter Proteo C-12 column (250 × 10 mm) using gradient elution, consisting of 0.1% TFA in water (eluent A) and 0.1% TFA in acetonitrile/water = 80/20 (v/v) (eluent B).
Purified peptides were analyzed by LC-MS using a Thermo Scientific Q Exactive Focus Hybrid Quadrupole-Orbitrap Mass Spectrometer. For the separation, a Waters Acquity UPLC BEH C18 (1.7 µm, 150 × 2.1 mm) column was used with a flow rate of 0.3 ml/min.
Residual TFA and TFA counter-ion was removed by using an acetate-exchange resin. For the detailed description, see Supplementary Information 1.1).

First, we selected expressed mutations as follows: FPKM ≥ 30 and RNA variant allele frequency (VAF) ≥ 0.04. Then 8-, 9-, and 10-mer epitopes containing the mutated amino acid were inspected using NetMHCpan2.8 [8 (link)] and NetMHCpan4.1 [9 (link)] for prediction of IC₅₀ and eluted ligand (EL) rank to H-2D^b and H-2K^b. Presentation percentiles of MHCflurry (ver.2.0.1) [10 (link)] were predicted using 21-mer sequences with the mutated amino acid in the middle. Epitopes with IC₅₀ of NetMHCpan ≤ 250 nM, EL rank of NetMHCpan ≤ 0.5 and presentation percentile of MHCflurry ≤ 0.5 were selected. Peptides were synthesized by standard solid-phase synthesis using a Syro I (Biotage, Uppsala, Sweden) as described previously [7 (link)].

Peptides were prepared on TGS Wang resin (250 mg, capacity = 0.27 mmol/g) in an automated peptide synthesizer (Syro-I, Biotage, Uppsala, Sweden) using Fmoc/tBu strategy with DIC/HOBt coupling reagents. Palmitic acid was coupled on the N-terminus of the peptides using DIC/HOBt coupling reagents. After the synthesis was complete, peptides were cleaved from the resin with TFA in the presence of scavengers (H₂O and TIS, 3–3 v/v%). Crude products were precipitated in cold diethyl ether, centrifuged (4000 rpm, 5 min), and lyophilized from H₂O/AcN. Palmitoylated peptides were then purified by RP-HPLC on a Phenomenex Jupiter Proteo C12 column (10 μm, 90 Å, 10 mm × 250 mm) with linear gradient elution using 0.1% TFA in H₂O (eluent A) and 0.1% TFA in AcN:H₂O = 80:20 (v/v) (eluent B) on an UltiMate 3000 Semiprep HPLC (Thermo Fisher Scientific, Waltham, MA, USA). Purified peptides were analyzed by RP-HPLC using an LC-40 HPLC System (Shimadzu, Kyoto, Japan) on an analytical Phenomenex Jupiter Proteo C12 column (10 μm, 90 Å, 4.6 mm × 150 mm). The flow rate was 1 mL/min, and the gradient was 5–100 B% in 20 min (UV detection at λ = 220 nm). High-resolution mass spectra were acquired by direct injection to a Thermo Scientific QExactive Focus Hybrid Quadrupole-Orbitrap Mass Spectrometer (Waltham, MA, USA). Data were analyzed by Xcalibur program (Thermo Fisher Scientific, Waltham, MA, USA).

Peptide E was synthesized
on a Biotage Syro I fully automated parallel peptide synthesizer using
standard Fmoc chemistry. Rink amide resin with a loading of 0.55 mmol/g
(Sigma-Aldrich) was used as a support. Coupling reactions were performed
with 0.5 M HCTU (Novabiochem) in DMF (Biosolve), 2 M DIPEA (Carl Roth)
in a 1:1 mixture of NMP (Biosolve) and DMF, and 0.5 M Fmoc-protected
amino acid (Novabiochem) in DMF. Deprotection steps were carried out
with 40% piperidine (Biosolve) in DMF. All solutions contained 1 g/L
LiCl (Sigma-Aldrich). Up from the 15th coupling step, amino acids
were coupled to the peptide using double coupling steps. The N-terminus
was acetylated with 0.5 M acetic anhydride (Biosolve) and 0.125 M
DIPEA in NMP for 2 h. The peptide was cleaved from the resin using
a mixture of 2.5% triisopropylsilane (Sigma-Aldrich), 2.5% water,
and 95% TFA (Biosolve) and subsequently precipitated in cold diethyl
ether (Honeywell). The precipitate was collected by centrifugation,
dissolved in water, and lyophilized. The peptide was purified by preparative
reverse phase HPLC and the purity was confirmed with LC–MS
(Figures S1–S2).
E-OVA323
and E-OVA323-GC were synthesized on a CEM Liberty Blue automated peptide
synthesizer using similar methods to those described above.

Peptides (Table S1) were synthesized by Fmoc-chemistry using solid-phase peptide synthesis (SPPS) on an automatic peptide synthesizer (Syro I, Biotage). The resin-bound peptides were cleaved and deprotected with TFA containing 10% scavenger mixture H₂O/TIA/EDT/TIS (1:3:3:3) at room temperature for 1.5 h. The peptides were then precipitated from cold diethyl ether, recovered by centrifugation at 4 °C, washed three times with cold ether, dried under nitrogen, dissolved in 0.1% aqueous TFA, and lyophilized.