Peptide-binding assay

The efficiency of the shortlisted epitopes to stabilize the HLA-A 02 allele was measured by HLA stabilization assay using TAP-deficient human cell line that express HLA-A2 (T2 cell line) according to our previously described methodology with certain modifications⁴⁹. Briefly, T2 cells (2 × 10⁵/well) were cultured with 20 μg/mL of individual peptides in serum-free RPMI medium for 20 hours at 37 °C in 5% CO₂. The HLA stabilization assay was performed in triplicate for each peptide. The tumor HLA A2 bound 10mer peptide was used as positive control. The expression of HLA-A 02 on T2 cells was measured by PE-conjugated mouse anti-human HLA-A 02 monoclonal antibody (clone: BB7.2; Pharmingen), the samples were acquired by FACS Calibur and analyzed by CellQuest software (BD Biosciences). The binding affinity of each peptide was measured by a fluorescence index: FI = (mean PE fluorescence with the given peptide−mean PE fluorescence without peptide)/(mean FITC fluorescence without peptide). The epitope with a fluorescence index (FI) more than 1 was considered as strong binding affinity epitopes¹⁷.