Abstract 1574: Quantitation of HLA-A2-MAGE3271-279 complexes on the DC surface improves anticancer vaccine designs

Abstract Background: Dendritic cells (DC) loaded with tumor antigen (TA)-derived peptides are used as vehicles for anti-cancer vaccines. HLA class-I restricted short synthetic peptides are commonly used to load DC for therapy. However, randomized clinical trials have failed to confirm the efficacy of DC-based therapies. We hypothesized that new methods for the quantification of peptide loading of DC are needed to improve the overall response rate to anti-tumor vaccines. Methods: An unlabeled monoclonal antibody (Ab), 12B6 Ab, recognizing HLA-A2-MAGE3271-279 complexes was generated by S. Ferrone. Monocyte-derived DC were generated from HLA-A2+ buffy coats of healthy donors in media containing IL-4 and GM-CSF. DC harvested on day 5 of culture were matured by further 48-h exposure to LPS or Interferon-α (IFN-γ). The HLA-A2+ cell line (T2) deficient for LMP2, TAP1 and TAP2 was maintained in a complete RPMI medium. Peptides were synthesized at the University of Pittsburgh Peptide Synthesis facility. An HLA-A2+ CTL clone specific for MAGE3271-279 was used in ELISPOT assays. Cells were stained with 12B6 Ab followed by a PE-labeled secondary Ab. Flow cytometry was used to measure mean fluorescence intensity (MFI) of the HLA class-I-peptide complexes and the loading kinetics. HLA class-I-peptide complexes were dissociated by treatment of cells with citric acid, pH 3.0. Results: The 12B6 Ab reacted with HLA-A2+DC or T2 cells loaded with MAGE3271-279 but not with HLA-A2− DC loaded with MAGE3271-279 or cells loaded with irrelevant peptides. The MFI obtained for the complexes positively correlated with the concentration of MAGE3271-279 used for loading. Maximum levels of HLA-A2-MAGE3271-279 were detectable on DC after 30min loading, while optimal loading of T2 cells required 4h incubation. The levels of detectable complexes correlated with the total expression levels of HLA-A2 molecules on the DC surface. However, donors with comparable levels HLA-A2 expression showed different levels HLA-A2-MAGE3271-279 expression after loading with the same peptide concentration. The MFI for HLA-A2-MAGE3271-279 on DC, but not on T2 cells, was increased when peptide loading was performed after low pH treatment. This treatment did not affect expression of co-stimulatory molecules on DC. Importantly, the peptide-specific stimulation of CTL generated with mDC loaded after low pH treatment resulted in a significantly increased percentage of IFN-γ secreting T cells. Conclusion: The 12B6 Ab is specific for HLA-A2-MAGE3271-279 complexes and allows for their quantification on the surface of DC. Loading of DC was increased by the removal of HLA class-I-self antigen complexes prior to the addition of the TA-derived exogenous peptide. The treated DC showed a higher density of HLA-A2-MAGE3271-279 by flow cytometry and were more effective in priming MAGE3271-279 specific CTL. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1574. doi:1538-7445.AM2012-1574