OR8 Quantitative assessment of HLA class I presentation in ICT-107, a dendritic cell-based immunotherapy

Aim Dendritic Cells (DC) mediate anti-tumor immunity, and DC-based immunotherapies represent a promising way to fight cancer. However, questions about the optimal manufacturing process and delivery strategies still remain. So far, DC-based immunotherapies have only been tested for maturational state and expression of select CD markers. However, these characteristics do not establish successful peptide loading or actual presentation of peptides by HLA. To date, class I HLA presentation of peptides on loaded DC has only been confirmed using indirect immune monitoring techniques such as ELISPOT and tetramer staining. Here, we present the first quantitative assay designed for ICT-107, a DC immunotherapy being tested in glioblastoma, that directly assesses peptide presentation on pulsed DC. This assay may prove valuable for monitoring quality and potency of DC-based immunotherapies via a reliable, reproducible and straightforward method. Methods We generated a T cell receptor mimic monoclonal antibody (TCRm), RL13A, specific for HLA-A∗01:01 in complex with MAGE-1 peptide (EADPTGHSY; “EAD9”). Next, mature DC from three different HLA-A1+ patients were pulsed with 20 μg/ml of EAD9 peptide. Loaded DC were then stained with PE-labeled RL13A. QuantiBRITE PE-Beads were used to establish a standard curve so that the number of HLA-A1/EAD9 complexes could subsequently be determined. Results Flow cytometric staining of peptide-pulsed DC with RL13A showed a clear shift in MFI compared to patient-matched, unpulsed DC. Using the QuantiBRITE PE-bead standard curve, the number of HLA-A1/EAD9 on the surface of ICT-107 was determined. All 3 lots of ICT-107 presented a comparable number of HLA-A1/EAD complexes (1200–2500 copies) on the surface. Conclusions The data from ICT-107 provide proof of concept that an HLA-specific, quantitative assay can be used to directly confirm presentation of the peptides on DC-based immunotherapies. TCRm staining enables detection as well as quantification of HLA/peptide complexes presented by peptide-pulsed DC, and the assay can now be used to characterize the DC immunotherapy, ICT-107. Future applications for this assay include both the optimization of the manufacturing process and serving as a release assay for HLA-A1+ DC-based therapeutics containing the MAGE-1 EAD9 peptide. R. Buchli: Employee; Company/Organization; Pure MHC LLC. W.H. Hildebrand: Scientific/Medical Advisor; Company/Organization; Pure MHC LLC.