Highly Efficient Presentation of Endogenously Processed Class I Peptides by Artificial APC for the Generation of Effective Anti-Tumor Responses.
Authors: Hirano Naoto , Butler Marcus O. , Xia Zhinan , Nadler Lee M. 
Published: April 2020

Abstract
Appropriate processing and presentation of tumor associated antigens (TAA) by antigen presenting cells (APC) is absolutely required for the development of clinically relevant anti-tumor T cell responses. One common approach which utilizes the exogenous pulsing of synthetic peptides onto APC can sometimes generate ineffective immune responses. This failure may, in part, be the consequence of erroneous conformations of HLA/synthetic pulsed peptides which can differ from conformations formed with HLA/endogenous peptides that are derived from intracellular proteins. Also, endogenously processed peptides sometimes undergo post-translational modifications during transport to the cell surface, a process that does not occur with exogenously loaded peptides. Since our goal is to induce immunity that can recognize TAA that are endogenously presented by tumors, it is logical that the ideal APC would not only express the required immunoaccessory molecules, but would also endogenously process and appropriately present target antigenic peptides. In this report, we employed our artificial APC (aAPC) that expresses HLA-A2, CD80, and CD83 and is capable of priming and supporting the prolonged expansion of peptide specific CD8+ cytotoxic T cells (CTL). We hypothesized that aAPC can endogenously process and present HLA class I peptides and can induce functional T cell immunity. To test this, aAPC was transduced with an EGFP-mini MP1 (aa 55–66) fusion gene containing the sequence for the influenza derived peptide MP58 (aa 58–66). We observed that this HLA-A2 restricted peptide is processed and presented by aAPC by demonstrating that MP58 specific CTL are able to recognize aAPC/mini MP1 target cells. This was completely abrogated by treating aAPC/mini MP1 with proteasome inhibitors, suggesting that MP58 is endogenously processed by the proteasome. This was confirmed by the elevation of EGFP-mini MP1 fusion protein and the accumulation of ubiquitinated forms as detected by flow cytometry and Western blot analysis, respectively. At the protein level, aAPC was shown to express all proteasome subunits examined and to upregulate immunoproeosome subunits with exposure to IFN-γ. We biochemically confirmed the presence of MP58 in the A2 groove on the surface of aAPC/mini MP1, by performing reverse phased HPLC, mass spectrometry and peptide sequencing of peptides directly acid stripped from the cell surface. Since aAPC expresses only one HLA allele, A2, this finding provides strong support that MP58 is processed and presented in the groove of the A2 molecule on aAPC/mini MP1. We next evaluated the density of MP58 presented by HLA-A2 on aAPC/mini MP1. A2 positive CD8+ T cells were stimulated at weekly intervals by either aAPC/mini MP1 or parental aAPC exogenously pulsed with graded concentrations of synthetic MP58. After three stimulations, peptide specificity of generated CTL was examined by tetramer analysis. The comparison of tetramer staining revealed that the density of endogenously processed and presented MP58 corresponded to pulsing aAPC with 100 μg/ml. In order to extend this strategy to a TAA, we transduced aAPC with EGFP-mini MART1 (aa 27–35) mini gene. We have demonstrated that MART1 peptide is processed and presented on the cell surface and have induced the expansion of MART1 peptide specific T cells. These results suggest that our APC can naturally process and present class I restricted peptides, resulting in the efficient priming and expansion of clinically relevant antigen specific CTL.