Background: Tumor-associated antigens and neoantigens serve as primary targets for cancer immunotherapies such as vaccines and T-cell based therapy. However, identifying pancreatic ductal adenocarcinoma (PDAC) associated T-cell epitopes have been challenging due to its low genomic mutational burden. In this study, we attempted to directly identify PDAC T-cell epitopes by using mass spectrometry. Methods: The protein lysate from PDAC specimens and cell lines were subjected to the antibody affinity purification of human major histocompatibility complexes (MHC) including both HLA Class I and Class II complexes. Peptides bound to the MHC were eluted and identified through LC-MS/MS. Peptide sequences were analyzed with MAXQUANT and Novor Denovo. HLA-binding affinity of peptides were predicted using NetMHC4.0 and validated by in vitro T2 binding assays. Their ability to induce T cell response were measured in a cytokine-Flurospot assay. TCRs specific for selected peptides were cloned by single-cell TCR sequencing and their anti-PDAC activity were tested in vivo on the patient-derived xenograft(PDX) models. Results: We identified 6553 unique HLA-I bound 9-mer peptides from eight PDAC specimens and two PDAC cell lines (Panc10.05 and Pan06.03). Among them, 1163 peptides and 1354 proteins were found in two or more PDAC specimens. We identified 8 potentially immunogenic peptides that bind strongly to matched and nonmatched HLA molecules and induced T cell response in peripheral T cells from both HLA-type matched and non-matched patients. We also identified HLA-II bound peptides in six PDAC tissues and found that the HLA-I and HLA-II peptides isolated from the same patient are highly overlapped. These overlapped peptides were able to induce polyfunctional cytokine response (IFN-γ, TNF-α, and IL-2) in peripheral T cells from patient PBMC. We further investigated the anti-tumor capability of T cell receptors (TCR) for an LAMC-2 derived HLA-class I epitope and a TMSB10 derived peptide eluted from both HLA-Class I and Class II affinity purification. Immunohistochemistry revealed both proteins to be more highly expressed in PDAC tissue compared to paranormal normal tissue. We stimulated HLA-type matched patient’s PBMC with LAMC-2 and TMSB10 peptides to induce clonal expansion of epitope-specific CD8+ and CD4+ T-cells, respectively. We subsequently performed single-cell TCR sequencing of these expanded T cells and infected Jurkat cells with the lentivirus expressing TCR of interest. Mice with orthotopically implanted PDAC tumors that were treated with Jurkat cells expressing LAMC-2 specific cells showed significant slowdown tumor growth. We are currently in the processing of testing TMSB10 peptide targeting TCRs. Conclusion: This study provides a novel venue for identifying T cell epitopes in a nonimmunogenic tumor such as PDAC for the design and development of cancer vaccine and T cell therapy. Citation Format: Tengyi Zhang, Jianxin Wang, Yingkuan Shao, Pan Li, Nan Niu, Brian Herbst, Jessica Gai, Juan Fu, Pingbo Zhang, Jun Yu, Kenji Fujiwara, Lei Zheng. Direct identification of MHC class I and class II epitopes for TCR-based T cell therapy for pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB099.
