Tumor antigens are crucial targets for T-cell-based therapy to induce tumor-specific rejection. However, identifying pancreatic ductal adenocarcinoma (PDAC)-specific T-cell epitopes has been challenging. Using advanced mass spectrometry (MS) analysis, we previously identified cancer-associated, class I MHC-bound epitopes shared by multiple PDAC patients with different HLA-A types. Here, we investigated one of these epitopes, LAMC2203-211, a naturally occurring nonmutated epitope on the LAMC2 protein. Following stimulation with the LAMC2203-211 peptide, we cloned T-cell receptors (TCRs) and transduced them into the Jurkat human T-cell line using a lentiviral vector. We found that Jurkat cells expressing LAMC2203-211-specific TCRs resulted in potent, LAMC2 specific, in vitro cytotoxic effects on PDAC cells. Furthermore, in mice that harbored either subcutaneously or orthotopically implanted tumors originating from both HLA-A allele-matched and unmatched PDAC patients, tumor growth was suppressed in a LAMC2-dependent manner following the infusion of LAMC2-targeting T cells. We have therefore developed a LAMC2-specific TCR-based T-cell therapy strategy likely suitable for many PDAC patients. This is the first study to adopt MS analysis to identify natural CD8+ T-cell epitopes in PDAC that could potentially serve as targets for PDAC immunotherapy.

The human gut microbiota represents a complex ecosystem that is composed of bacteria, fungi, viruses, and archaea. It affects many physiological functions including metabolism, inflammation, and the immune response. The gut microbiota also plays a role in preventing infection. Chemotherapy disrupts an organism's microbiome, increasing the risk of microbial invasive infection; therefore, restoring the gut microbiota composition is one potential strategy to reduce this risk. The gut microbiome can develop colonization resistance, in which pathogenic bacteria and other competing microorganisms are destroyed through attacks on bacterial cell walls by bacteriocins, antimicrobial peptides, and other proteins produced by symbiotic bacteria. There is also a direct way. For example, Escherichia coli colonized in the human body competes with pathogenic Escherichia coli 0157 for proline, which shows that symbiotic bacteria compete with pathogens for resources and niches, thus improving the host's ability to resist pathogenic bacteria. Increased attention has been given to the impact of microecological changes in the digestive tract on tumor treatment. After 2019, the global pandemic of novel coronavirus disease 2019 (COVID-19), the development of novel tumor-targeting drugs, immune checkpoint inhibitors, and the increased prevalence of antimicrobial resistance have posed serious challenges and threats to public health. Currently, it is becoming increasingly important to manage the adverse effects and complications after chemotherapy. Gastrointestinal reactions are a common clinical presentation in patients with solid and hematologic tumors after chemotherapy, which increases the treatment risks of patients and affects treatment efficacy and prognosis. Gastrointestinal symptoms after chemotherapy range from nausea, vomiting, and anorexia to severe oral and intestinal mucositis, abdominal pain, diarrhea, and constipation, which are often closely associated with the dose and toxicity of chemotherapeutic drugs. It is particularly important to profile the gastrointestinal microecological flora and monitor the impact of antibiotics in older patients, low immune function, neutropenia, and bone marrow suppression, especially in complex clinical situations involving special pathogenic microbial infections (such as clostridioides difficile, multidrug-resistant Escherichia coli, carbapenem-resistant bacteria, and norovirus).

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.