PURPOSE:Developing T-cell or vaccine therapies for pancreatic ductal adenocarcinoma (PDAC) has been challenging because of a lack of knowledge regarding immunodominant, cancer-specific antigens as PDAC are characterized by a scarcity of genomic mutation-associated neoepitopes, and effective approaches to discover them are limited. EXPERIMENTAL DESIGN:An advanced mass spectrometry approach was employed to compare the immunopeptidome of PDAC tissues and matched normal tissues from the same patients. RESULTS:This study identified HLA class I-binding variant peptides derived from canonical proteins, which had single amino-acid substitutions not attributed to genetic mutations or RNA editing. These amino-acid substitutions appeared to result from translational errors. The variant peptides were predominantly found in tumor tissues, with certain peptides common among multiple patients. Importantly, several of these variant peptides were more immunogenic than their wild-type counterparts. CONCLUSIONS:The shared noncanonical neoepitopes identified in this study offer promising candidates for vaccine and T-cell therapy development, potentially providing new avenues for immunotherapy in PDAC. See related commentary by Yuan et al., p. 1821.

Background: Pancreatic ductal adenocarcinoma (PDAC) is predicted to be the second leading cause of cancer-related death by 2030. This is driven by a high case-fatality rate with most patients even with radiologically localized PDAC at diagnosis ultimately relapsing with metastatic disease. KRAS mutations present in 90% to 95% of PDAC drive these poor statistics through its role in driving cellular growth, inhibition of apoptosis, and immunosuppression. The recent development of KRAS inhibitors has increased interest in understanding key molecular differences between the different KRAS codon changes seen in PDAC and other malignancies and how this might alter therapeutic decision making. Methods: To understand how mutant KRAS influences the PDAC tumor microenvironment (TME) and cytokine signaling, we evaluated patients enrolled on NCT02451982 (A Platform Study of Combination Immunotherapy for the Neoadjuvant and Adjuvant Treatment of Patients with Surgically Resectable Adenocarcinoma of the Pancreas). Interleukin 8 levels were measured using ELISA, these levels were compared with previously determined KRAS mutation status using next generation sequencing, tumor immune microenvironment populations quantified using multiplex immunohistochemistry, and survival outcomes. Results: We identified a total of 30 patients from cohorts A: GVAX, an allogeneic whole cell cancer vaccine (n=16) and B: GVAX + anti-PD-1 (nivolumab) (n=14) with known KRAS mutation status and survival outcomes. Twenty-six of these tumors were KRAS mutant (G12C: 1, G12D: 11, G12R: 4, G12V: 10) and four were KRAS wild type. As KRAS G12D was the most commonly identified mutation and has been associated in some cohorts with worse outcomes, this was evaluated as a separate subgroup. KRAS G12D mutant PDAC had decreased disease-free survival (P=0.01) and a trend towards inferior overall survival in patients treated with GVAX alone (P=0.14) or GVAX plus anti-PD-1 (P=0.17) which became significant when combining both treatment groups (P=0.04). Looking at the relationship between KRAS status and the immune composition of the TME, patients with KRAS mutant PDAC had a trend towards decreased CD8+ T lymphocyte (P=0.06) following treatment with GVAX compared to KRAS wild type tumors. With the addition of anti-PD-1 in Arm B, patients with KRAS G12D mutant disease had a lower ratio of CD8+ GZMB+/CD8+ T lymphocytes (P=0.005). Conclusions: KRAS G12D mutated PDAC represents a unique subtype of disease with decreased survival and lower ratio of activated CD8+ T lymphocytes as denoted by granzyme B (GZMB) positivity following GVAX/aPD-1 treatment.

Radiotherapy is hypothesized to have an immune-modulating effect on the tumor microenvironment (TME) of pancreatic ductal adenocarcinoma (PDAC) to sensitize it to anti–PD-1 antibody (a–PD-1) treatment. We collected paired pre- and posttreatment specimens from a clinical trial evaluating combination treatment with GVAX vaccine, a–PD-1, and stereotactic body radiation (SBRT) following chemotherapy for locally advanced PDACs (LAPC). With resected PDACs following different neoadjuvant therapies as comparisons, effector cells in PDACs were found to skew toward a more exhausted status in LAPCs following chemotherapy. The combination of GVAX/a–PD-1/SBRT drives TME to favor antitumor immune response including increased densities of GZMB + CD8 + T cells, T H 1, and T H 17, which are associated with longer survival, however increases immunosuppressive M2-like tumor-associated macrophages (TAMs). Adding SBRT to GVAX/a–PD-1 shortens the distances from PD-1 + CD8 + T cells to tumor cells and to PD-L1 + myeloid cells, which portends prolonged survival. These findings have guided the design of next radioimmunotherapy studies by targeting M2-like TAM in PDACs.

Successful pancreatic ductal adenocarcinoma (PDAC) immunotherapy requires therapeutic combinations that induce quality T cells. Tumor microenvironment (TME) analysis following therapeutic interventions can identify response mechanisms, informing design of effective combinations. We provide a reference single-cell dataset from tumor-infiltrating leukocytes (TILs) from a human neoadjuvant clinical trial comparing the granulocyte-macrophage colony-stimulating factor (GM-CSF)-secreting allogeneic PDAC vaccine GVAX alone, in combination with anti-PD1, or with both anti-PD1 and CD137 agonist. Treatment with GVAX and anti-PD-1 led to increased CD8+ T cell activation and expression of cytoskeletal and extracellular matrix (ECM)-interacting components. Addition of CD137 agonist increased abundance of clonally expanded CD8+ T cells and increased immunosuppressive TREM2 signaling in tumor associated macrophages (TAMs), identified by comparison of ligand-receptor networks, corresponding to changes in metabolism and ECM interactions. These findings associate therapy with GVAX, anti-PD1, and CD137 agonist with enhanced CD8+ T cell function while inducing alternative immunosuppressive pathways in patients with PDAC.

4024 Background: PDAC is an aggressive cancer and refractory to immunotherapy due to its immunosuppressive tumor microenvironment (TME). Focal adhesion kinase (FAK) is a master regulator of the TME and associated with TME immune suppression. Our current randomized phase II trial evaluates the use of pembrolizumab, a programmed cell death 1 (PD-1) immune checkpoint inhibitor, with or without defactinib, a FAK inhibitor (FAKi), as sequential neoadjuvant and adjuvant therapy in patients with high risk resectable PDAC. We hypothesize that the patients receiving pembrolizumab and defactinib treatment will exhibit a decrease in immunosuppressive fibroblasts and myeloid subtype populations, leading to increased CD8+ T-cell infiltration into the TME compared to patients receiving pembrolizumab alone. Methods: We performed quantitative analysis of multiplex immunohistochemistry (mIHC) using 40 biomarkers, evaluating immune and stromal cell types on 14 pre-treatment biopsies and post-treatment resections of PDAC patients enrolled in our platform neoadjuvant clinical trial (NCT03727880). All patients received 2 cycles of gemcitabine+nab-paclitaxel neoadjuvant chemotherapy and underwent a biopsy after completion of chemotherapy. Patients randomized to Arm A received 2 cycles of pembrolizumab 200 mg IV every 3 weeks and defactinib 400 mg PO BID, and those randomized to Arm B received pembrolizumab alone, followed by surgical resection. Image cytometry was used to quantify immune cell populations and colocalize biomarker expression in distinct cell types. Cell populations were compared using unpaired T-tests. Results: Lower FAP+ fibroblast density was significantly associated with higher CD8+ T-Cell infiltration in Arm A (p=0.002), but not in Arm B. Patients in Arm A demonstrated a 5.44-fold average increase in CD8+ T-cell percentage between pre-immunotherapy treatment and post-immunotherapy treatment specimens compared to a 2.01-fold increase in patients in Arm B (p=0.02, p=0.09, respectively). Only patients in Arm A showed a significant increase in M1 macrophage cell density (p=0.02) after treatment. Both arms increased in CXCR4+ cell percentage (p=0.011, p=0.06) following neoadjuvant immunotherapy; however, the increase in Arm A was less than in Arm B (0.72 and 0.84-fold, respectively). Conclusions: In this analysis, pembrolizumab combined with defactinib was associated with lower fibroblast infiltration, higher anti-tumor M1 macrophage expression and increased CD8+ T-cell infiltration into the TME, versus pembrolizumab alone. The increased expression of CXCR4 across both treatment arms may represent a resistance mechanism and supports CXCR4 as an additional TME target. These preliminary findings warrant continued research into FAK inhibition and immune checkpoint combinatorial strategies. Clinical trial information: NCT03727880 .

Background:Pancreatic ductal adenocarcinoma (PDAC) has a poor prognosis and is highly metastatic. Our prior studies have demonstrated the critical role of axon guidance pathway genes in PDAC and the connection between neuronal development and the tumor microenvironment. A recent study newly identified 20 neuronal development genes [disks large homolog 2 (DLG2), neuron-glial-related cell adhesion molecule (NRCAM), neurexin3 (NRXN3), mitogen-activated protein kinase 10 (MAPK10), platelet-derived growth factor D (PDGFD), protein kinase C epsilon (PRKCE), potassium calcium-activated channel subfamily M alpha 1 (KCNMA1), polycystic kidney and hepatic disease 1 (PKHD1), neural cell adhesion molecule 1 (NCAM1), neuregulin-1 (NRG1), zinc finger protein 667 (ZNF667), cystic fibrosis transmembrane conductance regulator (CFTR), acyl-CoA medium-chain synthetase-3 (ACSM3), complement 6 (C6), protein tyrosine phosphatase receptor type M (PTPRM), hypoxia-inducible factor 1 alpha (HIF1A), adenylyl cyclase 5 (ADCY5), adherens junctions-associated protein 1 (AJAP1), neurobeachin (NBEA), sodium voltage-gated channel alpha subunit 9 (SCN9A)] that are associated with perineural invasion and poor prognosis of PDAC. The relationship between genetic alterations in these 20 genes and tumor immune microenvironment (TME) has not previously been investigated.Methods:We hence applied the sequential multiplex immunohistochemistry results of biopsy specimens from 63 PDAC patients to investigate this relationship.Results:We found that, except for PTPRM and NBEA, genetic alterations involving these 20 genes are associated with significant changes in the densities of major immune cell subtypes. Except for AJAP1, the copy number loss involving this panel of neuronal development genes is significantly associated with changes in immune cell infiltrates. In contrast, the copy number gain in fewer genes, including NRXN3, ZNF667, ACSM3, C6, ADCY5, SCN9A, and PRKCE, is significantly associated with changes in immune cell infiltrates.Conclusions:Our study suggested that neuronal development genes play a role in modulating TME in a pancreatic cancer setting.

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.

e16309 Background: The optimal management of BR-PDAC remains undefined due to the high rate of local progression and/or metastasis with upfront surgery alone. Given the limited activity of single agent PD-1 blockade in PDAC, combination strategies aimed at increasing high-avidity T-cells in the tumor microenvironment (TME) are under investigation. GVAX is an allogenic, whole cell, GM-CSF-secreting vaccine that activates T-cell immunity against tumor-associated antigens. Prior work has shown that GVAX in combination with low-dose cyclophosphamide (Cy) can inhibit T-regulatory cells and induce both PD-L1 expression and the formation of tertiary lymphoid aggregates. This multi-center phase II clinical trial evaluates the safety and clinical efficacy of the addition of GVAX/Cy/nivolumab and SBRT to neoadjuvant chemotherapy in patients with BR-PDAC. Methods: Patients received neoadjuvant mFOLFIRINOX or gemcitabine/abraxane if intolerant to mFOLFIRINOX. Two to six weeks after chemotherapy, the first dose combination immunotherapy with Cy (200 mg/m2), nivolumab (240mg) and GVAX (5 x 10 8 vaccine cells) was given. Three weeks later, patients received the second dose of combined immunotherapy concurrently with SBRT (6.6 Gy x 5 days) prior to surgical resection. The primary endpoint is CD8 + T-cell density (CD8) in surgical specimens compared with CD8 in historical control samples treated with neoadjuvant mFOLFIRINOX and SBRT only. Additional endpoints include pathologic response rates, adverse events (AEs) as graded by NCI CTCAE version 4.03, OS, and evaluation of immune changes to the TME. Results: 31 patients were enrolled from 2/2/2018 to 7/27/2021. Of these, 18 patients received at least one dose of combination immunotherapy. Following study treatment, one patient had progressive disease, three had occult intra-operative metastatic disease and 14 underwent definitive surgical resection (13 R0 resections and one R1 resection). The major pathologic response rate was 35% (defined as <10% residual viable tumor), including one pathologic complete response (pCR). At a median follow-up of 20.5 months, median OS was 20.4 months (95% CI 18.2, NA). There were two grade 3 or higher AEs (one case of nivolumab-related autoimmune hepatitis and one case of SBRT-related pancytopenia). Conclusions: The addition of combined immunotherapy and SBRT to chemotherapy was safe and feasible in BR-PDAC. Observed median OS, pCR and R0 resection rates were comparable to contemporary studies administering neoadjuvant mFOLFIRINOX and SBRT. Additional immune correlative data will be available and reported at the time of the conference, guiding future applications of novel vaccine approaches. Clinical trial information: NCT03161379 .

A neoadjuvant immunotherapy platform clinical trial allows for rapid evaluation of treatment-related changes in tumors and identifying targets to optimize treatment responses. We enrolled patients with resectable pancreatic adenocarcinoma into such a platform trial (NCT02451982) to receive pancreatic cancer GVAX vaccine with low-dose cyclophosphamide alone (Arm A; n = 16), with anti-PD-1 antibody nivolumab (Arm B; n = 14), and with both nivolumab and anti-CD137 agonist antibody urelumab (Arm C; n = 10), respectively. The primary endpoint for Arms A/B - treatment-related change in IL17A expression in vaccine-induced lymphoid aggregates - was previously published. Here, we report the primary endpoint for Arms B/C: treatment-related change in intratumoral CD8+ CD137+ cells and the secondary outcomes including safety, disease-free and overall survivals for all Arms. Treatment with GVAX+nivolumab+urelumab meets the primary endpoint by significantly increasing intratumoral CD8+ CD137+ cells ( p = 0.003) compared to GVAX+Nivolumab. All treatments are well-tolerated. Median disease-free and overall survivals, respectively, are 13.90/14.98/33.51 and 23.59/27.01/35.55 months for Arms A/B/C. GVAX+nivolumab+urelumab demonstrates numerically-improved disease-free survival (HR = 0.55, p = 0.242; HR = 0.51, p = 0.173) and overall survival (HR = 0.59, p = 0.377; HR = 0.53, p = 0.279) compared to GVAX and GVAX+nivolumab, respectively, although not statistically significant due to small sample size. Therefore, neoadjuvant and adjuvant GVAX with PD-1 blockade and CD137 agonist antibody therapy is safe, increases intratumoral activated, cytotoxic T cells, and demonstrates a potentially promising efficacy signal in resectable pancreatic adenocarcinoma that warrants further study.

Introduction: Pancreatic ductal adenocarcinomas (PDACs) are immunogenically “cold” tumors that lack effector T cell infiltration associated with immune checkpoint inhibitor (ICI) responsiveness. Neoadjuvant immunotherapy clinical trials facilitate understanding of the complexities of combination treatment strategies on immunosuppressive mechanisms. Our recent multi-omics analysis of a neoadjuvant trial comparing the GVAX vaccine alone or with nivolumab and supporting preclinical data uncovered crosstalk between CD8+CD137+ T cells and neutrophils as an axis for therapeutic intervention. These studies have formed the foundation for the addition of the CD137 agonist, urelumab, to our combination regimen to improve T cell activation in PDACs. Methods: Patients were enrolled on NCT02451982 and initiated treatment 2 weeks prior to surgical resection. We compared specimens from 3 treatment arms for downstream analysis: Arm A: an allogeneic whole cell vaccine, GVAX (n=7); Arm B: GVAX and nivolumab (n=4); Arm C: GVAX, nivolumab, and urelumab (n=4). Samples underwent dissociation and Percoll separation to enrich for immune cells, followed by single-cell RNA- and TCR-seq using 10X Genomics. Computational analysis was performed for cell type classification with Seurat, differential expression with DESeq2 of pseudobulk data, cell-cell communication with Domino, and TCR-seq analysis with scRepertoire. Results: Single-cell RNA-seq analysis identified changes in immune cell proportions and immune cell states across treatment arms. Alignment of scTCR-seq to RNA data showed greater CD8+ T cell expansion following triple agent therapy. Gene set enrichment identified changes in CD8+ T cell, tumor associated macrophage (TAM), and B cell responses to cytokine signaling, locomotion, amino acid metabolism, and cell adhesion pathways in Arm C. Domino analysis demonstrated increases in the naïve T cell signal CCR7 in Arm A and integrin ligand signal ICAM1 in Arm C between the CD8+ T cells and other cell types present in PDACs. CCR7 expression was significantly downregulated in CD8+ T cells in Arm C whereas CCL5, a T cell agonist cytokine, and LCP2, a TCR signaling activator and T cell receptor which is also required for integrin signaling, were significantly upregulated in CD8+ T cells in Arm C. The increased integrin ligand signaling via ICAM1 and LCP2 in Arm C is also associated with changes in CD8+ T cell motility observed from the differential expression analysis. Conclusions: Analysis of combination GVAX, PD-1 inhibition and CD137 agonist therapy demonstrated changes in multiple immune cell proportions and their functional pathways. These data provide new evidence that PDACs can become T cell rich and respond to combination immunotherapies. Citation Format: Joseph A. Tandurella, Jacob T. Mitchell, Janelle M. Montagne, Eric Christenson, Qingfeng Zhu, Ludmilla V. Danilova, Alexander V. Favorov, Won J. Ho, Luciane T. Kagohara, Melanie Loth, Su Jin Lim, Rui Zheng, Jessica Gai, Sarah Mitchell, Dimitrios N. Sidiropoulos, Wenpin Hou, Yao Xu, Hao Wang, Jacquelyn W. Zimmerman, Srinivasan Yegnasubramanian, Robert Anders, Elizabeth M. Jaffee, Lei Zheng, Elana J. Fertig. Single-cell RNA and TCR sequencing of tumor infiltrating lymphocytes identifies changes in pancreatic ductal adenocarcinomas following neoadjuvant treatment with combined anti-PD-1 blocking and anti-CD137 agonist therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3241.

Pancreatic ductal adenocarcinoma(PDAC) does not respond to single-agent immune checkpoint inhibitor therapy, including anti-PD-1 antibody(aPD-1) therapy. Higher plasma levels of IL-8 are associated with poorer outcomes in patients who receive aPD-1 therapies, providing a rationale for combination immunotherapy with an anti-IL-8 antibody(aIL-8) and aPD-1. We thus investigated whether human aIL-8 therapy can potentiate the antitumor activity of aPD-1 and further investigated how the combination affects the immune response by regulating myeloid cells in the tumor microenvironment in a humanized murine model of PDAC with a reconstituted immune system consisting of human T cells and a combination of CD14+ and CD16+ myeloid cells. The results show that the combination of aIL-8 and aPD-1 treatment significantly enhanced antitumor activity following the infusion of myeloid cells. Our results further showed that the target of IL-8 is mainly present in CD16+ myeloid cells and is likely to be granulocytes. FACS analysis showed that aIL-8 treatment increased granulocytic myeloid cells in tumors. Consistently, single-nuclear RNA-sequencing analysis of tumor tissue showed that the innate immune response and cytokine response pathways in the myeloid cell cluster were activated by aIL-8 treatment. This is the first preclinical study using a humanized mouse model for new combination immunotherapeutic development and supports the further clinical testing of aIL-8 in combination with aPD-1 for PDAC treatment. This study also suggests that peripherally derived myeloid cells can potentiate the antitumor response of T cells, likely through the innate immune response, and aIL-8 re-educates tumorinfiltrating myeloid cells by activating the innate immune response.

558 Background: Utilizing a vaccine that induces and activates host effector T cells and co-administering it with immune modulating agents that enhance anti-tumor T cell activity is a potential strategy for overcoming pancreatic adenocarcinoma’s (PDA) resistance to immunotherapy. Our prior clinical trial demonstrated a GM-CSF-secreting, allogeneic tumor cell vaccine (GVAX) increases infiltrating CD8+ T cells in PDA. Follow up preclinical work demonstrated therapeutic synergy between GVAX and PD-1inhibition (PD1) with efficacy further enhanced by CD137 agonism (CD137). Methods: This was a 3-arm trial of neoadjuvant & adjuvant GVAX-based therapy in resectable (r) PDA patients (pts). Adults with clinically resectable, untreated PDA were enrolled in 1 of 3 study treatments: Arm A (GVAX alone), Arm B (GVAX + PD1 [Nivolumab]), or arm C (GVAX + PD1 + CD137 [Urelumab]). Treatment was given as follows: Day 1 - Cyclophosphamide 200mg/m2 IV (All Arms), Nivolumab 480mg IV (Arms B, C), Urelumab 8mg IV (Arm C); Day 2 – GVAX ID (All Arms). Pts were treated at 3 timepoints: 1) once 2 weeks prior to surgery; 2) once post-surgical recovery prior to standard of care adjuvant chemotherapy (SOC); 3) every month (up to 4 mo) following completion of SOC (if disease-free). SOC regimes included (m)FOLFIRINOX, Gem +/- Cap/NAB-Paclitaxel. The study was powered for a primary biologic endpoint: treatment-related change in intratumoral CD8+CD137+ T cells. Clinical endpoints included disease-free survival (DFS: time from surgery to recurrence), overall survival (OS: time from surgery to death), and safety. Results: 38 pts (N = 15 [Arm A], N = 13 [Arm B], N = 10 [Arm C]) were eligible for efficacy analysis (had R0/R1 resection) and 45 pts (N = 17 [A], N = 17 [B], N = 11 [C]) were eligible for safety analysis (had ≥1 dose of study treatment). Demographics, surgical pathology features, and SOC durations were similar in all Arms. At median follow up of 23 mo [A], 26 mo [B], and 22 mo [C], median DFS (95% CI) was 14.82 mo (6.0, NA), 16.23 mo (7.49, NA) and not reached (16.33, NA) for Arms A, B, C, respectively. There was no DFS benefit to adding PD1 compared to GVAX alone (HR 0.98 [95% CI 0.42, 2.27], p = 0.96). Combination CD137 + PD1 + GVAX was associated with marginally significant improved DFS compared to GVAX alone (HR 0.38 [95%CI 0.12, 1.19], p = 0.097) and GVAX + PD1 (HR 0.38 [95%CI 0.12, 1.21], p = 0.103). Median OS (95% CI) was 25.0 mo (18.8, NA), 26.4 mo (20.3, NA), and not yet reached for Arms A, B, C, respectively. There were no serious adverse events. In Arm C, 1 pt had grade 3 rash that delayed treatment and there was 1 instance of grade 2 AST/ALT elevation. The biologic endpoint will be reported at the meeting. Conclusions: Despite a small sample size, combining GVAX with dual immune-targeting of PD-1 blockade and CD137 agonism was safe and may enhance DFS in rPDA pts treated in the perioperative and post-adjuvant settings. Clinical trial information: NCT02451982.

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.

Successful pancreatic ductal adenocarcinoma (PDAC) immunotherapy necessitates optimization and maintenance of activated effector T cells (Teff). We prospectively collected and applied multi-omic analyses to paired pre- and post-treatment PDAC specimens collected in a platform neoadjuvant study of granulocyte-macrophage colony-stimulating factor-secreting allogeneic PDAC vaccine (GVAX) vaccine ± nivolumab (anti-programmed cell death protein 1 [PD-1]) to uncover sensitivity and resistance mechanisms. We show that GVAX-induced tertiary lymphoid aggregates become immune-regulatory sites in response to GVAX + nivolumab. Higher densities of tumor-associated neutrophils (TANs) following GVAX + nivolumab portend poorer overall survival (OS). Increased T cells expressing CD137 associated with cytotoxic Teff signatures and correlated with increased OS. Bulk and single-cell RNA sequencing found that nivolumab alters CD4+ T cell chemotaxis signaling in association with CD11b+ neutrophil degranulation, and CD8+ T cell expression of CD137 was required for optimal T cell activation. These findings provide insights into PD-1-regulated immune pathways in PDAC that should inform more effective therapeutic combinations that include TAN regulators and T cell activators.