A paucity of effector T cells within tumors renders pancreatic ductal adenocarcinoma (PDAC) resistant to immune checkpoint therapies. While several under-development approaches target immune-suppressive cells in the tumor microenvironment, there is less focus on improving T cell function. Here we show that inhibiting vasoactive intestinal peptide receptor (VIP-R) signaling enhances anti-tumor immunity in murine PDAC models. In silico data mining and immunohistochemistry analysis of primary tumors indicate overexpression of the neuropeptide vasoactive intestinal peptide (VIP) in human PDAC tumors. Elevated VIP levels are also present in PDAC patient plasma and supernatants of cultured PDAC cells. Furthermore, T cells up-regulate VIP receptors after activation, identifying the VIP signaling pathway as a potential target to enhance T cell function. In mouse PDAC models, VIP-R antagonist peptides synergize with anti-PD-1 antibody treatment in improving T cell recruitment into the tumors, activation of tumor-antigen-specific T cells, and inhibition of T cell exhaustion. In contrast to the limited single-agent activity of anti-PD1 antibodies or VIP-R antagonist peptides, combining both therapies eliminate tumors in up to 40% of animals. Furthermore, tumor-free mice resist tumor re-challenge, indicating anti-cancer immunological memory generation. VIP-R signaling thus represents a tumor-protective immune-modulatory pathway that is targetable in PDAC.

Vasoactive intestinal peptide (VIP), an anti-inflammatory neuropeptide with pleiotropic cardio-vascular effects, induces differentiation of hematopoietic stem cells into regulatory dendritic cells that limit graft-versus-host disease (GvHD) in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We have previously shown that donor plasmacytoid dendritic cells (pDCs) in bone marrow (BM) donor grafts limit the pathogenesis of GvHD. In this current study we show that murine and human pDCs express VIP, and that VIP-expressing pDCs limit T cell activation and expansion using both in vivo and in vitro model systems. Using T cells or pDCs from transgenic luciferase+ donors in murine BMT, we show similar homing patterns of donor pDCs and T cells to the major sites for allo-activation of donor T cells: spleen, and gut. Co-transplanting VIP-KO pDCs with hematopoietic stem cells (HSCs) and T cells in MHC mis-matched allogeneic BMT led to lower survival, higher GvHD scores, and more colon crypt cell apoptosis than transplanting wild-type pDCs. BMT recipients of VIP-KO pDCs had more Th1 polarized T cells, and higher plasma levels of GM-CSF and TNF-alpha than recipients of wild-type pDCs. T cells from VIP-KO pDC recipients had increasing levels of bhlhe40 transcripts during the first two weeks post-transplant, and higher levels of CyclophilinA/Ppia transcripts at day 15 compared with T cells from recipients of wild type pDCs. Collectively, these data indicate paracrine VIP synthesis by donor pDCs limits pathogenic T cell inflammation, supporting a novel mechanism by which donor immune cells regulate T cell activation and GvHD in allogeneic BMT.

An experiment for the upper-division undergraduate biochemistry laboratory is described in which students investigate the influence of genetic variations of cytochrome P450 1A2 on drug metabolism, using caffeine as a model compound. Saliva samples from human subjects are characterized for a single-nucleotide polymorphism in the CYP1A2 gene (genotyping) and for the rate of caffeine clearance (phenotyping). This experiment has clinical significance in the area of personalized medicine.

In the past decades, advances in the use of adoptive cellular therapy to treat cancer have led to unprecedented responses in patients with relapsed/refractory or late-stage malignancies. However, cellular exhaustion and senescence limit the efficacy of FDA-approved T-cell therapies in patients with hematologic malignancies and the widespread application of this approach in treating patients with solid tumors. Investigators are addressing the current obstacles by focusing on the manufacturing process of effector T cells, including engineering approaches and ex vivo expansion strategies to regulate T-cell differentiation. Here we reviewed the current small-molecule strategies to enhance T-cell expansion, persistence, and functionality during ex vivo manufacturing. We further discussed the synergistic benefits of the dual-targeting approaches and proposed novel vasoactive intestinal peptide receptor antagonists (VIPR-ANT) peptides as emerging candidates to enhance cell-based immunotherapy.

BackgroundPancreatic ductal adenocarcinoma (PDAC), the fourth leading cause of cancer related death in the U.S, has a 5-year survival rate of only 10%.1 The paucity of T cells in the immune privileged tumor microenvironment (TME) is a major limitation in developing an effective immunotherapy against PDAC.2The cancer genome atlas (TCGA) shows that human PDAC tumors express high levels of vasoactive intestinal peptide (VIP), an immunosuppressive neuropeptide (figure 1A), that inhibits effector T cell responses.3 4 We hypothesized that paracrine secretion of VIP in the TME is a targetable mediator of immune paralysis in PDAC, and that pharmacological inhibition of VIP receptor signaling could enhance anti-tumor responses in PDAC.MethodsVIP levels in plasma or cell culture supernatant was determined via VIP-specific enzyme immunoassay. Luciferase transfected KPC (KPC.luc) cells were injected subcutaneously or orthotopically into the pancreas of C57BL/6, CD4KO, or CD8KO mice from Jackson Laboratories. C57BL/6 mice T cell subsets in were depleted post tumor implantation with anti-CD4 and/or anti-CD8 antibodies. Tumor-bearing mice were treated daily with ANT008, a novel VIP receptor antagonist peptide, and/or anti-PD1 monoclonal antibody (MoAb) for 10 days, starting 7-10 days after implantation. T cells isolated from peripheral blood of PDAC patients were expanded 9 days ex vivo in anti-CD3 MoAb coated plates with 30U/ml IL-2 and either control peptide (scrambled VIP sequence) or ANT008. Survival by VIP and VIP receptor expression from the TCGA was clinically correlated.ResultsIncreased human and mouse PDAC expression correlates with elevated blood levels (figure 1). While the PDAC cancer cell lines express VIP receptors, ANT008 does not have direct cytotoxic effect on cell growth in vitro (figure 2). However, in orthotopic KPC model, treatment with ANT008 & anti-PD-1 significantly decreased tumor growth rate and burden (figure 3) while increasing the intratumoral levels of CD4 and CD8 proliferating T cells (figure 4) via a T cell dependent mechanism (figure 5). Additionally, in ex vivo cultures of T cells isolated from PDAC patients, ANT008 improved the effector properties of T cells via decreasing expression levels of co-inhibitory molecules and decreasing frequency of regulatory T cells (figure 6). Clinically, VIPR1 receptor expression, but not VIP, provides a survival benefit (figure 7).Abstract 819 Figure 1Overexpression of VIP in PDAC tumors(A) VIP mRNA expression in several solid malignancies, with red arrow pointing at the levels in pancreatic cancer, as obtained from The Cancer Genome Atlas. (B) Blood collected from healthy volunteers (n=26) and consented untreated pancreatic cancer patients before surgery/chemotherapy (n=41) were quantified for levels of VIP. (C) Cell free supernatant collected from B16F10, SM1 and D4M (murine melanoma cell lines) or KPC, MT5 or Panc02 (murine PDAC cell lines) or BXPC3 and Panc1 (human PDAC cell lines), were quantified for VIP (D) C57BL/6 mice were implanted with 1 million B16F10 cells (n=4), D4M cells (n=4) or MT5 cells (n=3) subcutaneously, or with KPC cells in the tail of the pancreas after laparotomy (n=3). When tumor volumes reached 500mm3 for subcutaneous tumors or the tumor flux reached 2 x 1010 photons/sec for orthotopic KPC tumors, mice were sacrificed and the concentration of VIP in the blood was determined. P value for B was calculated using student t test and C and D were calculated by one-way ANOVA followed by Tukey’s post-test. Error bars show mean with standard deviation. *p<0.0001Abstract 819 Figure 2PDAC cell lines express receptors for VIP(A) Western blot analysis showing constitutive expression of VPAC1, VPAC2 and PACAP receptors in murine and human PDAC cell lines and B16F10, a murine melanoma cell line. GAPDH was used as the loading control. (B) Percentage viability of KPC and Capan02 cells treated with varying concentrations of ANT008 (0.5-5µM) relative to cells treated with 0µM ANT008 for 24, 48 and 72 hours as measured by MTT assay.Abstract 819 Figure 3Efficacy of ANT008+aPD-1 therapy in in-vivo model5x105 KPC.luc cells on a matrigel were implanted in the tail of pancreas of C57BL/6 mice. Seven days after tumor implantation, mice were randomized and treated with ANT008 and/or aPD-1 until day 25, when they were sacrificed. (A) From each treatment group, one mouse with biggest non-ulcerated tumor was imaged via IVIS imaging (top), sacrificed and imaged with 9.4T MRI scanning (MRI) and their paraffin embedded pancreatic tissues were stained via H&E (bottom). (B) The tumor flux measured via IVIS imaging is plotted with respect to days after tumor implantation. ‘o’ represent mice that were imaged via MRI on day 28 and ‘+’ represent mice that were sacrificed before day 25 due to ulceration. (C) On day 25 mice were sacrificed and the weight of the pancreas was plotted. The brown dashed line represent average weight of pancreas from naïve age matched mice. P value was calculated using one-way ANOVA followed by Tukey’s post-test. Error bars show mean with standard deviation with **p<0.01.Abstract 819 Figure 4ANT008+aPD-1 therapy increases T cell infiltration5x105 KPC.luc cells were implanted in the tail of the pancreas following laparotomy. Once the tumors were detectable via bioluminescent imaging, mice were randomized into four treatment groups and treated with isotype antibody and scrambled peptide or ANT008 and/or anti-PD-1 until day 25, when the mice were sacrificed, tumor tissues harvested, and formalin fixed. The tissues were stained for nucleus, CD4, CD8 and Ki67 via multiplex immunohistochemistry (A) A representative multiplex IHC image showing T cell infiltration in the different treatment groups .(B) Numbers of CD4, CD8, Ki67+ CD4 and Ki67+ CD8 T cells/mm2 with respect to weight of the pancreas is shown.Abstract 819 Figure 5ANT008+aPD-1 therapy is T cell dependent5x105 KPC.luc cells were subcutaneously implanted in C57BL/6 or CD4KO (A) and C57BL/6 or CD8KO (B) mice and treated with scrambled+IgG (control) or ANT008+aPD-1 (treated) for 10 days from day 5 or 7 when the tumors were palpable. Mice were monitored for tumor growth and sacrificed when the tumor volume reached 500mm3 or when the tumors ulcerated. P values were calculated using Log rank test. *p<0.0001.Abstract 819 Figure 6ANT008 improves effector properties of T cellsCryopreserved PBMCs from peripheral blood of consented PDAC patients were thawed and rested overnight at 37°C in a CO2 incubator followed by T cell isolated via negative magnetic sorting. Isolated T cells were seeded on human anti-CD3 coated plates and cultured in media supplemented with 30U/ml human recombinant IL-2 and 3uM of scrambled peptide (SCRAM) or ANT008 for 9 days. On day 9 the cells were counted and stained for CD3, CD4, CD8, Tim-3, Lag-3, PD-1, CD25, FoxP3 and with aqua live/dead viability stain and analyzed via flow cytometry. (A) The gating strategy, (B) yield on da 9 with respect to number of cells seeded on day 0 and C) the percentage of T cells expressing co-inhibitory molecules are shown. P values were calculated by student t test . *p<0.001.Abstract 819 Figure 7Survival correlates with VPAC1 expression (TCGA)Survival probability analysis and the respective Kaplan Meier curves from TCGA data sets for pancreatic ductal adenocarcinoma (PAAD), stomach adenocarcinoma (STAD), and colorectal adenocarcinoma (CAAD). (A) Combined survival analysis of multiple GI malignancies (PAAD, STAD and COAD) demonstrates increased VPAC1 expression is associated with a 1-year 50% survival benefit (75 vs 50%) (n=560 (high) & 557 (low)), (B) while VIP expression is not (n=540 (high) and 540 (low)). (C) In pancreatic adenocarcinoma, increased VIP receptor (VIPR1) expression trends toward an overall survival benefit (n=91 (high) & 92 (low)), while (D) VIP expression does not.ConclusionsVIP is a targetable mechanism of immune escape in PDAC. Inhibiting VIP receptor signaling improves effector properties of T cells and synergistically improves the anti-tumor response to checkpoint inhibitors in mouse PDAC models.ReferencesSiegel RL, Miller KD, Jemal A. Cancer statistics, 2020. CA Cancer J Clin2020;70(1):7-30.Sahin IH, et al. Immunotherapy in pancreatic ductal adenocarcinoma: an emerging entity?Ann Oncol 2017;28(12):2950-2961.Gonzalez-Rey E, Anderson P, Delgado M. Emerging roles of vasoactive intestinal peptide: a new approach for autoimmune therapy. Ann Rheum Dis 2007;66(Suppl 3):iii70-6.Anderson P, Gonzalez-Rey E. Vasoactive intestinal peptide induces cell cycle arrest and regulatory functions in human T cells at multiple levels. Mol Cell Biol 2010;30(10):2537-51.