CLDN18.2 BiTE Engages Effector and Regulatory T Cells for Antitumor Immune Response in Preclinical Models of Pancreatic Cancer.

BACKGROUND & AIMS: BiTE (bispecific T-cell engager) immune therapy has demonstrated clinical activity in multiple tumor indications, but its influence in the tumor microenvironment remains unclear. CLDN18.2 is overexpressed in solid tumors including gastric cancer (GC) and pancreatic ductal adenocarcinoma (PDAC), both of which are characterized by the presence of immunosuppressive cells, including regulatory T cells (Tregs) and few effector T cells (Teffs). METHODS: We evaluated the activity of AMG 910, a CLDN18.2-targeted half-life extended (HLE) BiTE molecule, in GC and PDAC preclinical models and cocultured Tregs and Teffs in the presence of CLDN18.2-HLE-BiTE. RESULTS: AMG 910 induced potent, specific cytotoxicity in GC and PDAC cell lines. In GSU and SNU-620 GC xenograft models, AMG 910 engaged human CD3+ T cells with tumor cells, resulting in significant antitumor activity. AMG 910 monotherapy, in combination with a programmed death-1 (PD-1) inhibitor, suppressed tumor growth and enhanced survival in an orthotopic Panc4.14 PDAC model. Moreover, Treg infusion enhanced the antitumor efficacy of AMG 910 in the Panc4.14 model. In syngeneic KPC models of PDAC, treatment with a mouse surrogate CLDN18.2-HLE-BiTE (muCLDN18.2-HLE-BiTE) or the combination with an anti-PD-1 antibody significantly inhibited tumor growth. Tregs isolated from mice bearing KPC tumors that were treated with muCLDN18.2-HLE-BiTE showed decreased T cell suppressive activity and enhanced Teff cytotoxic activity, associated with increased production of type I cytokines and expression of Teff gene signatures. CONCLUSIONS: Our data suggest that BiTE molecule treatment converts Treg function from immunosuppressive to immune enhancing, leading to antitumor activity in immunologically "cold" tumors.

Overcome the challenge for intratumoral injection of STING agonist for pancreatic cancer by systemic administration.

Due to the challenge for intratumoral administration, innate agonists have not made it beyond preclinical studies for efficacy testing in most tumor types. Pancreatic ductal adenocarcinoma (PDAC) has a hostile tumor microenvironment that renders T cells dysfunctional. Innate agonist treatments may serve as a T cell priming mechanism to sensitize PDACs to anti-PD-1 antibody (a-PD-1) treatment. Using a transplant mouse model with spontaneously formed liver metastasis, a genetically engineered KPC mouse model that spontaneously develops PDAC, and a human patient-derived xenograft model, we compared the antitumor efficacy between intrahepatic/intratumoral and intramuscular systemic administration of BMS-986301, a next-generation STING agonist. Flow cytometry, Nanostring, and cytokine assays were used to evaluate local and systemic immune responses. This study demonstrated that administration of STING agonist systemically via intramuscular injection is equivalent to its intratumoral injection in inducing both effector T cell response and antitumor efficacy. Compared to intratumoral administration, T cell exhaustion and immunosuppressive signals induced by systemic administration were attenuated. Nonetheless, either intratumoral or systemic treatment of STING agonist was associated with increased expression of CTLA-4 on tumor-infiltrating T cells. However, the combination of a-PD-1 and anti-CTLA-4 antibody with systemic STING agonist demonstrated the antitumor efficacy in the KPC mouse spontaneous PDAC model. The mouse pancreatic and liver orthotopic model of human patient-derived xenograft reconstituted with PBMC also showed that antitumor and abscopal effects of both intratumoral and intramuscular STING agonist are equivalent. Taken together, this study supports the clinical development of innate agonists via systemic administration for treating PDAC.

PanIN and CAF transitions in pancreatic carcinogenesis revealed with spatial data integration.

This study introduces a new imaging, spatial transcriptomics (ST), and single-cell RNA-sequencing integration pipeline to characterize neoplastic cell state transitions during tumorigenesis. We applied a semi-supervised analysis pipeline to examine premalignant pancreatic intraepithelial neoplasias (PanINs) that can develop into pancreatic ductal adenocarcinoma (PDAC). Their strict diagnosis on formalin-fixed and paraffin-embedded (FFPE) samples limited the single-cell characterization of human PanINs within their microenvironment. We leverage whole transcriptome FFPE ST to enable the study of a rare cohort of matched low-grade (LG) and high-grade (HG) PanIN lesions to track progression and map cellular phenotypes relative to single-cell PDAC datasets. We demonstrate that cancer-associated fibroblasts (CAFs), including antigen-presenting CAFs, are located close to PanINs. We further observed a transition from CAF-related inflammatory signaling to cellular proliferation during PanIN progression. We validate these findings with single-cell high-dimensional imaging proteomics and transcriptomics technologies. Altogether, our semi-supervised learning framework for spatial multi-omics has broad applicability across cancer types to decipher the spatiotemporal dynamics of carcinogenesis.

Phosphodiesterase-5 inhibition collaborates with vaccine-based immunotherapy to reprogram myeloid cells in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is highly lethal and resistant to immunotherapy. Although immune recognition can be enhanced with immunomodulatory agents including checkpoint inhibitors and vaccines, few patients experience clinical efficacy because the tumor immune microenvironment (TiME) is dominated by immunosuppressive myeloid cells that impose T cell inhibition. Inhibition of phosphodiesterase-5 (PDE5) was reported to downregulate metabolic regulators arginase and inducible NOS in immunosuppressive myeloid cells and enhance immunity against immune-sensitive tumors, including head and neck cancers. We show for the first time to our knowledge that combining a PDE5 inhibitor, tadalafil, with a mesothelin-specific vaccine, anti-programmed cell death protein 1, and anti-cytotoxic T lymphocyte-associated protein 4 yields antitumor efficacy even against immune-resistant PDAC. To determine immunologic advantages conferred by tadalafil, we profiled the TiME using mass cytometry and single-cell RNA-sequencing analysis with Domino to infer intercellular signaling. Our analyses demonstrated that tadalafil reprograms myeloid cells to be less immunosuppressive. Moreover, tadalafil synergized with the vaccine, enhancing T cell activation including mesothelin-specific T cells. Tadalafil treatment was also associated with myeloid/T cell signaling axes important for antitumor responses (e.g., Cxcr3, Il12). Our study shows that PDE5 inhibition combined with vaccine-based immunotherapy promotes pro-inflammatory states of myeloid cells, activation of T cells, and enhanced myeloid/T cell crosstalk to yield antitumor efficacy against immune-resistant PDAC.

Transfer Learning Reveals Cancer-Associated Fibroblasts Are Associated with Epithelial-Mesenchymal Transition and Inflammation in Cancer Cells in Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy characterized by an immunosuppressive tumor microenvironment enriched with cancer-associated fibroblasts (CAF). This study used a convergence approach to identify tumor cell and CAF interactions through the integration of single-cell data from human tumors with human organoid coculture experiments. Analysis of a comprehensive atlas of PDAC single-cell RNA sequencing data indicated that CAF density is associated with increased inflammation and epithelial-mesenchymal transition (EMT) in epithelial cells. Transfer learning using transcriptional data from patient-derived organoid and CAF cocultures provided in silico validation of CAF induction of inflammatory and EMT epithelial cell states. Further experimental validation in cocultures demonstrated integrin beta 1 (ITGB1) and vascular endothelial factor A (VEGFA) interactions with neuropilin-1 mediating CAF-epithelial cell cross-talk. Together, this study introduces transfer learning from human single-cell data to organoid coculture analyses for experimental validation of discoveries of cell-cell cross-talk and identifies fibroblast-mediated regulation of EMT and inflammation. SIGNIFICANCE: Adaptation of transfer learning to relate human single-cell RNA sequencing data to organoid-CAF cocultures facilitates discovery of human pancreatic cancer intercellular interactions and uncovers cross-talk between CAFs and tumor cells through VEGFA and ITGB1.

CD4 T cell-activating neoantigens enhance personalized cancer vaccine efficacy.

Personalized cancer vaccines aim to activate and expand cytotoxic antitumor CD8+ T cells to recognize and kill tumor cells. However, the role of CD4+ T cell activation in the clinical benefit of these vaccines is not well defined. We previously established a personalized neoantigen vaccine (PancVAX) for the pancreatic cancer cell line Panc02, which activates tumor-specific CD8+ T cells but required combinatorial checkpoint modulators to achieve therapeutic efficacy. To determine the effects of neoantigen-specific CD4+ T cell activation, we generated a vaccine (PancVAX2) targeting both major histocompatibility complex class I- (MHCI-) and MHCII-specific neoantigens. Tumor-bearing mice vaccinated with PancVAX2 had significantly improved control of tumor growth and long-term survival benefit without concurrent administration of checkpoint inhibitors. PancVAX2 significantly enhanced priming and recruitment of neoantigen-specific CD8+ T cells into the tumor with lower PD-1 expression after reactivation compared with the CD8+ vaccine alone. Vaccine-induced neoantigen-specific Th1 CD4+ T cells in the tumor were associated with decreased Tregs. Consistent with this, PancVAX2 was associated with more proimmune myeloid-derived suppressor cells and M1-like macrophages in the tumor, demonstrating a less immunosuppressive tumor microenvironment. This study demonstrates the biological importance of prioritizing and including CD4+ T cell-specific neoantigens for personalized cancer vaccine modalities.

Digitize your Biology! Modeling multicellular systems through interpretable cell behavior.

Cells are fundamental units of life, constantly interacting and evolving as dynamical systems. While recent spatial multi-omics can quantitate individual cells' characteristics and regulatory programs, forecasting their evolution ultimately requires mathematical modeling. We develop a conceptual framework-a cell behavior hypothesis grammar-that uses natural language statements (cell rules) to create mathematical models. This allows us to systematically integrate biological knowledge and multi-omics data to make them computable. We can then perform virtual "thought experiments" that challenge and extend our understanding of multicellular systems, and ultimately generate new testable hypotheses. In this paper, we motivate and describe the grammar, provide a reference implementation, and demonstrate its potential through a series of examples in tumor biology and immunotherapy. Altogether, this approach provides a bridge between biological, clinical, and systems biology researchers for mathematical modeling of biological systems at scale, allowing the community to extrapolate from single-cell characterization to emergent multicellular behavior.

Multi-omic analyses of changes in the tumor microenvironment of pancreatic adenocarcinoma following neoadjuvant treatment with anti-PD-1 therapy.

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.

Reduced synchroneity of intra-islet Ca2+ oscillations in vivo in Robo-deficient ß cells.

The spatial architecture of the islets of Langerhans is hypothesized to facilitate synchronized insulin secretion among ß cells, yet testing this in vivo in the intact pancreas is challenging. Robo ßKO mice, in which the genes Robo1 and Robo2 are deleted selectively in ß cells, provide a unique model of altered islet spatial architecture without loss of ß cell differentiation or islet damage from diabetes. Combining Robo ßKO mice with intravital microscopy, we show here that Robo ßKO islets have reduced synchronized intra-islet Ca2+ oscillations among ß cells in vivo. We provide evidence that this loss is not due to a ß cell-intrinsic function of Robo, mis-expression or mis-localization of Cx36 gap junctions, or changes in islet vascularization or innervation, suggesting that the islet architecture itself is required for synchronized Ca2+ oscillations. These results have implications for understanding structure-function relationships in the islets during progression to diabetes as well as engineering islets from stem cells.

Implantation of a neoantigen-targeted hydrogel vaccine prevents recurrence of pancreatic adenocarcinoma after incomplete resection.

Tumor involvement of major vascular structures limits surgical options in pancreatic adenocarcinoma (PDAC), which in turn limits opportunities for cure. Despite advances in locoregional approaches, there is currently no role for incomplete resection. This study evaluated a gelatinized neoantigen-targeted vaccine applied to a grossly positive resection margin in preventing local recurrence. Incomplete surgical resection was performed in mice bearing syngeneic flank Panc02 tumors, leaving a 1 mm rim adherent to the muscle bed. A previously validated vaccine consisting of neoantigen peptides, a stimulator of interferon genes (STING) agonist and AddaVaxTM (termed PancVax) was embedded in a hyaluronic acid hydrogel and applied to the tumor bed. Tumor remnants, regional lymph nodes, and spleens were analyzed using histology, flow cytometry, gene expression profiling, and ELISPOT assays. The immune microenvironment at the tumor margin after surgery alone was characterized by a transient influx of myeloid-derived suppressor cells (MDSCs), prolonged neutrophil influx, and near complete loss of cytotoxic T cells. Application of PancVax gel was associated with enhanced T cell activation in the draining lymph node and expansion of neoantigen-specific T cells in the spleen. Mice implanted with PancVax gel demonstrated no evidence of residual tumor at two weeks postoperatively and healed incisions at two months postoperatively without local recurrence. In summary, application of PancVax gel at a grossly positive tumor margin led to systemic expansion of neoantigen-specific T cells and effectively prevented local recurrence. These findings support further work into locoregional adjuncts to immune modulation in PDAC.