Betrixaban activates cGAS-STING to promote antitumor immunity without pathological inflammation

Advances in Immunogenic Cell Death for Cancer Immunotherapy

Immunogenicity of most cancer types is a result of either neoantigenic mutational load, which stokes up an adaptive immune response, or due to oncogenic stress pathways, which elicit an innate antitumor response. There have been exponential gains made in recent times using numerous strategies to reactivate the host antitumor immunity including the development of immune checkpoint inhibitors. Nonetheless, the promise of a cure and durable response in select patients does not refute the low response rates in advanced cases, most of which also relapse. These observations shore up the possibility of other mechanisms beyond the inactivation of local lymphocyte infiltrates that might also play in tumor cell evasion of antitumor immune response. Through comprehensive computational and follow-up experimental validations, we have found that a subset of cancers (~15-20% of all cancers) are characterized by severe defects in almost the entire epigenetic and transcriptional apparatus. These defects result in genome-wide deregulation of histone modifications, mRNA transcription elongation, and mRNA processing and nuclear export, especially in the long genes which we term as Transcriptional Elongation Deficient: TEdef. As such, cancer cells with TEdef suppressed the expression of the pathways enriched for long genes, such as proinflammatory signaling pathways (FasL response, TNF/NF-kB signaling, interferon signaling) at both mRNA and protein levels, and had diminished response to interferon and TNF stimuli. Remarkably, in renal cell carcinoma and metastatic melanoma patients in four cohorts, the TEdef phenotype significantly correlated with poor response and unfavorable outcome to immunotherapy, but not to chemo- or targeted therapy. Importantly, forced induction of TEdef in tumor cells impaired the expression of, and signaling through, the proinflammatory pathways, and imposed a resistance to the innate and adaptive antitumor immune responses and to immune checkpoint inhibitor therapy in vivo. Tumor lymphocyte infiltration (TIL) and somatic neoepitope load (SNL) are some of the best markers of immunotherapy response in the clinic. However, TEdef tumors were characterized by a higher rate of immune cell infiltration, but paradoxically, less immune-mediated local tumor cell lysis, further supporting the notion that TEdef is a tumor cell-autonomous mechanism of immune resistance. As such, combining TIL or SNL with TEdef had superior power in predicting the progression-free and overall survival of melanoma patients treated with the anti-CTLA4 and anti-PD-1 therapy. Overall, TEdef is a novel epigenetic mechanism of resistance to antitumor immune attack, which warrants its assessment in cancer patients undergoing immunotherapy. Citation Format: Vishnu Modur. Defective transcription elongation in a subset of cancers confers immunotherapy resistance [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B083.

Ethics Approval This study was approved by C.E.I.M. reference 20/1615 (E.C.P.S.) (Spain); CUHK-NTEC CREC 2021.294-T (Hong Kong); CMO Arnhem-Nijmegen Region NL-number: NL76406.091.21 (Netherlands). Consent The patient was identified as an...

SummaryProtein phosphatase 1 regulatory subunit 15A (PPP1R15A) is an important factor in the integrated stress response (ISR) in mammals and may play a crucial role in tumorigenesis. In our studies, we found an inhibitor of PPP1R15A, Sephin1, plays a protumorigenic role in mouse tumor models. By analyzing the single-cell transcriptome data of the mouse tumor models, we found that in C57BL/6 mice, Sephin1 treatment could lead to higher levels of ISR activity and lower levels of antitumor immune activities. Specifically, Sephin1 treatment caused reductions in antitumor immune cell types and lower expression levels of cytotoxicity-related genes. In addition, T cell receptor (TCR) repertoire analysis demonstrated that the clonal expansion of tumor-specific T cells was inhibited by Sephin1. A special TCR + macrophage subtype in tumor was identified to be significantly depleted upon Sephin1 treatment, implying its key antitumor role. These results suggest that PPP1R15A has the potential to be an effective target for tumor therapy.

Removing the B (cell) STING to improve cancer immunotherapy.

Background: One cause for the failure of checkpoint inhibitors is the immunosuppressive nature of the tumor microenvironment. LILRB1 (ILT2) and LILRB2 (ILT4) are ITIM-containing inhibitory receptors that recognize HLA Class 1 and nonclassical ligands (e.g., HLA-A, HLA-G, etc.). LILRB1 is expressed on myeloid cells and subsets of B, NK, and T cells, while LILRB2 expression is mostly restricted to myeloid cells. Interaction of LILRB1 and LILRB2 receptors with HLA ligands promotes an inhibitory milieu that prevents T cells from attacking cancer cells. The distinct pattern of expression and function of these lymphoid and myeloid checkpoints suggests complementary targeting approaches for cancer immunotherapy. Dual blockade of LILRB1 and LILRB2 receptors by a single antibody that restores both innate and adaptive immune responses is a promising strategy to enhance efficacy of checkpoint inhibitors. Methods: Dual LILRB1 and LILRB2 targeting antibodies were cloned from B cells derived from rabbits immunized with human LILRB2 recombinant protein, and subsequently humanized. Antibodies were evaluated for binding to human LILRB1 and LILRB2 proteins. Dual targeting antibodies were evaluated in a panel of functional and phenotypic assays. Selected antibodies were further tested for efficacy in a humanized NSG-SGM3 tumor model. Results: Dual antibodies were selected based on binding to recombinant human LILRB1 and LILRB2 protein, as well as blocking of HLA-G binding. These antibodies demonstrated binding to cells expressing LILRB1 and LILRB2, with no appreciable binding to other family members. Lead antibodies demonstrated activity in functional cell-based assays modeling LILRB1- or LILRB2-mediated immunosuppression. Dual antibodies also enhanced IFN-γ production by LPS-stimulated human PBMC. Selected clones restored T-cell function from M2c macrophage-mediated suppression in coculture with CD8+ T cell, and enhanced the tumoricidal activity of NK cells. Importantly, the lead antibody demonstrated in vivo efficacy with significant tumor growth inhibition and tumor regression in an SK-MEL-5 tumor model in humanized NSG-SGM3 mice. Conclusions: We have identified dual antagonist antibodies targeting both LILRB1 and LILRB2 antibodies that restore both innate and adaptive immune responses. Additionally, dual antibodies restored CD8+ T cell activation from macrophage-mediated suppression and enhanced NK cell cytotoxic activity. These data provide a strong rationale for further development of dual antibodies as an anti-cancer immunotherapy. Citation Format: Meghan Zuck, Myriam Bouchlaka, Huyen Dinh, Kevin Green, Francisco Zapata, Ramya Chandrasekaran, Tatyana Pisarenko, Lauren Loh, Gajendra S. Naika, Meilyn Sylvestre, Jacob Heit, Raymond Fox, Darbie Whitman, Tom Graddis, Kamal D. Puri, Peter Probst. Discovery and preclinical characterization of dual antagonist antibodies targeting both LILRB1 and LILRB2 that enhance innate and adaptive anti-cancer immune responses [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 6376.

The adaptive immune checkpoints such as PD-1(programmed death-1)/PD-L1 (programmed death-ligand 1) play an important role in cancer immunotherapy, whereas increasing evidence suggests that cancer cell evades immune surveillance by innate immune checkpoints such as SIRPα (signal-regulatory protein α)/CD47 (cluster of differentiation 47). In multiple types of cancer cells and solid tumor tissues, highly expressed CD47 protein level has been observed, which is triggered by some transcription factors including NFκB, Myc, and HIF. As a transmembrane protein, the binding of CD47 to SIRPα ligand on phagocytes results in phagocytosis resistance and cancer cell immune escape. In contrast, CD47-SIRPα interaction blockade enhances cancer cell clearance by phagocytes such as macrophages and dendritic cells (DCs) to activate an innate immune response, whereas this process could promote antigen cross-presentation by antigen present cells (APCs) leading to T cell priming, consequently, activates an adaptive antitumor immune response. In this review, we discussed the current SIRPα-CD47 axis-mediated cancer cell immune escape and immunotherapy, which could provide an effective antitumor strategy by the innate and adaptive immune response.

The innate immune system serves as the first line of defense against viral infections. Type I interferon (IFN-I) signaling, in particular, plays a crucial role in mediating antiviral immunity. Here, we identify Betrixaban (BT), a novel small-molecule compound that activates innate immune responses, leading to broad-spectrum antiviral effects. BT induces IFN-β production and upregulates interferon-stimulated genes (ISGs), effectively suppressing the replication of multiple viruses, including vesicular stomatitis virus (VSV), herpes simplex virus type 1 (HSV-1), murine hepatitis virus strain A59 (MHV-A59), encephalomyocarditis virus (EMCV), and influenza A virus (IAV). BT’s antiviral activity relies on innate immune activation, with IRF3 playing a key role. The antiviral effect was significantly reduced upon loss of ISGs induction, including Mx1 and Mx2. In vivo, BT treatment markedly induced IFNB1 expression across multiple mouse tissues and significantly inhibited viral replication in VSV-infected wild-type mice, confirming the essential role of innate antiviral immune activation. These findings establish BT as a potent stimulator of the innate immune system, demonstrating broad-spectrum antiviral potential and highlighting its promise as a therapeutic agent.

Ex vivo effects of high‐density lipoprotein exposure on the lipopolysaccharide‐induced inflammatory response in patients with severe cirrhosis†

Gene vaccines against defined antigens represent a novel and promising immunization approach for cancer immunotherapy and for battling infectious diseases. Immunization with plasmid DNA is the simplest gene-based approach. In order to induce protective antitumor immunity, gene vaccines are designed to deliver one or several genes encoding tumor-associated antigens, thereby eliciting or augmenting antigen-specific immune responses. The efficacy of gene vaccines can be significantly improved through integration of advances in immunology and molecular biology. Recent evidences point out the central role of dendritic cells and show the importance of innate immune responses in the induction and enhancement of antigen-specific adaptive immune responses. Hence, manipulations that integrate both tumorassociated antigens and danger signals in the vaccine design, can achieve activation of both innate and adaptive immune responses, thereby overcoming the self-tolerance towards many tumor antigens. Coadministration of genetic adjuvants and optimized prime-boost strategies enhance the efficacy of gene vaccines. In this context, strategies that target antigens of choice to dendritic cells and induce, in vivo, antitumor immune responses are discussed. This review highlights vaccine strategies based on transfer of nucleic acid sequences encoding well-defined tumor-associated antigens and genetic adjuvants to the host in vivo in order to induce successful antitumor immunity. Keywords: gene vaccines, dna vaccination, rna, prime-boost, cancer immunotherapy

B cells are emerging as key players in anti-tumor immunity. Their ability to produce antibodies against tumor-associated antigens makes them potent agents in cancer immunotherapy. However, B cell-based therapies remain underutilized compared with T cell-focused approaches. Tumor-infiltrating B cells can form tertiary lymphoid structures (TLS) that resemble lymphoid follicles of inflamed lymph nodes. Significant evidence indicates that TLS are associated with enhanced anti-tumor immunity, and the presence of TLS correlates with improved prognosis and better therapeutic responses across various cancers. However, the development of B cell-dependent anti-tumor immunity might be generated mainly in the tumor-draining lymph nodes, and TLS formation may be a bystander effect of the strong local immune responses. Thus, direct evidence for TLS-B cells contributing to anti-tumor immunity is lacking. Recently, our laboratory discovered that simultaneous activation of innate immune effectors, stimulator of interferon genes (STING) and lymphotoxin beta receptor (LTβR), by their agonists induces mature TLS with germinal center B cell responses in various mouse tumor models. In the current study, we investigated the role of TLS using CD79a knockout mice, which cannot develop TLS due to B cell deficiency. We also leveraged hybridoma technology to generate monoclonal antibodies from TLS-B cells for potential tumor targeting. We show that, in wild type mice, the STING activation improved the fitness of LTβR-induced TLS with B cell maturation to IgG+ long-lived plasma cells and memory cells. This treatment reduced the tumor size and heightened the efficacy of anti-PD-1 immune checkpoint inhibition, leading to complete cure. When used as neoadjuvant therapy, it prevented tumor recurrence and metastasis with 100% survival post-tumor rechallenge, indicating the effective immunization against tumors. Functional TLS formed in different tumor types and anatomical sites using this approach. Hybridoma clones developed from TLS-B cells of these tumors generated monoclonal IgG antibodies targeting mouse pancreatic cancer cells, which effectively induced NK cell-mediated cytotoxicity. In contrast, B cell-deficient CD79a KO mice failed to develop TLS while still being capable of developing high endothelial venules in primary tumors. T cell infiltration did not appear to be reduced by the B cell deficiency. However, these mice showed significantly reduced survival (40%) after tumor rechallenge, underscoring the importance of TLS-B cells for coordinated anti-tumor immune responses. These findings underscore the critical role of B cells in the development of functional TLS and strong anti-tumor immunity. The STING and LTβR agonist combination therapeutically induces TLS to produce tumor-specific high-affinity antibodies with significant tumor-killing potential. This approach highlights a promising strategy for targeting immune-cold tumors that are refractory to standard immunotherapies, paving the way for innovative immunotherapies that harness the power of B cells. Citation Format: Maxwell Duah, Yasuhiro Kikuchi, Tomoko Stansel, Krisztian Csomos, Nobuyoshi Hiraoka, Jolan Walter, Carl F Ware, Masanobu Komatsu. Intratumoral B cells are essential for anti-tumor immune responses of tertiary lymphoid structures: Impact of STING and LTβR activation in pancreatic cancer [abstract]. In: Proceedings of the AACR IO Conference: Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2025 Feb 23-26; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2025;13(2 Suppl):Abstract nr PR001.

Target-based immunocytokine approaches have been reported to be efficacious in the control of tumor growth in preclinical and clinical studies. By targeting cytokines-activated immune effectors to local tumor sites, an antibody-based immunocytokine is able to achieve antitumor immunity in the tumor microenvironment while reducing cytokines-mediated systemic side effects. Recently, immune checkpoint antagonists to PD-1/PD-L1 have shown success in certain clinical settings across multiple cancer types. However, the full potential of the checkpoint inhibitor is limited due to impaired overall antitumor immunity. It is therefore desirable to develop immunotherapeutics with the capacity of simultaneously inhibiting immunosuppressive pathways and stimulating immune effector cells to potentiate innate and adaptive immune responses against tumor growth. To this end, we generated a bifunctional fusion protein, KD033, composed of an antibody specific for PD-L1 and IL-15 as a novel immunocytokine for achieving better immunotherapeutic efficacy against tumors. Previously, we demonstrated that KD033 has an enhanced immunological activity and stronger antitumor efficacy in some syngeneic mouse tumor models in comparison to single agents. In the present report, we show that the mechanisms of actions of the bifunctional protein in the enhancement of antitumor immune responses results from an increase in Th1 cytokine secretion, the expansion and cytotoxicity of CD8 T-cells and NK cells and a decrease in immunosuppressive cells, i.e. regulatory T cells and myeloid derived suppressive cells in a number of preclinical experimental models. In the preclinical studies, KD033 regimens, with the unique immunological properties, led to stronger anti-tumor efficacy in controlling primary tumor growth and prolonging the survival of tumor bearing mice in a number of mouse tumor models including PDX and GEMM tumor models. Importantly, the PD-L1-targeted IL-15 bifunctional protein had significantly less cytokine-related toxicity when compared to non-targeted full IgG antibody-IL-15 fusion protein in vivo. These results further elucidate the capacity of targeting IL-15-stimulated innate and adaptive immune effector cells into tumor microenvironment, thereby effectively controlling tumor progression while having minimized adverse effect in vivo. These encouraging preclinical results of the novel immunotherapeutics suggest further advancement of this innovative therapeutic candidate towards clinical development for cancer treatment. Citation Format: Yan Wu, Zhaojing Zhong, Stella Martomo, Dan Lu, Haifan Zhang, Zhanna Polonskaya, Xenia Luna, Zhikai Zhang, Zhun Wang, Leo Liu, Jeegar Patel, James Tonra, Henry Li, Larry Witte, Sam Waksal, Zhenping Zhu. Novel anti-PD-L1/IL-15 bifunctional immunotherapeutics potentiates antitumor immunity. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4997.

Toll-like Receptors (TLRs) are a class of Pattern Recognition Receptors (PRRs) capable of recognizing pathogen-associated molecular patterns (PAMPs) (1) and damage-associated molecular patterns (DAMPs) (2) to initiate immune responses. TLRs are crucial for the detection of infections, and activating downstream signaling pathways that lead to the production of pro-inflammatory cytokines and interferons, thereby initiating host defense mechanisms against a wide range of pathogens (3). Moreover, TLRs are involved in the crosstalk between the innate and adaptive immune systems, as their activation can influence the development of antigenspecific adaptive immune responses (4). TLR ligands differentially regulate the function of dendritic cells that play a central role during priming and activation of naive T cells (5). Understanding the complex interplay of TLRs in immune regulation not only contributes to our comprehension of host defense mechanisms but also has implications for the development of vaccines, immunotherapies, and treatments for various autoimmune and inflammatory disorders such as cancer.Recent developments in the field of TLRs and cancer immunity underscore the dual role of TLRs in cancer development: promoting or inhibiting tumor growth. TLR agonists are being explored as adjuvants in cancer vaccines and immunotherapies, thereby enhancing the activation of dendritic cells and adaptive immune responses against cancer. However, the intricate interplay between TLRs and the tumor microenvironment has been increasingly recognized, with some TLRs implicated in supporting an immunosuppressive milieu that benefits tumor growth. Velasco et al. demonstrated that TLR signaling blockade significantly decrease COPDlike inflammation dependent tumor onset in the lung using k-ras driven lung adeno-carcinoma model (6).Researchers are now delving into the specific TLR subtypes, their differential effects on immune cells, and the development of TLR-targeted therapies that could either enhance the immune response against cancer or attenuate immunosuppressive signals, all of which hold significant promise in advancing cancer immunotherapy strategies (7). Interestingly, Liu et al. showed that LPS signaling enhanced tumor apoptotic activity of IAP targeting therapy, suggesting TLR signaling may use "synthetic lethal" approach for reducing its toxicity (8).In recent years much scientific interest has focused on improving the efficacy of cancer immunotherapy. In the realm of cancer immunotherapy, TLRs are garnering increased attention as potential therapeutic targets. TLR agonists are being explored for their capacity to stimulate innate immune responses, such as the activation of dendritic cells and natural killer cells, which can bolster antitumor immunity. Combining TLR agonists with other immunotherapies like checkpoint inhibitors or adoptive cell therapies holds promise in creating synergistic effects to enhance cancer treatment outcomes (7). However, there is ongoing research to better understand the nuances of TLR signaling in the context of different cancer types and patient populations to optimize their use in personalized cancer immunotherapies. Furthermore, efforts to mitigate potential side effects and enhance the specificity of TLR-targeted treatments are also underway, indicating a dynamic and evolving field within cancer immunotherapy. Ota et al. tried to achieve efficacy-safety margin by utilizing small molecule TLR7 specific agonist which shows rapid clearance from the body (9).The therapeutic prospects for TLRs are exceptionally promising, as they represent a critical avenue in both the understanding and application of immunotherapy. In this topic there are 3 review articles discussing the strategy of clinical application by targeting TLR signaling, especially in the oncology field. Hoden et al. focuses on TLR agonists for use in lung cancer (7). More broadly, Yang et al. focuses more on the mechanisms and cutting-edge technologies of various tumor types (10) and Mukheerjee et al. explores cancer type specific strategies (11). Ongoing research is likely to uncover novel TLR subtypes and ligands, shedding light on their nuanced roles in diverse disease contexts, including cancer, infectious diseases, and autoimmune disorders. Additionally, the development of more refined TLR-targeted therapies, such as selective agonists or antagonists, offers the potential for precise modulation of immune responses, minimizing unwanted side effects. Harnessing TLRs as adjuvants in combination with emerging immunotherapies and personalized medicine approaches may further revolutionize the treatment of various conditions, ultimately paving the way for more effective and tailored therapeutic interventions. This evolving field holds great promise for the continued advancement of immunotherapy and precision medicine in the years to come.

Chlamydia trachomatisinfection is the most common bacterial sexually transmitted infection worldwide and a leading cause of inflammatory reproductive tract disease and infertility in women. Much of the tissue damage associated with genital chlamydial infection arises from host inflammatory responses rather than direct bacterial cytotoxicity. Epithelial cells lining the female reproductive tract represent the primary host cells infected during chlamydial infection and play key roles in initiating innate immune responses. Among the cytokines produced by infected epithelial cells, type-I interferons have emerged as important regulators of host defense and inflammatory signaling; however, the specific contribution of interferon-β (IFN-β) to epithelial transcriptional responses during chlamydial infection remains incompletely defined.In the present study, we investigated the role of IFN-β in coordinating epithelial immune signaling networks during infection with Chlamydia muridarum. Using wild-type murine oviduct epithelial cells (OE-WT) and IFN-β-deficient epithelial cells (OE-IFNβ-KO), we performed pathway-focused RT² Profiler PCR array analyses examining transcriptional responses across four biological pathways: (1) innate and adaptive immune responses, (2) type-I interferon signaling, (3) inflammatory and autoimmune responses, and (4) fibrosis-associated pathways. Infection of OE-WT cells resulted in coordinated induction of cytokines, chemokines, and interferon-stimulated genes associated with antimicrobial defense and immune cell recruitment. In contrast, IFN-β deficiency resulted in widespread dysregulation of these transcriptional programs, including reduced induction of interferon-responsive chemokines such as CCL5 and CXCL10, altered inflammatory cytokine expression, and transcriptional signatures consistent with enhanced tissue remodeling responses.Notably, IFN-β deficiency resulted in increased TNF expression accompanied by reduced IL-6 induction, suggesting disruption of balanced inflammatory signaling networks. Pathway analyses further revealed dysregulated expression of fibrosis-associated genes includingSerpine1, Ctgf,andEngin IFN-β-deficient epithelial cells, indicating potential mechanisms linking interferon signaling to tissue remodeling during infection. Collectively, these findings identify IFN-β as a central regulator of epithelial immune networks during chlamydial infection and suggest that disruption of IFN-β signaling may promote inflammatory and fibrotic pathology within the female reproductive tract.Author SummarySexually transmitted infections caused byChlamydia trachomatisare a major cause of infertility worldwide. Although antibiotic treatment can eliminate the bacteria, damage to the reproductive tract often results from the body’s own immune response to infection. The epithelial cells lining the reproductive tract are the first cells infected and play an important role in initiating immune responses. In this study, we investigated how a specific immune signaling molecule, interferon-β (IFN-β), regulates the gene expression programs activated in epithelial cells during chlamydial infection. Using pathway-focused gene expression arrays, we found that IFN-β coordinates multiple immune pathways, including interferon signaling, inflammatory cytokine networks, and genes associated with tissue remodeling. When IFN-β was absent, many of these pathways became dysregulated, resulting in altered inflammatory signaling and gene expression patterns linked to fibrosis. These findings suggest that IFN-β functions as a key regulator that helps balance protective immune responses with inflammatory processes that can damage reproductive tissues during infection.

Coadministration of Telomerase Genetic Vaccine and a Novel TLR9 Agonist in Nonhuman Primates