Neutralizing Antibodies Impair the Oncolytic Efficacy of Reovirus but Permit Effective Combination with T cell-Based Immunotherapies.

Reovirus type 3 Dearing (Reo), manufactured for clinical application as pelareorep, is an attractive anticancer agent under evaluation in multiple phase 2 clinical trials for the treatment of solid tumors. It elicits its anticancer efficacy by inducing both oncolysis and intratumoral T-cell influx. Because most people have been preexposed to Reo, neutralizing antibodies (NAb) are prevalent in patients with cancer and might present a barrier to effective Reo therapy. Here, we tested serum of patients with cancer and healthy controls (n = 100) and confirmed that Reo NAbs are present in >80% of individuals. To investigate the effect of NAbs on both the oncolytic and the immunostimulatory efficacy of Reo, we established an experimental mouse model with Reo preexposure. The presence of preexposure-induced NAbs reduced Reo tumor infection and prevented Reo-mediated control of tumor growth after intratumoral Reo administration. In B cell-deficient mice, the lack of NAbs provided enhanced tumor growth control after Reo monotherapy, indicating that NAbs limit the oncolytic capacity of Reo. In immunocompetent mice, intratumoral T-cell influx was not affected by the presence of preexposure-induced NAbs and consequently, combinatorial immunotherapy strategies comprising Reo and T-cell engagers or checkpoint inhibitors remained effective in these settings, also after a clinically applied regimen of multiple intravenous pelareorep administrations. Altogether, our data indicate that NAbs hamper the oncolytic efficacy of Reo, but not its immunotherapeutic capacity. Given the high prevalence of seropositivity for Reo in patients with cancer, our data strongly advocate for the application of Reo as part of T cell-based immunotherapeutic strategies.

Combination of pembrolizumab and pelareorep promotes anti-tumour immunity in advanced pancreatic adenocarcinoma (PDAC).

BACKGROUND: We previously reported activity of pelareorep, pembrolizumab and chemotherapy. Patients developed new T-cell clones and increased peripheral T-cell clonality, leading to an inflamed tumour. To evaluate a chemotherapy-free regimen, this study assesses if pelareorep and pembrolizumab has efficacy by inducing anti-tumour immunological changes (NCT03723915). METHODS: PDAC patients who progressed after first-line therapy, received iv pelareorep induction with pembrolizumab every 21-days. Primary objective is overall response rate. Secondary objectives included evaluation of immunological changes within tumour and blood. RESULTS: Clinical benefit rate (CBR) was 42% amongst 12 patients. One patient achieved partial response (PR) and four stable disease (SD). Seven progressed, deemed non-responders (NR). VDAC1 expression in peripheral CD8+ T cells was higher at baseline in CBR than NR but decreased in CBR upon treatment. On-treatment peripheral CD4+ Treg levels decreased in CBR but not in NR. Analysis of tumour demonstrated PD-L1+ cells touching CD8+ T cells, and NK cells were more abundant post-treatment vs. baseline. A higher intensity of PD-L1 in tumour infiltrates at baseline, particularly in CBR vs. NR. Finally, higher levels of soluble (s)IDO, sLag3, sPD-1 observed at baseline among NR vs. CBR. CONCLUSION: Pelareorep and pembrolizumab showed modest efficacy in unselected patients, although potential immune and metabolic biomarkers were identified to warrant further evaluation.

The differential expression of toll like receptors and RIG-1 correlates to the severity of infectious diseases.

The toll like receptors (TLRs) and RIG-1 are proteins involved in the initial reaction of the innate immune system to infectious diseases and, thus, can provide much information to the surgical pathologist in terms of the molecular dynamics of the infection. The TLRs (TLR1, 2, 3, 4, 7, 8) and RIG-1 distribution as determined by immunohistochemistry was examined in the following diseases: human papillomavirus (n = 30 including 15 squamous intraepithelial lesions (SIL), 5 cancers, and 10 controls); molluscum contagiosum (n = 8 including 4 controls), SARS-CoV2 (n = 52 including 20 mild, 5 fatal, and 27 controls) and reovirus infection as oncolytic therapy. Mild, regressing infection (molluscum contagiosum, mild SARS-CoV2 and low grade SIL) each showed the same pattern: marked up regulation of at least three of the TLRs/RIG-1 with decreased expression of none compared to the controls. Severe infection (fatal SARS-CoV2, and cervical cancer) each showed marked decrease expression in at least three of the TLRs/RIG-1. We recently documented an equivalent marked decrease expression of the TLRs/RIG-1 in the placenta in fatal in utero infections. The reoviral infected tissues showed an overall pattern of marked increase expression of TLRs/RIG-1, consistent with a strong anti-viral response. Thus, the in situ testing of infectious diseases by a panel of these early infectious disease recognition proteins may allow the surgical pathologist to predict the outcome of the disease which, in turn, may assist in the understanding of the role of the TLRs/RIG-1 in determining the fate of a given infectious process.

GOBLET: a phase I/II study of pelareorep and atezolizumab +/- chemo in advanced or metastatic gastrointestinal cancers.

Most gastrointestinal (GI) cancers have microsatellite-stable (MSS) tumors, which have an immunologically 'cold' phenotype with fewer genetic mutations, reduced immune cell infiltration and downregulated immune checkpoint proteins. These attributes make MSS tumors resistant to conventional immunotherapy including checkpoint blockade therapy. Pelareorep is a naturally occurring, nongenetically modified reovirus. Upon intravenous administration, pelareorep selectively kills tumor cells and promotes several immunologic changes that prime tumors to respond to checkpoint blockade therapy. Given its demonstrated synergy with checkpoint blockade, as well as its encouraging efficacy in prior GI cancer studies, pelareorep plus atezolizumab will be evaluated in the GOBLET study in multiple GI cancer indications.

The GOBLET study is investigating a new drug combination for gastrointestinal tumors, specifically pancreatic, colorectal and anal cancers, which have already spread or might spread to the body. Currently, the standard treatment in most gastrointestinal tumors still consists of chemotherapy. Newer drugs (immune checkpoint inhibitors [ICIs]), which activate the body's natural defenses (immune system) and consequently increase the triggering of the immune system against tumor cells, have been developed and are commonly used as a single agent or in combination with chemotherapy. Yet, these are only effective in a small subset of patients. Certain drugs can also make tumors respond better to ICIs. One such drug being tested is pelareorep. Pelareorep is a safe virus that detects and kills only cancer cells and has shown promising results by increasing the activity of the patient's immune system toward the tumor in combination with ICIs in previous studies. The new drug combination (ICI plus virus) is used together with or without chemotherapy in this study. The aim of the GOBLET study is to investigate the safety of the new drug combination and assess improvements in tumor size related to treatment. Eudra-CT Number: 2020-003996-16.

Oncolytic virus-mediated expansion of dual-specific CAR T cells improves efficacy against solid tumors in mice.

Oncolytic viruses (OVs) encoding a variety of transgenes have been evaluated as therapeutic tools to increase the efficacy of chimeric antigen receptor (CAR)-modified T cells in the solid tumor microenvironment (TME). Here, using systemically delivered OVs and CAR T cells in immunocompetent mouse models, we have defined a mechanism by which OVs can potentiate CAR T cell efficacy against solid tumor models of melanoma and glioma. We show that stimulation of the native T cell receptor (TCR) with viral or virally encoded epitopes gives rise to enhanced proliferation, CAR-directed antitumor function, and distinct memory phenotypes. In vivo expansion of dual-specific (DS) CAR T cells was leveraged by in vitro preloading with oncolytic vesicular stomatitis virus (VSV) or reovirus, allowing for a further in vivo expansion and reactivation of T cells by homologous boosting. This treatment led to prolonged survival of mice with subcutaneous melanoma and intracranial glioma tumors. Human CD19 CAR T cells could also be expanded in vitro with TCR reactivity against viral or virally encoded antigens and was associated with greater CAR-directed cytokine production. Our data highlight the utility of combining OV and CAR T cell therapy and show that stimulation of the native TCR can be exploited to enhance CAR T cell activity and efficacy in mice.

CD248 enhances tissue factor procoagulant function, promoting arterial and venous thrombosis in mouse models.

BACKGROUND: CD248 is a pro-inflammatory, transmembrane glycoprotein expressed by vascular smooth muscle cells (VSMC), monocytes/macrophages, and other cells of mesenchymal origin. Its distribution and properties are reminiscent of those of the initiator of coagulation, tissue factor (TF). OBJECTIVE: We examined whether CD248 also participates in thrombosis. METHODS: We evaluated the role of CD248 in coagulation using mouse models of vascular injury, and by assessing its functional interaction with the TF-factor VIIa (FVIIa)-factor X (FX) complex. RESULTS: The time to ferric chloride-induced occlusion of the carotid artery in CD248 knockout (KO) mice was significantly longer than in wild-type (WT) mice. In an inferior vena cava (IVC) stenosis model of thrombosis, lack of CD248 conferred relative resistance to thrombus formation compared to WT mice. Levels of circulating cells and coagulation factors, prothrombin time, activated partial thromboplastin time, and tail bleeding times were similar in both groups. Proximity ligation assays revealed that TF and CD248 are <40 nm apart, suggesting a potential functional relationship. Expression of CD248 by murine and human VSMCs, and by a monocytic cell line, significantly augmented TF-FVIIa-mediated activation of FX, which was not due to differential expression or encryption of TF, altered exposure of phosphatidylserine or differences in tissue factor pathway inhibitor expression. Rather, conformation-specific antibodies showed that CD248 induces allosteric changes in the TF-FVIIa-FX complex that facilitates FX activation by TF-FVIIa. CONCLUSION: CD248 is a newly uncovered protein partner and potential therapeutic target in the TF-FVIIa-FX macromolecular complex that modulates coagulation.

Exploring traditional and nontraditional roles for thrombomodulin.

Thrombomodulin (TM) is an integral component of a multimolecular system, localized primarily to the vascular endothelium, that integrates crucial biological processes and biochemical pathways, including those related to coagulation, innate immunity, inflammation, and cell proliferation. These are designed to protect the host from injury and promote healing. The "traditional" role of TM in hemostasis was determined with its discovery in the 1980s as a ligand for thrombin and a critical cofactor for the major natural anticoagulant protein C system and subsequently for thrombin-mediated activation of the thrombin activatable fibrinolysis inhibitor (also known as procarboxypeptidase B2). Studies in the past 2 decades are redefining TM as a molecule with many properties, exhibited via its multiple domains, through its interacting partners, complex regulated expression, and synthesis by cells other than the endothelium. In this report, we review some of the recently reported diverse properties of TM and how these may impact on our understanding of the pathogenesis of several diseases.