Catherine Bedard

Fibrinogen-induced perivascular microglial clustering is required for the development of axonal damage in neuroinflammation

Blood-brain barrier disruption, microglial activation and neurodegeneration are hallmarks of multiple sclerosis. However, the initial triggers that activate innate immune responses and their role in axonal damage remain unknown. Here we show that the blood protein fibrinogen induces rapid microglial responses toward the vasculature and is required for axonal damage in neuroinflammation. Using in vivo two-photon microscopy, we demonstrate that microglia form perivascular clusters before myelin loss or paralysis onset and that, of the plasma proteins, fibrinogen specifically induces rapid and sustained microglial responses in vivo. Fibrinogen leakage correlates with areas of axonal damage and induces reactive oxygen species release in microglia. Blocking fibrin formation with anticoagulant treatment or genetically eliminating the fibrinogen binding motif recognized by the microglial integrin receptor CD11b/CD18 inhibits perivascular microglial clustering and axonal damage. Thus, early and progressive perivascular microglial clustering triggered by fibrinogen leakage upon blood-brain barrier disruption contributes to axonal damage in neuroinflammatory disease.

Blood coagulation protein fibrinogen promotes autoimmunity and demyelination via chemokine release and antigen presentation

Autoimmunity and macrophage recruitment into the central nervous system (CNS) are critical determinants of neuroinflammatory diseases. However, the mechanisms that drive immunological responses targeted to the CNS remain largely unknown. Here we show that fibrinogen, a central blood coagulation protein deposited in the CNS after blood–brain barrier disruption, induces encephalitogenic adaptive immune responses and peripheral macrophage recruitment into the CNS leading to demyelination. Fibrinogen stimulates a unique transcriptional signature in CD11b+ antigen-presenting cells inducing the recruitment and local CNS activation of myelin antigen-specific Th1 cells. Fibrinogen depletion reduces Th1 cells in the multiple sclerosis model, experimental autoimmune encephalomyelitis. Major histocompatibility complex (MHC) II-dependent antigen presentation, CXCL10- and CCL2-mediated recruitment of T cells and macrophages, respectively, are required for fibrinogen-induced encephalomyelitis. Inhibition of the fibrinogen receptor CD11b/CD18 protects from all immune and neuropathologic effects. Our results show that the final product of the coagulation cascade is a key determinant of CNS autoimmunity.

Early detection of thrombin activity in neuroinflammatory disease

Although multiple sclerosis (MS) has been associated with the coagulation system, the temporal and spatial regulation of coagulation activity in neuroinflammatory lesions is unknown. Using a novel molecular probe, we characterized the activity pattern of thrombin, the central protease of the coagulation cascade, in experimental autoimmune encephalomyelitis. Thrombin activity preceded onset of neurological signs, increased at disease peak, and correlated with fibrin deposition, microglial activation, demyelination, axonal damage, and clinical severity. Mice with a genetic deficit in prothrombin confirmed the specificity of the thrombin probe. Thrombin activity might be exploited for developing sensitive probes for preclinical detection and monitoring of neuroinflammation and MS progression. Ann Neurol 2014;75:303–308

Abstract B029: OMX: An oxygen carrier biotherapeutic that ameliorates the hypoxic tumor microenvironment and promotes anticancer T cell activity

A hypoxic microenvironment is a hallmark of cancer that has been shown in numerous cancer types to drive tumor progression and poor patient outcomes. Hypoxia promotes tumor evasion of the host immune responses by generating an immunosuppressive tumor microenvironment through activation of multiple pathways mediated predominantly, but not exclusively, by hypoxia inducible factor-1 (HIF-1) signaling. We have previously shown that Omniox9 lead anti-cancer immunotherapeutic, OMX, is well tolerated in small and large animals, efficiently accumulates in a variety of orthotopic and subcutaneous rodent tumor models and spontaneous canine melanomas and brain cancers, and effectively reduces tumor hypoxia as assessed by ex vivo immunoassays using hypoxia markers, in vivo FMISO PET imaging, and direct intratumor pO2 measurements with optical probes. Here, we used a combination of quantitative immunohistochemistry and flow cytometry to analyze the effects of OMX treatment and dosing regiment on leukocyte infiltration and activity in normoxic and hypoxic tumor regions in multiple syngeneic mouse tumor models (MC38, CT26, 4T1, B16F10). First, we confirmed in our models published findings that cytotoxic T cells (CTL) are predominantly excluded from hypoxic tumor areas. Next, we explored the effect of single and multi-dose OMX treatments on tumor immune cell populations, and demonstrated that a single iv administration of OMX reduces hypoxia and enhances T cell localization in previously hypoxic tumor areas labelled by two independent markers of hypoxia (pimonidazole and CAIX). Furthermore, 12h after a single OMX treatment we observed >85% intra-tumor reduction in immunosuppressive regulatory T cells (Treg). Tumor Treg reduction was maintained and even more pronounced with repeated dosing, resulting in long-term Treg depletion. Importantly, OMX treatment resulted in a 5-10 fold higher CTL/Treg ratio concomitant with a 3-fold increase in the fraction of activated effector T lymphocytes, with no effect on overall leukocyte populations within the tumor. Taken together, our data suggest that OMX treatment changes the tumor microenvironment from an immunosuppressive to an immunopermissive state in multiple tumor types. Results from ongoing OMX+checkpoint inhibitor combination studies will also be presented. In conclusion, by delivering oxygen specifically to the hypoxic tumor microenvironment, OMX may restore anti-cancer immune responses in cancer patients. Given that OMX is well-tolerated and that its mechanism of action is upstream of major immunosuppressive pathways, OMX holds the potential to synergize with multiple immunotherapeutic agents in enhancing tumor control and improving patient outcomes in solid tumors. Citation Format: Kevin G. Leong, Natacha Le Moan, Yuqiong Pan, Philberta Leung, Catherine Bedard, Jon Winger, Stephen PL Cary, Ana Krtolica. OMX: An oxygen carrier biotherapeutic that ameliorates the hypoxic tumor microenvironment and promotes anticancer T cell activity [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B029.

Abstract 3003: OMX-4.80P, a novel H-NOX oxygen carrier that oxygenates hypoxic tumors in multiple tumor models and canine cancer patients, downregulates HIF-1 pathway and increases response to radiation therapy leading to cures

BACKGROUND: Omniox has engineered OMX-4.80P, a PEGylated H-NOX oxygen carrier, as a long-acting therapeutic candidate to enhance radiotherapy (RT) in the treatment of glioblastoma and other solid tumors. Here, we describe the pre-clinical profile of OMX-4.80P, demonstrating it is well tolerated, long-lasting in circulation and tumors, and it penetrates deep into tumor tissue reducing hypoxia and altering hypoxic phenotype by downregulating HIF-1 pathway. Furthermore, it dramatically enhances RT leading to tumor cures. METHODS: We assessed the ability of OMX-4.80P to penetrate tumor tissue and reduce hypoxia in multiple orthotopic and immunocompetent mouse and rat models of glioblastoma and other tumors as well as in spontaneous canine brain tumors in veterinary patients. We measured the efficacy of OMX-4.80P in NSCLC tumors (H460 and Calu 6), and its activity in intracranial glioblastoma models in nude mice (U251), immunocompetent rats (F98) and in spontaneous canine brain tumors. We assessed exogenous hypoxia markers (pimonidazole and CCI-103F) and hypoxia inducible transcriptional factor HIF-1 by IHC and ELISA, and HIF-1 downstream targets by IHC and qRT PCR. We also conducted toxicology and pharmacokinetic studies in mice, rats and in naive and oncology patient dogs. RESULTS: In xenograft studies of large, hypoxic, radioresistant tumors, single doses of OMX-4.80P in combination with RT result in apparent tumor cures in ∼30-50% of tumors compared to 0% cures in RT-only groups. We observed good penetration into mouse and rat intracranial and subcutaneous tumors (∼1 cm3), and into spontaneous canine brain tumors, that resulted in hypoxia reduction, as assessed by OxyLite pO2 probe and pimonidazole and CCI-103F, leading to downregulation of the HIF-1 pathway. Observed dramatic drop in HIF-1α, VEGF, GLUT-1 and PDL-1 levels suggests OMX-4.80P has profound effects on tumor cell phenotype beyond radiosensitization. Pharmacokinetic and toxicology studies using single or multiple supratherapeutic and therapeutic doses of OMX-4.80P in rodents and dogs demonstrated that it has a circulation half-life of ∼20h in rats and ∼30-40h in dogs, and that it is well tolerated. Finally, OMX-4.80P has no detectable immunogenic response. CONCLUSIONS: The preclinical data demonstrating hypoxia reduction, HIF-1 pathway downregulation and radiation enhancement, and promising PK and toxicology profile of OMX-4.80P support its clinical development as a radiosensitizer for multiple types of hypoxic tumors. Furthermore, its ability to alter key downstream effectors of the HIF-1 pathway suggest it may have potential to alter tumor biology and enhance patient responses to variety of targeted and chemo therapies by affecting tumor drug resistance, immune responsiveness, angiogenesis, metabolism and invasion. Citation Format: Ana Krtolica, Natacha Le Moan, Jen Getz, Tina Davis, Sarah Ng, Catherine Bedard, Andrew Davis, Philberta Leung, Laura Serwer, Kevin Tanaka, Tim Keating, Feng Yan, Teri Guerrero, Michael Kent, Peter Dickinson, Jonathan Winger, Stephen P. L. Cary. OMX-4.80P, a novel H-NOX oxygen carrier that oxygenates hypoxic tumors in multiple tumor models and canine cancer patients, downregulates HIF-1 pathway and increases response to radiation therapy leading to cures. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3003. doi:10.1158/1538-7445.AM2015-3003