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BACKGROUND: Hypoxic cells in solid tumors are resistant to radiotherapy. Omniox has developed a novel class of oxygen carrier proteins termed H-NOX, engineered to deliver oxygen selectively into hypoxic tissues. Previous studies have shown that one of our H-NOX candidates, OMX-4.80, penetrates intracranial glioblastoma (GB) tumors in mice, and in a dose dependent manner decreases tumor hypoxia and extends survival when combined with radiotherapy. However, we discovered that the circulation half-life of OMX-4.80 is ∼1h in dogs, which may limit clinical utility. To improve the circulation half-life, we developed a PEGylated version of OMX-4.80 (OMX-4.80P). The aim of this study was to characterize the pharmacokinetic and safety profile of OMX-4.80P and examine its effect on tumor hypoxia. METHODS: After intravenous administration of OMX-4.80P to mice and rats bearing GB tumors, we collected tumors, blood and plasma, and analyzed OMX-4.80P pharmacokinetics, immunogenicity and its effects on blood cell counts and chemistries. We evaluated OMX-4.80P bio-distribution by immunohistochemistry and its effect on tumor hypoxia by ELISA of endogenous (HIF-1a) and exogenous (pimonidazole) hypoxia markers. RESULTS: Compared to OMX-4.80, OMX-4.80P has an increased stability and circulation half-life (30h vs. 1h), significantly longer tumor accumulation (48h vs. 4h), and is not immunogenic. Furthermore, OMX-4.80P crosses the blood tumor barrier in several different intracranial rodent GB models and reduces HIF-1a and pimonidazole accumulation by ∼50%. Pharmacokinetic and toxicology studies in rodents and dogs showed that OMX-4.80P is well tolerated, and has a circulation half-life of ∼52h in dogs. The resulting exposure is expected to be sufficient for OMX-4.80P to accumulate in human GB tumors and reduce hypoxia. CONCLUSIONS: In summary, the increased retention of OMX-4.80P in circulation, the longer exposure achieved in solid tumors and efficiency in delivering oxygen into hypoxic tissues suggest OMX-4.80P is a promising IND candidate for radiation therapy enhancement in GB.

BACKGROUND: Hypoxia in solid tumors blunts the tumor cytotoxicity of radiotherapy (RT) and is a major independent predictor of poor patient outcomes in glioblastoma (GB). To increase tumor oxygenation, Omniox has engineered a novel oxygen delivery protein, OMX-4.80P (OMX), tuned to release oxygen specifically in hypoxic tumor tissue. When given with radiotherapy, OMX significantly enhances RT efficacy in rodent tumor models. Since spontaneous canine brain cancers, including GB, share similarities with human brain cancers in terms of morphology, disease progression, and response to approved therapies, a Phase 0 clinical trial was conducted in patient dogs to assess OMX safety, PK/PD and signs of efficacy to inform human clinical strategy. METHODS: Dogs with presumed brain cancers (MRI) underwent either resection surgery with radiotherapy (Sx + RT) or stereotactic radiosurgery (SRS). In the Sx + RT group, OMX was administered once before surgery (4–48 hours pre-Sx) and twice weekly in conjunction with RT (20x2.5Gy). In the SRS group, OMX was dosed on 2 consecutive days in combination with SRS (3x8Gy). PK, CBC, blood chemistry, coagulation, and immune responses were assessed. Additionally, resected tumors were analyzed for OMX accumulation and hypoxia levels (ELISA, IHC). RESULTS: To date, 14 dogs have completed the trial (10 Sx, 5 Sx + RT, 4 SRS): Five dogs show no signs of tumor regrowth at 3 months and 2 dogs had no visible tumors at 6 months. OMX induced no dose-limiting adverse events and median T1/2 is 35h. Resected tumors show a strong correlation between OMX concentration in tumor tissue and reduced hypoxia with OMX penetrating deep into tumor tissue. CONCLUSIONS: When given in conjunction with RT regimens equivalent to human standard of care, OMX is safe and well-tolerated. Tumor tissue analysis suggests that OMX-4.80P decreases tumor hypoxia, thus supporting its further clinical development for the enhancement of radiotherapy efficacy in GB patients.

BACKGROUND: Hypoxia is a hallmark of glioblastoma (GB) and is correlated with tumor progression and decreased overall survival. OMX-4.80P is a protein oxygen carrier engineered for the treatment of GB in combination with radiotherapy. Prior studies in rodent GB models demonstrated that OMX-4.80P accumulates within intracranial tumors and reduces tumor hypoxia—as assessed by immunohistochemical and ELISA methods—and enhances tumor killing by radiotherapy. To support clinical evaluation of OMX-4.80P in patients, this report describes non-invasive positron emission tomography (PET) imaging to assess both OMX-4.80P tumor accumulation and oxygen delivery. METHODS: To assess its biodistribution, OMX-4.80P was labelled with 89Zr using a desferrioxamine (DFO) chelator. Either 89Zr-DFO-OMX-4.80P or free 89Zr was administered intravenously to both nude mice bearing H460 subcutaneous xenografts, as well as to Fischer rats bearing intracranial F98 tumors. OMX-4.80P distribution was assessed by PET, and by measuring radioactivity in major organs using a gamma counter. To assess OMX-4.80P-mediated changes in tumor hypoxia in the same tumor models, tumor:brain signal ratio of the hypoxia marker fluoromisonidazole (18F-FMISO) was assessed before and after treatment with OMX-4.80P by performing scans on two consecutive days. RESULTS: While free 89Zr was present mainly in the bones, 89Zr-DFO-OMX-4.80P was not, suggesting that the label is stable in vivo. As expected, 89Zr-DFO-OMX-4.80P accumulated in both subcutaneous and intracranial tumors, and in the clearance organs (liver, kidneys, spleen). Furthermore, 18F-FMISO levels, as measured by tumor:brain ratios, were decreased by >2 fold after either intravenous or intratumoral injection of OMX-4.80P, in both subcutaneous and intracranial tumors demonstrating OMX-4.80P-mediated tumor oxygenation activity. CONCLUSIONS: PET-based approaches are a feasible method for assessing OMX-4.80P distribution and oxygenation. These methods will be developed as biomarkers of OMX-4.80P activity in a planned Phase 1 clinical trial in newly diagnosed GB patients.

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 naïve 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

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 (GB) and other solid tumors. We have previously described the un-PEGylated OMX-4.80 variant, which can penetrate intracranial mouse GB tumors, release oxygen exclusively into hypoxic tumor tissue, and enhance tumor growth delay and survival when combined with RT. However, OMX-4.80 has a short circulation half-life that limits its clinical potential. Here, we describe the pre-clinical profile of PEGylated OMX-4.80 (OMX-4.80P), demonstrating it is well tolerated, long-lasting in circulation and tumors, and is significantly more efficacious than OMX-4.80. METHODS: We PEGylated OMX-4.80 to increase its circulation half-life while retaining its ability to preferentially accumulate in tumors and selectively release oxygen to hypoxic microenvironments. We characterized the efficacy of OMX-4.80P in large, hypoxic, radioresistant tumors, and its activity in intracranial GB models in nude mice and immunocompetent rats. We also conducted toxicology and pharmacokinetic studies in multiple animal models including naïve 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. Interestingly, the larger molecular size of OMX-4.80P (∼120kDa) did not prevent its passing the blood-tumor barrier in mouse and rat intracranial tumors and resulted in hypoxia reduction. 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 ∼24h in rats and ∼52h in dogs, compared to 1-2h for OMX-4.80, and that it is well tolerated. Finally, OMX-4.80P has no detectable immunogenic response. CONCLUSIONS: The preclinical efficacy data, and promising pharmacokinetic and toxicology profile, of OMX-4.80P support its clinical development for the enhancement of radiotherapy in GB patients.