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Tumor-associated macrophages (TAMs) play a role in cancer progression and are associated with Sorafenib resistance in hepatocellular carcinoma (HCC)1. D-4559 is a new potent macrophage switching nanomedicine technology that selectively inhibits VEGF receptor tyrosine kinases (VEGFR1, 2, 3) in TAMs leading to a functional reprogramming of TAMs toward a pro-inflammatory activated phenotype. Here, we evaluate the effect of D-4559 on the M1 and M2 polarization of TAMs and its anti-tumor efficacy in a murine HCC tumor model. In vivo efficacy of D-4559 was examined in the subcutaneous Hepa 1-6 liver tumor model in C57BL/6 mice. Animals (n=15/group) were treated with D-4559 (i.p., 200 mg/kg daily) and free drug Sorafenib (p.o., 40 mg/kg daily) as a positive control for 4 weeks. The treatment started when the mean tumor size reached approximately 100 mm3, then the animals were randomly allocated into study groups. The day of randomization and treatment was denoted as day 0. Tumor sizes were measured using a caliper for 27 days, M1/M2 macrophage polarization was examined by flow cytometry at day 16, and cytokine biomarkers were evaluated with MSD Cytokine Multiplex Assay at day 16 and 27.D-4559 significantly reduced Hepa 1-6 tumor growth (**p References: 1. Wang et al, Journal of Biomedical Science (2022) 2. Hayashi, T., et al. BMC Cancer 17, 870 (2017) 3. Li, H., et al., Translational Cancer Research 8, 2 (2019) Citation Format: Naze G. Avci, Tony Wu, Susan E. Alters, Emily Zhang, Jinping Liu, Dong Huang, Natacha Le Moan, Jeffrey L. Cleland. A potent macrophage switching drug D-4559 reduces tumor GROWTH in a hepatocellular carcinoma mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB208.

BACKGROUND: Intratumoral hypoxia is associated with resistance to chemo- and radio-therapies and poor patient outcomes. In addition, hypoxia promotes the immune escape of tumors by altering the recruitment and function of innate and adaptive immune effector and suppressor cells. Therefore, reversing tumor hypoxia to create an immunopermissive microenvironment may improve anti-tumor response, and combined with immunotherapy approaches such as checkpoint inhibitors (CPI) may increase therapeutic efficacy. OMX, an anti-cancer therapy designed to reverse tumor hypoxia, efficiently accumulates in orthotopic rodent GB and spontaneous canine brain tumors, reduces tumor hypoxia and enhances immunotherapeutic efficacy. METHODS: We used in vivo bioluminescence imaging of tumor, immunohistochemistry, flow cytometry, and cytokine multiplex assays to evaluate OMX’s ability to immunosensitize the GL261 brain tumor microenvironment and promote tumor cures. RESULTS: Following intravenous administration in brain tumor-bearing mice, OMX reduces tumor hypoxia, modulates the IFNg signaling pathway, enhances the infiltration of tumor-specific CX3CR1(+) CD8 T cells into the tumor (using the EphA2 as a GL261-specific tumor antigen), increases the activation of cytotoxic T lymphocytes (CTLs), decreases Tim3 and Lag3 exhaustion markers on CD8 T cells, and reduces the number of immunosuppressive cells such as MDSCs and Tregs in the tumor. Similar immunological changes are observed when OMX is combined with anti-PD-1. In late-stage tumor-bearing mice, we observed a 40% tumor cure rate for the combination of OMX with anti-PD-1, while anti-PD-1 alone resulted only in 5% tumor cures. Following rechallenge with GL261 tumor cells injected on the other side of the brain, all mice treated with the combination of OMX with anti-PD-1 survived, indicating the presence of long-term immunological memory against glioma cells. CONCLUSION: By delivering oxygen specifically to the hypoxic tumor microenvironment, OMX may restore anti-cancer immune responses in GB patients and synergize with radiotherapy and immunotherapy to enhance tumor control and improve patient outcomes.

Background: Hypoxia, a common feature in solid tumors such as glioblastoma (GB), is associated with resistance to chemo- and radio-therapies and poor patient outcomes. In addition, hypoxia promotes the immune escape of tumors. Therefore, reversing tumor hypoxia to create an immunopermissive microenvironment can improve antitumor response, and combined with immunotherapy approaches such as checkpoint inhibitors (CPI), may increase therapeutic efficacy. OMX is an oxygen carrier well tolerated in small (rats and mice) and large (sheep and dogs) animals. Following intravenous administration, OMX extravasates through leaky tumor vasculature and efficiently accumulates in orthotopic rodent GB and spontaneous canine brain tumors. Consequently, OMX significantly reduces hypoxia and improves the efficacy of radiotherapy and CPI. Methods: We used in vivo bioluminescence imaging of tumor, immunohistochemistry, flow cytometry, and cytokine multiplex assays to evaluate OMX's ability to immunosensitize the GL261 brain tumor microenvironment and promote tumor cures. Results: A single dose of OMX in brain tumor-bearing mice reduces tumor hypoxia, enhances the recruitment and infiltration of tumor-specific CX3CR1+ CD8 T cells into the tumor (using the EphA2 as a GL261-specific tumor antigen), decreases Tregs and increases activation and proliferation of cytotoxic T lymphocytes (CTLs). Specifically, OMX increases the Teff/Treg ratio by ~3-fold, indicating a switch from an immunosuppressive to an immunopermissive microenvironment. Similarly, when combined with anti-PD-1, OMX decreases Tregs, increases CTL infiltration, proliferation and cytotoxic activity, and modulates IFNg and IFNg-inducible cytokines that polarize T cells towards a Th1 phenotype. Treatment with OMX alone resulted in a 55% tumor cure rate, comparable to anti-PD-1 treatment. Furthermore, in late-stage tumor-bearing mice, we observed a 40% tumor cure rate for the combination of OMX with anti-PD-1, while anti-PD-1 alone resulted only in 5% tumor cures. In symptomatic mice with very high tumor burden, in which the combination of anti-PD-1 with anti-CTLA4 does not provide tumor cures, the addition of OMX resulted in a 20% tumor cure rate. Following rechallenge with GL261 tumor cells injected on the other side of the brain, all mice treated with OMX alone or in combination with CPI survived, indicating the presence of long-term immunologic memory against glioma cells. The survival benefit observed with OMX could be predicted with an identified circulating chemokine biomarker signature (post-hoc test). Conclusion: By delivering oxygen specifically to the hypoxic tumor microenvironment, OMX may restore anticancer immune responses in GB patients and synergize with radiotherapy and immunotherapy to enhance tumor control and improve patient outcomes. Citation Format: Natacha Le Moan, Philberta Leung, Sarah Ng, Tina Davis, Carol Liang, Jonathan Winger, Stephen P. Cary, Nicholas Butowski, Ana Krtolica. The oxygen carrier omx restores antitumor immunity and cures tumors as a single agent or in combination with checkpoint inhibitors in an intracranial glioblastoma mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4726A.

Chronic inflammation of the colon increases cancer development risk. Ulcerative colitis, characterized by excessive inflammation initiated by innate immune cells and exacerbated by a dysregulation in adaptive immunity, can give rise to colitis-associated colon cancer (CAC). Whereas overactivity of effector T cells and loss of immunosuppressive cells are hallmarks of ulcerative colitis, the opposite is true for CAC, with CAC tumors exhibiting a lack of effector T cell infiltration and a preponderence of immunosuppressive Treg cells and myeloid-derived suppressor cells (MDSCs). Recently, hypoxia has been identified as a potential driver in the pathogenesis of ulcerative colitis, with hypoxia persisting upon progression to CAC tumor formation. We have previously demonstrated that (i) hypoxia generates an immunosuppressive tumor microenvironment that limits effector T cell infiltration and activation, (ii) OMX, a first-in-class anti-cancer therapy designed to reverse tumor hypoxia to enhance immunotherapeutic efficacy, accumulates in preclinical rodent and spontaneous canine tumors and reduces tumor hypoxia, and (iii) OMX promotes effector T cell infiltration, reduces Treg cells, and enhances checkpoint inhibitor efficacy, resulting in greater tumor control. Given that CAC tumors are hypoxic and immunosuppressed, we hypothesized that hypoxia drives CAC tumor immunosuppression, and accordingly, that reversal of hypoxia with OMX may restore immunosensitivity and elicit an anti-tumor response. Here, using a chemically induced mouse model of CAC generated by administering azoxymethane (AOM) followed by repeated cycles of dextran sulfate sodium (DSS) exposure, we show that OMX treatment exhibits anti-tumor efficacy in advanced CAC tumors. We characterized CAC tumor progression from 8 to 12 weeks post-tumor induction, and confirmed previous reports that advanced CAC tumors are indeed hypoxic, and that immunosuppressive Treg cells and MDSCs are more abundant in CAC tumors relative to adjacent normal mucosa or control non-AOM/DSS-treated colons. Moreover, we observed a negative correlation between hypoxia and CD8+ T cell infiltration into CAC tumors. OMX single agent treatment reduced both CAC tumor number and total CAC tumor burden. Of note, OMX treatment reversed colon length shortening that was characteristic of tumor-bearing mice, indicative of a restoration of colon crypt regeneration and hence normal colon biology. Investigations into the immunological mechanism(s) responsible for OMX anti-tumor efficacy are currently underway. Taken together, our data suggest that OMX, by delivering oxygen to hypoxic CAC tumor regions, may be sufficient to induce an immunological change in the CAC tumor microenvironment from an immunosuppressive to an immunopermissive state, leading to tumor responses and a restoration of normal physiology. Citation Format: Kevin G. Leong, Yuqiong Pan, Changan Guo, Padmini Narayanan, Jonathan A. Winger, Stephen P. Cary, Natacha Le Moan, Ana Krtolica. Reversal of advanced colitis-associated colon cancer by OMX, a novel oxygen carrier that immunosensitizes the hypoxic tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1744.

Oxygen is one of the key modulators of tumor microenvironment whereby low oxygen or hypoxia is associated with resistance to chemo- and radio- therapies and poor patient outcomes. Hypoxia favors an immunosuppressive tumor microenvironment by promoting Treg recruitment and activation and suppressing T cell and NK cell proliferation and effector function and pro-inflammatory cytokine secretion. Therefore, reversing tumor hypoxia could create an immunopermissive microenvironment and improve the efficacy of several immunotherapies. Omniox has developed an oxygen carrier OMX that can specifically deliver oxygen to hypoxic tumor regions without affecting oxygenation of tissues within physiologic oxygen levels. Due to its biochemical features, OMX is well tolerated in small (rats and mice) and large (sheep and dogs) animals. Following intravenous administration, OMX extravasates through leaky tumor vasculature and accumulates within immunocompetent rodent orthotopic glioblastoma models as well as spontaneous canine brain tumors. Consequently, OMX decreases hypoxia levels in the tumor tissue measured directly using oxygen sensor probes and indirectly with exogenous hypoxia markers using ELISA, immunohistochemistry and flow cytometry methods. Here we evaluated OMX’ activity in reversing the immunosupressive tumor microenvironment using a combination of immunohistochemistry, flow cytometry and Luminex methods. Moreover, we investigated the efficacy of OMX in improving mouse survival and effectiveness of checkpoint inhibitors (CPI). Similar to previously published findings, we demonstrated that T lymphocytes are mostly excluded from hypoxic tumor areas in the GL261 model. A single OMX treatment in GL261 tumor-bearing mice reduces tumor hypoxia, enhances T cell localization in previously hypoxic tumor areas, and increases CD8 accumulation by ~4-fold. Specifically, OMX treatment increased the activated cytotoxic T lymphocytes (CTLs) fraction by ~2 fold and reduced the immunosuppressive Treg fraction by 2-fold, resulting in a 3-fold increase of Teff/Treg ratio, which indicates a switch from an immunosupressive to an immunopermissive microenvironment. When combined with CPI, OMX reverses the immunosuppressive tumor microenvironment by increasing CD8 T cell infiltration, proliferation and cytotoxic activity, and modulating IFNg and IFNg-inducible cytokines that may polarize T cells towards a Th1 phenotype. Furthermore, treatment of late-stage GL261 tumor-bearing mice with the combination of OMX-CPI increases mouse survival by 80%. By delivering oxygen specifically to the hypoxic tumor microenvironment, OMX may restore anti-cancer immune responses in glioblastoma patients and synergize with radiotherapy and immunotherapy to enhance tumor control and improve patient outcomes. Citation Format: Natacha Le Moan, Philberta Leung, Sarah Ng, Tina Davis, Carol Liang, Jonathan W. Winger, Stephen P. Cary, Nicolas Butowski, Ana Krtolica. Omx a hypoxia modulator reverses the immunosuppressive glioblastoma microenvironment by stimulating T cell infiltration and activation that results in increased number of long-term survivors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4686. doi:10.1158/1538-7445.AM2017-4686

Hypoxia is a hallmark of cancer and a driver of tumor progression and poor patient outcomes. By generating an immunosuppressive tumor microenvironment that limits cytotoxic T lymphocyte (CTL) infiltration and activation, hypoxia limits the effectiveness of cancer immunotherapy and thus promotes tumor cell evasion of the host immune response. Omniox has developed a first-in-class anti-cancer immunotherapeutic, OMX, specifically designed to reverse tumor hypoxia to enhance cancer immunotherapy efficacy. In preclinical models, we have demonstrated that OMX accumulates in rodent subcutaneous and orthotopic tumors, as well as spontaneous canine melanomas and brain tumors, resulting in significant tumor hypoxia reduction.Here, using multiple subcutaneous syngeneic mouse tumor models (MC38, CT26, 4T1), we assessed OMX effects on intratumoral CTLs and immunosuppressive regulatory T cells (Treg), as well as the anti-tumor potential of OMX as a single agent and in combination with established immunotherapies. Using quantitative immunohistochemistry, we confirmed reports that hypoxic tumor areas are devoid of CTLs. Accordingly, by flow cytometry we observed a negative correlation between tumor hypoxia and CTL infiltration. While OMX single agent treatment did not affect the overall CD45-positive leukocyte population, Treg cells were selectively depleted and the CTL:Treg ratio was substantially increased, suggesting that OMX induced a shift towards immunosensitization. Consistent with this finding, we observed OMX single agent anti-tumor efficacy in MC38 colon tumors. Impressively, anti-tumor effects of OMX single agent were equivalent to that of a single treatment of the checkpoint inhibitor anti-CTLA4. We next assessed whether OMX would enhance the efficacy of checkpoint inhibitors when used in combination. In CT26 colon tumors, OMX exhibited combination anti-tumor activity with anti-CTLA4, giving rise to faster cures and a greater number of complete and durable responders compared to anti-CTLA4 alone. Of note, this enhanced response was observed for both early-stage and late-stage CT26 tumors. In 4T1 breast tumors, known to be insensitive to checkpoint inhibitors, treatment of early-stage (~60mm3) tumors with combination OMX and anti-PD1 resulted in a 27% response rate, compared to a 0% response rate to anti-PD1 alone. Taken together, our data suggest that OMX, by delivering oxygen to hypoxic tumor areas, induces a microenvironmental change from an immunosuppressive to an immunopermissive state. Given that OMX is well-tolerated in both small and large animals, and that its mechanism of action is upstream of numerous major immunosuppressive pathways, OMX holds great clinical potential to synergize with multiple immunotherapeutic agents to enhance tumor control by restoring anti-cancer immune responses in cancer patients. Citation Format: Kevin G. Leong, Yuqiong Pan, Jonathan A. Winger, Stephen P. Cary, Natacha Le Moan, Ana Krtolica. Enhancement of anti-cancer immunity by OMX, a novel oxygen carrier immunotherapeutic that ameliorates the hypoxic tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1627. doi:10.1158/1538-7445.AM2017-1627

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.

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 Omniox' 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.

Hypoxia, or lack of oxygen, is a key modulator of the tumor microenvironment and is associated with immunosuppression, invasiveness, angiogenesis, radiotherapy resistance and poor patient outcomes. Methods to increase oxygen in the hypoxic tumor niche have failed due to the long distance oxygen must diffuse from dysfunctional vasculature to the hypoxic cells. To address this problem, we engineered OMX oxygen carriers that bind oxygen with high affinity and can specifically deliver oxygen to severely hypoxic tumor regions without afecting normoxic tissues. Here, we show that OMX extravasates through leaky tumor vasculature, penetrates into and delivers oxygen to hypoxic tumor tissue. This reduces hypoxia in the tumor microenvironment and sensitizes tumors to therapy. Specifically, OMX accumulation in tumors leads to an increase in tumor oxygenation in mouse human xenograft and immunocompetent rat orthotopic models, as assessed by direct oxygen measurement (oxygen probe), by18F-FMISO PET in vivo imaging and by IHC and ELISA assessment of hypoxia markers (pimonidazole, CAIX, CCI, HIF-1). Moreover, OMX-mediated tumor oxygenation increases efficacy of radiation therapy as demonstrated by ex-vivo clonogenic assay and tumor growth delay. Importantly, while radiation alone only moderately delays tumor growth, combination of OMX and radiation therapy leads to tumor eradication in >50% of tumors. In addition to the enhancement of radiation therapy with OMX, we are currently exploring the capacity of OMX to ameliorate immunosuppressive microenvironment caused by hypoxia. Results from a Phase 0 clinical trial in canine cancer patients indicate that OMX is well tolerated and safe to use in combination with standard of care radiation therapy even in aged and fragile canine patient populations. Similar to rodent tumor models, OMX penetrates the tumor tissue in spontaneous canine brain and melanoma tumors. Furthermore, OMX accumulation correlates with reduced hypoxia in the tumor microenvironment as assessed by multiple hypoxia markers using quantitative IHC and ELISA. Taken together, these preclinical safety and efficacy data in both rodents and canines strongly support our IND submission to initiate human clinical trials in newly diagnosed glioblastoma patients. Results from completed and ongoing studies support the potential of OMX as modulator of hypoxic tumor microenvironment that may significantly impact the effectiveness of radiotherapy, chemotherapy and immunotherapy in a variety of solid tumors where hypoxia is major contributor to therapeutic resistance. Citation Format: Ana Krtolica, Natacha Le Moan, Philberta Leung, Youngho Seo, Jonathan Winger, Henry Van Brocklin, Michael Kent, Stephen Cary. Sensitizing the hypoxic tumor microenvironment with OMX, a breakthrough oxygen delivery protein: From protein engineering to clinical trial. [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 2790.

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.