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ONCR-177 is an engineered recombinant oncolytic herpes simplex virus (HSV) with complementary safety mechanisms, including tissue-specific miRNA attenuation and mutant UL37 to inhibit replication, neuropathic activity, and latency in normal cells. ONCR-177 is armed with five transgenes for IL12, FLT3LG (extracellular domain), CCL4, and antagonists to immune checkpoints PD-1 and CTLA-4. In vitro assays demonstrated that targeted miRNAs could efficiently suppress ONCR-177 replication and transgene expression, as could the HSV-1 standard-of-care therapy acyclovir. Although ONCR-177 was oncolytic across a panel of human cancer cell lines, including in the presence of type I IFN, replication was suppressed in human pluripotent stem cell–derived neurons, cardiomyocytes, and hepatocytes. Dendritic cells activated with ONCR-177 tumor lysates efficiently stimulated tumor antigen–specific CD8+ T-cell responses. In vivo, biodistribution analyses suggested that viral copy number and transgene expression peaked approximately 24 to 72 hours after injection and remained primarily within the injected tumor. Intratumoral administration of ONCR-177 mouse surrogate virus, mONCR-171, was efficacious across a panel of syngeneic bilateral mouse tumor models, resulting in partial or complete tumor regressions that translated into significant survival benefits and to the elicitation of a protective memory response. Antitumor effects correlated with local and distant intratumoral infiltration of several immune effector cell types, consistent with the proposed functions of the transgenes. The addition of systemic anti–PD-1 augmented the efficacy of mONCR-171, particularly for abscopal tumors. Based in part upon these preclinical results, ONCR-177 is being evaluated in patients with metastatic cancer (ONCR-177-101, NCT04348916).

Supplemental Tables and Figures

Legends of Figs S1 to S8, unmarked version

Supplemental Table I: Antibodies used for Western blotting and immunohistochemistry (IHC); Supplemental Table II: Sequences used for qRT-PCR primers.

Figure S1: Generation of mAb428.2 antibody; Figure S2: Affinity of mAb428.2 for fibulin-3; Figure S3: mAb428.2 binds to cell-surface and interstitial fibulin-3; Figure S4: mAb428.2 does not cause acute toxicity; Figure S5: Locally-delivered mAb428.2 targets fibulin-3 signaling and tumor growth; Figure S6: IV-delivered mAb428.2 inhibits tumor growth; Figure S7: Effects of mAb428.2 against intracranial tumors; Figure S8: Biodistribution of mAb428.2 in naïve and tumor-bearing mice.

Cytomegalovirus (CMV) has been implicated in glioblastoma (GBM); however, a mechanistic connection in vivo has not been established. The purpose of this study is to characterize the effects of murine CMV (MCMV) on GBM growth in murine models. Syngeneic GBM models were established in mice perinatally infected with MCMV. We found that tumor growth was markedly enhanced in MCMV(+) mice, with a significant reduction in overall survival compared with that of controls (P < 0.001). We observed increased angiogenesis and tumor blood flow in MCMV(+) mice. MCMV reactivation was observed in intratumoral perivascular pericytes and tumor cells in mouse and human GBM specimens, and pericyte coverage of tumor vasculature was strikingly augmented in MCMV(+) mice. We identified PDGF-D as a CMV-induced factor essential for pericyte recruitment, angiogenesis, and tumor growth. The antiviral drug cidofovir improved survival in MCMV(+) mice, inhibiting MCMV reactivation, PDGF-D expression, pericyte recruitment, and tumor angiogenesis. These data show that MCMV potentiates GBM growth in vivo by increased pericyte recruitment and angiogenesis due to alterations in the secretome of CMV-infected cells. Our model provides evidence for a role of CMV in GBM growth and supports the application of antiviral approaches for GBM therapy.

Purpose: We sought a novel approach against glioblastomas (GBM) focused on targeting signaling molecules localized in the tumor extracellular matrix (ECM). We investigated fibulin-3, a glycoprotein that forms the ECM scaffold of GBMs and promotes tumor progression by driving Notch and NFκB signaling. Experimental Design: We used deletion constructs to identify a key signaling motif of fibulin-3. An mAb (mAb428.2) was generated against this epitope and extensively validated for specific detection of human fibulin-3. mAb428.2 was tested in cultures to measure its inhibitory effect on fibulin-3 signaling. Nude mice carrying subcutaneous and intracranial GBM xenografts were treated with the maximum achievable dose of mAb428.2 to measure target engagement and antitumor efficacy. Results: We identified a critical 23-amino acid sequence of fibulin-3 that activates its signaling mechanisms. mAb428.2 binds to that epitope with nanomolar affinity and blocks the ability of fibulin-3 to activate ADAM17, Notch, and NFκB signaling in GBM cells. mAb428.2 treatment of subcutaneous GBM xenografts inhibited fibulin-3, increased tumor cell apoptosis, and enhanced the infiltration of inflammatory macrophages. The antibody reduced tumor growth and extended survival of mice carrying GBMs as well as other fibulin-3–expressing tumors. Locally infused mAb428.2 showed efficacy against intracranial GBMs, increasing tumor apoptosis and reducing tumor invasion and vascularization, which are enhanced by fibulin-3. Conclusions: To our knowledge, this is the first rationally developed, function-blocking antibody against an ECM target in GBM. Our results offer a proof of principle for using “anti-ECM” strategies toward more efficient targeted therapies for malignant glioma. Clin Cancer Res; 24(4); 821–33. ©2017 AACR.

Although oncolytic viruses have shown clinical efficacy for local treatment of cancerous tumors, the ability to induce immune-mediated regression to visceral lesions is not robust. Immune checkpoint modulation has been efficacious in a fraction of cancers associated with an inflamed microenvironment but is associated with toxicity due to nonspecific T-cell activation. Therefore, combining these two strategies has the exciting potential to be an effective cancer therapy. Oncolytic virus can be genetically modified to decrease pathogenicity to normal cells and to increase their lytic potential and ability to stimulate antitumor immunity, thus improving the risk-benefit ratio for cancer patients. Oncorus oHSV (ONCR) are novel oncolytic herpes simplex virus type-1 vectors developed for the treatment of solid tumors. ONCR utilizes a unique conditional-lethal strategy in which tissue-specific miRs binding sites are inserted into early genes essential for viral replication and in the neurovirulence gene ICP34.5. This strategy curtails viral replication in normal cells, including neurons, and limits the expression of g34.5 to tumor cells, thus promoting potent viral replication in presence of type I interferon. Fifteen immunostimulatory transgenes and combination thereof were systematically evaluated in dual flank syngeneic mouse tumor models to test both for oncolysis and abscopal efficacy on the contralateral noninjected tumor. Among the selected transgenes, IL-12 was found to elicit the most potent efficacy on the ipsilateral and contralateral tumors. We demonstrated that the replication of ONCR and the expression of IL-12 was limited to the injected tumors. By contrast, induction of systemic immunity as assessed by IFNg production in contralateral tumors and in plasma was observed. ONCR expressing IL-12 elicited efficacy in multiple subcutaneous and metastatic models. Additional payloads designed to stimulate antigen presentation, recruitment of immune cells and inhibition of tumor immune suppression were identified as further enhancing IL-12 activity. Data supporting the selection of a suite of payloads cloned into a single ONCR vector will be presented. ONCR represents a new class of oncolytic virus that promote antitumor responses through a multiprong mechanism of action dependent on selective tumor cell killing, the induction of systemic antitumor immunity and reversion of immune suppression. Citation Format: Lorena Lerner, Edward M. Kennedy, Terry Farkaly, Allison Colthart, Caity Montagna, Prajna Behera, Judith Jacques, Peter Grzesik, Jennifer Lee, Agnieszka Denslow, Jacqueline Gursha, Brian Haines, Michael Ball, Daniel Wambua, Cecilia Kwong, Lingxin Kong, Michael Paglia, Laura Viggiano Salta, Lorenz Ponce, Caroline Webb, Mitchell Finer, Christophe Quéva. microRNA attenuated oHSV-1 armed with multiple immunomodulatory payloads mediates robust and selective antitumor immune response in preclinical tumor models [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B23.

Oncolytic viruses represent a new class of therapeutic agents for the treatment of cancers that poorly respond to immune checkpoint inhibitors. Oncorus is developing ONCR-177, an oncolytic herpes simplex virus engineered for enhanced oncolysis and antitumor immunity. ONCR-177 is replication competent in tumor cells, but its replication and neuropathic activities are attenuated in healthy cells using miRNA-dependent degradation of viral transcripts and by mutations in UL37 that prevent retrograde transport in neurons. Efficacy is enhanced by a gain of function in the fusogenic glycoprotein B to enhance viral entry and inactivation of US12 to restore antigen processing. ONCR-177 is armed with five immunostimulatory transgenes: IL-12, CCL4, FLT3LG, a PD-1 humanized antagonist VHH nanobody, and the CTLA4 blocking monoclonal antibody ipilimumab. The antitumor effect of ONCR-177 utilizes two distinct modalities of immune stimulation. First, ONCR-177 is specific for tumor cells where infection and replication result in direct tumor cell killing. Second, lysis of tumor cells and expression of ONCR-177 immune payloads stimulate the immune environment through multiple mechanisms: innate signaling through production of PAMP/DAMP signals, immune cell recruitment, exposure of tumor-specific antigens, and activation of the adaptive immune response. Tumor-specific immunogenicity was evaluated using mONCR-171, a mouse functional surrogate for ONCR-177, that is built on the same base vector ONCR-159 but expresses the immune transgenes suitable for murine studies. In a CT26 bilateral colon carcinoma tumor model, immune responses elicited by intratumoral injection of mONCR-171 and ONCR-159 were compared. Intratumoral T cells were evaluated for effector phenotype and function in response to a tumor-associated antigen. We observed a shift toward enhanced activation in the mONCR-171 treatment group. Treatment with mONCR-171 resulted in more tumor-associated CD8+ T cells, higher activation, and more effector cells. This group also had lower PD-1 expression, fewer Treg cells, and a favorable CD8:Treg ratio. The tumor-specific response was assessed using the CT26 tumor-associated antigen AH1, a peptide derived from envelope glycoprotein 70 (gp70) of endogenous murine leukemia virus (MuLV). Treatment with mONCR-171 elicited significantly more IFNγ and TNFα producing, intratumoral CD8+ T cells, producing significantly higher levels of IFNγ. The increase in antitumor immunogenicity with mONCR-171 treatment was further evidenced by the increase in polyfunctional CD8+ T cells, producing IFNγ, TNFα, and/or IL-2 in response to antigen stimulation. These results highlight the dual modality mechanism of action of mONCR-171 treatment though the direct tumor cell killing and immune stimulation through the antiviral response and their enhancement by the immune stimulatory transgenes. Citation Format: Melissa Hayes, Agnieska Denslow, Jacqueline Gurshaw, Daniel Wambua, Shreeya Khatiwada, Lingxin Kong, Jacob Spinale, Prajna Behera, Peter Grzesik, Jennifer Lee, Terry Farkaly, Edward Kennedy, Lorena Lerner, Christophe Quéva, Brian Haines, Sonia Feau. Dual mechanism of ONCR-177 enhances antitumor immunogenicity [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B74.

ONCR-177 is a highly modified recombinant oncolytic Herpes Simplex Virus (oHSV) designed to be a safe and efficacious therapy for the treatment of solid tumor indications. Complementary safety mechanisms, such as tissue-specific miR attenuation of essential viral genes and UL37 mutation, were introduced to reduce replication, neuropathic activity, and latency in normal cells, while preserving oncolytic ability in tumor cells. In addition, ONCR-177 is armed with five transgenes: the NK and T cell activating cytokine IL-12, the chemokines CCL4 and FLT3LG (extracellular domain) to allow for expansion and recruitment of classical dendritic cells, and antagonists to the clinically validated immune checkpoint targets PD-1 and CTLA-4 to counter T cell exhaustion. Therefore, ONCR-177 is proposed to have a dual mechanism of action whereby the miR attenuation strategy allows for selective oncolysis of tumors cells and the transgenes mediate potent stimulation of systemic anti-tumor immunity. Since human IL-12 and the anti-CTLA-4 antagonist do not cross-react with the mouse targets, most nonclinical pharmacology studies were conducted with the mouse surrogate mONCR-171, which expresses within the same base vector as ONCR-177 the mouse equivalent to the ONCR-177 transgenes. Intra-tumoral administration of mONCR-171 in the oHSV-sensitive A20 BALB/c lymphoma bilateral tumor model resulted in response rates (partial and complete tumor regressions) of 100% and 80%, respectively, on the injected (ipsilateral) and distant (contralateral) tumor. mONCR-171 was also highly efficacious in the B16F10 melanoma model, an oHSV-resistant C57BL/6 based tumor model engineered to be permissive to oHSV by introduction of Nectin-1, and in the oHSV-resistant MC38 C57BL/6 colon carcinoma model. Abscopal anti-tumor activity could not be explained by propagation of the virus since viral DNA and transgenes were only detectable in the injected tumor. Rather, mONCR-171 treatment resulted in increased numbers of activated NK cells, CD8 and CD4 T cells, and classical dendritic cells. The proportion of Tregs decreased, resulting in large CD8/Treg ratios. These changes in immune contexture occurred in both the ipsilateral and contralateral tumor and were more pronounced with mONCR-171 treatment compared to the base vector without transgenes, indicating that the observed abscopal effects were due to the elicitation of systemic anti-tumor immunity mediated in part by the transgenes. These encouraging preclinical data warrant the clinical investigation of ONCR-177 in patients with metastatic cancer. Citation Format: Brian B. Haines, Agnieszka Denslow, Michael S. Ball, Jacqueline Gursha, Daniel Wambua, Cecilia Kwong, Lingxin Kong, Prajna Behera, Peter Grzesik, Caitlin Goshert, Allison Colthart, Jennifer S. Lee, Terry Farkaly, Edward M. Kennedy, Lorena Lerner, Christophe Queva. Development of ONCR-177, an miR-attenuated oncolytic HSV-1 designed to potently activate systemic antitumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 940.

Oncolytic viruses, through cancer cell-selective lysis and accompanying release of danger signals that promote immune activation, have demonstrated antitumor efficacy in monotherapy of metastatic melanoma and have shown promising activity in combination with checkpoint inhibitors. We present here the activity of ONCR-148, a recombinant oncolytic Herpes Simplex Virus (oHSV) designed to be a safe and efficacious therapy for the treatment of solid tumors. Neuron-specific miR attenuation of ICP4 gene inhibits viral replication in neurons while preserving its potent oncolytic activity in tumor cells. The antitumor potency of the virus is enhanced by incorporating in its genome a transgene expressing fibroblast activation protein (FAP)- and CD3- bispecific antibody that recruits and re-directs the cytolytic activity of T cells toward FAP-expressing stromal cells. FAP is a cell-surface serine protease that is almost exclusively found in fibroblasts within tumor stroma. However, systemic administration of a FAP-CD3 bispecific antibody was not tolerated due to the toxicity toward FAP-expressing bone marrow mesenchymal cells (Tran et al., 2013. J.Exp.Med, 210(6), 1125-1135). Thus, the local intra-tumoral delivery of FAP-CD3 is an attractive approach to promote tumor lysis and T cell activation against cancerous lesions. To test the in vivo activity of ONCR-148, as FAP is poorly expressed in syngeneic mouse tumor models, we developed a bilateral mouse FAP-expressing MC38 syngeneic colon carcinoma model (MC38-FAP), allowing to test local antitumor activity in oHSV-injected tumors and abscopal efficacy mediated by immune cells in the non-injected tumors. Intra-tumoral administration of ONCR-148 in the MC38-FAP model resulted in tumor growth inhibition of 60% (p Citation Format: Agnieszka Denslow, Brian B. Haines, Michael S. Ball, Jacqueline Gursha, Daniel Wambua, Cecilia Kwong, Lingxin Kong, Allison Colthart, Prajna Behera, Peter Grzesik, Jennifer Lee, Terry Farkaly, Caitlin Goshert, Edward M. Kennedy, Lorena Lerner, Christophe Quéva. Development of ONCR-148, a miR-attenuated oncolytic HSV-1 designed to potently activate antitumor T cell response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1452.

Real-time, continuous monitoring of local oxygen contents at the cellular level is desirable both for the study of cancer cell biology and in tissue engineering. In this paper, we report the successful fabrication of polydimethylsiloxane (PDMS) nanofibers containing oxygen-sensitive probes by electrospinning and the applications of these fibers as optical oxygen sensors for both gaseous and dissolved oxygen. A protective ‘shell’ layer of polycaprolactone (PCL) not only maintains the fiber morphology of PDMS during the slow curing process but also provides more biocompatible surfaces. Once this strategy was perfected, tris(4,7-diphenyl-1,10-phenanthroline) ruthenium(II) (Ru(dpp)) and platinum octaethylporphyrin (PtOEP) were dissolved in the PDMS core and the resulting sensing performance established. These new core-shell sensors containing different sensitivity probes showed slight variations in oxygen response but all exhibited excellent Stern-Volmer linearity. Due in part to the porous nature of the fibers and the excellent oxygen permeability of PDMS, the new sensors show faster response (

Glioblastoma (GBM) is the most aggressive form of brain cancer, with a dismal prognosis and extremely low percentage of survivors. Novel therapies are in dire need to improve the clinical management of these tumors and extend patient survival. Genetic therapies for GBM have been postulated and attempted for the past twenty years, with variable degrees of success in pre-clinical models and clinical trials. Here we review the most common approaches to treat GBM by gene therapy, including strategies to deliver tumor-suppressor genes, suicide genes, immunomodulatory cytokines to improve immune response, and conditionally-replicating oncolytic viruses. The review focuses on the strategies used for gene delivery, including the most common and widely used vehicles (i.e., replicating and non-replicating viruses) as well as novel therapeutic approaches such as stem cell-mediated therapy and nanotechnologies used for gene delivery. We present an overview of these strategies, their targets, different advantages, and challenges for success. Finally, we discuss the potential of gene therapy-based strategies to effectively attack such a complex genetic target as GBM, alone or in combination with conventional therapy.

The entangled relationship of brain aging, mitochondrial dysfunction, and amyloid-β peptide (Aβ₄₂) toxicity occupies the center stage in the pathogenesis of Alzheimer's disease (AD). The present study examines some of the toxic effects of Aβ₄₂ on brain mitochondria and provides evidence that aged brain mitochondria are significantly more vulnerable to Aβ₄₂ toxicity. In particular, the study has shown that the aggregated, but not the monomeric, form of Aβ₄₂ in varying concentrations (10-40 μM) during in vitro incubation causes a loss of mitochondrial membrane potential, a decrease in phosphorylation capacity and ATP synthesis, and the release of cytochrome c from the mitochondria but without any noticeable change in the activities of respiratory chain complexes. Such effects of Aβ₄₂ are strikingly more conspicuous on aged rat (22-24 months) brain mitochondria compared to that on brain mitochondria of young rats (4-6 months). More interestingly is the observation that in contrast to young rat brain mitochondria, a significantly higher level of Aβ₄₂ remains associated with aged brain mitochondria under basal incubation condition as well as after exposure to exogenously added peptide. Extrapolated to an in vivo scenario, the results have clear implications in AD pathogenesis and also partly explain why brain aging is a dominant risk factor for this disease condition.

Although mitochondrial dysfunction has been well documented in obese people with type 2 diabetes mellitus, its presence or absence in nonobese subjects with type 2 diabetes mellitus has not been well studied so far. The aim of the present study was to assess the status of mitochondrial oxidative phosphorylation in subcutaneous adipose tissue of nonobese type 2 diabetes mellitus subjects in comparison to control, obese nondiabetic, and obese type 2 diabetes mellitus subjects. Mitochondria were isolated from subcutaneous white adipose tissue obtained from the abdominal region of control, obese nondiabetic, nonobese type 2 diabetes mellitus, and obese type 2 diabetes mellitus subjects. The activities of complex I, I to III, II to III, and IV; transmembrane potential; and inorganic phosphate utilization of mitochondria from different groups were measured. Mitochondrial transmembrane potential, inorganic phosphate utilization, and the activities of respiratory chain complexes were significantly reduced in obese nondiabetic and obese type 2 diabetes mellitus patients compared with those in control subjects. No detectable change in mitochondrial functional parameters was observed in case of nonobese type 2 diabetes mellitus subjects compared with control subjects. Furthermore, a significant difference was noticed in mitochondrial phosphate utilization and activities of respiratory complexes, for example, I, I to III, and II to III, between obese type 2 diabetes mellitus subjects and obese nondiabetic subjects. Obesity modulates mitochondrial dysfunction associated with type 2 diabetes mellitus.

A combination of antioxidants (N-acetyl cysteine, α-lipoic acid, and α-tocopherol) was selected for long term oral supplementation study in rats for protective effects on age-related mitochondrial alterations in the brain. Four groups of rats were chosen: young control (6-7 months); aged rats (22-24 months); aged rats (22-24 months) on daily antioxidant supplementation from 18 month onwards and young rats (6-7 months) on daily antioxidant supplementation from 2 month onwards. The brain mitochondrial functional parameters, status of antioxidant enzymes and accumulation of oxidative damage markers were measured in the four groups of rats. A significant decrease in complex IV activity and a loss of transmembrane potential and phosphorylation capacity along with an increased accumulation of oxidative damage markers and compromised antioxidant enzyme status were noticed in aged rat brain mitochondria as compared to that in young controls, but in aged rats supplemented with oral antioxidants the mitochondrial alterations were largely prevented. Antioxidant supplementation in young rats had no effect on mitochondrial parameters investigated in this study. The results have implications in biochemical and functional deficits of brain during aging as well as in neurodegenerative disorders.

Effective treatment of advanced stage cancer with oncolytic viruses requires potent cancer cell-specific oncolytic activity. We modified oncolytic HSV (oHSV) to utilize a novel conditional-lethal strategy that retains all viral genes to maintain potency of the oncolytic virus, yet block replication in normal cells. In addition, we armed the virus with extracellular matrix decreasing enzyme to promote viral spread and immunomodulatory payloads to increase recruitment and effector function of immune cells. To enhance the selectivity of viral replication to tumor cells, Oncorus engineered oHSVs containing microRNA (miRNA)-binding cassettes (miR-T) in essential genes required for viral replication. The first Oncorus clinical candidate, ONCR-1, under development for the treatment recurrent glioblastoma multiform (GBM), has been attenuated for replication in neurons, the most trophic cell type for HSV. Neuronal attenuation was achieved by inserting binding sites for a highly specific neuronal miRNA, miR-124, into the 3′UTR of the essential ICP4 gene. We demonstrate that these engineered miR-124 response elements can selectively inhibit the expression of ICP4 and VP5 as well as the expression of ONCR-1 payloads. This attenuation strategy eliminates viral replication in miR-124 expressing cells without compromising oncolytic activity in cancer cells, thereby providing a therapeutic window. We extended the design oHSV attenuation by profiling of miRNAs expression in panels of solid tumors and non-diseased tissue counterparts in other potentially high-risk tissues, prioritized to indications amenable to local or systemic administration. We leveraged our comprehensive normal and malignant tissue dataset to identify numerous highly expressed unique non-cancerous tissue-specific candidate miRNAs. Insertion of miR-T cassettes into HSV-1 genes collaborated to stall the early steps in the viral replication cycle and further increased the depth and breadth of oHSV attenuation in non-disease tissue relative to the single miR-T cassette in ICP4. We found that miR-122, a highly expressed miRNA in normal hepatocytes, is low or absent in many carcinoma cells. Insertion of miR-122 cassette into the 3′UTR of an immediate early gene, ICP27, inhibited completely viral replication in miR-122 expressing cells. oHSV with dual miR-124 and miR-122 was potently attenuated cells expressing one or both miRs while displaying a robust oncolytic activity in malignant cells. We have shown that insertion of miR-T cassettes in essential viral genes effectively attenuates oHSV replication in normal human cells, and most importantly, robust oncolysis was retained in cancer cells. This work extends the miR-T paradigm to multiple viral genes with diverse miRNA targets, thus expanding the protection of normal tissues and facilitating the application of oHSV to the treatment of multiple malignancies. Citation Format: Edward M. Kennedy, Terry Farkaly, Caitlin Goshert, Allison Colthart, Prajna Behera, Peter Grzesik, Kyle Grant, Michael Paglia, Paola Grandi, Christophe Quéva, Mitchell Finer, Lorena Lerner. microRNA control of an oHSV vector allows for robust oncolysis and selective control of viral replication in normal tissues [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 5929.

Glioblastomas (GBMs) are the most common primary tumors of the Central Nervous System and one of the most aggressive and difficult to treat forms of cancer. Our laboratory was the first to identify the extracellular protein fibulin-3 (EFEMP1) in GBMs and demonstrate the pro-tumoral functions of this protein in solid tumors. We have recently developed a function-blocking antibody ("mAb428.2") against fibulin-3, which inhibits the enhancing effect of this protein on ADAM17 activity and NFkB signaling (Nandhu et al., Clin Cancer Res, 2017). Using mAb428.2 we observed a significant anti-tumor effect and prolongation of survival in nude mice carrying patient-derived GBM xenografts implanted subQ or intracranially (antibody treatment: 8x 30 mg/kg, q24h, IV). Interestingly, reduction of tumor growth during mAb428.2 treatment was accompanied with significant tumor necrosis and increased infiltration of Iba1-positive tumor-associated macrophages (TAMs) both in subQ and orthotopic tumors. Remarkably, TAMs in mAb428.2-treated GBMs showed very low co-expression of Arginase-I, a bona-fide marker of M2 ("tumor promoting") TAM polarization. Further analysis of mouse-specific gene expression in the tumor mass and in recovered macrophages (CD11b-positive cells) showed that TAMs in mAb428.2-treated GBMs had significantly increased expression of inflammatory cytokines (IFN-gamma, IL-1b, IL-10) and downregulation of M2 markers (CD163, CD206, ARG1), suggesting that our anti-fibulin-3 treatment prevented the expected M2 polarization of TAMs. Accordingly, mAb428.2-treated tumors showed drastically decreased expression of CSF-1a, an M2-inducing cytokine that may be regulated by fibulin-3. Co-culture experiments of PMA-activated U937 macrophages with GBM stem-like cells did not affect tumor cell viability when a control IgG1 was added to the cultures. However, addition of mAb428.2 caused significant decrease of GBM cell viability, suggesting that anti-fibulin-3 treatment enables TAMs to attack tumor cells. Taken together, our results strongly suggest that anti-fibulin-3 and possibly other anti-ECM approaches may induce a marked inflammatory reaction driven by innate immune cells, potentiating anti-tumor therapy. Citation Format: Sharon L. Longo, Prajna Behera, Mariano S. Viapiano, Mohan Sobhana Nandhu. Inhibition of fibulin-3 reverses macrophage polarization in glioblastoma and increases anti-tumor inflammatory responses [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 1706.

Glioblastoma is an aggressive, invasive tumor of the central nervous system (CNS). There is a widely acknowledged need for anti-invasive therapeutics to limit glioblastoma invasion. BKM-120 is a CNS-penetrant pan-class I phosphatidyl-inositol-3 kinase (PI3K) inhibitor in clinical trials for solid tumors, including glioblastoma. We observed that BKM-120 has potent anti-invasive effects in glioblastoma cell lines and patient-derived glioma cells in vitro. These anti-migratory effects were clearly distinguishable from cytostatic and cytotoxic effects at higher drug concentrations and longer durations of drug exposure. The effects were reversible and accompanied by changes in cell morphology and pronounced reduction in both cell/cell and cell/substrate adhesion. In vivo studies showed that a short period of treatment with BKM-120 slowed tumor spread in an intracranial xenografts. GDC-0941, a similar potent and selective PI3K inhibitor, only caused a moderate reduction in glioblastoma cell migration. The effects of BKM-120 and GDC-0941 were indistinguishable by in vitro kinase selectivity screening and phospho-protein arrays. BKM-120 reduced the numbers of focal adhesions and the velocity of microtubule treadmilling compared with GDC-0941, suggesting that mechanisms in addition to PI3K inhibition contribute to the anti-invasive effects of BKM-120. Our data suggest the CNS-penetrant PI3K inhibitor BKM-120 may have anti-invasive properties in glioblastoma.

Fibulin-3 is a protein secreted by glioblastoma (GBM) cells that is virtually absent in normal brain. This protein promotes tumor invasion and, more unusually, apoptotic resistance, by paracrine activation of Notch and NFkB pathways (see companion abstract). Conditional knockdown of fibulin-3 reduces GBM growth, invasion and vascularization, and prolongs survival. Here we report initial validation of biologics generated against this unique target in GBM. We first identified the sequence of fibulin-3 that activates Notch signaling (aa25-47) and developed an immunizing peptide maintaining a required Cys-loop within this sequence. An IgG1k monoclonal antibody generated against this peptide (mAb428.2) showed high affinity for fibulin-3 (Kd 5 nM) and no cross-reactivity against highly homologous fibulin-4 or -5. mAb428.2 detected fibulin-3 in the stroma and capillaries of human GBM without cross-reactivity against normal brain. The antibody (50-250 µg/ml) blocked the activation of Notch and NFkB reporters induced by fibulin-3, and caused significant GBM cell cytotoxicity but had no effects on astrocytes or HEK293 cells, which do not express fibulin-3. IV injection of mAb428.2 (range: 0.1-30 mg/kg, evaluation: 15min to 14 days) did not cause adverse effects and showed no histological findings. Using Alexa750-labeled mAb428.2 (IV) we observed rapid elimination through urine and slow accumulation in liver and lungs. Therefore, we performed initial efficacy tests with a GBM stem cell model implanted subcutaneously. mAb428.2 (4 x 10 mg/kg) or control IgG1 were administered intratumorally and animals sacrificed at 1 days. Results indicated 45% median volume reduction in mAb428.2-injected tumors, with significant tumor necrosis. To further develop targeted reagents against fibulin-3 we have subcloned the Vh and Vl sequences of mAb428.2 and already generated a single-chain variable fragment (scFv) that recognizes fibulin-3. Our overall encouraging results suggest that fibulin-3 is a novel target with high potential for developing targeted agents against GBM.

Glioblastomas (GBM) are highly invasive brain tumors that resist cytotoxic and antiangiogenic therapies, resulting in a high rate of recurrence. Fibulin-3 is an extracellular matrix protein secreted by GBM cells but absent from normal brain. This protein activates Notch signaling to promote tumor invasion and resistance to apoptosis. Conditional knockdown of fibulin-3 reduces GBM growth, invasion and vascularization, thus extending survival. Here we report the initial validation of a function-blocking antibody against this unique GBM target. We first identified the Notch-activating motif of fibulin-3 and developed an immunizing peptide against a key sequence within this motif. A monoclonal antibody generated against this peptide (mAb428.2) showed high affinity for fibulin-3 (Kd 5 nM) and no cross-reactivity against highly homologous fibulin-4 or -5. Antibody mAb428.2 detected fibulin-3 in the stroma and capillaries of human GBM without cross-reactivity against normal brain. The antibody (50-250 μg/ml) blocked the activation of Notch induced by fibulin-3 in GBM cells and caused GBM cell cytotoxicity but had no effects on astrocytes or HEK293 cells. Treatment of mice carrying subcutaneous GBM stem-cell (GSC) derived tumors with mAb428.2 (30 mg/kg IV, q24h x 8 days) resulted in tumor slowdown and extended median survival (47% and 64% in two different GSC models). mAb428.2-treated tumors showed decreased BrdU uptake and increased inflammatory reaction in the tumor stroma (macrophage infiltration and cytokine levels). Mice bearing intracranial tumors did not respond to mAb428.2 when delivered by IV or IP routes, likely due to the inability of the antibody to cross the blood brain barrier. This deficiency was overcome by direct intraparenchymal delivery of the mAb428.2 using chronic infusion (0.3 mg of mAb in 200 μl delivered at 1 μl/h over 7d), which resulted in a 26% increase in median survival. These encouraging results suggest that targeted reagents against fibulin-3 may be of clinical importance for novel combination therapies against GBM. Citation Format: Mohan Sobhana Nandhu, Prajna Behera, Vivek Bhaskaran, Ennio Antonio Chiocca, Mariano S. Viapiano. Validation of a novel antibody against fibulin-3 for targeted therapy of glioblastoma. [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 3797.

Alzheimer’s disease (AD) and vascular dementia (VAD) are the major forms of dementia affecting elderly people, in which the levels of many metabolites are altered in cerebrospinal fluid (CSF) and serum. These metabolites could be risk factors or potential biomarkers, but the significance of some of these are not clearly understood in the context of the disease pathogenesis. In the present study serum levels of homocysteine, dehydroepiandrosterone sulphate (DHEA-S) and lipoprotein (a) or Lp(a) have been measured by ELISA using commercial kits in AD (n = 40), VAD (n = 40) and age matched control subjects (n = 40). The data are compared by ANOVA and post-hoc analysis. The serum homocysteine is markedly elevated compared to control both in AD and VAD subjects, but to a significantly higher extent in the latter. Lp(a) is increased in the serum of VAD subjects only compared to control. Likewise, serum DHEA-S level is lowered in AD but not in VAD compared to control. The analysis of the present data and those published by others suggest that alterations in homocysteine and Lp(a) in serum are indicators of vascular pathology in AD or VAD, while the lowering of serum DHEA-S is a consequence of AD pathology.