609 improving the lytic capacity of a conditionally replicative adenoviruses by incorporating promoter elements responsive to tumor environmental conditons

609 Improving the Lytic Capacity of a Conditionally Replicative Adenoviruses by Incorporating Promoter Elements Responsive to Tumor Environmental Conditons Molecular Therapy Volume 18, Supplement 1, M[.]

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CANCER-ONCOLYTIC VIRUSES II

with systemic delivery of oncolytic viruses to treat established

tumors Since this novel combinatorial approach targets the tumor

endothelium, these data have direct, wide-ranging and immediate

clinical applicability across a broad range of tumor types

607 The Type III Interferon IL-28 Mediates Anti

Tumor Ef cacy of Oncolytic VSV in a Fully Immune

Competent Model

Phonphimon Wongthida,1 Rosa Diaz,1,2 Feorillo Galivo,1 Timothy

Kottke,1 Jill Thompson,1 Jose Pulido,3 Kevin Pavelko,2 Larry

Pease,2 Alan Melcher,4 Richard Vile.1,2,4

1 Department of Molecular Medicine, Mayo Clinic, Rochester,

MN; 2 Department of Immunology, Mayo Clinic, Rochester,

MN; 3 Department of Ophthalmology and Ocular Oncology,

Mayo Clinic, Rochester, MN; 4 Institute of Molecular Medicine,

University of Leeds, Leeds, United Kingdom.

Several studies have suggested that activation of innate immune

effector mechanisms against oncolytic viruses, in addition to, or

even instead of, progressive viral-mediated replication and lytic

spread, contribute signi cantly to clearance of both infected and

uninfected tumor cells in an immunocompetent host Here, we

developed an in vitro tumor cell/bone marrow co-culture assay to

dissect the innate immune sensor/effector responses to intratumoral

VSV We established an assay in which host derived bone marrow

cells were cultured with B16ova tumor cells in the presence, or

absence, of virus Exposure of B16ova tumor cells to VSV, even at

low MOI, induced rapid and extensive cytotoxicity, which was almost

completely inhibited by co-culturing with anti-VSV neutralizing

serum These data suggested that exposure of bone marrow cells

to VSV induces bystander killing of B16ova tumor cells, distinct

from direct viral-mediated oncolysis Moreover, ELISA analysis of

supernatant from co-culturing showed the induction of the type III

interferon, IL28 Antibody mediated blockade of IL-28 signaling in

the in vitro bone marrow co-culture assay abrogated VSV-activated

bone marrow-mediated killing In addition, prior depletion of the bone

marrow cultures of both macrophages and GR1+ cells reproducibly

led to signi cant reductions in the levels of VSV-activated IL-28

secretion and cell killing Finally, we tested the role of IL-28 in VSV

treatment of tumors in vivo B16ova tumor lysates expressed IL-28

following exposure to VSV in vivo When this intratumoral IL-28 was

neutralized by antibody blockade, concomitant with treatment with

VSV, anti tumor ef cacy was signi cantly reduced to levels similar

to those seen with control treatment Taken together, these studies

open new avenues to understand the immuno-virological mechanisms

associated with oncolytic virotherapy in immune competent hosts

Finally, our data suggest novel approaches by which key immune

signals might be manipulated to enhance both viral replication,

and anti tumor immune activation, to improve ef cacy of oncolytic

virotherapies

608 A New Oncolytic Herpes Simplex Virus

That Synergizes with Small Molecule Inhibitors of

PI3K/Akt To Treat Malignant Glioma

Ryuichi Kanai,1 Matthew Gardner,1 Hiroaki Wakimoto,1 Robert L

Martuza,1 Samuel D Rabkin.1

1 Department of Neurosurgery, Massachusetts General Hospital,

Boston, MA.

The phosphatidylinositol 3-kinase (PI3K)/Akt/mTOR pathway has

been implicated as one of the central pathways contributing to the

formation, survival and recurrence of a variety of tumors, including

malignant gliomas, and cancer stem cells A number of inhibitors

targeting PI3K/Akt have been isolated, and some have successfully

entered or completed clinical trials in the US They exhibit anti-tumor

activity as single agents and possess anti-angiogenic properties

However, targeting only one signaling pathway often results in the emergence of resistant cancer cells, as frequently seen with molecularly targeted drugs

The HSV Us3 gene product inhibits apoptosis and therefore Us3 mutants (R7041) have been evaluated as oncolytic herpes simplex virus (oHSV) vectors In addition, HSV infection of cells induces Akt activation, which is limited by Us3 However, for clinical translation there are concerns about singly mutated oHSVs Here,

we describe a new ICP6-/Us3- double mutant oHSV, MG18L, that has strong oncolytic activity against not only established glioma

cell lines (U87, T98), but also glioma stem cells (GSCs) in vitro

In contrast to G207, which is a ICP6-/gamma34.5- double mutant, MG18L replicates well in GSCs Importantly, MG18L did not show pathogenicity after intracerebral injection of HSV sensitive A/J mice, whereas 7041 did

We previously reported that R7041 synergizes with small molecule inhibitors of PI3K/Akt to kill cancer cell lines MG18L synergizes with small molecule inhibitors of PI3K/Akt (LY294002 and Triciribine) to kill GSCs and glioma cell lines in vitro, while other oHSV vectors without Us3 mutations (G207, G47∆) only exhibit additive interactions MG18L infection of GSCs induced increased levels of activated Akt compared with G207 or G47∆ infection, and also induced apoptosis more rapidly In vivo, intratumoral administration of MG18L prolonged the survival of mice bearing intracerebral GSC (BT74EF) derived tumors We are currently testing the combination of MG18L with NVP-BEZ235, an orally available PI3K/mTor inhibitor in clinical trial MG18L has suf cient safety and ef cacy to be considered for the treatment of malignant gliomas In addition, the combination with PI3K/Akt/mTOR inhibitors provides a new multimodal strategy that may be particularly useful for targeting cancer stem cells

609 Improving the Lytic Capacity of a Conditionally Replicative Adenoviruses by Incorporating Promoter Elements Responsive to Tumor Environmental Conditons

Diego L Viale,1 Veronica M Lopez,1 Eduardo G Cafferata,1 David Gould,2 Yuti Chenajovsky,2 Osvaldo L Podhajcer.1

1 Laboratory of Molecular and Cellular Therapy, Leloir Institute-CONICET, Buenos Aires, Argentina; 2 Bone and Joint Research Unit, Queen Mary’s School of Medicine and Dentistry, University

of London, London, United Kingdom.

Human tumors are composed of a heterogeneous mass of malignant cells that also include tumor-associated stromal cells that might become a barrier for successful therapies Conditionally Replicative Adenoviruses (CRAds) are a new modality for cancer therapy

We have previously reported that a new CRAd (Ad-F512) where E1A transcription is regulated by a 0.5 kb fragment of the SPARC promoter (SPPr) exhibited strong killing effect over established human melanomas xenografted in nude mice However, its oncolytic effect was strongly reduced on established tumors made of a mix

of melanoma cells and human  broblasts (Lopez MV et al, PLOS One, 2009)

In order to improve the therapeutic efficacy of Ad-F512 we hypothesized that addition of DNA sequences containing responsive elements to different patho-physiological conditions that characterize tumor tissue, such as hypoxia and in ammation, would increase its replication and, therefore its oncolytic activity

We constructed twelve different chimeric promoters containing SPPr combined with Hypoxia Response Elements (HRE), NFκB-response elements (NFκB) or both and compared its transcriptional activity in luciferase expressing plasmids Based on their activity/

speci city ratio in different cell types we selected the chimeric promoter HRE-SPPr where three HREs were placed upstream of SPPr and a triple chimeric promoter NFκB-SP(HRE)Pr where twelve NFκB

response elements were placed upstream of SPPr and 3 HREs inside SPPr sequence We further evaluated the transcriptional activity of the chimeric promoters in an adenovirus backbone using luciferase as a reporter gene We observed 2-10 fold induction of luciferase activity under hypoxia of both chimeric promoters and a 5-fold induction under TNFα treatment of NFκB-SP(HRE)Pr in malignant cells

Based on these results, we constructed two CRAds (Ad-HRE-SPPr and Ad-NFκB-SP(HRE)Pr) where E1A transcription was regulated by the chimeric promoters Both CRAds exhibited 2 to 10 fold increased lytic effect under hypoxia on melanoma cells and  broblasts compared

to the parental Ad-F512

Finally, nude mice harboring tumors made of a mix of melanoma cells and  broblasts that were resistant to three administrations of Ad-F512 (Lopez et al, PlosONE 2009) were treated intratumorally with  ve administrations of 1010 vp/mouse with either of the CRAds

Ad-F512 treatment induced the elimination of tumors in 2 out of 4 mice, while the other 2 mice showed a slight tumor growth However,

in mice treated with Ad-NFκB-SP(HRE)Pr we observed a complete elimination of the tumor in all mice that did not recur even after a follow up period of more than 100 days These results indicate that addition of enhancer elements responsive to tumor-environmental conditions improved the therapeutic effect of a CRAd and might overcome the restriction imposed by the presence of stromal cells

610 De ning the Role of Innate Immune Mechanisms in the Ef cacy of Oncolytic VSV Virotherapy in a Fully Immune Competent Model

Phonphimon Wongthida,1 Rosa Diaz,1,2 Feorillo Galivo,1 Timothy Kottke,1 Jill Thompson,1 Jose Pulido,3 Alan Melcher,4 Richard Vile.1,2,4

1 Department of Molecular Medicine, Mayo Clinic, Rochester, MN; 2 Department of Immunology, Mayo Clinic, Rochester, MN; 3 Department of Ophthalmology and Ocular Oncology, Mayo Clinic, Rochester, MN; 4 Institute of Molecular Medicine, University of Leeds, Leeds, United Kingdom.

We have shown previously that VSV therapy depends on host CD8

and NK cells (Cancer Res 2007 15:2840) and that intra-tumoral

injection of VSV induces several pro-in ammatory cytokines and

chemokines in the B16ova tumor microenvironment (Galivo et

al,.Gene Ther 2010 In Press) We have also observed that virus

replication was, at best, minimal in the B16ova model and that therapy was as effective with a VSV that could only undergo a single cycle of replication as it was with a fully replication competent virus These data suggest that therapy of VSV in the B16ova model is, at least in part, immune mediated Therefore, we investigated the role of the immune system in more detail in this model MyD88 is a critical adaptor protein for IL-1 and TLR signaling in the innate immune response to viruses and other pathogens VSV-mediated therapy was signi cantly decreased in MyD88 knock out mice although levels of in ltrating CD8 T cells, anti virus antibody or virus replication were not signi cantly affected in these mice Therefore, innate immune signaling through the MyD88 pathway is critical for anti tumor therapy However, VSV therapy in TLR4 or IL-1 receptor knock out mice was not signi cantly different from that in C57Bl/6 control mice

We are currently testing the effects of other TLR signaling pathways

on the ef cacy of VSV therapy against B16ova tumors Furthermore,

we observed a highly signi cant increase in the levels of plasmacytoid

DC (CD11b-GR1+), known to be major mediators of IFN-α/β secretion

in response to viral infection, reaching the draining LN following VSV injection In this respect, following intratumoral injection with VSV

in IFN α/β receptor knock out mice, viral spread was uncontrolled leading to rapid toxicity Therapy was also lost when single cycle virus was administered Taken together, our data show that VSV acts

as a potent immune adjuvant in the immune competent setting and that therapeutic ef cacy – at least in this model – is immune based

Therefore, the balance between enhancing virus replication and reducing bene cial immune mediated rejection responses need to

be considered when using immunomodulatory strategies to improve the therapeutic ef cacy of oncolytic virotherapy

611 Differential Effects of VSV and Cyclophosphamide on NK Cells Leads to Antagonistic Effects on Anti-Tumor Therapy of a Murine Mesothelioma Model

Candice L Willmon,1 Jill Thompson,1 Timothy Kottke,1 Stephen Albelda,2 Richard Vile.1

1 Department of Molecular Medicine, Mayo Clinic, Rochester, MN; 2 Pulmonary, Allergy & Critical Care Division, University of Pennsylvania Medical Center, Philadelphia, PA.

Malignant mesothelioma is an aggressive neoplasm of the lining

of the pleura or peritoneum and, to date, conventional treatments for mesothelioma, including chemotherapy, radiation and surgical resection, tend to be more palliative than ef cacious However, recent evidence has indicated that mesothelioma is sensitive to immunotherapy This led us to hypothesize that the treatment of mesothelioma with a combination of chemo-immunotherapy and an oncolytic virus may provide both enhanced tumor cell killing and immunotherapeutic bene ts Using the oncolytic virus, vesicular stomatitis virus (VSV), in conjunction with cyclophosphamide (CPA),

we investigated the generation of both anti-viral and anti-tumor responses and how such immune responses can be manipulated to improve on the therapy of a murine mesothelioma model, AE17ova Treatment of AE17ova-bearing mice with CPA, with or without virus, prolongs survival (p<0.0001) and, in some cases, leads to complete cures (∼40%) when given in either a metronomic or single

dosing schedule Ex vivo FACS analysis showed that this therapy

was associated with a decrease in the number of T regulatory (Treg) cells (p=0.0009) and an increase in natural killer (NK) cells (p=0.0042), CD4+ (p=0.0002) and CD8+ T cells (p=0.0033), as well

as CD11b+Ly6G+ cells (p=0.0007) Surprisingly, however, treatment

with VSV in conjunction with CPA consistently decreased this

survival prolongation in comparison to mice treated with CPA alone (p=0.0023) Mice treated with both CPA and VSV showed both similar decreases in Tregs, as well as increases in NK, CD4+ and CD8+ T cells,

as CPA-alone treated mice In contrast, although treatment with CPA alone induced an increase in the number of IFN-γ-producing, non-antigen speci c-CD8+ T cells, this effect was signi cantly diminished

in mice treated with both CPA and virus Depletion studies showed that the treatment ef cacy associated with CPA, with or without VSV,

is dependent upon CD4+ T cells Notably, splenocytes from mice treated with CPA had high levels of cytolytic activity against target AE17ova tumor cells However, this activity was signi cantly reduced

in mice treated with both CPA and VSV (p=0.0007 compared to PBS; p=0.014 compared to CPA/VSV) Consistent with these data, survival prolongation induced by CPA treatment, alone or in combination with VSV, was dependent upon NK cells, as shown by depletion of

NK cells by asialo-GM-1 antibody treatment (p = 0.0044 CPA; p = 0.023 CPA/VSV) Taken together, these data suggest that CPA and VSV have differential effects on immune cell subsets, such as NK cells, in this system, which leads to antagonism between these two

agents when used in combination in vivo This contrasts with other

tumor models where the combination of oncolytic virus and CPA have bene cial additive effects on therapy and highlights the importance of understanding the mechanistic basis of interactions between viruses, chemotherapeutic drugs and immune components in the development

of combination therapies

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