425 translation of enhanced dendritic cell vaccines using a cid inducible cd40 receptor

To determine efficacy in an orthotopic model, syngeneic GL261-luciferase-stable tumor cells were implanted into the brains of C57BL/6 mice so that tumor progression and/or regression cou

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CANCER-IMMUNOTHERAPY: SIGNAL TRANSDUCTION AND ANTIGEN PRESENTATION

further improvements are needed to provide a breakthrough for

GBM treatment To overcome the restraint of transient expression

using nonviral vectors, we utilized Sleeping Beauty transposon

vectors to achieve integration and sustained expression in GBM In

parallel we used CpG oligodeoxynucleotides to stimulate potent

anti-tumor immune responses To determine efficacy in an orthotopic

model, syngeneic GL261-luciferase-stable tumor cells were implanted

into the brains of C57BL/6 mice so that tumor progression and/or

regression could be measured by bioluminescent imaging in vivo

(see progression in Figure 1) We used intratumoral

convection-enhanced delivery (CED) to infuse CpG oligodeoxynucleotides or

Sleeping Beauty-based INF-gamma transposons into mice bearing

intracranial GBM Either therapy alone minimally extended survival

(p>0.05), but the combination of CpG plus INF-gamma gene transfer

facilitated complete tumor regression and long-term survival in a

significant fraction of mice (p<0.05) Current studies are underway

to define the immune mechanisms responsible for tumor regression

and optimize treatment efficacy These results suggest that this

combined treatment approach may be useful for treating patients

with GBM

424 Myd88/TLR Signaling Is Required for

Immunotherapy-Mediated Glioblastoma

Regression

James F Curtin,1 Gwendalyn D King,1 Marianela Candolfi,1

Chunyan Liu,1 Kathrin S Michelsen,2 Moshe Arditi,2 Tamer M

Fakhouri,1 Begonya Comin-Anduix,3 Antoni Ribas,3 Pedro R

Lowenstein,1 Maria G Castro.1

1 GTRI and Medical and Molecular Pharmacology, Cedars-Sinai

Medical Center and UCLA, Los Angeles, CA; 2 Pediatrics

Infectious Diseases and Immunology, Cedars-Sinai Medical

Center, Los Angeles, CA; 3 Medicine Hematology and Oncology,

UCLA, Los Angeles, CA.

Glioblastoma multiforme (GBM) is the most common type of

brain tumor and is a leading cause of mortality with mean patient

survival 6-12 months following diagnosis We have recently shown

that combined treatment of glioma bearing rats with Ad-Flt3L and Ad-TK+GCV dramatically improved survival in an intracranial syngeneic rat model Ad-Flt3L encodes human soluble fms-like tyrosine kinase 3 ligand (Flt3L) and stimulates dendritic cell proliferation and improves antigen presentation Ad-TK expresses herpes simplex type I-thymidine kinase and selectively kills rapidly dividing cells when used in combination with the non-toxic prodrug ganciclovir (GCV) We now show that combined treatment with Ad-TK and Ad-Flt3L improves long-term survival in C57BL/6 mice bearing syngeneic intracranial GL26 tumors when all individual therapies completely fail (p<0.001) Survival is not improved in RAG1 deficient mice (RAG1-/-), deficient for mature B and T cells,

or mice deficient for either CD4+ and CD8+ cells, implicating that both CD4 and CD8 T cells play a critical role in tumor regression Tumor antigen Trp2 is expressed by GL26 cells and clonal expansion

of Trp2(180-188) reactive T cells was determined using ELISPOT, MHC I – Trp2(180-188) tetramers and T cell proliferation assays following treatment with Ad-Flt3L and Ad-TK+GCV Furthermore, Ad-Flt3L and Ad-TK did not improve survival in tumor bearing Myd88- or TLR2-deficient mice, indicating that TLR2 and Myd88 signaling was involved in the development of effective anti-tumor immune responses In summary, combined treatment with Ad-Flt3L and Ad-TK+GCV improves survival in a large syngeneic intracranial tumor model in mice This was dependent on the establishment of tumor antigen specific T cell anti tumor immune responses that was dependent, in turn, on TLR2 signaling via the adapter protein Myd88

425 Translation of Enhanced Dendritic Cell Vaccines Using a CID-Inducible CD40 Receptor

Natalia Lapteva,1 Brent A Hanks,1 Jianghong Jiang,1 Kevin M Slawin,2 David M Spencer.1

1 Immunology, Baylor College of Medicine, Houston, TX;

2 Urology, Baylor College of Medicine, Houston, TX; 3 Houston, TX.

Dendritic cell (DC)-based vaccination is a promising strategy to induce cancer immunity Nevertheless, clinical applications have been almost universally disappointing One potential limitation of current strategies is the use of maturation cocktails (MC) that include PGE2, which upregulate chemotaxis to lymph nodes, but may inhibit the ultimate activation state of the resulting DC product Herein, we describe a novel DC-based vaccine that circumvents the use of standard MC and instead utilizes a genetically engineered inducible CD40 (iCD40) receptor [1]

Our platform contains human monocyte-derived DCs (MoDCs) transduced with adenovector Ad5/f35-ihCD40 (iCD40-MoDC) and incubated for 2 days in the presence of monophosphoryl lipid A (MPL) and dimerizing drug, AP20187 Here we show that MoDC transduction with Ad5/f35-iCD40 effectively enhances co-stimulatory and migratory functions, including upregulation of

IL-6, activation marker CD83, chemokine receptor CCR7 and augmentation of chemotaxis to CCL19/MIP3β Furthermore, the migratory abilities of iCD40-MoDCs were comparable to MoDCs matured with standard maturation cocktail (i.e TNF-α, 1β,

IL-6 and PGE2) Unlike murine bone marrow-derived DCs expressing iCD40, IL-12 production by human MoDCs required additional TLR-4-ligand agonists, such as LPS or clinical analog MPL, which led to high (ng) levels of IL-12p70 in ihCD40-MoDCs, but not MC-stimulated or control MoDCs Moreover, iCD40 and TLR4 agonists additively enhanced activation marker expression

We further show that iCD40 and MPL enhanced the immunostimulatory functions of MoDC, such as improving the stimulation and tumor-specific killing activity of MAGE-3 tumor antigen-specific CTL (shown by IFN-γ ELISPOT and 51Cr-release assays) and facilitated Th1 polarization of nạve CD4+ T-helper cells in vitro Moreover, we have developed a novel in vivo system

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of tracking human DCs transduced with adenovirus expressing

red-shifted luciferase MPL-treated iCD40-MoDCs readily migrated to

the lymph nodes of SCID mice and were detectable for over 1 week

after inoculation In addition, we appraised the systemic toxicity of

the iCD40-DC vaccine in nạve C57BL/6 male mice Mouse DCs

were pulsed with TRP2 melanoma antigen, transduced with

Ad-iCD40 or control Ad-GFP and injected 3 times bi-weekly Mice

were monitored for 3 months and routine CBC and serum chemistry

(for liver and kidney functions) was tested regularly We detected no

significant toxicity of the iCD40-DCs vaccine and highly significant

long-lived elevation of tumor antigen specific CTLs

This data suggest the potency and safety of this

immunotherapeutic approach that combines non-inflammatory TLR

stimulation with synthetic activation of iCD40 in human MoDCs

Unlike standard DC maturation approaches, this combination of

reagents leads to high IL-12p70 levels, enhanced chemotactic

response to lymph node-derived chemokines and augmented tumor

antigen-specific CTL and Th1 responses Inducible CD40-enhanced

DCs represent a promising new strategy for cancer immunotherapy

[1] Hanks B.A et al (05) Nat Med, 11, 130-7

Starting a company (Bellicum Pharmaceuticals) to conduct

prostate cancer trials based partly on the work to be presented

426 dSLIM Immunomodulators Induce

Anti-Tumor Responses Both In Vitro and In Vivo

Manuel Schmidt,1 Javier Cristobal,2 Astrid Sander,1 Bernadette

Brzezicha,2 Sven A Konig Merediz,2 Burghardt Wittig.1,3

1 Mologen, AG, Berlin, Germany; 2 Vivotecnia, Madrid, Spain;

3 Institute for Molecular Biology and Bioinformatics, Charite

Universitätsmedizin, Campus Benjamin Franklin, Berlin,

Germany.

Cytosine-guanine (CpG) motifs containing oligonucleotides

(ODN) are commonly used for immunomodulatory purpose in

cancer therapy and for the treatment of allergic diseases since they

resemble bacterial DNA and serve as “danger signals” These

CpG-ODNs promote predominately a TH1-response with secretion of

IL-12 and IFN-γ, In addition their broad potential includes activation

of B-cell proliferation, monocyte stimulation and secretion of IgM

and IL-6, and stimulation of plasmacytoid DC to produce IFN-

α/-β and thus γδT-cells and NK-cells to express CD69 and secrete

IFN-γ Usually phosphorothioate (PS) modifications are to enhance

the stability, but these are leading to several side-effects, like severe

organ enlargements, morphological changes and immunosuppression

in mice We designed immunomodulatory molecules based on short

covalently-closed dumbbell-like structures (dSLIM) to stabilize the

DNA without the otherwise necessary PS-modification To evaluate

the anti-tumor effect of the dSLIM molecules we developed an in

vitro anti-tumor assay This assay uses supernatant from

dSLIM-activated human PBMCs for incubation with tumor cells in vitro.

We observed increased apoptosis and necrosis of the HT-29 tumor

cell line after incubation with supernatant from dSLIM-treated

PBMC which was significantly higher than the effect of supernatant

from non-treated PBMC In addition, supernatant from

dSLIM-treated PBMC increased the expression of HLA-ABC on the tumor

cells, a pre-requisite for tumor cell recognition by the immune system

These effects were confirmed with human HEK293 and murine

Renca cell lines Analyzing the effect with neutralizing antibodies to

various apoptosis-related cytokines, we observed a crucial role of

IFN-γ but not IFN-α or TNFα To investigate the anti-tumor effects

of dSLIM in vivo, we employed a SKH1 murine model which is

prone to spontaneous development of papillomas Using chemicals

for initiation and weekly promotion of de novo papilloma

development we compared groups of weekly s.c or i.p dSLIM

injections, respectively, with the PBS control group The number of

papilloma developing mice was significantly lower in the dSLIM groups and the total number of papillomas on all mice was reduced

by approximately 50% In conclusion, we showed that dSLIM

immunomodulators exhibit potent anti-tumor effects in vitro and in

vivo.

MS, AS, BW are employees of Mologen AG

427 Adenovirus Mediated GRP94/gp96 Expression in Treatment of Neuroblastoma

Shanling Liu,1,3 Jiangao Zhu,2 He Wang,3 Takashi Kon,1 Chuan-Yuan Li.1

1 Radiation Oncology, Duke University Medical Center, Durham, NC; 2 Immunology, Duke University Medical Center, Durham, NC; 3 West China Second University Hospital of Sichuan University, Chengdu, Sichuan, China.

Neuroblastoma is one of the most common solid tumors of childhood Response to conventional therapy in patients with advanced stage disease is low and mortality is high Novel therapeutic approaches that improve patient survival are clearly needed Tumor-derived glucose-regulated protein 94 (GRP94/gp96), a HSP90 family member, has shown great promise as a tumor vaccine In this study,

we explored the therapeutic efficacy of a combined GRP94/gp96-based genetic immunotherapy and radiation therapy strategy in a mouse neuroblastoma model: Neuro 2a

An adenovirus encoding a secretable form of GRP94 gene (AdsGRP94) was evaluated in various anti-tumor experiments Lethally irradiated, virus-infected cells were used as vaccines Adenoviral vectors were also injected directly into tumors in conjunction with tumor irradiation The results showed that inoculation of AdsGRP94 infected Neuro 2a cells in syngeneic A/J mouse could result in significant tumor growth delay Vaccination with lethally irradiated, AdsGRP94-infected Neuro 2a cells completely prevented subsequent tumor growth from challenge inoculations of as many as 107cells/mouse Splenocytes from mice immunized with AdsGRP94-infected tumor cells showed significant increase in specific tumor cell lytic ability as compared to splenocytes from mice immunized with control-virus infected cells In addition, intracellular cytokine staining results showed AdsGRP94-infected tumor cells immunization could increase T cell IFN-g generation, especially in CD8+ T cell group In established tumor models, when vaccination was combined with radiation therapy and intratumoral AdsGRP94 injections, tumor growth was markedly inhibited Our results indicate that combined AdsGRP94-based immunotherapy and radiation therapy may be a potentially effective strategy for neuroblastoma treatment

428 Development of an Effective Safety Switch for Selective Elimination of Human T Cells In Vivo after Adoptive Transfer

Tom van Meerten,1 Henk Rozemuller,1 Wendy Mackus,2 Paul Parren,2 Jan van de Winkel,2 Marie-Jose Claessen,1 Rozemarijn van Rijn,1 Anton Hagenbeek,1 Anton Martens,1 Saskia Ebeling.1

1 Hematology, University Medical Center Utrecht, Utrecht, Netherlands; 2 Genmab BV, Netherlands.

Adoptive transfer of T cells is frequently associated with unwanted side effects In order to tackle these effects one could introduce a

safety switch into the cells that permits their selective in vivo

elimination The human CD20 gene in combination with CD20 antibodies was recently proposed as a novel safety switch In such

a system, T cells may be genetically modified with a CD20-encoding vector prior to adoptive transfer If necessary, CD20-transgenic

cells can be eliminated in vivo through administration of CD20

antibodies, such as the chimeric antibody rituximab (RTX) that is currently used to treat CD20+ lymphoma cells RTX activates the