190 development of an HLA a0201 restricted t cell receptor that recognizes peptide epitopes from MAGE a3 and MAGE a12 for targeted adoptive t cell immunotherapy of multiple cancer types

190 Development of an HLA A*0201 Restricted T Cell Receptor That Recognizes Peptide Epitopes from MAGE A3 and MAGE A12 for Targeted Adoptive T Cell Immunotherapy of Multiple Cancer Types Molecular The[.]

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CANCER - IMMUNOTHERAPY I

its aim, we attempted to present interleukin (IL)-12 on the surface of

HVJ-E We constructed a mouse IgG constant region (Fc)-displayed

HVJ-E without HN (Fc-DHN-HVJ-E) and produced a single-chain

interleukin-12 (scIL12) fused with ZZ domain of Protein A

(ZZ-scIL12) Fc-DHN-HVJ-E was connected with ZZ-scIL12 by binding

Fc and ZZ domain, and it was possible to have present ZZ-scIL12

on the surface of HVJ-E ZZ-scIL12-presented

Fc-DHN-HVJ-E (scIL12-Fc-DHN-HVJ-E) could activate dendritic cells and splenocytes,

and induced secretion of IFN-g from them Moreover, the anti-tumor

effect of scIL12-HVJ-E was much more powerful than conventional

HVJ-E in mice bearing F10 melanoma and complete eradication was

achieved in approximately 30% of tumor-bearing mice by

scIL12-HVJ-E Systemic injection of scIL12-HVJ-E suppressed metastatic

melanoma nodules in lung In this study, we succeeded in enhancing

the anti-tumor effect of HVJ-E by means of the scIL12 presentation

The high-performance HVJ-E will be available for cancer treatment

by systemic administration in future

188 Combined Gene Therapy Using Adenovirus

Expressing Interleukin-12,

Granulocyte-Macrophage Colony-Stimulating Factor, and

Thymidine Kinase with Prodrug Ganciclovir

Ji-seong Kim,1,2 Kyoung-ju Choi,1,2 Chae-Ok Yun.1,2

1 Brain Korea 21 Project for Medical Science, Yonsei University

College of Medicine, Seoul, Republic of Korea; 2 Brain Korea

21 Project for Medical Science, Yonsei University College of

Medicine, Seoul, Republic of Korea.

Tumors exhibit immune escape properties by generating

immunosuppressive tumor microenvironment and subverting host

immune responses that  nally promote their survival Accordingly,

Immunotherapy has been investigated with various strategies

involving multiple elements such as combination with several

cytokines, co-stimulatory molecules and DC vaccination to improve

immune system in tumor microenvironment Despite of these efforts,

still cancer immunotherapy is limited by various huddles Especially,

lack of tumor susceptibility is one of the focused problems To further

investigate the ef cient and synergistic cancer immunotherapy by

improving tumor susceptibility and tumor cell killing, combined

cytokine and “suicide gene” therapy can be novel approach that

leads to stimulate tumor speci c immune system In this study, we

generated IL-12, GM-CSF and HSV-TK co-expressing oncolytic

Ads for additive and synergistic anti-tumor immune responses and

therapeutic effects through activating type-I immune response and

inducing DC activation via enhancing tumor susceptibility In vitro

assay, cell viability of RdB/IL12/GMCSF-TK Ads treated with

ganciclovir remarkably decreased in viral and ganciclovir

dose-dependent manner Besides, combinational administration of RdB/

IL12/GMCSF-TK Ads with ganciclovir showed signi cant antitumor

effect and prolonged survival rate were observed compared with

control virus in vivo Moreover, massive in ltration of CD4+, CD8+

T cells into tumor tissues were observed and also enhanced

tumor-speci c CTL and increased numbers of IFN-gamma secreting T cells

were induced in splenocytes of co-treated group Overall, our results

indicate that combined IL-12, GMCSF and suicide gene therapy is a

promising cancer immunotherapy by synergistic anti-tumor immune

response

189 Safety Data of MGN1601, a Tumor Vaccine, Made of Allogeneic, Transfected and Irradiated Tumor Cells in Combination with an Immunomodulator for the Treatment of Metastatic Renal Cell Carcinoma

Manuel Schmidt,1 Baerbel Volz,1 Matthias Schroff,1 Kerstin Kapp,1

Christiane Kleuss,1 Marina Tschaika,1 Burghardt Wittig.1,2

1 Mologen AG, Berlin, Germany; 2 Charite Universitaetsmedizin Campus Benjamin Franklin, Institute foe Molecular Biology and Bioinformatics, Berlin, Germany.

Background The cell-based tumor vaccine MGN1601 for the

treatment of renal cell carcinoma (RCC), consists of two active pharmaceutical ingredients: First, genetically modi ed allogeneic

(human) cells in-vitro transiently transfected with four different

MIDGE® vectors encoding IL-7, GM-CSF, CD80 and CD154 and second, the synthetic DNA-based immunomodulatory molecule dSLIM®-30L1, a TLR-9 agonist The cells originate from primary

RCC material Methods Studies of single-dose toxicity were

performed with MGN1601 in rats (heterologous setting) by either subcutaneous or intradermal application (up to 300-fold or 60-fold

of the amount of cells for patients/kg, respectively) Additionally, a single dose subcutaneous application of the murine homologue of MGN1601 in NMRI mice was performed (allogeneic setting, up to 2.900-fold of the amount of cells for patients/kg) Studies of repeated-dose toxicity (5- and 13-times, 28 days and 13 weeks, respectively) were performed in either the heterologous setting or the homologue setting Furthermore, the local tolerance of MGN1601 was tested in

rabbits Results The vaccine generally showed low to no toxicity

at the tested doses In all single-dose toxicity studies the NOEL was higher than the highest dose used In the repeated-dose 28 days toxicity studies (5-times) only slight hematological changes in some female rats from the high-dose group (decrease of neutrophils and increase of lymphocytes) as well as minor changes at the injection

site in some animals of all dose groups were observed Conclusions

The murine homologue of MGN1601 applied to mice as well as MGN1601 in the heterologoues setting in rats showed excellent safety characteristics and was extremely well tolerated in all tested models

up to very high multiples of the anticipated human therapeutic dose/

kg A clinical trial Phase I/II was initiated in November 2009 First patients will be included in the 1st Q/2010 The treatment duration will

be 3 months An extension treatment will last for further 2 years In this clinical study safety and ef cacy of MGN1601 in patients with metastatic RCC will be evaluated

190 Development of an HLA-A*0201 Restricted

T Cell Receptor That Recognizes Peptide Epitopes from MAGE-A3 and MAGE-A12 for Targeted Adoptive T Cell Immunotherapy of Multiple Cancer Types

Nachimuthu Chinnasamy,1 Jennifer A Wargo,2 Zhiya Yu,1

Timothy L Frankel,1 John P Riley,1 Maria R Parkhurst,1 Steven

A Feldman,1 Nicholas P Restifo,1 Paul F Robbins,1 Steven A

Rosenberg,1 Richard A Morgan.1

1 Surgery Branch, National Cancer Institute, Bethesda, MD;

2 Surgery Department, Massachusetts General Hospital, Boston, MA.

Adoptive cell transfer therapy using peripheral blood lymphocytes (PBL) genetically engineered to express tumor antigen-speci c T cell receptors (TCRs) is a promising strategy for treating patients with malignancies TCR engineered PBL targeting melanocyte differentiation antigens mediated objective tumor regression in patients with metastatic melanoma However, this therapy has also been shown to lead to severe on-target toxicity against skin, eye and ear, normal tissue that contain antigen-expressing melanocytes

CANCER-ONCOLYTIC VIRUSES I

(Johnson LA et al, Blood, 114: 535-546, 2009) In an attempt to avoid toxicity to normal tissue, TCRs were derived that recognize cancer testis antigens (CTA) These antigens are expressed in multiple tumors with no expression in normal tissue with the exception of immunoprivileged sites such as testis and placenta that lack expression

of MHC class I molecules The MAGE family of CTA represent attractive candidates for targeted adoptive immunotherapy of cancer and one of the members of this family MAGE-A3 is expressed in

>70% of metastatic melanomas, as well as a wide range of non-melanoma epithelial malignancies including lung, ovarian, bladder, head and neck carcinomas, multiple myeloma and hepatocellular carcinoma

Initially, transgenic mice that expresses the human class I HLA-A*0201 molecule were immunized with either of two candidate peptides, MAGE-A3: 112-120 (KVAELVHFL) or 271-279 (FLWGPRALV) with a helper peptide (HBVc: 128-140) Murine T cell clones were generated from the splenocytes following repeated

in vitro stimulation with peptides and MAGE-A3 speci c TCR α and

β chains were then isolated, tested for reactivity, and cloned into an MSGV1 based retroviral vector Two TCRs recognizing the epitopes were selected for detailed evaluation Expression of both TCRs in human PBL demonstrated antigen-speci c reactivity in the form of tumor cell lysis and cytokine secretion against a range of melanoma and non-melanoma tumor cell targets Based on the levels of antigen speci c reactivity against target cells, the TCR against MAGE-A3:

112-120 was selected for further development Peptide epitopes from both MAGE-A3 and A12 were ef ciently recognized by the TCR engineered PBL, thus potentially broadening the number of tumors that can be targeted by this TCR In an effort to improve the function of the TCR, single amino acid substitution variants of the CDR3 region in the α chain were generated Substitution of alanine

to threonine at position 118 in the CDR3 region of the α chain of MAGE-A3: 112-120 TCR improved its function Based on these results a clinical trial is planned in which PBL will be transduced with the optimized TCR and transferred to the autologous patient in

an attempt to treat MAGE-A3 and/or MAGE-A12 expressing tumors

of different histologies

Cancer-Oncolytic Viruses I

191 A Comparative Study of Stem Cell-Based Cell Carrier Systems for the Systemic Delivery of

an Oncolytic Adenovirus for Antiglioma Therapy

Matthew A Tayler,1 Atique U Ahmed,1 Ilya V Ulasov,1 Irina Y

Balyasnikova,1 Maciej S Lesniak.1

1 The University of Chicago Brain Tumor Center, The University of Chicago, Chicago, IL.

CRAd-S-pk7 is a survivin promoter regulated oncolytic adenovirus, which has shown optimistic anti-glioblastoma (GBM) activity in the preclinical setting However, successful application of this approach will require enhanced delivery of the therapeutic virus in order to treat disseminated tumor burden in the brain The discovery of the inherent tumor-tropic properties of the stem cells provides a unique opportunity for targeted therapy that employs stem cells as cellular vehicles to track metastatic tumor burden and also deliver the therapeutic payload

at the tumor sites without activating the host immune system Based on this, we optimized the stem cell based delivery system by comparing human neural stem cell (NSC) and mesenchymal stem cell (MSC)

as a cell carrier to deliver oncolytic adenovirus systemically for antiglioma therapy We  rst evaluated the direct migratory capacity

of the NSCs and the MSCs to glioma both in vitro and in vivo and

observed that NSCs have a signi cantly greater speci c migratory activity in response to two primary GBM samples and glioma cell

lines (P<0.0001) NSCs also exhibited higher expression of most

of the cell surface receptors required for adenovirus (Ad) infection

including the primary Ad binding receptor CAR (98% for NSCs vs 1% MSCs positive), avb3 (NSC 76% vs MSC 1%) and Syndecan (NSC 18% vs MSC 1%), one of many heparan sulfate proteoglycans targeted by pk-7 modi ed CRAds Both MSC and NSC infected with CRAd-S-pk7 virus showed dose-dependent increase in viral burst size with a log higher viral progeny produced by the NSC It took about 60-72h to complete one cycle of virus replication in the NSC

as compared to 84-96h for the MSC The tumor speci c migration

of both stem cells was signi cantly affected upon infection with CRAd-S-pk7 Finally, we compared the CRAd delivery capacity of

the two stem cells in vivo by directly injecting CRAd-S-pk7 loaded

stem cells into established intracranial glioma tissue and observed considerably higher number of stem cells and about one log more oncolytic virus recovered in the group that received neural stem cell loaded CRAd-S-pk7 Taken together, we conclude that NSC based carrier system is more effective in homing to tumor and supporting oncolytic adenovirus replication and therefore, more suitable as a cell carrier system to speci cally deliver oncolytic virus for treatment of malignant gliomas

192 A Novel Coxsackievirus B Shows Remarkable Oncolytic Capacity Against Lung Cancer

Shohei Miyamoto,1 Hiroyuki Inoue,1 Meiko Yamada,2 Xin Meng,1 Takafumi Nakamura,3 Yasuo Urata,2 Hiroyuki Shimizu,4

Kenzaburo Tani.1

1 Division of Molecular and Clinical Genetics, Medical Institute

of Bioregulation, Kyushu University, Fukuoka, Japan; 2 Oncolys BioPharma Inc., Tokyo, Japan; 3 Core Facility for Therapeutic Vectors, The Institute of Medical Science, Tokyo University, Tokyo, Japan; 4 Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan.

Recently, lung cancer is the most common cause of cancer death

in the world Three standard therapeutic approaches including surgery, chemotherapy and radiotherapy are currently used There are, however, still many patients suffered from recurrent or metastatic tumors refractory to these therapies Development of novel therapies is de nitely required to overcome such refractory cancers Oncolytic viral therapy is one of the promising candidates for such novel anticancer therapy A small number of enteroviruses, such as coxsackievirus A (CVA) and echovirus, were reported to have lytic activities for various human malignancies We found that coxsackievirus B (CVB) displayed a high level of tropism and lytic activities for human lung cancer cells after screening of representative human enteroviruses CVB targeted and destroyed lung cancer cells via surface virus receptors of coxsackievirus and adenovirus receptor (CAR) at a multiplicity of infection (MOI) of 0.001 in crystal violet assay, whereas CVB did not destroy normal lung cells at a MOI of

10 due to their lower expression of CAR MTS cell proliferation assay also supported the results By contrast, the expression levels

of decay-accelerating factor (DAF), well-known alternative receptor for CVB infection, were not associated with the entry of CVB into cancer cells Flow cytometric annexin V assay demonstrated that the main oncolytic effects of CVB for cancer cells were attributed not to apoptosis but cellular degenerative destruction caused by their viral proliferations Furthermore, in vivo study using human tumor-bearing nude mice showed that the intratumor injections of CVB inhibited substantial growth of subcutaneously pre-established lung tumors

In conclusion, our  ndings suggest that CVB can be a novel therapeutic modality for human lung cancer

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