358 systemic combination virotherapy for malignant melanoma with vesicular stomatitis virus and adoptive t cell transfer

358 Systemic Combination Virotherapy for Malignant Melanoma with Vesicular Stomatitis Virus and Adoptive T Cell Transfer Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American So[.]

Molecular Therapy Volume 19, Supplement 1, May 2011

CANCER-ONCOLYTIC VIRUSES

356 CD8:Class I MHC Interactions Infl uence

T Cell Activity Mediated by Chimeric Antigen

Receptors

David H Aggen,1 Jennifer D Stone,1 Adam S Chervin,1 Andrea

Schietinger,2 Karin Schreiber,2 Hans Schreiber,2 David M Kranz.1

1 Department of Biochemistry, University of Illinois

Urbana-Champaign, Urbana, IL; 2 Department of Pathology, Committee on

Cancer Biology, University of Chicago, Chicago, IL.

One approach to adoptive T cell therapy uses chimeric antigen

receptors (CARs) that contain an antibody (scFv) specifi c for a

tumor-associated antigen CARs derived from antibodies were

originally conceived to target non-MHC restricted antigens, and

unlike most full-length T cell receptors, should in principle be able

to redirect T cell activity independent of the coreceptors CD4 or

CD8 To examine the ability of a single-chain fragment variable

(scFv) CAR to redirect T cells to tumor antigen, we used a model

murine scFv, called 237, to redirect T cell activity against a

tumor-specifi c antigen The 237 antibody recognizes a mutated glycopeptide

expressed by an aggressive murine fi brosarcoma tumor cell line called

Ag104a The tumor-specifi c glycopeptide is generated as a result of

a mutated chaperone protein Cosmc that when repaired, ablates the

237 neo-epitope Using a CAR that consisted of the 237 scFv fused

to the intracellular signaling domains of CD28,CD3ζ, and Lck,

we demonstrate here that T cell specifi c activation occurred in the

presence of the Ag104a tumor line, but not in the presence of a cell

line with a repaired chaperone protein that lacks the tumor antigen

(ACosmc) To assess whether CD8 plays any role in the activation,

we generated a T cell hybridoma line that expressed the 237 CAR,

with and without CD8 The CD8- 237 and CD8+ 237 expressing cells

mediated similar activation with Ag104a tumor cells However, when

the 237 CAR lines were incubated with plate-bound glycopeptide

antigen, the CD8+ line was completely inactive, whereas the CD8-

line was stimulated effectively Thus, CD8 expression inhibited

antigen-specifi c activity when antigen was targeted that lacked MHC

Class I We propose that Lck sequestration by the co-receptors away

from the immunological synapse reduced signaling mediated by

interactions with non-MHC ligands Additional lines of evidence

included the inhibition of activity of the 237 CAR/CD8 line, in

the presence of the Ag104a APC, with an antibody to CD8 In the

normal activation of CD8+ T cells, CD8 with its associated Lck

co-localizes at the immunologic synapse with T cell receptors, facilitating

effi cient T cell responses CD8 contributed to CAR mediated activity,

presumably through interactions with class I MHC molecules that

cluster near the scFv antigen epitopes This observation that class I

MHC can facilitate the activity of a CAR through a CD8-dependent

mechanism has important implications for CARs directed against

non-MHC antigens in tumor immunotherapy For example, reduced

class I MHC levels on a patient’s tumor cells may reduce the effi cacy

of CAR-mediated T cell activity by allowing Lck to be sequestered

away form the synapse Support from NIH Program Project # P01

CA097296

357 Genetically Engineered T-Cells Targeting

Cancer Associated Fibroblasts for Cancer

Immunotherapy

Sunitha Kakarla,1,3 Lisa Wang,1 David Rowley,1 Klaus

Pfi zenmaier,2 Stephen Gottschalk.1,3

1 Translational Biology and Molecular Medicine, Baylor College of

Medicine, Houston, TX; 2 Institute of Cell Biology and Immunology,

University of Stuttgart, Stuttgart, Germany; 3 Center for Cell and

Gene Therapy, Baylor College of Medicine, Houston, TX.

Introduction: Adoptive T-cell therapy has had considerable

success in effectuating antitumor responses, however complete

eradication of bulky disease is rarely observed This limited effi cacy

is most likely due to the tumor stroma, which is not targeted by tumor-specifi c T cells Cancer associated fi broblasts (CAFs), the central component of the tumor stroma, secrete inhibitory factors and nutrient depleting enzymes that are detrimental to effector T-cell function In addition, CAFs promote angiogenesis and secrete extracelluar matrix components, which act as a physical barrier CAFs express fi broblast activation protein (FAP); a membrane bound serine protease, which is an attractive immunotherapeutic target The aim of this project was to generate FAP-specifi c T cells and determine if targeting the tumor stroma with FAP-specifi c T cells

has antitumor effects Methods: To generate FAP-specifi c T cells

we took advantage of chimeric antigen receptors (CARs), which consists of antigen-specifi c single chain variable fragments (scFv) linked to T-cell receptor signaling domains Using this approach we generated 2 CARs, which are specifi c for human FAP (hFAP) or human and murine FAP (mhFAP) T cells expressing hFAP-CARs

or mhFAP-CARs were generated by retroviral transduction (hFAP-

or mhFAP-T cells) Ex vivo, effi cacy of hFAP- and mhFAP-T cells was determined by their ability to 1) secrete cytokines in coculture experiments with FAP-positive tumor or stroma cells, and 2) kill

FAP-positive targets in cytotoxicity assays To test in vivo, if selective

targeting of FAP on tumor stroma prevents the development of human tumors in a xenograft model, we took advantage of lymphoblastoid

cell lines (LCL), which are FAP-negative Results: hFAP-CARs or

mhFAP-CARs were successfully expressed on T cells as judged by FACS analysis hFAP- T cells recognized FAP-positive human tumor and stroma cells as judged by cytokine production and cytotoxicity assays In addition to recognizing human FAP positive targets, the mhFAP-T cells also recognized and killed murine FAP targets To evaluate if targeting the tumor stroma, prevents the development of tumors, FAP-negative, luciferase-expressing LCLs, were mixed with hFAP, mhFAP-, or nontransduced T cells prior to the s.c injection into fl anks of SCID mice While LCLs tumor readily established

in mice injected with LCL/hFAP-T cells or LCL/NT-T cells; LCL tumor growths was 10 to 100 fold slower in mice injected with LCL/mhFAP-T cells as judged by serial bioluminescence imaging

Conclusions: Our results indicate that targeting FAP-positive stroma

with T cells delays the development of tumors in human xenograft models Thus, targeting the tumor stroma in addition to cancer cells with FAP-specifi c T cells has the potential to improve current immunotherapy approaches for cancer

Cancer-Oncolytic Viruses

358 Systemic Combination Virotherapy for Malignant Melanoma with Vesicular Stomatitis Virus and Adoptive T Cell Transfer

Diana M Rommelfanger,1,2 Phonphimon Wongthida,1 Karen K Kaluza,1,3 Rosa M Diaz,1,3 Jill M Thompson,1 Timothy J Kottke,1

Richard G Vile.1,3

1 Department of Molecular Medicine, Mayo Clinic, Rochester, MN; 2 Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Rochester, MN; 3 Department of Immunology, Mayo Clinic, Rochester, MN.

Our goal is to develop truly systemic virotherapy treatment regimens for metastatic cancer that do not necessitate direct intratumoral injection We have shown in the B16ova melanoma model that combining adoptively transferred OT-I T cells (1x10^6 nạve cells), which are specifi c for the surrogate tumor-associated antigen (TAA) OVA, with intratumoral VSV (5x10^8 PFU) encoding the OVA antigen (VSV-ova) results in regression of established tumors and an enhancement of survival compared to either treatment alone (p<0.00001, p=0.052 respectively) Comparable results were obtained when combination treatments targeting the endogenous melanoma TAA gp100 (gp100-specifi c PMEL T cells + VSV-gp100) were

Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy S140

CANCER-ONCOLYTIC VIRUSES

administered (p<0.00001 compared to PMEL T cells alone, p=0.016

compared to VSV-gp100 alone) We also showed that combining

adoptively transferred T cells with intratumoral virus results in

activation of a potent, tumor-specifi c T cell response that is able to

target and eliminate both the injected and, importantly, uninjected

tumors Therefore, we are currently developing a fully systemic

regimen that is designed to provide effi cacious anti-tumor therapy,

even for tumors that are inaccessible to the end of a needle In this

respect we have shown that combining PMEL T cells (1x10^6 nạve

cells) with intravenous VSV-gp100 (5x10^7 PFU) provides signifi cant

survival benefi t over the individual treatments (p=0.0001, p=0.0002

respectively) Presently, we are working to improve upon the number

of long-term regressions by refi ning the timing of the T cell and virus

injections and investigating further combinations targeting additional

tumor antigens Mechanistic studies are also ongoing and indicate

that intravenous, TAA-expressing virus promotes the survival and

activation of the adoptively transferred T cells in the tumor-draining

lymph nodes and spleen, which is critical for the observed therapy

More specifi cally, intravenous VSV-gp100 results in a signifi cantly

greater persistence of PMEL T cells than the control treatments of

intravenous and intratumoral VSV-GFP (p<0.001), intravenous and

intratumoral PBS (p<0.002) as well as intratumoral VSV-gp100

(p<0.01) These data will lead to clinically-relevant, completely

systemic virotherapy protocols for cancer patients, melanoma and

otherwise, that have a dismal prognosis with the currently available

treatments

359 Enhancing Therapeutic Index of Oncolytic

Vaccinia Virus through Combining MicroRNA

Regulation and Thymidine Kinase Deletion

Mina Hikichi,1 Minoru Kidokoro,2 Hisatoshi Shida,3 Hideaki

Tahara,1 Takafumi Nakamura.1,4

1 Institute of Medical Science, Tokyo University, Tokyo, Japan;

2 National Institute of Infectious Diseases, Musashimurayama,

Japan; 3 Institute for Genetic Medicine, Hokkaido University,

Sapporo, Japan; 4 PRESTO, Japan Science and Technology Agency,

Kawaguchi, Japan.

Vaccinia virus, once widely used for smallpox vaccine, has been

engineered and used as an oncolytic virus for cancer virotherapy

We have previously shown that microRNA (miRNA) regulation

enables tumor-specifi c viral replication by altering the expression of

a targeted viral gene of an attenuated vaccinia virus vaccine strain

LC16m8, which is an attractive backbone because of extremely

low neurovirulence profi le Since the deletion of viral glycoprotein

B5R not only decreases viral pathogenicity but also impairs the

oncolytic activity of vaccinia virus, we used miRNA-based gene

regulation to suppress B5R expression through let-7a, a miRNA that

is downregulated in many tumors In mice with s.c human pancreatic

cancer xenografts, intratumoral administration of miRNA-regulated

vaccinia virus (MRVV) with target sequences complementary to

let-7a in the 3’-untranslated region (3’UTR) of the B5R gene resulted

in tumor-specifi c viral replication and signifi cant tumor regression

without side effects, although all of the mice treated with the

control let-7a-mut virus containing insertion of the disrupted miRNA

target sequences died or were sacrifi ced on days 39–59 due to the

virus-associated toxicity, such as pock lesions on body surfaces and

weight loss On the other hand, our recent study has shown that

MRVV escapes from the cellular miRNA system in another therapy

model, where the control let-7a-mut virus causes death on days 24–43

earlier than the intratumoral therapy model Although intraperitoneal

injection of MRVV on day 0 (1 x 107 pfu per mouse) in mice with

the i.p xenografts signifi cantly prolonged survival compared with

that of the control let-7a-mut virus, all of the mice treated with MRVV

fi nally died or were sacrifi ced on days 32–52 due to the viral toxicity

Sequence analysis did not show any mutations in the target inserts

of MRVV which is recovered from pock lesions on their tail 35 days after virus administration, suggesting that there are other possibilities such as miRNA saturation In an effort to address this toxicity, we have developed a combined miRNA-regulated and thymidine kinase-deleted vaccinia virus (MDVV) and investigated its properties in the same intraperitoneal therapy model The expression cassette encoding luciferase and EGFP was inserted into the A56R or J2R locus of MRVV by homologous recombination, resulting in MRVV-LG or MDVV-LG respectively Intraperitoneal administration of MDVV-LG signifi cantly prolonged survival compared with that of MRVV-LG and mock therapy Furthermore, all of the mice treated with

MDVV-LG did not show any symptoms of viral toxicity at the end of the experiment Our study demonstrated that multi-mechanistic regulation

of oncolytic vaccinia viruses through combining incorporation of specifi c miRNA target sequences into the 3’UTR of B5R gene and insertional inactivation of vaccinia virus gene encoding thymidine kinase enhances the safety profi le, tumor specifi city and therapeutic index

360 Engineering Viruses To Enhance and Monitor Systemic Oncolytic Effi cacy

Shruthi Naik,1 Rebecca Nace,1 Glen Barber,2 Stephen J Russell.1

1 Department of Molecular Medicine, Mayo Clinic, Rochester, MN;

2 Department of Microbiology & Immunology, University of Miami

School of Medicine, Miami, FL.

The need for potent systemically delivered therapies prompted us

to engineer Vesicular stomatitis virus (VSV) to enhance both viral cytolytic activity and immunogenicity to treat Multiple Myeloma (MM), an incurable disseminated plasma cell malignancy VSV is

a rapidly replicating oncolytic vector that preferentially replicates

in and kills tumor cells We demonstrate here a single systemic administration of VSV coding for IFNβ (VSV-IFN) has potent activity

in treating both subcutaneous and disseminated 5TGM1 myeloma in syngeneic C57KaLwRij mice VSV administration in mice bearing disseminated myeloma transiently reduced myeloma burden, delayed overall myeloma progression and signifi cantly prolonged survival Expression of murine IFNβ significantly enhanced therapeutic activity We re-engineered VSV to induce increased expression of Interferon-β (IFN) and the Sodium Iodide symporter (NIS) transgenes,

to improve viral toxicity profi le, enhance IFNβ mediated oncolytic effi cacy and to allow noninvasive imaging of viral bio-distribution following systemic administration Similar to previous fi ndings, a single intravenous dose of VSV-IFN-NIS induced complete regression

of subcutaneous 5TGM1 myeloma tumors with a signifi cantly reduced tumor relapse rate of tumors in mice treated with VSV-mIFN-NIS (expressing murine IFNβ) Non-invasive SPECT-CT imaging using Tc-99m shows tumor specifi c viral gene expression by 24h and viral activity occurs within the fi rst week post viral administration The speed of viral activity indicated by in vivo imaging prompted further analysis of treated tumors providing detailed visualization of the key steps of viral entry and oncolysis Viral extravasation is distributed throughout the tumor parenchyma infecting clusters of tumor cells without infecting blood vessel endothelial cells Localized spread

of infectious centers forms rapidly expanding foci of infection that subsequently coalesce to destroy tumors by 48-72h following treatment Preliminary results have demonstrated similar ability of systemically administered to pervade and destroy MPC-11 myeloma tumors in immune competent Balb/c mice emphasizing the potent ability of VSV to effectively eradicate myeloma tumors in vivo by direct viral spread and oncolysis Further studies using antibodies

to suppress T-cells revealed T-cell activity mediated IFNβ-induced prevention of tumor relapse Overall, the data provides clear evidence that engineered VSV-IFN-NIS specifi cally and rapidly eliminates tumor cells following systemic administration Viral activity can