331 phase i study of the “triad″ autologous (FANGâ„¢) vaccine, incorporating bifunctional shRNAfurin and GMCSF transgene expression, in advanced cancer patients

331 Phase I Study of the “Triad� Autologous (FANG™) Vaccine, Incorporating Bifunctional shRNAfurin and GMCSF Transgene Expression, in Advanced Cancer Patients Molecular Therapy Volume 20, Supple[.]

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CANCER – IMMUNOTHERAPY: HARNESSING GENE THERAPY TO IMPROVE ANTI-TUMOR EFFECT

329 Redirection of TH17 Cells with a Car

Containing the ICOS Costimulatory Domain

Enhances Function, Antitumor Activity and

Persistence of TH17 Cells

Sonia Guedan,1 Carmine Carpenito,1 Shannon E McGettigan,1

Matthew J Frigault,1 John Scholler,1 Yangbing Zhao,1 Carl H

June.1

1 Abramson Family Cancer Research Institute and Department of

Pathology and Laboratory Medicine, University of Pennsylvania,

Philadelphia, PA.

Adoptive transfer of large numbers of Th17 cells polarized and

expanded in vitro is an attractive therapy for the treatment of cancer

CD278, the inducible costimulator (ICOS) has been shown to be

critical for the sustained expansion of human Th17 cells after their

primary activation We analyzed whether incorporation of the ICOS

intracellular domain in a chimeric antigen receptor can promote

Th17 phenotype after antigen priming and enhance the antitumor

activity of engineered T cell therapies Th17 polarized cells were

engineered to express a single-chain variable fragment that binds

mesothelin (SS1) fused to the T cell receptor- ζ signal transduction

domain in tandem with the CD28, CD137 (4-1BB) or CD278 (ICOS)

intracellular domains When stimulated with aAPC or tumor cells

expressing mesothelin, Th17 cells redirected with the SS1-ICOS-z

chimeric receptor secreted high amounts of IL17A, IL17F and CCL20

but nominal levels of IL-2 Moreover, Th17 activation through ICOS

induced the expression of CD161, consistent with a predominant

Th17 phenotype By contrast, Th17 cells redirected with the

SS1-28-z, secreted higher amounts of IL-2 and IFNg but nominal levels of

IL17A and IL17F, and had low CD161 expression When transferred

into NSG mice with large vascularized pre-established tumors,

Th17/Tc17 cells redirected with SS1-ICOS-z mediated enhanced

antitumor responses, with 70% of mice showing complete remission

Importantly, incorporation of the ICOS intracellular domain in the

CAR signifi cantly increased Th17 cell persistence post infusion when

compared with the incorporation of CD28 or 4-1BB intracellular

domains Incorporation of the ICOS signaling domain in CAR T cells

imparts novel functions compared to CARs encoding CD28 or 4-1BB

signaling domains Our studies indicate that redirection of Th17 cells

with a CAR encoding the ICOS intracellular domain is critical for

obtaining potent Th17 cells with enhanced function and persistence

The design of novel ICOS-based CARs has the potential to augment

antitumor effects in clinical trials

330 Tumor Pericytes: A Novel Target for

Immunotherapy in Renal Cell Carcinoma

Nina C Sabins,1 Anamaka Bose,1 Walter J Storkus.1

1 Dermatology, University of Pittsburgh, Pittsburgh, PA.

Tumor vasculature is classifi ed by leaky, chaotic blood vessels

consisting of several components including vascular endothelial

cells (VEC) and mural cells (smooth muscle cells and pericytes), as

well as immune regulatory cells, stem cells and vascular progenitors

An irregular tumor vasculature limits immune effector cell access

to the tumor, and may in part be responsible for the modest success

observed in many current anti-cancer immunotherapies Certain

inhibitory drugs targeting tyrosine kinase receptors expressed on

tumor stromal cells induce a Type-1 proinfl ammatory skewing of

the tumor microenvironment (TME) with a coordinate reduction

in the number of regulatory immune cell populations A transient

remodeling of the vasculature may also occur in association with

enhanced (pro-infl ammatory) leukocyte recruitment We have shown

by quantitative PCR that the cell-surface antigen delta-like kinase-1

(DLK1), a progenitor cell marker, is exclusively expressed by

tumor-associated pericytes, and is absent from other tumor stromal cell

populations, including VEC and the tumor cells themselves DLK1 is

also not expressed by vascular pericytes isolated from normal somatic tissues, such as kidney Little is currently known regarding pericyte biology in the TME, with no existing treatments specifi cally targeting this cell population We hypothesize that vaccination against antigens expressed preferentially by tumor pericytes will result in vascular remodeling and improved delivery of protective Type-1 T cells into the TME Using a prediction algorithm, we have identifi ed candidate peptide epitopes derived from DLK1 presented by H-2Kd or H-2Ld

to CD8+ T cells in BALB/c mice We have now shown that BALB/c mice bearing established RENCA (renal cell carcinomas) exhibit profoundly suppressed tumor growth after therapeutic vaccination with syngeneic dendritic cells pulsed with DLK1-derived peptides Mice receiving control vaccines exhibited progressive tumor growth indistinguishable from untreated, tumor-bearing animals We believe that immune-based targeting of the tumor pericytes as a consequence

of specifi c vaccination (against antigens like DLK1) has signifi cant translational merit for the treatment of vascularized, solid cancers

331 Phase I Study of the “Triad” Autologous (FANG™) Vaccine, Incorporating Bifunctional shRNAfurin and GMCSF Transgene Expression, in Advanced Cancer Patients

Neil Senzer,1,2,3,4 Minal Barve,1 Cynthia Bedell,1 Joseph Kuhn,5 Peter Beitsch,2 Robert Steckler,2 Robert Hebeler,6 Mitchell J Magee,2 Walton Taylor,2 Brian M Gogel,2 Padmasini Kumar,4 Phillip B Maples,4 John Nemunaitis.1,2,3,4

1 Mary Crowley Cancer Research Centers, Dallas; 2 Medical City HCA, Dallas; 3 Texas Oncology, P.A., Dallas; 4 Gradalis, Inc., Dallas; 5 WLS Surgery Associates, P.A., Dallas; 6 Baylor Medical Center, Dallas.

This non-randomized Phase I study of the autologous FANG™ vaccine expands the “triad” concept of de-tolerizing and augmenting the afferent arm of the immune response (i.e autologous tumor antigen incorporation, GMCSF enhanced antigen presentation and processing, and suppression of endogenous immunosuppression) It contains a plasmid encoding both GMCSF and an RNA interference moiety, bifunctional shRNAfurin, that targets and downregulates the proprotein convertase Furin resulting in knockdown of both TGFb1 and b2 The bi-shRNA technology is designed to maximize target inhibition via simultaneous mRNA cleavage, translational repression and p-body sequestration Briefl y, the vaccine was cGMP manufactured following autologous tumor tissue harvest from a medically indicated procedure at Gradalis, Inc Depending on cell yield of 2 dose levels of vaccine was produced (1 x 107 and 2.5 x 107 cells/injection) The vaccine was administered intradermally once a month (5-12 doses) gIFN ELISPOT was assessed following PBMC co-incubation with the patient’s irradiated, autologous FANG™ vaccine at baseline, Month 4, and individual follow up time points Thirty-one of 58 harvested patients with advanced, previously treated cancer were treated (27 were not for the following reasons: 5 vaccine failures, 2 insuffi cient tissue, 1 benign lesion, 17 pursued other options, 2 repeat harvest but not treated) Vaccine manufacturing success was 88% There were no treatment-related serious adverse events (SAE); Grade 1/2 AE were limited to local reactions Post-manufacturing quality control assays of vaccines assessed GMCSF, Furin, TGFb1 and -b2 expression pre-versus post-transfection Mean GMCSF production post transfection was 1108 pg/106 cells/mL The mean Furin knockdown was 85.09% and the mean TGFb1 and -b2 knockdown was 93.5% and 92.5%, respectively Median survival

of FANG™-treated versus non-treated patients was 554 and 132 days from time of procurement (p<0.0001), respectively, without evidence of dose-response relationship Of only those patients who survived ≥4 months from procurement, median survival was 554 days versus 255 days for FANG™ and non-treated patients (p=.006), respectively Nine (9) of 18 patients for whom sequential ELISPOT

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CANCER – IMMUNOTHERAPY: HARNESSING GENE THERAPY TO IMPROVE ANTI-TUMOR EFFECT

assays were available achieved a positive response at Month 4 which

was durable through at least 6 months of subsequent follow up There

was a signifi cant FANG™-correlated survival benefi t from time of

treatment (p=0.025) Based on this encouraging Phase I data, three

Phase II studies have been initiated

332 Sipuleucel-T in Metastatic

Castrate-Resistant Prostate Cancer (mCRPC): Clinical

Evidence of the Immunologic Mechanism of Action

(MOA)

John Nemunaitis.1

1 Mary Crowley Medical Research Center, Dallas, TX.

Introduction: Sipuleucel-T is an autologous cellular immunotherapy

designed to stimulate an immune response against prostate cancer,

and was approved in 2010 by the US FDA for treatment of patients

with asymptomatic or minimally symptomatic mCRPC Data from

three phase 3 trials have shown that sipuleucel-T significantly

improves overall survival (OS) vs control Materials and Methods:

Sipuleucel-T comprises 3 courses of treatment approximately 2 wks

apart During each course, autologous peripheral blood mononuclear

cells, including antigen presenting cells (APCs), are isolated via

leukaphereses and activated ex vivo with PA2024, a recombinant

fusion protein composed of prostatic acid phosphatase (PAP; an

antigen expressed in most prostate cancers) linked to

granulocyte-macrophage colony-stimulating factor (an immune cell activator)

The activated cells are then re-infused Lot release parameters (total

nucleated cell [TNC] counts, absolute CD54+ cell counts, and CD54

upregulation) were evaluated in all courses of sipuleucel-T for each

patient (pt) Baseline and post-treatment samples were assayed for

antibody responses as measured by ELISA and for cellular responses

as measured by T cell proliferation and IFNg ELISPOT Correlations

between OS and these lot release and immune monitoring parameters

were examined Results: APC activation (as shown by CD54+ cell

counts and CD54 upregulation) was evident in pts randomized to

sipuleucel-T, but not control The magnitude of APC activation

increased signifi cantly at the second and third infusions vs the fi rst,

suggesting an immunologic priming and boosting effect Similarly,

cellular immune responses to PA2024 and PAP during manufacture

were absent during the fi rst infusion, but became detectable at the

second and increased further at the third Cellular responses did not

occur to either antigen in control pts Antibody responses to PA2024

and PAP have also been observed with sipuleucel-T but not control,

with evidence of IgM to IgG class switch In patients treated with

sipuleucel-T, there were positive correlations (P<0.05) between OS

and all lot release parameters, which persisted after adjustment for the

baseline prognostic factors PSA and LDH In the IMPACT study (N =

512), there was evidence of correlations between OS and measures of

peripheral immune response to PAP2024 (ie, antibody response at Wk

6 [P=0.079], T cell proliferation at Wk 14 [P=0.057], and ELISPOT at

Wk 26 [P=0.049]) AEs most frequently associated with sipuleucel-T

include chills, fever and headache – symptoms commonly associated

with cytokine release Conclusions: Data generated continue to show

that APC activation ex vivo with sipuleucel-T stimulates robust,

persistent in vivo immune responses The pattern of APC activation

in sipuleucel-T products and cellular responses to the immunizing

antigen are consistent with immunologic priming and boosting

Correlations between OS, lot release parameters and peripheral

immune responses support antigen-specifi c immune activation as

the MOA of sipuleucel-T

333 A Rationally Designed Oncolytic Adenovirus, Ad-TD-IL-12, Cures Pancreatic Cancer

in the Immunocompetent Syrian Hamster

Pengju Wang,1 Guozhong Jiang,1 Louisa Chard,2 Jiwei Wang,1 Dongling Gao,1 Huan Cao,1 Nicholas R Lemoine,2 Yaohe Wang.2

1 Sino-British Research Centre for Molecular Oncology, Zhengzhou University, Zhengzhou, China; 2 Centre for Molecular Oncology, Barts Cancer Institute, Queen Mary University of London, United Kingdom.

Pancreatic cancer is one of the deadliest types of malignancy, with a

fi ve-year survival rate of less than 5%, and represents an area of unmet clinical need Oncolytic viruses (OVs) are a new class of therapeutic being developed for cancer treatment The fi rst generation of oncolytic adenovirus with deletion of E1B55k gene, H101 (Shanghai Sunway Biotech Co Ltd.), has been approved as the world’s fi rst oncolytic virus for head and neck cancer therapy The similar virus dl1520 has been used for locally advanced primary tumours in pancreatic cancer patients The treatments were well tolerated, but the therapeutic effi cacy was very limited even when the most effective chemotherapy for pancreatic cancer (Gemcitabine) was used in combination Based on our previous fi ndings of the effects of adenovirus E3B and E1B19K genes on the antitumour effi cacy of oncolytic adenovirus,

we created a new generation of oncolytic adenovirus, named Ad-TD,

in which there are triple deletions of E1ACR2, E1B19k and E3 gp19k and retention of the E3B genes The virus demonstrated a superior antitumour effi cacy compared to dl1520, while retaining equivalent safety In order to enhance the antitumour effi cacy further, the Ad-TD virus was armed with the immunomodulatory gene IL-12 through a simple one-step approach developed in our lab The new rationally designed Ad-TD-IL12 adenovirus demonstrated similar cytotoxicity and replication to Ad-TD and expressed IL-12 in a panel of human and Syrian hamster pancreatic cancer cell lines The Ad-TD-IL12 could eradicate 100% of subcutaneously established pancreatic cancer tumours in Syrian hamster in vivo and induce long-lasting tumour-specifi c immunity Intraperitoneal administration of Ad-TD-IL12 also signifi cantly prolonged the survival of Syrian hamsters bearing intraperitoneally spread pancreatic cancer cells without signifi cant toxicity These results suggest that Ad-TD-IL12 is a potential therapeutic for treatment of pancreatic cancer and other solid tumours

334 AAV-Mediated CNS Gene Transfer of Bevacizumab Reduces Glioblastoma Growth and Increases Survival in Mice

Martin J Hicks,1 Lan Wang,1 Eric Aronowitz,2 Jonathan P Dyke,2 Douglas J Ballon,2 Kosuke Funato,3 Vivianne S Tabar,3 David F Havlicek,1 Esther Z Frenk,1 Jianping Qiu,1 Dolan Sondhi,1 Stephen

M Kaminsky,1 Neil R Hackett,1 Ronald G Crystal.1

1 Department of Genetic Medicine, Weill Cornell Medical College, New York, NY; 2 Department of Radiology, Citigroup Biomedical Imaging Center, Weill Cornell Medical College, New York, NY;

3 Department of Surgery, Memorial Sloan-Kettering Cancer Center, New York, NY.

Glioblastoma multiforme (GBM), the most common CNS malignancy, has a median survival of only 14 months Although a great deal is known about the aberrant biology exhibited by GBM, applying therapies against these biologic processes is limited by the blood-brain barrier which restricts systemically administered therapies from reaching the brain We have developed a novel strategy

to bypass these barriers using CNS administration of adenoassociated virus (AAV) gene transfer vectors to deliver the genetic sequences for monoclonal antibodies, modifying normal CNS cells to chronically deliver therapeutic monoclonal antibodies in the local milieu To test this approach, AAVrh.10BevMab, an AAVrh.10-based vector coding for bevacizumab (Avastin®), an anti–vascular endothelial