635 human ovarian cancer derived ascitic fluid has mixed effects on CRAds efficacy

635 Human Ovarian Cancer Derived Ascitic Fluid Has Mixed Effects on CRAds Efficacy Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy S242 CANC[.]

Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy

S242

CANCER - ONCOLYTIC VIRUSES II

633 HLA Typing Identity Test for Patients and

Their FANG™ Autologous Cancer Vaccines –

Update

Yang Yu,1 Padmasini Kumar,1 Fabienne Norvell,1 Connor Phalon,1

Nicolas Taquet,1 Phillip B Maples.1

1 Gradalis, Inc, Dallas.

In the Phase I and II clinical development of the FANGTM

autologous cancer vaccine, we receive tumors from a number of

surgeons / hospitals Assuring the matching of Patient and Autologous

Vaccine is critically important We have taken a number of steps to

ensure the integrity of Patient – Vaccine tumor collection / cGMP

manufacturing / clinical treatment continuum One identity test we

have adopted is the HLA (Human Leukocyte Antigen) molecular

genotyping by SSP-PCR HLA typing applications include organ

and tissue transplantation, immunological control, and paternity

test, etc Molecular typing has been considered as “gold standard”

for HLA typing HLA molecular typing method is reliable, straight

forward, does not require expensive and high maintenance instrument

of gene sequencer like tissue identity test STR assay does, and with

short turnaround time The typing utilizes genomic DNA DNA

from vaccine cells was extracted from 4 million tumor cells which

were obtained during cGMP vaccine manufacture before plasmid

transfection The DNA represent the patient is extracted from one

yellow top vaccutainer The DNA extraction was performed by the

DNeasy kit (Qiagen) and DNA quantity and quality is subsequently

determined by Nano-Drop The ABDRDQ SSP-PCR HLA Typing

Kit, the Class I A Locus, B Locus and the Class II DR/DQ kits are

from One Lambda TAQ polymerase is from Promega We previously

reported an analysis of 31 vaccine-blood sample pairs from patients on

the following FANG vaccine trials (Phase I, 4 pt.s; Phase II Ovarian,

17 pt.s; Phase II Melanoma, 2 pt.s) and 2 patients from TAG Phase I

Since that time another 17 FANG vaccine-blood sample pairs have

been tested and analyzed Among these are 12 ovarian, 2 melanoma,

and 2 colon, all are from Phase II trials All newly tested HLA typing

results are complete matches between vaccines and their respective

bloods, without exception For the single case of mismatch previously

reported, we did further investigation by sending one original

vaccine QC vial and three different cryopreserved PBMC collected

on different dates to a third party for STR tissue identity testing

The results demonstrated that the vaccine matched two of the blood

samples, but not the PBMC originally tested Obviously there was a

mishandling of that particular blood sample Since the HLA typing

matched with the two blood samples drawn later from the patient, the

vaccine origin from the patient is confi rmed The positive outcome

of this incident is that it proved the sensitivity and reliability of the

HLA typing The STR tissue test by third party can always be utilized

as a backup as needed

Cancer - Oncolytic Viruses II

634 Therapeutic Targeting of

Chitosan-PEG-Folate-Complexed Oncolytic Adenovirus for Active

and Systemic Cancer Gene Therapy

Oh-Joon Kwon,1 Eunah Kang,1 Sung Wan Kim,1,2 Chae-Ok Yun.1

1 Department of Bioengineering, Hanyang University, Seoul,

Republic of Korea; 2 Department of Pharmaceutics and

Pharmaceutical Chemistry, University of Utah, Salt Lake City.

Adenovirus (Ad)-based cancer therapies have shown much

promise However, until now, Ad has only been delivered directly

to primary tumors because the therapeutic effi cacy of systemic

delivery is limited by the immune response of the host, short blood

circulation times, and non-specifi c liver uptake of Ad In order to

circumvent the issues regarding systemic delivery and to increase

the safety and effi cacy of Ad therapies, the surface of Hmt oncolytic

Ad was coated with cationic polymer chitosan via ionic crosslinking (Hmt/chitosan), after which polyethylene glycol (PEG) and/or folate (FA) was chemically conjugated onto the surface of Hmt/ chitosan, generating Hmt/chitosan-FA, Hmt/chitosan-PEG, and Hmt/chitosan-PEG-FA nanocomplex The FA-coordinated Hmt nanocomplexes (Hmt/chitosan-FA  Hmt/chitosan-PEG-FA) elicited

FR-selective cancer cell killing effi cacy In vivo administration of

Hmt/chitosan-PEG or Hmt/chitosan-PEG-FA into mice demonstrated that PEGylation greatly increased blood circulation time, resulting

in 9.0-fold and 48.9-fold increase at 24 hrs after injection compared with naked Hmt, respectively In addition, generation of Ad-specifi c neutralizing antibodies in mice treated with

Hmt/chitosan-PEG-FA was markedly decreased by 75.3% compared with naked Ad The Quantitative PCR assay results showed 285.0-fold increase in tumor tissues and 378-fold reduction of Hmt/chitosan-PEG-FA in liver tissues compared with naked Hmt Bioluminescence imaging study further supported the enhanced tumor-to-liver ratio of Hmt/ chitosan-PEG-FA Consequently, systemic delivery of Hmt/chitosan-PEG-FA signifi cantly inhibited the growth of FR-positive tumor, decreasing 52.8% compared to the Hmt-treated group Importantly, PEGylated oncolytic Ad nanocomplexes showed no elevation of both ALT and AST levels, demonstrating that systemically delivered Ad-related hepatic damage can be completely eliminated with PEG conjugation In sum, these results demonstrate that conjugation of chitosan-PEG-FA to oncolytic Ad signifi cantly improves antitumor effi cacy and safety profi les, suggesting that Hmt/chitosan-PEG-FA has potential as a therapeutic agent to target FR-positive cancer via systemic administration

635 Human Ovarian Cancer-Derived Ascitic Fluid Has Mixed Effects on CRAds Effi cacy

Veronica M Lopez,1 Nicasio Cuneo,2 Mariela A Gangemi,1 Leonardo Sganga,1 Marina Demonte,2 Alejandro Soderini,2 Osvaldo L Podhajcer.1

1 Lab of Molecular and Cellular Therapy, Instituto Leloir, Ciudad Atonoma de Buenos Aires, Buenos Aires, Argentina; 2 Servicio

de Ginecología-Departamento de Cirugía, Hospital Oncologico Marie Curie, Ciudad Atonoma de Buenos Aires, Buenos Aires, Argentina.

Ovarian cancer is one of the leading gynecologic malignancies and the 5-year survival rate for patients with advanced stage ovarian cancer is still low Most ovarian cancer patients are diagnosed at advanced stages and more than half of them have ascites which

is associated with poor prognosis and reduced quality of life The ascitic fl uid is a permissive reactive tumor-host microenvironment that maintains alive malignant cells previously to their homing to their metastatic site Beside, ascites is rich in cytokines and growth factors secreted by malignant and mesothelial cells lining in the peritoneal cavity that could act to directly stimulate malignant cell growth CRAds (Conditionally Replicative Adenoviruses) faces this microenvironment when directly injected into the peritoneal cavity

We have recently developed a stroma-targeted CRAd, AdF512v1 and improved variants whose replication is driven by a 0.5Kb fragment

of the SPARC promoter AdF512v1 was extremely effective in the remission of established human tumors disseminated in the peritoneal cavity of nude mice Moreover, AdF512v1 was also able to replicate

in tumor samples from patients In this work we improved AdF512v1

by adding DNA sequences containing responsive elements to different patho-physiological conditions that characterize tumor tissue, such

as hypoxia and infl ammation The new CRAd was named AdF512v4 and contain a chimeric promoter that combines the 0.5 Kb SPARC promoter, a Hypoxia-Response Element (HRE) and a NFkB-response element (NFkB) The chimeric promoter drives the expression of delta-RB E1A and the CRAd was pseudotyped with a chimeric fi ber 5/3 One of the indications for phase I clinical trials of ovarian cancer

Molecular Therapy Volume 21, Supplement 1, May 2013

Copyright © The American Society of Gene & Cell Therapy S243

CANCER - ONCOLYTIC VIRUSES II

with CRAds imply its administration into the peritoneal cavity We

collected ascitic fl uid from 6 patients and ascities-derived cells from

5 of them Protein arrays showed high expression levels of 6,

IL-8, MCP-1, osteopontin, GRO, TGF BP-1 and 2, and NAP-2 among

other cytokines and chemokines In in vitro assays we found that

ascitic fl uids blocked the lytic effect of AdF512v4 on the ovarian

cancer cell lines SKOV-3, PA1 and OV4, as well as on the fi ve

cells isolated from patients even at 1:50 dilution This effect was

observed in normoxia and hypoxia Ascities-derived cells were more

susceptible to Ad512v4 lytic activity than the ovarian cancer cell

lines Interestingly, the ascites was also able to stimulate the chimeric

promoter activity in an adenoviral context An in vivo assay aiming to

establish the ascites effect on CRAd effi cacy in xenografts models of

human ovary cancer in nude mice will be presented at the meeting

The identifi cation of the ascities-derived soluble factors responsible

for the effects described above might have important implications in

the search for improving CRAd effi cacy

636 The Oncolytic Virus ∆PK Lyses Cells

with Cancer Stem Cell (CSC) Properties through

Calpain-Mediated Clearance of the Selective

Autophagy Protein p62/SQSTM1

Aric Colunga,1 Dominique Bollino,1 Amanda Schech,1 Laure

Aurelian.1

1 Pharmacology, University of Maryland School of Medicine,

Baltimore, MD.

Oncolytic virotherapy is a new strategy to reduce tumor burden

through selective virus replication in rapidly proliferating cells

However, the ability of the oncolytic viruses to lyse the slowly

replicating cancer stem cells (CSC) that maintain neoplastic clonality

is still unclear We report that the oncolytic HSV-2 mutant PK lyses

breast cancer and melanoma cells that grow in soft agar and as 3-D

multi-cellular tumor spheroids and express unique CSC molecular

phenotypes, at very low titers (0.1pfu/cell) and in the absence of

resistance development Cell death was due to calpain activation and

was inhibited by PD150606 in both breast and melanoma CSC and

it included autophagy induction in melanoma CSC PK induced

accumulation of microtubule-associated protein 1 Light chain 3

(LC3-II) in melanoma spheroids and it increased autophagosome

formation in A2058 melanoma cells transduced with GFP-labeled

LC3-II The LC3-II/LC3-I ratio was further increased in infected

spheroids treated with PD150606 or PD150606 and chloroquine (CQ),

indicating that calpain activation contributes to autophagic fl ux PK

treatment caused the clearance of the selective autophagy protein

p62/SQSTM1, but its expression was restored by PD150606 and

partially by CQ The data demonstrate for the fi rst time that a

calpain-autophagy interaction contributes to the execution of autophagic cell

death through p62/SQSTM1 degradation This interaction appears

to be specifi c for CSC, underscoring the importance of developing

strategies that target specifi c CSC death pathways and the strong

therapeutic potential of PK

637 Demonstration of Anti-Metastasis Activity

of Oncolytic Measles Virus Retarging Urokinase

Receptor in Experimental Breast Cancer

Metastasis Model

Yuqi Jing,1 Krisztina Kovacs,1 Jaime Merchan.1

1 Sylverster Cancer Center, University of Miami, Miami, FL.

In view of the limited success of available treatment for metastatic

breast cancer, alternative and complementary strategies need to be

developed Oncolytic measles virus (MV) is a promising novel

therapeutic agent for the treatment of cancer The aim of this study was

to evaluate the antimetastatic activity of recombinant MV targeting

urokinase plasminogen receptor (MV-uPA) in experimental breast

cancer metastases models Recombinant MV-uPA targeting human or mouse uPA recepor was generated and characterized in vitro on human and murine breast cancer cells MV-h-uPA and MV-m-uPA infected and induce cell fusion and syncytia formation in uPAR overexpressing human breast cancer cells (MDA-MB231, MDA-MB436 and MCF-7) and murine mammary cancer cells (4T1) respectively Human breast cancer metastasis model was established by injection of 1×10e6 MDA-MB231-luc2 cells via tail vein into female nude mice Ten days after tumor cell injection, mice were treated with three IV injections (every other day) of PBS or MV-huPA (1×10e6 PFUs) To assess effectiveness, PBS or virus-treated tumor-bearing mice were followed for survival Metastases progression was measured by in vivo luciferase imaging of lung metastases at days 0, 7, 14, 21, 28 after treatment MV-h-uPA treatment was associated with signifi cant survival prolongation and inhibition of lung metastasis compared to controls Breast cancer metastases were also established in immune-competent Balb/c mice by intravenous injection of syngeneic 4T1 cells expressing fi refl y luciferase MV-m-uPA was administrated into the tumor-bearing mice via the tail vein Bioluminescence analysis was followed Oncolytic MV-m-uPA treatment was associated with signifi cant therapeutic benefi t for lung metastasis and prolongation of animal survival In conclusion, systemic administration of MV-uPA

is effective in the treatment of experimental breast cancer metastases

in immune-competent and immune-defi cient model, suggesting that further development of this approach may have potential for clinical application in patients

638 Development of Dual Targeted Oncolytic Adenovirus for Human Malignant Mesothelioma

Shuji Kubo,1 Misato Takagi-Kimura,1 Atsuko Tamamoto,1 Tomoko Hashimoto-Tamaoki,1 Noriyuki Kasahara,2 Masatoshi Tagawa.3

1 Genetics, Hyogo College of Medicine, Nishinomiya, Hyogo, Japan; 2 Medicine, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA; 3 Division of Pathology and Cell Therapy, Chiba Cancer Center Research Institute, Chiba, Japan.

Background: Malignant mesothelioma is highly aggressive and generally incurable, necessitating new treatment paradigms We have recently shown that the midkine (Mdk) promoter could confer tumor-selective transcriptional targeting to oncolytic adenoviruses, which proved to be highly effective in malignant mesothelioma models (J Gene Med 2010; 12:681-92) However, the low transduction effi ciency of adenovirus serotype 5 (Ad5)-based oncolytic vectors

is still a rate-limiting factor in cancer cells that down-regulate coxsackievirus/adenovirus receptor (CAR) As a potential solution,

we investigated the use of Ad35 fi ber for modifi cation of binding tropism in Mk-regulated oncolytic Ad5 vectors (Dual Targeted oncolytic Adenovirus; DT-Ad) Methods: We evaluated the expression

of CAR (receptor for Ad5 fi ber) and CD46 (receptor for Ad35

fi ber) in 6 human mesothelioma cell lines by fl ow cytometry, and assessed the relationship between their expression levels and Ad transduction effi ciency To enhance infectivity and replication of the Ad5-based oncolytic adenovirus Ad5-Mdk-E1, which contains

an Mdk promoter-driven E1 gene and CMV promoter-driven eGFP marker gene, we created the chimeric adenovirus DT-Ad (Ad5/35-Mdk-E1), which is further modifi ed with the Ad35 fi ber knob Selectivity of viral replication and cytolysis was characterized in normal versus malignant mesothelial cells in vitro, and intratumoral spread and antitumor effi cacy were evaluated in vivo Results: Among the mesothelioma cell lines tested, there was low CAR expression

in NCI-H2052 and MSTO-211H, whereas the other lines showed strong expression In contrast, CD46 was highly expressed in all mesothelioma cell lines The Ad35 fi ber-modifi ed DT-Ad vector showed approximately 10-fold higher in vitro cytocidal effect against NCI-H2052 and MSTO-211H cells, compared to Ad5-Mdk-E1