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[.]
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 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 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, pt.s; Phase II Ovarian, 17 pt.s; Phase II Melanoma, pt.s) and 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, melanoma, and 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 confirmed 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-PEGFolate-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 Department of Bioengineering, Hanyang University, Seoul, Republic of Korea; 2Department 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 efficacy of systemic delivery is limited by the immune response of the host, short blood circulation times, and non-specific liver uptake of Ad In order to circumvent the issues regarding systemic delivery and to increase the safety and efficacy of Ad therapies, the surface of Hmt oncolytic S242 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 efficacy 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-specific neutralizing antibodies in mice treated with Hmt/chitosan-PEGFA 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/chitosanPEG-FA significantly 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 significantly improves antitumor efficacy and safety profiles, 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 Efficacy Veronica M Lopez,1 Nicasio Cuneo,2 Mariela A Gangemi,1 Leonardo Sganga,1 Marina Demonte,2 Alejandro Soderini,2 Osvaldo L Podhajcer.1 Lab of Molecular and Cellular Therapy, Instituto Leloir, Ciudad Atonoma de Buenos Aires, Buenos Aires, Argentina; 2Servicio 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 fluid 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 inflammation 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 fiber 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 CANCER - ONCOLYTIC VIRUSES II with CRAds imply its administration into the peritoneal cavity We collected ascitic fluid from patients and ascities-derived cells from of them Protein arrays showed high expression levels of IL-6, IL8, 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 fluids blocked the lytic effect of AdF512v4 on the ovarian cancer cell lines SKOV-3, PA1 and OV4, as well as on the five 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 efficacy in xenografts models of human ovary cancer in nude mice will be presented at the meeting The identification of the ascities-derived soluble factors responsible for the effects described above might have important implications in the search for improving CRAd efficacy 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 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 Light chain (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 flux 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 first time that a calpainautophagy interaction contributes to the execution of autophagic cell death through p62/SQSTM1 degradation This interaction appears to be specific for CSC, underscoring the importance of developing strategies that target specific 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 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 Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy 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 significant survival prolongation and inhibition of lung metastasis compared to controls Breast cancer metastases were also established in immunecompetent Balb/c mice by intravenous injection of syngeneic 4T1 cells expressing firefly 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 significant therapeutic benefit 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-deficient 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 Genetics, Hyogo College of Medicine, Nishinomiya, Hyogo, Japan; 2Medicine, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA; 3Division 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 efficiency of adenovirus serotype (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 fiber for modification 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 fiber) and CD46 (receptor for Ad35 fiber) in human mesothelioma cell lines by flow cytometry, and assessed the relationship between their expression levels and Ad transduction efficiency 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 (Ad5/35Mdk-E1), which is further modified with the Ad35 fiber knob Selectivity of viral replication and cytolysis was characterized in normal versus malignant mesothelial cells in vitro, and intratumoral spread and antitumor efficacy 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 fiber-modified DT-Ad vector showed approximately 10-fold higher in vitro cytocidal effect against NCI-H2052 and MSTO-211H cells, compared to Ad5-Mdk-E1 S243 .. .CANCER - ONCOLYTIC VIRUSES II with CRAds imply its administration into the peritoneal cavity We collected ascitic fluid from patients and ascities -derived cells from of... effect on CRAd efficacy in xenografts models of human ovary cancer in nude mice will be presented at the meeting The identification of the ascities -derived soluble factors responsible for the effects. .. 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