- Email: [email protected]
48. Increased Potency of Engineered FasL-Expressing T Cells Is Modified in Primary Prostate Cancer Cells by Common Chemotherapeutic Agents
46. Adenoviral-Mediated Gene Transfer of Single Chain Fv 425:sTRAIL Fusion Protein Induces Target Cell Restricted Apoptosis in EGFR-Positive Tumor Cells and a Potent Bystander Effect without Toxicity In Vivo
Hidde 1. Haisma,' Marike H. Dijkstra,' Edwin Bremer.? Wijnand Helfrich.2
'Therapeutic Gene Modulation, University Center/or Pharmacy, Groningen, Netherlands; 2PatllOlogy and Laboratory Medicine, University Medical Center Groningen, Groningen, Netherlands.
Soluble TRAIL (sTRAIL), a recombinant form of tumor necrosis factor-related apoptosis-inducing ligand, has been shown to induce apoptosis in a wide variety of cancer cells in vitro and to suppress tumor growth in vivo. Unfortunately the specificity of sTRAIL for tumor cells is limited as several reports show toxicity of sTRAIL towards normal cells, Genetic linkage of sTRAIL to an antibody-fragment specific for a tumor-associated antigen increases the specificity of sTRAIL for tumor cells and simultaneously enhances thc apoptosis inducingcapacityof sTRAIL by cross linking the agonistic TRAIL-receptors. Wc hypothesized that an adenovirus expressing a fusion protein consisting of a Epidermal Growth Factor Receptor (EGFR)-specific single chain Fv antibody fused to sTRAIL: AdscFv425:sTRAIL would induce specific apoptosis in EGFR-positive tumor cells with a potent bystander effect AdscFv425:sTRAIL infected cells showed high expression and secretion of functional trimerized scFv425:sTRAIL. Infection of renal cell carcinoma cells with AdscFv425:sTRAILresulted in EGFR-restrictedand TRAIL-mediatedapoptosis induction in 80% of the tumor cells, strongly prevailing the percentage of transduced cells. The bystander effect was investigated in a transwell system withAdscFv425:sTRAIL infectedhumanumbilicalveinendothelial cells (HUVEC)cells and renal carcinoma bystandercells separated by a permeable membrane, In the cancer cells 60% apoptosis was observed without any sign of viral infection with concurrently no toxicity of AdscFv425:sTRAILtowards the infected HUVECcells. Intravenous injection of 101U v.p, AdscFv425:sTRAIL resulted in serum levels of 15 ug/ml TRAIL, which induced high levels of apoptosis in renal cancer cells. Liver samples showed high levels of Ad infectionwithout any sign of toxicity towards hepatocytes, as measured by caspase 3/7 activation.Conclusion: From these results we conclude that AdscFv425:sTRAIL is a vel)' potent treatment for EGFR-expressingtumor cells as it induces target cell-restricted apoptosis and massive bystander killing with no detectable toxicity towards normal cells in vitro and in vivo. This suggest a putative role ofadenoviral delivel)' of targeted sTRAIL-fusion proteins for treatment of several kinds of cancers.
47. Heat Shock Protein Inhibitors Increase the Efficacy of Measles Virotherapy Chunsheng Liu,' Charles Erlichman.' Cari J. Mclzonald,' James N. Ingle.! Paula Zollman; lanko lankov,' Stephen 1. Russell; Evanthia Galanis.' Molecular Medicine Program, Mayo Clinic, Rochester, MN; 2Division a/Medical Oncology; Mayo Clinic, Rochester, MN. I
Introduction: Oncolytic measles virus strains have activity againstmultipletumortypesand are currentlyin phaseI clinicaltesting.lnduction of the heatshock protein 70 (Hsp70)constitutesone of the earliest changes in cellular gene expression following infection with RNAviruses includingmeasles virus, and Hsp70 upregulation inducedby heatshock has been shown to result in increased measles virus infection rates and plaque recovery. Hsp90 inhibitors such as geldanamycin (GA) or 17-aminogeldanamycin (17-AAG) result in pharmacologic upregulation of Hsp70 and they arc currently in clinical testing as cancer therapeutics, We therefore investigated 820
the hypothesis that heat shock protein inhibitors could augment the measles virus inducedcytopathic effect. Methods and Results: We tested the MV-CEAand GAcombination in MDA-MB23I (breast), SKOV3.1P (ovarian) and TE67 I (rhabdomyosarcoma) cancer cell lines. Optimal synergy was accomplished when GA treatment was initiated6-24 hours followingMV infection.Combination treatment resulted in statistically significant increase in syncytia formation (p<0.05 as compared to MV-CEA infection alone) and increase in antitumor activity. Clonogenic assays demonstrated significant decrease in tumor colony formation in MV-CEAlGA combination treated cells. In addition there was increase in apoptosis by DAPI staining.Western immunoblotting for caspase-9, caspase-S, caspase3 and PARI', demonstrated increase in cleaved caspase-S, -3 and PARI'. The pan-caspase inhibitor Z-VAD-FMK and caspase-8 inhibitorZ-IETD-FMK,but not the caspase-9inhibitorZ-IEHD-FMK protectedtumor cells from MV-CEA/GA inducedRARPactivation, indicatingthat apoptosis in combination treated cells occurs via the extrinsiccaspasepathway. Treatmentof normalcells, such as normal human fibroblasts, however, with the MV-CEAlGA combination. did not result in cytopathic effect , indicating that GA did not alte~ the MV-CEA specificity for tumor cells. One-step growth curves, western blot for MV-N protein expression and QRT-PCR quantitation of MV-genome copy number indicate that the difference in cytopathic effect is not due to increased proliferation of MV-CEA in GA treated tumor cells. Rho activation assays and Western blot for total RhoA, a GTPase associated with the actin cytoskeleton, and inactive pRhoA demonstrated decrease in RhoA activation in combinationtreatedcells, whichhas beenshownto result in increase in paramyxovirus inducedcell-cell fusion(Schowalter, 2006). Conclusion: The combination of MV-CEA and GA treatment resulted in significant increase in syncytia formation and antitumor effect in tumor cells, but not in non-transformedcells, and increase in apoptosis mediated via the extrinsic easpase pathway. The mechanism of GA enhanced cell fusion appears to be GA mediated decrease in activation ofRhoA. These findings supportthe translational potential of this approach in the treatment of cancer. Supported by Prospect Creek Foundation and Breast SPORE CA 116201.
48. Increased Potency of Engineered FasLExpressing T Cells Is Modified in Primary Prostate Cancer Cells by Common Chemotherapeutic Agents
Julie Symes, I Neil Flcshncr,' Daniel H. Fowler," Jeffrey A. Medin.I.2
.~
I Department
a/Medical Biophysics , University a/Toronto, Toronto, ON, Canada; 21nstitute ofMedical Sciences, University a/Toronto. Toronto, ON, Canada; J Division 0/ Urology. Princess Margaret Hospital, Toronto, ON, Canada; "Experimental Transplantation and Immunology Branch, National Institutes 0/ Health, Bethesda, MD; 'Division a/Stem Cell and Developmental Biology, University Health Network, Toronto, ON, Canada.
The manipulation of death receptor pathways is an attractive approach to cancer therapy. While signaling through the apoptosis receptor Fas leads to apoptosis in several prostate cancer cell lines, the function of this pathway in primal)' prostate cancer tissue is unknown. We are developing a system to more effectively deliver apoptosis-inducing FasL to tumor sites by virus-mediated overexpression on T lymphocytes, which have the potential to deliver transgene to tumors in an antigen-dependentmanner. The apoptotic response of murine prostate cancer RM-I cells to these transduced, FasL-T cellswas firstassessed.In addition, certainchemotherapeutic agents are knownto increasethe susceptibilityof tumor cell lines to apoptosis by increasingFas expression. We investigatedthe ability of primary human prostate cancer cells to undergo Fas-rncdiatcd apoptosis before and after treatment with common chcmotheraMolecular Therapy Volume15.Supplement t••\by 2007 Copyright © The Americ.. m Society {I
t
Gene Therapy
peutic drugs. Effective genetic modification of primary murine 'I' cells was achieved by transductionwith eeotropieoneoretroviruses. Transductionefficiencies ofJO-60% were routinelyachieved. These FasL-expressingT lymphocytes induced apoptotic death of RM-I cells in in vitro "Cr-release assays, with up to 30% killing after 6 hrs. This killing was shown to be Fas-mediated,as blocking of the perforin/granzyme pathway by addition of EGTA to cultures had no effect on killing. To determine whether such an approach might apply to the clinical situation, a more pertinentmodel using primary human prostate cells was developed for further studies. Epithelial cells, derived from II tumors obtained from radical prostatectomy, were determined to express high levels of Fas and no surface FasL. When co-incubated with K562 cells engineered to express a membrane bound, non-cleavableFasL (ncFasL) in 6 hr ~ICr-release assays, between 12-87% of prostate cells were killed (average 34 ± 19%). We found that differences in susceptibility to apoptosis among patientcellscannotbe attributedto overexpression of c-FLIP or members of the lAP family. Inductionofapoptosis was partially inhibited by pre-treating a subset of cell lines for 24 hrs with 10 to IOOOnM docetaxel (up to 2-fold less killing compared to untreated cells), while apoptotic response was increasedby up to 3-fold when cells were pretreated for 48 hrs with 100nM mitoxantrone. This data demonstrates for the first time that primary human prostate eancercells are able to undergo Fas-mediated apoptosis, and that the potential for combined chemo- and Fasl.- gene therapies depends on the schedule of administration. Experiments are underway to investigatethe mechanismofthe varied responseof patientsamples to apoptosis.As well, we are testing this 'I' cell-based Fasl, delivery method on tumor growth in an in vivo mouse model. ONCOLITIC VIRUSES
49. Exponential Enhancement of Oncolytic VSV Potency by Vector-Mediated Suppression of Anti-Viral Inflammatory Responses In Vivo
Lan WU,l Jennifer Altomonte,':' Li Chen,' Marcia Meseck,' Oliver Ebert,' :' Adolfo Garcla-Sastrc.' John T. Fallon.' Savio L. C. Woo.I I Department ofGene and Cell Medicine, Mount Sinai School 0/ Medicine, New York. NY; 2Department cfMicrobiology; Mount Sinai School ofMedicine, New }ork, NY; 'Depanment 0/ Pathology, Mount Sinai School 0/ Medicine. New }ork, N}~' "Znd Medical Department. Klinikum Reclus del' lsar; Technical University 0/ Munich, Munich, Germany.
Oncolytic virotherapy is a promising strategy for treatment of malignancy including hepatocellular carcinoma (I·ICC). Vesicular Stomatitis Virus (VSV) is a particularly attractive oncolytic agent because of its short replication cycle and ability to reach high titers in most rodent and human tumor cells. We have previously reported a three-log elevation in intratumoral VSV titer at oneday after vector administration in rats bearing multi-focal HCC, which was followed by a logarithmic decline that correlated with an accumulation of inflammatory cells such as natural killer (NK) cells at the lesions. Upon depletion ofNK cells by intraperitoneal administration of rabbit anti-asialo GMI in tumor-bearingrats, the intratumoral virustitersandextentsoftumor necrosiswereenhanced by 2-logs and >2-fold, respectively. We then constructed a rVSV vector that expresses equine herpesvirus-I glycoprotein G, which is a broad spectrum viral chcmokine binding protein (rVSV-gG).ln vitro functional characterizationof rVSV-gG showed that this new vector had no significantdifferences in viral life cycle or viralyield, and it is equally effective in killing rat and human Hepatoma cells as a control VSV vector. Moreover,NK cell migration was significantly inhibited by conditioned media from rVSV-gG infected rat hepatoma cell as compared to that of a control-VSV vector. In vivo study showed that hepatic artery infusion ofrVSV-gG in immuneMolecular Therapy Volume 15.Supplement I. ~"r Copyright © The American Soc iety of Gene Therapy
2007
competentratsbearingsyngeneicand multi-focal HCC intheir livers resulted in the suppression ofNK and NKT cell chemotaxis to the tumors and a one-log enhancement of intratumoral virus titer over that of a control VSV vector, which Jed to elevated tumor necrosis andsubstantially prolongedanimalsurvivalwithouttoxicities. These results indicate that rVSV-gG could be developed as an effective and safe oncolytic agent to treat advanced HCC patients in the future. Furthermore, the novel concept that oncolytic potency can be substantially enhanced by vector-mediated suppression of host anti-viral inflammatory responses might be generally applicable in the field of oncolytic virotherapy for cancer.
50. Phase I Clinical Trial Study Ultilizing Oncolytic Adenovirus with HSP Gene Expression (H103) in Treating Patients with Advanced Solid Tumor Hongli Liu,' Yu Wang,' Yong Ben,' Datong Chu,' Fang Hu.'
Medical Affairs Department. Shanghai Sunway Biotech Co.. Ltd.. Shanghai. China; 2Cancer Institute and Hospital. Chinese Academy ofMedical Sciences and Peking Union Medical College. Peiking, China. I
Objective: HI 03 is a type 2-adenovirus with E I B-55kD deleted and human HSP70 (heat shock protein)gene inserted using a CMV promoter in viral genome, aiming to stimulate host's immune responsethroughmaximizingHSP-inducedtumor antigenpresenting. Toevaluatethe safety ofH 103when treatingpatientswith advanced solid tumor, a phase I study was conducted from Sep 2003 to April 2004. Methods: Total 27 patients were recruited.(Male 18 cases, female 9 cases; Median age 58; age 26-68). Tumor types included melanoma (II), renal carcinoma (5), lung cancer (5), colon cancer (I), breast cancer (I) and others (4). HI03 was administrated in an intratumoral mode. Viral dosage escalation is from 2.5x107vp to I.5xlO12vp in singledosageregimen (la, 10patients)and I.5x 1012vp, 3.0x1012vp in multiple dosage (lb, 9 patients). Samples of blood, urine, stool, pharyngeal swabs and injection site swabs were collectedat D1,2,3, 5, 7, 14,21 after injectionfor H I 03 biodistribution using PCR. Blood Samples were collected at D3, 7, 14,21 after injectionfor detectingneutralizingantibody, changeoflymphocytes panel and HSP70 expression level.A tissue sample from one patient was analyzed for HSP expression using immunohistochemistry staining.Results: Dose LimitedToxicity(DLT)was observedat one patient of l.5x I 012vp/patient and one patient on.ox I 012vp/patient, however, MaximumTolerated Dose (MTD) was not reached in this phase I study.The commonadverseevents were fever77.8%(21/27, I-II grades except one patient with III grade), local site pain 22.2% (6/27), leucopeniaand thrombopenia 18.5%(5/27, I-II grade except one patient with IV grade). Overall objective response (CR+PR) rate ofHl03-injceted tumor is 11.1%(3/27) and the caleulatcd CBR (CR+PR+MR+SD) is 77.8% among all patients, And the transient abseopalresponsesof distant tumor were observed in 3 pateintsduring the HI03 treatment.Administration ofI-II 03 trendedto stimulate the host's cell immuneresponseand antibodyresponseby increasing major immunecell (lymphocytes,total 'I' cells, 'I' helpercells, CTLs, even NK cells) and anti Ad2 antibody which is unrelated to anti cancer efficacy. HSPexpressionwas confirmedin tumortissue after HI03 injectionwithouteffectingother sites.QuantitativePCR failed in detecting replicating viral copies in the samples of blood, urine, stool, pharyngeal swabs and injection site swabs at D21 after 1-1103 administration.Conclusions: This phase I study showed that 1-1103 was safe when applied intratumorally to patients with advanced solid tumor,showing promising anti cancer efficacyand increasing host's immune response. Phase II study should be investigated for exploring efficacy and monitoring further safety.
S21
Hidde 1. Haisma,' Marike H. Dijkstra,' Edwin Bremer.? Wijnand Helfrich.2
'Therapeutic Gene Modulation, University Center/or Pharmacy, Groningen, Netherlands; 2PatllOlogy and Laboratory Medicine, University Medical Center Groningen, Groningen, Netherlands.
Soluble TRAIL (sTRAIL), a recombinant form of tumor necrosis factor-related apoptosis-inducing ligand, has been shown to induce apoptosis in a wide variety of cancer cells in vitro and to suppress tumor growth in vivo. Unfortunately the specificity of sTRAIL for tumor cells is limited as several reports show toxicity of sTRAIL towards normal cells, Genetic linkage of sTRAIL to an antibody-fragment specific for a tumor-associated antigen increases the specificity of sTRAIL for tumor cells and simultaneously enhances thc apoptosis inducingcapacityof sTRAIL by cross linking the agonistic TRAIL-receptors. Wc hypothesized that an adenovirus expressing a fusion protein consisting of a Epidermal Growth Factor Receptor (EGFR)-specific single chain Fv antibody fused to sTRAIL: AdscFv425:sTRAIL would induce specific apoptosis in EGFR-positive tumor cells with a potent bystander effect AdscFv425:sTRAIL infected cells showed high expression and secretion of functional trimerized scFv425:sTRAIL. Infection of renal cell carcinoma cells with AdscFv425:sTRAILresulted in EGFR-restrictedand TRAIL-mediatedapoptosis induction in 80% of the tumor cells, strongly prevailing the percentage of transduced cells. The bystander effect was investigated in a transwell system withAdscFv425:sTRAIL infectedhumanumbilicalveinendothelial cells (HUVEC)cells and renal carcinoma bystandercells separated by a permeable membrane, In the cancer cells 60% apoptosis was observed without any sign of viral infection with concurrently no toxicity of AdscFv425:sTRAILtowards the infected HUVECcells. Intravenous injection of 101U v.p, AdscFv425:sTRAIL resulted in serum levels of 15 ug/ml TRAIL, which induced high levels of apoptosis in renal cancer cells. Liver samples showed high levels of Ad infectionwithout any sign of toxicity towards hepatocytes, as measured by caspase 3/7 activation.Conclusion: From these results we conclude that AdscFv425:sTRAIL is a vel)' potent treatment for EGFR-expressingtumor cells as it induces target cell-restricted apoptosis and massive bystander killing with no detectable toxicity towards normal cells in vitro and in vivo. This suggest a putative role ofadenoviral delivel)' of targeted sTRAIL-fusion proteins for treatment of several kinds of cancers.
47. Heat Shock Protein Inhibitors Increase the Efficacy of Measles Virotherapy Chunsheng Liu,' Charles Erlichman.' Cari J. Mclzonald,' James N. Ingle.! Paula Zollman; lanko lankov,' Stephen 1. Russell; Evanthia Galanis.' Molecular Medicine Program, Mayo Clinic, Rochester, MN; 2Division a/Medical Oncology; Mayo Clinic, Rochester, MN. I
Introduction: Oncolytic measles virus strains have activity againstmultipletumortypesand are currentlyin phaseI clinicaltesting.lnduction of the heatshock protein 70 (Hsp70)constitutesone of the earliest changes in cellular gene expression following infection with RNAviruses includingmeasles virus, and Hsp70 upregulation inducedby heatshock has been shown to result in increased measles virus infection rates and plaque recovery. Hsp90 inhibitors such as geldanamycin (GA) or 17-aminogeldanamycin (17-AAG) result in pharmacologic upregulation of Hsp70 and they arc currently in clinical testing as cancer therapeutics, We therefore investigated 820
the hypothesis that heat shock protein inhibitors could augment the measles virus inducedcytopathic effect. Methods and Results: We tested the MV-CEAand GAcombination in MDA-MB23I (breast), SKOV3.1P (ovarian) and TE67 I (rhabdomyosarcoma) cancer cell lines. Optimal synergy was accomplished when GA treatment was initiated6-24 hours followingMV infection.Combination treatment resulted in statistically significant increase in syncytia formation (p<0.05 as compared to MV-CEA infection alone) and increase in antitumor activity. Clonogenic assays demonstrated significant decrease in tumor colony formation in MV-CEAlGA combination treated cells. In addition there was increase in apoptosis by DAPI staining.Western immunoblotting for caspase-9, caspase-S, caspase3 and PARI', demonstrated increase in cleaved caspase-S, -3 and PARI'. The pan-caspase inhibitor Z-VAD-FMK and caspase-8 inhibitorZ-IETD-FMK,but not the caspase-9inhibitorZ-IEHD-FMK protectedtumor cells from MV-CEA/GA inducedRARPactivation, indicatingthat apoptosis in combination treated cells occurs via the extrinsiccaspasepathway. Treatmentof normalcells, such as normal human fibroblasts, however, with the MV-CEAlGA combination. did not result in cytopathic effect , indicating that GA did not alte~ the MV-CEA specificity for tumor cells. One-step growth curves, western blot for MV-N protein expression and QRT-PCR quantitation of MV-genome copy number indicate that the difference in cytopathic effect is not due to increased proliferation of MV-CEA in GA treated tumor cells. Rho activation assays and Western blot for total RhoA, a GTPase associated with the actin cytoskeleton, and inactive pRhoA demonstrated decrease in RhoA activation in combinationtreatedcells, whichhas beenshownto result in increase in paramyxovirus inducedcell-cell fusion(Schowalter, 2006). Conclusion: The combination of MV-CEA and GA treatment resulted in significant increase in syncytia formation and antitumor effect in tumor cells, but not in non-transformedcells, and increase in apoptosis mediated via the extrinsic easpase pathway. The mechanism of GA enhanced cell fusion appears to be GA mediated decrease in activation ofRhoA. These findings supportthe translational potential of this approach in the treatment of cancer. Supported by Prospect Creek Foundation and Breast SPORE CA 116201.
48. Increased Potency of Engineered FasLExpressing T Cells Is Modified in Primary Prostate Cancer Cells by Common Chemotherapeutic Agents
Julie Symes, I Neil Flcshncr,' Daniel H. Fowler," Jeffrey A. Medin.I.2
.~
I Department
a/Medical Biophysics , University a/Toronto, Toronto, ON, Canada; 21nstitute ofMedical Sciences, University a/Toronto. Toronto, ON, Canada; J Division 0/ Urology. Princess Margaret Hospital, Toronto, ON, Canada; "Experimental Transplantation and Immunology Branch, National Institutes 0/ Health, Bethesda, MD; 'Division a/Stem Cell and Developmental Biology, University Health Network, Toronto, ON, Canada.
The manipulation of death receptor pathways is an attractive approach to cancer therapy. While signaling through the apoptosis receptor Fas leads to apoptosis in several prostate cancer cell lines, the function of this pathway in primal)' prostate cancer tissue is unknown. We are developing a system to more effectively deliver apoptosis-inducing FasL to tumor sites by virus-mediated overexpression on T lymphocytes, which have the potential to deliver transgene to tumors in an antigen-dependentmanner. The apoptotic response of murine prostate cancer RM-I cells to these transduced, FasL-T cellswas firstassessed.In addition, certainchemotherapeutic agents are knownto increasethe susceptibilityof tumor cell lines to apoptosis by increasingFas expression. We investigatedthe ability of primary human prostate cancer cells to undergo Fas-rncdiatcd apoptosis before and after treatment with common chcmotheraMolecular Therapy Volume15.Supplement t••\by 2007 Copyright © The Americ.. m Society {I
t
Gene Therapy
peutic drugs. Effective genetic modification of primary murine 'I' cells was achieved by transductionwith eeotropieoneoretroviruses. Transductionefficiencies ofJO-60% were routinelyachieved. These FasL-expressingT lymphocytes induced apoptotic death of RM-I cells in in vitro "Cr-release assays, with up to 30% killing after 6 hrs. This killing was shown to be Fas-mediated,as blocking of the perforin/granzyme pathway by addition of EGTA to cultures had no effect on killing. To determine whether such an approach might apply to the clinical situation, a more pertinentmodel using primary human prostate cells was developed for further studies. Epithelial cells, derived from II tumors obtained from radical prostatectomy, were determined to express high levels of Fas and no surface FasL. When co-incubated with K562 cells engineered to express a membrane bound, non-cleavableFasL (ncFasL) in 6 hr ~ICr-release assays, between 12-87% of prostate cells were killed (average 34 ± 19%). We found that differences in susceptibility to apoptosis among patientcellscannotbe attributedto overexpression of c-FLIP or members of the lAP family. Inductionofapoptosis was partially inhibited by pre-treating a subset of cell lines for 24 hrs with 10 to IOOOnM docetaxel (up to 2-fold less killing compared to untreated cells), while apoptotic response was increasedby up to 3-fold when cells were pretreated for 48 hrs with 100nM mitoxantrone. This data demonstrates for the first time that primary human prostate eancercells are able to undergo Fas-mediated apoptosis, and that the potential for combined chemo- and Fasl.- gene therapies depends on the schedule of administration. Experiments are underway to investigatethe mechanismofthe varied responseof patientsamples to apoptosis.As well, we are testing this 'I' cell-based Fasl, delivery method on tumor growth in an in vivo mouse model. ONCOLITIC VIRUSES
49. Exponential Enhancement of Oncolytic VSV Potency by Vector-Mediated Suppression of Anti-Viral Inflammatory Responses In Vivo
Lan WU,l Jennifer Altomonte,':' Li Chen,' Marcia Meseck,' Oliver Ebert,' :' Adolfo Garcla-Sastrc.' John T. Fallon.' Savio L. C. Woo.I I Department ofGene and Cell Medicine, Mount Sinai School 0/ Medicine, New York. NY; 2Department cfMicrobiology; Mount Sinai School ofMedicine, New }ork, NY; 'Depanment 0/ Pathology, Mount Sinai School 0/ Medicine. New }ork, N}~' "Znd Medical Department. Klinikum Reclus del' lsar; Technical University 0/ Munich, Munich, Germany.
Oncolytic virotherapy is a promising strategy for treatment of malignancy including hepatocellular carcinoma (I·ICC). Vesicular Stomatitis Virus (VSV) is a particularly attractive oncolytic agent because of its short replication cycle and ability to reach high titers in most rodent and human tumor cells. We have previously reported a three-log elevation in intratumoral VSV titer at oneday after vector administration in rats bearing multi-focal HCC, which was followed by a logarithmic decline that correlated with an accumulation of inflammatory cells such as natural killer (NK) cells at the lesions. Upon depletion ofNK cells by intraperitoneal administration of rabbit anti-asialo GMI in tumor-bearingrats, the intratumoral virustitersandextentsoftumor necrosiswereenhanced by 2-logs and >2-fold, respectively. We then constructed a rVSV vector that expresses equine herpesvirus-I glycoprotein G, which is a broad spectrum viral chcmokine binding protein (rVSV-gG).ln vitro functional characterizationof rVSV-gG showed that this new vector had no significantdifferences in viral life cycle or viralyield, and it is equally effective in killing rat and human Hepatoma cells as a control VSV vector. Moreover,NK cell migration was significantly inhibited by conditioned media from rVSV-gG infected rat hepatoma cell as compared to that of a control-VSV vector. In vivo study showed that hepatic artery infusion ofrVSV-gG in immuneMolecular Therapy Volume 15.Supplement I. ~"r Copyright © The American Soc iety of Gene Therapy
2007
competentratsbearingsyngeneicand multi-focal HCC intheir livers resulted in the suppression ofNK and NKT cell chemotaxis to the tumors and a one-log enhancement of intratumoral virus titer over that of a control VSV vector, which Jed to elevated tumor necrosis andsubstantially prolongedanimalsurvivalwithouttoxicities. These results indicate that rVSV-gG could be developed as an effective and safe oncolytic agent to treat advanced HCC patients in the future. Furthermore, the novel concept that oncolytic potency can be substantially enhanced by vector-mediated suppression of host anti-viral inflammatory responses might be generally applicable in the field of oncolytic virotherapy for cancer.
50. Phase I Clinical Trial Study Ultilizing Oncolytic Adenovirus with HSP Gene Expression (H103) in Treating Patients with Advanced Solid Tumor Hongli Liu,' Yu Wang,' Yong Ben,' Datong Chu,' Fang Hu.'
Medical Affairs Department. Shanghai Sunway Biotech Co.. Ltd.. Shanghai. China; 2Cancer Institute and Hospital. Chinese Academy ofMedical Sciences and Peking Union Medical College. Peiking, China. I
Objective: HI 03 is a type 2-adenovirus with E I B-55kD deleted and human HSP70 (heat shock protein)gene inserted using a CMV promoter in viral genome, aiming to stimulate host's immune responsethroughmaximizingHSP-inducedtumor antigenpresenting. Toevaluatethe safety ofH 103when treatingpatientswith advanced solid tumor, a phase I study was conducted from Sep 2003 to April 2004. Methods: Total 27 patients were recruited.(Male 18 cases, female 9 cases; Median age 58; age 26-68). Tumor types included melanoma (II), renal carcinoma (5), lung cancer (5), colon cancer (I), breast cancer (I) and others (4). HI03 was administrated in an intratumoral mode. Viral dosage escalation is from 2.5x107vp to I.5xlO12vp in singledosageregimen (la, 10patients)and I.5x 1012vp, 3.0x1012vp in multiple dosage (lb, 9 patients). Samples of blood, urine, stool, pharyngeal swabs and injection site swabs were collectedat D1,2,3, 5, 7, 14,21 after injectionfor H I 03 biodistribution using PCR. Blood Samples were collected at D3, 7, 14,21 after injectionfor detectingneutralizingantibody, changeoflymphocytes panel and HSP70 expression level.A tissue sample from one patient was analyzed for HSP expression using immunohistochemistry staining.Results: Dose LimitedToxicity(DLT)was observedat one patient of l.5x I 012vp/patient and one patient on.ox I 012vp/patient, however, MaximumTolerated Dose (MTD) was not reached in this phase I study.The commonadverseevents were fever77.8%(21/27, I-II grades except one patient with III grade), local site pain 22.2% (6/27), leucopeniaand thrombopenia 18.5%(5/27, I-II grade except one patient with IV grade). Overall objective response (CR+PR) rate ofHl03-injceted tumor is 11.1%(3/27) and the caleulatcd CBR (CR+PR+MR+SD) is 77.8% among all patients, And the transient abseopalresponsesof distant tumor were observed in 3 pateintsduring the HI03 treatment.Administration ofI-II 03 trendedto stimulate the host's cell immuneresponseand antibodyresponseby increasing major immunecell (lymphocytes,total 'I' cells, 'I' helpercells, CTLs, even NK cells) and anti Ad2 antibody which is unrelated to anti cancer efficacy. HSPexpressionwas confirmedin tumortissue after HI03 injectionwithouteffectingother sites.QuantitativePCR failed in detecting replicating viral copies in the samples of blood, urine, stool, pharyngeal swabs and injection site swabs at D21 after 1-1103 administration.Conclusions: This phase I study showed that 1-1103 was safe when applied intratumorally to patients with advanced solid tumor,showing promising anti cancer efficacyand increasing host's immune response. Phase II study should be investigated for exploring efficacy and monitoring further safety.
S21