- Email: [email protected]
34. Highly Attenuated Vaccinia Virus with microRNA-Regulated Oncolysis for Cancer Virotherapy
CANCER-ONCOLYTIC VIRUSES I 34. Highly Attenuated Vaccinia Virus with microRNA-Regulated Oncolysis for Cancer Virotherapy
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; 3Institute for Genetic Medicine, Hokkaido University, Sapporo, Japan; 4PRESTO, Japan Science and Technology Agency, Kawaguchi, Japan. A highly attenuated LC16m8 (m8) smallpox vaccine was administrated to >100,000 infants without any serious side effects from 1973 to 1975 in Japan. The m8 was isolated from Lister through intermediate strains, mO. The m8 has lost the function of B5R as the result of a single base deletion in the ORF, which encodes a 42-kDa glycoprotein that is essential for formation of extracellular enveloped virus. We recently found that m8∆, which is more genetically stable virus by deleting B5R from m8, has the potential of more selective and safer oncolytic agent than parental viruses. Based on the m8∆, we have developed microRNA (miRNA)-regulated vaccinia viruses which achieve enhancement of viral replication and oncolysis in cancer cells, but elimination of unwanted replication and associated toxicity in normal cells. For this purpose, we focused on let-7a which is one member of let-7 family of highly conserved miRNAs demonstrating decreased expression in cancer cells compared with normal cells. Multiple copies of miRNA complementary target sequences for let7a were incorporated into the 3’ UTR of the B5R gene of m8B5R which was constructed from m8∆ by introducing the complete B5R cloned from mO. Furthermore, the B5R gene was fused with the EGFP gene to evaluate B5R expressions following infection on HeLa cervix carcinoma cells that express let-7a, or A549 lung carcinoma cells with lower expression of let-7a. The m8B5Rgfp-let7a induced CPE following EGFP expression on A549 cells 3 days after infection, but not HeLa cells. In contrast, control virus m8B5Rgfp-let7a mut containing insertion of the disrupted miRNA target sequences resulted in CPE formation following EGFP expression on both cells. Next, we generated trackable virus m8B5R-let7a/LG which was constructed by inserting expression cassette encoding luciferase and EGFP into the A56R locus of the viral genome. Intratumoral injections of each virus on days 0, 3 and 6 (total 3 x 107 pfu per mouse) into nude mice with established s.c. A549 or BxPC-3 tumors were used to evaluate its oncolytic activity, toxicity and tumor specicity. In both models, m8B5R-let7a/LG showed much higher oncolytic activity than m8∆/ LG, and dramatically reduced toxicity compared with mO/LG and m8B5R-let7a mut/LG. Although the tumor regression in the m8B5R-let7a/LG treated mice was comparable to that observed in the mO/LG and m8B5R-let7a mut/LG, all mice treated with mO/LG and m8B5R-let7a mut/LG were dead or sacriced by 59 days postinjection because of pock lesions and weight loss. These results were supported by bioluminescence imaging, showing tumor-specic viral replication of m8B5R-let7a/LG. Thus, m8B5R-let7a/LG resulted in a highly signicant increase in survival compared with mock therapy or administration of other viruses. Our study demonstrated that incorporation of specic miRNA target sequences into the 3’ UTR of B5R gene is a novel strategy for engineering vaccinia virus to not only enhance oncolytic activity but also decrease viral pathogenicity.
35. A Phase 1 Trial of Intravesical (IVE) CG0070 in Patients with Supercial Bladder Cancer after BCG Failure
James M. Burke,1 Don Lamm,2 James Mckiernan,3 John Nemunaitis,4 Joe Stephenson,5 Max Meng,6 James Arsenau,4 Junko Aimi,7 Daniel Maslyar.7 1 Hematology and Oncology, Billings Clinic, Billings, MT; 2 Urology, BCG Oncology, Scottsdale, AZ; 3Urology, Columbia University, New York, NY; 4Oncology, Mary Crowley Medical Research Center, Dallas, TX; 5Oncology, Cancer Centers of the Carolinas, Greenville, SC; 6Urology, UC San Francisco, San Francisco, CA; 7Cell Genesys, Inc, South San Francisco, CA.
Background: Patients with superficial bladder cancer (T1, Ta, Tcis) failing intravesical (IVE) BCG have limited treatment options. Cystectomy is often the only therapeutic option. CG0070 is a replication competent oncolytic adenovirus genetically modied to express GM-CSF under control of the human E2F-1 promoter. E2F-1 is active in cells with Retinoblastoma (Rb) pathway defects. Design: This trial evaluated the safety and feasibility of single and multiple doses of CG0070. Multi-dose regimens were tested at two schedules: every 4 weeks or weekly. Clinical efcacy was determined by quarterly cystoscopy, biopsy, and/or urine cytology. In addition, serum and urine was tested for viral genomes, and GM-CSF after virus treatment. Results: 35 patients (pts) were treated with CG0070 at doses ranging from 1012 to 3 x 1013 viral particles (vp) in the single dose schedule, and from 1012 to 1013 vp per dose in the weekly x 6 and every 4 week x 3 cohorts. No SAEs and only one dose limiting toxicity was reported, gr 3 lymphopenia, at a dose of 1012 vp on the every 4 week schedule. Transient, local bladder related toxicities were the most common adverse events reported including dysuria, hematuria, urgency, bladder spasm, nocturia, and pain. Flu-like illness was also observed in 10-20% of patients including fever, chills, fatigue, and myalgias. The maximum tolerated dose was not reached in the single or multi-dose cohorts. A complete response rate (RR) of 46% was observed for all pts. Peak levels of urine GM-CSF were detected on D2 after the rst treatment for 94% of pts. Additionally, 58% of pts in the single dose cohort had an increase in CG0070 levels between D2 and D5, and 25% had at least a 10x increase in CG0070 levels suggesting in vivo viral replication. Conclusion: IVE CG0070 was well tolerated with local bladder related toxicities and u-like symptoms observed. RR was 46% in this dose escalation study. The majority of pts demonstrated elevated urine GM-CSF levels and a delayed “second peak” in CG0070 urine genomes suggesting infection and replication.
36. A Phase 1 Trial of the Novel InfectivityEnhanced Bicistronic Adenovirus Ad5.SSTR/ TK.RGD in Recurrent Gynecologic Cancer
Kenneth H. Kim,1 Kellie Schneider,1 Minghui Wang,1 Janis O’Malley,1 Wen Wan,1 Meredith A. Preuss,1 Raymond D. Harris,2 Rosemarie Aurigemma,3 Gene P. Siegal,1 Kurt Zinn,1 David T. Curiel,1 Ronald D. Alvarez.1 1 The University of Alabama at Birmingham, Birmingham, AL; 2 Biopharmaceutical Development Program – SAIC, National Cancer Institute, Frederick, MD; 3Biological Resources Branch, National Cancer Institute, Frederick, MD. Background: Adenoviral-based gene therapy has been evaluated as a potential therapeutic strategy for those ovarian and endometrial cancer patients with chemorefractory disease. Ad5.SSTR/TK.RGD is an infectivity-enhanced adenovirus that expresses a therapeutic thymidine kinase suicide gene and a somatostatin type 2 receptor, allowing for noninvasive assessment of gene transfer with nuclear imaging, and is given in combination with the prodrug ganciclovir (GCV). The purpose of this study was to identify the MTD, spectrum
Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy
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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; 3Institute for Genetic Medicine, Hokkaido University, Sapporo, Japan; 4PRESTO, Japan Science and Technology Agency, Kawaguchi, Japan. A highly attenuated LC16m8 (m8) smallpox vaccine was administrated to >100,000 infants without any serious side effects from 1973 to 1975 in Japan. The m8 was isolated from Lister through intermediate strains, mO. The m8 has lost the function of B5R as the result of a single base deletion in the ORF, which encodes a 42-kDa glycoprotein that is essential for formation of extracellular enveloped virus. We recently found that m8∆, which is more genetically stable virus by deleting B5R from m8, has the potential of more selective and safer oncolytic agent than parental viruses. Based on the m8∆, we have developed microRNA (miRNA)-regulated vaccinia viruses which achieve enhancement of viral replication and oncolysis in cancer cells, but elimination of unwanted replication and associated toxicity in normal cells. For this purpose, we focused on let-7a which is one member of let-7 family of highly conserved miRNAs demonstrating decreased expression in cancer cells compared with normal cells. Multiple copies of miRNA complementary target sequences for let7a were incorporated into the 3’ UTR of the B5R gene of m8B5R which was constructed from m8∆ by introducing the complete B5R cloned from mO. Furthermore, the B5R gene was fused with the EGFP gene to evaluate B5R expressions following infection on HeLa cervix carcinoma cells that express let-7a, or A549 lung carcinoma cells with lower expression of let-7a. The m8B5Rgfp-let7a induced CPE following EGFP expression on A549 cells 3 days after infection, but not HeLa cells. In contrast, control virus m8B5Rgfp-let7a mut containing insertion of the disrupted miRNA target sequences resulted in CPE formation following EGFP expression on both cells. Next, we generated trackable virus m8B5R-let7a/LG which was constructed by inserting expression cassette encoding luciferase and EGFP into the A56R locus of the viral genome. Intratumoral injections of each virus on days 0, 3 and 6 (total 3 x 107 pfu per mouse) into nude mice with established s.c. A549 or BxPC-3 tumors were used to evaluate its oncolytic activity, toxicity and tumor specicity. In both models, m8B5R-let7a/LG showed much higher oncolytic activity than m8∆/ LG, and dramatically reduced toxicity compared with mO/LG and m8B5R-let7a mut/LG. Although the tumor regression in the m8B5R-let7a/LG treated mice was comparable to that observed in the mO/LG and m8B5R-let7a mut/LG, all mice treated with mO/LG and m8B5R-let7a mut/LG were dead or sacriced by 59 days postinjection because of pock lesions and weight loss. These results were supported by bioluminescence imaging, showing tumor-specic viral replication of m8B5R-let7a/LG. Thus, m8B5R-let7a/LG resulted in a highly signicant increase in survival compared with mock therapy or administration of other viruses. Our study demonstrated that incorporation of specic miRNA target sequences into the 3’ UTR of B5R gene is a novel strategy for engineering vaccinia virus to not only enhance oncolytic activity but also decrease viral pathogenicity.
35. A Phase 1 Trial of Intravesical (IVE) CG0070 in Patients with Supercial Bladder Cancer after BCG Failure
James M. Burke,1 Don Lamm,2 James Mckiernan,3 John Nemunaitis,4 Joe Stephenson,5 Max Meng,6 James Arsenau,4 Junko Aimi,7 Daniel Maslyar.7 1 Hematology and Oncology, Billings Clinic, Billings, MT; 2 Urology, BCG Oncology, Scottsdale, AZ; 3Urology, Columbia University, New York, NY; 4Oncology, Mary Crowley Medical Research Center, Dallas, TX; 5Oncology, Cancer Centers of the Carolinas, Greenville, SC; 6Urology, UC San Francisco, San Francisco, CA; 7Cell Genesys, Inc, South San Francisco, CA.
Background: Patients with superficial bladder cancer (T1, Ta, Tcis) failing intravesical (IVE) BCG have limited treatment options. Cystectomy is often the only therapeutic option. CG0070 is a replication competent oncolytic adenovirus genetically modied to express GM-CSF under control of the human E2F-1 promoter. E2F-1 is active in cells with Retinoblastoma (Rb) pathway defects. Design: This trial evaluated the safety and feasibility of single and multiple doses of CG0070. Multi-dose regimens were tested at two schedules: every 4 weeks or weekly. Clinical efcacy was determined by quarterly cystoscopy, biopsy, and/or urine cytology. In addition, serum and urine was tested for viral genomes, and GM-CSF after virus treatment. Results: 35 patients (pts) were treated with CG0070 at doses ranging from 1012 to 3 x 1013 viral particles (vp) in the single dose schedule, and from 1012 to 1013 vp per dose in the weekly x 6 and every 4 week x 3 cohorts. No SAEs and only one dose limiting toxicity was reported, gr 3 lymphopenia, at a dose of 1012 vp on the every 4 week schedule. Transient, local bladder related toxicities were the most common adverse events reported including dysuria, hematuria, urgency, bladder spasm, nocturia, and pain. Flu-like illness was also observed in 10-20% of patients including fever, chills, fatigue, and myalgias. The maximum tolerated dose was not reached in the single or multi-dose cohorts. A complete response rate (RR) of 46% was observed for all pts. Peak levels of urine GM-CSF were detected on D2 after the rst treatment for 94% of pts. Additionally, 58% of pts in the single dose cohort had an increase in CG0070 levels between D2 and D5, and 25% had at least a 10x increase in CG0070 levels suggesting in vivo viral replication. Conclusion: IVE CG0070 was well tolerated with local bladder related toxicities and u-like symptoms observed. RR was 46% in this dose escalation study. The majority of pts demonstrated elevated urine GM-CSF levels and a delayed “second peak” in CG0070 urine genomes suggesting infection and replication.
36. A Phase 1 Trial of the Novel InfectivityEnhanced Bicistronic Adenovirus Ad5.SSTR/ TK.RGD in Recurrent Gynecologic Cancer
Kenneth H. Kim,1 Kellie Schneider,1 Minghui Wang,1 Janis O’Malley,1 Wen Wan,1 Meredith A. Preuss,1 Raymond D. Harris,2 Rosemarie Aurigemma,3 Gene P. Siegal,1 Kurt Zinn,1 David T. Curiel,1 Ronald D. Alvarez.1 1 The University of Alabama at Birmingham, Birmingham, AL; 2 Biopharmaceutical Development Program – SAIC, National Cancer Institute, Frederick, MD; 3Biological Resources Branch, National Cancer Institute, Frederick, MD. Background: Adenoviral-based gene therapy has been evaluated as a potential therapeutic strategy for those ovarian and endometrial cancer patients with chemorefractory disease. Ad5.SSTR/TK.RGD is an infectivity-enhanced adenovirus that expresses a therapeutic thymidine kinase suicide gene and a somatostatin type 2 receptor, allowing for noninvasive assessment of gene transfer with nuclear imaging, and is given in combination with the prodrug ganciclovir (GCV). The purpose of this study was to identify the MTD, spectrum
Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy
S15