818 New Adenoviral Vectors for Molecular Chemotherapy of Prostate Cancer Molecular Therapy �������� ��� ���� ���������������� �������� ��� ������®������������ �!����� ����"� �������� S315 ��������� ��[.]
ONCOLYTIC VIRUS AND SUICIDE GENE THERAPY FOR CANCER administration of the appropriate non-toxic pro-drug after a sufficient number of cells has been “seeded” with the replicating vector; this also represents a self-destructing safety mechanism that will terminate viral replication even in extratumoral tissues We have devised a novel RCR vector design which directly links transgene expression to viral gene expression by insertion of an internal ribosome entry site (IRES)-transgene cassette just 3’ of the env gene; this vector design exhibits far greater genomic structural stability over multiple serial replication cycles compared to all previously described MLV-based RCR vectors, which invariably employed unstable insertions in the LTR-U3 region After intratumoral injection of RCR vector supernatant at MOIs even as low as 0.001, over a period of several weeks it was possible to achieve up to 98.7% transduction throughout entire solid tumors in breast cancer, prostate cancer, and glioma models Treatment of pre-established intracranial U87 gliomas with RCR vector-mediated suicide gene therapy resulted in 100% survival over a follow-up period of more than 120 days, compared to 0% survival of control groups receiving vector alone or pro-drug alone with a median survival of 35 days Histological examination revealed that the groups treated with vector alone and pro-drug alone showed extensive growth of large tumors causing midline shifts and perturbation of normal brain architecture; in contrast, RCR-injected gliomas were completely eradicated with administration of pro-drug Notably, after intratumoral injection the vectors were undetectable by sensitive PCR assays in all normal tissues tested, presumably due to selectivity for rapidly dividing tumor cells provided by the intrinsic inability of MLV to infect quiescent cells and the limited amount of vector leakage from the tumor as cell-to-cell budding occurs Thus, RCR vectors can achieve highly efficient and tumorrestricted replication with concomitant delivery of inserted suicide genes throughout entire solid tumor masses in vivo, resulting in significant suppression of tumor growth and prolonged survival We are now also testing various targeting strategies to further enhance tumor selectivity and minimize the potential risk to normal cells 817 Circulating Oncolytic and Wild Type Adenovirus Levels in Clinical Trial Patients Treated with CG7870 Paul Husak,1 Ken Ho,1 Junko Aimi,1 Shian-Jiun Shih,1 Flavia Borellini,1 D C Yu,1 David Kirn,1 Dale Ando.1 Cell Genesys, Inc., South San Francisco, CA CG7870 is an attenuated, conditionally replication-competent, dual-promoter oncolytic adenovirus that preferentially replicates in prostate cells Both CG7870 and CG7060, a single-promoter oncolytic adenovirus, have been tested in Phase I/II trials in patients with prostate cancer by intraprostatic (CG7870 and CG7060) and intravenous (CG7870) administration Data collected from these trials were analyzed to determine the safety of CG7870 and CG7060 in this patient population Additional investigation was performed to determine the relationship between [1] levels of wild type adenovirus in GMP lots of oncolytic adenovirus administered to patients and estimate wild type exposure; [2] pharmacokinetics of wild type and oncolytic adenovirus genomes in the blood of treated patients; [3] clinical safety profiles associated with wild type adenovirus exposure during these Phase I/II trials Quantitative PCR was used to determine wild type adenovirus levels in clinical lots, in addition to circulating levels of oncolytic adenovirus and wild type adenovirus in patient serum or plasma samples Wild type genomes were detected in all clinical lots used to treat patients All patients tested (37 of 63 total) had various levels of circulating CG7870 or CG7060 genomes for up to four weeks post-treatment In approximately one-quarter of the 37 patients tested, low levels of wild type genomes were detected in the blood at baseline prior to treatment, consistent with environmental exposure Eleven percent Molecular Therapy Vol 7, No 5, May 2003, Part of Parts Copyright ® The American Society of Gene Therapy of analyzed patients (4/37) had circulating wild type adenovirus genomes above baseline threshold for approximately two weeks post-treatment, all of whom had received a theoretical wild type adenovirus dose >10E6 intraprostatically Wild type exposure below 10E6 did not result in circulating wild type adenovirus by the same route of administration Calculated wild type adenovirus exposure up to 10E4 (maximum tested) did not result in any reproducible amounts of circulating wild type following intravenous delivery Moreover, the clinical safety profile of all patients were similar regardless of adenovirus dose administered or oncolytic or wild type adenovirus detected post-treatment The adverse events and laboratory safety profiles of all patients indicated that both CG7870 and CG7060 were well-tolerated with no dose-limiting toxicity at doses up to 10E13 particles, the highest dose administered Most adverse events were mild to moderate, and none could be attributed to levels of circulating wild type adenovirus These clinical safety data provide a preliminary framework for the establishment of safety specifications for the continued clinical development of CG7870 in patients with early stage prostate cancer 818 New Adenoviral Vectors for Molecular Chemotherapy of Prostate Cancer Debbie L Della Manna,1 Masato Yamamoto,2 Victor Krasnykh,2 Kurt R Zinn,3 Julia Davydova,2 Scott Chiz,1 Donald J Buchsbaum.1 Department of Radiation Oncology, University of Alabama at Birmingham, Birmingham, AL, United States; 2Division of Human Gene Therapy, University of Alabama at Birmingham, Birmingham, AL, United States; 3Department of Radiology, University of Alabama at Birmingham, Birmingham, AL, United States Object The goal of this project is to develop novel two gene adenovirus (Ad) vectors expressing somatostatin receptor subtype (SSTr2) and cytosine deaminase (CD) under control of the cyclooxygenase-2 (cox2) promoter for radiolabeled peptide/ molecular chemotherapy of prostate cancer The tumor specific promoter cox2 was selected based on the fact that the liver is cox2 negative and is the predominant site of Ad vector localization after systemic administration, thus the Ad may infect normal liver cells but no transgene expression would occur Two forms of the cox2 promoter were compared and contrasted to the cytelomegalovirus (CMV) promoter; a “long” form (cox2L) and a truncated “medium” form (cox2M) Materials and Methods Ad were made using the AdEasy system and propagated in 911 cells Somatostatin binding and internalization assays were performed by incubating infected cells with 99mTc-P2045, a peptide specific for SSTr2, and gamma camera imaging after the surface bound peptide was removed CD conversion assays were performed with lysates from infected cells with the addition of ³H-5-fluorocytosine (5-FC) at various time points, and separation on thin layer chromatography plates in butanol and water Spots containing 5-FC and 5-fluorouracil (5-FU) were cut out, put into scintillation fluid, and counted in a beta counter Cytotoxicity assays were performed using the MTS assay (Promega) as described in the manufacturer’s protocol Results Six vectors expressing CD and SSTr2 were developed and evaluated using the prostate cancer cell lines DU145 and PC3: Adcox2LCDcox2LSSTr2, Adcox2LSSTr2cox2LCD,Adcox2LCDIRESSSTr2, Adcox2MCDcox2MSSTr2, Adcox2MSSTr2cox2MCD, and AdCMVCDCMVSSTr2 Based on CD conversion assays, cytotoxicity assays and SSTr2 binding assays, the vectors driven by cox2L proved to be superior to those driven by cox2M, however the cox2L vector constructed using IRES failed to bind 99mTc-P2045 Although AdCMVCDCMVSSTr2 infected cells converted more 5FC to 5-FU than Adcox2LCDcox2LSSTr2 and Adcox2LSSTr2cox2LCD (7.53 vs 2.95 and 2.51 pmol/min/mg, in S315 ONCOLYTIC VIRUS AND SUICIDE GENE THERAPY FOR CANCER DU145 cells; 2.46 vs 1.21 and 0.87 pmol/min/mg in PC3 cells), higher cytotoxicity was demonstrated with the cox2L vectors after exposure to 5-FC in both cell lines tested (IC50 of 13.1 and 21.4 nM for Adcox2LCDcox2LSSTr2 and Adcox2LSSTr2cox2LCD with DU145 cells, and 24.5 and 30.4 nM, respectively with PC3 cells) Conclusion The combination of the therapeutic genes CD and SSTr2 with the cox2L promoter should provide specificity for tumor uptake of radiolabeled peptides that bind to SSTr2 and selective 5FC molecular chemotherapy, which are to be evaluated in metastatic prostate cancer models Supported by DOD grant DAMD17-02-10001 819 Gene Therapy for Retinoblastoma Using AdV/TK Followed by Ganciclovir: Report of a Clinical Trial Richard L Hurwitz,1,2 Murali Chintagumpala,1 William F Mieler,2 Evelyn Paysse,2 Milton Boniuk,2 Mary Y Hurwitz,1 Patricia Chevez-Barrios,2,3 Texas Children’s Cancer Center, Center for Cell and Gene Therapy Pediatrics, Baylor College of Medicine, Houston, TX; Ophthalmology, Baylor College of Medicine, Houston, TX; Pathology, Baylor College of Medicine, Houston, TX Objective: To determine the safety and assess the efficacy of using an intraocular injection of an adenoviral vector containing the herpes thymidine kinase gene (ADV/TK) followed by systemic administration of ganciclovir to treat refractory retinoblastoma Methods: This was an IRB, RAC and FDA approved pilot study utilizing intrapatient dose escalation All patients had bilateral retinoblastoma with only one functional eye containing measurable retinoblastoma that was refractory to standard therapies; enucleation was the imminent treatment Each experimental treatment consisted of an injection of adenoviral vector via a single needle insertion through the cornea, iris, zonules, and into the vitreous and was confirmed by direct observation Each injection was followed by days of ganciclovir delivered intravenously every 12 hours Vitreous seeds were treated by intravitreous injection adjacent to the site of disease Results: Eight patients have been enrolled on the study The first patient received a single injection of 108 vp AdV/TK and the next patients received an initial injection of 109 vp followed by either one or injections of 1010 vp Two patients who received 1010 vp experienced mild intraocular inflammatory responses that were well-controlled with steroids One patient who had numerous vitreous seeds developed vitreous contraction One patient with vitreous seeds has had a complete response and is tumor free with excellent vision 22 months following the completion of therapy Three patients were enucleated The eyes of patients who were enucleated all contained necrotic tumor at and around the injection sites and marked decreases in the number of vitreous seeds Three subsequent patients received an injection of 1010 vp followed by one to four injections of 1011 vp while a fourth received a single injection of 1011 vp One patient was enucleated because of an increase in the number of vitreous opacities (seeds) after a total of one injection of 1010 vp and injections of 1011 vp Histopathologic examination revealed islands of inflammatory cells but no evidence of tumor The other patients (2 patients treated with one dose of 1010 vp and one dose of 1011 vp and one patient with a single dose of 1011 vp) achieved either a partial response and are being treated with standard therapies (one patient) or have achieved a complete response and are being observed for signs of recurrent disease (two patients) The major adverse event was moderate intraocular inflammation (responsive to steroids) seen in three patients treated with 1011 vp There was no evidence of extraocular spread of tumor through the needle tract in any patient Conclusions: AdV/TK followed by ganciclovir can be safely administered to children with retinoblastoma Patients with vitreous S316 seeds, a complication particularly difficult to treat with standard therapies, appeared to respond Suicide gene therapy may be able to contribute to the treatment of children with retinoblastoma 820 Phase I Study of Replication-Competent Adenovirus-Mediated Double Suicide Gene Therapy in Combination with Three-Dimensional Conformal Radiation Therapy for the Treatment of Locally Aggressive Prostate Cancer Svend O Freytag,1 Hans Stricker,1 Jan Pegg,1 Dell Paielli,1 Summer Xia,1 Steve Brown,1 Jae Ho Kim.1 Radiation Oncology and Urology, Henry Ford Health System, Detroit, MI Adenovirus-mediated suicide gene therapy may hold much promise in the treatment of human cancer Although originally conceived as a monotherapy, we were the first to propose using suicide gene therapy as a means to improve the effectiveness of radiation therapy We have developed a novel, trimodal approach that utilizes a lytic, replication-competent adenovirus (Ad5-CD/ TKrep) to selectively and efficiently deliver a CD/HSV-1 TK fusion gene to tumors Our preclinical studies have demonstrated that the Ad5-CD/TKrep virus itself, via its cytolytic activity, has potent anti-tumor activity The efficacy of Ad5-CD/TKrep viral therapy can be enhanced significantly by invoking the CD/5-FC and HSV-1 TK/GCV enzyme/prodrug systems, which render malignant cells sensitive to specific pharmacological agents, and importantly, sensitizes them to radiation Our preclinical work led to the first FDA-approved clinical trial (BB-IND 8436) in which a replicationcompetent virus was used to deliver a therapeutic gene to humans This trial was completed in 2001 with excellent results As a followup to this trial, we evaluated the safety and efficacy of our viral oncolytic/double suicide gene therapy approach in combination with three-dimensional conformal radiation therapy (3DCRT) in patients with newly diagnosed, intermediate to high risk prostate cancer (BB-IND 9852) Fifteen patients received a single intraprostatic injection (1012 vp) of the replication-competent Ad5-CD/TKrep virus followed by one to three weeks of 5-FC (150 mg/kg/day) and valganciclovir vGCV (1,800 mg/day) prodrug therapy and standard dose (70 Gy) 3DCRT The primary endpoint was toxicity six weeks after completion of 3DCRT The trial has been completed with excellent results There were no dose-limiting toxicities and the maximum tolerated dose duration of the 5-FC + vGCV prodrug therapy was not defined Ninety three percent (93%) of the adverse events observed were grade 1/2 in severity and there were no serious adverse events Prostate biopsies obtained at 2, and weeks postinjection demonstrated that CD/HSV-1 TK transgene expression can persist in the prostate for at least weeks Our early analyses have indicated that PSA response and post-treatment prostate biopsy results are better than what would be expected for patients receiving standard dose 3DCRT alone Based on these encouraging results, a randomized, two-arm, prospective Phase II study has been planned using a second-generation adenovirus that, in preclinical models, is markedly more efficacious than the prototype Ad5-CD/ TKrep virus Our work continues to give us hope that gene therapy may demonstrate value in the clinic, particularly when combined with conventional cancer therapies such as radiation therapy Molecular Therapy Vol 7, No 5, May 2003, Part of Parts Copyright ® The American Society of Gene Therapy ... provide specificity for tumor uptake of radiolabeled peptides that bind to SSTr2 and selective 5FC molecular chemotherapy, which are to be evaluated in metastatic prostate cancer models Supported... Children’s Cancer Center, Center for Cell and Gene Therapy Pediatrics, Baylor College of Medicine, Houston, TX; Ophthalmology, Baylor College of Medicine, Houston, TX; Pathology, Baylor College of Medicine,... injection of 1010 vp followed by one to four injections of 1011 vp while a fourth received a single injection of 1011 vp One patient was enucleated because of an increase in the number of vitreous