Scholarly article on topic 'Safety of Adenoviral Vectors: Results of Clinical Investigations in 445 Cancer Patients Treated with AdvexinS (Adenoviral p53) Gene Therapy'

Safety of Adenoviral Vectors: Results of Clinical Investigations in 445 Cancer Patients Treated with AdvexinS (Adenoviral p53) Gene Therapy Academic research paper on "Clinical medicine"

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Academic research paper on topic "Safety of Adenoviral Vectors: Results of Clinical Investigations in 445 Cancer Patients Treated with AdvexinS (Adenoviral p53) Gene Therapy"

the absence of enzyme replacement therapy. All the children are presently healthy, they did not experience any severe infections or adverse effect, with the longest follow up being of 40 months after gene therapy. A systematic analyses to identify and map vector integration sites by inverse-PCR and LM-PCR was undertaken. Cloned integrations were selected for analyses only if contained the correct retroviral and primer sequences and yielded a unique best hit with >95% identity to the human genome (NCBI34). Collective data from 160 mapped integrations showed a frequency of insertions inside RefSeq genes of 33.4% (n=53), similar to the one reported in cell lines infected in vitro with retroviral vectors. Genomic regions within 5 Kb upstream from transcription start site were favored sites of integrations (15.1%). There was no preferential target locus, with the exception that two independent integrations were found outside a gene on chromosome 3p13, within a distance of 120 Kb. Integrations in circulating T cells were highly polyclonal (>50 in Pt1 and Pt3), as shown by the random cloning of PCR products from peripheral blood T cells as well as by PCR analyses in T-cell clones generated ex vivo. Fewer integrations (3-15) were detected in myeloid cells from bone marrow and peripheral blood. Common integrants were identified in various lineages during the follow-up of patients, demonstrating the engraftment of transduced HSC with multilineage potential. Collectively, these data show the efficacy of gene therapy in correcting the immune and metabolic defect of ADA-SCID. In addition, these studies are providing new information on the safety and biology of retroviral vectors as well as on the in vivo dynamics of HSC and their progeny.

*MGR and CB equally contributed to the work

1005. Expression of Normal Dystrophin Following Myoblast Transplantation to Duchenne Muscular Dystrophy Patients

Jacques P. Tremblay,1 Daniel Skuk,1 Bouchard Jean-Pierre,2 Michel Sylvain,1 Roy Raynald,1 Goulet Marilyne,1 Roy Brigitte,1 Pierre Chapdelaine,1 Dugré Francine,1 Jean-Guy Lachance,1 Louise Deschènes,1 Hélène Senay.1

'Centre de Recherche du CHUQ, Centre Hospitalier Universitaire de Québec, Québec, QC, Canada; 2Neurologie, Centre Hospitalier Affilier de Québec, Québec, QC, Canada.

Three Duchenne muscular dystrophy (DMD) patients received injections of myogenic cells obtained from skeletal muscle biopsies of normal donors. Cells were injected in 1 cm3 of the Tibialis anterior by 25 parallel injections. We performed similar patterns of saline injections in the contralateral muscles as controls. The patients received tacrolimus for immunosuppression. Muscle biopsies were performed at the injected sites 4 weeks later. We observed dystrophin-positive myofibers in the cell-grafted sites: 9 % (patient 1), 6.8 % (patient 2) and 11 % (patient 3). Since patients 1 and 2 had identified dystrophin-gene deletions these results were obtained using mAbs specifically to epitopes coded by the deleted exons. Donor-dystrophin was absent in the control sites. Patient 3 had exon duplication and thus specific donor-dystrophin detection was not possible. However there was 4-fold more dystrophin-positive myofibers in the cell-grafted than in the control site. Donor-dystrophin transcripts were detected by RT-PCR (using primers reacting with a sequence in the deleted exons) only in the cell-grafted sites in patients 1 and 2. Dystrophin transcripts were more abundant in the cell-grafted than in the control site in patient 3. Therefore, significant dystrophin expression can be obtained in the skeletal muscles of DMD patients following specific conditions of cell delivery and immunosuppression.

I am president and part owner of CellGene inc.

1006. Preliminary Results of a Phase I Trial Using Retroviral Gene Transfer of G156A MGMT To Protect Hematopoiesis during BG and BCNU Therapy of Advanced Malignancies

Jane Reese,1 Karen Lingas,1 Pamela Ksenich,1 Colin Sweeney,1 Omer Koc,1 Stanton Gerson.1

'Hematology/Oncology, Center for Stem Cell and Regenerative Medicine, Case Comprehensive Cancer Center, Case Western Reserve University School of Medicine, Cleveland, OH.

We have extensively studied the G156A MGMT mutation which confers O6aLkylguanine DNA alkyltransferase (AGT) that is resistant to O6benzylguanine (BG). In a Phase I clinical trial at our institution, BG was shown to inhibit tumor AGT at 120mg/m2 and the maximum tolerated dose of BCNU was 40 mg/m2. In the laboratory, we have shown that the retrovirus MFG-G156A-MGMT confers BG and BCNU resistance ex vivo into human CD34+ cells. We have also reported that G156A-MGMT transduced murine hematopoietic progenitors repopulate the bone marrow and can be enriched up to 1000-fold after 2 cycles of BG and BCNU. These studies demonstrate the strong capacity of this model to confer BG and BCNU resistance and a selection advantage in vivo. Based on these data, we initiated a clinical trial using gene transfer into autologous CD34+ cells in patients with advanced malignancies. Patients undergo CD34+ mobilization with G-CSF and GM-CSF. CD34+ enriched cells (CliniMACS, Miltenyi) are transduced with MFG-G156A MGMT (produced in PG13 cells by the NGVL, K. Cornetta, Director) in the presence of the fibronectin fragment CH-296 (provided by Takara Bio Inc) and the cytokine combination SCF, Tpo, and Flt-3 ligand for 86 hours. The cell culture is harvested on day 4 and the entire culture is re-infused into the patient who received BG and BCNU 48 hours prior. Patients receive subsequent cycles of BG and BCNU every 6 weeks with analysis of marrow and blood cells for evidence of gene transfer prior to each cycle. In the first three patients, the frequency of gene transfer measured by proviral containing CFU in the post transduction culture was 20 ± 7% and 6.3 ± 3 % of the cells expressed G156A-AGT by flow cytometry. An average of 4.4 ± 2 x 107viable mononuclear cells with a CD34 purity of 93 ± 5% were infused. In one patient, 10% of bone marrow CFU showed presence of the provirus 5 weeks after infusion but prior to the second cycle of BG and BCNU. In this same patient, insertion site analysis by LAM-PCR showed a similar banding pattern in both the lymphocyte-monocyte and granulocyte fractions, indicating multilineage reconsitituion by a single transduced stem cell. Our preliminary results demonstrate efficient gene transfer into cytokine stimulated CD34+ cells using the MFG-G156A-MGMT vector and these cells can be infused without toxicity. Given the strong degree of selection observed in pre-clinical models, we anticipate enrichment for transduced cells following each cycle of chemotherapy.

1007. Safety of Adenoviral Vectors: Results of Clinical Investigations in 445 Cancer Patients Treated with Advexin® (Adenoviral p53) Gene Therapy

Louis A. Zumstein,1 Donna Call,1 James Merritt,1 Robert E.

Sobol,1 Kerstin Menander.1

'Introgen Therapeutics, Inc, Houston, TX.

Safety data were collected from 445 patients treated with Advexin® (adenoviral p53) gene therapy for cancer in 14 clinical trials (Phase I, II and III). Advexin gene therapy was used as monotherapy in 11 trials and in combination with chemotherapy or radiation in 3 clinical studies. The treated patients had advanced cancers, primarily lung and squamous cell head and neck cancers, but also included patients with prostate cancer, colorectal cancer, and other solid tumors. Most patients were treated with multiple

Molecular Therapy Volume 9, Supplement 1, May 2004 Copyright © The American Society of Gene Therapy

cycles of therapy by an intratumoral route of administration, while 17 patients were treated intravenously. Overall, greater than 3,000 injections of Advexin were administered.

As the patients had advanced cancers, most of the side-effects were attributed to the underlying condition. The most frequently reported side-effects considered to be possibly or probably related to treatment with Advexin were fever (in 44% of the patients), injection site pain (33%), chills (18%), nausea (13%), and asthenia (12%). Most side-effects were mild or moderate in severity. Only 58 side-effects attributed to the treatment with Advexin or Advexin with chemotherapy or radiation required medical intervention or hospitalization. Some of these side-effects were believed to be due to the concomitant chemotherapy or radiotherapy. The combination of Advexin with other cancer treatments did not increase the frequency or severity of side-effects expected from chemotherapy or radiation alone. There were no deaths attributed to Advexin gene therapy.

These results indicate that Advexin gene therapy is safe and well tolerated as a monotherapy or in combination with chemotherapy or radiation. The data support the safety of adenoviral p53 gene therapy, which exhibited an excellent safety profile compared to conventional cancer treatments such as chemotherapy and radiation.

The authors are employees of Introgen Therapeutics, and hold Introgen Stock.

1008. Intratumoral Injection of INGN 241, a Non-Replicating Adenovector Expressing the Melanoma-Differentiation Associated Gene-7 (MDA-7/IL-24): Biologic Outcome in Advanced Cancer Patients

Alex W. Tong,1,2 John Nemunaitis,2 Dan Su,1 Yuan Zhang,1 Casey Cunningham,2 Neil Senzer,1,2 George Netto,1 Dawn Rich,2 Abner Mhashilkar,3 Karen Parker,3 Keith Coffee,3 Rajagopal Ramesh,4 Suhendan Ekmekcioglu,5 Elizabeth A. Grimm,5 Jill van Wart Hood,3 James Merritt,3 Sunil Chada.3,5 'Baylor Sammons Cancer Center, Baylor University Medical Center, Dallas, TX; 2US Oncology, Dallas, TX; 3Introgen Therapeutics, Inc, Houston, TX; 4Dept. of Thoracic and Cardiovascular Surgery, the University of Texas M.D. Anderson Cancer Center, Houston, TX; 5Dept. of Bioimmunotherapy, the University of Texas M.D. Anderson Cancer Center, Houston, TX.

The mda-7 gene is a novel tumor suppressor gene with tumor-apoptotic and immune activating properties. We completed a Phase I dose-escalating clinical trial, in which a non-replicating adenoviral construct expressing the mda-7 transgene (Ad-mda7; INGN 241) was administered intratumorally to 22 patients with surgically resectable advanced cancers. Excised tumors were evaluated for vector-specific DNA, RNA, transgenic MDA-7 expression and biological effects. DNA PCR analyses demonstrated a dose-dependent penetration of INGN 241 (up to 4x108 copies/ug DNA at the 2x1012 vp dose). A parallel distribution of vector RNA, MDA-7 protein expression and apoptosis induction were observed; with signals decreasing with distance away from the injection site. P-catenin and iNOS expression were reduced after INGN 241 treatment. Transient increases (up to 20-fold) of serum IL-6, IL-10, and TNFa were observed. Significantly higher elevations of IL-6 and TNFa were observed in 3 patients who responded clinically to INGN 241. Patients also showed marked increases of CD3+ CD8+ T cells post-treatment, suggesting that INGN 241 increased systemic TH1 cytokine production and mobilized CD8+ T cells. Intratumoral delivery of INGN 241 induced apoptosis in a large volume of tumor and elicited tumor regulatory and immune activating events that are consistent with the preclinical features of MDA-7/IL-24.

A number of authors are employees and hold stock in Introgen Therapeutics

1009. A Phase I Study of the Clinical and Local Biological Effects of Intratumoral Injection of mda-7(INGN 241) in Patients with Advanced Carcinoma

C. Casey Cunningham,1 Sunil Chada,2 James Merritt,2 Alex Tong,3 Neil Senzer,1 Yuan Zhang,3 Abner Mhashilkar,2 Karen Parker,2 Sasha Vukelja,4 Donald Richards,4 Jill Hood,2 Keith Coffee,2 John J. Nemunaitis.1

'US Oncology, Mary Crowley Medical Research Center, Dallas, TX; 2Introgen Therapeutics, Inc., Houston, TX; 3Sammons Cancer Center, Baylor University Medical Center, Dallas, TX; 4Tyler Cancer Center, Tyler, TX.

The melanoma differentiation-associated gene-7 (mda-7) is a tumor suppressor gene whose expression causes apoptotic cell death in cells from a wide variety of solid tumor types, but has little effect in non-malignant cell cultures. To begin to characterize the safety and biologic activity of mda-7 gene transfer in a clinical setting, we conducted a phase I trial using intratumoral injections of an adenovirus containing the mda-7 construct (Ad-mda7; INGN 241) in patients with resectable solid tumor lesions. Sequential cohorts of patients received escalating doses of INGN 241 from 2 x 1010 to 2 x 1012 viral particles (vp) injected into the center of the accessible target tumor lesion. In the first 5 cohorts, the injected lesions were resected at 24 to 96 hours post injection, serially sectioned, then analyzed to determine the distribution and concentration of the viral agent, mRNA and protein expression, and assess the resultant biologic effects on the tumor cells. The last two cohorts had incisional biopsies performed pre-treatment and 30 days post treatment with the final cohort receiving 2 x 1012 vp injected twice weekly for 3 weeks of a 28 day cycle. All injected lesions demonstrated INGN 241 vector DNA and mRNA copies with the highest level near the injection site, then decreasing inversely with distance. Protein expression of MDA-7 determined by immunostaining paralleled the geographic and temporal pattern of expression of DNA and mRNA. Apoptosis staining by TUNEL reactivity also closely correlated with the expression pattern of the MDA-7 protein. Toxicity attributable to the injections consisted of transient low grade fever and mild injection site pain and erythema. Evidence of activity in two patients was seen with the repeat injections. One was a patient with metastatic melanoma who achieved a complete response in two separately injected lesions. In summary, INGN 241 delivered through intratumoral injection is well tolerated, induces apoptosis in a large percentage of tumor cells where protein expression is seen, and, with repeat injections, demonstrates evidence of clinically significant activity.

Some authors are or were employees of Introgen, including S. Chada, J. Merritt, A. Mhashilkar, K. Parker, J. Hood, and K. Coffee

Adenovirus: Toxicity and the Immune Response

1010. Interference with the IL-1 Signaling Pathway Significantly Improves the Toxicity Profile of Systemically Applied Adenovirus Vectors

Dmitry M. Shayakhmetov, Zong-Yi Li, Shaoheng Ni, Andre Lieber.

'Division of Medical Genetics, Department of Medicine, University of Washington, Seattle, WA.

Adenovirus vectors (Ad) are the second largest group of viral vectors that are extensively used in clinical trials in the US as prospective therapeutics against numerous inborn and acquired human diseases, including cancer. Most recently, interest in Ad has further expanded due to its potential as a vector for vaccination

Molecular Therapy Volume 9, Supplement 1, May 2004 Copyright © The American Society of Gene Therapy