Scholarly article on topic 'Interference with the IL-1 Signaling Pathway Significantly Improves the Toxicity Profile of Systemically Applied Adenovirus Vectors'

Interference with the IL-1 Signaling Pathway Significantly Improves the Toxicity Profile of Systemically Applied Adenovirus Vectors Academic research paper on "Biological sciences"

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Academic research paper on topic "Interference with the IL-1 Signaling Pathway Significantly Improves the Toxicity Profile of Systemically Applied Adenovirus Vectors"

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

against life threatening infectious agents such as anthrax. It is currently recognized that manifestations of virus-related systemic toxicity greatly impairs safety and efficacy of Ad vectors. To better understand the molecular mechanisms and primary mediators involved in the initiation of the innate host response toward systemically applied Ad, and to analyze the feasibility of pharmacological management of Ad-mediated toxicity, we administered Ad5-based vectors intravenously into both wild type C57BL/6 and genetically manipulated mice knocked out for specific signaling pathways involved in activating and maintaining an innate immune response. At different times post virus injection, the hepatic mRNAs and plasma levels of different cytokines/chemokines were evaluated using an RNAse protection assay and mouse inflammatory cytometric bead arrays. These analyses revealed that immediately following systemic Ad application, the transcription of IL-1 and MIP-2 genes in the liver was up-regulated more than 30 times over their baseline. Although TNFa gene transcription was slightly up-regulated at 30 min post virus injection, its maximum levels were not reached before 6 h post virus administration. Importantly, although TNFa signaling was clearly involved in the host response toward systemically applied Ad, our data demonstrate that TNF signaling mediates a delayed rather than an immediate response, and that TNFa is not a primary mediator, initiating anti-Ad host inflammatory responses. Thus, pre-injection of wild type mice with anti-TNFa antibody prior to virus administration could neither reduce the levels of immediate IL-1 and MIP-2 gene up-regulation nor alleviate liver pathology seen following Ad administration. Using capsid-modified Ad vectors which have a reduced capacity to transduce liver cells in vivo, we found that the level of MIP-2 gene up-regulation directly correlates with the level of IL-1 gene transcription. Furthermore, we found that in wild type mice, the level of IL-1 gene up-regulation depends on the level of Ad hepatocyte transduction. Interference with IL-1 signaling pathways by pre-injection of mice with anti-IL-1a/p antibody dramatically reduced both the levels of immediate cytokine gene up-regulation and liver damage caused by systemic Ad administration. Importantly, interference with IL-1 signaling pathways did not affect the ability to Ad vectors to efficiently transduce liver cells in vivo. Our data provide new insights into molecular mechanisms involved in host innate immune and inflammatory responses toward systemically applied Ad. They also provide a rational approach for pharmacological management of anti-Ad host responses, allowing for significant improvement of the safety profiles of existing Ad vectors while preserving their gene transfer efficiency.

1011. Tumor Specific Oncolytic Adenovirus Vector VRX-011 Which Overproduces ADP and Has the E4 Promoter Replaced by the hTERT Promoter

Mohan Kuppuswamy,1 Ann E. Tollefson,1 Karoly Toth,1 Jacqueline F. Spencer,1 Konstantin Doronin,1 Drew L. Lichtenstein,2 Lou Zumstein,3 William S. M. Wold.1,2 'Molecular Microbiology & Immunology, Saint Louis University School of Medicine, St. Louis, MO; 2VirRx, Inc., St. Louis, MO; 3Introgen Therapeutics, Inc., Houston, TX.

We have previously evaluated the conditionally replicating adenovirus (Ad) vector named KD3 for possible use in anti-cancer therapy. KD3 markedly overexpresses ADP, an Ad nuclear membrane glycoprotein required at late stages of infection for efficient cell lysis and release of Ad from cells. KD3 also has two E1A mutations that reduce its replication in primary cells but allow efficient replication in cancer cells. We have also reported on a replication-competent vector named VRX-007 which overexpresses ADP and has wild type E1A. VRX-007 showed remarkable early cytopathic effects, early cell lysis, large plaques, increased cell to cell dissemination in cultured cells, enhanced cytolysis of cultured

cancer cells, and efficient suppression of tumor growth in animal models. In this report we describe the vector VRX-011, which is similar to VRX-007 except that the Ad E4 promoter has been replaced by the promoter for human telomerase (hTERT). Telomerase is an enzyme involved in the maintenance of chromosomal termini known as telomeres. About 90% of cancer cells have elevated levels of telomerase activity, whereas telomerase activity is not detected in normal somatic cells. Since E4 proteins are required for Ad replication, VRX-011 is expected to replicate in most cancer cell types but not (well) in non-cancerous cells. VRX-011 replicated as well as Ad5 in the majority of cancer cell lines tested and was growth restricted in primary cells. Ad late protein expression was at normal levels in A549 human lung cancer cells but was markedly reduced in primary cells. Intratumor administration of VRX-011 effectively suppressed Hep3B liver carcinoma xenograft growth in nude mice. Systemic administration of VRX-011 by tail vein injection also efficiently suppressed Hep3B xenograft growth in nude mice. Also, intravenous administration of VRX-011 significantly reduced the number and size of A549 cancer cell metastases in nude mice lungs. Single tail vein administration of different PFU doses of VRX-011 in C57B/6 mice showed no toxicity as indicated by liver enzyme levels and animal weights. These in vitro and in vivo data show that VRX-011 has enhanced specific oncolytic efficacy and restriction in primary cells. VRX-011 may be useful in treating different types of human cancer.

1012. Ad Interactions with the Complement Systems of Humans and Mice

Haixiang Jiang,1 Ruth Everett,1 Anne Kiang,1 Zhaoxia Wang,1 Huame Zhang,1 Delila Serra,1 Michael M. Frank,1 Andrea Amalfitano.1

'Dept. of Pediatrics, Duke University Medical Center, Durham, NC.

Advanced generation Ad vectors can allow for long term persistence by avoiding activation of the adaptive arm of the immune response. Unfortunately, significant acute toxicities can be incurred after i.v. injection of Ads. Precious little is known about Ad interactions with the acellular defense systems present in the blood, including the complement system. The complement system has evolved to rapidly sequester invading pathogens, while rapidly initiating multiple downstream immune functions. However, inappropriate or excessive complement activation is also known to be associated with a number of serious clinical consequences. Intriguingly, many of these are known to also occur after high dose Ad injections, suggesting that Ads may be directly activating the complement cascade. We therefore set out to test this hypothesis. The exposure of Ad5 capsids to Ad-seropositive or sero-negative human serum resulted in activation of the complement cascade, suggesting that the presence of anti-Ad antibodies was not required for Ad induced complement activation. We found that complement consumption occurred despite specific blockade of the classical complement pathway (using the AP50 assay) implicating the alternative complement pathway as being directly activated by Ads. Ads also activated the alternative pathway when C1q depleted human serum (C1q is typically required for classical complement pathway activation) was utilized in the AP50 assay. Supporting these observations, we did not detect consumption of human C4 activity in either of these experiments. In vitro analyses confirmed that the human C3 protein could bind to immobilized Ad5 particles in the presence of EGTA (blocks classical pathway) but not EDTA (blocks both the classical and alternative complement pathways). This result was also achieved when Ad5 particles were incubated with C1q depleted serum. Again, the human C4 protein was not found to bind Ad particles under any of these assay conditions. We also found that 125-I labeled C3 bound to immobilized Ad particles,

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