Kilian Guse

Treatment of Cancer Patients With a Serotype 5/3 Chimeric Oncolytic Adenovirus Expressing GMCSF

Augmenting antitumor immunity is a promising way to enhance the potency of oncolytic adenoviral therapy. Granulocyte-macrophage colony-stimulating factor (GMCSF) can mediate antitumor effects by recruiting natural killer cells and by induction of tumor-specific CD8(+) cytotoxic T-lymphocytes. Serotype 5 adenoviruses (Ad5) are commonly used in cancer gene therapy. However, expression of the coxsackie-adenovirus receptor is variable in many advanced tumors and preclinical data have demonstrated an advantage for replacing the Ad5 knob with the Ad3 knob. Here, a 5/3 capsid chimeric and p16-Rb pathway selective oncolytic adenovirus coding for GMCSF was engineered and tested preclinically. A total of 21 patients with advanced solid tumors refractory to standard therapies were then treated intratumorally and intravenously with Ad5/3-D24-GMCSF, which was combined with low-dose metronomic cyclophosphamide to reduce regulatory T cells. No severe adverse events occurred. Analysis of pretreatment samples of malignant pleural effusion and ascites confirmed the efficacy of Ad5/3-D24-GMCSF in transduction and cell killing. Evidence of biological activity of the virus was seen in 13/21 patients and 8/12 showed objective clinical benefit as evaluated by radiology with Response Evaluation Criteria In Solid Tumors (RECIST) criteria. Antiadenoviral and antitumoral immune responses were elicited after treatment. Thus, Ad5/3-D24-GMCSF seems safe in treating cancer patients and promising signs of efficacy were seen.

Mitochondrial myopathy induces a starvation-like response

Mitochondrial respiratory chain (RC) deficiency is among the most common causes of inherited metabolic disease, but its physiological consequences are poorly characterized. We studied the skeletal muscle gene expression profiles of mice with late-onset mitochondrial myopathy. These animals express a dominant patient mutation in the mitochondrial replicative helicase Twinkle, leading to accumulation of multiple mtDNA deletions and progressive subtle RC deficiency in the skeletal muscle. The global gene expression pattern of the mouse skeletal muscle showed induction of pathways involved in amino acid starvation response and activation of Akt signaling. Furthermore, the muscle showed induction of a fasting-related hormone, fibroblast growth factor 21 (Fgf21). This secreted regulator of lipid metabolism was also elevated in the mouse serum, and the animals showed widespread changes in their lipid metabolism: small adipocyte size, low fat content in the liver and resistance to high-fat diet. We propose that RC deficiency induces a mitochondrial stress response, with local and global changes mimicking starvation, in a normal nutritional state. These results may have important implications for understanding the metabolic consequences of mitochondrial myopathies.

Oncolytic Adenovirus Coding for Granulocyte Macrophage Colony-Stimulating Factor Induces Antitumoral Immunity in Cancer Patients

Granulocyte macrophage colony-stimulating factor (GMCSF) can mediate antitumor effects by recruiting natural killer cells and by induction of tumor-specific cytotoxic T-cells through antigen-presenting cells. Oncolytic tumor cell-killing can produce a potent costimulatory danger signal and release of tumor epitopes for antigen-presenting cell sampling. Therefore, an oncolytic adenovirus coding for GMCSF was engineered and shown to induce tumor-specific immunity in an immunocompetent syngeneic hamster model. Subsequently, 20 patients with advanced solid tumors refractory to standard therapies were treated with Ad5-D24-GMCSF. Of the 16 radiologically evaluable patients, 2 had complete responses, 1 had a minor response, and 5 had disease stabilization. Responses were frequently seen in injected and noninjected tumors. Treatment was well tolerated and resulted in the induction of both tumor-specific and virus-specific immunity as measured by ELISPOT and pentamer analysis. This is the first time that oncolytic virus-mediated antitumor immunity has been shown in humans. Ad5-D24-GMCSF is promising for further clinical testing.

Proteoglycan 4 expression protects against the development of osteoarthritis.

Proteoglycan 4 protects from age-related and posttraumatic osteoarthritis development. Rubbing Arthritis the Wrong Way There are two main forms of joint inflammation (arthritis). Patients with rheumatoid arthritis develop joint inflammation because of an autoimmune reaction, whereas wear and tear is thought to contribute to the development of osteoarthritis. Indeed, most people have at least some symptoms of osteoarthritis by the age of 70. Treatment is mostly limited to reducing symptoms. Now, Ruan et al. provide a new mechanism for a potential new therapy for osteoarthritis. Proteoglycan 4 (lubrican, PRG4) has been previously thought to protect against osteoarthritis by acting as a lubricant between bones in a joint. Here, the authors show that not only does PRG4 protect against the development of osteoarthritis in both aging and trauma mouse models, but that its effect may extend beyond that of a mere lubricant. Indeed, PRG4 has a direct chondroprotective effect in these models, suggesting that it may form the basis for a new disease-altering therapy for osteoarthritis. Osteoarthritis (OA) is a common degenerative condition that afflicts more than 70% of the population between 55 and 77 years of age. Although its prevalence is rising globally with aging of the population, current therapy is limited to symptomatic relief and, in severe cases, joint replacement surgery. We report that intra-articular expression of proteoglycan 4 (Prg4) in mice protects against development of OA. Long-term Prg4 expression under the type II collagen promoter (Col2a1) does not adversely affect skeletal development but protects from developing signs of age-related OA. The protective effect is also shown in a model of posttraumatic OA created by cruciate ligament transection. Moreover, intra-articular injection of helper-dependent adenoviral vector expressing Prg4 protected against the development of posttraumatic OA when administered either before or after injury. Gene expression profiling of mouse articular cartilage and in vitro cell studies show that Prg4 expression inhibits the transcriptional programs that promote cartilage catabolism and hypertrophy through the up-regulation of hypoxia-inducible factor 3α. Analyses of available human OA data sets are consistent with the predictions of this model. Hence, our data provide insight into the mechanisms for OA development and offer a potential chondroprotective approach to its treatment.

Oncolytic Adenovirus ICOVIR-7 in Patients with Advanced and Refractory Solid Tumors

Purpose: Twenty-one patients with cancer were treated with a single round of oncolytic adenovirus ICOVIR-7. Experimental Design: ICOVIR-7 features an RGD-4C modification of the fiber HI-loop of serotype 5 adenovirus for enhanced entry into tumor cells. Tumor selectivity is mediated by an insulator, a modified E2F promoter, and a Rb-binding site deletion of E1A, whereas replication is optimized with E2F binding hairpins and a Kozak sequence. ICOVIR-7 doses ranged from 2 × 1010 to 1 × 1012 viral particles. All patients had advanced and metastatic solid tumors refractory to standard therapies. Results: ICOVIR-7 treatment was well tolerated with mild to moderate fever, fatigue, elevated liver transaminases, chills, and hyponatremia. One patient had grade 3 anemia but no other serious side effects were seen. At baseline, 9 of 21 of patients had neutralizing antibody titers against the ICOVIR-7 capsid. Treatment resulted in neutralizing antibody titer induction within 4 weeks in 16 of 18 patients. No elevations of serum proinflammatory cytokine levels were detected. Viral genomes were detected in the circulation in 18 of 21 of patients after injection and 7 of 15 of the samples were positive 2 to 4 weeks later suggesting viral replication. Conclusions: Overall, objective evidence of antitumor activity was seen in 9 of 17 evaluable patients. In radiological analyses, 5 of 12 evaluable patients had stabilization or reduction in tumor size. These consisted of one partial response, two minor responses and two cases of stable disease, all occurring in patients who had progressive disease before treatment. In summary, ICOVIR-7 treatment is apparently safe, resulting in anticancer activity, and is therefore promising for further clinical testing. Clin Cancer Res; 16(11); 3035–43. ©2010 AACR.

Targeted cancer immunotherapy with oncolytic adenovirus coding for a fully human monoclonal antibody specific for CTLA-4

Promising clinical results have been achieved with monoclonal antibodies (mAbs) such as ipilimumab and tremelimumab that block cytotoxic T lymphocyte-associated antigen-4 (CTLA-4, CD152). However, systemic administration of these agents also has the potential for severe immune-related adverse events. Thus, local production might allow higher concentrations at the target while reducing systemic side effects. We generated a transductionally and transcriptionally targeted oncolytic adenovirus Ad5/3-Δ24aCTLA4 expressing complete human mAb specific for CTLA-4 and tested it in vitro, in vivo and in peripheral blood mononuclear cells (PBMCs) of normal donors and patients with advanced solid tumors. mAb expression was confirmed by western blotting and immunohistochemistry. Biological functionality was determined in a T-cell line and in PBMCs from cancer patients. T cells of patients, but not those of healthy donors, were activated by an anti-CTLA4mAb produced by Ad5/3-Δ24aCTLA4. In addition to immunological effects, a direct anti-CTLA-4-mediated pro-apoptotic effect was observed in vitro and in vivo. Local production resulted in 43-fold higher (P<0.05) tumor versus plasma anti-CTLA4mAb concentration. Plasma levels in mice remained below what has been reported safe in humans. Replication-competent Ad5/3-Δ24aCTLA4 resulted in 81-fold higher (P<0.05) tumor mAb levels as compared with a replication-deficient control. This is the first report of an oncolytic adenovirus producing a full-length human mAb. High mAb concentrations were seen at tumors with lower systemic levels. Stimulation of T cells of cancer patients by Ad5/3-Δ24aCTLA4 suggests feasibility of testing the approach in clinical trials.

Prolonged systemic circulation of chimeric oncolytic adenovirus Ad5/3-Cox2L-D24 in patients with metastatic and refractory solid tumors

Eighteen patients with refractory and progressive solid tumors were treated with a single round of triple modified oncolytic adenovirus (Ad5/3-Cox2L-D24). Ad5/3-Cox2L-D24 is the first non-Coxsackie-adenovirus receptor-binding oncolytic adenovirus used in humans. Grades 1–2 flu-like symptoms, fever, and fatigue were seen in most patients, whereas transaminitis or thrombocytopenia were seen in some. Non-hematological grades 3–5 side effects were seen in one patient with grade 3 ileus. Treatment resulted in high neutralizing antibody titers within 3 weeks. Virus appeared in serum 2–4 days after treatment in 83% of patients and persisted for up to 5 weeks. One out of five radiologically evaluable patients had partial response (PR), one had minor response (MR), and three had progressive disease (PD). Two patients scored as PD had a decrease in tumor density. Tumor reductions not measurable with Response Evaluation Criteria In Solid Tumors (RECIST) were seen in a further four patients. PR, MR, stable disease, and PD were seen in 12, 23.5, 35, and 29.5% of tumor markers analyzed, respectively (N=17). Ad5/3-Cox2L-D24 appears safe for treatment of cancer in humans and extended virus circulation results from a single treatment. Objective evidence of anti-tumor activity was seen in 11/18 (61%) of patients. Clinical trials are needed to extend these findings.

Integrin targeted oncolytic adenoviruses Ad5‐D24‐RGD and Ad5‐RGD‐D24‐GMCSF for treatment of patients with advanced chemotherapy refractory solid tumors

The safety of oncolytic viruses for treatment of cancer has been shown in clinical trials while antitumor efficacy has often remained modest. As expression of the coxsackie‐adenovirus receptor may be variable in advanced tumors, we developed Ad5‐D24‐RGD, a p16/Rb pathway selective oncolytic adenovirus featuring RGD‐4C modification of the fiber. This allows viral entry through alpha‐v‐beta integrins frequently highly expressed in advanced tumors. Advanced tumors are often immunosuppressive which results in lack of tumor eradication despite abnormal epitopes being present. Granulocyte‐macrophage colony stimulating factor (GMCSF) is a potent activator of immune system with established antitumor properties. To stimulate antitumor immunity and break tumor associated immunotolerance, we constructed Ad5‐RGD‐D24‐GMCSF, featuring GMCSF controlled by the adenoviral E3 promoter. Preliminary safety of Ad5‐D24‐RGD and Ad5‐RGD‐D24‐GMCSF for treatment of human cancer was established. Treatments with Ad5‐D24‐RGD (N = 9) and Ad5‐RGD‐D24‐GMCSF (N = 7) were well tolerated. Typical side effects were grade 1‐2 fatigue, fever and injection site pain. 77% (10/13) of evaluable patients showed virus in circulation for at least 2 weeks. In 3 out of 6 evaluable patients, disease previously progressing stabilized after a single treatment with Ad5‐RGD‐D24‐GMCSF. In addition, 2/3 patients had stabilization or reduction in tumor marker levels. All patients treated with Ad5‐D24‐RGD showed disease progression in radiological analysis, although 3/6 had temporary reduction or stabilization of marker levels. Induction of tumor and adenovirus specific immunity was demonstrated with ELISPOT in Ad5‐RGD‐D24‐GMCSF treated patients. RGD modified oncolytic adenoviruses with or without GMCSF seem safe for further clinical development.

Oncolytic vaccinia virus for the treatment of cancer

Introduction: Gene therapy offers promising approaches for the development of anticancer agents with new modes of action. Among gene therapy vectors, vaccinia virus has emerged as an attractive agent especially when used as an oncolytic virus. Areas covered: This review describes the use of vaccinia virus in cancer therapy as a gene therapy vector, as an oncolytic virus and in the generation of oncolysates. The main achievements of each field are summarized with a special emphasis on vaccinia as an oncolytic vector and its combination therapies. The virus that has advanced furthest in clinical trials, GM-CSF expressing JX-594, is described in detail and its preclinical and clinical data are reviewed. Expert opinion: Vaccinia virus has great potential in cancer gene therapy, especially when used as an oncolytic virus. In particular, JX-594 has shown promising preclinical and clinical data, and a multi-continental randomized Phase III trial in hepatocellular carcinoma is expected to start soon.

Treatment of metastatic renal cancer with capsid-modified oncolytic adenoviruses

Renal cancer is a common and deadly disease that lacks curative treatments when metastatic. Here, we have used oncolytic adenoviruses, a promising developmental approach whose safety has recently been validated in clinical trials. Although preliminary clinical efficacy data exist for selected tumor types, potency has generally been less than impressive. One important reason may be that expression of the primary receptor, coxsackie-adenovirus receptor, is often low on many or most advanced tumors, although not evaluated in detail with renal cancer. Here, we tested if fluorescence-assisted cell sorting could be used to predict efficacy of a panel of infectivity-enhanced capsid-modified marker gene expressing adenoviruses in renal cancer cell lines, clinical specimens, and subcutaneous and orthotopic murine models of peritoneally metastatic renal cell cancer. The respective selectively oncolytic adenoviruses were tested for killing of tumor cells in these models, and biodistribution after locoregional delivery was evaluated. In vivo replication was analyzed with noninvasive imaging. Ad5/3-Δ24, Ad5-Δ24RGD, and Ad5.pK7-Δ24 significantly increased survival of mice compared with mock or wild-type virus and 50% of Ad5/3-Δ24 treated mice were alive at 320 days. Because renal tumors are often highly vascularized, we investigated if results could be further improved by adding bevacizumab, a humanized antivascular endothelial growth factor antibody. The combination was well tolerated but did not improve survival, suggesting that the agents may be best used in sequence instead of together. These results set the stage for clinical testing of oncolytic adenoviruses for treatment of metastatic renal cancer currently lacking other treatment options. [Mol Cancer Ther 2007;6(10):2728–36]