Development of Preclinical Models for Immunogene Therapy of Brain Cancer: It's Not Monkey Business!
Authors: Maciej S Lesniak
Journal: Molecular Therapy (2014)

The choice of an appropriate animal model for toxicology studies is one of the most important decisions in the US Food and Drug Administration (FDA)–directed investigational new drug process for gene therapies. A model should, as closely as possible, recapitulate the complexity of the human body, so that preclinical toxicity and safety can be established before any human clinical trials. In a study appearing in Molecular Therapy—Methods & Clinical Development, VanDerVeen et al. used marmoset monkeys to assess the safety of injection into the brain of an adenoviral vector expressing both herpes simplex type 1 thymidine kinase (TK) and an immune-stimulatory gene, the cytokine fms-like tyrosine kinase ligand 3 (Flt3L), under the control of a doxycycline (DOX)-inducible promoter.1VanDerVeen N Paran C Appelhans A Krasinkiewicz J Lemons R Appelman H et al.Marmosets as a preclinical model for testing “off-label” use of doxycycline to turn on Flt3L expression from high-capacity adenovirus vectors.Mol Ther Method Clin Dev. 2005; (in press)Google Scholar This is the first study to examine off-label use of DOX for immunogene therapy of malignant brain cancer, and it establishes a favorable safety profile that supports translation to a phase I clinical trial. To date, the treatment of malignant brain cancer, in particular glioblastoma multiforme (GBM), has met with limited success. The current standard of care, which includes surgical resection followed by temozolomide-based chemotherapy with radiation therapy, achieves a maximum survival rate of 26.5% at two years.2Stupp R Mason WP van den Bent MJ Weller M Fisher B Taphoorn MJ et al.Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma.N Engl J Med. 2005; 352: 987-996Crossref PubMed Scopus (14644) Google Scholar In light of this poor prognosis, much effort is being devoted to the development of innovative biological therapies. Among these, gene therapy—particularly immunogene therapy—is appealing for several reasons. First, GBMs rarely, if ever, metastasize outside of the central nervous system (CNS). Lack of systemic metastasis therefore allows one to focus on a single organ. Second, the presence of the blood–brain barrier protects the brain, as well as brain tumors, from a vast majority of systemically delivered chemotherapy drugs. Hence, local delivery of therapeutically targeted agents to the CNS has been an area of active research for many years. Third, the role of the immune system in the CNS, which historically has been considered immune-privileged but is more aptly described as an organ of finely regulated immune response, poses particular opportunities as well as challenges for immunogene therapies. Taken together, these characteristics lend themselves to the development of targeted and localized biological therapies for GBM that can effectively control tumor growth and spread. Several gene therapy protocols have been tested in brain cancer, and a full description of each has been summarized in several published reviews.3Tobias A Ahmed A Moon KS Lesniak MS The art of gene therapy for glioma: a review of the challenging road to the bedside.J Neurol Neurosurg Psychiatry. 2013; 84: 213-222Crossref PubMed Scopus (82) Google Scholar,4Mohyeldin A Chiocca EA Gene and viral therapy for glioblastoma: a review of clinical trials and future directions.Cancer J. 2012; 18: 82-88Crossref PubMed Scopus (36) Google Scholar,5Wollmann G Ozduman K van den Pol AN Oncolytic virus therapy for glioblastoma multiforme: concepts and candidates.Cancer J. 2012; 18: 69-81Crossref PubMed Scopus (133) Google Scholar Regardless of the vector (e.g., herpesvirus, adenovirus, reovirus, measles virus) or vector backbone (replication deficient or replication competent), clinical efficacy has been encouraging but limited. Thankfully, there have been no serious or lethal adverse consequences in any of the published studies, and a relative safety profile has been established for moving gene therapy for GBM forward. Researchers are now focusing on developing viral vectors that are more effective at infecting target cells, capable of enhanced replication in the case of oncolytic viruses, and better “armed” with genes that can modulate a variety of anticancer pathways. Indeed, Dr. Castro's laboratory6Muhammad AK Puntel M Candolfi M Salem A Yagiz K Farrokhi C et al.Study of the efficacy, biodistribution, and safety profile of therapeutic gutless adenovirus vectors as a prelude to a phase I clinical trial for glioblastoma.Clin Pharmacol Ther. 2010; 88: 204-213Crossref PubMed Scopus (31) Google Scholar has been developing a high-capacity adenoviral vector that constitutively expresses TK, with Flt3L under the control of a DOX-inducible promoter. In this setting the vector constitutively expresses TK to selectively kill proliferating brain cancer cells upon addition of ganciclovir or valacyclovir. The immune-stimulatory component, consisting of DOX-inducible Flt3L, mediates recruitment of dendritic cells—the most potent antigen-presenting cells—to the brain tumor microenvironment, where tumor antigens trigger specific anti-GBM immunity. This approach has been championed by the authors for the last several years, has been shown to have a favorable safety profile,6Muhammad AK Puntel M Candolfi M Salem A Yagiz K Farrokhi C et al.Study of the efficacy, biodistribution, and safety profile of therapeutic gutless adenovirus vectors as a prelude to a phase I clinical trial for glioblastoma.Clin Pharmacol Ther. 2010; 88: 204-213Crossref PubMed Scopus (31) Google Scholar and has led to impressive long-term survival in rodent models of GBM.7King GD Muhammad AK Larocque D Kelson KR Xiong W Liu C et al.Combined Flt3L/TK gene therapy induces immunological surveillance which mediates an immune response against a surrogate brain tumor neoantigen.Mol Ther. 2011; 19: 1793-1801Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar,8Puntel M Muhammad AK Candolfi M Salem A Yagiz K Farrokhi C et al.A novel bicistronic high-capacity gutless adenovirus vector that drives constitutive expression of herpes simplex virus type 1 thymidine kinase and Tet-inducible expression of Flt3L for glioma therapeutics.J Virol. 2010; 84: 6007-6017Crossref PubMed Scopus (32) Google Scholar,9King GD Muhammad AK Curtin JF Barcia C Puntel M Liu C et al.Flt3L and TK gene therapy eradicates multifocal glioma in a syngeneic glioblastoma model.Neuro Oncol. 2008; 10: 19-31Crossref PubMed Scopus (52) Google Scholar Despite impressive efficacy and safety data, rodent models do not always predict outcomes in humans. To address this concern, the authors have now examined the safety and biodistribution of their virus in a nonhuman primate, the marmoset monkey. As pointed out by the authors, marmosets are small, easy to handle, inbred, and can be used to model the human immune system and its response to human cytokines, an important parameter given the expression of Flt3L in the context of the proposed human clinical trial. Following injection of the virus into striatum, the authors show that astrocytes, microglia, neurons, and oligodendrocytes can be transduced with the adenoviral vector and express Flt3L following DOX administration. Fortunately, neither the transduction of these cells nor the expression of the gene resulted in gross tissue abnormalities. Activated astrocytes and oligodendrocytes were found along the injection site, as were microglia, macrophages, and CD3+, CD8+ cells. No systemic toxicities were shown, with only a rise in anti-adenovirus neutralizing antibodies a month following injection in a group treated with high-dose DOX. In summary, the study showed that clinically relevant doses of DOX activate Flt3L gene expression from the adenoviral vector and that there is no toxicity associated with the expression of the virus or the transgene in the monkey brain. Such results go a long way to characterize the safety profile of any gene therapy vector before its injection into the CNS. If there is a limitation to this work, it is that it does not incorporate a brain tumor within the model. Ideally, all FDA-directed safety and toxicity studies should be performed in a tumor-bearing model that closely resembles the human disease. This is by no means an absolute requirement, and many gene therapy protocols have in fact been approved without the need for primate studies or safety/toxicity studies involving tumor-bearing animals. One must consider, however, that the tumor and tumor microenvironment play an important role in any biological process. For example, it is a well-known fact that brain tumors are highly immunosuppressive, and at least one component responsible for this finding consists of regulatory T cells (Tregs). Although Flt3L acts on dendritic cells, it has been shown to facilitate the formation of Tregs in some clinical models.10Svensson MN Andersson SE Erlandsson MC Jonsson IM Ekwall AK Andersson KM et al.Fms-like tyrosine kinase 3 ligand controls formation of regulatory T cells in autoimmune arthritis.PLoS One. 2013; 8: e54884Crossref PubMed Scopus (19) Google Scholar,11Klein O Ebert LM Zanker D Woods K Tan BS Fucikova J et al.Flt3 ligand expands CD4+FoxP3+ regulatory T cells in human subjects.Eur J Immunol. 2013; 43: 533-539Crossref PubMed Scopus (43) Google Scholar One would not expect that such potential and additional expansion of Tregs would cause any toxicity, but it may limit the effectiveness of the proposed therapy. Ultimately, we will have to await the results of an upcoming human clinical trial, as no brain tumor model exists in monkeys that could help to answer this question. Does the current study pave the way for more safety and toxicity studies of gene therapy vectors using nonhuman primates? Given the current lack of precedence from the FDA for all such studies to be conducted in monkeys, the answer is most likely “No.” However, it is always wise and advisable to accumulate as many safety and efficacy data as possible. Although I suspect that most physicians would not have a problem with off-label use of DOX at the proposed doses to carry out the clinical trial, the current data are reassuring, and even the skeptics will find it easier to sleep at night. The true value of this work, in my opinion, is the lack of a robust inflammatory component following Flt3L expression, which clinically could manifest as cerebral edema, and in worst circumstances, death. None of the animals developed such clinical manifestations, and the inflammatory infiltrate was localized only to the injection site. There is tremendous value in establishing this safety profile in an immune-competent and clinically relevant model. Gene therapy—in particular, immunogene therapy—is clearly coming of age. Lessons learned from the past have guided the development of improved vectors and the delivery of more therapeutically relevant agents. When it comes to brain cancer, both are welcome, as we have a long way to go before we can make a significant difference in this deadly disease. We can only hope that Ad-Flt3L will help patients in an upcoming clinical trial; for now, we can rest more assured that it is unlikely to cause more harm than good.