“Man's best friend”: a new model system for cancer therapeutics?
Authors: John G Hay
Journal: Molecular Therapy (2003)

The use of chemotherapeutic agents has not led to high cure rates in the treatment of most solid tumors, lung cancer and pancreatic cancer being good examples. One major obstacle is the inability to deliver adequate levels of drug to the tumor without giving rise to serious toxic side effects in other organs. Conditionally replicating viruses are therapeutic agents that replicate specifically within the target tumor. Two main strategies are being followed to achieve tumor specific replication of adenoviral gene therapy vectors. The first relies on the use of tumor-specific promoter elements that can be inserted into the virus to limit the expression of viral genes controlling replication to tumor cells. The second strategy limits viral replication to dividing cells, by mutation or deletion of viral genes that encode proteins that interact with cell-cycle regulating proteins. Examples include deletion of the E1b-55kD gene that normally binds to p53, and modification of the E1a gene that interacts with Rb (see [1Curiel D.T. The development of conditionally replicative adenoviruses for cancer therapy.Clin. Cancer Res. 2000; 6: 3395-3399PubMed Google Scholar] for review). Animal models are essential tools to evaluate and approve new cancer therapies. However, the inability of the human adenovirus to replicate in nonhuman cells limits the utility of animal models in the study of replicating human adenoviruses. Murine cells are infected by the human adenovirus, viral proteins are synthesized, and viral DNA replicates, but the production of new infectious virus particles is severely restricted [2Eggerding F.A. Pierce W.C. Molecular biology of adenovirus type 2 semipermissive infections. I. Viral growth and expression of viral replicative functions during restricted adenovirus infection.Virology. 1986; 148: 97-113Crossref PubMed Scopus (34) Google Scholar]. This leads to difficulties in interpreting data from the two main model systems of experimental cancer therapy, the murine syngeneic tumor model and the nude mouse human tumor xenograft model. Tumors are composed not only of cancer cells, but also of supporting stromal cells and vascular cells [3Hanahan D. Weinberg R.A. The hallmarks of cancer.Cell. 2000; 100: 57-70Abstract Full Text Full Text PDF PubMed Scopus (22627) Google Scholar]. In the syngeneic model, the human adenovirus will not replicate in the tumor or supporting cells—a severe limitation. In human tumor xenografts established in nude mice, although the tumor cells are of human origin and support viral replication, the supporting mouse cells will not support viral replication. This is likely to alter the dynamics of viral spread throughout the tumor. In addition, the impact of immune responses directed against both the virus and against virus-infected tumor cells cannot be studied in an immune-incompetent nude mouse. This limits the evaluation of both the safety and efficacy of viral vectors in the nude mouse xenograft model. In this issue of Molecular Therapy, Hemminki and colleagues attempt to overcome some of these difficulties by developing conditionally replicating canine adenoviral vectors. The osteocalcin promoter was used to control the expression of the canine adenovirus E1a gene, restricting the replication of the canine adenovirus to dog osteosarcoma cells. This virus kills canine osteosarcoma cells in vitro, and a therapeutic effect in a nude mouse canine osteosarcoma cell xenograft model is reported. By establishing this vector system, it is the authors’ supposition that the spontaneously occurring canine osteosarcoma tumor now becomes a highly relevant model to study the importance of immune mechanisms when using replicating adenovirus for gene therapy. This tumor, which is frequently seen in dogs, has many similarities with its human counterpart, and similar treatment options are available. This is a novel strategy with substantial translational potential that overcomes many of the limitations just described, although several questions still remain. The Adenoviridae family of viruses is composed of two main genera, the Mastadenovirus, derived from animal hosts, and the Aviadenovirus, derived from avian hosts. The Mastadenovirus genus includes members with human, murine, simian, canine, and several other animal host origins. In attempting to translate findings from the canine model to the human, it must first be determined if the canine virus is in fact similar to the human adenovirus. Despite close homology of the human, murine, and canine adenoviral genomes, considerable variability in biology does occur. For instance, even within human adenoviruses, minor genomic changes are associated with major changes in viral behavior. Ad2 and Ad5 serotypes, the most frequently used serotypes for gene therapy applications, are not tumorgenic in animals. In contrast, the Ad12 serotype potently induces tumors in animals [4Sawada Y. Fohring B. Shenk T.E. Raska Jr., K. Tumorigenicity of adenovirus-transformed cells region E1A of adenovirus 12 confers resistance to natural killer cells.Virology. 1985; 147: 413-421Crossref PubMed Scopus (59) Google Scholar]. Expression of the E1a gene is of critical importance in the control of replication of the human virus and for the transactivation of other viral genes. In contrast, the murine adenovirus E1a gene is not essential for viral replication in murine fibroblasts [5Ying B. Smith K. Spindler K.R. Mouse adenovirus type 1 early region 1A is dispensable for growth in cultured fibroblasts.J. Virol. 1998; 72: 6325-6331PubMed Google Scholar]. This fact would create major problems in the evaluation of transcriptional targeting approaches in the mouse. Ad2 and Ad5 serotypes (but not all human serotypes) infect cells by two interactions, an interaction between the viral fiber protein and the coxsackievirus-ad receptor (CAR), and an interaction between RGD sequences within the viral penton protein and integrins on the cell surface [6Wickham T.J. Mathias P. Cheresh D.A. Nemerow G.R. Integrins alpha v beta 3 and alpha v beta 5 promote adenovirus internalization but not virus attachment.Cell. 1993; 73: 309-319Abstract Full Text PDF PubMed Scopus (1959) Google Scholar]. The canine adenoviruses also interact with CAR, and a putative CAR sequence has also been isolated [7Fechner H.A. et al.Expression of coxsackie adenovirus receptor and alphav-integrin does not correlate with adenovector targeting in vivo indicating anatomical vector barriers.Gene Ther. 1999; 6: 1520-1535Crossref PubMed Scopus (270) Google Scholar]. However, canine viruses, like some human serotypes, lack the RGD sequences in the penton proteins that mediate an interaction with cell surface integrins. Nevertheless, the canine viruses have many similarities to human serotypes [8Soudais C.S. et al.Canine adenovirus type 2 attachment and internalization coxsackievirus-adenovirus receptor, alternative receptors, and an RGD-independent pathway.J. Virol. 2000; 74: 10639-10649Crossref PubMed Scopus (99) Google Scholar] and appear worthy of further study. The next question is how similar are the immune responses induced against the adenovirus in the dog model compared to those in the human. Immunity may play three important roles in gene therapy. First, pre-existing immunity may affect the ability of the virus to infect the target cells. Second, immunity may have an effect on subsequent elimination of tumor and virus-infected tumor cells. Finally, immunity may play a role in viral toxicity. Most humans have been repeatedly exposed to adenovirus, have antibodies to various human adenoviral serotypes, and about half of the time possess neutralizing antibodies that may modify the efficacy of gene/viral delivery [9Chirmule N.K. et al.Immune responses to adenovirus and adeno-associated virus in humans.Gene Ther. 1999; 6: 1574-1583Crossref PubMed Scopus (618) Google Scholar]. Following the first exposure to an adenoviral vector, the development or an increase in neutralizing antibody titer is also seen [10Juillard V. et al.Long-term humoral and cellular immunity induced by a single immunization with replication-defective adenovirus recombinant vector.Eur. J. Immunol. 1995; 25: 3467-3473Crossref PubMed Scopus (140) Google Scholar]. However, neutralizing antibodies play only a partial role in viral elimination, and predominantly after intravenous but not intratumoral injection of virus [11Bramson J.L. Hitt M. Gauldie J. Graham F.L. Pre-existing immunity to adenovirus does not prevent tumor regression following intratumoral administration of a vector expressing IL-12 but inhibits virus dissemination.Gene Ther. 1997; 4: 1069-1076Crossref PubMed Scopus (110) Google Scholar]. Innate immune responses, including infiltration by macrophages and natural killer cells, are very important in the elimination of virus over the first few days following infection [12Worgall S. Wolff G. Falck-Pedersen E. Crystal R.G. Innate immune mechanisms dominate elimination of adenoviral vectors following in vivo administration.Hum. Gene Ther. 1997; 8: 37-44Crossref PubMed Scopus (431) Google Scholar]. Specific cytotoxic T-cell responses that could reduce tumor burden develop after a week or more. Dogs also frequently have immunity to adenovirus and are frequently immunized with CAdV-2 to induce some immunity against a more serious CAdV-1 infection. The canine model therefore mirrors many of the challenges seen in the human and will also allow study of the role that immunity may play on the therapeutic effect and toxicity of the virus in a way not possible at present with other model systems. The final question is how similar are the tumors that develop in other species to the tumors that occur in humans. The canine osteosarcoma appears to correlate quite well with the human compared to other species, in the terms of histology, available therapeutic options, animal size, and life expectancy. The work of Hemminki and colleagues is an important step towards evaluating conditionally replicating adenovirus in a model system that much more closely reflects human circumstances, and it should yield some interesting results with important translational potential for adenoviral-mediated gene therapy of human tumors.