Abstract: Cancer treatment has been marred by the fact that most drugs target cancer cells as well as normal cells. Gene therapy is one of a handful of methods that will make cancer cells "stand out," allowing drugs or the host's immune system to selectively target cancer cells.
The destructive capacity of the immune system is well demonstrated in autoimmune disorders such as arthritis and in the rejection of transplanted organs. Cancerous tumor cells have cell surface structures (tumor associated antigens), which should enable recognition and rejection of tumor tissue by the immune system. It is likely that many, if not most, tumors are rejected before they are even noticed. However, malignant cancers have developed ways to evade the immune response as part of the selective process during cancer growth.
Cancer cells are able to escape immune detection and/or rejection by a variety of measures. Cell surface molecules, which are required for the effective policing of tissues by the immune system, are often modified, reduced or eliminated. In addition cancer cells secrete soluble molecules that inhibit the patients’ ability to develop an immune response. The ability of the immune system to recognize and reject cancerous growths has been demonstrated in a series of experimental model systems. Efforts are now being made to use this knowledge for the treatment of cancer.
Described below are two different gene-based approaches to stimulate the rejection of an established cancer in patients. The first involves procedures which modify the tumor itself, render it a more attractive target to the immune system, and allow immune cells to penetrate the tumor and kill the cancerous cells. The second approach requires a very powerful vaccine to stimulate a strong immune response against the tumor associated antigens in patients with an established cancer.
Early efforts to harness the power of the immune system to eliminate cancer were made by Dr. William Coley very early in the 20th century (Coley, 1906). Dr. Coley injected cancerous tissue, usually sarcomas (tumors of the supportive tissues such as bone, cartilage fat or muscle), with a mix of bacteria and/or their toxins. This would result in an inflammatory response in the tumor and the influx of many immune cells. He went on to publish, in detail, the successes of his treatments. Tumor rejection was always accompanied by a very high fever. Despite his successes, his treatment concept was soon overshadowed by the development of radiation, and later, chemotherapy.
It wasn’t until the 1970’s when immunologists began unravelling the complexities of the immune system and hoped to use this knowledge to treat cancer, that the remarkable successes of William Coley’s treatments were appreciated. It has been shown that the injection of toxic biological substances, such as the bacteria in Coley’s treatment, results in the generation of a cascade of soluble protein molecules, known as cytokines, which stimulate and control immune responses, similar to the ways in which hormones control the endocrine system. Cytokines have been shown to control an astonishingly complex system of overlapping and interwoven biological functions.
Some of the first cytokines to be discovered are now used clinically. The clinical applications of interleukin-2 (IL-2) (Rosenberg et al., 1987) and interferon alpha (IFNα) (Vogelzang et al., 1993) have shown two important features:
2. Therapeutic doses of these two cytokines, which are currently injected systemically meaning that they circulate through the entire body of the patient, are very toxic. This toxicity limits the dose of cytokine that can be used.
Injection of the cytokine protein directly into the tumor results in its rapid elimination from the tumor and the spread of the cytokine into the circulation.
More recently, gene therapy experiments have shown that the gene that encodes a particular cytokine (or combinations of cytokines) can be inserted into tumor cells such that these cells now become miniature cytokine factories. Cytokines do diffuse out of the tumor, but always with a gradient favoring a higher concentration in the tumor. The goal is to maintain a high, therapeutic concentration of the cytokine in the tumor, which then results in the stimulation of an immune response to tumor associated antigens, so that not only is the injected tumor eventually eliminated but a tumor-specific immune response is also generated. The tumor-specific immune cells then circulate throughout the patient’s body and eliminate any metastatic cancer cells that have spread to other tissues. Diffusion of the cytokine out of the tumor, though causing toxicity, is also an important feature, since cytokine-based stimulation and regulation of the patient’s immune system play an important role in the control of cancer. These important immune activities have been demonstrated in various animal models.
In these days of very sophisticated surgery and chemotherapy approaches to the treatment of cancer, one could ask why we should treat a tumor by leaving it in place and injecting it with gene therapy agents. While it is true that some solid tumors are best removed as soon as possible, other tumors, such as lung cancer, very often cannot be removed due to their location. Treatment with chemotherapy is often effective at reducing tumor burden, but many cancers soon develop resistance to chemotherapeutic chemicals. In addition, once a cancer has spread by the process of metastasis, it is often impossible to remove all metastatic tumors that have also become resistant to chemotherapy. In these cases, immune-mediated destruction of cancer cells may represent the best hope.
The method used to transfer genes into tumor tissue is not trivial. Some groups have attempted to transfer genes into cancer cells by injecting DNA or lipid DNA complexes. This results in some gene expression in the tumor, but these “synthetic vectors” still cannot compete with the efficiency of Nature’s own method of transferring genes into a cell, notably, viruses. Enough is now known about the biology, functions and risks associated with various viruses to enable their use as gene transfer vectors. The viruses used for gene transfer are first crippled by removing the genes that are necessary for viral self replication and the removal of any genes which might be associated with risks such as tumor formation. Genes encoding the cytokine are inserted into the genome of the virus, under the control of a very powerful gene “promoter” such that a cell infected with this virus will not produce any progeny virus, but will produce the recombinant cytokine molecule for the life of that cell (Figure 1).
Transgene’s Cancer Gene Therapy Programs
The biotechnology company Transgene has undertaken a series of cancer immunotherapy clinical trials using crippled adenovirus vectors in which the genes encoding either interleukin-2 or interferon-gamma (IFNγ) have been inserted (Liu et al., 2004). These viruses are first produced on an industrial scale, purified and tested for any toxicity. Only after regulatory agency approval (the U.S. FDA) for clinical trials has been obtained, can clinical studies to determine the effectiveness of the treatment be undertaken. Transgene currently has both Adeno-IL2 and Adeno-IFNγ in clinical trials. Adeno-IL2 is being tested as a treatment for melanoma and Adeno-IFNγ for the treatment of cutaneous lymphomas (Dummer et al., 2004).
The second approach to the immunotherapy of cancer is to take advantage of our knowledge of the tumor associated antigens and to construct a vaccine in order to generate an immune response to the cancer cells. In this case, the vaccination occurs at a healthy part of the body, less likely to be subject to the effective immuno-suppression exerted by cancer cells within the tumor. Vaccination can be intramuscular (into the muscle), subcutaneous (under the skin) or intradermic (into the skin). Tumor-specific immune cells are stimulated at the site of vaccination and then travel throughout the patients’ body in search of cancer cells.
Transgene currently has two programs in cancer vaccine immunotherapy. The first involves tumor associated antigens which are, in fact, viral proteins. Some cancers are the result of cancer-causing viruses. In these cases, in order for the cancer cell to be able to continue dividing it must maintain the viral genes that caused the cancer. Even when other genes of the virus have been eliminated by the growing cancer cells, some viral genes are maintained to continue driving the cancer cell division. These “viral oncogenes” code for proteins. As such these viral proteins should be able to serve as targets for immune attack on the cancer cells.
One such cancer-associated virus is the human papilloma virus (HPV), which infects, and can cause cancer in, the cervical tissue of the uterus. Most often, the maintained viral proteins are derived from the HPV E6 and E7 genes. Transgene scientists have put slightly modified HPV E6 and E7 genes (modified to ensure that they are no longer causing cancer) into vaccinia virus. The vaccinia virus has been modified to render it even safer than the vaccinia virus that was used to vaccinate millions of people during the smallpox vaccination campaign, some thirty years ago. This modified vaccinia Ankara (MVA) virus is unable to replicate in most mammalian cells but maintains the characteristics of a powerful vaccine. The genes for E6 and E7 from the most common HPV strain, HPV-16, have been inserted into the genome of MVA. The gene encoding IL2 is also inserted in the MVA to provide an additional stimulus to the patients’ immune system. MVA-HPV-16 E6/E7-IL2 has been in clinical trials in women with cervical cancer and in women with pre-malignant cervical “neoplasia” (CIN II/III). Women, with CIN do not have cancer, but the cervical cells are infected with HPV and, therefore, these women can be at risk for the development of cervical cancer. The hope is to reduce the requirement for surgical intervention in these patients.
The second immunotherapeutic cancer vaccine under development by Transgene uses a tumor-associated antigen called MUC1. In contrast to the tumor-associated antigens vaccine described above, MUC1 is not a viral antigen, but a large glycoprotein molecule that can be found on some normal cells. However, in cancer, MUC1 is produced in large quantities and is modified (the protein is no longer hidden by complex sugar structures). Over-expression of modified MUC1 is associated with about one half of all cancer cases. These properties make MUC1 an interesting target for cancer vaccine immunotherapy. Transgene scientists have inserted the gene encoding MUC1 into the MVA vector, as described above. Again, the IL2 gene is also inserted into the same virus as an additional immunostimulant. The resulting vaccine (MVA-MUC1-IL2) is being tested in clinical trials in various cancers (Rochlitz et al., 2003).
The following points have been described in this article.
Clinical studies in the very early 1900s indicated that the immune system could be recruited and used to combat cancer.
Since the 1970s there has been an enormous amount of progress made in the understanding of how the immune system works.
Both cytokines, protein messengers which control the immune system, and tumor-associated antigens can be used to stimulate the immune rejection of tumors.
Recombinant adenoviruses have been produced which incorporate either interleukin-2 or interferon gamma into the viral DNA. The viruses cannot replicate so that they produce no progeny virus upon infection.
These recombinant adenoviruses are being tested clinically in cutaneous lymphoma and in melanoma.
Therapeutic cancer vaccines have also been produced and tested clinically. The replication-deficient Modified Vaccinia Ankara (MVA) has been used to carry the genes for HPV tumor-associated antigens plus IL2 or the MUC1 tumor-associated antigen plus IL2.
Immunotherapy with recombinant viruses is currently being evaluated for clinical effectiveness and could soon be part of the arsenal of treatments available to doctors to fight cancer.
References and Further Readings
Rosenberg SA, Lotze MT, Muul LM, Chang AE, Avis FP, Leitman S, Linehan WM, Robertson CN, Lee RE, Rubin JT, et al. A progress report on the treatment of 157 patients with advanced cancer using lymphokine-activated killer cells and interleukin-2 or high-dose interleukin-2 alone. New England Journal of Medicine 316(15):889-897, 1987.
Vogelzang NJ, Lipton A, Figlin RA. Subcutaneous interleukin-2 plus interferon alfa-2a in metastatic renal cancer: an outpatient multicenter trial. Journal of Clinical Oncology 11:1809-1816, 1993.
Liu M, Acres B, Balloul JM, Bizouarne N, Paul S, Slos P, Squiban P. Gene-based vaccines and immunotherapeutics. Proceedings of the National Academy of Sciences USA 101(2):14567-14571, 2004.
Dummer R, Hassel JC, Maier T, Slos P, Eichmüller S, Acres B, Bataille V, Squiban P, Burg G, Urosevic M. Adenovirus-mediated intralesional interferon-g gene transfer induces tumor regressions in cutaneous lymphomas. Blood 104(6):1631-1638, 2004.
Rochlitz C, Figlin R, Squiban P, Salzberg M, Pless M, Herrmann R, Tartour E, Zhao Y, Bizouarne N, Baudin B, Acres B. Phase I immunotherapy with a modified vaccinia virus (MVA) expressing human MUC1 as antigen-specific immunotherapy in patients with MUC1-positive advanced cancer. Journal of Gene Medicine 5:690-699, 2003.
[Discovery Medicine, 5(25):25-29, 2005]