Edward P Cohen, Edwin F de Zoeten, Morton Schatzman. American Scientist. Volume 87, Issue 4. Jul/Aug 1999).
In human society, the fear of foreigners is the source of many troubles. But within a living organism, a little xenophobia goes a long way. In fact an entire organ system—the immune system—is devoted to distinguishing “self” from “foreign” and eliminating the foreign. This distinction is the basis of vaccines that protect people from infectious viruses and bacteria: A vaccine introduces a harmless version of an infectious foreign agent to the body, familiarizing the immune system with the pathogen’s features. The immune system builds a defensive force ready to recognize and eliminate the pathogen, should the real thing ever show up.
But not all medical threats are external. In cancer, the body’s own cells gradually acquire the ability to grow out of control. As the disease progresses and tumor cells spread, or metastasize, from their origin to other organs, the danger grows. Known treatments are rarely effective against cancer that has metastasized. Radiation and chemotherapy kill cancer cells, but in most cases these highly toxic treatments fail to destroy all of them, leaving behind some nests of cancer too small to be detected. Other cancer cells develop resistance to chemotherapeutic drugs. Curing the cancer patient requires that every remaining cancer cell be killed. New forms of therapy that effectively eliminate cancer cells are urgently needed.
Fortunately, there is some hope that the power of the immune system can be marshaled with the use of vaccines to fight cancer. As it turns out, cancer cells are not entirely the same as the normal cells from which they derive—a distinction that suggests that vaccines against cancer may be effective. Cancer cells express on their surfaces a cluster of proteins, called tumor antigens, which are distinct from the proteins expressed by their counterpart normal cells. Strong evidence indicates that the immune system can recognize these tumor antigens as foreign and can mount a response. Unfortunately, this response is insufficient to destroy all of the tumor’s cells. Some escape destruction, and the cancer continues to grow. But the fact that the body initiates any immune response toward the cancer holds out a tantalizing therapeutic possibility: Cancer vaccines may help stimulate an immune response against the tumor in a patient with malignant disease.
Just as vaccines are used to introduce portions of viruses and bacteria to the immune system, they can also be used to present tumor antigens to the immune system. An anti-cancer therapy therefore may someday include a vaccine that contains the DNA encoding tumor antigens. Investigators are exploring the possibility that a vaccine derived from tumor DNA injected into the patient can augment the immune response to such a degree that it successfully eliminates most, if not all, cancerous cells. Of course, it is important to note that cancer vaccines differ from vaccines against pathogens in one important respect: Vaccines against pathogens seek to prevent disease before the person is exposed to a pathogen, whereas cancer vaccines cannot prevent cancer and would be used therapeutically after a person develops cancer.
If it works-and some recent animal studies suggest it might-vaccination with tumor DNA would produce an immune response directed only toward the cancer cells. Unlike radiation and chemotherapy, immunization with DNA for tumor antigens has not been found to be toxic.
Such an approach could have important advantages. The immune system could seek out and destroy malignant cells anywhere in the body. It could eliminate small nests of tumor, too small to be detected by the most sensitive diagnostic methods. It could kill tumor cells in the blood or lymph channels. In theory, the immune system could kill all the patient’s tumor cells, just as all influenza viruses are killed by the immune system as we recover from the flu.
This novel approach toward cancer therapy is in its earliest stages. It is the direct result of progress at the most fundamental level in scientists’ understanding of the cause of cancer and how it spreads in the body. Along with these discoveries has come greater knowledge of the nature of tumor antigens as well as the means for developing innovative and effective tumor vaccines. Someday, such vaccines may be an important adjunct to traditional chemotherapy and radiation in order to kill as many cancer cells as possible.
For generations, little was known about the cause of cancer. Many hormonal, environmental and dietary influences were known to be common in patients who developed malignant disease. Women who began menstruating at an earlier than average age, for example, and entered menopause later than most, had a higher risk of developing breast cancer. Cigarette smoking was found to be a cause of lung cancer and was often associated with bladder cancer. It was recognized that cancer of the head and neck was more common in patients who chewed tobacco and drank alcoholic beverages, and that excessive exposure to the sun resulted in melanoma and other types of skin cancer. How these and other factors led to cancer, what caused a normal cell to become transformed into a malignant cell, was unknown until relatively recently. The discovery of the causes of cancer will rank among the greatest achievements in biology. The results offer the promise of new types of highly effective treatments that attack the tumor cell at its most basic level.
Cancer results from damage to certain crucial genes in an individual cell of the body. Most often these genes code either for proteins that control cell division directly or for proteins that respond to molecules, called growth factors, that stimulate cell division. When these genes become damaged, the cell loses the controls on growth. Without precise controls, the cell begins to divide and becomes insensitive to the factors that, under normal circumstances, limit the extent of cell proliferation. The result is a malignant tumor, an accumulation of cancer cells that no longer function properly Many of the genes controlling cell proliferation have been identified, and the alterations responsible for their loss of function have been determined.
Once a normal cell that has become transformed into a malignant one divides, it yields daughter cells that inherit the damaged genes. The transformed cells continue to proliferate until a cluster of cells-the tumor-forms. The number of malignant cells increases progressively as the cancer develops, and all the while additional genetic changes take place. Genetic variants of the original tumor begin to appear. These and multiple other changes make it possible for the malignant cells to metastasize beyond the primary site and invade surrounding tissues. The invasive cells migrate into the blood and lymphatic vessels through which they spread to various organs throughout the body.
The genetic changes in different cells that make up the population of malignant cells take place randomly. Early in the 1990s, Richard Kolodner, who is now the director of the National Cancer Institute, found that genes within cancer cells are inherently unstable, giving rise to numerous genetic alterations. Genetic instability was found in the malignant cells of patients with colorectal cancer, bladder, lung, ovarian, prostate, endometrial and breast cancer. As a result, many genes in cancer cells differ from their counterparts in normal, nonmalignant cells from the same individual. The cells within the tumor differ genetically from each other as well, so there is a great deal of genetic diversity within the malignantcell population.
Most of the genetic changes that take place in cancer cells are “silent,” damaging genes that are not expressed in that cell type. But in some instances, the products of the randomly altered genes are expressed, and they are so different from their counterparts found in normal cells that, on the molecular level, the cell starts to look different from normal cells. It is viewed as “foreign” by the immune system, which responds by mounting an immune response. The malignant cells are killed by the immune system, just as the immune system destroys foreign bacteria or infectious viruses. The altered gene products are known as tumor antigens.
Discovery of the tumor antigens began with the classic studies of Richmond T. Prehn and Joan Main, tumor immunologists working at the National Cancer Institute in 1957. Prehn and Main induced a skin cancer in mice by “painting” the animal’s skin with the carcinogen methylcholanthrene. The mice developed a highly aggressive skin cancer that, if left untreated, killed them. The investigators discovered that they could transfer the skin-cancer cells to normal inbred mice of the same genetic type, or strain, as those in which the initial cancers had been induced and that the transferred cancer cells formed lethal tumors in the recipient mice. If, however, the animals were first immunized by an injection of killed cancer cells from the skin cancer, they did not form tumors. The immunized animals became immune to the tumor and rejected the cancer cells. This immunity was limited; it could be overcome if large numbers of cancer cells were injected.
Continuing their studies, the scientists found that animals that developed immunity to tumors after being immunized with cells from that tumor were nevertheless highly susceptible to the growth of cells from a tumor that arose independently in a different mouse. This was the case even if the two independently arising tumors looked the same under the microscope. From this observation, an important principle emerged. Each tumor has its own unique antigenic phenotype. This makes sense: Since tumor antigens result from random genetic changes, it would be highly unlikely that tumors arising independently in different animals would develop exactly the same set of tumor antigens.
This point was substantiated when genes specifying tumor antigens were cloned and sequenced by Thierry Boon and his colleagues at the Ludwig Institute for Cancer Research in Brussels. In 1991 Boon found that a population of mouse mastocytoma (breast cancer) cells included some that formed tumors when injected into other mice of the same strain, whereas other mastocytoma cells from the same tumor cell population were rejected. They reasoned that the difference between the cells that grew, which they called tum+ because they formed tumors, and those that were rejected, which they called tum—because they failed to grow, was the absence or presence of strong tumor antigens. Tum- cells expressed tumor antigens that were highly immunogenic; that is, they stimulated a vigorous immune response against the tumor. Tum+ cells, on the other hand, expressed weakly immunogenic tumor antigens and failed to stimulate an immune response to the tumor.
Analyzing many tum- cells, the researchers discovered considerable diversity among them. Despite the fact that all the cells stimulated an immune response, the specific tumor antigens they expressed were found to vary.
Boon made a culture from one tumcell and used it as a model. He transferred various portions of the tumDNA into tum+ cells, hoping to isolate a gene responsible for forming at least one tumor antigen. After searching thousands of colonies of cells that received the tumor DNA, Boon was able to find one that had been converted from tum+ to turn . Boon continued these experiments, transferring successively smaller and smaller portions of the tumor DNA until eventually he was able to isolate a gene for an individual tumor antigen. The gene specified a medium-sized protein that was homologous or highly similar to a protein found in normal cells, with a small mutation significant enough to convert it into a protein viewed as foreign by the immune system. When Boon transferred a mixture of tum- and tum+ cells to mice that were immunized with only tum cells, the animals developed a tumor, but the proliferating tumor consisted of cells that were all tum+. Thus the immune system had recognized and was able to kill the cells in the population that expressed the one tumor antigen that was strongly immunogenic. Cells that expressed “weak” antigens failed to be recognized by the immune system, escaped destruction and continued to proliferate.
Since those studies were done, a number of tumor antigens have been identified in both animal and human tumors. Joyce Taylor-Papadimitriou and her colleagues at the Imperial Cancer Research Fund Laboratories in London identified mucin as a tumor antigen associated with breast and ovarian cancer cells. Stephen Rosenberg and his associates at the National Institutes of Health found that an enzyme called tyrosinase, which is involved in pigment synthesis, is a tumor antigen associated with melanoma, a severe type of skin cancer.
Others, designated in various waysMelan-A/MART-1, MAGE-1, MAGE-3, gp1OO and GAGE-1-have been identified as tumor antigens associated with melanoma cells. P53 and HER-2/neu have also been identified as tumor antigens expressed by breast cancer cells. These are a few examples of the different tumor antigens expressed by different types of malignant cells. Weak and Strong
Boon’s discovery that a tumor can contain a mixture of strongly and weakly immunogenic cells has been borne out in studies of actual tumors. More often than not, cancer cells specify structures that are insufficiently foreign to provoke a response. The cells that express “weak” antigens fail to stimulate the immune system. Strongly antigenic cancer cells are killed off, leaving the cancer patient with a tumor populated with malignant cells that express “weak” tumor-associated antigens.
In principle, the fact that tumor antigens are expressed by cancer cells alone, and not by normal cells, makes it possible to develop a vaccine that can stimulate an anti-cancer immune response while sparing normal cells. But in order to do this, immunologists need to find some way to stimulate an immune response to otherwise weakly immunogenic tumor antigens.
In a few cancer patients, immunization with tumor cells mixed with adjuvants, substances (often of bacterial origin) that stimulate the immune system, generates an immune response to the cancer cells and results in the rejection of established tumors. Some patients treated in this way have survived longer than might be expected. In other cases, the injection of an adjuvant alone directly into a skin tumor leads to a severe inflammatory response at the site and regression of the cancer. However, the anti-tumor effects are local. Metastatic tumor cells in organs and at distant tissue sites continue to proliferate, and the tumors continue to grow. Moreover, for most types of cancer (a type of bladder cancer is a notable exception), adjuvants have not proved to be an effective treatment, and this form of cancer therapy has fallen into disuse. Nevertheless, the result is highly instructive. Since only the tumor cells are killed in patients treated with adjuvants, the result reaffirms the antigenic differences between normal and malignant cells of the same individual.
Tumor antigens can be identified and purified, so the next logical step would be to try to prepare vaccines containing specific tumor-antigen proteins. The purity of such a vaccine would diminish the likelihood of an untoward reaction, since the immune response should be directed against only those cells bearing the tumor antigens-that is, the patient’s cancer alone. The use of defined tumor antigens, however, or even genes specifying known tumor antigens to treat cancer patients, could be limited to special circumstances in which the patient’s tumor antigen has been identified. As we have seen, the tumor-cell population consists of multiple subpopulations of cancer cells that differ in important ways from each other.
Because of the heterogeneity of the tumor-cell population, some of the patient’s cancer cells may not express the defined tumor antigen chosen for use in the vaccine. Immunizations with defined antigens could fail to stimulate the immune system to kill cancer cells that fail to express the specific antigen or antigens used in the vaccine, or cancer cells that express a different set of antigens. A related question is whether or not the defined tumor antigen chosen for immunization of the patient is the most effective one in inducing the strongest anti-tumor response. It might not be. The only way to determine whether the “proper” antigen was chosen would be to conduct long-term clinical trials in cancer patients. Such trials often take years to complete.
Instead of preparing standard vaccines from a single tumor antigen, it seems logical to develop more broadly based vaccines, ones that stimulate immunity to a large array of different tumor antigens encompassing the patient’s total malignant cell population. Such an approach would, in theory, result in the killing of larger and larger numbers of the patient’s cancer cells.
To overcome many of the drawbacks of using purified defined antigens, we have been exploring ways to make a more comprehensively acting vaccine. We have been considering ways to transfer all of the DNA from tumor cells into a highly immunogenic cell lineone that, if successful, would stimulate a vigorous immune response to antigens that would otherwise be only weakly immunogenic.
The advantage of this approach is obvious. DNA from a patient’s tumor contains all the altered genes that specify tumor antigens expressed by different cells in the tumor. The expression of altered genes specifying “weak” tumor antigens by highly immunogenic cells could convert turn+ determinants, which fail to induce an anti-tumor immune response, into turn determinants, or antigens that lead to tumor rejection. The strategy is to capture the entire array of antigens expressed by the patient’s tumor and to convert it into a vaccine that can be used in patient therapy.
The notion that one can transfer the entire complement of tumor DNA into a highly immunogenic cell line is based on classic studies performed by Michael Wigler and colleagues at Columbia University College of Physicians and Surgeons. As early as the 1970s, before Boon did his work, Wigler and colleagues found that the genome, the total DNA content, of one cell type could be stably modified by transfer of DNA from another cell type. They found that the genome of cells deficient in an enzyme called adenine phosphoribosyltransferase could be modified to form the missing substance by transfection, or transfer of high-molecular-weight DNA from cells that possessed the gene for the enzyme. Some of the recipient cells took up various portions of the DNA, incorporated the gene and expressed the missing enzyme. A similar approach was used to convert cells lacking the gene for the enzyme thymidine kinase into cells that expressed the missing enzyme by transfer of genomic DNA from a variety of thymidine kinase-positive tissues and cultured cells. Transfection of genomic DNA also resulted in the expression of genes that specified membrane receptors, or proteins found in the membranes of cells. DNA from cells that contained the gene specifying the receptor for the polio virus, when transfected into receptor-negative cells resulted in cells that expressed the receptor.
In short, cells that take up DNA from another type of cell become modified genetically. They express properties that characterize the type of cell from which the DNA was obtained. This includes both the formation of enzymes as well as the expression of membrane-associated determinants. Furthermore, the transfections are surprisingly efficient. In some instances as many as one in 500 of the transfected cells expresses the products of individual genes.
Efficient as transfection may be, however, the question remains whether it is efficient enough to be clinically useful as a cancer-vaccination technique. Two recent studies suggest that it might. In the first, David Curiel and his colleagues at the University of Alabama at Birmingham found that intramuscular injection of experimental animals with DNA specifying carcinoembryonic antigen (CEA), a tumor antigen associated with colon carcinoma cells, resulted in an immune response directed toward colon carcinoma, one that prolonged the survival of tumorbearing mice. In the second, T. M. Pertmer and his colleagues at Auragen coated genes for tumor antigens onto microscopic gold beads that were then forced under pressure into cells in the skin. Quantities of DNA in the microgram (one-millionth of a gram) range introduced into the cells by this so-called “gene gun” were sufficient to induce a potent anti-tumor immune response.
In our own studies, we wanted to explore the possibility that weakly immunogenic tumor antigens could be expressed in a highly immunogenic form by the transfected cells. Using experimental animals to test this hypothesis, we extracted and purified DNA from a mouse melanoma, a highly aggressive, often lethal form of skin cancer, not only in mice but in humans as well. We then transferred the DNA into a cell line derived from a different mouse strain and used this DNAbased vaccine to treat animals with melanoma.
We chose the cell line into which we transferred the melanoma DNA quite deliberately. These recipient cells had already been modified to secrete interleukin-2, a chemical messenger molecule known to stimulate an immune response. In addition, the recipient cells bear surface proteins that the human immune system typically identifies as foreign. Hence, these recipient cells were preprogrammed to activate the immune system. When we transferred the melanoma DNA into these cells, we hoped to direct all of that immune activity toward the melanoma cells, even those bearing weak antigens. The vaccine contains these transfected, immunogenic cells, which are injected underneath the skin.
The results were striking. Mice with melanoma treated solely by this vaccine survived longer than did untreated mice. In the best instances, the treated animals appeared to have rejected the melanoma and survived significantly longer than the 30 days that untreated mice survived. In almost all instances, the animals eventually died from the tumor. The immune system was unable to eliminate all of the cancer cells.
We tested DNA-based tumor vaccines for mice with several different types of cancer, including breast cancer, and obtained similar results. The animals lived longer than untreated mice and, in some instances, they rejected the cancer cells. As predicted, the anti-tumor immune responses in the mice immunized with the DNAbased vaccines were directed toward the type of tumor from which the DNA was obtained, consistent with the formation of different tumor antigens by different tumors.
Our studies led us to conclude that cells transfected with tumor DNA incorporated and expressed genes that specified tumor antigens, and that the number was sufficient to induce the anti-tumor immune response. A large array of undefined antigens associated with the total population of cancer cells was expressed in a highly immunogenic form by cells that took up the tumor DNA.
A vaccine prepared by transfer of DNA from the tumor into a highly immunogenic cell line has important practical advantages. The technique is relatively simple and can be consistently and reliably carried out. Only a small quantity of tumor DNA is required to generate a vaccine that contains all of the antigens expressed by the patient’s cancer. Sufficient quantities of tumor DNA can be obtained from small surgical specimens (a needle biopsy could provide enough DNA for this purpose) or possibly from microscopic amounts of tumor. Since the transferred DNA is stably incorporated or integrated into the genome of recipient cells and is replicated as the cells divide, the number of cells in the vaccine can be expanded as might be required for repeated treatments. New vaccines that reflect the antigenic properties of the tumor can be prepared if the tumor recurs.
To date, studies testing DNA-based cancer vaccines have been carried out only in experimental animals. Clinical trials in cancer patients have not begun. However, encouraging results from multiple sources suggest that DNA-based vaccines will benefit cancer patients someday.
Few believe that cancer patients will be cured even if they are treated solely with an ideal cancer vaccine. The inherent genetic instability of the tumor cells along with the large diversity of tumor antigens present within the tumor-cell population may make a completely successful treatment with a tumor vaccine an unreachable goal. This is no less the case for immunization of cancer patients with DNA-based tumor vaccines than it is for other types of vaccine therapy.
The antigenic properties of a patient’s tumor are continually evolving. New antigenic variants are arising, in addition to those present at the time the treatment is given. In addition to the appearance of cells that form “new” tumor antigens, cells that can resist chemotherapy, cells that can invade and metastasize and cells that are able to inhibit killing mechanisms used by the immune system appear as the tumor progresses. As stated previously, the patient is cured only if each and every cancer cell in the patient is killed.
Nevertheless, a vaccine that induces immunity to the broad diversity of antigens formed by the patient’s tumor is likely to result in the killing of larger and larger numbers of malignant cells with important benefits to the cancer patient.
In the future, cancer patients will continue to receive multiple forms of therapy Each treatment will be designed to kill more and more tumor cells in the patient’s body. In addition to surgery to remove the primary tumor, radiation to kill identifiable tumor and chemotherapy to kill hidden cells, immunotherapy will find a place in the overall management of the patient’s disease. Just as patients may have multiple rounds of treatment with various forms of chemotherapy and undergo more than one course of radiation, they will likely receive multiple immunizations with a broadly based tumor vaccine. The vaccine will reflect to the extent possible the diversity of the patient’s tumor. Patients with small, microscopic cancer treated with a DNA-based cancer vaccine will receive the greatest benefit. The immune system is ideally designed to accomplish this goal.