1.1. Essentials of tumor immunology
The function of the immune system is to protect the body. This defensive function is performed by leukocytes (white blood cells) and a number of accessory cells distributed throughout the body. Lymphocytes are the key cells controlling the immune response. They specifically recognize “foreign” material and distinguish it from the body’s own “self” components.
There are two main types of lymphocytes: B cells which produce antibodies, and T cells which have a number of functions including:
helping B cells to make antibodies
recognizing and destroying virus-infected cells
activating phagocytes to destroy pathogens
controlling the level and quality of the immune response.
The essential role of T lymphocytes is to recognize antigen
1.2. Overview of the treatment limitations of patients with malignant gliomas
1.2.1. Treatment limitations of patients with malignant brain tumors
Although technical advances have resulted in marked improvements in the ability to diagnose and surgically treat primary brain tumors, the incidence and mortality rates of these tumors are increasing . Particularly affected are young adults and the elderly. Primary malignant brain tumors are the second leading cause of death in people under the age of 35. Furthermore in the elderly population, mortality rates from these tumors have increased more than 5-fold since 1970 . The present standard treatment modalities following surgical resection including cranial irradiation and systemic or local chemotherapy each have serious adverse side effects. The few long-term survivors are inevitably left with cognitive deficits and other disabilities [3,4]. The difficulties in treating malignant gliomas can be attributed to several factors. Glial tumors are inherently resistant to radiation and standard cytotoxic chemotherapies [5,6]. The existence of blood-brain and blood-tumor barriers impedes drug delivery to the tumor and adjacent brain infiltrated with tumor. Finally the low therapeutic index between tumor sensitivity and toxicity to normal brain severely limits the ability to systemically deliver therapeutic doses of drugs to the tumor.
1.2.2. Transfer of genomic DNA from one cell type to another alters both the genotype and the phenotype of cells that take up the exogenous DNA
Classic studies indicate that transfection of genomic DNA from one cell type to another results in integration of the transferred DNA and stable alteration of the genotype of the recipient cells. The transferred genes are replicated as the cells divide and are expressed. An analogous approach can theoretically be used to prepare a vaccine for use in patients with brain tumors . The malignant cell-population is characterized by the presence of numerous TAAs, some of which are unique and others are differentially expressed by cancer cells but all are strong potential targets of immune-mediated attack. Genes specifying tumor associated antigens (TAAs) that fail to provoke anti-tumor immunity can potentially become highly immunogenic antigenic determinants if they are expressed by immunogenic cells.
The most compelling reason for the vaccination strategy involving DNA-based cellular vaccines is the current lack of effective therapy for patients with a variety of brain tumors. This is verified by the dismal survival statistics, which have remained essentially unchanged for 30 years. Immunization with a vaccine that induces strong anti-tumor responses is an attractive addition or possibly even an alternative to conventional therapies. The DNA-based vaccines described in this chapter have shown remarkable therapeutic efficiency and survival benefits in some initial murine preclinical studies.
2. Preclinical experimental findings
2.1. Treatment of intracerebral glioma in C57Bl/6 mice by immunization with allogeneic cytokine-secreting fibroblasts
As an initial study, we measured the survival of C57Bl/6 mice injected intracerebrally (i.c.) with a mixture of Gl261 glioma cells and cytokine secreting LM cells . Gl261 cells are a glioma cell-line of C57Bl/6 mouse origin (H-2b). LM fibroblasts are derived from C3H/He mice and express H-2k determinants. We initially evaluated the immunotherapeutic effects of single cytokine-secreting LM-IL-2 cells and double cytokine-secreting LM-IL-2/interferon- cells in mice bearing an i.c. glioma. A mixture of G1261 cells and the single or double cytokine-secreting cells were injected i.c. into the right frontal lobe of C57BL/6 mice, syngeneic with G1261 cells. Mice injected i.c. with the mixture of glioma and LM-IL-2 cells survived significantly longer (P<0.025) than control mice injected i.c. with an equivalent number of glioma cells alone. Somewhat more dramatic results were obtained for mice injected i.c. with a mixture of glioma cells and LM-IL-2/interferon- double cytokine-secreting cells. No prolongation of survival was noted when allogeneic cytokine secreting fibroblasts mixed with tumor cells or tumor antigens were administered subcutaneously in mice with an intracerebral tumor even though a strong anti-tumor immune response was detected in the spleen cells of the treated animals. Of special interest, mice injected i.c. with an equivalent number of LM-IL-2 cells alone lived for more than three months and showed no evidence of ill effects or neurologic deficit. Immunocytotoxic studies (Figure 2) demonstrate a significantly elevated cromium release from Gl261 cells co-incubated with spleen cells from mice injected i.c. with glioma cells and the cytokine secreting fibroblasts. This indicates that a systemic anti-tumor response did develop in the mice injected intracerebrally with the cytokine secreting cells in the presence of tumor antigens.
2.2. Treatment of intracerebral breast cancer in C3H mice by immunization with syngeneic/allogeneic fibroblasts transfected with DNA from breast cancer cells
Whether results obtained by transfer of DNA from a tumor cell line into mouse fibroblasts can be applied to tumors that develop spontaneously is uncertain. Conclusions based on a model system involving tumor cell lines may not apply to neoplasms that arise spontaneously in patients. The appearance of spontaneous breast neoplasms in C3H mice provides an opportunity to investigate this question. DNA isolated from a breast neoplasm that arose in a C3H mouse (H-2K) was transferred into mouse fibroblasts (H-2k). To increase their immunogenic properties and to ensure rejection, the fibroblasts were modified to express H-2Kb determinants beforehand. H-2Kb determinants are allogeneic in C3H mice. The results indicated that C3H mice with intracerebral breast cancer treated solely by immunization with fibroblasts transfected with DNA from the same spontaneous breast neoplasm survived significantly longer (p0.005) than mice in various control groups .
2.3. T cell mediated toxicity toward intracerebral breast cancer in mice immunized with syngeneic/allogeneic transfected fibroblasts modified to secrete IL-2, GM-CSF or IL-18
An MTS cytotoxicity assay was used to detect the presence of T cells reactive with breast cancer cells in mice injected i.c. with the mixture of SB5b cells and the modified, DNA-transfected fibroblasts. The T cells obtained from the spleens of the injected mice were analyzed two weeks after the i.c. injection of the cell mixture. The results indicated that the cytotoxic response of greatest magnitude was in mice injected i.c. with the mixture of SB5b cells and transfected fibroblasts modified to secrete IL-2 or GM-CSF . Lesser cytotoxic effects were present in mice injected i.c. with SB5b cells and transfected fibroblasts modified to secrete IL-18.
2.4. The proportion of T cells responsive to tumor cells in mice bearing an intracerebral tumor immunized intracerebrally with syngeneic/allogeneic transfected fibroblasts modified to secrete IL-2, IL-18 or IL-2+IL-18
An ELISPOT-IFN- assay was used to determine the proportion of splenic T cells reactive with SB-5b cells in mice immunized with transfected fibroblasts modified to secrete IL-2, IL-18 or both IL-2 and IL-18. The animals were injected i.c. with a mixture of 1.0 X 104 SB-5b breast carcinoma cells and 1.0 X 106 treatment cells consisting of LMKbIL-2/SB5b, LMKbIL-18/SB5b, or a mixture of LMKbIL-2/SB5b and LMKbIL-18/SB5b cells. The animals were sacrificed at two weeks and an ELISPOT assay was done using the spleen cells to detect IFN- secretion in the presence of SB-5b tumor cells and antibodies against various T-cell subsets. The results indicate that the cellular anti-breast carcinoma immune response was mediated by CD4+, CD8+and NK/LAK cells . Although IL-18 secreting cells did not produce a significant anti-tumor immune response as detected with the ELISPOT assay, the combination of IL-2 with IL-18 secreting cells did result in an enhancement of the anti-tumor responses in comparison to animals that were treated with IL-2 secreting cells alone.
2.5. Increased numbers of responding T-cells were detected in the spleens and cervical lymph nodes of naïve mice or mice with i.c. breast cancer injected into the brain with cells from the immunohigh pool
An enrichment strategy for the vaccine was developed based on the hypothesis that if aliquots of a transfected cell population were divided into smaller populations, some populations by chance would contain more highly immunogenic cells than others. The populations with higher numbers of immunogenic cells could be identified by their stronger immunogenic response against SB5b cells in C3H/He mice. Two subpools that stimulated immunity to the greatest (immunohigh pool) and least (immunolow pool) extents after three rounds of enrichment were selected for further study.
To determine if systemic anti-tumor immunity was generated in tumor-free mice injected i.c. with cells from the immunohigh pool, cervical lymph node and spleen cells from the injected mice were analyzed by ELISPOT IFN- assays for responding T cells. Naïve C3H/He mice received 2 i.c. injections at weekly intervals of 1.0 X 106 cells from the immunohigh pool. One week after the second injection, mononuclear cells from the spleens and cervical lymph nodes of the immunized mice were analyzed for the presence of T cells responsive to the breast cancer cells. As controls, an equivalent number of cells from the non-selected master pool or cells from the immunolow pool were substituted for cells from the immunohigh pool. As additional controls, the same protocol was followed except that the mice were injected i.c. with equivalent numbers of SB5b cells, with LMKb cells or with media. Mice injected with SB5b tumor cells received only one injection. The results from the cervical lymph nodes (Figure 3) indicated that the highest number of responding cells was in mice injected i.c. with cells from the immunohigh pool (
ELISPOT IFN- assays were also used to determine the number of responding T cells in the spleens of mice with i.c. breast cancer injected into the tumor bed with cells from the immunohigh pool . A micro cannula was placed into the right frontal lobe of C3H/He mice. SB5b cells (1.0 X 104 in 10 l) were introduced into the brain through the cannula. On days two and nine following, the animals were injected through the cannula into the tumor bed with 1.0 X 106 cells from the immunohigh pool. As controls, the same procedure was followed except that the cells from the non-enriched master pool or cells from the immunolow pool were substituted for cells from the immunohigh pool. As additional controls, the tumor bearing mice were injected into the tumor bed with equivalent numbers of non DNA-transfected LMKb cells or the mice were injected with SB5b cells alone. The results indicate that the highest number of responding T cells were in the spleens of tumor-bearing mice injected i.c. with cells from the immunohigh pool (
The effect of antibodies against various T-cell subsets on the cytotoxic response was used to determine the types of cells activated for antitumor immunity in mice injected into the tumor bed with cells from the immunohigh pool. The greatest inhibitory effect was obtained when CD4+antibodies were added to the mixed cell cultures . Lesser effects were observed if the spleen cells were incubated in the media containing CD8+or NK/LAK antibodies.
2.6. T-reg cells are relatively deficient in the spleens of mice with i.c. breast cancer injected into the tumor bed with cells from the immunohigh pool
T-reg cells are potent inhibitors of natural antitumor immunity. The success of immunotherapeutic protocols may depend upon the relative numbers of T-reg cells and cytotoxic T lymphocytes in tumor-bearing animals and patients. Quantitative RT-PCR for Foxp3, a transcription factor characteristic of T-reg cells, was used to determine the relative proportions of T-reg cells in the spleens and brains of mice with i.c. breast cancer injected into the tumor bed with cells from the immunohigh pool of transfected cells. Naïve C3H/He mice were injected i.c. with 5.0 X 104 SB5b cells along with 1.0 X 106 cells from the immunohigh pool of transfected cells. One week later, the animals received a second i.c. injection of cells from the immunohigh pool through the same burr hole alone. As controls, the same procedure was followed except that the mice were injected with equivalent numbers of SB5b cells and cells from the non-enriched master pool or the immunolow pool. The results indicate that CD4+/CD25+/Foxp3+T-reg cells were relatively deficient in the spleens but not in the brains of animals injected with cells from the immunohigh pool . An analysis by FACS of the spleens of the injected animals revealed a relative deficiency of CD4+/CD25+T cells and a corresponding increase in the relative numbers of CD8+cells in the spleens of mice injected i.c. with cells from the immunohigh pool.
Despite standard therapeutic approaches, the survival of patients with primary or metastatic tumors to the brain has not improved significantly in more than thirty years. There is an urgent need for new and more effective forms of treatment. Immunotherapy, designed to stimulate immunity to the autologous tumor, is under active investigation for a number of different histologic types of cancer. The enhanced immunotherapeutic properties of a vaccine prepared by transfer of a cDNA expression library derived from breast cancer cells into a mouse fibroblast cell line appears to have great potential in treatment of intracerebral tumors. As the transferred cDNA integrates spontaneously into the genome of the recipient cells, replicates as the cells divide and is expressed, the vaccine could be prepared from small amounts of tumor tissue, enabling treatment at an early stage of the disease, when tumor tissue is available in only limited amounts and the tumor is most susceptible to immune-based therapy. However, like other cellular tumor vaccines, only a small proportion of the transfected cell population was expected to have incorporated cDNA fragments that specified tumor antigens. A novel enrichment strategy has also been developed to increase the proportion of immunotherapeutic cells in the vaccine.
A number of different strategies have been attempted to develop vaccines that generate enhanced anti-tumor immune responses in mice and patients with intracerebral neoplasms involving the central nervous system. Vaccines have been prepared by “feeding” antigen presenting (dendritic) cells apoptotic bodies from tumor cells or tumor cell lysates. Introduction of tumor cell-derived RNA into dendritic cells is another approach which has been developed. Immunization with dendritic cells “fed” derivatives of tumor cells or transfected with tumor-RNA can result in the induction of immune responses against the broad array of tumor antigens expressed by the population of malignant cells including tumors of neuroectodermal origin [10, 11]. In patients, immunization with autologous dendritic cells transfected with mRNA from malignant glioma elicited tumor-specific CD8+cytotoxic T-lymphocyte (CTL) responses against the patient’s malignant cells . Although results of dendritic cell immunotherapy have demonstrated promise in animal models, clinical trials have been disappointing thus far .
Other tumor vaccination strategies have been used including modification of neoplastic cells to generate anti-tumor immune responses. Immunization with tumor cells modified to secrete immune-augmenting cytokines such as IL-2 and GM-CSF has resulted in the development of generalized MHC-restricted anti-tumor immune responses in animal models [13, 14-22]. Selective tumor regression was observed in experimental animals and patients receiving immunotherapy alone, in support of the potential of this type of treatment for patients with malignant disease . The effects of cytokine expression by central nervous system tumors (CNS) were examined initially using glioma cells that were engineered to secrete IL-4 . In these studies it was demonstrated that IL-4 transduced glioma cells resulted in the development of anti-tumor immune responses. Delivery of an IFN-β expression plasmid by cationic liposomes to the CNS tumor site was also found to induce significant anti-CNS tumor immunity in pre-clinical models . Use of a high-titer adenoviral vector encoding IL-12 is another strategy that was reported to induce anti-tumor responses in a glioma model .
Previous studies indicated that transfection of genomic DNA from the malignant cells into a fibroblast cell line resulted in stable integration and expression of the transferred DNA. Both the genotype and the phenotype of the cells that took up the exogenous DNA were altered as portions of the transferred DNA were expressed. Immunization of tumor-bearing mice with the DNA-based vaccine resulted in the induction of cell mediated immunity directed toward the type of tumor from which the DNA was obtained, and prolongation of survival, consistent with the expression of an array of TAA by the transfected cells. This was the case for mice with melanoma, squamous cell carcinoma and in mice with breast cancer . Multiple undefined genes specifying TAA that characterize the malignant cell population were expressed by cells that took up DNA from the tumor. The number of vaccine cells could be expanded as required for multiple immunizations. In addition, the recipient cells can also be modified before DNA-transfer to increase their immunogenic properties, as for example, by the introduction of genes specifying immune-augmenting cytokines or allogeneic MHC-determinants, which act as strong immune adjuvants. In animal models, injection of cytokine-secreting allogeneic fibroblasts into the tumor bed of intracerebral neoplasms was partially effective in the treatment of mice with established brain tumors .
To be successful, every remaining tumor cell in the patient must be eliminated. It is unlikely that a single form of therapy is capable of achieving this goal. However immunotherapy in combination with surgery, radiation therapy and chemotherapy will likely find a place as a new and important means of treatment for patients with brain tumors. A major advantage of DNA-based vaccines is that they do not require protein purification or its production and yet they are able to elicit robust and long-lasting activation of the immune response, which results in tumor rejection. From a practical point of view, these vaccines are easy to prepare and they are relatively inexpensive. Only a limited quantity of tumor-derived DNA is required, which can be obtained from small surgical specimens. The enrichment strategy enables the generation of highly immunogenic pools of transfected cells with enhanced immunotherapeutic properties.
Thus DNA-based vaccines offer a number of advantages, which greatly encourage their further development for cancer immunotherapy in general and specifically for treatment of malignant brain tumors.
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