Glioma

Traditional therapies for the treatment of malignant glioma have failed to make appreciable gains regarding patient outcome in the last decade. Therefore, immunotherapeutic approaches have become increasingly popular in the treatment of this cancer. This article reviews general immunology of the central nervous system and the immunobiology of malignant glioma to provide a foundation for understanding the rationale behind current glioma immunotherapies. A review of currently implemented immunological treatments is then provided with special attention paid to the use of vaccines, gene therapy, cytokines, dendritic cells and viruses. Insights into future and developing avenues of glioma immunotherapy, such as novel delivery systems, are also discussed. [Neurol Res 2005; 27: 703-716]

Keywords: Cytokines; dendritic cells; gene therapy; glioma; immunotherapy; neuroimmunology; vaccine; virus

INTRODUCTION

Since the last decade, there has been a significant interest in developing novel immunotherapeutic approaches for malignant glioma. Given the lack of success with currently available therapeutic modalities, the appeal of this strategy is obvious-it relies solely on the ability of the immune system to recognize and destroy tumor cells while leaving normal and healthy tissue intact. To date, different immunological approaches have been utilized with various degrees of success. This review summarizes our current knowledge of immunology and glioma immunobiology and provides an overview into some of the currently tested immunotherapeutic regimens which are gaining increasing roles in clinical trials for malignant brain tumors.

GENERAL IMMUNOLOGY

The immune system is designed to recognize and eliminate pathogens. To achieve this, the system is comprised of innate and adaptive components-both mediating responses to foreign antigens.

The innate system is comprised of macrophages, granulocytes, natural killer cells and monocytes [microglia in the central nervous system (CNS)]. This system recognizes pathogen-associated molecular patterns (PAMPs) or general ligands (e.g. NKG2D ligand) that are upregulated during cellular stress, infection or transformation1-8. This recognition results in immunologie responses such as phagocytosis and the release of various cytokines (e.g. IL-1, IL-6, TNF-α) and inflammatory mediators (e.g. oxygen radicals, leukotrienes). These mediators aid in the localization and initiation of the adaptive immune response via their attractant and proliferative properties3,9-11 (Figure 7).

The adaptive immune system differs from the innate system with its specificity and capacity for the memory of foreign antigens. The specificity of this system is derived from lymphocyte receptors, which recognize unique foreign antigens presented to them in the context of a major histocompatibility complex (MHC). The action of this system typically begins with the ingestion and presentation of foreign antigens by antigen presenting cells (APCs) within the context of MHC class II molecules. If a CD4+ (helper) T cell receptor (TCR) recognizes this antigen then the leukocyte will become activated and release cytokines. Depending on specific helper T cell type (Th1 or Th2), released cytokines and the presence of required co-stimulatory molecular interactions, this results in the activation and proliferation of nearby CD8+ cytotoxic T lymphocytes (CTLs) or B cells. Activated CD8+ lymphocytes (effectors of the cell-mediated system) target cells presenting the previously mentioned antigens in the context of MHC class I complexes. These targeted cells are then eliminated by CTLs either by the initiation of apoptosis via Fas/Fas ligand interactions or by perforin-mediated cell lysis (Figure 2). Activated B cells release immunoglobulins (effectors of the humoral system) to neutralize specific antigens. However, because immunoglobulins interact with free or soluble antigen, the humoral system is poorly equipped to respond to the mostly cytoplasmic derived antigens of tumor cells. Also, because antibodies traverse the blood-brain barrier (BBB) inefficiently, their effectiveness against CNS tumors is further limited12. Lastly, in both the humoral and cell-mediated portions of the adaptive system, activated T or B cells may become long-lived memory cells that are ready to respond quickly to repeat immunological challenges.

IMMUNOLOGY OF THE CENTRAL NERVOUS SYSTEM-SPECIAL CONSIDERATIONS

The generation of an immunologic response to neoplasms in the brain has the same general requirements as that necessary for cancers in peripheral tissues. The CNS, however, has historically been viewed as 'immune privileged' with the assumption that an effective immune response is difficult to obtain13. The presence of a BBB, lack of organized lymphoid tissue or significant lymphatic drainage, paucity of MHC expression in normal brain parenchyma and presence of immunoregulatory factors all contribute to the perception of immune privilege12, 14. While the above properties contribute to the distinctive immunoreactivity of the CNS, they do not render it a completely immune privileged site. They do, however, necessitate an understanding of alterations in the traditional schema of peripheral immunobiology for one to grasp the dynamics of the CNS antitumor response.

As in the periphery, cell-mediated responses to neoplasms in the CNS require efficient antigen presentation. The precise cell type and mechanism responsible for antigen presentation in the brain has been debated; however, the requirements for APCs in the CNS are the same as for those in the periphery. They include the ability to ingest or phagocytose foreign antigens, expression of MHC complexes and the ability to induce T lymphocyte responses (probably via costimulatory molecules such as B7). Microglia have emerged as the likeliest candidates for antigen presentation in the brain15-17. In addition to their hematopoietic origin, microglial cells express MHC class I, MHC class II and certain costimulatory molecules. In vitro, these cells can induce alloreactive T cell responses and stimulate T cell lines to proliferate and secrete cytokines. In vivo, CD54 and B7 costimulatory molecules have been detected on reactive microglial cells in multiple sclerosis (MS) plaques18. Also, the CNS may participate in trafficking dendritic cells (highly effective peripheral antigen presenting cells) between normal or inflamed brain and immune system tissues. This mechanism is not entirely clear but is probably under tight environmental controls19. One possible mechanism may be through drainage of CNS antigens to cervical lymph nodes where they may interact with these APCs. Such pathways have been illustrated in primate models of experimental allergic encephalomyelitis (EAE) and human MS20. Furthermore, evidence from murine models with brain tumors suggests that tumor-specific T cells can be primed in cervical lymph nodes21.

The presence of a tight BBB also contributes to the distinctive immunoreactivity of the CNS and once was thought to effectively hinder the influx of immune cells into the brain. Despite presenting a formidable obstacle, immunologically functional cells (including activated T lymphocytes) have the ability to pass the BBB and enter brain parenchyma22-24. In fact, multiple types of lymphoid cells can be detected in the CNS during disease states25-29. Pro-inflammatory cytokines probably play critical roles in increasing BBB permeability along with enhancing the adherence and transendothelial migration of leukocytes19.

Another potential obstacle to CNS tumor reactivity is the relative lack of MHC expression and antigenicity in normal and malignant brain tissue; these molecules are indispensable for antigen recognition by TCRs. However, there is now experimental evidence that MHC antigens are upregulated at sites of brain injury, degenerative disease, tumor or after the exposure to cytokines such as interferon-γ30-35. In addition to antigen-presentation complexes, most human glioma cells have been shown to express Fas/APO-1 (CD 95) and Fas ligand, which allow these cells to undergo Fas/Fas ligand interactions with activated T lymphocytes resulting in apoptosis35,36. While no universal glioma-specific antigen presented on these MHC molecules has been found, several tumor-associated antigens shared by histogenetically related tumors have been identified (e.g. tenascin, gp240, gp1000, epidermal growth factor receptor, tyrosinase, tyrosinase-related proteins 1 and 2, MAGE-1 and MAGE-3)37-41. There is also evidence that many of these antigens are capable of generating immune responses experimentally42.

Lastly, the environment of the CNS is decidedly antiinflammatory under normal conditions. The need for such attenuation is underscored by examples of states of intense immune or inflammatory response that lead to diseases such as MS or post-infectious encephalomyelitis. Astrocytes have been shown to exert a number of important physiologic effects related to immune homeostasis, however, their exact function remains controversial43. While under normal conditions astrocytes lack MHC expression, they may be induced to express these molecules by interferon-γ44. The precise role of MHC class II positive astrocytes remains unclear; however, a prevailing view is that astroglia exert a negative immunoregulatory function that contributes to the anti-inflammatory state of the normal CNS15,45,46.

CNS TUMOR IMMUNOLOGY-HOW GLIOMAS ESCAPE THE IMMUNE RESPONSE

In addition to the typical restraints placed upon immune regulation by the CNS, malignant brain tumors also provide means to neutralize an effective immune response (Figure 3). As stated earlier, brain neoplasms typically express low levels of class I MHC antigens47. This lack of MHC class I expression has been theorized to be a potential mechanism by which gliomas can escape recognition and subsequent lysis by CD8+ T cells. The potential for upregulation of these MHC antigens by factors such as interferon-γ, however, raises questions about this simplified scheme. Furthermore, receptors on NK cells sense MHC down-regulation and can initiate NK lysis of MHC-deficient tumor cells48. As such, there probably exists a level of glioma MHC class I expression that balances T leukocyte and NK cell detection. The regulation of this balance has yet to be precisely elucidated; however, it almost certainly plays an important role in the ability of malignant brain tumors to evade the immune response49. Also, expression of altered MHC has been identified in a variety of malignancies including glioma cells of the brain50-53. HLA (human leukocyte antigen)-G is a non-classical MHC class I (class 1b) molecule that is structurally related to the classical MHC class I (class 1a) molecule. HLA-C, however, has been recently identified to have strong immunoregulatory functions and may render the glioma cells that express it to be less susceptible to antitumoral immune responses54.

In addition to evading host immune responses, malignant brain tumors can have a profound effect upon the function of immune system components. The maturation and function of APCs can be impaired by secreted tumor factors, thus limiting their ability to activate tumor-specific T cells or participate in active immunotherapy approaches55-57. T lymphocyte function in the setting of primary intracranial tumors has also been shown to be defective with abnormal TCR mediated signaling and depressed proliferative response to mitogen58,59. Additionally, these lymphocytes exhibit a propensity for apoptosis59,60. The partial reversibility of T cell suppression following brain tumor removal along with the demonstration of down-regulated function of T cells harvested from brain tumor patients or normal patients exposed to glioma supernatants provide strong evidence of glioma-produced immunosuppressive factors61-63. Transforming growth factor beta 2 (TGF-β2) has been one of the most extensively studied of all immunosuppressive factors identified thus far. It has been shown to be significantly over-expressed in patients with glioblastoma multiforme (GBM) while virtually absent in normal brain tissue64,65. Additionally, TGF-β2 has been shown to effectively limit T cell and B cell proliferation, IL-2 receptor induction, cytokine production, natural killer cell activity, cytotoxic T lymphocyte development, lymphokine activated killer (LAK) cell generation and the cytotoxic response of tumor infiltrating lymphocytes65-70. TGF-β has also been shown to down-regulate the expression of HLA-DR (a non-classical MHC antigen expressed on glioma cells) and has important effects on blood-neural barrier properties-both may be other mechanisms by which gliomas escape immune surveillance71,72. Lastly, TGF-β probably acts as a growth promoter for glioma cells (via TGF-β type I and type II surface receptors) leading to tumor angiogenesis and stromal growth, likely, through autocrine or paracrine loops73,74. As such, TGF-β suppression in human gliomas by retroviral gene transfection has resulted in the enhancement of tumor cell susceptibility to LAK cells75. Additionally, TGF-β antagonists have been shown experimentally to inhibit malignant glioma cell proliferation, motility and TGF-β mediated morphologic changes76.

Other factors produced by glioma cells that may play a role in immune regulation and tumor cell escape include prostaglandin E^sub 2^ (PGE^sub 2^) and IL-10. PGE2 has been shown to reduce LAK cell activity and may contribute to the ability of gliomas to escape immune surveillance via the down-regulation of HLA-DR77,78. The effects of IL-10 on tumor immunology are currently not as clearly defined. While some studies have suggested that IL-10 blunts the immune response by inhibiting IFN-γ release, TNF-α release and MHC class II expression, another has shown enhanced response to tumor in animal models79-81. Clearly, further investigation will be required to better identify the role of IL-10 in CNS tumor immunology.

CNS TUMOR IMMUNOTHERAPIES

Despite the unique anatomical and physiologic barriers posed by the CNS, evidence of brain-specific antigens resulting in the priming of T cells in peripheral immune tissues does exist (see section on CNS immunology). Furthermore, systemic immunizations with brain-specific antigens resulting in CNS manifestations have been demonstrated with EAE and paraneoplastic cerebellar degeneration (PCD)82,83. These data led to the investigation of various types of peripheral vaccinations as treatment strategies for brain tumors.

Cytokines

As described earlier, cytokines play critical roles in CNS tumor immunology by enhancing T cell activation and MHC antigen expression. Cytokine gene transfer in cancer models resulting in tumor rejection has been demonstrated with IL-2, IL-4, IL-12, IL-18, IL-23, IFN-α and granulocyte-macrophage colonystimulating factor (GM-CSF)84-93. The use of peripheral cytokine-secreting vaccines for intracranial cancers has, therefore, been explored in hopes of overriding the tumor-induced immunosuppressive microenvironment and enhancing the activation and infiltration of host-derived APCs and lymphocytes. As such, a panel of cytokine secreting vaccines was screened in a murine glioma model with IL-2, IL-4, IL-12 and CM-CSF being most effective in producing T cell inflammation94. As an excellent review exists regarding cytokine gene therapy for malignant glioma, we will highlight the salient points95 (Table 1).

Several studies have demonstrated the efficacy of peripheral vaccinations with GM-CSF transfected tumor cells against brain tumors in animal models28,96-99. Furthermore, some studies have suggested intensified antitumoral responses when GM-CSF vaccines were coupled with those expressing IL-4 or the costimulatory molecule B70 -2(97,100,101. Interestingly, antitumor cytotoxicity only exceeded that of peripheral blood mononuclear cells (PBMCs) stimulated with wild type tumor alone when PBMCs were stimulated with both wild type tumor and B7-2/GM-CSF (but not IL-12) transduced cells100. Lastly, peripheral tumor cell inoculation coupled with continuous, peripheral GM-CSF alone or with IL-2 or IL-12 resulted in improved survival rates in the treatment of intracranial glial tumors in rat models102,103. Presumably, the antitumor activity of GM-CSF is related to its potency in generating dendritic cells (DCs). Whether GM-CSF alone or in combination with other factors will result in CNS antitumor efficacy in humans remains to be seen.

The effectiveness of IL-2 in the treatment intracerebral tumors has been investigated using multiple methods. Vaccines composed of IL-2 secreting autologous fibroblasts and glioma cells have been shown to induce cytotoxic CD8+ T cells in the blood of a patient with malignant glioma104. A later study also demonstrated a vigorous systemic antitumor response following subcutaneous injections of IL-2 secreting cells in the presence of tumor antigens in mice105. Despite the systemic response, this did not result in prolonged survival. However, this study did demonstrate significantly longer survival following intratumoral injections of IL-2 secreting allogeneic cells alone. Intracerebral injection of IL-2 secreting fibroblasts has also been found to be effective as a protective treatment in preventing the development of murine brain tumors when the tumor cells are introduced into the same site where the fibroblasts were injected earlier106. Additionally, this treatment has proven effective for established intracerebral breast carcinoma or glioma in mice107. Both of these studies involving intracerebral injection of IL-2 secreting fibroblasts resulted in significantly increased survival rates in the animal subjects. Combining peripheral vaccination and intratumoral injection of IL-2 expressing cells or IL-2 activated NK cells has also shown promise in the elimination of brain tumors in animal models108,109. Also, injection of IL-2 generated LAK cells into tumor resection cavities of humans with histologically confirmed recurrent GBM was found to be safe and resulted in higher median survival rates than those reported in other published series of patients that underwent reoperation for CBM110. Given the promising results of intracranial IL-2 treatment, novel means of delivery (including the use of biodegradable microspheres) have also been explored. The use of microspheres for delivery of IL-2 and the chemotherapeutic agent carmustine (BCNU) has shown improved median survival when compared with controls in animal models111.

The use of IL-4 for the treatment of intracranial tumors has also been explored in multiple ways. In one important study, the effectiveness of various cytokines (IL-4, IL-12, GM-CSF and IFN-α) was compared using intracerebral or intradermal injections of transfected tumor cells as prophylaxis or therapy against intracerebral glial tumors in an animal model112. All cytokine transfected cell lines were rejected following their intradermal injection; conversely, all lead to tumor outgrowth and animal death following intracerebral injection. However, intradermal IL-4 expressing cell vaccinations were the only cell lines that were effective at providing immunity (90% long-term rat survival) against later challenge with intracranial tumor. This was compared with 40% long-term survival in rats vaccinated with GM-CSF or IFN-α secreting cells and 0% in IL-12 secreting vaccinations. Additionally, only IL-4 peripherally vaccinated rats had long-term survival benefits when used as therapy for existing tumors. These results corroborated data obtained earlier demonstrating the efficacy of subcutaneous tumor cell vaccines expressing IL-4 [with and without herpes simplex virus thymidine kinase (HSVtk)/ganciclovir] as therapy or prophylaxis against intracranial glial tumors in animals85. This led to a Phase I clinical trial of vaccinations with autologous glioma cells admixed with transgene derived IL-4 expressing fibroblasts in one human patient with recurrent right temporal lobe GBM113. A transient response to the vaccine was suggested in this trial and the patient survived 10 months after treatment. Clearly, further trials with peripheral IL-4 vaccines will be needed to better assess their effectiveness.

The subcutaneous immunization of rats with IL-12 secreting glial tumors has been demonstrated to suppress the growth of identical cell type brain tumors114. This cytotoxic activity was limited when compared with that of IL-2. The same study also showed reduced tumor growth rates when these transduced tumor cell lines were implanted intracerebrally, but again, growth retardation was greater with IL-2 secreting cells. The use of intracerebral cytokine delivery has also been explored by other means. Significantly prolonged survival times have been demonstrated in animals challenged intracranially with glioma cells after earlier intracranial paracrine delivery of IL-T2 with transduced glial cells115. Neural stem cells (NSC) that express IL-12 (another possible mechanism of intracranial cytokine delivery) have also shown effectiveness in prolonging survival time in mice and rats with existing tumors116,117. Clear evidence for tropism of injected NSCs into infiltrating or satellite portions of tumors was also exhibited117. Finally, systemic IL-12 has also shown effectiveness in eliciting immunologic responses against transplanted and endogenous murine CNS tumors118,119.

Dendritic cells

The rationale for immunotherapy regimens utilizing dendritic cells lies in their potency as antigen presenting cells. With the advancement of techniques allowing for their isolation and propagation in vitro, these therapies have become more feasible in the treatment of gliomas. Each of these therapy regimens, however, requires patient-derived tumor antigens owing to the lack of a universal glioma-specific antigen. There are several reports of peripheral vaccinations with dendritic cells loaded with glioma antigens (e.g. tumor cell extracts, lysates, RNA) or dendritic cells fused with glioma cells (with or without recombinant IL-12), leading to the generation or augmentation of antitumor responses in the brains of animal models120-123. The promising results of glioma DC vaccines in animal models have led to several clinical trials in adults and children124-129. Initially, phase I trials established the feasibility and safety of peptide pulsed dendritic cells vaccinations124,128. More recently, vaccination with tumor lysate pulsed dendritic cells was shown to elicit the generation of antigen-specific cytotoxic T cells in patients with malignant gliomas127. In this study, the median survival for patients (n=8) with recurrent glioblastoma who were treated with the vaccine was extended to 133 weeks. Together, these results suggest that DC vaccinations are safe and can generate intracranial antitumor effects and T cell infiltration.

Intracranial/intratumoral injection with DCs has also been explored as a therapeutic strategy for intracranial glioma130-132. Interestingly, such direct intratumoral DC therapy appeared to be more effective than peripheral subcutaneous vaccination at generating tumor regression, although other studies will need to confirm this finding130. This result was mediated by the migration of DCs to extracranial lymph nodes and resulted in systemic antitumor immunity against intracranial glioma cells. Furthermore, the activity of IL-12 or IL-18 appears to enhance the effectiveness of intratumoral injection of dendritic cells132,133. These results suggest that augmenting the local immune response with cytokines can further enhance the infiltration of T cells into the tumor parenchyma, thereby promoting the interaction of T cells with tumor pulsed antigen presenting cells.

In spite of the early successes with dendritic immunizations, several problems remain to be solved in order to fully optimize this form of therapy. These include (1) sources of dendritic cells or precursor cells, (2) methods of maturation, (3) antigen-loading strategies, (4) route of administration, (5) role of administering additional cytokines and (5) endpoints for clinical trials. To date, the major sources of DCs include direct isolation from peripheral blood or in vitro generation and a direct comparison of these DCs has not yet been made. Moreover, given the different immunological properties of immature versus mature DCs (antigen uptake and presentation, MHC expression, migratory capacity, expression of adhesion molecules or co-stimulatory activity), it will be important to determine which subset provides for an optimal vaccine. In a similar fashion, the elucidation of the best antigen source, i.e. peptides, proteins, DNA, mRNA, cell lysates, apoptoic bodies and fusions, awaits further investigation. While the majority of current trials employ subcutaneous or intratumoral immunization, the availability of other strategies (intravenous, intradermal, intranodal and intralymphatic) has not been compared, with or without the use of adjuvant cytokines. Finally, we do not have any established criteria for measuring the success of vaccine therapy although the increase in overall survival is commonly used as a study endpoint. Perhaps a better determinant of success would be delay in time to progression or recurrence, given that immunotherapeutic applications are most likely to be successful in clinical situations where the volume of residual disease is low.

Oncolytic viruses and viral gene delivery

A great deal of effort has been generated about the use of viral vectors for gene therapy of cancer. Viruses, in the form of herpes virus, adenovirus, vaccinia or reovirus, have been created to be either replication incompetent or replication competent. Initial studies utilizing replication incompetent viruses have met with limited clinical success given the lack of effective transgene expression in tissue beyond the injection site. As a result, replication-competent viruses have been created where the expression of the gene is regulated either by the introduction of tissue specific promoters or genetic modification of the viral binding site. Such oncolytic viruses have gained significant interest, given their ability to infect neoplastic cells, produce progeny and lyse the tumor cell. As such, several oncolytic viruses have been developed that exhibit antitumoral properties in both animal models and clinical studies (Table 2).

While an extensive discussion of the various replication competent viral vectors is beyond the scope of this review, several vectors are worth pointing out owing to their potential of enhancing immunotherapy. The HSV-1 mutant G207 has been shown to effectively reduce the growth of subcutaneous U87 glioma cells and prolong survival of mice injected with these cells intracranially134. A recent Phase I G207 clinical trial in humans with recurrent or progressive malignant glioma has shown that no maximally tolerated dose (MTD) or dose limiting toxicities could be established135. Additionally, while the goal of this study was to ascertain a safety profile for this treatment, a therapeutic benefit was suggested. Currently, Phase Ib/II trials have begun in hopes verifying safety and tolerability of intratumoral G207 injections for malignant glioma.

The adenovirus ONYX-015 is another oncolytic replication competent virus which is being investigated for the treatment of malignant glioma in human clinical trials. This virus selectively replicates within and lyses cells with defects in p53 or the p53 pathway136. ONYX-015 has demonstrated effective antitumor activity in animal studies and Phases I and II trials for treatment of head and neck cancers have shown safety and potential efficacy as well137-140. Additionally, the recently published results of a Phase I trial for the treatment of glioma have demonstrated safety with injection of ONYX-015 within resection cavities141.

Newcastle disease virus (NDV) is an enveloped paramyxovirus that has been found to have selectively increased replication in neoplastic (as compared with non-neoplastic) cells, prompting interest in its use as an oncolytic virus142. Several animal studies have demonstrated effective tumor regression in fibrosarcoma and neuroblastoma cells using the 73-T strain administered intratumorally and systemically143-145. Interestingly, the literature also reveals case reports of patients with GBM treated with intravenous NDV resulting in progressive tumor shrinkage and long-term survival146,147.

Lastly, reovirus, a non-enveloped virus associated with mild respiratory and gastrointestinal tract symptoms in humans, is known to infect and proliferate in cells with unregulated Ras pathway activity. Since Ras pathway dysregulation is common in gliomas (coupled with the cytolytic activity of reovirus at the end of its replication cycle), the use of this virus for oncolytic therapy has become attractive. SCID mice implanted in the flank with the glioblastoma cell line U87 had significantly decreased tumor growth compared with control following intratumoral injection of reovirus148. Intratumoral/intracerebral injection of reovirus into glioma-bearing SCID mice resulted in significant tumor regression-however, significant virus related toxicities were noted in these immunodeficient hosts149. Phase I dose-escalation trials with intratumoral/intracranial injection in patients with recurrent malignant glioma are in progress150.

The significance of viruses in the potential treatment of gliomas lies not only in their inherent oncolytic ability, but more importantly, in their ability to further enhance immunotherapy (Table 3). For instance, HSV has been used to deliver IL-4, IL-10 and IL-12 against experimental glioma151-153. In fact, the combination of HSV/IL-4 was shown to augment the oncolytic effect of HSV alone and improve the survival of mice with experimental brain tumors. Likewise, local expression of IL-12 via HSV was shown to increase Th1 response and significantly increase the survival of mice with intracranial tumors. The effects observed with HSV have also been documented with adenoviral vectors for the delivery of IL-4, IL-12 and TNF-α154-156 and vaccinia mediated delivery of IL-2 and IL-12117. In all instances, mice intracerebrally implanted with viral vectors producing IL-2, IL-4, IL-12 or TNF-α survived significantly longer than those implanted with non-cytokine secreting vectors. These results further confirm the use of local cytokine delivery against brain tumors and when used in combination with oncolytic therapy, suggest a powerful means for targeted brain tumor therapy.

FUTURE DIRECTIONS

Without a doubt, immunotherapeutic approaches for malignant glioma are here to stay. We now have significant preclinical evidence regarding the safety and efficacy of different immunologie approaches. Furthermore, a number of these immune based therapies are currently in different phases of human clinical trials. Two are worth particular attention, given their common origin, method of delivery, and potential for changing the treatment of malignant glioma.

The first study is based on the expression of IL-4 receptors on malignant gliomasistt. The observation that cultured human glioma cell lines, as well as a large proportion of GBMs in situ, express IL-4 receptors has led to the use of IL-4 molecules fused with cytotoxins as a treatment for these cancers158-160. Several studies using proteins with IL-4 receptor binding domains fused to Pseudomonas aeruginosa toxins have been investigated in human patients164-164. By means of convectionenhanced delivery, these cytotoxins have been shown to have acceptable safety and toxicity profiles. A newly proposed phase III trial will hopefully confirm the effectiveness of this antitumor strategy. The second, and more promising trial, is based on IL-13 receptor expression on malignant gliomas164. This receptor is not only over-expressed on high grade gliomas but, in fact, serves as a marker of tumor progression166. Convectionenhanced delivery of IL-13/Pseudomonas exotoxin has been tested in preliminary phase I/II trials with extremely favorable results16 . The drug has recently been designed by the FDA for fast track drug development and is currently undergoing a large-scale, international, prospective and randomized phase III study.

While convection-enhanced delivery of cytotoxins targeted against glioma-specific antigens represents an exciting approach against malignant brain tumors, our ability to identify unique antigens, which are uniformly expressed in all glial tumors, remains a significant challenge. Moreover, while the capacity of tumors to develop antigen variants in the setting of ongoing therapy has been shown to be responsible for tumor recurrence and treatment failure, our ability to target these cells has met with limited success. Future studies will have to aim not only at identifying new surface markers on high grade tumors but also at markers of tumor transformation. The ability of resident CNS antigen-presenting cells to effectively present these antigens and stimulate appropriate T cells constitutes an area of active interest. Whether via cytokine-mediated augmentation of tumor-specific T cells or via clonal expansion of virally transduced T cells with tumor- specific TCRs, it is becoming increasingly clear that our ability to destroy a large tumor mass depends on a sufficient number of primed CTLs. In addition, recent identification of regulatory T cells (Treg) and their capacity to inhibit the immune response within tumor stroma has opened a new area for glioma immunotherapy. Shifting the balance from tolerance to response will be critical in enhancing the antitumor response, and the ability to target Tregs will play an important role in this process. The promise of immunotherapy against malignant brain tumors is here; the capacity to effectively employ it remains a challenge which awaits further investigations and appropriate clinical trials.

ACKNOWLEDGEMENTS

We gratefully acknowledge the artistic assistance of Lydia M. Johns who prepared the figures which appear in this manuscript.

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Christian W. Sikorski and Maciej S. Lesniak

Division of Neurosurgery, The University of Chicago Pritzker School of Medicine, 584 7 South Maryland Avenue, MC 3026, Chicago, Illinois 60637, USA

Correspondence and reprints requests to: Maciej S. Lesniak, Division of Neurosurgery, The University of Chicago Pritzker School of Medicine, 5841 South Maryland Avenue, MC 3026, Chicago, Illinois 60637, USA. [mlesniak@surgery.bsd.uchicago.edu] Accepted for publication June 2005.

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