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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Neurosurg Clin N Am. Author manuscript; available in PMC 2011 January 1.
Published in final edited form as:
PMCID: PMC2786905
NIHMSID: NIHMS142685

Challenges in Clinical Design of Immunotherapy Trials for Malignant Glioma

Abstract

Glioblastoma multiforme (GBM) is the most common and lethal primary malignant brain tumor. The traditional treatments for GBM, including surgery, radiation, and chemotherapy, only modestly improve patient survival. Therefore, immunotherapy has emerged as a novel therapeutic modality. Immunotherapeutic strategies exploit the immune system's ability to recognize and mount a specific response against tumor cells, but not normal cells. Current immunotherapeutic approaches for glioma can be divided into three categories: immune priming (active immunotherapy), immunomodulation (passive immunotherapy), and adoptive immunotherapy. Immune priming sensitizes the patient's immune cells to tumor antigens using various vaccination protocols. In the case of immunomodulation, strategies are aimed at reducing suppressive cytokines in the tumor microenvironment or using immune molecules to specifically target tumor cells. Adoptive immunotherapy involves harvesting the patient's immune cells, followed by ex vivo activation and expansion prior to re-infusion. This review will provide an overview of the interactions between the central nervous system and the immune system and discuss the challenges facing current immunotherapeutic strategies.

Keywords: glioblastoma multiforme (GBM), immunotherapy, clinical trials, brain tumors, vaccines, dendritic cells, cytokines

Introduction

Glioblastoma multiforme (GBM) is the most common and lethal primary malignant brain tumor, with an incidence of 5 – 8/100,000 population and a median survival of 14 months [1]. The current standard of care for newly diagnosed GBM patients is a tripartite regimen of surgery, radiotherapy, and chemotherapy. The most meaningful improvement for the treatment of GBM has been the efficacy of temozolomide (TMZ). According to the study conducted by Stupp et al., the median survival rate with radiotherapy alone was 12.1 months compared to 14.6 months with radiotherapy plus TMZ [1]. In addition to the efficacy of TMZ, improvements in delivery have also greatly enhanced the treatment of GBM, including local delivery of chemotherapeutics to tumor cells and convection enhanced delivery (reviewed by Sampson) [2].

A major limitation in the treatment of GBM is its location within the brain and the blood brain barrier (BBB). Recently, evidence of immune surveillance within the CNS and a role of T cells within glioma have led to the development of novel immunotherapeutic strategies [3-6]. Immunotherapy seeks to exploit the immune system's ability to specifically recognize and mount a response against the tumor cells, while leaving the normal brain tissue intact. The success of immunotherapy is fueled by the growing understanding of the immune mechanisms in play within the CNS and glioma immunobiology. These immunotherapeutic strategies fall into three categories: immune priming, immunomodulation, and adoptive immune therapy. Additionally, antibodies or immune peptides fused to toxins have also been used to treat GBM.

The emergence of novel immunotherapeutic strategies has cultivated a renewed optimism for the treatment of GBM. Most of these strategies are focused on the induction of specific immune responses against tumor associated antigens (TAA). Currently, targeting two of these TAA, EGFRvIII (NCT00458601) and IL-13Rα2 (NCT00089427), are in clinical trials and we will discuss them in further detail. Another major immunotherapeutic strategy that has gathered a lot of attention is dendritic cell (DC) vaccination, albeit only demonstrating modest success in clinical trials.

Despite the fact that there are a number of immunotherapeutic strategies currently in clinical trials (Table 1), which were successful in animal models of glioma, convincing evidence of their efficacy remains unclear in patients. It has been difficult to study novel immunotherapeutic strategies in clinical trials because of the rarity of GBM in the population. Moreover, the design of clinical trials is often flawed, especially with regard to patient enrollment in targeted treatment studies. The eligibility criteria should include a screening to assess the expression of target molecules prior to enrollment. The emergence of better imaging protocols, end-point analyses, and substantial improvements in protocol design should further aid in the development of clinical trials to assess the efficacy of targeted tumor therapies.

Table 1
Current clinical trials for patients with malignant glioma.

Target identification

EGFRvIII

The epidermal growth factor receptor (EGFR) is frequently over-expressed in solid tumors. Glioma cells often express a mutated form of EGFR, referred to as EGFR variant III, which has an in-frame deletion from the extracellular domain of the EGFR (Fig. 1) [7-15]. This mutation results in increased tumorgenicity and migration, and confers radiation and chemotherapeutic resistance to tumor cells [16-24]. A retrospective analysis of Japanese patients with GBM enrolled in clinical trials determined that EGFR amplification was a negative prognostic factor, and in cases where EGFR amplification occurred with EGFRvIII, the prognosis was even worse [25]. The restriction of EGFRvIII expression to tumors makes it an ideal target for anti-tumor immunotherapy.

Figure 1
Schematic diagram of the EGFR wild-type protein showing the area of in-frame deletion which forms EGFRvIII. During the deletion amino acids 6 and 273 are split forming a novel glycine at the junction of amino acids 5 and 274. PEPvIII is a 13 amino acid ...

In experimental animal models, EGFRvIII-expressing cell lines or an EGFRvIII-specific 14-amino acid peptide (PEPvIII) chemically conjugated to keyhole limpet hemocyanin (KLH) (PEPvIII-KLH) have been used for the generation of EGFRvIII-specific antibodies and the induction of cellular immune responses [26-36]. EGFRvIII vaccination in mouse models of established intracerebral glioma showed tumor regression compared to controls [27]. EGFRvIII has also been shown to be immunogenic in humans [37, 38]. Purev et al., determined that patients with EGFRvIII-expressing breast adenocarcinomas and malignant gliomas developed EGFRvIII-specific antibodies [37, 38]. The authors also observed weak CTL epitopes restricted by major histocompatibility complex (MHC) Class I and Class II motifs which were sufficient to induce EGFRvIII-specific lymphocyte proliferation and cytokine production.

According to Heimberger et al., EGFRvIII peptide vaccination in animal models of intracerebral and subcutaneous glioma demonstrated significant efficacy over controls [27]. In phase II trials, patients were administered the EGFRvIII peptide vaccine along with temozolomide and radiation, following a complete surgical resection. This study demonstrated efficacy over historical controls. The observed time to progression was 12.8 months and the overall median survival was ≥ 18 months. A peptide vaccine directed against EGFRvIII is currently in phase II trials (NCT00458601). Tumor-specific mutation is targeted currently under a Phase I (conducted at Duke University, PI: John H. Sampson) and one multi-institutional Phase II immunotherapy trial (conducted at Duke University, PI: John H. Sampson; and the University of Texas, M.D. Anderson Cancer Center, PI: Amy B. Heimberger) demonstrating that vaccines targeting EGFRvIII are capable of inducing potent T- and B-cell immunity [38]. The authors surmise that the vaccine approach has been highly successful at eliminating tumor cells expressing EGFRvIII, very similar to the experimental animal model studies, without any evidence of toxicity [10, 38].

The limitations of clinical studies to evaluate the efficacy of peptide vaccines include patient selection and immune editing. First, expression of EGFRvIII should be confirmed prior to patient selection for efficacy studies. Second, immune editing was observed in 20/23 patients with recurrent tumor, as the tumor biopsies failed to express EGFRvIII (unpublished data from CDX-110 clinical trials). Therefore, on the basis of glioma restricted expression of EGFRvIII and mechanism of action in glioma, future trials should focus on EGFRvIII targeting in primary glioma patients to assess efficacy. Moreover, to circumvent immune editing in recurrent glioma, initial treatments should target multiple tumor associated antigens either using whole tumor lysates or personalized peptide vaccines.

IL-13Rα2

Similar to EGFR, IL-13Rα2 is highly expressed in glioma cells, but not normal brain cells, making it a suitable target for immune cell activation [39]. Despite the over-expression of IL-13Rα2 in glioma cells, its role in glioma cells remains undefined. According to a pre-clinical study conducted by Okano and colleagues, the IL-13Rα2 protein contains an antigenic peptide that activates CD8+ T cells to secrete IFNγ and lyse IL-13Rα2+ tumor cells [40]. This deserves further analysis to determine the benefits of IL-13Rα2 targeting in vivo. Furthermore, a fusion protein composed of human IL-13 and Pseudomonas exotoxin A (IL13-PE38QQR) showed limited efficacy in 50 patients that received localized intracerebral administration. Moreover, a phase III study in which the IL-13Rα2 fusion peptide was compared to carmustine wafers was completed and showed no significant benefits. As is the case with EGFRvIII studies, the major challenge facing IL-13Rα2 studies is prospective identification of patients that are likely to respond, based on the expression of IL-13Rα2.

IL-4R

IL-4R is over-expressed in primary tumor specimens and cell lines in a variety of human malignancies, including glioma [41-46]. According to Joshi et al., IL-4 signals via the heterodimeric IL-4Rα and IL-13Rα1 receptor in tumor cells [42]. Therapeutic strategies aimed at specifically targeting tumor cells have utilized IL-4R over-expression using IL-4 fused to Pseudomonas exotoxin (IL4(38-37)-PE38KDEL). In vitro studies using glioma cell lines found IL4(38-37)-PE38KDEL caused glioma cell death, similar to IL13-PE38QQR.[42] Furthermore, in animal models of glioma using human tumors, IL4(38-37)-PE38KDEL was toxic to glioma cells, but largely spared normal brain parenchyma. Phase I trials revealed that IL4(38-37)-PE38KDEL was well tolerated, with no incipient drug related toxicity. The most notable finding from the related dose-escalation study was a long-term survival of three years in a patient with recurrent malignant glioma treated with a single intratumoral dose of IL4(38-37)-PE38KDEL. The findings of these earlier trials were promising and as such, IL4(38-37)-PE38KDEL is still under further consideration.

Dendritic cell vaccination

Dendritic cells (DC) are hematopoietically derived cells that act as antigen presenting cells to activate innate and adaptive immune responses. DC based vaccination strategies seek to exploit the potent APC activity of these cells. The potential to generate large numbers of mature DC in vitro from patient blood or bone marrow has resulted in an abundance of DC based vaccination strategies. These studies have utilized DCs pulsed with either tumor peptides eluted from tumor cells or whole tumor lysates [26, 47-50]. In short, autologous DC are matured and loaded with tumor specific peptides or tumor lysate and then infused into the patient. A few key issues underlie the use of DC cell vaccines and must be resolved prior to the routine use of DC vaccines to treat GBM. These include the best source of DC, the in vitro maturation protocol, the route and dose of DC administration and the source of antigen.

In an early study conducted by Yu and colleagues, four out of seven patients that received DC pulsed with eluted MHC class I peptides had developed cytotoxic responses against the tumor, and at the time of re-operation, two out of those four patients had effector and memory CD8+ T cell infiltrates in the tumor [48]. In this study, DC vaccination was not associated with any adverse side effects. In a phase I trial, 16 patients with malignant glioma were immunized intradermally with autologous DC pulsed with KLH conjugated to EGFRvIII peptides [38]. This study showed promising results based on the increased time to progression and median survival time. Stable disease was observed in two out of three grade III patients. The mean time to progression was 46.9 weeks and the median survival was 110.8 weeks. A similar study conducted by Liau et al., in 12 GBM patients, showed that intradermal infusion of peptide-pulsed DC improved survival compared to historical controls. The median time to progression was 15.5 months and the median survival was 23.4 months [49]. Additionally, 100% survival was observed at six months, 75% at one year, and 50% at two years, with two patients surviving long-term (≥4 year). Presumably, the administration of DC intradermally allowed the DC to traffic to the lymph nodes, where they are able to activate tumor antigen-specific T cells.

One of the largest DC vaccine studies to date, the HGG-Immuno study, conducted by De Vleeschouwer and colleagues, assessed 56 patients with recurrent GBM [51]. The patients were separated into three groups and treated with autologous DC pulsed with autologous tumor lysate, followed by tumor lysate boosts every four weeks. The clinical response was minimal, with a median progression free survival of 3 months and overall median survival of 24 – 36 months. Overall, this treatment strategy was not significantly better than historical controls. Despite disappointing results from these clinical trials, multiple other clinical trials are under way.

A large multi-institutional randomized placebo control study is currently being sponsored by Northwest Biotherapeutics (DCVax-Brain, Phase II, NCT00045968). DCVax-Brain is a personalized (autologous) dendritic cell based vaccine. The vaccine is prepared from PBMC obtained from the patient and are then loaded with tumor lysate from surgically resected tumor tissue. According to the sponsors, in phase I trials, 8 out of 19 GBM patients treated with DCVax-Brain, in addition to the standard of care for GBM, were still alive with stable disease. The median overall survival was 33.6 months. The median time to progression was 18.1 months. In this study, 90% of the patients surpassed the standard of care median time to disease progression of 8.1 months and median overall survival time of 17.0 months. The two year survival rate is 68% and 42% of the patients have survived longer than 4 years (reviewed by Wheeler et al, 2009) [52].

Parajuli and colleagues investigated the best protocol for antigen preparation for DC vaccination strategies [53]. DC were isolated and matured from patient derived PBMC. The four conditions evaluated were: DC fused with glioma cells; DC pulsed with apoptotic tumor cells; DC pulsed with total tumor RNA; and DC pulsed with tumor lysate. All four conditions produced similar amounts of mature DC, however, DC pulsed with apoptotic tumor cells or total tumor RNA were the best at inducing CTL. Furthermore, DC pulsed with apoptotic tumor cells were also able to induce NKT cell activation. Collectively, these data suggest that DC pulsed with apoptotic cells are the best preparation for autologous DC vaccination strategies.

Heat Shock Proteins-tumor peptide vaccination

Heat shock proteins (HSP) are chaperone proteins which are localized to the ER and aid in nascent protein folding and also play a role in antigen presentation via MHC class I (reviewed by Srivastava et al) [54]. Recent studies have shown that at least two HSP, Gp96 and HSP70, have antigenic properties and are able to generate immune responses directed against the proteins to which they are associated [55, 56]. The benefits of using HSP-peptide complexes for vaccination is the potential to limit immune editing since HSP are associated with a broad range of the tumor peptide repertoire. Furthermore, HSP have been identified as potent activators of APCs, making them ideal candidates for tumor immunotherapy [57].

Gp96 has been shown to induce immunity specifically against antigens found in the cells from which it has been isolated. This has been exploited in the case of tumor cells to generate antitumor immune responses. According to Binder et al., one potential mechanism by which this may occur is through cross-presentation by DC via Gp96 binding CD91 expressed on DC [58]. In a study of 12 patients with recurrent high grade glioma, patients received 4 injections over 2-4 weeks. Seven out of 8 patients had a survival time of 10.5 months compared to the historical survival time of 6.5 months [59]. More importantly, Gp96 vaccination has garnered success in the treatment of malignant melanoma and renal cell carcinoma, and hopefully similar success will be obtained in malignant glioma [15, 60-62]. Currently, the Gp96-tumor peptide vaccination strategy is in phase I/II clinical trials (NCT00293423).

Immunomodulation

Cytokines

The cytokine milieu of the CNS ensures that primarily humoral immune responses are generated in order to prevent damage due to inflammation. The normal humoral response is further skewed in glioblastoma patients [63, 64]. Additionally, immunosuppressive cytokines, such as TGF-β2 and IL-10, are highly expressed in glioma cell lines and patient specimens [65-68]. These cytokines suppress T cell proliferation and IL-2 production, and also support glioma cell growth. To alter the cytokine milieu of glioma, studies have focused on supplementing the immuno-activating cytokine IL-2, or conversely, inhibiting the immunosuppressive cytokine TGFβ.

IL-2 is the cytokine most often associated with T cell activation and expansion. Recent studies have shown that IL-2 is required for differentiation of naïve T cells into cytokine producing effector cells. According to a study conducted by Colombo and colleagues, IL-2 was administered as a transgene in combination with herpes simplex virus tyrosine kinase in a retroviral vector to 12 patients with recurrent GBM [69]. Two out of the 12 patients had a partial response, four had a minor response, four had stable disease, and two had progressive disease. In another study, five patients with recurrent glioma were infused with IL-2 in combination with cytotoxic T cells [70]. Although, two patients with GBM died, the other patients showed no evidence of tumor at least 28 weeks post-treatment. These studies suggest that IL-2, either in combination with effectors cells or alone, may be beneficial in the treatment of glioma. However, it is important to note that these studies were small, and lacked adequate randomization or controls.

TGF-β2 was originally named for its ability to suppress T cell growth and IL-2 production, and was isolated and cloned from glioblastoma cell lines [67, 71]. The expression of TGF-β1 and -β2 in two glioblastoma cell lines and newly isolated patient samples was confirmed at the mRNA level [65]. However, only TGFβ-2 was detected in the supernatant of glioma cell lines and in the cerebral spinal fluid of patients with malignant glioma [66]. Primary glioma cells treated with antisense TGFβ-2 (Antisense Pharma, AP 12009) showed a significant reduction in TGFβ-2 expression from 73% positive cells to 49% positive cells, and glioma cell proliferation [72]. According to a phase I/II trial (NCT00844064), Hau and colleagues showed promising results in 24 patients with malignant glioma treated with antisense oligonucleotides (AP 12009) [72]. A complete remission was observed in two patients with anaplastic astocytoma (AA), and the overall survival in AA (146.6 weeks) and GBM (44 weeks) patients was increased relative to historical controls. The two year survival for the treatment group was 80%. This immunotherapeutic strategy is designed to improve the immune system's ability to mount antitumor immune responses and is currently in phase III trials (NCT00761280).

Interferons are normally expressed in response to altered cells. In animal models, IFN-α and -β inhibit glioma growth. On this basis, IFN has been investigated in multiple clinical trials for the treatment of malignant glioma. A phase I trial using IFN-α in combination with BCNU, as an initial treatment modality for high-grade glioma, found that five of nine patients had a partial response and a median survival of four years [73]. In a phase II trial of 21 patients with recurrent high-grade glioma, seven patients had partial response and six patients maintained stable disease following treatment with IFN-α and BCNU [74]. In contrast to these earlier trials, a phase III trial of 214 eligible patients with high-grade glioma, in which patients received BCNU in combination with IFNα, the response was no better than in patients that received BCNU alone, with regard to time to disease progression or overall survival [75]. A few caveats of the early studies involved patient selection and inconsistent endpoint analysis. It is important to note that in addition to being ineffective for the treatment of glioma, systemic IFN administration also causes severe adverse reactions, including neurocortical effects, fever, chills, and myalgias.

Depletion of regulatory T cells

Regulatory T cells (Tregs; CD4+CD25+FOXP3+) Tregs are a fraction of the T cell population that suppress immune activation and thereby maintain homeostasis and tolerance to self-antigens. Functional deletion of Tregs induce autoimmunity, facilitates transplantation tolerance and also increases immunity to tumors [3, 76, 77]. A lack of immune rejection of neoplastic cells is believed to be maintained by Tregs in many malignancies including colorectal, esophageal, pancreatic, breast, lung, ovarian and brain tumors [3, 78-81]. An increased fraction of regulatory T cells has been reported to infiltrate glioma contributing to the immunosuppressive status associated with glioma [3-5, 82, 83]. It is therefore very important to understand the biology and function of Tregs for its potential therapeutic potentials.

The precise mechanism(s) by which Tregs suppress effector T cell-mediated immune response have not been definitively characterized. Some studies highlight the importance of cytokines in the regulation, and others, cell-to-cell contact with effector T cells in which case membrane bound TGF-β and cytotoxic T-lymphocyte protein (CTLA-4) plays an important role.[84-86] Heme oxygenase-1 (HO-1), a rate limiting enzyme in heme metabolism also plays a role in Treg mediated immune suppression. HO-1 is constitutively expressed in human Tregs and is induced by FoxP3 expression [87, 88]. It is suggested that HO-1 suppresses effector T cell by carbon-monoxide production [89, 90].

In 2006, our group demonstrated tumor infiltration of Tregs in glioblastoma multiforme (GBM) patients [4]. The expression of FoxP3+ Tregs was significantly higher in patients with GBM than in controls, whereas these cells were absent from control brain specimens. Higher levels of FoxP3 expression were observed in regulatory T cells isolated from the tumor tissue in comparison to autologous patient blood and blood from control individuals. In an in vitro suppression assay, Tregs inhibited T cell proliferation in a dose-dependent manner. Among various markers analyzed, the expression of CD62L and CTLA-4 was elevated in the glioma infiltrating Tregs in comparison to that of the controls. We showed improved survival of mice with experimental brain tumors, following the depletion of Tregs with anti-CD25 monoclonal antibody (PC61) [3].

A prominent population of Tregs and a corresponding lack of effector/activated T cells was demonstrated in GBM patient specimens [82]. Absolute counts of both CD4+ T cells and FoxP3+CD45RO+ Tregs were greatly diminished in the peripheral pool of patients with malignant glioma, but the Tregs fraction was increased in the remaining CD4 compartment in 5 out of the 8 patients evaluated [5]. The proportion of Tregs in the peripheral blood of patients with GBM was 2.63 times higher than that found in the blood of normal volunteers. The patients with an elevated Tregs fraction showed significant CD4+ T cell lymphopenia, whereas the patients without Tregs elevation possessed normally proliferating CD4+ T cell levels. T cells from the patients bearing malignant gliomas regained their function after Tregs depletion in vitro, and proliferated to levels equivalent to those of normal controls.

The depletion of Tregs is normally achieved using anti-CD25 antibodies, which may also deplete activated T cells which express CD25. Curtin et al demonstrated the efficacy of immunotherapy using anti-CD25 depleting antibodies (PC61) in experimental animal of glioma. Interestingly, the efficacy of Tregs depletion was time dependent and greatly influenced by tumor burden [91]. Systemic depletion of Tregs 15 days after tumor implantation improved long-term survival, but Tregs depleted 24 days after tumor implantation showed no improvement in survival. It is very important to note this observation suggests that immunotherapy alone may not be the fail-safe therapeutic strategy. Moreover, Tregs depletion should be performed prior to immunotherapy to limit depletion of effector cells along with Tregs following the administration of anti-CD25 antibodies.

Small Molecule Inhibitors of STAT-3

Signal transducer and activator of transcription-3 (STAT-3) is a convergence point of several signaling pathways in quite a multiple malignancies including glioblastoma, breast, lung, ovarian, pancreatic, skin and prostate cancer [92, 93]. It has recently emerged as a potential target for glioma immunotherapy. The binding of STAT3 to its target genes affects proliferation, survival, differentiation and development. Receptor engagement by members of IL-6 cytokine family like IL-6, oncostatin M and Leukemia inhibitory factor, or growth factors like platelet-derived growth factor (PDGF), fibroblast growth factor (FGF) and epithelial growth factor (EGF) activate STAT-3. The activation of STAT-3 requires the activation of receptor-associated kinases like Janus kinase (JAK) family memebers, FHFR, EGFR, PDGFR or nonreceptor-associated kinases like Ret, Src or Bcl-Abl. STAT-3 activity is attenuated by suppressors of cytokine signaling (SOCS) by down-regulating its upstream kinase activity, while protein inhibitors of activated STAT (PIAS) and protein tyrosine phosphatases target STAT-3 directly [94-96]. Other than promoting oncogenesis, active STAT-3 also enables tumor growth by suppressing tumor recognition by the immune system.[97] STAT-3 promotes tumor immune evasion by inhibiting pro-inflammatory cytokine signaling and amplifying Tregs. STAT-3 activity in cancers other than glioblastoma has been targeted in several different therapeutic strategies. STAT-3 inhibition has been approached from two fronts either through RNA interference or chemical inhibitors, or through modulation of endogenous regulators such as PIAS3 and SOCS-3 (Fig. 2).

Figure 2
Inhibition of STAT-3 signal transduction. A variety of endogenous and pharmacologic inhibitors can attenuate STAT-3 signaling. SOCS-3, PIAS3, and various protein tyrosine phosphatases (PTP) inhibit STAT-3 activity endogenously. STAT-3 – specific ...

Several compounds block STAT-3 signaling by directly targeting the STAT-3 protein. Platinum compounds like CPA-1 and CPA-7 have been successfully used to block STAT-3 activity and induce apoptosis in breast, lung, and prostate cancer cell lines [98]. More recently, Zhang et al (2009) used CPA-7 to successfully block STAT-3 activation in glioma-associated microglia [99]. Decoy oligonucleotides like G-quartets or Transcription factor decoy (TFD) oligodeoxynucleotides, and inhibitors like S31-201 have been used by researchers for directly blocking STAT-3 signal transduction in human cancer cells [100-103]. Furthermore, the knock down of STAT-3 in human glioma cell lines by STAT-3 siRNA induced apoptosis and inhibited survival [104, 105]. STAT3 decoy ODN treatment in U251 and A172 glioma cell lines blocked STAT3 signaling and inhibited glioma proliferation by inducing apoptosis and cell-cycle arrest [103].

The pharmacological inhibitors of growth factor receptors and upstream tyrosine kinases have also been very successful at blocking STAT-3 activity. Inhibitors of JAK and Src showed potential STAT-3 inhibition and are in early stages of experimental testing [106, 107]. Preliminary in vivo studies showed that WP1066, a JAK inhibitor, has the potential to cross the blood-brain barrier, which is very important for glioma patients. WP1066 abrogated immune tolerance in glioblastoma patients and stimulated T cell proliferation by up-regulating secretion of costimulatory molecules and T-cell effector cytokines and improved immunogenic responses [108]. In an independent experiment, growth of glioma xenografts was restricted by decreased STAT-3–mediated expression of Bcl-xL, Mcl-1, and c-Myc when STAT-3 was inhibited with WP1066 [109]. The effects were also very tumor-specific as normal astrocytoma cells were not affected. Attenuation of upstream FGF signaling pathway by dobesilate, a vasoactive drug, in C6 glioma cells triggered apoptosis and growth arrest by inhibiting STAT-3 activation [110]. These observations illustrate a possible relationship between STAT-3 and glioblastoma. Pharmacologic inhibitors of individual kinases that are in command upstream of STAT-3 inhibitors therefore might be an ideal candidate for potential therapeutic intervention of glioma progression.

Active Immunotherapy

LAK cells

In vitro studies using tumor cells from a variety of malignancies, including glioma, showed lymphokine activated killer (LAK) cell lysis [111, 112]. Human studies conducted by Rosenberg et al., showed therapeutic benefits of LAK cells in multiple types of tumor cells and they were largely inefficient at lysing normal tissues [113]. A phase I study evaluated 10 patients with recurrent GBM following surgical resection and intratumoral injection of LAK and IL-2 [114]. In this study, steroids were restricted during treatment, unless required for the treatment of acute symptoms of IL-2 toxicity (edema and confusion). The therapeutic efficacy of LAK cells was characterized by a median survival of 53 weeks, with 53% of the patients still being alive after one year, compared to a median survival of 25.5 weeks for the chemotherapy alone group [115]. This study highlighted the potential benefits of LAK cell infusion for the treatment of glioma. To date, the mechanism of action of LAK cells remains unclear, thereby limiting their use in immunotherapy. Furthermore, LAK cells must be administered locally at the tumor site since they fail to effectively home to tumor lesions. In light of these factors, immunotherapeutic strategies have moved from LAK cells toward T cells. Moreover, T cells have been to be more lytic than LAK cells, on a per cell basis.

Effector T cells

Adoptive immunotherapy has emerged as a novel treatment modality for multiple cancers. The use of tumor specific T cells was based on the belief that tumor antigen specific T cells could traffic to tumor lesions and preferentially target tumor cells, over non-tumor cells. In many of these studies, autologous T cells are primed against tumor antigens and expanded in vitro prior to re-infusion. Using an animal model of glioma, adoptively transferred CTL were shown to effectively home to and reject tumors following intravenous administration [116]. According to Yamasaki et al., the mean survival time following intravenous administration of in vitro expanded CTLs was over 15 weeks (except for one animal that died at 10 weeks) compared to approximately 3.3 weeks in vehicle only or in vivo primed CTLs isolated from the draining lymph nodes (3.6 weeks) and spleen (2.0 weeks). Further analysis revealed that the CTL activity of the adoptively transferred cells was specific for tumor cells and not non-glia tumor cells. The ability to generate and maintain tumor specific T cells was a major advantage compared to LAK cells, and propelled it to prominence in the field of adoptive immunotherapy.

In one such study, Kitahara et al., generated CTL in vitro from the blood of five malignant glioma patients [117]. Briefly, the peripheral blood lymphocytes were cultured with autologous tumor cells plus IL-2, in order to generate CTL, which were later administered intracranially. The results from this study were largely poor. One patient showed a transient regression for 20 weeks prior to recurrence and one patient had a complete regression to at least 104 weeks. Three other patients progressed quickly and died from recurrent tumor. This study underscored the potential benefits of this treatment modality and served as a building block for future trials.

The use of autologous tumor cells to sensitize CTL in vitro requires the isolation and maintenance of tumor cells. Furthermore, in order to increase the amount of T cells harvested from peripheral blood, recent studies used BCG vaccination in combination with GM-CSF and IL-2 infusion. In an attempt to circumvent these issues, a more recent study of nine high-grade glioma patients utilized anti-CD3 for polyclonal T cell activation in combination with IL-2 [118]. Two patients with grade III disease had complete tumor regression to atleast five years, and one patient had a partial regression. This treatment strategy was not effective in the GBM patients. Plautz et al obtained encouraging results using autologous CTL [119]. Patients were infused with GM-CSF and T cells were isolated from the draining lymph nodes. Two patients showed tumor regression, one patient did not observe tumor growth out to 17 months, while the remaining 7 patients had progressive disease. Overall, all patients with GBM survived at least a year. Although polyclonal stimulation with anti-CD3 stimulates a large pool of T cells, which may include tumor specific T cells, the frequency of these cells may be relatively low in the entire T cell pool thus minimizing their therapeutic efficacy.

More recent studies sought to isolate and expand tumor infiltrating lymphocytes (TIL), however, no clear therapeutic benefits were observed. According to Quattrocchi et al., six patients with high grade glioma were treated with autologous TIL plus IL-2 in the tumor cavity following surgical resection [120]. Cerebral edema was the only adverse side effect noted. One in six patients demonstrated tumor regression and was tumor free at 45 months. A limitation of this study was the fact that the TIL were simply re-infused without depleting suppressor cells, which have been to be highly suppressive and abundant in TIL. Future trials should seek to deplete Tregs prior to re-infusion and may consider ex vivo activation and expansion to increase the cytotoxic function of TIL.

Conclusions

Novel immunotherapeutic strategies have emerged as the understanding of CNS immunobiology and gliomas has progressed. The anatomical location of glioma within the CNS is beneficial for tumor progression and limits the success of many treatment modalities. Multiple groups, including ours, have demonstrated the therapeutic efficacy of immunotherapy in pre-clinical models of glioma, but these have yet to show clinical efficacy. We suggest that the observed deficiencies of many of these treatment modalities are linked to the poor design of many of the clinical trials. Additionally, large randomized studies are often difficult to conduct since GBM is rare. Moreover, many preclinical trials are conducted in immune comprised animals making extrapolation to immune competent hosts difficult.

In order to truly realize the promise of immunotherapy modalities, there needs to be improvements in study design. To date, EGFRvIII has emerged as the key molecule for tumor targeting. As is the case with other targeted therapies, EGFRvIII vaccination has seen minimal successes in the clinic due to poor patient selection. Further, better end point analyses are required to determine treatment efficacy. In brief, the induction of an immune response does is not always correlative with improved time to tumor progression or overall survival. Therefore, studies should clearly define enrollment criteria and result interpretation prior to study initiation, so that we are able to truly realize the therapeutic efficacy of immunotherapy.

Footnotes

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