New Perspectives in Glioma Immunotherapy

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New Perspectives in Glioma Immunotherapy Antonio Daga*, Cristina Bottino§, Roberta Castriconi°, Rosaria Gangemi* and Silvano Ferrini* *Department of Translational Oncology, National Institute for Cancer Research, 16132 Genova, Italy, °Department of Experimental Medicine (Di.Me.S), University of Genova, 16132 Genova, Italy, §Giannina Gaslini Institute, 16147 Genova, Italy Abstract: Glioblastoma (GBM) is a deadly tumor, which in spite of surgery and radio/chemotherapy frequently undergoes relapses related to the infiltration of the normal parenchyma and to resistance to cytotoxic and radiation therapy. Immunotherapy may represent a promising approach, which may complement existing therapies with the aim of eliminating residual tumor cells, through their selective targeting by immune effector cells or antibodies. This goal can be achieved through different approaches, based either on the induction of an immune response of the host, or by the injection of in vitro generated effector cells or monoclonal antibodies. Recent advances in the immunobiology of GBM and of its stem cell compartment will help in the development of more effective immunotherapy protocols. To this aim, the identification of antigens and receptors involved in GBM/immune cell interactions and of GBM immune escape mechanisms will provide new targets and tools. In this review we will discuss active immunotherapy approaches, including molecular-defined, GBM cell-based and dendritic-cell based vaccines. In addition, cytokines such as interferons and several interleukins can be used to enhance the immune response, both as recombinant molecules and by gene transfer technologies. Monoclonal antibodies or other ligands specific for GBM- or neovasculature-associated targets are now available in different genetically modified formats and can be used as such or for the targeted delivery of active compounds. Finally the in vitro activation and expansion of specific or innate immunity effector cells endowed with anti-GBM properties may provide an additional weapon for adoptive imunotherapy approaches.

Keywords: Glioblastoma, immune regulation, interleukin, interferon, T cells, NK cells, dendritic cells, vaccines, monoclonal antibodies. INTRODUCTION GBM is the most malignant of all astrocytic tumors and consists of poorly differentiated neoplastic astrocytes. Histopathological features include nuclear atypia, presence of frequent mitotic figures, cellular polymorphism, prominent microvascular proliferation associated with vascular thrombosis and/or necrosis and formation of pseudo-palisades. Regional heterogeneity and highly invasive growth are typical of GBM, which preferentially affects adults and locates in the cerebral hemispheres. GBM may develop from diffuse WHO grade II astrocytomas or anaplastic astrocytomas (secondary GBM). However, in most instances GBM present as primary tumor, without previous evidence of a less malignant precursor lesion. About 87% of patients with GBM are between 55 and 84 years of age and their prognosis is very poor, with relative survival rates of less than 34% at one year and 4.5% at five years [1]. Indeed, in spite of progresses in surgery, radiotherapy and chemotherapy, most patients relapse. Surgery is the first therapeutic modality for GBM and its optimal result would be complete resection. However, GBM is a highly infiltrative tumor, whose complete resection is virtually impossible, and relapse is almost inevitable. Therefore the only feasible goal of surgery is the bulk reduction and decompression of the brain, which leads to alleviation of cranial hypertension, to a transient improvement of quality of life and, possibly, to increased survival [2]. Fractionated external-beam radiotherapy has been the standard post-operative treatment, as it approximately doubled the overall survival. Indeed, two phase III randomized trials demonstrated a significant prolongation of survival for patients receiving postoperative radiotherapy [3, 4]. Several randomized clinical trials have studied the role of adjuvant chemotherapy in the survival of brain tumor patients, but limited effects have been reported using different regimens [5-7], until a phase III randomized study of the alkylating drug temozolomide *Address correspondence to this author at the Immunotherapy Unit Istituto Nazionale per la Ricerca sul Cancro, C/o CBA Torre C2 Largo R. Benzi 10, 16132 Genoa, Italy: Tel: 39-010-5737-372; Fax: 374: E-mail: [email protected] 1381-6128/11 $58.00+.00

(TMZ) was completed (EORTC 22981/26981). This study, which compared radiotherapy alone vs. TMZ administered concomitantly and after radiotherapy, demonstrated a significant improvement in median survival from 12.1 to 14.6 months, and in 2-year survival from 10% to 26%, respectively [8] [9]. In view of the minimal additional toxicity of TMZ, this treatment has become the actual standard for newly diagnosed GBM. Further studies showed that the expression of the direct reversal repair protein O6-methylguanine-DNA methyltransferase (MGMT) by GBM mediates resistance to TMZ, by removing alkyl groups from the O6 position of guanine. Instead, GBM showing methylation of the MGMT promoter region, are more sensitive to TMZ treatment [10]. Indeed, patients with methylated MGMT promoter treated with TMZ and radiotherapy had a median survival of 22 months and a 2-year survival rate of 46%, which was significantly higher as compared to patients with an unmethylated promoter (median survival time of 13 months and a 2-year survival rate of 14%). In spite of the improvement of survival in a subset of patients, in general the prognosis of GBM patients is still very poor and the development of new treatment modalities such as immunotherapy is urgently needed. Immunotherapy seems particularly attractive as it acts through different mechanisms than those of standard cytotoxic treatments, it may lead to different toxicities and may therefore complement existing therapies, with the specific goal of eliminating residual tumor cells after standard treatments. GLIOMA IMMUNOBIOLOGY CNS as Immune Privileged Site: the Blood Brain Barrier In the past, the CNS has been described as an immune-privileged organ, where serum antibodies or immune cells are not allowed to enter [11]. Indeed, the Blood Brain Barrier (BBB) blocks the passage of molecules greater than 500 daltons [12] due to the occluding tight junctions between adjacent cerebral endothelial cells, the closely associated pericytes [13, 14] and glial cells [15]. As a result, the brain is isolated from most peripheral immune cells, soluble factors, and plasma proteins. Moreover, the CNS lacks a conventional lymphatic system, which allows the trafficking of dendritic and lymphoid cells. Neurons also contribute to the im-

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mune-privilege of the CNS, as they are an important source of TGF-beta, a pleiotropic cytokine, that is involved in immune regulation [16]. Furthermore, TGF-beta down-regulates the expression of adhesion molecules and chemoattractant proteins by both astrocytes and endothelial cells, preventing lymphocyte entry into the brain under physiological conditions [17] [18]. Finally, cells of the CNS express low levels of major histocompatibility complex (MHC) molecules, also known as human-leukocyte antigens (HLA), which are required for antigen presentation to the T cell compartment [19]. However, more recent data indicate that the immune privilege of the brain is not absolute and, despite the factors minimizing CNS immune function, effective immune responses can occur in the CNS [20] [21]. For example a particular subpopulation of T helper (Th) cells named Th17, because of its unique ability to produce interleukin (IL)-17 [22], has been found in lesions from patients with Multiple Sclerosis (MS) [23]. Moreover, in Experimental Autoimmune Encephalomyelitis (EAE), the mouse model that resembles human MS, it has been demonstrated that CCR6 is required for the migration of Th17 cells into the CNS and that mouse and human epithelial cells of the choroid plexus constitutively express CCL20, the CCR6 ligand. These data strongly suggest BBB as a new front door for inflammatory cells into the CNS [24]. In addition, different districts within the CNS display variable levels of immune competence. The immune response in meninges and choroid plexus of the ventricles is similar to that of other organs, whereas the brain parenchyma shows a lower level of immune reactivity. Although GBM develops in the parenchyma, it must be considered that in GBM the BBB appears to be disorganized due to the abnormal structure of brain capillaries into the tumor and a dysfunction of tight junctions between endothelial cell [25] [26]. Indeed, it is well known that contrast agents used for imaging techniques, such as Magnetic resonance imaging (MRI) and Computed Tomography (CT), accumulate particularly in the peripheral area of GBM due to the rich neovascularization, which lacks of a functional BBB. Glioma-mediated Immune Suppression and Escape It is well established that tumors display several mechanisms of immune evasion or suppression and can also recruit immuneregulatory subsets of lymphoid or myelomonocytic cells, which limit the onset of an efficient immune response [27]. These mechanisms are particularly active in the tumor microenvironment, although they may lead to systemic effects, particularly at advanced tumor stages. These general concepts also apply to GBM, which express a variety of immune suppressive molecules such as cytokines, surface proteins, enzymes and gangliosides. Among suppressive cytokines VEGF, IL-10, IL-6 and TGF-beta are expressed in GBM, which also produce chemokines such as CCL2 and CCL22 involved in the recruitment of immune regulatory cells. VEGFs are pro-angiogenic cytokines that bind to specific tyrosine-kinase receptors (VEGF-R) present on endothelial cells [28], on myelomonocytic cells and on their hematopoietic precursors [29]. GBMs are highly vascularized tumors and a high VEGF expression and microvessel density have been related to recurrence and poor survival [30] [31]. Besides its crucial role in neoangiogenesis, VEGF also mediates immune suppression, by inhibiting the differentiation of dendritic cell (DC) precursors to antigenpresenting cells (APC), capable of priming efficient T cell responses [32] [33] [34] [35]. This defect in DC maturation has been associated to an enhanced signaling via the Jak2/STAT-3 pathway [36] and to an impairment of the NF-kB pathway [33]. IL-6 belongs to a family of structurally related proteins, which also include Oncostatin M (OSM), ciliary neurotrophic factor (CNF), and leukemia inhibitory factor (LIF). IL-6 expression is induced by several stimuli, such as hypoxia, inflammatory mediators, and IL-6 cytokines themselves, which are up-regulated in GBM [37] [38] [39]. Both IL-6 and OSM are expressed in GBM cells in

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vivo and in vitro [40, 41] [42], are involved in activation of STAT-3 in GBM, through autocrine pathways [43] and are required for GBM development. Indeed neutralizing antibodies to IL-6 reduced STAT-3 activation and inhibited GBM growth [44] and IL-6-/transgenic mice (GFAP–v-src heterozygous), failed to develop spontaneous glioma [45]. IL-6 has been involved in STAT-3-dependent induction of proinvasive and angiogenic molecules, such as matrix metalloproteinase-9 and VEGF [39], and similarly to VEGF, it inhibits T cell responses by blocking DC maturation. IL-10, initially defined Cytokine Synthesis Inhibitory Factor, is an immune-suppressive and anti-inflammatory cytokine, which is produced by several tumor cell types, including GBM cells [46] [47] [48] and cells with immune-suppressive activity such as tumorassociated macrophages (TAM) or tumor-infiltrating or peripheral blood lymphocytes from cancer patients [49] [50]. In this context, GBM cells have been shown to induce the differentiation of monocytes to Myeloid-Derived Suppressor Cells (MDSC) [51], which produce IL-10 and TGF-beta. Wu et al. recently reported that the GBM stem cell cultures producing TGF-beta, CSF-1 and macrophage-inhibitory cytokine-1 (MIC-1) mediate the differentiation of microglial cells towards IL-10- and TGF-beta-producing M2 type macrophages [52]. Similarly to VEGF, IL-10 alters DC differentiation and function, as it inhibits their IL-12 and CD86 co-stimulatory molecule expression [53] [54] [55] and induces their differentiation towards “tolerogenic DC”, thus preventing the induction of a T cell response [53] [54] [55-57]. In addition, IL-10, inhibits the production of Th1 cytokines [58] and of pro-inflammatory cytokines in macrophages [59] [60]. Also the chemokine CCL2, previously known as macrophagechemoattractive protein-1 (MCP-1) is produced by GBM cells and has been originally purified from the supernatant of a human glioma cell line [61]. CCL2 is an important factor in the recruitment of TAMs [62], which then undergo M2 polarization in the tumor microenvironment [63]. Therefore CCL2 is involved in tumor immune suppression, but also displays pro-angiogenic activity [64] and has been involved in GBM cell proliferation [65] and invasiveness [66]. TGF-beta, which is produced by normal neurons, is highly expressed also in GBM, where it has been involved in tumor progression and in tumor-related immunosuppression [67] [68] [69]. TGFbeta directly suppresses T cell-mediated immunity by limiting T cell proliferation [70] and by inhibiting the expression of IFNgamma, Perforin, Granzyme A and B, and FASL in CD8+ T cells, thus reducing their anti-tumor cytotoxic activity [71]. Differently from T cells, GBM cells are resistant to the anti-proliferative effect of TGF-beta due to mutations of TGF-beta signaling components, including the loss or promoter methylation of the p15 (Ink4b) gene [72, 73] [74]. TGF-beta also inhibits IFN-gamma production by natural killer (NK) cells and T cells [75] and affects NK cell cytotoxicity [76-78]. In particular, TGF-beta strongly inhibits the surface expression of NKp30 and NKG2D, triggering receptors that are involved in the interaction of NK cells with DC and tumors [7981]. In addition, TGF-beta is an essential factor for the maintenance of thymic-derived Treg cell phenotype and functions in the periphery [82]. Moreover, it induces the peripheral conversion of CD4+CD25-FoxP3- precursor cells into immune suppressive CD4+CD25+FoxP3+ Treg cells [83] [84]. Indeed, Treg cells increase in peripheral blood of GBM patients in the context of decreased CD4+ T cell counts and infiltrate and accumulate in GBM tumors [85] [86] [87]. A recent report indicates that the percentage of tumor infiltrating Treg cells is correlated with the histological grade of brain tumors and suggests an inverse correlation between Tregs counts and survival [88]. Moreover, GBM secrete the CCL22 chemokine, which contributes to the accumulation of Tregs, which express CCR4, a CCL22-interacting chemokine receptor [89]. Therefore Tregs may participate in the generation of a immune

Glioma Immunotherapy

suppressive microenvironment in GBM, as well as in the multiple immune system dysfunctions found in GBM patients, such as reduced antibody and delayed type hypersensitivity and CTL responses [90]. GBM cells also display surface Fas-L and CD70, which contribute to tumor-related immune suppression. These molecules are transmembrane type II proteins belonging to the TNF family ligands and bind to the receptors of the TNF-R family FAS and CD27, respectively. Fas-L mediates T cell apoptosis through the receptor FAS, which activates the caspase cascade and is involved in the control of tolerance to self-antigens [91]. GBM cells expressing Fas-L may therefore mediate death signals to FAS+ T lymphocytes [92], whereas the tumor cells are resistant to FAS-mediated apoptosis. Similarly surface expression of CD70 on GBM cells may mediate apoptotic signals to CD27+ T cells [93]. Similar to Fas-L and CD70, also Receptor-Binding Cancer Antigen Expressed on SiSo cells (RCAS-1) is expressed on the GBM cell surface and mediates induction of apoptosis on T, B and NK lymphocytes through the interaction with still unknown surface receptors [94]. Interestingly the expression of RCAS-1 in GBM correlates with histological grade, apoptosis of tumor-infiltrating lymphocytes and prognosis, thus suggesting a potential role in tumor aggressiveness [95]. Another surface molecule, expressed by several tumors including GBM [96] [97] and by tolerogenic DC, is B7-H1 (CD274), also known as Programmed cell death 1 ligand 1 (PD-L1), a member of the growing B7 family of cell-surface ligands, which includes also the well known B7-1 (CD80) and B7-2 (CD86) molecules [98]. B7H1 interacts with the Programmed cell death 1 (PD-1) receptor, which is expressed by T lymphocytes and mediates cell apoptosis [99] [100]. A recent study shows that freshly isolated NK cells from patients with Multiple myeloma (MM), but not from healthy donors, express PD-1. Moreover, PD-1 engagement by PD-L1 present on tumor cells results in down-modulation of the NK cell cytotoxicity against MM. Importantly, the disruption of the PD-1/PD-L1 signaling axis through the use of a novel anti–PD-1 antibody, restored the NK cell function against autologous, primary MM cells, thus representing a promising novel therapy for MM [101]. Another member of B7 family is represented by B7-H3, which is characterized in humans by 4 or 2 Ig like domains. Functional data suggest that T and NK lymphocytes express a B7-H3 receptor displaying inhibitory function. In particular, 4Ig-B7-H3 is expressed by neuroblastoma cells infiltrating the bone marrow of stage 4 patients and exerts a protective role from NK-mediated lysis [102]. Moreover, the high expression of 4Ig-B7-H3 has been shown to correlate with worsening clinicopathologic features and poor prognosis in neuroblastoma [103] as well as in prostate [104], pancreatic [105] and breast [106] cancers. High expression of 4Ig-B7-H3 was detected also in GBM cells cultured under stem cell conditions (unpublished data). However, its role in NK/GBM interactions remains to be investigated. Lack or downregulation of classical HLA-class I antigens can mediate immune escape by preventing CTL-mediated antigen recognition in several tumors, including glioma [107] [108]. However, an unusual immune escape mechanism is related to the spontaneous or IFN-gamma induced expression of the HLA-G or HLA-E antigens on GBM [109] [110] or on glioma-associated microglial cells [111]. Human leukocyte antigen (HLA) –G and -E are non classic HLA class I molecules displaying very low polymorphism as compared to classic HLA class I molecules (HLA-A, -B and –C). HLAE is expressed in all HLA class I+ tissues since it assembles at the ER with peptides derived from the signal sequences of classical HLA or of HLA-G molecules. HLA-E is recognized by the CD94/NKG2A heterodimer, a receptor complex displaying inhibitory function that is expressed by NK cells and CTL [112]. Interestingly, as demonstrated for HLA-A, -B, and –C, soluble HLA-E

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molecules can be generated by a membrane-bound metalloproteinase [113]. Different from HLA-E, HLA-G shows a restricted tissue expression comprising trophoblast, thymus, cornea, erythroid and endothelial precursors. However, HLA-G expression can be induced in pathological situations such as autoimmune, inflammatory and viral infectious diseases, cancer and transplantation. HLA-G shows a complex splicing pattern: different alleles yield at alternatively spliced isoforms including membrane-bound (HLA-G1, -G2, -G3, G4) and soluble HLA-G5, -G6 and -G7 proteins. Moreover, soluble HLA-G can further be generated by metalloproteinase-mediated release of surface membrane-bound HLA-G [114]. HLA-G is the sole ligand of KIR2DL4 (CD158d), a receptor belonging to the Killer cell Immunoglobulin-like Receptor (KIR) family whose expression is mainly restricted to CD56bright CD16- NK cells, which represent a minor subset in peripheral blood, while representing the majority of uterine NK cells. Additional HLA-G binding receptors are represented by ILT2 (CD85j, LILRB1) and ILT4 (CD85d, LILRB2). Both lymphoid and myeloid cells express ILT2, while ILT4 is present in myeloid cells only. ILT2 and ILT4 are clearly inhibitory receptors, whereas KIR2DL4 seems capable to transduce both inhibitory and activating signals, having both an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic tail, and a positively charged arginine in the transmembrane region [114]. Functionally, the membrane-bound and soluble HLA-G molecules as well as HLA-E are capable of blocking the cytolytic function of NK and T lymphocytes. Moreover, HLA-G was proved to contribute to the expansion of Tregs. In successful human pregnancy the balance between active immunity and tolerance at the decidua, i.e. the site of contact between mother and child, is of critical importance. Thus, effective immunity must be maintained to protect the mother from harmful pathogens. On the other hand, the process of pregnancy must also include mechanisms capable of preventing rejection of the fetus, which can be considered as semiallograft. Indeed, most decidual NK cells express CD94/NKG2A, ILT2 and KIR2DL4 receptors and their interactions with HLA-G and –E expressed by extravillous cytotrophoblasts would play a crucial role in protecting the fetus from maternal uterine natural killer cytolysis [115]. Interestingly HLA-G was first identified in choriocarcinoma cells and HLA-G is expressed/released by a variety of hematological and non-hematological malignances including glioma [116]. Tumors are subjected to hypoxia, which leads to the induction of HIF-1alpha and express the enzyme indoleamine 2,3dioxygenase (IDO) at high levels which leads to tryptophan depletion and kynurenine accumulation. HIF-1alpha and kynurenine have been shown to induce HLA-G expression. Moreover, HLA-G (and HLA-E) is expressed also by tumor infiltrating cells such as activated microglia/macrophages present in GBMs [111]. In different tumors HLA-G expression was associated with high-grade histology and advanced stage of the disease and poor prognosis [117]. Altogether, experimental and clinical evidences propose HLA-G and HLA-E expression/release in the tumor microenvironment as a mechanism of tumor immune escape and as a prognostic factor. Also some small immunosuppressive molecules such as IDOgenerated catabolites and Prostaglandins (PGs) have been involved in GBM immune suppression. IDO catalyzes the degradation of tryptophan to N-formylkynurenine, which subsequently deformylates to L-kynurenine. Lkynurenine and its catabolites picolinate and quinolinate, are immunosuppressive, and their activity is increased by the concomitant tryptophan depletion [118] . While IDO activity is fundamental in tolerizing the mother toward the semi-allogeneic fetus [119], IDOdependent immune regulation contributes to GBM immune escape as it is expressed in the majority of GBMs [120]. IDO is generally absent or inactive in cells of the immune system, but it can be in-


duced or activated in macrophages and DC subsets by cytokines such as IFN-gamma. IDO has been found in various tumors of different histotypes and correlates with tumor progression. IDO enzymatic activity has been shown to induce tolerance or immunosuppression by inhibiting T-cell proliferation [121]. Moreover, the Lkynurenine is able to affect NK-cell activation mediated by different cytokines. In particular, it interferes with the IL-2–driven upregulation of NKp46- and NKG2D-triggering receptors. This modulation affects both surface expression and function of these receptors and consequently renders NK cells unable to kill different tumor targets [122]. Also Prostaglandins (PGs) such as PGE2, produced by the enzymatic activity of cycloxygenase (COX-2), are expressed in GBM, and may contribute to both progression and immune suppression [123] [124] [125]. PGE2 inhibits T- and B-cell proliferation, NK-cell-mediated cytotoxicity, DC differentiation and function and tumor necrosis factor (TNF) production, while it induces the production of IL-10 [49]. PGs can be produced by tumor cells or by TAMs and has been involved in tumor neo-angiogenesis through the induction of metalloproteases, enhancement of endothelial cell survival and promotion of vascular sprout formation [126]. Moreover, PGE2 may mediate in the differentiation of Treg cells from CD25- precursors [127] [128] or in the induction of Tr1 immunosuppressive cells [129]. Finally also gangliosides produced by human GBM and shed by the cell surface exert multiple inhibitory effect on adaptive or innate immunity. Indeed, gangliosides can inhibit T cell proliferation in vitro [130] [131], DC functions [132] and generation of cytolytic T cells [133]. In addition, GM1 and GM3 gangliosides highly expressed by human GBM and neuroblastoma cells efficiently block NK cell activity [134]. Glioma Antigenic Profile Genetic and epigenetic modifications make cancer cells antigenically distinct from normal human cells. Several antigens recognized by auto-antibodies, CTLs and/or Th cells have been molecularly identified and may therefore be used for eliciting antigenspecific anti-glioma immune responses for therapeutic purposes [135]. CD8+ CTLs and CD4+ Th cells recognize through clonally distributed T cell receptors molecules sequential epitopes of about 8-12a aminoacids, bound to a hydrophobic cleft within specific HLA class I or class II molecules, respectively. Tumor Associated Antigens (TAA) can be divided into several categories: (1) Tumorspecific antigens are proteins produced by mutated genes expressed uniquely by tumors; (2) tissue-specific antigens, differentiation or lineage antigens; (3) cancer-testis antigens, proteins expressed by spermatocytes/ spermatogonia and tumor cells; (4) oncofetal antigens, typically expressed during fetal development only; (5) overexpressed antigens, normal proteins whose expression is upregulated in tumor; (6) Tumor-matrix related antigens; (7) Viral oncoproteins, responsible for the malignant transformation of infected cells. Tumor Specific Antigens In primary GBM the most frequent genetic alteration is the amplification of the EGFR gene [136] [137], and consequent over expression of EGFR, which has been found in 40-60% of GBM patients. Moreover, 60-70% of these patients express also the molecular variant EGFRvIII [138], which is characterized by the deletion of exons 2–7, resulting in a truncated extracellular domain lacking the ligand-binding domain [139] [140]. Although EGFRvIII is unable to bind the EGF family of ligands, it has been shown to be constitutively tyrosine-phosphorylated and therefore acquires prooncogenic effects. The in frame deletion of the EGFR gene splits a codon and introduces a novel glycine at the fusion junction creating a new epitope that can be recognized by monoclonal antibodies or CTLs [141].

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EGFRvIII is of particular interest because it is a true tumor specific antigen absent in normal tissues. Thus, a specific immune response to this antigen will be concentrated on GBM cells limiting the systemic toxicity. On the other hand, targeting a single antigen should select for escape variants, as already reported in peptide vaccination against EGFRvIII antigen in GBM patients [142] or adoptive transfer of T cells targeted against melanoma specific antigens [143]. Tissue-specific Differentiation Antigens GBM express the squamous cell carcinoma-associated reactive antigen for cytotoxic T cells (SART)1 [144] and SART3 [145], described in different tumors including squamous and renal cell carcinoma. SART proteins are capable of inducing HLA-restricted CTL responses and may be used for peptide immunization. Other possible targets include cell differentiation antigens, present both in tumor cells and their normal cell counterpart. Several differentiation antigens have been identified in brain tumors, such as ADP ribosylation factor 4-like protein (ARF4L) [146], which expression increases after loss of PTEN [147], the tyrosinaserelated protein-1 and 2 (TRP-1 and TRP2) [148] and gp100, also expressed by melanoma cells. Several antigen-derived peptides are able to induce HLA-A2 restricted CTLs that lyse glioma cells [149]. Another glioma antigen is the UDP-Gal: bG1cNAc b1, 3 galactosyltransferase, polypeptide 3 (GALT3), in which several CTLdefined epitopes have been identified [150]. Cancer-Testis Antigens Cancer testis antigens (CTA) are a large group, which are expressed in several cancer cell types and in spermatogonia or in placental tissue only. In all the other tissues the CTA genes are shutdown by promoter methylation, whereas their re-expression in cancer cells is related to de-methylation events. The discovery of these antigens led to the theory that re-expression of germline genes in cancer is related to the activation of the silenced gametogenic program in neoplastic cells, and may represent one of the driving forces of tumorigenesis [151] [152]. Thus in view of their tumorrestricted expression, and their ability to induce spontaneous cellular and/or humoral immune responses, CTA are considered as suitable antigenic targets for cancer immunotherapy in several tumors. CTA genes, most of which are located on the X-chromosome, comprise at least 44 different families, such as the MAGE family, each composed by several members. Most studies of CTA expression in human GBM and other brain tumors were performed by RT-PCR analyses, whereas CTA protein expression and induction of CTAspecific immune responses have been rarely demonstrated [153]. Some reports indicated the expression of MAGE-1, MAGE-3, MAGE-E1 or GAGE-1 genes in GBM, possibly in relationship to the common ectodermal origin of gliomas and melanomas, where these antigens were initially identified [154] [153, 155] [156] [157]. Other studies showed weak expression of the NY-ESO-1, a CTA detected by both CTLs and antibodies, in human GBM. However, NY-ESO-1 expression could be greatly increased in GBM cells, but not in normal cells by the use of DNA demethylating agents such as 5’aza-2’deoxycytidine [158], which act synergistically with the histone deacetylase inhibitor valproic acid [159]. In addition, the CTA Homo sapiens testis (HOM-TES)-14 and –85 and Synovial sarcoma X breakpoint (SSX)-1, -2 and –4 were detected in malignant gliomas [157]. Moreover, CTA were detected in cancer stem cells with a level of expression higher than in differentiated cells [160]. Oncofetal Antigens Oncofetal antigens are expressed primarily in fetal tissues and in cancer cells, and could represent potentially interesting targets for glioma therapy. L-type amino acid transporter 1 (LAT1), also known as tumor-associated gene-1 (TA1), is an oncofetal antigen, which functions as a neutral amino acid transporter. LAT1 was found in human glioma cells and immunohistochemical analyses showed stronger reactivity in infiltrating glioma cells at the border zone between tumors and normal brain tissues [161].


A fibronectin (FN) isoform (B-FN), generated by alternative splicing of 3’ regions, containing the oncofetal extra domain B, is expressed in the tumor-remodeled extracellular matrix and in fetal tissues [162]. The B-FN isoform is undetectable in normal adult tissues not undergoing physiologic remodeling or wound healing. In GBM it is expressed around newly forming tumoral blood vessels and can be used to differentiate high-grade from low-grade astrocytomas [163]. Overexpressed Antigens Besides the tumor-specific variant EGFRvIII, GBM cells also express unmutated EGFR, a transmembrane glycoprotein with an extracellular domain ligand-binding site for either EGF or TGF-alpha. EGFR is expressed in an wide variety of normal tissues and frequently overexpressed in GBM [136] [137]. A large panel of monoclonal antibodies (mAbs) against EGFR has been developed that can be used to target GBM cells [164] [165] HER2 is another transmembrane tyrosine/kinase receptor member of the EGF-R family. HER2 is absent on normal glial cells, but it is present on glioma, where its expression increases with the degree of anaplasia. Elevated HER2 expression was found in primary GBM, while secondary GBM, deriving from the progression of low-grade gliomas, had low levels of HER2 expression. Moreover, the survival time was shorter in HER2high as compared with HER2low GBMs [166]. HER2 is regarded as a suitable antigen for immunotherapy, as it can be targeted by specific monoclonal antibodies and by Her2-peptide specific CD8+ T cells [153]. Interestingly, recent reports indicate that HER2 is highly expressed on human GBM stem cells cultured in vitro, which are sensitive to Her2based immunotherapy [166]. IL-13 receptor alpha chain 2 (IL-13Ralpha2) is a subunit of the IL-13R complex and binds IL-13, which is then internalized. IL13Ralpha2 is expressed at low levels by several tissues and is overexpressed by a significant proportion of gliomas [167] [168], renal cell carcinoma and other cancers [169]. The Sry-related HMG box domain that mediates sequencespecific DNA binding characterizes the SOX gene family. GBM and GBM stem cells express very high levels of several SOX genes, including SOX2 [170], SOX4 [171], SOX6 [172], SOX9 [173], SOX10 [174] and SOX11 [175]. SOX2 is overexpressed in the vast majority of malignant gliomas, whereas it is undetectable in normal brain. Silencing this gene in GBM stem cells by RNAi causes the loss of tumorigenicity, demonstrating that SOX2 is fundamental for the maintenance of the self-renewal capacity of GBM stem cells [176]. An immunogenic HLA-A*0201-restricted peptide derived from SOX2 was recently identified and was effective in activating CTL responses [177]. Recent reports demonstrated the presence of IgG antibodies against SOX5 and SOX6 in one-third of glioma patient sera [178, 179]. The transcription factor Wilm’s Tumor 1 (WT1) is an oncogene overexpressed in various types of leukemia and solid tumor cells [180] [181]. Increased expression of WT1 protein was found in the majority GBM specimens [182] [183] and related with the proliferation index [184]. Moreover, peptides derived from WT1 induce antigen specific CTLs [185]. Survivin, a member of the family of inhibitor of apoptosis proteins, plays an important physiological role during development, but is absent in differentiated adult tissues. Survivin gene expression is transcriptionally repressed by wild-type p53 and aberrant expression is found in most human cancers as a consequence of gene amplification, promoter hypomethylation and loss of p53 function. GBM express high levels of Survivin [186] and its nuclear localization predict poorer prognosis [187]. Many T-cell epitopes are present in the protein [188] and anti-survivin antibodies have been found in some GBM patients [189]. The IL-4 receptor (IL-4R) is another cell surface receptor overexpressed in gliomas and selected solid tumors [190]. IL-4R is a


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potential target for the delivery of IL-4PE, a chimeric protein formed by IL-4 and the pseudomonas exotoxin, The IL-4-toxin conjugate internalizes after binding with IL-4R, and the exotoxin block protein synthesis in tumor cells [191] [192]. EphrinA2 (EPHA2), ligand of the Eph receptors, is a GPIlinked surface protein expressed in the CNS during embryonic development and, at low levels, on the surface of proliferating adult epithelial cells. EPHA2 activation decreases cell-extracellular matrix contacts, playing an important role in invasiveness and angiogenesis. The EPHA2 overexpression in GBM is inversely related to survival in GBM patients [193, 194]. EPHA2 is able to induce a spontaneous immune response, which has been originally detected in a long-term survivors [194]. Telomerase (TERT) is a eukaryotic reverse transcriptase enzyme that helps to stabilize telomere length in human stem cells. Many tumors, including the vast majority of GBMs [195], are known to depend upon telomerase for survival, whereas the TERT gene expression in normal tissue is confined to some rapidly dividing cells. TERT-derived peptides are able to elicit CTL responses [196], suggesting that TERT can be a therapeutic target for several tumors including GBM [197]. Tumor-Matrix Related Antigens Besides the fetal isoform of FN, Tenascin (TN) is another component of the extracellular matrix that mediates the interaction between neurons and glia. TN is expressed in different isoforms, generated by alternative splicing, and the large one (TN-C) is preferentially expressed in neoplastic tissues [198]. TN-C is expressed at high levels in up to 90% of GBM [199] [200] [201] and has been studied as a target using specific antibodies. Viral Oncoproteins Although this is a controversial issue, GBM has been reported to express different viral oncoproteins such as the large T-antigen of SV-40 [202], SV-40-related JC virus [203, 204], and CMVderived [205, 206] antigens. Besides the potential role of viralderiving oncoproteins in gliomagenesis, these studies suggest that viral-related antigens associated to GBM could serve as an immunotherapeutic target. Indeed, the induction of an immune response against viral antigens should be highly facilitated, as they are highly immunogenic exogenous proteins, compared to self-deriving TAA. Role of NK Cells It’s becoming more and more evident that the success of a host defense against tumor transformation is the result of complex mechanisms of resistance that involve cells of the innate as well as of the adaptive immunity. Highly specialized lymphocytes such as T lymphocytes are crucial for successful immune responses. However, they are unable to attack tumor cells that downregulate HLA class I molecules, a common strategy used by tumors to evade T cell recognition [207]. HLA down-regulation however, promotes the attack of lymphocytes e.g. Natural Killer (NK) cells. It is widely accepted that NK cells play an active role in immune responses against cancer and pathogen infections. Upon activation they exert potent cytolytic activity and produce large amounts of Th1 cytokines such as IFN-gamma and TNF–alpha. Unlike T lymphocytes, HLA class I molecules provides the major turn off signal that inactivate NK cell-mediated activity. Indeed, NK cells express families of surface receptors that recognize self-HLA class I molecules on potential target cells and transduce potent inhibitory signals [208, 209]. These include the CD94/NKG2A heterodimer, which recognizes HLA-E, [210], and the Killer Ig-like Receptor (KIR, CD158) family, which is composed of clonally distributed receptors specific for determinants shared by groups of classical HLA class I alleles [211]. On the other hand, other molecular mechanisms are responsible for NK cell activation. NK cell function is linked to type 1 cytokines such as IL-12, IL-15 and IL18 [212] and to the interaction between ligands on target cells and a


large array of activating receptors that are expressed at the NK cell surface. In humans, these include the NKp46 (CD335), NKp30 (CD337) and NKp44 (CD336) receptors (collectively termed natural cytotoxicity receptors, NCR) mostly expressed by NK cells, DNAM-1 (CD226) and NKG2D (CD314), which are also present in T cell subsets [208, 213, 214] as well as other activating molecules, NKp80, 2B4 (CD244) and NTBA, which are referred generally as co-receptors since they were shown to support the function of the key receptors [208, 215]. Different cellular ligands specific for activating NK cell receptors have been identified. DNAM-1 recognizes PVR (CD155) and Nectin-2 (CD112) [216], two members of the Nectin family, which are over-expressed in tumors of different histotypes [217, 218]; NKG2D interacts with MICA/B and ULBPs, MHC class I-related, stress inducible molecules expressed after tumor transformation and virus infection [81, 219]; 2B4 interacts with CD48 [215]; NKp80 recognizes the Activation-Induced C-type lectin (AICL) [220], while NTBA displays homophilic recognition [221]. While NKp46 and NKp44 still represent orphan receptors, recently NKp30 has been shown to interact with B7-H6, a tumorassociated surface molecule that represents a novel member of the B7 family [222]. The survival or death of potential cellular targets depends on the type and the number of receptor/ligand interactions occurring at the NK/target immune synapse. In an autologous setting, normal cells are spared from NK-mediated killing because of the expression of high (“protective”) amounts of self-HLA class I molecules and of the lack or low expression of ligands for activating NK receptors. On the contrary, most tumor cells become susceptible to killing mediated by autologous NK cells because defective (“non protective”) expression of HLA class I molecules is accompanied by de novo expression or up-regulation of ligands for activating NK receptors [81, 170, 217, 218, 222, 223]. NK cells represent the most potent effector cells against hematological malignances [224] and preliminary studies suggest that they may be efficient also in eliminating solid tumors and tumor metastases. In this context, GBM cells displaying stem cell-like properties are highly susceptible to lysis by autologous (and allogeneic) cytokine conditioned NK cells [170]. Indeed, GBM cells express low (“non protective”) levels of HLA class I molecules while expressing several ligands recognized by activating NK receptors. In particular, NKp46 and DNAM-1 receptors were involved in NKmediated recognition and killing of tumor cells, while other activating receptor such as NKp30 and NKG2D did not play a significant role in the process. Accordingly, GBM cells analyzed (as well as primary tumors) expressed PVR and Nectin-2, i.e. the DNAM-1 specific ligands but does not express NKG2D ligands. In this context, GBM have been shown to release TGF-beta, an immunomodulatory cytokine that is capable of inducing downregulation of the NKp30 and NKG2D activating receptors (on NK cells) and of MICA on cancer cells [76, 225]. On the other hand, according to that observed in GBM, TGF-beta does not affect the expression of DNAM-1 and NKp46 (on NK cells) and of their ligands on tumor cells. Downregulation of MICA and ULBPs represent classical examples of tumor escape from NK cell-mediated recognition. On the contrary, PVR has been detected in most cancer cell lines and exvivo derived tumors including GBM, neuroblastomas, medulloblastomas, carcinomas and leukemias [217, 218, 226, 227]. Moreover, PVR expression/up-regulation has been shown to correlate with the invasive and migratory capabilities of GBM cells [226, 228] and its interaction with the counter-receptor expressed in platelets, appears to be critical for efficient metastasis of cancer cells [229]. Thus, PVR appears to represent a molecule essential for tumor survival and most tumor variants could be obliged to preserve its expression. Even PVR however could miss its role in cancer cell biology at certain stages of tumor progression. For example, metastatic cells isolated from bone marrow aspirates of neuroblastoma patients at stage 4 of the disease in most cases lacked the expression of PVR and this

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clearly correlated with a very poor susceptibility to lysis mediated by activated NK cells [217]. Despite the encouraging demonstration of T and NK anti-tumor immunity in patients diagnosed with cancer, immunotherapy has not advanced to become a common treatment in cancer patients due to the lack of indisputable evidence showing the in vivo clinical relevance of this therapeutic strategy. The reasons for such failure are probably multiple, but many data point to a relevant intervention of tumor microenvironment in restraining both motility and functions of the immune system. Various reports have provided evidence that cancer cells acquire the ability to shape the hosting tissue toward microenvironment conditions favoring the survival and spreading of the tumor. Indeed, cancer cells can release soluble mediators supporting tumor growth in an autocrine manner, promoting angiogenesis as well as lymphangiogenesis. Moreover, although able to release chemokines that favor the recruitment of cells of the immune system, cancer cells also produce cytokines that hijack immune responses thus favoring tumor survival/progression. For example there is a local production of immunosuppressive molecules that inhibit effector T cell responses and promote the recruitment of CD4+CD25+FoxP3+ regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs) that suppress immune responses [230, 231]. Moreover, cancer cells while secreting a wide spectrum of soluble factors that attract circulating monocytes promote their differentiation to macrophages while blocking that to DC [232]. Importantly, NK cells do not act alone but cooperate with other cell types during immune responses. Indeed, during inflammatory events NK cells are recruited into tissues in response to chemokine gradients [233] and interact with other immune cell types that are either resident or recruited during inflammation [234]. It is likely that these multiple interactions could play a critical role not only during early innate immune responses but also in the initiation, amplification and polarization of adaptive responses. For example, it has been demonstrated that a bidirectional crosstalk exists between NK and monocyte-derived dendritic cells (DC) [235-238]. During inflammation immature DC (iDC) undergo a process of maturation characterized by upregulation of the expression of HLA class I and II molecules, co-stimulatory molecules and CCR7, a chemokine receptor which confer DC with the ability to migrate toward lymph nodes [239]. Importantly, Maturing DC (mDC) induce in resting NK cells the expression of activation markers such as CD69 and enhance their cytolytic activity [240, 241]. mDC-mediated activation of NK cells leads to killing of autologous iDC that unlike mDC, express low (“non protective”) amounts of HLA class I molecules [241]. This process termed “DC editing” might represent a crucial step that leads to the selection of DC that during maturation express amounts of HLA molecules suitable for optimal T cell priming [239]. Following the interaction with mDC, NK cells also upregulate their ability to release IFNgamma that would be crucial for skewing immune responses towards Th1 polarization [242] and de novo express CCR7, which confers to NK the ability to migrate toward secondary lymphoid organs [243, 244]. As DC, also macrophages are crucial components of both innate and acquired immune responses. In response to microenvironmental factors macrophages (M0) polarize toward either a classical (M1) or an alternative (M2) functional phenotype [245, 246]. M1 and M2 display different cytokines’ and chemokines’ gene expression profiles and functional properties. M1 are characterized by the IL-12high, IL-23high, IL-10low transcription profile while M2 macrophages have an IL-12-low, IL-23low, IL10high, high mannose and scavenger receptor transcription profile. M1 macrophages have immunostimulatory Th1-orienting properties and tumor suppressor activity. On the contrary, M2 polarized cells suppress Th1 adaptive immunity, support TH2 responses and display tumor promoting function. Macrophages are the most represented leukocytes in cancer tissues. Importantly however, the tumor microenvironment twists the polarization of tumor-associated macrophages (TAM) towards an M2-like phenotype that would


display strong pro-tumoral activity [245]. In this context, it has been demonstrated that malignant gliomas by releasing chemoattractants such as CC chemokine ligand 2 (CCL2) and soluble colony-stimulating factor 1 (sCSF-1) recruit microglia and macrophages at the tumor site were they adopt M2 tumor-promoting phenotypes capable of mediating immunosuppression and invasion [52]. Recently, the molecular pathways involved and the functional outcome of the interaction between different types of macrophages and autologous NK cells has been analyzed [247]. Macrophages polarizing toward M1 induce strong activation of resting NK cells resulting in enhancement of cytolytic activity, release of high amounts of IFN-gamma and expression of CCR7. These data suggest that NK cells may play a role in amplifying Type 1-oriented immune responses [246], as a result of their interaction not only with maturing DC but also with M1 polarizing macrophages. On the contrary, M2 macrophages were unable to activate NK cell function [247]. Thus, it is tempting to speculate that TAM, which in established progressing tumors generally have M2-like phenotype [232], might also be unable to co-operate with NK cells. It is noteworthy however that M2 are neither terminally differentiated nor “exhausted” cells. Indeed, microbial products such as BCG reverted the functional “immunomodulatory” M2 phenotype toward an “immunostimolatory” M1-oriented, which resulted in strong NK cell activation [247]. Re-programming TAM versus M1 phenotype and function represents a novel strategy to restore anti-tumor responses. For example the use of CpG and anti-IL-10R antibodies promoted innate responses in tumor-bearing mice [248] and BCG immunotherapy provided substantial clinical benefits in ovarian and bladder cancer patients [249, 250]. Interestingly, once activated NK cells kill M2 macrophages, which express low, “non protective” amounts of HLA class I molecules while M1 macrophages (HLAclass I high) similarly to mDC, are resistant to NK cells [247]. Thus, the restoration of M1 polarization of cancer-propelling TAM might enhance the NK cell-mediated cytolytic activity not only against cancer cells but also against residual TAM. DIFFERENT APPROACHES FOR GLIOMA IMMUNOTHERAPY Active immunization to glioma antigens, adoptive transfer of effector cells, immunomodulatory cytokines, TLR-agonists, and antibodies have been proposed as tools for GBM therapy. In addition, antibodies directed to GBM-associated antigens or cytokines binding to cytokine receptors, are regarded as suitable vehicles for the targeted delivery of toxic compounds or radionuclides. Cytokine-Based Therapy Several immune-enhancing cytokines have been successfully used to treat tumors, in preclinical models and some of them were also tested in clinical trials in GBM patients. Different procedures of cytokine administration have been used in GBM, including systemic or local administration of recombinant molecules, cytokine gene-transfer systems or targeted delivery. The use of systemic administration of high doses of recombinant cytokines may have several limitations, due to the side effects related to the use of high dosages. In addition, if the aim of the cytokine therapy is the activation of an anti-GBM response by adaptive or innate immunity, it must be considered that the CNS is an immune-privileged site and that GBM elicits several suppressive mechanisms, which may hamper the immune response. Among immune-enhancing cytokines IL2, IL-4, IL-7, IL-12, IL-21, IL-23, IL-27 and IFNs have been used for GBM therapy in pre-clinical and/or clinical settings. Besides activation of the immune system, other cytokines such as IFNs, TNF-alpha or TRAIL may affect glioma cell viability or proliferation or exert anti-angiogenic effects, as in the case of IL-12 and IFNs. IL-2


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IL-2 is the first discovered member of the cytokine family that also includes IL-4, IL-7, IL-9, IL-15 and IL-21. These cytokines have common structural characteristics, consisting of a four alphahelix bundle structure, and share the usage of the cytokine receptor common subunit gamma (IL-2Rg) in their surface receptor complexes [251]. IL-2 was originally identified as a “T cell growth factor” for activated T cells, and has several immune enhancing activities on T, B and NK cells. In addition, short-term culture of peripheral blood lymphocytes with IL-2 generated “lymphokine-activated killer” (LAK) cells, which display potent anti-tumor activity in vitro and in vivo in animal models [252]. Further studies indicated that IL-2-activated NK cells were mainly responsible for LAK cytotoxic activity against tumor cells, with a minor contribution of a subset of T cells [253]. IL-2 is considered as a milestone in the history of tumor immunotherapy [254] and is still in use for the treatment of melanoma and renal carcinoma. Indeed, the occurrence of long-lasting complete remissions in some metastatic patients, treated with IL-2 alone or in combination with LAK cells provided the proof of principle that activation of the immune system may result in the eradication of tumors at advanced stage [252] [254] [255]. Nonetheless, these effects were observed in a minority of patients and high-dose IL-2 showed a remarkable toxicity. Early studies in cancer patients with no brain pathology showed that intravenous injection of IL-2 resulted in the appearance of IL-2 in CSF, which persisted over time, suggesting the possibility that IL-2 disrupts the BBB upon systemic administration [256] . However intravenous IL-2 is not tolerated by patients with gliomas due to the increase in peritumoral edema [257], in relationship to the “vascular-leak” induced by high-dose IL-2. Other studies in animal models pursued the use of gene transfer strategies for a prolonged low-dose IL-2 delivery at the GBM site, in the attempt to reduce side effects. The intratumoral injection of IL-2-gene transduced allogeneic fibroblasts in glioma-bearing mice produced an increase of survival related to the activation of a local immune response by paracrine IL-2 [258]. Another report showed that C6 rat glioma cells genetically engineered to secrete IL-2 grew more slowly than wild-type tumors when orthotopically implanted in vivo and were eventually rejected. In addition, longlasting immunity was observed by re-challenging surviving rats with wild-type C6 cells in the brain [259]. The possibility of a direct gene delivery to human brain tumors was first examined using stereotactic injection of adeno-associated virus (AAV)-based vectors in orthotopically-implanted nude mice. To increase the effects of IL-2, the herpes simplex thymidine kinase (HSVtk) suicide gene, which mediates ganciclovir (GCV) prodrug activation, was combined with IL-2 gene in an AAV-bicistronic vector (AAV-tk-IRESIL2). Transduction of glioma cells rendered them sensitive to GCV cytotoxicity and also mediated IL-2 secretion. Stereotactic delivery of AAV-tk-IRES-IL2 particles into established glioma followed by administration of GCV strongly reduced the tumor volume [260]. A similar approach was attempted through the use of a retroviral vector simultaneously expressing HSVtk and IL-2 [261] to produce both direct and immune-mediated tumor cell killing. A pilot study showed that the implant of retroviral packaging cells in GBM postsurgical cavity followed by GCV treatment showed no significant toxicity and no generation of replication competent retroviruses. MRI and CT demonstrated in one out of four patients a significant reduction of the tumor mass with clinical benefit and disease stabilization [262]. The safety of this treatment and the induction of some clinical responses were confirmed in a further study on 12 patients, which also showed an increase of intratumor and plasma Th1 cytokines after treatment [263]. Altogether these data suggest that HSVtk/IL-2 gene therapy approaches are feasible in GBM and warrant further investigation. Several data indicated that IL-2, besides its immune enhancing activities, also displays immune-regulatory functions as it supports the proliferation of immunosuppressive Treg cells. The hemagglu-


tinating virus of Japan envelope (HVJ-E) vector, derived from a defective Sendai virus strain, has inhibitory activity on Tregs and was therefore considered suitable for IL-2 gene therapy. Intratumoral injection of HVJ-E vector containing the IL-2 gene significantly inhibited the growth of intracranial murine gliomas and the expansion of Treg cells, while it induced a marked infiltration of T cells into tumors and prolonged survival [264]. An additional approach to minimize systemic toxicity and maximize anti-tumor effect is based on the targeted delivery of cytokines, for example through the construction of recombinant antibody/cytokine chimeras, also defined as immunocytokines. An immunocytokine (F16-IL2) consisting of human IL-2 fused to a recombinant antibody specific for the tenascin-C matrix protein of glioma was generated and used in preclinical testing. F16-IL2 selectively localized to subcutaneous or intracranial U87MG GBM cell line xenografted in nude mice and, in combination with TMZ, induced complete remission or consistent size reduction of subcutaneous or intracranial xenografts, respectively [265]. These data suggest that TMZ with F16-IL2 deserves clinical investigations. Other Members of the IL-2 Family IL-4 plays an important role in Th2-related defense mechanisms against parasitic infections and in allergy, which is strictly dependent of Th2 hyper reactivity [266]. Two different receptor complexes can initiate IL-4 signaling: a type I receptor composed of IL-4Ralpha and the IL-2Rgamma and a type II receptor formed by IL-4Ralpha and IL-13Ralpha1. Both receptors signal through the STAT6 pathway, which is essential for Th2-mediated responses [267]. Transduction of IL-4 gene into human glioma cells showed impaired tumor growth upon implantation in nude mice [268]. In another preclinical model, injection into intracranial GBM tumors of a retroviral producer cells, releasing IL-4 and IL-4-encoding retroviruses, resulted in tumor regression in 50% of rats and the cured rats developed immunity to GBM antigens [269]. The gene for IL-4 was also transduced into mouse or rat primary neural progenitor cells, which were then injected into syngeneic mouse GBM or in rat GBM, respectively [270]. The survival of progenitor cells was lasting for several weeks and this treatment cured large tumors, thus indicating that neural stem cells genetically engineered to release therapeutic molecules are an optimal approach for gene therapy of brain tumors. Altogether, preclinical models suggested that IL-4 gene transfer is a promising approach for treating GBM. However, other findings indicated that IL-4 mediates an aberrant signaling in human GBM cells, which may sustain GBM cell survival [271]. Although the type II IL-4 receptor is expressed in GBM cells, IL-4 fails to activate their STAT6 signaling. This finding is related to the expression of the IL-13Ralpha2 decoy receptor in GBM cells [272], which blocks IL-4 signaling via STAT6 in GBM cells. Moreover, IL-13Ralpha2 mediates the activation of STAT3, which could be inhibited by IL-13Ralpha2 silencing [271]. Through this pathway IL-4 up-regulates the levels of the anti-apoptotic molecules Bcl-2, Bcl-xL and Mcl-1 expression in GBM cells, suggesting a potential role of the IL-4/IL-13Ralpha2 system in GBM pathogenesis. In view of these findings, the use of IL-4 in GBM therapy in humans should be considered with great caution. IL-7 is produced by stromal cells of the bone marrow and thymus and plays a central role in lymphoid cell development [273]. Murine glioma cells genetically engineered to secrete IL-7 showed reduced tumorigenicity in vivo due to the activation of a CTL response [274]. In addition, mice immunized with IL-7-producing glioma cells showed a specific immune response to glioma. A recent study in a rat glioma [275] combined peripheral immunization by IFN-gamma-transduced glioma cells with intratumoral injection of IL-7-producing mesenchymal stromal cells. IL-7 delivery alone inhibited tumor growth and this effect was increased by vaccination with IFN-gamma producing glioma cells.

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IL-21 is the lastly discovered member of the IL-2 family and, similar to IL-2, it displays immune enhancing functions. Indeed, IL21 co-stimulates T cell proliferation, augments NK and CTL activities [276] and also regulates B cell responses [277]. However, different from IL-2, IL-21 does not support Treg cell expansion in vitro [278] and may counteract Treg cell-mediated immune suppression [279]. In view of its functional activities, IL-21 has been considered as a potential candidate for cancer immunotherapy. Data obtained in several tumor animal models indicated that IL-21 enhances anti-tumor immune responses mediated by CTLs or NK cells resulting in tumor inhibition [280]. In addition, intratumoral injection of IL-21-transduced cells or of recombinant IL-21 in an orthotopic GBM model produced tumor rejection in syngeneic mice. Mice that had been cured by IL-21 therapy developed immunity to GBM, as demonstrated by rejection of GBM cell implants in the contralateral hemisphere. The protective immunity and the therapeutic effect of IL-21 were mediated predominantly by cytotoxic anti-GBM antibodies and were abrogated in antibodydeficient mice [281]. In view of its anti-tumoral activity in preclinical models, IL-21 has been administered intravenously in metastatic melanoma or renal carcinoma patients in Phase I-II clinical studies. These studies showed that in some patients IL-21 enhances T and NK cell activation, induces clinical responses or disease stabilizations and has a limited toxicity [282] [283] [284]. The finding that IL-21 did not mediate the vascular leak syndrome typical of IL2, together with its activity in a preclinical model of GBM, supports its potential application in human GBM immunotherapy. IL-12 Cytokine Family IL-12 is a member of a heterodimeric cytokine family, which also comprises IL-23 and IL-27 [285]. IL-12 plays a crucial role in Th1 cell differentiation and mediates IFN-gamma production by T and NK cells [286]. IL-12 showed antitumor activity in several preclinical models, in relationship to IFN-gamma secretion and activation of T or NK cell responses [287]. IL-12 also displays antiangiogenic properties, which are mediated by IFN-gammainducible antiangiogenic chemokines CXCL-8, -9 and -10 [288], although IFN-gamma-independent mechanisms have been reported [289]. The structurally related cytokines IL-23 [290] and IL-27 [291] display non-redundant functions and have different effects on tumor growth. The antitumor effects of IL-27 are similar to those of IL-12, as they are mediated via the IFN-gamma/CXCL-8-9 axis and induction of CTL activity. In addition, IL-27 can also directly mimic the effects of IFN-gamma, through the activation of the STAT-1 pathway [292]. A recent study showed that serum IL-12p40 and IL-27p28 levels were decreased in glioma patients as compared with healthy controls and that an IL-12 gene polymorphism (16974 A/C) may regulate expression of the serum IL-12p40 and IL-27p28, and associate with increased risk of glioma [293]. These data suggest that IL-12 and/or IL-27 may contribute to inhibit glioma development. In spite of the anti-tumor activity of IL-12 in several preclinical tumor models, the use of IL-12 in clinical oncology has been initially hampered by elevated toxicity encountered in a phase II trial, which led to withdrawal of IL-12 clinical trials for several years. Therefore strategies of local delivery of this cytokine were developed, particularly by gene transfer technologies, with the specific aim of achieving prolonged local concentrations and minimize systemic toxic effects. Different IL-12 gene transfer approaches have been used in preclinical glioma models, including the use of adeno-associated [294], semliki forest [295], herpes simplex [296] or adenoviral vectors and plasmids encapsulated in DNA/PPC (polyethylenimine covalently modified with methoxypolyethyleneglycol and cholesterol) complexes [297]. Altogether these preclinical models showed that IL-12 gene transfer in GBM cells results in strong anti-tumor effects, which are frequently followed by immunity to GBM-associated antigens, mediated by T cells.


Other preclinical studies used neural stem cells that are capable of tracking migrating glioma cells. Genetically engineered IL-12producing stem cells inoculated intratumorally in glioma-bearing mice prolonged survival and induced persistent antitumor immunity, as the result of a T-cell infiltration in tumor microsatellites [298]. In addition, IL-12 has been used as an adjuvant to enhance responses to glioma vaccines. The safety and clinical response to immunotherapy using fusions of dendritic and glioma cells combined with recombinant IL-12 was tested in 15 patients with malignant glioma. No serious adverse effects were observed and MRI showed a >50% reduction in tumor size in 4 patients, suggesting that this treatment may result in clinical antitumor effects in glioma patients [299]. The anti-tumor activity of IL-23 has been rather controversial [285], but a few reports indicate anti-tumor activity of IL-23 in GBM models. The intratumoral injection of bone marrow-derived neural stem-like cells, genetically engineered to express IL-23, showed protective effects in glioma-bearing syngeneic mice. CTLs were crucial in the anti-tumor effects of IL-23 and mice cured by this treatment showed immunity to GBM re-challenge in relationship with IFN-gamma expression in the brain [300]. Similarly, bone marrow-derived DC transduced by a single-chain mouse IL-23 construct implanted intratumorally into GBM-bearing mice produced a CTL-mediated protective effect, which was followed to anti-GBM immunity [301]. Interferons Interferons (IFNs) are a family of cytokines with anti-viral properties, which comprises a large number of members including type I IFNs (in humans: thirteen IFN-alpha species, IFN-beta, IFNepsilon, IFN-kappa, and IFN-omega), type II IFN (IFN-gamma) and the most recently discovered type III IFNs (IL-28A, IL-28B and IL-29, also designated as IFN-lamda2, -lamba3 and -lamda1, respectively) [302]. All members have a common secondary structure consisting of six or seven a-helices assembled in an antiparallel conformation, although aminoacidic sequence identity may be low. Their biological effects are mediated through heterodimeric transmembrane receptor complexes, which signals predominantly through receptor-associated JAK family tyrosine kinases and downstream STAT molecules. In addition to their antiviral activity, IFNs have immune-modulatory functions, exert anti-proliferative effects on different tumor cell types and inhibit angiogenesis [303] [304] [305]. Among immune-modulatory functions, IFNs promote the differentiation of human monocytes into DCs and support their ability to induce Th1 differentiation and to mediate CTL cross-priming [306]. Further, type I IFNs increase the clonal expansion of CD8+ T-cells, their survival and the establishment of a CTL memory [307]. Type I IFNs also enhance the IgG responses to soluble antigens, induce immunological B-cell memory [308] and potentiate NK cytotoxic activity. Type I and II IFNs show pro-apoptotic or antiproliferative effects on tumor cells, whereas the tumor growth-inhibiting activity of type III IFN seems more limited. In particular, only a minority of GBM cell lines were sensitive to the anti-viral and cytostatic effect of IFN-lambda1 [309]. In addition to their anti-proliferative effects, IFNs also increase the expression of HLA molecules on tumor cells and renders them more susceptible to T cell mediated cytolysis. Immunostimolatory functions of type I IFNs promote an increase in tumor immunogenicity and activate an efficient immune response that mediates tumor rejection [310] [311] [312] [313]. Indeed, the hematopoietic compartment of the host is necessary for IFNs antitumor activity, suggesting that the crucial target of IFNs is the immune system [312]. Very recent data indicate that the type I IFNs play a relevant role in controlling gliomagenesis by inducing CTL responses and downregulating suppressor cells. In fact, gliomagenesis induced by intracerebroventricular transfection of NRas and a


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short hairpin RNA against P53 through a transposon system, was increased in type 1 IFN receptors deficient (Ifnar1-/-) mice as compared to wild-type mice. Ifnar1-/- mice also showed increased numbers of glioma-infiltrating Treg and myeloid-suppressor cells and decreased numbers of CD8+ T cells. In addition, the analysis of single nucleotide polymorphisms (SNP) in type 1 IFN-related genes IFNAR1 and IFNA8 were associated with altered overall survival of patients with grade 2 to 3 gliomas, further suggesting a role for the IFNs system in glioma natural history in humans [314]. IFN-alpha was the first cytokine to show therapeutic activity in different types of cancer, such as melanoma, renal cancer, chronic myelogenous leukemia, hairy cell leukemia and others. However, high dosages of IFN-alpha are required to achieve responses in cancer patients, thus resulting in toxicity [310] [311] [315]. The subcutaneous administration of IFN-alpha in a phase III randomized clinical study combining radiotherapy and Carmustine (or BCNU, e.g. bis-chloronitrosourea) did not show significant clinical benefit in high-grade glioma [316]. However, two out of nine patients with recurrent GBM showed complete responses in a phase I study that combined escalating doses of IFN-alpha with locally implanted BCNU wafers [317]. Two single-arm phase II studies assessed the toxicity and clinical benefit of TMZ combined with IFN-alpha2b or long-acting pegylated IFN-alpha2b in recurrent GBM patients. Grade 3 or 4 toxicities were common, as leucopoenia and thrombocytopenia occurred in 35-38% and 18-21% of patients receiving IFN-alpha and pegylated IFN-alpha, respectively. Both studies however showed improved efficacy when compared to historical controls [318]. These results suggest a possible use of IFN-alpha or pegylated IFNalpha combined with standard therapies. Another clinical study with pegylated IFN-alpha2b administered after standard radiation therapy is ongoing in children with diffuse pontine gliomas. The study will compare the 2-year survival vs. historical controls receiving radiotherapy only and will define toxicities in pediatric patients (NCT00036569). In view of the anti-angiogenic activity of IFN-alpha, a clinical study was designed to test the efficacy in recurrent high-grade gliomas of a combination of the anti-angiogenic drug Thalidomide and pegylated IFN-alpha2b. The study, which compared progression-free survival compared vs. historical controls, has been recently completed (NCT00047879). To limit systemic toxicity of IFN-alpha, local delivery strategies have been studied in preclinical studies. Tumor-homing monocytes express Tie2, a cell-surface receptor, which bind and is activated by the angiopoietins. Tie-2-expressing monocytes have been genetically engineered with a Tie2 promoter/enhancer-driven Ifna1 gene. The engineered monocytes efficiently targeted the IFN response to orthotopic human gliomas and produced significant antitumor responses, though inhibition of angiogenesis and activation of innate and adaptive immunity, without systemic side effects [319]. These results suggest that cell-based IFN-alpha delivery may represent a new potential tool to treat GBM or other cancers. In an early phase I study the intravenous administration of escalating doses of IFN-beta showed no clinical benefit in patients with recurrent gliomas [320]. A subsequent multicenter phase I-II trial performed in 21 children with recurrent or progressive primary brain and spinal cord tumors, showed partial responses in 19% of the patients and 38% displayed disease stabilizations for a median of 5+ (2 to 14+) months. Dose-limiting toxicities were hematologic, hepatic, and CNS and the MTD was established at 500 mlU/m2. The study suggests that IFN-beta in a dose-intensive regimen has antineoplastic activity in children with high-grade astrocytomas and brainstem gliomas [321]. In addition, a phase I study based on intrathecal infusion of IFN-beta in recurrent GBM showed low toxicity and disease stabilizations in 3 out of 12 patients [322].


An Integrated Japanese Multicenter Clinical Phase I study is been conducted in 16 newly diagnosed and 7 recurrent high-grade glioma patients to evaluate the safety, feasibility and clinical effectiveness of IFN-beta in combination with TMZ and Radiotherapy (INTEGRA Study) [323]. Overall 40% of patients showed objective responses to treatment and patients with newly diagnosed GBM had a 17.1 months overall survival. Grade 3-4 leukocytopenia and neutropenia were observed in a minority of patients. A further phase II clinical trial may corroborate these findings. The effectiveness of a combined treatment with IFN-beta and TMZ was also analyzed in six human malignant glioma cell lines. The TMZ-mediated cell growth inhibition was dose-dependent, and the sensitivity to TMZ of the various glioma cell lines correlated with the expression of the DNA repair protein MGMT. IFN-beta synergized with TMZ by down-regulating MGMT mRNA levels in resistant cell lines. These data suggest that the clinical therapeutic efficacy of TMZ might be improved by a combination with IFNbeta in gliomas with unmethylated MGMT gene [324]. A recent preclinical study evaluated the effects in a xenotransplant model of the continuous delivery of IFN-beta, mediated by IFN-beta liver gene transfer through an adenoviral vector. Although IFN-beta had no direct effect on glioma stem cells in vitro, in vivo IFN-beta decreased the glioma size and the number of stem cells in glioma xenografts by disrupting the vascular niche, essential for stem cell survival [325]. Another recent study showed that IFN-beta markedly reduced miR-21 expression in glioma cells, and in mice IFN-beta administration suppressed the growth of intracranial tumors derived from glioma-initiating cells. Since miR-21 is involved in gliomagenesis, the downregulation of miR-21 clearly contributes to the antitumor effects of IFN-beta [326]. Toll-like Receptor Agonists Toll-like receptors (TLR) are a family of evolutionaryconserved receptors, which recognize pathogen-associated molecular patterns (PAMPS). PAMPs are small molecular motifs that are conserved within certain classes of pathogens and mediate the activation of innate immunity through pattern-receptors, including the TLR. PAMPs comprise the bacterial products lipopolysaccaride, flagellin, lipoteichoic acid, peptidoglycans, DNA containing unmethylated CpG motifs, and viral double-stranded RNA. TLR, upon agonist binding, activate cells of the immune system or other cell types to produce cytokines and co-stimulatory molecules. TLR signaling elicits the production of molecules involved in inflammation, inhibition of viral replication, activation of innate immunity, antibody production and enhanced antigen presentation to the T cells [327]. In view of these properties, TLR agonists can increase anti-tumor responses in different preclinical tumor models. Synthetic TLR agonists that mimic PAMPs have been designed as non-specific immune-enhancing agents, such as imiquimod, polyinosinic-polycytidilic acid (poly-ICLC) and synthetic olygodeoxynucleotydes bearing CpG motifs (CpG-ODN). Poly-ICLC is a synthetic double-stranded RNA, which mimics the structure of viral RNAs and binds to TLR3. Poly-ICLC mediates the production of type I IFNs but also had IFN-independent immune-enhancing activities resulting in increased antibody production in response to antigen stimulation, activation of macrophages, T and NK cells. In particular, Poly-ICLC (as well as CpG-ODNs) has been shown to activate human NK cells that release high amounts of IFN-gamma, TNF-alpha and up-regulates their cytolytic activity against tumor cells [328]. Altogether these effects contributed to the anti-tumor activity of poly-ICLC in pre-clinical models [329, 330]. Early clinical trials were based on the use of poly-ICLC at high doses and showed a remarkable toxicity [331, 332]. However, further reports showed that poly-ICLC at low doses was sufficient to elicit anti-viral and

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anti-tumoral effects [333, 334], thus leading to re-examine the potential benefit of poly-ICLC in clinical studies. An early pilot study with low-dose intramuscular poly-ICLC was performed in patients with GBM or astrocytoma at different clinical stages (newly diagnosed or recurrent). Poly-ICLC was used either alone or in combination with lomustine (CCNU). Sixty-six% of the poly-ICLC-treated patients showed tumor regression or stabilization and newly diagnosed GBM had a mean survival time of 19 months [335]. In view of these results a phase II clinical trial with poly-ICLC plus radiotherapy was started in newly diagnosed supratentorial GBM (North American Brain Tumor Consortium, NABTC01-05). As the trial was started before the introduction of TMZ in glioma therapy, it was discontinued when the new standard care for GBM was established [8]. However, the evaluation of the available cases showed that poly-ICLC + radiotherapy was well tolerated and the overall survival was of 15 months, similar to that described using TMZ + radiotherapy (16 months) [336] [337]. Recently, a multi-institutional phase II study that treated newly diagnosed GBM patients with poly-ICLC + radiotherapy together with concurrent and adjuvant TMZ, has been concluded. Ninety-six patients were enrolled and 14 of them did not start adjuvant treatment. An overall median survival of 17.2 months was found, which reached 18.3 months for subjects 18-70 years old, as compared with 14.6 reported for standard treatment alone. These results demonstrate that the use of poly-ICLC in combination with radiation and TMZ is safe and may improve the efficacy of standard treatment [338]. In addition a recent study indicates that poly-ICLC can be effectively incorporated into vaccine-based immunotherapy involving peptide antigen-loaded type 1 polarized DC [339]. Synthetic CpG-ODNs mimic the structure of bacterial DNA that differs from eukaryotic DNA and is rich in unmethylated CpG motifs. Bacterial DNA and CpG-ODNs interact with TLR-9, which activates both innate and specific immunity and drives the Th1 cell polarization. In a preclinical study, local injection of CpG-ODN was combined with radiotherapy to treat rat gliomas. The combined treatment induced tumor regression in most of the animals and was superior to either treatment alone [340]. A very recent study showed that multiple intratumoral injections of low-dose CpGODN eradicated gliomas in 70% of syngeneic mice, which showed durable remission and immunity to a subsequent re-challenge. NK cells played an important role in the initial CpG-ODN-mediated tumor rejection, while both CD8+ T cells and NK cells were involved in long-term immunity [341]. Based on pre-clinical studies, a phase I trial of intratumoral CpG-ODN through convection-enhanced delivery was performed in recurrent GBM. An MTD of 20 mg per dose was established and adverse effects were fever, transient lymphopenia and worsening of neurologic conditions. Two out of 24 patients showed minor responses and the overall median survival time of 7.2 months [342]. In a further phase II trial 31 patients with recurrent GBM received 20 mg of intratumoral CpG-ODN (CpG-28) after radiotherapy and chemotherapy. The patients’ progression-free survival at 6 months was 19%, and one partial and 3 minor responses were observed. The median overall survival was 28 weeks. Twenty-four % and 15% of the patients were alive 1 year and 2 years after, respectively [343]. Although GPG-ODN did not reach the target survival benefit in patients with recurrent GBM, the few long-term survivors suggest that a subset of GBM patients could be responsive to the treatment. Therefore a randomized phase II trial is ongoing in earlydiagnosed GBM in combination with surgery, radiotherapy and TMZ (NCT00190424). Adoptive Transfer: T Cells vs. NK or LAK Cells Adoptive T cell transfer is aimed to increase the anti-glioma immune response by the administration of tumor-specific autologous T cells that have been expanded in vitro. Different protocols have been utilized for ex vivo stimulation, activation and expansion


of T cells. In addition various routes of administration have been used such as systemic infusion or local intracerebral inoculation. Early approaches to GBM adoptive cell therapy involved the administration of autologous peripheral blood mononuclear cells (PBMC), using either intrathecal [344] or intratumoral [345] administration, establishing the feasibility and safety of this procedure and suggesting a clinical benefit in some patients. Further studies combined the administration of autologous lymphocytes with interferon [346], but these strategies did not yield significant differences in survival time. Subsequent investigations focused on the adoptive transfer of specific T-cell populations. The demonstration that IL-2-expanded tumor-infiltrating lymphocytes (TILs) produced a specific tumoricidal response, induced initial attempts to therapeutically utilize this subset of T-cells presensitized in vivo by the contact with tumor cells. However, lymphoid cells infiltrating GBMs may suffer of several defects as a result of glioma immunosuppressive factors, of Treg cells and M2 macrophages in the microenvironment. Accordingly, in spite of one clinical report describing some encouraging results [347], the use of TILs did not obtained much success in GBM. Further investigations involved the inoculation of lymphocytes isolated ex vivo from tumor-draining lymph nodes or from PBMCs. In order to generate mono/oligoclonal CD8+ T cells able to recognize specific TAA in the context of HLA class I molecules, lymphocytes were expanded in vitro in the presence of exogenous IL-2 and repeated antigenic stimulation, e.g. by co-cultivation with autologous irradiated glioma cells. Activated T cells were infused back in the tumor bed of five GBM patients. In two cases out of five tumor regressed more than 50% and one patient survived more than 104 weeks [348]. Similar results were obtained by Tsuboi using the same protocol [349]. The generation of tumor-specific CD8+ T-cell clones requires not only the in vitro supply of IL-2, which substitutes CD4+ helper T cells, but also optimal levels of HLA class I expression by glioma cells. Most GBM cells express low level of HLA class I making them inefficient antigen-presenting cells that may induce T-cell inactivation through anergizing or tolerizing mechanisms. Optimization of the protocol requires the antigens presentation by professional antigen presenting cells (APCs) that express high levels of HLA class I as well as co-stimulatory signals. Moreover, because lymphocytes isolated from glioma patients could be functionally defective, an alternative strategy involves the intratumoral transplantation of alloreactive T cells from healthy donors sensitized in vitro by the patient HLA through a Mixed Lymphocyte Reaction (MLR). This approach exploit the fact that normal CNS cells express lower levels of HLA than GBM cells and therefore locally infused alloreactive T cells would target only tumor cells sparing normal brain cells. In a clinical trial two of the five patients that have been treated with intracavitary alloreactive cytotoxic T lymphocytes and IL-2 had an increased survival [350]. An additional dose escalation trial using intratumoral injection of alloreactive CTLs and IL-2 is currently ongoing at UCLA for the treatment of recurrent Malignant Gliomas (NCT01144247). Cytotoxic T cells generated against TAAs Besides the ability of GBM cells to produce immune suppressive factors, another obvious limitation of using GBM cells to generate tumor specific CD8+ T cells is their heterogeneous antigenic composition, which includes normal self-antigens, thus leading to the generation of potential autoimmunity. These limitations may be overcome by the use of selected TAAs to induce the proliferation of CD8+ specific T-cells. Advances in knowledge of human T-cell biology and molecular biology allowed the ex vivo expansion of TAA-specific CD8 Tcells or the generation of genetically engineered T-cells.


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TAA-specific CD8+ T cells for use in adoptive immunotherapy have been obtained in vitro by co-cultivation of CD8+ cells, purified from PBMC, with APCs pulsed with synthetic TAA peptides or transfected with the cDNA coding for the selected TAA. Repetitive stimulation of CD8+ cells with peptide-pulsed APCs allows the generation of peptide-specific T-cell lines, which can be expanded in vitro and purified using peptide-HLA class I tetramers [351]. The effectiveness of this approach has been demonstrated mainly in melanoma patients. After infusion, the CD8+ TAA specific cell clone or oligoclonal populations infiltrate and destroy melanoma and melanocytes [352]. The therapeutic efficacy of adoptive transfer of antigen-specific CD8+ cells has been improved by preventive lymphodepletion of the patients [353]. A Phase I trial is evaluating the safety, persistence and the effects of escalating doses of autologous CMV-specific CTL in patients with CMV-positive GBM. To generate CMV-T cells, in vitrodifferentiated DC are transduced by an adenovirus carrying one CMV gene and are then used to stimulate autologous T cells. In vitro expanded CMV-T cells are injected i.v. to the patient (NCT01205334). Another Phase I trial is evaluating the safety of CMV pp65activated T-cells in adult patients with newly diagnosed GBMs during recovery from therapeutic TMZ-induced lymphopenia. Six CMV seropositive patients with newly diagnosed GBM are randomized to receive autologous CMV- specific T cells with or without CMV-DCs. After GBM resection in vitro generated DCs are pulsed with pp65 mRNA and used to activate pp65-specific T cells. Patients will then receive RT and concurrent TMZ at a standard dose (NCT00693095). The potential therapeutic efficacy of adoptive T cell transfer is limited by the rapid disappearance of CD8+ T cells, due to the fact that most of the in vitro expanded CD8+ T cells are short-lived effector cells. Such cells can effectively kill targets cells, but, in the absence of long term surviving memory cells, the immune response is transient. To solve this problem, a recent study in a mouse model showed that activation of the Wnt–Beta catenin pathway, using an inhibitor of glycogen synthase kinase-3 beta, generated CD8+ memory T stem cells (TSCM) from a subset of in vitro expanded antigen-specific CD8+ T cells [354]. The generation of TSCM allows the use of fewer cells for adoptive immunotherapy and to produces a long-lasting response. To circumvent the difficulty of expanding autologous antigen-specific T cell from GBM Peripheral Blood Lymphocytes (PBL), an alternative approach is represented by the use of semiallogeneic T cells, which share with the patient a common HLA-class I allele required for GBM-antigen presentation [355]. An ongoing clinical trial evaluates the safety and response to systemic infusion of partially matched, allogeneic, CMV-specific CTL for patients with CMV+ GBM that have failed primary therapy (NCT00990496). Genetically Engineered T Cells Genetic modification of T cells to redirect antigen specificity is an attractive strategy compared to the process of growing autologous T cell lines specific for each patient. High-avidity T cells that are reactive against GBM antigens have been used to clone the genes coding for the TCR alpha and beta chain. The cloned TCR genes were inserted into retro- or lenti-viruses and used to transduce autologous CD8+ T cells of patients that share the restricting HLA allele. The engineered T-cells can be expanded in vitro to obtain therapeutically effective numbers of T cells, which express the specific TCR redirecting lymphocyte effector functions to GBM. T cells engineered in this way, however, express both the endogenous TCR alpha/beta as well as the antigen specific TCR alpha/beta genes. The co-expression of four different molecules results in reciprocal pairing between introduced TCR alpha/beta with endogenous TCR alpha/beta chains, creating new unknown immune specificities. The mispairing of the four chains can be reduced by


genetic modification of the transduced chains, inserting the murine constant region sequences that increase pairing of only the transduced chains. Engineered T cells are reported to gain stable reactivity against specific TAA [356]. Adoptive transfer of engineered T cells in 15 melanoma patients produced stable engraftment for one year and two patients had regression of metastatic melanoma lesions [357]. A limitation revealed by this study was low level of expression of the transgenic TCRs by the transferred T cells. Another problem of TCR engineering is the relatively low affinity of TCR for their antigen. Phage display techniques have been used as an alternative procedure to generate TCRs with high affinity. Using this approach TCRs with a million fold improvement in affinity compared to the parent TCR have been produced [358]. Since many tumors cells downregulate HLA class I, rendering them invisible to the adaptive immune system, an alternative approach to the use of transgenic TCR is the generation of chimeric antigen receptors (Tbodies). T-bodies are artificial T cell receptors that combine the ability of antibodies to bind antigens with high affinity, with the cytotoxic abilities of T cells. The chimeric receptors are composed by the extracellular single-chain variable fragment (scFv) of an antibody fused to an intracellular T-cell signalling chains, such as CD3Z [359]. When expressed on T cells, the receptor bypasses the need for antigen presentation on HLA since the scFv binds directly to cell surface antigens. Recent studies focused on the development of scFv receptor constructs that incorporate the CD28 costimulatory domain linked with the TCRZ signaling domain [360], have shown increased antitumor function in vitro and in experimental murine models of colon carcinoma and GBM [361, 362] [363]. A phase I/II study to evaluate the safety and biological activity of escalating doses of autologous CMV-specific cytotoxic T-lymphocytes (CTL) genetically modified to express chimeric TCR specific for the HER2 molecule is planned in patients with HER2-positive GBM (NCT01109095). Another attractive possibility to redirect T cell cytotoxicity against IL-13Ralpha2+ GBM cells is based on the generation of an IL-13/CD3z chimeric construct (zetachine) [364]. In Zetachine, IL13 has an E13Y mutation, which increases its affinity of IL-13 for the IL-13Ralpha2 of GBM, while decreasing that for the conventional IL-13Ralpha1. A clinical study to test the safety and efficacy of allogeneic zetachine-expressing T cells in combination with IL-2 administered through convection-enhanced delivery is ongoing (NCT01082926). To avoid possible autoimmune adverse effects, the engineered T cells also express a suicide gene (HSV-TK), which allows selective elimination of the T cells by ganciclovir. LAK and NK Cells As T cells, NK cells are considered suitable effectors for immunotherapy of cancer patients, particularly those affected by hematological malignances. Hematopoietic stem cell transplantation (HSCT) led to major achievements in the cure of high-risk leukemia. However, only ~60% of patients have matched siblings or unrelated donors, and among the latter there is an important delay related to donor search. On the other hand, most patients have a relative identical for one HLA haplotype and fully mismatched for the other, who may rapidly serve as HSC donor. It is of note that in haploidentical HSC transplantation (Haplo HSCT), all cases were at high risk of T cell-mediated alloreaction in the graft vs. host (GvHD) direction. This side effect has been controlled by an extensive T cell depletion of the graft. Unfortunately, the benefit of GvHD prevention was offset by a markedly increased rate of relapses due to the lack of the T cell-mediated graft vs. leukemia (GvL) effect. Recent studies show that in Haplo HSCT, NK cells can mediate the GvL effect. However, since both myeloid and lymphoid leukemia express high “protective” amounts of HLA class I molecules, a “KIR-ligand mismatched” must be present in the donor vs. host direction; e.g. NK cells derived from HSC of the donor must express KIRs that do not recognize HLA-class I molecules expressed by the patient (“alloreactive” NK cells). Notably, allore-

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active NK cells do not mediate GvHD since they predominantly attack neoplastic hematopoietic cells of the patient while sparing other tissues. At present, Haplo HSCT from donors capable of generating alloreactive NK cells represents a major breakthrough in the cure of otherwise fatal leukemias. For example, the estimated survival rate in children with acute lymphoblastic leukemia (ALL) transplanted from an NK-alloreactive relative is now >75% [365]. Thus, a huge number of studies proved that NK cells play a central role in eradicating leukemic cells and Haplo HSCT from NK alloreactive donors (and possibly alloreactive NK cell infusion) entered the clinical practice. On the contrary, the clinical benefit of an NK cell-based immunotherapy in non-hematologic malignances is far to be demonstrated. Epidemiological findings however, show that infiltration of NK cells into human tumors might be associated with better prognosis [366] and that NK activity inversely correlates with cancer incidence [367]. NK cells efficiently kill tumors of different histotype including carcinoma, melanoma and tumor of neuroectodermal origin and have been demonstrated to play an important role in controlling the growth of various tumor cell lines injected in mice [368]. For example, NK cells exerted anti-tumor activity in a preclinical mouse model of neuroblastoma (NB) [369]. NOD/SCID mice were injected i.v. with HTLA-230, a human (HLA class I negative) NB cell line. Mice developed a disease reminiscent of that observed in Stage 4 patients, which was characterized by abdominal masses involving the kidney and/or the adrenal glands and bone marrow metastasis leading to the death of all the animals within 60 days. When infused in NB mice, in vitro activated human NK cells localized in different tissues including bone marrow. Moreover, with respect to untreated mice, NK-treated animals had reduced bone marrow infiltration and prolonged survivals, which increased according to the number of NK cell injections performed. On the above premises, various preclinical model of human solid cancers have been developed to evaluate the efficacy of NKcell based immunotherapy, clinical grade purifications of human NK cells have been set up and a phase I trial have been started in patients with various types of cancer including glioma (NCT00909558). A phase I/II trial enrolling 48 patients is studying the safety and clinical efficacy (in terms of tumor response, response duration, and survival) of donor stem cell transplant followed by donor NK cell infusion in patients with different advanced cancers including Brain Tumors (NCT00823524). In addition, also trials using IL-2-generated LAK cells, predominantly exploit the effect of IL-2-activated NK cells, although LAK cells are a heterogeneous mixture of NK and polyclonal T cells. Rosenberg [252] demonstrated that PBMC short-term cultured in presence of IL-2 generates lymphokine-activated killer cells (LAKs) able to lyse a wide variety of autologous and allogeneic tumors but with little cytotoxicity against normal tissues. The use of LAK cells for the treatment of glioma has been investigated, generally with transplantation of these cells into the post resection surgical cavity. Jacobs [370] infused LAK cells and IL-2 locally in the tumor with minimal toxicity and a small increase in the mean progression-free survival. Hayes [371] reported an increase in the survival of 19 GBM or Anaplastic Astrocytomas patients treated with 12 doses of LAK cells plus IL-2, establishing the IL-2 maximal tolerated dose. Despite these encouraging results, other trials did not produce similar success [372], and two clinical trials using intratumoral administration of IL-2 in combination with LAK cells or with IFN-alpha in glioma patients showed a remarkable toxicity related to increased edema and no significant clinical benefits [373] [374]. However, in another clinical study the intratumoral administration of IL-2-induced LAK cells resulted in a median survival of 9 months in 40 patients with recurrent GBM and the treatment was judged safe and feasible [375]. Intriguingly, it appeared that inclusion of IL-2 at the cell administration did not correlate with outcome. In a more recent study Dillman [376] treated 33 newly diag-


nosed GBM patients, that completed primary therapy for GBM without disease progression, with intralesional injection of adjuvant LAK cells. This study demonstrated a correlation between the number of LAK cells injected and survival, with a median overall survival of 20.5 months, with 75% of patients alive at one year, thus suggesting that intralesional LAK cell therapy may result in clinical benefit and warrants further investigation in randomized trials. A Phase II Randomized Trial with 80 enrolled patients will compare the side effects (including infections, abnormal healing at the surgery site) and overall survival of intralesional LAK cells vs. polifeprosan 20 plus carmustine implant in patients with newly diagnosed resectable glioblastoma (NCT00814593). A Phase II Trial has been completed, which analyzed the side effects and toxicity, progression-free survival and overall survival of grade IV anaplastic astrocytoma patients receiving rhIL-2-stimulated LAK cells intracranially at the time of therapeutic craniotomy or via an Ommaya reservoir placed during craniotomy (NCT00331526). Active Immunotherapy Immunization of patients against GBM-associated antigens has been attempted by different approaches, such as the use of cellbased vaccines, GBM-extracted heat-shock proteins, antigenic peptides or antigen-loaded DC. Vaccination with Synthetic Peptides Synthetic peptides are non-biological chemicals that can be produced on a large scale under the current good manufacturing practice (GMP) conditions, are safe and easy to administrate and suitable for multicenter clinical trials. However, several limitations affect the use of short peptides encompassing single antigenic T cell epitopes. Their main disadvantage is the HLA allele restriction, which allows their use only in the patients that possess the appropriate allele. Moreover, targeting a single antigen confer a growth advantage to neoplastic cells not expressing this epitope or the relevant HLA allele. This limitation could be potentially overcome by the simultaneous targeting of multiple epitopes and/or multiple tumor antigens. Other problems may arise as a consequence of suboptimal binding of peptides to specific HLA allele(s). In addition, peptide predicted in silico by specific algorithms may not necessarily be able to induce anti-tumor response in patients. Peptides used as cancer vaccines usually consist of ninefourteen amino acids capable of binding to a particular HLA class I antigen that can be recognized by specific CTLs, which are then activated and generate an expanded progeny capable to lyse tumor cells. Nonetheless, responses induced by CTL epitopes are usually short-lived due to the lack of a concomitant Th cell response. To increase the efficacy of the response, synthetic CTL epitopes can be mixed with an oil-adjuvant and/or linked to a carrier protein such as keyhole limpet hemocyanin (KLH), which possess multiple Th epitopes. Recent findings indicate that also a pool of longer synthetic peptides (20-30 aminoacids) of HPV viral oncoproteins E6 and E7 can activate both CD8 and CD4 T cell responses and cured a significant portion of patients with intraepithelial vulvar carcinoma [377]. Regarding GBM, preclinical studies demonstrated that a peptide (PEPvIII) derived from the fusion junction of the EGFRvIII variant, chemically conjugated to KLH, induced CTL responses against cells stably transfected with EGFRvIII [378]. To confirm these promising early pre-clinical results, a Phase II clinical trial evaluating the efficacy of intradermal vaccination with PEPvIII-KLH without DCs, was recently conducted at the Duke University and Anderson Cancer Center. Eighteen newly diagnosed EGFRvIIIpositive GBM patients treated by surgical resection, radiation therapy and concurrent TMZ, were treated every two weeks with a vaccine dose followed by monthly vaccinations. The vaccination produced a specific humoral response and induction of EGFRvIII specific CD8+ T cells. The OS of vaccinated patients was 26 months


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compared with 15 months of control group [142]. The titer of EGFRvIII-specific antibodies and the positivity for DTH skin tests was evaluated. The median OS for patients that developed EGFRvIIIspecific antibodies was 47.7 months, compared with 22 months for patients without antibody response. Patients that developed PEPvIII-specific DTH response had a median OS of more than 50 months, compared with 23,1 months for patients that did not developed DHT response. At recurrence, tumor material obtained from 11 patients was evaluated for EGFRvIII expression. The analysis showed that 9 patients had lost the expression and 1 had less than 1% of residual expression. Although results are encouraging, a blinded, placebo-controlled, randomized phase III study would be required to provide a definitive evidence of efficacy. Additional patients are recruited in trials that use intradermal vaccination with PEPvIII-KLH. A Phase II ongoing Study, enrolling 65 patients is evaluating the clinical activity of CDX-110 (PEPvIII-KLH) vaccination plus GM-CSF given together with standard therapy (NCT00458601). A randomized phase I/II trial is studying the side effects and best schedule of chemotherapy plus radiation therapy followed by vaccine therapy with PEP-3-KLH and anti-CD25 mAbs (basiliximab or daclizumab) used to target Treg cells in patients with GBM (NCT00626015). Izumoto reported a Phase II clinical trial vaccinating 21 patients with recurrent GBM with a single WT1 peptide. Patients received weekly intra-dermal injections of a modified 9-mer WT1 peptide for 12 weeks. The study reports partial responses in 2 patients, stable disease in 10 patients, and progressive disease in 9 patients, a median PFS of 20 weeks and a possible association between the WT1 expression levels and clinical responses [379]. Several other clinical phase I or phase I/II studies employing different types of antigenic peptides are currently ongoing, including IMA950 a mixture of 11 tumor-associated peptides (NCT01222221), telomerase peptide (aa 540-548) plus GM-CSF (NCT00069940) and survivin peptide (NCT01250470). Cellular Vaccines An alternative to overcome the several limitations connected with the use of selected individual TAAs is the vaccination with autologous or allogeneic whole tumor cells. Tumor cells express both characterized and uncharacterized TAAs, containing epitopes of both CD8+ cytotoxic T cells and CD4+ T helper cells. The presentation of both HLA class I and II-restricted antigens helps to generate a stronger anti-tumor response and long-term CD8+ T-cell memory via CD4+ T-cells. In addition, the chance of tumor escape, deriving from the use of single epitope, is greatly diminished. The simpler method to deliver antigens for GBM immunotherapies involves the use of allogeneic tumor cell lines that can be easily expanded in GMP facilities, ensuring reproducibility. On the other hand, autologous tumor cells potentially carry gene mutations coding for unique TAAs. Allogeneic or autologous tumor cells inactivated by radiation, sometimes genetically modified or infected with a virus, or applied together with cytokine-secreting fibroblasts to boost the immune reaction, have been used in several clinical trials. In one of the earliest trials, Mahaley treated 20 postoperative GBM patients with monthly subcutaneous inoculations of a human glioma cell line (U-251MG) together with levamisole plus 500 micrograms of bacillus Calmette-Guerin cell wall (BCG-CW) at the first inoculation. Vaccinated patients had prolonged survival when compared with historic controls without evidence of encephalomyelitis [380]. Since its publication, numerous immunotherapy studies have followed. Results from a Phase I/IIa trial using autologous formalinfixed tumor vaccine for newly diagnosed GBM have been recently reported. In this study 24 patients, after radiotherapy, were injected with the vaccine intradermally weekly for 3 times. One patient had dermal toxicity, no other toxicities were observed. The median overall survival was 21.4 months, and 7.6 months the median progression-free survival. The PFS of the patients with greater DTH reaction (13.9 months) was longer than that of the patients with


weaker DTH reaction (4.3 months) [381]. Several protocols used autologous tumor cells genetically modified to produce immunostimulants are described elsewhere in this review. Recent clinical trials use GBM stem cells as source of antigens. Some trials combine active and passive approaches to treat GBM patients. GM-CSF is a cytokine, which can act on myelomonocytic precursors of DC and initiate their differentiation into DC. Therefore it is considered as a possible adjuvant for vaccines. An ongoing Phase II trial is designed to study the effectiveness of vaccination with irradiated, autologous tumor cells plus GM-CSF administered intradermally in GBM patients. Enlarged draining lymph nodes are then removed 7-10 days later and activated with staphylococcal enterotoxin A (SEA) and IL-2. T cells are expanded ex vivo and infused i.v. after 1-2 days from a single dose of cyclophosphamide (NCT00003185). GM-CSF-producing cells mixed with syngeneic tumor cells have been shown to activate capture of TAA by DC, antigen presentation and induction of an anti-tumor response. A Phase I Study determines the safety and biological activity of subcutaneous and intradermal injection of irradiated autologous malignant glioma cells mixed with an irradiated GM-CSF producing GM-K562 cell line in 25 patient with recurrent malignant glioma (NCT00694330). An alternative to cellular vaccines consists in the isolation of Heat shock proteins (HSP) from tumor specimens. HSP function as molecular chaperones ensuring folding, assembly, and transport of nascent peptides and binds to tumor antigens forming an immunogenic complex capable of activating CTL. A Phase I/II trial is studying the side effects and best dose of gp96 heat shock proteinpeptide complex vaccine in treating patients with recurrent GBM is enrolling 50 patients (NCT00293423). Dendritic Cells Dendritic cells, (DCs) known as professional antigen presenting cells (APCs), are the most potent initiators of the immune response. DCs have the capacity of capturing antigens from the environment, which is followed by antigen processing and presentation to the T cells. In addition, DCs have also a role in B, NK and NK-T cell responses, bridging natural and adaptive immunity. Immature DC are characterized by high antigen uptake and sequestration but not by antigen processing and presentation nor by immunostimulatory cytokines secretion. Danger signals, including endogenous stressrelated molecules (heat shock proteins, DNA and ATP) or PAMPs (LPS and bacterial or viral nucleic acids) are detected by Toll-like, NOD-like, RIG-1-like receptors expressed by DCs. Upon activation by danger signals, immature DCs undergo a maturation process, which leads to the upregulation of HLA class I and II molecules required for antigen presentation as well as of T cell co-stimulatory cell surface molecules and release of immunostimulatory cytokines. CD80 and CD86 provide an essential co-stimulatory signal to the T cells through the CD28 molecule, which amplifies activation signals delivered via the TCR complex engaged by the HLA + antigen peptide complex (Fig. 1). DCs activation triggers not only maturation but also migration toward secondary lymphoid organs where mature DCs prime naive CD8+ T cells and CD4+ T cells by present antigenic peptides in the context of HLA class I and II molecules, respectively. The establishment of an immunological synapse between DC and the CD4+ Th cell mediates a cross-talk between the two cells through a series of receptor/ligand pairs. Activated CD4+ Th cells license DCs for CTL cross-priming through CD40 ligand (CD40L)–CD40 interactions and stimulate CTL proliferation and differentiation through the production of cytokines such as IL-2 and other cytokines (Fig. 1) [382]. Maturation is a complex process triggered by agonists receptors which may be differentially expressed by functionally district subsets of DC and promote different patterns of cytokine secretions and T cells immunostimulatory effects. Depending on the signal and their functional (mature or immature) status, DC are able to produce IL-12 that polarize naive

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CD4+ T lymphocytes toward the Th1 phenotype, the most important for generation of anti-tumor CTL responses. In addition, DCs undergoing maturation induce activation of NK cells which results in IFN-gamma release and enhancement of the cytolytic activity [235] [236] [237] [238]. Tumor associated DC are probably ineffective in mounting an anti-tumor response because immunomodulatory cytokines, IL-10, TGF-beta, VEG-F as well as Treg and myeloid suppressor cells present in the microenvironment would alter DC maturation [232] [231]. To overcome the inefficiency of tumor resident DCs it seemed therefore reasonable to vaccinate patients with DCs loaded with tumor antigens and matured ex vivo. Animal models that explored the effectiveness and safety of dendritic cell vaccination [383] [384] were developed since late 1990s. Early animal studies using different strategies based on glioma antigens loaded–DC, showed a potential efficacy and also alleviated concerns about the possible development of experimental allergic encephalomyelitis as side effect of vaccination [385]. Glioma lysates, synthetic peptides, acid eluted glioma peptides, tumorextracted RNA, antigen gene containing vectors, loaded on DC or DC fused with glioma cells were used as immunogens [386] [387] [388] [389] [390]. More recent evidences suggest that only a small fraction of tumor cells, defined as cancer stem cells (CSC) is able to perpetuate the tumor and to be tumorigenic upon transplantation in mice. It is conceivable that this highly malignant cancer subpopulation should be therefore primarily targeted. In a syngeneic mouse model of glioblastoma vaccination with DCs pulsed with GL261 neurospheres cell lysates (CSC enriched cells) injected 1 week after intracranial glioma transplantation, cured 60% of CSC inducedtumors and 80% of not CSC induced-tumors. Whereas vaccination with DCs pulsed with GL261 adherent cell lysates cured only 50% of not CSC induced-tumors and none of the CSC induced-tumors [391]. These results suggest that CSC targeting by DCs vaccine may offer a therapeutic advantage against glioma. Altogether most animal studies showed that vaccination with antigen pulsed DCs resulted in increased overall survival, infiltration of CD8+ CTL in the tumor and strong in vitro anti-tumor cytotoxicity of splenocytes derived from immunized animals, paving the way for further clinical studies. DCs may be isolated from peripheral blood though usually in small number. Many clinical trials therefore obtained DCs from hematopoietic CD34+ cells or from monocytes by short-term culture in medium containing cytokines such as GM-CSF and IL-4 [392]. To further activate DCs most clinical trials use an inflammatory cytokine cocktail. However, optimal DCs maturation and IL-12 secretion occur through TLR signaling, which results in an effective Th1 response [393]. The use of TLR receptor agonists such as polyICLC has therefore been considered [394]. Other groups prefer using immature DC, characterized by a greater ability to antigen uptake and processing. These immature DC when injected into adjuvant-pretreated sites would then mature in a more physiologic manner [395]. Antigen loading has been performed through antigens exposure, transfection with antigen encoding RNA and cell fusion [396] [397] [398]. A phase I clinical trial enrolling 9 patients with newly diagnosed anaplastic astrocytoma and GBM, used DCs loaded with autologous GBM-acid-eluted peptides injected subcutaneously. The study reported an increased median survival, 455 days versus 257 days of control group. Tumors infiltration of CD8+ CTL and memory T cells was demonstrated in two patients that had second surgery, and systemic anti-tumor CTL activity was detected in four of seven patients [396]. Okada et al recently proposed a novel protocol of DCs vaccination, in a phase I/II clinical trial. DCs obtained from peripheral blood monocytes cultured in the presence of GM-CSF and IL4, were matured with IL-1beta, TNF-alpha, IFN-alpha, IFN-



15

Fig. (1). Dendritic cells are professional APC, which establish a dynamic cross-talk with T cells during antigen presentation. TLRs provide an activation/maturation signal to DC, which produce cytokines such as IL-12 mediating Th1 polarization and upregulate HLA molecule and co-stimulatory molecule expression. Mature DC presents peptide antigens in the context of HLA-class II molecules to the Th cell. CD80/CD86 expressed on mature DC co-stimulate Th cell activation via CD28. Th cell activation results in secretion of Th cytokines and in CD40L expression. CD40L binding to CD40 licenses DC for cross-priming of CTLs, which recognize antigenic peptides complexed with HLA class I. CD80-86, besides providing a co-stimulus via CD28, also provide a negative signal through the CTLA4 molecule, which limits CTL activation. Blockade of CTLA-4 by specific antibodies releases T cells from the inhibitory signal and potentiates their response.4

gamma and the TLR-agonist poly-ICLC to mediate Th1 polarization. Twenty-two patients received intranodal vaccination with DCs loaded with four different glioma associated peptides, and intramuscular injections of immunoadjuvant poly-ICLC. This study demonstrated safety and immunogenicity as well as preliminary efficacy of this vaccination protocol with nine patients progressionfree for at least 12 months, of which five patients are currently progression-free [399]. A dose escalation study was performed in a phase I clinical trial with one, five and ten millions DCs loaded with acid-eluted autologous tumor peptides per injection in 12 recurrent or newly diagnosed GBM patients. Six out of 12 patients developed peripheral antitumor CTL responses, with no relationship to the DC dose administered. The detection of CTL responses in the peripheral blood of immunized patients was not in itself predictive of objective clinical response and/or prolonged survival. However, there was a significant correlation between intracranial CD8+ and CD4+ T-cell infiltration within the local tumor and prolonged survival. Interestingly, intratumoral infiltration of T lymphocytes negatively correlated with the expression of the immunosuppressive factor TGFbeta2 thus supporting a possible synergistic antitumor effect of TGF-beta inhibitors in active dendritic cell vaccination [400]. A phase I-II clinical trial used DC fused with glioma cells plus recombinant human IL-12 (rhIL-12) for the treatment of 15 patients with malignant glioma. Autologous dendritic cells were derived from peripheral blood, activated in vitro with GM-CSF, IL-4 and TNF-alpha and injected intradermally. Recombinant IL-12 was then administered subcutaneously at the same site. Also in this study no serious adverse effects were observed. Although anti-tumor cytotoxic activity, as well as IFN-gamma production was found in few patients, clinical and radiographic response was observed in half of

them. This study shows again that systemic peripheral blood antitumor response does not necessarily translate to vaccine-induced responses within the tumor [299]. In a recent phase II clinical study, 34 patients with newly diagnosed or recurrent GBM were treated with subcutaneous injection of autologous tumor lysate-loaded DCs. More than 50% of tested patients showed an IFN-gamma response in PBMC that correlated with survival. Advantage in survival was not observed in patients with recurrent disease. Moreover, both responders and not responders experienced a prolonged time to progression when DCs vaccination was followed by adjuvant chemotherapy. This study suggests that the effectiveness of DCs vaccination is negatively influenced by tumor burden and recurrence, whereas chemotherapy following vaccination may be beneficial [401]. Cytomegalovirus related peptides may be considered as potent immunogens against glioma, in fact an increase in circulating CMV-reactive T lymphocytes, has been reported in GBM patients treated with tumor lysate-pulsed DCs [402]. At the Duke University phase I/II clinical trial (NCT00626483) recruited patients with newly diagnosed GBM to determine if the anti-CD25 mAb daclizumab, an antibody targeting regulatory Tregs, inhibits recovery of T-regulatory cells after TMZ-induced lymphopenia in the context of vaccination. The vaccination protocol is complex and includes CMV pp65-lysosomal-associated membrane protein mRNA-loaded DCs and autolymphocyte therapy in patients who are seropositive and seronegative for CMV. Results of this study will add informations about the influence of regulatory Tregs on therapeutic DCs vaccination. In addition the combination of passive and active immunization after TMZ treatment might improve the final therapeutic effect.


Another ongoing phase I clinical trial conducted by the Duke University (NCT00890032) enrolled so far 50 patients with recurrent GBM and use CD133-positive brain tumor stem cells mRNApulsed autologous DC vaccine. Primary objective of this study is the comparison of the proportion of vaccinated patients alive at 6 months from the time of surgery with matched historical controls and the assessment of humoral and cellular immune responses to vaccination. A similar phase I/II study is ongoing at Ullevaal University Hospital (Oslo), NCT00846456, which uses Tumor Stem Cell derived mRNA-Transfected Dendritic Cells in patients receiving standard therapy for GBM. The aim of this study is the evaluation of immunological response, time to progression and survival. DCs vaccination, in preclinical and clinical studies, is having encouraging results, not only for the absence of major toxicity but also for evidences of induction of an immune response and some clinical benefit. Many issues about the type of dendritic cells to be used, route and times of administration, and type of antigen loaded will be addressed by ongoing trials. It must be stressed that in spite of a general skepticism on cancer vaccines, the demonstration of clinical benefit of the autologous DC-based sipuleucel-T vaccine by a recent phase III clinical trial in castration-resistant metastatic prostate cancer patients, has led to the FDA approval for clinical use of the first therapeutic cancer vaccine [403]. Antibody-based Therapy Monoclonal antibodies have been originally produced by somatic hybridization of B cells from immunized mice with a nonsecreting myeloma [404]. MAbs produced by a cloned hybridoma are identical molecules, homogeneous for their antigen binding specificity, affinity and effector functions. Murine mAbs have been initially thought to represent suitable “magic bullets” to selectively eliminate unwanted neoplastic cell populations in cancer patients. Although some murine antibodies are still used in cancer therapy, it became soon evident that xenogenic mAbs are immunogenic and can generate anaphylactic reactions and a host anti-mouse antibody (HAMA) response. HAMA binding to mouse mAbs may reduce their half-life or inactivate the antibody and leads to formation of immune complexes, resulting in side effects, such as serum sickness. Therefore chimeric human-mouse antibodies [405] or humanized antibodies [406], which contain only the mouse Ig portions necessary for antigen recognition have been generated by genetic engineering to reduce mAb immunogenicity. Fully human antibodies can be also generated in human Ig-gene-Knock-in mice, by procedures identical to those used for producing murine mAbs [407]. Another possibility to generate human antibody fragments is based on the screening of large combinatorial phage-display libraries of single-chain Fv fragments (ScFv) [408]. Whole antibody molecules possess antigen recognition sites and effector functions dependent on the Fc portion of the antibody, which is determined by the Ig isotype. Most therapeutic antibodies generated for tumor targeting have the human IgG1 scaffold, as this isotype displays a high capacity to activate the complement system and Antibody-Dependent Cell-mediated Cytotoxicity (ADCC). Innate immunity effectors, such as NK cells that express CD16 (Fcgamma RIII), i.e. the low affinity Fc-gamma receptor mediate ADCC. IgG immune complexes on the surface of target cells bind to CD16, which activates the cytotoxic functions of NK cells. Clinical studies found a relationship between Fc-gammaRIII gene polymorphisms and response to different therapeutic mAbs, as in the case of the anti-EGFR cetuximab [409], thus supporting a role of ADCC in clinical efficacy. Besides these cytotoxic properties therapeutic antibodies can also functionally block surface receptors essential for tumor cell growth or survival, or block soluble growth factors, thus preventing their interaction with receptor molecules. In addition, antibodies (either whole molecules or ScFV fragments) can also be used as vehicle to selectively target toxic compounds, isotopes or immune-enhancing cytokines to the tumor. As

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anticipated in the previous section, gliomas display surface molecules including EGF-R, its variant EGFRvIII and HER2 and extracellular matrix antigenic targets such as tenascin-C, which can be targeted by antibodies. Moreover, antibodies that block angiogenesis-related growth factors such as VEGF-A or the hepatocytegrowth factor (HGF) are currently studied for GBM therapy. Finally antibodies can also be used for eliminating immune-system checkpoints that downregulate the immune response: such as antiCD25 antibodies targeting Treg cells, or anti-CTLA-4 blocking antibodies. The CTLA-4 is a molecule expressed on activated T cells and binds to CD80 and CD86 molecules expressed by antigen presenting cells (Fig. 1). Whereas CD80 and CD86 provide a costimulatory signal to T cells through their interaction with CD28, their binding with CTLA-4 mediates an inhibitory signal, which limits T cell activation. Blockade of CTLA-4 molecule by specific antibodies such as Ipilimumab or Tremelimumab releases T cells from the inhibitory signal and potentiates T cell-mediated immune responses. In addition, CTLA-4 is expressed on Treg cells and plays a role in their immunosuppressive activity. Anti-CTLA-4 mAbs have shown significant effects in phase I-II trials in a subset of melanoma and renal cancer patients, through the activation of an immune response [410] [411]. A recent phase III study on 676 HLA-A*0201-positive patients with metastatic melanoma showed that the anti-CTLA4 mAb Ipilimumab improved overall survival, irrespective of its use as single agent or in combination with a gp100 peptide vaccine (NCT00094653) [412]. This study led to the very recent approval of Ipilumumab for the treatment of metastatic melanoma. In addition, the efficacy of anti-CTLA-4 mAbs is currently being tested in several studies in different types of tumors [413]. CTLA-4 blockade by a specific mAb increased survival in 80% of GBM-bearing syngeneic mice, and re-established normal CD4 counts while abrogating the increase in Treg cells induced by GBM [414]. Nonetheless anti-CTLA4 antibodies have not yet entered clinical trials in GBM patients. Anti-EGFR antibodies have been used to target GBM, as the EGFR is overexpressed in the majority of GBM. The anti-EGFR chimeric mAb cetuximab has been shown to block EGFR-ligand interaction [415] and also prevents EGFR dimerization and ligandindependent activation [416]. In preclinical studies cetuximab showed anti-proliferative and pro-apoptotic effects on human GBM cell lines overexpressing the EGFR in vitro and also inhibited GBM xenograft growth [417]. Moreover, the mAb enhanced the cytotoxic effects of radiation in GBM cells both in vitro and in vivo [418]. In view of its activity in pre-clinical testing, cetuximab has entered clinical studies in GMB patients. A phase I/II trial, is studying in primary GBM the combination of cetuximab, intravenously administered weekly, with the standard treatment (radiotherapy + TMZ) (“GERT”) [419]. A preliminary report indicates that this combination is well tolerated and 87% of treated patients were still alive at 12 months [420]. A two-arm, open-label, phase II study of cetuximab monotherapy was performed in recurrent high-grade glioma after failure of surgery, radiation therapy, and chemotherapy. Cetuximab was administered intravenously weekly. This treatment was well tolerated but only 3 (5.5%) out of 55 patients (28 with and 27 without EGFR amplification) had a partial response, 16 patients (29.6%) had stable disease and the median OS was 5.0 months. Therefore cetuximab monotherapy had limited activity in progressive high-grade glioma. In addition, there was no correlation between response or survival and EGFR gene copy number [421]. Another possibility of using mAbs is radioimmunotherapy where mAbs were used to selectively target isotopes to the tumor. A Phase I clinical study evaluated the toxicity and clinical effect of an intracavitary administration of a single dose of the humanized anti-EGFR mAb Nimotuzumab labeled with 128Re. Three patients


with anaplastic astrocytoma (AA) and 8 with GBM were treated. MTD of the radioconjugate was 3 mg labeled with 10 mCi of 188Re. One out of eight GBM patient had a partial response for more than 1 year and 2 patients (1 GBM and 1 AA) were asymptomatic and in complete response after 3 years of treatment, suggesting that this immunoconjugate may represent a potentially interesting approach for high-grade gliomas [422]. A Phase II study tested the efficacy of adjuvant radioimmunotherapy with 125I-labeled murine anti-EGFR mAb-425 in patients with newly diagnosed GBM. A total of 192 patients with GBM were treated by 3 weekly intravenous injections of 1.8 GBq 125ImAb-425 following surgery and radiation therapy, whereas a subgroup also received TMZ. The radioimmunotherapy was well tolerated. The overall median survival was 15.7 months, and the median survival was 14.5 and 20.2 months for the subgroups receiving 125ImAb-425 only or 125I-mAb-425+TMZ, respectively [423]. Several other trials using anti-EGFR antibodies are currently ongoing. For example a phase 2, single-arm, multi-center study has been designed to evaluate the efficacy and safety of nimotuzumab in pediatric patients with recurrent diffuse intrinsic pontine glioma (NCT00600054). Another phase II study will test the activity and response rate of a combination of the panitumumab with the topoisomerase 1 inhibitor irinotecan in malignant glioma patients (NCT01017653). Mabs specific for the GBM-associated EGFRvIII variant, such as mAb 806 and Y10 have been also generated and have shown activity in preclinical models [141] [424] [425]. However no data on clinical testing are yet available. Bevacizumab, a humanized mAb that specifically blocks the VEGF-A, is the first anti-angiogenesis drug approved for colorectal cancer therapy and shows therapeutic activity in different tumors [426]. A clinical trial of bevacizumab combined with irinotecan in 23 recurrent GBM patients showed a response rate of 61% with a median progression-free survival of 23 weeks [427]. Two phase II clinical trials of Bevacizumab alone and in combination with irinotecan in recurrent glioblastoma showed an increased response rate and prolongations of the 6-months progression-free survival [Bevacizumab alone and in combination with irinotecan in recurrent glioblastoma [428] [429]. These data indicated that bevacizumab and irinotecan are an effective treatment for recurrent GBM, and led to accelerated approval of bevacizumab for progressive GBM by FDA. A phase II trial studied the combination of Cetuximab, bevacizumab and irinotecan in patients with recurrent GBM in progression after standard radiotherapy and TMZ. Bevacizumab and irinotecan were administered intravenously every 2 weeks and cetuximab weekly. Imaging responses (2 complete and 9 partial) were observed in 34% of 32 evaluable patients. The median OS was 29 weeks. Cetuximab in combination with bevacizumab and irinotecan in recurrent GBM was in general well tolerated, except for skin toxicity, and showed an encouraging response rate, which however seemed not superior to that of bevacizumab and irinotecan alone [430]. Altogether the studies of bevacizumab in recurrent gliomas showed high response rates, based on radiographic imaging, and on increased 6-months progression-free survival. The real benefit of bevacizumab-based therapies in GBM and other high-grade gliomas is now being addressed in several ongoing clinical studies in combination with other treatments, such as metronomic TMZ (NCT00501891), irinotecan + carboplatin (NCT00953121) AMG 102 (NCT01113398), and others. A very recent study of bevacizumab + TMZ during and after radiation therapy in seventy patients with newly diagnosed GBM showed an improved progression-free survival without improved overall survival compared to a control cohort [431]. Two phase III studies are currently testing bevacizumab in combination with standard treatment in newly diagnosed glioblastoma multiforme (NCT00884741; NCT00943826). Thus,


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further studies are warranted to verify whether addition of bevacizumab to standard first-line therapy really improves survival. AMG 102 is a fully human, IgG2, neutralizing antibody that selectively targets the hepatocyte growth factor/scatter factor (HGF). The rationale for its use in glioma is that the HGF and its receptor c-Met are involved in gliomagenesis through an autocrine pathway, which regulates cancer cell survival, invasion, migration, and angiogenesis [432]. A phase II study is ongoing to assess safety and efficacy of AMG 102 infusion in 40 patients with recurrent GBM. A preliminary report on 20 patients who received AMG 102 at 10 mg/kg showed 1 complete response, 1 partial response and two disease-stabilizations out of 18 evaluable patients. Five patients reported grade 3 or 4 toxicities, but no treatment-related deaths were reported. These results suggest that a subset of recurrent GBM may be dependent on the c-MET/HGF axis. Among antibodies directed to the extracellular matrix proteins of GBM, the antitenascin-C murine antibody 81C6 [200] and BC4 [433] showed strong reactivity against GBM tissue. These antibodies were then conjugated with 131I and tested in different clinical Phase I and II trials, which suggested clinical benefit in GBM [434]. A Phase III randomized clinical trial termed GLASS-ART (GBM Locoregional Agent Survival Study -Anti-tenascin Radiolabeled antibody Therapy) will compare the efficacy of a combination of the radiolabeled 81C6 mAb (Neuradiab) plus standard treatment, with standard therapy alone (NCT00615186). CONCLUDING REMARKS Several approaches of immunotherapy have been developed and have shown efficacy in pre-clinical models of GBM (Table I). Many of these therapies have been translated into clinical studies, either in patients with recurrent disease or in newly diagnosed GBM, and in several instances a potential clinical benefit was found in phase I-II trials (Table 2). Nonetheless, most early trials have been performed before the new standard treatment of post-operative radiotherapy and TMZ has been adopted [8]. Therefore recently designed clinical studies, including some phase III randomized trials are now evaluating the potential benefit of adding immunotherapy to standard therapy in newly diagnosed GBM. These trials will define whether immunotherapy approaches such as monoclonal antibodies (anti-EGFR, anti-VEGFA and anti-tenascin), immune enhancing cytokines (IFNs), TLR agonists (CPG-ODN and PolyICLC), adoptive T or NK cell transfer, or cancer vaccines (peptides or DC-based vaccines) will hold promise and enhance the effect of standard treatment. As far as active immunization towards GBM-associated antigens is concerned, it must be said that there have been many doubts over the efficacy of cancer vaccines for many years. However, in 2010 three studies have shown clear effects of vaccine approaches in cancer. Indeed, the autologous DC-based sipuleucel-T vaccine has shown to increase overall survival in castration-resistant metastatic prostate cancer patients in a recent phase III clinical trial, leading to the FDA approval of the first therapeutic cancer vaccine [403]. Another study reported a high response rate in intraepithelial carcinoma of the vulva by vaccination with long synthetic peptides derived from HPV oncogenic proteins [377]. A very recent phase II study of EGFRvIII synthetic peptide vaccine showed a potential clinical benefit in GBM [142], supporting that this type of vaccines should be further tested in clinical trials. The development of immunotherapy involving active immunization to GBM antigens raises the critical issue of how would standard treatment affect the immune response and which is the optimal timing for immunotherapy initiation. This does not necessarily mean that standard treatment will hamper active immunization, as transient removal of GBM-related immune suppression may favor the induction of an immune response. In this context, cyclophosphamide, an alkylating agent of the same class of TMZ, has shown immune-modulatory effects and may diminish Treg cell numbers


Table 1.

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Representative Preclinical Models of Glioma Immunotherapy Deserving Further Investigation

Ref. #

Animal Model

Treatment

Results: % Survival Treated/Control or Reduction in Tumor Size

U87 human glioma xenografted in nude mice (sc or ic)

F16 (anti-tenascin)-IL-2 fusion protein + TMZ

sc: no growth at 160 days

[275]

Adult N32 glioma in syngenic Fisher 344 rats (ic)

N32-IFNgamma ip+ Stromal-IL-7 it

Over 90% reduction in size

[281]

GL261 glioma in syngenic mice (ic)

IL-21 transduced tumor cells or recombinant IL-21 it

100%/0 survival

[297]

GL261 glioma in syngeneic mice (ic)

IL-12plasmid/PPC it+ with adjuvant local biodegradable carmustine

100%/0 survival

[298]

GL26 glioma in syngenic mice (ic)

IL-12 transduced neurospheres it

30%/0 survival at 60 days

[301]

GL26 glioma in syngeneic mice (ic)

Syngenic DC transduced with IL-23adenovirus vector it

80%/20% survival at 120 days

[319]

U87 human glioma in athymic mice (ic)

Targeted expression of IFN-alpha in Tie-2 macrophages

85% reduction of tumor size by MRI

[391]

GL261 stem or adherent cells in syngenic mice (ic)

Sc vaccination with DC loaded with lysates from GL261 stem cells (DC-NS)

80%/0 survival with DC-NS

[413]

SMA-560 glioma in syngenic mice (ic)

anti-CTLA-4 ip

80% /0 survival Restoration of CD4 counts. Abrogation of Treg increase

[265]

ic: 55%/0 survival at 160 days

sc: subcutaneous; ic: intracranial; iv: intravenous; it: intratumoral; PPC: polymeric vehicle; DC: dendritic cells; ip: intraperitoneal.

Table 2. Ref. # [142]

[323]

Representative Clinical Trials that Showed Potential Benefits in GBM Patients and Warrant Further Studies. Treatment

Phase

EGFRvIII PEP-3-KLH / intradermal

II

IFN-beta/ iv

I

Patients 18 newly diagnosed GBM EGFRvIII

Concurrent Treatment Standard therapy

Results OS 26 vs. 15 mo

+

16 newly diagnosed GBM

Related Studies NCT00458601 NCT00626015

Standard therapy

OS 17.1

NCT00031083

40% OR [338]

poly-ICLC / im

II


Standard therapy

OS 17.2 mo

NCT00058123 NCT00058123

[343]

CpG-ODN /

II

31 recurrent GBM

none

intracerebral

OS 6.5 mo, few

NCT00190424

long-term survivors

[376]

LAK/ intralesional

II


Standard therapy

OS 20.5 vs. 15 mo

NCT00814593

[381]

Formalin fixed autologous tumor cells / intradermal

I/II


Radiotherapy

OS 21.4 mo

NCT00003185

[399]

DC1 + syntetic peptides/intranodal + CpG-ODN/im

I/II

22 recurrent glioma

None

PFS>12mo 41%

NCT00612001 NCT01204684 NCT01213407 NCT00323115 NCT01006044

[422]

anti-EGFR

125

I-mAb-425/ iv

II

192 newly diagnosed high grade glioma

Radiation or

OS 14.5 or

Standard therapy

20.2 mo

NCT01317888



19

(Table 2) Contd….

Ref. #

Treatment

[428]

Bevacizumab alone or + irinotecan/ iv

Phase II

Patients

Concurrent Treatment

167 recurrent GBM

none

Results PFS>6mo 42.6 or 50.3%

Related Studies Ref [431] NCT00890786 NCT00501891 NCT01113398 NCT00884741 NCT00943826

[434]

Anti-tenascin intracavitary

131

I-81C6 mAb

II

33 newly diagnosed glioma (27 GBM)

Followed by radiotherapy and chemotherapy

OS 20.2 mo

NCT00002753 NCT00615186 NCT00003478 NCT00002752

OS: overall survival. PFS: progression free survival. OR: objective response rate

and functions, thus enhancing the effects of vaccines [435]. Production of CCL2 and CCL22 by glioma cells is responsible for accumulation of Treg cells in the tumor environment, and this effect can be mitigated by TMZ [436]. In addition, Treg depletion can be achieved in GBM-bearing rats by a low-dose metronomic TMZ regimen, which additionally decreased suppressive function of the remaining Treg cells [437]. Moreover, transient lymphodepletion by alkylating agents may result in a wave of endogenous homeostatic cytokines, which may help not only the replenishment of the lymphocyte pool, but also the enhancement of an immune response [438]. Collectively, these data suggest that indeed chemotherapy could augment active immunotherapy for GBM. It must also be stressed that immunotherapy acts through different mechanisms as compared radiotherapy and chemotherapy, thus suggesting potential additive therapeutic effects without increasing toxicities of each treatment. Finally, several molecules, which have shown efficacy in preclinical models, have not yet entered clinical trials. This is the case of antibodies specific for the EGFRvIII variant, or immuneenhancing antibodies, which block mechanisms of immune regulation such as anti-CTLA4 or anti-TGF-beta antibodies. The recent FDA approval of Ipilimumab for the treatment of metastatic melanoma is leading to its clinical testing in different tumors. Also some cytokines such as the IL-21, IL-12 and IL-23 have shown antitumor effects in pre-clinical models and may warrant clinical investigation. In this context the delivery modalities of cytokines seem very important as systemic administration may have side effects and result in low effects at the tumor site. Thus, approaches allowing to achieve local sustained concentrations at the tumor site could be more effective; these include local administration by convection enhanced delivery, gene transfer strategies or the use of genetically engineered cytokine-producing cells. Other possibilities are related to the generation of immunocytokines, chimeric molecules formed by an scFV antibody linked to an active cytokine, which may concentrate the cytokine activity on GBM cells or on its extracellular matrix. For example, this is the case of the L19-IL-2 immunocytokine, designed to deliver IL-2 to a tumor-specific fetal isoform of fibronectin, also expressed in GBM [439]. Finally, in view of the crucial role of tumor stem cells in resistance to therapy and in relapses, it would be essential that target molecules were expressed in this cell compartment. For example, Her2 has been reported as a good target to eliminate this subset of tumorigenic cells [361], while other approaches utilizes antigen mixture extracted from GBM stem cells to load DCs for vaccine therapy. Unfortunately cancer stem cell markers identified so far (such as CD133) are shared by normal stem cells and are therefore

unsuitable for selective tumor targeting. It is hoped that a better knowledge of GBM stem cells may provide new suitable targets for immunotherapy. ACKNOWLEDGEMENTS This work was supported by Investigator Grant (10643 and 8915) and special project 5x1000 (9962) from Associazione Italiana per la Ricerca sul Cancro (A.I.R.C.), Ministero dell'Istruzione, dell'Università e della Ricerca (M.I.U.R), Ministero della Salute and Compagnia di San Paolo. REFERENCES [1]

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Received: June 27 2011

Accepted: July 12, 2011

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