Glioblastoma multiforme targeted therapy: The Chlorotoxin story

Journal of Clinical Neuroscience xxx (2016) xxx–xxx

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Glioblastoma multiforme targeted therapy: The Chlorotoxin story Or Cohen-Inbar a,b,c,d,⇑, Menashe Zaaroor a,b,c a

Department of Neurological Surgery, Rambam Health Care Center, Haifa, Israel Molecular Immunology Laboratory, Technion Israel Institute of Technology, Haifa, Israel c Faculty of Medicine, Technion Israel Institute of Technology, Haifa, Israel d Department of Neurological Surgery and Gamma-Knife Radiosurgical Center, University of Virginia Health Care Centre, 1215 Lee St, Charlottesville, VA 22908, USA b

a r t i c l e

i n f o

Article history: Received 3 March 2016 Accepted 2 April 2016 Available online xxxx Keywords: Annexin-2 Chlorotoxin Cl-Channel Glioblastoma Immunotherapy MMP-2

a b s t r a c t Glioblastoma multiforme (GBM) is the most common malignant primary brain neoplasm having a mean survival of <24 months. Scorpion toxins are considered promising cancer drug candidates, primarily due to the discovery of hlorotoxin, derived from the venom of the Israeli yellow scorpion. This intriguing short peptide of only 36 amino-acids length and tight configuration, possess the ability to bind to GBM cells in a grade-related manner with 100% of GBM cells staining positive and no cross reactivity to normal brain. Chlorotoxin has an anti-angiogenic effect as well. Molecular targets for Chlorotoxin include voltage gated chloride channels (GCC), calcium-dependent phospholipid-binding protein Annexin-2, and the inducible extracellular enzyme Matrix Metalloproteinase-2 (MMP-2). Of all its targets, MMP-2 seems to bear the most anti-neoplastic potential. Chlorotoxin is a promising tumortargeting peptide. Its small size and compact shape are convenient for intracranial delivery. We present a short discussion on Chlorotoxin. The structure, biological activity, molecular targets and possible clinical role of Chlorotoxin are discussed. Chlorotoxin can be utilized as a targeting domain as well, attaching different effector functions to it. Clinical applications in GBM therapy, intraoperative imaging, nano-probes and nano-vectors based technology; targeted chemotherapy and immunotherapy are discussed as well. Chlorotoxin is likely to play a significant role in effective GBM immunotherapy in the future. Ó 2016 Elsevier Ltd. All rights reserved.

1. Introduction Grade-IV astrocytoma, better known as glioblastoma multiforme (GBM) is unfortunately ranked as both the most common and the most malignant glioma in adults. GBM is associated with a median overall survival of 1–2 years and a 5-year survival rate of less than 10% [1–3]. The unique nature of Glioblastoma multiforme (GBM), featuring both local and distant aggressive recurrence, and its inherent challenging features was evident as early as 80 years ago. Patients with GBM exhibit altered and suppressed function spanning different elements of the immune system [4–21]. These reports set forth the basic premise that GBM cells have cloaking abilities in addition to suppressive mechanism which allow them to persevere. Another premise laid by these reports, is that homing of immune elements to the tumor site may prove sufficient for an effective anti-tumor response. Targeted immunotherapy is rapidly becoming one of the pillars of anti-cancer therapy. Its targeted nature and reduced treatment ⇑ Corresponding author at: 137 Yellowstone Drive, Charlottesville, VA 22903, USA. Tel.: +972 54 6660565. E-mail addresses: [email protected], [email protected], or_coheni@ rambam.health.gov.il (O. Cohen-Inbar).

related toxicity, stemming from recruiting and activating own cells, selective cytotoxic mechanisms, and effector responses, makes it intuitively an attractive option [22,23]. Metastatic melanoma treatment is one such example. FDA approval of cytokine based therapy with interleukin-2 in 1998 and checkpoint blockade with Ipilimumab in 2011 were major developments in the treatment of melanoma [24,25]. Ipilimumab was shown to increase survival in patients with unresectable stage III/IV melanoma [25–27]. GBM has not received similar clinical successes as of yet [4–8]. This may be attributed to its relative inaccessibility (its location beyond the confines of the blood–brain barrier), any of the multiple mechanisms of active or passive immunosuppression reported [4,20,21] which result in overall poor immunogenicity, or the lack of characterized cancer antigens. One should note that the immune response against tumors is limited by the fact that these arise from the organism’s own tissue, and, therefore, mainly express selfantigens. The patient’s T cells are rendered tolerant to these selfantigens via either central or peripheral tolerance [4–8,28,29], and thus do not act to eradicate these cells. Preclinical studies suggest that targeted therapies can elicit significant antitumor responses in GBM, overcoming some of the barriers and tumor-related escape mechanisms, while others fail at specific points [4]. We will next briefly describe the structure,

http://dx.doi.org/10.1016/j.jocn.2016.04.012 0967-5868/Ó 2016 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Cohen-Inbar O, Zaaroor M. Glioblastoma multiforme targeted therapy: The Chlorotoxin story. J Clin Neurosci (2016), http://dx.doi.org/10.1016/j.jocn.2016.04.012

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biological activity. Molecular target and possible clinical role of Chlorotoxin (ClTx), an intriguing and short peptide derived from the venom of the Israeli Yellow Scorpion. We will present both pre-clinical laboratory data as well as clinical data from our own laboratory as well as from other colleagues, supporting the premise that ClTx will play a significant role in effective GBM targeted therapy in the future. ClTx is a promising tumortargeting peptide. Its small size and compact shape are convenient for intracranial delivery. 2. Classification, structure and synthesis of Chlorotoxin Scorpion toxins are considered promising cancer drug candidates [30,31], primarily due to the discovery of ClTx, a peptide from the venom of the giant Israeli yellow scorpion Leiurus quinquestriatus [32], which can preferentially bind to cancer cells [30]. Its venom is known to inhibit reconstituted smallconductance chloride channels, when applied to the cytoplasmic surface [33] (discussed later). Of note, this scorpion’s venom is rich in many potent physiologically active substances in addition to ClTx. An array of bio-polysaccharides, hyaluronic acid derivate, serotonin, histamine, histamine-releasing factors and protease inhibitors are known to comprise the crude venom [34,35]. These bioactive polypeptides selectively bind to and modulate specific ion channels of excitable cell membranes. Scorpion-derived peptide toxins can be classified according to their specificity in inhibiting various ion channel receptors. So far scorpion-derived toxins identified specifically target either Na+ [36,37], K+ [38], Cl [39] and Ca2+ channels [40]. Since its original description in 1992, a number of Chlorotoxin related peptides have been isolated and identified [32]. ClTx is 4070 Da and 36 amino acids basic peptide with considerable sequence homology to the class of small insectotoxins [30,32,41,42]. Its amino acids sequence includes eight cysteines and four disulfide bonds, and it is therefore classified as a shortchain, disulfide-containing peptide. Other scorpion peptides with a similar primary structure to Chlorotoxin have been collectively referred to as Chlorotoxin-like peptides (i.e. ClTx-a, -b, -c, -d, BmKCL1, Lqh-8:6, Be I5A, BeI1, AmmP2 and GaTx1) [43–45]. The secondary structure of ClTx was first determined using nuclear magnetic resonance (NMR) spectroscopy in 1995 [46]. The NMR data showed that ClTx comprises an a-helix, formed by amino acids 11–21, and three b-sheets, formed by amino acids 1–4, 26–29, and 32 –36 (Fig. 1) [46,47]. The amount of ClTx present in venom is limited. Thus, an alternative or synthetic source of the peptide is necessary for

biochemical and biophysical studies, as well as for potential therapeutic applications. Chlorotoxin has been successfully synthesized and folded in vitro, using classical molecular biology laboratory techniques. Ojeda et al. [30] described synthesis using a solid-phase peptide synthesis (SPPS). An alternative and simple method utilized in our laboratory entailed cloning the ClTx gene [7]. The widely published and available amino-acid sequence of the 36-amino-acids ClTx, was converted into a nucleic acids sequence empirically. Next, this sequence was commercially optimized for expression in Escherichia coli (GENEART Inc.). In the optimization process, the codon usage is adapted to the codon bias of E. coli genes. In addition, regions of very high (>80%) or very low (<30%) GC (Guanine-thymine) has been avoided when possible. The expressed protein can then be purified and refolded in vitro. This allows for a cheap, easy and highly reliable method of ClTx production. It also allows for unique chimeric molecular designs to be planned and produced from the DNA level and then expressed in E. coli (discussed later) [7]. 3. Chlorotoxin activity ClTx is assigned two clinical effects; a tumor binding activity and an antiangiogenic activity. Soroceanu et al. [48] first described in 1998 the tumor binding activity of ClTx using a 125I-labeled peptide (125I-ClTx) [48]. The authors showed that upon 125I-Cltx administration to GBM tumor-bearing mice, the peptides accumulated within the tumor. Soroceanu et al. [48] also showed the specific binding of 125I-Cltx to glioma cells, sparing normal rat astrocytes or neurons [48]. ClTx was initially thought to bind only to the so-called glioma-specific chloride ion channel (GCC) [49–53] which was found to be abundantly expressed in glioma cells but absent in normal brain tissue. Ullrich et al. [49] reported a ClTx-sensitive Cl current, present in human astrocytoma (glioma) cell lines [49]. From then, ClTx has been widely used as a Cl -channel blocker to analyze Cl channels [50–52] and has been proposed as a glioma-specific marker with diagnostic and therapeutic potential [53,54]. GCC expression in situ using labeled ClTx was found to correlate with the tumor’s grade, with only 40–45% of low-grade astrocytoma’s (World Health Organization [WHO] grade I–II) binding it, as opposed to over 90% of high-grade tumors (WHO grade III) and essentially all of the GBM samples, binding labeled ClTx. Comparison tissues including normal human brain, kidney, and colon were consistently negative for ClTx immunostaining [51,53,54]. Sontheimer et al. [55] also showed ClTx binding to tumors of neuro-ectodermal descent (sharing a common embryonic origin

Fig. 1. Chlorotoxin (Cltx). (A) The three-dimensional structure of Cltx. a-Helix is in red, b-sheets are in yellow arrows. The disulfide bonds are not shown. Toxicon 38 (2000). (B) Amino-acids sequence of Cltx. The Disulfide bones are shown as black lines, the cysteines are labeled with roman letters. The cyclic configuration is allowed using an extra amino-acids linker sequence, marked in purple. Adapted from Ojeda et al. [14].

Please cite this article in press as: Cohen-Inbar O, Zaaroor M. Glioblastoma multiforme targeted therapy: The Chlorotoxin story. J Clin Neurosci (2016), http://dx.doi.org/10.1016/j.jocn.2016.04.012

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with glial cells), in addition to GBM’s, such as melanoma, neuroblastomas, meduloblastomas, and small cell lung carcinomas in over 200 human surgical biopsy samples [55]. Biotinylated ClTx did not bind to normal tissues from brain, skin, kidney and lung, showing impressive selectivity. In vivo studies, using ClTx conjugated to a fluorescent dye (Cy5.5, BLZ-100, 800CW), have further confirmed the specific binding of ClTx to GBM’s [56–58]. Xu et al. [59] reported on two new ClTx derivatives (termed CA4 and Cltx-23) that show highly selective binding to malignant glioma cells [59]. A much less pronounced, yet proven ClTx feature is an antiangiogenic property. ClTx has been shown in vitro to inhibit the migration and invasion of glioma cells and human umbilical vein endothelial cell (HUVEC) [53,60]. ClTx was shown to inhibit the migration of endothelial cells when these were stimulated with VEGF and bFGF (vascular related growth factors implicated in the proliferation, migration and differentiation of endothelial cells) [60]. This antiangiogenic effect was noted in the absence of any other ClTx-related toxic effects, suggesting that inhibition of migration of HUVECs was due to a mechanism other than cell death [60]. 4. Molecular targets of Chlorotoxin 4.1. Chloride channels ClTx was first described, as the name suggests, as a chloride channel inhibitor [32]. In this report, Strichartz et al. [32] showed an inhibitory activity only when ClTx was applied to the intracellular face of reconstituted small-conductance chloride ion channels on rat epithelia using patch-clamping technique [32]. The inhibitory effect of ClTx on human GBM associated chloride channels was described [50,61]. Cheng et al. [62] recently described the scorpion venom blocking activity on single-channel as a firstorder binding reaction and revealed the identification of ClTx amongst others as a single Cl specific peptide blocker [62]. The cell cycle-dependent expression of a GCC current was suggested to be linked to cellular cytoskeletal changes [51], shape glioma cell morphology, foster proliferation and migration, and regulates apoptosis [51,63–65].

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extracellular matrix during cancer invasion of normal tissue [79,80]. MMPs are active at neutral pH and can therefore catalyze the normal turnover of extracellular matrix (ECM) macromolecules such as the interstitial and basement membrane collagens, proteoglycans such as aggrecan, decorin, biglycan, fibromodulin and versican as well as accessory ECM proteins (i.e. fibronectin). Members of the MMP family include the ‘‘classical” MMPs, the membrane-bound MMPs (MT-MMPs) the ADAMs (a disintegrin and metalloproteinase; adamlysins) and the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motif) [81]. Most members are secreted as a latent zymogen requiring activation. Once activated, the latent 72-kDa MMP-2 is converted via a 64-kDa activation intermediate to a 62-kDa mature protease [82,83]. Efficient activation of the pro-enzyme to the mature enzyme has shown to contribute to the invasive potential of tumor cells. The mechanism of activation and regulation of MMP-2 is tightly regulated by several other proteins that form a macromolecular complex [84]. Specifically, this involves interactions with membrane-associated MT1-MMP and avb3 integrin, matrix proteins, and its endogenous inhibitor TIMP-2. These proteins are stoichiometrically balanced in equimolar ratios to facilitate tumor cell migration, invasion, as well as control and maintenance of ECM proteolysis [84]. Glioma cells require the activation of MMP-2, which degrades the ECM during invasion and migration [85,86]. In the central nervous system, membrane type MMP-1 (MT1-MMP) has a more important role than MMP-2 during ECM remodeling, migration, infiltration, and invasion of gliomas [87]. MT1-MMP on cell surfaces is replenished by auto-degradation or clathrin-dependent internalization, and its concentration is stabilized by the tissue inhibitor of MMP (TIMP)-2 [88,89]. Malignant human gliomas express membrane-anchored MMPs and their endogenous TIMPs [90–92]. MMP-2 is specifically up-regulated in gliomas as well as in other tumors of neuroectodermal origin, but is not normally expressed in the central nervous system (Fig. 2, 3) [55,93]. Additionally, MMP-2 expression was shown to be in direct correlation to the tumor’s aggressiveness and grade [20,52,55]. Sontheimer et al. [93] demonstrated in 2003, using a recombinant His-Cltx, that principal ClTx-receptor on the surface of glioma cells was cell surface Matrix Metalloproteinase-2 (MMP-2) [93].

4.2. Annexin A2 The Annexin protein family is a group of calcium-dependent phospholipid-binding proteins that are found implicated in both sensing and repairing plasma membrane lesions triggered by various stimuli [66–68]. Besides their role in membrane repair, Annexin family members bind anionic phospholipids and regulate various cellular functions including vesicle trafficking, vesicle fusion, and membrane segregation, in a Ca2+-dependent manner [69,70]. Furthermore, Annexins and their binding partners (the S100 proteins) are known regulators of the cellular actin cytoskeleton [71–73]. Annexin-A2 was proposed as a molecular target for ClTx in tumor and vascular endothelial cells [74]. Using AnnexinA2 knockdown cells, the binding of ClTx to the surface of the cells was abolished [74]. The A2-complex, comprised of annexin-A2 and the protein p11 [75,76], was shown to be over-expressed at the cell surface in GBM as well as in many other human cancers and is correlated with poor prognosis [77,78]. 4.3. Matrix Metalloproteinase-2 Matrix Metalloproteinases (MMPs) are members of an enzyme family that require a zinc ion in their active site for catalytic activity (zinc-dependent endopeptidases). MMPs are critical for maintaining tissue allostasis, able to degrade structural proteins of the

Fig. 2. Glioblastoma multiforme (GBM) cell lines express Matrix Metalloproteinase2 (MMP-2). A Reverse-Transcriptase Polymerase-Chain-Reaction (RT-PCR) for the presence of MMP-2 intracellularly. GBM cell lines tested are U118, D392. First, messenger-RNA (mRNA) was extracted from the cells, followed by the production of cloned-DNA (cDNA) from the mRNA template. Both cell lines show intracellular presence of MMP-2 [4]. GAP-DH is a positive control. 100 base pairs (100 bp) marker is shown on the left. NTC = no template control.

Please cite this article in press as: Cohen-Inbar O, Zaaroor M. Glioblastoma multiforme targeted therapy: The Chlorotoxin story. J Clin Neurosci (2016), http://dx.doi.org/10.1016/j.jocn.2016.04.012

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ClTx was shown to inhibit the enzymatic activity of MMP-2 in a dose-dependent fashion and reduced MMP-2 surface expression. The ClTx MMP-2 binding was shown specific, with MMP-1, MMP-3, or MMP-9 (also expressed by glioma cells) unaffected or bound by ClTx [93]. Veiseh et al. [58] showed that ClTx binding to GBM cells was reduced in the presence of a MMP-2 inhibitor. 5. Can Chlorotoxin penetrate through the blood–brain barrier? The short answer to this critical question is yes. ClTx analogues (i.e., conjugated to Cy5.5, 800CW and BLZ-100) were shown to bind to GBM’s tumors in mice when delivered via tail injection [56–58]. Xiang et al. [94] described the used of ClTx as a carrier for delivering levodopa for the treatment of Parkinson’s disease, and showed an increased distribution of dopamine in the brain of Parkinson’s disease mice model [94]. Still, in studies quoted, the integrity of the blood–brain barrier (BBB) has not been proven conclusively. This is an extension of a general discussion on the local integrity of the BBB in brain tumors, and its evolution during the course of the disease [95]. Adding to the complexity of the question, even if we assume that ClTx in its basic form can cross through the BBB, any chimeric molecular structure, in which ClTx serves as a targeting domain, will not necessarily preserve such as ability. 6. Therapeutic applications of Chlorotoxin: present and future The clinical applications of ClTx in GBM therapy are only limited by our imagination. ClTx serves as an optimal ‘‘magic bullet”, specifically targeting the GBM cells, while posing no danger to

normal cells of the brain or the body. One could argue that administering ClTx by itself may have some tumorocidic effect, binding to MMP-2 and inhibiting infiltration as well binding to GCC and influencing membrane potential. Still, multiple applications using a chimeric molecule that utilizes the ClTx as a targeting domain and bringing an effector domain to the tumor microenvironment were reported [96] and are summarized briefly next. These molecules differ in function based on the action of its effector domain only. In addition, it seems that potential side effects, toxicity and effectiveness are also governed by the effector domain molecule rather than by ClTx. One such application is in optical imaging [97], or an intraoperative ‘‘tumor paint” [58]. ClTx conjugated to a fluorescent molecular probe (Cy5.5) [58], which absorbs light in the near infrared spectrum (poorly absorbed by water or haemoglobin), makes it compatible with intra-operative imaging. This probe was proven to be the most sensitive probe for intraoperative visualization of GBM foci, having approximately 500-times sensitivity than normal MRI [58]. ClTx has been used to deliver magnetic resonancece imaging (MRI) contrast agents or nano-probes to tumorigenic tissue [98]. ClTx nanoparticles, i.e. iron oxide nanoparticles conjugated to both a therapeutic molecule or an imaging agent and ClTx were first published in 2005, using ClTx and Cy5.5 [99]. Radionuclide iodine–131–labeled, chemically synthesized ClTx (commercial name 131I–TM601) has been designated as an orphan drug for the treatment of malignant gliomas [100] and melanomas by the U.S. Food and Drug Administration (FDA) [100–102]. In a phase I clinical trial, a single dose of 131I–TM601, delivered directly to the brain (via a catheter located in the tumor bed), was shown to be well tolerated and was eliminated from the body within

Fig. 3. Human glioblastoma multiforme samples, Immunohistochemistry. (A) Chlorotoxin (ClTx) staining. Cltx was detected using a primary (BB7.2) antibody and a secondary fluorescent-labeled anti-mouse antibody. A granular membranal staining is detected in high power (100). (B) Positive control. Commercially available anti-MMP-2 antibodies and fluorescent-labeled anti-rabbit secondary antibody. A similar granular staining pattern is shown. (100) (C–E) Negative controls to samples presented in A and B (40). (C) Primary and secondary antibody without Cltx, (D) Secondary anti-mouse antibody and (E) anti-rabbit antibody, all showing negative staining [4].

Please cite this article in press as: Cohen-Inbar O, Zaaroor M. Glioblastoma multiforme targeted therapy: The Chlorotoxin story. J Clin Neurosci (2016), http://dx.doi.org/10.1016/j.jocn.2016.04.012

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24–48 h [100]. Testing of 131I–TM601 has now moved into phase III trials. ClTx nano-probes have also been used to deliver potent chemotherapeutic drugs or biologics to GBM cells. Sun et al. [103] reported that the conjugation of ClTx to Methotrexate (a potent chemotherapeutic agent used for intracerebral administration), was shown to have increased cytotoxicity towards cancer cells when compared to the effect of the drug alone [103]. Similarly, Graf et al. [104] reported the conjugation of ClTx to platinum chemotherapy (cisplatin) [104] and for alisertib, which is currently in Phase II clinical trials for relapsed and refractory aggressive B/T-cell non-Hodgkin lymphomas [105,106]. Cytotoxicity of the nano-probe on these cancer cells was shown to increase as compared to the drug alone. Mu et al. [107] reported the development of a ClTx-gemcitabine conjugated iron oxide nanoparticle for GBM therapy, having a small size (32 nm) and uniform size distribution, stable in biological medium. This nanoparticle was shown to effectively enter GBM cells without losing potency compared to free drug. Significantly, these conjugate nanoparticles showed a prolonged blood half-life and the ability to cross the BBB in wild type mice [107]. ClTx homing can has also been used to deliver siRNA or DNA to cancer cells as a targeting gene delivery system [45,96,108–114]. Veiseh et al. [108] reported the development of a nano-vector construct comprised of a super-paramagnetic iron oxide nanoparticle core coated with polyethylene glycol (PEG)-grafted chitosan, and polyethylenimine (PEI). The construct was then functionalized with siRNA and a ClTx to improve its tumor specificity and potency. The authors concluded that ClTx enabled nanoparticle carrier may be well suited for delivery of RNAi therapeutics to brain cancer cells. Yue et al. [114] reported the use of OX26/CTX-conjugated PEGylated liposome as a dual-targeting gene delivery system for GBM. The authors concluded that with the addition of ClTx, these liposomes were ‘‘able to significantly promote cell transfection, increase the transport of plasmid DNA across the BBB and afterwards target the brain glioma cells in vitro and in vivo, exhibiting the most significant therapeutic efficacy” [114]. Tamborini et al. [115] reported a combined approach employing ClTx nano-vectors and low dose ionizing radiation to reach infiltrating tumor niches in a GBM model. Poly (lactic–co-glycolic acid) (PLGA) nanoparticles (PNP) were conjugated to ClTx. Silver nanoparticles were entrapped inside the functionalized nanoparticles (Ag-PNP-CTX), to allow detection and quantification of the cellular uptake by confocal microscopy, both in vitro and in vivo. In vitro experiments showed higher cytoplasmic uptake of Ag-PNP-CTX, with respect to non-functionalized nanoparticles. Utilizing a single whole brain X-ray irradiation, performed 20 h before nanoparticle injection, served to enhance the expression of the ClTx targets (MMP-2 and ClC-3), and through BBB permeabilization, potently increases the amount of internalized Ag-PNP-CTX. Notably, the application of Ag-PNP-CTX to irradiated tumor cells decreases the extracellular activity of MMP-2 [115]. A novel immunotherapeutic concept involves the use of targeting domains, such as the ClTx for the delivery of highly potent immunesystem components [cytotoxic T-cells (CTL’s) or NK-cells] to the tumor milieu. This assisted targeting of immune-competent cells helps circumvent some of the GBM’s immune-evasion mechanisms [4–8]. These immune cells, serving the effector function of the construct, carry the potential benefit of minimal toxicity, able to discern self from non-self in their effector functions [4].

7. Summary and conclusions GBM patients exhibit a mean survival of less than 2 years. GBM tumors are highly aggressive and infiltrative to healthy tissue,

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rendering current multimodality treatment less effective. We present herein a new immunotherapeutic approach for GBM patients. This approach utilizes the targeting of different effector molecules and vectors to the tumor site using the specific binding of chlorotoxin. Chlorotoxin, a short 36-amino-acid compact folded peptide, has been shown to specifically bind neuroectodermal originated neoplasms including malignant gliomas via specific recognition of MMP-2, GCC Chloride Channels and Annexin-2. This GBM targeting moiety can be safely used to deliver specifically to GBM cells, and not healthy tissues, different effector mechanism, circumventing some of the GBM immune-evasion mechanisms. It seems that in current day of targeted selective immune-mediated therapy, chlorotoxin will play a key role in the battle against GBM.

Conflicts of Interest/Disclosures The authors declare that they have no financial or other conflicts of interest in relation to this research and its publication.

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Please cite this article in press as: Cohen-Inbar O, Zaaroor M. Glioblastoma multiforme targeted therapy: The Chlorotoxin story. J Clin Neurosci (2016), http://dx.doi.org/10.1016/j.jocn.2016.04.012