• Users Online: 170
  • Print this page
  • Email this page


 
 
Table of Contents
REVIEW
Year : 2019  |  Volume : 2  |  Issue : 2  |  Page : 88-95

Chimeric antigen receptor T-cells for glioblastoma: The journey ahead


Center for Cell and Gene Therapy, Texas Children’s Hospital, Houston Methodist Hospital, Baylor College of Medicine; Departments of Pediatrics, Baylor College of Medicine; Texas Children's Cancer and Hematology Centers, Texas Children's Hospital, Houston, TX, USA

Date of Web Publication27-Jun-2019

Correspondence Address:
Dr. Meenakshi Hegde
Departments of Pediatrics, Baylor College of Medicine, 1102 Bates Avenue, Houston, TX 77030
USA
Dr. Sujith Kurian Joseph
Departments of Pediatrics, Baylor College of Medicine, 1102 Bates Avenue, Houston, TX 77030
USA
Login to access the Email id

Source of Support: None, Conflict of Interest: None


DOI: 10.4103/glioma.glioma_6_19

Get Permissions

  Abstract 

Chimeric antigen receptors (CARs) are genetically engineered transmembrane cell receptors consisting of an antigen-binding ectodomain fused to an activating intracellular T-cell signaling chain. Grafting CAR molecules on T-cells enables targeted killing of tumors. The gene therapy approach of modifying autologous patient immune cells with CARs has now moved from the research bench to early-phase clinical trials in patients with refractory, recurrent, and nonresectable glioblastoma. This is a review of the state of the science in the field of CAR T-cells for glioblastoma and an update on the completed and ongoing clinical trials available at the Clinical Trials Registry (www.clinicaltrials.gov). Here, we also discuss insights gained from the clinical trials of CAR T-cells against glioblastoma and innovative approaches to improve their efficacy.

Keywords: Chemokine, chimeric antigen receptor T-cells, chimeric antigen receptors, clinical trials, glioblastoma, immune cells, immunotherapy, microenvironment


How to cite this article:
Joseph SK, Ahmed NM, Hegde M. Chimeric antigen receptor T-cells for glioblastoma: The journey ahead. Glioma 2019;2:88-95

How to cite this URL:
Joseph SK, Ahmed NM, Hegde M. Chimeric antigen receptor T-cells for glioblastoma: The journey ahead. Glioma [serial online] 2019 [cited 2019 Aug 23];2:88-95. Available from: http://www.jglioma.com/text.asp?2019/2/2/88/261676


  Introduction Top


Chimeric antigen receptors (CARs) are synthetic fusion molecules consisting of an ectodomain that binds to a tumor-associated antigen and a signal transducing endodomain that triggers an intracellular T-cell signaling cascade.[1] The most abundantly used antigen-recognizing ectodomains are single-chain variable fragments of antibodies.[2] Multiple preclinical studies have also successfully used peptide ligands, muteins, and centyrins, though of these ectodomains, only interleukin (IL) 13 receptor alpha 2-targeted mutein has been tested in humans (NCT03288493).[3],[4],[5],[6] In most iterations, the ζ chain of the T-cell receptor complex provides signal 1 for phosphorylation of immunoreceptor tyrosine-based activation motifs, which is required for T-cell activation and killing.[1] To provide a co-stimulatory T-cell activation signal (Signal 2) in order to prevent T-cell anergy when engaging the target antigen, improved CARs have incorporated co-stimulatory signaling endodomains such as CD28 (phosphoinositide 3-kinase pathway), CD134/OX40, and CD137/4-1BB (tumor necrosis factor receptor-associated factor pathway) alone or in combination.[7],[8] Further improvements to provide Signal 3 using constitutive cytokine expression of IL-2, IL-7, or IL-15 have also been developed. The cytokine-expressing CAR T-cells are referred to as TRUCK T-cells, indicating that these cells deliver a payload to the target site.[9]

CAR T-cells, directed against the pan B-cell antigen CD19, Kymriah™ (tisagenlecleucel) and Yescarta™ (axicabtagene ciloleucel), recently became the first US Food and Drug Administration-approved gene therapy in the clinic. A relatively large cohort, long-term persistence Phase I/IIA study of CD19 CAR T-cells against refractory/relapsed acute lymphoblastic leukemia reported complete remission in 27/30 patients (90%), with a 6-month event-free survival rate of 67% and an overall survival rate of 78%.[10],[11] This trial was followed by the successful use of CD19 CAR T-cells against other advanced B-cell malignancies, including diffuse large B-cell lymphoma, advanced lymphomas, and chronic lymphocytic leukemia. In this study, 8/15 patients achieved complete remission, four achieved partial remission, and one had stable disease at the time of reporting.[12]

The antitumor efficacy of CAR T-cells against solid tumors to date is not as impressive as against hematological malignancies. A Phase 1 study testing Epstein–Barr virus-specific T-cell receptor T-cells or activated T-cells engrafted with CARs targeting the surface disialoganglioside GD2 in neuroblastoma patients demonstrated the therapy's safety and moderate efficacy (NCT00085930). A single infusion of the GD2-specific CAR T-cells led to complete remission in three out of 11 patients with active disease (27%); however, the expansion of infused cells at the periphery could not be detected even in responders.[13] In another Phase 1 study in sarcoma patients, human epidermal growth factor receptor 2 (HER-2)-targeted CAR T-cells demonstrated safety in all patients, but the best response was stable disease in 4 out of 19 evaluable patients for 12–14 months (NCT00902044).[14] CAR T-cells targeting carcinoembryonic antigen (CEA) in colorectal cancers, carcinoembryonic antigen related cell adhesion molecule 5 (CEACAM5) in CEACAM5+ malignancies, PMSA in prostate cancer, c-Met in metastatic breast cancer, carboxy-anhydrase-IX in metastatic renal cell carcinoma, tumor-associated glycoprotein-72 in colorectal cancer, and mesothelin in pancreatic ductal adenocarcinoma have demonstrated variable safety and antitumor efficacy profiles in early clinical trials.[15],[16],[17],[18],[19]

In glioblastoma (GBM), CAR T-cells redirected against antigens such as the epidermal growth factor receptor variant III (EGFRvIII), HER-2 (ErbB2/neu), and IL-13 receptor alpha 2 (IL-13Rα2) have shown safety and some efficacy in early-phase clinical trials.[20],[21],[22],[23] A Phase 1 trial of CAR-modified cytomegalovirus-specific T-cells targeting HER-2 by Ahmed et al.[21] demonstrated safety and objective response in 8 out of 16 evaluable GBM patients (9 adults and 7 children; NCT01109095). Of these patients, one had a partial response for over 9 months, 7 had stable disease (including three patients that survived >3 years), and 8 progressed after T-cell infusion. Similarly, Brown et al.[22] demonstrated that multiple infusions of IL-13Rα2 CAR T-cells were safe and led to a complete response in one patient with recurrent multifocal GBM that lasted for 7.5 months (NCT02208362). A summary of completed and ongoing clinical trials against GBM using CAR T-cells is provided in [Table 1].
Table 1: List of chimeric antigen receptor T-cell clinical trials registered at the Clinical Trials Registry for patients with glioblastoma

Click here to view


CAR T-cell therapy still faces multiple challenges against GBM. While the challenges posed by the unique anatomical location and presence of the blood–brain barrier (BBB) prevent most infused CAR T-cells from reaching their destination, tumor-intrinsic challenges such as antigen heterogeneity and escape reduce the effectiveness of CAR T-cells at their destinations. A suppressive tumor microenvironment (TEM) further adds to the poor performance of these cells at target sites. In this review, we will address advancements that will allow the CAR T-cells to reach the destination and boost their performance at the glioma site.


  Database Search Strategy Top


The literature selected for this review was obtained from articles indexed on MEDLINE®/PubMed®, a US National Library of Medicine database of abstracts and citations (https://www.ncbi.nlm.nih.gov/). All completed and ongoing clinical trials cited for this review were registered in ClinicalTrials.gov, a US government database registry of all clinical trials funded both privately and publicly around the world (https://clinicaltrials.gov/). The medical subject headings used for searching the databases included Glioblastoma AND CAR T cells AND clinical trials, CNS tumors and blood brain barrier, GBM AND microenvironment, CNS AND T cells, GBM AND heterogeneity, CNS AND Immune cells, CAR AND cytokine release syndrome (CRS), CAR AND safety, GBM AND chemokine, transendothelial migration AND GBM access. The selection of peer-reviewed literature was based on direct relevance to the topics covered in this review and on the expert judgment of the authors as to the impact of the research article in the field of GBM immunotherapy.


  Navigating Chimeric Antigen Receptor T-Cells to the Tumor Site Top


The efficacy of CAR T-cell therapy is dependent on the ability of adoptively transferred cells to reach brain and induce a potent antitumor response. The central nervous system compartment tightly controls lymphocyte infiltration because any inflammation is highly detrimental to its function. The glia limitans and BBB tightly regulate lymphocyte traffic, allowing activated T-cells alone and not naïve T-cells to enter the brain parenchyma and its interstitial fluid.[24],[25] Moreover, the brain lacks a conventional lymphatic system and hence poses a major challenge to infiltrating cells of the adaptive immune system, in turn preventing effective antigen presentation and antitumor response.[26],[27]

Locoregional delivery of CAR T-cells is one promising strategy to overcome this limitation.[2],[28],[29] Along with overcoming trafficking restrictions across the BBB, this approach may also reduce toxicities associated with systemic delivery of CAR T-cells and achieve antitumor effects with a lower cell dose. Locoregional delivery was safely demonstrated in the clinical trial using IL-13Rα2 CAR T-cells in patients with recurrent or refractory malignant glioma. In this trial, the T-cells were administered through the intratumoral and/or intraventricular route (NCT02208362).[22] Additional trials utilizing this strategy with CARs targeting EGFRvIII (NCT03283631) and HER-2 (NCT02442297) are ongoing. Patients who have an intracranial catheter (Rickham or Ommaya) in place will benefit this approach especially because multiple infusions are likely necessary for effective antitumor response. While this approach has clear advantages over delivering CAR T-cells through the systemic route, delivery to the ventricles or thecal sac does not necessarily ensure that these cells reach the brain parenchyma where tumors are present.

A second strategy is to engineer T-cells to bind and migrate through the postcapillary vessels into the perivascular space to reach the tumor site. Transendothelial migration involves binding of cell adhesion molecules such as integrins α4 β1 and lymphocyte-associated antigen-1 on the T-cells and their cognate receptors, namely vascular cell adhesion molecule 1 and intracellular cell adhesion molecule 1 on the endothelial cells in a coordinated manner.[30],[31] The binding leads to rolling and arrest of the T-cells on the vascular endothelium, followed by conformational changes that allow the T-cells to transmigrate and reach the perivascular space. Understanding the changes in pathology-associated binding molecule expression such as activated cell adhesion molecule and signaling activation can be utilized to preferentially home CAR T-cells to the tumor sites.[31],[32] Alternately, genetically engineering T-cells to express chemokine receptor CCR4 may help direct these T-cells to the brain in response to high chemokine (C-C motif) ligand 17 or chemokine (C-C motif) ligand 22 chemokine secretion by GBM, originally intended to promote T regulatory cell infiltration in GBM.[33],[34],[35],[36],[37] Others have demonstrated a role for C-X-C chemokine receptor 3, along with its ligands C-X-C chemokine ligand 9 and C-X-C chemokine ligand 10, in T-cell homing to the tumor site and thus can be useful to redirect CAR T-cells to the brain.[38]


  Steering Chimeric Antigen Receptor T-Cells Through a Heterogeneous Tumor Landscape Top


Intrapatient and interpatient tumor heterogeneity is a hallmark of GBM.[39] Along with causing variability and resistance in treatment responses to standard therapy, this heterogeneity has also made targeted approaches difficult in GBM. The altered expression or downregulation of tumor-associated antigens, often termed antigen escape, following CAR T-cell treatment is another mechanism tumors use in immune evasion.[20],[40] The immune escape observed in GBM following CAR T-cell treatment occurs at epigenetic, genetic, and transcriptional levels with variations observed in O[6]-methylguanine-DNA methyltransferase (MGMT) methylation status, DNA repair mechanisms, and gene expression.[41],[42] Preclinically, researchers have demonstrated antigen downregulation in xenograft models of GBM where CAR T-cells specific for only one antigen were used.[40],[43] In the Phase 1 trial using EGFRvIII-specific CAR T-cells, the only neoantigen-based therapy to date in GBM, a decrease in EGFRvIII-expression likely accounted for the low objective responses, in spite of the presence of CAR T-cells in the tumor biopsies (NCT02209376).[20]

CAR T-cells targeting two or more antigens can be an effective way to overcome this limitation. Hegde et al.[40],[44] previously demonstrated that simultaneously targeting HER-2 and IL-13Rα2 using CAR T-cells where the binding CAR ectodomains to the tumor-associated antigens are arranged in tandem (called tandem CAR T-cells) can circumvent escape in a xenograft model of GBM. Further, the same team demonstrated that including a third antigen, Ephrin type A receptor 2, with HER-2 and IL-13Rα2 could overcome the interpatient heterogeneity by broadening the target spectrum.[45] Trivalent CAR T-cell-targeting multiple antigens may be well poised to be effective against a heterogeneous GBM target profile, making them applicable to a wider cohort of patients. An alternate method developed by Cho et al.[46] uses a split, universal, and programmable (SUPRA) CAR system for T-cell therapy, where exodomains can be switched based on the affinity between leucine zipper pairs that attach the single-chain variable fragment portion to the CAR molecule. While this approach overcomes the limitation of manufacturing different CAR T-cells based on antigen variability in the patient, it still poses the bioavailability challenge of the zip-Fvs at the glioma site. Finally, epigenetic mechanisms responsible for antigen loss may be inhibited by concomitant treatment with epigenetic modifiers, such as DNA methyltransferase inhibitors or histone deacetylase inhibitors, which are already approved for other disease indications.[47]


  Armoring Chimeric Antigen Receptor T-Cells to Overcome Microenvironment Inhibition Top


The use of CAR T-cells overcomes one of the major concerns encountered during cell-mediated immune response, the low expression of human leukocyte antigen Class I molecules on the surface of tumor cells. The human leukocyte antigen Class I downregulation or loss of function mutation allows GBM to escape detection by immune cells.[48] Here, CAR T-cells offer advantage over autologous tumor lysate-pulsed dendritic cell vaccine (DCVax®-L), where antitumor responses depend on CD8+ cells in a Class I major histocompatibility complex-restricted manner.[49] Still, the highly suppressive TME poses major challenges to the function of CAR T-cells, including the recruitment of T regulatory cells, the presence of suppressive myeloid cell population, and upregulation of immune checkpoint molecules and immunosuppressive cytokines.[50] For instance, myeloid-derived suppressor cells enable tumor growth and support gliomagenesis.[51],[52] Tumor-infiltrating T-cells also express checkpoint inhibitors such as programmed cell death protein 1 (PD-1), cytotoxic T-lymphocyte-associated protein 4, lymphocyte-activation gene 3, T-cell immunoreceptor with immunoglobulin and ITIM domains, and T-cell immunoglobulin and mucin domain containing-3.[53] Upregulation of these molecules occurs in response to the high expression of programmed death ligand receptor-1 (PD-L1), indoleamine-2,3-deoxygenase 1, and other suppressive molecules on GBM and other components of the TEM, functionally exhausting CAR T-cells that reach tumor site. In the EGFRvIII clinical trial, the authors observed increased expression of immunosuppressive molecules such as indoleamine-2,3-deoxygenase 1 and FoxP3 in the TEM. Further, patients whose tumors were surgically resected after CAR T-cell infusion showed loss of EGFRvIII expression.[20] Moreover, GBMs can generate extracellular vesicles in patients that express PD-L1 on their surface that can cause profound local and systemic immunosuppression.[54]

The use of checkpoint inhibitor antibodies targeted against PD-1 (pembrolizumab, nivolumab, and cemiplimab), PD-L1 (atezolizumab, avelumab, durvalumab, and ipilimumab), and cytotoxic T-lymphocyte-associated protein 4 (ipilimumab) alone or in combination has demonstrated varying degrees of efficacy against GBM and other solid tumors.[55],[56],[57] Along this line, a Phase 1 trial at the University of Pennsylvania (NCT03726515) is currently testing pembrolizumab (a PD-1 blocking antibody) with EGFRvIII CAR T-cells in GBM patients whose tumors have EGFRvIII expression and unmethylated MGMT. Two major challenges associated with using antibodies directly are ensuring their bioavailability at the tumor site due to low BBB penetrance and the associated systemic toxicity observed with the treatment. Using PD-1/PD-L1 blocking antibody-secreting T-cells or T-cells grafted with PD-1-dominant negative receptor or a checkpoint reversal receptor is one way to circumvent these issues. In the checkpoint reversal strategy, the PD-1 signals are truncated or reversed by a co-stimulatory signaling domain such as CD28 or 4-1BB.[58],[59] A Phase 1 clinical trial is ongoing at the Beijing Sanbo Brain Hospital, China, where the T-cells carry a chimeric switch receptor consisting of PD-1 extracellular domain and a CD28 endodomain (NCT02937844). The same effect could also be achieved by knocking out PD-1 in CAR T-cells by genome editing techniques that use zinc finger nucleases, transcription activator-like effector nucleases, or Cas9 nucleases.[60] As the checkpoint inhibition is enabled by redundant pathways, blocking or knocking out one receptor alone may not achieve the desired effects, rather blocking nodes of regulation would prove beneficial with this approach. Similar to the concept of dominant negative receptors for PD-1, a dominant negative transforming growth factor beta (TGF-β) type II receptor can render CAR T-cells resistant to tumor-secreted TGF-β, improving the antitumor function of these cells.[61],[62] A recent study demonstrated that CRISPR/Cas9-mediated knockout of diacylglycerol kinase, an enzyme metabolizing diacylglycerol to phosphatidic acid, rendered CAR T-cells resistant to TGF-β and prostaglandin E2, released to the TEM.[63]

Low-dose radiation has been shown to be beneficial in a preclinical model against pancreatic adenocarcinoma with heterogeneous expression of sialyl Lewis-A (sLeA) recently.[64] The study demonstrated that low-dose radiation made both sLeA + and sLeA-cells susceptible to CAR therapy, reducing antigen-negative tumor relapse through a TRAIL-receptor 2 (also known as death receptor 5)-mediated mechanism. Moreover, increased leakage through the BBB and microvascular collapse have been observed flowing radiation in mice and humans and should allow more lymphocyte infiltration to the tumor site.[64] Preconditioning with lymphodepletion chemotherapy with agents such as cyclophosphamide and fludarabine has been shown to improve the expansion and persistence of infused CD19 CAR T-cells.[11],[12] There are two Phase 1 trials currently recruiting that have incorporated lymphodepletion chemotherapy with fludarabine and cyclophosphamide into the treatment regimen before introducing EGFRvIII targeting CAR T-cells against GBM (NCT02844062 and NCT03170141). Cyclophosphamide can cross the BBB and may allow lymphatic space for the infused CAR T-cells to expand along with depleting suppressive cells at the tumor site.[65] A similar approach using an intensified dose of temozolomide, a drug currently included in the standard of care for GBM, demonstrated improved engraftment and proliferation of EGFRvIII-targeted CAR T-cells. Currently, this observation is being tested in a Phase 1 trial at Duke University (NCT02664363).[66]


  Torqueing Chimeric Antigen Receptor T-Cell Performance and Endurance Top


The GBM microenvironment is composed of glial cells, vascular cells, and immune cells, especially tumor-associated macrophages of the M2 phenotype.[50],[67] Tumor-associated macrophages make up ~30%–40% of cells in GBM and can promote tumor growth and progression. Additionally, tumor-associated macrophages can secrete immunosuppressive cytokines such as IL-10 and TGF-β that hinder the function of CAR T-cells and limit their persistence at the tumor site.[68]

CAR T-cells armed with stimulatory cytokines such as IL-12, IL-15, and IL-21 can counteract the suppressive milieu of the TEM and provide the boost necessary to induce a potent antitumor response alone or in combination. The cytokine IL-12 can improve the activation and proliferation of CD8+ T-cells and provide a type I signal that can impact the innate immune cell infiltrates.[69] Further, IL-12 can also improve the cytotoxicity associated with natural killer cells through interferon-gamma production and subsequent Fas (CD95) and Fas-L upregulation on tumor cells. Preclinical evaluation of IL-12-secreting CAR T-cells indicated improved activation and persistence that translated to better antitumor effects compared to regular CAR T-cells.[70] A recent preclinical study evaluating IL-13Rα2 targeting CAR T-cells that co-express IL-15 demonstrated superior persistence and antitumor activity against GBM xenografts when compared to IL-13Rα2 CAR T-cells.[43] Additionally, IL-15 can activate natural killer cells through components of the IL-2 receptor.[71] IL-21 is another cytokine that can be co-expressed on CAR T-cells because it mediates Foxp3 suppression and enhances the generation of CD8+ cytotoxic T-cells.[72] However, systemic toxicities have been associated with intravenous administration of recombinant human IL-12 or other cytokines. Thus, a high degree of caution is recommended when administering CAR T-cells with constitutive cytokine expression to the brain.[73] An alternate approach would be to “backpack” cytokines such as IL-15 or IL-21 on T-cells using protein nanogels and release them gradually to reduce bystander activation or use membrane tethering to prevent secretion.[74]

The use of central memory T-cells has demonstrated improved persistence for infused CD19 CAR T-cells in patients with relapsed B-cell non-Hodgkin lymphoma.[75] Currently, a Phase 1 clinical trial is ongoing at the City of Hope Medical Center where HER-2-directed CAR T-cells with central memory phenotype are infused in patients with recurrent GBM (NCT03389230).


  Enhancing Chimeric Antigen Receptor T-Cell Safety Top


Any enhancement to CAR T-cells should not increase the toxicity associated with the therapy. The safety challenge of treating brain tumors with CAR T-cells includes the fact that elements of a strong immune response such as inflammatory infiltration and edema near the neural tissues can be detrimental. There have been several reported adverse events associated with CRS and neurotoxicity after infusion of CD-19 CAR T-cells.[10],[11],[76] Symptoms associated with CRS included high fever, hypotension, and respiratory difficulties caused by serum IL-6, and those with neurotoxicity included encephalopathy, somnolence, and aphasia. High-grade adverse events after CAR T-cell infusion have not been reported to date in clinical trials for GBM, possibly due to the low expansion and persistence observed. In the study administering HER-2 CAR T-cells for GBM, the researchers did not observe any dose-limiting toxicity, except for Grade-II toxicity in two of the 17 patients treated.[21]

CAR T-cells can be selectively depleted at the tumor site or in systemic circulation through incorporation of a dimerizable death molecule-inducible Fas or Caspase 9 (iCasp9), a safety switch that can be turned on using inert bivalent small molecules.[77],[78] The dimerization of the components of the binding protein activates downstream caspases, inducing T-cell apoptosis. Studies in murine CRS models have elegantly demonstrated that the severity of CRS can be attributed to IL-6, IL-1, and nitric oxide produced by macrophages and not by the CAR T-cells themselves.[79] This study highlights IL-1 as an actionable target, and use of anakinra, an IL-1 receptor antagonist, or a soluble IL-1RA-secreting cell modification to block the effects of IL-1, which can prevent CRS without affecting the antitumor activity of the CD19 CAR T-cells. Other approaches to manage and mitigate the symptoms of CRS include IL-6 antibody, steroids, and prophylactic anti-epileptics.


  Customizing Manufacturing and Delivery of Chimeric Antigen Receptor T-Cells Top


CAR T-cell therapy entails manufacturing and distribution of large numbers of patient-specific products. The major step in this manufacturing involves gene modification of the patients' cells, starting with T-cell selection to cell formulation. Manufacturing further requires dedicated clean rooms and highly trained personnel in a good manufacturing practice environment. Recent advances such as CliniMACS® Prodigy Platform have made possible automation of all cell processing steps, additionally ensuring a highly reproducible and standardized manufacturing process.[80],[81] An alternate approach to clean rooms could be the use of modular hood systems, with independent programmable control of oxygen, carbon dioxide, and temperature in modular incubator and cell-handling chambers like the Xvivo System manufactured by BioSpherix (Lacona, NY, USA). These closed manufacturing methods can reduce open process steps, minimizing the risks for cross-contamination and can be cost-effective in the long run.

Currently, CAR T-cell therapy is restricted to transplant centers that have the expertise, professional teams, accreditation, and infrastructure to administer the therapy safely and effectively. While CAR T-cells for GBM presently remain in an experimental phase, if cell therapy is to go frontline, it will require collaboration between oncologists, neurosurgeons, and transplant specialists to decide on an algorithm for treating patients. An automated manufacturing platform can decentralize manufacturing to or near hospitals and transplant centers where patients are treated. The decentralized manufacturing process for patient T-cell modification can use viral vectors and other nonpatient-specific components made using the currently approved pharmaceutical manufacturing models. Such decentralization may reduce distribution logistics and make planning of treatment infusions easier.


  Conclusion Top


Adoptive cell therapy with autologous CAR T-cells holds great promise in treating refractory or recurring brain tumors, unresectable and residual GBMs, as well as secondary neoplasms that have metastasized to the brain from other primary tumors. While there is no one strategy that can enable CAR T-cells to reach tumor site and enhance their cytotoxicity once there, a combination of targeted delivery along with checkpoint reversal and cytokine boost should allow multitargeted T-cells to persist and produce robust responses in treated patients. The successful journey of CAR T-cells to the clinic will not be a lone one, but will occur in the company of new technological advancements and standard of care approaches.

Acknowledgments

We thank Catherine Gillespie for language editing and proofreading of the manuscript.

Financial support and sponsorship

The authors received funding from the Alex's Lemonade Stand Foundation for Childhood Cancer, Cookies for Kids' Cancer™ Childhood Cancer Foundation, and St. Baldrick's-Stand Up to Cancer Dream Team Translational Research Grant (SU2C-AACR-DT-27-17). Stand Up to Cancer is a division of the Entertainment Industry Foundation. Research Grants are administered by the American Association for Cancer Research, the Scientific Partner of SU2C.

Conflicts of interest

NMA and MH have patent applications in the field of T-cell and gene-modified T-cell therapy for cancer. NMA is on the scientific advisory board of Adaptimmune LLC. SKJ has no conflict of interest.



 
  References Top

1.
Eshhar Z, Waks T, Gross G, Schindler DG. Specific activation and targeting of cytotoxic lymphocytes through chimeric single chains consisting of antibody-binding domains and the gamma or zeta subunits of the immunoglobulin and T-cell receptors. Proc Natl Acad Sci U S A 1993;90:720-4.  Back to cited text no. 1
    
2.
Ahmed N, Salsman VS, Kew Y, Shaffer D, Powell S, Zhang YJ, et al. HER2-specific T cells target primary glioblastoma stem cells and induce regression of autologous experimental tumors. Clin Cancer Res 2010;16:474-85.  Back to cited text no. 2
    
3.
Zhang T, Lemoi BA, Sentman CL. Chimeric NK-receptor-bearing T cells mediate antitumor immunotherapy. Blood 2005;106:1544-51.  Back to cited text no. 3
    
4.
Kahlon KS, Brown C, Cooper LJ, Raubitschek A, Forman SJ, Jensen MC. Specific recognition and killing of glioblastoma multiforme by interleukin 13-zetakine redirected cytolytic T cells. Cancer Res 2004;64:9160-6.  Back to cited text no. 4
    
5.
Krebs S, Chow KK, Yi Z, Rodriguez-Cruz T, Hegde M, Gerken C, et al. Tcells redirected to interleukin-13Rα2 with interleukin-13 mutein – Chimeric antigen receptors have anti-glioma activity but also recognize interleukin-13Rα1. Cytotherapy 2014;16:1121-31.  Back to cited text no. 5
    
6.
Davies DM, Foster J, Van Der Stegen SJ, Parente-Pereira AC, Chiapero-Stanke L, Delinassios GJ, et al. Flexible targeting of erbB dimers that drive tumorigenesis by using genetically engineered T cells. Mol Med 2012;18:565-76.  Back to cited text no. 6
    
7.
Savoldo B, Ramos CA, Liu E, Mims MP, Keating MJ, Carrum G, et al. CD28 costimulation improves expansion and persistence of chimeric antigen receptor-modified T cells in lymphoma patients. J Clin Invest 2011;121:1822-6.  Back to cited text no. 7
    
8.
Imai C, Mihara K, Andreansky M, Nicholson IC, Pui CH, Geiger TL, et al. Chimeric receptors with 4-1BB signaling capacity provoke potent cytotoxicity against acute lymphoblastic leukemia. Leukemia 2004;18:676-84.  Back to cited text no. 8
    
9.
Chmielewski M, Kopecky C, Hombach AA, Abken H. IL-12 release by engineered T cells expressing chimeric antigen receptors can effectively muster an antigen-independent macrophage response on tumor cells that have shut down tumor antigen expression. Cancer Res 2011;71:5697-706.  Back to cited text no. 9
    
10.
Maude SL, Frey N, Shaw PA, Aplenc R, Barrett DM, Bunin NJ, et al. Chimeric antigen receptor T cells for sustained remissions in leukemia. N Engl J Med 2014;371:1507-17.  Back to cited text no. 10
    
11.
Lee DW, Kochenderfer JN, Stetler-Stevenson M, Cui YK, Delbrook C, Feldman SA, et al. Tcells expressing CD19 chimeric antigen receptors for acute lymphoblastic leukaemia in children and young adults: A phase 1 dose-escalation trial. Lancet 2015;385:517-28.  Back to cited text no. 11
    
12.
Abramson JS, McGree B, Noyes S, Plummer S, Wong C, Chen YB, et al. Anti-CD19 CAR T cells in CNS diffuse large-B-cell lymphoma. N Engl J Med 2017;377:783-4.  Back to cited text no. 12
    
13.
Louis CU, Savoldo B, Dotti G, Pule M, Yvon E, Myers GD, et al. Antitumor activity and long-term fate of chimeric antigen receptor-positive T cells in patients with neuroblastoma. Blood 2011;118:6050-6.  Back to cited text no. 13
    
14.
Ahmed N, Brawley VS, Hegde M, Robertson C, Ghazi A, Gerken C, et al. Human epidermal growth factor receptor 2 (HER2)-specific chimeric antigen receptor-modified T cells for the immunotherapy of HER2-positive sarcoma. J Clin Oncol 2015;33:1688-96.  Back to cited text no. 14
    
15.
Zhang C, Wang Z, Yang Z, Wang M, Li S, Li Y, et al. Phase I escalating-dose trial of CAR-T therapy targeting CEA+ metastatic colorectal cancers. Mol Ther 2017;25:1248-58.  Back to cited text no. 15
    
16.
Thistlethwaite FC, Gilham DE, Guest RD, Rothwell DG, Pillai M, Burt DJ, et al. The clinical efficacy of first-generation carcinoembryonic antigen (CEACAM5)-specific CAR T cells is limited by poor persistence and transient pre-conditioning-dependent respiratory toxicity. Cancer Immunol Immunother 2017;66:1425-36.  Back to cited text no. 16
    
17.
Junghans RP, Ma Q, Rathore R, Gomes EM, Bais AJ, Lo AS, et al. Phase I trial of anti-PSMA designer CAR-T cells in prostate cancer: Possible role for interacting interleukin 2-T cell pharmacodynamics as a determinant of clinical response. Prostate 2016;76:1257-70.  Back to cited text no. 17
    
18.
Tchou J, Zhao Y, Levine BL, Zhang PJ, Davis MM, Melenhorst JJ, et al. Safety and efficacy of intratumoral injections of chimeric antigen receptor (CAR) T cells in metastatic breast cancer. Cancer Immunol Res 2017;5:1152-61.  Back to cited text no. 18
    
19.
Beatty GL, O'Hara MH, Lacey SF, Torigian DA, Nazimuddin F, Chen F, et al. Activity of mesothelin-specific chimeric antigen receptor T cells against pancreatic carcinoma metastases in a phase 1 trial. Gastroenterology 2018;155:29-32.  Back to cited text no. 19
    
20.
O'Rourke DM, Nasrallah MP, Desai A, Melenhorst JJ, Mansfield K, Morrissette JJ, et al. Asingle dose of peripherally infused EGFRvIII-directed CAR T cells mediates antigen loss and induces adaptive resistance in patients with recurrent glioblastoma. Sci Transl Med 2017;9. pii: eaaa0984.  Back to cited text no. 20
    
21.
Ahmed N, Brawley V, Hegde M, Bielamowicz K, Kalra M, Landi D, et al. HER2-specific chimeric antigen receptor-modified virus-specific T cells for progressive glioblastoma: A phase 1 dose-escalation trial. JAMA Oncol 2017;3:1094-101.  Back to cited text no. 21
    
22.
Brown CE, Alizadeh D, Starr R, Weng L, Wagner JR, Naranjo A, et al. Regression of glioblastoma after chimeric antigen receptor T-cell therapy. N Engl J Med 2016;375:2561-9.  Back to cited text no. 22
    
23.
Brown CE, Badie B, Barish ME, Weng L, Ostberg JR, Chang WC, et al. Bioactivity and safety of IL13Rα2-redirected chimeric antigen receptor CD8+ T cells in patients with recurrent glioblastoma. Clin Cancer Res 2015;21:4062-72.  Back to cited text no. 23
    
24.
Galea I, Bernardes-Silva M, Forse PA, van Rooijen N, Liblau RS, Perry VH. An antigen-specific pathway for CD8 T cells across the blood-brain barrier. J Exp Med 2007;204:2023-30.  Back to cited text no. 24
    
25.
Engelhardt B. Regulation of immune cell entry into the central nervous system. Results Probl Cell Differ 2006;43:259-80.  Back to cited text no. 25
    
26.
Aspelund A, Antila S, Proulx ST, Karlsen TV, Karaman S, Detmar M, et al. Adural lymphatic vascular system that drains brain interstitial fluid and macromolecules. J Exp Med 2015;212:991-9.  Back to cited text no. 26
    
27.
Carare RO, Bernardes-Silva M, Newman TA, Page AM, Nicoll JA, Perry VH, et al. Solutes, but not cells, drain from the brain parenchyma along basement membranes of capillaries and arteries: Significance for cerebral amyloid angiopathy and neuroimmunology. Neuropathol Appl Neurobiol 2008;34:131-44.  Back to cited text no. 27
    
28.
Choi BD, Suryadevara CM, Gedeon PC, Herndon JE 2nd, Sanchez-Perez L, Bigner DD, et al. Intracerebral delivery of a third generation EGFRvIII-specific chimeric antigen receptor is efficacious against human glioma. J Clin Neurosci 2014;21:189-90.  Back to cited text no. 28
    
29.
Mount CW, Majzner RG, Sundaresh S, Arnold EP, Kadapakkam M, Haile S, et al. Potent antitumor efficacy of anti-GD2 CAR T cells in H3-K27M+ diffuse midline gliomas. Nat Med 2018;24:572-9.  Back to cited text no. 29
    
30.
Baron JL, Madri JA, Ruddle NH, Hashim G, Janeway CA Jr. Surface expression of alpha 4 integrin by CD4 T cells is required for their entry into brain parenchyma. J Exp Med 1993;177:57-68.  Back to cited text no. 30
    
31.
Cayrol R, Wosik K, Berard JL, Dodelet-Devillers A, Ifergan I, Kebir H, et al. Activated leukocyte cell adhesion molecule promotes leukocyte trafficking into the central nervous system. Nat Immunol 2008;9:137-45.  Back to cited text no. 31
    
32.
Lécuyer MA, Saint-Laurent O, Bourbonnière L, Larouche S, Larochelle C, Michel L, et al. Dual role of ALCAM in neuroinflammation and blood-brain barrier homeostasis. Proc Natl Acad Sci U S A 2017;114:E524-33.  Back to cited text no. 32
    
33.
Jacobs JF, Idema AJ, Bol KF, Grotenhuis JA, de Vries IJ, Wesseling P, et al. Prognostic significance and mechanism of treg infiltration in human brain tumors. J Neuroimmunol 2010;225:195-9.  Back to cited text no. 33
    
34.
Oelkrug C, Ramage JM. Enhancement of T cell recruitment and infiltration into tumours. Clin Exp Immunol 2014;178:1-8.  Back to cited text no. 34
    
35.
Brown CE, Vishwanath RP, Aguilar B, Starr R, Najbauer J, Aboody KS, et al. Tumor-derived chemokine MCP-1/CCL2 is sufficient for mediating tumor tropism of adoptively transferred T cells. J Immunol 2007;179:3332-41.  Back to cited text no. 35
    
36.
Zhou M, Bracci PM, McCoy LS, Hsuang G, Wiemels JL, Rice T, et al. Serum macrophage-derived chemokine/CCL22 levels are associated with glioma risk, CD4 T cell lymphopenia and survival time. Int J Cancer 2015;137:826-36.  Back to cited text no. 36
    
37.
Crane CA, Ahn BJ, Han SJ, Parsa AT. Soluble factors secreted by glioblastoma cell lines facilitate recruitment, survival, and expansion of regulatory T cells: Implications for immunotherapy. Neuro Oncol 2012;14:584-95.  Back to cited text no. 37
    
38.
Mikucki ME, Fisher DT, Matsuzaki J, Skitzki JJ, Gaulin NB, Muhitch JB, et al. Non-redundant requirement for CXCR3 signalling during tumoricidal T-cell trafficking across tumour vascular checkpoints. Nat Commun 2015;6:7458.  Back to cited text no. 38
    
39.
Soeda A, Hara A, Kunisada T, Yoshimura S, Iwama T, Park DM. The evidence of glioblastoma heterogeneity. Sci Rep 2015;5:7979.  Back to cited text no. 39
    
40.
Hegde M, Mukherjee M, Grada Z, Pignata A, Landi D, Navai SA, et al. Tandem CAR T cells targeting HER2 and IL13Rα2 mitigate tumor antigen escape. J Clin Invest 2016;126:3036-52.  Back to cited text no. 40
    
41.
Parker NR, Hudson AL, Khong P, Parkinson JF, Dwight T, Ikin RJ, et al. Intratumoral heterogeneity identified at the epigenetic, genetic and transcriptional level in glioblastoma. Sci Rep 2016;6:22477.  Back to cited text no. 41
    
42.
Parker JJ, Canoll P, Niswander L, Kleinschmidt-DeMasters BK, Foshay K, Waziri A. Intratumoral heterogeneity of endogenous tumor cell invasive behavior in human glioblastoma. Sci Rep 2018;8:18002.  Back to cited text no. 42
    
43.
Krenciute G, Prinzing BL, Yi Z, Wu MF, Liu H, Dotti G, et al. Transgenic expression of IL15 improves antiglioma activity of IL13Rα2-CAR T cells but results in antigen loss variants. Cancer Immunol Res 2017;5:571-81.  Back to cited text no. 43
    
44.
Grada Z, Hegde M, Byrd T, Shaffer DR, Ghazi A, Brawley VS, et al. TanCAR: A novel bispecific chimeric antigen receptor for cancer immunotherapy. Mol Ther Nucleic Acids 2013;2:e105.  Back to cited text no. 44
    
45.
Bielamowicz K, Fousek K, Byrd TT, Samaha H, Mukherjee M, Aware N, et al. Trivalent CAR T cells overcome interpatient antigenic variability in glioblastoma. Neuro Oncol 2018;20:506-18.  Back to cited text no. 45
    
46.
Cho JH, Collins JJ, Wong WW. Universal chimeric antigen receptors for multiplexed and logical control of T cell responses. Cell 2018;173:1426-38.e11.  Back to cited text no. 46
    
47.
Xu J, Sampath D, Lang FF, Prabhu S, Rao G, Fuller GN, et al. Vorinostat modulates cell cycle regulatory proteins in glioma cells and human glioma slice cultures. J Neurooncol 2011;105:241-51.  Back to cited text no. 47
    
48.
Facoetti A, Nano R, Zelini P, Morbini P, Benericetti E, Ceroni M, et al. Human leukocyte antigen and antigen processing machinery component defects in astrocytic tumors. Clin Cancer Res 2005;11:8304-11.  Back to cited text no. 48
    
49.
Michalek MT, Grant EP, Gramm C, Goldberg AL, Rock KL. A role for the ubiquitin-dependent proteolytic pathway in MHC class I-restricted antigen presentation. Nature 1993;363:552-4.  Back to cited text no. 49
    
50.
Charles NA, Holland EC, Gilbertson R, Glass R, Kettenmann H. The brain tumor microenvironment. Glia 2012;60:502-14.  Back to cited text no. 50
    
51.
Qi L, Yu H, Zhang Y, Zhao D, Lv P, Zhong Y, et al. IL-10 secreted by M2 macrophage promoted tumorigenesis through interaction with JAK2 in glioma. Oncotarget 2016;7:71673-85.  Back to cited text no. 51
    
52.
Fujita M, Kohanbash G, Fellows-Mayle W, Hamilton RL, Komohara Y, Decker SA, et al. COX-2 blockade suppresses gliomagenesis by inhibiting myeloid-derived suppressor cells. Cancer Res 2011;71:2664-74.  Back to cited text no. 52
    
53.
Moon EK, Wang LC, Dolfi DV, Wilson CB, Ranganathan R, Sun J, et al. Multifactorial T-cell hypofunction that is reversible can limit the efficacy of chimeric antigen receptor-transduced human T cells in solid tumors. Clin Cancer Res 2014;20:4262-73.  Back to cited text no. 53
    
54.
Ricklefs FL, Alayo Q, Krenzlin H, Mahmoud AB, Speranza MC, Nakashima H, et al. Immune evasion mediated by PD-L1 on glioblastoma-derived extracellular vesicles. Sci Adv 2018;4:eaar2766.  Back to cited text no. 54
    
55.
Clarke J, Neil E, Terziev R, Gutin P, Barani I, Kaley T, et al. Multicenter, phase 1, dose escalation study of hypofractionated stereotactic radiation therapy with bevacizumab for recurrent glioblastoma and anaplastic astrocytoma. Int J Radiat Oncol Biol Phys 2017;99:797-804.  Back to cited text no. 55
    
56.
Johnson DR, Omuro AM, Ravelo A, Sommer N, Guerin A, Ionescu-Ittu R, et al. Overall survival in patients with glioblastoma before and after bevacizumab approval. Curr Med Res Opin 2018;34:813-20.  Back to cited text no. 56
    
57.
Omuro A, Vlahovic G, Lim M, Sahebjam S, Baehring J, Cloughesy T, et al. Nivolumab with or without ipilimumab in patients with recurrent glioblastoma: Results from exploratory phase I cohorts of checkMate 143. Neuro Oncol 2018;20:674-86.  Back to cited text no. 57
    
58.
Cherkassky L, Morello A, Villena-Vargas J, Feng Y, Dimitrov DS, Jones DR, et al. Human CAR T cells with cell-intrinsic PD-1 checkpoint blockade resist tumor-mediated inhibition. J Clin Invest 2016;126:3130-44.  Back to cited text no. 58
    
59.
Suarez ER, Chang de K, Sun J, Sui J, Freeman GJ, Signoretti S, et al. Chimeric antigen receptor T cells secreting anti-PD-L1 antibodies more effectively regress renal cell carcinoma in a humanized mouse model. Oncotarget 2016;7:34341-55.  Back to cited text no. 59
    
60.
Rupp LJ, Schumann K, Roybal KT, Gate RE, Ye CJ, Lim WA, et al. CRISPR/Cas9-mediated PD-1 disruption enhances anti-tumor efficacy of human chimeric antigen receptor T cells. Sci Rep 2017;7:737.  Back to cited text no. 60
    
61.
Bruna A, Darken RS, Rojo F, Ocaña A, Peñuelas S, Arias A, et al. High TGFbeta-smad activity confers poor prognosis in glioma patients and promotes cell proliferation depending on the methylation of the PDGF-B gene. Cancer Cell 2007;11:147-60.  Back to cited text no. 61
    
62.
Foster AE, Dotti G, Lu A, Khalil M, Brenner MK, Heslop HE, et al. Antitumor activity of EBV-specific T lymphocytes transduced with a dominant negative TGF-beta receptor. J Immunother 2008;31:500-5.  Back to cited text no. 62
    
63.
Jung IY, Kim YY, Yu HS, Lee M, Kim S, Lee J. CRISPR/Cas9-mediated knockout of DGK improves antitumor activities of human T cells. Cancer Res 2018;78:4692-703.  Back to cited text no. 63
    
64.
DeSelm C, Palomba ML, Yahalom J, Hamieh M, Eyquem J, Rajasekhar VK, et al. Low-dose radiation conditioning enables CAR T cells to mitigate antigen escape. Mol Ther 2018;26:2542-52.  Back to cited text no. 64
    
65.
Yule SM, Price L, Pearson AD, Boddy AV. Cyclophosphamide and ifosfamide metabolites in the cerebrospinal fluid of children. Clin Cancer Res 1997;3:1985-92.  Back to cited text no. 65
    
66.
Suryadevara CM, Desai R, Abel ML, Riccione KA, Batich KA, Shen SH, et al. Temozolomide lymphodepletion enhances CAR abundance and correlates with antitumor efficacy against established glioblastoma. Oncoimmunology 2018;7:e1434464.  Back to cited text no. 66
    
67.
Badie B, Schartner JM. Flow cytometric characterization of tumor-associated macrophages in experimental gliomas. Neurosurgery 2000;46:957-61.  Back to cited text no. 67
    
68.
Hambardzumyan D, Gutmann DH, Kettenmann H. The role of microglia and macrophages in glioma maintenance and progression. Nat Neurosci 2016;19:20-7.  Back to cited text no. 68
    
69.
Curtsinger JM, Lins DC, Mescher MF. Signal 3 determines tolerance versus full activation of naive CD8 T cells: Dissociating proliferation and development of effector function. J Exp Med 2003;197:1141-51.  Back to cited text no. 69
    
70.
Kerkar SP, Muranski P, Kaiser A, Boni A, Sanchez-Perez L, Yu Z, et al. Tumor-specific CD8+ T cells expressing interleukin-12 eradicate established cancers in lymphodepleted hosts. Cancer Res 2010;70:6725-34.  Back to cited text no. 70
    
71.
Carson WE, Giri JG, Lindemann MJ, Linett ML, Ahdieh M, Paxton R, et al. Interleukin (IL) 15 is a novel cytokine that activates human natural killer cells via components of the IL-2 receptor. J Exp Med 1994;180:1395-403.  Back to cited text no. 71
    
72.
Li Y, Yee C. IL-21 mediated Foxp3 suppression leads to enhanced generation of antigen-specific CD8+ cytotoxic T lymphocytes. Blood 2008;111:229-35.  Back to cited text no. 72
    
73.
Leonard JP, Sherman ML, Fisher GL, Buchanan LJ, Larsen G, Atkins MB, et al. Effects of single-dose interleukin-12 exposure on interleukin-12-associated toxicity and interferon-gamma production. Blood 1997;90:2541-8.  Back to cited text no. 73
    
74.
Tang L, Zheng Y, Melo MB, Mabardi L, Castaño AP, Xie YQ, et al. Enhancing T cell therapy through TCR-signaling-responsive nanoparticle drug delivery. Nat Biotechnol 2018;36:707-16.  Back to cited text no. 74
    
75.
Wang X, Popplewell LL, Wagner JR, Naranjo A, Blanchard MS, Mott MR, et al. Phase 1 studies of central memory-derived CD19 CAR T-cell therapy following autologous HSCT in patients with B-cell NHL. Blood 2016;127:2980-90.  Back to cited text no. 75
    
76.
Davila ML, Riviere I, Wang X, Bartido S, Park J, Curran K, et al. Efficacy and toxicity management of 19-28z CAR T cell therapy in B cell acute lymphoblastic leukemia. Sci Transl Med 2014;6:224ra25.  Back to cited text no. 76
    
77.
Thomis DC, Marktel S, Bonini C, Traversari C, Gilman M, Bordignon C, et al. Afas-based suicide switch in human T cells for the treatment of graft-versus-host disease. Blood 2001;97:1249-57.  Back to cited text no. 77
    
78.
Tey SK, Dotti G, Rooney CM, Heslop HE, Brenner MK. Inducible caspase 9 suicide gene to improve the safety of allodepleted T cells after haploidentical stem cell transplantation. Biol Blood Marrow Transplant 2007;13:913-24.  Back to cited text no. 78
    
79.
Giavridis T, van der Stegen SJ, Eyquem J, Hamieh M, Piersigilli A, Sadelain M. CAR T cell-induced cytokine release syndrome is mediated by macrophages and abated by IL-1 blockade. Nat Med 2018;24:731-8.  Back to cited text no. 79
    
80.
Zhu F, Shah N, Xu H, Schneider D, Orentas R, Dropulic B, et al. Closed-system manufacturing of CD19 and dual-targeted CD20/19 chimeric antigen receptor T cells using the CliniMACS prodigy device at an academic medical center. Cytotherapy 2018;20:394-406.  Back to cited text no. 80
    
81.
Mock U, Nickolay L, Philip B, Cheung GW, Zhan H, Johnston IC, et al. Automated manufacturing of chimeric antigen receptor T cells for adoptive immunotherapy using CliniMACS prodigy. Cytotherapy 2016;18:1002-11.  Back to cited text no. 81
    



 
 
    Tables

  [Table 1]



 

Top
 
  Search
 
    Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
    Access Statistics
    Email Alert *
    Add to My List *
* Registration required (free)  

 
  In this article
Abstract
Introduction
Database Search ...
Navigating Chime...
Steering Chimeri...
Armoring Chimeri...
Torqueing Chimer...
Enhancing Chimer...
Customizing Manu...
Conclusion
References
Article Tables

 Article Access Statistics
    Viewed212    
    Printed16    
    Emailed0    
    PDF Downloaded32    
    Comments [Add]    

Recommend this journal