**7. Immunotherapy clinical trials for brain tumors**


• Vaccine Therapy, Temozolomide, and Radiation Therapy in Treating Patients With Newly Diagnosed Glioblastoma Multiforme

• Dendritic Cell Vaccine for Patients With Brain Tumors

Resection

• Dendritic Cell Vaccine Therapy With In Situ Maturation in Pediatric Brain Tumors • Dendritic Cell Vaccine With Imiquimod for Patients With Malignant Glioma

• Immunotherapy for Patients With Brain Stem Glioma and Glioblastoma

• Study of a Drug [DCVax®-L] to Treat Newly Diagnosed GBM Brain Cancer

• Tumor Lysate Pulsed Dendritic Cell Immunotherapy for Patients With Brain Tumors

• Vaccination-Dendritic Cells With Peptides for Recurrent Malignant Gliomas • Vaccine for Patients With Newly Diagnosed or Recurrent Low-Grade Glioma

• Vaccine Therapy in Treating Patients With Malignant Glioma

Glioblastoma That Has Progressed on Bevacizumab

Multiforme That Has Been Removed by Surgery

**Table 3.** Dendritic Cell Vaccine

above.

antibody)

• Proteome-based Personalized Immunotherapy of Glioblastoma

• Study of DC Vaccination Against Glioblastoma

• Efficacy & Safety of Autologous Dendritic Cell Vaccination in Glioblastoma Multiforme After Complete Surgical

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199

• Surgical Resection With Gliadel Wafer Followed by Dendritic Cells Vaccination for Malignant Glioma Patients

• Vaccination With Dendritic Cells Loaded With Brain Tumor Stem Cells for Progressive Malignant Brain Tumor

• Vaccine Immunotherapy for Recurrent Medulloblastoma and Primitive Neuroectodermal Tumor • Vaccine Therapy and Temozolomide in Treating Patients With Newly Diagnosed Glioblastoma • Vaccine Therapy in Treating Patients Undergoing Surgery for Recurrent Glioblastoma Multiforme

• Vaccine Therapy in Treating Young Patients Who Are Undergoing Surgery for Malignant Glioma

• Vaccine Therapy With or Without Sirolimus in Treating Patients With NY-ESO-1 Expressing Solid Tumors

The above table lists several current clinical trials using dendritic cells combined with tumor antigen as a method of delivering peripheral vaccination against CNS tumors. See section 4.1

• A Phase 2 Evaluation of TRC105 in Combination with Bevacizumab for the Treatment of Recurrent or Progressive

• Chemotherapy, Radiation Therapy, and Vaccine Therapy With Basiliximab in Treating Patients With Glioblastoma

• Use of Racotumomab in Patients With Pediatric Tumors Expressing N-glycolylated Gangliosides (anti-idiotype

• Vaccine Therapy in Treating Patients With Newly Diagnosed Glioblastoma Multiforme

• Phase II Feasibility Study of Dendritic Cell Vaccination for Newly Diagnosed Glioblastoma Multiforme

• Safe Study of Dendritic Cell (DC) Based Therapy Targeting Tumor Stem Cells in Glioblastoma

**Table 1.** Tumor Antigen Vaccine

www.clinicaltrials.gov

The above table lists several current clinical trials using tumor-derived antigens for developing peripheral vaccination against CNS tumors. See section 4.1 above.

• Chemotherapy and Vaccine Therapy Followed by Bone Marrow or Peripheral Stem Cell Transplantation and Interleukin-2 in Treating Patients With Recurrent or Refractory Brain Cancer


• Vaccination With Lethally Irradiated Glioma Cells Mixed With GM-K562 Cells in Patients Undergoing Craniotomy For Recurrent Tumor

**Table 2.** Tumor Lysate or Cell Vaccine

The above table lists several current clinical trials using whole tumor cells to develop periph‐ eral vaccines against multiple antigens found on CNS tumors. See section 4.1 above.


<sup>•</sup> A Study of ICT-107 Immunotherapy in Glioblastoma Multiforme (GBM)

• Dendritic Cell Vaccine for Patients With Brain Tumors

• Phase III Study of Rindopepimut/GM-CSF in Patients With Newly Diagnosed Glioblastoma (uses EGFR)

• Vaccine Therapy, Temozolomide, and Radiation Therapy in Treating Patients With Newly Diagnosed Glioblastoma

The above table lists several current clinical trials using tumor-derived antigens for developing

• Chemotherapy and Vaccine Therapy Followed by Bone Marrow or Peripheral Stem Cell Transplantation and

• Phase I/II Study To Test The Safety and Efficacy of TVI-Brain-1 As A Treatment For Recurrent Grade IV Glioma

• Study To Test the Safety and Efficacy of TVI-Brain-1 As A Treatment for Recurrent Grade IV Glioma

• Study to Evaluate the Effects of Imiquimod and Tumor Lysate Vaccine Immunotherapy in Adults With High Risk or

• Vaccination With Lethally Irradiated Glioma Cells Mixed With GM-K562 Cells in Patients Undergoing Craniotomy For

The above table lists several current clinical trials using whole tumor cells to develop periph‐

eral vaccines against multiple antigens found on CNS tumors. See section 4.1 above.

• Daclizumab in Treating Patients With Newly Diagnosed Glioblastoma Multiforme Undergoing Targeted

• Dendritic Cell Vaccine For Malignant Glioma and Glioblastoma Multiforme in Adult and Pediatric Subjects

• Poliovirus Vaccine for Recurrent Glioblastoma Multiforme (GBM)

198 Tumors of the Central Nervous System – Primary and Secondary

Multiforme

Recurrent Tumor

**Table 1.** Tumor Antigen Vaccine

• Derivation of Tumor Specific Hybridomas

**Table 2.** Tumor Lysate or Cell Vaccine

www.clinicaltrials.gov

• Vaccine Therapy and Sargramostim in Treating Patients With Malignant Glioma • Vaccine Therapy and Sargramostim in Treating Patients With Sarcoma or Brain Tumor • Vaccine Therapy in Treating Patients With Newly Diagnosed Glioblastoma Multiforme

peripheral vaccination against CNS tumors. See section 4.1 above.

Interleukin-2 in Treating Patients With Recurrent or Refractory Brain Cancer

• Imiquimod/Brain Tumor Initiating Cell (BTIC) Vaccine in Brain Stem Glioma

• A Study of ICT-107 Immunotherapy in Glioblastoma Multiforme (GBM) • Biological Therapy in Treating Patients With Glioblastoma Multiforme

Immunotherapy and Temozolomide-Caused Lymphopenia • Dendritic Cell Cancer Vaccine for High-grade Glioma

• Pilot Immunotherapy Trial for Recurrent Malignant Gliomas

Recurrent/Post-Chemotherapy WHO Grade II Gliomas


**Table 3.** Dendritic Cell Vaccine

The above table lists several current clinical trials using dendritic cells combined with tumor antigen as a method of delivering peripheral vaccination against CNS tumors. See section 4.1 above.

<sup>•</sup> A Phase 2 Evaluation of TRC105 in Combination with Bevacizumab for the Treatment of Recurrent or Progressive Glioblastoma That Has Progressed on Bevacizumab

<sup>•</sup> Chemotherapy, Radiation Therapy, and Vaccine Therapy With Basiliximab in Treating Patients With Glioblastoma Multiforme That Has Been Removed by Surgery

<sup>•</sup> Use of Racotumomab in Patients With Pediatric Tumors Expressing N-glycolylated Gangliosides (anti-idiotype antibody)

• Vaccine Therapy With Bevacizumab Versus Bevacizumab Alone in Treating Patients With Recurrent Glioblastoma Multiforme That Can Be Removed by Surgery

• Evaluation of Recovery From Drug-Induced Lymphopenia Using Cytomegalovirus-specific T-cell Adoptive Transfer • Phase I Study of Cellular Immunotherapy for Recurrent/Refractory Malignant Glioma Using Intratumoral Infusions of GRm13Z40-2, An Allogeneic CD8+ Cytolitic T-Cell Line Genetically Modified to Express the IL 13-Zetakine and HyTK

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The above table lists several current clinical trials using adoptive immunotherapy as a way of applying passive immunotherapy to return autologous tumor-antigen-specific T cells back to

Gene therapy as it relates to malignant gliomas is based on tumor-specific introduction of genetic material for the purpose of treatment. It involves direct injection of a gene transfer vector or vector producing cells (VPC) into the tumor itself or into the cavity left after resection. Although preclinical studies have been quite promising, unfortunately therapeutic response to gene therapy clinical trials remains low (Tobias, Ahmed et al. 2013). Three classes of genetic therapy treatment have taken center stage over the last several decades: prodrug/suicide genes, oncolytic viruses, and gene immunotherapy. Although each is its own distinct entity, they all facilitate delivery of genetic material through the use of one or more vectors as described

Retroviruses and retroviral vector producing cells (RVPCs) may be used to deliver specific genes to glioma cells; they are perhaps the most widely-studied class of vectors for treatment of GBM. This class of virus is advantageous in that its transduction is limited to rapidly dividing cells, meaning that normal brain cells remain unaltered. However, the transduction rate is low secondary to rapid inactivation of free retroviral vectors by complement as well as a lack of movement of virus to sites distant to the injection. It should be noted that transduction of circulating cells by vectors may occur, thus putting the patient at risk of cancer initiation

Adenoviruses belong to a family of 90-100 nm non-enveloped viruses made up of a nucleo‐ capsid and double-stranded linear DNA. They account for roughly one tenth of all upper

and to be Resistant to Glucocorticoids, in Combination With Interleukin-2

via insertional mutagenesis (Barzon, Zanusso et al. 2006).

• White Blood Cells With Anti-EGFR-III for Malignant Gliomas

**Table 7.** Adoptive Immunotherapy

**8. Gene therapy**

below.

**8.1. Vectors**

*8.1.1. Retroviral vectors*

*8.1.2. Adenoviral vectors*

• Safety and Effectiveness Study of Autologous Natural Killer and Natural Killer T Cells on Cancer

the patient as a means of targeting CNS tumors. See section 4.3.3 above.

**Table 4.** Vaccine with monoclonal antibody

The above table lists several current clinical trials using passive immunotherapy by means of delivering monoclonal antibodies directed at tumor cells. See section 4.3.1 above.

• Convection-Enhanced Delivery of 124I-8H9 for Patients With Non-Progressive Diffuse Pontine Gliomas Previously Treated With External Beam Radiation Therapy

• Intrathecal Radioimmunotherapy, Radiation Therapy, and Chemotherapy After Surgery in Treating Patients with Medullolblastoma

• Radiolabeled Monoclonal Antibody Therapy in Treating Patients with Primary or Metastatic Brain Tumors

• Radiosurgery Plus Bevacizumab in Glioblastoma

**Table 5.** Radioimmunotherapy with monoclonal antibody

The above table lists current clinical trials using cytotoxic radiation coupled to monoclonal antibodies to kill tumor cells. See section 4.3.1 above.

• IL-4 (38-37)-PE38KDEL Immunotoxin in Treating Patients With Recurrent Malignant Astrocytoma

• Imaging Study of the Distribution of IL13-PE38QQR Infused Before and After Surgery in Adult Patients With Recurrent Malignant Glioma

• NBI-3001 Followed by Surgery in Treating Patients with Recurrent Glioblastoma Multiforme

• TP-38 Toxin in Treating Young Patients with Recurrent or Progressive Supratentorial High-Grade Glioma

**Table 6.** Transfer of Ligands

The above table lists current clinical trials using molecules fused with toxins as a means of killing tumor cells.

• A Phase I Study to Investigate Tolerability and Efficacy of ALECSAT Administered to Glioblastoma Multiforme Patients

• Autologous Natural Killer T Cells Infusion for the Treatment of Cancer


• Evaluation of Recovery From Drug-Induced Lymphopenia Using Cytomegalovirus-specific T-cell Adoptive Transfer

• Phase I Study of Cellular Immunotherapy for Recurrent/Refractory Malignant Glioma Using Intratumoral Infusions of GRm13Z40-2, An Allogeneic CD8+ Cytolitic T-Cell Line Genetically Modified to Express the IL 13-Zetakine and HyTK and to be Resistant to Glucocorticoids, in Combination With Interleukin-2


#### **Table 7.** Adoptive Immunotherapy

• Vaccine Therapy With Bevacizumab Versus Bevacizumab Alone in Treating Patients With Recurrent Glioblastoma

The above table lists several current clinical trials using passive immunotherapy by means of

• Convection-Enhanced Delivery of 124I-8H9 for Patients With Non-Progressive Diffuse Pontine Gliomas Previously

• Intrathecal Radioimmunotherapy, Radiation Therapy, and Chemotherapy After Surgery in Treating Patients with

The above table lists current clinical trials using cytotoxic radiation coupled to monoclonal

• Radiolabeled Monoclonal Antibody Therapy in Treating Patients with Primary or Metastatic Brain Tumors

• IL-4 (38-37)-PE38KDEL Immunotoxin in Treating Patients With Recurrent Malignant Astrocytoma

• NBI-3001 Followed by Surgery in Treating Patients with Recurrent Glioblastoma Multiforme

• Imaging Study of the Distribution of IL13-PE38QQR Infused Before and After Surgery in Adult Patients With

• TP-38 Toxin in Treating Young Patients with Recurrent or Progressive Supratentorial High-Grade Glioma

The above table lists current clinical trials using molecules fused with toxins as a means of

• Cellular Adoptive Immunotherapy Using Genetically Modified T-Lymphocytes in Treating Patients With Recurrent or

• CMV-specific Cytotoxic T Lymphocytes Expressing CAR Targeting HER2 in Patients with GBM (HERT-GBM)

• A Phase I Study to Investigate Tolerability and Efficacy of ALECSAT Administered to Glioblastoma Multiforme

delivering monoclonal antibodies directed at tumor cells. See section 4.3.1 above.

Multiforme That Can Be Removed by Surgery

200 Tumors of the Central Nervous System – Primary and Secondary

**Table 4.** Vaccine with monoclonal antibody

Treated With External Beam Radiation Therapy

• Radiosurgery Plus Bevacizumab in Glioblastoma

**Table 5.** Radioimmunotherapy with monoclonal antibody

antibodies to kill tumor cells. See section 4.3.1 above.

• Autologous Natural Killer T Cells Infusion for the Treatment of Cancer

• Cellular Adoptive Immunotherapy in Treating Patients With Glioblastoma Multiforme

Medullolblastoma

Recurrent Malignant Glioma

**Table 6.** Transfer of Ligands

killing tumor cells.

Refractory High-Grade Malignant Glioma

• Cellular Immunotherapy Study for Brain Cancer

Patients

The above table lists several current clinical trials using adoptive immunotherapy as a way of applying passive immunotherapy to return autologous tumor-antigen-specific T cells back to the patient as a means of targeting CNS tumors. See section 4.3.3 above.

#### **8. Gene therapy**

Gene therapy as it relates to malignant gliomas is based on tumor-specific introduction of genetic material for the purpose of treatment. It involves direct injection of a gene transfer vector or vector producing cells (VPC) into the tumor itself or into the cavity left after resection. Although preclinical studies have been quite promising, unfortunately therapeutic response to gene therapy clinical trials remains low (Tobias, Ahmed et al. 2013). Three classes of genetic therapy treatment have taken center stage over the last several decades: prodrug/suicide genes, oncolytic viruses, and gene immunotherapy. Although each is its own distinct entity, they all facilitate delivery of genetic material through the use of one or more vectors as described below.

#### **8.1. Vectors**

#### *8.1.1. Retroviral vectors*

Retroviruses and retroviral vector producing cells (RVPCs) may be used to deliver specific genes to glioma cells; they are perhaps the most widely-studied class of vectors for treatment of GBM. This class of virus is advantageous in that its transduction is limited to rapidly dividing cells, meaning that normal brain cells remain unaltered. However, the transduction rate is low secondary to rapid inactivation of free retroviral vectors by complement as well as a lack of movement of virus to sites distant to the injection. It should be noted that transduction of circulating cells by vectors may occur, thus putting the patient at risk of cancer initiation via insertional mutagenesis (Barzon, Zanusso et al. 2006).

#### *8.1.2. Adenoviral vectors*

Adenoviruses belong to a family of 90-100 nm non-enveloped viruses made up of a nucleo‐ capsid and double-stranded linear DNA. They account for roughly one tenth of all upper respiratory tract infections in children, infecting the host via introduction of their genome into the nucleus of the host organism's cells where the viral DNA remains free. This is in opposition to the retroviral mechanism involving incorporation of genetic material into the host cell's genomic structure.

phosphorylates the prodrug of GCV into its active compound, whose mechanism of action involves DNA cross-linking, which leads to cell death. Following treatment with GCV, there may also be an observed "bystander effect" which involves the killing of non-transduced adjacent cells or even distant cells via immune response (T Cells, NK Cells) and toxic metab‐ olites received via gap junctions (Ram, Culver et al. 1997; Floeth, Shand et al. 2001; Matuskova, Hlubinova et al. 2010). In a xenograft glioma model, a significant therapeutic effect was found when only approximately 10% of tumor cells were transduced with HSV-tk (Chen, Chang et al. 1995; Sandmair, Loimas et al. 2000). Introduction of HSV-tk/GCV may also increase response to standard measures such as radio- and chemotherapy (Rainov, Fels et al. 2001; Chiocca, Broaddus et al. 2004). This method has also been hypothesized to stimulate an immune response and provide an anti-angiogenic effect (Culver, Ram et al. 1992; Ayala, Satoh et al. 2006; Chiocca, Aguilar et al. 2011). Although there have been numerous enzyme-prodrug clinical trials ranging from Phase I to Phase III, endpoints such as median survival have not been overly impressive (Iwami, Natsume et al. 2010; Kroeger, Muhammad et al. 2010).

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Intratumor injection of RVPCs has shown a high percentage of tumor regression in some studies (Ram, Culver et al. 1997; Pulkkanen and Yla-Herttuala 2005). Rainov et al. conducted a Phase III, multicenter, open-label, randomized trial of newly diagnosed GBM comparing standard therapy vs. standard therapy with adjuvant gene therapy of the tumor bed by HSVtk. Although this mode of treatment was shown to be safe, there was no significant difference in 12-month survival rates or progression-free median survival (Rainov 2000). A recent Phase I head-to-head trial of intra-operative HSV-tk introduction via retrovirus vs. adenovirus showed promising results for adenoviral vectors in a small number of patients (Sandmair,

It should also be noted that unlike retroviral vectors, adenovirus can transduce both dividing and non-dividing cells. The majority of adenoviruses used for this purpose are E1-deleted adenoviral vectors, which may be injected at a higher titer than RVPCs; however high doses may indeed lead to serious side effects, including confusion, seizures, fever, leukocytosis, and hyponatremia that appear to be secondary to immune response to the vector (Trask, Trask et al. 2000). This same immune response lowers the yield of viral delivery but also aids in tumor reduction (Trask, Trask et al. 2000; Lang, Bruner et al. 2003). Notably, the adenoviral vector may be found transiently in blood but has not been found as a replication-competent entity.

Preliminary clinical data suggest that adenoviral mediated gene transfer of suicide genes (AdvHSV-tk) may have clinical utility (Germano, Fable et al. 2003; Immonen, Vapalahti et al. 2004). A Phase IIB randomized controlled trial of patients with malignant gliomas reported a significant increase in OS from 37.7 weeks in the control arm (n=19) to 62.4 weeks in the adenoviral treated arm (AdvHSV-tk, n=17) (Immonen, Vapalahti et al. 2004). A recent Phase 1B trial showed treatment with adenovirus-HSV-tk followed by Valacyclovir, when paired

*8.2.2. Retrovirally-mediated therapy*

*8.2.3. Adenovirally-mediated therapy*

Loimas et al. 2000).

Adenovirus enters the host cell by way of 2 distinct sets of interactions. Firstly, the knob domain of the virus's fiber protein binds to the cell receptor (either CD46 or coxsackievirus adenovirus receptor). This is followed by the interaction of a specialized motif in the penton base protein with an integrin molecule, which prompts internalization of the virus via an endosome. Thereafter the capsid components dissociate and the virion is released into the cytoplasm. Viral DNA enters the nucleus via the nuclear pore, later associating with histones. Following nuclear invasion, the viral genome is reproduced along with the host cell's DNA. However, the progeny of the original host cell will not carry the newly-introduced viral DNA. This neces‐ sitates numerous rounds of viral introduction in the treatment of cancer (Doloff and Waxman 2013).

#### *8.1.3. Reoviral vectors*

The genome of Reoviridae is segmented, double-stranded RNA, and the virus has the ability to make use of a non-functional protein kinase R (PKR) pathway in glioma cells to allow for viral replication. This is advantageous as the virus does not require genetic engineering. Other advantages include small size (70-80nm) and an absence of known consequent encephalitis in humans (Clarke, Debiasi et al. 2005).

#### *8.1.4. Nonviral vectors*

There are several nonviral vectors either currently in use or being considered for use in gene therapy such as synthetic vectors, nanoparticles, and stem cells/progenitor cells. From this group, perhaps the most studied is the liposome (included in the category of nanoparticles). Cationic Liposomes are easy to produce, have relatively low immunogenicity and toxicity, and typically exhibit long-term stability (Tobias, Ahmed et al. 2013).

#### **8.2. Gene therapy strategies**

#### *8.2.1. Prodrug activating genes/suicide genes*

Prodrug/suicide genes represent an ingenious wing of gene therapy. The basis of this antitumor modality is introduction of genes, either into the host genome or the intranuclear milieu, which imparts susceptibility to a subsequent therapeutic agent. The vectors themselves are genetically modified to produce an enzyme which converts a prodrug, given systemically, into toxic metabolites which act specifically on the malignancy.

Perhaps the earliest/most-studied example of prodrug/suicide gene utility when addressing gliomas is that of Herpes Simplex Type 1 Thymidine Kinase (HSV-tk). After incorporation of this gene into tumor cells (often residual cells status-post resection) and the endothelium of their vasculature, the host is treated with an antiviral such as gancyclovir (GCV). HSV-TK phosphorylates the prodrug of GCV into its active compound, whose mechanism of action involves DNA cross-linking, which leads to cell death. Following treatment with GCV, there may also be an observed "bystander effect" which involves the killing of non-transduced adjacent cells or even distant cells via immune response (T Cells, NK Cells) and toxic metab‐ olites received via gap junctions (Ram, Culver et al. 1997; Floeth, Shand et al. 2001; Matuskova, Hlubinova et al. 2010). In a xenograft glioma model, a significant therapeutic effect was found when only approximately 10% of tumor cells were transduced with HSV-tk (Chen, Chang et al. 1995; Sandmair, Loimas et al. 2000). Introduction of HSV-tk/GCV may also increase response to standard measures such as radio- and chemotherapy (Rainov, Fels et al. 2001; Chiocca, Broaddus et al. 2004). This method has also been hypothesized to stimulate an immune response and provide an anti-angiogenic effect (Culver, Ram et al. 1992; Ayala, Satoh et al. 2006; Chiocca, Aguilar et al. 2011). Although there have been numerous enzyme-prodrug clinical trials ranging from Phase I to Phase III, endpoints such as median survival have not been overly impressive (Iwami, Natsume et al. 2010; Kroeger, Muhammad et al. 2010).

#### *8.2.2. Retrovirally-mediated therapy*

respiratory tract infections in children, infecting the host via introduction of their genome into the nucleus of the host organism's cells where the viral DNA remains free. This is in opposition to the retroviral mechanism involving incorporation of genetic material into the host cell's

Adenovirus enters the host cell by way of 2 distinct sets of interactions. Firstly, the knob domain of the virus's fiber protein binds to the cell receptor (either CD46 or coxsackievirus adenovirus receptor). This is followed by the interaction of a specialized motif in the penton base protein with an integrin molecule, which prompts internalization of the virus via an endosome. Thereafter the capsid components dissociate and the virion is released into the cytoplasm. Viral DNA enters the nucleus via the nuclear pore, later associating with histones. Following nuclear invasion, the viral genome is reproduced along with the host cell's DNA. However, the progeny of the original host cell will not carry the newly-introduced viral DNA. This neces‐ sitates numerous rounds of viral introduction in the treatment of cancer (Doloff and Waxman

The genome of Reoviridae is segmented, double-stranded RNA, and the virus has the ability to make use of a non-functional protein kinase R (PKR) pathway in glioma cells to allow for viral replication. This is advantageous as the virus does not require genetic engineering. Other advantages include small size (70-80nm) and an absence of known consequent encephalitis in

There are several nonviral vectors either currently in use or being considered for use in gene therapy such as synthetic vectors, nanoparticles, and stem cells/progenitor cells. From this group, perhaps the most studied is the liposome (included in the category of nanoparticles). Cationic Liposomes are easy to produce, have relatively low immunogenicity and toxicity, and

Prodrug/suicide genes represent an ingenious wing of gene therapy. The basis of this antitumor modality is introduction of genes, either into the host genome or the intranuclear milieu, which imparts susceptibility to a subsequent therapeutic agent. The vectors themselves are genetically modified to produce an enzyme which converts a prodrug, given systemically, into

Perhaps the earliest/most-studied example of prodrug/suicide gene utility when addressing gliomas is that of Herpes Simplex Type 1 Thymidine Kinase (HSV-tk). After incorporation of this gene into tumor cells (often residual cells status-post resection) and the endothelium of their vasculature, the host is treated with an antiviral such as gancyclovir (GCV). HSV-TK

typically exhibit long-term stability (Tobias, Ahmed et al. 2013).

toxic metabolites which act specifically on the malignancy.

genomic structure.

202 Tumors of the Central Nervous System – Primary and Secondary

2013).

*8.1.3. Reoviral vectors*

*8.1.4. Nonviral vectors*

**8.2. Gene therapy strategies**

*8.2.1. Prodrug activating genes/suicide genes*

humans (Clarke, Debiasi et al. 2005).

Intratumor injection of RVPCs has shown a high percentage of tumor regression in some studies (Ram, Culver et al. 1997; Pulkkanen and Yla-Herttuala 2005). Rainov et al. conducted a Phase III, multicenter, open-label, randomized trial of newly diagnosed GBM comparing standard therapy vs. standard therapy with adjuvant gene therapy of the tumor bed by HSVtk. Although this mode of treatment was shown to be safe, there was no significant difference in 12-month survival rates or progression-free median survival (Rainov 2000). A recent Phase I head-to-head trial of intra-operative HSV-tk introduction via retrovirus vs. adenovirus showed promising results for adenoviral vectors in a small number of patients (Sandmair, Loimas et al. 2000).

#### *8.2.3. Adenovirally-mediated therapy*

It should also be noted that unlike retroviral vectors, adenovirus can transduce both dividing and non-dividing cells. The majority of adenoviruses used for this purpose are E1-deleted adenoviral vectors, which may be injected at a higher titer than RVPCs; however high doses may indeed lead to serious side effects, including confusion, seizures, fever, leukocytosis, and hyponatremia that appear to be secondary to immune response to the vector (Trask, Trask et al. 2000). This same immune response lowers the yield of viral delivery but also aids in tumor reduction (Trask, Trask et al. 2000; Lang, Bruner et al. 2003). Notably, the adenoviral vector may be found transiently in blood but has not been found as a replication-competent entity.

Preliminary clinical data suggest that adenoviral mediated gene transfer of suicide genes (AdvHSV-tk) may have clinical utility (Germano, Fable et al. 2003; Immonen, Vapalahti et al. 2004). A Phase IIB randomized controlled trial of patients with malignant gliomas reported a significant increase in OS from 37.7 weeks in the control arm (n=19) to 62.4 weeks in the adenoviral treated arm (AdvHSV-tk, n=17) (Immonen, Vapalahti et al. 2004). A recent Phase 1B trial showed treatment with adenovirus-HSV-tk followed by Valacyclovir, when paired with resection, chemotherapy and radiotherapy, was safe and without dose-limiting toxicity (Chiocca, Aguilar et al. 2011).

further tumor reduction. This method is tumor-specific as it makes use of either attenuated viruses containing inactivated genes which replicate in tumor cells only, or viruses with replication-essential genes in tumor-specific promoters (Chiocca 2002). This method employs herpes simplex virus (HSV), Adenovirus, Reovirus, Poliovirus, Newcastle Disease Virus

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Herpes Simplex is an enveloped, doubled-stranded DNA virus which exhibits inherent action upon the human nervous system; it can replicate in both active and quiescent cells. Conse‐ quently, safety was an original concern with this viral vector. Approximately 8 different HSV-1 genes have been altered or deleted to promote tumor specificity and lower collateral CNS damage (Tobias, Ahmed et al. 2013). There are two strains of replication-competent HSV-1 which have been significantly studied: G207 and HSV1716. G207 is the more widely-examined of the two and possesses a mechanism of action involving alteration of the gene which produces ribonucleotide reductase. In a recent phase 1B clinical trial, patients received injections of this virus both before and after tumor resection. Although viral replication was observed, treatment efficacy was sparse (Markert, Liechty et al. 2009). Additional studies have

likewise shown adequate safety but minimal efficacy (Todo, Martuza et al. 2001).

performed months after treatment (Rampling, Cruickshank et al. 2000).

G207 overcomes host defenses mediated by protein kinase R (PKR), which normally shuts down translation in infected cells through phosphorylation of eIF-2 alpha (Barzon, Zanusso et al. 2006). In a Phase I study by Markert et al., conditionally replicating G207 virus (given by stereotactic intratumor injection) was not found to lead to the development of herpes ence‐ phalitis (Markert, Medlock et al. 2000). Additionally, replication-competent HSV1716 admin‐ istration in a Phase 1 dose-escalation study by Rampling et al. did not lead to encephalitis. Furthermore, no viral shedding was noted and no viral genome was found in tumor biopsies

Adenoviruses carrying mutations in E1A or E1B can also act on GBM via oncolysis. Their mechanism of action involves tumor-specific binding and inactivation of apoptotic proteins like pRB family and p53. Of note, adenovirus is inherently non-neurotropic, which may lend itself to superior safety versus HSV. One adenovirus, *ONYX-015,* has been found to preferen‐ tially replicate in p53 deficient cells secondary to its deletion for p53-inactivating protein E1B-55K. In one clinical trial it was injected into the surgical cavity after resection and found to have no serious adverse effects; however; almost all patients involved in the trial had progression of their GBM (Chiocca, Abbed et al. 2004). It should also be noted that Geoerger et al found human xenografts to be responsive to ONYX-015 without correlation to their p53

(NDV), and Measles virus.

*8.3.2. Oncolytic adenoviruses*

status (Geoerger, Grill et al. 2003).

*8.3.1. Oncolytic HSV-1*

Despite the aforementioned promising results from a small number of patients, the Phase III international open-label, randomized ASPECT clinical trial, which studied the intra-operative administration of adenoviral-HSV-tk followed by GCV (n=124) as compared to resection and standard of care alone (n=126), was not positive. Unfortunately, the data revealed no difference between the groups in terms of OS; furthermore, more patients in the experimental group had one or more treatment-related adverse events than those in the control group (88 [71%] vs 51 [43%]) (Westphal, Yla-Herttuala et al. 2013).

#### *8.2.4. Nanopartical/Neural stem cell-mediated therapy*

Synthetic vectors, including nanoparticles have been applied to deliver DNA plasmids, RNA and siRNA (Jin and Ye 2007; Germano and Binello 2009; Jin, Bae et al. 2011). Liposomes are perhaps the most-researched of all nanoparticles (Tobias, Ahmed et al. 2013). Given through convection-enhanced delivery via stereotactically-placed catheters a liposome-DNA complex has been used to deliver HSV-tk in a small number of patients. The treatment was welltolerated without major side effects (Jacobs, Voges et al. 2001; Voges, Reszka et al. 2003).

Pleuripotent neural stem cells procured from the subgranular zone of the hippocampus and the areas surrounding the lateral ventricles have the ability to migrate to areas of parenchymal damage (Luskin 1993). Neural stem cell clones may migrate to areas of tumor infiltration and thus were examined as vehicles for delivery of suicide genes, cytokines, or tumor necrosis factor-related apoptosis-inducing ligand (TRAIL); there is evidence of potential efficacy in animal models but no clinical utility data yet (Aboody, Brown et al. 2000; Marsh, Goldfarb et al. 2013).

#### *8.2.5. Tumor suppressor gene replacement*

A well-documented characteristic of GBM is its inherent inactivation of the p53 tumor suppressor gene. Animal trials have shown that re-introduction of the wild-type p53 gene is pro-apoptotic leading to increased sensitivity to current modalities of treatment such as chemo- and radiotherapy. A Phase 1 trial of adenoviral gene transfer of intra-tumoral wildtype p53 in recurrent malignant glioma proved to be safe, but the transfected cells were not found in a radius large enough to be therapeutically effective (Lang, Bruner et al. 2003).

#### **8.3. Oncolytic gene therapy**

The realm of oncolytic virus therapy involves the use of replication-competent viruses with the ability to selectively replicate and kill cancer cells, with or without gene transfer. This is in opposition to prodrug/suicide gene therapy which makes use of replication-incompetent modalities. In order to combat the inefficiency of suicide gene therapy, oncolytic treatment employs tumor-specific, conditionally replicating viral vectors (Tobias, Ahmed et al. 2013). The mechanism of action involves viral replication which eventually leads to lysis of the host tumor cell and subsequent release of additional copies of competent virus which may lead to further tumor reduction. This method is tumor-specific as it makes use of either attenuated viruses containing inactivated genes which replicate in tumor cells only, or viruses with replication-essential genes in tumor-specific promoters (Chiocca 2002). This method employs herpes simplex virus (HSV), Adenovirus, Reovirus, Poliovirus, Newcastle Disease Virus (NDV), and Measles virus.
