**6. Immunotherapy strategies and targets**

Immunotherapy for malignant gliomas is based on various strategies aimed at the induction of anti-tumor immunity. Nevertheless, though curing a mouse from a brain tumor using immunotherapy is rather easy, this goal has proven to be more challenging in humans especially when coupled with the globally impaired immune response and increased tumor tolerance in patients with GBM (Luptrawan, Liu et al. 2008). Because of the aforementioned data, the goal of immunotherapy for gliomas should be not only to activate the cytotoxic T cell response, but also to counteract the active immunological depressive effects by the tumor itself. We will not list an exhaustive search of all immunotherapeutic strategies but will instead discuss an outline of the approaches than can be used. A thorough discussion can be found in Okada et al. (Okada, Kohanbash et al. 2009). Here, we will emphasize the different categories and discuss limitations of immunotherapy.

### **6.1. Priming in the periphery**

pression (Yang, Han et al. 2010). Pathological examination typically reveals a large numbers of microglia dispersed within the tumor and not just in necrotic tissue (Yang, Han et al. 2010). The data of Okada et al. suggest that the glioma-infiltrating cells may compose up to 30% of the glioma tumor, correlating in volume with degree of malignancy (Okada, Kohanbash et al. 2009). The lack of phagocytosis by the microglia is thought be related to decreased expression of MHC II and co-stimulatory molecules CD80/86 and CD40, thus prohibiting appropriate T cell activation (Okada, Kohanbash et al. 2009; Yang, Han et al. 2010). Glioma cells appear to attract microglia by secreting chemoattractants and growth factors including Macrophage Chemoattractive Protein-1 (MCP-1), which binds to the microglial MCP-1 receptor, as well as colony stimulating factor-1, Granulocyte-CSF, and hepatocyte growth factor/scatter factor (Yang, Han et al. 2010). Microglial secretion of epidermal growth factor (EGF), VEGF and MCP-1 promote tumor propagation and angiogenesis (Okada, Kohanbash et al. 2009; Yang, Han et al. 2010). Additionally, the release by microglia of MMPs assists in tumor dispersal (Yang, Han et al. 2010). Interestingly, tumors depleted of microglia actually become less

In addition to altering the response of microglial cells, gliomas take an active role in downregulating the immune response. Recent data has shown that reduced phagocytic activity by glioma-associated microglia stems from defective antigen presentation for T cell activation due to decreased MHC II expression as well as suppression of pro-inflammatory cytokine (TNFα) release, especially in high-grade gliomas (Yang, Han et al. 2010). Instead, glioma cells favor TGF- β, IL-10, and PGE2 secretion, which inhibits both cytotoxic function of T cells and IFNγ-induced MHC II expression in microglial cells (Luptrawan, Liu et al. 2008; Okada, Kohan‐ bash et al. 2009; Yang, Han et al. 2010). PGE2 specifically inhibits T cell activation, suppresses natural killers cell activity, and favors a Th2 response by increasing cytokines Il-4, Il-10, and Il-6 while suppressing the Th1 cytokines Il-2, IFN-gamma, and TNF-α (Luptrawan, Liu et al. 2008). Additionally, glioma cells do not express adequate co-stimulatory molecules required for appropriate T cell activation, potentiating anergy through tolerance (Luptrawan, Liu et al. 2008). A homologue to the B7 family (B71/2 (CD80/86)), B7-H1 expression on the surface of glioma cells inhibits CD4+ and CD8+ T cell activation. IFN- γ not only enhances antigen processing but also promotes increased B7-H1 expression, ultimately reducing T lymphocyte effectiveness in the presence of gliomas (Okada, Kohanbash et al. 2009). Additionally, some gliomas display Fas-L leading to apoptosis of Fas-labeled T cells contacting the tumor cells

Immunotherapy for malignant gliomas is based on various strategies aimed at the induction of anti-tumor immunity. Nevertheless, though curing a mouse from a brain tumor using immunotherapy is rather easy, this goal has proven to be more challenging in humans especially when coupled with the globally impaired immune response and increased tumor tolerance in patients with GBM (Luptrawan, Liu et al. 2008). Because of the aforementioned data, the goal of immunotherapy for gliomas should be not only to activate the cytotoxic T cell

invasive (Okada, Kohanbash et al. 2009).

192 Tumors of the Central Nervous System – Primary and Secondary

(Okada, Kohanbash et al. 2009).

**6. Immunotherapy strategies and targets**

Initiating an immune response against tumors is typically difficult due to poor antigen presentation and the active immunosuppressive effects by tumor cells (Luptrawan, Liu et al. 2008). Peripheral vaccination has been performed using purified antigen and irradiated genetically modified tumor cells. Through vaccination with a tumor antigen, one hopes to induce an immune response peripherally, which translates to CNS immunity as activated T cells cross the BBB. This goal may be achieved by processing the antigen via APCs at the subcutaneous injection site, migration to lymph nodes, and priming naïve T cells. Neverthe‐ less, choosing an appropriate antigen is crucial so as to avoid an autoimmune response causing encephalitis (Okada, Kohanbash et al. 2009).

Peptide-based vaccines (see Table 1) for glioma epitopes are synthetically derived for specific antigens and run less risk of autoimmune encephalitis. This process has the potential to be individually tailored based on assessment of the patient's peripheral blood for positive response to the various antigens (Okada, Kohanbash et al. 2009). Many antigen epitopes exist and will be briefly covered. Il-13Rα2 appears as a membrane protein in more than 80% of gliomas but not in normal brain tissue, making it a target for immunotherapy (Debinski, Gibo et al. 1999). The tyrosine kinase receptor EphA2, which is involved in cell-cell contact in normal cells, contributes to malignant nature of tumor cells (Kinch, Moore et al. 2003). T-cell epitopes of Survivin, an apoptosis inhibitor protein present in several human cancers, have shown promise via vaccination for patients with pancreatic cancer and melanoma (Otto, Andersen et al. 2005; Wobser, Keikavoussi et al. 2006). These proteins are found in 100% of astrocytomas but not in normal brain tissue (Uematsu, Ohsawa et al. 2005; Okada, Kohanbash et al. 2009). Wilm's Tumor 1 gene, a transcription factor oncogene, is also present in many tumor types, including the majority of GBM but not in normal glial cells (Sugiyama 2002). The transcrip‐ tional cofactor family SOX, Sry-Related High-Mobility Group Box, is present in normal tissue development and is upregulated in various tumors, including gliomas. Vaccinations with SOX have been shown to be therapeutic in mice with gliomas (Ueda, Kinoshita et al. 2008; Okada, Kohanbash et al. 2009). HER-2/neu, in the EGFR family, promotes tumor growth by inhibiting apoptosis and stimulating migration, adhesion, and angiogenesis in many tumor-types, most notably breast, ovarian, colorectal, pancreatic, renal-cell, and GBM (Meric-Bernstam and Hung 2006; Okada, Kohanbash et al. 2009). Additional epitopes have been identified involving EGFR variant III, found in 30-50% of GBMs, Squamous Cell Carcinoma Antigen Recognized by T Cells 1 (SART-1), a gene-coding tumor antigen in many cancer types, including glioma but not in normal tissue, and Cytomegalovirus, which infects a large number of gliomas and may contribute to glioma pathogenesis (Cobbs, Harkins et al. 2002; Saikali, Avril et al. 2007; Okada, Kohanbash et al. 2009).

In addition to using purified antigen as above, whole glioma cells may be used for vaccination (See Table 2). In this process, tumor cells, either autologous or allogeneic, are grown *in vitro*, irradiated, and injected back into the patient (Wikstrand and Bigner 1980; Zhang, Eguchi et al. 2007). The benefit of whole cell vaccinations is the availability of multiple associated antigens and, specifically, the ones expressed by the individual patient's glioma (Okada, Kohanbash et al. 2009).

than patients with intradermal administration only (Yamanaka, Homma et al. 2005; Luptra‐ wan, Liu et al. 2008). Another method, which has shown promise in animal models was used by Choi et al and involves the injection of chimeric antigen receptors-transduced T cells targeting EGFR variant III into mice gliomas. The results show a dose-dependent increase in survival, while at the same time sparing cytotoxicity to normal brain tissue (Choi, Suryadevara

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In passive immunotherapy, the patient is given *effector* cells or molecules. Such therapies include monoclonal antibodies, radio-nucleotides that are conjugated to monoclonal antibod‐

The use of monoclonal antibodies (see Table 4) for CNS targets necessitates overcoming important barriers (Okada, Kohanbash et al. 2009); for instance, the size of monoclonal antibodies, around 150kDa, impairs their diffusion into the CNS. However, evidence suggests that the BBB both in normal patients and those with malignancy tolerates the entry of mono‐ clonal antibodies (Chen and Mitchell 2012). Additionally, antibodies bound to the tumor boundary layer create a concentration gradient that makes it difficult for additional antibodies to permeate against a concentration gradient, essentially not being able to reach the core of the tumor. This option may be more valid for use in conjunction with surgical resection and convection enhanced delivery (CED) where the agent of choice is given at high pressure and in bulk through an intracranial catheter into the brain tumor and parenchyma (Okada, Kohanbash et al. 2009). As opposed to using diffusion, this method uses bulk flow and has been implemented in several clinical trials. While bypassing the BBB and limiting systemic toxicities, a limitation of this method is that it can be slow and thus difficult to deliver high volumes of molecules (Bobo, Laske et al. 1994; Ferguson and Lesniak 2007; Okada, Kohanbash

Several targets for monoclonal antibodies have been investigated in clinical trials. Epidermal growth factor receptor (EGFR) antibodies target the EGFR on glioma cells, over-expressed on 40-50% of tumors (Rivera, Vega-Villegas et al. 2008; Okada, Kohanbash et al. 2009). EGFR is a transmembrane receptor responsible for initiating gene transcription and thus increased tumor growth and spread(Baselga 2001). A variant of EGFR, EGFR variant III, is often found in GBM (Batra, Castelino-Prabhu et al. 1995). The monoclonal antibody Cetuximab inhibits this EGFR pathway, including glioma cells expression variant EGFR (Fukai, Nishio et al. 2008). Nimotu‐

Radio-immunotherapy (RIT) via radionucleotides conjugated to monoclonal antibodies is another technique for targeting specific tumor antigens (see Table 5). This technique delivers localized, cytotoxic radiation to tumor cells resulting in cell death. This method is used concurrently with surgical resection into the surgical cavity. Specifically, antitenascin has been most studied for RIT due to high prevalence of the glycoprotein tenascin on the surface of high-

zumab works similarly (Ramos, Figueredo et al. 2006).

et al. 2013).

et al. 2009).

**6.3. Passive transfer of immunity**

ies, coupled toxins, and T cells.

*6.3.1. Transfer of monoclonal antibodies*

As a means of bypassing local antigen presentation at the site of the tumor, DC vaccination has also been a source for many clinical trials with various techniques of uniting the DC with the antigen (See Table 3). Some have used DCs pulsed with autologous glioma cell peptides and have shown promise when the DC vaccines were given both into the tumor and subcu‐ taneously (Yamanaka, Homma et al. 2005; Okada, Kohanbash et al. 2009; D'Agostino, Gott‐ fried-Blackmore et al. 2012). Through loading autologous DCs, one can use either tumor lysates, apoptotic tumor cells or tumor-based cDNA (D'Agostino, Gottfried-Blackmore et al. 2012). DC-glioma cell fusion, to create a multinucleated cell such that the DC can present tumor antigen, has also shown potential (D'Agostino, Gottfried-Blackmore et al. 2012). The results of DC vaccinations are encouraging; in one study, repeat surgical resection showed infiltration into the tumor of appropriate CD8+ T cells (Luptrawan, Liu et al. 2008). Furthermore, DC vaccination was well tolerated by 12 GBM patients; the median OS was 23.4 months as compared to 18.3 months in controls. In addition to best method of preparing the vaccine, several questions remain unanswered including the best DC subtypes to use, ideal conditions and co-stimulation, prime route of administration, and the correct vaccination dosing and frequency (Okada, Kohanbash et al. 2009). Additional obstacles include the initial immune state of the host prior to vaccination; for example, patients with increased tumor burden have elevated levels of TGF- β and Il-10, which inhibit entry into a cytotoxic response (Luptrawan, Liu et al. 2008).

#### **6.2. Priming in the brain**

Fathallah-Shaykh et al. showed that priming in the brain elicits an anti-tumor response leading to destruction of the brain tumor as well as to anti-tumor systemic immunity in animals (Fathallah-Shaykh, Gao et al. 1998). The basic mechanisms for eliciting such an immune response in the CNS are detailed above. One possible method consists of injecting DCs directly into the tumor; the goal is to enhance local antigen processing followed by glymphatic drainage and priming in cervical lymph nodes (Luptrawan, Liu et al. 2008). Early preliminary results in humans are encouraging. In a study of 10 patients with glioma, half received subcutaneous vaccination of pulsed DC with autologous tumor lysate and the other half received both subcutaneous vaccine and intra-tumoral injection of immature autologous DC. On follow-up imaging, the patients who received both therapies showed diminution of contrast-enhancing tumor (Yamanaka, Yajima et al. 2003; Luptrawan, Liu et al. 2008). A phase I/II trial including 24 patients with Grade III or IV glioma at first recurrence evaluated the safety and benefits of DC immunotherapy given either via subcutaneous injection near a cervical lymph node or both subcutaneously and intra-tumorally via an Ommaya reservoir. The study revealed that patients with both intratumoral and intradermal administrations had a longer survival times than patients with intradermal administration only (Yamanaka, Homma et al. 2005; Luptra‐ wan, Liu et al. 2008). Another method, which has shown promise in animal models was used by Choi et al and involves the injection of chimeric antigen receptors-transduced T cells targeting EGFR variant III into mice gliomas. The results show a dose-dependent increase in survival, while at the same time sparing cytotoxicity to normal brain tissue (Choi, Suryadevara et al. 2013).

#### **6.3. Passive transfer of immunity**

In addition to using purified antigen as above, whole glioma cells may be used for vaccination (See Table 2). In this process, tumor cells, either autologous or allogeneic, are grown *in vitro*, irradiated, and injected back into the patient (Wikstrand and Bigner 1980; Zhang, Eguchi et al. 2007). The benefit of whole cell vaccinations is the availability of multiple associated antigens and, specifically, the ones expressed by the individual patient's glioma (Okada, Kohanbash et

As a means of bypassing local antigen presentation at the site of the tumor, DC vaccination has also been a source for many clinical trials with various techniques of uniting the DC with the antigen (See Table 3). Some have used DCs pulsed with autologous glioma cell peptides and have shown promise when the DC vaccines were given both into the tumor and subcu‐ taneously (Yamanaka, Homma et al. 2005; Okada, Kohanbash et al. 2009; D'Agostino, Gott‐ fried-Blackmore et al. 2012). Through loading autologous DCs, one can use either tumor lysates, apoptotic tumor cells or tumor-based cDNA (D'Agostino, Gottfried-Blackmore et al. 2012). DC-glioma cell fusion, to create a multinucleated cell such that the DC can present tumor antigen, has also shown potential (D'Agostino, Gottfried-Blackmore et al. 2012). The results of DC vaccinations are encouraging; in one study, repeat surgical resection showed infiltration into the tumor of appropriate CD8+ T cells (Luptrawan, Liu et al. 2008). Furthermore, DC vaccination was well tolerated by 12 GBM patients; the median OS was 23.4 months as compared to 18.3 months in controls. In addition to best method of preparing the vaccine, several questions remain unanswered including the best DC subtypes to use, ideal conditions and co-stimulation, prime route of administration, and the correct vaccination dosing and frequency (Okada, Kohanbash et al. 2009). Additional obstacles include the initial immune state of the host prior to vaccination; for example, patients with increased tumor burden have elevated levels of TGF- β and Il-10, which inhibit entry into a cytotoxic response (Luptrawan,

Fathallah-Shaykh et al. showed that priming in the brain elicits an anti-tumor response leading to destruction of the brain tumor as well as to anti-tumor systemic immunity in animals (Fathallah-Shaykh, Gao et al. 1998). The basic mechanisms for eliciting such an immune response in the CNS are detailed above. One possible method consists of injecting DCs directly into the tumor; the goal is to enhance local antigen processing followed by glymphatic drainage and priming in cervical lymph nodes (Luptrawan, Liu et al. 2008). Early preliminary results in humans are encouraging. In a study of 10 patients with glioma, half received subcutaneous vaccination of pulsed DC with autologous tumor lysate and the other half received both subcutaneous vaccine and intra-tumoral injection of immature autologous DC. On follow-up imaging, the patients who received both therapies showed diminution of contrast-enhancing tumor (Yamanaka, Yajima et al. 2003; Luptrawan, Liu et al. 2008). A phase I/II trial including 24 patients with Grade III or IV glioma at first recurrence evaluated the safety and benefits of DC immunotherapy given either via subcutaneous injection near a cervical lymph node or both subcutaneously and intra-tumorally via an Ommaya reservoir. The study revealed that patients with both intratumoral and intradermal administrations had a longer survival times

al. 2009).

194 Tumors of the Central Nervous System – Primary and Secondary

Liu et al. 2008).

**6.2. Priming in the brain**

In passive immunotherapy, the patient is given *effector* cells or molecules. Such therapies include monoclonal antibodies, radio-nucleotides that are conjugated to monoclonal antibod‐ ies, coupled toxins, and T cells.

#### *6.3.1. Transfer of monoclonal antibodies*

The use of monoclonal antibodies (see Table 4) for CNS targets necessitates overcoming important barriers (Okada, Kohanbash et al. 2009); for instance, the size of monoclonal antibodies, around 150kDa, impairs their diffusion into the CNS. However, evidence suggests that the BBB both in normal patients and those with malignancy tolerates the entry of mono‐ clonal antibodies (Chen and Mitchell 2012). Additionally, antibodies bound to the tumor boundary layer create a concentration gradient that makes it difficult for additional antibodies to permeate against a concentration gradient, essentially not being able to reach the core of the tumor. This option may be more valid for use in conjunction with surgical resection and convection enhanced delivery (CED) where the agent of choice is given at high pressure and in bulk through an intracranial catheter into the brain tumor and parenchyma (Okada, Kohanbash et al. 2009). As opposed to using diffusion, this method uses bulk flow and has been implemented in several clinical trials. While bypassing the BBB and limiting systemic toxicities, a limitation of this method is that it can be slow and thus difficult to deliver high volumes of molecules (Bobo, Laske et al. 1994; Ferguson and Lesniak 2007; Okada, Kohanbash et al. 2009).

Several targets for monoclonal antibodies have been investigated in clinical trials. Epidermal growth factor receptor (EGFR) antibodies target the EGFR on glioma cells, over-expressed on 40-50% of tumors (Rivera, Vega-Villegas et al. 2008; Okada, Kohanbash et al. 2009). EGFR is a transmembrane receptor responsible for initiating gene transcription and thus increased tumor growth and spread(Baselga 2001). A variant of EGFR, EGFR variant III, is often found in GBM (Batra, Castelino-Prabhu et al. 1995). The monoclonal antibody Cetuximab inhibits this EGFR pathway, including glioma cells expression variant EGFR (Fukai, Nishio et al. 2008). Nimotu‐ zumab works similarly (Ramos, Figueredo et al. 2006).

Radio-immunotherapy (RIT) via radionucleotides conjugated to monoclonal antibodies is another technique for targeting specific tumor antigens (see Table 5). This technique delivers localized, cytotoxic radiation to tumor cells resulting in cell death. This method is used concurrently with surgical resection into the surgical cavity. Specifically, antitenascin has been most studied for RIT due to high prevalence of the glycoprotein tenascin on the surface of highgrade gliomas, including 90% of GBMs (Zalutsky 2004). Duke University has developed the specific antibody 81C6, which has shown promise when given into the tumor cavity concur‐ rently with resection (Zalutsky, Moseley et al. 1989; Okada, Kohanbash et al. 2009). Several other clinical trials have used similar approaches with RIT and glycoprotein tenascin. Other targets include the DNA/Histone H1 complex, the extra domain B of fibronectin, and the alpha chain of the IL-2 receptor (Okada, Kohanbash et al. 2009).

**6.4. Limitations of immunotherapy**

the immune system.

Astrocytoma or Oligodendroglioma

The limitations of immunotherapy for malignant gliomas include: 1) physical obstacles of drug administration due to the BBB, 2) direct and indirect down-regulation of the immune response by gliomas, 3) the high mutation rate of the tumor, which will select for tumor cells that do not express the target of the immune response. In fact, cancer genomes are unstable as evidenced by microsatellite instability of the tumor cells, which aides in tumor evolution and progression (van de Kelft and Verlooy 1994; Yip, Miao et al. 2009; Milinkovic, Bankovic et al. 2012). Additional limitations include difficulty in monitoring the tumor response to treatment because inducing an inflammatory response may create MRI changes that mimic tumor growth. Immunotherapy is also complicated by the common use of steroids, which suppress

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197

In our opinion, the most significant limitation of immunotherapy is the limited understanding of the dynamics of the interactions of cytotoxic T lymphocytes with the tumor microenviron‐ ment. Clinical trials using immunotherapy have failed to show a clinically significant thera‐ peutic response despite demonstrating the presence of circulating tumor-specific CTL (Lasalvia-Prisco, Garcia-Giralt et al. 2008; Leffers, Lambeck et al. 2009). The key obstacle that we need to overcome is not the induction of a systemic anti-tumor immune response, but

making that immune response effective within the tumor microenvironment

• A Pilot Study of Glioma Associated Antigen Vaccines in Conjunction With Poly-ICLC in Pediatric Gliomas

• Biological Therapy Following Surgery and Radiation Therapy in Treating Patients With Primary or Recurrent

• Effects of Vaccinations With HLA-A2-Restricted Glioma Antigen-Peptides in Combination With Poly-ICLC for Adults

• GP96 Heat Shock Protein-Peptide Complex Vaccine in Treating Patients With Recurrent or Progressive Glioma • HLA-A2-Restricted Glioma Antigen-Peptides Vaccinations With Poly-ICLC for Recurrent WHO Grade II Gliomas

• Immunotherapy for Recurrent Ependymomas in Children Treatment for Recurrent Ependymomas Using HLA-A2

• A Study of Rindopepimut/GM-CSF in Patients With Relapsed EGFRvIII-Positive Glioblastoma

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

With High-Risk WHO Grade II Astrocytomas and Oligo-Astrocytomas

Restricted Tumor Antigen Peptides in Combination With Imiquimod • Peptide Vaccine for Glioblastoma Against Cytomegalovirus Antigens • Peptide-based Glioma Vaccine IMA950 in Patients With Glioblastoma

• Phase I Study of Safety and Immunogenicity of ADU-623

• HSPPC-96 Vaccine With Temozolomide in Patients With Newly Diagnosed GBM

• Phase I/II Trial of IMA950 Multi-peptide Vaccine Plus Poly-ICLC in Glioblastoma • Phase II Study of Rindopepimut (CDX-110) in Patients With Glioblastoma Multiforme

#### *6.3.2. Transfer of ligands (cytokines)*

Via coupled targeted toxins, cytokines fused with toxins can be delivered to tumor cells (see Table 6). Specifically, IL-4R and IL-13Rα2 expression is increased in high-grade gliomas making them ideal targets for chimeric fused proteins. For these chimeras, pseudomonas exotoxin is fused to IL-4 and IL-13, creating IL4-PE and IL13-PE, respectively (Debinski, Obiri et al. 1995; Joshi, Leland et al. 2001). These proteins are then delivered via CED (Okada, Kohanbash et al. 2009). By combining toxins with cytokines, one can target tumor receptors and induce cytotoxicity. Additional chimeras have been made using diphtheria toxin, which bonds to transferring, and TGFα, which binds to Pseudomonas exotoxin.

#### *6.3.3. Transfer of cells*

For the adoptive transfer of tumor-reactive autologous cytotoxic T lymphocytes (see Table 7), various techniques are used to create an antigen-specific receptor on a CD8+ T cell that can prompt T cell activation (Okada, Kohanbash et al. 2009). This process has previously been used in conjunction with IL-2 infusion for the treatment of melanoma. Antigen-specific cytotoxic T cells from peripheral blood or from tumor nodules are isolated from the patient. The T cells will then undergo clonal expansion *in vitro* with specificity for tumor antigen, possibly with the aid of IL-2. These cells are then returned to the patient where they would in theory perform cytotoxic responses upon recognition of tumor-associated antigen in the brain parenchyma (Okada, Kohanbash et al. 2009). In terms of usage in gliomas, the first steps would be creating a library of highly avid cytotoxic T cell clones from which to build highly selective TCR gene pairs to create transgenic cytotoxic T cells. Adoptive therapy is not limited to CD8+ T cells and has been also tried using NK cells and CD4+ T cells, both of which can be similarly removed, expanded, and injected back into the patient. Blancher et al treated 13 GBM by recombinant IL-2, with and without lymphokine activated killer cells, given directly via a catheter into the tumor resection bed. Unfortunately, the treatment had no effects on tumor progression. The adverse reactions included cerebral edema, confusion, and fever (Blancher, Roubinet et al. 1993).

Some obstacles with the adoptive process include creating T cells with TCR of appropriate avidity (Okada, Kohanbash et al. 2009). Further difficulties arise with T cell reproduction; many of these specialized T cell populations are thought to be terminally differentiated and thus unable to propagate long-term existence (Wherry, Teichgraber et al. 2003). Additionally, these transgenic T cells must also overcome the immunosuppressive features of GBM and, in fact, do so better than natural T cells due to the ability to manipulate them and strengthen them with specific chemokines and integrin receptors (Okada, Kohanbash et al. 2009).

#### **6.4. Limitations of immunotherapy**

grade gliomas, including 90% of GBMs (Zalutsky 2004). Duke University has developed the specific antibody 81C6, which has shown promise when given into the tumor cavity concur‐ rently with resection (Zalutsky, Moseley et al. 1989; Okada, Kohanbash et al. 2009). Several other clinical trials have used similar approaches with RIT and glycoprotein tenascin. Other targets include the DNA/Histone H1 complex, the extra domain B of fibronectin, and the alpha

Via coupled targeted toxins, cytokines fused with toxins can be delivered to tumor cells (see Table 6). Specifically, IL-4R and IL-13Rα2 expression is increased in high-grade gliomas making them ideal targets for chimeric fused proteins. For these chimeras, pseudomonas exotoxin is fused to IL-4 and IL-13, creating IL4-PE and IL13-PE, respectively (Debinski, Obiri et al. 1995; Joshi, Leland et al. 2001). These proteins are then delivered via CED (Okada, Kohanbash et al. 2009). By combining toxins with cytokines, one can target tumor receptors and induce cytotoxicity. Additional chimeras have been made using diphtheria toxin, which

For the adoptive transfer of tumor-reactive autologous cytotoxic T lymphocytes (see Table 7), various techniques are used to create an antigen-specific receptor on a CD8+ T cell that can prompt T cell activation (Okada, Kohanbash et al. 2009). This process has previously been used in conjunction with IL-2 infusion for the treatment of melanoma. Antigen-specific cytotoxic T cells from peripheral blood or from tumor nodules are isolated from the patient. The T cells will then undergo clonal expansion *in vitro* with specificity for tumor antigen, possibly with the aid of IL-2. These cells are then returned to the patient where they would in theory perform cytotoxic responses upon recognition of tumor-associated antigen in the brain parenchyma (Okada, Kohanbash et al. 2009). In terms of usage in gliomas, the first steps would be creating a library of highly avid cytotoxic T cell clones from which to build highly selective TCR gene pairs to create transgenic cytotoxic T cells. Adoptive therapy is not limited to CD8+ T cells and has been also tried using NK cells and CD4+ T cells, both of which can be similarly removed, expanded, and injected back into the patient. Blancher et al treated 13 GBM by recombinant IL-2, with and without lymphokine activated killer cells, given directly via a catheter into the tumor resection bed. Unfortunately, the treatment had no effects on tumor progression. The adverse reactions included cerebral edema, confusion, and fever (Blancher, Roubinet et al.

Some obstacles with the adoptive process include creating T cells with TCR of appropriate avidity (Okada, Kohanbash et al. 2009). Further difficulties arise with T cell reproduction; many of these specialized T cell populations are thought to be terminally differentiated and thus unable to propagate long-term existence (Wherry, Teichgraber et al. 2003). Additionally, these transgenic T cells must also overcome the immunosuppressive features of GBM and, in fact, do so better than natural T cells due to the ability to manipulate them and strengthen them

with specific chemokines and integrin receptors (Okada, Kohanbash et al. 2009).

bonds to transferring, and TGFα, which binds to Pseudomonas exotoxin.

chain of the IL-2 receptor (Okada, Kohanbash et al. 2009).

196 Tumors of the Central Nervous System – Primary and Secondary

*6.3.2. Transfer of ligands (cytokines)*

*6.3.3. Transfer of cells*

1993).

The limitations of immunotherapy for malignant gliomas include: 1) physical obstacles of drug administration due to the BBB, 2) direct and indirect down-regulation of the immune response by gliomas, 3) the high mutation rate of the tumor, which will select for tumor cells that do not express the target of the immune response. In fact, cancer genomes are unstable as evidenced by microsatellite instability of the tumor cells, which aides in tumor evolution and progression (van de Kelft and Verlooy 1994; Yip, Miao et al. 2009; Milinkovic, Bankovic et al. 2012). Additional limitations include difficulty in monitoring the tumor response to treatment because inducing an inflammatory response may create MRI changes that mimic tumor growth. Immunotherapy is also complicated by the common use of steroids, which suppress the immune system.

In our opinion, the most significant limitation of immunotherapy is the limited understanding of the dynamics of the interactions of cytotoxic T lymphocytes with the tumor microenviron‐ ment. Clinical trials using immunotherapy have failed to show a clinically significant thera‐ peutic response despite demonstrating the presence of circulating tumor-specific CTL (Lasalvia-Prisco, Garcia-Giralt et al. 2008; Leffers, Lambeck et al. 2009). The key obstacle that we need to overcome is not the induction of a systemic anti-tumor immune response, but making that immune response effective within the tumor microenvironment
