*2.1.2 Multipeptide vaccines*

To identify multiple tumor-associated peptides for immunotherapy, a study set out to assess the potential of using HLA-associated tumor peptidomes as a source of tumor-associated antigens to be used in immunotherapy [48]. The components found gave rise to the multipeptide vaccine IMA950. A phase I/II set out to assess IMA950 and its 11 tumor-associated peptides which include brevican (BCAN); chondroitin sulfate proteoglycan 4 (CSPG4); fatty acid-binding protein 7, brain (FABP7); insulin-like growth factor 2 messenger mRNA-binding protein 3 (IGF2BP3), neuroligin 4, X-linked (NLGN4X); neuronal cell adhesion molecule (NRCAM), protein tyrosine phosphatase, receptor-type, z polypeptide 1 (PTPRZ1); tenascin C (TNC); Met proto-oncogene (MET); baculoviral IAP repeat-containing 5 (BIRC5); and hepatitis B virus core antigen [48]. In this study, IMA950 was adjuvanted with poly-ICLC (polyinosinic-polycytidylic acid stabilized with polylysine and carboxymethylcellulose) [49]. The multi-peptide vaccine was used in 19 patients, 16 with GBM and 3 with grade III astrocytoma. Results showed a median overall survival of 19 and 17 months for the whole cohort and GBM patients-only, respectively, with a PFS of 68% at 6 months for the whole cohort and 69% for GBM patients only when calculated from the study entry [50]. There was no mention of a historical control group used as a comparator in this study. Due to the findings in this study, a follow-up trial is actively recruiting patients with recurrent GBM to test IMA950/poly-ICLC alone or in combination with pembrolizumab, a checkpoint inhibitor that will be discussed later [51].

Another multi-peptide vaccine was generated based on observations of three tumor-associated antigens that were observed to be highly expressed in pediatric gliomas. This vaccine targets the peptide epitopes of EPH receptor A2 (EphA2; a tyrosine kinase), interleukin-13 receptor alpha 2 (IL-13Rα2), and survivin. This study was conducted in 26 pediatric patients with diffuse brainstem gliomas (BSG) or high-grade gliomas (HGG) [52]. Results showed a median survival of 13.3 months from diagnosis in the overall cohort with a median survival of 12.7 months in the BSG group and a median survival of 25.1 months in the HGG group. Though no historical control group was discussed in this phase I study, the authors mentioned that for children with BSGs, current therapies at the time failed to increase median overall survival beyond 9–12 months [53].

Though these studies are showing promising results, the lack of clear indication of efficacy and eventual tumor progression in these phase I-III trials may be attributed to the multiple obstacles in place by brain cancers including the high degree of heterogeneity of antigenic expression, an outgrowth of subclones not expressing the antigens, lack of major histocompatibility complex molecules and/or an immunosuppressive tumor microenvironment.

#### *2.1.3 Dendritic cell (DC) vaccines*

The aforementioned peptide cancer vaccines require uptake and activation of endogenous antigen-presenting cells (APCs) such as DCs. These DCs then present antigens to tumor-specific T cells leading to T cell activation. To circumvent the reliance of endogenous DC antigen loading and activation, some studies utilize DC vaccines and load DCs *ex vivo* with a variety of tumor antigens including autologous tumor lysates, tumor-associated peptides, and tumor-associated viral antigens. DC vaccines have a variety of advantageous characteristics making them an ideal choice for antitumor vaccines. They are considered to be the professional APC and most effective in sensitizing naïve T cells to specific antigens. They also are able to cross-prime, allowing them to present exogenous antigens for presentation on major histocompatibility complex (MHC) class I molecules, activating cytotoxic T lymphocytes.

A phase I trial of the DC vaccine DCVax-L was completed which loads autologous DCs with tumor lysate from newly diagnosed or recurrent GBM participants [54]. In this trial that enrolled 23 patients, the 1-year survival rate was 91% with a median OS of 31.4 months from the time of initial surgical diagnosis. The authors compared this median OS to the median OS of 18.6 months found in a large study of GBM patients who underwent tumor resection and chemoradiotherapy [55]. However, the study noted that it was unclear whether the extended survival of participants is a direct result of the vaccine effects or good responses to follow-up therapies after failing the vaccine [56]. DCVax-L has since gone on to a large phase III clinical trial with 331 participants with the primary endpoint of PFS and the secondary endpoint of OS [57]. Preliminary results of the study reveal a median OS of 23.1 months from surgery in the overall intention-to-treat population (ITT) and 34.7 months from surgery in patients with a methylated O6 -methylguanine-DNAmethyltransferase (MGMT) gene promoter. The authors compared the median OS in the ITT population to a median OS of 15–17 months from surgical intervention typically achieved with a standard of care in past studies. The PFS was not evaluated in this interim analysis. In this blinded interim survival analysis, the authors found that patients were living longer than expected and that this warrants further follow-up and analyses [58].

ICT-107 is another DC vaccine loaded with synthetic tumor-associated peptides of antigens commonly overexpressed in CD133-positive cancer stem cells that

#### *Immunotherapy against Gliomas DOI: http://dx.doi.org/10.5772/intechopen.101386*

includes Erbb2 (HER2), second tyrosinase-related protein (TRP-2), glycoprotein 100 (gp100), melanoma-associated antigen 1 (MAGE-1), IL-13Rα2, and absent in melanoma 2 (AIM-2). In a phase I trial of 21 participants who were HLA-A1 or HLA-A2-positive and with newly diagnosed GBM (n = 17), recurrent GBM (n = 3), or with a brain stem glioma (n = 1), the median PFS was 16.9 months with a median OS of 38.4 months. These results suggest a correlation with prolonged OS and PFS though no comparator group or historic controls were mentioned [59]. The same group then conducted a phase II randomized, double-blind, placebo-controlled study using ICT-107 in 124 participants with newly diagnosed GBM following resection and radiotherapy with concomitant temozolomide [60]. The primary endpoint of median OS was not increased but a significant increase in the PFS by 2.2 months was observed in the intent-to-treat population treated with ICT-07 (11.2 months versus 9 months) [61]. A phase III trial was halted due to insufficient financial resources [62].

Another pair of studies made use of the immunodominant cytomegalovirus (CMV) antigen phosphoprotein 65 (pp65) in their DC vaccines. This antigen is expressed in GBM but not in normal brains [63]. The first was a randomized blinded phase I clinical trial in 12 patients with newly diagnosed GBM who received pre-conditioning in the form of tetanus/diptheria toxoid (a potent recall antigen) or unpulsed mature DCs before bilateral vaccinations with DCs pulsed with CMV pp65 RNA [64]. Td pre-conditioning led to a significant increase in both median PFS and median OS compared to the DC alone cohort which had a median PFS and OS of 10.8 and 18.5 months (consistent with patients treated with standard of care) [65]. A later study from the same group evaluated DCs pulsed with CMV pp65 RNA along with dose-intensified temozolomide (TMZ) and adjuvant GM-CSF [64]. Here they observed a median PFS of 25.3 months and a median OS of 41.1 months in the treatment group compared to 8.0 months and 19.2 months in historical controls, respectively [66]. A phase II randomized, blinded, placebo-controlled trial of DCs pulsed with CMV pp65 and Td is underway with a target of 120 patients [67]. Another phase II trial utilizing DCs pulsed with CMV pp65 was recently completed with results pending which is assessing whether basiliximab, a monoclonal anti-CD25 antibody, may inhibit the functional and quantitative recovery of T-regulatory cells after TMZ-induced lymphopenia in newly diagnosed GBM [68].

The potential for DC vaccines is vast in their ability to generate antitumor immunity however, to date, they have provided suboptimal and overall unsatisfactory clinical benefits in large trials. Work now includes methods to improve *in vitro* APC generation [69, 70], improve DC vaccine activity with additional treatments [65], and increase inflammation at the vaccine site [56, 66, 71]. It is now thought that the next major advances in DC vaccines will come from their combination with other immunotherapies such as checkpoint inhibitors [20].

## **2.2 Immune checkpoint inhibitors**

The principal breakthrough in cancer treatment over the last 15 years is the introduction of immune checkpoint inhibitors (ICIs) blocking the immune checkpoints programmed death 1 (PD-1), programmed death-ligand 1 (PD-L1), and cytotoxic lymphocyte antigen 4 (CTLA-4). Immune checkpoints are negative regulators of T-cell immune function and are central for the modulation of physiological immune responses and the maintenance of self-tolerance. T cells are created in the thymus where they undergo positive and negative selection and undergo apoptosis if they fail to recognize self-MHC or bind too strongly to MHC with self-peptides. This process is called central tolerance [72]. T cells that appropriately respond to MHC molecules are then sent into the circulation where they eventually interact

with APCs displaying mutated self-proteins (in cancers) or foreign antigens (in infection) [73]. However, central tolerance is sometimes incomplete and some T cells escape and become autoreactive. To prevent autoreactivity, there are multiple inhibitory checkpoint pathways that regulate the activation of T cells at multiple levels during an immune process called peripheral tolerance [74].

Central to cancer immunotherapy is that tumor cells can take advantage of peripheral tolerance and hijack these checkpoint mechanisms, inhibiting T cells from attacking. The arrival of checkpoint inhibitors in 2011 introduced a new mechanism to treat cancer and revolutionized cancer management in a variety of solid tumors [75–78]. There are now several FDA-approved monoclonal antibodies against solid tumors including ipilimumab targeting CTLA-4, pembrolizumab, and nivolumab targeting PD-1, and atezolizumab and durvalumab targeting PD-L1. However, despite numerous articles describing preclinical efficacy of checkpoints in central nervous system (CNS) tumors, activity against brain metastases from melanoma and non-small-cell lung cancer [79, 80], and multiple studies describing increased PD-L1 expression in GBM [81, 82], no FDA approval has occurred for immune checkpoints in GBM. Here, we will discuss some of the phase III trials that have occurred with immune checkpoint inhibitors, what has been learned, and where the research is going.

#### *2.2.1 Phase III trials*

One randomized phase III study assessed the effect of nivolumab versus bevacizumab (anti-vascular endothelial growth factor A) in 439 patients with recurrent glioblastoma [83]. The study found no statistical difference between the median OS of nivolumab monotherapy (9.8 months) and bevacizumab (10.0 months) [84]. Interestingly, this study observed that corticosteroid use at baseline seemed to be associated with worse outcomes in the nivolumab group. This may be due to the direct effects of corticosteroids on T cell function which may abrogate activation or priming of the immune system.

Additionally, a phase III study compared nivolumab versus temozolomide in newly diagnosed patients with unmethylated MGMT GBM [85]. In 2019, Bristol-Myers Squibb announced that the study did not meet its primary endpoint, which assessed overall survival [86].

Another randomized phase III single-blind study set out to compare TMZ plus radiation therapy combined with nivolumab or placebo in newly diagnosed patients with MGMT-methylated glioblastoma [87]. In 2019, Bristol-Myers Squibb provided an update that the nivolumab group did not meet one of its primary endpoints, progression-free survival, but that the data monitoring committee recommended continuing the trial to allow the other primary endpoint, overall survival, to mature [88]. The final results are pending.

It remains to be seen whether the lack of demonstrated efficacy of checkpoint therapeutic efficacy is due to difficulty getting to the tumor site or the tumor itself. Though it has been shown that T cells can traffick to the CNS, the relatively immune-privileged CNS may prove to be a limitation if checkpoint inhibition must enter into these tumors to be effective [20]. However, at least one study demonstrated clinically meaningful intracranial efficacy with ipilimumab combined with nivolumab in patients with melanoma with untreated brain metastasis, suggesting that immune checkpoint strategies can target tumors located intracranially [80]. Lack of effective checkpoint strategies in primary CNS tumors could be due to a variety of challenges that interplay with one another. First, glioblastomas generally are considered cold tumors, lacking intratumoral inflammatory cells though this is also considered to be heterogenous. Lack of efficacy could also be due to

#### *Immunotherapy against Gliomas DOI: http://dx.doi.org/10.5772/intechopen.101386*

the relatively low mutational burden since it has been consistently shown that malignancies with a high burden of clonal neoantigens have a higher response rate to checkpoint inhibition [89]. Also, the high degree of heterogeneity found within gliomas, makes specific immunological targeting difficult. Lastly, the observed systemic T cell dysfunction and sequestration imposed by an intracranial tumor remain another domineering challenge as this singly does away with the requirement of a viable T cell compartment for immune checkpoints to act on [90].

Though multiple challenges must be overcome for immune checkpoint inhibitors to overcome glioblastoma specifically, a better understanding of treatment resistance in addition to many promising synergistic combinatorial approaches will provide important incremental advances to efficacy. Finally, as seen in other solid tumors, resistance to immune checkpoint blockade leads to upregulation of a host of alternative inhibitory immune checkpoint molecules that are currently also being targeted in ongoing clinical trials. These new inhibitory immune checkpoint targets potentially offer increased therapeutic targets to be used as single agents or in combination with other immunotherapies [91].

#### **2.3 Adoptive cellular therapy (ACT) immunotherapy**

Immunotherapy can be considered active or passive. The difference between each centers on how they modulate the immune system. Active immunotherapy, such as the aforementioned vaccines, relies on the process of endogenous immune cells activation, producing a durable response and generation of immunological memory. Passive immunotherapy, however, produces an immediate response due to the administration of cytokines, antibodies, or immune cells. A form of passive immunotherapy is adoptive cellular therapy (ACT) which specifically allows for the *ex vivo* generation and expansion of autologous immune cells that can then be given back to patients. This section will first discuss the non-specific adoptive cellular therapies such as lymphokine-activated killer (LAK) cells and natural killer (NK) cells followed by adoptive T cell therapies.

#### *2.3.1 Lymphokine-activated killer (LAK) cells*

LAK cells were thought to be a promising candidate for adoptive cellular therapies due to their ease of generation (culturing peripheral blood lymphocytes in the presence of IL-2), rapid expansion, the long shelf life *in vitro*, and tumor lysing capabilities [92]. These characteristics and favorable results in other cancers led to a phase I/II clinical trial in adult patients with recurrent or progressive supratentorial malignant glioma who were candidates for reoperative surgery. In this study, 19 eligible patients underwent craniotomy with debulking and placement of LAK cells and IL-2 in a reservoir inserted in the tumor resection cavity. Compared to an institutional historical control group of GBM after reoperation with a median OS of 28 weeks, LAK-treated patients had a median OS of 53 weeks. After treatment, the 1-year survival was 53% compared to less than 6% in a control contemporary chemotherapy group after reoperation suggesting improved long-term survival [93].

Another phase I/II trial was initiated in 40 patients with GBM who had autologous LAK cells placed in the tumor cavity. Findings from this study showed a median survival from the original diagnosis of 17.5 months compared to 13.6 months in a contemporary age-matched group [94]. The same group conducted a phase II trial with LAK cell treatment in 33 GBM patients who had not experienced clinical or radiographic evidence of progressive disease during or shortly after completion of initial therapy which showed a median survival from diagnosis of 20.5 months with a 1-year survival of 75%. The authors stated that 20.5 months

median survival is 88% longer than the 12-month survival associated with GBM and 33% longer than the 15-month median survival observed in the clinical trials that established the benefit of temozolomide therapy [95].

Overall, the use of LAK has since fallen out of favor [20, 96]. In phase III randomized trial of IL-2 with or without LAK in the treatment of patients with advanced renal cell carcinoma (RCC), the addition of LAK did not improve the response rate against RCC [97]. It is thought the efficacy of LAK cell ACT is due to the amplification of a subset of therapeutic cells found in the peripheral blood that are reactive against tumors [96]. Thus, the use of tumor-infiltrating lymphocytes (TILs; discussed later), which are more specific to the target tumor, might have better potential.

#### *2.3.2 Natural killer (NK) cells*

The NK cell ACT field is rapidly expanding in both biological understanding of NK cells, including their distinct immune checkpoints [98, 99] in addition to clinical development of NK cell ACT. These cytotoxic cells are part of the innate immune system and have many advantageous characteristics which include rapid *ex vivo* activation and expansion without the use of autologous tumor cells and are not MHC restricted [100]. It is recognized that NK cells target other cells types based on a lack of MHC-I expression [101]. Glioblastoma is known to employ immune evasion tactics including downregulation of MHC-I [102–104] which may make it amenable to ACTs using NK cells.

An early preliminary trial was conducted in nine patients with recurrent malignant gliomas using autologous NK cells injected into the tumor cavity, using a reservoir system, and intravenously. This study found that NK cell therapy was safe with some clinical benefit demonstrating three patients with partial response (50% decrease in tumor volume), two with a minor response (25% decrease in tumor volume), seven with progressive disease (increase of 25% in tumor volume), and four with no change [100].

Currently, there is at least three phase I trials in the process utilizing NK cells in high-grade gliomas [105–107].

#### *2.3.3 Tumor-infiltrating lymphocytes (TILs)*

As mentioned before, ACT allows for *ex vivo* generation and expansion. During expansion, several modifications and enhancements can occur to confer advantageous characteristics in antitumor activity. T cells can be positively selected based on specificity to tumor antigens and increased effector function. Or, they can also be transduced to express specific tumor-associated T cell receptors (TCRs) that, though MHC-restricted and MHC-dependent, can target intracellular antigens. Alternatively, T cells can be modified to express chimeric antigen receptors (CARs) for specific tumor cell surface proteins.

As the name implies, tumor-infiltrating lymphocytes are thought to have undergone *in vivo* recognition of their cognate antigen and migration into the tumor. Thus, the administration of autologous TILs have produced durable objective responses in patients with advanced melanoma [108]. However, TILs are less feasible in GBM owing to the difficulty in isolating and expanding them [109] and T cell exhaustion while within the tumor microenvironment [110]. A more feasible approach is the aforementioned targeting of the ubiquitously expressed human CMV antigen pp65 in GBM tissue [111]. This approach was conducted in an early phase clinical trial and was able to successfully expand CMV-specific T cells from 13 out of 19 patients of which 11 received all four T-cell infusions and found that

#### *Immunotherapy against Gliomas DOI: http://dx.doi.org/10.5772/intechopen.101386*

the median overall survival of these patients since the first recurrence was 403 days. The overall median OS in this study was >57 weeks (a range of 19–345 weeks) and a median PFS of >35 weeks (a range between 15.4–254 weeks). No comparator group or historic controls were mentioned in this early phase trial. Interestingly, molecular profiling of CMV-specific T cells from the patients revealed distinct gene expression signatures which correlated with their clinical response [111]. Another phase I randomized study was initiated in 22 CMV-seropositive, newly diagnosed GBM patients. This study assessed CMV pp65-specific T cells that were generated *ex vivo* with autologous CMV pp65 RNA-transfected DCs with or without a CMV-DC vaccination [112]. Though this study was not powered to detect differences between cohorts with regard to PFS and OS, the study found an association between higher IFNγ<sup>+</sup> , TNFα<sup>+</sup> , and CCL3<sup>+</sup> polyfunctional, CMV-specific CD8<sup>+</sup> T cells and OS [113].
