Against the Resilience of High-Grade Gliomas: The Immunotherapeutic Approach (Part I)

The resilience of high-grade gliomas (HGGs) against conventional chemotherapies is due to their heterogeneous genetic landscape, adaptive phenotypic changes, and immune escape mechanisms. Innovative immunotherapies have been developed to counteract the immunosuppressive capability of gliomas. Nevertheless, further research is needed to assess the efficacy of the immuno-based approach. The aim of this study is to review the newest immunotherapeutic approaches for glioma, focusing on the drug types, mechanisms of action, clinical pieces of evidence, and future challenges. A PRISMA (Preferred Reporting Items for Systematic Review and Meta-Analysis)-based literature search was performed on PubMed/Medline and ClinicalTrials.gov databases using the keywords “active/adoptive immunotherapy,” “monoclonal antibodies,” “vaccine,” and “engineered T cell.”, combined with “malignant brain tumor”, “high-grade glioma.” Only articles written in English published in the last 10 years were selected, filtered based on best relevance. Active immunotherapies include systemic temozolomide, monoclonal antibodies, and vaccines. In several preclinical and clinical trials, adoptive immunotherapies, including T, natural killer, and natural killer T engineered cells, have been shown to be potential treatment options for relapsing gliomas. Systemic temozolomide is considered the backbone for newly diagnosed HGGs. Bevacizumab and rindopepimut are promising second-line treatments. Adoptive immunotherapies have been proven for relapsing tumors, but further evidence is needed.


Results
The literature search returned a total of 216 articles and 75 clinical trials. After the removal of duplicates and implementation of the exclusion criteria, a total of 135 articles and 69 clinical trials were assessed for eligibility and included in the review. Figure 1 presents the PRISMA flow chart for the literature selection process (Figure 1) and Table 2 summarizes the main clinical trials on immunotherapies for HGGs (Table 2).

Classification of Immunotherapies
Immunotherapies for brain gliomas can be classified based on the molecular mechanism and type of drug involved [27][28][29][30][31]. Two potential strategies were outlined: active immunotherapy, aimed at directly inducing the host antitumoral immune response, and adoptive (or passive) immunotherapy, resulting in the transduction of autologous/allogeneic immune cells aimed at the introduction or restoration of immune functions [29,32,33]. Alkylating agents, monoclonal antibodies, and vaccines are used for active immunotherapy, whereas engineered T cells, natural killer (NK) cells, and natural killer T (NKT) cells are used for adoptive immune strengthening. Table 3 reports the classification of novel immunotherapies for HGGs.

Classification of Immunotherapies
Immunotherapies for brain gliomas can be classified based on the molecular mechanism and type of drug involved [27][28][29][30][31]. Two potential strategies were outlined: active immunotherapy, aimed at directly inducing the host antitumoral immune response, and adoptive (or passive) immunotherapy, resulting in the transduction of autologous/allogeneic immune cells aimed at the introduction or restoration of immune functions [29,32,33]. Alkylating agents, monoclonal antibodies, and vaccines are used for active immunotherapy, whereas engineered T cells, natural killer (NK) cells, and natural killer T (NKT) cells are used for adoptive immune strengthening. Table 3 reports the classification of novel immunotherapies for HGGs.   [6,7]. Alkylating agents are basically chemotherapy drugs. They target tumor cells and block the cell cycle by damaging DNA double strands [34,35]. In prodrug status, TMZ crosses the blood-brain barrier (BBB) owing to its lipophilic properties and it reaches the tumor cells at therapeutic-relevant concentrations [36][37][38]. It is spontaneously converted into the active compound 5-(3-methyltriazen-1-yl)-imidazole-4-carboxamide (MTIC), which can methylate DNA bases. MTIC transfers alkyl groups to guanine or adenine at the N7-/O6-or N3-position, respectively. The methylation results in base pair mismatch and chromatin structure remodeling, induces G2/M cell cycle arrest, and finally leads to cell apoptosis [37,[39][40][41]. As a result, TMZ therapeutic success is closely dependent on DNA repair pathways, which leads to glioma therapeutic resistance [42,43]. The demethylating enzyme MGMT acts directly in repairing O6-methylguanine, consequently avoiding gene mutation and glioma cell death [44,45]. The methylation status of the MGMT promoter and the enzyme expression predict the response to TMZ and are also prognostic factors of survival [46][47][48][49]. Figure 2 describes the TMZ mechanism of action and its effects in the glioma cells (Figure 2).

Monoclonal Antibodies
Monoclonal antibodies (MAbs) are purified immunoglobulin with monovalent affinity. MAbs bind specific molecular epitopes and enlist the host immune system against tumors. Bevacizumab (BVZ) is the most widely tested, especially for the treatment of recurrent glioblastoma [50][51][52][53][54][55][56][57]. It directly targets the vascular endothelial growth factor A (VEGF-A) inhibiting the interaction with VEGF tyrosine kinase receptor, which is overexpressed on the surface of endothelial cells as a result of tissue hypoxia, and interrupts aberrant tumor angiogenesis ( Figure 3A). The antiangiogenic effect exerted by BVZ extends to the reduction of microvessel density and vascular permeability, resulting in the antiedema effect [51,58,59]. Furthermore, recent studies have investigated the potential role of BVZ in modulating the immunosuppressive tumor microenvironment [60][61][62]. Two phase III clinical trials, the Avastin in Glioblastoma (AVAglio92) [63] (#NCT00943826) and the Radiation Therapy Oncology Group (RTOG)-082593 [64] (#NCT00884741), studied the combination of BVZ with standard therapy, demonstrating promising results for recurrent HGGs.

Monoclonal Antibodies
Monoclonal antibodies (MAbs) are purified immunoglobulin with monovalent affinity. MAbs bind specific molecular epitopes and enlist the host immune system against tumors. Bevacizumab (BVZ) is the most widely tested, especially for the treatment of recurrent glioblastoma [50][51][52][53][54][55][56][57]. It directly targets the vascular endothelial growth factor A (VEGF-A) inhibiting the interaction with VEGF tyrosine kinase receptor, which is overexpressed on the surface of endothelial cells as a result of tissue hypoxia, and interrupts aberrant tumor angiogenesis ( Figure 3A). The antiangiogenic effect exerted by BVZ extends to the reduction of microvessel density and vascular permeability, resulting in the antiedema effect [51,58,59]. Furthermore, recent studies have investigated the potential role of BVZ in modulating the immunosuppressive tumor microenvironment [60][61][62]. Two phase III clinical trials, the Avastin in Glioblastoma (AVAglio92) [63] (#NCT00943826) and the Radiation Therapy Oncology Group (RTOG)-082593 [64] (#NCT00884741), studied the combination of BVZ with standard therapy, demonstrating promising results for recurrent HGGs. The AVAglio92 documented a significant improvement in the median progression free survival (PFS) to 10.6 months in the BVZ group, versus 6.2 months in the placebo one (p < 0.001). Overall survival (OS) proved to be higher only during the first two years of treatment in the BVZ group (72.4% versus 66.3%) [63]. The RTOG-082593 reported an increase in PFS in the BVZ group compared to the control group (10.7 months versus 7.3 months, p = 0.007), but equally failed to demonstrate a better OS [64].
Nivolumab, a human IgG4, directly binds programmed cell death protein 1 (PD-1), exposed on activated T cells. Physiologically, PD-1 interacts with its ligands, namely PD-L1 and PD-L2, downregulating the immune cascade. PD-Ls are overexpressed on HGG The AVAglio92 documented a significant improvement in the median progression free survival (PFS) to 10.6 months in the BVZ group, versus 6.2 months in the placebo one (p < 0.001). Overall survival (OS) proved to be higher only during the first two years of treatment in the BVZ group (72.4% versus 66.3%) [63]. The RTOG-082593 reported an increase in PFS in the BVZ group compared to the control group (10.7 months versus 7.3 months, p = 0.007), but equally failed to demonstrate a better OS [64].

Vaccine
Anticancer vaccination was designed to strengthen host immune response by stimulating the production of self-antibodies against tumor antigens. Advanced research in translational medicine has allowed the isolation of specific glioma immunogens, i.e., epidermal growth factor receptor variant III (EGFRvIII). EGFRvIII is an active splice variant, found in 30% of primary glioblastoma (GBM), which enhances cell proliferation and survival [75][76][77][78][79][80]. Rindopepimut (Rintega ® , Celldex Therapeutics, Inc., Phillipsburg, NJ, USA), a 14-mer injectable peptide vaccine against EGFRvIII, was projected to activate CD4+ and CD8+ T cells against malignant brain tumor cells (Figure 4). It was also evaluated in some preclinical studies. The first phase I trial, VICTORI, showed an excellent safety profile, a PFS of 10.2 months and an OS of 22.8 months. Some phase II trials (ACTIVATE, ACT II-III, and ReACT) confirmed the low toxicity and demonstrated an increase in median progression-free and overall survival for recurrent gliomas. The ACTIVATE phase II trial reported a PFS of 14.2 and 6.4 months, and an OS of 26 and 15.2 months in the vaccinated patients and control group, respectively. In ACT II and III studies, the PFS was 15.2 and 12.3 for the rindopepimut group, respectively, while the PFS was 6.4 months in both control groups. The OS was 23.6 and 24.6 months in ACT II and III trials, respectively, compared to the control groups (15.2 months). ReACT showed a PFS at 6 months of 28% for rindopepimut, compared to 16% in the control group [79,[81][82][83][84].
ACT IV, a multicentric phase III trial, tested the combination of rindopepimut and standard chemotherapy with TMZ in newly diagnosed GBM, showing no significant difference in OS between the two groups (#NCT01480479) [85].
The TCR complex is exposed on the surface of T cells and interacts with the major histocompatibility complex (MHC), leading to activation of the cellular immune cascade. TCR transgenic T cells are genetically engineered to express receptors against specific tumor MHC and stimulate the immune response against malignant brain tumors [108][109][110][111]. CAR and TCR engineered T cells are expanded ex vivo and injected, cross the BBB, bind the tumor cells, and activate the antitumoral immune cascade, thus promoting apoptosis ( Figure 5) [94,[112][113][114][115]. The TCR complex is exposed on the surface of T cells and interacts with the major histocompatibility complex (MHC), leading to activation of the cellular immune cascade. TCR transgenic T cells are genetically engineered to express receptors against specific tumor MHC and stimulate the immune response against malignant brain tumors [108][109][110][111]. CAR and TCR engineered T cells are expanded ex vivo and injected, cross the BBB, bind the tumor cells, and activate the antitumoral immune cascade, thus promoting apoptosis ( Figure 5) [94,[112][113][114][115].

NK Cells
NK cells are involved in the treatment of malignant brain tumors by means of different strategies [116]. The first strand can exploit allogeneic NK cells and target tumor cells by the lack of MHC and, without being inactivated, exert oncolytic effects [117][118][119][120]. Another approach includes the NK immunoglobulin-like receptor antibodies (anti-KIR Abs). They block the link between NK cells and MHC class I, avoid the immunosuppression response, and increase the NK-mediated tumor apoptosis [116,121]. Furthermore, the antibodydependent cell-mediated cytotoxicity (ADCC) is a mechanism wherein specific antibodies, directed to HGG antigens, interact with NK cells, promoting tumor lyses. The Fab portion binds antigens on the tumor cell surface, whereas the fragment crystallizable (Fc) region recognizes the NK cell receptors, such as CD16/FcγRIII, mediating the antitumoral immune response. Among ADCC, EGFR tumor antigen and CD16 (FcγIIIA), KIR2DS2, and NK Group 2D (NKG2D) receptors are the most researched [121].
In 2017, Yvon and colleagues proved the efficacy of cord blood NK cells that were retrovirally transduced and manipulated to express the TGF-β-dominant-negative receptor II (DNRII) [121,122]. DNRII makes NK cells immune to the TGF-β that is overexpressed in the tumor microenvironment as an immune evasion promoter. Other techniques under development involve the exosomes as a vector for NK cells and a new sort of CAR (CAR-KHYG-1) targeting EGFRvIII [123][124][125].

NKT Cells and Hybrid Therapies
NKT cells are a subgroup of T cells with concomitant properties of T and NK cells, both expressing the type of molecular surfaces. Particularly, CD1d-restricted NKT cells were found to play an important role as immunoregulatory cells within the glioma microenvironment [126]. Strategies to overcome the immune tolerance of glioma include the expansion in vitro of autologous mature dendritic cells (DCs) with NKT ligand α-galactosyl ceramide, which enhances NKT cell cytotoxic activity [127,128]. Another integrated approach is the autologous lymphoid effector cells specific against tumor cell (ALECSAT) therapy (Cytovac A/S, Hørsholm, Denmark). ALECSAT is a 26-day immunization protocol. Autologous lymphocytes and monocytes were isolated and differentiated in vitro. The mature DCs and nonactivated lymphocytes are cultured with CD4+ T cells and 5-aza-2 -deoxycytidine, a DNA-demethylation agent, which induces the expression of tumor antigens. The activated CD4+ T cell and CD8+ cytotoxic lymphocyte selectively target tumor cells, whereas the glioma cells that do not express the antigen are targeted by activated NK cells, reducing the chance of cancer immune evasion (#NCT02060955) [129].

Discussion
The present study reviews current immunotherapeutic approaches to malignant brain tumors, specifically focusing on classification, immunomechanisms, evidence from clinical trials, limitations, and future challenges.
The current first-line multimodal treatment involves the combination of gross total surgical resection, followed by the use of adjuvant alkylating agents and whole-brain radiotherapy [6,7,130]. Despite TMZ being the only systemic agent approved as of now, glioma cells have often been found to be resistant [131][132][133]. One of the major disadvantages of TMZ is the rapid in vivo hydrolytic degradation. Several studies seek to overcome the quick drug clearance through polymer scaffold molecules, simultaneously upgrading therapeutic efficacy and reducing adverse events [134,135]. TMZ also frequently induces lymphopenia and myelosuppression, as do many other chemotherapy drugs. The immunodepletion leads to less immune surveillance and inhibition of antitumor immune response [136][137][138]. Another downside is the tumor expression of DNA repair enzymes, such as MGMT, which nullify the alkylating agents' therapeutic effects. The MGMT promoter status heavily influences overall survival, which is almost 21 months for methylated MGMT HGGs rather than 13 months for the unmethylated ones [46,49,139,140].
The failure of current therapeutic approaches, combined with advances in translational medicine, has led to the development of new strategies. The first turning point was the use of MAbs and BVZ. The Food and Drug Administration approved the use of BVZ based on the encouraging results achieved by the phase II BRAIN study (#NCT00345163), conducted on 167 patients affected by recurrent GBMs [141]. The initial enthusiasm around BVZ approval waned due to the lack of an effective overall survival improvement. The decrease of enhancement on MRI T1, which occurred in up to 60% of BVZ patients and was defined as a radiographic pseudo-response, is nothing more than an antiangiogenic effect without real tumor shrinkage. BVZ treatment stops blood vessel proliferation and reduces BBB permeability, resulting in an immediate contrast-enhancement decrease. However, these microvascular changes do not affect tumor proliferation or overall survival [142][143][144][145]. The third therapeutic option, within active immunotherapies, is the antitumoral vaccination. Peptide vaccines are designed to activate host immunity versus tumor-specific antigens. They have proven to be feasible and safe, but are still ineffective [80,146]. HGGs' intrinsic heterogeneity, mechanisms of immune escape, and loss of antigenic variants mediate patient immunosuppression and invalidate the potency of the vaccine. Personalized multipeptide vaccines that are customized to target more than one tumor antigen should be developed in the future [147,148].
The glioma microenvironment, populated by cancer stem cells, immunosuppressive molecules, and interleukins, plays a pivotal role in tumor growth and immune resistance [20,[149][150][151][152][153][154]. The rationale of adoptive immunotherapies is to modulate the tumor setting through the activation of cell immunity. These technologies are based on the manipulation of autologous/allogenic immunological cells and the engineering of CD8+ and CD4+ T lymphocytes [155]. Preliminary studies have considered CAR T cells as a valid and safe tool, which are tested intravenously and through intracranial infusion. However, the limited survival of T CAR, mechanisms of antigen escape, and ineffective homing to tumor cells do not make this a strategy yet achievable as a second-line treatment. The emerging NK-cell-based approach still has its limitations, such as the high expression of MHC I and HLA type A on glioma cells, which tie the KIRs and impede NK cells' antitumoral activity.

Limitations and Future Perspectives
Several aspects are responsible for the resilience of glioma, such as the lack of tumor antigen, loss of immunological phenotype, and immune evasive molecule production, which all together still limit the success of both active and adoptive immunotherapies.
Furthermore, the main limitation lies in the need to overcome the BBB, the reason why the administration route is pivotal. Several studies have proposed innovative methods, such as the implanted intracerebral convection-enhanced deliveries or the superselective intra-arterial cerebral infusion [156][157][158][159][160][161]. The intra-arterial administration is additionally facilitated by the previous destruction of BBB, carried out through osmotic agents, hypertonic solutions and mannitol, or ultrasounds [156,162,163]. Low frequency ultrasounds are a non-invasive strategy which lead to BBB disruption resulting in increased drug penetration and faster time to reach the tumor site [162]. An innovative emerging technique exploits the vascularized temporoparietal fascial flaps with the aim to bypass the BBB. These flaps are vascularized by the external carotid artery system, free from the BBB system. They can be transposed into the surgical cavity, after glioma resection, and could allow effective drug penetration and residual tumor cell targeting [164].
Another key aspect is the engineering of biocompatible and non-toxic small carriers able to improve drug diffusion and tissue distribution. Viral vehicles, nanoparticles, and liposomes are currently the most investigated [165][166][167][168].
The development of new administration routes and the advances in engineering more efficient and safe carriers will allow the implementation of standard therapeutic protocols with concomitant tailored immunotherapies.

Conclusions
Immunotherapeutic strategies are designed to overcome the immune escape pathways and the immunosuppressive glioma microenvironment, which are responsible for the resilience of HGGs.
Antitumoral immunotherapies involve active immune products along with adoptive engineered T, NK, and NKT cells.
BVZ is indicated in the treatment of recurrent HGGs, but not yet within the upfront protocol for newly diagnosed HGGs. Vaccines have been proven to be safe and feasible, although their treatment efficacy needs to be evaluated further. CAR T cells against EGFRvIII and engineered TCR T, NK, and hybrid cells have demonstrated a promising potential in several preclinical studies.
Although they are still not considered to be the first-line treatment against malignant gliomas, immunotherapies have shown excellent results in improving PFS.
Further clinical studies are focusing on validating antitumoral immunotherapeutic approaches such as personalized second-line strategies.