Glioblastoma Immunotherapy: A Systematic Review of the Present Strategies and Prospects for Advancements

Immune checkpoint inhibitors are monoclonal antibodies designed to counteract negative regulatory pathways that impede T-cell activation. These antibodies target surface receptors known as immune checkpoints. Under normal circumstances, immune checkpoint molecules can dampen cytotoxic T-cell function. However, when ICIs are employed, they disrupt this normal activation of immune checkpoints, restoring T-cell function and enhancing the immunotherapeutic effect. Presently, the primary focus of immune checkpoint inhibition revolves around two key receptors: programmed cell death protein 1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4). Notable advancements have been made in treating challenging cancers, such as melanoma, lung cancer, and renal cancer, using anti-PD-1 and anti-PD-L1 antibodies [75,93,118,119,120,121,122].

The NCT02017717 trial, a pioneering randomized phase-I clinical study for recurrent GBM, assessed the efficacy and tolerability of nivolumab (a PD-1 inhibitor) as monotherapy or in combination with ipilimumab (a CTLA-4 inhibitor). All patients underwent surgical resections, RT, and TMZ treatments before being allocated to three different treatment arms. Intriguingly, nivolumab monotherapy demonstrated a superior median overall survival (10.4 months) compared to the NIVO1 + IPI3 or NIVO3 + IPI1 combinations (9.2 and 7.3 months, respectively). Phase III of this trial involved 369 recurrent GBM patients randomized to receive either nivolumab or bevacizumab. While mOS and toxicity were comparable, bevacizumab-treated patients exhibited a shorter duration of radiologic responses [118].

A phase-II clinical trial assessed bevacizumab alone or combined with notecan for efficacy in recurrent GBM patients, with both treatment arms showing tolerable side effects and median overall survival rates of 9.2 and 8.7 months, respectively.

At present, two ongoing phase-III trials, NCT02667587 and NCT02617589, are investigating nivolumab’s potential as a treatment for MGMT-unmethylated glioblastoma. The NCT02667587 clinical trial compares SOC with nivolumab or placebo, while NCT02617589 compares nivolumab versus TMZ, each in combination with RT.

Pembrolizumab, another anti-PD1 checkpoint inhibitor, is being investigated as a glioma treatment. A phase-II trial involving neoadjuvant pembrolizumab administration before surgery, followed by post-surgery adjuvant treatment, demonstrated increased survival in recurrent GBM patients.

CTLA-4, an immune checkpoint that competes with CD80 and CD86 for binding, thereby suppressing T-cell function, is under evaluation in clinical trials (NCT02311920, NCT02829931) using ipilimumab therapies.

Another immune checkpoint receptor, LAG-3, inhibits T-cell activity while enhancing the suppressive function of Tregs. A phase-I trial (NCT02658981) is currently assessing BMS-986,016, a LAG-3 inhibitor, alone and in combination with nivolumab in recurrent GBM patients. TIM-3, an additional lymphocyte-expressed receptor capable of inducing T-cell exhaustion and immune response suppression, can lead to unfavorable outcomes [4,92,118,119,120,121,122].

Vaccines hold promise as tumor treatment, leveraging tumor antigens. They activate immune surveillance against GBM, fortifying the adaptive immune system. GBM vaccines fall into four categories: peptides, DNA, cells, and mRNA. Peptide and DNA vaccines administer tumor-specific antigens or DNA to provoke an adaptive immune response. Cell vaccines, specifically DC vaccines derived from PBMCs, prime with tumor antigens. mRNA vaccines employ viral vectors loaded with mRNA, generating potent immune responses. Despite years of development, vaccines remain under investigation. Of these, only three have advanced to phase-III clinical trials: Rindopepimut, DCvax, and PPV.

Rindopepimut, a peptide-based vaccine, targets EGFRvIII mutation exclusively in GBM, minimizing off-tumor toxicity risk. However, GBM’s tumor heterogeneity poses challenges, as EGFRvIII expression varies. Phase-II trials demonstrated improved progression free survival (PFS) and median survival, compared to historical controls. ACT IV, a double-blind phase-III trial, examined Rindopepimut’s effect on GBM patients with minimal residual disease (MRD). MRD was defined as <2 cm² of enhancing tumor tissue post-surgery and chemoradiotherapy. The trial showed no significant difference in overall survival for MRD patients. IDH1 peptide vaccines, targeting the IDH1R132 H mutation, are in phase-I trials following promising preclinical results. Survivin, highly expressed in GBM, led to the development of SurVaxM, a peptide vaccine. Early results from a phase-II study combining SurVaxM with TMZ show improved PFS and OS. An antisense oligodeoxynucleotide against the IGF type-I receptor (IMV-001) demonstrated improved PFS in phase-I trials. PPV immunotherapy displayed safety and efficacy in a phase-I trial for recurrent GBM patients. However, a phase-III trial comparing PPV to best supportive care yielded unfavorable results. Neoantigens, identified through DNA and RNA sequencing, offer a personalized approach to GBM treatment. Two clinical trials demonstrated the potential of personalized GBM vaccination.

In the GAPVAC study, 16 newly diagnosed GBM patients received two synthesized vaccines: one targeting unmutated peptides (APVAC1) and the other neoantigens (APVAC2). Both vaccines elicited CD4+ and CD8+ T-cell responses, with varying immunogenicity. APVAC1 induced 50% immunogenicity, while APVAC2 achieved 84.7%. APVAC1 primarily stimulated CD8+ T-cell responses, while APVAC2 focused on CD4+ T-cell responses. Median PFS and OS were encouraging.

Another study administered a neoantigen vaccine (NeoVax) to newly diagnosed and MGMT unmethylated GBM patients. Two patients displayed immunogenicity and both CD4+ and CD8+ T-cell responses. Median PFS reached 7.6 months, with a median OS of 16.8 months. DCVax-L, a personalized peptide vaccination, demonstrated potential in a phase-III trial when combined with standard GBM therapy. mRNA-transfected DC vaccines were well-tolerated and significantly extended PFS compared to controls in a phase-II trial. Autologous DCs, pulsed with autologous whole tumor lysate and combined with standard chemoradiotherapy, proved feasible and safe in newly diagnosed GBM patients. In contrast, a multicentric phase-II study involving tumor lysate-charged autologous DCs (Audencel) failed to improve clinical outcomes for newly diagnosed GBM patients. AV-GBM-1, autologous DCs loaded with tumor-associated antigens from short-term autologous tumor cell cultures, is being evaluated in an ongoing phase-II clinical trial.

Dendritic cells (DCs), capable of promoting adaptive antitumor immune responses, are ideal for cellular vaccination. DC vaccines demonstrated effectiveness in preclinical and early stage clinical trials, showing significantly longer overall survival in GBM patients. Intratumoral and intradermal administrations yielded better results than intradermal alone. ICT-107, a DC-based vaccine designed for newly diagnosed GBM patients, proved safe with encouraging median PFS and OS in phase-I trials. A phase-III trial comparing ICT-107 to standard care was suspended due to funding issues.

The landscape of GBM treatment is evolving, with vaccines offering a promising avenue for intervention. While many vaccine candidates are in development, only a select few have progressed to phase-III trials. The complex nature of GBM, including its heterogeneity, presents both challenges and opportunities in vaccine development. Neoantigens, identified through advanced sequencing techniques, offer a personalized approach that shows significant potential. Additionally, DC-based vaccines have demonstrated effectiveness in the preclinical models and early stage clinical trials, with some promising results for patients. The ongoing quest for effective GBM vaccines continues, with researchers exploring various strategies to harness the power of the immune system in the fight against this devastating disease [24,46,53,55,56,64,123,124,125,126,127,128,129,130,131,132,133].

In recent years, OVs have emerged as a novel therapeutic approach in treating various solid tumors, including GBM. OVs offer a dual mechanism of antitumor action, involving the direct killing of tumor-specific cells and the induction of systemic antitumor immunity, encompassing both innate and adaptive responses. OVs trigger immunogenic cell death in tumor cells, leading to the release of tumor-associated antigens (TAAs), damage-associated molecular patterns (DAMPs), and pathogen-associated molecular patterns (PAMPs). DAMPs and PAMPs serve as potent stimulators of innate immunity by activating pattern recognition receptors, such as Toll-like receptors. Furthermore, these molecules enhance antigen cross-presentation and adaptive immune responses.

OVs also elicit a proinflammatory immune response, increasing the production of CXCL9, CXCL10, and CXCL11, which promote the trafficking and infiltration of T cells into tumors. Various OVs, including adenovirus, herpes simplex virus, measles virus, parvovirus, poliovirus, and zika virus, have demonstrated efficacy against GBM in preclinical studies. Encouraging data from clinical trials have shown OVs to have a favorable safety profile and promising efficacy, with evidence of intratumoral viral replication and lymphocyte infiltration. For instance, a phase-I clinical trial (NCT01470794) demonstrated the safety and efficacy of Toca 511 in treating 56 recurrent high-grade glioma patients. In a follow-up phase-III study involving 23 eligible patients, the median overall survival (OS) reached 14.4 months, with one- and two-year survival rates of 65.2% and 34.8%, respectively. Notably, five patients achieved complete responses and survived for extended periods after Toca 511 treatment.

DNX-2401, a tumor-selective oncolytic adenovirus, exhibited antiglioma efficacy in preclinical studies. In a phase-I dose-escalation trial involving 37 patients with recurrent malignant gliomas, DNX-2401 demonstrated both safety and promising responses across different dose levels. Approximately 20% of patients survived beyond three years post-treatment, and some patients experienced over a 95% reduction in tumor size with a progression free survival exceeding three years. Analyses of post-treatment tumor specimens revealed viral replication and spread within the tumor, along with the induction of intratumoral CD8+ and T-bet+ T-cell infiltration and a reduced expression of transmembrane immunoglobulin mucin-3.

Both Toca 511 and DNX-2401 trials reported that approximately 20% of GBM patients exhibited complete responses after intratumoral OV administration, with rare virotherapy-associated severe adverse events. Oncolytic H-1 parvovirus, although showing only a slight improvement in the median OS when administered intratumorally to GBM patients, displayed an increase in infiltrating lymphocytes and IFN-γ levels.

PVSRIPO, a live attenuated poliovirus type-1 vaccine with a modified internal ribosome entry site, received a breakthrough therapy designation from the FDA based on a phase-I study in recurrent GBM patients. PVSRIPO recognizes the poliovirus receptor CD155, which is upregulated on malignant and antigen-presenting cells within the tumor microenvironment. The phase-I study demonstrated safety and sustained survival rates, with about 20% of patients remaining alive for 57–70 months after PVSRIPO injection. A phase-II randomized trial of PVSRIPO alone or in combination with lomustine in patients with recurrent grade-IV malignant glioma (NCT02986178) is ongoing.

Herpes simplex virus-1 (HSV-1), a double-stranded DNA virus, has been extensively explored as a treatment for various solid tumors, including GBM. Genetically engineered variants, such as G207 and G47 delta, have shown promise. In a phase-I clinical trial involving children and adolescents with recurrent or progressive high-grade gliomas, G207 demonstrated safety and enhance the immunological response.

G47 delta (DELYTACT), another oncolytic HSV-1 variant, was evaluated in adult patients with residual or recurrent GBM in a single-arm phase-II clinical trial in Japan. Minimal side effects were observed, and the survival rate after one year reached an impressive 84.4%. G47 delta (Delytact/Teserpaturev) received conditional approval from the Japan Ministry of Health, Labor and Welfare (MHLW) for the treatment of malignant gliomas in Japan [49,54,55,56,66,68,134,135].

Several ongoing clinical trials are exploring the use of OVs as therapeutic agents for recurrent high-grade gliomas. While these results are promising, further clinical trials are needed to establish the safety and efficacy of OVs as a therapy for GBM.

CAR T-cell therapy presents a promising strategy for overcoming the formidable challenges posed by the BBB and the intricate tumor microenvironment (TME) within the context of adoptive cell T-cell therapy (ACT). ACT involves the reintroduction of autologous or allogenic anti-tumor T cells engineered to target tumor-specific antigens highly expressed on tumor cells while sparing normal cells. In a meticulously orchestrated process, these T cells are meticulously engineered in vitro using a lentiviral vector, thereby expressing a high-affinity single-chain fragment variable (scFv) tailored to the target antigen. This scFv is intricately fused with transmembrane regions, co-stimulatory domains, and an intracellular signaling domain derived from the CD3 molecule of the T-cell receptor (TCR). This engineering empowers autologous T cells to keenly recognize antigens upon scFv binding, initiating CAR molecule clustering and ensuing activation. This activation, in turn, triggers a cascade of events, including cytokine release, proliferation, cytotoxicity, and metabolic shifts, thereby enabling CAR T cells to chiefly exert their anti-tumor functions through cytokine release and the involvement of the granzyme and perforin axis, as well as the Fas and Fas ligand axis, thus equipping them to effectively surmount TME-related immunosuppression.

Notably, CAR T cells meticulously engineered to target IL13 Rα2 have demonstrated a heightened degree of selectivity for this receptor over IL13Ra1/IL4Ra. Intracranial injections of these IL13-zetakine CAR T cells, executed within a glioma xenograft model, have yielded auspicious improvements in median overall survival. A groundbreaking first-in-human pilot safety and feasibility trial conducted in 2015 evaluated the application of IL13-zetakine CAR T cells in three patients with recurrent GBM. These CAR T cells were methodically delivered via an implanted reservoir/catheter system, directly into the resection cavity, thereby resulting in a precisely controlled, transient bout of brain inflammation. Although the CAR T cells were met with overall favorable tolerability, certain adverse events, including grade-3 headaches and transient grade-3 neurologic events, were observed. A subsequent analysis of tumor tissue indicated a tangible reduction in IL13 Rα2 expression, while an MRI analysis suggested an increase in tumor necrotic volume, thereby potentially extending overall survival. Following this pioneering trial, a second-generation IL13-zetakine CAR T cell was painstakingly developed, featuring a 4-1BB costimulatory domain and an ingeniously mutated IgG4-Fc linker, all designed to amplify antitumor potential while curbing off-target interactions. A patient afflicted with recurrent GBM subsequently received intracavitary infusions of these CAR T cells, resulting in a pronounced inhibition of local tumor progression. However, the emergence of novel intracranial tumors and spinal lesions was noted, with the fifth intraventricular infusion precipitating a substantial reduction in tumor size. Regrettably, this regression proved transient, lasting merely 7.5 months, and recurrence subsequently manifested at new locations, characterized by diminished IL13 Rα2 expression, thereby underscoring the indispensable need for combinatory therapeutic modalities aimed at addressing antigen-loss relapse.

EGFRvIII, a frequently encountered GBM mutation, was initially associated with abbreviated survival periods. In a phase-I clinical trial, 18 patients afflicted with recurrent GBM received third-generation EGFRvIII-directed CAR T cells, endowed with CD28 and 4-1BB costimulatory domains. Patients, upon infusion of these CAR T cells, expeditiously developed respiratory symptoms, particularly evident at higher dose levels, a phenomenon indicative of dose-limiting toxicity, despite the absence of discernible clinical benefits. The median overall survival rate reached 6.9 months, with select patients achieving survival exceeding a year. To effectively address instances of EGFRvIII-negative yet EGFR-positive glioblastomas, a novel approach surfaced, involving the fusion of EGFRvIII scFv with a bispecific T-cell engager (BiTE). These BiTEs, as synthetic bispecific antibodies, serve to heighten immune interactions while simultaneously augmenting antibody specificity. Recent investigations have explored SynNotch-CAR T cells, meticulously designed to target multiple antigens, thus yielding improvements in specificity, comprehensiveness, and longevity in comparison to conventional T-cell therapy, as employed in the context of glioblastoma.

B7-H3, alternatively known as CD276, plays a pivotal role in regulating T-cell functions and, notably, is found to be overexpressed across a diverse array of human cancer cells, a correlation that frequently coincides with negative clinical outcomes. This distinct profile renders it an enticing target for immunotherapeutic interventions. Nevertheless, significant challenges persist, not the least of which is the full elucidation of its receptor and the intricacies of immune regulation associated therewith.

HER2, conspicuously expressed in various CNS tumors, including GBM, yet conspicuously absent in normal CNS tissue, emerges as an attractive and distinctively selective target. Clinical trials employing HER2-specific CAR T cells have borne witness to well-tolerated treatments, despite the lack of substantial and tangible survival benefits. Recent preclinical inquiries have embarked upon the exploration of trivalent CAR T-cell therapy, an innovative approach that targets HER2, IL13 Rα2, and ephrin-A2, demonstrating encouraging improvements in terms of cytokine release and cytotoxicity when contrasted with the outcomes achieved using monospecific or bispecific CAR T cells, thereby strongly hinting at the potential utility of this multifaceted therapeutic approach within the context of glioblastoma therapy.

CAR T-cell therapy, undoubtedly, occupies a paramount and promising position in the realm of glioblastoma immunotherapy. Recent strides and advancements, such as the development of second-generation CAR T cells with heightened specificity and reduced off-target effects, serve as a testament to the adaptability and burgeoning potential of this therapeutic modality. Strikingly promising results have emerged from studies targeting antigens, such as IL13 Rα2 and EGFRvIII, as reflected in the significant tumor regression and improved patient survival rates. Moreover, the visionary concept of trivalent CAR T cells augurs a future where combination therapies that adeptly target multiple antigens may yield even more favorable treatment outcomes. Though formidable challenges persist, encompassing the phenomena of antigen loss and the imperative [74,136,137,138,139,140,141,142,143,144,145,146,147,148,149].