Immune checkpoints are surface molecules on immune cells that can regulate host immunity when they bind to the corresponding ligands or receptors on tumor cells or other cells (9). And tumor cells are proficient at employing this strategy to avoid the lethal effect exerted by immune cells (mainly T cells) (17). Immune checkpoint blockade (ICB) primarily refers to blocking the immunosuppressive immune checkpoints such as PD-1/PD-L1, and CTLA-4, thus inhibiting their corresponding immunosuppressive effects and playing an antitumor effect. And ICB has been proven feasible and effective in treating many cancers (18). However, there are no successful phase III clinical trials or marketing authorizations of ICBs in GBM treatment all over the world. Nevertheless, the exploration of this field is continuous and active, which can be indicated in Table 1 . With a deeper understanding of the TME, some co-stimulatory checkpoints and their specific agonists have also been studied. Unlike other cancers, such as melanoma and lung cancer, the application of ICBs seems unfavorable in treating gliomas (19). Nevertheless, the combination therapy of ICB and other treatments, such as standard chemoradiotherapy, targeted therapies, or other different kinds of immunotherapies, may find a way to success (9).

There are several completed clinical trials associated with ICBs for GBM treatment. All three clinical trials used anti-PD-1 monoclonal antibodies. PD-1 is a co-repressor molecule of the CD2 family and is expressed on the activated immune cells constitutively (20). After PD-1 binding to its ligands (mainly PD-L1) on tumor cells or antigen-presenting cells (APCs), T cells’ impotence, failure, or even apoptosis can be induced (21). In addition, PD-1 can also stimulate the proliferation of regulatory T cells (Tregs) and reduce the immune responses of natural killer cells and B cells (22).

Pembrolizumab (KEYTRUDA) is a commonly used monoclonal anti-PD-1 antibody. In 2019, satisfactory results of a randomized, multi-institutional trial of neoadjuvant pembrolizumab in patients with recurrent, surgically resectable GBM were published (23). Compared with patients receiving adjuvant, post-surgical administration of pembrolizumab alone, patients receiving neoadjuvant pembrolizumab and sustained adjuvant therapy after surgery had substantially improved OS and progression-free survival (PFS). The median OS of patients in the adjuvant-only group and the neoadjuvant group were 228 days (7.5 months) and 417 days (13.7 months), respectively, with a hazard ratio (neoadjuvant/adjuvant) of 0.39 (P = 0.04). And the median PFS of patients in the adjuvant-only group and the neoadjuvant group were 72.5 days (2.4 months) and 99.5 days (3.3 months), respectively, with a hazard ratio (neoadjuvant/adjuvant) of 0.43 (P = 0.03). The frequency of the focal upregulation of PD-L1 in the TME, the downregulation of PD-1 on T cells in the peripheral blood, and the intensive clonal expansion of T cells was higher in the neoadjuvant group compared with the adjuvant-only group. And the functional activation of tumor-infiltrating lymphocytes (TILs) was induced by neoadjuvant PD-1 blockade, which produced an interferon response within the TME (23).

The other two clinical trials applied another anti-PD-1 antibody, nivolumab (OPDIVO). Nivolumab is one of the most studied anti-PD-1 antibodies in treating gliomas. CheckMate 143 was the first phase III randomized clinical trial to test the efficacy of nivolumab in patients with recurrent GBM (rGBM) (24). But this trial’s primary endpoint (median OS, mOS) was not reached. Compared with the control group (bevacizumab) (10.0 months), the mOS was similar in the nivolumab group (9.8 months) (P = 0.76). And the PFS and the ORR (overall response rate) of the bevacizumab group were better than those of the nivolumab group. Besides, the post-subgroup analysis indicated that the potential benefits from nivolumab are more probable to be obtained in patients with methylated MGMT promoters (24). NCT02550249 was another crucial phase II clinical trial of nivolumab for GBM treatment. In this single-arm phase II clinical trial, twenty-seven patients with relapsed GBM and three patients with primary GBM were included to explore the feasibility, safety, and antitumor effects of neoadjuvant nivolumab in patients with resectable GBM (25). The neoadjuvant nivolumab was found to increase the expression of chemokines, infiltration of immune cells, and clonal diversity of T cell receptors in the TME (25).

There were also two completed randomized phase III clinical trials to test the efficacy of nivolumab in patients with newly diagnosed GBM (nGBM) (26, 27). The Checkmate 498 trial aimed to test the effectiveness of nivolumab plus radiotherapy, compared with TMZ plus radiotherapy, in patients with nGBM characterized as unmethylated MGMT promoters. However, its recently published results did not reach the primary endpoint (mOS). The mOSs of the nivolumab plus radiotherapy group and TMZ plus radiotherapy were 13.4 months and 14.9 months, respectively (27). The CheckMate 548 trial was a similar phase III study to evaluate the effectiveness of nivolumab plus the Stupp regime (radiotherapy plus TMZ), compared with the Stupp regime plus placebo, in patients with nGBM with methylated MGMT promoter (26). However, the results also showed a failure in adding nivolumab to the Stupp regime could not improve the OS and PFS of the patients. The mPFSs of nivolumab plus the Stupp regime group and the Stupp regime plus placebo group were 10.6 months and 10.3 months, respectively. And the mOSs were 28.9 months and 32.1 months, respectively.

In addition, some other immune checkpoints in gliomas, such as CTLA-4 (28–30), CD47 (31–33), CD73 (34, 35), TIGIT (36), and CD137 (37), were also studied in either clinical trials and preclinical research. It is worth noting that TILs in GBM are proven to highly express a number of co-inhibitory immune checkpoints, such as TIM-3, PD-1, and LAG-3, as a result of severe exhaustion of T cells (38). And this supports the necessity of exploring other feasible immune checkpoints and combined therapies to increase the effectiveness of the ICB (26, 39).

Vaccines have a long history of use in cancer treatment (40, 41). Several types of vaccines are used in cancer treatment, and peptide and dendritic cell (DC) vaccines are the main strategies for glioma treatment (42–44). In addition, the TAA and TSA can be used as the immune targets of vaccines to stimulate adaptive immunity (45). Table 2 lists the recent phase II/III clinical trials of vaccine therapy for GBM treatment from ClinicalTrials.gov.

Peptide vaccines comprise 8-25 amino acids and have epitopes acting as antigenic targets (43). Moreover, they are usually conjugated to a carrier protein to increase immunogenicity. These vaccines with simple structures are relatively easy to manufacture and store and have relatively lower variability when compared to other vaccines (43, 46). The frequently mutated or highly expressed proteins or antigens in GBM mainly include EGFR, EGFRvIII, NF1, TERT, PDGFRA, PTEN, RB1, IDH1, TP53, PIK3R1, and PIK3CA, some of which are regarded as ideal vaccine targets (43, 47). EGFRvIII is a deletion mutation of EGFR, which can be detected in approximately 20% of GBM (48, 49). And EGFRvIII is proven to enhance tumor growth and chemotherapy resistance. Currently, EGFRvIII is one of the most studied TSAs and an important target in vaccines for GBM treatment (50). There was an important phase III clinical trial that used the peptide vaccine rindopepimut (51). However, the results of this phase III trial of rindopepimut in combination with TMZ in patients with EGFRvIII-positive nGBM did not show a not disappoint. Compared with the TMZ group, the rindopepimut plus TMZ group did not increase the mOS. The mOS of TMZ and rindopepimut plus TMZ groups were 20.1 months and 20.0 months, respectively (P=0.93). Besides, the result also indicated the necessity of multi-peptide vaccines against several targets to overcome the antigenic heterogeneity in GBM (43, 51).

DC vaccine is another studied type of vaccine for glioma treatment (52). Dendritic cells’ function as antigen-presenting cells serves as the foundation for the main mechanism of DC vaccines (53). When immune cells, especially T cells, are activated by DCs, they can cross BBB and enter the brain tumor site to play the antitumor effects (53, 54). And DCs are believed to trigger both innate and adaptive immune responses to facilitate the transformation of immunologically cold gliomas into immunologically hot gliomas (44, 53, 55). Currently, about half of the current phase II and III trials involving vaccines are cell-based strategies, especially DC vaccines (56).

In 2023, a phase III trial of an autologous tumor lysate-loaded DC vaccine (DCVax-L) plus TMZ in patients with nGBM and rGBM reported encouraging findings (10, 11). This study’s primary and secondary endpoints were the mOSs in patients with nGBM and rGBM, respectively (10). In the nGBM part, the mOSs in the DCVax-L plus TMZ group and the TMZ control group were 19.3 and 16.5 months, respectively (P = 0.002). And the survival rates of two and five years in DCVax-L plus TMZ group and TMZ control group were 15.7% vs 9.9% and 13.0% vs 5.7%, respectively (10). In the rGBM part, the mOSs in DCVax-L plus TMZ group and TMZ control group were 13.2 and 7.8 months, respectively (P < 0.001). And the survival rates of two years and 30 months of DCVax-L plus TMZ group and TMZ control group were 15.7% vs. 9.9% and 13.0% vs. 5.7%, respectively (10). Besides, DCVax-L-treated patients with nGBM with methylated MGMT promoter survived longer (21.3 months) than those in the external control group (P = 0.03) (10). Obviously, this phase III trial advances the pace of vaccine therapy for gliomas. However, the individual patient-level data of the external control populations are not accessible. And a more credible and reasonable investigation of DCVax-L in GBM treatment is needed.

CAR-T cell therapy is a typical type of adoptive T-cell therapies (57). This therapeutic approach involves collecting T cells from a patient’s peripheral blood, followed by their modification, amplification, and activation to express CAR molecules on the cell membranes. These genetically engineered T cells are then administered to the patient through injection, allowing them to target specific tumor cell antigens (57). The significance of CAR-T cell therapy in treating gliomas has been identified, although it has yet to exhibit big success (58). Table 3 lists the recent phase II/III clinical trials of CAR-T therapy for GBM treatment from ClinicalTrials.gov.

The phase I clinical trials associated with CAR-T cell therapy in GBM treatment shows that scientists are also actively trying in this field. In these clinical trials, several commonly used targeted antigens, including EGFRvIII (59), IL13Rα2 (60), and HER2 (61), had also been indicated. NCT02208362 was a phase I clinical trial of IL13Rα2-targeted CAR-T cell therapy in GBM treatment (60). In this study, two intracranial approaches were applied to deliver CAR-T cells to a patient with rGBM, including infusions into the resected tumor cavity and the ventricular system. And it was found that intraventricular therapy can achieve the wide regression of central nervous system tumors. In comparison, the intracavitary treatment seemed only to control the local tumor recurrence. Moreover, the CAR-T cell therapy provided the patient with a response for 7.5 months. Overall, the result proved that CAR-T cell therapy can show the antitumor activity in GBM treatment, and IL13Rα2 is a valid immunotherapeutic target for CAR-T cell therapy (60). NCT02209376 was another phase I study of CAR-T cell therapy in patients with EGFRvIII-positive rGBM (59). This first-in-human experiment of EGFRvIII-targeting CAR-T cells by intravenous administration proved that CAR-T cells could transfer to the brain tumor site. It is worth noting that the EGFRvIII level on tumor cells was observed to be reduced or eliminated after the CAR-T cell administration. Moreover, the effect of CAR-T cells on the TME in GBM was also emphasized in this study. The immunosuppressive Tregs were the dominant T cell type for TILs in the TME. And many immunosuppressive molecules, including PD-L1, IL-10, and IDO1, were also observed to increase. Besides, the study also indicated the combination therapy of CAR-T cell therapy and the inhibition of immunosuppressive pathways (59). In another phase I study (NCT01109095), researchers investigated the use of HER2-targeting CAR-modified virus-specific T cells (HER2-CAR VSTs) in patients with rGBM (61). Participants in this trial were administered one or more autologous HER2-CAR VST injections across five different dosage levels. Of the 17 patients, eight exhibited clinical benefits, with one showing a partial response and seven maintaining stable disease. The median overall survival (mOS) was 11.1 months following HER2-CAR VSTs administration and 24.5 months from the time of diagnosis (61).

CAR-T cell therapy has made a step forward in both hematologic and solid tumors. However, its use for GBM treatment remains limited due to the BBB, antigen escape, tumor heterogeneity, and TME (62). Though the mentioned three phase I clinical trials indicated firm hopes for GBM treatment, more clinical trials with larger samples and more reliable examinations are needed.

Oncolytic virus (OV) therapy has emerged as a significant treatment strategy and has become a focus of research in the field of oncology (63). In 2021, the Japanese Ministry of Health, Labor, and Welfare (MHLW) granted conditional and time-limited approval for G47Δ to treat patients with malignant glioma in Japan (12). Table 4 presents a summary of recent phase II/III clinical trials for OV therapy in the treatment of glioblastoma (GBM), as found on ClinicalTrials.gov.

OVs are genetically modified, weakly pathogenic viruses that enhance the antitumor effects without harming normal cells (63). OV therapy hasadvantage in this era of mature genetic engineering. There are three purposes for genetic modification of OVs: 1) delete virulence genes to improve the safety of OVs, 2) enhance the tumor cell tropism and targeting of OVs to tumor cells, and 3) modify OVs with different therapeutic genes to enhance anti-tumor effects (64). On the one hand, OVs can self-replicate in host cancer cells, leading to direct lysis of the cancer cells (63). On the other hand, OVs help activate innate and adaptive immune responses by the releases of damage-related molecular patterns (DAMPs), viral pathogen-associated molecular patterns (PAMPs), and TAAs, to improve the immunosuppressive TME (64–67). Besides, OVs can selectively target and inhibit glioma stem cells (GSCs), which is an essential factor for drug resistance, tumor blood vessel formation, and immunosuppressive glioma microenvironment (68–70).

Since gliomas develop predominantly in the brain and lack distant metastases, which allows for the viruses that need an active cell cycle for reproduction, gliomas are particularly well-suited for OV treatment (71). The viruses used for OV therapy in gliomas include Oncolytic H-1 Parvovirus (72), Oncolytic Reovirus (73, 74), Oncolytic measles virus (75), Newcastle disease virus (74), Oncolytic vaccinia virus (76), Poliovirus (77), Oncolytic adenovirus (78), Oncolytic zika virus (79), Oncolytic herpes simplex virus (64, 80). Moreover, of all the OVs, oHSV progresses furthest in the clinical practice, including G47Δ, G207, HSV1716, and rQNestin-34.5 (80).

UMIN000015995 was a phase II trial of G47Δ in Japan for patients with residual or recurrent GBM (12). The third-generation as well as the triple-mutated oncolytic herpes simplex virus type 1 (HSV-1) G47 was created by deleting the US11 promoter from its parental G207 and overlapping the US11 gene (81). The primary endpoint (1-year survival rate) was achieved ahead of schedule, which was 84.2%. Additionally, the secondary endpoints of OS and PFS were 20.2 months and 4.7 months, respectively, after the G47 initiation (12). Based on the exciting results, G47Δ obtained conditional and time-limited approval in Japan for malignant glioma patients (12).

As a promising cancer treatment approach, OV therapy has a significant effect on gliomas. Moreover, the successful application of G47Δ in Japan also indicates the huge potential of OV therapy. Nevertheless, further research and phase III trials with large samples are needed to verify the real efficacy of this novel treatment approach.