The Application of PD-1 Inhibitors in Immunotherapy for Glioblastoma.

1
Structure and Function of PD‐1
Programmed cell death protein‐1 (PD‐1) is a key immune inhibitory receptor expressed on activated T lymphocytes, B lymphocytes, and myeloid cells [7]. Programmed cell death‐ligand 1 (PD‐L1) is primarily expressed on tumor cells and antigen‐presenting cells (APCs), while programmed cell death‐ligand 2 (PD‐L2) is mainly expressed on dendritic cells and macrophages [8, 9]. Antigen‐presenting cells, such as microglia and macrophages, can activate T cells, which then travel through perivascular spaces to the central nervous system to recognize tumor cells. This process is regulated by the PD‐1/PD‐L1 pathway [10]. When PD‐1 binds PD‐L1, its immunoreceptor tyrosine‐based inhibitory motif (ITIM) and the immunoreceptor tyrosine‐based switch motif (ITSM) motifs are phosphorylated and recruit Src homology 2 domain‐containing phosphatase 2 (SHP‐2), which dephosphorylates key T cell receptor (TCR) signaling molecules (e.g., PI3K/AKT, ZAP70), reducing T‐cell proliferation, cytokine secretion (e.g., interferon‐γ, IFN‐γ), and cytotoxicity [11]. PD‐1/PD‐L1 engagement dampens MHC‐mediated antigen presentation and blocks costimulatory signaling (e.g., CD80/CD86 to CD28), further suppressing T‐cell activation and antigen recognition. The binding of PD‐1 to PD‐L1 prevents T cells from recognizing tumor cells, allowing tumor cells to evade immune surveillance by T cells. This leads to the phenomenon of “immune escape,” enabling the tumor to gain unchecked proliferative capacity [12] (Figure 1).

2
PD‐1 Inhibitor
Lwai et al. were the first to report the significance of the PD‐1/PD‐L1 pathway in tumor immunity. They found that blocking the PD‐1/PD‐L1 interaction can significantly restore anti‐tumor immune responses and promote lymphocyte attacks on tumor cells. Targeting the PD‐1/PD‐L1 axis with immune checkpoint blockade therapy has clearly become a focal point in tumor immunotherapy, attracting significant attention [13]. Anti‐PD‐1 monoclonal antibodies have become one of the most promising strategies for treating various solid tumors. Many anti‐PD‐1 antibodies have been approved to date, including nivolumab, pembrolizumab, and cemiplimab, which received FDA approval and were launched in September 2014, December 2014, and September 2018, respectively [12, 14, 15, 16]. PD‐1 inhibitors have shown significant efficacy in various cancers, including malignant melanoma, breast cancer, lung cancer, and multiple myeloma, one of the most promising immune checkpoint inhibitors (ICIs) currently available [17, 18, 19].

3
The Mechanism of Action of PD‐1 Inhibitors and Their Relationship With GBM
PD‐1 inhibitors restore T cell function by blocking the interaction between PD‐1 and its ligands (PD‐L1 and PD‐L2), thereby eliminating the immune suppression against anti‐tumor T cells. This enhances their sensitivity to mediated cytotoxic effects and indirectly promotes the activation of other immune cells. Additionally, the T cells that are freed from immune suppression can proliferate and infiltrate the tumor microenvironment, inhibiting the proliferation of tumor cells while enhancing the body's immune response against the tumor [20, 21, 22, 23] (Figure 2).
Although the central nervous system has long been considered to possess immune privilege, increasing evidence suggests that immune cells can cross the blood–brain barrier and elicit robust immune responses within the brain [24, 25]. GBM alters the immune microenvironment, preventing T cells from recognizing tumor cells and allowing them to evade immune system surveillance. Due to the high expression of PD‐L1 in primary GBM (with expression levels correlating with glioma grade and clinical prognosis), and the tendency of GBM cells to migrate beyond the tumor margins, this facilitates immune cells in accurately locating and eliminating tumor cells. This provides a rationale for exploring anti‐PD‐1 therapies for this disease [24, 26, 27, 28].

4
Current Research Status of PD‐1 Inhibitors in Glioblastoma
A substantial amount of clinical research has driven the development of immune checkpoint blockade therapy for glioblastoma. At present, immunotherapy has become one of the main treatment strategies for glioblastoma.
In mouse GBM models, PD‐1/PD‐L1 blockade alone can induce durable tumor‐free survival [29]. In contrast, retrospective analyses in recurrent GBM show nivolumab salvage therapy offers no clear survival advantage after bevacizumab failure [30]. Similarly, pembrolizumab has also shown no survival advantage in patients with recurrent GBM [31]. Additionally, compared to mice that underwent tumor resection and received anti‐PD‐1 treatment, mice that did not receive tumor resection and anti‐PD‐1 treatment exhibited an increase in tumor‐specific T cells. However, these T cells demonstrated poorer effector functions, and the expansion of tumor‐specific T cells was associated with whether surgical treatment was performed and the duration of the disease. Thus, it is evident that the failure of anti‐PD‐1 treatment in glioblastoma may also be attributed to this mechanism [32, 33].
A subsequent clinical trial evaluated the efficacy of the anti‐PD‐1 antibody pembrolizumab in patients with recurrent GBM. The results showed significant improvements in overall survival and progression‐free survival, with pembrolizumab enhancing both local and systemic anti‐tumor immune responses [34]. Melero et al. further observed in a single‐arm phase II study (NCT02550249) that some newly diagnosed or recurrent patients on nivolumab survived beyond 2 years, with similar systemic immune changes [35]. Overall, using solely immune checkpoint blockade strategies for GBM treatment has its limitations [36].
Another randomized clinical trial aimed to explore whether nivolumab could improve survival rates in patients with recurrent GBM compared to bevacizumab. The results showed that the incidence of O6‐methylguanine‐DNA methyltransferase (MGMT) promoter methylation was 22.7% in the bevacizumab group and 23.4% in the nivolumab group; the objective response rates (ORR) were 23.1% for the bevacizumab group and 7.8% for the nivolumab group; the 12‐month overall survival (OS) was 42% for both groups, with the rates of grade ≥ 3 treatment‐related adverse events (TRAE) being 15.2% and 18.1%, respectively [37]. Compared to patients receiving bevacizumab, although nivolumab did not improve survival rates in recurrent GBM patients and the treatment effect was not significant, those with MGMT promoter methylation who were not dependent on corticosteroids could benefit from treatment with immune checkpoint inhibitors.
Although PD‐1 inhibitors are effective against several solid tumors, their anti‐tumor activity against GBM is limited. The fact that monotherapy has poor efficacy against GBM is becoming increasingly evident, leading to a rise in combined treatment strategies.

5
PD‐1 Inhibitors in Combination With Chemotherapy
Glioblastoma is a tumor that is insensitive to immunotherapy, diminishing the efficacy of immune checkpoint inhibitors for this disease. Simply blocking the PD‐1/PD‐L1 axis is insufficient to stimulate an effective anti‐tumor immune response. Combination therapies may be a crucial approach to enhancing the effectiveness of PD‐1 inhibitors and boosting anti‐tumor activity [38]. A clinical trial found that localized chemotherapy combined with PD‐1 inhibitors promoted anti‐tumor immune responses in GBM and improved survival rates [39]. Immunotherapy is typically administered after the failure of initial chemotherapy regimens. Although upfront chemotherapy can suppress immunity, careful sequencing and dosing with PD‐1 inhibitors may enhance anti‐tumor activity in GBM.
In a mouse model of glioma, the group receiving temozolomide in combination with anti‐PD‐1 antibodies exhibited significantly reduced tumor size and improved OS compared to the group treated with temozolomide alone [40]. Subsequently, research by Wang et al. also found that the combination of temozolomide and PD‐1 inhibitors significantly reduced tumor size and improved survival rates in animal models of GBM. These findings provide strong preclinical evidence to support this treatment approach [41].
CheckMate 143 (NCT02017717) is the first phase I clinical trial to evaluate the use of immune checkpoint inhibition in the first‐line treatment of GBM. The results indicate that nivolumab can be safely combined with temozolomide ± radiotherapy in newly diagnosed GBM patients. In those with unmethylated MGMT promoters, the mOS was similar to that of patients not receiving temozolomide. Additionally, survival rates were found to correlate with the methylation status of the MGMT promoter. The combination of PD‐1 inhibitors and temozolomide holds significant promise for the treatment of GBM, but further trials are needed to explore this approach in greater depth.

6
PD‐1 Inhibitors in Combination With Radiotherapy
Another standard treatment for GBM is radiotherapy. However, the combination of radiotherapy with PD‐1 inhibitors is likely to emerge as a new strategy for controlling GBM. In a mouse model of GBM, it was confirmed that the combination of anti‐PD‐1 immunotherapy with stereotactic radiotherapy resulted in reduced local immune suppression and significantly extended survival time [42]. Additionally, in a clinical trial involving six patients with GBM (NCT02313272), treatment with stereotactic radiotherapy, pembrolizumab, and the anti‐vascular endothelial growth factor (VEGF) antibody bevacizumab resulted in durable disease control for three patients. Among them, one achieved a complete response (CR), and two had stable disease (SD) [43]. The trial demonstrated good tolerability and effective tumor treatment outcomes.

7
Combination of PD‐1 Inhibitors With Oncolytic Virus Therapy
Oncolytic virus therapy typically involves directly injecting oncolytic viruses into the tumor site or administering them systemically. These viruses are designed to specifically target glioblastoma cells, selectively infecting cancer cells, leading to their apoptosis and inducing an immunogenic response [44]. In a recent trial assessing the efficacy of oncolytic virus DNX‐2401 combined with pembrolizumab for patients with recurrent GBM, the ORR was 10.4% (not statistically significant). The 12‐month OS rate was 52.7% (95% CI 40.1%–69.2%), with a median OS of 12.5 months. Additionally, 56.2% (95% CI 41.1%–70.5%) of patients had stable disease, indicating good clinical benefit. Three patients who underwent long‐term treatment remained alive at 45, 48, and 60 months [45]. The study confirmed that the combination of DNX‐2401 and pembrolizumab provides significant survival benefits for patients with recurrent GBM and has a favorable safety profile. Meanwhile, a research team developed a next‐generation oncolytic virus, C5252, which expresses both anti‐PD‐1 antibodies and interleukin‐12 (IL‐12). Intracranial administration of C5252 in GBM models was found to be safe and effective. Notably, C5252, which also expresses anti‐PD‐1 antibodies, represents a promising new generation of combination therapy for GBM, providing strong support for upcoming clinical trials [46].

8
Combination of PD‐1 Inhibitors With Vaccines
Recently, a neoantigen‐based cancer vaccine has been tested in patients with GBM. This neoantigen induces effective antigen‐specific T cell responses in GBM patients and demonstrates a certain degree of clinical activity [47, 48]. The combination therapy of anti‐PD‐1 antibodies and personalized cancer vaccines has demonstrated good clinical activity in other cancer types, including non‐small cell lung cancer, melanoma, and bladder cancer [49, 50, 51]. The efficacy of this combination therapy is gradually being validated in clinical trials for GBM. In one case involving a patient treated with a dendritic cell (DC) vaccine, anti‐PD‐1 antibodies, and poly‐ICLC, there was no disease progression for 69 months, and no immune‐related adverse events were observed during the treatment [52]. In the latest mouse model study, a targeted DNA vaccine combined with PD‐1 checkpoint blockade was used to treat mice bearing intracranial tumors expressing TRP‐2 and gp100. The results showed a significant extension in the survival time of the mice [53]. The combination of PD‐1 inhibitors and cancer vaccines offers a promising new approach for treating GBM, and it is essential to conduct large‐scale trials to validate these results.

9
Combination of PD‐1 Inhibitors With Angiogenesis Inhibitors
Angiogenesis and immune escape are considered hallmarks of cancer, as they are interdependent processes that often occur simultaneously, promoting tumor development [54, 55]. Currently, the combination of anti‐angiogenic agents with PD‐1/PD‐L1 inhibitors has become a first‐line treatment strategy for non‐small cell lung cancer, endometrial cancer, and primary liver cancer [56]. Moreover, this combination therapy is gradually being applied to the treatment of GBM. An initial multicenter phase II clinical trial report indicated that pembrolizumab combined with bevacizumab is beneficial for controlling the progression of GBM and improving survival rates [57]. Subsequently, another phase II clinical trial further confirmed that the combination of these two drugs for treating GBM achieves good efficacy and extends survival time [58]. Di et al. discovered in mouse models that the combination of angiogenesis inhibitors and PD‐1 inhibitors in a dual blockade therapy significantly improved overall survival rates [59]. This finding is consistent with previously reported literature results [60, 61].
A case report indicated that a patient with unmethylated MGMT promoter GBM who was intolerant to temozolomide received a combination therapy of the PD‐1 inhibitor camrelizumab, the epidermal growth factor receptor tyrosine kinase inhibitor axitinib, and the anti‐angiogenic agent anlotinib during radiotherapy. The results showed a progression‐free survival (PFS) and OS of nearly 11 and 18 months, respectively, significantly surpassing historical data, and the patient tolerated the treatment well, with no severe adverse events reported [62]. PD‐1 inhibitors combined with anti‐angiogenic agents show promise in GBM, but their efficacy still requires confirmation in larger trials.

10
Combination of PD‐1 Inhibitors With Cytotoxic T‐Lymphocyte‐Associated Protein 4 (CTLA‐4) Inhibitors
The combination of cytotoxic T‐lymphocyte‐associated protein 4 (CTLA‐4) inhibitors with PD‐1 inhibitors has been shown to improve tumor response rates and survival compared to monotherapy with either agent. However, this combination therapy is associated with a higher incidence of immune‐related adverse events (irAEs). In the first reported phase I clinical trial, perioperative injection of the CTLA‐4 inhibitor ipilimumab and the PD‐1 inhibitor nivolumab after resection of recurrent GBM was found to be feasible and safe, resulting in long‐term survival rates [63]. Recent studies involving newly diagnosed GBM patients showed that 32 participants received combination therapy with ipilimumab and nivolumab after completing standard chemotherapy. For all patients undergoing combination treatment, the mOS and PFS were 20.7 and 16.1 months, respectively. The treatment was well tolerated, with a grade 4 TRAE incidence of 16%, and there was no unexpected increase in toxicity with the combined therapy [64]. These findings further confirm that ipilimumab and nivolumab are safe and well tolerated in newly diagnosed GBM patients, providing support for future trials.

11
Combination of PD‐1 Inhibitors With CAR‐T Cell Therapy
Chimeric antigen receptors (CARs) are artificially synthesized receptors that re‐target T cells to tumor surface antigens, thereby inducing a specific anti‐tumor immune response against cancer cells [65]. CAR‐T cell immunotherapy is a promising approach, and combining it with immune checkpoint blockade strategies can help overcome CAR‐T cell exhaustion in patients with solid malignancies, including GBM; this combination therapy has the potential to significantly enhance anti‐tumor efficacy [66]. One study first investigated the anti‐tumor effects of CAR‐T cell therapy targeting the epidermal growth factor receptor variant III (EGFRvIII) in combination with PD‐1 checkpoint blockade in a mouse model of GBM. The study found that PD‐1 checkpoint blockade enhanced the functionality of tumor‐infiltrating lymphocytes (TILs) in the mouse model, significantly improving the anti‐tumor activity of anti‐EGFRvIII CAR‐T cells and markedly extending the survival of tumor‐bearing mice [67]. This combination therapy demonstrated significant tumor‐suppressive effects in vitro, confirming its therapeutic efficacy. Additionally, ganglioside GD2 is also emerging as a promising target for CAR‐T cell therapy in GBM. Research suggests that GD2 CAR‐T cell therapy combined with nivolumab may offer an improved treatment strategy for GBM [68]. In a recent phase I trial (NCT03726515), seven newly diagnosed GBM patients with the epidermal growth factor receptor variant III (EGFRvIII) received combination therapy with CAR‐T cell therapy and the PD‐1 inhibitor pembrolizumab. The results confirmed the safety of the treatment, with no observed dose‐limiting toxicities; however, the efficacy appeared to be limited [69]. Clinical research on the combination of immune checkpoint blockade and CAR‐T cell therapy for GBM is just beginning and requires ongoing exploration in numerous clinical studies.

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Challenges and Future Perspectives
Current challenges in PD‐1 therapy for glioblastoma (GBM) are driven by several key factors. The existing evidence is constrained by small, heterogeneous cohorts, inconsistent endpoints (PFS vs. OS), and the confounding effect of corticosteroid use, which impairs immune responses. Monotherapy trials have demonstrated limited benefits, with contradictory findings across studies. GBM‐specific barriers—such as the blood–brain barrier, a highly immunosuppressive tumor microenvironment, intratumoral heterogeneity, and T‐cell exhaustion—continue to hinder the efficacy of PD‐1 inhibitors. Additionally, pseudoprogression complicates the accurate assessment of treatment response. Combination therapies offer potential but raise significant concerns regarding safety and feasibility, including immune‐related adverse events (irAEs), neurotoxicity, and edema. The optimal sequencing of PD‐1 blockade with other treatments, including chemotherapy, radiotherapy, anti‐angiogenics, vaccines, oncolytic viruses, CTLA‐4 blockade, or CAR‐T therapies, remains uncertain. There are also persistent biomarker gaps: PD‐L1 expression, MGMT methylation, tumor mutational burden (TMB), immune infiltrates, and steroid exposure are not consistently incorporated into trial designs or used for patient selection, limiting the ability to predict response and resistance. Future research should prioritize biomarker‐driven, adaptive trial designs and optimize the timing (including neoadjuvant strategies) and dosing of combination therapies. Exploring locoregional or intratumoral delivery methods to bypass the blood–brain barrier, pairing PD‐1 blockade with strategies to reprogram the tumor microenvironment, and incorporating robust translational correlative studies (such as multi‐omics and spatial profiling) are crucial for identifying true responders and understanding resistance mechanisms.

13
Conclusion
Currently, anti‐PD‐1 antibody immunotherapy is gaining significant attention in the treatment of GBM, with both domestic and international clinical trials investigating the use of PD‐1 inhibitors as monotherapy or in combination therapy for GBM (as shown in Table 1). Monotherapy remains limited, but rational combinations with PD‐1 inhibitors can boost anti‐tumor activity and improve PFS/OS. Such regimens may evolve into future first‐line options for GBM pending robust safety and efficacy data. However, further in‐depth research is needed to evaluate its safety and efficacy, and additional combination therapies should be actively explored.

Author Contributions

Ming‐zhen Dong: writing – review and editing, writing – original draft. Hui‐ying Che: writing – review and editing. Ming Cui: writing – review and editing. Lin‐zhuo Qu: writing – review and editing. Hong‐jian Guan: writing – review and editing, supervision, project administration, investigation, funding acquisition.

Funding
This work was supported in part by grants from the Research Project of Provincial Department of Education (JJKH20240692KJ), the Research Fund of Yanbian University (Yanbian University Science and Technology Agreement No. 20(2021)) and the Changbai Elite Talent Program (202434024).

Conflicts of Interest
The authors declare no conflicts of interest.

Supporting information

Data S1: fsb271589‐sup‐0001‐Supinfo1.pdf.

Data S2: fsb271589‐sup‐0002‐Supinfo2.pdf.