In DIPG patients, Hwang et al. (2018) found that inhibition of PD-1, an immunosuppressive checkpoint molecule, using pembrolizumab resulted in a worsened condition, while Cacciotti et al. (2020) and Dunkel et al. (2023) (NCT03130959) found no significant improvements in survival as well as minimal to no adverse effects. Overall results for the use of anti-PD1 in DIPG do not seem promising (Hwang et al., 2018; Cacciotti et al., 2020; Dunkel et al., 2023). Recent studies have also investigated other checkpoint molecules such as TIM3, B7-H3, and CD40. TIM3 is an inhibitory checkpoint expressed on DIPG cells and immune cells such as microglia and macrophages. Ausejo-Mauleon et al. (2023) reported that using TIM-3 blockades promotes anti-tumour effects in immunocompetent DIPG murine models and found enhanced microglia- and CD8⁺ T cell-mediated pro-inflammatory immune responses, which ultimately improved survival (Ausejo-Mauleon et al., 2023). The differences in efficacy between anti-PD1 and anti-TIM3 may be attributed to the tumour-immune microenvironment (TIME) composition. Past research indicates that the DIPG TIME has low PD-1 expression, while there is a comparatively higher TIM3 expression (Lin et al., 2018; Ausejo-Mauleon et al., 2023; Chen et al., 2023). This highlights the role that macrophages may play in the TIME and is a potential area for exploration (Lin et al., 2018; Lieberman et al., 2019). B7-H3, also known as CD276, is expressed on both tumour and antigen-presenting cells and inhibits the anti-tumour functions of T cells (Zhang and Zheng, 2020). In the DIPG context, studies have utilized convection-enhanced delivery in combination with the monoclonal B7-H3 antibody, 8H9, as well as the development of anti-B7-H3 CAR-T cells (Souweidane et al., 2018; Vitanza et al., 2023) (NCT04185038). Clinical trials are currently ongoing for various forms of B7-H3-targeting CAR-T cells, such as the single target B7-H3 and the quad-target B7-H3, EGFR806, HER2, and IL13-Zetakine (Quad) CAR-T Cell (NCT04185038, NCT05768880). Preliminary results of the anti-B7-H3 CAR-T cell study showed improved survival (Vitanza et al., 2023). Extended survival was also observed in the 8H9 antibody study, although selection bias may be present due to the study’s design (Souweidane et al., 2018).

The indoleamine 2,3-dioxygenase (IDO) pathway is primarily involved in the conversion of tryptophan into kynurenines, but indoleamine 2,3-dioxygenase 1 (IDO1), an enzyme in the pathway, also acts as an immune checkpoint by modulating immunosuppressive responses (Zhai et al., 2018). A clinical trial (NCT02502708) evaluating indoximod, an IDO pathway inhibitor, combined with the chemotherapy drug temozolomide and radiation recently published its results. Overall, the authors concluded that the combination treatment can be tolerated, with variable disease and immune responses observed among the participants. Further studies must be conducted to evaluate treatment efficacy (Johnson et al., 2023). Contrary to the previously mentioned immune checkpoints, CD40 is typically involved in the stimulatory pathway and is expressed on immune cells to promote tumour-killing activity. An agonistic CD40 antibody, APX005M, has been developed to enhance immune activity against the tumour and induce tumour cell apoptosis. Studies have confirmed preliminary safety, with efficacy against DIPG being tested (Lindsay et al., 2020) (NCT03389802).

Tumour-associated antigens (TAAs) are also potential targets in immunotherapy, ranging from antigens seen across different cancer types to DIPG-specific TAAs. GD2 is a disialoganglioside that is highly expressed in solid tumours, including DIPG, small cell lung cancer, and melanoma; GD2 expression in normal tissue is limited, making GD2 an ideal target for immunotherapy with minimal off-target effects (Mount et al., 2018; Nazha et al., 2020). Clinical trials are ongoing for anti-GD2 CAR-T cells (NCT04196413) and C7R-coexpressing anti-GD2 CAR-T cells (NCT04099797), with clinical and radiological improvements observed in DIPG/DMG patients enrolled in the anti-GD2 CAR-T cells trial (Majzner et al., 2022). Anti-GD2 CAR-T cells and anti-GD2 CAR NK-92 cells are effective in in vivo assays using patient-derived DIPG cells and DIPG cell lines (Mount et al., 2018; Mount et al., 2018; Zuo et al., 2023). The Wilms’ tumor 1 gene (WT1) is an oncogene in various cancers including leukemia, breast cancer, glioblastoma, and DIPG. Overexpression of WT1 is associated with these cancers, and targeted protein knockdown inhibits cancer growth (Qi et al., 2015). A completed clinical trial that studied DSP-7888, a cancer vaccine that induces WT1-specific T cells, showed improved survival in DIPG patients compared to controls (Fujisaki et al., 2018) (NCT02750891). Separately, an ongoing clinical trial investigates vaccination with WT1 mRNA-loaded autologous monocyte-derived dendritic cells (DCs) as a potential therapeutic for DIPG. It is hypothesized that the DC vaccine will act as an adjuvant to boost anti-tumour immune activity with minimal side effects, given its specificity to WT1-expressing cells (NCT04911621). Epidermal growth factor (EGFR) is another common TAA expressed in tumour cells with limited expression in normal tissue. EGFR mutations leads to constitutive activation of the gene and results in cancer phenotypes, with EGFR overexpression observed in DIPG (Li et al., 2012). More recent studies focus on using nimotuzumab, an anti-EGFR, combined with radiation or radiochemotherapy (Fleischhack et al., 2019) (NCT04532229). While radiation with nimotuzumab does not significantly improve survival compared to radiochemotherapy alone, side effects and adverse events were significantly reduced. Overall, the DIPG patients had an improved quality of life, albeit no changes in survival (Fleischhack et al., 2019). The HER2 protein is a member of the EGFR family, and its overexpression is seen in many solid tumours, including DIPG, with breast cancer being the most notable (Oh and Bang, 2020). Wang et al. (2023a) investigated the use of anti-HER2 CAR-T cells as a therapeutic in DIPG and other DMGs. Results showed HER2-specific immune targeting and cytokine release when co-cultured in vitro with patient-derived DIPG cells. Anti-HER2 CAR-T cells also reduce tumour size in in vivo DIPG xenografts. The authors concluded that the CAR-T cells may show efficacy in DIPG patients, although a clinical trial is needed to confirm their hypothesis (Wang et al., 2023a). Vascular endothelial growth factor (VEGF) is a molecule regulating angiogenesis. However, in a tumour context, this is harnessed by the tumour cells to improve tumour angiogenesis and blood vessel accessibility, ultimately supporting tumourigenesis (Shibuya, 2011). A recently completed clinical trial with results published in 2020 showed that the anti-VEGF, bevacizumab, in combination with valproic acid and radiation, was well tolerated but did not improve survival (Su et al., 2020) (NCT00879437). In addition, an ongoing clinical trial will evaluate the survival of DIPG patients when treated with bevacizumab in combination with low-dose radiation (NCT04250064).

Survivin is an inhibitor of the apoptotic pathway and can be found in most cancers (Chen et al., 2016). It is also involved in various cell cycle and signalling pathways such as p53, Wnt, and Notch. Mutations in survivin allow the tumour cells to persist as apoptosis is inhibited (Chen et al., 2016). SurVaxM, a vaccine that immunizes patients with survivin proteins, has been developed. This allows the immune system to recognize survivin-expressing cells as “harmful” and kill the cells, leading to an anti-tumour effect. The clinical trial for SurVaxM is currently in progress and is combined with adjuvant Montanide ISA 51 administration to boost immune responses (NCT04978727). As the hallmark mutation in DIPG, many therapeutic developments have also focused on H3K27M-targetted therapies. This includes the use of H3K27M antigen vaccines (NCT04749641) (Zhang et al., 2023), CAR-T cells (Wang et al., 2023b), and combination therapies with immune checkpoint blockades (NCT02960230) (Grassl et al., 2023). Preliminary data from the NCT04749641 trial testing H3K27M antigen vaccines indicates that survival may be improved compared to other therapies (Zhang et al., 2023). Anti-H3.3K27M CAR-T cells that are specific to HLA-A∗02:01 have recently been developed (Wang et al., 2023b). HLAs, also known as human leukocyte antigens, are proteins that bind to peptides and subsequently present them on the cell surface for T cell recognition (Kulski et al., 2022). In this case, the CAR-T cells would only be able to recognize the H3.3K27M peptide in the context of the HLA-A∗02:01 complex. Wang et al. (2023b) evaluated the binding and recognition capacity of the CAR-T cells using DIPG cell lines. No binding was detected, and upon further investigation, they found that H3.3K27M peptides were not endogenously presented on HLA-A∗02:01 complexes in the DIPG cell lines. The authors concluded that anti-H3.3K27M CAR-T cells specific to HLA-A∗02:01 would not be a feasible immunotherapy for DIPG (Wang et al., 2023b). An ongoing clinical trial is investigating the safety and immune activity of a combination therapy using the H3.3.K27M peptide vaccine, poly-ICLC, and the PD-1 blockade antibody nivolumab. Poly-ICLC acts as an adjuvant to boost immune activity. At the same time, nivolumab can block immunosuppressive signalling, which should enhance the anti-tumour activity induced by the H3.3.K27M peptide vaccine (NCT02960230). Separately, Grassl et al. (2023) evaluated the combination therapy of H3K27M peptide vaccines with anti-PD-1 in DMG; some participants had tumours in the pons, indicating DIPG. Due to regulations, the authors could not conduct their study using a consistent anti-PD-1 treatment. Thus, the drugs used were based on availability. Overall, results indicate that the combination therapy is both safe and immunogenic. Additionally, peripheral immune activity decreased over time, along with the observed tumour regression, and tumour progression coincided with decreased immune responses (Grassl et al., 2023).

Autologous cellular vaccines and cell transfers allow for targeting of tumour- and patient-specific antigens. In cellular vaccines, the immune cells and tumour lysate containing the antigens are derived from patients. The cells are then incubated or “pulsed” with the lysate to improve tumour antigen-specificity and re-infused into the patient (Alaniz et al., 2014; Yan et al., 2020). Currently, the immunogenicity and safety of cellular vaccines have been shown in non-DIPG gliomas (Yan et al., 2020). An ongoing clinical trial (NCT04837547) is focusing on vaccination with autologous dendritic cells that have been pulsed with total tumour messenger ribonucleic acid (TTRNA-DC) derived from the patient, this allows the dendritic cells to become loaded with the antigens and can present them to T and B cells when re-infused into the patient (Yan et al., 2020). The same trial will stimulate autologous T lymphocytes ex vivo using total tumour messenger ribonucleic acid (TTRNA-xALT) and similarly transfer back into the patient. This clinical trial will primarily assess the safety and feasibility of these therapies. Separately, another clinical trial also focuses on vaccination with TTRNA-DC, but in combination with the cytokine GM-CSF as an adjuvant to boost immune response (NCT03396575). Benitez-Ribas et al. (2018) also published preliminary results demonstrating the safety of autologous dendritic cells pulsed with tumour lysates derived from allogenic DIPG cell lines (Benitez-Ribas et al., 2018).

There has also been interest in natural killer (NK) cells for cell transfers other than CAR-T cells. NK cells have increased anti-tumour activity compared to T cells in glioblastoma due to T cell targeting mutations in the tumour (Lieberman et al., 2019; Galat et al., 2023). Galat et al., 2023 differentiated human pluripotent stem cells into NK cells and assessed their cytotoxicity in vitro using DIPG cell lines. They found that the cells could successfully engraft in peripheral blood samples and are cytotoxic against DIPG cells. A separate study is focusing on AloCELYVIR, a novel therapy where oncolytic Adenovirus-infected bone marrow-derived allogeneic mesenchymal stem cells are transfused into the patient. These cells can enhance anti-tumour responses and transport oncolytic molecules to various tumour sites, enabling the killing of cancer cells in locations that are more difficult to reach with conventional therapy. Assessments for the safety and efficacy of this therapy in DIPG patients are currently in progress (NCT04758533).

There are also studies focusing on combination therapies, with the majority involving immune checkpoint blockades and cancer vaccines. A clinical trial evaluated the efficacy of nivolumab (anti-PD-1) with ipilimumab (anti-CTLA-4). However, no significant improvements in survival compared were noted (NCT03130959) (Dunkel et al., 2023). A novel neo-antigen heat shock protein vaccine (rHSC-DIPGVax) targeting different DIPG/DMG neo-epitopes has also been developed. Investigation into the vaccine’s safety and tolerability in combination with balstilimab (anti-PD-1) and zalifrelimab (anti-CTLA-4) is currently ongoing (NCT04943848). Dual checkpoint inhibitors have been shown to improve objective response rates in cervical cancer compared to standard treatments, although only in patients with PD-1-expressing tumours (O'Malley et al., 2022). Several clinical trials have also investigated combination therapies such as bevacizumab (anti-VEGF) with cetuximab (anti-EGF) (NCT01884740), as well as nivolumab (anti-PD-1) with bempegaldesleukin, which is an immunostimulatory IL2 pathway agonist (NCT04730349), but have since been terminated due to low accrual (NCT01884740) and change in business objectives (NCT04730349). Nivolumab with bempegaldesleukin showed efficacy in various solid tumours regardless of the PD-1 expression levels (Diab et al., 2020); this may be a potential area to explore as DIPG typically has low PD-1 presence (Lin et al., 2018).