Advancements and challenges in CAR T cell therapy for pediatric brain tumors: A review

Abstract

Chimeric Antigen Receptor (CAR) T cell therapy represents a groundbreaking advancement in immunotherapy, initially gaining FDA approval for treating hematological malignancies. This therapy has shown promising results in solid tumors, particularly in pediatric brain tumors, which are the leading cause of cancer-related death in children. CAR T cells are engineered to target specific antigens on tumor cells, thereby reducing off-target effects and increasing the cytotoxic impact on cancer cells. Over the years, CAR T cell technology has evolved through five generations, each enhancing the structure, functionality, and safety of these cells. Despite these advancements, the application of CAR T cells in solid tumors, especially within the central nervous system (CNS), faces significant challenges. These include the physical barrier posed by the blood-brain barrier (BBB), the immunosuppressive tumor microenvironment (TME), and the heterogeneity of tumor antigens. The review discusses several promising antigenic targets for CAR T cells in pediatric brain tumors, such as HER2, EphA2, IL-13Rα2, and Survivin, which have been explored in recent clinical trials. These trials have shown early promise in improving patient outcomes, though the risks of cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS) remain concerns. The future of CAR T cell therapy lies in overcoming these barriers through innovative approaches like “Armored CARs” or TRUCKs, designed to modulate the TME and improve CAR T cell efficacy in solid tumors. Additionally, combination therapies and safety switches in next-generation CAR T cells are being explored to enhance therapeutic potential while minimizing adverse effects.

Keywords

T-cells chimeric antigen receptor pediatric brain tumors solid tumor tumor microenvironment

Introduction

Chimeric antigen receptor (CAR) T cells are an example of adoptive cell therapy used in immunotherapy. CAR T cells were first licensed by the FDA in 2017 for hematological malignancies; however, numerous preclinical and clinical studies have demonstrated their efficacy in solid tumors such as glioblastoma, medulloblastoma, and ependymoma.^1–3

Pediatric central nervous system (CNS) tumors continue to be the largest cause of cancer-related death in children, highlighting the urgent need for novel therapeutics.⁴ To reduce recurrence, these new medicines must be directed particularly to malignant cells, restrict off-target cytotoxicity inherent in chemotherapeutics, and have a powerful, prolonged cytotoxic effect on cancer cells. CAR-T cells have the ability to achieve these goals.

The evolution of Chimeric Antigen Receptor (CAR) T cells has progressed through four distinct generations, each improving upon the previous in terms of structure and functionality.

First-generation CARs consisted of a simple design with an antigen-binding domain (scFv), a transmembrane region, and an intracellular signaling domain (CD3ζ). These CARs could activate the T cell receptor (TCR) but had limited activity and persistence of T cells.^5,6

Second-generation CARs introduced an additional co-stimulatory domain (such as CD28 or 4-1BB) alongside CD3ζ. This enhancement led to better proliferation and persistence of T cells, as well as improved cytotoxicity and cytokine secretion.^7–11

Third-generation CARs further advanced this concept by incorporating two co-stimulatory domains (CD28 and 4-1BB) in addition to CD3ζ. The synergy between these co-stimulatory signals resulted in even greater persistence and activity of T cells.^12–14

Fourth-generation CARs, also known as “Armored CARs” or TRUCKs (T cells Redirected for Universal Cytokine Killing), built upon the second or third-generation structures by adding genes that encode cytokines or other immunomodulatory molecules. These CARs can secrete cytokines locally to modulate the tumor microenvironment, significantly improving efficacy, especially in solid tumors.^15,16

Fifth-generation CARs incorporate elements from previous generations and include a cytokine receptor-based signaling domain. This addition enables the CAR T cells to respond not only to antigen recognition but also to cytokine signaling, further enhancing their proliferation, survival, and anti-tumor activity. These advanced CARs are designed to have improved control over the immune response, making them more effective and safer for clinical use.¹⁷

This evolutionary journey reflects the continuous advancements in CAR T cell technology aimed at enhancing the therapeutic potential and effectiveness of cancer immunotherapy . The development of CAR-T cell products is presented in Figure 1.

Overview of CAR-T cell targeting

The concept of engineering chimeric antigen receptors (CAR) has been around for over 25 years. It involves combining a single-chain variable fragment (scFv) of an antibody with the T cell receptor (TCR) signaling domain CD3, resulting in antibody-like antigen recognition and T cell cytolytic activity.^19,20 This ingenuity allows for the recognition of a target antigen without presentation by major histocompatibility complex (MHC).¹⁹ However, co-stimulation is still required for T cells to carry out their cytolytic function, multiply, and persist in the local microenvironment.²¹ These co-stimulatory signals are normally delivered by the antigen presenting cell, but in designed CAR T cell constructions, several co-stimulatory domains can be introduced to improve T cell activity.^22–24

For CAR T cell applications in cancer treatment, the modified target should only be present on tumor cells and not on normal cells, reducing off-target therapeutic effects.¹ In 2017, the FDA approved CAR T cell treatment for B cell malignancies that target CD19.²⁵ CAR T cell uses continue to increase in the clinical setting, particularly in the treatment of hematological malignancies.²⁶ Multiple clinical trials are underway to assess the efficacy of CAR T cell therapy in solid tumors, however the FDA has not yet approved CAR therapy for this indication. Many difficulties exist that impede the efficacy of CAR T treatment in solid tumors, including but not limited to the difficulty in trafficking the tumor site, presence of an immunosuppressive environment, toxicity, and tumor antigen heterogeneity^.²⁷

Solid tumors in the brain pose additional challenges due to the semipermeable features of the blood brain barrier (BBB), which impede the administration of many treatments.²⁸ The blood-brain barrier is made up of specialized endothelial cells that block big hydrophobic substances and undesirable cells from entering the brain. Brain cancers alter the BBB, resulting in the blood-tumor barrier (BTB), which has heterogeneous perfusion and permeability throughout the tumor and impedes therapeutic delivery.²⁹ CAR T cell infusion into the brain can occur through the blood, cerebral spinal fluid (CSF), or locally in the tumor cavity. The difference between the two barriers is illustrated in Figure 2.

Brain tumors are now the most prevalent solid tumor forms under clinical trial testing for CAR T cell effectiveness, and they have showed early promise in the treatment of glioblastoma (GBM).³¹ In this review, we focus on pediatric brain tumors as novel interventions are needed given the grim prognoses for many patients.

Pediatric brain tumors

Medulloblastoma

Medulloblastoma is the most frequent malignant brain tumor in children (10–20% of all pediatric brain tumors), with an incidence rate of 6.0 per million individuals aged 1 to 9 years.³² Until recently, medulloblastoma prognosis and categorization were mostly determined by histology, as well as age and metastatic status.³³ With improved access to advanced molecular genetic tools, medulloblastoma has been further categorized into various molecular subtypes (WNT, SHH, Group 3, and Group 4), throwing fresh light on possible treatment targets.³⁴ These tumors originate in the cerebellar vermis and consequently are only found in the posterior fossa.³⁵

Antigenic targets

Tyrosine-protein kinase receptor Though HER2 expression is present in about 40% of medulloblastomas, ERBB2 (HER2) expression is more commonly associated with a subset of breast malignancies.³⁶ ERBB2 protein is an appealing target for CAR T cell treatment since it is not found in the normal brain.³⁷

Pediatric ependymoma

Ependymomas rank third in frequency among pediatric patients with central nervous system (CNS) malignancies, accounting for 5.2% of all pediatric CNS cancers.^38,39 These tumors develop from cells that line the central canal of the spinal cord or the cerebral ventricles. The spinal column, the posterior fossa, and the supratentorium are the three main regional compartments into which ependymomas have been divided in recent studies. Roughly two-thirds of pediatric ependymomas originate in the posterior fossa.

Antigenic targets

Research has indicated that ependymomas exhibit elevated expressions of EphA2, IL-13Rα2, HER2, and Survivin.^40,41 As a result, these antigens could be useful therapeutic targets for CAR T cell mediated treatment. In xenograft models of ependymomas, CAR-T cells with trivalent targeting to EPHA2, HER2, and IL13Rα2 shown effectiveness.³ Even though some young patients with ependymomas may benefit from traditional therapy, recurrence is frequently fatal, requiring more research into innovative treatments and immunotherapy.

Pediatric high-grade gliomas

Pediatric High-Grade Gliomas (pHGGs) make up less than 20% of pediatric brain tumors. Based on 2021 WHO classification, they are classified to four types, including Diffuse midline glioma (DMG) H3 K27-altered, Diffuse hemispheric glioma H3 G34-mutant, Diffuse pediatric-type high-grade glioma H3-wildtype and IDH-wildtype, and infant-type hemispheric glioma.⁴² DMG H3 K27-altered arise in midline regions such as thalamus, brainstem, and spinal cord, lending to their inoperability. Thus far, no standard therapy for DMG has been proven to be beneficial, though radiotherapy, targeted chemotherapy, and several strategies with mechanism of cell cycle inhibitor or anti-angiogenesis are treatment options.^43,44

Antigenic targets

Recently, both GD2 and B7-H3 were found to be highly expressed in pediatric diffuse midline glioma (DIPG). Interestingly, Haydar et al. established a hierarchy of antigens expressions in pediatric brain tumors showing that, despite a high heterogeneity, GD2 and B7-H3 maintain the highest expression as compared to IL-13Rα2, HER2 and EphA2.⁴⁵ These data suggest the importance of focusing on these targets for pHGGs rather than antigens mostly relevant in adult gliomas.

Notable clinical trials

Three clinical trials are actively recruiting pediatric patients:

Phase 1 study of HER2-specific CAR T cell locoregional immunotherapy for HER2 positive recurrent/refractory pediatric central nervous system tumors (NCT03500991)

This is a Phase 1 study of central nervous system (CNS) locoregional adoptive therapy with autologous CD4 and CD8 T cells lentivirally transduced to express a HER2-specific chimeric antigen receptor (CAR) and EGFRt, delivered by an indwelling catheter in the tumor resection cavity or ventricular system in children and young adults with recurrent or refractory HER2-positive CNS tumors. A child or young adult with a refractory or recurrent CNS tumor will have their tumor tested for HER2 expression by immunohistochemistry (IHC) at their home institution or at Seattle Children's Hospital. If the tumor is HER2 positive and the patient meets all other eligibility criteria, including having a CNS catheter placed into the tumor resection cavity or into their ventricular system, and meets none of the exclusion criteria, then they can be apheresed, meaning T cells will be collected. The T cells will then be bioengineered into a second-generation CAR T cell that targets HER2-expressing tumor cells. The patient's newly engineered T cells will then be administered via the indwelling CNS catheter for two courses. In the first course they will receive a weekly dose of CAR T cells for three weeks, followed by a week off, an examination period, and then another course of weekly doses for three weeks. Following the two courses, patient's will undergo a series of studies including MRI to evaluate the effect of the CAR T cells and may have the opportunity to continue receiving additional courses of CAR T cells if the patient has not had adverse effects and if more of their T cells are available.⁴⁶

GD2-CAR T cell therapy for H3K27M-mutated diffuse midline gliomas (NCT04196413)

This phase I dose-escalation trial of autologous GD2-CAR T cells (containing a GD2 binding domain, a 4-1BB co-stimulatory domain and a CD3Ζ signalling domain) in children and young adults with pontine and spinal cord DMG characterized by a K27 M mutation in genes encoding histone H3 (H3K27 M) was designed with the primary objectives of assessing feasibility of manufacturing, safety and tolerability, and identifying the maximally tolerated dose or recommended phase II dose.⁴⁸ Assessment of clinical activity was a secondary objective and identifying biomarkers of response was an exploratory objective. We anticipated the development of neurological symptoms related to CAR T cell-mediated inflammation in sites of central nervous system (CNS) disease,⁴⁷ which we have termed tumour inflammation-associated neurotoxicity (TIAN). To mitigate risks associated with TIAN, we excluded patients with bulky thalamic or cerebellar tumours, required placement of an Ommaya reservoir in patients with DIPG to monitor intracranial pressure (ICP), and instituted a TIAN toxicity management algorithm incorporating the removal of cerebrospinal fluid (CSF) via Ommaya, hypertonic saline, anti-cytokine agents and corticosteroids.⁴⁸

Locoregional delivery of IL-13Rα2-targeting CAR-T cells in recurrent high-grade glioma: a phase 1 trial (NCT02208362)

This is a phase I study to evaluate memory-enriched IL-13Rα2-CAR-T cells for recurrent HGG (rHGG).⁴⁹ This trial enrolled heavily pretreated patients with no enrollment restrictions for tumor size, multifocal disease, prior bevacizumab or number of recurrences.⁵⁰ Trial eligibility criteria included confirmed IL-13Rα2 tumor expression, Karnofsky Performance Score (KPS) ≥ 60 and life expectancy >4 weeks.

Patients were treated at one of three dose schedules of weekly infusions and evaluated for 1 week after the third cycle for dose-limiting toxicities (DLTs). Additional infusions were allowed, and patients were followed for toxicities, response and survival until they progressed or required disallowed therapy. After patients went off protocol therapy, they were only followed for toxicities and survival, and not disease-response, since other therapies were allowed.⁵¹

CAR-T therapy continues to offer hope for long-term disease-free survival, however the final results will most likely be delayed due to the tumors’ rarity and lethality.⁵² A general view of the three clinical trials is presented in the Table 1.

Table 1.

General view summarizing the three clinical trials.

ClinicalTrials.gov Identifier	Study Title	CAR-T Cell Type	Summary	Results
NCT03500991	Phase I Study of HER2-Specific CAR T Cells in Pediatric Patients with HER2-Positive CNS Tumors	HER2-Targeted	This trial aims to evaluate the safety and feasibility of administering HER2-specific CAR-T cells in children with recurrent or refractory CNS tumors expressing HER2.	Early-phase results have shown that HER2-specific CAR-T cells can be safely administered with manageable side effects, and some patients have shown partial responses.
NCT04196413	GD2-Specific CAR T Cells for Diffuse Intrinsic Pontine Glioma (DIPG)	GD2-Targeted	This trial investigates the safety and preliminary efficacy of GD2-specific CAR-T cells in patients with DIPG, a highly aggressive pediatric brain tumor.	Preliminary results indicate that GD2-targeted CAR-T cells can infiltrate the tumor and may lead to tumor reduction in some patients.
NCT02208362	Phase I Study of IL13Rα2-Specific CAR T Cells in Pediatric Brain Tumors	IL13Rα2-Targeted	This trial tests the safety and efficacy of IL13Rα2-specific CAR-T cells in children with recurrent or refractory high-grade gliomas.	Early-phase data suggest that IL13Rα2-specific CAR-T cells can be administered safely and show potential for anti-tumor activity.

Treatment toxicity and risks

CAR T cell therapy can cause treatment-related toxicity. Cytokine release syndrome (CRS) is one of the most prevalent and severe side effects of CAR T cell therapy, resulting from systemic immune activation. While elevated inflammatory cytokines are to be expected, and they frequently cause mild flu-like symptoms, CRS has been linked to multiorgan system failure and mortality.⁵³ The most commonly observed cytokines are IL-10, IL-6, and IFN-γ. Corticosteroids and interleukin-6 inhibition are effective treatments for CRS.^54,55 Corticosteroids can have a deleterious impact on T-cell proliferation, hence doctors should seek alternative modalities of CRS therapy. Despite the availability of many treatment options, CRS can still be fatal and must be diagnosed early.⁵⁶

Another issue is neurotoxicity, often known as immune effector cell-associated neurotoxicity syndrome (ICANS). ICANS is the second most prevalent adverse event after CAR T-cell infusion. ICANS symptoms range from mild disorientation to comatose.⁵⁷ ICANS, like CRS, can be fatal, hence any neurological symptom that occurs after CAR T cell infusion should be considered probable ICANS unless proven otherwise. In a meta-analysis of CAR T cell trials for cancer, 55.3 and 37.2% of all patients had CRS and neurotoxicity, respectively.⁵⁸

Future Directions of CAR-T Cells

Tumor immunosuppressive microenvironment

The future of CAR-T cell therapy seems promising, with considerable developments aimed at addressing present limits and expanding its usefulness. One crucial area of exploration is the targeting of solid tumors, which have proven to be more resistant to CAR-T therapy than hematologic malignancies. Researchers are working to overcome the physical and immunosuppressive hurdles in the tumor microenvironment by developing CARs with improved homing capabilities and resilience to these barriers.

The intricate immune inhibitory network within the TME, known as the tumor immunosuppressive microenvironment, is comprised of various immune cells, secretions and inhibitory signals, all working together to facilitate tumor initiation and progression.⁵⁹ The suppressive immune cells within the TME, including tumor-associated macrophages (TAMs), regulatory T cells (Tregs), and myeloid-derived suppressor cells (MDSCs), exert their inhibitory effects on proliferation and effective antitumor response of effector cells.⁶⁰ These immune cells and tumor cells promote the production of immune-suppressive-related cytokines, like TGF-β, IL-10, and IL-4, which accelerate the exhaustion of T cells and CAR-T cells.⁶¹ CAR-T cells may exhibit increased PD-1 expression due to the TME. Consequently, the binding of PD-1 on CAR-T cells to PD-L1 on tumor cells initiates inhibitory signals, leading to compromised functionality of CAR-T cells⁶² and facilitating immune evasion by tumor cells.⁶³

MDSCs have been implicated in the poor response of solid tumor patients to immunotherapy. MDSCs suppress the immune response of effector T cells through diverse mechanisms, which involve the induction of Tregs, generation of reactive oxygen species, release of anti-inflammatory cytokines such as IL-10 and TGFβ, and exhaustion of essential amino acids required for T cell proliferation by inducing arginase and indoleamine 2,3-dioxygenase.⁶⁴ Furthermore, by producing matrix metalloproteinase-9 (MMP-9), MDSCs remodel the extracellular matrix (ECM), which promotes angiogenesis, tumor aggression, and spread.⁶⁵ Given these facts, researchers have been devoted to finding strategies to eliminate MDSCs from the TME.

Recent studies have indicated that constructing TRUCKs or armored CARs, which secrete cytokines or express ligands in a constitutive or inducible manner, is an appealing option for enhancing the effectiveness of CAR-T cells by engaging in endogenous immune responses or creating an immune-supportive environment.⁶⁶

Challenges and coping strategies of toxicities

To avoid long-term adverse outcomes caused by low-dose infusion and intense toxic reactions associated with high-dose infusion, a stepwise dosing regimen is recommended for the administration of CAR-T cells. The approach includes a sequence of CAR-T cell administrations with escalating doses, and halts the infusion if early clinical signs of CRS are detected, which shows promise in achieving a balance between the efficacy and safety of CAR-T cell treatment.⁶⁷

Furthermore, enhancing the CAR framework has been shown to mitigate toxic reactions. Research has shown that CAR-T cells containing the 4-1BB co-stimulatory region demonstrated lower levels of toxicity in comparison to CAR-T cells possessing the CD28 co-stimulatory domain.⁶⁸

Another interesting strategy is that the Next-generation CAR-T cells are being designed with built-in safety switches and dual-targeting capabilities to reduce severe side effects such as cytokine release syndrome (CRS) and neurotoxicity.

Combination therapies to enhance CAR T cells efficacy

Currently, numerous studies have suggested that the use of CAR-T cell therapy alone has shown limited efficacy in treating solid tumors. The combination of therapies presents possible approaches to enhance the efficacy of CAR-T cell therapy:

Combination with chemotherapy

TME is a significant barrier to CAR-T cell therapy efficacy. Chemotherapy has the ability to alter TME and enhance the effectiveness of CAR-T cell therapy. Docetaxel⁶⁹ and gemcitabine,^70,71 employed as neoadjuvant therapies, have shown the ability to enhance the effectiveness of GD2 CAR-T cells by decreasing MDSCs in the tumor. Studies have also demonstrated that oxaliplatin altered the TME by regulating the chemokine profile, resulting in increased recruitment of ROR1-CAR T cells.⁷² Furthermore, in a phase I clinical trial,⁷³ the exploration of a combined therapy involving CAR-T cells along with paclitaxel and cyclophosphamide revealed noteworthy clinical improvements in 21 of the 28 patients who had previously experienced unsuccessful paclitaxel therapy.

Combination with radiotherapy

A key issue in the progression and immune evasion of solid tumors is the polarization of the TME towards an immune-suppressive and tolerant phenotype,⁷⁴ leading to the transition of “hot” tumors to “cold” tumors. In addition to hindering the movement of CAR-T cells towards the center of the tumor, cold tumors also attract immune-suppressing cells, leading to the depletion of effector cells.

In the past few years, numerous studies have demonstrated that radiotherapy (RT) could transform “cold” tumors into “hot” tumors, allowing immune cells to penetrate tumor tissues and reshaping the immunosuppressive barriers in the local TME.⁷⁵ Hence, the fusion of RT and CAR-T treatment arises as a possibly advantageous approach to enhance the anti-cancer impact. Real-time imaging analysis in a glioblastoma model has shown that RT enabled the swift movement of CAR-T cells from the vascular system into the TME, as well as their amplification within the TME, leading to enhanced and prolonged immune responses.⁷⁶

Combination with oncolytic viruses

Oncolytic viruses (OVs) are regarded as a type of tumor immunotherapy due to their potential impact on immune activation and virus-mediated tumor cell death.⁷⁷ OVs therapies, including oncolytic adenovirus (OAV), face several challenges, such as poor infiltration ability, antiviral immune response, off-target infection, inhibitory TME, and lack of specific targets.⁷⁸ Given these limitations, the benefits of OV monotherapy remain limited and insufficient for tumor clearance. Nevertheless, it can function as a supplementary approach to enhance the anti-cancer impacts of alternative therapies, such as CAR-T cell therapy. Conceptually, OVs can alleviate the obstacles of CAR-T cell therapy by many ways including: direct tumor cells lysis and release of tumor neoantigens; activation of local host innate immune responses, and subsequent cytokine release and recruitment of various immune cells; carrying viral-encoded transgenes to ‘reprogram’ TME into a pro-inflammatory environment.⁷⁹

Conclusion

Over the last decade, tremendous progress has been achieved in the development of CAR-T cell therapy for brain tumors, providing fresh hope for treating these difficult illnesses. While the outlook for many children brain tumors remains bleak, CAR-T cell treatment shows great potential. Ongoing preclinical research is critical for closing current gaps, such as refining delivery modalities, understanding the tumor microenvironment, discovering new targets, and boosting overall efficacy. Despite these obstacles, the future of CAR-T cells in pediatric brain tumor management is hopeful, with more research expected to improve therapeutic outcomes.

Footnotes

Authors contributions

YG Proposed the subject and outlined the structure of the review. AM Contributed to data collection and analysis and assisted with the literature search. YG Synthesized the information from the literature, and drafted the manuscript. ZA and LY revised the last article version. YR and MB checked initial version of the study and approved the final version.

Acknowledgements

Thanks to all Ibn Sina Hospital Center Pharmaceutical staff.

Authors’ note

The views expressed in this article are those of the authors alone and don’t necessarily reflect those of their respective employers.

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The authors received no financial support for the research, authorship, and/or publication of this article.

ORCID iD

Yasmina Gaoual

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