CAR T cells for pediatric CNS tumors: Clinical experience and reverse translational opportunities.

Introduction

Central nervous system (CNS) tumors are one of the leading causes of cancer-related mortality in children (0–14 years) and the second most leading cause of cancer-related death in adolescents and young adults (15–39 years). 1 In pediatric patients, gliomas and embryonal tumors account for 50% of CNS tumors. Diffuse intrinsic pontine glioma (DIPG), a diffuse midline glioma (DMG) originating in the pons, is the most common brainstem tumor 2 with median survival of 11–13 months and medulloblastoma is the most common embryonal tumor with median survival of 10–27 months in relapsed disease. 3 , 4 , 5 Despite decades of research, effective treatments for high-grade gliomas (HGGs) are still lacking and new therapeutic options are urgently needed.

Immunotherapy has revolutionized the treatment of oncologic diseases and chimeric antigen receptor (CAR) T cell therapies have transformed the outcomes in hematologic malignancies. Hence, multiple CAR T cell products are now FDA-approved, including CD19 CAR T cells for refractory adult leukemia/lymphomas, 6 , 7 , 8 , 9 , 10 BCMA CAR T cells for multiple myeloma, 11 , 12 and one CD19 CAR T cell product (tisagenlecleucel) for pediatric leukemia patients. 13 Given these successes in hematologic malignancies, CAR T cell therapy for solid and CNS tumors has become a major research priority, with multiple clinical trials now demonstrating initial safety. However, CAR T cells must overcome many challenges when it comes to solid and CNS tumors. These include tumor intrinsic factors, including tumor heterogeneity and antigen escape, T cell intrinsic factors, including T cell trafficking, expansion, and persistence, and the immune environment, including suppressive immune cells, metabolic, and cytokine milieu. 14

In CNS tumors, there is inherent tumor heterogeneity, 15 , 16 which makes optimal antigen selection a challenge in CAR T cell therapy and provides a mechanism for resistance through potential loss of the targeted antigen. 17 , 18 Beyond inherent tumor-intrinsic barriers, there are also T cell-specific challenges, such as CAR T cell trafficking to the tumor, 18 which is partially being addressed by multiple delivery approaches (intravenous [IV], intracerebroventricular/intraventricular [ICV], intratumoral (iTu), or a combination). Despite tumor trafficking, CAR T cell expansion and persistence remain challenging in the CNS environment, with many clinical trials administering repeated CAR T cell infusions. Finally, CNS tumors exist in a distinct CNS immune environment that is generally suppressive, with a paucity of infiltrating T cells 19 and may also augment systemic immunosuppression by sequestrating naive T cells in the bone marrow. 20 The CNS immune environment is distinct with specialized immune cells at brain border immunologic niches, tightly regulated bi-directional communication, and regulation between the CNS and the immune system. 21 Together these potential contributors to CAR T cell function in patients must be understood to optimize durable activity of CAR T cells for patients.

To define the drivers of CAR T cell success or failure in patients, the field has begun to integrate reverse translation into pediatric and adult clinical trials, applying clinical research insights from bedside-to-bench to understand the mechanisms associated with CAR T cell function in patients. Reverse translational studies can offer valuable insights into the drivers of CAR T cell activity in patients. Specifically in CNS tumors, where the CNS environment adds to the complexity of CAR T cell function, reverse translational studies provide a unique opportunity to define the dynamics of CNS immune biology in the context of CAR T cells.

In this review, we will discuss current CAR T cell clinical trials with a focus on trials in the pediatric CNS tumor space. In addition, we will examine insights gleaned from reverse translational studies in these trials, which is central to iterative improvements in CAR T cell therapies for all patients. Employing the full cycle of translational research, novel CAR T cell therapies are being tested in the clinic with a goal to improve patient outcomes. Combining clinical experience with reverse translational investigations, the CNS CAR T cell field is poised to accelerate discoveries from each patient to advance CAR T cell therapies for patients with CNS tumors.

Initial CAR T cell trials in adult CNS tumors

The first published CAR T cell trials for CNS tumors were in adult patients with HGGs. The most common malignant adult brain tumor is glioblastoma (GBM), which comprises most adult HGGs and has a dismal median overall survival (OS) of 15–21 months. 1 , 22 , 23 These early trials demonstrated feasibility of CAR T cell administration in CNS tumor patients and provided initial insights into mechanisms of resistance to CAR T cell treatments. We will review the initial adult CAR T cell trials for CNS tumors to provide context for the development of CAR T cells in pediatric CNS tumors.

IL-13Rα2

One of the first trials tested interleukin (IL)-13Rα2 CAR T cells in WHO grade IV glioma patients 24 ( NCT02208362 ). IL-13Rα2 is a promising target for CAR T cell therapy since it is highly expressed in approximately 60% of WHO grade IV gliomas with low expression in normal brain tissue. 25 , 26 In addition, preclinical models showed robust anti-tumor activity in mouse models with intracranial administration. 24 The first 3 HGG patients were treated via iTu infusions with no preceding lymphodepleting (LD) chemotherapy. There were no dose-limiting toxicities (DLTs) in the first 3 patients although one patient had grade 3 neurotoxicity with shuffling gait and tongue deviation that resolved with dexamethasone. Tumor tissue after treatment showed decreased IL-13Rα2 expression, indicating potential on-target effect of the CAR T cell and hinting at potential antigen loss as a resistance mechanism. A subsequent case report from the same trial treated a recurrent GBM patient using repeated intracranial IL-13Rα2 CAR T cell infusions, initially iTu then switching to ICV due to concern of leptomeningeal disease spread, resulting in a durable complete response (CR) for 7.5 months. 27 The final results of the phase 1 trial with IL-13Rα2 CAR T cells included 65 patients with recurrent HGG 28 ( NCT02208362 ). The study consisted of 5 separate arms: arm 1, iTu after biopsy; arm 2, iTu after maximal resection; arm 3, ICV; and arm 4/5, dual iTu/ICV Arms 1–4 used a manufacturing platform consisting of CD62L + CD45RA-enriched central memory T cells (Tcm), while arm 5 utilized CD62L + enriched naive, stem cell memory and central memory T cells (Tn/mem) for manufacturing CAR T cell product. Of the 58 evaluable patients, 29 (50%) had stable disease (SD), 3 patients with IDH-mutant tumors had 2 partial response (PR), and 1 CR. Arm 5 showed an improved OS of 10.2 months as compared to 7.5 months in arms 1–4, with ongoing studies evaluating the recommended phase 2 dose. The initial phases of the trial generated much excitement for CAR T cells as a feasible approach to CNS tumors, especially with one patient demonstrating CR for 7 months. Key insights from the IL-13Rα2 CAR T cell trial highlighted the early promise of IL-13Rα2-targeted CAR T cells, providing rationale for further clinical investigation.

EGFRvIII

Another target of initial CAR T cell trials was epidermal growth factor receptor variant III (EGFRvIII), a variant EGFR protein formed due to in-frame deletion of exons 2–7 in the extracellular domain creating a new glycine junction between exons 1 and 8. EGFRvIII is highly expressed in 30%–50% of GBMs with low expression in normal brain. 29 , 30 , 31 Preclinical mouse models showed an 80% decrease in tumor reduction. 30 The subsequent clinical trial for adult patients with recurrent GBM tested EGFRvIII CAR T cells infusion IV with no LD chemotherapy ( NCT02209376 ). 17 While patients had no DLTs defined on the study, 1 patient had grade 3 seizures, 1 patient had grade 3 cerebral edema, and 1 patient had post-operative iTu hemorrhage. A subset of patients had early surgery due to symptomatic progression, and another subset had late surgery due to concern for recurrent disease. EGFRvIII expression decreased in 5/7 patients with tumor tissue sampling, suggesting potential tumor-intrinsic mechanisms of antigen escape. EGFRvIII CAR T cells were detected in brain tumor specimens up to 2 months post-infusion and up to 3 months in peripheral blood by qPCR, suggesting T cell trafficking to tumor tissue and persistence in blood months after IV infusion. One patient survived 36 months with persistent CAR T cells detected in the peripheral blood up to 29 months post-infusion. 32 Tumor tissue evaluated 104 days after CAR T cell infusion demonstrated upregulation of immunosuppressive molecules such as indoleamine 2,3-dioxygenase 1 (IDO1), tryptophan 2,3-dioxygenase (TDO), programmed cell death ligand 1 (PD-L1), transforming growth factor β (TGF-β), IL-10, and FoxP3, suggesting changes in the CNS immune environment that may contribute to resistance mechanisms. While there were no DLTs and some hints of potential resistance mechanisms on the initial trial with EGFRvIII, another trial utilizing EGFRvIII CAR T cells unfortunately demonstrated severe fatal toxicity. 33 In that trial, EGFRvIII CAR T cells were infused IV after LD chemotherapy, and IL-2 was administered 24 h after cell infusion. Of the 18 treated patients, no patients experienced an objective response and the median progression-free survival (PFS) was 1.3 months. Two patients developed hypoxia, with one patient at the highest dose level unfortunately dying from pulmonary edema, suggesting congestion of pulmonary vasculature by activated T cells. 33 Together, these two trials demonstrate the range of potential activity and toxicity in CAR T cell trials and highlight the need to understand how dose, route, and supporting medications associate with CAR T cell toxicity.

As a whole, initial adult experience with CAR T cells for CNS tumors ( Table 1 ) has demonstrated feasibility of administering this therapy, while highlighting the importance of understanding and managing toxicities. They also provide some evidence of CAR T cell activity against tumor cells and suggest potential resistance mechanisms, with contributions from both tumor intrinsic and CNS immune environment factors. Tumor assessment identified loss of IL-13Rα2 24 and EGFRvIII 17 antigen expression in patient tumor samples 17 , 28 after treatment as well as increased immunosuppressive mechanisms that may contribute to limited durable response for patients. 17

Based on these data, more recent trials have implemented optimized tactics to address potential immunosuppressive mechanisms of resistance. EGFRvIII CAR T cells have been combined with a PD-1 immune checkpoint inhibitor, pembrolizumab, to treat newly diagnosed GBM patients ( NCT03726515 ). 34 While no DLTs were reported, one patient experienced liver toxicity related to pembrolizumab. The post-treatment tumor tissue demonstrated antigen loss in 6/7 patients and CAR T cells were identified in 1 patient tumor sample. Post-treatment tissue showed increased activation of the myeloid cells and inflammation, but also increased exhaustion markers. However, there was a lack of clinical efficacy, with median PFS) of 5.2 months and OS of 10.2 months. 34 As a result, another approach utilized a dual-targeting bivalent CAR T cell, targeting EGFRvIII and IL-13Rα2 in patients with recurrent GBM via ICV infusions. 35 Of the 18 patients treated, there was 1 DLT (anorexia, weakness, and fatigue >7 days) at the maximum tolerated dose (MTD, 2.5 × 10 7 cells) and all patients experienced neurotoxicity that was manageable with steroids, anakinra, and supportive care. Median OS was not reached at the data cutoff of 8.1 months with 1 patient showing PR, 8 patients with SD, and 4 patients with progressive disease (PD). Another optimization strategy tested EGFRvIII CAR T cells that secreted a T cell-engaging antibody against wild-type EGFR (CARv3-TEAM-E). 36 The first 3 patients treated via ICV infusion had no DLTs. One patient demonstrated tumor reduction that was durable at 150 days, and 2 patients had initial radiographic reduction with subsequent disease progression. Overall, each optimized CAR T cell trial attempted to address specific identified mechanisms of tumor resistance and showed the potential for reverse translational insights to guide iterative improvements of these therapies for patients.

While initial CAR T cell trials were predominantly in the adult space, CAR T cells have now been tested across many trials in the pediatric space. Given the pace of new and active phase 1 trials in both adult and pediatric population, the field is poised to establish shared lessons learned between pediatric and adult trials.

Pediatric CNS CAR T cell trials

Multiple trials are active for the pediatric population across many CNS tumor types, including DMG, medulloblastoma, and ependymoma ( Table 2 ). Similar to adult CAR T cell studies, challenges of tumor heterogeneity and the immunosuppressive microenvironment apply to pediatric CNS tumors. Pediatric and adult HGGs predominantly differ in terms of tumor histology and genetic mutations, for example PDGFRA mutations are more common in pediatric brain tumors, while TERT mutations are predominantly in adult brain tumors. 37 , 38 While research on tumor microenvironment and its immunosuppressive nature is ongoing, there are hints that the pediatric tumor immune landscape may also differ from adult brain tumors, such as higher number of myeloid cells in pediatric tumors compared with adult tumors. 39 The CNS tumor immune environment may also differ based on disease type, for example increased tumor associated macrophages (TAM) in pediatric low and high grade gliomas compared to pediatric DIPG. 40 In addition, pediatric patients pose unique clinical challenges including difficulty obtaining consistent serial neurologic examinations in younger children. As a result, some pediatric trials enrolled initial cohorts targeting patients in their teens. 41 Similar to adult studies, pediatric CNS CAR T cell trials began with single-target CAR T cells, but are now optimizing CAR T cell function using tumor microenvironment targeting strategies or multi-valent CAR T cells to address the immune microenvironment or antigen escape, respectively.

HER2

HER2 CAR T cell therapy was also examined early in the field of CAR T cell trials with initial results published in 2017 that included both pediatric and adult patients. 42 HER2 serves as an appealing target given lack of expression in normal tissue and high expression across multiple CNS tumors including GBM and medulloblastoma. 43 , 44 Virus-specific T cells (VST) transduced with CAR-T cells 42 ( NCT01109095 ) were employed to treat 7 pediatric and 10 adult patients with recurrent GBM. Patients could receive up to 6 IV infusions with no preceding LD chemotherapy. Of the 16 evaluable patients, 7 had SD and one had PR. HER2-CAR VSTs were detected in peripheral blood for up to 12 months by qPCR. Magnetic resonance imaging (MRI) at 6 weeks detected some peritumoral edema, suggesting either inflammatory response or disease progression. HER2 CAR T cells were also assessed in a phase 1 trial specifically in pediatric patients with recurrent/refractory CNS tumors. 41 The initial report treated 3 pediatric/young adult patients (1 anaplastic astrocytoma, WHO grade III and 2 ependymoma, WHO grade III) with repeated ICV or iTu infusions without LD chemotherapy. Patients received weekly ICV or iTu infusions on an intra-patient dose escalation regimen up to 18 doses. Toxicities included grade 2–3 headaches as well as grade 1–2 transient worsening of neurologic changes, but the therapy was generally well tolerated with no DLTs. In terms of response, 1 patient had SD, and 2 patients had PD. Initial studies with HER2 CAR T cells in CNS tumors appear safe and may have a wide applicability across diverse range of CNS tumors, with further studies necessary to determine efficacy against CNS tumors.

B7-H3

B7-H3, an immune checkpoint molecule, is another antigen of interest expressed across pediatric and adult gliomas 45 , 46 without significant expression on normal adult brain tissue. 47 Use of B7-H3 CAR T cells in preclinical orthotopic mouse models showed efficacy for GBM 47 as well as in pediatric HGGs. 45 As a result B7-H3 CAR T cells have been investigated in a phase 1 trial for DIPG with the initial 3 patients described in an initial report 48 , 49 followed by a description of the full phase 1 cohort of 21 patients. 23

This phase 1 trial enrolled pediatric patients (age 1–26), treated with repeated ICV infusions without preceding LD chemotherapy every 14 days over 8 weeks. Patients were allowed to be on a stable dose of dexamethasone ≤2.5 mg/m 2 and could receive additional infusions every 2–4 weeks if they continued to be eligible. Of the 21 patients, 15 had molecular confirmation of H3K27M DMG diagnosis. Patients in the initial cohort received 1 × 10 7 cells and cohorts 2–4 used an intra-patient dose escalation of 2.5 × 10 7 cells, 5 × 10 7 cells, and 10 × 10 7 cells. There was a DLT in the 2nd cohort due to an intra-tumoral hemorrhage. The most frequent adverse events (AEs) attributable to therapy included headaches, nausea/vomiting, fatigue and fever, which was thought to be due to local inflammation rather than cytokine release syndrome (CRS). One patient developed hydrocephalus, grade 3. No patients had immune effector cell associated-neurotoxicity syndrome (ICANS). Median survival from diagnosis to death was 19.8 months and 10.7 months from initial CAR T infusion to death. Three patients were still alive at 44.5, 45.6, and 52.5 months from diagnosis. Of 18 patients evaluable, best imaging responses were 1 PR, 15 SD, and 2 PD. These responses to B7-H3 CAR T cells in DIPG patients represent meaningful improvement over historical controls (median survival 11 months), warranting continued investigation to optimize durable responses.

GD2

GD2, a disialoganglioside, is highly expressed in both adult and pediatric CNS tumors. 45 , 50 , 51 In preclinical models, GD2 CAR T cells demonstrated robust killing of DMG tumor cells in murine models 50 and tumor reduction in GBM mouse models, 51 resulting in clinical translation for patients. For DMG, a phase 1 trial treated patients with preceding LD chemotherapy, followed by an IV infusion and subsequent repeated ICV infusions. 52 In the initial report of 4 patients, one patient in active progression did not have a response, but the other 3 patients demonstrated clinically and radiographic benefit. 52 A follow-up report of the initial 11 patients used two dose levels, with DLT at the higher dose of 3 × 10 6 kg −1 CAR T cells IV due to CRS. 53 Four patients had major radiographic reductions in tumor volume (50%–100%) with 1 patient achieving a CR that was durable for >30 months. Median OS was 20.6 months, and 9/11 patients had neurologic benefit. This study demonstrated consistent clinically meaningful benefit for DMG patients, but requires validation in larger studies.

Another approach utilizing GD2 CAR T cells also aimed to target the tumor microenvironment utilizing constitutively active IL-7 receptor (C7R-GD2 CAR T cells), but at slightly lower doses compared to the prior study. 54 The phase 1 trial described 11 patients (age 4–18) in an initial cohort of GD2 CAR T alone at 1 × 10 7 cells followed by C7R-GD2 CAR T cells in two dose levels (1 × 10 7 and 3 × 10 7 cells). Patients were treated with standard LD chemotherapy followed by IV administration of GD2 CAR T cells and could be eligible for subsequent infusions. Patients who received C7R-GD2 CAR T cells demonstrated transient improvement of neurologic deficits with partial responses in 2/7 patients. These GD2 CAR T cells for DMG illustrate the promise of these therapies, with a hope for iterative optimization to enhance durable responses.

CAR T cells for pediatric CNS tumors have demonstrated initial safety with activity seen in a subset of patients. Ultimately, while the majority of active CAR T cell trials remain phase I clinical trials, phase II clinical trials will be necessary to achieve a complete understanding of efficacy and implementation. Phase II clinical trials will likely necessitate new infrastructure, including support for multi-site trials. As CAR T cells advance into multi-site trials, it will be imperative to standardize toxicity management, evaluate efficacy, and understand mechanisms of tumor resistance, within and across trials.

Challenges in clinical application of CAR T cells for CNS tumors

Toxicities

As clinical experience with CAR T cells in CNS tumors expands, there is a growing need to define the grading and management of unique toxicities in these patients. Toxicities of CAR T cell therapy were originally defined in hematologic malignancies, including CRS, immune-effector cell-associated neurotoxicity syndrome (ICANS), 55 and immune effector cell-associated hemophagocytic lymphohistiocytosis-like syndrome (IEC-HS). 56 In CAR T cell therapy for CNS tumors, systemic toxicities such as CRS have been described, but are potentially more common with IVIV CAR T cell administration. 53 , 54 On the contrary, IEC-HS has not been commonly described in CNS CAR T cell trials, particularly in ICV/iTu infusions, but vigilance for such toxicities will be important as more patients are treated. Patients with CNS tumors can be uniquely challenging since almost all patients have baseline neurologic deficits, which further complicate delineation of neurotoxicity, pseudo-progression, and PD. However, these are important toxicities to understand and manage as further clinical trials are explored in the CNS space.

Distinct neurologic toxicities have been evaluated in CAR T cell therapies for CNS tumors, including ICANS and a new neurologic toxicity termed tumor inflammation-associated neurotoxicity (TIAN). 57 While ICANS was first described in early CAR T cell trials for hematologic malignancies, the exact pathophysiology continues to be evaluated, with the driving hypothesis suggesting blood-brain barrier (BBB) dysfunction as well as endothelial dysregulation. 58 Increased BBB permeability may lead to enhanced cytokine trafficking to the CNS space, thereby contributing to the manifestation of ICANS. Some studies identified CD19 expression on mural cells, which line blood vessels and maintain the BBB, suggesting on-target off-tumor toxicity. 59 Other studies are investigating the role of pre-existing neurologic injury in ICANS 60 and the role of CXCL16/CXCR6 in recruitment of cytotoxic T cells that can contribute to ICANS. 61 On the contrary, TIAN is the result of localized tumor inflammation and edema, which increases risk of intracranial pressure due to space constraints in the skull (type 1) and neurologic dysfunction leading to worsening of pre-existing neurologic symptoms depending on tumor location (type 2). 57 As a newly described toxicity, the pathophysiology of TIAN has yet to be determined with further investigations ongoing to understand the mechanistic drivers of this unique toxicity.

Clinically, most patients with CNS tumors develop neurologic symptoms after CAR T cells, but distinguishing ICANS versus TIAN can be difficult depending on tumor location and symptoms. TIAN is based on location of tumor and may vary based on tumor location or mimic symptoms of ICANS. For example, if language areas of the brain are involved, aphasia, a known symptom of ICANS, may develop with TIAN. Additionally, multifocal disease may lead to encephalopathy with TIAN and increased intracranial pressure from TIAN can lead to somnolence, both of which are clinical symptoms of ICANS. Differentiating the etiology of toxicity symptoms in patients can help to direct treatment approaches, which may include cerebrospinal fluid (CSF) diversion, dexamethasone, and anakinra. Tocilizumab, traditionally used for CRS, does not have CNS penetration and thus is not an ideal management for TIAN, which is localized inflammation from CAR T cell therapy. Conversely, ICANS is thought to originate from a systemic inflammatory state that traffic into the brain. 55 Depending on the pathophysiology of these toxicities, there is hope in the field to identify more targeted treatments for neurotoxicity management that will improve the safety profile of these therapies. Finally, the field is moving toward standardizing TIAN grading, and incorporating this assessment up-front in clinical trial design. Implementing standardized grading criteria across clinical trials will enhance identification of this toxicity, providing context for establishing management guidelines and evaluating mechanistic insights from patients.

Lymphodepleting chemotherapy

As demonstrated in the summary of pediatric clinical trials ( Table 2 ), there is variability between studies in both LD chemotherapy administration and route of CAR T cell delivery. Standard LD chemotherapy, consisting of cyclophosphamide and fludarabine, has been established for all FDA commercial products for hematologic malignancies 62 and has been shown to be important in peripheral solid tumors. 63 However, the role of LD chemotherapy in CNS CAR T cell therapy is an active question under investigation and studies vary in terms of addition or omission of LD chemotherapy regimen ( Table 2 ). One major question is understanding the role of LD chemotherapy in CNS tumors. Future research comparing LD versus no LD chemotherapy with CNS tumor CAR T cells will be required to provide insights into this question. Understanding whether LD chemotherapy creates the cytokine milieu for CAR T cell expansion or resets the immune microenvironment can help to guide potential alternative approaches in the future.

Route of CAR T cell delivery

In addition, the route of delivery of CAR T cells to CNS tumors is another consideration, including IV, ICV, iTu, or a combination. Most CAR T cell trials in CNS tumors are administering repeated infusions of CAR T cells to patients, but the spacing of these infusions is not standardized. As multiple infusions are being prepared, methods such as thaw-and-dilute have been found to be easier to implement compared to traditional thaw-and-wash techniques. 64 Route of delivery also varies across trials. In hematologic and peripheral solid tumors, IV CAR T cell infusion would be logical. However, for CNS tumors, both IV and ICV CAR T cell infusions have shown clinical activity. Interestingly, preclinical studies have indicated ICV delivery may lead to more efficient trafficking to the tumor site. 65 Which approach is optimal may be different based on CAR T cell and tumor type, but can only be investigated through consistent assessment of CAR T cell kinetics and functional activity within and across trials. As clinical trial experience expands, questions on route of CAR T cell delivery may be clarified using collaborative analyses of data across trials.

Target antigen identification

Another challenge for CNS CAR T cells is the target antigen for CNS tumors. Since the mutational burden of CNS tumors is low compared to other solid tumors, 66 neoantigens are less prevalent for target antigen identification. Fortunately, despite this potential limitation, a number of promising targets are being evaluated as targets for CAR T cells. To identify new targets for CNS tumors, researchers must find targets highly expressed on tumor cells with low or negative expression on normal tissue. In pediatric CNS tumors, a promising new target under clinical trial investigation is GPC2, an oncofetal protein with preclinical data demonstrating expression in medulloblastoma and embryonal tumors and limited expression in normal tissues after birth ( NCT07087002 ). 67 In GBM, ephrin type A receptor 2 (EphA2) demonstrates high expression in GBM and low expression in normal brain tissue. 57 Additionally, in pediatric HGG, CD70 CAR T cells are currently under investigation in an active clinical trial ( NCT06946680 ). 68 While single-antigen targeting CAR T cells represented the initial experience, a multivalent quadrivalent B7-H3, EGFR806, HER2, and IL-13-Zetakine CAR T cell clinical trial is evaluating approaches to address tumor antigen heterogeneity ( NCT05768880 ). These studies will broaden potential antigen targeting in CAR T cell therapy and provide increased opportunity for treatment options for patients.

While initial feasibility and safety were demonstrated for CAR T cells for pediatric and adult CNS tumors, the field now confronts the complex challenge of optimizing multiple variables simultaneously to achieve consistent, durable responses. Critical questions include determining the role of LD chemotherapy, establishing the optimal route of delivery, standardizing dosing protocols, and expanding target selection. As trials develop experience with unique CNS toxicities, standardized guidelines for toxicity grading and management will lay the foundation for expanding access to these therapies. Initial limited reverse translational insights have identified antigen escape and suppressive CNS immune environments, thereby providing rationale for optimizing CAR T cell trials. As described below, harmonizing and expanding reverse translational assessments into clinical trials will be invaluable to establish generalizable lessons from larger numbers of patients. Indeed, as more clinical evidence accumulates across different trials, reverse translation can provide important insight into mechanisms of toxicity, response, and progression in patients.

Reverse translation in CAR T cells for CNS tumors

To date, CAR T cell trials in CNS tumors have shown glimmers of clinical activity in a subset of patients, with select individuals achieving dramatic responses. However, inconsistency of responses across patients highlights key unanswered questions about the immune biology governing CAR T cell success or failure in patients. Each patient enrolled in these clinical trials represents a valuable opportunity to understand the mechanisms governing CAR T cell activity, tumor response, and the complex CNS immune environment. Recent advances in multi-dimensional assays are enabling researchers to systematically dissect the underlying biology driving therapeutic outcomes through reverse translation ( Figure 1 ). Reverse translation provides the most insight when systematically integrated into clinical trials up-front with consistent pre-defined and pre-specified trigger time points of collection. Applied across CNS tumor CAR T cell trials, reverse translational studies could define generalizable principles of the immune biology of CAR T cells in CNS tumors. Here, we will discuss these reverse translational correlative analyses across adult and pediatric studies, considering both widely accepted and more uniquely applied approaches.

Sample handling

The first step to reverse translation is high quality sample collection and processing to allow for downstream analyses. In CNS tumors, the tissues of patient sample collection include peripheral blood, CSF, and tumor ( Figure 1 ). The processing of each of these tissue types allows for different superimposed assays to be utilized. In the field, harmonizing sample handling and assays provides the opportunity to conduct individual trial analyses and inter-trial comparisons and meta-analyses. 69 Divided by tissue type, peripheral blood can be processed for plasma/serum and viable peripheral blood mononuclear cells (PBMCs), both of which can be cryopreserved for downstream analyses. CSF processing, however, is much more complex. While CSF supernatant can be processed and stored, the cellular compartment is limited in cell number and therefore must be processed carefully for adequate yield. To address unique CSF issues, researchers have incorporated fresh processing of CSF for single-cell RNA sequencing (scRNA-seq), an approach that is setting the stage for more in-depth understanding of the CNS immune biology surrounding CAR T cell infusions in patients. 52 , 70 , 71 As methods improve, it is possible that cryopreservation of CSF cells may be feasible with adequate yield for downstream assays. 72 Finally, tumor samples can be processed fresh, frozen, or preserved in formalin-fixed paraffin-embedded (FFPE). Depending on how samples are processed, different assays can be employed to evaluate cellular, cell-free, and tumor tissue compartments. 69 Superimposing these data with clinical metadata collected surrounding treatment can provide valuable insights into T cell intrinsic, CNS immune environment, and tumor-intrinsic factors that contribute to CAR T cell activity in CNS tumors.

T cell intrinsic factors

To evaluate T cell intrinsic mechanisms contributing to CAR T cell function in CNS malignancies, reverse translational studies assess CAR T cell kinetics, phenotype, and function. CAR T cell persistence has been a well-documented correlate to clinical outcome in hematologic malignancies and has been associated with prolonged time to progression and improved patient outcomes in solid tumor CAR T cell clinical trials. 73 , 74 , 75 , 76 In CNS tumors, CAR T cell clinical trials often utilize multiple repeated infusions, suggesting the need for ongoing immune pressure from CAR T cells to maintain tumor control.

To evaluate CAR T cell expansion and persistence, clinical trials utilize qPCR and flow cytometry assays on the cellular compartment of peripheral blood and CSF. 17 , 24 , 33 , 41 , 42 , 48 , 49 , 52 , 53 , 54 , 77 Learning from hematologic malignancies, while flow cytometry is useful to track the initial expansion kinetics of CAR T cells in the first month after treatment, qPCR provides more sensitive tracking of CAR T cells after that initial expansion. 13 , 78 In CNS tumors, a similar kinetic difference has been identified. By applying qPCR and flow cytometry to both peripheral blood and CSF samples, studies have demonstrated initial expansion and signals of CAR T cell persistence.

Since CNS CAR T cells can be delivered IV, ICV, or iTu, qPCR and flow cytometry of CAR T cells have provided important information about the expansion, persistence, and trafficking of CAR T cells to CNS tumors based on route of administration. 17 , 53 IVgr CAR T cell infusions demonstrate expansion and persistence in the blood, with increased sensitivity of qPCR over flow cytometry at later time points. 17 , 33 , 42 , 77 While some studies detected CAR T cell persistence up to 6 weeks post infusion, 17 , 79 others tracked CAR T cell persistence up to a year later. 42 Although dose level has not consistently been associated with persistence, one trial suggested a correlation of CAR T cell persistence with cell dose. 33 Interestingly, resected tumor tissue samples after IV CAR T cell treatment evaluated by RNAscope in situ hybridization (RNAscope ISH) or qPCR have demonstrated evidence of CAR T cell infiltrations in EGFRvIII, 17 IL-13-Zetakine, 24 and GD2 52 CAR T cell trials. Together, these data demonstrate that IV CAR T cell infusions can support expansion, persistence, and CAR T cell trafficking to tumors in patients.

In CAR T cell clinical trials using ICV or iTu infusions, CAR T cell expansion and persistence have been demonstrated in peripheral blood and CSF samples using qPCR and flow cytometry 41 , 48 , 49 , 52 , 53 with different kinetics compared to IV infusion. For example, in the B7-H3 CAR T cell trial for DIPG patients, EGFRt+ CAR T cells were detected in serial CSF, but not peripheral blood of patients. 48 , 49 In a GD2 CAR T cell trial for DMG where the first infusion was IV followed by repeated ICV infusions, 52 , 53 GD2 CAR T cells were detected in blood after IV infusions, but were detected at higher levels in CSF after ICV infusions. 52 , 53 In this trial, loss of GD2 CAR T cells in blood associated with clinical or imaging progression. 53 Together, this work suggests that while IV CAR T cell infusions provide better visibility into CAR T cell expansion and persistence in peripheral blood, CSF CAR T cell expansion kinetics remain robust when administering CAR T cells ICV or iTu.

While the kinetics of CAR T cell expansion are an essential starting point for CAR T cell functionality, the phenotypic characteristic of T cells over time may provide important insights into durable CAR T cell activity. Employing reverse translational assays including multiparametric flow cytometry or mass cytometry by time-of-flight (CyTOF) provide proteomic evaluation of immune cell compartments. 24 , 33 , 41 , 52 , 53 , 73 , 74 , 80 , 81 , 82 , 83 Applying this approach to pre-manufacturing apheresis, CAR T cell product, or peripheral blood and CSF samples from patients, researchers have characterized both T cell and myeloid determinants of CAR T cell function in patients. 49 , 52 , 84 For example, in a B7-H3 CAR T cell DIPG trial, analyzed CAR T cell products demonstrated high expression of CD45RA, CXCR3, Ki-67, and CD127 markers, and a stem cell-like phenotype (CD69-CD39-), suggesting the proliferative potential and engraftment fitness of CAR T cell products. 49 , 85 Based on this and preclinical data, some trials enrich for central memory T cells to achieve prolonged CAR T cell persistence. 27 , 65 As multiparametric flow cytometry and CyTOF technologies advance, these approaches can provide insight into T cell phenotype and functional activation capacity and can provide meaningful insight into CAR T cell activity in patients.

In addition to single-cell proteomic evaluations, single-cell transcriptomic evaluations through paired scRNA-seq and single-cell T cell receptor sequencing (scTCR-seq) can provide additional orthogonal assessment of T cell phenotypes. 24 , 41 , 83 However, different clinical trials have provided varying insights based on assay approach, study design, and analysis approach. For example, in IL-13Rα2 CAR T cells for GBM, T cells demonstrated an effector phenotype, 24 while other clinical trials demonstrate increased memory and decreased effector phenotypes with reduced expression of exhaustion markers. 33 , 73 , 79 , 80 , 83 Further studies will be required to analyze these data in the context of patient responses. Combining these types of analyses across many different trials, while accounting for the known variables, may provide a more comprehensive understanding of T cell transcriptomic signatures in CNS tumor patients treated with CAR T cells.

CNS immune environment

The CNS immune environment poses a uniquely challenging landscape that fundamentally differs from the rest of the body. As CAR T cells encounter this complex environment—whether delivered systemically or locoregionally—they must navigate not only the immunosuppressive tumor microenvironment but also the unique regulatory mechanisms that govern CNS immunity. Understanding how CAR T cells interact with and potentially reshape this specialized immune landscape is critical for optimizing therapeutic efficacy. By combining systematic analysis of the cell-free and cellular compartments of CSF and peripheral blood, researchers are beginning to decode the inflammatory signatures, cytokine networks, and cellular dynamics that determine whether CAR T cells can establish productive anti-tumor immunity in the CNS or succumb to the suppressive forces that have evolved to protect this vital organ system.

To assess the inflammatory milieu in patients surrounding CAR T cells, multiplex cytokine assays have been integrated into clinical trials for peripheral blood and CSF samples before and after CAR T cell infusions. 17 , 27 , 41 , 48 , 49 , 54 , 80 , 82 , 83 Head-to-head comparisons have been conducted between cytokine and chemokine assays such as Luminex, Olink, and Nucleic acid Linked Immuno-Sandwich Assay (NULISA), 86 with each approach providing slightly different analytes, sensitivities, and readouts. Using these platforms, the field is gaining perspective on CAR T cell activation and suppression through evaluating pro-inflammatory and suppressive cytokines in the context of toxicity, medication, and response data.

Looking across CAR T cell studies, specific pro-inflammatory signatures are consistently identified across trials, but vary in kinetics based on route of CAR T cell administration. In an ICV IL-13Rα2 CAR T cell GBM trial, one patient experienced intracranial and spinal tumor regression after serial intracranial CAR T cell infusions, which correlated with increased IFN-γ-inducible chemokines such as CXCL9 and CXCL10 in CSF, but not peripheral blood. 27 Similarly, infusing HER2 CAR T cells locoregionally in children with CNS tumors demonstrated increased CXCL10 and CCL2 levels across all time points after infusion in CSF, but not in peripheral blood. 41 Trials testing locoregional B7-H3 CAR T cells in DIPG patients found similar increased cytokines in CSF, including CCL2, CXCL10, IFN-γ, GM-CSF, and TARC (thymus and activation-regulated chemokine). 48 , 49 Conversely, when GD2 or C7R-GD2 CAR T cells were administered IV, granzyme B and IFN-γ levels were elevated in peripheral blood of patients treated with C7R-GD2 CAR T cells compared to GD2 CAR T cells alone. 54 Both GD2 and C7R-GD2 CAR T cell-treated patients exhibited increased IL-6 and CXCL10 in blood that were associated with CRS toxicity. Similarly, IV administration of EGFRvIII-CAR T cells led to more than 10-fold increase in IL-6 levels in peripheral blood, with two subjects also showing increase in serum levels of IL-5 or IL-10. In a trial with both IV and ICV GD2-CAR T cells administration to DMG patients, IV CAR T cells led to pro-inflammatory cytokines in the blood and CSF, while ICV CAR T cell administration led to pro-inflammatory cytokines predominantly in the CSF, including IFN-γ, TNF, IL-2 and IL-6. 52 , 53 For these patients, grade 2 + CRS correlated with increased CCL2 (MCP-1) in plasma, while grade 2 + TIAN had increased IL-10 and CCL2 (MCP-1) in CSF. 53 Furthermore, the immunosuppressive cytokine TGF-β1 increased in patient CSF after CAR T cell infusion. 52 , 53 Together, these cytokine data across multiple CNS CAR T cell trials demonstrate that cytokines change in CSF and peripheral blood based on route of CAR T cell administration.

While cytokines/chemokines provide important insights into the CNS tumor inflammatory milieu and are more consistently characterized across CAR T cell studies, additional assays can provide information about cell-free proteomic signatures. Specifically, in the clinical trial testing B7-H3 CAR T cells against DIPG, patient CSF and serum samples were evaluated using a targeted proteomics assay called multiple reaction monitoring-mass spectrometry (MRM-MS) assay, which quantitatively measures specific peptides of interest in a digested protein lysate using a multiplexed panel of validated antibodies. 49 Evaluating 50 CSF and 59 serum proteins that were detected, CSF post-treatment samples demonstrated a sharp increase in several immunoregulatory peptides, including BCL10, CXCL13, HAVCR2 (TIM-3), ICOS ligand, PD-L2, TNFRSF14, and VCAM-1, while serum samples had fewer fluctuations. 49 Several markers of macrophage maturation and proteins involved in immune cell recruitment, including CD163, CD44, CD14, CXCL13, CSF-1 and VCAM-1, were consistently expressed in patient CSF. 49 Interestingly, CSF MRM-MS analysis demonstrated increased CD276 (B7-H3) protein in a patient with PD, while the patient with clinical improvement had lower CD276 protein, suggesting a potential biomarker of response to evaluate in future studies.

Over the last decade, advancement in scRNA-seq assays has provided additional insight into CNS tumor patients treated with CAR T cells, especially with regards to lymphocyte and myeloid phenotypes in the CNS immune environment. In the GD2 CAR T cell trial for DMG, patient CSF identified increased regulatory T cells (Tregs) after IV as compared to ICV infusion, 52 aligning with the FoxP3+ Tregs identified by immunohistochemistry (IHC) of tumor tissue in the EGFRvIII CAR T cell study for GBM after treatment. 17 Additionally, myeloid cell states fluctuated from suppressive to pro-inflammatory after ICV CAR T cell infusion, whereas suppressive CD163+ myeloid cells were identified in tumor tissue of a non-responding patient. 52 Similar suppressive myeloid cell states have been identified in the IL-13Rα2 CAR T cell clinical trial, where SPP1-expressing myeloid cells with increased suppressive receptor ligand interactions between SPP1-CD44, MIF-CD44+CD74, and TREM2-APOE correlated with poor prognosis and survival in patients. 70 Conversely, scRNA-seq of fresh pre-treatment tumor samples demonstrated increased CD3+ immune infiltrates with upregulated MHC class I and MHC class II presentation pathways in responding patients compared to non-responding patients, suggesting a more permissive immune milieu. 70 Together, these data suggest suppressive myeloid cells emerge following CAR T cell treatment and that pro-inflammatory or suppressive immune milieus could affect durable responses in patients.

To deepen understanding of the CNS tumor immune environment, larger spatial proteomic and transcriptomic platforms have been developed in the field. 87 , 88 , 89 Transcriptomic analysis of patient tumor tissue have incorporated an autologous natural killer (NK) and T cell study for recurrent GBM patients. 82 Using spatial transcriptomic analyses, 85 genes were identified to be significantly associated with PFS, with the majority of these genes upregulated in good responders and linked to either chemokines, like CCL4, CCL3, CCL1, and CCL20, or were linked to extravasation, namely ICAM2, ITGA1, and PECAM1. 82 In CNS tumors, like DIPG, spatial platforms are beginning to be evaluated with encouraging insights that can contextualize the findings from CAR T cell-treated patients. 90 While spatial transcriptomic and proteomic platforms have yet to be broadly studied in the context of CNS tumor CAR T cell therapies, they represent an important area for growth in the field and will be central to enhancing understanding of the immune biology associated with clinical outcomes in patients.

Tumor-intrinsic resistance to CAR T cells

While understanding the T cell and CNS immune environment, contributors to CAR T cell function are crucial, the ultimate measure of CAR T cell efficacy lies in their ability to recognize, engage, and eliminate tumor cells. Tumor-intrinsic resistance represents a formidable challenge in CNS CAR T cell therapy. Initial clinical trials have provided early evidence of antigen escape with decreased target antigen expression in post-treatment tumor samples. The challenge of understanding tumor-intrinsic resistance in CNS tumors is compounded by the practical limitations of obtaining serial tumor biopsies, particularly in locations like the brainstem where repeat surgical sampling is not feasible. As a result, researchers have turned to innovative approaches including analysis of autopsy tissues, liquid biopsy techniques to track circulating tumor DNA, and advanced spatial technologies to define the molecular changes that occur within tumors following CAR T cell therapy. As these approaches are implemented, insights can guide the rational design of next-generation CAR T cell approaches to overcome these resistance mechanisms.

Imaging modalities and neurologic evaluations provide clinically important response information. However, in parallel the field has looked for alternative approaches to track tumor burden, response, and progression. While these approaches are nascent, some studies have incorporated assays like digital droplet PCR (ddPCR) to track cell-free tumor DNA in serial CSF or plasma samples. 52 , 53 , 91 In the GD2 CAR T cell trial for DMG patients, CSF samples demonstrated decreased tumor H3K27M mutation by ddPCR in some patients associated with tumor regression, while increased levels of tumor H3K27M mutation ddPCR associated with peak inflammation or progression. 53 While these assays may provide valuable insight into tumor kinetics, further studies will be needed to fully understand the value of integrating these assays into disease monitoring and treatment response assessment.

To understand mechanisms of tumor resistance, both solid and CNS tumors can benefit from evaluation of the tumor and its immune environment. While serial tumor biopsies would be very helpful for this endeavor, the reality is that this may not be feasible in patients. For example, in adult GBM studies, serial tumor acquisition is feasible, but serial biopsies are not an option in other tumors like DIPG. In those cases, autopsy tissue, generously donated by patients and their families, may be the only window into tumor biology after CAR T cells. In some CAR T cell clinical trials, qPCR, IHC, and flow cytometry have been used to assess antigen expression levels in recurrent tumor or autopsy samples. Across multiple trials, tumor samples after CAR T cell treatment have demonstrated decreased antigen expression, suggesting this will be an important mechanism of resistance to overcome for durable response in patients. 17 , 24 , 33 , 41 , 52 , 53 , 54 , 80 , 83 Based on these data many centers are beginning to explore targeting multiple antigens simultaneously. This demonstrates the potential of reverse translational data supporting iterative improvements of CAR T cell therapies for patients.

Discussion and future directions

CAR T cell therapy for pediatric CNS tumors represents a novel approach to treating some of the most challenging malignancies in children, yet significant obstacles remain before these therapies can achieve their full therapeutic potential. Early clinical trials have established the feasibility and safety of administering CAR T cells to pediatric patients with CNS tumors, while simultaneously revealing unique toxicities such as TIAN that require specialized management strategies. The initial glimpses of clinical activity across multiple targets, including B7-H3, GD2, HER2, and IL-13Rα2, provide encouraging evidence that CAR T cells can mediate anti-tumor effects in the CNS environment. However, the inconsistent and often transient responses highlight the complex interplay between T cells, tumors, and the CNS immune environment that must be overcome to achieve durable clinical benefit.

The integration of robust reverse translational platforms into clinical trials has proven invaluable for understanding the mechanisms underlying CAR T cell success or failure in patients. To accelerate discovery from patients, clinical trials must integrate systematic analysis of peripheral blood, CSF, and tumor samples. This approach will identify key insights including the role of CAR T cell persistence, the effects of suppressive myeloid cells, and the evidence of antigen loss as resistance mechanisms. Initial discoveries from reverse translational evaluations have already guided iterative improvements in CAR T cell design, including developing dual-targeting constructs, optimizing delivery routes, and overcoming immunosuppressive barriers. As the field advances, harmonizing reverse translational analyses across trials will be essential to establish generalizable principles for the field and inform the next generation of treatment approaches to improve patient responses.

Realizing the full potential of CNS CAR T cell therapies will require addressing fundamental questions, including optimal delivery routes, the role of LD chemotherapy, toxicity management, and mechanisms of resistance. Some of these questions will require cross-trial comparisons of studies already underway and others will require direct assessments within clinical trials. As clinical trials transition from single-institution Phase I trials to multi-center studies, maintaining these questions at the forefront will allow for the field to continue to improve these therapies for patients. By employing the full cycle of translational research—from bench to bedside and back—and fostering strong collaboration between pediatric centers to enable cross-trial learning, the field is positioned to iteratively optimize CAR T cell therapies for pediatric CNS tumors. The foundation laid by early trials, particularly the dramatic responses seen in subsets of children with universally fatal diseases, provides hope that CAR T cells will evolve from experimental therapies into transformative treatments that meaningfully improve outcomes for both pediatric and adult patients with CNS tumors.

Acknowledgments

S.R. is a project member of the Parker Institute for Cancer Immunotherapy, which supports the Stanford University Cancer Immunotherapy Program. S.R. was supported by K08CA267057. S.R. and A.K. are supported by the Chadtough Defeat DIPG New Investigator Grant and by Pray for Dominic, a St. Baldrick’s Hero Fund.

Author contributions

K.S., A.K., and S.R. have conceptualized, written, and edited this manuscript.

Declaration of interests

None of the authors have conflicts of interest to disclose related to this work.

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