Highlights in IO: next-generation CAR-T therapy for glioblastoma.

Introduction
Glioblastoma (GBM) is a malignant brain tumor with an extremely poor prognosis that exhibits resistance to various treatments, representing a huge clinically unmet need. Chimeric antigen receptor (CAR)-T therapy has demonstrated remarkable efficacy in hematologic cancers. However, its effectiveness against solid tumors, including GBM, remains limited. Resistance to CAR-T cell therapy in GBM arises from multiple factors, including immunosuppressive cell populations, particularly myeloid-derived suppressor cells, antigen heterogeneity, hypoxia, metabolic constraints, and the anatomically isolated environment created by the blood–brain barrier. Additionally, standard-of-care treatments, such as corticosteroids and chemoradiotherapy, may further compromise CAR-T cell efficacy. These challenges have driven active efforts to develop next-generation CAR-T cells, which are engineered to overcome these barriers. Over the past 2 years, we have gained new insights from several clinical trials, and clinical trials of next-generation CAR-T have been initiated. In this report, we summarize the current state of CAR-T therapy development for GBM.

Overview of the latest published results of clinical trials of CAR-T therapy for GBM
In phase 1 clinical trial of locoregional delivery of CAR-T cells targeting interleukin-13 receptor alpha 2 (IL-13Rα2) for recurrent high-grade glioma, 56 patients received treatment (NCT02208362).1 Notably, this trial evaluated three delivery routes and two product platforms. The product platforms were developed based on experience from clinical trials in hematologic malignancies, enriching either central memory T cells (Tcm) or naïve/stem cell memory and central memory T cells (Tn/mem). Regarding safety, no dose-limiting toxicities (DLTs) were observed, and grade ≥3 adverse events with possible or higher attribution to CAR-T therapy occurred in 35% of patients. In this study, 72% of enrolled patients had recurrent GBM (rGBM), and the best responses assessed by modified response assessment in neuro-oncology (RANO) in rGBM cases were 19 cases of stable disease (SD) and 23 cases of progression disease (PD). One patient with rGBM with meningeal dissemination achieved a complete response (CR) through a single-subject protocol.2 Importantly, the Tn/mem product showed a significantly longer overall survival compared with the Tcm product. Although this was a result of a non-randomized trial, it provides an important insight—namely, the use of a less differentiated T-cell subset for manufacturing logistics.
In another phase 1 clinical trial targeting IL-13Rα2, the safety of intrathecal delivery of allogeneic CAR-T cells was evaluated in patients with recurrent high-grade glioma (ChiCTR2000028801).3 In this study, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology was used to disrupt the endogenous T-cell receptor and human leukocyte antigen class I molecules, enabling the manufacture of off-the-shelf CAR-T cells independent of patient-specific factors and prior treatment history. This strategy is expected to reduce CAR-T cell exhaustion and preserve effector function, thereby addressing key logistical challenges associated with autologous CAR-T therapy. However, natural killer cell-mediated rejection was not completely eliminated. Although only five patients were enrolled, transient antitumor efficacy was observed, including one CR, three partial responses (PR), and one SD, with a median duration of response of 3.8 months. Notably, clinical responses were observed following intrathecal delivery via lumbar puncture. Regarding safety, grade 1 cytokine release syndrome (CRS) was reported, but no grade ≥3 adverse events occurred. Importantly, no cases of graft-versus-host disease were reported.
In a separate phase 1 clinical trial, single intracerebroventricular delivery of bivalent CAR-T cells targeting epidermal growth factor receptor (EGFR) and IL-13Rα2 was evaluated in EGFR-amplified rGBM, and 18 patients were enrolled (NCT05168423).4 Regarding safety profiling, grade 1–2 CRS was observed in all patients, and grade 3 immune effector-associated neurotoxicity was observed in 56% (10/18) of patients. Among the 13 patients with measurable disease, the modified RANO-based response assessment showed one PR, eight cases of SD, and four cases of PD, resulting in an objective response rate of 8%. The median progression-free survival (PFS) from initial study treatment to disease progression was 1.9 months, and yet durable disease control was observed in two patients, with PFS of 7.7 months and 16.6 months, both ongoing at the time of data cut-off. Quantitative PCR-based assays revealed that the CAR transgene in cerebrospinal fluid (CSF) declined between days 14 and 28 after infusion in most cases. However, in one patient with a durable response, the CAR transgene remained detectable even 12 months post-infusion, suggesting the need to consistently enhance CAR-T cell persistence in the CSF.

Ongoing clinical trial of next-generation CAR-T therapy for GBM

Chlorotoxin-directed CAR-T cell (NCT04214392)
Chlorotoxin (CLTX) is a peptide derived from scorpion venom that selectively binds to glioma cells via matrix metalloproteinase-2 (MMP-2) and does not bind to non-tumor cells. CAR constructs incorporating CLTX as the antigen-recognition domain represent a unique approach and are considered a novel strategy for GBM, where ideal target antigens are limited. A phase 1 clinical trial is currently ongoing to evaluate the safety of intracavitary/intratumoral delivery of CLTX-directed CAR-T cells in patients with rGBM.5 Regarding safety, no cases of CRS were reported. However, two of the four treated patients developed grade 3 brain edema that was possibly attributable to the therapy. Although local tumor control was achieved at the sites of CLTX-directed CAR-T cell administration, tumor recurrence occurred at a distant site in one patient despite persistent MMP-2 expression, highlighting challenges related to the route of delivery. Given that CLTX is a non-human peptide, immunogenicity is a theoretical concern. However, no immune responses against CLTX-directed CAR-T cells have been detected to date.

CARv3-TEAM-E T cell (NCT05660369)
The first-in-human trial of CAR-T cells targeting EGFRvIII for GBM revealed that treatment resistance was associated with decreased EGFRvIII expression and upregulation of wild-type EGFR.6 To overcome this resistance mechanism, a next-generation CAR-T cell targeting EGFRvIII was developed to secrete a wild-type EGFR–CD3 T-cell engaging antibody molecule (TEAM). Because wild-type EGFR is also expressed in normal tissues, the locally acting TEAM secreted within the tumor microenvironment is expected to minimize on-target toxicity while enhancing antitumor efficacy mediated by endogenous T cells. A phase 1 clinical trial is currently ongoing to evaluate the safety of intraventricular delivery of CARv3-TEAM-E T cells in patients with rGBM.7 A notable safety profile was that all seven patients experienced grade 1 CRS, with one patient developing grade 2 CRS, and all patients developed grade 1 tumor-inflammation-associated neurotoxicity. The immunotherapy RANO-based response assessment showed five out of seven patients achieved SD as the best response (table 1 #6). Notably, in patients who showed diminished CAR-T cell persistence with repeat infusions, anti-CARv3-TEAM-E antibodies were identified in both the CSF and serum (table 1 #4). This finding suggested that humoral responses against CARv3-TEAM-E represent one of the mechanisms of resistance.

8R-70 CAR-T (NCT05353530)
To enhance infiltration into GBM, CAR-T cells targeting CD70 have been engineered to express the interleukin (IL)-8 receptor, thereby using tumor-secreted IL-8 as a homing signal (8R-70 CAR-T cell).8 In addition, they aim to remodel the tumor microenvironment by reducing IL-8 and CD70, both of which play tumor-promoting roles. A phase 1 clinical trial is currently ongoing to evaluate the safety of intravenous administration of 8R-70 CAR-T cells in patients with newly diagnosed GBM with CD70 positivity (>20%) following completion of standard-of-care therapy. This treatment period is also based on the observation that both IL-8 production and CD70 expression in the tumor increase following radiotherapy. No DLTs have been reported to date (table 1 #10).

E-SYNC CAR-T (NCT06186401)
The choice of target antigen is a critical determinant of the efficacy of CAR-T cell therapy; however, no target antigen is known that is truly tumor-specific and homogeneously expressed. Thus, it is challenging to find a single ideal surface GBM antigen that can address the dual challenges of off-target toxicity and incomplete killing. To overcome this challenge, E-SYNC CAR-T cells were designed using a synNotch receptor-based “prime-and-kill” circuit that recognizes GBM-specific EGFRvIII and subsequently induces the expression of a tandem CAR-Targeting EphA2 and IL-13Rα2—antigens that are not truly tumor-specific but are expressed in the majority of GBMs—specifically within the tumor microenvironment.9 Furthermore, because CAR expression is regulated by the synNotch system, tonic signaling of the CAR is reduced, leading to improved in vivo persistence of the engineered T cells. A phase 1 clinical trial is currently ongoing to evaluate the safety of intravenous administration of E-SYNC CAR-T cells in EGFRvIII-positive GBM.

TGFβ receptor-knock out CAR-T (NCT06815029)
Transforming growth factor beta receptor (TGFβR)-knock out (KO) CAR-T cells are CAR-T cells targeting IL-13Rα２ in which the receptor for TGFβ, a cytokine abundantly present and immunosuppressive in the GBM tumor microenvironment, has been knocked out using CRISPR technology. Preclinical studies have demonstrated that disruption of TGFβR signaling renders CAR-T cells less prone to exhaustion, increases the memory subset within circulating CAR-T cells, and enhances their in vivo antitumor efficacy.10 A phase 1 clinical trial is currently ongoing to evaluate the safety of intracranial administration of TGFβR-KO CAR-T cells in recurrent or progressive GBM or grade 3 or 4 isocitrate dehydrogenase (IDH)-mutant astrocytoma.
In addition to the knockout approach, CAR-T cells expressing a decoy-type dominant-negative TGFβR (dnTGFβR) have also been developed to block the TGFβ pathway. Beyond improving antitumor efficacy via TGFβ signaling inhibition, dnTGFβR has been reported to function as a “sink” that reduces local TGFβ concentrations in the tumor microenvironment, thereby improving the fitness of bystander T cells.11
The latest updates from the major scientific meetings on clinical trials of CAR-T therapy for GBM were summarized in table 1.

Major considerations in the field
The results of phase 1 clinical trials of CAR-T therapies for GBM revealed the need to improve both safety and durable efficacy. Although direct central nervous system delivery of CAR-T cells has been employed in many recent studies, it often resulted in acute and sometimes severe neurotoxicity that is not typically observed with intravenous infusion. This highlights the importance of carefully selecting antigens, delivery routes, and designing functional CARs to minimize toxicity. Moreover, clinical and radiographic responses have often been transient, and with few exceptions, the infused CAR-T cells have not persisted long-term in vivo. Given the correlation between long CAR-T cell persistence and antitumor efficacy,12 it is essential to optimize functional CAR design, delivery strategies, and manufacturing logistics to enhance in vivo persistence.

Summary
The clinical trials of CAR-T therapy for GBM revealed a high incidence of neurotoxicity that was disproportionate to their limited antitumor efficacy. The clinical trial outcome of next-generation CAR-T cells, engineered to address the challenges revealed by prior CAR-T therapy for GBM trials, is expected to become the highlights of CAR-T therapy for GBM in the coming 5 years.

Supplementary material
10.1136/jitc-2025-014670online supplemental file 1