The safety and efficacy of bispecific T-cell engagers (TCEs) in patients with glioma.

Introduction

Overview of glioma and GBM
Gliomas are a broad category of brain tumors that are believed to originate from neural stem or progenitor cells that possess tumor-initiating genetic mutations [1]. The most prevalent and aggressive variety of glioma is GBM. Globally, the incidence of GBM is 3.19 per 100,000 individuals, with an estimated 30% of all brain tumors and 80% of malignant tumors affected [2]. The maximum incidence of GBM occurs in individuals aged 45–70 years, with the highest rates observed in those aged 75–84 years, where it peaks at approximately 15 per 100,000 people before declining after age 85; reports indicate that primary GBM diagnoses are more common than secondary GBM, and the median age at diagnosis is approximately 64 years [3, 4]. Glioma incidence varies by ethnicity, with American and northern European populations having higher rates than Asians. From 2000 to 2014, GBM constituted 80% of astrocytic tumors in North America, Europe, and Oceania, whereas GBM constituted only 60% or less in Central and South America [5]. Non-Hispanic Whites have higher incidence rates, possibly due to genetic or environmental factors [6]. Despite the declining incidence since 1987, it slightly increased after 1992 in some regions, promoting concerns about underlying risk factors and the need for improved surveillance and management strategies [7]. A substantial portion of glioma-related morbidity and mortality are attributed to high-grade gliomas, particularly GBM [8]. The current standard of care for GBM includes surgical resection, radiotherapy, and chemotherapy (typically with temozolomide). However, complete surgical resection is often not feasible because of the infiltrative nature of the tumor, leading to suboptimal outcome. Poor prognostic factors, such as age at diagnosis, performance status, and tumor genetics (e.g., MGMT methylation status), influence survival. GBM exhibits remarkable treatment resistance due to a resilient tumor microenvironment and immune evasion [9]. Despite aggressive treatment approaches, the median survival of GBM patients is approximately 15 months [10]. The aggressive nature of GBM impacts patients’ quality of life (QoL), leading to increased caregiver burden and decreased well-being.

Immunotherapy in glioma
Immunotherapy for gliomas has evolved significantly over the past few decades, with early approaches focusing on nonspecific immunostimulants and vaccines [11]. Current strategies include ICIs such as nivolumab and pembrolizumab, chimeric antigen receptor (CAR) T-cell therapy, oncolytic virus therapy, and cancer vaccines [12]. However, successes have been limited, particularly in GBM [13]. T-cell-based therapies aim to harness the body’s immune system to target and destroy glioma cells, targeting tumor-specific antigens [14].
Unique challenges in brain tumors include the blood‒brain barrier (BBB), an immunosuppressive microenvironment, and tumor heterogeneity [15]. The selective permeability of the BBB limits the entry of systemic therapies, and the immunosuppressive microenvironment inhibits effective antitumor immune responses. The unique immune regulatory mechanisms and potential neurotoxicity of the central nervous system raise concerns regarding its safety and tolerability [16]. Combination strategies and further research are needed to overcome these challenges [17].

Mechanism of action of TCEs
TCEs are a broad class of bispecific (or multispecific) molecules designed to redirect CTLs to tumor cells. By simultaneously binding CD3 on T cells and a TAA on malignant cells, TCEs physically bridge T cells to cancer cells and trigger formation of an immunologic synapse. This synapse induces T cell activation, release of cytotoxic granules and inflammatory cytokines, resulting in targeted tumor cell lysis [18, 19]. Among TCEs, the classical Bi-specific T-cell engager (BiTE) format refers to tandem single-chain variable fragments (scFvs) without an Fc region; however, modern clinical-stage TCEs also include IgG-like architectures with Fc domains, trispecific constructs, half-life–extended (HLE) molecules or constructs incorporating costimulatory or conditional activation modules. Recent advances have expanded the TCE platform beyond classical BiTE. Next-generation TCEs are being engineered with dual-targeting capacity (e.g., recognizing two distinct TAAs to improve specificity and reduce antigen escape), conditional activation (e.g., masked antibodies that become active only in the tumor microenvironment), and inclusion of costimulatory domains or Fc for half-life extension features designed to enhance efficacy, improve pharmacokinetics, and mitigate toxicity [20–22].

TCEs and the bite subclass
T-cell retargeting for cancer therapy has been a concept since the 1970 s because of its robust cytotoxic responses, immunologic memory, and ability to attack tumors from the outside and infiltrate into the tumor. However, the requirement for a second stimulatory signal to achieve full T-cell activation and prevent anergy has been challenging [23]. In the 1980 s, bispecific antibodies (BsAbs) were developed to meet this requirement, starting with the first T-cell-engaging trispecific antibodies. However, concerns have been raised about the potential undesirable effects of Fc receptor interactions, leading to the development of bispecific TCEs [24–26].
TCEs are a heterogeneous class of molecules that redirect T cells toward tumor cells by simultaneously binding CD3 and a TAA. Among TCEs, the canonical TCEs format represents a specific subclass composed of two tandem single-chain variable fragments (scFvs) without an Fc domain. Most clinically approved TCEs are IgG-like molecules containing an Fc region rather than classical BiTE structures (20, 22). Typically, one arm of the BiTE molecule binds to CD3 on T cells, whereas the other arm is made to detect a particular TAA found on cancer cells [27]. This dual-binding ability allows BiTE to bridge T cells and tumor cells, facilitating targeted immune responses against the tumor, leading to T-cell proliferation, cytokine release, and tumor cell lysis [28]. TCEs, including the canonical BiTE subclass, recognize specific antigens on tumor cells and allowing precise targeting of cancer cells while sparing normal tissues [29, 30] (Fig. 1).

TCEs technology offers advantages over conventional immunotherapies, including dual specificity, rapid activation of T cells, broad application, and a reduced risk of CRS [31]. It targets multiple tumor antigens, making it adaptable to different cancers and reducing the risk of severe CRS [28].
This review evaluates the safety and efficacy of TCEs therapies across gliomas, utilizing clinical trial data to identify patient populations benefiting the most. This study focused on histology-based outcomes and analyzed the responses of various tumor types to TCEs therapy. This review also discusses the clinical implications of TCEs technology in oncology practice, explores potential combinations, optimal treatment protocols, and future research prospects to improve patient outcomes in GBM treatment.

TCEs in oncology
In recent decades, the development of BsAbs for the treatment of hematologic malignancies has progressed at a remarkable pace. Currently, there are over 100 different formats of BsAbs, among which TCEs are particularly well engineered, with new structures continuously emerging [32]. The initial concept of BsAbs was introduced in the early 1960 s, culminating in the first constructed example in 1985 [23]. TCEs are specifically designed to engage both CD3 and tumor-specific antigens, thereby enhancing T-cell cytotoxicity. Following the approval of blinatumomab, a prototypical CD3/CD19 BiTE by the FDA in December 2014 for adult patients with Philadelphia chromosome-negative (Ph-) relapsed or refractory (R/R) B-cell progenitor acute lymphoblastic leukemia (B-ALL), there has been swift advancement in the development of TCEs for hematologic malignancies [33, 34]. Figure 2 depicts GBM-relevant target antigens explored for TCE development.

The promising outcomes associated with TCEs therapy in hematological cancers have led to the development of various TCEs antibodies aimed at TAA present in solid tumors, as detailed in Table 1. Prostate-specific antigen (PSA), a member of the type II transmembrane protein family, is predominantly found in prostate tissues and is frequently overexpressed in prostate cancer, making it an optimal target for tumor antigen therapy [35]. Pasotuxizumab (AMG 212) serves as a representative TCEs antibody that specifically targets PSA [36]. The antitumor efficacy, indicated by a reduction in PSA levels, was observed to be dose dependent. Notably, two patients sustained long-term PSA responses exceeding one year while receiving doses of 40 µg/d and 80 µg/d. Adverse events (AEs) of Grade 3 were reported in 81% of the patients, with the most prevalent being decreased lymphocyte counts (44%) and infections (44%). Only one patient experienced a treatment-related serious AEs. This study represents a significant advancement in demonstrating the effectiveness of TCEs immunotherapy for patients with solid tumors. Furthermore, at the 2020 ESMO conference, HLE-TCEs AMG 160 exhibited a favorable safety profile and preliminary evidence of efficacy in patients with metastatic castration-resistant prostate cancer (mCRPC) [37]. Additionally, another PSMA-targeting T-cell engager, HPN424, has commenced a phase 1 clinical trial, showing early indications of clinical activity in mCRPC patients [38].

Clinical experience of TCEs in solid tumors
The clinical landscape of TCEs in oncology has recently expanded beyond hematologic malignancies into solid tumors. Notably, tebentafusp, a bispecific T-cell engager targeting the gp100–HLA-A 02:01 complex, received regulatory approval in January 2022 for the treatment of unresectable or metastatic uveal melanoma, providing the first TCE demonstrating OS benefit in a solid tumor [39]. More recently, tarlatamab-dlle was approved in 2024 for extensive-stage small cell lung cancer after platinum-based chemotherapy, marking a significant milestone as a TCE for a major thoracic solid malignancy [40]. These approvals underscore the translational success of TCEs in diverse solid malignancies. Moreover, emerging TCE programs targeting prostate cancer via antigens such as STEAP1 are under early clinical investigation, suggesting a potential further expansion of the TCE therapeutic frontier [22, 41]. While these breakthroughs in solid tumors outside the CNS are encouraging, translating TCE strategies to GBM remains challenging primarily due to BBB constraints, tumor heterogeneity, antigen escape, and immunosuppressive tumor microenvironment. Nonetheless, the clinical success of TCEs in non-CNS solid tumors supports the rationale for continued preclinical and early-phase exploration of TCEs in GBM.

BiTE and CAR-T-cell therapies represent promising frontiers in the treatment of cancer, particularly in the context of GBM. Although substantial research has been undertaken, considerable challenges remain before these innovative therapies can be integrated into standard clinical practice. Preclinical studies have demonstrated the potential efficacy of BsAbs in GBM; however, numerous obstacles persist. Enhancing the delivery mechanisms to effectively traverse the BBB and prolong the therapeutic half-life is critical. Strategies under investigation include local secretion by resident or immune cells, the use of oncolytic viruses, and the development of trispecific antibodies that target multiple antigens. Additionally, employing conditional designs that depend on the presence of various tumor markers may improve safety profiles, while combining these approaches with other immunotherapies, such as CAR-T cells and oncolytic viruses, shows promise [42].
In contrast, CAR-T-cell therapies have a more extensive body of clinical data, albeit derived from small-scale studies with heterogeneous methodologies and outcomes. Key challenges include determining the optimal delivery method, whether intracranially or systemically. The issue of antigen escape can potentially be mitigated by increasing the number of targets or by integrating immunotherapies, such as BiTE-secreting CAR-T cells. As living therapeutics, CAR-T cells offer numerous opportunities for adaptable designs; however, issues related to T-cell persistence, trafficking, and exhaustion remain significant hurdles. Furthermore, these therapies necessitate a costimulatory signal, and the selection of this signal can markedly influence T-cell function. Next-generation designs that incorporate cytokine or chemokine activity, as well as engineering CARs for intermittent rather than continuous activity, may help address these challenges. Targeting components of the tumor microenvironment or combining CAR-T cells with ICIs or oncolytic viruses could provide additional strategies to increase treatment efficacy. While toxicity remains a major concern, innovative designs such as SynNotch, universal CARs, and inhibitory CARs are being explored to mitigate these risks [42] (Fig. 3).

The evolving understanding of T cells and their contributions to anticancer immunity has led to significant advancements in T-cell-based immunotherapies over the past decade. Notably, TCEs have demonstrated promising clinical results in relapsed/refractory hematologic malignancies and have shown preliminary evidence of efficacy in solid tumors. Nevertheless, a substantial proportion of patients exhibit resistance to TCEs therapy, resulting in a lack of durable responses. Research has identified two primary mechanisms contributing to treatment failure: antigen loss and the influence of immunosuppressive factors, particularly the upregulation of inhibitory immune checkpoint molecules. Consequently, ongoing investigations are focused on enhancing TCEs constructs and creating novel T-cell-engager antibodies that possess increased antigen avidity and target multiple antigens. These efforts are being explored through various preclinical and clinical studies, alongside combination therapies that integrate TCEs antibodies with other treatment modalities. Furthermore, interest in innate cell or innate-like cell engagers that target innate immunity, which have demonstrated significant antitumor efficacy across a range of cancers, is increasing, indicating a promising direction for future research.

TCEs in glioma: preclinical to clinical evidence

Preclinical studies

EGFRvIII
Preclinical studies have demonstrated the remarkable antitumor efficacy of TCEs in GBM. A significant preclinical investigation involved a TCEs designed to target the EGFRvIII mutation, which is frequently expressed in GBM. A significant preclinical study conducted by Choi et al. (2013) demonstrated the potential of bscEGFRvIIIxCD3, a TCEs that targets EGFRvIII, for glioma immunotherapy. This study revealed that bscEGFRvIIIxCD3 effectively activated T cells to mediate potent, antigen-specific lysis of EGFRvIII-expressing glioma cells in vitro at very low concentrations (10 ng/mL) and low effector-to-target ratios (2.5:1). Furthermore, systemic administration of this TCE significantly prolonged survival in murine glioma models and achieved complete tumor eradication in up to 75% of cases. Importantly, blocking EGFRvIII binding abolished these effects, emphasizing the necessity of antigen specificity for TCEs efficacy. These findings establish bscEGFRvIIIxCD3 as a promising candidate for GBM immunotherapy, highlighting the feasibility of TCEs -based approaches in overcoming the challenges of CNS-targeted treatments [43].
In parallel, Ellwanger et al. (2017) introduced EGFRvIII/CD3 TandAbs, a tetravalent format that also targets EGFRvIII-positive GBM. These TandAbs exhibited high specificity for EGFRvIII with no cross-reactivity to wild-type EGFR. In vitro experiments demonstrated strong cytotoxicity toward EGFRvIII-expressing glioma cells, with EC50 values in the picomolar range. Additionally, in vivo studies confirmed dose-dependent tumor growth inhibition in murine GBM models, underscoring the clinical relevance of TCEs -based immunotherapy [44].
In addition, Gardell et al. (2020) introduced an innovative TCEs delivery strategy using genetically engineered macrophages (GEMs) capable of secreting an EGFRvIII-targeted BiTE. These GEMs successfully infiltrated GBM tumors, sustained local TCEs secretion, and induced robust T-cell activation and tumor cell lysis both in vitro and in vivo. Importantly, combination therapy with BiTE-secreting GEMs and IL-12 further enhances T-cell responses and leads to complete tumor growth suppression in murine GBM models [45].
Building on these findings, Sun et al. (2021) introduced a novel EGFRvIII-targeting BsAb via the “BAPTS” platform, which was designed to recruit and activate T cells for GBM immunotherapy. This BsAb demonstrated high specificity for EGFRvIII, with in vitro experiments showing efficient T-cell-mediated cytotoxicity and cytokine release. In vivo studies using NOD/SCID and BALB/c mouse models confirmed the long circulation time, potent tumor elimination, and reduced immunogenicity of TCEs compared with conventional TCEs [46].
More recently, Park et al. (2023) explored an in vivo DNA-encoded TCEs (DBTE) approach, where EGFRvIII-DBTE was designed for prolonged expression and tumor clearance. Their study demonstrated that a single administration of DBTE resulted in durable TCEs production for several months, enabling strong tumor control and clearance in both peripheral and orthotopic GBM models. Furthermore, a multivalent DBTE strategy targeting both EGFRvIII and HER2 resulted in enhanced tumor suppression and mitigated antigen escape, addressing the challenge of GBM heterogeneity [47].

IL13R 2
Innovative BiTE-based approaches have shown promise in GBM treatment. Pitucha et al. (2021) utilized neural stem cells (NSCs) to deliver BiTELLON, a TCEs targeting IL-13Rα2, demonstrating its ability to stimulate T-cell activation, promote cytokine release (IFNγ, TNFα), and induce selective tumor cytotoxicity. In vivo, BiTELLON-secreting NSCs exhibited strong tumor tropism, persisted in the tumor microenvironment for more than a week, and significantly extended survival in glioma-bearing mice. This study revealed that NSC-mediated TCEs delivery is a viable method for enhancing TCEs stability and effectiveness [48].
To expand on this concept, Bhojnagarwala et al. (2022) introduced an in vivo DNA-launched TCEs (dBTE) that targets IL-13Rα2, allowing continuous TCEs expression without the need for infusion. In an orthotopic GBM model, dBTE treatment enhanced T-cell activation, triggered potent tumor lysis, and prolonged survival. Notably, their findings confirmed that systemically delivered dBTEs can cross the BBB, marking a significant advancement in TCEs therapy by improving CNS penetration and therapeutic durability ​ [49].

Fn14-targeted TCEs
Recent research has explored Fn14 as a novel target for GBM immunotherapy. Li et al. (2021) developed Fn14-targeted TCEs (Fn14×CD3) and Fn14-specific CAR-T cells, which demonstrated their potent preclinical activity against GBM​. Their study confirmed high Fn14 expression in GBM tissues and cell lines, while it was undetectable in normal brain samples, making Fn14 a promising tumor-specific target. In vitro experiments revealed that Fn14×CD3 TCEs effectively redirected T cells to eliminate Fn14-expressing GBM cells, leading to strong antigen-specific cytotoxicity. In vivo, systemic administration of Fn14×CD3 TCEs resulted in significant tumor regression in xenograft models, highlighting its therapeutic potential. Additionally, Fn14 CAR-T cells engineered to secrete IL-15 exhibited enhanced persistence and antitumor effects, further improving treatment outcomes [50].

NKG2D
Baugh et al. (2024) developed a TCEs targeting NKG2DLs designed to engage CD3-positive T cells with GBM cells expressing NKG2DLs​. Their study demonstrated that NKG2D BiTE-mediated T-cell activation led to significant tumor cell death, cytokine release, and antigen-independent immune responses. A key innovation in this study was the combination of TCEs therapy with oncolytic herpes simplex virus-1 (G207). Researchers engineered G207 to express and secrete NKG2D BiTE, enhancing local TCEs production within the tumor microenvironment. In preclinical models, this dual-therapy approach—integrating virotherapy and BiTE-mediated T-cell activation—has led to increased tumor cytotoxicity and elimination of glioma stem-like cells (GSCs), which are typically resistant to conventional treatment. Furthermore, pretreatment with sublethal doses of radiation and temozolomide (TMZ) increased NKG2DL expression on GBM cells, thereby improving NKG2D TCEs efficacy and enhancing tumor sensitivity to T-cell cytotoxicity [51].

Clinical studies
In preclinical research, AMG 596, a novel TCEs that targets EGFRvIII, has advanced into clinical evaluation. A phase 1, first-in-human, open-label trial (NCT03296696) is currently assessing its safety, tolerability, pharmacokinetics, and pharmacodynamics (PK/PD) in patients with recurrent or newly diagnosed GBM. AMG 596 is administered via continuous intravenous infusion and is designed to engage CD3-positive T cells and induce targeted tumor cell lysis. As of the latest available data, the study is ongoing, and the final results have not yet been published.
As of June 2019, 15 rGBM patients had been enrolled, with 14 receiving at least one dose of AMG 596. SAEs were reported in 50% of patients, although no dose-limiting toxicities (DLTs) were identified. The most frequent high-grade adverse effects included headache and altered consciousness.
Among eight patients with sufficient follow-up, one achieved a PR, two had stable disease (SD), and four experienced disease progression. Additionally, EGFRvIII expression analysis revealed variable antigen levels across patients.
However, preliminary findings indicate that AMG 596 is generally well tolerated, with early signs of a tumor response observed in some patients. The study continues to enroll patients, aiming to further evaluate its therapeutic potential and clinical benefits in EGFRvIII-positive GBM [52].

Mechanistic insights
BsAbs have diverse applications, including immune cell recruitment and blocking immune checkpoint receptors, inflammatory mediators, or signaling pathways. One of their key functions is engaging immune cells, particularly CTLs. Under normal conditions, CTLs require multiple activation signals: antigen presentation via MHC molecules and costimulation through CD28. The TCR recognizes antigen fragments presented by MHC I on tumor cells or MHC II on APCs, triggering intracellular signaling via the CD3 subunit, with CD8 acting as a coreceptor. Without proper costimulation, CD8 + T cells remain inactive and undergo apoptosis [42, 53].
Most immune-engaging BsAbs circumvent MHC-dependent activation by directly linking CD3 on T cells to a TAA on cancer cells, resulting in the formation of a cytolytic synapse. This interaction stimulates T-cell activation, leading to the release of perforin and granzyme-B (GzmB), which induce tumor cell apoptosis [54].

BBB penetration studies
TCEs are emerging as promising therapeutic approaches for GBM, largely because of their small molecular size (~ 55 kDa), which enhances BBB penetration [55]. Unlike IgG-like BsAbs, TCEs lack the Fc region, reducing their overall size compared with that of conventional antibodies (~ 130 kDa) [32]. This structural modification not only improves tissue permeability but also eliminates Fc-mediated immune responses, such as antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity. As a result, TCEs help mitigate the risk of CRS, a serious adverse effect observed with certain IgG-like BsAbs, such as Catumaxomab, making them safer and more effective options for GBM treatment [56, 57].
This characteristic is particularly advantageous in treating GBM, where effective delivery of therapeutic agents to the brain is a significant challenge. For example, TCEs targeting EGFRvIII have demonstrated efficacy when administered systemically in intracranial tumor models, indicating their potential to reach and impact tumor sites within the brain [43].

Novel approaches
Recent advancements in GBM immunotherapy have focused on novel strategies to increase TCEs efficacy. One promising approach involves combining TCEs with ICIs, such as PD-1/PD-L1 blockers, to overcome T-cell exhaustion, a key challenge in the immunosuppressive environment of tumors. Preclinical studies indicate synergistic effects when these therapies are used together [58, 59]. Another innovative strategy is localized TCEs delivery through NSCs or oncolytic viruses, which can increase therapeutic concentrations at the tumor site while minimizing systemic toxicity [48]. For example, studies have shown that NSCs that secrete IL13Rα2-targeting TCEs significantly improve survival in glioma models [48]. Additionally, to address tumor heterogeneity, researchers are developing dual-target TCEs (e.g., EGFRvIII + HER2) that broaden therapeutic coverage by targeting multiple tumor cell populations [60, 61]. A study investigating a bispecific variable heavy domain antibody targeting both EphA2 and EphA3, two Eph receptor family members present in GBM and GBM stem cells but not in adult tissues, demonstrated reduced tumorigenesis and enhanced differentiation in a recurrent brain metastasis model. Notably, blocking only one receptor failed to elicit a significant therapeutic response, highlighting the necessity of dual targeting [62].

T-cell targets and Tumor-Specific antigens
The CD3 receptor, which is integral to the T-cell receptor (TCR) complex, is crucial for T-cell activation and function. Recent research has indicated that TCEs targeting CD3 may serve as an effective strategy for activating the immune system to oppose and eliminate cancer cells [22]. TCEs possess unique capabilities to bind to cancer cells, featuring two arms: one arm specifically binds to CD3 on T cells, while the other arm targets a specific tumor antigen, leading to robust T-cell activation, which results in the release of factors such as cytokines and granzymes, ultimately inducing the death of cancer cells. Nonetheless, this treatment method presents obstacles, requiring a design that achieves the intended therapeutic benefit while reducing immune system overstimulation and associated side effects. A significant issue in the application of TCEs is the overactivation of T cells, potentially resulting in a “cytokine storm” that triggers a severe immunological response and may cause perilous adverse effects, including systemic shock [63]. Researchers have endeavored to increase the binding precision of TCEs to CD3 and cancer tumor markers to increase their effectiveness. These modifications involve engineering molecules with suitable linker lengths and adjusting the binding affinity to mitigate the risk of unintended T-cell activation and adverse effects. Researchers are also investigating structural modifications to prolong the lifespan of these molecules in the patient’s system and diminish the requirement for repeated doses [64]. In addition to CD3, other molecules have been found on T-cell surfaces that may serve as potential targets for TCEs design. Research indicates that these objectives have proven more efficacious in certain studies and are correlated with a reduction in adverse effects [65]. CD28 is an alternative molecule that has been investigated as a target in T cells, as it is crucial for the activation process and enhances T-cell activation signals. Additionally, 1BB-4, another molecule with a regulatory function in immune responses, has been considered in the development of novel TCEs [66, 67]. Certain studies indicate that these alternative targets are more effective than CD3 in specific instances, particularly in cancers exhibiting greater resistance to immunotherapy interventions. Nonetheless, additional clinical studies are needed to precisely evaluate the efficacy of these targets [68, 69]. Future advancements in TCEs targeting a broader range of T-cell antigens may enhance therapeutic success for glioma patients. Furthermore, the integration of these molecules with additional medicines, such as immunosuppressants, can result in more personalized and efficacious treatments. The use of alternative molecules to target T cells may mitigate side effects, as these molecules play a regulatory role in the immune system, enhancing the immune response while averting severe reactions [42, 70]. The appropriate identification of therapeutic targets is a critical aspect of the efficacy of TCEs. This decision should be predicated on considerations such as the prevalence of antigen expression in the tumor, the safety of targeting, and the capacity to elicit a robust immune response [71]. The identification of tumor-specific antigens is critical for the efficacy of TCEs in glioma treatment. Antigens uniquely expressed in tumor cells can serve as appropriate targets in these therapies [43]. EGFRvIII is a mutant variant of the epidermal growth factor receptor (EGFR) prevalent in numerous people with GBM. This mutation is exclusively expressed in cancer cells and serves as a primary target in the development of TCEs [72]. HER2 is an antigen frequently associated with breast cancer; however, it is also present in certain glioma variants. This antigen may serve as an adjunctive target in certain patients [73]. The IL-13Rα2 receptor is selectively expressed in certain gliomas and has been suggested as a novel target in the development of TCEs. This receptor is a promising target for immunotherapy owing to its elevated expression in brain malignancies and absence in normal tissues [74]. In addition to the established antigens, novel antigens that may be efficacious in glioma treatment are being identified. These antigens may enhance the efficacy and specificity of TCEs treatments. The expression of several antigens in gliomas may vary according to histological subtype. Compared with other glioma variants, GBM has greater expression of EGFRvIII. These disparities may influence the selection of treatment targets [75]. Examining the correlation between antigen expression and other biological variables, including genetic and epigenetic alterations in tumors, can enhance the understanding of the mechanisms underlying tumor resistance and sensitivity to treatment. This information may enhance the optimization of treatment regimens for individuals [76]. Some antigens are suggested as both therapeutic targets and prognostic indicators. The expression of certain antigens is correlated with accelerated disease development and diminished patient survival, potentially serving as risk flags [77].

Subgroup analysis on the basis of histology and molecular features

Histological classification
The genetic diversity of glioma is associated with the expression of various tumor-specific antigens. EGFRvIII is the most investigated target for immunotherapy and is expressed by approximately 30% of GBM patients [78, 79]. Interleukin-13 receptor subunit alpha-2 (IL13Rα2), HER2, and GD2 are potential alternative targets for EGFRvIII [61, 80, 81]. However, GBM expresses a highly heterogeneous pattern of antigens and escapes from the immune system by downregulating or completely deleting antigens, mutation, and selective survival of tumor cells that are negative for specific antigens. As a consequence, the efficacy of TCEs is highly influenced by the abundance of cells expressing the target antigen [47, 82].
IDH-mutant gliomas constitute 90% of all secondary GBMs. This mutation results in the progression of low-grade glioma to high-grade tumors [83]. IDH mutation is associated with alterations in the expression of various genes, specifically immune-related genes [84]. NKG2D ligands, including MICA, MICB, and ULBP1-6, are highly important in NK-mediated immunity [84]. NKG2DLs, including MICA, MICB, and ULBP1–6, are upregulated on GBM cells and are therefore potential targets for TCEs [85]. However, the expression of the NKG2DLs is markedly lower in IDH-mutant gliomas than in IDH-wild-type gliomas. Low expression of the NKG2DLs may result in resistance to TCEs that target this ligand [86]. In addition, IDH-mutant gliomas produce (R)−2-hydroxyglutarate (R-2-HG), which is a T-cell suppressor oncometabolite. Thus, IDH-mutant gliomas contain fewer T cells in their microenvironment, which might restrict the ability of TCEs to activate T cells in tumoral cell lines [87].
The upregulation of PD-L1 and CD86 in glioma cells, the proliferation of T regs and myeloid-derived suppressor cells (MDSCs), and the differentiation of macrophages into the M2 phenotype, which release immunosuppressive cytokines, including IL-10 and TGF-β, are the major factors leading to the immunosuppressive tumor microenvironment (TME) of glioma and resulting in a decreased number of tumor-infiltrating lymphocytes [88, 89]. This immunosuppressive TME might result in the limited efficacy of TCEs, which mainly relies on T-cell activation.

Response patterns
Loss of the targeted antigen is the major obstacle influencing the efficacy of TCEs. It has been hypothesized that targeting alternative antigens (e.g., IL13Rα2, HER2, and GD2) may reduce the risk of tumor escape and enhance the ability to target different cell lines [61, 80, 81]. An animal study by Park et al. (2023) demonstrated that the use of EGFRvIII, which targets DNA-encoded bispecific TCEs in combination with HER2, which targets DNA-encoded bispecific TCEs, is associated with increased tumor control and decreased tumor evasion [47]. Moreover, DNA-encoded tri-specific TCEs targeting EGFRvIII, IL-13Rα2, and T cells have exhibited sustained expression and potential clinical efficacy in animal models of GBM [80].
In addition, combination therapy with HER2-targeting TCEs and NKG2D-based CAR-NK and T cells has been shown to effectively induce antitumor immunity and result in tumor control [90].
Simultaneous administration of erythropoietin-producing human hepatocellular carcinoma A2 receptor (EphA2)-targeting TCEs and EphA2 oncolytic adenoviruses has shown promising efficacy in preclinical studies [91].
Considering the T-cell-privileged TME of GBM, simultaneous administration of activated T cells may increase the efficacy of BiTE. However, this hypothesis requires careful investigation in preclinical studies [55]. The combination of EGFRvIII, which targets BiTE, and monoclonal antibodies blocking PD-1 has also been suggested to improve the immunosuppressive TME of GBM, and its efficacy is being investigated in a phase 1 clinical trial (NCT03296696) [55].
Preclinical studies have demonstrated that CAR-T cells engineered to secrete EGRvIII-targeting TCEs are effective at controlling tumor growth despite the heterogeneous expression of EGFRvIII [92].

Safety considerations

General safety profile
The safety profile of TCEs, particularly for glioma treatment, includes a range of AEs that vary in frequency and severity. The key AEs associated with blinatumomab include CRS, neurotoxicity, fever, and fatigue. In clinical studies, CRS occurs in approximately 4.9% of patients, whereas neurotoxic events are observed in 9% of cases [93, 94]. Other common AEs include headaches (47%), tremors (36%), and leukopenia (19%) [34].
Certain patient characteristics, such as a high disease burden or altered B/T-cell ratios, increase the risk of severe AEs, particularly CRS and neurotoxicity [34, 94]. Preexisting conditions, including infections or compromised immunity, may also increase the risk of AE [93]. Managing AEs requires close monitoring and supportive care. CRS management includes corticosteroids or tocilizumab to control symptoms, while neurotoxicity may necessitate dose adjustments or treatment interruptions [94].
Prophylactic dexamethasone is sometimes administered to prevent severe reactions. Careful patient selection, dose titration, and patient education are essential to mitigate risks and promote early intervention [94].
Neurotoxicity remains a significant concern with TCEs, manifesting as mild cognitive issues to severe events such as seizures or coma. The incidence of grade ≥ 3 neurotoxicity varies between 5.5% and 24% in clinical studies, with symptoms often appearing in the first treatment cycle. Dizziness, tremors, confusion, and encephalopathy are common presentations [55, 95].
Activated T cells can adhere to cerebral vessels and infiltrate cerebrospinal fluid, impairing microcirculation and leading to local ischemia [34]. The BBB complicates glioma treatment by limiting the effectiveness of TCEs. Although smaller TCEs molecules can penetrate the BBB more easily than monoclonal antibodies can, rapid clearance from the circulation reduces their therapeutic window [55]. Inflammatory cytokines such as IL-6 and IL-1β, which are released by T cells that cross the BBB, can further disrupt T cells, exacerbating neurotoxic symptoms and leading to immune effector cell-associated neurotoxicity syndrome [55, 93].

Special considerations
CRS is a significant AE associated with TCEs therapies, particularly in the treatment of gliomas, which require close management to optimize outcomes. Its severity depends on factors such as immune status, tumor burden, and drug dosage. Higher tumor burdens or preexisting inflammation increase the likelihood of severe CRS. Effective monitoring is crucial for the early detection and management of CRS. This involves the regular assessment of vital signs and laboratory tests to evaluate cytokine levels and organ function. Patients should be monitored closely during the first few cycles of treatment when CRS is most likely to occur. Symptoms such as fever, hypotension, tachycardia, and neurological changes should be tracked continuously [96, 97].
Management strategies for CRS typically follow a tiered approach:

Mild cases: Supportive care (e.g., hydration, antipyretics).

Moderate to severe cases: Corticosteroids or tocilizumab are used to control inflammation.

Life-threatening CRS: Immediate intensive care may be needed [96, 97].

Preventive strategies include premedicating with corticosteroids and using gradual dose escalation to minimize severe reactions [96, 97].

Long-term safety and QoL
Long-term safety is essential for glioma patients receiving TCEs therapy. Although early AEs such as CRS are concerning, many patients have favorable long-term safety profiles, with most not developing chronic toxicities. Data suggest favorable survival rates, with median overall survival reaching 23 months in some studies. However, chronic neurocognitive deficits and immune-related issues may arise, affecting patient outcomes [98–100].
QoL assessments indicate that many patients maintain stable QoL posttreatment, but cognitive impairments from the disease or treatment may diminish well-being. Factors such as age, sex, and psychological status also influence QoL outcomes [101].
Long-term surveillance includes regular neurological evaluations, cognitive assessments, and imaging to monitor for late-onset complications. Structured follow-up protocols enable early detection and management of chronic effects, ensuring better long-term outcomes [100].

Clinical experience of TCEs in GBM
Although clinical development of TCEs in GBM remains limited compared with hematologic malignancies, several early-phase studies have begun to evaluate the feasibility, safety and preliminary activity of TCE-based approaches. The most advanced programs include EGFRvIII-targeting TCEs such as AMG 596, IL-13Rα2-directed constructs, and Fn14-targeted or NKG2DLs -targeted agents, which have demonstrated manageable safety profiles and early signals of biological activity in phase 0–1 trials. However, response rates remain modest, and challenges such as antigen heterogeneity, immunosuppressive tumor microenvironment and restricted drug penetration across the BBB continue to limit clinical efficacy. Despite these barriers, the emerging clinical experience provides a foundation for next-generation TCE strategies and combinatorial regimens [42, 102–104].
An interim analysis of a phase 1 clinical trial (NCT03296696) evaluating the safety and efficacy of AMG 596 in patients with recurrent GBM showed that one PR (12.5%) and two cases of SD (25%) among eight patients had sufficient follow-up. Among the 14 patients eligible for evaluation, serious AEs were reported in 50% of the cohort, with the most observed treatment-related grade ≥ 3 AEs of headache and altered consciousness, each occurring in 14.3% of patients [105].
AMG 596 represents a prototypical TCEs molecule that specifically targets the class III variant of the EGFRvIII. EGFR is a transmembrane receptor that is often overexpressed in GBM patients and plays a significant role in tumor development. Notably, EGFRvIII is the predominant mutation of EGFR found in EGFR-positive GBM patients. An interim analysis from a phase 1 clinical trial (NCT03296696) assessing the safety and efficacy of AMG 596 in recurrent GBM revealed one partial response (PR) (12.5%) and two SD outcomes (25%) among eight patients with adequate follow-up. Among the 14 evaluable patients, 50% reported serious AEs, with the most frequently observed treatment-related grade ≥ 3 AEs being headache and altered consciousness, each occurring in 14.3% of patients [106, 107].

Future directions
TCEs comprise multiple structural formats, including tandem-scFv TCEs molecules and IgG-like Fc-containing designs. BiTEs are the smallest and most compact TCE subclass; however, most next-generation TCEs entering clinical development employ IgG-like or multivalent architectures. TCEs These fragments are connected to each other by a short linker and form the characteristic structure of a TCEs molecule. scFvs are composed of immunoglobulin (Ig) heavy chain (VH) and light chain (VL) variable regions that serve as antigen-binding domains [20, 108]. As a result, TCEs is characterized by its small size and flexibility. It enables efficient diffusion and rapid transport from the site of administration to the target lesions, thereby facilitating the redirection of cytotoxic T cells to cancer cells with high specificity [109] (Fig. 4).

Although belinatumumab has shown clinical efficacy in the treatment of B-cell malignancies, several challenges remain, including cumbersome administration methods, the emergence of treatment resistance, and limited efficacy in solid tumors. In response to these challenges, considerable research has focused on structural modification of the traditional TCEs structure and the creation of multifunctional T-cell engaging antibodies, with several candidates progressing toward clinical trials [109] (Fig. 5).

Half-life extended-bispecific T-cell engager (HLE- TCEs)
Canonical TCEs antibodies consist of two single-chain variable fragments (scFvs) and lack the Fc domain, which leads to a limited half-life necessitating continuous intravenous infusion [110]. To address this challenge and reduce treatment expenses, HLE-TCEs were engineered by incorporating an Fc domain, enabling weekly administration [111]. Nonetheless, the increased serum concentration associated with HLE-TCEs may increase the risk of toxicity in comparison with the rapidly eliminated canonical TCEs. Ongoing clinical trials are investigating HLE- TCEs antibodies across various cancer types. Initial findings from the HLE- TCEs AMG 673 trial in patients with relapsed/refractory acute myeloid leukemia (R/R AML) indicated that a weekly short intravenous infusion resulted in grade ≥ 3 AEs in 50% of participants, with a 13.3% occurrence of grade 3 CRS [112]. Additional dose escalation studies are currently underway.

CiTE and smite
The upregulation of immune checkpoints, particularly PD-1/PD-L1, is recognized as a mechanism contributing to resistance to TCEs therapy. Research has indicated that the expression of PD-L1 is elevated when AMG330 is introduced into acute myeloid leukemia (AML) cells in vitro. Inhibition of the PD-1/PD-L1 pathway has been shown to augment the cytotoxic effects of AMG 330 [113]. To specifically target CD33 + PD-L1 + cells while minimizing immune-related AEs, a bifunctional CiTE antibody was engineered by combining a low-affinity extracellular domain of PD-1 with an anti-CD33 TCEs construct [114].
TCEs antibodies facilitate TCR signaling through their binding to CD3 and TAAs, thereby establishing an immunological synapse between T cells and tumor cells without the need for costimulatory signals [115, 116]. The incorporation of CD137 or CD28 has been demonstrated to further enhance the cytotoxic potential of TCEs antibodies. Additionally, SMITE, which consists of two distinct TCEs antibodies targeting TAAs, CD3, and CD28, offers supplementary costimulatory activation and has the potential to counteract adaptive immune evasion [117, 118].

Secreted bite
The administration of BiTE and other T-cell-engaging antibodies is currently performed via intravenous infusion, necessitating continuous delivery owing to their limited half-life. If these antibodies are produced by living cells within the body, they could sustain consistent therapeutic concentrations and yield prolonged antitumor responses. Genetically engineered CAR-T cells and oncolytic viruses (OVs) have been designed to express BiTE antibodies, thereby combining complementary cancer immunotherapies to bolster antitumor efficacy, particularly in the context of solid tumors [119].
Choi et al. introduced CART. TCEs cells that specifically target EGFRvIII and secrete BiTE antibodies aimed at EGFR demonstrated significant antitumor effects in murine models of GBM. BiTE-armed OVs are capable of inducing localized oncolysis and activating T cells directly at the tumor site, which helps reduce systemic toxicity. Additionally, these OVs can target tumor-promoting cells within the tumor microenvironment (TME) [120].
Moreover, OVs can be engineered to express genes that encode cytokines, ICIs, and other therapeutic agents. For example, OVs that produce TCEs targeting CD44v6, along with IL-12 and anti-PD-L1, when combined with CAR-T cells targeting HER2, have demonstrated enhanced disease control in tumors exhibiting heterogeneous HER2 expression. The integration of TCEs therapy with other cancer immunotherapies, such as CAR-T cells and OVs, represents a promising avenue for optimizing therapeutic outcomes while minimizing systemic toxicity [121].

TCEs with a silenced Fc domain
The engagement of the Fc domain with its corresponding receptor on immune cells can result in unintended immune activation and potential toxicity. Nevertheless, the Fc domain plays a crucial role in extending the half-life of antibodies. To address this issue, antibodies featuring silent Fc domains have been engineered through specific point mutations, diminishing undesirable immune activities while preserving a prolonged half-life. An illustrative example is GEN3013, a human IgG1 bispecific antibody designed to target CD3 and CD20, which incorporates silent Fc mutations (L234F, L235E, D265A) and is currently in clinical trials for relapsed/refractory B-cell lymphoma. Recent research has suggested that bispecific antibodies with silent Fc domains promote T-cell infiltration and enhance antitumor responses, in contrast to bispecific antibodies with functional Fc domains that do not effectively facilitate T-cell trafficking [57].

Multivalent and multispecific TCEs
TCEs antibodies are characterized by their bispecific and bivalent nature, allowing for enhancement through the use of multivalent or multispecific T-cell engager antibodies. These antibodies possess multiple antigen-binding sites, which increase their binding affinity and enable them to target various TAAs and molecules that play a role in T-cell activation [122, 123]. Notably, multivalent T-cell engager molecules, such as AMV 564, have demonstrated encouraging preliminary clinical outcomes. AMV 564 is a tetravalent bispecific molecule that simultaneously targets CD3 on T cells and CD33 on MDSCs, thereby facilitating T-cell activation and promoting antitumor immune responses. Phase 1 clinical trials have indicated its efficacy and manageable toxicity profile [124].
The development of multispecific TCEs is focused on enhancing therapeutic efficacy while minimizing toxicity and addressing treatment resistance. An example of this is a tri-specific T-cell engager antibody that targets CD3, CD28, and CD38, which serves to amplify T-cell activation and specifically addresses hematologic malignancies. Although clinical trials for this trispecific antibody are still forthcoming, it holds significant promise as a next-generation immunotherapeutic approach [125].
AMG 596 represents the first TCEs molecule that specifically targets the EGFRvIII. EGFR is a transmembrane receptor that is frequently overexpressed in GBM patients and plays an important role in tumorigenesis. Notably, EGFRvIII has been identified as the predominant EGFR mutation in EGFR-positive GBM patients [106].
Research is currently underway to amplify TCEs constructs and develop new T-cell-engaging antibodies that exhibit greater antigenic affinities and target multiple antigens. This exploration includes a range of preclinical and clinical studies, as well as investigations of combination therapies that integrate TCEs antibodies with other therapies. Furthermore, interest in approaches that target innate immune cells or innate-like cells, which have shown significant antitumor effects in various types of cancer, is increasing, indicating a promising direction for future therapeutic strategies [95].

Future perspectives
The future of TCEs therapy in glioma depends on overcoming the unique problems posed by tumor heterogeneity, immunosuppression, and limited drug delivery across the BBB. Creating multivalent or trivalent TCEs that can target more than one antigen at the same time is promising. This method could lower the chances of immune evasion and improve long-term tumor control. Moreover, innovative drug delivery systems, such as DNA-encoded TCEs, NSCs, and oncolytic viruses, are being explored to achieve stable and localized TCEs expression directly within the tumor microenvironment. Another important area of progress is combining TCEs with other immunotherapy methods. TCEs can work together with ICIs, CAR-T cells, and oncolytic viral therapies to fight gliomas, which suppress the immune system and increase T-cell activation. Moreover, advancements in molecular engineering, such as Fc-silenced constructs and TCEs with longer half-lives, are being used to improve therapeutic exposure while lowering toxicity, especially neurotoxicity and CRS. In the future, it will be very important to make TCEs therapy more personal. Tailoring therapy to specific genetic subtypes, antigen expression patterns, and tumor immune profiles may enhance its efficacy and safety, especially in distinguishing IDH-mutated gliomas from wild-type gliomas. TCEs are expected to evolve from experimental treatment to a crucial element of comprehensive glioma strategies through continuous clinical validation and innovation, thereby providing renewed hope to patients suffering from this highly aggressive disease.

Limitations, challenges and next steps for TCEs therapy in GBM
TCE-based immunotherapy for GBM faces several unique challenges. First, target antigen heterogeneity and rapid antigen loss promote immune escape and limit durable responses. Second, physical barriers such as the BBB constrain adequate drug exposure, especially for systemically administered IgG-like TCEs. Third, the profoundly immunosuppressive tumor microenvironment characterized by T-cell exhaustion, suppressive myeloid populations and low effector T-cell infiltration reduces the functional capacity of redirected T cells. Next steps should include the development of dual-targeting TCEs to mitigate antigen escape, conditional TCEs activated only within the tumor microenvironment to reduce systemic toxicity, and TCEs incorporating costimulatory domains to enhance T-cell persistence. Combining TCEs with radiotherapy, ICIs, or myeloid-targeting agents may further augment anti-tumor immunity. Key limitations of current evidence include the scarcity of clinical trials, small patient numbers, lack of randomized data, and incomplete understanding of optimal delivery routes. Addressing these barriers will be essential for translating the growing preclinical promise of TCEs into meaningful clinical benefit for patients with GBM.

Conclusion
Bispecific T-cell receptors constitute a novel immunotherapy strategy for gliomas because they offer potent T-cell homing against highly resistant tumors and antigen-specific targeting. According to preclinical and early clinical studies, they can induce tumor regression while preserving acceptable safety levels. However, significant challenges such as limited durability, tumor heterogeneity, and treatment-related toxicity must be addressed before widespread clinical acceptance. Examples of innovations that could improve efficacy and expand use include logical immunotherapy combinations, advanced drug delivery techniques, and multiantigen targeting. When all is said and done, TCEs have a great deal of potential to improve or even transform the therapeutic landscape for GBM, which would ultimately lead to better patient outcomes in a condition with a large unmet medical need.