Exploration of Neuroscience

Table 1. Summary of bi-, tri-, and multi-specific T-cell engager therapies in preclinical GBM models and their effects against key immunological barriers.

Study, year	Construct, model type (in vivo, in vitro), delivery method	Antigen target	Antigen heterogeneity	Immune escape	Blood-brain barrier	Tumor microenvironment
Choi et al. [25], 2025	Construct: BiTE (encoded by oncolytic adenoviruses) + CAR-T cells Model (in vitro + in vivo): in vitro (U87, U251 cell lines, 2D/3D, BBB spheroid); in vivo (subcutaneous GBM xenograft, NSG mice) Delivery: intratumoral injection of BiTE followed by systemic infusion of CAR-T	IL13Rα2 (BiTE), EGFR, and EGFRvIII (CAR-T)	√ Multimodal BiTE & CAR-T targeting multiple antigens	× Significant reductions in tumor luminescence intensity; however, no evidence of durable control, memory, or bystander T-cell recruitment	× The subcutaneous tumor model is not able to accurately assess the BBB	√ Increased intratumoral CD3⁺ T-cell recruitment and infiltration
Zannikou et al. [24], 2025	Construct: BiTE Model (in vivo + in vitro): immunocompetent mice (genetically engineered mouse model, orthotopic GL261-IL13Rα2) + murine glioma cell lines (GL261, SMA560, and CT2A) Delivery: systemic (IV)	IL13Rα2	× Single target	√ Enhanced memory T-cell formation, sustained T-cell activity	√ BiTE detected in the brain following systemic IV administration	√ Increased intracranial CD8⁺ T-cells, memory T-cells, and regulatory T-cells (Tregs). Reduced glioma volume and viability, reduced intracranial immunosuppressive myeloid cells
Brosius et al. [16], 2024	Construct: BiTE Model (in vivo + in vitro): orthotopic high-grade glioma xenografts in nude mice + in vitro human/mouse co-cultures Delivery: injected intracranially	EGFR	× Single target	× Not specifically addressed	√ Bypassed via local delivery of migratory cortical inhibitory interneuron precursors (MCIPs), which migrated intracranially to tumors and secreted BiTEs within the CNS	√ In vitro, killing of GBM cells was induced by CD8⁺ T-cells, but no clear evidence of TME modulation in vivo
Park et al. [21], 2024	Construct: TriTE Model (in vivo + in vitro): immunocompromised mice, as well as a patient in vitro assay using patient peripheral blood mononuclear cells (PBMCs) Delivery: intramuscular injection	EGFRvIII and IL13Rα2	√ Targets both EGFRvIII and IL13Rα2 in a single construct	√ Durable tumor clearance, sustained expression (up to 105 days), and effective tumor control in a heterogeneous GBM model, including post-radiotherapy and post-chemotherapy PBMC	× Unclear if crosses BBB or acts via peripheral immune activation	√ Induces activation of CD4⁺, CD8⁺, and natural killer T-cells; promotes antitumor cytokine release (IFN-γ, TNF-α, IL-2)
Baugh et al. [1], 2024	Construct: BiTE (delivered via oncolytic HSV-1 G207) Model (in vitro only): GBM cell lines, patient-derived mesenchymal glioma stem cells Delivery: no in vivo models	NKG2DL	× Single target	× Not addressed—no demonstration of durable tumor control, memory T-cell formation, or bystander activation	× Entirely in vitro	√ CD4⁺ and CD8⁺ T-cell activation with increased CD25, CD69, IFN-γ, granzyme B, perforin, and CD107a in the presence of GBM cells; activity synergized with sublethal radiation and temozolomide, enhancing antigen expression and T-cell activation
Park et al. [22], 2023	Construct: multivalent BiTEs Model (in vivo + in vitro): mice Delivery: intramuscular injection plus electroporation	EGFRvIII and HER2	√ Targeting both EGFRvIII and HER2 resulted in enhanced cytotoxicity and 80% tumor clearance	√ Potent and durable CD4⁺ and CD8⁺ T-cell activation; mitigated immune escape in 80% of the challenged mice	× Not directly addressed	√ Activated CD4⁺ and CD8⁺ T-cells with increased secretion of IFN-γ, TNF-α, IL-2, and activation of CD107a (marker for degranulation). Tumor regression in orthotopic models
Bhojnagarwala et al. [15], 2022	Construct: BiTE Model (in vivo + in vitro): immunodeficient NSG mice; In vitro: U87, U251, U373 GBM lines Delivery: systemic (IV) via DNA electroporation	IL13Rα2	× Single target	× Not specifically addressed	√ Peripherally delivered DNA-based BiTE crossed the BBB and controlled orthotopic GBM growth	√ CD4⁺ and CD8⁺ T-cell activation, cytokine release (IFN-γ, IL-2, TNF-α), granule secretion (perforin, granzyme A and B), and tumor cytolysis
Huynh et at. [28], 2022	Construct: dual antigen T-cell engager Model (in vitro only): 3D GBM spheroid models (GBM08, BT935) Delivery: no in vivo models; in vitro used hydrogel-based local release system	CD133	× Single target	√ Small increase in CD45RO⁺ effector memory T-cells	× Not addressed (no in vivo or BBB-relevant model)	× TME effects are not specifically addressed, as no in vivo or stromal component
Yin et al. [11], 2022	Construct: BiTE Model (in vivo + in vivo): orthotopic mice Delivery: infusion (route not specified)	EGFR and IL13Rα2	√ Dual antigen targeting (EGFR and IL13Rα2) using bivalent BiTE constructs	× No evidence demonstrated	× Unclear if systemic delivery crossed the BBB	√ Enhanced T-cell activation (CD69), cytokine production (IFN-γ, TNF-α, IL-2), tumor infiltration, and tumor suppression
Li et al. [29], 2021	Construct: BiTE Model (in vivo + in vitro): mice; U87/U251/A172/T98G cell lines Delivery: local intratumoral injection	Fn14	× Single target	× No evidence demonstrated	√ Injected directly into the lesion and suppressed tumor growth	√ Increased CD3⁺ T-cell infiltration and tumor suppression
Pituch et al. [18], 2021	Construct: BiTE (secreted by neural stem cells) Model (in vivo + in vitro): in vivo (mice), in vitro (human PBMCs, GBM6, GBM12, GBM39) Delivery: local intratumoral injection	IL13Rα2	× Single target	× Partially addressed as it engages local CD3⁺ T-cells and produces granzyme B, but no durable control, memory T-cells, bystander cell recruitment	√ Local intracranial NSC delivery enables CNS access, where it persists in the tumor	√ Increased CD3⁺ infiltration, IFN-γ/TNF-α/IL-2 cytokine production, activation of exhausted tumor-infiltrating T-cells
Arnone et al. [30], 2021	Construct: BiTE (encoded by oncolytic adenoviruses) Model (in vitro + in vivo): in vitro human GBM cancer cell lines (U373, U87) and an in vivo xenograft mouse model Delivery: local intratumoral injection	EphA2	× Single target	√ Increase memory T cells, increase activation of CD4 and CD8 T cells	√ Injected directly into the lesion with detection of infiltrating T-cells in the tumor	√ Enhanced intratumoral T-cell infiltration, activation of T-cell effector function, including Th1 cytokines (IFN-γ), and increased chemokine production
Gardell et al. [19], 2020	Construct: BiTEs (secreted by genetically engineered human macrophages) Model (in vivo + in vitro): mice (subcutaneous and intracranial GBM U87 EGFRvIII xenografts); in vitro (human T-cells + GEMs + EGFRvIII⁺ GBM cells) Delivery: local intratumoral injection	EGFRvIII	× Single target	× Tumor rebound observed in both models; no survival benefit from BiTE GEMs alone; modest effect enhanced by IL-12 co-secretion. In vitro upregulation of memory-associated gene (PRDM1) and cytokines associated with T-cell survival (IL-2, IL-7, IL-15), but not formally assessed in vivo	√ Bypasses BBB via direct intracranial injection; GEMs enable local BiTE secretion and CNS-targeted immune activation	√ Increased CD3⁺, CD8⁺ T-cell infiltration (↑ CD25, CD69, CD107a, IFN-γ, granzyme B), increased cytokines (IFN-γ, TNF-α, IL-2/7/15), chemokines (CXCL9/13), cytotoxic markers (GZMB, LAMP3); downregulation of immunosuppressant TGFB1
Choi et al. [31], 2019	Construct: BiTEs (secreted by CAR-T cells) Model (in vivo + in vitro): mice (orthotopic and heterogenous GBM xenografts), in vivo (primary human T-cells and GBM cells) Delivery: local intraventricular injection	EGFRvIII	√ Able to mitigate antigen escape by redirecting bystander T-cells against EGFR-positive, EGFRvIII-negative tumor cells	√ Redirect non-specific bystander T-cells and Tregs to exert cytotoxicity; reverses exhaustion when CAR + BiTE co-stimulation used (reduced PD-1, TIM-3 and LAG-3)	√ Intraventricular delivery enables local BiTE production within the CNS. BiTE was not detected systemically	√ Increased T-cell infiltration and cytokine secretion (IFN-γ, TNF-α)

Return to article view

×: Does not fulfill criteria; √: fulfills criteria. BBB: blood-brain barrier; BiTE: bispecific T-cell engager; CAR-T: chimeric antigen receptor T-cell; CNS: central nervous system; EGFR: epidermal growth factor receptor; EGFRvIII: epidermal growth factor receptor variant III; EphA2: erythropoietin-producing human hepatocellular carcinoma A2 receptor; Fn14: fibroblast growth factor-inducible 14; GBM: glioblastoma multiforme; GEMs: genetically engineered macrophages; IL13Rα2: interleukin 13 receptor alpha 2; NKG2DL: natural killer group 2 member D ligands; TME: tumor microenvironment.

Declarations

Author contributions

AHL: Conceptualization, Investigation, Writing—original draft, Writing—review & editing. MA: Conceptualization. Both authors read and approved the submitted version.

Conflicts of interest

Both authors declare that they have no conflicts of interest.

Ethical approval

Not applicable.

Consent to participate

Not applicable.

Consent to publication

Not applicable.

Availability of data and materials

All datasets analyzed for this study are included in the main text and the references.

Funding

Not applicable.

Copyright

Publisher’s note

Open Exploration maintains a neutral stance on jurisdictional claims in published institutional affiliations and maps. All opinions expressed in this article are the personal views of the author(s) and do not represent the stance of the editorial team or the publisher.

References

Baugh R, Khalique H, Page E, Lei-Rossmann J, Wan PK, Johanssen T, et al. Targeting NKG2D ligands in glioblastoma with a bispecific T-cell engager is augmented with conventional therapy and enhances oncolytic virotherapy of glioma stem-like cells. J Immunother Cancer. 2024;12:e008460. [DOI] [PubMed] [PMC]

Stupp R, Mason WP, Bent MJvd, Weller M, Fisher B, Taphoorn MJB, et al.; National Cancer Institute of Canada Clinical Trials Group. Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. N Engl J Med. 2005;352:987–96. [DOI] [PubMed]

Reardon DA, Brandes AA, Omuro A, Mulholland P, Lim M, Wick A, et al. Effect of Nivolumab vs Bevacizumab in Patients With Recurrent Glioblastoma: The CheckMate 143 Phase 3 Randomized Clinical Trial. JAMA Oncol. 2020;6:1003–10. [DOI] [PubMed] [PMC]

Ameratunga M, Coleman N, Welsh L, Saran F, Lopez J. CNS cancer immunity cycle and strategies to target this for glioblastoma. Oncotarget. 2018;9:22802–16. [DOI] [PubMed] [PMC]

Tiwari S, Han Z. Immunotherapy: Advancing glioblastoma treatment-A narrative review of scientific studies. Cancer Rep (Hoboken). 2023;7:e1947. [DOI] [PubMed] [PMC]

Liau LM, Ashkan K, Tran DD, Campian JL, Trusheim JE, Cobbs CS, et al. First results on survival from a large Phase 3 clinical trial of an autologous dendritic cell vaccine in newly diagnosed glioblastoma. J Transl Med. 2018;16:142. [DOI] [PubMed] [PMC]

Choi BD, Gerstner ER, Frigault MJ, Leick MB, Mount CW, Balaj L, et al. Intraventricular CARv3-TEAM-E T Cells in Recurrent Glioblastoma. N Engl J Med. 2024;390:1290–8. [DOI] [PubMed] [PMC]

Lassman AB, Pugh SL, Wang TJC, Aldape K, Gan HK, Preusser M, et al. Depatuxizumab mafodotin in EGFR-amplified newly diagnosed glioblastoma: A phase III randomized clinical trial. Neuro Oncol. 2023;25:339–50. [DOI] [PubMed] [PMC]

Sterner RC, Sterner RM. CAR-T cell therapy: current limitations and potential strategies. Blood Cancer J. 2021;11:69. [DOI] [PubMed] [PMC]

10.

Rolin C, Zimmer J, Seguin-Devaux C. Bridging the gap with multispecific immune cell engagers in cancer and infectious diseases. Cell Mol Immunol. 2024;21:643–61. [DOI] [PubMed] [PMC]

11.

Yin Y, Rodriguez JL, Li N, Thokala R, Nasrallah MP, Hu L, et al. Locally secreted BiTEs complement CAR T cells by enhancing killing of antigen heterogeneous solid tumors. Mol Ther. 2022;30:2537–53. [DOI] [PubMed] [PMC]

12.

Ahn M, Cho BC, Felip E, Korantzis I, Ohashi K, Majem M, et al.; DeLLphi-301 Investigators. Tarlatamab for Patients with Previously Treated Small-Cell Lung Cancer. N Engl J Med. 2023;389:2063–75. [DOI] [PubMed]

13.

Hassel JC, Piperno-Neumann S, Rutkowski P, Baurain J, Schlaak M, Butler MO, et al. Three-Year Overall Survival with Tebentafusp in Metastatic Uveal Melanoma. N Engl J Med. 2023;389:2256–66. [DOI] [PubMed] [PMC]

14.

Dowlati A, Hummel H, Champiat S, Olmedo ME, Boyer M, He K, et al. Sustained Clinical Benefit and Intracranial Activity of Tarlatamab in Previously Treated Small Cell Lung Cancer: DeLLphi-300 Trial Update. J Clin Oncol. 2024;42:3392–9. [DOI] [PubMed] [PMC]

15.

Bhojnagarwala PS, O’Connell RP, Park D, Liaw K, Ali AR, Bordoloi D, et al. In vivo DNA-launched bispecific T cell engager targeting IL-13Rα2 controls tumor growth in an animal model of glioblastoma multiforme. Mol Ther Oncolytics. 2022;26:289–301. [DOI] [PubMed] [PMC]

16.

Brosius SN, Manley W, Harvey K, Gallanaugh J, Rohacek D, Anderson S, et al. Interneurons that BiTE: Harnessing the migratory capacity of cortical inhibitory interneuron precursors to treat high-grade glioma. bioRxiv [Preprint]. 2024 [cited 2025 Sep 15]. Available from: https://www.biorxiv.org/content/10.1101/2024.04.29.591530v1

17.

Singh K, Hotchkiss KM, Mohan AA, Reedy JL, Sampson JH, Khasraw M. For whom the T cells troll? Bispecific T-cell engagers in glioblastoma. J Immunother Cancer. 2021;9:e003679. [DOI] [PubMed] [PMC]

18.

Pituch KC, Zannikou M, Ilut L, Xiao T, Chastkofsky M, Sukhanova M, et al. Neural stem cells secreting bispecific T cell engager to induce selective antiglioma activity. Proc Natl Acad Sci U S A. 2021;118:e2015800118. [DOI] [PubMed] [PMC]

19.

Gardell JL, Matsumoto LR, Chinn H, DeGolier KR, Kreuser SA, Prieskorn B, et al. Human macrophages engineered to secrete a bispecific T cell engager support antigen-dependent T cell responses to glioblastoma. J Immunother Cancer. 2020;8:e001202. [DOI] [PubMed] [PMC]

20.

Subklewe M. BiTEs better than CAR T cells. Blood Adv. 2021;5:607–12. [DOI] [PubMed] [PMC]

21.

Park DH, Bhojnagarwala PS, Liaw K, Bordoloi D, Tursi NJ, Zhao S, et al. Novel tri-specific T-cell engager targeting IL-13Rα2 and EGFRvIII provides long-term survival in heterogeneous GBM challenge and promotes antitumor cytotoxicity with patient immune cells. J Immunother Cancer. 2024;12:e009604. [DOI] [PubMed] [PMC]

22.

Park DH, Liaw K, Bhojnagarwala P, Zhu X, Choi J, Ali AR, et al. Multivalent in vivo delivery of DNA-encoded bispecific T cell engagers effectively controls heterogeneous GBM tumors and mitigates immune escape. Mol Ther Oncolytics. 2023;28:249–63. [DOI] [PubMed] [PMC]

23.

Sharma P, Aaroe A, Liang J, Puduvalli VK. Tumor microenvironment in glioblastoma: Current and emerging concepts. Neurooncol Adv. 2023;5:vdad009. [DOI] [PubMed] [PMC]

24.

Zannikou MZ, Duffy JT, Procissi D, Najem H, Levine RN, Hambardzumyan D, et al. A Bi-Specific T Cell-Engaging Antibody Shows Potent Activity, Specificity, and Tumor Microenvironment Remodeling in Experimental Syngeneic and Genetically Engineered Models of GBM. bioRxiv [Preprint]. 2025 [cited 2025 Sep 15]. Available from: https://www.biorxiv.org/content/10.1101/2024.12.18.628714v2 [PubMed] [PMC]

25.

Choi MJ, So EY, Akosman B, Lee YE, Raufi AG, Bertone P, et al. Enabling CAR-T Cell Immunotherapy in Glioblastoma by Modifying Tumor Microenvironment via Oncolytic Adenovirus Encoding Bispecific T Cell Engager. bioRxiv [Preprint]. 2025 [cited 2025 Sep 15]. Available from: https://www.biorxiv.org/content/10.1101/2025.07.30.667708v1

26.

Rosenthal MA, Balana C, van Linde ME, Sayehli C, Fiedler WM, Wermke M, et al. ATIM-49 (LTBK-01). AMG 596, a novel anti-EGFRvIII bispecific T cell engager (BITE®) molecule for the treatment of glioblastoma (GBM): planned interim analysis in recurrent GBM (RGBM). Neuro-Oncol. 2019;21:vi283–5. [DOI] [PMC]

27.

Sternjak A, Lee F, Thomas O, Balazs M, Wahl J, Lorenczewski G, et al. Preclinical Assessment of AMG 596, a Bispecific T-cell Engager (BiTE) Immunotherapy Targeting the Tumor-specific Antigen EGFRvIII. Mol Cancer Ther. 2021;20:925–33. [DOI] [PubMed]

28.

Huynh V, Tatari N, Marple A, Savage N, McKenna D, Venugopal C, et al. Real-time evaluation of a hydrogel delivery vehicle for cancer immunotherapeutics within embedded spheroid cultures. J Control Release. 2022;348:386–96. [DOI] [PubMed]

29.

Li G, Zhang Z, Cai L, Tang X, Huang J, Yu L, et al. Fn14-targeted BiTE and CAR-T cells demonstrate potent preclinical activity against glioblastoma. Oncoimmunology. 2021;10:1983306. [DOI] [PubMed] [PMC]

30.

Arnone CM, Polito VA, Mastronuzzi A, Carai A, Diomedi FC, Antonucci L, et al. Oncolytic adenovirus and gene therapy with EphA2-BiTE for the treatment of pediatric high-grade gliomas. J Immunother Cancer. 2021;9:e001930. [DOI] [PubMed] [PMC]

31.

Choi BD, Yu X, Castano AP, Bouffard AA, Schmidts A, Larson RC, et al. CAR-T cells secreting BiTEs circumvent antigen escape without detectable toxicity. Nat Biotechnol. 2019;37:1049–58. [DOI] [PubMed]

32.

García-Montaño LA, Licón-Muñoz Y, Martinez FJ, Keddari YR, Ziemke MK, Chohan MO, et al. Dissecting Intra-tumor Heterogeneity in the Glioblastoma Microenvironment Using Fluorescence-Guided Multiple Sampling. Mol Cancer Res. 2023;21:755–67. [DOI] [PubMed] [PMC]

33.

Coy S, Lee JS, Chan SJ, Woo T, Jones J, Alexandrescu S, et al. Systematic characterization of antibody-drug conjugate targets in central nervous system tumors. Neuro Oncol. 2024;26:458–72. [DOI] [PubMed] [PMC]

34.

Barish ME, Weng L, Awabdeh D, Zhai Y, Starr R, D’Apuzzo M, et al. Spatial organization of heterogeneous immunotherapy target antigen expression in high-grade glioma. Neoplasia. 2022;30:100801. [DOI] [PubMed] [PMC]

35.

Philippova J, Shevchenko J, Sennikov S. GD2-targeting therapy: a comparative analysis of approaches and promising directions. Front Immunol. 2024;15:1371345. [DOI] [PubMed] [PMC]

36.

Brown CE, Alizadeh D, Starr R, Weng L, Wagner JR, Naranjo A, et al. Regression of Glioblastoma after Chimeric Antigen Receptor T-Cell Therapy. N Engl J Med. 2016;375:2561–9. [DOI] [PubMed] [PMC]

37.

O’Rourke DM, Nasrallah MP, Desai A, Melenhorst JJ, Mansfield K, Morrissette JJD, et al. A single dose of peripherally infused EGFRvIII-directed CAR T cells mediates antigen loss and induces adaptive resistance in patients with recurrent glioblastoma. Sci Transl Med. 2017;9:eaaa0984. [DOI] [PubMed] [PMC]

38.

Einsele H, Borghaei H, Orlowski RZ, Subklewe M, Roboz GJ, Zugmaier G, et al. The BiTE (bispecific T-cell engager) platform: Development and future potential of a targeted immuno-oncology therapy across tumor types. Cancer. 2020;126:3192–201. [DOI] [PubMed]

39.

Iwahori K, Kakarla S, Velasquez MP, Yu F, Yi Z, Gerken C, et al. Engager T cells: a new class of antigen-specific T cells that redirect bystander T cells. Mol Ther. 2015;23:171–8. [DOI] [PubMed] [PMC]

40.

Hart E, Odé Z, Derieppe MPP, Groenink L, Heymans MW, Otten R, et al. Blood-brain barrier permeability following conventional photon radiotherapy - A systematic review and meta-analysis of clinical and preclinical studies. Clin Transl Radiat Oncol. 2022;35:44–55. [DOI] [PubMed] [PMC]

41.

Woroniecka KI, Rhodin KE, Chongsathidkiet P, Keith KA, Fecci PE. T-cell Dysfunction in Glioblastoma: Applying a New Framework. Clin Cancer Res. 2018;24:3792–802. [DOI] [PubMed] [PMC]

42.

Tawbi HA, Forsyth PA, Algazi A, Hamid O, Hodi FS, Moschos SJ, et al. Combined Nivolumab and Ipilimumab in Melanoma Metastatic to the Brain. N Engl J Med. 2018;379:722–30. [DOI] [PubMed] [PMC]

43.

Reardon DA, Kim TM, Frenel J, Simonelli M, Lopez J, Subramaniam DS, et al. Treatment with pembrolizumab in programmed death ligand 1-positive recurrent glioblastoma: Results from the multicohort phase 1 KEYNOTE-028 trial. Cancer. 2021;127:1620–9. [DOI] [PubMed]

44.

Reardon DA, Kaley TJ, Dietrich J, Clarke JL, Dunn G, Lim M, et al. Phase II study to evaluate safety and efficacy of MEDI4736 (durvalumab) + radiotherapy in patients with newly diagnosed unmethylated MGMT glioblastoma (new unmeth GBM). J Clin Oncol. 2019;37:2032. [DOI]

45.

Arvedson T, Bailis JM, Urbig T, Stevens JL. Considerations for design, manufacture, and delivery for effective and safe T-cell engager therapies. Curr Opin Biotechnol. 2022;78:102799. [DOI] [PubMed]