EphA3-targeted chimeric antigen receptor T cells are effective in glioma and generate curative memory T cell responses.

BACKGROUND: High-grade gliomas including glioblastoma (GBM) and diffuse midline gliomas (DMG) represent the most lethal and aggressive brain cancers where current treatment modalities offer limited efficacy. Chimeric antigen receptor (CAR) T cell therapies have emerged as a promising strategy, boasting tumor-specific targeting and the unique ability to penetrate the blood-brain barrier. However, the effective clinical application hinges on the optimal choice of antigen, with a limited number, currently under investigation. METHODS: We employed cell surface proteomic analysis of primary human high-grade glioma samples from both adult and pediatric patients. This led to the identification of Ephrin type-A receptor 3 (EphA3) as a prevalently expressed target. We engineered a second-generation EphA3-targeted CAR T cell and assessed function using in vitro and in vivo models of GBM and DMG. RESULTS: EphA3-targeted CAR T cells demonstrated robust antigen-specific killing of human GBM and DMG cell lines in vitro. In an orthotopic xenograft NSG mouse model, EphA3-targeted CAR T cells not only effectively eradicated tumors but also established a functional T cell population protective on rechallenge. Remarkably, mice rechallenged with a second contralateral orthotopic tumor implantation achieved complete tumor clearance and maintained a sustained complete response 6 months following initial treatment. CONCLUSION: Building on the proven safety profile of EphA3 antibodies in clinical settings, our study provides compelling preclinical evidence supporting the efficacy of EphA3-targeted CAR T cells against high-grade gliomas. These findings underscore the potential for transitioning this innovative therapy into clinical trials, aiming to revolutionize the treatment landscape for patients afflicted with these formidable brain cancers.

HER2 chimeric antigen receptor T cell immunotherapy is an effective treatment for diffuse intrinsic pontine glioma.

Background: Diffuse intrinsic pontine glioma (DIPG) and other diffuse midline gliomas (DMG) of the thalamus and spinal cord are rare but devastating high-grade glial tumors of childhood with no curative treatment. Despite aggressive treatment attempts the prognosis has remained poor. Chimeric antigen receptor (CAR) T cell therapy has been identified as a promising new approach in the treatment of DMG tumors; however, additional targets are urgently required given known tumor heterogeneity and the prospect of antigen escape of this cancer. Methods: Using cell surface mass spectrometry, we detected high HER2 cell surface protein across a panel of patient-derived DIPG cells, thereby identifying an existing CAR T cell therapy for use in DIPG. Primary human T cells were transduced to express a second-generation HER2 CAR and interrogated for efficacy against patient-derived DIPG cells. Results: HER2 CAR T cells demonstrated potent and antigen-specific cytotoxicity and cytokine secretion when co-cultured with patient-derived DIPG cells. Furthermore, HER2 CAR T cells provided a significant regression in intracranial DIPG xenograft tumors. Conclusions: HER2 CAR T cells are already in clinic development and are well tolerated in pediatric patients. Here we provide strong preclinical evidence for the inclusion of DIPG patients in future pediatric CNS tumor HER2 CAR T cell clinical trials.

Human EGFRvIII chimeric antigen receptor T cells demonstrate favorable safety profile and curative responses in orthotopic glioblastoma.

Objectives: Glioblastoma is a highly aggressive and fatal brain malignancy, and effective targeted therapies are required. The combination of standard treatments including surgery, chemotherapy and radiotherapy is not curative. Chimeric antigen receptor (CAR) T cells are known to cross the blood-brain barrier, mediating antitumor responses. A tumor-expressed deletion mutant of the epidermal growth factor receptor (EGFRvIII) is a robust CAR T cell target in glioblastoma. Here, we show our de novo generated, high-affinity EGFRvIII-specific CAR; GCT02, demonstrating curative efficacy in human orthotopic glioblastoma models. Methods: The GCT02 binding epitope was predicted using Deep Mutational Scanning (DMS). GCT02 CAR T cell cytotoxicity was investigated in three glioblastoma models in vitro using the IncuCyte platform, and cytokine secretion with a cytometric bead array. GCT02 in vivo functionality was demonstrated in two NSG orthotopic glioblastoma models. The specificity profile was generated by measuring T cell degranulation in response to coculture with primary human healthy cells. Results: The GCT02 binding location was predicted to be located at a shared region of EGFR and EGFRvIII; however, the in vitro functionality remained exquisitely EGFRvIII specific. A single CAR T cell infusion generated curative responses in two orthotopic models of human glioblastoma in NSG mice. The safety analysis further validated the specificity of GCT02 for mutant-expressing cells. Conclusion: This study demonstrates the preclinical functionality of a highly specific CAR targeting EGFRvIII on human cells. This CAR could be an effective treatment for glioblastoma and warrants future clinical investigation.

BH3 mimetic drugs cooperate with Temozolomide, JQ1 and inducers of ferroptosis in killing glioblastoma multiforme cells.

Glioblastoma multiforme (GBM) is the most common and aggressive form of brain cancer, with treatment options often constrained due to inherent resistance of malignant cells to conventional therapy. We investigated the impact of triggering programmed cell death (PCD) by using BH3 mimetic drugs in human GBM cell lines. We demonstrate that co-targeting the pro-survival proteins BCL-XL and MCL-1 was more potent at killing six GBM cell lines compared to conventional therapy with Temozolomide or the bromodomain inhibitor JQ1 in vitro. Enhanced cell killing was observed in U251 and SNB-19 cells in response to dual treatment with TMZ or JQ1 combined with a BCL-XL inhibitor, compared to single agent treatment. This was reflected in abundant cleavage/activation of caspase-3 and cleavage of PARP1, markers of apoptosis. U251 and SNB-19 cells were more readily killed by a combination of BH3 mimetics targeting BCL-XL and MCL-1 as opposed to dual treatment with the BCL-2 inhibitor Venetoclax and a BCL-XL inhibitor. The combined loss of BAX and BAK, the essential executioners of intrinsic apoptosis, rendered U251 and SNB-19 cells refractory to any of the drug combinations tested, demonstrating that apoptosis is responsible for their killing. In an orthotopic mouse model of GBM, we demonstrate that the BCL-XL inhibitor A1331852 can penetrate the brain, with A1331852 detected in both tumour and healthy brain regions. We also investigated the impact of combining small molecule inducers of ferroptosis, erastin and RSL3, with BH3 mimetic drugs. We found that a BCL-XL or an MCL-1 inhibitor potently cooperates with inducers of ferroptosis in killing U251 cells. Overall, these findings demonstrate the potential of dual targeting of distinct PCD signalling pathways in GBM and may guide the utility of BCL-XL inhibitors and inducers of ferroptosis with standard of care treatment for improved therapies for GBM.

Erratum: Novel high-affinity EGFRvIII-specific chimeric antigen receptor T cells effectively eliminate human glioblastoma.

[This corrects the article DOI: 10.1002/cti2.1283.].

Novel high-affinity EGFRvIII-specific chimeric antigen receptor T cells effectively eliminate human glioblastoma.

OBJECTIVES: The increasing success of Chimeric Antigen Receptor (CAR) T cell therapy in haematological malignancies is reinvigorating its application in many other cancer types and with renewed focus on its application to solid tumors. We present a novel CAR against glioblastoma, an aggressive, malignant glioma, with a dismal survival rate for which treatment options have remained unchanged for over a decade. METHODS: We use the human Retained Display (ReD) antibody platform (Myrio Therapeutics) to identify a novel single-chain variable fragment (scFv) that recognises epidermal growth factor receptor mutant variant III (EGFRvIII), a common and tumor-specific mutation found in glioblastoma. We use both in vitro functional assays and an in vivo orthotopic xenograft model of glioblastoma to examine the function of our novel CAR, called GCT02, targeted using murine CAR T cells. RESULTS: Our EGFRvIII-specific scFv was found to be of much higher affinity than reported comparators reverse-engineered from monoclonal antibodies. Despite the higher affinity, GCT02 CAR T cells kill equivalently but secrete lower amounts of cytokine. In addition, GCT02-CAR T cells also mediate rapid and complete tumor elimination in vivo. CONCLUSION: We present a novel EGFRvIII-specific CAR, with effective antitumor functions both in in vitro and in a xenograft model of human glioblastoma.