Kollis, P. M. | Search bioRxiv

bioRxiv2025

A Characterization of the Immune Cells in Immunocompetent and Immunodeficient Mice with Orthotopic Brain Tumors.

BackgroundGlioblastoma is characterized by poor survival with few treatment advances for over 20 years. Active research is being conducted in immunotherapies; however, this may be hampered by the lack of detailed characterization of the immune compartments of orthotopic murine brain tumor models. MethodsWe used cell lines derived from glioblastoma patients and murine glioma cell lines to produce intracranial xenograft and syngraft brain tumors in NSG or C57BL/6 mice, respectively. High-parameter flow cytometry and tissue immunofluorescence staining were used to characterize the immune cell compartment in each model. We investigated brain, spleen, lymph node, and bone marrow for the frequencies of different immune cells. Further, we investigated the effect of sub-lethal whole-body irradiation, commonly used for lymphodepletion, on immune cells in immunocompetent mice. ResultsIn our study of non-tumor-bearing mice, we observed dramatic differences between NSG and C57BL/6 mice, not only of T and B cells but also other immune cells, including decreased monocytes, dendritic cells, and eosinophils. In immunocompetent brain tumor-bearing mice, we observed significant differences in the numbers of immune cells compared to non-tumour-bearing controls, primarily in the brain; however, significant differences were also in the spleen, lymph node and bone marrow, particularly in the subsets of monocytes, T cells and dendritic cells. Sub-lethal irradiation caused lasting changes in immune composition. ConclusionsOur quantitative characterization of different immune compartments in orthotopic murine models of glioblastoma may allow for a better-informed selection of tumor models that are appropriate for the pre-clinical investigation of immunotherapies for glioblastoma.

cancer biology↗

bioRxiv2022

GD2-targeting CAR-T cells enhanced by transgenic IL-15 expression are an effective and clinically feasible therapy for glioblastoma

BackgroundAggressive primary brain tumors such as glioblastoma are uniquely challenging to treat. The intracranial location poses barriers to therapy, and the potential for severe toxicity. Effective treatments for primary brain tumors are limited, and 5-year survival rates remain poor. Immune checkpoint inhibitor therapy has transformed treatment of some other cancers but has yet to significantly benefit patients with glioblastoma. Early phase trials of CAR-T cell therapy have demonstrated that this approach is safe and feasible, but with limited evidence of its effectiveness. The choices of appropriate target antigens for CAR-T cell therapy also remain limited. MethodsWe profiled an extensive biobank of patients biopsy tissues and patient-derived early passage glioma neural stem cell lines for GD2 expression using immunomicroscopy and flow cytometry. We then employed an approved clinical manufacturing process to make CAR-T cells from peripheral blood of glioblastoma and diffuse midline glioma patients and characterized their phenotype and function in vitro. Finally, we tested intravenously administered CAR-T cells in an aggressive intracranial xenograft model of glioblastoma and used multicolor flow cytometry, multicolor whole-tissue immunofluorescence and next-generation RNA sequencing to uncover markers associated with effective tumor control. ResultsHere we show that the tumor-associated antigen GD2 is highly and consistently expressed in primary glioblastoma tissue removed at surgery. Moreover, despite glioblastoma patients having perturbations in their immune system, highly functional GD2-specific CAR-T cells can be produced from their peripheral T cells using an approved clinical manufacturing process. Finally, after intravenous administration, GD2-CAR-T cells effectively infiltrated the brain and controlled tumor growth in an aggressive orthotopic xenograft model of glioblastoma. Tumor control was further improved using CAR-T cells manufactured with a clinical retroviral vector encoding an IL-15 transgene alongside the GD2-specific CAR. These CAR-T cells achieved a striking 50% complete response rate by bioluminescence imaging in established intracranial tumors. Markers associated with tumor control included those related to T-cell homing, infiltration, and cytotoxicity. ConclusionsTargeting GD2 using a clinically deployed CAR-T therapy has a sound scientific and clinical rationale as a treatment for glioblastoma and other aggressive primary brain tumors. What is already known on this topicGD2 is a tumor antigen of significant interest for targeting immunotherapy. A single preclinical study has shown the effectiveness of GD2-CAR-T cell therapy in an orthotopic xenograft model of diffuse midline glioma. Similarly, there is one previous preclinical study of GD2-CAR-T therapy in a orthotopic glioblastoma xenograft model but tumor control was achieved only following intracranial injection of CAR-T cells. Given that GD2-CAR-T therapy is already being evaluated clinically for other tumor indications, it is important to establish whether there is an acceptable rationale for its use in brain tumors. What this study addsThis is the first description of a GD2-targeted CAR-T cell therapy that shows antitumor effectiveness in a preclinical model of human glioblastoma following intravenous administration. It is also the first study to investigate the potential effects that the immune profile of glioblastoma patients may have on the feasibility of CAR-T cell manufacturing. How this study might affect research, practice, or policyThe results of this study have led to the initiation of an Australian phase 1 clinical trial program aiming to test GD2-specific CAR-T cells for the treatment of childhood and adult primary brain tumors. The study provides valuable insights into the microenvironmental factors that influence the effectiveness of CAR-T cell therapy for this type of tumor, paving the way for further optimization of CAR-T cell technology for treatment of aggressive primary brain tumors such as glioblastoma.

immunology↗