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FIGURE 3 from CAR T-cell Design-dependent Remodeling of the Brain Tumor Immune Microenvironment Modulates Tumor-associated Macrophages and Anti-glioma Activity

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posted on 2023-12-01, 02:20 authored by Dalia Haydar, Jorge Ibañez-Vega, Jeremy Chase Crawford, Ching-Heng Chou, Clifford S. Guy, Michaela Meehl, Zhongzhen Yi, Scott Perry, Jonathan Laxton, Trevor Cunningham, Deanna Langfitt, Peter Vogel, Christopher DeRenzo, Stephen Gottschalk, Martine F. Roussel, Paul G. Thomas, Giedre Krenciute

CAR structural design significantly impacts anti-glioma efficacy of mB7-H3 CAR T-cells in the GL261 immunocompetent model. Albino C57BL/6 mice were transplanted with 1 × 105 GL261 cells orthotopically, followed 7 days later by intratumoral injection of 3 × 106 mB7-H3-CAR T-cells transduced with different constructs and adjusted to 40% CAR expression. A, Axial brain MRI images from 3 representative mice per treatment group at days 16 and 29 after tumor implantation. B, Summary plots showing percentage of survival and deceased mice within each treatment group at days 16, 29, and 45 after tumor implantation. C, Kaplan–Meier survival curve (n = 11, log-rank Mantel–Cox test with Bonferroni correction for multiple comparisons, *, P < 0.05; ***, P < 0.001). Experiments were repeated twice with CAR T-cells generated from 2 different T-cell donors. D, Summary table for performance of different mB7-H3 CAR designs from in vitro and in vivo data [(−) means no response, increasing number of (+) signs mean better response].

Funding

HHS | NIH | National Institute of Neurological Disorders and Stroke (NINDS)

HHS | NIH | National Cancer Institute (NCI)

the Katzen Foundation

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ARTICLE ABSTRACT

Understanding the intricate dynamics between adoptively transferred immune cells and the brain tumor immune microenvironment (TIME) is crucial for the development of effective T cell–based immunotherapies. In this study, we investigated the influence of the TIME and chimeric antigen receptor (CAR) design on the anti-glioma activity of B7-H3–specific CAR T-cells. Using an immunocompetent glioma model, we evaluated a panel of seven fully murine B7-H3 CARs with variations in transmembrane, costimulatory, and activation domains. We then investigated changes in the TIME following CAR T-cell therapy using high-dimensional flow cytometry and single-cell RNA sequencing. Our results show that five out of six B7-H3 CARs with single costimulatory domains demonstrated robust functionality in vitro. However, these CARs had significantly varied levels of antitumor activity in vivo. To enhance therapeutic effectiveness and persistence, we incorporated 41BB and CD28 costimulation through transgenic expression of 41BBL on CD28-based CAR T-cells. This CAR design was associated with significantly improved anti-glioma efficacy in vitro but did not result in similar improvements in vivo. Analysis of the TIME revealed that CAR T-cell therapy influenced the composition of the TIME, with the recruitment and activation of distinct macrophage and endogenous T-cell subsets crucial for successful antitumor responses. Indeed, complete brain macrophage depletion using a CSF1R inhibitor abrogated CAR T-cell antitumor activity. In sum, our study highlights the critical role of CAR design and its modulation of the TIME in mediating the efficacy of adoptive immunotherapy for high-grade glioma. CAR T-cell immunotherapies hold great potential for treating brain cancers; however, they are hindered by a challenging immune environment that dampens their effectiveness. In this study, we show that the CAR design influences the makeup of the immune environment in brain tumors, underscoring the need to target specific immune components to improve CAR T-cell performance, and highlighting the significance of using models with functional immune systems to optimize this therapy.

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