Abstract Outcomes for pediatric patients with diffuse midline glioma (DMG) and relapsed/refractory CNS malignancies remain poor. Loc3CAR is an ongoing, first-in-human, phase I clinical trial (NCT05835687) evaluating intracranial delivery of B7-H3-CAR T cells expressing 41BB ligand (B7-H3-CAR-T) for patients ≤21 years old with i) relapsed/refractory B7-H3+ CNS tumors (Cohort A), or ii) DMG post-radiation (Cohort B). Treatment included 6 intracerebroventricular B7-H3-CAR-T infusions administered over 7 weeks. Primary and secondary outcomes were safety and disease response, respectively. To date, 84% (21/25) of tumors screened were B7-H3+ (median H-score 200; range 0-300). CAR-T products were successfully manufactured for all patients enrolled on the collection/manufacture phase of the trial (n=11). Eight patients (7 cohort A; 1 cohort B) were treated with a total of 42 infusions on dose level 1 (1x107/3x107 CAR+ T cells/dose). Post-infusion, multiple participants exhibited neurologic findings suggestive of on-tumor CAR-T activation, including seizure, dysphasia, and localized pain. One patient, with a large tumor burden and high B7-H3 expression (H-score = 300), experienced a dose limiting toxicity with unresponsiveness and decorticate posturing approximately 16 hours post-infusion #1. Their clinical status rapidly improved following CSF removal and intravenous dexamethasone. Common adverse events attributable to B7-H3-CAR-T included headache (8/8), fever (7/8), and nausea/vomiting (5/8). Two of 7 (29%) evaluable patients achieved clinical benefit with stable disease at week 8. B7-H3-CAR-T and inflammatory cytokines were detected in the CSF from all patients evaluated. Circulating tumor DNA (ctDNA) was measured using a novel DNA methylation-based sequencing pipeline and detected in the CSF from 6/8 patients. For 2 patients, ctDNA abundance increased post-infusion, followed by clearance, suggesting subclinical antitumor activity. Single cell RNAseq of immune cells in the CSF from 1 patient analyzed to date, demonstrated abundant Tregs post-infusion #4, suggesting B7-H3-CAR-T may induce local anti-inflammatory responses. Overall, intracranially administered B7-H3-CAR-T have an acceptable safety profile and warrant continued exploration for pediatric patients with CNS tumors.

Background We are conducting a Phase 1 clinical study to evaluate the safety and efficacy of escalating doses of autologous CD123-CAR T cells for pediatric patients with r/r CD123-positive AML or ALL. We previously reported (Naik et al, Blood 2022, 140 (Supplement 1): 4584-5) that infusion of CD123-CAR T cells after lymphodepleting chemotherapy with fludarabine and cyclophosphamide was well tolerated, with transient fevers representing grade 1 cytokine release syndrome (CRS) and without dose limiting toxicities. We observed anti-AML activity as evidenced by responses in two out of six infused patients albeit with limited CAR T-cell expansion. Mechanistic studies revealed that CD123-CAR T cells were predominantly effector memory and expressed markers associated with T cell exhaustion (TIM3, PD1 or CD39). This phenotype was completely reversed by generating CD123-CAR T cells in the presence of dasatinib (CD123-CAR.dasa T cells), a multikinase inhibitor known to transiently inhibit CAR signaling. Based on these findings, we modified the CD123-CAR T cell production for our protocol and now report on an extended comparison of CD123-CAR and CD123-CAR.dasa T cells and on the clinical course of patients who were infused with CD123-CAR.dasa T cells. Methods and Results Single cell RNA seq analysis revealed that CD123-CAR.dasa T cells had upregulated IFNa signaling pathways in comparison to CD123-CAR T cells. In addition, they were in a less differentiated state as judged by a methylation-based multipotency index, exhibited preferential oxphos metabolism, and had increased apoptosis after initial antigen exposure as evidenced by caspase 3/7 activity. Six patients (5 AML, 1 ALL) were infused with CD123-CAR.dasa T cells on dose level (DL) 3 (3x106/kg) or 4 (1x107/kg). All patients developed high-grade fevers within 3 hours of infusion. All 3 patients on DL3 developed grade 2 CRS that responded to a single dose of tocilizumab. On DL4, all 3 patients presented with grade 4-5 CRS with evidence of immune effector cell-associated hemophagocytic lymphohistiocytosis-like syndrome (IEC-HS). All DL4 patients required multiple doses of immune modulatory agents including tocilizumab, steroids and emapalumab. One patient also received ruxolitinib, anakinra and etanercept without benefit, and died of cardiorespiratory failure. Autopsy results were consistent with CRS and no evidence of on target/off cancer toxicity. Patients post CD123-CAR.dasa T-cell infusion had significantly higher levels of circulating chemokines and cytokines, including IFNg, GMCSF, IL10, IL6 and TNFa, in comparison to patients who had received CD123-CAR T cells. Peak expansion of CD123-CAR.dasa T cells, as judged by qPCR analysis, was higher in comparison to CD123-CAR T cells. However, this did not translate into improved anti-leukemia activity as no patients had a response. Conclusions CD123-CAR T cells generated in the presence of dasatinib induced high grade CRS and/or IEC-HS without improved anti-leukemia activity. Our findings are in contrast with CAR.dasa T-cell products that target other antigens. While additional mechanistic studies are in progress, our results highlight that dasatinib cannot be considered a universal agent to improve the effector function of CAR T cells for hematological malignancies.

TIME heterogeneity after CAR T-cell treatment. A, Experimental scheme. Albino C57BL/6 mice were transplanted with 1 × 105 GL261 cells orthotopically, followed 25 days later by intratumoral injection of 3 × 106 mB7-H3-CAR T-cells (28.mζ, BBL-28.mζ,CD8tmBB.ζ, or Ctrl). Tumors were collected at 4 days after treatment and processed for scRNA-seq. Scheme created with BioRender.com. B, UMAP with major cell subsets in all tumor samples. C, Bar graph showing the percentage of each major cell type per treatment group. D, UMAP dimensionality reduction of single-cell data from all tumors clustered into 21 Seurat clusters annotated by number. E, UMAP visualization of the 21 Seurat clusters by treatment group from the best to the worst functioning CARs. Mac, macrophages; Mono, monocytes; MG, microglia; DC, dendritic cells.

Successful responses with 28.mζ CAR T-cells are associated with balanced proinflammatory and anti-inflammatory myeloid cell responses. Seurat clusters 0, 7, 10, 14 were reclustered into 10 Mac/MG subclusters to further define the diversity of myeloid responses after CAR T-cell treatment. A, UMAP plots of the Mac/MG subclusters visualized by treatment group. B, Summary plot of Mac/MG subcluster 2 and 7 frequencies per treatment. Volcano plots showing differentially upregulated and downregulated genes in macrophage subclusters C2 in C, C7 in D as compared with all other myeloid subclusters. E, Dot plot depicting expression of myeloid lineage markers, genes associated with protumorigenic responses, antitumorigenic, and differentiation genes. Dot size represents the percentage of cells expressing each gene and dot color represents mean expression level with a gradient of lowest expression in blue to highest expression in red. F, Dot plot depicting differentially expressed genes associated with immunosuppression, invasion, recruitment, M1-like and M2-like macrophage responses per treatment group.

Supplementary Figure S9 shows cluster frequency per treatment group for each cluster.

Successful responses with 28.mζ CAR T-cells are associated with balanced proinflammatory and anti-inflammatory myeloid cell responses. Seurat clusters 0, 7, 10, 14 were reclustered into 10 Mac/MG subclusters to further define the diversity of myeloid responses after CAR T-cell treatment. A, UMAP plots of the Mac/MG subclusters visualized by treatment group. B, Summary plot of Mac/MG subcluster 2 and 7 frequencies per treatment. Volcano plots showing differentially upregulated and downregulated genes in macrophage subclusters C2 in C, C7 in D as compared with all other myeloid subclusters. E, Dot plot depicting expression of myeloid lineage markers, genes associated with protumorigenic responses, antitumorigenic, and differentiation genes. Dot size represents the percentage of cells expressing each gene and dot color represents mean expression level with a gradient of lowest expression in blue to highest expression in red. F, Dot plot depicting differentially expressed genes associated with immunosuppression, invasion, recruitment, M1-like and M2-like macrophage responses per treatment group.

Supplementary Figure S7 shows initial analysis of scRNAseq data.

Global Mac/MG depletion abrogates effective CAR T-cell responses. GL261 glioma-bearing mice were treated with BLZ945 at 200 mg/kg starting 5 days after tumor implantation. A, Experimental scheme of BLZ945 macrophage depletion kinetics experiment. Daily drug dosing via oral gavage was for 2 weeks and tumors were harvested for FACS analysis at days 9, 16, and 20 after tumor implantation. B, Summary plot showing frequency of TAMs infiltrating tumors as percentage of live CD45+ immune cells. C, Experimental scheme for combination study. Glioma-bearing mice were treated with BLZ945 at 200 mg/kg starting 5 days after tumor implantation and continued daily for 3 weeks. B7-H3 CAR T-cells with Ctrl or 28.mζ constructs were then injected intratumorally at day 16. D, Kaplan–Meier survival curve (n = 11, log-rank Mantel–Cox test with Bonferroni correction for multiple comparisons, ***, P < 0.001). E, Representative images from immunostaining for T-cell and macrophage markers from tumors at endpoint showing CD3 and Iba1 staining in brain samples from each treatment group at 40x magnification (scale bar = 100 µm). F, H-scores depicting quantitative analysis of CD3 and Iba1 staining in brain tumor samples at endpoint from E as evaluated by blinded pathologist.

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].

Supplementary Figure S4 shows phenotype and cytotoxicity of murine B7-H3 CAR T cells.

Supplementary Figure S8 provides cluster identities.

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. Significance: 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.

Surface expression of 41BBL on CD28-based mB7-H3-CAR T-cells enhances effector cytokines release in repeat stimulation assay. Culture supernatants were collected at 24 hours after repeated stimulation with GL261 tumor cells at 2:1 ratio and analyzed using Multiplex assay. Summary plots of cytokines and chemokines produced by CAR T-cells after first stimulation (A) and fourth stimulation (B) against GL261 tumor cells (n = 4, mean ± SEM, two-way ANOVA with Tukey test for multiple comparisons). C–E, CAR T-cell production of IFNγ, IL2, and GMCSF at 24 hours’ after stimulations one and four (n = 4, mean ± SEM, two-way ANOVA with Tukey test for multiple comparisons).

Supplementary Figure S6 shows experimental pipeline for single cell RNAseq experiment.

Supplementary Figure S3 shows expression of murine B7-H3 CARs in producer cells and T cells.

Supplementary Figure S5 shows murine CAR T cell expansion in media and after exposure to B7-H3-negative tumor cells.

Supplementary Figure S1 shows murine CAR T cell efficacy in vivo and Cd276 IHC post-treatment.