Investigating α- and β-emitting radionuclides targeting myeloid cells in a preclinical glioblastoma model.

Introduction: Myeloid cells are the key mediators of immunosuppression and treatment resistance in primary brain tumors including Glioblastoma (GBM). We previously published that targeting myeloid cells with β emitting radionuclide ¹⁷⁷Lu radiolabeled anti-CD11b antibody moderately reduces tumor- infiltrating myeloid cells and sensitizes preclinical GBM to check-point immunotherapy. We have attempted to improve myeloid cells targeting with an α-emitting radionuclide ²²⁵Ac-radiolabeled CD11b antibody. Herein we establish the relative impact of α versus β radionuclide therapy targeting myeloid cells using anti-CD11b antibody.

Methods: An anti-CD11b antibody (αCD11b) was modified for radiolabeling with Actinium-225. Short-term survival studies(120-day post treatment) with and without immune-checkpoint immunotherapy(ICI) were performed at an optimized eMTA dose in orthotropic syngeneic glioma mice models 10 day post tumor inoculation for[²²⁵Ac]Ac-αCD11b (0.5kBq/100μg protein dose). Single cell RNA sequencing studies (scRNAseq) were performed in a separate cohort to assess the Tumor Associated Myeloid cell (TAMC) phenotypes, T-cell clonality, DNA damage/repair in Tumor Microenvironment as a result of [²²⁵Ac]Ac-αCD11b at 0.5kBq/100μg (α-therapy) and [¹⁷⁷Lu]Lu-αCD11b at 7.4MBq/100μg (β-therapy) with respect to untreated controls. scRNAseq studies were performed on day13 post treatment resected tumor tissues using 10X Genomics platform. Data analysis, gene expression matrices, integration of data, quality control, demultiplexing was performed using CellRanger, Seurat, and V(D)J loupe browser. A combined UMAP projection to define general cell types was created and changes in cell frequency between groups were quantified.

Results: Anti-CD11b antibody was radiolabeled with Ac-225 with >95% radiolabeling yields. Therapeutic efficacy of [²²⁵Ac]Ac-αCD11b in combination with ICIs (anti-PD1/anti-CTLA4) demonstrated the most promising results with a median survival of 82 days, and 5/10 mice surviving to the endpoint of 120 days post treatment. The [²²⁵Ac]Ac-αCD11b alone had 1/10 mice survive to 120 days with a median survival of 14-days. The vehicle control had a median survival of 13-days and ICI alone had a median survival of 20-days. Single cells from post treatment dissociated brain tumor were sequenced and analyzed. [¹⁷⁷Lu]Lu-αCD11b therapy demonstrated a more robust recruitment of immune cells into the tumor as compared to [²²⁵Ac]Ac-αCD11b. We identified and annotated 7 clusters from tumor infiltrating myeloid cells which showed varying degrees of change between the treatments [Figure1A]. The T-cell frequency in the control group was 4.7%, [²²⁵Ac]Ac-αCD11b group was 8.0%, and the [¹⁷⁷Lu]Lu- αCD11b was 11.9%. While the [²²⁵Ac]Ac-αCD11b showed significant expansion of few T-cell clonotypes, the [¹⁷⁷Lu]Lu-αCD11b group showed expansion across a range of T-cell clonotypes[Figure1B].

Conclusions: The [²²⁵Ac]Ac-αCD11b, demonstrated promising results in combination with ICIs with a dramatic improvement in overall survival in a GBM mouse model. Our data indicates preclinical efficacy of [²²⁵Ac]Ac-αCD11b in reducing tumor-infiltrating myeloid cells and sensitizing gliomas to immunotherapy. Additionally, we demonstrate that α- and β-emitting radionuclides conjugated to anti-CD11b each have a unique impact on anti-tumor immunity.

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