675 Radiopharmaceutical therapy enhances CAR T-cell activity against neuroblastoma by tumor sensitization and tumor microenvironment remodeling

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Background Chimeric antigen receptor (CAR) T-cell therapy remains less effective against solid tumors such as neuroblastoma (NB) than hematological malignancies. Key obstacles for CAR T cell efficacy include extensive tumor burden and the presence of an immunosuppressive tumor microenvironment (TME). 1 Therefore, we propose a combination approach with [67Cu]Cu-LLP2A, a radiopharmaceutical therapy (RPT) targeting the very late antigen-4 (VLA-4, α4β1 integrin). VLA-4 is broadly expressed in tumors, including NB, and lymphoid tissue (figure 1).2 The β-emitter radionuclide 67Cu (half-life=60h) enables effective radiation delivery and sufficient decay before CAR infusion.2–3 We hypothesize that RPT can enhance CAR engraftment and efficacy by reducing tumor burden and remodeling the TME before T-cell infusion.Methods NSG mice were engrafted with NB cell lines with different radio-sensitivities: SK-N-AS ( TP53 mutant radio-resistant) or IMR-5 (TP53 wildtype radio-sensitive). Tumor-bearing mice were treated with non-radioactive cold LLP2A, 0.5 or 1 mCi [67Cu]Cu-LLP2A. After 4 days, either untransduced (mock) or two clinically relevant CAR-T cells (anti-GD2 or anti-B7-H3) were injected intravenously. Tumor burden was evaluated weekly, and tumor weights were assessed at the endpoint for both models. Single-cell RNA sequencing (scRNA-seq) and multiplex imaging were performed on tumor samples at day 7 post-CAR T-cells infusion to assess TME modulation. For further mechanistic studies, cell viability and bulk RNA-seq analyses were performed in 67Cu-treated NB cells in vitro. Results Mice treated with 0.5 mCi [ 67Cu]Cu-LLP2A+CAR T-cells showed an increased number of complete remissions (CR) compared to CAR T-cells or RPT alone in both NB models. Increased RPT dose did not improve efficacy (figure 2A-B). In IMR-5 but not SK-N-AS, RPT reduced cell viability. Transcriptomic analysis showed induction of pro-inflammatory gene pathways (TNFα) after 72h only for IMR-5, related to T-cell activation in a paracrine manner. In contrast, in SK-N-AS, scRNA-seq RPT+CAR group showed that pre-treatment with [67Cu]Cu-LLP2A increased CAR T-cell infiltration into the tumor and reduced M2-like myeloid cell proportions in the TME. Multiplex imaging of tumors resected at that time confirmed increased CD8+ GZMB+ T-cells in the TME with the combination therapy. Neighborhood analysis showed increased T cell aggregates within the tumors in both SK-N-AS and IMR-5.Conclusions [ 67Cu]Cu-LLP2A RPT improves CAR T-cell efficacy by direct tumoricidal effects and T-cell activation via TNFα in radio-sensitive IMR-5 model. In contrast, in the radio-resistant SK-N-AS model, the synergistic effects of RPT+CAR is due to the remodeling of the TME, thereby enhancing T-cell trafficking to the tumor and reducing immunosuppressive tumor-associated myeloid cells.References Trautmann T, Yakobian N, Nguyen R. CAR T-cells for pediatric solid tumors: where to go from here? Cancer Metastasis Rev. 2024;43:1445-1461.Reyes-Gonzalez JM, et al. Evaluation of VLA-4 (integrin alpha4beta1) as a shared target for radiopharmaceutical therapy across solid tumors. Mol Cancer Ther. (2025).Salerno KE, et al. A primer on radiopharmaceutical therapy. Int J Radiat Oncol Biol Phys 2023;115;48–59.Ethics Approval This study was approved by the NIH institutional IACUC; study number protocol POB-003-2-B.Abstract 675 Figure 1[67Cu]Cu-LLP2A radiopharmaceutical therapyAbstract 675 Figure 2Low dose RPT synergizes with CAR T-cells in preclinical models of NB. Treatment schema and tumor growth assessment for IMR-5 (A) and SK-N-AS (B) injected mice under RPT and CAR therapy, measured by caliper and IVIS, respectively. The number of mice with complete remission at endpoint (CR) is shown in each plot

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