Abstract PS3-12-30: Targeting dendritic cells with CD40 agonist-based triplet immunotherapy induces IL12B-driven tumor control in triple-negative breast cancers

Abstract Background: Although immunotherapies deliver lasting remissions in many solid cancers, triple-negative breast cancers demonstrate limited immunity. One potential reason for this is functional impairment within the antigen-presenting cell (APC) compartment. Cross-presenting conventional dendritic cells (cDC1s) are essential for initiating effective CD8+ T cell responses but are frequently scarce or suppressed in breast tumors. We evaluated a rationally designed triplet regimen combining immunogenic anthracycline chemotherapy, Flt3 ligand to expand dendritic cell populations, and a CD40 agonist to activate them, aiming to reprogram the tumor microenvironment and promote durable anti-tumor T cell responses. Methods: EO771, AT3, or 4T1 tumors were implanted orthotopically in female mice. Once tumors reached ∼50 mm3, mice received pegylated liposomal doxorubicin (Doxil, day 1), recombinant Flt3 ligand (Flt3L, days 1-5), and a CD40 agonist antibody (CD40a, days 11, 14, 17) or monotherapy or doublet combinations. Tumor burden and survival were monitored longitudinally. Tumors and draining lymph nodes were analyzed using flow cytometry, single-cell RNA sequencing (scRNA-seq), and TCR sequencing (TCR-seq). Mechanistic dependencies were assessed through genetic knockout models, antibody-mediated depletion, cytokine or chemokine blockade, and lymphocyte trafficking inhibition. Results: Triplet treatment significantly delayed tumor progression and prolonged survival compared to monotherapy or doublet combinations. To elucidate the underlying immune mechanisms, flow cytometry and scRNA-seq revealed robust activation across all three major APC lineages: dendritic cells (DCs), macrophages, and B cells. Specifically, Flt3L expanded cDC1s, cDC2s, and mature dendritic cells enriched in immunoregulatory features (mregDCs), while CD40a upregulated CD80, CD86, β2-microglobulin, and Il12b. Inflammatory macrophages expressing high levels of Cxcl9 were enriched, and B cells exhibited increased markers of activation and memory differentiation. Consistent with APC activation, triplet therapy increased intra-tumoral CD8+ T cell infiltration, reduced expression of exhaustion markers (LAG-3, TIM-3), and promoted stem-like features. TCR-seq demonstrated clonal expansion of T cells without changes in Shannon diversity, indicating predominant expansion of pre-existing T cell clones. Mechanistic studies demonstrated that cDC1s are essential for therapeutic efficacy, as tumor control was lost in Batf3-/- mice but remained intact following depletion of CSF1R+ macrophages or CD20+ B cells. CD8+ T cell depletion partially reduced efficacy, while co-depletion of CD8+ and CD4+ T cells resulted in complete loss of tumor control, indicating a cooperative role for both subsets. Removal of regulatory T cells further enhanced treatment response, suggesting they act as a barrier to optimal efficacy. Importantly, therapy remained effective despite FTY720-mediated inhibition of lymphocyte trafficking from the tumor draining lymph node, pointing to a role for pre-existing intra-tumoral T cells. Finally, neutralization of IL-12 completely abolished tumor control, whereas CXCL9 or CXCR3 blockade had little impact, underscoring the central role of IL-12 in mediating therapeutic response. Conclusion: This CD40 agonist-based triplet immunotherapy reprograms the tumor microenvironment through coordinated APC activation and IL-12-driven CD4+ and CD8+ T cell responses. cDC1s are essential mediators of therapeutic efficacy, while macrophages and B cells adopt inflammatory and activated states but are dispensable for treatment effect. Tregs constrain treatment potency and are a rational co-target to improve responses in breast cancer. Citation Format: P. L. Mylabathula, R. Ali, A. Alkhani, X. Qi, S. Hebbale, J. Zhu, I. Chan, S. M. Reddy. Targeting dendritic cells with CD40 agonist-based triplet immunotherapy induces IL12B-driven tumor control in triple-negative breast cancers abstract. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr PS3-12-30.