Abstract LB-B008: cDC expansion and CD40 activation after immunogenic chemotherapy shift the breast tumor microenvironment toward functional T cell immunity and IL-12-dependent tumor control

Abstract Background: Metastatic breast cancers often derive limited benefit from immunotherapy, potentially due to impaired antigen presentation and limited cross-presenting cDC1 activity. We tested a rational triplet designed to induce immunogenic antigen release (pegylated liposomal doxorubicin; Doxil), expand dendritic cell populations including cDC1s (Flt3 ligand; Flt3L), and license antigen-presenting cells (APC) by CD40 agonism (CD40a) to increase co-stimulation T cell immunity. Methods: Female mice bearing orthotopic EO771, AT3, or 4T1 tumors (∼50 mm3) received Doxil D1, Flt3L D4-8, and an agonistic CD40 antibody D11, 14, 17, with monotherapy and doublet controls. Tumors and draining lymph nodes (TDLN) were analyzed by immunohistochemistry, flow cytometry, scRNA-seq, and TCR-seq. Mechanistic requirements were assessed using Batf3-/- mice, immune cell depletion, IL-12 neutralization, and inhibition of lymphocyte egress (FTY720). Results: Triplet therapy improved tumor control and survival across models. Flt3L expanded cDC1, cDC2, and mature DC states, while CD40a increased co-stimulation and antigen-presentation programs (Cd86/B2m) and induced Il12b. APC remodeling was associated with increased intratumoral CD8+ T cell infiltration, reduced LAG-3/TIM-3 exhaustion markers, and enrichment of stem-like features. TCR-seq demonstrated clonal expansion with addition of Flt3L and CD40a to Doxil. Tumor control required cDC1s as efficacy was lost in Batf3-/- mice, while remaining intact after depletion of the other 2 primary antigen presenting cells (macrophages and B cells). CD8 depletion partially reduced efficacy, whereas combined CD4/CD8 depletion eliminated tumor control. Benefit persisted with FTY720, supporting reinvigoration of pre-existing intratumoral T cells. We found efficacy was dependent on IL-12, as neutralization abolished tumor control. Conclusion: Anthracycline chemotherapy plus Flt3L-driven DC expansion and CD40 licensing remodels murine breast tumors toward cDC1 and IL-12 dependent T cell control. This framework is being evaluated in a phase I pilot combining Doxil with CDX-301 (recombinant Flt3L) and CDX-1140 (CD40 agonist) in metastatic or unresectable locally advanced HER2-negative breast cancer (NCT05029999). Two lead-in cohorts randomize participants 2:1 to one cycle of triplet therapy vs Doxil alone with paired biopsies at baseline and post-cycle 1 lead-in; all patients then transition to triplet therapy. The primary endpoint is recommended phase II dose for CDX-1140 based on DLTs; secondary endpoints include CD8+ T cell infiltration (triplet vs Doxil alone) and clinical activity. Citation Format: Preteesh L. Mylabathula, Riyasat Ali, Skanda Hebbale, Xinyi Qi, Shruthi Nooka, Sangeetha M. Reddy. cDC expansion and CD40 activation after immunogenic chemotherapy shift the breast tumor microenvironment toward functional T cell immunity and IL-12-dependent tumor control abstract. In: Proceedings of the AACR Immuno-Oncology Conference (AACR IO): Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2026 Feb 18-21; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2026;14(2 Suppl):Abstract nr LB-B008.