Abstract 5314: Improved breast cancer tumor control with addition of CD40 agonist and Flt3 ligand to pegylated liposomal doxorubicin is only partially mediated by CD8 T cells
Authors: Mylabathula Preteesh L. , Nooka Shruthi , Alkhani Anas , Zhu James , Vu Ashley , Ali Riyasat , Balasubramanian Varshini Ramasamy , Chan Isaac , Reddy Sangeetha M. 
Published: March 2024

Abstract
Background: There is a need for improved immunotherapy strategies beyond current FDA approved treatments for triple negative breast cancer. Defective antigen presentation has been shown to play a contributory role in deficient anti-tumor immunity in breast cancers. Flt3 ligand (Flt3L) is a growth factor that increases differentiation into DC1 dendritic cells. CD40 agonist (CD40a) activates all 3 classes of antigen presenting cells - DCs, B cells, and macrophages. Synergy with chemotherapy and CD40a has been seen in other cancers but not explored in breast cancers.
Methods: C57BL/6 mice (aged 6-8 weeks) were injected with 150,000 E0771 breast cancer cells within the mammary fat pad. Tumors were allowed to grow to a volume of ~50 mm3 before randomization into treatment groups. Mice were treated with triplet therapy of pegylated liposomal doxorubicin (PLD) via tail vein followed by Flt3L intraperitoneal (IP) daily for 5 days and CD40a IP 1 week later as well as monotherapy and doublet therapy controls. Tumor growth and survival were monitored throughout the experiment. Tumors were harvested for single cell RNA sequencing (scRNAseq) and immunohistochemistry. To further study subset specific effects on efficacy of the immunotherapy, CD8 and CD4 T-cells were systemically depleted with anti-CD8 and anti-CD4 antibodies administered IP every 4 days starting 2 days before PLD treatment.
Results: Triplet therapy led to improved tumor control compared to no treatment, monotherapy, and doublet controls (p&lt;0.0001). Increased tumor infiltration with CD8 T cells was observed with triplet therapy via immunohistochemistry. scRNAseq showed increased cDC1 subset with Flt3L treatment. Contrastingly, CD40a decreased DC subsets likely due to activation and emigration out of the tumor, remodeled macrophage populations, and decreased T regulatory cells. CD8 depletion led to faster tumor growth whereas CD4 depletion reduced tumor growth. Triplet therapy maintained efficacy over PLD with both CD8 and CD4 depletion but showed reduced magnitude of benefit with CD8 T cell depletion suggesting a partial CD8 mediated process. CD4 T cell depletion did not hinder triplet therapy efficacy compared to PLD but depletion further enhanced tumor control in combination with triplet therapy, with 20% (2/10) of animals achieving complete regression.
Conclusion: Triplet therapy with PLD, Flt3L, and CD40a improved tumor control and survival in syngeneic E0771 breast cancer mouse model. Added benefit of immunotherapy to PLD is partially CD8 T cell mediated. CD4 depletion further enhances tumor control in combination with triplet therapy. Experiments to explore the interactive effect of triplet therapy with other pertinent immune subsets are ongoing. A clinical trial with this combination in metastatic triple negative breast cancer patients is open and recruiting (NCT05029999).
Citation Format: Preteesh L. Mylabathula, Shruthi Nooka, Anas Alkhani, James Zhu, Ashley Vu, Riyasat Ali, Varshini Ramasamy Balasubramanian, Isaac Chan, Sangeetha M. Reddy. Improved breast cancer tumor control with addition of CD40 agonist and Flt3 ligand to pegylated liposomal doxorubicin is only partially mediated by CD8 T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5314.