Abstract 4197: Treatment with chemotherapy, CD40 agonist, and Flt3 ligand triplet combination enhances antigen presentation and leads to cures in triple negative breast cancer
Authors: Ramani Vijay , Nooka Shruthi , Zhang Yu-An , Gibbs Serena , Huang Hai-Cheng , Hullings Melanie , Conzen Suzanne , Arteaga Carlos L. , Chan Isaac , Reddy Sangeetha M. 
Published: June 2022

Introduction: Triple negative breast cancers (TNBC) have shown limited responses to immune checkpoint blockade (ICB). Breast cancer is associated with defects in antigen presentation which may contribute to resistance to ICB. Flt3 ligand (Flt3L) is a growth factor that increases differentiation of DC1 dendritic cells, critical mediators of antigen presentation. CD40 agonist activates all 3 classes of antigen presenting cells - dendritic cells, B cells, and macrophages. Synergy has been demonstrated between Flt3L and CD40 agonist as well as between CD40 agonist and chemotherapy in other cancers, however the combination of all three has not been studied in breast cancer.

Methods: 6-8 week old Balb/c mice were injected with 4T1 triple negative breast cancer cells. When tumors were 50 mm3, mice were first treated with intraperitoneal pegylated liposomal doxorubicin (PLD) once and subcutaneous Flt3L daily for 5 days in different sequencing schedules (PLD first followed by Flt3L immediately or 4 days later or Flt3L first followed by PLD 7 days or 10 days later). Based on optimal scheduling of PLD and Flt3L, mice were treated with PLD alone, Flt3L alone, CD40 agonist alone, PLD + Flt3L, PLD + CD40 agonist, PLD + PD-1 blockade, or PLD + CD40 agonist + Flt3L. In addition to serial tumor measurements, mice were sacrificed and tumors and lymphoid organs harvested for flow cytometry analyses.

Results: Treatment with PLD at least 4 days before Flt3L led to an optimal increase in intra-tumoral DC1 cells (p=0.0002), increase in polyfunctional CD8 T cell response (p&lt;0.0001), reduced PD-L1 expression on DC1 cells (p&lt;0.0001), and reduced PD-1 expression on CD8 T cells (p&lt;0.0001) compared to other sequencing schedules. Therefore, this regimen was advanced for further study. Treatment with novel triplet combination of PLD, Flt3L, and CD40 agonist resulted in improved tumor control and survival compared to PLD alone (p&lt;0.0001 and p&lt;0.0001, respectively), Flt3L alone (p&lt;0.0001 and p&lt;0.0001), CD40 agonist alone (p &lt;0.0001 and p&lt;0.0001), PLD + Flt3L (p&lt;0.0001 and p&lt;0.0001), PLD + CD40 agonist (p&lt;0.0001 and p&lt;0.0001), and PLD + PD-1 blockade (p&lt;0.0001 and p&lt;0.0001). 22% of mice were tumor-free in the triplet therapy group only and were resistant to tumor re-challenge. Treatment with triplet therapy was associated with an increase in antigen specific CD8 T cells. Additional mechanistic studies will also be presented.

Conclusions: Novel triplet combination with PLD, CD40 agonist, and Flt3L leads to enhanced tumor control in the 4T1 TNBC mouse model. A clinical trial with this combination in metastatic TNBC patients is expected to begin recruitment soon (NCT05029999).

Citation Format: Vijay Ramani, Shruthi Nooka, Yu-An Zhang, Serena Gibbs, Hai-Cheng Huang, Melanie Hullings, Suzanne Conzen, Carlos L. Arteaga, Isaac Chan, Sangeetha M. Reddy. Treatment with chemotherapy, CD40 agonist, and Flt3 ligand triplet combination enhances antigen presentation and leads to cures in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4197.