Abstract 2267: MEDI5083, a novel CD40L-Fc fusion protein, activates the CD40 pathway on antigen-presenting cells and promotes robust anti-tumor immune responses in both hot and cold murine tumor models
Authors: Malhotra Deepali , Turman Sean , McGlinchey Kelly , Wang Yaya , Wang Yue 
Published: September 2020

Abstract
Background: While diverse strategies may boost anti-tumor immunity, of significant interest are therapeutic approaches that are efficacious in tumors with high and low immune cell infiltration. Targeting CD40 with its natural ligand (CD40L) augments the immune-stimulatory function of antigen-presenting cells and enhances T cell priming, making this an attractive means of promoting anti-tumor responses. MEDI5083 is a novel fusion protein containing a hexameric recombinant human CD40L structure covalently linked to human IgG4p Fc. Here, we present preclinical functional characterization of MEDI5083. Methods MEDI5083 functionality was first established using several in vitro approaches with human cell lines and primary human cells. To examine anti-tumor activity in vivo, we constructed a murine surrogate (mCD40L-Fc) with similar physical and target-binding properties to those of MEDI5083. The effects of mCD40L-Fc on immune cell activation and tumor growth were determined in the immunologically cold B16F10 model of melanoma and the immunologically hot CT26 model of colon carcinoma.
Results: MEDI5083 specifically bound to human CD40, triggered its internalization, and robustly stimulated CD40 signaling as measured by NF-κB activation in Ramos and THP-1 cells. MEDI5083 potently upregulated expression of MHCII and the co-stimulatory molecules CD80 and CD86 on primary human B cells and myeloid cells, while inducing secretion of pro-inflammatory cytokines. MEDI5083 also enhanced secretion of IFN-γ by memory CD8+ T cells in an antigen-recall assay.
In the B16F10 model, repeated mCD40L-Fc dosing significantly decreased tumor growth. Consistent with enhancing immune cell activation and T cell priming, mCD40L-Fc treatment elevated serum levels of IL-12 and IFN-γ, and increased numbers of tumor-infiltrating CD8+ T cells. While this model responds poorly to checkpoint blockade, co-administration of mCD40L-Fc with anti-PD-L1 antibody demonstrated additivity, consistent with PD-1/PD-L1-mediated regulation of mCD40L-Fc-driven adaptive immunity.
Repeated dosing of mCD40L-Fc in the CT26 model inhibited tumor growth. Treatment was associated with diminished tumor-infiltrating Treg numbers, increased CD8+ T cell:Treg ratios, and phenotypic changes in tumor-infiltrating immune cells. Tumor supernatants exhibited increased IFN-γ, consistent with enhanced anti-tumor immunity. Rechallenge of mice with complete tumor regression confirmed generation of durable memory.
Conclusions: We have demonstrated that stimulation of CD40 signaling with MEDI5083 or mCD40L-Fc leads to robust activation of immune cells and enhanced adaptive immune responses. In vivo, this translates to enhanced control of both poorly- and well-infiltrated syngeneic tumors and generation of durable memory in mice with complete tumor regression.
Citation Format: Deepali Malhotra, Sean Turman, Kelly McGlinchey, Yaya Wang, Yue Wang. MEDI5083, a novel CD40L-Fc fusion protein, activates the CD40 pathway on antigen-presenting cells and promotes robust anti-tumor immune responses in both hot and cold murine tumor models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2267.