Abstract 5178: MEDI7526: a novel bispecific antibody that activates the CD40 pathway and down-regulates cell surface PD-L1 expression
Authors: Wang Yaya , Turman Sean , Malhotra Deepali , Gilbreth Ryan , Wang Yue 
Published: September 2020

Abstract
CD40 stimulation on antigen-presenting cells (APCs) promotes activation of CD8(+) cytotoxic T cells and several CD40 agonistic antibodies have shown promising anti-tumor activities in initial clinical trials. However, agonistic CD40 stimulation also leads to PD-L1 upregulation on APCs, thus creating a negative feedback loop to dampen the anti-tumor immune response. We hypothesized that the combination of PD-L1 antagonism with CD40 agonists would achieve stronger antitumor responses than with anti-CD40 alone. Here we report a novel bispecific antibody MEDI7526, which is composed of an anti-PD-L1 antibody, two trimer forms of CD40 ligand (CD40L) fusion protein (FP) and a human IgG4p Fc. MEDI7526 has been designed to stimulate the CD40 pathway and suppress PD-L1 function to improve T cell activation and potentially overcome the limitations associated with conventional CD40 agonists. The binding characteristics and functional activity of MEDI7526 were analyzed in vitro using flow cytometry and cell-based reporter assays. Internalization of MEDI7526, CD40, and PD-L1 were examined by flow cytometry and immunofluorescence microscopy. Effects of MEDI7526 on cytokine production from human PBMC were analyzed with MSD multiplex assay. Anti-tumor efficacy was determined by a murine surrogate version of MEDI7526 in a B16F10 syngeneic model.
We confirmed that MEDI7526 can bind CD40 and PD-L1 simultaneously. MEDI7526 induced robust activation of CD40 signaling and triggered fast internalization of CD40. Interestingly, MEDI7526 also triggered rapid and robust down-regulation of PD-L1 on the cell surface of multiple types of cells that express CD40, including THP1, MDA-MB-231 and human monocytes, whereas PD-L1 expression was not affected by the treatment of either anti-PD-L1 antibody or CD40L FP. Prolonged treatment of MEDI7526 on MDA-MB-231 cells induced sustained down-regulation of PD-L1 for up to 4 days. When THP1 cells were transiently treated with MEDI7526, cell surface CD40 returned after 24 hours but levels of PD-L1 on the cell membrane remained low, indicating re-expression of cell surface CD40 and PD-L1 following distinct pathways. We demonstrated that internalized PD-L1 undergoes lysosome-dependent, proteasome-independent degradation, resulting in sustained down-regulation of PD-L1 from cell surface. Moreover, MEDI7526 induced robust IFNγ production in antigen recall assays, and the murine surrogate of MEDI7526 effectively inhibited tumor growth in the B16F10 syngeneic model. These studies demonstrate that MEDI7526 drives the internalization and degradation of PD-L1 and exhibits robust in vitro and in vivo activities. Building this novel bispecific molecule brings two molecules together, resulting in novel mechanisms of action that are different from
combinations of parental reagents. MEDI7526, through its unique function of simultaneously stimulating the CD40 pathway and down-regulating PD-L1 expression, represents a promising therapeutic approach against cancer.
Citation Format: Yaya Wang, Sean Turman, Deepali Malhotra, Ryan Gilbreth, Yue Wang. MEDI7526: a novel bispecific antibody that activates the CD40 pathway and down-regulates cell surface PD-L1 expression [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 5178.