BACKGROUND: Avelumab has recently been approved by the Food and Drug 
Administration for the therapy of Merkel cell carcinoma and urothelial 
carcinoma. M7824 is a novel first-in-class bifunctional fusion protein 
comprising a monoclonal antibody against programmed death-ligand 1 (PD-L1, 
avelumab), fused to the extracellular domain of human transforming growth factor 
beta (TGFβ) receptor 2, which functions as a TGFβ "trap." Advanced urothelial 
tumors have been shown to express TGFβ, which possesses immunosuppressive 
properties that promote cancer progression and metastasis. The rationale for a 
combined molecule is to block the PD-1/PD-L1 interaction between tumor cells and 
immune cell infiltrate and simultaneously reduce or eliminate TGFβ from the 
tumor microenvironment. In this study, we explored the effect of M7824 on 
invasive urothelial carcinoma cell lines.
METHODS: Human urothelial (transitional cell) carcinoma cell lines HTB-4, HTB-1, 
and HTB-5 were treated with M7824, M7824mut (M7824 that is mutated in the 
anti-PD-L1 portion of the molecule and thus does not bind PD-L1), anti-PD-L1 
(avelumab), or IgG1 isotype control monoclonal antibody, and were assessed for 
gene expression, cell-surface phenotype, and sensitivity to lysis by TRAIL, 
antigen-specific cytotoxic T lymphocytes and natural killer cells.
RESULTS: M7824 retains the ability to mediate antibody-dependent cellular 
cytotoxicity of tumor cells, although in some cases to a lesser extent than 
anti-PD-L1. However, compared to anti-PD-L1, M7824 increases (A) gene expression 
of molecules involved in T-cell trafficking in the tumor (e.g., CXCL11), (B) 
TRAIL-mediated tumor cell lysis, and (C) antigen-specific CD8+ T-cell-mediated 
lysis of tumor cells.
CONCLUSIONS: These studies demonstrate the immunomodulatory properties of M7824 
on both tumor cell phenotype and immune-mediated lysis. Compared to anti-PD-L1 
or M7824mut, M7824 induces immunogenic modulation of urothelial carcinoma cell 
lines, rendering them more susceptible to immune-mediated recognition and lysis. 
These findings show the relevance of the dual blockade of PD-L1 and TGFβ in 
urothelial carcinoma cell lines and thus support the rationale for future 
clinical studies of M7824 in patients with urothelial cancer.