Abstract 2405: B7H4 as a T cell inhibitory regulator in bladder cancer
Authors: Meeks Joshua , Glaser Alexander P. , Fantini Damiano , Yu Yanni , Eaton Valerie , Podojil Joseph R. , Miller Stephen D. 
Published: August 2019

Abstract
Despite a high number of neoantigens and T cell infiltration, most locally advanced and metastatic bladder cancers (BC) (&gt;70%) do not respond to anti-PD1 treatment. Thus, targeting additional checkpoints may play an important role in treatment of BC. We sought to identify negative regulators of T cell activity and evaluate their function using a previously validated pre-clinical carcinogen-induced mouse model of BC.
To identify regulators of T cell activity, we first investigated the timing and infiltration of T cells during tumor development in the BBN mouse BC model. We found that numbers of CD4+ and CD8+ T cell increased dramatically within the first 1-2 weeks, but then decreased over the next six weeks decreasing to the lowest levels at 8 weeks before increasing again coordinating with increasing tumor volume. During the decrease in T cell numbers, macrophages increased and by transcriptome profiling we identified increased levels of the T cell inhibitory protein B7-H4. B7-H4+CD11b+ macrophages increased over 4 weeks with opposite expression of CD4+ and CD8+ T cells by FACS. By IHC we localized B7-H4 expression in macrophages adjacent to tumor. A bioinformatic analysis of the human TCGA identified highest B7-H4 expression in luminal infiltrated subtypes of MIBCs, but did not correlate with CD4, CD8 or overall tumor mutation burden. MIBCs with elevated B7-H4 expression had significantly worse survival. In vitro, addition of anti-B7-H4 significantly increased proliferation and IFN-γ production by human T cells co-cultured with B7-H4 expressing APCs. To determine the role of B7-H4 in BC, we treated mice with overt BC (beginning at 4 months of carcinogen administration) with vehicle, anti-PD1 or anti-B7-H4 antibody. Compared to vehicle treated animals, anti-B7-H4 treated mice had a reduction in the number of pT3 tumors (50% vs. 73%), but less than anti-PD1 treated tumors (27%). However, anti-B7-H4 treated mice had significantly increased tumor infiltrating CD8+ T cells per histological field (77.1+34.3 vs. 11.5+7.8; p=0.0121), as well as a significant increase splenic CD8+IFN-γ+ T cells and levels of secreted IFN-γ upon anti-CD3 stimulation. Transcriptome profiling of tumors that responded to anti-B7-H4 antibody by RNA-Seq identified a decrease in mitotic cell cycle, cell cycle checkpoints, RHO GTPases and mitotic spindle checkpoint pathways in responsive tumors.
In summary, we confirm the expression of B7-H4 in macrophages found in a murine model of BC and correlate expression to poor survival in the TCGA of MIBC. Inhibition of B7-H4 results in lymphocyte proliferation in vitro and inhibition of B7-H4 in mice resulted in decreased tumor stage and increased CD8+ TILs. This data suggests that B7-H4 may be a candidate alternative checkpoint that can be targeted in humans with BC unresponsive to PD1 therapy.
Citation Format: Joshua Meeks, Alexander P. Glaser, Damiano Fantini, Yanni Yu, Valerie Eaton, Joseph R. Podojil, Stephen D. Miller. B7H4 as a T cell inhibitory regulator in bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2405.