Stabilized Recombinant Trefoil Factor 2 (Tff2-Ctp) Enhances Anti-Tumor Activity Of Pd-1 Blockade In Mouse Models Of Colorectal Cancer

Despite remarkable responses to immune checkpoint blockade across multiple tumor types, the clinical benefit in colorectal cancer (CRC) is limited to microsatellite unstable tumors. PD-L1 expression is a negative prognostic marker in CRC but correlates with a better response to PD-1 blockade. Here, we investigated the role of PD-L1 in colorectal tumorigenesis and evaluated the utility of targeting myeloid-derived suppressor cells (MDSCs) in combination with PD-1 blockade in mouse models of CRC. We generated knockin mice that conditionally express the murine Pdl1 gene (R26-LSL-Pdl1-EGFP) and crossed them with LysM-Cre mice to overexpress PD-L1 specifically in the myeloid lineage. Mice received azoxymethane (AOM; 10 mg/kg i.p.) followed one week later with 2.5% dextran sodium sulfate (DSS) in the drinking water for 7 days. AOM/DSS-treated control mice formed tumors at 10 weeks and developed adenocarcinoma at 17 weeks post-AOM. LysM-Cre; R26-PD-L1 mice treated with AOM/DSS showed markedly enhanced colorectal tumorigenesis, with a significant increase in tumor number and size and enlarged spleen. In both groups, AOM/DSS treatment led to a significant expansion of myeloid cells, particularly CD11b+Gr-1+ MDSCs, in the colon and spleen, along with decreased NK cells compared with untreated mice. Notably, AOM/DSS-treated LysM-Cre; R26-PD-L1 mice showed decreased intratumoral CD8+ T cell infiltration compared to control tumors. Furthermore, there was a significant decrease in the percentage of Ki-67+ cells in intratumoral CD8+ T cells, indicating attenuated anti-tumor immunity. Trefoil factor 2 (TFF2), a secreted anti-inflammatory peptide, inhibits colon tumor growth by suppressing the expansion of CD11b+Gr-1+ MDSCs. TFF2 fused with two carboxyl-terminal peptide and three Flag motifs (TFF2-CTP-Flag) prolonged the circulation time in blood but retained bioactivity. We induced tumors in R26-PD-L1 mice with AOM/DSS, administered fusion recombinant TFF2-CTP-Flag (300 ug i.p.) and/or anti-PD-1 (RMP1-14; 200 ug i.p.) three times a week starting at 10 weeks and 14 weeks, respectively, and examined tumors at 18 weeks post-AOM. R26-PD-L1 mice treated with anti-PD-1 + TFF2-CTP showed a marked reduction in tumor growth while anti-PD-1 monotherapy failed to suppress growth. The combination of anti-PD-1 and TFF2-CTP significantly increased tumor-infiltrating CD8+ T cells and concomitantly decreased intratumoral regulatory T cells and CD11b+Gr-1+ myeloid cells. These early findings suggest that TFF2 augments the response rate of CRC to PD-1 blockade, possibly through suppressing MDSC expansion, supporting the potential of TFF2-CTP in combination I-O treatment for CRC. We are currently testing the efficacy of combined TFF2-CTP and anti-PD-1 therapy in the AOM/DSS model with PD-L1-overexpressing LysM-Cre; R26-PD-L1 mice. Citation Format: Woosook Kim, Na Fu, Phaneendra Duddempudi, Zinaida Dubeykovskaya, Steven Almo, Chandan Guha, Seth Lederman, Timothy Wang. Stabilized recombinant trefoil factor 2 (TFF2-CTP) enhances anti-tumor activity of PD-1 blockade in mouse models of colorectal cancer [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 6640.