Abstract 2208: Combination of the EP4 antagonist CR6086 and anti-PD-1 monoclonal antibody inhibits tumor growth in a microsatellite stable colorectal cancer in mice
Authors: Caselli Gianfranco , Ferrari Flora , Piepoli Tiziana , Grotti Adriana , Cavagnoli Rosanna , Montagna Giuseppe M. , Romanelli Pierpaolo , Bonazzi Albino , Lanza Marco , Recordati Camilla , Rovati Lucio C. 
Published: September 2020

Abstract
BACKGROUND: Immune checkpoint inhibitors (ICIs) have marked therapeutic effects in many cancer patients. This is not the case for the large majority (~85%) of colorectal cancers (CRC), i.e. those classified as microsatellite stable (MSS) tumors. Cancers that do not respond to immunotherapies, such as this predominant CRC form, have a T cell non-infiltrated phenotype and are known as “cold” tumours. They are poorly immunogenic, as opposed to ICI-responsive “hot” tumours that are associated with high T cell infiltration. A typical immunosuppressive mechanism used by tumors is the production of prostaglandin E2 (PGE2), which binds four EP receptor subtypes on tumor and immune cells to promote tumor survival. The EP4 receptor plays a major role in PGE2-induced immunosuppression. CR6086 is a clinical stage EP4 receptor antagonist acting as a targeted immunomodulator. This study tested the hypothesis that the EP4 antagonist CR6086 could turn cold into hot tumors, favoring the response to ICI therapy.
METHODS: We investigated the efficacy of combining CR6086 with an ICI (i.e. anti-mouse PD-1 (CD279), clone RMP1-14 [mAb]) in a syngeneic model of CRC resistant to ICI therapy. CT26 colon carcinoma cells (1 × 106) were inoculated subcutaneously in the right flank of female BALB/c mice. Treatments started on day 7 post-inoculation, when all tumors were within the target volume range of 50-100 mm3. CR6086 (30 mg/kg) was administered orally once daily for 14 days (QDx14); anti-PD-1 mAb (150 μg/mouse) was administered intraperitoneally on days 7, 10, 14 and 17.
RESULTS: The combination of CR6086 and anti-PD-1 significantly decreased tumor growth vs vehicle-treated mice (mean tumor volume: 721±166 mm3 vs 1457±219 mm3; P&lt;0.05, Two-way RM ANOVA followed by Dunnett's test, n=11/group), as measured after 14 days of treatment. The anti-PD-1 alone did not affect tumor progression, similarly to CR6086 monotherapy. RT-PCR analysis on tumor tissues showed that combined treatment increased (≥2-fold change) the gene expression of key factors for lymphocyte recruitment and activation. They include specific chemokines (CCL4, CCL5, CXCL10) responsible for increased T cell and dendritic cell infiltrate; CD8α, a major indicator for the presence of cytotoxic T lymphocytes; the antigen processing marker H2-Eb1s; INFγ, a T cell activation factor; TNFα, a cytokine produced by M1 macrophages and involved in tumor cell death; PD-L1, a prognostic biomarker with predictive value for the response of patients under anti-PD-1 therapy. These findings on gene expression are well reflected at the cellular level, because combined treatment increased general and T cell infiltrate, as assessed by haematoxylin/eosin staining and CD3+ immunohistochemistry. Conversely, the combination of CR6086 and anti-PD-1 reduced the gene expression of MMP-9, a matrix metalloproteinase involved in tumor growth, invasion, metastasis, extracellular matrix remodeling, and angiogenesis.
CONCLUSION: These results suggest that the EP4 receptor antagonist CR6086 may turn a cold MSS murine colorectal cancer, non-responsive to ICI treatment, into a hot tumor responsive to ICIs.
Citation Format: Gianfranco Caselli, Flora Ferrari, Tiziana Piepoli, Adriana Grotti, Rosanna Cavagnoli, Giuseppe M. Montagna, Pierpaolo Romanelli, Albino Bonazzi, Marco Lanza, Camilla Recordati, Lucio C. Rovati. Combination of the EP4 antagonist CR6086 and anti-PD-1 monoclonal antibody inhibits tumor growth in a microsatellite stable colorectal cancer in mice [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 2208.