THE CLINICALLY RELEVANT STING AGONIST, E7766, INDUCES IMMUNOGENIC TUMOUR CLEARANCE AND PROTECTION AGAINST RECURRENT DISEASE IN THE KP MODEL OF UNDIFFERENTIATED PLEOMORPHIC SARCOMA

Soft tissue sarcomas (STS) are rare malignancies that arise from tissues of mesenchymal lineage[1]. One of the most common, aggressive, and metastatic STS in adults in undifferentiated pleomorphic sarcoma (UPS) [2]. Once metastatic, there are no curative therapies available. Generally, UPS are not responsive to immunotherapies, and this largely attributed to the tumor microenvironment (TME) of these tumors containing few tumor infiltrating lymphocytes (TILs) and an abundance of immunosuppressive myeloid cells [3-5]. Previous studies have revealed that intratumoural (i.t.) activation of the STimulator of INterferon Genes (STING) pathway, using the murine specific agonist DMXAA, recruited TILs into the TME in the murine KP model of UPS and induced survival in 50-75% of treated mice [6]. To assess the clinical translation feasibility of STING immunotherapy, we will investigate the anti-UPS properties of the human and murine STING agonist, E7766 in the KP model. The objectives of this study are 1) to evaluate the anti-tumour potential of and TME modulation of E7766, 2) to determine if E7766 therapy can induce anti-UPS protection, 3) to characterize CD8+ T-cell contributions to memory immune responses in surviving mice, and 4) to determine if E7766 can sensitize KP UPS tumors to anti-PD1 ICB therapy. Immune competent C57Bl/6 mice were engrafted with a KP UPS cells in the hindlimb muscle. Once palpable, mice were treated with E7766 or DMXAA i.t. Following treatment, mice were divided into STING monotherapy or STING + anti-PD1 ICB therapy groups and followed for survival. Another cohort of DMXAA or E7766 treated mice were sacrificed at following STING therapy and tumors were assessed via flow cytometry. Mice who survived and eradicated their primary UPS tumors following STING monotherapy were re-challenged with an additional inoculation of UPS cells in the contralateral limb to assess the extent of anti-UPS protection obtained from STING therapy. E7766 survivor mice who survived and rejected UPS tumor development following re-challenge were divided into; i) a CD8+ depleting antibody group or ii) an isotype control group. Following depletion, survivor mice were re-challenged a second time with UPS cells, followed for survival, and sacrificed to confirm depletion via flow cytometry (Fig1). Thirty-eight percent of mice in the E7766 monotherapy group eradicated their primary UPS tumors (Fig2A). Flow cytometry studies of tumors 1-week following STING therapy displayed that E7766 induces similar increases in CD8+ TILs to DMXAA relative to control mice (Fig2B). All mice who survived STING therapy rejected the first contralateral UPS re-challenge (Fig2C). Surprisingly, re-challenged CD8+ T-cell depleted survivors maintained their ability to reject UPS tumor engraftment, indicating that protection against UPS recurrence is independent of this cell type (Fig2D-E). To confirm the importance of CD8+ T-cells when mounting an initial anti-tumor response following STING therapy, an experiment with naive mice receiving CD8+ depleting antibodies prior to UPS engraftment and STING therapy is ongoing. Excitingly, in the E7766 + anti-PD1 combination therapy group, survival increased to 54% of treated mice eradicated their UPS tumors (Fig1F), which is significantly greater than anti-PD1 monotherapy in this model. We have shown that E7766 as a monotherapy and in combination with anti-PD1 are promising immunotherapeutic strategies in the KP model of UPS. Further investigation into which cell types are required for anti-UPS protection and elucidating cell types that engage in STING signaling in the UPS TME through scRNAseq are ongoing and will provide mechanistic understanding of STING immunotherapy that will be valuable for clinical applications. For any figures or tables, please contact the authors directly.