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Myeloid-derived suppressor cells (MDSCs) expand in response to malignancy and suppress responsiveness to immunotherapy, including checkpoint inhibitors (CPIs). Within the liver, MDSCs have unique immunosuppressive features. While TLR9 agonists have shown promising activities in enhancing CPI responsiveness in superficial tumors amenable to direct needle injection, clinical success for liver tumors with TLR9 agonists has been limited by delivery challenges. Here, we report that regional intravascular infusion of ODN2395 into mice with liver metastasis (LM) partially eliminated liver MDSCs and reprogrammed residual MDSC. TLR9 agonist regional infusion also induced an increase in the M1/M2 macrophage ratio. Enhanced TLR9 signaling was demonstrated by an increased activation of in NFκB (pP65) and production of IL6 compared with systemic infusion. Further, PBMC-derived human MDSCs express TLR9, and treatment with class C TLR9 agonists (ODN2395 and SD101) reduced the expansion of MDSC population. TLR9 stimulation induced MDSC apoptosis and increased the M1/M2 macrophage ratio. Regional TLR9 agonist infusion along with systemic anti-PD-1 therapy improved control of LM. With effective delivery, TLR9 agonists have the potential to favorably reprogram the liver TME through reduction of MDSCs and favorable macrophage polarization, which may improve responsiveness to systemic CPI therapy.

CpG oligodeoxynucleotides (ODNs) are TLR9 agonists (TLR9As) that stimulate pDC and B cells. Yet, multiple other cell types in the liver express TLR9. The effect of TLR9A in reshaping the tumor microenvironment (TME), particularly in the myeloid-derived suppressor cells (MDSC) compartment, is yet to be determined. Clinical success of TLR9As has been limited by toxicity when given systemically and delivery limitations when administered by direct needle injection. Here, we hypothesized that regional intra-vascular infusion of TRL9As would reprogram the liver MDSC compartment to enable immune control of liver metastases (LM). In this study, we evaluated the effect of class C ODN-2395 as TLR9A in inhibiting LM progression and its impact on the liver MDSC population. C57/BL6 mice were challenged with 2.5e6 MC38-CEA-Luc cells via intra-splenic route. After a week, mice were treated with 1, 3, 10 or 30 µg ODN2395 via PV or IV (30 µg). Bioluminescence was measured at 24 and 48 hrs post-ODN administration. CD45+ cells were isolated, and FACS analysis was performed to quantify MDSCs, monocytic MDSCs (M-MDSC), and M1-macrophage subsets, along with downstream signaling. We found that 30 µg ODN2395 delivered via PV compared to IV reduced tumor burden at 24 hrs and persisted up to 48 hrs. Also, 30 µg ODN2395 infused via PV was optimal in controlling MDSC, M-MDSC cells and polarized CD11b+F4/80+ monocytic cells towards pro-inflammatory/anti-tumorigenic M1 macrophage. Using an NFκB-dependent soluble alkaline phosphatase assay (SEAP), we determined that ODN2395 dose-dependently enhanced NFκB transcription factor activity (p Table:Readout30µg IV30µg PVP valuenBioluminescence-24 h (photon/s)4e71e7 Citation Format: Chandra Ghosh, Kara Heatherton, Kyle O'Connell, Jason Laporte, Prajna Guha, David Jaroch, Bryan Cox, Steven C. Katz. Regional administration of class C CpG Oligodeoxynucleotides results in superior intrahepatic TLR9 activation and immunomodulation compared to systemic infusion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1793.

e15016 Background: Colorectal cancer liver metastases (CRCLM) are a major source of morbidity and mortality. Historically, curative therapy has been limited to surgical resection, but only a small fraction of patients are eligible. Cellular immunotherapy has shown promise in hematologic cancers, but challenges related to solid tumor therapy remain with optimal cell trafficking, elevated interstitial fluid pressures (IFP), and immunosuppression. We hypothesized that engineered natural killer (NK) cells expressing a natural killer group 2, member D (NKG2D) activating chimeric receptor (ACR) and membrane bound IL-15 (NKX101) would increase anti-tumor activity in vitro and in vivo utilizing our established regional delivery strategies. Methods: In vitro cytotoxicity and cytokine release of NKX101 cells or non-transduced NK cells (NT-NK) derived from the same donor were determined by co-culture systems with HCT116 cells that endogenously express NKG2D ligands. CRCLM-bearing NSG™ mice were treated with NKX101, NT-NK, or vehicle (CTRL) via portal vein (PV) for regional delivery (RD) or tail vein (TV) for systemic delivery (SD). Tumor burden (TB) was measured via tumor bioluminescence (TBL) and histopathology (HP). Flow cytometry (FC) determined the quantity of cells delivered. Student’s t-test and Mann-Whitney tests were performed for statistical comparisons. Results: NKX101 transduction efficiencies ranged between 63.5 – 75.6% across 3 separate healthy donors. EC50 values derived from a 4-hour cytotoxicity assay for NKX101 vs. NT-NK were 3-4 fold lower with the greatest difference observed at the 1:1 effector-to-target (E:T) ratio (mean percent cytotoxicity: 72% vs. 20%, p = 0.001). In vitro cytokine assessment revealed 2.0-2.6 fold increases in IFN-γ, GM-CSF, and TNF-α levels compared to NT-NK cells (p < 0.0001 across all groups). In vivo, FC showed 2.89-fold increase in cell delivery using RD vs. SD on PTD1 (n = 3, p = 0.006). TBL was improved with 5 x 106 cells via PV vs. TV (n = 6) from post-treatment day (PTD) 1-7, with greatest difference seen on PTD7 (12.9 vs. 42.6, p = 0.07). HP analysis showed reduction of TB at PTD7 with PV treatment. Conclusions: NKX101 demonstrated improvements in in vitro cytotoxicity and pro-inflammatory cytokine release. RD techniques in vivo revealed increased cell delivery and improved tumor control. Further studies are underway to confirm our initial findings and understand NKX101 cellular kinetics and susceptibility to immunosuppression in the liver, along with planned clinical evaluation in Phase 1 trials.