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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.

Effective treatment of solid tumors requires multi-modality approaches. In many patients with stage IV liver disease, current treatments are not curative. Chimeric antigen receptor T cells (CAR-T) are an intriguing option following success in hematological malignancies, but this has not been translated to solid tumors. Limitations include sub-optimal delivery and elevated interstitial fluid pressures. We developed a murine model to test the impact of high-pressure regional delivery (HPRD) on trafficking to liver metastases (LM) and tumor response.CAR-T were generated from CD45.1 mice and adoptively transferred into LM-bearing CD45.2 mice via regional or systemic delivery (RD, SD). Trafficking, tumor growth, and toxicity were evaluated with flow cytometry, tumor bioluminescence (TB, photons/sec logRD of CAR-T was more effective at controlling tumor growth versus SD from post-treatment days (PTD) 2-7 (P = 0.002). HPRD resulted in increased CAR-T penetration versus low-pressure RD (LPRD, P = 0.004), suppression of tumor proliferation (P = 0.03), and trended toward improved long-term control at PTD17 (TB=3.7 versus 6.1, P = 0.47). No LFT increase was noted utilizing HPRD versus LPRD (AST/ALT P = 0.65/0.84) while improved LFTs in RD versus SD groups suggested better tumor control (HPRD AST/ALT P = 0.04/0.04, LPRD AST/ALT P = 0.02/0.02).Cellular immunotherapy is an emerging option for solid tumors. Our model suggests RD and HPRD improved CAR-T penetration into solid tumors with improved short-term tumor control. Barriers associated with SD can be overcome using RD techniques to maximize therapeutic delivery and HPRD may further augment efficacy without increased toxicity.

Metastatic liver tumors have presented challenges with the use of checkpoint inhibitors (CPIs), with only limited success. We hypothesize that regional delivery (RD) of CPIs can improve activity in the liver and minimize systemic exposure, thereby reducing immune-related adverse events (irAE). Using a murine model of colorectal cancer liver metastases (LM), we confirmed high levels of PD-L1 expression on the tumor cells and liver myeloid-derived suppressor cells (L-MDSC). In vivo, we detected improved LM response at 3 mg/kg on PTD7 via portal vein (PV) regional delivery as compared to 3 mg/kg via tail vein (TV) systemic delivery (p = 0.04). The minimal effective dose at PTD7 was 5 mg/kg (p = 0.01) via TV and 0.3 mg/kg (p = 0.02) via PV. We detected 6.7-fold lower circulating CPI antibody levels in the serum using the 0.3 mg/kg PV treatment compared to the 5 mg/kg TV cohort (p &lt, 0.001) without increased liver toxicity. Additionally, 3 mg/kg PV treatment resulted in increased tumor cell apoptotic signaling compared to 5 mg/kg TV (p &lt, 0.05). Therefore, RD of an anti-PD-1 CPI therapy for CRCLM may improve the therapeutic index by reducing the total dose required and limiting the systemic exposure. These advantages could expand CPI indications for liver tumors.

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.

Introduction Efficacy of cellular immunotherapy for liver metastases (LM) has been limited by poor trafficking and elevated intra-tumoral pressures. In a phase 1b study, we found that regional, pressure-enabled drug delivery (PEDD, TriSalus Life Sciences, Inc.) improved LM CAR-T delivery. We hypothesized that PEDD of CAR-T in a murine model can improve LM control. Methods LM bearing CD45.2 mice were treated with CD45.1 anti-CEA CAR-T via systemic delivery (SD, tail vein) or regional delivery (RD, portal vein) at low pressure (LPRD) vs. high pressure (HPRD) to model PEDD. Flow rates were calibrated to achieve LPRD or HPRD. Tumor bioluminescence (TB), flow cytometry (FC), immunofluorescence (IF), & quantitative polymerase chain reaction (qPCR) were assessed on various post-treatment days (PTD). Results FC of liver non-parenchymal cells on PTD7 showed increased CAR-T density in HPRD & LPRD (9.4% & 11.0%, p = 0.03 & 0.002) vs. SD (3.7%). qPCR of the CAR transgene supported this observation, but was not significant (HPRD 135.8, p = 0.76, LPRD 108.6, p = 0.40 vs. SD 69.8). TB was lower in RD vs. SD up to PTD7 (log2 fold-change HPRD −1.3 & LPRD −0.9 vs. SD 4.3, p = 0.005 & 0.006). Tumor proliferation by Ki67 IF was significantly reduced with RD vs. control (HPRD & LPRD, p < 0.001 & p = 0.05) with HPRD superior to LPRD (p = 0.03). Serum liver enzymes were not significantly elevated using HPRD vs. LPRD at PTD7 (AST/ALT 154.7/36.3 vs. 139.1/29.1, p = 0.84/0.67), but did rise at PTD17, correlating with tumor progression (AST/ALT 800.7/229.3 vs. 387.5/139.5, p = 0.10/0.28). Conclusions RD increased LM CAR-T delivery and HPRD showed evidence of improved CAR-T trafficking and tumor control relative to LPRD. Further clinical testing of PEDD is planned for LM cellular therapy.