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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.

729 Background: Systemic immunotherapy has had limited clinical benefit in pancreatic ductal adenocarcinoma (PDAC). This is thought to be due to its desmoplastic immunosuppressive tumor microenvironment in addition to high intratumoral pressures that limit drug delivery. Recent pre-clinical cancer models and early-phase clinical trials have demonstrated the efficacy of toll-like receptor 9 agonists (TLR9a), including the synthetic CpG oligonucleotide SD101, to stimulate innate and adaptive immune cells and eliminate suppressive myeloid cells. We hypothesized that Pressure Enabled Drug Delivery (PEDD) via Pancreatic Retrograde Venous Infusion (PRVI) of a TLR9a would improve responsiveness to systemic anti-programmed death receptor-1 (PD-1) checkpoint inhibitor therapy in a murine orthotopic PDAC model. Methods: Murine PDAC (KPC4580P) tumors were implanted into the pancreatic tails of C57BL/6J mice and treated 8 days after implantation. Mice were assigned to PRVI saline (pSAL, n=9), systemic anti-PD1 100 mcg/mouse on Day 0, 2 and 4 (sAPD1, n = 6), 30mcg PRVI TLR9a (pTLR9a, n=9), 30mcg systemic TLR9a (sTLR9a, n=7) on Day 0, and combination 30mcg PRVI TLR9a on Day 0 and systemic anti-PD1 100 mcg/mouse on Day 0, 2 and 4 (COMBO, n =9). Fluorescently-labeled TLR9a (radiant efficiency [RE]) was measured on day 1. Blood and tumors were collected at necropsy 12 days after infusion. Results: All mice survived to necropsy. Site of tumor fluorescence measurements revealed higher intensity fluorescence in pTLR9a compared to sTLR9a (7.5x10^5 vs. 2.4x10^5 RE, p= 0.048). Significantly lower MDSCs in the COMBO vs. pSAL are shown. Tumor weights were significantly lower in the COMBO group compared to pSAL (400 vs. 964 mg, p=0.003), and were also lower, although not significantly, than in the pTLR9a (400 vs. 518mg, p=0.50), and sAPD1 (400 vs. 645 mg, p = 0.70) groups. Conclusions: PEDD of TLR9a by PRVI with systemic anti-PD-1 demonstrated improved PDAC tumor control. These results support study of this combination therapy in PDAC patients and expansion of ongoing PEDD clinical trials. [Table: see text]

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.

Introduction We have demonstrated safety and clinical activity with anti-CEA CAR-T (Sorrento/TNK Therapeutics, Inc.) regional infusions for treatment of liver metastases (LM) using a pressure-enabled drug delivery device (PEDD™, TriSalus™ Life Sciences, Inc.) to increase CAR-T penetration. To further explore the potential benefit of PEDD, we developed a pre-clinical murine LM model that enables correlation of infusion pressure with response. Methodology Mice with CEA+ LM were infused with anti-CEA CAR-T into the portal vein with specified rate and pressure levels. Flow cytometry (FC), bioluminescence imaging (BLI), and serum liver function tests (LFTs) were conducted at multiple time points. Results Flow-pressure relationship was established with ΔP of 7.23 mmHg vs 2.33 mmHg (p=0.01) in high-pressure (10 mL/min, HP) and low-pressure (1 mL/min, LP) cohorts. FC assessment of hepatic non-parenchymal cells 1d after infusion demonstrated 15.9% CD3+CAR+ in HP, compared to 5.1% in LP (p=0.0004). Tumor BLI at day 3 (geometric mean relative to baseline) showed significantly enhanced therapeutic effect in HP, −18% relative signal intensity (RSI), compared to +148% RSI in LP (p=0.05) or +178% RSI in controls (p=0.04). LFTs were similar in LP and HP groups with AST 302 vs 437 U/L (p=0.60) and total bilirubin 0.25 vs 0.7 mg/dL (p=0.33), respectively. Conclusions HP delivery correlated with enhanced CAR-T delivery and control of tumor growth, without an increase in liver toxicity. Follow up to this proof of concept study will investigate multiple infusions with combinatorial approaches with the goal of durable tumor eradication. An ongoing clinical study will further explore CAR-T PEDD in patients.

Introduction Checkpoint inhibitors (CI) have greatly impacted the treatment of solid tumors. Success in the liver has been more limited. When intravenously infused, the CI is delivered predominantly to extrahepatic sites, which results in systemic side effects. Previously, we reported on regional delivery of cellular immunotherapy for liver tumors using pressure-enabled drug delivery (PEDD™, TriSalus™ Life Sciences, Inc.). We hypothesized that PEDD of CI would improve the therapeutic index (TI). Methods In a murine model of colorectal liver metastases (CRLM), we infused high (HP) or low pressure (LP) anti-PD-1 antibodies (RMP1-14) via the portal vein and compared to systemic tail vein delivery (SD) at 0, 0.3, 1.0 and 3.0 mg/kg. Tumor bioluminescence (TB), histopathology, and serologic analysis informed treatment effects and toxicity. Results At a dose of 3mg/kg, LP resulted in similar IVIS TB growth kinetics compared to SD 3 mg/kg at 7 days post-treatment (geometric mean, p = 0.94). In animals treated with a 10-fold lower dose (0.3 mg/kg), tumor growth was significantly lower with HP vs. LP (7.5-fold improvement, p = 0.04). Circulating serum anti-PD-1 levels were similar when we compared SD and LP at 1 and 3 mg/kg (Mean 2512.0 vs. 2250.2 ng/mL, p = 0.25). Comparatively, systemic exposure was significantly lower at 0.3 mg/kg LP (Mean 508.1 ng/mL, p < 0.0001). No increases in hepatotoxicity were seen with PEDD (AST p = 0.57, ALT p = 0.43). Conclusion PEDD of anti-PD-1 CI improved liver tumor control while also limiting systemic exposure. These findings support PEDD for anti-PD-1 therapy to improve TI. Phase 1 exploration of anti-PD-1 PEDD for patients with hepatic malignancy is thus warranted.