564 - Porcine-based Platform for Image-guided Delivery of Allogeneic Natural Killer Cell Immunotherapy

Purpose: Murine models are widely used in cancer immunology, but their small size and dissimilar immune limit clinical relevance for catheter-based cell therapies {1}. In contrast, pigs have similar body size, liver structure, vascular anatomy {2,3}, and immune system compared to humans {4,5}, making them suitable for image-guided adoptive cell research. This study develops a clinically relevant image-guided pig allogeneic natural killer (NK) cell immunotherapy delivery platform by combining pig NK cell isolation and activation, a PD-L1 checkpoint assay, and hepatic arterial delivery in an Oncopig liver tumor model.

Materials and Methods: Peripheral blood from wild-type pigs was processed via Ficoll, magnetic negative selection (Triple negative (TN): CD21⁻SWC3⁻CD3⁻), and FACS to isolate CD3⁻CD8α⁺±CD16⁺ NK cells. NK cells were activated with IL-2 and assessed for cytotoxicity against an Oncopig liver tumor cell line (272, with/without PD-L1 overexpression). PD-L1 blockade assay was used to evaluate checkpoint effects on NK cell cytotoxicity. Allogeneic IL-2 activated TN NK cells (3x10⁶) were infused intra-arterially into liver tumors via hepatic arteries of Oncopigs (n=2) under CT guidance, followed by serial imaging to monitor tumors. Statistical analysis used unpaired t-tests and two-way ANOVA with Tukey correction (p=0.05).


Results: IL-2 activated CD3⁻CD8α⁺ NK cells showed significantly faster killing than bulk TN cells (KT20: 3.1 ± 0.1 vs 43.8 ± 6.2 h, p=0.008). CD8α⁺CD16⁺ cells mediated the greatest cytolysis, followed by CD8α^-CD16⁺ and then CD8α⁺CD16⁻subsets. PD-L1 overexpression on 272 cells reduced CD3⁻CD8α⁺CD16⁺NK cell cytolysis (WT: 97.3 ± 2.5% vs PDL1: 70.4 ± 2.4%, p< 0.0001). Anti PD-L1 partially restored NK cytotoxicity (rescue trend present but not found to be statistically significant). In vivo intra-arterial NK infusion was feasible and safe. CT scans showed rapid tumor reduction (week 1: 68.0%, week 2: 90.5%, week 3: 100%) in treated animals compared with historical tumor alone controls (n=11); however, this study was not powered for efficacy.

Conclusion: This feasibility study shows that pig NK cell immunotherapy can be evaluated using in vitro benchmarking and checkpoint modulation and delivered via catheter directed approaches. In vivo results showed the safety of allogeneic arterial NK cell infusion in Oncopig liver tumors, highlighting the potency and cross-talk between the PD-1/PD-L1 axis of porcine NK cells. This platform can potentially be used for image-guided studies on cell-based immunotherapy delivery in an immunocompetent large animal tumor model.