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Cancer Technologies

Cancer Technologies - Poster Session B

Poster H3 - Creating a Multi-Modal CAR-NK-92 Cell Therapy Platform through LNP-mediated mRNA Delivery and Metabolic Glycoengineering

Thursday, October 24, 2024
2:45 PM – 3:45 PM EST
Location: Exhibit Hall E, F & G

Introduction:  Chimeric antigen receptor T-cell (CAR-T) therapy excels in treating hematological malignancies but faces challenges like cytokine release syndrome, antigen escape, and ineffectiveness in solid tumors. Natural killer (NK) cells' inherent cancer-targeting abilities led to CAR-NK therapies, leveraging potent cytotoxicity with reduced graft-versus-host disease risk. Before clinical use, however, CAR-NK therapy must improve anticancer activity and efficacy in solid tumor microenvironments. Here, we propose engineering NK-92 cells into a potent multi-modal therapy platform. First, lipid nanoparticles (LNPs), known for transient mRNA delivery (e.g., COVID-19 mRNA vaccines), were designed for NK-92 cells, revealing a top performer, C8i-200. This LNP was then modified to incorporate DBCO groups that could conjugate to sugar-modified NK-92 cells. C8i-200 with DBCO, however, experienced disrupted transfection into cells, possibly because the LNPs were attaching to the surface of the cells rather than internalizing. This finding supports a possible ex vivo procedure where NK-92 cells are first treated with non-DBCO LNPs carrying CAR mRNA to transform them into CAR-NK cells and then treated with DBCO-LNPs carrying a cytotoxic payload that will only attach to the surface of the CAR-NK cells. These CAR-NK cells can then be delivered in vivo to target tumor cells through different modes of action: NK-cell cytotoxicity and LNP-mediated delivery of a cytotoxic payload. Overall, this therapy would minimize off-target genetic effects and combat tumor cell immune escape, yielding a safer and more effective immunotherapy. Future studies aim to build off the data from this study and confirm this proposed cellular engineering platform.

Materials and

Methods:  Materials:
Non-IL lipid excipients were from Avanti Polar Lipids. Firefly luciferase and mCherry mRNA, modified with N1pseudoU substitutions, were from TriLink Biotechnologies.

LNP Formulation and Characterization:
The organic phase for unmodified LNPs included IL, DOPE, cholesterol, and C14-PEG2000 in a molar ratio of 35:16:46.5:2.5. For DBCO LNPs, DBCO PEG2000 was added in varying molar ratios. The aqueous phase contained mRNA dissolved in 10 mM citrate buffer (pH 3). The weight ratio of mRNA to IL was 1:10, and the volume ratio between organic and aqueous phases was 1:3. Using Hamilton syringes and a microfluidic device, both phases were mixed at specific flow rates.

Encapsulation efficiency and mRNA concentration were assessed using the Quant-iT RiboGreen assay. LNP samples were diluted and treated with 1X TE buffer and 1% Triton X-100 to lyse LNPs. Fluorescence intensity was measured using an Infinite 200 Pro plate reader. RNA content was determined via linear regression to calculate encapsulation efficiency.

In Vitro Studies:
NK-92 cells were cultured and screened for mycoplasma contamination. Cells were seeded at 60,000 cells per well and incubated overnight. LNPs with FLuc mRNA were added, and luciferase expression was measured after 24 hours. Cell viability was assessed using CellTiter-Glo.

Azido Sugar Labeling:
Ac4ManNAz was added to NK-92 cells at various concentrations for toxicity and flow cytometry assays.

Flow Cytometry:
DBCO-Cy5 conjugation on labeled NK-92 cells was evaluated using a BD LSR II flow cytometer and analyzed with FlowJo software. The gating strategy included FSC-A/SSC-A and FSC-A/FSC-H.

Results, Conclusions, and Discussions: In our study, C8i-200 LNPs demonstrated threefold higher transfection efficiency in NK-92 cells compared to the gold-standard C12-200 LNPs, with over 95% cell viability, indicating no toxicity at a 200-ng mRNA dose. Azido sugars at concentrations above 250 µM were cytotoxic to NK-92 cells, but concentrations of 25 µM and above achieved over 90% cell labeling without toxicity. However, adding DBCO to LNPs significantly disrupted delivery, particularly at concentrations above 0.5%, with almost no transfection at 1.0% or higher. This disruption was observed both with and without sugar azido pre-treatment, suggesting that DBCO interferes with LNP cell entry and might cause LNPs to stick to azido sugars on the cell surface, preventing internalization and transfection. Moreover, the decreased encapsulation efficiencies of LNPs with higher DBCO percentages indicate potential issues with LNP quality and mRNA carrying capacity.

Future studies aim to further investigate these mechanisms, using LNPs carrying both mCherry and dye to track their distribution in and out of NK-92 cells. The goal is to confirm whether LNPs stick to the sugar azido groups on the cell surface, supporting the proposed ex vivo cellular engineering platform for multi-modal CAR-NK therapy. Additionally, future experiments will involve transfecting NK-92 cells with C8i-200 LNPs carrying CD19 CAR mRNA to assess delivery and anti-tumor efficacy. CAR-NK cells will then be treated with DBCO-LNPs carrying a cytotoxic payload, with efficacy tested in mouse solid tumor models.

This approach aims to overcome the current limitations of CAR-T therapies, offering a potentially safer and more effective treatment for solid tumors. By addressing these mechanistic challenges, such as optimizing LNP composition and understanding their interactions with NK cells, we hope to enhance the therapeutic potential of CAR-NK cells and improve outcomes for patients with solid tumors. Overall, this multi-modal cell engineering platform will pave the way for more precise and targeted immunotherapies in the future, ultimately benefiting patients worldwide by minimizing off-target genetic effects and combatting tumor cell immune escape, yielding a safer and more effective immunotherapy.

Acknowledgements (Optional):