Abstract A030: Striking Gold: CpG gold (Au) lipid nanoparticles (CAL) promote toll-like receptor 9 mediated killing in mouse and human lymphomas
Isabella M. Stathas, Jonathan S. Rink, Colby S. Thaxton, Leo I. Gordon, Adam Y. LinAbstract
Introduction:
B cell lymphomas express toll-like receptor 9 (TLR9), located in endosomes and activated by CpG oligodeoxynucleotides (CpGs). Previous clinical studies yielded promising results but had limitations. Systemic class B CpGs combined with radioimmunotherapy achieved 93% overall responses (PMID 23619698), though the contribution of CpGs remains unclear. In contrast, intratumor class C CpGs, combined with radiation, resulted in partial responses in 7 of 29 patients (PMID 30154192), indicating a potential role for CpGs in lymphoma treatment. A systemic delivery method is needed. Since effective CpG delivery is a bottleneck and TLR9 resides in endosomes accessible via nanoparticles. We previously found that modified CpGs (tmCpG) can form on 15 nm gold nanoparticles, enhancing lymphoma cell killing compared to free CpGs (PMID 32798731). However, manufacturing these at scale and ensuring stability is challenging. Therefore, we shifted to a more scalable platform: lipid bilayer-coated 5 nm gold nanoparticles (PMID: 33208460). This study assesses the impact of a CpG gold lipid nanoparticle (CAL) platform on lymphoma cells.
Methods:
Class B (1826/7909) and class C (2395) CpG with amine modifications were conjugated to 16:0 Ptd thioethanol. Lipids and CpG conjugates were added to 5 nm gold nanoparticle solutions. Target cells included two EZH2-mutant mouse lymphoma lines: P1 (follicular lymphoma) and P6 (DLBCL), as well as aggressive lymphomas like double-hit lines (RC, OCI-Ly1, Ly8, Ly18) and TP53 mutated mantle cell lymphoma (JeKo-1, MAVER-1).
Results:
Particle characterization showed CALs increased size (6.97nm to13.68 nm) and became more negative in zeta potential (-4.9mV to -45.54mV) after adding phospholipids/CpGs. CALs contained ∼0.2µg/ml CpG per nM. Using HEK-Blue TLR9 cells, CALs activated TLR9 1.5 times more than free CpG at equal DNA amounts. This enhanced activation correlated with improved killing of lymphoma cells. After 3 days, B CALs reduced the viability of A20 murine lymphoma to 36.5%, compared with 67% with free CpG. The IC50 for B CALs was 1.5 µg/ml DNA (7.5nM NPs), versus 6.47 µg/ml for free CpG. Control sequences in lipid bilayers and CALs had no effect. Class C CpGs, whether free or on CALs, were less effective than class B. This pattern was consistent across EZH2-mutant mouse lines and extended to human lymphoma lines. CALs showed low IC50s in several models: RC (0.21nM), OCI-Ly1 (18.83nM), Ly8 (4.83nM), Ly18 (60.45nM), JeKo-1 (5.92nM), MAVER-1 (3.9nM). Both CpG and CAL treatments increased Caspase 1 and Caspase 3/7 activity without affecting CellRox levels.
Conclusion:
We present a scalable gold-lipid nanoparticle platform for delivering TLR9 agonist CpGs, that works with class B and C sequences. CALs provided superior TLR9 activation and lymphoma cell killing, even in difficult cases like double hit lymphomas and TP53 mutated MCLs. Grammarly was used in the editing of this abstract.
Citation Format:
Isabella M. Stathas, Jonathan S. Rink, Colby S. Thaxton, Leo I. Gordon, Adam Y. Lin. Striking Gold: CpG gold (Au) lipid nanoparticles (CAL) promote toll-like receptor 9 mediated killing in mouse and human lymphomas [abstract]. In: Proceedings of the Fifth AACR International Meeting on Advances in Malignant Lymphoma: From Discovery to Clinical Impact; 2026 Jun 24-27; Philadelphia, PA. Philadelphia (PA): AACR; Blood Cancer Discov 2026;7(3_Suppl):Abstract nr A030.