(W1030-03-15) Controlled Release of TLR7/8 Agonist-Manganese from PLGA Microparticles for Enhanced Cancer Immunotherapy

Purpose: Toll-like receptors (TLR) are key regulators of innate immune sensing of cancer and serve as a promising target for cancer immunotherapy. TLR agonists can promote dendritic cell (DC) activation and T-cell recruitment, converting “cold” tumors to “hot” tumors. However, clinical translation of TLR agonists has been challenging due to poor pharmacokinetic properties and off-target toxicities. We have previously demonstrated that nutritional metal ions, such as manganese, can synergize with innate sensing inflammatory pathways and elicit an amplified IFN-I response that triggers innate and adaptive immune responses against cancer [1]. In this work, we sought to expand on this strategy and evaluated whether a combination of a TLR7/8 agonist and manganese can achieve synergy. To test this idea, we have examined DC activation after co-treatment with a TLR7/8 agonist (3M-052, [2]) and manganese. We further developed poly(lactic-co-glycolic acid) (PLGA) microparticles (MS) co-loaded with 3M-052 and manganese as a controlled release formulation (Figure 1). Using this strategy, we hope to reduce dosing and treatment frequency for eventual translation into a post-operative setting and/or the treatment of hard-to-reach tumors.

Methods: Bone marrow-derived dendritic cells (BMDCs) were treated with either 3M-052/manganese alone or combinations of 3M-052 and manganese to evaluate modulation of immune response. IFN-I/proinflammatory markers were quantified using ELISA. 3M-052-Mn PLGA microparticles were formulated using a single emulsion-solvent evaporation method. Briefly, Resomer 503H, 3M-052, and manganese carbonate were dissolved or suspended in dichloromethane, homogenized with polyvinyl alcohol, stirred until hardened, sieved between 20–63 microns, washed and freeze-dried. The resulting particles were characterized for drug loading using ultra-performance liquid chromatography (UPLC) with UV detection at 214 nm. Manganese content was assessed using inductively coupled plasma mass spectrometry (ICP-MS). Size and morphology were evaluated by Mastersizer and scanning electron microscopy. Lastly, cytokine analysis was also performed on BMDCs treated with 3M-052-Mn²⁺ PLGA MS for up to seven days to evaluate the ability of the particles to sustain IFN-I release in vitro.

Results: BMDCs treated with both 3M-052 and manganese ion increased expression of IFN-β, CXCL9, CXCL10, and TNF-α, compared with either 3M-052 or Mn alone (Figure 2), suggesting that manganese synergizes with TLR7/8 pathways. PLGA microparticles were successfully co-loaded with 3M-052 and manganese, with an encapsulation efficiency of 97% for 3M-052 and 45% for manganese (Figure 3). Microspheres had a D₅₀ of 41.7 microns. BMDCs treated with 3M-052-Mn²⁺ PLGA MS showed sustained expression of proinflammatory markers for up to seven days. In vivo studies are ongoing to assess the therapeutic efficacy of a single dose intratumoral administration of 3M-052-Mn PLGA MS in reducing tumor growth and improving survival rates in murine tumor models as compared to single/multi-dose treatment of free 3M-052 + Mn²⁺.

Conclusion: Our findings show that 3M-052 combined with manganese can boost IFN-I response and can be successfully co-loaded into PLGA microparticles, thus further expanding our increasing list of “metalloimmunotherapies”. The success of this therapy in vivo could broaden clinical indications of TLR-targeted cancer immunotherapy and may provide a patient-centric solution that can promote patient comfort and adherence.

References: [1] Sun, X., Zhang, Y., Li, J., Park, K. S., Han, K., Zhou, X., Xu, Y., Nam, J., Xu, J., Shi, X., Wei, L., Lei, Y. L., & Moon, J. J. (2021). Amplifying STING activation by cyclic dinucleotide-manganese particles for local and systemic cancer metalloimmunotherapy. Nature Nanotechnology, 16(11), 1260–1270.
[2] Smirnov DV, Zhao G, Beaurline JM, Johnson KE, Capecchi JT, Harrison LI, Tomai MA, Elvecrog JM, Klinman DM, Vasilakos JP. Intratumoral immunotherapy with the TLR7/8 agonist 3M-052. J Immunother Cancer. 2013 Nov 7;1(Suppl 1):P138.

Acknowledgements: This work was supported in part by a Bristol Myers Squibb Doctoral Fellowship, NIH (R01DE030691, R01DE031951, R01CA271799, R01NS122536), and the Bioinnovations in Brain Cancer (BIBC) program at the University of Michigan.

Figure 1. 3M-052-Mn2+ PLGA MS Proposed Mechanism of Action

Figure 2. 3M-052 + Mn2+ Combination Can Amplify IFN-I & Proinflammatory Response

Figure 3. 3M-052-Mn2+ PLGA MS Characterization