Reprogramming tumor microenvironment via systemic delivery of TLR3 agonist/Mn2+coordination nanomedicine

Introduction:: Toll-like receptor (TLR) agonists play role in activation of innate immunity as well as in initiation of adaptive immune responses. Recent studies have demonstrated a potential strategy to exploit various TLR agonists as cancer immunotherapy adjuvants for their ability to orchestrate anti-tumor immunity and promote anti-tumor immune responses. However, numerous immunosuppressive circuits in the tumor microenvironment limits the efficacy of TLR agonists as monotherapy. Moreover, poor pharmacokinetic properties of small molecule TLR agonists and suboptimal delivery to the targets are critical hurdles to overcome in the development of effective therapies against solid cancers.

Previously, we have reported that Manganese ion (Mn2⁺) can potentiate the stimulator of interferon genes (STING) pathway and boost type I interferon (IFN) production when co-delivered through cyclic dinucleotide (CDN) containing lipid nanoparticle [1]. Here, we have screened several TLR agonists both in vitro and in vivo and report our discovery that Mn2⁺ synergizes with TLR3 agonist, low-molecular weight poly(inosinic:cytidylic acid) (LMW-polyIC), leading to potent anti-tumor immune activities. We further developed new lipid nanoparticles loaded with LMW-polyIC and Mn2⁺ via metal coordination for systemic administration. LMW-polyIC/Mn²⁺-loaded lipid nanoparticles (PLCMP) administered intravenously elicited strong anti-tumor immune responses and improved the therapeutic efficacy of immune-checkpoint blockaders (ICB) with minimal adverse events.




Materials and Methods:: In vitro evaluation of TLR agonists plus Mn²⁺ were conducted by measuring the level of cytokines (IFN-𝛽, IL-6, TNF-𝛼, CXCL-9, and CXCL-10) released from mouse bone marrow-derived dendritic cells (BMDCs). PLCMP was prepared by quickly mixing the aqueous LMW-polyIC phase and lipid solvent phase. After thorough mixing of two phases, MnCl₂ was added to the mixture, dialyzed, followed by ultrafiltration. Drug loading was quantified by UV-absorbance. Size and zeta potential of PLCMP were measured by Zetasizer. In vivo anti-tumor efficacy of PLCMP were evaluated in CT26, B16F10, or Nooc1 tumor bearing mice. PLCMP were administered intravenously were administered every three days, for a total of four injections. The percentages of tumor antigen-specific CD8⁺ T cells among PBMCs were analyzed using the tetramer (AH1 peptide-MHC tetramer, NIH) staining assay. Tumor and spleen tissues were collected and processed after the treatment for phenotypic and functional assessments of T cells and myeloid cells.




Results, Conclusions, and Discussions:: Results

Among various TLR agonists, LMW-polyIC demonstrated the most potent synergistic efficacy with Mn²⁺ in inducing pro-inflammatory cytokines and generating anti-tumor activities both in vitro and in vivo. LMW-polyIC and Mn²⁺ coordinated their self-assembly and were successfully loaded into the core of lipid nanoparticles. The resulting PLCMP exhibited average hydrodynamic diameter of 35 to 59 nm, negative surface charges, and drug loading efficiency of 75 to 80%. The therapeutic effect of systemically delivered PLCMP was evaluated in CT26 or B16F10 tumor-bearing mice. PLCMP monotherapy resulted in significant inhibition of tumor growth, and combination with anti-PD-1 therapy achieved 50% of complete response rate (Figure 1). Tetramer staining using PBMCs showed that tumor antigen-specific CD8 T cells were significantly upregulated in the PLCMP treatment groups. Tumor microenvironment analysis revealedthat systemic delivery of PLCMP significantly promoted M2-to-M1 repolarization of intratumoral macrophages and induced robust maturation of dendritic cells. Lastly, safetyof intravenously administered PLCMP was validated from the hematological assessment.



Discussion and Conclusion

We have developed a systemically applicable self-assembled coordination nanomedicinebased on Mn²⁺ and a TLR3 agonist, LMW-polyIC and. The co-delivery of LMW-polyIC and Mn²⁺ achieved a potent synergism in augmenting pro-inflammatory immune responses and transforming immunosuppressive tumor milieu into more immune favorable environment for enhanced ICB therapy. Based on the robust anti-tumor efficacy with minimal adverse events observed with PLCMP therapy, we surmise that our strategy for metalloimmunotherapy will broaden the applications of TLR agonists in cancer immunotherapy.


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References (Optional): :
1. Sun, X., Zhang, Y., Li, J. et al. Amplifying STING activation by cyclic dinucleotide–manganese particles for local and systemic cancer metalloimmunotherapy. Nat. Nanotechnol. 16, 1260–1270 (2021).