Intranasal delivery of immunotherapies enhances survival in Lymphangioleiomyomatosis

Introduction: Lymphangioleiomyomatosis (LAM) is a rare disease occurring in women of reproductive age that is characterized by development of pulmonary cysts and eventual respiratory failure.1 The only FDA-approved treatment for LAM, rapamycin, is stabilizing but not curative, and many patients cannot tolerate life-long use.2–4 Thus there is a need to develop new LAM treatments. Recent work identified immunosuppression in LAM,5,6 and checkpoint inhibition showed improved survival in mouse models.6,7 Our current work investigates immune activation through administration of toll-like receptor (TLR) agonists. We have observed significant improvement in disease outcomes following intranasal administration of TLR9 agonist CpG (Fig. 1).7 Given prior success with checkpoint inhibition and existing clinical treatment of LAM with rapamycin, we here investigated combination therapy of rapamycin and α-PD1 checkpoint inhibition with CpG.
Research has also shown that targeting adjuvant immunotherapies to the lymph nodes (LNs) improves their effectiveness. The lung draining LNs emerge as a prime target for CpG treatment in LAM, as lung lymph nodes contain antigens from LAM cells and thus adaptive immune cells specific to LAM can be activated in the LNs.8 LN targeting can be achieved by delivering nanoparticles (NPs) 10-250nm in size to the lymphatic vasculature that transports materials from peripheral tissues to the lymph nodes.9 Our prior work has investigated NP surface chemistry to maximize targeting the skin LNs via lymphatic vessels. Here we investigated targeted nanoparticle delivery to the lung LNs via lymphatic vessels to develop a novel nanoparticle-based CpG treatment platform for improving LAM outcomes.

Materials and

Methods: CpG administration studies were performed on C57Bl/6J mouse LAM model, wherein LAM tumor formation is induced via IV administration of TTJ cells. 5μg doses of CpG are administered intranasally twice weekly for duration of survival study. Co-administration of either rapamycin or α-PD1 was performed simultaneously with CpG administrations. In addition to evaluating survival, histology was performed on tissue to evaluate effects on physiological structure, as well as lung tissue digestion and flow cytometry to identify relevant immune cells present. For nanoparticle work, surface chemistry of model polystyrene NPs was modified to investigate the properties to optimize lung LN targeting. Specifically, PLGA NPs with low (Rf/D < 2) and high (Rf/D > 4) density polyethylene glycol (PEG) coatings were evaluated. PEG density was evaluated via zeta potential of NPs, with more negative zeta potential corresponding to less PEG surface coverage (ζ = -10mV very low density, ζ > -5mV high density). For biodegradable nanoparticle work, we formulated PLGA-PEG nanoparticles using nanoprecipitation to yield high and mid-low density PEG coatings. We assessed nanoparticle transport across lymphatics in an in vitro transport model for biodegradable nanoparticles. Model nanoparticles were then either administered intradermally or intranasally/intratracheally in C57Bl/6J mice. Quantification of nanoparticle LN accumulation was performed via imaging using an in-vitro imaging system (IVIS) to visualize nanoparticle accumulation via fluorescence followed by tissue digestion and quantification of nanoparticle mass in LNs via fluorescence reading on a microplate reader.

Results, Conclusions, and Discussions: CpG combination therapy performed with 5ug CpG and rapamycin showed significant improvement in survival and decrease in LAM nodule count when compared to the untreated group and the groups treated with rapamycin and CpG alone (Fig 2A). A similar trend was observed with combination α-PD1 CpG therapy, wherein the combination therapy had significantly improved survival outcomes when compared to the untreated group and the groups treated with α-PD1 or CpG alone (Fig. 2B). These results indicate that the therapeutics have additive effects, improving disease outcomes overall. However, significant inflammation was observed in mice administered CpG. When compared to untreated mice, those treated with 10 ug CpG had high T-cell count comparable to the untreated group (Fig. 3), but also showed decreases in LAM nodule count compared 5ug CpG (Fig. 1), suggesting that higher doses of CpG reduce disease burden but cause significant side effects that reduce survival.
In our efforts to develop a biodegradable CpG platform, we found that nanoparticles with high density PEG coatings (5:10 PGA-PEG: PLGA) maximized transport across lymphatics in vitro and we expect that densely PEG coated nanoparticles will also accumulate to greater extent in the lymph nodes compared to those with little or no PEGylation (Fig. 4).

Conclusions: This work identified CpG as a LAM immunotherapy with significant treatment potential, as it was shown to significantly improve disease outcomes. CpG has increased effectiveness when combined with the current standard of care, rapamycin, or checkpoint inhibitor αPD1. This significant potential, however, is hindered by the side effects of the treatment, in that free CpG was found to be highly inflammatory. In order to reduce these side effects, we are developing a LN targeted nanoparticle-based CpG treatment that is likely to both increase CpG efficacy and reduce unwanted side effects. We identified dense PEGylation as a driving factor for passive LN targeting via lymphatic vessels. This work represents significant steps towards developing LAM immunotherapies, identifying CpG as LAM treatment with significant effectiveness, as well as beginning the work required to develop a targeted NP delivery system for CpG delivery to the LNs.

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