Combining Synthetic Biology and Nanoparticle Engineering to Selectively Deplete Mast Cells

Introduction:: Mast cells (MCs) are granulocytes of the innate immune system and are primary drivers of allergies. In addition to these associations, various malignant diseases of MCs can occur, such as aggressive systemic mastocytosis. A major unmet need exists for drugs that selectively reduce MC populations. One of the first drugs approved to treat systemic mastocytosis is Midostaurin. This drug is a multiple tyrosine kinase inhibitor (TKI) that disrupts MC proliferation, but it also causes side effects such as severe thrombocytopenia due to non-specific interactions with a broad range of leukocytes. Sialic acid-binding immunoglobulin-type receptor 6 (Siglec-6) is selectively expressed on MCs and has inhibitory and endocytic functions. Synthetically dimerized Fab (sdFab) fragments are antibody variable region fragments that have been synthesized using cell-free biochemistry techniques. This method of fabrication allows for the customization of protein components and incorporation of non-canonical amino acids to allow for easier chemical conjugation. We have previously demonstrated the use of poly(ethylene glycol)-block-poly(propylene sulfide) (PEG-bl-PPS) nanocarriers across an array of applications designed to improve drug delivery and selectivity. Thus, we hypothesized that anti-Siglec-6 sdFab conjugated to PEG-bl-PPS micelles loaded with midostaurin could increase selective depletion of mast cells in mice.


Materials and Methods:: PEG₄₅-bl-PPS₂₀ was dissolved in tetrahydrofuran with 2 wt% DSPE-PEG2K-DBCO and 5 wt% midostaurin and allowed to dry into a thin film in a class scintillation vial. After vacuum desiccation, the polymer-drug film was rehydrated with milliQ water and placed on a shaker until the solution was clear. Synthesis of anti-Siglec-6 sdFabs containing an azido reactive group was accomplished using cell-free lysate from an engineered E. coli strain and a plasmid sequence for the protein. After purification, the azido-sdFabs were reacted with DBCO on micelles at a ratio of at least 4:1, overnight at 4°C in PBS, pH 7.5. Particles were purified using spin filtration and physically characterized using dynamic light scattering and cryo-TEM. Quantification of sdFab loading and drug loading was done using a combination of UV-vis spectroscopy and fluorescence spectroscopy with fluorescently labeled DBCO or azide.

Results, Conclusions, and Discussions:: Anti-Siglec-6/midostaurin micelles showed potent cytotoxic and antiproliferative effects on malignant MCs in vitro. However more importantly, these particles demonstrated selective depletion of mast cells and decreased off-target toxicity compared to free drug in a humanized mouse model of systemic mastocytosis and in transgenic Siglec-6 mast cell knock-in mice. Notably, in KI mice, we saw a reduction to roughly 0.5% mast cells remaining per 100,000 cells in peritoneal lavage when treated with Fab-drug micelles, vs around 10% in PBS controls, whereas free drug and other controls (Fab-micelles and drug-micelles) had about 4% remaining mast cells, a reduction of only 5-6% at most. This highlights a potent targeted delivery of midostaurin to mast cells in vivo.


PEG-bl-PPS micelles are multifaceted tools able to potently regulate MC populations through receptor-mediated drug delivery. The enhancement of drug activity against mast cells indicates that this method could be applied to improve existing mast cell therapies, and more broadly the generation of sdFabs could be used to make more selective therapies for different diseases.




Acknowledgements (Optional): : Funded by NIAID (NIH grant no. 1R21AI159586-02)

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