(I-328) Depletion of Tuberculosis-Associated Myeloid-Derived Suppressor Cells with Synthetic Nanoparticle Antibodies (SNAbs)

Myeloid-derived suppressor cells (MDSCs) play a crucial role in the regulation of immune responses. They are known to accumulate in various pathological conditions such as cancer, autoimmune disorders, and infectious diseases.¹ In particular, MDSCs have been implicated in causing immunosuppression during tuberculosis (TB) infection.² Therefore, targeting and depleting MDSCs could be an effective strategy to improve immune responses against TB. We have developed synthetic nanoparticle antibodies (SNAbs) for the targeted depletion of MDSCs by exploiting the overexpression of multiple MDSC markers that are upregulated during TB. Our approach involved the use of solid-phase synthesis to create bifunctional Janus nanoparticles with anisotropic surface chemistry, allowing for spatially separated conjugation of two different ligands.³ The SNAbs were designed to bind to multiple MDSC markers, including S100A8/A9, CD124, and CD206, which are highly expressed on MDSCs during TB. SNAbs pair MDSCs with immune effector cells, triggering MDSC killing. In this work, we aim to evaluate SNAbs that bind three different MDSC receptors for their ability to deplete MDSCs in vitro.

All procedures were approved by the Georgia Tech and Emory IACUCs. SNAbs were fabricated using solid-phase synthesis by modification of our previously published work.³ Specifically, we hydrated an aminomethyl resin and reacted it with a reducible crosslinker (sulfo-NHS-SS-biotin) to bind streptavidin-coated NPs (30-50nm). The crosslinker's disulfide bonds were cleaved with tris(2-carboxyethyl) phosphine (TCEP), releasing Janus NPs with anisotropic surface chemistry, one “face” having free streptavidin for biotin binding and the other with free thiols for maleimide reaction, providing spatially separated conjugation of two different ligands. The domains were functionalized with biotinylated MDSC-targeting ligands^4,5,6 and maleimide-terminated Fc-mimicking ligands⁷.

Bone marrow was isolated from C57Bl/6 mice and seeded onto 100mm petri dishes at 1x10⁷ cells/plate.⁸ To enrich for MDSCs, cells were supplemented with RPMI 1640 media containing GMCSF and IL6 for 4 days. For macrophages, cells were enriched with DMEM F12 media containing MCSF and GMCSF for 7 days. The bone marrow-derived MDSCs and macrophages were harvested and cocultured in a 24-well-plate at a 1:1 ratio. They were then incubated for 24 hours, with each well receiving a distinct type of SNAb or PBS as a control. Following treatment, cells were harvested and stained for flow cytometry. MDSC populations (CD11b⁺Ly6G⁺/Ly6C⁺) were analyzed for depletion.

Our results demonstrate the effective targeting and depletion of MDSCs by three different types of SNAbs in vitro. The comprehensive depletion across monocytic and granulocytic subpopulations of MDSCs highlights the potency and specificity of SNAbs in modulating MDSC activity. These findings suggest that SNAbs could be a promising immunotherapeutic strategy for TB by modulating MDSC activity and improving immune responses against the pathogen.

To further explore the potential of these novel SNAbs, we plan to conduct in vivo experiments using a mouse model of TB. We will monitor biodistribution, assess cytotoxicity, and evaluate the impact on lung bacterial burden. Additionally, we will investigate the effects of SNAb treatment on the innate and adaptive immune landscapes within this mouse model. Understanding the molecular pathways involved in the interaction between SNAbs and MDSCs will enable the development of more refined and tailored immunotherapeutic approaches to combat TB. These studies will establish a platform to investigate MDSCs as a target for host-directed therapies that improve TB treatment efficacy.

This project is supported by NIH Grant No. R01AI155023, and the Robert A. Milton Endowed Chair funds to KR.

1. Gabrilovich, D. I. Myeloid-Derived Suppressor Cells. doi:10.1158/2326-6066.CIR-16-0297.


2. Knaul, J. K. et al. Lung-residing myeloid-derived suppressors display dual functionality in murine pulmonary tuberculosis. Am J Respir Crit Care Med 190, 1053–1066 (2014).


3. Liu, J. et al. Bifunctional Janus Particles as Multivalent Synthetic Nanoparticle Antibodies (SNAbs) for Selective Depletion of Target Cells. Nano Lett 21, 875–886 (2021).


4. Qin, H. et al. Generation of a new therapeutic peptide that depletes myeloid-derived suppressor cells in tumor-bearing mice. Nat Med 20, 676–681 (2014).


5. Vadevoo, S. M. P. et al. IL4 Receptor–Targeted Proapoptotic Peptide Blocks Tumor Growth and Metastasis by Enhancing Antitumor Immunity. Mol Cancer Ther 16, 2803–2816 (2017).


6. Ghebremedhin, A. et al. A Novel CD206 Targeting Peptide Inhibits Bleomycin Induced Pulmonary Fibrosis in Mice Title: CD206 targeted peptide reduces lung fibrosis. doi:10.1101/2020.07.27.218115.


7. McEnaney, P. J. et al. Chemically Synthesized Molecules with the Targeting and Effector Functions of Antibodies. J Am Chem Soc 136, 18034–18043 (2014).


8. Bailey, J. D. et al. Isolation and culture of murine bone marrow-derived macrophages for nitric oxide and redox biology. Nitric Oxide 100–101, 17 (2020).