Background:Rheumatoid arthritis (RA) is an immune mediated inflammatory disease with autoimmune features, including antibodies to citrullinated proteins and peptides (ACPAs). Several in vitro studies have suggested a pathogenic role of ACPAs in RA. However, in vivo proof of this concept has been hampered by the lack of therapeutic strategies to reduce or deplete ACPA in serum and synovial fluid. Previously, we constructed a chitosan-hyaluronic acid nanoparticle formulation with the ability to use neutrophil recruitment as a delivery mechanism to inflamed joints. Specifically, nanoparticles got phagocytosed and then released to synovial fluid upon death of the short-lived neutrophilsObjectives:We hypothesized that reducing ACPA levels would have a therapeutic effect by blocking cytokine production. In this study, we prepared and tested a series of therapeutic nanoparticles for specific targeting of ACPA in synovial fluid.Methods:Nanoparticles were prepared by the microdroplet method and then decorated with synthetic cyclic citrullinated peptide aptamer PEP2, PEG/hexanoic acid and fluorophore (Cy5.5). Nanoparticles were characterized by dynamic light scattering (DLS), scanning electron microscopy (SEM) and high-performance liquid chromatography (HPLC). Nanoparticles were then used in a series of in vitro assays, including cell uptake with flow cytometry (FACS) detection, and in vivo studies including disease activity scores, cytokine measurements and near-infrared imaging.Results:We screened a series of citrullinated peptide epitopes and identified a fibrinogen-derived 21-amino-acid-long citrullinated peptide showing high selectivity toward autoantibodies in RA samples. We incorporated this aptamer in the chitosan-hyaluronic acid nanoparticle formulation previously described. Average nanoparticle size was 230 nm ± 10 nm by DLS and SEM; z potential was -0.0012. Purity by HPLC was over 95%. Attachment efficiency of the aptamer was 92% by HPLC. FACS study showed selective uptake of Cy5.5 labelled aptamer-nanoparticle conjugates by neutrophils in the concentration range 0.5-4 nM. Similar to previous studies,1there was no apparent immunogenicity for this nanoparticle formulation measured by cytokine secretion from human peripheral blood leukocytes. In vivo, over 50% reduction of disease activity was achieved in three weeks treatment using as little as 1 nM drug candidate (dosed every 48 hours) in the collagen-induced (CIA) mouse model of RA (N=30; p<0.001 for treated vs placebo). Same was observed in the serum transfer model (N=10). The aptamer-nanoparticle conjugate significantly reduced IL-6 and TNFα levels in the mouse sera (p<0.01). The effects were not inferior to tocilizumab treated controls (N=30). To confirm mode of action, we applied Cy5.5-labelled aptamer-nanoparticles in the collagen-induced mouse model (N=10) and analyzed the resulting uptake by near-infrared imaging. We confirmed over 6-fold higher signal accumulation in inflamed vs healthy joints (p<0.01), which strongly supports the fact that the aptamer is highly specific to the inflammatory process.Conclusion:Overall, we have designed a first-in-class therapeutic nanoparticle drug for specific targeting of anti-citrullinated protein antibodies. The marked effect of this nanoparticle observed in vivo holds promise for targeting ACPAs as a therapeutic option in RA.References:[1]Khatri S, Hansen J, Mendes AC, Chronakis IS, Hung S-C, Mellins ED, Astakhova K. Bioconjug Chem. 2019 Oct 16;30(10):2584–259Disclosure of Interests:Sangita Khatri: None declared, Jonas Hansen: None declared, Mads Hartvig Clausen Shareholder of: iBio Tech ApS, Tue Wenzel Kragstrup Shareholder of: iBio Tech ApS, Consultant of: Bristol-Myers Squibb, Speakers bureau: TWK has engaged in educational activities talking about immunology in rheumatic diseases receiving speaking fees from Pfizer, Bristol-Myers Squibb, Eli Lilly, Novartis, and UCB., Shu-Chen Hung: None declared, Elisabeth Mellins: None declared, Kira Astakhova: None declared
CalDAG-GEFI deficiency protects mice in a novel model of FcγRIIA-mediated thrombosis and thrombocytopenia
