Date: Sunday, November 8, 2020
Session Type: Abstract Session
Session Time: 10:00AM-10:50AM
Background/Purpose: 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.1 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. Attachment efficiency of 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 nM-4 nM. In vitro, the aptamer-nanoparticles dramatically decreased Ly6C expression in mouse bone marrow cells analyzed by FACS. 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). The aptamer-nanoparticle conjugate, significantly reduced IL-6 and TNFα levels in the mouse sera (p< 0.01). Importantly, this effect was confirmed in the serum transfer arthritis model (N=10). The effects was not inferior compared with 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 accumulation of the signal 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.
To cite this abstract in AMA style:Khatri S, Hansen J, Clausen M, Kragstrup T, Hung S, Mellins E, Astakhova K. A First in Class Therapeutic Nanoparticle for Specific Targeting of Anti-citrullinated Protein Antibody Ameliorates Serum Transfer and Collagen Induced Arthritis [abstract]. Arthritis Rheumatol. 2020; 72 (suppl 10). https://acrabstracts.org/abstract/a-first-in-class-therapeutic-nanoparticle-for-specific-targeting-of-anti-citrullinated-protein-antibody-ameliorates-serum-transfer-and-collagen-induced-arthritis/. Accessed January 28, 2022.
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ACR Meeting Abstracts - https://acrabstracts.org/abstract/a-first-in-class-therapeutic-nanoparticle-for-specific-targeting-of-anti-citrullinated-protein-antibody-ameliorates-serum-transfer-and-collagen-induced-arthritis/