FRI0011 Targeting neutrophil microvesicles to damaged cartilage using antibodies to post translationally modified collagen ii
Authors: LM Topping, H Rhys, L Norling, A Nissim
Journal: Annals of the Rheumatic Diseases (2017)

<h3>Background</h3> Microvesicles (MV) are double membrane-bound extracellular vesicles released from the plasma membrane of cells. MV derived from polymorphonuclear neutrophils (PMN) promote tissue protection, and have been demonstrated to penetrate cartilage during inflammatory arthritis and provide protection to the tissue <sup>1</sup>. Collagen type II (CII) is the most abundant protein found in cartilage. We have produced a single chain variable fragment (scFv) antibodies specific to CII modified by reactive oxygen species (ROS), namely anti-ROS-CII<sub>scFv</sub>. Previously, we have demonstrated the ability of anti-ROS-CII<sub>scFv</sub> to localise exclusively and deliver payload drugs to the arthritic joint in mice models of rheumatoid arthritis<sup>2</sup>. <h3>Objectives</h3> To test our hypothesis that anti-ROS-CII association with MV might i) target delivery of MV to inflamed joint and/or ii) enhance the avidity of the scFv (several scFv can be loaded in each MV) and may thus increase localisation and enhance therapeutic efficacy. <h3>Methods</h3> Cy5.5 labeled Anti-ROS-CII<sub>scFv</sub> were loaded on fluorescently labelled human PMN MV by aqueous energy dissemination using a sonic dismembrator <sup>3–4</sup>. Anti-ROS-CII<sub>scFv</sub> MV incorporation was confirmed by Imagestream<sup>X</sup> analysis. Anti-ROS-CII<sub>scFv</sub> MV were tested by ELISA to assess the retention of antibody binding capabilities. <h3>Results</h3> Positive incorporation of Anti-ROS-CII<sub>scFv</sub> upon MV was observed by flow cytometric analysis. ELISA demonstrated the ability of the anti-ROS-CII loaded MV to bind strongly to ROS-CII following incorporation into MV. <h3>Conclusions</h3> In this study, we have demonstrated a simple, efficient and cost effective way of antibody targeting that retains antibody function. Such technology has the potential to increase efficacy of existing therapies by ensuring specific targeting. Future <i>in vivo</i> studies will assess the ability of the Anti-ROS-CII<sub>scFv</sub> MV to localise to arthritic joints. <h3>References</h3> Headland, S. E. et al. Neutrophil-derived microvesicles enter cartilage and protect the joint in inflammatory arthritis. Science Translational Medicine, 7(315), (2015). Hughes, C. et al. Human single-chain variable fragment that specifically targets arthritic cartilage. Arthritis and Rheumatism 62(4), 1007–1016 (2010). Dalli, J. et al. Microparticle alpha-2-macroglobulin enhances pro-resolving responses and promotes survival in sepsis. EMBO Molecular Medicine 6(1), 27–42 (2014). Norling, L.V. et al. Cutting Edge: Humanized Nano-Proresolving Medicines Mimic Inflammation-Resolution and Enhance Wound Healing. The Journal of Immunology 186(10), 5543–5547 (2011). <h3>Acknowledgements</h3> The author would like to acknowledge Timothy Harrison and Amit Gupta for their contrabutions to preliminary results. The author would also like to acknowledge Dr Jesmond Dalli for his guidance on methodology. <h3>Disclosure of Interest</h3> None declared