FRI0011 Targeting neutrophil microvesicles to damaged cartilage using antibodies to post translationally modified collagen ii

Background Microvesicles (MV) are extracellular vesicles released from the plasma membrane of cells. MV derived from neutrophils (PMN) have been shown to penetrate cartilage and exert chondro-protective effects in inflammatory arthritis 1–2 . Collagen type II (CII) is the most abundant protein found in cartilage. Inflammation in the joint results in post-translational modification of collagen II (CII) by reactive oxygen species (ROS). We have produced a single chain variable fragment (scFv) antibody specific to CII modified by ROS, namely anti-ROS-CII 3 . We hypothesise that loading anti-ROS-CII upon MV will localise them to the arthritic cartilage. Objectives The main objectives of this study were: i) Load anti-ROS-CII upon MV using aqueous energy dissemination; ii) Assess incorporation of anti-ROS-CII upon MV in vitro and iii) Validate the localisation and therapeutic potential of the enriched microvesicles in an in vivo model of arthritis. Methods Anti-ROS-CII were produced in house. Human PMN MV from healthy donors were isolated as previously described 4 . Anti-ROS-CII were intercalated upon MV using 100 ug 1,2-dioleoyl- sn- glycero-3-phospho-l-serine by aqueous energy dissemination, as previously described 4–5 . Incorporation was assessed by Imagestream X (IS x ), ELISA, NanoSight and immunofluorescence. In vivo experiments were performed in an Antigen Induced Arthritis (AIA) mouse model. Localisationwas assessed by IVIS Imaging following intravenous treatment of enriched MV. Ex vivo IVIS imaging was performed to confirm in vivo data. In vivo experiments were also conducted using MV enriched with anti-ROS-CII fused to vIL-10 and anti-mTNF therapies. Clinical scores were assessed throughout. Knee joints were harvested and snap frozen to detect the presence of enriched MV in the arthritic cartilage by confocal microscopy, and for qPCR analysis. Results Incorporation of anti-ROS-CII upon MV was observed by Imagestream. Antibodies present on the MV retained their binding capabilities, as shown by ELISA. Strong microvesicle fluorescence was detected in the arthritic cartilage of mouse tissue sections when using anti-ROS-CII enriched MV as a primary incubation. In vivo studies exhibited the ability of the enriched MV to localise specifically in the arthritic joint. Ex vivo IVIS knee imaging confirmed in vivo results. Ex vivo knee cryosections showed the presence of enriched MV specifically within the arthritic cartilage. In vivo treatment studies using anti-ROS-CII fused to anti-inflammatory therapeutics led to a significant reduction in knee swelling. Conclusions In this study we have demonstrated the ability to use aqueous energy dissemination to successfully enrich MV with antibodies. These enriched MV are able to localise in the arthritic joint and deliver anti-inflammatory therapeutics. Overall, this study demonstrates the attainability of targeting a biological scaffold to the arthritic joint. The potential of co-delivering MV alongside anti-inflammatory therapeutics is paramount to simultaneously protect cartilage and reduce inflammation. References [1] Dalli J, et al. Blood2008;112:2512–9. [2] Headland SE, et al. Science Translational Medicine 2015;7(315). [3] Hughes C, et al. Arthritis and Rheumatism2010;62(4):1007–1016. [4] Norling LV, et al. The Journal of Immunology 2011;186(10):5543–5547. [5] Dalli J, et al. EMBO Molecular Medicine2014;6(1):27–42. Disclosure of Interest None declared