Background/Purpose: In the Phase 1/2 clinical study, E6011, a novel humanized anti-fractalkine (FKN) mAb demonstrated a promising efficacy in active RA patients who were inadequately controlled by MTX and/or TNF-α inhibitors. In preclinical study, anti-FKN mAb significantly lowered arthritis score of CIA model, and reduced infiltration of inflammatory cells and bone erosion in the synovium (Nanki et al., J Immunol 2004). Furthermore, anti-FKN mAb reduced the migration of adoptively transferred splenic monocytes/macrophages into the inflamed synovium. FKN is expressed on endothelial cells and fibroblast-like synoviocytes and also expressed on osteoblasts. CX3CR1, the receptor for FKN, is expressed on monocytes/macrophages and osteoclast precursors. Moreover, intravascular monocytes contribute vascular inflammation. However, the roles of FKN and CX3CR1 in the pathogenesis of RA, especially the behavior of intravascular monocytes, remain unclear. In this study, we evaluated the role of FKN-CX3CR1 axis on intravascular monocyte behavior in synovium of CIA model by intravital imaging and tissue-clearing techniques.

Methods: For the induction of CIA, mice were immunized with bovine type II collagen. The effect of anti-FKN mAb on synovium of MCP joint was examined by intravital imaging and three-dimensional (3D) tissue analysis with tissue-clearing technique. In intravital imaging, we established a system for MCP joint observation by two-photon laser scanning microscope (TPLSM), and the behavior of intravascular monocytes visualized by anti-CD115 mAb were compared in the presence or absence of anti-FKN mAb or control IgG. 3D tissue analysis was performed by tissue-clearing reagent LUCID (WO 2014115206 A1) and TPLSM. One hour after anti-FKN mAb or control IgG administration, the mice underwent to low-speed perfusion fixation so that the cells attached to the blood vessels did not detach. Before fixation, intravascular monocytes and blood vessels were visualized by anti-CD115 mAb and RCA-1, respectively. The number of intravascular monocytes were quantified. In order to clarify the difference of point of action with other biologics, the same experiment was carried out with TNF-α inhibitor or CTLA4-Ig administration.

Results: In intravital imaging, angiogenesis in the synovium of CIA occurred actively, and adhered and/or slow-paced monocytes appeared in the vasculature. At thirty minutes after anti-FKN mAb administration, the intravascular monocytes were reduced, but control IgG did not change compared to before administration. In tissue transparency analysis, the number of intravascular monocytes was markedly reduced by anti-FKN mAb as compared with control IgG. When the same experiments were performed with TNF-α inhibitor or CTLA4-Ig, intravascular monocytes were not reduced.

Conclusion: Intravital imaging and tissue-clearing techniques revealed that anti-FKN mAb dislodges intravascular monocytes in inflamed synovium. These results suggest that the FKN–CX3CR1 axis regulates intravascular monocyte behavior in inflamed synovium, such as enhancement of vascular inflammation and infiltration of osteoclast precursors. FKN–CX3CR1 blockade may be a novel attractive strategy for the treatment of RA.

« Back to 2018 ACR/ARHP Annual Meeting

ACR Meeting Abstracts - https://acrabstracts.org/abstract/anti-fractalkine-monoclonal-antibody-dislodges-intravascular-monocytes-involved-in-exacerbation-of-synovial-inflammation-in-collagen-induced-arthritis-model/