Background:Rheumatoid Arthritis (RA) is a systemic autoimmune disease with a prevalence of 0.5-1% worldwide1. Anti-cytokine antibodies, especially anti-Tumor Necrosis Factor (TNF) antibodies are considered the gold standard for RA therapy. However, there are some concerns regarding their lack of therapeutic efficacy in a significant proportion of patients2 and their potential systemic implications such as the risk of serious infections3. Developing novel agents with synovial targeting specificity might help to increase the therapeutic index while reducing systemic side effects of RA therapeutics.Objectives:Our work aims to develop a novel bispecific tandem single-chain variable fragment (scFv)-Fc fusion protein combining synovium specificity with anti-TNFα activity. The potential advantage of this construct is a reduced systemic TNF-binding activity and increase delivery and activation of the TNF-neutralising capacity at the inflamed joints.Methods:The therapeutic tandem scFv-Fc fusion protein comprises two external arms with synovium specific targeting ability linked through a metalloproteinase (MMP) cleavable linker to the anti-TNFα variable fragments of Adalimumab fused to the CH2 and CH3 domains of IgG1 (Figure 1). The external scFv regions with synovium specificity were previously identified by in vivo phage display using a SCID mouse model transplanted with human synovium4. The construct was tested for its ability to bind and neutralize in vitro and ex vivo targets.Results:The fusion protein was tested by immunohistochemistry staining on RA synovium biopsies and an array of non-inflamed human tissues showing specific targeting of synovial microvasculature without no reactivity to the non-inflamed tissues. The TNFα binding and blocking capacity of the fusion protein was measured respectively by ELISA and cell assays measuring NF-κB activation, and it showed a two-fold decreased activity compared to the control antibody Adalimumab prior to detachment of the cleavable targeting fragment shielding the active anti-TNF fragment. Human synovial fluid and recombinant human MMP-1 efficiently cleaved the external arms of the antibody, releasing the anti-TNF scFv-Fc. The cleaved construct, detached from the synovium targeting arms, showed the same binding and anti-TNF inhibitory capacity/potency as Adalimumab.Conclusion:The novel bispecific tandem scFv-Fc demonstrated specific synovium targeting ability and intended reduced anti-TNF activity in its intact form prior to reaching the joint. Following MMPs-induced cleavage present in RA synovial fluid the therapeutic activity was restored to the same level as Adalimumab. Overall, this construct has the potential of decreasing the anti-TNF off-site activity and consequently, reduce systemic toxicity while maintaining high on-site activity. Also, the presence of a synovium targeting domain has the advantage of increasing the delivery and retention within the inflamed synovium and possibly increase the therapeutic index of this anti-TNF therapeutics.Figure 1.Schematic diagram of the bispecific tandem scFv-Fc fusion protein.References:[1]Silman, A. J. & Pearson, J. E. Epidemiology and genetics of rheumatoid arthritis. 265–272 (2002).[2]Balogh, E. et al. Comparison of remission criteria in a tumour necrosis factor inhibitor treated rheumatoid arthritis longitudinal cohort: patient global health is a confounder. Arthritis Res. Ther.15, R221 (2013).[3]Galloway, J. B. et al. Anti-TNF therapy is associated with an increased risk of serious infections in patients with rheumatoid arthritis especially in the first 6 months of treatment: updated results from the British Society for Rheumatology Biologics Register with special emp. Rheumatology (Oxford).50, 124–131 (2011).[4]Kamperidis, P. et al. Development of a novel recombinant biotherapeutic with applications in targeted therapy of human arthritis. Arthritis Rheum.63, 3758–3767 (2011).Disclosure of Interests:None declared
POS0206 DESIGN AND CHARACTERISATION OF A THERAPEUTIC BISPECIFIC TANDEM scFv-Fc FUSION PROTEIN WITH ANTI-TNF AND SYNOVIUM-TARGETING SPECIFICITY FOR THE TREATMENT OF RHEUMATOID ARTHRITIS
