Despite the success of current biological agents, achievement of broader efficacy and improved safety profile remains an unmet need in rheumatoid arthritis therapy. Neovasculogenesis plays a vital role in the progression and perpetuation of rheumatoid arthritis and significant evidence has demonstrated molecular heterogeneity within the endothelium (MVE) of different tissues. The heterogeneity of the synovial MVE can be exploited for the development of organ-specific therapeutic and diagnostic reagents. A novel recombinant antibody fragment, scFv A7, with specificity for human arthritic synovium, was isolated in our laboratory following in vivo phage display. The aim of the project described in this thesis is to characterise the antibody reactivity and develop a novel tissue specific therapeutic. The scFv A7 antibody proved to specifically target the microvasculature of human arthritic synovium with no detectable reactivity in a comprehensive range of normal tissues. Furthermore, the detected reactivity was not a common feature of chronic inflammatory conditions. Hence, the A7 antibody represents a unique and versatile tool with great potential for the development of diagnostic and/or therapeutic agents. The unique properties of A7 were combined with the anti-TNF Adalimumab, forming a bispecific antibody with neutralising activity and synovial homing properties. The new construct was able to retain the synovial specificity and showed comparable TNF binding kinetics and biological activity to the parent Adalimumab antibody in vitro. In conclusion, these results demonstrate that scFv A7 reactivity is specific to the microvasculature of human arthritic synovium, suggesting that the target molecule may have potential as a biomarker in arthritis and applications as an immunotherapeutic target. The bispecific antibody format developed showed unaltered TNF blocking capacity and synovial specificity that may allow reduction in the dosage and/or administration frequency, with the ultimate goal to reduce the systemic exposure, achieve a better therapeutic index and decreasing health care costs.

The ubiquitous ɣ-herpesvirus Epstein-Barr virus (EBV) infects B cells and modifies their differentiation programme leading to B cell activation and immortalization. Although different evidences support a link between EBV infection and rheumatoid arthritis (RA) and Sjogren’s syndrome (SS), the exact role of EBV in RA and SS pathogenesis remain elusive. Recently ectopic lymphoid structures (ELS) have been identified as preferential niches for EBV persistence and reactivation in patients with multiple sclerosis and myasthenia gravis. Independent studies demonstrated that around 50% of RA synovia and 30% of SS salivary glands are characterised by the development of functional ELS, capable to promote local differentiation of autoreactive plasma cells. In this PhD project I explored the potential role of EBV in RA and SS pathogenesis by analysing EBV infection in the RA synovium and SS salivary glands and its relationship with ELS, in situ autoreactive plasma cell differentiation, pathogenic autoantibodies production and cytotoxic immune response. In this work I demonstrated that: i) markers of EBV latent and lytic infection are consistently associated with the presence of ELS in the RA synovium and SS salivary glands; ii) latent EBV proteins are preferentially expressed by B cells, while viral reactivation occurs in plasma cells; iii) a large subset of autoreactive plasma cells is EBV lytically infected in the RA synovia and SS salivary glands; iv) antibodies specific for unmodified and citrullinated EBV peptides, known to cross-recognize ACPA, are produced within ectopic lymphoid structures as 8 demonstrated in vivo in human RA/SCID chimeras; v) SS salivary gland grafts transplanted into SCID mice release human IgG against EBV antigens, whose production correlates with the level of SS-associated auto-antibodies and vi) analysis of CD8+ and CD4+ T-cell localization and granzyme B expression indicated that EBV persistence in ELS-containing RA synovia and SS salivary glands may be favoured by exclusion of CD8+ T cells from B-cell follicles and impaired CD8-mediated cytotoxicity. Overall, these results redefine a novel and pathogenically relevant role for EBV in B-cell dysregulation and chronic inflammation in RA synovium and SS salivary glands.