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A1.54 Development of a novel bispecific therapeutic for rheumatoid arthritis
  1. Mathieu Ferrari1,
  2. Shimobi Onuoha1,
  3. Daniele Sblattero2,
  4. Costantino Pitzalis1
  1. 1WHRI, Queen Mary University of London
  2. 2University of Eastern Piedmont, Italy

Abstract

Background and Objectives Despite the obvious success of current biological agents for treatment of rheumatoid arthritis (RA), achievement of broader efficacy and improved safety profile remains an unmet need in RA therapy. There is significant evidence demonstrating molecular heterogeneity within the vascular endothelium of different tissues, conferring organ tropism to migrating lymphocytes subsets by interaction with specific homing receptors. We have previously reported the isolation of a single-chain Fv (scFvA7) antibody with specificity for synovial arthritic microvasculature, following an in vivo phage display selection on SCID mice model of RA. The aim of the present study was to develop a tissue specific therapeutic for Rheumatoid Arthritis.

Materials and Methods The scFvA7 was subcloned in fusion with the CH2-CH3 domain of human IgG (A7 scFv-Fc) and coupled with the anti-TNF antibody Adalimumab via a monocistronic RNA approach, to form a bispecifc antibody (BsAb) using the Knobs-into-Holes technology (A7/Adalimumab). The reactivity in frozen and paraffin embedded tissue sections was investigated using immunohistochemistry and immunofluorescence analysis. In-vitro functionality and biological activity was assessed in Biacore, TNF-ELISA and TNF cytotoxicity assay on L-929 cell line.

Results The scFv-Fc fusion protein of Adalimumab showed analogous anti-TNF properties with the parent antibody, EC50 0.04nM (Adalimumab 0.025nM), confirming the validity of the scFv format. The BsAb A7/Adalimumab achieved a high degree of efficient heterodimerisation showing only 3% Adalimumab homodimers and was able to selectively bind TNFα in vitro with similar efficacy to the TNF blocker, EC50 0.06nM, with a KD of 31pM. Despite the monovalency for the anti-TNF activity, the BsAb showed a dose dependent rescue of TNF induced cytotoxicity in L-929 cell line with a 0.5nM IC50 (Adalimumab 0.17nM), demonstrating the biological functionality of the construct. In addition, the A7/Adalimumab antibody proved to efficiently and specifically target the stromal compartment of RA synovial microvasculature, maintaining unaltered the organ tropism of the original scFvA7.

Conclusions Our results demonstrate the development of a bispecific antibody showing unaltered TNF blocking capacity and optimal synovial specificity, suggesting that the target molecule for A7 may have applications as a biomarker and immunotherapeutic target, potentially allowing reduction in the dosage and/or administration frequency, with the ultimate goal to reduce the systemic exposure and achieve a better therapeutic index and decreasing health care costs.

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