Session Type: Abstract Submissions (ACR)
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 endothelium of different tissues, conferring organ tropism to migrating lymphocytes subsets by direct interaction with specific homing receptors. We have previously reported the isolation of a single chain Fv (scFv A7) antibody with specificity for synovial arthritic microvasculature, following an in vivo phage display selection on SCID mice grafted with human arthritic synovium¹. The aim of the present study was to develop the scFv A7 as a tissue specific therapeutic in arthritic disease conditions.
In order to obtain a bivalent molecule with increased avidity and serum half-life, the scFvA7 was subcloned in fusion with the CH2-CH3 domain of the human IgG (scFv A7-Fc). In addition, scFv A7-Fc was coupled with the scFv-Fc of the Adalimumab anti-TNF antibody, via a monocistronic RNA approach, to form a bispecifc antibody (BsAb) using the Knobs-into-Holes technology² (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 TNF-ELISA and TNF cytotoxicity assay on L-929 cell line.
The scFv-Fc fusion protein of Adalimumab showed analogous anti-TNF properties with the parent antibody, EC50 0.008nM (Adalimumab 0.006nM), proving the validity of the scFv format. The BsAb A7/Adalimumab achieved a high degree of efficient heterodimerisation showing only 3% Adalimumab homodimer and was able to selectively bind TNFα in vitro with similar efficacy to the TNF blocker with a 0.01nM EC50. Despite the monovalency for the anti-TNF activity, the BsAb showed a dose dependent rescue of TNF induced cytotoxicity in L-929 cell line comparable to Adalimumab with a 0.4nM IC50 (Adalimumab 0.16nM), demonstrating the biological functionality of the construct. In addition, the A7/Adalimumab antibody proved to efficiently and specifically target the stromal compartment of human arthritic synovial microvasculature, maintaining unaltered the organ tropism of the original scFv A7.
Our results demonstrate that the reactivity of scFv A7 is specific to the microvasculature of human arthritic synovium. This specific reactivity suggests that the target molecule for scFv A7 may have potential as a biomarker in arthritis and also have 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 and achieve a better therapeutic index and decreasing health care costs.
1. Kamperidis P, Kamalati T et al. 2011. Arthritis Rheum. 63:3758-67.
2. Ridgway JBB et al. 1996. Protein Engineering. 9(7):617-621.
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ACR Meeting Abstracts - https://acrabstracts.org/abstract/development-of-a-novel-bispecific-therapeutic-for-arthritic-diseases/