Abstract
Upregulation of the expression of tumor necrosis factor (TNF-α, TNF) has a significant role in the development of autoimmune diseases. The fluorescent antibodies binding TNF may be used for personalized therapy of TNF-dependent diseases as a tool to predict the response to anti-TNF treatment. We generated recombinant fluorescent proteins consisting of the anti-TNF module based on the variable heavy chain (VHH) of camelid antibodies fused with the far-red fluorescent protein Katushka (Kat). Two types of anti-TNF VHH were developed: one (BTN-Kat) that was bound both human or mouse TNF, but did not neutralize their activity, and a second (ITN-Kat) that was binding and neutralizing human TNF. BTN-Kat does not interfere with TNF biological functions and can be used for whole-body imaging. ITN-Kat can be evaluated in humanized mice or in cells isolated from humanized mice. It is able to block human TNF (hTNF) activities both in vitro and in vivo and may be considered as a prototype of a theranostic agent for autoimmune diseases.
Highlights
Therapeutic neutralization of the inflammatory cytokines, in particular TNF, has revolutionized the treatment of autoimmune diseases including rheumatoid arthritis (RA), Crohn’s disease, spondyloarthritis (SpA), and others
Two genetically encoded fluorescent sensors based on two distinct TNF-binding modules and far-red protein Katushka (Figure 1A) were designed and successfully expressed in prokaryotic system (Result S1, Figure S2)
The small size and the absence of Fc-fragment in variable heavy chain (VHH) modules may reduce side effects caused by interactions with receptors of immunocompetent cells and with complement system, as it usually happens with classical antibodies [34]
Summary
Therapeutic neutralization of the inflammatory cytokines, in particular TNF, has revolutionized the treatment of autoimmune diseases including rheumatoid arthritis (RA), Crohn’s disease, spondyloarthritis (SpA), and others. Several attempts to monitor the disease activity and the localization of therapeutic antibodies in the inflamed joints using molecular imaging with radiolabeled monoclonal anti-TNF antibodies (certolizumab pegol, adalimumab, and infliximab) in rheumatic diseases were reported [6,7,8,9,10]. Real-time images could be captured in vivo using a fairly simple equipment and appropriate fluorescent proteins [11]. The use of far-red fluorescent proteins, such as Katushka [12], allows for in vivo imaging of fluorescence in the deep tissues.
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