FRI0021 Alx-0061, an anti-IL-6r nanobody® for therapeutic use in rheumatoid arthritis, demonstrates in vitro a differential biological activity profile as compared to tocilizumab
Authors: M. Van Roy, A. Van de Sompel, K. De Smet, J. Jacobs, T. Denayer, H. Ulrichts, J. Baumeister, J.-B. Holz
Journal: Annals of the Rheumatic Diseases (2013)

<h3>Background</h3> Interleukin-6 (IL-6) is a pleiotropic cytokine inducing a wide range of biological activities via its receptor, which can either be soluble (sIL-6R) or membrane-bound (mIL-6R). Blocking of IL-6R results in clinical benefit in rheumatoid arthritis as demonstrated by the marketed IL-6R inhibitor tocilizumab (TCZ). Signalling via the mIL-6R ("classical pathway") is confined to selected cell types due to the restricted expression of mIL-6R. However, IL-6 can also activate cells through sIL-6R in a process known as trans-signalling. Unwanted pharmacology associated with IL-6 pathway inhibition has been linked to inhibition of mIL-6R. Preferential inhibition of sIL-6R could therefore provide higher therapeutic efficacy with a better side effect profile compared to equivalent inhibition of both IL-6R forms (1). Nanobodies are therapeutic proteins based on the smallest functional fragments of heavy chain-only antibodies, naturally occurring in the <i>Camelidae</i> family. ALX-0061 is a bispecific anti-IL-6R Nanobody engineered to have an extended half-life <i>in vivo</i> by targeting human serum albumin (HSA), in combination with a high target affinity and potency using a single anti-IL-6R building block. <h3>Objectives</h3> ALX-0061 was extensively characterised using <i>in vitro</i> systems: biological activity and affinity for both sIL-6R and mIL-6R were assessed and compared to TCZ. <h3>Methods</h3> Biological activity of ALX-0061 and TCZ was analysed in a cell-based assay for mIL-6R, ELISA-based neutralisation assays for sIL-6R, and cell-binding and cell-signalling (mIL-6R) experiments in whole blood from human donors using flow cytometry. Due to very tight target binding, the affinity of ALX-0061 for sIL-6R could not be accurately determined via surface plasmon resonance. Consequently, the more sensitive Gyrolab<sup>TM</sup> platform was used to assess affinity for both receptors. For the K<sub>D</sub> determination on mIL-6R, free compound concentrations were measured in the supernatant, after pre-incubation of mIL-6R-transfected cells with a constant compound concentration. <h3>Results</h3> Flow cytometry experiments demonstrated that ALX-0061 binds to mIL-6R expressed on peripheral blood leukocyte populations with expected pharmacology. ALX-0061 specifically neutralised sIL-6R with a 10-fold higher <i>in vitro</i> potency compared to TCZ, while the (apparent) affinity of ALX-0061 for sIL-6R (0.20 pM) was 2000-fold superior compared to TCZ (462 pM). In the mIL-6R-driven cell-based assay, however, <i>in vitro</i> potencies were similar for ALX-0061 and TCZ, with the latter one showing avid binding due to its bivalency. In addition, TCZ showed a 3-fold higher affinity for mIL-6R (160 pM) compared to sIL-6R, while the affinity of ALX-0061 was 45-fold lower for mIL-6R (9 pM) compared to sIL-6R. <h3>Conclusions</h3> ALX-0061 demonstrates <i>in vitro</i> a preferential biological activity profile for sIL-6R with a lesser activity for mIL-6R, while TCZ has a higher preference for mIL-6R. Preferential inhibition of sIL-6R trans-signalling by ALX-0061 could provide superior therapeutic efficacy with a better side effect profile than TCZ. ALX-0061 is currently in clinical development. Analysis of a phase I/II study demonstrated a strong efficacy and an attractive safety profile. <h3>References</h3> Waetzig G.H. &amp; Rose-John S., Expert Opin Ther Targets (2012) 16(2) <h3>Disclosure of Interest</h3> None Declared