Background 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 Camelidae family. ALX-0061 is a bispecific anti-IL-6R Nanobody engineered to have an extended half-life in vivo by targeting human serum albumin (HSA), in combination with a high target affinity and potency using a single anti-IL-6R building block.
Objectives ALX-0061 was extensively characterised using in vitro systems: biological activity and affinity for both sIL-6R and mIL-6R were assessed and compared to TCZ.
Methods 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 GyrolabTM platform was used to assess affinity for both receptors. For the KD 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.
Results 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 in vitro 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, in vitro 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.
Conclusions ALX-0061 demonstrates in vitro 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.
Waetzig G.H. & Rose-John S., Expert Opin Ther Targets (2012) 16(2)
Disclosure of Interest None Declared