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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
  1. M. Van Roy1,
  2. A. Van de Sompel1,
  3. K. De Smet1,
  4. J. Jacobs1,
  5. T. Denayer1,
  6. H. Ulrichts1,
  7. J. Baumeister1,
  8. J.-B. Holz1
  1. 1Ablynx NV, Zwijnaarde, Belgium

Abstract

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.

References

  1. Waetzig G.H. & Rose-John S., Expert Opin Ther Targets (2012) 16(2)

Disclosure of Interest None Declared

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