Session Type: Abstract Submissions (ACR)
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). In rheumatoid arthritis, blocking of IL-6R results in clinical benefit as demonstrated by the 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 safety profile compared to equivalent inhibition of both IL-6R forms (Waetzig & Rose-John, Expert Opin Ther Targets, 2012). 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 strong target binding using a single anti-IL-6R building block. 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.
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. The affinity of ALX‑0061 for sIL‑6R could not be accurately determined via surface plasmon resonance due to its very tight target binding. Consequently, the more sensitive GyrolabTM platform was used to assess affinity for both receptors. The KDfor mIL-6R was determined after pre-incubation of mIL-6R-transfected cells with constant compound concentrations and subsequent quantification of free compound in the supernatant.
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 (0.19 ± 0.08 pM) was about 2400-fold superior compared to TCZ (462 ± 138 pM). In the mIL-6R-driven cell-based assay, however, in vitropotencies 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 (462 ± 138 pM) compared to sIL-6R (154 ± 16 pM), while the affinity of ALX-0061 was about 50-fold lower for mIL-6R compared to sIL-6R (9.1 ± 3.6 pM).
ALX-0061 demonstrates in vitro a preferential binding 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 improved therapeutic efficacy with a better safety profile compared to TCZ.
M. Van Roy,
A. Van De Sompel,
K. De Smet,
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ACR Meeting Abstracts - https://acrabstracts.org/abstract/alx-0061-an-anti-il-6r-nanobody-for-use-in-rheumatoid-arthritis-demonstrates-a-different-in-vitro-profile-as-compared-to-tocilizumab/