title: The preclinical pharmacology of the high affinity anti-IL-6R Nanobody® ALX-0061 supports its clinical development in rheumatoid arthritis.
abstract: BACKGROUND
  The pleiotropic cytokine interleukin-6 (IL-6) plays an important role in the pathogenesis of different diseases, including rheumatoid arthritis (RA). ALX-0061 is a bispecific Nanobody® with a high affinity and potency for IL-6 receptor (IL-6R), combined with an extended half-life by targeting human serum albumin. We describe here the relevant aspects of its in vitro and in vivo pharmacology.
  
  
  METHODS
  ALX-0061 is composed of an affinity-matured IL-6R-targeting domain fused to an albumin-binding domain representing a minimized two-domain structure. A panel of different in vitro assays was used to characterize the biological activities of ALX-0061. The pharmacological properties of ALX-0061 were examined in cynomolgus monkeys, using plasma levels of total soluble (s)IL-6R as pharmacodynamic marker. Therapeutic effect was evaluated in a human IL-6-induced acute phase response model in the same species, and in a collagen-induced arthritis (CIA) model in rhesus monkeys, using tocilizumab as positive control.
  
  
  RESULTS
  ALX-0061 was designed to confer the desired pharmacological properties. A 200-fold increase of target affinity was obtained through affinity maturation of the parental domain. The high affinity for sIL-6R (0.19 pM) translated to a concentration-dependent and complete neutralization of sIL-6R in vitro. In cynomolgus monkeys, ALX-0061 showed a dose-dependent and complete inhibition of hIL-6-induced inflammatory parameters, including plasma levels of C-reactive protein (CRP), fibrinogen and platelets. An apparent plasma half-life of 6.6 days was observed after a single intravenous administration of 10 mg/kg ALX-0061 in cynomolgus monkeys, similar to the estimated expected half-life of serum albumin. ALX-0061 and tocilizumab demonstrated a marked decrease in serum CRP levels in a non-human primate CIA model. Clinical effect was confirmed in animals with active drug exposure throughout the study duration.
  
  
  CONCLUSIONS
  ALX-0061 represents a minimized bispecific biotherapeutic of 26 kDa, nearly six times smaller than monoclonal antibodies. High in vitro affinity and potency was demonstrated. Albumin binding as a half-life extension technology resulted in describable and expected pharmacokinetics. Strong IL-6R engagement was shown to translate to in vivo effect in non-human primates, demonstrated via biomarker deregulation as well as clinical effect. Presented results on preclinical pharmacological properties of ALX-0061 are supportive of clinical development in RA.
authors.affiliation: Ablynx NV, Technologiepark 21, 9052, Zwijnaarde, Belgium. maarten.vanroy@ablynx.com.
authors.forename: Maarten
authors.initials: M
authors.lastname: Van Roy
authors.affiliation: Ablynx NV, Technologiepark 21, 9052, Zwijnaarde, Belgium. Cedric.Ververken@ablynx.com.
authors.forename: Cedric
authors.initials: C
authors.lastname: Ververken
authors.affiliation: Ablynx NV, Technologiepark 21, 9052, Zwijnaarde, Belgium. beirnaerte@gmail.com.|VIB, Rijvisschestraat 120, 9052, Zwijnaarde, Belgium. beirnaerte@gmail.com.
authors.forename: Els
authors.initials: E
authors.lastname: Beirnaert
authors.affiliation: Ablynx NV, Technologiepark 21, 9052, Zwijnaarde, Belgium. Sven.Hoefman@ablynx.com.
authors.forename: Sven
authors.initials: S
authors.lastname: Hoefman
authors.affiliation: Ablynx NV, Technologiepark 21, 9052, Zwijnaarde, Belgium. JKolkman@its.jnj.com.|Crucell, Archimedesweg 4-6, 2333, CA, Leiden, The Netherlands. JKolkman@its.jnj.com.
authors.forename: Joost
authors.initials: J
authors.lastname: Kolkman
authors.affiliation: Department of Immunobiology, Biomedical Primate Research Centre, Lange Kleiweg 161, 2288 GJ, Rijswijk, The Netherlands. vierboom@bprc.nl.
authors.forename: Michel
authors.initials: M
authors.lastname: Vierboom
authors.affiliation: Department of Immunobiology, Biomedical Primate Research Centre, Lange Kleiweg 161, 2288 GJ, Rijswijk, The Netherlands. breedveld@bprc.nl.
authors.forename: Elia
authors.initials: E
authors.lastname: Breedveld
authors.affiliation: Department of Immunobiology, Biomedical Primate Research Centre, Lange Kleiweg 161, 2288 GJ, Rijswijk, The Netherlands. hart@bprc.nl.|Department of Neuroscience, University of Groningen, University Medical Center, Hanzeplein 1, 9700 RB, Groningen, The Netherlands. hart@bprc.nl.
authors.forename: Bert
authors.initials: B
authors.lastname: 't Hart
authors.affiliation: Ablynx NV, Technologiepark 21, 9052, Zwijnaarde, Belgium. Sofie.Poelmans@ablynx.com.
authors.forename: Sofie
authors.initials: S
authors.lastname: Poelmans
authors.affiliation: Ablynx NV, Technologiepark 21, 9052, Zwijnaarde, Belgium. Lieselot.Bontinck@ablynx.com.
authors.forename: Lieselot
authors.initials: L
authors.lastname: Bontinck
authors.affiliation: Ablynx NV, Technologiepark 21, 9052, Zwijnaarde, Belgium. ahemeryc@gmail.com.
authors.forename: Alex
authors.initials: A
authors.lastname: Hemeryck
authors.affiliation: Ablynx NV, Technologiepark 21, 9052, Zwijnaarde, Belgium. Sandy.Jacobs@ablynx.com.
authors.forename: Sandy
authors.initials: S
authors.lastname: Jacobs
authors.affiliation: Ablynx NV, Technologiepark 21, 9052, Zwijnaarde, Belgium. Judith.Baumeister@ablynx.com.
authors.forename: Judith
authors.initials: J
authors.lastname: Baumeister
authors.affiliation: Ablynx NV, Technologiepark 21, 9052, Zwijnaarde, Belgium. hans.ulrichts@ablynx.com.
authors.forename: Hans
authors.initials: H
authors.lastname: Ulrichts
mesh_terms: D000818:Animals; D018033:Antibodies, Bispecific / Q000494:pharmacology*; D018501:Antirheumatic Agents / Q000494:pharmacology*; D001169:Arthritis, Experimental / Q000188:drug therapy*; D001172:Arthritis, Rheumatoid / Q000188:drug therapy; D004195:Disease Models, Animal; D004797:Enzyme-Linked Immunosorbent Assay; D006207:Half-Life; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000276:immunology; D015850:Interleukin-6 / Q000276:immunology; D008252:Macaca fascicularis; D008253:Macaca mulatta; D019947:Receptors, Interleukin-6 / Q000037:antagonists & inhibitors*; D012709:Serum Albumin / Q000276:immunology; D061905:Single-Domain Antibodies / Q000494:pharmacology*
chemical_list: C000606096:ALX-0061; D018033:Antibodies, Bispecific; D018501:Antirheumatic Agents; C508600:IL6 protein, human; D007143:Immunoglobulin Heavy Chains; D015850:Interleukin-6; D019947:Receptors, Interleukin-6; D012709:Serum Albumin; D061905:Single-Domain Antibodies