A GH antagonist fusion with GH binding protein is biologically active, shows delayed clearance and inhibits growth in rabbits Ian Wilkinson, Sarbhendra Pradhananga, Jon Sayers, Pete Artymiuk, Michael Phipps & Richard Ross Sheffield University, UK 1

Action of GH and Pegvisomant B2036 PEG GHBP 1 2 JAK2 1* GHBP GHBP X X Ross et al. J Clin Endocrinol Metab. 2001 ,86(4):1716-23

Growth Hormone Antagonist - The Unmet Need Acromegaly: Current Treatment Only 50% cured by surgery Only 70% controlled by SMS analogue therapy Pegvisomant Controls >95% and well tolerated Majority of patients require daily dosing Requires high dose of 20-60mg per day High cost of goods & very expensive: £55k / year Challenges Generate a potent long-acting antagonist Weekly injections Define an animal model to test human GH antagonist

Background: LR-Fusion Technology Linking GH to GHBP (extracellular domain GHR) creates a long acting agonist [Ref: Wilkinson et al., Nature Medicine 2007] Our AFTTM fusion protein 1 2 3 Extracellular receptor Intermolecular interaction Flexible GHR N-terminus Ligand ProFuseTM constituents (expressed as single construct) Physiological pool of protected ligand (dynamic equilibrium) Ligand available to bind to natively expressed receptor (long acting in vivo effect)

LR-Fusion GH antagonist: Expressed & Purified Two LR-fusions made with and without Gly-Ser linkers Site two mutant with G120R Site two and site one SDS-PAGE gel of purified LR-fusions kDa 100 75 50 37 25 20 Key: M: Markers 1: Site 2 (+ linker) 2: Site 2 (no linker) 3: Site 1 & 2 (+ linker) 4: Site 1 & 2 (no linker

Site 2 mutant is an antagonist in dual Luciferase bioassay

Site 2 mutant antagonist shows delayed clearance in rat Given at 1nmol/kg subcutaneously (sc) to 6 rats the LR fusion antagonist (site 2 only) showed delayed clearance with plasma half-life of 21.0hrs compared to GH at 1.2hrs LR fusion antagonist can still be detected 10 days post injection sc injection iv injection

Site 2 mutant antagonist more potent than PEGvisomant in vivo A single sc dose of GH antagonist resulted in decreased weight gain in growing NZ white rabbits over 12 days compared to vehicle control Decrease in weight gain was greater than Pegvisomant given as 5 separate injections (Days 1, 2, 6, 7 & 8) Pegvisomant 5 x sc dosing regime

Summary The LR fusion antagonist Proof of concept : is an antagonist in vitro and in vivo Is more potent than Pegvisomant in vivo Proof of concept : Delayed clearance demonstrated in rat Block GH activity in bioassay Growth inhibition demonstrated in rabbit growth model Potential for weekly dosing Preclinical work required: Candidate optimisation in progress Further develop rabbit model for proof of concept using IGF-I

Acknowledgements Berlin Group Christian Strasburger Zida Wu Work colleagues Richard Ross Sarbendra Pradhananga Pete Artymiuk Jon Sayers Sue Justice Michael Phipps Munich Group Max Bielohuby Martin Bidlingmaier