title: Increased brain bio-distribution and chemical stability and decreased immunogenicity of an engineered variant of GDNF.
abstract: Several lines of evidence indicate that Glial cell line-derived neurotrophic factor (GDNF) is a trophic factor for dopaminergic neurons. Direct parenchymal administration of GDNF is robustly neuroprotective and neurorestorative in multiple neurotoxin-based animal models (rat and non-human primate (NHP)) of Parkinson's Disease (PD), suggesting its potential as a therapeutic agent. Although small, open-label clinical trials of intra-putamenal administration of bacteria-derived, full length, wild type GDNF (GDNFwt) were efficacious in improving standardized behavioral scores, a double-blinded, randomized controlled trial failed to do so. We hypothesize that the lack of clinical efficacy of GDNFwt in the larger randomized trial was due to poor bio-distribution in the putamen and/or poor chemical stability while in the delivery device for prolonged time periods at 37°C. The development of neutralizing antibodies in some patients may also have been a contributing factor. GDNFv is an engineered form of GDNFwt, expressed and purified from mammalian cells, designed to overcome these limitations, including removal of the N-terminal heparin-binding domain to improve its diffusivity in brain parenchyma by reducing its binding to extracellular matrix (ECM), and key amino acid substitutions to improve chemical stability. Intra-striatal administration of a single injection of GDNFv in the rat produced significantly greater brain distribution than GDNFwt, consistent with reduced binding to ECM. Using liquid chromatography/mass spectrometry (LS/MS) methods GDNFv was shown to have improved chemical stability compared to GDNFwt when stored at 37°C for 4weeks. In addition, GDNFv resulted in lower predicted clinical immunogenicity compared to GDNFwt, as demonstrated by reduced CD4+ T cell proliferation and reduced IL-2-induced secretion in peripheral blood mononucleated cells collected from volunteers representing the world's major histocompatibility complex (MHC) haplotypes. GDNFv was demonstrated to be pharmacologically equivalent to GDNFwt in the key parameters in vitro of GFRα1 receptor binding, c-Ret phosphorylation, neurite outgrowth, and in vivo in its ability to increase dopamine turnover (DA). GDNFv protected dopamine nerve terminals and neurons in a 6-hydroxy-dopamine (6-OHDA) rat model. In summary, we empirically demonstrate the superior properties of GDNFv compared to GDNFwt through enhanced bio-distribution and chemical stability concurrently with decreased predicted clinical immunogenicity while maintaining pharmacological and neurotrophic activity. These data indicate that GDNFv is an improved version of GDNF suitable for clinical assessment as a targeted regenerative therapy for PD.
authors.affiliation: Eli Lilly & Co., Biotechnology Discovery Research, Lilly Corporate Center, Indianapolis, IN 46285, USA. Electronic address: smith_ros@lilly.com.
authors.forename: Rosamund C
authors.initials: RC
authors.lastname: Smith
authors.affiliation: Eli Lilly & Co., Biotechnology Discovery Research, Lilly Corporate Center, Indianapolis, IN 46285, USA. Electronic address: o'bryan_linda_maureen@lilly.com.
authors.forename: Linda M
authors.initials: LM
authors.lastname: O'Bryan
authors.affiliation: Eli Lilly & Co., Biotechnology Discovery Research, Lilly Corporate Center, Indianapolis, IN 46285, USA. Electronic address: mitchell_pamela@lilly.com.
authors.forename: Pamela J
authors.initials: PJ
authors.lastname: Mitchell
authors.affiliation: Eli Lilly & Co., Lilly Biotechnology Center, 10300 Campus Point Dr, Suite 200, San Diego, CA 92121, USA. Electronic address: leung_donmienne@lilly.com.
authors.forename: Donmienne
authors.initials: D
authors.lastname: Leung
authors.affiliation: Eli Lilly & Co., Biotechnology Discovery Research, Lilly Corporate Center, Indianapolis, IN 46285, USA. Electronic address: mahmoud.ghanem@siemens.com.
authors.forename: Mahmoud
authors.initials: M
authors.lastname: Ghanem
authors.affiliation: Eli Lilly & Co., Tailored Therapeutics, Lilly Corporate Center, Indianapolis, IN 46285, USA. Electronic address: wilson_jonathan_matthew@lilly.com.
authors.forename: Jonathan M
authors.initials: JM
authors.lastname: Wilson
authors.affiliation: Eli Lilly & Co., Information Technology, Lilly Corporate Center, Indianapolis, IN 46285, USA. Electronic address: hanson_jeffrey_c@lilly.com.
authors.forename: Jeff C
authors.initials: JC
authors.lastname: Hanson
authors.affiliation: Eli Lilly & Co. Ltd, Erl Wood Manor, Windlesham, Surrey GU20 6PH, UK. Electronic address: sossick_sandra@lilly.com.
authors.forename: Sandra
authors.initials: S
authors.lastname: Sossick
authors.affiliation: Eli Lilly & Co. Ltd, Erl Wood Manor, Windlesham, Surrey GU20 6PH, UK. Electronic address: cooper_jane_x1@lilly.com.
authors.forename: Jane
authors.initials: J
authors.lastname: Cooper
authors.affiliation: Eli Lilly & Co., Bioproduct Research and Development, Indianapolis, IN 46285, USA. Electronic address: huang_lihua@lilly.com.
authors.forename: Lihua
authors.initials: L
authors.lastname: Huang
authors.affiliation: Eli Lilly & Co., Tailored Therapeutics, Lilly Corporate Center, Indianapolis, IN 46285, USA. Electronic address: merchant.transthera@gmail.com.
authors.forename: Kalpana M
authors.initials: KM
authors.lastname: Merchant
authors.affiliation: Eli Lilly & Co., Biotechnology Discovery Research, Lilly Corporate Center, Indianapolis, IN 46285, USA. Electronic address: lu_jirong@lilly.com.
authors.forename: Jirong
authors.initials: J
authors.lastname: Lu
authors.affiliation: Eli Lilly & Co. Ltd, Erl Wood Manor, Windlesham, Surrey GU20 6PH, UK. Electronic address: oneill_michael_j@lilly.com.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: O'Neill
mesh_terms: D000818:Animals; D001921:Brain / Q000187:drug effects / Q000378:metabolism*; D004195:Disease Models, Animal; D004305:Dose-Response Relationship, Drug; D005343:Fibrinolytic Agents / Q000494:pharmacology; D051100:Glial Cell Line-Derived Neurotrophic Factor / Q000235:genetics* / Q000378:metabolism*; D006493:Heparin / Q000494:pharmacology; D006801:Humans; D055261:Immunogenetic Phenomena / Q000235:genetics*; D008297:Male; D009154:Mutation / Q000235:genetics*; D009419:Nerve Tissue Proteins / Q000378:metabolism; D016501:Neurites / Q000187:drug effects; D016627:Oxidopamine / Q000633:toxicity; D016716:PC12 Cells; D010300:Parkinson Disease / Q000209:etiology / Q000378:metabolism / Q000473:pathology; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding / Q000187:drug effects / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D005343:Fibrinolytic Agents; D051100:Glial Cell Line-Derived Neurotrophic Factor; D009419:Nerve Tissue Proteins; D016627:Oxidopamine; D006493:Heparin
keywords: 6-OHDA; Brain bio-distribution; Dopamine turnover; GDNF; GFRα1; Glial cell line-derived neurotrophic factor; Heparin binding; Neurite outgrowth; Parkinson's disease; Putamen; c-Ret