Plates were coated with 2.5?g/ml muNGF and blocked with 5% BSA/PBS. to dogs, to determine its pharmacokinetic properties and to evaluate its efficacy in a model of inflammatory pain in Proglumide sodium salt vivo. Results Starting with a rat anti-NGF mAb, we used a novel algorithm based on expressed canine immunoglobulin sequences to design and characterise recombinant caninised anti-NGF mAbs. Construction with only 2 of the 4 canine IgG heavy chain isotypes (A and D) resulted in stable antibodies which bound and inhibited NGF with high-affinity and potency but did not bind complement C1q or the high-affinity Fc receptor gamma R1 (CD64). One of the mAbs (NV-01) was selected for scale-up manufacture, purification and pre-clinical evaluation. When administered to dogs, NV-01 was well tolerated, had a long serum half-life of 9?days, was not overtly immunogenic following repeated dosing in the dog and reduced signs of lameness in a kaolin model of inflammatory pain. Conclusions The combination of stability, high affinity and potency, no effector activity and long half-life, combined with safety and activity in the Proglumide sodium salt model of inflammatory pain in vivo suggests that further development of the caninised anti-NGF mAb NV-01 as a therapeutic agent for the treatment of chronic pain in dogs is usually warranted. Keywords: Nerve growth factor, Analgesia, Companion animals, Monoclonal antibody, Pharmacokinetics, Chronic pain, Veterinary bio-therapeutic Background Current therapeutic options for pain management in dogs are limited to a few classes of drugs including non-steroidal anti-inflammatory drugs (NSAID), narcotics and polysulphated glycosaminoglycans (PSGAG) [1,2]. Alternative therapeutic options are desirable, in particular for the management of chronic pain. Recently, a new class of antibody drugs have been developed which provide effective analgesia in rodents and man through interference of binding of NGF to its cellular receptors on nociceptive neurons. Whereas during mammalian development, NGF is essential for the survival of sensory and sympathetic neurons [3,4] in the adult it is expressed locally at sites of injury and inflammation and is a major factor promoting pain and hyperalgesia [5,6]. NGF is usually produced by a variety of inflammatory and immune cells, joint chondrocytes and has also been detected in nerve and neuroma preparations [5]C[7]. Following binding to its receptor trkA on nociceptors, NGF causes immediate and long-termexcitability through activation of ion channels, the transient receptor potential vanilloid receptor (TRPV1) and secondary neurotransmitters including material P and brain-derived neurotrophic factor (BDNF) [5]C[7]. NGF also causes the sprouting of nerve endings into the site of inflammation but does not appear to play a role in inflammation vitro characteristics of NV-01, together with preliminary studies investigating its safety and effectiveness are described herein. Collectively they show that NV-01 is usually a potent inhibitor of NGF, is usually well tolerated and non-immunogenic and shows promise as an analgesic in dogs. These preliminary data support our hypothesis that NV-01 might be useful as a treatment for pain in dogs (e.g. treatment of joint pain associated with osteoarthritis, cancer pain and post-surgical pain) and suggest that its further development as a veterinary medicine is warranted. Methods Sources of NGF A cDNA sequence encoding the amino acid sequence of canine pre-pro beta NGF (Physique?1A) with a C-terminal poly-His tag was synthesized from oligonucleotides, cloned into pcDNA3.1+ expression vector and transiently transfected into HEK293 cells at Geneart AG (Life Technologies, Regensberg, Germany). The supernatant was harvested and purified by Ni-HiTrap chromatography (GE Healthcare, Upsalla, Sweden). Purified mouse NGF (muNGF) was purchased from Biosensis (Thebarton, Australia). Open in a separate window Physique 1 NGF and anti-NGF antibody sequences. A) Alignment of the mature peptide sequence of NGF from human, mouse & doggie. Identical amino acids are indicated by dots and comparable amino acids are underlined. B) Variable heavy &C) variable light chain sequences of the anti-NGF antibody experiments, NV-01 antibody was expressed in CHO cells (Lonza Biologics plc, Cambridge, UK). Stable pooled transfections of CHO cells with cDNA encoding NV-01 heavy & light chains were cultured in a fed batch system for 13?days, before harvesting of supernatant containing NV-01. Clarified supernatant was diluted 1:2 with 50?mM Tris pH?8.0. The protein was captured on a HiTrap 5?ml anion exchange Q FF column (GE Healthcare) and impurities removed by washing the column with 50?mM Tris, 100?mM NaCl, pH?8.0. The protein was eluted with 50?mM Tris, 200?mM NaCl, pH?8.0. Anion exchange fractions made up of antibody were concentrated and diluted 1:10 with 50?mM sodium phosphate, 1?M ammonium sulphate, pH?7.0. The protein was captured on a Rabbit Polyclonal to OR10C1 HiTrap hydrophobic conversation Phenyl HP column (GE Healthcare) and impurities removed by washing the Proglumide sodium salt column with 50?mM sodium phosphate, 1?M ammonium sulphate, pH?7.0 (loading buffer). The protein was eluted with a linear gradient from loading buffer to 50?mM sodium phosphate,.