title: Generation of mutant leukaemia inhibitory factor (LIF)-IgG heavy chain fusion proteins as bivalent antagonists of LIF.
abstract: Two leukaemia inhibitory factor (LIF) mutants, designated MH35-BD and LIF05, have been shown to have a capacity to inhibit the biological activities of not only human LIF (hLIF) but also other interleukin-6 (IL-6) subfamily cytokines such as human oncostatin M (hOSM). These cytokines share the same receptor complex in which the glycoprotein 130 (gp130) subunit is a common constituent. However, at low concentrations and in their monomeric forms, such molecules have a relatively short plasma half-life due to rapid clearance from the kidneys. Here, to prolong their serum half-lives, we have used a multi-step polymerase chain reaction (PCR) to fuse each of the LIF05 and MH35-BD cDNA fragments to a sequence encoding the Fc portion, and the hinge region, of the human immunoglobulin G (hIgG) heavy chain. The linking was achieved through an oligomer encoding a thrombin-sensitive peptide linker thus generating MH35-BD:Fc and LIF05:Fc, respectively. Both Fc fusion constructs were expressed in insect cell Sf21 and the proteins were purified by two successive affinity chromatography steps using nickel-nitrilotriacetic acid (Ni-NTA) agarose and protein A beads. The Ba/F3 cell-based proliferation assay was used to confirm that the proteins were biologically active. In addition, preliminary pharmacokinetics indicates that the Fc fusion constructs have a longer serum half-life compared to their non-fusion counterparts.
authors.affiliation: Department of Pharmaceutical Biology, Victorian College of Pharmacy, Monash University, Parkville, Melbourne, Victoria 3052, Australia. jalal.jazayeri@vcp.monash.edu.au
authors.forename: Jalal A
authors.initials: JA
authors.lastname: Jazayeri
authors.forename: Nicole
authors.initials: N
authors.lastname: De Weerd
authors.forename: Warren
authors.initials: W
authors.lastname: Raye
authors.forename: Tony
authors.initials: T
authors.lastname: Velkov
authors.forename: Lanie
authors.initials: L
authors.lastname: Santos
authors.forename: David
authors.initials: D
authors.lastname: Taylor
authors.forename: Graeme J
authors.initials: GJ
authors.lastname: Carroll
mesh_terms: D000818:Animals; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D006207:Half-Life; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000096:biosynthesis* / Q000494:pharmacology*; D007074:Immunoglobulin G / Q000494:pharmacology; D053680:Leukemia Inhibitory Factor Receptor alpha Subunit / Q000037:antagonists & inhibitors*; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000493:pharmacokinetics*; D014162:Transfection
chemical_list: D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; C507840:LIFR protein, human; D053680:Leukemia Inhibitory Factor Receptor alpha Subunit; D011993:Recombinant Fusion Proteins