Generation of mutant leukaemia inhibitory factor (LIF)-IgG heavy chain fusion proteins as bivalent antagonists of LIF.
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.
Jazayeri
Jalal A
JA
Department of Pharmaceutical Biology, Victorian College of Pharmacy, Monash University, Parkville, Melbourne, Victoria 3052, Australia. jalal.jazayeri@vcp.monash.edu.au
De Weerd
Nicole
N
Raye
Warren
W
Velkov
Tony
T
Santos
Lanie
L
Taylor
David
D
Carroll
Graeme J
GJ
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
D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; C507840:LIFR protein, human; D053680:Leukemia Inhibitory Factor Receptor alpha Subunit; D011993:Recombinant Fusion Proteins