Generation of mutant leukaemia inhibitory factor (LIF)-IgG heavy chain fusion proteins as bivalent antagonists of LIF

Jalal Jazayeri, Nicole Anne De Weerd, Warren Sean Raye, Tony Velkov, Leilani Llanes Santos, David Taylor, Graeme James Carroll

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6 Citations (Scopus)

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 Fe fusion constructs have a longer serum half-life compared to their non-fusion counterparts.
Original languageEnglish
Pages (from-to)1 - 10
Number of pages10
JournalJournal of Immunological Methods
Volume323
Issue number1
Publication statusPublished - 2007

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