Receptor-binding determinant from leukaemia inhibitory factor

Details Receptor-binding determinant from leukaemia inhibitory factor Receptor-binding determinant from leukaemia inhibitory factor

1995-09-27

2001-02-06

Mertz, Prema (Department: 1646)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C530S351000, C435S069500, C435S069700, C435S071100, C435S071200, C435S471000, C435S325000, C435S252300, C435S320100, C536S023500

Reexamination Certificate

active

06184370

ABSTRACT:

The present invention relates generally to molecules carrying one or more binding determinants for the &agr;-chain of human Leukaemia Inhibitory Factor binding receptor and to genetic sequences encoding same.
Bibliographic details of the publications referred to in this specification are collected at the end of the description. Sequence Identity Numbers (SEQ ID NOs.) for amino acid sequences referred to in the specification are defined following the bibliography.
Throughout this specification, unless the context requires otherwise, the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.
Certain aspects of the present invention relate to leukaemia inhibitory factor (hereinafter referred to as “LIF”) of human (h) (SEQ. ID. No:2) or murine (m) (SEQ. ID. NO:1) origin or to combinations thereof. Where reference is made to amino acid residues in these molecules, the residues are numbered consecutively from the first serine residue of the mature, native protein (see FIG.
4
). In certain circumstances, the production of recombinant LIF molecules may involve a fusion protein with glutathione-S-transferase (GST) in an expression vector system. Agents such as thrombin may be used to cleave the GST portion of the LIF-GST molecule which can result in an additional glycine residue at position −1 (FIG.
4
). Murine and human LIF hybrids (MH) referred to in the specification are defined in Table 1 and FIG.
1
. The legend to Table 1 describes the nomenclature used to define the hybrid structures. It should be noted that the numbering system of amino acid residues referred to in Australian Provisional Application No. PL7102 filed on Feb. 3, 1993 and from which the present application claims priority starts at the initiating methione of the immature, native protein. Accordingly, the serine at position +1 described in the present specification corresponds to position +24 in the aforementioned provisional application.
LIF is a glycoprotein that was originally purified and cloned on the basis of its ability to induce terminal macrophage differentiation of the M1 myeloid leukaemic cell line (Hilton et al. 1988a). It has since been shown to have a variety of activities on a wide range of cell types including megakaryocytes, osteoblasts, hepatocytes, adipocytes, neurons, embryonal stem cells and primordial germ cells (Metcalf, 1991).
It is proposed that LIF transduces its biological signal via a multi-subunit membrane-bound receptor. The receptor for LIF is a member of the haemopoietin or cytokine family of receptors, which generally have roles in cell growth and differentiation. These receptors are characterised by their extracellular domain, which contains at least one copy of an approximately 200 amino acid haemopoietin domain (Cosman, 1990). A number of primary amino acid sequence motifs distinguish this domain, including pairs of disulfide-bonded cysteine residues and the Trp-Ser-X-Trp-Ser motif (where X is any amino acid). The overall secondary and tertiary fold of the haemopoietin domain is predicted to be similar in each member of the receptor family (Bazan, 1990a). Like the extracellular domain of the growth hormone receptor (de Vos et al, 1992), the 200 amino acids of the haemopoietin domain form 14 anti-parallel &bgr;-strands, folded into two barrel structures. The LIF receptor consists of two known subunits, both of which are members of the haemopoietin receptor family. The LIF receptor &agr;-chain binds LIF with low-affinity and contains two haemopoietin domains (Gearing et al, 1991). The &bgr;-chain of the LIF receptor has been identified as gp130 (Gearing et al, 1992) which is also a component of the interleukin (IL)-6, IL-11, oncostatin M (OSM) and ciliary neurotrophic factor receptor (CNTF) complexes.
The ligands for this family of receptors are unrelated at the primary amino acid sequence level, but secondary and tertiary structural predictions indicate that all known ligands for haemopoietin receptors have a similar overall fold, suggesting evolution from a common ancestor (Bazan, 1990b). Members of this family of ligands form an anti-parallel, four &agr;-helical bundle (the helices are designated A, B, C and D ordered from the N-terminus), that is characterised by one short and two long connecting loops (labelled by the helices they join). This has been shown for several ligands that are members of this family, including granulocyte colony-stimulating factor (G-CSF) (SEQ. ID. NO:4) (Hill et al, 1993), granulocyte-macrophage CSF (GM-CSF) (Diederichs et al, 1991), growth hormone (GH) (de Vos et al, 1992; Abdel-Meguid et al, 1987), IL-2 (Brandhuber et al, 1987), IL-4 (Powers et al, 1992) and IL-5 (Milburn et al, 1993), whose structures have been solved, either by x-ray crystallography or by NMR studies. The three-dimensional structure of LIF is predicted to be most similar to the structures of OSM, CNTF, IL-6 and G-CSF, amongst others.
Whilst murine LIF (mLIF) is unable to bind to the human LIF (hLIF) receptor (hLIF-R), hLIF (SEQ. ID. NO:2) is able to bind to both high- and low-affinity mouse LIF receptors (mLIF-R SEQ. ID. NO:1), and is fully biologically active on mouse cells. Intriguingly, hLIF (SEQ. ID. NO:2) binds to both the naturally occurring soluble form of the mLIF-R &agr;-chain, mLIF-binding protein (mLBP) (Layton et al, 1992), and the high-affinity mLIF-R on PC.13 cells with a higher affinity than mLIF, due to markedly different dissociation kinetics. Competitive displacement curves showed that unlabelled mLIF (SEQ. ID. NO:1) and hLIF (SEQ. ID. NO:2) had a similar ability to complete with [
125
I]mLIF, for binding to mLBP, while unlabelled hLIF was consistently 1000- to 5000-fold more effective than mLIF (SEQ. ID. NO:1) in competing with [
125
I]hLIF for binding to mLBP (Layton et al, 1992). Mouse LBP is also able to act as a competitive inhibitor of LIF binding to its cellular receptor, leading to inhibition of biological responses to LIF. Again, mLBP was an approximately 1000-fold more potent inhibitor of hLIF (SEQ. ID. NO:2) than mLIF (SEQ. ID. NO:1) in this system. Thus, at least two features of hLIF (SEQ. ID. NO:2) distinguish it from mLIF (SEQ. ID. NO:1): first, its capacity to bind to the hLIF-R where mLIF (SEQ. ID. NO:1) cannot and second, its capacity to bind to the mLIF-R with higher affinity than does mLIF (SEQ. ID. NO:1).
Understanding the way in which a cytokine interacts with its receptor at a molecular level is required for the rational design of agonists and antagonists to growth factor and cytokine action as well as the design of growth factor and cytokine analogues. In work leading up to the present invention, the inventors exploited the differences in binding characteristics of murine (SEQ. ID. NO:1) and human LIF (SEQ. ID. NO:2) to identify a major determinant responsible for the binding of human LIF (hLIF) to the &agr;-chain of its receptor. The present invention provides, for the first time, a rational approach to the generation of a new range of therapeutics based on LIF by providing non-naturally occurring molecules capable of binding to hLIF receptor. Such therapeutics may be both proteinaceous and non-proteinaceous molecules.
Accordingly, one aspect of the present invention contemplates a non-naturally occurring molecule comprising a tertiary structure which presents a functional binding face for the &agr;-chain of hLIF binding receptor.
More particularly, the present invention is directed to a molecule which is non-naturally occurring and which comprises a carrier portion and an active portion wherein the tertiary structure of said active portion comprises non-contiguous amino acid residues or chemical equivalents thereof selected to constitute a binding determinant for the &agr;-chain of human leukaemia inhibitory factor (hLIF) binding receptor.
This aspect of the present invention is predicated in par

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