Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
1994-11-10
2002-05-14
Kunz, Gary L. (Department: 1647)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C514S008100, C514S013800, C514S021800
Reexamination Certificate
active
06387875
ABSTRACT:
The present invention relates to a Leukaemia Inhibitory Factor-Binding Protein (LBP) and more particularly to a soluble LBP, uses thereof and compositions containing same.
Leukaemia Inhibitory Factor (LIF) is a polyfunctional glycoprotein with actions on a broad range of tissue and cell types, including induction of differentiation in a number of myeloid leukaemic cell lines, suppression of differentiation in normal embryonic stem cells, stimulation of proliferation of osteoblasts and DA-1 haemopoietic cells and potentiation of the proliferative action of interleukin-3 (IL-3) on megakaryocyte precursors. Functionally, it is able to switch autonomic nerve signalling from adrenergic to cholinergic mode, stimulate calcium release from bones, stimulate the production of acute phase proteins by hepatocytes and induce loss of fat deposits by inhibiting lipoprotein lipase-mediated lipid transport into adipocytes
1
.
This array of actions is puzzling since it is difficult to conceive of any situation that would require a coordinated response in all the known target tissues of LIF. Actions of LIF, therefore, are probably designed to be restricted by co-localisation of LIF-producing cells and LIF-responsive cells, with tight regulation of LIF production. However, such an arrangement is likely to result in some release of LIF into the circulatory system including blood and other bodily fluids.
In work leading up to the present invention, the inventors discovered a LIF-binding protein in serum which is capable of inhibiting the biological activity of LIF. The identification of this LIF antagonist will now permit greater control in LIF therapy and to prevent any systemic effects of locally administered LIF which are not therapeutically desirable. It also provides a new agent useful in the treatment of LIF associated diseases or conditions. In a particular embodiment, the inventors have discovered that the inhibitory effect of the LIF-binding protein may be more pronounced in heterologous systems, i.e. where the LIF-binding protein from one mammal is used to inhibit LIF in another mammal.
Accordingly, one aspect of the present invention provides a LIF-binding protein (LBP) in soluble form and isolatable from a mammal.
More particularly, the present invention is directed to an isolated LBP in soluble form and obtainable from a first mammalian species, said LBP capable of inhibiting the ability of LIF from a second mammalian species to induce differentiation of M1 myeloid leukaemic cells in vitro to a greater extent when compared to its ability to inhibit LIF from said first mammalian species.
The isolated LBP is preferably biologically pure meaning that it represents at least 20%, preferably at least 50%, even more preferably at least 70% and still more preferably at least 85% of the molecule in a solution or composition as determined by weight, biological activity or other convenient means of measurement. Notwithstanding that the LBP is isolated, it may also be in the form of a composition. According to this aspect of the present invention there is contemplated a composition comprising an LBP in soluble form and obtainable from a first mammalian species, said LBP capable of inhibiting the ability of LIF from a second mammalian species to induce differentiation of M1 myeloid leukaemic cells in vitro to a greater extent when compared to its ability to inhibit LIF from said first mammalian species, said composition substantially free of protein molecules not having LBP properties.
The isolated LBP in soluble form and obtainable from the first mammalian species is further characterised in that the LBP has at least a 100 fold higher binding affinity for a LIF from the second mammalian species compared to the binding affinity for a LIF from said first mammalian species.
In accordance with the present invention, the first mammal is preferably a human, mouse or rat or other rodent, pig, cow, sheep or other ruminant, goat, horse or primate. The second mammal may also be a human, mouse or rat or other rodent, pig, cow, sheep or other ruminant, goat, horse or primate. Preferably, the first mammal is a non-human mammal and the second mammal is a human. Most preferably, the first mammal is a mouse and the second mammal is a human.
Accordingly, in a preferred embodiment, the present invention is directed to a LBP in soluble form isolatable from a murine animal. More particularly, the present invention provides a LBP in soluble form isolatable from a murine animal, said LBP capable of greater inhibition of human LIF compared to murine LIF.
The isolated LBP may be the naturally occurring molecule, a naturally occurring derivative, part or fragment thereof or may be a recombinant or synthetic form of the molecule including any recombinant or synthetic derivatives, parts or fragments thereof. The LBP may be naturally glycosylated, partially glycosylated or unglycosylated or may have an altered glycosylation pattern from the naturally occurring molecule. The molecule may, for example, undergo treatment with N-glycanase resulting in a deglycosylated or substantially deglycosylated molecule. A “derivative” of LBP is considered herein to generally comprise a single or multiple amino acid insertion, deletion and/or substitution of amino acid residues relative to the naturally occurring sequence or an insertion, deletion and/or substitution of molecules associated with LBP such as carbohydrate moieties. A “derivative” is also considered to be a molecule with at least 45% amino acid sequence similarity to the amino acid sequence of the LBP.
Amino acid insertional derivatives of LBP include amino and/or carboxyl terminal fusions as well as intra-sequence insertions of single or multiple amino acids. Insertional amino acid sequence variants are those in which one or more amino acid residues are introduced into a predetermined site in the protein although random insertion is also possible with suitable screening of the resulting product. Deletional variants are characterised by the removal of one or more amino acids from the sequence. Substitutional amino acid variants are those in which at least one residue in the sequence has been removed and a different residue inserted in its place. Typical subsitutions are those made in accordance with the following Table 1:
TABLE 1
Suitable residues for amino acid substitutions
Original Residue
Exemplary Substitutions
Ala
Ser
Arg
Lys
Asn
Gln; His
Asp
Glu
Cys
Ser
Gln
Asn
Glu
Ala
Gly
Pro
His
Asn; Gln
Ile
Leu; Val
Leu
Ile; Val
Lys
Arg; Gln; Glu
Met
Leu; Ile
Phe
Met; Leu; Tyr
Ser
Thr
Thr
Ser
Trp
Tyr
Tyr
Trp; Phe
Val
Ile; Leu
Where the LBP is derivatised by amino acid substitution, the amino acids are generally replaced by other amino acids having like properties, such as hydrophobicity, hydrophilicity, electronegativity, bulky side chains and the like. Amino acid substitutions are typically of single residues. Amino acid insertions will usually be in the order of about 1-10 amino acid residues and deletions will range from about 1-20 residues. Preferably, deletions or insertions are made in adjacent pairs, i.e. a deletion of two residues or insertion of two residues.
The amino acid variants referred to above may readily be made using peptide synthetic techniques well known in the art, such as solid phase peptide synthesis
(14)
and the like, or by recombinant DNA manipulations. Techniques for making substitution mutations at predetermined sites in DNA having known or partially known sequence are well known and include, for example, M13 mutagenesis. The manipulation of DNA sequences to produce variant proteins which manifest as substitutional, insertional or deletional variants are conveniently described, for example, in Maniatis et al
(15)
.
Other examples of recombinant or synthetic mutants and derivatives of the LBP of the present invention include single or multiple substitutions, deletions and/or additions of any molecule associated with the LBP such as carbohydrates, lipids and/or proteins or polypeptides.
In one embodiment, the LBP is truncated at its carboxy terminal end por
Layton Meredith
Metcalf Donald
Nicola Nicos Anthony
Simpson Richard J
Amrad Corporation Limited
Gucker Stephen
Kunz Gary L.
Scully Scott Murphy & Presser
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