Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Hormone or other secreted growth regulatory factor,...
Reexamination Certificate
1998-05-28
2001-11-27
Eyler, Yvonne (Department: 1646)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Hormone or other secreted growth regulatory factor,...
C424S085100, C514S002600, C530S399000, C530S324000, C530S351000
Reexamination Certificate
active
06322791
ABSTRACT:
The present invention relates generally to variant recombinant forms of haemopoietic growth factors useful as antagonists to the corresponding native haemopoietic growth factor and their use in ameliorating aberrant effects caused by the native molecules and in the treatment of tumours and cancers and inflammation.
Sequence Identity Numbers (SEQ ID NOs.) for the nucleotide and amino acid sequences referred to in the specification are defined following the description.
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.
The rapidly increasing sophistication of recombinant DNA technology is greatly facilitating research and development in a range of industries. The medical and allied health fields in particular have, and continue to, benefit from this developing technology. An area of substantial interest is the field of growth factors and cytokines.
Granulocyte-macrophage colony-stimulating factor (GM-CSF) is one member of a family of haemopoietic growth factors (HGFs) and exhibits a range of important activities such as supporting survival of normal leukaemic and haemopoietic cells by suppressing programmed cell death, which is also known as apoptosis. GM-CSF binds to a heterodimeric receptor composed of a GM-CSF specific a chain and a &bgr;c chain which is shared with the receptors for interleukins (IL) 3 and 5. Both chains are required for GM-CSF mediated signaling.
In International Patent Application No. PCT/AU94/00432 filed on Jul. 28, 1994, which is incorporated herein by reference, a series of HGF antagonists was described. It has now been surprisingly discovered that certain forms or types of these antagonists induce apoptosis in cells expressing HGF receptors. This property of the HGF antagonists provides inter alia a novel approach to the treatment of a range of tumours and cancers and in particular myeloid leukaemias.
Accordingly, one aspect of the present invention contemplates a method for inducing apoptosis in cells carrying an HGF heterodimeric receptor comprising an HGF-specific &agr;-chain and a &bgr;c chain, said method comprising contacting said cells with an effective amount of an HGF antagonist for a time and under conditions sufficient to induce apoptosis wherein said HGF antagonist comprises a sequence of amino acids within a first &agr;-helix of HGF wherein one or more exposed amino acids in said first &agr;-helix of HGF having acidic properties is/are substituted with a basic amino acid residue or a non-acidic amino acid residue.
The HGFs are preferably GM-CSF, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, EL-11, IL-13, IL-14, IL-15 and others of this family yet to be discovered, granulocyte colony-stimulating factor (G-CSF), erythropoietin (EPO) and thrombopoietin (TPO). Most preferably, the HGF is GM-CSF.
According to this aspect of the present invention there is provided a method for inducing apoptosis in cells carrying an HGF heterodimeric receptor comprising an HGF-specific &agr;-chain and a &bgr;c chain, said method comprising contacting said cells with an effective amount of an GM-CSF antagonist for a time and under conditions sufficient to induce apoptosis wherein said GM-CSF antagonist comprises a sequence of amino acids within a first &agr;-helix of HGF wherein one or more exposed amino acids in said first &agr;-helix of HGF having acidic properties is/are substituted with a basic amino acid residue or a non-acidic amino acid residue.
The HGF antagonists are preferably produced in recombinant or synthetic form and, with the exception of the amino acid substitution(s) in the first &agr;-helix, the amino acid sequence of the HGF may be the same as the naturally occurring molecule (i.e. native molecule) or may carry single or multiple amino acid substitutions, deletions and/or additions to the native sequence.
In one particular embodiment, the HGF antagonists are:
(i) in unglycosylated form;
(ii) lack post-translational modification;
(iii) produced in prokaryotic microorganisms; and/or
(iv) produced by chemical synthesis.
The antagonists may be derivatives of an HGF or may be any other molecule which selectively binds to the &agr;-chain of the HGF receptor such as, but not limited to, antibodies or other small or large molecules resulting in apoptosis. The present invention is hereinafter described with reference to GM-CSF antagonists and in particular human GM-CSF antagonists or mammalian GM-CSF antagonists capable of functioning in humans. This is done, however, with the understanding that the present invention extends to any HGF antagonist which is capable of inducing cell apoptosis by interaction with an HGF-specific &agr;-chain of an HGF receptor. The present invention also extends to HGF antagonists which interact with the &bgr; chain.
Although not intending to limit the present invention to any one theory or mode of action, it is proposed that the GM-CSF antagonist either blocks the action of wild-type GM-CSF by interacting selectively with the receptor &agr;-chain or induces apoptosis by interacting with the &agr;- and &bgr;-chains of the receptor in a manner that leads to abnormal stimulation of the &bgr;-chain either qualitatively or quantitatively. Qualitative differences to wild-type GM-CSF include recruitment of signalling molecules (for example, type of kinase or state); quantitative differences include intensity of duration of signalling.
The present invention is particularly directed to the use of a GM-CSF antagonist carrying a substitution of amino acid 21 (Glu) of human GM-CSF by Arg or Lys or any other basic or non-acidic amino acid residue. Such mutants are designated herein “E21R” and “E21K”, respectively, which is based on a single letter designation of the amino acids involved in the substitution and the position of the substitution. The construction of vectors expressing E21R is described in the Examples. E21R is a fusion protein expressed in inclusion bodies as GM-CSF(E21R) preceded by a twelve amino acid leader sequence, namely, MFATSSSTGNDG (SEQ ID NO:3)
In accordance with the present invention, it has been surprisingly discovered that GM-CSF antagonist E21R binds to the GM-CSF-specific &agr;-chain of the GM-CSF receptor and that such binding directly induces apoptosis of normal and malignant myeloid cells expressing the GM-CSF receptor. Apoptosis occurs even in the presence of the survival factors such as G-CSF and stem cell factor (SCF) but is prevented by engaging receptor chain &bgr;c with IL-3.
Accordingly, in a particularly preferred embodiment, the present invention contemplates a method for inducing apoptosis of myeloid cells said method comprising contacting said cells with an apoptotic effective amount of a GM-CSF antagonist having a basic amino acid residue substituted at position 21 of the GM-CSF amino acid sequence for a time and under conditions sufficient for programmed cell death to initiate.
This method has particular relevance in the treatment of cancers such as myeloid leukaemias and inflammation, for example, rheumatoid arthritis and allergic conditions such as asthma. Preferred GM-CSF antagonists comprise amino acids Arg or Lys at position 21 of GM-CSF. The most preferred antagonist comprises Arg at position 21 of GM-CSF and is referred to herein as E21R.
Accordingly, a further aspect of the present invention contemplates a method of inducing apoptosis in a cell carrying receptors for GM-CSF, said method comprising contacting said cell with a molecule capable of binding to the &agr;-chain of the GM-CSF receptor or binding both to the &agr;-chain and &bgr;-chain for a time and under conditions sufficient for programmed cell death to initiate.
Preferably, the molecule is a derivative of GM-CSF with amino residue 21 substituted by a basic amino acid.
Preferably, the basic amino acid residue is Arg or Lys. Most preferabl
Bastiras Stan
Cheah Keat-Chye
Lopez Angel Francisco
Robins Allan
Senn Carol Ruth
Andres Janet L.
BreasaGen Limited
Eyler Yvonne
Knobbe Martens Olson & Bear LLP
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