Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues
Reexamination Certificate
1999-04-12
2002-10-15
Romeo, David S. (Department: 1646)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
C530S326000, C514S002600
Reexamination Certificate
active
06465616
ABSTRACT:
The present invention relates to modified and variant forms of Interleukin-5 molecules capable of antagonizing or reducing the activity of IL-5 and their use in ameliorating, abating or otherwise reducing the aberrant effects caused by native or mutant forms of IL-5.
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 the medical and allied health fields. This is particularly important in the area of haemopoietic growth factor research where a number of disease conditions are predicated on the aberrant effects of native or mutant forms of growth factors.
One particularly important haemopoietic growth factor is IL-5. This molecule is a lymphokine secreted by T-cells and mast cells and is a disulfide-linked homodimeric glycoprotein. The human form of this molecule comprises 114 amino acids per monomer. IL-5 consists of a bundle of four a-helices in an up-up, down-down array. The phenomenon of D-helix swapping whereby one bundle is built up of three helices coming from one monomer and a fourth helix which is contributed by the second monomer is unique to IL-5. The IL-5 molecule also contains two short anti-parallel &bgr;-strands located between helices A and B and helices C and D.
Human and murine IL-5 receptors comprise two different chains, the &agr; and &bgr;-subunits.
Human IL-5 binds to the &agr;-subunit but the binding affinity is increased upon association with the &bgr;-chain. The &bgr;-chain is shared by other cytokines such as GM-CSF and IL-3.
IL-5 is a haemopoietic growth factor with selectivity for production and activation of human eosinophils. There is a need, therefore, to develop antagonists of IL-5 to act as therapeutic agents for chronic asthma or other disease states with demonstrated eosinophilia or other conditions associated with IL-5. It is also important for the IL-5 antagonist not to interfere with the activities of other cytokines, such as GM-CSF or IL-3.
Accordingly, one aspect of the present invention contemplates a modified IL-5 comprising a sequence of amino acids within a first a-helix wherein one or more exposed amino acids in said first a-helix having acidic or acidic-like properties are substituted with a basic amino acid residue or a non-acidic amino acid residue.
The IL-5 which is subject to modification is generally of mammalian origin such as from humans, primates, livestock animals (eg. sheep, cows, pigs, horses), laboratory test animals (eg. mice, rats, guinea pigs, rabbits), companion animals (eg. dogs, cats) and captive wild animals (eg. kangaroos, foxes, deer). Most preferably, the IL-5 is of human origin. The modified IL-5 of the present invention may be glycosylated or unglycosylated and does not interfere with GM-CSF or IL-3 activity.
Even more particularly, the present invention is directed to a modified human IL-5 molecule comprising a sequence of amino acids wherein Glu at amino acid position 13 (or its equivalent position) in a first a-helix is replaced by Arg or Lys or a chemical equivalent or derivative thereof. An alternative substitution may also be made using non-acidic amino acid residues such as but not limited to Gln and Asn or their chemical equivalent or derivatives. A “derivative” may be a naturally occurring or synthetic amino acid residue.
In accordance with the present invention, it is proposed that the modified IL-5 molecules defined above act as antagonists of the native form of IL-5. The term “modified” is considered herein synonymous with terms such as “variant”, “derivative” and “mutant”. The present invention is particularly directed to a modified IL-5 which exhibits specific antagonism of IL-5 mitogenic effects such as observed in vitro. The modified IL-5 molecules may be glycosylated or unglycosylated and do not interfere with GM-CSF or IL-3 activity.
Accordingly, another aspect of the present invention is directed to an IL-5 antagonist said antagonist comprising an IL-5 molecule having an amino acid sequence in its first at-helix wherein one or more exposed amino acids in said first &agr;-helix having acidic or acidic-like properties are substituted with a basic amino acid residue or a non-acidic amino acid residue.
More particularly, the present invention provides an antagonist of IL-5 said antagonist comprising an IL-5 molecule with Gln at position 13 (or its equivalent position) in a first &agr;-helix substituted by Arg or Lys or a chemical equivalent or derivative thereof An alternatively substitution may also be made using non-acidic amino acid residues such as but not limited to Gln and Asn or their chemical equivalents or derivatives.
The modified IL-5 molecule of the present invention is preferably in recombinant or synthetic form and, with the exception of the amino acid substitution(s) in the first a-helix, the amino acid sequence of the IL-5 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 amino acid sequence. It is then referred to as a “mutant” IL-5. The structure of the first &agr;-helix of IL-5 has been determined at 2.4 angstrom resolution by X-ray crystallography and comprises amino acid residues 7 to 27 or their equivalents (see Milburn et al.
Nature
363: 172-176, 1993). The modified IL-5 of the present invention may or may not comprise a leader sequence.
The nucleotide and corresponding amino acid sequence for the modified IL-5 having Arg in position 13 is shown in SEQ ID NOs: 1 and 2 and FIG.
1
. The leader sequence is shown as amino acids 1 to 6 (Met His Tyr His His His [SEQ ID NO:3]). Consequently, amino acids 7 to 27 are shown as amino acids 13-33 in SEQ ID NOs: 1 and 2 and in FIG.
1
. Reference to amino acids 7 to 27 is taken as amino acid residue numbers in a molecule without a leader sequence. The amino acid sequence for amino acids 7 to 27 is shown as SEQ ID NO:4 except that amino acid 13 is represented as Xaa. In accordance with the present invention Xaa is preferably a basic amino acid residue or a non-acidic amino acid residue.
Reference to “unglycosylated form” herein means that the molecule is completely unglycosylated such as when expressed in recombinant form in a prokaryotic organism (e.g.
E. coli
). Alternatively, a glycosylation-deficient mammalian cell may be used or complete deglycosylation may occur in vitro using appropriate enzymes. Different glycosylation patterns are encompassed by the present invention such as when recombinant molecules are produced in different mammalian cells.
An “exposed” amino acid is taken herein to refer to an amino acid on a solvent-exposed or outer portion of an &agr;-helix compared to those amino acids orientated towards the inside of the molecule.
An acidic amino acid includes, for example, Glu and Asp. Preferred basic amino acids are Arg and Lys. Preferred non-acidic amino acids are Gln and Asn.
According to another aspect of the present invention there is provided a modified IL-5 characterised by:
(i) comprising a sequence of amino acids within a first &agr;-helix,
(ii) having one or more exposed amino acids in said &agr;-helix which have acidic or acidic-like properties being substituted by a basic amino acid residue or a non-acidic amino acid residue;
(iii) being in recombinant or synthetic form; and
(iv) being capable of antagonising IL-5 mitogenic activity in vitro.
In a related embodiment, the present invention provides an IL-5 antagonist characterised by:
(i) comprising a sequence of amino acids within a first &agr;-helix;
(ii) having one or
Bastiras Stan
Hey Allan William
Lopez Angel
Shannon Frances
Vadas Matthew
BresaGen Limited
Murphy Joseph F.
Romeo David S.
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