Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Lymphokines – e.g. – interferons – interlukins – etc.
Reexamination Certificate
1998-11-18
2003-08-12
Ponnaluri, Padmashri (Department: 1639)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Lymphokines, e.g., interferons, interlukins, etc.
C530S350000, C530S324000, C530S325000
Reexamination Certificate
active
06605702
ABSTRACT:
The present invention relates to a chemotactic cytokine.
The accumulation of eosinophil leukocytes is a characteristic feature of IgE-mediated allergic reactions such as allergic asthma, rhinitis and eczema. Eosinophil accumulation also occurs in non-allergic asthma. The immediate bronchoconstriction in response to a provoking stimulus in the asthmatic involves mast cell activation and the release of constrictor mediators. This is followed after several hours in some individuals by a late bronchoconstrictor response associated with a massive influx of eosinophils (1). Repeated provocation results in chronic inflammation in the airways and a marked hyper-responsiveness to constrictor mediators. The magnitude of both the late response and the chronic hyper-responsiveness-correlates with the numbers of eosinophils present in the lung (2,3).
The present invention provides a chemoattractant protein capable of attracting eosinophils and of inducing eosinophil accumulation and/or activation in vitro and in vivo. The chemoattractant protein of the present invention is designated “eotaxin”.
Eotaxins are proteins of the C—C branch of the platelet factor 4 superfamily of chemotactic cytokines. Within the C—C branch of the platelet factor 4 superfamily of chemotactic cytokines, or chemokines, certain members have the property of attracting eosinophils in vitro and some may induce eosinophil accumulation in vivo. For example, the chemokines RANTES and MIP-1&agr; attract eosinophils in vitro while MCP-1 and MIP-1&bgr; do not. (“RANTES” denotes Regulated upon Activation in Normal T cells Expressed and Secreted, “MIP” denotes Macrophage Inflammatory Protein, and “MCP” denotes Monocyte Chemo-attractant Protein.)
Naturally-occurring cytokines within the platelet factor
4
superfamily of chemotactic cytokines may have marked inter-species variations in the amino acid sequence of the protein, and in the carbohydrate modifications of the protein, while retaining the same characteristic functional properties. Similar variations in structure may occur in cytokines obtained from different individuals within the same species. Many chemokines within the C—C branch of the platelet factor
4
superfamily show promiscuity of receptor binding, and the ability of different chemokines to bind to the same receptor is not necessarily dependent on a high degree of homology at the amino acid level. Accordingly, both interspecies and intraspecies variations in protein length, amino acid sequence and carbohydrate modifications are generally to be expected for eotaxins.
The ability to attract eosinophils and to induce eosinophil accumulation and/or activation in vitro and in vivo is a characteristic property of eotaxins. Furthermore, eotaxins generally show substantially no attractive effect for neutrophils in vivo. The eosinophil chemoattractant effect may be an inter-species effect, for example, guinea-pig eotaxin appears to be potent in inducing chemotaxis of human eosinophils in vitro.
An eotaxin may be obtained from an appropriate body fluid, for example, from bronchoalveolar lavage fluid obtained from a human or non-human subject, particularly an allergic subject after an allergen challenge, either experimentally induced or naturally incurred. Other sources of eotaxins are, for example, inflammatory exudate fluids and in vitro cultures of macrophages, lymphocytes, neutrophils, mast cells, airway epithelial cells, connective tissue cells, vascular endothelial cells and eosinophils themselves
For example, an eotaxin may be obtained from a sensitised guinea-pig after allergen challenge. Guinea-pig models are useful as they share many common features with the asthmatic response in man. Eotaxin obtainable from bronchoalveolar lavage fluid of a sensitised guinea-pig by sequential HPLC purification generally has a molecular weight in the range of from 6-16 kDa. (As indicated above, intraspecies molecular weight variations of this order of magnitude are observed in members of the platelet factor 4 superfamily.)
The amino acid sequence of a guinea-pig eotaxin is set out in SEQ. ID. NO. 1, SEQ. ID. NO. 2 and in
FIGS. 7 and 8
of the accompanying drawings. Other guinea-pig eotaxins will generally have at least 50% overall homology with the sequence shown in SEQ. ID. NO. 1 (
FIG. 7
) at the amino acid level. The homology may be at least 60%, for example at least 70%, for example at least 80% with the sequence set out in SEQ. ID. NO. 1 and in FIG.
7
.
Percentage homology in the present case is calculated on the basis of amino acids that are identical in corresponding positions in the two sequences under investigation. Conservative substitutions are not taken into account. In the calculation of percentage homology of a putative eotaxin molecule under investigation with the sequence shown in SEQ. ID. NO. 1 (
FIG. 7
) or with SEQ. ID. NO. 2 (
FIG. 8
) if the molecule under investigation has a different length from the eotaxin set out in SEQ. ID. NO. 1 or SEQ. ID. NO. 2 (
FIG. 7
or FIG.
8
), then the calculation is based on the amino acids in the portion of the molecule under investigation that overlaps with the sequence shown in SEQ. ID. NO. 1 (
FIG. 7
) or SEQ. ID. NO. 2 (FIG.
8
). Software packages for the alignment of amino acid sequences and the calculation of homology are available commercially, for example, the “Bestfit” program available from Genetics Computer Group Sequence Analysis Software, Madison, Wis., U.S.A.
Unless specified otherwise, the specific values of percentage homology between eotaxin and other chemotactic cytokines given in the present specification have been calculated on the basis of the eotaxin set out in SEQ. ID. NO. 1 (FIG.
7
).
As indicated above, eotaxins obtainable from species other than guinea-pigs, for example humans, will exhibit inter-species differences of the type demonstrated by other members of the C—C branch of the platelet factor 4 superfamily of chemokines, for example, differences in protein length, amino acid sequence and carbohydrate modifications. There may, for example, be variations in the C- and/or N-terminal residues. For example, it is expected that the molecular weight of an eotaxin from a species other than guinea-pig will generally fall within the range of from 6 kDa to 16 kDa, but in some cases an eotaxin may have a molecular weight less than 6 kDa or more than 16 kDa.
Similarly, it is expected that in general an eotaxin from a species other than guinea-pig will have at least 40% overall homology with the sequence set out in SEQ. ID. NO. 1 and in
FIG. 7
The homology may be at least 50%, for example at least 60%, for example at least 70%, for example at least 80% with the sequence set out in SEQ. ID. NO. 1 and in
FIG. 7
There may, however, be eotaxins from species other than guinea-pigs that have less than 40% homology with SEQ. ID. NO. 1 (FIG.
7
).
Eotaxins may be identified by any one or more of the characteristics set out above, in particular by their ability to attract and/or actuate eosinophils in vitro and cause their accumulation and/or activation in vivo. A characteristic that assists the identification of a molecule as an eotaxin is the lack of attractive effect on neutrophils.
The present invention provides a method of determining the ability of a substance to induce eosinophil accumulation and/or activation in vivo, that is to say, a method for testing putative eotaxins, which comprises administering the substance, generally intradermally, to a test animal previously treated with labelled, for-example
111
In-labelled, eosinophils and subsequently determining the number of labelled eosinophils at a skin site.
One in vitro method that may be used to test a putative eotaxin for the ability to attract and/or activate eosinophils in vitro is the ability of the substance to increase eosinophil intracellular calcium levels. Other general methods for determining chemotactic activity in vitro may be used to test putative eotaxins in vitro.
Confirmation that an eosinophil attractant is an eotaxin may also be made by consideration of sequence homology of t
Griffiths-Johnson David A.
Hsuan John J.
Jose Peter J.
Williams Timothy J.
Imperial College of Science Technology & Medicine
Nixon & Vanderhye
Ponnaluri Padmashri
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