Chemistry: natural resins or derivatives; peptides or proteins; – Peptides of 3 to 100 amino acid residues – Peptides containing saccharide radicals – e.g. – bleomycins – etc.
Reexamination Certificate
1998-06-10
2001-03-20
Mertz, Prema (Department: 1647)
Chemistry: natural resins or derivatives; peptides or proteins;
Peptides of 3 to 100 amino acid residues
Peptides containing saccharide radicals, e.g., bleomycins, etc.
C530S324000, C530S397000, C530S412000, C530S413000, C530S416000, C514S008100, C514S012200
Reexamination Certificate
active
06204359
ABSTRACT:
The present invention relates to the field of wound healing factors. More particularly, the present invention relates to a novel form of amphiregulin produced by keratinocytes, as well as analogs thereof, methods for their production, their uses, more particularly the use thereof in treatment of a variety of conditions including wound healing, skin disorders and cancer.
BACKGROUND OF THE INVENTION
After wounding of the skin, one of the objectives is to close the defect as quickly as possible. This is especially important in the case of extensive burns, in order to limit infection risk and fluid loss. Apart from several dressing and grafting techniques developed over the past decades, the use of cultured epidermal sheets has gained increasing attention. This technology allows the covering of the wound with a stratified sheet of living keratinocytes, derived either from the patients' own skin (autologous sheets) or from other donors (allogeneic sheets). Interestingly, although it has been shown that allogeneic keratinocyte sheets have only a limited survival time on the acceptor wound, they have the same clinical effect as autologous sheets when used in combination with split thickness meshed autografts or when applied on partial thickness wounds. This has been explained by hypothesising the existence of wound healing factors produced by these keratinocytes. These factors enhance the wound repair process by stimulating the proliferation and/or migration of the various cell types present in the wound. A new autologous epidermis can thus be formed by the outgrowth from keratinocyles present in the meshed skin autografts or epidermal appendages (e.g. sweat glands, hair follicles).
Although clinically successful in many cases, and sometimes lifesaving, cultured keratinocyte sheets suffer from a number of obvious disadvantages. They are extremely expensive, time-consuming to prepare and difficult to transport, apply and preserve for prolonged periods. Therefore, it would be desirable to develop a wound therapeutic based directly on keratinocyte-derived wound repair factors, without the need of using the living cells themselves.
Several growth factors with potential therapeutic interest in the field of wound healing have been identified or isolated already in keratinocytes. These include, among others, TGF-&agr;, bFGF, amphiregulin and HB-EGF. However, to date, none of these factors has proven to provide the same clinical potential as the living keratinocyte cultures.
One factor of particular interest is amphiregulin (AR). This is a heparin-binding glycoprotein of approximately 20 kDa which was originally purified from phorbol ester-treated MCF-7 human breast carcinoma cells. The factor belongs to the EGF-family of growth factors and stimulates the proliferation of several cell types (including keratinocytes and some fibroblast cell lines), while inhibiting the proliferation of other cells (including many carcinoma cell lines) (Shoyab et al., Proc. Natl. Acad. Sci. USA 85, 6528-6532 (1988); Shoyab et al., Science 243, 1074-1076 (1989)). Sequencing of AR revealed the existence of two forms, containing 78 and 84 amino acids, respectively (Shoyab et al, Science 243, 1074-1076 (1989), Plowman et al., Mol. Cell. Biol. 10, 1969-1981 (1990)). The N-termini of these two subforms were reported to be located at residues 101 and 107 of the preprotein sequence (taking the initiation methionine as residue No.1), whereas the C-terminus was reported to be located at residue 184 of the preprotein sequence. Later studies revealed however that the C-terminal processing site is located probably at least 2 amino acid residues downstream from the originally reported one (Adams et al., Biochim. Biophys. Acta 1266, 83-90 (1994); Thompson et al., J. Biol. Chem. 271, 17927-17931 (1996)). This means that the total length of the published amphiregulin subforms should be at least 80 and 86 amino acids, respectively. To avoid confusion, we will henceforth designate the subforms with their respective N-terminal- and C-terminal endpoints (taking the initiation methionine of the preprotein as residue No. 1). Glycosylation seems not to be important for biological activity of the molecule (Shoyab et al., Proc. Natl. Acad. Sci. USA 85, 6528-6532 (1988)). Later studies revealed that a major keratinocyte autocrine factor is also amphiregulin (Cook et al., Mol. Cell. Biol., 11, 2547-2557 (1991); Cook et al., In Vitro Cell. Dev. Biol. 28A, 218-222 (1992); Piepkorn et al., J. Cell. Physiol. 159, 114-120 (1994)). In cultured keratinocyte conditioned media also, two subforms starting at residues 101 and 107 of the preprotein were detected. A special feature of AR is that its biological activity is completely blocked in the presence of heparin sulphate. It has been reported to be the only growth factor displaying this property (Cook et al., Mol. Cell. Biol., 11, 2547-2557 (1991); Cook et al., In Vitro Cell. Dev. Biol. 28A, 218-222 (1992), Piepkorn et al., J. Cell. Physiol. 159, 114-120 (1994)). The isolation, properties, cloning and potential therapeutical use of AR are disclosed in U.S. Pat. No. 5,115,096 (May 19, 1992), assigned to Oncogen (Seattle).
AIMS OF THE INVENTION
It is an aim of the present invention to isolate and characterize a novel form of amphiregulin.
It is also an aim of the present invention to provide analogs of this novel form of amphiregulin.
It is also an aim of the present invention to provide a composition comprising said novel form of amphiregulin or its analogs.
It is also an aim of the present invention to provide methods for producing said novel form of amphiregulin.
It is further an aim of the present invention to provide pharmaceutical compositions comprising as an active ingredient the novel form of amphiregulin of the present invention.
It is further an aim of the present invention to provide methods of treating diseases comprising the use of said novel form of amphiregulin.
All the aims of the present invention have been met by the following embodiments as set out below.
SUMMARY OF THE INVENTION
The present invention relates to the discovery of a new form of amphiregulin, referred to as AR97-187. The new form differs from the hitherto described amphiregulin forms in that it contains 4 additional amino acids at its N-terminal end. It also differs from already described forms with respect to its interaction with heparin in that its biological activity is only marginally reduced in the presence of heparin. Possibly, AR97-187 is produced by differentiating keratinocytes. AR97-187 is of therapeutical interest for the treatment of a variety of conditions, including wound healing, skin disorders and cancer.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates more particularly to a polypeptide representing a novel form of amphiregulin referred to as AR97-187 (or AR88) and characterized by the novel N-terminal amino amino sequence extension: NH
2
-IVDD (SEQ ID NO 14).
As is described in detail in the examples section, a novel form of amphiregulin most probably containing at least 90 amino acids, and with the N-terminal end starting at residue 97 of the preprotein sequence, was identified by the present inventors. In the prior art, two other forms (starting respectively at residues 101 and 107 of the preprotein sequence) have been isolated. The novel form is produced by stratified keratinocyte cultures, cultured in a high calcium, serum-containing medium. These cultures are substantially different from those used in the prior art, particularly with respect to the differentiation state of the cells, and to the fact that they form multilayered, stratified sheets. Possibly, these differences constitute one reason why these cultures produce the presently identified novel form of amphiregulin.
The term “stratified” refers to sheets of keratinocytes comprising at least two cell layers, preferably at least three cell layers, more preferably at least 4 cell layers.
The medium which is used to obtain said stratified keratinocyte cultures essentially contains the following components: DMEM/H
Delaey Bernard
Raymackers Jos
Van Heuverswyn Hugo
Bierman, Muserlian and Lucas
Hamud Fozia
Innogenetics N.V.
Mertz Prema
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