AU775027B2 - Keratinocyte growth factor-2 products - Google Patents

Description

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AUSTRALIA

PATENTS ACT 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT

ORIGINAL

*e Name of Applicant: Amgen Inc.

Actual Inventors: Timothy Osslund and Linda Owens Narhi Address for Service: BALDWIN SHELSTON WATERS MARGARET STREET SYDNEY NSW 2000 Invention Title: 'KERATINOCYTE GROWTH FACTOR-2 PRODUCTS' Details of Original Application No. 47576/97 dated 15 Oct 1997 The following statement is a full description of this invention, including the best method of performing it known to me/us:- File: 31187AUP00 ~1 -la- KERATINOCYTE GROWTH FACTOR-2 PRODUCTS FIELD OF THE INVENTION The present invention relates to recombinant DNA technology and protein engineering. Specifically, the present invention relates to variants and derivatives of keratinocyte growth factor-2 (KGF-2), and to uses thereof.

BACKGROUND OF THE INVENTION Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common general knowledge in the field.

The complex process of tissue generation and regeneration is mediated by a number of protein factors sometimes referred to as soft tissue growth factors.

These molecules are generally released by one cell type and act to influence proliferation of other cell types (Rubin et al. (1989), Proc. Nat'l. Acad. Sci. USA, 86:802-806). There are also some growth factors released from cells that themselves have the capacity to respond to such growth factors. Some soft tissue growth factors are secreted by particular cell types and influence the proliferation, differentiation, and/or maturation of responsive cells in the development of: multicellular organisms (Finch et al. (1989), Science, 25 2I:752-755). In addition to their roles in developing organisms, some soft tissue growth factors are significant in the continued health and maintenance of more mature systems. For instance, in mammals there are many systems where rapid cell turnover occurs. Such systems include the skin and the gastrointestinal tract, both of which are comprised of epithelial cells.

Included within this group of soft tissue growth factors is a protein family of fibroblast growth factors (FGFs).

The fibroblast growth factor (FGF) family is now known to consist of at least fourteen members, namely GF-1 to FGF-10 and homologous factors PEF-1 to 2 FHF-4, which share a relatedness among primary structures: basic fibroblast growth factor, bFGF (Abraham et al. (1986), EMBO 5:2523-2528); acidic fibroblast growth factor, aFGF (Jaye et al. (1986), Science, 233:541-545); int-2 gene product, int-2 (Dickson Peters (1987), Nature, 326:833); hst/kFGF (Delli-Bovi et al. (1987), Cell, .5:729-737 and Yoshida et al. (1987), Proc. Natl. Acad. Sci. USA, 84:7305- 7309); FGF-5 (Zhan et al. (1988), Mol. Cell. Biol., a:3487-3495); FGF-6 (Marics et al. (1989), Oncogene, A:335-340); keratinocyte growth factor, KGF (Finch et al. (1989), Science, 21:752-755); hisactophilin ;(Habazzettl et al. (1992), Nature, 59:855-858); FGF-9 (Miyamoto et al. (1993), Mol. Cell Biol., 117):-.4251- 15 4259); and fibroblast growth factor-10, also known as keratinocyte growth factor-2, KGF-2 (PCT patent application WO 96/25422), the disclosures of which are hereby incorporated by reference. More recently, four homologous factors (or "FHFs") were identified from the human retina by a combination of random cDNA sequencing, searches of existing sequence databases and homology- !based polymerase chain reactions (Smallwood et al.

(1996), Proc. Natl. Acad. Sci. USA, 9:9850-9857). It has been proposed that FHF-1, FHF-2, FHF-3 and FHF-4 25 should be designated as FGF-11, FGF-12, FGF-13 and FGF- 14, respectively, in accordance with the recommendation of the Nomenclature Committee (Coulier et al. (1997), Journal of Molecular Evolution, 4A:43-56, the disclosure of which is hereby incorporated by reference).

WO 96/25422 describes the cloning, expression and purification of full-length (with signal sequence, residues Met 1 to Thr 3 6 of SEQ ID NO:2) and mature (without signal sequence, residues Cys 3 7 to Ser 2 0 8 of SEQ ID NO:2) XGF-2 in a bacterial expression system E. coli) and eukaryotic expression systems

II

baculovirus and COS cells). This reference further teaches that KGF-2 might be useful to stimulate cell growth and proliferation for new blood vessel growth or angiogenesis, the prevention of hair loss, the healing of dermal wounds and the differentiation of muscle cells, nervous tissue, prostate cells and lung cells.

Much remains to be learned regarding KGF-2, including modifications which can be made thereto to generate variant(s) and derivatives which retain some or all of the biological activity of KGF-2. Generally, the effects of any specific amino acid change or chemical derivatization upon biological activity of a protein will vary depending upon a number of factors, including whether or not modifications affect the three-dimensional structure or the receptor binding region of the protein. As neither the three-dimensional structure nor the receptor binding region of KGF-2 has been published, the knowledge within the art does not permit generalisation about the effects of specific amino acid modification or chemical derivatization to KGF-2.

a SUMMARY OF THE INVENTION It is an aim of one or more aspects of the present invention to provide variants and derivatives of KGF-2 that retain some of all of the biological activity of KGF-2.

The present invention is directed to KGF-2 protein product(s), as defined below.

These KGF-2 protein product(s) have general applicability and may retain some or all of the biological activity of KGF-2. In particular, there are provided variants of KGF-2.

Accordingly, in one aspect of the present invention there is provided a variant of a protein comprising amino acid residues Cys 37 to Ser 208 of SEQ ID NO:2 (KGF-2), said variant selected from a variant comprising the amino acid sequence of R-[Asn 71 -Pro 2 03

]-R

2 wherein R represents a methionylated or nonmethionylated amine group of Asn 71 or of aminoterminus amino acid residue(s) selected from the group: Tyr Ser-Tyr Arg-Ser-Tyr Val-Arg-Ser-Tyr (SEQ ID NO:9), His-Val-Arg-Ser-Tyr, (SEQ ID Arg-His-Val-Arg-Ser-Tyr (SEQ ID NO: 11), Gly-Arg-His-Val-Arg-Ser-Tyr (SEQ ID NO:12), -4- Ala-Gly-Arg-His-Val-Arg-Ser-Tyr (SEQ ID NO: 13), Ser-Ala-Gly-Arg-His-Val-Arg-Ser-Tyr (SEQ IID NO: 14), Ser-Ser-Ala-Gly-Arg-His-Val-Arg-Ser-Tyr (SEQ ID NO: Pro-Ser-Ser-Ala-Gly-Arg-His-Val-Arg-Ser-Tyr (SEQ ID NO: 16), Ser-Pro-Ser-Ser-Ala-Gly-Arg-His-Val-Arg-Ser-Tyr (SEQ ID NO: 17), Ser-Ser-Pro-Ser-Ser-Ala-Gly-Arg-His-Val-Arg-Ser-Tyr (SEQ IID NO: 18), Phe-Ser-Ser-Pro-Ser-Ser-Ala-Gly-Arg-His-Val-Arg-Ser-Tyr (SEQ ID NO: 19), Ser-Phe-Ser-Ser-Pro-Ser-Ser-Ala-Gly-Arg-His-Val-Arg-Ser-Tyr (SEQ ID NO Ser-Ser-Phe-Ser-Ser-Pro-Ser-Ser-Ala-Gly-Arg-His-Val-Arg-Ser-Tyr (S EQ ID NO:2 1), Ser-Ser-Ser-Phe-Ser-Ser-Pro-Ser-Ser-Ala-Gly-Arg-His-Val-Arg-Ser-Tyr (SEQ ID NO:22), Ser-Ser-Ser-Ser-Phe-Ser-Ser-Pro-Ser-Ser-Ala-Gly-Arg-His-Val-Arg-Ser-Tyr (SEQ ID NO:23), Ser-Ser-Ser-Ser-Ser-Phe-Ser-Ser-Pro-Ser-Ser-Ala-Gly-Arg-His-Val-Arg-Ser-Tyr (SEQ ID NO:24), Asn-Ser-Ser-Ser-Ser-Ser-Phe-Ser-Ser-Pro-Ser-Ser-Ala-Gly-Arg-His-Val-Arg-Ser-Tyr (SEQ ID Thr-Asn-Ser-Ser-Ser-Ser-Ser-Phe-Ser-Ser-Pro-Ser-Ser-Ala-Gly-Arg-His-Val-Arg-Ser- Tyr (SEQ ID NO:26), Ala-Thr-Asn-Ser-Ser-Ser-Ser-Ser-Phe-Ser-Ser-Pro-Ser-Ser-Ala-Gly-Arg-His-Va-Arg- Ser-Tyr (SEQ ID NO:27), Glu-Ala-Thr-Asn-Ser-Ser-Ser-Ser-Ser-Phe-Ser-Ser-Pro-Ser-Ser-Ala-Gly-Arg-His-Val- Arg-Ser-Tyr (SEQ ED NO:28), Pro-Glu-Ala-Thr-Asn-Ser-Ser-Ser-Ser-Ser-Phe-Ser-Ser-Pro-Ser-Ser-Ala-Gly-Arg-His- Val-Arg-Ser-Tyr (SEQ ID NO:29), Ser-Pro-Glu-Ala-Thr-Asn-Ser-Ser-Ser-Ser-Ser-Phe-Ser-Ser-Pro-Ser-Ser-Ala-Gly-Arg- His-VaI-Arg-Ser-Tyr (SEQ ID Val-Ser-Pro-Glu-Ala-Thr-Asn-Ser-Ser-Ser-Ser-Ser-Phe-Ser-Ser-Pro-Ser-Ser-Ala-Gly- Arg-H-is-Val-Arg-Ser-Tyr (SEQ ID NO:3 1), Met-Val-Ser-Pro-Glu-Ala-Thr-Asn-Ser-Ser-Ser-Ser-Ser-Phe-Ser-Ser-Pro-Ser-Ser-Ala- Gly-Arg,-His-Val-Arg-Ser-Tyr (SEQ ID NO:32), Asp- Met-V al-S er-Pro-G Iu-A la-Thr-Asn-S er-S er- Ser- Ser-S er-Phe-S er-S er-Pro-S er-S er- Ala-Gly-Arg-His-Val-Arg-Ser-Tyr (SEQ ID NO:33), Gln-Asp-Met-Val-Ser-Pro-Glu-Ala-Thr-Asn-Ser-Ser-Ser-Ser-Ser-Phe-Ser-Ser-Pro-Ser- Ser-Ala-Gly-Arg-His-Val-Arg-Ser-Tyr (SEQ IID NO:34), Gly-Gln-Asp-Met-Val-Ser-Pro-Glu-Ala-Thr-Asn-Ser-Ser-S er-Ser-Ser-Phe-Ser-Ser-Pro- Ser-Ser-Ala-Gly-Arg-His-Val-Arg-Ser-Tyr (SEQ DD NO: Leu-Gly-Gln-Asp-Met-Val-Ser-Pro-Glu-Ala-Thr-Asn-Ser-Ser-Ser-Ser-Ser-Phe-Ser-Ser- Pro-Ser-Ser-Ala-Gly-Axrg-His-Val-Arg-Ser-Tyr (SEQ ID NO:36), Ala-Leu-Gly-Gln-Asp-Met-Val-Ser-Pro-Glu-Ala-Thr-Asn-Ser-S er-Ser-Ser-Ser-Phe-Ser- Ser-Pro-Ser-Ser-Ala-Gly-Arg-His-Val-Arg-Ser-Tyr (SEQ ID NO:37), Gln-Ala-Leu-Gly-Gln-Asp-Met-Val-Ser-Pro-Glu-Ala-Thr-Asn-Ser-Ser-Ser-Ser-Ser-Phe- S er-S er-Pro-S er-S er-Al a-G ly-Arg-Hi s-Val- Arg,-S er-Tyr (SEQ DD NO:38), or Cys-Gln-Ala-Leu-Gly-Gln-Asp-Met-Val-Ser-Pro-Glu-Ala-Thr-Asn-Ser-Ser-Ser-Ser-Ser- Phe- Ser-S er-Pro- Ser-S er-Al a-Gly-Arg-Hi s-ValI-Arg-Ser-Tyr (SEQ ID NO:39), :and, wherein R 2 represents a carboxy group of Pro 23or of carboxy-termninal amino acid residues of: Met Met-Val Met-Val-Val Met-Val-Val-His (SEQ ID or Met-Val-Val-His-Ser (SEQ ID NO:4 provided however that R, and R 2 are not selected so as to reconstruct Cys 3 7 to Ser 20 8 of SEQ ID NO:2; a variant comprising at least one amino acid residue within Asn 6 to Met'7 of SEQ ID NO:2 being deleted or substituted with a non-native amino acid; a variant comprising at least one non-native amino acid residue being added within Asn 6 to Met 16of SEQ ID NO:2, and chemical derivatives thereof, a variant comprising at least one neutral or positively charged amino acid residue within amino acid residues 85-198 of SEQ ID NO:2 being deleted or substituted with a neutral residue or negatively charged residue, whereby a charge-change protein with reduced positive charge is generated, and chemical derivatives thereof, a variant comprising the substitution of at least one amino acid residue having a higher loop forming potential for an amino acid having a lower loop forming potential within a putative loop-forming region of amino acid residues 160-164 of SEQ ID NO:2, and chemical derivatives thereof; a variant comprising at least one naturally-occurring cysteine at position 37, 106 or 150 of SEQ ID NO:2 being deleted or substituted with a non-native amino acid residue, and chemical derivatives thereof; a variant comprising at least one amino acid within an N-linked or O-linked glycosylation site being deleted or substituted with a non-native amino acid, whereby the N-linked or O-linked glycosylation site is modified, and derivatives thereof; a variant comprising the addition or substitution of at least one non-native amino acid to generate an N-linked or O-linked glycosylation site, and chemical derivatives thereof; or a variant comprising a C-terminal addition of at least one domain of the constant region of a heavy chain of a human immunoglobulin, and chemical derivatives thereof.

The variant(s) of KGF-2 may be produced by recombinant genetic engineering techniques. In an alternative embodiment, a variant(s) of KGF-2 is synthesized by chemical techniques, or a combination of the recombinant and chemical techniques. A variant(s) of KGF-2 may be made in glycosylated or non-glycosylated form.

In one aspect, the present invention provides a protein selected from an amino acid sequence consisting of N-terminally methionylated or non-methionylated NH 2 -Ser-Tyr- [Asn-Ser 208 ]-COOH or NH 2 -Ala-Gly-Arg-His-Val-Arg-Ser-Tyr-[Asn'-Ser 208

]-COOH,

wherein [Asn 71 -Ser 208 represents residues 71 to 208 of SEQ ID NO.2.

In yet another aspect of the present invention there is provided a polynucleotide encoding a protein of the invention. Each such nucleic acid sequence may be used in the expression of a variant(s) of KGF-2 in a eukaryotic or prokaryotic host cell. The polynucleotides may also be used in cell therapy or gene therapy applications.

In still another aspect of the present invention there is provided a vector comprising a polynucleotide of the invention operatively linked to an amplification and/or expression control sequence.

*In yet another aspect of the present invention there is provided a prokaryotic or 30 eukaryotic host cell containing a polynucleotide or vector of the invention.

In another aspect of the present invention there is provided a method of producing a protein of the invention comprising culturing a host cell of the invention in a suitable culture medium. Typically, the variant of KGF-2 will be expressed by the cells and optionally, isolated from the host cells and/or the nutrient medium.

In a further aspect, the present invention provides a method for producing the protein selected from an amino acid sequence consisting of N-terminally methionylated or non-methionylated NH 2 -Ser-Tyr-[Asn 7 1 -Ser 2 0 8 ]-COOH or NH 2 -Ala-Gly-Arg-His-Val- Arg-Ser-Tyr-[Asn 7 1 -Ser2 0 8 ]-COOH, wherein [Asn 71 -Ser 208 represents residues 71 to 208 of SEQ ID NO.2 comprising growing a host cell in accordance with the invention in a suitable nutrient medium to express the encoded protein and isolating said protein from said cells or said nutrient medium.

In still a further aspect of the present invention there is provided KGF-2 protein(s), as defined below, attached to a water soluble polymer. For example, a variant(s) of KGF-2 may be conjugated to one or more polyethylene glycol molecules in order to improve pharmacokinetic performance by increasing the molecule's apparent molecule weight.

In another aspect of the present invention there is provided a pharmaceutical composition comprising a protein of the invention, or chemical derivative(s) of KGF-2 protein Typically, a variant(s) of KGF-2 may be formulated in association with a pharmaceutically acceptable vehicle. A variety of other formulation materials may be used to facilitate the manufacture, storage, handling, delivery and/or efficacy of a 20 variant(s) of KGF-2.

There are also provided methods of modulating the growth and differentiation of epithelial cells. Specifically, a patient in need of stimulation (including cytoprotection, proliferation and/or differentiation) of epithelial cells will be administered a therapeutically-effective or prophylactically-effective amount of a variant(s) of KGF-2 and/or a chemical derivative of KGF-2 protein(s).

Accordingly, in another aspect of the present invention there is provided a method of stimulating the production of epithelial cells comprising contacting such cells with an effective amount of a protein of the invention.

"i In still a further aspect of the present invention there is provided a method for the 30 prevention or treatment of a condition in a patient suffering from a condition selected from gastric ulcers, duodenal ulcers, erosions of the stomach and esophagus, inflammatory bowel disease, radiation- or chemotherapy-induced gut toxicity, hepatic cirrhosis, fulminant liver failure, acute viral hepatitis, a toxic insult to the liver, and diabetes, comprising administering to said patient a therapeutically effective amount of a protein of the invention.

In a further aspect of the invention there is provided the use of a protein of the invention in the manufacture of a medicament for stimulating the production of epithelial cells.

In another aspect of the present invention there is provided the use of a protein of the invention in the manufacture of a medicament for stimulating production of epithelial cells in a patient for the prevention or treatment of a condition, wherein said condition is selected from gastric ulcers, duodenal ulcers, erosions of the stomach and esophagus, inflammatory bowel disease, radiation- or chemotherapy-induced gut toxicity, hepatic cirrhosis, fulminant liver failure, acute viral hepatitis, a toxic insult to the liver, and diabetes.

In another aspect of the present invention there is provided use of a protein selected from an amino acid sequence consisting of N-terminally methionylated or nonmethionylated NH 2 -Ser-Tyr-[Asn 7 '-Ser2 0 8 ]-COOH or NH 2 -Ala-Gly-Arg-His-Val-Arg- Ser-Tyr-[Asn 71 -Ser 2 08 ]-COOH, wherein [Asn 71 -Ser 208 represents residues 71 to 208 of SEQ ID NO.2 for the preparation of a medicament for preventing or reducing gut toxicity in radiation and chemotherapy treatment regimes.

20 In still a further aspect of the present invention there is provided use of a protein selected from an amino acid sequence consisting of N-terminally methionylated or non- Smethionylated NH 2 -Ser-Tyr-[Asn 7 1 -Ser 2 0 8 ]-COOH or NH 2 -Ala-Gly-Arg-His-Val-Arg- Ser-Tyr-[Asn 7 -Ser 20 8 ]-COOH, wherein [Asn 7 -Ser 208 represents residues 71 to 208 of SEQ ID NO.2 for the preparation of a medicament for proliferation and/or differentiation of adnexal structures such as hair follicles, sweat glands, and sebaceous glands in patients with bums and other partial and full-thickness injuries.

In a related embodiment there is provided a variant of a protein comprising amino acid residues Cys 37 to Ser 208 of SEQ ID NO:2 (KGF-2), said variant selected from the i group consisting of a variant comprising AN41 KGF-2, AN40 KGF-2, AN39 KGF-2, AN38 KGF-2, AN37 KGF-2, AN36 KGF-2 and AN35 KGF-2, or R'-[Asn 7-Pro203]-R 2 proteins, wherein Ans 71 -Pro20 3 represents residues 71 through 203 of SEQ ID No: 2; and wherein 5d R1 represents a methionylated or nonimethionylated amine group of Asn 7 1 or of aminoterminus amino acid residue(s) selected from the group: Ser-Tyr Arg-Ser-Tyr Val-Arg-Ser-Tyr His-Val-Arg-Ser-Tyr, Arg-His-Val-Arg-Ser-Tyr Gly-Arg-His-Val-Arg-Ser-Tyr Ala-Gly-Arg-His-Val-Arg-Ser-Tyr Ser-Ala-Gly-Arg-His-Val-Arg-Ser-Tyr Ser-Ser-Ala-Gly-Arg-His-Val-Arg-Ser-Tyr Pro-Ser-Ser-Ala-Gly-Arg-His-Val-Arg-Ser-Tyr Ser-Pro-Ser-Ser-Ala-Gly-Arg-His-Val-Arg-Ser-Tyr Ser-Ser-Pro-Ser-Ser-Ala-Gly-Arg-His-Val-Arg-Ser-Tyr Phe-Ser-Ser-Pro-Ser-Ser-Ala-Gly-Arg-His-Val-Arg-Ser-Tyr Ser-Phe-Ser-Ser-Pro-Ser-Ser-Ala-Gly-Arg-His-VaI-Arg-Ser-Tyr Ser-Ser-Phe-Ser-Ser-Pro-Ser-Ser-Ala-Gly-Arg-His-Val-Arg-Ser-Tyr (SEQ ID NO:9), (SEQ ID NO: (SEQ ID NO: 11), (SEQ ID NO: 12), (SEQ ID NO: 13), (SEQ ID NO: 14), (SEQ ID NO: (SEQ ID NO: 16), (SEQ ID NO: 17), (SEQ ID NO: 18), (SEQ ID NO: 19), (SEQ ID (SEQ ID NO:2 1), Ser-Ser-Ser-Phe-Ser-Ser-Pro-Ser-Ser-Ala-Gly-Arg-His-Val-Arg-Ser-Tyr (SEQ ID NO:22), Ser-Ser-Ser-Ser-Phe-Ser-Ser-Pro-Ser-Ser-Ala-Gly-Arg-His-Val-Arg-Ser-Tyr (SEQ ID NO:23), Ser-Ser-Ser-Ser-Ser-Phe-Ser-Ser-Pro-Ser-Ser-Ala-Gly-Arg-His-Va-Arg-Ser-Tyr (SEQ ID NO:24), Asn-Ser-Ser-Ser-Ser-Ser-Phe-Ser-Ser-Pro-Ser-Ser-Ala-Gly-Arg-His-Val-Arg-Ser-Tyr (SEQ ID Thr-Asn-Ser-Ser-Ser-Ser-Ser-Phe-Ser-Ser-Pro-Ser-Ser-Ala-Gly-Arg-His-Va-Arg-Ser- Tyr (SEQ ID NO:26), Ala-Thr-Asn-Ser-Ser-Ser-Ser-Ser-Phe-Ser-Ser-Pro-Ser-Ser-Ala-Gly-Arg-Hi s-Val-Arg- Ser-Tyr (SEQ ID NO:27), Glu-Ala-Thr-Asn-Ser-Ser-Ser-Ser-Ser-Phe-Ser-Ser-Pro-Ser-Ser-Ala-Gly-Arg-His-Val- Arg-Ser-Tyr (SEQ ID NO:28), 5e Val-Arg-Ser-Tyr (SEQ ID NO:29), His-Val-Arg-Ser-Tyr (SEQ ED Val- Ser-Pro-G] u-Ala-Thr-Asn- Ser- Ser-Ser-Ser-S er-Phe-S er-S er-Pro-S er- Ser-Ala-Gly- Arg-His-Val-Arg-Ser-Tyr (SEQ ID NO:3 1), Met-Val-Ser-Pro-Glu-Ala-Thr-Asn-Ser-Ser-Ser-Ser-Ser-Phe-Ser-Ser-Pro-Ser-Ser-Ala- Gly-Arg-His-Val-Arg-Ser-Tyr (SEQ ID NO:32), Asp-Met-Val-Ser-Pro-Glu-Ala-Thr-Asn-Ser-Ser-Ser-Ser-Ser-Phe-Ser-Ser-Pro-Ser-Ser- Ala-Gly-Arg-His-Val-Arg-Ser-Tyr (SEQ lID NO:33), Gln-Asp-Met-Val-Ser-Pro-Glu-Ala-Thr-Asn-Ser-Ser-Ser-Ser-Ser-Phe-Ser-Ser-Pro-Ser- Ser-Ala-Gly-Arg-His-Val-Arg-Ser-Tyr (SEQ ID NO:34), Gly-Gin-Asp-Met-Val-Ser-Pro-Glu-Ala-Thr-Asn-Ser-Ser-Ser-Ser-Ser-Phe-Ser-Ser-Pro- Ser-Ser-Ala-Gly-Arg-His-Val-Arg-Ser-Tyr (SEQ ID NO:3 Leu-Gly-Gln-Asp-Met-Val-Ser-Pro-Glu-Ala-Thr-Asn-Ser-Ser-Ser-Ser-Se-Phe-Ser-Ser- Pro-Ser-Ser-Ala-Gly-Arg-His-Val-Arg-Ser-Tyr (SEQ IlD NO:36), Ala-Leu-Gly-Gln-Asp-Met-Val-Ser-Pro-Glu-Ala-Thr-Asfl-Ser-Ser-Ser-Ser-Ser-Phe-Ser- Ser-Pro-Ser-Ser-Ala-Gly-Arg-His-VaI-Arg-Ser-Tyr (SEQ ID NO :37), Gln-Ala-Leu-Gly-Gln-Asp-Met-Val-Ser-Pro-Glu-Ala-Thr-Asn-Ser-Ser-Ser-Ser-Ser-Phe- Ser-Ser-Pro-Ser-Ser-Ala-Gly-Arg-His-Val-Arg-Ser-Tyr (SEQ ED NO:38), or Cys-Gln-Ala-Leu-Gly-Gln-Asp-Met-Val-Ser-Pro-Glu-Ala-Thr-Asn-Ser-Ser-Ser-Ser-Ser- Phe-Ser-Ser-Pro-Ser-Ser-Ala-Gly-Arg-His-VaI-Arg-Ser-Tyr (SEQ ID NO:39), and, wherein R 2 represents a carboxy group of Pro20 or of carboxy-terminal amino acid residues of: Met Met-Val Met-Val-Val Met-Val-Val-His (SEQ ID or Met-Val-Val-His-Ser (SEQ ID NO:4 1), provided however, that R, and R 2 are not selected so as to reconstruct Cys 37 to Ser 208 of SEQ ID NO:2; a variant comprising at least one amino acid residue within Asn 168 to Met 176 of SEQ ID NO:2 being deleted or substituted with a non-native amino acid; a variant comprising at least one non-native amino acid being added within Asn' 68 to Met 7 6 of SEQ ID NO:2, and chemical derivatives thereof; a variant comprising at least one neutral or positively charged amino acid residue within amino acids 85-198 of SEQ ID NO:2 being deleted or substituted with a neutral residue or negatively charged residue, whereby a charge-change protein with reduced positive charge is generated, and chemical derivatives thereof; a variant comprising the substitution of at least one amino acid residue having a higher loop forming potential for an amino acid having a lower loop forming potential within a putative loop-forming region of amino acid residues 160-164 of SEQ ID NO:2, and chemical derivatives thereof; a variant comprising at least one naturally-occurring cysteine at position 37, 106 or 150 of SEQ ID NO:2 being deleted or substituted with a non-native amino acid residue, and chemical derivatives thereof; a variant comprising at least one amino acid within an N-linked or O-linked glycosylation site being deleted or substituted with a non-native amino acid, whereby the N-linked or O-linked glycosylation site is modified, and derivatives thereof; a variant comprising the addition or substitution of at least one non-native amino 20 acid to generate an N-linked or O-linked glycosylation site, and chemical derivatives •thereof; and a variant comprising a C-terminal addition of at least one domain of the constant region of a heavy chain of a human immunoglobulin, and chemical derivatives thereof.

Unless the context clearly requires otherwise, throughout the description and the claims, the words 'comprise', 'comprising', and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of"including, but not limited to".

Additional aspects and advantages of the invention will be apparent to those g skilled in the art upon consideration of the following description, which details the 30 practice of the present invention.

BRIEF DESCRIPTION OF THE FIGURES Numerous aspects and advantages of the present invention will become apparent upon review of the Figures, wherein: Figure 1 depicts a cDNA sequence (SEQ ID NO:1) encoding full-length, recombinant human KGF-2. Also depicted is the amino acid sequence (SEQ ID NO:2) of full-length, recombinant human KGF-2. The initial 36 amino acid residues (Met' to Thr 36 represent the putative leader sequence of full-length KGF-2, and residues Cys 37 to Ser 208 of SEQ ID NO:2 represent mature KGF-2. The full-length and mature forms are collectively termed "KGF-2".

Figure 2 depicts a cDNA sequence (SEQ ID NO:3) encoding dN29 Also depicted is the amino acid sequence (SEQ ID NO:4) of dN29 o 6 Figure 3 depicts a cDNA sequence (SEQ ID encoding dN20 hFGF10. Also depicted is the amino acid sequence (SEQ ID NO:6) of dN20 Figure 4 depicts a cDNA sequence (SEQ ID NO:7),encoding hFGF10 R149Q. Also depicted is the amino acid sequence (SEQ ID NO:8) of hFGF10 R149Q.

DETAILED DESCRIPTION OF THE INVENTION KGF-2 protein(s) In accordance with the terms of this invention, by the term "KGF-2 protein(s)" is meant the *";protein defined by amino acids Cys 37 to Ser 2 0 8 of SEQ ID NO:2 (mature KGF-2) and variant proteins thereof.

15 The term "KGF-2 protein(s)" thus includes a protein in which one or more amino acid residues have been deleted from ("deletion variant(s)"), inserted into ("addition variant(s)"), and/or substituted for ("substitution variant(s)") residues within the amino acid sequence of 20 SEQ ID NO:2 and which retains biological activity.

Thus, while the descriptions below of protein modifications refer to mature KGF-2, it does not preclude additional modifications thereto.

The term "biological activity" as used herein means that a KGF-2 protein(s) possesses some but not necessarily all the same properties of (and not necessarily to the same degree as) mature KGF-2. The S* selection of the particular properties of interest depends upon the desired use of the desired KGF-2 protein(s).

It will be appreciated by those skilled in the art that many combinations of deletions, insertions, and substitutions can be made, provided that the final protein is biologically active. There are two principal variables in the construction of amino acid sequence 7 variant(s): the location of the mutation site and the nature of the mutation. In designing a variant(s), the location of the mutation site and the nature of the mutation will depend on the biochemical characteristic(s) to be modified. Mutation sites can be modified individually or in series, by deleting the target amino acid residue, inserting amino acid residues adjacent to the located site or substituting first with conservative amino acid choices and, depending upon the results achieved, then with more radical selections.

Amino acid sequence deletions generally range from about 40 amino acid residues, from about 30 amino acids, from about 20 amino acids, and typically from about 1 to 10 residues. Deletions within the amino acid sequence of mature KGF-2 may be made, for example, in regions of low homology with the sequences of other members of the FGF family. Deletions within the amino acid sequence of mature KGF-2 in areas of substantial homology with the sequences of other members of the FGF -family will be more likely to significantly modify the biological activity. The number of total deletions and/or consecutive deletions preferably will be selected so as to preserve the tertiary structure of mature KGF-2 (amino acids Cys 37 to Ser 2 0 8 of SEQ ID NO:2) in the affected domain, cysteine crosslinking.

•Amino acid sequence additions may include amino- and/or carboxyl-terminal fusions ranging in length from one residue to one hundred or more residues, as well as internal intrasequence insertions of single or multiple amino acid residues. Internal additions may range preferably from about 1 to 10 amino acid residues, more preferably from about 1 to 5 amino acid residues, and most preferably from about 1 to 3 amino acid residues. Additions within the amino acid sequence of 8 mature KGF-2 may be made in regions of low homology with the sequences of other members of the FGF family.

Additions within the amino acid sequence of mature KGF-2 in areas of substantial homology with the sequences of other members of the FGF family will be more likely to significantly modify the biological activity.

Insertions or additions preferably include amino acid sequences derived from the sequences of other FGF family members.

An amino-terminus addition is contemplated to include the addition of a methionine (for example, as an artifact of the direct expression in bacterial recombinant cell culture) or an amino acid residue or sequence of mature KGF-2. A further example of an N- S 15 terminal addition includes the fusion of a signal S.sequence to the N-terminus of mature KGF-2 in order to facilitate the secretion of protein from recombinant host cells. Such signal sequences generally will be obtained from and thus be homologous to the intended 20 host cell species. Included within the scope of this invention is the native signal sequence, for example, the native signal sequence of the protein defined by amino acids Met 1 to Thr 3 6 of SEQ ID NO:2 or a heterologous signal sequence. A heterologous signal sequence selected should be one that is recognized and processed cleaved by a signal peptidase) by the host cell. For prokaryotic host cells that do not .recognize and process the native signal sequence, the signal sequence may be substituted by a prokaryotic signal sequence selected, for example, from the group of the alkaline phosphatase, penicillinase or heat-stable enterotoxin II leaders. For yeast secretion, the signal sequence may be selected, for example, from the group of the yeast invertase, alpha factor or acid phosphatase leader sequences. In mammalian cell expression 9 specifically, signal sequences of full-length KGF-2 or of other FGF family members KGF) may be suitable.

An example of a carboxy-terminus addition includes chimeric proteins comprising the fusion of KGF- 2 with all or part of the constant domain of the heavy or light chain of human immunoglobulin. Such chimeric polypeptides are preferred wherein the immunoglobulin portion comprises all the domains except the first domain of the constant region of the heavy chain of a human immunoglobulin such as IgG, IgA, IgM or IgE, especially IgG, IgG1 or IgG3. A skilled artisan will appreciate that any amino acid of the immunoglobulin portion can be deleted or substituted by one or more amino acids, or one or more amino acids can 15 be added as long as the KGF-2 portion still stimulates epithelial cells and the immunoglobulin portion shows one or more of its characteristic properties.

Another group of variant(s) is amino acid substitution variant(s) of the amino acid sequence of 20 mature KGF-2. These variant(s) have at least one amino acid residue in the sequence of Cys 37 to Ser 208 of SEQ ID NO:2 removed and a different residue inserted in its place. Substitution variant(s) include allelic variant(s), which are characterized by naturally-occurring nucleotide sequence changes in the species population that may or may not result in an amino acid change. One skilled in the art can use any information known about the binding or active site of the polypeptide in the selection of possible mutation sites.

One method for identifying amino acid residues or regions for mutagenesis is called "alanine scanning mutagenesis", as described by Cunningham and Wells (1989), Science, 244:1081-1085, the disclosure of which is hereby incorporated by reference. In this 10 method, an amino acid residue'or group of target residues is identified charged residues such as Arg, Asp, His, Lys, and Glu) and replaced by a neutral or negatively-charged amino acid (most preferably alanine or polyalanine) to effect the interaction of the amino acids with the surrounding aqueous environment in or outside the cell. Those domains demonstrating functional sensitivity to the substitutions are then refined by introducing additional or alternate residues at the sites of substitution. Thus, the site for introducing an amino acid sequence modification is predetermined and, to optimize the performance of a mutation at a given site, alanine scanning or random mutagenesis may be conducted and the variant(s) may be 15 screened for the optimal combination of desired activity and degree of activity.

The sites of greatest interest for substitutional mutagenesis include sites in which particular residues within amino acids Cys 37 to Ser 208 20 of SEQ ID NO:2 are substantially different from various species or other FGF family members in terms of side-chain bulk, charge, and/or hydrophobicity. Other sites of interest include those in which particular residues within amino acids Cys 37 to Ser 2 0 8 of SEQ ID NO:2, are identical among various species or other FGF family members. Such positions are generally important S for the biological activity of a protein. Accordingly, a skilled artisan would appreciate that initially these sites should be modified by substitution in a relatively conservative manner.

Such conservative substitutions are shown in Table 1 under the heading of "Preferred Substitutions".

If such substitutions result in a change in biological activity, then more substantial changes (Exemplary Substitutions) may be introduced and/or other 11 additions/deletions may be made and the resulting products screened.

TABLE 1: Amino Acid Substitutions original Preferred Exemplary Residue Substitutions Substitutions Ala Val Val; Leu; Ile Arg Lys Lys; Gin; Asn Asn Gi-n Gin; His; Lys; Arg Asp Giu Giu Cys Ser Ser Gin Asfl Asn Giu Asp Asp Giy Pro Pro His Arg Asn; Gin; Lys; Arg Ile Leu Leu; Vai; Met; Ala; Phe; norleucine Leu Ile norleucine; Ile; Vai; Met; Ala; Phe Lys Arg Arg; Gin; Asn Met Leu Leu; Phe; Ile Phe Leu Leu; Val; Ile; Ala Pro Giy Giy Ser Thr Thr Thr Ser Ser Trp Tyr Tyr Tyr Phe Trp; Phe; Thr; Ser Val Leu Ile; Leu; Met; Phe; Ala; norieucine In making such changes of an equivalent nature, the hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index in conferring interactive biologicai function on a protein is generally understood in the art (Kyte and Doolittie (1982), J. Mol. Biol., lL7:105-i31, 12 the disclosure of which is incorporated herein by reference). It is known that certain amino acids may be substituted for other amino acids having a similar hydropathic index or score and still retain a similar biological activity.

It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity, particularly where the biological functional equivalent protein or peptide thereby created is intended for use in immunological embodiments, as in the present case. U.S. Patent 4,554,101, the disclosure of which is incorporated herein by reference, states that the greatest local average hydrophilicity of a protein, as governed by the 15 hydrophilicity of its adjacent amino acids, correlates with its immunogenicity and antigenicity, with a biological property of the protein.

U.S. Patent 4,554,101 also teaches the identification and preparation of epitopes from primary 20 amino acid sequences on the basis of hydrophilicity.

Through the methods disclosed in U.S. Patent 4,554,101 a skilled artisan would be able to identify epitopes, for :example, within the amino acid sequence of KGF-2. These regions are also referred to as "epitopic core regions".

Numerous scientific publications have been devoted to the prediction of secondary structure, and to the identification of epitopes, from analyses of amino acid sequences (Chou and Fasman (1974), Biochemistry, 13(2):222-245; Chou and Fasman (1974), Biochemistry, 13(2):211-222; Chou and Fasman (1978), Adv. Enzymol.

Relat. Areas Mol. Biol., 41:45-148; Chou and Fasman (1978), Ann. Rev. Biochem., 47:251-276 and Chou and Fasman (1979), Biophys. 26:367-384, the disclosures of which are incorporated herein by reference).

13 Moreover, computer programs are currently available to assist with predicting antigenic portions and epitopic core regions of proteins. Examples include those programs based upon the Jameson-Wolf analysis (Jameson and Wolf (1988), Comput. Appl. Biosci., 4lI):181-186 and Wolf et al. (1988), Comput. Appl. Biosci., 4(l):187-191, the disclosures of which are incorporated herein by reference); the program PepPlot® (Brutlag et al. (1990), CABS, 6_:237-245 and Weinberger et al. (1985), Science, 228:740-742, the disclosures of which are incorporated herein by reference); and other new programs for protein tertiary structure prediction (Fetrow and Bryant (1993), 00 BIOTEChNOLOGY, U1:479-483, the disclosure of which is 0 incorporated herein by reference).

In contrast, substantial modifications in the functional and/or chemical characteristics of the amino .o acids Cys 3 7 to Ser 2 0 8 of SEQ ID NO:2 may be accomplished by selecting substitutions that differ significantly in their effect on maintaining the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, the relative charge or hydrophobicity of the protein at the *target site or the bulk of the side chain.

Naturally-occurring residues are divided into groups based on common side chain properties: 1 hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; 2) neutral hydrophilic: Cys, Ser, Thr; 3) acidic: Asp, Glu; 4) basic: Asn, Gln, His, Lys, Arg; residues that influence chain orientation: Gly, Pro; and 6) aromatic: Trp, Tyr, Phe.

14 Non-conservative substitutions may involve the exchange of a member of one of these groups for another. Such substituted residues may be introduced into regions of amino acids Cys 3 7 to Ser 2 0 8 of SEQ ID NO:2 that, for example, are homologous with regions of other FGF family members or into the non-homologous regions of the protein.

In a specific embodiment, a variant polypeptide will preferably be substantially homologous to amino acids Cys 3 7 to Ser 2 0 8 of SEQ ID NO:2. The term "substantially homologous", as used herein, means having Sa degree of homology identity of amino acid residues) to amino acids Cys 3 7 to Ser 2 0 8 of SEQ ID NO:2 in excess of eighty percent preferably, in excess 15 of ninety percent more preferably, in excess of ninety-five percent and most preferably, in excess of ninety-nine percent The percentage of homology as described herein is calculated as the percentage of amino acid residues found in the smaller of the two sequences which align with identical amino acid residues in the sequence being compared when four .o gaps in a length of 100 amino acids may be introduced to assist in that alignment, as set forth by Dayhoff (1972), in Atlas of Protein Sequence and Structure, 25 5:124, National Biochemical Research Foundation, Washington, the disclosure of which is hereby incorporated by reference. Also included as substantially homologous are variant(s) of the amino acids Cys 3 7 to Ser 208 of SEQ ID NO:2 which may be isolated by virtue of cross-reactivity with antibodies to amino acids Cys 37 to Ser 2 0 8 of SEQ ID NO:2, or whose genes may be isolated through hybridization with the DNA of SEQ ID NO:1 or with segments thereof.

A first class of variant(s) is a group of deletion variants of Cys 3 7 to Ser 2 0 8 of SEQ ID NO:2.

15 These variants include R2-(Asn 7 1 -Pro 2 0 3 J-COOH proteins, and further include an amino acid sequence comprising

NH

2 (His* 72 -Ser 2 0 8 J]-COOH (also referred to as AN35 KGF-2),

NH

2 [Leu 7 3 -Ser 2 0 8 _COOH (also referred to as AN36 KGF-2),

NH

2 fGln 7 4 -Ser 2 0 8 -COOH (also referred to as AN37 KGF-2),

NH

2 -[CGly 7 5 Ser 2 0 8 ]-COOH (also referred to as AN38 KGF-2),

NH

2 -[(Asp 7 6 -Ser 2 0 8 J -COOH (also referred to as AN39 KGF-2), N1 2 -([Val 7 7 -Ser 2 0 8 ]J-COOH (also referred to as AN40 KGF-2) and NH 2 (Arg 7 8 -Ser 20 8 -COON (also referred to as AN41 KGF-2), in which each may be N-terminally .*:.methionylated or non-methionylated, provided however that Cys37 to Ser 2 0 8 of SEQ ID NO:2 is excluded.

By "Rj-[Asn 71 -Pro 20 3 ]_COOH. is meant a group of deletion variant(s), wherein [Asn 7 1 _pro 2

O

3 15 represents residues 71 through 203 of SEQ ID NO:2; wherein R 1 represents a methionylated or nonmethionylated amine group of Asn 7 1 or of aminoterminus amino acid residue(s) selected from the group: Tyr Ser-Tyr V. Arg- Ser-Tyr Val-Arg-Ser-Tyr (SEQ ID NO:9), His-Val-Arg-Ser-Tyr (SEQ ID NO:lO), Arg-His-Val-Arg-Ser-Tyr (SEQ ID NO:11), *Gly-Arg- His-Val-Arg-Ser-Tyr (SEQ ID NO:12), Ala-Gly-Arg-His-Val--Arg-Ser-Tyr (SEQ ID NO: 13), Ser-Ala-Gly-Arg-His-Val-Arg-Ser-Tyr (SEQ ID NO: 14), Ser-Ser-Ala-Gly-Arg-His-Val-Arg-Ser-Tyr (SEQ ID Pro-.Ser-Ser-Ala-Gly-Arg-Hiis-Val -Arg-Ser -Tyr (SEQ ID NO:16), Ser-Pro-Ser-Sex-Ala-Gly-Arg-His-Val-Arg-Ser-Tyr (SEQ ID NO:17), 16 Ser-Ser-Pro-Ser-Ser-Ala-Gly-Arg-His-Val-Arg-Ser-Tyr (SEQ 11D NO:18), Phe -Ser -Ser Pro- Ser -Ser-Ala-Gly-Arg-His -Val -Arg-Ser-Tyr (SEQ ID NO:19), Ser-Phe-Ser-Ser-Pro-Ser-Ser-Ala-Gly-Arg-His-val-Arg- Ser-Tyr (SEQ ID Ser-Ser-Phe-Ser-Ser--Pro-Ser-Ser-Ala-Gly-Arg-His-Val- .Arg-Ser-Tyr SEQ ID NO:21), Ser-Ser-Ser-Phe-Ser-Ser-Pro-Ser-Ser-AJla-Gly-Arg-His- Val-Arg-Ser-Tyr SEQ ID NO:22), Ser-Ser-Ser-Ser-Phe-Ser-Ser-Pro-Ser-Ser-Ala-Gly-Arg- His-Val-Arg-Ser-Tyr (SEQ ID NO:23), Ser-Ser-Ser-Ser-Ser-Phe-Ser-Ser-Pro-Ser-Ser-Ala-Gly- **Arg-His-Val-Arg-Ser-Tyr (SEQ ID NO:24), Asn-Ser-Ser-Ser-Ser-Ser-Phe-Ser-Ser-Pro-Ser-Ser-Ala- **Gly-Arg-His-Val-Arg-Ser-Tyr (SEQ ID NO:2 Thr-Asn-Ser-Ser-Ser-Ser-Ser-Phe-Ser-Ser-Pro-Ser-Ser- Ala-Gly--Arg-His-Val-Arg-Ser-Tyr (SEQ ID NO:26), Ala-Thr-Asn-Ser- Ser-Ser-Ser-Ser-Phe-Ser--Ser-Pro-Ser- Ser-Ala-Gly-Arg-His-Val-Arg-Ser-Tyr (SEQ ID NO:27), Glu-Ala-Thr-Asn-Ser- Ser-Ser-Ser-Ser-Phe-Ser-Ser-Pro- Ser-Ser-Ala-Gly-Arg-Hi s-Val-Arg-Ser-Tyr (SEQ ID INO:28), Pro-Glu-Ala-Thr-Asn-Ser-Ser-Ser-Ser-Ser-Phe-Ser-Ser- Pro-Ser-Ser-Ala-Gly-Arg-His -Val -Arg-Ser-Tyr (SEQ ID NO:29), Ser-Pro-Glu-Ala-Thr-Asn-Ser-Ser-Ser-Ser-Ser-Phe- Ser- Ser- Pro-Ser-Ser-Ala-Gly-Arg-His -Val -Arg-Ser-Tyr (SEQ ID NO:3O), Val-Ser-Pro-Glu-Akla-Thr-Asn-Ser-Ser-Ser-Ser-Ser-Phe- Ser-Ser-Pro-Ser-Ser-Ala-Gly-Arg-His -Val -Arg-Ser-Tyr (SEQ ID NO:31), Met-Val-Ser-Pro-Glu-Ala-Thr-Asn-Ser-Ser-Ser-Ser-Ser- Phe-Ser-Ser-Pro-Ser-Ser-Ala-Gly-Arg-His-Val-Arg-Ser-Tyr (SEQ ID NO:32), 17 Asp-Met-Val-Ser-Pro-Glu-Ala-Thr-Asn-Ser-Ser-Ser-Ser- Ser-Phe-Ser-Ser-Pro-Ser-Ser-Ala-Gly-Arg-His -Val-Arg- Ser-Tyr (SEQ ID NO:33), Gln-Asp-Met-Val-Ser-Pro-Glu-Ala-Thr-Asn- Ser-Ser-Ser- Ser-Ser-Phe-Ser-Ser-Pro-Ser-Ser-Ala-Gly-Arg-H.s -Val- Arg-Ser-Tyr (SEQ ID NO:34), Gly-Gln-Asp-Met-Val -Ser-Pro-Glu-Ala-Thr-Asn-Ser-Ser- .Ser-Ser-Ser- Phe-Ser-Ser-Pro-Ser-Ser-Ala-Gly-Arg-His Val-Arg-Se.r-Tyr (SEQ ID Leu-Gly-Glh-;-Asp-Met -Val -Ser- Pro -Glu-Ala-Thr--Asn-Ser- Ser--Ser-Ser-Ser- Phe-Ser-Ser-Pro-Ser-Ser-Ala-Gly-Arg- His-Val-Arg-Ser-Tyr (SEQ ID NO:36), Ala-Leu-GJly-Gln-Asp-Met-Val -Ser-Pro--Glu-Ala-Thr-Asn- Ser-Ser-Ser--Ser-Ser-Phe-Ser-Ser-Pro-Ser-Ser-Ala-Gly- **Arg-His-val-Arg-Ser-Tyr (SEQ ID NO:37), Gln-Ala-Leu-Gly-Gln-Asp-Met-Val -Ser-Pro -Glu-Ala-Thr- Asn-Ser-Ser-Ser-Ser-Ser-Phe-Ser-Ser-Pro-Ser-Ser-Ala- Gly-Arg-His--Val-Arg-Ser-Tyr (SEQ TD NO:38), or Cys-Gln-Ala-Leu-Gly-Gln-Asp-Met-Val -Ser-Pro-Glu-Ala- Thr-Asn-Ser-Ser-Ser-Ser-Ser-Phe-Ser-Ser-Pro-Ser-Ser- Ala-Gly-Arg-His-Val-Arg-Ser-Tyr (SEQ ID NO:39), and, wherein R2 represents a carboxy group of Pro 203 or of carboxy-terininal amino acid residues of: Met Met -Val Met -Val -Val Met-Val-Val-His (SEQ ID or Met-Val--Val-His-Ser (SEQ ID lqO:41), provided however, that R 1 and R 2 are not selected so as to reconstruct Cys 3 7 to Ser 2 0 8 of SEQ ID NO:2.

Preferred variant(s) within this class include the following molecules: AN36 KGF-2; AN35 KGF-2; is 2 [Asn 7 l -Ser 2 0 8 J]-COOH (also referred to as AN3 4 KGF-2);

NH

2 -Tyr- [Asn 7 1 _Ser 20 8 ]-COOH (also referred to as AN33 KGF-2) NH 2 -Ser-Tyr- [Asn 7 l-Ser 20 8 -COOH (also referred to as AN32 KGF-2); NH2-Arg-Ser-Tyr-[Asn 7 l-Ser 2 O8]-COOH (also referred to as AN31 KGF-2); NH2-Val-Arg-Ser-Tyr-[Asn 7 1_ Ser 208 -COOH (also referred to as AN30 KGF-2); NH 2 -His- .Val -Arg-Ser-Tyr- [Asn 7 1 Ser 2 8 ]-COOH (also referred to as zN29 KGF-2); NH 2 -Arg-His -Val -Arg- Ser-Tyr- [Asn 7 1 -Ser 2 0 8 COGH (also, referred to as AN28 KGF-2) LNH2-Gly-Arg-His- OV 10 Val -Arg-Ser-Tyr- Asn 7 -Ser 2 8 COOH (also referred to as AN27 KGF-2); and NH2-Ala-Gly-Arg-His-Val-Arg-Ser-Tyr- (Asn 7 1 -5er 2 0 8 ]-COOH (also referred to as A1426 KGF-2), *either methionylated or nonmethionylated.

A second class of variant(s) is a group of substitution, deletion or addition variant(s)*of KGF-2 and/or of the first class of KGF-2 variant(s), described above, having a region corresponding to Asn 1 6 8 to Met 1 7 6 of SEQ ID NO:2 wherein at least one amino acid residue within the region corresponding to Asn 1 6 8 to Met 1 7 6 of SEQ ID NO:2 is deleted or substituted with a non-native amino acid, or a non-native amino acid is added within the region corresponding to Asn 1 6 8 to Met 1 7 6 of SEQ ID NO:'2; this region is unique among the FGF family and *:contains residues (Trp- 1 6 9 and His' 7 1 which may predominantly confer binding specificity and residues (Gly 1 7 3 and Met' 7 6 which may predominantly stabilize the structure to the region. In a specific embodiment, the region corresponding to Asn 1 6 8 to Met 1 7 6 is deleted or replaced with the following sequence: NH2-Ala-Lys-Trp- Thr-His-Asn-Gly-Gly-Glu-Met-COOH, which is the sequence of a putative receptor binding region of KGF.

A third class of variant(s) is a group of deletion or substitution variant(s) of KGF-2 and/or of 19 the first class of KGF-2 variant(s) and/or of the second class of KGF-2 variant(s), described above, having a region corresponding to Phe 85 to Ser 1 9 8 of SEQ ID NO:2 wherein at least one neutral or positively charged amino acid residue within the region corresponding to Phe 85 to Ser 198 of SEQ ID NO:2 is deleted or substituted with a neutral residue or negatively charged residue selected to effect a charge-change protein with a reduced positive charge. Preferred residues for modification are residues corresponding to Phe 85 Thr 86 Asn 1 5 9 Gly 182 ArgI 87 Asn1 96 Thr 197 Ser 198 of SEQ ID NO:2, .with residues Thr 8 6 Gly 1 82 Arg 1 8 7 and Asn 1 9 6 being more preferred. Preferred amino acids for substitution include alanine, glutamic acid, aspartic acid, 15 glutamine, asparagine, glycine, valine, leucine, isoleucine, serine and threonine; with alanine, glutamic acid, glutamine, aspartic acid and asparagine being more preferred; and with alanine being most preferred.

A fourth class of variant(s) is a group of 20 substitution variant(s) of KGF-2 and/or of the first class of KGF-2 variant(s) and/or of the second class of KGF-2 variant(s) and/or of the third class of KGF-2 variant(s), described above, having a region corresponding to a putative surface loop-forming region, Asn 1 6 0 to Thr 1 6 4 of SEQ ID NO:2, wherein at least one S" amino acid having a higher loop forming potential is substituted for at least one amino acid having a lower loop forming potential within the region corresponding to Asn 1 6 0 to Thr 1 6 4 of SEQ ID NO:2. The non-native amino acid is selected for its higher loop-forming potential in order to stabilize this area of the protein. Amino acids having relatively high loop-forming potential include glycine, proline, tyrosine, aspartic acid, asparagine, and serine. Leszcynski et al, Science, 234, 849-855 (1986) (relative values of loop-forming 20 potential assigned on the basis of frequency of.

appearance in loop structures of naturally occurring molecules). Preferably, a different amino acid having higher loop-forming potential replaces a threonine residue corresponding to Thr 164 of SEQ ID NO:2 in the loop-forming sequence.

A fifth class of variant(s) is a group of substitution variant(s) of KGF-2 and/or of the first class of KGF-2 variant(s) and/or of the second class of KGF-2 variant(s) and/or of the third class of KGF-2 variant(s) and/or of the fourth class of KGF-2 variant(s), described above, having amino acid residues corresponding to Cys 3 7 Cys 1 0 6 or Cys 150 of SEQ ID NO:2 wherein at least one naturally-occurring cysteine 15 residue at a position corresponding to position 37, 106 or 150 of SEQ ID NO:2 is deleted or substituted with a non-native amino acid residues Ala, Leu, or Ser).

A sixth class of variant(s) is a group of substitution or deletion variant(s) of KGF-2 and/or of 20 the first class of KGF-2 variant(s) and/or of the second class of KGF-2 variant(s) and/or of the third class of KGF-2 variant(s) and/or of the fourth class of KGF-2 variant(s) and/or of the fifth class of KGF-2 variant(s), described above, having at least one N- 25 linked or O-linked glycosylation site corresponding to :an N-linked or 0-linked glycosylation site within Cys 37 to Ser 2 0 8 of SEQ ID NO:2. Such variant(s) have at least one amino acid within the N-linked or O-linked glycosylation site within the region corresponding to an N-linked or 0-linked glycosylation site within Cys 37 to Ser 2 0 8 of SEQ ID NO:2 deleted or substituted with a non-native amino acid, to modify the N-linked or O-linked glycosylation site and generate a protein with altered glycosylation. An asparagine-linked glycosylation recognition site comprises a tripeptide 21 sequence which is specifically recognized by appropriate cellular glycosylation enzymes. These tripeptide sequences are either Asn-Xaa-Thr or Asn-Xaa-Ser, where Xaa can be any amino acid other than Pro. Proven or predicted asparagine residues exist at positions 51 and 196 of amino acids Cys 3 7 to Ser 2 0 8 of SEQ ID NO:2. A variety of amino acid substitutions or deletions may be made to modify N-linked or O-linked glycosylation sites.

A seventh class of variant(s) is a group of addition variant(s) of KGF-2 and/or of the first class of KGF-2 variant(s) and/or of the second class of KGF-2 Svariant(s) and/or of the third class of KGF-2 variant(s) and/or of the fourth class of KGF-2 variant(s) and/or of the fifth class of KGF-2 variant(s) and/or of the sixth 15 class of KGF-2 variant(s), described above, wherein putative cleavage sites Asn"a-Gly"' and/or Asn L -Gly are modified by a substitution of an amino acid Gln or Ser) for the Asn"' and/or Asn 41 SA eighth class of variant(s) is a group of 20 addition variant(s) of KGF-2 and/or of the first class of KGF-2 variant(s) and/or of the second class of KGF-2 variant(s) and/or of the third class of KGF-2 variant(s) and/or of the fourth class of KGF-2 variant(s) and/or of the fifth class of KGF-2 variant(s) and/or of the sixth class of KGF-2 variant(s) and/or of the seventh class of KGF-2 variant(s), described above, wherein fused to the C-terminus of one of the aforementioned proteins is an immunoglobulin portion comprising at least one domain of the constant region of the heavy chain of human immunoglobulin (however, generally excluding the first domain) such as IgG, IgA, IgM or IgE, especially IgG, IgGl or IgG3.

Exemplary substitutions of KGF-2 and of variant(s) of KGF-2 (particularly R 1 -[Asn 7 1 -Pro 2 0 3] -COOH proteins, and more particulary AN36 KGF-2, AN35 KGF-2, 22 AN34 KGF-2, AN33 KGF-2, &N32 KGF-2, AN31 KGF-2, KGF-2, aN29 KGF-2, AN28 KGF-2, AN27 KGF-2 and 6~N26 KGF- 2, either methionyjlated or nonxnethionyiated) are set forth in the following table: TABLE 2

S

S

Oriainal residue Asn 7 l Leu 82 Phe8 5 Thr 86 Glu 93

LYS

102 LyS 103 Glu 1 0 4 Cys 106 Pro 107 TyX 1 0 8 Leu 111 Tbx 114 VaI 123 Asn 1 2 7 TIyr 13 0 Gly 1 4 2 Ser 1 4 3 Phe 146 Leu 1 52 Asn 1 5 9 Gly 1 6 0 Phe 1 6 7 Gin 1 7 0 Arg 174 TIyr 1 77 Gly1 8 2 Arg 187 ArgB 8 8 Arg, Asp, Glu, Lys Gly Arg, .Tyr Ala, Ap Glu, Gly, Ser Asp Gin, Glu Glu, Gin Met Ser, Ala, Met, Asn Ala, Asn, Gly Ala, Phe, Ser Ala, Met, Ser Ala, Arg, Lys, Ser Ile, Leu Asp, Giu, Lys Phe Ala, Ser Ala, Glu, Lys Tyr, Ser, Met Ala, Ile, Met, Phe Ala, Asp, Gin Gly, Ile, Lys, Met Ala, His, Ser, Tyr Ala, Ser, Tyr Arg, Glu, Ser, Tbr Gly, Ala, Ser Phe, Leu Ala, Asp, Glu, Ser Ala, Gfu, Gly, Ser Gin Glu, 23 Lys 195 Glu, Gin Asn 1 9 6 Ala, Arg, Asp, Gin, Glu, Gly, Lys Thr 1 9 7 Ala, Arg, Asp, Lys, Glu, Gly Ser 198 Ala, Asp, Glu, Gly, Thr Va1 206 Ala, lie, Leu, Val His 207 Leu, Ser, Thr, Tyr It will be appreciated by those skilled in the art that many combinations of deletions, insertions and substitutions can be made, provided that the final 5 KGF-2 protein(s) are biologically active. A variant(s) of KGF-2 may be rapidly screened to assess its physical properties. For example, the level of biological activity receptor binding and/or affinity, mitogenic, cell proliferative and/or in vivo activity) may be tested using a variety of assays. One such assay includes a mitogenic assay to test the ability of a protein to stimulate DNA synthesis (Rubin et al. (1989), supra, the disclosure of which is hereby incorporated by reference). Another such assay includes a cell proliferative assay to test the ability of a protein to stimulate cell proliferation (Falco et al. (1988), Oncogene, 2:573-578, the disclosure of which is hereby incorporated by reference).

PolvoeDtide Derivatives Chemically-modified derivatives of KGF-2 protein(s) in which the polypeptide is linked to a polymer in order to modify properties (referred herein as "derivatives"), are included within the scope of the present invention. Chemically-modified derivatives of KGF-2 protein(s) may be prepared by one skilled in the art given the disclosures herein. Conjugates may be prepared using glycosylated, non-glycosylated or de- 24 glycosylated KGF-2 protein(s). Typically, nonglycosylated KGF-2 protein(s) will be used.

Suitable chemical moieties for derivatization include water soluble polymers. Water soluble polymers are desirable because the protein to which each is attached will not precipitate in an aqueous environment, such as a physiological environment. Preferably, the polymer will be pharmaceutically acceptable for the preparation of a therapeutic product or composition.

One skilled in the art will be able to select the desired polymer based on such considerations as whether the polymer/protein conjugate will be used therapeutically and, if so, the therapeutic profile the duration of sustained release; resistance to 15 proteolysis, the effects, if any, on dosage, biological S: activity; the ease in handling; the degree or lack of antigenicity and other known effects of a water soluble polymer on a therapeutic protein).

Suitable, clinically acceptable, water 20 soluble polymers include, but are not limited to, polyethylene glycol (PEG), polyethylene glycol propionaldehyde, copolymers of ethylene glycol/propylene glycol, monomethoxy-polyethylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol (PVA), polyvinyl pyrrolidone, poly-1, 3-dioxolane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, poly (P-amino acids) (either homopolymers or random copolymers), poly(n-vinyl pyrrolidone)polyethylene glycol, polypropylene glycol homopolymers (PPG) and other polyalkylene oxides, polypropylene oxide/ethylene oxide copolymers, polyoxyethylated polyols (POG) glycerol) and other polyoxyethylated polyols, polyoxyethylated sorbitol, or polyoxyethylated glucose, colonic acids or other carbohydrate polymers, Ficoll or dextran and 25 mixtures thereof. As used herein, polyethylene glycol is meant to encompass any of the forms that have been used to derivatize other proteins, such as mono-(C1-C10) alkoxy- or aryloxy-polyethylene glycol. Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water.

The water soluble polymers each may be of any molecular weight and may be branched or unbranched.

Generally, the higher the molecular weight or the more branches, the higher the polymer:protein ratio. The water soluble polymers each typically have an average molecular weight of between about 2kDa to about 100kDa (the term "about" indicating that in preparations of a water soluble polymer, some molecules will weigh more, some less, than the stated molecular weight). The average molecular weight of each water soluble polymer preferably is between about 5kDa and about 40kDa, more preferably between about 10kDa and about 35kDa and most preferably between about 15kDa and about There are a number of attachment methods available to those skilled in the art, including acylation reactions or alkylation reactions (preferably to generate an N-terminal chemically modified protein) with a reactive water soluble molecule. See, for example, EP 0 401 384, the disclosure of which is hereby S" incorporated by reference; see also, Malik et al.

(1992), Exp. Hematol., 2&:1028-1035; Francis (1992), Focus on Growth Factors, 3(2):4-10,published by Mediscript, Mountain Court, Friern Barnet Lane, London N20 OLD, UK; EP 0 154 316; EP 0 401 384; WO 92/16221; WO 95/34326; WO 95/13312; WO 96/11953; PCT International Application No. US96/19459; and the other publications cited herein that relate to pegylation, the disclosures of which are.hereby incorporated by reference.

26 A specific embodiment of the present invention is an unbranched monomethoxy-polyethylene glycol aldehyde molecule having an average molecular weight of about 20kDa conjugated via reductive alkylation to the N-terminus of a KGF-2 protein(s).

Polvvalent Forms Polyvalent forms, molecules comprising more than one active moiety, may be constructed. In one embodiment, the molecule may possess multiple KGF-2 protein(s). Additionally, the molecule may possess at least one KGF-2 protein(s) and, depending upon the Sdesired characteristic of polyvalent form, at least one other molecule.

15 In one embodiment, KGF-2 protein(s) may be chemically coupled. For example, KGF-2 protein(s) may be chemically coupled to a divalent water soluble molecule via the pegylation technology described above.

Additionally, KGF-2 protein(s) may be chemically coupled to biotin, and the biotin/KGF-2 protein(s) which are conjugated are then allowed to bind to avidin, resulting *4 in tetravalent avidin/biotin/KGF-2 protein(s). KGF-2 .protein(s) may also be covalently coupled to dinitrophenol (DNP) or trinitrophenol (TNP) and the 25 resulting conjugates precipitated with anti-DNP or anti- TNP-IgM to form decameric conjugates.

S In yet another embodiment, a recombinant fusion protein may also be produced having KGF-2 protein(s) wherein each recombinant chimeric molecule has a KGF-2 protein(s) sequence, as described above, substituted for the variable domains of either or both of the immunoglobulin molecule heavy and light chains and having all or parts of the constant domains, but at least one constant domain, of the heavy or light chain of human immunoglobulin. For example, each such 27 chimeric KGF-2 protein(s)/IgGl fusion protein may be produced from two chimeric genes: KGF-2 protein(s)/human kappa light chain chimera (KGF-2 protein(s)/Ck) and KGF- 2 protein(s)/human gamma-i heavy chain chimera (KGF-2 protein(s)/Cg-1). Following transcription and translation of the two chimeric genes, as described below, the gene products may be assembled into a single chimeric molecule having a KGF-2 protein(s) displayed bivalently. Additional details relating to the construction of such chimeric molecules are disclosed in United States Patent 5,116,964, PCT Publication No. WO 089/09622, PCT Publication No. WO 91/16437 and EP 315062, the disclosures of which are hereby incorporated by reference.

0 15 In yet a further embodiment, recombinant fusion proteins may also be produced wherein each 00 recombinant chimeric molecule has at least one KGF-2 protein(s), as described above, and at least a portion fe 0 a of the region 186-401 of osteoprotegerin (OPG), as ••0o 20 described in European Patent Application No. 96309363.8.

The production of KGF-2 protein(s) are described in further detail below. Such proteins may be 0 *se prepared, for example, by recombinant techniques or by in vitro chemical synthesis of the desired KGF-2 25 protein(s).

oo Polvnucleotides Based upon the present description and using the universal codon table, one of ordinary skill in the art can readily determine all of the nucleic acid sequence which encodes an amino acid sequence of a KGF-2 protein(s).

Recombinant expression techniques conducted in accordance with the descriptions set forth below may be followed to produce these polynucleotides to express 28 the encoded proteins. For example, by inserting a nucleic acid sequence which encodes a KGF-2 protein(s) into an appropriate vector, one skilled in the art can readily produce large quantities of the desired nucleotide sequence. The sequences can then be used to generate detection probes or amplification primers.

Alternatively, a polynucleotide encoding a KGF-2 protein(s) can be inserted into an expression vector.

By introducing the expression vector into an appropriate host, the desired KGF-2 protein(s) may be produced in large amounts.

As further described herein, there are numerous host/vector systems available for the propagation of desired nucleic acid sequences and/or the 15 production of KGF-2 protein(s). These include, but are not limited to, plasmid, viral and insertional vectors, S* and prokaryotic and eukaryotic hosts. One skilled in the art can adapt a host/vector system which is capable of propagating or expressing heterologous DNA to produce 20 or express the sequences of the present invention.

Furthermore, it will be appreciated by those skilled in the art that, in view of the present disclosure, the nucleic acid sequences include the nucleic acids 109 to 624 of SEQ ID NO:1, as well as degenerate nucleic acid sequences thereof, nucleic acid sequences which encode variant(s) of mature KGF-2 and those nucleic acid sequences which hybridize (under hybridization conditions disclosed in the cDNA library screening section below, or equivalent conditions or more stringent conditions) to complements of nucleic acids 109 to 624 of SEQ ID NO:1.

Also provided by the present invention are recombinant DNA constructs involving vector DNA together with the DNA sequences encoding KGF-2 protein(s). In each such DNA construct, the nucleic acid sequence 29 encoding a KGF-2 protein(s) (with or without signal peptides) is in operative association with a suitable expression control or regulatory sequence capable of directing the replication and/or expression of the KGF-2 protein(s) in a selected host.

Preparation of Polvnucleotides A nucleic acid sequence encoding a KGF-2 protein(s) can readily be obtained in a variety of waysincluding, without limitation, chemical synthesis, cDNA or genomic library screening, expression library screening, and/or PCR amplification of cDNA. These methods and others which are useful for isolating such nucleic acid sequences are set forth in Sambrook et al.

15 (1989), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; S"by Ausubel et al. (1994), eds Current Protocols in Molecular Biology, Current Protocols Press; and by •Berger and Kimmel (1987), Methods in Enzymology: Guide S 20 to Molecular Cloning Techniques, Vol. 152, Academic Press, Inc., San Diego, CA, the disclosures of which are hereby incorporated by reference.

Chemical synthesis of nucleic acid sequences which encode desired proteins can be accomplished using methods well known in the art, such as those set forth by Engels et al. (1989), Angew. Chem. Intl. Ed., 28.:71.6-734 and Wells et al. (1985), Gene, 14:315, the disclosures of which are hereby incorporated by reference. These methods include, inter alia, the phosphotriester, phosphoramidite and H-phosphonate methods of nucleic acid sequence synthesis. Large nucleic acid sequences, for example those larger than about 100 nucleotides in length, can be synthesized as several fragments. The fragments can then be ligated together to form a suitable nucleic acid sequence. A

II

30 preferred method is polymer-supported synthesis using standard phosphoramidite chemistry.

Alternatively, a suitable nucleic acid sequence may be obtained by screening an appropriate cDNA library a library prepared from one or more tissue sources believed to express the protein) or a genomic library (a library prepared from total genomic DNA). The source of the cDNA library is typically a tissue from any species that is believed to express a desired protein in reasonable quantities. The source of the genomic library may be any tissue or tissues from any mammalian or other species believed to harbor a gene encoding a KGF-2 protein(s).

Hybridization mediums can be screened for the S 15 presence of a DNA encoding a KGF-2 protein(s) using one or more nucleic acid probes (oligonucleotides, cDNA or genomic DNA fragments that possess an acceptable level of homology to the cDNA or gene to be cloned) that will hybridize selectively with cDNA(s) or gene(s) present in 20 the library. The probes typically used for such screening encode a small region of DNA sequence from the same or a similar species as the species from which the library is prepared. Alternatively, the probes may be degenerate, as discussed herein.

Hybridization is typically accomplished by annealing the oligonucleotide probe or cDNA to the S* clones under conditions of stringency that prevent non-specific binding but permit binding of those clones that have a significant level of homology with the probe or primer. Typical hybridization and washing stringency conditions depend in part on the size number of nucleotides in length) of the cDNA or oligonucleotide probe and whether the probe is degenerate. The probability of identifying a clone is also considered in 31 designing the hybridization medium whether a cDNA or genomic library is being screened).

Where a DNA fragment (such as cDNA) is used as a probe, typical hybridization conditions include those as set forth in Ausubel et al. (1994), eds., supra. After.hybridization, the hybridization medium is washed at a suitable stringency, depending on several factors such as probe size, expected homology of probe to clone, the hybridization medium being screened, the number of clones being screened, and the like.

Exemplary stringent hybridization conditions are hybridization in 4 x SSC at 62-67 0 C, followed by washing in 0.1 x SSC at 62-67C for approximately an hour.

Alternatively, exemplary stringent hybridization 15 conditions are hybridization in 45-55% formamide, 4 x SSC at 40-45"C. Also included are DNA sequences which hybridize to the nucleic acid sequences set forth in Figure 1 under relaxed hybridization conditions and which encode KGF-2 protein(s). Examples of such relaxed 20 stringency hybridization conditions are 4 x SSC at or hybridization with 30-40% formamide at 40-45 0

C.

See Maniatis et al. (1982), Molecular Cloning (A Laboratory Manual), Cold Spring Harbor Laboratory, pages 387 to 389.

There are also exemplary protocols for S" stringent washing conditions where oligonucleotide probes are used to screen hybridization mediums. For example, a first protocol uses 6 X SSC with 0.05 percent sodium pyrophosphate at a temperature of between about 35"C and 63 0 C, depending on the length of the probe. For example, 14 base probes are washed at 35-40 0 C, 17 base probes at 45-50 0 C, 20 base probes at 52-57C, and 23 base probes at 57-63 0 C. The temperature can be increased 2-3 0 C where the background non-specific binding appears high. A second protocol uses 32 tetramethylammonium chloride (TMAC) for washing. One such stringent washing solution is 3 M TMAC, Tris-HCl, pH 8.0 and 0.2% SDS.

Another method for obtaining a suitable nucleic acid sequence is the polymerase chain reaction (PCR). In this method, cDNA is prepared from poly(A)+RNA or total RNA using the enzyme reverse transcriptase. Two primers, typically complementary to two separate regions of cDNA (oligonucleotides) encoding a KGF-2 protein(s), are then added to the cDNA along with a polymerase such as Taq polymerase, and the polymerase amplifies the cDNA region between the two S: primers.

The oligonucleotide sequences selected as 15 probes or primers should be of adequate length and sufficiently unambiguous so as to minimize the amount of non-specific binding that may occur during screening or PCR amplification. The actual sequence of the probes or primers is usually based on conserved or highly 20 homologous sequences or regions. Optionally, the probes or primers can be fully or partially degenerate, can contain a mixture of probes/primers, all encoding the same amino acid sequence but using different codons to do so. An alternative to preparing 25 degenerate probes is to place an inosine in some or all of those codon positions that vary by species. The oligonucleotide probes or primers may be prepared by chemical synthesis methods for DNA, as described above.

Vectors DNA encoding a KGF-2 protein(s) may be inserted into vectors for further cloning (amplification of the DNA) or for expression. Suitable vectors are commercially available, or the vector may be specifically constructed. The selection or construction 33 of an appropriate vector will depend on whether it is to be used for DNA amplification or for DNA expression, the size of the DNA to be inserted into the vector, and the intended host cell to be transformed with the vector.

The vectors each involve a nucleic acid sequence which encodes a desired protein operatively linked to one or more of the following expression control or regulatory sequences capable of directing, controlling or otherwise affecting the expression of a desired protein by a selected host cell. Each vector contains various components, depending on its function (amplification of DNA or expression of DNA) and its compatibility with the intended host cell. The vector 15 components generally include, but are not limited to, :one or more of the following: a signal sequence, an origin of replication, one or more selection or marker genes, promoters, enhancer elements, a transcription termination sequence and the like. These components may 20 be obtained from natural sources or be synthesized by known procedures.

Examples of suitable prokaryotic cloning vectors include bacteriophages, such as lambda derivatives, or plasmids from E. coli pBR322, col 25 El, pUC, the F-factor and Bluescript® plasmid derivatives (Stratagene, LaJolla, Other appropriate expression vectors, of which numerous types are known in the art for the host cells described below, can also be used for this purpose.

Sianal Secuence The nucleic acid encoding a signal sequence may be inserted 5' of the sequence encoding a desired protein, e.g, it may be a component of a vector or it may be a part of a nucleic acid encoding a desired 34 protein. The nucleic acid encoding the native signal sequence of KGF-2 protein(s) is known (WO 96/25422).

Origin of Replication Expression and cloning vectors each generally include a nucleic acid sequence that enables the vector to replicate in one or more selected host cells. In a cloning vector, this sequence is typically one that enables the vector to replicate independently of the host chromosomal DNA and includes origins of replication or autonomously replicating sequences. Such sequences .are well known. The origin of replication from the plasmid pBR322 is suitable for most Gram-negative bacteria, and various origins SV40, polyoma, 15 adenovirus, VSV or BPV) are useful for cloning vectors in mammalian cells. Generally, the origin of replication is not needed for mammalian expression vectors (for example, the SV40 origin is often used only because it contains the early promoter).

Selection Gene S* The expression and cloning vectors each typically contain a selection gene. This gene encodes a "marker" protein necessary for the survival or growth of 25 the transformed host cells when grown in a selective culture medium. Host cells that are not transformed with the vector will not contain the selection gene and, therefore, they will not survive in the culture medium.

Typical selection genes encode proteins that confer resistance to antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate or tetracycline; complement auxotrophic deficiencies; or supply critical nutrients not available from the culture medium.

35 Other selection genes may be used to amplify the genes to be expressed. Amplification is the process wherein genes which are in greater demand for the production of a protein critical for growth are reiterated in tandem within the chromosomes of successive generations of recombinant cells. Examples of suitable selectable markers for mammalian cells include dihydrofolate reductase (DHFR) and thymidine kinase. The cell transformants are placed under selection pressure which only the transformants are uniquely adapted to survive by virtue of the marker present in the vector. Selection pressure is imposed by culturing the transformed cells under conditions in S* which the concentration of selection agent in the medium 15 is successively changed, thereby leading to amplification of both the selection gene and the DNA that encodes the desired protein. As a result, increased quantities of the desired protein are synthesized from the amplified DNA.

For example, cells transformed with the DHFR selection gene are first identified by culturing all of the transformants in a culture medium that contains methotrexate, a competitive antagonist of DHFR. An appropriate host cell when wild-type DHFR is used is the 25 Chinese hamster ovary cell line deficient in DHFR activity (Urlaub and Chasin (1980), Proc. Natl. Acad.

Sci., USA, 77(7):4216-4220, the disclosure of which is hereby incorporated by reference). The transformed cells are then exposed to increased levels of methotrexate. This leads to the synthesis of multiple copies of the DHFR gene and, concomitantly, multiple copies of other DNA present in the expression vector, such as the DNA encoding the desired protein.

36 Expression and cloning vectors each will typically contain a promoter that is recognized by the host organism and is operably linked to a nucleic acid sequence encoding the desired protein. A promoter is an untranslated sequence located upstream to the start codon of a structural gene (generally within about 100 to 1000 bp) that controls the transcription and 10 translation of a particular nucleic acid sequence.

A promoter may be conventionally grouped into one of two classes, inducible promoters and constitutive promoters.

An inducible promoter initiates increased levels of transcription from DNA under its control in response to some change in culture conditions, such as the presence or absence of a nutrient or.a change in temperature.

A large number of promoters, recognized by a variety of potential host cells, are well known. A promoter may be operably linked to DNA encoding the desired protein by 20 removing the promoter from the source DNA by restriction enzyme digestion and inserting the desired promoter sequence. The native KGF-2 promoter sequence may be used to direct amplification and/or expression of DNA encoding a desired protein. A heterologous promoter is 25 preferred, however, if it permits greater transcription and higher yields of the expressed protein as compared to the native promoter and if it is compatible with the host cell system that has been selected for use. For example, any one of the native promoter sequences of other FGF family members may be used to direct amplification and/or expression of the DNA encoding a desired protein.

Promoters suitable for use with prokaryotic hosts include the beta-lactamase and lactose promoter systems; alkaline phosphatase, a tryptophan (trp) 37 promoter system; a bacterial luminescence (luxR) gene system; and hybrid promoters such as the tac promoter.

Other known bacterial promoters are also suitable.

Their nucleotide sequences have been published, thereby enabling one skilled in the art to ligate them to the desired DNA sequence(s) using linkers or adaptors as needed to supply any required restriction sites.

Suitable promoting sequences for use with yeast hosts are also well known in the art. Suitable promoters for use with mammalian host cells are well known and include those obtained from the genomes of viruses such as polyoma virus, fowlpox virus,

S*

adenovirus (such as Adenovirus bovine papilloma •virus, avian sarcoma virus, cytomegalovirus, a 15 retrovirus, hepatitis-B virus and, most preferably, t Simian Virus 40 (SV40). Other suitable mammalian promoters include heterologous mammalian promoters, heat-shock promoters and the actin promoter.

Enhancer Element The expression and cloning vectors each will Stypically contain an enhancer sequence to increase the transcription by higher eukaryotes of a DNA sequence encoding a desired protein. Enhancers are cis-acting 25 elements of DNA, usually from about 10-300 bp in length, that act on the promoter to increase its transcription.

Enhancers are relatively orientation and position independent. They have been found 5' and 3' to the transcription unit. Yeast enhancers are advantageously used with yeast promoters. Several enhancer sequences available from mammalian genes are known globin, elastase, albumin, alpha-fetoprotein and insulin). Additionally, viral enhancers such as the SV40 enhancer, the cytomegalovirus early promoter enhancer, the polyoma enhancer and adenovirus 38 enhancers are exemplary enhancing elements for the activation of eukaryotic promoters. While an enhancer may be spliced into a vector at a position 5' or 3' to a DNA encoding a desired protein, it is typically located at a site 5' from the promoter.

Transcription Termination Expression vectors used in eukaryotic host cells each will typically contain a sequence necessary for the termination of transcription and for stabilizing the mRNA. Such sequences are commonly available from the 5' and occasionally 3' untranslated regions of eukaryotic DNAs or cDNAs. These regions contain nucleotide segments transcribed as polyadenylated 15 fragments in the untranslated portion of the mRNA encoding a desired protein.

The construction of a suitable vector containing one or more of the above-listed components (together with the desired coding sequence) is 20 accomplished by standard ligation techniques. Isolated plasmids or DNA fragments are cleaved, tailored and religated in the desired order to generate the required vector. To confirm that the correct sequence has been constructed, the ligation mixture may be used to transform E. coli, and successful transformants may be S•selected by known techniques as described above.

S* Quantities of the vector from the transformants are then prepared, analyzed by restriction endonuclease digestion, and/or sequenced to confirm the presence of the desired construct.

A vector that provides for the transient expression of DNA encoding a desired protein in mammalian cells may also be used. In general, transient expression involves the use of an expression vector that is able to replicate efficiently in a host cell, such 39 that the host cell accumulates many copies of the expression vector and, in turn, synthesizes high levels of the desired protein encoded by the expression vector.

Each transient expression systems, comprising a suitable expression vector and a host cell, allows for the convenient positive identification of proteins encoded by cloned DNAs, as well as for the rapid screening of such proteins for desired biological or physiological properties.

Host Cells Any of a variety of recombinant host cells, each of which contains a nucleic acid sequence for use in expressing a desired protein, is also provided by the 15 present invention. Exemplary prokaryotic and eukaryotic host cells include bacterial, mammalian, fungal, insect, yeast or plant cells.

Prokaryotic host cells include but are not limited to eubacteria such as Gram-negative or Gram-positive organisms E. coli (HB101, and MC1061); Bacilli such as B. subtilis; .Pseudomonas species, such as P. aeruginosa; Streptomyces spp.; Salmonella typhimurium; or Serratia marcescans.

As a specific embodiment, a KGF-2 protein(s) may be expressed in E. coli.

In addition to prokaryotic host cells, a KGF protein(s) may be expressed in glycosylated form by any one of a number of suitable host cells derived from multicellular organisms. Such host cells are capable of complex processing and glycosylation activities. In principle, any higher eukaryotic cell culture might be used, whether such culture involves vertebrate or invertebrate cells, including plant and insect cells.

Eukaryotic microbes such as filamentous fungi or yeast may be suitable hosts for the expression of a 40 KGF-2 protein(s). Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used among lower eukaryotic host microorganisms, but a number of other genera, species and strains are well known and commonly available.

Vertebrate cells may be used, as the propagation of vertebrate cells in culture (tissue culture) is a well-known procedure. Examples of useful mammalian host cell lines include but are not limited to monkey kidney CV1 line transformed by SV40 (COS-7), human embryonic kidney line (293 cells or 293 cells subcloned for growth in suspension culture), baby hamster kidney cells and Chinese hamster ovary cells.

Other suitable mammalian cell lines include but are not limited to HeLa, mouse L-929 cells, 3T3 lines derived from Swiss, Balb-c or NIH mice, and BHX or HaK hamster cell lines. As a specific embodiment, a KGF-2 protein(s) may be expressed in COS cells or in baculovirus cells.

S 20 A host cell may be transfected and preferably transformed with a desired nucleic acid under appropriate conditions permitting expression of the nucleic acid. The selection of suitable host cells and methods for transformation, culture, amplification, screening and product production and purification are well known in the art (Gething and Sambrook (1981), Nature, 293:620-625 or, alternatively, Kaufman et al.

(1985), Mol. Cell. Biol., 5(7):1750-1759, or U.S. Pat.

No. 4,419,446, the disclosures of which are hereby incorporated by reference). For example, for mammalian cells without cell walls, the calcium phosphate precipitation method may be used. Electroporation, microinjection and other known techniques may also be used.

41 It is also possible that a desired protein may be produced by homologous recombination or with recombinant production methods utilizing control elements introduced into cells already containing DNA encoding a KGF-2 protein(s). Homologous recombination is a technique originally developed for targeting gene to induce or correct mutations in transcriptionally .active genes (Kucherlapati (1989), Prog. in Nuci. Acid Res. and Mol. Biol., U:301, the disclosure of which is hereby incorporated by reference). The basic technique was developed as a method for introducing specific mutations into specific regions of the mammnalian genome (Thomas et al. (1986), Cell, AA.:419-428; Thomas and Capecchi (1987), Cell, U5.:503-512 and Doetscbman et al.

(1988), Proc. Natl. Acad. Sdi., :8583-8587, the disclosures of which are hereby incorporated by reference) or to correct specific mutations within defective genes (Doetschman et al. (1987), Nature, .jjQ:576-578, the disclosure of which is hereby incorporated by reference). Exemplary techniques are described in U.S. Patent No. 5,272,071; WO 92/01069; WO 93/03183; WO 94/12650 and WO 94/31560, the disclosures of which are hereby incorporated by reference.

Culturingr the Host Cells The method for culturing each of the one or more recombinant host cells for production of a desired protein will vary depending upon many factors and considerations; the optimum production procedure for a given situation will be apparent to those skilled in the art through minimal experimentation. Such recombinant host cells are cultured in suitable medium and the expressed protein is then optionally recovered, isolated and purified from the culture medium (or from the cell, 42 if expressed intracellularly) by appropriate means known to those skilled in the art.

Specifically, each of the recombinant cells used to produce a desired protein may be cultured in media suitable for inducing promoters, selecting suitable recombinant host cells or amplifying the gene encoding the desired protein. The media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium and phosphate), buffers (such as HEPES), nucleosides (such as adenosine and *thymidine), antibiotics (such as gentamicin), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or another energy source. Other supplements may also be included, at appropriate concentrations, as will be appreciated by those skilled in the art. Suitable culture conditions, such as temperature, pH and the 20 like, are also well known to those skilled in the art for use with the selected host cells.

The resulting expression product may then be purified to near homogeneity using procedures known in the art. Exemplary purification techniques are taught in published PCT Application Nos. WO 90/08771 and WO 96/11952, the disclosures of which are hereby incorporated by reference.

Uses Variant(s) of KGF-2 described herein, and chemically-modified derivatives of KGF-2 and variant(s) of KGF-2 protein (collectively, "KGF-2 protein product(s)") may be used as research reagents and as therapeutic and diagnostic agents. Thus, a KGF-2 protein product(s) may be used in in vitro and/or in 43 vivo diagnostic assays to quantify the amount of KGF-2 in a tissue or organ sample.

For example, a KGF-2 protein product(s) can be used for identification of the receptor(s) for a KGF-2 protein(s) in various body fluids and tissue samples using techniques known in the art (WO 90/08771).

This invention also contemplates the use of a .KGF-2 protein product(s) in the generation of antibodies made against the KGF-2 protein product(s), including native KGF-2. One of ordinary skill in the art can use well-known, published procedures to obtain monoclonal and polyclonal antibodies or recombinant antibodies.

Such antibodies may then be used to purify and characterize KGF-2 protein product(s), including native KGF-2.

S* Pharmaceutical Compositions The present invention encompasses pharmaceutical preparations each containing therapeutically- or 20 prophylatically-effective amounts of a KGF-2 protein product(s).

Pharmaceutical compositions each will :generally include a therapeutically-effective or prophylatically-effective amount of a KGF-2 protein product(s) in admixture with a vehicle. The vehicle preferably includes one or more pharmaceutically and physiologically acceptable formulation materials in admixture with the KGF-2 protein product(s).

The primary solvent in a vehicle may be either aqueous or non-aqueous in nature. In addition, the vehicle may contain other pharmaceutically acceptable excipients for modifying or maintaining the pH buffers such as citrates, phosphates, and amino acids such as glycine); osmolarity mannitol and sodium chloride); viscosity; clarity; color; 44 sterility; stability sucrose and sorbitol); odor of the formulation; rate of dissolution solubilizers or solubilizing agents such as alcohols, polyethylene glycols and sodium chloride); rate of release; as well as bulking agents for lyophilized formulation mannitol and glycine); surfactants polysorbate 20, polysorbate 80, triton, and pluronics); antioxidants sodium sulfite and sodium hydrogen-sulfite); preservatives benzoic acid and salicylic acid); flavoring and diluting agents; emulsifying agents; suspending agents; solvents; fillers; delivery vehicles; diluents and/or pharmaceutical adjuvants. Other effective administration forms such as parenteral slow-release 15 formulations, inhalant mists, orally-active formulations, or suppositories are also envisioned.

The composition may also involve particulate preparations of polymeric compounds such as bulk erosion polymers poly(lactic-co-glycolic acid) (PLGA) copolymers, PLGA polymer blends, block copolymers of PEG, and lactic and glycolic acid, poly(cyanoacrylates)); surface erosion polymers poly(anhydrides) and poly(ortho esters)); hydrogel esters pluronic polyols, poly(vinyl alcohol), poly(vinyipyrrolidone), maleic anhydride-alkyl vinyl ether copolymers, cellulose, hyaluronic acid derivatives, alginate, collagen, gelatin, albumin, and starches and dextrans) and composition systems thereof; or preparations of liposomes or microspheres. Such compositions may influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the present proteins and derivatives. The optimal pharmaceutical formulation for a desired protein will be determined by one skilled in the art depending upon the route of administration and desired dosage.

45 Exemplary pharmaceutical compositions are disclosed in Remington's Pharmaceutical Sciences, 18th Ed. (1990), Mack Publishing Co., Easton, PA 18042, pages 1435-1712; Gombotz and Pettit (1995), Bioconjugate Chem., .:332- 351; Leone-Bay, et al. (1995), Journal of Medicinal Chemistry, .:4263-4269; Haas, et al. (1995), Clinical Immunology and Immunopathology, 76(11:93; WO 94/06457; ,WO 94/21275; FR 2706772 and WO 94/21235, the disclosures of which are incorporated herein by reference.

Specific sustained release compositions are available from the a variety of suppliers including Depotech Corp. (Depofoam T M a multivesicular liposome); Alkermes, Inc. (ProLease T M a PLGA microsphere). As used herein, hyaluronan is intended to include hyaluronan, hyaluronic acid, salts thereof (such as sodium hyaluronate), esters, ethers, enzymatic derivatives and cross-linked gels of hyaluronic acid, and chemically modified derivatives of hyaluronic acid (such as hylan). Exemplary forms of hyaluronan are disclosed in U.S. Patent Nos. 4,582,865, 4,605,691, 4,636,524, 4,713,448, 4,716,154, 4,716,224, 4,772,419, 4,851,521, 4,957,774, 4,863,907, 5,128,326, 5,202,431, 5,336,767, 5,356,883; European Patent Application Nos. 0 507 604 A2 and 0 718 312 A2; and WO 96/05845, the disclosures of which are hereby incorporated by reference. Suppliers of hyaluronan include BioMatrix, Inc. Ridgefield, NJ; Fidia Abano Terme, Italy; Kaken Pharmaceutical Co., Ltd., Tokyo, Japan; Pharmacia AB, Stockholm, Sweden; Genzyme Corporation, Cambridge, MA; Pronova Biopolymer, Inc. Portsmouth, NH; Calbiochem- Novabiochem AB, Lautelfingen, Switzerland; Intergen Company, Purchase, NY and Kyowa Hakko Kogyo Co., Ltd., Tokyo, Japan.

For treatment and/or prevention of oral indications, a liquid solution or suspension can be used 46 in a manner similar to a mouthwash, where the liquid is swished around in the mouth so as to maximize treatment of lesions (United States Patent 5,102,870, the teachings of which are incorporated by reference).

Longer contact with the mucosal surface can be attained by selecting a suitable vehicle which is capable of coating mucosa. Typical examples are pectin containing formulations such as Orabase Registered" (Colgate-Hoyt Laboratories, Norwood, MA), sucralfate suspensions, Kaopectate and Milk of Magnesia. The formulation can also be a spreadable cream, gel, lotion or ointment :having a pharmaceutically acceptable non-toxic vehicle or carrier. KGF-2 protein product(s) can also be :incorporated into a slow dissolving lozenge or troche, a 15 chewing gum base, or a buccal or slow delivery prosthesis hooked onto a back molar, for example.

Therapeutic agents such as analgesics and anesthetics can be administered to alleviate pain and such as antiinfectictives, anti-bacterials, anti-fungals and 20 antiseptics can be administered to prevent and/or treat secondary infection of the lesions.

Once the pharmaceutical composition has been formulated, it may be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or a dehydrated or lyophilized powder. Such formulations may be stored either in a ready-to-use form or in a form lyophilized) requiring reconstitution prior to administration.

In a specific embodiment, the present invention is directed to kits for producing a single-dose administration unit. The kits may each contain both a first container having a dried protein and a second container having an aqueous formulation. Kits included within the scope of this invention are single and multi-chambered pre-filled 47 syringes; exemplary pre-filled syringes liquid syringes, and lyosyringes such as Lyo-Ject®, a dualchamber pre-filled lyosyringe) are available from Vetter GmbH, Ravensburg, Germany.

It should be noted that KGF-2 protein product(s) formulations described herein may be used for veterinary as well as human applications and that the term "patient" should not be construed in a limiting manner.

The frequency of dosing the KGF-2 protein product(s) to a patient will depend on the disease and the condition of the patient, as well as the pharmacokinetic parameters of KGF-2 protein product(s) as formulated, and the route of administration. The 15 KGF-2 protein product(s) may be administered once, administered daily, or administered with an initial bolus dose followed by a continuous dose or sustained delivery. It is also contemplated that other modes of a continuous or near-continuous dosing may be practiced.

For example, chemical derivatization may result in sustained release forms of the protein which have the effect of a continuous presence in the bloodstream, in predictable amounts, based on a determined dosage regimen.

A patient in need of stimulation (including cytoprotection, proliferation and/or differentiation) of epithelial cells may be administered an effective amount of a KGF-2 protein product(s) to elicit the desired response in the patient and will, thus, generally be determined by the attending physician. The dosage regimen involved in a method of preventing or treating a specific condition will be determined by the attending physician, considering various factors which modify the action of drugs, the age, condition, body weight, sex and diet of the patient, the severity of any

II

48 infection, the time of administration and other clinical factors. Appropriate dosages may be ascertained through use of established assays for determining dosages utilized in conjunction with appropriate dose-response data. Typical dosages will range from 0.001 mg/kg body weight to 500 mg/kg body weight, preferably up to 200 mg/kg body weight, more preferably 100 mg/kg body weight.

The KGF-2 protein product(s) may be administered via topical, enteral or parenteral administration including, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, 15 subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, and intrasternal injection and infusion.

The KGF-2 protein product(s) may be administered via oral administration or administered through mucus membranes, that is, intranasally, sublingually, buccally 20 or rectally for systemic delivery. The KGF-2 protein product(s) may be used once or administered repeatedly, depending on the disease and the condition of the patient. In some cases, the KGF-2 protein product(s) may be administered as an adjunct to other therapy and also with other pharmaceutical preparations In another embodiment, cell therapy implantation of cells producing KGF-2 protein(s) is also contemplated. This embodiment of the present invention may include implanting into patients cells which are capable of synthesizing and secreting a biologically-active form of KGF-2 protein(s). Such cells producing KGF-2 protein(s) may be cells which do not normally produce KGF-2 protein(s) but which have been modified to produce KGF-2 protein(s), or which may be cells whose ability to produce KGF-2 protein(s) have 49 been augmented by transformation with a polynucleotide suitable for the expression and secretion of such protein. In order to minimize a potential immunological reaction in patients being administered KGF-2 protein(s) of a foreign species, it is preferred that the cells be of the same species as the patient human) or that the cells may be encapsulated with material that provides a barrier against immune recognition, or that cells be placed into an immunologically-privileged anatomical location, such as in the testis, eye and central nervous system.

Human or non-human animal cells may be implanted in patients in biocompatible, semi-permeable Spolymeric enclosures or membranes to allow release of a 15 KGF-2 protein(s), but prevent destruction of the cells by the patient's immune system or by other detrimental *9 factors from the surrounding tissue. Alternatively, the patient's own cells, transformed ex vivo to produce KGF-2 protein(s), could be implanted directly into the 20 patient without such encapsulation. The methodology for the membrane encapsulation of living cells is familiar to those of ordinary skill in the art, and the preparation of the encapsulated cells and their implantation in patients may be accomplished with known techniques Patent Nos. 4,892,538; 5,011,472; and 5,106,627, the disclosures of which are hereby incorporated by reference).

In yet another embodiment, in vivo gene therapy is also envisioned, wherein a nucleic acid sequence encoding a KGF-2 protein(s) is introduced directly into a patient. Efficient and long lasting gene transfer to hepatocytes is required for effective gene therapy for local expression of the protein to prevent and/or treat liver diseases and/or for secretion 50 of the protein to prevent and/or treat diseases in other organs or tissues.

The DNA construct may be directly injected into the tissue of the organ to be treated, where it can be taken up in viva and expressed, provided that the DNA is operable linked to a promoter that is active in such tissue. The DNA construct may also additionally include vector sequence from such vectors as an adenovirus vector, a retroviral3vector, papilloma virus and/or a herpes virus vector, to aid uptake in the cells.

Physical transfer may be achieved in viva by.local injection of the desired nucleic acid construct or other appropriate delivery vector containing the desired nucleic acid sequence, such as liposome-mediated 15 transfer, direct injection (naked DNA), receptormediated transfer (ligand-DNA complex), or microparticle bombardment (gene gun). For the in vivo regeneration of hepatocytes in the liver, the use of Moloney retroviral vectors may be especially effective (Bosch, et al.

20 (1996), Cold Spring Harbor, Gene Therapy Meeting, September 25-29, 1996; and Bosch, et al. (1996), Journal of Clinical Investigation, 98(12):2683-2687).

A KGF-2 protein product(s) may be applied in therapeutically- and prophylactically-effective amounts to organs or tissues specifically characterized by :having damage to or clinically insufficient numbers of epithelium cells. It should be noted that a KGF-2 protein product(s) may be used for veterinary as well as human applications and that the term "patient" should not be construed in a limiting manner.

In accordance with the present invention, a KGF-2 protein product(s) may be used in vivo to induce stimulation (including cytoprotection, proliferation and/or differentiation), proliferation and/or differentiation of epithelial cells including, but not 51 limited to, the eye, ear, gums, hair, lung, skin, pancreas (endocrine and exocrine), thymus, thyroid, urinary bladder, liver and gastrointestinal tract including cells in the oral cavity, in the esophagus, in the glandular stomach and small intestine, in the colon and the intestinal mucosa, in the rectum and in the anal canal. Indications in which a KGF-2 protein product(s) -may be successfully administered include, but are not limited to: burns and other partial and full-thickness injuries in need of stimulation of adnexal structures such as hair follicles, sweat glands, and sebaceous glands; lesions caused by epidexmolysis bullosa, which is a defect in adherence of the epidermis to the underlying dermis, resulting in frequent open, painful 15 blisters which can cause severe morbidity; chemotherapyinduced alopecia and male-pattern baldness, or the progressive loss of hair in men and women; gastric and duodenal ulcers; gut-toxicity in radiation- and chemotherapy-treatment regimes; erosions of the gastrointestinal tract esophagus, stomach and intestines) include erosive gastritis, esophagitis, esophageal ref lux or inflammatory bowel diseases, such as Crohn's disease (affecting primarily the small intestine) and ulcerative colitis (affecting primarily the large bowel); disorders or damage to salivary gland tissue including radiation/chemotherapy effects, autoimmune diseases such as Sjogren's Syndrome which can cause salivary gland insufficiency (sicca syndrome); insufficient production of mucus throughout the gastrointestinal tract; adult respiratory distress syndrome (ARDS), pneumonia, hyaline membrane disease infant respiratory distress syndrome and bronchopulmonary dysplasia) in premature infants; acute or chronic lung damage or insufficiency due to inhalation injuries (including high oxygen levels), 52 emphysema, lung damage from chemotherapeutics, ventilator trauma or other lung damaging circumstances; hepatic cirrhosis, fulminant liver failure, damage caused by acute viral hepatitis and/or toxic insults to the liver and/or bile duct disorders, and viral-mediated gene transfer to liver; corneal abrasion and/or corneal ulcerations due to chemicals, bacteria or viruses; progressive gum disease; eardrum damage; ulcerations and/or inflammations including conditions resulting from chemotherapy and/or infection; pancreatic disorders and pancreatic insufficiencies including diabetes (Type I and Type II), pancreatitis, cystic fibrosis, and as an adjunct in islet cell transplantation.

This invention thus has significant 15 implications in terms of enabling the application of KGF-2 protein product(s) specifically characterized by the prophylactic and/or therapeutic use of KGF-2 to reduce, delay and/or block the onset of damage to or deficiencies in these particular types of cells. The 20 following is a more specific description of diseases and medical conditions which can be treated with KGF-2 protein product(s) in accordance with the invention.

Specific uses of the KGF-2 protein product(s) are disclosed in PCT Patent Application No.

filed on the same date herewith by Lacey, Ulich, Danilenko and Farrell, entitled on the Application transmittal letter as "USES OF KERATINOCYTE GROWTH FACTOR-2" (Attorney Docket No. A-422C-PCT), the disclosure of which is hereby incorporated by reference.

KGF-2 protein product(s) are useful to increase cytoprotection, proliferation and/or differentiation of hepatocytes in order to increase liver function. KGF-2 protein product(s) are useful to treat and/or prevent hepatic cirrhosis, fulminant liver 53 failure, damage caused by acute viral hepatitis, toxic insults to the liver and/or bile duct disorders.

Hepatic cirrhosis, secondary to viral hepatitis and chronic alcohol ingestion, is a significant cause of morbidity and mortality. KGF-2 protein product(s) are useful to treat and/or prevent the development of cirrhosis. A standard in vivo model of hepatic cirrhosis is known (Tomaszewski et al.

(1991), J. Appl. Toxicol., 11:229-231, the disclosure of which is hereby incorporated by reference).

Fulminant liver failure is a life-threatening condition which occurs with end-stage cirrhosis and which is presently treatable only with liver 0 transplantation. KGF-2 protein product(s) are useful to 15 treat and/or prevent fulminant liver failure. Standard in vivo models of fulminant liver failure are known (Mitchell. et al. (1973), J. Pharmacol. Exp. Ther., 187:185-194; Thakore and Mehendale (1991), Toxicologic Pathol., 12:47-58; and Havill et al. (1994), FASEB Journal, 8(4-5):A930, Abstract 5387, the disclosures of which are hereby incorporated by reference).

Acute viral hepatitis is frequently subclinical and self-limiting. However, in a minority of 'ease, patients severe liver damage can result over several weeks. KGF-2 protein product(s) are useful in *;.-;preventing and/or treating viral hepatitis. Standard in vivo models of hepatocyte proliferation are known r* (Housley et al. (1994), Journal of Clinical Investigation, 94(5):1764-1777; and Havill et al.

(1994), supra, the disclosures of which are hereby incorporated by reference).

Toxic insults to the liver caused by acetaminophen, halothane, carbon tetrachloride and other toxins may be prevented and/or treated by KGF-2 protein product(s). Standard in vivo models of liver toxicity 54 are known (Mitchell et al. (1973), supra; Thakore and Mehendale (1991), supra; and Havill et al. (1994), supra, the disclosures of which are hereby incorporated by reference).

KGF-2 protein product(s) are useful to increase cytoprotection, proliferation and/or differentiation of epithelial cells in the gastrointestinal tract the oral cavity, esophagus, stomach, small intestine, colon, rectum and anal-canal). The terms "gastrointestinal tract", as defined herein, and "gut" are art-recognized terms and are used interchangeably herein. Specifically, KGF-2 protein product(s) are useful to treat and/or prevent gastric ulcers, duodenal ulcers, inflammatory bowel 15 disease, gut toxicity and erosions of the Sgastrointestinal tract.

Gastric ulcers cause significant morbidity, have a relatively high recurrence rate, and heal by scar formation on the mucosal lining. KGF-2 protein 20 product(s) are useful to prevent degeneration of glandular mucosa and to regenerate glandular mucosa more o" rapidly, offering a significant therapeutic improvement in the treatment of gastric ulcers.

Standard in vivo models of gastric ulcers are known (Tarnawski et al. (1991), "Indomethacin Impairs Quality of Experimental Gastric Ulcer Healing: A Quantitative Histological and Ultrastructural Analysis", In:Mechanisms of Injury, Protection and Repair of the Upper Gastrointestinal Tract, (eds) Garner and O'Brien, Wiley Sons; Brodie (1968), Gastroenterology, .5:25; and Ohning et al. (1994), Gastroenterology, 106(4 Supol.):A624, the disclosures of which are hereby incorporated by reference).

Duodenal ulcers, like gastric ulcers, cause significant morbidity and have a relatively high 55 recurrence rate. KGF-2 protein Product(s) are useful to prevent degeneration of the mucosal lining of the duodenum and to rapidly regenerate the mucosa. lining of the duodenum to heal those ulcers and decrease their recurrence. Standard In viva models of duodenal ulcers are known (Berg et al. (1949), Proc. Soc. Exp. Biol.

Med., 2:374-376; Szabo and Pihan, Chr-onobiol. Inc.

(1987), 5:31-42; and Robert et al.. (1970), Gastroenterology, U.S:95-102, the disclosures of which are hereby incorporated by reference).

Gut toxicity is a major limiting factor associated with cancer treatment, both in radiation (abdominal, total body or local, eg. head and neck) and chemotherapy. Of primary concern are those patients undergoing: chemotherapy f or cancer such as leukemia, breast cancer or as an adjuvant to tumor removal; radiotherapy for head and neck cancer; and combined chemotherapy and radiotherapy for bone marrow transplants. The severity of damage is related to the type and dose of chemotherapeutic agent(s) and concomitant therapy such as radiotherapy.

Mucositis in portions of the gastrointestinal tract may account for significant pain and discomfort for these patients, and range in severity from redness and swelling to frank ulcerative lesions. The lesions :often become secondarily infected and become much harder to heal. Standard in viva models of radiation-induced gut toxicity are known (Withers and Elkind (1970), Int.

Radiat., 17(3).:261-267, the disclosure of which is hereby incorporated by reference). Standard in viva models of chemotherapy-induced gut toxicity are known (Farrell et al., The American Society of Hematology, 38th Annual Meeting (Orlando, FL), December 6-8, 1996; Sonis et al. (1990), Oral Surg. Oral Med Oral Pathol., 69(4:437-443; and Moore (1985), Cancer Chemotherapy 56 Pharmacol., 15:11-15, the disclosures of which are hereby incorporated by reference).

Exemplary chemotherapeutic agents include, but are not limited to, BCNU, busulfan, carboplatin, cyclophosphamide, cisplatin, cytosine arabinoside,daunorubicin, doxorubicin, etoposide, gemcytabine, ifosphamide, irinotecan, melphalan, methotrexate, navelbine, topotecan, taxol and taxotere, and exemplary treatment regimes include, but are not limited to, BEAM (busulfan, etoposide, cytosine arabinoside, methotrexate); cyclophosphamide and total body irradiation; cyclophosphamide, total body irradiation and etoposide; cyclophosphamide and busulfan; and 5-fluorouracil with leucovorin or 15 levamisole.

Treatment, pretreatment and/or posttreatment, with KGF-2 protein product(s) are useful to generate a cytoprotective effect or regeneration or both, for example, on the small intestinal mucosa, allowing increased dosages of such therapies while reducing potential fatal side effects of gut toxicity.

KGF-2 protein product(s) may preferentially be adninistered in the following settings. Colorectal patients routinely are administered 5-fluorouracil with 25 leucovorin on days 1 to 5; KGF-2 protein product(s) may be administered on days -1 and 0. Head and neck cancer patients routinely are administered hypofractionated radiation therapy, plus and cisplatin over a seven week period; KGF-2 protein product(s) may be administered on days -1 and 0 and thereafter once per week until the end of the radiation therapy. In lymphoma transplantation patients are frequently administered BEAM therapy for 6 days (days 1 to KGF protein product(s) may be administered on 57 days -1 and 0 and as a three day post-treatment (days 7 to 9).

In specific embodiments, KGF-2 protein product(s) may be administered prophylactically and/or therapeutically to reduce, delay and/or block the onset of mucositis (due to chemotherapy and/or radiotherapy), in combination with one or more cytokines to delay -and/or block the onset of cytopenia.

Typically, bone marrow, peripheral blood progenitor cells or stem cells (McNiece et al. (1989), Blood, 74:609-612 and Moore et al. (1979), Blood Cells, ":297-311, the disclosures of which are hereby incorporated by reference) are removed from a patient •prior to myelosuppressive cytoreductive therapy (chemotherapy alone or with radiation therapy) and are then readministered to the patient concurrent with or following cytoreductive therapy in order to counteract the myelosuppressive effects of such therapy.

Many different approaches have been undertaken to protect an organism from the side effects of radiation or toxic chemicals. One approach is to replace bone marrow cells before toxicity has developed.

Another approach is to use progenitor cells from the peripheral blood (PBPC). These PBPC can be collected by 25 apheresis or phlebotomy following cytokine therapy alone (G-CSF or GM-CSF), or with chemotherapy or cytokines.

They can be given back fresh or cryopreserved. If desired, the cells may be CD34+ selected, Tn-cell depleted, tumor cell depleted, or the progenitor cells can be expanded (caused to multiply) by means known in the art, prior to administration. The benefits of reinfusion of autologous or allogeneic progenitors following myelosuppressive therapy have been described in the literature (Morse et al. (1992), Ann. Clin. Lab.

Sci., 22:221-225; Kessinger and Armitage (1991), Blood, 58 77:211-213; Kessinger et al. (1989), Blood, IA:1260- 1265; Takam et al. (1989), Blood, A:1245-1251 and Kessinger et al. (1988), Blood, 122:723-727).

As used herein, the term *cytokine" is a generic term for proteins released by one cell population which act on another cell as intercellular mediators. Examples of such cytokines are lymphokines, monokines and traditional polypeptide hormones.

Included among the cytokines are insulin-like growth factors; human growth hormone; N-methionyl human growth hormone; bovine growth hormone; parathyroid hormone; S.thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH) and leutinizing hormone hemopoietic growth factor; hepatic growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor alpha and -beta; mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin; nerve growth factors such as NGF-beta; platelet-growth factors such as TPO and MGDF; transforming growth factors (TGFs) such as TGF-alpha and TGF-beta; insulin-like growth factor-I and -II; erythropoietin; osteoinductive factors; interferons **25 such as interferon-alpha, -beta and -ganmma; colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF), granulocyte-macrophage-CSF (GM-CSF), and granulocyte-CSF G-CSF); interleukins (ILs) such as IL-, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15 and IL-16; and other polypeptide factors. The cytokines can be used alone or in combination to protect against, mitigate and/or reverse myeloid or hematopoietic toxicity associated wi h cytotoxic agents.

59 The mode of administration of KGF-2 protein product(s), as well as of the cytokine, should be coordinated and optimized. Depending upon the circumstances, an appropriate dose of KGF-2 protein product(s) can be administered prior to or subsequent to administration of the therapeutic agent(s). For example, a parameter to be considered is whether the cytokine is administered in a single dose or in multiple doses during the course of therapy. Certain cytokines are cleared rapidly from the body and will require periodic or continuous administration in order for their efficacy to be maximized. The manner of administration can differ, depending on whether a pre-treatment or •post-treatment of the cytokine is given. For example, if the cytokine is given prior to the cytotoxic agent, it is preferable to administer the cytokine by intravenous bolus injection for several hours and, optionally, to repeat such administration on one or more days during and after completion of the cytotoxic therapy.

In a specific embodiment, KGF-2 protein S* product(s) are administered intraveneously) at 0.1 to 500 micrograms/kg/dose, preferably up to about 200 micrograms/kg/dose, prior to 1 to 3 days) 25 and/or after chemotherapy or radiation therapy, and G-CSF (Neupogen TM or Lenograstim) or GM-CSF (Sargramostimar) is administered subcutaneously) at 5 micrograms/kg/dose for 1 to 10 days (preferably 7 to. 10 days) after chemotherapy.

Erosions of the gastrointestinal tract esophagus, stomach and intestine) include erosive gastritis, esophagitis, esophageal reflux and inflammatory bowel diseases. Inflammatory bowel diseases, such as Crohn's disease (affecting primarily the small intestine) and ulcerative colitis (affecting 60 primarily the large bowel), are chronic diseases of unknown etiology which result in the destruction of the mucosal surface, inflammation, scar and adhesion formation during repair, and significant morbidity to the affected individuals. KGF-2 protein product(s) are useful to regenerate the mucosal lining and decrease the recurrence of these erosions, resulting in faster healing, and may be of benefit in controlling progression of the disease. Standard in vivo models of erosion of the gastrointestinal tract are known (Geisinger et al. (1990), Mod-Pathol., 3(5)J:619-624; Carlborg et al. (1983), Laryngoscope, 93i2):184-187; Carlborg et al. (1980), Eur-Surg-Res., 12(4):270-282; •Keshavarzian et al. (1991), Alcohol-Clin-Exp-Res., 15(il.):116-121; Katz et al. (1988), Dig-Dis-Sci:, 33(2):217-224; and Zeeh et al. (1996), Gastroenterology, 110(4):1077-1083, the disclosures of which are hereby incorporated by reference). Standard in vivo models of inflammatory bowel disease are well known (Morris et al.

~20 (1989), Gastroenterology, U:795-803; Rachmilewitz et al. (1989), Gastroenterology, 297:326-327; Allgayer et al. (1989), Gastroenterology, 96:1290-1300; and Kim and Borstad (1992), Scand. J. Gastroenterol, 27(7):529-537, the disclosures of which are hereby 25 incorporated by reference).

Animal studies have established the relationship between total parenteral nutrition (TPN) and intestinal mucosal atrophy (Buchman et al. (1995), Journal of Parenteral and Enteral Nutrition, 12:453- 460). The decrease in intestinal villus height is attributed to the lack of growth stimulus provided through oral intake of nutrients. This is reflected in a reduction in the labeling index, a measure of growth.

Decreases in villus height are also correlated with decreases in specific activities of enzymes involved in 61 nutrient absorption. KGF-2 protein product(s) are useful to either protect against atrophy during the fasting and/or facilitate regrowth upon reintroduction of oral nutrients.

Hyaline membrane disease of premature infants results in the absence of surfactant production by type II pneumocytes within the lung, resulting in the collapse of the alveoli. KGF-2 protein product(s) are useful to treat and/or prevent hyaline membrane disease.

Smoke inhalation is a significant cause of morbidity and mortality in the week following a burn injury, due to necrosis of the bronchiolar epithelium and the alveoli. KGF-2 protein products are useful treat and/or prevent inhalation injuries.

Emphysema results from the progressive loss of alveoli. KGF-2 protein product(s) are useful to treat and/or prevent emphysema.

Disorders of the pancreas may be endocrinerelated such as Type I or Type II diabetes, or may be exocrine-related such as pancreatitis and pancreatic inefficiencies or cystic fibrosis. Patients with diagnosed Type I diabetes require constant exogenous insulin administration. Patients with diagnosed Type II diabetes progress through varying stages of insulin 25 resistance/insufficiency to ultimately also require exogenous insulin administration. KGF-2 protein product(s) are useful to ameliorate, delay and/or circumvent permanent manifestation of diabetes mellitus or as an adjunct in the setting of islet cell transplantation by inducing pancreatic beta cell function in order to normalize blood glucose levels during varying metabolic demands, yet avoid frequent or profound hypoglycemia. Standard models of diabetes are known (Junod et al. (1967), Proc. Soc. Exp. Bio. Med.

126(11:201-205; Rerup (1970), Pharm. Rev., 22:485-518; 62 Rossini et al. (1977), 74:2485-2489; and Ar'Rajab and Ahren (1993), Pancreas, a:50-57, the disclosures of which are hereby incorporated by reference). A standard model of pancreatic cell S proliferation is known (Yi et al. (1994), American Journal of Pathology, 145(i):80-85, the disclosure of which is hereby incorporated by reference).

Corneal cells may be damaged by corneal abrasion and/or corneal ulcerations due to chemicals, bacteria or viruses. KGF-2 protein product(s) are *.useful treat and/or prevent corneal degeneration.

Standard in vivo models of corneal cell regeneration are known (Inatomi et al. (1994), Investigative Opthalmology and Visual Science, 35(4):1318, Abstract 299; Sotozono et al. (1994), Investigative Opthalmology and Visual Science, 35(4):1941, Abstract 317; Wilson et al. (1994), Investigative Opthalmology and Visual Science, 35(4):1319, Abstract 301; Wilson et al. (1993), The FASEB Journal, 7(3):A493, Abstract 2857; Inatomi et al.

(1994), Investigative Ophthalmology Visual Science, 35(4):1318; Wilson et al. (1994), Experimental Eye Research, 59(6):665-678; and Sotozono et al. (1995), Investigative Ophthalmology Visual Science, 36(8):1524-1529, the disclosures of which are hereby incorporated by reference).

KGF-2 protein product(s) are useful to treat and/or prevent gum disease. Standard in vivo models of gum disease are known.

KGF-2 protein product(s) are useful to treat and/or prevent ulcerating and/or inflammatory conditions including conditions related to chemotherapy (as discussed above) and/or infection. Standard in vivo models of urinary bladder damage are known (Ford and Hess (1976), Arch. Intern. Med., 13:616-619 and Droller, et al. (1982), Urol., 20:256-258, the 63 disclosures of which are hereby incorporated by reference).

KGF-2 protein product(s) are useful to treat and/or prevent eardrum damage. Standard in vivo models of tympanic membrane perforations are known (Clymer et al. (1996), Laryngoscope (USA), 10631:280-285, the disclosure of which is hereby incorporated by reference).

KGF-2 protein product(s) are useful to treat and/or prevent disorders or damage to salivary gland tissue, including radiation/chemotherapy effects (as .discussed above) and autoimmune diseases such as SSjogren's Syndrome which can cause salivary glandinsufficiency (sicca syndrome). Standard in vivo 15 models of salivary gland tissue damage are'-known.

*The following examples are included to more fully illustrate the present invention. It is understood that modifications can be made in the procedures set forth without departing from the spirit of the invention.

EXAMPLES

*0 Standard methods for many of the procedures described in the following examples, or suitable 25 alternative procedures, are provided in widely recognized manuals of molecular biology such as, for example, Sambrook et al. (1989), supra and Ausubel et al. (1990), supra. All chemicals are either analytical grade or USP grade.

Example 1: Protein Production The following example teaches the production of the following KGF-2 protein(s): dN29 hFGF10, hFGF10, hFGF10 and hFGF10 R149Q.

64 Please note that t4e numbering of the -dNdesignation is based on the number of amino acids deleted from the normal N-terminal of the rat fulllength sequence. Human FGF10 has fewer amino acids at the N-terminal than rat FGFlO. The number does not include the methionine added by E. coi expression. The amino acid sequence of dN37 rFGF1O is identical to dN29 hFGF1O. The amino acid sequence of dN28 rFGFlO is identical to dN2O hFGF1O.

A. Freoaration ofDN pAMG2l dN29 rFGF1O: The plasmid pAMG2l dN29 hFGF10 contains DNA *.15 encoding the amino acid sequence set forth in Figure 2 (dN29 h.FGFlO). The plasmid pAMG2l dN29 hFGFlO contains a truncation of the DNA encoding the 37 amino-terminal residues from the mature rFGF10 sequence, with the truncation having the following N-terminal amino acid sequence: MSYNH{LQ.... (beginning at residue #76, Figure 2, Yamasaki et al., (1996), supra). Thus, dN29 hFGF1O has the sequence of Ser 6 9 to Ser 2 0 8 of SEQ ID NO:2 (AN32 GF-2). pAMG2l dN29 hFGFlO was created as follows.

First, plasmid pAM2l dN6 rFGFlO was constructed. For this construction, PCR was performed using mature rat FGFl0 cDNA (Yamasaki et al. (1996), J.

Biol. Chem., 271(27)L:15918-15921, rFGF) in the vector pGEM-T (Promega, Madison, WI), termed pGEM-T rFGFlO, as a Itemplate with the following 51 oligonucleotide primer (OLIGO#l), which incorporates an NdeI site, and 3' oligonucleotide primer (OLIGO#2) which incorporates a BamHI site: OLIGO~l: (SEQ ID NO:42) 5' -AAA CAA CAT ATG GTT TCT CCG GAG GCT ACC AAC TCC-3' 65 OLIGO#2: (SEQ ID NO:43) CAA GGA TCC TTT ATG AGT GGA CCA CCA TGG GG-3' The PCR product generated in this reaction was purified and digested with restriction endonucleases Ndel and BamHI. The 525 base pair (bp) restriction-digested PCR product was purified from an agarose gel and ligated with a similarly purified 6 Kilobase (Kb) BamHI to NdeI pAMG21 vector DNA fragment. [The expression vector pAMG21 (ATCC accession no. 98113) contains appropriate restriction sites for insertion of genes downstream from a luxPR promoter (see U.S. Patent No. 5,169,318 for description of the lux expression system).] The *0 15 resultant encoded rFGF10 protein differs from rFGF10 by S. deletion of the first 6 amino-terminal amino acid residues to a naturally occurring methionine residue, with the protein having the following amino-terminal (Nterminal) amino acid sequence: MVSPEAT.... (beginning at residue #43, Figure 2, Yamasaki et al.(1996), supra).

Next, the plasmid pAMG21 His rFGF10 was constructed using a pAMG21 His vector. The pAMG21 His vector differs from pAMG21 as follows: between the initiating methionine codon of pAMG21 (ATG) and the 25 sequence that follows it (GTTAACG...), the following sequence is inserted "AAA CAT CAT CAC CAT CAC CAT CAT GCT AGC" which codes for "KHHHHHHHAS". The addition of the codons for Ala and Ser after the 7x His tag afford a convenient restriction site, Nhel, for cloning.

The 4.7 Kb BstXI-NheI fragment of pAMG21 His plasmid vector was then ligated with the 1.8Kb BspEI- BstXI fragment of pAMG21 dN6 rFGF10 and the following oligonucleotide linkers OLIGO#3 and OLIGO#4 (Nhel to BspEI).

66 OLIGO*3: (SEQ ID NO:44) '-CTA GCG ATG ACG ATG ATA AAC AGG CTC TGG GTC AGG ACA TGG T11' CT-3' OLIGO#4: (SEQ ID -CCG GAG AAA CCA TGT CCT GAC CCA GAG CCT GT'r TAT CAT CGT CAT CG-3' The resultant encoded protein differs from dN6 rFGF1O by having a histidine tag (7x His) followed by an enterokinase cleavage site with the full-length (mature) N-terminal sequence (beginning at residue #37, Figure 1,.

Yamasaki et al. (1996), supra). Twenty-two amtino acids were added to the amino-terminus of the dN6 rFGF1O as follows: MKHHHHHHlIIHASDDDDKQALGQD (MVSPEAT. .:pAMG21 His rFGFIG was used as a template for PCR amplification using the following 5' oligonucleotide primer (OLIGO#5) which incorporates an NdeI site, and 3' oligonucleotide primer (OLIGO#6) which incorporates a BaxnHI site: OLIGO#S: (SEQ ID NO:46) -GGA GGA ATA ACA TAT GTC CTA CAA TCA CCT GCA GGG AGA TOGT CCG-31 *OLIGO#6: (SEQ ID NO:47) -AAA CAA GGA TCC 'Tr ATG AGT GGA CCA CCA TOG GG-31 The PCR product generated in this reaction was purified and then used as a template for subsequent PCR amplification-with the following 5' oligonucleotide primer (OLIGO#7) which incorporates an XbaI site, and 3' oligonucleotide primer (OLIGO#8) which incorporates a BamHI site: 67 OLIGO#7: (SEQ ID NO:48) GAT TCT AGA TTT GTT TTA ACT AAT TAA AGG AGG AAT AAC ATA TG-3' OLIGO#8: (SEQ ID NO:49) CAA GGA TCC TTT ATG AGT GGA CCA CCA TGG GG-3' The PCR product generated in this reaction was purified and digested with restriction endonucleases BamHI and Xbal. The 465 bp restriction-digested product was purified from an agarose gel and ligated with a similarly purified 6 Kb pAMG21 BamHI-Xbal DNA fragment to form pAMG21 dN29 E. coli host strain GM120 (ATCC accession *no.55764) has the lacIQ promoter and lacI gene integrated into a second site in the host chromosome of a prototrophic E cjli K12 host. Transformation of GM120 E. coli host with this ligation mixture and plating on Luria agar plates containing kanamycin yielded recombinant bacterial colonies. A bacterial clone containing the correct recombinant plasmid was identified by PCR screening. Plasmid DNA was purified and sequenced to confirm the insert sequence. Growth of recombinant bacterial cultures to express the gene product is described below.

pAMG21 dN20 The plasmid pAMG21 dN20 hFGF10 contains DNA encoding the amino acid sequence set forth in Figure 3 hFGF10). The plasmid pAMG21 dN20 hFGF10 contains a deletion of the DNA encoding the first 28 amino acids of the mature rFGF10 sequence resulting in the following N-terminal amino acid sequence: MSSPSSA..... (beginning at residue #65, Figure 2, Yamasaki et al. (1996), 68 supra). plasmid. Thus dN20 hFGF10 has the sequence of Ser 58 to Ser 208 of SEQ ID NO:2 (AN21 KGF-2).

pAMG21 dN20 hFGF10 was constructed as follows.

The 6 Kb BamHI-NdeI pAMG21 vector fragment was ligated to an NdeI-BamHI dN20 hFGF10 PCR product generated as follows: PCR was carried out using pGEM-T as the template and the following oligonucleotide primer (OLIGO#9) which incorporates an NdeI site at the 5' end of the rFGF10 gene and deletes codons for the first 28 amino acids, and 3' oligonucleotide primer (OLIGO#10) which incorporates a BamHI site at the 3' end of the rFGF10 gene: OLIGO#9: (SEQ ID AAA CAA CAT ATG TCT TCT CCT TCC TCT GCA GGT AGG CAT GTG CGG AGC TAC AA -3' (SEQ ID NO:51) AAA CAA GGA TCC TTT ATG AGT GGA CCA CCA TGG GG -3' This PCR product was purified, digested with restriction endonucleases Ndel and BamHI and, as described above, ligated to the 6 Kb BamHI-NdeI pAMG21 vector fragment.

25 Transformation of GM120 E. coli host with this pAMG21 dN20 hFGF10 ligation product and plating on Luria agar plates containing 40pg/ml kanamycin yielded recombinant bacterial colonies. A bacterial clone containing the correct recombinant plasmid was identified by PCR screening. Plasmid DNA was purified and sequenced to confirm the insert sequence. Growth of recombinant bacterial cultures to express the gene product is described below.

69 pAMG2l hFGF1O R149Q: The plasmid pAMG2l h.FGF10 R149Q replaces an arginine residue at position 149 in hFGF1O (Leu"O to Ser 2 "a of SEQ ID NO:2) with a glutamine residue (Figure pAMG2l hFGFlO R149Q was constructed as follows.

The plasmid pAMG2l rFGFlO was created by ligation of the 6.5 Kb BspEI-NdeI fragment of pAMG2l His .rFGF1O with the following oligonucleotide linkers OLIGO#il and OLIGO#12 (NdeI to BspEI).

OLIGO#1l: (SEQ ID NO:52) TAT GCT GGG TCA GGA CAT GGT ?I'C T -3' OLIGO#12: (SEQ ID NO:53) CCG GAG AAA CCA TGT CCT GAC CAGCA -31 The resultant encoded protein differs from His rFGF1O by deletion of the 7x Histidine tag and restoration of the original mature amino terminal protein sequence (MLGQDM. A 4.8Kb BstXI-BspEI fragment of pAMG2l rFGFlO was ligated with the 1.8Kb fragment of pAMG21 dN2O hFGF1O PstI (introduced)-BstXI and the following OLIGO#13 and OLIGO#14 oligonucleotide linkers (PstI to BspEl) to delete eight serine codons from the rat sequence.

OLIGO#13: (SEQ ID NO:54) CCG GAG GCT ACC AAC TCT AGC TCC AGC AGC TTC TCC TCT CCT AGC TCT GCA -3' OLIGO#14: (SEQ ID GAG CTA GGA GAG GAG AAG CTG CTG GAG CTA GAG TTG GTA GCC T -3' 70 pAMG21 h.FGFlO R149Q was constructed by the ligation of the 6.1 Kb pAMG21 hPGFlO BaflHI-PstI fragment with a bPGFlO IR149Q PstI-BamHI PCR product. This PCR product was created as follows: PCR A was performed using pAMG21 dN29 hFrGF1O as the template with the following S5 oligonucleotide -primer (OLIGO#15) and oligonucleotide primer (CLIGO#16), which introduces a codon change AA3A-->CAG: OLIGO#15: (SEQ ID NO:56) AAC ACC TAT GCA TCT TII' AAC TGG C -3' OLIGO#16: (SEQ ID NO:57) 15 GTC CCT GCC TGG GAG CTC CTTi 17M CAT TC -31 PCR B was performed using pAMG21 dN29 hFIGFlO as the template with the following 5' oligonucleotide primer (OLIGO#l7), which introduces a codon-change, and 3' oligonucleotide primer (OLIGO#l8), which incorporates a BamHI site: OLIGO#17: (SEQ ID NO:58) 51- GCT CCC AGG CAG GGA CAA AAA ACA AGA AGG -3' OLIGO#l8: (SEQ ID NO:59) AAC AAA GGA TCC TTTw ATG AGT GGA CCA CC -3' The products of PCR amplifications A and B above were purified and subsequent PCR was performed using them as template with the following 5' oligonucleotide primer OLIGO#19, and OLIGO#20, which incorporates a BainHI site.

OLIGO#19: (SEQ ID AAC ACC TAT GCA TCT 'rrT AAC TGG C -3' 71 (SEQ ID NO:61) AAC AAA GGA TCC TTT ATG AGT GGA CCA CC -3' The product of that reaction was also purified and subsequent PCR was performed using it as template with the following 5' oligonucleotide primer OLIGO#21, which incorporates a BamHI site, and OLIGO#22: OLIGO#21: (SEQ ID NO:62) AAC AAA GGA TCC TTT ATG AGT GGA CCA CC -3' OLIGO#22: (SEQ ID NO:63) CCG GAG GCT ACC AAC TCT AGC TCC AGC AGC TTC TCC TCT 15 CCT AGC TCT GCA -3' That final PCR product was purified and digested with restriction endonucleases PstI and BamHI. Following restriction digestion, the 440 bp DNA fragment was gel purified and ligated as described above.

Transformation of GM120 E. coli host with this ligation and plating on Luria agar plates ~containing 40pg/ml kanamycin yielded recombinant bacterial colonies. A bacterial clone containing the 25 correct recombinant plasmid was identified by PCR screening. Plasmid DNA was purified and sequenced to confirm the insert sequence. Growth of recombinant bacterial cultures to express the gene product is described below.

B. Production in E. coli: Cultures of recombinant GM120 E. coli cells containing the DNA sequence-confirmed plasmid of interest (pAMG21 dN29 rFGF10 and pAMG21 hFGF10 R149Q, 72 respectively) are each grown to optimize expression of the introduced gene, as follows: Five hundred milliliter flasks of Luria Broth plus Kanamycin were seeded with cells and grown at 30 0

C

degrees from 10 to 16 hours. All 500 mL was added to a 9 L to 11 L of NZ amine-based media in a 15 L fermentor.

All batches were grown at a pH of 7 and a dissolved oxygen level of Batches of cells containing pAMG21 dN29 rFGF10 were grown and induced at 37 0 C and of cells containing pAMG21 hFGF10 R149Q were grown and induced at 30 0 C. The batches were grown at a pH of 7 and a dissolved oxygen level of When optical cell density reached 10 2, autoinducer was added to the fermentor and cells were allowed to grow for 12 hours.

15 After 12 hours, the broth was chilled to less than the fermentor was drained and the cells were collected by centrifugation. The cell paste was frozen.

C. Purification dN29 dN29 hFGF10 was purified using three chromatography steps: S-Sepharose at pH 7.5, Heparin- Sepharose at pH 7.5, and hydroxyapatite. One hundred 25 grams E. coli cell paste containing dN29 hFGF10 was homogenized and disrupted exactly as described above.

Following centrifugation at 15,300 x g for 3 hours, the supernatant-containing soluble dN29 hFGF10 was adjusted to 40 mM Tris-HCl, pH 7.5, by addition of 1 M Tris-HCl, pH 7.5, then applied to a 300 mL S-Sepharose FF column equilibrated in 40 mM Tris-HCl, pH 7.5. After washing the column with equilibration buffer to remove unbound protein, the column was eluted with a 40-volume gradient from 0 to 2 M NaCl in 40 mM Tris-HCl, pH 7.5. Fractions eluting between 0.9 M and 1.1 M NaCl contained dN29 73 which was detected as a 16 kDa band on SDS-PAGE.

The identity of this band was confirmed by N-terminal sequencing. These fractions were pooled, diluted with mM Tris-HCl, pH 7.5, to reduce the NaC1 concentration to 0.4 M, and applied to a 60 mL Heparin-Sepharose column equilibrated in 40 mM Tris-HCl, pH 7.5. The column was washed with equilibration buffer to remove unbound protein, then eluted with an 80-volume gradient from 0 to 3 M NaCl in the same buffer. dN29 eluted between 1.0 M and 1.35 M NaC1. These fractions were pooled and dialyzed against 40 mM Tris-HCl, pH The dialyzed sample was applied to a 50 mL hydroxyapatite column equilibrated in 40 mM Tris-HCl, pH 7.5. Following sample application the column was washed 15 with equilibration buffer to remove unbound protein, then eluted with a 40-volume gradient from 0 to 0.5 M NaCl. Fractions eluting between 0.24 M and 0.44 M NaCl contained dN29 hFGF0O. These fractions were pooled, concentrated, and buffer-exchanged to PBS. Sample purity was estimated to be greater than 97% by Coomassie Blue-stained SDS-gels. The yield of purified dN29 was 90 mg from 100 g cell paste.

R149Q: 25 hFGF10 R149Q was purified exactly as described above for dN29 hFGF10. Human R149Q FGF10 had a lower binding affinity for Heparin-Sepharose than dN29 eluting between 0.5-0.8 M NaCl. The identity and purity of hFGF10 R149Q was analyzed by N-terminal sequencing, and SDS-gel electrophoresis.

Example 2: In vitro bioassav The bioactivity of purified dN29 hFGF10 was assessed by the .Balb/MK mouse keratinocyte proliferation assay, which is designed to measure specific activity.

74 For the Balb/MK keratinocyte assay, 0.5 mg/mL stock solutions of dN29 hFGF10 in PBS was prepared.

These samples were serially diluted into assay medium and 50 mL of each dilution was added to tissue culture wells containing Balb/MK cells in 180 mL assay medium.

The final concentration of dN29 hFGF0l ranged from 1.2 x 10-1 ng/mL to 2.2 x 104 ng/mL. Cell proliferation was measured by uptake of tritiated thymidine.

The estimated ED,, value for dN29 hFGF10 was 46 ng/mL. The results show that dN29 hFGFl0 is effective in this assay.

Example 3: Exploratory Studies in Normal Mice In the first study, 18 female BDF1 mice were 15 divided into 6 groups of 3 mice each (1 treated and 1 control group at each of 3 time points). The first two groups received 5 mg/kg dN29 hFGF10 or the buffer control IV for 1 day, the second two groups received mg/kg dN29 hFGFIO or the buffer control IV daily for 3 days, and the third two groups received 5 mg/kg dN29 hFGFlO or the buffer control IV daily for 7 days. All mice were injected with 50 mg/kg BrdU one hour prior to harvest, radiographed, and sacrificed. Body and selected organ weights (including all segments of.small 25 intestine) were taken, blood was drawn for hematology and serum chemistries, and organs were harvested for histologic analysis and BrdU labeling. There was some elevation of stomach on day 7, liver on day 3, jejenum on day 7. There was no effect on thymus. The serum chemistries were variable and normalize very rapidly.

Example 4: Chemotherapy-induced Pulmonary Fibrosis Male Lewis rats weighing approximately 225 grams received an i.v. injection or intratracheal instillation of 5 mg/kg of dN29 hFGFIO or vehicle 72 and 75 48 hours prior to receiving 2.5U of bleomycin via the intratracheal route. Rat weight was monitored over the course of the following 15 days at which time pulmonary function tests were performed in the rats that had received the dN29 hFGF10 via the intratracheal route.

For histology, catheters was placed in the trachea of each rat and the lungs were filled with 3 ml of formalin via hydrostatic pressure. Following fixation for 48 hours and the lungs were processed into paraffin for sectioning and staining.

Rats receiving saline administration lost 42% "of their body weight while those treated with dN29 hFGF10 were are 129% of their weight compared to the day of bleomycin administration. One rat in the saline group died before the time-of sacrifice, this death is assumed to be from the insult of bleomycin to the lung.

There was a significant difference in pulmonary respiratory rate and tidal volume between the two groups. Rats treated with saline had a respiration rate 20 of 286 breaths per minute compared with 247 for the dN29 group. Untreated control rats had respiratory rate of 216 breaths per minute which is significant vs.

the dN29 hFGF10 group at the p<0.05 level.

ooo Histologically, there was gross and microscopic changes to the saline treated group. The •lungs in this group were deformed, as was observed at the time of sacrifice, and had excessive inflammation and fibrosis. Lungs of the dN29 hFGFO0 group were very similar to those of the untreated controls with the exception of having focal mild microscopic inflammation.

There was no distinguishable gross difference between the dN29 hFGF10 and normal rats.

76 Example 5: Radiation-induced Mucositis Model Mucositis is induced in mice with 12 Gy of whole-body radiation. Mice are treated daily with mg/kg/day of recombinant human KGF-2 (prepared generally in accordance with the teachings of WO 96/25422, rhuKGF-2) beginning on the day before radiation and continuing to day three post-radiation.

Four days after radiation, the mice are necropsied and the number of proliferating crypts (containing BUdRpositive cells) are counted.

rhuKGF-2 treatment increases the number of proliferating crypts in the duodenum, proximal and distal jejunum of the small intestine relative to nonrhuKGF-2 treated animals. rhuKGF-2 is also able to 15 decrease the body weight loss in the irradiated mice.

Example 6: Adriamvcin-induced Mucositis Model Mucositis is induced in mice with a single intraperitoneal dose of Adriamycin at 24 mg/kg. Mice 20 are treated daily with 1 mg/kg/day of rhuKGF-2 beginning on the day before radiation and continuing to day three post-radiation. Four days after radiation, the mice are necropsied and the number of proliferating crypts (containing BUdR-positive cells) are counted.

rhuKGF-2 treatment increases the number of Sproliferating crypts in the duodenum, jejunum and ileum relative to non-rhuKGF-2 treated animals.

Example 7: 5-fluorouracil-induced Mucositis Model Mice are injected with 5-fluorouracil mg/kg/day x 4 days), a regimen that in non-treated animals leads to a survival ranging only between 20-50%.

rhuKGF-2 pretreatments (5 mg/kg/day x 3 days), but not post-treatments, increase survival relative to nonrhuKGF-2 treated animals and improvements in survival

II

77 are seen at doses as low as 0.5 mg/kg/day. Hepatic abscesses are commonly found in control, but not rhuKGF- 2-pretreated, surviving mice indicating that toxicity is in part due to loss of the GI barrier function. In addition, rhuKGF-2-pretreated mice lose less weight and consume more food and water during the treatment period. rhuKGF-2 pretreatments also ameliorate weight loss in mice following carboplatin (125 mg/kg x 1 day) exposure, excluding the possibility that rhuKGF's effects are 5-FU specific. rhuKGF-2 pretreatments also improve survival and weight loss nadirs in chemotherapy/radiation combination experiments when mice are injected with a single dose of 150 or 300 mg/kg of methotrexate followed by irradiation (6 Gy) 15 1 hour later.

Example 8:Colitis Model In two groups of 10 animals each, colitis is induced by colonic instillation of 2,4,6trinitrobenzenesulfonic acid in ethanol at a dose of mg/kg body weight. To determine if rhuKGF-2 acts through a protective mechanism, one group of rats (group A) is precreated with rhuKGF-2 or vehicle at 24 hours and at 1 hour prior to induction of colitis at a dose of 5 mg/kg and the animals are sacrificed •8 hours after injury. To assess potential healing effects, rhuKGF-2 or vehicle (same dosage, is injected in a second group (group B) 24 hours after induction of colitis and treatment is continued daily for 1 week. Tissue damage is examined microscopically and is expressed as percentage of ulcerations or erosions.

Animals which are treated with rhuKGF-2 after induction of colitis (group B) show significantly less ulcerations compared to the control group (group A).

78 In animals treated prior to induction of colitis, there are erosions, but no ulcers are seen due to the short study period of 8 hours, and the erosions are not significantly different.from those seen in the control group (group A).

Example 9:Dextran sulfate-induced Colitis Model Study 1: Rats are fed 4% and 6% DSS in water for 1 week. At the end of the second week the distal 4 cm of the colon is preserved. Eight sections at 0.5 cm intervals are prepared and stained with H E. The percent of each colon section with necrosis (destruction of the glandular structure) is assessed in a randomized and coded fashion.

15 Study 2: Rats are given IP vehicle or rhuKGF-2 (1 mg/kg/day) and fed either water or 4% dextran sulfate sodium for 14 days. The colonic sections are stained with PAS.

Dextran sulfate sodium appears to induce a 20 dose-related increase in colonic mucosal necrosis.

rhuKGF-2 administered at 1 mg/kg/day for 14 days increases colonic mucin production in the control group as well as in the dextran sulfate sodium-treated rats.

25 Example 10: Rat Cirrhosis Model S: Male Sprague-Dawley rats weighing between 150 and 175 gms are used. Animals are exposed to phenobarbitol (0.35 mg/ml) in their drinking water for the duration of the study. Animals are dosed weekly with CC14 in a corn oil vehicle while under light isoflurance anesthesia. The initial dose of CC14 is gl per rat. The dose is adjusted weekly, up or down in 40 gl increments based on weight gain. Ten control animals are exposed to phenobarbitol in their drinking water and lavaged weekly with corn oil vehicle. Liver

I;

79 function is assessed by measurement of bromosulphopthylein (BSP) clearance, serum transaminase levels and serum albumin levels. At the time of sacrifice, livers are removed, weighed and processed for determination of hydroxyproline levels as an indicator of collagen deposition and fibrosis.

Animals are maintained on the above cirrhosis induction protocol for 11 weeks. In the eleventh week, animals are randomized into control and rhuKGF-2 treatment groups. rhuKGF-2 is given once per day by subcutaneous injection at a dose of 1 mg/kg, for a total of 15 days. After 15 days of rhuKGF-2 treatment, the animals are euthanized.

Rats in which cirrhosis is induced with CCl4 show an elevation in serum BSP concentration, reflecting impaired liver clearance of this agent. Rats treated with rhuKGF-2 have a lower BSP serum level than untreated animals, suggesting an improved liver function. Rats made cirrhotic by CC14 show an elevation in SGPT which is reversed by rhuKGF-2 treatment.

rhuKGF-2 treatment is able to elevate serum albumin.

rhuKGF-2 treatment results in an increased liver-to-body weight ratio, reflecting compensatory liver growth.

Example 11: Henatectomv Model Rats subjected to a 70% partial hepatectomy recover their original liver mass more quickly when treated with 1 mg/kg/d rhuKGF-2 than when compared to untreated animals.

Example 12: Acute heDatotoxicity Models In acute hepatotoxicity models, rhuKGF-2 treatment (1 mg/kg either prior to or 3 hr after the inciting agent) blunts increases in serum transaminase levels in rats with acute hepatic failure induced with 80 either carbon tetrachloride, acetaminophen or galactosamine. Pretreatment with rhuKGF-2 also prevents a decrease in liver clearance functions after acetaminophen, as measured by sulfobromophthalein (BSP) clearance.

Example 13: In Vivo Retro-viral-Mediated Gene Transfer Model Mice are intraveneously administered with rhuKGF-2 (1-5 mg/kg). 48 hours after intravenous injection of rhuKGF-2, murine hepatocyte proliferation increases, compared to non-stimulated livers, and o. returns to normal proliferative levels. No modules or microscopic abnormalities are noted either acutely or 15 after 5 months.

When rhuKGF-2 treatment is followed by intravenous injection of high titer E. coli LacZ expressing Moloney retroviral vectors (1 x 108 cfu.ml), -galactosidase expression increases with a percentage of the hepatocytes being transduced. Several months later, a portion of the transduced hepatocytes remain Xgal positive.

Example 14: In Vivo Model of Diabetes Male rats weighing 200 to 260 grams at study initiation are used in this model (WO 9611950). Diabetes is induced by a single intravenous injection of streptozotocin at 50 mg of streptozotocin in sodium citrate buffer per kg of body weight. Non-diabetic control rats receive a single intravenous injection of sodium citrate buffer for control purposes. rhuKGF-2 is administered daily as a subcutaneous injection. The rhuKGF-2 dose is 3 or 5 mg/kg/day, depending upon the experiment.

81 In the first experiment, rhuKGF-2 therapy is initiated two days before diabetes, is induced and continued after the induction of diabetes for a total of eight injections. Those diabetic rats which are treated with rhuKGF-2 prior to diabetes induction, and for which rhuKGF-2 is also continued after the induction, show symptoms indicative of a milder form of diabetes. Thus, rhuKGF-2 therapy either partially prevents induction of the disease or restores insulin-producing islet cells after streptozotocin-induced beta cell destruction.

In the second and third experiments, rhuKGF-2 therapy administered subcutaneously is initiated one day after the induction of diabetes with streptozotocin. In the second study, fasting water intake and urine output 15 are significantly less in the rhuKGF-2-treated diabetic rats when compared to diabetic rats on day 9, which is further indicative of amelioration of the disease condition. In the third study, rhuKGF-2 therapy is able to increase the total content of insulin and C-peptide in 20 the pancreas of diabetic rats when compared to diabetic rats treated with sodium chloride solution.

In the fourth experiment, a 7-day course of rhuKGF-2 therapy is initiated 7 days after streptozotocin treatment and the animals are then followed for an additional 12 weeks. In all experiments, except for the fourth experiment, blood glucose levels, urine glucose levels and urine volume are used as end points for analysis. Additionally, water intake, urine C-peptide levels, or total pancreatic insulin and C-peptide content are measured in some experiments. In the fourth experiment, the only assessed endpoint is blood glucose.

Because a large fraction of insulin is removed from the circulation by the liver, measurement of peripheral insulin concentrations reflects post-hepatic metabolism events rather than insulin secretion from the 82 pancreas. Therefore, measurements of C-peptide are often made and used as a peripheral marker of insulin secretion. C-peptide is produced from the processing of pro-insulin to insulin. Insulin and C-peptide are secreted from the beta cells in equimolar amounts, and only a small amount of C-peptide is extracted by the liver.

STZ-treated animals from both groups receiving rhuKGF-2 have significant declines in blood glucose during the rhuKGF-2 dosing period.

Example 15: Hvyeroxia-induced Mortality Model To determine the effect of rhuKGF-2 administration on hyperoxia-induced lung injury, rats are treated by intratracheal instillation and exposed to >98% oxygen for up to 120 hours. At necropsy, after 120 hours of hyperoxia exposure, the lungs of rhuKGF-2 treated animals appear grossly normal, with few scattered areas of puncture hemorrhage on the pleural 20 surface, compared with the grossly hemorrhagic lungs of untreated rats dying between 55 and 80 hours of hyperoxia exposure.

Histopathologically, the lungs of untreated animals demonstrate large areas of hemorrhage and interstitial edema. The intraaveolar space contains red blood cells, inflammatory cells, and proteinaceous exudate. In contrast, there is no intraaveolar exudate and minimal evidence of hemorrhage in the lungs of the animals treated with rhuKGF-2 who survive for 120 hours in hyperoxia.

At doses of 5 and 1 mg/kg, rhuKGF-2 significantly decreases hyperoxia-induced mortality.

_1 83 Example 16: Acute Lunr Iniurv Model Acute permeability pulmonary edema is induced with an injection of a-naphthylthiourea, and lung leak is assessed in an isolated perfused lung model over 180 minutes. Leakage is confirmed with wet/dry lung weight ratios, and the alveolar fluid protein concentration is measured after bronchoalveolar lavage.

SThe effect of pretreatment with rhuKGF-2 (injected intratracheally 48 hours before the experiment) on a-naphthylthiourea-induced pulmonary edema is assessed (rhuKGF-2/a-naphthylthiourea group). Control groups (Control and rhuKGF-2/Control) are also studied.

istopathology is performed for each of the four groups.

The a-naphthylthiourea produces an acute 15 permeability pulmonary edema detected by lung leak over the 180-minute ex vivo period of monitoring the isolated perfused lung. Pretreatment with rhuKGF-2 significantly *o attenuates these parameters which are not significantly different from the control group and the rhuKGF- 2/control group. Histopathology shows abundant type II pneumocyte hyperplasia in the lungs of animals **pretreated with rhuKGF-2, and marked pulmonary edema in animals pretreated with a-naphthylthiourea. Less edema is apparent in the rhuKGF-2/ a-naphthylthiourea group.

25 While the present invention has been described above both generally and in terms of preferred embodiments, it is understood that other variations and modifications will occur to those skilled in the art in light of the description above.

DATED this 21st March, 2001 AMGEN, INC Attorney: DAVID A. ADAMTHWAITE Fellow Institute of Patent and Trade Mark Attorneys of Australia of BALDWIN SHELSTON WATERS 84 SEQUENCE LISTING GENERAL INFORMATION: APPLICANT: Amgen Inc.

(ii) TITLE OF INVENTION: KERATINOCYTE GROWTH FACTOR-2 PRODUCTS (iii) NUMBER OF SEQUENCES: 63 (iv) CORR.ESPONDENCE ADDRESS: ADDRESSEE: Amgen Inc.

STREET: 1840 De Havilland Drive CITY: Thousand Oaks STATE: California COUNTRY: US ZIP: 91320-1789 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk C014PUTDER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.30 CURRENT APPLICATION DATA: APPLICATION NUMBER: FILING DATE:

CLASSIFICATION:

(vii) PRIOR APPLICATION DATA: APPLICATION NUMBER: US 60/028,493 FILING DATE: 15-OCT-1996 (vii) PRIOR APPLICATION DATA: APPLICATION NUMBER: US 60/032,781 FILING DATE: 06-DEC-1996 (vii) PRIOR APPLICATION DATA: CA) APPLICATION NUMBER: US 60/033,046 FILING DATE: 10-DEC-1996 INFORMATION FOR SEQ ID NO:l: Ci) SEQUENCE CHARACTERISTICS: CA) LENGTH: 627 base pairs TYPE: nucleic acid STRANDEDNESS: unkniown TOPOLOGY: unkniown (ii) MOLECULE TYPE: cDNA Cix) FEATURE: CA) NAME/ KEY: COS CB) LOCATION: 1.-627 85 (xi) SEQUENCE DESCRIPTION: SEQ.ID NO: 1: ATG TOG AAA TOG ATA CTG ACA CAT TGT GCC TCA GCCTTT CCC CAC Met Trp Lys Trp, 1 CCC GGC TGC TGC Pro Gly Cys Cys GTC CCT GTC ACC Val Pro Val Thr GCC ACC AAC TCT Ala Th~r Asn Ser so AGO CAT GTO CGG Arg His Val Arg -AAG CTA ?TC TCT Lys Leu Phe Ser AAG GTC AGC 000 Lys Val Ser Gly 100 ATA ACA TCA GTA Ile Thr Set Val 115 AAC TAT TAC TTA Asn Tyr Tyr Leu 130 GAA T TT AAC AAT Glu Phe Asn Asa 145 TAC AAT ACC TAT Tyr Asn Thr Tyr TAT GTG GCA TTG Tyr Val Ala Leu.

180 CGA AGG AAA AAC Arg Arg Lys Asn 195

TAG

Ile

TOC

Cys Cys

TCT

Ser

AGC

Ser TrC Phe as

ACC

Thr

GAA

Glu

GCC

Ala

GAC

Asp

GCA

Ala 165

AAT

Asn

ACC

Thr TOC T GC TTr Cys Cys Phe CAA 0CC CTT Gin Ala Leu 40 TCC TCC TOc Ser Ser Ser 55 TAC AAT CAC Tyr Asn His 70 ACC AAG TAC Thr Lys Tyr AAG AAG GAG Lys Lys Giu ATC GGA GTT Ile Gly Val 120 ATG AAC AAG Met Asn Lys 135 TOT AAG CTG Cys Lys Leu 150 TCA TTT AAC Ser ?he Asa GGA AAA OGA Gly Lys Gly TCT OCT CAC Set Ala His 200 Leu 25

GT

Oly

TTC

Phe Leu Phe

AAC

Asn 105 Val

AAG

Lys

AAG

Lys

TG

Trp

OCT

Ala 185 Phe Leu

CAG

Gin

TCC

Ser

CAA

Gin

CTC

Leu 90

TGC

Cys

GCC

Ala

GGO

Giy

GAG

Glu

CAG

Gin 170

CCA

Pro Leu

CTG

Leu

GAC

Asp

TCT

Set

GGA

Gly 75

AAG

Lys

CCG

Pro

GTC

Val

AAA

Lys

AGO

Arg 155

CAT

His

AGO

Arg

CCA

Pro

TTC

Phe

ATG

Met

CCT

Pro

GAT

Asp

ATT

Ile

TAC

Tyr

AAA

Lys

CTC

Leu 140

ATA

Ile

AAT

Asa

AGA

Arg

ATG

Met TGG'iG is TcT TICC c 2 Leu Val Set Set O'TG TCA CCA GAG Vai Set Pro Giu TCC AGC GCG GGA Set Ser Ala Giy GTC CGC TOG AGA Val Arg Trp Arg G3AG AAG AAC 000 G1u Lys Asn Gly P.00 ATC CTO GAG Ser Ile Leu Oiu 110 GCC ATT A6AC AGC kia Ile Asa Set 125S T GGC 'rCA AAA L'yr Gly Set Lys 3AG GAA AAT GGA 1lu Oiu Asn Gly 160 iGG AGO CAA ATO ;iy Arg Gin Met 175 ;GA CAG AAA ACA ;iy Gin Lys Thr 190 ,M GTA CAC TCA rai Val His Ser 144 192 240 288 336 384 432 480 528 576 624 Leu Thr His Cys Ala Set Ala Phe Pro His 86 INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 209 amino acids TYPE: amiuno acid TOPOLO0GY: unknown (ii) MOLECULE TYPE: protein Met Pro Val Ala Arg

LYS

LYS

Ile Asn Glu 145 Tyr (xi) Ti-p Lys Gly Cys Pro Val Thr Asrn so His Val Leu. Phe Val Ser Th- Ser 115 Tyr Tyr 130 Phe Asn Asn Thr Val Ala

SEQUENCE

5Ti-p Ile

S

aCys Cys Thr Cys Ser Ser Arg Ser Ser Phe 85 Giy Thr 100 Val Glu Leu Ala Asn Asp 'Tyr Ala 165 Leu Asn 180 Phe Leu Leu 25 Leu Gly Gin Phe Leu Phe 105 Val Lys Lys Ti-p Ala 185 Ser Ser Gin Gly 75 Leu Lys 90 Cys Pro Ala Val Gly Lys Giu Arg 155 Gin His 170 Pro Arg Leu Val S30 Val Ser Ser Ser Val Arg Giu Lys Ser Ile 110 Ala Ile 125 Tyr Gly Glu Giu Gly Arg Gly Gin DESCRIPTION: SEQ ID NO:2; Leu Thr His Cys Ala Ser Al1a Phe Pro His Leu 10 Ser Pro Ala T-p, Asn Leu Asn Ser Asn Gin .175 Lys Ser Glu Gly Arg so Gly Giu Ser Lys Gly 160 Met Th- 190 Arg Arg Lys 195 Asn Thr Ser Ala PeLuPr Phe Leu Pro Met Val Val His Ser 205 87 INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 425 base pairs TYPE: nucleic acid STRANDEDNESS: unknxown TOPOLOGY: unknxown (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 1. .426 (xi) SEQUENCE DESCRIPTIION: SEQ ID NO:3.

ATG TCC TAC AAT CAC CTG CAG GGA GAT GTC CGC TGG AGA AAG CTG TTC Met Ser Tyr Asn His Leu Gin Gly Asp Val. Arg Trp Arg Lys Leu Phe 1. 5 TCC TTC ACC AAG TAC Ser Phe Thx Lys Tyr GGG ACC AAG AAG GAA Gly Thr Lys Lys Glu GTG GAA ATC GGA G?1' Val Glu Ile Gly Val TTA GCC ATG AAC AAG Leu Ala Met Asn Lys AAT GAC TGT AAA CTG Asn Asp Cys Lys Leu TAT GCA TCT TTT AAC Tyr Ala Ser Phe Asn 100 TTG AAT GGA AAA GGA Leu Asn Gly Lys Gly 115 10~ is GAA AAG AAC GGC Glu Lys Asn Gly AGT ATC CTA GAG Ser Ile Leu Glu GCC ATIT AAC AGC Ala Ile Asn Ser TAT GGC TCA AAA Tyr Gly Ser Lys GAG GAA A.AT GGA Glu Glu Asn Gly GGC AGG CAA ATG Gly Arg Gin Met GGA CAA AAA ACA Gly Gin Lys Thr 125 96 144 192 240 288 336 384 AAC ACC TCC GCT CAC ?1'C CTC CCC ATG GTG GTC CAC TCA TAA- Asn Thr Ser Ala His ?he 130 Leu. Pro Met Val Val His Ser 135 140 88 INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 142 amino acids TYPE: amino acid TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: Met Ser Tyr Asn His Leu Gin Gly Asp Val Arg Trp Arg Lys Leu Phe 1 5 10 Ser Phe Thr Lys Tyr Phe Leu Lys Ile Glu Lys Asn Gly Lys Val Ser 25 Gly Thr Lys Lys Glu Asn Cys Pro Tyr Set Ile Leu Glu Ile Thr Ser 40 Val Glu Ile Gly Val Val Ala Val Lys Ala Ile Asn Ser Asn Tyr Tyr S: 50 55 Leu Ala Met Asn Lys Lys Gly Lys Leu Tyr Gly Ser Lys Glu Phe Asn 65 70 75 9 Asn Asp Cys Lys Leu Lys Glu Arg Ile Glu Glu Asn Gly Tyr Asn Thr 85 90 Tyr Ala Ser Phe Asn Trp Gin His Asn Gly Arg Gin Met Tyr Val Ala I 100 105 110 Leu Asn Gly Lys Gly Ala Pro Arg Arg Gly Gin Lys Thr Arg Arg Lys 115 120 125 Asn Thr Ser Ala His Phe Leu Pro Met Val Val His Ser S130 135 140 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 459 base pairs TYPE: nucleic acid STRANDEDNESS: unknown (D TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..459 89 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:S: ATG TCT TCT CCT TCC TCT GCA GGT AGG CAT GTG CGG AGC TAC AAT CAC Met Ser Ser Pro Ser Ser Ala Gly Axg His Val Axg Ser Tyr Asn His 1

CTC

Leu Phe

AAC

Asn GT'r Val

AAG

Lys

AAA

Lys

TGG

Trp

GCT

Ala

TTC

Phe 145 CAG GGA GAT Gin Gly Asp CTC AAG ATT Leu Lys Ile TGT CCG TAC Cys Pro Tyr GCC GTC AAA Ala Val Lys GGG AAA CTC Gly Lys Leu GAG AGG ATA Glu Arg Ile 100 CAG CAC AAC Gin His Asn 115 CCC AGG AGA Pro Arg .Arg 130 CTC CCC ATG Leu Pro Met CGC TOG AGA Arg Trp Arg AAG, AAC GGC Lys Asn Gly 40 ATC CTA GAG Ile Leu Glu 55 ATT AAC AGC Ile Asn Ser 70 GOC TCA AAA Gly Ser Lys GAA AAT GGA Giu Asn Gly AGG CAA ATG Arg Gin Met 120 CAA AAA ACA Gin Lys Thx 135 GTC CAC TCA Val His Ser 150 10 AAG CTG Lys Leu 25 AAG GTC Lys Vat ATA ACA Ile Thr AAC TAT Asn Tyr GAA TTT Glu Phe 90 TAC AAC Tyr Asn 105 TAT GTG Tyr Val TTC TCC Phe Ser AGC 000 Ser Gly TCA GTG Ser Vat TAC TTA Tyr Leu AAC AAT Asn Asn ACC TAT Thr Tyr GCA TTG Ala Leu TTC ACC AAG Phe Thr Lys ACC AAG AAG Thr Lys Lys GAA ATC GGA, Glu Ile Gty GCC ATG AAC Ala Met Asn GAC TOT AAA Asp Cys Lys GCA TCT T Ala Ser Phe 110 AAT GGA AAA Asn Gly Lys 125 AGG AAA AAC Arg Lys Asn 140 ACC TCC GCT CAC Tbx Sex Ala His INFORMATION FOR SEQ ID NO:6: SEQUENCE CHARACTERISTICS: LENGTH.: 153 amino acids TYPE: amino acid TOPOLOGY: unknown (ii) MOLECULE TYPE: protein 90 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: Met Ser 5cr Pro Ser Ser Ala Gly Arg His Val Ar; Ser Tyr Asa His 1 5 10 Leu Gin Gly Asp Val Ar; Trp, Ar; Lys Leu Phe Ser Phe Thr Lys Tyr 25 Phe Leu Lys Ile Giu Lys Asn Gly Lys Vai Ser Giy Thr Lys Lys Giu 40 Asn Cys Pro Tyr Ser Ile Leu Giu Ile Thr Ser Val Giu Ile Gly Val 55 Val Ala Val Lys Ala Ile Asn Ser Asn Tyr Tyr Leu Ala Met Asn Lys 70 75 Lys Gly Lys Leu Tyr Gly 5cr Lys Glu Phe Asn Asn Asp Cys Lys Leu, 90 Lys Glu Ar; Ile Glu Giu Asn Gly Tyr Asn Thr Tyr Ala Ser Phe Asn 100 105 110 Trp Gin His Asn Gly Arg Gin Met Tyr Val Ala Leu Asn Giy Lys Gly *115 120 125 *Ala Pro Arg Ar; Giy Gin Lys Thr Arg Arg Lys Asn Thr Ser Ala His *130 135 140 Phe Leu Pro Met Val Val His Ser* 145 150 INFORMATION FOR SEQ ID NO:7: SEQUENCE CHARACTERISTICS: LENGTH:.513 base pairs TYPE: nucleic acid STRANDEDNESS: unkn~own TOPOLOGY: unkn~own (ii) MOLECULE TYPE: cDNA (ix) FEATURE.

NAME/KEY: CDS LOCATION: 1. .513 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: ATO CTG GGT CAG GAC ATG GTT TCT CCG GAG OCT ACC AAC TCT AGC TCC 48 Met Leu Gly Gin Asp Met Vai Ser Pro Glu Ala Thr Asn Ser Ser Ser 1 S 10 AGC AGC TTC TCC TCT CCT AGC TCT GCA GOT AGG CAT OTG CGG AGC TAC 96 Ser Ser Phe 5cr Ser Pro Ser 5cr Ala Gly Arg His Val Ar; 5cr Tyr 25 91 AAT CAC CTC CAG GGA GAT GTC A-sn His Leu Gin Gly Asp Val CGC TGG A-GA AAG CTG TTC TCC TTC ACC A-rg Trp Ag Lys Leu Phe Ser Phe Tbx A-AG TAC T'!r CTC A-AG ATT GAA A-AG AAC GGC A-AG Lys TIyr Phe Leu AAG OAA AAC TOT Lys Giu A-sn Cys OGA OTT OTT 0CC OGly Val Val Ala A-AC A-AG A-AG 000 A-sn Lys Lys Gly 100 A-A-A CTG AAA GAG Lys Leu Lys Glu 115 ?1'T A-AC TOG CA-G Phe A-sn Trp Gin 130 AAA GGA GCT CCC Lys Giy A-la Pro 145 OCT CAC TTC CTC A-la His Phe Lou Lys Ile Oiu Lys A-sn Gly Lys 55 CCG TA-C AGT ATC CTA GAG A-TA Pro Tyr Ser Ile Lou Glu Ile 70 75 OTC A-A-A 0CC ATT A-AC A-GC A-AC Val Lys Ala Ile A-sn Ser A-sn 90 AA-A CTC TAT GGC TCA AAA GAA Lys Lau Tyr Gly Ser Lys Giu 105 AGO A-TA GAG GAA A-AT OGA TAC A-rg Ile Glu Oiu A-sn Oly Tyr 120 CAC A-AC 0CC AGO CAA A-TO TAT His A-sn Oly A-rg Gin Met Tyr 135 A-GG CAG OGA CAA A-AA A-CA A-GA A-rg Gin Gly Gin Lys Thr A-rg 150 155 CCC A-TO OTO OTC CA-C TCA TAA Pro Met Vai Val His Ser 165 170 GTC A-OC 000 A-CC A-AG Val Ser Oly Thr Lys A-CA TCA OTG GAA A-TC Thr Ser 'Ial Olu Ile TAT TA-C TTA 0CC A-TO Tyr Tyr Lou A-ia Met ?1'T AAC A-AT GAC TOT Phe A-sn A-sn Asp Cys 110 A-AC A-CC TAT OCA TCT A-sn Thr Tyr A-ia, Ser 125 OTO OCA TTG AAT GGA Val Ala Lou A-sn Gly 140 A-GO AAA AAC A-CC TCC Arg Lys A-sn Thr Ser 160 144 192 240 288 336 384 432 480 INFORMATION FOR SEQ ID NO:8: SEQUENCE CHARACTERISTICS: LENGTH: 171 amino acids TYPE: amino acid TOPOLOGY: unkn~own (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: Met Leu Oly Gin Asp Met Val Ser Pro Olu A-la Thr A-sn Ser Ser Ser 1 S 10 Ser Ser Phe Ser Ser Pro Ser Ser A-ia Oly Arg His Vai Arg Ser Tyr 25 A-sn His Lou Gin Oly Asp Val Arg Trp Arg Lys Lou Phe Ser Phe Thr 40 92 Lys Lys Gly Asn Lys Phe Lys 145 Ala (2) Tyr Phe Leu Lys Ile Glu Lys Asn Gly LN so Glu Asn Cys Pro Tyr Ser Ile Leu Giu Il 70 Vail Val Ala Val Lys Ala Ile Asn Ser As 90 Lys Lys Gly Lys Leu Tyr Gly Ser Lys Gi 100 105 Leu. Lys Giu Arg Ile Glu Giu Asn Gly Ty .115 120 Asn Trp Gin His Asn Gly Arg Gin Met Ty 130 135 Gly Ala Pro Arg Gin Giy Gin Lys Thr Ar 150 15 His Phe Leu Pro Met Val Val His Ser 165 170 INFORMATION FOR SEQ ID NO:9: SEQUENCE CHARACTERISTICS: LENGTH: 4 amino acids TYPE: Amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: protein Ser Gly Thr Lys Ser Val Glu Ile Tyr Leu Ala Met Asn Asn Asp Cys 110 Thr Tyr Ala Ser 125 Ala Leu Asn Gly Lys Asn Tbhr Ser 160 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: Val Arg Ser Tyr INFORMATION FOR SEQ TD (W SEQUENCE CHARACTERISTICS: LENGTH: 5 amino acids TYPE: amino acid STRANDEDNESS:.unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:iO: His Val Arg Ser Tyr 1 93 INFORMATION FOR SEQ ID NO:11: SEQUENCE CHARACTERISTICS: LENGTH: 6 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: Arg His Val Arg Ser Tyr 1 INFORMATION FOR SEQ ID NO:12: Ci) SEQUENCE CHARACTERISTICS: LENGTH: 7 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: Gly Arg His Val Arg Ser Tyr 1 INFORMATION FOR SEQ =f NO:13: Ci) SEQUENCE CHARACTERISTICS: LENGTH: 8 amino acids TYPE: amino acid STRAZ4DEDNESS: unknown C(D) TOPOLOGY unknown (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ, ID NO:13: Ala Gly Arg His Val Arg Ser Tyr 1 94 INFORMATION FOR SEQ ID NO:14: i)SEQUENCE CHARACTERISTICS: LENGTH: 9 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: Ser Ala Gly Arg His Val Azg Ser Tyr 1 S INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 10 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID Ser Ser Ala Gly Arg His Val Arg Ser Tyr 1 5 INFORMATION FOR SEQ ID NO:16: Ci) SEQUENCE CHARACTERISTICS: LENGTH: 11 amino acids TYPE: amino acid STP.ANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: Pro Ser Ser Ala Gly Arg His Val. Arg Ser Tyr 1 5 95 INFORMATION FOR SEQ ID NO:17: Ci) SEQUENCE CHARACTERISTICS: LENGTH: 12 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: Ser Pro Ser Ser A-la Gly A-rg His Val Arg Ser Tyr 1 INFORMATION FOR SEQ ID NO:18: SEQUENCE CHARACTERISTICS: LENGTH: 13 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: protein SEQUENCE DESCRIPTION: SEQ ID NO:18: Ser Ser P ro Ser Ser Ala Gly Arg His Val Arg Ser Tyr 1 5 INFORMATION FOR SEQ ID NO:19: SEQUENCE CHARACTERISTICS: LENGTH: 14 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ, ID NO:19: Phe Ser Ser Pro Ser Ser Ala Gly Arg His Val Arg Ser Tyr 1 5 96 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 15 amino acids TYPE: amino acid STRANDF.DNE.SS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ, ID Ser Phe Ser Ser Pro Ser Ser Ala Gly Ar; His Val Arg Ser Tyr 1 5 10 INFORMATION FOR SEQ ID NO:21: SEQUENCE CHARACTERISTICS: LENGTH: 16 amino acids -TYPE: amino acid CC) STRANDEDNESS: unknown TOPOLOGY: unknown MOLECULE TYPE: protein SEQUENCE DESCRIPTION: SEQ ID NO:21: Ser Ser Phe Ser Ser Pro Ser Ser Ala Gly Ar; His Val Arg Ser Tyr 1 5 10 INFORMATION FOR SEQ-ID NO:22: i)SEQUENCE CHARACTERISTICS: LENGTH: 17 amino acids *sot TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE:. protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: Ser Ser Ser Phe Ser Ser Pro Ser Ser Ala Gly Ar; His Val Ar; Ser 1 5 10 1s 97 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 18 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: Ser Ser Ser Sex Phe Ser Ser Pro Ser Ser Ala Gly Arg His Val Arg 1 5 10 Ser Tyr INFORMATION FOR SEQ ID NO:24: S* I(i) SEQUENCE CHARACTERISTICS: LENGTH: 19 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: protein S*oo: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: Ser Ser Ser Ser Ser Phe Ser Ser Pro Ser Ser Ala Gly Arg His Val 1 5 10 Arg Ser Tyr INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID Asn Ser Ser Ser Ser Ser Phe Ser Ser Pro Ser Ser Ala Gly Arg His 1 5 10 Val Arg Ser Tyr 98 INFORMATION FOR SEQ ID NO:26: SEQUENCE CHARACTERISTICS: LENGTH: 21 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: -Thr Asn Ser Ser Ser Ser Ser Phe Ser Ser Pro Ser Ser Ala Gly Arg 1 5 10 His Val Arg Ser Tyr INFORMATION FOR SEQ ID NO:27: SEQUENCE CHARACTERISTICS: LENGTH: 22 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: Ala Thr Asn Ser Ser 5cr Ser Ser Phe Ser 5cr Pro Ser 5cr Ala Gly 1 5 10 Arg His Val Arg Ser Tyr INFORMATION FOR SEQ ID NO:28: Ci) SEQUENCE CHARACTERISTICS: LENGTH: 23 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: Glu. Ala Thr.Asn Ser Ser Ser 5cr Scr Phe Ser Ser Pro Ser Scr Ala 1 5 10 Gly Arg His Val Arg Ser Tyr 99 INFORMATION FOR SEQ ID NO:29: SEQUENCE CHARACTERISTICS: LENGTH 24 amino acids TYPE: Amino acid STRA2NDEDNESS: unknown TOPOLOGY: unknown- (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: Pro Glu Ala Thr Asn Ser Ser Ser Ser Ser Phe Ser Ser Pro Ser Ser 1 5 10 Ala Gly Arg His Val Arg Ser Tyr INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 25 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID Ser Pro Glu Ala Thr Asn Ser Ser Ser Ser Ser Phe Ser Ser Pro Ser 1 5 10 1 *Ser Ala Gly Arg His Val Arg Ser Tyr INFORMATION FOR SEQ ID NO:31: SEQUENCE CHARACTERISTICS: LENGTH: 26 amino acids TYPE: amino acid STRANDEDNESS: unknown CD) TOPOILOGY:.unknown (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31: Val Ser Pro Glu Ala Thr Asn Ser Ser Ser Ser Ser Phe Ser Ser Pro 1 5 10 Ser Ser Ala Gly Arg His Val Az-g Ser Tyr 100 INFORMATION FOR SEQ, ID NO:32: SEQUENCE CHARACTER.ISTICS: LENGTH: 27 amino acids TYPE: Amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLE CUL E TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32: Met Val. Sex Pro Glu Ala Thr Asn Ser Ser Ser Ser Ser Phe Ser Sex 1 5 10 1 Pro Sex Ser Ala Gly Arg His Val Arg Ser Tyr INFORMATION FOR SEQ ID NO: 33: Ci) SEQUENCE CHARACTERISTICS: LENGTH: 28 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33: Asp Met Val. Sex Pro Glu Ala Thr Asn Sex Ser Ser Ser Ser Phe Sex 1 5 10 1 Sex Pro Sex Sex Ala Gly Axg His Val. Arg Sex Tyr INFORMATION FOR SEQ ID NO:34: i) SEQUENCE CHARACTERISTICS: LENGTH: 29 amino acids TYPE: amino acid STRANDEDNESS: unknown CD) TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34: Gin Asp Met Val Sex Pro Glu Ala Thr Asn Sex Sex Sex Sex Sex Phe 1 5 10 Ser Sex Pro Sex Ser Ala Gly Arg His* Val Axg Ser Tyr 101 INFORMATION FOR SEQ Ifl SEQUENCE CHARACTERISTICS: LENGTH: 30 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID Gly Gin Asp Met Val Ser Pro Glu Ala Thx Asn Ser Ser Ser Ser Ser 1 5 10 Phe Set Ser Pro Ser Ser Ala Gly Arg His Val Arg Ser Tyr 25 INFORMATION FOR SEQ ID NO:36: Ci) SEQUENCE CHARACTERISTICS: LE-NGTH: 3i amino acids CB) TYPE: amino acid CC) STPRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36: Leu Gly Gin Asp Met Val Ser Pro Giu Aia Thr Asn Ser Ser Ser Ser 1 5 10 Ser Phe Se~r Ser Pro Ser Ser Ala Gly Arg His Val Arg Ser Tyr INFORMATION FOR SEQ ID NO:37: SEQUENCE CHARACTERISTICS: CA) LENGTH: 32 amino acids TYPE: amino acid STRANDEDNIESS: unknown CD) TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37: Ala Leu Gly Gin Asp Met Val Ser Pro Giu Ala Thr Asn Ser Ser Ser 1 S 10 Ser Ser Phe Ser Ser Pro Ser Ser Ala Gly Arg His Val Arg Ser Tyr 25 102 INFORMATION FOR SEQ ID NO:38: SEQUENCE CHARACTERISTICS: LENGTH: 33 amino acids TYPE: amriino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38: Gin Ala Leu Gly Gin Asp Met Val Ser Pro Glu Ala Thr Asn Ser Ser 1 5 10 Ser Ser Ser Phe Ser Ser Pro Ser Ser Ala Gly Arg His Val Arg Ser 25 3 Tyr INFORMATION FOR SEQ ID NO:39: SEQUENCE CHARACTERISTICS: LENGTH: 34 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39: Cys Gin Ala Leu Gly Gin Asp Met Val Ser Pro Glu Ala Thr Asn Ser *1 5 10 Ser Ser Ser Ser Phe Ser Ser Pro Ser Ser Ala Gly Arg His Val Arg 25 Ser Tyr INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 4 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: protein 103 (xi) SEQUENCE DESCRIPTION: SEQ ID Met Val Val His 1 INFORMATION FOR SEQ ID NO:41: SEQUENCE CHARACTERISTICS: LENGTH: 5 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (1i) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41: Met Val Val His Ser *1 INFORMATION FOR SEQ ID NO:42:, i) SEQUENCE CHARACTERISTICS.

LENGTH: 36 base pairs TYPE: nucleic acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ, ID NO:42: AAAcAAcATA TGGTccc GGAGGCTACC- AACTCC 36 INFORMATION FOR SEQ ID NO:43: i) SEQUENCE CHARACTERISTICS: (A LENGTH: 35 base pairs TYPE: nucleic acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43: AAACAAGGAT CCTTTATGAG TGGACCACCA TGGGG 104 INFORMATION FOR SEQ ID NO:44: SEQUENCE CHARACTERISTICS: LENGTH: 47 base pairs TYPE: nucleic acid STRANDEDNESS: unknown TOPOLOGY: unknCown (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44: CTAGCGATGA CGATGATAAA CAGGCTCTGG GTCAGGACAT GGTTTCT 47 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 47 base pairs TYPE: nucleic acid STRANDEDNESS: unknown TOPOLOGY: unknown 0 MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ, ID CCGGAGAAAC CATGTCCTGA CCCAGAGCCT GTTTATCATC GTCATCG 47 INFORMATION FOR SEQ ID NO:46: SEQUENCE CHARACTERISTICS: LENGTH: 45 base pairs TYPE: nucleic acid STRANDEDNESS: unknown TOPOLO0GY: unknown (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46: *GGAGGAATAA CATATGTCCT ACAATCACCT GCAGGGAGAT GTCCG INFORMATION FOR SEQ ID NO:47: SEQUENCE CHARACTERISTICS: LENGTH: 35 base pairs TYPE: nucleic acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47: AAACAAGGAT CCTTTATGAG TGGACCACCA TGGGG 105 INFORMATION FOR SEQ TD NO:48: SEQUENCE CHARACTERISTICS: LENGTH: 47 base pairs TYPE: nucleic acid STRANDEDNESS: unknown CD) TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48: TTAGATTCTA GATTrGTTTr AACTAATTAA AGGAGGAATA ACATATG 47 INFORMATION FOR SEQ ID NO:49: SEQUENCE CHARACTERISTICS: LENGTH: 35 base pairs TYPE: nucleic acid C(C) STRAIIDEDNESS: unknown CD) TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ, ID NO:49: AAACAAGGAT CCTTTATGAG TGGACCACCA TGGGG INFORMATION FOR SEQ 11D SEQUENCE CHARACTERISTICS: LENGTH: 53 base pairs TYPE: nucleic acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID AAACAACATA TGTCTTCTCC TTCCTCTGC.A GGTAGGCATG TGCGGAGCTA CAA 53 INFORMATION FOR SEQ ID Ci) SEQUENCE CHARACTERISTICS: CA) LENGTH: 35 base pairs CB) TYPE: nucleic acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA 106 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:Sl: AAACAAGGAT CCTTTATGAG TGGACCACCA TGGGG INFORMATION FOR SEQ ID NO:52: Ci) SEQUENCE CHARACTERISTICS: LENGTH: 25 base pairs TYPE: nucleic acid STRAjIDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA SEQUENCE DESCRIPTION: SEQ ID NO:52: TATGCTGGGT CAGGACATGG TTTCT INFORMATION FOR SEQ ID NO:53: 009 Ci SEQUENCE CHARACTERISTICS: LENGTH: 27 base pairs TYPE: nucleic acid STRANDEDNESS: unknown TOPOLOGY: unknown MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53: @00CCGGAGAAAC CATGTCCTGA. CCCAGCA 27 INFORMATION FOR SEQ ID NO:54: SEQUENCE CHARACTERISTICS: LENWGTH: 51 base pairs TYPE,: nucleic acid 0 STRANDEDNESS: unknown CD) TOPOLOGY: unknown (ii) MCLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ 1D NO:54: CCGGAGGCTA CCAACTCTAG CTCCAGCAGC TTCTCCTCTC CTAGCTCTGC A 51 INFORMATION FOR SEQ ID Ci) SEQUENCE CHARACTERISTICS: LENGTH: 43 base pairs TYPE: nucleic acid STRANDEDNESS: unknown TOPOLOGY: unknown 107 (ii) MOLECULE TYPE: cUNA (xi) SEQUENCE DESCRIPTION: SEQ ID GAGCTAGGAG AGGAGAAGCT GCTGGAGCTA GAGTTGGTAG CCT 43 INFORMATION FOR SEQ ID NO:56: SEQUENCE CHARACTERISTICS: LENGTH: 25 base pairs TYPE: nucleic acid STRAZ4DEnNESS: unknxown TOPOLOGY: unknxown (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56: *AACACCTATG CATCTTAA CTGGC INFORMATION FOR SEQ ID NO: 57: SEQUENCE CHARACTERISTICS: LENGTH: 29 base pairs TYPE: nucleic acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: cONA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:57: GTCCCTGCCT GGGAGCTCCT TTTCCATTC 29 INFORMATION FOR SEQ ID NO:58: SEQUENCE CHARACTERISTICS: LENGTH: 30 base pairs TYPE: nucleic acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:58: GCTCCCAGGC AGGGACAAAA AACAAGAAGG 108 INFORMATION FOR SEQ ID NO:59: Ci) SEQUENCE CHARACTERISTICS: LENGTH: 29 base pairs TYPE: nucleic acid STRANDEDNESS: unknxown TOPOLOGY: unknown (ii) MOLECULE TYPE: cOMA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:59: AACAAAGGAT CCTTATGAG TGGACCACC 29 INFORMATION FOR SEQ ID Ci) SEQUENCE CHARACTERISTICS: LENGTH: 25 base pairs TYPE: nucleic acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: cONA (xi) SEQUENCE DESCRIPTION: SEQ ID AACACCTATG CATCTTTAA C7GGC INFORMATION FOR SEQ ID NO:61: SEQUENCE CHARACTERISTICS: LENGTH: 29 base pairs TYPE: nucleic acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: clM (xi) SEQUENCE DESCRIPTION: SEQ ID NO:61: AACAAAGGAT CCTTTATG.A4 TGGACCAcc 29 INFORMATION FOR SEQ ID NO:62: Ci) SEQUENCE CHARACTERISTICS: LENGTH: 29 base pairs CB) TYPE: nucleic acid STRANDEDNESS: unknown CD) TOPOLOGY: unknown (ii) MOLECULE TYPE: cD:NA 109 (xi) SEQUENCE DESCRIPTION: SEQ ID) NO:62: AACAAAGGAT CCTTTATGAG TGGACCACC 29 INFORMATION FOR SEQ ID NO:63: SEQUENCE CHAACTRISTICS: LENXGTH: 51 base pairs TYPE: nucleic acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA *e~e (xi) SEQUENCE DESCRIPTION: SEQ ID NO:63: CCGGAGGCTA CCAACTCTAG CTCCAGCAGC TCTCCTCTC CTAGCTCTGC A 51

Claims (10)

1. 3. Jun. 2004 12:07 No. 0501 P. 6 -110- THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:- 1. A prokaryotic or eukaryotic host cell comprising a vector, the vector comprising a polynucleotide encoding a protein selected from an amino acid sequence consisting of N-terminally methionylated or non-methionylated NH 2 -Ser-Tyr-[Asn-Ser?']-COOH, wherein [Asn 71 -Se?8] represents residues 71 to 208 of SEQ ID NO.2, the polynucleotide being operatively linked to an expression control sequence, wherein said host cell is an insect cell. 2. A method of producing a protein selected from an amino acid sequence consisting of N-terminally methionylated or non-methionylated NH-Ser-Tyr-[Asn 7 -Ser2 8 COOH, wherein [Asn -Ser 208 represents residues 71 to 208 of SEQ ID NO.2 comprising culturing a host cell of Claim 1 in a suitable nutrient medium. 3. The method according to Claim 2, wherein said host cell is an E. coli cell.
2. 4. The method according to Claim 2, wherein said host cell is selected from baculovirus cells, COS cells or Chinese hamster ovary cells. A method according to any one of Claims 2 to 4 further comprising the step of isolating a variant of KGF-2 protein from said cultured host cell.
3. 6. The method according to Claim 5 comprising the step of modifying the isolated variant of KGF-2 to generate a compound capable of stimulating the production of epithelial cells.
4. 7. A method of stimulating the production of epithelial cells comprising contacting such cells with an effective amount of a protein selected from an amino acid sequence consisting of N-terminally methionylated or non-methionylated NH2-Ser-Tyr-[Asn Ser?]-COOH, wherein [Asn 7 -Ser 208 represents residues 71 to 208 of SEQ ID NO.2. 25 8. A method according to Claim 7, wherein said epithelial cells are selected from cells within the pancreas, liver and gastrointestinal tract.
5. 9. A method for the prevention or treatment of a patient suffering from a condition selected from gastric ulcers, duodenal ulcers, erosions of the stomach and esophagus, inflammatory bowel disease, radiation- or chemotherapy-induced gut toxicity, hepatic 30 cirrhosis, fulminant liver failure, acute viral hepatitis, a toxic insult to the liver, and diabetes, comprising administering to said patient a therapeutically effective amount of a S• protein selected from an amino acid sequence consisting of N-terminally methionylated *000 COMS ID No: SBMI-00777411 Received by IP Australia: Time 12:09 Date 2004-06-03 3. Jun. 2004 12:07 No. 0501 P. 7 -111 or non-methionylated NH2-Ser-Tyr-[Asn t -Seos]-COOH, wherein [Asn"-Ser? 8 represents residues 71 to 208 of SEQ ID NO.2.
6. 10. Use of a protein selected from an amino acid sequence consisting of N-terminally methionylated or non-methionylated NH2-Ser-Tyr-[Asn 7 '-Sero -COOH, wherein [Asn 7 -Ser 08 represents residues 71 to 208 of SEQ ID NO.2 in the manufacture of a medicament for stimulating the production of epithelial cells.
7. 11. Use of an effective amount of a protein selected from an amino acid sequence consisting of N-terminally methionylated or non-methionylated NH 2 -Ser-Tyr-[Asn t Ser 08 ]-COOH, wherein [Asn 71 -Se 2 08 represents residues 71 to 208 of SEQ ID NO.2 in the manufacture of a medicament for stimulating production of epithelial cells in a patient for the prevention or treatment of a condition, wherein said condition is selected from gastric ulcers, duodenal ulcers, erosions of the stomach and esophagus, inflammatory bowel disease, radiation- or chemotherapy-induced gut toxicity, hepatic cirrhosis, fulminant liver failure, acute viral hepatitis, a toxic insult to the liver, and diabetes.
8. 12. A method for the prevention or treatment of a condition in a patient suffering from a condition selected from gastric ulcers, duodenal ulcers, erosions of the stomach and esophagus, inflammatory bowel disease, radiation- or chemotherapy-induced gut toxicity, hepatic cirrhosis, fulminant liver failure, acute viral hepatitis, a toxic insult to the liver, and diabetes, comprising administering to the patient a therapeutically effective amount of a protein selected from an amino acid sequence consisting of N-terminally methionylated or non-methionylated NH 2 -Ser-Tyr-[Asn 71 -Ser 0 ]-COOH, wherein S 55 [Asn 7t -Ser s represents residues 71 to 208 of SEQ ID NO.2, substantially as *hereinbefore described with reference to any one or more of the examples but excluding comparative examples.
9. 13. Use of a protein selected from an amino acid sequence consisting of N-terminally methionylated or non-methionylated NH2-Ser-Tyr-Asn T 1 -Ser 0 8 O-COOH, wherein 60 [Asn 7 '-Ser 08 represents residues 71 to 208 of SEQ ID NO.2 in the manufacture of a medicament for stimulating production of epithelial cells, substantially as hereinbefore described with reference to any one or more of the examples but excluding comparative examples.
10. 14. Use of a protein selected from an amino acid sequence consisting of N-terminally 65 methionylated or non-methionylated NHz-Ser-Tyr-[Asn 71 -Ser 0 8 -COOH, wherein [Asn"'-Ser2o] represents residues 71 to 208 of SEQ ID NO.2 for the preparation of a COMS ID No: SBMI-00777411 Received by IP Australia: Time 12:09 Date 2004-06-03 3. Jun. 2004 12:07 No. 0501 P. 8 -112- medicament for proliferation and/or differentiation of adnexal structures, substantially as hereinbefore described with reference to any one or more of the examples but excluding comparative examples. DATED this 3"' d day of June 2004 BALDWIN SHELSTON WATERS Attorneys for: AMGEN INC. g.e. S S S 0@ 0. S S 0 S J S 0* 0 0 Se S* S S 0. 0 005* 0 .U. 0 CS ES @0 0 S 000 S 0050 COMS 0ID No: SBMI-00777411 Received by IP Australia: Time 12:09 Date 2004-06-03