MXPA02000152A - Keratinocyte growth factor-2. - Google Patents

Abstract

This invention relates to newly identified polynucleotides, polypeptides encoded by such polynucleotides, the use of such polynucleotides and polypeptides, as well as the production of such polynucleotides and polypeptides. More particularly, the polypeptide of the present invention is a Keratinocyte Growth Factor, sometimes hereinafter referred to as "KGF-2" also formerly known as Fibroblast Growth Factor 12 (FGF-12). This invention further relates to the therapeutic use of KGF-2 to promote or accelerate wound healing. This invention also relates to novel mutant forms of KGF-2 that show enhanced activity, increased stability, higher yield or better solubility.

Description

n / FACTOR 2 GROWTH OF KERATINOCYTES BACKGROUND OF THE INVENTION FIELD OF THE INVENTION This invention relates to the newly identified polynucleotides, to the polypeptides encoded by such polynucleotides, to the use of such polynucleotides and polypeptides, as well as to the production of such polynucleotides and polypeptides. More particularly, the polypeptide of the present invention is a Keratinocyte Growth Factor, sometimes referred to herein as "KFG-2" also formerly known as Fibroblast Growth Factor 12 (FGF-12). This invention also relates to the therapeutic use of KFG-2 to promote or accelerate wound healing. This invention also relates to the novel mutant forms of KGF-2 which show improved activity, increased stability, higher yield or better solubility. In addition, this invention relates to a method for purifying the KGF-2 polypeptide.

REF: 135456 RELATED TECHNIQUE The fibroblast growth factor family has emerged as a large family of growth factors involved in the development and regeneration of soft tissues. Currently, it includes several members that share a varying degree of homology at the level of proteins, and that, with one exception, appear to have a broad, similar mitogenic spectrum, for example, they promote the proliferation of a variety of cells of mesodermal origin and neuroectodermal and / or promote angiogenesis. The expression pattern of different family members is very different, ranging from extremely restricted expressions of some stages of development, to rather ubiquitous expression in a variety of tissues and organs. All members appear to bind to heparin and proteoglycans and glycosaminoglycans of heparin sulfate and are strongly concentrated in the extracellular matrix. KGF was originally identified as a member of the FGF family by sequential homology or purification and cloning of the factor. Keratinocyte growth factor (KGF) was isolated as a mitogen for a cultured murine keratinocyte line (Rubin, J.S. et al., Proc. Nati, Acad. Sci. USA 86: 802-806 (1989)).

Contrary to the other members of the FGF family, it has little activity on cells derived from the mensenchyme, but stimulates the development of epithelial cells. The keratinocyte growth factor gene codes for a 194 amino acid polypeptide (Finch, P.. Et al., Science 245: 752-755 (1989)). The N-terminal 64 amino acids are unique, but the rest of the protein has approximately 30% homology to bFGF. KGF is the most divergent member of the FGF family. The molecule has a hydrophobic signal sequence and is efficiently secreted. Post-translational modifications include cleavage of the signal sequence and N-linked glycosylation at one site, resulting in a 28 kDa protein. The keratinocyte growth factor is produced by fibroblasts derived from the skin and from the fetal lung (Rubin et al., (1989)). Keratinocyte growth factor mRNA was found to be expressed in adult kidney, colon and ileum, but not brain or lung (Finch, P. W. et al., Science 245: 752-755 (1989)). KGF shows conserved regions within the FGF family of proteins. KGF binds to the FGF-2 receptor with high affinity. The deteriorated healing of wounds is a significant source of morbidity and can result in complications such as dehiscence, anastomotic rupture and wounds that do not heal. In normal individuals, wound healing is carried out without complications. In contrast, impaired healing is associated with several conditions such as diabetes, infection, immunosuppression, obesity and poor nutrition (Cruse, P.J. and Foord, R., Arch. Surg. 107-206 (1973)).; Schrock, T.R., and collaborators, Ann. Surg. 177: 513 (1973); Poole, G.U. , Jr., Surgery 97: 631 (1985); Irvin, G.L. and collaborators, Am. Surg. 51: 418 (1985)). The repair of wounds is the result of complex interactions and biological processes. Three phases have been described in the normal healing of wounds: the acute inflammatory phase, the synthesis of collagen and extracellular matrix, and the remodeling (Peacock, EE, Jr., Wound Repair, 2nd Edition, WB Saunders, Philadelphia (1984 )). The process involves the interaction of keratinocytes, fibroblasts and inflammatory cells at the site of the wound. The regeneration of the tissue seems to be controlled by specific peptide factors that regulate the migration and proliferation of the cells involved in the repair process (Barrett, TB et al, Proc. Nati, Acad. Sci. USA, 81-6772-6774 (1985 ), Collins, T. et al., Nature 316: 748-750 (1985)): Thus, growth factors can be promising therapeutic products in the treatment of wounds, burns and other skin disorders (Rifkin, DB and Moscatelli, J. "Cell. Biol. 109: 1-6 (1989); Sporn, MB et al., J. Cell. Biol. 105: 1039-1045 (1987); Pierce, GF et al., J. Cell. Biochem 45: 319-326 (1991)) The sequence of the healing process is initiated during an acute inflammatory phase with provisional tissue deposition, followed by reepithelialization, synthesis and deposition of collagen, proliferation of fibroblasts, and neovascularization, all of which ultimately define the rem phase odelation (Clark, R.A.F., J. Am. Acad. Dermatol. 13: 701 (1985)). These events are influenced by growth factors and cytokines secreted by inflammatory cells or by cells located at the edges of the wound (Assoian, RK et al., Nature (Lond.) 309: 804 (1984); Nemeth, GG et al. "Growth Factors and Their Role in Wound and Fracture Healing ", Growth Factors and Other Aspects of Wound Healing in Biological and Clinical Implications, New York (1988), pp. 1-17. Several polypeptide growth factors have been identified as being involved in wound healing, including keratinocyte growth factor (KGF) (Antioniades, H. et al, Proc. Nati, Acad. Sci. USA 88: 565 (1991)) , the platelet derived growth factor (PDGF) (Antioniades, H. et al., Proc.

Nati Acad. Sci. USA 88: 565 (1991); Staiano-Coico, L. et al., Jour. Exp. Med. 178: 865-878 (1993)), the basic fibroblast growth factor (bFGF) (Golden, MA et al., J. Clin.Research 87: 406 (1991)), the growth factor of acid fibroblasts (aFGF) (Mellin, TN et al., J. Invest. Dermatol. 104: 850-855 (1995)), epidermal growth factor (EGF) (Whitby, DJ and Ferguson, WJ Dev. Biol. 147: 207 (1991)); transforming growth factor (TGF-a) (Gartner, MH et al., Surg. Forum 42: 643 (1991); Todd, R. et al., Am. J. Pathol., 138; 307 (1991)), the factor ß transforming growth (TGF-ß) (Wong, DTW et al., Am. J. Pathol. 143: 622 (1987)), neu differentiation factor (rNDF) (Danilenko, DM et al., J. Clin. Invest. 95; 842-851 (1995)), insulin-like growth factor I (IGF-I), and insulin-like growth factor II (IGF-II) (Cromack, DT et al., J. Surg. Res. 42: 622 (1987)). It has been reported that rKGF-I in the skin stimulates the epidermal keratinocytes, the keratinocytes within the hair follicles and the sebaceous glands (Pierce, G. F. et al., J. "Exp. Med. 179: 831-840 (1994)).

BRIEF DESCRIPTION OF THE INVENTION The present invention provides isolated nucleic acid molecules comprising a polynucleotide encoding keratinocyte growth factor (KGF-2) having the amino acid sequence as shown in Figure 1 [SEQ ID No. 2] or the amino acid sequence encoded by the cDNA clones deposited as ATCC Deposit Number 75977 on December 16, 1994. The nucleotide sequence determined by the sequencing of deposited KGF-2 clone, which is shown in Figure 1 [SEQ ID NO. 1], contains an open reading frame that codes for a polypeptide of 208 amino acid residues, including a start codon at positions 1-3, with a predicted leader sequence of about 35 or 36 amino acid residues, and a molecular weight deduced from approximately 23.4 kDa. The amino acid sequence of mature KGF-2 is shown in Figure 1, the amino acid residues about 36 or 37 to 208 [SEQ ID NO. 2] . The polypeptide of the present invention has been putatively identified as a member of the FGF family, more particularly the polypeptide has been putatively identified as KGF-2 as a result of homology in the amino acid sequence with other members of the FGF family.

In accordance with one aspect of the present invention, novel mature polypeptides which are KGF-2 as well as biologically active and diagnostically or therapeutically useful fragments, analogs and derivatives thereof are provided. The polypeptides of the present invention are of human origin. According to yet another aspect of the present invention, isolated nucleic acid molecules encoding human KGF-2 are provided, including mRNAs, DNAs, cDNAs, genomic DNA, as well as antisense analogs thereof, and biologically active fragments. and diagnostically or therapeutically useful thereof. According to yet another aspect of the present invention, there is provided a process for producing such a polypeptide by recombinant techniques through the use of recombinant vectors, such as the cloning and expression plasmids useful as reagents in the recombinant production of KGF-proteins. 2, as well as recombinant prokaryotic and / or eukaryotic host cells comprising a human KGF-2 nucleic acid sequence. According to yet another aspect of the present invention, there is provided a process for the use of such a polypeptide, or the polynucleotide encoding such a polypeptide for therapeutic purposes, for example, to stimulate the proliferation of epithelial cells and basal keratinocytes for of healing of wounds, and to stimulate the production of the hair follicle and the healing of the dermal wounds. KGF-2 may be clinically useful in the stimulation of wound healing, including surgical wounds, excision wounds, deep wounds involving damage to the dermis and epidermis, wounds to the ocular tissue, wounds to dental tissue, wounds in the oral cavity, diabetic ulcers, skin ulcers, ulcer ulcers, arterial ulcers, venous stasis ulcers, burns resulting from exposure to heat or chemicals, and other abnormal wound healing conditions such as uremia, poor nutrition, vitamin deficiencies and complications associated with systemic treatment with steroids, radiation therapy, and antineoplastic and antimetabolite drugs. KGF-2 can be used to promote skin re-establishment subsequent to skin loss. KGF-2 can be used to increase the adherence of skin grafts to a wounded bed and to stimulate re-epithelialization from the wound bed. The following are types of KGF-2 grafts that could be used to increase adherence to a wound bed: autografts, artificial skin, allografts, autodermal grafts, autoepidermal grafts, aortic grafts, Blair-Brown grafts, bone grafts, grafts. Brephoplasts, skin grafts, delayed grafts, dermal grafts, epidermal grafts, fascia grafts, full thickness grafts, heterologous grafts, xenografts, homologous grafts, hyperplastic grafts, lamellar grafts, mesh grafts, mucosal grafts, Ollier-Thiersch grafts , omental grafts, patch grafts, pedicle grafts, penetration grafts, split skin grafts, or thick skin grafts. KGF-2 can be used to promote skin resistance and to improve the appearance of aged skin. It is believed that KGF-2 will also produce changes in the proliferation of hepatocytes, and the proliferation of epithelial cells in the lung, in the chest, in the pancreas, in the stomach, in the small intestine, and in the large intestine. KGF-2 can promote the proliferation of epithelial cells such as sebocytes, hair follicles, hepatocytes, type II pneumocytes, mucin-producing globular cells, and other epithelial cells and their progenitors contained within the skin, lung, liver, and gastrointestinal tract. KGF-2 can promote the proliferation of endothelial cells, keratinocytes, and basal keratinocytes. KGF-2 can also be used to reduce the side effects of toxicity to the intestine that results from radiation, chemotherapeutic treatments or viral infections. KGF-2 can have a cytoprotective effect on the mucosa of the small intestine. KGF-2 can also stimulate the healing of mucositis (ulcers of the mouth) resulting from chemotherapy and viral infections. KGF-2 can also be used in the complete regeneration of the skin and in full and partial thickness skin defects, including burns, (for example, repopulation of hair follicles, sweat glands, and sebaceous glands) treatment of other defects of the skin such as psoriasis. KGF-2 can be used to treat epidermolysis bullosa, a defect in adhesion of the epidermis to the underlying dermis that results in frequent, open and painful blisters due to the acceleration of re-epithelialization of these lesions. KGF-2 can also be used to treat gastric and duodenal ulcers and help the healed by the formation of mucosal lining and regeneration of the glandular mucosa and lining of the duodenal mucosa., more quickly. Inflammatory bowel diseases, such as Crohn's disease and ulcerative colitis, are diseases that result in the destruction of the mucosal surface of the small or large intestine, respectively. In this way, KGF-2 could be used to promote surface regeneration of the mucosal surface to aid in faster healing and to prevent the progression of inflammatory bowel disease. Treatment with KGF-2 is expected to have a significant effect on mucosal production throughout the gastrointestinal tract, and could be used to protect the intestinal mucosa from harmful substances that are ingested or after surgery. KGF-2 can be used to treat diseases associated with the sub expression of KGF-2. In addition, KGF-2 can be used to prevent and heal damage to the lungs due to various pathological conditions. A growth factor such as KGF-2 that could stimulate proliferation and differentiation and promote repair of the alveoli and bronchiolar epithelium to prevent or treat acute or chronic lung damage. For example, emphysema, which results in the progressive loss of the alveoli and damage by inhalation, for example, resulting from the inhalation of smoke and burns, which cause necrosis of the bronchiolar epithelium and the alveoli, could be effectively treated. with KGF-2. Also, KGF-2 could be used to stimulate the proliferation of and differentiation of type II pneumocytes, which can help treat or prevent diseases such as hyaline membrane diseases, such as infant respiratory distress syndrome. and bronchopulmonary dysplasia, in premature infants. KGF-2 could stimulate the proliferation and differentiation of hepatocytes and, in this way, could be used to alleviate or treat liver diseases and pathologies such as fulminating hepatic failure caused by cirrhosis, liver damage caused by viral hepatitis and toxic substances (for example, acetaminophen, carbon tetrachloride, and other hepatotoxins known in the art). In addition, KGF-2 could be used to treat or prevent the onset of diabetes mellitus. In patients with newly diagnosed Type I and II diabetes, where some islet formula function remains, KGF-2 could be used to maintain islet formation to alleviate, slow or prevent the permanent manifestation of the disease. Also, KGF-2 could be used as an assistant in the transplantation of islet cells to improve or promote the function of islet cells. According to yet another aspect of the present invention, antibodies against such polypeptides are provided. According to yet another aspect of the present invention, nucleic acid probes comprising nucleic acid molecules of sufficient length to specifically hybridize to human KGF-2 sequences are provided. According to a further aspect of the present invention, the mimetic peptides of KGF-2 that can be used as therapeutic peptides are provided. The mimetic KGF-2 peptides are short peptides that mimic the biological activity of the KGF-2 protein by binding to and activating the cognate KGF-2 receptors. The KGF-2 peptides can also bind to and inhibit the cognate KGF-2 receptors. According to yet another aspect of the invention, antagonists are provided for such polypeptides, which can be used to inhibit the action of such polypeptides, for example, to reduce scar formation during the wound healing process and to prevent and / or treat tumor proliferation, diabetic retinopathy, rheumatoid arthritis, osteoarthritis and tumor development. KGF-2 antagonists can also be used to treat diseases associated with overexpression of KGF-2. According to yet another aspect of the present inventionDiagnostic assays are provided to detect diseases or susceptibility to diseases related to mutations in KGF-2 nucleic acid sequences or overexpression of polypeptides encoded by such sequences. According to yet another aspect of the present invention, there is provided a process for the use of such polypeptides, or the polynucleotides encoding such polypeptides, for purposes related to scientific research, DNA synthesis, and manufacture of DNA vectors. Thus, one aspect of the invention provides an isolated nucleic acid molecule comprising a polynucleotide having a nucleotide sequence selected from the group consisting of (a) a nucleotide sequence encoding the KGF-2 polypeptide having the sequence complete of amino acids in Figure 1 [SEQ ID NO. 2]; (b) a nucleotide sequence encoding the mature KGF-2 polypeptide having the amino acid sequence at positions 36 or 37 to 208 in Figure 1 [SEQ ID NO. 2]; (c) a nucleotide sequence encoding the KGF-2 polypeptide having the complete amino acid sequence encoded for the cDNA clone contained in ATCC Deposit No. 75977; (d) a nucleotide sequence encoding the mature KGF-2 polypeptide having the amino acid sequence encoded by the cDNA clone contained in the ATCC Deposit No. 75977; and (e) a nucleotide sequence complementary to any of the nucleotide sequences in (a), (b), (c) or (d) above. Additional embodiments of the present invention include isolated nucleic acid molecules comprising a polynucleotide having at least an 80% identical nucleotide sequence, and more preferably at least 85%, 90%, 91%, 92%, 93%, 94 %, 95%, 97%, 98%, or 99% identical, to any of the nucleotide sequences in (a), (b), (c), (d) or (e), above, or a polynucleotide that is hybrid under stringent hybridization conditions to a polynucleotide in (a), (b), (c), (d) or (e), above. This hybridizing polynucleotide does not hybridize under stringent hybridization conditions to a polynucleotide having a nucleotide sequence consisting only of residues A or only of residues T. A further embodiment of nucleic acid of the invention refers to an acid molecule Isolated nucleic acid comprising a polynucleotide encoding the amino acid sequence of an epitope-possessing portion of a KGF-2 having an amino acid sequence in (a), (b), (c), or (d), above. The invention further provides an isolated KGF-2 polypeptide having the amino acid sequence selected from the group consisting of: (a) the amino acid sequence of the KGF-2 polypeptide having the complete sequence of 208 amino acids, including the leader sequence shown in Figure 1 [SEQ ID NO. 2]; (b) the amino acid sequence of the mature KGF-2 polypeptide (without the leader) having the amino acid sequence at positions 36 or 37 to 208 in Figure 1 [SEQ ID No. 2]; (c) the amino acid sequence of the KGF-2 polypeptide having the complete amino acid sequence, including the leader, encoded by the cDNA clone contained in the ATCC Deposit No. 75977; and (d) the amino acid sequence of the mature KGF-2 polypeptide having the amino acid sequence encoded by the cDNA clone contained in the ATCC Deposit No. 75977. The polypeptides of the present invention also include polypeptides having a sequence of amino acids with at least 80% similarity, and more preferably at least 90%, 95%, 96%, 97%, 98% or 99% similarity to those described in (a), (b), (c), or (d) above, as well as polypeptides having at least 80% identical amino acid sequence, more preferably at least 85% identical, and still more preferably 90%, 91%, 92%, 93%, 94%, 95% , 97%, 98% or 99% identical to those previously mentioned. A further aspect of the invention relates to a peptide or polypeptide having the amino acid sequence of an epitope-possessing portion of a KGF-2 polypeptide having an amino acid sequence described in (a), (b), (c) or (d), previous. Peptides and polypeptides having the amino acid sequence of an epitope-possessing portion of a KGF-2 polypeptide of the invention, include portions of such polypeptides with at least six or seven, preferably at least nine, and more preferably at least about 30. amino acids up to about 50 amino acids, although epitope-possessing polypeptides of any length up to or including the complete amino acid sequence of a polypeptide of the invention described above, are also included in the invention. In yet another embodiment, the invention provides an isolated antibody that specifically binds to a KGF-2 polypeptide having an amino acid sequence described in (a), (b), (c) or (d), above. According to yet another aspect of the present invention, the novel variants of KGF-2 are described. These can be produced by the deletion or substitution of one or more amino acids of KGF-2. Natural mutations are called allelic variations. The allelic variations can be silent (without change in the encoded polypeptide) or they can have altered amino acid sequence. In order to try to improve or alter the characteristics of native KGF-2, the genetic manipulation of proteins can be employed. The recombinant DNA technology known in the art can be used to create new polypeptides. Muteins and deletion mutations can show, for example, increased activity or increased stability. In addition, these could be purified with a higher yield and show better solubility at least under certain purification and storage conditions. These and other aspects of the present invention should be apparent to those skilled in the art from the teachings herein.

BRIEF DESCRIPTION OF THE FIGURES The following drawings are illustrative of the embodiments of the invention and are not intended to limit the scope of the invention as encompassed by the claims. Figures 1A-1C illustrate the cDNA and the corresponding deduced amino acid sequence of the polypeptide of the present invention. The 35 or 36 initial amino acid residues represent the putative guiding sequence (underlined). Standard one-letter abbreviations for amino acids are used. The sequencing inaccuracies are a common problem when trying to determine the polynucleotide sequences. Sequencing was performed using an automated DNA sequencer 373 (Applied Biosystems, Inc.). The sequencing accuracy is predicted to be greater than 97% accuracy. [SEQ ID NO. 1] Figures 2A-2D are an illustration of a comparison of the amino acid sequence of the polypeptide of the present invention and other fibroblast growth factors. [SEQ ID NOS. 13-22] Figures 3A-3D show the full-length mRNA and the amino acid sequence for the KGF-2 gene. [SEQ ID NOS. 23 and 24] Figures 4A-4E show an analysis of the amino acid sequence of KGF-2. The alpha, beta, back and helix regions; hydrophilicity and hydrophobicity; the unfriendly regions; the flexible regions; the antigenic index and the superficial probability are also shown. In the graph of "Antigenic Index - Jameson-Wolf" the amino acid residues 41-109 in Figure 1 [SEQ ID NO. 2) correspond to the highly antigenic regions shown of the KGF-2 protein. The hydrophobic regions (Hopp-Woods plot) fall below the midline (negative values) while hydrophilic regions (Kyte-Doolittle plot) are above the midline (positive values, for example, amino acid residues 41-109 ). The graph is about the complete ORF of 208 amino acids.

Figure 5 shows the evaluation of KGF-2 on wound closure in diabetic mice. The wounds were measured immediately after the wound was performed and every day for 5 consecutive days on day 8. The percentage of wound closure was calculated using the following formula: [Area on day 1] - [Area on the day 8] / [Area on day 1]. The statistical analysis performed used an unpaired t test (mean +/- SEM, n = 5). Figure 6 shows the evaluation of KGF-2 on wound closure in non-diabetic mice. The wounds were measured immediately after the wound was performed and every day for 5 consecutive days and on day 8. The percent of wound closure was calculated using the following formula: [Area on day 1] - [Area on on day 8] / [Area on day 1]. The statistical analysis was performed using a non-paired t test (mean +/- SEM, n = 5). Figure 7 shows a time course of wound closure in diabetic mice. The wound areas were measured immediately after the wound was made and every day for 5 consecutive days and on day 8. The values are presented as the total area (mm2). The statistical analysis was performed using a non-paired t test (mean +/- SEM, n = 5). Figure 8 shows a time course of wound closure in non-diabetic mice. The wound areas were measured immediately after the wound was made and every day for 5 consecutive days and on day 8. The values are presented as the total area (mm2). The statistical analysis was performed using a non-paired t test (mean +/- SEM, n = 5). Figure 9 shows a histopathological evaluation on KGF-2 on diabetic mice. The ratings were given by a "blind" observer. The statistical analysis was performed using an unpaired t test (mean +/- SEM, n = 5). Figure 10 shows a histopathological evaluation on KGF-2 on non-diabetic mice. The ratings were given by a "blind" observer. Statistical analysis was performed using an unpaired t test (mean +/- SEM, n = 5). Figure 11 shows the effect of keratinocyte growth in diabetic mice. The ratings were given by a "blind" observer. The statistical analysis was performed using an unpaired t test (mean +/- SEM, n = 5). Figure 12 shows the effect of keratinocyte growth in non-diabetic mice. The ratings were given by a "blind" observer. The statistical analysis was performed using an unpaired t test (mean +/- SEM, n = 5).

Figure 13 shows the effect of skin proliferation in diabetic mice. The ratings were given by a "blind" observer. The statistical analysis was performed using an unpaired t test (mean +/- SEM, n = 5). Figure 14 shows the effect of skin proliferation in non-diabetic mice. The ratings were given by a "blind" observer. The statistical analysis was performed using an unpaired t test (mean +/- SEM, n = 5). Figure 15 shows the DNA sequence and the protein expressed from the pQE60-Cys37 construct [SEQ ID NOS. 29 and 30]. The expressed KGF-2 protein contains the sequence from cysteine at position 37 to serine at position 208 with a 6X tag (His) linked to the N-terminus of the protein. Figure 16 shows the effect of methyl-prednisolone on the healing of wounds in rats. SD male adult rats (n = 5) were injected on the day of the wound with 5 mg of methylprednisolone. The animals received skin puncture wounds (8 mm) and were treated daily with buffer solution or KGF-2 solution in 50 μl of buffer for 5 consecutive days. Wounds were measured daily on days 1-5 and on day 8 with a calibrated Jameson meter. The values represent the measurements taken on day 8. (Mean +/- SEM). Figure 17 shows the effect of KGF-2 on wound closure. SD adult male rats (n = 5) received dermal puncture wounds (8 mm) and 5 mg of methyl-prednisolone on the day of wounding. The animals were treated daily with a buffer solution or KGF-2 in 50 μl of buffer for 5 consecutive days, starting on the day of the wounding. The measurements were taken daily for 5 consecutive days and on day 8. The wound closure was calculated by the following formula: [Area on day 8] - [Area on day l] / [Area on day 1] . The area on day 1 was determined as 64 mm2, the area made by the dermal puncture. The statistical analysis was performed using a non-paired t-test. (Average +/- SEM). Figure 18 shows the time course of wound healing in the impaired glucocorticoid model of wound healing. Adult SD male rats (n = 5) received dermal puncture wounds (8 mm) on day 1 and were treated daily for 5 consecutive days with a buffer solution or a KGF-2 solution in 50 μl. The animals received 5 mg of methylprednisolone on the day of the wounding. The wounds were measured daily for 5 consecutive days starting on the day of the wound and on day 8 with a calibrated Jameson meter. The statistical analysis was performed using a non-paired t-test. (Average +/- SEM). Figure 19 (A) shows the effect of KGF-2 on the wound area in a rat model of wound healing without methylprednisolone on day 5 after the wound was made. SD male rats (n = 5) received dermal puncture wounds (8 mm) on day 1 and were treated daily with either a buffer or KGF-2 in 50 μl of a solution on the day of the wound realization and after that for 5 consecutive days. Wounds were measured daily using a calibrated Jameson meter. The statistical analysis was performed using a non-paired t-test. (Average +/- SEM). (B) Evaluation of PDGF-BB and KGF-2 in SD male rats (n = 6). All rats received 8 mm back injuries and methylprednisolone (MP) (17 mg / kg) to deteriorate wound healing. Wounds were treated daily with buffer or various concentrations of PDGF-BB and KGF-2. Wounds were measured on days 2, 4, 6, 8 and 10 using a calibrated Jameson meter. The statistical analysis was performed using an unpaired t-test (Average +/- SE) * Compared with shock absorber. ** PDGF-BB 1 μg versus KGF-2 / E3 1 μg.

Figure 20 shows the effect of KGF-2 on the distance of wounds in a damaged model with glucocorticoid wound healing. SD adult male rats (n = 5) received dermal puncture wounds (8 mm) and 17 mg / kg methyl prednisolone on the day of wounding. The animals were treated daily with a buffer solution or KGF-2 in 50 μl of buffer for 5 consecutive days on day 8. The distance of the wound was measured under light microscopy with a calibrated micrometer. The statistical analysis was performed using a non-paired t-test. (Average +/- SEM). Figure 21 (A) shows the stimulation of the proliferation of primary, normal, epidermal keratinocytes by KGF-2. (B) shows the stimulation of the proliferation of primary, normal epidermal keratinocytes by KGF-2? 33. (C) shows the stimulation of the proliferation of primary, normal epidermal keratinocytes by KGF-2? 28. The normal, normal, human epidermal keratinocytes were incubated with various concentrations of KGF-2, KGF-2? 33 or KGF-2? 28 for three days. For the three experiments, Alamar blue was then added for 16 hours and the intensity of the red color converted from Blue alamar by the cells was measured by the difference between D.O. 570 nm and D.O. 600 nm. For each of the KGF-2 proteins, a positive control was included with the complete keratinocyte growth medium (KGM) and a negative control with the basal keratinocyte medium (KBM) on the same assay plate. Figure 22 (A) shows the stimulation of thymidine incorporation by KGF-2 and FGF7 in Baf3 cells transfected with FGFRlb and FGFR2. The effects of KGF-2 (right panel) and FGF7 (left panel) on the proliferation of Baf3 cells transfected with FGFRliiib (blank circle) or FGFR2iiib / FGFR (dark circle) were examined. The Y axis represents the amount of [3H] thymidine incorporation (cpm) in the DNA of Baf3 cells. The X axis represents the final concentration of KGF-2 or FGF7 added to the tissue culture media. (B) shows the stimulation of thymidine incorporation by KGF-2Δ 33 in Baf3 cells transfected with FGFR2iiib. (C) shows the stimulation of thymidine incorporation by KGF-2 (white bar), KGF-2? 33 (black bar) and KGF-2? 28 (gray bar) in Baf3 cells transfected with FGFR2iiib. Figure 23 shows the DNA and protein sequence [SEQ ID NOS. 38 and 39] for optimized full-length KGF-2 from E. coli. Figures 24A and B show the DNA and protein sequences [SEQ ID NOS. 42, 43, 54 and 55] for mature, optimized KGF-2 from E. coli.

Figure 25 shows the DNA and the encoded sequence of proteins [SEQ ID NOS. 65 and 66] for the KGF-2 suppression construct comprising amino acids 36 to 208 of KGF-2. Figure 26 shows the DNA and the encoded sequence of proteins [SEQ ID NOS. 67 and 68] for the KGF-2 suppression construct comprising amino acids 63 to 208 of KGF-2. Figure 27 shows the DNA and the encoded sequence of proteins [SEQ ID NOS. 69 and 70] for the construction of KGF-2 suppression comprising amino acids 77 to 208 of KGF-2. Figure 28 shows the DNA and the encoded sequence of proteins [SEQ ID NOS. 71 and 72] for the construction of KGF-2 deletion comprising amino acids 93 to 208 of KGF-2. Figure 29 shows the DNA and the encoded sequence of proteins [SEQ ID NOS. 73 and 74] for the KGF-2 suppression construct comprising amino acids 104 to 208 of KGF-2. Figure 30 shows the DNA and the encoded sequence of proteins [SEQ ID NOS. 75 and 76] for the KGF-2 suppression construct comprising amino acids 123 to 208 of KGF-2.

Figure 31 shows the DNA and the encoded sequence of proteins [SEQ ID NOS. 77 and 78] for the KGF-2 suppression construct comprising amino acids 138 to 208 of KGF-2. Figure 32 shows the DNA and the encoded sequence of proteins [SEQ ID NOS. 79 and 80] for the construction of KGF-2 suppression comprising amino acids 36 to 153 of KGF-2. Figure 33 shows the DNA and the encoded sequence of proteins [SEQ ID NOS. 81 and 82] for the KGF-2 suppression construct comprising amino acids 63 to 153 of KGF-2. Figure 34 shows the DNA sequence for the mutant construction of cysteine 37 to serine from KGF-2 [SEQ ID NO. 83]. Figure 35 shows the DNA sequence for the mutant construction of cysteine 37 / cysteine 106 to serine of KGF-2 [SEQ ID NO. 84]. Figure 36 shows the evaluation of the effects of KGF-2? 33 on wound healing in SD male rats (n = 5). The animals received 6 mm back injuries and were treated with various concentrations of buffer, or KGF-2? 33 for 4 consecutive days. Wounds were measured daily using a calibrated Jameson meter.

The statistical analysis was performed using a non-paired t-test. (Average +/- SE). * Compared with shock absorber. Figure 37 shows the effect of KGF-2? 33 on wound healing in normal rats. Male SD rats of 250-300 g (n = 5) were subjected to 6 mm full thickness back injuries. The wounds were measured with a calibrator and treated with various concentrations of KGF-2? 33 and shock absorber for four days, starting on the day of surgery. On the final day, the wounds were harvested. The statistical analysis was performed using a non-paired t-test. * The value is compared to the control without treatment. The value is compared to the Control with Shock Absorber Figure 38 shows the effect of KGF-2 33 on the breaking force in the incisional wounds Adult SD male rats (n = 10) received full-thickness incisional wounds. 2.5 cm on day 1 and were intra-incisionally treated post-wound with either a buffer application or KGF-2 (Delta 33) (1, 4 and 10 μg) .The animals were sacrificed on day 5 and wound specimens from 0.5 cm were excised for routine histology and analysis of the breaking force.The biomechanical test was carried out using an Instron skin tension meter with a force applied through the wound.The resistance to breakage was defined as the most Large sample supported by each wound before rupture.The statistical analysis was performed using an unpaired t test (Mean +/- SE) Figure 39 shows the effect of KGF-2 (Delta 33) on the epidermal thickness in the incisional wounds. Male adult SD rats (n = 10) received incisional wounds of full thickness of 2.5 cm on day 1 and were intra-incisionally treated after performing the wound with either a buffer application or KGF-2 (Delta 33) (1 , 4 and 10 μg). The animals were sacrificed on day 5 and wound specimens of 0.5 cm were excised for routine histology analysis of breaking strength. The epidermal thickness was determined by taking the average of 6 measurements taken around the wound site. The measurements were taken by a "blind" observer in sections stained with Masson Trichrome under light microscopy using a calibrated lens micrometer. The statistical analysis was performed using a non-paired t-test. (Average +/- SE). Figure 40 shows the effect of KGF-2 (Delta 33) on the epidermal thickness after a simple intradermal injection. Adult SD male rats (n = 18) received 6 intradermal injections of either buffer or KGF-2 at a concentration of 1 and 4 μg in 50 μl on day 0. Animals were sacrificed 24 and 48 hours after injection . The epidermal thickness was measured from the granular layer towards the bottom of the basal layer. Approximately 20 measurements were made along the injection site and the average thickness was quantified. The measurements were determined using a micrometer calibrated on sections stained with Masón Trichrome under light microscopy. The statistical analysis was performed using a non-paired t-test. (Average +/- SE). Figure 41 shows the effect of KGF-2 (Delta 33) on the BrdU rating. Male adult SD rats (n = 18) received 6 intradermal injections of either placebo or KGF-2 at a concentration of 1 and 4 μg in 50 μl on day 0. Animals were sacrificed 24 and 48 hours after injection. The animals were injected with 5-2'-bromo-deoxyrudine (100 mg / kg intraperitoneally) two hours before slaughter. The qualification was performed by a "blind" observer under light microscopy using the following rating system: 0-3 none at minimal cells marked with BrdU; 4-6 moderate dialing; 7-10 cells marked intensely. The statistical analysis was performed using a non-paired t-test. (Average +/- SE). Figure 42 shows the anti-inflammatory effect of KGF-2 on foot edema induced by PAF.

Figure 43 shows the anti-inflammatory effect of KGF-2? 33 on leg edema induced by PAF in Lewis rats. Figure 44 shows the anti-inflammatory effect of KGF-2? 33 on the survival of irradiated Balb / c mice in the whole body. Male Balb / c mice (n = 5), 22.1 g were irradiated with 519 RADS. The animals were treated with buffer or KGF-2 (1 and 5 mg / kg, subcutaneously) 2 days before irradiation and daily thereafter for 7 days. Figure 45 shows the effect of KGF-2? 33 on the body weight of irradiated mice. Male Balb / c mice (n = 5), weighing 22.1 g were injected with either KGF-2Δ33 buffer (1 and 5 mg / kg) for 2 days before irradiation with 519 Rad / minute. The animals were weighed daily and injected for 7 days after irradiation. Figure 46 shows the effect of KGF-2? 33 on the survival rate of irradiated whole-body Balb / c mice. Male Balb / c mice (n = 7) of 22.1 g were irradiated with 519 RADS. The animals were treated with buffer or KGF-2 (1 and 5 mg / kg, subcutaneously) 2 days before irradiation and daily thereafter for 7 days.

Figure 47 shows the effect of KGF-2? 33 on wound healing in a rat model impaired with glucocorticoid. Figure 48 shows the effect of KGF-2? 33 on cell proliferation as determined using BrdU labeling. Figure 49 shows the effect of KGF-2? 33 on the collagen content located in the anastomotic surgical sites in the colonies of rats. Figure 50 shows a schematic representation of the expression vector pHE4-5 (SEQ ID NO: 147) and the coding sequence of the subcloned KGF-2 cDNA. The locations of the kanamycin resistance marker gene, the KGF-2 coding sequence, the oriC sequence, and the laclq coding sequence are indicated. Figure 51 shows the nucleotide sequence of the regulatory elements of the pHE promoter (SEQ ID No. 148.).

The two sequences of the lac operator are indicated, the sequence Shine-Dalgarno (S / D), and the terminal restriction sites Hip lII and Ndel (in italics). Figure 52 shows the profile of the bladder epithelium after interperitoneal or subcutaneous administration of KGF-2? 33.

Figure 53 shows the proliferation of prostatic epithelial cells after systemic administration of KGF-2? 33. Figure 54 shows the effect of KGF-2? 33 on ulceration of the bladder wall in a model of hemorrhagic cystitis induced by cyclophosphamide, in rat. Fig. 55 shows the effect of KGF-2? 33 on the thickness of the bladder wall in a rat model with cystitis induced by cyclophosphamide. Figure 56 provides an overview of the study design to determine if KGF-2Δ 33 induces normal epithelial proliferation in rats when administered systemically using SC and IP routes. Figure 57. Normal Sprague Dawley rats were injected daily with KFG-2Δ33 (5 mg / kg, HG03411-E2) or buffer and sacrificed one day after the final injection. A blinded observer counted the proliferating cells in ten fields randomly chosen by animals at a magnification of 10 x. The SC administration of KGF-2? 33 caused a significant proliferation after one day, which then returned to normal for 2 days. KGF-2? 33 administered ip stimulated the proliferation of 1-3 days but only the results of days 1 and 3 were statistically significant.

Figure 58. Normal Sprague Dawley rats were injected daily with KFG-2Δ33 (5 mg / kg, HG03411-E2) or with buffer and sacrificed one day after the final injection. A "blind" observer counted the proliferating cells in ten randomly chosen fields per animal at a magnification of 10 x. KGF-2? 33 administered ip stimulated proliferation in the entire study period while administration of KGF-2? 33 did not increase proliferation at any time point. Figure 59. Normal Sprague Dawley rats were injected daily with KFG-2? 33 (5 mg / kg; HG03411-E2) or buffer and sacrificed one day after the final injection. A "blind" observer counted the proliferating cells in a cross-section per animal at a magnification of 10 x. KGF-2? 33 administered sc caused a significant increase in proliferation after 1, 2 and 3 days of daily administration. When KGF-2? 33 was administered ip, proliferation was observed after 2 and 3 days only. Figure 60 demonstrates the proliferation induced by KGF-2? 33 in normal rat lung.

DETAILED DESCRIPTION According to one aspect of the present invention, there is provided an isolated nucleic acid (polynucleotide) encoding the polypeptide having the deduced amino acid sequence of Figure 1 (SEQ ID NO: 2) or for the polypeptide encoded by the cDNA of the clone deposited with ATCC No. 75977 on December 16, 1994 in the Patent Depository of the American Type Culture Collection, 10801 Uníversity Boulevard, Manassas, VA 20110-2209 or the polypeptide encoded by the cDNA of the clone deposited as ATCC Deposit No. 75901 on September 29, 1994 in the Patent Depository of the American Type Culture Collection, 10801 University Boulevard, Manassas, VA 20110-2209.

Nucleic Acid Molecules Unless otherwise indicated, all nucleotide sequences determined by sequencing a DNA molecule herein were determined using an automatic DNA sequencer (such as model 373 from Applied Biosystems, Inc.), and all the amino acid sequences of polypeptides encoded with the DNA molecules determined herein were predicted by translation of a DNA sequence determined as described above. Therefore, as is known in the art for any DNA sequence determined by this automated method, any nucleotide sequence determined herein may contain some errors. The nucleotide sequences determined by automation are typically at least 90% identical, more typically at least about 95% to at least about 99.9% identical to the effective nucleotide sequence of the sequenced DNA molecule. The reactive sequence can be more precisely determined by other methods including manual DNA sequencing methods well known in the art. As is also known in the art, a simple insertion or deletion in a given nucleotide sequence in comparison to the effective sequence will cause a structural shift in the translation of the nucleotide sequence, such that the predicted amino acid sequence encoded by a given nucleotide sequence will be completely different from the amino acid sequence effectively encoded by the sequenced DNA molecule, beginning at the point of such insertion or deletion. Unless indicated otherwise, each "nucleotide sequence" described herein is presented as a deoxyribonucleotide sequence. (abbreviated A, G, C and T). However, by "nucleotide or nucleotide sequence" of a nucleic acid molecule or a polynucleotide is meant, for a DNA molecule or polynucleotide, a deoxyribonucleotide sequence, and for an RNA or polynucleotide molecule, the corresponding sequence of ribonucleotides (A, G, C and U), where each thymidine deoxyribonucleotide (T) in the specified deoxyribonucleotide sequence is replaced by the ribonucleotide uridine (U). For example, reference to an RNA molecule having the sequence of SEQ ID NO. 1 described using the abbreviations of deoxyribonucleotides, is intended to indicate an RNA molecule having a sequence in which each deoxyribonucleotide A, G or C of SEQ ID NO. 1 has been replaced by the corresponding ribonucleotide A, G or C, and each deoxyribonucleotide has been replaced by a U-ribonucleotide. The "isolated" nucleic acid molecule (s) is intended to indicate a nucleic acid, DNA or RNA, which has been removed from its native environment. For example, recombinant DNA molecules contained in a vector are considered isolated for purposes of the present invention. Additional examples of isolated DNA molecules include recombinant DNA molecules maintained in heterologous host cells or purified DNA molecules (partially or substantially) in solution. Isolated RNA molecules include RNA transcripts in vivo or in vi tro of the DNA molecules of the present invention. The isolated nucleic acid molecules according to the present invention further include such synthetically produced molecules. The isolated nucleic acid molecules of the present invention include DNA molecules comprising an open reading frame (ORF) with a start codon at positions 1-3 of the nucleotide sequence shown in Figure 1 (SEQ ID NO. 1); the DNA molecules comprising the coding sequence for the KFG-2 protein, mature shown in Figure 1 (last 172 or 173 amino acids) (SEQ ID No. 2); and DNA molecules comprising a sequence substantially different from those described above but which, due to the degeneracy of the genetic code, still code for the KGF-2 protein. Of course, the genetic code is well known in the art. In this way, it could be routine for a person skilled in the art to generate the degenerate variants described above. A polynucleotide encoding a polypeptide of the present invention can be obtained from a human or fetal lung prostate. A fragment of the cDNA encoding the polypeptide was initially isolated from a library derived from a normal human prostate. The open reading frame encoding the full-length protein was substantially isolated from a human fetal lung cDNA genome, randomly primed. This is structurally related to the FGF family. It contains an open reading frame which codes for a protein of 208 amino acid residues, of which approximately the first 35 or 36 amino acid residues are the putative leader sequence such that the mature protein comprises 173 or 172 amino acids. The protein shows the highest degree of homology to human keratinocyte growth factor with 45% identity and 82% similarity over a stretch of 206 amino acids. It is also important that the sequences that are conserved throughout the FGF family be found as conserved in the protein of the present invention. In addition, the results from the nested PCR of the KGF-2 cDNA from the libraries showed that there were spliced, alternative, potential forms of KGF-2. Specifically, using the flanking primers of the N-terminus of the open reading structure of KGF-2, the 0.2 kb and 0.4 kb PCR products of various cDNA libraries were obtained. A size of 0.2 kb was the expected product for KGF-2 while the size of 0.4 kb could result in an alternatively spliced form of KGF-2. The 0.4 kb product was observed in libraries from stomach cancer, adult testes, duodenum and pancreas. The polynucleotide of the present invention may be in the form of RNA or in the form of DNA, with the DNA including cDNA, genomic DNA and synthetic DNA. The DNA can be double-stranded or single-stranded, and if it is single-stranded it can be the coding strand or the non-coding strand (anti-sense). The coding sequence coding for the mature polypeptide may be identical to the coding sequence shown in Figure 1 (SEQ ID NO. 1) or that of the deposited clone or it may be a different coding sequence whose coding sequence, as a result of the redundancy or degeneracy of the genetic code, it codes for the same mature polypeptide as the DNA of Figure 1 (SEQ ID NO. 1) or deposited cDNA. The polynucleotide encoding the predicted mature polypeptide of Figure 1 (SEQ ID NO: 2) or for the predicted mature polypeptide encoded by the deposited cDNA may include: only the coding sequence for the mature polypeptide; the coding sequence carries the mature polypeptide and the additional coding sequence such as a guiding or secretory sequence or a pro-protein sequence; the coding sequence for the mature polypeptide (and optionally the additional coding sequence) and the non-coding sequence, such as the 5 'and / or 3' intron or non-coding sequence of the coding sequence for the predicted mature polypeptide. In addition, a full length mRNA has been obtained which contains the 5 'and 3' untranslated regions of the gene (Figure 3 (SEQ ID NO.23)). As a person of ordinary skill in the art would appreciate, due to the possibilities of the sequencing errors discussed above, as well as the variability of the cleavage sites for the guides in different known proteins, the effective KGF-2 polypeptide encoded by the Deposited cDNA, comprises approximately 208 amino acids, but can be anywhere in the range of 200 to 220 amino acids; and the effective leader sequence of this protein is about 35 or 36 amino acids, but it can be anywhere in the range of about 30 to about 40 amino acids. Thus, the term "polynucleotide encoding a polypeptide" encompasses a polynucleotide that includes only the coding sequence for the polypeptide as well as a polynucleotide that includes the additional coding and / or non-coding sequence. The present invention further relates to the variants of the polynucleotides described hereinabove, which code for fragments, analogs and derivatives of the polypeptide having the deduced amino acid sequence of Figure 1 (SEQ ID NO: 2) or the polypeptide encoded by the cDNA of the deposited clone. The variant of the polynucleotide can be an allelic variant of natural origin of the polynucleotide or a non-natural variant of the polynucleotide. Thus, the present invention includes polynucleotides encoding the same predicted mature polypeptide as shown in Figure 1 (SEQ ID NO 2) or the same predicted mature polypeptide encoded by the cDNA of the deposited clone as well as variants of such polypeptides whose variants code for a fragment, derivative or analog of the polypeptide of Figure 1 (SEQ ID NO: 2) or the polypeptide encoded by the cDNA of the deposited clone. Such nucleotide variants include deletion variants, substitution variants and addition or insertion variants. The present invention includes the polynucleotides that code for the KGF-2 mimetic peptides that can be used as therapeutic peptides. The mimetic KGF-2 peptides are short peptides that mimic the biological activity of the KGF-2 protein by binding to and activating the cognate KGF-2 receptors. The KGF-2 mimetic peptides can also bind to and inhibit the cognate KGF-2 receptors. KGF-2 receptors include, but are not limited to, FGFR2iiib and FGFRliiib. Such mimetic peptides are obtained by methods such as, but not limited to, visual representation of the phage or combinatorial chemistry. For example, the method described by Wrigthton et al., Science 273: 458-463 (1996) to generate the mimetic KGF-2 peptides. As indicated hereinabove, the polynucleotide may have a coding sequence which is a naturally occurring allelic variant of the coding sequence shown in Figure 1 (SEQ ID NO: 1) or the coding sequence of the deposited clone. As is known in the art, an allelic variant is an alternate form of a polynucleotide sequence which may have a substitution, deletion or addition of one or more nucleotides, which does not substantially alter the function of the encoded polypeptide. The present invention also includes polynucleotides, wherein the coding sequence for the mature polypeptide can be fused in the same reading frame to a polynucleotide sequence that aids in the expression and secretion of a polypeptide from a host cell, e.g. , a guiding sequence that functions as a secretory sequence to control the transport of a polypeptide from the cell. The polypeptide having a leader sequence is a preprotein and may have the leader sequence cleaved by the host cell to form the mature form of the polypeptide. The polynucleotides can also code for the proprotein which is the mature protein plus the additional 5 'amino acid residues. A mature protein that has a prosequence is a proprotein and is an inactive form of the protein. Once the prosequence is cleaved, an active mature protein remains. Thus, for example, the polynucleotide of the present invention can code for a mature protein, or for a protein having a prosequence or for a protein having the prosequence and a presequence (guide sequence). The polynucleotides of the present invention may also have the coding sequence fused intrainstructurally to a marker sequence that permits purification of the polypeptide of the present invention. The marker sequence can be a hexahistidine tag supplied by a pQE-9 vector to provide for purification of the mature polypeptide fused to the tag in the case of a bacterial host, or for example, the tag sequence can be a hemagglutinin tag (HA) when a mammalian host is used, for example, COS-7 cells. The HA marker corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson, I. et al., Cell 37: 767 (1984)). The term "gene" means the segment of DNA involved in the production of a polypeptide chain; it includes the regions that precede and follow the coding region (guide and subsequent) as well as the intervening sequences (introns) between the individual coding segments (exons). Fragments of the full-length gene of the present invention can be used as a hybridization probe for a cDNA genot to isolate the full length cDNA and to isolate other cDNAs which have a high sequence similarity to the gene or a similar biological activity . Probes of this type preferably have at least 30 bases and may contain, for example, 50 or more bases. The probe can also be used to identify a cDNA clone corresponding to a full-length transcript and a clone or genomic clones containing the complete gene including the regulatory and promoter regions, the exons and the introns. An example of a selection comprises isolating the coding region of the gene, by using the known DNA sequence to synthesize an oligonucleotide probe. The labeled oligonucleotides having a sequence complementary to that of the gene of the present invention are used to select a library of human cDNA, genomic DNA or cDNA to determine which members of the library the probe hybridizes to. Additional embodiments of the invention include isolated nucleic acid molecules comprising a polynucleotide having at least an 80% identical nucleotide sequence, and more preferably at least 95%, 90%, 91%, 92%, 93%, 94% 95%, 97%, 98% or 99% identical to (a) a nucleotide sequence encoding the full length KGF-2 polypeptide having the complete amino acid sequence in Figure 1 (SEQ ID NO. 2), including the predicted guide sequence; (b) a nucleotide sequence that encodes the mature KGF-2 polypeptide (full length polypeptide with the leader removed) having the amino acid sequence at positions approximately 36 or 37 to 208 in Figure 1 (SEQ ID NO: 2); (c) a nucleotide sequence encoding the full-length KGF-2 polypeptide having the complete amino acid sequence including the leader encoded by the cDNA clone contained in the ATCC Deposit No. 75977; (d) a nucleotide sequence encoding the mature KGF-2 polypeptide having the amino acid sequence encoded by the cDNA clone contained in the ATTC Deposit No. 75977; (e) a nucleotide sequence encoding any of the KGF-2 analogs or deletion mutants described below; or (f) a nucleotide sequence complementary to any of the nucleotide sequences in (a), (b), (c), (d) or (e). For a polynucleotide having at least one nucleotide sequence, for example 95% "identical" to a reference nucleotide sequence coding for a KGF-2 polypeptide, the nucleotide sequence of the polynucleotide is intended to be identical to the reference sequence, except that the polynucleotide sequence can include up to five point mutations per 100 nucleotides of the reference nucleotide sequence encoding the KGF-2 polypeptide. In other words, to obtain a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence can be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence can be inserted into the reference sequence. These mutations of the reference sequence can occur at the 5 'or 3' terminal positions of the reference nucleotide sequence or at any site between those terminal positions, interspersed either individually between the nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.

As a practical matter, if any particular nucleic acid molecule is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98% or 99% identical to, for example, the nucleotide sequence shown in Figure 1 (SEQ ID NO.1) or the nucleotide sequence of the deposited cDNA clone, this can be determined conventionally using known computer programs such as the Bestfit program (Wisconsin Sequential Analysis Package). , Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wl 537111). Bestfit uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathe atics 2: 482-489 (1981), to find the best homology segment between two sequences. When Bestfit or any other sequence alignment program is used to determine if a particular sequence is, for example, 95% identical to a reference sequence according to the present invention, the parameters are set, of course, such that the percentage of identity is calculated on the full length of the reference nucleotide sequence and that empty spaces in homology of up to 5% of the total number of nucleotides in the reference sequence are allowed. A preferred method for determining the best match or complete match between a search sequence (a sequence of the present invention) and an objective sequence, also referred to as a global sequential alignment, can be determined using the FASTDB computer program on the algorithm of Brutlag et al. (Comp. App. Biosci. 1 (990) 6: 237-245). In a sequential alignment, the search and target sequences are both DNA sequences. An RNA sequence can be compared by converting the U's to T's. The result of the alignment of the global sequence is in the percentage identity. The preferred parameters used in a FASTDB alignment of the DNA sequences to calculate the percentage identity are: Matrix = Unitary, k-tuple = 4, Penalty for mal-coupling = l, Penalty per union = 30, Length of Randomization Group = 0 , Cutoff rating = l, Penalty for empty space = 5, Penalty for empty space size = 0.05, Window size = 500 or the length of the target nucleotide sequence, whichever is shorter. If the target sequence is shorter than the search sequence due to the deletion of 5 'or 3 not due to internal deletions, a manual correction to the results must be made. This is because the FASTDB program does not explain the 5 'and 3' truncations of the target sequence, when the percent identity is calculated. For target sequences truncated at the 5 'and 3' ends, relative to the search sequence, the percentage identity is corrected by calculating the number of bases of the search sequence that is 5 'and 3' of the target sequence , which are not coupled / aligned, as a percentage of the total bases of the search sequence. Whether a nucleotide is coupled or not aligned, this is determined by the results of the sequential FASTDB alignment. This percentage is then subtracted from the percentage identity, calculated by the aforementioned FASTDB program using the specified parameters, to arrive at a final percentage identity score. This corrected grade is that which is used for purposes of the present invention. The only bases outside the 5 'and 3' bases of the target sequence, as shown by the FASTDB alignment, which are not coupled / aligned with the search sequence, are calculated for purposes of manually adjusting the percentage identity rating. For example, a target sequence of 90 bases is aligned to a search sequence of 100 bases to determine the percent identity. Deletions occur at the 5 'end of the target sequence and therefore, the FASTDB alignment does not show a coupling / alignment of the first 10 bases at the 5 'end. The 10 unpaired bases represent 10% of the sequence (number of bases at the 5 'and 3' ends not coupled / total number of bases in the search sequence) so that 10% is subtracted from the calculated percent identity rating for the FASTDB program. If the remaining 90 bases were perfectly coupled, the final procentual identity would be 90%. In yet another example, a target sequence of 90 bases is compared to a search sequence of 100 bases. This time the deletions are internal deletions so that there are no bases on the 5 'or 3' end of the target sequence that are not coupled / aligned with the search. In this case, the percentage identity calculated by FASTDB is not manually corrected. Again, only the 5 'and 3' bases of the target sequence that are not coupled / aligned with the search sequence are manually corrected. No other manual corrections are made for purposes of the present invention. The present application is directed to the nucleic acid molecules at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98% or 99% identical to the sequence of nucleic acid shown in Figure 1 [SEQ ID NO. 1) or the nucleic acid sequence of the deposited ANDc, regardless of whether they code for a polypeptide having KGF-2 activity. This is because even where a particular nucleic acid molecule does not code for a polypeptide having KGF-2 activity, a person skilled in the art would still know how to use the nucleic acid molecule, for example, as a probe. hybridization, or a primer of the polymerase chain reaction (PCR). The uses of the nucleic acid molecules of the present invention, which do not code for a polypeptide having KGF-2 activity include, among others, (1) isolation of the KGF-2 gene or allelic variants thereof in a cDNA library; (2) hybridization in itself (eg, "FISH") to metaphase chromosomal diffusions to provide accurate chromosomal localization of KGF-2, as described in Verma et al., Human Chromoso is: A Manual of Basic Techniques . Pergamon Press, New York (1988); and Northern blot analysis to detect the expression of KGF-2 mRNA in specific tissues. However, nucleic acid molecules having sequences of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98% or 99% identical to those of the invention are preferred. the nucleic acid sequence shown in Figure 1 [SEQ ID NO. 1] or to the nucleic acid sequence of the deposited cDNA, which, in fact, encode a polypeptide having KGF-2 protein activity. By "a polypeptide having KGF-2 activity" is meant polypeptides that show activity similar, but not necessarily identical, to an activity of the wild-type KGF-2 protein of the invention, or an activity that is enhanced over that of the wild-type KGF-2 protein (either the full-length protein or, preferably, the mature protein), as measured in a particular biological assay. Tests of KGF-2 activity are described, for example, in Examples 10 and 11 below. These assays can be used to measure the KGF-2 activity of the purified or partially purified native or recombinant protein. KGF-2 stimulates the proliferation of epidermal keratinocytes but not mesenchymal cells such as fibroblasts. Thus, "a polypeptide having KGF-2 protein activity" includes polypeptides that show KGF-2 activity, in the keratinocyte proliferation assay described in Example 10 and will bind to isoforms 1-iiib and 2 -iiib of the FGF receptor (Example 11). Although the degree of activity need not be identical to that of the KGF-2 protein, preferably, "a polypeptide having KGF-2 protein activity" will show substantially similar activity compared to the KGF-2 protein (e.g., the polypeptide candidate will show greater activity or no more than about ten times less and preferably, no more than about two times less activity relative to the reference KGF-2 protein).

Of course, due to the degeneracy of the genetic code, a person of ordinary skill in the art will immediately recognize that a large number of nucleic acid molecules having a sequence at least 80%, 85%, 90%, 91%, 92 %, 93%, 94%, 95%, 97%, 98% or 99% identical to the nucleic acid sequence of the deposited cDNA or the nucleic acid sequence shown in Figure 1 [SEQ ID NO. 1] will code for a polypeptide "having KGF-2 protein activity". In fact, since the degenerate variants of these nucleotide sequences all code for the same polypeptide, this will be clear to the person skilled in the art, even without performing the comparison test described above. It will be further recognized in the art that, for such nucleic acid molecules that are not degenerate variants, a reasonable number will encode a polypeptide having KGF-2 protein activity. This is because the person skilled in the art is fully aware of amino acid substitutions that are either less likely or unlikely to significantly affect the function of the protein (eg, the replacement of an aliphatic amino acid with a second amino acid). aliphatic). For example, a guide concerning how to make substitutions of phenotypically silent amino acids is provided in Bowie, J.U. and collaborators, "Deciphering the message in Protein Sequences: Tolerance to Amino Acid Substitutions", Science 247: 1306-1310 (1990), where the authors indicate that there are two main procedures for studying the tolerance of an amino acid sequence to be changed. The first method relies on the process of evolution, in which mutations are either accepted or rejected by natural selection. The second procedure used the manipulation by genetic engineering to introduce amino acid changes at specific positions of a cloned gene and selections to identify sequences that maintain functionality. As established by the authors, these studies have revealed that proteins are surprisingly tolerant of amino acid substitutions. The authors also indicate which amino acid changes are likely to be permissive at a certain position of the protein. For example, the most buried amino acid residues require non-polar side chains, while few features of the surface side chains are generally conserved. Other phenotypically silent substitutions are described in Bowie, J.U. and collaborators, supra, and the references cited therein. The present invention further relates to polynucleotides that hybridize to the sequences described above if there is at least 70%, preferably at least 80%, and more preferably at least 85%, and still more preferably 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98% or 99% identity between the sequences. The present invention relates particularly to polynucleotides that hybridize under stringent conditions to the polynucleotides described hereinabove. As used herein, the term "stringent conditions" means that hybridization will occur only if there is at least 95% and preferably at least 97% identity between the sequences. The polynucleotides that hybridize to the polynucleotides described above in a preferred embodiment code for polypeptides that retain either substantially the same function or biological activity as the mature polypeptide encoded by the cDNAs of Figure 1 (SEQ ID NO. Deposited cDNAs. An example of "stringent hybridization conditions" includes overnight incubation at 42 ° C in a solution comprising: 50% formamide, 5 x SSC (150 mM sodium chloride, 15 mM trisodium citrate), 50 sodium phosphate mM (pH 7.6), Denhardt 5x solution, 10% dextran sulfate, and 20 μg / ml sperm DNA from cut salmon, denatured, followed by washing the filters 0.1 x SSC at approximately 65 ° C. Alternatively, the polynucleotide may have at least 20 bases, preferably 30 bases, and more preferably at least 50 bases which hybridize to a polynucleotide of the present invention, and which have an identity thereto, as described hereinabove, and who may or may not keep the activity. For example, such polynucleotides can be used as probes for the polynucleotide of SEQ ID NO. 1, for example, for the recovery of the polynucleotide or as a diagnostic probe or as a PCR primer. Nucleic acid molecules that hybridize to KGF-2 polynucleotides at hybridization conditions of moderately high requirement are also contemplated. Changes in the requirement for hybridization and in the detection of the signal are mainly achieved through the manipulation of the formamide concentration (lower percentages of formamide result in decreased requirement); the salt conditions, or the temperature. For example, conditions of moderately high requirement include an overnight incubation at 3 ° C in a solution comprising 6X SSPE (20X SSPE = 3 M sodium chloride, 0.2 M sodium diacid phosphate, 0.02 M EDTA, pH 7.4) , 0.5% SDS, 30% formamide, 100 μg / ml DNA blocking salmon sperm; followed by washing at 50 ° C with IXSSPE, 0.1% SDS. In addition, to achieve the even lower requirement, washes made after stringent hybridization can be performed at higher salt concentrations (eg, 5X SSC).

Note that variations in the above conditions can be achieved through the inclusion and / or substitution of alternative blocking reagents used to suppress the background in the hybridization experiments. Typical blocking reagents include Denhardt reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercially available proprietary formulations. The inclusion of specific blocking reagents may require modification of the hybridization conditions described above, due to problems with compatibility. Of course, polynucleotides that hybridize to a larger portion of the reference polynucleotide (e.g., the deposited cDNA clone), e.g., a 50-750 nucleotide length portion, or even the full length of the polynucleotide of reference, are also useful as probes according to the present invention, as are the polynucleotides corresponding to most, if not all, the nucleotide sequence of the deposited cDNA or the nucleotide sequence as shown in Figure 1 [SEQ. ID NO. 1] . For a portion of a polynucleotide of "at least 20 nucleotides in length", for example, it is intended to mean 20 or more contiguous nucleotides from the nucleotide sequence of the reference polynucleotide (eg, the deposited cDNA or the nucleotide sequence). as shown in Figure 1 [SEQ ID NO. 1]). As indicated, such portions are diagnostically useful either as a probe according to conventional DNA hybridization techniques or as primers for the purification of an objective sequence by the polymerase chain reaction (PCR), as described, by example, in Molecular Cloning, A Laboratory Manual, 2a. edition, edited by Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989), Cold Spring Harbor Laboratory Press, the full description of which is incorporated by reference herein. Since a cDNA clone of KGF-2 has been deposited and its determined nucleotide sequence is provided in Figure 1 [SEQ ID NO. 1), the generation of polynucleotides that hybridize to a portion of the KGF-2 cDNA molecule could be routine for the person skilled in the art. For example, cleavage or restriction endonuclease cleavage by sonication of the KGF-2 cDNA clone could be easily used to generate the DNA portions of various sizes which are polynucleotides that hybridize to a portion of the KGF cDNA molecule. -2. Alternatively, the hybridization polynucleotides of the present invention could be generated synthetically according to known techniques. Of course, a polynucleotide that hybridizes only to a poly A sequence (such as the terminal poly (A) 3 'stretch of the KGF-2 cDNA shown in Figure 1 [SEQ ID NO. 1]), or to a complementary stretch of residues T (or U), could not be included in a polynucleotide of the invention, used to hybridize to a portion of a nucleic acid of the invention, since such a polynucleotide could hybridize to any nucleic acid molecule that contains a poly stretch (A) or the complement thereof (for example, practically any double-stranded cDNA clone). The invention further provides isolated nucleic acid molecules comprising a polynucleotide that codes for an epitope-possessing portion of the KGF-2 protein. In particular, the isolated nucleic acid molecules are provided by coding for the polypeptides comprising the following amino acid residues in Figure 1 (SEQ ID NO: 2), which the present inventors have determined, are antigenic regions of the KGF-2 protein : 1. Gly41-Asn71: GQDMVSPEATNSSSSSFSSPSSAGRHVRSYN [SEQ ID NO. 25]; 2. Lys91-Serl09: KIEKNGKVSGTKKENCPYS [SEQ ID NO. 26]; 3. Asnl35-Tyrl64: NKKGKLYGSKEFNNDCKLKERIEENGYNTY [SEQ ID NO. 27]; and 4. Asnl81-Alal99: NGKGAPRRGGQKTRRKNTSA [SEQ ID NO. 28] Also, there are two additional, shorter, predicted antigenic areas, Gln74-Arg78 of Figure 1 (SEQ ID DO NOT. 2) and Glnl70-Glnl75 of Figure 1 (SEQ ID NO.2). The methods for generating such epitope-possessing portions of KGF-2 are described in detail below. The deposits herein will be maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for purposes of Patent Procedure. These deposits are provided merely as convenience to those skilled in the art and are not an admission that a deposit under 35 U.S.C. Section 112. The sequence of the polynucleotides contained in the deposited materials, as well as the amino acid sequence of the polypeptides encoded thereby, are hereby incompatible, by reference, and are control in the case of any conflict with any description of the sequences in the present. A license may be required to develop, use or sell the deposited materials, and this license is not granted.

KGF-2 Polypeptides and Fragments The present invention further relates to a polypeptide having the deduced amino acid sequence of Figure 1 (SEQ ID NO 2), or which has the amino acid sequence encoded by the cDNA, deposited as well as fragments, analogs and derivatives of such a polypeptide. As a person skilled in the art would appreciate, due to the possibilities of the sequencing errors discussed above, as well as the variability of the cleavage sites for the guides in different known proteins, the effective KGF-2 polypeptide, encoded by the deposited cDNA comprises approximately 208 amino acids, but can be anywhere in the range of 200-220 amino acids; the effective leader sequence of this protein is about 35 or 36 amino acids, but it can be anywhere in the range of about 30 to about 40 amino acids. The term "fragment", "derivative" and "analogue" when referring to the polypeptide of Figure 1 (SEQ ID NO 2) or that encoded by the deposited cDNA, means a polypeptide that retains essentially the same biological function or activity than such a polypeptide. Thus, an analog includes a proprotein that can be activated by cleavage of the proprotein portion to produce an active mature polypeptide. The polypeptide of the present invention can be a recombinant polypeptide, a natural polypeptide or a synthetic polypeptide, preferably a recombinant polypeptide. The fragment, derivative or analog of the polypeptide of Figure 1 (SEQ ID NO 2) or that encoded by the deposited cDNA can be (i) one in which one or more of the amino acid residues are substituted with an amino acid residue conserved or non-conserved (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid residues includes a substituent group, or (iii) one in which the mature polypeptide is fused to another compound, such as a compound to increase the half-life of the polypeptide (eg, polyethylene glycol), or (iv) one in which additional amino acids are fused to the polypeptide mature, such as a guiding or secretory sequence or a sequence that is employed for the purification of the mature polypeptide or a proprotein sequence. Such fragments, derivatives and the like are considered within the scope of those skilled in the art from the teachings herein. The terms "peptide" and "oligopeptide" are considered synonymous (as is commonly recognized) and each term can be used interchangeably as required by the context to indicate a chain of at least two amino acids coupled by peptidyl bonds. The word "polypeptide" is used herein for chains containing more than ten amino acid residues. All oligopeptide and polypeptide formulas or sequences herein are written from left to right and in the direction from the amino terminus to the carboxyl terminus. It will be recognized in the art that some amino acid sequences of the KGF-2 polypeptide can be varied without significant effect of the structure or function of the protein. If such differences in the sequence are contemplated, it must be remembered that there will be critical areas on the protein which determine the activity. In general, it is possible to replace the waste that forms the tertiary structure, with the condition that waste that performs a similar function is used. In other cases, the type of residue may be completely trivial if the alteration occurs in a non-critical region of the protein. Thus, the invention further includes variations of the KGF-2 polypeptide that show substantial activity of the KGF-2 polypeptide or that include regions of the KGF-2 protein such as the protein portions discussed below. Such mutants include deletions, insertions, inversions, repeats, and substitutions of type (for example, the substitution of a hydrophilic residue for another, but not strongly hydrophilic for strongly hydrophobic as a rule). Small changes or such substitutions of "neutral" amino acids will generally have little effect on activity. Typically, observed as conservative substitutions are the replacements, one for another, between the aliphatic amino acids Ala, Val, Leu e lie; the exchange of the hydroxyl residues Ser and Thr, the exchange of the acid residues Asp and Glu, the substitution between the amide residues Asn and Gln, the exchange of the basic residues Lys and Arg and the replacements between the aromatic residues Phe and Tyr . As indicated in detail above, additional guidance concerning which amino acid changes are likely to be phenotypically silent (eg, not likely to have a significant deleterious effect on a function) can be found in Bowie, J.U. and collaborators, "Deciphering the Message in Protein Sequences: Tolerance to Amino Acid Substitutions," Sci en 247: 1306-1310 (1990). The present invention includes the mimetic peptides of KGF-2 that can be used as therapeutic peptides. The mimetic KGF-2 peptides are short peptides that mimic the biological activity of the KGF-2 protein by binding to and activating the KGF-2 cognate receptors. The KGF-2 mimetic peptides can also bind to and inhibit the cognate KGF-2 receptors. KGF-2 receptors include, but are not limited to, FGFR2iiib and FGRFliiib. Such mimetic peptides are obtained from methods such as, but not limited to, phage display or combinatorial chemistry. For example, the method described by Wrighton et al., Science 273: 458-463 (1996) can be used to generate the mimetic peptides of KGF-2. The polypeptides and polynucleotides of the present invention are preferably provided in an isolated form and are preferably purified to homogeneity. The polypeptides of the present invention are preferably in an isolated form. By "isolated polypeptide" is meant a polypeptide removed from its native environment. Thus, a polypeptide produced and / or contained within a recombinant host cell, is considered isolated for purposes of the present invention. Polypeptides that have been purified, either partially or substantially, from a recombinant host cell or from a native source are also considered. The polypeptides of the present invention include the polypeptide of SEQ ID NO. 2 (in particular the mature polypeptide) as well as polypeptides having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98% or 99% similarity (more preferably at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98% or 99% identity) to the polypeptide of SEQ ID NO. 2 and also include portions of such polypeptides with such a portion of the polypeptide (such as the deletion mutants described below) generally containing at least 30 amino acids and more preferably at least 50 amino acids. As is known in the art, "similarity" between two polypeptides is determined by comparing the amino acid sequence and its amino acid substitutes conserved from a polypeptide to the sequence of a second polypeptide. By "% similarity" for two polypeptides, we mean a similarity score produced by comparing the amino acid sequences of the two polypeptides using the Bestfit program (Sequence Analysis Package, Wisconsin, Version 8 for Unix, Genetics Computer Group , University Research Park, 575 Science Drive, Madison, Wl 53711) and the default settings to determine similarity. Bestfit uses the local homology algorithm of Smith and Waterman (Advances in Applied Mathematics 2: 482-489, 1981) to find the best segment of similarity between two sequences. For a polypeptide having at least one amino acid sequence, for example, 95% "identical" to an amino acid sequence preferably of a KGF-2 polypeptide, the amino acid sequence of the polypeptide is intended to be identical to the reference sequence except that the polypeptide sequence can include up to five amino acid alterations per 100 amino acids of the reference amino acid of the KGF-2 polypeptide. In other words, To obtain a polypeptide having an amino acid sequence at least 95% identical to an amino acid sequence reference, up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 5% of the total amino acid residues in the reference sequence can be inserted into the reference sequence. These alterations of the reference sequence may occur at the amino- or carboxyl-terminal positions of the reference amino acid sequence or at any site between those terminal positions, interspersed either individually between the residues in the reference sequence or in one or more contiguous groups within the reference sequence. As a practical matter, if any particular polypeptide is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98% or 99% identical to, for example, the amino acid sequence shown in Figure 1, [SEQ ID NO. 2] or to the amino acid sequence encoded by the deposited cDNA clone, can be determined conventionally using known computer programs such as the Bestfit program (Sequence Analysis Package, Wisconsin, Version 8 for Unix, Genetics Computer Group, University Research Park , 575 Science Drive, Madison, Wl 53711). When Bestfit or any other sequence alignment program is used to determine if a particular sequence is, for example 95% identical to, a reference sequence according to the present invention, the parameters are set, of course, such that the percentage of Identity is calculated on the full length of the reference amino acid sequence and that empty spaces in homology of up to 5% of the total number of amino acid residues in the reference sequence are allowed. A preferred method for determining the best complete match between a search sequence (a sequence of the present invention) and an objective sequence, also referred to as a global sequential alignment, can be determined using the FASTDB computer program based on the Brutlag algorithm. and collaborators (Comp. App. Biosci. (1990) 6: 237-245). In a sequential alignment, the search and target sequences are either both nucleotide sequences or both amino acid sequences. The result of this global sequential alignment is in percentage identity. Preferred parameters used in an amino acid alignment FASTDB are: Matrix = PAM 0, k-tuple = 2, Penalty bad coupling = l, Penalty Union = 20, Length Randomization Group = 0, Rating Court = l, Window Size = length of sequence, Penalty for Empty Space = 5, Penalty for Empty Space Size = 0.05, Window Size = 500 or the length of the target amino acid sequence, whichever is shorter. If the target sequence is shorter than the search sequence due to N- or C-terminal deletions, not due to internal deletions, a manual correction to the results must be made. This is because the FASTDB program does not explain the N- and C-terminal truncations of the target sequence when calculating the overall percent identity. For objective sequences truncated at the N- and C- ends, relative to the search sequences, the percentage identity is corrected by calculating the number of residues of the search sequence that are N- and C-terminal of the target sequence , which are not coupled / aligned with a corresponding target residue, as a percentage of the total bases of the search sequence. If a residue is coupled / aligned this is determined by the results of the FASTDB sequential alignment. This percentage is then subtracted from the percentage identity, calculated by the previous FASTDB program, using the specified parameters, to arrive at a final percentage identity grade. This final percentage identity grade is that used for purposes of the present invention. Only the residues of the N- and C-termini of the target sequence, which are not coupled / aligned with the search sequence, are considered for purposes of manually adjusting the percentage identity rating. That is, only the positions of the search residue outside the N- and C-terminal residues furthest from the target sequence. For example, a target sequence of 90 amino acid residues is aligned with a search sequence of 100 residues to determine percent identity. Deletion occurs at the N-terminus of the target sequence and therefore, the FASTDB alignment does not show a coupling / alignment of the first 10 residues at the N-terminus. The 10 unpaired residues represent 10% of the sequence (number of residues at the non-coupled N and C ends / total number of residues in the search sequence) so that 10% is subtracted from the percentage identity rating calculated by the FASTDB program. If the remaining 90 residues were perfectly coupled, the final percentage identity would be 90%. In yet another example, an objective sequence of 90 residues is compared to a search sequence of 100 residues. This time the deletions are internal deletions so that there are no residues at the N or C ends of the target sequence that are not coupled / aligned with the search. In this case, the percentage identity calculated by FASTDB is not manually corrected. Again, only the residue positions outside the N- and C-terminal ends of the target sequence, as visually shown in the FASTDB alignment, which are not coupled / aligned with the search sequence are manually corrected. No other manual corrections are made for purposes of the present invention. As described in detail below, the polypeptides of the present invention can be used to give rise to polyclonal and monoclonal antibodies, which are useful in diagnostic assays for detecting the expression of KGF-2 protein as described below. or as agonists and antagonists capable of increasing or inhibiting the function of the KGF-2 protein. In addition, such polypeptides can be used in the yeast two-hybrid system to "capture" the KGF-2 protein that binds to proteins that are also candidate agonists and antagonists according to the present invention. The yeast two-hybrid system is described in Fields and Song, Nature 340: 245-246 (1989). In still another aspect, the invention provides a peptide or polypeptide comprising a portion that possesses epitope of a polypeptide of the invention. The epitope of this polypeptide portion is an immunogenic or antigenic epitope of a polypeptide of the invention. An "immunogenic epitope" is defined as a part of a protein that promotes an antibody response when the entire protein is the immunogen. It is believed that these immunogenic epitopes are to be confined to a few loci on the molecule. On the other hand, a region of a protein molecule to which an antibody can be linked is defined as an "antigenic epitope". The number of immunogenic epitopes of a protein is generally less than the number of antigenic epitopes. See for example, Geysen et al., Proc. Nati Acad. Sci. USA 81: 3998-4002 (1983). For the selection of peptides or polypeptides possessing an antigenic epitope (eg, containing a region of a protein molecule to which an antibody can be linked) it is well known in the art that relatively short synthetic peptides that mimic part of A protein sequence is routinely capable of producing an antiserum that reacts with the partially imitated protein. See, for example, Sutcliffe, J.G., Shinnick, T.M., Green, N and Learner, R.A. (1983) Antibodies that react with predetermined sites on proteins. Sciences 219: 660-666. Peptides capable of producing serums reactive to the protein are frequently represented in the primary sequence of a protein, they can be characterized by a group of simple chemical rules, and they are not confined either to the immunodominant regions of the intact proteins (for example, immunogenic epitopes) or to the amino or carboxyl termini. Peptides that are extremely hydrophobic and those with six or less residues are generally ineffective in inducing antibodies that bind to the mimicked protein; Soluble peptides larger, especially those that contain protein residues are usually effective. Sutcliffe et al., Supra, 661. For example, 18 of 20 peptides designated according to these guidelines, containing 8-39 residues that cover 75% of the HA1 polypeptide chain sequence of the influenza virus hemagglutinin, induced antibodies that reacted with the HAI protein or with the intact virus; and 12/12 peptides from the MuLV polymerase and 18/18 from the antibodies induced by the rabies glycoprotein that precipitated the respective proteins.

The peptides and polypeptides possessing the antigenic epitope of the invention are therefore useful for producing antibodies, including monoclonal antibodies, which specifically bind to a polypeptide of the invention. Thus, a high proportion of hybridomas obtained by fusion of spleen cells from donors immunized with a peptide having an antigenic epitope, generally secrete the antibody reactive with the native protein. Sutcliffe et al., Supra, 663. Antibodies produced by peptides or polypeptides possessing an antigenic epitope are useful for detecting the mimicked or imitated protein, and antibodies for different peptides can be used to track the fate of the various regions of a precursor. of protein that undergoes post-translational processing. Peptides and anti-peptide antibodies can be used in a variety of qualitative or quantitative assays for the mimicked protein, for example, in competition assays since it has been shown that even short peptides (eg, about 9 amino acids) can be linked and displace the larger peptides and the immunoprecipitation assays. See for example, Wilson et al., Cell 37: 767-778 (1984) at 777. The anti-peptide antibodies of the invention are also useful for the purification of the mimicked protein, for example, by adsorption chromatography using well-known methods. in the technique. Peptides and polypeptides possessing the antigenic epitope of the invention, designed according to the above guidelines preferably contain a sequence of at least seven, preferably at least nine, and most preferably between about 15 to about 30 amino acids contained within the sequence of amino acids of a polypeptide of the invention. However, peptides or polypeptides comprising a larger portion of an amino acid sequence of a polypeptide of the invention, containing about 30, 40, 50, 60, 70, 80, 90, 100 or 150 amino acids, or any length up to and including the complete amino acid sequence of a polypeptide of the invention, are also considered epitope-possessing peptides or polypeptides of the invention, and are also useful for inducing antibodies that react with the mimicked protein. Preferably, the amino acid sequence of the epitope-possessing peptide is selected to provide substantial solubility in aqueous solvents (for example the sequence includes relatively hydrophilic residues, and highly hydrophobic sequences are preferably avoided); and sequences containing proline residues are particularly preferred.

Non-limiting examples of antigenic polypeptides or peptides that can be used to generate KGF-2 antibodies include the following: 1. Gly41-Asn71: GQDMVSPEATNSSSSSFSSPSSAGRHVRSYN [SEQ ID NO. 25]; 2. Lys91-Serl09: KIEKNGKVSGTKKENCPYS [SEQ ID NO. 26]; 3. Asnl35-Tyrl64: NKKGKLYGSKEFNNDCKLKERIEENGYNTY [SEQ ID NO. 27]; and 4. Asnl81-Alal99: NGKGAPRRGQKTRRKNTSA [SEQ ID NO. 28] Also, there are two additional, shorter, predicted antigenic areas, Gln74-Arg78 of Figure 1 (SEQ ID NO.2) and Glnl70-Glnl75 of Figure 1 (SEQ ID NO.2). The epitope-possessing peptides and polypeptides of the invention can be produced by any conventional means for the preparation of peptides or polypeptides, including the recombinant media using nucleic acid molecules of the invention. For example, a short, amino acid sequence possessing an epitope can be fused to a larger polypeptide that acts as a carrier during recombinant production and purification, as well as during immunization to produce anti-peptide antibodies. The epitope-possessing peptides can also be synthesized using known methods of chemical synthesis. For example, Houghten has described a simple method for synthesizing large numbers of peptides, such as 10-20 mg of 248 different 13 residue peptides representing simple amino acid variants of a HA1 polypeptide segment that were prepared and characterized (by linkage studies). ELISA type) in less than four weeks. Houghten, R.A. (1985) General method for the rapid solid-phase synthesis of large numbers of peptides: specificity of antigen-antibody interaction at the level of individual amino acids. Proc. Nati Acad. Sci. USA 82: 5131-5135. This process of "Simultaneous Synthesis of Multiple Peptides (SMPS)" is further described in U.S. Patent No. 4,631,211 to Houghten et al. (1986). In this process the individual resins for the solid phase synthesis of various peptides are contained in separate solvent permeable packages, which make possible the optimal use of the many identical repetitive steps involved in the solid phase methods. A completely manual procedure allows 500-1000 or more synthesis to be conducted simultaneously. Houghten et al., Supra, 5134. The present invention encompasses polypeptides comprising, or alternatively consisting of, an epitope of the polypeptide having an amino acid sequence of SEQ ID NO. 2, or an epitope of the polypeptide sequence encoded by a polynucleotide sequence contained in the ATCC Deposit No. 75977 or encoded by a polynucleotide that hybridizes to the complement of the sequence of SEQ ID NO. l or contained in the ATCC Deposit No. 75977 under stringent hybridization conditions or low stringency hybridization conditions as defined above. The present invention further encompasses the polynucleotide sequences that code for an epitope of a polypeptide sequence of the invention (such as, for example, the sequence described in SEQ ID NO: 1) the polynucleotide sequences of the complementary strand of a polynucleotide sequence that encodes for an epitope of the invention, and polynucleotide sequences that hybridize to the complementary strand under stringent hybridization conditions or low stringency hybridization conditions defined above. The term "epitopes" as used herein, refers to portions of a polypeptide that have antigenic or immunogenic activity in an animal, preferably a mammal, and more preferably in a human. In a preferred embodiment, the present invention encompasses a polypeptide comprising an epitope, as well as the polynucleotide encoding this polypeptide. An "immunogenic epitope", as used herein, is defined as a portion of a protein that promotes an antibody response in an animal, as determined by any method known in the art, for example, by methods for generation of antibodies described later. (See, for example, Geysen et al., Proc. Nati, Acad. Sci. USA 81: 3998-4002 (1983)). The term "antigenic epitope", as used herein, is defined as a portion of a protein to which an antibody can immunospecifically bind to its antigen, as determined by any method well known in the art, for example, by the immunoassays described herein. Immunospecific binding excludes non-specific binding but does not necessarily exclude cross-reactivity with other antigens. Antigenic epitopes do not necessarily need to be immunogenic. Fragments that function as epitopes can be produced by any conventional means. (See, for example, Houghten, Proc. Nati, Acad. Sci. USA 82: 5131-5135 (1985), further described in U.S. Patent No. 4,631,211). In the present invention, the antigenic epitopes preferably contain a sequence of at least 4at least 5, at least 6, at least 7, more preferably at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20 , at least 25, at least 30, at least 40, at least 50, and, more preferably, from about 15 to about 30 amino acids.

Preferred polypeptides comprising immunogenic or antigenic epitopes are at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 Amino acid residues in length. Further preferred antigenic epitopes comprise, or alternatively consist of, the amino acid sequence of the residues: M-1 to H-15; W-2 to L-16; K-3 to P-17; W-4 to G-18; 1-5 to C-19; L-6 to C-20; T-7 to C-21; H-8 to C-22; C-9 to C-23; A-10 to F-24; S-ll to L-25; A-12 to L-26; F-13 to L-27; P-14 to F-28; H-15 to L-29; L-16 to V-30; P-17 to S-31; G-18 to S-32; C-19 to V-33; C-20 to P-34; C-21 to V-35; C-22 to T-36; C-23 to C-37; F-24 to Q-38; L-25 to A-39; L-26 to L-40; L-27 to G-41; F-28 to Q-42; L-29 to D-43; V-30 to M-44; S-31 to V-45; S-32 to S-46; V-33 to P-47; P-34 to E-48; V-35 to A-49; T-36 to T-50; C-37 to N-51; Q-38 to S-52; A-39 to S-53; L-40 to S-54; G-41 to S-55; Q-42 to S-56; D-43 to F-57; M-44 to S-58; V-45 to S-59; S-46 to P-60; P-47 to S-61; E-48 to S-62; A-49 to A-63; T-50 to G-64; N-51 to R-65; S-52 to H-66; S-53 to V-67; S-54 to R-68; S-55 to S-69; S-56 to Y-70; F-57 to N-71; S-58 to H-72; S-59 to L-73; P-60 to Q-74; S-61 to G-75; S-62 to D-76; A-63 to V-77; G-64 to R-78; R-65 to W-79; H-66 to R-80; V-67 to K-81; R-68 to L-82; S-69 to F-83; Y-70 to S-84; N-71 to F-85; H-72 to T-86; L-73 to K-87; Q-74 to Y-88; G-75 to F-89; D-76 to L-90; V-77 to K-91; R-78 to 1-92; W-79 to E-93; R-80 to K-94; K-81 to N-95; L-82 to G-96; F-83 to K-97; S-84 to V-98; F-85 to S-99; T-86 to G-100; K-87 to T-101; Y-88 to K-102; F-89 to K -103; L-90 to E-104; K-91 to N-105; 1-92 to C-106; E- • 93 a P-107; K-94 to Y-108; N-95 to S-109; G-96 to 1-110; K-97 to L- 111 V -98 to E-112; S-99 to 1-113; G-100 to T-114; T-101 to S-115 K -102 to V-116 K - 103 to E - 117 E - 104 to I - 118 N-105 to G- 119 • C -106 to V-120 P-- 107 to V - 121 Y - 108 to A - 122 S - 109 to V - 123 • I -110 to K - 124 L - 111 to A - 125 E - 112 to I - 126 1- 113 to N- 127 • T -114 to S-128 S - 115 to N - 129 V - 116 to Y - 130 E-117 to Y- 131 • I -118 to L-132 G - 119 a A - 133 V - 120 to M - 134 V - 121 to N - 135 • A • 122 to K - 136 V - 123 to K - 137 K - 124 to G - 138 A - 125 to K- 139 I -126 to L-140 N - 127 to Y - 141 s - 128 to G - 142 N - 129 to S - 143 and -130 to K - 144 Y - 131 to E - -145 L - 132 to F - 146 A-133 to N- 147 M • 134 to N-148 N - 135 to D - 149 K - 136 to C - 150 K-137 to K- 151 G -138 to L-152 K - 139 to K - 153 L - 140 to E - 154 Y-141 to R- 155 G- -142 to 1-156 S - 143 to E - 157 K --144 to E - 158 E-145 to N- 159 F- -146 to G-160 N - 147 to Y - 161 N - 148 to N - 162 D - 149 to T - 163 C - 150 to Y-164 K - 151 to A - 165 L - 152 to S - 166 K-153 to F- 167 E -154 to N-168 R - 155 to W - 169 I - 156 a Q - 170 E-157 to H- 171 E -158 to N-172 N - 159 to G - 173 G - 160 to R - 174 Y-161 to Q- 175 N 162 to M-176 T - 163 to Y - 177 Y - 164 to V - 178 A-165 to A- 179 S 166 to L-180 F - 167 to N- • 181 N - 168 to G - 182 W-169 to K- 183, Q • 170 to G-184 H - 171 to A - 185 N - 172 to P - 186 G-173 to R-187, R-174 to R-188 Q - 175 to G - 189 M-- 176 to Q - 190 Y-177 to K- 191 V- 178 to T-192 A - 179 to R- 193 L- 180 to R - 194 N-181 to K- 195 G- • 182 to N- 196 K- 183 to T- 197 G- 184 to S - 198 A-185 to A- 199 P- 186 to H-200 R- 187 to F- 201 R- 188 to L - 202 G-189 to P -203; Q-190 to M-204, K-191 to V-205; T-192 to V-206, R-193 to H-207; and / or R-194 to S-208 of SEQ ID NO. 2. The polynucleotides encoding these polypeptide fragments are also encompassed by the invention. Additional, non-exclusive, preferred antigenic epitopes include the antigenic epitopes described herein, as well as portions thereof. Antigenic epitopes are useful, for example, to produce antibodies, including monoclonal antibodies, which specifically bind to the epitope. Preferred antigenic epitopes include the antigenic epitopes described herein, as well as any combination of two, three, four, five, or more of these antigenic epitopes. Antigenic epitopes can be used as the target molecules in immunoassays. (See, for example, Wilson et al., Cell 37: 767-778 (1984); Sutcliffe et al., Science 219: 660-666 (1983)). similarly, immunogenic epitopes can be used, for example, to induce antibodies according to methods well known in the art (See, for example, Sutcliffe et al., supra).; Wilson et al., Supra; Chow et al., Proc. Nati Sci. USA 82: 910-914; and Bittle et al., J. Gen. Virol. 66: 2347-2354 (1985). Preferred immunogenic epitopes include the immunogenic epitopes described herein, as well as any combination of two, three, four, five or more of these immunogenic epitopes. Polypeptides comprising one or more immunogenic epitopes can be presented to promote an antibody response together with a carrier protein, such as an albumin, to an animal system (such as rabbit or mouse), or if the polypeptide is of sufficient length ( at least about 25 amino acids), the polypeptide can be presented without a carrier. However, immunogenic epitopes comprising as few as 8 to 10 amino acids have been shown to be sufficient to produce antibodies capable of binding at least linear epitopes to a denatured polypeptide (eg, Western blot). The epitope-possessing peptides and polypeptides of the invention are used to induce antibodies according to methods well known in the art. See for example, Sutcliffe et al., Supra; Wilson et al., Supra; Chow, M. et al., Proc. Nati Acad. Sci. USA 82: 910-914; and Bittle, F.J. and collaborators, J. Gen. Virol. 66: 2347-2354 (1985). In general, animals can be immunized with a free peptide, however, the anti-peptide antibody titer can be enhanced by coupling the peptide to a macromolecular carrier, such as lapa hemacyanin (KLH) or tetanus toxoid. For example, peptides containing cysteine can be coupled to the carrier using a linker such as the ester of m-maleidobenzoyl-N-hydroxysuccinimide (MBS), while other peptides can be coupled to the carrier using a more general binding agent such as glutaraldehyde Animals such as rabbits, rats and mice are immunized, either with the free peptides coupled to the carrier, for example, by intraperitoneal and / or intradermal injection of the emulsions containing approximately 100 μg of peptide or protein carrier and Freund's adjuvant. Several reinforcing injections may be necessary, for example, at intervals of about two weeks, to provide a useful titer of the anti-peptide antibody that can be detected, for example, by ELISA assay using the free peptide adsorbed to a solid surface. The titre of the anti-peptide antibodies in the serum from an immunized animal can be increased by the selection of anti-peptide antibodies, for example, by adsorption to the peptide on a solid support, and the elution of the selected antibodies according to to methods well known in the art. The immunogenic, epitope-possessing peptides of the invention, for example, those portions of a protein that promote an antibody response when the entire protein is the immunogen, are identified according to methods known in the art. For example, Geysen et al., Supra, describes a method for the rapid concurrent synthesis on solid supports of hundreds of peptides of sufficient purity to react in an enzyme-linked immunosorbent assay. The interaction of the peptides synthesized with the antibodies is then easily detected without removing them from the support. In this manner, a peptide that possesses an immunogenic epitope of a desired protein can be routinely identified by a person of ordinary skill in the art. For example, the immunologically important epitope in the coat protein of foot and mouth disease virus was localized by Geysen et al., With a resolution of seven amino acids by synthesis of an overlapping group of all 208 possible hexapeptides that cover the sequence Complete of 213 amino acids of the protein. Then, a complete peptide replacement group in which all 20 amino acids were substituted in turn at each position within the epitope, were synthesized, and the particular amino acids that confer specificity for the reaction with the antibody were determined. Thus, peptide analogs of the epitope-possessing peptides of the invention can be routinely made by this method. U.S. Patent No. 4,708,781 to Geysen (1987) further discloses this method of identifying a peptide that possesses an immunogenic epitope of a desired protein. Still further, U.S. Patent No. 5,194,392 to Geysen (1990) discloses a general method for detecting or determining the sequence of monomers (amino acids or other compounds) that is a topological equivalent of the epitope (e.g., a "mimotope"). ) which is complementary to a particular paratope (antigen binding site) of an antibody of interest. More generally, U.S. Patent No. 4,433,092 to Geysen (1989) discloses a method for detecting or determining a monomer sequence that is a topographic equivalent of a ligand that is complementary to the ligand binding site of a particular receptor. of interest. Similarly, U.S. Patent No. 5,480,971 to Houghten, R.A. et al. (1996) on Peralkylated Oligopeptide Mixtures, describes the peralkylated oligopeptides with linear alkyl of 1 to 7 carbon atoms and the groups and libraries of such peptides, as well as the methods for using such oligopeptide groups and the libraries for determining the sequence of a peralkylated oligopeptide that is preferably linked to an acceptor molecule of interest. Thus, non-peptide analogues of the peptides possessing the epitope of the invention can also be routinely made by these methods. As will be appreciated by a person skilled in the art, the KGF-2 polypeptides of the present invention and the epitope-possessing fragments thereof described above, can be combined with portions of the constant domain of immunoglobulins (IgG), resulting in chimeric polypeptides. . These fusion proteins facilitate purification and show an increased half-life in vivo. This has been shown, for example, for chimeric proteins consisting of the first two domains of the human CD4 polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins (EPA 394,827; Traunecker et al., Nature 331: 84-86 (1988)). The fusion proteins that have a dimeric structure linked by disulfide bridges due to the IgG part, can also be more efficient in the binding and neutralization of other molecules than the monomeric KGF-2 protein or the protein fragment alone (Fountoulakis et al. , J. Biochem 270: 3958-3964 (1995)). In accordance with the present invention, the novel KGF-2 variants are also described. These can be produced by the deletion or substitution of one or more amino acids of KGF-2. Natural mutations are called allelic variations. The allelic variations can be silent (no change in the encoded polypeptide) or they can have altered amino acid sequence. In order to try to improve or alter the characteristics of the native KGF-2 protein, manipulation by genetic engineering can be employed. The recombinant RNA technology known to those skilled in the art can be used to create new polypeptides. Muteins and deletions may show, for example, increased activity or increased stability. In addition, these could be purified with higher yield and show better solubility at least under certain purification and storage conditions. The examples of mutations that can be constructed are described below. The KGF-2 polypeptides of the invention may be in monomers or in multimers (eg, dimers, trimers, tetramers, and higher multimers). Accordingly, the present invention relates to the monomers and multimers of the KGF-2 polypeptides of the invention, their preparation, and (preferably therapeutic) compositions containing them. In specific embodiments, the polypeptides of the invention are monomers, dimers, trimers or tetramers. In further embodiments, the multimers of the invention are at least dimers, at least trimers, or at least tetramers. The multimers encompassed by the invention can be homomers or heteromers. As used herein, the term "homomer," refers to a multimer containing only polypeptides corresponding to the amino acid sequence of SEQ ID NO. 2 or encoded by the cDNA contained in the deposited clone (including fragments, variants, splice variants, and fusion proteins, corresponding to these as described herein). These homomers may contain the KGF-2 polypeptides having identical or different amino acid sequences. In a specific embodiment, a homomer of the invention is a multimer containing only KGF-2 polypeptides having an identical amino acid sequence. In another specific embodiment, a homomer of the invention is a multimer containing the KGF-2 polypeptides having different amino acid sequences. In specific embodiments, the multimer of the invention is a homodimer (e.g., containing KGF-2 polypeptides having identical or different amino acid sequences) or a homotrimer (e.g., containing the KGF-2 polypeptides having amino acid sequences) identical and / or different). In further embodiments, the homomeric multimer of the invention is at least one homodimer, at least one homotrimer, or at least one homotetramer. As used herein, the term "heteromer" refers to a multimer containing one or more heterologous polypeptides (e.g., polypeptides of different proteins) in addition to the KGF-2 polypeptides of the invention. In a specific embodiment, the multimer of the invention is a heterodimer, a heterotrimer, or a heterotetramere. In additional embodiments, the heteromeric multimer of the invention is at least one heterodimer, at least one heterotrimer, or at least one heterotetramer. The multimers of the invention can be the result of hydrophobic, hydrophilic, ionic and / or covalent associations, and / or can be indirectly linked, for example, by liposome formation. Thus, in one embodiment, the multimers of the invention such as, for example, the homodimers or homotrimers, are formed when the polypeptides of the invention make contact with one another in solution. In yet another embodiment, the heteromultimers of the invention, such as, for example, the heterotrimers or heterotetramers, are formed when the polypeptides of the invention make contact with the antibodies to the polypeptides of the invention (including the antibodies to the heterologous polypeptide sequence). in a fusion protein of the invention) in solution. In other embodiments, the multimers of the invention are formed by covalent associations with and / or between the KGF-2 polypeptides of the invention. Such covalent associations may involve one or more amino acid residues contained in the polypeptide sequence (for example, that indicated in SEQ ID NO.; or contained in the polypeptides encoded by clone HPRCC57 or the clone contained in the ATCC Deposit No. 75977 or 75901). In one case, the covalent associations are the cross-linking between the cysteine residues located within the polypeptide sequences that interact in the nastive polypeptide (e.g., of natural origin). In another case, covalent associations are the consequence of chemical or recombinant manipulation. Alternatively, such covalent associations may involve one or more amino acid residues contained in the heterologous polypeptide sequence in a KGF-2 fusion protein. In one example, the covalent associations are between the heterologous sequence contained in a fusion protein of the invention (see for example, U.S. Patent No. 5,478,925). In a specific example, the covalent associations are between the heterologous sequence contained in a KGF-2-Fc fusion protein of the invention (as described herein). In another specific example, the covalent associations of the fusion proteins of the invention are between the heterologous polypeptide sequence from another protein that is capable of forming covalently associated multimers, such as for example, osteoprotegerin (see for example, International Publication No WO 98/49305, the content of which is hereby incorporated by reference in its entirety). In yet another embodiment, two or more polypeptides of the invention are linked through peptide bonds. Examples include those peptide linkers described in U.S. Patent No. 5,073,627 (incorporated by reference herein). The proteins comprising the multiple polypeptides of the invention separated by peptide linkers can be produced using conventional recombinant DNA technology. Yet another method for the preparation of the multimeric polypeptides of the invention involves the use of polypeptides of the invention fused to a polypeptide zipper sequence of leucine or zipper isoleucine. The zipper and leucine zipper domains of isoleucine are polypeptides that promote multimerization of the proteins in which they are found. Leucine zippers were originally identified in several proteins that bind to DNA (Landschulz et al., Science 240: 1759, (1988)), and have since been found in a variety of different proteins. Among the known leucine zippers are peptides of natural origin and derivatives thereof which are dimerized or trimerized. Examples of leucine zipper domains, suitable for producing soluble multimeric proteins of the invention are those described in PCT Application No. WO 94/10308, incorporated by reference herein. Recombinant fusion proteins comprising a polypeptide of the invention fused to a polypeptide sequence that is dimerized or trimerized in solution, are expressed in suitable host cells, and the resulting soluble multimeric fusion protein is recovered from the culture supernatant using techniques known in the field. The trimeric polypeptides of the invention may offer the advantage of increased biological activity. Preferred portions of leucine zipper and portions and isoleucine are those which preferably form trimers. One example is a leucine zipper derived from the lung surfactant protein D (SPD), as described in Hoppe et al. (FEBS Letters 344: 191, (1994)) and in the United States patent application No. of Series 08 / 446,922, incorporated by reference herein. Other peptides derived from trimeric proteins of natural origin can be used in the preparation of the trimeric polypeptides of the invention.

In yet another example, the proteins of the invention are associated by interactions between the Flag® polypeptide sequence contained in the fusion proteins of the invention containing the Flag® polypeptide sequence. In a further embodiment, the association proteins of the invention are associated by interactions between the heterologous polypeptide sequence contained in the Flag® fusion proteins of the invention, and the anti-Flag® antibody. The multimers of the invention can be generated using chemical techniques known in the art. For example, the desired polypeptides to be contained in the multimers of the invention may be chemically crosslinked using linker molecules and the techniques for optimizing the length of the linker molecule, known in the art (see for example, United States number 5,478,925, which is incorporated herein by reference, in its entirety). In addition, the multimers of the invention can be generated using techniques known in the art to form one or more inter-molecular cross-links between the cysteine residues located within the sequence of the desired polypeptides to be contained in the multimer (see example, U.S. Patent Number 5,478,925, which is incorporated by reference herein, in its entirety). In addition, the polypeptides of the invention can be routinely modified by the addition of cysteine or biotin to the C-terminus or N-terminus of the polypeptide, and techniques known in the art can be applied to generate multimers containing one or more of these modified polypeptides. (see for example, U.S. Patent Number 5,478,925, which is incorporated by reference herein, in its entirety). Additionally, techniques known in the art can be applied to generate liposomes containing the desired polypeptide components, to be contained in the multimer of the invention (see for example, U.S. Patent Number 5,478,925, which is incorporated by reference in the present, in its entirety). Alternatively, the multimers of the invention can be generated using genetic engineering techniques known in the art. In one embodiment, the polypeptides contained in the multimers of the invention are recombinantly produced using fusion protein technology, described herein or otherwise known in the art (see for example, U.S. Patent Number 5,478,925, which is incorporated by reference herein, in its entirety). In a specific embodiment, the polynucleotides encoding a homodimer of the invention are generated by ligation of a polynucleotide sequence encoding a polypeptide of the invention to a sequence encoding a linker polypeptide and then further to a synthetic polynucleotide encoding a the translated product of the polypeptide in the reverse orientation from the original C-terminus to the N-terminus (lacking the leader sequence) (see for example, U.S. Patent Number 5,478,925, which is incorporated by reference herein, in its entirety). In another embodiment, the recombinant techniques described herein or otherwise known herein or other known in the art are applied to generate the recombinant polypeptides of the invention that contain a transmembrane domain (or the hydrophobic or signal peptide) and which can be incorporated by membrane reconstitution techniques into liposomes (see for example, U.S. Patent Number 5,478,925, which is incorporated by reference herein, in its entirety).

Polynucleotide and Polypeptide Fragments The present invention is further directed to the fragments of the isolated nucleic acid molecules described herein. For a fragment of an isolated nucleic acid molecule having, for example, the nucleotide sequence of the deposited cDNA (clone HPRCC57), a nucleotide sequence coding for the polypeptide sequence encoded by the deposited cDNA, a nucleotide sequence coding for the sequence polypeptide described in Figure 1 (SEQ ID NO: 2), the nucleotide sequence shown in Figure 1 (SEQ ID NO: 1), or the strand complementary thereto, is meant to mean the fragments of at least 15 nucleotides, and more preferably at least about 20 nucleotides, still more preferably at least 30 nucleotides, and even more preferably, at least about 40, 50, 100, 150, 200, 250, 300, 325, 350, 375, 400, 450 , 500, 550, or 600 nucleotides in length. These fragments have numerous uses including, but not limited to, diagnostic probes and primers as discussed herein. Of course, larger fragments, such as those of 500-1500 nucleotides in length are also useful according to the present invention as are the fragments corresponding to most, if not all, of the nucleotide sequences of the deposited cDNA ( clone HPRCC57) or as shown in Figure 1 (SEQ ID No. 1). A fragment of at least 20 nucleotides in length, for example, means fragments that include 20 or more contiguous bases, for example, from the nucleotide sequence of the deposited cDNA, or the nucleotide sequence as shown in Figure 1 (SEQ ID No. 1). In addition, representative examples of fragments of the KGF-2 polynucleotide include, for example, fragments having a sequence of about a nucleotide number of 1-50, 51-100, 101-150, 151-200, 201-250. , 251-300, 301-350, 351-400, 401-450, 451-500, 501-550, 551-600, 651-700, 701-750, 751-800, 800-850, 851-900, 901 -950, 951-1000, 1001-1050, 1051-1100, 1101-1150, 1151-1200, 1201-1250, 1251-1300, 1301-1350, 1351-1400, 1401-1450, 1451-1500, 1501-1550 , 1551-1600, 1601-1650, 1651-1700, 1701-1750, 1751-1800, 1801-1850, 1851-1900, 1901-1950, 1951-2000 and / or 2001 until the end of SEQ ID NO. 1 or the complementary strand to it, or the cDNA contained in the deposited clone. In tcontext "approximately" includes the intervals particularly indicated, larger or smaller by several (5, 4, 3, 2 or 1) nucleotides, at either end or at both ends. Preferably, the polynucleotide fragments of the invention code for a polypeptide that demonstrates a functional activity of KGF-2. By a polypeptide demonstrating a "functional activity" of KGF-2 is meant a polypeptide capable of displaying one or more known functional activities associated with a full length (full length) KGF-2 protein. Such functional activities include, but are not limited to, biological activity, antigenicity [ability to bind (or compete with a KGF-2 polypeptide for binding) to an anti-KGF-2 antibody], immunogenicity (ability to generate antibody which binds to a KGF-2 polypeptide), the ability to form multimers with the KGF-2 polypeptides of the invention, and the ability to bind to a receptor or a ligand for a KGF-2 polypeptide. The functional activity of the KGF-2 polypeptides, and the fragments, variants, derivatives and analogues thereof, can be evaluated by various methods. For example, in a modality where the ability to bind to or compete with the full-length KGF-2 polypeptide for binding to the anti-KGF-2 antibody is being evaluated, various immunoassays known in the art, including but not limited to, can be used. a, competitive and non-competitive testing systems using techniques such as radioimmunoassays, ELISA (enzyme-linked immunosorbent assay), "sandwich" immunoassays, immunoradiometric assays, gel diffusion precipitation reactions, immunodiffusion assays, immunoassays in themselves (using colloidal gold, enzymes or radioisotopic markers, for example), staining of Western, precipitation reactions, agglutination assays (e.g., gel agglutination assays, hemagglutination assays), complement fixation assays, immunofluorescence assays, protein A assays, and immunoelectrophoresis assays, etc. In one embodiment, the binding to the antibody is detected by the detection of a marker on the primary antibody. In yet another embodiment, the primary antibody is detected by detecting the binding of a secondary antibody or reagent for the primary antibody. In a further embodiment, the secondary antibody is labeled. Many means are known in the art to detect binding in an immunoassay and are within the scope of the present invention. In yet another embodiment, where a KGF-2 ligand is identified, or the ability of a polypeptide fragment, variant or derivative of the invention to be multimerized is being evaluated, the link can be evaluated, for example, by means well known in the art. , such as, for example, reducing or non-reducing gel chromatography, protein affinity chromatography, and affinity staining or transfer. See in general, Phizicky, E. et al., Microbiol. Rev. 59: 94-123 (1995). In another modality, the physiological correlations of KGF-2 binding to its substrates (signal transduction) can be evaluated.

In addition, the assays described herein (see Examples) and otherwise known in the art can be routinely applied to measure the ability of KGF-2 polypeptides and fragments, variants, derivatives and analogs thereof to promote activity. related to KGF-2 (either in vi tro or in vivo). Other methods will be known to the person skilled in the art and are within the scope of the invention. The present is further directed to the fragments of the KGF-2 polypeptide described herein. For a fragment of an isolated KGF-2 polypeptide, for example, encoded by the deposited cDNA (clone HPRCC57), the polypeptide sequence encoded by the deposited cDNA, the polypeptide sequence described in Figure 1 (SEQ ID NO. it is intended to encompass the polypeptide fragments contained in SEQ ID NO. 2 or encoded by the cDNA contained in the deposited clone. The protein fragments may be "free standing", or comprised within a larger polypeptide, of which the fragment forms a part or region, more preferably as a single contiguous region. Representative examples of the polypeptide fragments of the invention include, for example, fragments of about one amino acid number of 1-20, 21-40, 41-60, 61-80, 81-100, 102-120, 121-140. , 141-160, 161-180, 181-200, 201-220, 221-240, 241-260, 261-280 or 281 until the end of the coding region. In addition, the polypeptide fragments can be at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140 or 150 amino acids in length. In this context "approximately" includes the intervals particularly indicated, larger or smaller by several (5, 4, 3, 2 or 1) amino acids, at either end or at both ends. Even if the deletion of one or more amino acids from the N-terminus of a protein results in the modification of the loss of one or more biological functions of the protein, other functional activities (eg, biological activities, the ability to multimerize, the ability to bind to the KGF-2 ligand). For example, the ability of shortened KGF-2 muteins to induce and / or bind antibodies that recognize full or mature forms of polypeptides in general will be retained when at least the majority of the complete or mature polypeptide residues are removed. from the N-terminus. If a particular polypeptide lacking the N-terminal residues of a complete polypeptide retains such immunological activities, this can be easily determined by routine methods described herein and otherwise known in the art. It is not unlikely that a KGF-2 mutein with a large number of N-terminal amino acid residues can retain some biological or immunogenic activities. In fact, peptides composed of as few as six amino acid residues of KGF-2 can often evoke an immune response. Accordingly, the polypeptide fragments include the secreted KGF-2 protein as well as the mature form. Additional preferred polypeptide fragments include the secreted KGF-2 protein or the mature form having a continuum of deleted residues from the amino terminus to the carboxyl terminus, or both. For example, any number of amino acids in the range of 1 to 60 can be deleted from the amino terminus of the secreted KGF-2 polypeptide or the mature form. Similarly, any number of amino acids, in the range of 1 to 30, can be deleted from the carboxyl terminus of the secreted KGF-2 protein or the mature form. In addition, any combination of the above deletions at the amino terminus and at the carboxyl terminus are preferred. Similarly, polynucleotide fragments encoding these fragments of the KGF-2 polypeptide are also preferred. Particularly, N-terminal deletions of the KGF-2 polypeptide can be described by the general formula m-208, where m is an integer from 2 to 207, where m corresponds to the position of the amino acid residue identified in SEQ ID. No. 2. More particularly, the invention provides the polynucleotides that encode the polypeptides comprising, or consisting alternatively of, the amino acid sequence of residues from W-2 to S-208; K-3 to S-208; W-4 to S-208; 1-5 to S-208; L-6 to S-208; T-7 to S-208; H-8 to S-208; C-9 to S-208; A-10 to S-208; S-ll to S-208; A-12 to S-208; F-13 to S-208; P-14 to S-208; H-15 to S-208; L-16 to S-208; P-17 to S-208; G-18 to S-208; C-19 to S-208; C-20 to S-208; C-21 to S-208; C-22 to S-208; C-23 to S-208; F-24 to S-208; L-25 to S-208; L-26 to S-208; L-27 to S-208; F-28 to S-208; L-29 to S-208; V-30 to S-208; S-31 to S-208; S-32 to S-208; V-33 to S-208; P-34 to S-208; V-35 to S-208; T-36 to S-208; C-37 to S-208; Q-38 to S-208; A-39 to S-208; L-40 to S-208; G-41 to S-208; Q-42 to S-208; D-43 to S-208; M-44 to S-208; V-45 to S-208; S-46 to S-208; P-47 to S-208; E-48 to S-208; A-49 to S-208; T-50 to S-208; N-51 to S-208; S-52 to S-208; S-53 to S-208; S-54 to S-208; S-55 to S-208; S-56 to S-208; F-57 to S-208; S-58 to S-208; S-59 to S-208; P-60 to S-208; S-61 to S-208; S-62 to S-208; A-63 to S-208; G-64 to S-208; R-65 to S-208; H-66 to S-208; V-67 to S-208; R-68 to S-208; S-69 to S-208; Y-70 to S-208; N-71 to S-208; H-72 to S-208; L-73 to S-208; Q-74 to S-208; G-75 to S-208; D-76 to S-208; V-77 to S-208; R-78 to S-208; W-79 to S-208; R-80 to S-208; K-81 to S-208; L-82 to S-208; F-83 to S-208; S-84 to S-208; F-85 to S-208; T-86 to S-208; K-87 to S-208; Y-88 to S-208; F-89 to S-208; L-90 to S-208; K-91 to S-208; 1-92 to S- -208; K-94 to S -208; N-95 to S 208; G- 96 to S-208; V-98 to S-208; S-99 to S-208 G-100 to S-208 T-101 to S-208 K-102 to S-208; K-103 to S-208 E-104 to S-208 N-105 to S-208 C-106 to S-208; P-107 to S-208 Y-108 to S-208 S-109 to S-208 1-110 to S-208; -lll to S-208 E-112 to S-208 I-113 to S-208 T-114 to S-208; S-115 to S-208 V-116 to S-208 E-117 to s-208 1-118 to S-208; G-119 to S-208 V-120 to S-208 V-121 to s-208 A-122 to S-208; V-123 to S-208 K-124 to S-208 A-125 to s-208 1-126 to S-208; N-127 to S-208 S-128 to S-208 N-129 to s-208 Y-130 to S-208; Y-131 to S-208 L-132 to S-208 A-133 to s-208 M-134 to S-208; N-135 to S-208 K-136 to S-208 K-137 to s-208 G-138 to S-208; K-139 to S-208 L-140 to S-208 Y-141 to s-208 G-142 to S-208; S-143 to S-208 K-144 to S-208 E-145 to s-208 F-146 to S-208; N-147 to S-208 N-148 to S-208 D-149 to s-208 C-150 to S-208; K-151 to S-208 L-152 to S-208 K- 153 to s-208 E-154 to S-208; R-155 to S-208 1-156 to S-208 E-157 to s-208 E-158 to S-208; N-159 to S-208 G-160 to S-208 Y-161 to s-208 N-162 to S-208; T-163 to S-208 Y-164 to S-208 A-165 to s-208 S-166 to S-208; F-167 to S-208 N-168 to S-208 W-169 to s-208 Q-170 to S-208; H-171 to S-208 N-172 to S-208 G-173 to s-208 R-174 to S-208; Q-175 to S-208 M-176 to S-208 Y-177 to s-208 V-178 to S-208; A-179 to S-208 L-180 to S-208 N-181 to s-208 G-182 to S-208; K-183 to S-208 G-184 to S-208 A-185 to s-208 P-186 to S-208; R-187 to S-208 R-188 to S-208 G-189 to s-208 Q-190 to S-208; K-191 to S-208 T-192 to S-208 R-193 to S-208; R-194 to S-208; K-195 to S-208; N-196 to S-208; T-197 to S-208; S-198 to S-208; A-199 to S-208; H-200 to S-208; F-201 to S-208; L-202 to S-208; P-203 to S-208; of SEQ ID No. 2. The polynucleotides encoding these polypeptides are also encompassed by the invention. Particularly preferred are fragments comprising or consisting of: S69-S208; A63-S208; Y70-S208; V77-S208; E93-S208; E104-S208; V123-S208; G138-S208; R80-S208; A39-S208; S69-V178; S69-G173; S69-R188; S69-S198; S84-S208; V98-S208; A63-N162; S69-N162; and M35-N162. Also as mentioned above, even if the deletion of one or more amino acids from the C-terminus of a protein results in the modification of the loss of one or more biological functions of the protein, other functional activities (e.g., biological activities, the ability to multimerize, the ability to bind to the KGF-2 ligand) can still be conserved. For example, the ability of the shortened KGF-2 mutein to induce and / or bind to antibodies that recognize the full or mature forms of the polypeptide will generally be retained when at least the majority of the complete or mature polypeptide residues are removed. of the C-terminus. If a particular polypeptide lacking the C-terminal residues of a complete polypeptide retains such immunological activities, this can be easily determined by routine methods described herein and otherwise known in the art. It is not unlikely that a KGF-2 mutein with a large number of deleted C-terminal amino acid residues can retain some biological or immunogenic activities. In fact, peptides composed of as few as six KGF-2 amino acid residues can often evoke an immune response. Accordingly, the present invention further provides polypeptides having one or more residues deleted from the carboxyl terminus of the amino acid sequence of the KGF-2 polypeptide shown in Figure 1.

(SEQ ID No. 2), as described by general formula 1-n, where n is an integer from 2 to 207, where n corresponds to the position of the amino acid residue identified in SEQ ID No. 2. More in particular, the invention provides polynucleotides that encode polypeptides comprising, or consisting alternatively of, the amino acid sequence of residues Ml to H-207; M-1 to V-206; M-1 to V-205; M-1 to M-204; M-1 to P-203; M-1 to L-202; M-1 to F-201; M-1 to H-200; M-l to A-199; M-1 to S-198; M-l to T-197; M-1 to N-196; M-1 to K-195; M-1 to R-194; M-1 to R-193; M-l to T-192; M-1 to K-191; M-1 to Q-190; M-1 to G-189; M-1 to R-188; M-1 to R-187; M-1 to P-186; M-1 to A-185; M-1 to G-184; M-l to K-183; M-1 to G-182; M-1 to N-181; M-1 to L-180; M-1 to A-179; M-1 to V-178; M-l to Y-177; M-1 to M-176; M-1 to Q-175; M-1 to R-174; M-1 to G-173; M-l to N-172; M-1 to H-171; M-1 to Q-170; M-1 to W-169; M-1 to N-168; M-1 to F -167; M-1 to S-166; M-1 to A-165; M-1 to Y- • 164; M-l to T-163; M-1 to N-162; M-l to Y-161; M-1 to G-160; M-1 to N-159; M-1 to E-158; M-1 to E-157; M-1 to 1-156; M-1 to R-155; M-1 to E-154; M-1 to K -153; M-1 to L-152; M-1 to K-151; M-1 to C- • 150; M-1 to D-149; M-l to N-148; M-l to N-147; M-1 to F-146; M-1 to E-145; M-1 to K-144; M-1 to S-143; M-1 to G-142; M-l to Y-141; M-1 to L-140; M-l to K -139; M-1 to G-138; M-1 to K-137; M-1 to K- • 136; M-1 to N-135; M-1 to M-134; M-l to A-133; M-1 to L-132; M-1 to Y-131; M-1 to Y-130; M-1 to N-129; M-1 to S-128; M-1 to N-127; M-1 to I-126; M-1 to A-125; M-1 to K-124; M-1 to V-123; M-1 to A- • 122; M-1 to V-121; M-1 to V-120; M-1 to G-119; M-1 to 1-118; M-1 to E-117; M-1 to V-116; M-1 to S-115; M-1 to T-114; M-1 to 1-113; M-1 to E-112; M-1 to L -111; M-1 to I-110; M-1 to S-109; M-1 to Y- • 108; M-1 to P-107; M-1 to C-106; M-1 to N-105; M-1 to E-104; M-1 to K-103; M-1 to K-102; M-l to T-101; M-1 to G-100; M-1 to S-99; M-1 to V-98; M-1 to K-97 M-1 to G-96 M-1 to N-95 M-1 to K-94; M-1 to E-93; M-1 to 1-92 M-1 to K-91 M-1 to L-90 M-1 to F-89; M-1 to Y-88; M-1 to K-87 M-1 to T-86 M-1 to F-85 M-1 to S-84; M-1 to F-83; M-1 to L-82 M-1 to K-81 M-1 to R-80 M-1 to W-79; M-1 to R-78; M-1 to V-77 M-1 to D-76 M-1 to G-75 M-1 to Q-74; M-1 to L-73; M-1 to H-72 M-1 to N-71 M-1 to Y-70 M-1 to S-69; M-1 to R-68; M-1 to V-67 M-1 to H-66 M-1 to R-65 M-1 to G-64; M-1 to A-63; M-1 to S-62 M-1 to S-61 M-1 to P-60 M-1 to S-59; M-1 to S-58; M-1 to F-57; M-1 to S-56; M-1 to S-55; M-1 to S-54; M-1 to S-53; M-1 to S-52; M-1 to N-51; M-l to T-50; M-l to A-49; M-1 to E-48; M-l to P-47; M-1 to S-46; M-1 to V-45; M-l to M-44; M-l to D-43; M-1 to Q-42; M-1 to G-41; M-1 to L-140; M-l to A-39; M-l to Q-38; M-1 to C-37; M-l to T-36; M-1 to V-35; M-1 to P-34; M-1 to V-33; M-1 to S-32; M-1 to S-31; M-1 to V-30; M-1 to L-29; M-1 to F-28; M-1 to L-27; M-1 to L-26; M-1 to L-25; M-1 to F-24; M-1 to C-23; M-1 to C-22; M-1 to C-21; M-1 to C-20; M-1 to C-19; M-1 to G-18; M-l to P-17; M-1 to L-16; M-1 to H-15; M-1 to P-14; M-1 to F-13; M-1 to A-12; M-1 to S-11; M-l to A-10; M-l to C-9; M-1 to H-8; M-l to T-7; of SEQ ID No. 2. The polynucleotides encoding these polypeptides are also encompassed by the invention. Similarly, the C-terminal deletions of the KGF-2 polypeptide of the invention, shown as SEQ ID No. 2 include the polypeptides comprising the amino acid sequence of the residues: S-69 to H-207; S-69 to V-206; S-69 to V-205; S-69 to M-204; S-69 to P-203; S-69 to L-202; S-69 to F-201; S-69 to H-200; S-69 to A-199; S-69 to S-198; S-69 to T-197; S-69 to N-196; S-69 to K-195; S-69 to R-194; S-69 to R-193; S-69 to T-192; S-69 to K-191; S-69 to Q-190; S-69 to G-189; S-69 to R-188; S-69 to R-187; S-69 to P-186; S-69 to A-185; S-69 to G-184; S-69 to K-183; S-69 to G-182; S-69 to N-181; S-69 to L-180; S-69 to A-179; S-69 to V-178; S-69 to Y-177; S-69 to M-176; S-69 to Q-175; S-69 to R-174; S-69 to G-173; S-69 to N-172; S-69 to H-171; S-69 to Q-170; S-69 to W-169; S-69 to N-168; S-69 to F-167; S-69 to S-166; S-69 to A-165; S-69 to Y-164; S-69 to T-163; S-69 to N-162; S-69 to Y-161; S-69 to G-160; S-69 to N-159; S-69 to E-158; S-69 to E-157; S-69 to 1-156; S-69 to R-155; S-69 to E-154; S-69 to K-153; S-69 to L-152; S-69 to K-151; S-69 to C-150; S-69 to D-149; S-69 to N-148; S-69 to N-147; S-69 to F-146; S-69 to E-145; S-69 to K-144; S-69 to S-143; S-69 to G-142; S-69 to Y-141; S-69 to L-140; S-69 to K-139; S-69 to G-138; S-69 to K-137; S-69 to K-136; S-69 to N-135; S-69 to M-134; S-69 to A-133; S-69 to L-132; S-69 to Y-131; S-69 to Y-130; S-69 to N-129; S-69 to S-128; S-69 to N-127; S-69 to 1-126; S-69 to A-125; S-69 to K-124; S-69 to V-123; S-69 to A-122; S-69 to V-121; S-69 to V-120; S-69 to G-119; S-69 to 1-118; S-69 to E-117; S-69 to V-116; S-69 to S-115; S-69 to T-114; S-69 to 1-113; S-69 to E-112; S-69 to L-111; S-69 to I-110; S-69 to S-109; S-69 to Y-108; S-69 to P-107; S-69 to C-106; S-69 to N-105; S-69 to E-104; S-69 to K-103; S-69 to K-102; S-69 to T-101; S-69 to G-100; S-69 to S-99; S-69 to V-98; S-69 to K-97; S-69 to G-96; S-69 to N-95; S-69 to K-94; S-69 to E-93; S-69 to I-92; S-69 to K-91; S-69 to L-90; S-69 to F-89; S-69 to Y-88; S-69 to K-87; S-69 to T-86; S-69 to F-85; S-69 to S-84; S-69 to F-83; S-69 to L-82; S-69 to K-81; S-69 to R-80; S-69 to W-79; S-69 to R-78; S-69 to V-77; S-69 to D-76; S-69 to G-75; of SEQ ID No. 2. In addition, any of the N- or C-terminal deletions listed above can be combined to produce a deleted KGF-2 polypeptide at the N-terminus and at the C-terminus. The invention also provides the polypeptides that they have one or more amino acids deleted from both ends, amino and carboxyl, which can be described in general as having the residues mn of SEQ ID No. 2, where n and m are integers as described above. In addition, the N- or C-terminal deletion mutants may also contain the site-specific amino acid substitutions. The polynucleotides encoding these polypeptides are also encompassed by the invention. Also included is a nucleotide sequence encoding a polypeptide consisting of a portion of the complete KGF-2 amino acid sequence, encoded by the cDNA clone contained in the ATCC Deposit No. 75977, where this portion excludes any whole number of residues. of amino acids from one to about 198 amino acids from the amino terminus of the complete amino acid sequence encoded by the cDNA clone contained in the ATCC Deposit No. 75977, or any integer number of the amino acid residues from 1 to about 198 amino acids from the carboxyl terminus, or any combination of the above amino-terminal and carboxyl-terminal deletions, of the complete amino acid sequence encoded by the cDNA clone contained in the ATCC Deposit No. 75977. The polynucleotides that encode all forms of the mutant polypeptide of suppression, above, are also provided. The present application is also directed to the proteins containing the polypeptides at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98% or 99% identical to the sequence of the KGF-2 polypeptide described in the present mn. In the preferred embodiments, the request is directed to the proteins containing the polypeptides at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98% or 99% identical to the polypeptides having the amino acid sequence of the specific KGF-2 and C-terminal deletions indicated herein. The polynucleotides encoding these polypeptides are also encompassed by the invention. Among the especially preferred fragments of the invention are fragments characterized by structural or functional attributes of KGF-2. Such fragments include amino acid residues comprising the regions of the alpha helix and alpha helix-forming ("alpha regions"), the beta-sheet and beta-sheet-forming regions ("beta regions"), the regions of return and the spinning machines ("spin regions"), winding and rollforming regions ("winding regions"), hydrophilic regions, hydrophobic regions, antipathetic alpha regions, beta antipatic regions, surface forming regions, and regions of high antigenic index (eg, containing four or more contiguous amino acids that have an antigenic index greater than or equal to 1.5, as identified using the default parameters of the Jameson-Wolf program) of complete KGF-2 (e.g., full-length) (SEQ ID No. 2). Certain preferred regions are those described in Figure 4 and include, but are not limited to, the regions of the aforementioned types identified by analysis of the amino acid sequence described in Figure 1 (SEQ ID No. 2), such preferred regions include; the predicted alpha regions of Garnier-Robson, the beta regions, the return regions, and the winding regions; the predicted alpha regions of Chou-Fasman, the beta regions, the return regions, and the winding regions; the hydrophilic and hydrophobic regions predicted by Kyte-Doolittle; the alpha and beta antiseptic regions of Eisenberg; the surface forming regions of Emini; and regions of high antigenic index of Jameson-Wolf, as predicted using the default parameters of these computer programs. The polynucleotides encoding these polypeptides are also encompassed by the invention. In additional embodiments, the polynucleotides of the invention code for functional attributes of KGF-2.

Preferred embodiments of the invention in this regard include fragments comprising the alpha helix and alpha helix forming regions ("alpha regions"), the beta sheet and beta sheet formation regions ("beta regions"), the regions back and back formation ("back regions"), winding and coiling regions ("winding regions"), hydrophilic regions, hydrophobic regions, alpha antipatic regions, beta antipatic regions, flexible regions, surface forming regions, and regions of high antigenic index of KGF-2. The data representing the structural or functional attributes of KGF-2 described in Figure 1 and / or Table I, as described above, were generated using the various DNA * STAR algorithms and modules set in the default parameters. In a preferred embodiment, the data presented in columns VIII, IX, XIII and XIV of Table I can be used to determine regions of KGF-2 that show a high degree of potential for antigenicity. The regions of high antigenicity are determined from the data presented in columns VIII, IX, XIII, and / or IV by the choice of the values representing regions of the polypeptide that are likely to be exposed on the surface of the polypeptide in a environment in which the recognition of the antigen in the process of initiation of an immune response can occur. Certain preferred regions in this respect are described in Figure 4, but can, as shown in Table I, be represented or identified by the use of tabular representations of the data presented in Figure 4. The DNA * STAR computer algorithm used to generate Figure 4 (described in the original default parameters) was used to present the data in Figure 4 in a tabular format (see Table I). The tabular format of the data in Figure 4 can be used to easily determine the specific limits of a preferred region. The above-mentioned preferred regions described in Figure 4 and Table I include, but are not limited to, regions of the aforementioned types identified by analysis of the amino acid sequence described in Figure 1. As described in Figure 4 and in Table I, such preferred regions include the Garnier-Robson alpha regions, the beta regions, the return regions, and the winding regions, the Chou-Fas an alpha regions, the beta regions, and the regions of winding, the Kyte-Doolittle hydrophilic regions and the hydrophobic regions, the alpha- and beta-antipathetic regions of Eisenberg; the flexible regions of Karplus-Schulz, the surface-forming regions of Emini; and the regions of Jameson-Wolf of high antigenic index. The columns are marked with the headings "Res", "Position", and the Roman numerals I-XIV. The headings of the columns refer to the following characteristics of the amino acid sequence presented in Figure 3, and Table I: "Res": amino acid residue of SEQ ID No. 2 and Figures IA and IB; "Position": position of the corresponding residue within SEQ ID No. 2 and Figures IA and IB; I: Alpha regions - Garnier-Robson; II: Alpha regions - Chou Fasman, III: Alpha regions - Garnier-Robson; IV: Beta regions - Chou-Fasman; V: Regions of return - Garnier-Robson; VI: Regions of return - Chou-Fasman; VII: Winding regions - Garnier-Robson; VIII: Hydrophilicity chart - Kyte-Doolittle; IX: Hydrophobicity chart - Hopp-Woods; X: alpha antiseptic regions -Eisenberg; Beta antipatic regions - Eisenberg; XII: Flexible regions - Karplus-Schulz; XIII: antigenic index - Jameson-Wolf; and XIV: Surface probability graph -Emini.

Cp or Cn p Table I Res Position I II III IV V VI VII VIII X XI XII XIII XIV Met 1 AA -0.08 0.73 * -0.60 0.82 Tf 2 AA -0.50 0.99 * -0.60 0.45 Lys 3 AA -0.42 1.24 * -0.60 0.29 Tf 4 AA - 0.07 1.30 * -0.60 0.42 lie 5 AA -0.34 1.19 * -0.60 0.55 Leu 6 AA -0.33 0.84 * -0.60 0.15 Thr 7 AB -0.34 1.34 * -0.60 0.14 His 8 A r -0.98 0.81 * -0.20 0.27 Cys 9 A r -1.39 0.63. . -0.20 0, 33 Wing 10 A r -0.71 0.73 * -0.20 0.20 Ser 11 A G 0.07 0.67 * -0.20 0.23 Wing 12 A G -0.43 0.67 * -0.20 0.57 Phe 13 A B -0.61 0.79. . -0.60 0.47 Pro 14 G -0.29 0.71. . 0.00 0.54 His 15 G -0.37 0.76. . 0.00 0.53 Leu 16 G r -0.73 0.83. . 0.20 0.33 Pro 17 G r -0.81 0.61. . 0.20 0.1 1 Gly 18 G r -0.78 0.76. . 0.20 0.04 Cys 19 G G -1.23 0.83. . 0.20 0.03 tsj IV) p Cp or Cp C Table I, continued Res Position I 1 11 III IV V VI - VII HIV IX X XI XII XIII XIV Cys 20 T 'r -1.90 0.71. . 0.20 0.01 Cys 21 B G -1.90 1.07. . -0.20 0.01 Cys 22 B G -2.50 1.33. . -0.20 0.01 Cys 23 B G -2.97 1.44. . -0.20 0.02 Phe 24 B B -3.00 1.56. . -0.60 0.03 Leu 25 B B -3.14 1.77. . -0.60 0.05 Leu 26 B B -3.33 1.89. . -0.60 0.08 Leu 27 B B -2.97 1.96. . -0.60 0.07 Phe 28 B B -2.60 1.56. . -0.60 0.11 Leu 29 B B -2.76 1.26. . -0.60 0.18 Val 30 B B -2.16 1.21. . -0.60 0.16 Ser 31 B T -2.20 0.96. . -0.20 0.29 Ser 32, B < C -1.70 0.81. . -0.40 0.26 Val 33 B B -1.67 0.61. . -0.60 0.51 Pro 34 B B -0.86 0.54. • -0.60 0.20 Val 35 B B -0.59 0.56. . -0.60 0.26 Thr 36 B B -1.10 0.67. * -0.60 0.36 Cys 37 B B -1.14 0.71. * -0.60 0.19 GIn 38 B B -0.29 0.71. * -0.60 0.25 Wing 39 B B -0.08 0.47. . -0.60 0.30 Leu 40 B B 0.18 -0.01. . 0.30 0.95 Gly 41 B T -0.37 0.03. . F 0.25 0.54 Gln 42 B T 0.00 0.27 *. F 0.25 0.40 Cp O cp cp Table I, continued Res Position I II III IV V VI VII HIV IX X XI XII XIII XIV Asp 43 B T -0.21 0.16. F 0.25 0.65 Met 44 B T 0.38 -0.10. F 1.00 1.01 Val 45 B 0.60 -0.53. 0.95 1.01 Ser 46 B T 0.63 -0.43. . F 0.85 0.61 Pro 47 B T 0.63 0.06. F 0.49 0.89 Glu 48 A B T 0.33 -0.16. F 1.48 1.93 Wing 49 A T 0.63 -0.41. F 1.72 1.93 Thr 50 A. 1.19 -0.41. F 1.76 1.67 Asn 51 T c 1.19 -0.46. F 2.40 1.29 N3 Ser 52 T c 1.10 -0.07. F 2.16 1.72 Ser 53 T c 0.40 -0.19. F 1.92 1.59 Ser 54 T T 0.69 0.11. F 1.13 0.86 Ser 55 T c 0.70 0.10. F 0.69 0.86 Ser 56 T T 0.49 0.10. F 0.65 0.86 Phe 57 T T 0.49 0.14. F 0.65 0.99 Ser 58 T c 0.49 0.14. F 0.69 0.99 Ser 59 T c 0.20 0.14. F 0.93 0.99 Pro 60 T c 0.16 0.26 * F 1.32 1.15 Ser 61 T c 0.57 -0.10 * F 2.01 0.85 Ser 62 T c 1.23 -0.49 * F 2.40 1.25 Wing 63. c 0.68 -0.37 * F 1.96 1.10 Gly 64 B. 1.09 -0.16 * F 1.37 0.61 Arg 65 B 1.00 -0.54 * F 1.43 0.89 Table I, continued Res Position I 11 III IV V VI VII HIV IX X XI XII XIII XIV His 66 B 1.06 -0.54 * 1.19 1.18 Val 67 B 1.36 -0.29 * 0.65 1.86 Arg 6S B r 1.91 -0.31 *. 0.85 1.53 Ser 69 B r 1.44 0.19 * * 0.25 1.53 Tyr 70 B G 1.33 0.37 * * 0.25 1.70 Asn 71 T G 1.02 0.13 * * 0.65 1.50 His 72 < C 1.88 0.56 * * -0.05 1.11 Leu 73 r i C 0.91 0.17 * * 0.45 1.18 Gln 74 B r 1.32 0.06 * * F 0.25 0.55 Gly 75 B r 1.28 -0.34. * F 0.85 0.79 Asp 76 B r 1.39 0.07. * F 0.40 1.00 Val 77 B B 1.47 -0.61. * F 0.90 1.13 Arg 78 B B 1.47 -1.01 * * 0.75 2.29 T 79 B B 0.77 -0.76 * * 0.75 1.13 Arg 80 B B 0.81 0.03 * * -0.15 1.32 Lys 81 B B 0.1 1 -0.23. * 0.30 0.90 Leu 82 B B 0.66 0.56 * * -0.60 0.74 Phe 83 B B 0.59 0.13 * * -0.30 0.55 Ser 84 B B 0.63 0.13 * -0.30 0.55 Phe 85 A. B -0.18 0.89 * -0.45 1.04 Thr 86 A. B: -1.03 0.99 * -0.45 1.04 Lys 87 A A B -0.18 0.89 * * -0.60 0.64 Tyr 88 A A B -0.37 0.50 * • -0.45 1.48 Cp O cp cp Table I, continued Res Position I II III IV V VI VIII HIV IX X XI XII XIII XIV Phe 89 A A B -0.07 0.40 *. -0.30 0.72 Leu 90 A A B. 0.68 -0.09 * *. 0.30 0.62 Lys 91 A A B. 0.99 -0.09 * * F 0.45 0.79 lie 92 AA 0.60 -0.44 * * F 0.60 1.48 Glu 93 AT 0.89 -0.80 * * F 1.30 1.77 Lys 94 AT 0.73 -1.49 * * F 1.30 1.77 Asn 95 AT 1.24 -0.84 * F 1.30 1.88 Gly 96 AT 0.86 -1.14 * * F 1.64 1.45 Lys 97 A. 1.43 -0.71 * * F 1.63 0.72 Val 98 A 1.48 -0.23 * F 1.67 0.64 -O Be 99 C 1.48 -0.63 F 2.66 1.30 Gly 100 T T 1.48 -1.06 * F 3.40 1.30 Thr 101. B T 1.82 -1.06 * F 2.66 3.04 Lys 102. B T 1.1 1 -1.30 * F 2.49 3.65 Lys 103 T T 1.76 -1.1 1 F 2.72 1.98 Glu 104 T 1.81 -1.1 1 F 2.35 2.12 Asn 105 T 1.86 -0.84 F 2.18 1.66 Cys 106 1 3. T 1.28 -0.46 1.70 l l l Pro 107 T T 0.42 0.23 1.18 0.45 Tyr 108 T T 0.38 0.91. 0.71 0.23 Ser 109 1 3. T -0.51 0.51 *. 0.14 0.75 lie 110 1 3 I B. -0.82 0.63 *. -0.43 0.34 Leu 1 11 1 3 1 3. -0.46 0.69 -0.60 0.31 Cp O Cp cp Table I, continued Res Position 1 11 111 IV V VI VII HIV IX X XI XII XIII XIV Glu 112 BB -1.10 0.31 -0.30 0.31 lie 1 13 1 BB -0.86 0.57 -0.60 0.33 Thr 114 BB -1.44 -0.11 F 0.45 0.69 Ser 1 15 BB -0.90 -0.11 F 0.45 0.28 Val 116 AB -0.94 0.31 -0.30 0.40 Glu 117 AB -1.80 0.27 -0.30 0.20 He 1 18 AB -1.50 0.43 -0.60 0.11 Gly 1 19 AB -2.04 0.54 -0.60 0.15 Val 120 \ B - 1.70 0.54 -0.60 0.07 Val 121. \ B -1.43 0.54 -0.60 0.19 N3 Wing 122 \ B -2.32 0.36 -0.30 0.19 Val 123 BB -1.43 0.61 -0.60 0.18 Lys 124 BB -1.39 0.37 -0.30 0.39 Wing 125 B -0.53 0.1 1 -0.10 0.52 lie 126 B 0.08 0.01 0.05 1.13 Asn 127 BT 0.42 0.13 F 0.25 0.88 Ser 128. B T 0.47 0.89 F 0.10 1.37 Asn 129. B T -0.17 1.07 -0.05 1.61 Tyr 130. B T -0.18 0.89 -0.05 1.01 Tyr 131 A A 0.71 1.10 -0.60 0.75 Leu 132 A A 0.76 1.11 -0.60 0.75 Wing 133 A A 1.10 0.71 -0.60 0.95 Met 134 A A 0.76 -0.04 0.45 1.22 cp or cp or cp Table I, continued Res Position 1 II 111 IV V VI VIII HIV IX X XI XII XIII XIV Asn 135 A T 1.04 -0.37. * 0.85 1.46 Lys 136 A T 0.48 -1.06. < F 1.30 2.89 Lys 137 A T 1.04 -0.87. «'F 1.30 2.41 Gly 138 A T 1.29 -0.73. * 'F 1.30 2.34 Lys 139 A. 1.59 -0.70 * '* F 1.10 1.16 Leu 140 B 1.63 -0.31. < "F 0.65 0.78 Tyr 141 BT 1.59 -0.31. 'F 1.00 1.57 Gly 142 BT 0.84 -0.74.' F 1.30 1.36 Ser 143 BT 1.19 0.04. * F 0.40 1.43 Lys 144 BT 1.14 -0.24. * F 1.00 1.47 Glu 145 A 1.96 -0.60 * F 1.10 2.38 Phe 146 A. 1.53 -1.03 *> F 1.10 2.97 Asn 147 AT 1.92 -0.84 * 'F 1.15 0.80 Asn 148 AT 1.41 -0.84. * F 1.15 0.92 Asp 149 AT 1.41 -0.16. * F 0.85 0.88 Cys 150 AT 1.41 -0.94 * »F 1.30 1.09 Lys 151 AA 2.22 -1.34 * * F 0.90 1.17 Leu 152 AA 1.33 -1.74 * • F 0.90 1.37 Lys 153 AA 1.33 -1.06 *» F 0.90 1.80 Glu 154 AA 1.33 -1.63 * * F 0.90 1.56 Arg 155 A, A 2.00 -1.63 * 'F 0.90 3.27 He 156 A. A 1.61 -1.91 * * F 1.24 2.63 Glu 157 A -A 2.18 -1.49 * • F 1.58 1.50 ro Cp or cp or cp Table I, continued Res Position 1 II III IV V VI HIV VIII X XI XII XIII XIV Glu 158 2.13 -0.73 < '* F 1.92 1.20 Asn 159 T T 1.82 -0.33 «' * F 2.76 2.76 Gly 160 T T 1.47 -0.53 < * * F 3.40 2.30 Tyr 161 T T 1.77 0.23. F 2.16 2.08 Asn 162 T c 1.47 0.73. F 1.32 1.31 Thr 163 c 0.77 0.71. 0.63 1.77 Tyr 164 B 0.77 1.07. -0.06 0.98 Wing 165 B 0.82 0.71. -0.40 0.98 Ser 166 B T 1.07 1.23. -0.20 0.71 Phe 167 B T 1.03 1.14. -0.20 0.79 Asn 168 T T 1.34 0.89. 0.35 1.06 T 169 T T 1.24 0.79. 0.35 1.27 Gln 170 c 1.94 0.83 '0.11 1.45 His 171 T c 2.24 0.04 < 0.77 1.77 Asn 172 T c 2.34 0.04 < > * F 1.08 2.92 Gly 173 T T 2.10 -0.26 < > * F 2.04 1.67 Arg 174 T T 1.53 0.10 < "* F 1.60 1.92 Gln 175 BB 0.94 0.24 '0.34 0.89 Met 176 BB 0.17 0.34 <. 0.18 0.90 Tyr 177 BB 0.17 0.60 <> -0.28 0.38 Val 178 BB 0.17 1.00. * -0.44 0.35 Wing 179 BB 0.10 1.03. * -0.60 0.35 Leu 180 BB -0.24 0.41. * -0.30 0.45 > Cp or Cp O cp Table I, continued Res Position I II III IV V vi - VIII HIV IX X Kl XII XIII XIV Asn 181 r T -0.23 0.09. 'F 1.25 0.60 Gly 182 GT -0.20 -0.06 * * F 2.15 0.60 Lys 183 GT 0.77 -0.13 * * F 2.60 1.13 Gly 184 T (C 1.47 -0.81 * * F 3.00 1.37 Wing 185 1 C 1.93 -1.21 * * F 2.50 2.72 Pro 186 BT 1.93 -1.21 * F 2.20 1.35 Arg 187 BT 2.32 -0.81 * F 1.90 2.35 Arg 188 BT 1.97 -1.24 * F 1.60 4.66 Gly 189 BT 2.42 -1.26 * F 1.30 4.35 oo Gln 190 B 3.12 -1.69 * F 1.10 4.35 Lys 191 B 3.38 -1.69 * F 1.10 4.35 Thr 192 B 3.27 -1.69 * F 1.44 8; 79 Arg 193 B 2.84 -1.71. F 1.78 8.16 Arg 194 G 2.89 -1.63 * F 2.52 5.89 Lys 195 T 2.30 -1.24 * F 2.86 5.47 Asn 196 T T 2.22 -1.23. * F 3.40 2.82 Thr 197 T C 1.83 -0.73. F 2.86 1.96 Ser 198 T C 0.91 0.06. F 1.47 0.85 Wing 199 B T 0.59 0.74. . 0.48 0.44 His 200 B. -0.06 0.77. . -0.06 0.47 Phe 201 B B -0.91 0.90 *. -0.60 0.34 Leu 202 B B -1.46 1.16. . -0.60 0.25 Pro 203 B B -1.19 1.30. . -0.60 0.14 ? > K) Cp O cp Cp Table I, continued Res Position 1 II III IV V VI VIII HIV IX X XI XII XIII XIV Met 204 B B -0.90 1.30 * -0.60 0.22 Val 205 A B -1.26 0.90 *. -0.60 0.35 Val 206 A. B -0.94 0.64. . -0.60 0.29 His 207 A. B -0.52 0.64. . -0.60 0.38 Ser 208 A B -0.70 0.46. . -0.60 0.65 DO NOT Among the highly preferred fragments in this regard are those comprising the KGF-2 regions that combine various structural features, such as several of the features described above. In addition, the techniques of gene intermixing, intermingling of portions, intermixing of exons, and / or intermixing codons (collectively referred to as "DNA intermixing") can be employed to modulate the activities of KGF-2 whereby they are effectively generated the agonists and antagonists of KGF-2. See in general, United States Patents Nos. 5,605,793; 5,811,238; 5,830,721; 5,834,252; and 5,837,458; and Patten, P.A. and collaborators, Curr. Opinion Biotechnol. 8: 724-33 (1997); Harayama, S., Trends Biotechnol. 16 (2): 76-82 (1998); Hansson, L.O. and collaborators, J. Mol. Biol. 287: 265-76 (1999); and Lorenzo, M. M. and Blasco, R., Biotechniques 24 (2): 308-13 (1998) (each of these patents and publications are incorporated by reference herein). In one embodiment, the alteration of the KGF-2 polynucleotides and the corresponding polypeptides can be achieved by mixing DNA. DNA intermixing involves the assembly of two or more segments of DNA into a desired KGF-2 molecule, by homologous or site-specific recombination. In yet another embodiment, the KGF-2 polynucleotides and the corresponding polypeptides can be altered by being subjected to random mutagenesis by error prone PCR, random insertion of nucleotides and other methods prior to recombination. In yet another embodiment, one or more components, portions, sections, parts, domains, fragments, etc., of KGF-2 can be recombined with one or more components, portions, sections, parts, domains, fragments, etc., of one or more heterologous molecules. In preferred embodiments, the heterologous molecules are members of the KGF-2 family. In additional preferred embodiments, the heterologous molecule is a growth factor such as, for example, platelet derived growth factor (PDGF), insulin-like growth factor (IGF-I), transforming growth factor (TGF). -alpha, epidermal growth factor (EGF), fibroblast growth factor (FGF), TGF-beta, bone morphogenetic protein (BMP) -2, MBP-4, MBP-5, BMP-6, BMP-7 , activins A and B, decapentaplégico (dpp), 60A, OP-2, dorsalina, factors of growth differentiation (GDFs), nodal, MIS, inhibin-alpha, TGF-betal, TGF-beta2, TGF-beta3, TGF- beta5, and neurotrophic factor derived from the glia (GDNF). Other preferred fragments are biologically active fragments of KGF-2. The biologically active fragments are those that show activity similar, but not necessarily identical, to an activity of the KGF-2 polypeptide. The biological activity of the fragments may include a desired, improved activity, or a decreased undesirable activity. In addition, this invention provides a method for selecting the compounds to identify those that modulate the action of the polypeptide of the present invention. An example of such an assay comprises the combination of a mammalian fibroblast cell, the polypeptide of the present invention, the compound to be selected and 3 [H] -thymidine under cell culture conditions where the fibroblast cell could normally proliferate. A control assay can be performed in the absence of the compound to be selected, and compared to the amount of proliferation of fibroblasts in the presence of a compound to determine whether the compound stimulates proliferation, by determining the uptake of 3 [H ] -thymidine in each case. The amount of proliferation of fibroblast cells is measured by liquid scintillation chromatography which measures the incorporation of 3 [H] -thymidine. The agonist and antagonist compounds can be identified by this method. In still another method, a mammalian cell or a membrane preparation expressing a receptor for a polypeptide of the present invention is incubated with a labeled polypeptide of the present invention in the presence of the compound. The ability of the compound to improve or block this interaction could then be measured. Alternatively, the response of a known second messenger system, after the interaction of a compound to be selected and the KGF-2 receptor, is measured and the ability of the compound to bind to the receptor and promote a response is also measured. of second messenger, to determine if the compound is a potential agonist or antagonist. Such second messenger systems include but are not limited to, the cAMP guanylate cyclase, the ion channels or the hydrolysis with phosphoinositide. All previous trials can be used as diagnostic or prognostic markers. The molecules discovered using these assays can be used to treat diseases or to give rise to a particular result in a patient (e.g., the development of blood vessels) by activating or inhibiting the KGF-2 molecule. In addition, the assays can discover agents that can inhibit or increase the production of KGF-2 from properly manipulated tissue cells. Therefore, the invention includes a method for identifying compounds that bind to KGF-2, comprising the steps of: (a) incubating a candidate binding compound with KGF-2; and (b) determine if the link has occurred. In addition, the invention includes a method for identifying agonists / antagonists comprising the steps of: (a) incubating a candidate compound with KGF-2, (b) evaluating a biological activity, and (c) determining whether a biological activity of KGF -2 has been altered. Also, molecules that bind to KGF-2 could be identified experimentally by using the folded beta sheet regions described in Figure 4 and Table I. Accordingly, the specific embodiments of the invention are directed to the polynucleotides. encoding the polypeptides comprising, or consisting alternatively of, the amino acid sequence of each of the folded beta sheet regions described in Figure 3 / Table 1. Additional embodiments of the invention are directed to the polynucleotides encoding for KGF-2 polypeptides comprising, or consisting alternatively of, any combination or all regions of folded beta sheet described in Figure 4 / Table 1. Additional preferred embodiments of the invention are directed to polypeptides comprising, or which consist alternatively of the amino acid sequence of KGF-2 from each of the plate regions beta-folded proteins described in Figure 4 / Table 1. Additional embodiments of the invention are directed to KGF-2 polypeptides comprising, or consisting alternatively of, any combination or all of the folded beta sheet regions described in Figure 4 / Table 1. Other preferred embodiments of the invention are fragments of KGF-2 that bind to the KGF-2 receptor. Fragments that bind to the KGF-2 receptor may be useful as agonists or antagonists of KGF-2. For example, fragments of KGF-2 that bind to the receptor can prevent binding to KGF-2 and active portions thereof. Other fragments can bind to the receptor and specifically deactivate the receptor and the receptor activation, or they can be specifically antibodies that recognize the receptor-ligand complex and, preferably, do not specifically recognize the unbound receptor or the unbound ligand. Similarly, fragments that activate the receptor are included in the invention. These fragments can act as receptor agonists, for example, they potentiate or activate either all or a subset of the biological activities of receptor activation mediated by the ligand, for example, by induction of receptor dimerization. The fragments can be specified as agonists, antagonists or inverse agonists for the biological activities comprising the specific biological activities of the peptides of the invention described herein.Non-limiting examples of the KGF-2 fragments that bind to the KGF-2 receptor include amino acids 147-155, 95-105, 78-94, 119-146, 70-94, 78-105, 114-146 , 70-105, 86-124, 100-139, 106-146, 160-209, and / or 156-209 of SEQ ID No. 2. Polynucleotides encoding such polypeptides are also preferred. Other preferred fragments are the biologically active KGF-2 fragments. The biologically active fragments are those that show similar activity, but not necessarily identical to an activity of the KGF-2 polypeptide. The biological activity of the fragments may include a desired, improved activity, or a desirable, decreased activity. However, many polynucleotide sequences, such as EST sequences, are publicly available and accessible through the sequence databases. Some of these sequences are related to SEQ ID No. 1 and may have been publicly available prior to the conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. Listing each related sequence could be problematic. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 and 613 of SEQ ID No. 1; b is an integer from 15 to 627, where a and b correspond to the positions of the nucleotide residues shown in SEQ ID No. 1, and where b is greater than or equal to + 14.

Amino-Terminals and Carboxyl-Terminals Suppressions Various media of the FGF family have been modified using recombinant DNA technology. Positively charged molecules have been replaced or deleted in aFGF and bFGF that are important for binding to heparin. The modified molecules resulted in reduced activity in heparin binding. Accordingly, it is known that the amount of the modified molecule sequestered by heparin in a patient could be reduced, increasing the potency as more FGF could reach the appropriate receptor (EP 0 298 723). Native KGF-2 is relatively unstable in the aqueous state and undergoes chemical and physical degradation, resulting in the loss of biological activity during processing and storage. Native KGF-2 is also prone to aggregation in aqueous solution, at elevated temperatures, and becomes inactivated under acidic conditions.

In order to improve or alter one or more of the characteristics of native KGF-2, protein manipulation can be employed. Ron et al., J ". Biol. Chem. 268 (4): 2984-2988 (1993) reported modified KGF proteins that had heparin binding activity even if the 3, 8 or 27 amino-terminal amino acid residues were The suppression of 3 and 8 amino acids had complete activity.More suppressions of KGF-2 have been described in PCT / IB95 / 00971. The suppression of carboxyl-terminal amino acids can improve the activity of proteins. gamma showing up to ten times greater activity by deletion of ten amino acid residues from the carboxyl end of the protein (Dobeli et al., "of Biotechnology 7: 199-216 (1988)). Thus, one aspect of the invention is to provide the polypeptide analogues of KGF-2 and the nucleotide sequences encoding such analogues, which show enhanced activity (eg when exposed to typical conditions of pH, heat or other conditions of storage) relative to the native KGF-2 polypeptide. Particularly preferred KGF-2 polypeptides are shown below (numbering begins with the first amino acid in the protein (Met) (Figure 1 (SEQ ID No. 2)): Thr (residue 36) - Arg (65) - Ser (208) Ser (residue (208) Cys (37) - Ser (208) Val (67) - Ser (208) Gln (38) - Ser (208) Ser (69) - Ser (208) Ala (39) - Ser (208) Val (77) - Ser (208) Leu (40) - Ser (208) Arg (80) - Ser (208) Gly (41) - Ser (208) Met (1 Thr (36¡ Cys (37) - His (207) Gln (42) - Ser (208 ) Met (1 Thr (36) Cys (37) - Val (206) Asp (43) - Ser (208) Met (1 Thr (36¡ Cys (37) - Val (205) Met (44) - Ser (208 ) Met (1 Thr (36¡ Cys (37) - Met (204) Val (45) - Ser (208) Met (1 Thr (36¡ Cys (37) - Pro (203) Ser (46) - Ser (208 ) Met (1 Thr (36) Cys (37) - Leu (202) Pro (47) - Ser (208) Met (1 Thr (36¡ Cys (37) - Phe (201) Glu (48) - Ser (208 ) Met (1 Thr (36¡ Cys (37) - His (200) Ala (49) - Ser (208) Met (1 Thr (36) Cys (37) - Ala (199) Thr (50) - Ser (208 ) Met (1 Thr (36) Cys (37) - Ser (198) Asn (51) - Ser (208) Met (1 Thr (36¡ Cys (37) - Thr (197) Ser (52) - Ser (208 ) Met (1 Thr (36¡ Cys (37) - Asn (196) Ser (53) - Ser (208) Met (1 Thr (36; Cys (37) - Lys (195) Ser (54) - Ser (208) Met (1 Thr (36; Cys (37) - Arg (194) Ser (55) - Ser (208) Met (1 Thr (36) Cys (37) - Arg (193) Ser (56) - Ser (208) Met (1 Thr (36; Cys (37) - Thr (192) Phe (57) - Ser (208) Met (1 Thr (36) Cys (37) - Lys (191) Ser (59) - Ser (208) Met (1 Thr (36) Cys (37) - Arg (188) Ser (62) - Ser (208) Met (1 Thr (36; Cys (37) - Arg (187) Ala (63) - Ser (208) Met (l), Thr (36), or Cys (37) - Lys (183) Gly (64) - Ser (208) Preferred embodiments include the N-terminal deletions Ala (63) -Ser (208) (KGF-2? 28) (SEQ ID No. 68) and Ser (69) -Ser (208 (KGF-2? 33) (SEQ ID No. 96) Other preferred N-terminal and C-terminal deletion mutants are described in Examples 13 and 16 (c) of the specification and include: Ala (39) -Ser (208) (SEQ ID NO. ); Pro (47) - Ser (208) of Figure 1 (SEQ ID No. 2); Val (77) - Ser (208) (SEQ ID No. 70); Glu (93) - Ser (208) ( SEQ ID No. 72); Glu (104) -Ser (208) (SEQ ID No. 74); Val (123) -Ser (208) (SEQ ID No. 76); and Gly (138) -Ser (208 (SEQ ID No. 78) Other preferred C-terminal deletion mutants include: Met (l), Thr (36), or Cys (37) -Lys (153) of Figure 1 (SEQ ID No. 2) Also included by the present invention are the deletion mutants having amino acids deleted from the N-terminus and the C-terminus .. Such mutants include all combinations of the N-terminal deletion mutants and C-terminal deletion mutants described. previously, for example, Ala (39) -His (200) of Figure 1 (SEQ ID No. 2); Met (44) -Arg (193) of Figure 1 (SEQ ID No. 2); Ala (63) - Lys (153) of Figure 1 (SEQ ID No. 2); Ser (69) - Lys (53) of Figure 1 (SEQ ID No. 2), etc. These combinations can be made using recombinant techniques known to those skilled in the art. Thus, in one aspect, the N-terminal deletion mutants are provided by the present invention. Such mutants include those comprising the amino acid sequence shown in Figure 1 (SEQ ID No. 2) except for a deletion of at least the first 38 N-terminal amino acid residues (e.g., a deletion of at least Met ( ) - Gln (38)) but not more than the first 147 N-terminal amino acid residues of Figure 1 (SEQ ID No. 2). Alternatively, the deletion will include at least the first 38 N-terminal amino acid residues (eg, a deletion of at least Met (l) -Gln (38)) but not more than the first 137 N-terminal amino acid residues of the Figure 1 (SEQ ID No. 2). Alternatively, the deletion will include at least the first 46 N-terminal amino acid residues, but not more than the first 137 N-terminal amino acid residues of Figure 1 (SEQ ID No. 2). Alternatively, the deletion will include at least the first 62 N-terminal amino acid residues but not more than the first 137 N-terminal amino acid residues of Figure 1 (SEQ ID No. 2). Alternatively, the deletion will include at least the first 68 N-terminal amino acid residues but not more than the first 137 N-terminal amino acid residues of Figure 1 (SEQ ID No. 2). Alternatively, the deletion will include at least the first 76 N-terminal amino acid residues but not more than the first 137 N-terminal amino acid residues of Figure 1 (SEQ ID No. 2). Alternatively, the deletion will include at least the first 92 N-terminal amino acid residues but not more than the first 137 N-terminal amino acid residues of Figure 1 (SEQ ID No. 2). Alternatively, the deletion will include at least the first 103 N-terminal amino acid residues but not more than the first 137 N-terminal amino acid residues of Figure 1 (SEQ ID No. 2). Alternatively, the deletion will include at least the first 122 N-terminal amino acid residues but not more than the first 137 N-terminal amino acid residues of Figure 1 (SEQ ID No. 2). In addition to the ranges of the N-terminal deletion mutants described above, the present invention is also directed to all combinations of the ranges described above, for example, deletions of at least the first 62 N-terminal amino acid residues but not more than the first 68 N-terminal amino acid residues of Figure 1 (SEQ ID No. 2); deletions of at least the first 62 N-terminal amino acid residues but not more than the first 76 N-terminal amino acid residues of Figure 1 (SEQ ID No. 2); the deletions of at least the first 62 N-terminal amino acid residues but not more than the first 92 N-terminal amino acid residues of Figure 1 (SEQ ID No. 2); deletions of at least the first 62 N-terminal amino acid residues but not more than the first 103 N-terminal amino acid residues of Figure 1 (SEQ ID No. 2); deletions of at least the first 68 N-terminal amino acid residues but not more than the first 76 N-terminal amino acid residues of Figure 1 (SEQ ID No. 2); deletions of at least the first 68 N-terminal amino acid residues but not more than the first 92 N-terminal amino acid residues of Figure 1 (SEQ ID No. 2); deletions of at least the first 68 N-terminal amino acid residues but not more than the first 103 N-terminal amino acid residues of Figure 1 (SEQ ID No. 2); the deletions of at least the first 46 N-terminal amino acid residues but not more than the first 62 N-terminal amino acid residues of Figure 1 (SEQ ID No. 2), - the deletions of at least the first 46 residues of N-terminal amino acids but not more than the first 68 N-terminal amino acid residues of Figure 1 (SEQ ID No. 2); the deletions of at least the first 46 N-terminal amino acid residues but not more than the first 76 N-terminal amino acid residues of Figure 1 (SEQ ID No. 2), - etc.

In yet another aspect, the C-terminal deletion mutants are provided by the present invention. Preferably, the N-terminal amino acid residue of the C-terminal deletion mutants is amino acid residue 1 (Met), 36 (Thr), or 37 (Cys) of Figure 1 (SEQ ID No. 2). Such mutants include those comprising the amino acid sequence shown in Figure 1 (SEQ ID No. 2), except for a deletion of at least the last C-terminal amino acid residue (Ser (208)) but not more than the latter 55 C-terminal amino acid residues (eg, a deletion of amino acid residues Glu (154) Ser (208) of Figure 1 (SEQ ID No. 2) Alternatively, the deletion will include at least the last amino acid residue C-terminals but not more than the last 65 C-terminal amino acid residues of Figure 1 (SEQ ID No. 2) Alternatively, the deletion will include at least the last 10 C-terminal amino acid residues but not more than the last 55 C-terminal amino acid residues of Figure 1 (SEQ ID No. 2) Alternatively, the deletion will include at least 20 C-terminal amino acid residues but not more than the last 55 C-terminal amino acid residues of the Figure 1 (SEQ ID No. 2) Alternatively, the deletion will include at least 30 C-terminal amino acid residues but no more than the last 55 C-terminal amino acid residues of Figure 1 (SEQ ID No. 2) . Alternatively, the deletion will include at least 40 C-terminal amino acid residues but not more than the last 55 C-terminal amino acid residues of Figure 1 (SEQ ID No. 2). Alternatively, the deletion will include at least 50 C-terminal amino acid residues but not more than the last 55 C-terminal amino acid residues of Figure 1 (SEQ ID No. 2). In addition to the ranges of the C-terminal deletion mutants described above, the present invention is also directed to all combinations of the ranges described above, for example the deletions of at least the last C-terminal amino acid residue but not more than the last 10 C-terminal amino acid residues of Figure 1 (SEQ ID No. 2); the deletions of at least the last C-terminal amino acid residue but not more than the last C-terminal amino acid residues of Figure 1 (SEQ ID No. 2); the deletions of at least the last C-terminal amino acid residue but not more than the last 30 C-terminal amino acid residues of Figure 1 (SEQ ID No. 2); deletions of at least the last C-terminal amino acid residue but not more than the last 40 C-terminal amino acid residues of Figure 1 (SEQ ID No. 2); deletions of at least the last 10 C-terminal amino acid residues but not more than the last 20 C-terminal amino acid residues of Figure 1 (SEQ ID No. 2); deletions of at least the last 10 C-terminal amino acid residues but not more than the last 30 C-terminal amino acid residues of Figure 1 (SEQ ID No. 2); deletions of at least the last 10 C-terminal amino acid residues but not more than the last 40 C-terminal amino acid residues of Figure 1 (SEQ ID No. 2); deletions of at least the last 20 C-terminal amino acid residues but not more than the last 30 C-terminal amino acid residues of Figure 1 (SEQ ID No. 2); etc. In yet another aspect, also included by the present invention are the deletion mutants having the amino acids deleted from the N-terminal and C-terminal residues. Such mutants include all combinations of the N-terminal deletion mutants and the C-terminal deletion mutants described above. Such mutants include those comprising the amino acid sequence shown in Figure 1 (SEQ ID No. 2) except for a deletion of at least the first 46 N-terminal amino acid residues but not more than the first 137 N-amino acid residues. terminals of Figure 1 (SEQ ID No. 2) and a deletion of at least the last C-terminal amino acid residue but not more than the last 55 C-terminal amino acid residues of Figure 1 (SEQ ID No. 2) . Alternatively, a deletion may include at least the first 62, 68, 76, 92, 103 or 122 N-terminal amino acids, but not more than the first 137 N-terminal amino acid residues of Figure 1 (SEQ ID No. 2) and a deletion of at least the last 10, 20, 30, 40 or 50 C-terminal amino acid residues, but not more than the last 55 C-terminal amino acid residues of Figure 1 (SEQ ID No. 2). All combinations of all the ranges described above are also included.

Substitution of Amino Acids A further aspect of the present invention also includes the substitution of amino acids. Native mature KGF-2 contains 42 charged residues, 32 of which have a positive charge. Depending on the location of such residues in the three-dimensional structure of the protein, the substitution of one or more of these residues in groups with the amino acids that have a negative charge or a neutral charge, can alter the electrostatic interactions of the adjacent residues and can be useful to achieve the increased stability and reduced aggregation of the protein. The aggregation of the proteins can not only result in a loss of activity but is problematic when preparing pharmaceutical formulations, because they can be immunogenic (Pinckard et al., Clin.Exp.Immunol.23: 331-340 ( 1967), Robbins et al., Diabetes 36: 838-845 (1987), Cleland et al., Crit., Rev. Therapeutic Drug Carrier Systems 10: 307-377 (1993)). Any modification should give consideration to minimize the load repulsion in the tertiary structure of the protein molecule. Thus, of special interest are substitutions of the charged amino acid, with another charge, or with neutral or negatively charged amino acids. The latter results in proteins with a reduced positive charge, to improve the characteristics of KGF-2. Such improvements include the increased stability and reduced aggregation of the analog, as compared to the native KGF-2 protein. The replacement of amino acids can also change the selectivity of the binding to cell surface receptors. Ostade et al., Nature 361: 266-268 (1993), described certain TNF alpha mutations that result in the selective binding of TNF alpha to only one of the two known TNF receptors. A further embodiment of the invention relates to a polypeptide comprising the amino acid sequence of a KGF-2 polypeptide having an amino acid sequence containing at least one amino acid substitution, but not more than 50 amino acid substitutions, even more preferably , no more than 40 amino acid substitutions, still more preferably, no more than 30 amino acid substitutions, and still more preferably, no more than 20 amino acid substitutions. Of course, in order to increase the preference, it is highly preferable that a peptide or polypeptide has an amino acid sequence comprising the amino acid sequence of a KGF-2 polypeptide, which contains at least one, but not more than 20, 19 , 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid substitutions. In specific embodiments, the number of additions, substitutions and / or deletions in the amino acid sequence of Figure 1 or fragments thereof (eg, the mature form and / or other fragments described herein) is 1-5. , 5-10, 5-25, 5-50, 10-50 or 50-150, conservative amino acid substitutions that are preferable. The KGF-2 molecules can include one or more amino acid substitutions, deletions or additions thereof, either from the natural mutation or from human manipulation. Mutations can be performed in full-length KGF-2, mature KGF-2, and any other appropriate fragments of KGF-2, eg, A63-S208, S69-208, V77-S208, R80-S208 or E93-S208 . Examples of some preferred mutations are: Ala (49) Gln, Asn (51) Ala, Ser (54) Val, Ala (63) Pro, Gly (64) Glu, Val (67) Thr, Trp (79) Val, Arg (80) Lys, Lys (87) Arg, Tyr (88) Trp, Phe (89) Tyr, Lys (91) Arg, Ser (99) Lys, Lys (102) Gln, Lys 103 5) Lys, Pro (107) Asn, Ser (109) Asn, Leu (111 Met, Thr (114) Arg, Glu (117 Ala, Val (120) He, Val (123 He, Ala (125) Gly, He (126 Val, Asn (127) Glu, Asn (127 Gln, Tyr (130) Phe, Met (134 Thr, Lys (136) Glu, Lys (137 Glu, Gly (142) Ala, Ser (143 Lys, Phe (146) Ser, Asn (148 Glu, Lys (151) Asn, Leu (152 Phe, Glu (154) Gly, Glu (154 Asp, Arg (155) Leu, Glu (157 Leu, Gly (160) His, Phe (167 Wing, Asn (168) Lys, Gln (170 Thr, Arg (174) Gly, Tyr (177 Phe, Gly (182) Gln, Wing (185 Val, Wing (185) Leu, Ala (185 He, Arg (187) Gln (190) Lys r Lys (195) Glu, Thr (197) Lys Ser (198) Thr i Arg (194) Glu, Arg (194) Gln, Lys (191) Glu Lys (191) Gln, Arg (188) Glu, Arg (188) Gln, Lys (183) Glu Arg (187) Wing «Arg (188) Wing, Arg 174 (Wing), Lys (183) Ala Lys (144) Ala, Lys (151) Ala, Lys (153) Ala, Lys (136) Ala, Lys (137) Ala, and Lys (139) Ala. By the designation, for example, Ala (49) Gln it is intended that Ala at position 49 of Figure 1 (SEQ ID No. 2) be replaced by Gln. Additionally, the following mutants are particularly preferred: S69-S208 with a point mutation in R188E; S69-S208 with a point mutation in K191E; S69-S208, with a point mutation in K149E; S69-S208 with a point mutation in K183Q; S69-S208 with a point mutation in K183E; A63-S208 with a point mutation in R68G; A63-S208 with a point mutation in R68S; A63-S208 with a point mutation in R68A; A63-S208 with point mutations in R78A, R80A and R81A; A63-S208 with point mutations in K81A, K87A, and K91A; A63-S208 with point mutations in R78A, R80A, K81A, K87A and K91A; A63-S208 with point mutations in K136A, K137A, K139A and K144A; A63-S208 with point mutations in K151A, K153A and K155A; A63-S208 with point mutations in R68G, R78A, R80A, and K81A; A63-S208 with point mutations in R68G, K81A, K87A and K91A; A63-S208 with point mutations in R68G, R78A, R80A, K81A, K87A and K91A; A63-S208 with point mutations in R68G, K136A, K137A, K139A, and K144A; A62-A208 with point mutations in R68G, K151A, K153A, and R155A; A63-S208 with point mutations in R68S, R78A, R80A, and K81A; A63-S208 with point mutations in R68S, K81A, R87A and K91A; A63-S208 with point mutations in R68S, R78A, R80A, K81A, K87A and K91A; A63-S208 with point mutations in R68S, K136A, K137A, K139A, and K144A; A63-208 with point mutations in R68S, K151A, K153A, and R155A; A63-S208 with point mutations in R68A, R78A, R80A, and K81A; A63-S208 with point mutations in R68A, K81A, K87A, and K91A; A63-S208 with point mutations in R68A, R78A, R80A, K81A, K87A, and K91A; A63-S208 with point mutations in R68A, K136A, K137A, K139A and K144A; and A63-208 with point mutations in R68A, K151A, K153A and R155A. Also preferred are: A63-S208 with positively charged residues between and including R68 to K91 which are replaced with alanine [A63-S208 (R68-K91A)]; Full-length KGF-2 with positively charged residues between and including R68 to K91 replaced with alanine [KGF-2 (R68-K91A)]; A63-S208 with positively charged residues between and including R68 to K91 replaced with neutral residues, such as G, S and / or A; Full-length KGF-2 with positively charged residues between and including R68 to K91 replaced with neutral residues, such as G, S and / or A; A63-S208 with positively charged residues between and including R68 to K91 replaced with negatively charged acid residues, such as D and / or E; Full-length KGF-2 with positively charged residues between and including R68 to K91 replaced with negatively charged acid residues, such as D and / or E; Full-length KGF-2 with point mutations in R78A, R80A, and K81A; Full-length KGF-2 with point mutations in K81A, K87A and K91A; Full-length KGF-2 with a point mutation in R68G, full-length KGF-2 with a point mutation in R68S; Full-length KGF-2 with a point mutation in R68A; A63-S208 with point mutations in R174A and K183A; and A63-S208 with point mutations in R187A and R188A.

A63-S208 with a point mutation in R188E, K191E, K149E, K183Q or K183E is also preferred; A63-S208 with point mutations in R78A, R80A and K81A; A63-S208 with point mutations in K81A, K87A and K91A; A63-S208 with point mutations in R174A and K183A; A63-S208 with point mutations in R187A and R188A; V77-S208 with a point mutation in R188E, K191E, K149E, K183Q, or K183E; V77-S208 with point mutations in R78A, R80A and K81A; V77-S208 with point mutations in K81A, K87A and K91A; V77-S208 with point mutations in R174A and K183A; V77-S208 with point mutations in R187A and R188A; R80-S208 with a point mutation in R188E, K191E, K149E, K183Q or K183E; R80-S208 with point mutations in R174A and K183A; R80-S208 with point mutations in R187 and R188A; E93-S208 with point mutations in R188E, K191E, K149E, K183Q, or K183E; E93-S208 with point mutations in R174A and K183A; E9-S208 with point mutations in R187A and R188A. All the above point mutations can also be performed on the full-length KGF-2, on the mature KGF-2, or any other fragment of KGF-2 described herein. For the designation, for sample, R188E it is intended that the arginine in position 188 be replaced with a glutamic acid. In addition, mutations directed to the site can be performed at each of the amino acids of KGF-2, preferably between amino acids A63 to E93. Each amino acid can be replaced with any other of the remaining 19 amino acids. For example, preferred mutations include: A63 replaced with C, D, E, F, G, I, K, L, M, N, P, Q, R, S, T, V, or Y; G64 replaced with A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W or Y; R65 replaced with A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W or Y; H66 replaced with A, C, D, E, F, G, I, K, L, M, N, P, Q, R, S, T, V, W or Y; V67 replaced with A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W or Y; R68 replaced with A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, or Y; S69 replaced with A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, or Y; Y70 replaced with A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or; N71 replaced with A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, or Y; H72 replaced with A, C, D, E, F, G, I, K, L, M, N, P, Q, R, S, T, V, or Y; L73 replaced with A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, or Y; Q74 replaced with A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W or Y; G75 replaced with A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; D76 replaced with A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; V77 replaced with A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, or Y; R78 replaced with A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, or Y; 79 replaced with A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V or Y; R80 replaced with A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, or Y; K81 replaced with A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, or Y; L82 replaced with A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, or Y; F83 replaced with A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; S84 replaced with A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W or Y; F85 replaced with A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; T86 replaced with A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, or Y; K87 replaced with A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W or Y; Y88 replaced with A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V or W; F89 replaced with A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; L90 replaced with A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, or Y; K91 replaced with A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, or Y; 192 replaced with A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, or Y; and / or E93 replaced with A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; These mutations can be carried out in the N-terminal deletion constructions previously described, particularly the constructions starting with amino acids Ml, T36, C37 or A63. In addition, more than one amino acid (for example, 2, 3, 4, 5, 6, 7, 8, 9 and 10) can be replaced in this region (A63 to E93) with other amino acids. The resulting constructs can be selected for loss of heparin binding, loss of KGF-2 activity, and / or loss of enzymatic cleavage between amino acids R68 and S69. Preferred mutations are located at amino acid positions R68 and S69 in the N-terminal, M1, T36, C37 and A63 deletion constructs, as well as mutations in the heparin binding domain, of all N-terminal mutants previously listed, especially T36, C37, A63, S69, V77, R80 or E93. The binding domain to heparin is between Argl74 and Lysl83. Preferred Arg68 mutants replace arginine with Gly, Ser or Ala; the preferred Argl87 mutants replace the arginine with alanine. Two ways in which mutations can be performed either through site-directed mutagenesis or accelerated mutagenesis (Kuchner and Arnold, Tibtech 5: 523-530 (1997), Crameri et al., Nature (1998), and Christians et al. , Nature Biotechnology 17: 259264 (1999)). These methods are well known in the art. The changes are preferably of a minor nature, such as conservative substitutions of amino acids that do not significantly affect the folding or activity of the protein. Examples of conservative amino acid substitutions known to those skilled in the art are described below: Aromatics Phenylalanine Tryptophan Tyrosine Hydrophobic: Leucine Isoleucine Valine Polar: Glutamine Asparagine Basics Arginine Lysine Histidine Acids: Aspartic acid Glutamic acid Small: Alanine Serine Threonine Methionine Glycine Of course, the number of amino acid substitutions that a person skilled in the art could make, depends on many factors, including those described above. Generally speaking, the number of substitutions for any given KGF-2 polypeptide will be more than 50, 40, 30, 20, 10, 5 or 3, depending on the objective. For example, a number of substitutions that can be made at the C-terminus of KGF-2 to improve stability are described above and in Example 22. Particularly preferred are KGF-2 molecules with conservative amino acid substitutions, including: Ml replaced with A, G, I, L, S, T or V; 2 replaced with F or Y; K3 replaced with H or R; 4 replaced with F or Y; 15 replaced with A, G, L, S, T, M or V; L6 replaced with A, G, I, S, T, M or V; T7 replaced with A, G, I, L, S, M or V; H8 replaced with K or R; AlO replaced with G, I, L, S, T, M, or V; Sil replaced with A, G, I, L, T, M or V; A12 replaced with G, I, L, S, T, M or V; F13 replaced with or Y; H15 replaced with K or R; L16 replaced with A, G, I, S, T, M or V; G18 replaced with A, I, L, S, T, M or V; F24 replaced with or Y; L25 replaced with A, G, I, S, T, M or V; L26 replaced with A, G, I, S, T, M or V; L27 replaced with A, G, I, S, T, M or V; F28 replaced with or Y; L29 replaced with A, G, I, S, T, M or V; V30 replaced with A, G, I, L, S, T or M; S31 replaced with A, G, I, L, T, M or V; S32 replaced with A, G, I, L, T, M or V; V33 replaced with A, G, I, L, S, T or M; V35 replaced with A, G, I, L, S, T or M; T36 replaced with A, G, I, L, S, M or V; Q38 replaced with N; A39 replaced with G, I, L, S, T, M or V; L40 replaced with A, G, I, S, T, M or V; G41 replaced with A, I, L, S, T, M or V; Q42 replaced with N; D43 replaced with E; M44 replaced with A, G, I, L, S, T or V; V45 replaced with A, G, I, L, S, T or M; S46 replaced with A, G, I, L, T, M or V; E48 replaced with D; A49 replaced with G, I, L, S, T, M or V; T50 replaced with A, G, I, L, S, M or V, N51 replaced with Q; S52 replaced with A, G, I, L, T, M or V; S53 replaced with A, G, I, L, T, M or V; S54 replaced with A, G, I, L, T, M or V; S55 replaced with A, G, I, L, T, M or V; S56 replaced with A, G, I, L, T, M or V, F57 replaced with or Y; S58 replaced with A, G, I, L, T, M or V; S59 replaced with A, G, I, L, T, M or V; S61 replaced with A, G, I, L, T, M or V; S62 replaced with A, G, I, L, T, M or V; A63 replaced with G, I, L, S, T, M or V; G64 replaced with A, I, L, S, T, M or V; R65 replaced with H or K; H66 replaced with K or R; V67 replaced with A, G, I, L, S, T or M; R68 replaced with H or K; S69 replaced with A, G, I, L, T, M or V; Y70 replaced with F o; N71 replaced with Q; H72 replaced with K or R; L73 replaced with A, G, I, S, T, M or V; Q74 replaced with N; G75 replaced with A, I, L, S, T, M or V; D76 replaced with E; V77 replaced with A, G, I, L, S, T or M; R78 replaced with H or K; W79 replaced with F or Y; R80 replaced with H or K; K81 replaced with H or R; L82 replaced with A, G, I, S, T, M or V; F83 replaced with or Y; S84 replaced with A, G, I, L, T, M or V; F85 replaced with or Y; T86 replaced with A, G, I, L, S, M or V; K87 replaced with H or R; Y88 replaced with F o; F89 replaced with or Y; L90 replaced with A, G, I, S, T, M or V; K91 replaced with H or R; 192 replaced with A, G, L, S, T, M or V; E93 replaced with D; K94 replaced with H or R; N95 replaced with Q; G96 replaced with A, I, L, S, T, M or V; K97 replaced with H or R; V98 replaced with A, G, I, L, S, T or M; S99 replaced with A, G, I, L, T, M or V; G100 replaced with A, I, L, S, T, M or V; T101 replaced with A, G, I, L, S, M or V; K102 replaced with H or R; K103 replaced with H or R; E104 replaced with D; N105 replaced with Q; Y108 replaced with F o; S109 replaced with A, G, I, L, T, M or V; 1110 replaced with A, G, L, S, T, M or V; Lili replaced with A, G, I, S, T, M or V; E112 replaced with D; 1113 replaced with A, G, L, S, T, M or V; T114 replaced with A, G, I, L, S, M or V; S115 replaced with A, G, I, L, T, M or V; V116 replaced with A, G, I, L, S, T or M, E117 replaced with D; 1118 replaced with A, G, L, S, T, M or V; G119 replaced with A, I, L, S, T, M or V; V120 replaced with A, G, I, L, S, T or M; V121 replaced with A, G, I, L, S, T or M; A122 replaced with G, I, L, S, T, M or V; V123 replaced with A, G, I, L, S, T or M; K124 replaced with H or R; A125 replaced with G, I, L, S, T, M or V; 1126 replaced with A, G, L, S, T, M or V; N127 replaced with Q; S128 replaced with A, G, I, L, T, M 0 V; N129 replaced with Q; Y130 replaced with F o; Y131 replaced with F or W; L132 replaced with A, G, I, S, T, M or V; A133 replaced with G, I, L, S, T, M or V; M134 replaced with A, G, I, L, S, T or V; N135 replaced with Q; K136 replaced with H or R; K137 replaced with H or R; G138 replaced with A, I, L, S, T, M or V; K139 replaced with H or R; L140 replaced with A, G, I, S, T, M or V; Y141 replaced with F o; G142 replaced with A, I, L, S, T, M or V; S143 replaced with A, G, I, L, T, M or V; K144 replaced with H or R; E145 replaced with D; F146 replaced with W or Y; N147 replaced with Q; N148 replaced with Q; D149 replaced with E; K151 replaced with H or R; L152 replaced with A, G, I, S, T, M or V; K153 replaced with H O R; E154 replaced with D; R155 replaced with H or K, 1156 replaced with A, G, L, S, T, M or V; E157 replaced with D; E158 replaced with D; N159 replaced with Q; G160 replaced with A, I, L, S, T, M or V; Y161 replaced with F o; N162 replaced with Q; T163 replaced with A, G, I, L, S, M or V; Y164 replaced with F o; A165 replaced with G, I, L, S, T, M or V; S166 replaced with A, G, I, L, T, M or V; F167 replaced with or Y; N168 replaced with Q; 169 replaced with F or Y; Q170 replaced with N; H171 replaced with K or R; N172 replaced with Q; G173 replaced with A, I, L, S, T, M or V; R174 replaced with H or K; Q175 replaced with N; M176 replaced with A, G, I, L, S, T or V; Y177 replaced with F or W; V178 replaced with A, G, I, L, S, T or M; A179 replaced with G, I, L, S, T, M or V; L180 replaced with A, G, I, S, T, M or V; N181 replaced with Q; G182 replaced with A, I, L, S, T, M or V; K183 replaced with H or R; G184 replaced with A, I, L, S, T, M or V; Al85 replaced with G, I, L, S, T, M or V; R187 replaced with H or K; R188 replaced with H or K; G189 replaced with A, I, L, S, T, M or V; Q190 replaced with N; K191 replaced with H or R; T192 replaced with A, G, I, L, S, M or V; R193 replaced with H or R; R194 replaced with H or K; K195 replaced with H or R; N196 replaced with Q; T197 replaced with A, G, I, L, S, M or V; S198 replaced with A, G, I, L, T, M or V; A199 replaced with G, I, L, S, T, M or V; H200 replaced with K or R; F201 replaced with or Y; L202 replaced with A, G, I, S, T, M or V; M204 replaced with A, G, I, L, S, T or V, V205 replaced with A, G, I, L, S, T or M; V206 replaced with A, G, I, L, S, T or M; H207 replaced with K or R; or S208 replaced with A, G, I, L, T, M or V. However, KGF-2 molecules with non-conservative amino acid substitutions are also preferred, including: Ml replaced with D, E, H, K, R, N, Q, F,, Y, P or C; 2 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; K3 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F,, Y, P or C; 4 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; 15 replaced with D, E, H, K, R, N, Q, F,, Y, P or C; L6 replaced with D, E, H, K, R, N, Q, F,, Y, P or C; T7 replaced with D, E, H, K, R, N, Q, F,, Y, P or C; H8 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; C9 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F,, Y or P; AlO replaced with D, E, H, R, N, Q, F,, Y, P or C; Sil replaced with D, E, H, K, R, N, Q, F,, Y, P or C; A12 replaced with D, E, H, K, R, N, Q, F,, Y, P or C; F13 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; P14 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F,, Y or C; H15 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F,, Y, P or C; L16 replaced with D, E, H, K, R, N, Q, F,, Y, P or C; P17 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F,, Y or C; G18 replaced with D, E, H, K, R, N, Q, F,, Y, P or C; C19 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or P; C20 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F,, Y or P; C21 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F,, Y or P; C22 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F,, Y or P; C23 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F,, Y or P; F24 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; L25 replaced with D, E, H, K, R, N, Q, F,, Y, P or C; L26 replaced with D, E, H, K, R, N, Q, F,, Y, P or C; L27 replaced with D, E, H, K, R, N, Q, F,, Y, P or C; F28 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; L29 replaced with D, E, H, K, R, N, Q, F,, Y, P or C; V30 replaced with D, E, H, K, R, N, Q, F,, Y, P or C; S31 replaced with D, E, H, K, R, N, Q, F,, Y, P or C; S32 replaced with D, E, H, K, R, N, Q, F,, Y, P or C; V33 replaced with D, E, H, K, R, N, Q, F,, Y, P or C; P34 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F,, Y or C; V35 replaced with D, E, H, K, R, N, Q, F,, Y, P or C; T36 replaced with D, E, H, K, R, N, Q, F,, Y, P or C; C37 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F,, Y or P; Q38 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F,, Y, P or C; A39 replaced with D, E, H, K, R, N, Q, F,, Y, P or C; L40 replaced with D, E, H, K, R, N, Q, F,, Y, P or C; G41 replaced with D, E, H, K, R, N, Q, F,, Y, P or C; Q42 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F,, Y, P or C; D43 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F,, Y, P or C; M44 replaced with D, E, H, K, R, N, Q, F, W, Y, P or C; V45 replaced with D, E, H, K, R, N, Q, F,, Y, P or C; S46 replaced with D, E, H, K, R, N, Q, F,, Y, P or C; P47 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F,, Y or C; E48 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; A49 replaced with D, E, H, K, R, N, Q, F,, Y, P or C; T50 replaced with D, E, H, K, R, N, Q, F,, Y, P or C; N51 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F,, Y, P or C; S52 replaced with D, E, H, K, R, N, Q, F,, Y, P or C; S53 replaced with D, E, H, K, R, N, Q, F,, Y, P or C; S54 replaced with D, E, H, K, R, N, Q, F,, Y, P or C; S55 replaced with D, E, H, K, R, N, Q, F,, Y, P or C; S56 replaced with D, E, H, K, R, N, Q, F,, Y, P or C; F57 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; S58 replaced with D, E, H, K, R, N, Q, F,, Y, P or C; S59 replaced with D, E, H, K, R, N, Q, F,, Y, P or C; P60 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F,, Y or C; S61 replaced with D, E, H, K, R, N, Q, F,, Y, P or C; S62 replaced with D, E, H, K, R, N, Q, F,, Y, P or C; A63 replaced with D, E, H, K, R, N, Q, F,, Y, P or C; G64 replaced with D, E, H, K, R, N, Q, F,, Y, P or C; R65 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F,, Y, P or C; H66 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F,, Y, P or C; V67 replaced with D, E, H, K, R, N, Q, F,, Y, P or C; R68 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F,, Y, P or C; S69 replaced with D, E, H, K, R, N, Q, F, W, Y, P or C; Y70 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; N71 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F,, Y, P or C; H72 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F,, Y, P or C; L73 replaced with D, E, H, K, R, N, Q, F,, Y, P or C; Q74 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F,, Y, P or C; G75 replaced with D, E, H, K, R, N, Q, F, W, Y, P or C; D76 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; V77 replaced with D, E, H, K, R, N, Q, F,, Y, P or C; R78 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F,, Y, P or C; 79 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; R80 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F,, Y, P or C; K81 replaced with D, E, A, G, I, L, S, T, M, N, Q, F,, Y, V, P or C; L82 replaced with D, E, H, K, R, N, Q, F,, Y, P or C; F83 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; S84 replaced with D, E, H, K, R, N, Q, F,, Y, P or C; F85 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; T86 replaced with D, E, H, K, R, N, Q, F, W, Y, P or C; K87 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F,, Y, P or C; Y88 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; F89 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; L90 replaced with D, E, H, K, R, N, Q, F,, Y, P or C; K91 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F,, Y, P or C; 192 replaced with D, E, H, K, R, N, Q, F, W, Y, P or C; E93 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F,, Y, P or C; K94 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F,, Y, P or C; N95 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F,, Y, P or C; G96 replaced with D, E, H, K, R, N, Q, F,, Y, P or C; K97 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F,, Y, P or C; V98 replaced with D, E, H, K, R, N, Q, F,, Y, P or C; S99 replaced with D, E, H, K, R, N, Q, F,, Y, P or C; G100 replaced with D, E, H, K, R, N, Q, F,, Y, P or C; T101 replaced with D, E, H, K, R, N, Q, F, W, Y, P or C; K102 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F,, Y, P or C; K103 replaced with D, E, H, A, G, I, L, S, T, M, V, N, Q, F,, Y, P or C; E104 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F,, Y, P or C; N105 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F,, Y, P or C; C106 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or P; P107 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F,, Y or C; Y108 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; S109 replaced with D, E, H, K, R, N, Q, F,, Y, P or C; 1110 replaced with D, E, H, K, R, N, Q, F, W, Y, P or C; Lili replaced with D, E, H, K, R, N, Q, F,, Y, P or C; E112 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; 1113 replaced with D, E, H, K, R, N, Q, F,, Y, P or C; T114 replaced with D, E, H, K, R, N, Q, F,, Y, P or C; S115 replaced with D, E, H, K, R, N, Q, F,, Y, P or C; V116 replaced with D, E, H, K, R, N, Q, F,, Y, P or C; E117 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F,, Y, P or C; 1118 replaced with D, E, H, K, R, N, Q, F,, Y, P or C; G119 replaced with D, E, H, K, R, N, Q, F, W, Y, P or C; V120 replaced with D, E, H, K, R, N, Q, F,, Y, P or C; V121 replaced with D, E, H, K, R, N, Q, F,, Y, P or C; A122 replaced with D, E, H, K, R, N, Q, F,, Y, P or C; V123 replaced with D, E, H, K, R, N, Q, F, W, Y, P or C; K124 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F,, Y, P or C; A125 replaced with D, E, H, K, R, N, Q, F,, Y, P or C; 1126 replaced with D, E, H, K, R, N, Q, F, W, Y, P or C; N127 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F,, Y, P or C; S128 replaced with D, E, H, K, R, N, Q, F,, Y, P or C; N129 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; Y130 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; Y131 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; L132 replaced with D, E, H, K, R, N, Q, F,, Y, P or C; A133 replaced with D, E, H, K, R, N, Q, F,, Y, P or C; M134 replaced with D, E, H, K, R, N, Q, F, W, Y, P or C; N135 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F,, Y, P or C; K136 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F,, Y, P or C; K137 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F,, Y, P or C; G138 replaced with D, E, H, K, R, N, Q, F,, Y, P or C; K139 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F,, Y, P or C; L140 replaced with D, E, H, K, R, N, Q, F,, Y, P or C; Y141 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; G142 replaced with D, E, H, K, R, N, Q, F,, Y, P or C; S143 replaced with D, E, H, K, R, N, Q, F,, Y, P or C; K144 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F,, Y, P or C; E145 replaced with H, K, A, G, I, L, S, T, M, V, N, Q, F,, Y, P or C; F146 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; N147 replaced with D, E, H, K, A, G, I, L, S, T, M, V, F,, Y, P or C; N148 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F,, Y, P or C; D149 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F,, Y, P or C; C150 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F,, Y or P; K151 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F,, Y, P or C; L152 replaced with D, E, H, K, R, N, Q, F,, Y, P or C; K153 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; E154 replaced with H, K, A, G, I, L, S, T, M, V, N, Q, F,, Y, P or C; R155 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F,, Y, P or C; 1156 replaced with D, E, H, K, R, N, Q, F, W, Y, P or C; E157 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F,, Y, P or C; E158 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F,, Y, P or C; N159 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F,, Y, P or C; G160 replaced with D, E, H, K, R, N, Q, F,, Y, P or C; Y161 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; N162 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F,, Y, P O C; T163 replaced with D, E, H, K, R, N, Q, F,, Y, P or C; Y164 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; A165 replaced with D, E, H, K, R, N, Q, F,, Y, P or C; S166 replaced with D, E, H, K, R, N, Q, F,, Y, P or C; F167 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; N168 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F,, Y, P or C; 169 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; Q 170 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F,, Y, P or C; H171 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F,, Y, P or C; N172 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F,, Y, P or C; G173 replaced with D, E, H, K, R, N, Q, F, W, Y, P or C; R174 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F,, Y, P or C; Q175 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F,, Y, P or C; M176 replaced with D, E, H, K, R, N, Q, F,, Y, P or C; Y177 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; V178 replaced with D, E, H, K, R, N, Q, F,, Y, P or C; A179 replaced with D, E, H, K, R, N, Q, F,, Y, P or C; L180 replaced with D, E, H, K, R, N, Q, F,, Y, P or C; N181 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; G182 replaced with D, E, H, K, R, N, Q, F,, Y, P or C; K183 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F,, Y, P or C; G184 replaced with D, E, H, K, R, N, Q, F,, Y, P or C; A185 replaced with D, E, H, K, R, N, Q, F,, Y, P or C; P186 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F,, Y, or C; R187 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F,, Y, P or C; R188 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F,, Y, P or C; G189 replaced with D, E, H, K, R, N, Q, F,, Y, P or C; Q190 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; K191 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F,, Y, P or C; T192 replaced with D, E, H, K, R, N, Q, F,, Y, P or C; R193 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F,, Y, P or C; R194 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F,, Y, P or C; K195 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F,, Y, P or C; N196 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F,, Y, P or C; T197 replaced with D, E, H, K, R, N, Q, F,, Y, P or C; S198 replaced with D, E, H, K, R, N, Q, F,, Y, P or C; A199 replaced with D, E, H, K, R, N, Q, F,, Y, P or C; H200 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; F201 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; L202 replaced with D, E, H, K, R, N, Q, F,, Y, P or C; P203 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; M204 replaced with D, E, H, K, R, N, Q, F,, Y, P or C; V205 replaced with D, E, H, K, R, N, Q, F,, Y, P or C; V206 replaced with D, E, H, K, R, N, Q, F,, Y, P or C; H207 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F,, Y, P or C; or S208 replaced with D, E, H, K, R, N, Q, F,, Y, P or C. Substitution mutants can be tested in any of the assays described herein for activity. Particularly preferred are KGF-2 molecules with conservative substitutions that maintain the activities and properties of the wild-type protein; have an improved activity or property compared to the wild-type protein, while all other activities or properties are maintained; or have more than one activity or improved property compared to the wild-type protein. In contrast, KGF-2 molecules with non-conservative substitutions preferably lack an activity or property of the wild-type protein, while maintaining all other activities and properties; or lack more than one activity or property of the wild-type protein. For example, the activities or properties of KGF-2 can be altered in KGF-2 molecules with conservative or non-conservative substitutions include, but are not limited to: growth stimulation of keratinocytes, epithelial cells, hair follicles, hepatocytes, cells kidney, breast tissue, bladder cells, prostate cells, pancreatic cells; stimulation of the differentiation of muscle cells, nervous tissue, prostate cells, lung cells, hepatocytes, renal cells, breast tissue; promotion of wound healing; stimulation of angiogenesis; reduction of inflammation; cytoprotection; link to heparin; link to the ligand; stability; solubility; and / or properties that affect purification. The amino acids in KGF-2 that are essential for function can be identified by methods well known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (Cunningham and Wells, Science 244: 1081-1085 ( 1989)). The last procedure introduces simple alanine mutations in each residue in the molecule. The resulting mutant molecules are then tested for biological activity such as receptor binding or proliferative activity in vi tro and in vivo. (See for example, Examples 10 and 11). Sites that are critical for ligand-receptor binding can also be determined by structural analysis such as crystallization, nuclear magnetic resonance or photoaffinity labeling. (See for example: Smith et al., J. "Mol. Biol. 224: 899-904 (1992), and de Vos et al., Science, 255: 306-312 (1992).) Another aspect of the substitutions of the present invention of serine by cysteine at amino acid positions 37 and 106 and 150. A non-uniform number of cysteines means that at least one cysteine residue is available for intermolecular crosslinks or linkages that can cause the protein to adopt a tertiary structure The new KGF-2 proteins that have one or more cysteines replaced by serine or for example alanine are generally purified at a higher yield of the correctly folded, soluble protein.Although it is not tested, it is believed that the cysteine residue at position 106 it is important for function.This cysteine residue is highly conserved among all other members of the FGF family.An additional aspect of the present invention are the K mergers. GF-2 with other proteins or fragments thereof, such as fusions or hybrids with other FGF proteins, for example KGF (FGF-7), bFGF, aFGF, FGF-5, FGF-6, etc. Such a hybrid has been reported for KGF (FGF-7). In the published PCT application number 90/08771, a chimeric protein consisting of the first 40 amino acid residues of KGF and the C-terminal portion of aFGF has been produced. It has been reported that the chimera targets keratinocytes such as KGF, but lacked the susceptibility to heparin, a characteristic of aFGF but not of KGF. Fusions with parts of the constant domain of immunoglobulins (IgG) often show an increased half-life in vivo. This has been shown, for example, for chimeric proteins consisting of the first two domains of the human CD4 polypeptide with several domains of the constant regions of the heavy and light chains of mammalian immunoglobulins (European Patent Application, Publication No. 394827, Traunecker et al., Nature 331: 84-86 (1988) .Fusion proteins having a dimeric structure linked by disulfide bridges can also be more efficient in binding to monomeric molecules alone (Fountoulakis et al., J ". of Biochemistry, 270: 3958-3964 (1995).) Additional fusion proteins of the invention can be generated through the techniques of gene intermixing, intermixing of portions, intermixing of exons, and / or intermixing of codons (collectively referred to as "DNA intermixing") DNA intermixing can be employed to modulate the activities of the polypeptides of the invention, Methods can be used to generate the polypeptides with altered activity, as well as the agonists and antagonists of the polypeptides. See in general, U.S. Patent Nos. 5,605,793; 5,811,238; 5,830,721; 5,834,252; and 5,837,458, and Patten et al., Curr. Opinion Biotechnol. 8: 724-33 (1997); Harayama, Trends Biotechnol. 16 (2): 76-82 (1998); Hansson et al., J.

Mol. Biol. 287: 265-76 (1999); and Lorenzo and Blasco, Biotechnologies 24 (2): 308-13 (1998) (each of these patents and publications are incorporated by reference herein, in their entirety). In one embodiment, the alteration of the polynucleotides corresponding to SEQ ID No. 1 and the polypeptides encoded by these polynucleotides can be achieved by DNA intermixing. DNA intermixing involves the assembly of two or more DNA segments by homologous or site-specific recombination, to generate variation in the polynucleotide sequence. In yet another embodiment, the polynucleotides of the invention, or the encoded polypeptides, can be altered by being subjected to random mutagenesis by error prone PCR, random insertion of nucleotides or other methods prior to recombination. In yet another embodiment, one or more components, portions, sections, parts, domains, fragments, etc., of a polynucleotide encoding a polypeptide of the invention can be recombined with one or more components, portions, sections, parts, domains , fragments, etc., of one or more heterologous molecules.

Antigenic / hydrophilic parts of KGF-2 As shown in Figure 4A-4E, there are 4 major highly hydrophilic regions in the KGF-2 protein. The amino acid residues Gly41-Asn71, Lys91-Ser 109, Asnl35-Tyrl64 and Asnl81-Alal99 [SEQ ID Nos. 25-28]. There are two additional, shorter, predicted antigenic areas, Gln74-Arg78 of Figure 1 (SEQ ID No. 2) and Glnl70-Glnl75 of Figure 1 (SEQ ID No. 2). The hydrophilic parts are known to be mainly on the outside (surface) of the proteins and, therefore, available for recognition by antibodies from these regions. It is likely that these regions are also involved in the link of KGF-2 to his or her receivers. Synthetic peptides derived from these areas can interfere with the binding of KGF-2 to its or its receptors and, therefore, block the function of the protein. Synthetic peptides from hydrophilic portions of the protein can also be agonists, for example mimic the function of KGF-2. Thus, the present invention is further directed to isolated polypeptides comprising a hydrophilic region of KGF-2 wherein said polypeptide is not greater than 150 amino acids in length, preferably not greater than 100, 75 or 50 amino acids in length, comprising one or more of the hydrophilic regions of KGF-2 described above.

Portions of KGF-2 that possess Epitope In still another aspect, the invention provides the peptides and polypeptides comprising the epitope-possessing portions of the polypeptides of the present invention. These epitopes are immunogenic or antigenic epitopes of the polypeptides of the present invention. An "immunogenic epitope" is defined as a part of a protein that promotes an antibody response in vivo when the complete polypeptide of the present invention, or fragment thereof, is the immunogen. On the other hand, the region of a polypeptide to which an antibody can be linked is defined as an "antigenic determinant" or "antigenic epitope". The number of immunogenic epitopes in vivo of a protein is generally less than the number of antigenic epitopes. See, for example, Geysen et al., Proc. Nati Acad. Sci. USA 81: 3998-4002 (1983). However, the antibodies can be made for any antigenic epitope, regardless of whether it is an immunogenic epitope, by using methods such as phage display. See, for example, Petersen G. et al., Mol. Gen Genet 249: 425-431 (1995). Therefore, immunogenic epitopes and antigenic epitopes are included in the present invention. A list of exemplified amino acid sequences comprising the immunogenic epitopes are in Table 1 below. It is noted that Table 1 only lists the amino acid residues comprising epitopes that are predicted to have the highest degree of antigenicity using the algorithm of Jameson and Wolf (1988) Comp. Appl. Biosci. 4: 181-186 (said references are incorporated by reference in the present, In its whole) . The antigenic analysis of Jameson-Wolf was performed using the PROTEAN computer program, using the default parameters (Version 3.11 for Power Macintosh, DNASTAR, Inc., 1228 South Park Street Madison, Wl). Table 1 and portions of the polypeptides not listed in Table 1 are not considered non-immunogenic. The immunogenic epitopes of Table 1 are an exemplified list, not an exhaustive list, because other immunogenic epitopes are merely unrecognized such as the particular algorithm used. Amino acid residues comprising other immunogenic epitopes can be routinely determined using algorithms similar to Jameson-Wolf analysis or by in vivo testing for an antigenic response using methods known in the art. See, for example, Geysen et al., Supra; U.S. Patent Nos. 4,708,781; 5,194,394; 4,433,092; and 5,480,971 (such references are incorporated by reference in their entirety). The peptides and polypeptides possessing the antigenic epitope of the invention preferably contain a sequence of at least seven, more preferably at least nine and most preferably between about 15 to about 30 amino acids contained within the amino acid sequence of a polypeptide of the invention . Non-limiting examples of antigenic polypeptides or peptides that can be used for KGF-2 specific antibodies include: a polypeptide comprising the amino acid residues in SEQ ID No. 2 of about Gly41-Asn71; Lys91-Serl09; Asnl35-Tyrl64; Asnl81-Alal99; Gln74-Arg78; and Glnl70-Glnl75. These polypeptide fragments have been determined to possess antigenic epitopes of the KGF-2 protein by analysis of the Jameson-Wolf antigenic index, as shown in Figure 4 above. It is particularly noted that the amino acid sequences of Table 1 comprise immunogenic epitopes. Table 1 lists only the critical residues of the immunogenic epitopes determined by the Jameson-Wolf analysis. Thus, additional flanking residues on the N-terminus, C-terminus or both, and the C-terminal ends can be added to the sequences of Table 1 to generate a polypeptide possessing epitope of the present invention. Therefore, the immunogenic epitopes of Table 1 may include additional N-terminal or C-terminal amino acid residues. Additional flanking amino acid residues may be flanking, N-terminal and / or C-terminal sequences, contiguous from the polypeptides of the present invention, heterologous polypeptide sequences, or may include contiguous flanking sequences from the polypeptides herein invention and the heterologous polypeptide sequences. The polypeptides of the present invention comprising the immunogenic or antigenic epitopes are at least 7 amino acid residues in length. "At least" means that a polypeptide of the present invention comprising an immunogenic or antigenic epitope can be 7 amino acid residues in length or any whole number between 7 amino acids and the number of amino acid residues of the full length polypeptides of the invention. Preferred polypeptides comprising the immunogenic or antigenic epitopes are at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 amino acid residues in length. However, it is pointed out that each and every one of the integers between 7 and the number of amino acid residues of the full-length polypeptide are included in the present invention. The immunogenic and antigenic epitope-possessing fragments can be specified either by the number of contiguous amino acid residues, as described above, or further specified by the N-terminal and C-terminal positions of these fragments on the amino acid sequence of the SEQ ID No. 2. Each combination of an N-terminal and C-terminal position that a fragment for example of at least 7 or at least 15 contiguous amino acid residues in length could occupy on the amino acid sequence of SEQ ID No. 2, is included in the invention. Again, "at least 7 contiguous amino acid residues in length" means 7 amino acid residues in length or any integer among 7 amino acids and the number of amino acid residues of the full length polypeptide of the present invention. Specifically, each and every one of the integers between 7 and the number of amino acid residues of the full-length polypeptide is included in the present invention. Polypeptides possessing immunogenic and antigenic epitopes of the invention are useful, for example, to make antibodies that specifically bind to the polypeptides of the invention, and in immunoassays to detect the polypeptides of the present invention. The antibodies are useful, for example, in affinity purification of the polypeptides of the present invention. The antibodies can also be routinely used in a variety of qualitative or quantitative immunoassays, specifically for the polypeptides of the present invention using methods known in the art. See, for example, Harlow et al., Antibodies: A Laboratory Manual, Col Spring Harbor Laboratory Press; 2a. Ed, Cold Spring Harbor, NY (1988). The epitope-possessing polypeptides of the present invention can be produced by any conventional means for the manufacture of polypeptides including synthetic and recombinant methods known in the art. For example, epitope-possessing peptides can be synthesized using the known methods of chemical synthesis. For example, Houghten has described a simple method for the synthesis of large numbers of peptides, such as 10-20 mgs of 248 individual peptides and 13 distinct residues, representing simple amino acid variants of a HAl polypeptide segment, all of which were prepared and characterized (by ELISA-type linkage studies) in less than four weeks (Houghten, RA, Proc. Nati. Acad. Sci. USA 82: 5131-5135 (1985)). This process of "Simultaneous Multiple Peptide Synthesis (SMPS)" is further described in U.S. Patent No. 4,631,211 to Houghten et al. (1986). In this process, the individual resins for the solid phase synthesis of various peptides are contained in separate solvent permeable packages, which make possible the optimal use of the many identical repetitive steps involved in the solid phase methods. A completely manual procedure allows 500-1000 or more synthesis to be conducted simultaneously (Houghten et al. (1985) Proc. Nati. Acad. Sci. 82: 5131-5135 in 5134). The epitope-possessing polypeptides of the present invention can be used to induce antibodies according to methods well known in the art including, but not limited to, in vivo immunization, in vi tro immunization, and phage display methods. See for example, Sutcliffe et al., Supra, Wilson et al., Supra, and Bittle et al., J ". Gen. Virol., 66: 2347-2354 (1985) .If immunization is used in vivo, animals can be immunized. with the free peptide, however, the anti-peptide antibody titer can be enhanced by the coupling of the peptide to a macromolecular carrier, such as the lame hemocyanin.

(KLH) or tetanus toxoid. For example, peptides containing cysteine residues can be coupled to a carrier using a linker such as the m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other peptides can be coupled to the carriers, using a carrier agent. more general link such as glutaraldehyde. Animals such as rabbits, rats and mice are immunized either with the free peptides or coupled to the carrier, for example, by intraperitoneal and / or intradermal injection of the emulsions comprising approximately 100 μg of the peptide or carrier protein and Freund's adjuvant or any other adjuvant known to stimulate an immune response. Various reinforcement injections may be necessary, for example, at intervals of about two weeks, to provide a useful titer of the anti-peptide antibody that can be detected, for example, by ELISA assay using the free peptide adsorbed to a solid surface. . The anti-peptide antibody titre in serum from an immunized animal can be increased by the selection of the anti-peptide antibodies, for example, by adsorption to the peptide on a solid support and elution of the antibodies selected according to the methods well known in the art. As a person skilled in the art will appreciate, as discussed above, polypeptides of the present invention comprising an immunogenic or antigenic epitope can be fused to other polypeptide sequences. For example, the polypeptides of the present invention can be fused to the constant domain of immunoglobulins (IgA, IgE, IgG, IgM), or portions thereof (CH1, CH2, CH3, or any combination thereof and portions thereof). the same) resulting in chimeric polypeptides. Such fusion proteins can facilitate purification and can increase the half-life in vivo. This has been shown for chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins. See for example, European Patent 394,827; Traunecker et al., Nature 331: 84-86 (1988). The improved distribution of an antigen through the epithelial barrier to the immune system has been demonstrated for antigens (eg, insulin) conjugated to an FcRn binding partner such as IgG or Fe fragments (see for example, PCT Publications WO 96/22024 and WO 99/04813). IgG fusion proteins that have a dimeric structure linked by disulfide sources due to the disulfide bonds of the IgG portion, have also been found to be more efficient in binding and neutralizing other molecules than monomeric polypeptides or fragments thereof alone See, for example, Fountoulakis et al., J. "Biochem., 270: 3958-3964 (1995) .The nucleic acids encoding the above epitopes can also be recombined with a gene of interest as an epitope tag (e.g. label or marker of hemagglutinin ("HA) or flag tag) to aid in the detection and purification of the expressed polypeptide. For example, a system described by Janknech et al. Allows the easy purification of non-denatured fusion proteins expressed in human cell lines (Janknecht et al., 1991, Proc. Nati, Acad. Sci. USA 88: 8972-897). . In this system, the gene of interest is subcloned into a vaccinia recombination plasmid such that the open reading structure of the gene is translationally fused to an amino-terminal tag or label consisting of six histidine residues. The tag serves as a binding domain to the matrix for the fusion protein. Extracts from the cells infected with the recombinant vaccinia virus are loaded onto the column of nitriloacetic acid Ni2 + -agarose and the proteins marked with histidine can be selectively eluted with imidazole-containing buffers.

Chemical modifications The wild-type KGF and the analogs can also be modified to contain additional chemical portions not normally part of the protein. Those derivatized portions can improve the solubility, the biological half-life or the absorption of the protein. The portions may also reduce or eliminate any desirable side effects of the proteins and the like. A general overview for those portions can be found in REMINGTON'S PHARMACEUTICAL SCIENCES, 18th ed. , Mack Publishing Co., Easton, PA (1990). Polyethylene glycol (PEG) is such a chemical moiety that has been used for the preparation of therapeutic proteins. Coupling of PEG to proteins has been shown to protect against proteolysis, Sada et al., J. Fermentation Bioengineering 71: 137-139 (1991). Various methods are available for coupling certain portions of PEG. For a review, see Abuchowski et al., In Enzymes as Drugs. (Holcerberg and Roberts, eds.) Pp. 367-383 (1981). Many published patents describe PEG derivatives and processes of how to prepare them, for example Ono et al., U.S. Patent No. 5,342,940; Nitecki et al., U.S. Patent No. 5,089,261; Delgado et al., United States Patent No. 5,349,052. In general, the PEG molecules are connected to the protein via a reactive group found on the protein. Amino groups, for example, on lysines or the amino terminus of the protein are suitable for this coupling, among others. The full description of each document cited in this section in "Polypeptides and Peptides" is incorporated by reference herein. In addition, the polypeptides of the invention can be chemically synthesized using techniques known in the art (for example, see Creighton, 1983, Proteins: Structures and Molecular Principles, WH Freeman and Co., NY, and Hunkapiller et al., Nature, 310: 105-111 (1984)) . For example, a polypeptide corresponding to a fragment of a KGF-2 polypeptide can be synthesized by the use of a peptide synthesizer. In addition, if desired, non-classical amino acids or chemical analogues of amino acids can be introduced as a substitution or addition within the polypeptide sequence of KGF-2. Non-classical amino acids include, but are not limited to, the D-isomers of the common amino acids, 2,4-diaminobutyric acid, α-aminoisobutyric acid, 4-aminobutyric acid, Abu, 2-aminobutyric acid, g- Abu, e-Ahx, 6-aminohexanoic acid, Aib, 2-aminoisobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulin, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, b-alanine, fluoroamino acids, designer amino acids such as b-methylamino acids, Ca-methylamino acids, Na-methylamino acids, and amino acid analogs in general. In addition, the amino acid can be D (dextrorotatory) or L (levorotatory). The invention encompasses KGF-2 polypeptides that are differentially modified during or after translation, for example, by glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, binding to an antibody molecule or other cellular ligand, etc. Any of the numerous chemical modifications can be carried out by known techniques, including but not limited to, the specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH4; acetylation, formylation, oxidation, reduction; metabolic synthesis in the presence of tunicamycin; etc. Additional post-translational modifications encompassed by the invention include, for example, the N-linked or O-linked carbohydrate chains, the processing of the N-terminal or C-terminal ends), the coupling of chemical portions to the main amino acid chain, chemical modifications of the N-linked or O-linked carbohydrate chains, and addition or deletion of an N-terminal methionine residue as a result of expression in prokaryotic host cells. The polypeptides can also be modified with a detectable label, such as an enzymatic, fluorescent, isotopic or affinity tag to allow detection and isolation of the protein. Also, provided by the invention are the chemically modified derivatives of the polypeptides of the invention which may provide additional advantages such as increased solubility, stability and circulation time of the polypeptide, or decreased immunogenicity (See United States Patent No. 4,179,337). The chemical portions for derivatization can be selected from water-soluble polymers such as polyethylene glycol, ethylene glycol / propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol and the like. The polypeptides can be modified at random positions within the molecule, or at predetermined positions within the molecule and can include one, two, three or more linked chemical moieties. The polymer can be of any molecular weight, and can be branched or unbranched. For polyethylene glycol, the preferred molecular weight is between about 1 kDa and about 100 kDa (the term "about" indicates that in polyethylene glycol preparations, some molecules will weigh more, some less, than molecular weight) for ease of handling and in manufacturing. Other sizes may be used, depending on the desired therapeutic profile (for example, the duration of the desired sustained release, the effects, if any, on biological activity, ease of handling, degree or lack of antigenicity and other effects). known polyethylene glycol to a therapeutic or analogous protein). The polyethylene glycol molecules (or other chemical moieties) must be coupled to the protein with consideration of the effects on the functional or antigenic domains of the protein. There are a number of linking or coupling methods available to those skilled in the art, see for example, European Patent 0 401 384, incorporated by reference herein (coupling of PEG to G-CSF), see also Malik et al. Exp. Hematol. 20: 1028-1035 (1992) (which reports pegylation of GM-CSF using tresyl chloride). For example, the p-ethylene glycol can be covalently linked through amino acid residues via a reactive group, such as a free amino or carboxyl group. The reactive groups are those to which an activated polyethylene glycol molecule can be linked. The amino acid residues having a free amino group can include lysine residues and the N-terminal amino acid residues; those having a free carboxyl group may include aspartic acid residues, glutamic acid residues and the C-terminal amino acid residue. The sulfhydryl groups can also be used as a reactive group for coupling the polyethylene glycol molecules. Preferred for therapeutic purposes is the linkage to an amino group, such as the N-terminal bond or the lysine group. Someone may specifically desire the chemically modified proteins at the N-terminus. Using polyethylene glycol as an illustration of the present composition, one may select from a variety of polyethylene glycol molecules (by molecular weight, branching, etc.), the proportion of molecules of polyethylene glycol to the protein (polypeptide) molecules in the reaction mixture, the type of pegylation reaction to be performed, and the method of obtaining the N-terminally selected pegylated protein. The method for obtaining the N-terminally pegylated preparation (e.g., separation of this portion from other monopegylated portions if necessary) can be by purification of the N-terminally pegylated material from a population of pegylated protein molecules. The proteins selectively chemically modified at the N-terminus can be obtained by reductive alkylation which exploits the differential reactivity of different types of primary amino groups (lysine versus the N-terminus) available for derivatization in a particular protein. Under the appropriate reaction conditions, the highly selective derivatization of the N-terminal protein with a carbonyl group containing the polymer is achieved.

Antibodies Additional polypeptides of the invention refer to antibodies and T-cell antigen (TCR) receptors that immunospecifically bind to a polypeptide, polypeptide fragment, or variant of SEQ ID No. 2, and / or an epitope, of the present invention (as determined by immunoassays well known in the art for evaluating specific antibody-antigen binding). Antibodies of the invention include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized or chimeric antibodies, single chain antibodies, Fab fragments, F (ab ') fragments, fragments produced by a Fab expression library. , anti-idiotypic antibodies (anti-Id) (including, for example, anti-Id antibodies for the antibodies of the invention), and fragments of binding to the epitope of any of the foregoing. The term "antibody" as used herein, refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, for example, molecules that contain an antigen binding site that binds immunospecifically to an antigen. . The immunoglobulin molecules of the invention can be of any type (eg, IgG, IgE, IgM, IgD, IgA and IgY), class (eg, IgG1, IG2, IgG3, IgG4, IgA1 and IgA2) or subclass of molecule of immunoglobulin. More preferably, the antibodies are the antigen binding human antibody fragments of the present invention and include, but are not limited to, Fab, Fab 'and F (ab') 2, Fd, single chain Fvs (scFv) , single chain antibodies, Fvs linked by disulfide bridges (sdFv) and frames comprising either a VL or a VH domain. The antigen binding antibody fragments, including the single chain antibodies, may comprise the variable regions or regions alone or in combination with all or a portion of the following: the hinge region, the CH1, CH2 and CH3 domains. Also included in the invention are the antigen binding fragments which also comprise any combination of one or more variable regions with a hinge region, or CH1, CH2 and CH3 domains. The antibodies of the invention can be from any animal origin including birds and mammals. Preferably, the antibodies are human, murine (e.g., mouse and rat), donkey, sheep, rabbit, goat, cobalt, camel, horse, or chicken. As used herein, "human" antibodies include antibodies that have the amino acid sequence of a human immunoglobulin, and include antibodies isolated from human immunoglobulin libraries or from animals transgenic for one or more human immunoglobulins, and which do not express endogenous immunoglobulins, as described below and, for example, in U.S. Patent No. 5,939,598 by Kucherlapati et al. The antibodies of the present invention can be monospecific, bispecific, trispecific or of a greater multispecificity. The multispecific antibodies may be specific for different epitopes of a polypeptide of the present invention or may be specific for a polypeptide of the present invention as well as for a heterologous epitope, such as a heterologous polypeptide or a solid support material. See, for example, PCT publications WO 93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt et al., J. "Immunol., 147: 60-69 (1991), U.S. Patents Nos. 4,474,893, 4,174,681, 4,925,648, 5,573,920, 5,601,819, Kostenly et al., J". Immunol. 148: 1547-1553 (1992).

The antibodies of the present invention can be described or specified in terms of the epitope (s) or the polypeptide portions (s) of the present invention, which they recognize or specifically bind to. The epitope (s) or the polypeptide portion (s) can be specified as described herein, for example, by the N-terminal and C-terminal positions, by the size in the contiguous amino acid residues, or listed in the Tables and in the Figures. Preferred epitopes of the invention include: amino acids 41-71, 91-109, 135-164, 181-199, 74-78, and 170-175 of SEQ ID No. 2, as well as the polynucleotides encoding these epitopes . Antibodies that bind specifically to any epitope or polypeptide of the present invention can also be excluded. Therefore, the present invention includes antibodies that specifically bind to the polypeptides of the present invention, and allow the exclusion thereof. The antibodies of the present invention can also be described or specified in terms of their cross-reactivity. Antibodies that do not bind to any other analog, ortholog, or homolog of a polypeptide of the present invention are included. Antibodies that bind to the polypeptides with at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at less 55%, and at least 50% identity (as calculated using methods known in the art and described herein) to a polypeptide of the present invention, are also included in the present invention. In specific embodiments, the antibodies of the present invention cross-react with murine, rat and / or rabbit homologs of human proteins and corresponding epitopes thereof. Antibodies that do not bind to polypeptides with less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, and less than 50% identity (as calculated using methods known in the art and described herein) to a polypeptide of the present invention, are also included in the present invention. In a specific embodiment, the cross-radioactivity described above is with respect to any single, specific, antigenic or immunogenic polypeptide, or one or more combinations of 2, 3, 4, 5 or more of the specific antigenic and / or immunogenic polypeptides described in I presented. Also included in the present invention are antibodies that bind to polypeptides encoded by polynucleotides that hybridize to a polynucleotide of the present invention, under stringent hybridization conditions (as described herein). The antibodies of the present invention can also be described or specified in terms of their binding affinity to a polypeptide of the invention. Preferred binding affinities include those with a constant dissociation or Kd of less than 5xl0"2 M, 10" 2 M, 5xl0 ~ 3 M, 10"3 M, 5xl0 ~ 4 M, 10 ~ 4 M, 5xl0" 5 M, 105 M, 5 x 10 ~ 6 M, 10"6 M, 5 x 10" 7 M, 10"7 M, 5 x 10" 8 M, 10"8 M, 5 x 10" 9 M, 10 ~ 9 M, 5 x 10"10 M, 10" 10 M, 5X10"11 M, 10" 11 M, 5 x 10"12 M, 10 ~ 12 M, 5 x 10" 13 M, 10"13 M, 5 x 10 ~ 14 M, 10" 14 M, 5 x 10"15 M, or 10 ~ 15 M. The invention also provides antibodies that competitively inhibit the binding of an antibody to an epitope of the invention, as determined by any method known in the art, for the determination of competitive binding, for example, the immunoassays described in In preferred embodiments, the antibody competitively inhibits binding to the epitope by at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, or at least 50% The antibodies of the present invention have uses that include, but are not limited to, the methods all known in the art for purifying, detecting, and sending the polypeptides of the present invention, including diagnostic and therapeutic methods in vi tro and in vivo. For example, antibodies have use in immunoassays to qualitatively and quantitatively measure the levels of the polypeptides of the present invention in biological samples. See, for example, Harlow et al., ATNIBODIES: A LABORATORY MANUAL, (Cold Spring Harbor Laboratory Press, 2nd Ed. 1988) (incorporated by reference herein). The antibodies of the present invention can be used either alone or in combination with other compositions. The antibodies can also be recombinantly fused to a heterologous polypeptide at the N-terminus or C-terminus, or chemically conjugated (including covalent and non-covalent conjugations) to polypeptides or other compositions. For example, the antibodies of the present invention may be recombinantly fused or conjugated to molecules useful as labels in detection assays and effector molecules such as heterologous polypeptides, drugs, or toxins. See, for example, WO 92/08495; WO 91/14438; WO 89/12624; U.S. Patent No. 5,314,995; and European Patent No. 0,396,387. The antibodies of the present invention can be prepared by any suitable method known in the art. For example, a polypeptide of the present invention or an antigenic fragment thereof can be administered to an animal for the purpose of inducing the production of sera containing polyclonal antibodies. The term "monoclonal antibody" is not limited to the antibodies produced through the hybridoma technology. The term "monoclonal antibody" refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced. Monoclonal antibodies can be prepared using a wide variety of techniques known in the art, including the use of hybridoma, recombinant, and phage visualization technology. Hybridoma techniques include those known in the art and taught in Harlow et al., ANTIBODIES: A LABORATORY MANUAL (Cold Spring Harbor Laboratory Press, 2nd Ed., 1988); Hammerling et al., In: MONOCLONAL ANTIBODIES AND T-CELL HYBRIDOMAS 563-681 (Elsevier, N.Y., 1981) (said references are incorporated by reference in their entirety). The Fab and F (ab ') 2 fragments can be produced by proteolytic cleavage, using enzymes such as papain (to produce the Fab fragments) or pepsin (to produce the F (ab') 2 fragments). Alternatively, the antibodies of the present invention can be produced through the application of recombinant DNA technology and visual phage display, or through synthetic chemistry using methods known in the art. For example, the antibodies of the present invention can be prepared using various methods of phage display, known in the art. In phage display methods, functional antibody domains are displayed on the surface of a phage particle that possesses polynucleotide sequences that encode them. The phage with a desired binding property is selected from a combinatorial repertoire or library of antibodies (eg, human or murine) by direct selection with the antigen, typically the antigen bound or captured to a solid surface or sphere. Phage used in these methods are typically filamentous phage including fd and M13 with Fab, Fv or Fv antibody domains disulfide stabilized, recombinantly fused to either protein gene III or VIII of the phage. Examples of phage display methods that can be used to make the antibodies of the present invention include those described in Brinkman U. et al. (1995), J. Immunol. Methods 182: 41-50; Ames, R.S. et al. (1995) J. "Immunol. Methods 184: 177-186; Kettleborough, CA et al. (1994) Eur. J. Immunol., 24: 952-958; Persic, L. et al., (1997) Gene 187 : 9-18; Burton, DR et al (1994) Advances in Immunology 57: 191-280; PCT / GB91 / 01134; WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93 / 11236; WO 95/15982; WO 95/20401;. and US Patent 5,698,426 We, 5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821.047, 5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727 and 5,733,743 (these references are incorporated by reference in their entirety.) As described in the references above, after phage selection, antibody coding regions from phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen that binds to the fragment, and expressed at any desired host, including mammalian cells, insect cells, plant cells, yeast and bacteria. For example, techniques for recombinantly producing the Fab, Fab 'and F (ab') 2 fragments can also be employed using methods known in the art such as those described in WO 92/22324; Mullinax, R.L. et al. (1992) BioTechniques 12 (6): 864-869, - and Sawai, H. and collaborators (1995) AJRI 34: 26-34; and Better, M. et al., (1988) Science 240: 1041-1043 (said references are incorporated by reference in their entirety). Examples of techniques that can be used to produce single chain Fvs and antibodies include those described in U.S. Patent Nos. 4,496,778 and 5,258,498; Huston et al. (1991) Methods in Enzymology 203: 46-88; Shu, L. and collaborators (1993) PNAS 90: 7995-7999; and Skerra, A. et al. (1988) Science 240: 1038-1040. For some uses, including in vivo use of antibodies in humans, and in vitro detection assays, it may be preferable to use chimeric, humanized, or human antibodies. Methods for producing chimeric antibodies are known in the art. See, for example, Morrison, Science 229: 1202 (1985); Oi et al., BioTechniques 4: 214 (1986); Gillies, S.D. et al., (1989) J. I munol. Methods 125: 191-202 and U.S. Patent No. 5,807,715. The antibodies can be humanized using a variety of techniques including CDR grafting (European Patent 0,239,400; International Patent WO 91/09967; US Patent Nos. 5,530,101; and 5,585,089), coating or reshaping (European Patent Nos. 0,592,106;; 0.519.596; Padlan EA, (1991) Molecular I munology 28 (4/5): 489-498; Studnicka GM et al. (1994) Protein Engineering 7 (6): 805-814; Roguska MA et al. (1994) PNAS 91 : 969-973), and interspersed with chains (U.S. Patent No. 5,565,332). Human antibodies can be made by a variety of methods known in the art, including the phage display methods described above. See also, U.S. Patent Nos. 4,444,887, 4,716,111, 5,545,806 and 5,814,318; WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741 (said references are incorporated by reference in their entirety). The antibodies of the present invention can act as agonists or antagonists of the polypeptides of the present invention. For example, the present invention includes antibodies that perturb receptor / ligand interactions with the polypeptides of the invention, either partially or completely. Preferably, the antibodies of the present invention bind to an antigenic epitope described herein, or a portion thereof. The invention characterizes receptor-specific antibodies and ligand-specific antibodies. The invention also characterizes receptor-specific antibodies that do not prevent binding to the ligand but prevent activation of the receptor. Activation of the receptor (eg, signaling) can be determined by techniques described herein or otherwise known in the art. For example, receptor activation can be determined by detecting the phosphorylation (eg, tyrosine or serine / threonine) of the receptor or its substrate by immunoprecipitation followed by spotting or western blot analysis (eg, as described above). ). In specific embodiments, antibodies are provided by inhibiting ligand activity or receptor activity by at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at less 60%, or at least 50% of the activity in the absence of the antibody. The invention also characterizes receptor-specific antibodies which prevent ligand binding and receptor activation, as well as antibodies that recognize the receptor-ligand complex, and preferably do not specifically recognize the unbound receptor or unbound ligand. Likewise, included in the invention are the neutralizing antibodies that bind to the ligand and prevent the binding of the ligand to the receptor, as well as the antibodies that bind to the ligand, thereby preventing activation of the receptor, but do not prevent the ligand is linked to the receptor. Also included in the invention are the antibodies that activate the receptor. These antibodies can act as receptor agonists, for example, potentiate or activate all or a subset of the biological activities of receptor activation mediated by the ligand, for example, by inducing the dimerization of the receptor. The antibodies can be specified as agonists, antagonists or inverse agonists for the biological activities comprising the specific biological activities of the polypeptides of the present invention, described herein. The aforementioned antibody agonists can be made using methods known in the art. See, for example, PCT publication WO 96/40281; U.S. Patent No. 5,811,097; Deng et al., Blood 92 (6): 1981-1988 (1998) Chen et al. Cancer Res. 58 (16): 3688-3678 (1998) Harrop et al., J ". Immunol. 161 (4): 1786-1794 (1998) Zhu et al., Cancer Res. 58 (15): 3209-3214 (1998) Yoon et al., J. I munol, 160 (7): 3170-3179 (1998) Prat et al., J. Cell, Sci. 111 (Pt2) -. 237-247 (1998) Pitard et al., J. Immunol. 205 (2) 177-190 (1997), Liautard et al, Cytokine 9 (4): 233-241 (1997), Carlson et al., J ". Biol. Chem. 272 (17); 11295-11301 (1997); Taryman et al., Neuron 14 (4): 755-762 (1995); Muller et al., Structure 6 (9): 1153-1167 (1998); Bartunek et al., Cytokine 8 (l): 14-20 (1996) (all of which are incorporated by reference herein, in their entirety). The antibodies of the present invention can be used, for example, but not limited to, to purify, detect, and direct the polypeptides of the present invention, including diagnostic and therapeutic methods in vi tro and in vivo. For example, antibodies have use in immunoassays to qualitatively and quantitatively measure the levels of the polypeptides of the present invention in biological samples. See, for example, Harlow et al., Antiboides: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd Ed. 1998) (incorporated by reference herein, in its entirety). In a preferred embodiment, KGF-2 levels are detected in a purified sample using goat and chicken antibodies (see for example 50, below). As discussed in more detail below, the antibodies of the present invention may be used either alone or in combination with other compositions. The antibodies can also be recombinantly fused to a heterologous polypeptide at the N-terminus or C-terminus, or chemically conjugated (including the conjugations covalently and non-covalently) to the polypeptides or other compositions. For example, antibodies of the present invention can be recombinantly fused or conjugated to molecules useful as markers in detection assays and effector molecules such as heterologous polypeptides, drugs, radionuclides, or toxins. See, for example, PCT publications WO 92/08495; WO 91/14438; WO 89/12624; U.S. Patent No. 5,314,995; and European Patent No. 396,387.

The antibodies of the invention include derivatives that are modified, for example, by the covalent coupling of any type of molecule to the antibody, such that the covalent bond does not prevent the antibody from generating an anti-idiotypic response. For example, but not by way of limitation, antibody derivatives include antibodies that have been modified, for example, by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, binding to a ligand cellular or another protein, etc. Any of the numerous chemical modifications can be carried out by known techniques, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Additionally, the derivative may contain one or more non-classical amino acids. The antibodies of the present invention can be generated by any suitable method known in the art. Monoclonal antibodies to an antigen of interest can be produced by various methods well known in the art. For example, a polypeptide of the invention can be administered to various host animals including, but not limited to, rabbits, mice, rats, etc., to induce the production of sera containing polyclonal antibodies specific for the antigen. Various adjuvants can be used to increase the immune response, depending on the host species, which include but are not limited to, Freund's adjuvant (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oily emulsions, limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (Bacillus Calmette-Guerin) and Corynebacterium parvum. Such adjuvants are also well known in the art. Monoclonal antibodies can be prepared using a wide variety of techniques known in the art, including the use of hybridoma, recombinant, and phage visualization technologies, or a combination thereof. For example, monoclonal antibodies can be produced using hybridoma techniques including those known in the art and shown, for example, in Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 1988); Hammerling and collaborators, in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981) (said references are incorporated by reference herein, in their entirety). The term "monoclonal antibody" as used herein is not limited to the antibodies produced through the hybridoma technology. The term "monoclonal antibody" refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced. Methods for producing and selecting specific antibodies using the hybridoma technology are routine and well known in the art, and are discussed in detail in the Examples. In a non-limiting example, mice can be immunized with a polypeptide of the invention or a cell expressing such a peptide. Once an immune response is detected, for example, antibodies specific for the antigen are detected in the mouse serum, the spleen of the mouse is harvested and the splenocytes are isolated. The splenocytes are then fused by well-known techniques to any suitable myeloma cells, for example, cells from the SP20 cell line available from the ATCC. Hybridomas are selected and cloned by limited dilution. Hybridoma clones are then evaluated by methods known in the art for cells that secrete antibodies capable of binding to a polypeptide of the invention. The ascites fluid, which generally contains high levels of antibodies, can be generated by immunizing the mice with positive hybridoma clones. Accordingly, the present invention provides the methods for generating the monoclonal antibodies as well as the antibodies produced by the method comprising the culture of a hybridoma cell that secretes an antibody of the invention, wherein, preferably, the hybridoma is generated by the fusion of splenocytes isolated from a mouse immunized with an antigen of the invention, with myeloma cells and then selecting the hybridomas resulting from the fusion for the hybridoma clones that secrete an antibody capable of binding to a polypeptide of the invention. Antibody fragments that recognize specific epitopes can be generated by known techniques. For example, the Fab and F (ab ') 2 fragments of the invention can be produced by the proteolytic cleavage of the immunoglobulin molecules, using enzymes such as papain (to produce the Fab fragments) or pepsin (to produce the F fragments ( ab ') 2. The F (ab') 2 fragments contain the variable region, the light chain constant region and the CH1 domain of the heavy chain, for example, the antibodies of the present invention can also be generated using various methods phage display methods known in the art: In phage display methods, functional antibody domains are shown on the surface of the phage particles carrying the polynucleotide sequences encoding them. phage can be used to display the antigen binding domains expressed from a combinatorial antibody repertoire or library (e.g. The phage that expresses a binding domain to the antigen that binds to the antigen of interest can be selected or identified with antigen, for example, using the labeled antigen or the antigen bound or captured to a solid surface or a sphere. . The phages used in these methods are typically filamentous phages that include the fd and M13 binding domains expressed from phage with Fab, Fv or Fv antibody domains stabilized with disulfide bridges, recombinantly fused to either the gene III protein or the gene VIII of the phage. Examples of phage display methods that can be used to make the antibodies of the present invention include those described in Brinkman et al., J. Immunol. Methods 182: 41-50 (1995); Ames et al., J. I munol. Methods 184: 177-186 (1985); Kettleborough et al., Eur. J. Immunol. 24: 952-958 (1994); Persic et al., Gene 187: 9-18 (1997); Burton et al., Advances in Immunology 57: 191-280 (1994); PCT application No. PCT / GB91 / 01134; PCT publications WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982 WO 95/20401; and U.S. Patent Nos. 5,698,426 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225 5,658,727; 5,733,743 and 5,969,108; each of which is incorporated by reference herein in its entirety). As described in the above references, after phage selection, antibody coding regions from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, for example, as described in detail below. For example, techniques for recombinantly producing Fab, Fab 'and F (ab') 2 fragments can also be employed using methods known in the art such as those described in PCT publication WO 92/22324; Mullinax et al., BioTechniques 12 (6): 864-869 (1992); and Sawai et al., AJRI 34: 26-34 (1995); and Better et al., Science 240: 1041-1043 (1988) (such references are incorporated by reference herein, in their entirety). Examples of techniques that can be used to produce single chain Fvs and antibodies include those described in U.S. Patent Nos. 4,946,778 and 5,258,498; Huston et al., Methods in Enzimology 203: 46-88 (1991); Shu et al., PNAS 90: 7995-7999 (1993) and Skerra et al., Science 240: 1038-1040 (1998) For some uses, including the in vivo use of antibodies in humans and in vitro detection assays. it may be preferable to use chimeric, humanized, or human antibodies A chimeric antibody is a molecule in which different portions of the antibody are derived from different species of animals such as antibodies having a variable region derived from a murine monoclonal antibody and a region human immunoglobulin constant Methods for producing chimeric antibodies are known in the art See, for example, Morrison, Science 229: 1202 (1985); Oi et al., BioTechniques 4: 214 (1986); Gillies et al., (1989) J. Immunol, Methods 125: 191-202, U.S. Patent Nos. 5,807,715, 4,816,567, and 4,816,397, which are incorporated herein by reference, in their entirety. antibody molecules of non-human species, which bind to the desired antigen having one or more regions of complementarity determination (CDRs) from non-human species and structural regions derived from a human immunoglobulin molecule. Often, the structural residues in the human structural regions will be substituted with the corresponding residue from the CDR donor antibody to alter, preferably to improve, the binding to the antigen. These structural substitutions are identified by methods well known in the art, for example, by modeling the interactions of the CDR and structural residues to identify the structural residues important for antigen binding, and the sequential comparison to identify the residues. Unusual structural changes in particular positions (See, for example, Queen et al., United States Patent No. 5,585,089; Riechmann et al., Nature 332: 323 (1988), which are incorporated by reference herein, in their entirety). The antibodies can be humanized using a variety of techniques known in the art including, for example, CDR grafting (European Patent 239,400, PCT publication WO 91/09967, US Patent Nos. 5,225,539, 5,530,101, and 5,585,098), coating or remodeling (European Patents Nos. 592,106; 519,596; Padlan, Molecular I munology 28 (4/5): 489-498 (1991); Studnicka et al., Protein Engineering 7 (6): 805-814 (1994); Roguska et al., PNAS 91: 969-973 (1994)), and interspersed with chains (U.S. Patent No. 5,565,332). Fully human antibodies are particularly desirable for the therapeutic treatment of human patients. Human antibodies can be made by a variety of methods known in the art including the phage display methods described above using antibody libraries derived from the human immunoglobulin sequences. See also, U.S. Patent Nos. 4,444,887 and 4,716,111; and PCT publications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741; each of which is incorporated by reference herein, in its entirety). Human antibodies can also be produced using transgenic mice that are unable to express functional endogenous immunoglobulins, but can express human immunoglobulin genes. For example, the heavy chain and human light chain immunoglobulin gene complexes can be introduced randomly or by homologous recombination into mouse embryonic pluripotent cells. Alternatively, the human variable region, the constant region, and the diversity region can be introduced into mouse embryonic pluripotent cells in addition to the human heavy and light chain genes. The heavy chain and light chain immunoglobulin genes of mouse can be made non-functional separately or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. In particular, the homozygous deletion of the JH region prevents the production of the endogenous antibody. Embryonic pluripotent cells modified with expanded and microinjected into blasts to produce chimeric mice. The chimeric mice are then reproduced to produce homozygous progeny expressing the human antibodies. The transgenic mice are immunized in a normal manner with a selected antigen, for example, all or a portion of a polypeptide of the invention. Monoclonal antibodies directed against the antigen can be obtained from transgenic, immunized mice, using conventional hybridoma technology. The human immunoglobulin transgenes harbored by the gransgenic mice are rearranged during the differentiation of the B cells and subsequently undergo class change and somatic mutation. Thus, using such a technique, it is possible to produce therapeutically useful IgG, IgA, and IgE antibodies. For a review of this technology to produce human antibodies, see Lonberg and Huszar, Int. Rev. Im unol. 13: 65-93 (1995). For a detailed discussion of such technology for producing human antibodies and human monoclonal antibodies, and protocols for producing such antibodies, see for example, PCT publications WO 98/24893; WO 92/01047; WO 96/34096; WO 96/33735; European Patent No. 0,598,877; U.S. Patent Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318, 5,885,793; 5,916,771; and 5,939,598, which are incorporated by reference herein, in their entirety). In addition, companies such as Abgenix, Inc. (Freemont, CA) and GenPharm (San Jose, CA) can be contracted to provide human antibodies directed against a selected antigen, using technology similar to that described above. Fully human antibodies that recognize a selected epitope can be generated using the technique called "guided selection". In this method, a non-human monoclonal antibody selected, for example, a mouse antibody, is used to guide the selection of a fully human antibody, which recognizes the same epitope (Jaspers et al., Bio / technology 12 - 899-903). (1988)). In addition, antibodies to the polypeptides of the invention can, in turn, be used to generate anti-idiotype antibodies that "mimic" the polypeptides of the invention using techniques well known to those skilled in the art. (See for example, Greenspan and Bona, FASEB J. 7 (5): 437-444; (1989) and Nissinoff, J. "Immunol 147 (8): 2429-2438 (1991).) For example, antibodies that bind to and competitively inhibit the multimerization of the polypeptide and / or the binding of a polypeptide of the invention. to a ligand, can be used to generate anti-idiotypes that "mimic" the multimerization of the polypeptide and / or the binding domain, and as a consequence bind to and neutralize the polypeptide and / or its ligand. Fab fragments of such anti-idiotypes can be used in therapeutic regimens to neutralize the polypeptide ligand For example, such anti-idiotypic antibodies can be used to bind to a polypeptide of the invention and / or bind to its ligands / receptors, and This invention also relates to antibodies that act as agonists or antagonists of the polypeptides of the present invention. agonists or antagonists of the polypeptides of the present invention include, for example, antibodies that perturb receptor / ligand interactions with the polypeptides of the invention, either partially or completely. For example, the present invention includes antibodies that disrupt the ability of the proteins of the invention to multimerize. In yet another example, the present invention includes antibodies that allow the proteins of the invention to multimerize, but disturb the ability of the proteins of the invention to bind to one or more KGF-2 receptors / ligands. In yet another example, the present invention intuits antibodies that allow the proteins of the invention to multimerize, and bind to the KGF-2 receptor (s) / ligands, but block the biological activity associated with the KGF-2 / receptor complex / 1igando Antibodies that act as agonists or antagonists of the polypeptides of the present invention also include, the receptor-specific antibodies and the ligand-specific antibodies. This includes receptor-specific antibodies that do not prevent binding to the ligand but prevent receptor activation. Activation of the receptor (eg, signaling) can be determined by techniques described herein or otherwise known in the art. Also included are receptor-specific antibodies which prevent ligand binding and receptor activation. Likewise, neutralizing antibodies are included, which bind to the ligand and prevent the binding of the ligand to the receptor, as well as antibodies that bind to the ligand, thereby preventing receptor activation, but do not prevent the ligand from binding to the receptor. It is linked to the receiver. Also included are antibodies that activate the receptor. These antibodies can act as agonists for all or less than all the biological activities affected by receptor activation mediated by the ligand. These antibodies can be specified as agonists or antagonists for the biological activities comprising the specific activities described herein. The aforementioned antibody agonists can be made using methods known in the art. See, for example, WO 96/40281; U.S. Patent No. 5,811,097; Deng, B. et al., Blood 92 (6): 1981-1988 (1988); Chen, Z. et al., Cancer Res. 58 (16): 3668-3678 (1998); Harrop, J.A. and collaborators, J. I munol. 16 (4): 1786-1794 (1998); Zhu, Z. et al., Cancer Res. 58 (15): 3209-3214 (1998); Yoon, D.Y. and collaborators, J. I munol. 160 (7): 3170-3179 (1988); Prat, M. et al., J. Cell. Sci. 111 (Pt2): 237-247 (1998); Pitard, V. and collaborators, J. Immunol. Methods 205 (2): 177-190 (1997); Liautard, J. et al., Cytokinde 9 (4): 233-241 (1997); Carlson, N.G. and collaborators, J. Biol. Chem. 272 (17): 11295-11301 (1997); Taryman, R.E. and collaborators, Neuron 14 (4): 755-762 (1995); Muller, Y.A. and collaborators, Structure 69 (9): 1153-1167 (1998); Bartunek, P. et al., Cytokine 8 (l): 14-20 (1996) (these references are incorporated by reference in their entirety). As discussed above, the antibodies to the KGF-2 proteins of the invention can, in turn, be used to generate anti-idiotype antibodies that "mimic" KGF-2 using techniques well known to those skilled in the art (See example, Greenspan and Bona, FASEB J. 7 (5): 437-444 (1989) and Nissinoff, J. Immunol. 147 (8): 2429-2438 (1991)). For example, antibodies that bind to KGF-2 and competitively inhibit KGF-2 multimerization and / or ligand binding can be used to generate anti-idiotypes that "mimic" the multimerization of KGF-2 and / or the link to the domain and, as a consequence, bind to and neutralize KGF-2 and / or its ligand. Such neutralizing anti-idiotypes or Fab fragments of such anti-idiotypes can be used in therapeutic regimens to neutralize the KGF-2 ligand. For example, such anti-idiotypic antibodies can be used to bind to KGF-2, or to bind to the KGF-2 ligands / receptors, and thereby block the biological activity of KGF-2.

Polynucleotides that Code for Antibodies The invention further provides polynucleotides comprising a nucleotide sequence encoding an antibody of the invention, and fragments thereof. The invention also encompasses polynucleotides that hybridize under stringent or low-stringency hybridization conditions, for example, as defined above, to polynucleotides that encode an antibody, preferably, that specifically bind to a polypeptide of the invention, preferably , an antibody that binds to a polypeptide having the amino acid sequence of SEQ ID No. 2. The polynucleotides can be obtained, and the nucleotide sequence of the polynucleotides determined, by any method known in the art. For example, if the nucleotide sequence of the antibody is known, a polynucleotide encoding the antibody can be assembled from chemically synthesized oligonucleotides (for example, as described in Kutmeier et al., BioTechniques 17: 242 (1994)), which, briefly, involves the synthesis of overlapping oligonucleotides containing portions of the sequence encoding the antibody, annealing and ligating those oligonucleotides, and then amplifying the ligated oligonucleotides by PCR. Alternatively, a polynucleotide encoding an antibody can be generated from the nucleic acid from a suitable source. If a clone containing a nucleic acid encoding a particular antibody is not available, but the sequence of the antibody molecule is known, a nucleic acid encoding the immunoglobulin can be chemically synthesized or obtained from a suitable source (eg, example, a cDNA library of antibodies, or a cDNA library generated from, or the nucleic acid, preferably poly A + RNA, isolated from, any tissue or cells expressing the antibody, such as selected hybridoma cells for expressing an antibody of the invention) by PCR amplification using synthetic primers hybridizable to the 3 'and 5' ends of the sequence or by cloning using an oligonucleotide probe specific for the particular gene sequence to identify eg a cDNA clone from of a cDNA library encoding the antibody. The amplified nucleic acids generated by PCR can then be cloned into replicable cloning vectors using any method well known in the art.

Once the nucleotide sequence and the corresponding amino acid sequence of the antibody is determined, the nucleotide sequence of the antibody can be manipulated using methods well known in the art for manipulation of nucleotide sequences, for example, recombinant DNA techniques, site-directed mutagenesis, PCR, etc. (see, for example, the techniques described in Sambrook et al., 1990, Molecular Cloning, A Laboratory Manual, 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY and Ausubel et al., 1998, Current Protocols in Molecular Biology, John Wiley and Sons, NY, which are incorporated by reference herein, in their entirety) to generate antibodies having a different amino acid sequence, for example to create amino acid substitutions, deletions and / or insertions thereof . In a specific embodiment, the amino acid sequences of the heavy and / or light chain variable domains can be inspected to identify the sequences of complementarity determination regions (CDRs) by methods that are well known in the art, for example , by comparison to the known amino acid sequences of other heavy chain and light chain variable regions, to determine regions of sequence hypervariability. Using recombinant DNA techniques, routine, one or more of the CDRs can be inserted into the structural regions, eg, within the human framework regions, to humanize a non-human antibody, as described above. The structural regions may be of natural origin or consensus structural regions, and preferably human structural regions (see, for example, Clothia et al., J. Mol.-Biol. 278: 457-479 (1998) for a listing of the structural regions human) Preferably, the polynucleotide generated by the combination of the structural regions and the CDRs codes for an antibody that specifically binds to a polypeptide of the invention. Preferably, as discussed above, one or more amino acid substitutions may be made within the framework regions, and preferably, the amino acid substitutions improve the binding of the antibody to its antigen. Additionally, such methods can be used to make amino acid substitutions or deletions of one or more variable region cysteine residues that participate in an intrachain disulfide bond to generate antibody molecules lacking one or more intrachain disulfide bridges. Other alterations to the polynucleotide are encompassed by the present invention, and are within the skill in the art. In addition, the techniques developed for the production of "chimeric antibodies" (Morrison et al., Proc. Nati. Acad. Sci. 81: 851-855 (1984); Neuberger et al., Nature 312: 604-608 (1984); Takeda et al., Nature 314: 452-454 (1985)) by splicing genes from a mouse antibody molecule of appropriate antigenic specificity, together with genes from a human antibody molecule of appropriate biological activity, can be used. As described above, a chimeric antibody is a molecule in which different portions are derived from different species of animals, such as those having a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region, for example, humanized antibodies. Alternatively, the techniques described for the production of single chain antibodies (U.S. Patent No. 4,946,778; Bird, Science 242: 423-42 (1988); Huston et al., Proc. Nati. Acad. Sci. USA. 85: 5879-5883 (1988); and Ward et al., Nature 334: 544-54 (1989)) can be adapted to produce single chain antibodies. Single chain antibodies are formed by binding the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide. Techniques for the assembly of functional Fv fragments in E. coli can also be used (Skerra et al., Science 242: 1308-1041 (1988)).

Methods of Antibody Production The antibodies of the invention can be produced by any method known in the art for the synthesis of the antibodies, in particular, by chemical synthesis or preferably, by recombinant expression techniques. The recombinant expression of an antibody of the invention, or fragment, derivative or analogue thereof, (for example, a heavy or light chain of an antibody of the invention or a single chain antibody of the invention), requires the construction of a expression vector that contains a polynucleotide that codes for the antibody. Once a polynucleotide encoding an antibody molecule or a light or heavy chain of an antibody, or a portion thereof (preferably containing the variable domain of the heavy or light chain) of the invention has been obtained, the vector for the production of the antibody molecule can be produced by recombinant DNA technology using techniques well known in the art. Thus, methods for preparing a protein by expressing a polynucleotide containing a nucleotide sequence encoding the antibody are described herein. Methods that are well known to those skilled in the art can be used to construct expression vectors containing the antibody coding sequences, and appropriate transcriptional and translational control signals. These methods include, for example, recombinant DNA techniques in vi tro, synthetic techniques, and genetic recombination in vivo. The invention thus provides replicable vectors comprising a nucleotide sequence encoding an antibody molecule of the invention, or a heavy or light chain thereof, or a variable domain of the heavy or light chain, operably linked to a promoter. . Such vectors can include the nucleotide sequence coding for the constant region of the antibody molecule (see for example, PCT Publication WO 86/05807; PCT Publication WO 89/01036; and U.S. Patent No. 5,122,464) and the domain The antibody variable can be cloned into such a vector for the expression of the complete heavy or light chain.

The expression vector is transferred to a host cell by conventional techniques and the transfected cells are then cultured by conventional techniques to produce an antibody of the invention. Thus, the invention includes host cells that contain a polynucleotide that encodes an antibody of the invention, or a heavy or light chain thereof, or a single chain antibody of the invention, operably linked to a heterologous promoter. In preferred embodiments for the expression of the double-chain antibodies, the vectors encoding the heavy and light chains can be co-expressed in the host cell for the expression of the complete immunoglobulin molecule, as detailed below. A variety of host expression vector systems can be used to express the antibody molecules of the invention. Such expression systems in the host or host represent vehicles by which the coding sequences of interest can be produced and subsequently purified, but also represent cells which can, when transformed or transfected with the appropriate nucleotide coding sequences, express an antibody molecule of the invention in si tu. These include but are not limited to microorganisms such as bacteria (e.g., E. coli, B. subtilis) transformed with the recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing the antibody coding sequences; yeasts (eg, Saccharomyces, Pichia) transformed with yeast expression vectors, recombinants, which contain antibody coding sequences; insect cell systems, infected with recombinant viral expression vectors (eg, baculovirus) that contain the antibody coding sequences; plant cell systems infected with recombinant virus expression vectors (eg, cauliflower mosaic virus, CaMV, tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (eg, Ti plasmid) which contain the antibody coding sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., the metallothionein promoter) or from mammalian viruses (for example, the late adenovirus promoter, the 7.5K promoter of vaccinia virus). Preferably, bacterial cells such as Escherichia coli, and more preferably, eukaryotic cells, especially for the expression of the complete recombinant antibody molecule, used for the expression of a recombinant antibody molecule. For example, mammalian cells such as Chinese hamster ovary (CHO) cells, in conjunction with a vector such as the promoter element of the early or early greater early gene, from human cytomegalovirus, is an effective expression system for antibodies (Foecking et al., Gene 45: 101 (1086); Cockett et al., Bio / Technology 8: 2 (1990)). In bacterial systems, a number of expression vectors can be advantageously selected depending on the intended use for the antibody molecule that is expressed. For example, when a large amount of such a protein is to be produced, for the generation of pharmaceutical compositions of an antibody molecule, vectors that direct the expression of high levels of the fusion protein products that easily purified, can be desirable Such vectors include, but not limited to, the E. coli expression vector, pUR278 (Ruther et al., EMBO J. 2: 1791 (1983)), in which the antibody coding sequence can be ligated individually within of the vector intrastructurally with the lac Z coding region, so that a fusion protein is produced; pIN vectors (Inouye and Inouye, Nucleic Acids Res. 13: 3101-3109 (1985); Van Heeke and Schuster, J. "Biol. Chem. 24: 5503-5509 (1989)), and the like.PGEX vectors can also be used to express foreign polypeptides as fusion proteins with glutathione-S-transferase (GST). In general, such fusion proteins are soluble and can be easily purified from lysed cells by adsorption and binding to glutathione-agarose matrix spheres, followed by elution in the presence of free glutathione.PGEX vectors are designed to include the cleavage sites with thrombin or the factor Xa protease, so that the cloned target gene product can be released from the GST portion In an insect system, the Autographa californica nuclear polyhedrosis virus (AcNPV) is used As a vector to express foreign genes, the virus develops in Spodoptera frugiperda cells.The coding sequence of the antibody can be cloned individually into regions not that (for example the polyhedrin gene) of the virus, and placed under the control of an AcNPV promoter (for example the polyhedrin promoter). In mammalian host cells, a number of virus-based expression systems can be used. In cases where an adenovirus is used as an expression vector, the coding sequence of the antibody of interest can be ligated to an adenovirus transcription / translation control complex, eg, the late promoter and the tripartite leader sequence. This chimeric gene can then be inserted into the adenovirus genome by in vitro or in vivo recombination. Insertion into a non-essential region of the viral genome (eg, the El or E3 region) will result in a recombinant virus that is viable and capable of expressing the antibody molecule in infected hosts (eg, see Logan and Shenk, Proc. Nati, Acad. Sci. USA 81: 355-359 (1984)). Specific start signals may also be required for efficient translation of the inserted sequences encoding the antibody. These signals include the ATG start codon and the adjacent sequences. In addition, the start codon must be in phase with the reading structure of the desired coding sequence, to ensure translation of the complete insert. These exogenous translational control signals and the start codons can be from a variety of origins, both natural and synthetic. The efficiency of expression can be improved by the inclusion of appropriate transcription-enhancing elements, transcription terminators, etc. (See Bittner et al., Methods in Enzymol, 153: 51-544 (1987)).

In addition, a host cell strain can be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in a specific desired manner. Such modifications (eg, glycosylation) and processing (eg, cleavage) of the protein products may be important for the function of the protein. Different host cells have characteristic and specific mechanisms for the processing and post-translational modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure correct modification and processing of the foreign protein expressed. For this purpose, eukaryotic host cells possessing the cellular machinery for the proper processing of the primary transcript, glycosylation and phosphorylation of the gene product, can be used. Such mammalian host cells include, but are not limited to CHO, VERA, BHK, Hela, COS, MDCK, 293, 3T3, W138, and in particular, breast cancer cell lines, such as, for example, BT483, Hs578T , HTB2, BT20 and T47D, and the normal mammary gland cell line, such as, for example, CRL7030 and Hs578Bst. For the production of high-performance long-term recombinant proteins, stable expression is preferred. For example, cell lines that stably express the antibody molecule can be engineered. Instead of using expression vectors that contain viral origins of replication, the host cells can be transformed with the DNA controlled by the appropriate expression control elements (e.g., the promoter, the enhancer, the sequences, the terminators of transcription, polyadenylation sites, etc.), and a selectable marker. After the introduction of the foreign DNA, the engineered cells can be left to develop for 1 to 2 days in an enriched medium, and then they are changed to a selective medium. The selectable marker in the recombinant plasmid confers resistance to selection and allows the cells to stably integrate the plasmid into their chromosomes and develop to form foci which in turn can be cloned and expanded into the cell lines. This method can be advantageously used to engineer the cell lines expressing the antibody molecule by genetic engineering. Such cell lines engineered can be particularly useful in the selection and evaluation of compounds that interact directly or indirectly with the antibody molecule.

A number of selection systems, including but not limited to, the thymidine kinase gene of the herpes simplex virus (Wigler et al., Cell 11: 223 (1977)), hypoxanthine-guanine phosphoribosyltransferase (Szylbalska and Szylbalski, Proc. Nati, Acad. Sci. USA 48: 202 (1992)), and the adenine phosphoribosyltransferase gene (Lowy et al., Cell 22: 817 (1980)) can be used in tk-, hgprt cells - or aprt, respectively. Also, anti-metabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., Nati, Acad. Sci. USA 77: 357 (1980); Hare et al., Proc. Nati, Acad. Sci USA 78: 1527 (1981)), gpt, which confers resistance to mycophenolic acid (Mulligan and Berg, Proc. Nati, Acad. Sci. USA 78: 2072 (1981); neo, which confers resistance to aminoglycoside G-418 (Goldspiel et al., Clinical Pharmacy 12: 488-505 (1993); Wu and Wu, Biotherapy 3: 87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol. 32: 573-596 (1993), Mulligan, Science 260: 926-932 (1993), and Morgan and Anderson, Ann. Rev. Biochem. 61: 191-217 (1993); May, 1993, TIB TECH 11 (5): 155-215); and hygro, which confers resistance to hygromycin (Santerre et al., Gene 30: 147 (1984) ) . Methods commonly known in the art of recombinant DNA technology can be routinely applied to select the desired recombinant clone, and such methods are described for example, in Ausubel et al. (Eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990); and in Chapters 12 and 13 of Dracopoli et al. (eds) Current Protocols in Human Genetics, John Wiley & Sons, NY (1994); Colberre-Garapin et al., J. Mol. Biol. 150: 1 (1981), which are incorporated by reference herein, in their entirety. The expression levels of an antibody molecule can be increased by the amplification of the vector (for a review, see Bebbington and Hentschel) The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in the cloning of DNA, Vol. 3 (Academic Press, New York, 1987)). When a marker in the vector system expressing the antibody is amplifiable, the increase in the level of inhibitor present in the culture of the host cell will increase the copy number of the marker gene. Since the amplifier region is associated with the antibody gene, the production of the antibody will also be increased (Crouse et al., Mol.Cell. Biol. 3: 257 (1983)). The host cell can be con-transfected with the expression vectors of the invention, the first vector encoding a polypeptide derived from the heavy chain and the second vector encoding a polypeptide derived from the light chain. The two vectors may contain identical selectable markers that make possible the equal expression of heavy and light chain polypeptides. Alternatively, a simple vector can be used which encodes, and is capable of expressing, the heavy and light chain polypeptides. In such situations, the light chain must be placed before the heavy chain to avoid an excess of heavy chain free of toxicity (Proudfoot, Nature 322: 52 (1986); Kohler, Proc. Nati. Acad. Sci. USA 77: 2197 (1980)). The coding sequences for the heavy and light chains can comprise AD? C or AD? genomic Once an antibody molecule of the invention has been produced by an animal, chemically synthesized, or recombinantly expressed, it can be purified by any method known in the art for the purification of an immunoglobulin molecule, for example, by chromatography ( for example, ion exchange, of affinity, particularly by affinity for the specific antigen after protein A, and column chromatography for size adjustment), centrifugation, differential solubility, or by any other standard technique for purification of proteins. In addition, the antibodies of the present invention or fragments thereof can be fused to heterologous polypeptide sequences described herein or otherwise known in the art, to facilitate purification. The present invention encompasses recombinantly fused or chemically conjugated antibodies (including covalently and non-covalently conjugated) to a polypeptide (or portions thereof, preferably at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acids of the polypeptide) of the present invention, to generate the fusion proteins. The fusion does not necessarily need to be direct, but can occur through linker sequences. The antibodies may be specific for antigens other than the polypeptides (or portions thereof, preferably at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acids of the polypeptide) of the present invention. For example, the antibodies can be used to direct the polypeptides of the present invention to particular cell types, either in vitro or in vivo, by fusion or conjugation of the polypeptides of the present invention to the specific antibodies for the particular receptors. of the cell surface. Antibodies fused or conjugated to the polypeptides of the present invention can also be used in in vitro immunoassays and purification methods using methods known in the art. See for example, Harbor et al., Supra and PCT publication WO 93/21232; European Patent 439,095; Naramura et al., Immunol. Lett. 39: 91-99 (1994); U.S. Patent 5,474,981; Gillies et al., PNAS 89: 1428-1432 (1992); Fell et al., J. Im unol. 146: 2446-2452 (1991), which are incorporated by reference herein, in their entirety. The present invention further includes compositions comprising the polypeptides of the present invention fused or conjugated to the antibody domains other than the variable regions. For example, the polypeptides of the present invention can be fused or conjugated to an Fe region of the antibody, or a portion thereof. The antibody portion fused to a polypeptide of the present invention may comprise the constant region, the hinge region, the CH1 domain, the CH2 domain, and the CH3 domain or any combination of the entire domains or portions thereof. The polypeptides can also be fused or conjugated to the aforementioned portions of antibodies, to form multimers. For example, Fe portions fused to the polypeptides of the present invention can form dimers through the disulfide bond between Fe portions. Higher multimeric forms can be made by fusing the polypeptides to IgA and IgM portions. Methods for fusing or conjugating the polypeptides of the present invention to the antibody portions are known in the art. See for example, U.S. Patent No. 5,336,603; 5,622,929; 5,359,046; 5,349,053; 5,447,851; 5,112,946; European Patent 307,434; European Patent 367,166; PCT publications WO 96/04388; WO 91/06570; Ashkenazi et al., Proc. Nati Acad. Sci. USA 88: 10535-10539 (1991); Zheng et al., J. Immunol. 154: 5590-5600 (1995); and Vil et al., Proc.

Nati Acad. Sci. USA 89: 11337-11341 (1992) (said references are incorporated by reference in their entirety). As discussed above, polypeptides corresponding to a polypeptide, polypeptide fragment, or a variant of SEQ ID No. 2, can be fused or conjugated to the aforementioned antibody portions to increase the in vivo half-life of the polypeptides or for use in immunoassays using methods known in the art. In addition, the polypeptides corresponding to SEQ ID No. 2, can be fused or conjugated to the aforementioned antibody portions to facilitate purification. One reported example describes chimeric proteins consisting of the first two domains of the human CD4 polypeptide and several domains of the constant regions of the heavy or light chains of mammalian immunoglobulins (European Patent 394,827; Traunecker et al., Nature 331: 84- 86 (1988) The polypeptides of the present invention fused or conjugated to an antibody having dimeric structures linked by disulfide bridge (due to IgG) may also be more efficient in binding and neutralizing other molecules, than the secreted monomeric protein. or the protein fragment alone (Fountoulakis et al., J. Biochem 270: 3958-3964 (1995)). In many cases, the Fe part in a fusion protein is beneficial in therapy and in diagnosis and thus can resulting in, for example, improved pharmacokinetic properties (European Patent 232,262) Alternatively, the deletion of the Fe part after the Fusion protein has been expressed, detected, and purified, it could be desired. For example, the Fe moiety can prevent therapy and diagnosis if the fusion protein is used as an antigen for immunizations. In drug discovery, for example, human proteins, such as the hIL-5 receptor, have been fused with Fe portions for purposes of high throughput screening assays, to identify hIL-5 antagonists. (See, Bennett et al., J. Molecular Recognition 8: 52-58 (1995); Johanson et al., J. Biol. Chem. 270: 9459-9471 (1995)).

In addition, antibodies or fragments thereof of the present invention can be fused to marker sequences such as a peptide to facilitate purification. In preferred embodiments, the amino acid marker sequence is a hexa-histidine peptide, such as the marker provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA, 91311), among others, many of which They are commercially available. As described in Gentz et al., Proc. Nati Acad. Sci. USA 86: 821-824 (1989), for example, hexa-histidine provides convenient purification of the fusion protein. Other peptide markers useful for purification include, but are not limited to, the "HA" marker, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., Cell 37: 767 (1984)) and the "flag" marker. The present invention also encompasses antibodies or fragments thereof, conjugated to a diagnostic or therapeutic agent. Antibodies can be used diagnostically, for example, to periodically check the development or progression of a tumor as part of a clinical test procedure, for example to determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emission metals using various positron emission tomographies, and non-radioactive paramagnetic metal ions. The detectable substance can be coupled or conjugated either directly to the antibody (or fragment thereof) or indirectly, through an intermediary (such as, for example, a linker known in the art) using techniques well known in the art. See for example, U.S. Patent No. 4,741,900 for metal ions that can be conjugated for antibodies, for use as diagnostic reagents according to the present invention. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamino-fluorescein, dansyl chloride, or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin; and examples of suitable radioactive material include 125I, 131I, X11I or 99Tc. In addition, an antibody or fragment thereof can be conjugated to a therapeutic portion such as a cytotoxin, for example, a cytostatic or cytocidal agent, a therapeutic agent or a radioactive metal ion, for example, alpha emitters such as, for example, 213Bi. A cytotoxin or cytotoxic agent includes any agent that is harmful to the cells. Examples include paclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine colchicine, doxorubicin, daunorubicin, dihydroxy-anthracdendione, mitoxantrone, mitramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine , tetracaine, lidocaine, propranolol, and puromycin and analogues or homologs thereof. Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil-decarbazine), alkylating agents (e.g., mechlorethamine, thioepa, chlorambucil, melphalan, carmustine).

(BNSU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiaminoplatinum (II) (DDP) cisplatin), anthracyclines (eg daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (eg example, dactinomycin (formerly actinocymin), bleomycin, mithramycin, and anthramycin (AMC), and anti-mitotic agents (eg, vincristine and vinblastine) The conjugates of the invention can be used to modify a given biological response, the therapeutic agent or the portion of the drug is not to be considered as limited to the classical, chemical therapeutic agents For example, the drug portion may be a protein or polypeptide having a desired biological activity Such proteins may include, for example, a toxin such as abrin, ricin A, Pseudomonas exotoxin, or diphtheria toxin, a protein such as tumor necrosis factor, α-interferon, β-in terferon, nerve growth factor, platelet-derived growth factor, tissue plasminogen activator, an apoptotic agent, e.g., TNF-α, TNF-β, AIM I (See, International Publication No. WO 97/33899), AIM II (See, International Publication No. WO 97/34911), Fas ligand (Takahashi et al. Int. Immunol. 6: 1567-1574 (1994)), VEGI (See, International Publication No. WO 99/23105), a thrombotic agent or an anti-angiogenic agent, for example, angiostatin or endostatin; or biological response modifiers such as, for example, lymphokines, interleukin-1 ("IL-1"), interleukin-2 ("IL-2"), interleukin-6 ("IL-6"), colony stimulating factor of granulocytes-macrophages ("GM-CSF"), granulocyte colony-stimulating factor ("G-CSF"), or other growth factors. The antibodies can also be adhered to the solid supports, which are particularly useful for immunoassays or purification of the target antigen. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene. Techniques for conjugating such a therapeutic moiety to antibodies are well known, see for example, Arnon et al., "Monoclonal Antibodies for Immunotherapy of Drugs in Cancer Therapy," in Monoclonal Antibodies and Cancer Therapy, Reisfeld et al., (Eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom and collaborators, "Antibodies For Drug Delivery", in Controlled Drug Delivery (2nd ed.) Robinson et al. (Eds.) Pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, "Antibody Carriers of Cytotoxic Agents in Cancer Therapy: A Review", in Monoclonal Antibodies '84: Biological and Clinical Applications, Pinchera et al., (Eds.), Pp. 475-506 (1985); "Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal Antibodies for Cancer Detection and Therapy, Baldwin et al., (Eds.), Pp. 303-16 (Academic Press 1985), and Thorpe et al., "The Preparation and Cytotoxic Properties of Antibody-Toxin Conjugates", Immunol. Rev. 62: 119-58 (1982). Alternatively, an antibody can be conjugated to a Second antibody to form an antibody heteroconjugate as described by Segal in U.S. Patent No. 4,676,980, which is incorporated herein by reference in its entirety. An antibody, with or without a therapeutic portion conjugated thereto, administered alone or in combination with the cytotoxic factor (s) and / or the cytokine (s), can be used as a therapeutic agent.

Immuno fenot ipi fication The antibodies of the invention can be used for the immunophenotyping of cell lines and biological samples. The translation product of the gene of the present invention may be useful as a specific cellular marker, or more specifically as a cellular marker that is differentially expressed at various stages of differentiation and / or maturation of particular types of cells. Monoclonal antibodies directed against a specific epitope, or combination of epitopes, will allow the selection of cell populations that express the marker. Various techniques can be used using monoclonal antibodies to select cell populations expressing the marker (s), and include magnetic separation using magnetic spheres coated with antibody, "panning" with the antibody bound to a solid matrix (e.g., plaque) , and flow cytometry (for example, see United States Patent No. 5,985,660; and Morrison et al., Cell, 96: 737-49 (1999)). These techniques allow the selection of particular populations of cells, such as can be found with haematological malignancies (for example, minimal residual disease (MRD) in patients with acute leukemia) and "non-self" or "foreign" cells in transplants to prevent Graft Disease. versus Guest (GVHD). Alternatively, these techniques allow the selection of hematopoietic and progenitor stem cells capable of undergoing proliferation and / or differentiation, as can be found in human umbilical cord blood.

Tests for the Antibody Link The antibodies of the invention can be evaluated for immunospecific binding by any method known in the art. Immunoassays that can be used include but are not limited to competitive and non-competitive assay systems, using techniques such as western spotting or transfer, radioimmunoassays, ELISA (enzyme-linked immunosorbent assay), "sandwich" immunoassays, immunoprecipitation, precipitin reactions, precipitin reactions by gel diffusion, immunodiffusion assays, agglutination assays, complement fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays, to name but a few. Such assays are routine and well known in the art (see for example, Ausubel et al., Eds., 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley and Sons, Inc., New York, (which is incorporated by reference herein, in their entirety.) Exemplary immunoassays are briefly described below (but are not designed by way of limitation.) Immunoprecipitation protocols generally comprise the lysis of a population of cells in a lysis buffer such as the RIPA buffer (1% NP-40 or Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.15 M sodium chloride, 0.01 M sodium phosphate at pH 7.2, 1% Trasilol) supplemented with protein phosphatase and / or protease inhibitors (e.g., EDTA, PMSF, aprotinin, sodium vanadate), adding the antibody of interest to the cell lysate, incubating for a period of time (eg, 1-4 hours) at 4 hours. ° C, adding protein A sepharose spheres and / or prot Ein G to the cell lysate, incubating for approximately one hour or more at 4 ° C, washing the spheres in lysis buffer and resuspending the spheres in SDS / sample buffer. The ability of the antibody of interest to immunoprecipitate a particular antigen can be evaluated by, for example, spotting or western blot analysis. A person skilled in the art could be aware of the parameters that can be modified to increase the binding of the antibody to an antigen and decrease the background (for example, pre-clearance of the cell lysate with sepharose beads). For further discussion regarding immunoprecipitation protocols see, for example, Ausubel et al., Eds. 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley and Sons, Inc., New York on 10.16.1. The western blot analysis generally comprises the preparation of the protein samples, the electrophoresis of the protein samples in a polyacrylamide gel (for example, 8% -20% SDS-PAGE depending on the molecular weight of the antigen), transfer of the protein sample from the polyacrylamide gel to a membrane such as nitrocellulose, PVDF or nylon, blocking the membrane in the blocking solution (e.g., PBS with 3% BSA or skim milk), washing the membrane in buffer wash (for example, PBS-Tween 20), blocking the membrane with primary antibody (the antibody of interest) diluted in the blocking buffer, washing the membrane in the wash buffer, blocking the membrane with a secondary antibody (which recognizes the primary antibody, for example, an anti-human antibody) conjugated to an enzymatic substrate (eg, horseradish peroxidase or alkaline phosphatase) or a radioactive molecule (eg. lo, 32p Q 125j) d luida in the blocking buffer, washing the membrane in the wash buffer, and detecting the presence of the antigen. A person skilled in the art could know about the parameters that can be modified to increase the detected signal and reduce background noise. For further discussion regarding staining or western blotting protocols see for example Ausubel et al., Eds., 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & amp; amp;; Sons, Inc. New York on 10.8.1. ELISAs comprise the preparation of the antigen, the well coating of a 96-well microtiter plate with the antigen, the addition of the antibody of interest conjugated to a detectable compound such as an enzyme substrate (eg, horseradish peroxidase or alkaline phosphatase). ) to the well, and incubating for a period of time, and detecting the presence of the antigen. In ELISAs, the antibody of interest does not have to be conjugated to a detectable compound; rather, a second antibody (which recognizes the antibody of interest) conjugated to a detectable compound can be added to the well. In addition, instead of coating the well with the antigen, the antibody can be coated in the well. In this case, a second antibody conjugated to a detectable compound can be added after the addition of the antigen of interest to the coated well. A person skilled in the art could know the parameters that can be modified to increase the detected signal, as well as other variations of the ELISAs known in the art. For further discussion regarding ELISAs see for example, Ausubel et al., Eds., 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York in 11.2.1. The binding affinity of an antibody to an antigen and the proportion of an antibody-antigen interaction can be determined by competitive binding assays. An example of a competitive binding assay is a radioimmunoassay comprising the incubation of the labeled antigen (eg, 3H or 125I) with the antibody of interest in the presence of increasing amounts of the unlabeled antigen, and detection of the antibody bound to the antibody. labeled antigen. The affinity of the antibody of interest for a particular antigen and the binding ratios can be determined from the data by the scatchard graphic analysis. The competition with a second antibody can also be determined using radioimmunoassays. In this case, the antigen is incubated with the antibody of interest conjugated to a labeled compound (e.g., 3 H or 125 I) in the presence of increasing amounts of a second unlabeled antibody.

Vectors and Host Cells or Hosts The present invention also relates to vectors that include the isolated DNA molecules of the present invention, the host cells or hosts, which are genetically engineered with the recombinant vectors, and the production of KGF-2 polypeptides or fragments of the same, by means of recombinant techniques. Fragments or portions of the polypeptides of the present invention can be employed to produce the corresponding full length polypeptide by peptide synthesis; therefore, the fragments can be used as intermediates to produce the full-length polypeptides. Fragments or portions of the polynucleotides of the present invention can be used to synthesize full-length polynucleotides of the present invention. The present invention also relates to vectors that include polynucleotides of the present invention, host cells that are engineered with vectors of the invention, and production of polypeptides of the invention by recombinant techniques. The host cells are engineered (transduced or transformed or transfected) with the vectors of this invention which can be, for example, a cloning vector or an expression vector. The vector can be, for example, in the form of a plasmid, a viral particle, a phage, etc. Genetically engineered host cells can be cultured in conventional nutrient media, modified as appropriate to activate the promoters, by selecting the transformants or amplifying the KGF-2 genes. The culture conditions, such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to the person of ordinary skill in the art. The polynucleotides of the present invention can be used to produce polypeptides by recombinant techniques. Thus, for example, the polynucleotide can be included in any of a variety of expression vectors for the expression of a polypeptide. Such vectors include chromosomal, non-chromosomal and synthetic DNA sequences, for example, SV40 derivatives; bacterial plasmids; Phage DNA; baculovirus; yeast plasmids; vectors derived from combinations of plasmids and phage DNA, viral DNA such as vaccinia virus, adenovirus, chickenpox virus, and pseudorabies. However, any other vector can be used, as long as it is replicable and viable in the host. The appropriate DNA sequence can be inserted into the vector by a variety of methods. In general, the DNA sequence is inserted into one or several appropriate restriction endonuclease sites by procedures known in the art. Such procedures and others are considered within the scope of those skilled in the art. The DNA sequence in the expression vector is operably linked to one or more appropriate expression control sequences (promoters) to direct DNA synthesis. As representative examples of such promoters, there may be mentioned: the LTR or SV40 promoter, lac or trp of E. coli, the P promoter of lambda phage and other promoters that are known to control the expression of genes in prokaryotic or eukaryotic cells or their virus. The expression vector also contains a ribosome binding site for the start of translation and a transcription terminator. The vector may also include appropriate sequences for the amplification of expression. In addition, expression vectors preferably contain one or more selectable marker genes to provide a phenotypic trait for the selection of transformed host cells, such as dihydrofolate reductase or resistance to neomycin., for the culture of eukaryotic cells, or such as resistance to tetracycline or to ampicillin in E. coli. The vector containing the appropriate DNA sequence as described hereinabove, as well as an appropriate promoter or an appropriate control sequence, can be employed to transform an appropriate host, to allow the host to express the protein. As indicated, the expression vectors will preferably include at least one selectable marker. Such labels include dihydrofolate reductase, G418 or neomycin resistance for the culture of eukaryotic cells and the tetracycline resistance genes, to kanamycin or ampicillin, for culture in E. coli and other bacteria. Representative examples of suitable hosts include, but are not limited to, bacterial cells such as E. coli, Streptomyces and Salmonella typhimurium, - fungal cells, such as yeast cells (eg, Saccharomyces cerevisiae or Pichia pastoris (ATCC Access No. 201178)); insect cells such as Drosophila S2 and Spodoptera Sf9; animal cells such as CHO, COS, 293, and Bowes melanoma cells; plant cells and adenovirals. The appropriate culture media and conditions for the host cells described above are known in the art. In addition to the use of expression vectors in the practice of the present invention, the present invention further includes novel expression vectors comprising operator elements and promoters operably linked to the nucleotide sequences encoding a protein of interest. An example of such a vector is pHE4-5, which is described in detail below. As summarized in Figures 50 and 51, the components of the vector pHE4-5 (SEQ ID No. 147) include: 1) a neomycin phosphotransferase gene as a selection marker, 2) an E. coli origin of replication, 3) a promoter sequence of phage T5, 4) two sequences of the lac operator, 5) a Shine-Delgarno sequence, 6) the repressor gene of the lactose operon (laclq). The origin of replication (oriC) is derived from pUC19 (LTI, Gaithersburg, MD). The promoter sequence and the operator sequences were synthetically prepared. The synthetic production of the nucleic acid sequences is well known in the art. CLONTECH 95/96 Catalog, pages 215-216, CLONTECH, 1020 East Meadow Circle, Palo Alto, CA 94303. A nucleotide sequence coding for KGF-2 (SEQ ID No. 1), is operatively linked to the promoter and the operator by the insertion of the nucleotide sequence between the Ndel and Asp718 sites of the vector pHE4-5. As noted above, vector pHE4-5 contains a laclq gene. Laclq is an allele of the lacl gene that confers strong regulation of the lac operator. Amann, E. et al., Gene 69: 301-315 (1988); Stark, M., Gene 51: 255-267 (1987). The laclq gene encodes a repressor protein that binds to the lac operator sequences and blocks the transcription of the downstream sequences (eg, 3 '). However, the product of the laclq gene dissociates from the lac operator in the presence of either lactose or certain lactose analogs, for example, isopropyl-B-D-thiogalactopyranoside (IPTG). KGF-2 is thus not produced in appreciable amounts in uninduced host cells containing the vector pHE4-5. Induction of these host cells by the addition of an agent such as IPTG, however, results in the expression of the sequence encoding KGF-2.

The promoter / operator sequences of the vector pHE4-5 (SEQ ID No. 148) comprises a T5 phage promoter and two lac operator sequences. One operator is located 5 'to the transcription start site, and the other is located 3' to the same site. These operators, when present in combination with the product of the laclq gene, confer strong repression of the sequences downstream (3 ') in the absence of an inducer of the lac operon, for example IPTG. The expression of the operably linked sequences, located downstream (3 ') of the lac operators can be induced by the addition of an inducer of the lac operon, such as IPTG. The binding of a lac inducer to the laclq proteins results in their release from the lac operator sequences and the initiation of transcription of the operably linked sequences. The regulation of the lac operon of gene expression is reviewed in Devlin, T., TEXTBOOK OF BIOCHEMISTRY ITU CLINICAL CORRELATIONS, 4a. Edition (1997), pages 802-807. The pHE4 series of vectors contain all the components of the vector pHE4-5 except the sequence encoding KGF-2. Characteristics of the pHE4 vectors include the optimized synthetic T5 phage promoter, the lac operator, and the Shine-Delagarno sequences. In addition, these sequences are also optimally spaced, so that the expression of an inserted gene can be strongly regulated and high-level expression occurs after induction. Among the known bacterial promoters suitable for use in the production of proteins of the present invention, are included the lacl and lacZ promoters of E. coli, the T3 and T7 promoters, the gpt promoter, the PR and PL lambda promoters, and the trp promoter. Suitable eukaryotic promoters include the immediate early promoter of CMV, the HSV thymidine kinase promoter, the SV40 early and late promoters, the retroviral LTR promoters, such as those of Rous Sarcoma virus (RSV), and promoters of metallothionein such as the mouse metallothionein-I promoter. The vector pHE4-5 also contains a 5 'Shine-Delgarno sequence to the AUG start codon. The Shine-Delgarno sequences are short sequences generally located approximately 10 nucleotides upstream (eg, 5 ') of the AUG start codon. These sequences essentially direct the prokaryotic ribosomes towards the AUG start codon. Thus, the present invention is also directed to the expression vector useful for the production of the proteins of the present invention. This aspect of the invention is simplified by the vector pHE4-5 (SEQ ID No. 147). The vector pHE4-5 containing a cDNA insert encoding KGF-2Δ33 was deposited with the ATCC on January 9, 1998 as ATCC No. 209575. More particularly, the present invention also includes recombinant constructs comprising a or more of the sequences as broadly described above. The constructs comprise a vector, such as a plasmid or viral vector, within which a sequence of the invention has been inserted, in a forward or inverse orientation. In a preferred aspect of this embodiment, the construct further comprises the regulatory sequences including, for example, a promoter, operably linked to the sequence. Large numbers of suitable vectors and promoters are known to those skilled in the art, and are commercially available. The following vectors are provided by way of example; Bacterials: pQE70, pQE60, pQE-9 (Qiagen), pBS, pDlO, phagescript, psiX174, pbluescript SK, pbsks, pNH8A, pNH16a, pNHldA, pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia); Eukaryotic: pWLNEO, pSV2CAT, pOG44, pXTl, pSG (Stratagene), pSVK3, pBPV, pMSG, pSVL (Pharmacia). However, any other plasmid or vector can be used, as long as they are replicable and viable in the host. Preferred vectors for use in bacteria include pQE70, pQE60 and pQE-9, available from QIAGEN, Inc.; pBluescript vectors, Phagescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available from Stratagene Cloning Systems, Inc .; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia Biotech, Inc. Among the preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia. Preferred expression vectors for use in yeast systems include, but are not limited to pYES2, pYDl, pTEFl / Zeo, pYES2 / GS, pPICZ, pGAPZ, pGAPZalph, pPIC9, pPIC3.5, pHIL-D2, pHIL- Sl, pPIC3.5K, pPIC9K, and PA0815 (all available from Invitrogen, Carlbad, CA). Other suitable vectors will be readily apparent to those skilled in the art. The promoter regions can be selected from any desired gene using the CAT vectors (chloramphenicol transferase) or other vectors with selectable markers. Two appropriate vectors are pKK232-8 and pCM7.

The named, particular bacterial promoters include lacl, lacZ, T3, T7, gpt, lambda PR, PL and trp. The eukaryotic promoters include the immediate early promoter of CMV, the thymidine kinase of HSV, the early and late SV40, the LTRs of the retroviruses, and the mouse metallothionein-I. The selection of the appropriate vector and promoter is well within the level of ordinary skill in the art.

The introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-dextran-mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al., Basic Methods in Molecular Biology (1986). It is specifically contemplated that the KGF-2 polypeptides can in fact be expressed by a host cell lacking a recombinant vector. In a further embodiment, the present invention relates to host cells containing the constructions described above. The host cell can be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell. The introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-dextran-mediated transfection, or electroporation (Davis, L. et al., Basic Methods in Molecular Biology (1986)). Constructs in the host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence.

Alternatively, the polypeptides of the invention can be synthetically produced by conventional peptide synthesizers. Mature proteins can be expressed in mammalian cells, yeast cells, bacteria, or other cells under the control of appropriate promoters. Cell-free translation systems can also be employed to produce such proteins, using the RNAs derived from the DNA constructs of the present invention. Suitable cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y. (1989), the description of which is incorporated by reference herein. The transcription of the DNA encoding the polypeptides of the present invention by higher eukaryotes is increased by the insertion of an enhancer sequence within the vector. Augmentators are cis-acting elements of DNA, usually about 10 to 300 base pairs acting on a promoter, to increase its transcription. Examples include the SV40 enhancer on the late side of the replication origin of 100 to 270 base pairs, an early cytomegalovirus promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenoviral enhancers. For the secretion of the translated protein towards the lumen of the endoplasmic reticulum, towards the periplasmic space or towards the extracellular environment, appropriate secretion signals can be incorporated within the expressed polypeptide. The signals may be endogenous to the polypeptide or these may be heterologous signals. The polypeptide can be expressed in a modified form, such as a fusion protein, and can include not only secretion signals, but also additional, heterologous functional regions. For example, a region of additional amino acids, particularly charged amino acids, can be added to the N-terminus of the polypeptide to improve stability and persistence in the host cell, during purification, or during subsequent handling and storage. Also, the peptide portions can be added to the polypeptide to facilitate purification. Such regions can be removed before the final preparation of the polypeptide. The addition of the peptide portions to the polypeptides to engender secretion or excretion, to improve stability and to facilitate purification, among others, are familiar and routine techniques in the art. A preferred fusion protein comprises a heterologous region from the immunoglobulin, which is useful for solubilizing the receptors. For example, European Patent A-0,464,533 (Canadian counterpart 2045869) describes the fusion proteins comprising various portions of the constant region of the immunoglobulin molecules, together with another human protein or part thereof. In many cases, the Fe part in the fusion protein is completely advantageous for use in therapy and diagnosis and thus, results in, for example, improved pharmacokinetic properties (European Patent A-0232262). On the other hand, for some uses it might be desirable to make it possible to suppress the Fe part after the fusion protein has been expressed, detected and purified in an advantageous manner, described. This is the case when the Fe portion proves to be an impediment for use in therapy and diagnosis, for example, when the fusion protein is to be used as an antigen for immunizations. In drug discovery, for example, human proteins, such as the shIL-5 receptor has been fused with Fe portions for the purpose of high throughput screening assays, to identify hIL-5 antagonists. See, D. Bennett et al., J. Mol. Recognition, Vol. 8 52-58 (1995) and K. Johanson et al., J. Biol. Chem. 270 (16): 9459-9471 (1995).

In general, recombinant expression vectors will include origins of replication and selectable markers that allow the transformation of the host cell, for example, the E. coli ampicillin resistance gene and the TRP1 gene of S. cerevisiae, and a promoter. derived from a gene highly expressed to direct the transcription of a structural sequence downstream (3 '). Such promoters can be derivatives of operons that code for glycolytic enzymes such as 3-phosphoglycerate kinase (PGK), factor a, acid phosphatase, or heat shock proteins, among others. The heterologous structural sequence is assembled in the appropriate phase with the translation initiation and termination sequences, and preferably, a guiding sequence capable of directing secretion in the translated protein into the periplasmic space or into the extracellular medium. Optionally, the heterologous sequence can encode a fusion protein that includes an N-terminal identification peptide that imparts desired characteristics, for example, stabilization or simplified purification of expressed recombinant product. Expression vectors useful for use in bacteria are constructed by inserting a structural DNA sequence encoding a desired protein, together with suitable translation initiation and termination signals, in the reading phase operable with a promoter. functional. The vector will comprise one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector, and if desirable, to provide amplification within the host. Suitable prokaryotic hosts for transformation include E. coli, Bacillus subtilis, Salmonella typhimurium and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus, although others may be employed as a matter of choice. As a representative but not limiting example, expression vectors useful for use in bacteria may comprise a selectable marker and the bacterial origin of replication derived from commercially available plasmids, comprise genetic elements of the well-known pBR322 cloning vector (ATCC 37017) .

Such commercial vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEMÍ (Promega Biotec, Madison, Wl, USA). These "backbone" sections of pBR322 are combined with an appropriate promoter and the structural sequence to be expressed. After transformation of a suitable host strain and development of the host strain to an appropriate cell density, the selected promoter is induced by appropriate means (e.g., temperature change or chemical induction) and the cells are cultured for an additional period . The cells are typically harvested by centrifugation, disintegrated by physical or chemical means, and the resulting crude extract retained for further purification. The microbial cells employed in the expression of proteins can be disintegrated by any convenient method, including freeze-thaw cycles, sonication, mechanical disintegration, or the use of cell lysis agents, such methods are well known to those skilled in the art. technique. Various mammalian cell culture systems can also be employed to express the recombinant protein. Examples of mammalian expression systems include the COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell 23: 175 (1981), and other cell lines capable of expressing a compatible vector, eg, cell lines. C127, 3T3, CHO, HeLa and BHK. The mammalian expression vectors will comprise an origin of replication, a suitable promoter and an enhancer, and also any necessary sites of binding to the ribosome, the polyadenylation site, the splice donor and acceptor sites, the transcription termination sequences, and the 5 'non-transcribed flanking sequences. The DNA sequences derived from the SV40 junction, and the polyadenylation sites can be used to provide the required non-transcribed genetic elements. KGF-2 polypeptides can be recovered and purified from recombinant cell cultures by well-known methods including precipitation with ammonium sulfate or ethanol, acid extraction, anionic or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography and lectin chromatography. More preferably, high performance liquid chromatography ("HPLC") is used for the purification. The polypeptides of the present invention may be a naturally purified product, or a product of synthetic chemical processes, or produced by recombinant techniques from a prokaryotic or eukaryotic host (for example, by bacterial, yeast, higher plant, insect and mammal, in culture). Depending on the host employed in a recombinant production method, the polypeptides of the present invention can be glycosylated or can be non-glycosylated. The polypeptides of the invention may also include an initial methionine amino acid residue. The KGF-2 polypeptides, and preferably the secreted form, can also be recovered from: the products purified from natural sources, including fluids, tissues and body cells, either directly isolated or cultured; products of chemical synthesis procedures; and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect, and mammalian cells. Depending on the host employed in a recombinant production process, the KGF-2 polypeptides can be glycosylated or can be non-glycosylated. In addition, the KGF-2 polypeptides may also include an initial, modified methionine residue, in some cases as a result of the host-mediated processes. Thus, it is well known in the art that the N-terminal methionine encoded by the translation start codon is, in general, removed with high efficiency of any protein after translation in all eukaryotic cells. While the N-terminal methionine in most proteins is also efficiently removed in most prokaryotes, for some proteins, this process of prokaryotic elimination is inefficient, depending on the nature of the amino acid to which methionine N is covalently bound. -terminal. In one embodiment, the yeast Pichia pastoris is used to express the KGF-2 protein in a eukaryotic system. Pichia pastoris is a methylotrophic yeast which can metabolize methanol as its sole carbon source. A major step in the methanol metabolism pathway is the oxidation of methanol to formaldehyde using 02. This reaction is catalyzed by the enzyme alcohol oxidase. In order to metabolize methanol as its sole carbon source, Pichia pastoris must generate high levels of alcohol -oxidase due in part to the relatively low affinity of alcohol -oxidase for 02. Consequently, in a growth medium depending on methanol, as a main source of carbon, the promoter region of one of the two alcohol -oxidase genes (A0X1) is highly active. In the presence of methanol, the alcohol -oxidase produced from the AOX1 gene comprises up to about 30% of the total soluble protein in Pichia pastoris. See Ellis, S.B. and collaborators, Mol. Cell. Biol. 5: 1111-21 (1985); Koutz, P.J. and collaborators, Yeast 5: 167-77 (1989); Tschopp, J.F. and collaborators, Nucí. Acids Res. 15: 3859-76 (1987). Thus, a heterologous coding sequence, such as for example a KGF-2 polynucleotide of the present invention, under the transcriptional regulation of all or part of the regulatory sequence A0X1 is expressed at exceptionally high levels in Pichia yeast grown in the presence of methanol. In one example, the plasmid vector pPIC9K is used to express the DNA encoding a KGF-2 polypeptide of the invention, as described herein, in a Pichia yeast system, essentially as described in "Pichia Protocols: Methods in Molecular Biology ", DR Higgins and J. Cregg, eds. The Humana Press, Totowa, NJ, 1998. This expression vector allows the expression and secretion of a KGF-2 protein of the invention, by virtue of the strong AOX1 promoter, linked to the alkaline phosphatase-secreting signal (PHO) peptide. of Pichia pastoris (eg, guide) located upstream (5 ') of a multiple cloning site. Many other yeast vectors could be used in place of pPIC9K, such as, pYES2, pYDl, pTEFl / Zeo, pYES2 / GS, pPICZ, pGAPZ, pGAPZalpha, pPIC9, pPIC3.5, pHIL-D2, pHIL-Sl, pPIC3 .5K, and PA0815, as a person skilled in the art would readily appreciate, as long as the proposed expression construct provides the appropriately localized signals for transcription, translation, secretion (if desired), and the like, including an intrastructural AUG , As required.

In a further embodiment, the high level expression of a heterologous coding sequence such as, for example, a KGF-2 polynucleotide of the present invention, can be achieved by cloning the heterologous polynucleotide of the invention in such an expression vector. as, for example, pGAPZ or pGAPzalfa, and developing the yeast culture in the absence of methanol. In addition to encompassing the host cells containing the vector constructs discussed herein, the invention also encompasses primary, secondary, and immortalized host cells of vertebrate origin, particularly of mammalian origin, which have been engineered to suppress or replace the endogenous genetic material (for example, the sequence encoding KGF-2), and / or to include the genetic material (e.g., heterologous polynucleotide sequences) that is operably associated with the KGF-2 polynucleotides of the invention, and which activates, alters, and / or amplifies the endogenous KGF-2 polynucleotides. For example, techniques known in the art can be used to operably associate the heterologous control regions (eg, promoters and / or augmentations) and the endogenous KGF-2 polynucleotide sequences, via homologous recombination, resulting in the formation of a new transcription unit (see for example, U.S. Patent No. 5,641,670, issued June 24, 1997, U.S. Patent No. 5,733,761, issued March 31, 1998; WO 96/29411, published September 26, 1996; International Publication No. WO 94/12650, published August 4, 1994; Koller et al., Proc. Nati, Acad. Sci. USA 86: 8932-8935 ( 1989), and Zijlstra et al., Nature 342: 435-438 (1989), the descriptions of each of which are incorporated by reference herein, in their entirety).

Diagnostic and Therapeutic Applications of KGF-2 As used in the following section, "KGF-2" is intended to refer to the full-length and mature forms of KGF-2 described herein and to the KGF-2 analogues, the derivatives of the mutants thereof. , described in the present. This invention is also related to the use of the KGF-2 gene as part of a diagnostic assay for detecting diseases or susceptibility to diseases related to the presence of mutations in the nucleic acid sequences of KGF-2. Individuals that have mutations in the gene of KGF-2 can be detected at the DNA level within a variety of techniques. The nucleic acids for diagnosis can be obtained from the cells of a patient, such as blood, urine, saliva, tissue biopsy and autopsy material. Genomic DNA can be used directly for detection or can be amplified enzymatically by the use of PCR (Saiki et al., Nature 324: 163-166 (1986)) before analysis. The AR? or the AD? c can also be used for the same purpose. As an example, PCR primers complementary to the nucleic acid encoding KGF-2 can be used to identify and analyze KGF-2 mutations. For example, deletions and insertions can be detected by a change in the size of the amplified product, compared to the normal genotype. Point mutations can be identified by the hybridization of AD? amplified to AR? of radiolabelled KGF-2 or alternatively, the sequences of AD? antisense of KGF-2, radiolabeled. The perfectly coupled sequences can be distinguished from mismatched duplexes by digestion with RA? Asa A or by differences in melting temperatures. The genetic test based on the differences in the sequence of AD? can it be carried out by detecting the alteration in the electrophoretic mobility of the AD fragments? in gels with and without denaturing agents. Suppressions and small sequential insertions can be visualized by high resolution gel electrophoresis. The DNA fragments of different sequences can be distinguished on denaturing formamide gradient gels, in which the mobilities of the different DNA fragments are delayed in the gel at different positions, according to their specific melting or partial melting temperatures. (see for example, Myers et al., Science, 230: 1242 (1985)). Changes in sequence at specific sites can also be revealed by nuclease protection assays such as RNase and SI protection or the chemical cleavage method (eg, Cotton et al., PNAS, USA, 85-4397-4401). (1985)). Thus, detection of a specific DNA sequence can be achieved by methods such as hybridization, RNase protection, chemical cleavage, direct DNA sequencing or the use of restriction enzymes (eg, Fragment Length Polymorphisms). of Restriction (RFLP)) and spotting or Southern blotting of genomic DNA. In addition to more conventional gel electrophoresis and DNA sequencing, mutations can also be detected by in situ analysis. The present invention also relates to a diagnostic assay for detecting altered levels of the KGF-2 protein in various tissues, since an overexpression of the proteins compared to normal control tissue samples can detect the presence of a disease or susceptibility to a disease, for example, a tumor. Assays used to detect levels of the KGF-2 protein in a sample derived from a host are well known to those skilled in the art and include radioimmunoassays, competitive binding assays, stain or Western blot analysis, ELISA assays and "sandwich" trials. An ELISA assay (Coligan et al., Current Protocols in Imunology, 1 (2), Chapter 6, (1991)) comprises initially the preparation of an antibody specific for the KGF-2 antigen, preferably a monoclonal antibody. In addition, a reporter antibody is prepared against the monoclonal antibody. A detectable reagent such as radioactivity, fluorescence or, in this example, a horseradish peroxidase enzyme is coupled to the reporter antibody. A sample is removed from a host and incubated on a solid support, for example, a polystyrene box that binds the proteins in the sample. Any free binding sites of the protein on the box are then covered by incubation with a non-specific protein such as bovine serum albumin. Right away, the monoclonal antibodies adhere to any KGF-2 proteins adhered to the polystyrene box. All unbound monoclonal antibody is washed with buffer. The reporter antibody linked to horseradish peroxidase is now placed in the box, resulting in the binding of the reporter antibody to any monoclonal antibody bound to KGF-2. The uncoupled reporter antibody is then washed. The peroxidase substrates are then added to the box and the amount of color developed in a given period of time is a measurement of the amount of the KGF-2 protein present in a given volume of the patient sample when compared against a curve standard. A competition assay can be employed, wherein antibodies specific for KGF-2 that are coupled to a solid support and labeled KGF-2 and a sample derived from the host, are passed over the solid support and the amount of the detected marker, for example, by liquid scintillation chromatography, it can be correlated to an amount of KGF-2 in the sample. The "sandwich" test is similar to an ELISA assay. In a "sandwich" assay, KGF-2 is passed over a solid support and bound to the antibody coupled to a solid support. A second antibody is then bound to KGF-2. A third antibody that is labeled and specific for the second antibody is then passed over the solid support and bound to the second antibody, and an amount can then be quantified. The polypeptides, their fragments or other derivatives, or analogs thereof, or cells expressing them can be used as an immunogen to produce antibodies thereto. These antibodies can be, for example, polyclonal or monoclonal antibodies. The present invention also includes chimeric, single chain, and humanized antibodies as well as Fab fragments, or the product of a Fab expression library. Various methods known in the art can be used for the production of such antibodies and fragments. The antibodies generated against the polypeptides corresponding to a sequence of the present invention can be obtained by direct injection of the polypeptides in an animal or by administration of the polypeptides to an animal, preferably a non-human one. The antibody thus obtained will also bind to the polypeptides themselves. In this way, even a sequence encoding only a fragment of the polypeptides can be used to generate antibodies that bind to the entire native polypeptides. Such antibodies can then be used to isolate the polypeptide from the tissue expressing that polypeptide.

For the preparation of monoclonal antibodies, any technique that provides antibodies produced by cultures of continuous cell lines can be used. Examples include the hybridoma technique (Kohler and Milstein, Nature, 256: 495-497 (1975)), the trioma technique, the human B-cell hybridoma technique (Kozbor et al., Im unology Today 4:72 (1983). )), and the EBV hibrhythm technique for producing human monoclonal antibodies (Colé et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-86 (1985)). The techniques described for the production of single chain antibodies (U.S. Patent No. 4,946,778) can be adapted to produce single chain antibodies for immunogenic polypeptide products of this invention. Also, transgenic mice can be used to express humanized antibodies to immunogenic polypeptide products of this invention. The polypeptides of the present invention have been shown to stimulate epithelial development. In this way, the polypeptides of the present invention can be used to stimulate the development of the epithelium. "Epithelium" refers to the covering of the internal and external surfaces of the body, including the lining of vessels and other small cavities. This consists of cells joined by small amounts of cementing substances. The epithelium is classified into several types, based on the number of depth to the layers and the shape of the superficial cells. Epithelial cells include the anterior cornea, Barrett's epithelium, capsular epithelium, ciliated epithelium, columnar epithelium, corneal epithelium, cubital epithelium, semicircular epithelial ducts, enamel epithelium, false epithelium, germinal epithelium, gingival epithelium, glandular epithelium, glomerular epithelium , laminated epithelium, lens epithelium, mesenchymal epithelium, olfactory epithelium, pavement epithelium, pigmentary epithelium, protective epithelium, pseudostratified epithelium, pyramidal epithelium, respiratory epithelium, rod epithelium, seminiferous epithelium, sensory epithelium, simple epithelium, squamous epithelium, stratified epithelium, subcapsular epithelium, sulcular epithelium, tessellated epithelium, transitional epithelium, and epithelial cells of the eye, tongue, glands, oral mucosa, duodenum, ileum, jejunum, caecum, nasal passages , the esophagus, the colon, the mammary glands, and reproductive systems masculine and feminine. The "glands" refer to an aggregation of cells, specialized to secrete or excrete materials unrelated to their ordinary metabolic needs. Examples of glands that may include epithelial cells include: absorbent glands, accessory glands, acinar glands, acid glands, maxillary glands, adrenal glands, aggregated glands, Albarran glands, anal glands, alveolar glands, anteprostatic glands, aortic glands, apical glands of the tongue, apocrine glands, areolar glands, arterial glands, arteriococcygeal glands, arytenoid glands, Aselli glands, Avicenna glands, atrial glands, axillary glands, Bartholin's glands, Bauhin's glands, Baumgarten's glands, glands of the biliary mucosa , Blandin glands, blood vessel glands, Boerhaave glands, Bonnot glands, Bowman glands, brachial glands, bronchial glands, Bruch glands, Brunner glands, buccal glands, bulbocavernosal glands, cardiac glands, glands carotids, celiac glands, ceruminous glands, cervical glands of the uterus, choroid glands, glands of Ciaccio, ciliary glands of the conjunctiva, circumanales glands, cloquet glands, Cobelli glands, coccygeal glands, reel glands, compound glands, conglobated glands, conjunctival glands, Cowper's glands, cutaneous glands, cytogenic glands, glands without conduit, duodenal glands, Duverney's glands, Ebner's glands, eccrine glands, Eglis glands, endocrine glands, endoepithelial glands, esophageal glands, excretory glands , exocrine glands, follicular duct glands, fundus glands, gastric glands, gastroepiploic glands, Gay glands, genital glands, gingival glands, Gley glands, globular glands, agglomerated glands, glossopalatin glands, Guerin glands, guttural glands, glands of Haller, Harder's glands, Haversian glands, hedonic glands, hema glands, hepatic lymphatic glands, hematopoietic glands, hemolymphatic glands, glands of Henle, hepatic glands, heterocrine glands, hibernation glands, holocrine glands and incremental glands. Additional examples of glands include intercarotid glands, intermediate glands, intercapsular glands, interstitial glands, intestinal glands, intraepithelial glands, intramuscular glands of the tongue, jugular glands, Krause glands, labial glands of the mouth, lacrimal glands, accessory lacrimal glands , lactiferous glands, large intestine glands, large sweat glands, laryngeal glands, lenticular glands of the stomach and tongue, Lieberkuhn's glands, lingual glands, anterior lingual glands, Littre's glands, Luschka's glands, lymphatic glands, extrapatrid lymphatic glands , malar glands, mammary glands, accessory mammary glands, mandibular glands, Manz glands, Mehlis glands, meibomian glands, merocrine glands, mesenteric glands, mesocolic glands, mixed glands, molar glands, Molí glands, monoptíficas glands, Montgomery glands, Morgagni glands, glands of the mouth, mucilaginous glands, muciparous glands, mucous glands, lingual mucous glands, mucous glands of the auditory tube, mucous glands of the duodenum, mucous glands of the Eustachian tube, multicellular glands, myometrial glands, Naboth glands, Naboth glands, nasal glands, neck glands, odoriferous foreskin glands, oily glands, olfactory glands, oxyntic glands, pachyonian glands, glands, palatinas, pancreaticosplenic glands, parafrenal glands , parathyroid glands, paraurethral glands, parotid glands, accessory parotid glands, pectoral glands, peptic glands, respiratory glands, Peyre's glands, pharyngeal glands, Philip's glands, pineal glands, and the pituitary gland. Other examples of glands include Poirier glands, poliptichich glands, preen glands, pregnancy glands, prehyoid glands, foreskin glands, prostate gland, puberty glands, pyloric glands, racemose glands, retroling glands, retromolar glands, Rivinus glands, Rosenmuller's gland, saccular gland, salivary glands, abdominal salivary glands, external salivary glands, internal salivary glands, Sandstrom's glands, Schuller's glands, sebaceous glands, sebaceous glands of the conjunctiva, sentinal glands, seromucous glands, glands serous, Serres glands, Sigmunds glands, Skene glands, simple gland, small intestine glands, solitary glands of the large intestine, splenoid glands, Stahr's gland, staropiline glands, subauricular glands, sublingual glands, submannanal glands dibular, suborphic glands, adrenal glands, accessory adrenal glands, Suzanne gland, sweat glands, synovial glands, tarsal glands, Theile glands, thymus gland, thyroid gland, accessory thyroid glands, tongue glands, tracheal glands, glands of the tacoma, tubular glands, tubuloacinar glands, tympanic glands, Tyson glands, unicellular glands, urethral glands, urethral glands, urethral glands of the female urethra, uropigial gland, uterine glands, utricular glands, vaginal glands, vascular glands, vestibular glands (major and minor), Virchow's glands, vitelline gland, bulbovaginal gland, Waldeyer's glands, Weber's glands, Wolfring's glands, Zeis's glands and Zuckerkandl's glands. In this way, KGF-2 can be used to stimulate the development of any of these cells, or cells within these glands. The polypeptides of the present invention can be used to stimulate the development of new blood vessels or angiogenesis. Particularly, the polypeptides of the present invention can stimulate the growth and proliferation of keratinocyte cells. Accordingly, the present invention provides a process for using such polypeptides, or polynucleotides that encode such polypeptides for therapeutic purposes, for example, to stimulate the proliferation of basal epithelial and keratinocyte cells for purposes of wound healing, and to stimulate production. of the hair follicle and the healing of the dermal wounds. As noted above, the polypeptides of the present invention can be used to heal skin wounds by stimulating the proliferation of epithelial cells. These wounds may be superficial in nature or may be deep and involve damage to the dermis and epidermis of the skin. Thus, the present invention provides a method for the promotion of wound healing that involves the administration of an effective amount of KGF-2 to an individual. The individual to whom KGF-2 is administered can heal their wounds at a normal speed or may be healing in a deteriorated manner. When administered to an individual who has no deteriorated healing, KGF-2 is administered to accelerate the normal healing process. When administered to an individual who has deteriorated healing, KGF-2 is administered to facilitate the healing of wounds that might otherwise heal slowly or not at all. As noted below, a number of afflictions and conditions can result in deterioration of the healing. These conditions and conditions include diabetes (for example, diabetes mellitus type II), treatment with steroids and other pharmacological agents, and blockage or ischemic damage. Steroids that have been shown to impair wound healing include cortisone, hydrocortisone, dexamethasone, and methylprednisolone. Non-steroidal compounds, for example, octreotide acetate, have also been shown to deteriorate wound healing. Waddell, B. and collaborators, Am. Surg. 63: 446-449 (1997). It is believed that the present invention promotes the healing of wounds in individuals undergoing treatment with non-steroidal agents.

A number of growth factors have been shown as promoters of wound healing in individuals with impaired healing. See, for example, Steed, D. and collaborators, J. Am. Coll. Surg. 183: 61-64 (1996); Richard, J. et al., Diabetes Care 18: 64-69 (1995); Steed, D., J. Vasc. Surg. 21: 71-78 (1995); Kelley, S. et al., Proc. Soc. Exp. Biol. 194: 320-326 (1990). These growth factors include the growth hormone releasing factor, platelet derived growth factor, and basic fibroblast growth factor. Thus, the present invention also encompasses the administration of KGF-2 in conjunction with one or more additional growth factors or another agent that promotes wound healing. The present invention also provides a method for promoting the healing of anastomotic wounds and other wounds, caused by surgical procedures in individuals who heal their wounds at a normal speed and have deteriorated healing. This method involves the administration of an effective amount of KGF-2 to an individual, before, after, and / or during anastomotic surgery or other surgery. The anastomosis is the connection of two tubular structures, as happens, for example, when an intermediate section of the intestine is removed and the remaining portions are linked together to reconstitute the intestinal tract. Conversely with skin healing, the healing process of anastomotic wounds is generally obscured from sight. In addition, the healing of wounds, at least in the gastrointestinal tract, occurs rapidly in the absence of complications; however, complications often require correction for additional surgery. Thornton, F. and Barbul, A., Surg. Clin. North Am. 77: 549-573 (1997). As shown in Examples 21 and 28, treatment with KGF-2 causes a significant decrease in peritoneal leak and anastomotic constriction after colonic anastomosis. It is believed that KGF-2 causes these results by accelerating the healing process thereby decreasing the likelihood of complications arising after such procedures. Thus, the present invention also provides a method for accelerating healing after anastomosis or other surgical procedures in an individual, who heals their wounds at a normal speed or has deteriorated healing, compromising the administration of an effective amount of KGF. -2. The polypeptides of the present invention can also be used to stimulate the differentiation of cells, for example, muscle cells, cells that constitute nervous tissue, prostate cells, and lung cells.

KGF-2 may be clinically useful for stimulating the healing of wounds including surgical wounds, removal wounds, deep wounds involving damage to the dermis and epidermis, ocular tissue wounds, dental tissue wounds, wounds of the oral cavity, ulcers diabetics, dermal ulcers, ulcers of the ulna, arterial ulcers, venous stasis ulcers, and burns resulting from exposure to heat or chemicals, in normal individuals and those subject to conditions that induce abnormal healing of wounds such as uremia, poor nutrition , vitamin deficiencies, obesity, infection, immunosuppression and complications associated with systemic treatment with steroids, radiotherapy, and antineoplastic drugs, and antimetabolites. KGF-2 is also useful to promote the healing of wounds associated with ischemia and ischemic damage, for example, venous ulcers of the chronic legs, caused by a deterioration of the return of the venous circulatory system and / or insufficiency thereof. KGF-2 can also be used to promote skin re-establishment subsequent to skin loss. In addition, KGF-2 can be used to increase the tensile strength of the epidermis and the epidermal thickness. KGF-2 can be used to increase the adherence of skin grafts to a wound bed and to stimulate re-epithelialization from the wound bed. The following are types of grafts in which KGF-2 could be used to increase adherence to a wound bed: autografts, artificial skin, allografts, autodermal graft, autoepidermal grafts, aortic grafts, Blair-Brown grafts, bone grafts, brephoplastic grafts, skin grafts, delayed grafts, dermal grafts, epidermal grafts, fascia grafts, full thickness grafts, grafts heterologous, xenografts, homologous grafts, hyperplastic grafts, lamellar grafts, mesh grafts, mucosal grafts, Ollier-Thiersch grafts, omenpal grafts, patch grafts, pedicle grafts, penetration grafts, split skin grafts, thick split grafts . KGF-2 can be used to promote skin resistance and to improve the appearance of aged skin. It is believed that KGF-2 will also produce changes in the proliferation of hepatocytes, and the proliferation of epithelial cells in the lung, breast, pancreas, stomach, small intestine, and large intestine. KGF-2 can promote the proliferation of epithelial cells such as sebocytes, hair follicles, hepatocytes, type II pneumocytes, mucin-producing globular cells, and other epithelial cells and their progenitors, contained within the skin, lung, liver, Kidney and the gastrointestinal tract. As shown in Example 31, KGF-2 stimulates the proliferation of hepatocytes. Thus, KGF-2 can also be used prophylactically or therapeutically to prevent or attenuate acute or chronic viral hepatitis as well as hepatic or fulminating or sub-terminal insufficiency caused by diseases such as acute viral hepatitis, cirrhosis, drug-induced hepatitis and toxins. (eg, acetaminophen, carbon tetrachloride, methotrexate, organic arsenicals, and other hepatotoxins known in the art), autoimmune chronic active hepatitis, liver transplantation, and partial hepatectomy (Cotran et al., Pathologic basis of disease (5th Ed. ), Philadelphia, WB Saunders Company, 1994). KGF-2 can also be used to stimulate or promote liver regeneration and in patients with alcoholic liver disease. KGF-2 can be used to treat fibrosis of the liver. Approximately 80% of cases of acute pancreatitis are associated with biliary tract disease and alcoholism (Rattner D.W., Scand J. Gastroenterol 31: 6-9 (nineteen ninety six); Cotran et al., Pathologic basis of disease (5th Ed.), Philadelphia, W.B. Saunders Company, 1994). Acute pancreatitis is a major clinical problem with significant morbidity and mortality (Banerjee et al., Bri tish Journal of Surgery 81: 1096-1103 (1994)). The pathogenesis of this disease is still somewhat unresolved but it is widely recognized that pancreatic enzymes are released into the pancreas leading to proteolysis, interstitial inflammation, fat necrosis, and hemorrhage. Acute pancreatitis can lead to disseminated intravascular coagulation, adult respiratory distress syndrome, stroke, and acute renal tubular necrosis (Cotran et al., Pathologic basis of diesease (5th ed.), Philadelphia, W.B. Saunders Company, 1994). Despite palliative measures, approximately 5% of these patients die of shock during the first week of the clinical course. In surviving patients, sequelae may include pancreatic abscess, pseudocysts, and duodenal obstruction (Cotran et al, Pathologic basic of disease (5th ed.), Philadelphia, W.B. Saunders Company, 1994). Chronic pancreatitis is often a progressive destruction of the pancreas caused by repeated outbreaks of acute pancreatitis. Chronic pancreatitis appears to incur a modestly increased risk of pancreatic carcinoma (Cotran et al., Pathologic basis of disease (5th ed.), Philadelphia, W.B. Saunders Company, 1994). As indicated above and in Example 31, KGF-2 also promotes the proliferation of pancreatic cells. Thus, in a further aspect, KGF-2 can be used prophylactically or therapeutically to prevent or attenuate acute or chronic pancreatitis. KGF-2 can also be used to reduce the side effects of bowel toxicity that result from the treatment of viral infections, radiation therapy, chemotherapy or other treatments. KGF-2 can have a cytoprotective effect on the mucosa of the small intestine. KGF-2 can also be used prophylactically or therapeutically to prevent or attenuate mucositis and to stimulate the healing of mucositis (eg, oral, esophageal, intestinal, colonic, rectal, and anal ulcers) resulting from chemotherapy, other agents and viral infections. Thus, the present invention also provides a method for preventing or treating diseases or pathological events of the mucosa, including ulcerative colitis, Crohn's disease, and other diseases where the mucosa is damaged, comprising the administration of an effective amount of KGF- 2. The present invention similarly provides a method for preventing or treating oral mucositis (including odynophagia associated with mucosal damage in the pharynx and hypopharynx), esophageal, gastric, intestinal, colonic and rectal, regardless of the people or modality which causes this damage. In addition, KGF-2 could be used to treat and / or prevent: blisters and burns due to chemicals; damage to the ovaries, for example, due to treatment with chemotherapeutic products or treatment with cyclophosphamide; cystitis induced by radiation or by chemotherapy; or intestinal damage induced by high-dose chemotherapy. KGF-2 could be used to promote internal healing, the healing of the donor site, the healing of internal surgical wounds, or the healing of incisional wounds made during cosmetic surgery. KGF-2 can promote the proliferation of endothelial cells, keratinocytes, and basal keratinocytes. Thus, the present invention also provides a method for stimulating the proliferation of such cell ty which involves contacting the cells with an effective amount of KGF-2. KGF-2 can be administered to an individual in an effective amount to stimulate cell proliferation in vivo, or KGF-2 can be contacted with such cells in vi tro. The present invention further provides a method for promoting urothelial healing comprising the administration of an effective amount of KGF-2 to an individual. Thus, the present invention provides a method for accelerating the healing or treatment of a variety of pathologies involving uroepithelial cells. (for example, cells that line the urinary tract). The tissue layers comprising such cells can be damaged by numerous mechanisms including catheterization, surgery, or bacterial infection (for example, infection by an agent that causes a sexually transmitted disease, such as gonorrhea). The present invention also encompasses methods for the promotion of tissue healing in the female genital tract, comprising the administration of an effective amount of KGF-2. Tissue damage in the female genital tract can be caused by a wide variety of conditions including Candida infections, trichomoniasis, Gardnerella infections, gonorrhea, chlamydia, mycoplasma and other sexually transmitted diseases. As shown in Examples 10, 18, and 19, KGF-2 stimulates the proliferation of epidermal keratinocytes and increases epidermal thickening. In this way, KGF-2 can be used in the complete regeneration of the skin; in full and partial thickness skin defects, including burns (eg, repopulation of hair follicles, sweat glands, and sebaceous glands); and the treatment of other skin defects such as psoriasis. KGF-2 can be used to treat epidermolysis bullosa, a defect in the adherence of the epidermis to the underlying dermis, which results in frequent, open and painful ampullae due to the acceleration of the re-epithelialization of these lesions. KGF-2 can also be used to treat gastric and duodenal ulcers and helps to heal the mucosal lining and regeneration of the glandular mucosa, and the duodenal mucosal lining more quickly. Inflammatory bowel diseases, such as Crohn's disease and ulcerative colitis, are diseases that result in the destruction of the mucosal surface of the small or large intestine, respectively. In this way, KGF-2 could be used to promote surface reconstruction of the mucosal surface to aid in faster healing and to prevent or attenuate the progression of inflammatory bowel disease. It is expected that treatment with KGF-2 will have a significant effect on mucus production throughout the gastrointestinal tract and could be used to protect the intestinal mucosa from harmful substances that are ingested, or after surgery. As noted above, KGF-2 could also be used to promote the healing of intestinal or colonic anastomosis. KGF-2 can also be used to treat diseases associated with underexpression of KGF-2. As shown in Example 32 below, KGF-2 stimulates the proliferation of lung epithelial cells.

In this way, KGF-2 can be administered prophylactically to reduce or prevent damage to the lungs, caused by various pathological conditions. KGF-2 can also be administered during or after a damaging event that occurs to promote healing. For example, KGF-2 can stimulate proliferation and differentiation, and promote repair of the alveoli and bronchiolar epithelium, to prevent, attenuate, or treat lung damage, acute or chronic. Emphysema, which results in progressive loss of the alveoli, and damage by inhalation, for example, resulting from the inhalation of smoke and burns, which cause necrosis of the bronchiolar epithelium and the alveoli, could be effectively treated using KGF- 2, as it could be the damage attributable to chemotherapy, radiation treatment, lung cancer, asthma, black lung and other conditions that damage the lung. Also, KGF-2 could be used to stimulate the proliferation and differentiation of type II pneumocytes, which can help treat or prevent disease such as hyaline membrane diseases, such as infant respiratory distress syndrome and bronchopulmonary dysplasia, in premature infants. The three causes of acute renal failure are prerenal (for example, heart failure), intrinsic (for example, nephrotoxicity induced by chemotherapeutic agents) and postrenal (for example, obstruction of the urinary tract) that lead to the death of renal tubular cells, obstruction of the tubular lumens, and backflow of the filtrate to the glomeruli (reviewed by Thadhani et al., N. Engl. J. Med. 334: 1448-1460 (1996)). Growth factors such as insulin-like growth factor I, osteogenic protein I, hepatocyte growth factor, and epidermal growth factor have shown potential to improve kidney disease in animal models. Taub et al., Cytokine 5: 175-179 (1993); Vukicevic et al., J. Am. Soc. Nephrol. 7: 1867 (1996). As shown in Example 31 below, KGF-2 stimulates the proliferation of renal epithelial cells and, thus, is useful for alleviating or treating kidney diseases or pathologies such as acute and chronic renal failure and stage kidney disease. terminal. KGF-2 could stimulate the proliferation and differentiation of breast tissue and therefore could be used to promote healing of breast tissue damage due to surgery, trauma, or cancer. In addition, KGF-2 could be used to treat or prevent the onset of diabetes mellitus. In newly diagnosed type I and II diabetes patients, where some islet cell function remains, KGF-2 could be used to maintain islet function to alleviate, slow or prevent the permanent manifestation of the disease. Also, KGF-2 could be used as an assistant in the transplantation of islet cells to improve or promote the function of islet cells. In addition, the anti-inflammatory property of KGF-2 could be beneficial for the treatment of acute and chronic conditions in which inflammation is a key pathogenesis of diseases including, but not limited to, psoriasis, eczema, dermatitis and / or arthritis. Thus, the present invention provides a method for preventing or attenuating inflammation, and diseases involving inflammation, in an individual comprising administering an effective amount of KGF-2. KGF-2 can be used to promote healing and relieve brain tissue damage due to trauma, surgery or chemical damage. In addition, since KGF-2 increases the thickness of the epidermis, the protein could be used to improve aging skin, reduce wrinkles in the skin, and reduce scarring after surgery. Scarring of wound tissues often involves hyperproliferation of dermal fibroblasts. As noted in Example 10, the proliferation of the fibroblasts is not stimulated by KGF-2. Therefore, KGF-2 appears to be a specific mitogen for epidermal keratinocytes and induces healing of wounds with minimal scarring. Thus, the present invention provides a method for promoting healing of wounds with minimal scarring that involves the administration of an effective amount of KGF-2 to an individual. KGF-2 can be administered before, during, and / or after the process that produces the wound (for example, cosmetic surgery, accidental or deliberate tissue trauma caused by an acute object). As noted above, KGF-2 also stimulates the proliferation of keratinocytes and hair follicles, and therefore can be used to promote the healing of wounds from the bald scalp, and in patients with hair transplantation. Thus, the present invention further provides a method for promoting hair growth, which comprises the administration of a sufficient amount of KGF-2 to stimulate the production of hair follicles. The present invention also provides a method for protecting an individual from the effects of ionizing radiation, chemotherapy, or treatment with antiviral agents, which comprises the administration of an effective amount of KGF-2. The present invention further provides a method for treating tissue damage that results from exposure to ionizing radiation, chemotherapeutic agents, or antiviral agents, which comprises the administration of an effective amount of KGF-2. An individual may be exposed to ionizing radiation for a number of reasons, including for therapeutic purposes (for example, for the treatment of hyperproliferative disorders), as a result of an accidental release of a radioactive isotope into the environment or during the procedures of non-invasive medical diagnosis (for example, X-rays) . In addition, a substantial number of individuals are exposed to radioactive radon at their work sites and in their homes. Long-term environmental exposure has been used to calculate the estimated life expectancy. Johnson, W. and Kearfott, K. Heal th Phys. 73: 312-319 (1997). As shown in Example 23, the proteins of the present invention increase the survival of animals exposed to radiation. In this way, KGF-2 can be used to increase the survival rate of individuals suffering from radiation-induced damage, to protect individuals from sub-lethal doses of radiation, and to increase the therapeutic proportion of irradiation in the treatment of conditions such as hyperproliferative disorders. KGF-2 can also be used to protect individuals against radiation doses, chemotherapeutic drugs or antiviral agents that normally would not be tolerated. When used in this manner, or as otherwise described herein, KGF-2 can be administered before, after, and / or during radiation therapy / exposure, chemotherapy or treatment with antiviral agents . High doses of radiation and chemotherapeutic agents can be especially useful when treating an individual having an advanced stage of an affliction such as a hyperproliferative disorder. In yet another aspect, the present invention provides a method for preventing or treating conditions such as oral and gastrointestinal damage induced by radiation, mucositis, intestinal fibrosis, proctitis, radiation-induced pulmonary fibrosis, radiation-induced pneumonitis, radiation-induced pleural retraction. , radiation induced hemopoietic syndrome, radiation induced myelotoxicity, comprising administering an effective amount of KGF-2 to an individual. KGF-2 can be used alone or in conjunction with one or more additional agents that confer protection to radiation or other agents. A number of cytokines (eg, IL-1, TNF, IL-6, IL-12) have been shown to confer such protection. See for example, Neta, R. and collaborators, J. Exp. Med. 173: 1177 (1991). In addition, IL-11 has been shown to protect mucosal cells of the small intestine after combined irradiation and chemotherapy, Du, X.X. and collaborators, Blood 83:33 (1994), and radiation-induced thoracic damage. Redlich, C.A. and collaborators, J. Im a. 157: 1705-1710 (1996). Various growth factors have also been shown to confer protection against radiation exposure, for example, fibroblast growth factor and transforming growth factor beta-3. Ding, I. et al., Acta Oncol. 36: 337-340 (1997); Potten, C. et al., Br. J. Cancer 75: 1454-1459 (1997). Hemorrhagic cystitis is a syndrome associated with certain disease states as well as exposure to drugs, viruses, and toxins. This manifests as diffuse bleeding of the endothelial lining of the bladder. Known treatments include intravesical, systemic, and non-pharmacological therapies (West, NJ Pharmacotherapy 17: 696-706 (1997).) Some clinically used cytotoxic agents have side effects that result in the inhibition of normal epithelial proliferation in the bladder, leading to life-threatening ulceration, and rupture in the epithelial lining.For example, cyclophosphamide is a cytotoxic agent that is biotransformed mainly in the liver to activate the alkylating metabolites by a mixed function microsomal oxidase system.These metabolites interfere with the growth of rapidly proliferating, susceptible malignant cells The mechanism of action is believed to involve the cross-linking of tumor cell DNA (Phisicians' Desk reference, 1997) Cyclophosphamide is an example of a cytotoxic agent that causes cystitis hemorrhagic in some patients, a complication This can be severe and in some cases fatal. Fibrosis of the urinary bladder can also develop with or without cystitis. It is thought that this damage is caused by the metabolites of cyclophosphamide excreted in the urine. The hematuria caused by cyclophosphamide is usually present for several days, but may persist. In severe cases, medical or surgical treatment is required. Cases of severe hemorrhagic cystitis result in discontinuous therapy with cyclophosphamide. In addition, malignancies of the urinary bladder generally occur within two years of treatment with cyclophosphamide and occur in patients who previously had hemorrhagic cystitis (CYTOXAN package insert (cyclophosphamide)). Cyclophosphamide has toxic effects on the prostate and male reproductive systems. Treatment with cyclophosphamide can result in the development of sterility, and results in some degree of testicular atrophy.

As shown in Figures 52 and 53, systemic administration of KGF-2 to an individual stimulates the proliferation of prostatic and bladder epithelial cells. Thus, in one aspect, the present invention provides a method for stimulating the proliferation of prostatic epithelial and bladder cells. Thus, in one aspect, the present invention provides a method for stimulating the proliferation of bladder epithelium and prosthetic epithelial cells, by administering to an individual an effective amount of a KGF-2 polypeptide. More importantly, as shown in Figures 54 and 55, KGF-2 can be used to reduce the damage caused by cytotoxic agents that have side effects that result in the inhibition of the proliferation of bladder epithelial cells and the prostate To reduce such damage, KGF-2 can be administered either before, after, or during treatment with or exposure to the cytotoxic agent. As a consequence, in a further aspect, there is provided a method for reducing the damage caused by an inhibition of the normal proliferation of the bladder or prostate epithelial cells, by administering to an individual an effective amount of KGF-2. As indicated, inhibitors of normal proliferation of the prostatic epithelium or bladder include radiation therapy (which causes acute or chronic radiation damage) and cytotoxic agents such as chemotherapeutic and antineoplastic drugs including, but not limited to, cyclophosphamide, busulfan, and ifosfamide In a further aspect, KGF-2 is administered to produce or prevent fibrosis and ulceration of the urinary bladder. Preferably, KGF-2 is administered to reduce or prevent hemorrhagic cystitis. Suitable dosages, formulations, and suitable administration routes are described below. As used herein, "individual" is intended to mean an animal, preferably a mammal (such as apes, cows, horses, pigs, boars, sheep, rodents, goats, dogs, cats, chickens, monkeys, rabbits, urones, whales, and dolphins), and more preferably a human. The signal sequence of KGF-2 coding for amino acids 1 to 35 or 36 can be used to identify secreted proteins in general by hybridization and / or computational search algorithms. The nucleotide sequence of KGF-2 can be used to isolate the 5 'sequences by hybridization. Plasmids comprising the KGF-2 gene under the control of their native promoter / enhancer sequences could then be used in in vi tro studies directed to the identification of endogenous cellular and viral transactivators of the expression of the KGF-2 gene. The KGF-2 protein can also be used as a positive control in experiments designed to identify peptide mimetics that act on the KGF-2 receptor. According to yet another aspect of the present invention, there is provided a process for using such polypeptides, or the polynucleotides encoding such polypeptides, for in vi tro purposes related to scientific research, DNA synthesis, manufacturing of DNA vectors and for the purpose of providing diagnostic and therapeutic products for the treatment of human diseases. Full-length KGF-2 gene fragments can be used as a hybridization probe for a cDNA library to isolate full-length KGF-2 genes, and to isolate other genes that have high sequential similarity to these genes or similar biological activity. Probes of this type generally have at least 20 bases. Preferably, however, the probes have at least 30 bases and generally do not exceed 50 bases, although these may have a higher number of bases. The probe can also be used to identify a cDNA clone corresponding to a full-length transcript and a clone or genomic clones containing the complete KGF-2 gene including the regulatory and promoter regions., the exons, and the introns. An example of a selection comprises isolating the coding region of the KGF-2 gene by using the known DNA sequence to synthesize an oligonucleotide probe. The labeled oligonucleotides having a sequence complementary to that of the gene of the present invention are used to select a library of human cDNA, genomic DNA or cDNA to determine which members of the library the probe hybridizes to. This invention provides a method for the identification of the receptors for the KGF-2 polypeptide. The gene encoding the receptor can be identified by numerous methods known to those skilled in the art, for example, panoramic representation of the ligand, and FACS sorting (Coligan et al., Current Protocols in I mun., 1 (2), Chapter 5 (1991)). Preferably, expression cloning is employed wherein the polyadenylated RNA is prepared from a cell responsive to the polypeptides, and a cDNA library created from RNA is divided into pools, and used to transfect COS cells or other cells that do not respond to polypeptides. Transfected cells that are developed on glass slides are exposed to the labeled polypeptides. The polypeptides can be labeled by a variety of means including iodination or inclusion of a recognition site for a site-specific protein kinase. After fixation and incubation, the slides are subject to autoradiographic analysis. The positive combinations are identified and the sub-combinations are prepared and re-transfected using a process of sub-combination and iterative reselection, eventually producing a simple clone that codes for the putative receptor. As an alternative method for identification of the receptor, the labeled polypeptides can be ligated by photoaffinity with the cell membrane or with extract preparations expressing the receptor molecule. The crosslinked material is resolved by PAGE analysis and exposed to X-ray film. The labeled complex containing the polypeptide receptors can be excised, resolved into peptide fragments, and subject to protein microsequencing. The amino acid sequence obtained from the microsequencing could be used to design a group of oligonucleotide probes originated to select a cDNA library to identify the genes encoding the putative receptors.

This invention provides a method for screening compounds to identify those that are agonists of the action of KGF-2 or block the function of KGF-2. An example of such an assay comprises the combination of a mammalian keratinocyte cell, the compound to be selected and 3 [H] -thymidine under cell culture conditions, where the keratinocyte cell could proliferate normally. A control assay can be performed in the absence of the compound to be selected, and compared to the amount of keratinocyte proliferation in the presence of the compound to be determined, if the compound stimulates the proliferation of keratinocytes. For the selection of antagonists, the same assay can be prepared in the presence of KGF-2 and the ability of the compound to prevent keratinocyte proliferation is measured, and the determination of the antagonist skill is performed. The amount of keratinocyte cell proliferation is measured by liquid scintillation chromatography which measures the incorporation of 3 [H] -thymidine. In yet another method, a mammalian cell or a membrane preparation expressing the KGF-2 receptor could be incubated with the labeled KGF-2 in the presence of the compound. The compound's ability to increase or block this interaction could then be measured. Alternatively, the response of a known second messenger system after the interaction of KGF-2 and the receptor could be measured and compared in the presence or absence of the compound. Such second messenger systems include but are not limited to, cAMP-guanylate cyclase, ion channels and phosphoinositide hydrolysis. Examples of potential KGF-2 antagonists include an antibody, or in some cases, an oligonucleotide, which binds to the polypeptide. Alternatively, a potential KGF-2 antagonist may be a mutant form of KGF-2 that binds to KGF-2 receptors, however, no second messenger response is promoted and therefore the action of KGF-2 is effectively blocked. Another potential KGF-2 antagonist is an antisense construct prepared using the antisense technology. The antisense technology can be used to control the expression of the gene through the formation of a triple helix or the antisense DNA or RNA, whose methods are based on the binding of a polynucleotide to DNA or RNA. For example, the 5 'coding portion of the polynucleotide sequence, which codes for the mature polypeptides of the present invention, is used to design an antisense RNA oligonucleotide of about 10 to 40 base pairs in length. A DNA oligonucleotide is designed to be complementary to a region of the gene involved in transcription (triple helix - see Lee et al., Nucí Acids Res. 6: 3073 (1979); Cooney et al., Science 241: 456 (1988); and Dervan et al., Science 51: 1360 (1991)), thereby preventing transcription and production of KGF-2. The antisense RNA oligonucleotide hybridizes to the cDNA in vivo and blocks translation of the cDNA molecule into the KGF-2 polypeptide (Antisense-Okano, J., Neurochem, 56: 560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression. , CRC Press, Boca Raton, FL (1988).) Oligonucleotides described above can also be distributed to cells such that RNA or antisense DNA can be expressed in vivo to inhibit the production of KGF-2. -2 potentials include small molecules that bind to and occupy the binding site of the KGF-2 receptor whereby the receptor is made inaccessible to KGF-2, such that normal biological activity is prevented.Examples of small molecules include, but are not limited to, small peptides or molecules similar to the peptide.KGF-2 antagonists can be used to prevent the induction of the growth of new blood vessels or the angiog enesis in tumors KGF-2-stimulated angiogenesis also contributes to various pathologies that can also be treated by antagonists of the present invention including diabetic retinopathy, and the inhibition of the growth of pathological tissues, such as rheumatoid arthritis. KGF-2 antagonists can also be used to treat glomerulonephritis, which is characterized by the marked proliferation of glomerular epithelial cells that form a cell mass that fills Bowman's space. Antagonists can also be employed to inhibit the overproduction of the healing tissue observed in keloid formation after surgery, fibrosis after myocardial infarction or fibrotic lesions associated with pulmonary fibrosis and restenosis. KGF-2 antagonists can also be used to treat other proliferative diseases which are stimulated by KGF-2, including cancer and Kaposi's sarcoma. KGF-2 antagonists can also be used to treat keratitis which is a chronic infiltration of the deep layers of the cornea with uveal inflammation characterized by the proliferation of epithelial cells.

Antagonists can be employed in a composition with a pharmaceutically acceptable carrier, for example, as described hereinafter. The polypeptides, agonists and antagonists of the present invention can be used in combination with a suitable pharmaceutical carrier to comprise a pharmaceutical composition. Such compositions comprise a therapeutically effective amount of the polypeptide, agonist or antagonist and a pharmaceutically acceptable carrier or excipient. Such a carrier includes but is not limited to saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The formulation must be adapted to the mode of administration. The invention also provides a pharmaceutical package or equipment comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Associated with such containers may be a notice in the form prescribed by a government agency that regulates the manufacture, use or sale of pharmaceutical or biological products, whose notice reflects approval by the agency regarding the manufacture, use or sale for human administration. In addition, the polypeptides, agonists and antagonists of the present invention can be used in conjunction with other therapeutic compounds.

The polypeptide having KGF-2 activity can be administered in pharmaceutical compositions in combination with one or more pharmaceutically acceptable excipients. It will be understood that, when administered to a human patient, the total daily use of the pharmaceutical compositions of the invention will be decided by the physician attending, within the scope of sound medical judgment. The therapeutically effective dose level, specific to any particular patient, will depend on a variety of factors, including the type and degree of the response that will be achieved; the specific composition and another agent, if any, employed; age, body weight, general health, sex and the patient's diet; the time of administration, the route of administration, and the rate of excretion of the composition; the duration of the treatment; the drugs (such as a chemotherapeutic agent) used in combination or coincident with the specific composition; and similar factors well known in the medical arts. Suitable formulations, known in the art, can be found in Remington's Pharmaceutical Sciences (latest edition), Mack Publishing Company, Easton, PA. The composition of KGF-2 that will be used in therapy will be formulated and dosed in a manner consistent with good medical practice, taking into account the clinical condition of the individual patient (especially the side effects of treatment with KGF-2 alone) , the site of administration of the composition of KGF-2, the method of administration, the administration program, and any other factors known to practitioners. The "effective amount" of KGF-2 for purposes of this is thus determined by such considerations. The pharmaceutical compositions can be administered in a convenient manner such as by the oral, topical, intravenous, intraperitoneal, intramuscular, intraarticular, subcutaneous, intranasal, intrathecal or intradermal routes. The pharmaceutical compositions are administered in an amount that is effective for the treatment and / or prophylaxis of the specific indication. In most cases, the dosage is from about 1 μg / kg to about 30 mg / kg of body weight daily, taking into account the routes of administration, symptoms, etc. However, the dose can be as low as 0.001 μg / kg. For example, in the specific case of topical administration the doses are preferably administered from about 0.01 μg to 9 mg per cm. As a general proposition, the total pharmaceutically effective amount of KGF-2 administered parenterally per dose more preferably will be in the range of about 1 μg / kg / day to 100 mg / kg / day of the patient's body weight, although, as noted above, this will be Subject to therapeutic discretion. If administered continuously, KGF-2 is typically administered at a dose rate of about 1 μg / kg / hour to about 50 μg / kg / hour, either for 1 to 4 injections per day or for continuous subcutaneous infusions, for example , using a mini pump. An intravenous bag solution or bottle solution can also be used. A course of treatment with KGF-2 to affect the fibrinolytic system seems to be optimal if it continues longer than a certain minimum number of days, 7 days in the case of mice. The duration of the treatment, necessary to observe changes, and the interval of the next treatment for the responses to occur, seems to vary depending on the desired effect. Such treatment lengths are indicated in the following Examples. The KGF-2 polypeptide is also suitably administered by sustained release systems. Suitable examples of sustained release compositions include semipermeable polymer matrices in the form of shaped articles, e.g., films, or microcapsules. Sustained-release matrices include polylactides (U.S. Patent No. 3,773,919, European Patent No. 5881), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (U. Sidman et al., Biopolymers 22: 547 -556 (1983)), poly- (2-hydroxyethyl methacrylate) (R. Langer et al., J. Bio ed. Mater. Res. 15: 167-277 (1981), and R. Langer, Chem. Tech. 12 : 98-105 (1982)), ethylene vinyl acetate (R. Langer et al., Id.) Or poly-D- (-) - 3-hydroxybutyric acid (European Patent No. 133,988). Sustained-release KGF-2 compositions also include liposomally entrapped KGF-2. Liposomes containing KGF-2 are prepared by methods known per se: German Patent No. 3,218,121; Epstein et al., Proc. Nati Acad. Sci. USA 82: 3688-3692 (1985); Hwang et al., Proc. Nati Acad. Sci. USA 77: 4030-4034 (1980); European Patent Nos. 52,322; 36,676; 88,046; 143,949; 142,641; Japanese Patent Application No. 83-118008; U.S. Patent Nos. 4,485,045 and 4,544,545; and European Patent No. 102,324. Ordinarily, the liposomes are of the small unilamellar type (approximately 200-800 Angstroms) in which the lipid content is greater than about 30 mol percent of cholesterol, the selected proportion being adjusted for optimal KGF-2 therapy. For parenteral administration, in one embodiment, KGF-2 is formulated in general by mixing it, to the desired degree of purity, in an injectable unit dosage form (solution, suspension, or emulsion) with a pharmaceutically acceptable carrier, by example, one that is non-toxic to recipients at the doses and concentrations employed, and is compatible with other ingredients of the formulation. For example, the formulation preferably does not include oxidizing agents and other compounds that are known to be harmful to the polypeptides. In general, the formulations are prepared by contacting the KGF-2 uniformly and intimately with liquid carriers or finely divided solid carriers, or both. Then, if necessary, the product is shaped into the desired formulation. Preferably the carrier is a parenteral carrier, more preferably a solution that is isotonic with the blood of the container. Examples of such carriers include water, saline, Ringer's solution, and dextrose solution. Non-aqueous vehicles such as fixed oils and ethyl oleate are also useful herein, as well as liposomes. Suitable formulations, known in the art, can be found in Remington's Pharmaceutical Sciences (latest edition), Mack Publishing Company, Easton, PA. The carrier suitably contains minor amounts of additives such as substances that increase isotonicity and chemical stability. Such materials are non-toxic to the containers at the doses and concentrations employed, and include buffers such as phosphate, citrate, succinate, acetic acid, and other organic acids or their salts; antioxidants such as ascorbic acid; low molecular weight polypeptides (less than about ten residues), eg, polyarginine or tripeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids, such as glycine, glutamic acid, aspartic acid, or arginine; monosaccharides, disaccharides, and other carbohydrates including cellulose or its derivatives, glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; counterions such as sodium; and / or nonionic surfactants such as polysorbates, poloxamers, or PEG. KGF-2 is typically formulated in such vehicles at a concentration of about 0.01 μg / ml to 100 mg / ml, preferably 0.01 μg / ml up to 10 mg / ml, at a pH of about 3 to 8. It will be understood that the use of certain above-mentioned excipients, carriers or stabilizers, will result in the formation of KGF-2 salts. KGF-2 that is going to be used for therapeutic administration must be sterile. Sterility is easily achieved by filtration through sterile filtration membranes (e.g., 0.2 micrometer membranes). Therapeutic compositions of KGF-2 are generally placed in a container having a sterile access port, for example, an intravenous solution bag or a bottle having a plug pierceable by a hypodermic injection needle. KGF-2 will ordinarily be stored in single-dose or multi-dose containers, for example, sealed vials or vials, as an aqueous solution or as a lyophilized formulation for reconstitution. As an example of a lyophilized formulation, 10 ml bottles are filled with 5 ml of 1% (w / v) aqueous KGF-2 solution sterilized by filtration, and the resulting mixture is lyophilized. The infusion solution is prepared by reconstituting lyophilized KGF-2 using water for bacteriostatic injection. The dosage can also be accommodated in a patient-specific manner, to provide a predetermined concentration of a KGF-2 activity in the blood, as determined by an RIA technique, for example. In this way, the dosage to the patient can be adjusted to achieve regular blood levels, as measured by RIA, in the order of 50 to 1000 ng / ml, preferably 150 to 500 ng / ml.

The pharmaceutical compositions of the invention can be administered orally, rectally, parenterally, intracisternally, intradermally, intravaginally, intraperitoneally, topically (as by powders, ointments, gels, creams, drops or transdermal patch), buccally, as an oral or nasal spray. By "pharmaceutically acceptable carrier" is meant a non-toxic, semi-solid or liquid filler, diluent, encapsulation or auxiliary material of the formulation, of any type. The term "parenteral" as used herein refers to administration modalities that include infusion and intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection. Preferred formulations of KGF-2 are described in U.S. Provisional Application No. 60/068493, filed December 22, 1997, which is incorporated by reference herein. Polypeptides, agonists and antagonists of KGF-2 that are polypeptides can also be employed according to the present invention by the expression of such polypeptides in vivo, which is often referred to as "gene therapy". Thus, for example, cells from a patient can be genetically engineered with a polynucleotide (DNA or RNA) that codes for an ex vivo polypeptide, with cells engineered that are provided to a patient who is to be treated with the polypeptide. Such methods are well known in the art. For example, cells can be manipulated by methods known in the art, by the use of a retroviral particle containing RNA encoding a polypeptide of the present invention. In addition, before the cells are reintroduced into the patient, they can be seeded on cellular carriers, including biodegradable matrices (eg, polyglycolic acid), substitutes or tissue equivalents (eg, artificial skin), artificial organs, and derived matrices. of collagen, etc. Similarly, cells can be engineered in vivo for the expression of a polypeptide in vivo by, for example, methods known in the art. As is known in the art, a producer cell for producing a retroviral particle containing RNA encoding the polypeptide of the present invention can be administered to a patient to engineer the cells in vivo and the expression of the polypeptide in vivo. . These and other methods for the administration of a polypeptide of the present invention by such a method should be apparent to those skilled in the art from the teachings of the present invention. For example, the expression vehicle for manipulating the cells can be different from a retrovirus, for example, an adenovirus that can be used to manipulate the cells in vivo after combination with a suitable delivery vehicle. Examples of other delivery vehicles include a HSV-based vector system, adeno-associated viral vectors, and inert carriers, for example, ferrite particles coated with dextran. Retroviruses from which the aforementioned retroviral plasmid vectors can be derived herein include but are not limited to Moloney Murine Leukemia virus, splenic necrosis virus, retroviruses such as the Sarcoma virus of Rous, Harvey's Sarcoma virus, avian leukosis virus, gibbon monkey leukemia virus, human immunodeficiency virus, adenovirus, myeloproliferative sarcoma virus, and mammary tumor virus. In one embodiment, the retroviral plasmid vector is derived from the Moloney Murine Leukemia Virus. The vector includes one or more promoters. Suitable promoters that can be employed include, but are not limited to, the retroviral LTR; the SV40 promoter; and the human cytomegalovirus (CMV) promoter described in Miller et al., Biotechniques Vol. 7, No. 9: 980-990 (1989), or any other promoter (e.g., cellular promoters such as eukaryotic cell promoters including, but not limited to, histone, promoters of pol III, and of β-actin). Other viral promoters that may be employed include, but are not limited to, adenoviral promoters, thymidine kinase (TK) promoters, and parvovirus B19 promoters. The selection of a suitable promoter will be apparent to those skilled in the art from the teachings contained herein. The nucleic acid sequence encoding the polypeptide of the present invention is under the control of a suitable promoter. Suitable promoters that can be employed include, but are not limited to, adenoviral promoters, such as the late adenoviral major promoter; or heterologous promoters, such as the cytomegalovirus (CMV) promoter; the respiratory syncytial virus (RSV) promoter; inducible promoters, such as the MMT promoter, the metallothionein promoter; heat shock promoters; albumin promoters; the ApoAI promoter, promoters of human globin; viral thymidine kinase promoters, such as the Herpes Simplex thymidine kinase promoter; Retroviral LTRs (including modified retroviral LTRs, previously described herein); the β-actin promoter; and the promoters of human growth hormone. The promoter may also be the native promoter that controls the gene encoding the polypeptide. The retroviral plasmid vector is used to transduce the packaging cell lines to form producer cell lines. Examples of packaging cell lines that can be transfected include, but are not limited to, the cell lines PE501, PA317,? -2,? -AM, PA12, T19-14X, VT-19-17-H2,? CRE ,? CRIP, GP + E-86, GP + envAml2, and DAN as described in lines as described in Miller, Human Gene Therapy 1: 5-14 (1990), which is incorporated by reference herein in its entirety . The vector can transduce the packaging cells through any means known in the art. Such means include, but are not limited to, electroporation, the use of liposomes, and calcium phosphate precipitation. In an alternative, the retroviral plasmid vector can be encapsulated in a liposome, or coupled to a lipid, and then administered to a host. The producer cell line generates infectious retroviral vector particles that include the nucleic acid sequence (s) encoding the polypeptides. Such retroviral vector particles can then be used to transduce eukaryotic cells, either in vitro or in vivo. The transduced eukaryotic cells will express the nucleic acid sequence (s) encoding the polypeptide. Eukaryotic cells that can be transduced include, but are not limited to, embryonic pluripotent cells, embryonic carcinoma cells, as well as hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts, keratinocytes, endothelial cells, and bronchial epithelial cells. The invention provides methods of treatment, inhibition and prophylaxis by administering to a subject an effective amount of a compound or pharmaceutical composition of the invention, preferably an antibody of the invention. In a preferred aspect, the compound is substantially purified (eg, substantially free of substances that limit its effect or produce undesirable side effects.) The subject is preferably an animal, including but not limited to animals such as cows, pigs, horses, chickens, cats, dogs, etc., and is preferably a mammal, and more preferably a human The formulations and methods of administration that can be employed when the compound comprises a nucleic acid or an immunoglobulin, are described above; Additional appropriate formulations and routes of administration may be selected from those described hereinafter.

The various delivery systems are known, and can be used to administer a compound of the invention, for example, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compound, receptor-mediated endocytosis (see for example, Wu and Wu, J. "Biol. Chem. 262: 4429-4432 (1987)), the construction of a nucleic acid as part of a retroviral vector or other vector, etc. The methods of introduction include but are not limited to, the intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes The compounds or compositions can be administered by any convenient route, for example by infusion or bolus injection, by absorption through the epithelial or mucocutaneous coatings (for example, oral mucosa, rectal and intestinal mucosa, etc.), and can be administered together with other biologically active agents. The administration can be systemic or local. In addition, it may be desirable to introduce the compounds or pharmaceutical compositions of the invention into the central nervous system by any suitable route, including intraventricular and intrathecal injection, intraventricular injection may be facilitated by an intraventricular catheter, for example, coupled to a reservoir, such as an Ommaya deposit. The pulmonary administration can also be employed for example, by the use of an inhaler or nebulizer, and the formulation with an aerosolizing agent. In a specific embodiment, it may be desirable to administer the compounds or pharmaceutical compositions of the invention locally to the area in need of treatment; this can be achieved by, for example, and not by way of limitation, local infusion during surgery, topical application, for example, in conjunction with a wound dressing after surgery, by injection, by means of a catheter, by means of a suppository, or by means of an implant, said implant being a porous, non-porous, or gelatinous material, including membranes such as sialastic membranes, or fibers. Preferably, when administering a protein, or including an antibody, of the invention, care must be taken in using materials to which the protein is not absorbed. In yet another embodiment, the compound or composition can be distributed in a vesicle, in particular a liposome (see Langer, Science 249: 1527-1533 (1990); Treat et al., In Liposomes in the Therapy of Infectious Disease and Cancer, Lopez -Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989), Lopez-Berestein, ibid., Pp. 317-327, see in general ibid.).

In yet another embodiment, the compound or composition can be administered in a controlled release system. In one embodiment, a pump may be used (see Langer, supra; Sefton, CRS Cri. Ref Biomed. Eng. 14: 201 (1987); Buchwaid et al., Surgery 88: 507 (1980); Saudek et al. N. Engl. J. Med. 321: 574 (1989)). In yet another embodiment, polymeric materials may be used (see Medical Applications of Controlled Relay, Langer and Wise (eds.), CRC Pres., Boca Raton, Florida (1974), Controlled Drug Bioavailabiblity, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley,? Ew York (1984), Ranger and Peppas, J. "Mac romo 1. Sci. Rev. Macromol. Chem. 23:61 (1983), see also Levy et al., Science 228: 190 (1985), During et al, Ann Neurol 25: 351 (1989), Howard et al, J "Neurosurg 71: 105 (1989)). In yet another embodiment, a controlled release system can be placed in proximity to the therapeutic target, for example, the brain, thus requiring only a fraction of the systemic dose (see for example, Goodson, in Medical Applications of Controlled Relay, supra, vol 2, pp. 115-138 (1984)). Other controlled-release systems are discussed in the review by Langer (Science 249: 1527-1533 (1990)).

In a specific embodiment wherein the compound of the invention is a nucleic acid encoding a protein, the nucleic acid can be administered in vivo to promote the expression of its encoded protein, by constructing it as part of an expression vector of suitable nucleic acid and its administration so that it becomes intracellular, for example, by the use of a retroviral vector (see U.S. Patent No. 4,980,296), or by direct injection, or by the use of bombardment microparticles (for example, a gene gun; Biolistic, Dupont), or coating with lipids or other cell surface receptors, or transfection agents, or by administering it in connection with a peptide similar to a homeobox that is known enters the nucleus (see for example, Joliot et al, Proc. Nati, Acad. Sci. USA 88: 1864-1868 (1991)), etc. Alternatively, a nucleic acid can be introduced intracellularly and incorporated into the DNA of the host cell for expression, by homologous recombination. The present invention also provides the pharmaceutical compositions. Such compositions comprise a therapeutically effective amount of a compound, and a pharmaceutically acceptable carrier. In a specific embodiment, the term "pharmaceutically acceptable" means approved by a regulatory agency of the federal or state government or listed in the United States Pharmacopoeia or other pharmacopoeia generally recognized for use in animals, and more particularly in humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic agent is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous solutions of dextrose and glycerol can also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, gypsum, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene glycol, water, ethanol and the like. The composition, if desired, may also contain minor amounts of wetting agents or emulsifiers, or pH buffering agents. These compositions may take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained release formulations and the like. The composition can be formulated as a suppository, with binders and traditional carriers such as triglycerides. The oral formulation may include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin. Such compositions will contain a therapeutically effective amount of the compound, preferably in purified form, together with a suitable amount of carrier to provide the form for proper administration to the patient. The formulation must be adapted to the mode of administration. In a preferred embodiment, the composition is formulated according to routine procedures as a pharmaceutical composition adapted for intravenous administration to humans. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to relieve pain at the site of injection. In general, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or a water-free concentrate in a hermetically sealed container such as a vial or sack, indicating the amount of active agent. Where the composition is to be administered by infusion, it can be supplied with an infusion bottle containing water or saline in pharmaceutical grade, sterile. Where the composition is administered by injection, a vial of sterile water for injection or saline can be provided so that the ingredients can be mixed before administration. The compounds of the invention can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc. The amount of the compound of the invention that will be effective in the treatment, inhibition and prevention of a disease or disorder associated with the activity and / or aberrant expression of a polypeptide of the invention, can be determined by standard clinical techniques. In addition, in vitro trials may be optionally employed to help identify optimal dose ranges. The precise dose that will be used in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and the circumstances of each patient. Effective doses can be extrapolated from the dose-response curves derived from model test systems in vivo or in animals. For antibodies, the dose administered to a patient is typically 0.1 mg / kg to 100 mg / kg of the patient's body weight. Preferably, the dose administered to a patient is between 0.1 mg / kg and 20 mg / kg of the patient's body weight, more preferably 1 mg / kg to 10 mg / kg of the patient's body weight. In general, human antibodies have a longer half-life within the human body than antibodies from other species, due to the immune response to foreign polypeptides. In this way, low doses of human antibodies and less frequent administration, is often possible. In addition, the dose and frequency of administration of the antibodies of the invention can be reduced by increasing the uptake and penetration into the tissue (eg, in the brain) of the antibodies by modifications such as, for example, lipidation.

The invention also provides a pharmaceutical package or equipment comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Optionally associated with such or such containers may be a notice in the form prescribed by a governmental agency that regulates the manufacture, use or sale of pharmaceutical or biological products, whose notice reflects the approval by the agency, with respect to manufacturing, use or sale for human administration.

Therapeutic Uses Based on Antibody The present invention is further directed to antibody-based therapies, which involve the administration of antibodies of the invention to an animal patient, preferably a mammal, and more preferably a human, for the treatment of one or more of the disorders, diseases or diseases. conditions described above. The therapeutic compounds of the invention include, but are not limited to, antibodies of the invention (including fragments, analogs and derivatives thereof as described herein) and nucleic acids encoding the antibodies of the invention (including fragments) , analogs and derivatives thereof and anti-idiotypic antibodies as described herein). The antibodies of the invention can be used to treat, inhibit or prevent diseases, disorders or conditions associated with aberrant expression and / or activity of a polypeptide of the invention, including, but not limited to, one or more of the diseases, disorders or conditions described above. The treatment and / or prevention of diseases, disorders, or conditions associated with the expression and / or aberrant activity of a polypeptide of the invention includes, but is not limited to, relief of symptoms associated with those diseases, disorders or conditions. The antibodies of the invention can be provided in pharmaceutically acceptable compositions as are known in the art, as described herein. A summary of the ways in which the antibodies of the present invention can be used, therapeutically includes the polynucleotides or binding polypeptides of the present invention, either locally or systemically in the body or by direct cytotoxicity of the antibody, for example as is mediated by complement (CDC) or by effector cells (ADCC). Some of these procedures are described in more detail later. Armed with the teachings provided herein, persons of ordinary skill in the art will know how to use the antibodies of the present invention for diagnostic, periodic verification or therapeutic purposes without undue experimentation. The antibodies of this invention can be advantageously used in combination with other monoclonal or chimeric antibodies, or with lymphokines or hematopoietic growth factors (such as, IL-2, IL-3 and IL-7), for example, which serve to increase the number or activity of the effector cells that interact with the antibodies. The antibodies of the invention can be administered alone or in combination with other types of treatments (e.g., radiotherapy, chemotherapy, hormone therapy, immunotherapy and anti-tumor agents). In general, the administration of the products of a species origin or species reactivity (in the case of antibodies) which is the same species as that of the patient, is preferred. Thus, in a preferred embodiment, human antibodies, fragments, derivatives, analogs, or nucleic acids, are administered to a human patient for therapy or prophylaxis. It is preferred to use inhibitory and / or neutralizing antibodies in vivo, of high affinity and / or potent against the polypeptides or polynucleotides of the present invention, fragments or regions thereof, for the immunoassays directed to and the therapy of the disorders related to the polynucleotides. or polypeptides, including fragments thereof, of the present invention. Such antibodies, fragments, or regions will preferably have an affinity for the polynucleotides or polypeptides of the invention, including fragments thereof. Preferred binding affinities include those with a dissociation constant or Kd less than 5 x 10 ~ 2 M, 10.2 M, 5 x 10"3 M, 10" 3 M, 5 x 10 ~ 4 M, 10 ~ 4 M, 5 x 10"5 M, 10 ~ 5 M, 5 x 10" 6 M, 10"6 M, 5 x 10 ~ 7 M, 10" 7 M, 5 x 10"8 M, 10" 8 M, 5 x 10"9 M, 10 ~ 9 M, 5 x 10" 10 M, 10"10 M, 5 x 10" 11 M, 10"11 M, 5 X 10 ~ 12 M, 10" 12 M, 5 x 10 ~ 13 M, 10"13 M, 5 x 10" 14 M, 10"14 M, 5 x 10" 15 M, and 10"15 M.

Chromosome assays The sequences of the present invention are also valuable for the identification of chromosomes. The sequence is specifically targeted to and can hybridize to a particular site on an individual human chromosome. In addition, there is a current need to identify particular sites on the chromosome. Few chromosome labeling reagents based on effective sequential data (repeated polymorphisms) are currently available for chromosome site labeling. The mapping of the DNAs to the chromosomes according to the present invention is an important first step in the correlation of those sequences with the genes associated with the disease. In summary, the sequences can be mapped to the chromosomes by preparation of PCR primers (preferably 15-25 base pairs) from the cDNA. Computer analysis of the 3 'untranslated region is used to rapidly select primers that do not span more than one exon in the genomic DNA, thereby complicating the amplification process. These primers are then used for the selection by PCR of somatic cell hybrids containing individual human chromosomes. Only those hybrids that contain the human gene corresponding to the primer, will produce an amplified fragment. The mapping of the somatic cell hybrids by PCR is a rapid procedure for assigning a particular DNA to a particular chromosome. Using the present invention with the same oligonucleotide primers, sublocalization with panels of fragments from specific or combined chromosomes of large genomic clones can be achieved in an analogous manner. Other map mapping strategies can be similarly used to map your chromosome including hybridization in itself, preselecting with flow-sorted chromosomes, markers, and pre-selection by hybridization to build chromosome-specific cDNA libraries. Fluorescence hybridization in si tu (FISH) from a cDNA clone to a metaphase chromosome reproduction can be used to provide a precise chromosomal location in one step. This technique can be used with the cDNA as short as 50 or 60 bases. For a review of this technique, see Verma et al., Human Chromosomes: a Manual of Basic Techniques, Pergamon Press, New York (1988). Once a sequence has been plotted on a map to an accurate chromosome site, the physical position of the sequence on the chromosome can be correlated with the data of the genetic map. Such data are found, for example, in V. McKusick, Mendelian Inheri tance in Man (available online through Johns Hopkins University Welch Medical Library). The relationship between genes and diseases that have been mapped to the same chromosomal region are then identified through linkage analysis (coherence of physically adjacent genes). Next, it is necessary to determine the differences in the cDNA or the genomic sequence between affected and unaffected individuals. If a mutation is observed in some or all of the affected individuals but not in any of the normal individuals, then the mutation is likely to be the causative agent of the disease. With the current resolution of the techniques of mapping the physical map and mapping the genetic map, a cDNA located precisely to a chromosomal region associated with the disease could be one of between 50 and 500 potential causal genes. (This assumes a mapped resolution of 1 megabase and one gene per 20 kb). The present invention will be further described with reference to the following examples; however, it should be understood that the present invention is not limited to such examples. All parts or quantities, unless otherwise specified, are by weight. In order to facilitate the understanding of the following examples, certain methods and / or terms that appear frequently will be described. "Plasmids" are designated by a lowercase letter p preceded and / or followed by uppercase letters and / or numbers. The initial plasmids herein are either commercially available, publicly available on an unrestricted basis, or can be constructed from available plasmids according to published procedures. In addition, plasmids equivalent to those described are known in the art and will be apparent to the person of ordinary skill in the art.

"Digestion" of DNA refers to the catalytic cleavage of DNA with a restriction enzyme that acts only on certain sequences in DNA. The various restriction enzymes used herein are commercially available and their reaction conditions, cofactors and other requirements were used as would be known to the person of ordinary skill in the art. For analytical purposes, typically 1 μg of the plasmid or the DNA fragment is used with about 2 units of enzyme in about 20 μl of buffer. For purposes of isolating the DNA fragments for the construction of plasmids, typically 5 to 50 μg of DNA are digested with 20 to 250 units of enzyme in a larger volume. The appropriate buffers and the amounts of substrate for the particular restriction enzymes are specified by the manufacturer. Incubation times of approximately 1 hour at 37 ° C are ordinarily used, but may vary according to the supplier's instructions. After digestion, the reaction is subjected to electrophoresis directly on a prolyacrylamide gel to isolate the desired fragment. Size separation of the excised fragments is performed using 8 percent polyacrylamide gel described by Goeddel, D., et al., Nucleic Acids Res. , 8: 4057 (1980). "Oligonucleotides" refers to either a single-stranded polydeoxynucleotide or two complementary polydeoxynucleotide strands that can be chemically synthesized. Such synthetic oligonucleotides do not have 5 'phosphate and thus will not bind to another oligonucleotide without adding a phosphate with an ATP in the presence of a kinase. A synthetic oligonucleotide will be ligated to a fragment that has not been dephosphorylated. "Ligation" refers to the process of forming phosphodiester bonds between two double-stranded nucleic acid fragments (Maniatis, T. et al., Id., P.146). Unless otherwise provided, the ligation can be carried out using known buffers and known conditions with 10 units of T4 DNA ligase ("ligase") per 0.5 μg of approximately equimolar amounts of the DNA fragments that go to be linked. A cell has been "transformed" by exogenous DNA when such exogenous DNA has been introduced into the cell membrane. The exogenous DNA may or may not be the integrated interchromosomal DNA (covalently linked) that makes up the genome of the cell. Procaryote and yeast DNA, for example, exogenous DNA can be maintained on an episomal element, such as a plasmid. With respect to eukaryotic cells, a stably transformed or transfected cell is one in which the exogenous DNA has become integrated into the chromosome so that it can be inherited by daughter cells through chromosomal replication. This ability is demonstrated by the ability of eukaryotic cells to establish cell lines or clones comprised of a population of daughter cells containing exogenous DNA. An example of transformation is shown in Graham, F. and Van der Eb, A., Virology, 52: 456-457 (1973). "Transduction" or "transduced" refers to a process by which cells pick up foreign DNA and integrate that foreign DNA into its chromosome. Transduction can be achieved, for example, by transfection, which refers to various techniques by which cells collect DNA, or infection, by which viruses are used to transfer DNA to cells.

Gene Therapy Methods Yet another aspect of the present invention relato methods of gene therapy for treating disorders, diseases and conditions. Gene therapy methods refer to the introduction of the nucleic acid sequences (DNA, RNA and antisense DNA or RNA) into an animal to achieve expression of the KGF-2 polypeptide of the present invention. This method requires a polypeptide that encodes a KGF-2 polypeptide operably linked to a promoter and any other genetic elements necessary for the expression of the polypeptide by the target tissue. Such gene therapy and administration techniques are known in the art, see for example, WO 90/11092, which is incorporated by reference herein. Thus, for example, cells from a patient can be genetically engineered with a polynucleotide (DNA or RNA) comprising a promoter operably linked to an ex vivo KGF-2 polynucleotide, with the cells engineered which are then provided to a patient to be treated with the polypeptide. Such methods are well known in the art. See for example, Belldegrun, A. and collaborators, J. Nati. Cancer Inst. 85: 207-216 (1993); Ferrantini, M. et al., Cancer Research 53: 1107-1112 (1993); Ferrantini, M. et al., J ". Immunology 153: 4604-4615 (1994); Kaido, T. et al., Int. J. Cancer 60: 221-229 (1995); Ogura, H. et al., Cancer Research 50: 5102-5106 (1990), Santodonato, L. et al, Human Gene Therapy 7: 1.10 (1996), Santodonato, L. et al, Gene Therapy 4: 1246-1255 (1997), and Zhang, JF et al. , Cancer Gene Therapy 3: 31-38 (1996)), which are incorporated by reference herein In one embodiment, the cells that are genetically engineered are arterial cells, the arterial cells can be reintroduced into the patient through direct injection into the artery, the tissues surrounding the artery, or through catheter injection.As discussed in more detail below, KGF-2 polynucleotide constructs can be administered by any method that distributes injectable materials to the cells of an animal, such as by injection into the interstitial space of tissues (heart, muscle, skin, lung, liver, and the like). The KGF-2 polynucleotide constructs can be distributed in a pharmaceutically acceptable liquid or aqueous carrier. In one embodiment, the KGF-2 polypeptide is distributed as a naked polynucleotide. The term "naked" polynucleotide, DNA or RNA refers to sequences that are free of any delivery vehicle that acts to aid, promote or facilitate entry into the cell, including viral sequences, viral particles, liposomal formulations, lipofectin or precipitating agents and the like. However, KGF-2 polynucleotides can also be administered in liposomal formulations and lipofectin formulations, and the like, and can be prepared by methods well known to those skilled in the art. Such methods are described, for example, in U.S. Patent Nos. 5,593,972, 5,589,466 and 5,580,859, which are incorporated by reference herein. The vector constructs of the KGF-2 polynucleotide, used in the gene therapy method, are preferably constructs that will not integrate into the host genome nor contain sequences that allow replication. Suitable vectors include pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available from Stratagene; pSVK3, pBPV, pMSG and pSVL available from Pharmacia; and pEFI / V5, pcDNA3.1, and pRc / CMV2 available from Invitrogen. Other suitable vectors will be readily apparent to those skilled in the art. Any strong promoter known to those skilled in the art can be used to boost the expression of KGF-2 DNA. Suitable promoters include adenoviral promoters, such as the adenoviral major late promoter; or heterologous promoters, such as the cytomegalovirus (CMV) promoter; the respiratory syncytial virus (RSV) promoter; inducible promoters, such as the MMT promoter, the metallothionein promoter; the heat shock promoters; the albumin promoter; the ApoAI promoter; the human globin promoters; the promoters of the viral thymidine kinase, such as the herpes simplex thymidine kinase promoter; the retroviral LTRs; the β-actin promoter; and the promoters of human growth hormone. The promoter can also be the native promoter for KGF-2. Contrary to other gene therapy techniques, a major advantage of introducing naked nucleic acid sequences within the target cells is the transient nature of the synthesis of the polynucleotide in the cells. Studies have shown that DNA sequences that do not replicate can be introduced into cells to provide production of the desired polypeptide for periods of up to six months. The construction of the KGF-2 polynucleotide can be administered to the interstitial space of tissues within an animal, including muscle, skin, brain, lungs, liver, spleen, bone marrow, thymus, heart , lymph nodes, blood, bone, cartilage, pancreas, kidney, gall bladder, stomach, intestine, testes, ovaries, uterus, rectum, nervous system, eyes, glands, and connective tissue. The interstitial space of the tissues comprises the mucopolysaccharide matrix, fluid, intercellular, between the reticular fibers of the tissues of organs, the elastic fibers in the walls of the vessels or in the chambers, the collagen fibers of the fibrous tissues, or that same matrix within the connective tissue that lines the muscle cells or in the gaps of the bone. It is similarly the space occupied by the plasma of the circulation and the lymphatic fluid of the lymphatic channels. The administration of the interstitial space of muscle tissue is preferred for the reasons discussed below. These can be conveniently administered by injection into the tissues comprising these cells. These are preferably administered to and expressed in non-dividing cells, persistent, which are differentiated, although the distribution and expression can be achieved in undifferentiated or less completely differentiated cells such as, for example, pluripotent cells of the blood or skin fibroblasts. Muscle cells in vivo are particularly competent in their ability to capture and express the polynucleotides. For injection of the nude acid sequence, an effective dose amount of DNA or RNA will be in the range of about 0.5 mg / kg of body weight to about 50 mg / kg of body weight. Preferably, the dose will be from about 0.005 mg / kg to about 20 mg / kg and more preferably from about 0.05 mg / kg to about 5 mg / kg. Of course, as the person skilled in the art will appreciate, this dosage will vary according to the tissue injection site. The appropriate and effective dose of the nucleic acid sequence can be readily determined by those of ordinary skill in the art and may depend on the condition being treated and the route of administration. The preferred route of administration is through the parenteral route of injection into the interstitial space of the tissues. However, other parenteral routes may also be used such as inhalation of an aerosol formulation particularly for administration to the lungs or bronchial tissues, throat or mucous membranes of the nose. In addition, the naked KGF-2 DNA constructs can be delivered to the arteries during angioplasty by the catheter used in the procedure. The naked polynucleotides are administered by any method known in the art, including, but not limited to, direct needle injection at the site of administration, intravenous injection, topical administration, catheter infusion, and so-called "gene guns". These methods of administration are known in the art. As evidenced in the Examples, the naked nucleic acid sequences of KGF-2 can be administered in vivo, and result in the successful expression of the KGF-2 polypeptide in the femoral arteries of rabbits. The constructs can also be administered with administration vehicles such as viral sequences, viral particles, liposome formulations, lipofectin, precipitation agents, etc. Such methods of administration are well known in the art. In certain embodiments, constructs of the KGF-2 polynucleotide are complexed in a liposomal preparation. The liposomal preparation for use in the present invention include cationic preparations (positively charged), anionic (negatively charged) and neutral. However, cationic liposomes are particularly preferred because a tight charge complex can be formed between the cationic liposome and the polyanionic nucleic acid. Cationic liposomes have been shown to mediate the intracellular administration of plasmid DNA (Felgner et al, Proc.Nat.Acid.Sci.U.A. (1987) 84: 7413-7416, which is incorporated by reference herein), mRNA ( Malone et al., Proc. Nati, Acad. Sci. USA (1989) 86: 6077-6081, which is incorporated by reference herein); and purified transcription factors (Debs et al., J. Biol. Chem. (1990) 265: 10189-10192, which is incorporated by reference herein), in functional form. Cationic liposomes are readily available. For example,? - [1-2,3-dioleyloxy) propyl] -?,?,? - triethylammonium (DOTMA) liposomes are particularly useful and are available under the trademark Lipofectin, from GIBCO BRL, Grand Island,? .and (See also, Felgner et al., Proc. Nati. Acad. Sci. USA (1987) 84: 7413-7416, which is incorporated by reference herein. Other commercially available liposomes include Transfectase (DDAB / DOPE) and DOTAP / DOPE (Boehringer). Other cationic liposomes can be prepared from readily available materials using techniques well known in the art. See for example, the Publication? O. WO 90/11092 (which is incorporated by reference herein) for a description of the synthesis of liposomes of DOTAP (1,2-bis (oleoyloxy) -3- (trimethylammonio) propane). The preparation of liposomes from DOTMA is explained in the literature, see for example, P.

Felgner et al., Proc. Nati Acad. Sci. USA 84: 7413-7417, which is incorporated by reference herein. Similar methods can be used to prepare liposomes from other cationic lipid materials. Similarly, anionic and neutral liposomes are readily available, such as from Avanti Polar Lipids (Birmingham, Ala.), Or can be readily prepared using readily available materials. Such materials include phosphatidyl, choline, cholesterol, phosphatidylethanolamine, dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylglycerol (DOPG), dioleoylphosphatidylethanoimine (DOPE), among gold. These materials can also be mixed with the initial materials of DOTMA and DOTAP in appropriate proportions. Methods for making liposomes using these materials are well known in the art. For example, dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylglycerol (DOPG), and commercially available dioleoylphosphatidylethanolamine (DOPE) can be used in various combinations to make conventional liposomes, with or without the addition of cholesterol. Thus, for example, DOPG / DOPC vesicles can be prepared by drying 50 mg of each of DOPG and DOPC under a stream of nitrogen gas in a sonification bottle. The sample is placed under a vacuum pump overnight and hydrated the next day with deionized water. The sample is then sonicated for 2 hours in a bottle with a lid, using a Heat Systems model 350 sonicator equipped with an inverted cup probe (bath type) at the maximum setting while the bath is circulated at 15EC. Alternatively, negatively charged vesicles can be prepared without sonication to produce multilamellar vesicles or by extrusion through nucleophore membranes to produce unilamellar vesicles of discrete size. Other methods are known and available to those skilled in the art. Liposomes may comprise multilamellar vesicles (MLVs), small unilamellar vesicles (SUVs), or large unilamellar vesicles (LUVs), with the SUVs that are preferred. The various liposome-nucleic acid complexes are prepared using methods well known in the art. See for example, Straubinger et al., Methods of Immunology (1983), 101: 512-527, which is incorporated by reference herein. For example, MLVs containing nucleic acid can be prepared by depositing a thin film of phospholipid on the walls of a glass tube and subsequently hydrating with a solution of the material to be encapsulated. SUVs are prepared by prolonged sonication of MLVs to produce a homogenous population of unilamellar liposomes. The material to be trapped is added to a suspension of the preformed MLVs and then sonicated. When liposomes containing cationic lipids are used, the dried lipid film is resuspended in an appropriate solution such as sterile water or an isotonic buffer solution such as 10 mM Tris / NaCl, sonicated, and then the preformed liposomes are directly mixed with the DNA. The liposome and DNA form a very stable complex due to the binding of positively charged liposomes to the cationic DNA. SUVs find use with small nucleic acid fragments. The LUVs are prepared by a number of methods, well known in the art. Commonly used methods include chelation with Ca2 + -EDTA (Papahadjopoulos et al., Biochim, Biophys. Acta (1975) 394-483).; Wilson et al., Cell (1979) 17:77); injection with ether (Deamer, D. and Bangham, A., Biochim, Biophys. Acta (1976) 443: 629; Ostro et al., Biochem. Biophys, Res.

Commun. (1977) 76: 836; Fraley et al., Proc. Nati Acad. Sci. USA (1979) 76: 3348), dialysis with detergent (Enoch, H. and Strittmatter, P., Proc. Nati, Acad. Sci. USA (1979) 76: 145); and reverse phase evaporation (REV) (Fraley et al., J. Biol. Chem. (1980) 255: 10431; Szoka, F. and Papahadjopoulos, D., Proc. Nati. Acad. Sci. USA (1978) 75: 145; Schaefer-Ridder et al., Science (1982) 215: 166), which are incorporated by reference herein.

In general, the ratio of DNA to liposomes will be from about 10: 1 to about 1:10. Preferably, the ratio will be from about 5: 1 to about 1: 5. More preferably, the ratio will be from about 3: 1 to about 1: 3. Even more preferably, the ratio will be about 1: 1. U.S. Patent No. 5,676,954 (which is incorporated by reference herein) reports on the injection of genetic material, formed in complex with cationic liposomal carriers, in mice. U.S. Patents Nos. 4,897,355, 4,946,787, 5,049,386, 5,459,127, 5,589,466, 5,693,622, 5,580,859, 5,703,055, and international publication no. WO 94/9469 (which are incorporated by reference herein) provide cationic lipids for use in the transfection of DNA within cells and mammals. U.S. Patent Nos. 5,589,466, 5,693,622, 5,580,859, 5,703,055 and international publication no. WO 94/9469 (which are incorporated by reference herein) provide methods for administering cationic DNA-lipid complexes to mammals. In certain embodiments, the cells are engineered, ex vivo or in vivo, using a retroviral particle containing RNA comprising a sequence encoding KGF-2. Retroviruses from which retroviral plasmid vectors can be derived include, but are not limited to, the Moloney murine leukemia virus, splenic necrosis virus, Rous sarcoma virus, Harvey sarcoma virus, virus. of avian leukemia, gibbon monkey leukemia virus, human immunodeficiency virus, myoloproliferative sarcoma virus, and mammary tumor virus. The retroviral plasmid vector is used to transduce the packaging cell lines to form the producer cell lines. Examples of packaging cells that can be transfected include, but are not limited to, the PE501, PA317, R-2, R-AM, PA12, T19-14X, VT-19-17-H2, RCRE, RCR cell lines. , GP + E-86, GP + envAml2, and DAN as described in Miller, Human Gene Therapy 1: 5-14 (1990), which is incorporated by reference herein, in its entirety. The vector can transduce the packaging cells through any means known in the art. Such means include but are not limited to, electroporation, the use of liposomes, and calcium phosphate precipitation. In an alternative, the retroviral plasmid vector can be encapsulated within a liposome, or coupled to a lipid, and then administered to a host. The producer cell line generates infectious retroviral vector particles that include the polynucleotide encoding KGF-2. Such retroviral vector particles can then be employed to transduce the eukaryotic cells either in vitro or in vivo. The transduced eukaryotic cells will express KGF-2. In other certain embodiments, the cells are engineered, ex vivo or in vivo, with the KGF-2 polynucleotide contained in an adenoviral vector. The adenovirus can be manipulated such that it encodes and expresses KGF-2, and at the same time is inactivated in terms of its ability to replicate in a normal lytic viral life cycle. The expression of the adenovirus is achieved without the integration of the viral DNA into the chromosome of the host cell, whereby the problems regarding the insertion mutagenesis are solved. In addition, adenoviruses have been used as live enteric vaccines for many years with an excellent safety profile (Schwartz, A. R. et al., (1974) Am. Rev. Respir.

Dis. 109: 233-238). Finally, adenovirus-mediated gene transfer has been demonstrated in a number of cases including the transfer of alpha-1-antitrypsin and CFTR to the lungs of cotton rats (Rosenfeld, MA et al. (1991) Science 252: 431-434). Rosenfeld et al., (1992) Cell 68: 143-155). In addition, extensive studies to try to establish adenovirus as a causative agent in human cancer were uniformly negative (Green, M. et al., (1979) Proc. Nati, Acad. Sci. USA 76: 6606). Suitable adenoviral vectors useful in the present invention are described, for example, in Kozarsky and Wilson, Curr. Opin. Genet Devel. 3: 499-503 (1993); Rosenfeld et al., Cell 68: 143-155 (1992); Engelhardt et al., Human Genet. Ther. 4: 759-769 (1993); Yang and collaborators, Nature Genet. 7: 362-369 (1994); Wilson et al., Nature 365: 691-692 (1993); U.S. Patent No. 5,652,224, which are incorporated by reference herein. For example, the adenoviral vector Ad2 is useful and can be developed in human 293 cells. These cells contain the El region of the adenovirus and constitutively express Ela and Elb, which complement the defective adenovirus by providing the products of the genes deleted from the vector. In addition to Ad2, other varieties of adenoviruses (e.g., Ad3, Ad5 and Ad7) are also useful in the present invention. Preferably, the adenoviruses used in the present invention are replication deficient. The replication-deficient adenoviruses require the aid of an auxiliary virus and / or a packaging cell line to form the infectious particles. The resulting virus is capable of infecting the cells and can express a polynucleotide of interest which is operably linked to a promoter, eg, the HARP promoter of the present invention, but can not replicate in most cells. Replication-deficient adenoviruses can be deleted in one or more of all or a portion of the following genes: Ela, Elb, E3, E4, E2a, or Li to L5. In other certain modalities, the cells are engineered by genetic engineering, ex vivo or in vivo, using an adeno-associated virus (AAV). AAVs are naturally occurring defective viruses that require helper viruses to produce infectious particles (Muzyczka, N., Curr, Topics in Microbiol, Immunol., 158: 97 (1992)). This is also one of the few viruses that can integrate their DNA into cells that do not divide. Vectors containing as few as 300 base pairs of AAV can be packaged and can be integrated, but the space for the exogenous DNA is limited to approximately 4.5 kb. Methods for producing and using such AAVs are known in the art. See, for example, U.S. Patent Nos. 5,139,941, 5,173,414, 5,354,678, 5,436,146, 5,474,935, 5,478,745, and 5,589,377. For example, an AAV vector suitable for use in the present invention will include all sequences necessary for DNA replication, encapsidation, and integration within the host or host cell. The polynucleotide construct of KGF-2 is inserted into the AAV vector using standard cloning methods, such as those found in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press (1989). The recombinant vector AAV is then transfected into packaging cells that are infected with an auxiliary virus, using any standard technique, including lipofection, electroporation, calcium phosphate precipitation, etc. Suitable helper viruses include adenovirus, cytomegalovirus, vaccinia virus, or herpes virus. Once the packaging cells are transfected and infected, they will produce infectious AAV viral particles which contain the construction of the KGF-2 polynucleotide. These viral particles are then used to transduce eukaryotic cells, either ex vivo or in vivo. The transduced cells will contain the KGF-2 polynucleotide construct integrated into their genome, and will express KGF-2. Yet another method of gene therapy involves operably associating the heterologous control regions and endogenous polynucleotide sequences (e.g., coding for KGFF-2) via homologous recombination (see for example, U.S. Patent No. 5,641,670, issued on June 24, 1997; International Publication No. WO 96/29411, published September 26, 1996; International Publication No. WO 94/12650, published August 4, 1994; Koller et al., Proc. Nati Acad. Sci. USA 86: 8932-8935 (1989), and Zijlstra et al., Proc. Nati Acad. Sci. USA 86: 8932-8935 (1989); and Zijlstra et al., Nature 342: 435-438 (1989). This method involves the activation of a gene that is present in the target cells, but that is not normally expressed in the cells, or is expressed at a lower level than desired. The polynucleotide constructs are made using standard techniques known in the art, which contain the promoter with the target sequences flanking the promoter. Suitable promoters are described herein. The target sequence is sufficient to complement an endogenous sequence to allow homologous recombination of the targeting sequence to the promoter with the endogenous sequence. The targeting sequence will be sufficiently close to the 5 'end of the desired endogenous polynucleotide sequence of KGF-2 so that the promoter will be operably linked to the endogenous sequence after homologous recombination. The promoter and targeting sequences can be amplified using PCR. Preferably, the amplified promoter contains different restriction enzyme sites on the 5 'and 3' ends. Preferably, the 3 'end of the first targeting sequence contains the same restriction enzyme site as the 5' end of the amplified promoter and the 5 'end of the second target sequence contains the same restriction site as the 3' end. 'of the amplified promoter. The amplified promoter and targeting sequences are digested and ligated together. The targeting promoter-sequence construct is distributed to the cells, either as a naked polynucleotide, or in conjunction with the agents that facilitate transfection, such as liposomes, viral sequences, viral particles, whole viruses, lipofection, precipitation, etc., described in more detail above. The targeting promoter-sequence can be administered by any method, including direct needle injection, intravenous injection, topical administration, catheter infusion, particle accelerators, etc. The methods are described in more detail later. The targeting promoter-sequence construct is picked up by the cells. The homologous recombination between the construct and the endogenous sequence takes place, such that an endogenous KGF-2 sequence is placed under the control of the promoter. The promoter then promotes the expression of the endogenous sequence of KGF-2.

The polynucleotides encoding KGF-2 can be administered together with other polynucleotides that code for other angiogenic proteins. Angiogenic proteins include, but are not limited to, growth factors of acidic and basic fibroblasts, VEGF-1, epidermal growth factor alpha and beta, platelet-derived endothelial cell growth factor, platelet-derived growth factor, factor tumor necrosis alpha, hepatocyte growth factor, insulin-like growth factor, colony stimulating factor, macrophage colony stimulating factor, granulocyte / macrophage colony stimulating factor, and nitric oxide synthase. Preferably, the polynucleotide encoding KGF-2 contains a secretory signal sequence that facilitates the secretion of the protein. Typically, the signal sequence is placed in the coding region of the polynucleotide to be expressed towards or at the 5 'end of the coding region. The signal sequence can be homologous or heterologous to the polynucleotide of interest, and can be homologous or heterologous to the cells to be transfected. In addition, the signal sequence can be chemically synthesized using methods known in the art.

Any mode of administration of any of the above-described polynucleotide constructs can be used as long as the mode results in the expression of one or more molecules in an amount sufficient to provide a therapeutic effect. This includes direct needle injection, systemic injection, catheter infusion, biolistic injectors, particle accelerators (eg, "gene gun"), sponge gel foam deposits, other commercially available reservoir materials, osmotic pumps ( for example, Alza minipumps), solid oral or suppository pharmaceutical formulations (tablets or pills), and decanting or topical application during surgery. For example, direct injection of the naked plasmid precipitated with calcium phosphate into rat liver and rat spleen or a plasmid coated with protein into the portal vein, have resulted in gene expression of the foreign gene in the rat livers (Kaneda et al., Sci en 243: 375 (1989)). A preferred method of local administration is by direct injection. Preferably, a recombinant molecule of the present invention formed the complex with a delivery vehicle is administered by direct injection into or locally within the area of the arteries. The administration of a composition locally within the area of the arteries refers to the injection of the composition to centimeters, and preferably, to millimeters within the arteries. Another method of local administration is to contact a polynucleotide construct of the present invention in or around a surgical wound. For example, a patient may undergo surgery and the polynucleotide construct may be coated on the surface of the tissue within the wound or the construct may be injected into the tissue areas within the wound. Therapeutic compositions useful in systemic administration include the recombinant molecules of the present invention, complexed to a target delivery vehicle, of the present invention. Suitable administration vehicles for use with systemic administration comprise liposomes which include ligands for directing the vehicle to a particular site. Preferred methods of systemic administration include intravenous injection, aerosol, oral and percutaneous (topical) distribution. Intravenous injections can be performed using standard methods in the art. The aerosol distribution can also be performed using standard methods in the art (see, for example, Stribling et al., Proc. Nati. Acad.

Sci. USA 189: 11277-11281, 1992, which is incorporated by reference herein). Oral administration can be performed by complex formation of a polynucleotide construct of the present invention to a carrier capable of resisting degradation by digestive enzymes in the intestine of an animal. Examples of such carriers include plastic capsules or tablets, such as those known in the art. Topical administration can be accomplished by mixing a polynucleotide construct of the present invention with a lipophilic reagent (e.g., DMSO) that is capable of passing into the skin. The determination of an effective amount of substance to be distributed may be dependent on a number of factors including, for example, the chemical structure and biological activity of the substance, the age and weight of the animal, the precise condition it requires treatment and its severity, and the route of administration. The frequency of treatments depends on a number of factors, such as the amount of polynucleotide constructs administered per dose, as well as the health and history of the subject. The precise amount, the number of doses, and the timing of doses will be determined by the attending physician or veterinarian.

The therapeutic compositions of the present invention can be administered to any animal, preferably to mammals and birds. Preferred mammals include humans, dogs, cats, mice, rats, rabbits, sheep, cattle, horses and pigs, with humans being particularly preferred. In a specific embodiment, nucleic acids comprising sequences encoding the antibodies or functional derivatives thereof, are administered to treat, inhibit or prevent a disease or disorder, associated with aberrant expression and / or the activity of a the invention, by means of gene therapy. Gene therapy refers to therapy performed by administering a expressed or expressible nucleic acid to a subject. In this embodiment of the invention, the nucleic acids produce their encoded protein that mediates a therapeutic effect. Any of the methods for gene therapy available in the art can be used according to the present invention. Exemplary methods are described below. For general reviews of gene therapy methods, see Goldspiel et al., Clinical Pharmacy 12: 488-505 (1993); Wu and Wu, Biotherapy 3: 87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol 32: 573-596 (1993); Mulligan, Science 260: 926-932 (1993); and Morgan and Anderson, Ann. Rev. Biochem. 62: 191-217 (1993); May, TIBTECH 11 (5): 155-215 (1993). Methods commonly known in the art of recombinant DNA technology that can be used are described in Ausubel et al. (Eds.), Current Protocols in Molecular Biology, John Wiley and Sons, NY (1993); and Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990). In a preferred aspect, the compound comprises the nucleic acid sequences encoding an antibody, said nucleic acid sequences being part of the expression vectors expressing the antibody or the fragments or the chimeric proteins or the heavy or light chains of the themselves, in a suitable host. In particular, such nucleic acid sequences have promoters operably linked to the antibody coding region, said promoter being inducible or constitutive, and, optionally, tissue-specific. In another particular embodiment, nucleic acid molecules are used in which the sequences encoding the antibody and any other desired sequences are flanked by regions that promote homologous recombination at a desired site in the genome, thus providing the expression of the nucleic acids encoding the antibody (Koller and Smithies, Proc. Nati, Acad. Sci. USA 86: 8932-8935 (1989); Zijlstra et al., Nature 342: 435-438 (1989). In specific embodiments, the expressed antibody molecule is a single chain antibody; alternatively, the nucleic acid sequences include sequences encoding the heavy and light chains, or fragments thereof, of the antibody. The distribution of the nucleic acids in a patient can be direct, in which case the patient is directly exposed to the nucleic acid or to the vectors that possess the nucleic acid, or indirectly, in which case, the cells are first transformed with the nucleic acids in vi tro, then transplanted to the patient. These two methods are respectively known as in vivo or ex vivo gene therapy. In a specific embodiment, the nucleic acid sequences are directly administered in vivo, where they are expressed to produce the encoded product. This may be accomplished by any of the numerous methods known in the art, for example, by constructing them as part of an appropriate nucleic acid expression vector and administering them so that they become intracellular, for example, by infection using retroviral vectors or defective or attenuated viral (see U.S. Patent No. 4,980,286), or by direct injection of the naked DNA, or by the use of microparticle bombardment (e.g., a gene gun; Biolistic, Dupont); lipids or cell surface receptors or transfection agents, encapsulation in liposomes, microparticles, or microcapsules, or by administering them in binding to a peptide known to enter the nucleus, administering them in binding to a ligand subject to receptor-mediated endocytosis (see for example , Wu and Wu, J. Biol. Chem. 262: 4429-4432 (1987) (which can be used to direct cell types which specifically express the receptors), etc. In yet another embodiment, nucleic acid-ligand complexes can be formed, in which the ligand comprises a fusogenic viral peptide to disorganize the endosomes, allowing the nucleic acid to prevent lysosomal degradation. In yet another embodiment, the nucleic acid can be directed in vivo for the uptake and specific expression of the cells by targeting a specific receptor (see, for example, PCT publications WO 92/06180, WO 92/22635, WO 92 / 20316; WO 93/14188, WO 93/20221). Alternatively, the nucleic acid can be introduced intracellularly and incorporated into the DNA of the host cell for expression, by homologous recombination (Koller and Smithies, Proc.Nat.Acid.Sci.U.A. 86: 8932-8935 (1989); Zijlstra et al. collaborators, Nature 342: 435-438 (1989)).

In a specific embodiment, viral vectors containing the nucleic acid sequences encoding an antibody of the invention are used. For example, a retroviral vector can be used (see Miller et al., Meth. Enzymol, 217: 581-599 (1993)). These retroviral vectors contain the necessary components for the correct packaging of the viral genome and integration into the DNA of the host cell. The nucleic acid sequences encoding the antibody to be used in gene therapy are cloned into one or more vectors, which facilitates the distribution of the gene to a patient. More details regarding retroviral vectors can be found in Boesen et al., Biotherapy 6: 291-302 (1994), which describes the use of a retroviral vector to distribute the mdrl gene to hematopoietic stem cells in order to make the pluripotent cells more resistant to chemotherapy. Other references that illustrate the use of retroviral vectors in gene therapy are: Clowes et al., J. "Clin. Invest. 93: 644-651 (1994); Kiem et al., Blood 83: 1467-1473 (1994); Gunzberg, Human Gene Therapy 4: 129-141 (1993) and Grossman and Wilson, Curr Opin., In Genetics and Devel.3: 110-114 (1993) Adenoviruses are other viral vectors that can be used in gene therapy Adenoviruses are especially attractive vehicles for the distribution of genes to the respiratory epithelium, and adenoviruses naturally infect the respiratory epithelium, where they cause mild disease.Other targets for adenovirus-based distribution systems are the liver, the system Central nervous system, endothelial cells, and muscle Adenoviruses have the advantage of being able to infect non-dividing cells Kozarsky and Wilson, Current Opinion in Genetics and Development 3: 499-503 (1993) present a review ion of adenovirus-based gene therapy. Bout et al., Human Gene Therapy 5: 3-10 (1994) demonstrated the use of adenoviral vectors to transfer genes to the respiratory epithelium of rhesus monkeys. Other cases of the use of adenoviruses in gene therapy can be found in Rosenfeld et al., Science 252: 431-434 (1991); Rosenfeld et al., Cell 68: 143-155 (1992); Mastrangeli et al., J. Clin. Invest. 91: 225-234 (1993); PCT Publication WO 94/12649; and Wang et al., Gene Therapy 2: 775-783 (1995). In a preferred embodiment, the adenoviral vectors are used. Adeno-associated virus (AAV) has also been proposed for use in gene therapy (Walsh et al., Proc. Soc. Exp. Biol. Med. 204: 289-300 (1993), U.S. Patent No. 5,436,146).

Another method for gene therapy involves the transfer of a gene to cells in tissue culture by methods such as electroporation, lipofection, calcium phosphate mediated transfection, or viral infection. Usually, the transfer method includes the transfer of a selectable marker to the cells. The cells are then placed under selection to isolate those cells that have captured and are expressing the transferred gene. Those cells are distributed to a patient. In this embodiment, the nucleic acid is introduced into a cell prior to in vivo administration of the resulting recombinant cells. Such introduction can be carried out by any method known in the art, including but not limited to, transfection, electroporation, microinjection, infection with a viral or bacteriophage vector containing the nucleic acid sequences, cell fusion, gene transfer mediated by chromosomes, the transfer of genes mediated by micro cells, the fusion of spheroplasts, etc. Numerous techniques are known in the art for the introduction of foreign genes into cells (see, for example, Loeffler and Behr, Meth. Enzimol, 217: 599-618 (1993)).; Cohen and collaborators, Meth. Enzymol. 217: 618-644 (1993); Cline, Phar ac. Ther. 29: 69-92, (1985) and may be used in accordance with the present invention, with the proviso that the functions necessary for the physiological and development of recipient cells or containers, are not disturbed. The technique should provide stable transfer of the nucleic acid to the cell, so that the nucleic acid is expressible by the cell and preferably heritable and expressible by its cell line. The resulting recombinant cells can be distributed to a patient by methods known in the art. Recombinant blood cells (e.g., totipotent hematopoietic or progenitor cells) are preferably administered intravenously). The amount of cells considered for use depends on the desired effect, the condition of the patient, etc., and can be determined by a person skilled in the art. Cells into which a nucleic acid can be introduced for gene therapy purposes, encompass any available, desired cell type, and include but are not limited to epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes; blood cells such as T lymphocytes, B lymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, granulocytes; various pluripotent or progenitor cells, in particular hematopoietic or progenitor stem cells, for example, as obtained from bone marrow, umbilical cord blood, peripheral blood, fetal liver, etc. In a preferred embodiment, the cell used for gene therapy is autologous to the patient. In an embodiment in which the recombinant cells are used in gene therapy, the nucleic acid sequences encoding an antibody are introduced into the cells such that they are expressible by the cells or their progeny, and the recombinant cells are then administered. in vivo for the therapeutic effect. In a specific embodiment, pluripotent or progenitor cells are used. Any pluripotent and / or progenitor cells that can be isolated and maintained in vi tro can potentially be used in accordance with this embodiment of the present invention (see for example, PCT Publication WO 94/08598; Stemple and Anderson, Cell 71: 973 -985 (1992), Rheinwald, Meth Cell Bio 21A: 229 (1980), and Pittelkow and Scott, Mayo Clinic Proc. 61: 771 (1986) In a specific embodiment, the nucleic acid to be introduced for The purpose of gene therapy comprises an inducible promoter operably linked to the coding region, such that the expression of the nucleic acid is controllable by controlling the presence or absence of the appropriate inducer of transcription.

Demonstration of the Therapeutic or Prophylactic Activity.

The compounds or pharmaceutical compositions of the invention are preferably tested in vi tro, and then in vivo for the desired therapeutic or prophylactic activity, before use in humans. For example, in vitro tests to demonstrate the therapeutic or prophylactic utility of a compound or pharmaceutical composition include, the effect of a compound on a patient's cell line or tissue sample. The effect of the composition or compound on the cell line and / or tissue sample can be determined using techniques known to those skilled in the art including, but not limited to, rosetting assays and cell lysis assays. According to the invention, the in vi tro assays that can be used to determine whether the administration of a specific compound is indicated or not, include in vitro cell culture assays, in which a tissue sample from the patient is developed in culture, and exposed to or otherwise administered with a compound, and the effect of such a compound on the tissue sample is observed.

Immune activity KGF-2 polynucleotides or polypeptides, or KGF-2 agonists or antagonists, may be useful in the treatment of deficiencies or disorders of the immune system, by activating or inhibiting the proliferation, differentiation, or mobilization (chemotaxis) of cells immune Immune cells develop through a process called hematopoiesis, producing myeloid cells (platelets, red blood cells (erythrocytes), neutrophils, and macrophoids) and lymphoid cells (B and T lymphocytes), from stem cells. The etiology of these immune deficiencies or disorders can be genetic, somatic, such as cancer or some autoimmune disorders, acquired (for example, by chemotherapy or toxins), or infectious. In addition, KGF-2 polynucleotides or polypeptides, or KGF-2 agonists or antagonists, can be used as a marker or detector of a particular disease or disorder of the immune system. KGF-2 polynucleotides or polypeptides, or KGF-2 agonists or antagonists may be useful in the treatment or detection of deficiencies or disorders of hematopoietic cells. KGF-2 polynucleotides or polypeptides, or KGF-2 agonists or antagonists could be used to increase the differentiation and proliferation of hematopoietic cells, including stem cells, in an effort to treat those disorders associated with a decrease in certain types (or many) of hematopoietic cells. Examples of immune deficiency syndromes include, but are not limited to: blood protein disorders (eg, agammaglobulinemia, disgammaglobulinemia), ataxia telangiectasia, common variable immunodeficiency, Digeorge syndrome, HIV infection, HTLV-BLV infection, deficiency syndrome due to leukocyte adhesion, lymphopenia, phagocytic bactericidal dysfunction, severe combined immunodeficiency (SCIDs), Wiskott-Aldrich disorder, anemia, thrombocytopenia, or hemoglobinuria. In addition, KGF-2 polynucleotides or polypeptides, or KGF-2 agonists or antagonists can be used to mote hemostatic activity (the arrest of bleeding) or thrombolytic activity (clot formation). For example, by increasing haemostatic or thrombolytic activity, KGF-2 polynucleotides or polypeptides, or KGF-2 agonists or antagonists, could be used to treat blood coagulation disorders (e.g., afibrinogenemia, factor deficiencies). ), blood platelet disorders (eg, thrombocytopenia), or injuries resulting from trauma, surgery, or other causes. Alternatively, the KGF-2 polynucleotides or polypeptides, or the KGF-2 agonists or antagonists, which can decrease hemostatic or thrombolytic activity could be used to inhibit or dissolve the clot, important in the treatment of heart attacks (infarction), strokes, or scarring. KGF-2 polynucleotides or polypeptides, or KGF-2 agonists or antagonists may also be useful in the treatment or detection of autoimmune disorders. Many autoimmune disorders result from the inappropriate recognition of one's own as foreign material by immune cells. This inappropriate recognition results in an immune response that leads to tissue destruction of the host or host. Therefore, the administration of KGF-2 polynucleotides or polypeptides, or KGF-2 agonists or antagonists, which can inhibit an immune response, particularly the proliferation, differentiation, or chemotaxis of T cells, can be an effective therapy in the prevention of autoimmune disorders. Examples of autoimmune disorders that can be treated or detected include, but are not limited to: Adison's disease, hemolytic anemia, antiphospholipid syndrome, rheumatoid arthritis, dermatitis, allergic encephalomyelitis, glomerulonephritis, Goodpasture's syndrome, Graves' disease, multiple sclerosis, myasthenia gravis, neuritis, ophthalmia, bullous pemphigoid, pemphigus, polyendocrinopathies, purpura, Reiter's disease, rigid man syndrome, autoimmune thyroiditis, systemic lupus erythematosus, autoimmune pulmonary inflammation, Guillain-Barre syndrome, insulin-dependent diabetes mellitus, and disease inflammatory autoimmune ophthalmic. Similarly, allergic reactions and conditions, such as asthma (particularly allergic asthma) or other respiratory problems, can also be treated by KGF-2 polynucleotides or polypeptides, or KGF-2 agonists or antagonists. In addition, these molecules can be used to treat anaphylaxis, hypersensitivity to an antigenic molecule, or incompatibility of blood groups. The KGF-2 polynucleotides or polypeptides, or KGF-2 agonists or antagonists, can also be used to treat and / or prevent organ rejection or graft versus host disease (GVHD). Organ rejection occurs by the destruction of host immune cells from the transplanted tissue through an immune response. Similarly, an immune response is also involved in GVHD, but, in this case, foreign, transplanted immune cells destroy host tissues. The administration of KGF-2 polynucleotides or polypeptides, or agonists or antagonists of KGF-2, which inhibits an immune response, particularly the proliferation, differentiation, or chemotaxis of T cells, can be an effective therapy in the prevention of rejection of organs or GVHD. Similarly, polynucleotides or polypeptides KGF-2, or agonists or antagonists of KGF-2, can also be used to modulate inflammation. For example, KGF-2 polynucleotides or polypeptides, or KGF-2 agonists or antagonists, can inhibit the proliferation and differentiation of cells involved in an inflammatory response. These molecules can be used to treat inflammatory conditions, chronic and acute conditions, including inflammation associated with infection (for example, septic shock, sepsis, or other systemic inflammatory syndrome (SIRS)), ischemia-reperfusion injury, endotoxin lethality, arthritis, hyperacute complement-mediated rejection, nephritis, lung injury induced by cytokine or chemokine, inflammatory bowel disease, Crohn's disease, or resulting from overproduction of cytokines (eg, TNF, IL-1).

Hyperproliferative disorders KGF-2 polynucleotides or polypeptides, or KGF-2 agonists or antagonists, can be treated to treat or detect hyperproliferative disorders, including neoplasms. KGF-2 polynucleotides or polypeptides or KGF-2 agonists or antagonists can inhibit the proliferation of the disorder through direct or indirect interactions. Alternatively, KGF-2 polynucleotides or polypeptides or KGF-2 agonists or antagonists can proliferate other cells that can inhibit hyperproliferative disorder. For example, by increasing an immune response, particularly by increasing the antigenic qualities of the hyperproliferative disorder or by proliferation, differentiation, or mobilization of T cells, hyperproliferative disorders can be treated. This immune response can be increased either by increasing an existing immune response, or by initiating a new immune response. Alternatively, the decrease in an immune response may also be a method of treating hyperproliferative disorders, such as a chemotherapeutic agent. Examples of hyperproliferative disorders that can be treated or detected by KGF-2 polynucleotides or polypeptides, or KGF-2 agonists or antagonists, include, but are not limited to, neoplasms located in: the abdomen, bone, breast , the digestive system, the liver, the pancreas, the peritoneum, the endocrine glands (adrenal, parathyroid, pituitary, testes, ovaries, thymus, thyroid), the eye, the head and neck, the nervous system (central and peripheral) , the lymphatic system, the pelvis, the skin, the soft tissue, the spleen, the thorax, and the urogenital system. Similarly, other hyperproliferative disorders can also be treated or detected by KGF-2 polynucleotides or polypeptides, or KGF-2 agonists or antagonists. Examples of such hyperproliferative disorders include, but are not limited to: hypergammaglobulinemia, lymphoproliferative disorders, paraproteinemias, purpura, sarcoidosis, Sezary's syndrome, Waldenstron's macroglobulinemia, Gaucher's disease, histiocytosis, and any other hyperproliferative disease, in addition to the neoplasm, located in an organ system listed above.

Cardiovascular Disorders KGF-2 polynucleotides or polypeptides, or KGF-2 agonists or antagonists, which code for KGF-2, can be used to treat cardiovascular disorders, including peripheral arterial disease, such as limb ischemia.

Cardiovascular disorders include cardiovascular abnormalities, such as arterio-arterial fistula, arteriovenous fistula, cerebral arteriovenous malformations, congenital heart defects, pulmonary atresia, and Scimitar syndrome. Congenital heart defects include aortic coarctation, heart with three cavities, anomalies of the coronary vessels, heart with crossed vessels, dextrocardia, patent arteriosis of the ducts, Ebstein's anomaly, Eisenmenger's complex, hypoplastic left heart syndrome, levocardia, tetralogy of Fallot, transposition of the large vessels, double right ventricular outflow, attrition of the tricuspid valve, persistent arteriosis of the trunk, and cardiac septal defects, such as such as the aortopulmonary septal defect, endocardiac cushion defects, Lutembacher syndrome, Fallot trilogy, and ventricular cardiac septal defects. Cardiovascular disorders also include heart disease, such as arrhythmias, carcinoid heart disease, high cardiac output, low cardiac output, cardiac tamponade, endocarditis (including bacterial), cardiac aneurysm, cardiac arrest, congestive heart failure, congestive cardiomyopathy, dyspnea paroxysmal, cardiac edema, cardiac hypertrophy, congestive cardiomyopathy, left ventricular hypertrophy, right ventricular hypertrophy, post-infarction cardiac rupture, ventricular septal rupture, diseases of the heart valves, myocardial diseases, myocardial ischemia, pericardial effusion, pericarditis (including constrictive and tuberculous ), pneumopericardium, postpericardiotomy syndrome, cardiopulmonary disease, cardiac rheumatic disease, ventricular dysfunction, hyperemia, cardiovascular complications of pregnancy, Scimitar syndrome, cardiovascular syphilis, and cardiovascular tuberculosis. Arrhythmias include sinus arrhythmia, atrial fibrillation, atrial flutter, bradycardia, extrasystole, Adams-Stokes syndrome, bundle-bundle block, sinus-atrial block, long QT syndrome, parasystole, Lown-Ganong-Levine syndrome, pre-syndrome. Mahaim type excitation, Wolff-Parkinson-White syndrome, sick sinus syndrome, tachycardia, and ventricular fibrillation. Tachycardias include paroxysmal tachycardia, supraventricular tachycardia, accelerated idioventricular rhythm, atrioventricular nodal reentry tachycardia, ectopic atrial tachycardia, ectopic union tachycardia, sinoatrial nodal reentry tachycardia, sinus tachycardia, Torsades de Pointes, and ventricular tachycardia. Heart valve diseases include aortic valve failure, aortic valve stenosis, cardiac murmurs, prolapse of the aortic valve, mitral valve prolapse, tricuspid valve prolapse, mitral valve insufficiency, valve stenosis mitral, pulmonary atresia, pulmonary valve insufficiency, pulmonic valve stenosis, tricuspid valve atresia, tricuspid valve insufficiency, and tricuspid valve stenosis. Myocardial diseases include alcoholic cardiomyopathy, congestive cardiomyopathy, hypertrophic cardiomyopathy, aortic subvalvular stenosis, pulmonary subvalvular stenosis, restrictive cardiomyopathy, Chagas cardiomyopathy, endocardial fibroelastosis, endomyocardial fibrosis, Kearns syndrome, myocardial reperfusion injury, and myocarditis. Myocardial ischemias include coronary disease, such as angina pectoris, coronary aneurysm, coronary arteriosclerosis, coronary thrombosis, coronary vasospasm, myocardial infarction, and myocardial fainting. Cardiovascular diseases also include vascular diseases such as aneurysms, angiodysplasia, angiomatosis, bacillary angiomatosis, Hippel-Lindau disease, Klippel-Trenaunay-Weber syndrome, Sturge-Weber syndrome, angioneurotic edema, aortic diseases, Takayasu arteritis, aortitis, Leriche syndrome, arterial occlusive diseases, arteritis, enarteritis, polyarteritis nodosa, cerebrovascular disorders, diabetic angiopathies, diabetic retinopathy, embolisms, thrombosis, erythromelalgia, hemorrhoids, hepatic occlusive disease, hypertension, hypotension, ischemia, peripheral vascular diseases, phlebitis, veno-occlusive disease pulmonary, Reynaud's disease, CREST syndrome, retinal vein occlusion, Scimitar syndrome, superior vena cava syndrome, telangiectasia, atacia telangiectasia, hereditary hemorrhagic telangiectasia, varicocele, varicose veins, varicose ulcer, vasculitis, and venous insufficiency. Aneurysms include dissection aneurysms, false aneurysms, infected aneurysms, ruptured aneurysms, aortic aneurysms, cerebral aneurysms, coronary aneurysms, cardiac aneurysms, and iliac aneurysms. Arterial occlusive diseases include arteriosclerosis, intermittent claudication, stenosis of the carotids, fibromuscular dysplasias, mesenteric vascular occlusion, Moyamoya's disease, obstruction of the renal arteries, occlusion of the retinal arteries, and thromboangiitis obliterans. Cerebrovascular disorders include diseases of the carotid artery, cerebral amyloid angiopathy, cerebral aneurysm, cerebral anoxia, cerebral arteriosclerosis, cerebral arteriovenous malformation, cerebral artery diseases, cerebral embolism and thrombosis, carotid artery thrombosis, breast thrombosis, Wallenberg, cerebral hemorrhage, epidural hematoma, subdural hematoma, subarachnoid hemorrhage, cerebral infarction, cerebral ischemia (including transient), subclavicular theft syndrome, periventricular leukomalacia, vascular headache, cluster headache, migraine, and vertebrobasilar insufficiency. Embolisms include air embolisms, embolisms of amniotic fluids, cholesterol embolisms, blue toe syndrome, fat embolisms, pulmonary embolisms, and thromboembolisms. Thromboses include coronary thrombosis, hepatic vein thrombosis, retinal vein occlusion, carotid artery thrombosis, sinus thrombosis, Wallenberg syndrome, and thrombophlebitis. Ischemia includes cerebral ischemia, ischemic colitis, compartment syndromes, anterior compartment syndrome, myocardial ischemia, reperfusion injury, and ischemia of the peripheral limbs. Vasculitis includes aortitis, arteritis, Behcet's syndrome, Churg-Strauss syndrome, mucocutaneous lymph node syndrome, thromboangiitis obliterans, hypersensitivity vasculitis, Schoenlein-Henoch purpura, allergic cutaneous vasculitis, and Wegener's granulomatosis. The KGF-2 polynucleotides or polypeptides or the agonists or antagonists of KGF-2, are especially effective for the treatment of critical limb ischemia and coronary artery disease. As shown in the Examples, administration of the KGF-2 polynucleotides and polypeptides to an experimentally induced ischemic rabbit hind limb, can restore the ratio of blood pressure, blood flow, angiographic rating, and capillary density. The KGF-2 polypeptides can be administered using any method known in the art, including, but not limited to, direct needle injection at the site of administration, intravenous injection, topical administration, catheter infusion, biolistic injectors, particle accelerators. , gel foam sponge deposits, other commercially available reservoir materials, osmotic pumps, oral or solid pharmaceutical formulations for suppository, decantation or topical applications during surgery, aerosol administration. Such methods are known in the art. The KGF-2 polypeptides can be administered as part of a pharmaceutical composition, described in more detail below. Methods for administering KGF-2 polynucleotides are described in more detail herein.

Anti-Angiogenic Activity The balance of natural origin between endogenous stimulators and inhibitors of angiogenesis is one in which the inhibitory influences predominate. Rastinejad et al., Cell 56: 345-355 (1989). In those rare cases in which neovascularization occurs under normal physiological conditions, such as wound healing, organ regeneration, embryonic development, and female reproductive processes, angiogenesis is strictly regulated and spatially and temporally demarcated. Under conditions of pathological angiogenesis such as that which characterizes solid tumor development, these regulatory controls fail. Unregulated angiogenesis becomes pathological and supports the progression of many neoplastic and non-neoplastic diseases. A number of serious diseases are dominated by abnormal neovascularization including the development of solid tumors and metastases, arthritis, some types of ophthalmic disorders, and psoriasis. See, for example, reviews by Moses et al., Biotech. 9: 630-634 (1991); Folkman et al., N. Engl. J. Med. 333: 1757-1763 (1995); Auerbach et al., J ". Microvasc. Res. 29: 401-411 (1985); Folkman, Advances in Cancer Research, eds. Klein and Weinhouse, Academic Press, New York, pp. 175-203 (1985); , Am. J ". Opthalmol. 94: 715-743 (1982); and Folkman et al., Science 221: 719-725 (1983). In a number of pathological conditions, the process of angiogenesis contributes to the disease state. For example, significant data have been accumulated that suggest that the development of solid tumors is dependent on angiogenesis. Folkman and Klagsbrun, Science 235: 442-447 (1987). The present invention provides the treatment of diseases or disorders associated with neovascularization by the administration of the KGF-2 polynucleotides and / or polypeptides of the invention, as well as KGF-2 agonists or antagonists. Malignant and metastatic conditions that can be treated with the polynucleotides and polypeptides, or the agonists and antagonists of the invention include, but are not limited to, malignancies, solid tumors, and cancers described herein and otherwise known in the art. (for a review of such disorders, see Fishman et al., Medicine, 2nd Ed., JB Lippincott Co., Philadelphia (1985)). Ocular disorders associated with neovascularization that can be treated with the polynucleotides and KGF-2 polypeptides of the present invention (including KGF-2 agonists and / or antagonists) include, but are not limited to: neovascular glaucoma, diabetic retinopathy , retinoblastoma, retrolental fibroplasia, uveitis, retinopathy of premature macular degeneration, neovascularization by corneal graft, as well as other ophthalmic inflammatory diseases, ocular tumors and diseases associated with choroidal or iris neovascularization. See, for example, reviews by Waltman et al., Am. J. Ophthal. 85: 704-710 (1978) and Gartner et al., Surv. 10 Ophthal. 22: 291-312 (1978). In addition, disorders that can be treated with the KGF-2 polynucleotides and polypeptides of the present invention (including KGF-2 agonists and / or antagonists) include, but are not limited to, hemangioma, arthritis, 15 psoriasis, angiofibroma, atherosclerotic plaques, delayed wound healing, granulations, hemophilic joints, hypertrophic scars, non-union fractures, Osler-Weber syndrome, pyogenic granuloma, scleroderma, trachoma, and vascular adhesions. In addition, disorders and / or conditions, which can be treated with the KGF-2 polynucleotides and polypeptides of the present invention (including KGF-2 agonists and / or antagonists), include, but are not limited to, solid tumors. , blood-borne tumors such as leukemias, 25 tumor metastasis, Kaposi's sarcoma, benign tumors, by ^^ ¿Ü £ ^ example hemangiomas, acoustic neuromas, neurofibromas, trachoma, and pyogenic granulomas, rheumatoid arthritis, psoriasis, ocular angiogenic diseases, for example, diabetic retinopathy, retinopathy of prematurity, macular degeneration, rejection of corneal graft, neovascular glaucoma, retrolental fibroplasia, rubeosis, retinoblastoma, and uveitis, delayed healing of wounds, endometriosis, vasculogenesis, granulations, hypertrophic scars (keloids), fractures without union, scleroderma, trachoma, vascular adhesions, myocardial angiogenesis, coronary collaterals, cerebral collaterals, arteriovenous malformations, ischemic limb angiogenesis, Osler-Webber, neovascularization of plaque, telangiectasia, hemophilic joints, angiofibroma, fibromuscular dysplasia, granulation of wounds, Crohn's disease, atherosclerosis, birth control agent for the prevention of vascularization required for the implantation of the embryo, c control of menstruation, diseases that have angiogenesis as a pathological consequence such as cat scratch disease (Róchele minalia quintosa), ulcers (Helicobacter pylori), Bartonellosis and bacillary angiomatosis.

Digestive diseases KGF-2 has been shown to stimulate the proliferation of tract cells. gastrointestinal. In this way, the polynucleotides, polypeptides, agonists and / or antagonists of KGF-2 can be used to treat and / or detect digestive diseases. Examples of digestive diseases that can be treated or detected include: diseases of the biliary tract (such as diseases of the bile duct including bile duct neoplasms, obstructions of the bile duct, Caroli's disease, cholangitis, common bile duct diseases such as cyst choledochal, stones of the common bile duct, and neoplasms of the common bile duct, - bile reflux, biliary atresia, biliary dyskinesia, biliary fistula, neoplasms in the biliary tract, gallbladder neoplasms, cholelithiasis such as common bile duct stones, cholestasis obstruction of the bile duct, Alagille syndrome and liver cirrhosis, gallbladder diseases such as cholecystitis, cholelithiasis and gallbladder neoplasms, haemobilia and postcholecystectomy syndrome), digestive system abnormalities (such as imperforate anus, Barrett's esophagus) , biliary atresia, diaphragmatic eventration ca, esophageal atresia, Hirschsprung's disease, intestinal atresia, Meckel's diverticulum), digestive system fistula (which includes biliary fistula and esophageal fistula such as tracheoesophageal fistula, gastric fistula, intestinal fistula such as rectal fistula), fistula of the digestive system (such as intestinal fistula, such as rectal fistula including rectovaginal fistula and pancreatic fistula), neoplasms of the digestive system (such as neoplasms of the biliary tract including neoplasms of the common bile duct, neoplasms of the gallbladder), esophageal neoplasms, gastrointestinal neoplasms , such as intestinal neoplasms such as cecal neoplasms that include appendiceal neoplasms, such as colonic polyps such as adenomatous polyposis coli, colorectal neoplasms such as hereditary and non-polyposis colorectal neoplasms, sigmoid neoplasms, duodenal neoplasms, ileal neoplasms, intemal polyps such as colonic polyps such as polyposis adenomatous coli, Gardner syndrome and Peutz-Jeghers syndrome, neoplasms of the jejunum, rectal neoplasms such as neoplasms of the anus), neoplasms of the digestive system (such as gastrointestinal neoplasms, such as intestinal neoplasms, such as rectal neoplasms that include anal neoplasms and neoplasms of the anal glands, stomach neoplasms, pancreatic neoplasms and peritoneal neoplasms), esophageal diseases such as Barrett's esophagus, esophageal and gastric varices, esophageal atresia, esophageal cyst, esophageal diverticulum such as Zenker's diverticulum, esophageal motility disorders such as CREST Syndrome, swallowing disorders such as Plummer-Vinson Syndrome, esophageal achalasia, diffuse esophageal spasm and gastroesophageal reflux, esophageal neoplasms, esophageal perforation such as Mallory's Syndrome Weiss, esophageal stenosis, esophagitis such as peptic esophagitis, diaphragmatic hernia such as traumatic diaphragmatic hernia, hiatal hernia). Examples of gastrointestinal diseases that can be treated or detected include gastroenteritis such as cholera morbus, gastrointestinal hemorrhage (such as hematemesis, melena and peptic ulcer), hernia (such as diaphragmatic hernia that includes traumatic diaphragmatic hernia and hiatal hernia, femoral hernia, hernia inguinal, obturator hernia, umbilical hernia and ventral hernia), intestinal diseases (such as caecal diseases which include appendicitis, cecal neoplasms such as appendiceal neoplasms, colonic diseases such as colitis that include ischemic colitis, ulcerative colitis such as toxic megacolon, enterocolitis such as pseudomembranous enterocolitis, proctocolitis, functional colonic diseases such as colonic pseudo-obstruction, colonic neoplasms such as colonic polyps such as polyposis adenomatous coli, colorectal neoplasms such as hereditary and non-polyposis colorectal neoplasms, sigmoid neoplasms, colonic diverticulitis, colonic diverticulosis , megacolon such as Hirschsprung's disease and toxic megacolon, sigmoid diseases such as proctocolitis and sigmoid neoplasms, constipation, Crohn's disease, diarrhea such as infantile diarrhea, dysentery such as amoebic dysentery and bacillary dysentery, duodenal diseases such as duodenal neoplasms, duodenal obstruction such as superior mesenteric artery syndrome, duodenal ulcer such as Curling ulcer and duodenitis, enteritis such as enterocolitis including pseudomembranous enterocolitis, ileal diseases such as neoplasm s ileal and ileitis, immunoproliferative disease of the small intestine, inflammatory bowel diseases such as ulcerative colitis and Crohn's disease, intestinal atresia, parasitic intestinal diseases such as anisakiasis, balantidiasis, blastocystis infections, cryptosporidiosis, dientamoebiasis, amoebic dysentery and giardiasis, fistula intestinal such as rectal fistula including rectovaginal fistula, intestinal neoplasms such as cecal neoplasms including appendiceal neoplasms, colonic neoplasms such as colonic polyps including adenomatous polyposis coli, colorectal neoplasms such as hereditary and non-polyposis colorectal neoplasms, sigmoid neoplasms, duodenal neoplasms, ileal neoplasms, intestinal polyps such as colonic polyps such as polyposis adenomatous coli, Gardner syndrome, Peutz-Jeghers syndrome, intestinal obstruction such as afferent loop syndrome, obstr duodenal uction, impacted feces, intestinal pseudo-obstruction such as colonic pseudo-obstruction, intussusception, intestinal perforation, intestinal polyps such as colonic polyps including adenomatous polyposis coli, diseases of the jejunum such as jejunal neoplasms, malabsorption syndromes, such as blind loop syndrome, celiac disease, lactose intolerance, intestinal lipodystrophy, short bowel syndrome, tropical sprue, mesenteric vascular occlusion, pneumatosis cystoid intestinalis, protein loss enteropathies such as intestinal lymphangiectasis, rectal diseases such as diseases of the anus that include anal neoplasms such as neoplasms of the anal glands, fissure in the anus, anal itching, stool incontinence, hemorrhoids, proctitis such as proctocolitis, rectal fistula such as rectovaginal fistula, rectal neoplasms such as anus neoplasms such as neoplasms of the s anal glands, rectal diseases such as rectal prolapse, peptic ulcer, peptic esophagitis, marginal ulcer, peptic ulcer hemorrhage, perforation by peptic ulcer, stomach ulcer, Zollinger-Ellison syndrome, post-gastrectomy syndromes, such as the emptying syndrome rapid, stomach diseases such as achlorhydria, duodenogastric reflux such as bile reflux, gastric fistula, prolapse of the gastric mucosa, obstruction of the gastric outlet such as pyloric stenosis, gastritis such as atrophic gastritis and hypertrophic gastritis, gastroparesis, stomach dilation, stomach diverticulum , stomach neoplasms, stomach rupture, stomach ulcer and stomach volvulus, gastrointestinal tuberculosis, visceroptosis, vomiting such as hematemesis and hyperemesis gravidarum), pancreatic diseases such as cystic fibrosis, pancreatic cyst such as pancreatic pseudocyst, pancreatic fistula , pancreatic insufficiency, pancreatic neoplasms and pancreatitis), peritoneal diseases such as chyloperitoneum, hemoperitoneum, mesenteric cyst, mesenteric lymphadenitis, mesenteric vascular occlusion, peritoneal panniculitis, peritoneal neoplasms, peritonitis, pneumoperitoneum, subphrenic abscess, and peritoneal tuberculosis. Digestive diseases that can be treated or detected also include liver diseases. Liver diseases include acute yellow atrophy, intrahepatic cholestasis such as Alagille syndrome and biliary liver cirrhosis, fatty liver such as fatty alcoholic liver and Reye's syndrome, hepatic vein thrombosis, hepatic veno-occlusive disease, hepatitis such as alcoholic hepatitis, animal hepatitis such as viral animal hepatitis such as infectious canine hepatitis and Rift Valley Fever, toxic hepatitis, human viral hepatitis such as delta infection, hepatitis A, hepatitis B, hepatitis C, chronic active hepatitis and hepatitis E, hepatolenticular degeneration, hepatomegaly, hepatorenal syndrome, portal hypertension such as Cruveilhier-Baumgarten syndrome and esophageal and gastric varices, liver abscess such as amoebic liver abscess, liver cirrhosis such as alcoholic liver cirrhosis, hepatic biliary cirrhosis and experimental liver cirrhosis, alcoholic liver diseases such as liver alcoholic fatty, alcoholic hepatitis and alcoholic liver cirrhosis, parasitic liver diseases such as hepatic echinococcosis, fascioliasis, and amoebic liver abscess, liver failure such as hepatic encephalopathy and acute liver failure, hepatic neoplasms, peliosis hepatitis, erythrohepatic porphyria, and hepatic porphyria such as acute intermittent porphyria and delayed cutaneous porphyria, liver tuberculosis and Zellweger syndrome). Examples of stomatognathic diseases that can be treated or detected include jaw diseases (such as cysts, giant cell granuloma, jaw abnormalities such as cleft palate, micrognathism, Pierre Robin syndrome, prognathism and retrognathism, cysts in the jaw such as non-odontogenic cysts, odontogenic cysts such as basal cell nevus syndrome, dentigerous cyst, calcifying odontogenic cyst, periodontal cyst such as radicular cyst, edentulous jaw such as the partially edentulous jaw, neoplasms of the jaw such as mandibular neoplasms, neoplasms maxillary and neoplasms of the palate, mandibular diseases such as craniomandibular disorders that include diseases of the temporomandibular joint such as temporomandibular joint syndrome, mandibular neoplasms, prognathism and retrognathism, maxillary diseases such it is like maxillary neoplasms), diseases of the mouth (such as Behcet's Syndrome, Burnt Mouth Syndrome, oral candidiasis, dry alveolus, focal epithelial hyperplasia, oral leucoedema, oral lichen planus, lip diseases such as cheilitis, cleft lip , cold sores and lip neoplasms, Ludwig's angina, Melkersson-Rosenthal syndrome, mouth abnormalities such as cleft lip, cleft palate, fibromatose gingiva, macroglossia, macrostomy, microstomia and velopharyngeal insufficiency, edentulous mouth such as edentulous jawbone such as partially edentulous mandible, neoplasms of the mouth such as gingival neoplasms such as gingival neoplasms, oral leukoplakia such as hairy leukoplakia, neoplasms of the lips, neoplasms of the palate, neoplasms of the salivary glands such as neoplasms of the parotids, neoplasms of the sublingual glands and neoplasms of the submandibular glands and neoplasms of the tongue, noma, oral fistula such as dental fistula, oroantral fistula and fistula of salivary glands, oral bleeding such as gingival bleeding, oral manifestations, fibrosis of oral submucosis, periapical periodontitis such as periapical abscess and periapical granuloma and radicular cyst), periodontal diseases (such as alveolar bone loss, furcation defects such as gingival hemorrhage, gingival hyperplasia, gingival hypertrophy, gingival neoplasms, gingival recession, gingivitis such as gingival crevicular fluid, gingival pouch, necrotizing ulcerative gingivitis, giant cell granuloma and pericoronitis, loss of periodontal adhesion, periodontal cyst, periodontitis such as periodontal abscess, periodontal pocket and periodontitis, exfoliation of teeth, loss of teeth, migration of teeth such as mesial movement of teeth and mobility of teeth), ranula, diseases of the salivary glands (such as Mikulicz disease, parotid diseases such as parotid neoplasms and parotitis such as parotitis, salivary gland stones such as salivary duct stones, salivary gland fistula, neoplasms of the salivary glands such as neoplasms of the parotids, neoplasms of the sublingual glands and neoplasms of the submandibular glands), sialadenitis, necrotizing sialometaplasia, sialorrhea, diseases of the submandibular glands such as neoplasms of the submandibular gland, xerostomia such as Sjogren's syndrome, stomatitis (such such as Stevens-Johnson Syndrome, aphthous stomatitis, stomatitis of the dentition and herpetic stomatitis), diseases of the tongue (such as glossiness, glossitis such as benign migratory glossitis), macroglossia, tongue diseases (such as fissured tongue, tongue pilose and neoplasms of the tongue, and oral tuberculosis), pharyngeal diseases (such as pharyngeal diseases such as nasopharyngeal diseases, such as nasopharyngeal neoplasms and nasopharyngitis), peritonsillar abscess, pharyngeal neoplasms such as hypopharyngeal neoplasms, nasopharyngeal neoplasms and oropharyngeal neoplasms including neopl tonsillar asthma, pharyngitis, retropharyngeal abscess, tonsillitis and velopharyngeal insufficiency), abnormalities of the stomatognathic system, diseases of the temporomandibular joint such as temporomandibular joint syndrome, tooth diseases (such as bruxism, dental deposits including dental calculus and plaque dental, dental fugue, dental pulp diseases that include autolysis of the dental pulp, calcification of the dental pulp, exposure of the dental pulp, gangrene of the dental pulp, secondary dentin and pulpitis, sensitivity of the dentin, dental focal infection, hypercementosis, malocclusion such as traumatic dental occlusion, diastema, class I angle malocclusion, class II angle malocclusion, class III angle malocclusion, speckle enamel, tooth abnormalities such as amelogenesis imperfecta such as dental enamel hypoplasia, anodonitis, dens in dente, dysplasia of dentin, dentinogenesis imperfecta, fused teeth, odontodysplasia and supernumerary teeth, abrasion of teeth, demineralization of teeth such as dental caries including tooth fissures and root caries, discoloration of teeth, erosion of teeth, ectopic eruption of the teeth, impacted teeth, damage to the teeth such as fracture of the teeth, such as the syndrome of the cracked tooth and dislocation of the teeth, loss of the teeth, resorption of the teeth such as resorption of the root and tooth not sprouted and tooth pain).

Eye diseases It has been shown that KGF-2 stimulates the proliferation of eye cells. Thus, KGF-2 polynucleotides, polypeptides, agonists and / or antagonists can be used to treat and / or detect ocular diseases. Examples of eye diseases that can be treated or detected include asthenopia, conjunctival diseases, conjunctival neoplasms, conjunctivitis (allergic, bacterial, inclusion, neonatal ophthalmia, trachoma, viral, acute hemorrhagic), keratoconjunctivitis, keratoconjunctivitis (infectious or dry) ), Reiter's disease, Pterygium, xerophthalmia, corneal diseases, corneal dystrophies (hereditary), Fuchs endothelial dystrophy, corneal edema, corneal neovascularization, corneal opacity, senile arch, keratitis, acanthamoeba keratitis, corneal ulcer, herpetic keratitis, Dendritic keratitis, keratoconjunctivitis, keratoconus, trachoma, eye abnormalities (aniridia, WAGR syndrome, Anophtamos, blepharophimosis, coloboma, ectopia lentis, hydroftalmos, microphthalmos, retinal dysplasia), hereditary eye diseases (albinism, ocular albinism, oculocutaneous albinism, choroideremia, hereditary corneal dystrophies, rotated atrophy, hereditary optic atrophy, retinal dysplasia, retinitis pigmentosa), ocular hemorrhage (choroidal hemorrhage, hyphema, retinal hemorrhage, vitreous body hemorrhage), ocular infections (corneal ulcer, bacterial eye infections, bacterial conjunctivitis, inclusion conjunctivitis, neonatal ophthalmia, trachoma, hordeolum, infectious keratoconjunctivitis, ocular tuberculosis), eye fungal infections, eye infections by parasites (acanthamoeba keratitis, ocular onchocerciasis, ocular toxoplasmosis), viral eye infections (viral conjunctivitis, acute hemorrhagic conjunctivitis, cytomegalovirus retinitis, Herpes Zoster Ophthalmicus, herpetic keratitis, dendritic keratitis), suppurative uveitis (endophthalmitis, panophthalmitis), eye damage (burns to the eyes, foreign bodies in the eyes, damage by penetrac ion to eyes), ocular manifestations, ocular neoplasms (conjunctival neoplasms, eyelid neoplasms, orbital neoplasms, uveal neoplasms (neoplasms of the choroid, neoplasms of the iris), eyelid diseases (blepharitis, blepharophimosis, blepharoptosis, blepharospasm, chalazion, ectropion , entropion, neoplasms of the eyelid, hordeolum), diseases of the lacrimal apparatus (dacryocystitis, dry eye syndromes, dry keratoconjunctivitis, Sjogren's syndrome, xerophthalmia, obstruction of the lacrimal duct), lens diseases (aphacia, postcatarata affacia, cataracts, subluxation of the crystalline, ectopia lentis, ocular hypertension, glaucoma (closure of the Angle, neovascular, open-angle, hydrophthalmos), ocular hypotension, ocular motility disorders (amblyopia, nystagmus, oculomotor nerve palsy, ophthalmoplegia) (Duane syndrome, Horner's syndrome, chronic progressive external ophthalmoplegia, Kearn syndrome), strabismus (esotropia), optic nerve diseases (optic atrophy, hereditary optic atrophy, optic disk drusen, optic neuritis, optic neuromyelitis, papilledema), orbital diseases (enophthalmos, exophthalmos, Graves' disease, cell granuloma) orbital plasma, orbital neoplasms), abnormal pupillary functions (anisocoria, tonic pupil, Adié syndrome, miosis, mydriasis, Horner's syndrome), refractive errors (aniseiconia, anisometropia, astigmatism, hyperopia, myopia, presbyopia), retinal diseases (angioid stripes, diabetic retinopathy, retinal artery occlusion, retinal degeneration, macular degeneration, cystoid macular edema, retinal drusen, retinitis pigmentosa, Kearns syndrome, detachment of the retina, retinal dysplasia, retinal hemorrhage, retinal neovascularization, retinal perforations, retinal vein occlusion, retinitis (chorioretinitis, cytomegalovirus retinitis, acute retinal necrosis syndrome), retinopathy of prematurity, proliferative vitreoretinopathy), sclerotic diseases (scleritis), uveal diseases (choroidal diseases, choroidal hemorrhage, choroidal neoplasms, choroideremia, choroiditis, chorioretinitis, pars lanitis, rotated atrophy), iris diseases (exfoliation syndrome, iridocyclitis, iris neoplasms), uveitis (panuveitis, sympathetic ophthalmia, anterior Behcet syndrome, iriocyclitis, iritis, posterior uveitis, choroiditis, chorioretinitis, pars planitis, intermediate uveitis, pars planitis, suppurative uveitis (endophthalmitis, panophthalmitis), uveomeningoencephalitic syndrome), vision disorders (amblyopia, blindness, hemianopsia, color vision defects, diplopia, night blindness, scotoma, subnormal vision), and proliferative vitreoretinopathy .

Diseases of the Skin and Connective Tissue KGF-2 stimulates the proliferation of skin and connective tissue cells. Therefore, polynucleotides, polypeptides, agonists and / or antagonists can be used to treat and / or detect diseases of the skin and / or connective tissue. Examples of connective tissue diseases include: cartilage diseases, such as polychondritis relaxant and Tietze syndrome; cellulitis; collagen diseases, such as Ehlers Danlos syndrome, keloids (including keloid acne), mucopolysaccharidosis I, necrobiotic disorders (including granuloma annulare, necrobiosis lipoidica), and osteogenesis imperfecta; loose skin; dermatomyositis; Dupytren's contracture; homocystinuria; lupus erythematosus (including cutaneous, discoid, panniculitis, systemic and nephritis; Marfan syndrome; connective tissue disease; mucinosis, including follicular, mucopolysaccharidosis (I, II, UU, IV, IV, and VII), myxedema, adult scleroderma; synovial cysts, connective tissue neoplasms, Noonan syndrome, osteopoikilia, panniculitis, including erythema induratum, nodular nonsuppurative and peritoneal, induration of the penis, pseudoxanthoma elasticum, rheumatic diseases, including arthritis (rheumatoid, juvenile rheumatoid, Caplan syndrome, Felty, rheumatoid nodule, ankylosing spondylitis, and Still's disease), hyperostosis, polymyalgia rheumatica, circumscribed scleroderma, and systemic scleroderma (CREST syndrome) Examples of skin diseases include angiolymphoid hyperplasia with eosinophilia, scar (including hypertrophic); cutaneous fistula, loose skin, dermatitis, including acrodermatitis, atopic dermatitis , contact dermatitis (allergic by contact, photoallergic, by toxicodendron), irritant dermatitis (phototoxic, diaper rash), occupational dermatitis; exfoliative dermatitis, dermatitits herpetiformis, seborrheic dermatitis, drug rash (such as toxic epidermal necrolysis, erythema nodosum, serum sickness), eczema, including dyshidrotic, intertrigo, neurodermatitis, and radiodermatitis; dermatomyositis; erythema, including chronicum migrans, indurated, infectious, multiform (Stevens-Johnson syndrome), and nodose (Sweet's syndrome); rash, including sudden; facial dermatosis, including acneiform eruptions (keloid, rosacea, vulgaris, and Favre-Racouchot syndrome), foot dermatitis, including tinea pedis; dermatosis of the hand; keratoacanthoma; keratosis, including calluses, cholesteatoma (including middle ear), ichthyosis (including congenital ichthyosiform erythroderma, epidermolytic hyperkeratosis, laminar ichthyosis, ichthyosis vulgaris, X-linked ichthyosis, and Sjogren-Larsson syndrome), blennorrhagic keratoderma, palmoplantar keratoderma, follicular keratosis, seborrheic keratosis, parakeratosis and porokeratosis; dermatosis of the legs, mastocytosis (urticaria pigmentosa), necrobiotic disorders (granuloma annulare and necrobiosis lipoidica), disorders of photosensitivity (photoallergic or phototoxic dermatitis, hidroa vacuniforme, solar erythema, and xeroderma pigmentosum); disorders of pigmentation, including argyria, hyperpigmentation, melanosis, acontosis nigricans, lentigo, Peutz-Jeghers syndrome, hypopigmentation, albinism, pibaldism, vitiligo, incontinentia pigmenti, urticaria pigmentosa, and xeroderma pigmentosum. Additional examples of skin disorders include prurigo; pruritis (including the anus and vulva); Hypoderma, including ecthyma and pyoderma gangrenosum; dermatosis of the scalp; scleroderma of the adult; scleroderma of the neonate; diseases of appendages of the skin, including hair diseases (alopecia, folliculitis, hirsutism, hyperticosis, Kinky hair syndrome), diseases of the nails (nail-patella syndrome, buried or malformed nails, onychomycosis, paronychia), the sebaceous glands (rhinophyma, neoplasms), diseases of the sweat glands (hydradentitis, hyperhidrosis, hypohidrosis, miliara, Fox-Fordyce disease, neoplasms); genetic diseases of the skin, including alpinism, lax skin, benign familial pemphigus, porphyria, acrodermatitis, ectodermal dysplasia, Ellis-Van Creveld syndrome, focal dermal hypoplasia, Ehlers-Danlos syndrome, epidermolysis bulosa, ichthyosis; infectious diseases of the skin, including dermatomycosis, blastomycosis, candidiasis, chromoblastomycosis, maduromycosis, paracoccidioidomycosis, sporotrichosis, tinea; bacterial diseases of the skin, such as cervicofacial actinomycosis, bacillary angiomatosis, ecthyma, erysipelas, chronic erythema migrans, erythrasma, inguinal granuloma, hidradenitis suppurativa, maduromycosis, paronychia, pinto, rinoscleroma, staphylococcal infections of the skin (furuncolosis, carbuncle, impetigo, scalded skin syndrome), cutaneous syphilis, cutaneous tuberculosis, yaws; parasitic diseases of the skin, including larva migrans, Leishmaniasis, pediculosis, and scabies; viral diseases of the skin, including infectious erythema, sudden rash, herpes simplex, contagious moolusum, and warts. Additional examples of skin diseases include metabolic diseases of the skin, such as painful adiposis, lipodystrophy, lipoid necrobiosis, porphyria, juvenile xanthogranuloma, xanthomatosis (Wolman's disease); papulosquamous diseases of the skin, including lichenoid rash, parpasoriasis, pityriasis, and psoriasis; vascular diseases of the skin, such as Behcet's syndrome, mucocutaneous lymph node syndrome, polyarteritis nodosa, pyoderma gangrenosum, Takayasu's arteritis; vascular diseases of the skin; including acantholysis, bladders, gestational herpes, hycuba vacciniforme, pemphigoid, pemphigus; skin neoplasms; skin ulcers, such as decubitus ulcers, leg ulcers, foot ulcers, diabetic foot ulcers, varicose ulcers and pyoderma gangrenosum.

Diseases and urogenital disorders KGF-2 can stimulate the proliferation of cells of the uro-genital tract. Therefore, KGF-2 polynucleotides, polypeptides, agonists and / or antagonists can be used to treat and / or detect diseases and / or male and female genital disorders, and complications of pregnancy. Examples of urological and male genital diseases that can be treated or detected include epididymitis, male genital neoplasms, penile neoplasms, prosthetic neoplasms, testicular neoplasms, hematocele, genital herpes, hydrocele, male infertility, oligospermia, penile diseases including balanitis, hypospadias , penile induration, neoplasms of the penis, phimosis, paraphimosis, priapism, prosthetic diseases such as hypertrophy, neoplasms, and prostatitis, sexual disorders such as impotence and vasculogenic impotence, spermatic duct torsion, spermatocele, testicular diseases including cryptorchidism, orchitis, and Testicular neoplasms, male genital tuberculosis, varicocele, urogenital tuberculosis (male genital, renal), urogenital abnormalities, exstrophy of the bladder, cryptorchidism, epispadias, hypospadias, polycystic kidney (autosomal dominant and autosomal recessive), nephritis disorders, disorders of sexual differentiation, gonadal dysgenesis, mixed gonadal dysgenesis, hermaphroditism, pseudohermaphroditism, Kallman syndrome, Klinefelter syndrome, testicular feminization, WAGR syndrome, urogenital neoplasms, male genital neoplasms (of the penis, prostate and testes), urological neoplasms (from the bladder, kidney, urethra, ureter), bladder diseases (stones, exstrophy, fistula, vesicovaginal fistula, neck obstruction, neoplasms, neurogenic cystitis, vesico-ureteral reflux), hematuria, hemoglobinuria, nephropathy associated with AIDS, anuria, oliguria, diabetic nephropathies, Fanconi syndrome, hepatorenal syndrome, hydronephrosis, primary hyperoxaluria, renal hypertension, renovascular hypertension, kidney stones, necrosis of the renal cortex, cystic kidney, polycystic kidney, (autosomal dominant, autosomal recessive), spongy kidney, renal failure (nephrogenic insipid diabetes, acute renal failure, renal papillary necrosis), nephritis (glomerulonephritis (IGA, membronoproliferative, membranous, focal, Goodpasture syndrome, Lupus nephritis), hereditary nephritis, interstitial nephritis, Balkan nephropathy, pyelonephritis, xanthogranulomatous pyelonephritis , nephrocalcinosis, nephrosclerosis, nephrosis, lipoid nephrosis, nephrotic syndrome, perinephritis), pyelitis (pyelocystitis, pyelonephritis, xanthogranulomatous pyelonephritis), renal artery obstruction, renal osteodystrophy, fetal errors in renal tubular transport, renal tubular acidosis, renal aminoaciduria, cyst inuria, Hartnup's disease, Cystinosis, Franconi's syndrome, renal glucosuria, familial hypophosphatemia, oculocerebrorenal syndrome, pseudohypoaldosteronism, renal tuberculosis, uremia, hemolytic-uremic syndrome, Wegener's granulomastosis, Zellweger syndrome, proteinuria, albuminuria, ureteral diseases including ureteral stones ureteral neoplasms, ureteral obstruction, ureterocele, urethral diseases including epispadias, urethral neoplasms, urethral obstruction, urethral constriction, urethritis (Reiter's disease), urinary stones (bladder, kidney, ureteral), urinary fistula (bladder fistula (vesicovaginal fistula) )); urinary tract infections (bacteruria, pyuria, schistosomiasis haematobia), and urination disorders (enuresis, polyuria, urinary incontinence, stress-related urinary incontinence, urinary retention). Examples of female genital diseases and pregnancy complications that can be treated or detected include adnexal diseases, including adnexitis (oophoritis, parametritis, salpingitis), fallopian tube diseases such as fallopian tube neoplasms and salpingitis, diseases of the ovaries (anovulation, oophoritis, ovarian cysts, polycystic ovarian syndrome, premature ovarian failure, ovarian hyperstimulation syndrome, ovarian neoplasms, Meig's syndrome), Parovarian cyst, endometriosis, female genital neoplasms, ovarian neoplasms, uterine neoplasms, neoplasms of the cervix, neoplasms endometrial, vaginal neoplasms, vulvar neoplasms, ginatresia, hematocolpos, hematometra, genital herpes, female infertility, menstruation disorders including amenorrhea, dysmenorrhea, menorrhagia, oligomenorrhea and premenstrual syndrome, pseudopregnancy, sexual disorders such as dipareunia and f stiffness, urogenital tuberculosis, female genital tuberculosis, urogenital diseases including bladder extrophy, epispadias, polycystic kidney (autosomal dominant and autosomal recessive), hereditary nephritis, disorders of sexual differentiation including gonad dysgenesis (46 XY, Mixed), Syndrome de Turner, hermaphroditism, pseudohermaphroditism, Kallmann syndrome, WAGR syndrome, urogenital neoplasms, urological neoplasms (bladder, urethra, ureter), uterine diseases including diseases of the cervix (cervicitis, erosion of the cervix, hypertrophy of the cervix, incompetence of the cervix, neoplasms of the cervix), endometrial hyperplasia, endometritis, uterine hemorrhage, menorrhagia, metrorrhagia, uterine neoplasms including neoplasms of the cervix and endometrial neoplasms, uterine prolapse, uterine rupture, uterine perforation, vaginal diseases including vulvovaginal candidiasis, disparenunia, hematocolpos, leucorrhoea, fistula vaginal, rectovaginal fistula, vesicovaginal fistula, vaginal neoplasms, vaginitis (vaginal trichomonias, bacterial vaginosis, vulvovaginitis), complications of pregnancy including habitual abortion, incompetence of the cervix, incomplete abortion, failed abortion, septic abortion, threat of abortion, veterinary abortion, death fetal, resorption of the embryo, fetal resorption, fetal diseases (chorioamnionitis, fetal erythroblastosis, hydrops fetalis, fetal alcohol syndrome, fetal anoxia, fetal distress, fetal growth retardation, fetal macrosomia, and meconium aspiration, herpes gestationis, labor complications including premature detachment of placenta, dystocia, uterine inertia, premature rupture of fetal membranes, chorioamnionitis, placenta accreta, placenta previa, postpartum haemorrhage, uterine rupture, preterm delivery, oligohydramnios, maternal phenylketonurine, placental diseases (premature placental abruption , corioamnioni tis, placenta accreta, retained placenta, placental insufficiency), polyhydramnios, cardiovascular complications of pregnancy, embolism of the amniotic fluid, hematological complications of pregnancy, infectious complications of pregnancy (septic abortion, parasitic complications of pregnancy, puerperal infection), neoplastic complications of pregnancy (trophoblastic neoplasms, choriocarcinoma, hydatidiform mole, sive hydatidiform mole, placental site trophoblastic tumor), ectopic pregnancy, abdominal pregnancy, tubal pregnancy, pregnancy in diabetes, gestational diabetes, fetal macrosomia, pregnancy product, pregnancy toxemias (eclampsia, HELLP syndrome, pre-eclampsia, EPH Gestsis, hyperemesis gravidarum), puerperal disorders, lactation disorders such as Chiari-Frommel syndrome, galactorrhea, and mastitis, postpartum hemorrhage, and puerperal infection.

Infertility As stated above, the polynucleotides, polypeptides, variants, antibodies, agonists and / or antagonists of KGF-2 can be used to treat male or female infertility. Thus, in one embodiment of the invention, there is provided a method using the KGF-2 polynucleotides, polypeptides, variants, antibodies, agonists and / or antagonists to treat and / or prevent male infertility. In yet another embodiment, a method is provided that utilizes KGF-2 polynucleotides, polypeptides, variants, antibodies, agonists and / or antagonists to treat and / or prevent female infertility. Preferred KGF-2 polypeptides used to treat infertility include KGF-2Δ33, full-length and mature KGF-2, KGF-2Δ28, and polypeptides comprising amino acids 77 to 208, 80 to 208, and 93 to 208 of KGF-2; as well as any mutant of KGF-2 described herein. Also preferred are polynucleotides encoding these polypeptides. For the treatment or prevention of infertility, preferred modes of administration of KGF-2 include orally, rectally, parenterally, intracisternally, intradermally, intravaginally, intraperitoneally, topically (by powders, ointments, gels, creams, drops or transdermal patch), orally, or as an oral or nasal spray. Other modes of administration are described herein. Preferably, the KGF-2 polynucleotide, polypeptide, variant, antibody, agonist and / or antagonist is administered with a pharmaceutical carrier as part of a pharmaceutical composition. Suitable carriers are described herein. The polynucleotides, polypeptides, variants, antibodies, agonists and / or antagonists of KGF-2 can be used to treat infertility treated by any factor, including environmental causes such as coffee, MSG, plastics, Nutrasweet, alcohol, food additives, chemical products, cigars, pesticides, vehicle leaks, and pollution; age; congenital infertility; low sperm count; infectious diseases, such as mumps, tuberculosis, influenza, smallpox, cytomegalovirus (CMV) infection, chlamydia, mycoplasma, gonorrhea, syphilis and other sexually transmitted diseases; endocrine diseases such as diabetes; neurological diseases, such as paraplegia; high fevers; endometriosis; toxins such as lead in paints, varnishes and automotive manufacturing agents, ethylene oxide, substances found in the chemical and materials industries, such as papermaking; chemotherapy; low weight or excessive weight loss; obesity or extreme weight gain; stress; ovulatory disorders; hormonal imbalances, Cushings syndrome; blockage of the fallopian tubes; pelvic infection; surgical adhesions; intrauterine devices (IUD); cervical disorders, such as anatomical problems, cervical infections, and mucus quality; cervical stenosis; uterine disorders, such as intrauterine adhesions, trauma and / or infection of the uterine lining, Asherman's Syndrome, uterine fibroids, ovarian scar tissue; ovarian cysts, including chocolate cyst; asthenospermia; maturation arrest; Hypospermia; Sertoli cell syndrome; gonadotropin deficiency, including that arising from expanded pituitary tumors that compromise the secretion of LH and FSH, surgical damage, or external trauma to the skull with damage to the portal blood supply; anabolic steroids; nicotine; illicit drugs such as marijuana, heroin and cocaine; alkaline agents, procarbazine, some halogenated hydrocarbons used in pesticides, and frequent exposure to large quantities of ethanol; pelvic inflammatory disease (PID); epididymitis; exposure to toxic or hazardous substances, such as lead, cadmium, mercury, ethylene oxide, vinyl chloride, radioactivity, and X-rays; prescription drugs for ulcers or psoriasis; exposure to DES in utero; exposure of male genitalia to high temperatures - hot baths, whirlpool tubs, steam rooms; hernia repair, testicles not descended; vitamin deficiency; previous abortions; and cyclophosphamide. The polynucleotides, polypeptides, variants, antibodies, agonists and / or antagonists of KGF-2 can be used to treat or prevent primary or secondary infertility. KGF-2 can also be used to treat temporary or permanent infertility.

The polynucleotides, polypeptides, variants, antibodies, agonists and / or antagonists of KGF-2 can be administered together with other fertility-promoting substances, such as clomiphene citrate (clomid, serofen), progesterone, and / or 17β-estradiol. KGF-2 can be used to treat infertility in women during natural conception or during assisted reproduction. Assisted reproduction techniques include in vitro fertilization (IVF), embryo transfer (ET), gamete intrafallopian transfer (GIFT), zygote intrafallopian transfer (ZIFT), IVF with donor eggs, donor sperm, and embryos of donor, and micromanipulation of eggs and embryos. In IVF-ET, an oocyte is surgically removed, fertilized in vi tro, and placed in the uterus or in the fallopian tube of the same woman. In the donation of oocytes, the oocyte is retrieved from a donor and after IVF it is transferred to an infertile recipient as in ET. This procedure requires synchronization between the donor and the recipient, which is generally achieved by administering steroid hormones to the recipient. In regular IVF-ET, the treatments given to induce the development of multiple follicles frequently lead to insufficient luteal function. Therefore, implantation may not take place without supplemental treatment with molecules such as KGF-2. A preferred method of administering KGF-2 for the treatment or prevention of infertility in a woman is through a sustained release system via a vaginal ring, as described in U.S. Patent No. 5,869,081, the description of which is incorporated by reference herein. Polysiloxane carriers have been used for the distribution of progesterone as a contraceptive for lactating women (Croxatto et al., 1991, in "Female Contraception and Male Fertility Regulation, Advances in Gynecological and Obstetric Research Series", Reinnebaum et al., Eds. .) and for the administration of estradiol in postmenopausal women (Stumpf et al., (1982), J. Clin Endocrinol, Metab., 58: 208). Simon et al. (1986), Fertility and Sterility, 46: 619 describe vaginal rings of polysiloxane impregnated with 17β-estradiol and / or with progesterone and cylinders for endometrial preparation in functionally agonal women. The ring and cylinder system was used to reach serum levels of 17β-estradiol and progesterone within the normal range for a complete menstrual cycle. U.S. Patent No. 4,816,257 describes the use of polysiloxane rings containing 17β-estradiol or 17β-estradiol and progesterone to mimic the levels of normal steroid hormones in a functionally agonadal female. The present invention provides a method for administering KGF-2 for the establishment and maintenance of pregnancy. The method of the invention comprises the insertion of a carrier containing KGF-2 into the vagina of the woman and maintaining the carrier intravaginally for approximately 1-28 days. In a preferred embodiment, the carrier is a polysiloxane ring having an in vitro release rate of about 1 μg / day to 1000 mg / day, although this amount is subject to therapeutic discretion. In addition, the method can be used to treat or prevent infertility in a woman suffering from assisted reproduction. The method comprises inserting a carrier containing KGF-2 into the vagina of a woman and maintaining the carrier intravaginally until about the seventh to twelfth week of pregnancy. In a preferred embodiment, the carrier is a polysiloxane ring having an in vitro release rate of about 1 μg / day to 1000 mg / day of KGF-2. The present invention relates to methods for the administration of KGF-2 to women with functional ovaries and functionally agonal women. Women with functional ovaries who are infertile or can not conceive because their partner is infertile can become pregnant through assisted reproduction techniques. However, the hormonal treatments used to induce the development of multiple follicles cause insufficient production of progesterone by the corpus luteum. In this way, the start and maintenance of the implantation is impaired. Functionally agonational women are infertile as a result of undeveloped or inadequately developed ovaries, surgical removal of the ovaries, or other failure or ovarian dysfunction. Assisted reproduction techniques such as OD, IVF and ET allow functionally agonadal women to become pregnant. However, hormonal supplementation is necessary in assisted reproduction techniques in order to prepare the endometrium for the establishment and continuation of pregnancy. Thus, in accordance with the present invention, KGF-2 can be used to treat or prevent infertility through, among others, the promotion of the embryo implant. The present invention provides a method of administration of KGF-2 for the establishment and maintenance of pregnancy by assisted reproduction techniques in a normogonadal woman and a functionally agonadal woman. The method comprises inserting a carrier containing KGF-2 into the vagina of a normogonadal woman or a functionally agonadal woman, and maintaining the carrier intravaginally for at least about twenty-eight days. The present invention also provides a method of hormone replacement therapy for a woman suffering assisted reproduction. The method comprises inserting a carrier containing KGF-2 into the vagina of a woman undergoing assisted reproduction, and maintaining the carrier intravaginally until about the seventh to the twelfth week of pregnancy. The physiologically acceptable KGF-2 containing carriers useful in the method of the present invention are preferably ring-shaped solid carriers made of silicone rubber, also referred to herein as polysiloxane, or other suitable material. The administration of steroid hormones by vaginal rings of polysiloxane is known in the art. The rate of passage of KGF-2 from a polysiloxane ring is dependent on factors that include the surface area of the ring. Accordingly, the amount of KGF-2 in the ring is conveniently described in terms of the rate of in vitro release of KGF-2 from the ring. In vitro release rates are routinely used in the art to characterize the hormone-containing polysiloxane rings. Polysiloxane rings containing KGF-2 having release rates of from about 0.001 to about 1000 mg of KGF-2 per day are contemplated for use in the present method. In a preferred embodiment, the polysiloxane rings have an in vitro release rate of from about 0.01 to about 100 mg of KGF-2 per day. In a more preferred embodiment, the polysiloxane rings have an in vitro release rate of about 0.1 to about 10 mg of KGF-2 per day. The polysiloxane carriers containing KGF-2 are administered by insertion into the vagina. The rings are inserted inside the vagina and placed around the cervix. The ring can be inserted and removed by the female subject in a manner similar to that of the commonly used diaphragm, thus providing yet another advantage of the present invention. The carrier containing KGF-2 can be administered approximately two to seven days, and preferably three days, before the embryo transfer, and can be supplemented by the administration of another hormone, for example the oral administration of 17β-estradiol or progesterone . In a preferred embodiment, the carrier is a ring and is inserted three days before the embryo transfer. The carrier is removed and replaced by another carrier after approximately twenty-eight days. If pregnancy occurs, the carrier allows enough KGF-2 for pregnancy maintenance until the luteal-placental displacement, at which time the administration can be discontinued. In a preferred embodiment, the ring is maintained continuously in the vagina, and the administration is discontinued at about twelve weeks of pregnancy.

Injuries, Occupational Diseases KGF-2 has been shown to stimulate the proliferation of a variety of tissues. Therefore, KGF-2 polynucleotides, polypeptides, agonists and / or antagonists can be used to treat occupational injuries or diseases. Examples of injuries, occupational diseases and poisoning that can be treated or detected include occupational diseases such as diseases of agricultural workers that include the farmer's lung and silo filler disease, the lung of the bird fan, Occupational dermatitis, high pressure nervous syndrome, inert gas narcosis, laboratory infection, pneumoconiosis such as asbestosis, berylliosis, byssinosis, Caplan syndrome, siderosis, silicosis such as anthracosilicosis and silicotuberculosis, poisoning such as alcohol intoxication including alcoholism such as alcoholic cardiomyopathy, alcoholic fetal syndrome, fatty alcoholic liver, alcoholic hepatitis, alcoholic liver cirrhosis, alcoholic psychosis such as alcoholic amnesic disorder, delirium due to alcohol withdrawal, argyria, bites and stings such as arachnoidism, bites and stings insect, snake bites, tick toxicosis such as tick paralysis, cadmium poisoning, carbon tetrachloride poisoning, drug toxicity such as drug-induced akathisia, drug rash such as toxic epidermal necrolysis, erythema nodosum and serum sickness, drug-induced dyskinesia and neuroleptic malignant syndrome, ergotism, fluoride poisoning, food poisoning such as botulism, favism, fungal poisoning, food poisoning with salmonella and food poisoning with staphylococci, gas poisoning such as carbon monoxide poisoning, narcotic inert gas, toxic hepatitis, lead poisoning, mercury poisoning, mycotoxicosis such as ergotism and fungal poisoning, overdose, plant poisoning such as ergotism, favism, lathyrism, and milk disease , psychosis ind ucidas by substances, wounds and injuries such as abdominal injuries that include traumatic diaphragmatic hernia, splenic rupture such as splenosis, rupture of the stomach, traumatic amputation, injuries of the arms such as injuries to the forearms including fractures of the radius and fractures of the ulna, humerus fractures, shoulder dislocation, shoulder fractures, tennis elbow and wrist injuries, asphyxia, athletic injuries, barotrauma such as burst injuries and decompression sickness, birth injuries such as obstetric paralysis, bites and stings such as human bites, burns such as chemical burns, electric burns, inhalation burns such as smoke inhalation injury, eye burns and sunburn, bruises, dislocations such as shoulder and hip dislocations, drowning such as drowning , electrical burns and injury it is by rays, esophageal perforation, extravasation of diagnostic and therapeutic materials, foreign bodies such as bezoar, foreign bodies in the eyes, migration of foreign bodies, reaction to foreign bodies such as granuloma by foreign bodies, fractures such as femoral fractures such as hip fractures including fractures of the femoral neck, closed fractures, shredded fractures, mal-linked fractures, open fractures, spontaneous fractures, stress fractures, unbound fractures such as pseudoarthrosis, humeral fractures, radius fractures such as Colles fractures, knee fractures, shoulder fractures, skull fractures such as jaw fractures such as jaw and jaw fractures, orbital fractures and zygomatic fractures, spinal fractures, tibial fractures, cubital fractures such as Monteggia fractures, frostbite such as chilblains, hand injuries such as injuries to the fingers, head injuries such as injuries to the brain which include cerebral concussion, cerebrospinal otorrhea, cerebrospinal rhinorrhea, closed head injuries, maxillofacial injuries such as facial injuries that include eye injuries such eye burns, foreign bodies in the eyes and eye penetration injuries, jaw fractures such as mandibular and maxillary fractures, mandibular injuries such as mandibular fractures, and zygomatic fractures, maxillary fractures, pneumocephalus, skull fractures such as fractures in the jaws, including mandibular and maxillary fractures, orbital fractures and zygomatic fractures, exhalation of the head such as apoplex by sun exposure, leg injuries such as ankle injuries, femoral fractures such as hip fractures that include femoral neck fractures, foot injuries, hip dislocation, knee injuries, and fractures of the hip. lukewarm, motion sickness, such as spacious movement disease, multiple trauma, radiation injuries such as radiation-induced abnormalities, radiation-induced leukemia, radiation-induced neoplasms, osteoradionecrosis, experimental radiation injury, radiation pneumonitis and radiodermatitis, retropneumoperitoneum , rupture such as aortic rupture, splenic rupture such as splenosis, rupture of the stomach and uterine rupture, such as uterine perforation, self-mutilation, traumatic stroke such as crush syndrome, soft tissue injuries, spinal cord injuries such as compression of the spinal cord, spinal injuries ta such as spinal injuries and whiplash injuries, dislocations and sprains such as repeated sprain injury, tendon injuries, thoracic injuries such as shaken chest, cardiac injuries and rib fractures, injuries to teeth such as tooth fractures that include cracked tooth, dislocation of teeth, perforation of the tympanic membrane, infection by wounds, non-penetrating wounds such as concussion, and closed head injuries, and penetrating wounds such as penetration damage to the eyes, injuries by firearm and injuries from stabbing injuries such as needle-bite injuries.

Hemic and Lymphatic Diseases The KGF-2 polynucleotides, polypeptides, agonists and / or antagonists can be used to treat and / or detect hemic and / or lymphatic diseases. Examples of Hemic and Lymphatic Diseases that can be treated or detected include aplastic anemia (such as Fanconia anemia), hemolytic anemia (such as autoimmune hemolytic anemia and congenital hemolytic anemia, including congenital dyserythropoietic anemia, congenital non-spherocytic hemolytic anemia, anemia due to false cells, such as SC disease of hemoglobin and trait of counterfeit cells, hereditary elliptocytosis and deficiency of glucose phosphate dehydrogenase, such as favism, hemoglobin C disease, hereditary spherocytosis, thalassemia, such as alpha-thalassemia including fetal hydrops and beta-thalassemia, favism, hemoglobinuria, such as paroxysmal hemoglobinuria, and haemolytic-uremic syndrome), hypochromic anemia (such as iron deficiency anemia), macrocytic anemia (such as megaloblastic anemia, including pernicious anemia), myelophthic anemia, anemia neonatal (such as transfusion s etofatal and fetomaternal transfusion), refractory anemia (such as refractory anemia with excess blasts), sideroblastic anemia, pure red cell aplasia, fetal erythroblastosis (such as fetal hydrops and kernicterus), Rh isoimmunization, abetalipoproteinemia, agammaglobulinemia, disgammaglobulinemia (such as IgA deficiency and IgG deficiency), hypergammaglobulinemia (such as benign monoclonal gammopathies), hyperproteinemia, paraproteinemias (such as amyloidosis, including amyloid neuropathies and cerebral amyloid angiopathy, cryoglobulinemia, heavy chain disease, such as immunoproliferative small bowel disease, Multiple Myeloma, POEMS Syndrome, Waldenstrom Macroglobulinemia), Protein S Deficiency. Additional examples of hemic and lymphatic diseases that can be treated or detected include diseases of the bone marrow such as aplastic anemia, myelodysplastic syndromes (including or refractory anemia such as refractory anemia with excess blasts, sideroblastic anemia, paroxysmal hemoglobinuria, and myeloid leukemia), myeloproliferative disorders (including myelophthic anemia, acute erythroblastic leukemia, leukoid reaction, myelofibrosis, myeloid metaplasia, polycythemia vera, hemorrhagic thrombocytotemia, and thrombocytosis ), intravascular aggregation of erythrocytes, hemoglobinopathies such as falsiform cell anemia (including SC hemoglobin disease and sickle cell trait), hemoglobin SC disease, thalassemia (including alpha-thalassemia such as fetal dropsy and beta-thalassemia), hemorrhagic diathesis such as abrinogenemia, Christmas disease, disseminated intravascular coagulation, factor VII deficiency, factor XI deficiency, factor XII deficiency, factor XIII deficiency, haemophilia, hypoprothrombinemia (including factor V deficiency and factor X deficiency), Sch wartzman, Bernard-Soulier syndrome, hemolytic-uremic syndrome, platelet storage deficiency, thrombasthenia, hemorrhagic thrombocytopenia (including thrombocytopenic purpura such as idiopathic thrombocytopenic purpura, thrombotic thrombocytopenic purpura, and Wiskott-Aldrich syndrome), hyperglobulinemic purpura, purpura Schoenlich-Henoch, thrombocytopenic purpura (idiopathic thrombocytopenic purpura), thrombotic thrombocytopenic purpura, Wiskott-Aldrich syndrome, hereditary hemorrhagic telangiectasia, vitamin K deficiency (including neonatal hemorrhagic disease), and Willebrand's disease, leukocyte disorders such as eosinophilia (including angiolymphoid hyperplasia with eosinophilia, eosinophilia-myalgia syndrome, eosinophilic granuloma, and hypereosinophilic syndrome such as pulmonary eosinophilia), infectious mononucleosis, leukocytosis (including leucamoid reaction and lymphocytosis), leukopenia (including agranulocytosis such as neutropenia and lymphopenia such as positive idiopathic T-lymphopenia to CD4), Pelger-Huet abnormality, phagocytic bactericidal dysfunction (including Chediak-Higashi syndrome, Granulomatosis disease Chronic, Job syndrome), methemoglobinemia, pancytopenia, polycythemia, haematological preleukemia, and sulfohemoglobinemia. Additional examples of hemic and lymphatic diseases that can be treated or detected include lymphatic diseases such as lymphadenitis (including cat spider disease and mesenteric lymphadenitis), lymphangiectasis, lymphangitis, lymphedema (including elephantiasis and filarial elephantiasis), lymphocele, lymphoproliferative disorders ( including agammaglobulinemia, amyloidosis such as amyloid neuropathies and cerebral amyloid angiopathy, giant lymph node hyperplasia, heavy chain disease such as immunoproliferative small bowel disease, immunoblastic lymphadenopathy, infectious mononucleosis, hairy cell leukemia, lymphocytic leukemia, myeloid leukemia ( including acute non-lymphocytic leukemia and acute myelocytic leukemia), lymphangiomyoma (including lymphangiomyomatosis), and lymphoma (including Hodgkin's disease, non-Hodgkin's lymphoma, such as B-cell lymphoma including Burk's lymphoma) itt, AIDS-related lymphoma, mucosal-associated lymphoid tissue lymphoma, small-cell lymphoma, diffuse lymphoma including diffuse large-cell lymphoma, large-cell immunoblastic lymphoma, lymphoblastic lymphoma, diffuse mixed-cell lymphoma, small lymphocytic lymphoma, and Uncleaved small cell lymphoma, follicular lymphoma including large cell follicular lymphoma, mixed cell follicular lymphoma, and small excised follicular lymphoma, high grade lymphoma including large cell immunoblastic lymphoma, lymphoblastic lymphoma, and small cell lymphoma no excised such as Burkitt's lymphoma, intermediate grade lymphoma including diffuse large cell lymphoma, large cell follicular lymphoma, diffuse mixed cell lymphoma, and diffuse small cell excised lymphoma, large cell lymphoma including diffuse large cell lymphoma, follicular lymphoma of g cells radicles, large-cell immunoblastic lymphoma, Ki-1 large cell lymphoma, and large-cell immunoblastic lymphoma, low-grade lymphoma including mixed cell follicular lymphoma, mucosal-associated lymphoid tissue, small cell follicle excised lymphoma, and lymphoma small lymphocytic, mixed cell lymphoma including diffuse mixed cell lymphoma and mixed cell follicular lymphoma, small cell lymphoma, including diffuse small cell excised lymphoma, small excised follicular cell lymphoma, small lymphocytic lymphoma, and small cell lymphoma no excised, T-cell lymphoma including lymphoblastic lymphoma, cutaneous T-cell lymphoma, such as Ki-1 large cell lymphoma, mycosis fungoides, and Sezary syndrome, and peripheral T-cell lymphoma, undifferentiated lymphoma including diffuse cell lymphoma large, and small undissolved cell lymphoma such such as Burkitt's lymphoma, lymphomatoid granulomatosis), Marek's disease, sarcoidosis (including pulmonary sarcoidosis and uveoparotic fever), tumor lysis syndrome, mucocutaneous lymph node syndrome, reticuloendotheliosis (including Gaucher's disease, histiocytosis such as malignant histiocytic disorders including malignant histiocytosis, acute monocytic leukemia, large cell lymphoma such as Ki-1 large cell lymphoma, histiocytosis of Langerhans cells such as eosinophilic granuloma, Hand-Scheller-Christian syndrome, and Letterer-Siwe disease, cell histiocytosis no Langerhans such as breast histiocytosis, Niemann-Pick disease, navy blue histiocyte syndrome, and juvenile xanthogranuloma, mast cell sarcoma), splenic diseases (including hypersplenism, myeloid metaplasia, splenic infarction, splenic neoplasms, splenic rupture such as splenosis, splenomegaly, and splenic tuberculosis) ), thymus hyperplasia, thymus neoplasms, tuberculosis of lymph nodes such as King's disease.

Diseases and Neonatal Abnormalities The KGF-2 polynucleotides, polypeptides, agonists and antagonists can be used to treat, prevent, and / or detect diseases and / or neonatal abnormalities. Examples of diseases and neonatal abnormalities that can be treated or detected include drug-induced abnormalities, multiple abnormalities including Alagille syndrome, Angelman syndrome, basal cell nevus syndrome, Beckwith-Widemann syndrome, Bloom syndrome, Bonnevie syndrome. -Ulrich, Cockayne syndrome, Cri-du-Chat syndrome, De Lange syndrome, Down syndrome, ectodermal dysplasia such as Ellis-Van Creveld syndrome and focal dermal hypoplasia, Gardner syndrome, holoprosencephaly, pigmentosa incontinence, syndrome of Laurence-Moon Biedl, Marfan syndrome, Nail-Patella syndrome, oculocerebrandrenal syndrome, orofaciodigital syndromes, Prader-Willi syndrome, Proteus syndrome, Prune Belly syndrome, congenital rubella syndrome, Rubenstein-Taybi syndrome, syndrome of the short rib-polydactyly, Waardenburg syndrome, Wolfram syndrome, Zelweger syndrome, Radiation-induced abnormalities, chromosomal abnormalities including Angelman syndrome, Beckwith-Wiedemann syndrome, Cri-du-Chat syndrome, Down syndrome, holoprosencephaly, Prader-Willi syndrome, abnormalities in sex chromosomes such as Bonnevie syndrome. Ulrich, ectodermal dysplasia including focal dermal hypoplasia, fragile X syndrome, gonadal dysgenesis 46, XY, mixed gonadal dysgenesis, Kallman syndrome, Klinefelter syndrome, oculocerebronial syndrome, orofaciodigital syndromes, Turner syndrome, and XYY karyotype, and abnormalities in the digestive system.

Respiratory diseases KGF-2 has also been shown to stimulate the proliferation of cells of the respiratory tract. Thus, KGF-2 polynucleotides, polypeptides, agonists and / or antagonists can be used to treat and / or detect respiratory diseases. Examples of diseases of the respiratory tract that can be treated or detected include bronchial diseases, such as asthma (including asthma induced by exercise and asthmatic state), bronchial fistula, bronchial hyperreactivity, bronchial neoplasms, bronchial spasm, bronchiectasis, bronchitis (including bronchiolitis, bronchiolitis obliterans, organizational pneumonia, viral bronchiolitis, bronchogenic cyst, bronchopneumonia, tracheobronchomegaly), ciliary motility disorders such as Kartagener syndrome, laryngeal diseases (such as laryngeal granuloma, laryngeal edema, laryngeal neoplasms, laryngeal pericondritis, laryngism, laryngitis such as croup, laryngostenosis, laryngeal tuberculosis, vocal cord paralysis, vocal cord disorders, voice disorders such as aphonia, and hoarseness), diseases of the lungs, such as atelectasis that include intermediate lobe syndrome, bronchial dysplasia opulmonary, congenital cystic adenomatoid malformation of the lung, cystic fibrosis, pulmonary plasma cell granuloma, hemoptysis, pulmonary abscesses, fungal diseases of the lungs such as allergic bronchopulmonary aspergillosis and Pneumocystis carinii pneumonia, interstitial diseases of the lungs such as extrinsic allergic alveolitis such as lung of the bird fan, farmer's lung, Goodpasture syndrome, histiocytosis of langerhans cells, pneumoconiosis such as asbestosis, berylliosis, byssinosis, Caplan syndrome, siderosis, silicosis such as anthracosilicosis and silicotuberculosis, pulmonary fibrosis, pneumonitis by radiation, pulmonary sarcoidosis, Wegener's granulomatosis), obstructive pulmonary diseases, viral bronchiolitis, pulmonary emphysema, parasitic diseases of the lungs such as pulmonary echinococcosis, pulmonary neoplasms such as bronchogenic carcinoma, lung coin injury and Pancoast syndrome, meconium aspiration, pneumonia (such as bronchopneumonia, pleuropneumonia, aspiration pneumonia such as lipid pneumonia, bacterial pneumonia such as lobar pneumonia, mycoplasma pneumonia, Rickettsia pneumonia and staphylococcal pneumonia, Pneumocystis carinii pneumonia, viral pneumonia), pulmonary alveolar proteinosis, pulmonary edema, pulmonary embolism, pulmonary eosinophilia, pulmonary veno-occlusive disease, respiratory distress syndrome such as hyaline membrane disease, adult respiratory distress syndrome, of Scimitar, Silo filler syndrome, pulmonary tuberculosis such as silicotuberculosis; diseases of the nose, such as choanal atresia, epistaxis, lethal midline granuloma, nasal obstruction, nasal polyps, acquired deformities of the nose, nasal neoplasms such as nasal polyps, paranasal sinus neoplasms such as maxillary sinus neoplasms, neoplasms of the nose paranasal sinuses such as maxillary sinus neoplasms, sinusitis such as ethmoid sinusitis, frontal sinusitis, maxillary sinusitis and sphenoid sinusitis, rhinitis such as hay fever, perennial allergic rhinitis, atrophic rhinitis and vasomotor rhinitis, rhinoscleroma). Respiratory diseases that can be treated and / or diagnosed also include pleural diseases, such as chylothorax, pleural empyema (such as tuberculous empyema), hemoneumotorax, hemothorax, hydroneuomotorax, hydrotorax, pleural effusion such as malignant pleural effusion, pleural neoplasms such as malignant pleural effusion, pleurisy such as pleuropneumonia, pneumothorax, pleural tuberculosis such as tuberculous empyema, respiratory disorders such as apnea such as sleep apnea syndromes that include Pickwick's syndrome, Cheyne-Stokes respiration, cough, dyspnea as paroxysmal dyspnea, hoarseness, hyperventilation such as respiratory alkalosis, laryngism, meconium aspiration, mouth breathing, respiratory insufficiency syndrome such as hyaline membrane disease, adult respiratory distress syndrome, respiratory failure such as respiratory acidosis, obstruction of the V Respiratory tracts such as nasal obstruction, laryngeal granuloma, hantavirus pulmonary syndrome, hypoventilation, intrinsic positive-pressure breathing and respiratory paralysis, respiratory hypersensitivity such as extrinsic allergic alveolitis such as the lung of the bird fan and farmer's lung, allergic bronchopulmonary aspergillosis , asthma such as asthma induced by exercise and asthmatic status, hay fever, perennial allergic rhinitis, respiratory system abnormalities such as bronchogenic cyst, bronchopulmonary sequestration, choanal atresia, congenital cystic adenomatoid malformation of the lung, Kartagener syndrome, Scimitar syndrome, tracheobronchomegaly, respiratory tract fistula such as bronchial fistula including tracheoesophageal fistula), respiratory tract infections (such as bronchitis including bronchiolitis such as viral bronchiolitis, common cold, pleural empyema such as empyema tub eric, influenza, laryngitis such as epiglottitis, legionellosis such as Legionnaires' disease, lung abscess, pleurisy such as pleuropneumonia, pneumonia such as bronchopneumonia, pleuropneumonia, aspiration pneumonia such as lipid pneumonia, bacterial pneumonia such as lobar pneumonia, pneumonia microplasma, Rickettsia pneumonia and Staphylococcal pneumonia, Pneumocystis carinii pneumonia, viral pneumonia, rhinitis, rhinoscleroma, sinusitis such as ethmoid sinusitis, frontal sinusitis, maxillary sinusitis and sphenoid sinusitis, tonsillitis such as peritonsillar abscess, tracheitis, laryngeal tuberculosis, pleural tuberculosis such as tubercular empyema, pulmonary tuberculosis such as silicotuberculosis, whooping cough, respiratory tract neoplasms such as bronchial neoplasms, laryngeal neoplasms, pulmonary neoplasms such as bronchogenic carcinoma, lung coin injury, and Pancoast syndrome, nasal neoplasms ta such as nasal polyps, paranasal sinus neoplasms such as maxillary sinus neoplasms, pleural neoplasms such as malignant pleural effusion, tracheal neoplasms, tracheal diseases such as tracheal neoplasms, tracheal stenosis, tracheitis, tracheobronchomegaly, and tracheoesophageal fistula. Examples of otorhinolaryngological diseases that can be treated or detected include ciliary motility disorders such as Kartagener's syndrome, ear diseases such as cholesteatoma of the middle ear, ear acquired deformities, ear neoplasms, earache, hearing disorders such as deafness that include sudden deafness, partial hearing loss such as bilateral loss of ear, conductive ear loss, functional hearing loss, hearing loss high frequency, loss of the neural sensory ear such as loss of the central ear, loss of ear induced by noise and presbycusis, volume exacerbation, tinnitus, herpes zoster oticus, labyrinth diseases such as cochlear diseases, endolymphatic dropsy such as disease of Meniere, labyrinthitis, vestibular diseases such as motion sickness that includes space movement disease, vertigo, otitis such as otitis externa, otitis media such as mastoiditis, otitis media with effusion and suppurative otitis media, otosclerosis, retrocochlear diseases such as nerve diseases acoustics that include ac neuroma such as neurofibromatosis 2, central auditory diseases such as perceptual auditory disorders and loss of the central ear, perforation of the tympanic membrane), laryngeal diseases such as laryngeal granuloma, laryngeal edema, laryngeal neoplasms, laryngeal pericondritis, laryngism, laryngitis such as croup , laryngostenosis, laryngeal tuberculosis, vocal cord paralysis, voice disorders such as aphonia and hoarseness, nasal diseases (such as choanal atresia, epistaxis, lethal midline granuloma, nasal obstruction, nasal polyps, acquired deformities of the nose, nasal neoplasms such as nasal polyps, paranasal sinus neoplasms such as maxillary sinus neoplasms, paranasal sinus diseases such as paranasal sinus neoplasms including maxillary sinus neoplasms, sinusitis such as ethmoid sinusitis, frontal sinusitis, maxillary sinusitis and sphenoid sinusitis, rhinitis such as fieb hayfever, perennial allergic rhinitis, atropic rhinitis and vasomotor rhinitis, rhinoscleroma), otorhinolaryngological neoplasms such as ear neoplasms, laryngeal neoplasms, acoustic neuroma such as neurofibromatosis 2, nasal neoplasms such as nasal polyps, paranasal sinus neoplasms such as neoplasms of the maxillary sinus, pharyngeal neoplasms such as hypopharyngeal neoplasms, nasopharyngeal neoplasms, oropharyngeal neoplasms such as tonsillar neoplasms, pharyngeal neoplasms such as hypopharyngeal neoplasms, nasopharyngeal neoplasms, oropharyngeal neoplasms including tonsillar neoplasms, pharyngitis, retropharyngeal abscess, tonsillitis, and velopharyngeal insufficiency.Neurological diseases The KGF-2 polynucleotides, polypeptides, agonists and / or antagonists can be used to treat and / or detect neurological diseases. Examples of neurological diseases that can be treated or detected include brain diseases (such as metabolic brain diseases including phenylketonuria such as maternal phenylketonuria, pyruvate-carboxylase deficiency, pyruvate-dehydrogenase complex deficiency, Wernicke encephalopathy, cerebral edema, cerebral neoplasms such as cerebellar neoplasms including infratentorial neoplasms, cerebral ventricle neoplasms such as choroidal plexus neoplasms, hypothalamic neoplasms, supratentorial neoplasms, canavan disease, cerebellar diseases such as cerebellar ataxia including spinocerebellar degeneration such as ataxia telangiectasia, cerebellar dyssynergia, Friederich's ataxia, Machado-Joseph's disease, olivopontocerebellar atrophy, cerebellar neoplasms such as infratentorial neoplasms, diffuse cerebral sclerosis such as periaxialis encephalitis, cell leukodystrophy as globoids, metachromatic leukodystrophy and subacute sclerosing panencephalitis, cerebrovascular disorders (such as carotid artery diseases that include carotid artery thrombosis, carotid stenosis and Moyamoya's disease, cerebral amyloid angiopathy, cerebral aneurysm, cerebral anoxia, cerebral arteriosclerosis, cerebral arteriovenous malformations, cerebral artery diseases, cerebral embolism and cerebral thrombosis such as carotid artery thrombosis, sinus thrombosis and Wallenberg syndrome, cerebral hemorrhage such as epidural hematoma, subdural hematoma and subarachnoid hemorrhage, cerebral infarction, cerebral ischemia such as transient cerebral ischemia, subclavicular Steal syndrome, and vertebrobasilar insufficiency, vascular dementia such as multi-infarct dementia, periventricular leukomalacia, vascular headache such as cluster headache, migraine, dementia such as dementia complex. to AIDS, presenile dementia such as Alzheimer's disease and Creutzfeldt-Jakob syndrome, senile dementia such as Alzheimer's disease and progressive supranuclear palsy, vascular dementia such as multi-infarct dementia, encephalitis including periaxial encephalitis, viral encephalitis such as encephalitis epidemic, Japanese encephalitis, St. Louis, encephalitis carried by ticks and West Nile fever, acute disseminated encephalomyelitis, meningoencephalitis such as uveomeningoencephalitic syndrome, post-encephalitic Parkinson's disease and subacute sclerosing pancencephalitis, encephalomalacia such as periventricular leukomalacia, epilepsy such as generalized epilepsy that includes infantile spasms, epilepsy absence, myoclonic epilepsy including MERRF syndrome, tonic-clonic epilepsy, partial epilepsy such as complex partial epilepsy, frontal lobe epilepsy and temporal lobe epilepsy, post-traumatic epilepsy, epileptic status such as continuous partial epilepsy, Hallervorden's syndrome Spatz, hydrocephalus such as Dandy-Walker syndrome and normal-pressure hydrocephalus, hypothalamic diseases such as hypothalamic neoplasms, cerebral malaria, narcolepsy including cataplexy, bulbar polio, pseudotumor cerebri, Rett syndrome, yes Reye syndrome, thalamic diseases, cerebral toxoplasmosis, intracranial tuberculoma and Zellweger syndrome, central nervous system infections such as AIDS dementia complex, cerebral abscess, subdural empyema, encephalomyelitis such as equine encephalomyelitis, Venezuelan equine encephalomyelitis, necrotizing hemorrhagic encephalomyelitis, visna, cerebral malaria, meningitis such as arachnoiditis, aseptic meningitis such as viral meningitis that includes lymphocytic choriomeningitis. Bacterial meningitis that includes Haemophilus meningitis, meningitis due to Listeria, meningococcal meningitis such as Waterhouse-Friderichsen syndrome, pneumococcal meningitis and meningeal tuberculosis, fungal meningitis such as cryptococcal meningitis, subdural effusion, meningoencephalitis such as uvemeningoencephalitic syndrome, myelitis such as transverse myelitis, neurosyphilis as dorsal tabes, poliomyelitis that includes bulbar polio and post-polio syndrome, previous diseases (such as Creutzfeldt-Jakob syndrome, bovine spongiform encephalopathy, Gerstmann-Straussler syndrome, Kuru, Scrapie), cerebral toxoplasmosis, neoplasms of the central nervous system such as neoplasms Cerebrals that include cerebellar neoplasms such as infratentorial neoplasms, neoplasms of the cerebral ventricle such as choroidal plexus neoplasms, hypothalamic neoplasms and supratentorial neoplasms, meningeal neoplasms, neoplasms of the spinal cord including epidural neoplasms ales, demyelinating diseases such as Canavan diseases, diffuse cerebral sclerosis including adrenoleukodystrophy, periaxialis encephalitis, globoid cell leukodystrophy, diffuse cerebral sclerosis such as metachromatic leukodystrophy, allergic encephalomyelitis, necrotizing hemorrhagic encephalomyelitis, progressive multifocal leukoencephalopathy, multiple sclerosis, pontine myelinolysis central, transverse myelitis, neuromyelitis optica, Scrapie, enzootic ataxia, chronic fatigue syndrome, visna, high pressure nervous syndrome, meningism, spinal cord diseases such as congenital amyotonia, amyotrophic lateral sclerosis, spinal muscular atrophy such as Werdnig's disease -Hoffmann, compression of the spinal cord, neoplasms of the spinal cord such as epidural neoplasms, syringomyelia, tabes dorsalis, rigid man syndrome, mental retardation such as Angelman syndrome, Cri-du-Chat syndrome, syndrome d e De Lange, Down syndrome, gangliosidosis such as gangliosidosis G (M1), Sandhoff's disease, Tay-Sachs disease, Hartnup's disease, hemocystinuria, Laurence-Moon-Biedl syndrome, Lesch-Nyhan syndrome, maple syrup urine, mucolipidosis such as fucosidosis, neuronal ceroid lipofucsinosis, oculocerebrorenal syndrome, phenylketonuria such as maternal phenylketonuria, Prader-Willi syndrome, Rett syndrome, Rubinstein-Taybi syndrome, tuberous sclerosis, WAGR syndrome, system abnormalities such as holoprosencephaly, neural tube defects such as anacephaly including hydrangencephaly, Arnold-Chairi deformity, encephalocele, meningocele, meningomyelocele, spinal dysraphism such as spina bifida cystica and occult spina bifida, hereditary sensory and motor neuropathies including Charcot disease -Marie, hereditary optic atrophy, Refsum's disease, hereditary spastic paraplegia , Werdnig-Hoffmann disease, hereditary sensory and autonomic neuropathies such as congenital analgesia and familial dysautonomia, neurological manifestations (such as agnosia including Gerstmann syndrome, amnesia such as retrograde amnesia, apraxia, neurogenic bladder, cataplexy, communication disorders such such as hearing disorders that include deafness, partial hearing loss, volume aggravation and tinnitus, language disorders such as aphasia that include agraphy, anomia, broca aphasia and Wernicke's aphasia, dyslexia such as acquired dyslexia, language development disorders , voice disorders such as aphasia including anomia, drill bit aphasia and Wernicke aphasia, joint disorders, communication disorders such as voice disorders, which include dysarthria, echolalia, mutism and stuttering, voice disorders such like aphonia and hoarseness, brainless state, delirium, fasciculation n, hallucinations, meningismus, movement disorders such as Angelman, ataxia, athetosis, chorea, dystonia, hypokinesia, muscular hypotonia, myoclonus, tic, torticollis and tremor, muscular hypertonia such as muscle stiffness such as rigid man syndrome, muscle spasticity, paralysis such as facial paralysis including herpes zoster oticus, gastroparesis, hemiplegia, ophthalmoplegia such as diplopia, Duane syndrome, Horner syndrome, chronic progressive external ophthalmoplegia such as Kearns syndrome, bulbar paralysis, tropical spastic paraparesis, paraplegia such as Brown-Sequard syndrome, quadriplegia, respiratory paralysis and paralysis of the vocal cords, paresis, phantom limbs, taste disorders such as ageusia and dysgeusia, vision disorders such as amblyopia, blindness, color vision defects, diplopia, hemianopsia, scotoma and subnormal vision, disorders of the sleep such as hypersomnia, which includes Kleine-Levin syndrome, insomnia, and sleepwalking, spasm such as trismus, unconssness such as coma, persistent vegetative state and syncope and vertigo, neuromuscular diseases such as congenital amyotonia, amyotrophic lateral sclerosis, Lambert-Eaton myasthenic syndrome, motor neuron disease, muscle atrophy such as spinal muscular atrophy, Charcot-Marie disease and Werdnig-Hoffmann disease, post-polio syndrome, muscular dystrophy, myasthenia gravis, atrophic myotonia, congenital myotonia, nemaline myopathy, familial periodic paralysis, multiple paramiloclonus, tropical spastic paraparesis and rigid man syndrome, systemic diseases Peripheral nervous system such as acrodynia, amyloid neuropathies, autonomic nervous system diseases such as Adè syndrome, Barre-Lieou syndrome, familial dysautonomia, Horner syndrome, reflex sympathetic dystrophy and Shy-Drager syndrome, cranial nerve diseases such as sickness of the acoustic nerve such as acoustic neuroma including neurofibromatosis 2, facial nerve diseases such as facial neuralgia, Melkersson-Rosenthal syndrome, ocular motility disorders including amblyopia, nystagmus, oculomotor nerve palsy, ophthalmoplegia such as Duane, Horner's syndrome, chronic progressive external ophthalmoplegia including Kearns syndrome, strabismus such as esotropia and exotropia, oculomotor nerve palsy, optic nerve diseases such as optic atrophy including hereditary optic atrophy, optic disc drusen, optic neuritis such as neuromyelitis optics, papilledema, trigeminal neuralgia, vocal cord paralysis, demyelination diseases such as neuromyelitis optics and enzootic apaxia, diabetic neuropathies such as diabetic foot, compression syndromes of the nerves such as carpal tunnel syndrome, tunnel syndrome tars al, thoracic outlet syndrome such as cervical rib syndrome, ulnar nerve compression syndrome, neuralgia such as causalgia, cervical-brachial neuralgia, facial neuralgia and trigeminal neuralgia, neuritis such as experimental allergic neuritis, optic neuritis, polyneuritis , polyradiculoneuritis and radiculitis such as polyradiculitis, hereditary motor and sensory neuropathies such as Charcot-Marie's disease, hereditary optic atrophy, Refsum's disease, hereditary spastic paraplegia and Werdnig-Hoffmann's disease, hereditary sensory and autonomic neuropathies that include congenital analgesia and Family dysautonomia, POEMS syndrome, sciatica, gustatory sweating and tetany.

Metabolic and Endocrine Diseases The KGF-2 polynucleotides, polypeptides, agonists and / or antagonists can be used to treat and / or diagnose metabolic or endocrine diseases.

Examples of nutritional and metabolic diseases that can be treated or detected include achlorhydria, acid-base imbalance, acidosis (including lactic, renal or respiratory tubular), diabetic ketoacidosis, ketosis, alkalosis, respiratory alkalosis, calcium metabolism disorders, calcinosis, calciphylaxis, CREST syndrome, nephrocalcinosis, pathological decalcification, hypercalcemia, hypocalcemia, tetany, osteomalacia, pseudohypoparathyroidism, Ricketts, diabetes insipidus, nephrogenic diabetes insipidus, Wolfram syndrome, diabetes mellitus (including experimental and insulin-dependent, lipoatrophic, non-insulin dependent ), diabetic angiopathies, diabetic foot, gestational diabetes, fetal macrosomia, glucose intolerance, glucosuria, renal glucosuria, hypergiucemia, hyperlipidemia, hypercholesterolemia, hyperlipoproteinemia, hypertriglyceridemia, hyperprolactinemia, hypervitaminosis A, hypoglycaemia, insulin coma, syndrom it is malabsorption (including blind loop syndrome, celiac disease, lactose intolerance, intestinal lipodystrophy, tropical sprue), birth errors in the metabolism (including birth errors in amino acid metabolism, ocular albinism, oculocutaneous albinism, piebaldism), alkaptonuria, ochronosis, renal aminoaciduria, cystinuria, Hartnup's disease, homocystinuria, Maple Syrup urine disease, multiple carboxylase deficiency , phenylketonuria, maternal phenylketonuria, amyloidosis, amyloid neuropathies, cerebral amyloid angiopathy, neonatal errors in carbohydrate metabolism such as errors in the neonate in fructose metabolism (deficiency in fructose- 1, 6-diphosphatase, intolerance to fructose), galactosemia, glucose intolerance, glycogen storage disease (Types I, II, III, IV, V, VI, VII, VIII), hyperoxaluria, primary hyperoxaluria, mannosidosis, mucopolysaccharidosis, (I, II, III, IV, VI, VII), multiple carboxylase deficiency, neonatal errors in pyruvate metabolism, Leigh's disease, pyruvate-carboxylase deficiency, deficiency in the pyruvate-dehydrogenase complex, deficiency in glucose phosphate dehydrogenase, hereditary hyperbilirubinemia, Crigler-Najjar syndrome, Gilbert's disease, chronic idiopathic jaundice, errors in the newborn in lipid metabolism such as hyperlipoproteinemia, hypercholesterolemia familial, combined familial hyperlipidemia, hypercholesterolemia (familial type III, IV, V), familial deficiency in lipoprotein-lipase, hypolipoproteinemia (abetalipoproteinemia, hypobetalipoproteinemia, deficiency in lecithin-acyltransferase, Tangier's disease), lipoidosis (ester storage disease) of cholesterol, lipoidoproteinosis, neuronal ceroid lipofucsinosis, Refsum disease, Sjo-Larsson syndrome, sphingolipidosis (adrenoleukodystrophy, Fabry disease, ganglisidosis, Sandhoff's disease, Tay-Sachs disease, Gaucher's disease, globoid cell leukodystrophy, metachromat leukodystrophy ica, Niemann-Pick disease, Dark blue histiocyte syndrome, Wolman's disease, mitochondrial myopathies, mitochondrial encephalomyopathies, MELAS syndrome MERRF syndrome, external chronic prosive ophthalmoplegia, lysosomal storage diseases such as cholesterol ester storage disease ,, Mannosidosis, mucolipidosis, fucosidosis, mucopolisacaridosis (I, II, III, IV, VI and VII), errors in the neonate in the metabolism of metals including hemacromatosis, hepatolenticular degeneration, hypophosphatasia, familial hypophosphatemia, kinky hair syndrome, paralysis periodic family, and pseudohypoparathyroidism, mucolipidosis, fucosidosis, porphyria, (erythrohepatic, erythropoietic, hepatic, acute intermittent, late cutaneous), errors in the newborn in the purine-pyrimidine metabolism such as gout, gouty arthritis, and Lesch-Nyhan syndrome , errors in the neonate in renal tubular transport such as renal tubular acidosis, renal aminoaciduria, cystinuria, hartnup's disease, cystinosis, Fanconi syndrome, renal glucosuria, familial hypophosphatemia, oculocerebrorenal syndrome, and pseudohypoaldosteronism, disorders in phosphorus metabolism , hypophosphatemia, protein loss enteropathies, intestinal lymphangiectasis, water electrolyte imbalance (dehydration, hypercalcemia, hyperkalemia, hypernatraemia, hypocalcemia, hyponatremia, inappropriate adh syndrome, water intoxication), xanthomatosis, Wolman's disease, childhood nutrition disorders such as infant nutrition disorders e, deficiency diseases such as vitamin deficiency, ascorbic acid deficiency, scurvy, vitamin A deficiency, vitamin B deficiency, choline deficiency, folic acid deficiency, pellagra, pyridoxine deficiency, riboflavin deficiency, thiamine deficiency, beriberi, Wernicke encephalopathy, vitamin B12 deficiency (pernicious anemia), vitamin D deficiency (osteomalacia, steatitis), vitamin E deficiency (steatitis), vitamin K deficiency, magnesium deficiency, potassium deficiency, protein deficiency (poor nutrition by protein energy, kwashiorkor), enzootic ataxia, obesity in diabetes, morbid obesity, Pickwickian syndrome, Prader-Willi syndrome, and starvation. Examples of endocrine diseases that can be treated or detected include diseases of the adrenal glands (diseases of the cortex, neoplasms of nortex), hyperfunction of the adrenal gland (Cushing's syndrome, hyperaldosteronism, Bartter's disease), hypofunction of the adrenal gland (Addison's disease, adrenoleukodystrophy, hypoaldosteronism), neoplasms of the adrenal gland, neoplasms of the adrenal cortex, congenital adrenal hyperplasia, Waterhause-Friderichsen syndrome, breast neoplasms, male breast neoplasms, fibrocystic disease of the breast, gynecomastia, disorders of lactation such as Chiari-Frommel syndrome and galactorrhea, mastitis, Bowie mastitis, diabetes mellitus (experimental, insulin-dependent, Wolfram syndrome.}, lipoatrophic, and non-insulin dependent), diabetic angiopathies, diabetic foot, , diabetic retinopathy, diabetic coma, non-ketotic hyperosmolar hyperglu coma seizure, diabetic ketoacidosis, diabetic nephropathies and those associated with diabetic foot, obesity in diabetes, gestational diabetes, fetal macrosomia, dwarfism (Cockayne syndrome, pituitary, thanatophoric dysplasia), neoplasms of the endocrine glands such as neoplasm of the adrenal cortex , multiple endocrine neoplasia (types 1, 2a 2b), neoplastic pseudoendocrine syndromes, ACTH syndrome (ectopic), Zollinger-Ellison syndrome, ovarian neoplasms, Meig's syndrome, parathyroid neoplasms, pituitary neoplasms, Nelson's syndrome, neoplasms testicles, thymus neoplasms, thyroid neoplasms, thyroid nodules, gonadal disorders such as adrenal hyperplasia (congenital), feminization, testicular feminization, hyperandrogenism, hypogonadism, eunuchism, Kallmann syndrome, Klinefelter syndrome, ovarian diseases such as anovulation , oophoritis, ovarian cysts, poliq ovarian syndrome UTIs, premature ovarian failure, ovarian hyperstimulation syndrome, ovarian neoplasms, Meigs syndrome, delayed puberty, and precocious puberty, disorders in sexual differentiation such as gonadal dysgenesis (46, XY, mixed) and Turner syndrome, hermaphroditism, pseudohermaphroditism, Kallmann syndrome, Klinefelter, testicular feminization, testicular diseases such as cryptorchidism, orchitis, testicular neoplasms, virilism, hirsutism, hyperinsulinism, neoplastic pseudoendocrine syndromes such as ACTH syndrome (ectopic) and Zollinger-Ellison syndrome, parathyroid diseases including hyperparathyroidism (secondary), renal osteodystrophy, hyperparathyroidism, tetany, parathyroid neoplasms, pituitary diseases, Empy Sella syndrome, hyperpituitarism, acromegaly, gigantism, hyperpituitarism (diabetes insipidus, nephrogenic diabetes insipidus, Wolfram syndrome, pituitary dwarfism), inappropriate ADH syndrome, pituitary apoplexy, pituitary neoplasms, Nelson's syndrome, autoimmune polyendocrinopathies, progeria, Werner syndrome, thymic hyperplasia, the thyroid gland such as euthyroid disease syndrome, goiter, (endemic, nodular, substernal, Graves disease), hyperthyroidism and that associated with Graves disease, hyperthyroxinemia, hypothyroidism (cretinism and myxedema), resistance syndrome thyroid hormone, thyroid neoplasms, thyroid nodule, thyroiditis (autoimmune, subacute, suppurative), thyrotoxicosis, thyroid crisis, and endocrine tuberculosis.

Diseases at the cellular level Diseases associated with increased cell survival or inhibition of apoptosis that could be treated or detected by KGF-2 polynucleotides or polypeptides, as well as KGF-2 antagonists or agonists, include cancers (such as follicular lymphocytes, carcinomas with p53 mutations, and hormone-dependent tumors, including, but not limited to, colon cancer, cardiac tumors, pancreatic cancer, melanoma, retinoblastoma , glioblastoma, lung cancer, intestinal cancer, testicular cancer, stomach cancer, neuroblastoma, myxoma, myoma, lymphoma, endothelioma, osteoblastoma, osteoclastoma, osteosarcoma, chondrosarcoma, adenoma, breast cancer, prostate cancer, Kaposi's sarcoma, and ovarian cancer), autoimmune disorders (such as, multiple sclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliary cirrhosis, Behcet's disease, Crohn's disease, polymyositis, systemic lupus erythematosus and immune-related glomerulonephritis, and rheumatoid arthritis), and viral infections (such as herpes virus, smallpox virus and adenovirus), inflammation, graft disease versus h ugra, acute rejection of the graft, and chronic rejection of the graft. In the preferred embodiments, the KGF-2 polynucleotides, polypeptides and / or antagonists of the invention are used to inhibit the development, progression, and / or metastasis of the cancers, in particular those listed above. Additional diseases or conditions associated with increased cell survival that could be treated or detected by KGF-2 polynucleotides or polypeptides, or KGF-2 agonists or antagonists, include but are not limited to, progression and / or metastasis. of malignancies and related disorders such as leukemia (including acute leukemia (eg, acute lymphocytic leukemia, myelocytic leukemia (including myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia)) and chronic leukemias (eg, chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia), polycythemia vera, lymphomas (eg, Hodgkin's disease and non-Hodgkin's disease) , multiple myeloma, Waldenstrom's macroglobulidemia, heavy chain disease, and solid tumors including, but not limited to, sarcomas and carcinomas, such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendoteliosarcoma , synovitis, mesothelioma, Ewing tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, carcinoma of the glands sweat, carcinoma of the sebaceous glands, papilloma carcinoma ar, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, testicular cancer, lung carcinoma, lung carcinoma, small cells, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, and retinoblastoma.

Diseases associated with increased apoptosis that could be treated or detected by KGF-2 polynucleotides or polypeptides, as well as by KGF-2 agonists or antagonists, include AIDS; neurodegenerative disorders (such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, retinitis pigmentosa, cerebellar degeneration and brain tumor or previous associated disease); autoimmune disorders (such as multiple sclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliary cirrhosis, Behcet's disease, Crohn's disease, polymyositis, systemic lupus erythematosus and glomerulonephritis related to the immune system and rheumatoid arthritis) myelodysplastic syndromes (such as aplastic anemia) , graft versus host disease, ischemic damage (such as that caused by myocardial infarction, stroke and reperfusion injury), liver damage (eg, liver damage related to hepatitis, ischemia / reperfusion injury, cholestosis (damage to the duct bile) and liver cancer); hepatic disease induced by toxins (such as those caused by alcohol), septic shock, cachexia and anorexia.

Healing of wounds and proliferation of epithelial cells.

According to yet another aspect of the present invention, there is provided a process for using the KGF-2 polynucleotides or polypeptides, as well as KGF-2 agonists or antagonists for therapeutic purposes, for example to stimulate the proliferation of epithelial cells and proteins. basal keratinocytes for wound healing purposes, and to stimulate the production of the hair follicle and the healing of dermal wounds. KGF-2 polynucleotides or polypeptides, as well as KGF-2 agonists or antagonists, may be clinically useful in the stimulation of wound healing, including surgical wounds, excision wounds, deep wounds involving damage to the dermis and epidermis , ocular tissue injuries, dental tissue wounds, oral cavity wounds, diabetic ulcers, dermal ulcers, elbow ulcers, venous stasis ulcers, burns resulting from exposure to heat or chemicals, and other abnormal conditions of healing wounds such as uremia, malnutrition, vitamin deficiencies and complications associated with systemic treatment with steroids, radiotherapy and antineoplastic drugs and antimetabolites. The KGF-2 polynucleotides or polypeptides, as well as KGF-2 agonists or antagonists, could be used to promote skin re-establishment subsequent to skin loss. KGF-2 polynucleotides or polypeptides, as well as KGF-2 agonists or antagonists, could be used to increase the adherence of skin grafts to a wound bed, and to stimulate re-epithelialization from the bed of the wound. wound. The following are types of grafts in which KGF-2 polynucleotides or polypeptides, or KGF-2 agonists or antagonists, could be used to increase adherence to a wound bed: autografts, artificial skin, allografts, autodermal graft, grafts autoepidermal, Avicular grafts, Blair-Brown grafts, bone grafts, brephoplastic grafts, skin graft, delayed graft, dermal graft, epidermal graft, fascia graft, full thickness graft, heterologous graft, xenograft, homologous graft, hyperplastic graft, lamellar graft, mesh graft, mucosal graft, Ollier-Thiersch graft, epiploic graft, patch graft, pedicle graft, penetration graft, split skin graft, and thick skin graft. KGF-2 polynucleotides or polypeptides, as well as KGF-2 agonists or antagonists, could also be used to promote skin reinforcement and to improve the appearance of aged skin.

It is believed that KGF-2 polynucleotides or polypeptides, as well as KGF-2 agonists or antagonists, will also produce changes in the proliferation of hepatocytes, and proliferation of epithelial cells in the lung, breast, pancreas, stomach , the small intestine, and the large intestine. KGF-2 polynucleotides or polypeptides, as well as KGF-2 agonists or antagonists, could promote the proliferation of epithelial cells such as sebocytes, hair follicles, hepatocytes, type II pneumocytes, mucin-producing goblet cells, and others. epithelial cells and their progenitors, contained within the skin, lung, liver, and gastrointestinal tract. KGF-2 polynucleotides or polypeptides, as well as KGF-2 agonists or antagonists, can promote the proliferation of endothelial cells, keratinocytes, and basal keratinocytes. KGF-2 polynucleotides or polypeptides, as well as KGF-2 agonists or antagonists, could also be used to reduce the side effects of bowel toxicity resulting from radiation, chemotherapy treatments or viral infections. The KGF-2 polynucleotides or polypeptides, as well as KGF-2 agonists or antagonists, can have a cytoprotective effect on the mucosa of the small intestine. KGF-2 polynucleotides or polypeptides, as well as KGF-2 agonists or antagonists, can also stimulate the healing of mucositis (mouth ulcers) resulting from chemotherapy and viral infections. The KGF-2 polynucleotides or polypeptides, as well as KGF-2 agonists or antagonists, could also be used in the complete regeneration of the skin in full and partial thickness skin defects, including burns (eg, repopulation of the hair follicles, sweat glands, and sebaceous glands), treatments of other skin defects such as psoriasis. KGF-2 polynucleotides or polypeptides, as well as KGF-2 agonists or antagonists, could be used to treat bullous epidermolysis, a defect in adhesion of the epidermis to the underlying dermis, which results in frequent, open bladders and painful by the acceleration of the reepititelialization of these lesions. The KGF-2 polynucleotides or polypeptides, as well as the KGF-2 agonists or antagonists, could be used to treat gastric and duodenal ulcers and help the healed by the formation of the scar of the mucosal lining, and regeneration of the glandular mucosa and the mucosa. mucosal doudenal lining, more quickly. Inflammatory bowel diseases, such as Crohn's disease and ulcerative colitis, are diseases that result in the destruction of the mucosal surface of the small or large intestine, respectively. Thus, KGF-2 polynucleotides or polypeptides, as well as KGF-2 agonists or antagonists, could be used to promote surface reshaping of the mucosal surface, to aid in faster healing and to prevent the progression of the disease Inflammatory bowel Treatment with KGF-2 polynucleotides or polypeptides, or KGF-2 agonists or antagonists, is expected to have a significant effect on mucus production throughout the gastrointestinal tract, and could be used to protect the intestinal mucosa. of harmful substances that are ingested or after surgery. KGF-2 polynucleotides or polypeptides, as well as KGF-2 agonists or antagonists, could be used to treat diseases associated with low expression of KGF-2. In addition, KGF-2 polynucleotides or polypeptides, as well as KGF-2 agonists or antagonists, could be used to prevent and heal damage to the lungs due to various disease states. A growth factor such as KGF-2 polynucleotides or polypeptides, as well as KGF-2 agonists or antagonists, which could stimulate proliferation and differentiation, and promote repair of the alveoli and bronchiolar epithelium to prevent or treat acute or chronic lung damage. For example, emphysema, which results in progressive loss of the alveoli, and damage by inhalation, resulting from the inhalation of smoke and burns, which cause necrosis of the bronchiolar epithelium and the alveoli could effectively be treated using the polynucleotides or KGF-2 polypeptides, KGF-2 agonists or antagonists. Also, KGF-2 polynucleotides or polypeptides, as well as KGF-2 agonists or antagonists, could be used to stimulate the proliferation of and differentiation of type II pneumocytes, which can help to treat or prevent diseases such as diseases. of the hyaline membrane, such as the syndrome of respiratory failure of the infant and bronchopulmonary dysplasia, in premature infants. KGF-2 polynucleotides or polypeptides, as well as KGF-2 agonists or antagonists, could stimulate the proliferation and differentiation of hapatocytes and, thus, could be used to alleviate or treat liver diseases and liver pathologies such as fulminant hepatic insufficiency. caused by cirrhosis, liver damage caused by viral hepatitis and toxic substances (for example, acetaminophen, carbon tetrachloride and other hepatotoxins known in the art). In addition, KGF-2 polynucleotides or polypeptides, as well as KGF-2 agonists or antagonists, could be used to treat or prevent the onset of diabetes mellitus. In newly diagnosed Type I and II diabetes patients, where some islet cell function remains, the KGF-2 polynucleotides or polypeptides, as well as the KGF-2 agonists or antagonists, could be used to maintain the function of the cells. islets to alleviate, delay or prevent the permanent manifestation of the disease. Also, KGF-2 polynucleotides or polypeptides, as well as KGF-2 agonists or antagonists, could be used as an adjunct in the transplantation of islet cells to improve or promote the function of islet cells.

Infectious Disease The KGF-2 polynucleotides or polypeptides, as well as KGF-2 agonists or antagonists, can be used to treat or detect infectious agents. For example, by increasing the immune response, particularly by increasing the proliferation and differentiation of B and / or T cells, infectious diseases can be treated. The immune response can be increased by increasing an existing immune response, or by initiating a new immune response. Alternatively, the KGF-2 polynucleotides or polypeptides, as well as KGF-2 agonists or antagonists, can also directly inhibit the infectious agent, without necessarily promoting an immune response. Viruses are an example of an infectious agent that can cause disease or symptoms that can be treated or detected by KGF-2 polynucleotides or polypeptides, as well as KGF-2 agonists or antagonists. Examples of viruses include, but are not limited to, the following viral and RNA viral families: Arboviruses, Adenoviridae, Arenaviridae, Arterivirus, Birnaviridae, Bunyaviridae, Caliciviridae, Circoviridae, Coronaviridae, Flaviviridae, Hepadnaviridae (Hepatitis), Herpesviridae (such as Cytomegalovirus, Herpes Simple, Herpes Zoster), Mononegaviruses (for example, Paramyxoviridae, Morbillivirus, Rhabdoviridae, Orthomyxoviridae (for example, Influenza), Papovaviridae, Parvoviridae, Picornaviridae, Poxviridae, (such as smallpox or Vaccinia), Reoviridae (for example, Rotavirus), Retroviridae (HTLV-I, HTLV-II, Lentivirus), and Togaviridae (for example, Rubivirus). The viruses that fall within these families can cause a variety of diseases or symptoms, including, but not limited to: arthritis, bronchiolitis, encephalitis, eye infections (eg, conjunctivitis, keratitis), chronic fatigue syndrome, hepatitis (A, B, C, E, Chronic Active, Delta), meningitis, opportunistic infections (eg, AIDS), pneumonia, Burkitt's lymphoma, chicken pox , hemorrhagic fever, measles, mumps, parainfluenza, rabies, the common cold, polio, leukemia, rubella, sexually transmitted diseases, skin diseases (eg Kaposi, wrinkles), and viremia. KGF-2 polynucleotides or polypeptides, as well as KGF-2 agonists or antagonists, can be used to treat or detect any of these symptoms or diseases. Similarly, bacterial or fungal agents that can cause disease or symptoms and that can be treated or detected by KGF-2 polynucleotides or polypeptides, or KGF-2 agonists or antagonists, include, but are not limited to, the following families of Gram-negative and Gram-positive bacteria and fungi: Actinomycetales (for example, Corynebacterium, Mycobacterium, Norcardia), Aspergillosis, Bacillaceae (for example, Anthrax, Clostridium), Bacteroidaceae, Blastomycosis, Bordetella, Borrelia, Brucellosis, Candidiasis, Campylobacter, Coccidioidomycosis, Cryptococcosis, Dermatocicosis, Enterobacteriaceae (Klebsiella, Salmonella, Serratia, Yersinia), Erysipelothrix, Helicobacter, Legionellosis, Leptospirosis, Listeria, Mycoplasmatales, Neisseriaceae (for example, Acinetobacter, Gonorrhea, Meningococci), Pasteurellaceae infections (for example, Actinobacillus, Heamophilus, Pasteurella), Pseudomonas, Rickettsiaceae, Chlamydiaceae, syphilis and Staphylococcus . These families of bacteria and fungi can cause the following diseases or symptoms, including, but not limited to: bacteremia, endocarditis, eye infections (conjunctivitis, tuberculosis, uveitis), gingivitis, opportunistic infections (for example, infections related to AIDS) , paronychia, prosthetic-related infections, Reiter's disease, respiratory tract infections, such as whooping cough or empyema, sepsis, Lyme disease, cat scratch disease, dysentery, paratyphoid fever, food poisoning, typhoid, pneumonia, gonorrhea, meningitis, chlamydia, syphilis, diphtheria, leprosy, paratuberculosis, tuberculosis, lupus, botulism, tetanus gangrene, impetigo, rheumatic fever, scarlet fever, sexually transmitted diseases, skin diseases (for example, cellulitis, dermatocicosis), toxemia, urinary tract infections and wound infections. The KGF-2 polynucleotides or polypeptides, or KGF-2 agonists or antagonists, can be used to treat or detect any of these symptoms or diseases. In addition, parasitic agents that cause disease or symptoms that can be treated or detected by KGF-2 polynucleotides or polypeptides, or agonists or antagonists of KGF-2, include, but are not limited to, the following families: Amibiasis, Babesiosis , Coccidiosis, Cryptosporidiosis, Dientamoebiasis, Durina, Ectoparasitosis, Giardiasis, Helminthiasis, Leishmaniasis, Theileriasis, Toxoplasmosis, Trypanosomiasis, and Trichomoniasis. These parasites can cause a variety of diseases or symptoms, including, but not limited to: Scabies, Trombiculiasis, eye diseases, intestinal disease (e.g., dysentery, giardiasis), liver disease, lung disease, opportunistic infections (e.g., related to AIDS), Malaria, complications of pregnancy, and toxoplasmosis. Polynucleotides or KGF-2 polypeptides or KGF-2 agonists or antagonists can be used to treat or detect any of these symptoms or diseases. Preferably, the treatment using the KGF-2 polynucleotides or polypeptides, or KGF-2 agonists or antagonists, could be either by administering an effective amount of the KGF-2 polypeptide to the patient, or by removing the cells from the patient, supplying the cells with the KGF-2 polynucleotide, and returning the engineered cells to the patient (ex vivo therapy). In addition, the KGF-2 polypeptide or polynucleotide can be used as an antigen in a vaccine, to give rise to an immune response against the infectious disease.

Regeneration KGF-2 polynucleotides or polypeptides, or KGF-2 agonists or antagonists, can be used to differentiate, proliferate, and attract cells, leading to the regeneration of tissues. (See, Science 276: 59-87 (1997)). Tissue regeneration could be used to repair, replace, or protect tissue damaged by congenital defects, trauma (wounds, burns, incisions, or ulcers), age, disease (eg, osteoporosis, osteoarthritis, periodontal disease) , liver failure), surgery, including cosmetic plastic surgery, fibrosis, reperfusion injury, or systemic cytokine damage. The tissues that could be regenerated using the present invention include organs (eg, pancreas, liver, intestine, kidney, skin, endothelium), muscle (smooth, skeletal or cardiac), vasculature (including vascular and lymphatic vessels), nervous, hematopoietic, and skeletal (bone, cartilage, tendon, and ligament). Preferably, regeneration occurs without or with diminished healing. Regeneration may also include angiogenesis. In addition, KGF-2 polynucleotides or polypeptides, or KGF-2 agonists or antagonists, can increase regeneration of difficult-to-heal tissues. For example, increased regeneration of the tendons / ligaments could accelerate the recovery time after damage. The KGF-2 polynucleotides or polypeptides, or the KGF-2 agonists or antagonists of the present invention could also be used prophylactically in an effort to prevent damage. Specific diseases that could be treated include tendonitis, carpal tunnel syndrome, and other tendon or ligament defects. A further example of tissue regeneration from wounds that do not heal includes pressure ulcers, ulcers associated with vascular insufficiency, surgical and traumatic wounds. Similarly, nerve and brain tissue could also be regenerated by the use of KGF-2 polynucleotides or polypeptides, or KGF-2 agonists or antagonists, to proliferate and differentiate nerve cells. Diseases that could be treated using this method include central and peripheral nervous system disease, neuropathies, or mechanical and traumatic disorders (e.g., spinal cord disorders, head trauma, cerebrovascular disease, and stroke). Specifically, diseases associated with peripheral nerve damage, peripheral neuropathy (eg, resulting from chemotherapy or other medical therapies), localized neuropathies, and central nervous system diseases (eg, Alzheimer's disease, Parkinson's disease, Huntington's disease, Amyotrophic lateral sclerosis, and Shy-Drager syndrome), could all be treated using the KGF-2 polynucleotides or polypeptides, or the KGF-2 agonists or antagonists.

Chemotaxis The KGF-2 polynucleotides or polypeptides, or the KGF-2 agonists or antagonists, can have chemotaxis activity. A chemotactic molecule attracts or mobilizes cells (e.g., monocytes, fibroblasts, neutrophils, T cells, mast cells, eosinophils, epithelial and / or endothelial cells) to a particular site in the body, such as inflammation, infection, or site of hyperproliferation. The mobilized cells can then fight and / or heal the particular trauma or the particular abnormality. KGF-2 polynucleotides or polypeptides, or agonists or antagonists of KGF-2 can increase the chemotaxic activity of particular cells. These chemotactic molecules can then be used to treat inflammation, infection, hyperproliferative disorders, or any disorder of the immune system by increasing the number of cells directed to a particular site in the body. For example, chemotactic molecules can be used to treat wounds and other traumas to tissues, by attracting immune cells to the damaged site. As a chemotactic molecule, KGF-2 could also attract fibroblasts, which can be used to treat wounds. It is also contemplated that KGF-2 polynucleotides or polypeptides or KGF-2 agonists or antagonists can inhibit chemotactic activity. These molecules could also be used to treat disorders. Thus, KGF-2 polynucleotides or polypeptides, or KGF-2 agonists or antagonists, can be used as an inhibitor of chemotaxis.

Link Activity The KGF-2 polypeptides can be used to select the molecules that bind to KGF-2 or for the molecules to which KGF-2 binds. The binding of KGF-2 and the molecule can activate (agonists), increase, inhibit (antagonists), or decrease the activity of KGF-2 or the bound molecules. Examples of such molecules include antibodies, oligonucleotides, proteins (e.g., receptors), or small molecules.

Preferably, the molecule is closely related to the natural ligand of KGF-2, for example, a fragment of the ligand, or a natural substrate, a ligand, a structural or functional mimetic. (See, Coligan et al., Current Protocols in Immunology 1 (2): Chapter 5 (1991)). Similarly, the molecule can be closely related to the natural receptor to which KGF-2 binds, or at least, a fragment of the receptor capable of being linked by KGF-2 (e.g., active site). In any case, the molecule can be rationally designed using known techniques. Preferably, selection for these molecules involves the production of appropriate cells that express KGF-2, either as a secreted protein or on the cell membrane. Preferred cells include cells from mammals, yeast, Drosophila, or E coli. Cells expressing KGF-2 (or the cell membrane containing the expressed polypeptide) are then preferably contacted with a test compound that potentially contains the molecule, to observe binding, stimulation, or inhibition of activity already Be it KGF-2 or the molecule. The assay can simply prove the binding of a candidate compound to KGF-2, where the link is detected by a marker, or in an assay that involves competition with a marked competitor. In addition, the assay can test whether the candidate compound results in a signal generated by the binding to KGF-2. Alternatively, the assay can be carried out using cell-free preparations, polypeptide / molecule attached to a solid support, chemical libraries, or mixtures of natural products. The assay may also simply comprise the steps of mixing a candidate compound with a solution containing KGF-2, measuring the activity of KGF-2 / molecule or the link, and comparing the activity of KGF-2 / molecule or the link to a standard. Preferably, an ELISA assay can measure the level or activity of KGF-2 in a sample (eg, biological sample) using a monoclonal or polyclonal antibody. The antibody can measure the level or activity of KGF-2 either by binding, directly or indirectly, to KGF-2 or by competition with KGF-2 for a substrate. In addition, the receptor to which KGF-2 is linked can be identified by numerous methods known to those skilled in the art, for example, the panoramic representation of the ligand and the FACS classification (Coligan et al., Current Protocols In Immun., 1 (2) .Chapter 5, (1991)). For example, expression cloning is employed wherein the polyadenylated RNA is prepared from a cell that responds to the polypeptides, e.g., NIH3T3 cells known to contain multiple receptors for the FGF family proteins, and the cells SC-3, and a cDNA library created from this RNA is divided into pools and used to transfect COS cells or other cells that do not respond to the polypeptides. Transfected cells that are developed on glass slides are exposed to the polypeptide of the present invention, after they have been labeled. The polypeptides can be labeled by a variety of means including iodination or inclusion of a recognition site for a site-specific protein kinase. After fixation and incubation, the slides are subject to autoradiographic analysis. The positive combinations are identified, and the subcombinados are prepared and retransfected using a process of subcombination and iterative reselection, eventually producing a simple clone that codes for the putative receptor. As an alternative method for identification of the receptor, the tagged polypeptides can be linked by photoaffinity with the cell membrane or extraction preparations expressing the receptor molecule. The crosslinked material is resolved by PAGE analysis and exposed to X-ray film. The labeled complex containing the polypeptide receptors can be excised, resolved into peptide fragments, and subject to protein microsequencing. The amino acid sequence obtained from the microsequencing could be used to design a group of degenerate oligonucleotide probes to select a cDNA library to identify the genes encoding the putative receptors. In addition, the techniques of gene intermixing, intermingling of portions, intermixing of exons, and / or intermixing codons (collectively referred to as "DNA intermixing") can be employed to modulate the activities of KGF-2 thereby generating effectively agonists and antagonists of KGF-2. See in general, U.S. Patent Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252, and 5,837,458, and Patten P.A., et al., Curr. Opinion Biotechnol. 8: 724-33 (1997); Harayama, S. Trends Biotechnol. 16 (2); 76-82 (1998); Hansson, L.O., et al., J. Mol. Biol. 287: 265-76 (1999); and Lorenzo, M. M. and Blasco, R. Biotechniques 24 (2): 308-13 (1998) (each of these patents and publications are incorporated by reference herein). In one embodiment, the alteration of the KGF-2 polynucleotides and the corresponding polypeptides can be achieved by DNA intermixing. DNA intermixing involves the assembly of two or more segments of DNA into a desired KGF-2 molecule by homologous or site-specific recombination. In yet another embodiment, the KGF-2 polynucleotides and the corresponding polypeptides can be altered by subjecting them to random mutagenesis by error-prone PCR, random insertion of nucleotides or other methods prior to recombination. In yet another embodiment, one or more components, portions, sections, parts, domains, fragments, etc. of KGF-2 can be recombined with one or more components, portions, sections, parts, domains, fragments, etc. of one or more heterologous molecules. In preferred embodiments, the heterologous molecules are members of the fibroblast growth factor family. In additional preferred embodiments, the heterologous molecule is a growth factor such as, for example, platelet derived growth factor (PDGF), insulin-like growth factor (IGF-I), transforming growth factor (TGF). ) -alpha, epidermal growth factor (EGF), fibroblast growth factor (FGF), TGF-beta, bone morphogenetic protein (BMP) -2, BMP-4, BMP-5, BMP-6, BMP-7, activins A and B, decapentagic (ddp), 60A, OP-2, dorsalin, growth differentiation factors (GDFs), factor nodal, MIS, inhibin-alpha, TGF-betal, TGF-beta2, TGF-beta3, TGF-beta5, and glia-derived neurotrophic factor (GDNF). Other preferred fragments are the biologically active KGF-2 fragments. The biologically active fragments are those that show activity similar, but not necessarily identical, to an activity of the KGF-2 polypeptide. The biological activity of the fragments may include a desired, improved activity, or a decreased undesirable activity. Additionally, this invention provides a method for screening compounds to identify those that modulate the action of the polypeptide of the present invention. An example of such an assay comprises the combination of a mammalian fibroblast cell, the polypeptide of the present invention, the compound to be selected and 3 [H] -thymidine under cell culture conditions where the fibroblast cell could proliferate normally . A control assay can be performed in the absence of the compound to be selected and compared to the amount of proliferation of fibroblasts in the presence of the compound to determine whether the compound stimulates proliferation by determining the uptake of 3 [H] -thymidine in each case. The amount of fibroblast cell proliferation is measured by liquid scintillation chromatography which measures the incorporation of 3 [H] -thymidine. The agonist and or antagonist compounds can be identified by this procedure. In yet another method, a mammalian cell or a membrane preparation that expresses a receptor for a polypeptide of the present invention is incubated with a labeled polypeptide of the present invention, in the presence of the compound. The ability of the compound to improve or block this interaction could then be measured. Alternatively, the response of a known second messenger system, after the interaction of a compound to be selected and the KGF-2 receptor, is measured, and the ability of the compound to bind to the receptor is also measured, and promote a second messenger response, to determine if the compound is a potential agonist or antagonist. Such second messenger systems include, but are not limited to, cAMP guanylate cyclase, ion channels or phosphoinositide hydrolysis. All previous trials can be used as diagnostic or prognostic markers. The molecules discovered using these assays can be used to treat diseases or to give rise to a particular result in a patient (e.g., development of blood vessels) by activation or inhibition of KGF-2 / molecule. In addition, assays can detect agents that can inhibit or enhance the production of KGF-2 from properly manipulated cells or tissues. Therefore, the invention includes a method for identifying compounds that bind to KGF-2, comprising the steps of: (a) incubating a candidate binding compound with KGF-2; and (b) the determination of whether the link has occurred. In addition, the invention includes a method for identifying agonists / antagonists, comprising the steps of: (a) incubating a candidate compound with KGF-2, (b) evaluating a biological activity, and (c) determining whether a biological activity of KGF-2 has been altered. Also, molecules that bind to KGF-2 could be identified experimentally by using the folded beta sheet regions described in Figure 4 and Table 1. Accordingly, the specific embodiments of the invention are directed to the polynucleotides that encode for the polypeptides comprising, or consisting alternatively of, the amino acid sequence of each of the folded beta sheet regions described in Figure 4 / Table 1. Additional embodiments of the invention are directed to the polynucleotides encoding the KGF-2 polypeptides comprising, or alternatively consisting of, any combination or all regions of folded beta sheet described in Figure 4 / Table 1. Additional preferred embodiments of the invention are directed to polypeptides comprising, or consisting alternatively of the amino acid sequence of KGF-2 from each of the folded beta sheet regions described in Figure 4 / Table 1. Additional embodiments of the invention are directed to KGF-2 polypeptides which comprise, or consist alternatively of, any combination or all the folded beta sheet regions described in Figure 4 / Table 1.

Antisense and Ribozyme Antagonists In specific embodiments, the antagonists according to the present invention are nucleic acids corresponding to the sequences contained in SEQ ID NO. 1, or the complementary strand thereof and / or the nucleotide sequences contained in the deposited clone 75977. In one embodiment, the antisense sequence is generated internally by the organism, in another embodiment, the antisense sequence is separately administered (see, for example, O'Connor, J., Neurochem, 56: 560 (1991), Oligodeoxynucleotides as gene expression antisense inhibitors, CRC Press, Boca Raton, FL (1988) .The antisense technology can be used to control the expression of the gene. through DNA or antisense RNA, or through the formation of the triple helix Antisense techniques are discussed, for example, in Okano, J., Neurochem, 56: 560 (1991); Oligodeoxynucleotides as antisense inhibitors of gene expression, CRC Press , Boca Raton, FL (1988) .The formation of the triple helix is discussed, for example, in Lee et al., Nucleic Acid Research 6: 3073 (1979), Cooney et al., Science 241: 456 (1988); Dervan et al ., Science 251: 1300 (1991). The methods are based on the binding of a polynucleotide to a complementary DNA or RNA. For example, the 5 'coding portion of a polynucleotide encoding the mature polypeptide of the present invention can be used to design an antisense RNA oligonucleotide of about 10 to 40 base pairs in length. A DNA oligonucleotide is designed to be complementary to a region of the gene involved in transcription, thereby preventing transcription and receptor production. The antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks the translation of the mRNA molecule into the receptor polypeptide. In one embodiment, the KGF-2 antisense nucleic acid of the invention is produced intracellularly by transcription from an exogenous sequence. For example, a vector or a portion thereof is transcribed, producing an antisense nucleic acid (RNA) of the invention. Such a vector could contain a sequence encoding the KGF-2 antisense nucleic acid. Such a vector can remain episomal or become chromosomally integrated, as long as it can be transcribed to produce the desired antisense RNA. Such vectors can be constructed by the methods of recombinant DNA technology, standard in the art. The vectors can be plasmid, viral, or others known in the art, used for replication and expression in vertebrate cells. The expression of the sequence encoding KGF-2, or fragments thereof, can be by any promoter known in the art to act on vertebrates, preferably on human cells. Such promoters may be inducible or constitutive. Such promoters include, but are not limited to, the SV40 early promoter region (Bernoist and Chambon, Nature 29: 304-310 (1981), the promoter contained in the 3 'long terminal repeat of the Rous sarcoma virus (Yamamoto et al. al., Cell 22: 787-797 (1980), the herpes thymidine promoter (Wagner et al., Proc. Nati, Acad. Sci. USA 78: 1441 (1981), the regulatory sequences of the metalathionionein gene ( Brinster, et al., Nature 296: 39-42 (1982)), etc. The antisense nucleic acids of the invention comprise a sequence complementary to at least a portion of an RNA transcript of a KGF-2 gene. , absolute complementarity, although preferred, is not required A sequence "complementary to at least one portion of RNA", referred to herein, means a sequence that has sufficient complementarity to be able to hybridize with RNA, forming a duplex stable, in the case of the KGF-2 antisense nucleic acids of double strand, a single strand of duplex DNA can thus be tested, or the formation of the triple strand can be evaluated. The ability to hybridize will depend on the degree of complementarity and the length of the antisense nucleic acid. In general, the larger the hybridization nucleic acid, the more base mismatches with the KGF-2 RNA can contain and still form a stable duplex (or triples as the case may be). A person of skill in the art can evaluate a tolerable degree of mis-coupling by using stable procedures to determine the melting point of the hybridized complex. Oligonucleotides that are complementary to the 5 'end of the message, for example, the 5' untranslated sequence up to and including the AUG start codon, must function more efficiently by inhibiting translation. However, the sequences complementary to the 3 'untranslated sequences of the mRNAs have shown that they are effective at t-to inhibit the translation of mRNAs too. See generally, Wagner R., 1994, Nature 372: 333-335. Thus, oligonucleotides complementary to the non-coding 5 'or 3' untranslated regions of KGF-2, shown in Figures 1A-B, could be used in an antisense method to inhibit translation of KGF-2 mRNA. endogenous. Oligonucleotides complementary to the 5 'untranslated region of the mRNA must include the complement of the AUG start codon. The antisense oligonucleotides complementary to the mRNA coding regions are less efficient inhibitors of translation but could be used according to the invention. Whether designed to hybridize to the 5 ', 3' or coding region of KGF-2 mRNA, the antisense nucleic acids must be at least 6 nucleotides in length, and are preferably oligonucleotides in the range of 6 to about 50 nucleotides in length. In specific aspects, the oligonucleotide is at least 10 nucleotides, at least 17 nucleotides, at least 25 nucleotides or at least 50 nucleotides. The polynucleotides of the invention may be DNA or RNA or chimeric mixtures or derivatives or modified versions thereof, single-stranded or double-stranded. The oligonucleotide can be modified in the base portion, in the sugar portion, or in the phosphate backbone, for example, to improve the stability of the molecule, the hybridization, etc. The oligonucleotide may include other adjoining groups such as peptides (for example, to target receptors of the host cell in vivo), or agents that facilitate transport across the cell membrane (see for example Letsinger et al., 1989, Proc, Nati, Acad. Sci. USA 86: 6553-6556; Lemaitre et al., 1987, Proc, Nati, Acad. Sci. 86: 648-652; PCT Publication No. WO88 / 09810, published on December 15. 1988) or the blood-brain barrier (see for example PCT Publication No. WO89 / 10134, published April 25, 1988), cleavage agents triggered by hybridization (see for example Krol et al., 1988, Bio Techniques 6: 958-976) or intercalating agents (see for example Zon, 1988, Pharm. Res. 5: 539-549). For this purpose, the oligonucleotide can be conjugated to another molecule, for example, a peptide, the crosslinking agent triggered by the hybridization, the transport agent, the cleavage agent triggered by hybridization, etc. The antisense oligonucleotide may comprise at least one modified base portion which is selected from the group including, but not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5- (carboxyhydroxymethyl) -uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-arabinose, 2-fluoroarabinose, xylulose, and hexose. In yet another embodiment, the antisense oligonucleotide comprises at least one modified phosphate backbone selected from the group including, but not limited to, phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a phosphoniamidate, a methylphosphonate, an alkyl phosphotriester. , and formacet to or analogous thereof.

In yet another embodiment, the antisense oligonucleotide is an α-anomeric oligonucleotide. An α-anomeric antisense oligonucleotide forms specific double-stranded hybrids with complementary RNA er. which, contrary to the usual units, the strands run parallel to one another (Gautier et al., FL987, Nucí, Acid Res. 15: 6625-6641). The oligonucleotide is uh 2 ' O-methylribonucleotide (Inoue et al., 1987, Nucí Acids Res. 15: 6131-6148), or a chimeric RNA-DNA analogue (Inoue et al., 1987, FEB Lett 215: 327-330). The polynucleotides of the invention can be synthesized by standard methods known e: the art, for example, by the use of an automated DNA synthesizer (such as are commercially available from Biosearch, Applied Biosystems, etc.). As examples, the phosphorothioate oligonucleotides can be synthesized by the method of Stein et al., (1988), Nucí. Acids Res. 16: 3209), the methylphosphonate oligonucleotides can be prepared by the use of controlled pore glass polymeric supports J (Sarin et al., 1988, Proc. Nati Acad. Sci. USA 85: 7448-7451) , etc., While antiseiitide nucleotides complementary to the sequence of the coding region of KGF-2 could be used, those complementary to the transcribed untranslated region, are most preferred. Potential antagonists according to the invention also include catalytic RNA. , or a ribozyme (see, for example, the PCT International publication WO 90/11364, published on October 4, 1990; Server et al., Science 247: 1222-1225 (1990). While ribozymes that break the mRNA in site-specific recognition sequences can be used to destroy KGF-2 mRNAs, the use of ribozymes in the form of a hammerhead is preferred. Hammerhead-shaped ribozymes break mRNAs at sites dictated by the flanking regions that form the base pairs complementary to the target mRNA. The only requirement is that the target mRNA has the following sequence of two bases: 5'-UG-3 '. The construction and production of ribozymes in the form of a hammerhead is well known in the art and is described more fully in Haseloff and Gerlach, Nature 334: 585-591 (1988). There are numerous potential hammerhead ribozyme cleavage sites within the nucleotide sequence of KGF-2 (Figures 1A-B). Preferably, the ribozyme is engineered so that the cleavage recognition site is located near the 5 'end of the KGF-2 mRNA; for example to increase efficiency and minimize the intracellular accumulation of non-functional mRNA transcripts.

As in the antisense procedure, the ribozymes of the invention can be composed of modified oligonucleotides (eg, for improved stability, targeting, etc.) and must be distributed to cells expressing KGF-2 in vivo. The DNA constructs encoding the ribozyme can be introduced into the cell in the same manner as described above for the introduction of the antisense coding DNA. A preferred method of administration involves the use of a DNA construct "encoding" for the ribozyme under the control of a strong constitutive promoter, such as, for example, the pol III or pol II promoter, so that the transfected cells will produce sufficient amounts of ribozyme to destroy the endogenous KGF-2 messages and inhibit translation. Since ribozymes in a manner contrary to antisense molecules are catalytic, a lower intracellular concentration is required for efficiency. The antagonist / agonist compounds can be used to inhibit cell growth and the proliferation effects of the polypeptides of the present invention on neoplastic cells and neoplastic tissues, for example the stimulation of tumor angiogenesis, and therefore, retard or prevent abnormal cell development and proliferation, for example, in the formation or development of tumors. The antagonist / agonist can also be used to prevent hypervascular diseases, and prevent the proliferation of epithelial lens cells after extracapsular cataract surgery. The prevention of mitogenic activity of the polypeptides of the present invention may also be desired in cases such as restenosis after balloon angioplasty. The antagonist / agonist can also be used to prevent the development of scar tissue during wound healing. The antagonist / agonist can also be used to treat the diseases described herein.

Other activities The polypeptide of the present invention, as a result of the ability to stimulate the development of vascular endothelial cells, can be employed in the treatment to stimulate revascularization of ischemic tissue due to various disease conditions such as thrombosis, arteriosclerosis and other conditions cardiovascular These polypeptides can also be used to stimulate angiogenesis and regeneration of the extremities, as discussed above. The polypeptide can also be used to treat wounds due to trauma, burns, post-operative tissue repair, and ulcers, since these are mitogenic for various cells of different origins, such as fibroblast cells and skeletal muscle cells, and for therefore they facilitate the repair or replacement of damaged or diseased tissue. The polypeptide of the present invention can also be used to stimulate neuronal development and to treat and prevent neuronal damage that occurs in certain neuronal disorders or neurodegenerative conditions such as Alzheimer's disease, Parkinson's disease, and AIDS-related complex. . KGF-2 may also have the ability to stimulate the development of chondrocytes, therefore, chondrocytes may be used to improve bone and periodontal regeneration, and aid in tissue transplants or bone grafts. The polypeptide of the present invention can also be employed to prevent skin aging due to sunburn by stimulating the growth of keratinocytes. The KGF-2 polypeptide can also be used to prevent hair loss, since members of the FGF family activate the hair-forming cells and promote the growth of melanocytes. Along the same lines, the polypeptides of the present invention can be used to stimulate the development and differentiation of hematopoietic cells and cells of the bone marrow., when used in combination with other cytokines. The KGF-2 polypeptide can also be used to maintain the organs before transplantation or to support the culture of primary tissue cells. The polypeptide of the present invention can also be used to induce tissue of mesodermal origin to differentiate into early embryos. The KGF-2 polynucleotides or polypeptide, or KGF-2 agonists or antagonists, can also increase or decrease the differentiation or proliferation of embryonic stem cells, in addition to, as discussed above, the hematopoietic line. KJGF-2 polynucleotides or polypeptides, or KGF-2 agonists or antagonists, can also be used to modulate the characteristics of mammals, such as body weight, body stature, hair color, eye color , the skin, the percentage of adipose tissue, pigmentation, size, and 1 to form (for example, cosmetic surgery). Similarly, KGF-2 polynucleotides or polypeptides, or KGF-2 agonists or antagonists, can be used to modulate mammalian metabolism, which affects catabolism, anabolism, processing, utilization, and energy storage. Polynucleotides or KGF-2 polypeptides, or KGF-2 agonists or antagonists, can be used to change a mental state or physical state of the mammal by influencing biorhythms, heart rhythms, depression (including depressive disorders) , the tendency to violence, tolerance to pain, reproductive capacities (preferably due to activity of activin or similar to inhibin), hormonal or endocrine levels, appetite, libido, memory, stress, or other cognitive qualities . KGF-2 polynucleotides or polypeptides, or KGF-2 agonists or antagonists, can also be used as a food additive or preservative, such as to increase or decrease storage capacities, fat content, lipids, proteins, carbohydrates, vitamins, minerals, cofactors or other nutritional components. The above-mentioned applications have uses in a wide variety of guests. Such hosts include, but are not limited to, humans, murines, rabbits, goats, guinea pigs, camels, horses, mice, rats, hamsters, pigs, micro-culprits, chickens, goats, cows, sheep, dogs, cats, non-human primates, and humans. In specific modalities, the host is a mouse, rabbit, goat, guinea pig, chicken, hamster, pig, sheep, dog or cat. In preferred embodiments, the host is a mammal. In the most preferred embodiments, the host is a human.

Diagnosis and image formation The labeled antibodies, and the derivatives and analogs thereof, which specifically bind to a polypeptide of interest, may be used for diagnostic purposes to detect, diagnose, or periodically verify diseases, disorders, and / or conditions. associated with the aberrant expression and / or the activity of a polypeptide of the invention. The invention provides for the detection of aberrant expression of a polypeptide of interest, comprising (a) evaluating the expression of the polypeptide of interest in cells or in an individual's body fluid, using one or more antibodies specific for the polypeptide of interest and ( b) comparing the level of expression of the gene with a standard gene expression level, whereby an increase or decrease in the level of expression of the polypeptide gene evaluated, compared to the level of standard expression, is indicative of an aberrant expression. The invention provides a diagnostic assay for diagnosing a disorder, comprising (a) evaluating the expression of the polypeptide of interest in the cells or in the body fluid of an individual, using one or more antibodies specific for the polypeptide of interest and (b) ) comparing the level of expression of the gene with a level of expression of the standard gene, whereby an increase or decrease in the level of expression in the gene of the polypeptide evaluated, compared to the level of standard expression is indicative of a particular disorder. With respect to cancer, the presence of a relatively high amount of the transcript in biopsy tissue from an individual may indicate a predisposition for the development of the disease, or may provide a means to detect the disease prior to the appearance of the disease. Real clinical symptoms. A more definitive diagnosis of this type may allow health professionals to use preventive measures or aggressive treatment earlier, thus preventing the further development or progression of the cancer. The antibodies of the invention can be used to evaluate protein levels in a biological sample using classical immunohistochemical methods known to those skilled in the art (for example see Jalkanen, et al., J. Cell, Biol. 101: 976-985 (1985), Jalkanen, et al., J. Cell, Biol. 105: 3087-3096 (1987)). Other antibody-based methods useful for detecting the expression of the protein gene include immunoassays, such as enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA). Suitable antibody assay labels are known in the art and include enzymatic labels, such as glucose oxidase; radioisotopes, such as iodine (125 I, 121 I), carbon (14 C), sulfur (35 S), tritium (3 H), indium (112 In), and technetium (99 Tc); luminescent markers, such as luminol; and fluorescent labels, such as fluorescein and rhodamine, and biotin. One aspect of the invention is the detection and diagnosis of a disease or disorder associated with the aberrant expression of a polypeptide of interest in an animal, preferably a mammal and most preferably a human. In one embodiment, the diagnosis comprises: a) administration (eg, parenterally, subcutaneously, or intraperitoneally) to a subject of an effective amount of a labeled molecule that specifically binds to the polypeptide of interest; b) waiting a time interval after administration, to allow the labeled molecule to be concentrated preferentially at sites in the subject where the polypeptide is expressed (and so that the labeled, unbound molecule is cleared at the background level); c) determine the antecedent level; d) detecting the labeled molecule in the subject, such that detection of the marked molecule above the background level indicates that the subject has a particular disease or a particular disorder associated with the aberrant expression of the polypeptide of interest. The antecedent level can be determined by various methods including, the comparison of the amount of the labeled molecule detected to a predetermined standard value for a particular system. It will be understood in the art that the size of the subject and the image formation system used will determine the amount of the portion of image formation necessary to produce diagnostic images. In the case of a radioisotope portion, for a human subject, the amount of radioactivity injected will normally be in the range of about 5 to 20 millicuries of 99mTc. The labeled antibody or antibody fragment will then accumulate preferentially at the site of the cells containing the specific protein. Tumor imaging in vivo is described in S.W. Burchiel et al., "Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments" (Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, SWBurchiel and BA Rodees, eds., Masson Publishing Inc. (1982) .Depending on various variables, including the type of marker used and the mode of administration, the time interval after administration to allow the labeled molecule to be concentrated preferentially at sites in the subject and for the unbound labeled molecule to be cleared at the background level, is 6 at 48 hours or from 6 to 24 hours, or from 6 to 12 hours In another modality, the time interval after administration is from 5 to 20 days or from 5 to 10 days. of the disease or disorder is carried out by repeating the method for diagnosis of the disease, for example, one month after the initial diagnosis, 6 months after the initial diagnosis, one year after of the initial diagnosis, etc. The presence of the labeled molecule can be detected in the patient using methods known in the art for in vivo screening. These methods depend on the type of marker used. Those skilled in the art will be able to determine the appropriate method for detecting a particular marker. Methods and devices that can be used in diagnostic methods of the invention include, but are not limited to, computed tomography (CTC), whole-body scanning such as positron emission tomography (PET), image imaging magnetic resonance imaging (MRI), and sonography. In a specific modality, the molecule is labeled with a radioisotope and is detected in the patient using a surgical instrument that responds to radiation (Thurston et al., United States Patent No. 5,441,050). In yet another embodiment, the molecule is labeled with a fluorescent compound and is detected in the patient using a scanning instrument that responds to fluorescence. In yet another embodiment, the molecule is labeled with a positron emitting metal and is detected in the patient using positron emission tomography. In yet another embodiment, the molecule is labeled with a paramagnetic marker and detected in a patient using magnetic resonance imaging (MRI).

Equipment The present invention provides the equipment that can be used in the above methods. In one embodiment, the kit comprises an antibody of the invention, preferably a purified antibody, in one or more containers. In a specific embodiment, the kits of the present invention contain a substantially isolated polypeptide comprising an epitope that is specifically immunoreactive with an antibody included in the kit. Preferably, the kits of the present invention further comprise a control antibody that does not react with the polypeptide of interest. In another specific embodiment, the kits of the present invention contain a means for detecting the binding of an antibody to a polypeptide of interest (eg, the antibody can be conjugated to a detectable substrate such as a fluorescent compound, an enzyme substrate, a radioactive compound or a luminescent compound, or a second antibody that recognizes the first antibody, can be conjugated to a detectable substrate). In another specific embodiment of the present invention, the kit is a diagnostic kit for use in the selection of serum containing specific antibodies against proliferative and / or cancerous polynucleotides and polypeptides. Such a kit may include a control antibody that does not react with the polypeptide of interest. Such a kit can include a substantially isolated polypeptide antigen comprising an epitope that is specifically immunoreactive with at least one anti-polypeptide antigen antibody. In addition, such equipment includes means for detecting the binding of the antibody to the antigen (for example, the antibody can be conjugated to a fluorescent compound such as fluoroscein or rhodamine which can be detected by flow cytometry). In specific embodiments, the kit can include a recombinantly produced or chemically synthesized polypeptide antigen. The polypeptide antigen of the kit can also be linked to a solid support. In a more specific embodiment, the detection means of the above described equipment includes a solid support to which the polypeptide antigen is linked. Such equipment may also include an anti-human antibody, labeled with the reporter, not linked. In this embodiment, the binding of the antibody to the polypeptide antigen can be detected by linking the labeled antibody with the reporter. In a further embodiment, the invention includes a diagnostic kit for use in the selection of serum containing antigens of the polypeptide of the invention. The diagnostic kit includes a substantially isolated antibody, specifically immunoreactive with the polypeptide or polynucleotide antigens, and means for detecting the binding of the polynucleotide or polypeptide antigen to the antibody. In one embodiment, the antibody is bound to a solid support. In a specific embodiment, the antibody can be a monoclonal antibody. The detection means of the kit can include a second monoclonal antibody, labeled. Alternatively, or in addition, the detection means may include a competing, labeled antigen. In a diagnostic configuration, the test serum is reacted with a solid phase reagent having an antigen bound to the surface, obtained by the methods of the present invention. After binding with the specific antigen antibody to the reagent, and by removing the unbound serum components, by washing, the reagent is reacted with the anti-human antibody, labeled with the reporter, to bind the reporter to the reagent in proportion to the amount of the bound anti-antigen antibody, on the solid support. The reagent is again washed to remove the labeled, unbound antibody, and the amount of reporter associated with the reagent is determined. Typically, the reporter is an enzyme that is detected by incubating the solid phase in the presence of a suitable fluorometric, luminescent or colorimetric substrate (Sigma, St. Louis, MO). The solid surface reagent in the above test is prepared by known techniques for binding the protein material to the solid support material, such as polymer spheres, dip bars, 96-well plate or filter material. These binding methods generally include non-specific adsorption of the protein to the support or covalent linkage of the protein, typically through a free amino group, to a chemically reactive group on the solid support, such as a carboxyl, hydroxyl group , or aldehyde activated. Alternatively, streptavidin coated plates can be used in conjunction with the biotinylated antigen (s). In this way, the invention provides a system or test equipment for carrying out this diagnostic method. The kit includes, in general, a support with recombinant antigens linked to the surface, and an anti-human antibody, labeled with a reporter, to detect the anti-antigen antibody bound to the surface. Having generally described the invention, it will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended to be limiting.

Example 1 Bacterial Expression and Purification of KGF-2 The DNA sequence encoding KGF-2, ATCC # 75977, is initially amplified using the oligonucleotide PCR primers corresponding to the sequences of the 5 'and 3' ends of the processed KGF-2 cDNA (including the peptide sequence) of signal). The 5 'oligonucleotide primer has the sequence: 5' CCCCACATGTGGAAATGGATACTGACACATTGTGCC 3 '(SEQ ID No. 3) containing an AflIII restriction enzyme site including and followed by 30 nucleotides of the KGF-2 coding sequence, starting from the presumed presumed start codon. The 3 'sequence: 5' CCCAAGCTTCCACAAACGTTGCCTTCCTCTATGAG 3 '(SEQ ID No. 4) contains the sequences complementary to the HindIII site and is followed by 26 nucleotides of KGF-2. The restriction enzyme sites are compatible with the restriction enzyme sites on the bacterial expression vector pQE-60 (Qiagen, Inc. Chatsworth, California). pQE-60 codes for antibiotic resistance (Ampr), a bacterial origin of replication (ori), an operator promoter that can be regulated by IPTG (P / 0), a ribosome binding site (RBS), a 6-His marker and restriction enzyme sites. pQE-60 is then digested with Ncol and HindIII. The amplified sequences are ligated into pQE-60 and inserted intra-structurally. The ligation mixture is then used to transform E. coli strain M15 / rep 4 (Qiagen, Inc.) by the procedure described in Sambrook, J., et al. , Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory Press, (1989). M15 / rep4 contains multiple copies of plasmid pREP4, which expresses the lacl repressor and also confers resistance to kanamycin (Kanr). Transformants are identified by their ability to grow on LB plates and colonies resistant to ampicillin / kanamycin are selected. The plasmid DNA is isolated and confirmed by restriction analysis. Clones containing the desired constructs are grown overnight (0 / N) in liquid culture in LB medium supplemented with Amp (100 μg / ml) and Kan (25 μg / ml). O / N culture is used to inoculate a large crop at a ratio of 1: 100 to 1: 250. The cells are developed at an optical density 600 (O./D.600) of between 0.4 and 0.6. IPTG ("isopropyl-B-D-thiogalactopyranoside") is then added to a final concentration of 1 mM. IPTG interacts with the lacl repressor to cause it to dissociate itself from the operator, forcing the promoter to direct transcription. The cells are developed from 3 to 4 extra hours. The cells are then harvested by centrifugation. The cellular button is solubilized in the chaotropic agent Guanidine 6 Molar hydrochloride. After clarification, solubilized KGF-2 is purified from this solution by chromatography on a Heparin affinity column under conditions that allow strong binding of the proteins (Hochuli E et al., J. Chromatography 411: 177-184 (1984) .KGF-2 (75% pure) is eluted from the column with high salinity buffer.

Example 2 Bacterial Expression and Purification of a Truncated Version of KGF -2 The DNA sequence that codes for KGF-2, ATCC # 75977, is initially amplified using the oligonucleotide PCR primers corresponding to the 5 'and 3' sequences of the truncated version of the KGF-2 polypeptide. The truncated version comprises the polypeptide minus the 36 amino acid signal sequence, with a methionine and alanine residue that is added just before the cysteine residue comprising amino acid 37 of the full-length protein. The 5 'oligonucleotide primer has the 5' sequence CATGCCATGGCGTGCCAAGCCCTTGGTCAGGACATG 3 '(SEQ ID No. 5) contains a Ncol restriction enzyme site including and followed by 24 nucleotides of the KGF-2 coding sequence. The 3 'sequence of 5' CCCAAGCTTCCACAAACGTTGC CTTCCTC TATGAG 3 '(SEQ ID No. 6) contains the sequences complementary to the Hind III site and is followed by 26 nucleotides of the KGF-2 gene. The restriction enzyme sites are compatible with the restriction enzyme sites on the bacterial expression vector pQE-60 (Qiagen, Inc., Chatsworth, CA). pQE-60 codes for antibiotic resistance (Ampr), a bacterial origin of replication (ori), a promoter operator that can be regulated by * _ÍA iá? IPTG (P / 0), a ribosome binding site (RBS), a 6-His tag and the restriction enzyme sites. pQE-60 is then digested with Ncol and HindIII. The amplified sequences are ligated into pQE-60 and are inserted into the structure. The ligation mixture is then used to transform E. coli strain M15 / rep4 (Qiagen, Inc.) by the procedure described in Sambrook, J., et al., Molecular Cloning: A Laboratory Manual, Cold, Spring Laboratory Press, (1989 ). M15 / rep4 contains multiple copies of plasmid pREP4, expresses the lacl repressor and also confers resistance to kanamycin (Kanr). Transformants are identified by their ability to grow on LB plates and colonies resistant to ampicillin / kanamycin are selected. The plasmid DNA is isolated and confirmed by restriction analysis. Clones containing the desired constructs are grown overnight (O / N) in liquid culture in LB medium supplemented with Amp (100 μg / ml) and Kan (25 μg / ml). O / N culture is used to inoculate a large crop at a ratio of 1: 100 to 1: 250. The cells are developed at an optical density 600 (O.D.600) of between 04 and 0.6. IPTG ("Isopropyl-B-D-thiogalactopyranoside") is then added to a final concentration of 1 mM. IPTG induces by activating the laci repressor, clearing P / 0 leading to increased gene expression. The cells are developed 3 to 4 extra hours. The cells are then harvested by centrifugation. The cell button is solubilized in the chaotropic agent Guanidine 6 Molar hydrochloride. After clarification, the solubilized KGF-2 is purified from this solution by chromatography on a heparin affinity column under conditions that allow strong binding of the proteins (Hochuli, E. et al, J ". Chromatography 411: 177 - 184 (1984)) The KGF-2 protein is eluted from the column by the high-salinity buffer.

Example 3 Cloning and Expression of KGF-2 Using the Baculoviral Expression System The DNA sequence encoding the full-length KGF-2 protein, ATCC # 75977, is amplified using the oligonucleotide PCR primers corresponding to the 5 'and 3' sequences of the gene: The 5 'primer has the sequence 5' GCGGGATCCGCCATCATGTGGAAATGGATACTCAC3 '(SEQ ID No. 7) and contains a BamHI restriction enzyme site (in bold) followed by 6 nucleotides that resemble an efficient signal for the initiation of translation in eukaryotic cells (Kozak, M., J. Mol. Biol., 196: 947-950 (1987)) and just behind the first 17 nucleotides of the KGF-2 gene (the start codon for the translation "ATG" is underlined). The 3 'primer has the sequence 5' GCGCGGTACCACAAACGTTGCCTTCCT 3 '(SEQ ID No. 8) and contains the cleavage site for the restriction endonuclease Asp718 and 19 nucleotides complementary to the 3' untranslated sequence of the KGF-2 gene. The amplified sequences are isolated from a 1% agarose gel using a commercially available equipment from Qiagen, Inc., Chatsworth, CA. The fragment is then digested with the BamHI and Asp718 endonucleases and then purified again on a 1% agarose gel. This fragment is designated F2. Vector pA2 (modification of vector pVL941, discussed below) is used for the expression of KGF-2 protein using the baculoviral expression system (for review see Summers, M.D. &Smith, G. E., A manual of methods for baculovirus vectors and insect cell culture procedures, Texas Agricultural Experimental Station Bulletin No. 1555 (1987)). This expression vector contains the strong polyhedrin promoter of Autographa californica nuclear polyhidrosis virus (AcMNPV) followed by the recognition sites for the restriction endonucleases BamHI and Asp718. The polyadenylation site of simian virus (SV) 40 is used for efficient polyadenylation. For an easy selection of recombinant viruses, the beta-galactosidase gene from E. coli is inserted in the same orientation as the pilihedrine promoter., followed by the polyadenylation signal of the polyhedrin gene. The polyhedrin sequences are flanked on both sides by the viral sequences for the cell-mediated homologous recombination of the wild-type, cotransfected viral DNA. Many other baculoviral vectors could be used such as pAc373, pVL941 and pAcIMl (Luckow, V.A. & amp;; Summers, M.D. Virology; 170: 31-39). The plasmid is digested with the restriction enzymes BamHI and Asp718. The DNA is then isolated from a 1% agarose gel using commercially available equipment (Qiagen, Inc., Chatsworth, CA). This vector DNA is designated V2. The fragment F2 and the plasmid V2 are ligated with the T4 DNA ligase. The E. coli cells of HB101 are then transformed and the bacteria containing the plasmid (pBacKGF-2) are identified with the KGF-2 gene using PCR with the cloning oligonucleotides. The sequence of the cloned fragment is confirmed by DNA sequencing. 5 μg of plasmid pBacKGF-2 are cotransfected with 1.0 μg of commercially available linearized baculovirus (BaculoGold ™ Baculoviral DNA Pharmingen, San Diego, CA) using the lipofection method (Felgner, et al., Proc. Nati. Acad. Sci , USA, 84: 7413-7417 (1987)). 1 μg of BaculoGold ™ viral DNA and 5 μg of plasmid pBacKGF-2 are mixed in a sterile well of a microtiter plate containing 50 μl of serum-free Grace's medium (Life Technologies Inc., Gaithersburg, MD). After this 10 μl of lipofectin plus 90 μl of Grace's medium are added, mixed and incubated for 15 minutes at room temperature. Then the transfection mixture is added dropwise to the Sf9 insect cells (ATCC CRL 1711) seeded in a 35 mm tissue culture plate with 1 ml Grace's medium without serum. The plate is swung back and forth to mix the newly added solution. The plate is then incubated for 5 hours at 27 ° C. After 5 hours, the transfection solution is removed from the plate and 1 ml of Grace's insect medium supplemented with 10% fetal calf serum is added. The plate is then put back into an incubator and the culture is continued at 27 ° C for four days. After four days, the supernatant is harvested and plaque assay performed similarly as described by Summers and Smith (supra). As an amendment, an agarose gel with "Blue Gal" is used (Life Technologies Inc., Gaithersburg) that allows easy isolation of blue-stained plates. (A detailed description of a "plaque assay" can also be found in the user guide for the culture of insect and baculovirology cells distributed by Life Technologies Inc., Gaithersburg, pages 9-10). Four days after serial dilution, the viruses are added to the cells and the blue-stained plates are picked up with the tip of an Eppendorf pipette. The agar containing the recombinant viruses is then resuspended in an Eppendorf tube containing 200 μl of Grace's medium. The agar is removed by a brief centrifugation and the supernatant containing the recombinant baculovirus is used to infect the Sf9 cells seeded in 35 mm boxes. Four days later the supernatants of these culture boxes are harvested and then stored at 4 ° C. Sf9 cells are developed in Grace's medium supplemented with 10% FBS inactivated by heat. The cells are infected with the recombinant baculovirus V-KGF-2 at a multiplicity of infection (MOI) of 2. Six hours later, the medium is removed and replaced with the SF900 II medium minus methionine and cysteine (Life Technologies Inc., Gaithersburg ). 42 hours later, 5 μCi of 35S-methionine and 5 μCi of 35S-cysteine (Amersham) are added. The cells are subsequently incubated for 16 hours before they are harvested by centrifugation and the labeled proteins are visualized by SDS-PAGE and autoradiography.

Example 4 Most of the vectors used for the transient expression of the KGF-2 protein gene sequence in mammalian cells must carry the SV40 origin of replication. This allows replication of the vector at high copy numbers in the cells (e.g. COS cells) that express the T antigen required for the initiation of viral DNA synthesis. Any other mammalian cell line can also be used for this purpose. A typical mammalian expression vector contains the promoter element, which mediates the initiation of transcription of the mRNA, the coding sequence of the protein, and the signals required for the termination of transcription and polyadenylation of the transcript. Additional elements include enhancers, Kozak sequences and intervening sequences flanked by the donor and acceptor sites for RNA splicing. Highly efficient transcription can be achieved with the SV40 early and late promoters, the long terminal repeats (LTRs) from retroviruses, for example RSV, HTLVI, HIVI and the cytomegalovirus immediate early promoter (CMV). However, cellular signals can also be used (for example, the human actin promoter). Expression vectors suitable for use in the practice of the present invention include, for example, vectors such as pSVL and pMSG (Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37151), pSV2dhfr (ATCC 37146) and pBC12MI (ATCC 67109). Mammalian host cells that could be used include, for example, the human Hela, 283, H9 and Jurkart cells, NIH3T3 and C127 mouse cells, the African green monkey cells Cos 1, Cos 7 and CV1, the QC1 quail cells. -3, 293T cells, mouse L cells and Chinese hamster ovary cells. Alternatively, the gene can be expressed in stable cell lines that contain the gene integrated within a chromosome. Cotransfection with a selectable marker such as dhfr, gpt, neomycin, hygromycin follows the identification and isolation of the transfected cells. The transfected gene can also be amplified to express large amounts of the encoded protein. DHFR (dihydrofolate reductase) is a useful marker for developing cell lines that carry several hundred or even several thousand copies of the gene of interest. Another useful selection marker is the enzyme glutamine synthase (GS) (Murphy et al., Biochem J. 227: 279 (1991); Bebbington et al., Bio / Technology 10: 1 (59-175 (1992)). Using these markers, the mammalian cells are developed in selective medium and the cells with the highest resistance are selected. These cell lines contain the amplified gene (s) integrated within a chromosome. Chinese hamster ovary (CHO) cells are frequently used for the production of proteins. The pCI and pC4 expression vectors contain the strong promoter (LTR) of the Rous sarcoma virus (Cullen et al., Molecular and Cellular Biology, 438-447 (March 1985)) plus a fragment of the CMV enhancer (Bosha.rt et al. , Cell 41: 521-530 (1985)). Multiple cloning sites, for example, with the restriction site cleavage sites of BamHI, Xbal and Asp718, facilitate the cloning of the gene of interest. The vectors also contain the 3 'intron, the polyadenylation and termination signal of the rat preproinsulin gene.

A. Expression of Recombinant KGF-2 in COS Cells Plasmid expression, KGF-2 HA was derived from a pcDNAI / Amp vector (Invitrogen) containing: 1) the SV40 replication origin, 2) the ampicillin resistance gene, 3) the E. replication origin. coli, the CMV promoter followed by a polylinker region, an SV40 intron and the polyadenylation site. The HA marker corresponds to an epitope derived from the influenza hemagglutinin protein as previously described (Wilson, I., et al., 37: 767, (1984)). Infusion of the HA marker to the target protein allows easy detection of the recombinant protein with an antibody that recognizes the HA epitope. A DNA fragment encoding the precursor HA marker of the complete KGF-2 was intrastructurally fused with the HA marker, therefore, the expression of the recombinant protein is directed under the CMV promoter. The construction strategy of the plasmid is described as follows: The DNA sequence encoding KGF-2, ATCC # 75977, is constructed by PCR using two primers: the 5 ': 5' primer TAACGAGGATCCGCCATCATGTGGAAATGGATACTGACAC 3 '(SEQ ID No. 9) contains a BamHI site followed by 22 nucleotides of the KGF-2 coding sequence starting from the codon of start; the 3 'sequence: 5' TAAGCACTCGAGTGAGTGTACCACCATTGGAAGAAATG3 '(SEQ ID No. 10) contains the sequences complementary to an Xhol site, the HA marker and the last 26 nucleotides of the KGF-2 coding sequence (not including the stop codon) .

Thus, the PCR product containing a BamHI site, the KGF-2 coding sequence followed by an Xhol site, an HA marker intrastructurally fused, and a stop codon of translation termination next to the HA marker. The DNA fragment amplified by PCR and the vector, pcDNA-3'HA, are digested with the BamHI and Xhol restriction enzymes and ligated, resulting in pcDNA-3 'HA-KGF-2. The ligation mixture is transformed into E. coli strain XLI Blue (Stratagene Cloning Systems, La Jolla, CA) the transformed culture is seeded in plates on plates with ampicillin medium and the resistant colonies are selected. The plasmid DNA was isolated from the transformants and examined by PCR and restriction analysis for the presence of the correct fragment. For expression of recombinant KGF-2, COS cells were transfected with the expression vector by the DEAE-DEXTRAN method (Sambrook, J., et al Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory Press, (1989)) . Expression of the HA protein of KGF-2 was detected by radiolabelling and the immunoprecipitation method (Harlow, E. &Lane, D., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, (1988)). The cells were labeled for 8 hours with 35S-cysteine two days after transfection. The culture media was collected and the cells were lysed with detergent (RIPA buffer (150 mM NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 50 mM Tris, pH 7.5) (Wilson, I., et al, Id. 37: 767 (1984).) The cell lysate and the culture media were precipitated with a monoclonal antibody specific for HA.The precipitated proteins were analyzed on SDS-PAGE gels. to 15%.

B. Expression and Purification of human KGF-2 protein using a CHO Expression system The pCl vector is used for the expression of the KGF-2 protein. Plasmid pCl is a derivative of plasmid pSV2-dhfr [Access ATCC No. 37146]. Both plasmids contain the mouse DHFR gene under the control of the SV40 early promoter. Chinese hamster ovary cells or other cells lacking the hydrofolate activity that are transfected with these plasmids can be selected by developing the cells in a selective medium (alpha nebis MEM, Life Technologies), supplemented with the chemotherapeutic agent methotrexate The amplification of DHFR genes in methotrexate-resistant cells (MTX) has been well documented (see for example Alt, FW, Kellems, RM, Bertino, JR and Schimke, RT, 1978, J. Biol. Chem. 253: 1357 -1370, Hamlin, JL and Ma, C. 1990, Biochem et Biophys, Acta, 1097: 107-143, Page, MJ and Sydenham, MA 1991, Biotechnology Vol. 9: 64-68). Cells developed in increasing concentrations of MTX develop resistance to the drug by overproduction of the target enzyme, DHFR, as a result of the amplification of the DHFR gene. If a second gene is linked to the DHFR gene, it is usually co-amplified and overexpressed. It is in the state of the art to develop cell lines that have more than 1,000 copies of the genes. Subsequently, when methotrexate is removed, the cell lines contain the integrated amplified gene of the chromosome (s). Plasmid pCl contains, for the expression of the gene of interest, a strong promoter of the long terminal repeat (LTR) of the Rouse Sarcoma virus (Cullen, et al., Molecular and Cellular Biology, March 1985: 438-4470) plus an isolated fragment of the immediate early human cytomegalovirus (CMV) enhancer (Boshart et al., Cell 41: 521-530, 1985). Downstream (3 ') of the promoter are the following cleavage sites with simple restriction enzyme that allow integration of the genes: BamHI, Pvull, and Nrul. Behind these cloning sites the plasmid contains the stop codons of the translation in the three reading structures, followed by the 3 'intron and the polyadenylation site of the rat preproinsulin gene. Other highly efficient promoters can also be used for the expression, for example, the human β-actin promoter, the early or late SV40 promoters or the long terminal repeats from other retroviruses, for example HIV and HTLVI. For polyadenylation of mRNA other signals, for example, from human growth hormone or globin genes can also be used. Stable cell lines that possess a gene of interest integrated within the chromosomes can also be selected after cotransfection with a selectable marker such as gpt, G418 or hygromycin. It is advantageous to use more than one selectable marker at the beginning, for example, G418 plus methotrexate. The plasmid pCl is digested with the restriction enzyme BamHI and then dephosphorylated using the intestinal calf phosphatases by methods known in the art. The vector is then isolated from a 1% agarose gel. The DNA sequence encoding KGF-2, ATCC No. 75977, is amplified using the oligonucleotide PCR primers corresponding to the 5 'and 3' sequences of the gene: The 5 'primer has the sequence: 5' TAACGAGGATCCGCCATCATGTGGAAATGGATACTGACAC3 '(SEQ ID No. 9) containing the BamHI restriction enzyme site underlined, followed by 21 bases of the KGF-2 sequence of Figure 1 (SEQ ID No. 1). Inserted into an expression vector, as described below, the 5 'end of the amplified fragment encoding KGF-2 provides an efficient signal peptide. An efficient signal for the start of translation in eukaryotic cells, as described in Kozak, M., J. Mol. Biol. 196: 947-950 (1987) is appropriately located in the vector portion of the construction. The 3 'primer has the sequence: 5' TAAGCAGGATCCTGAGTGTACCACCATTGGAAGAAATG3 '(SEQ ID No. 10) containing the BamHI restriction followed by the nucleotides complementary to the last 26 nucleotides of the KGF-2 coding sequence described in Figure 1 (SEQ. ID No. 1), not including the stop codon. The amplified fragments are isolated from a 1% agarose gel as described above and are then digested with the BamHI endonuclease and then purified again on a 1% agarose gel. The isolated fragment and the dephosphorylated vector are then ligated with the T4 DNA ligase. The E. coli HB101 cells are then transformed and the bacteria containing the plasmid pCl are identified. The sequence and orientation of the inserted gene is confirmed by DNA sequencing.

Transfection of CHO-DHFR cells Chinese hamster ovary cells lacking an active DHFR enzyme are used for transfection. 5 μg of the expression plasmid Cl are cotransfected with 0.5 μg of the pSVneo plasmid using the lipofection method (Felgner et al., Supra). Plasmid pSV2-neo contains a selectable, dominant marker, the neo gene from Tn5 that codes for an enzyme that confers resistance to a group of antibiotics, including G418. The cells are seeded in alpha minus MEM supplemented with 1 mg / ml of G418. After 2 days, the cells are trypsinized and seeded in the cloning plates of hybridomas (Greiner, Germany) and cultured for 10-14 days. After this period, the single clones are trypsinized and then seeded in 6-well petri dishes using different concentrations of methotrexate (25 nM, 50 nM, 100 nM, 200 nM, 400 nM). The clones that develop at the highest concentrations of methotrexate are then transferred to the new 6-well plates containing even higher concentrations of methotrexate (500 nM, 1 μM, 2 μm, 5 μm). The same procedure is repeated until the clones develop at a concentration of 100 μm. The expression of the desired gene product is analyzed by Western blot analysis and SDS-PAGE.

Example 5 Transcription and Translation of KGF -2 Recombinant in vi tro A PCR product is derived from the cDNA cloned in the pA2 vector used for the expression of KGF-2 in insect cells. The primers used for this PCR were: 5 ' ATTAACCCTCACTAAAGGGAGGCCATGTGGAAATGGATACTGACACATTGTGCC 3 ' (SEQ ID No. 11) and 5 'CCCAAGCTTCCACAAACGTTGCCTTCCTCTATGAG 3' (SEQ ID No. 12). The first primer contains the sequence of a 5 'T3 promoter to the ATG start codon. The second primer is complementary to the 3 'end of the open reading structure of KGF-2, and codes for the reverse complement of a stop codon. The resulting PCR product is purified using commercially available Qiagen equipment, 0.5 μg of this DNA is used as a template for an in vitro transcription-translation reaction. The reaction is carried out with a commercially available equipment from Promega under the name of TNT. The test is performed as described in the instructions for the equipment, using the radioactively labeled methionine as a substrate, with the exception that only 1/2 of the indicated volumes of reagents are used and that the reaction is allowed to proceed at 33 °. C for 1.5 hours. Five μl of the reaction are separated electrophoretically on a denaturing 10 to 15% polyacrylamide gel. The gel is fixed for 30 minutes in a mixture of water: methanol: acetic acid at volumes of 6: 3: 1 respectively. The gel is then dried under heat and vacuum and subsequently exposed to an X-ray film for 16 hours. The film is revealed showing the presence of a radioactive protein band corresponding in size to the conceptually translated KGF-2, strongly suggesting that the cDNA cloned for KGF-2 contains an open reading frame that encodes a protein of the expected size.

Example 6 Expression Via Gene Therapy Fibroblasts are obtained from a subject by skin biopsy. The resulting tissue is placed in tissue culture medium and separated into small pieces. The small pieces of tissue are placed on a wet surface of a tissue culture flask, about ten pieces are placed in each flask. The flask is turned upside down, hermetically sealed and left at room temperature overnight. After 24 hours at room temperature, the flask is inverted and the pieces of tissue remain fixed to the bottom of the flask and fresh medium (for example Ham's F12 medium, with 10% PBS, penicillin and streptomycin) is added. This is then incubated at 37 ° C for about a week. For this end, fresh media is added and subsequently it is changed every several days. After two additional weeks in culture, a monolayer of fibroblasts emerges. The monolayer is triptinized and transferred to larger flasks. pMV-7 (Kirschmeier, PT et al., DNA, 7: 219-25 (1988) flanked by long terminal repeats of Moloney murine sarcoma virus, digested with EcoRI and HindIII and subsequently treated with calf intestinal phosphatase The linear vector is fractionated on agarose gel and purified, using glass beads The cDNA encoding a polypeptide of the present invention is amplified using the PCR primers corresponding to the 5 'and 3' sequences respectively. 5 'containing an EcoRI site and the 3' primer that also includes a HindIII site.

Equal amounts of the linear backbone of the Moloney murine sarcoma virus and the amplified EcoRI and HindIII fragment are aggregated together in the presence of the T4 DNA ligase. The resulting mixture is maintained under conditions appropriate for the ligation of the two fragments. The ligation mixture is used to transform HB101 bacteria, which are then plated onto agar containing kanamycin for purposes of confirming that the vector had the gene of interest properly inserted. The amphotropic amplicon pA317 or GP + aml2 packaging cells are grown in tissue culture to a confluent density in the Dulbecco Modified Eagle Medium (DMEM) with 10% calf serum (CS), penicillin and streptomycin. The MSV vector containing the gene is then added to the media and the packaging cells are transduced with the vector. The packaging cells now produce infectious viral particles that contain the gene (the packaging cells are now referred to as producer cells). The fresh medium is added to the transducer producing cells, and subsequently, the medium is harvested from a 10 cm plate of confluent producer cells. The spent medium, which contains the infectious viral particles, is filtered through a millipore filter to eliminate the detached production cells, and this medium is then used to infect fibroblast cells. The medium is removed from a subconfluent plate of fibroblasts and rapidly replaced with the medium from the producer cells. This medium is removed and replaced with fresh medium. If the virus titer is high, then virtually all fibroblasts will be infected and selection is not required. If the titer is very low, then it is necessary to use a retroviral vector that has a selectable marker, such as neo or his. Genetically engineered fibroblasts are then injected into the host or host, either alone or after being developed for confluence on cytodex 3 microcarrier spheres. Fibroblasts now produce the protein product.

Example 7 KGF-2 Stimulated Wound Healing in the Diabetic Mouse Model To demonstrate that keratinocyte growth factor 2 (KGF-2) could accelerate the healing process, the genetically diabetic mouse model of wound healing was used. The full-thickness wound healing model in the db + / db + mouse is a well-characterized, clinically relevant and reproducible model of deteriorated wound healing. Healing of the diabetic wound is dependent on granulation tissue formation and re-epithelialization, instead of contraction (Gartner MH et al., J. Surg. Res. 52: 389 (1992); Greenhalgh, DG et al., Am. J. Pathol., 136: 1235 (1990).) Diabetic animals have many of the distinctive characteristics observed in type II diabetes mellitus.Multi-homozygous mice (dg + / db +) are obese compared to their normal heterozygous relatives. (db + / + m). Diabetic mutant mice (db + / db +) have a simple autosomal recessive mutation on chromosome 4 (db +) (Coleman et al, Proc. Nati, Acad. Sci USA 77: 283-293 (1982)). The animals show polyphagia, polydipsia and polyuria. Diabetic mutant mice (db + / db +) have high blood glucose, increased or normal levels of insulin, and cell-mediated immunity, suppressed (Mandel et al., J. "Immunol., 120: 1375 (1978); Debray-Sachs, M. et al., Immunol., 51 (1): 1-7 (1983), Leiter et al., Am. J. of Pathol 114: 46-55 (1985).) Peripheral neuropathy, myocardial complications and injuries. Microvascular diseases, thickening of the basement membrane and abnormalities in glomerular filtration have been described in these animals (Norido, F. et al., Exp. Neurol. 83 (2) -.221-232 (1984)).; Robertson et al., Diabetes 29 (l): 60-67 (1980); Giacomelli et al., Lab Invest. 40 (4).-460-473 (1979); Coleman, D. L. Diabetes 31 (Suppl) -. 1-6 (1982)). These homozygous diabetic mice develop hypergiukaemia that is resistant to insulin analogs for human type II diabetes (Mandel et al., J. Immunol 120: 1375-1377 (1978)). The characteristics observed in these animals suggest that the healed in this model may be similar to the healed observed in human diabetes (Greenhalgh, et al., Am. J. of Pathol, 136: 1235-1246 (1990)). The results of this study showed that KGF-2 has a potent stimulating effect on the healing of full-thickness wounds in diabetic and non-diabetic heterozygous litters. The marked effects on re-epithelialization and an increase in collagen fibrils in the granulation tissue within the dermis were observed in animals treated with KGF-2. The exogenous application of growth factors can accelerate the formation of granulation tissue by extracting inflammatory cells towards the wound.

Animals Female C57BL / KsJ (db + / db +) mice, genetically diabetic and their non-diabetic heterozygous litters (db + / + m) were used in this study (Jackson Laboratories). The animals were acquired at 6 weeks of age and were 8 weeks old at the beginning of the study. The animals were individually housed and received food and water ad libitum. All manipulations were performed using aseptic techniques. The experiments were conducted according to the rules and guidelines of the Human Genome Sciences, Inc. Committee on the Care and Use of Institutional Animals and Guidelines for the Care and Use of Laboratory Animals.

KGF -2 The recombinant human KGF-2 used for the wound healing studies was overexpressed and purified from pQE60-Cys37, an E. coli expression vector system (pQE-9, Qiagen). The protein expressed from this construct is KGF-2 from cysteine at position 37 to Serine at position 208 with a 6X tag (His) linked to the N-terminus of the protein (SEQ ID Nos. 29-30) ( Figure 15). Fractions containing more than 95% pure recombinant materials were used for the experiment. Growth factor-2 of keratin) cytes was formulated in a vehicle containing 100 mM Tris, 8.0 and 600 M sodium chloride. The final concentrations were 80 μg / ml and 8 μg / ml of the reserve solution. Dilutions were made from the reserve solution using the same vehicle.

Surgical wound The wound protocol was performed according to previously reported methods (Tsuboi, R. and Rifkin, D. B., J. Exp. Med. 172: 245-251 (1990)). In summary, on day 1 of the wound, the animals were anesthetized with an intraperitoneal injection of Avertin (0.01 mg / ml), 2:, 2,2-tribromoethanol and 2-methyl-2-butanol dissolved in deionized water. The dorsal region of the animal was shaved and the skin was washed with 70% ethanol and iodine solution. The surgical area was dried with a sterile gauze before inflicting the wound. A full thickness wound of 8 mm was then created using a Keyes tissue punch. Immediately after the wound, the neighboring area was gently stretched to eliminate the expansion of the wound. The wounds were left open for the entire duration of the experiment. The application of the treatment was given topically for 5 consecutive days starting on the day of the wound. Before treatment, the wounds were gently cleansed with sterile saline and gauze sponges. The wounds were visually examined and photographed at a fixed distance on the day of surgery, and at intervals of two days after this. The wound closure was determined by daily measurement on days 1-5 and on day 8. The wounds were measured horizontally and vertically using a calibrated Jameson meter. The wounds were considered healed if the granulation tissue was no longer visible and the wound was covered by a continuous epithelium. KGF-2 was administered using two different doses of KGF-2, one at 4 μg per wound per day, for 8 days and the second at 40 μg per wound per day, for 8 days, in 50 μl of vehicle. The control groups with vehicle received 50 μl of the vehicle solution. The animals were sacrificed on day 8 with an intraperitoneal injection of sodium pentobarbital (300 mg / kg). Wounds and surrounding skin were then harvested for histology and immunohistochemistry studies. Tissue specimens were placed in 10% neutral buffered formalin in tissue cassettes between biopsy sponges for further processing.

Experimental design Three groups of 10 animals each (5 diabetics and 5 non-diabetic controls) were evaluated: 1) control with vehicle placebo, 2) KGF-2 4 μg / day and 3) KGF-2 40 μg / day. This study was designed as follows: Wound Area Measurement and Closure The closure of the wound was analyzed by measuring the area on the vertical and horizontal axis, and obtaining the total square area of the wound. The contraction was then estimated by establishing the differences between the initial wound area (day 0) and that of post-treatment (day 8). The wound area on day 1 was 64 mm2, the corresponding size of the dermal puncture. The calculations were made using the following formula: [Open area on day 8] - [Open area on day l] / [Open area on day 1] Histology The specimens were fixed in 10% buffered formalin and the embedded paraffin blocks were sectioned perpendicular to the wound surface (5 μm) and cut using a Reichert-Jung microtome. Routine hematoxylin-eosin staining (H & E) was performed on cross sections of the bisected wounds. The histological examination of the wounds was used to evaluate if the healing process and the morphological appearance of the repaired skin was altered by the treatment with KGF-2. This evaluation included verification of the presence of accumulation of cells, inflammatory cells, capillaries, fibroblasts, re-epithelialization and epidermal maturity (Greenhalgh, DG et al., Am. J. Pathol. 136: 1235 (1990)) (Table 1 ). A calibrated lens micrometer was used by an observer in the "blind" mode.

Immunohistochemistry Re-epithelialization Tissue sections were stained immunohistochemically with a polyclonal rabbit anti-human keratin antibody using an ABC Elite detection system. Human skin was used as a positive tissue control while non-immune IgG was used as a negative control. The growth of keratinocytes was determined by evaluating the degree of re-epithelialization of the wound, using a calibrated lens micrometer.

Cell Proliferation Marker The proliferating cell / cyclin nuclear antigen (PCNA) in skin specimens was demonstrated by the use of the anti-PCNA antibody (1:50) with an ABC Elite detection system. Human colon cancer served as a positive tissue control and human brain tissue was used as a negative tissue control. Each specimen included a section with omission of the primary antibody and substitution with non-immune mouse IgG. The classification of these sections was based on the degree of proliferation on a scale of 0 to 8, the lower limit of the scale reflects slight proliferation to the upper limit that reflects intense proliferation.

Statistic analysis The experimental data were analyzed using an unpaired t-test. A value of p < 0.05 was considered significant. The data were expressed as the mean ± SEM.

Results Effect of KGF-2 on Wound Closure Diabetic mice showed impaired healing compared to normal heterozygous animals. The dose of 4 μg of KGF-2 per site seemed to produce maximum response in diabetic and non-diabetic animals (Figures 5, 6). These results were statistically significant (p = 0.002 and p <0.0001) when compared to the control groups with buffer. Treatment with KGF-2 resulted in a final average closure of 60.6% in the group that received 4 μg / day and 34.5% in the group of 40 μg / day. Wounds in the control group with shock absorber only had a 3.8% closure by day 8. Repeated measurements of wounds on days 2-5 after inflicting the wound and on day 8, taken from the db + / db + mice treated with KGF-2, showed a significant improvement in the total wound area (mm2) by day 3 after inflicting the wound, when compared to the control group with buffer. This improvement continued and by the end of the experiment, statistically significant results were observed (Figure 7). The animals in the db / + m groups that received KGF-2 also showed a greater reduction in the wounded area compared to the control groups with buffer in repetitive measurements (Figure 8). These results confirmed a higher speed of wound closure in the animals treated with KGF-2.

Effect of KGF-2 on Histological Qualification The histopathological evaluation of KGF-2 in the diabetic model (db + / db +) on day 8 demonstrated a statistically significant improvement (p <0.0001) in the wound score when compared to the control with shock absorber. The pharmacological effects observed with the doses of 4 μg and 40 μg of KGF-2 were not significantly different from one another. The control group with buffer showed minimal cell accumulation without tissue granulation or epithelial trip while doses of 4 μg and 40 μg of KGF-2 (p <0.0001 and p = 0.06 respectively) showed wound epithelial coverage, neovascularization, formation of granulation tissue and deposition of fibroblasts and collagen (Figure 9). The histopathological evaluation of the skin wounds was performed on samples stained with hematoxylin-eosin. The qualification criteria included a scale of 1-12, a rating of one that represents the minimum accumulation of cells, with little to no granulation, and a score of 12 represents the abundant presence of fibroblasts, collagen deposition and new epithelial coverage of the wound (Table 1). 10-12 Coarse vascular granulation tissue, dominated by fibroblasts and extensive collagen deposition. Partial epithelium completely covering the wound.

The evaluation of the non-diabetic litters, after both doses of KGF-2, showed no significant activity compared to the control group with buffer for all the evaluated measurements (Figure 10). The control group with buffer showed immature granulation tissue, inflammatory cells, and capillaries. The mean score was higher than the diabetic group, indicating deteriorated healing in the diabetic mice (db + / db +).

Effect of KGF-2 on re-epithelialization Immunostaining with cytokeratin was used to determine the degree of re-epithelialization. The ratings were given based on the degree of closure on a scale of 0 (without closure) to 8 (complete closure). In the groups that received 4 μg / day, there was a statistically significant improvement over the re-epithelialization score when compared to the control group with buffer p < 0.001 (Figure 11). In this group, the keratinocytes were observed located in the newly formed epidermis covering the wound. Both doses of KGF-2 also showed mitotic figures in several stages. The evaluation of the non-diabetic groups at both doses of KGF-2 also significantly improved the re-epithelialization range (p = 0.006 and 0.01 respectively) (Figure 12).

Effect of KGF-2 on Cell Proliferation Immunostaining of the proliferating cell nuclear antigen showed significant proliferation in the 4 μg and 40 μg groups (Figure 13). The non-diabetic group showed similar results as both groups that received both doses of KGF-2, which showed significantly higher rating compared to the control group with buffer (Figure 14). Epidermal proliferation was observed especially on the basal layer of the epidermis. In addition, cells marked with high density PCNA were observed in the dermis, especially in hair follicles. conclusion The results show that KGF-2 specifically stimulates the development of primary epidermal keratinocytes. In addition, these experiments demonstrate that recombinant human, topically applied KGF-2 markedly accelerates the healing rate of full-thickness, excised dermal wounds in diabetic mice. Histological evaluation shows that KGF-2 induces the proliferation of keratinocytes with epidermal thickening. This proliferation is localized in the basal layer of the epidermis as demonstrated by the proliferating cell nuclear antigen (PCNA). At the level of the dermis, the deposition of collagen, the proliferation of fibroblasts, and neovascularization re-established the normal structure of the skin. The high density of PCNA-labeled cells on animals treated with KGF-2 in contrast to the buffer group, which had fewer cells labeled with PCNA, indicates the stimulation of keratinocytes at the dermal-epidermal level, fibroblasts and hair follicles . The increase in the healing process by KGF-2 was consistently observed in this experiment. This effect was statistically significant in the evaluated parameters (percentage re-epithelialisation and wound closure). Importantly, keratinocytes labeled with PCNA were mainly observed in the lower basal layer of the epidermis. The dermis showed normalized tissue with fibroblasts, collagen, and granulation tissue.

The activity observed in non-diabetic animals indicates that KGF-2 had a significant pharmacological response in the percentage of wound closure on day 8, as well as during the course of the experiment, based on daily measurements. Although the histopathological evaluation was not significantly different when compared to the control with buffer, the development of keratinocytes and the PCNA scores demonstrated significant effects. In summary, these results demonstrated that KGF-2 shows significant activity in impaired and normal excision models using the db + / db + mouse model and therefore may be useful in the treatment of wounds including surgical wounds, diabetic ulcers, Venous stasis ulcers, burns and other skin conditions.

Example 8 Wound Healing Mediated by KGF-2 in Rat Model Deteriorated with Steroids Inhibition of wound healing by steroids has been well documented in various in vitro and in vivo systems (Wahl, SM Glucocorticoids and Wound healing, In Anti-Inflammatory Steroid Action: Basic and Clinical Aspects, 280-302 (1989); , SM et al, J. Immunol 115: 476 -481 (1975); Werb, Z. and collaborators, J. "Exp. Med. 147: 1684-1694 (1978).) Glucocorticoids delay wound healing by inhibiting angiogenesis, decreasing vascular permeability (Ebert, RH et al, An. Intern Med. 37: 701-705 (1952)), proliferation of fibroblasts and collagen synthesis (Beck, LS et al., Growth Factors. : 295-304 (1991); Haynes, BF et al., J. Clin. Invest. 61: 703-797 (1978)) and producing a transient reduction of circulating monocytes (Haynes, BF et al., J. Clin. Invest. 61: 703-797 (1978), Wahl, SM Glucocorticoids and wound healing.In Antiinflammatory Steroid Action: Basic and Cli nical Aspects. Academic Press. New York, pp. 280-302 (1989)). Systemic administration of steroids to impaired wound healing is a well-established phenomenon in rats (Beck, LS et al, Growth Factors 5: 295-304 (1991); Haynes, BF et al., J. Clin. Invest. : 703-797 (1978), Wahl, SM Glucocorticoids and wound healing, Antiinflammatory Steroid Action: Basic and Clinical Aspects, Academic Press, New York, pp. 280-302 (1989), Pierce, GF et al., Proc. Acad. Sci. USA 86: 2229-2233 (1989)). To demonstrate that KGF-2 could accelerate the healing process, the effects of multiple topical applications of KGF-2 on full-thickness, excised skin wounds were evaluated in rats in which the healing was impaired by the systemic administration of methylprednisolone. In vitro studies have shown that KGF-2 specifically stimulates the development of primary human epidermal keratinocytes. This example demonstrates that recombinant, topically applied human KGF-2 accelerates the healing rate of full-length, excised skin wounds in rats, by measuring the wound void space with a calibrated Jameson meter and by histomorphometry and immunohistochemistry. Histological evaluation demonstrates that KGF-2 accelerates re-epithelialization and subsequently, wound repair.

Animals Adult, young Sprague Dawley male rats weighing 250-300 g (Charles River Laboratories) were used in this example. The animals were acquired at 8 weeks of age and were 9 weeks old at the beginning of the study. The response to the healing of the rats was impaired by the systemic administration of methylprednisolone (17 mg / kg / rat, intramuscularly) at the time of wounding. The animals were individually housed and received water and food ad libi tum. All manipulations were performed using aseptic techniques. This study was conducted according to the rules and guidelines of the Human Genome Sciences, Inc. Committee for the Care and Use of Institutional Animals and the Guidelines for the Care and Use of Laboratory Animals.

KGF -2 Recombinant human KGF-2 was overexpressed and purified from pQE60-Cys37, an E. coli expression vector system (pQE-9, Qiagen). The protein expressed from this construct is KGF-2 from Cysteine at position 37 to Serine at position 208 with a 6X tag (His) linked to the N-terminus of the protein (Figure 15) (SEQ ID Nos. 29-30). Fractions containing more than 95% pure recombinant materials were used for the experiment. KGF-2 was formulated in a vehicle containing IX of PBS. The final concentrations were 20 μg / ml and 80 μg / ml of the stock solution. The dilutions were made from the reserve solution using the same vehicle. KGF-2-2? 28 was overexpressed and purified from an E. coli expression vector system. The fractions containing more than 95% pure recombinant materials were used for the experiment. KGF-2 was formulated in a vehicle containing IX of PBS. The final concentrations were 20 μg / ml and 80 μg / ml reserve solution. The dilutions were made from the reserve solution using the same vehicle.

Surgical wound The wound-carrying protocol was followed according to Example 7 above. On the day of the wounding, the animals were anesthetized with an intramuscular injection of ketamine (50 mg / kg) and xylazine (5 mg / kg). The dorsal region of the animal was shaved and the skin was washed with 70% ethanol and iodine solutions. The surgical area was dried with a sterile gauze before inflicting the wound. A full thickness wound of 8 mm was created using a Keyes tissue punch. The wounds were left open for the entire duration of the experiment. The applications of the test materials were administered topically once a day for 7 consecutive days, beginning on the day of the wounding and subsequent to the administration of methylprednisolone. Before the treatment, the wounds were gently cleansed with sterile saline and gauze sponges. The wounds were visually examined and photographed at a fixed distance on the day the wound was made and at the end of the treatment. The closure of the wound was determined by daily measurement on days 1-5 and on day 8 for the Figure. Wounds were measured horizontally and vertically using a calibrated Jameson meter. The wounds were considered healed if the granulation tissue was no longer visible and the wound was covered with a continuous epithelium. A dose-response curve was performed using two different doses of KGF-2, one at 1 μg per wound per day, and the second at 4 μg per wound per day, for 5 days in 50 μl of vehicle. The control groups with vehicle received 50 μl of IX PBS. The animals were sacrificed on day 8 with an intraperitoneal injection of sodium pentobarbital (300 mg / kg). The wounds and the neighboring skin were then harvested for histology. Tissue specimens were placed in 10% neutral buffered formalin in tissue cassettes between the biopsy sponges for further processing.

Experimental design Four groups of 10 animals each (5 with methylprednisolone and 5 without glucocorticoid) were evaluated: 1) untreated group 2), vehicle control with placebo, 3) KGF-2 1 μg / day and 4) KGF-2 4 μg / day day. This study was designed as follows: n Treatment Group • Treated with Glucocorticoid N = 5 Not treated N = 5 Vehicle 50 μl N = 5 KGF-2 (1 μg) 50 μl N = 5 KGF-2 (4 μg) 50 μl • Without glucocorticoid N = 5 Not treated -N = 5 Vehicle 50 μl N = 5 KGF-2 (1 μg) 50 μl N = 5 KGF-2 (4 μg) 50 μl Area Measurement and Wound Closure The closure of the wound was analyzed by measuring the area on the vertical and horizontal axis, and obtaining the total area of the wound. The closure was then estimated by establishing the differences between the initial wound area (day 0) and that after treatment (day 8). The wound area on day 1 was 64 mm2, the corresponding size of the dermal punch. The calculations were made using the following formula: [Open area on day 8] - [Open area on day l] / [Open area on day 1] Histology The specimens were fixed in 10% buffered formalin and the embedded paraffin blocks were sectioned perpendicular to the wound surface (5 μm) and cut using an Olympus microtome. Routine hematoxylin-eosin staining (H & E) was performed on cross sections of the bisected wounds. The histological examination of the wounds allowed to evaluate if the healing process and the morphological appearance of the repaired skin was improved by the treatment with KGF-2. A calibrated lens micrometer was used by a "blind" type observer to determine the distance of the empty space of the wound.

Statistic analysis The experimental data were analyzed using an unpaired t-test. A p-value of < 0.05 was considered significant. The data were expressed as the mean ± SEM.

Results A comparison of wound closure of untreated control groups with and without methylprednisolone demonstrates that rats treated with methylprednisolone have significant deterioration of wound healing at 8 days after wounding, compared to normal rats. The total area of the wound measured 58.4 mm2 in the group injected with methylprednisolone and 22.4 mm2 in the group that did not receive glucocorticoid (Figure 16).

Effect of KGF-2 on wound closure Systemic administration of methylprednisolone in rats at the time of wounding delayed wound closure (p = 0.002) in normal rats. Measurements of wound closure of the deteriorated groups with methylprednisolone at the end of the experiment on day 8 showed that wound closure with KGF-2 was significantly statistically higher (1 μg p = 0.002 and 4 μg p = 0.005) when compared with the untreated group (Figure 16). The percentage of wound closure was 60.2% in the group that received 1 μg of KGF-2 (p = 0.002) and 73% in the group that received 4 μg of KGF-2 (p = 0.0008). In contrast, wound closure of the untreated group was 12.5% and the group with placebo vehicle was 28.6% (Figure 17). The longitudinal analysis of wound closure in the glucocorticoid groups from day 1 to day 8 shows a significant reduction in wound size from day 3 to day 8 after wounding, in both doses of KGF-2 in the treated groups (Figure 18). The results demonstrate that the group treated with 4 μg of KGF-2 had statistically significant accelerated wound closure (p = 0.05) compared to the untreated group (Figure 19A). Although it is difficult to evaluate the ability of a protein or other compounds to accelerate wound healing in normal animals (due to rapid recovery), however, KGF-2 was shown to accelerate wound healing in this model.

Histopathological Evaluation of Wounds Treated with KGF-2 The histomorphometry of the wound space indicated a reduction in the distance of the wound from the group treated with KGF-2. The wound space observed for the untreated group was 5336 μ while the group treated with 1 μg of KGF-2 had a reduction in wound space to 2972 μ; and the group treated with 4 μg of KGF-2 (p = 0.04) had a reduction in wound space to 3086 μ (Figure 20).

Effects of KGF-2A28 on Wound Healing The evaluation of KGF-2? 28 and PDGF-BB in wound healing in the rat model impaired with methylprednisolone was also examined. The experiment was carried out as for the previous KGF-2 protein, except that the KGF-2? 28 protein was not labeled with His and the wound healing was measured on days 2, 4, 6, 8 and 10 The vehicle with buffer for the proteins was 40 mM NaOAc and 150 mM sodium chloride, pH 6.5 for all except the full-length "E2" preparation of KGF-2. The vehicle with buffer for the preparation of KGF-2"E2" was 20 mM NaOAc and NaOAc and 400 mM NaCl, pH 6.4. The results shown in Figure 19B demonstrate that KGF-2Δ28 has accelerated wound closure, statistically significant compared to the untreated group, and has reversed the effects of methylprednisolone on wound healing.

Conclusions This example demonstrates that KGF-2 reversed the effects of methylprednisolone on wound healing. The exogenous application of growth factors can accelerate the formation of granulation tissue by removing inflammatory cells to the wound. A similar activity was also observed in animals that did not receive methylprednisolone, indicating that KGF-2 had a significant pharmacological response in the percentage of wound closure on day 5, based on daily measurements. The model of damaged wound healing with glucocorticoid, in rats, was shown to be an appropriate and reproducible model for measuring the effectiveness of KGF-2 and other compounds in the area of wound healing. In summary, the results show that KGF-2 shows significant activity in the impaired models with glucocorticoids and normal excised wound. Therefore, KGF-2 may be clinically useful in the stimulation of wound healing, including surgical wounds, diabetic ulcers, venous stasis ulcers, burns, and other conditions of abnormal wound healing, such as uremia, poor nutrition, vitamin deficiencies and systemic treatment with steroids and antineoplastic drugs.

Example 9 Tissue Distribution of KGF-2 mRNA Expression Staining or Northern blot analysis is carried out to examine the expression levels of the gene encoding the KGF-2 protein in human tissues, using the methods described by, among others Sambrook et al., Cited above. A probe corresponding to the complete open reading frame of KGF-2 of the present invention (SEQ ID No. 1) was obtained by PCR and was labeled with 32P using the J? And iprime ™ DNA labeling system (Amersham Life Science) , according to the manufacturer's instructions. After labeling, the probe was purified using a CHROMA SPIN-100MR column (Clontech Laboratories, Inc.), according to the manufacturer's protocol number PT1200-1. The labeled, purified probe was then used to screen various human tissues for the expression of the gene encoding KGF-2. Multi-Tissue Northern blotting (MTN) containing poly-A RNA from various human tissues (H) or tissues of the human immune system (IM) were obtained from Clontech and were examined with the labeled probe using the ExpressHybMR hybridization solution (Clontech) according to the manufacturer's protocol number PT1190-1. After hybridization and washing, the stains are mounted and exposed to film at -70 ° C overnight, and the films were developed according to standard procedures. A mRNA species greater than about 4.2 kb was observed in most human tissues. KGF-2 mRNA was relatively abundant in the heart, pancreas, placenta and ovary. A species of mRNA less than about 5.2 kb was also observed ubiquitously. The identity of this species of 5.2 kb mRNA was not clear. It is possible that the 5.2 kb transcript codes for an alternately spliced form of KGF-2 or a third member of the KGF family. The KGF-2 cDNA was 4.1 kb, consistent with the size of the 4.2 kb mRNA.

EXAMPLE 10 Keratinocyte Proliferation Assays Dermal keratinocytes are cells in the epidermis of the skin. The development and diffusion of keratinocytes in the skin is an important process in the healing of wounds. A keratinocyte proliferation assay is therefore a valuable indicator of protein activities in the stimulation of keratinocyte growth and, consequently, in wound healing. However, keratinocytes are difficult to develop in vi tro. There are few keratinocyte cell lines. These cell lines have different cellular and genetic defects. In order to avoid complications in this assay for cellular defects such as the loss of the key growth factor receptors or the dependence of key growth factors for development, the primary dermal keratinocytes are chosen for this assay. These primary keratinocytes are obtained from Clonetics, Inc., (San Diego, CA).

Keratinocyte Proliferation Assay with Alamar Blue The alamar blue is a viable blue dye that is metabolized by mitochondria when added to the culture medium. The dye then becomes red in the tissue culture supernatants. The quantities of the red dye can be directly quantified by the difference in reading at the optical densities between 570 nm and 600 nm. This reading reflects cellular activities and the number of cells. Normal primary dermal keratinocytes (CC-0255, NHEK-Neo combined) were purchased from Clonetics, Inc. These cells are passage 2. Keratinocytes are grown in complete keratinocyte growth medium (CC-3001, KGM; Clonetics; Inc.) until they reach 80% confluence. The cells are trypsinized according to the manufacturer's specification. Briefly, the cells were washed twice with Hank's balanced salt solution. 2 to 3 ml of trypsin was added to the cells for approximately 3-5 minutes at room temperature. The trypsin neutralization solution was added and the cells were harvested. The cells were centrifuged at 600 xg for 5 minutes at room temperature and plated in fresh flasks at 3,000 cells per square centimeter using the preheated medium. For the proliferation test, they were seeded 1,000-2,000 keratinocytes per well of the 96-well flat-bottomed plates, Corning, in complete medium, except for the rows of the outermost part. The external wells were filled with 200 μl of sterile water. This helps keep the temperature and humidity fluctuations of the wells to a minimum. The cells are grown overnight at 37 ° C with 5% C02. The cells are washed twice with the basal keratinocyte medium (CC-3101, KBM, Clonetics, Inc.) and 100 μl of KBM is added to each well. They are incubated for 24 hours. The growth factors are diluted in KBM in serial dilution and 100 μl is added to each well. KGM is used as a positive control and KBM is used as a negative control. 6 wells are used for each concentration point. It is incubated for two to three days. At the end of the incubation, the cells are washed once with KBM and 100 μl of KBM with 10% alamar v / v blue premixed in the medium is added. Incubate for 6 to 16 hours until the color of the medium begins to turn red in the positive control of KGM. The D.O. 570 nm less D.O 600 nm directly by placing the plates in the plate reader.

Results Proliferation of Keratinocyte Proliferation by KGF-2 To demonstrate that KGF-2 (for example starting at the amino acid Cys37 as described in Examples 7 and 8 above), and the N-terminal deletion mutants KGF-2? 33 and KGF-2? 28 were active in the stimulation of the growth of epidermal keratinocytes, the normal, primary human epidermal keratinocytes were incubated with purified KGF-2 protein expressed in E. coli (lot number E3) (SEQ ID No. 2), KGF-2? 33 (number of lot El) and KGF-2? 28 (lot number E2). The KGF-2 protein stimulated the development of epidermal keratinocytes with an EC50 of approximately 5 ng / ml, equivalent to that of FGF7 / KGF-1 (Figure 21A). In contrast, other FGFs such as FGF-1 and FGF-2 did not stimulate the development of primary keratinocytes. The EC50 for KGF-2? 33 was 0.2 ng / ml and that of KGF-2? 28 2 ng / ml (See Figures 21B and C). Thus, KGF-2 appeared to be as potent as FGF7 / KGF in stimulating the proliferation of primary epidermal keratinocytes. However, KGF-2? 33 is more potent in stimulating the proliferation of keratinocytes than KGF-2"Cys (37)" described in Examples 7 and 8 above and KGF-2? 28. The healing of wounded tissues involves hyperproliferation of dermal fibroblasts. To determine if the stimulatory effects of KGF-2 were specific for keratinocytes but not for fibroblasts, Balb / c mouse 3T3 fibroblasts and human lung fibroblasts were tested. None of the fibroblast types responded to KGF-2 in the proliferation assays. Therefore, KGF-2 appeared to be a specific mitogen for epidermal keratinocytes but not for mesenchymal cells such as fibroblasts. This suggested that the likelihood of KGF-2 causing scarring of wounded tissues was low.

Example 11 A. Mitogenic Effects of KGF -2 on Cells Transfected with Specific FGF Receptors To determine which isoforms of the FGF receptor are mediators of the proliferative effects of KGF-2, the effects of KGF-2 on the cells expressing the isoforms of the specific FGF receptor were tested according to the method described by Santos-Ocampo and collaborators, J. Biol. Chem. 271: 1726-1731 (1996). It was known that FGF7 / KGF induces the mitogenesis of epithelial cells by binding to and specifically activating the FGFR2iiib form (Miki et al., Science 251: 72-75 (1991)). Therefore, the proliferative effects of KGF-2 in the mitogenesis assays were tested using cells expressing one of the following isoforms of the FGF receptor: FGFRiiib, FGFR2iiib, FGFR3iiib, and FGFR4.

Myogenesis assay of cells expressing FGF receptors Thymidine incorporation of BaF3 cells expressing specific FGF receptors was performed as described by Santos-Ocampo et al., J. "Biol. Chem. 271: 1726-1731 (1996). Briefly, BaF3 cells expressing the specific FGF receptors were washed and resuspended in Dulbecco's modified Eagle medium, 10% neonatal bovine serum, and L-glutamine. Approximately 22,500 cells were seeded per well in a 96-well assay plate in medium containing 2 μg / ml Heparin. The test reagents were added to each well for a volume of 200 μl per well. The cells were incubated for 2 days at 37 ° C. To each well, 1 μCi of 3H-thymidine in a volume of 50 μl was then added. The cells were harvested after 4-5 hours by filtration through glass fiber paper. The incorporated 3H-thymidine was counted in a Wallac beta plate scintillation counter.

Results The results revealed that the KGF-2 protein (Thr (36) -Ser (208) of Figure 1 (SEQ ID No. 2) with an N-terminal Met added to it) strongly stimulated the proliferation of Baf3 cells expressing the KGF receptor, the FGFR2iiib isoform, as indicated by the incorporation of 3H-thymidine (Figure 22A). Interestingly, a slight stimulatory effect of KGF-2 was observed on the proliferation of Baf3 cells expressing the FGFRiiib isoform. KGF-2 had no effect on the cells expressing the FGFR3iiib or the FGFR4 forms of the receptor. FGF7 / KGF stimulated the proliferation of cells expressing the KGF receptor, FGFR2iiib but not the FGFRliiib isoform. The difference between KGF-2 and FGF7 / KGF was intriguing. In the control experiments, aFGF stimulated its receptors, FGFRliiib and iiic and bFGF stimulated its FGFR2ÜIC receptor. Thus, these results suggested that KGF-2 binds to the FGFR2iiib isoform and stimulates mitogenesis. In contrast to FGF7 / KGF, KGF-2 also binds to the FGFRliiib isoform and stimulates mitogenesis.

B. Mitogenesis effects of KGF-2? 33 on Cells Transfected with the FGF Specific Receptors As shown previously, FGFs or KGF-1 and -2 both bind to and activate the FGF 2iiib receptor (FGFR2iiib). The proliferative effects of KGF-2? 33 in the mitogenesis assays were tested using cells expressing one of the following FGF receptor isoforms: FGFR2iiib or FGFR2iiic (cells transfected with the 2iiic receptor are included as a negative control) . The experiments were performed as described above in part A of this example. In summary, BaF3 cells were developed in RPMl containing 10% fetal bovine serum (BCS - not fetal serum), 10% conditioned medium from WEHI3 cell cultures (developed in MPI containing 5% BCS) , 50 nM of β-mercaptoethanol, L-glu (2% of a 100X reserve) and pen / strep (1% of a 100X reserve). For the assay, BaF3 cells were rinsed twice in the RPMI medium containing 10% BCS and 1 μg / ml heparin. BaF3 cells (22, 000 / well) were seeded in a 96-well plate in 150 μl of the RPMl medium containing 10% BCS and 1 μg / ml heparin. The acid FGF, basic FGF, KGF-1 (HG15400) or KGF-2 (HG03400, 03401, 03410 or 03411) proteins were added at concentrations from about 0 to 10 nm. The cells were incubated in a final volume of 200 μl for 48 hours at 37 ° C. All the tests were performed in triplicate. Tritiated thymidine (0.5 μCi) was added to each well for 4 hours at 37 ° C and the cells were then harvested by filtration through a glass fiber filter. The total amount of the incorporated radioactivity was then determined by the liquid scintillation count. The following positive controls were used: basic FGF and FGF acid for FGFR2iiic cells; FGF acid and KGF-1 for FGFR2iiib cells. The following negative controls were used: basal medium (RPMl medium containing 10% BCS and 1 μg / ml heparin).

Resulted: The results revealed that KGF-2 (Thr (36) -Ser (208) with N-terminal Met added), KGF-2? 33 and KGF-2? 28 proteins strongly stimulated the proliferation of BaF3 cells expressing the receptor of KGF, the isoform of FGFR2iiib, as indicated by the incorporation of 3H-thymidine (Figures 22A-C). The KGF-2 proteins had no effect on the cells expressing the FGFR2iiic forms of the receptor. These results suggested that KGF-2 proteins bind to the FGFR2iiib isoform and stimulate mitogenesis. In addition, it appears that KGF-2? 33 was able to stimulate the proliferation of BaF3 cells better than KGF-2 (Thr (36) -Ser (208)).

Example 12 A. Construction of KGF -2 of Longi tud Complete Optimized with E. coli In order to increase expression levels of full-length KGF-2 in an expression system of E. coli, the codons of the amino-terminal portion of the gene were optimized to highly used E. coli codons. For synthesis of the optimized region of KGF-2, a series of six oligonucleotides were synthesized: numbers 1-6 (sequences described below). These overlapping oligos were used in a PCR reaction for seven rounds under the following conditions: Denaturing 95 degrees 20 seconds Annealing 58 degrees 20 seconds Extension 72 degrees 60 seconds A second PCR reaction was established using 1 μl of the first PCR reaction with the synthetic primer 6 of KGF-2 as the 3 'primer and the synthetic 5' BamHI primer of KGF-2 as the 5 'primer, using the same conditions that are described above for 25 cycles. The product produced by this final reaction was restricted with Avall and BamHI. The KGF-2 construct of Example 1 was restricted with AvalI and HindIII and the fragment was isolated. These two fragments were cloned into pQE-9 restricted with BamHI and HindIII in a three fragment ligation. The primers used for the construction of optimized synthetic KGF-2 1/208: Synthetic Primer 1 of KGF-2: ATGTGGAAATGGATACTGACCCACTGCGCTTCTGCTTTCCCGCACC TGCCGGGTTGCTGCTGCTTCCTGCTGCTGTTC (SEQ ID No. 31) Synthetic Primer 2 of KGF-2: CCGGAGAAACCATGTCCTGACCCAGAGCCTGGCAGGTAACCGGAA CAGAAGAAACCAGGAACAGCAGCAGGAAGCAGCAGCA (SEQ ID No. 32) Synthetic Primer 3 of KGF-2: GGGTCAGGACATGGTTTCTCCGGAAGCTACCAACTCTTCTTCTTCTT CTTTCTCTTCTCCGTCTTCTGCTGGTCGTCACG (SEQ ID No. 33) Synthetic Primer 4 of KGF-2: GGTGAAAGAGAACAGTTTACGCCAACGAACGTCACCCTGCAGGTG GTTGTAAGAACGAACGTGACGACCAGCAGAAGACGG (SEQ ID No. 34) Synthetic Primer 5 of KGF-2: CGTTGGCGTAAACTGTTCTCTTTCACCAAATACTTCCTGAAAATCGA AAAAAACGGTAAAGTTTCTGGGACCAAA (SEQ ID No. 35) Synthetic Primer 6 of KGF-2: TTTGGTCCCAGAAACTTTACCGTTTTTTCGATTTTCAG (SEQ ID No. 36) 5 'BamHII Synthetic of KGF-2: AAAGGATCCATGTGGAAATGGATACTGACCCACTGC (SEQ ID No. 37) The resulting clone is shown in Figure 23 (SEQ ID Nos. 38 and 39).

B. Construction of KGF -2 Mature Optimized with E. coli In order to further increase the expression levels of the mature form of KGF-2 in an E. coli expression system, the codons of the amino-terminal portion of the gene were optimized to the highly used E. coli codons. To match the mature form of KGF-1, a truncated form of KGF-2 was constructed starting at threonine 36. A synthetic KGF-2 from E. coli from Example 12A was used as a template in a PCR reaction using BspHI 5 'KGF-2 as the 5' primer (sequence given below) and HindIII 3 'KGF-2 as the 3' primer (sequence given below). The amplification was performed using standard conditions as given above in Example 12A for 25 cycles. The resulting product was restricted with BspHI and HindII and cloned into the pQE60 expression vector of E. coli, digested with Ncol and HindIII. 5 'BspHI Primer of KGF-2: TTTCATGACTTGTCAAGCTCTGGGTCAAGATATGGTTC (SEQ ID No. 40) 3' HindIII Primer of KGF-2: GCCCAAGCTTCCACAAACGTTGCCTTCC (SEQ ID No. 41) The resulting clone is shown in Figure 24A (SEQ ID No. 42 and 43).

C. Construction of an Optimized Mature KGF-2, from E. coli, alternative In order to further increase the expression levels of the mature form of KGF-2 in an E. coli expression system, the codons of 53 amino acids in the amino-terminal portion of the optimized E. coli gene were changed to alternate the codons of E. coli highly used. For synthesis of the optimized region of KGF-2, a series of six oligonucleotides were synthesized: numbers 18062, 18061, 18058, 18064, 18059, and 18063 (sequences described below). These overlapping oligos were used in a PCR region for seven rounds under the following conditions: Denaturing 95 degrees 20 seconds Annealing 58 degrees 20 seconds Extension 72 degrees 60 seconds After seven rounds of synthesis, a 5 'primer to this region, 18169 and a 3' primer to this complete region, 18060, were added to a PCR reaction, containing 1 microliter of the initial reaction of the six oligonucleotides. This product was purified for 30 rounds using the following conditions: Denaturation 95 degrees 20 seconds Annealing 55 degrees 20 seconds Extension 72 degrees 60 seconds A second PCR reaction was established to amplify the 3 'region of the gene using primers 18066 and 18065 under the same conditions as described above for 25 rounds. The resulting products were separated on an agarose gel. The gel slices containing the product were diluted in 10 mM Tris, 1 mM EDTA, pH 7.5. One microliter of each of the diluted gel slices was used in an additional PCR reaction using primer 18169 as the 5 'primer, and primer 18065 as the 3' primer. The product was amplified for 25 cycles using the same conditions as described above. The product produced by this final reaction was restricted with EcoRI and HindIII, and cloned into pQE60, which was also cut with EcoRI and HindIII (pQE6 now).

Sequences of the synthetic primers 5 ': 18169 KGF2 5'EcoRl / RBS: TCAGTGAATTCATTAAAGAGGAGAAATTAATCATGACTTGCCAGG [SEQ ID No. 44] Rl new synthetic 18062 KGF2 in sense: TCATGACTTGCCAGGCACTGGGTCAAGACATGGTTTCCCCGGAAGCTA [SEQ ID No. 45] Synthetic R2 18061 KGF2 in sense: GCTTCAGCAGCCCATCTAGCGCAGGTCGTCACGTTCGCTCTTACAACC [ SEQ ID No. 46] Synthetic R3 18058 KGF2 in direction: GTTCGTTGGCGCAAACTGTTCAGCTTTACCAAGTACTTCCTGAAAATC [SEQ ID No. 47] Ava II 20 base pairs 18066 KGF-2, in the sense: TCGAAAAAAACGGTAAAGTTTCTGGGAC [SEQ ID No. 48] Synthetic Fl 18064 KGF2, antisense : GATGGGCTGCTGAAGCTAGAGCTGGAGCTGTTGGTAGCTTCCGGGG AA [SEQ ID No. 49] F2 18059 KGF2 synthetic antisense: AACAGTTTGCGCCAACGAACATCACCCTGTAAGTGGTTGTAAGAG [SEQ ID No. 50] 18063 KGF2 synthetic F3 antisense: TTCTTGGTCCCAGAAACTTTACCGTTTTTTTCGATTTTCAGGAAGTA [SEQ ID No. 51] Avall 18060 KGF2, antisense: TTCTTGGTCCCAGAAACTTTACCG [SEQ ID No. 52] HindIII 3 '18065 KGF2, Detention: AGATCAGGCTTCTATTATTATGAGTGTACCACCATTGGAAGAAAG [SEQ ID No. 53] The sequence of the synthetic KGF-2 gene and its corresponding amino acid is shown in Figure 24B (SEQ.

ID No. 54 and 55) Example 13 Construction of the Suppression Mutants of KGF-2 Deletion mutants were constructed from the 5 'end and the 3' end of the KGF-2 gene using the optimized KGF-2 construct of Example 12A as a template. The deletions were selected based on the regions of the gene that can negatively affect expression in E. coli. For 5 'deletion, the primers listed below were used as the 5' primer. These primers contain the indicated restriction site and an ATG to code for the initiating methionine. The HindIII 3 '208 amino acid primer of KGF-2 (FGF-12) was used for the 3' primer. PCR amplification for 25 rounds was performed using standard conditions as described in Example 12. Products for deletion mutant KGF-2 36aa / 208aa were restricted with BspHI for the 5 'site and HindIII for the 3' site and cloned within pQE60 that has been digested with BspHI and HindIII. All other products were restricted with Ncol for the 5 'restriction enzyme and HindIII for the 3' site, and cloned into pQE60 that had been digested with Ncol and HindIII. For KGF-2 (FGF-12), 36aa / 153aa and 128aa 3 'HindIII was used as the 3' primer with FGF-12 36aa / 208aa as the 5 'primer. For FGF-12 62aa / 153aa, 128aa 3 'HindIII was used as the 3' primer with FGF-12 62aa / 208aa as the 5 'primer. The nomenclature of the resulting clones indicates the first and last amino acid of the polypeptide resulting from the deletion. For example, KGF-2 36aa / 153aa indicates that the first amino acid of the deletion mutant is amino acid 36 and the last amino acid is amino acid 153 of KGF-2. In addition, as indicated in Figures 25-33, each mutant has the N-terminal Met added to it. Sequences of Suppression Primers: FGF12 36aa / 208aa: 5 'Bsphl GGACCCTCATGACCTGCCAGGCTCTGGGTCAGGAC [SEQ ID No. 56] FGF12 63aa / 208aa: 5 'Ncol GGACAGCCATGGCTGGTCGTCACGTTCG [SEQ ID No. 57] FGF12 77aa / 208aa: 5' Ncol GGACAGCCATGGTTCGTTGGCGTAAACTG [SEQ ID No. 58] FGF12 93aa / 208aa: 5 'Ncol GGACAGCCATGGAAAAAAACGGTAAAGTTTC [SEQ ID No. 59] FGF12 104aa / 208aa: 5 'Ncol GGACCCCATGGAGAACTGCCCGTAGAGC [SEQ ID No. 60] FGF12 123aa / 208aa: 5' Ncol GGACCCCCATGGTCAAAGCCATTAACAGCAAC [SEQ ID No. 61] FGF12 138aa / 208aa: 5 'Ncol GGACCCCCATGGGGAAACTCTATGGCTCAAAAG [SEQ ID No. 62] FGF12 3' HindIII : (Used for all previous deletion clones) CTGCCCAAGCTTATTATGAGTGTACCACCATTGGAAG [SEQ ID No. 63] FGF12 36aa / l53aa: 5 'Bsphl (as described above) 3' HindIII CTGCCCAAGCTTATTACTTCAGCTTACAGTCATTGT [SEQ ID No. 64] FGF12 63aa / l53aa: 5 'Ncol and 3'HindIII, as described above The sequences for the resulting deletion mutations are as described in Figures 25-33. [I KNOW THAT ID No. 65-82]. When KGF-2? 28 (amino acids 63-208) is expressed in E. coli, a protease inhibitor, such as guanidine hydrochloride (Gu-HCl), is used to prevent degradation of the protein. For example, the E. coli paste resuspended in 50 mM Tris-Acetate, 10 mM EDTA-NA2, pH 7.7 ± 0. 2 followed by lysis. The lysed suspension is treated with an equal volume of 1.0 M Gu-HCl solution and stirred gently for 2-4 hours at 2-8 ° C. The suspension is then centrifuged and filtered before loading on the first column for purification. The initial purification takes place on an SP-Sepharose FF column where the bound KGF-2 is eluted with a salt gradient. The resulting SP-Sepharose elution pool is diluted and filtered on a 0.2 μm filter and loaded onto a Fractogel COO "(S) column.The elution is carried out through a saline gradient and the elution pool is diafiltered and concentrates on a shock absorber.

Example 14 Construction of Cysteine Mutants of KGF-2 Construction of the mutation primers C-37, 5457 5 'Bsphl and 5258 173aa 3' HindIII were used to amplify the KGF-2 template (FGF-12) of Example 12A. Primer 5457 5 'Bsphl changes cysteine 37 to a serine. The amplification was performed using the standard conditions described above in Example 12A for 25 cycles. The resulting product was restricted with BspHI and HindIII and cloned into the E. coli expression vector, PQE60, digested with BspHI and HindIII. (Figure 34) [SEQ ID No. 83]. For the mutation of cysteine 106 to serine, two PCR reactions were established for site-directed mutagenesis of the oligonucleotide, of this cysteine. In one reaction, 5453 BspHI was used as the 5 'primer, and 5455 was used as the 3' primer in the reaction. In a second reaction, 5456 was used as the 5 'primer, and 5258 HindIII was used as the 3' primer. The reactions were amplified for 25 rounds under standard conditions as described in Example 12. One microliter of each of these PCR reactions was used as the template in a subsequent reaction using, as a 5 'primer, 5453 BspHI, and as a 3 'primer, 5258 HindIII. The amplification by 25 rounds was performed using standard conditions as described in Example 12. The resulting product was restricted with BspHI and HindIII and was cloned into the expression vector pQE60, which was restricted with Ncol and HindIII. Two PCR reactions were required to make the mutant C-37 / C-106. Primers 5457 Bsphl and 5455 were used to create the 5 'region of the mutant containing the substitution of cysteine 37 to serine, and primer 5456 and 5258 HindIII were used to create the 3 'region of the mutant containing the substitution of cysteine 106 to serine. In the second reaction, primer 5457 Bsphl was used as the 5 'primer and primer 5258 HindIII was used as the 3' primer to create mutant C-37 / C-106 using 1 μl of each of the initial reactions together with the template. This PCR product was restricted with Bsphl and HindIII, and cloned into pQE60 that had been restricted with Ncol and HindIII. The resulting clone is shown in Figure 35 (SEQ ID No. 84) Sequences of the Cysteine Mutant Primers: 5457 BspHI: GGACCCTCATGACCTCTCAGGCTCTGGGT (SEQ ID No. 85) 5456: AAGGAGAACTCTCCGTACAGC (SEQ ID No. 86) 5455: GCTGTACGGTCTGTTCTCCTT (SEQ ID No. 87) 5453: BspHI: GGACCCTCATGACCTGCCAGGCTCTGGGTCAGGAC (SEQ ID No. 88) 5258 HindIII: CTGCCCAAGCTTATTATGAGTGTACCACCATTGGAAG (SEQ ID No. 89) Example 15 Production and Purification of KGF -2 (FGF-12) The DNA sequence encoding the optimized mature protein described in Example 12B (eg, amino acids T36 to S208 of KGF-2) was cloned into the plasmid pQE-9 (Qiagen). E. coli (M15 / rep4; Qiagen) was grown to the stationary phase overnight at 37 ° C in LB containing 100 μg / ml ampicillin and 25 μg / ml kanamycin. This culture was used to inoculate medium Fresh LB containing 100 μg / ml ampicillin and 25 μg / ml kanamycin at a dilution of 1:50. Cells were grown at 37 ° C to D.0.595 of 0.7, induced by the addition of 1-thio-b-D-galactopyranoside isopropyl (IPTG) to a final concentration of 1 mM. After 3-4 hours, the cells were harvested by centrifugation, and resuspended in a buffer containing 60 mM sodium phosphate, and 360 mM sodium chloride at a ratio of 5 volumes of buffer: 1 volume of cell paste. After disintegration in a Mautin Gaulin, the extract was adjusted to pH 8.0 by the addition of sodium hydroxide and clarified by centrifugation. The rinse soluble extract was applied to a Poros HS-50 column (2.0X10.0 cm; PerSeptive Biosystems, Inc.) and the bound proteins were gradually eluted with 50 mM sodium phosphate pH 8.0, containing 0.5 M sodium chloride, 1.0 M, and 1.5 M. The KGF-2 eluted in the 1.5 M salt fraction was then diluted five times with 50 mM sodium phosphate pH 6.5 to a final salt concentration of 300 mM. This fraction containing KGF-2 was then sequentially passed over a Poros HQ-20 column (2.0X7.0 cm; PerSeptive Biosystems, Inc.) and then linked to a Poros CM-20 column (2.0X9.0 cm; PerSeptive Biosystems, Inc.). Fractions containing KGF-2 (FGF-12) that eluted at about 500 mM to about 750 mM sodium chloride were combined, diluted and reapplied to a CM-20 column to concentrate. Finally, the protein was separated on a gel filtration column (S-75; Pharmacia) in 40 mM NaOAC; pH 6.5; 150 mM sodium chloride (Lot E-5). Alternatively, the gel filtration column was run in Phosphate Buffered Saline (PBS, Lot E-4). The fractions containing KGF-2 were combined and the protein concentration was determined by the Bio-Rad Protein Assay. The proteins were judged as > 90% pure by SDS-PAGE. Finally, it was found that the endotoxin levels determined by the Limulus Amebocyte Lysate assay (Cape Cod Associates) was < 1 Eu / mg. Proteins prepared in this way were able to bind to heparin which is a hallmark of members of the FGF family.

Example 16 TO . Construction of the N-terminal deletion mutant of KGF-2? 33 To increase the expression level of KGF2 in E. coli, and to increase the solubility and stability properties of KGF2 expressed in E. coli, a KGF-2? 33 suppression variant (KGF-2 aa 69-208) was generated. (SEQ ID No. 96) that eliminates the first 68 amino acids of pre-processed KGF2. The reason for creating this deletion variant was based on the following observations. First, mature KGF2 (KGF-2 aa 36-208) contains an odd number (three) of cysteine residues that can lead to aggregation due to intramolecular disulfide bridge formation. The deletion variant KGF? 33 contains only two cysteine residues, which reduces the potential for intramolecular disulfide bridge formation and subsequent aggregation. A decrease in aggregation could lead to an increase in the yield of the active KGF2 protein. Second, the deletion variant KGF? 33 removes a stretch of poly-serine that is not present in KGF-1 and does not appear to be important for activity, but may prevent the expression of the protein in E. coli. In this way, the elimination of the poly-serine stretch can increase the expression levels of the active KGF-2 protein. Third, the expression of KGF? 33 in E. coli results in the natural cleavage of KGF-2 between residues 68 and 69. Thus, it anticipates that KGF2? 33 will be processed efficiently and will be stable in E. coli Construction of KGF2? 33 in pQE6 To allow for amplification directed by the polymerase chain reaction and subcloning of KGF2Δ 33 into the E. coli protein expression vector, pQE6, two oligonucleotide primers (5952 and 19138) complementary to the desired region of KGF2 were synthesized. , with the following sequence of bases. Primer 5952: 5 'GCGGCACATGTCTTACAACCACCTGCAGGGTG 3' (SEQ ID No. 91) Primer 19138: 5 'GGGCCCAAGCTTATGAGTGTACCACCAT 3' (SEQ ID No. 92) In the case of the N-terminal primer (5952), an AflIII restriction site was incorporated, whereas in the case of the N-terminal primer (19138), a HindIII restriction site was incorporated. Primer 5952 also contains an adjacent ATG sequence and within the structure with the coding region of KGF2 to allow translation of the cloned fragment in E. coli, while primer 19138 contains two restriction codons (preferably used in E. coli). ) adjacent and intra-structural with the coding region of KGF2, which ensures the correct translational termination in E. coli. The polymerase chain reaction was performed using the standard conditions well known to those skilled in the art and the nucleotide sequence for mature KGF-2 (aa 36-208) (constructed in Example 12C) as a template. The resulting amplicon was restriction digested with AflIII and HindIII and subcloned into the protein expression vector pQE6 digested with Ncol / HindIII.

Construction of KGF2? 33 in pHEl To allow the amplification directed by the polymerase chain reaction and the subcloning of KGF2? 33 into the expression vector of E. coli, pHEl, two oligonucleotide primers (6153 and 6150) were synthesized. the desired region of KGF2, with the following base sequence. Primer 6153: 5 'CCGGCGGATCCCATATGTCTTACAACCACCTGCAGG 3' (SEQ ID No. 93) Primer 6150: 5 'CCGGCGGTACCTTATTATGAGTGTACCACCATTGG 3' (SEQ ID No. 94) In the case of the N-terminal primer (6153), an Ndel restriction site was incorporated, whereas in the case of the C-terminal primer (6150) an Asp718 restriction site was incorporated. Primer 6153 also contains an adjacent and intrastructural ATG sequence with the coding region of KGF2 to allow translation of the cloned fragment in E. coli, while primer 6150 contains two stop codons (preferably used in E. coli) adjacent to it. intranstructural with the coding region of KGF2, which ensures the correct translational termination in E. coli. The polymerase chain reaction was performed using standard conditions well known to those skilled in the art, and the nucleotide sequence for mature KGF-2 (aa 36-208) (constructed in Example 12C) as a template. The resulting amplicon was digested by restriction with Ndel and Asp718 and subcloned into the pHEl protein expression vector digested with Ndel / Asp718.

Nucleotide sequence of KGF2 33: ATGTCTTACAACCACCTGCAGGGTGACGTTCGTTGGCGTAAACTGT TCTCTTTCACCAAATACTTCCTGAAAATCGAAAAAAACGGTAAAGT TTCTGGGACCAAGAAGGAGAACTGCCCGTACAGCATCCTGGAGAT AACATCAGTAGAAATCGGAGTTGTTGCCGTCAAAGCCATTAACAGC AACTATTACTTAGCCATGAACAAGAAGGGGAAACTCTATGGCTCAA AAGAATTTAACAATGACTGTAAGCTGAAGGAGAGGATAGAGGAAA ATGGATACAATACCTATGCATCATTTAACTGGCAGCATAATGGGAG GCAAATGTATGTGGCATTGAATGGAAAAGGAGCTCCAAGGAGAGG ACAGAAAACACGAAGGAAAAACACCTCTGCTCACTTTCTTCCAATTG GTGGTACACTCATAA (SEQ ID No. 95) Amino acid sequence of KGF2 33: MSYNHLQGDVRWRKLFSFTKYFLKIEKNGKVSGTKKENCPYSILEITS VEIGWAVKAINSNYYLAMNKKGKLYGSKEFNNDCKLKERIEENGYN TYASFNWQHNGRQMYVALNGKGAPRRGQKTRRKNTSAHFLPMWHS (SEQ ID No. 96) B. Construction of a KGF-2? 33 Optimized In order to increase the expression levels of KGF2? 33 in E. coli, the codons of the complete gene were optimized to match those most highly used in E. coli. Since the template used to generate KGF2? 33 was optimized by codon, within the N-terminal region, the C-terminal amino acids (84-208) required optimization. First, amino acids 172-208 were optimized by codon to generate KGF2Δ 33 (sl72-208). This 10 was achieved through an overlapping PCR strategy. The oligonucleotides PM07 and PM08 (corresponding to amino acids 172-208) were combined and annealed together by heating them to 70 ° C and allowing them to cool to 37 ° C. The annealed oligonucleotides were then 15 used as template for a standard PCR reaction which was directed by primers PM09 and PM10. In a separate PCR reaction, following standard conditions well known to those skilled in the art using KGF2Δ33 as a template, oligonucleotides PM05 20 (which overlaps with the PstI site within the coding region of KGF2) and PM11 were used to amplify the KGF2 region corresponding to amino acids 84-172. In a third PCR reaction, the product of the first PCR reaction (corresponding to the 25 amino acids 172-208 optimized by codon) and the product of ? ta t ÉÉuHMMß? the second PCR reaction (corresponding to amino acids 84-172 not optimized by codon) were combined and used as a template for a standard PCR reaction directed by oligonucleotides PM05 and PM10. The resulting amplicon was digested with PstI / HindIII and subcloned into pQE6KGF2? 33 digested with PstI / HindIII, effectively replacing the corresponding non-optimized region by codon, and creating pQE6KGF2? 33 (sl72-208). To complete the codon optimization of KGF2, a codon of the synthetic gene optimized for the KGF2 region corresponding to amino acids 84-172 was generated using overlapping oligonucleotides. First, four oligonucleotides (PM31, PM32, PM33 and PM34) were combined and seven cycles of the following PCR were performed: 94 ° C 30 seconds; 46.5 ° C 30, seconds and 72 ° C, 30 seconds. A second PCR reaction directed by primers PM35 and PM36 was then performed following standard procedures, using 1 μl of the first PCR reaction as template. The resulting codon optimized gene fragment was then digested with PstI / SalI and subcloned into pQE6KGF2Δ33 digested with PstI / Sall (S172-208) to create a fully optimized KGF2 coding gene, pQE6KGF2Δ33s. To create an alternative E. coli protein expression vector, KGF2? 33s was amplified by PCR using primers PM102 and PM130 on pQE6KGF2? 33s. The resulting amplicon was digested with Ndel and EcoRV and subcloned into the pHEl expression vector which had been digested with Ndel and Asp718 (blunt end) to create pHEl? 33s. ? Oligonucleotide Sequences used in construction of codon optimized KGF2 33s: PM05: CAACCACCTGCAGGGTGACG (SEQ ID No. 97) PM07: AACGGTCGACAAATGTATGTGGCACTGAACGGTAAAGGTGCTCCAC GTCGTGGTCAGAAAACCCGTCGTAAAAACACC (SEQ ID No. 98) PM08: GGGCCCAAGCTTAAGAGTGTACCACCATTGGCAGAAAGTGAGCAG AGGTGTTTTTACGACGGGTTTTCTGACCACG (SEQ ID No. 99) PM09: GCCACATACATTTGTCGACCGTT ( SEQ ID No 100) PM10. GGGCCCAAGCTTAAGAGTG (SEQ ID No 101) PM11. GCCACATACATTTGTCGACCGTT (SEQ ID No. 102) PM31: CTGCAGGGTGACGTTCGTTGGCGTAAACTGTTCTCCTTCACCAAAT ACTTCCTGAAAATCGAAAAAAACGGTAAAGTTTCTGGTACCAAG (SEQ ID No. 103) PM32: AGCTTTAACAGCAACAACACCGATTTCAACGGAGGTGATTTCCAGG ATGGAGTACGGGCAGTTTTCTTTCTTGGTACCAGAAACTTTACC (SEQ ID No. 104) PM33: GGTGTTGTTGCTGTTAAAGCTATCAACTCCAACTACTACCTGGCTAT GAACAAGAAAGGTAAACTGTACGGTTCCAAAGAATTTAACAAC (SEQ ID No. 105) PM34: GTCGACCGTTGTGCTGCCAGTTGAAGGAAGCGTAGGTGTTGTAACC GTTTTCTTCGATACGTTCTTTCAGTTTACAGTCGTTGTTAAATTCTTT GGAACC (SEQ ID No. 106) PM35: GCGGCGTCGACCGTTGTGCTGCCAG (SEQ ID No. 107) PM36: GCGGCCTGCAGGGTGACGTTCGTTGG (SEQ ID No. 108) PM102: CCGGCGGATCCCATATGTCTTACAACCACCTGCAGG (SEQ ID No. 109) PM130: CGCGCGATATCTTATTAAGAGTGTACCACCATTG (SEQ ID No. 110) Nucleotide sequence of KGF2 33 (sl72-208): ATGTCTTACAACCACCTGCAGGGTGACGTTCGTTGGCGTAAACTGT TCTCCTTCACCAAATACTTCCTGAAAATCGAAAAAAACGGTAAAGT TTCTGGTACCAAGAAAGAAAACTGCCCGTACTCCATCCTGGAAATC ACCTCCGTTGAAATCGGTGTTGTTGCTGTTAAAGCTATCAACTCCA ACTACTACCTGGCTATGAACAAGAAAGGTAAACTGTACGGTTCCAA AGAATTTAACAACGACTGTAAACTGAAAGAACGTATCGAAGAAAA CGGTTACAACACCTACGCTTCCTTCAACTGGCAGCACAACGGTCGA CAAATGTATGTGGCACTGAACGGTAAAGGTGCTCCACGTCGTGGTC AGAAAACCCGTCGTAAAAACACCTCTGCTCACTTTCTGCCAATGGT GGTACACTCTTAA (SEQ ID No. 111.) Amino acid sequence of KGF2? 33 (sl72-208): MSYNHLQGDVRWRKLFSFTKYFLKIEKNGKVSGTKKENCPYSILEITS VEIGWAVKAINSNYYLAMNKKGKLYGSKEFNNDCKLKERIEENGYN TYASFNWQHNGRQMYVALNGKGAPRRGQKTRRKNTSAHFLPMWHS SEQ ID No. 112) C. construction of the N-terminal deletion mutant, KGF-2Á4 To increase the level of expression of KGF2 in E. coli and increase stability and solubility properties in KGF2 expressed in E. coli, a variant of KGF2? 4 suppression (amino acids 39-208) that eliminates the first 38 amino acids of KGF2 preprocessed, it was constructed, including the cysteine at position 37. Since the resulting KGF2 suppression molecule contains an even number of cysteines, the problems due to aggregation caused by the formation of the intramolecular disulfide bridge should be reduced, resulting in a increased level of expression of the active protein. To allow the amplification and subcloning directed by the Polymerase Chain Reaction, of KGF2Δ4 in the protein pQE6 expression vector of E. coli, two oligonucleotide primers (PM61 and 19138) were synthesized with the following sequence of bases. PM61: CGCGGCCATGGCTCTGGGTCAGGACATG (SEQ ID No. 113) 19138: GGGCCCAAGCTTATGAGTGTACCACCAT (SEQ ID No. 114) In the case of the N-terminal primer (PM61), an Ncol restriction site was incorporated, whereas in the case of the C-terminal primer (19138) a HindIII restriction site was incorporated. PM61 also contains an adjacent and intrastructural ATG sequence with the coding region of KGF2 to allow translation of the cloned fragment in E. coli, while 19138 contains a stop codon (preferably used in E. coli) adjacent to and within the structure with the coding region of KGF2, which ensures the correct translational termination in E. coli. The Polymerase Chain Reaction was performed using standard conditions well known to those skilled in the art and full-length KGF2 (aa 36-208) as a template (constructed in Example 12C). The resulting amplicon was digested by restriction with Ncol and HindIII and subcloned into the protein expression vector pQE6 digested with NcoI / HindIII.

Nucleotide sequence of KGF2? 4: ATGGCTCTGGGTCAAGATATGGTTTCTCCGGAAGCTACCAACTCTT CCTCTTCCTCTTTCTCTTCCCCGTCTTCCGCTGGTCGTCACGTTCGTT CTTACAACCACCTGCAGGGTGACGTTCGTTGGCGTAAACTGTTCTC TTTCACCAAATACTTCCTGAAAATCGAAAAAAACGGTAAAGTTTCT GGGACCAAGAAGGAGAACTGCCCGTACAGCATCCTGGAGATAACA TCAGTAGAAATCGGAGTTGTTGCCGTCAAAGCCATTAACAGCAACT ATTACTTAGCCATGAACAAGAAGGGGAAACTCTATGGCTCAAAAG AATTTAACAATGACTGTAAGCTGAAGGAGAGGATAGAGGAAAATG GATACAATACCTATGCATCATTTAACTGGCAGCATAATGGGAGGCA AATGTATGTGGCATTGAATGGAAAAGGAGCTCCAAGGAGAGGACA GAAAACACGAAGGAAAAACACCTCTGCTCACTTTCTTCCAATGGTG GTACACTCATAA (SEQ ID No. 115) Amino acid sequence of KGF2 4: MALGQDMVSPEATNSSSSSFSSPSSAGRHVRSYNHLQGDVRWRKLFSF TKYFLKIEKNGKVSGTKKENCPYSILEITSVEIGWAVKAINSNYYLAM NKKGKLYGSKEFNNDCKLKERIEENGYNTYASFNWQHNGRQMYVAL NGKGAPRRGQKTRRKNTSAHFLPMWHS (SEQ ID No. 116) Example 17 KGF-2Á33 Stimulated Healing of Wounds in Normal Rats To demonstrate that KGF-2? 33 could accelerate the healing process, wound healing from excision wounds was examined using the following model. A 6mm, dorsal extirpation wound was created on Sprague Dawley rats (n = 5) with a Keyes skin punch. The wounds are left open and treated topically with various concentrations of KGF-2? 33 (in 40 mM sodium acetate and 150 mM sodium chloride, pH 6.5, as a buffer) and buffer (40 mM sodium acetate and 150 sodium chloride mM, pH 6.5) for 4 days beginning on the day of the wounding. Wounds were measured daily using a calibrated Jameson meter. The size of the wound is expressed in square millimeters. On the final day, the wounds were measured and harvested for further analysis. The statistical analysis was performed using a non-paired t test (mean ± SE). The parameters of evaluation include the percentage closing of the wound, the histological qualification (1-3 minimal accumulation of cells, without granulation; 4-6 immature granulation; inflammatory cells, capillaries; 7-9 granulation tissue, cells, fibroblasts, new epithelium; 10-12 mature dermis with fibroblasts, collagen, epithelium), re-epithelialization and immunohistochemistry. Three days after performing the wound, treatment with KGF-2? 33 showed a decrease in wound size (30.4 mm2 at 4 μg, p = 0.006, 33.6 mm2 at 1 μg, p = 0.0007) when compared to the control with shock absorber of 38.9 mm2. At day four after wounding, treatment with KGF-2? 33 showed a decrease in wound size (27.2 mm2 at 0.1 μg p = 0.02, 27.9 mm2 at 0.4 μg p = 0.04) when compared to the control with shock absorber of 33.8 mm2. On day five after wounding, treatment with KGF-2? 33 showed a decrease in wound size (18.1) mm2 at 4 μg p = 0.02, when compared to control with 25.1 mm2 cushion . See Figure 36. After harvesting the wound on day 5, additional parameters were evaluated. KGF-2? 33 showed an increase in the percentage of wound closure at 4 μg (71.2%, p = 0.02) when compared to the control with buffer, of 60.2%. The administration of KGF-2? 33 also results in an improvement in the histological rating at 1 and 4 μg (8.4 to 1 μg p = 0.005, 8.5 to 4 μg p = 0.04) compared to the shock absorber control of 6.4. The re-epithelialization was also improved to 1 and 4 μg of KGF-2? 33 (1389 μm to 1 μg p = 0.007, 1220 μm to 4 μg p = 0.02) in relation to the control with buffer of 923 μm. See Figure 37. This study demonstrates that daily treatment with KGF-2? 33 accelerates the speed of wound healing in normal animals, as shown by a decrease in the gross area of the wound, in addition, the histological evaluation of the Wound samples and re-epithelialization evaluation also show that KGF-2? 33 improves the healing rate in this normal rat model.

Example 18 Effect of KGF-2? 33 on Traction Resistance and Epidermal Thickness in Normal Rat To demonstrate that KGF-2? 33 could increase the tensile strength and epidermal thickness of wounds, the following experiment was performed.

A full-thickness, 2.5-cm intermediate incisional wound is created on the back of male Sprague Dawley rats (n = 8 or 9). The incision in the skin is closed using 3 metal clamps, equidistant. They were applied topically at the time of performing the wound, buffer (40 mM sodium acetate and 150 mM sodium chloride, pH 6.5) or KGF-2? 33 (in 40 M sodium acetate and 150 mM sodium chloride, buffer of pH 6.5). Four wound strips measuring 0.5 cm wide were excised on day 5. The specimens are used for the study of the resistance to rupture using an Instron ™ skin tensiometer, hydroxyproline determination and histopathological evaluation. Resistance to breakage was defined as the greatest force resisted by each wound before rupture. The statistical analysis was performed using a non-paired t test (mean ± SE). In an incised skin rat model, topically applied KGF-2? 33 showed a statistically significant increase in breakage resistance, tensile strength and epidermal thickness, as a result of a simple intraincisional application, subsequent to the performance of the wound. In one study, resistance to breakage of wounds treated with KGF-2 at 4, and 10 μg was significantly higher when compared to controls with buffer (107.3 g at 1 μg p = 0.0006, 126.4 g at 4 μg p <0.0001 , 123.8 g to 10 μg p <0.0001). See Figure 38. The epidermal thickness was evaluated using light microscopy on Masson Trichome sections. The wounds treated with KGF-2? 33 showed increased epidermal thickening (60.5 μ to 1 μg, 66.51 μ to 4 μg p = 0.01, 59.6 μ to 10 μg) in contrast to the control with 54.8 μ buffer. See Figure 39. These studies demonstrate that a simple intraincisional application of KGF-2 increases and accelerates the process of wound healing characterized by an increased resistance to breakage and epidermal thickness of incisional wounds.

Example 19 Effect of KGF-2? 33 on Normal Rat Skin In order to determine the effect of KGF-2? 33 on normal rat skin after intradermal injection, the following experiment was performed. Adult SD male rats (n = 3) received six intradermal injections of either placebo or KGF-2β3 (in 40 mM sodium acetate and 150 mM sodium chloride, buffer 6.5) at a concentration of 1 and 4 μg in 50 μl on day 0. The animals were injected with 5,2'-bromo-deoxyuridine (BrdU) (100 mg / kg ip) two hours before slaughter at 24 and 48 hours. The epidermal thickness was measured from the granular layer towards the bottom of the basal layer. Approximately 20 measurements were made along the injection site and the average thickness was quantified. The measurements were determined using a micrometer calibrated on sections stained with Masson Trichome under light microscopy. The BrdU score was performed by two observers (in blind) under light microscopy using the following rating system: 0-3 none at a minimum of cells marked with BrdU; 4-6 moderate dialing; 7-10 intense labeled cells. The animals were sacrificed 24 and 48 hours after the injection. The statistical analysis was performed using a non-paired t test (mean ± SE). Skin treated with KGF-2? 33 showed increased epidermal thickening at 24 hours (32.2 μ to 1 μg p <0.001, 35.4 μ to 4 μg p <0.0001) in contrast to the control with 27.1 μ buffer. At 48 hours the skin treated with KGF-2? 33 showed increased epidermal thickening (34.0 μ to 1 μg p = 0.0003, 42.4 μ to 4 μg p <; 0.0001) compared to the control with 27.8 μ buffer. See Figure 40. Skin treated with KGF-2? 33 also showed immunostaining with BrdU increased at 48 hours (4.73 at 1 μg p = 0.07, 6.85 at 4 μg p <0.0001) compared to control with 3.33 buffer. See Figure 41. These studies demonstrate that an intradermal injection of KGF-2 increases and accelerates epidermal thickening. In this way, KGF-2 could have applications to prevent or alleviate wrinkles, improve aged skin and reduce healing or improve healing from cosmetic surgery. In addition, KGF-2 can be used prophylactically to prevent or reduce oral mucosistis (mouth ulcers), intestinal inflammation in response to chemotherapy or other agents.

Example 20 Anti-inflammatory effect of KGF-2 on Paw-induced Edema in PAF To demonstrate an anti-inflammatory effect of KGF-2, the following experiment was performed using the swelling model of edema in the paw, induced by PAF. Groups of four lewis rats (190 to 210 gm) were injected subcutaneously into the sole of the foot of the right hind foot with 120 μl of a solution containing 2.5 nMol of PAF, together with the following reagents: 125 μg of Ckb-10 (B5), 24 μg of LPS, 73 μg of KGF-2 (Thr (36) -Ser (208) of Figure 1 (SEQ ID No. 2) with an N-terminal Met) or without protein. The left hind legs were administered with the same amount of shock absorber to be used as a parallel control. The volume of the paw was quantified immediately before, or 30 and 90 minutes after the injection of PAF using a plethysmograph system. The percentage change (%) of the leg volume was calculated.

Test reagents in experiment No. 1 and No. 2 Groups PAF (R.) C ß-10 (R.) LPS (R.) KGF-2 (R.) Shock absorber (N = 4) 2. 5 nMol 1.04 mg / ml 200 μg / ml 0.73 mg / ml 1 20 μl 100 μl 2 20 μl 100 μl 3 20 μl 100 μl 4 20 μl 100 μl As shown in Figure 42, the right hind legs injected with PAF alone resulted in a significant increase in paw volume (75 or 100% for experiment No. 1 or No. 2, respectively) at 0.5 hours after the injection, as expected; while the left posterior legs received shock or the right posterior legs that received LPS or SEB only show little sign of edema (data not shown). However, when KGF-2 was administered together with PAF locally, there was a substantial reduction (25 or 50% for experiment No. 1, No. 2, respectively) in paw volume compared to paws challenged only with PAF. . The reduction in foot edema was not observed in the animal that received PAF together with Ckb-10 (a different protein), LPS or SEB (two inflammatory mediators). These results suggest that the anti-inflammatory effect of KGF-2 is specific and not due to any specific nature of the protein.

Effect of KGF-2? 33 on paw edema induced by PAF in rats Following the experiments described above with KGF-2? 33 to confirm its in vitro biological activities to stimulate the proliferation of keratinocytes and its effect in vivo on wound healing, KGF-2? 33 was also evaluated in the model of edema in paw , induced by PAF, in rats. Groups of four Lewis rats (190-210 gm) were injected subcutaneously into the footplate of the right hind foot with 120 μl of a solution containing 2.5 nMol of PAF, together with 210 μg of KGF-2α 33 or albumin. The left hind legs were administered with the same amount of buffer, albumin or KGF-2? 33 alone, to be used as a parallel control. The volume of the paw was quantified at different intervals after the injection of PAF using a plethysmograph system. The percentage change (%) of the leg volume was calculated. As shown in Figure 43, right posterior legs injected with PAF and albumin resulted in a significant (75%) increase in paw volume at 0.5 hours post-injection, as expected; while the left posterior legs that received shock absorber, albumin or KGF-2? 33 only showed little sign of edema. However, when KGF-2? 33 was administered together with PAF locally, there was a substantial reduction (average 20%) in the volume of the paw, when compared with the legs challenged with PAF plus albumin, throughout the experiment complete, which was completed in 4 hours. These results confirm the anti-inflammatory property of KGF-2? 33.

Test reagents Thus, KGF-2 is useful for treating acute and chronic conditions in which inflammation is a key pathogenesis of diseases, including but not limited to psoriasis, eczema, dermatitis and / or arthritis.

Example 21 Effect of KGF-2? 33 on the Rat Model with End-to-End Colonic Anastomosis This example demonstrates that KGF-2Δ 33 will increase the rate or rate of intestinal repair in an intestinal or colonic anastomosis model in Wistar or Sprague Dawley rats. The use of the rat in experimental anastomosis is a well characterized, relevant and reproducible model of the healing of surgical wounds. This model can also be extended to study the effects of chronic steroid treatment or the effects of various chemotherapeutic regimens on the quality and speed of healing of surgical wounds in the colon and small intestine (Mastboom WJB et al., Br. J. Surg. 78: 54-56 (1991), Salm R. et al. J Surg. Oncol. 47: 5-11, (1991), Weiber S. et al., Eur. Surg. Res. 26: 173-178 (1994)). The healing of the anastomosis is similar to that of healed wounds elsewhere in the body. The first stages of healing are characterized by acute inflammation, followed by proliferation of fibroblasts and synthesis of collagen. Collagen is gradually modeled and the wound is reinforced as the new collagen is synthesized (Koruda MJ and Rolandelli, RHJ Surg, Res 48: 504-515 (1990).) Most postoperative complications such as anastomotic leak occur during the first a few days after surgery - a period during which resistance of the colon is mainly ensured by the ability of the wound margin to maintain the sutures.The suture retention capacity of the gastrointestinal tract has been reported to decrease as much as 80% during the first days after the operation (Hogstrom H and Haglund U. Acta Chir Scand 151: 533-535 (1985), Jonson K, and collaborators Am J. Surg. 145: 800-803 (1983)). Adult SD (n = 5) were anesthetized with a combination of ketamine (50 mg / kg) and xylazine (5 mg / kg) intramuscularly, the abdominal cavity was opened with a midline incision of 4 cm in length. d e 1 cm wide of the left colon was excised 3 cm proximal to the peritoneal reflection, while the marginal vessels were preserved. An end-to-end simple layer anastomosis was performed with 8 to 10 inverted Vicryl 5-0 sutures, interrupted, to restore intestinal continuity. The anastomosis was then topically treated by means of a syringe with either buffer or KGF-2? 33 at concentrations of 1 and 4 μg. The incision wound was closed with 3-0 silk suture run for the muscular layer and surgical staples for the skin. The treatments were then administered daily after this and consisted of buffer or KGF-2? 33 and 1 and 5 mg / kg subcutaneously. Weights were taken on the day of surgery and daily after it. The animals were sacrificed 24 hours after the last treatment (day 5). The animals were anesthetized and received barium enemas and were subjected to X-rays at a fixed distance. Radiological analysis after intracolonic administration by 2 observers (in the cecum) revealed that the groups treated with KGF-2? 33 had 1) a decreased rate of leakage of barium at the surgical site, 2) lower degree of constriction at the site surgical, and 3) an increase in the presence of faecal pellets distal to the surgical site.

Radiological Analysis of the Colonic Anastomosis Groups Stool Constriction Distension Fugue present Proximal anastomotic Per.Ltoneal No treatment (N = 5) 20% 80% 80% 60% Shock absorber (N = 5) 40% 60% 80% 75% KGF-2? 33 [1 mg / kg] 60% 20% 100% 20% (N = 5) KGF-2? 33 [5 mg / kg] 100% 0% 75% 25% (N = 4) Example 22 Construction of Carboxyl-Terminal Mutations in KGF -2 The carboxyl terminus of KGF-2 is highly charged. The density of these charged residues can affect the stability and consequently the solubility of the protein. To produce muteins that can stabilize the protein in solution, a series of mutations were created in this region of the gene. To create point mutations 194 R / E, 194 R / Q, 191 K / E, 191 K / Q, 188R / E, 188R / Q, the primer 5952 KGF-2? 33, 5 'Afl III 5' was used with the indicated 3 'primers, which contain the appropriate point mutations for KGF-2, in PCR reactions using standard conditions well known to those skilled in the art, with KGF-2β33 as template. The resulting products were restricted with AflIII and HindIII and cloned into the expression vector of E. coli, pQE60 restricted with Ncol and HindIII.

Construction of KGF-2? 33, 194 R / E: The following primers were used: 5952 KGF? 33 5 'AflIII: 5' GCGGCACATGTCTTACAACCACCTGCAGGGTG 3 '(SEQ ID No. 117) KGF2 3'HindIII 194aa R a E: 5' CTGCCCAAGCTTTTATGAGTGTACCACCATTGGAAGAAAGTGAGC AGAGGTGTTTTTTTCTCGTGTTTTCTGTCC 3 '(SEQ ID No. 118) Nucleotide sequence of KGF-2? 33, 194 R / E: ATGTCTTACAACCACCTGCAGGGTGACGTTCGTTGGCGTAAACTGT TCTCTTTCACCAAATACTTCCTGAAAATCGAAAAAAACGGTAAAGT TTCTGGGACCAAGAAGGAGAACTGCCCGTACAGCATCCTGGAGAT AACATCAGTAGAAATCGGAGTTGTTGCCGTCAAAGCCATTAACAGC AACTATTACTTAGCCATGAACAAGAAGGGGAAACTCTATGGCTCAA AAGAATTTAACAATGACTGTAAGCTGAAGGAGAGGATAGAGGAAA ATGGATACAATACCTATGCATCATTTAACTGGCAGCATAATGGGAG GCAAATGTATGTGGCATTGAATGGAAAAGGAGCTCCAAGGAGAGG ACAGAAAACACGAGAAAAAAACACCTCTGCTCACTTTCTTCCAATG GTGGTACACTCATAG (SEQ ID No. 119) Amino acid sequence of KGF-2? 33, 194 R / E: MSYNHLQGDVRWRKLFSFTKYFLKIEKNGKVSGTKKENCPYSILEITS VEIGWAVKAINSNYYLAMNKKGKLYGSKEFNNDCKLKERIEENGYN TYASFNWQHNGRQMYVALNGKGAPRRGQKTREKNTSAHFLPMWHS (SEQ ID No. 120) Construction of KGF-2? 33, 194 R / Q: The following primers were used: 5952 KGF? 33 5 'Afl III: 5' GCGGCACATGTCTTACAACCACCTGCAGGGTG 3 '(SEQ ID No. 121) KGF2 3 'HindIII 194 aa R a Q: 5' CTGCCCAAGCTTTTATGAGTGTACCACCATTGGAAGAAAGTGAGC AGAGGTGTTTTTCTGTCGTGTTTTCTGTCC 3 '(SEQ ID No. 122) Nucleotide sequence of KGF-2 33 194 R / Q: ATGTCTTACAACCACCTGCAGGGTGACGTTCGTTGGCGTAAACTGT TCTCTTTCACCAAATACTTCCTGAAAATCGAAAAAAACGGTAAAGT TTCTGGGACCAAGAAGGAGAACTGCCCGTACAGCATCCTGGAGAT AACATCAGTAGAAATCGGAGTTGTTGCCGTCAAAGCCATTAACAGC AACTATTACTTAGCCATGAACAAGAAGGGGAAACTCTATGGCTCAA AAGAATTTAACAATGACTGTAAGCTGAAGGAGAGGATAGAGGAAA ATGGATACAATACCTATGCATCATTTAACTGGCAGCATAATGGGAG GCAAATGTATGTGGCATTGAATGGAAAAGGAGCTCCAAGGAGAGG ACAGAAAACACGACAGAAAAACACCTCTGCTCACT TTCTTCCAATGGTGGTACACTCATAG (SEQ ID No. 123) Amino Acid Sequence of KGF-2? 33, 194 R / Q: MSYNHLQGDVRWRKLFSFTKYFLKIEKNGKVSGTKKENCPYSILEITS VEIGWAVKAINSNYYLAMNKKGKLYGSKEFNNDCKLKERIEENGYN TYASFNWQHNGRQMYVALNGKGAPRRGQKTRQKNTSAHFLPMWHS (SEQ ID No. 124) Construction of KGF-2? 33, 191 K / E: The following primers were used: 5952 KGF? 33 5 'Afl III: 5' GCGGCACATGTCTTACAACCACCTGCAGGGTG 3 '(SEQ ID No. 125) KGF2 3' HindIII 191aa K to E 5 ' CTGCCCAAGCTTTTATGAGTGTACCACCATTGGAAGAAAGTGAGC AGAGGTGTTTTTCCTTCGTGTTTCCTGTCCTCTCCTTGG 3 '(SEQ ID No. 1261 Nucleotide sequence of KGF-2 33, 191 K / E: ATGTCTTACAACCACCTGCAGGGTGACGTTCGTTGGCGTAAACTGT TCTCTTTCACCAAATACTTCCTGAAAATCGAAAAAAACGGTAAAGT TTCTGGGACCAAGAAGGAGAACTGCCCGTACAGCATCCTGGAGAT AACATCAGTAGAAATCGGAGTTGTTGCCGTCAAAGCCATTAACAGC AACTATTACTTAGCCATGAACAAGAAGGGGAAACTCTATGGCTCAA AAGAATTTAACAATGACTGTAAGCTGAAGGAGAGGATAGAGGAAA ATGGATACAATACCTATGCATCATTTAACTGGCAGCATAATGGGAG GCAAATGTATGTGGCATTGAATGGAAAAGGAGCTCCAAGGAGAGG ACAGGAAACACGAAGGAAAAACACCTCTGCTCACT TTCTTCCAATGGTGGTACACTCATAG (SEQ ID NO. 127) Amino acid sequence of KGF-2? 33, 191 K / E: MSYNHLQGDVRWRKLFSFTKYFLKIEKNGKVSGTKKENCPYSILEITS VEIGWAVKAINSNYYLAMNKKGKLYGSKEFNNDCKLKERIEENGYN TYASFNWQHNGRQMYVALNGKGAPRRGQETRRKNTSAHFLPMWHS (SEQ ID No. 128) ? 5952 KGF 33 5 'Afl III: 5' GCGGCACATGTCTTACAACCACCTGCAGGGTG3 '(SEQ ID NO. 129) KGF2 3' HindIII 191aa K to Q 5 'CTGCCCAAGCTTTTATGAGTGTACCACCATTGGAAGAAAGTGAGC AGAGGTGTTTTTCCTTCGTGTCTGCTGTCCTCTCCTTGG 3' Construction of KGF2 33, 191 K / Q The following primers were used? (SEQ ID NO 1 30: Nucleotide sequence of KGF2 33, 191 K / Q: ATGTCTTACAACCACCTGCAGGGTGACGTTCGTTGGCGTAAACTGT TCTCTTTCACCAAATACTTCCTGAAAATCGAAAAAAACGGTAAAGT TTCTGGGACCAAGAAGGAGAACTGCCCGTACAGCATCCTGGAGAT AACATCAGTAGAAATCGGAGTTGTTGCCGTCAAAGCCATTAACAGC AACTATTACTTAGCCATGAACAAGAAGGGGAAACTCTATGGCTCAA AAGAATTTAACAATGACTGTAAGCTGAAGGAGAGGATAGAGGAAA ATGGATACAATACCTATGCATCATTTAACTGGCAGCATAATGGGAG GCAAATGTATGTGGCATTGAATGGAAAAGGAGCTCCAAGGAGAGG ACAGCAGACACGAAGGAAAAACACCTCTGCTCACT TTCTTCCAATGGTGGTACACTCATAG (SEQ ID NO. 131) Amino acid sequence of KGF-2? 33, 191 K / Q: MSYNHLQGDVRWRKLFSFTKYFLKIEKNGKVSGTKKENCPYSILEITS VEIGWAVKAINSNYYLAMNKKGKLYGSKEFNNDCKLKERIEENGYN TYASFNWQHNGRQMYVALNGKGAPRRGQQTRRKNTSAHFLPMWHS (SEQ ID No. 132) Construction of KGF-2? 33, 188 R / E: The following primers were used: 5952 KGF? 33 5 'Afl III: 5' GCGGCACATGTCTTACAACCACCTGCAGGGTG 3 '(SEQ ID No. 133) KGF2 3' HindIII 188aa R to E: 5 'CTGCCCAAGCTTTTATGAGTGTACCACCATTGGAAGAAAGTGAGC AGAGGTGTTTTTCCTTCGTGTTTTCTGTCCTTCCCTTGGAGCTCCTTT 3' (SEQ ID No. 134) Nucleotide sequence of KGF2 33, 188R / E: ATGTCTTACAACCACCTGCAGGGTGACGTTCGTTGGCGTAAACTGT TCTCTTTCACCAAATACTTCCTGAAAATCGAAAAAAACGGTAAAGT TTCTGGGACCAAGAAGGAGAACTGCCCGTACAGCATCCTGGAGAT AACATCAGTAGAAATCGGAGTTGTTGCCGTCAAAGCCATTAACAGC AACTATTACTTAGCCATGAACAAGAAGGGGAAACTCTATGGCTCAA AAGAATTTAACAATGACTGTAAGCTGAAGGAGAGGATAGAGGAAA ATGGATACAATACCTATGCATCATTTAACTGGCAGCATAATGGGAG GCAAATGTATGTGGCATTGAATGGAAAAGGAGCTCCAAGGGAAGG ACAGAAAACACGAAGGAAAAACACCTCTGCTCACTTTCTTCCAATG GTGGTACACTCATAG (SEQ ID No. 135) Amino acid sequence of KGF2? 33, 188R / E: MYNHLQGDVRWRKLFSFTKYFLKIEKNGKVSGTKKENCPYSILEITSV EIGWAVKAINSNYYLAMNKKGKLYGSKEFNNDCKLKERIEENGYNT YASFNWQHNGRQMYVALNGKGAPREGQKTRRKNTSAHFLPMWHS (SEQ ID No. 136) Construction of KGF2? 33, 188 R / Q: The following primers were used: 5952 KGF? 33 5 'Afl III: 5' GCGGCACATGTCTTACAACCACCTGCAGGGTG 3 '(SEQ ID No. 137) KGF2 3 'HindIII 188aa R a Q: 5' CTGCCCAAGCTTTTATGAGTGTACCACCATTGGAAGAAAGTGAGC AGAGGTGTTTTTCCTTCGTGTTTTCTGTCCCTGCCTTGGAGCTCCTTT 3 '(SEQ ID No. 138) Nucleotide sequence of KGF2? 33, 188 R / Q ATGTCTTACAACCACCTGCAGGGTGACGTTCGTTGGCGTAAACTGT TCTCTTTCACCAAATACTTCCTGAAAATCGAAAAAAACGGTAAAGT TTCTGGGACCAAGAAGGAGAACTGCCCGTACAGCATCCTGGAGAT AACATCAGTAGAAATCGGAGTTGTTGCCGTCAAAGCCATTAACAGC AACTATTACTTAGCCATGAACAAGAAGGGGAAACTCTATGGCTCAA AAGAATTTAACAATGACTGTAAGCTGAAGGAGAGGATAGAGGAAA ATGGATACAATACCTATGCATCATTTAACTGGCAGCATAATGGGAG GCAAATGTATGTGGCATTGAATGGAAAAGGAGCTCCAAGGCAGGG ACAGAAAACACGAAGGAAAAACACCTCTGCTCACTTTCTTCCAATG GTGGTACACTCATAG (SEQ ID No. 139) Amino acid sequence of KGF2? 33, 188 R / Q: MSYNHLQGDVRWRKLFSFTKYFLKIEKNGKVSGTKKENCPYSILEITS VEIGWAVKAINSNYYLAMNKKGKLYGSKEFNNDCKLKERIEENGYN TYASFNWQHNGRQMYVALNGKGAPRQGQKTRRKNTSAHFLPMWHS (SEQ ID No. 140) Construction of KGF2? 33, 188K / E: For the 183 K / E mutation, two PCR reactions were established for site-directed mutagenesis of this lysine oligonucleotide. In one reaction, 5952 KGF was used? 33 5 'AflIII as the 5' primer, and KGF 183aa K to E antisense was used as the 3 'primer in the reaction. In a second reaction, KGF2 5 '183aa K to E was used in sense, as the 5' primer, and the KGF2 3 'HindIII TAA stop was used as the 3' primer. KGF-2? 33 was used as a template for these reactions. The reactions were amplified using standard conditions well known to those skilled in the art. One microliter of each of these PCR reactions was used as the template in a subsequent reaction using, as a 5 'primer, 5453 BspHI, and as a 3' primer, 5258 HindIII. The amplification was performed using standard conditions well known to those skilled in the art. The resulting product was restricted with Afl III and HindIII, and cloned into the expression vector in E. coli, pQE60, which was restricted with Ncol and HindIII. The following primers were used: 5952 KGF? 33 5 'Afl III: 5' GCGGCACATGTCTTACAACCACCTGCAGGGTG 3 '(SEQ ID No. 141) KGF2 5' 183aa KAE in direction: 5 'TTGAATGGAGAAGGAGCTCCA 3' (SEQ ID No. 142) KGF2 183aa K a E antisense: 5 'TGGAGCTCCTTCTCCATTCAA 3' (SEQ ID No. 143) KGF2 3 'HindIII TAA arrest: 5' CTGCCCAAGCTTTTATGAGTGTACCACCATTGG 3 '(SEQ ID No. 144) Nucleotide sequence KGF2 33, 183K / E: ATGTCTTACAACCACCTGCAGGGTGACGTTCGTTGGCGTAAACTGT TCTCTTTCACCAAATACTTCCTGAAAATCGAAAAAAACGGTAAAGT TTCTGGGACCAAGAAGGAGAACTGCCCGTACAGCATCCTGGAGAT AACATCAGTAGAAATCGGAGTTGTTGCCGTCAAAGCCATTAACAGC AACTATTACTTAGCCATGAACAAGAAGGGGAAACTCTATGGCTCAA AAGAATTTAACAATGACTGTAAGCTGAAGGAGAGGATAGAGGAAA ATGGATACAATACCTATGCATCATTTAACTGGCAGCATAATGGGAG GCAAATGTATGTGGCATTGAATGGAGAAGGAGCTCCAAGGAGAGG 5 ACAGAAAACACGAAGGAAAAACACCTCTGCTCACTTTCTTCCAATG GTGGTACACTCATAG (SEQ ID NO. 145) Amino acid sequence of KGF2? 33, 183K / E: MSYNHLQGDVRWRKLFSFTKYFLKIEKNGKVSGTKKENCPYSILEITS 10 VEIGWAVKAINSNYYLAMNKKGKLYGSKEFNNDCKLKERIEENGYN TYASFNWQHNGRQMYVALNGEGAPRRGQKTRRKNTSAHFLPMWHS (SEQ ID No. 146) Example 23 15 Effect of KGF-2 on Survival After Total Body Irradiation in Balb / c Mice Ionizing radiation is commonly used to treat many malignancies, including lung and breast cancer, lymphoid and pelvic tumors (Ward, WF et al., CRC Handbook of Animal Models of Pulmonary Disease, CRC Press, pp. 165-195 ( 1989)). However, damage induced by radiation (lung, intestine, etc.) limits the intensity and success of radiation therapy (Morgan, G. * ^ *** ~ < ~ * * ^. * ^ * ^ * Mi ^? Ai ^^^ ^ W. and collaborators, Int. J. Radiat. Oncol. Biol. Phys. 31: 361 (1995)). The gastrointestinal mucosa has a rapid cell cycle and is particularly sensitive to cytotoxic agents (Potten, C. S. et al., In: Cytotoxic Insul t to Tissue, Churchill Livingstone, pp. 105-152 (1983)). Some of the manifestations of intestinal radiation damage include acute proctitis, intestinal fibrosis, narrowing or fistula formation (Anseline, D.F. et al, Ann.Surgery 194: 716-724 (1981)). A treatment that protects the normal structures of radiation without altering the radiosensitivity of the tumor, could be beneficial in the management of these disorders. Despite the irradiated area, the radiation dose is limited by the radiosensitivity of normal tissue. Complications after total or partial irradiation to the body include pneumonitis, fibrosis, gastrointestinal damage and disorders of the bone marrow. Several cytokines, including IL-1, TNF, IL-6, IL-12, have demonstrated radioprotective effects after TBI (Neta, R. et al, J. "Exp. Med. 173: 1177 (1991)). that IL-11 protects the mucosal cells of the small intestine after combined radiotherapy and chemotherapy (Du, XX et al., Blood 83: 33 (1994)) and radiation-induced thoracic damage (Redlich, CA et al., The Journal of Immunology 157: 1705-1710 (1996)).

Animals All experiments were performed using Balb / c mice. The animals were acquired at 6 weeks of age and were 7 weeks old at the beginning of the study. All manipulations were performed using aseptic techniques. This study was conducted according to the guidelines described by the Institutional Animal Care and Use Committee of Human Genome Sciences, Inc., which reviewed and approved the experimental protocol.

KGF -2 The protein consists of a 141-amino acid human protein called KGF-2? 33. This protein is a truncated isoform of KGF-2 that lacks the first 33 amino-terminal residues of the mature protein. The gene encoding this protein has been cloned into an E. coli expression vector. Fractions containing more than 95% pure recombinant materials were used for the experiment. KGF-2 was formulated in a vehicle containing 40 mM sodium acetate + 150 mM sodium chloride, pH 6.5. The dilutions were made from the reserve solution using the same vehicle.

Whole Body Irradiation and Experimental Design Mice were irradiated with 519 RADS (5.19 Gy) using a Cesium 68 Mark I Shepherd irradiator. KGF-2? 33 was administered daily subcutaneously, beginning 2 days before irradiation and continuing for 7 days after irradiation. Daily, weights were obtained in all mice. Groups of mice were randomly divided to receive one of the treatments: Whole body irradiation (TBI) plus buffer, TBI plus KGF-2? 33 (1 mg / kg subcutaneous), TBI plus KGF-2? 33 (5 mg / subcutaneous kg). Two independent experiments were performed.

Results Two studies were performed using irradiated animals. In the first study, the animals were irradiated with 519 RADS (5.19 Gy). The animals were treated with buffer KGF-2? 33 to 1 and 5 mg / kg subcutaneously two days before irradiation and daily thereafter for 7 days. On day 25 after the total irradiation of the body 1/5 animals survived in the group with shock absorber. In contrast, the groups treated with KGF-2 had 5/5 animals at 1 mg / kg and 4/5 at 5 mg / kg (Figure 44). In addition, animals treated with KGF-2 showed 0. 9% and 5.3% gain in weight on day 20 after TBI. In contrast, the group treated with shock absorber had a loss of 4.2% by weight on day 20. Normal, non-irradiated, normal control animals showed 6.7% gain in weight over the same period of time (Figure 45). The animals in the second study were also irradiated with 519 RADS (5.19 Gy). These animals were treated with buffer KGF-2? 33 to 1 and 5 mg / kg subcutaneously, two days before irradiation and daily thereafter for 7 days. On day 15 after total irradiation of the body all the animals in the group with buffer were sacrificed. KGF-2 at 1 mg / kg had 30% survival and 60% survival at 5 mg / kg. On day 25 after TBI the group of 1 mg / kg showed survival of 20% and the group of 5 mg / kg, 50% survival (Figure 46).

» Conclusions In summary, these results demonstrate that KGF-2 has protective effect after TBI. The ability of KGF-2 to increase the survival rate of animals subject to TBI suggests that it could also be useful in radiation-induced damage and to increase the therapeutic proportion of irradiation in the treatment of malignancies.

Example 24 Evaluation of KGF-2 in the TPA model of Cutaneous Inflammation in Mice To demonstrate that KGF-2 could attenuate the progression of contact dermatitis, the cutaneous inflammation model induced by tetradecanoylphorbol acetate (TPA) in mice was used. The use of female BALB / c mice and Swiss Webster male in experimental skin inflammation are well characterized, relevant and reproducible models of contact dermatitis. It has been shown that these strains of mice develop a long-lasting inflammatory response, after topical application of TPA, which is comprised of local hemodynamics, vascular permeability and local migration of leukocytes, and these pathological changes are similar to those of the human dermatitis (Rao et al 1993, Inflammation 17 (6): 723; Rao et al., 1994, J. Kipid Mediators Cell Signaling 10: 213). Groups of mice receive either the vehicle or KGF-2 intraperitoneally, subcutaneously, or intravenously 60 minutes after the topical application of TPA (4 μg / ear) applied as a solution in acetone (200 μg / ml), 10 μl of each one to the inner and outer surface of the ear. The control group receives 20 μl of acetone as a topical application. Four hours after the application of TPA, the increase in the thickness of the ear is measured and the ears are excised for histology. To determine the vascular permeability in response to TPA, the mice are intravenously injected through the tail veins Evans blue (300 mg / kg) at selected times after topical application of TPA, and the mice are sacrificed. minutes later. The ears are excised and removed, then extracted in dimethylformamide and centrifuged. The absorbance readings are spectrophotometrically measured at 590 nm.

Example 25 Effect of KGF-2? 33 on Wound Healing The biological effects of KGF-2? 33 on the skin were examined based on the initial in vitro data demonstrating the ability of KGF-2 to stimulate primary human epidermal keratinocytes, as well as murine BaF3 pro-B cells transfected with the isoform 2iiib of FGFR. Initial experiments were performed to determine the biological effects of KGF-2? 33 after intradermal administration. After intradermal studies, KGF-2? 33 was screened in a variety of wound healing models (including puncture, full thickness, and incision wounds) to determine its potential as a wound healing agent. .

Effect of KGF-2? 33 on Rat Model Impaired with Glucocorticoid, Wound Healing The healed of impaired wound is an important clinical problem associated with a variety of pathological conditions such as diabetes and is a complication of the systemic administration of steroids or antimetabolites. Systemic treatment with glucocorticoids is known to impair wound healing in humans and in animal models of tissue repair. A decrease in circulating monocyte levels and in the inhibition of procollagen synthesis has been observed subsequent to glucocorticoid administration. The inflammatory phase of the heal and the matrix synthesis are therefore important factors involved in the complex process of tissue repair. In the present study, the effects of multiple topical applications of KGF-2 on skin wounds by extirpation, full thickness, in rats in which the healing was impaired by the systemic administration of methylprednisolone were evaluated. Sprague Dawley rats (n = 5 / treatment group) received 8 mm back injuries and methylprednisolone (17 mg / kg, intramuscular) to deteriorate healing. The wounds were treated topically each day with buffer by KGF-2 at doses of 0.1, 0.5 and 1.5 μg in a volume of 50 μl. Wounds were measured on days 2, 4, 6 and 8 using a calibrated Jameson meter. On day 6 (data not shown), and on day 8 (Figure 47) the groups treated with KGF-2 showed a statistically significant reduction in wound closure when compared to control with buffer.

Effect of KGF-2? 33 on Wound Healing in a Diabetic Mouse Model Female homozygous, genetically diabetic (db + / db +) mice 6 weeks of age (n = 6) weighing 30-35 g were subjected to a full thickness dorsal wound with a 6 mm biopsy punch. The wounds were left open and treated daily with placebo or KGF-2 at 0.1, 0.5 and 1.5 μg. The closure of the wound was determined using a Jameson caliper. The animals were sacrificed on day 10 and the wounds were harvested for histology. KGF-2 showed a significant improvement in percent wound closure at 0.1 μg (p = 0.02) when compared to placebo or the untreated group. The administration of KGF-2 also resulted in an improvement in histological score at 0.1 μg (p = 0.03) when compared to placebo or the untreated group (p = 0.01) and 1.5 μg (p = 0.05) compared to the untreated group.

Conclusions Based on the results presented above, KGF-2 shows significant activity in impaired conditions such as the administration of glucocorticoids and diabetes. Therefore, KGF-2 may be clinically useful in stimulating wound healing after surgery, chronic ulcers in patients with diabetes or poor circulation (eg, venous insufficiency and venous ulcers), burns and other abnormal conditions of healing. of wounds, such as uremia, poor nutrition, vitamin deficiencies and systemic treatment with steroids and antineoplastic drugs.

Example 26 Effects of KGF-2? 33 on the Oral Mucosa Clinically used cytotoxic agents have the unfortunate effect of inhibiting the proliferation of normal epithelia at some sites, such as the oral mucosa, leading to life-threatening disturbances at the mucosal barrier. Studies have been conducted to realize the efficacy of KGF-2 in this clinical area. The data support a therapeutic effect of KGF-2 in mucositis models.

Effects of KGF-2? 33 on the Hamster Oral Mucosa We sought to determine if KGF-2 can induce the proliferation of the epithelium of the normal oral mucosa. The effect of KGF-2 on the oral mucosa was evaluated in Golden Syrian hamster. The hamster cheek pouch was treated daily with buffer or KGF-2? 33 (at 0.1, 1 and 10 μg / cheek) which was applied topically to the anesthetized hamster cheeks, in a volume of 100 μl per cheek. The compound was in contact with the cheek for a minimum of 60 seconds and was subsequently swallowed. After 7 days of treatment, the animals were injected with BrdU and sacrificed as described above. The proliferating cells were labeled using the anti-BrdU antibody. Figure 48 shows that there was a significant increase in BrdU labeling (cell proliferation) when the animals were treated with 1 μg and 10 μg of KGF-2Δ33 (when compared to treatment with buffer). Topical treatment with KGF-2 induced the proliferation of normal mucosal epithelial cells. Based on these results, KGF-2 may be clinically useful in the prevention of oral mucositis caused by any chemotherapeutic agents (or other toxic drug regimens), radiation therapy, or any combination chemotherapy-radiotherapy regimen. In addition, KGF-2 can be useful as a therapeutic agent by decreasing the severity of damage to the oral mucosa, as a result of toxic agents (chemotherapy) or radiotherapy.

Example 27 The Effect of KGF-2? 33 on the Healing of Ischemic Wounds in Rats The aim of the experiments presented in this example was to determine the efficacy of KGF-2 in wound healing using an ischemic wound healing model. The blood supply of local skin was partially interrupted by raising a random, full thickness, single pedicle (3x4 cm) flap. A full-thickness wound was made on the local skin, which was composed of the myocutaneous flap. Sixty adult Sprague Dawley rats were used and randomized into treatment groups with KGF-2? 33 and placebo, for this study (5 animals / group / time point). The wounds were harvested respectively on day 1, 3, 5, 7, 10 and 15 after the wound was made.

The strength of wound breakage did not show a significant difference between KGF-2 and the groups treated with buffer at the early time points until day 10 and 15 after the performance of the wound. The results indicated that KGF-2 significantly improved the resistance to wound breakage in the repair of the ischemic wound after 10 days post-wound. These results also suggest that ischemia delays the healing process in both groups, compared to the data previously obtained in studies of normal wound healing. This model of myocutaneous flap provides data and information in an ischemic situation resulting from venous return. These results suggest that KGF-2 could be used in the treatment of chronic venous leg ulcers, caused by a deterioration of venous return and / or venous insufficiency.

Example 28 Evaluation of KGF -2 in the Healing of Colonic Anastomosis in Rats The results of the present experiment demonstrate that KGF-2? 33 increases the rate of intestinal repair in an intestinal or colonic anastomosis model in Wistar or Sprague Dawley rats. In addition, this model can be used to demonstrate that KGF-2 and its isoforms increase the ability of the gastrointestinal wall or the colon to join the sutures. The use of the rat in experimental anastomosis is a well characterized, relevant and reproducible model of surgical wound healing. This model can also be extended to study the effects of chronic steroid treatment or the effects of various chemotherapeutic regimens on the quality and proportion of surgical wound healing in the colon and small intestine.

(Mastboom, W. J. B. et al., Br. J. Surg. 78: 54-56 (1991); Salm, R. and collaborators, J. Surg. Oncol. 47: 5-11 (1991); Weiber, S. et al., Eur. Surg. Res. 26: 173-178 (1994)). The healing of the anastomosis is similar to that of healed wounds elsewhere in the body. The early stages of healing are characterized by acute inflammation followed by proliferation of fibroblasts and synthesis of collagen. Collagen is gradually modeled and the wound is reinforced as the new collagen is synthesized (Koruda, MJ and Rolandelli, RH J. Surg. Res. 48: 504-515 (1990).) Most postoperative complications such as Anastomotic leakage occurs during the first few days after surgery-a period during which resistance of the colon is mainly ensured by the ability of the wound margin to maintain the sutures.It has been reported that the suture retaining capacity of the Gastrointestinal tract decreases as much as 80% during the first days after the operation (Hogstrom, H. and Haglund, U., Acta Chir Scand 151: 533-535 (1985); Jonsson, K. et al., Am. J. Surg. 145: 800-803 (1983)) Rats were anesthetized with a combination of ketamine (50 mg / kg) and xylazine (5 mg / kg) intramuscularly.The animals were kept on a heating pad during the disinfection of the foot l, surgery and post-surgery. The abdominal cavity was opened with an incision of intermediate line of 4 cm in length. A 1 cm wide segment of the left colon was resected 3 cm proximal to the peritoneal reflection, while the marginal blood vessels were preserved. An end-to-end simple layer anastomosis was performed with 8 to 10 interrupted reversed sutures, 8-0 propylene, which were used to restore intestinal continuity. This incisional wound was closed with a 3-0 running silk suture for the muscular layer and surgical staples for the skin. Daily clinical evaluations were conducted on each animal, consisting of individual body weight, body temperature, and food consumption patterns.

Treatment with KGF-2? 33 and with placebo were administered daily subcutaneously, topical, intraperitoneal, intramuscular, intragastric, or intracolonic, immediately after surgery and continued after this until the day of sacrifice, on day 7. There was an untreated control, a group with placebo, and groups with KGF-2 33 Two hours before euthanasia, the animals were injected with 100 mg / kg BrdU i.p. The animals were sacrificed 24 hours after the last treatment (day 5). An intermediate line incision was made on the anterior abdominal wall and a colon segment 1 cm long, including the anastomosis, was removed. A third segment in the surgical site was taken for the total collagen analysis. In a series of two experiments, adult male SD rats (n = 5) were anesthetized and received an end-to-end single-layer anastomosis of the distal colon with 8-10 inverted 6-0 proleno sutures interrupted. The anastomotic site was then topically treated via a syringe either with buffer or KGF-2Δ33 at concentrations of 1 and 4 μg. The animals were then treated daily after this either with buffer or KGF-2? 33 at concentrations of 1 mg / kg or 5 mg / kg ip. The animals were sacrificed on day 5 and the colon excised and frozen in liquid nitrogen, lyophilized and subjected to collagen determinations. The concentration of the collagen is expressed with μg of collagen / mg of tissue in dry weight. The statistical analysis was performed using a non-paired t-test. Mean ± SE. On day 5 the rats were anesthetized and subjected to barium enemas followed by radiographic analysis. Radiological evaluation with end-to-end left colon colonic anastomosis with barium enema from two experiments showed a consistent reduction in peritoneal leak with animals treated with KGF-2 at 1 and 5 mg / kg. This data is shown in the following Table. In addition, the breaking force at the site of the surgery was also examined using a tensiometer. No significant differences were observed between the groups with KGF-2? 33 and with buffer. As shown in Figure 49, significant increases in collagen content at the surgical site were demonstrated at 1 mg / kg of KGF-2? 33 (p = 0.02) and 5 mg / kg (p = 004) relative to to the controls with shock absorber.

Radiological Analysis of Colonic Anastomosis Table Groups Stool Constriction Leak present Anastomotic * peritoneal No treatment (N = 8) 50% 2.0 75% Shock absorber (N = 7) 57% 1.0 50% KGF-2? 33 [1 mg / kg] (N = 8) 50% 1.3 37% KGF-2? 33 [5 mg / kg] (N = 9) 77% 1.6 11% * Anastomotic Constriction Rating: 0 without constriction; 1-5 minimum to severe constriction. Male adult SD rats (n = 5) were anaesthetized with a combination of ketamine (50 mg / kg) and xylazine (5 mg / kg) intramuscularly. The abdominal cavity was opened with an incision of intermediate line of 4 cm in length. A 1 cm wide segment of the left colon was resected 3 cm proximal to the peritoneal reflection while the marginal vessels were preserved. An end-to-end single-layer anastomosis was performed with 8 to 10 interrupted inverted 6-0 prolene sutures to restore intestinal continuity. The anastomosis was then topically treated by means of a syringe either with buffer or KGF-2 at concentrations of 1 and 4 μg. The incisional wound was closed with a 3-0 running silk suture for the muscular layer and surgical staples for the skin. The treatments were then administered daily after this and consisted of buffer or KGF-2? 33 to 1 and 5 mg / kg subcutaneously. The weights were taken on the day of surgery and daily after it. The animals were sacrificed 24 hours after the last treatment (day 5). The animals were anesthetized and received barium enemas and were subjected to X-rays at a fixed distance. The anastomosis was then excised for histopathological and biomechanical analysis.

Example 29 Evaluation of KGF-2 in a Inflammatory Inflammatory Inflammatory Inflammatory Model KGF-2 is a protein that induces the proliferation of keratinocytes in vi tro and is active in a variety of models of wound healing in vivo. The purpose of this study was to determine if KGF-2 was effective in a model of murine colitis induced by ad libitum exposure to sodium dextran sulfate in drinking water. Female Swiss Webster mice from six to eight weeks of age (20-25 g, Charles River, Raleigh, NC) were used in a model of inflammatory bowel disease, induced with a 4% solution of sodium sulfate (DSS, molecular weight 36,000-44,000, American International Chemistry, Natick, MA)) administered ad libitum for one week. KGF-2 was administered by parenteral daily administration (n = 10). Three parameters were used to determine the efficacy: 1) clinical rating, based on the evaluation of the feces; 2) histological qualification, based on the evaluation of the colon; and 3) change in weight. The clinical score was comprised of two parts that total a maximum of four. The consistency of the evacuations was graded as: 0 firm; 1 = loose; 2 = diarrhea. The blood in the stools was also evaluated on a scale of 0 to 2, with 0 = no blood; 1 = occult blood; and 2 = abundant rectal bleeding. An average group score above 3 indicated probable lethality, and the decrease had progressed beyond its treatable stage. Clinical scores were taken on day 0, 4, 5, 6 and 7. To reach a histological score, ascending, transverse and descending colon slices were evaluated in a blind fashion, based on the inflammation rating (0 -3) and qualification of the crypt (0-4). Body weight was measured daily. The data were expressed as the mean + SEM. An unpaired Student t test was used to determine the significant differences compared to the control with disease (* p <0.05; ** p <0.01; *** p <0.001). When mice treated with DSS were administered with a daily intraperitoneal (IP) injection of KGF-2Δ33 at a dose of 1, 5 or 10 mg / kg for 7 days, KGF-2 significantly reduced the clinical score, 28, 38 and 50 percent, respectively. The histological evaluation was in parallel closely with the dose-dependent inhibition of the clinical score, with the dose of 1, 5 and 10 mg / kg which reduces the histological qualification a significant percentage of 26, 48 and 51. KGF-2 also significantly reduced the weight loss associated with the colitis induced with DSS. In a second study, a comparison was made of the relative efficacy of KGF-2? 33 (10 mg / kg) when administered PI or subcutaneously (SC) daily. By the end of the experiment on day 7, animals injected IP with KGF-2 had a significant reduction of 34 percent in the clinical score, while injected KGF-2 SC resulted in a significant reduction of 46 percent. The SC dose also significantly reduced weight loss over controls with DSS. Based on the measurement of the clinical score and body weight, the SC administration of KGF-2 is at least as effective as the IP administration.

EXAMPLE 30 Effects of KGF-2? 33 on Normal Urinary Tissue and Normal Prostate in Hemorrhagic Cystitis Induced by Cyclophosphamide, in Rats The purpose of this example is to show that KGF-2? 33 is able to stimulate the proliferation of the urinary bladder in normal rats and that there is a therapeutic effect of KGF-2? 33 in a rat model of cyclophosphamide-induced hemorrhagic cystitis . Some clinically used cytotoxic agents have side effects that result in the inhibition of normal epithelial proliferation in the bladder, leading to life-threatening ulceration, and rupture in the epithelial lining of the bladder. For example, cyclophosphamide causes hemorrhagic cystitis in some patients, a complication that can be severe and in some cases fatal. Fibrosis of the urinary bladder can also develop with or without cystitis. It is thought that this damage is caused by the metabolites of cyclophosphamide excreted in the urine. The hematuria caused by cyclophosphamide is usually present for several days, but may persist. In severe cases, medical or surgical treatment is required. Cases of severe hemorrhagic cystitis result in discontinuous therapy with cyclophosphamide. In addition, malignancies of the urinary bladder generally occur within two years of treatment with cyclophosphamide and occur in patients who previously had hemorrhagic cystitis (CYTOXAN package insert (cyclophosphamide).) Cyclophosphamide has toxic effects on the prostate and male reproductive systems Treatment with cyclophosphamide can result in the development of sterility, and results in some degree of testicular atrophy.

Effects of KGF-2? 33 on the Normal Bladder, Testicles and Prostate Experimental Design Male Sprague-Dawley rats (160 to 200 g), (n = 4 to 6 / treatment group) were used in these studies. KGF-2? 33 was administered at a dose of 5 mg / kg / day. Daily or sc injections of recombinant KGF-2Δ 33 or buffer (40 mM sodium acetate + 150 mM sodium chloride, at pH 6.5) were administered daily for a period of 1 to 7 days and the rats were sacrificed the next day. To examine the reversibility of the effects induced with KGF-2? 33, the additional animals were injected ip daily for 7 days with KGF-2? 33 or buffer, and sacrificed after a treatment-free period of 7 days. On the day of sacrifice, the rats were injected ip with 100 mg / kg BrdU. Two hours later, the rats were overdosed with ether and the selected organs were removed. The tissue samples were fixed in 10% neutral buffered formalin for 24 hours and embedded in paraffin. To detect the incorporation of BrdU in replicating cells, sections of five micras were subjected to immunohistological procedures using an anti-BrdU mouse monoclonal antibody and the ABC Elite detection system. The sections were slightly counterstained with hematoxylin. The sections were read by blind observers. The number of proliferating cells was counted in 10 randomized fields per animal at an amplification of 10x for the prostate. To evaluate the effects of KGF-2? 33 in the bladder, cross sections of these tissues were prepared and the number of proliferating and non-proliferating cells were counted in 10 randomized fields at a 20x amplification. The results are expressed as the percentage of cells marked to those not marked. The data is presented as the mean + SEM. The statistical analyzes (unpaired two-tailed t-test) were performed with the StatView computer software package and the statistical significance is defined as p < 0.05.

Results Vej iga: The intraperitoneal injection of KGF-2? 33 induced the proliferation of bladder epithelial cells by day 7 in the study period (dark squares, Figure 52) but this did not influence the weight of the organ. Subcutaneous administration promoted a small increase in proliferation, but this failed to achieve statistical significance (dark circles, Figure 52).

Prostate and Testicles: The administration sc and ip of KGF-2? 33 induced significant proliferation of the prostate (Figure 53) but this was normalized after two injections. Prolonged ip treatment with KGF-2? 33 did not increase the weight of the prostate or testes.

Effects of KGF-2? 33 on the Experimental Design of Hemorrhagic Cystitis Induced by Cyclophosphamide Male Sprague Dawley rats (300-400 g) (n = 5 / group) were injected i.v. via the tail vein with placebo with buffer or KGF-2? 33 at concentrations of 1 or 5 mg / kg, 24 hours before injection of 200 mg / kg i.p. of cyclophosphamide. On the final day, 48 hours after the cyclophosphamide injection, the rats were injected ip with 100 mg / kg BrdU. Two hours later, the rats were sacrificed by administration of C02. Bladder fixation was performed by direct injection of 10% formalin into the lumen of the bladder, and rinsing the outside of the bladder with formalin. After 5 minutes, the bladder and prostate were removed. The urinary bladder and the prosthetic gland were embedded in paraffin, transected and stained with H & E and a mouse monoclonal anti-BrdU antibody. The degree of urothelial damage was assessed using the following rating system: bladders were scored by two independent observers to describe the degree of urothelial loss. (The urothelial damage was rated as 0, 25%, 50%, 75% and 100% urothelial loss). In addition, the thickness of the bladder wall was measured at 10 random sites per section and expressed in μm.

Results Macroscopic observations In rats treated with placebo and cyclophosphamide, the bladders were thick and stiff. After the 10% injection of formalin, very little expansion of the bladders was noted. However, in the groups pretreated with KGF-2? 33, a greater elasticity of the bladder was observed after direct injection with formalin, suggesting a lower degree of fibrosis.

Microscopic Observations Figure 54 shows the results of pretreatment with KGF-2? 33 on the degree of ulceration in the bladder. In normal rats treated with saline i.p. (saline control), the bladders appeared histologically normal and no ulceration of the urothelium was observed. The administration of 200 mg / kg i.p. of cyclophosphamide resulted in ulceration of the bladder epithelium that was between 25 and 50% of the total epithelial area (with an average of 37%). The administration of KGF-2? 33 24 hours before cyclophosphamide resulted in a significant reduction in the degree of ulceration (1 mg / kg 0. 4% p = 0.0128, and 5 mg / kg 5%, p = 0.0338) when compared to animals treated with placebo who received cyclophosphamide.

Figure 55 shows the effects of KGF-2? 33 on the thickness of the urinary bladder wall that includes the epithelium, smooth muscle layers and the serous surface. In groups treated with shock absorber alone, the thickness of the bladder wall is approximately 40 μm. Treatment with cyclophosphamide results in a 5-fold increase in the thickness of the bladder wall to 210 μm. Pretreatment with KGF-2? 33 of the animals treated with cyclophosphamide resulted in a significant inhibition of the bladder wall enlargement, by cyclophosphamide (1 mg / kg 98.6 μm (p = 0.007) and 5 mg / kg 52.3 μm (p <0.0001)) when compared to treatment with cyclophosphamide alone.

Microscopic Observations Prosthetic gland: In rats receiving shock absorber and cyclophosphamide, a marked atrophy of the prosthetic glands (acne) was observed accompanied by enlarged interstitial spaces with marked edema when compared to normal. In addition, it was observed that the epithelial cell lining of the prosthetic glands was much shorter and less dense than in the corresponding normal prosthetic tissue. Pretreatment with KGF-2? 33 to 1 mg / kg and 5 mg / kg showed a normal histological appearance of the prosthetic gland. No increase in the interstitial spaces or edema was observed, and the epithelial cells that line the prosthetic glands were similar in size and density to normal prosthetic tissue. conclusion The results show that KGF-2 specifically induces the proliferation of epithelial cells of the bladder and the epithelial cells that line the prostate glands. The results also demonstrate that KGF-2 specifically results in a significant reduction in the degree of ulceration in hemorrhagic cystitis induced by cyclophosphamide.

Example 31 Effect of KGF-2 on Proliferation of Cells in Normal Rats Introduction KGF-2, a member of the FGF family, induces the proliferation of normal human and rat keratinocytes.

It has approximately 57% homology to KGF-1 (a member of the FGF family). It has been reported that KGF-1 induces the proliferation of the epithelia of many organs (Housley et al, keratinocyte growth factor induces the proliferation of hepatocytes and epithelial cells throughout the rat gastrointestinal tract J. Clin. 94: 1764-1777 (1994) Ulich et al, keratinocyte growth factor is a growth factor for type II pneumocytes in vivo J. Clin Invest 93: 1298-1306 (1994); Ulich et al. , the keratinocyte growth factor is a growth factor for mammary epithelium in vivo The mammary epithelium of the lactating cells is resistant to the proliferative action of the keratinocyte growth factor Am J Pathol 144: 862-888 (1994); Nguyen et al., The expression of the keratinocyte growth factor in the embryonic liver of transgenic mice causes changes in the development and epithelial differentiation resulting in ri Polycystic ones and other organ malformations. Oncogene 12: 2109-2119 (1996); Yi et al., The growth factor of keratinocytes induces epithelial proliferation of the pancreatic ducts. Am J Pathol 145: 80-85 (1994); and Yi et al., the keratinocyte growth factor causes proliferation of the urothelium in vivo. J. Urology 154: 1566-1570 (1995)). Similar experiments were performed with KGF-2 to determine if this induces normal epithelial proliferation in rats when administered systemically using the sc and ip routes.

Methods: Male Sprague-Dawley rats, weighing 160-220 g, were obtained from Harlan Sprague Dawley for these studies. KGF-2? 33 (HG03411-E2) was administered at a dose of 5 mg / kg / day. Daily ip or sc injections of KGF-2? 33 0 recombinant buffer (40 mM sodium acetate + 150 mM sodium chloride, at pH 6.5) were administered for a period of 1 to 7 days and the rats were sacrificed the next day (see below). To examine the reversibility of the effects induced with KGF-2? 33, additional animals were injected ip daily for 7 days with KGF-2? 33 or with buffer and sacrificed after a treatment-free period of 7 days. On the day of sacrifice, the rats were injected ip with 100 mg / kg BrdU. Two hours later, the rats were overdosed with ether and the selected organs were removed. The tissue samples were fixed in 10% neutral buffered formalin for 24 hours and embedded in paraffin. To detect the incorporation of BrdU within the cells in replication, sections of five microns were subjected to immunohistochemical procedures using an anti-BrdU mouse monoclonal antibody (Boehringer Mannheim) and the ABC Elite detection system (Vector Laboratories). The sections were slightly counterstained with hematoxylin. The sections were read by blind observers. The number of proliferating cells was counted in 10 randomized fields per animal at an IOx amplification for the following tissues: liver, pancreas, prostate, and heart. Ten random fields were used for the analysis of the lungs, except that the proliferation was quantified at a 20x amplification. Since the kidney has many functionally discrete areas, proliferation was evaluated in a coronal cross section taken through the center of one kidney per animal. To evaluate the effects of KGF-2? 33 on the esophagus and bladder, cross sections of these tissues were prepared and the number of proliferating and non-proliferating cells was counted in ten random fields at an amplification of lOx and 20x, respectively. The results are expressed as the percentage of cells marked to those not marked. The data are presented as the mean ± SEM.

Statistical analysis (unpaired two-tailed t test) were performed with the StatView Program Package (Abacus Concepts, Inc., Berkeley, CA) and statistical significance is defined as p < 0.05.

Results Figure 56 shows an overview of the experimental protocol. Six animals were used per group. However, during the analysis by the "blind" observers it became clear that occasionally the injection with BrdU was unsuccessful. Before the results were decoded, data from 8 rats of 116 rats (or 7% of the animals) were excluded from the study and the sizes of the resulting groups are shown in the following Table: Group sizes used in these studies n = Treatment Time ip sc KGF-2? 33 1 day 6 5 shock absorber 1 day 6 6 KGF-2? 33 2 days 6 4 Shock absorber 2 days 6 6 KGF-2? 33 3 days 5 5 Shock absorber 3 days 5 5 KGF-2? 33 7 days 6 6 Shock absorber 7 days 6 5 KGF-2? 33 7 days + 7 days 6 ND free of treatment Shock absorber 7 days + 7 days 6 ND free of treatment Liver. When ip was administered, KGF-2Δ 33 induced a rapid proliferation of the hepatocytes (dark squares) (Figure 57) after 1 injection and this increased the mitotic activity that persisted for three days, returning to normal after 7 days of daily injections. In contrast to the dramatic effect that the ip administration of KGF-2 exerted on the liver, when sc was administered (dark circle, Figure 57) This growth factor showed less effects. Proliferation was elevated after one day of treatment but returned to normal after two daily injections.

Pancreas. In contrast to the rapidly reversible effects of KGF-2? 33 administered ip on the liver, such injections induced proliferation of the pancreas, which continued in the study period of 14 days (dark squares, Figure 58). Surprisingly, subcutaneous administration of KGF-2? 33 (dark circles) failed to induce proliferation at any time point.

Kidney and Bladder. KGF-2? 33 induced proliferation of the renal epithelium when administered either by the sc or ip route, but the former induced a greater effect. The SC administration induced a rapid increase in proliferation (dark circles) that were at maximum after 2 days, after which it returned to normal after 7 daily treatments (Figure 59). When KGF-2? 33 was administered ip (dark squares), there was a modest but significant increase in proliferation observed on days 2 and 3 only. Intraperitoneal injection of KGF-2? 33 also induced the proliferation of bladder epithelial cells over the 7-day study period (dark squares, Figure 52). Subcutaneous administration promoted a small increase in proliferation, but this failed to achieve statistical significance (dark circles, Figure 52).

Prostate. The administration sc and ip of KGF-2? 33 induced significant proliferation of the prostate (Figure 53) but normalized after two injections.

Esophagus. KGF-2? 33 administered sc or ip promoted a short-lived increase, early, in the proliferation of esophageal cells (1 and 2 days, respectively) that returned rapidly to normal (results not shown).

Other organs Systemic administration of KGF-2? 33 by the ip and sc routes failed to promote the proliferation of lung epithelia in a 7-day dosing period (results not shown).

Discussion When administered in a sc route, stimulation of normal epithelial proliferation was observed in some organs (liver, kidney, esophagus, and prostate) but these effects, for the most part, were short-lived and all were reversible. The proliferation in these organs was reversed even during the daily sc administration of KGF-2. The route of administration had dramatic effects on the observed proliferation. While daily ip administration increased the rate of proliferation in the liver in a period of 3 days, the animals administered with KGF-2 sc daily showed high proportions after 1 day of treatment only. Even more surprising was the response of the pancreas. When the animals were administered ip KGF-2, the pancreas showed a significantly high level of proliferation in the 14-day study period. However, sc administration of KGF-2 did not induce increased mitotic activity in the pancreas. Similarly, the ip treatment, but not sc with KGF-2, promoted a proliferation of the bladder mucosa. The ip administration of KGF-2 promoted a small burst of proliferation, short-lived in the kidney, which focused on the region containing the collecting ducts. Daily sc treatment induced an exaggerated, prolonged proliferation in this area.

Example 32 Effects of KGF-2? 33 on Proliferation of Pulmonary Cells After Intratracheal Administration The purpose of this example is to show that KGF-2? 33 is capable of stimulating pulmonary proliferation in normal rats after intratracheal administration (administration of KGF-2? 33 directly to the lung).

Methods: Male Lewis rats (220-270 g), (n = 5 / treatment group) were used in these studies. KGF-2? 33 or placebo (40 mM sodium acetate + 150 mM sodium chloride at pH 6.5) were intratracheally administered at a dose of 1 and 5 mg / kg in a volume of 0.6 ml followed by 3 ml of air.

The treatments were administered on day 1 and on day 2 of the experimental protocol. On day 3, the day of sacrifice, the rats were injected ip with 100 mg / kg of BrdU. Two hours later the rats were sacrificed by asphyxia with C02. Lungs were inflated with 10% buffered formalin via an intratracheal catheter, and sagittal sections of the lung were embedded with paraffin. To detect the incorporation of BrdU in cells in replication, five micron sections were subjected to immunohistochemical procedures using a mouse anti-BrdU monoclonal antibody and the ABC Elite detection system. The sections were slightly counterstained with hematoxylin. The sections were read by two observers "in blind". The number of proliferating cells was counted in 10 random fields per section at a 20x amplification. The results are expressed as the number of BrdU positive cells per field. The data are presented as the mean ± SEM. The statistical analyzes (unpaired t test) were performed with Instat v2.0.1 and the statistical significance is defined as p < 0.05. Results: Intratracheal injection of KGF-2? 33 to 1 and 5 mg / kg resulted in an increase in the proliferation of lung epithelial cells as shown in Figure 60. Treatment with KGF-2? 33 resulted in increases statistically significant in the number of cells positive to BrdU / field at 1 mg / kg, 23.4 cells / field (p = 0.0002) and at 5 mg / kg of 10.3 cells / field (p = 0.0003) relative to the buffer controls of 1.58 cells per field.

Example 33 KGF-2 Topical in Infected Incisional Wounds Bacterial wound infection continues to be of crucial clinical importance. Under normal situations, the complex process of wound healing progresses without difficulty. However, the inoculation of a wound by bacteria causes an imbalance of the cellular mediators in the inflammatory response, resulting in delayed wound healing. Contamination of the open wound inhibits the healing process of the wound as characterized by decreased contraction of the wound, less than the normal collagen content of the wound and decreased tensile strength. Male Sprague Dawley rats (n + 10 / group) were anesthetized with a combination of ketamine (53 mg / kg im) and xylazine (5.3 mg / kg im) on day 1. The dorsal region was shaved and disinfected with alcohol 70% A surgical wound of 2.5 cm of full thickness was created (through the epidermis, the dermis to the subcutaneous layer) started approximately below the shoulder blades using a sterile scalpel no. 10. The wounds were covered with 3 equidistant skin staples. The incisions were then inoculated intraincisionally with Staphylococcus aureus (107 cfu / 50 μl) in PBS. KGF-2? 33 was applied topically at the time of wounding (day 0) at doses of 0.1, 1 and 10 μg per wound in a volume of 50 μl. The wounds were then covered with a gas permeable occlusion bandage (Tegaderm). The animals were sacrificed on day 5 by anesthesia with ketamine / xylazine, followed by lethal intracardiac administration of sodium pentobarbital (300 mg / kg). The intermediate segment 0.5 cm of the wound was excised and frozen for the determination of collagen. Two additional strips of the wound measuring 0.5 cm wide were excised. The excised strips of the wound were used for the study of the resistance to rupture using an Instron skin tensiometer. The breaking strength was defined as the highest force supported by each wound before rupture using a load cell of 4,989 kg (11 lb) at a speed of 0 mm / sec. Two values for each animal were averaged to provide a mean value of resistance to wound breakage. The statistical analysis was performed using a non-paired t test (mean ± SE). The intraincisional application of Staphylococcus aureus in the wound resulted in a significant deterioration in the healing of the wound, as measured by resistance to breakage (uninfected wound, treated with bacterial vehicle 136 ± 6 g; infected wound 87 ± 6 g; p < 0.001 in an experiment; uninfected wound treated with bacterial vehicle 200 ± 14 g, - infected wound 154 and 10 g p = 0.01 in another experiment). Topical administration of KGF-2 caused an increase in breakdown resistance that was statistically significant at doses of 0.1 and 10 μg when compared to KGF-2 + S aureus control buffer (KGF-2 0.1 μg 152 ± 16 g (p = 0.002), 1 μg 135 ± 12 g (p = 0.003), 10 μg 158 ± 10 g (p <0.0001) in one experiment, 0.1 μg 185 ± 10 g, (p = 0.03), 1 μg 186 ± 11 g (p = 0.03), 10 μg 190 ± 7 g p + 0.009) in another experiment). The analysis of the collagen of the intermediate strip of the wound, of 0.5 cm, revealed that there was an increased content of collagen in the wounds treated with KGF-2. However, when compared to the buffer controls, no statistically significant increase in collagen content was observed.

Example 33 Proliferative effect of the dosage i. v. every third day with 1 mg / kg of KGF-2? 33 Male Sprague Dawley rats were intravenously injected with either KGF-2? 33 at a dose of 1 mg / kg, or with buffer. The animals were injected either daily or every other day. Each treatment group was injected for a week and sacrificed at the end of the week. On the day of sacrifice, the animals were injected i.p. with 100 mg / kg of BrdU. Two hours later, the animals were sacrificed, and the serum was collected. Several tissues were collected and fixed in 10% neutral buffered formalin. The tissues were processed for histological evaluation. The tissues were stained with hematoxylin and eosin, periodic acid-Schiff, or alcian blue. Additional sections were subjected to immunohistochemical staining with an anti-BrdU antibody. Proliferation was quantified using an image analysis spectrum, the IPlab Spectrum. The analysis of the serum chemistry was performed using an automated chemical analyzer. The following parameters were quantified: weight of the thyroid gland, proliferation of globet cells in the small intestine (duodenum, jejunum and ileum); proliferation of globet cells in the colon; proliferation in the parotid and submandibular glands, and serum chemistry analytes (glucose, BUN, calcium, total protein, albumin, alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, cholesterol, and triglycerides). In the small intestine and colon, daily treatment with KGF-2 caused a significant increase in the number of globet cells. The treatment every third day did cause a slight increase in the number of globet cells, however, it did not reach a statistically significant level. In the salivary gland, an increase in the cells was observed in the parotid gland only. There was no difference between the treatment groups. There was an enlargement in the thyroid gland due to both dosing regimens. The magnitude of this increase was greater in the daily treatment group. Daily treatment with KGF-2 resulted in a statistically significant increase in the following analytes: triglycerides, alkaline phosphatase, calcium, albumin, and total protein. The treatment every third day had no effect on these analytes. The cholesterol levels were high in both treatment groups. However, the magnitude of the increase was greater in the daily treatment group. Cell damage markers, such as ALT and AST, were similarly reduced in both treatment groups.

Example 34 Formulation of a Polypeptide The composition of KGF-2 will be formulated and dosed in a manner consistent with good medical practice, taking into account the clinical condition of the individual patient (especially the side effects of treatment with the KGF-2 polypeptide alone), the site of administration, the method of administration, the administration program, and other factors known to practitioners. The "effective amount" for purposes herein is thus determined by such considerations. As a general proposition, the pharmaceutically effective total amount of KGF-2 administered parenterally per dose will be in the range of about 1 μg / kg / day to 10 mg / kg / day of the patient's body weight, although, as noted above , this will be subject to therapeutic discretion. More preferably, this dose is at least 0.01 mg / kg / day, and most preferably for humans between about 0.01 and 1 mg / kg / day for the hormone. If administered continuously, KGF-2 is typically administered at a dose rate of about 1 μg / kg / hour to about 50 μg / kg / hour, either by 1 to 4 injections per day or by continuous subcutaneous infusions, for example, using a mini pump. An intravenous bag solution may also be employed. The length of treatment necessary to observe changes in the interval after treatment for the responses to occur seems to vary depending on the desired effect. Pharmaceutical compositions containing KGF-2 are administered orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (as by powders, ointments, gels, drops or transdermal patch), buccally, or as an oral or nasal spray. "Pharmaceutically acceptable carrier" refers to a solid, semi-solid or liquid, non-toxic filler, diluent, encapsulation or auxiliary material of the formulation, of any type. The term "parenteral" as used herein refers to modes of administration that include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion. KGF-2 is also properly administered by sustained release systems. Suitable examples of sustained release compositions include semipermeable polymer matrices in the form of shaped articles, e.g., films, or microcapsules. Sustained-release matrices include polylactides (U.S. Patent No. 3,773,919, European Patent 58,881), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (Sidman, U. et al., Biopolymers 22: 547-556 (1983)), poly (2-hydroxyethyl methacrylate) (R. Langer et al., J. Biomed, Mater. Res. 15: 167-277 (1981), and R.

Langer, Chem. Tech. 12: 98-105 (1982)), ethylene vinyl acetate (R. Langer et al.) Or poly-D- (-) - 3-hydroxybutyric acid (European Patent No. 133,988). Sustained-release compositions also include liposomally entrapped KGF-2 polypeptides. Liposomes containing KGF-2 are prepared by methods known per se: German Patent DE 3,218,121; Epstein et al., Proc. Nati Acad. Sci. USA 82: 3688-3692 (1985); Hwang et al., Proc. Nati Acad. Sci. USA 77: 4030-4034 (1980); European Patent Nos. 52,322; 36,676; 88,046; 143,949; 142,641; Japanese Patent Application No. 83-118008; U.S. Patent Nos. 4,485,045 and 4,544,545; and European Patent No. 102,324. Ordinarily, the liposomes are of the small unilamellar type (approximately 200 to 800 Angstroms) in which the lipid content is greater than about 30 mol percent of cholesterol, the selected proportion being adjusted for optimal secreted polypeptide therapy. For parenteral administration, in one embodiment, KGF-2 is generally formulated by blending it to the desired degree of purity, in a unit dose injectable form (solution, suspension, or emulsion), with a pharmaceutically acceptable carrier, for example, one that It is non-toxic for patients at the doses and concentrations used, and is compatible with other ingredients of the formulation. For example, the formulation preferably does not include oxidizing agents and other compounds known to be harmful to the polypeptides. In general, the formulations are prepared by contacting KGF-2 uniformly and intimately with liquid carriers or finely divided solid carriers, or both. Then, if necessary, the product is shaped into the desired formulation. Preferably, the carrier is a parenteral carrier, more preferably a solution that is isotonic with the patient's blood. Examples of such carriers include water, saline, Ringer's solution, and dextrose solution. Aqueous vehicles such as fixed oils and ethyl oleate are also useful herein, as well as liposomes.

The carrier suitably contains minor amounts of additives such as substances that increase isotonicity and chemical stability. Such materials are non-toxic to patients at the doses and concentrations employed, and include buffers such as phosphate, citrate, succinate, acetic acid, and other organic acids or their salts; antioxidants such as ascorbic acid; low molecular weight polypeptides (less than about 10 residues), for example, polyarginine or tripeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids, such as glycine, glutamic acid, aspartic acid, or arginine; monosaccharides, disaccharides, and other carbohydrates including cellulose or its derivatives, glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; counter ions such as sodium; and / or nonionic surfactants such as polysorbates, poloxamers, or PEG. KGF-2 is typically formulated in such vehicles at a concentration of about 1 mg / ml, up to 100 mg / ml, preferably at 1-10 mg / ml, at a pH of about 3 to 8. It will be understood that the use of certain excipients, carriers or stabilizers mentioned above will result in the formation of polypeptide salts. KGF-2, used for therapeutic administration can be sterile. Sterility is easily achieved by filtration through sterile filtration membranes (eg 0.2 micrometer membranes). Therapeutic polypeptide compositions are generally placed in a container having a sterile access gate, for example, a bag for intravenous solution or a bottle having a plug pierceable by a hypodermic injection needle. The KGF-2 polypeptides will ordinarily be stored in unit dose or multi-dose containers, for example, sealed vials or flasks, as an aqueous solution or as a lyophilized formulation for reconstitution. As an example of a lyophilized formulation, 10 ml bottles are filled with 5 ml of sterile filtered 1% (w / v) aqueous KGF-2 polypeptide solution, and the resulting mixture is lyophilized. The infusion solution is prepared by reconstituting the lyophilized KGF-2 polypeptide using water for injection, bacteriostatic. The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Associated with such or such containers may be a notice in the form prescribed by a governmental agency, which regulates the manufacture, use or sale of pharmaceutical products or biological products, whose notice or note reflects the approval by the agency of manufacture, use or sale for human administration. In addition, KGF-2 can be used in conjunction with other therapeutic compounds. The compositions of the invention can be administered alone or in combination with other therapeutic agents. Therapeutic agents that can be administered in combination with the compositions of the invention include, but are not limited to, other members of the TNF family, chemotherapeutic agents, antibiotics, anti-inflammatory steroids and non-steroids, conventional immunotherapeutic agents, cytokines and / or growth factors. The combinations can be administered either concomitantly, for example, as a mixture, separately but simultaneously or concurrently; or sequentially. This includes presentations in which the combined agents are co-administered as a therapeutic mixture, and also the methods in which the combined agents are administered separately but simultaneously, for example as via separate intravenous lines to the same individual. Administration "in combination" further includes the separate administration of one of the compounds or agents given first, followed by the second. In one embodiment, the compositions of the invention are administered in combination with other members of the TNF family. TNF, TNF-related or TNF-like molecules that can be administered with the compositions of the invention include, but are not limited to, soluble forms of TNF-alpha, lymphotoxin alfa (LT-alpha, also known as TNF-beta) , LT-beta (found in the LT-alpha2-beta heterotrimer complex), OPGL, FasL, CD27L, CD30L, CD40L, 4-1BBL, DcR3, OX40L, TNF-gamma (International Publication No. WO 96/14328), AIM-I (International Publication No. WO 97/33899), alpha endotoxin (International Publication No. WO 98/07880), TR6 (Publication International No. WO 98/30694), OPG, and neutrocin alfa (International Publication No. WO 98/18921, OX40, and nerve growth factor (NGF), and soluble forms of Fas, CD30, CD27, CD40 and 4-IBB, TR2 (International Publication No. WO 96/34095), DR3 (International Publication No. WO 97/33904), DR4 (International Publication No. WO 98/32856), TR5 (International Publication No. WO 98/30693), TR6 (Publication International No. WO 98/30694), TR7 (Publication International No. WO 98/41629), TRANK, TR9 (International Publication No. WO 98/56892), TRIO (International Publication No. WO 98/54202), 312C2 (International Publication No. WO 98/06842), and TR12, and the soluble forms CD154, CD70, and CD153. Conventional non-specific immunosuppressive agents that can be administered in combination with the compositions of the invention include, but are not limited to, steroids, cyclosporine, cyclosporin analogs, cyclophosphamide methylprednisone, prednisone, azathioprine, KF-506, 15-deoxyspergualin , and other immunosuppressive agents that act by suppressing the function of responder T cells. In a further embodiment, the compositions of the invention are administered in combination with an antibiotic agent. Antibiotic agents that can be administered with the compositions of the invention include, but are not limited to, tetracycline, metronidazole, amoxicillin, beta-lactamases, aminoglycosides, macrolides, quinolones, fluoroquinolones, cephalosporins, erythromycin, ciprofloxacin, and streptomycin. In a further embodiment, the compositions of the invention are administered alone or in combination with an anti-inflammatory agent. Anti-inflammatory agents that can be administered with the compositions of the invention include, but are not limited to, glucocorticoids and non-steroidal anti-inflammatories, aminoarilcarboxylic acid derivatives, arylacetic acid derivatives, arylbutyric acid derivatives, arylcarboxylic acids, derivatives of arylpropionic acid, pyrazoles, pyrazolones, salicylic acid derivatives, thiazinecarboxamides, e-acetamidocaproic acid, S-adenosylmethionine, 3-amino-4-hydroxybutyric acid, amixetrin, bendazac, benzydamine, bucoloma, diphenpiramide, ditazole, emorfazone, guaiazulene, nabumetone , nimesulide, orgoteína, oxaceprol, paranilina, perisoxal, pifoxime, procuazona, proxazol, and tenidap. In yet another embodiment, the compositions of the invention are administered in combination with a chemotherapeutic agent. Chemotherapeutic agents that can be administered with the compositions of the invention include, but are not limited to, antibiotic derivatives (for example doxorubicin, bleomycin, daunorubicin and dactinomycin); antiestrogens (for example tamoxifen); antimetabolites (for example fluorouracil, 5-FU, methotrexate, floxuridine, interferon alfa-2b, glutamic acid, plicamycin, mercaptopurine, and 6-thioguanine), cytotoxic agents (eg carmustine, BCNU, lomustine, CCNU, cytosine arabinoside, cyclophosphamide, estramucine, hydroxyurea, procarbazine, mitomycin, busulfan, cis-platinum, and vincristine sulfate), - hormones (eg, medroxyprogesterone, sodium estramustine phosphate, ethinylestradiol, estradiol, megestrol acetate, methyltestosterone, diethylethylbestrol diphosphate, chlorotrianisene, and testolactone); nitrogen mustard derivatives (e.g., mephalen, chlorambucil, mechlorethamine (nitrogen mustard) and thiotepa); steroids and combinations (for example betamethasone sodium phosphate); and others (e.g., dicarbazine, asparaginase, mitotane, vincristine sulfate, vinblastine sulfate and etoposide). In an additional mode, the compositions of the invention are administered in combination with cytokines. Cytokines that can be administered with the compositions of the invention include, but are not limited to IL2, IL3, IL4, IL5, IL6, IL7, IL10, IL12, IL13, IL15, anti-CD40, CD40L, IFN-gamma and TNF -alpha. In a further embodiment, the compositions of the invention are administered in combination with angiogenic proteins. Angiogenic proteins that can be administered with the compositions of the invention include, but are not limited to, Glioma Derived Growth Factor (GDGF), as described in European Patent Number EP-399816; Platelet Derived Growth Factor A (PDGF-A), as described in European Patent Number EP-682110; Platelet Derived Growth Factor B (PDGF-B), as described in European Patent Number EP-282317; Placental Growth Factor (PIGF), as described in International Publication Number WO 92/06194; Factor 2 of Placental Growth (PIGF-2), as described in Hauser et al., Growth Factors, 4: 259-268 (1993); Vascular Endothelial Growth Factor (VEGF), as described in International Publication Number WO 90/13649; Vascular Endothelial Growth Factor A (VEGF-A), as described in European Patent Number EP-506477; Vascular Endothelial Growth Factor 2 (VEGF-2), as described in International Publication Number WO 96/39515; Vascular Endothelial Growth Factor B-186 (VEGF-B186), as described in International Publication Number WO 96/26736; Vascular Endothelial Growth Factor D (VEGF-D), as described in International Publication Number WO 98/02543; Vascular Endothelial Growth Factor D (VEGF-D), as described in International Publication Number WO 98/07832; and Vascular Endothelial Growth Factor E (VEGF-E), as described in German Patent Number DE19639601. The aforementioned references are incorporated herein by reference. In a further embodiment, the compositions of the invention are administered in combination with Fibroblast Growth Factors. Fibroblast Growth Factors that can be administered with the compositions of the invention include, but are not limited to, FGF-1, FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF-7. , FGF-8, FGF-9, FGF-10, FGF-11, FGF-12, FGF-13, FGF-14, and FGF-15. In additional embodiments, the compositions of the invention are administered in combination with other therapeutic or prophylactic regimens, such as, for example, radiotherapy.

Example 35 Method of Treatment of Decreased Levels of KGF -2 The present invention also relates to a method for treating an individual in need of an increased level of KGF-2 activity in the body, which comprises administering to such an individual a composition comprising a therapeutically effective amount of KGF-2 or a agonist thereof. In addition, it will be appreciated that conditions caused by a decrease in the standard or normal expression level of KGF-2 in an individual can be treated by administration of KGF-2, preferably in the secreted form. Thus, the invention also provides a method of treating an individual in need of an increased level of KGF-2 polypeptide, comprising administering to such an individual a pharmaceutical composition comprising an amount of KGF-2, to increase the activity level of KGF-2 in such an individual. For example, a patient with decreased levels of the KGF-2 polypeptide receives a daily dose of 0.1-100 μg / kg of the polypeptide for six consecutive days. Preferably, the polypeptide is in the secreted form. The exact details of the dosing scheme, based on the administration and formulation, are provided in Example 24.

Example 36 Treatment Method of Increased Levels of KGF-2 The present invention relates to a method for the treatment of an individual in need of a decreased level of KGF-2 activity in the body, comprising the administration to such an individual of a composition that includes a therapeutically effective amount of the KGF-2 antagonist in the body. KGF-2. Preferred antagonists for use in the present invention are antibodies specific for KGF-2. The antisense technology is used to inhibit the production of KGF-2. This technology is an example of a method of decreased levels of KGF-2 polypeptide, preferably a secreted form, due to a variety of etiologies, such as cancer. For example, a patient diagnosed with abnormally increased levels of KGF-2 is administered intravenously with antisense polynucleotides at 0.5, 1.0, 1.5, 2.0 and 3.0 mg / kg / day for 21 days. This treatment is repeated after a rest period of 7 days if the treatment was well tolerated. The formulation of the antisense polynucleotide is provided in Example 24.

Example 37 Treatment Method Using Gene Therapy-Ex Vivo A method of gene therapy transplants fibroblasts, which are capable of expressing KGF-2 polypeptides, to a patient. In general, fibroblasts are obtained from a subject by skin biopsy. The resulting tissue is placed in the tissue culture medium and separated into small pieces. Small pieces of tissue are placed on a wet surface of a tissue culture flask, approximately ten pieces are placed in each flask. The flask is turned upside down, hermetically sealed and left at room temperature overnight. After 24 hours at room temperature, the flask is inverted and the pieces of tissue remain fixed to the bottom of the flask and fresh medium is added (for example Ham's F12 medium, with 10% FBS, penicillin and streptomycin). The flasks are then incubated at 37 ° C for about a week. At this time, the fresh medium is added and subsequently changed every several days. After two additional weeks in culture, a monolayer of fibroblasts emerges. The monolayer is trypsinized and raised in scale to larger flasks. pMV-7 (Kirschemeier, P. T. et al., DNA, 7: 219-25 (1988)), flanked by the long terminal repeats of Moloney murine sarcoma virus, digested with EcoRI and HindIII, and subsequently treated with calf intestinal phosphatase. The linear vector is fractionated on agarose gel and purified, using glass spheres. The cDNA encoding KGF-2 can be amplified using the PCR primers corresponding to the 5 'and 3' end sequences respectively described in Example 1. Preferably, the 5 'primer contains an EcoRI site, and the 3' primer includes a HindIII site. Equal amounts of the linear backbone of Moloney murine sarcoma virus and the amplified EcoRI and HindIII fragment are added together in the presence of the T4 DNA ligase. The resulting mixture is maintained under conditions appropriate for the ligation of the two fragments. The ligation mixture is then used to transform HB101 bacteria, which are then plated on agar containing kanamycin, for purposes of confirming that the vector contains KGF-2 suitably inserted. The amphotrophic repackaging cells pA317 or GP + aml2 are developed in tissue culture to confluent density in the Dulbecco Modified Eagle Medium (DMEM) with 10% calf serum (CS), penicillin and streptomycin. The MSV vector containing the KGF-2 gene is then added to the medium and the packaging cells transduced with the vector. The packaging cells now produce infectious viral particles that contain the KGF-2 gene (the packaging cells are now referred to as the producer cells). Fresh medium is added to the transduced producer cells, and subsequently, the medium is harvested from a 10 cm plate of confluent producer cells. The spent medium, which contains the infectious viral particles, is filtered through a millipore filter to eliminate the detached production cells, and this medium is then used to infect fibroblast cells. The medium removed from a subconfluent plate of fibroblasts and quickly replaced with the medium from the producer cells. This medium is removed and replaced with fresh medium. If the virus titer is high, then virtually all fibroblasts will be infected and selection is not required. If the titer is very low, then it is necessary to use a retroviral vector that has a selectable marker, such as neo or his. Once the fibroblasts have been efficiently infected, the fibroblasts are analyzed to determine if the KGF-2 protein is produced. The fibroblasts manipulated by genetic engineering are then transplanted on the host, either alone or after being developed to confluence on cytodex 3 microcarrier spheres.

Example 38 Gene Therapy Using the Endogenous KGF-2 Gene Yet another method of gene therapy according to the present invention involves the operable association of the endogenous KGF-2 sequence with a promoter, via homologous recombination as described, for example, in U.S. Patent No. 5,641,670, issued on June 24, 1997; International Publication WO 96/29411, published September 26, 1996; International Publication No. WO 94/12650, published August 4, 1994; Koller et al., Proc. Nati Acad. Sci. USA 86: 8932-8935 (1989); and Zijlstra et al., Nature 342: 435-438 (1989). This method involves the activation of a gene that is present in target cells, but that is not expressed in cells, or is expressed at a lower level than desired. The polynucleotide constructs are elaborated, which contain a promoter and targeting sequences, which are homologous to the 5 'sequence of non-coding endogenous KGF-2, flanking the promoter. The targeting sequence will be sufficiently close to the 5 'end of KGF-2, so that the promoter will be operably linked to the endogenous sequence after homologous recombination. The promoter and targeting sequences can be amplified using PCR. Preferably, the amplified promoter contains distinct restriction enzyme sites on the 5 'and 3' ends. Preferably, the 3 'end of the first targeting sequence contains the same restriction enzyme site as the 5' end of the amplified promoter, and the 5 'end of the second targeting sequence contains the same restriction site. than the 3 'end of the amplified promoter. The amplified promoter and amplified targeting sequences are digested with the appropriate restriction enzymes and subsequently treated with calf intestinal phosphatase. The digested promoter and digested targeting sequences are aggregated together in the presence of the T4 DNA ligase. The resulting mixture is maintained under conditions appropriate for the ligation of the two fragments. The construction is fractionated by size on an agarose gel, then purified by extraction with phenol and precipitation with ethanol. In this Example, polynucleotide constructs are administered as naked polynucleotides via electroporation. However, polynucleotide constructs can also be administered with agents that facilitate transfection, such as liposomes, viral sequences, viral particles, precipitation agents, etc. Such distribution methods are known in the art. Once the cells are transfected, homologous recombination will take place, which results in the promoter being operably linked to the endogenous KGF-2 sequence. This results in the expression of KGF-2 in the cell. The expression can be detected by immunological staining, or any other method known in the art.

Fibroblasts are obtained from a subject by skin biopsy. The resulting tissue is placed in DMEM + 10% fetal calf serum. The exponential development or fibroblasts almost in stationary phase are trypsinized and rinsed from the plastic surface with nutrient medium. An aliquot of the cell suspension is removed for counting, and the remaining cells are subjected to centrifugation. The supernatant is aspirated and the button is resuspended in 5 ml of electroporation buffer (20 mM HEPES pH 7.3, 137 mM sodium chloride, 5 mM potassium chloride, 0.7 mM disodium hydrogen phosphate, 6 mM dextrose). The cells are again centrifuged, the supernatant is aspirated, and the cells are resuspended in the electroporation buffer containing 1 mg / ml of acetylated bovine serum albumin. The final cell suspension contains approximately 3X106 cells / ml. Electroporation should be immediately after resuspension. The plasmid DNA is prepared according to standard techniques. For example, to construct a plasmid for the address to the KGF-2 locus, the pUCld plasmid (MBI Fermentas, Amherst, NY) is digested with HindIII. The CMV promoter is amplified by PCR with a Xbal site on the 5 'end, and a BamHI site on the 3' end. Two non-coding sequences of KGF-2 are amplified via PCR: a non-coding sequence of KGF-2 (fragment 1 of KGF-2) is amplified with a HindIII site at the 5 'end, and an Xba site at the end 3 ', - the other non-coding sequence of KGF-2 (fragment 2 of KGF-2) is amplified with a BamHI site at the 5' end, and a HindIII site at the 3 'end. The CMV promoter and the KGF-2 fragments are digested with the appropriate enzymes (CMV promoter - Xbal and BamHI); fragment 1 of KGF-2-Xbal; fragment 2 of KGF-2 -BamHI) and ligated together. The resulting ligation product is digested with HindIII, and ligated with the plasmid pUC18 digested with H ndIII. The plasmid DNA is added to a sterile cuvette with 0.4 cm electrode gap (Bio-Rad). The final concentration of DNA is generally at least 120 μg / ml. 0.5 ml of the cell suspension (containing approximately 1.5 X 106 cells) is then added to the cuvette, and the cell suspension and DNA solutions are gently mixed. Electroporation is performed with a Gene-Pulser device (Bio-Rad). Capacitance and voltage are adjusted to 960 μF and 250-300 V, respectively. As the voltage increases, cell survival decreases, but the percentage of surviving cells that stably incorporate the introduced DNA into their genome increases dramatically. Given these parameters, a pulsed time of approximately 14-20 msec should be observed. The cells subjected to electroporesis are kept at room temperature for approximately 5 minutes, and the contents of the cuvette are then gently removed with a sterile transfer pipette. The cells are added directly to 10 ml of preheated nutrient medium (DMEM with 15% calf serum) in a 10 cm box and incubated at 37 ° C. The next day, the medium is aspirated and replaced with 10 ml of fresh medium and incubated for an additional 16 to 24 hours. The genetically engineered fibroblasts are then injected into the host, either alone or after they have grown to confluence on cytodex 3 microcarrier spheres. Fibroblasts now produce the protein product. The fibroblasts can then be introduced into a patient as described above.

Example 39 Treatment Method Using Gene Therapy - In Vivo Advances in technical research have resulted in the development of techniques to distribute and express genes in human cells. The ideal target for gene therapy is the distribution of normal genes in order to generate active proteins and compensate for the lack of endogenous production (Gorecki, DC, et al., Arch. Immunol. Ther. Exp. 45 (5-6): 375-381 (1997)). The distribution of genes that code for the cytokines or growth factors involved in the different phases of wound healing and tissue repair have the potential to modify the outcome of wound healing (Taub, PJ, and collaborators, J.). Microsur 14 (6): 387-390 (1998)) The use of cDNA for growth factors or other cytokines for wound healing and tissue repair has been extensively described.

(Tchorzewski, M. T. et al., J. Surg. Res. 77: 99-103. (1998)). Genes transferred by a vector can be used to generate new cell lines, identify transplanted cells and express growth factors or enzymes. One of the advantages of gene therapy is to achieve therapeutic concentrations of the protein derived from the gene, locally within the site of the lesion. Human recombinant KGF-2 protein has been shown to stimulate the healing of wounds of the skin, the gastrointestinal tract and other organs that contain cells of epithelial origin. The use of the KGF-2 gene is expected to have a similar pharmacological profile as the recombinant protein. The KGF-2 gene can be involved in events related to tissue repair such as cell proliferation, migration and extracellular matrix formation.

The transcribed and translated cDNA has been used to distribute the genes to sites of interest. Some examples of genes used in this manner include aFGF, BMP-7 (Breitbart, A.S., et al., Ann Plast, Surg 24 (5) -488-495 (1999)). These cells have also been seeded in cell carriers including biodegradable matrices (eg polyglycolic acid), tissue equivalents or substitutes (eg artificial skin), artificial organs, collagen derived matrices, etc. Liposomes have been used to carry the cDNA. The PDGF-BB cDNA in the Japanese suspension haemagglutination virus (HVJ) -liposome was studied in patellar ligament healing (Nkamura et al., Gene Ther 5 (9): 1165-1170 (1998)). The genes can also be distributed directly to the site of action by direct injection (for example to the heart). Thus, yet another aspect of the present invention is the use of in vivo gene therapy methods to treat disorders, diseases and conditions. The gene therapy method refers to the introduction of the naked nucleic acid (DNA, RNA and antisense DNA or RNA) of the KGF-2 sequences into an animal, to increase or decrease the expression of the KGF-2 polypeptide. The KGF-2 polypeptide can be operably linked to a promoter or to any other genetic elements necessary for the expression of the KGF-2 polypeptide by the target tissue. Such gene therapy and distribution techniques and methods are known in the art, see for example WO90 / 11092, W098 / 11779; U.S. Patent No. 5693622, 5705151, 5580859; Tabata H., et al., Cardiovasc. Res. 35 (3): 470-479 (1997), Chao, J., et al., Pharmacol. Res. 35 (6): 517-522 (1997), Wolf, J.A. , Neuromuscul. Disord 7 (5): 314-318 (1997), Schwartz B., et al., Gene Ther. 3 (5): 405-411 (1996), Tsurumi, Y., et al., Circulation 94 (12): 3281-3290 (1996) (incorporated by reference herein). The polynucleotide constructs of KGF-2 can be distributed by any method that administers the injectable materials to the cells of an animal, such as, injection into the interstitial space of the tissues (heart, muscle, skin, lung, liver, intestine and the like). The KGF-2 polynucleotide constructs can be distributed in a pharmaceutically acceptable liquid or aqueous carrier. The term "naked" polynucleotide, DNA or RNA refers to sequences that are free of any delivery vehicle that acts to assist, promote, or facilitate entry into the cell, including viral sequences, viral particles, liposomal formulations, polilipofectina or agents of precipitation and similars. However, KGF-2 polynucleotides can also be distributed in liposomal formulations (such as those shown in Felgner PL et al., Ann. NY Acad Sci 772: 126-139 (1995) and Abdallah B. et al., Biol. Cell 85 (1) -. 1-7 (1995)) which can be prepared by methods well known to those skilled in the art. The vector constructs of the KGF-2 polynucleotide, used in the gene therapy method, are preferably constructs that will not integrate into the host genome nor contain sequences that allow replication. Any strong promoter known to those skilled in the art can be used to boost the expression of AD ?. Contrary to other gene therapy techniques, a major advantage of introducing naked nucleic acid sequences within the target cells is the transient nature of the synthesis of the polynucleotide in the cells. Studies have shown that the sequences of AD? of non-replication can be introduced into the cells to provide production of the desired polypeptide for periods up to six months. The construction of the KGF-2 polynucleotide can be distributed to the interstitial space of the tissues within an animal, including the muscle, the skin, the brain, the lung, the liver, the spleen, the bone marrow, the thymus, the heart, lymph nodes, blood, bone, cartilage, pancreas, kidney, gall bladder, stomach, intestine, testes, ovaries, uterus, rectum, nervous system, eye, glands , and the connective tissue. The interstitial space of the tissues comprises the intercellular fluid, the mucopolysaccharide matrix between the reticular fibers of the tissues of organs, the elastic fibers in the walls of the vessels or chambers, the collagen fibers of the fibrous tissues, or that same matrix within the connective tissue that lines muscle cells or in bone gaps. This is similarly the space occupied by the plasma of the circulation and the lymphatic fluid of the lymphatic channels. The distribution in the interstitial space of muscle tissue is preferred for the reasons discussed below. These can be conveniently distributed by injection into the tissues comprising these cells. These are preferably distributed to and expressed in non-dividing persistent cells, which are differentiated, although the distribution and expression can be achieved in undifferentiated or less completely differentiated cells, such as, for example, blood pluripotent cells or fibroblasts of the skin. Muscle cells in vivo are particularly competent in their ability to collect and express the polynucleotides. For the injection of the naked KGF-2 polynucleotide, an amount of effective dose of the DNA or RNA will be in the range of about 0.05 g / kg of body weight to about 50 mg / kg of body weight. Preferably, the dose will be from about 0.005 mg / kg to about 20 mg / kg and more preferably from about 0.05 mg / kg to about 5 mg / kg. Of course, as the ordinarily skilled artisan will appreciate, this dosage will vary according to the injection site in the tissue. The appropriate and effective dose of the nucleic acid sequence can be readily determined by those of ordinary skill in the art and may depend on the condition being treated and the route of administration. The preferred route of administration is by the parenteral route of injection into the interstitial space of the tissues. However, other parenteral routes may also be used, such as, inhalation of an aerosol formulation, particularly for distribution to the lungs or bronchial tissues, to the throat or to the mucous membranes of the nose. In addition, constructions of the naked KGF-2 polynucleotide can be distributed to the arteries during angioplasty by the catheter used in the procedure.

The dose-response effects of the KGF-2 polynucleotide injected into the muscle, in vivo, are determined as follows. The template DNA of KGF-2, suitable for the production of the mRNA encoding the KGF-2 polypeptide, is prepared according to a standard methodology of recombinant DNA. The template DNA, which can be either circular or linear, is either used as naked DNA or complexed with liposomes. The quadriceps muscles of the mice are then injected with various amounts of the template DNA. Male and female Balb / c mice from five to six weeks of age are anesthetized by intraperitoneal injection with 0.3 ml of 2.5% Avertin. An incision is made in 1.5 cm on the anterior thigh, and the quadriceps muscle is directly visualized. The DNA template of KGF-2 is injected in 0.1 ml carrier in a 1 cm3 syringe through a 27-gauge needle in one minute, approximately 0.5 cm from the distal muscle insertion site in the knee and approximately 0.2 cm of depth. A suture is placed over the injection site for future location, and the skin is closed with stainless steel staples. After an appropriate incubation time (for example 7 days) the muscle extracts are prepared by removing the complete quadriceps. Each 15 μm cross section of the individual quadriceps muscles is histochemically stained for the expression of KGF-2 protein. A time course for the expression of the KGF-2 protein can be performed in a similar manner, except that the quadriceps of different mice are harvested at different times. The persistence of KGF-2 DNA in the muscle after injection can be determined by Southern blot analysis after preparation of the total cellular DNA and HIRT supernatants from the injected and control mice. The results of previous experimentation in mice can be used to extrapolate the appropriate doses and other treatment parameters in humans and in other animals using naked KGF-2 DNA.

Example 40 KGF-2 Therapy for Inflammatory Bowel Disease In this example, the inhibition of pathological changes in mouse colons, caused by exposure to sodium dextran sulfate (DSS) in drinking water is determined by systemic (intranasal) and intraperitoneal administration of the KGF-2 polypeptides.

Intranasal Administration. A polynucleotide encoding KGF-2Δ33 is introduced into the nasal passages of anesthetized Swiss Webster female mice (n = 10 / group) through a 26 gauge blunt needle at a dose of 1, 10 or 100 μg of polynucleotide . The control polynucleotide is administered to a separate group of mice. Five days after the intranasal administration of the polynucleotide, 5% DSS is added to the drinking water. Mice are checked periodically for body weight, hematocrit, and stool rating. After seven days of exposure to DSS in the drinking water, the mice are sacrificed. The histopathological evaluation of the colon and small intestine is performed. The RT-PCR analysis is performed to determine the expression of KGF-2 in the liver, in the spleen and in the colon.

Intraperitoneal administration. A polynucleotide encoding KGF-2Δ 33 is injected intraperitoneally into female Swiss Webster mice (n = 10 / group) through a blunt 26 gauge needle at a dose of 1, 10 or 100 μg of polynucleotide on days 0 and 3. The control polynucleotide is administered to a separate group of mice using an identical regimen. On day 7, 5% DSS is added to the drinking water. Mice are checked periodically for body weight, hematocrit, and stool rating. On day 14, the mice are sacrificed. The histopathological evaluation of the colon and small intestine is performed. The RT-PCR analysis is performed to determine the expression of KGF-2 in the liver, in the diaphragm and in the colon. The studies described in this example prove the activity in the KGF-2? 33 polynucleotides. However, a person skilled in the art could easily modify the exemplified studies to test the activity of other KGF-2 polynucleotides, including full-length and mature KGF-2, KGF-2Δ28, and the polynucleotides encoding the amino acids 77 to 208, 80 to 208 and 93 to 208 of KGF-2; and the KGF-2 polypeptides, variants, fragments, agonists, and / or antagonists; and any KGF-2 mutant described herein.

Example 41 Therapy with KGF-2 for Ocular Surface Disease In this example, the effect of the subconjunctival administration of KGF-2? 33 polynucleotides on the conjunctiva, cornea or lacrimal gland of rats is determined. A polynucleotide encoding KGF-2Δ 33 is injected into the subconjunctival space of anesthetized female Sprague Dawley rats (150-200 g body weight, 6 / treatment group) at a dose of 1, 10 or 100 μg. The control polynucleotide is injected in a manner similar to a separate group of control rats. The separate groups of rats are sacrificed at 3, 7 and 14 days after the injection. BrdU is administered intraperitoneally to some of the rats 30 minutes before euthanasia. The eye and surrounding tissues are removed for histological analysis. The degree of incorporation of BrdU in the epithelial cells of the cornea, the conjunctiva and the lacrimal glands is measured. The thickness of the epithelial layer in the cornea and the conjunctiva is evaluated. The number of goblet cells in the conjunctiva is measured. The studies described in this example test the activity of KGF-2? 33 polynucleotides. However, a person skilled in the art could easily modify the exemplified studies to test the activity of the other KGF-2 polynucleotides, including mature and full-length KGF-2, KGF-2Δ28, and the polynucleotides they encode for amino acids 77 to 208, 80 to 208, and 93 to 208 of KGF-2; and KGF-2 polypeptides, variants, fragments, agonists and / or antagonists thereof; as well as any mutant of KGF-2 described herein.

Example 42 Therapy with KGF-2 for Salivary Gland Dysfunction In this example, the effect of the administration of the KGF-2 polynucleotide on the papillae of the parotid salivary gland duct of normal rats on the epithelial cells of the ducts and the acini of those glands is determined. Female Sprague Dawley rats (150-250 grams 6 / treatment group) are anesthetized by intramuscular injection of ketamine and xylazine. A polynucleotide encoding KGF-2Δ33 is introduced into the papillae of the parotid salivary gland using a 30 gauge steel priming needle, at a dose of 1, 10 or 100 μg. The polynucleotide is infused over a period of 10 minutes at a rate of 1 μl per minute. The control polynucleotide is administered to a separate group of rats. The separate groups of rats were sacrificed at 3, 7 and 14 days after the infusion. BrdU is administered intraperitoneally 30 minutes before euthanasia. The salivary glands are heavy, and the number of cells stained with BrdU is counted on the histological section. In a separate experiment, the saliva secretion stimulated by pilocarpine in rats was measured 7 days after the infusion.

The studies described in this example test the activity of KGF-2? 33 polynucleotides. However, a person skilled in the art could easily modify the exemplified studies to test the activity of the other KGF-2 polynucleotides, including mature and full-length KGF-2, KGF-2Δ28, and the polynucleotides they encode for amino acids 77 to 208, 80 to 208, and 93 to 208 of KGF-2; and KGF-2 polypeptides, variants, fragments, agonists and / or antagonists thereof; as well as any mutant of KGF-2 described herein.

Example 43 Therapy with KGF-2 for Healing of Dermal Wounds In this example, the ability of the KGF-2 polynucleotide to stimulate wound healing in normal rats and diabetic mice is determined.

Normal rats Female anesthetized Sprague Dawley rats (175-250 gm, 6 / treatment group) are wounds with 8 mm biopsy punches. The KGF-2? 33 polypeptide (1, 10 or 30 μg) is administered intradermally at 4 different sites throughout the wound. The control polypeptide is administered in a manner similar to a separate group of rats. The wounds are covered with ventilated cloth pads, sterile. After the pad is placed, the waterproof adhesive tape is wrapped around the middle section of the rat. The separate groups of rats are sacrificed at 2 and 5 days after the wound is made. Wound tissues are fixed in 10% formalin and embedded in paraffin. The incorporation of BrdU in proliferating epithelial cells in the pre-existing and new epidermis is measured, and it measures the length and thickness of the new epithelial tongue. Diabetic mice. Diabetic mice (db + / db +, 10 / treatment group) and non-diabetic mice (db / + m +, 10 / treatment group) are injured with a 6 mm punch on the back. The KGF-2? 33 polynucleotide (1, 10 or 30 μg) is administered intradermally at 4 different sites along the wound. The control polynucleotide is administered in a manner similar to a separate group of mice. The wounds are covered with Tegaderm (diabetic mice) or Tegaderm plus adhesive tape (non-diabetic mice). The wounds are photographed on days 0, 3, 7, 10 and 14 after the wound is made. The surface area of the wounds is measured by image analysis.

The studies described in this example test the activity of KGF-2? 33 polynucleotides. However, a person skilled in the art could easily modify the exemplified studies to test the activity of the other KGF-2 polynucleotides, including mature and full-length KGF-2, KGF-2Δ28, and the polynucleotides they encode for amino acids 77 to 208, 80 to 208, and 93 to 208 of KGF-2; and KGF-2 polypeptides, variants, fragments, agonists and / or antagonists thereof; as well as any mutant of KGF-2 described herein.

Example 44 Constructions for the distribution of KGF-2 One construction suitable for therapy with the KGF-2 gene is pVGI. O-KGF-2. This construct contains the native open reading frame, complete with KGF-2 cloned into the expression vector pVGI .0. pVGI .0 contains a kanamycin resistance gene, a CMV enhancer, and an RSV promoter. pVGI.O-KGF-2 was deposited at the American Type Culture Collection Patent Depository, 10801 University Boulevard, Manassas, VA 20110-2209, on June 30, 1999, and was given the ATCC PTA290 deposit number. This construct was elaborated by subcloning ORF of KGF-2 from a previously verified KGF-2 construct of the sequence into the expression vector pVGI.O, using methods well known in the art. Another construction suitable for the administration of KGF-2 is pVGI-0-MPIFspKGF-2? 33. This construct contains the native KGF-2Δ33 sequence fused to the heterologous MPIF signal peptide (CKβ8) cloned into the expression vector pVGI .0. pVGI .0-MPIFspKGF-2? 33 was deposited at the American Type Culture Collection patent depository, 1081 University Boulevard, Manassas, VA 20110-2209, on June 30, 1999, and was given the ATCC deposit number PTA289. This construct was made by methods known in the art and the following primers: 5 'Primer: GAGCGCGGATCCGCCACCATGAAGGTCTCCGTGGCTGCCCTCTCC TGCCTCATGCTTGTTACTGCCCTTGGATCTCAGGCCAGCTACAATCA CCTTCAAGGAGATG (SEQ ID No. 149) 3 'Primer: GAGCGC GGATCC CTATGAGTGTACCACCATTGGAAG (SEQ ID No. 150) Example 45 Angiogenesis During Gene Therapy with KGF-2 The characterization of the multiple aspects of microvascular physiology in transparent window systems in mice has provided valuable data on angiogenesis, inflammation, microvascular transport, tissue rejection and tumor physiology. In this example, the development of the vasculature is evaluated during a response to wound healing in implanted collagen gels, through direct observation of the tissue and the associated microvascular bed, through a window implanted in the skin. This model is used to determine if therapy with the KGF-2 gene can simultaneously induce accelerated tissue regrowth and revascularization. Skin biopsies from nude mice are digested in collagenase, the resulting cell suspensions are washed and then cultured in DMEM with 10% FBS to obtain dermal fibroblasts. The cultures of confluent fibroblasts are transfected with KGF-2 or the control polynucleotide and then harvested and washed in PBS. 106 cells were suspended in 20 μl of collagen matrix. Samples of the cell suspension are removed for confirmation by Western blotting of KGF-2 production. A 2 mm punch biopsy is performed on an existing dorsal skin window, and the skin is walled between two glass coverslips. The mixture of cells and collagen is placed in the circular wound and the chamber is sealed. The implanted gels were observed at regular intervals for the development of the vessels. The tissue growth in the wound is checked periodically as changes in the optical density of the collagen gel over a period of three weeks. The tissue from the dorsal chambers is removed after the conclusion of the study for histological evaluation. The control experiments involve the addition of the KGF-2 polypeptide or buffer in the collagen gels instead of the fibroblasts.

Preparation of mice. Surgical procedures are performed on nude Swiss mice. For surgical procedures, animals (20-30 g) are anesthetized with a subcutaneous injection of a 90 mg cocktail of ketamine and 9 mg of xylazine per kg of body weight. All surgical procedures are performed under aseptic conditions in a horizontal laminar flow hood, with all equipment that is sterilized by steam, gas or chemically. During surgery, the body temperature of the animals is kept constant by means of a hot work surface. All mice are housed individually in micro-insulating cages and all manipulations are performed in laminar flow hoods. Buprenorphine (0.1 mg / kg for 12 hours) is administered as an analgesic for 3 days after implantation. The mice are placed such that the chamber is walled between a double layer of skin that extends above the dorsal surface. A layer of skin is removed in a circular area approximately 15 mm in diameter. The second layer (consisting of epidermis, fascia, and striated muscle) is placed on the chamber structure and covered with a sterile glass coverslip. The camera is held in place with nylon poles that pass through the extended skin and the holes along the top of the chamber. After 3 days, the coverslip is carefully removed and the gel inserted. A new sterile coverslip is then placed on the observation surface. Measurements are made by morphometric analysis using an intensified CCD camera, video cassette recorder, S-VHS and direct digital image acquisition. Mice with the implanted chambers were observed for 28 days.

Measurements The mice were anesthetized with a subcutaneous injection of a 90 mg cocktail of ketamine and 9 mg of xylazine per kg body weight, then placed on a sterile plastic platform assembly. The vascular maps of the window are made using translumination (dorsal skin window) or after an injection of 100 μl of BSA-FITC (1 mg / ml, i.v.) and epi-illumination. Videorecordings of vascular beds are made at a range of amplifications (from IX to 40X) as well as digital structures for off-line analysis. The determinations of the angiogenesis of the implanted gels are made from the offline analysis of the video tapes. The studies described in this example test the activity of KGF-2? 33 polynucleotides. However, a person skilled in the art could easily modify the exemplified studies to test the activity of the other KGF-2 polynucleotides, including mature and full-length KGF-2, KGF-2Δ28, and the polynucleotides they encode for amino acids 77 to 208, 80 to 208, and 93 to 208 of KGF-2; and KGF-2 polypeptides, variants, fragments, agonists and / or antagonists thereof; as well as any mutant of KGF-2 described herein.

Example 46 Transgenic Animals KGF-2 The KGF-2 polypeptides can also be expressed in transgenic animals. Animals of any species, including, but not limited to, mice, rats, rabbits, hamsters, guinea pigs, pigs, micro-nerds, goats, sheep, cows and non-human primates, for example baboons, monkeys, and chimpanzees can be used to generate transgenic animals. In a specific embodiment, techniques described herein or otherwise known in the art, are used to express the polypeptides of the invention in humans, as part of a gene therapy protocol. Any technique known in the art can be used to introduce the transgene (e.g., polynucleotides of the invention) into animals to produce the founder lines of transgenic animals. Such techniques include, but are not limited to, pronuclear microinjection (Paterson et al., Microbiol, Biotechnol., 40: 691-698 (1994)).; Carver et al., Biotechnology (NY) 11: 1263-1270 (1993); Wright et al., Biotechnology (NY) 9: 830-834 (1991); and Hoppe et al., U.S. Patent No. 4,873,191 (1989)); gene transfer mediated by retroviruses, within germ lines (Van der Putten et al., Proc. Nati, Acad. Sci. USA 82: 6148-6152 (1985)), blasts or embryos; direction of genes in embryonic pluripotent cells (Thompson et al., Cell 56: 313-321 (1989)); electroporation of cells or embryos (Lo, Mol, Cell, Biol. 3: 1803-1814 (1983)); introduction of the polynucleotides of the invention using a gene gun (see, for example, Ulmer et al., Science 259: 1745 (1993); introduction of nucleic acid constructs into embryonic pluripotent cells and transfer of pluripotent cells back to blasts and sperm-mediated gene transfer (Lavitrano et al., Cell 57: 717-723 (1989), etc. For a review of such techniques, see Gordon, "Transgenic Animáis," Int. Rev. Cytol. 115: 171- 229 (1989), which is hereby incorporated by reference in its entirety, Any technique known in the art can be used to produce transgenic clones containing the polynucleotides of the invention, for example, nuclear transfer in enucleated oocytes from originating nuclei. of embryonic, fetal, or adult cells, cultured, induced to rest (Campell et al., Nature 380: 64-66 (1996); Wilmut et al. Radores, Na ture 385: 810-813 (1997)).

The present invention provides the transgenic animals that carry the transgene in all its cells, as well as the animals that carry the transgene in some, but not in all its cells, for example, the mosaic or chimeric animals. The transgene can be integrated as a single transgene or as multiple copies such as in concatamers, for example, head-to-head tandems or head-to-tail tandems. The transgene can also be selectively introduced into and activated in a particular cell type following for example the teachings of Lasko et al. (Lasko et al., Proc Nati, Acad. Sci. USA 89: 6332-6236 (1992)). The regulatory sequences required for such cell-type specific activation will depend on a particular cell type of interest, and will be apparent to those skilled in the art. When it is desired that the transgene of the polynucleotide be integrated into the chromosomal site of the endogenous gene, targeting of the gene is preferred. In summary, when such a technique is to be used, vectors containing some nucleotide sequences homologous to the endogenous gene are designed for integration purposes, via homologous recombination with chromosomal sequences, within and disturbing the function of the nucleotide sequence of the endogenous gene . The transgene can also be selectively introduced into a particular cell type, thereby inactivating the endogenous gene only in this cell type, following for example the teaching of Gu et al. (Gu et al., Science 265: 103-106 (1994)) . The regulatory sequences required for such specific inactivation of the cell type will depend on the particular cell type of interest, and will be apparent to those skilled in the art. The contents of each of the documents indicated in this paragraph are incorporated by reference herein, in their entirety. Once the transgenic animals have been generated, the expression of the recombinant gene can be evaluated using standard techniques. The initial selection can be achieved by spotting or Southern blot analysis or PCR techniques to analyze the animal tissues, to verify that the integration of the transgene has taken place. The level of expression of the transgene mRNA in the tissues of the transgenic animals can also be evaluated using techniques that include, but are not limited to, staining or Northern blot analysis of tissue samples obtained from the animal, in situ hybridization analysis , Reverse transcriptase PCR (rt-PCR). Tissue samples expressing the transgenic gene can also be evaluated immunocytochemically or immunohistochemically using antibodies specific for the transgenic product.

Once the founding animals are produced, they can be crossed endogamously, exogamically, or cross-linked to produce colonies of the particular animal. Examples of such crossing strategies include, but are not limited to: the production of founder animals with more than one integration site, in order to establish separate lines; the inbreeding crossing of separate lines in order to produce compound transgenic animals that express the transgene at higher levels due to the effects of the additive expression of each transgene; the crossing of heterozygous transgenic animals to produce animals homozygous for a given integration site, in order to increase expression and eliminate the need for animal selection by DNA analysis; crosses it with separate homozygous lines to produce heterozygous or homozygous compound lines; and crosses it to place the transgene in a different antecedent that is appropriate for an experimental model of interest. The transgenic animals of the invention have uses that include, but are not limited to, animal model systems useful in the elaboration of the biological function of the KGF-2 polypeptides, study of the conditions and / or disorders associated with the aberrant expression of KGF-2, and in the selection for effective compounds in the improvement of such conditions and / or disorders.

Example 47 Animals Deleted in the KGF -2 gene Expression of the endogenous KGF-2 gene can also be reduced by inactivation or "deletion in a gene" of the KGF-2 gene and / or its promoter using targeted homologous recombination (for example see Smithies et al., Nature 317: 320 -234 (1985), Thomas and Capecchi, Cell 51: 503-512 (1987), Thompson et al., Cell 5: 313-321 (1989), each of which is incorporated by reference herein, in its entirety. ). For example, polynucleotide nonfunctional mutant of the invention (or a sequence completely unrelated DNA) flanked by DNA homologous to the endogenous polynucleotide sequence (either the coding regions or regulatory regions of the gene) can be used, with or without a selectable marker and / or a negative selectable marker, to transfect the cells expressing the polypeptides of the invention in vivo. In yet another embodiment, techniques known in the art are used to generate suppressed organisms in a gene, in cells that contain, but do not express the gene of interest. The insertion of the DNA construct, via directed homologous recombination, results in the inactivation of the targeted gene. Such methods are particularly suitable in the fields of research and agriculture where modifications to embryonic pluripotent cells can be used to generate the animal progeny with an inactive directed gene (for example see Thomas &Capecchi 1987 and Thomson 1989, supra). However, this method can be routinely adapted for use in humans, with the proviso that the recombinant DNA constructs are directly administered or directed to the required site in vivo using appropriate viral vectors that will be apparent to those skilled in the art. In further embodiments of the invention, cells that are genetically engineered to express the polypeptides of the invention, or alternatively, that are engineered to not express the polypeptides of the invention (for example, deleted in a gene) are administered to a patient in vivo. Such cells can be obtained from the patient (e.g., animal, including humans) or an MHC-compatible donor and can include, but are not limited to, fibroblasts, bone marrow cells, blood cells (e.g., lymphocytes), adipocytes, muscle cells, endothelial cells, etc. The cells are engineered by in vitro using recombinant DNA techniques to introduce the coding sequence of the polypeptides of the invention into the cells, or alternatively, to disrupt the coding sequence and / or the endogenous regulatory sequence associated with polypeptides of the invention, for example, by transduction (using viral vectors, and preferably vectors that integrate the transgene into the cell genome) or transfection procedures, including, but not limited to, the use of plasmids, cosmids , YACs, naked DNA, electroporation, liposomes, etc. The coding sequence of the polypeptides of the invention can be placed under the control of a strong constitutive or inducible promoter, or a promoter / enhancer to achieve expression, and preferably secretion, of the KGF-2 polypeptides. Genetically engineered cells that express and preferably secrete the polypeptides of the invention can be introduced into the patient systemically, for example, in the circulation or intraperitoneally. Alternatively, the cells can be incorporated into a matrix and implanted in the body, for example, engineered fibroblasts can be implanted as part of a skin graft; Endothelial cells manipulated by genetic engineering can be implanted as part of a lymphatic or vascular graft. (See, for example, Anderson et al., United States Patent No. 5,399,349; and Mulligan and Wilson, U.S. Patent No. 5,460,959 each of which is incorporated by reference herein, in its entirety). When the cells to be administered are non-autologous or cells not compatible with MHC, they can be administered using well-known techniques that prevent the development of a host immune response against the introduced cells. For example, the cells can be introduced in an encapsulated form the cuai, while allowing an exchange of the components with the immediate extracellular environment, does not allow the introduced cells to be recognized by the host's immune system. The deleted animals in some gene, of the invention, have uses that include, but are not limited to, animal model systems useful in the elaboration of the biological function of the KGF-2 polypeptides, the study of the conditions and / or associated disorders with the aberrant expression of KGF-2, and in the selection for the effective compounds in the improvement of such conditions and / or disorders.

Example 48 Construction of KGF-2 mutants To create point mutations, the primers indicated in the PCR reactions were used using standard conditions well known to those skilled in the art. The resulting products were restricted with either Nde and Asp718 and cloned into pHE4; or with BamHI and Xba, and cloned into pcDNA3; as indicated. Any of the described KGF-2 variants can be produced in other vectors, or by themselves, using methods well known in the art. pHE4: KGF2: R80-S208 was constructed using the following primers: 5 'primer: CCGGC CATATG CGTAAACTGTTCTCTTTCACC (SEQ ID No. 151) 3 'primer: CCGGC GGTACC TTATTATGAGTGTACCACCATTGG (SEQ ID No. 152) pHE4: KGF2: A63-S208 (R68G) was constructed using the following primers: 5 'primer: GATCGC CATATG GCTGGTCGTCACGTTCGTTC (SEQ ID No. 153) 3 'primer: GATCGC GGTACC TTATTATGAGTGTACCACCATTGGAAG (SEQ ID No. 154) pHE4: KGF2: A63-S208 (R68S) was constructed using the following primers: 5 'primer: GATCGC CATATG GCTGGTCGTCACGTTCGTTC (SEQ ID No. 155) 3' primer: GATCGC GGTACC TTATTATGAGTGTACCACCATTGGAAG (SEQ ID No. 156) pHE4 : KGF2: A63-S208 (R68A) was constructed using the following primers: 5 'primer: GATCGC CATATG GCTGGTCGTCACGTTCGTTC (SEQ ID No. 157) 3' primer: GATCGC GGTACC TTATTATGAGTGTACCACCATTGGAAG (SEQ ID No. 158) pHE4: KGF2: A63- S208 (R78R80K81A) was constructed using the following primers: 5 'primer: GATCGC CATATG GCTGGTCGTCACGTTCGTTC (SEQ ID No. 159) 3' primer: GATCGC GGTACC TTATTATGAGTGTACCACCATTGGAAG (SEQ ID No. 160) pcDNA3: KGF2 (K136137139144A) was constructed using the following primers: 5 'primer: GATCGCGGATCCGCCACCATGTGGAAATGGATACTGACACATTGTGC (SEQ ID No. 161) 3' primer: GATCGCTCTAGATTATGAGTGTACCACCATTGGAAGAAAG (SEQ ID No. 162) pcDNA3: KGF2 (K151153R155A) was constructed using the following primers: 5 'primer: GATCGCGGATCCG CCACCATGTGGAAATGGATACTGACACATTGTGC (SEQ ID No. 163) 3 'Primer: GATCGCTCTAGATTATGAGTGTACCACCATTGGAAGAAAG (SEQ ID No. 164) pcDNA3: KGF2 (R174K183A) was constructed using the following primers: 5' primer: GATCGCGGATCCGCCACCATGTGGAAATGGATACTGACACATTGTGC (SEQ ID No. 165) 3 'primer: GATCGCTCTAGATTATGAGTGTACCACCATTGGAAGAAAG (SEQ ID No. 166) pcDNA3: KGF2 (R187R188A) was constructed using the following primers: 5 'primer: GATCGCGGATCCGCCACCATGTGGAAATGGATACTGACACATTGTGC (SEQ ID No. 167) 3' primer: GATCGCTCTAGATTATGAGTGTACCACCATTGGAAGAAAG (SEQ ID No. 168) pHE4: KGF2.A63 ( K136137139144A) was constructed using the following primers: 5 'primer: GATCGCCATATGGCTGGTCGTCACGTTCGTTC (SEQ ID No. 169) 3' primer: GATCGCGGTACCTTATTATGAGTGTACCACCATTGGAAG (SEQ ID No. 170) pHE4: KGF2: A63 (K151153R155A) was constructed using the following primers: primer 5 ': GATCGCCATATGGCTGGTCGTCACGTTCGTTC (SEQ ID No. 171) 3' primer: GATCGCGGTACCTTATTATGAGTGTACCACCATTGGAAG (SEQ ID No 172) Example 49 Use of KGF-2 for the Treatment and / or Prevention of Infertility Implantation is the simplest critical factor in a successful pregnancy, and it is clinically and economically important. In humans, the largest fraction of the 70% loss in embryonic life occurs at implantation. The mouse is the model of choice to study implantation in mammals. Three essential cell lines are differentiated and divided into the peri-implantation mouse embryo: the embryonic, placental and amniotic sac precursors. The growth factor of fibroblasts (FGF) -4 is essential for the development of the three cell lines. It has been found, using a "transient transgenic" procedure to distribute the FGF receptor genes for gain of function and loss of function (dominant negative), that endogenous FGF signaling is necessary for the cell division of all pluripotent cells to the embryo and placenta lines in the mouse embryo beginning in the fifth cell division two days before implantation. Interestingly, it has been found that the null mutant for fgfr-2 and fgf4 dies in utero within a day after implantation and ICM dies. Before the embryo is implanted in the uterus, the cells in the embryonic line and in the placental line require FGF to continue proliferating. It is possible that one or several other 19 FGF ligands are transiently expressed in the preimplantation mouse embryo and this ligand delays the effect of the null mutants on fgfr-2 and fgf4 until after implantation. Six FGF ligands have been tested using RT-PCR. To date, KGF-2 and FGF-8 are the only FGF ligands, in addition to FGF-4, detected in the preimplantation embryo. KGF-2 mRNA is detected in the embryo after two cellular stages and through early post-implantation. The null mutants in KGF-2 suggest that KGF-2 is not essential for survival during the expression of KGF-2 in pre-implantation mouse embryos (Min et al., 1998; Sekine et al., 1999). However, other members of the FGF family can compensate or be redundant for KGF-2 during pre-implantation embryonic development. Many redundant genetic effects have been observed during the analysis of null mutants in mice and compensation within a family of genes has also been observed (Thomas et al., 1995; Stein et al., 1994). KGF-2 may be more important in early development than what is suggested by null mutants in KGF-2. The best way to detect if KGF-2 may have a role in early development at a time when null mutants do not suggest any essential function, is to perform gain-of-function experiments. These experiments test whether KGF-2 has an influence on the development of pre-implantation embryos (Rappolee et al., 1994), on placental cells / trophoblasts in blastocyst development (Chai et al., 1998) and in cells of the endodermal line in growths of internal callus mass (ICM) (Rappolee and collaborators 1994). Loss-of-function tests can be performed in a limited manner by the use of antisense oligonucleotides (Rappolee et al., 1992) or in blocking antibodies (LaFleur et al., 1996). It is known that embryos undergo size regulation, large positive and negative changes in the number of cells that are regulated homeostatically, shortly after implantation (Rappolee 1998). This suggests that the sublethal, small KGF-2 dependent effects can be completely eliminated in the null mutants in KGF-2. The loss and gain-of-function experiments are used to test pre-implantation mouse embryos for the effects of KGF-2. To date, the detection of mRNA for a growth factor in the pre-implantation mouse embryo has led universally to the detection of the corresponding protein. (Rappolee et al, 1998, 1992, 1994, reviewed in Rappolee 1998, 1999). To determine whether the KGF-2 protein is present or not (and where) in the embryos where the KGF-2 mRNA was detected, an antibody for KGF-2 suitable for immunocytochemistry is used.

Example 50 Detection of KGF-2 in a clinical sample Goat PAb, purified is diluted to 2 μg / ml in the coating buffer (sodium carbonate 0.05 M, pH 9.5). 100 μl of the diluted antibody is added per well to an Immuno 4 microplate. The microplate is stored overnight at 4 ° C. The antibody solution is decanted from the plate. 200 μl of the blocking buffer (1% milk powder (BioRad) in coating buffer) are added to each well. The plate is allowed to incubate at room temperature for 2 hours. The blocking damper is decanted from the plate. The plate is vacuumed and allowed to dry completely in a vacuum chamber at 32 ° C for 1.5 hours. The plate is removed from the vacuum chamber and sealed in a mylar bag with 3 desiccant packs. The plate is stored at 4 ° C until it is ready to be used. KGF-2 is diluted to 16 ng / ml with the diluent (0.1% Tween 20, lxPBS, 1% BSA, and 0.001% Thimerosal), then a subsequent dilution of 2.5x is performed for the next 7 dilutions. The concentration range from 16 ng / ml to 0.026 ng / ml is used as the standard. The antecedent wells consist of the diluent without protein. The unknown samples are diluted 10X, 50X and 250X with the diluent 1. 100 μl per well of the diluted standard solution, in series and the unknown samples are added to the coated ELISA plate. The plate is stored at 4 ° C overnight. The solutions are decanted from the plate. The plate is washed with the wash buffer (0.1% Tween 20 and lx PBS) five times, using the Wheaton Instrument at 1.6 ml (each well receives 200 μl per wash). 15 seconds of incubation of the wash buffer are allowed between each wash. The detector, the biotinylated chicken anti-KGF-2, is diluted to 0.5 μg / ml in diluent 1. 100 μl of the diluted detector is added to each well. The plate is incubated for 2 hours at room temperature. The solution is decanted and the plate is washed with the wash buffer 5 times, as described above. A time of 15 seconds of incubation is allowed between each wash. The peroxidase-streptavidin is diluted 1: 2000 in the diluent 1. 100 μl per well of peroxidase-streptavidin diluted to the plate is added, and it is allowed to incubate at room temperature for 1 hour. The plate is decanted and washed with the wash buffer five times. 15 seconds of incubation of the wash buffer are allowed between each wash. The plate is not allowed to dry. Equal amounts of the TMB peroxidase substrate at room temperature and the peroxidase B solution (from TMB Peroxidase Microwell Substrate System, KPL) are mixed. 100 μl of the mixed solution is added to each well, and the color is allowed to develop at room temperature for 10 minutes. The development of color is stopped by the addition of 50 μl of 1 M sulfuric acid to each well. The plate is read at 450 nm.

Example 51 Construction of optimized, truncated KGF-2 from E. coli In order to increase the expression levels of a truncated KGF-2 in an E. Coli expression system, the codons of the gene were optimized to the highly used E. coli codons. For example, the following construction was made called pHE4: KGF-2.A63-S608 5 'CATATGGCTGGTCGTCACGTTCGTTCTTACAACCACCTGCAGGGT GACGTTCGTTGGCGTAAACTGTTCTCTTTCACCAAATACTTCCTGAA AATCGAAAAAAACGGTAAAGTTTCTGGGACCAAGAAGGAGAACTG CCCGTACAGCATCCTGGAGATAACATCAGTAGAAATCGGAGTTGTT GCCGTCAAAGCCATTAACAGCAACTATTACTTAGCCATGAACAAGA AGGGGAAACTCTATGGCTCAAAAGAATTTAACAATGACTGTAAGCT GAAGGAGAGGATAGAGGAAAATGGATACAATACCTATGCATCATT TAACTGGCAGCATAATGGGAGGCAAATGTATGTGGCATTGAATGG AAAAGGAGCTCCAAGGAGAGGACAGAAAACACGAAGGAAAAACA CCTCTGCTCACTTTCTTCCAATGGTGGTACACTCATAATAAGGTACC 3' (SEQ ID No. 173) A plasmid comprising a cDNA having the nucleotide sequence of SEQ ID No. 173 was deposited as ATCC Deposit No. on July 3, 2000, in the American Type Culture Collection, Patent Depository, 10801 University Boulevard, Manassas, VA 20110-2209.

Another construct, designated pHE4: KGF-2.A63-S208 cod.opt, was constructed using the following primers: In the 5 'direction GACTACATATGGCTGGTCGTCACGTTCGTTCTTACAACC ACCTGCA GG3' (SEQ ID No. 174) Antisense 5 'CTAGTCTCTAGATTATTATGAGTGTACAACCATCG GCAGGAAGTGAG 3' (SEQ ID No 175) The nucleotide sequence pHE4: KGF-2.A63-208 cod.opt is as follows: 5 'ATGGCTGGTCGTCACGTTCGTTCTTACAACCACCTGCAGGGTG ACGTTCGTTGGCGTAAACTGTTCTCTTTCACCAAATACTTCCTGAAA ATCGAAAAGAACGGTAAAGTTTCTGGTACCAAGAAAGAAAACTGC CCGTACTCTATCCTGGAAATCACCTCCGTTGAAATCGGTGTTGTAG CCGTTAAAGCCATCAACTCCAACTATTACCTGGCCATGAACAAAAA GGGTAAACTGTACGGCTCTAAAGAATTCAACAACGACTGCAAACTG AAAGAACGTATCGAAGAGAACGGTTACAACACCTACGCATCCTTC AACTGGCAGCACAACGGTCGTCAGATGTACGTTGCACTGAACGGTA AAGGCGCTCCGCGTCGCGGTCAGAAAACCCGTCGCAAAAACACCT CTGCTCACTTCCTGCCGATGGTTGTACACTCATAATAA 3' (SEQ ID No. 176) A plasmid comprising a cDNA having the nucleotide sequence of SEQ ID No. 176 was deposited as ATCC Deposit No. on July 3, 2000, in the American Type Culture Collection, Patent Depository, 10801 University Boulevard, Manassas, VA 20110-2209.

Both constructs described in this example are useful in the production of KGF-2 polypeptides, for example as described in Example 13. Nucleotides 4 to 444 of SEQ ID No. 173 and nucleotides 1 to 441 of SEQ ID No 176 encode amino acids 63 to 208 of SEQ ID No. 2, plus an N-terminal methionine. It will be clear that the invention can be practiced in another way than is particularly described in the above description and in the examples. Numerous modifications and variations of the present invention are possible in light of the foregoing teachings and, therefore, within the scope of the appended claims, the invention may be practiced in another manner than is particularly described. The complete description of all publications (including patents, patent applications, journal articles, laboratory manuals, books, or other documents) cited herein are incorporated by reference herein.

It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention is that which is clear from the present description of the invention LIST OF SEQUENCES < 110 > Rubén, Steven M. Jiménez, Pablo Duan, D. Roxanne Rampy, Mark A. Mendrick, Donna Zhang, Jun Ni, Jian Moore, Paul A. Coleman, Timothy A Gruber, Joachim R. Dillon, Patrick J. Gentz, Reiner L. < 120 > Keratinocyte growth factor 2 < 130 > 1488.036PC0K < 140 > < 141 > < 150 > 60 / 142,343 < 151 > 1999-07-02 < 150 > 60 / 143,548 < 151 > 1999-07-14 < 150 > 60 / 144,024 < 151 > 1999-07-15 < 150 > 60 / 148,628 < 151 > 1999-08-12 < 150 > 60 / 149,935 < 151 > 1999-08-19 < 150 > 60 / 163,375 < 151 > 1999-11-03 < 150 > 60 / 171,677 < 151 > 1999-12-22 < 150 > 60 / 205,417 < 151 > 2000-05-19 < 150 > 60 / 198,322 < 151 > 2000-04-19 < 160 > 176 < 170 > Patentln Ver. 2.1 < 210 > 1 < 211 > 627 < 212 > DNA < 213 > Homo sapiens < 220 > < 221 > CDS < 222 > (1) .. (624) < 400 > 1 atg tgg aaa tgg ata ctg here cat tgt gcc tea gcc ttt ecc falls ctg 48 Met Trp Lys Trp He Leu Thr His Cys Wing Wing Wing Phe Pro His Leu 1 5 10 15 ecc ggc tgc tgc tgc tgc tgc ttt ttg ttg ttc ttc ttg gtg tet tcc 96 Pro Gly Cys Cys Cys Cys Cys Phe Leu Leu Leu Phe Leu Val Ser Ser 20 25 30 gtc ect gtc acc tgc ca gcc ctt ggt cag gac atg gtg tea cea gag 144 Val Pro Val Thr Cys Gln Ala Leu Gly Gln Asp Met Val Ser Pro Glu 35 40 45 gcc acc aac tet tet tcc tcc tcc ttc tcc tet ect tcc age gcg gga 192 Wing Thr Asn Being Ser Being Being Phe Being Pro Pro Being Wing Gly 50 55 60 agg cat gtg cgg age tac aat falls ctt ca gga gat gtc cgc tgg aga 240 Arg His Val Arg Ser Tyr Asn His Leu Gln Gly Asp Val Arg Trp Arg 65 70 75 80 aag cta ttc tet ttc acc aag tac ttt ctc aag att gag aag aac ggg 288 Lys Leu Phe Ser Phe Thr Lys Tyr Phe Leu Lys He Glu Lys Asn Gly 85 90 95 aag gtc age ggg acc aag aag gag aac tgc ceg tac age ate ctg gag 336 Lys Val Ser Gly Thr Lys Lys Glu Asn Cys Pro Tyr Ser He Leu Glu 100 105 110 ata here tea gta gaa ate gga gtt gtt gcc gtc aaa gcc att aac age 384 He Thr Ser Val Glu He Gly Val Val Wing Val Lys Ala He Asn Ser 115 120 125 aac tat tac tta gcc atg aac aag aag ggg aaa ctc tat ggc tea aaa 432 Asn Tyr Tyr Leu Ala Met Asn Lys Lys Gly Lys Leu Tyr Gly Ser Lys 130 135 140 gaa ttt aac aat gac tgt aag ctg aag gag agg ata gag gaa aat gga 480 Glu Phe Asn Asn Asp Cys Lys Leu Lys Glu Arg He Glu Glu Asn Gly 145 150 155 160 tac aat tat tat gca tea ttt aac tgg cag cat aat ggg agg caa atg 528 Tyr Asn Thr Tyr Wing Being Phe Asn Trp Gln His Asn Gly Arg Gln Met 165 170 175 tat gtg gca ttg aat gga aaa gga gct cea agg aga gga cag aaa here 576 Tyr Val Ala Leu Asn Gly Lys Gly Ala Pro Arg Arg Gly Gln Lys Thr 180 185 190 cga agg aaa aac acc tet gct falls ttt ctt cea atg gtg gta falls tea 624 Arg Arg Lys Asn Thr Ser Ala His Phe Leu Pro Met Val Val His Ser 195 200 205 tag 627 < 210 > 2 < 211 > 208 < 212 > PRT < 213 > Homo sapiens < 400 > 2 Met Trp Lys Trp He Leu Thr His Cys Wing Being Wing Phe Pro His Leu 1 5 10 15 Pro Gly Cys Cys Cys Cys Cys Phe Leu Leu Leu Phe Leu Val Ser Ser 20 25 30 Val Pro Val Thr Cys Gln Ala Leu Gly Gln Asp Met Val Ser Pro Glu 35 40 45 Wing Thr Asn Being Ser Being Ser Phe Ser Ser Pro Being Wing Gly 50 55 60 Arg His Val Arg Ser Tyr Asn His Leu Gln Gly Asp Val Arg Trp Arg 65 70 75 80 Lys Leu Phe Ser Phe Thr Lys Tyr Phe Leu Lys He Glu Lys Asn Gly 85 90 95 Lys Val Ser Gly Thr Lys Lys Glu Asn Cys Pro Tyr Ser He Leu Glu 100 105 110 He Thr Ser Val Glu He Gly Val Val Ala Val Lys Ala He Asn Ser 115 120 125 Asn Tyr Tyr Leu Ala Met Asn Lys Lys Gly Lys Leu Tyr Gly Ser Lys 130 135 140 Glu Phe Asn Asn Asp Cys Lys Leu Lys Glu Arg He Glu Glu Asn Gly 145 150 155 160 Tyr Asn Thr Tyr Wing Being Phe Asn Trp Gln His Asn Gly Arg Gln Met 165 170 175 Tyr Val Wing Leu Asn Gly Lys Gly Wing Pro Arg Arg Gly Gln Lys Thr 180 185 190 Arg Arg Lys Asn Thr Ser Wing His Phe Leu Pro Met Val Val His Ser 195 200 205 < 210 > 3 < 211 > 36 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of Artificial Sequence: oligonucleotide < 400 > 3 ccccacatgt ggaaatggat actgacacat tgtgcc 36 < 210 > 4 < 211 > 35 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence; oligonucleotide < 400 > 4 cccaagcttc cacaaacgtt gccttcctct atgag 35 < 210 > 5 < 211 > 36 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: oligonucleotide < 400 > 5 catgccatgg cgtgccaagc ccttggtcag gacatg 36 < 210 > 6 < 211 > 35 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: oligonucleotide < 400 > 6 cccaagcttc cacaaacgtt gccttcctct atgag 35 < 210 > 7 < 211 > 35 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: oligonucleotide < 400 > 7 gcgggatccg ccatcatgtg gaaatggata ctcac 35 < 210 > 8 < 211 > 27 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: oligonucleotide < 400 > 8 gcgcggtacc acaaacgttg ccttcct 27 < 210 > 9 < 211 > 40 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: oligonucleotide < 400 > 9 taacgaggat ccgccatcat gtggaaatgg atactgacac 40 < 210 > 10 < 211 > 38 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: oligonucleotide < 400 > 10 taagcactcg agtgagtgta ccaccattgg aagaaatg 38 < 210 > 11 < 211 > 54 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: oligonucleotide < 400 > 11 attaaccctc actaaaggga ggccatgtgg aaatggatac tgacacattg tgcc 54 < 210 > 12 < 211 > 35 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: oligonucleotide < 400 > 12 cccaagcttc cacaaacgtt gccttcctct atgag 35 < 210 > 13 < 211 > 206 < 212 > PRT < 213 > Homo sapiens < 400 > 13 Met Ser Gly Pro Gly Thr Wing Wing Val Wing Leu Leu Pro Wing Val Leu 1 5 10 15 Leu Wing Leu Leu Wing Pro Trp Wing Gly Arg Gly Wing Wing Wing Pro 20 25 30 Thr Wing Pro Asn Gly Thr Leu Glu Wing Glu Leu Glu Arg Arg Trp Glu 35 40 45 Ser Leu Val Ala Leu Ser Leu Ala Arg Leu Pro Val Ala Ala Gln Pro 50 55 60 Lys Glu Ala Ala Val Gln Ser Gly Ala Gly Asp Tyr Leu Leu Gly He 65 70 75 80 Lys Arg Leu Arg Arg Leu Tyr Cys Asn Val Gly He Gly Phe His Leu 85 90 95 Gln Ala Leu Pro Asp Gly Arg He Gly Gly Ala His Wing Asp Thr Arg 100 105 110 Asp Ser Leu Leu Glu Leu Ser Pro Val Glu Arg Gly Val Val Ser He 115 120 125 Phe Gly Val Wing Ser Arg Phe Phe Val Wing Met Ser Ser Lys Gly Lys 130 135 140 Leu Tyr Gly Ser Pro Phe Phe Thr Asp Glu Cys Thr Phe Lys Glu He 145 150 155 160 Leu Leu Pro Asn Asn Tyr Asn Wing Tyr Glu Ser Tyr Lys Tyr Pro Gly 165 170 175 Met Phe He Wing Leu Ser Lys Asn Gly Lys Thr Lys Lys Gly Asn Arg 180 185 190 Val Ser Pro Thr Met Lys Val Thr His Phe Leu Pro Arg Leu 195 200 205 < 210 > 14 < 211 > 198 < 212 > PRT < 213 > Homo sapiens < 400 > 14 Met Ser Arg Gly Wing Gly Arg Leu Gln Gly Thr Leu Trp Wing Leu Val 1 5 10 15 Phe Leu Gly He Leu Val Gly Met Val Val Pro Pro Pro Wing Gly Thr 20 25 30 Arg Wing Asn Asn Thr Leu Leu Asp Ser Arg Gly Trp Gly Thr Leu Leu 35 40 45 Ser Arg Ser Arg Ala Gly Leu Ala Gly Glu He Ala Gly Val Asn Trp 50 55 60 Glu Ser Gly Tyr Leu Val Gly He Lys Arg Gln Arg Arg Leu Tyr Cys 65 70 75 80 Asn Val Gly He Gly Phe His Leu Gln Val Leu Pro Asp Gly Arg He 85 90 95 Ser Gly Thr His Glu Glu Asn Pro Tyr Ser Leu Leu Glu He Ser Thr 100 105 110 Val Glu Arg Gly Val Val Ser Leu Phe Gly Val Arg Ser Ala Leu Phe 115 120 125 Val Ala Met Asn Ser Lys Gly Arg Leu Tyr Ala Thr Pro Ser Phe Gln 130 135 140 Glu Glu Cys Lys Phe Arg Glu Thr Leu Leu Pro Asn Asn Tyr Asn Ala 145 150 155 160 Tyr Glu Ser Asp Leu Tyr Gln Gly Thr Tyr He Wing Leu Ser Lys Tyr 165 170 175 Gly Arg Val Lys Arg Gly Ser Lys Val Ser Pro He Met Thr Val Thr 180 185 190 His Phe Leu Pro Arg He 195 < 210 > 15 < 211 > 268 < 212 > PRT < 213 > Homo sapiens < 400 > 15 Met Ser Leu Ser Phe Leu Leu Leu Leu Phe Phe Ser His Leu He Leu 1 5 10 15 Be Wing Trp Wing His Gly Glu Lys Arg Leu Wing Pro Lys Gly Gln Pro 20 25 30 Gly Pro Wing Wing Thr Asp Arg Asn Pro Arg Gly Being Being Arg Gln 35 40 45 Being Being Being Wing Met Being Being Wing Being Ser Ser Pro Ala 50 55 60 Wing Ser Leu Gly Ser Gln Gly Ser Gly Leu Glu Gln Ser Ser Phe Gln 65 70 75 80 Trp Ser Pro Ser Gly Arg Arg Thr Gly Ser Leu Tyr Cys Arg Val Gly 85 90 95 He Gly Phe His Leu Gln He Tyr Pro Asp Gly Lys Val Asn Gly Ser 100 105 110 His Glu Wing Asn Met Leu Ser Val Leu Glu He Phe Wing Val Ser Gln 115 120 125 Gly He Val Gly He Arg Gly Val Phe Ser Asn Lys Phe Leu Wing Met 130 135 140 Ser Lys Lys Gly Lys Leu His Wing Being Wing Lys Phe Thr Asp Asp Cys 145 150 155 160 Lys Phe Arg Glu Arg Phe Gln Glu Asn Ser Tyr Asn Thr Tyr Wing Ser 165 170 175 Wing He His Arg Thr Glu Lys Thr Gly Arg Glu Trp Tyr Val Wing Leu 180 185 190 Asn Lys Arg Gly Lys Wing Lys Arg Gly Cys Ser Pro Arg Val Lys Pro 195 200 205 Gln His He Ser Thr His Phe Leu Pro Arg Phe Lys Gln Ser Glu Gln 210 215 220 Pro Glu Leu Ser Phe Thr Val Thr Val Pro Glu Lys Lys Asn Pro Pro 225 230 235 240 Ser Pro He Lys Ser Lys He Pro Leu Ser Ala Pro Arg Lys Asn Thr 245 250 255 Asn Ser Val Lys Tyr Arg Leu Lys Phe Arg Phe Gly 260 265 < 210 > 16 < 211 > 155 < 212 > PRT < 213 > Homo sapiens < 400 > 16 Met Wing Glu Gly Glu He Thr Thr Phe Thr Wing Leu Thr Glu Lys Phe 1 5 10 15 Asn Leu Pro Pro Gly Asn Tyr Lys Lys Pro Lys Leu Leu Tyr Cys Ser 20 25 '30 Asn Gly His Phe Leu Arg He Leu Pro Asp Gly Thr Val Asp Gly 35 40 45 Thr Arg Asp Arg Ser Asp Gln His He Gln Leu Gln Leu Ser Ala Glu 50 55 60 Ser Val Gly Glu Val Tyr He Lys Ser Thr Glu Thr Gly Gln Tyr Leu 65 70 75 80 Wing Met Asp Thr Asp Gly Leu Leu Tyr Gly Ser Gln Thr Pro Asn Glu 85 90 95 Glu Cys Leu Phe Leu Glu Arg Leu Glu Glu Asn His Tyr Asn Thr Tyr 100 105 110 He Ser Lys Lys His Wing Glu Lys Asn Trp Phe Val Gly Leu Lys Lys 115 120 125 Asn Gly Ser Cys Lys Arg Gly Pro Arg Thr His Tyr Gly Gln Lys Wing 130 135 140 He Leu Phe Leu Pro Leu Pro Val Ser Ser Asp 145 150 155 < 210 > 17 < 211 > 155 < 212 > PRT < 213 > Homo sapiens < 400 > 17 Met Wing Wing Gly Ser He Thr Thr Leu Pro Wing Leu Pro Glu Asp Gly 1 5 10 15 Gly Ser Gly Wing Phe Pro Pro Gly His Phe Lys Asp Pro Lys Arg Leu 20 25 30 Tyr Cys Lys Asn Gly Gly Phe Phe Leu Arg He His Pro Asp Gly Arg 35 40 45 Val Asp Gly Val Arg Glu Lys Ser Asp Pro His He Lys Leu Gln Leu 50 55 60 Gln Wing Glu Glu Arg Gly Val Val Ser He Lys Gly Val Cys Wing Asn 65 70 75 80 Arg Tyr Leu Wing Met Lys Glu Asp Gly Arg Leu Leu Wing Ser Lys Cys 85 90 95 Val Thr Asp Glu Cys Phe Phe Phe Glu Arg Leu Glu Ser Asn Asn Tyr 100 105 110 Asn Thr Tyr Arg Ser Arg Lys Tyr Thr Ser Trp Tyr Val Wing Leu Lys 115 120 125 Arg Thr Gly Gln Tyr Lys Leu Gly Ser Lys Thr Gly Pro Gly Gln Lys 130 135 140 Ala He Leu Phe Leu Pro Met Ser Ala Lys Ser 145 150 155 < 210 > 18 < 211 > 208 < 212 > PRT < 213 > Homo sapiens < 400 > 18 Met Ala Pro Leu Gly Glu Val Gly Asn Tyr Phe Gly Val Gln Asp Ala 1 5 10 15 Val Pro Phe Gly Asn Val Pro Val Leu Pro Val Asp Ser Pro Val Leu 20 25 30 Leu Ser Asp His Leu Gly Gln Ser Glu Wing Gly Gly Leu Pro Arg Gly 35 40 45 Pro Wing Val Thr Asp Leu Asp His Leu Lys Gly He Leu Arg Arg Arg 50 55 60 Gln Leu Tyr Cys Arg Thr Gly Phe His Leu Glu He Phe Pro Asn Gly 65 70 75 80 Thr He Gln Gly Thr Arg Lys Asp His Ser Arg Phe Gly He Leu Glu 85 90 95 Phe He Ser He Wing Val Gly Leu Val Ser He Arg Gly Val Asp Ser 100 105 110 Gly Leu Tyr Leu Gly Met Asn Glu Lys Gly Glu Leu Tyr Gly Ser Glu 115 120 125 Lys Leu Thr Gln Glu Cys Val Phe Arg Glu Gln Phe Glu Glu Asn Trp 130 135 140 Tyr Asn Thr Tyr Ser Ser Asn Leu Tyr Lys His Val Asp Thr Gly Arg 145 150 155 160 Arg Tyr Tyr Val Wing Leu Asn Lys Asp Gly Thr Pro Arg Glu Gly Thr 165 170 175 Arg Thr Lys Arg His Gln Lys Phe Thr His Phe Leu Pro Arg Pro Val 180 185 190 Asp Pro Asp Lys Val Pro Glu Leu Tyr Lys Asp He Leu Ser Gln Ser 195 195 205 < 210 > 19 < 211 > 194 < 212 > PRT < 213 > Homo sapiens < 400 > 19 Met His Lys Trp He Leu Thr Trp He Leu Pro Thr Leu Leu Tyr Arg 1 5 10 15 Ser Cys Phe His He He Cys Leu Val Gly Thr He Ser Leu Wing Cys 20 25 30 Asn Asp Met Thr Pro Glu Gln Met Ala Thr Asn Val Asn Cys Ser Ser 35 40 45 Pro Glu Arg His Thr Arg Ser Tyr Asp Tyr Met Glu Gly Asp He 50 55 60 Arg Val Arg Arg Leu Phe Cys Arg Thr Gln Trp Tyr Leu Arg He Asp 65 70 75 80 Lys Arg Gly Lys Val Lys Gly Thr Gln Glu Met Lys Asn Asn Tyr Asn 85 90 95 He Met Glu He Arg Thr Val Wing Val Gly He Val Wing He Lys Gly 100 105 110 Val Glu Ser Glu Phe Tyr Leu Ala Met Asn Lys Glu Gly Lys Leu Tyr 115 120 125 Wing Lys Lys Glu Cys Asn Glu Asp Cys Asn Phe Lys Glu Leu He Leu 130 135 140 Glu Asn His Tyr Asn Thr Tyr Ala Ser Wing Lys Trp Thr His Asn Gly 145 150 155 160 . -.

Gly Glu Met Phe Val Ala Leu Asn Gln Lys Gly He Pro Val Arg Gly 165 170 175 Lys Lys Thr Lys Lys Glu Gln Lys Thr Wing His Phe Leu Pro Met Wing 180 185 190 He Thr < 210 > 20 < 211 > 208 < 212 > PRT < 213 > Homo sapiens < 400 > 20 Met Trp Lys Trp He Leu Thr His Cys Wing Ser Wing Phe Pro His Leu 1 5 10 15 Pro Gly Cys Cys Cys Cys Cys Phe Leu Leu Leu Phe Leu Val Ser Ser 20 25 30 Val Pro Val Thr Cys Gln Ala Leu Gly Gln Asp Met Val Ser Pro Glu 35 40 45 Wing Thr Asn Being Ser Ser Ser Phe Ser Ser Pro Ser Ser Ala Gly 50 55 60 Arg His Val Arg Ser Tyr Asn His Leu Gln Gly Asp Val Arg Trp Arg 65 70 75 80 Lys Leu Phe Ser Phe Thr Lys Tyr Phe Leu Lys He Glu Lys Asn Gly 85 90 95 Lys Val Ser Gly Thr Lys Lys Glu Asn Cys Pro Tyr Ser He Leu Glu 100 105 110 He Thr Ser Val Glu He Gly Val Val Ala Val Lys Ala He Asn Ser 115 120 125 Asn Tyr Tyr Leu Ala Met Asn Lys Lys Gly Lys Leu Tyr Gly Ser Lys 130 135 140 Glu Phe Asn Asn Asp Cys Lys Leu Lys Glu Arg He Glu Glu Asn Gly 145 150 155 160 Tyr Asn Thr Tyr Ala Ser Phe Asn Trp Gln His Asn Gly Arg Gln Met 165 170 175 Tyr Val Wing Leu Asn Gly Lys Gly Wing Pro Arg Arg Gly Gln Lys Thr 180 185 190 Arg Arg Lys Asn Thr Ser Wing His Phe Leu Pro Met Val Val His Ser 195 200 205 < 210 > 21 < 211 > 239 < 212 > PRT < 213 > Homo sapiens < 400 > 21 Met Gly Leu He Trp Leu Leu Leu Leu Ser Leu Leu Glu Pro Gly Trp 1 5 10 15 Pro Wing Wing Gly Pro Gly Wing Arg Leu Arg Arg Asp Wing Gly Gly Arg 20 25 30 Gly Gly Val Tyr Glu His Leu Gly Gly Wing Pro Arg Arg Arg Lys Leu 35 40 45 Tyr Cys Wing Thr Lys Tyr His Leu Gln Leu His Pro Ser Gly Arg Val 50 55 60 Asn Gly Ser Leu Glu Asn Ser Wing Tyr Ser He Leu Glu He Thr Wing 65 70 75 80 Val Glu Val Gly He Val Wing He Arg Gly Leu Phe Ser Gly Arg Tyr 85 90 95 Leu Wing Met Asn Lys Arg Gly Arg Leu Tyr Wing Ser Glu His Tyr Ser 100 105 110 Wing Glu Cys Glu Phe Val Glu Arg He His Glu Leu Gly Tyr Asn Thr 115 120 125 Tyr Ala Ser Arg Leu Tyr Arg Thr Val Ser Ser Thr Pro Gly Ala Arg 130 135 140 Arg Gln Pro Ser Wing Glu Arg Leu Trp Tyr Val Ser Val Asn Gly Lys 145 150 155 160 Gly Arg Pro Arg Arg Gly Phe Lys Thr Arg Arg Thr Gln Lys Ser Ser 165 170 175 Leu Phe Leu Pro Arg Val Leu Asp His Arg Asp His Glu Met Val Arg 180 185 190 Gln Leu Gln Ser Gly Leu Pro Arg Pro Pro Gly Lys Gly Val Gln Pro 195 200 205 Arg Arg Arg Arg Gln Lys Gln Ser Pro Asp Asn Leu Glu Pro Ser His 210 215 220 Val Gln Wing Ser Arg Leu Gly Ser Gln Leu Glu Wing Ser Wing His 225 230 235 < 210 > 22 < 211 > 268 < 212 > PRT < 213 > Homo sapiens 5 < 400 > 22 Met Gly Ser Pro Arg Ser Ala Leu Ser Cys Leu Leu Leu His Leu Leu 1 5 10 15 10 Val Leu Cys Leu Gln Wing Gln Val Arg Wing Wing Gln Lys Arg Gly 20 25 30 Pro Gly Wing Gly Asn Pro Wing Asp Thr Leu Gly Gln Gly His Glu Asp 35 40 45 15 Arg Pro Phe Gly Gln Arg Ser Arg Ala Gly Lys Asn Phe Thr Asn Pro 50 55 60 Wing Pro Asn Tyr Pro Glu Glu Gly Ser Lys Glu Gln Arg Asp Ser Val 20 65 70 75 80 Leu Pro Lys Val Thr Gln Arg His Val Arg Glu Gln Ser Leu Val Thr 85 90 95 25 Asp Gln Leu Be Arg Arg Leu He Arg Thr Tyr Gln Leu Tyr Ser Arg 100 105 110 Thr Ser Gly Lys His Val Gln Val Leu Wing Asn Lys Arg He Asn Wing 115 120 125 30 Met Wing Glu Asp Gly Asp Pro Phe Wing Lys Leu He Val Glu Thr Asp 130 135 140 Thr Phe Gly Ser Arg Val Arg Val Arg Gly Ala Glu Thr Gly Leu Tyr 35 145 150 155 160 He Cys Met Asn Lys Lys Gly Lys Leu He Wing Lys Ser Asn Gly Lys 165 170 175 40 Gly Lys Asp Cys Val Phe Thr Glu He Val Leu Glu Asn Asn Tyr Thr 180 185 190 Wing Leu Gln Asn Wing Lys Tyr Glu Gly Trp Tyr Met Wing Phe Thr Arg 195 200 205 45 Lys Gly Arg Pro Arg Lys Gly Ser Lys Thr Arg Gln His Gln Arg Glu 210 215 220 Val His Phe Met Lys Arg Leu Pro Arg Gly His His Thr Thr Glu Gln 50 225 230 235 240 Ser Leu Arg Phe Glu Phe Leu Asn Tyr Pro Pro Phe Thr Arg Ser Leu 245 250 255 afttaArt ^ fc Arg Gly Ser Gln Arg Thr Trp Wing Pro Glu Pro Arg 260 265 < 210 > 23 < 211 > 4177 < 212 > DNA < 213 > Homo sapiens < 220 > < 221 > CDS < 222 > (593) .. (1216) < 400 > 23 ggaattccgg gaagagaggg aagaaaacaa cggcgactgg gcagctgcct ccacttctga 60 caactccaaa gggatatact tgtagaagtg gctcgcaggc tggggctccg cagagagaga 120 ccagaaggtg ccaaccgcag aggggtgcag atatctcccc ctattcccca ccccacctcc 180 cttgggtttt gttcaccgtg ctgtcatctg tttttcagac ctttttggca tctaacatgg 240 tgaagaaagg agtaaagaag agaacaaagt aactcctggg ggagcgaaga gcgctggtga 300 ccaacaccac caacgccacc accagctcct gctgctgcgg ccacccacgt ccaccattta 360 ccgggaggct ccagaggcgt aggcagcgga tccgagaaag gagcgagggg agtcagccgg 420 cttttccgag gagttatgga tgttggtgca ttcacttctg gccagatccg cgcccagagg 480 gcagccacca gagctaacca cctcgagctc tctccttgcc ttgcatcggg tcttaccctt 540 ccagtatgtt ccttctgatg agacaatttc atg tgg ca cagtgccgag agtttcagta Met Trp 598 aaa tgg ata i ctg gcc tgt cat here tea gcc ttt ctg ecc ecc falls ggc 646 Lys Trp He Leu Thr His Cys Wing Being Wing Phe Pro His Leu Pro Gly 5 10 15 tgc tgc tgc tgc tgc ttt ttg ttg ctg ttc ttg gtg tet tcc gtc ect 694 Cys Cys Cys Cys Cys Phe Leu Leu Leu Phe Leu Val Ser Ser Val Pro 20 25 30 gtc acc tgc ca gcc ctt ggt cag gac atg gtg tea cea gag gcc acc 742 Val Thr Cys Gln Ala Leu Gly Gln Asp Met Val Ser Pro Glu Ala Thr 35 40 45 50 aac tet tet tcc tcc ttc tcc tet ect tcc age gcg gga agg cat 790 Asn Being Being Being Being Phe Being Pro Pro Being Being Wing Gly Arg His 55 60 65 gtg cgg age tac aat falls ctt ca gga gat gtc cgc tgg aga aag cta 838 Val Arg Ser Tyr Asn His Leu Gln Gly Asp Val Arg Trp Arg Lys Leu 70 75 80 ttc tet ttc acc aag tac ttt ctc aag att gag aag aac ggg aag gtc 886 Phe Ser Phe Thr Lys Tyr Phe Leu Lys He Glu Lys Asn Gly Lys Val 85 90 95 age ggg acc aag aag gag aac tgc ceg tac age ate ctg gag ata here 934 Ser Gly Thr Lys Lys Glu Asn Cys Pro Tyr Ser He Leu Glu He Thr 100 105 110 tea gta gaa ate gga gtt gtt gcc gtc aaa gcc att aac age aac tat 982 Ser Val Glu He Gly Val Val Ala Val Lys Ala He Asn Ser Asn Tyr 115 120 125 130 tac tta gcc atg aac aag aag ggg aaa ctc tat ggc tea aaa gaa ttt 1030 Tyr Leu Ala Met Asn Lys Lys Gly Lys Leu Tyr Gly Ser Lys Glu Phe 135 140 145 aac aat gac tgt aag ctg aag gag agg ata gag gaa aat gga tac aat 1078 Asn Asn Asp Cys Lys Leu Lys Glu Arg He Glu Glu Asn Gly Tyr Asn 150 155 160 acc tat gca tea ttt aac tgg cag cat aat ggg agg caa atg tat gtg 1126 Thr Tyr Ala Ser Phe Asn Trp Gln His Asn Gly Arg Gln Met Tyr Val 165 170 175 gca ttg aat gga aaa gga gct cea agg aga gga cag aaa here cga agg 1174 Wing Leu Asn Gly Lys Gly Wing Pro Arg Arg Gly Gln Lys Thr Arg Arg 180 185 190 aaa aac acc tet gct falls ttt ctt cea atg gtg gta cae tea 1216 Lys Asn Thr Ser Ala His Phe Leu Pro Met Val Val His Ser 195 200 205 tagaggaagg caacgtttgt ggatgcagta aaaccaatgg ctcttttgcc aagaatagtg 1276 atgaagacag gatattette tagattgaaa ggcaaagaca cgttgcagat gtctgcttgc 1336 ttaaaagaaa gccagccttt gaaggttttt gtattcactg ctgacatatg atgttctttt 1396 aattagttct gtgtcatgtc ttataatcaa gatataggca gategaatgg gatagaagtt 1456 attcccaagt gaaaaacatt gtggctgggt tttttgttgt tgttgtcaag tttttgtttt 1516 taaacctctg agatagaact taaaggacat agaacaatct gttgaaagaa cgatcttcgg 1576 gaaagttatt tatggaatac gaaetcatat caaagaette attgetcatt caagectaat 1636 gaatcaatga acagtaatac gtgcaagcat ttactggaaa gcacttgggt catatcatat 1696 gcacaaccaa aggagttctg gatgtggtct ttgaatagaa catggaataa tttaaaaata 1756 taaacatgtt agtgtgaaac tgttctaaca atacaaatag tatggtatgc ttgtgcattc 1816 tgccttcatc cctttctatt tctttctaag ttatttattt aataggatgt taaatatctt 1876 ttggggtttt aaagagtatc tcagcagctg tcttctgatt tatcttttct ttttattcag 1936 cacaccacat gcatgttcac gacaaagtgt ttttaaaact tggcgaacac ttcaaaaata 1996 ggagttggg to ttagggaagc agtatgagtg cccgtgtgct atcagttgac ttaatttgca 2056 cttctgcagt aataaccatc aacaataaat atggcaatgc tgtgccatgg cttgagtgag 2116 agatgtctgc tatcatttga aaacatatat tactctcgag gcttcctgtc tcaagaaata 2176 gaccagaagg ccaaattctt ctctttcaat acatcagttt gcctccaaga atatactaaa 2236 ttaattgcta aaaaggaaaa aatacattta aatagcctag cctcattatt tactcatgat 2296 ttcttgccaa atgtcatggc ggtaaagagg ctgtccacat ctctaaaaac cctctgtaaa 2356 ttccacataa tgcatctttc ccaaaggaac tataaagaat ttggtatgaa gcgcaactct 2416 cccaggggct taaactgagc aaatcaaata tatactggta tatgtgtaac catatacaaa 2476 aacctgttct agetgtatga tctagtcttt acaaaaccaa ataaaacttg ttttctgtaa 2536 atttaaagag ctttacaagg ttccataatg taaccatatc aaaattcatt ttgttagagc 2596 acgtatagaa aagagtacat aagagtttac caatcatcat cacattgtat tccactaaat 2656 aaatacataa gccttatttg cagtgtctgt agtgatttta aaaatgtaga aaaatactat 2716 ttgttctaaa tacttttaag caataactat aatagtatat tgatgctgca gttttatctt 2776 catatttett gttttgaaaa agcattttat tgtttggaca cagtattttg gtacaaaaaa 2836 taaa aaagaetcae tgtgtc ttactaaagt ttaacctttg gaaatgctgg cgttctgtga 2896 ttctccaaca aacttatttg tgtcaatact taaccagcac ttccagttaa tctgttattt 2956 ttaaaaattg ctttattaag aaattttttg tataatecca taaaaggtca tatttttccc 3016 attcttcaaa aaaactgtat ttcagaagaa acacatttga ggcactgtct tttggcttat 3076 agtttaaatt gcatttcatc atactttgct tccaacttgc tttttggcaa atgagattat 3136 aaaaatgttt aatttttgtg gttggaatct ggatgttaaa atttaattgg taactcagtc 3196 tgtgagctat aatgtaatgc attcctatcc aaactaggta tctttttttc ctttatgttg 3256 aaataataat ggcacctgac acatagacat agaccaccca caacctaaat taaatgtttg 3316 gtaagacaaa tacacattgg atgaccacag taacagcaaa cagggcacaa actggattct 3376 tatttcacat agacatttag attactaaag agggctatgt gtaaacagtc atcattatag 3436 tactcaagac actaaaacag aatatattaa cttctagcca agcttgcaga ggccaaaaat 3496 agaaaacatc tcccctgtct ctcccacatt tccctcacag aaagacaaaa aacctgcctg 3556 gtgcagtagc tcacacctgt aatcccagca gtttgggaga ctgtgggaag atggcttgag 3616 tccaggagtt ctagacaggc ctgagaaacc tagtgagaca tccttctctt aaacaaaaca 3676 aatgtagcca aaacaaaaca tgcgtggtgg catatacctg tggtcccaac tactcaggag 3736 gctgaaacgg aaggatctct tgggccccag gagtttgagg ctgcagtgag ctataatctt 3796 gccattgcac tccagcctgg gtgaaaaaga gccagaaaga aaggaaagag agaaaagaga 3856 agaaaagaca aaagaaagag gaaagacagg aaggaaggaa ggaaggaagg aaggaaggaa 3916 ggaagcaagg aaagaa GGAA aagggaggga ggaaggaaag gagaaagaaa aggaaggaga 3976 taaggagtaa gattgtttgg tgacattctc ttgcatttaa tttgcttgaa aagtggcata 4036 atggaaatag aattctggtc ccttttgcaa ctactgaaga aaaaaaaaag cagtttcagc gtagatttga 4096 cctgaatgtt aaaaaaaaaa aaaaaaactc gagggggggc ccgtacccaa ttcgccctat 4156 to 4177 agtgagtcgt < 210 > 24 < 211 > 208 < 212 > PRT < 213 > Homo sapiens < 400 > 24 Met Trp Lys Trp He Leu Thr His Cys Wing Ser Wing Phe Pro His Leu 1 5 10 15 Pro Gly Cys Cys Cys Cys Cys Phe Leu Leu Leu Phe Leu Val Ser Ser 20 25 30 Val Pro Val Thr Cys Gln Ala Leu Gly Gln Asp Met Val Ser Pro Glu 35 40 45 Wing Thr Asn Being Ser Being Ser Phe Ser Ser Pro Being Wing Gly 50 55 60 Arg His Val Arg Ser Tyr Asn His Leu Gln Gly Asp Val Arg Trp Arg 65 70 75 80 Lys Leu Phe Ser Phe Thr Lys Tyr Phe Leu Lys He Glu Lys Asn Gly 85 90 95 Lys Val Ser Gly Thr Lys Lys Glu Asn Cys Pro Tyr Ser He Leu Glu 100 105 110 He Thr Ser Val Glu He Gly Val Val Ala Val Lys Wing He Asn Ser 115 120 125 Asn Tyr Tyr Leu Wing Met Asn Lys Lys Gly Lys Leu Tyr Gly Ser Lys 130 135 140 Glu Phe Asn Asn Asp Cys Lys Leu Lys Glu Arg He Glu Glu Asn Gly 145 150 155 160 Tyr Asn Thr Tyr Wing Ser Phe Asn Trp Gln His Asn Gly Arg Gln Met 165 170 175 Tyr Val Wing Leu Asn Gly Lys Gly Wing Pro Arg Arg Gly Gln Lys Thr 180 185 190 Arg Arg Lys Asn Thr Ser Wing His Phe Leu Pro Met Val Val His Ser 195 200 205 < 210 > 25 < 211 > 31 < 212 > PRT < 213 > Homo sapiens < 400 > 25 Gly Gln Asp Met Val Ser Pro Glu Ala Thr Asn Ser Ser Ser Ser Ser 1 5 10 15 Phe Be Ser Pro Pro Be Wing Gly Arg His Val Arg Ser Tyr Asn 20 25 30 < 210 > 26 < 211 > 19 < 212 > PRT < 213 > Homo sapiens < 400 > 26 Lys He Glu Lys Asn Gly Lys Val Ser Gly Thr Lys Lys Glu Asn Cys 1 5 10 15 Pro Tyr Ser < 210 > 27 < 211 > 30 < 212 > PRT < 213 > Homo sapiens < 400 > 27 Asn Lys Lys Gly Lys Leu Tyr Gly Ser Lys Glu Phe Asn Asn Asp Cys 1 5 10 15 Lys Leu Lys Glu Arg He Glu Glu Asn Gly Tyr Asn Thr Tyr 20 25 30 < 210 > 28 < 211 > 19 < 212 > PRT < 213 > Homo sapiens < 400 > 28 Asn Gly Lys Gly Ala Pro Arg Arg Gly Gln Lys Thr Arg Arg Lys Asn 1 5 10 15 Thr Ser Ala < 210 > 29 < 211 > 555 < 212 > DNA < 213 > Artificial Sequence < 220 > < 221 > CDS < 222 > (1) .. (552) < 220 > < 223 > Description of Artificial Sequence: construction pQE60-Cys37 < 400 > 29 atg aga gga teg cat falls cat falls cat falls gga tcc tgc cag gct ctg 48 Met Arg Gly Ser His His His His His His Gly Ser Cys Gln Ala Leu 1 5 10 15 ggt cag gac atg gtt tet ceg gaa gct acc aac tet tcc tet tet tet 96 Gly Gln Asp Met Val Ser Pro Glu Wing Thr Asn Being Ser Being Ser 20 25 30 ttc tet tcc tg tg cct gct ggt cgt gtt ct tc tac aac ct fall 144 Phe Ser Ser Pro Ser Wing Gly Arg His Val Arg Ser Tyr Asn His 35 40 45 ctg cag ggt gac gtt cgt tgg cgt aaa ctg ttc tet ttc acc aaa tac 192 Leu Gln Gly Asp Val Arg Trp Arg Lys Leu Phe Ser Phe Thr Lys Tyr 50 55 60 ttc ctg aaa ate gaa aaa aac ggt aaa gtt tet ggg acc aag aag gag 240 Phe Leu Lys He Glu Lys Asn Gly Lys Val Ser Gly Thr Lys Lys Glu 65 70 75 80 aac tgc ceg tac age ate ctg gag ata here tea gta gaa ate gga gtt 288 Asn Cys Pro Tyr Ser He Leu Glu He Thr Ser Val Glu He Gly Val 85 90 95 gtt gcc gtc aaa gcc att aac age aac tat tac tta gcc atg aac aag 336 Val Ala Val Lys Ala He Asn Ser Asn Tyr Tyr Leu Ala Met Asn Lys 100 105 110 aag ggg aaa ctc tat ggc tea aaa gaa ttt aac aat gac tgt aag ctg 384 Lys Gly Lys Leu Tyr Gly Ser Lys Glu Phe Asn Asn Asp Cys Lys Leu 115 120 125 aag gag agg ata gag gaa aat gga tac aat tat tat gca tea ttt aac 432 Lys Glu Arg He Glu Glu Asn Gly Tyr Asn Thr Tyr Wing Ser Phe Asn 130 135 140 tgg cag cat aat ggg agg cag atg tat gtg gca ttg aat gga aaa gga 480 Trp Gln His Asn Gly Arg Gln Met Tyr Val Wing Leu Asn Gly Lys Gly 145 150 155 160 gct cea agg aga gga cag aaa here cga agg aaa aac acc tet gct drops 528 Wing Pro Arg Arg Gly Gln Lys Thr Arg Arg Lys Asn Thr Ser Ala His 165 170 175 ttt ctt cea atg gtg gta falls tea tag 555 Phe Leu Pro Met Val Val His Ser 180 < 210 > 30 < 211 > 184 < 212 > PRT < 213 > Artificial Sequence < 223 > Description of Artificial Sequence: construction pQE60-Cys37 < 400 > 30 Met Arg Gly Ser His His His His His His His Gly Ser Cys Gln Ala Leu 1 5 10 15 Gly Gln Asp Met Val Ser Pro Glu Ala Thr Asn Ser Ser Ser Ser 20 25 30 Phe Ser Ser Pro Ser Ser Ala Gly Arg His Val Arg Ser Tyr Asn His 35 40 45 Leu Gln Gly Asp Val Arg Trp Arg Lys Leu Phe Ser Phe Thr Lys Tyr 50 55 60 Phe Leu Lys He Glu Lys Asn Gly Lys Val Ser Gly Thr Lys Lys Glu 65 70 75 80 Asn Cys Pro Tyr Ser He Leu Glu He Thr Ser Val Glu He Gly Val 85 90 95 Val Wing Val Lys Wing He Asn Ser Asn Tyr Tyr Leu Wing Met Asn Lys 100 105 110 Lys Gly Lys Leu Tyr Gly Ser Lys Glu Phe Asn Asn Asp Cys Lys Leu 115 120 125 Lys Glu Arg He Glu Glu Asn Gly Tyr Asn Thr Tyr Wing Ser Phe Asn 130 135 140 Trp Gln His Asn Gly Arg Gln Met Tyr Val Wing Leu Asn Gly Lys Gly 145 150 155 160 Wing Pro Arg Arg Gly Gln Lys Thr Arg Arg Lys Asn Thr Ser Wing His 165 170 175 Phe Leu Pro Met Val Val His Ser 180 < 210 > 31 < 211 > 84 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: synthetic primer < 400 > 31 atgtggaaat ggatactgac ccactgcgct tctgctttcc cgcacctgcc gggttgctgc 60 tgctgctgct tcctgctgct gttc 84 < 210 > 32 < 211 > 82 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: synthetic primer < 400 > 32 ccggagaaac catgtcctga cccagagcct ggcaggtaac cggaacagaa gaaaccagga 60 acagcagcag gaagcagcag ca 82 < 210 > 33 < 211 > 80 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: synthetic primer < 400 > 33 gggtcaggac atggtttctc cggaagctac caactcttct tettettett tctcttctcc 60 gtcttctgct ggtcgtcacg 80 < 210 > 34 < 211 > 81 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: synthetic primer < 400 > 34 ggtgaaagag aacagtttac gccaacgaac gtcaccctgc aggtggttgt aagaaegaac 60 gtgacgacca gcagaagacg g 81 < 210 > 35 < 211 > 75 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: synthetic primer < 400 > 35 cgttggcgta aactgttetc tttcaccaaa tacttcctga aaatcgaaaa aaaeggtaaa 60 gtttctggga ccaaa 75 < 210 > 36 < 211 > 39 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: synthetic primer < 400 > 36 tttggtccca gaaactttac cgtttttttc gattttcag 39 < 210 > 37 < 211 > 36 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: synthetic primer < 400 > 37 aaaggatcca tgtggaaatg gatactgacc cactgc 36 < 210 > 38 < 211 > 627 < 212 > DNA < 213 > Escherichia coli < 220 > < 221 > CDS < 222 > (1) .. (627) < 400 > 38 atg tgg aaa tgg ata ctg acc fall tgc gct tet gct ttc ceg cae ctg 48 Met Trp Lys Trp He Leu Thr His Cys Ala Be Wing Phe Pro Hís Leu 1 5 10 15 ceg ggt tgc tgc tgc tgc tgc ttc ctg ctg ctg ttc ctg gtt tet tet 96 Pro Gly Cys Cys Cys Cys Cys Phe Leu Leu Leu Phe Leu Val Ser Ser 20 25 30 gtt ceg gtt acc tgc cag gct ctg ggt cag gac atg gtt tet ceg gaa 144 Val Pro Val Thr Cys Gln Ala Leu Gly Gln Asp Met Val Ser Pro Glu 35 40 45 gct acc aac tet tcc tet tet tet tet tcc tccct act tct gct ggt 192 Wing Thr Asn Being Ser Being Be Phe Be Ser Pro Thr Be Ala Gly 50 55 60 cgt fall gtt cgt tet tac aac fall ctg cag ggt gac gtt cgt tgg cgt 240 Arg His Val Arg Ser Tyr Asn His Leu Gln Gly Asp Val Arg Trp Arg 65 70 75 80 aaa ctg ttc tet ttc acc aaa tac ttc ctg aaa ate gaa aaa aac ggt 288 Lys Leu Phe Ser Phe Thr Lys Tyr Phe Leu Lys He Glu Lys Asn Gly 85 90 95 aaa gtt tet ggg acc aag aag gag aac tgc cec tac age ate ctg gag 336 Lys Val Ser Gly Thr Lys Lys Glu Asn Cys Pro Tyr Ser He Leu Glu 100 105 110 ata here tea gta gaa ate gga gtt gtt gcc gtc aaa gcc att aac age 384 He Thr Ser Val Glu He Gly Val Val Ala Val Lys Ala He Asn Ser 115 120 125 aac tat tac tta gcc atg aac aag aag ggg aaa ctc tat ggc tea aaa 432 Asn Tyr Tyr Leu Wing Met Asn Lys Lys Gly Lys Leu Tyr Gly Ser Lys 130 135 140 gaa ttt aac aat gac tgt aag ctg aag gag agg ata gag gaa aat gga 480 Glu Phe Asn Asn Asp Cys Lys Leu Lys Glu Arg He Glu Glu Asn Gly 145 150 155 160 tac aat acc tat gca tea ttt aac tgg cag cat aat ggg agg caa atg 528 Tyr Asn Thr Tyr Ala Ser Phe Asn Trp Gln His Asn Gly Arg Gln Met 165 170 175 tat gtg gca ttg aat gga aaa gga gct cea agg aga gga cag aaa here 576 Tyr Val Ala Leu Asn Gly Lys Gly Ala Pro Arg Arg Gly Gln Lys Thr 180 185 190 cga agg aaa aac acc tet gct cae ttt ctt cea atg gtg gta cae tea 624 Arg Arg Lys Asn Thr Ser Ala His Phe Leu Pro Met Val Val His Ser 195 200 205 tag 627 < 210 > 39 < 211 > 208 < 212 > PRT < 213 > Escherichia coli < 400 > 39 Met Trp Lys Trp He Leu Thr His Cys Wing Ser Wing Phe Pro His Leu 1 5 10 15 Pro Gly Cys Cys Cys Cys Cys Phe Leu Leu Leu Phe Leu Val Ser Ser 20 25 30 Val Pro Val Thr Cys Gln Ala Leu Gly Gln Asp Met Val Ser Pro Glu 35 40 45 Wing Thr Asn Being Ser Being Ser Phe Ser Ser Thr Ser Wing Gly 50 55 60 Arg His Val Arg Ser Tyr Asn His Leu Gln Gly Asp Val Arg Trp Arg 65 70 75 80 Lys Leu Phe Ser Phe Thr Lys Tyr Phe Leu Lys He Glu Lys Asn Gly 85 90 95 Lys Val Ser Gly Thr Lys Lys Glu Asn Cys Pro Tyr Ser He Leu Glu 100 105 110 He Thr Ser Val Glu He Gly Val Val Ala Val Lys Ala He Asn Ser 115 120 125 Asn Tyr Tyr Leu Ala Met Asn Lys Lys Gly Lys Leu Tyr Gly Ser Lys 130 135 140 Glu Phe Asn Asn Asp Cys Lys Leu Lys Glu Arg He Glu Glu Asn Gly 145 150 155 160 Tyr Asn Thr Tyr Wing Being Phe Asn Trp Gln His Asn Gly Arg Gln Met 165 170 175 Tyr Val Wing Leu Asn Gly Lys Gly Wing Pro Arg Arg Gly Gln Lys Thr 180 185 190 Arg Arg Lys Asn Thr Ser Wing His Phe Leu Pro Met Val Val His Ser 195 200 205 < 210 > 40 < 211 > 38 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Artificial Sequence Description: Primer < 400 > 40 tttcatgact tgtcaagctc tgggtcaaga tatggttc 38 < 210 > 41 < 211 > 28 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Artificial Sequence Description: Primer < 400 > 41 gcccaagctt ccacaaacgt tgccttcc 28 < 210 > 42 < 211 > 525 < 212 > DNA < 213 > Escherichia coli < 220 > < 221 > CDS < 222 > (1) .. (522) < 400 > 42 atg acc tgc cag gct ctg ggt cag gac atg gtt tet ceg gaa gct acc 48 Met Thr Cys Gln Ala Leu Gly Gln Asp Met Val Ser Pro Glu Ala Thr 1 5 10 15 aac tet tcc tet tcc tet ttc tet tcc ceg tet tcc gct ggt cgt falls 96 Asn Being Being Being Ser Phe Being Ser Pro Pro Being Wing Gly Arg His 20 25 30 gtt cgt tet tac aac falls ctg cag ggt gac gtt cgt tgg cgt aaa ctg 144 Val Arg Ser Tyr Asn His Leu Gln Gly Asp Val Arg Trp Arg Lys Leu 35 40 45 ttc tet ttc acc aaa tac ttc ctg aaa ate gaa aaa aac ggt aaa gtt 192 Phe Ser Phe Thr Lys Tyr Phe Leu Lys He Glu Lys Asn Gly Lys Val 50 55 60 tet ggg acc aag aag gag aac tgc ceg tac age ate ctg gag ata here 240 Ser Gly Thr Lys Lys Glu Asn Cys Pro Tyr Be He Leu Glu He Thr 65 70 75 80 tea gta gaa ate gga gtt gtt gcc gtc aaa gcc att aac age aac tat 288 Ser Val Glu He Gly Val Val Ala Val Lys Ala He Asn Ser Asn Tyr 85 90 95 tac tta gcc atg aac aag aag aag agg aaa ctc tat ggc tea aaa gaa ttt 336 Tyr Leu Ala Met Asn Lys Lys Gly Lys Leu Tyr Gly Ser Lys Glu Phe 100 105 110 aac aat gac tgt aag ctg aag gag agg ata gag gaa aat gga tac aat 384 Asn As n Asp Cys Lys Leu Lys Glu Arg He Glu Glu Asn Gly Tyr Asn 115 120 125 acc tat gca tea ttt aac tgg cag cat aat ggg agg caa atg tat gtg 432 Thr Tyr Ala Ser Phe Asn Trp Gln His Asn Gly Arg Gln Met Tyr Val 130 135 140 gca ttg aat gga aaa gga gct cea agg aga gga cag aaa here cga agg 480 Wing Leu Asn Gly Lys Gly Wing Pro Arg Arg Gly Gln Lys Thr Arg Arg 145 150 155 160 aaa aac ac tet gct cae ttt ctt cea atg gtg gta falls tea tag 525 Lys Asn Thr Ser Ala His Phe Leu Pro Met Val Val His Ser 165 170 < 210 > 43 < 211 > 174 < 212 > PRT < 213 > Escherichia coli < 400 > 43 Met Thr Cys Gln Wing Leu Gly Gln Asp Met Val Ser Pro Glu Wing Thr 1 5 10 15 Asn Being Ser Being Being Phe Being Pro Pro Being Wing Gly Arg His 20 25 30 Val Arg Being Tyr Asn His Leu Gln Gly Asp Val Arg Trp Arg Lys Leu 35 40 45 Phe Ser Phe Thr Lys Tyr Phe Leu Lys He Glu Lys Asn Gly Lys Val 50 55 60 Ser Gly Thr Lys Lys Glu Asn Cys Pro Tyr Ser He Leu Glu He Thr 65 70 75 80 Ser Val Glu He Gly Val Val Wing Val Lys Wing He Asn Ser Asn Tyr 85 90 95 Tyr Leu Wing Met Asn Lys Lys Gly Lys Leu Tyr Gly Ser Lys Glu Phe 100 105 110 Asn Asn Asp Cys Lys Leu Lys Glu Arg He Glu Glu Asn Gly Tyr Asn 115 120 125 Thr Tyr Wing Ser Phe Asn Trp Gln His Asn Gly Arg Gln Met Tyr Val 130 135 140 Wing Leu Asn Gly Lys Gly Wing Pro Arg Arg Gly Gln Lys Thr Arg Arg 145 150 155 160 Lys Asn Thr Ser Ala His Phe Leu Pro Met Val Val His Ser 165 170 < 210 > 44 < 211 > 45 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: synthetic primer < 400 > 44 tcagtgaatt cattaaagag gagaaattaa tcatgacttg ccagg 45 < 210 > 45 < 211 > 48 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: synthetic primer < 400 > 45 tcatgacttg ccaggcactg ggtcaagaca tggtttcccc ggaagcta 48 < 210 > 46 < 211 > 48 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: synthetic primer < 400 > 46 gcttcagcag cccatctagc gcaggtcgtc acgttcgctc ttacaacc 48 < 210 > 47 < 211 > 48 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: synthetic primer < 400 > 47 gttcgttggc gcaaactgtt cagctttacc aagtacttcc tgaaaatc 48 < 210 > 48 < 211 > 28 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: synthetic primer < 400 > 48 tcgaaaaaaa cggtaaagtt tctgggac 28 < 210 > 49 < 211 > 48 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: synthetic primer < 400 > 49 gatgggctgc tgaagctaga getggagetg ttggtagctt ceggggaa 48 < 210 > 50 < 211 > 45 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: synthetic primer < 400 > 50 aacagtttgc gccaacgaac atcaccctgt aagtggttgt aagag 45 < 210 > 51 < 211 > 47 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: synthetic primer < 400 > 51 ttcttggtcc cagaaacttt accgtttttt tcgattttca ggaagta 47 < 210 > 52 < 211 > 24 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: synthetic primer < 400 > 52 ttcttggtcc cagaaacttt accg 24 < 210 > 53 < 211 > 45 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: synthetic primer < 400 > 53 agatcaggct tctattatta tgagtgtacc accattggaa gaaag 45 < 210 > 54 < 211 > 525 < 212 > DNA < 213 > Escherichia coli < 220 > < 221 > CDS < 222 > (1) .. (522) < 400 > 54 atg act tgc cag gca ctg ggt caa gac atg gtt tcc ceg gaa gct acc 48 Met Thr Cys Gln Ala Leu Gly Gln Asp Met Val Ser Pro Glu Ala Thr 1 5 10 15 aac age tc age tet age ttc age age cea tet age gca ggt cgt falls 96 Asn Being Being Being Ser Phe Being Ser Pro Pro Being Wing Gly Arg His 20 25 30 gtt cgc tet tac aac falls tta cag ggt gat gtt cgt tgg cgc aaa ctg 144 Val Arg Ser Tyr Asn His Leu Gln Gly Asp Val Arg Trp Arg Lys Leu 35 40 45 ttc age ttt acc aag tac ttc ctg aaa ate gaa aaa aac ggt aaa gtt 192 Phe Ser Phe Thr Lys Tyr Phe Leu Lys He Glu Lys Asn Gly Lys Val 50 55 60 tet ggg acc aag aag gag aac tgc ceg tac age ate ctg gag ata here 240 Ser Gly Thr Lys Lys Glu Asn Cys Pro Tyr Ser He Leu Glu He Thr 65 70 75 80 tea gta gaa ate gga gtt gtt gcc gtc aaa gcc att aac age aac tat 288 Ser Val Glu He Gly Val Val Ala Val Lys Ala He Asn Ser Asn Tyr 85 90 95 tac tta gcc atg aac aag aag ggg aaa ctc tat ggc tea aaa gaa ttt 336 Tyr Leu Wing Met Asn Lys Lys Gly Lys Leu Tyr Gly Ser Lys Glu Phe 100 105 110 aac aat gac tgt aag ctg aag gag agg ata gag gaa aat gga tac aat 384 Asn Asn Asp Cys Lys Leu Lys Glu Arg He Glu Glu Asn Gly Tyr Asn 115 12 0 125 acc tat gca tea ttt aac tgg cag cat aat ggg agg caa atg tat gtg 432 Thr Tyr Ala Ser Phe Asn Trp Gln His Asn Gly Arg Gln Met Tyr Val 130 135 140 gca ttg aat gga aaa gga gct cea agg aga gga cag aaa here cga agg 480 Wing Leu Asn Gly Lys Gly Wing Pro Arg Arg Gly Gln Lys Thr Arg Arg 145 150 155 160 aaa aac acc tet gct falls ttt ctt cea atg gtg gta falls tea tag 525 Lys Asn Thr Ser Ala His Phe Leu Pro Met Val Val His Ser 165 170 < 210 > 55 < 211 > 174 < 212 > PRT < 213 > Escherichia coli < 400 > 55 Met Thr Cys Gln Wing Leu Gly Gln Asp Met Val Ser Pro Glu Wing Thr 1 5 10 15 Asn Being Ser Being Being Phe Being Pro Pro Being Wing Gly Arg His 20 25 30 Val Arg Being Tyr Asn His Leu Gln Gly Asp Val Arg Trp Arg Lys Leu 35 40 45 Phe Ser Phe Thr Lys Tyr Phe Leu Lys He Glu Lys Asn Gly Lys Val 50 55 60 Ser Gly Thr Lys Lys Glu Asn Cys Pro Tyr Ser He Leu Glu He Thr 65 70 75 80 Ser Val Glu He Gly Val Val Wing Val Lys Wing He Asn Ser Asn Tyr 85 90 95 Tyr Leu Wing Met Asn Lys Lys Gly Lys Leu Tyr Gly Ser Lys Glu Phe 100 105 110 Asn Asn Asp Cys Lys Leu Lys Glu Arg He Glu Glu Asn Gly Tyr Asn 115 120 125 Thr Tyr Wing Ser Phe Asn Trp Gln His Asn Gly Arg Gln Met Tyr Val 130 135 140 Wing Leu Asn Gly Lys Gly Wing Pro Arg Arg Gly Gln Lys Thr Arg Arg 145 150 155 160 Lys Asn Thr Ser Ala His Phe Leu Pro Met Val Val His Ser 165 170 < 210 > 56 < 211 > 35 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Artificial Sequence Description: Primer < 400 > 56 ggaccctcat gacctgccag gctctgggtc aggac 35 < 210 > 57 < 211 > 28 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Artificial Sequence Description: Primer < 400 > 57 ggacagccat ggctggtcgt cacgttcg 28 < 210 > 58 < 211 > 29 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Artificial Sequence Description: Primer < 400 > 58 ggacagccat ggttcgttgg cgtaaactg 29 < 210 > 59 < 211 > 31 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Artificial Sequence Description: Primer < 400 > 59 ggacagccat ggaaaaaaac ggtaaagttt c 31 < 210 > 60 < 211 > 29 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Artificial Sequence Description: Primer < 400 > 60 ggacccccat ggagaactgc ccgtagagc 29 < 210 > 61 < 211 > 32 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Artificial Sequence Description: Primer < 400 > 61 ggacccccat ggtcaaagcc attaacagca ac 32 < 210 > 62 < 211 > 33 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Artificial Sequence Description: Primer < 400 > 62 ggacccccat ggggaaactc tatggctcaa aag 33 < 210 > 63 < 211 > 37 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Artificial Sequence Description: Primer < 400 > 63 ctgcccaagc ttattatgag tgtaccacca ttggaag 37 < 210 > 64 < 211 > 36 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Artificial Sequence Description: Primer < 400 > 64 ctgcccaagc ttattacttc agcttacagt cattgt 36 < 210 > 65 < 211 > 525 < 212 > DNA < 213 > Homo sapiens < 220 > < 221 > CDS < 222 > (1) . [522) < 400 > 65 atg acc tgc cag gct ctg ggt cag gac atg gtt tet ceg gaa gct acc 48 Met Thr Cys Gln Ala Leu Gly Gln Asp Met Val Ser Pro Glu Ala Thr 1 5 10 15 aac tet tcc tet tcc tet ttc tet tcc ceg tet tcc gct ggt cgt falls 96 Asn Being Being Being Ser Phe Being Ser Pro Pro Being Wing Gly Arg His 20 25 30 gtt cgt tet tac aac falls ctg cag ggt gac gtt cgt tgg cgt aaa ctg 144 Val Arg Ser Tyr Asn His Leu Gln Gly Asp Val Arg Trp Arg Lys Leu 35 40 45 ttc tet ttc acc aaa tac ttc ctg aaa ate gaa aaa aac ggt aaa gtt 192 Phe Ser Phe Thr Lys Tyr Phe Leu Lys He Glu Lys Asn Gly Lys Val 50 55 60 tet ggg acc aag aag gag aac tgc cec tac age ate ctg gag ata aca 240 Be Gly Thr Lys Lys Glu Asn Cys Pro Tyr Be He Leu Glu He Thr 65 70 75 80 tea gta gaa ate gga gtt gtt gcc gtc aaa gcc att aac age aac tat 288 Ser Val Glu He Gly Val Val Ala Val Lys Ala He Asn Ser Asn Tyr 85 90 95 tac tta gcc atg aac aag aag ggg aaa ctc tat ggc tea aaa gaa ttt 336 Tyr Leu Wing Met Asn Lys Lys Gly Lys Leu Tyr Gly Ser Lys Glu Phe 100 105 110 aac aat gac tgt aag ctg aag gag agg ata gag gaa aat gga tac aat 384 Asn Asn Asp Cys Lys Leu Lys Glu Arg He Glu Glu Asn Gly Tyr Asn 115 120 125 acc tat gca tea ttt aac tgg cag cat aat ggg agg caa atg tat gtg 432 Thr Tyr Ala Ser Phe Asn Trp Gln His Asn Gly Arg Gln Met Tyr Val 130 135 140 gca ttg aat gga aaa gga gct cea agg aga gga cag aaa here cga agg 480 Wing Leu Asn Gly Lys Gly Wing Pro Arg Arg Gly Gln Lys Thr Arg Arg 145 150 155 160 aaa aac acc tet gct falls ttt ctt cea atg gtg gta falls tea tag 525 Lys Asn Thr Ser Ala His Phe Leu Pro Met Val Val His Ser 165 170 < 210 > 66 < 211 > 174 < 212 > PRT < 213 > Homo sapiens < 400 > 66 Met Thr Cys Gln Wing Leu Gly Gln Asp Met Val Ser Pro Glu Wing Thr 1 5 10 15 Asn Being Ser Being Being Phe Being Pro Pro Being Wing Gly Arg His 20 25 30 Val Arg Being Tyr Asn His Leu Gln Gly Asp Val Arg Trp Arg Lys Leu 35 40 45 Phe Ser Phe Thr Lys Tyr Phe Leu Lys He Glu Lys Asn Gly Lys Val 50 55 60 Ser Gly Thr Lys Lys Glu Asn Cys Pro Tyr Ser He Leu Glu He Thr 65 70 75 80 Ser Val Glu He Gly Val Val Ala Ala Lys Ala He Asn Ser Asn Tyr 85 90 95 Tyr Leu Ala Met Asn Lys Lys Gly Lys Leu Tyr Gly Ser Lys Glu Phe 100 105 110 Asn Asn Asp Cys Lys Leu Lys Glu Arg He Glu Glu Asn Gly Tyr Asn 115 120 125 Thr Tyr Wing Ser Phe Asn Trp Gln His Asn Gly Arg Gln Met Tyr Val 130 135 140 Wing Leu Asn Gly Lys Gly Wing Pro Arg Arg Gly Gln Lys Thr Arg Arg 145 150 155 160 Lys Asn Thr Ser Ala His Phe Leu Pro Met Val Val His Ser 165 170 < 210 > 67 < 211 > 444 < 212 > DNA < 213 > Homo sapiens < 220 > < 221 > CDS < 222 > (1) .. (444) < 400 > 67 atg gct ggt cgt falls gtt cgt tet tac aac falls ctg cag ggt gac gtt 48 Met Ala Gly Arg His Val Arg Ser Tyr Asn His Leu Gln Gly Asp Val 1 5 10 15 cgt tgg cgt aaa ctg ttc tet ttc acc aaa tac ttc ctg aaa ate gaa 96 Arg Trp Arg Lys Leu Phe Ser Phe Thr Lys Tyr Phe Leu Lys He Glu 20 25 30 aaa aac ggt aaa gtt tet ggg acc aag aag gag aac tgc ceg tac age 144 Lys Asn Gly Lys Val Ser Gly Thr Lys Lys Glu Asn Cys Pro Tyr Ser 35 40 45 ate ctg gag ata here tea gta gaa ate gga gtt gtt gtt gcc gtc aaa gcc 192 He Leu Glu He Thr Ser Val Glu He Gly Val Val Ala Val Lys Wing 50 55 60 att aac age aac tat tac tta gcc atg aac aag aag agg ggg aaa ctc tat 240 He Asn Ser Asn Tyr Tyr Leu Ala Met Asn Lys Lys Gly Lys Leu Tyr 65 70 75 80 ggc tea aaa gaa ttt aac aat gac tgt aag ctg aag gag agg ata gag 288 Gly Ser Lys Glu Phe Asn Asn Asp Cys Lys Leu Lys Glu Arg He Glu 85 90 95 gaa aat gga tac aat acc tat gca tea ttt aac tgg cag cat aat ggg 336 Glu Asn Gly Tyr Asn Thr Tyr Ala Ser Phe Asn Trp Gln His Asn Gly 100 105 110 agg cag atg tat gtg gca ttg aat gga aaa gga gct cea agg aga gga 384 Arg Gln Met Tyr Val Ala Leu Asn Gly Lys Gly Ala Pro Arg Arg Gly 115 120 125 cag aaa here cga agg aaa aac acc tet gct falls ttt ctt cea atg gtg 432 Gln Lys Thr Arg Arg Lys Asn Thr Ser Ala His Phe Leu Pro Met Val 130 135 140 gta falls tea tag 444 Val His Ser 145 < 210 > 68 < 211 > 147 < 212 > PRT < 213 > Homo sapiens < 400 > 68 Met Wing Gly Arg His Val Arg Ser Tyr Asn His Leu Gln Gly Asp Val 1 5 10 15 Arg Trp Arg Lys Leu Phe Ser Phe Thr Lys Tyr Phe Leu Lys He Glu 5 20 25 30 Lys Asn Gly Lys Val Ser Gly Thr Lys Lys Glu Asn Cys Pro Tyr Ser 35 40 45 He Leu Glu He Thr Ser Val Glu He Gly Val Val Wing Val Lys Wing 50 55 60 10 He Asn Ser Asn Tyr Tyr Leu Wing Met Asn Lys Lys Gly Lys Leu Tyr 65 70 75 80 Gly Ser Lys Glu Phe Asn Asn Asp Cys Lys Leu Lys Glu Arg He Glu 85 90 95 Glu Asn Gly Tyr Asn Thr Tyr Ala Ser Phe Asn Trp Gln His Asn Gly 15 100 105 110 Arg Gln Met Tyr Val Ala Leu Asn Gly Lys Gly Wing Pro Arg Arg Gly 115 120 125 Gln Lys Thr Arg Arg Lys Asn Thr Ser Wing His Phe Leu Pro Met Val 130 135 140 20 Val His Ser 145 < 210 > 69 25 < 211 > 402 < 212 > DNA < 213 > Homo sapiens < 220 > 30 < 221 > CDS < 222 > (1) .. (402) < 400 > 69 atg gtt cgt tgg cgt aaa ctg ttc tet ttc acc aaa tac ttc ctg aaa 48 35 Met Val Arg Trp Arg Lys Leu Phe Ser Phe Thr Lys Tyr Phe Leu Lys 1 5 10 15 ate gaa aaa aac ggt aaa gtt tet ggg acc aag aag gag aac tgc ceg 96 He Glu Lys Asn Gly Lys Val Ser Gly Thr Lys Lys Glu Asn Cys Pro 40 20 25 30 tac age ate ctg gag ata here tea gta gaa ate gga gtt gtt gcc gtc 144 Tyr Ser He Leu Glu He Thr Ser Val Glu He Gly Val Val Wing Val 35 40 45 45 aaa gcc att aac age aac tat tac tta gcc atg aac aag aag ggg aaa 192 Lys Wing He Asn Being Asn Tyr Tyr Leu Wing Met Asn Lys Lys Gly Lys 50 55 60 ctc tat ggc tea aaa gaa ttt aac aat gac tgt aag ctg aag gag agg 240 50 Leu Tyr Gly Ser Lys Glu Phe Asn Asn Asp Cys Lys Leu Lys Glu Arg 65 70 75 80 * £ &g & >&m >aga gga aat gga tac aat tat tat gca tea ttt aac tgg cag cat 288 He Glu Glu Asn Gly Tyr Asn Thr Tyr Ala Ser Phe Asn Trp Gln His 85 90 95 aat ggg agg ca atg tat gtg gca ttg aat gga aaa gga gct cea agg 336 Asn Gly Arg Gln Met Tyr Val Ala Leu Asn Gly Lys Gly Ala Pro Arg 100 105 110 aga gga cag aaa here cga agg aaa aac acc tet gct cae ttt ctt cea 384 Arg Gly Gln Lys Thr Arg Arg Lys Asn Thr Ser Wing His Phe Leu Pro 115 120 125 atg gtg gta falls tea tag 402 Met Val Val His Ser 130 < 210 > 70 < 211 > 133 < 212 > PRT < 213 > Homo sapiens < 400 > 70 Met Val Arg Trp Arg Lys Leu Phe Ser Phe Thr Lys Tyr Phe Leu Lys 1 5 10 15 He Glu Lys Asn Gly Lys Val Ser Gly Thr Lys Lys Glu Asn Cys Pro 20 25 30 Tyr Ser He Leu Glu He Thr Ser Val Glu He Gly Val Val Ala Ala 35 40 45 Lys Ala He Asn Ser Asn Tyr Tyr Leu Ala Met Asn Lys Lys Gly Lys 50 55 60 Leu Tyr Gly Ser Lys Glu Phe Asn Asn Asp Cys Lys Leu Lys Glu Arg 65 70 75 80 He Glu Glu Asn Gly Tyr Asn Thr Tyr Wing Being Phe Asn Trp Gln His 85 90 95 Asn Gly Arg Gln Met Tyr Val Wing Leu Asn Gly Lys Gly Wing Pro Arg 100 105 110 Arg Gly Gln Lys Thr Arg Arg Lys Asn Thr Ser Wing His Phe Leu Pro 115 120 125 Met Val Val His Ser 130 < 210 > 71 < 211 > 354 < 212 > DNA < 213 > Homo sapiens < 220 > < 221 > CDS < 222 > (1) . (354) < 400 > 71 atg gaa aaa aac ggt aaa gtt tet ggg acc aag aag gag aac tgc ceg 48 Met Glu Lys Asn Gly Lys Val Ser Gly Thr Lys Lys Glu Asn Cys Pro 1 5 10 15 tac age ate ctg gag ata here tea gta gaa ate gga gtt gtt gcc gtc 96 Tyr Ser He Leu Glu He Thr Ser Val Glu He Gly Val Val Wing Val 20 25 30 aaa gcc att aac age aac tat tac tta gcc atg aac aag aag ggg aaa 144 Lys Ala He Asn Ser Asn Tyr Tyr Leu Ala Met Asn Lys Lys Gly Lys 35 40 45 ctc tat ggc tea aaa gaa ttt aac aat gac tgt aag ctg aag gag agg 192 Leu Tyr Gly Ser Lys Glu Phe Asn Asn Asp Cys Lys Leu Lys Glu Arg 50 55 60 ata gag gaa aat gga tac aat acc tat gca tea ttt aac tgg cag cat 240 He Glu Glu Asn Gly Tyr Asn Thr Tyr Wing Ser Phe Asn Trp Gln His 65 70 75 80 aat ggg agg caa atg tat gtg gca ttg aat gga aaa gga gct cea agg 288 Asn Gly Arg Gln Met Tyr Val Wing Leu Asn Gly Lys Gly Wing Pro Arg 85 90 95 aga gga cag aaa ac cga agg aaa aac acc tet gct falls ttt ctt cea 336 Arg Gly Gln Lys Thr Arg Arg Lys Asn Thr Ser Wing His Phe Leu Pro 100 105 110 atg gtg gta falls tea tag 354 Met Val Val His Ser 115 < 210 > 72 < 211 > 117 < 212 > PRT < 213 > Homo sapiens < 400 > 72 Met Glu Lys Asn Gly Lys Val Ser Gly Thr Lys Lys Glu Asn Cys Pro 1 5 10 15 Tyr Ser He Leu Glu He Thr Ser Val Glu He Gly Val Val Wing Val 20 25 30 Lys Wing He Asn Ser Asn Tyr Tyr Leu Wing Met Asn Lys Lys Gly Lys 35 40 45 Leu Tyr Gly Ser Lys Glu Phe Asn Asn Asp Cys Lys Leu Lys Glu Arg 50 55 60 He Glu Glu Asn Gly Tyr Asn Thr Tyr Wing Ser Phe Asn Trp Gln His 65 70 75 80 Asn Gly Arg Gln Met Tyr Val Wing Ala Leu Asn Gly Lys Gly Wing Pro Arg 85 90 95 Arg Gly Gln Lys Thr Arg Arg Lys Asn Thr Ser Wing His Phe Leu Pro 100 105 110 Met Val Val His Ser 115 < 210 > 73 < 211 > 321 < 212 > DNA < 213 > Homo sapiens < 220 > < 221 > CDS < 222 > (1) .. (321) < 400 > 73 atg gag aac tgc ceg tac age ate ctg gag ata here tea gta gaa ate 48 Met Glu Asn Cys Pro Tyr Ser He Leu Glu He Thr Ser Val Glu He 1 5 10 15 gga gtt gtt gcc gtc aaa gcc att aac age aac tat tac tta gcc atg 96 Gly Val Val Ala Val Lys Ala He Asn Ser Asn Tyr Tyr Leu Ala Met 20 25 30 aac aag aag agg ggg aaa ctc tat ggc tea aaa gaa ttt aac aat gac tgt 144 Asn Lys Lys Gly Lys Leu Tyr Gly Ser Lys Glu Phe Asn Asn Asp Cys 35 40 45 aag ctg aag gag agg ata gag gaa aat gga tac aat tat tat gca tea 192 Lys Leu Lys Glu Arg He Glu Glu Asn Gly Tyr Asn Thr Tyr Ala Ser 50 55 60 ttt aac tgg cag cat aat ggg agg ca atg tat gtg gca ttg aat gga 240 Phe Asn Trp Gln His Asn Gly Arg Gln Met Tyr Val Ala Leu Asn Gly 65 70 75 80 aaa gga gct cea agg aga gga cag aaa here cga agg aaa aac acc tet 288 Lys Gly Wing Pro Arg Arg Gly Gln Lys Thr Arg Arg Lys Asn Thr Ser 85 90 95 gct falls ttt ctt cea atg gtg gta falls tea tag 321 Ala His Phe Leu Pro Met Val Val His Ser 100 105 < 210 > 74 < 211 > 106 < 212 > PRT < 213 > Homo sapiens < 400 > 74 Met Glu Asn Cys Pro Tyr Ser He Leu Glu He Thr Ser Val Glu He 1 5 10 15 Gly Val Val Ala Val Lys Ala He Asn Ser Asn Tyr Tyr Leu Ala Met 20 25 30 Asn Lys Lys Gly Lys Leu Tyr Gly Ser Lys Glu Phe Asn Asn Asp Cys 35 40 45 Lys Leu Lys Glu Arg He Glu Glu Asn Gly Tyr Asn Thr Tyr Wing Ser 50 55 60 Phe Asn Trp Gln His Asn Gly Arg Gln Met Tyr Val Ala Leu Asn Gly 65 70 75 80 Lys Gly Wing Pro Arg Arg Gly Gln Lys Thr Arg Arg Lys Asn Thr Ser 85 90 95 Wing His Phe Leu Pro Met Val Val His Ser 100 105 < 210 > 75 < 211 > 264 < 212 > DNA < 213 > Homo sapiens < 220 > < 221 > CDS < 222 > (1) .. (261) < 400 > 75 atg gtc aaa gcc att aac age aac tat tac tta gcc atg aac aag aag 48 Met Val Lys Ala He Asn Ser Asn Tyr Tyr Leu Ala Met Asn Lys Lys 1 5 10 15 ggg aaa ctc tat ggc tea aaa gaa ttt aac aat gac tgt aag ctg aag 96 Gly Lys Leu Tyr Gly Ser Lys Glu Phe Asn Asn Asp Cys Lys Leu Lys 20 25 30 gag agg ata gag gaa aat gga tac aat acc tat gca tea ttt aac tgg 144 Glu Arg He Glu Glu Asn Gly Tyr Asn Thr Tyr Ala Ser Phe Asn Trp 35 40 45 cag cat aat ggg agg caa atg tat gtg gca ttg aat gga aaa gga gct 192 Gln His Asn Gly Arg Gln Met Tyr Val Ala Leu Asn Gly Lys Gly Ala 50 55 60 cea agg aga gga cag aaa here cga agg aaa aac acc tet gct cae ttt 240 Pro Arg Arg Gly Gln Lys Thr Arg Arg Lys Asn Thr Ser Wing His Phe 65 70 75 80 ctt cea atg gtg gta falls tea tag 264 Leu Pro Met Val Val His Ser 85 < 210 > 76 < 211 > 87 < 212 > PRT < 213 > Homo sapiens < 400 > 76 Met Val Lys Ala He Asn Ser Asn Tyr Tyr Leu Wing Met Asn Lys Lys 1 5 10 15 Gly Lys Leu Tyr Gly Ser Lys Glu Phe Asn Asn Asp Cys Lys Leu Lys 20 25 30 Glu Arg He Glu Glu Asn Gly Tyr Asn Thr Tyr Wing Ser Phe Asn Trp 35 40 45 Gln His Asn Gly Arg Gln Met Tyr Val Wing Leu Asn Gly Lys Gly Wing 50 55 60 Pro Arg Arg Gly Gln Lys Thr Arg Arg Lys Asn Thr Ser Wing His Phe 65 70 75 80 Leu Pro Met Val Val His Ser 85 < 210 > 77 < 211 > 219 < 212 > DNA < 213 > Homo sapiens < 220 > < 221 > CDS < 222 > (1) .. (219) < 400 > 77 atg ggg aaa ctc tat ggc tea aaa gaa ttt aac aat gac tgt aag ctg 48 Met Gly Lys Leu Tyr Gly Ser Lys Glu Phe Asn Asn Asp Cys Lys Leu 1 5 10 15 aag gag agg ata gag gaa aat gga tac aat tat tat gca tea ttt aac 96 Lys Glu Arg He Glu Glu Asn Gly Tyr Asn Thr Tyr Wing Ser Phe Asn 20 25 30 tgg cag cat aat ggg agg caa atg tat gtg gca ttg aat gga aaa gga 144 Trp Gln His Asn Gly Arg Gln Met Tyr Val Wing Leu Asn Gly Lys Gly 35 40 45 gct cea agg aga gga cag aaa here cga agg aaa aac acc tet gct cae 192 Wing Pro Arg Arg Gly Gln Lys Thr Arg Arg Lys Asn Thr Ser Ala His 50 55 60 ttt ctt cea atg gtg gta falls tea tag 219 Phe Leu Pro Met Val Val His Ser 65 70 < 210 > 78 < 211 > 72 < 212 > PRT < 213 > Homo sapiens < 400 > 78 Met Gly Lys Leu Tyr Gly Ser Lys Glu Phe Asn Asn Asp Cys Lys Leu 1 5 10 15 Lys Glu Arg He Glu Glu Asn Gly Tyr Asn Thr Tyr Wing Ser Phe Asn 20 25 30 Trp Gln His Asn Gly Arg Gln Met Tyr Val Wing Leu Asn Gly Lys Gly 35 40 45 Wing Pro Arg Arg Gly Gln Lys Thr Arg Arg Lys Asn Thr Ser Wing His 50 55 60 Phe Leu Pro Met Val Val His Ser 65 70 < 210 > 79 < 211 > 357 < 212 > DNA < 213 > Homo sapiens < 220 > < 221 > CDS < 222 > (1) .. (357) < 400 > 79 atg acc tgc cag gct ctg ggt cag gac atg gtt tet ceg gaa gct acc 48 Met Thr Cys Gln Ala Leu Gly Gln Asp Met Val Ser Pro Glu Ala Thr 1 5 10 15 aac tet tcc tet tcc tet ttc tet tcc tc cct tcct gct gctt cgt falls 96 Asn Being Being Being Ser Phe Being Ser Pro Pro Being Wing Gly Arg His 20 25 30 gtt cgt tet tac aac falls ctg cag ggt gac gtt cgt tgg cgt aaa ctg 144 Val Arg Ser Tyr Asn His Leu Gln Gly Asp Val Arg Trp Arg Lys Leu 35 40 45 ttc tet ttc acc aaa tac ttc ctg aaa ate gaa aaa aac ggt aaa gtt 192 Phe Ser Phe Thr Lys Tyr Phe Leu Lys He Glu Lys Asn Gly Lys Val 50 55 60 tet ggg acc aag aag gag aac tgc ceg tac age ate ctg gag ata here 240 Be Gly Thr Lys Lys Glu Asn Cys Pro Tyr Be He Leu Glu He Thr 65 70 75 80 tea gta gaa ate gga gtt gtt gcc gtc aaa gcc att aac age aac tat 288 Ser Val Glu He Gly Val Val Ala Val Lys Ala He Asn Ser Asn Tyr 85 90 95 tac tta gcc atg aac aag aag ggg aaa ctc tat ggc tea aaa gaa ttt 336 Tyr Leu Wing Met Asn Lys Lys Gly Lys Leu Tyr Gly Ser Lys Glu Phe 100 105 110 aac aat gac tgt aag ctg aag 357 Asn Asn Asp Cys Lys Leu Lys 115 < 210 > 80 < 211 > 119 < 212 > PRT < 213 > Homo sapiens < 400 > 80 Met Thr Cys Gln Wing Leu Gly Gln Asp Met Val Ser Pro Glu Wing Thr 1 5 10 15 Asn Being Ser Being Being Phe Being Pro Pro Being Wing Gly Arg His 20 25 30 Val Arg Being Tyr Asn His Leu Gln Gly Asp Val Arg Trp Arg Lys Leu 35 40 45 Phe Ser Phe Thr Lys Tyr Phe Leu Lys He Glu Lys Asn Gly Lys Val 50 55 60 Ser Gly Thr Lys Lys Glu Asn Cys Pro Tyr Ser He Leu Glu He Thr 65 70 75 80 Ser Val Glu He Gly Val Val Wing Val Lys Wing He Asn Ser Asn Tyr 85 90 95 Tyr Leu Wing Met Asn Lys Lys Gly Lys Leu Tyr Gly Ser Lys Glu Phe 100 105 110 Asn Asn Asp Cys Lys Leu Lys 115 < 210 > 81 < 211 > 276 < 212 > DNA < 213 > Homo sapiens < 220 > < 221 > CDS < 222 > (1) .. (276) < 400 > 81 atg gct ggt cgt falls gtt cgt tet tac aac falls ctg cag ggt gac gtt 48 Met Ala Gly Arg His Val Arg Ser Tyr Asn His Leu Gln Gly Asp Val 1 5 10 15 cgt tgg cgt aaa ctg ttc tet ttc acc aaa tac ttc ctg aaa ate gaa 96 Arg Trp Arg Lys Leu Phe Ser Phe Thr Lys Tyr Phe Leu Lys He Glu 20 25 30 aaa aac ggt aaa gtt tet ggg acc aag aag gag aac tgc ceg tac age 144 Lys Asn Gly Lys Val Ser Gly Thr Lys Lys Glu Asn Cys Pro Tyr Ser 35 40 45 ate ctg gag ata here tea gta gaa ate gga gtt gtt gtt gcc gtc aaa gcc 192 He Leu Glu He Thr Ser Val Glu He Gly Val Val Ala Val Lys Wing 50 55 60 att aac age aac tat tac tta gcc atg aac aag aag agg ggg aaa ctc tat 240 He Asn Ser Asn Tyr Tyr Leu Ala Met Asn Lys Lys Gly Lys Leu Tyr 65 70 75 80 ggc tea aaa gaa ttt aac aat gac tgt aag ctg aag 276 Gly Ser Lys Glu Phe Asn Asn Asp Cys Lys Leu Lys 85 90 < 210 > 82 < 211 > 92 < 212 > PRT < 213 > Homo sapiens < 400 > 82 Met Ala Gly Arg His Val Arg Ser Tyr Asn His Leu Gln Gly Asp Val 1 5 10 15 Arg Trp Arg Lys Leu Phe Ser Phe Thr Lys Tyr Phe Leu Lys He Glu 20 25 30 Lys Asn Gly Lys Val Ser Gly Thr Lys Lys Glu Asn Cys Pro Tyr Ser 35 40 45 He Leu Glu He Thr Ser Val Glu He Gly Val Val Wing Val Lys Wing 50 55 60 He Asn Ser Asn Tyr Tyr Leu Wing Met Asn Lys Lys Gly Lys Leu Tyr 65 70 75 80 Gly Ser Lys Glu Phe Asn Asn Asp Cys Lys Leu Lys 85 90 < 210 > 83 < 211 > 525 < 212 > DNA < 213 > Homo sapiens < 400 > 83 atgacctctc aggetetggg tcaggacatg gtttctccgg aagetaccaa ctcttcctct 60 tcctctttct cttccccgtc ttccgctggt cgtcacgttc gttettacaa ccacctgcag 120 gttggcgtaa ggtgaegtte actgttctct ttcaccaaat acttcctgaa aategaaaaa 180 aacggtaaag tttctgggac caagaaggag aactctccgt acagcatcct ggagataaca 240 tcggagttgt tcagtagaaa tgccgtcaaa gccattaaca gcaactatta cttagccatg 300 aacaagaagg ggaaacteta tggctcaaaa gaatttaaca atgactgtaa gctgaaggag 360 aaaatggata aggatagagg caataectat gcatcattta actggcagca taatgggagg 420 caaatgtatg tggcattgaa tggaaaagga gctccaagga gaggacagaa aacacgaagg 480 aaaaacacct ctgctcactt tettecaatg gtggtacact catag 525 < 210 > 84 < 211 > 525 < 212 > DNA < 213 > Homo sapiens < 400 > 84 atgacctgcc aggetetggg tcaggacatg gtttctccgg aagetaccaa ctcttcctct 60 tcctctttct cttccccgtc ttccgctggt cgtcacgttc gttettacaa ccacctgcag 120 gttggcgtaa ggtgaegtte actgttctct ttcaccaaat acttcctgaa aategaaaaa 180 aacggtaaag tttctgggac caagaaggag aactctccgt acagcatcct ggagataaca 240 tcggagttgt tcagtagaaa tgccgtcaaa gccattaaca gcaactatta cttagccatg 300 aacaagaagg ggaaacteta tggctcaaaa gaatttaaca atgactgtaa gctgaaggag 360 aaaatggata aggatagagg caataectat gcatcattta actggcagca taatgggagg 420 caaatgtatg tggcattgaa tggaaaagga gctccaagga gaggacagaa aacacgaagg 480 aaaaacacct ctgctcactt tettecaatg gtggtacact catag 525 < 210 > 85 < 211 > 29 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Artificial Sequence Description: Primer < 400 > 85 ggaccctcat gacctctcag gctctgggt 29 < 210 > 86 < 211 > 21 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Artificial Sequence Description: Primer < 400 > 86 aaggagaact ctccgtacag c 21 < 210 > 87 < 211 > 21 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Artificial Sequence Description: Primer < 400 > 87 gctgtacggt ctgttctcct t 21 < 210 > 88 < 211 > 35 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Artificial Sequence Description: Primer < 400 > 88 ggaccctcat gacctgccag gctctgggtc aggac 35 < 210 > 89 < 211 > 37 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Artificial Sequence Description: Primer < 400 > 89 ctgcccaagc ttattatgag tgtaccacca ttggaag 37 < 210 > 90 < 211 > 33 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Artificial Sequence Description: Primer < 400 > 90 aaaggatcct gccaggctct gggtcaggac atg 33 < 210 > 91 < 211 > 32 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Artificial Sequence Description: Primer < 400 > 91 gcggcacatg tcttacaacc acctgcaggg tg 32 < 210 > 92 < 211 > 28 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Artificial Sequence Description: Primer < 400 > 92 gggcccaagc ttatgagtgt accaccat 28 < 210 > 93 < 211 > 36 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Artificial Sequence Description: Primer < 400 > 93 ccggcggatc ccatatgtct tacaaccacc tgcagg 36 < 210 > 94 < 211 > 35 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Artificial Sequence Description: Primer < 400 > 94 ccggcggtac cttattatga gtgtaccacc attgg 35 < 210 > 95 < 211 > 426 < 212 > DNA < 213 > Homo sapiens < 400 > 95 accacctgca atgtcttaca gggtgacgtt cgttggcgta aactgttetc tttcaccaaa 60 aaatcgaaaa tacttcctga gtttetggga aaaeggtaaa ccaagaagga gaactgcccg 120 tacagcatcc tggagataac atcagtagaa atcggagttg ttgccgtcaa agecattaac 180 agcaactatt acttagccat gaacaagaag gggaaactct atggctcaaa agaatttaac 240 agctgaagga aatgactgta gaggatagag gaaaatggat acaataecta tgcatcattt 300 aactggcagc ataatgggag gcaaatgtat gtggcattga atggaaaagg agetecaagg 360 agaggacaga aaacacgaag gaaaaacacc tctgctcact ttcttccaat ggtggtacac 420 tcataa 426 < 210 > 96 < 211 > 141 < 212 > PRT < 213 > Homo sapiens < 400 > 96 Met Ser Tyr Asn His Leu Gln Gly Asp Val Arg Trp Arg Lys Leu Phe 1 5 10 15 Ser Phe Thr Lys Tyr Phe Leu Lys He Glu Lys Asn Gly Lys Val Ser 20 25 30 Gly Thr Lys Lys Glu Asn Cys Pro Tyr Ser He Leu Glu He Thr Ser 35 40 45 Val Glu He Gly Val Val Ala Val Lys Ala He Asn Ser Asn Tyr Tyr 50 55 60 Leu Ala Met Asn Lys Lys Gly Lys Leu Tyr Gly Ser Lys Glu Phe Asn 65 70 75 80 Asn Asp Cys Lys Leu Lys Glu Arg He Glu Glu Asn Gly Tyr Asn Thr 85 90 95 Tyr Wing Ser Phe Asn Trp Gln His Asn Gly Arg Gln Met Tyr Val Wing 100 105 110 Leu Asn Gly Lys Gly Wing Pro Arg Arg Gly Gln Lys Thr Arg Arg Lys 115 120 125 Asn Thr Ser Wing His Phe Leu Pro Met Val Val His Ser 130 135 140 < 210 > 97 < 211 > 20 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: Oligonucleotide < 400 > 97 caaccacctg cagggtgacg 20 < 210 > 98 < 211 > 78 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: oligonucleotide < 400 > 98 aacggtcgac aaatgtatgt ggcactgaac ggtaaaggtg ctccacgtcg tggtcagaaa 60 acccgtcgta aaaacacc 78 < 210 > 99 < 211 > 76 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: oligonucleotide < 400 > 99 gggcccaagc ttaagagtgt accaccattg gcagaaagtg ageagaggtg tttttacgac 60 gggttttctg accacg 76 < 210 > 100 < 211 > 23 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: oligonucleotide < 400 > 100 gccacataca tttgtcgacc gtt 23 < 210 > 101 < 211 > 19 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: oligonucleotide < 400 > 101 gggcccaagc ttaagagtg 19 < 210 > 102 < 211 > 23 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: oligonucleotide < 400 > 102 gccacataca tttgtcgacc gtt 23 < 210 > 103 < 211 > 90 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: oligonucleotide < 400 > 103 ctgcagggtg acgttcgttg gcgtaaactg ttctccttca ccaaatactt cctgaaaatc 60 gaaaaaaacg gtaaagtttc tggtaccaag 90 < 210 > 104 < 211 > 90 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: oligonucleotide < 400 > 104 agctttaaca gcaacaacac cgatttcaac ggaggtgatt tccaggatgg agtacgggca 60 gttttctttc ttggtaccag aaactttacc 90 < 210 > 105 < 211 > 90 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: oligonucleotide < 400 > 105 ggtgttgttg ctgttaaage tatcaactcc aactactacc tggetatgaa caagaaaggt 60 aaactgtacg gttccaaaga atttaacaac 90 < 210 > 106 < 211 > 100 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of Artificial Sequence: oligonucleotide < 400 > 106 gtcgaccgtt gtgctgccag ttgaaggaag cgtaggtgtt gtaaccgttt tettegatac 60 gttctttcag tttacagtcg ttgttaaatt ctttggaacc 100 < 210 > 107 < 211 > 25 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: oligonucleotide < 400 > 107 gcggcgtcga ccgttgtgct gccag 25 < 210 > 108 < 211 > 26 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: oligonucleotide < 400 > 108 gcggcctgca gggtgacgtt cgttgg 26 < 210 > 109 < 211 > 36 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: oligonucleotide < 400 > 109 ccggcggatc ccatatgtct tacaaccacc tgcagg 36 < 210 > 110 < 211 > 34 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence; oligonucleotide < 400 > 110 cgcgcgatat cttattaaga gtgtaccacc attg 34 < 210 > 111 < 211 > 426 < 212 > DNA < 213 > Homo sapiens < 400 > 111 accacctgca atgtcttaca gggtgacgtt cgttggcgta aactgttetc cttcaccaaa 60 aaatcgaaaa tacttcctga aaaeggtaaa gtttctggta ccaagaaaga aaactgcccg 120 tactccatcc tggaaatcac ctccgttgaa atcggtgttg ttgctgttaa agetatcaac 180 tccaactact acctggctat gaacaagaaa ggtaaactgt acggttccaa agaatttaac 240 aacgactgta aactgaaaga gaaaacggtt acgtatcgaa acaacaccta cgcttccttc 300 aactggcagc acaacggtcg acaaatgtat gtggcactga acggtaaagg tgctccacgt 360 cgtggtcaga aaacccgtcg taaaaacacc tctgctcact ttctgccaat ggtggtacac 420 tcttaa 426 < 210 > 112 < 211 > 141 < 212 > PRT < 213 > Homo sapiens < 400 > 112 Met Ser Tyr Asn His Leu Gln Gly Asp Val Arg Trp Arg Lys Leu Phe 1 5 10 15 Ser Phe Thr Lys Tyr Phe Leu Lys He Glu Lys Asn Gly Lys Val Ser 20 25 30 Gly Thr Lys Lys Glu Asn Cys Pro Tyr Ser He Leu Glu He Thr Ser 35 40 45 Val Glu He Gly Val Val Ala Val Lys Ala He Asn Ser Asn Tyr Tyr 50 55 60 Leu Ala Met Asn Lys Lys Gly Lys Leu Tyr Gly Ser Lys Glu Phe Asn 65 70 75 80 Asn Asp Cys Lys Leu Lys Glu Arg He Glu Glu Asn Gly Tyr Asn Thr 85 90 95 Tyr Wing Ser Phe Asn Trp Gln His Asn Gly Arg Gln Met Tyr Val Wing 100 105 110 Leu Asn Gly Lys Gly Wing Pro Arg Arg Gly Gln Lys Thr Arg Arg Lys 115 120 125 Asn Thr Ser Wing His Phe Leu Pro Met Val Val His Ser 130 135 140 < 210 > 113 < 211 > 28 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: oligonucleotide < 400 > 113 cgcggccatg gctctgggtc aggacatg 28 < 210 > 114 < 211 > 28 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: oligonucleotide < 400 > 114 gggcccaagc ttatgagtgt accaccat 28 < 210 > 115 < 211 > 516 < 212 > DNA < 213 > Homo sapiens < 400 > 115 atggctctgg gtcaagatat ggtttctccg gaagetacca actcttcctc ttcctctttc 60 tcttccccgt cttccgctgg tcgtcacgtt cgttcttaca accacctgca gggtgacgtt 120 cgttggcgta aactgttetc tacttcctga tttcaccaaa aaatcgaaaa aaaeggtaaa 180 gtttetggga ccaagaagga gaactgcccg tacagcatcc tggagataac atcagtagaa 240 atcggagttg ttgccgtcaa agecattaac ageaactatt aettagecat gaacaagaag 300 gggaaactct atggctcaaa agaatttaac aatgactgta agctgaagga gaggatagag 360 gaaaatggat acaataecta tgcatcattt aactggcagc ataatgggag gcaaatgtat 420 gtggcattga atggaaaagg agetecaagg agaggacaga aaacacgaag gaaaaacacc 480 tctgctcact ttcttccaat ggtggtacac tcataa 516 < 210 > 116 < 211 > 171 < 212 > PRT < 213 > Homo sapiens < 400 > 116 Met Ala Leu Gly Gln Asp Met Val Ser Pro Glu Ala Thr Asn Ser Ser 1 5 10 15 Ser Ser Phe Ser Ser Pro Ser Ser Ala Gly Arg His Val Arg Ser 20 25 30 Tyr Asn His Leu Gln Gly Asp Val Arg Trp Arg Lys Leu Phe Ser Phe 35 40 45 Thr Lys Tyr Phe Leu Lys He Glu Lys Asn Gly Lys Val Ser Gly Thr 50 55 60 Lys Lys Glu Asn Cys Pro Tyr Ser He Leu Glu He Thr Ser Val Glu 65 70 75 80 He Gly Val Val Ala Val Lys Ala He Asn Ser Asn Tyr Tyr Leu Wing 85 90 95 Met Asn Lys Lys Gly Lys Leu Tyr Gly Ser Lys Glu Phe Asn Asn Asp 100 105 110 Cys Lys Leu Lys Glu Arg He Glu Glu Asn Gly Tyr Asn Thr Tyr Ala 115 120 125 Ser Phe Asn Trp Gln His Asn Gly Arg Gln Met Tyr Val Wing Leu Asn 130 135 140 Gly Lys Gly Wing Pro Arg Arg Gly Gln Lys Thr Arg Arg Lys Asn Thr 145 150 155 160 Ser Ala His Phe Leu Pro Met Val Val His Ser 165 170 < 210 > 117 < 211 > 32 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Artificial Sequence Description: Primer < 400 > 117 gcggcacatg tcttacaacc acctgcaggg tg 32 < 210 > 118 < 211 > 75 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Artificial Sequence Description: Primer < 400 > 118 ctgcccaagc ttttatgagt gtaccaccat tggaagaaag tgageagagg tgtttttttc 60 tcgtgttttc tgtcc 75 < 210 > 119 < 211 > 426 < 212 > DNA < 213 > Homo sapiens < 400 > 119 accacctgca atgtcttaca gggtgacgtt cgttggcgta aactgttetc tttcaccaaa 60 aaatcgaaaa tacttcctga aaaeggtaaa gtttetggga ccaagaagga gaactgcccg 120 tacagcatcc tggagataac atcagtagaa atcggagttg ttgccgtcaa agecattaac 180 ageaactatt aettagecat gaacaagaag gggaaactet atggctcaaa agaatttaac 240 agctgaagga aatgactgta gaggatagag gaaaatggat acaataecta tgcatcattt 300 aactggcagc ataatgggag gcaaatgtat gtggcattga atggaaaagg agetecaagg 360 aaacaegaga agaggacaga aaaaaacacc tctgctcact ttcttccaat ggtggtacac 420 tcatag 426 < 210 > 120 < 211 > 141 < 212 > PRT < 213 > Homo sapiens < 400 > 120 Met Ser Tyr Asn His Leu Gln Gly Asp Val Arg Trp Arg Lys Leu Phe 1 5 10 15 Ser Phe Thr Lys Tyr Phe Leu Lys He Glu Lys Asn Gly Lys Val Ser 20 25 30 Gly Thr Lys Lys Glu Asn Cys Pro Tyr Ser He Leu Glu He Thr Ser 35 40 45 Val Glu He Gly Val Val Ala Val Lys Ala He Asn Ser Asn Tyr Tyr 50 55 60 Leu Ala Met Asn Lys Lys Gly Lys Leu Tyr Gly Ser Lys Glu Phe Asn 65 70 75 80 Asn Asp Cys Lys Leu Lys Glu Arg He Glu Glu Asn Gly Tyr Asn Thr 85 90 95 Tyr Wing Ser Phe Asn Trp Gln His Asn Gly Arg Gln Met Tyr Val Wing 100 105 110 Leu Asn Gly Lys Gly Wing Pro Arg Arg Gly Gln Lys Thr Arg Glu Lys 115 120 125 Asn Thr Ser Wing His Phe Leu Pro Met Val Val His Ser 130 135 140 < 210 > 121 < 211 > 32 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Artificial Sequence Description: Primer < 400 > 121 gcggcacatg tcttacaacc acctgcaggg tg 32 < 210 > 122 < 211 > 75 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Artificial Sequence Description: Primer < 400 > 122 ctgcccaagc ttttatgagt gtaccaccat tggaagaaag tgageagagg tgtttttctg 60 tcgtgttttc tgtcc 75 < 210 > 123 < 211 > 426 < 212 > DNA < 213 > Homo sapiens < 400 > 123 accacctgca atgtcttaca gggtgacgtt cgttggcgta aactgttetc tttcaccaaa 60 aaatcgaaaa tacttcctga gtttetggga aaaeggtaaa ccaagaagga gaactgcccg 120 tacagcatcc tggagataac atcagtagaa atcggagttg ttgccgtcaa agecattaac 180 ageaactatt aettagecat gaacaagaag gggaaactet atggetcaaa agaatttaac 240 agctgaagga aatgactgta gaggatagag gaaaatggat acaataecta tgcatcattt 300 aactggcagc ataatgggag gcaaatgtat gtggcattga atggaaaagg agetecaagg 360 aaacaegaca agaggacaga gaaaaacacc tctgctcact ttcttccaat ggtggtacac 420 tcatag 426 < 210 > 124 < 211 > 141 < 212 > PRT < 213 > Homo sapiens < 400 > 124 Met Ser Tyr Asn His Leu Gln Gly Asp Val Arg Trp Arg Lys Leu Phe 1 5 10 15 Ser Phe Thr Lys Tyr Phe Leu Lys He Glu Lys Asn Gly Lys Val Ser 20 25 30 Gly Thr Lys Lys Glu Asn Cys Pro Tyr Ser He Leu Glu He Thr Ser 35 40 45 Val Glu He Gly Val Val Ala Val Lys Ala He Asn Ser Asn Tyr Tyr 50 55 60 Leu Ala Met Asn Lys Lys Gly Lys Leu Tyr Gly Ser Lys Glu Phe Asn 65 70 75 80 Asn Asp Cys Lys Leu Lys Glu Arg He Glu Glu Asn Gly Tyr Asn Thr 85 90 95 Tyr Wing Ser Phe Asn Trp Gln His Asn Gly Arg Gln Met Tyr Val Wing 100 105 110 Leu Asn Gly Lys Gly Wing Pro Arg Arg Gly Gln Lys Thr Arg Gln Lys 115 120 125 Asn Thr Ser Wing His Phe Leu Pro Met Val Val His Ser 130 135 140 < 210 > 125 < 211 > 32 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Artificial Sequence Description: Primer < 400 > 125 gcggcacatg tcttacaacc acctgcaggg tg 32 < 210 > 126 < 211 > 84 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Artificial Sequence Description: Primer < 400 > 126 ctgcccaagc ttttatgagt gtaccaccat tggaagaaag tgageagagg tgtttttcct 60 tcgtgtttcc tgtcctctcc ttgg 84 < 210 > 127 < 211 > 426 < 212 > DNA < 213 > Homo sapiens < 400 > 127 accacctgca atgtcttaca gggtgacgtt cgttggcgta aactgttetc tttcaccaaa 60 aaatcgaaaa tacttcctga aaaeggtaaa gtttetggga ccaagaagga gaactgcccg 120 tacagcatcc tggagataac atcagtagaa atcggagttg ttgccgtcaa agecattaac 180 ageaactatt aettagecat gaacaagaag gggaaactet atggetcaaa agaatttaac 240 agctgaagga aatgactgta gaggatagag gaaaatggat acaataecta tgcatcattt 300 aactggcagc ataatgggag gcaaatgtat gtggcattga atggaaaagg agetecaagg 360 agaggacagg aaacacgaag gaaaaacacc tctgctcact ttcttccaat ggtggtacac 420 tcatag 426 < 210 > 128 < 211 > 141 < 212 > PRT < 213 > Homo sapiens < 400 > 128 Met Ser Tyr Asn His Leu Gln Gly Asp Val Arg Trp Arg Lys Leu Phe 1 5 10 15 Ser Phe Thr Lys Tyr Phe Leu Lys He Glu Lys Asn Gly Lys Val Ser 20 25 30 Gly Thr Lys Lys Glu Asn Cys Pro Tyr Ser He Leu Glu He Thr Ser 35 40 45 Val Glu He Gly Val Val Ala Val Lys Ala He Asn Ser Asn Tyr Tyr 50 55 60 Leu Ala Met Asn Lys Lys Gly Lys Leu Tyr Gly Ser Lys Glu Phe Asn 65 70 75 80 Asn Asp Cys Lys Leu Lys Glu Arg He Glu Glu Asn Gly Tyr Asn Thr 85 90 95 Tyr Wing Ser Phe Asn Trp Gln His Asn Gly Arg Gln Met Tyr Val Wing 100 105 110 Leu Asn Gly Lys Gly Wing Pro Arg Arg Gly Gln Glu Thr Arg Arg Lys 115 120 125 Asn Thr Ser Wing His Phe Leu Pro Met Val Val His Ser 130 135 140 < 210 > 129 < 211 > 32 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Artificial Sequence Description: Primer < 400 > 129 gcggcacatg tcttacaacc acctgcaggg tg 32 < 210 > 130 < 211 > 84 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Artificial Sequence Description: Primer < 400 > 130 ctgcccaagc ttttatgagt gtaccaccat tggaagaaag tgageagagg tgtttttcct 60 tcgtgtctgc tgtcctctcc ttgg 84 < 210 > 131 < 211 > 426 < 212 > DNA < 213 > Homo sapiens < 400 > 131 accacctgca atgtcttaca gggtgacgtt cgttggcgta aactgttetc tttcaccaaa 60 aaatcgaaaa tacttcctga aaaeggtaaa gtttetggga ccaagaagga gaactgcccg 120 tacagcatcc tggagataac atcagtagaa atcggagttg ttgccgtcaa agecattaac 180 ageaactatt aettagecat gaacaagaag gggaaactet atggetcaaa agaatttaac 240 agctgaagga aatgactgta gaggatagag gaaaatggat acaataecta tgcatcattt 300 aactggcagc ataatgggag gcaaatgtat gtggcattga atggaaaagg agetecaagg 360 agaggacagc agacaegaag gaaaaacacc tctgctcact ttcttccaat ggtggtacac 420 tcatag 426 < 210 > 132 < 211 > 141 < 212 > PRT < 213 > Homo sapiens < 400 > 132 Met Ser Tyr Asn His Leu Gln Gly Asp Val Arg Trp Arg Lys Leu Phe 1 5 10 15 Ser Phe Thr Lys Tyr Phe Leu Lys He Glu Lys Asn Gly Lys Val Ser 20 25 30 Gly Thr Lys Lys Glu Asn Cys Pro Tyr Ser He Leu Glu He Thr Ser 35 40 45 Val Glu He Gly Val Val Ala Val Lys Ala He Asn Ser Asn Tyr Tyr 50 55 60 Leu Ala Met Asn Lys Lys Gly Lys Leu Tyr Gly Ser Lys Glu Phe Asn 65 70 75 80 Asn Asp Cys Lys Leu Lys Glu Arg He Glu Glu Asn Gly Tyr Asn Thr 85 90 95 Tyr Wing Ser Phe Asn Trp Gln His Asn Gly Arg Gln Met Tyr Val Wing 100 105 110 Leu Asn Gly Lys Gly Wing Pro Arg Arg Gly Gln Gln Thr Arg Arg Lys 115 120 125 Asn Thr Ser Wing His Phe Leu Pro Met Val Val His Ser 130 135 140 < 210 > 133 < 211 > 32 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Artificial Sequence Description: Primer < 400 > 133 gcggcacatg tcttacaacc acctgcaggg tg 32 < 210 > 134 < 211 > 93 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Artificial Sequence Description: Primer < 400 > 134 ctgcccaagc ttttatgagt gtaccaccat tggaagaaag tgageagagg tgtttttcct 60 tcgtgttttc tgtccttccc ttggagctcc ttt 93 < 210 > 135 < 211 > 426 < 212 > DNA < 213 > Homo sapiens < 400 > 135 accacctgca atgtcttaca gggtgacgtt cgttggcgta aactgttetc tttcaccaaa 60 aaatcgaaaa tacttcctga gtttetggga aaaeggtaaa ccaagaagga gaactgcccg 120 tacagcatcc tggagataac atcagtagaa atcggagttg ttgccgtcaa agecattaac 180 ageaactatt aettagecat gaacaagaag gggaaactet atggetcaaa agaatttaac 240 agctgaagga aatgactgta gaggatagag gaaaatggat acaataecta tgcatcattt 300 aactggcagc ataatgggag gcaaatgtat gtggcattga atggaaaagg agetecaagg 360 gaaggacaga aaacacgaag gaaaaacacc tctgctcact ttcttccaat ggtggtacac 420 tcatag 426 < 210 > 136 < 211 > 140 < 212 > PRT < 213 > Homo sapiens < 400 > 136 Met Tyr Asn His Leu Gln Gly Asp Val Arg Trp Arg Lys Leu Phe Ser 1 5 10 15 Phe Thr Lys Tyr Phe Leu Lys He Glu Lys Asn Gly Lys Val Ser Gly 20 25 30 Thr Lys Lys Glu Asn Cys Pro Tyr Ser He Leu Glu He Thr Ser Val 35 40 45 Glu He Gly Val Val Ala Val Lys Ala He Asn Ser Asn Tyr Tyr Leu 50 55 60 Wing Met Asn Lys Lys Gly Lys Leu Tyr Gly Ser Lys Glu Phe Asn Asn 65 70 75 80 Asp Cys Lys Leu Lys Glu Arg He Glu Glu Asn Gly Tyr Asn Thr Tyr 85 90 95 Wing Ser Phe Asn Trp Gln His Asn Gly Arg Gln Met Tyr Val Wing Leu 100 105 110 Asn Gly Lys Gly Wing Pro Arg Glu Gly Gln Lys Thr Arg Arg Lys Asn 115 120 125 Thr Ser Wing His Phe Leu Pro Met Val Val His Ser 130 135 140 < 210 > 137 < 211 > 32 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Artificial Sequence Description: Primer < 400 > 137 gcggcacatg tcttacaacc acctgcaggg tg 32 < 210 > 138 < 211 > 93 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Artificial Sequence Description: Primer < 400 > 138 ctgcccaagc ttttatgagt gtaccaccat tggaagaaag tgageagagg tgtttttcct 60 tcgtgttttc tgtccctgcc ttggagctcc ttt 93 < 210 > 139 < 211 > 426 < 212 > DNA < 213 > Homo sapiens < 400 > 139 accacctgca atgtcttaca gggtgacgtt cgttggcgta aactgttetc tttcaccaaa 60 aaatcgaaaa tacttcctga gtttetggga aaaeggtaaa ccaagaagga gaactgcccg 120 tacagcatcc tggagataac atcagtagaa atcggagttg ttgccgtcaa agecattaac 180 ageaactatt aettagecat gaacaagaag gggaaactet atggetcaaa agaatttaac 240 agctgaagga aatgactgta gaggatagag gaaaatggat acaataecta tgcatcattt 300 aactggcagc ataatgggag gcaaatgtat gtggcattga atggaaaagg agetecaagg 360 cagggacaga aaacacgaag gaaaaacacc tctgctcact ttcttccaat ggtggtacac 420 tcatag 426 < 210 > 140 < 211 > 141 < 212 > PRT < 213 > Homo sapiens < 400 > 140 Met Ser Tyr Asn His Leu Gln Gly Asp Val Arg Trp Arg Lys Leu Phe 1 5 10 15 Ser Phe Thr Lys Tyr Phe Leu Lys He Glu Lys Asn Gly Lys Val Ser 20 25 30 Gly Thr Lys Lys Glu Asn Cys Pro Tyr Ser He Leu Glu He Thr Ser 35 40 45 Val Glu He Gly Val Val Ala Val Lys Ala He Asn Ser Asn Tyr Tyr 50 55 60 Leu Ala Met Asn Lys Lys Gly Lys Leu Tyr Gly Ser Lys Glu Phe Asn 65 70 75 80 Asn Asp Cys Lys Leu Lys Glu Arg He Glu Glu Asn Gly Tyr Asn Thr 85 90 95 Tyr Wing Ser Phe Asn Trp Gln His Asn Gly Arg Gln Met Tyr Val Wing 100 105 110 Leu Asn Gly Lys Gly Wing Pro Arg Gln Gly Gln Lys Thr Arg Arg Lys 115 120 125 Asn Thr Ser Wing His Phe Leu Pro Met Val Val His Ser 130 135 140 < 210 > 141 < 211 > 32 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: primer < 400 > 141 gcggcacatg tcttacaacc acctgcaggg tg 32 < 210 > 142 < 211 > 21 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Artificial Sequence Description: Primer < 400 > 142 10 ttgaatggag aaggagctcc a 21 < 210 > 143 < 211 > 21 15 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Artificial Sequence Description: Primer 20 < 400 > 143 tggagctcct tctccattca to 21 25 < 210 > 144 < 211 > 33 < 212 > DNA < 213 > Artificial Sequence 30 < 220 > < 223 > Artificial Sequence Description: Primer < 400 > 144 ctgcccaagc ttttatgagt gtaccaccat tgg 33 35 < 210 > 145 < 211 > 426 < 212 > DNA 40 < 213 > Homo sapiens < 400 > 145 atgtcttaca accacctgca gggtgacgtt cgttggcgta aactgttetc tttcaccaaa 60 tacttcctga aaatcgaaaa aaaeggtaaa gtttetggga ccaagaagga gaactgcccg 120 45 tacagcatcc tggagataac atcagtagaa atcggagttg ttgccgtcaa agecattaac 180 ageaactatt aettagecat gaacaagaag gggaaactet atggetcaaa agaatttaac 240 agctgaagga aatgactgta gaggatagag gaaaatggat acaataecta tgcatcattt 300 aactggcagc ataatgggag gcaaatgtat gtggcattga atggagaagg agetecaagg 360 agaggacaga aaacacgaag gaaaaacacc tctgctcact ttcttccaat ggtggtacac 420 50 tcatag 426 < 210 > 146 < 211 > 141 < 212 > PRT < 213 > Homo sapiens ^^^ s ^^^^^^^^^ 2 ^ < 400 > 146 Met Ser Tyr Asn His Leu Gln Gly Asp Val Arg Trp Arg Lys Leu Phe 1 5 10 15 Ser Phe Thr Lys Tyr Phe Leu Lys He Glu Lys Asn Gly Lys Val Ser 20 25 30 Gly Thr Lys Lys Glu Asn Cys Pro Tyr Ser He Leu Glu He Thr Ser 35 40 45 Val Glu He Gly Val Val Ala Val Lys Ala He Asn Ser Asn Tyr Tyr 50 55 60 Leu Ala Met Asn Lys Lys Gly Lys Leu Tyr Gly Ser Lys Glu Phe Asn 65 70 75 80 Asn Asp Cys Lys Leu Lys Glu Arg He Glu Glu Asn Gly Tyr Asn Thr 85 90 95 Tyr Wing Ser Phe Asn Trp Gln His Asn Gly Arg Gln Met Tyr Val Wing 100 105 110 Leu Asn Gly Glu Gly Wing Pro Arg Arg Gly Gln Lys Thr Arg Arg Lys 115 120 125 Asn Thr Ser Wing His Phe Leu Pro Met Val Val His Ser 130 135 140 < 210 > 147 < 211 > 3974 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: vector pHE4-5 < 400 > 147 ggtacctaag tgagtagggc gtcegatega cggacgcctt ttttttgaat tcgtaatcat 60 ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag 120 ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagetaaetc acattaattg 180 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa 240 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca 300 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg 360 atecacagaa taatacggtt tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc 420 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc 480 gcatcacaaa cccctgacga aategaeget caagtcagag gtggcgaaac ccgacaggac 540 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc 600 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata 660 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc 720 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca 780 acccggtaag acaegaetta tcgccactgg cagcagccac tggtaacagg attagcagag 840 cgaggtatgt aggcgg TGCT acagagttct tgaagtggtg gcctaactac ggetacacta 900 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg 960 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc 1020 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt 1080 gtggaacgaa ctgacgctca aactcacgtt aagggatttt ttatcgtcga ggtcatgaga 1140 gcgaaggcga caattcgcgc agcggcatgc atttacgttg atggtgcaaa acaccatcga 1200 acctttcgcg gtatggcatg atagcgcccg gaagagagtc aattcagggt ggtgaatgtg 1260 cgttatacga aaaccagtaa tgtcgcagag tatgccggtg tctcttatca gaccgtttcc 1320 cgcgtggtga accaggccag ccacgtttct gcgaaaacgc gggaaaaagt ggaagcggcg 1380 atggcggagc tgaattacat tcccaaccgc gtggcacaac aactggcggg caaacagtcg 1440 ttgctgattg gcgttgccac ctccagtctg gccctgcacg cgccgtcgca aattgtcgcg 1500 gcgattaaat ctcgcgccga tcaactgggt gccagcgtgg tggtgtcgat ggtagaacga 1560 agcggcgtcg aagcctgtaa agcggcggtg cacaatcttc tcgcgcaacg cgtcagtggg 1620 ctgatcatta actatccgct ggatgaccag gatgccattg ctgtggaagc tgcctgcact 1680 aatgttccgg cgttatttct tgatgtctct gaccagacac ccatcaacag tattattttc 1740 tcccatgaag acggtacgcg actgggcgtg gagcatctgg tcgcattggg tcaccagcaa 1800 atcgcgctgt tagcgggccc attaagttct gtctcggcgc gtctgcgtct ggctggctgg 1860 cataaatatc tcactcgcaa tcaaattcag ccgatagcgg aacgggaagg cgactggagt 1920 gccatgtccg gttttcaaca aaccatgcaa atgctgaatg agggcatcgt tcccactgcg 1980 atgctggttg ccaacgatca gatggcgctg ggcgcaatgc gcgccattac cgagtccggg 2040 ctgcgcgttg gtgcggatat ctcggtagtg ggatacgacg ataccgaaga cagctcatgt 2100 tatatcccgc cgttaaccac catcaaacag gattttcgcc tgctggggca aaccagcgtg 2160 gaccgcttgc tgcaactctc tcagggccag gcggtgaagg gcaatcagct gttgcccgtc 2220 tcactggtga aaagaaaaac caccctggcg cccaatacgc aaaccgcctc tccccgcgcg 2280 cattaatgca ttggccgatt gctggcacga caggtttccc gactggaaag cgggcagtga 2340 g cgcaacgca gttagcgcga attaatgtaa attgtcgacc aaagcggcca tcgtgcctcc 2400 ccactcctgc agttcggggg catggatgcg cggatagccg ctgctggttt cctggatgcc 2460 gacggatttg cactgccggt agaactccgc gaggtcgtcc agcctcaggc agcagctgaa 2520 ccaactcgcg aggggatcga gcccggggtg ggcgaagaac tccagcatga gatccccgcg 2580 ctggaggatc atccagccgg cgtcccggaa aacgattccg aagcccaacc tttcatagaa 2640 ggcggcggtg gaatcgaaat ctcgtgatgg caggttgggc gtcgcttggt cggtcatttc 2700 gtcccgctca gaaccccaga gaagaactcg tcaagaaggc gatagaaggc gatgcgctgc 2760 gaatcgggag cggcgatacc gtaaagcacg aggaagcggt cagcccattc gccgccaagc 2820 tcttcagcaa tatcacgggt agccaacgct atgtcctgat agcggtccgc cacacccagc 2880 cggccacagt cgatgaatcc agaaaagcgg ccattttcca ccatgatatt cggcaagcag 2940 gcatcgccat gggtcacgac gagatcctcg ccgtcgggca tgcgcgcctt gagcctggcg 3000 aacagttcgg ctggcgcgag cccctgatgc tcttcgtcca gatcatcctg atcgacaaga 3060 ccggcttcca tccgagtacg tgctcgctcg atgcgatgtt tcgcttggtg gtcgaatggg 3120 caggtagccg gatcaagcgt atgcagccgc cgcattgcat cagccatgat ggatactttc 3180 tcggcag gag caaggtgaga tgacaggaga tcctgccccg gcacttcgcc caatagcagc 3240 cagtcccttc ccgcttcagt gacaacgtcg agcacagctg cgcaaggaac gcccgtcgtg 3300 gccagccacg atagccgcgc tgcctcgtcc tgcagttcat tcagggcacc ggacaggtcg 3360 gtcttgacaa aaagaaccgg gcgcccctgc gctgacagcc ggaacacggc ggcatcagag 3420 cagccgattg tctgttgtgc ccagtcatag ccgaatagcc tctccaccca agcggccgga 3480 gaacctgcgt gcaatccatc ttgttcaatc atgcgaaacg atcctcatcc tgtctcttga 3540 tcagatcttg atcccctgcg ccatcagatc cttggcggca agaaagccat ccagtttact 3600 ttgcagggct tcccaacctt accagagggc gccccagctg gcaattccgg ttcgcttgct 3660 gtccataaaa ccgcccagtc tagctatcgc catgtaagcc cactgcaagc tacctgcttt 3720 ctctttgcgc ttgcgttttc ccttgtccag atagcccagt agctgacatt catccggggt 3780 cagcaccgtt tctgcggact ggctttctac gtgttccgct tcctttagca gcccttgcgc 3840 cctgagtgct tgcggcagcg tgaagcttaa aaaactgcaa aaaatagttt gacttgtgag 3900 agcggataac aatttcacac ttaagatgta cggataacaa cccaattgtg attaaagagg TATG 3960 3974 <agaaattaca; 210 > 148 < 211 > 112 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: promoter sequence pHE4-5 < 400 > 148 aagcttaaaa aactgcaaaa aatagtttga cttgtgagcg gataacaatt aagatgtacc 60 caattgtgag cggataacaa tttcacacat taaagaggag aaattacata tg 112 < 210 > 149 < 211 > 106 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Artificial Sequence Description: Primer < 400 > 149 gagcgcggat ccgccaccat gaaggtctcc gtggctgccc tctcctgcct catgcttgtt 60 actgcccttg gatctcaggc cagctacaat caccttcaag gagatg 106 < 210 > 150 < 211 > 36 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Artificial Sequence Description: Primer < 400 > 150 gagcgcggat ccctatgagt gtaccaccat tggaag 36 < 210 > 151 < 211 > 32 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Artificial Sequence Description: Primer < 400 > 151 ccggccatat gcgtaaactg ttctctttca cc 32 < 210 > 152 < 211 > 35 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Artificial Sequence Description: Primer < 400 > 152 ccggcggtac cttattatga gtgtaccacc attgg 35 < 210 > 153 < 211 > 32 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Artificial Sequence Description: Primer < 400 > 153 gatcgccata tggctggtcg tcacgttcgt te 32 < 210 > 154 < 211 > 39 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Artificial Sequence Description: Primer < 400 > 154 gatcgcggta cettattatg agtgtaccac cattggaag 39 < 210 > 155 < 211 > 32 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Artificial Sequence Description: Primer < 400 > 155 gatcgccata tggctggtcg tcacgttcgt te 32 < 210 > 156 < 211 > 39 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Artificial Sequence Description: Primer < 400 > 156 gatcgcggta cettattatg agtgtaccac cattggaag 39 < 210 > 157 < 211 > 32 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Artificial Sequence Description: Primer < 400 > 157 gatcgccata tggctggtcg tcacgttcgt te 32 < 210 > 158 < 211 > 39 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Artificial Sequence Description: Primer < 400 > 158 gatcgcggta cettattatg agtgtaccac cattggaag 39 < 210 > 159 < 211 > 32 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Artificial Sequence Description: Primer < 400 > 159 gatcgccata tggctggtcg tcacgttcgt te 32 < 210 > 160 < 211 > 39 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Artificial Sequence Description: Primer < 400 > 160 gatcgcggta cettattatg agtgtaccac cattggaag 39 < 210 > 161 < 211 > 47 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Artificial Sequence Description: Primer < 400 > 161 gatcgcggat ccgccaccat gtggaaatgg atactgacac attgtgc 47 < 210 > 162 < 211 > 40 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Artificial Sequence Description: Primer < 400 > 162 gatcgctcta gattatgagt gtaccaccat tggaagaaag 40 < 210 > 163 < 211 > 47 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Artificial Sequence Description: Primer < 400 > 163 gatcgcggat ccgccaccat gtggaaatgg atactgacac attgtgc 47 < 210 > 164 < 211 > 40 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Artificial Sequence Description: Primer < 400 > 164 gatcgctcta gattatgagt gtaccaccat tggaagaaag 40 < 210 > 165 < 211 > 47 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Artificial Sequence Description: Primer < 400 > 165 gatcgcggat ccgccaccat gtggaaatgg atactgacac attgtgc 47 < 210 > 166 < 211 > 40 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Artificial Sequence Description: Primer < 400 > 166 gatcgctcta gattatgagt gtaccaccat tggaagaaag 40 < 210 > 167 < 211 > 47 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Artificial Sequence Description: Primer < 400 > 167 gatcgcggat ccgccaccat gtggaaatgg atactgacac attgtgc 47 < 210 > 168 < 211 > 40 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Artificial Sequence Description: Primer < 400 > 168 gatcgctcta gattatgagt gtaccaccat tggaagaaag 40 < 210 > 169 < 211 > 32 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Artificial Sequence Description: Primer < 400 > 169 gatcgccata tggctggtcg tcacgttcgt te 32 < 210 > 170 < 211 > 39 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Artificial Sequence Description: Primer < 400 > 170 gatcgcggta cettattatg agtgtaccac cattggaag 39 < 210 > 171 < 211 > 32 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Artificial Sequence Description: Primer < 400 > 171 10 gatcgccata tggctggtcg tcacgttcgt te 32 < 210 > 172 < 211 > 39 15 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Artificial Sequence Description: Primer 20 < 400 > 172 gatcgcggta cettattatg agtgtaccac cattggaag 39 25 < 210 > 173 < 211 > 456 < 212 > DNA < 213 > Escherichia coli 30 < 400 > 173 catatggctg gtcgtcacgt tcgttcttac aaccacctgc agggtgacgt tcgttggcgt 60 aaactgttct ctttcaccaa atacttcctg aaaatcgaaa aaaacggtaa agtttctggg 120 accaagaagg agaactgccc gtacageate ctggagataa catcagtaga aatcggagtt 180 aagccattaa gttgccgtca cagcaactat tacttagcca tgaacaagaa ggggaaactc 240 35 tatggctcaa aagaatttaa caatgactgt aagctgaagg agaggataga ggaaaatgga 300 tacaatacct atgeatcatt taactggcag cataatggga ggcaaatgta tgtggcattg 360 aatggaaaag gagetecaag gagaggacag aaaacacgaa ggaaaaacac ctctgctcac 420 tttettecaa tggtggtaca etcataataa ggtacc 456 40 < 210 > 174 < 211 > 48 < 212 > DNA < 213 > Artificial Sequence 45 < 220 > < 223 > Artificial Sequence Description: Primer < 400 > 174 50 gactacatat ggctggtcgt cacgttcgtt cttacaacca cctgcagg 48 < 210 > 175 < 211 > 47 • rp-ri-ifty --'- * • < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Artificial Sequence Description: Primer < 400 > 175 ctagtctcta gattattatg agtgtacaac catcggcagg aagtgag 47 < 210 > 176 < 211 > 447 < 212 > DNA < 213 > Escherichia coli < 400 > 176 atggctggtc gtcacgttcg ttcttacaac cacctgcagg gtgacgttcg ttggcgtaaa 60 tcaccaaata ctgttctctt cttcctgaaa atcgaaaaga acggtaaagt ttctggtacc 120 actgcccgta aagaaagaaa ctctatcctg gaaatcacct ccgttgaaat cggtgttgta 180 gccgttaaag ccatcaactc caactattac ctggccatga acaaaaaggg taaactgtac 240 ggctctaaag aattcaacaa cgactgcaaa ctgaaagaac gtatcgaaga gaacggttac 300 aacacctacg catccttcaa ctggcagcac aacggtcgtc agatgtacgt tgcactgaac 360 ggtaaaggcg ctccgcgtcg cggtcagaaa acccgtcgca aaaacacctc tgctcacttc 420 ctgccgatgg ttgtacactc ataataa 447

Claims (12)

CLAIMS Having described the invention as above, the content of the following claims is claimed as property:

1. A polynucleotide comprising a nucleotide sequence encoding a polypeptide, characterized in that the polypeptide is identical to a reference polypeptide of SEQ ID No. 2, except for one or more mutations selected from the group consisting of: R68G, R68S, R68A , R78A, R80A, K81A, K87A, K91A, K136A, K137A, K139A, K144A, K148E, K149E, K159A, K151A, K153A, K155A, R174A, K183A, K183Q, K183E, R187A, R188A, R188E, K191E, the positively charged residues between and including R68 to K91 replaced with alanine, positively charged residues between and including R68 to K91 replaced with neutral residues, and residues positively charged between and including R68 to K91 replaced with negatively charged residues.

2. The polynucleotide according to claim 1, characterized in that the reference polypeptide comprises amino acids 63 to 208, 69 to 208, 69 to 208, 77 to 208, 80 to 208 or 93 to 208 of SEQ ID No. 2 .

3. A vector, characterized in that it comprises the polynucleotide according to claim 1.

4. A host cell, characterized in that it comprises the polynucleotide according to claim 1.

5. A method for producing a polypeptide, characterized in that it comprises the culture of the cell host according to claim 4 under conditions such that the polypeptide is expressed, and the recovery of said polypeptide.

6. A polypeptide, characterized in that it is encoded by the polynucleotide according to claim 1.

7. A method for stimulating the proliferation of epithelial cells in a patient, characterized in that it comprises the administration to the patient of the polypeptide according to claim 6. The method according to claim 7, characterized in that the patient has a disorder selected from the group consisting of a wound, mucositis, ulcer, inflammatory bowel disease, liver disorder, lung damage, diabetes, oral injury, injury gastrointestinal, bowel toxicity, epidermolysis bullosa, skin graft, skin disorder, kidney failure, brain damage, breast tissue damage, urothelial damage, female reproductive tract disorder, intestinal fibrosis, proctitis, pulmonary fibrosis, pneumonitis, pleural retraction , hematopoietic syndrome, and myelotoxicity. 9. A method for treatment or prevention of ovarian injury, infertility, or fibrosis of the liver in a patient, characterized in that it comprises administering an effective amount of a polypeptide of SEQ ID No. 2 or an active fragment or variant thereof to said patient. 10. A method for the promotion of internal healing, the healing of the donor site, the healing of the internal surgical wound, or the healing of incision wounds made during cosmetic surgery in a patient, characterized the method because it comprises the administration of a effective amount of a polypeptide of SEQ ID No. 2 or an active fragment or variant thereof to the patient. 11. A method for producing a polypeptide, characterized in that it comprises: (a) the insertion of a polynucleotide encoding amino acids 63 to 208 into a vector; (b) transfection of said vector into a host cell; (c) culturing the host cell under conditions such that the polypeptide is expressed; (d) lysis of the cells in the presence of guanidine hydrochloride; and (e) recovering the polypeptide. 12. A polynucleotide, characterized in that it comprises nucleotides 4 to 444 of SEQ ID No. 173 or l to 441 of SEQ ID No. 176.