WO1999055865A1 - Polynucleotides isolated from skin cells and methods for their use - Google Patents

Polynucleotides isolated from skin cells and methods for their use Download PDF

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Publication number
WO1999055865A1
WO1999055865A1 PCT/NZ1999/000051 NZ9900051W WO9955865A1 WO 1999055865 A1 WO1999055865 A1 WO 1999055865A1 NZ 9900051 W NZ9900051 W NZ 9900051W WO 9955865 A1 WO9955865 A1 WO 9955865A1
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Prior art keywords
seq
sequences
polypeptide
amino acid
identity
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PCT/NZ1999/000051
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French (fr)
Inventor
Lorna Strachan
Matthew Sleeman
James Douglas Watson
Rene Onrust
Anand Kumble
James Greg Murison
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Genesis Research And Development Corporation Limited
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Priority claimed from US09/188,930 external-priority patent/US6150502A/en
Application filed by Genesis Research And Development Corporation Limited filed Critical Genesis Research And Development Corporation Limited
Priority to NZ507728A priority Critical patent/NZ507728A/en
Priority to AU39614/99A priority patent/AU758074B2/en
Priority to KR1020007011932A priority patent/KR20010074494A/en
Priority to EP99922665A priority patent/EP1073740A4/en
Priority to CA002327317A priority patent/CA2327317A1/en
Priority to JP2000546009A priority patent/JP2002512798A/en
Publication of WO1999055865A1 publication Critical patent/WO1999055865A1/en
Priority to US09/866,050 priority patent/US20030040471A1/en
Priority to US10/152,661 priority patent/US20030022835A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/485—Epidermal growth factor [EGF], i.e. urogastrone
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to polynucleotides encoding polypeptides, polypeptides expressed in skin cells, and their use in therapeutic methods.
  • the skin is the largest organ in the body and serves as a protective cover.
  • the loss of skin as occurs in a badly burned person, may lead to death owing to the absence of a barrier against infection by external microbial organisms, as well as loss of body temperature and body fluids.
  • Skin tissue is composed of several layers.
  • the outermost layer is the epidermis which is supported by a basement membrane and overlies the dermis. Beneath the dermis is loose connective tissue and fascia which cover muscles or bony tissue.
  • the skin is a self-renewing tissue in that cells are constantly being formed and shed.
  • the deepest cells of the epidermis are the basal cells, which are enriched in cells capable of replication. Such replicating cells are called progenitor or stem cells. Replicating cells in turn give rise to daughter cells called 'transit amplifying cells'. These cells undergo differentiation and maturation into keratinocytes (mature skin cells) as they move from the basal layer to the more superficial layers of the epidermis.
  • keratinocytes become cornified and are ultimately shed from the skin surface.
  • Other cells in the epidermis include melanocytes which synthesize melanin, the pigment responsible for protection against sunlight.
  • the Langerhans cell also resides in the epidermis and functions as a cell which processes foreign proteins for presentation to the immune system.
  • the dermis contains nerves, blood and lymphatic vessels, fibrous and fatty tissue.
  • fibroblasts Within the dermis are fibroblasts, macrophages and mast cells. Both the epidermis and dermis are penetrated by sweat, or sebaceous, glands and hair follicles. Each strand of hair is derived from a hair follicle. When hair is plucked out, the hair re-grows from epithelial cells directed by the dermal papillae ofthe hair follicle.
  • the stem cells proliferate and daughter keratinocytes migrate across the wound to reseal the tissues.
  • the skin cells therefore possess genes activated in response to trauma.
  • the products of these genes include several growth factors, such as epidermal growth factor, which mediate the proliferation of skin cells.
  • the genes that are activated in the skin, and the protein products of such genes, may be developed as agents for the treatment of skin wounds. Additional growth factors derived from skin cells may also influence growth of other cell types.
  • proteins derived from skin that regulate cellular growth may be developed as agents for the treatment of skin cancers. Skin derived proteins that regulate the production of melanin may be useful as agents which protect skin against unwanted effects of sunlight.
  • Keratinocytes are known to secrete cytokines and express various cell surface proteins. Cytokines and cell surface molecules are proteins which play an important role in the inflammatory response against infection and also in autoimmune diseases affecting the skin. Genes and their protein products that are expressed by skin cells may thus be developed into agents for the treatment of inflammatory disorders affecting the skin.
  • Hair is an important part of a person's individuality. Disorders of the skin may lead to hair loss. Alopecia areata is a disease characterized by the patchy loss of hair over the scalp. Total baldness is a side effect of drug treatment for cancer.
  • the growth and development of hair are mediated by the effects of genes expressed in skin and dermal papillae. Such genes and their protein products may be usefully developed into agents for the treatment of disorders ofthe hair follicle. New treatments are required to hasten the healing of skin wounds, to prevent the loss of hair, enhance the re-growth of hair or removal of hair, and to treat autoimmune and inflammatory skin diseases more effectively and without adverse effects. More effective treatments of skin cancers are also required. There thus remains a need in the art for the identification and isolation of genes encoding proteins expressed in the skin, for use in the development of therapeutic agents for the treatment of disorders including those associated with skin. Summary of the Invention
  • the present invention provides polypeptides expressed in skin cells, together with polynucleotides encoding such polypeptides, expression vectors and host cells comprising such polynucleotides, and methods for their use.
  • isolated polynucleotides comprise a DNA sequence selected from the group consisting of: (a) sequences recited in SEQ ID NO: 1-14, 45-48, 64-68, 77-89, 118, 119, 198-231, 239-249, 254-274, 349-372 and 399- 405; (b) complements ofthe sequences recited in SEQ ID NO: 1-14, 45-48, 64-68, 77-89, 118, 119, 198-231, 239-249, 254-274, 349-372 and 399-405; (c) reverse complements of the sequences recited in SEQ ID NO: 1-14, 45-48, 64-68, 77-89, 118, 119, 198-231, 239- 249, 254-274, 349-372 and 399-405; (d) reverse sequences of the sequences recited in SEQ ID NO: 1-14, 45-48, 64-68, 77-89, 118,
  • the present invention provides isolated polypeptides comprising an amino acid sequence selected from the group consistmg of: (a) sequences provided in SEQ ID NO: 120-197, 275-348, 373-398 and 406-409; and (b) sequences having at least 50%, 75% or 90%) identity to a sequence provided in SEQ ID NO: 120- 197, 275-348, 373-398 and 406-409, together with isolated polynucleotides encoding such polypeptides.
  • Isolated polypeptides which comprise at least a functional portion of a polypeptide comprising an amino acid sequence selected from the group consisting of: (a) sequences provided in SEQ ID NO: 120-197, 275-348, 373-398 and 406-409; and (b) sequences having 50%, 75% or 90% identity to a sequence of SEQ ID NO: 120-197, 275-348, 373-398 and 406-409 are also provided.
  • the present invention provides expression vectors comprising the above polynucleotides, together with host cells transformed with such vectors.
  • the present invention provides a method of stimulating keratinocyte growth and motility, inhibiting the growth of epithelial-derived cancer cells, inhibiting angiogenesis and vascularization of tumors, or modulating the growth of blood vessels in a subject, comprising administering to the subject a composition comprising an isolated polypeptide, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of: (a) sequences provided in SEQ ID NO: 187, 196, 342, 343, 395, 397, and 398; and (b) sequences having at least 50%, 75% or 90% identity to a sequence provided in SEQ ID NO: 187, 196, 342, 343, 395, 397, and 398.
  • Methods for modulating skin inflammation in a subject comprising administering to the subject a composition comprising an isolated polypeptide, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of: (a) sequences provided in SEQ ID NO: 338 and 347; and (b) sequences having at least 50%, 75% or 90%) identity to a sequence provided in SEQ ID NO: 338 and 347.
  • the present invention provides methods for stimulating the growth of epithelial cells in a subject.
  • Such methods comprise administering to the subject a composition comprising an isolated polypeptide including an amino acid sequence selected from the group consisting of: (a) sequences provided in SEQ ID NO: 129 and 348; and (b) sequences having at least 50%, 75% or 90% identity to a sequence provided in SEQ ID NO: 129 and 348.
  • methods for inhibiting the binding of HIV-1 to leukocytes, for the treatment of an inflammatory disease or for the treatment of cancer in a subject comprising administering to the subject a composition comprising an isolated polypeptide including an amino acid sequence selected from the group consisting of: (a) sequences provided in SEQ ID NO: 340, 344, 345 and 346; and (b) sequences having at least 50%, 75% or 90% identity to a sequence provided in SEQ ID NO: 340, 344, 345 and 346.
  • isolated polynucleotides and polypeptides of the present invention may be usefully employed in the preparation of therapeutic agents for the treatment of skin disorders.
  • Fig. 1 shows the results of a Northern analysis of the distribution of huTRl mRNA in human tissues. Key: He, Heart; Br, Brain; PI, Placenta; Lu, Lung; Li, Liver; SM, Skeletal muscle; Ki, Kidney; Sp, Spleen; Th, Thymus; Pr, Prostate; Ov, Ovary.
  • Fig. 2 shows the results of a MAP kinase assay of muTRla and huTRla.
  • MuTRla 500ng/ml
  • huTRla lOOng/ml
  • LPS 3pg/ml
  • Fig. 3 shows the stimulation of growth of neonatal foreskin keratinocytes by muTRla.
  • Fig. 4 shows the stimulation of growth of the transformed human keratinocyte cell line HaCaT by muTRla and huTRla.
  • Fig. 5 shows the inhibition of growth of the human epidermal carcinoma cell line A431 by muTRla and huTRla.
  • Fig. 6 shows the inhibition of IL-2 induced growth of concanavalin A-stimulated murine splenocytes by KS2a.
  • Fig. 7 shows the stimulation of growth of rat intestinal epithelial cells (IEC-18) by a combination of KS3a plus apo-transferrin.
  • Fig. 8 illustrates the oxidative burst effect of TR-1 (100 ng/ml), muKSl (100 ng/ml), SDFl ⁇ (100 ng/ml), and fMLP (10 ⁇ M) on human PBMC.
  • Figure 9 shows the chemotactic effect of muKSl and SDF-l ⁇ on THP-1 cells.
  • Figure 10 shows the induction of cellular infiltrate in C3H/HeJ mice after intraperitoneal injections with muKSl (50 ⁇ g), GV14B (50 ⁇ g) and PBS.
  • Figure 11 demonstrates the induction of phosphorylation of ERK1 and ERK2 in CV1/EBNA and HeLa cell lines by huTRla.
  • Figure 12 shows the huTRl mRNA expression in HeLa cells after stimulation by muTRl, huTRl, huTGF ⁇ and PBS (100 ng/ml each).
  • Figure 13 shows activation of the SRE by muTRla in PC-12 (Fig. 13a) and HaCaT (Fig. 13b) cells.
  • Figure 14 shows the inhibition of huTRla mediated growth on HaCaT cells by an antibody to the EGF receptor.
  • the present invention provides polynucleotides that were isolated from mammalian skin cells.
  • polynucleotide means a single or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases and includes DNA and RNA molecules, both sense and anti-sense strands.
  • the term comprehends cDNA, genomic DNA, recombinant DNA and wholly or partially synthesized nucleic acid molecules.
  • a polynucleotide may consist of an entire gene, or a portion thereof.
  • a gene is a DNA sequence that codes for a functional protein or RNA molecule.
  • Operable anti-sense polynucleotides may comprise a fragment ofthe corresponding polynucleotide, and the definition of "polynucleotide” therefore includes all operable anti-sense fragments.
  • Anti-sense polynucleotides and techniques involving anti-sense polynucleotides are well known in the art and are described, for example, in Robinson- Benion et al., "Anti-sense Techniques," Methods in Enzymol. 254(23):363-375, 1995; and Kawasaki et al., Artific. Organs 20 (8):836-848, 1996.
  • DNAs can be accomplished by standard DNA/DNA hybridization techniques, under appropriately stringent conditions, using all or part of a cDNA sequence as a probe to screen an appropriate library.
  • PCR techniques using oligonucleotide primers that are designed based on known genomic DNA, cDNA and protein sequences can be used to amplify and identify genomic and cDNA sequences.
  • Synthetic DNAs corresponding to the identified sequences and variants may be produced by conventional synthesis methods. All the polynucleotides provided by the present invention are isolated and purified, as those terms are commonly used in the art.
  • the polynucleotides of the present invention comprise a DNA sequence selected from the group consisting of sequences provided in SEQ ID NO: 1-119, 198-274, 349-372 and 399-405, and variants of the sequences of SEQ ID NO: 1-119, 198-274, 349-372 and 399-405.
  • Polynucleotides that comprise complements of such DNA sequences, reverse complements of such DNA sequences, or reverse sequences of such DNA sequences, together with variants of such sequences, are also provided.
  • the definition of the terms "complement,” “reverse complement,” and “reverse sequence,” as used herein, is best illustrated by the following example.
  • the complement, reverse complement, and reverse sequence are as follows: complement 3 ' TCCTGG 5 ' reverse complement 3' GGTCCT 5' reverse sequence 5' CCAGGA 3'.
  • the present invention provides isolated polypeptides encoded, or partially encoded, by the above polynucleotides.
  • polypeptide encompasses amino acid chains of any length, including full length proteins, wherein the amino acid residues are linked by covalent peptide bonds.
  • polypeptide encoded by a polynucleotide includes polypeptides encoded by a polynucleotide which comprises a partial isolated DNA sequence provided herein.
  • inventive polypeptides comprise an amino acid sequence selected from the group consisting of sequences provided in SEQ ID NO: 120-197, 275-348, 373-398 and 406-409, as well as variants of such sequences.
  • Polypeptides of the present invention may be produced recombinantly by inserting a DNA sequence that encodes the polypeptide into an expression vector and expressing the polypeptide in an appropriate host. Any of a variety of expression vectors known to those of ordinary skill in the art may be employed. Expression may be achieved in any appropriate host cell that has been transformed or transfected with an expression vector containing a DNA molecule that encodes a recombinant polypeptide. Suitable host cells include prokaryotes, yeast, and higher eukaryotic cells. Preferably, the host cells employed are E. coli, insect, yeast, or a mammalian cell line such as COS or CHO. The DNA sequences expressed in this manner may encode naturally occurring polypeptides, portions of naturally occurring polypeptides, or other variants thereof.
  • polypeptides are provided that comprise at least a functional portion of a polypeptide having an amino acid sequence selected from the group consisting of sequences provided in SEQ ID NO: 120-197, 275-348, 373-398, 406-409, and variants thereof.
  • the "functional portion" of a polypeptide is that portion which contains the active site essential for affecting the function of the polypeptide, for example, the portion of the molecule that is capable of binding one or more reactants.
  • the active site may be made up of separate portions present on one or more polypeptide chains and will generally exhibit high binding affinity.
  • Functional portions of a polypeptide may be identified by first preparing fragments of the polypeptide by either chemical or enzymatic digestion of the polypeptide, or by mutation analysis of the polynucleotide that encodes the polypeptide and subsequent expression of the resulting mutant polypeptides. The polypeptide fragments or mutant polypeptides are then tested to determine which portions retain biological activity, using, for example, the representative assays provided below.
  • Portions and other variants of the inventive polypeptides may also be generated by synthetic or recombinant means.
  • Synthetic polypeptides having fewer than about 100 amino acids, and generally fewer than about 50 amino acids may be generated using techniques well known to those of ordinary skill in the art.
  • such polypeptides may be synthesized using any of the commercially available solid-phase techniques, such as the Merrifield solid-phase synthesis method, where amino acids are sequentially added to a growing amino acid chain. See Merrifield, J. Am. Chem. Soc. 85:2149-2146, 1963. Equipment for automated synthesis of polypeptides is commercially available from suppliers such as Perkin Elmer/ Applied BioSystems, Inc.
  • Variants of a native polypeptide may be prepared using standard mutagenesis techniques, such as oligonucleotide-directed site-specific mutagenesis (Kunkel, T., Proc. Natl. Acad. Sci. USA 82:488-492, 1985). Sections of DNA sequence may also be removed using standard techniques to permit preparation of truncated polypeptides.
  • polypeptides disclosed herein are prepared in an isolated, substantially pure, form.
  • the polypeptides are at least about 80% pure, more preferably at least about 90% pure, and most preferably at least about 99%) pure.
  • the isolated polypeptides are incorporated into pharmaceutical compositions or vaccines for use in the treatment of skin disorders.
  • variant comprehends nucleotide or amino acid sequences different from the specifically identified sequences, wherein one or more nucleotides or amino acid residues is deleted, substituted, or added. Variants may be naturally occurring allelic variants, or non-naturally occurring variants. Variant sequences (polynucleotide or polypeptide) preferably exhibit at least 50%o, more preferably at least 75%, and most preferably at least 90%> identity to a sequence of the present invention. The percentage identity is determined by aligning the two sequences to be compared as described below, determining the number of identical residues in the aligned portion, dividing that number by the total number of residues in the inventive (queried) sequence, and multiplying the result by 100.
  • Polynucleotide or polypeptide sequences may be aligned, and percentage of identical nucleotides in a specified region may be determined against another polynucleotide or polypeptide, using computer algorithms that are publicly available.
  • Two exemplary algorithms for aligning and identifying the similarity of polynucleotide sequences are the BLASTN and FASTA algorithms.
  • the alignment and similarity of polypeptide sequences may be examined using the BLASTP and algorithm.
  • BLASTX and FASTX algorithms compare nucleotide query sequences translated in all reading frames against polypeptide sequences.
  • the BLASTN, BLASTP and BLASTX algorithms are available on the NCBI anonymous FTP server (ftp ://ncbi .nlm.nih.
  • the FASTA and FASTX algorithms are available on the Internet at the ftp site ftp://ftp.virginia.edu/pub/.
  • the FASTA algorithm set to the default parameters described in the documentation and distributed with the algorithm, may be used in the determination of polynucleotide variants.
  • the readme files for FASTA and FASTX vl.Ox that are distributed with the algorithms describe the use of the algorithms and describe the default parameters.
  • FASTA and FASTX algorithms are also described in Pearson, WR and Lipman, DJ, "Improved Tools for Biological Sequence Analysis," PNAS 85:2444-2448, 1988; and Pearson WR, "Rapid and Sensitive Sequence Comparison with FASTP and FASTA," Methods in Enzymology 183:63-98, 1990.
  • the BLASTN algorithm version 2.0.4 [Feb-24-1998], set to the default parameters described in the documentation and distributed with the algorithm, is preferred for use in the determination of polynucleotide variants according to the present invention.
  • the BLASTP algorithm version 2.0.4 set to the default parameters described in the documentation and distributed with the algorithm, is preferred for use in the determination of polypeptide variants according to the present invention.
  • BLAST family of algorithms including BLASTN, BLASTP and BLASTX is described at NCBI's website at URL http://www.ncbi.nlm.nih.gov/BLAST/newblast.html and in the publication of Altschul, Stephen F., et al, "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs," Nucleic Acids Res. 25:3389-3402, 1997.
  • the following running parameters are preferred for determination of alignments and similarities using BLASTN that contribute to the E values and percentage identity for polynucleotides: Unix running command with default parameters thus: blastall -p blastn - d embldb -e 10 -G 0 -E 0 -r 1 -v 30 -b 30 -i queryseq -o results; and parameters are: -p Program Name [String]; -d Database [String]; -e Expectation value (E) [Real]; -G Cost to open a gap (zero invokes default behavior) [Integer]; -E Cost to extend a gap (zero invokes default behavior) [Integer]; -r Reward for a nucleotide match (blastn only) [Integer]; -v Number of one-line descriptions (V) [Integer]; -b Number of alignments to show (B) [Integer]; -i Query File [F
  • the following running parameters are preferred for determination of alignments and similarities using BLASTP that contribute to the E values and percentage identity for polypeptides: blastall -p blastp -d swissprotdb -e 10 -G 1 -E 11 -r 1 -v 30 -b 30 -i queryseq -o results; and the parameters are: -p Program Name [String]; -d Database [String]; -e Expectation value (E) [Real]; -G Cost to open a gap (zero invokes default behavior) [Integer]; -E Cost to extend a gap (zero invokes default behavior) [Integer]; -v Number of one-line descriptions (v) [Integer]; -b Number of alignments to show (b) [Integer]; -I Query File [File In]; -o BLAST report Output File [File Out] Optional.
  • the "hits" to one or more database sequences by a queried sequence produced by BLASTN, BLASTP, FASTA, or a similar algorithm align and identify similar portions of sequences.
  • the hits are arranged in order of the degree of similarity and the length of
  • Hits to a database sequence generally represent an overlap over only a fraction ofthe sequence length ofthe queried sequence.
  • the percentage similarity of a polynucleotide or polypeptide sequence is determined by aligning polynucleotide and polypeptide sequences using appropriate algorithms, such as BLASTN or BLASTP, respectively, set to default parameters; identifying the number of identical nucleic or amino acids over the aligned portions; dividing the number of identical nucleic or amino acids by the total number of nucleic or amino acids of the polynucleotide or polypeptide of the present invention; and then multiplying by 100 to determine the percentage similarity.
  • a queried polynucleotide having 220 nucleic acids has a hit to a polynucleotide sequence in the EMBL database having 520 nucleic acids over a stretch of 23 nucleotides in the alignment produced by the BLASTN algorithm using the default parameters.
  • the 23 nucleotide hit includes 21 identical nucleotides, one gap and one different nucleotide.
  • the percentage identity of the queried polynucleotide to the hit in the EMBL database is thus 21/220 times 100, or 9.5%.
  • the similarity of polypeptide sequences may be determined in a similar fashion.
  • the BLASTN and BLASTX algorithms also produce "Expect" values for polynucleotide and polypeptide alignments.
  • the Expect value (E) indicates the number of hits one can "expect” to see over a certain number of contiguous sequences by chance when searching a database of a certain size.
  • the Expect value is used as a significance threshold for determining whether the hit to a database indicates true similarity. For example, an E value of 0.1 assigned to a polynucleotide hit is interpreted as meaning that in a database of the size of the EMBL database, one might expect to see 0.1 matches over the aligned portion of the sequence with a similar score simply by chance.
  • the aligned and matched portions of the sequences then have a probability of 90%) of being the same.
  • the probability of finding a match by chance in the EMBL database is 1%) or less using the BLASTN algorithm.
  • E values for polypeptide sequences may be determined in a similar fashion using various polypeptide databases, such as the SwissProt database.
  • "variant" polynucleotides and polypeptides with reference to each of the polynucleotides and polypeptides of the present invention, preferably comprise sequences having the same number or fewer nucleic or amino acids than each of the polynucleotides or polypeptides of the present invention and producing an E value of 0.01 or less when compared to the polynucleotide or polypeptide of the present invention.
  • a variant polynucleotide or polypeptide is any sequence that has at least a 99%> probability of being the same as the polynucleotide or polypeptide of the present invention, measured as having an E value of 0.01 or less using the BLASTN or BLASTX algorithms set at the default parameters.
  • a variant polynucleotide is a sequence having the same number or fewer nucleic acids than a polynucleotide of the present invention that has at least a 99%o probability of being the same as the polynucleotide of the present invention, measured as having an E value of 0.01 or less using the BLASTN algorithm set at the default parameters.
  • a variant polypeptide is a sequence having the same number or fewer amino acids than a polypeptide of the present invention that has at least a 99%> probability of being the same as the polypeptide of the present invention, measured as having an E value of 0.01 or less using the BLASTP algorithm set at the default parameters.
  • Variant polynucleotide sequences will generally hybridize to the recited polynucleotide sequences under stringent conditions.
  • stringent conditions refers to prewashing in a solution of 6X SSC, 0.2%o SDS; hybridizing at 65°C, 6X SSC, 0.2% SDS overnight; followed by two washes of 30 minutes each in IX SSC, 0.1% SDS at 65 °C and two washes of 30 minutes each in 0.2X SSC, 0.1% SDS at 65 °C.
  • x-mer refers to a polynucleotide or polypeptide, respectively, comprising at least a specified number (" ") of contiguous residues of: any of the polynucleotides provided in SEQ ID NO: 1-119, 198-274, 349-372 and 399-405; or any of the polypeptides set out in SEQ ID NO: 120-197, 275-348, 373-398 and 406-409.
  • the value of x may be from about 20 to about 600, depending upon the specific sequence.
  • Polynucleotides of the present invention comprehend polynucleotides comprising at least a specified number of contiguous residues (x-mers) of any of the polynucleotides identified as SEQ ID NO: 1-119, 198-274, 349-372 and 399-405, or their variants.
  • Polypeptides of the present invention comprehend polypeptides comprising at least a specified number of contiguous residues (x-mers) of any of the polypeptides identified as SEQ ID NO: 120-197, 275-348, 373-398, and 406-409.
  • the value of x is at least 20, more preferably at least 40, more preferably yet at least 60, and most preferably at least 80.
  • polynucleotides of the present invention include polynucleotides comprising a 20-mer, a 40-mer, a 60-mer, an 80-mer, a 100-mer, a 120-mer, a 150-mer, a 180-mer, a 220-mer, a 250-mer; or a 300-mer, 400-mer, 500-mer or 600-mer of a polynucleotide provided in SEQ ID NO: 1-119, 198-274, 349-372 and 399-405 or a variant of one of the polynucleotides provided in SEQ ID NO: 1-119, 198-274, 349-372, and 399-405.
  • Polypeptides of the present invention include polypeptides comprising a 20-mer, a 40-mer, a 60-mer, an 80-mer, a 100-mer, a 120-mer, a 150-mer, a 180-mer, a 220-mer, a 250-mer; or a 300-mer, 400-mer, 500-mer or 600-mer of a polypeptide provided in SEQ ID NO: 120-197, 275-348, 373-398, and 406-409, or a variant of one of the polynucleotides provided in SEQ ID NO: 120-197, 275-348, 373-398, and 406-409.
  • the inventive polynucleotides may be isolated by high throughput sequencing of cDNA libraries prepared from mammalian skin cells as described below in Example 1.
  • oligonucleotide probes based on the sequences provided in SEQ ID NO: 1-119, 198-274, 349-372, and 399-405 can be synthesized and used to identify positive clones in either cDNA or genomic DNA libraries from mammalian skin cells by means of hybridization or polymerase chain reaction (PCR) techniques. Probes can be shorter than the sequences provided herein but should be at least about 10, preferably at least about 15 and most preferably at least about 20 nucleotides in length.
  • Hybridization and PCR techniques suitable for use with such oligonucleotide probes are well known in the art (see, for example, Mullis, et al., Cold Spring Harbor Symp. Quant. Biol., 51 :263, 1987; Erlich, ed., PCR Technology, Stockton Press: NY, 1989; (Sambrook, J, Fritsch, EF and Maniatis, T, eds., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring
  • Positive clones may be analyzed by restriction enzyme digestion, DNA sequencing or the like.
  • DNA sequences of the present invention may be generated by synthetic means using techniques well known in the art.
  • Equipment for automated synthesis of oligonucleotides is commercially available from suppliers such as Perkin Elmer/Applied Biosystems Division (Foster City, California) and may be operated according to the manufacturer's instructions.
  • polynucleotide sequences of the present invention have been derived from skin, they likely encode proteins that have important roles in growth and development of skin, and in responses of skin to tissue injury and inflammation as well as disease states. Some of the polynucleotides contain sequences that code for signal sequences, or transmembrane domains, which identify the protein products as secreted molecules or receptors. Such protein products are likely to be growth factors, cytokines, or their cognate receptors. Several of the polypeptide sequences have more than 25% similarity to known biologically important proteins and thus are likely to represent proteins having similar biological functions.
  • the inventive polypeptides have important roles in processes such as: induction of hair growth; differentiation of skin stem cells into specialized cell types; cell migration; cell proliferation and cell-cell interaction.
  • the polypeptides are important in the maintenance of tissue integrity, and thus are important in processes such as wound healing.
  • Some of the disclosed polypeptides act as modulators of immune responses, especially since immune cells are known to infiltrate skin during tissue insult causing growth and differentiation of skin cells.
  • many polypeptides are immunologically active, making them important therapeutic targets in a whole range of disease states not only within skin, but also in other tissues ofthe body.
  • Antibodies to the polypeptides of the present invention and small molecule inhibitors related to the polypeptides of the present invention may also be used for modulating immune responses and for treatment of diseases according to the present invention.
  • the present invention provides methods for using one or more of the inventive polypeptides or polynucleotides to treat disorders in a patient.
  • a "patient” refers to any warm-blooded animal, preferably a human.
  • the polypeptide or polynucleotide is generally present within a pharmaceutical composition or a vaccine.
  • Pharmaceutical compositions may comprise one or more polypeptides, each of which may contain one or more of the above sequences (or variants thereof), and a physiologically acceptable carrier.
  • Vaccines may comprise one or more of the above polypeptides and a non-specific immune response amplifier, such as an adjuvant or a liposome, into which the polypeptide is incorporated.
  • a vaccine or pharmaceutical composition of the present invention may contain DNA encoding one or more polypeptides as described above, such that the polypeptide is generated in situ.
  • the DNA may be present within any of a variety of delivery systems known to those of ordinary skill in the art, including nucleic acid expression systems, and bacterial and viral expression systems. Appropriate nucleic acid expression systems contain the necessary DNA sequences for expression in the patient (such as a suitable promoter and terminator signal).
  • Bacterial delivery systems involve the administration of a bacterium (such as Bacillus-Calmette-Guerin) that expresses an immunogenic portion of the polypeptide on its cell surface.
  • the DNA may be introduced using a viral expression system (e.g., vaccinia or other poxvirus, retro virus, or adenovirus), which may involve the use of a non-pathogenic, or defective, replication competent virus.
  • a viral expression system e.g., vaccinia or other poxvirus, retro virus, or adenovirus
  • Techniques for incorporating DNA into such expression systems are well known in the art.
  • the DNA may also be "naked,” as described, for example, in Ulmer, et al., Science 259:1745-1749, 1993 and reviewed by Cohen, Science 259:1691-1692, 1993.
  • the uptake of naked DNA may be increased by coating the DNA onto biodegradable beads, which are efficiently transported into the cells.
  • compositions and vaccines may be administered by injection (e.g., mtradermal, intramuscular, intravenous, or subcutaneous), intranasally (e.g., by aspiration) or orally.
  • amount of polypeptide present in a dose ranges from about 1 pg to about 100 mg per kg of host, typically from about 10 pg to about 1 mg per kg of host, and preferably from about 100 pg to about 1 ⁇ g per kg of host. Suitable dose
  • 15 sizes will vary with the size of the patient, but will typically range from about 0.1 ml to about 5 ml.
  • the type of carrier will vary depending on the mode of administration.
  • the carrier preferably comprises water, saline, alcohol, a lipid, a wax, or a buffer.
  • any of the above carriers or a solid carrier such as mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, sucrose, and magnesium carbonate, may be employed.
  • Biodegradable microspheres e.g., polylactic galactide
  • suitable biodegradable microspheres are disclosed, for example, in U.S. Patent Nos. 4,897,268 and 5,075,109.
  • adjuvants may be employed in the vaccines derived from this invention to non-specifically enhance the immune response.
  • Most adjuvants contain a substance designed to protect the antigen from rapid catabolism, such as aluminum hydroxide or mineral oil, and a non-specific stimulator of immune responses, such as lipid A, Bordetella pertussis, or M. tuberculosis.
  • Suitable adjuvants are commercially available as, for example, Freund's Incomplete Adjuvant and Freund's Complete Adjuvant (Difco Laboratories, Detroit, Michigan), and Merck Adjuvant 65 (Merck and Company, Inc., Rahway, New Jersey).
  • Other suitable adjuvants include alum, biodegradable microspheres, monophosphoryl lipid A, and Quil A.
  • polynucleotides of the present invention may also be used as markers for tissue, as chromosome markers or tags, in the identification of genetic disorders, and for the design of oligonucleotides for examination of expression patterns using techniques well known in the art, such as the microarray technology available from Synteni (Palo Alto, California). Partial polynucleotide sequences disclosed herein may be employed to obtain full length genes by, for example, screening of DNA expression libraries using hybridization probes or PCR primers based on the inventive sequences.
  • polypeptides provided by the present invention may additionally be used in assays to determine biological activity, to raise antibodies, to isolate corresponding Hgands or receptors, in assays to quantitatively determine levels of protein or cognate
  • the cDNA sequences of the present invention were obtained by high-throughput sequencing of cDNA expression libraries constructed from specialized rodent or human skin cells as shown in Table 1.
  • RNA Dermal papilla cells from rat hair vibrissae (whiskers) were grown in culture and the total RNA extracted from these cells using established protocols.
  • Total RNA isolated using TRIzol Reagent (BRL Life Technologies, Gaithersburg, Maryland), was used to obtain mRNA using a Poly(A) Quik mRNA isolation kit (Stratagene, La Jolla, California), according to the manufacturer's specifications.
  • a cDNA expression library was then prepared from the mRNA by reverse transcriptase synthesis using a Lambda
  • Keratinocytes obtained from human neonatal foreskins were grown in serum-free
  • KSFM (BRL Life Technologies) and harvested along with differentiated cells (10 cells). Keratinocytes were allowed to differentiate by addition of fetal calf serum at a final
  • RNA was isolated from the two cell populations using TRIzol Reagent (BRL Life Technologies) and used to obtain mRNA using a Poly(A) Quik mRNA isolation kit (Stratagene). cDNAs expressed in differentiated keratinocytes were enriched by using a PCR-Select cDNA Subtraction Kit (Clontech, Palo Alto, California). Briefly, mRNA was obtained from either undifferentiated keratinocytes ("driver mRNA”) or differentiated keratinocytes (“tester mRNA”) and used to synthesize cDNA. The two populations of cDNA were separately digested with Rsal to obtain shorter, blunt-ended molecules.
  • driver mRNA undifferentiated keratinocytes
  • tester mRNA differentiated keratinocytes
  • RNA Human neonatal fibroblast cells were grown in culture from explants of human neonatal foreskin and the total RNA extracted from these cells using established protocols.
  • Total RNA isolated using TRIzol Reagent (BRL Life Technologies, Gaithersburg, Maryland), was used to obtain mRNA using a Poly(A) Quik mRNA isolation kit (Stratagene, La Jolla, California), according to the manufacturer's specifications.
  • a cDNA expression library was then prepared from the mRNA by reverse transcriptase synthesis using a Lambda ZAP cDNA library synthesis kit (Stratagene).
  • cDNA library from mouse embryonic skin (MEMS) Embryonic skin was micro-dissected from day 13 post coitum Balb/c mice.
  • Embryonic skin was washed in phosphate buffered saline and mRNA directly isolated from the tissue using the Quick Prep Micro mRNA purification kit (Pharmacia, Sweden). The mRNA was then used to prepare cDNA libraries as described above for the DEPA library.
  • cDNA library from mouse stem cells (KSCL) and transit amplifying (TRAM) cells
  • Pelts obtained from 1-2 day post-partum neonatal Balb/c mice were washed and incubated in trypsin (BRL Life Technologies) to separate the epidermis from the dermis. Epidermal tissue was disrupted to disperse cells, which were then resuspended in growth medium and centrifuged over Percoll density gradients prepared according to the manufacturer's protocol (Pharmacia, Sweden). Pelleted cells were labeled using Rhodamine 123 (Bertoncello I, Hodgson GS and Bradley TR, Exp Hematol. 13:999- 1006, 1985), and analyzed by flow cytometry (Epics Elite Coulter Cytometry, Hialeah, Florida).
  • Single cell suspensions of rhodamine-labeled murine keratinocytes were then labeled with a cross reactive anti-rat CD29 biotin monoclonal antibody (Pharmingen, San Diego, California; clone Ha2/5). Cells were washed and incubated with anti-mouse CD45 phycoerythrin conjugated monoclonal antibody (Pharmingen; clone 30F11.1, lOug/ml) followed by labeling with streptavidin spectral red (Southern Biotechnology, Birmingham, Alabama).
  • Sort gates were defined using listmode data to identify four populations: CD29 bright rhodamine dull CD45 negative cells; CD29 bright rhodamine bright CD45 negative cells; CD29 dull rhodamine bright CD45 negative cells; and CD29 dull rhodamine dull CD45 negative cells.
  • Cells were sorted, pelleted and snap frozen prior to storage at -80°C. This protocol was followed multiple times to obtain sufficient cell numbers of each population to prepare cDNA libraries.
  • Skin stem cells and transit amplifying cells are known to express CD29, the integrin ⁇ l chain.
  • CD45 a leucocyte specific antigen, was used as a marker for cells to be excluded in the isolation of skin stem cells and transit amplifying cells.
  • Keratinocyte stem cells expel the rhodamine dye more efficiently than transit amplifying cells.
  • the CD29 bright, rhodamine dull, CD45 negative population (putative keratinocyte stem cells; referred to as KSCL), and the CD29 bright, rhodamine bright, CD45 negative population (keratinocyte transit amplifying cells; referred to as TRAM) were sorted and mRNA was directly isolated from each cell population using the Quick Prep Micro mRNA purification kit (Pharmacia, Sweden). The mRNA was then used to prepare cDNA libraries as described above for the DEPA library. cDNA sequences were obtained by high-throughput sequencing of the cDNA libraries described above using a Perkin Elmer/ Applied Biosystems Division Prism 377 sequencer.
  • Example 2 CHARACTERIZATION OF ISOLATED cDNA SEQUENCES
  • the isolated cDNA sequences were compared to sequences in the EMBL DNA database using the computer algorithms FASTA and or BLASTN.
  • the corresponding predicted protein sequences (DNA translated to protein in each of 6 reading frames) were compared to sequences in the SwissProt database using the computer algorithms FASTX and/or BLASTP.
  • Comparisons of DNA sequences provided in SEQ ID NO: 1-119 to sequences in the EMBL DNA database (using FASTA) and amino acid sequences provided in SEQ ID NO: 120-197 to sequences in the SwissProt database (using FASTX) were made as of March 21, 1998.
  • Isolated cDNA sequences and their corresponding predicted protein sequences were computer analyzed for the presence of signal sequences identifying secreted molecules.
  • Isolated cDNA sequences that have a signal sequence at a putative start site within the sequence are provided in SEQ ID NO: 1-44, 198-238, 349-358, and 399.
  • the cDNA sequences of SEQ ID NO: 1-6, 198-199, 349-352, 354, and 356-358 were determined to have less than 75% identity (determined as described above), to sequences in the EMBL database using the computer algorithms FASTA or BLASTN, as described above.
  • the predicted amino acid sequences of SEQ ID NO: 120-125, 275-276, 373-380, and 382 were determined to have less than 75% identity (determined as described above) to sequences in the SwissProt database using the computer algorithms FASTX or BLASTP, as described above.
  • Isolated cDNA sequences that have either one or more transmembrane domain(s) within the sequence are provided in SEQ ID NO: 45-63, 239-253, 359-364, 400-402.
  • the cDNA sequences of SEQ ID NO: 45-48, 239-249, 359-361, and 363 were found to have less than 75%o identity (determined as described above) to sequences in the EMBL database, using the FASTA or BLASTN computer algorithms.
  • Their predicted amino acid sequences provided in SEQ ID NO: 164-167, 316-326, 383, 385-388 and 407-408 were found to have less than 75% identity, determined as described above, to sequences in the SwissProt database using the FASTX or BLASTP database.
  • cDNA sequences isolated as described above in Example 1 were determined to encode predicted protein sequences that appear to be family members of known protein families.
  • a family member is here defined to have at least 25%o identity in the translated polypeptide to a known protein or member of a protein family.
  • These cDNA sequences are provided in SEQ ID NO: 64-76, 254-264, 365-369, and 403, with the conespondmg predicted amino acid sequences being provided in SEQ ID NO: 183-195, 331-341, 389-393 and 409, respectively.
  • the cDNA sequences of SEQ ID NO: 64-68, 254-264, and 365-369 show less than 75% identity (determined as described above) to sequences in the EMBL database using the FASTA or BLASTN computer algorithms.
  • the amino acid sequences of SEQ ID NO: 183-195, 331-341, and 389-393 show less than 75% identity to sequences in the SwissProt database.
  • Transforming growth factor alpha a protein which binds epidermal growth factor receptor and stimulates growth and mobility of keratinocytes
  • Interleukin-12 alpha subunit component of a cytokine that is important in the immune defense against intracellular pathogens.
  • IL-12 also stimulates proliferation and differentiation of TH1 subset of lymphocytes
  • EGF Epidermal growth factor family proteins which stimulate growth and mobility of keratinocytes and epithelial cells. EGF is involved in wound healing. It also inhibits gastric acid secretion.
  • Fibronectin Type III receptor family 74 193 Fibronectin Type III receptor family.
  • the fibronectin III domains are found on the extracellular regions of cytokine receptors
  • STK2_HUMAN Serine/threonine kinases
  • EGF Epidermal growth factor family proteins which stimulate growth and mobility of keratinocytes and epithelial cells. EGF is involved in wound healing. It also inhibits gastric acid secretion.
  • STK2_HUMAN Serine/threonine kinases
  • Notch family proteins which are receptors involved in cellular differentiation.
  • Fibronectin Type III receptor family The fibronectin III domains are found on the extracellular regions of cytokine receptors.
  • Mouse CXC chemokine family members are regulators of epithelial, lymphoid, myeloid, stromal and neuronal cell migration and cancers, agents for the healing of cancers, neurodegenerative diseases, wound healing, inflammatory autoimmune diseases like psoriasis, asthma, Crohns disease and as agents for the prevention of HIV- 1 of leukocytes
  • TGF-betas Transforming growth factor betas
  • LTBPs latent TGF-beta-binding proteins
  • Integrins are Type I membrane proteins that function as laminin and collagen receptors and play a role in cell adhesion.
  • Integrins are Type I membrane proteins that function as laminin and collagen receptors and play a role in cell adhesion.
  • 369 393 HT protein is a secreted glycoprotein with an EGF-like domain. It functions as a modulator of cell growth, death or differentiation.
  • polynucleotide sequences of SEQ ID NO: 77-117, 265-267, and 404-405 are differentially expressed in either keratinocyte stem cells (KSCL) or in transit amplified cells (TRAM) on the basis of the number of times these sequences exclusively appear in either one of the above two libraries; more than 9 times in one and none in the other (Audic S. and Claverie J-M, Genome Research, 7:986-995, 1997).
  • the sequences of SEQ ID NO: 77-89, 265-267, and 365-369 were determined to have less than 75% identity to sequences in the EMBL and SwissProt databases using the computer algorithm FASTA or BLASTN, as described above.
  • proteins encoded by these polynucleotide sequences have utility as markers for identification and isolation of these cell types, and antibodies against these proteins may be usefully employed in the isolation and enrichment of these cells from complex mixtures of cells.
  • Isolated polynucleotides and their corresponding proteins exclusive to the stem cell population can be used as drug targets to cause alterations in regulation of growth and differentiation of skin cells, or in gene targeting to transport specific therapeutic molecules to skin stem cells.
  • Example 1 was isolated by screening 50,000 pfu's of an oligo dT primed HeLa cell cDNA library. Plaque lifts, hybridization, and screening were performed using standard molecular biology techniques (Sambrook, J, Fritsch, EF and Maniatis, T, eds., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor: New York, 1989). The determined cDNA sequence of the isolated human homolog (huTRl) is provided in SEQ ID NO: 118, with the corresponding predicted amino acid sequence being provided in SEQ ID NO: 196.
  • the library was screened using an [ ⁇ P]-dCTP labeled double stranded cDNA probe corcesponding to nucleotides 1 to 459 ofthe coding region within SEQ ID NO: 118.
  • the polypeptide sequence of huTRl has regions similar to Transforming Growth
  • EGF epidermal growth factor
  • This EGF-like protein will serve to stimulate keratinocyte growth and motility, and to inhibit the growth of epithelial-derived cancer cells.
  • This novel gene and its encoded protein may thus be used as agents for the healing of wounds and regulators of epithelial- derived cancers.
  • mRNA for huTRl was 3.5-4kb in size and was observed to be most abundant in heart and placenta, with expression at lower levels being observed in spleen, thymus prostate and ovary (Fig. 1).
  • huTRl The high abundance of mRNA for huTRl in the heart and placenta indicates a role for huTRl in the formation or maintenance of blood vessels, as heart and placental tissues have an increased abundance of blood vessels, and therefore endothelial cells, compared to other tissues in the body. This, in turn, demonstrates a role for huTRl in angiogenesis and vascularization of tumors.
  • Polynucleotides 177-329 of muTRI (SEQ ID NO: 268), encoding amino acids 53-103 of muTRI (SEQ ID NO: 342), and polynucleotides 208-360 of huTRl (SEQ ID NO: 269), encoding amino acids 54-104 of huTRl (SEQ ID NO: 343), were cloned into the bacterial expression vector pProEX HT (BRL Life Technologies), which contains a bacterial leader sequence and N-terminal ⁇ xHistidine tag. These constructs were transformed into competent XLl-Blue E. coli as described in Sambrook et al., Ibid. Starter cultures of these recombinant XLl-Blue E. coli were grown overnight at
  • bacterial cell pellets were re-suspended in lysis buffer (20 mM Tris-HCl pH 8.0, 10 mM beta mercaptoethanol, 1 mM PMSF). To the lysed cells, 1% NP40 was added and the mix incubated on ice for 10 minutes. Lysates were further disrupted by sonication on ice at 95W for 4 x 15 seconds and then centrifuged for 15 minutes at 14,000 rpm to pellet the inclusion bodies.
  • lysis buffer 20 mM Tris-HCl pH 8.0, 10 mM beta mercaptoethanol, 1 mM PMSF.
  • the resulting pellet was re-suspended in lysis buffer containing 0.5%> w/v CHAPS and sonicated on ice for 5-10 seconds. This mix was stored on ice for 1 hour, centrifuged at 14,000 rpm for 15 minutes at 4 °C and the supernatant discarded. The pellet was once more re-suspended in lysis buffer containing 0.5% w/v CHAPS, sonicated, centrifuged and the supernatant removed as before.
  • the pellet was re-suspended in solubilizing buffer (6 M Guanidine HC1, 0.5 M NaCl, 20 mM Tris HC1, pH 8.0), sonicated at 95 W for 4 x 15 seconds and then centrifuged for 20 minutes at 14,000 rpm and 4 °C to remove debris. The supernatant was stored at 4 °C until use.
  • solubilizing buffer (6 M Guanidine HC1, 0.5 M NaCl, 20 mM Tris HC1, pH 8.0)
  • Polypeptides muTRla and huTRla were purified by virtue of the N-terminal 6x Histidine tag contained within the bacterial leader sequence, using a Nickel-Chelating Sepharose column (Amersham Pharmacia, Uppsala, Sweden) and following the manufacturer's recommended protocol.
  • the protein solution was added to 5x its volume of refolding buffer (1 mM EDTA, 1.25 mM reduced glutathione, 0.25 mM oxidised glutathione, 20 mM Tris-HCl, pH 8.0) over a period of 1 hour at 4 °C.
  • the refolding buffer was stirred rapidly during this time, and stirring continued at 4 °C overnight.
  • the refolded proteins were then concentrated by ultrafiltration using standard protocols.
  • huTRl act as ligands for a cell surface receptor that activates the MAP kinase signaling pathway, possibly the EGF receptor.
  • huTRla was also demonstrated to induce the phosphorylation of ERK1 and ERK2 in CV1/EBNA kidney epithelial cells in culture, as compared with the negative control. These assays were conducted as described above. This indicates that huTRla acts as a ligand for a cell surface receptor that activates the MAP kinase signaling pathway, possibly the EGF receptor in HeLa and CV1/EBNA cells.
  • NF keratinocytes neonatal foreskin (NF) keratinocytes
  • NF keratinocytes derived from surgical discards were cultured in KSFM (BRL Life Technologies) supplemented with bovine pituatary extract (BPE) and epidermal growth factor (EGF).
  • BPE bovine pituatary extract
  • EGF epidermal growth factor
  • the assay was performed in 96 well flat-bottomed plates in 0.1 ml unsupplemented KSFM.
  • MuTRla, human transforming growth factor alpha (huTGF ⁇ ) or PBS-BSA was titrated into the plates and 1 x 10 3 NF keratinocytes were added to each well. The plates were incubated for 5 days in an atmosphere of 5%o CO 2 at 37°C.
  • the ability of muTRla and huTRla to stimulate the growth of a transformed human keratinocyte cell line, HaCaT was determined as follows. The assay was performed in 96 well flat-bottomed plates in 0.1 ml DMEM (BRL Life Technologies) supplemented with 0.2% 0 FCS. MuTRla, huTRla and PBS-BSA were titrated into the plates and 1 xlO 3 HaCaT cells were added to each well. The plates were incubated for 5 days in an atmosphere containing 10%> CO at 37°C. The degree of cell growth was determined by MTT dye reduction as described previously (J. Imm. Meth. 93:157-165, 1986). As shown in Fig. 4, both muTRla and huTRla stimulated the growth of HaCaT cells, whereas the negative control PBS-BSA did not.
  • muTRla and huTRla were determined as follows. Polypeptides muTRla (SEQ ID NO: 342) and huTRla (SEQ ID NO: 343) and PBS-BSA were titrated as described previously (J. Cell Biol. 93:1-4, 1982) and cell death determined using the MTT dye reduction as described previously
  • muTRI and huTRl stimulate keratinocyte growth and motility, inhibit the growth of epithelial-derived cancer cells, and play a role in angiogenesis and vascularization of tumors.
  • This novel gene and its encoded protein may thus be developed as agents for the healing of wounds, angiogenesis and regulators of epithelial-derived cancers. Upregulation ofhuTRl and mRNA expression
  • HeLa cells human cervical adenocarcinoma
  • media was removed and replaced with media containing 100 ng/ml of muTRI, huTRl, huTGF ⁇ , or PBS as a negative control.
  • media was removed and the cells lysed in 2 ml of TRIzol reagent (Gibco BRL Life Technologies, Gaithersburg, Maryland).
  • Total RNA was isolated according to the manufacturer's instructions. To identify mRNA levels of huTRl from the cDNA samples, 1 ⁇ l of cDNA was used in a standard PCR reaction.
  • both mouse and human TRl up-regulate the mRNA levels of huTRl as compared with cells stimulated with the negative control of PBS. Furthermore, TGF ⁇ can also up-regulate the mRNA levels of huTRl .
  • TRl is able to sustain its own mRNA expression and subsequent protein expression, and thus is expected to be able to contribute to the progression of diseases such as psoriasis where high levels of cytokine expression are involved in the pathology of the disease. Furthermore, since TGF ⁇ can up-regulate the expression of huTRl, the up-regulation of TRl mRNA may be critical to the mode of action of TGF ⁇ . Serum response element reporter gene assay
  • the serum response element is a promoter element required for the regulation of many cellular immediate-early genes by growth. Studies have demonstrated that the activity of the SRE can be regulated by the MAP kinase signaling pathway.
  • PC 12 rat pheochromocytoma - neural tumor
  • HaCaT human transformed
  • keratinocytes containing eight SRE upstream of an SV40 promotor and luciferase reporter gene were developed in-house. 5 x 10 3 cells were aliquoted per well of 96 well plate and grown for 24 hours in their respective media.
  • HaCaT SRE cells were grown in 5%> fetal bovine serum (FBS) in D-MEM supplemented with 2mM L-glutamine (Sigma, St.
  • PC12 SRE cells were grown in 5% fetal bovine serum in Ham F12 media supplemented with 0.4 mg/ml geneticin (BRL Life Technologies). Media was then changed to 0.1% FBS and incubated for a further 24 hours. Cells were then stimulated with a titration of TRl from 1 ⁇ g/ml.
  • muTRI activates the SRE in both PC-12 (Fig. 13a) and HaCaT (Fig. 13b) cells.
  • this is a growth response.
  • PC- 12 cells this may be a growth, a growth inhibition, differentiation, or migration response.
  • TRl may be important in the development of neural cells or their differentiation into specific neural subsets. TRl may also be important in the development and progression of neural tumors. Inhibition by the EGF receptor assay
  • the HaCaT growth assay was conducted as previously described, except that modifications were made as follows. Concurrently with the addition of EGF and TRl to the media, anti-EGF Receptor (EGFR) antibody (Promega, Madison, Wisconsin) or
  • mouse IgG (PharMingen, San Diego, California) were added at a concentration of 62.5 ng/ml.
  • an antibody which blocks the function of the EGFR inhibits the mitogenicity of TRl on HaCaT cells. This indicates that the EGFR is crucial for transmission of the TRl mitogenic signal on HaCaT cells.
  • TRl may bind directly to the EGF receptor. TRl may also bind to any other members of the EGFR family - ErbB-2, -3, and/or -4 - that are capable of heterodimerizing with the EGFR. Sequence of splice variant ofhuTRl, huTRl ⁇
  • huTRl A variant of huTRl was isolated from the same library as huTRl (SEQ ID NO: 118), following the same protocols.
  • This sequence is a splice variant of huTRl and consists of the ORF of huTRl minus amino acids 87 to 137. This has the effect of deleting the third cysteine residue of the EGF motif and the transmembrane domain.
  • cysteine residue 147 (huTRl ORF numbering) may replace the deleted cysteine and thus the disulphide bridges are likely not affected. Therefore, huTRl ⁇ is a secreted form of huTRl. It functions as an agonist or an antagonist to huTRl or other EGF family members, including EGF and TGF ⁇ .
  • the determined nucleotide sequence of the splice variant of TRl, refened to as huTRl ⁇ is given in SEQ ID NO: 371 and the corresponding predicted amino acid sequence is SEQ ID NO: 3
  • a partial cDNA fragment referred to as DP3, was identified by differential display RT-PCR (modified from Liang P and Pardee AB, Science 257:967-971, 1992) using mRNA from cultured rat dermal papilla and footpad fibroblast cells, isolated by standard cell biology techniques.
  • This double stranded cDNA was labeled with [ ⁇ 32 P]- dCTP and used to identify a full length DP3 clone by screening 400,000 pfu's of an oligo dT-primed rat dermal papilla cDNA library.
  • the determined full-length cDNA sequence for DP3 is provided in SEQ ID NO: 119, with the coreesponding amino acid sequence being provided in SEQ ID NO: 197. Plaque lifts, hybridization and screening were performed using standard molecular biology techniques.
  • mRNA for muKSl was 1.6 kb in size and was observed to be most abundant in brain, lung, muscle, and heart. Expression could also be detected in lower intestine, skin, and kidney. No detectable signal was found in testis, spleen, liver, thymus, stomach.
  • Human homologue ofmuKSl MuKSl was used to search the EMBL database (Release 50, plus updates to June, 1998) to identify human EST homologues.
  • the top three homologies were to the following ESTs: accession numbers AA643952, HS1301003 and AA865643. These showed 92.63%o identity over 285 nucleotides, 93.64% over 283 nucleotides and 94.035% over 285 nucleotides, respectively.
  • Frame shifts were identified in AA643952 and HS1301003 when translated.
  • Combination of all three ESTs identified huKSl (SEQ ID NO: 270) and translated polypeptide SEQ ID NO: 344. Alignment of muKSl and huKSl polypeptides indicated 95%> identity over 96 amino acids.
  • Polynucleotides 269-502 of muKSl (SEQ ID NO: 271), encoding amino acids 23-99 of polypeptide muKSl (SEQ ID NO: 345), and polynucleotides 55-288 of huKSl (SEQ ID NO: 272), encoding amino acids 19-95 of polypeptide huKSl (SEQ ID NO: 346), were cloned into the bacterial expression vector pET-16b (Novagen, Madison, Wisconsin), which contains a bacterial leader sequence and N-terminal ⁇ xHistidine tag. These constructs were transformed into competent XLl-Blue E. coli as described in Sambrook et al., Ibid.
  • MuKSla and huKSla were expressed in insoluble inclusion bodies.
  • bacterial cell pellets were re-suspended in lysis buffer (20 mM Tris-HCl pH 8.0, 10 mM ⁇ Mercaptoethanol, 1 mM PMSF).
  • lysis buffer 20 mM Tris-HCl pH 8.0, 10 mM ⁇ Mercaptoethanol, 1 mM PMSF.
  • 1% NP-40 was added and the mix incubated on ice for 10 minutes. Lysates were further disrupted by sonication on ice at 95 W for 4 x 15 seconds and then centrifuged for 10 minutes at 18,000 rpm to pellet the inclusion bodies.
  • the pellet containing the inclusion bodies was re-suspended in lysis buffer containing 0.5%> w/v CHAPS and sonicated for 5-10 seconds. This mix was stored on ice for 1 hour, centrifuged at 14000 rpm for 15 minutes at 4°C and the supernatant discarded. The pellet was once more re-suspended in lysis buffer containing 0.5%) w/v CHAPS, sonicated, centrifuged, and the supernatant removed as before.
  • the pellet was resuspended in solubilizing buffer (6 M guanidine HC1, 0.5 M NaCl, 20 mM Tris-HCl pH 8.0), sonicated at 95W for 4 x 15 seconds and centrifuged for 10 minutes at 18000 rpm and 4°C to remove debris. The supernatant was stored at 4°C.
  • MuKSla and huKSla were purified by virtue of the N-terminal 6x histidine tag contained within the bacterial leader sequence, using a Nickel-Chelating sepharose column (Amersham Pharmacia, Uppsala, Sweden) and following the manufacturer's protocol. Proteins were purified twice over the column to reduce endotoxin contamination.
  • the protein solution was dialysed in a 4 M-2 M urea gradient in 20 mM tris-HCl pH 7.5 + 10%> glycerol overnight at 4°C. The protein was then further dialysed 2x against 2 litres of 20 mM Tris-HCl pH 7.5 + 10% glycerol.
  • Peptide sequencing ofmuKSl and huKSl Bacterially expressed muKSl and huKSl were separated on polyacrylamide gels and induced bands of 15 kDa were identified. The predicted size of muKSl is 9.4 kDa.
  • the determined amino acid sequences for muKS 1 and huKS 1 are given in SEQ ID NOS: 397 and 398, respectively. These amino acid sequences confirmed that the determined sequences are identical to that predicted from the cDNA sequences. The size discrepancy has previously been reported for other chemokines (Richmond A, Balentien E, Thomas HG, Flaggs G, Barton DE, Spiess J, Bordoni R, Francke U, Derynck R, "Molecular characterization and chromosomal mapping of melanoma growth stimulatory activity, a growth factor structurally related to beta-thromboglobulin," EMBO J.
  • Oxidative burst assays were used to determine responding cell types. 1 x 10 PBMC cells were resuspended in 5 ml HBSS, 20mM HEPES, 0.5% BSA and incubated for 30 minutes at 37°C with 5 ⁇ l 5 mM dichloro-dihydrofluorescein diacetate (H 2 DCFDA, Molecular Probes, Eugene, Oregon). 2 x 10 5 H 2 DCFDA-labeled cells were loaded in each well of a flat-bottomed 96 well plate. 10 ⁇ l of each agonist was added simultaneously into the well of the flat-bottomed plate to give final concentrations of 100 ng/ml (fMLP was used at 10 ⁇ M). The plate was then read on a Victor 2 1420 multilabel counter (Wallac, Turku, Finland) with a 485 nm excitation wavelength and 535 nm emission wavelength. Relative fluorescence was measured at 5 minute intervals over 60 minutes.
  • H 2 DCFDA
  • agonists were diluted in HBSS, 20mM HEPES, 0.5% BSA and added to the bottom wells of the chemotactic chamber.
  • THP-1 cells were re-suspended in the same buffer at 3 x 10 5 cells per 50 ⁇ l.
  • Top and bottom wells were separated by a PVP- free polycarbonate filter with a 5 ⁇ m pore size for monocytes or 3 ⁇ m pore size for lymphocytes.
  • muKSl was tested against T cells and THP-1 cells. MuKSl induced a titrateable chemotactic effect on THP-1 cells from 0.01 ng/ml to 100 ng/ml (Fig. 9). Human SDFl ⁇ was used as a positive control and gave an equivalent migration. MuKSl was also tested against IL-2 activated T cells. However, no migration was evidence for muKSl even at high concentrations, whereas SDF-l ⁇ provided an obvious titrateable chemotactic stimulus. Therefore, muKSl appears to be chemotactic for THP-1 cells but not for IL-2 activated T cells at the concentrations tested.
  • RNA for huKSl was 1.6 kb in size and was observed to be most abundance in kidney, liver, colon, small intestine, and spleen. Expression could also be detected in pancreas, skeletal muscle, placenta, brain, heart, prostate, and thymus. No detectable signal was found in lung, ovary, and testis. Analysis of human RNA transcripts in tumor tissue by Northern blotting
  • mice Two nude mice were anaesthetised intraperitoneally with 75 ⁇ l of 1/10 dilution of Hypnorm (Janssen Pharmaceuticals, Buckinghamshire, England) in phosphate buffered saline. 20ug of bacterially expressed muKSla (SEQ ID NO: 345) was injected subcutaneously in the left hind foot, ear and left-hand side of the back. The same volume of phosphate buffered saline was injected in the same sites but on the right-hand side of the same animal. Mice were left for 18 hours and then examined for inflammation. Both mice showed a red swelling in the ear and foot sites injected with the bacterially expressed protein. No obvious inflammation could be identified in either back site.
  • mice were culled and biopsies taken from the ear, back and foot sites and fixed in 3.7% formol saline. Biopsies were embedded, sectioned and stained with Haemotoxylin and eosin. Sites injected with muKSla had a marked increase in polymorphonuclear granulocytes, whereas sites injected with phosphate buffered saline had a low background infiltrate of polymorphonuclear granulocytes. Injection of bacterially recombinant muKSl into C3H/HeJ mice Eighteen C3H/HeJ mice were divided into 3 groups and injected intraperitoneally with muKSl, GV14B, or phosphate buffered saline (PBS). GV14B is a bacterially
  • mice 37 expressed recombinant protein used as a negative control.
  • Group 1 mice were injected with 50 ⁇ g ofmuKSl in 1 ml of PBS;
  • Group 2 mice were injected with 50 ⁇ g of GV14B in 1 ml of PBS; and Group 3 mice with 1 ml of PBS.
  • the cells in the peritoneal cavity ofthe mice were isolated by intraperitoneal lavage with 2 x 4 ml washes with harvest solution (0.02%> EDTA in PBS). Viable cells were counted from individual mice from each group.
  • Mice injected with 50 ⁇ g of muKSl had on average a 3-fold increase in cell numbers (Fig. 10).
  • mice 20 ⁇ g of bacterial recombinant muKSl was injected subcutaneously into the left hind foot of three C3H/HeJ mice. The same volume of PBS was injected into the same site on the right-hand side of the same animal. After 18 hours, mice were examined for inflammation. All mice showed a red swelling in the foot pad injected with bacterially recombinant KS1. From histology, sites injected with muKSl had an inflammatory response of a mixed phenotype with mononuclear and polymorphonuclear cells present.
  • Chemokines are a large superfamily of highly basic secreted proteins with a broad number of functions (Baggiolini, et al, Annu. Rev. Immunol, 15:675-705, 1997; Ward, et al, Immunity, 9: 1-11, 1998; Horuk, Nature, 393:524-525, 1998).
  • the polypeptide sequences ofmuKSl and huKSl have similarity to CXC chemokines, suggesting that this protein will act like other CXC chemokines.
  • the in vivo data from nude mice supports this hypothesis.
  • This chemokine-like protein may therefore be expected to stimulate leukocyte, epithelial, stromal, and neuronal cell migration; promote angiogenesis and vascular development; promote neuronal patterning, hemopoietic stem cell mobilization, keratinocyte and epithelial stem cell patterning and development, activation and proliferation of leukocytes; and promotion of migration in wound healing events. It has recently been shown that receptors to chemokines act as co-receptors for HIV-1 infection of CD4+ cells (Cairns, et al., Nature Medicine, 4:563-568, 1998) and that high circulating levels of chemokines can render a degree of immunity to those exposed to the HIV virus (Zagury, et al, Proc.
  • This novel gene and its encoded protein may thus be usefully employed as regulators of epithelial, lymphoid, myeloid, stromal, and neuronal cells migration and cancers; as agents for the treatment of cancers, neuro-degenerative diseases, inflammatory autoimmune diseases
  • muKSl can promote a quantifiable increase in cell numbers in the peritoneal cavity of C3H/HeJ mice injected with muKSl. Furthermore, we have shown that muKSl can induce an oxidative burst in human peripheral blood mononuclear cells and migration in the human monocyte leukemia cell line, THP-1, suggesting that monocyte/macrophages are one of the responsive cell types for KS 1. In addition to this, we demonstrated that huKSl was expressed at high levels in a number of non-lymphoid tissues, such as the colon and small intestine, and in breast tumors. It was also expressed in normal uterine and cervical tissue, but was completely down-regulated in their respective tumors.
  • IP- 10 and Mig two non-ELR chemokines, have previously been shown to be up-regulated during regression of tumors (Tannenbaum CS, Tubbs R, Armstrong D, Finke JH, Bukowski RM, Hamilton TA, "The CXC Chemokines IP- 10 and Mig are necessary for IL-12-mediated regression of the mouse RENCA tumor," J. Immunol.
  • KS1 is involved in cell migration showing that one of the responsive cell types is monocyte/macrophage.
  • the human expression data in conjunction with the in vitro and in vivo biology demonstrates that this molecule may be a useful regulator in cell migration, and as an agent for the treatment of inflammatory diseases, such as Crohn's disease, ulcerative colitis, and rheumatoid arthritis; and cancers, such as cervical adenocarcinoma, uterine leiomyoma, and breast invasive ductal carcinoma.
  • KS2 contains a transmembrane domain and may function as either a membrane- bound ligand or a receptor.
  • KS2 the extracellular domain was fused to the amino terminus of the constant domain of immunoglobulinG (Fc) that had a C-terminal ⁇ xHistidine tag.
  • This construct was transformed into competent XLl-Blue E. coli as described in Sambrook et al., Ibid.
  • the Fc fusion construct of KS2a was expressed by transfecting Cos-1 cells in 5 x T175 flasks with 180 ⁇ g of KSla using DEAE-dextran. The supernatant was harvested after seven days and passed over a Ni-NTA column. Bound KS2a was eluted from the column and dialysed against PBS.
  • Fc fusion polypeptide of KS2a to inhibit the IL-2 induced growth of concanavalin A stimulated murine splenocytes was determined as follows.
  • a single cell suspension was prepared from the spleens of BALB/c mice and washed into DMEM (GIBCO-BRL) supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 0.77 mM L-asparagine, 0.2 mM L-arganine, 160 mM penicillin G, 70 mM dihydrostreptomycin sulfate, 5 x 10 "2 mM beta mercaptoethanol and 5% FCS (cDMEM).
  • Splenocytes (4 x 10 6 /ml) were stimulated with 2 ug/ml concanavalin A for 24 hrs at 37°C in 10%) CO .
  • the cells were harvested from the culture, washed 3 times in cDMEM and resuspended in cDMEM supplemented with 10 ng/ml rhuIL-2 at 1 x 10 5 cells/ml.
  • the assay was performed in 96 well round bottomed plates in 0.2 ml cDMEM.
  • the Fc fusion polypeptide of KS2a, PBS, LPS and BSA were titrated into the plates and 1 x 10 4 activated T cells (0.1 ml) were added to each well.
  • the plates were incubated for 2 days in an atmosphere containing 10%) CO 2 at 37°C.
  • the degree of proliferation was
  • KS2a determined by pulsing the cells with 0.25 uCi/ml tritiated thymidine for the final 4 hrs of culture after which the cells were harvested onto glass fiber filtermats and the degree of thymidine incorporation determined by standard liquid scintillation techniques.
  • the Fc fusion polypeptide of KS2a was found to inhibit the IL-2 induced growth of concanavalin A stimulated murine splenocytes, whereas the negative controls PBS, BSA and LPS did not.
  • KS2 is expressed in skin keratinocytes and inhibits the growth of cytokine induced splenocytes. This suggests a role for KS2 in the regulation of skin inflammation and malignancy.
  • KS3 encodes a polypeptide of 40 amino acids (SEQ ID NO: 129).
  • KS3 contains a signal sequence of 23 amino acids that would result in a mature polypeptide of 17 amino acids (SEQ ID NO: 348; referred to as KS3a).
  • KS3a was prepared synthetically (Chiron Technologies, Victoria, Australia) and observed to enhance transfercin-induced growth of the rat intestinal epithelial cells IEC-18 cells.
  • the assay was performed in 96 well flat-bottomed plates in 0.1 ml DMEM (GIBCO-BRL Life Technologies) supplemented with 0.2% FCS.
  • KS3a SEQ ID NO: 348
  • apo-Transferrin media
  • PBS-BSA were titrated either alone, with 750 ng/ml Apo-transferrin or with 750 ng/ml BSA, into the plates and 1 xlO 3 IEC-18 cells were added to each well.
  • the plates were incubated for 5 days at 37°C in an atmosphere containing 10%> CO .
  • KS3a plus Apo-transferrin was found to enhance transferrin-induced growth of IEC-18 cells, whereas KS3a alone or PBS-BSA did not, indicating that KS3a and Apo- transferrin act synergistically to induce the growth of IEC-18 cells.
  • KS3 is epithelial derived and stimulates the growth of epithelial cells of the intestine. This suggests a role for KS3 in wound healing, protection from radiation- or drug-induced intestinal disease, and integrity of the epithelium of the intestine.
  • SEQ ID NOS: 1-409 are set out in the attached Sequence Listing.
  • the codes for polynucleotide and polypeptide sequences used in the attached Sequence Listing confirm to WIPO Standard ST.25 (1988), Appendix 2.

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Abstract

Isolated polynucleotides encoding polypeptides expressed in mammalian skin cells are provided, together with expression vectors and host cells comprising such isolated polynucleotides. Methods for the use of such polynucleotides and polypeptides are also provided.

Description

POLYNUCLEOTIDES ISOLATED FROM SKIN CELLS AND METHODS FOR
THEIR USE
Technical Field ofthe Invention
This invention relates to polynucleotides encoding polypeptides, polypeptides expressed in skin cells, and their use in therapeutic methods.
Background ofthe Invention
The skin is the largest organ in the body and serves as a protective cover. The loss of skin, as occurs in a badly burned person, may lead to death owing to the absence of a barrier against infection by external microbial organisms, as well as loss of body temperature and body fluids.
Skin tissue is composed of several layers. The outermost layer is the epidermis which is supported by a basement membrane and overlies the dermis. Beneath the dermis is loose connective tissue and fascia which cover muscles or bony tissue. The skin is a self-renewing tissue in that cells are constantly being formed and shed. The deepest cells of the epidermis are the basal cells, which are enriched in cells capable of replication. Such replicating cells are called progenitor or stem cells. Replicating cells in turn give rise to daughter cells called 'transit amplifying cells'. These cells undergo differentiation and maturation into keratinocytes (mature skin cells) as they move from the basal layer to the more superficial layers of the epidermis. In the process, keratinocytes become cornified and are ultimately shed from the skin surface. Other cells in the epidermis include melanocytes which synthesize melanin, the pigment responsible for protection against sunlight. The Langerhans cell also resides in the epidermis and functions as a cell which processes foreign proteins for presentation to the immune system. The dermis contains nerves, blood and lymphatic vessels, fibrous and fatty tissue.
Within the dermis are fibroblasts, macrophages and mast cells. Both the epidermis and dermis are penetrated by sweat, or sebaceous, glands and hair follicles. Each strand of hair is derived from a hair follicle. When hair is plucked out, the hair re-grows from epithelial cells directed by the dermal papillae ofthe hair follicle.
When the skin surface is breached, for example in a wound, the stem cells proliferate and daughter keratinocytes migrate across the wound to reseal the tissues. The skin cells therefore possess genes activated in response to trauma. The products of these genes include several growth factors, such as epidermal growth factor, which mediate the proliferation of skin cells. The genes that are activated in the skin, and the protein products of such genes, may be developed as agents for the treatment of skin wounds. Additional growth factors derived from skin cells may also influence growth of other cell types. As skin cancers are a disorder of the growth of skin cells, proteins derived from skin that regulate cellular growth may be developed as agents for the treatment of skin cancers. Skin derived proteins that regulate the production of melanin may be useful as agents which protect skin against unwanted effects of sunlight.
Keratinocytes are known to secrete cytokines and express various cell surface proteins. Cytokines and cell surface molecules are proteins which play an important role in the inflammatory response against infection and also in autoimmune diseases affecting the skin. Genes and their protein products that are expressed by skin cells may thus be developed into agents for the treatment of inflammatory disorders affecting the skin.
Hair is an important part of a person's individuality. Disorders of the skin may lead to hair loss. Alopecia areata is a disease characterized by the patchy loss of hair over the scalp. Total baldness is a side effect of drug treatment for cancer. The growth and development of hair are mediated by the effects of genes expressed in skin and dermal papillae. Such genes and their protein products may be usefully developed into agents for the treatment of disorders ofthe hair follicle. New treatments are required to hasten the healing of skin wounds, to prevent the loss of hair, enhance the re-growth of hair or removal of hair, and to treat autoimmune and inflammatory skin diseases more effectively and without adverse effects. More effective treatments of skin cancers are also required. There thus remains a need in the art for the identification and isolation of genes encoding proteins expressed in the skin, for use in the development of therapeutic agents for the treatment of disorders including those associated with skin. Summary of the Invention
The present invention provides polypeptides expressed in skin cells, together with polynucleotides encoding such polypeptides, expression vectors and host cells comprising such polynucleotides, and methods for their use.
In specific embodiments, isolated polynucleotides are provided that comprise a DNA sequence selected from the group consisting of: (a) sequences recited in SEQ ID NO: 1-14, 45-48, 64-68, 77-89, 118, 119, 198-231, 239-249, 254-274, 349-372 and 399- 405; (b) complements ofthe sequences recited in SEQ ID NO: 1-14, 45-48, 64-68, 77-89, 118, 119, 198-231, 239-249, 254-274, 349-372 and 399-405; (c) reverse complements of the sequences recited in SEQ ID NO: 1-14, 45-48, 64-68, 77-89, 118, 119, 198-231, 239- 249, 254-274, 349-372 and 399-405; (d) reverse sequences of the sequences recited in SEQ ID NO: 1-14, 45-48, 64-68, 77-89, 118, 119, 198-231, 239-249, 254-274, 349-372 and 399-405; (e) sequences having a 99% probability of being the same as a sequence of (a)-(d); and (f) sequences having at least 50%o, 75% or 90% identity to a sequence of (a)-(d).
In further embodiments, the present invention provides isolated polypeptides comprising an amino acid sequence selected from the group consistmg of: (a) sequences provided in SEQ ID NO: 120-197, 275-348, 373-398 and 406-409; and (b) sequences having at least 50%, 75% or 90%) identity to a sequence provided in SEQ ID NO: 120- 197, 275-348, 373-398 and 406-409, together with isolated polynucleotides encoding such polypeptides. Isolated polypeptides which comprise at least a functional portion of a polypeptide comprising an amino acid sequence selected from the group consisting of: (a) sequences provided in SEQ ID NO: 120-197, 275-348, 373-398 and 406-409; and (b) sequences having 50%, 75% or 90% identity to a sequence of SEQ ID NO: 120-197, 275-348, 373-398 and 406-409 are also provided.
In related embodiments, the present invention provides expression vectors comprising the above polynucleotides, together with host cells transformed with such vectors. In a further aspect, the present invention provides a method of stimulating keratinocyte growth and motility, inhibiting the growth of epithelial-derived cancer cells, inhibiting angiogenesis and vascularization of tumors, or modulating the growth of blood vessels in a subject, comprising administering to the subject a composition comprising an isolated polypeptide, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of: (a) sequences provided in SEQ ID NO: 187, 196, 342, 343, 395, 397, and 398; and (b) sequences having at least 50%, 75% or 90% identity to a sequence provided in SEQ ID NO: 187, 196, 342, 343, 395, 397, and 398.
Methods for modulating skin inflammation in a subject are also provided, the methods comprising administering to the subject a composition comprising an isolated polypeptide, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of: (a) sequences provided in SEQ ID NO: 338 and 347; and (b) sequences having at least 50%, 75% or 90%) identity to a sequence provided in SEQ ID NO: 338 and 347. In an additional aspect, the present invention provides methods for stimulating the growth of epithelial cells in a subject. Such methods comprise administering to the subject a composition comprising an isolated polypeptide including an amino acid sequence selected from the group consisting of: (a) sequences provided in SEQ ID NO: 129 and 348; and (b) sequences having at least 50%, 75% or 90% identity to a sequence provided in SEQ ID NO: 129 and 348. In yet a further aspect, methods for inhibiting the binding of HIV-1 to leukocytes, for the treatment of an inflammatory disease or for the treatment of cancer in a subject are provided, the methods comprising administering to the subject a composition comprising an isolated polypeptide including an amino acid sequence selected from the group consisting of: (a) sequences provided in SEQ ID NO: 340, 344, 345 and 346; and (b) sequences having at least 50%, 75% or 90% identity to a sequence provided in SEQ ID NO: 340, 344, 345 and 346.
As detailed below, the isolated polynucleotides and polypeptides of the present invention may be usefully employed in the preparation of therapeutic agents for the treatment of skin disorders.
The above-mentioned and additional features of the present invention, together with the manner of obtaining them, will be best understood by reference to the following more detailed description. All references disclosed herein are hereby incorporated herein by reference in their entirety as if each was incorporated individually. Brief Description ofthe Drawings
Fig. 1 shows the results of a Northern analysis of the distribution of huTRl mRNA in human tissues. Key: He, Heart; Br, Brain; PI, Placenta; Lu, Lung; Li, Liver; SM, Skeletal muscle; Ki, Kidney; Sp, Spleen; Th, Thymus; Pr, Prostate; Ov, Ovary.
Fig. 2 shows the results of a MAP kinase assay of muTRla and huTRla. MuTRla (500ng/ml), huTRla (lOOng/ml) or LPS (3pg/ml) were added as described in the text.
Fig. 3 shows the stimulation of growth of neonatal foreskin keratinocytes by muTRla.
Fig. 4 shows the stimulation of growth ofthe transformed human keratinocyte cell line HaCaT by muTRla and huTRla.
Fig. 5 shows the inhibition of growth of the human epidermal carcinoma cell line A431 by muTRla and huTRla. Fig. 6 shows the inhibition of IL-2 induced growth of concanavalin A-stimulated murine splenocytes by KS2a.
Fig. 7 shows the stimulation of growth of rat intestinal epithelial cells (IEC-18) by a combination of KS3a plus apo-transferrin.
Fig. 8 illustrates the oxidative burst effect of TR-1 (100 ng/ml), muKSl (100 ng/ml), SDFlα (100 ng/ml), and fMLP (10 μM) on human PBMC.
Figure 9 shows the chemotactic effect of muKSl and SDF-lα on THP-1 cells.
Figure 10 shows the induction of cellular infiltrate in C3H/HeJ mice after intraperitoneal injections with muKSl (50 μg), GV14B (50 μg) and PBS.
Figure 11 demonstrates the induction of phosphorylation of ERK1 and ERK2 in CV1/EBNA and HeLa cell lines by huTRla.
Figure 12 shows the huTRl mRNA expression in HeLa cells after stimulation by muTRl, huTRl, huTGFα and PBS (100 ng/ml each).
Figure 13 shows activation of the SRE by muTRla in PC-12 (Fig. 13a) and HaCaT (Fig. 13b) cells. Figure 14 shows the inhibition of huTRla mediated growth on HaCaT cells by an antibody to the EGF receptor. Detailed Description ofthe Invention
In one aspect, the present invention provides polynucleotides that were isolated from mammalian skin cells. As used herein, the term "polynucleotide" means a single or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases and includes DNA and RNA molecules, both sense and anti-sense strands. The term comprehends cDNA, genomic DNA, recombinant DNA and wholly or partially synthesized nucleic acid molecules. A polynucleotide may consist of an entire gene, or a portion thereof. A gene is a DNA sequence that codes for a functional protein or RNA molecule. Operable anti-sense polynucleotides may comprise a fragment ofthe corresponding polynucleotide, and the definition of "polynucleotide" therefore includes all operable anti-sense fragments. Anti-sense polynucleotides and techniques involving anti-sense polynucleotides are well known in the art and are described, for example, in Robinson- Benion et al., "Anti-sense Techniques," Methods in Enzymol. 254(23):363-375, 1995; and Kawasaki et al., Artific. Organs 20 (8):836-848, 1996.
Identification of genomic DNA and heterologous species DNAs can be accomplished by standard DNA/DNA hybridization techniques, under appropriately stringent conditions, using all or part of a cDNA sequence as a probe to screen an appropriate library. Alternatively, PCR techniques using oligonucleotide primers that are designed based on known genomic DNA, cDNA and protein sequences can be used to amplify and identify genomic and cDNA sequences. Synthetic DNAs corresponding to the identified sequences and variants may be produced by conventional synthesis methods. All the polynucleotides provided by the present invention are isolated and purified, as those terms are commonly used in the art.
In specific embodiments, the polynucleotides of the present invention comprise a DNA sequence selected from the group consisting of sequences provided in SEQ ID NO: 1-119, 198-274, 349-372 and 399-405, and variants of the sequences of SEQ ID NO: 1-119, 198-274, 349-372 and 399-405. Polynucleotides that comprise complements of such DNA sequences, reverse complements of such DNA sequences, or reverse sequences of such DNA sequences, together with variants of such sequences, are also provided.
The definition of the terms "complement," "reverse complement," and "reverse sequence," as used herein, is best illustrated by the following example. For the sequence 5' AGGACC 3', the complement, reverse complement, and reverse sequence are as follows: complement 3 ' TCCTGG 5 ' reverse complement 3' GGTCCT 5' reverse sequence 5' CCAGGA 3'. In another aspect, the present invention provides isolated polypeptides encoded, or partially encoded, by the above polynucleotides. As used herein, the term "polypeptide" encompasses amino acid chains of any length, including full length proteins, wherein the amino acid residues are linked by covalent peptide bonds. The term "polypeptide encoded by a polynucleotide" as used herein, includes polypeptides encoded by a polynucleotide which comprises a partial isolated DNA sequence provided herein. In specific embodiments, the inventive polypeptides comprise an amino acid sequence selected from the group consisting of sequences provided in SEQ ID NO: 120-197, 275-348, 373-398 and 406-409, as well as variants of such sequences.
Polypeptides of the present invention may be produced recombinantly by inserting a DNA sequence that encodes the polypeptide into an expression vector and expressing the polypeptide in an appropriate host. Any of a variety of expression vectors known to those of ordinary skill in the art may be employed. Expression may be achieved in any appropriate host cell that has been transformed or transfected with an expression vector containing a DNA molecule that encodes a recombinant polypeptide. Suitable host cells include prokaryotes, yeast, and higher eukaryotic cells. Preferably, the host cells employed are E. coli, insect, yeast, or a mammalian cell line such as COS or CHO. The DNA sequences expressed in this manner may encode naturally occurring polypeptides, portions of naturally occurring polypeptides, or other variants thereof.
In a related aspect, polypeptides are provided that comprise at least a functional portion of a polypeptide having an amino acid sequence selected from the group consisting of sequences provided in SEQ ID NO: 120-197, 275-348, 373-398, 406-409, and variants thereof. As used herein, the "functional portion" of a polypeptide is that portion which contains the active site essential for affecting the function of the polypeptide, for example, the portion of the molecule that is capable of binding one or more reactants. The active site may be made up of separate portions present on one or more polypeptide chains and will generally exhibit high binding affinity.
Functional portions of a polypeptide may be identified by first preparing fragments of the polypeptide by either chemical or enzymatic digestion of the polypeptide, or by mutation analysis of the polynucleotide that encodes the polypeptide and subsequent expression of the resulting mutant polypeptides. The polypeptide fragments or mutant polypeptides are then tested to determine which portions retain biological activity, using, for example, the representative assays provided below.
Portions and other variants of the inventive polypeptides may also be generated by synthetic or recombinant means. Synthetic polypeptides having fewer than about 100 amino acids, and generally fewer than about 50 amino acids, may be generated using techniques well known to those of ordinary skill in the art. For example, such polypeptides may be synthesized using any of the commercially available solid-phase techniques, such as the Merrifield solid-phase synthesis method, where amino acids are sequentially added to a growing amino acid chain. See Merrifield, J. Am. Chem. Soc. 85:2149-2146, 1963. Equipment for automated synthesis of polypeptides is commercially available from suppliers such as Perkin Elmer/ Applied BioSystems, Inc. (Foster City, California), and may be operated according to the manufacturer's instructions. Variants of a native polypeptide may be prepared using standard mutagenesis techniques, such as oligonucleotide-directed site-specific mutagenesis (Kunkel, T., Proc. Natl. Acad. Sci. USA 82:488-492, 1985). Sections of DNA sequence may also be removed using standard techniques to permit preparation of truncated polypeptides.
In general, the polypeptides disclosed herein are prepared in an isolated, substantially pure, form. Preferably, the polypeptides are at least about 80% pure, more preferably at least about 90% pure, and most preferably at least about 99%) pure. In certain preferred embodiments, described in detail below, the isolated polypeptides are incorporated into pharmaceutical compositions or vaccines for use in the treatment of skin disorders.
As used herein, the term "variant" comprehends nucleotide or amino acid sequences different from the specifically identified sequences, wherein one or more nucleotides or amino acid residues is deleted, substituted, or added. Variants may be naturally occurring allelic variants, or non-naturally occurring variants. Variant sequences (polynucleotide or polypeptide) preferably exhibit at least 50%o, more preferably at least 75%, and most preferably at least 90%> identity to a sequence of the present invention. The percentage identity is determined by aligning the two sequences to be compared as described below, determining the number of identical residues in the aligned portion, dividing that number by the total number of residues in the inventive (queried) sequence, and multiplying the result by 100.
Polynucleotide or polypeptide sequences may be aligned, and percentage of identical nucleotides in a specified region may be determined against another polynucleotide or polypeptide, using computer algorithms that are publicly available. Two exemplary algorithms for aligning and identifying the similarity of polynucleotide sequences are the BLASTN and FASTA algorithms. The alignment and similarity of polypeptide sequences may be examined using the BLASTP and algorithm. BLASTX and FASTX algorithms compare nucleotide query sequences translated in all reading frames against polypeptide sequences. The BLASTN, BLASTP and BLASTX algorithms are available on the NCBI anonymous FTP server (ftp ://ncbi .nlm.nih. ov) under /blast/executables/. The FASTA and FASTX algorithms are available on the Internet at the ftp site ftp://ftp.virginia.edu/pub/. The FASTA algorithm, set to the default parameters described in the documentation and distributed with the algorithm, may be used in the determination of polynucleotide variants. The readme files for FASTA and FASTX vl.Ox that are distributed with the algorithms describe the use of the algorithms and describe the default parameters. The use of the FASTA and FASTX algorithms is also described in Pearson, WR and Lipman, DJ, "Improved Tools for Biological Sequence Analysis," PNAS 85:2444-2448, 1988; and Pearson WR, "Rapid and Sensitive Sequence Comparison with FASTP and FASTA," Methods in Enzymology 183:63-98, 1990. The BLASTN algorithm version 2.0.4 [Feb-24-1998], set to the default parameters described in the documentation and distributed with the algorithm, is preferred for use in the determination of polynucleotide variants according to the present invention. The BLASTP algorithm version 2.0.4, set to the default parameters described in the documentation and distributed with the algorithm, is preferred for use in the determination of polypeptide variants according to the present invention. The use of the BLAST family of algorithms, including BLASTN, BLASTP and BLASTX is described at NCBI's website at URL http://www.ncbi.nlm.nih.gov/BLAST/newblast.html and in the publication of Altschul, Stephen F., et al, "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs," Nucleic Acids Res. 25:3389-3402, 1997.
The following running parameters are preferred for determination of alignments and similarities using BLASTN that contribute to the E values and percentage identity for polynucleotides: Unix running command with default parameters thus: blastall -p blastn - d embldb -e 10 -G 0 -E 0 -r 1 -v 30 -b 30 -i queryseq -o results; and parameters are: -p Program Name [String]; -d Database [String]; -e Expectation value (E) [Real]; -G Cost to open a gap (zero invokes default behavior) [Integer]; -E Cost to extend a gap (zero invokes default behavior) [Integer]; -r Reward for a nucleotide match (blastn only) [Integer]; -v Number of one-line descriptions (V) [Integer]; -b Number of alignments to show (B) [Integer]; -i Query File [File In]; -o BLAST report Output File [File Out] Optional. The following running parameters are preferred for determination of alignments and similarities using BLASTP that contribute to the E values and percentage identity for polypeptides: blastall -p blastp -d swissprotdb -e 10 -G 1 -E 11 -r 1 -v 30 -b 30 -i queryseq -o results; and the parameters are: -p Program Name [String]; -d Database [String]; -e Expectation value (E) [Real]; -G Cost to open a gap (zero invokes default behavior) [Integer]; -E Cost to extend a gap (zero invokes default behavior) [Integer]; -v Number of one-line descriptions (v) [Integer]; -b Number of alignments to show (b) [Integer]; -I Query File [File In]; -o BLAST report Output File [File Out] Optional.
The "hits" to one or more database sequences by a queried sequence produced by BLASTN, BLASTP, FASTA, or a similar algorithm, align and identify similar portions of sequences. The hits are arranged in order of the degree of similarity and the length of
10 sequence overlap. Hits to a database sequence generally represent an overlap over only a fraction ofthe sequence length ofthe queried sequence.
The percentage similarity of a polynucleotide or polypeptide sequence is determined by aligning polynucleotide and polypeptide sequences using appropriate algorithms, such as BLASTN or BLASTP, respectively, set to default parameters; identifying the number of identical nucleic or amino acids over the aligned portions; dividing the number of identical nucleic or amino acids by the total number of nucleic or amino acids of the polynucleotide or polypeptide of the present invention; and then multiplying by 100 to determine the percentage similarity. By way of example, a queried polynucleotide having 220 nucleic acids has a hit to a polynucleotide sequence in the EMBL database having 520 nucleic acids over a stretch of 23 nucleotides in the alignment produced by the BLASTN algorithm using the default parameters. The 23 nucleotide hit includes 21 identical nucleotides, one gap and one different nucleotide. The percentage identity of the queried polynucleotide to the hit in the EMBL database is thus 21/220 times 100, or 9.5%. The similarity of polypeptide sequences may be determined in a similar fashion.
The BLASTN and BLASTX algorithms also produce "Expect" values for polynucleotide and polypeptide alignments. The Expect value (E) indicates the number of hits one can "expect" to see over a certain number of contiguous sequences by chance when searching a database of a certain size. The Expect value is used as a significance threshold for determining whether the hit to a database indicates true similarity. For example, an E value of 0.1 assigned to a polynucleotide hit is interpreted as meaning that in a database of the size of the EMBL database, one might expect to see 0.1 matches over the aligned portion of the sequence with a similar score simply by chance. By this criterion, the aligned and matched portions of the sequences then have a probability of 90%) of being the same. For sequences having an E value of 0.01 or less over aligned and matched portions, the probability of finding a match by chance in the EMBL database is 1%) or less using the BLASTN algorithm. E values for polypeptide sequences may be determined in a similar fashion using various polypeptide databases, such as the SwissProt database.
11 According to one embodiment, "variant" polynucleotides and polypeptides, with reference to each of the polynucleotides and polypeptides of the present invention, preferably comprise sequences having the same number or fewer nucleic or amino acids than each of the polynucleotides or polypeptides of the present invention and producing an E value of 0.01 or less when compared to the polynucleotide or polypeptide of the present invention. That is, a variant polynucleotide or polypeptide is any sequence that has at least a 99%> probability of being the same as the polynucleotide or polypeptide of the present invention, measured as having an E value of 0.01 or less using the BLASTN or BLASTX algorithms set at the default parameters. According to a preferred embodiment, a variant polynucleotide is a sequence having the same number or fewer nucleic acids than a polynucleotide of the present invention that has at least a 99%o probability of being the same as the polynucleotide of the present invention, measured as having an E value of 0.01 or less using the BLASTN algorithm set at the default parameters. Similarly, according to a preferred embodiment, a variant polypeptide is a sequence having the same number or fewer amino acids than a polypeptide of the present invention that has at least a 99%> probability of being the same as the polypeptide of the present invention, measured as having an E value of 0.01 or less using the BLASTP algorithm set at the default parameters.
Variant polynucleotide sequences will generally hybridize to the recited polynucleotide sequences under stringent conditions. As used herein, "stringent conditions" refers to prewashing in a solution of 6X SSC, 0.2%o SDS; hybridizing at 65°C, 6X SSC, 0.2% SDS overnight; followed by two washes of 30 minutes each in IX SSC, 0.1% SDS at 65 °C and two washes of 30 minutes each in 0.2X SSC, 0.1% SDS at 65 °C. As used herein, the term "x-mer," with reference to a specific value of " ," refers to a polynucleotide or polypeptide, respectively, comprising at least a specified number (" ") of contiguous residues of: any of the polynucleotides provided in SEQ ID NO: 1-119, 198-274, 349-372 and 399-405; or any of the polypeptides set out in SEQ ID NO: 120-197, 275-348, 373-398 and 406-409. The value of x may be from about 20 to about 600, depending upon the specific sequence.
12 Polynucleotides of the present invention comprehend polynucleotides comprising at least a specified number of contiguous residues (x-mers) of any of the polynucleotides identified as SEQ ID NO: 1-119, 198-274, 349-372 and 399-405, or their variants. Polypeptides of the present invention comprehend polypeptides comprising at least a specified number of contiguous residues (x-mers) of any of the polypeptides identified as SEQ ID NO: 120-197, 275-348, 373-398, and 406-409. According to preferred embodiments, the value of x is at least 20, more preferably at least 40, more preferably yet at least 60, and most preferably at least 80. Thus, polynucleotides of the present invention include polynucleotides comprising a 20-mer, a 40-mer, a 60-mer, an 80-mer, a 100-mer, a 120-mer, a 150-mer, a 180-mer, a 220-mer, a 250-mer; or a 300-mer, 400-mer, 500-mer or 600-mer of a polynucleotide provided in SEQ ID NO: 1-119, 198-274, 349-372 and 399-405 or a variant of one of the polynucleotides provided in SEQ ID NO: 1-119, 198-274, 349-372, and 399-405. Polypeptides of the present invention include polypeptides comprising a 20-mer, a 40-mer, a 60-mer, an 80-mer, a 100-mer, a 120-mer, a 150-mer, a 180-mer, a 220-mer, a 250-mer; or a 300-mer, 400-mer, 500-mer or 600-mer of a polypeptide provided in SEQ ID NO: 120-197, 275-348, 373-398, and 406-409, or a variant of one of the polynucleotides provided in SEQ ID NO: 120-197, 275-348, 373-398, and 406-409.
The inventive polynucleotides may be isolated by high throughput sequencing of cDNA libraries prepared from mammalian skin cells as described below in Example 1. Alternatively, oligonucleotide probes based on the sequences provided in SEQ ID NO: 1-119, 198-274, 349-372, and 399-405 can be synthesized and used to identify positive clones in either cDNA or genomic DNA libraries from mammalian skin cells by means of hybridization or polymerase chain reaction (PCR) techniques. Probes can be shorter than the sequences provided herein but should be at least about 10, preferably at least about 15 and most preferably at least about 20 nucleotides in length. Hybridization and PCR techniques suitable for use with such oligonucleotide probes are well known in the art (see, for example, Mullis, et al., Cold Spring Harbor Symp. Quant. Biol., 51 :263, 1987; Erlich, ed., PCR Technology, Stockton Press: NY, 1989; (Sambrook, J, Fritsch, EF and Maniatis, T, eds., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring
13 Harbor Laboratory Press, Cold Spring Harbor: New York, 1989). Positive clones may be analyzed by restriction enzyme digestion, DNA sequencing or the like.
In addition, DNA sequences of the present invention may be generated by synthetic means using techniques well known in the art. Equipment for automated synthesis of oligonucleotides is commercially available from suppliers such as Perkin Elmer/Applied Biosystems Division (Foster City, California) and may be operated according to the manufacturer's instructions.
Since the polynucleotide sequences of the present invention have been derived from skin, they likely encode proteins that have important roles in growth and development of skin, and in responses of skin to tissue injury and inflammation as well as disease states. Some of the polynucleotides contain sequences that code for signal sequences, or transmembrane domains, which identify the protein products as secreted molecules or receptors. Such protein products are likely to be growth factors, cytokines, or their cognate receptors. Several of the polypeptide sequences have more than 25% similarity to known biologically important proteins and thus are likely to represent proteins having similar biological functions.
In particular, the inventive polypeptides have important roles in processes such as: induction of hair growth; differentiation of skin stem cells into specialized cell types; cell migration; cell proliferation and cell-cell interaction. The polypeptides are important in the maintenance of tissue integrity, and thus are important in processes such as wound healing. Some of the disclosed polypeptides act as modulators of immune responses, especially since immune cells are known to infiltrate skin during tissue insult causing growth and differentiation of skin cells. In addition, many polypeptides are immunologically active, making them important therapeutic targets in a whole range of disease states not only within skin, but also in other tissues ofthe body. Antibodies to the polypeptides of the present invention and small molecule inhibitors related to the polypeptides of the present invention may also be used for modulating immune responses and for treatment of diseases according to the present invention.
In one aspect, the present invention provides methods for using one or more of the inventive polypeptides or polynucleotides to treat disorders in a patient. As used herein, a "patient" refers to any warm-blooded animal, preferably a human.
14 In this aspect, the polypeptide or polynucleotide is generally present within a pharmaceutical composition or a vaccine. Pharmaceutical compositions may comprise one or more polypeptides, each of which may contain one or more of the above sequences (or variants thereof), and a physiologically acceptable carrier. Vaccines may comprise one or more of the above polypeptides and a non-specific immune response amplifier, such as an adjuvant or a liposome, into which the polypeptide is incorporated.
Alternatively, a vaccine or pharmaceutical composition of the present invention may contain DNA encoding one or more polypeptides as described above, such that the polypeptide is generated in situ. In such vaccines and pharmaceutical compositions, the DNA may be present within any of a variety of delivery systems known to those of ordinary skill in the art, including nucleic acid expression systems, and bacterial and viral expression systems. Appropriate nucleic acid expression systems contain the necessary DNA sequences for expression in the patient (such as a suitable promoter and terminator signal). Bacterial delivery systems involve the administration of a bacterium (such as Bacillus-Calmette-Guerin) that expresses an immunogenic portion of the polypeptide on its cell surface. In a preferred embodiment, the DNA may be introduced using a viral expression system (e.g., vaccinia or other poxvirus, retro virus, or adenovirus), which may involve the use of a non-pathogenic, or defective, replication competent virus. Techniques for incorporating DNA into such expression systems are well known in the art. The DNA may also be "naked," as described, for example, in Ulmer, et al., Science 259:1745-1749, 1993 and reviewed by Cohen, Science 259:1691-1692, 1993. The uptake of naked DNA may be increased by coating the DNA onto biodegradable beads, which are efficiently transported into the cells.
Routes and frequency of administration, as well as dosage, will vary from individual to individual. In general, the pharmaceutical compositions and vaccines may be administered by injection (e.g., mtradermal, intramuscular, intravenous, or subcutaneous), intranasally (e.g., by aspiration) or orally. In general, the amount of polypeptide present in a dose (or produced in situ by the DNA in a dose) ranges from about 1 pg to about 100 mg per kg of host, typically from about 10 pg to about 1 mg per kg of host, and preferably from about 100 pg to about 1 μg per kg of host. Suitable dose
15 sizes will vary with the size of the patient, but will typically range from about 0.1 ml to about 5 ml.
While any suitable carrier known to those of ordinary skill in the art may be employed in the pharmaceutical compositions of this invention, the type of carrier will vary depending on the mode of administration. For parenteral administration, such as subcutaneous injection, the carrier preferably comprises water, saline, alcohol, a lipid, a wax, or a buffer. For oral administration, any of the above carriers or a solid carrier, such as mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, sucrose, and magnesium carbonate, may be employed. Biodegradable microspheres (e.g., polylactic galactide) may also be employed as carriers for the pharmaceutical compositions of this invention. Suitable biodegradable microspheres are disclosed, for example, in U.S. Patent Nos. 4,897,268 and 5,075,109.
Any of a variety of adjuvants may be employed in the vaccines derived from this invention to non-specifically enhance the immune response. Most adjuvants contain a substance designed to protect the antigen from rapid catabolism, such as aluminum hydroxide or mineral oil, and a non-specific stimulator of immune responses, such as lipid A, Bordetella pertussis, or M. tuberculosis. Suitable adjuvants are commercially available as, for example, Freund's Incomplete Adjuvant and Freund's Complete Adjuvant (Difco Laboratories, Detroit, Michigan), and Merck Adjuvant 65 (Merck and Company, Inc., Rahway, New Jersey). Other suitable adjuvants include alum, biodegradable microspheres, monophosphoryl lipid A, and Quil A.
The polynucleotides of the present invention may also be used as markers for tissue, as chromosome markers or tags, in the identification of genetic disorders, and for the design of oligonucleotides for examination of expression patterns using techniques well known in the art, such as the microarray technology available from Synteni (Palo Alto, California). Partial polynucleotide sequences disclosed herein may be employed to obtain full length genes by, for example, screening of DNA expression libraries using hybridization probes or PCR primers based on the inventive sequences.
The polypeptides provided by the present invention may additionally be used in assays to determine biological activity, to raise antibodies, to isolate corresponding Hgands or receptors, in assays to quantitatively determine levels of protein or cognate
16 corresponding ligand or receptor, as anti-inflammatory agents, and in compositions for skin, connective tissue and/or nerve tissue growth or regeneration.
Example 1 ISOLATION OF CDNA SEQUENCES FROM SKIN CELL EXPRESSION LIBRARIES
The cDNA sequences of the present invention were obtained by high-throughput sequencing of cDNA expression libraries constructed from specialized rodent or human skin cells as shown in Table 1.
Table 1 Library Skin cell type Source
DEPA dermal papilla rat
SKTC keratinocytes human
HNFF neonatal foreskin fibroblast human
MEMS embryonic skin mouse
KSCL keratinocyte stem cell mouse
TRAM transit amplifying cells mouse
These cDNA libraries were prepared as described below. cDNA Library from Dermal Papilla (DEPA)
Dermal papilla cells from rat hair vibrissae (whiskers) were grown in culture and the total RNA extracted from these cells using established protocols. Total RNA, isolated using TRIzol Reagent (BRL Life Technologies, Gaithersburg, Maryland), was used to obtain mRNA using a Poly(A) Quik mRNA isolation kit (Stratagene, La Jolla, California), according to the manufacturer's specifications. A cDNA expression library was then prepared from the mRNA by reverse transcriptase synthesis using a Lambda
ZAP cDNA library synthesis kit (Stratagene). cDNA Library from Keratinocytes (SKTC)
Keratinocytes obtained from human neonatal foreskins (Mitra, R and Nikoloff, B in Handbook of Keratinocyte Methods, pp. 17-24, 1994) were grown in serum-free
KSFM (BRL Life Technologies) and harvested along with differentiated cells (10 cells). Keratinocytes were allowed to differentiate by addition of fetal calf serum at a final
17 concentration of 10%> to the culture medium and cells were harvested after 48 hours. Total RNA was isolated from the two cell populations using TRIzol Reagent (BRL Life Technologies) and used to obtain mRNA using a Poly(A) Quik mRNA isolation kit (Stratagene). cDNAs expressed in differentiated keratinocytes were enriched by using a PCR-Select cDNA Subtraction Kit (Clontech, Palo Alto, California). Briefly, mRNA was obtained from either undifferentiated keratinocytes ("driver mRNA") or differentiated keratinocytes ("tester mRNA") and used to synthesize cDNA. The two populations of cDNA were separately digested with Rsal to obtain shorter, blunt-ended molecules. Two tester populations were created by ligating different adaptors at the cDNA ends and two successive rounds of hybridization were performed with an excess of driver cDNA. The adaptors allowed for PCR amplification of only the differentially expressed sequences which were then ligated into T-tailed pBluescript (Hadjeb, N and Berkowitz, GA, BioTechniques 20:20-22 1996), allowing for a blue/white selection of cells containing vector with inserts. White cells were isolated and used to obtain plasmid DNA for sequencing. cDNA library from human neonatal fibroblasts (HNFF)
Human neonatal fibroblast cells were grown in culture from explants of human neonatal foreskin and the total RNA extracted from these cells using established protocols. Total RNA, isolated using TRIzol Reagent (BRL Life Technologies, Gaithersburg, Maryland), was used to obtain mRNA using a Poly(A) Quik mRNA isolation kit (Stratagene, La Jolla, California), according to the manufacturer's specifications. A cDNA expression library was then prepared from the mRNA by reverse transcriptase synthesis using a Lambda ZAP cDNA library synthesis kit (Stratagene). cDNA library from mouse embryonic skin (MEMS) Embryonic skin was micro-dissected from day 13 post coitum Balb/c mice.
Embryonic skin was washed in phosphate buffered saline and mRNA directly isolated from the tissue using the Quick Prep Micro mRNA purification kit (Pharmacia, Sweden). The mRNA was then used to prepare cDNA libraries as described above for the DEPA library. cDNA library from mouse stem cells (KSCL) and transit amplifying (TRAM) cells
Pelts obtained from 1-2 day post-partum neonatal Balb/c mice were washed and incubated in trypsin (BRL Life Technologies) to separate the epidermis from the dermis. Epidermal tissue was disrupted to disperse cells, which were then resuspended in growth medium and centrifuged over Percoll density gradients prepared according to the manufacturer's protocol (Pharmacia, Sweden). Pelleted cells were labeled using Rhodamine 123 (Bertoncello I, Hodgson GS and Bradley TR, Exp Hematol. 13:999- 1006, 1985), and analyzed by flow cytometry (Epics Elite Coulter Cytometry, Hialeah, Florida). Single cell suspensions of rhodamine-labeled murine keratinocytes were then labeled with a cross reactive anti-rat CD29 biotin monoclonal antibody (Pharmingen, San Diego, California; clone Ha2/5). Cells were washed and incubated with anti-mouse CD45 phycoerythrin conjugated monoclonal antibody (Pharmingen; clone 30F11.1, lOug/ml) followed by labeling with streptavidin spectral red (Southern Biotechnology, Birmingham, Alabama). Sort gates were defined using listmode data to identify four populations: CD29 bright rhodamine dull CD45 negative cells; CD29 bright rhodamine bright CD45 negative cells; CD29 dull rhodamine bright CD45 negative cells; and CD29 dull rhodamine dull CD45 negative cells. Cells were sorted, pelleted and snap frozen prior to storage at -80°C. This protocol was followed multiple times to obtain sufficient cell numbers of each population to prepare cDNA libraries. Skin stem cells and transit amplifying cells are known to express CD29, the integrin βl chain. CD45, a leucocyte specific antigen, was used as a marker for cells to be excluded in the isolation of skin stem cells and transit amplifying cells. Keratinocyte stem cells expel the rhodamine dye more efficiently than transit amplifying cells. The CD29 bright, rhodamine dull, CD45 negative population (putative keratinocyte stem cells; referred to as KSCL), and the CD29 bright, rhodamine bright, CD45 negative population (keratinocyte transit amplifying cells; referred to as TRAM) were sorted and mRNA was directly isolated from each cell population using the Quick Prep Micro mRNA purification kit (Pharmacia, Sweden). The mRNA was then used to prepare cDNA libraries as described above for the DEPA library. cDNA sequences were obtained by high-throughput sequencing of the cDNA libraries described above using a Perkin Elmer/ Applied Biosystems Division Prism 377 sequencer.
19 Example 2 CHARACTERIZATION OF ISOLATED cDNA SEQUENCES The isolated cDNA sequences were compared to sequences in the EMBL DNA database using the computer algorithms FASTA and or BLASTN. The corresponding predicted protein sequences (DNA translated to protein in each of 6 reading frames) were compared to sequences in the SwissProt database using the computer algorithms FASTX and/or BLASTP. Comparisons of DNA sequences provided in SEQ ID NO: 1-119 to sequences in the EMBL DNA database (using FASTA) and amino acid sequences provided in SEQ ID NO: 120-197 to sequences in the SwissProt database (using FASTX) were made as of March 21, 1998. Comparisons of DNA sequences provided in SEQ ID NO: 198-274 to sequences in the EMBL DNA database (using BLASTN) and amino acid sequences provided in SEQ ID NO: 275-348 to sequences in the SwissProt database (using BLASTP) were made as of October 7, 1998. Comparisons of DNA sequences provided in SEQ ID NO: 349-372 to sequences in the EMBL DNA database (using BLASTN) and amino acid sequences provided in SEQ ID NO: 373-398 to sequences in the SwissProt database (using BLASTP) were made as of January 23, 1999.
Isolated cDNA sequences and their corresponding predicted protein sequences were computer analyzed for the presence of signal sequences identifying secreted molecules. Isolated cDNA sequences that have a signal sequence at a putative start site within the sequence are provided in SEQ ID NO: 1-44, 198-238, 349-358, and 399. The cDNA sequences of SEQ ID NO: 1-6, 198-199, 349-352, 354, and 356-358 were determined to have less than 75% identity (determined as described above), to sequences in the EMBL database using the computer algorithms FASTA or BLASTN, as described above. The predicted amino acid sequences of SEQ ID NO: 120-125, 275-276, 373-380, and 382 were determined to have less than 75% identity (determined as described above) to sequences in the SwissProt database using the computer algorithms FASTX or BLASTP, as described above.
Further sequencing ofthe some ofthe isolated partial cDNA sequences resulted in the isolation of the full-length cDNA sequences provided in SEQ ID NO: 7-14, 200-231, and 372. The corresponding predicted amino acid sequences are provided in SEQ ID NO: 126-133, 277-308, and 396, respectively. Comparison of the full length cDNA
20 sequences with those in the EMBL database using the computer algorithm FASTA or BLASTN, as described above, revealed less than 75%0 identity (determined as described above) to known sequences. Comparison ofthe predicted amino acid sequences provided in SEQ ID NO: 126-133 and 277-308 with those in the SwissProt database using the computer algorithms FASTX or BLASTP, as described above, revealed less than 75% identity (determined as described above) to known sequences.
Comparison of the predicted amino acid sequences corresponding to the cDNA sequences of SEQ ID NO: 15-23 with those in the EMBL using the computer algorithm FASTA database showed less than 75% identity (determined as described above) to known sequences. These predicted amino acid sequences are provided in SEQ ID NO: 134-142.
Further sequencing of some ofthe isolated partial cDNA sequences resulted in the isolation of full-length cDNA sequences provided in SEQ ID NO: 24-44 and 232-238. The corresponding predicted amino acid sequences are provided in SEQ LD NO: 143-163 and 309-315, respectively. These amino acid sequences were determined to have less than 75%o identity, determined as described above to known sequences in the SwissProt database using the computer algorithm FASTX.
Isolated cDNA sequences having less than 75% identity to known expressed sequence tags (ESTs) or to other DNA sequences in the public database, or whose corresponding predicted protein sequence showed less than 75% identity to known protein sequences, were computer analyzed for the presence of transmembrane domains coding for putative membrane-bound molecules. Isolated cDNA sequences that have either one or more transmembrane domain(s) within the sequence are provided in SEQ ID NO: 45-63, 239-253, 359-364, 400-402. The cDNA sequences of SEQ ID NO: 45-48, 239-249, 359-361, and 363 were found to have less than 75%o identity (determined as described above) to sequences in the EMBL database, using the FASTA or BLASTN computer algorithms. Their predicted amino acid sequences provided in SEQ ID NO: 164-167, 316-326, 383, 385-388 and 407-408 were found to have less than 75% identity, determined as described above, to sequences in the SwissProt database using the FASTX or BLASTP database.
21 Comparison of the predicted amino acid sequences corresponding to the cDNA sequences of SEQ ID NO: 49-63 and 250-253 with those in the SwissProt database showed less than 75% identity (determined as described above) to known sequences. These predicted amino acid sequences are provided in SEQ ID NO: 168-182 and 327-330.
Using automated search programs to screen against sequences coding for molecules reported to be of therapeutic and or diagnostic use, some of the cDNA sequences isolated as described above in Example 1 were determined to encode predicted protein sequences that appear to be family members of known protein families. A family member is here defined to have at least 25%o identity in the translated polypeptide to a known protein or member of a protein family. These cDNA sequences are provided in SEQ ID NO: 64-76, 254-264, 365-369, and 403, with the conespondmg predicted amino acid sequences being provided in SEQ ID NO: 183-195, 331-341, 389-393 and 409, respectively. The cDNA sequences of SEQ ID NO: 64-68, 254-264, and 365-369 show less than 75% identity (determined as described above) to sequences in the EMBL database using the FASTA or BLASTN computer algorithms. Similarly, the amino acid sequences of SEQ ID NO: 183-195, 331-341, and 389-393 show less than 75% identity to sequences in the SwissProt database.
The likely utility for each of the proteins encoded by the DNA sequences of SEQ ID NO: 64-76, 254-264, 365-369, and 403, based on similarity to known proteins, is provided below:
22 Table 2 FUNCTIONS OF NOVEL PROTEINS
P/N A/A
SEQ SEQ. SIMILARITY TO KNOWN PROTEINS
ID ID
NO: NO.
64 183 Slit, a secreted molecule required for central nervous system
372 396 development
65 184 Immunoglobulin receptor family. About 40%> of leucocyte membrane polypeptides contain immunoglobulin superfamily domains
66 185 RIP protein kinase, a serine/threonine kinase that contains a death
403 409 domain to mediate apoptosis
67 186 Extracellular protein with epidermal growth factor domain capable of stimulating fibroblast proliferation
68 187 Transforming growth factor alpha, a protein which binds epidermal growth factor receptor and stimulates growth and mobility of keratinocytes
69 188 DRS protein which has a secretion signal component and whose expression is suppressed in cells transformed by oncogenes
70 189 A33 receptor with immunoglobulin-like domains and is expressed in greater than 95% of colon tumors
71 190 Interleukin-12 alpha subunit, component of a cytokine that is important in the immune defense against intracellular pathogens. IL-12 also stimulates proliferation and differentiation of TH1 subset of lymphocytes
72 191 Tumor Necrosis Factor receptor family of proteins that are involved in the proliferation, differentiation and death of many cell types including B and T lymphocytes.
73 192 Epidermal growth factor family proteins which stimulate growth and mobility of keratinocytes and epithelial cells. EGF is involved in wound healing. It also inhibits gastric acid secretion.
74 193 Fibronectin Type III receptor family. The fibronectin III domains are found on the extracellular regions of cytokine receptors
75 194 Serine/threonine kinases (STK2_HUMAN) which participate in cell cycle progression and signal transduction
76 195 Immunoglobulin receptor family
254 331 Receptor with immunoglobul in-like domains and homology to A33 receptor which is expressed in greater than 95%> of colon tumors
255 332 Epidermal growth factor family proteins which stimulate growth and mobility of keratinocytes and epithelial cells. EGF is
Figure imgf000025_0001
involved in wound healing. It also inhibits gastric acid secretion.
23 P/N A/A
SEQ SEQ. SIMILARITY TO KNOWN PROTEINS
ID ID
NO: NO.
256 333 Serine/threonine kinases (STK2_HUMAN) which participate in cell cycle progression and signal transduction
257 334 Contains protein kinase and ankyrin domains. Possible role in cellular growth and differentiation.
258 335 Notch family proteins which are receptors involved in cellular differentiation.
259 336 Extracellular protein with epidermal growth factor domain capable of stimulating fibroblast proliferation.
260 337 Fibronectin Type III receptor family. The fibronectin III domains are found on the extracellular regions of cytokine receptors.
261 338 Immunoglobulin receptor family
339
262 ADP/ATP transporter family member containing a calcium binding site.
263 340 Mouse CXC chemokine family members are regulators of epithelial, lymphoid, myeloid, stromal and neuronal cell migration and cancers, agents for the healing of cancers, neurodegenerative diseases, wound healing, inflammatory autoimmune diseases like psoriasis, asthma, Crohns disease and as agents for the prevention of HIV- 1 of leukocytes
264 341 Nucleotide-sugar transporter family member.
365 389 Transforming growth factor betas (TGF-betas) are secreted covalently linked to latent TGF-beta-binding proteins (LTBPs). LTBPs are deposited in the extracellular matrix and play a role in cell growth or differentiation.
366 390 Integrins are Type I membrane proteins that function as laminin and collagen receptors and play a role in cell adhesion.
367 391 Integrins are Type I membrane proteins that function as laminin and collagen receptors and play a role in cell adhesion.
368 392 Cell wall protein precursor. Are involved in cellular growth or differentiation.
369 393 HT protein is a secreted glycoprotein with an EGF-like domain. It functions as a modulator of cell growth, death or
Figure imgf000026_0001
differentiation.
These isolated sequences thus encode proteins that influence the growth, differentiation and activation of several cell types. They may usefully be developed as
24 agents for the treatment and diagnosis of skin wounds, cancers, growth and developmental defects, and inflammatory disease.
The polynucleotide sequences of SEQ ID NO: 77-117, 265-267, and 404-405 are differentially expressed in either keratinocyte stem cells (KSCL) or in transit amplified cells (TRAM) on the basis of the number of times these sequences exclusively appear in either one of the above two libraries; more than 9 times in one and none in the other (Audic S. and Claverie J-M, Genome Research, 7:986-995, 1997). The sequences of SEQ ID NO: 77-89, 265-267, and 365-369 were determined to have less than 75% identity to sequences in the EMBL and SwissProt databases using the computer algorithm FASTA or BLASTN, as described above. The proteins encoded by these polynucleotide sequences have utility as markers for identification and isolation of these cell types, and antibodies against these proteins may be usefully employed in the isolation and enrichment of these cells from complex mixtures of cells. Isolated polynucleotides and their corresponding proteins exclusive to the stem cell population can be used as drug targets to cause alterations in regulation of growth and differentiation of skin cells, or in gene targeting to transport specific therapeutic molecules to skin stem cells.
Example 3 ISOLATION AND CHARACTERIZATION OF THE HUMAN HOMQLOG OF MUTRI The human homolog of muTRI (SEQ ID NO: 68), obtained as described above in
Example 1, was isolated by screening 50,000 pfu's of an oligo dT primed HeLa cell cDNA library. Plaque lifts, hybridization, and screening were performed using standard molecular biology techniques (Sambrook, J, Fritsch, EF and Maniatis, T, eds., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor: New York, 1989). The determined cDNA sequence of the isolated human homolog (huTRl) is provided in SEQ ID NO: 118, with the corresponding predicted amino acid sequence being provided in SEQ ID NO: 196. The library was screened using an [α P]-dCTP labeled double stranded cDNA probe corcesponding to nucleotides 1 to 459 ofthe coding region within SEQ ID NO: 118. The polypeptide sequence of huTRl has regions similar to Transforming Growth
Factor- alpha, indicating that this protein functions like an epidermal growth factor (EGF).
25 This EGF-like protein will serve to stimulate keratinocyte growth and motility, and to inhibit the growth of epithelial-derived cancer cells. This novel gene and its encoded protein may thus be used as agents for the healing of wounds and regulators of epithelial- derived cancers. Analysis of RNA transcripts by Northern Blottins
Northern analysis to determine the size and distribution of mRNA for huTRl was performed by probing human tissue mRNA blots (Clontech) with a probe comprising nucleotides 93-673 of SEQ ID NO: 118, radioactively labeled with [α32P]-dCTP. Prehybridization, hybridization, washing and probe labeling were performed as described in Sambrook, et al, Ibid. mRNA for huTRl was 3.5-4kb in size and was observed to be most abundant in heart and placenta, with expression at lower levels being observed in spleen, thymus prostate and ovary (Fig. 1).
The high abundance of mRNA for huTRl in the heart and placenta indicates a role for huTRl in the formation or maintenance of blood vessels, as heart and placental tissues have an increased abundance of blood vessels, and therefore endothelial cells, compared to other tissues in the body. This, in turn, demonstrates a role for huTRl in angiogenesis and vascularization of tumors. This is supported by the ability of Transforming Growth Factor-alpha and EGF to induce de novo development of blood vessels (Schreiber, et al, Science 232:1250-1253, 1986) and stimulate DNA synthesis in endothelial cells (Schreiber, et al, Science 232:1250-1253, 1986), and their over- expression in a variety of human tumors. Purification ofmuTRl and huTRl
Polynucleotides 177-329 of muTRI (SEQ ID NO: 268), encoding amino acids 53-103 of muTRI (SEQ ID NO: 342), and polynucleotides 208-360 of huTRl (SEQ ID NO: 269), encoding amino acids 54-104 of huTRl (SEQ ID NO: 343), were cloned into the bacterial expression vector pProEX HT (BRL Life Technologies), which contains a bacterial leader sequence and N-terminal όxHistidine tag. These constructs were transformed into competent XLl-Blue E. coli as described in Sambrook et al., Ibid. Starter cultures of these recombinant XLl-Blue E. coli were grown overnight at
37°C in Terrific broth containing 100 μg/ml ampicillin. This culture was spun down and
26 used to inoculate 500 ml culture of Terrific broth containing 100 μg/ml ampicillin. Cultures were grown until the OD595 of the cells was between 0.4 and 0.8, whereupon IPTG was added to 1 mM. Cells were induced overnight and bacteria were harvested by centrifugation. Both the polypeptide of muTRI (SEQ ID NO: 342; referred to as muTRla) and that of huTRl (SEQ ID NO: 343; referred to as huTRla) were expressed in insoluble inclusion bodies. In order to purify the polypeptides muTRla and huTRla, bacterial cell pellets were re-suspended in lysis buffer (20 mM Tris-HCl pH 8.0, 10 mM beta mercaptoethanol, 1 mM PMSF). To the lysed cells, 1% NP40 was added and the mix incubated on ice for 10 minutes. Lysates were further disrupted by sonication on ice at 95W for 4 x 15 seconds and then centrifuged for 15 minutes at 14,000 rpm to pellet the inclusion bodies.
The resulting pellet was re-suspended in lysis buffer containing 0.5%> w/v CHAPS and sonicated on ice for 5-10 seconds. This mix was stored on ice for 1 hour, centrifuged at 14,000 rpm for 15 minutes at 4 °C and the supernatant discarded. The pellet was once more re-suspended in lysis buffer containing 0.5% w/v CHAPS, sonicated, centrifuged and the supernatant removed as before. The pellet was re-suspended in solubilizing buffer (6 M Guanidine HC1, 0.5 M NaCl, 20 mM Tris HC1, pH 8.0), sonicated at 95 W for 4 x 15 seconds and then centrifuged for 20 minutes at 14,000 rpm and 4 °C to remove debris. The supernatant was stored at 4 °C until use.
Polypeptides muTRla and huTRla were purified by virtue of the N-terminal 6x Histidine tag contained within the bacterial leader sequence, using a Nickel-Chelating Sepharose column (Amersham Pharmacia, Uppsala, Sweden) and following the manufacturer's recommended protocol. In order to refold the proteins once purified, the protein solution was added to 5x its volume of refolding buffer (1 mM EDTA, 1.25 mM reduced glutathione, 0.25 mM oxidised glutathione, 20 mM Tris-HCl, pH 8.0) over a period of 1 hour at 4 °C. The refolding buffer was stirred rapidly during this time, and stirring continued at 4 °C overnight. The refolded proteins were then concentrated by ultrafiltration using standard protocols.
27 Biological Activities of Polypeptides muTRla and huTRla muTRI and huTRl are novel members of the EGF family, which includes EGF, TGFα, epiregulin and others. These growth factors are known to act as ligands for the EGF receptor. The pathway of EGF receptor activation is well documented. Upon binding of a ligand to the EGF receptor, a cascade of events follows, including the phosphorylation of proteins known as MAP kinases. The phosphorylation of MAP kinase can thus be used as a marker of EGF receptor activation. Monoclonal antibodies exist which recognize the phosphorylated forms of 2 MAP kinase proteins - ERK1 and ERK2. In order to examine whether purified polypeptides of muTRla and huTRla act as a ligand for the EGF receptor, cells from the human epidermal carcinoma cell line A431 (American Type Culture Collection, No. CRL-1555, Manassas, Virginia) were seeded into 6 well plates, serum starved for 24 hours, and then stimulated with purified muTRla or huTRla for 5 minutes in serum free conditions. As a positive control, cells were stimulated in the same way with 10 to 100 ng/ml TGF-alpha or EGF. As a negative control, cells were stimulated with PBS containing varying amounts of LPS. Cells were immediately lysed and protein concentration of the lysates estimated by Bradford assay. 15 μg of protein from each sample was loaded onto \2% SDS-PAGE gels. The proteins were then transferced to PVDF membrane using standard techniques. For Western blotting, membranes were incubated in blocking buffer (lOmM Tris-
HCl, pH 7.6, 100 mM NaCl, 0.1% Tween-20, 5% non-fat milk) for 1 hour at room temperature. Rabbit anti-Active MAP kinase pAb (Promega, Madison, Wisconsin) was added to 50 ng/ml in blocking buffer and incubated overnight at 4 °C. Membranes were washed for 30 mins in blocking buffer minus non-fat milk before being incubated with anti rabbit IgG-HRP antibody, at a 1 :3500 dilution in blocking buffer, for 1 hour at room temperature. Membranes were washed for 30 minutes in blocking buffer minus non-fat milk, then once for 5 minutes in blocking buffer minus non-fat milk and 0.1 % Tween-20. Membranes were then exposed to ECL reagents for 2 min, and then autoradiographed for 5 to 30 min. As shown in Fig. 2, both muTRla and huTRla were found to induce the phosphorylation of ERKl and ERK2 over background levels, indicating that muTRI and
28 huTRl act as ligands for a cell surface receptor that activates the MAP kinase signaling pathway, possibly the EGF receptor. As shown in Fig. 11, huTRla was also demonstrated to induce the phosphorylation of ERK1 and ERK2 in CV1/EBNA kidney epithelial cells in culture, as compared with the negative control. These assays were conducted as described above. This indicates that huTRla acts as a ligand for a cell surface receptor that activates the MAP kinase signaling pathway, possibly the EGF receptor in HeLa and CV1/EBNA cells.
The ability of muTRla to stimulate the growth of neonatal foreskin (NF) keratinocytes was determined as follows. NF keratinocytes derived from surgical discards were cultured in KSFM (BRL Life Technologies) supplemented with bovine pituatary extract (BPE) and epidermal growth factor (EGF). The assay was performed in 96 well flat-bottomed plates in 0.1 ml unsupplemented KSFM. MuTRla, human transforming growth factor alpha (huTGFα) or PBS-BSA was titrated into the plates and 1 x 103 NF keratinocytes were added to each well. The plates were incubated for 5 days in an atmosphere of 5%o CO2 at 37°C. The degree of cell growth was determined by MTT dye reduction as described previously (J. Imm. Meth. 93:157-165, 1986). As shown in Fig. 3, both muTRla and the positive control human TGFα stimulated the growth of NF keratinocytes, whereas the negative control, PBS-BSA, did not.
The ability of muTRla and huTRla to stimulate the growth of a transformed human keratinocyte cell line, HaCaT, was determined as follows. The assay was performed in 96 well flat-bottomed plates in 0.1 ml DMEM (BRL Life Technologies) supplemented with 0.2%0 FCS. MuTRla, huTRla and PBS-BSA were titrated into the plates and 1 xlO3 HaCaT cells were added to each well. The plates were incubated for 5 days in an atmosphere containing 10%> CO at 37°C. The degree of cell growth was determined by MTT dye reduction as described previously (J. Imm. Meth. 93:157-165, 1986). As shown in Fig. 4, both muTRla and huTRla stimulated the growth of HaCaT cells, whereas the negative control PBS-BSA did not.
The ability of muTRla and huTRla to inhibit the growth of A431 cells was determined as follows. Polypeptides muTRla (SEQ ID NO: 342) and huTRla (SEQ ID NO: 343) and PBS-BSA were titrated as described previously (J. Cell Biol. 93:1-4, 1982) and cell death determined using the MTT dye reduction as described previously
29 (J. Imm. Meth. 93:157-165, 1986). Both muTRla and huTRla were found to inhibit the growth of A431 cells, whereas the negative control PBS-BSA did not (Fig. 5).
These results indicate that muTRI and huTRl stimulate keratinocyte growth and motility, inhibit the growth of epithelial-derived cancer cells, and play a role in angiogenesis and vascularization of tumors. This novel gene and its encoded protein may thus be developed as agents for the healing of wounds, angiogenesis and regulators of epithelial-derived cancers. Upregulation ofhuTRl and mRNA expression
HeLa cells (human cervical adenocarcinoma) were seeded in 10 cm dishes at a concentration of 1 x 106 cells per dish. After incubation overnight, media was removed and replaced with media containing 100 ng/ml of muTRI, huTRl, huTGFα, or PBS as a negative control. After 18 hours, media was removed and the cells lysed in 2 ml of TRIzol reagent (Gibco BRL Life Technologies, Gaithersburg, Maryland). Total RNA was isolated according to the manufacturer's instructions. To identify mRNA levels of huTRl from the cDNA samples, 1 μl of cDNA was used in a standard PCR reaction. After cycling for 30 cycles, 5 μl of each PCR reaction was removed and separated on a 1.5% agarose gel. Bands were visualized by ethidium bromide staining. As can be seen from Fig. 12, both mouse and human TRl up-regulate the mRNA levels of huTRl as compared with cells stimulated with the negative control of PBS. Furthermore, TGFα can also up-regulate the mRNA levels of huTRl .
These results indicate that TRl is able to sustain its own mRNA expression and subsequent protein expression, and thus is expected to be able to contribute to the progression of diseases such as psoriasis where high levels of cytokine expression are involved in the pathology of the disease. Furthermore, since TGFα can up-regulate the expression of huTRl, the up-regulation of TRl mRNA may be critical to the mode of action of TGFα. Serum response element reporter gene assay
The serum response element (SRE) is a promoter element required for the regulation of many cellular immediate-early genes by growth. Studies have demonstrated that the activity of the SRE can be regulated by the MAP kinase signaling pathway. Two cell lines, PC 12 (rat pheochromocytoma - neural tumor) and HaCaT (human transformed
30 keratinocytes), containing eight SRE upstream of an SV40 promotor and luciferase reporter gene were developed in-house. 5 x 103 cells were aliquoted per well of 96 well plate and grown for 24 hours in their respective media. HaCaT SRE cells were grown in 5%> fetal bovine serum (FBS) in D-MEM supplemented with 2mM L-glutamine (Sigma, St. Louis, Missouri), ImM sodium pyruvate (BRL Life Technologies), 0.77mM L-asparagine (Sigma), 0.2mM arginine (Sigma), 160mM penicillin G (Sigma), 70mM dihydrostreptomycin (Roche Molecular Biochemicals, Basel, Switzerland), and 0.5 mg/ml geneticin (BRL Life Technologies). PC12 SRE cells were grown in 5% fetal bovine serum in Ham F12 media supplemented with 0.4 mg/ml geneticin (BRL Life Technologies). Media was then changed to 0.1% FBS and incubated for a further 24 hours. Cells were then stimulated with a titration of TRl from 1 μg/ml. A single dose of basic fibroblast growth factor at 100 ng/ml (R&D Systems, Minneapolis, Minnesota) or epidermal growth factor at 10 ng/ml (BRL Life Technologies) was used as a positive control. Cells were incubated in the presence of muTRI or positive control for 6 hours, washed twice in PBS and lysed with 40 μl of lysis buffer (Promega). 10 μl was transferred to a 96 well plate and 10 μl of luciferase substrate (Promega) added by direct injection into each well by a Victor fluorimeter (Wallac), the plate was shaken and the luminescence for each well read at 3x1 sec Intervals. Fold induction of SRE was calculated using the following equation: Fold induction of SRE = Mean relative luminescence of agonist/Mean relative luminescence of negative control.
As shown in Fig. 13, muTRI activates the SRE in both PC-12 (Fig. 13a) and HaCaT (Fig. 13b) cells. This indicates that HaCaT and PC-12 cells are able to respond to muTRI protein and elicit a response. In the case of HaCaT cells, this is a growth response. In the case of PC- 12 cells, this may be a growth, a growth inhibition, differentiation, or migration response. Thus, TRl may be important in the development of neural cells or their differentiation into specific neural subsets. TRl may also be important in the development and progression of neural tumors. Inhibition by the EGF receptor assay
The HaCaT growth assay was conducted as previously described, except that modifications were made as follows. Concurrently with the addition of EGF and TRl to the media, anti-EGF Receptor (EGFR) antibody (Promega, Madison, Wisconsin) or
31 negative control antibody, mouse IgG (PharMingen, San Diego, California), were added at a concentration of 62.5 ng/ml.
As seen in Fig. 14, an antibody which blocks the function of the EGFR inhibits the mitogenicity of TRl on HaCaT cells. This indicates that the EGFR is crucial for transmission of the TRl mitogenic signal on HaCaT cells. TRl may bind directly to the EGF receptor. TRl may also bind to any other members of the EGFR family - ErbB-2, -3, and/or -4 - that are capable of heterodimerizing with the EGFR. Sequence of splice variant ofhuTRl, huTRlβ
A variant of huTRl was isolated from the same library as huTRl (SEQ ID NO: 118), following the same protocols. This sequence is a splice variant of huTRl and consists of the ORF of huTRl minus amino acids 87 to 137. This has the effect of deleting the third cysteine residue of the EGF motif and the transmembrane domain. However, cysteine residue 147 (huTRl ORF numbering) may replace the deleted cysteine and thus the disulphide bridges are likely not affected. Therefore, huTRl β is a secreted form of huTRl. It functions as an agonist or an antagonist to huTRl or other EGF family members, including EGF and TGFα. The determined nucleotide sequence of the splice variant of TRl, refened to as huTRlβ, is given in SEQ ID NO: 371 and the corresponding predicted amino acid sequence is SEQ ID NO: 395.
Example 4 IDENTIFICATION. ISOLATION AND CHARACTERIZATION OF DP3
A partial cDNA fragment, referred to as DP3, was identified by differential display RT-PCR (modified from Liang P and Pardee AB, Science 257:967-971, 1992) using mRNA from cultured rat dermal papilla and footpad fibroblast cells, isolated by standard cell biology techniques. This double stranded cDNA was labeled with [α32P]- dCTP and used to identify a full length DP3 clone by screening 400,000 pfu's of an oligo dT-primed rat dermal papilla cDNA library. The determined full-length cDNA sequence for DP3 is provided in SEQ ID NO: 119, with the coreesponding amino acid sequence being provided in SEQ ID NO: 197. Plaque lifts, hybridization and screening were performed using standard molecular biology techniques.
32 Example 5
ISOLATION AND CHARACTERIZATION OF THE
HUMAN HOMOLOG OF MUKSI
Analysis of RNA transcripts by Northern Blotting
Northern analysis to determine the size and distribution of mRNA for muKSl (SEQ ID NO: 263) was performed by probing murine tissue mRNA blots with a probe consisting of nucleotides 268-499 of muKSl, radioactively labeled with [α32P]-dCTP. Prehybridization, hybridization, washing, and probe labeling were performed as described in Sambrook, et al, Ibid. mRNA for muKSl was 1.6 kb in size and was observed to be most abundant in brain, lung, muscle, and heart. Expression could also be detected in lower intestine, skin, and kidney. No detectable signal was found in testis, spleen, liver, thymus, stomach. Human homologue ofmuKSl MuKSl (SEQ ID NO: 263) was used to search the EMBL database (Release 50, plus updates to June, 1998) to identify human EST homologues. The top three homologies were to the following ESTs: accession numbers AA643952, HS1301003 and AA865643. These showed 92.63%o identity over 285 nucleotides, 93.64% over 283 nucleotides and 94.035% over 285 nucleotides, respectively. Frame shifts were identified in AA643952 and HS1301003 when translated. Combination of all three ESTs identified huKSl (SEQ ID NO: 270) and translated polypeptide SEQ ID NO: 344. Alignment of muKSl and huKSl polypeptides indicated 95%> identity over 96 amino acids. Bacterial expression and purification ofmuKSl and huKSl
Polynucleotides 269-502 of muKSl (SEQ ID NO: 271), encoding amino acids 23-99 of polypeptide muKSl (SEQ ID NO: 345), and polynucleotides 55-288 of huKSl (SEQ ID NO: 272), encoding amino acids 19-95 of polypeptide huKSl (SEQ ID NO: 346), were cloned into the bacterial expression vector pET-16b (Novagen, Madison, Wisconsin), which contains a bacterial leader sequence and N-terminal όxHistidine tag. These constructs were transformed into competent XLl-Blue E. coli as described in Sambrook et al., Ibid.
33 Starter cultures of recombinant BL 21 (DE3) E. coli (Novagen) containing SEQ ID NO: 271 (muKSla) and SEQ ID NO: 272 (huKSla) were grown in NZY broth containing 100 μg/ml ampicillin (Gibco-BRL Life Technologies) at 37°C. Cultures were spun down and used to inoculate 800 ml of NZY broth and 100 μg/ml ampicillin. Cultures were grown until the OD5 5 of the cells was between 0.4 and 0.8. Bacterial expression was induced for 3 hours with 1 mM IPTG. Bacterial expression produced an induced band of approximately 15kDa for muKSla and huKSla.
MuKSla and huKSla were expressed in insoluble inclusion bodies. In order to purify the polypeptides, bacterial cell pellets were re-suspended in lysis buffer (20 mM Tris-HCl pH 8.0, 10 mM βMercaptoethanol, 1 mM PMSF). To the lysed cells, 1% NP-40 was added and the mix incubated on ice for 10 minutes. Lysates were further disrupted by sonication on ice at 95 W for 4 x 15 seconds and then centrifuged for 10 minutes at 18,000 rpm to pellet the inclusion bodies.
The pellet containing the inclusion bodies was re-suspended in lysis buffer containing 0.5%> w/v CHAPS and sonicated for 5-10 seconds. This mix was stored on ice for 1 hour, centrifuged at 14000 rpm for 15 minutes at 4°C and the supernatant discarded. The pellet was once more re-suspended in lysis buffer containing 0.5%) w/v CHAPS, sonicated, centrifuged, and the supernatant removed as before. The pellet was resuspended in solubilizing buffer (6 M guanidine HC1, 0.5 M NaCl, 20 mM Tris-HCl pH 8.0), sonicated at 95W for 4 x 15 seconds and centrifuged for 10 minutes at 18000 rpm and 4°C to remove debris. The supernatant was stored at 4°C. MuKSla and huKSla were purified by virtue of the N-terminal 6x histidine tag contained within the bacterial leader sequence, using a Nickel-Chelating sepharose column (Amersham Pharmacia, Uppsala, Sweden) and following the manufacturer's protocol. Proteins were purified twice over the column to reduce endotoxin contamination. In order to re-fold the proteins once purified, the protein solution was dialysed in a 4 M-2 M urea gradient in 20 mM tris-HCl pH 7.5 + 10%> glycerol overnight at 4°C. The protein was then further dialysed 2x against 2 litres of 20 mM Tris-HCl pH 7.5 + 10% glycerol. Peptide sequencing ofmuKSl and huKSl Bacterially expressed muKSl and huKSl were separated on polyacrylamide gels and induced bands of 15 kDa were identified. The predicted size of muKSl is 9.4 kDa.
34 To obtain the amino acid sequence of the 15 kDa bands, 20 μg recombinant muKSl and huSKl was resolved by SDS-PAGE and electroblotted onto Immobilon PVDF membrane (Millipore, Bedford, Massachusetts). Internal amino acid sequencing was performed on tryptic peptides of muKSl and huKSl by the Protein Sequencing Unit at the University of Auckland, New Zealand.
The determined amino acid sequences for muKS 1 and huKS 1 are given in SEQ ID NOS: 397 and 398, respectively. These amino acid sequences confirmed that the determined sequences are identical to that predicted from the cDNA sequences. The size discrepancy has previously been reported for other chemokines (Richmond A, Balentien E, Thomas HG, Flaggs G, Barton DE, Spiess J, Bordoni R, Francke U, Derynck R, "Molecular characterization and chromosomal mapping of melanoma growth stimulatory activity, a growth factor structurally related to beta-thromboglobulin," EMBO J. 7:2025-2033, 1988; Liao F, Rabin RL, Yannelli JR, Koniaris LG, Vanguri P, Farber JM, "Human Nig chemokine: biochemical and functional characterization," J. Exp. Med. 182:1301-1314, 1995). The isoelectric focusing point of these proteins was predicted to be 10.26 using DNASIS (HITACHI Software Engineering, San Francisco, California). Oxidative burst assay
Oxidative burst assays were used to determine responding cell types. 1 x 10 PBMC cells were resuspended in 5 ml HBSS, 20mM HEPES, 0.5% BSA and incubated for 30 minutes at 37°C with 5 μl 5 mM dichloro-dihydrofluorescein diacetate (H2DCFDA, Molecular Probes, Eugene, Oregon). 2 x 105 H2DCFDA-labeled cells were loaded in each well of a flat-bottomed 96 well plate. 10 μl of each agonist was added simultaneously into the well of the flat-bottomed plate to give final concentrations of 100 ng/ml (fMLP was used at 10 μM). The plate was then read on a Victor2 1420 multilabel counter (Wallac, Turku, Finland) with a 485 nm excitation wavelength and 535 nm emission wavelength. Relative fluorescence was measured at 5 minute intervals over 60 minutes.
A pronounced respiratory burst was identified in PBMC with a 2.5 fold difference between control treated cells (TRl) and cells treated with 100 ng/ml muKSl (Fig. 8).
35 Human stromal derived factor- lα (SDFlα) (100 ng/ml) and 10 μM formyl-Met-Leu-Phe (f LP) were used as positive controls. Chemotaxis assay
Cell migration in response to muKSl was tested using a 48 well Boyden's chamber (Neuro Probe Inc., Cabin John, Maryland) as described in the manufacturer's protocol. In brief, agonists were diluted in HBSS, 20mM HEPES, 0.5% BSA and added to the bottom wells of the chemotactic chamber. THP-1 cells were re-suspended in the same buffer at 3 x 105 cells per 50 μl. Top and bottom wells were separated by a PVP- free polycarbonate filter with a 5 μm pore size for monocytes or 3 μm pore size for lymphocytes. Cells were added to the top well and the chamber incubated for 2 hours for monocytes and 4 hours for lymphocytes in a 5% CO humidified incubator at 37°C. After incubation, the filter was fixed and cells scraped from the upper surface. The filter was then stained with Diff-Quick (Dade International Inc., Miami, Florida) and the number of migrating cells counted in five randomly selected high power fields. The results are expressed as a migration index (the number of test migrated cells divided by the number of control migrated cells).
Using this assay, muKSl was tested against T cells and THP-1 cells. MuKSl induced a titrateable chemotactic effect on THP-1 cells from 0.01 ng/ml to 100 ng/ml (Fig. 9). Human SDFlα was used as a positive control and gave an equivalent migration. MuKSl was also tested against IL-2 activated T cells. However, no migration was evidence for muKSl even at high concentrations, whereas SDF-lα provided an obvious titrateable chemotactic stimulus. Therefore, muKSl appears to be chemotactic for THP-1 cells but not for IL-2 activated T cells at the concentrations tested. Full length sequence ofmuKSl clone The nucleotide sequence of muKSl was extended by determining the base sequence of additional ESTs. Combination of all the ESTs identified the full-length muKSl (SEQ ID NO: 370) and the corresponding translated polypeptide sequence in SEQ ID NO: 394. Analysis of human RNA transcripts by Northern blotting Northern blot analysis to determine the size and distribution of mRNA for the human homologue of muKS 1 was performed by probing human tissue blots (Clontech,
36 Palo Alto, California) with a radioactively labeled probe consisting of nucleotides 1 to 288 of huKSl (SEQ ID NO: 270). Prehybridization, hybridization, washing, and probe labeling were performed as described in Sambrook, et al, Ibid. mRNA for huKSl was 1.6 kb in size and was observed to be most abundance in kidney, liver, colon, small intestine, and spleen. Expression could also be detected in pancreas, skeletal muscle, placenta, brain, heart, prostate, and thymus. No detectable signal was found in lung, ovary, and testis. Analysis of human RNA transcripts in tumor tissue by Northern blotting
Northern blot analysis to determine distribution of huKSl in cancer tissue was performed as described previously by probing tumor panel blots (Invitrogen, Carlsbad, California). These blots make a direct comparison between normal and tumor tissue. MRNA was observed in normal uterine and cervical tissue but not in the respective tumor tissue. In contrast, expression was up-regulated in breast tumor and down-regulated in normal breast tissue. No detectable signal was found in either ovary or ovarian tumors. Injection of bacteriallv expressed muKSla into nude mice
Two nude mice were anaesthetised intraperitoneally with 75 μl of 1/10 dilution of Hypnorm (Janssen Pharmaceuticals, Buckinghamshire, England) in phosphate buffered saline. 20ug of bacterially expressed muKSla (SEQ ID NO: 345) was injected subcutaneously in the left hind foot, ear and left-hand side of the back. The same volume of phosphate buffered saline was injected in the same sites but on the right-hand side of the same animal. Mice were left for 18 hours and then examined for inflammation. Both mice showed a red swelling in the ear and foot sites injected with the bacterially expressed protein. No obvious inflammation could be identified in either back site. Mice were culled and biopsies taken from the ear, back and foot sites and fixed in 3.7% formol saline. Biopsies were embedded, sectioned and stained with Haemotoxylin and eosin. Sites injected with muKSla had a marked increase in polymorphonuclear granulocytes, whereas sites injected with phosphate buffered saline had a low background infiltrate of polymorphonuclear granulocytes. Injection of bacterially recombinant muKSl into C3H/HeJ mice Eighteen C3H/HeJ mice were divided into 3 groups and injected intraperitoneally with muKSl, GV14B, or phosphate buffered saline (PBS). GV14B is a bacterially
37 expressed recombinant protein used as a negative control. Group 1 mice were injected with 50 μg ofmuKSl in 1 ml of PBS; Group 2 mice were injected with 50 μg of GV14B in 1 ml of PBS; and Group 3 mice with 1 ml of PBS. After 18 hours, the cells in the peritoneal cavity ofthe mice were isolated by intraperitoneal lavage with 2 x 4 ml washes with harvest solution (0.02%> EDTA in PBS). Viable cells were counted from individual mice from each group. Mice injected with 50 μg of muKSl had on average a 3-fold increase in cell numbers (Fig. 10).
20 μg of bacterial recombinant muKSl was injected subcutaneously into the left hind foot of three C3H/HeJ mice. The same volume of PBS was injected into the same site on the right-hand side of the same animal. After 18 hours, mice were examined for inflammation. All mice showed a red swelling in the foot pad injected with bacterially recombinant KS1. From histology, sites injected with muKSl had an inflammatory response of a mixed phenotype with mononuclear and polymorphonuclear cells present.
Chemokines are a large superfamily of highly basic secreted proteins with a broad number of functions (Baggiolini, et al, Annu. Rev. Immunol, 15:675-705, 1997; Ward, et al, Immunity, 9: 1-11, 1998; Horuk, Nature, 393:524-525, 1998). The polypeptide sequences ofmuKSl and huKSl have similarity to CXC chemokines, suggesting that this protein will act like other CXC chemokines. The in vivo data from nude mice supports this hypothesis. This chemokine-like protein may therefore be expected to stimulate leukocyte, epithelial, stromal, and neuronal cell migration; promote angiogenesis and vascular development; promote neuronal patterning, hemopoietic stem cell mobilization, keratinocyte and epithelial stem cell patterning and development, activation and proliferation of leukocytes; and promotion of migration in wound healing events. It has recently been shown that receptors to chemokines act as co-receptors for HIV-1 infection of CD4+ cells (Cairns, et al., Nature Medicine, 4:563-568, 1998) and that high circulating levels of chemokines can render a degree of immunity to those exposed to the HIV virus (Zagury, et al, Proc. Natl Acad. Sci. USA 95:3857-3861, 1998). This novel gene and its encoded protein may thus be usefully employed as regulators of epithelial, lymphoid, myeloid, stromal, and neuronal cells migration and cancers; as agents for the treatment of cancers, neuro-degenerative diseases, inflammatory autoimmune diseases
38 such as psoriasis, asthma and Crohn's disease for use in wound healing; and as agents for the prevention of HIV-1 binding and infection of leukocytes.
We have also shown that muKSl can promote a quantifiable increase in cell numbers in the peritoneal cavity of C3H/HeJ mice injected with muKSl. Furthermore, we have shown that muKSl can induce an oxidative burst in human peripheral blood mononuclear cells and migration in the human monocyte leukemia cell line, THP-1, suggesting that monocyte/macrophages are one of the responsive cell types for KS 1. In addition to this, we demonstrated that huKSl was expressed at high levels in a number of non-lymphoid tissues, such as the colon and small intestine, and in breast tumors. It was also expressed in normal uterine and cervical tissue, but was completely down-regulated in their respective tumors. It has recently been shown that non-ELR chemokines have demonstrated angiostatic properties. IP- 10 and Mig, two non-ELR chemokines, have previously been shown to be up-regulated during regression of tumors (Tannenbaum CS, Tubbs R, Armstrong D, Finke JH, Bukowski RM, Hamilton TA, "The CXC Chemokines IP- 10 and Mig are necessary for IL-12-mediated regression of the mouse RENCA tumor," J. Immunol. 161 : 927-932, 1998), with levels of expression inversely coreelating with tumor size (Kanegane C, Sgadari C, Kanegane H, Teruya-Feldstine J, Yao O, Gupta G, Farber JM, Liao F, Liu L, Tosato G, "Contribution of the CXC Chemokines LP-10 and Mig to the antitumor effects of IL-12," J. Leuko. Biol. 64: 384-392, 1998). Furthermore, neutralizing antibodies to IP- 10 and Mig would reduce the anti-tumor effect, indicating the contribution these molecules make to the anti-tumor effects. Therefore, it is expected that in the case of cervical and uterine tumors, KS 1 would have similar properties.
The data demonstrates that KS1 is involved in cell migration showing that one of the responsive cell types is monocyte/macrophage. The human expression data in conjunction with the in vitro and in vivo biology demonstrates that this molecule may be a useful regulator in cell migration, and as an agent for the treatment of inflammatory diseases, such as Crohn's disease, ulcerative colitis, and rheumatoid arthritis; and cancers, such as cervical adenocarcinoma, uterine leiomyoma, and breast invasive ductal carcinoma.
39 Example 6 CHARACTERIZATION OF KS2 KS2 contains a transmembrane domain and may function as either a membrane- bound ligand or a receptor. Northern analysis indicated that the mRNA for KS2 was expressed in the mouse keratinocyte cell line, Pam212, consistent with the cDNA being identified in mouse keratinocytes. Mammalian Expression
To express KS2, the extracellular domain was fused to the amino terminus of the constant domain of immunoglobulinG (Fc) that had a C-terminal όxHistidine tag. This was performed by cloning polynucleotides 20-664 of KS2 (SEQ ID NO: 273), encoding amino acids 1-215 of polypeptide KS2 (SEQ ID NO: 347), into the mammalian expression vector pcDNA3 (Invitrogen, NV Leek, Netherlands), to the amino terminus of the constant domain of immunoglobulinG (Fc) that had a C-terminal 6xHistidine tag. This construct was transformed into competent XLl-Blue E. coli as described in Sambrook et al., Ibid. The Fc fusion construct of KS2a was expressed by transfecting Cos-1 cells in 5 x T175 flasks with 180 μg of KSla using DEAE-dextran. The supernatant was harvested after seven days and passed over a Ni-NTA column. Bound KS2a was eluted from the column and dialysed against PBS.
The ability of the Fc fusion polypeptide of KS2a to inhibit the IL-2 induced growth of concanavalin A stimulated murine splenocytes was determined as follows. A single cell suspension was prepared from the spleens of BALB/c mice and washed into DMEM (GIBCO-BRL) supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 0.77 mM L-asparagine, 0.2 mM L-arganine, 160 mM penicillin G, 70 mM dihydrostreptomycin sulfate, 5 x 10"2 mM beta mercaptoethanol and 5% FCS (cDMEM). Splenocytes (4 x 106/ml) were stimulated with 2 ug/ml concanavalin A for 24 hrs at 37°C in 10%) CO . The cells were harvested from the culture, washed 3 times in cDMEM and resuspended in cDMEM supplemented with 10 ng/ml rhuIL-2 at 1 x 105 cells/ml. The assay was performed in 96 well round bottomed plates in 0.2 ml cDMEM. The Fc fusion polypeptide of KS2a, PBS, LPS and BSA were titrated into the plates and 1 x 104 activated T cells (0.1 ml) were added to each well. The plates were incubated for 2 days in an atmosphere containing 10%) CO2 at 37°C. The degree of proliferation was
40 determined by pulsing the cells with 0.25 uCi/ml tritiated thymidine for the final 4 hrs of culture after which the cells were harvested onto glass fiber filtermats and the degree of thymidine incorporation determined by standard liquid scintillation techniques. As shown in Fig. 6, the Fc fusion polypeptide of KS2a was found to inhibit the IL-2 induced growth of concanavalin A stimulated murine splenocytes, whereas the negative controls PBS, BSA and LPS did not.
This data demonstrates that KS2 is expressed in skin keratinocytes and inhibits the growth of cytokine induced splenocytes. This suggests a role for KS2 in the regulation of skin inflammation and malignancy.
Example 7 Characterization of KS3 KS3 encodes a polypeptide of 40 amino acids (SEQ ID NO: 129). KS3 contains a signal sequence of 23 amino acids that would result in a mature polypeptide of 17 amino acids (SEQ ID NO: 348; referred to as KS3a).
KS3a was prepared synthetically (Chiron Technologies, Victoria, Australia) and observed to enhance transfercin-induced growth of the rat intestinal epithelial cells IEC-18 cells. The assay was performed in 96 well flat-bottomed plates in 0.1 ml DMEM (GIBCO-BRL Life Technologies) supplemented with 0.2% FCS. KS3a (SEQ ID NO: 348), apo-Transferrin, media and PBS-BSA were titrated either alone, with 750 ng/ml Apo-transferrin or with 750 ng/ml BSA, into the plates and 1 xlO3 IEC-18 cells were added to each well. The plates were incubated for 5 days at 37°C in an atmosphere containing 10%> CO . The degree of cell growth was determined by MTT dye reduction as described previously (J. Imm. Meth. 93:157-165, 1986). As shown in Fig. 7, KS3a plus Apo-transferrin was found to enhance transferrin-induced growth of IEC-18 cells, whereas KS3a alone or PBS-BSA did not, indicating that KS3a and Apo- transferrin act synergistically to induce the growth of IEC-18 cells.
This data indicates that KS3 is epithelial derived and stimulates the growth of epithelial cells of the intestine. This suggests a role for KS3 in wound healing, protection from radiation- or drug-induced intestinal disease, and integrity of the epithelium of the intestine.
41 SEQ ID NOS: 1-409 are set out in the attached Sequence Listing. The codes for polynucleotide and polypeptide sequences used in the attached Sequence Listing confirm to WIPO Standard ST.25 (1988), Appendix 2.
All references cited herein, including patent references and non-patent references, are hereby incorporated by reference in their entireties.
Although the present invention has been described in terms of specific embodiments, changes and modifications can be carried out without departing from the scope of the invention which is intended to be limited only by the scope of the appended claims.
42

Claims

We claim:
1. An isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of: (1) the sequences recited in SEQ ID NO: 1-119, 198-274, 349-372, and 399-405; (2) complements of the sequences recited in SEQ ID NO: 1-119, 198-274, 349-372, and 399-405; (3) reverse complements of the sequences recited in SEQ ID NO: 1-119, 198-274, 349-372, and 399-405; (4) reverse sequences of the sequences recited in SEQ ID NO: 1-119, 198-274, 349-372, and 399-405; (5) sequences having at least a 99%> probability of being the same as a sequence selected from any of the sequences in (l)-(4), above, as measured by the computer algorithm BLASTP using the running parameters described above; and (6) nucleotide sequences having at least 50%) identity to any of the sequences in (l)-(4), above, as measured by the computer algorithm BLASTP using the running parameters and identity test defined above.
2. An expression vector comprising an isolated polynucleotide of claim 1.
3. A host cell transformed with an expression vector of claim 2.
4. An isolated polypeptide comprising an amino acid sequence selected from the group consisting of: (1) sequences provided in SEQ ID NO: 120-197, 275-348,
373-398, and 406-409; (2) sequences having at least a 99%> probability of being the same as a sequence of SEQ ID NO: 120-197, 275-348, 373-398, and 406-409, as measured by the computer algorithm BLASTP using the running parameters described above; and (3) sequences having at least 50% identity to a sequence provided in SEQ ID NO: 120-197, 275-348, 373-398, and 406-409, as measured by the computer algorithm BLASTP using the running parameters and identity test defined above.
5. An isolated polynucleotide encoding a polypeptide of claim 4.
6. An expression vector comprising an isolated polynucleotide of claim 5.
43
7. A host cell transformed with an expression vector of claim 6.
8. An isolated polypeptide comprising at least a functional portion of a polypeptide having an amino acid sequence selected from the group consisting of: (1) sequences provided in SEQ ID NO: 120-197, 275-348, 373-398, and 406-409; (2) sequences having at least a 99%> probability of being the same as a sequence of SEQ ID NO: 120-197, 275-348, 373-398, and 406-409, as measured by the computer algorithm BLASTP using the running parameters described above; and (3) sequences having at least 50% identity to a sequence provided in SEQ ID NO: 120-197, 275-348, 373-398, and 406-409, as measured by the computer algorithm BLASTP, using the running parameters and identity test defined above.
9. A method for stimulating keratinocyte growth and motility in a patient, comprising administering to the patient a composition comprising an isolated polypeptide, the polypeptide comprising an amino acid sequence of claim 4.
10. The method of claim 9, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of: (1) a sequence provided in SEQ ID NO: 187, 196, 342, 343, 397 and 398; (2) sequences having at least about 50% identity to a sequence of SEQ ID NO: 187, 196, 342, 343, 397 and 398 as measured by the computer algorithm BLASTP, using the running parameters and identity test defined above.
11. A method for inhibiting the growth of cancer cells in a patient, comprising administering to the patient a composition comprising an isolated polypeptide, the polypeptide comprising an amino acid sequence of claim 4.
12. The method of claim 11, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of: (1) a sequence provided in SEQ ID NO: 187, 196, 342, 343, 397 and 398; and (2) sequences having at least 50% identity to a sequence of SEQ ID NO: 187, 196, 342, 343, 397, and 398, as measured by the computer algorithm BLASTP, using the running parameters and identity test defined above.
44
13. A method for modulating angiogenesis in a patient, comprising administering to the patient a composition comprising an isolated polypeptide, the polypeptide comprising an amino acid sequence of claim 4.
14. A method of claim 13, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of: (1) a sequence provided in SEQ ID NO: 187, 196, 342, 343, 397 and 398; and (2) sequences having at least 50% identity to a sequence of SEQ ID NO: 187, 196, 342, 343, 397 and 398 as measured by the computer algorithm BLASTP, using the running parameters and identity test defined above.
15. A method for inhibiting angiogenesis and vascularization of tumors in a patient, comprising administering to a patient a composition comprising an isolated polypeptide, the polypeptide comprising an amino acid sequence of claim 4.
16. The method of claim 15, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of: (1) a sequence provided in SEQ ID NO: 187, 196, 342, 343, 397, and 398; and (2) sequences having at least 50% identity to a sequence of SEQ ID NO: 187, 196, 340, 342-346, 397, and 398, as measured by the computer algorithm BLASTP, using the running parameters and identity test defined above.
17. A method for modulating skin inflammation in a patient, comprising administering to the patient a composition comprising an isolated polypeptide, the polypeptide comprising an amino acid sequence of claim 4.
18. The method of claim 17, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of: (1) a sequence provided in SEQ ID NO: 338 and 347; and (2) sequences having at least 50% identity to a sequence of SEQ ID NO: 338 and 347 as measured by the computer algorithm BLASTP, using the running parameters and identity test defined above.
45
19. A method for stimulating the growth of epithelial cells in a patient, comprising administering to the patient a composition comprising an isolated polypeptide, the polypeptide comprising an amino acid sequence of claim 4.
20. The method of claim 19, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of: (1) a sequence provided in SEQ ID NO: 129 and 348; and (2) sequences having at least 50% identity to a sequence of SEQ ID NO: 129 and 348 as measured by the computer algorithm BLASTP, using the running parameters and identity test defined above.
21. A method for inhibiting the binding of HIV- 1 to leukocytes in a patient, comprising administering to the patient a composition comprising an isolated polypeptide, the polypeptide comprising an amino acid sequence of claim 4.
22. A method of claim 21, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of: (1) a sequence provided in SEQ ID NO: 340, 344, 345 and 346; (2) sequences having at least 50% identity to a sequence of SEQ ID NO: 340, 344, 345 and 346 as measured by the computer algorithm BLASTP, using the running parameters and identity test defined above.
23. A method for treating an inflammatory disease in a patient, comprising administering to the patient a composition comprising an isolated polypeptide, the polypeptide comprising an amino acid sequence of claim 4.
24. The method of claim 23, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of: (1) a sequence provided in SEQ ID NO: 340, 344, 345 and 346; and (2) sequences having at least 50% identity to a sequence of SEQ ID NO: 340, 344, 345 and 346 as measured by the computer algorithm BLASTP, using the running parameters and identity test defined above.
46
25. A method for treating cancer in a patient, comprising administering to the patient a composition comprising an isolated polypeptide, the polypeptide comprising an amino acid sequence of claim 4.
26. The method of claim 25, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of: (1) a sequence provided in SEQ ID NO: 340, 344, 345 and 346; and (2) sequences having at least 50%o identity to a sequence of SEQ ID NO: 340, 344, 345 and 346 as measured by the computer algorithm BLASTP, using the running parameters and identity test defined above.
27. A method for treating neurological disease in a patient, comprising administering to the patient a composition comprising an isolated polypeptide, the polypeptide comprising an amino acid sequence of claim 4.
28. The method of claim 27, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of: (1) a sequence provided in SEQ ID NO: 187, 196, 340, 342-346, and 395; and (2) sequences having at least 50% identity to a sequence of SEQ ID NO: 187, 196, 340, 342-346, and 395, as measured by the computer algorithm BLASTP, using the running parameters and identity test defined above.
47
PCT/NZ1999/000051 1998-04-29 1999-04-29 Polynucleotides isolated from skin cells and methods for their use WO1999055865A1 (en)

Priority Applications (8)

Application Number Priority Date Filing Date Title
NZ507728A NZ507728A (en) 1998-04-29 1999-04-29 Information from a cDNA library created from keratinocytes, dermal papilla, neonatal foreskin fibroblast or embryonic skin cells is used to treating cancer, angiogenesis, skin inflammation, inflammatory diseases or stimulating keratinocyte growth
AU39614/99A AU758074B2 (en) 1998-04-29 1999-04-29 Polynucleotides isolated from skin cells and methods for their use
KR1020007011932A KR20010074494A (en) 1998-04-29 1999-04-29 Polynucleotides isolated from skin cells and methods for their use
EP99922665A EP1073740A4 (en) 1998-04-29 1999-04-29 Polynucleotides isolated from skin cells and methods for their use
CA002327317A CA2327317A1 (en) 1998-04-29 1999-04-29 Polynucleotides isolated from skin cells and methods for their use
JP2000546009A JP2002512798A (en) 1998-04-29 1999-04-29 Polynucleotides isolated from skin cells and methods of use
US09/866,050 US20030040471A1 (en) 1998-04-29 2001-05-24 Compositions isolated from skin cells and methods for their use
US10/152,661 US20030022835A1 (en) 1998-04-29 2002-05-20 Compositions isolated from skin cells and methods for their use

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US09/188,930 US6150502A (en) 1998-04-29 1998-11-09 Polypeptides expressed in skin cells

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