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WO2001090357A1 - Compositions isolees des cellules cutanees et methodes d'utilisation de ces compositions - Google Patents

Compositions isolees des cellules cutanees et methodes d'utilisation de ces compositions Download PDF

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Publication number
WO2001090357A1
WO2001090357A1 PCT/NZ2001/000099 NZ0100099W WO0190357A1 WO 2001090357 A1 WO2001090357 A1 WO 2001090357A1 NZ 0100099 W NZ0100099 W NZ 0100099W WO 0190357 A1 WO0190357 A1 WO 0190357A1
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seq
sequences
identity
nos
sequence
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PCT/NZ2001/000099
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English (en)
Inventor
James D. Watson
Lorna Strachan
Matthew Sleeman
Rene Onrust
James Greg Murison
Krishanand D. Kumble
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Genesis Research & Development Corporation Limited
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Priority to AU2001260847A priority Critical patent/AU2001260847A1/en
Publication of WO2001090357A1 publication Critical patent/WO2001090357A1/fr

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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
    • C07K14/46—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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

  • This invention relates to polynucleotides, polypeptides, polypeptides expressed in skin cells, and various methods for treating a patient involving administration of a polypeptide or polynucleotide of the present 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.
  • keratinocytes become comified 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-gr ⁇ ws from epithelial cells directed by the dermal papillae of the 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 is 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 of the 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.
  • the present invention provides polypeptides and functional portions of polypeptides, which may be expressed in skin cells, together with polynucleotides encoding such polypeptides or functional portions thereof, expression vectors and host cells comprising such polynucleotides, and methods for their use.
  • isolated polynucleotides comprise a polynucleotide selected from the group consisting of: (a) sequences recited in SEQ ID NO: (a) sequences recited in SEQ ID NO: (a) sequences recited in SEQ ID NO: (a) sequences recited in SEQ ID NO: (a) sequences recited in SEQ ID NO: (a) sequences recited in SEQ ID NO: (a) sequences recited in SEQ ID NO:
  • the present invention provides isolated polypeptides comprising an amino acid sequence selected from the group consisting of: (a) sequences provided in SEQ ID NOS: 120-197, 275-348, 373-398, 406-409, 413-415, 417, 456-463, 465, 488-509, 512, 513 and 624-725; and (b) sequences having at least 50%, 75%, 90% or 95%) identity to a sequence provided in SEQ ID NOS: 120-197, 275-348, 373-398, 406-409, 413-415, 417, 456-463, 465, 488-509, 512, 513 and 624-725, 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 NOS: 120-197, 275-348, 373-398, 406-409, 413-415, 417, 456-463, 465, 488-509, 512, 513 and 624-725; and (b) sequences having 50%, 75% or 90% identity to a sequence of SEQ ID NOS: 120-197, 275-348, 373-398, 406-409, 413-415, 417, 456-463, 465, 488-509, 512, 513 and 624-725, 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 NOS: 187, 196, 342, 343, 395, 397 and 398; and (b) sequences having at least 50%, 75%, 90% or 95% identity to a sequence provided in SEQ ID NOS: 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 NOS: 338 and 347; and (b) sequences having at least 50%, 75%, 90% or 95% identity to a sequence provided-in SEQ ID NOS: 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 NOS: 129 and 348; and (b) sequences having at least 50%, 75%, 90% or 95% identity to a sequence provided in SEQ ID NOS: 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 NOS: 340, 344, 345 and 346; and (b) sequences having at least 50%, 75%, 90% or 95% identity to a sequence provided in SEQ ID NOS: 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 huTRl a.
  • 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.
  • Figure 15A shows the nucleotide sequence of KS1 cDNA (SEQ ID NO: 464) along with the deduced amino acid sequence (SEQ ID NO: 465) using single letter code.
  • the 5' UTR is indicated by negative numbers.
  • the underlined NH 2 -terminal amino acids represent the predicted leader sequence and the stop codon is denoted by ***.
  • the polyadenylation signal is marked by a double underline.
  • Figure 15B shows a comparison of the complete open reading frame of KS1 (referred to in Fig.
  • 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 of the 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 DNADNA 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 sequence selected from the group consisting of sequences provided in SEQ ID NOS: -1- 119, 198-274, 349-372, 399-405, 410-412, 416, 418-455, 464, 466-487, 510, 51k and 514-623, and variants of the sequences of SEQ ID NOS: 1-119, 198-274, 349-372, 399- 405, 410-412, 416, 418-455, 464, 466-487, 510, 511 and 514-623.
  • polynucleotides that comprise complements of such sequences, reverse complements of such sequences, or reverse sequences of such 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'.
  • the present invention provides isolated polypeptides and functional portions of 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 NOS: 120-197, 275-348, 373-398, 406-409, 413-415, 417, 456-463, 465, 488-509, 512, 513 and 624-725, 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.
  • polypeptides 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 NOS: 120-197, 275-348, 373-398, 406-409, 413-415, 417, 456-463, 465, 488-509, 512-513 and 624-725, 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.
  • variants 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. In certain preferred embodiments, variants of the inventive sequences retain certain, or all, of the functional characteristics of the inventive sequence. Variant sequences (polynucleotide or polypeptide) preferably exhibit at least 50%, more preferably at least 75%, and most preferably at least 90% or 95% 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 percentages 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.gov) under /blast/executables/ and are available from the National Center for Biotechnology Information (NCBI), National Library of Medicine, Building 38A, Room 8N805, Bethesda, MD 20894 USA.
  • NCBI National Center for Biotechnology Information
  • the FASTA and FASTX algorithms are available on the Internet at the ftp site ftp://ftp.Virginia.edu/pub/.
  • the FASTA software package is also available from the University of Virginia by contacting David Hudson, Assistant Provost for Research, University of Virginia, PO Box 9025, Charlottesville, VA 22906-9025.
  • 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, and Lipman, Proc. Natl. Acad. Sci. USA 85:2444-2448, 1988; and Pearson, Methods in Enzymol. 183:63-98, 1990.
  • 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 sequence overlap. Hits to a database sequence generally represent an overlap over only a fraction of the sequence length of the queried sequence.
  • the percentage identity 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 identity.
  • 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 identity 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% 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% 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 ("x") of contiguous residues of: any of the polynucleotides provided in SEQ ID NO: 1-119, 198-274, 349-372, 399-405, 410-412, 416, 418-455, 464, 466-487, 510, 511 and 514-623; or any of the polypeptides set out in SEQ ID NO: 120-197, 275-348, 373-398, 406-409, 413-415, 417, 456-463, 465, 488-509, 512, 513 and 624-725.
  • 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, 399-405, 410-412, 416, 418-455, 464, 466-487, 510, 511 and 514-623, 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, 406-409, 413-415, 417, 456-463, 465, 488,-509, 512, 513 and 624-725.
  • 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 NOS: 1-119, 198-274, 349-372, 399-405, 410-412, 416, 418-455, 464, 466-487, 510, 511 and 514-623, or of a variant of one of the polynucleotides provided in SEQ ID NO: 1-119, 198-274, 349-372, 399-405, 410-412, 416, 418-455, 464, 466-487, 510, 511 and 514-623.
  • 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 NOS: 120-197, 275-348, 373-398, 406-409, 413-415, 417, 456-463, 465, 488-509, 512, 513 and 624-725, or of a variant of one of the polypeptides provided in SEQ ID NOS: 120-197, 275-348, 373-398, 406-409, 413-415, 417, 456-463, 465, 488- 509, 512, 513 and 624-725.
  • 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 NOS: 1-119, 198-274, 349-372, 399-405, 410-412, 416, 418-455, 464, 466-487, 510, 511 and 514-623 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.
  • PCR polymerase chain reaction
  • 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; Erlieh, ed., PCR Technology, Stockton Press: NY, 1989; (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). 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 of the 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 or immunogenic composition.
  • 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.
  • Immunogenic compositions 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 pharmaceutical or immunogenic 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, retrovirus, 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, retrovirus, 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.
  • the pharmaceutical and immunogenic compositions may. be administered by injection (e.g., intradermal, intramuscular, intravenous, or subcutaneous), intranasally (e.g., by aspiration) or orally.
  • the 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 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 immunogenic compositions of the 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 Mycobacterium 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 Affymetrix (Santa Clara, CA). 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 ligands or receptors, in assays to quantitatively determine levels of protein or cognate corresponding ligand or receptor, as anti-inflammatory agents, and in compositions for skin, connective tissue and/or nerve tissue growth or regeneration.
  • the present invention further provides methods for modulating expression of the inventive polypeptides, for example by inhibiting translation of the relevant polynucleotide.
  • Translation of the relevant polynucleotide may be inhibited, for example, by introducing anti-sense expression vectors; by introducing antisense oligodeoxyribonucleotides or antisense phosphorothioate oligodeoxyribonucleotides; by introducing antisense oligoribonucleotides or antisense phosphorothioate oligoribonucleotides; or by other means which are well known in the art.
  • Cell permeation and activity of antisense oligonucleotides can be enhanced by appropriate chemical modifications, such as the use of phenoxazine-substituted C-5 propynyl uracil oligonucleotides (Flanagan et al., (1999) Nat. Biotechnol. 17 (1): 48-52) or 2'-0-(2-methoxy) ethyl (2'-MOE)-oligonucleotides (Zhang et al., (2000) Nat. Biotechnol. 18: 862-867).
  • the use of techniques involving antisense polynucleotides is well known in the art and is described, for example, in Robinson-Benion et al.
  • 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.
  • HNKA NK cells human
  • 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, Gaifhersburg, Maryland), was used to obtain mRNA using a Poly(A) Quik mRNA isolation kit (Stratagene, La JoUa, 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).
  • Keratinocytes obtained from human neonatal foreskins were grown in serum-free KSFM (BRL Life Technologies) and harvested along with differentiated cells (10 8 cells). Keratinocytes were allowed to differentiate by addition of fetal calf serum at a final 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.
  • driver mRNA undifferentiated keratinocytes
  • tester mRNA differentiated keratinocytes
  • 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 ⁇ broblasts 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).
  • HNFF human neonatal ⁇ broblasts
  • 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.
  • KSCL mouse stem cells
  • TAM transit amplifying
  • 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 leukocyte 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.
  • RNA isolated using TRIzol Reagent (BRL Life Technologies, Gaithersburg, MD), was used to obtain mRNA using a Poly(A)Quik mRNA isolation kit (Stratagene, La Jolla, CA), according to the manufacturer's specifications.
  • a cDNA expression library (referred to as the MFSE library) was then prepared from the mRNA by Reverse Transcriptase synthesis using a Lambda ZAP Express cDNA library synthesis kit (Stratagene, La Jolla, CA).
  • HLEA andHLEB Human Small Airway Epithelial Cells
  • Human small airway epithelium cells SAEC (Cell line number CC-2547, Clonetics Normal Human Cell Systems, Cambrex Corporation, East Rutherford NJ) transformed with human papilloma virus E6E7 that was infected with the bacterium Yersinia enterocolitica (ATCC No. 51871, American Type Culture Collection, Manassas VA) and the long form of the Respiratory Syncytial Virus (RSV, ATCC No. VR26), were used as source of RNA to construct the libraries called HLEA and HLEB. Cells from the twelfth passage of SAEC cells were infected with Y. enterocolitica for 2 hours at an initial seed of 12.5 bacteria per cell.
  • the cells were disinfected with gentamycin (100 ⁇ g/ml) for 2 hours and harvested 4 hours after infection. The cells were then infected with RSV at a moiety of infection of 0.7 for 1 hour and incubated for 6 and 24 hours. Cells were harvested and the RNA extracted following standard protocols. • -
  • RNA 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, CA), according to the manufacturer's specifications. Two cDNA expression libraries were then prepared from the mRNA by reverse transcriptase synthesis using a Lambda ZAP cDNA library synthesis kit (Stratagene). cDNA Library from Epithelial Cells (HNKA)
  • HNKA human natural killer (NK) cells
  • a NK library was first constructed using pooled RNA extracted from primary NK cells from multiple donors, stimulated for 4 or 20 hours with IL-2 (10 ng/ml), EL-12 (1 ng/ml), IL-15 (50 ng/ml), interferon alpha (IFN- ; 1,000 U/ml) immobilized anti-CD 16 or immobilized anti-NAIL antibody, or from unstimulated cells.
  • RNA was extracted following standard procedures.
  • cDNA was prepared using a TimeSaver kit (Pharmacia, Uppsala, Sweden) following the manufacturer's protocol.
  • the cDNA was ligated to BgUL adaptors and size-selected using cDNA sizing columns (Gibco BRL, Gaithersburg MD).
  • the size-selected NK cDNA was ligated into a pDc 409 vector and transformed into E. coli DH105 cells.
  • Single-stranded DNA was prepared from the plasmid library using a helper phage (Stratagene)
  • a second cDNA library (referred to as FF cDNA library) was constructed using fetal foreskin tissue. RNA was extracted and cDNA prepared following standard protocols. The cDNA was ligated into the plasmid pBluescript following standard protocols. 10 ⁇ g of the FF cDNA library was linearized with the restriction endonuclease Notl and used as template to synthesize biotin-labeled cRNA using SP6 polymerase.
  • the subtracted NK cell library was constructed as follows.
  • the biotinylated FF cRNA was mixed with the NK library, ethanol precipitated and resuspended in 5 ⁇ l buffer (50 mM HEPES pH 7.4, 10 mM EDTA, 1.5 M NaCl, 0.2% SDS). After addition of 5 ⁇ l formamide and heating to 95° for 1 min, the material was left to hybridize for 24 hours at 42°C. 90 ⁇ l of 10 mM HEPES pH 7.3, 1 mM EDTA and 15 ⁇ l streptavidin was added followed by an incubation for 20 min at 50°C. This step was repeated again after extraction with phenol/chloroform.
  • 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 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 BLASTX. 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 polypeptide 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, 399, 418-434, 440-449 and 466-471, 516, 519, 520, 523-527, 531, 532, 535-537, 548, 555, 574-580, 585-587, 589, 593, 595, 596, 598-601, 605-607, 609, 612, 613, 615, 616 and 622.
  • polypeptide sequences of SEQ ID NO: 120-125, 275-276, 373-380, 382, 456, 457, 460-462, 488-493, 633, 637, 642, 683, 685, ' 691, 693, 703, 706, 710, 714, 717, 718, 720, 721 and 725 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.
  • the cDNA sequences of SEQ ID NO: 418-422 encode the same amino acid sequences as the cDNA sequences of SEQ ID NO: 7 and 11-14, namely SEQ ID NO: 126 and 130-133, respectively.
  • polypeptide sequences encoded by the cDNA sequences of SEQ ID NO: 24-44, 232-238, 429, 466, 468-470, 475, 476 and 484 are provided in SEQ ID NOS: 143-163, 309-315, 456, 488, 490-492, 497, 498 and 506, respectively.
  • the cDNA sequences of SEQ ID NO: 423-428, 430-434 and 449 encode the same polypeptide sequences as the cDNA sequences of SEQ ID NO: 27-29, 34, 35, 37, 40-44 and 238, namely SEQ ID NO: 146-148, 153, 154, 156, 159-163 and 315, respectively.
  • These polypeptide sequences were determined to have less than 75% 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 polypeptide 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 one or more transmembrane domain(s) within the sequence are provided in SEQ ID NOS: 45- 63, 239-253, 359-364, 400-402, 435, 436, 450-452, 455, 470-472, 542, 553-555, 573, 576, 581, 592, 593, 595 and 606.
  • the cDNA sequences of SEQ ID NOS: 45-48, 239- 249, 359-361, 363, 450, 451, 455, 472, 473, 553-555, 573, 576 and 592 were found to have less than 75% identity (determined as described above) to sequences in the EMBL database, using the FASTA or BLASTN computer algorithms.
  • polypeptide sequences encoded by the cDNA sequences of SEQ ID NO: 45-48, 239-249, 359-361, 363, 450, 451, 472, 473, 553-555, 573 and 606 (provided in SEQ ID NOS: 164-167, 316- 326, 383, 385-388, 407-408, 460, 461, 494, 495, 662, 663, 664, 679, 682 and 711 respectively) were found to have less than 75% identity, determined as described above, to sequences in the SwissProt database using the FASTX or BLASTP database.
  • the cDNA sequence of SEQ ID NO: 455 encodes the same polypeptide sequence as the cDNA sequence of SEQ ID NO: 359, namely SEQ ID NO: 383.
  • polypeptide sequences corresponding to the cDNA sequences of SEQ ID NOS: 49-63, 250-253, 436 and 452 with those in the SwissProt database showed less than 75% identity (determined as described above) to known sequences.
  • These polypeptide sequences are provided in SEQ ID NOS: 168-182, 327-330, 457 and 462, respectively.
  • cDNA sequences isolated as described above in Example 1 were determined to encode polypeptides that are family members of known protein families.
  • a family member is here defined to have at least 25% identity in the translated polypeptide to a known protein or member of a protein family.
  • cDNA sequences are provided in SEQ ID NOS: 64-76, 254-264, 365-369, 403, 437-439, 453, 454, 475-487, 510, 511, 514-527, 529-531, 533-536, 538-546, 548, 549, 553-559, 562, 564, 565, 567, 569-575, 577-589, 591-602, 604-612, 616-618, 621 and 622.
  • the cDNA sequences of SEQ ID NO: 437 and 454 encode the same amino acid sequences as the cDNA sequences of SEQ ID NO: 68 and 262, namely SEQ ID NO: 187 and 339, respectively.
  • the isolated cDNA sequences encode proteins that influence the growth, differentiation and activation of several cell types, and that may usefully be developed as agents for the treatment and diagnosis of skin wounds, cancers, growth and developmental defects, and inflammatory disease.
  • the utility for certain of the proteins of the present invention, based on similarity to known proteins, is provided in Table 2 below, together with the location of signal peptides and transmembrane domains for certain of the inventive sequences:
  • ORFs open reading frames
  • the cDNA sequences of SEQ ID NO: 514, 515, 516, 557, 558, 559, 560, 561, 567, 568, 619 and 621 are extended sequences of SEQ ID NO: 479, 480, 353, 91, 108, 82, 92, 81, 105, 90, 362 and 360, respectively.
  • polynucleotide sequences of SEQ ED NOS: 77-117, 265-267, 404-405 and 557-611 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 NOS: 77-89, 265-267 and 365-369 were determined to have less than 75% identity to sequences in the EMBL database using the computer algorithm FASTA or BLASTN, as described above.
  • polypeptide sequences encoded by the cDNA sequences of SEQ ID NO: 77-117, 265-267, 404-405 and 557-611 are provided in SEQ ID NOS: 666-718.
  • the amino acid sequences of SEQ ID NOS: 666, 668, 669, 671-673, 675, 676, 679, 682, 683, 685, 688, 690, 691, 693, 694, 702, 703, 706-708, 710, 711, 713 and 714 show less than 75% identity to sequences . in the SwissProt database.
  • polypeptides 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 HOMOLOG 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 polypeptide sequence being provided in SEQ ID NO: 196.
  • the library was screened using an [ ⁇ 32 P]-dCTP labeled double stranded cDNA probe corresponding to nucleotides 1 to 459 of the coding region within SEQ ID NO: 118.
  • EGF epidermal growth factor
  • SEQ ID NO: 196 Alignment of the functional peptides of the EGF family with SEQ ID NO: 196 revealed that an internal segment of SEQ ID NO: 196 (amino acids 54-104) shows greater than 40% identity to the active peptides of EGF, TGF-alpha and Epiregulin.
  • the active peptides of the EGF family are sufficient for activity and contain several conserved residues critical for the maintenance of this activity. These residues are retained in huTRl.
  • 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 he 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 XLI-Blue E. coli as described in Sambrook et al., Ibid.
  • muTRla 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.
  • lysis buffer (20 mM Tris-HCl pH 8.0, 10 mM beta mercaptoethanol, 1 mM PMSF).
  • 1% NP40 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 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.
  • 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.
  • Biological Activities of Polypeptides muTRla and huTRla muTRI and huTRl are novel members of the EGF family, which includes EGF, TGF , 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.
  • muTRla and huTRla were found to induce the phosphorylation of ERKl and ERK2 over background levels, indicating that muTRI and huTRl act as ligands for a cell surface receptor that activates the MAP kinase signaling pathway, possibly the EGF receptor.
  • huTRla was also found to induce the phosphorylation of ERKl and ERK2 over background levels, indicating that muTRI and huTRl act as ligands for a cell surface receptor that activates the MAP kinase signaling pathway, possibly the EGF receptor.
  • huTRla was also
  • huTRla acts as a ligand for a cell surface receptor that activates the MAP kinase signaling pathway, possibly the EGF receptor in HeLa and CVl/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% C0 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% 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% C0 2 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 was determined using the MTT dye reduction as described previously (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).
  • muTRI and huTRl stimulate keratinocyte growth arid 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 TR1 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.
  • TR1 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 TR1 mRNA may be critical to the mode of action of TGF ⁇ .
  • 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.
  • Two cell lines, PC 12 (rat pheochromocytoma - neural tumor)- and 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. 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).
  • Fold induction of SRE Mean relative luminescence of agonist/Mean relative luminescence of negative control.
  • TR1 may be important in the development of neural cells or their differentiation into specific neural subsets. TR1 may also be important in the development and progression of neural tumors.
  • the HaCaT growth assay was conducted as previously described, with the following modifications. Concurrently with the addition of EGF and TR1 to the media, anti-EGF Receptor (EGFR) antibody (Promega, Madison, Wisconsin) or the negative control antibody, mouse IgG (PharMingen, San Diego, California), were added at a concentration of 62.5 ng/ml.
  • EGFR anti-EGF Receptor
  • mouse IgG mouse IgG
  • an antibody which blocks the function of the EGFR inhibited 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
  • TRl may also bind to any other members of the EGFR family (for example, ErbB-2, -3, and/or -4) that are capable of heterodimerizing with the EGFR.
  • huTRl-1 also known as TRl ⁇
  • TRl ⁇ The sequence referred to as TRl-1
  • huTRl-1 is a splice variant of huTRl and consists of the ORF of huTRl minus amino acids 15 to 44 and 87 to 137.
  • These deletions have the effect of deleting part of the signal sequence and following amino terminal linker sequence, residues following the second cysteine residue of the EGF motif and the following transmembrane domain.
  • cysteine residue 147 may replace the deleted cysteine and thus the disulphide bridges are likely not affected.
  • huTRl-1 is an intracellular 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 huTRl-1 is given in SEQ ID NO: 412, with the corresponding amino acid sequence being provided in SEQ ID NO: 415.
  • TR1-2 also known as TRl ⁇
  • TRl -3 also known as TRl ⁇
  • TRl ⁇ TRl ⁇
  • TRl ⁇ TRl ⁇
  • TR1-2 consists of the ORF of huTRl minus amino acids 95 to 137. This deletion has the effect of deleting the transmembrane domain. Therefore TR1-2 is a secreted form ofhuTRl and binds with equal or greater affinity to the TRl receptor as huTRl, since the EGF domain remains intact. It functions as an agonist or an antagonist to huTRl or other EGF family members, including EGF and TGF ⁇ .
  • the determined cDNA sequence of TR1-2 is given in SEQ JJD NO: 410 and the corresponding amino acid sequence in SEQ ID NO: 413.
  • TR1-3 consists of the ORF ofhuTRl minus amino acids 36 to 44 and amino acids 86 to 136. These deletions have the effect of deleting part of the amino terminal linker sequence, residues following the second cysteine of the EGF motif and the following transmembrane domain. However, cysteine residue 147 (huTRl ORF numbering) may replace the deleted cysteine and thus the disulphide bridges are likely not affected. Therefore, TR1-3 is also a secreted form of huTRl and functions as an agonist or an antagonist to huTRl or other EGF family members, including EGF and TGF ⁇ .
  • the determined cDNA sequence of TRl -3 is given in SEQ ID NO: 411 and the corresponding amino acid sequence is SEQ ID NO: 414.
  • TRl ⁇ consists of the ORF of huTRl minus amino acids 86 to 136. This deletion has the effect of deleting residues following the second cysteine 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, TRl ⁇ is also a secreted form of huTRl and functions as an agonist or an antagonist to huTRl or other EGF family members, including EGF and TGF ⁇ .
  • the determined cDNA sequence of TRl ⁇ is given in SEQ ID NO: 371 and the corresponding polypeptide sequence in SEQ ID NO: 395.
  • TRl ⁇ consists of the ORF of huTRl minus amino acids 36 to 44 and amino acids 95 to 136. These deletions have the effect of deleting part of the amino terminal linker sequence and the transmembrane domain. Therefore TRl ⁇ is a secreted form of huTRl and binds with equal or greater affinity to the TRl receptor as huTRl, since the EGF domain remains intact. It functions as an agonist or an antagonist to huTRl or other EGF family members, including EGF and TGF ⁇ .
  • the determined nucleotide sequence of TRl ⁇ is given in SEQ ID NO: 416 and the corresponding polypeptide sequence in SEQ ID NO: 417.
  • 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 [ ⁇ 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 corresponding 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, or any muscle, and heart. Expression could also be detected in lower intestine, skin, bone marrow, and kidney. No detectable signal was found in testis, spleen, liver, thymus, stomach.
  • 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% identity over 285 nucleotides, 93.64% over 283 nucleotides and 94.035% over 285 nucleotides, respectively. Frame shifts were identified in AA643952 and HS 1301003 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.
  • a directionally cloned cDNA library was constructed from immature murine keratinocytes and submitted for high-throughput sequencing. Sequence data from a clone designated KDCL009274 showed 35% identity over 72 amino acids with rat macrophage inflammatory protein-2B (MIP-2B) and 32% identity over 72 amino acids with its murine homologue.
  • the insert of 1633bp (SEQ ID NO: 464; Fig. 15A) contained an open reading frame of 300bp with a 5' untranslated region of 202bp and a 3' untranslated region of 1161bp.
  • a poly-adenylation signal of AATAAA is present 19 base-pairs upstream of the poly-A tail.
  • the mature polypeptide (SEQ ID NO: 465) is 77 amino acids in length containing 4 conserved cysteines with no ELR motif.
  • This putative chemokine was identical to KS1.
  • the full length sequence was screened against the EMBL database using the BLAST program and showed some identity at the nucleotide level with human EST clones AA643952, AA865643, and HS 1301003, respectively.
  • a recently described human CXC chemokine, BRAK has some identity with KSl at the protein level.
  • the alignment of KSl (referred to in Fig.
  • KSl and BRAK demonstrate a high degree of divergence from the other ⁇ -chemokines, supporting the relatively low homology shown in the multiple alignment.
  • 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.
  • 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% (w/v) glycerol.
  • Preparations obtained were greater than 95% pure as determined by SDS-PAGE. Endotoxin contamination of purified proteins were determined using a limulus amebocyte lysate assay kit (BIO Whittaker, Walkersville, MD). Endotoxin levels were ⁇ 0.1 ng/ ⁇ g of protein. Internal amino acid sequencing was performed on tryptic peptides of KSl.
  • Fc fusion protein was produced by expression in HEK 293 T cells. 35 ⁇ g of KLF-lplGFc DNA to transfect 6 x 10 6 cells per flask, 200 mis of Fc containing supernatant was produced. The Fc fusion protein was isolated by chromatography using an Affiprep protein A resin (0.3 ml column, Biorad). After loading, the columrr'was washed with 15 mis of PBS, followed by a 5 ml wash of 50 mM Na citrate pH 5.0. The protein was then eluted with 6 column volumes of 50 mM Na citrate pH 2.5, collecting 0.3 ml fractions in tubes containing 60 ⁇ l of 2M Tris-HCl pH 8.0. Fractions were analyzed by SDS-PAGE.
  • the determined amino acid sequences for muKSl and huKSl are given in SEQ ID NOS: 397 and 398, respectively. These amino acid sequences confirmed that the determined sequences are identical to those established on the basis of 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. Flow cytometric binding studies
  • THP-1 or Jurkat cells were resuspended in 3 mis of wash buffer (2% FBS and 0.2% sodium azide in PBS) and pelleted at 4°C, 200 x g for 5 minutes. Cells were then blocked with 0.5% mouse and goat sera for 30 minutes on ice. Cells were washed, pelleted, resuspended in 50 ⁇ l of KLF-lFc at 10 ⁇ g/ml and incubated for 30 minutes on ice.
  • the cells were prepared as before and resuspended in 50 ⁇ l of goat anti-human IgG biotin (Southern Biotechnology Associates, AL) at 10 ⁇ g/ml and incuated for 30 minutes on ice. Finally, cells were washed, pelleted and resuspended in 50 ⁇ l of streptavidin-RPE (Southern Biotechnology Associates, AL) at 10 ⁇ g/ml and incuabated for a further 30 minutes on ice in the dark. Cells were washed and resuspended in 250 ⁇ l of wash buffer and stained with l ⁇ l of 10 ⁇ g/ml propidium iodide (Sigma) to exclude any dead cells.
  • Purified Fc fragment (10 ⁇ g/ml) was used as a negative control in place of KLF-lFc to determine non-specific binding.
  • Ten thousand gated events were analyzed on log scale using PE filter arrangement with peak transmittance at 575 nm and bandwidth of 10 nm on an Elite cell sorter (Coulter Cytometry).
  • KSl Fc The respiratory burst and migration assays indicated that KSl is active on monocytes and not T cells; therefore, the KSl Fc fusion protein was tested in a binding study with THP-1 and Jurkat T cells. KSl Fc showed a marked positive shift on THP-1 cells compared with the Fc fragment alone. In contrast, KSl demonstrated no positive binding with Jurkat cells in an identical experiment.
  • 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 muKSl was performed by probing human tissue blots (Clontech, 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.
  • Northern blot analysis to determine distribution of huKSl in cancer tissue was performed as described previously by probing tumor panel blots (Invifrogen, 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.
  • mice Eighteen C3H/HeJ mice were divided into 3 groups and injected intraperit ⁇ neally with muKSl, GV14B, or phosphate buffered saline (PBS).
  • GV14B is a bacterially expressed recombinant protein used as a negative control.
  • Group 1 mice were injected with 50 ⁇ g of muKSl in 1 ml of PBS;
  • Group 2 mice were, injected with 50 ⁇ g of GV14B in 1 ml of PBS;
  • Group 3 mice with 1 ml of PBS.
  • the cells in the peritoneal cavity of the 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 KSl. From histology, sites injected with muKSl had an inflammatory response of a mixed phenotype with mononuclear and polymorphonuclear cells present.
  • T cells were required for the inflammatory response.
  • 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.
  • 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.
  • 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 of muKSl 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.
  • 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 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.
  • muKSl promotes a quantifiable increase in cell numbers in the peritoneal cavity of C3H/HeJ mice injected with muKSl. Furthermore, we have shown that muKSl induces 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 KSl. 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.
  • KSl 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 6xHistidine tag.
  • This construct was transformed into competent XLI-Blue E.
  • KS2a was expressed by transfecting Cos-1 cells in 5 x T175 flasks with 180 ⁇ g of KSla using DEAE-dexfran. 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 ⁇ g/ml concanavalin A for 24 hrs at 37°C in 10% C0 2 .
  • 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% C0 2 at 37°C.
  • the degree of proliferation was 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 indicates 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 transferrin-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% C0 .
  • 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: 1-725 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

L'invention concerne des polynucléotides isolés codant pour des polypeptides exprimés dans des cellules cutanées de mammifères, ainsi que des vecteurs d'expression et des cellules hôtes comprenant ces polynucléotides isolés. L'invention concerne également des méthodes d'utilisation de ces polynucléotides et de ces polypeptides.
PCT/NZ2001/000099 2000-05-24 2001-05-24 Compositions isolees des cellules cutanees et methodes d'utilisation de ces compositions WO2001090357A1 (fr)

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