AU781763B2 - Human CUB-domain-containing protein and gene encoding the same - Google Patents

Description

WO 01/29219 PCT/US00/28798 HUMAN CUB-DOMAIN-CONTAINING PROTEIN AND GENE ENCODING THE SAME The present invention claims the benefit of U.S.

Provisional Application Numbers 60/160,285 and 60/183,583 which were filed October 19, 1999 and February 18, 2000 respectively and are herein incorporated in their entirety.

1. INTRODUCTION The present invention relates to the discovery, identification, and characterization of novel human polynucleotides encoding proteins sharing sequence similarity with mammalian proteins having CUB domains. The invention encompasses the described polynucleotides, host cell expression systems, the encoded proteins, fusion proteins, polypeptides and peptides, antibodies to the encoded proteins and peptides, and genetically engineered animals that either lack or over express the disclosed sequences, antagonists and agonists of the proteins, and other compounds that modulate the expression or activity of the proteins encoded by the disclosed polynucleotides that can be used for diagnosis, drug screening, clinical trial monitoring, or the treatment of physiological disorders, or diseases.

2. BACKGROUND OF THE INVENTION The CUB domain is an extracellular domain (ECD) present in variety of diverse proteins such as bone morphogenetic protein 1, proteinases, spermadhesins, complement subcomponents, and neuronal recognition molecules. Given the importance of these functions, CUB proteins have been associated with, inter alia, regulating development, modulating cellular processes, and preventing infectious disease.

3. SUMMARY OF THE INVENTION The present invention relates to the discovery, identification, and characterization of nucleotides that encode novel human proteins, and the corresponding amino acid WO 01/29219 PCT/US00/28798 sequences of these proteins. The novel human proteins (NHPs) described for the first time herein share structural similarity with animal CUB domain proteins, coagulation factors V and XIII, milk fat globule-EGF factor 8, transcriptional repressor AE-binding protein-1, and neuropilins 1 and 2 (which, like the presently described protein, contain both CUB and discoidin domains).

The novel human nucleic acid (cDNA) sequences described herein, encode proteins/open reading frames (ORFs) of 487, 586, and 539 amino acids in length (see SEQ ID NOS: 2, 4, and 6 respectively).

The invention also encompasses agonists and antagonists of the described NHPs, including small molecules, large molecules, mutant NHPs, or portions thereof that compete with native NHPs, NHP peptides, and antibodies, as well as nucleotide sequences that can be used to inhibit the expression of the described NHPs antisense and ribozyme molecules, and gene or regulatory sequence replacement constructs) or to enhance the expression of the described NHPs expression constructs that place the described gene under the contrul of a strong promoter system) and transgenic animals that express a NHP transgene, or "knock-outs" (which can be conditional) that do not express a functional NHP.

Several knockout ES cell lines have been produced that contain a gene trap mutation in a murine ortholog/homolog of the disclosed NHPs.

Further, the present invention also relates to processes for identifying compounds that modulate, act as agonists or antagonists, of NHP expression and/or NHP activity that utilize purified preparations of the described NHPs and/or NHP products, or cells expressing the same. Such compounds can be used as therapeutic agents for the treatment of any of a wide variety of symptoms associated with biological disorders or imbalances.

WO 01/29219 PCT/US00/28798 4. DESCRIPTION OF THE SEQUENCE LISTING AND FIGURES The Sequence Listing provides the sequences of several NHP ORFs encoding the described NHP amino acid sequences. SEQ ID NO:7 describes a NHP ORF and flanking sequences.

DETAILED DESCRIPTION OF THE INVENTION The NHPs described for the first time herein are novel proteins that are expressed in, inter alia, human cell lines, and human prostate, pituitary, fetal brain, brain, thymus, spleen, lymph node, trachea, kidney, fetal liver, thyroid, adrenal gland, salivary gland, stomach, small intestine, colon, muscle, heart, mammary gland, adipose, skin, esophagus, bladder, cervix, rectum, and testis cells.

The described sequences were compiled from gene trapped cDNAs, genomic sequence, and clones isolated from human brain, adipose, testis, and placenta cDNA libraries (Edge Biosystems, Gaithersburg, MD, and Clontech, Palo Alto, CA). The present invention encompasses the nucleotides presented in the Sequence Listing, host cells expressing such nucleotides, the expression products of such nucleotides, and: nucleotides that encode mammalian hic.logs of the dscribed genes; including the specifically described NHPs, and NHP products; nucleotides that encode one or more portions of a NHP that correspond to functional domains, and the polypeptide products specified by such nucleotide sequences, including but not limited to the novel regions of any active domain(s); (c) isolated nucleotides that encode mutant versions, engineered or naturally occurring, of the described NHPs in which all or a part of at least one domain is deleted or altered, and the polypeptide products specified by such nucleotide sequences, including but not limited to soluble proteins and peptides in which all or a portion of the signal sequence is deleted; (d) nucleotides that encode chimeric fusion proteins containing all or a portion of a coding region of a NHP, or one of its domains a receptor or ligand binding domain, accessory protein/self-association domain, etc.) fused to another peptide or polypeptide; or therapeutic or diagnostic WO 01/29219 PCT/USOO/28798 derivatives of the described polynucleotides such as oligonucleotides, antisense polynucleotides, ribozymes, dsRNA, or gene therapy constructs comprising a sequence first disclosed in the Sequence Listing.

As discussed above, the present invention includes: the human DNA sequences presented in the Sequence Listing (and vectors comprising the same) and additionally contemplates any nucleotide sequence encoding a contiguous NHP open reading frame (ORF), or a contiguous exon splice junction first described in the Sequence Listing, that hybridizes to a complement of a DNA sequence presented in the Sequence Listing under highly stringent conditions, hybridization to filter-bound DNA in 0.5 M NaHPO 4 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65 0 C, and washing in 0.1xSSC/0.1% SDS at 68 0 C (Ausubel F.M. et al., eds., 1989, Current Protocols in Molecular Biology, Vol. I, Green Publishing Associates, Inc., and John Wiley sons, Inc., New York, at p. 2.10.3) and encodes a functionally equivalent gene product. Additionally contemplated are any nucleotide sequences that hybridize to the complement of the DNA sequence that encode and express an amino acid sequence presented in the Sequence Listing under moderately stringent conditions, washing in 0.2xSSC/0.1% SDS at 42 0 C (Ausubel et al., 1989, supra), yet still encode a functionally equivalent NHP product. Functional equivalents of a NHP include naturally occurring NHPs present in other species and mutant NHPs whether naturally occurring or engineered (by site directed mutagenesis, gene shuffling, directed evolution as described in, for example, U.S. Patent No. 5,837,458). The invention also includes degenerate nucleic acid variants of the disclosed NHP polynucleotide sequences.

Additionally contemplated are polynucleotides encoding a NHP ORF, or its functional equivalent, encoded by a polynucleotide sequence that is about 99, 95, 90, or about percent similar or identical to corresponding regions of the nucleotide sequences of the Sequence Listing (as measured by WO 01/29219 PCT/US00/28798 BLAST sequence comparison analysis using, for example, the GCG sequence analysis package using standard default settings).

The invention also includes nucleic acid molecules, preferably DNA molecules, that hybridize to, and are therefore the complements of, the described NHP gene nucleotide sequences. Such hybridization conditions may be highly stringent or less highly stringent, as described above. In instances where the nucleic acid molecules are deoxyoligonucleotides ("DNA oligos"), such molecules are generally about 16 to about 100 bases long, or about 20 to about 80, or about 34 to about 45 bases long, or any variation or combination of sizes represented therein that incorporate a contiguous region of sequence first disclosed in the Sequence Listing. Such oligonucleotides can be used in conjunction with the polymerase chain reaction (PCR) to screen libraries, isolate clones, and prepare cloning and sequencing templates, etc.

Alternatively, such NHP oligonucleotides can be used as hybridization probes for screening libraries, and assessing gene expression patterns (particularly using a micro array or high-throughput "chip" format). Additionally, a series of the described NHP oligonucleotide sequences, or the complements thereof, can be used to represent all or a portion of the described NHP sequences. The oligonucleotides, typically between about 16 to about 40 (or any whole number within the stated range) nucleotides in length may partially overlap each other and/or a NHP sequence may be represented using oligonucleotides that do not overlap. Accordingly, the described NHP polynucleotide sequences shall typically comprise at least about two or three distinct oligonucleotide sequences of at least about 18, and preferably about nucleotides in length that are each first disclosed in the described Sequence Listing. Such oligonucleotide sequences may begin at any nucleotide present within a sequence in the Sequence Listing and proceed in either a sense orientation vis-a-vis the described sequence or in an antisense orientation.

WO 01/29219 P~UOf89 PCTIUSOOn8798 For oligonucleotide probes, highly stringent conditions may refer, to washing in 6xSSC/0.05% sodium pyrophosphate at 37'C (for 14-base oligos), 48'C (for 17-base oligos), 55'C (for 20-base oligos), and 60*C (for 23-base oligos). These nucleic acid molecules may encode or act as NHiP gene antisense molecules, useful, for example, in NI{P gene regulation (for and/or as antisense primers in amplification reactions of NHP gene nucleic acid sequences). With respect to NHP gene regulation, such techniques can be used to regulate biological functions. Further, such sequences may be used as part of ribozyme and/or triple helix sequences that are also useful for NHP gene regulation.

Inhibitory antisense or double stranded oligonucleotides can additionally comprise at least one modified base moiety which is selected from the group including but not limited to 5-bromouracil, 5-chiorouracil, hypoxanthine, xantine, 4-acetylcytosine, (carboxyhydroxylmethyl) uracil, 2-thiouridine, dihydrouracil, beta-D-galactosylqueosine, inosine, NE-i~Annr~v1 enie. -methvlcruanine, 1-methylinosine, 2, 2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracilacid 5-methyl-2-thiouracil, 3-(3-amino-3-N-2carboxypropyl) uracil, (acp3 and 2, 6-diaminopurine.

The antisense oligonucleotide can also comprise at least one modified sugar moiety selected from the group including but not limited to arabinose, 2-f luoroarabinose, xylulose, and hexose.

WO 01/29219 PCT/US00/28798 In yet another embodiment, the antisense oligonucleotide will comprise at least one modified phosphate backbone selected from the group consisting of a phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl phosphotriester, and a formacetal or analog thereof.

In yet another embodiment, the antisense oligonucleotide is an a-anomeric oligonucleotide. An a-anomeric oligonucleotide forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual 3-units, the strands run parallel to each other (Gautier et al., 1987, Nucl. Acids Res. 15:6625-6641). The oligonucleotide is a methylribonucleotide (Inoue et al., 1987, Nucl. Acids Res.15:6131-6148), or a chimeric RNA-DNA analogue (Inoue et al., 1987, FEBS Lett. 215:327-330). Alternatively, double stranded RNA can be used to disrupt the expression and function of a targeted NHP.

Oligonucleotides of the invention can be synthesized by standard methods known in the art, e.g. by use of an automated DNA synthesizer (such as are commercially available from Biosearch, Applied Biczystems, etc.) A? examples, phosphorothioate oligonucleotides can be synthesized by the method of Stein et al. (1988, Nucl. Acids Res. 16:3209), and methylphosphonate oligonucleotides can be prepared by use of controlled pore glass polymer supports (Sarin et al., 1988, Proc. Natl. Acad. Sci. U.S.A. 85:7448-7451), etc.

Low stringency conditions are well known to those of skill in the art, and will vary predictably depending on the specific organisms from which the library and the labeled sequences are derived. For guidance regarding such conditions see, for example, Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual (and periodic updates thereof), Cold Springs Harbor Press, and Ausubel et al., 1989, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y.

Alternatively, suitably labeled NHP nucleotide probes can be used to screen a human genomic library using appropriately WO 01/29219 PCT/US00/28798 stringent conditions or by PCR. The identification and characterization of human genomic clones is helpful for identifying polymorphisms (including, but not limited to, nucleotide repeats, microsatellite alleles, single nucleotide polymorphisms, or coding single nucleotide polymorphisms), determining the genomic structure of a given locus/allele, and designing diagnostic tests. For example, sequences derived from regions adjacent to the intron/exon boundaries of the human gene can be used to design primers for use in amplification assays to detect mutations within the exons, introns, splice sites splice acceptor and/or donor sites), etc., that can be used in diagnostics and pharmacogenomics.

Further, a NHP homolog can be isolated from nucleic acid .from an organism of interest by performing PCR using two degenerate or "wobble" oligonucleotide primer pools designed on the basis of amino acid sequences within the NHP products disclosed herein. The template for the reaction may be total RNA, mRNA, and/or cDNA obtained by reverse transcription of mRNA prepared from human or non-human cell lines or tissue known or suspected to express an allele of a NHP gene. Th PCR product can be subcloned and sequenced to ensure that the amplified sequences represent the sequence of the desired NHP gene. The PCR fragment can then be used to isolate a full length cDNA clone by a variety of methods. For example, the amplified fragment can be labeled and used to screen a cDNA library, such as a bacteriophage cDNA library. Alternatively, the labeled fragment can be used to isolate genomic clones via the screening of a genomic library.

PCR technology can also be used to isolate full length cDNA sequences. For example, RNA can be isolated, following standard procedures, from an appropriate cellular or tissue source one known, or suspected, to express a NHP gene, such as, for example, testis tissue). A reverse transcription (RT) reaction can be performed on the RNA using an oligonucleotide primer specific for the most 5' end of the amplified fragment for the priming of first strand synthesis.

WO 01/29219 PCT/US00/28798 The resulting RNA/DNA hybrid may then be "tailed" using a standard terminal transferase reaction, the hybrid may be digested with RNase H, and second strand synthesis may then be primed with a complementary primer. Thus, cDNA sequences upstream of the amplified fragment can be isolated. For a review of cloning strategies that can be used, see e.g., Sambrook et al., 1989, supra.

A cDNA encoding a mutant NHP gene can be isolated, for example, by using PCR. In this case, the first cDNA strand may be synthesized by hybridizing an oligo-dT oligonucleotide to mRNA isolated from tissue known or suspected to be expressed in an individual putatively carrying a mutant NHP allele, and by extending the new strand with reverse transcriptase. The second strand of the cDNA is then synthesized using an oligonucleotide that hybridizes specifically to the 5' end of the normal gene. Using these two primers, the product is then amplified via PCR, optionally cloned into a suitable vector, and subjected to DNA sequence analysis through methods well known to those of skill in the art. By comparing the DNA sequence of the mutant NHP allele to that of a corresponding normal NiP allele, the mutation(S) responsible for the loss or alteration of function of the mutant NHP gene product can be ascertained.

Alternatively, a genomic library can be constructed using DNA obtained from an individual suspected of or known to carry a mutant NHP allele a person manifesting a NHPassociated phenotype such as, for example, obesity, high blood pressure, connective tissue disorders, infertility, etc.), or a cDNA library can be constructed using RNA from a tissue known, or suspected, to express a mutant NHP allele. A normal NHP gene, or any suitable fragment thereof, can then be labeled and used as a probe to identify the corresponding mutant NHP allele in such libraries. Clones containing mutant NHP gene sequences can then be purified and subjected to sequence analysis according to methods well known to those skilled in the art.

WO 01/29219 PTUO/89 PCTIUSOO/28798 Additionally, an expression library can be constructed utilizing cDNA synthesized from, for example, RNA isolated from a tissue known, or suspected, to express a mutant NHP allele in an individual suspected of or known to carry such a mutant allele. In this manner, gene products made by the putatively mutant tissue can be expressed and screened using standard antibody screening techniques in conjunction with antibodies raised against normal NHP product, as described below. (For screening techniques, see, for example, Harlow, E. and Lane, eds., 1988, "Antibodies: A Laboratory Manual", Cold Spring Harbor Press, Cold Spring Harbor.) Additionally, screening can be accomplished by screening with labeled NIIP fusion proteins, such as, for example, alkaline phosphatase-NHP or NHP-alkaline phosphatase fusion proteins.

In cases where a NEP mutation results in an expressed gene product with altered function as a result of a missense or a frameshift mutation), polyclonal antibodies to NHP are likely to cross-react with a corresponding mutant NHP gene product. Library clones detected via their reaction with such labeled antibodies can be purified and subjected to sequence analysis according to methods well knuwi Jin the art.

The invention also encompasses DNA vectors that contain any of the foregoing NHP coding sequences and/or their complements antisense); DNA expression vectors that contain any of the foregoing NHP coding sequences operatively associated with a regulatory element that directs the expression of the coding sequences (for example, baculo virus as described in U.S. Patent No. 5,869,336 herein incorporated by reference); genetically engineered host cells that contain any of the foregoing NHP coding sequences operatively associated with a regulatory element that directs the expression of the coding sequences in the host cell; and (d) genetically engineered host cells that express an endogenous NHP gene under the control of an exogenously introduced regulatory element gene activation). As used herein, regulatory elements include, but are not limited to, inducible and non-inducible promoters, enhancers, operators and other WO 01/29219 PCT/US00/28798 elements known to those skilled in the art that drive and regulate expression. Such regulatory elements include but are not limited to the human cytomegalovirus (hCMV) immediate early gene, regulatable, viral elements (particularly retroviral LTR promoters), the early or late promoters of adenovirus, the lac system, the trp system, the TAC system, the TRC system, the major operator and promoter regions of phage lambda, the control regions of fd coat protein, the promoter for 3-phosphoglycerate kinase (PGK), the promoters of acid phosphatase, and the promoters of the yeast a-mating factors.

The present invention also encompasses antibodies and anti-idiotypic antibodies (including Fab fragments), antagonists and agonists of a NHP, as well as compounds or nucleotide constructs that inhibit expression of a NHP gene (transcription factor inhibitors, antisense and ribozyme molecules, or gene or regulatory sequence replacement constructs), or promote the expression of a NHP expression constructs in which NHP coding sequences are operatively associated with expression control elements such as promoters, promoter/enhancers, eLc.).

The NHPs or NHP peptides, NHP fusion proteins, NHP nucleotide sequences, antibodies, antagonists and agonists can be useful for the detection of mutant NHPs or inappropriately expressed NHPs for the diagnosis of disease. The NHPs or NHP peptides, NHP fusion proteins, NHP nucleotide sequences, host cell expression systems, antibodies, antagonists, agonists and genetically engineered cells and animals can be used for screening for drugs (or high throughput screening of combinatorial libraries) effective in the treatment of the symptomatic or phenotypic manifestations of perturbing the normal function of NHP in the body. The use of engineered host cells and/or animals may offer an advantage in that such systems allow not only for the identification of compounds that bind to the endogenous receptor for a NHP, but can also identify compounds that trigger NHP-mediated activities or pathways.

WO 01/29219 PCT/US00/28798 Finally, the NHP products can be used as therapeutics.

For example, soluble derivatives such as NHP peptides/domains corresponding to NHP, NHP fusion protein products (especially NHP-Ig fusion proteins, fusions of a NHP, or a domain of a NHP, to an IgFc), NHP antibodies and anti-idiotypic antibodies (including Fab fragments), antagonists or agonists (including compounds that modulate or act on downstream targets in a NHP-mediated pathway) can be used to directly treat diseases or disorders. For instance, the administration of an effective amount of soluble NHP, or a NHP-IgFc fusion protein or an anti-idiotypic antibody (or its Fab) that mimics a NHP could activate or effectively antagonize the endogenous NHP receptor. Nucleotide constructs encoding such NHP products can be used to genetically engineer host cells to express such products in vivo; these genetically engineered cells function as "bioreactors" in the body delivering a continuous supply of a NHP, a NHP peptide, or a NHP fusion protein to the body. Nucleotide constructs encoding functional NHP, mutant NHPs, as well as antisense and ribozyme molecules can also be used in "gene therapy" approaches for the modulation of HP expression- Thus, the invention also encompasses pharmaceutical formulations and methods for treating biological disorders.

Various aspects of the invention are described in greater detail in the subsections below.

5.1 THE NHP SEQUENCES The cDNA sequences and corresponding deduced amino acid sequences of the described NHPs are presented in the Sequence Listing. SEQ ID NO:7 describes a NHP ORF as well as flanking regions. The NHP nucleotides were obtained from human cDNA libraries using probes and/or primers generated from human gene trapped sequence tags, and genomic sequence. Expression analysis has provided evidence that the described NHP can be expressed a variety of human cells as well as gene trapped human cells.

WO 01/29219 PCT/US00/28798 5.2 NHPs AND NHP POLYPEPTIDES NHPs, polypeptides, peptide fragments, mutated, truncated, or deleted forms of the NHPs, and/or NHP fusion proteins can be prepared for a variety of uses. These uses include, but are not limited to, the generation of antibodies, as reagents in diagnostic assays, for the identification of other cellular gene products related to a NHP, as reagents in assays for screening for compounds that can be as pharmaceutical reagents useful in the therapeutic treatment of mental, biological, or medical disorders and disease.

The Sequence Listing discloses the amino acid sequences encoded by the described NHP polynucleotides. The NHPs display an initiator methionines in DNA sequence contexts consistent with a translation initiation site, and several of the ORFs display a consensus signal sequence which can indicate that the described NHP ORFs are secreted proteins, or can be membrane associated.

The NHP amino acid sequences of the invention include the amino acid sequences presented in the Sequence Listing as well as analogues and derivatives thereof. Further, corresponding NHP h1c-- olgue from other speCb are encompassed by the invention. In fact, any NHPs encoded by a NHP nucleotide sequence described above are within the scope of the invention, as are any novel polynucleotide sequences encoding all or any novel portion of an amino acid sequence presented in the Sequence Listing. The degenerate nature of the genetic code is well known, and, accordingly, each amino acid presented in the Sequence Listing, is generically representative of the well known nucleic acid "triplet" codon, or in many cases codons, that can encode the amino acid. As such, as contemplated herein, the amino acid sequences presented in the Sequence Listing, when taken together with the genetic code (see, for example, Table 4-1 at page 109 of "Molecular Cell Biology", 1986, J. Darnell et al. eds., Scientific American Books, New York, NY, herein incorporated by reference) are generically representative of all the WO 01/29219 PCT/US00/28798 various permutations and combinations of nucleic acid sequences that can encode such amino acid sequences.

The invention also encompasses proteins that are functionally equivalent to the NHPs encoded by the presently described nucleotide sequences as judged by any of a number of criteria, including, but not limited to, the ability to bind and cleave a substrate of a NHP, or the ability to effect an identical or complementary downstream pathway, or a change in cellular metabolism proteolytic activity, ion flux, tyrosine phosphorylation, etc.). Such functionally equivalent NHP proteins include, but are not limited to, additions or substitutions of amino acid residues within the amino acid sequence encoded by the NHP nucleotide sequences described above, but which result in a silent change, thus producing a functionally equivalent gene product. Amino acid substitutions can be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; polar nieutral amino acid include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids include arginine, lysine, and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

A variety of host-expression vector systems can be used to express the NHP nucleotide sequences of the invention.

Where, as in the present instance, a NHP peptide or NHP polypeptide is thought to be a soluble or secreted molecule, the peptide or polypeptide can be recovered from the culture media. Such expression systems also encompass engineered host cells that express NHP, or functional equivalent, in situ.

Purification or enrichment of a NHP from such expression systems can be accomplished using appropriate detergents and lipid micelles and methods well known to those skilled in the art. However, such engineered host cells themselves may be used in situations where it is important not only to retain WO 01/29219 PCTUSOO/28798 the structural and functional characteristics of a NHP, but to assess biological activity, in drug screening assays.

The expression systems that may be used for purposes of the invention include but are not limited to microorganisms such as bacteria E. coli, B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing NHP nucleotide sequences; yeast Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing NHP encoding nucleotide sequences; insect cell systems infected with recombinant virus expression vectors baculovirus) containing NHP sequences; plant cell systems infected with recombinant virus expression vectors cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors Ti plasmid) containing NHP nucleotide sequences; or mammalian cell systems COS, CHO, BHK, 293, 3T3) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells metallothionein promoter) or from mammalian viruses the adenovirus late promoter; the vaccinia virus 7 .5K promoter).

In bacterial systems, a number of expression vectors may be advantageously selected depending upon the use intended for the NHP product being expressed. For example, when a large quantity of such a protein is to be produced for the generation of pharmaceutical compositions of and/or containing a NHP, or for raising antibodies to a NHP, vectors that direct the expression of high levels of fusion protein products that are readily purified may be desirable. Such vectors include, but are not limited, to the E. coli expression vector pUR278 (Ruther et al., 1983, EMBO J. 2:1791), in which a NHP coding sequence may be ligated individually into the vector in frame with the lacZ coding region so that a fusion protein is produced; pIN vectors (Inouye Inouye, 1985, Nucleic Acids Res. 13:3101-3109; Van Heeke Schuster, 1989, J. Biol. Chem.

264:5503-5509); and the like. pGEX vectors (Pharmacia or American Type Culture Collection) can also be used to express WO 01/29219 PCT/US00/28798 foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione. The PGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.

In an insect system, Autographa californica nuclear polyhidrosis virus (AcNPV) is used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. A NHP coding sequence can be cloned individually into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter). Successful insertion of NHP coding sequence will result in inactivation of the polyhedrin gene and production of non-occluded recombinant virus virus lacking the proteinaceous coat coded for by the polyhedrin gene). These recombinant viruses are then used to infect Spodoptera frugiperda cells in which the inserted sequence is expressed see Smith ct al., 1983. J.

Virol. 46: 584; Smith, U.S. Patent No. 4,215,051).

In mammalian host cells, a number of viral-based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, the NHP nucleotide sequence of interest may be ligated to an adenovirus transcription/translation control complex, the late promoter and tripartite leader sequence. This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome region El or E3) will result in a recombinant virus that is viable and capable of expressing a NHP product in infected hosts See Logan Shenk, 1984, Proc. Natl. Acad. Sci. USA 81:3655-3659). Specific initiation signals may also be required for efficient translation of inserted NHP nucleotide sequences. These signals include the ATG initiation codon and adjacent sequences. In cases where WO 01/29219 PCT/US0O/28798 an entire NHP sequence or cDNA, including its own initiation codon and adjacent sequences, is inserted into the appropriate expression vector, no additional translational control signals may be needed. However, in cases where only a portion of a NHP coding sequence is inserted, exogenous translational control signals, including, perhaps, the ATG initiation codon, must be provided. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (See Bittner et al., 1987, Methods in Enzymol. 153:516-544).

In addition, a host cell strain may be chosen that modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications glycosylation) and processing cleavage) of protein products may be important for the function uf the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. To this end, eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used. Such mammalian host cells include, but are not limited to, CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, WI38, and in particular, human cell lines.

For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines which stably express the NHP sequences described above can be engineered. Rather than using expression vectors which contain viral origins of replication, host cells can be WO 01/29219 PCT/US00/28798 transformed with DNA controlled by appropriate expression control elements promoter, enhancer sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Following the introduction of the foreign DNA, engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media.

The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines.

This method may advantageously be used to engineer cell lines which express a NHP product. Such engineered cell lines may be particularly useful in screening and evaluation of compounds that affect the endogenous activity of a NHP product.

A number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler, et al., 1977, Cell 11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska Szybalski, 1962, Proc.

Natl. Acad. Sci. USA 48:2026), and adenine phYphoribosy transferase (Lowy. et al. 1980. Cell 22:817) genes can be employed in tk-, hgprt or aprt- cells, respectively. Also, antimetabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler, et al., 1980, Natl. Acad. Sci. USA 77:3567; O'Hare, et al., 1981, Proc.

Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance to mycophenolic acid (Mulligan Berg, 1981, Proc. Natl. Acad.

Sci. USA 78:2072); neo, which confers resistance to the aminoglycoside G-418 (Colberre-Garapin, et al., 1981, J. Mol.

Biol. 150:1); and hygro, which confers resistance to hygromycin (Santerre, et al., 1984, Gene 30:147).

Alternatively, any fusion protein can be readily purified by utilizing an antibody specific for the fusion protein being expressed. For example, a system described by Janknecht et al. allows for the ready purification of non-denatured fusion proteins expressed in human cell lines (Janknecht, et al., WO 01/29219 PCT/US00/28798 1991, Proc. Natl. Acad. Sci. USA 88:8972-8976). In this system, the gene of interest is subcloned into a vaccinia recombination plasmid such that the gene's open reading frame is translationally fused to an amino-terminal tag consisting of six histidine residues. Extracts from cells infected with recombinant vaccinia virus are loaded onto Ni 2 nitriloacetic acid-agarose columns and histidine-tagged proteins are selectively eluted with imidazole-containing buffers.

5.3 ANTIBODIES TO NHP PRODUCTS Antibodies that specifically recognize one or more epitopes of a NHP, or epitopes of conserved variants of a NHP, or peptide fragments of a NHP are also encompassed by the invention. Such antibodies include but are not limited to polyclonal antibodies, monoclonal antibodies (mAbs), humanized or chimeric antibodies, single chain antibodies, Fab fragments, fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above.

The antibodies of the invention may be used, for example, in the detection of a NHP in a biolugical sample and may, therefore, be utilized as part of a diagnostic or prognostic technique whereby patients may be tested for abnormal amounts of NHP. Such antibodies may also be utilized in conjunction with, for example, compound screening schemes for the evaluation of the effect of test compounds on expression and/or activity of a NHP sequence product. Additionally, such antibodies can be used in conjunction gene therapy to, for example, evaluate the normal and/or engineered NHP-expressing cells prior to their introduction into the patient. Such antibodies may additionally be used as a method for the inhibition of abnormal NHP activity. Thus, such antibodies may, therefore, be utilized as part of treatment methods.

For the production of antibodies, various host animals may be immunized by injection with a NHP, an NHP peptide one corresponding to a functional domain of a NHP), truncated NHP polypeptides (NHP in which one or more domains WO 01/29219 PCT/US00/28798 have been deleted), functional equivalents of a NHP or mutated variants of a NHP. Such host animals may include but are not limited to pigs, rabbits, mice, goats, and rats, to name but a few. Various adjuvants may be used to increase the immunological response, depending on the host species, including but not limited to Freund's adjuvant (complete and incomplete), mineral salts such as aluminum hydroxide or aluminum phosphate, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum.

Alternatively, the immune response could be enhanced by combination and or coupling with molecules such as keyhole limpet hemocyanin, tetanus toxoid, diptheria toxoid, ovalbumin, cholera toxin or fragments thereof. Polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of the immunized animals.

Monoclonal antibodies, which are homogeneous populations of antibodies to a particular antigen, can be obtained by any technique which provides for the production of antibody molecules by continuous cell lines in culture. Th--e include but are not limited to, the hybridoma technique of Kohler and Milstein, (1975, Nature 256:495-497; and U.S. Patent No.

4,376,110), the human B-cell hybridoma technique (Kosbor et al., 1983, Immunology Today 4:72; Cole et al., 1983, Proc.

Natl. Acad. Sci. USA 80:2026-2030), and the EBV-hybridoma technique (Cole et al., 1985, Monoclonal Antibodies And Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclass thereof. The hybridoma producing the mAb of this invention may be cultivated in vitro or in vivo.

Production of high titers of mAbs in vivo makes this the presently preferred method of production.

In addition, techniques developed for the production of "chimeric antibodies" (Morrison et al., 1984, Proc. Natl.

Acad. Sci., 81:6851-6855; Neuberger et al., 1984, Nature, 312:604-608; Takeda et al., 1985, Nature, 314:452-454) by WO 01/29219 PCT/USO0/28798 splicing the genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity can be used. A chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine mAb and a human immunoglobulin constant region. Such technologies are described in U.S. Patents Nos. 6,075,181 and 5,877,397 and their respective disclosures which are herein incorporated by reference in their entirety.

Alternatively, techniques described for the production of single chain antibodies Patent 4,946,778; Bird, 1988, Science 242:423-426; Huston et al., 1988, Proc. Natl. Acad.

Sci. USA 85:5879-5883; and Ward et al., 1989, Nature 334:544- 546) can be adapted to produce single chain antibodies against NHP sequence products. Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide.

Antibody fragments which recognize specific epitopes may be generated by known techniques. For exmple, such fragments include, but are not limited to: the F(ab')2 fragments which can be produced by pepsin digestion of the antibody molecule and the Fab fragments which can be generated by reducing the disulfide bridges of the F(ab') 2 fragments. Alternatively, Fab expression libraries may be constructed (Huse et al., 1989, Science, 246:1275-1281) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity.

Antibodies to a NHP can, in turn, be utilized to generate anti-idiotype antibodies that "mimic" a given NHP, using techniques well known to those skilled in the art. (See, Greenspan Bona, 1993, FASEB J 7(5):437-444; and Nissinoff, 1991, J. Immunol. 147(8):2429-2438). For example antibodies which bind to a NHP domain and competitively inhibit the binding of NHP to its cognate receptor can be used to generate anti-idiotypes that "mimic" a NHP and, therefore, bind and activate or neutralize a receptor. Such anti- WO 01/29219 PCT/US00/28798 idiotypic antibodies or Fab fragments of such anti-idiotypes can be used in therapeutic regimens involving a NHP signaling pathway.

The present invention is not to be limited in scope by the specific embodiments described herein, which are intended as single illustrations of individual aspects of the invention, and functionally equivalent methods and components are within the scope of the invention. Indeed, various modifications of the invention, in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims. All cited publications, patents, and patent applications are herein incorporated by reference in their entirety.

EDITORIAL NOTE APPLICATION NUMBER 10945/01 The following Sequence Listing pages 1/7 to 7/7 are part of the description. The claims pages follow on page 23.

WO 01/29219 WOOI/9219PCT/US00128798 SEQUENCE LISTING <110> Donoho. Gregory Turner. C. Alexander Jr.

Nehis, Michael Friedrich, Glenn Zaxnbrowicz, Brian Sands, Arthur T.

<120> Novel Humian Proteins and Polynucleotides Encoding the Same <130> LEX-0071--PCT <150> US 60/160,285 <151> 1999-10-19 <150> US 60/183,583 <151> 2000-02-18 <160> 7 <170> FastSEQ for Windows Version <210> 1 <211> 1464 <212> DNA <213> homo sapiens <400> 1 atgacatcta acagtaccaa acctgtgctt ygaay La tga gagagtggat ccagatttaa t tc tgcccag tatagagata c tagg Lggcc gccaatggtg aatggttgca tggcagtcgg caggaccaag tggc tggaga acacagtcga tctaagtgga tctaactttc cgggttgtcc cagattacac tcaactaaga ggaataaaca ggaatgggga gcggaggctc tcagctgagt atcacaagtg agaattatcc aggggaaaag c tgac tatc t cccacatttc taacatgttt ctggttgtag cctctttatt agatcagtgt ttctttcgag gcagatcctt tcaatgagag gcccatcatg tcgatttggg acttcaactt agacctataa gggacccagt c ccagac at g aaggtaatga aagaagatga ttacaacggt tctttgcagc agaaaacaga ttaccatcag atatggcagg cgggacctac actgattctg tctcttcacc tggccggggt ggaacgagc t agacgtagca gtgcaaagct gcttcagcgc ggatggttcc gagttttgaa tggagaccaa ggc ttcgggC ggagaaaaag ttatgttaag aggaattgtg gcaaaacaat gcaccagagg ttcattggtg gacaatcaca ggctattcca ctttagaaag ctgttggaag ctatgataat t taa cccaatcaca aggttgggag agctcttcag 1 rIrgacaaa tttttgctga agccattatt ggagacattt gccatccatg aaagggatca ctgtcagaca cctgacgggc gttcactggt gacagtagca aaaataacag agttttgtga aataatgaag ttcacccctc atagccttga tggcgcaaga aggcccatcc ttggtgctcC aagaagaaga caga ttaaa t gagaaggaga ctgtttgcga atttggatat atcaatatgg ataaagtaac cctatgcgag tgaagacaga ctgggaatat caggaataat gtcgatatga agcgatttct aaatcagagc ctcctggcca acaaccacaa gaattaggac tgaacttcaa aaaaggtgtt ccatcgtggc aggtggagc t caagtcaaag cctcggaaga ttgttgtcct aaggaagtcc atccctttgc tgacacaaaa aaagacaat t cgaatcccag tccatactgt cgtccgcttt cagcgaccat atacagcaaa ggtagatgga tgctgatgaa agggattctg gtttacctcc ttcttcctca agcccgactt accacgagag cacaggatct aaacaataat tcagggtaac cagatatgtg cattggttgc caccagtgtt aacatccaca ggtgtttgct gtatggatca cagacatcag gttagatctc 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1464 <210> 2 <211> 487 <212> PRT <213> homo sapiens <400> 2 1/ 7 WO 01129219 PCT1US00128798 Met Thr Ser Lys Asn Tyr Pro Giy Thr Tyr Pro Asn His Thr Val Cys 1 5 10 Giu Lys Thr Ile Thr Val Pro Lys Gly Lys Arg Leu Ile Leu Arg Leu 25 Gly Asp Leu Asp Ile Giu Ser Gin Thr Cys Ala Ser Asp Tyr Leu Leu 40 Phe Thr Ser Ser Ser Asp Gin Tyr Gly Pro Tyr Cys Gly Ser Met Thr 55 Val Pro Lys Giu Leu Leu Leu Asn Thr Ser Giu Val Thr Val Arg Phe 70 75 Giu Ser Giy Ser His Ilie Ser Gly Arg Gly Phe Leu Leu Thr Tyr Ala 90 Ser Ser Asp His Pro Asp Leu Ilie Thr Cys Leu Giu Arg Ala Ser Hi-s 100 105 110 Tryr Leu Lys Thr Giu Tyr Ser Lys Phe Cys Pro Ala Gly Cys Arg Asp 115 120 125 Val Ala Gly Asp Ile Ser Gly Asn Met Val Asp Gly Tyr Arg Asp Thr 130 135 140 Ser Leu Leu Cys Lys Ala Ala Ile His Aia Gly Ile Ile Ala Asp Giu 145 150 155 160 Leu Gly Gly Gin Ilie Ser Val Leu Gin Arg Lys Gly Ile Ser Arg Tyr 165 170 175 Giu Gly Ile Leu Ala Asn Gly Vai Leu Ser Arg Asp Gly Ser Leu Ser 180 185 190 Asp Lys Arg Phe Leu Phe Thr Ser Asn Gly Cys Ser Arg Ser Leu Ser 195 200 205 Phe Giu Pro Asp Gly Gin Ilie Arg Ala Ser Ser Ser Trp Gin Ser Val 210 215 220 Asn Giu Ser Gly Asp Gin Val His Trp Ser Pro Gly Gin Ala Arg Leu 225 230 235 240 Gin Asp Gin Gly Pro Ser Trp, Ala Ser Gly Asp Ser Ser Asn Asn His 245 250 255 Lys Pro Arg Giu Trp, Leu Giu Ile Asp Leu Giy Giu Lys Lys Lys Ile 260 265 270 Thr Gv Ile Arg Thr Thr Gly Ser Thr Gin Ser Asn Phe Asn Phe Tyr 275 280 285 Val Lys Ser Phe Val Met Asn Phe Lys Asn Asn Asn Ser Lys Trp Lys 290 295 300 Thr Tyr Lys Giy Ile Val Asn Asn Giu Giu Lys Val Phe Gin Gly Asn 305 310 315 320 Ser Asn Phe Arg Asp Pro Val Gin Asn Asn Phe Ile Pro Pro Ilie Val 325 330 335 Ala Arg Tyr Val Arg Val Val Pro Gin Thr Trp His Gin Arg Ile Ala 340 345 350 Leu Lys Val Giu Leu Ile Gly Cys Gin Ile Thr Gin Gly Asn Asp Ser 355 360 365 Leu Val Trp Arg Lys Thr Ser Gin Ser Thr Ser Val Ser Thr Lys Lys 370 375 380 Giu Asp Giu Thr Ile Thr Arg Pro Ile Pro Ser Giu Giu Thr Ser Thr 385 390 395 400 Gly Ilie Asn Ilie Thr Thr Val Ala Ile Pro Leu Val Leu Leu Val Val 405 410 415 Leu Val Phe Ala Gly met Gly Ile Phe Ala Ala Phe Arg Lys Lys Lys 420 425 430 Lys Lys Giy Ser Pro Tyr Gly Ser Ala Glu Ala Gin Lys Thr Asp Cys 435 440 445 Trp, Lys Gin Ile Lys Tyr Pro Phe Ala Arg His Gin Ser Ala Giu Phe 450 455 460 Thr Ile Ser Tyr Asp Asn Giu Lys Giu Met Thr Gin Lys Leu Asp Leu 465 470 475 480 Ile Thr Ser Asp Met Ala Gly 485 2 /7 WO 01129219 <210> 3 <211> 1761 <212> DNA <213> hoino sapiens PCTIUSOO/28798 <400> 3 atgggattcg gaggcggccc aagctggcgc gctgccgggc gcggaggagc acatc taaga gtaccaaagg tgtgcttctg agtatgactg agtggatccc gatttaataa tgcccagcig agagatacct ggtggccaga aatggtgttc ggttgcagca cagtcggtca gaccaaggcc ctggagatcg cagtcgaac t aagtggaaga aactttcggg gttgtccccc attacacaag actaagaaag ataaacatta atggggatct gaggctcaga ,-rnnav r acaagtgata gtgcggggca ggcccgggca ccagcggggt ggggcctcct tgggtgatgg attatcccgg ggaaaagact actatcttct ttcccaaaga acatttctgg catg tt igga gttgtagaga ctttattgtg tcagtgtgct tttcgaggga gatccttgag atgagagtgg catcatgggc atttggggga tcaactttta cctataaagg acccagtgca agacatggca gtaatgattc aagatgagac caacggtggc ttgcagcctt aaacagac tg tggcaggtta gcgac tgcgc gctgcggctc catggtgccc ggctttgctg ctgtggacac gacctacccc gattctgagg cttcaccagc actcttgttg ccggggtttt acgagctagc cgtagcagga caaagctgcc tcagcgcaaa tggttCcctg ttttgaacct agaccaagtt ttcgggcgac gaaaaagaaa tgttaagagt aattgtgaat aaacaatttc ccagaggata attggtgtgg aatcacaagg tattccattg tagaaagaag t tggaagcag taataatqag a cccgtcccgg gggatccgtc ggcgcccgcg ctcgcggtct ctagtgactt aatcacactg ttgggagatt tcttcagatc aacacaagtg t tgc tgac ct cattatttga gacatttctg atccatgcag gggatcagtc tcagacaagc gacgggcaaa cactggtctc agtagcaaca ataacaggaa tttgtgatga aa tgaagaaa atccctccca gcct tgaagg cgcaagacaa cccatcccct gtgctccttg aagaagaaag attaaatatc aaggagatga cgccgcgctc gaggggaggc gcggcggcgC ccgccccgc t atcaggatag tttgcgaaaa tggatatcga aatatggtcc aagtaaccgt atgcgagcag agacagaata ggaatatggt gaataattgc gatatgaagg gatttctgtt tcagagcttc c tggccaagc accacaaacc ttaggaccac acttcaaaaa aggtgtttca tcgtggccag tggagctcat gtcaaagcac cggaagaaac ttgtcctggt gaagtccgta cctttgccag cacaaaagtt gtccgcagag cgagcttgcc actggcgcgg ccggctgcag tggcacaatg gacaattaca a tc ccagacc atactgtgga ccgctttgag cgaccatcca cagcaaattc agatggatat tgatgaacta gattctggcc tacctccaat t tcc tca tgg ccgacttcag acgagagtgg aggatctaca caataattct gggtaactct atatgtgcgg tggttgccag cagtgtttca atccacagga gtttgctgga tgga tcagcg acatcagtca agatctcatc 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1761 <210> 4 <211> 586 <212> PRT <213> homo sapiens <400> 4 Met Gly Phe Gly Ala Ala Giu Giu Gly Gin Arg Leu Arg Pro Val Pro Ser Ala Ala Arg Gin Leu Arg Ala Pro Arg Leu Gly Ile Gly Val Met Arg Arg Gly Val Pro Gly Gly Leu Leu Glu Ala Glu Leu Ala Ala Arg Ala Leu 40 Gly Gly Gly Ala 55 Leu Leu Ala Val Asp Gly Cys Gly Ala Glu Giu Leu Gly Ser Gly Thr Met Thr Leu Ala Pro Ser Leu Ala Arg Ala Ser Ala Pro Leu His Leu Val Thr 90 Pro Gly Thr T1yr Pro Lys Gly Lys 125 Ser Gin Thr Cys Ser Lys Asn Ala Gly Arg Leu Tyr Gin Asp Pro Asn His 110 Arg Leu Ile Ala Ser Asp Thr Val Cys 115 Leu Arg Leu Lys Thr Ile Gly Asp Leu Ile Glu 130 Tyr Leu 145 Leu Phe Thr 140 Ser Asp Gin Tyr Gly 155 Pro Tyr Cys 3,/7 WO 01129219 WO 0129219PCTIUSOO/28798 Ser Val Thr Ala Cys 225 Arg Ala Ser Ser Ser 305 Gin Ala Asn Lys Asri 385 Lys Gin Arg Asn 465 Thr Thr Leu Lys Thr 545 Ala Leu Glu Ser His Thr 215 Asp Cys Gin Leu Phe 295 Asp Gly Giy Giu Arg 375 Phe Gly Arg Val Giu 455 Arg Thr Ile Ala Ser 535 Ile Tryr Asp Leu Ile 185 Asp Tyr Ser Ala Ser 265 Asn Phe Gin Gin Ser 345 Leu Thr Met Val1 Pro 425 Val Ile Thr Thr Thr 505 Met Tyr Tyr Asn Ala 585 Asn Thr Gly Arg Ile Thr Lys Phe 220 Asn Met 235 Ile His Leu Gin Val Leu Ser Asn 300 Arg Ala 315 His Trp Ala Ser Ile Asp Ser Thr 380 Phe Lys 395 Asn Giu Gin Asn Pro Gin Cys Gin 460 Gin Ser 475 Pro Ile Ala Ile Ile Phe Ser Ala 540 Phe Ala 555 L.ys Giu Giu Phe 190 Leu Pro Asp Gly Lys 270 Arg Cys Ser Pro Asp 350 Gly Ser Asn Lys Phe 430 Trp Thr Ser Ser Leu 510 Ala Ala His Thr Thr Leu Arg Giy Tiyr 240 Ile Ile Gly Arg Trp, 320 Gin Ser Lys Phe Ser 400 Phe Pro Gin Gly Ser 480 Giu Leu Arg Lys Ser 560 Lys <210> <2ii> 1620 <212> DNA <213> homo sapiens <400> atggtgcccg gcgcccgcgg cggcggcgca ctggcgcggg ctgccgggcg gggcctcCtg gctttgctgc tcgcggtctc cgccccgctc cggctgcagg cggaggagct gggtgatggc 4 /7 WOO01/29219 tgtggacacc acctacccca attctgaggt ttcaccagct ctcttgttga cggggttttt cgagctagcc gtagcaggag aaagctgcca cagcgcaaag ggttccctgt tttgaacctg gaccaagttc tcgggCgaca aaaaagaaaa gttaagagtt attgtgaata aacaatttca cagaggatag t tggtgtggc atcacaaggc at tccat tgg agaaagaaga tggaagcaga gataatgaga PCT1USOO/28798 tagtgactta atcacactgt tgggagattt cttcagatca acacaagtga tgctgaccta attatttgaa acatttctgg tccatgcagg ggatcagtcg cagacaagcg acgggcaaat actggtctcc gtagcaacaa taacaggaat ttgtgatgaa atgaagaaaa tccctcccat ccttgaaggt gcaagacaag ccatcccctc tgCtccttgt agaagaaagg ttaaatatcc aggagatgac tcaggatagt ttgcgaaaag ggatatcgaa atatggtcca agtaaccgtc tgcgagcagc gacagaatac gaatatggta aataattgct atatgaaggg atttctgttt cagagcttct tggccaagcc ccacaaacca taggaccaca cttcaaaaac ggtgtttcag cgtggccaga ggagctcatt tcaaagcacc ggaagaaaca tgtcctggtg aagtccgtat ctttgccaga acaaaagtta ggcacaatga acaattacag tcccagacct tactgtggaa cgctttgaga gaccatccag agcaaattct gatggatata gatgaactag attctggcca acctccaatg tcctcatggc cgacttcagg cgagagtggc ggatctacac aataattcta ggtaactcta tatgtgcggg ggttgccaga agtgt ttcaa tccacaggaa tttgctggaa ggatcagcgg catcagtcag gatctcatca catctaagaa taccaaaggg gtgcttctga gtatgactgt gtggatccca atttaataac gcccagctgg gagatacctc gtggccagat atggtgttct gttgcagcag agtcggtcaa accaaggccc tggagatcga agtcgaactt agtggaagac actttcggga ttgtccccca ttacacaagg ctaagaaaga taaacattac tggggatctt aggctcagaa ctgagtttac caagtgatat ttatcccggg gaaaagactg ctatcttctc tcccaaagaa catttctggc atgtttggaa ttgtagagac tttattgtgc cagtgtgctt ttcgagggat atccttgagt tgagagtgga atcatgggct t ttgggggag caacttttat ctataaagga cccagtgcaa gacatggcac taatgattca agatgagaca aacggtggct tgcagccttt aacagactgt catcagctat ggcaggt taa 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 <210> 6 <211> 539 <212> PRT <213> homo sapiens <400> 6 Met Val Pro Gly Ala Arg Gly Gly T.4-1.1 Leu Leu Gin Ala Glu Asgp Ser Gly Leu Ala Leu Gly Thr Met Thr Asp Gly 40 Ser Lys 55 Thr Ile Gly Ala 10 Ala Val Cys Gly Asn Tyr Thr Val Leu Ala Arg Ser Ala Pro His Leu Val Pro Gly Thr Pro Lys Gly 75 Ser Gln Thr Gln Tyr Gly His Thr Ile Leu Val Cys Glu Ala Ala Gly Leu Arg Leu Thr Tyr Gln Tyr Pro Asn Lys Arg Leu Cys Ala Ser Pro Tyr Cys 110 Ser Glu Val Gly Phe Leu Arg Leu Gly Asp Phe Thr Leu Asp Ile Asp Tyr Leu Gly Ser Met 115 Thr Val Arg Ser Ser Val Pro Lys Leu Leu Asn Thr 125 Arg Phe Glu Ser His Ile Ser Tyr Ala Ser Ala Ser His Leu Lys Ala Gly His Pro Asp Thr Glu Tyr 170 Asp Ile Ser Thr Cys Leu Lys Phe Gly Cys Arg Gly Asn Met Cys Pro 175 Val Asp 190 Ala Gly Arg Asp 195 Ala Asp 210 Ser Arg Ser Leu Leu Ala Ala Ile Glu Leu Gly Gln Ile Ser Val A.rg Lys Gly Tyr Glu Leu Ala Asn Leu Ser Arg Ser Leu Ser Asp Lys Arg Phe Leu Phe Ser Asn Gly Cys 7 WO 01/29219 WO 0129219PcTIUSOO/mS98 Arg Ser Leu Ser 260 Trp Gin Ser Val 275 Gin Ala Arg Ueu Giu Pro Asp Gly 265 Ile Arg Ala 255 Ser Ser Ser 270 Ser Pro Giy Gly Asp Ser Asn Glu Ser Asp Gin Val His Pro Ser Trp, Ala Gin Asp Asn His Lys Pro Giu Trp Leu Asp Leu Gly Lys Lys Ile Gly Ile Arg Thr Ser Thr Gin Phe Asn Phe Ser Lys Trp 355 Phe Gin Gly 370 Pro Pro Ilie Lys Ser Phe Tyr Lys Gly 360 Asn Phe Lys Lys Thr Asn Ser Val Ala Val Asn Asn Asn Asn Asn 350 Giu Lys Val Asn Phe Ile Asn Phe 375 Arg Tyr 390 Lys Val Vai Trp Arg Asp Pro Val Val Arg Val Val 395 Giu Leu Ile Gly 410O Arg Lys Thr Ser Arg Ile Ala Pro Gin Thr Cys Gin Ile Gin Ser Thr Trp His 400 Thr Gin 415 Ser Val Gly Asn Asp Ser Ser Thr Lys 435 Giu Thr Ser Giu Asp Glu Thr 440 Asn Ile 455 Thr Arg Pro Pro Ser Giu Pro Leu Val Thr Gly Ile Thr Thr Val 450 Leu Leu Val Val Leu Phe Ala Gly Met Giy 475 Gly Ser Pro Tyr Gly Phe Ala Ala Lys Lys Lys Ser Ala Glu Ala Gin 495 Lys Thr Asp Ser Ala Giu 5.15 Lys Leu Asp 530 Cys Trp 500 Phe Thr Leu Ile Lys Gin Ile Ile Ser Tyr Thr Ser Asp Pro Phe Ala Arg His Gin 510 Met Thr Gin Asn Giu Lys Met Ala Gly <210> 7 <211> 1768 <212> DNA <2i3> homo sapiens <400> 7 ggcggaggag gacatctaag agtaccaaag ctgtgcttct aagtatgact gagtggatcc agatttaata c tgcccagct tagagatacc aggtggccag caatggtgtt tggttgcagc gcagtcggtc ggaccaaggc gctggagatc acagtcgaac taagtggaag taactttcgg ggttgtcccc ctgggtgatg aattatcccg gggaaaagac gactatcttc gttcccaaag cacatttctg acatgtttgg ggttgtagag tctttattgt accagtgtgc ctttcgaggg agatccttga aatgagagtg ccatcatggg gatttggggg ttcaactttt acctataaag gacccagtgc cagacatggc gctgtggaca ggacctaccc tgattctgag tcttcaccag aactcttgtt gccggggttt aacgagctag acgtagcagg gcaaagctgc ttcagcgcaa atggttccct gttttgaacc gagaccaagt cttcgggcga agaaaaagaa atgttaagag gaattgtgaa aaaacaattt accagaggat cctagtgact caatcacact gttgggagat ctcttcagat gaacacaagt tttgctgacc ccattatttg agacatttct catccatgca agggatcagt gtcagacaag tgacgggcaa tcac tgg tc t cagtagcaac aataacagga ttttgtgatg taatgaagaa catccctccc agccttgaag tatcaggata g tt tgcgaaa ttggatatcg caatatggtc gaagtaaccg tatgcgagca aagacagaat gggaatatgg ggaataattg cgatatgaag cgatttctgt atcagagctt cctggccaag aaccacaaac attaggacca aacttcaaaa aaggtgtttc atcgtggcca gtggagctca gtggcacaat agacaattac aatcccagac catactgtgg tccgctttga gcgaccatcc acagcaaatt tagatggata ctgatgaact ggattctggc ttacctccaa cttcctcatg cccgacttca cacgagagtg caggatctac acaataattc agggtaac tc gatatgtgcg ttggttgcca 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 6/ 7

I

WOO01129219 gattacacaa aac taagaaa aataaacatt aatggggatc ggaggc tcag agctgagttt cacaagtgat cctccgcatc tgttctttcc cagttaccga atgctgtctc PCT/tUSOO/28798 ggtaatgatt gaagatgaga acaacggtgg tttgcagcct aaaacagac t accatcagct atggcaggtt tatcagcagg caccctaaca ttaatctaga tatgcaaaaa cattggtgtg caatcacaag ctattccatt ttagaaagaa gttggaagca atgataatga aactccgttg ttgccccgga acaacaaagg gataaaatat aaaaaaaa gcgcaagaca gcccatcccc ggtgctcc tt gaagaagaaa gattaaatat gaaggagatg ac tgccaaaa tggatctcag gcagtaaatt tttcttaaaa agtcaaagca tcggaagaaa gttgtcctgg ggaagtccgt ccctttgcca acacaaaag t tagcatcccc agatgaggat aaagtactct atatatttca ccagtgtttc catccacagg tgtttgctgg atggatcagc gacatcagtc tagatctcat aacgtgcagc tggaacacca ttgtaaggta ttaaacacct 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1768 7 /7

Claims (4)

1. An isolated nucleic acid molecule comprising SEQ ID NO: 3.

2. An isolated nucleic acid molecule comprising a nucleotide sequence that: encodes the amino acid sequence shown in SEQ ID NO: 4; and hybridizes under stringent conditions to the nucleotide sequence of SEQ ID NO: 3 or the complement thereof.

3. An isolated cDNA comprising SEQ ID NO: 3.

4. An isolated nucleic acid molecule encoding the amino acid sequence presented in SEQ ID NO: 2. An isolated nucleic acid molecule encoding the amino acid sequence presented in SEQ ID NO: 6. a DATED THIS FIFTH DAY OF APRIL 2002 20 LEXICON GENETICS INCORPORATED BY PIZZEYS PATENT AND TRADE MARK ATTORNEYS o o ooo°*