AU2005239714A1 - Human kinase proteins and polynucleotides encoding the same - Google Patents

Description

Regulation 3.2

AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION STANDARD PATENT

APPLICANT:

Invention Title: Lexicon Genetics Incorporated HUMAN KINASE PROTEINS AND POLYNUCLEOTIDES ENCODING THE SAME The following statement is a full description of this invention, including the best method of performing it known to me: HUMAN KINASE PROTEINS AND POLYNUCLEOTIDES ENCODING THE SAME SThe present application claims the benefit of U.S.

Provisional Application Number 60/156,511 which was filed on September 28, 1999 and is herein incorporated by reference in its entirety.

h 1. INTRODUCTION C1 The present invention relates to the discovery, C 0 identification, and characterization of novel human C1 polynucleotides encoding proteins that share sequence similarity with animal kinases. 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 genes, antagonists and agonists of the proteins, and other compounds that modulate the expression or activity of the proteins encoded by the disclosed genes that can be used for diagnosis, drug screening, 0 clinical trial monitoring and the treatment of physiological disorders.

2. BACKGROUND OF THE INVENTION Kinases mediate phosphorylation of a wide variety of proteins and compounds in the cell. Along with phosphatases, kinases are involved in a wide range of regulatory pathways. Given the physiological importance of kinases, they have been subject to intense scrutiny and are proven drug targets.

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 sequences of these proteins. The novel human proteins (NHPs) described for the first time herein share structural similarity with animal

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kinases, and more particularly serine/threonine protein kinases. As such, the novel sequences represent a new family of proteins having homologues and orthologs across a range of phyla and species.

The novel human nucleic acid sequences described herein, encode alternative proteins/open reading frames (ORFs) of 187, 356, 324, 198, 347, and 315 amino acids in length (see SEQ ID NOS: 2, 4, 6, 8, 0\ 10, and 12) 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 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 NHP sequences expression constructs that place the described gene under the control of a strong promoter system). The present invention also includes both transgenic animals that express a NHP transgene, and NHP "knock-outs" (which can be conditional) that do not express a functional NHP.

Further, the present invention also relates to processes for identifying compounds that modulate, act as agonists or antagonists, of NHP expression and/or NHP product activity that utilize purified preparations of the described NHPs and/or NHP product, 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.

In the specification the term "comprising" shall be understood to have a broad meaning similar to the term "including" and will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. This definition also applies to variations on the term "comprising" such as "comprise" and "comprises".

S4. DESCRIPTION OF THE SEQUENCE LISTING AND FIGURES CI The Sequence Listing provides the sequence of novel human ORFs that encode the described NHP kinase-like proteins.

5. 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 S human brain, pituitary, spinal cord, spleen, trachea, kidney, C- prostate, testis, and adrenal gland cells. The described .0 sequences were compiled from gene trapped cDNAs, and human testis C-i cDNA libraries, (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 homologs of the described sequences, including the specifically described NHPs, and the NHP products; nucleotides that encode one or more portions of the NHPs 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); 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 in deleted; nucleotides that encode chimeric fusion proteins containing all or a portion of a coding region of an NHP, or one of its domains a receptor/ligand binding domain, accessory protein/self-association domain, etc.) fused to another peptide or polypeptide; or therapeutic or diagnostic derivatives of the described polynucleotides such as oligonucleotides, antisense polynucleotides, ribozymes, dsRNA, or S gene therapy constructs comprising a sequence first disclosed in Cl the Sequence Listing.

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SAs discussed above, the present invention includes: the human DNA sequences presented in the Sequence Listing (and vectors C 5 comprising the same) and additionally contemplates any nucleotide sequence encoding a contiguous NHP open reading frame (ORF) that hybridizes to a complement of a DNA sequence presented in the Sequence Listing under highly stringent conditions, e.g., C- hybridization to filter-bound DNA in 0.5 M NaHPO 4 7% sodium .0 dodecyl sulfate (SDS), 1 mM EDTA at 65 0 C, and washing in C-i 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 NHP ORFs, or their functional equivalents, encoded by polynucleotide sequences that are about 99, 95, 90, or about 85 percent similar to corresponding regions of SEQ ID NO:1 (as measured by BLAST sequence comparison analysis using, for example, the GCG sequence analysis package using default parameters).

0 The invention also includes nucleic acid molecules, Ci preferably DNA molecules, that hybridize to, and are therefore the 8 complements of, the described NHP nucleotide sequences. Such hybridization conditions may be highly stringent or less highly 5 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 to about 80, or about 34 to about 45 bases long, or any variation or combination of sizes represented therein that 0 incorporate a contiguous region of sequence first disclosed in the S 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 highthroughput "chip" format). Additionally, a series of the described NHP oligonucleotide sequences, or the complements 3 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 the 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 25, 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.

For oligonucleotide probes, highly stringent conditions may CI refer, to washing in 6xSSC/0.05% sodium pyrophosphate at S 37 0 C (for 14-base oligos), 48'C (for 17-base oligos), 55 0 C (for oligos), and 60 0 C (for 23-base oligos). These nucleic acid molecules may encode or act as NHP sequence antisense molecules, useful, for example, in NHP sequence regulation (for and/or as antisense primers in amplification reactions of NHP gene nucleic acid sequences) With respect to NHP sequence regulation, Ci such techniques can be used to regulate biological functions.

Further, such sequences can be used as part of ribozyme and/or Ci triple helix sequences that are also useful for NHP sequence 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-chlorouracil, hypoxanthine, xantine, 4-acetylcytosine, uracil, 5-carboxymethylaminomethyl-2-thiouridine, dihydrouracil, beta-Dgalactosylqueosine, inosine, N6-isopentenyladenine, 1-methyiguanine, 1-methylinosine, 2, 2 -dimethylguanine, 2-methylade ae, 2-methylguanine, 3-methylcytosine, N6-adenine, 7-methylguanine, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 2-methylthio-N6-isopentenyladenine, oxyacetic acid wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, uracil-5-oxyacetic acid methylester, uracil- 5-oxyacetic acid 5-methyl-2-thiouracil, 3-(3-amino-3-N-2carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine.

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

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, S a phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a S methylphosphonate, an alkyl phosphotriester, and a formacetal or analog thereof.

S.0- 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 p-units, the strands run parallel to each other (Gautier et al., 1987, Nucl. Acids Res. 15:6625-6641).

The oligonucleotide is a 2'-0-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.

0 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 Biosystems, etc.). As 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 S periodic updates thereof), Cold Springs Harbor Press, and CN 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 stringent conditions or by PCR. The identification and characterization of human genomic clones is helpful for Ch identifying polymorphisms (including, but not limited to, CI nucleotide repeats, microsatellite alleles, single nucleotide 0 LO polymorphisms, or coding single nucleotide polymorphisms), CI 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 sequence 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 Sthe basis of -amino acid sequences within the NHP producs disclosed herein. The template for the reaction may be total RNA, mRNA, and/or cDNA obtained by reverse transcription of mRNA prepared from, for example, human or non-human cell lines or tissue, such as prostate, rectum, colon, or adrenal gland, known or suspected to express an allele of a NHP gene. The PCR product can be subcloned and sequenced to ensure that the amplified sequences represent the sequence of the desired NHP. 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.

0 PCR technology can also be used to isolate full length cDNA CI sequences. For example, RNA can be isolated, following standard 1) procedures, from an appropriate cellular or tissue source one known, or suspected, to express a NHP sequence, such as, for 0 5 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

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0C priming of first strand synthesis. The resulting RNA/DNA hybrid C-i may then be "tailed" using a standard terminal transferase 3 reaction, the hybrid may be digested with RNase H, and second C-i 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 Sambrook et al., 1989, supra.

A cDNA encoding a mutant NHP sequence 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 NHP allele, the mutation(s) responsible for the loss or alteration of function of the mutant NHP sequence 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 NHP-associated phenotype such as, for example, immune disorders, obesity, high S blood pressure, etc.), or a cDNA library can be constructed using C RNA from a tissue known, or suspected, to express a mutant NHP allele. A normal NHP sequence, 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 sequences can then be purified and subjected S to sequence analysis according to methods well known to those skilled in the art.

c Additionally, an expression library can be constructed .0 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 may be expressed and screened using standard antibody screening techniques in conjunction with antibodies raised against a normal NHP product, as described below. (For screening techniques, see, for example, Harlow, E. and Lane, eds., 1988, "Antibodies:

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Laboratory Manual", Cold Spring Harbor Press, Cold Spring Harbor.) Additionally, screening can be accomplished by screening with labeled NHP fusion proteins, such as, for example, alkaline phosphatase-NHP or NHP-alkaline phosphatase fusion proteins. In cases where a NHP mutation results in an expressed gene product with altered function as a result of a missense or a frameshift mutation), polyclonal antibodies to a 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 known in the art.

An additional application of the described novel human polynucleotide sequences is their use in the molecular mutagenesis/evolution of proteins that are at least partially encoded by the described novel sequences using, S for example, polynucleotide shuffling or related S methodologies. Such approaches are described in U.S.

Patents Nos. 5,830,721 and 5,837,458 which are herein incorporated by reference in their entirety.

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 S regulatory element that directs the expression of the coding f 0 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 genetically engineered host cells that express an endogenous NHP sequence 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 0 other 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 SV40 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.

Where, as in the present instance, some of the described NHP peptides or polypeptides are thought to be cytoplasmic proteins, expression systems can be engineered that produce soluble derivatives of a NHP (corresponding to a NHP extracellular and/or S intracellular domains, or truncated polypeptides lacking one or CI more hydrophobic domains) and/or NHP fusion protein products (especially NHP-Ig fusion proteins, fusions of a NHP domain to an IgFc), NHP antibodies, and anti-idiotypic antibodies 0 5 (including Fab fragments) that can be used in therapeutic applications. Preferably, the above expression systems are engineered to allow the desired peptide or polypeptide to be \h recovered from the culture media.

The present invention also encompasses antibodies and anti- 0 idiotypic antibodies (including Fab fragments), antagonists and S agonists of the NHP, as well as compounds or nucleotide constructs that inhibit expression of a NHP sequence (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, etc.).

The NHPs or NHP peptides, NHP fusion proteins, NHP nucleotide sequences, antibodies, antagonists and agonists can be useful for !0 the detection of mutant NHPs or inappropriately expressed NHPs for the diagnosis of disease. The NHP prot.eins or 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 can offer an advantage in that such systems allow not only for the identification of compounds that bind to the endogenous receptor/ligand of a NHP, but can also identify compounds that trigger NHP-mediated activities or pathways.

O Finally, the NHP products can be used as therapeutics. For C( example, soluble derivatives such as NHP peptides/domains U corresponding the NHPs, NHP fusion protein products (especially NHP-Ig fusion proteins, fusions of a NHP, or a domain of a 0 5 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 NHPmediated pathway) can be used to directly treat diseases or M disorders. For instance, the administration of an effective S0 amount of soluble NHP, or a NHP-IgFc fusion protein or an antiidiotypic antibody (or its Fab) that mimics the NHP could activate or effectively antagonize the endogenous NHP or a protein interactive therewith. 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 NHPs, mutant NHPs, as well as antisense and ribozyme molecules can also be used in "gene Stherapy" approaches for the modulation of NHP 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 the corresponding deduced amino acid sequences of the described NHPs are presented in the Sequence Listing. The NHP nucleotide sequences were obtained from human cDNA libraries using probes and/or primers generated from human gene trapped sequence tags.

Expression analysis has provided evidence that the described NHPs can be expressed in human tissues as well as gene trapped human cells. In addition to the serine/threonine kinases, the S described NHPs also share significant similarity to a range of additional kinase families such as cell division protein kinases, cyclin dependent kinase, etc. from a range of phyla and species.

Given the physiological importance of protein kinases, they have been subject to intense scrutiny as exemplified and discussed in U.S. Patent No. 5,817,479 herein incorporated by reference in its 0\ entirety.

Cl During the generation of the described sequences, a 0 .0 polymorphism was identified in the 3' UTR reported in SEQ ID NO:13 CI (which includes a complete NHP ORF flanked by 5' and 3' sequences.

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 !0 products related to a NHP, as reagents in assays for screening for compounds that can-be as pharmaceutical reagents useful in thetherapeutic treatment of mental, biological, or medical disorders and disease.

The Sequence Listing discloses the amino acid sequences encoded by the described NHP sequences. The NHPs have initiator methionines in DNA sequence contexts consistent with eucaryotic translation initiation sites.

The NHP amino acid sequences of the invention include the amino acid sequence presented in the Sequence Listing as well as analogues and derivatives thereof. Further, corresponding NHP homologues from other species are encompassed by the invention.

In fact, any NHP protein encoded by the NHP nucleotide sequences described above are within the scope of the invention, as are any S novel polynucleotide sequences encoding all or any novel portion S of an amino acid sequence presented in the Sequence Listing. The U 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 S sequences presented in the Sequence Listing, when taken together M with the genetic code (see, for example, Table 4-1 at page 109 of V) 0 "Molecular Cell Biology", 1986, J. Darnell et al. eds., Scientific 0 American Books, New York, NY, herein incorporated by reference) are generically representative of all the 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 3 complementary downstream pathway, or a change in cellular Smetabolism 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 may 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 neutral amino acids include glycine, serine, threonine,- cysteine, tyrosine, asparagine, and Sglutamine; positively charged (basic) amino acids include C-1 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 the NHP peptide or polypeptide can exist, or has been engineered to exist, as a soluble or secreted molecule, the soluble NHP peptide or polypeptide can be recovered from the culture media. Such expression systems also encompass engineered host cells that Vf 0 express a NHP, or functional equivalent, in situ. Purification or S 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 the structural and functional characteristics of the 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 0 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 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 S viruses the adenovirus late promoter; the vaccinia virus C 7.5K promoter).

8 In bacterial systems, a number of expression vectors may be advantageously selected depending upon the use intended for the S NHP product being expressed. For example, when a large quantity of such a protein is to be produced for the generation of Spharmaceutical compositions of or containing 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.

(C 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 may also be used to express 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 may 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 S proteinaceous coat coded for by the polyhedrin gene). These C- recombinant viruses are then used to infect Spodoptera frugiperda cells in which the inserted sequence is expressed see Smith et al., 1983, J. Virol. 46: 584; Smith, U.S. Patent No.

c- 0 5 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 10 complex, the late promoter and tripartite leader sequence.

C-i This chimeric sequence may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a nonessential 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 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 C the expression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications glycosylation) and processing cleavage) of protein products may be important for the function of the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell C-i lines or host systems can be chosen to ensure the correct S0 modification and processing of the foreign protein expressed. To C-i 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, humnan 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 0 engineered. Rather than using expression vectors which contain viral origins of replication, host cells can be 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 the NHP product. Such engineered cell 0 lines may be particularly useful in screening and evaluation of C compounds that affect the endogenous activity of the NHP product.

8 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 l\ phosphoribosyltransferase (Lowy, et al., 1980, Cell 22:817) genes C can be employed in tk-, hgprt- or aprt- cells, respectively. Also, 0 0 antimetabolite resistance can be used as the basis of selection C 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., 1991, Proc.

Natl. Acad. Sci. USA 88:8972-8976). In this system, the sequence 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 Ni2' nitriloacetic acid-agarose columns and histidinetagged proteins are selectively eluted with imidazole-containing buffers.

0 5.3 ANTIBODIES TO NHP PRODUCTS CI Antibodies that specifically recognize one or more epitopes S of a NHP, or epitopes of conserved variants of a NHP, or peptide fragments of a NHP are also encompassed by the invention. Such 0 5 antibodies include but are not limited to polyclonal antibodies, monoclonal antibodies (mAbs), humanized or chimeric antibodies, S single chain antibodies, Fab fragments, F(ab') 2 fragments, S fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the 0 above.

The antibodies of the invention may be used, for example, in the detection of NHP in a biological 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 0 engineered NHP-expressing cells prior to their introduction into the patient. Such antibodies may additionally belused 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 the NHP, an NHP peptide one corresponding the a functional domain of an NHP), truncated NHP polypeptides (NHP in which one or more domains have been deleted), functional equivalents of the NHP or mutated variant of the 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), S mineral salts such as aluminum hydroxide or aluminum 0 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 toxoid f .0 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. These 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 Bcell hybridoma technique (Kosbor et al., 1983, Immunology Today 0 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 splicing the genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate S biological activity can be used. A chimeric antibody is a Cy- 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.

C 5 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, V' 0 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 example, 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 0 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, e.g., 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/ligand can be used to generate anti-idiotypes that "mimic" the NHP and, therefore, bind, activate, or neutralize a NHP, NHP receptor, or NHP ligand. Such anti-idiotypic t3 antibodies or Fab fragments of such anti-idiotypes can be used in S therapeutic regimens involving a NHP mediated pathway.

SThe present invention is not to be limited in scope by the specific embodiments described herein, which are intended as CI 5 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 tn 0 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.

Claims (8)

1. An isolated nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:9.

2. An isolated nucleic acid molecule comprising a nucleotide sequence that: encodes the amino acid sequence shown in SEQ ID NO:10; and Cc hybridizes under stringent conditions to the nucleotide sequence of SEQ ID NO:9 or the complement thereof. (N 3. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:12.

4. The isolated nucleic acid molecule of claim 3, wherein said molecule comprises the nucleotide sequence of SEQ ID NO:ll. A recombinant expression vector comprising the isolated nucleic acid molecule of claim 2.

6. The expression vector of claim 5, wherein said vector comprises the nucleotide sequence of SEQ ID NO:9.

7. A recombinant expression vector comprising the isolated nucleic acid molecule of claim 3.

8. The expression vector of claim 7, wherein said vector comprises the nucleotide sequence of SEQ ID NO: 11.

9. A host cell comprising the expression vector of claim A host cell comprising the expression vector of claim 7. SEQUENCE LISTING <110> Donoho, Gregory Turner, C. Alexander Jr. Nehis, Michael Friedrich, Glenn Zainbrowicz, Brian Sands, Arthur T. <120> Novel Human Kinase Proteins and Polynucleotides Encoding the Same <130> LEX-0046-PCT <150> US 60/156,511 <151> 1999-09-28 <160> 13 <170> FastSEQ for Windows Version <210> <211> <212> <213> 1 561 DNA homo sapiens <400> 1 atggaaaagt tgcagaaaca gatcctgttg ccaaatcttg gaatactgtg ggagtgatca aactgtattc aagatttgtg tccttgattg ctcctggctt atgaaaaatt aaacctctgg ttaagaaaat tgaacctcat atcatacact aaagcgtatt acagagatat acttcgggtt gcttaatagt ggatccattg agctaagact acaagtagta agcactaaga cgaggtgttc tttaaatgag atggcaaaca aaaacctgaa tgcacaaatt tgaccttctg ggagaagggt gctgttaaaa gaaatacgta aggagaaaaa ctggaaagaa cttcaagctc aat~ttctaa ctgagttgga aattcttttt cttatggggt tgtattcaaa aatttgtgga atctgaagat tgttgaagca attaaaacat ggaaaatgca tttagttttt acccaaatgg agttgctgat ttaatttctg tcatatacat taactaagca aggaataatc cttcatcttt' ctctggtgcc ctgccaattc agagattttt 120 180 240 300 360 420 48 O 540 561 <210> 2 <211> 187 <212> PRT <213> hoino sapiens <400> 2 Met Glu Lys Tyr Glu Lys Leu 1 5 Val Val Phe Lys Cys Arg Asn Lys Lys Phe Val Glu Ser Glu Leu Arg Giu Ile Arg Met Leu 55 Asn Leu Ile Glu Val Phe Arg 70 Glu Tyr Cys Asp His Thr Leu Ala Lys Thr 10 Ser Gly Glu Gly Ser Tyr Gly Lys Asp Thr 25 Asp Gly Gin Val Val Ala Val Lys Ile Ala Asn Leu Val Pro Val Val Lys Pro Lys Gln Leu Lys His Met Arg Lys Arg Leu Asn Glu Lys 75 Leu His Leu Val Phe Asn Glu Arg Asn Pro 1/ 9 Gly Val Ala Asp Gly Val Ile Lys 100 Ala Leu Asn Phe Cys His Ile His 115 120 Pro Giu Asn Ile Leu Ilie Thr Lys 130 135 Phe Giy Phe Ala Gin Ile Leu Ser 145 150 Ser Leu Ile Gly Leu Ilie Val Asp 165 Ser Giu Ile Phe Leu Leu Ala Trp 180 90 Val Ser 105 Asn Leu Trp Gin Thr Leu Gin 110 Asp Ile Lys Ile Cys Asp Cys Ile His Gin Gly Ile Ile 140 Ser Trp Thr Ser 155 Phe Ser Giy Leu Ile 185 Leu Asn 170 His Cys Ser Phe Ser Ala 175 <210> 3 <211> 1068 <212> DNA <213> homo sapiens <400> 3 atggaaaagt tgcagaaaca gatcctgttg ccaaatcttg gaatac tgtg ggagtgatca aactgtattc aagat ttgtg gtagctacga tcagtcgata tggcc tggaa gggtttcgc gtagcctc tc aaaagtaacg gaggaaaagt atgaatccag tttcaagagg caggtcagag atgaaaaat t aaacctctgg ttaagaaaat tgaacctcat atcatacac t aaagcgtatt acagagatat acttcgggtt gatggtaccg tatgggctat aatcagatgt atgttgacca aaagtgctgg ggtttttCca tctcagatgt atgacagatt cccaaattaa gctgtgtttg agctaagact acaagtagta agcac taaga cgaggtgttc tttaaatgag atggcaaaca aaaacctgaa tgcacaaatt agctcctgaa tggttgtgtt ggaccaactt ggctggtCtc aattacagga tggcatcagt tcatcctgtg aacctg'ttcc aagaaaagca gctgctgcag ggagaagggt gctgttaaaa gaaatacgta aggagaaaaa ctggaaagaa cttcaagctc aatattctaa ctgattccag cttcttgtgg tttgcagagc tatc tgataa gaactcttga aaattaatcc atacctgagc gctctgaact caactcctgg cgtaatgaag ctctgctcca cttatggggt aatttgtgga tgttgaagca ggaaaatgca acccaaatgg ttaatttctg taactaagca gagatgccta. gagatac tca tcctgacagg tcagaacact cgtcaagtga caagacatca. cagaagacat tcatgaaggg agagc tccta gaagaaacag ggctgcat tgtattcaaa atctgaagat attaaaacat tttagttttt agttgc tgat tcatatacat aggaataatc caccgattat gtatggttct ccagccactg agtagagacg tccacctgcc atcaatcttt ggaaactctt gtgtctgaag ctttgattct aagacgccaa 120 180 240 300 360 420 480 540 600 660 720 840 900 960 1020 1068 <210> 4 <211> 356 <212> PRT <213> homo sapiens <400> 4 Met Glu Lys Tyr Giu Lys Leu Ala Lys Thr Giy Giu Giy Ser Tyr Gly Vai Val Phe Lys Cys Arg Asn Lys Thr Gly Gin Vai Val Ala Val Lys Ile Ala Lys Lys Phe Val Glu Ser Giu Asp Asp Pro Val Vai Lys Leu Arg Glu Ile Arg Asn Leu Ile Giu Vai Giu Tyr Cys Asp His Met Leu Lys Gin Leu Lys His Met Pro Asn Leu Val Phe Arg Arg Lys Arg Lys His Leu Val Thr Leu Leu Asn Giu Leu Giu Arg Asn Pro 2 /9 Gly Val Ala Asp Gly Vai Ile 100 Ala Leu Asn Phe 115 Pro Giu Asn Ile Lys Ser 105 His Asn Val Leu Trp Gin Cys His Ile Leu Ile Thr Cys Ile His Thr Leu Gin 110 Asp Ile Lys Ile Cys Asp Arg 125 Lys Gin Gly Ile 130 Phe Giy 135 Leu Ile 140 Ala Phe Ala Gin 145 Val Ile 150 Tyr Ile Pro Giy Tyr Thr Asp Tyr 160 Ala Thr Arg Trp 165 Ser Arg Ala Pro Giu 170 Ala Leu Val Giy Asp Thr 175 Gin Tyr Gly Giu Leu Leu 195 Gin Leu Tyr Val Asp Ile Trp 185 Trp, Ile Gly Cys Giy Gin Pro Leu 200 Thr Pro Gly Lys Ser 205 Gly Val Phe Ala 190 Asp Val Asp Phe Arg His Leu Ile Ile Leu Val Glu 210 Val Asp Thr 220 Ser Gin Ala Gly 225 Val Leu 230 Ala Leu Leu Thr Asp Pro Pro Ala 240 Ala Ser Gin Gly Ile Thr Gly 250 Pile Leu Ile Pro Arg His 255 Gin Ser Ile Glu Pro Giu 275 Pro Val Ala Phe 260 Asp Ser Asn Giy Phe 265 Glu His Gly Ile Met Glu Thr Leu 280 Lys Glu Lys Phe Ser 285 Met Ser Ile Pro 270 Asp Val His Asn Pro Asp Leu Asn Phe 290 Asp Arg Met 295 Gin Gly Cys Leu Lys 300 Ser Leu Thr Cys Leu Leu Glu Tyr Phe Asp 305 Phe Gin Giu Ala Gin 325 Lys Arg Lys Al a 330 Val Asn Giu Gly Arg Asn 335 Leu Cys Arg Arg Arg Gin Val Arg Gly Cys 345 Trp Leu Leu Gin 350 Ser Arg Leu His 3.55 <210> <211> 972 <212> DNA <213> homo sapiens <400> atggaaaagt tgcagaaaca gatcctgttg ccaaatcttg gaatactgtg ggagtgatca aactgtattc aagatttgtg gtagctacga tcagtcgata tggcctggaa a tc ccaagac gagccagaag aacttcatga atgaaaaatt aaacctctgg ttaagaaaat tgaacctcat atcatacact aaagcgtatt acagagatat acttcgggtt gatggtaccg tatgggctat aatcagatgt atcaatcaat acatggaaac aggggtgtct agctaagac t acaagtagta agcactaaga cgaggtgttc tttaaatgag atggcaaaca aaaacc tgaa tgcacaaatt agc tcc tgaa tggttgtgtt ggaccaactt ctttaaaagt tct tgaggaa gaagatgaat ggagaagggt gctgttaaaa gaaatacgta aggagaaaaa ctggaaagaa cttcaagctc aatattctaa ctgattccag cttcttgtgg tttgcagagc tatctgataa aacgggtttt aagttctcag ccagatgaca cttatggggt aatttgtgga tgttgaagca ggaaaatgca acccaaatgg ttaatttctg taac taagca gagatgcc ta gagatactca tcctgacagg tcagaacact tccatggcat atgttcatcc gattaacctg tgtattcaaa atctgaagat attaaaacat tttagttttt agttgctgat tcatatacat aggaataatc caccgattat gtatggttct ccagccactg aggaaaatta cagtatacct tgtggctctg ttcccaactc

120. 180 240 300 360 420 480 540 600 660 720 780 840 3,/9 ctggagagct cctactttga ttcttttcaa gaggcccaaa ttaaaagaaa agcacgtaat gaaggaagaa acagaagacg ccaacaggtc agaggctgtg tttggctgct gcagctctgc tccaggctgc at <210> 6 <211> 324 <212>.PRT <213> homo sapiens <400> 6 900 960 972 Met Val Lys Leu Asn Glu Gly Al a Pro Phe 145 Val Gln Glu Gin Gin 225 Giu Pro Asp Phe Arg 305 Ser Giu Val Lys Arg Leu Tyr Val Leu Giu 130 Gly Al a Tyr Leu Leu 210 Ser Pro Val Arg Gln 290 Arg Arg Lys Tyr Phe Lys Phe Vai Glu Ile Ile Glu Cys Asp Ala Asp 100 Asn Phe 115 Asn Ile Phe Ala Thr Arg Gly Ser 180 Leu Thr 195 Tlyr Leu Ile Phe Giu Asp Ala Leu 260 Leu Thr 275 Glu Ala Arg Gin Leu His Giu Cys Giu Arg Val His Gly Cys Leu Gin Trp 165 Ser Gly Ile Lys Met 245 Asn Cys Gin Lys Leu Ala Lys Thr Gly Glu Gly Ser Tyr Gly 10 Arg Set Met Phe 70 Thr Val His Il e Ile 150 Tyr Val Gin Ile Ser 230 Glu Phd Ser Ile Asn Glu Leu 55 Arg Leu Ile Ile Thr 135 Leu Arg Asp Pro Arg 215 Asn Thr Met Gin Lys 295 Lys Asp 40 Lys Arg Le'j Lys His 120 Lys Ile Ala Ile Leu 200 Thr Gly Leu Lys Leu 280 Arg Thr 25 Asp Gin Lys Asn Ser 105 Asn Gin Pro Pro Trp 185 Trp Leu Phe Glu Gly 265 Leu Lys Ser Pro Leu At g Giu 90 Val Cys Gly Gly Glu 170 Ala Pro Gly Phe Giu 250 Cys Giu Ala Gly Val Lys Lys 75 Leu Leu Ile Ile Asp 155 Leu Ile Gly Lys His 235 Lys Leu Ser Arg Gin Val His Met Glu Trp His Ile 140 Ala Leu Giy Lys Leu 220 Giy Phe Lys Ser Asn 300 Val Lys Pro His Arg Gin Arg 125 Lys Tyr Val Cys Ser 205 Ile Ile Ser Met Tyr 285 Giu Val Lys Asn Leu Asn Thr 110 Asp Ile Thr Giy Val 190 Asp Pro Ser Asp Asn 270 Phe Gly Al a Ile Leu Val. Pro Leu Ile Cys Asp Asp 175 Phe Val Arg Ile Val 255 Pro Asp Arg Val Ala Val Phe Asn Gin Lys Asp 160 Thr Ala Asp His Pro 240 His Asp Ser Asn Gin Val Arg Gly Cys Val Trp Leu Leu Gin Leu Cys 310 315 320 <210>. 7 <211> 594 <212> DNA 4 /9 <213> homo sapiens <400> 7 atggaaaagt tgcagaaaca gatcctgttg ccaaatcttg gaatactgtg ggagtgatca aactgtattc aagatttgtg atgaaaaatt aaacctctgg ttaagaaaat tgaacctcat atcatacact aaagcgtatt acagagatat acttcgggtt agc taagac t acaagtagta agcactaaga cgaggtgttc tttaaatgag atggcaaaca aaaacctgaa tgcacaaatt tgaccttctg ctggaaaatt ggagaagggt gctgttaaaa gaaatacgta aggagaaaaa ctggaaagaa cttcaagctc aatattctaa c tgagttgga aattcttttt aatcccaaga cttatggggt aatttgtgga tgttgaagca ggaaaatgca acccaaatgg ttaatttctg taactaagca cttcatcttt ctgccaattc catcaatcaa tgtattcaaa atctgaagat attaaaacat tttagttttt agttgctgat tcatatacat aggaataatc ctctggtgcc agagattttt tctt tccttgattg gcttaatagt ctcctggctt ggatccattg <210> 8 <211> 198 <212> PRT <213> homo sapiens <400> 8 Met Glu 1 Lys Tyr Glu 5 Lys Leu Ala Lys Thr 10 Ser Gly Glu Gly Ser Tyr Gly Val Val Phe Lys Lys Phe Leu Arcr Glu Lys Val Cys Arg Asn Lys Gly Gin Val Glu Ser *Giu Asp Lys Pro Val Val Val Ala Val Lys Ile Ala Asn Leu Val Ile Arg Met Asn Leu Leu 55 Arg Gin Leu Lys His Met Ile Giu Val Giu Phe 70 Thr Arg Lys Arg Lys 75 Leu His Leu Val Phe Tyr Cys Asp Leu Leu Asn Giu Val Giu Arg Asn Pro Asn Gly Val Ala Ala Leu Asn Asp 100 Phe Val Ile Lys Leu Trp Gin. Cys His Ile His 120 Lys Cys Ile His Thr Leu Gin 110 A sp Ile Lys Ile Cys Asp Pro Giu 130 Ile Leu Ile Thr 135 Leu Gin Giy Ile Ile 140 Ser Phe Gly Phe 145 Ser Leu Ile Ser Giu Ile Lys Thr Ser 195 Ala Gin Giy Leu 165 Phe Leu Ile 150 Ile Ser Trp Thr Ser 155 Asn Phe Ser Gly Al a 160 Vai Asp Leu Leu 170 His Ser Phe Ser Ala Asn 175 Asn Pro Leu Ala Trp 180 Ile Asn Leu Ile 185 Cys Trp Lys Ile 190 <210> 9 <211> 1041 <212> DNA <213> homo sapiens <400> 9 atggaaaagt atgaaaaatt agctaagact ggagaagggt cttatggggt tgtattcaaa .tgcagaaaca aaacctctgg acaagtagta gctgttaaaa aatttgtgga atctgaagat gatcctgttg ttaagaaaat agcactaaga gaaatacgta tgttgaagca attaaaacat 120 180 9 ccaaatcttg tgaacctcat cgaggtgttc gaatactgtg atcatacact tttaaatgag ggagtgatca aaagcgtatt atggcaaaca aactgtattc acagagatat aaaacctgaa aagatttgtg acttcgggtt tgcacaaatt gtagctacga gatggtaccg agctcctgaa tcagtcgata tatgggctat tggttgtgtt tggcctggaa aatcagatgt ggaccaactt gggtttcgcc atgttgacca ggctggtctc gtagcctctc aaagtgctgg aattacagga aaaagtaacg ggtttttcca tggcatcagt gaggaaaagt tctcagatgt tcatcctgtg atgaatccag atgacagatt aacctgttcc tttcaagagg cccaaattaa aagaaaagca caggtacttc cgctcaaaag t aggagaaaaa ctggaaagaa cttcaagctc aatattctaa ctgattccag cttcttgtgg tttgcagagc tatctgataa gaactcttga aaattaatcc atacctgagc gctctgaact caactcctgg cgtaatgaag ggaaaatgca acccaaatgg ttaatttctg taactaagca gagatgccta gagatactca tcctgacagg tcagaacact cgtcaagtga caagacatca cagaagacat tcatgaaggg agagctccta gaagaaacag tttagttttt agttgctgat tcatatacat aggaataatc caccgattat gtatggttct ccagccac tg agtagagacg tccacctgcc atcaatcttt ggaaactctt gtgtctgaag ctttgattct aagacgccaa 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1041 <210> <211> 347 <212> PRT <213> homo sapiens <400> Met Glu Lys Tyr 1 Val Val Phe Lys Glu Lys Leu Ala Lys Thr Gly Giu Gly 10 Ser Cys Arg Asn Lys Gly Gin Val Ser Tyr Gly Val Ala Val Lys Ile Ala Asn Leu Val Lys Lys Phe Leu Arg Giu Asn Leu Ile Glu Tyr Cys Gly Val Ala Ala Leu Asn 115 Pro Giu Asn Val Glu Ser Giu Asp Lys Pro Val Val Ile Arg Met Leu Arg Gin Leu Lys Giu Val Phe 70 Thr Arg Lys Arg Lys 75 Leu His Leu Val Phe Asp Asp 100 Phe His Gly Leu Leu Asn Glu 90 Val Giu Arg Asn Pro Asn Val Ile Lys S er 105 Asn Leu Trp Gin Cys His Ile His 120 Lys Cys Ile His Arg 125 Lys Thr Leu Gin Asp Ile Lys Ile Cys Asp Ile Leu Ile 130 Phe Gly Thr 135 Leu Gin Gly Ile Phe Ala Gin 145 Val Ile 150 Tyr Ile Pro Giy Asp 155 Leu Tyr Thr Asp Tyr 160 Ala Thr Arg Trp 165 Ser Arg Ala Pro Giu 170 Ala Leu Val Giy Asp Thr 175 Gin Tyr Gly Giiu Leu Leu 195 Gin Leu Tyr 210 Ser 180 Thr Val Asp Ile Trp 185 Trp Ile Gly Cys Gly Gin Pro Leu 200 Thr Pro Gly Lys Ser 205 Gly Val Phe Ala 190 Asp Val Asp Phe Arg His Leu Ile Ile Arg 215 Leu Val Giu Thr 220 Ser Val Asp 225 Val Ala Gin Ser Gin Ala Gly Leu Giu Leu 230 Ser Gin Ser Ala Gly Ile 245 Ile Phe Lys Ser Asn Gly Leu Thr Thr Gly 250 Phe Phe 265 Ser 235 Asp Pro Pro Al a 240 Lys Leu Ile His Gly Ile Pro Arg His 255 Ser Ile Pro 270 260 6/ 9 Glu Pro Giu Asp Met Giu Thr 275 Leu 280 Lys Giu Glu Lys Phe Ser Asp Val His Pro Val Ala Leu 290 Asp Arg Leu Thr 305 Phe Gin Giu Ala Arg Arg Arg Gin 340 AsnPhe Met Gly Cys Leu Lys 300 Ser Asn Pro Asp Cys Gin 325 Gin Ser 310 Ile Gin Leu Leu Glu Lys Arg Lys Ala Ser 315 Arg Ser Tyr Phe Asp Ser 320 Asn Asn Giu Gly 330 Lys Arg 335 Val Leu Pro Leu 345 <210> 11 <211> 945 <212> DNA <213> homo sapiens <400> 11 atggaaaagt tgcagaaaca gatcctgttg ccaaatcttg gaatac tgtg ggagtgatca aactgtattc aagatttgtg gtagctacga tcagtcgata tggcc tggaa atcccaagac gagccagaag aacttcatga c tggagagc t gaaggaagaa atgaaaaatt aaac ct Ctgg ttaagaaaat tgaacctcat atcatacact aaagcgtatt acagagatat acttcg'ggtt gatggtaccg tatgggctat aatcagatgt atcaatcaat acatggaaac aggggtgtct cctactttga acagaagacg agctaagact acaa gtagta agcac taaga cgaggtgttc tttaaatgag atggcaaaca aaaacctgaa tgcacaaat t agctcctgaa tggttgtgtt ggaccaactt ctttaaaagt tcttgaggaa gaagatgaat ttcttttcaa ccaacaggta ggagaagggt gctgttaaaa gaaatacgta aggagaaaaa c tggaaagaa cttcaagctc aatattctaa ctgattccag Cttcttgtgg tttgcagagc tatctgataa aacgggtttt aagttctcag ccagatgaca gaggcccaaa Cttccgctca cttatggggt aat ttgtgga tgttgaagca ggaaaatgca acccaaatgg ttaatttctg taactaagca gagatgccta gagatactca tcc tgacagg tcagaacact tccatggcat atgttcatcc gattaacctg ttaaaagaaa aaagt tgtattcaaa atctgaagat attaaaacat tttagttttt agttgctgat tcatatacat aggaataat c caccgattat gtatggttct ccagccactg aggaaaatta cagtatacct tgtggc tctg ttcccaactc agcacgtaat 120 180 240 300 360 420 480 540 600 660 720 780 840 900 945 <210> 112 <211> 315 <212> PRT <213> homo sapiens <400> 12 Met Giu Lys Tyr Giu Lys Leu Ala Lys Thr Ser Gly Giu Gly Ser Tyr Gly Val Val Phe Lys Cys Lys Lys Phe Val Glu Leu Arg Giu Arg Asn Lys Ser Glu Asp 40 Met Leu Lys 55 Thr 25 Asp Pro Val Val Lys Pro Gly Gin Val Val Ala Val Lys Ile Ala Asn Leu Val Ile Arg Asn. Leu Ile Giu Val Phe 70 Giu Tyr Cys Asp His Thr Gly Val Ala Asp Gly Val 100 Ala Leu Asn Phe Cys His i115 Arg Arg Gin Leu Lys Lys Arg Lys 75 His Met His Leu Val Phe Leu Leu Asn Giu 90 Ile Lys Ser Val 105 Leu Leu Giu Arg Asn Pro Asn Trp Gin Thr Leu Gin Ile His Asn 120 110 Asp Ile Lys Cys Ile His Arg 125 7 /9 Pro Giu Asn 130 Phe Gly Phe 145 Val Ala Thr Gin TyrGly Giu Leu Leu 195 Gin Leu Tyr Ile Leu Ile Thr Lys Gin Gly Ile Ile Lys Ile Cys Asp 135 Ala Gin Ile Leu Ile 140 Pro Gly Asp Ala Tyr Arg Trp 165 Ser Ser 180 Thr Giy 150 Tyr Arg Ala Pro 155 Giu Leu 170 Ala Ile Thr Asp Tyr Leu Val Gly Asp Thr 175 Val Asp Ile Trp 185 Trp, Gly Cys Gin Pro Leu 200 Thr Pro Gly Lys Ser 205 Ile Vai Phe Ala 190 Asp Val Asp Pro Arg His Leu Ile Ile 210 Gin Ser Arg 215 Asn Leu Gly Lys. Leu 220 Gly Ile Phe Lys Gly Phe Phe 225 Giu His 235 Lys Ile Ser Ile Pro 240 Pro Giu Asp Met 245 Asn Thr Leu Giu Glu 250 Cys Phe Ser Asp Vai His 255 Pro Val Ala Asp Arg Leu 275 Phe Gin Giu 290 Leu 260 Thr Phe Met Lys Gly 265 Leu Leu Lys met Cys Ser Gin Giu Ser Ser Tyr 285 Giu Asn Pro Asp 270 Phe Asp.Ser Gly Arg Asn Ala Gin Ile Lys 295 Lys Ala Arg Asn 300 Arg 305 Arg Arg Gin Gin Val 310 Leu Pro Leu Lys Ser 315 <210> 13 <211> 1819 <212> DNA <213> homo sapiens <400> 13 Ctccgagcga caccgtcgcc aagtcacttc atggggttgt ttgtggaatc tgaagcaatt aaatgcattt caaatggagt atttctgtca ctaagcaagg catctttctc ccaattcaga gcccaggctc gctggattgc ag-ctcacaag caggagatgc tgggagatac agc tcctgac taatcagaac tgacgtcaag tcccaagaca agccagaaga act tcatgaa tggagagctc aaggaagaaa cacgcgcggg ctgtgggtgc agctataatg attcaaatgc tgaagatgat aaaacatcca agtt t ttgaa tgctgatgga tatacataac aataatcaag tg'gtgcCtcc gatttttctc atctcgaact aagtgtgagc attgttttta ctacaccgat tcagtatggt aggccagcca actagtagag tgatccacc t tcaatcaatc catggaaact ggggtgtctg ctactttgat *agctggggct agtgcagcat gaaaagtatg agaaacaaaa cctgttgtta aatcttgtga tac tgtgatc gtgatcaaaa tgtattcaca atttgtgact ttgattggct ctggcttgga tctggcctca caccgtgccc gtgggaacac tatgtagcta tcttcagtcg ctgtggcctg acggggtttc gccgtagcct tttaaaagta cttgaggaaa aagatgaatc tcttttcaag ggggctgttc tgtactgcaa aaaaattagc cctctggaca agaaaatagc acctcatcga atacactttt gcgtattatg gagatataaa tcgggtttgc taatagttga tccattgctg agtg'atcctt agccaga ttt aatttcgaac cgagatggta atatatgggc gaaaatcaga gccatgttga o tcaaagtgc acgggttttt agttctcaga cagatgacag aggcccaaat gaggctgtgt ggcgctgc to gtcaatcgat taagactgga agtagtagct actaagagaa ggtgttcagg aaatgagctg gcaaacactt acctgaaaat acaaattctg ccttctgaat acacagtgtt. ccacctcggc ttcaaacaat aaattcttga ccgagctcct tat tggt tgt tgtggaccaa ccaggctggt tggaattaca ccatggcatc tgttcatcct attaacctgt taaaagaaaa ttggctgctg gaagcttcgt acaataattt gaagggtctt gttaaaaaat atacgtatgt agaaaaagga gaaagaaacc caagctctta attctaataa agttggactt tctttttctg tcaccatggg ctcccaaagt aactactgag gaacgcattc gaac ttot tg gtttttgCag ctttatctga ctcgaactct ggaaaat taa agtatacctg gtggctctga tcccaactcc gcacgtaat~g cagctctgct 120 180 240 300 360 420 480 540 600 660 720 780 -840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 cagaagacgc caacaggtca 8 /9 ccaggctgca agctcatatg tgtcaggagt ttgrttggct tgtagtgatt aatatcaaaa ttgagaatcg atttcgagtg tcttctcatt cagggaccca gccaaggagc ggaaatatgc ccttctcatg gcagaggccc gaacttgaat acctaatgtc gacttccgct caaaagttaa agtgctataa aaataattcc ttttttgttt gcctttcaaa gtgagacaag gtggacacca agacctttca tttgtactgg gctagcttaa taaatattgg gaattgatgt ataaaaccct agactatgaa aaaaaaaaa 1560 1620 1680 1740 1800 1819 9,/9