AU783374B2 - Novel human kinase protein and polynucleotides encoding the same - Google Patents

Description

WO 01/34783 PCTUSOOI3038 NOVEL HUMAN KINASE PROTEIN AND POLYNUCLEOTIDES ENCODING THE SAME 1. INTRODUCTION The present application claims the benefit of U.S.

Provisional Application Number 60/164,289 which was filed on November 8, 1999 and is herein incorporated by reference in its entirety.

The present invention relates to the discovery, identification, and characterization of novel human polynucleotides encoding a protein that shares 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, 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 range of regulatory pathways. Given the physiological importance of kinases, they have been subject to intense scrutiny and are proven drug targets.

The discussion of documents, acts, materials, devices, articles and the like is included in this specification solely for the purpose of providing a context for the present invention.

It is not suggested or represented that any or all of these matters formed part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed in Australia before the priority date of each claim of :this application.

go• 3. SUMMARY OF THE INVENTION The present invention relates to the discovery, identification and characterisation of nucleotides that encode a novel human protein, and the corresponding amino acid sequences of this protein. The novel human protein (NHP) described for the first time herein shares structural similarity with animal kinases, including, but not limited to serine/threonine protein kinases. As such, the novel polynucleotides encode a new kinase protein having homologues and orthologs across a range or phyla and species.

The novel human polynucleotides described herein, encode an open reading frame (ORF) encoding a protein of 893 amino acids in length (see SEQ ID NO: 2).

Thus, the present invention provides an isolated nucleic acid molecule comprising at least 24 contiguous bases of nucleotide sequence first disclosed in the NHP gene described in SEQ ID NO: 1.

The present invention also provides an isolated nucleic acid molecule comprising a nucleotide sequence that encodes the amino acid sequence shown in SEQ ID NO:2, and hybridises under stringent conditions to the nucleotide sequence of SEQ ID NO:1 or the complement thereof. The present invention further provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:2.

o 25 The invention also encompasses agonists and antagonists of the described NHP, 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 describe NHP antisense and ribozyme molecules, and gene or regulatory sequence replacement constructs) or to enhance the expression of the described NHP polynucleotides expression constructs that place the described gene under the control of a strong promoter system). The present invention also includes both transgenic .gi, 35 animals that express a NHP transgene, and NHP "knock-outs" (which can be conditional) that do not express a functional NHP.

can be conditional) that do not express a functional NHP.

oo* w:F 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 utilise purified preparations of the described NHPs and/or NHP product, or cells expressing the same. Such compound can be used as therapeutic agents for the treatment of any of a wide variety of symptoms associated with biological disorders or imbalances.

Throughout the description and the claims of this specification the word "comprise" and variations of the word, such as "comprising" and "comprises" is not intended to exclude other additives, components, integers or steps.

4. DESCRIPTION OF THE SEQUENCE LISTING AND FIGURES The Sequence Listing provides the sequence of a novel human ORF that encodes the described novel human kinase-like protein.

DETAILED DESCRIPTION OF THE INVENTION The NHP, described for the first time herein, is a novel protein that is expressed in, inter alia, human cell lines, and human brain, pituitary, cerebellum, spinal cord, thymus, lymph 25 node, bone marrow, trachea, kidney, liver, prostate, *.aa *e oo* oooo oo o WAF Besl68519M8519_Stped .oc WO 01/34783 PCT/US00/30380 testis, thyroid, adrenal gland, pancreas, stomach, small intestine, colon, skeletal muscle, uterus, placenta, mammary gland, adipose, esophagus, bladder, cervix, rectum, pericardium, hypothalamus, ovary, fetal kidney, and fetal lung cells. The described sequences were compiled from gene trapped cDNAs, ESTs, a and human brain cDNA library, (Edge Biosystems, Gaithersburg, MD).

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 genes, including the specifically described NHP, and the NHP products; nucleotides that encode one or more portions of the 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); isolated nucleotides that encode mutant versions, engineered or naturally occurring, of the described NHP 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 a NHP, or one of its domains a receptor/ligand binding domain, accessory protein/selfassociation 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 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) that hybridizes to a complement of a DNA sequence presented in the Sequence Listing WO 01/34783 PCT/US00/30380 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 NHP ORFs, or their functional equivalents, encoded by polynucleotide sequences that are about 99, 95, 90, or about 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).

The invention also includes nucleic acid molecules, preferably DNA molecules, that hybridize to, and are therefore the complements of, the described NHP encoding polynucleotides. Such hybridization conditions can 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 WO 01/34783 PCT/US00/30380 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 the NHP sequence may be represented using oligonucleotides that do not overlap. Accordingly, the described NHP polynucleotide sequence 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 refer, to washing in 6xSSC/0.05% sodium pyrophosphate at 37'C (for 14-base oligos), 48 0 C (for 17-base oligos), 55°C (for 20-base oligos), and 60 0 C (for 23-base oligos). These nucleic acid molecules may encode or act as NHP gene antisense molecules, useful, for example, in NHP 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 can be used as part of ribozyme and/or triple helix sequences that are also useful for NHP gene regulation.

WO 01/34783 WO 0134783PCT/USOO/30380 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, (carboxyhydroxylmethyl) uracil, 2-thiouridine, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2, 2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-rethoxyaminomethyl-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, uracil-5-oxyacetic acid methylester, uracilacid 5-methyl-2-thiouracil, 3-(3-amino-3-N-2carboxypropyl) uracil, (acp3)w, 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.

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 methyiphosphonate, an alkyl phosphotriester, and a formacetal or analog thereof.

In yet another embodiment, the antisense oligonucleotide is an c-anomeric oligonucleotide. An oa-anomeric oligonucleotide forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual O-units, the strands run parallel to each other (Gautier et al. 1987, Nucl. Acids Res. 15:6625-6641). The oligonucleotide is methylribonucleotide (Inoue et al., 1987, Nucl. Acids WO 01/34783 PCTIUS00/30380 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 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 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 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.

WO 01/34783 PCT/US00/30380 Further, a NHP gene 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, 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 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.

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 WO 01/34783 PCT/USOO/30380 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 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, immune disorders, obesity, high blood pressure, 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.

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 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: A 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.

WO 01/34783 PCTIUS00/30380 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, for example, polynucleotide shuffling or related 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 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 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, WO 01/34783 PCT/USOO/30380 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 intracellular domains, or truncated polypeptides lacking one or 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 (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 recovered from the culture media.

Also encompassed by the present invention are novel protein constructs engineered in such a way that they facilitate transport of the NHP to the target site, to the desired organ, across the cell membrane and/or to the nucleus where the NHP can exert its function activity. This goal can be achieved by coupling of the NHP to a cytokine or other ligand that would direct the NHP to the target organ and facilitate receptor mediated transport across the membrane into the cytosol. Conjugation of NHPs to antibody molecules or their Fab fragments could be used to target cells bearing a particular epitope. Attaching the appropriate signal sequence to the NHP would also transport the NHP to the desired location within the cell. Alternatively targeting of NHP or its nucleic acid sequence can be achieved using liposome or lipid complex based delivery systems. Such technologies are described in U.S. Patents Nos. 4,594,595, 5,459,127, 5,948,767 and 6,110,490 and their respective disclosures which are herein incorporated by reference in their entirety.

WO 01/34783 P'CT/US00/30380 The present invention also encompasses antibodies and anti-idiotypic antibodies (including Fab fragments), antagonists and agonists of the 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, etc.).

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 NHP proteins 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.

Finally, the NHP products can be used as therapeutics.

For example, soluble derivatives such as NHP peptides/domains corresponding the NHPs, 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 antiidiotypic 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 WO 01/34783 PCT/US00/30380 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 therapy" 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 sequence and the corresponding deduced amino acid sequence of the described NHP are presented in the Sequence Listing. The NHP nucleotide sequences were obtained from a human cDNA library 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 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 entirety.

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 WO 01134783 PCT/US00/30380 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 sequence encoded by the described NHP-encoding polynucleotides. The NHP has an initiator methionine in a DNA sequence context consistent with eucaryotic translation initiation site.

The NHP amino acid sequence 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 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 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 modify a NHP substrate, or the ability to effect an identical or complementary downstream pathway, or a change in WO 01/34783 PCTIUSOO/30380 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 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 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 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 express a 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 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 bacteria E. coli, B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing NHP nucleotide sequences; yeast WO 01/34783 PCT/US00/30380 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 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 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. 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.

WO 01/34783 PCT/US0O/30380 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 encoding polynucleotide 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 gene coding sequence will result in inactivation of the polyhedrin gene and production of nonoccluded 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 gene is expressed see Smith et 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 can 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 an entire NHP gene 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 WO 01/34783 PCT/US00/30380 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 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 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 NTHP sequences described above can be 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 WO 01/34783 PCT/US00/30380 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 lines may be particularly useful in screening and evaluation of compounds that affect the endogenous activity of the NHP product.

A number of selection systems can 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 phosphoribosyltransferase (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., 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 WO 01/34783 PCT/US0/30380 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, F(ab') 2 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 can 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 gene 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 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 WO 01/34783 PCTUS0/30380 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. 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 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 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 WO 01/34783 PCTIUSOO/30380 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 gene 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 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/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 antibodies or Fab fragments of such anti-idiotypes can be used in therapeutic regimens involving a NHP mediated 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 WO 01/34783 PCT/US00/30380 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 13602/01 The following Sequence Listing pages 1/3 to 3/3 are part of the description. The claims page follows on page 24.

WO 01/34783 WO 0134783PCT/USOO/30380 SEQUENCE LISTING <110> LEXICON GENETICS INCORPORATED <120> Novel Human Kinase Protein and Polynucleotides Encoding the Same <130> LEX-0083-PCT <150> US 60/164,289 <151> 1999-11-08 <160> 2 <170> FastSEQ for Windows Version <210> 1 <211> 2682 <212> DNA <213> bomo sapiens <400> 1 atgtttcccc catgatgttc cgctttcaaa aggggaaagc t tgc tt cgag ct tcagggc t aatatcctgt acagctcatg gtaattgcac tc tggcccca agaaaattat tgtgtagatg gagcttcctg aggc ca accc acccccttta ctgcctgagg tctattgaag gtcaacaagg gaggatggtg gtgacattgt tacccattac tctgcagctg agaattattc aaagaagcaa ggagttgagg acagatagac tcaccagaag gaticttgtgt aataatgaag ttcttcttaa attgcatttc ctctatgcca ttccacctct gcaattcttc ctcttctccg ttttgttgga tctgacagca ccaaagacag tgaaggacgc gtggaagcaa tccttaaaac atgaacgact aaaggaaacc tgcagtatat tggaccgaaa gtgatgatgt agggaatttt aa tcaga tg t ttcagagctt acactttaat aaactgtgat cagatgaatt ccaaacc tgc atatcagtca aagtgtatta aaatcattcg aaagctttgg cgttatgcca t tgaagatga ccacgctccc tcttcgacag gagttgacat gagctattca aaattgaagt gtcatgcaaa attggcaagg ccttggctta aagacaactc atgatccaga tcccttggtt gggatacctt agcagctgcg atttaccaga ctcctaaaag gtggaggcag atctgtcaag tgaaatggga tggacttcct catcacccat agtggtcgtg tgtgagctgt gaacaaacat gggacatatt tgatttccca caaaaccact atggtctctt ggatatttct agttctggct agatcttttg aatgaaggac cagtctgttt gttgtgtaaa cctttggtgt atccaaacca acaaggtcga gctaagaaat ccagtctaat tttaatcatc gctgctaaag tcctcctctt tgccaagtac ggatattcct atttaggcgt tcttgactca tgcatgtatg acatgtaata gctgagtaat tcttaccatg actacttggg ggaccggctt aattgacatt tgctacttac aagttcggca agaatccatc gcctttacct ctcacaccaa cccagactct gctgaacatt tcaacggttt ggtatagtac aaattggcta atagggtatc gatcacatgc ggaatcatt t gaaagac taa gaagagcatg aataagtgcc aaagtattca tcatcttctc gatataaata ttggctggag cctatctgca gatagaagct agattgaaag t tact cat t agagagaagg gcttatccat atgagaggtt gatgcaattg cgctgtcatc g tattaaaag ctttgtgctc tctgctttta caagagta tc catctcaatg tttactcatg aattcctctt ttggctaatg gaacgctgtg agacagcatg ccttatttct ccattaaatg tctttgcctc attccatcaa gccagtatgt gtgaacgtag tgtgtatagc acagggcatt aatttggact cctcgtactt caagtaaaaa tatttgagct aatttttgct gttgtttgga ttaccttcca gtgaggtatc tgagatgtgc atgattacct gtgacttgga cactccccaa cgcttttaga atgttggtgg caaacagcaa atacagagta ataaaaaaaa taacctgggc ataaagacac agtacgatga cctgggtagt cattcctata ttcccaaata tgactgtctt agattggttt tatttccact tcccattctg gctt taatga tgagagaa tc ctcaacctcc ctgctgagtg act tga agt c ggcttgcca aattttaggg gga tat ttc t tctggaagac atttgaggtt gtctcctcat t tatcacatg ggcccctgag accat tgcct ttgtgtggga tactttggat cat tataaag tccttctaag acctttatat tgatttaact ggcagaaaga gaaagagct t ttttttcttt tgataccact agaagcattt taatgagttg ccaactaaat ccaaatctgg tgctcttctg tccaaitcct actgt tatca gtctcatctt tctaaacttc cctgtataac ctctcagatg cattccagat acacaaaatt tattggagta gtgtattctt tat caa cc tg aaagccatct tccagatcct agaagtatca 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 1800 1860 1920 1980 2040 2100 2160 2220 2280 1/3 WO 01/34793 WO 0134783PCT[USOO/30380 ccacggattt cagcagagga cctgattgac ttgtgtgagc aaaacacca gcaagaaaac aaagtccagt aaaccaaagc aatagtgaag actttattcg tggtcacatt tcaggaagca gccttcactg cagaagggga gcttacccag ggcccttaca aaagggaagg tcattgtcat cgtggggcat gtggcaaaac caccttgtga agatgaaata tCCaagaatc tgtattctag aagccaacag gcctcctcac catcccatct cctcaaatat tcacagtgac aggccacttc tcctggtggt~ tgacatccgg tcaacattcc attcagtgct ctgctatgct ccagaacttc acacagctga gtttgcagct atggtggcat taataaaata ga 2340 2400 2460 2520 2580 2640 2682 <210> 2 <211> 893 <212> PRT <213> homo sapiens <400> 2 Met Phe Pro Leu by: 1 5 Ser Ala Leu Pro Hi, Pro Asn Ser Ile Ly.

Thr His Pro Arg Le, Glu Arg Leu Val Va Leu Leu Arg Giu Ar Ala Phe Glu Val Lel 100 Val His Arg Ala Le 115 His Ilie Lys Leu Al 130 Asp Asp Val Asp Ph 145 Val Ile Ala Gin Gi 16 Lys Pro Leu Pro Se 180 Ile Leu Phe Glu Le 195 Ile Ser Glu Arg Le 210 Thr Leu Ile Val be 225 Giu Leu Pro Glu Th 24 His Pro Ser Lys Ar 260 Phe Ser Glu Val Se 275 Leu Phe Ser Ser Se 290 Ile Ser Gin Leu CY 305 Ser lie Giu Giu Va 32 Giu Lys Glu Leu Va 340 Cys Thr Leu Pro As 355 Gly Arg Asp Arg Se 370 Leu Cys Gin Leu Ar Asp Asp Ile Cys Val 70 Lys Gin Ser Lys Pro 150 Ile Gly Cys Lys Ala 230 Val1 Pro Pro Leu bys 310 Tyr Asn Phe Ser Asn Glu Cys Gly 40 Tyr Giu Val1 Leu His 120 Gly Gly Lys Lys Gly 200 Leu Glu Asp Pro Tyr 280 Cys Ile Leu Giu Phe 360 Leu Leu Gly 10 Ser Phe Asp Cys Cys 90 Tyr Ile Tyr Pro Thr 170 Asp Lys Thr Gly Leu 250 Glu Pro Asp Asn Cys 330 Ile Asp Asp Asp Ala Phe Thr Asn Gly Leu Gin Ile Leu Ile Ser Arg Glu Arg Ser 75 Ser Thr Val Met Asn Lys beu Leu Asp 125 His Met Thr 140 Ser Tyr Leu 155 Asp His Met Val Trp Ser Leu Phe Gin 205 Leu Asp Cys 220 Cys Leu Asp 235 Asn Lys Cys Leu Met Lys Phe Thr Lys 285 Leu Thr Leu 300 Asp Tyr Leu 315 Leu Ala Gly Arg Ser Lys Gly Glu Ser 365 Thr Thr Val 380 Val Gly Gly Phe Pro Lys Gly beu Leu His 110 Arg Ala Ala Pro Leu 190 Ser Val Ile Leu Asp 270 Pro Pro Ala Gly Pro 350 Phe Thr Phe Ala Leu Thr Thr Ile Lys His Giu Asp Cys Ile Gly Ile Lys Gly His Gly Pro Glu 160 Ser Lys 175 Gly Ile beu Asp Asp Asp Ile bys 240 'r Phe 255 Lys Val Ala Ser Glu Asp Giu Arg 320 Asp beu 335 Pro Ile Gly Gin Leu Ser Glu Ala Phe 2/3 WO 01/34783 WO 0134783PCTIJSOO/30380 Giu Asp Asp Gin Ser Asn Leu Pro His Ser Asn Ser 3/3

Claims (3)

1. An isolated nucleic acid molecule comprising a nucleotide sequence first disclosed in the NHP gene described in SEQ ID NO: 1.

2. An isolated nucleic acid molecule comprising a nucleotide sequence that: encodes the amino acid sequence shown in SEQ ID NO:2, and hybridises under stringent conditions to the nucleotide sequence of SEQ ID NO:1 or the complement thereof.

3. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:2. Dated: 23 August 2005 PHILLIPS ORMONDE FITZPATRICK 25 Attorneys for: LEXICON GENETICS INCORPORATED T:FBes\8651 9s66519_Spe.doc