AU781078B2 - Phosphatidylinositol 3-kinase P110 delta catalytic subunit - Google Patents

Description

P/00/011 28/5/91 Regulation 3.2

AUSTRALIA

Patents Act 1990

ORIGINAL

COMPLETE SPECIFICATION STANDARD PATENT Name of Applicant: Actual Inventor Icos Corporation David H CHANTRY Douglas A HOLTZMAN Merl F HOEKSTRA WRAY ASSOCIATES 239 Adelaide Terrace Perth, WA 6000 Address for service is: Attorney code: WR Invention Title: "Phosphatidylinositol 3-kinase P110 Delta Catalytic Subunit" The following statement is a full description of this invention, including the best method of performing it known to me:- 1/2- PHOSPHATIDYLINOSITOL 3-KINASE P110 DELTA CATALYTIC SUBUNIT This application is a continuation-in-part application of U.S.

Application Serial No. 08/777,405 filed November 25, 1996.

FIELD OF THE INVENTION The present invention relates generally to the identification and isolation of a novel lipid kinase and more particularly to the discovery of a novel catalytic subunit related to phosphatidylinositol 3-kinase, herein designated p1106.

BACKGROUND OF THE INVENTION 10 Phosphatidylinositol 3-kinase (PI 3-kinase) was originally identified as an activity associated with viral oncoproteins and growth factor receptor tyrosine kinases which phosphorylates phosphatidylinositol (PI) and phosphorylated derivatives of PI at the 3'-hydroxyl of the inositol ring [Panayotou et al., Trends in Cell Biol., 2:358-360 (1992)]. The initial purification and molecular cloning of PI 3-kinase revealed that it was a heterodimer consisting of p85 and p110 subunits [Otsu et al., Cell, 65:91-104 (1992); Hiles er al., Cell, 70:419-429 (1992)].

The p85 subunit acts to localize PI 3-kinase to the plasma membrane by the interaction of its SH2 domain with phosphorylated tyrosine residues (present in an appropriate sequence context) in target proteins [Rameh et al. Cell, 83:821-830 (1995)]. Two isoforms of p85 have been identified, which is ubiquitously expressed, and p853, which is primarily found in brain and lymphoid tissues [Volinia et al., Oncogene, 7:789-793 (1992)].

The p11 0 subunit contains the catalytic domain of PI 3-kinase and three isoforms P and y) of p110 have thus far been identified. p11 0 a and P associate with p8 5 whereas p110y which is activated by G protein py subunits, does not [Stoyanov et al., Science, 269:690-693 (1995)]. The cloning of p11lOy revealed additional complexity within this family of enzymes. p1lOy is closely related to pllOa and P (45-48 identity in the catalytic domain), but does not make use of p85 as a targeting subunit, instead p11Oy contains an additional domain termed a pleckstrin homology domain near its amino terminus. This domain allows interaction with the py subunits of heterotrimeric G proteins and it appears that it is this interaction that regulates its activity [Stoyanov et al., 1995]. Thus PI 3-kinases are defined by their amino acid identity or their activity. Additional members of this growing gene family include more distantly related lipid and protein kinases including Vps34, 10 TORI and TOR2 of Saccharomyces cerevisiae (and their mammalian homologous such as FRAP and mTOR), the ataxia telangiectasia gene product, and the catalytic subunit of DNA dependent protein kinase. [See, generally, *"the review of Hunter, Cell, 83:1-4 (1995).] The levels of phosphatidylinositol 4, 5) triphosphate (PIP 3 15 the primary product of PI 3-kinase activation, increase upon treatment of cells with a wide variety of agonists. PI 3-kinase activation is therefore believed to be involved in a range of cellular responses including cell growth, differentiation and apoptosis [Parker et al., Current Biology, 5:577-579 (1995); Yao et al., Science, 267:2003-2005 (1995)]. The downstream targets of the 20 phosphorylated lipids generated following PI 3-kinase activation have not been well characterized. In vitro, some isoforms of protein kinase C (PKC) are directly activated by PIP 3 and the PKC related protein kinase PKB has been shown to be activated by PI 3-kinase through an as-yet-undetermined mechanism [Burgering and Coffer, Nature, 376:599-602 (1995)].

PI 3-kinase also appears to be involved in a number of aspects of leukocyte activation. A p85 associated PI 3-kinase activity has been shown to physically associate with the cytoplasmic domain of CD28, an important costimulatory molecule for the activation of T cells in response to antigen [Pages et al., Nature, 369:327-329 (1994); Rudd, Immunity, 4:527-534 (1996)].

Activation of T cells through CD28 lowers the threshold for activation by antigen and increases the magnitude and duration of the proliferative response.

These effects are linked to increases in the transcription of a number of genes including the T cell growth factor interleukin 2 (IL-2) [Fraser et al., Science, 251:313-316 (1992)]. Mutation of CD28 such that it can no longer interact with PI 3-kinase leads to a failure to initiate IL-2 production, suggesting a critical role for PI 3-kinase in T cell activation [Pages et al. 1994]. Based on studies using the PI 3-kinase inhibitor, wortmannin, there is evidence that PI 3-kinase(s) are also required for some aspects of leukocyte signalling through G protein-coupled receptors [Thelen et al., Proc. Natl. Acad. Sci. USA., 91:4960-4964 (1994)].

There thus continues to exist a need in the art for further insights into the nature, function and distribution of PI 3-kinase providing means for effecting beneficial modulation of PI 3-kinase effects.

15 SUMMARY OF THE INVENTION The present invention provides novel purified and isolated polynucleotides DNA and RNA both sense and antisense strands) encoding a heretofore unknown catalytic member of the PI 3-kinase family, designated p 1 105, which is expressed predominantly in leukocytes and thus 20 likely plays a role in PI 3-Kinase mediated signaling in the immune system.

Preferred DNA sequences of the invention include genomic and cDNA sequences as well as wholly or partially chemically synthesized DNA sequences. The DNA sequence encoding p 1 105 that is set out in SEQ ID NO: 1 and DNA sequences which hybridize to the noncoding strand thereof under standard stringent conditions (or which would hybridize but for the redundancy of the genetic code) are contemplated by the invention. Exemplary stringent hybridization conditions are as follows: hybridization at 65°C in 3X SSC, NaPO 4 pH 6.8 and washing at 65*C in 0.2X SSC. It is understood by those of skill in the art that variation in these conditions occurs based on the length and GC nucleotide base content of the sequences to be hybridized.

Formulas standard in the art are appropriate for determining exact hybridization conditions. See Sambrook et al., 9.47-9.51 in Molecular Cloning, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989).

DNAIDNA

hybridization procedures carried out with DNA sequences of the invention under stringent conditions are expected to allow the isolation of DNAs encoding allelic variants of p11 0 8 non-human species enzymes homologous to p 1 105; and other structurally related proteins sharing one or more of the enzymatic 10 activities, or abilities to interact with members or regulators, of the cell pathways in which p 10 5 participates.

S. Also contemplated by the invention are biological replicas copies of isolated DNA sequences made in vivo or in vitro) of DNA sequences of the invention. Autonomously replicating recombinant constructions such as 15 plasmid and viral DNA vectors incorporating p1105 sequences and especially vectors wherein DNA encoding p110 6 is operatively linked to an endogenous or exogenous expression control DNA sequence and a transcription terminator are also provided. The skilled worker will understand the various components of vectors promoter(s), selectable marker(s), origin of replication(s), 20 multiple cloning site(s), etc.], methods for manipulating vectors and the uses of vectors in transforming or transfecting host cells (prokaryotic and eukaryotic) and expressing p 0 6 of the present invention.

According to another aspect of the invention, procaryotic or eukaryotic host cells are stably or transiently transformed with DNA sequences of the invention in a manner allowing the expression of pl105. Host cells expressing p110 6 or p110 6 along with a binding partner thereof can serve a variety of useful purposes. Such cells constitute a valuable source of immunogen for the development of antibody substances specifically immunoreactive with p1105. Host cells of the invention are also useful in methods for the large scale production of p110 8 wherein the cells are grown in a suitable culture medium and the desired polypeptide products are isolated from the cells or from the medium in which the cells are grown by, for example, immunoaffinity purification.

As described herein, p 1 108 is a polypeptide which possess kinase catalytic activity.

In one aspect, the present invention provides p 10 6 polypeptides.

The catalytic domain of p110 6 polypeptide (amino acid residues 723-1044 of SEQ ID NO: 2) exhibits greater than 72% identity to the catalytic domain of 10 p 1 10P. Preferably, the polypeptides of this invention exhibit identity to the catalytic domain of pll0 of 75% or greater. Even more preferably, the polypeptides comprise the amino acid residues according to SEQ ID NO: 2.

Yet another aspect of this invention provides polypeptide fragments or analogs of p110 6 The fragments of p110 6 are useful in modulating the binding of p 1 10 and a binding partner p85, Ras, and growth factor receptors). Analogs are polypeptides in which additions, substitutions, including conservative substitutions, or deletions of amino acid residues have been made in order to increase or decrease the binding affinity of the analog and a binding partner. These analogs of p1106 may be useful for modulating blocking, inhibiting, or stimulating) the interaction between p1106 and a binding partner.

The polypeptides of this invention may be modified to facilitate passage into the cell, such as by conjugation to a lipid soluble moiety. For example, p1106 (or fragments or analogs thereof) may be conjugated to myristic acid. The peptides may be myristoylated by standard techniques as described in Eichholtz et al., J. Biol. Chem. 268:1982-1986 (1993), incorporated herein by reference. Alternatively, the peptides may be packaged in liposomes that may fuse with cell membranes and deliver the peptides into the cells. Encapsulation of the peptides in liposomes may also be performed by standard techniques as generally described in U.S. Patent Nos. 4,766,046; 5,169,637; 5,180,713; 5,185,154; 5,204,112; and 5,252,263 and PCT Patent Application No. 92/02244, each of which is incorporated herein by reference.

Another aspect of this invention provides antibody substances polyclonal and monoclonal antibodies, antibody fragments, single chain antibodies, chimeric antibodies, CDR-grafted antibodies, humanized antibodies and the like) specifically immunoreactive with p1108. Antibody substances can be prepared by standard techniques using isolated naturally-occurring or recombinant p1108. Specifically illustrating monoclonal antibodies of the *10 present invention is the monoclonal antibody produced by hybridoma cell line 208F which was deposited with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, MD 20852 on October 8, 1996 and was assigned Accession No. HB 12200. The antibody substances are useful in modulating blocking, inhibiting, or stimulating) the binding between 15 p110 6 and its binding partner. Antibody substances are also useful for purification of p110 6 and are also useful for detecting and quantifying p11 0 in biological samples by known immunological procedures. In addition, cell lines hybridomas) or cell lines transformed with recombinant expression constructs which produce antibody substances of the invention are 20 contemplated.

In another aspect, methods of identifying a modulator that inhibits or activates the kinase activity of p110 6 are contemplated. In a preferred method, kinase activity of p1106 in the presence and absence of a potential modulator compound is determined and compared. A reduction in the kinase activity observed in the presence of the test compound indicates that the test compound is an inhibitor. An increase in the kinase activity observed in the presence of the test compound indicates that the test compound is an activator.

In another aspect, this invention provides methods of identifying a modulator that affects the binding of p110 5 and a binding partner Ras and growth factor receptors) and thereby increases or decreases the effective specific subcellular concentration of p1106. In this method, p1106 and its binding partner are incubated in the presence and absence of a putative modulator under conditions suitable for binding. The observed binding in the presence and absence of the modulator compound is compared. A reduction in the observed binding indicates that the compound inhibits binding. An increase in the observed binding indicates that the compound increases binding. These modulators are useful in affecting localization of p1106 to a specific subcellular S 10 location.

Modulators contemplated by the invention, for example, include polypeptides, polypeptide fragments of p110 5 and other organic and inorganic chemical compounds.

This invention further provides a method of detecting the presence of p 1 10 in a biological sample. The method comprises exposing a p1106 specific antibody to a biological sample to be tested. The binding of the p1105 specific antibody to p110 5 in the biological sample is detected by wellknown means. For example, a second antibody conjugated to horseradish peroxidase (HRP) that specifically recognizes anti-pi 106 antibody is used to detect anti-pll06 antibody. A positive color reaction catalyzed by HRP indicates that p1106 is present in the biological sample.

Yet another aspect of this invention provides a diagnostic reagent for detecting the presence of polynucleotides that encode p 1 106 in biological samples. The diagnostic reagent is a detectably labeled polynucleotide encoding part or all of the amino acid residues of p 105 set out in SEQ ID NO: 2. The presence of the polynucleotide in the biological sample is determined by hybridization of the diagnostic reagent to the polynucleotide encoding p1108.

Exemplary biological samples include chromosomes and chromosomal DNA.

The diagnostic reagent is detectably labeled with well-known-labels, including radioactive, enzymatic or other ligands, such as avidin/biotin, and fluorescent tags which are capable of providing a detectable signal.

The DNA sequence information provided by the present invention also makes possible the development, by homologous recombination or "knockout" strategies [see e.g. Capecchi, Science 244:1288-1292 (1989)] of mammals that fail to express a functional p110 6 or that express a variant analog of p 1 106. The mammals of the present invention comprise a disrupted p1108 gene or a disrupted homolog of the p110 6 gene. The general strategy utilized to produce the mammals of the present invention involves the preparation of a 10 targeting construct comprising DNA sequences homologous to the endogenous gene to be disrupted. The targeting construct is then introduced into embryonic stem cells (ES cells) whereby it integrates into and disrupts the endogenous gene or homolog thereof. After selecting cells which include the desired disruption, the selected ES cells are implanted into an embryo at the blastocyst 15 stage. Exemplary mammals include rabbits and rodent species.

Polynucleotides of the invention are also expected to be useful in chromosomal localization studies potentially useful in detection of inappropriate and/or over expression of p110 8 in abnormal cell types.

****Also made available by the invention are antisense 20 polynucleotides relevant to regulating expression of p1 10 by those cells which ordinarily express the same.

Numerous additional aspects and advantages of the present invention will be apparent from the following detailed description of illustrative embodiments thereof.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 presents an alignment of the predicted catalytic domain of pl 10 with the corresponding domain of other members of the PI 3-kinase family. The alignment was performed using Geneworks (Intelligenetics, Inc., Mountain View, CA).

Figure 2 presents an alignment of the predicted Ras regulatory region of p110 5 with the corresponding region of other members of the PI 3kinase family. The conserved lysine which is essential for interaction with Ras is indicated by the symbol below the consensus line.

DETAILED DESCRIPTION OF THE INVENTION The present invention is illustrated by the following examples.

Example 1 describes the cloning and characterization of cDNA encoding p 1 106. p1108 was obtained by combining three separate cDNA clones spanning the full length p 1 105 cDNA. Example 2 describes the expression and kinase activity of recombinant p1108. Example 3 describes the isolation of a mouse genomic pI 108 clone. Baculovirus expression of p 1 10 is described in Example 4. Example 5 assesses the ability of recombinant p 1 105 to associate with p85 in transfected mammalian cells. The expression of p1105 in various 15 human tissues is disclosed in Example 6. Example 7 provides monoclonal antibodies specific for p 1 105. Example 8 describes experiments directed to chromosomal localization of p 1 105. Example 9 describes experiments related to the association of p1105 and growth factor receptors. Example 10 discusses S". the use of transgenic animals which are engineered to include a disruption in the 20 p1105 gene.

.o Example 1 Degenerate oligonucleotide primers were designed for use in a PCR reaction based on sequences conserved in the catalytic domain of known PI 3-kinases. The sense primer was GCAGACGGATCCGGIGAYGAYHKIAGRCARGA (SEQ IDNO: 3) encoding the sequence GDDLRQD (SEQ ID NO: and the anti-sense primer was GCAGACGAATC RWRICCRAARTCIRYRTG (SEQ ID NO: encoding the amino acid sequence HIDFGH (SEQID NO: Barn HI and Eco RI restriction sites are underlined. PCR reactions consisted of 100 ng of cDNA template [from human peripheral blood mononuclear cells (PBMC) activated for 18 hours with 10 ng/ml phorbol myristate and 250 ng/ml calcium ionophore (Sigma)], 10 pg/ml oligonucleotide primers, 50mM KC1, 10mM Tris HCI (pH 1.5mM MgCl 2 200mM dNTPs, and 1U of Taq polymerase in a final volume of 100 il. Reactions were performed using denaturation for 1 minute at 94°C, annealing at 60°C for 2 minutes and extension for at 72°C for 1 minutes for 3 cycles. The procedure was then repeated using 56°C annealing temperature for 3 cycles, 52"C annealing temperature for 3 cycles and annealing temperature for 30 cycles. Amplified products were gel purified, digested with Bar HI and Eco RI, and subcloned into the vector pBSSKII+ (Stratagene, La Jolla, CA) for sequencing. All DNA for sequencing was prepared using the Wizard Miniprep DNA Purification System (Promega, Madison, WI). Sequencing was performed on the Applied Biosystems Model 15 373 automated sequencer. Data bank searches were made using the BLAST program, and protein and DNA alignments were made using the Geneworks program (Intelligenetics Inc. Mountain View Ca.) One clone contained a 399 bp insert that encoded a 133 amino acid open reading frame showing similarity to p1l10. This clone was a partial clone of a new catalytic subunit of PI 3kinase designated p1105.

To identify a cDNA encoding p 1 106, specific oligonucleotide primers were designed based on the sequence of the 399 bp PCR product. The forward primer was CATGCTGACCCTGCAGATGAT (SEQ ID NO: 7) and the reverse primer was AACAGCTGCCCACCTCTTCGG (SEQ ID NO: These primers were used to screen a cDNA library from human PBMC stimulated with PMA and ionomycin (as described above) in the mammalian expression vector pRc-CMV.

Successive rounds of PCR were performed initially on pools of 100,000 clones and subsequently on smaller pools until a single clone termed PBMC #249 was isolated by colony hybridization using the PCR product labelled by random -11priming as a probe. This cDNA was not full length. Therefore to identify longer cDNA clones the same approach was used to screen a cDNA library from human macrophages (also in the vector pRcCMV). This led to the isolation of an additional cDNA clone (M#928) which extended the cDNA sequence by 1302 bp.

The remaining 5' end of the cDNA encoding p110 8 was obtained by 5' RACE PCR (Clonetech, Palo Alto, CA.) Two anti-sense gene-specific oligonucleotide primers were designed based on the 5' end of cDNA M#928 for RACE PCR reactions. The primary RACE primer was GGGCCACATGTAGAGGCAGCGTrCCC (SEQ ID NO: 9) and the nested RACE primer was GGCCCAGGCAATGGGGCAGTCCGCC (SEQ ID NO: 10). Marathon-Race reactions were set up using Marathon-ready cDNA template from Human Leukocytes and the Advantage Core PCR Reaction kit (Clonetech, Palo Alto, CA) following the manufacturer's protocol. Touchdown PCR cycling 15 conditions were modified to-improve the specificity of the Marathon-RACE PCR primary reaction as follows: denaturation at 94°C for 2 minutes, followed by 5 cycles of denaturation at 94°C for 30 seconds and annealing and extension at 72°C for 3 minutes; 5 cycles of denaturation at 94°C for 30 seconds and annealing and extension at 70°C for 3 minutes; and 25 cycles of denaturation 20 at 94°C for 30 seconds and annealing and extension at 68 0 C for 3 minutes.

Amplified products were used as templates in a nested PCR reaction using the previously described cycling parameters. The reampiified products were then analyzed by Southern blotting using oligonucleotide probes specific for p 108. Probes (100ng each) were end-labelled with "P-yATP, and 25 hybridized and washed under standard conditions (Frisch and Sambrook). The sequences of the two probes were GATGCGGAACGGCTGCTCCAGGG (SEQ ID NO: 11) and CCAGGGACCACAGGGACACAGAG (SEQ ID NO: 12).

The specific 5' RACE PCR products identified in this manner were gel purified and subcloned into the TA vector PCRII (Invitrogen, San -12- Diego, CA) according to the manufacturer's instructions. Three independent clones were sequenced to ensure the veracity of the 5' sequence.

A full length cDNA for p1108 was assembled from clones #249, M#928 and the 5' RACE PCR products. The 5' RACE product was used as a template in PCR using the

AGTTACGGATCCGGCACCATG(GACTACAAGGACGACGATGACAAG)CCCCCTGGGGTGGA

CTGCCC (SEQ ID NO: 13) and the 3' primer CCACATGTAGAGGCAGCG-TCC (SEQ ID NO: 14). The 5' primer includes a Barn HI site (underlined), and sequences that encode the FLAG peptide sequence DYKDDDDK (SEQ ID NO: (shown in parenthesis) which is recognized by the M2 anti-FLAG monoclonal antibody (Kodak Scientific Imaging Systems, New Haven, CT). The resulting PCR product was digested with Bar HI and Afl II, and was ligated along with an Afl H/Pvu I fragment derived from the clone M#928 and a Pvu II/Xba I fragment derived from PBMC clone #249 into the Barn H/Xba I sites of the 15 mammalian expression vector pcDNA3 (Invitrogen, San Diego, CA). The vector containing the FLAG-tagged composite pl10 6 cDNA is designated *pCDNA3:pl 10FLAG. In the FLAG-tagged p1105, the FLAG-tag is located immediately after the initiating methionine.

A full-length composite cDNA encoding p110 8 is shown in SEQ 20 ID NO: 1. The sequence of p 1 108 includes an open reading frame of 3135 nucleotides which is predicted to encode a protein of approximately 114 KD.

In addition, there are 197 bp of 5' and 1894 bp of 3' untranslated sequence.

The sequence around the predicted initiating methionine is in good agreement with that required for optimal translational initiation [Kozak, J. Cell Biol., 25 115:887-992 (1991)] and the presence of stop codons in the 5' untranslated sequence is consistent with the isolation of the complete coding region of pl106.

Comparison of the deduced amino acid sequence of pl 10 (SEQ ID NO: 2) with other PI 3-kinases reveals that it is most closely related to Similar to pl 101, the catalytic domain of p1105 is found in the Cterminus of the protein and is believed to be reside within amino acid residues 723-1044 of SEQ ID NO: 2. An alignment of the predicted carboxyl terminal catalytic domains of the PI 3-kinase family (including p1106 residues 723 through 1044 of SEQ ID NO: 2) is shown in Figure 1. Table 1 shows the identity of p110 8 to other members of the PI 3-kinase family. p1106 is 72% identical to p110 in this region but is less closely related to p110a and p1lOy Table 1 also shows that p1108 shows low identity to and the yeast Vps 34 protein, 31 and 32% respectively.

TABLE 1 p1106 pl0l1 pll0a pl10y cpk/p170 Vps34 p110 6 72 49 45 31 32 pl1OP 49 48 37 31 Spll0a 45 39 29 39 31 15 cpk/pl70 28 Vps34 distinct sub-branch of the PI 3-kinase family. The distantly related ATM gene and the catalytic subunit of DNA dependent protein kinase have been included for comparison.

:It has been demonstrated that PI 3-kinase is an important intermediate in the Ras pathway [Hu et al. 1993; Rodriguez-Viciana et al., EMBO Journal, 15:2442-2451 (1996)]. A constitutively active form of PI 3-kinase has been shown to increase transcription of the c-fos gene, activate the protein kinase Raf, and stimulate oocyte maturation [Hu et-al., 1995]. The effects of PI 3-kinase in these systems can be blocked by co-expression of a dominant negative form of Ras indicating that PI 3-kinase acts upstream of Ras.

Additional studies have shown that Ras can physically interact with PI 3-kinase in vitro and stimulate its kinase activity [Rodriguez-Viciana et al., 1996]. Thus PI 3-kinase can either act as an effector of Ras-dependent signalling or be directly activated by interaction with Ras. A specific region at the amino terminus of the p110 subunits termed the Ras regulatory domain is responsible for this interaction [Rodriguez-Viciana et al. 1996]. Comparison of the sequence of p 1 108 with other p110 subunits indicates that this region is also conserved in p110 8 including a lysine residue which has been shown to be essential for physical association with Ras (Rodriguez-Viciana et al., 1996).

Thus p110 8 is also likely to interact with the Ras pathway. Figure 2 presents an alignment of the proposed Ras binding sites of four p 1 0 subunits including p110 8 residues 141 through 310 of SEQ ID NO: 2.

Example 2 The FLAG-tagged p 1 108 was expressed by transfecting into COS cells using DEAE dextran. Three days after transfection, expression of p 1 0 8 was determined by immunoprecipitations and S- western blotting using the M2 monoclonal antibody (Kodak Scientific Imaging Systems) according to the manufacturer's instructions. PI 3-kinase activity was determined as described [Hu et al., Mol. Cell. Biol., 13:7677-7688 (1993)].

~To determine the PI 3-kinase activity of p 1 108, 5l1 of immunoprecipitated p110 8 was mixed with 1/ 1 l of PI/EGTA and incubated at room temperature for 10 minutes [PI/EGTA is l0mg/ml PI(Sigma) in CHCI 3 which has been dried under a vacuum, resuspended in 20 mg/ml DMSO in th presence or absence of various concentrations of the PI3 kinase inhibitor wortmannin and diluted 1:10 in 5mM EGTA] and added to 11l 10X HM buffer (200mM HEPES pH 7 2 50mM MnCI 2 0.5 Il y 32 PATP 300Ci/mmol), 1p1 100/zM ATP, and 1.5txl H 2 0 and incubated at 30 0 C for minutes. The reactions were terminated by addition of 100li 1M HC1. Lipids were extracted with 2 0 0 tl CHCI 3 /MeOH by vortexing for 1 minute followed by centrifugation at 16,000 x g for 2 minutes at room temperature.

The lipids were further extracted with 80zl 1M HC1/MeOH by vortexing for 1 minutes, followed by centrifugation at 16,000 x g for 2 minutes at room temperature. The lipids were dried under vacuum, resuspended in CHCl3/MeOH and spotted 2cm from the bottom of a dry Silica gel chromatography plate (VWR) that had been pre-impregnated with 1 K 2

C

2 0 4 in HO0. 250/g of crude phosphoinositides (Sigma) were spotted as markers.

The products were resolved- by chromatography for 2 hours in SCHCl 3 /MeOH/4N NH 4 OH allowed to dry and placed in an Iodine vapor tank for 5 minutes in order to visualize the crude standards. The position of the standards was marked with a pencil and the plate was autoradiographed.

.15 Phosphorylated lipids were generated in the kinase assays. The major product was phosphatidyl inositol phosphate (PIP). Furthermore, the generation of these phosphorylated lipids was inhibited in a dose dependent manner by wortmannin (approximately 50% of the activity was inhibited at 100 nM wortmannin) demonstrating that p1106 is a functional PI3 kinase.

Example 3 A mouse genomic clone encoding p1106_was isolated as described below.

A mouse 129 SvEv lambda genomic library (Stratagene, La Jolla, Ca.) was screened using a fragment of the human cDNA clone for p1106 (corresponding to amino acids 739 to 1044 of SEQ ID NO.: 2) labelled to high specific activity 1 x 109 dpm/ug DNA) by random priming using the Random Primed DNA labelling Kit (Boehringer-Mannheim). Hybridization was performed for sixteen hours at 42°C in buffer containing 50% formamide, 5X SSC, 5 X Denhardts, 0.05M Na phosphate, and 100 ug/ml salmon sperm DNA. Filters were washed -16in 0.2 XSSC/0.1 SDS at 50C. A single clone was isolated. Purified phage DNA was digested with Not I and inserts were subcloned into the vector pBSSKII+ (Stratagene, La Jolla, Ca.) for sequencing. This clone was approximately 16kb and included the entire catalytic region of p 1 106.

Example 4 Recombinant p110 6 may be expressed in SF9 insect cells using a baculovirus expression system.

As discussed in Example 1, FLAG-tagged p110 8 encoding sequences are useful in expressing the kinases of this invention. Upon expression in insect cells, a monoclonal antibody that recognizes the FLAG tag (Eastman Kodak, Rochester, New York) is used to purify large quantities of the FLAG-PIK-related kinase fusion protein. Infected insect cells are incubated for 48 hours and lysed in lysis buffer (25mM 2-glycerolphosphate, 50mM sodium phosphate pH 7.2, 0.5% Triton-X 100, 2mM EDTA, 2mM EGTA, 25 mM 15 sodium fluoride, 100pM sodium vanadate, ImM PMSF, lgg/ml leupeptin, 14g/ml pepstatin, ImM benzamidine, and 2mM DT). Expressed FLAG fusion proteins are purified over a column containing anti-FLAG antibody M2 affinity resin (Eastman Kodak). The column is washed with 20 column volumes of lysis buffer, then 5 column volumes of 0.5M lithium chloride, 50mM Tris pH 7.6, 1mM DTT, and then eluted either with 0.1M glycine pH 3.0 followed by immediate neutralization or by competitive elution with the FLAG peptide. For- histidine tagged proteins, Ni-NTA agarose (Qiagen) is used for protein purification.

Plasmids for expression of p85 and pl10 6 in the baculovirus expression sytstem were prepared as follows.

The plasrnid pcDNA3:p85 DNA as described in Example 5 was digested with BamHI and EcoRI and the 2.5 kb FLAG-p85 band containing the entire p85 coding region with the FLAG tag was gel purified and inserted in -17- BamHI-EcoRI site of pFastbac Dual (Gibco BRL). The ligation mixture was transformed into E. coli XL-1 blue (Stratagene) and plated on ampicillin containing plate. A clone was purified that carries the plasmid.

The pFastbac-Dual-p85 plasmid was transformed into E. coli Bac cells and white colonies were selected on plates containing kanomycin, gentamycin, tetracyoline, X-gel and IPTG. One white colony was restreaked on a similar plate for repurification. Recombinant p85-bacmid DNA was purified from this clone.

The plasmid pcDNA3:p1108 containing the entire p110 8 coding region with the FLAG tag was digested with BamHI and XbaI, gel purified and inserted into the BamHI-XbaI site of pFastbac HTb (Gibco BRL) such that the coding region of FLAG-tagged p110 5 was in frame with the coding sequences of the histidine-tag present in the vector. The ligation mixture was then 15. transformed into E. coli XL-1 blue (Stratagene). A clone carrying pFast-bac Htb p 1 105 was isolated and the plasmid DNA was isolated and the plasmid DNA was purified. P1106-bacmid DNA was prepared by transforming E. coli

C

bac cells as described for To prepare virus stocks, the p85-bacmid and the pi 105-bacmid .o 20 DNAs were separately transfected into SF-9 cells according to the Gibco BRL suggested protocol. Forty-eight hours after transfection, the SF9 cell pellet and baculovirus produced by the transfected cells were harvested. The virus was stored at 4°C in Grace's Complete media containing 10% FBS, pennicillinstreptomycin, and gentamicin. This viral prep was used to make a high titer (P2) virus stock. The P2 virus stock was used to infect a 50 ml culture of SF9 cells. The cells were collected 48 hours after infection and centrifuged at low speed to pellet the cells'without lysis. The cell pellet was stored at -20 0 C for 24 hours before lysis. The cells were lysed in 5 ml of lysis buffer (50 mM Tris, pH 8.0; 500 mM NaCI; 1% NP40; 100 jm PMSF). Expression of and p110 8 was confirmed by immunoblot using the M2 antibody anti-FLAG as a probe. The SF-9 transfected cells produced an approximately 85 kDa protein and a 110 kDa protein which were immunoreactive with anti-FLAG antibodies.

The P2 virus stock were also used to co-infect a 2 liter culture of SF9 cells. The cells were collected 48 hours after infection, centrifuged at low speed to pellet the cells without lysis and stored at -20"C. A cell pellet from 150 mls of this culture was lysed in 7.5 ml of lysis buffer (50mM NaPO 4 pH7.2; 0.5% NP-40; 10mM imidazole, 25mM NaF, 100/M Na 3

VO

4 AEBSF; 1 xg/ml leupeptin; lg/ml pepstatin A) and incubated on ice for minutes. The lysate was then centrifuged for 30 minutes at 10,000 x g. The supernatant was removed and any DNA in the lysate resulting from broken nuclei was sheared by aspirating through an 20 gauge needle. Particulate matter was then removed by filtering through a 0.8 micron filter followed by a 0.2 micron filter. This cleared lysate was adjusted to contain 5 mM P- *i 15 mercaptoethanol and 0.4 M NaCl. A 1 ml Ni-NTA-agarose column (Qiagen) was equilibrated in Buffer A (0.4 M NaCI; 5 mM P-mercaptoethanol; 0.1% Triton X-100; 50 mM NaPO, 10 mM imidazole; 25 mM NaF, 100 IM Na 3

VO

4 0.5 mM AEBSF; 1 xg/ml leupeptin; 1 zg/ml pepstatin A) prior to loading the cleared lysate. The sample was loaded at a flow rate of 0.25 20 ml/minute, washed 5 ml of Buffer A and then eluted in 10 ml of a gradient of to 500 mM imidazole in Buffer A.

*o .Example The ability of p110 5 to associate with p85 was assessed by Western blot analysis. COS cells were transiently transfected with p 1 105 (see Example 2) and association with endogenous p85 was determined by coimmunoprecipitation.' As controls, cells were also transfected with FLAGtagged p85 DNA or empty vector. The cDNA encoding the p85 subunit was isolated from human leukocyte cDNA by Marathon-race PCR. The cDNA sequence of p85 was described in Otsu, Cell, 65:91-104 (1992). The cDNA was modified for expression as a FLAG-tagged protein (pcDNA3: in a manner similar to the protocols described herein for p 1 108.

COS cells were lysed in 3ml Buffer R Triton X-100, 150mM NaC1, 10mM Tris pH7.5, ImM EGTA, 0.5% NP-40, 0.2mM Na 3

VO

4 0.2mM PMSF, IX aprotinin, IX leupeptin, IX pepstatin After minutes at 4°C, the lysates were sheared by passing through a 27G needle several times. The lysates were clarified by centrifugation at 16,000 x g for minutes at 4°C, and immunoprecipitated for 2 hours at 4°C with either 14zg anti-p1l10P (Santa Cruz Laboratories, Santa Cruz, CA), 10zg anti-FLAG-M2 (Eastman Kodak), or 1;xg anti-p85 (Santa Cruz Laboratories). Immune complexes were bound to 60p1 of Protein G-sepharose (Pharmacia) for minutes at 4°C then washed 3 times in 3 0 0 ,l of Buffer R and resuspended in PAN (100mM NaCI, 10mM PIPES pH7.0, 20pg/ml Aprotinin). 5/zl of each immunoprecipitate was resolved by 8% SDS-PAGE (Novex), transferred to Immobilon-P (Millipore), blocked one hour at room temperature in 5 non- .fat dried milk in TBS, and detected by Western blotting using either rabbit polyclonal antibodies (Santa Cruz Laboratories) at l1xg/ml followed by goat anti-rabbit IgG HRP conjugated secondary antibody (Boehringer) or anti- FLAG-M2 monoclonal antibody at 10jg/ml followed by goat anti-mouse IgG HRP conjugated secondary antibody (Boehringer).

The Westerns showed that anti-FLAG-M2 antibody recognized immune complexes including FLAG-tagged p85 and FLAG-tagged p1108.

Example 6 25 While the activation of PI 3-kinase in a wide range of biological systems has been extensively studied, less is known concerning the cell type specific expression of particular p 1 10 isoforms. The expression of p110 8 in human heart, brain, placenta, lung, liver, skeletal muscle, kidney, pancreas, spleen, thymus, prostate, testis, uterus, small intestine, colon, and PBMC was determined by Northern blot analysis.

3 P-labelled cDNA probes were prepared by PCR using 10ng of plasmid DNA template encoding p1106, as described previously [Godiska et al, J. Neuroimmun., 58:167-176 (1995)]. The forward primer was CTGCCAT'GTTGCTCTTGTTGA (SEQ ID NO: 16) and the reverse primer was GAGTTCGACATCAACATC (SEQ ID NO: 17). Reactions were heated for 4 minutes at 94°C, followed by 15 cycles of denaturation for 1 minutes at 94 0

C,

annealing for 1 minutes at 55°C and extension for 2 minutes at 72 0

C.

Unincorporated nucleotides were removed by passing the reaction over a sephadex G50 column (Boehringer Mannheim Biochemicals).

A Multiple Tissue Northern blot (Clontech, Palo Alto, CA) was probed and washed under stringent conditions according to the manufacturer's recommendations. The autoradiograph was exposed for 1-4 days at -80"C with 15 intensifying screens.

ma. Northern blot analysis revealed a single transcript of approxi- "mately 5.4 kb (consistent with the size of the composite cDNA). The highest i levels of expression were seen in peripheral blood mononuclear cells (PBMC) and in spleen and thymus. On prolonged exposure of the autoradiograph, 20 expression of p1106 could also be detected in testes, uterus, colon, and small intestine, but not in other tissues examined including prostate, heart, brain, and liver. In contrast, p1 103 is expressed at high levels in brain, heart, kidney and liver, but canfiot be readily detected in lymphoid tissues such as spleen. p110 is expressed at high levels in the transformed Jurkat T cell line (Hu et al.

25 1993). The expression of the p l10a isoform has not been well documented.

p 10 isdforms have been shown to differ with respect to their preferred substrate specificities [Stephens et al., Current Biology, 4:203-214 (1994)]. In view of their potential for interaction with a common p85 adaptor -21protein, it is likely that the nature of the phosphorylated lipids generated in response to a particular agonist may be regulated at least in part by the cell/tissue specific expression of the different isoforms of the kinase enzymatic activity. The abundant expression of p110 8 in PBL and lymphoid tissues such as spleen and thymus suggests that this isoform may be involved in aspects of leukocyte activation.

Example 7 Monoclonal antibodies were generated against the carboxy terminal portion of p110 8 (amino acids 740-1044 of SEQ ID NO: 2) expressed as a fusion protein with glutathione S transferase (GST) [Pharmacia, Alameda, CA]. Five BaIb/c mice (Charles River Biotechnical Services, Inc., Wilmington, Massachusetts, IACUC #901103) were immunized subcutaneously with of antigen in complete Freund's adjuvant [CFA] (Sigma), a second immunization of 30ug of antigen in incomplete Freunds adjuvant (IFA) (Sigma) was administered on day 22. A third immunization with 30ug of antigen in IFA was administered on day 44. Immune serum was collected via retro-orbital bleeding on day 55 and tested by western blotting to determine reactivity to i p 10 8 All animals showed reactivity towards the immunogen and were immunized a fourth time on day 66 with 30ug of antigen in IFA. Immune :20 serum was collected via retro-orbital bleeding on day 76 and tested by western blotting to determine its reactivity, animal -#2321 showed the highest level of immunoreactivity and was chosen for fusion. On day 367 and 368 mouse S#2321 was injected intraperitoneally with 50ug of antigen in PBS and a fusion was performed on day 371.

25 The spleen was removed sterilely and a single-cell suspension was formed by grindirig the spleen between the frosted ends of two glass microscope slides submerged in serum free RPMI 1640, supplemented with 2mM L-glutamine, 1mM sodium pyruvate, 100 units/ml penicillin, and 100 tzg/ml streptomycin (RPMI) (Gibco, Canada). The cell suspension was filtered through sterile 70-mesh Nitex cell strainer (Becton Dickinson, Parsippany, New Jersey), and washed twice by centrifuging at 200 g for 5 minutes and resuspending the pellet in 20 ml serum free RPMI. Thymocytes taken from 3 naive Balb/c mice were prepared in the same manner.

Two x 10' spleen cells were combined with 4 x 107 NS-1 cells (kept in log phase in RPMI with 11 fetal bovine serum (FBS) for three days prior to fusion), centrifuged and the supernatant was aspirated. The cell pellet was dislodged by tapping the tube and 2 ml of 37 0 C PEG 1500 (50% in 75 mM Hepes, pH 8.0) (Boehringer Mannheim) was added with stirring over the course of 1 minute, followed by adding 14 ml of serum free RPMI over 7 minutes.

An additional 16 ml RPMI was added and the cells were centrifuged at 200 g for 10 minutes. After discarding the supernatant, the pellet was resuspended in 200 ml RPMI containing 15% FBS, 100 mM sodium hypoxanthine, 0.4 mM aminopterin, 16 mM thymidine (HAT) (Gibco), 25 units/ml IL-6 (Boehringer Mannheim) and 1.5 x 10 6 thymocytes/ml. The suspension was dispensed into ten 96-well flat bottom tissue culture plates (Coming, United Kingdom) at 200 l /well. Cells were fed on days 2, 4, and 6 days post-fusion by aspirating 100 .pl from each well with an 18 G needle (Becton Dickinson), and adding 100 l/well plating medium containing 10 U/ml IL-6 and lacking thymocytes.

S. :When cell growth reached 60-80% confluence (day 8-10), culture supernatants were taken from each well and screened for reactivity to p 1 106 by ELISA. Immulon 4 plates (Dynatech, Cambridge, Massachusetts) were coated at 4 0 C with 50 tl/well with 100ng/well of p 106:GST or GST in 50 mM carbonate buffer, pH 9.6. Plates were washed 3X with PBS with 0.05 Tween 20 (PBST), blocked 30 minutes at 37 0 C with 0.5 Fish Skin Gelatin. Plates were washed as described above and 50 pl culture supernatant was added. After incubation at 37 0 C for 30 minutes, 50 pl of horseradish peroxidase conjugated goat anti-mouse IgG(fc) (Jackson ImimunoResearch, -23- West Grove, PA) [diluted 1:10,000 in PBST] was added. Plates were incubated at 37C for 30 minutes, washed 4X with PBST and 100 pl of substrate, consisting of 1 mg/ml TMB (Sigma) and 0.15ml/ml 30% H 2 0, in 100 mM Citrate, pH 4.5, was added. The color reaction was stopped in 3 minutes with the addition of 50 ml of 15% H 2

SO

4

A

450 was read on a plate reader (Dynatech).

Thirty-six wells showed preferential reactivity to p 1 106 versus GST. Supernatants from these wells were then screened for reactivity to recombinant p1106 by Western blotting. Ten wells (208A, 208B, 208C, 208D, 208E, 208F, 208G, 208H, 2081, and 208J) showed reactivity by Western blotting and were cloned twice by limiting dilution. Selected wells were tested by ELISA 7-10 days later. Activity was retained in all ten lines. Monoclonal antibodies produced by the cell lines were isotyped by ELISA assay. 208A, 208C, 208D, 208E, 208G, 208H, 2081 were IgGz, while 208J was IgG, and 208B was IgG2b. An exemplary monoclonal antibody, produced by hybridoma S cell line 208F (ATCC HB 12200), showed high reactivity with p1105 and recognized a 110 kD protein in PBMC by Western analysis. The molecular weight of the 110 kD protein is consistent with the molecular weight of p1108.

Example 8 20 Elevated levels of 3' phosphorylated phosphoinositides have been detected in cells transformed with viral oncoproteins. This observation suggests that PI 3-kinases may play a role in carcinogenesis. Chromosomal localization of p 1 10 provides insights into the role of PI 3-kinase in carcinogenesis.

Chromosomal localization studies of p1106 of cancerous cells may identify 25 inappropriate and/or over expression of p1108.

For example, in 90-95 of chronic myelogenous leukaemia there is a reciprocal chromosomal translocation which leads to the transfer of the tyrosine kinase c-abl from chromosome 9 into the ber gene on chromosome 22.

The resultant inappropriate expression of c-abl tyrosine kinase activity is critical for cell transformation and tumorigenesis. Chromosomal localization of p1106 is determined by fluorescence in situ hybridization (FISH) using the complete cDNA for p1106 as a probe. In this manner, the role of p110 8 in chromosomal translocations observed during tumorigenesis leukemogenesis) is identified.

Example 9 PI 3-kinase activity has been reported to be associated with a number of growth factor receptors. In addition, it has been observed that PI 3-kinase activity increases following cell activation. The antibodies to p110 8 disclosed in Example 5 are utilized to determine by Western blotting and immunoprecipitation the nature of the receptors with which p 1 108 associates.

These antibodies are also useful in elucidating the regulation of PI 3-kinase 15 enzymatic activity and cellular localization during cell activation. In view of the high levels of expression of p1108 in the immune system, it is likely that growth factor receptors involved in immune activation may associate with or be regulated by p 1 108. These receptors include T-cell receptors CD28 and CD2 and cytokine receptors such as IL-1 and IL-4, and tyrosine kinase coupled 20 receptors such as CSF-1 R.

Example To determine the functional role of p1108 in vivo, the p110 8 gene is inactivated in the germline of mammals by homologous recombination.

Animals in which an endogenous gene has been inactivated by homologous S* 25 recombination are also known as "knockout" animals. Exemplary mammals include rabbits and rodent species such as mice. "Knockout" animals can be prepared by homologous recombination methods using the p1106 genomic clone of Example 3.

These "knockout" animals allow for the determination of the role of p1105 in immune and proliferative responses. The role of p1108 in immune and proliferative response is determined by analysis of the development of the immune system in these animals (as determined by FACS analysis of cell populations at different stages of development), characterization of the effector function of the mature lymphoid populations of these animals both in vivo (as determined by antibody responses to injected antigens, cytotoxic T cell responses to viruses and or injected tumor cell lines, and the ability to reject allografts) and in vivo (as determined by proliferation of lymphocytes in response to allo-antigen, polyclonal activation by mitogens/superantigens, and the ability to elaborate cytokines).

While the present invention has been described in terms of specific embodiments, it is understood that variations and modifications will occur to those skilled in the art. Accordingly, only such limitations as appear in the appended claims should be placed on the invention.

Throughout the specification, unless the context requires otherwise, the word "comprise" or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or group of integers but not the 20 exclusion of any other integer or group of integers.

S

S

i EDITORIAL NOTE APPLICATION NUMBER 18826/02 The following Sequence Listing pages 26 to 41 are part of the description. The claims pages follow on pages "43" to 44 -26- SEQUENCE LISTING GENERAL INFORMATION: APPLICANT: Chantry, David Hoekstra, Merl F.

Holtzman, Douglas A (ii) TITLE OF INVENTION: Novel Lipid Kinase (iii) NUMBER OF SEQUENCES: 17 (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: Marshall O'Toole Gerstein Murray Borun STREET: 6300 Sears Tower/233 South Wacker Drive CITY: Chicago STATE: Illinois COUNTRY: USA ZIP: 60606 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.30 (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: FILING DATE:

CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION: NAME: Noland, Greta E.

REGISTRATION NUMBER: 35,302 REFERENCE/DOCKET NUMBER: 27866/33441 (ix) TELECOMMUNICATION INFORMATION: TELEPHONE: (312) 474-6300 TELEFAX: (312) 474-0448 TELEX: 25-3856 INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 5220 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 196..3327 -27- (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: C-AGTCGCTCC GAGCGGCCGC GAGCAGAGCC GCCCAGCCCT GTCAGCTGCG CCGGGACGAT AAGGAGTCAG GCCAGGGCGG GATGACACTC ATTGATTCTA AAGCATCTr AATCTGCCAG GCGGAGGGGG C FTTGCTGGT CTrTC!TTGGA CTATTCCAGA GAGGACAACT GTCATCTGGG AAGTAACAAC GCAGG ATG CCC CCT GGG GTG GAC TGC CCC ATG GAA TTC TGG Met Pro Pro Gly Val Asp Cys Pro Met Giu Phie Trp 120 1.80 231 ACC AAG GAG Thr Lys Giu GAG AAT CAG Giu Asn Gin AGC GTT Ser Val 20 GTG GTI' GAC TTC Val Val Asp Phe

CTG

Leu CTG CCC AC-A Leu Pro Thr GGG GTC Gly Val TAC CTG AAC TTC Tyr Leu Asn Phe

CCT

Pro 35 GTG TCC CGC AAT GCC AAC CTC AGC ACC Val Ser Arg Asn Ala Asn Leu Ser Thr

ATC

Ile AAG CAG CTG CTG Lys Gin Leu Leu CAC CGC GCC CAG His Arg Ala Gin

TAT

Tyr 55 GAG CCG CTC TTC Giu Pro Leu Phe

CAC

His 375 ATG CTC AGT GGC Met Leu Ser Gly GAG GCC TAT GTG Giu Ala Tyr Val 'ITC ACC Phe Thr TGC ATC AAC Cys Ile Asn C.AG ACA Gin Thr a.

a.

GCG GAG CAkG Ala Glu Gin CAG CCC TTC Gin Pro Phe

C.AA

Gin 80 GAG CTG GAG GAC Giu Leu Giu Asp

GAG

Giu 85 CAA CGG CGT CTG Gin Arg Arg Leu TGT GAC GTG Cys Asp Val GGC GAC CGC Gly Asp Arg 471 CTG CCC GTC CTG Leu Pro Val Leu CTG GTG GCC CGT Leu Val Ala Arg

GAG

Giu 105 519 GTG AAG Val Lys 110 AAG CTC ATC AAC TCA CAG ATC AGC CTC Lys Leu Ile Asn Ser Gin Ile Ser Leu

CTC

Leu 120 ATC GGC AAA GGC Ile Gly Lys Gly

CTC

Leu 125 CAC GAG TTr GAG His Giu Phe Asp

TCCTTG

Ser Leu 130 TGC GAC CCA Cys Asp Pro

GAA

Glu 135 GTG AAC GAC TIT Vai Asn Asp Phe

CGC

Arg 140 GCC AAG ATG TGC Ala Lys Met Cys

CAA

Gin 145 TTC TGC GAG GAG Phe Cys Glu Giu

GCG

Ala 150 GCC GCC CGC CGG Ala Ala Arg Arg CAG CAG, Gin Gin 155 CTG GGC TGG Leu Gly Trp

GAG

Glu 160 GCC TGG CtG CAG Ala Trp Leu Gin AGT TTC CCC CTG Ser Phe Pro Leu CAG CTG GAG Gin Leu Glu 170 -28- CCC TCG GCT Pro Ser Ala 175 CAA ACC TGG GGG CCT Gin Thr Trp Gly Pro 180 GGT ACC CTG CGG Gly Thr Leu Arg

CTC

Leu 185 CCG AAC CGG Pro Asn Arg GCC =T Ala Leu 190 CTG GTC AAC GT1' Leu Val Asn Val

AAG

Lys 195 TTr GAG GGC AGC Phe Glu Gly Ser

GAG

Giu 200 GAG AGC TrC ACC Glu Ser Phe Thr

TTC

Phe 205 CAG GTG TCC ACC Gin Val Ser Thr

AAG

Lys 210 GAC GTG CCG CTG Asp Val Pro Leu CTG ATG GCC TGT Leu Met Ala Cys

GCC

Al a 220 CTG CGG AAG AAG Leu Arg Lys Lys ACA GTG 7rC CGG Thr Val Phe Arg

CAG

Gin 230 CCG CTG GTG GAG Pro Leu Val Giu GAG, CCG Gin Pro 235 GAA GAC TAC Glu Asp Tyr AAC TAC CCG Asn TIyr Pro 255

ACG

Thr 240 CTG GAG GTG AAC Leu Gin Vai Asn

GGC

Gly 245 AGG CAT GAG TAC Arg His Giu Tyr CTG TAT GGC Leu Tyr Gly 250 CTG CAC AGT Leu His Ser CTC TGC CAG TTC Leu Cys Gin Phe TAC ATC TGC AGC Tyr Ile Cys Ser

TGC

Cys 265 GGG TTG Gly Leu 270 ACC CCT CAC CTG Thr Pro His Leu

ACC

Thr 275 ATG GTC CAT TCC Met Val His Ser TCC ATC CTC GCC Ser Ile Leu Ala

ATG

Met 285 CGG GAT GAG GAG Arg Asp Giu Gin

AGC

Ser 290 AAC CCT GCC CCC Asn Pro Ala Pro GTC GAG AAA CCG Val Gin Lys Pro

CGT

Arg 300 1047 1095 1143 GCC AAA CCA CCT Ala Lys Pro Pro

CCC

Pro 305 ATT CCT GCG AAG Ile Pro Ala Lys

AAG

Lys 310 CCT TCC TCT GTG Pro Ser Ser Vai TCC CTG Ser Leu 315 TG G TCC CTG Trm Ser Leu GTG AAC GCC Val Asn Ala 335

GAG

Glu 320 G-AG CCG TTC CGC Gin Pro Phe Arg

ATC

Ile 325 GAG CTC ATC GAG Giu Leu Ile Gin GGC AGC AAA Gly Ser Lys 330 GGG cwr TIC Gly Leu Phe GAC GAG CGG ATG Asp Glu Arg Met

AAG

Lys 340 CTG GTG GTG GAG Leu Val Val Gin

GCC

Ala 345 1191 1239 1287 1335 CAC GGC His Gly 350 AAC GAG ATG CrG Asn Giu Met Leu

TGC

Cys 355 AAG ACG GTG TCC Lys Thr Val Ser

AGC

Ser 360 TCG GAG GTG AGC Ser Giu Val Ser TGC TCG GAG CCC Cys Ser Giu Pro

GTG

Val 370 TbG AAG GAG CGG Trp Lys Gin Arg

CTG

Leu 375 GAG TTC GAC ATC Giu Phe Asp Ile

A;LC

As n 380 ATC TGC GAC CTG Ile Cys Asp Leu CGC ATG GCC CGT Arg Met Ala Arg -29-

CTC

Leu 390 TGC 'ITT GCG CTG Cys Phe Ala Leu TAC GCC Tyr Ala 395 1383 GTG ATC GAG Val Ile Glu AAG GCG GAC Lys Ala Asp 415 AAA GCC Lys Ala 400 AAG AAG GCT Lys Lys Ala

CGC

A-rg 405 TCC ACC AAG AAG Ser Thr Lys Lys AAG TCC AAG Lys Ser Lys 410 =T GAC TAC Phe A sp Tyr 1431 1479 TGC CCC ATI GCC Cys Pro Ile Ala GCC AAC CTC ATG Ala Asn Leu Met

CTG

Leu 425 AAG GAC Lys Asp 430 CAG cTr AAG ACC Gin Leu Lys Thr

GGG

Gly 435 GAA CGC TGC CTC Giu Ar-g Cys Leu

TAG

440 ATG TGG-CCC TC Met Trp Pro Ser

GTC

Val 445 AGT Ser CCA GAT GAG AAG Pro Asp Glu Lys AAC CCC AAG ACG Asn Pro Asn Thr 465

GGC

Gly 450 GAG CTG CTG AAC Glu Leu Leu Asn

CCC

Pro 455 ACG GGC ACT GTG Thr Gly Thr Val

CGC

Arg 460 1527 1575 1623 GAT AGC GCC GCT Asp Ser Ala Ala

GCG

Al a 470 CTG GTC ATC TGC Lau Leu Ile Cys CTG CCC Leu Pro 475 GAG GTG GC Giu Val Aia GAG CTG GGG Glu Leu Gly 495 CAC CCC GTG TAG His Pro Val Tyr

TAC

485 CCC GCC CTG GAG Pro Ala Leu Giu AAG ATC TTG Lys Ile Leu 490 GAG GAG GAG Giu Glu Gin CGA GAG AGC GAG Arg His Ser Glu

TGT

Gys 500 GTG GAT GTC ACC Val His Val Thr

GAG

Giu 505 CTG GAG Leu Gin 510 CTG CGG GAA ATC Leu Arg Giu Ile

CTG

Leu 515 GAG CGG CGG GGG Giu Arg Arg Gly

TCT

Ser 520 GGG GAG CTG TAT Gly Giu Leu Tyr

GAG

Giu 525 CAC GAG AAG GAG His Giu Lys Asp GTG TGG AAG CTG Val Trp Lys Leu

CGG

Arg 535 CAT GAA GTC GAG His Giu Val Gin

GAG

Glu 540 1671 1719 1767 1815 1863 1911 1959 CAG TTC CCG GAG His Phe Pro Giu

GCG

Ala 545 CTA GCC GGG CTG Leu Ala Arg Leu

CTG

Leu 550 CTG GTC*ACC AAG Leu Val Thr Lys TGG AAC Trp Asn 555 AAG CAT GAG Lys His Giu GAG CTG CCC Giu Leu Pro 575

GAT

Asp 560 GTG GCC GAG ATG Val Ala Gin Met

CTC

Leu 565 TAG CTG CTG TGC Tyr Leu Leu Gys TCC TGG CCG Ser Trp Pro 570 AGC 'ITC CCC Ser Phe Pro GTC CTG AGC GC Val Leu Ser Ala

CTG

Leu 580 GAG CTG CTA GAC Giu Leu Leu Asp

TTC

Phe 585 GAT TGC CAC GTA GGC TCC Asp Cys His Val Gly Ser TTC GCC ATC AAG TCG CTG, CGG AAA CTG ACG 2007 Phe 595 Ala Ile Lys Ser 600 CAG GTG CTC AAG

GAC

Aso 605 GAT GAG CTG TTC Asp Glu Leu Phe

CAG

Gin 610 TAC CTG CTG CAG Tyr Leu Leu Gin CTG GTG Leu Val 615 2055 Gin Val Leu 620 TAC GAG TCC TAC Tyr Glu Ser Tyr GCC CTG GCC-AAC Ala Leu Ala Asn 640 TCC GAG ATG CAC Ser Glu Met His 655

CTG

Leu 625 GAC TGC GAG CTG Asp Cys Glu Leu

ACC

Thr 630 AAA TI'C CTG CTG Lys Phe Leu Leu GAC CGG Asp Arg 635 2103 CGC AAG ATC GGC Arg Lys Ile Gly TTC CTT TrC TGG Phe Leu Phe Trp CAC CTC CGC His Leu Arg 650 CTC ATC CTG Leu Ile Leu 2151 2199 GTG CCG TCG Val Pro Ser GCC CTG CGC TTC Ala Leu Arg Phe

GGC

Gly 665 GAG GCC Glu Ala 670 TAC TGC AGG GGC Tyr Cys Arg Gly

AGC

Ser 675 ACC CAC CAC ATG Thr His His Met

AAG

Lys 680 GTG CTG ATG AAG Val Leu Met Lys

CAG

Gin 685 GGG GAA GCA CTG Gly Giu Ala Leu

AGC

Ser 690 AAA CTG AAG GCC Lys Leu Lys Ala

CTG

Leu 695 AAT GAC TTC GTC Asn Asp Phe Val

AAG

Lys 700 *9 *5 S* S 5.55 S S CTG AGC TCT GAG Leu Ser Ser Gin

AAG

Lys 705 ACC CCC AAG CCC Thr Pro Lys Pro

CAG

Gin 710 ACC AAG GAG CTG Thr Lys Giu Leu ATG CAC Met His 7 TTG TGC ATG Leu Cys Met TCC CCA CTC Ser Pro Leu 735

CGG

Arg 720 CAG GAG GCC TAC Gin Glu Ala Tyr

CTA

Leu 725 GAG GCC CTC TCC Giu Aia Leu Ser CAC CTG GAG His Leu Gin 730 GTG GAG GAG Val Giu Gin 2247 2295 2343 2391 2439 2487 2535 2583 GAC CCC AGC ACC Asp Pro Ser Thr

CTG

Leu 740 CTG GCT GAA GTC Leu Ala Glu Val

TGC

Cys 745 TGC ACC Cys Thr 750 TTC ATG GAC TCC Phe Met Asp Ser

AAG

Lys 755 ATG AAG CCC CTG Met Lys Pro Leu

TGG

Trp 760 ATC ATG TAC AGC Ile Met Tyr Ser

AAC

Asn 765 GAG GAG GCA GGC Glu Giu Ala Giy

AGC

Ser 770 GGC GGC AGC GTG Gly Gly Ser Val

GGC

Gly 775 ATC ATC TFI' AAG Ile Ile Phe Lys

AAC

Asn 780 GGG GAT GAC CTC Gly Asp Asp Leu CGG GAG Arg Gin 785 GAC ATG CTG Asp Met Leu

ACC-

Thr 790 CTG GAG ATG ATC Leu Gin Met Ile GAG CTC Gin Leu 795 -31- ATG GAC GTC CTG TGG AAG CAG GAG GGG Met Asp Val TAT GGC TGC Tyr Giy Cys 815 Leu 800 Trp Lys Gin Glu Gly 805 CTG GAC CTG AGG ATG ACC CCC Leu Asp Leu Arg Met Thr Pro 810 2631.

CTC CCC ACC GGG Leu Pro Thr Gly CGC ACA GGC CTC Arg Thr Gly Leu Ile 825 GAG GTG GTA Giu Val Val 2679 CTC CGT Leu Arg 830 TCA GAC ACC ATC Ser Asp Thr Ile

GCC

Ala 835 AAC ATC CAA CTC Asn Ile Gin Leu AAC AAG AGC AAC ATG Asn Lys Ser Asn Met 840 CTC AAC TGG CTG AAG Leu Asn Trp Leu Lys 2727 2775

GCA

Al a 845 GCC ACA GCC GCC Ala Thr Ala Ala AAC AAG GAT GCC Asn Lys Asp Ala

CTG

Leu 855 TCC AAG AAC CCG Ser Lys Asn Pro

GGG

Gly 865 GAG GCC CTG GAT Glu Ala Leu Asp

CGA

Arg 870 GCC ATT GAG GAG Ala Ile Giu Giu TTC ACC Phe Thr 875 2823 CTC TCC TGT Leu Ser Cys GAT CGG CAC Asp Arg His 895

GCT

Al a 880 GGC TAT TGT GTG Gly Tyr Cys Val

GCC

Al a 885 ACA TAT GTG CTG Thr Tyr Val Leu GGC ATT GGC Gly Ile Gly 890 C.AG CTG TIC Gin Leu Phe AGC GAC AAC ATC Ser Asp Asn Ile ATC CGA GAG AGT Ile Arg Giu Ser

GGG

Gly 905

C*

C 9 C. I C 9S

S.

CC

C@

C C

CC

*9 9

C.

CAC ATT His Ile 910 GAT T= GGC CAC Asp Phe Gly His CTG GGG AAT TIC Leu Gly Asn Phe

AAG

Lys 920 ACC AAG TTT GGA Thr Lys Phe Gly 2871 2919 2967 3015 3063

ATC

Ile 925 AAC CGC GAG CGT Asn A-rg Glu Arg

GTC

Val 930 C CA TTC ATC CTC Pro Phe Ile Leu

ACC

Thr 935 TAT GAC. =I GTC Tyr Asp Phe Val

CAT

His 940 GTG ATT CAG CAG Val Ile Gln Gin

GGG

Gly 945 AAG ACT AAT AAT Lys Thr Asn Asn

AGT

Ser 950 GAG AAA TIT GAA Giu Lys Phe Giu CGG TIC Arg Phe 955 CGG GGC TAC Arg Gly Tyr CTC TTC CTC Leu Phe Leu 975

TGT

Cys 960 GAA AGG GCC TAC Giu Arg Ala Tyr ATC CTG CGG CGC Ile Leu Arg Arg CAC GGGCT His Gly Leu 970 CTG CCT GAG Leu Pro Giu 3111 3159 CAC CTC 'TTr GCC His Leu Phe Ala ATG CGG GCG GCA Met Arg Ala Ala

GGC

Giy 985 CTC AGC Leu Ser 990 TGC TCC AAA GAC Cys Ser Lys Asp AtC Ile 995 CAG TAT CTC AAG GAC TCC CTG GC-A CTG Gin Tyr Leu Lys Asp Ser Leu Ala Leu 1000 3207 -32- -GGG AAA ACA GAG GAG GAG GCA CTG AAG CAC TTC CGA GTG AAG =T1 AAC Gly Lys Thr Glu Glu Glu Ala Leu Lys His Phe Arg Val Lys Phe Asn 1005 1010 1015 1020 GAA GCC CTC CGT GAG AGC TGG AAA ACC AAA GTG AAC TGG CTG GCC CAC Glu Ala Leu Arg Glu Ser Trp Lys Thr Lys Val Asn Trp Leu Ala His 1025 1030 1035 AAC GTG TCC AAA GAC AAC AGG CAG TAGTGGCTCC TCCCAGCCCT GGGCCCAAGA Asn Val Ser Lys Asp Asn Arg Gin 1040 3255 3303 3357 0@ S S

COOS

*5 S .5 0S S S 55 @5 OS S *5 000055

S

S* 0 C @0

S@

0505

S

*0@5 0 S0 50 ego.

0 *000

OS

0 0 00 00 S 60

S.

GGAGGCGGCT

AACTGCACCT

TATG.ACTTGA

GCGGCGGTGC

GTCTTCCAGC

TCCCAGGCCT

GTCCGACAGG

TCTGGCAGCT

TTTTCAAGTG

GTTTGCAGGT

CCAG CC CCAT

CATGTAGCTC

ATTATCTCAA

CTGGAGTGCA

TCTCTI'GCC

TAA'T=GT

TCCTGACCTC

GCCACCACGC

AAAGGATGAG

TGAGGTACCA

TAGT7rGAAA

GCGGGTCGTG

AACGGGAAAG

AATAG'ITAA

TGGGCCCCCC

TGGTGGATCT

CCCGCCAGAC

ATGCCTCGAT

CCCCGAGGCA

GGTCTGGGT

AAGAAAATAA

AAAGGAGAAT

ACCCCGGTCA

AAATC-T

GTGGTGCAAT

TCAGCCTCCT

ATT=AGTAG

AGGTGATCCA

CCGGCCCACT

GCCAGAACTC

GAAGTGTGAG

TGGTGCAGGC

GGGACCAAGC

AACCGACATG

GGAGCTAAAC

GAGGCTGCAC

GGGCCCAGCA

TGCCTGGGTC

CCTCGTGCGA

GCCGGGGTAC

ACGAGAA'ITC

TAGATGACTC

CTACGCTGGT

CGCATGAAGG

CTITGGCTCAC

GAGTAGCTGG

AGACGGGGGT

CCCGCCTG.AG

CTGCCATTGT

TTCCAGAACC

AACGAGGGGG

TTIAGTCITTAA

ACATTGGtCC GCTGCC7FI

AGCCATAAAC

CTGGCTCTCG

AAGACTGTTC

CTGGCGCCTG

CCCACCCTGT

CCTCTAGATT

CCTCATCtI

ACCACACCTC

CCTCAGGACG

CAAAAGCAGG

TTGAGATGGG

TGTAACCTCC

GATTACAGGT

TTCACCATGT

CCTCCCAAAG

CTAAGCCACC

ATCACC=TG

CGTGCTGGGA

GCCTCCAAAG

TAAAGGGGCT

G'TIrACACTG

GGAAACGCCT

GCTGAGGATI'

TCCTCCCGAG

GCGGTCACCT

GTATCCTCCC

CAGGGATGCT

CTCTACTGTA

TACGGCTGGG

TGTTAAAGAG

TCAGAAGCGA

GTCIICCTCT

GCCTCCCAGG

GTGC-ACCACC

TGGCTGGGCT

TGCTGGGAT

TCTGAAAGCA

GGAACCTGCT

TCTTCTCTC

GCCTGGA'rr

GAAGAGCCTG

GTTAT1'T

CCTTCATITCA

GTCACCCCAA

GGAACC'ICT

GGTGCCTACT

TAGACTGAGT

TGCTCTCCAC

AAGTGATF1

GAGATCAGGC

ATCTGGGCCT

ATACTCTGCC

GTTGCCCAGG

TTICAAGTGAT

-CGTACCCAGC

GGTCTCGAAC

ACAGGCATGA

GG 'I=rAACA

GTGAGAGTGC

TGACTATACT

GAGCAGCI=

3417 3477 3537 3597 3657 3717 3777 3837 3897 3957 4017 4077 4137 4197 4257 4317 4377 4437 4497 4557 4617 -33-

AGAAATGCAG

GCTAGGACTG

AGGACCTGCC

AAGTAGCCGC

CAGTAGTCAT

TGAAACTCAC

TATTTAAGTG

TGGTTTTGGG

CATTTCATGT

CCAGCACTCT

GTTCTAGGGC

TGGGCTTI'TA

CGGTGATGCT

CCGCTCAGCG

TGCCACCCGC

TAGACCGTCT

CGCCTGTGTG

TGTACAGTCT

'FrCTGTCTGG

GTGAAACCTT

TTCTCCCAGC

GCAGCCCACA

GTTGATTCT

GCTCAGGTGC

GGGGCACCTC

GCCTGTITCG

CGCGGGTGTG

TGTGTGCCTG

GGAAGGCAAG

GAAATGAGAA

CTTCAGAAGC

GGTGATCCTA

CAAAGGTCTT

CAGCTCTGTT

C.AACTAACTC

ACATATCAGG

CCAAAACTCA

CTGATTCACC

CCTGGCCACA CGCCTGTTCC AAATGGGGAA AGCCGTGCGT GGAGCACACT TTGCAAAGCC GCGAGAAGAA TAT TTTCTAT TTAGTTAAGT ATCACTGATG GTAAAGGCAG ATGAAAAGAA

TGCAGATGGG

CC.ATGGACTC

ACAGAGCC.AG

AGGGGTCCGT

CAGCAAGTGC

GCGCGTTATI

ACAGCGTTC

I I I TAAGT

TGGGTTGAGA

4677 4737 4797 40~57 4917 4977 5037 5097 5157 5217 5220

AAA

INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 1044 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE (xi) SEQUENCE TYPE: protein DESCRIPTION: SEQ ID NO:2: Met Pro Pro Gly Val Asp Cys Pro Met Giu Phe Trp Thr Lys Giu. Giu Tyr Leu Asn Gin Ser Asn Phe Pro 35 Leu Trp His Val Val Asp Phe Lau Leu Pro Thr Gly Val Ser Arg Asn Asn Leu Ser Thr Ile Met Lys Gin Leu Leu Ser Gly Arg Ala Gin Tyr Pro Leu Phe Pro Giu Giu Ala Tyr Val Phe 70 Thr Cys Ile Asn Gin 75 Thr Ala Giu Gin Gin Leu Giu Asp Glu Gin Arg Arg Leu Asp Val Gin Pro Phe Leu Pro Val Leu Arg 100 Leu Val Ala Arg Giu 105 Gly Asp Arg Val Lys Lys Leu 110 -34- Ile Asn Ser Gin Ile Ser Leu Leu Ile Giy Lys Gly Leu His Giu Phe 115 120 125 Asp Gin 145 Al a Thr Asn Thr Aia 225 Leu Cys His Gin Pro 305 Gin Giu Met Pro Pro 385 Ser 130 Phe Trp Trp Vai Lys 210 Thr Gin Gin Leu Ser 290 Ilie Pro Arg Leu Val 370 Arg Leu Cys Leu Gly Lys 195 Asp Vai Val Phe Thr 275 Asn Pro Phe Met Cys 355 Trp Met Cys Giu Gin Pro 180 Phe Val Phe Asn Gin 260 Met Pro Al a Arg Lys 340 Lys Lys Al a Asp Pro Glu 135 Giu Ala Ala 150 Tyr Ser Phe 165 Gly Thr Leu Glu Gly Ser Pro Leu Ala 215 Arg Gin Pro 230 Gly Arg His 245 Tyr Ile Cys Val His Ser Ala Pro Gin 295 Lys Lys Pro 310 Ile Glu Leu 325 Leu Val Val Thr Val Ser Gin Arg Leu 375 Arg Leu Cys 390 Val Al a Pro Arg Glu 200 Leu Leu Glu Ser Ser 280 Vai Ser Ile Gin Ser 360 Giu Phe Asn Arg Leu Leu 185 Giu Met Val Tyr Cys 265 Ser Gin Ser Gin Al a 345 Ser Phe Ala Asp Phe Arg Gin 155 Gin Leu 170 Pro Asn Ser Phe Ala Cys Glu Leu 250 Leu Ile Lys Val Giy 330 Gly Giu Asp Leu Gin 235 Tyr His Leu Pro Ser 315 Ser Leu Val Ile Tyr 395 Arg 140 Gin Glu Arg Thr Al a 220 Pro Gly Ser Al a Arg 300 Leu Lys Phe Ser Asn 380 Ala Al a Leu Pro Ala Phe 205 Leu Giu Asn Gly Me t 285 Al a Trp Val His Val 365 Ile Val Lys Gly Ser Leu 190 Gin Arg Met Trp Ala 175 Leu Val Lys Asp Tyr Tyr pro 255 Leu Thr 270 Arg Asp Lys Pro Ser Leu Asn Ala 335 Gly Asn 350 Cys Ser Cys Asp Ile Glu Cysr Glu 160 Gin Val Ser Lys Thr 240 Leu Pro Giu Pro Giu 320 Asp Giu Giu Leu Lys 400 Ala Lys Lys Ala Arg Ser Thr Lys Lys Pro Lys Lys Thr 465 His His Glu Asp Al a 545 Val Leu Gly Phe Leu 625 Arg Val Ile Thr Gly 450 Asp Pro Ser Ile Leu 530 Leu Al a Ser Ser Gln 610 Asp Lys Pro Al a Gly 435 Glu Ser Val Glu Leu 515 Val Ala Gin Al a Phe 595 T-yr Cys Ile Ser Trp 420 Glu Leu Al a Tyr Cys 500 Glu Trp Arg Met Leu 580 Al a Leu Glu Gly Val 660 Ala Arg Leu Al a Tyr 485 Val Arg Lys Leu Leu 565 Glu Ile Leu Leu His 645 Al a Asn Cys Asn Al a 470 Pro His Arg Leu Leu 550 Tyr Leu Lys Gin Thr 630 Phe Leu Leu Leu Pro 455 Leu Al a Val Gly Arg 535 Leu Leu Leu Ser Leu 615 Lys Leu A!:g Met Tyr 440 Thr Leu Leu Thr Ser 520 His Val Leu Asp Leu 600 Val Phe Phe Phe Leu 425 Met Gly Ile Giu Glu 505 Gly Glu Thr Cys Phe 585 Arg Gin Leu Trp Gly 665 Lys 410 Phe Trp Thr Cys Lys 490 Glu Glu Val Lys Ser 570 Ser Lys Val Leu His 650 Leu Ser Asp Pro Val Leu 475 Ile Glu Leu Gin Trp 555 Trp Phe Leu Leu Asp 635 Leu Ile Lys Tyr Ser Arg 460 Pro Leu Gin Tyr Giu 540 Asn Pro Pro Thr Lys 620 Arg Arg Leu Lys Lys Val 445 Ser Giu Giu Leu Glu 525 His Lys Giu Asp Asp 605 Tyr Ala Ser Glu Al a Asp 430 Pro Asn Val Leu Gin 510 His Phe His Leu Cys 590 Asp Giu Leu Glu Al a 670 Asp Cys 415 Gin Leu Asp Giu Pro Asn Ala Pro 480 Gly Arg 495 Leu Arg Glu Lys Pro Giu Giu Asp 560 Pr6-Vai 575 His Val Giu Leu Ser Tyr Ala Asn 640 Met His 655 Tyr Cys Arg Giy Ser Thr His His met Lys Val Leu Met Lys Gin Gly Giu Ala 675

-"S

-36- Leu Lys 705 Gin Pro Asp Gly Arg 785 Trp Pro Thr Al a Gly 865 Gly Asp Gly Arg Gly 945 GJlu Ser Lys 690 Thr Pro Glu Ala Ser Thr Ser Lys 755 Ser Gly 770 Gin Asp Lys Gin Thr Gly Ile Ala 835 Phe Asn 850 Giu Ala Tyr Cys Asn le His Phe 915 Val Pro 930 Lys Thr Arg Ala Leu Lys Tyr Leu 740 Met Gly Met Glu Asp 820 Asn Lys Leu Val Met 900 Leu Phe Asn Tyr Lys Pro Leu 725 Leu Lys Ser Leu Gly 805 Arg Ile Asp Asp Aia 885 Ile Gly Ile Asn Thr 965 Ala Gin 710 Giu Ala Pro Val Thr 790 Leu Thr Gin Al a Arg 870 Thr Arg Asn Leu Ser 950 Ile ILeu Asn 695 Thr Lys Ala Leu Giu Val Leu Trp 760 Gly Ile 775 Leu Gin Asp Leu Gly Leu Leu Asn 840 Leu Leu 855 Ala Ile Tyr Vai Giu Ser Phe Lys 920 Thr Tyr 935 Gfu Lys Leu Arg Asp Giu Ser Cys 745 Ile Ile Met Arg Ile 825 Lys Asn Giu Leu Gly 905 Thr Asp Phe Arg Phe Leu His 730 Val Met Phe Ile Met 810 Giu Ser TrD Giu Gly 890 Gin Lys Phe Giu His 970 Val Met 715 Leu Giu Tyr Lys Gin 795 Thr Val Asn Leu Phe 875 Ile Leu Phe Val Arg 955 Gly Lys 700 His Gin Gin Ser Asn 780 Leu Pro Vai Met Lys 860 Thr Giy Phe Giy His 940 Phe Leu Leu Ser Leu Cys Ser Pro Cys Thr 750 Asn Gin 765 Giy Asp Met Asp Tyr Gly Leu A-rg 830 Ala Ala 845 Ser Lys Leu Ser Asp Arg His Ile 910 Ile Asn 925 Val Ile Arg Gly Leu Phe Ser Met Leu 735 Phe Gin Asp Val Cys 815 Ser Thr Asn Cys His 895 Asp Arg Gin Tyr Leu 975 Gin Arg 720 Asp Met Al a Leu Len 800 Len Asp Al a Pro Ala 880 Ser Phe Giu Gin Cys 960 His Leu Phe Ala Leu Met Arg Ala Ala Gly 980 985 Lys Asp Ile Gin Tyr Leu Lys Asp Ser 995 1000 Glu Glu Ala Leu Lys His Phe Arg Val 1010 1015 Glu Ser Trp Lys Thr Lys Val Asn Trp 1025 1030 Asp Asn Arg Gin -37- Leu Pro Glu Leu Ser Cys Ser 990 Leu Ala Leu Gly Lys Thr Glu 1005 Lys Phe Asn Glu Ala Leu Arg 1020 Leu Ala His Asn Val Ser Lys 1035 1040 r r r INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 32 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (ix) FEATURE: NAME/KEY: misc_feature LOCATION: OTHER INFORMATION: /note= "n=inosine" (ix) FEATURE: NAME/KEY: misc_feature LOCATION: 24 OTHER INFORMATION: /note= "n=inosine" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: GCAGACGGAT CCGGNGAYGA YHKNAGRCAR GA INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 7 amino acids TYPE: amino acid STPRANDEDNESS: not relevant TOPOLOGY: not relevant (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: Gly Asp Asp Leu Arg Gin Asp 1 -38r r r r r INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 30 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (ix) FEATURE: NAME/KEY: misc feature LOCATION: 16 OTHER INFORMATION: /note= "n inosine" (ix) FEATURE: NAME/KEY: miscfeature LOCATION: OTHER INFORMATION: /note= "n=inosine" (xi) SEQUENCE DESCRIPTION: SEQ ID GCAGACGAAT TCRWRNCCRA ARTCNRYRTG INFORMATION FOR SEQ ID NO:6: SEQUENCE CHARACTERISTICS: LENGTH: 6 amino acids TYPE: amino acid STRANDEDNESS: not relevant TOPOLOGY: not relevant (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: His Ile Asp Phe Gly His 1 INFORMATION FOR SEQ ID NO:7: SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: CATGCTGACC CTGCAGATGA T INFORMATION FOR SEQ ID NO:8: SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: AACAGCTGCC CACTCTCTCG G 21 INFORMATION FOR SEQ ID NO:9: SEQUENCE CHARACTERISTICS: LENGTH: 26 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: GGGCCACATG TAGAGGCAGC GTTCCC 26 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 25 base pairs TYPE: nucleic acid STRANDEDNESS: single D) TOPOLOGY: linear S(ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID GGCCCAGGCA ATGGGGCAGT CCGCC INFORMATION FOR SEQ ID NO:11: SEQUENCE CHARACTERISTICS: LENGTH: 23 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:ll: GATGCGGAAC GGCTGCTCCA GGG 23 INFORMATION FOR SEQ ID NO:12: SEQUENCE CHARACTERISTICS: LENGTH: 23 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: CCAGGGACCA CAGGGACACA GAG 23 INFORMATION FOR SEQ ID NO:13: SEQUENCE CHARACTERISTICS: LENGTH: 65 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: AGTTACGGAT CCGGCACCAT GGACTACAAG GACGACGATG ACAAGCCCCC TGGGGTGGAC TGCCC INFORMATION FOR SEQ ID NO:14: SEQUENCE CHARACTERISTICS: LENGTH: 22 base pairs o• TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid S"(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: CCACATGTAG AGGCAGCGTT CC 22 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 8 amino acids TYPE: amino acid STRANDEDNESS: not relevant TOPOLOGY: not relevant (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID Asp Tyr Lys Asp Asp Asp Asp Lys 1 INFORMATION FOR SEQ ID NO:16: SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: CTGCCATGTT GCTCTTGTTG A 21 INFORMATION FOR SEQ ID NO:17: SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: GAGTTCGACA TCAACATC 18 e

Claims (16)

1. A purified and isolated polynucleotde encoding the amino acid sequence of p1108 set out in SEQ ID NO:2.

2. The polynucleotide of claim 1 wherein said polynucleotide is a DNA.

3. The polynucleotide of claim 1 wherein said polynucleotide is selected from the group consisting of a genomic DNA, a cDNA, and a chemically synthesized DNA.

4. The polynucleotide of claim 2 comprising the DNA sequence set out in SEQ ID NO:1. .10

5. The polynucleotide of claim 1 wherein said polynucleotide is a RNA.

6. A vector comprising a DNA according to claim 2.

7. The vector of claim 6 wherein said DNA is operatively linked to an expression control DNA sequence. g

8. A host cell stably transformed or transfected with a DNA according to claim 2. 15

9. A method of producing p1105 comprising the steps of growing a host cell according to claim 8 in a suitable nutrient medium and isolating the expressed polypeptide from the cell or the nutrient medium.

A purified and isolated polypeptide comprising the p1108 amino acid sequence of SEQ ID NO:2.

11. Hybridoma cell line 208F (HB 12200).

12. The monoclonal antibody produced by the hybridoma cell line of claiml1. COMS ID No: SBMI-01158824 Received by IP Australia: Time 17:09 Date 2005-03-10 MAR. 2005 14:18 WRAY ANJD ASSOCIATES NO. 124 P. -44-

13. A vector according to claim 6 substantially as herein described with reference to any one of the examples.

14.A host cell according to claim 8 substantially as herein described with reference to any one of the examples.

15.A method according to claim 9 subs-tantially as herein described with reference to any one of the examples.

16.A polypeptide according to claim 10 substantially as herein described with reference to any one of the examples. Dated this TENTH day of MARCH 2005. ICOS Corporation Applicant Wray Associates Perth, Western Australia Patent Attorneys for the Applicant COMS ID No: SBMI-01 158824 Received by IP Australia: Time 17:09 Date 2005-03-10