AU745762B2 - Methods for modulating nerve cell function - Google Patents

Description

WO 99/25833 PCT/US98/24327 Methods for Modulating Nerve Cell Function The research carried out in the subject application was supported in part by NIH grant NS18366. The government may have rights in any patent issuing on this application.

INTRODUCTION

Field of the Invention The field of this invention is methods for modulating nerve cell function.

Background In the developing CNS, most growth cones confront the midline at one or multiple times during their journey and make the decision of whether to cross or not to cross. This decision is not a static one but rather changes according to the growth cone's history. For example, in the Drosophila ventral nerve cord, about 10% of the intemeurons project their axons only on their own side, in some cases extending near the midline without crossing it.

The other 90% of the interneurons first project their axons across the midline and then turn to project longitudinally on the other side, often extending near the midline. These growth cones, having crossed the midline once, never cross it again, in spite of their close proximity to the midline and the many commissural axons crossing it. This decision to cross or not to cross is not unique to Drosophila but is common to a variety of midline structures in all bilaterally symmetric nervous systems.

What midline signals and growth cone receptors control whether growth cones do or do not cross the midline? After crossing once, what mechanism prevents these growth cones from crossing again? A related issue concerns the nature of the midline as an intermediate target. If so many growth cones find the midline such an attractive structure, why do they cross over it rather than linger? Why do they leave the midline? One approach to find the genes encoding the components of such a system is to screen for mutations in which either too many or too few axons cross the midline. Such a large-scale mutant screen was previously conducted in Drosophila, and led to the identification of two key genes: commissureless (comm) and roundabout (robo) (Seeger et al., 1993; reviewed by Tear WO 99/25833 PCT/US98/24327 et al., 1993). In comm mutant embryos, commissural growth cones initially orient toward the midline but then fail to cross it and instead recoil and extend on their own side. robo mutant embryos, on the other hand, display the opposite phenotype in that too many axons cross the midline; many growth cones that normally extend only on their own side instead now project across the midline and axons that normally cross the midline only once instead appear to cross and recross multiple times (Seeger et al, 1993; present disclosure). Double mutants of comm and robo display a robo-like phenotype.

How do comm and robo function to control midline crossing? Neither the initial paper on these genes (Seeger et al., 1993) nor the cloning of comm (Tear et al., 1996) resolved this question, comm encodes a novel surface protein expressed on midline cells. In fact, the comm paper (Tear et al., 1996) ended with the hope that future work would help shed some light on the enigmatic function of Comm." A copending application (Robo, A Novel Family ofPolypeptides and Nucleic Acids, by inventors: Corey S. Goodman, Thomas Kidd, Kevin J. Mitchell and Guy Tear, and filed herewith) discloses the cloning and characterization of robo in various species including Drosophila. robo encodes a new class of guidance receptor with 5 immunoglobulin (Ig) domains, 3 fibronectin type III domains, a transmembrane domain, and a long cytoplasmic domain. Robo defines a new subfamily of Ig superfamily proteins that is highly conserved from fruit flies to mammals. The Robo ectodomains, and in particular the first two Ig domains, are highly conserved from fruit fly to human, while the cytoplasmic domains are more divergent. Nevertheless, the cytoplasmic domains contain three highly conserved short proline-rich motifs which may represent binding sites for SH3 or other binding domains in linker or signaling molecules.

For those axons that never cross the midline, Robo is expressed on their growth cones from the outset; for the majority of axons that do cross the midline, Robo is expressed at high levels on their growth cones only after they cross the midline. Transgenic rescue experiments in Drosophila reveal that Robo can function in a cell autonomous fashion, consistent with it functioning as a receptor. Thus, in Drosophila, Robo appears to function as the gatekeeper controlling midline crossing; growth cones expressing high levels of Robo are prevented from crossing the midline. Robo proteins in mammals function in a similar manner in controlling axon guidance.

Here we disclose ectopic and overexpression studies revealing that Comm down-regulates Robo expression, demonstrating that Comm functions to suppress the Robomediated midline repulsion. These results show that the levels of Comm at the midline and Robo on growth cones are tightly intertwined and dynamically regulated to assure that only certain growth cones cross the midline, that those growth cones that cross do not linger at the midline, and that once they cross they never do so again.

SUMMARY OF THE INVENTION The invention provides a method of promoting axon outgrowth of a human neuron determined to have expressed on its surface an amount of a natural human Robo and to be subject to an axon outgrowth repulsion mediated by the Robo, said method comprising the step of contacting the neuron with an amount of a natural Comm polypeptide sufficient to reduce the amount of Robo expressed on the 20 neuron, wherein the Comm is in an amount and under said conditions, sufficient to reduce the amount of Robo expressed on the neuron, wherein the Comm is provided to the neuron exogenously in a pharmaceutically acceptable composition and whereby the amount of Robo expressed on the surface is reduced, thereby reducing the outgrowth repulsion mediated by the Robo and whereby the axon outgrowth of the neuron is promoted.

The invention provides methods and compositions for S 30 modulating the amount of active Robo expressed on a cell.

The general method involves modulating the effective \A -p 3- -e\\meb_f ile\homne$\Pcabral\Keep\speci \14094 .99.doc 1/02/02 amount of a Comm polypeptide in contact with a cell expressing an amount of active Robo polypeptide, whereby the amount of expressed active Robo is modulated inversely with the modulation of the effective amount of Comm polypeptide in contact with the cell. For example, where the effective amount of the Comm polypeptide is increased, the amount of expressed Robo is decreased. The Robo polypeptide is preferably a human, mouse, C. elegans or Drosophila Robo I or II sequence or a polypeptide domain thereof having a Robo-specific activity, and the Comm polypeptide specifically modulates Robo expression and (a) comprises SEQ ID NO:14 or a deletion mutant thereof which specifically modulates Robo expression and/or is encoded by a nucleic acid comprising SEQ ID NO:13 or a nucleic acid which hybridizes with SEQ ID NO:13, preferably under stringent conditions. In particular embodiment, the Comm popypeptide is provided to the cell exogenously in a pharmaceutically acceptable composition.

In other aspect, the inventiojn provides methods of 20 screening for agents which modulate Robo-Comm interactions. These methods generally involve forming a mixture of a Robo-expressing cell, a Comm polypeptide and a candidate agent, and determining the effect of the agent on the amount of Robo expressed by the cell.

e* DETAILED DESCRIPTION OF THE INVENTION The subject methods involve modulating the effective amount of a Comm polypeptide in contact with a cell expressing an amount of active Robo polypeptide, whereby 30 the amount of expressed active Robo is modified inversely with the modulation of the effective amount of the Comm polypeptide in contact with the cell. Robo expression is found to regulate a wide 3a \\melb_files\home$\Pcabral\Keep\speci\14094.99.doc 1/02/02 PW WO 99/25833 PCT/US98/24327 variety of cell functions, including cell-cell interactions, cell mobility, morphology, etc.

Accordingly, the invention provides methods for modulating targeted cell function comprising the step of modulating Robo expression by contacting the cell with a Comm polypeptide.

The targeted Robo polypeptide is generally naturally expressed on the targeted cells.

The nucleotide sequences of exemplary natural cDNAs encoding drosophila 1, drosophila 2, C. elegans, human 1, human 2 and mouse 1 Robo polypeptides are shown as SEQ ID NOS:1, 3, 5, 7, 9 and 11, respectively, and the full conceptual translates are shown as SEQ ID NOS:2, 4, 6, 8, 10 or 12. The targeted Robo polypeptides comprise at least a functional domain of SEQ ID NOS:2, 4, 6, 8, 10 and 12, which domain has Robo-specific amino acid sequence and binding specificity or function. Preferred Robo domains comprise at least 8, preferably at least 16, more preferably at least 32, most preferably at least 64 consecutive residues of one of these SEQ ID NOS. In a particular embodiment, the domains comprise one or more structural/functional Robo immunoglobulin, fibronectin or cytoplasmic motif domains described herein. The subject domains provide Robo-specific antigens and/or immunogens, especially when coupled to carrier proteins. For example, peptides corresponding to Roboand human Robo-specific domains are covalently coupled to keyhole limpet antigen (KLH) and the conjugate is emulsified in Freunds complete adjuvant. Laboratory rabbits are immunized according to conventional protocol and bled. The presence of Robo-specific antibodies is assayed by solid phase immunosorbant assays using immobilized Robo polypeptides of SEQ ID NO:2, 4, 6, 8, 10 or 12. Generic Robo-specific peptides are readily apparent as conserved regions in the aligned Robo polypeptide sequences of Table 1.

Table 1. Sequence Alignment of Robo Family Members: The complete amino acid alignment of the predicted Robo proteins encoded by drosophila robo 1 (D1, SEQ ID NO:2) and Human robo 1 (H1, SEQ ID NO:8) are shown. The extracellular domain of C.elegans robo (CE, SEQ ID NO:6; Sax-3; Zallen et al., 1997), the extracellular domain of Drosophila robo 2 (D2, SEQ ID NO:4), and partial sequence of Human robo 2 (H2, SEQ ID NO:10) are also aligned. The D2 sequence was predicted by the gene-finder program Grail. The position of immunoglobulin domains fibronectin domains the transmembrane domain (TM), and conserved cytoplasmic motifs are indicated. The extracellular domain of rat robo 1 is nearly identical to HI.

WO 99/25833 PTU9/42 PCTIUS98/24327 PMHpENHAIaRSTSTTNNPSrSRSSRMWL1pAWLLLVLVASNGLP 47 Dl m.FNRKTL1CTi.liViQA............... 30 CE mKWKHVPF1VMiS11SS)3NHLFLaQLIPDPEDvErG. NDHGTPIpTSDNDDNSLGYTGS 59 Hi >IG #1 AVrGQYQSpriiehpTdiv-vKknepatinckVegKpEptiewf kdgepvStn. EKKshr 105 DI GENpriiehpMdTTvPknDpFtFncQaegNptptiQwfkdgRELKt. dTGshr D2 pViiehpldVvvsRgSpatincGaK. PStAKiTwykdgQpvltnkEQVNshr 82. CE RLrQEDFPpriVehpSdllvskgepatinckaegRptptiewykGgeRvEtDkDdPRshr 119 Hi >IG #2 VQFKDgAiffYriMQgkkeQ. .dGgEywcvaknRVgQavsrHasiqIavirddfrvepKd 163 Dl iMipAgGlfflkvIhSrReS. .dagTywcEakneFgVaRsrnaTiqlvavlrdEfrLepAN D2 iVlDTgslfLikvNSgkNGKDSdagAyYcvaSneHgeVKsNEGsly~aMlrEdfrvRpRT 141 CE MLlpSgslfflriVhgrkSRP .dEgVyVcvaRnYLgeavsHnasiEvaIlrddfrQwpSd 178 Hi trvaKgeTailecgppKgIpeptLIwIkdgVpddLmSFGASrVrivdggnlLiS~v 223 Dl trvaQgeVaimecgAprgSpepQiswrkNgQTiNL....... VGNKririvdggnlAiQEA D2 vQALGgeMaviecSpprg~pepVVswrkdDKEiRI.QDmP..rYTLHSDgnlIiDPv 195 CE vMvaVgePavrnecQpprgHpeptiswKkdgSpidd........KDEri .TIRggK1MiTYT 230 Hi >IG #3 EPldEgNyKclaQnLvgtresSYaKIivQvkpYfMkepkdqVMLYgQTaTfHcSvggdpP 283 Dl rQsdDgRyqcvVKnVvgtresATaFlKvHvrpFLIRGpQnqtAVvgSsvVf QcrIggdpL D2 DRsdSgTyqcvaNnmvgerVsNPaRiSvFekpKf EQepkdMtvDvgA~vLf DcrvTgdpQ 255 CE rKsdAgKyVcvGTnmvgeresEVaE1TvLerpSfVkRpSnLvTvDDsaEfKcEj~gdpV 290 Hl pKviwkk. .EEgnlpvsrA RiLHdEKsiEiSNltpTdegTyvceaHnNvg 331 Dl pDvlwrrTASGgnmpLRKFSWLHSASGRvHvi EdrsikLDDvtLEdmgeytceaDnAvg D2 pQITwkr. .KNEPmpvTra YiAKdNrGiRiERvQpSdegeyvcYaRnPAg 303 CE pTvRwrk. .DDgELpKsrY Ei RddHl~kiRKvtAGdmgSytcVaEnMvg 337 Hi >IG #,I QiSaRaSilvhappNfTKrpSnKKvGlNgVvQLPcMaSgnppSvfwTkegVSTlMfpn. 388 DI GiTaTGIitvhappKfvlrpKnqLvEIgDEvLfecoaNgHpRpTLYwsVegNSSllLpGy D2 TLeasaHiRvqappSfQTkpAdqSvPAggtAtfecTLVgQpSpaYfwskegoqDiifpsy 363 CE WO 99/25833 WO 9925833PCT/US98/24327 KAeasaTltvqEppHfvVkpRdqVvaigrtvtfQceaTgnpqpalfwRRegsqnllf sy 396 Hi qlvaoQrtvtfPceTKgnpqpavfwQkegsqnllfpn. H2 SsHGrQYvAADgtiQitDvrqedegyyv. cSaFsvvDssTVrVFlQvSS. vD 440 DI RDGRMEVTLTPEGRSV1SiARFAredSgKVvTcNalnAvgsVSsrTVVSvDt..QF D2 VSADGRTK. .vsptgtltiEEvrqVdegAyv. cAGMnSagsslskaAlKvttKAvTGNTP 420 CE qpPQsSsrFsvsQtgdltitnvqrsdVgyyi .cqTlrivagsiITkaYlevtd. .vIA. 450 Hi qpQQPNsrCsvsptgdltitnIqrsdAgyyi .cqalTvagsilAkaQlevtd. H2 >IG erpppiiQlgpAnqtlpKgsVaTipcratgNpSpRiKwFHdgHAvQA. GNRYSi. iqG. 496 Dl eLpppiieqgpvnqtipvKsIVvlpcrTLgTpvpQVswYLdgIpidVqEHERrN~LsDA.. D2 AKpppTieHgHQnqtlMvgsSallpcQaSgKpTpGiswlRdgLpidITd.. sri. sqHST 477 CE drpppViRqgpvnqtVavdgtFvlScVatgSpvpTiLwRkdgVLvSTqd. .sriK. qLeN 507 Hi drpppiiLqgpAnqtlavdgtabcKcKatgDpLpViswikEgFTFPGRd. .PrATiq. eQ H2 >FN #1 Ss1RVDdlq. lsdSgtytciasGeRgeTswAaTltveKpgs. TSLHraAdpstypAppg 553 Di gAlTiSdlqrHEdEgLytcvasnRNgKs swsGylRLDTptNpNiKf FrapEl stypgppg D2 gslHiAd kKPdtgVytciaKneDgestwsaSltveDHtsN .AqfVrMpdpsNFpsSpT 535 CE gvlqiR .YAklGdtgRytciasTPsgeatwsayIEvQeFgVp .VqPPrPTdpNSLIpsAps 565 Hi gTlqiKN rlsdtgtytcvaTSSsgeaswsaVlDvreSgAT. i. .SK1NYdsDLpgpps H2 TpKvLnvsrtsISiRwAKSqEKPGAVgpIi gyTVeyf spdlQTgwIVAaHrvGDtQVti 612 Di kpqMvEKGEnsvtisw... TRSNKVggSSLVgyVieMfGKUETDgwVAvGT-vQNttFtQ D2 QpIIvnvtDtEvEiHw... NAPSTsgaGpitgyiiQyYspdlgQTwFNIPDYvAstEyRi 592 CE kpEvtdvsrnTvtisw qpN'LNsgaTp. tSyiieaf sHASgSswqtvaEwvktEtSAi 62i Hi kpqvtdvtKnsvtlsw. qpGTPGTLpA. SAyiieafsQSVSNswqtvaNHvkttLytV H2 >FN #2 SgiTpgtsyvflvraenTQgisvpsGLsNViktIEA. DfDAASANdlsAarT. llTg 667 Dl TgiLpgVNyFfliraenSHgLsLpsPMsEpitVGrR.. YfNS. .gLdlsEarASilsg D2 kgikpSHsyMf ViraenEkgiGTpsVSsALvttSKPAAQVAlSDKNKv JAIaEK~lTsE 652 CE kglkpnAiylflvraAnAYgisDpsqlsDpvktQDV.1.PTSQgVdHKQVQRE. iGN 675 Hi RglRpntiylfMvralnPkV. svT. q H2 KSvelIDasAinAsavrlEwML-vSADEkyvegLRiHyK. .DaSVPSAQYHSITvMDAsa 725 Dl WO 99/25833 PTU9/42 PCT/US98/24327 DvveiSnasvVDstsMKiTwQI INGkyvegFyVYArQLpNPLNTKyRMLTILNGGGa D2 QLIKiEEVKTinstavriFwKKR. .KLEELiDgyyiKWrGPpRTNDNQyVN. .vTSpsT 707 CE AvLHiHnPTvLSsssIEVHwT.. vDQQSQyiQgyKiLyrPSGaNHGESDWLVFEvRTpAK 733 Hl >FN #3 esFvvGniKkytKyeffLTpf. fETiegQpsnskTaitYedvpsappDNIQiGmYn.. 780 Dl SsCTiTGlVQytLyeffIVpf... .YKsVegKpsnsRIaRtledvpsEApYgMEALLln.. D2 eNYxrvSnlMPFtnyeffVlpYHSGVHsiHgapsnsMDVitAeAPpsLppEDvRiRmlnL. 766 CE NsVviPDiRkGVnyeIKARpf. f NEFQgaDsElkFaKtieEApsappQgvTVSKNDGN 790 Hi QtaGWvRwTpppSQHHngNiYgykiEVSAgnTM..KVIAnMtLnaTtTsvLiNnltt 835 Dl SSaVFLKwkapELKDRHgV1LNyH .vivRgIDtAIHNFSRI1TnVtIdaASPTLvlAnltE D2 tTLRlswkapKAdGlngllKgFQiviv .gQAPNNNR..nItTnERAAsvT1FHiVt 819 CE GtaILvswQpppEdTongr4VQEykV. WCLgnEtR..YHInKtVdGStFsvvIPFiVP 844 Hi gAVysvrLNSFtKagDgpysKpIS1FMdpTHHTHPpR-AJPsGTHDGRHEGqDLTYHNgN 895 Dl gVMyTvGvaaGNnagvgpyCVpATlRldpITKRLDpFINQRDHVND.................. D2 gMTyKIrvAARSnGgvgv ShgTSEVIMNqDTlEKHL .AAQqENESFLYgL 868 CE gIRysvEvaaStGagSgvKsEpoFIQldAhgNPVSpEDqVsiAQQI................ 890 Hi

TM<

iPPGDINPTTHKKTTdYlSGpwLMViVCiVlLvVisAAIsM .vyFkrkhQmTKEiGHLS 954 Dl viTqpwFliiLgAiiaviMLs. .fGAMvFVkrkhmm. .MkQsAL D2 SHVpVIViVaILiIFvViiIAY.CYwRNS.rNSD. .gkDRSF 909 CE SdvVKqp. .AFiagiGAaCWiiLMVfsIwLyRHrkKR. .NgITsTY 932 Hi AflNSKESL.wIDHHRGwRTADTDKD.. 988 Di AGIRKVPSFTFTPTVTYQRGGEAVSSGGRPGLnSEPAQPwLADJ..TwPNTGNNHNDC 990 Hi SgLsEsKiLSHVNSSQ. DGGtDyAEvd.... .TRNL 1024 Di SISCCTAGNgNsDsNiTTYSRPADCIMXynnQLDNKQTNLMLPEStVyGDvdLSNKINEM 1050 Hi CYTOPLASMIC MOTIF #1 KSPDNptpyattMIiGTS......... SSETCTkT.TSISADkDSGT 1068 Dl KtfNSPNLKDGRFVNPSGQptpyattQLiQSNLSNNMNGsGDSGEkHWKPLGQQkQEVA 1110 Hi WO 99/25833 WO 9925833PCTIUS98/24327 DAFAGQVPAVpVV. 1097 D1 PVyIENLKYADVpIYQ~~NGSNSRSTGQHKA 1170 Hi.

CYTOPLASMIC MOTIF #2 InwSEFlppppEhppp. 1124 D1 TPKVPKQGGMnwADLppppAhpppHSNs EEyNISVDESyDqEMpCPVPPARMYLQQDEL 1230 Hi .eSSRKSSKSAGSgISTNQSILNAsIHsSSSGGFsAWGVSPQYAVAcp............. 1171 D1 EEeEDERGPTPPVRgAASS PAAVSYsHQs TATLTPs PQEELQPMLQDcpEETGHMQHQPD 1290 Hi pENVy... .sNp SAVAGGTQNRYQITPTNQHPPQ1 1203 D1 RRRQPVSPPPPPRPISpPHTyGYIsGp1VSDMDTDAPEEEEDEADMEVAKJMQTRR1LLRG 1350 Hi FATTGPGGAVPPNHLP.............. faTQRHaa 1230 D1 LEQTpaSSVGDLESSVTGSMINGWGSASEEDNISSGRSSVSSSDGSFFTDAfaQAVAaa 1410 HI cAQSRAC1~sCdALATPSPmq............... 1261 D1 Aey. agi KVarRQMQDAAGRRHFHASQc PRPTS PVs TdSNI'.SAAVmqKTRPAKKLKMQPG 1469 Hi CYTOPLASMIC MOTIF #3 ppppvpVpEGWYQPVHPNSH. PMHpTS .SNHQIYQCSSECsDHSRSsQS 1307 D1 HLRRETYTDDLppppvpPpAIKSPTAQSKTQLEVRpV\TVPKLPSMDARTDRsSDRKGsSY 1529 Hi QLEeHGSSAkQrgGHHRRrA. pVVQPCMESeN....... ENN D1 KGrEVLDGRQVVDMRTNPGDPREAQeQQNDGkGrgNKAAKrDLPAKMTHLIQeDILPYCRPTF Hi LAEYEQrQYTsDCCNssrEGDTC........... SCSeGSCl. .yAeAgePAPRQMTAKN~T 1395 D1 PTSNNPrDPSsSSSMssrGSGSRQREQANVGRRNIAeMQV1GGy. eRgeDNNEELEETES 1651 H1 Exemplary such Robo specific immunogenic and/or antigenic peptides are shown in Table 2.

Table 2. Immunogenic Robo polypeptides eliciting Robo-specific rabbit polyclonal antibody: Robo polypeptide-KLH conjugates immunized per protocol described below.

Robo Polypeptide, Sequence Immunogenicitv SEQ ID NO:2, residues 68-77 SEQ ID NO:2, residues 79-94 WO 99/25833 PCT/S98/24327 SEQ ID NO:2, residues 95-103 SEQ ID NO:2, residues 122-129 SEQ ID NO:2, residues 165-176 SEQ ID NO:2, residues 181-191 SEQ ID NO:2, residues 193-204 SEQ ID NO:2, residues 244-251 SEQ ID NO:2, residues 274-290 SEQ ID NO:2, residues 322-331 SEQ ID NO:2, residues 339-347 SEQ ID NO:2, residues 407-417 SEQ ID NO:2, residues 441-451 SEQ ID NO:2, residues 453-474 SEQ ID NO:2, residues 502-516 SEQ ID NO:2, residues 541-553 SEQ ID NO:2, residues 617-629 In addition, species-specific antigenic and/or immunogenic peptides are readily apparent as diverged extracellular or cytosolic regions in Table 1. Human Robo-specific antibodies are characterized as uncross-reactive with non-human Robo polypeptides (SEQ ID NOS:2, 4, 6 and 12). Exemplary such human specific peptides are shown in Table 3.

Table 3. Immunogenic Robo polypeptides eliciting human Robo-specific rabbit polyclonal antibody: Robo polypeptide-KLH conjugates immunized per protocol described below (some antibodies show cross-reactivity with corresponding mouse/rat Robo polypeptides).

Robo Polvpeptide, Sequence Immunogenicity SEQ ID NO:8, residues 1-12 SEQ ID NO:8, residues 18-28 SEQ ID NO:8, residues 31-40 SEQ ID NO:8, residues 45-65 SEQ ID NO:8, residues 106-116 SEQ ID NO:8, residues 137-145 WO 99/25833 PCTIUS98/24327 SEQ ID NO:8, residues 174-184 SEQ ID NO:8, residues 214-230 SEQ ID NO:8, residues 274-286 SEQ ID NO:8, residues 314-324 SEQ ID NO:8, residues 399-412 SEQ ID NO:8, residues 496-507 SEQ ID NO:8, residues 548-565 SEQ ID NO:8, residues 599-611 SEQ ID NO:8, residues 660-671 SEQ ID NO:8, residues 717-730 SEQ ID NO:8, residues 780-791 SEQ ID NO:8, residues 835-847 SEQ ID NO:8, residues 877-891 SEQ ID NO:8, residues 930-942 SEQ ID NO:8, residues 981-998 SEQ ID NO:8, residues 1040-1051 SEQ ID NO:8, residues 1080-1090 SEQ ID NO:8, residues 1154-1168 SEQ ID NO:8, residues 1215-1231 SEQ ID NO:8, residues 1278-1302 SEQ ID NO:8, residues 1378-1400 SEQ ID NO:8, residues 1460-1469 SEQ ID NO:8, residues 1497-1519 SEQ ID NO:8, residues 1606-1626 SEQ ID NO:8, residues 1639-1651 SEQ ID NO:10, residues 5-16 SEQ ID NO:10, residues 38-47 SEQ ID NO:10, residues 83-94 SEQ ID NO:10, residues 112-125 SEQ ID NO:10, residues 168-180 SEQ ID NO:10, residues 195-209 WO 99/25833 PCT/US98/24327 SEQ ID NO: 10, residues 222-235 SEQ ID NO:10, residues 241-254 The subject domains provide Robo domain specific activity or function, such as Robospecific cell, especially neuron modulating or modulating inhibitory activity, Robo-ligandbinding or binding inhibitory activity. Robo-specific activity or function may be determined by convenient in vitro, cell-based, or in vivo assays: e.g. in vitro binding assays, cell culture assays, in animals gene therapy, transgenics, etc.), etc. The binding target may be a natural intracellular binding target, a Robo regulating protein or other regulator that directly modulates Robo activity or its localization; or non-naturalbinding target such as a specific immune protein such as an antibody, or a Robo specific agent such as those identified in screening assays such as described below. Robo-binding specificity may be assayed by binding equilibrium constants (usually at least about 107M-', preferably at least about 108 more preferably at least about 10' by the ability of the subject polypeptide to function as negative mutants in Robo-expressing cells, to elicit Robo specific antibody in a heterologous host (e.g a rodent or rabbit), etc.

Similarly, the Comm polypeptide is conveniently selected from Comm polypeptides which specifically modulate Robo expression. Exemplary suitable Comm polypeptides (a) comprise SEQ ID NO:14 or a deletion mutant thereof which specifically modulates Comm expression and/or are encoded by a nucleic acid comprising SEQ ID NO:13 or a nucleic acid which hybridizes with SEQ ID NO:13 under stringent conditions. Suitable deletion mutants are readily screened in Robo down-regulations assays as described below. Preferred Comm domains comprise at least 8, preferably at least 16, more preferably at least 32, most preferably at least 64 consecutive residues of SEQ ID NO:14 and provide a Comm specific activity, such as Comm-specific antigenicity and/or immunogenicity, especially when coupled to carrier proteins as described above for Robo. Exemplary such Comm specific immunogenic and/or antigenic peptides are shown in Table 4.

Table 4. Immunogenic Comm polypeptides eliciting Comm-specific rabbit polyclonal antibody: Comm polypeptide-KLH conjugates immunized per protocol described above.

Comm Polypeptide. Sequence Immunogenicity WO 99/25833 PCT/US98/24327 SEQ ID NO:14, residues 1-11 SEQ ID NO:14, residues 6-17 SEQ ID NO:14, residues 18-34 SEQ ID NO:14, residues 35-44 SEQ ID NO: 14, residues 45-63 SEQ ID NO:14, residues 64-73 SEQ ID NO:14, residues 74-891 SEQ ID NO:14, residues 92-109 SEQ ID NO:14, residues 110-126 SEQ ID NO:14, residues 127-136 SEQ ID NO:14, residues 137-151 SEQ ID NO:14, residues 152-171 SEQ ID NO:14, residues 172-185 SEQ ID NO:14, residues 186-199 SEQ ID NO:14, residues 200-215 SEQ ID NO:14, residues 216-235 SEQ ID NO:14, residues 236-250 SEQ ID NO:14, residues 251-260 SEQ ID NO:14, residues 261-275 SEQ ID NO:14, residues 276-288 SEQ ID NO:14, residues 289-307 SEQ ID NO:14, residues 308-317 SEQ ID NO:14, residues 318-331 SEQ ID NO:14, residues 332-344 SEQ ID NO:14, residues 345-356 SEQ ID NO:14, residues 357-370 SEQ ID NO: 14, residues 41-153 SEQ ID NO:14, residues 117-329 The subject domains provide Comm domain specific activity or function, such as Comm-specific cell, especially neuron modulating or modulating inhibitory activity, Comm- WO 99/25833 PCT/US98/24327 ligand-binding or binding inhibitory activity. Comm-specific activity or function may be determined by convenient in vitro, cell-based, or in vivo assays: e.g. in vitro binding assays, cell culture assays, in animals gene therapy, transgenics, etc.), etc. The binding target may be a natural intracellular binding target, a Comm regulating protein or other regulator that directly modulates Comm activity or its localization; or non-natural binding target such as a specific immune protein such as an antibody, or a Comm specific agent such as those identified in screening assays such as described below. Comm-binding specificity may be assayed by binding equilibrium constants (usually at least about 10 M 1 preferably at least about 108 more preferably at least about 10' by the ability of the subject polypeptide to function as negative mutants in Comm-expressing cells, to elicit Comm specific antibody in a heterologous host (e.g a rodent or rabbit), etc.

In one embodiment, the Comm polypeptides are encoded by a nucleic acid comprising SEQ ID NO:13 or a nucleic acid which hybridizes with a full-length strand of SEQ ID NO:13, preferably under stringent conditions. Such nucleic acids are at least 36, preferably at least 72, more preferably at least 144 and most preferably at least 288 bases in length. Demonstrating specific hybridization generally requires stringent conditions, for example, hybridizing in a buffer comprising 30% formamide in 5 x SSPE (0.18 M NaCI, 0.01 M NaPO 4 pH7.7, 0.001 M EDTA) buffer at a temperature of 42 0 C and remaining bound when subject to washing at 42 0 C with 0.2 x SSPE (Conditions preferably hybridizing in a buffer comprising formamide in 5 x SSPE buffer at a temperature of 42°C and remaining bound when subject to washing at 42 0 C with 0.2 x SSPE buffer at 42 0 C (Conditions II). Exemplary nucleic acids which hybridize with a strand of SEQ ID NO:13 are shown in Table Table 5. Exemplary nucleic acids which hybridize with a strand of SEQ ID NO: 13 under Conditions I and/or II.

Comm Nucleic Acids Hybridization SEQ ID NO:13, nucleotides 1-47 SEQ ID NO: 13, nucleotides 58-99 SEQ ID NO:13, nucleotides 95-138 SEQ ID NO:13, nucleotides 181-220 SEQ ID NO:13, nucleotides 261-299 WO 99/25833 PCT/US98/24327 SEQ ID NO:13, nucleotides 274-315 SEQ ID NO:13, nucleotides 351-389 SEQ ID NO:13, nucleotides 450-593 SEQ ID NO:13, nucleotides 524-546 SEQ ID NO:13, nucleotides 561-608 SEQ ID NO:13, nucleotides 689-727 SEQ ID NO:13, nucleotides 708-737 SEQ ID NO:13, nucleotides 738-801 SEQ ID NO:13, nucleotides 805-854 SEQ ID NO:13, nucleotides 855-907 SEQ ID NO: 13, nucleotides 910-953 SEQ ID NO:13, nucleotides 1007-1059 A wide variety of cell types express Robo polypeptides subject to regulation by the disclosed methods, including many neuronal cells, transformed cells, infected virus) cells, etc. Ascertaining Robo expression is readily effected by antibody staining. Accordingly, indications for the subject methods encompass a wide variety of cell types and function, including axon outgrowth, tumor cell invasion or migration, etc. The target cell may reside in culture or in situ, i.e. within the natural host. For in situ applications, the compositions are added to a retained physiological fluid such as blood or synovial fluid. For CNS administration, a variety of techniques are available for promoting transfer of the therapeutic across the blood brain barrier including disruption by surgery or injection, drugs which transiently open adhesion contact between CNS vasculature endothelial cells, and compounds which facilitate translocation through such cells. Comm polypeptides may also be amenable to direct injection or infusion, topical, intratracheal/nasal administration e.g. through aerosol, intraocularly, or within/on implants e.g. fibers e.g. collagen, osmotic pumps, grafts comprising appropriately transformed cells, etc. A particular method of administration involves coating, embedding or derivatizing fibers, such as collagen fibers, protein polymers, etc. with therapeutic polypeptides. Other useful approaches are described in Otto et al. (1989) J Neuroscience Research 22, 83-91 and Otto and Unsicker (1990) J Neuroscience 10, 1912- 1921. Generally, the amount administered will be empirically determined, typically in the WO 99/25833 PCT/US98/24327 range of about 10 to 1000 tg/kg of the recipient and the concentration will generally be in the range of about 50 to 500 gg/ml in the dose administered. Other additives may be included, such as stabilizers, bactericides, etc. will be present in conventional amounts.

In one embodiment, the invention provides administering the subject Comm polypeptides in combination with a pharmaceutically acceptable excipient such as sterile saline or other medium, gelatin, an oil, etc. to form pharmaceutically acceptable compositions.

The compositions and/or compounds may be administered alone or in combination with any convenient carrier, diluent, etc. and such administration may be provided in single or multiple dosages. Useful carriers include solid, semi-solid or liquid media including water and nontoxic organic solvents. In another embodiment, the invention provides the subject compounds in the form of a pro-drug, which can be metabolically converted to the subject compound by the recipient host. A wide variety of pro-drug formulations for polypeptide-based therapeutics are known in the art. The compositions may be provided in any convenient form including tablets, capsules, troches, powders, sprays, creams, etc. As such the compositions, in pharmaceutically acceptable dosage units or in bulk, may be incorporated into a wide variety of containers. For example, dosage units may be included in a variety of containers including capsules, pills, etc. The compositions may be advantageously combined and/or used in combination with other therapeutic or prophylactic agents, different from the subject compounds. In many instances, administration in conjunction with the subject compositions enhances the efficacy of such agents, see e.g. Goodman Gilman's The Pharmacological Basis of Therapeutics, 9 th Ed., 1996, McGraw-Hill.

In another aspect, the invention provides methods of screening for agents which modulate Robo-Comm interactions. These methods generally involve forming a mixture of a Robo-expressing cell, a Comm polypeptide and a candidate agent, and determining the effect of the agent on the amount of Robo expressed by the cell. The methods are amenable to automated, cost-effective high throughput screening of chemical libraries for lead compounds.

Identified reagents find use in the pharmaceutical industries for animal and human trials; for example, the reagents may be derivatized and rescreened in in vitro and in vivo assays to optimize activity and minimize toxicity for pharmaceutical development. Cell and animal based neural guidance/repulsion assays are described in detail in the experimental section below.

For the purposes of this specification it will be clearly understood that the word "comprising" means "including but not limited to", and that the word "comprises" has a corresponding meaning.

The following experimental section and examples are offered by way of illustration and not by way of limitation.

EXPERIMENTAL

roundabout is Required to Prevent Ipsilateral Axons from Crossing the Midline.

Mutations in robo lead to an increase in the number of embryonic CNS axons in the commissures, coincident with a reduction of the number of axons in the longitudinal connectives as observed with MAb BP102. The two commissures are thicker than normal and partially fuse as they spill over into one another; the longitudinals are thinner and pulled closer together toward the midline. We analyzed the robo mutant phenotype in more detail using the 15 1D4 MAb (anti-Fas II) which at stage 13 stains a subset of growth cones (including aCC, pCC, i" vMP2, MP1, dMP2) and from stages 14-17 stains three major longitudinal axon tracts, including (from medial to lateral) the pCC pathway (pioneered by the pCC growth cone), the MP1 pathway (pioneered by the MP1 growth cone), and a 3rd lateral pathway (Lin et al., 1994; Hidalgo and Brand, 1997). Previous analysis (Seeger et al., 1993) with MAb 1D4 showed that the pCC growth cone, which normally projects anteriorly on its own side near the 20 midline to pioneer the pCC pathway, in robo mutant embryos projects across the midline, fasciculating with its contralateral homologue at the midline. The axon pathway it pioneers the pCC pathway which normally projects longitudinally on its own side near the midline, in robo mutant embryos projects back and forth across the midline. The pCC pathway takes on a circular pattern as it joins with the same pathway from the other side and whirls back and forth across the midline, thus defining the phenotype for which the gene was named.

The fuzzy commissure phenotype observed in robo mutant embryos does not appear to be due to changes in cell fates at the midline or elsewhere in the CNS. All of the midline cells are present, and their fates appear normal as assayed with a variety of different markers 3 (Seeger et al., 1993). All of the commissural and longitudinal axon pathways begin in their normal location, but the longitudinal pathways are pulled closer at the midline as axon bundles circle around the midline, and the commissures become fused and fuzzy as too many axons cross the midline. In contrast, in mutants in which all or some of the midline cells die or fail to properly differentiate, the longitudinal pathways either collapse onto the midline or from the tset form closer together than normal (Kliimbt et al., 1991; Mayer and Niisslein-Volhard, I 16 0F 6 SEP 2001 WO 99/25833 PCT/US98/24327 1988). Thus, the defects observed in robo mutant embryos are not due to changes in cell fates but rather result from defects in axon guidance.

We examined in greater detail the behavior of the pCC growth cone in robo mutant compared to wild type embryos. In wild type embryos, the vMP2 cell body lies embedded at the edge of the midline. The pCC growth cone extends anterior to a point just lateral to vMP2's cell body. The pCC growth cone is then met by the lateral extension ofvMP2's growth cone, and as pCC extends anteriorly and a bit laterally, the vMP2 growth cone wraps around pCC's axons and extends right behind it (Lin et al., 1994). This tight association of vMP2 and pCC is mediated by Fasciclin II (Fas II), a homophilic cell adhesion molecule (CAM) (Grenningloh et al., 1990, 1991) that is expressed from the beginning of axon outgrowth on the cell bodies, axons, and growth cones of a subset of neurons, including pCC and vMP2. In FasH mutant embryos, vMP2 and pCC no longer tightly associate, and their axons fail to fasciculate (Lin et al., 1994).

If the pCC and vMP2 neurons express Fas II, and their growth cones and axons are so attracted to each other in a Fas II-mediated fashion, why does not pCC's growth cone initially extend more medially toward vMP2's cell body which is a short distance away? The answer appears to be because vMP2's cell body is partly embedded in other midline cells, and thus vMP2's cell body is partly surrounded by the putative midline repellent. In robo mutant embryos, pCC's initial trajectory is directly toward vMP2's cell body, where it adheres to vMP2; pCC's growth cone then crosses the midline, fasciculating with its contralateral homologue at the midline.

roundabout is Required to Prevent Commissural Axons from Recrossing the Midline.

The circular pathway taken by the pCC pathway as it crosses back and forth across the midline (as visualized with the anti-Fas II MAb) led us to suggest that some axons were freely recrossing the midline. Although Fas II is expressed on a relatively small subset of axons in the early embryo, and thus we can use it to observe pCC's growth cone abnormal crossing of the midline in robo mutants, the resulting pattern of expression in older embryos becomes quite complicated and it is difficult to resolve precisely which axons are crossing the midline.

To confirm that axons cross and recross the midline freely in robo mutants, we examined the expression of Connectin (Nose et al., 1992), a CAM expressed on a more restricted subset of CNS axons than is Fas II. Connectin is also expressed on motor axons in WO 99/25833 PCT/US98/24327 the segmental nerve. We used the C1.427 MAb to follow Connectin expression (Meadows et al., 1994). Connectin is expressed on the SPI neuron whose cell body lies near the midline just anterior to the anterior commissure, and just medial to the longitudinal tracts. SPl's growth cone normally projects across the midline, fasciculating with the axon of its contralateral homologue. The growth cone then appears to adhere to the cell body of its contralateral homologue, grows around that cell body, and turns to project anteriorly in a medial sub-fascicle of the pCC pathway.

In robo mutant embryos as in wild type embryos, SPl's growth cone extends across the midline, adheres to the axon and then cell body of its contralateral homologue, and turns to project anteriorly. However, as it extends anteriorly into the next segment, it typically moves toward the midline, apparently attracted towards and adhering to the axon of its contralateral homologue just on the other side of the midline. The two SPI axons typically join together around the posterior commissure of the next anterior segment. Sometimes they extend together on the left side of the midline and sometimes on the right side, freely crossing and recrossing the midline while fasciculating with the SP 1 axons originating from both sides of neighboring segments. These results show that in addition to preventing ipsilaterally projecting axons from crossing the midline, Robo also functions to prevent contralaterally projecting axons from recrossing the midline.

roundabout Controls Crossing of the midline in a Dosage Sensitive Manner. Another axonal marker which labels a very small subset of axons is the Tau-f-galactosidase reporter gene expressed under control of the apterous promoter (called apC; Lundgren et al., 1995). In wild type embryos, the apC-tau-lacZ transgene labels three interneurons per abdominal hemisegment, here called the Ap neurons. The Ap neurons have lateral cell bodies and their growth cones initially project towards the midline. Upon nearing the midline, these growth cones then turn to project anteriorly on their own side along the edge of the midline, fasciculating with each other and with their homologues from neighboring segments; in wild type embryos, they never cross the midline in abdominal segments.

In robo mutant embryos, the Ap axons cross the midline in every segment, join up with their contralateral homologues, and often project anteriorly in one discrete longitudinal fasicle.

The Ap fascicle displays two behaviors, usually crossing and recrossing the midline multiple times as a single bundle, or occasionally separating into different bundles of axons which WO 99/25833 PCT/US98/24327 project on one side or the other and independently cross the midline.

We observed a partially penetrant Ap axon phenotype in robo heterozygous embryos.

In wild type, none of the 6 Ap axons in each segment ever cross the midline; in robo homozygous mutants, all 6 Ap axons cross the midline. In robo heterozygous mutant embryos, one of the Ap axons is observed crossing the midline in about 30% of segments, which accounts for a penetrance of about 5% of all Ap axons (Table This partially penetrant crossing with 50% of robo indicates a dose requirement for the robo gene product in these axons. Moreover, since the Ap axons extend midway through axonogenesis, once many axon pathways have already been pioneered, these results indicate that robo is required throughout axonogenesis, not just to establish the initial projections of the pioneer axons.

Underexpression of Comm Leads to Increased Levels of Robo Protein. The commissureless (comm) mutant has a complementary phenotype to that of robo in that too few axons cross the midline (Seeger et al., 1993). When visualised with MAb BP102, the axon commissures are noticeably absent. In certain hypomorphic comm alleles comm 7 Tear et al., 1996), the commissures are not completely absent, but rather partial and highly abnormal axon commissures do form in a few segments (particularly in the thorax). We examined the expression of Robo protein in these comm hypomorphic alleles using the 13C9 anti-Robo MAb (Kidd et al, 1997). Normally, Robo is expressed at very low levels on commissural axons and at high levels on longitudinal axons. In comm mutant embryos, Robo expression in the longitudinal tracts appears even higher than normal. Interestingly, in comm hypomorphic alleles, the occasional thin commissures express Robo protein at levels that are higher than normally seen in the commissures and closer to what is typically seen in the longitudinal tracts. This result was our first hint that Comm protein might function by suppressing Robo expression on commissural axons. Previous studies had shown that comm encodes a novel transmembrane protein that is expressed by the midline glia and that is apparently transferred to commissural axons (Tear et al., 1996). Given these results, we wondered whether expression of comm in all neurons might reduce Robo levels and lead to a robo phenotype.

Overexpression of Comm generates a robo-Like Phenotype. To test the hypothesis that increased expression of comm might lead to a robo-like phenotype, we used the UAS- GAL4 system (Brand and Perrimon, 1993) to change the pattern of comm expression. We WO 99/25833 PCT/US98/24327 generated UAS-comm transgenic lines and drove expression pan-neurally using the sca-GAL4 line. Since flies carrying a copy of both the driver and effector transgenes are viable, we used them as parents and examined their progeny. A continuous range of robo-like phenotypes was observed with MAbs BP102 and 1D4. The range of phenotypes reveals the comm gain-offunction phenotype to be dosage sensitive, as the severity increased in embryos carrying two copies of both transgenes as compared to embryos carrying only one copy of each.

Superficially, the robo phenotype can be mimicked by mutants causing inappropriate migration or cell death of the midline glia, both of which result in fuzzy commissures (Klmbt et al., 1991). However, such phenotypes are not visible until midway through axonogenesis, and are easily detected by examining early axon behavior. In addition, we stained the embryos with a MAb raised against 8H11, a protein expressed specifically by the midline glia, and confirmed that the midline glia are still present In the embryos ectopically expressing comm, Fas II positive axons, suich as pCC, were found to behave identical to how they behave in robo mutants. When Comm is overexpressed, the pCC growth cone extends towards the vMP2 cell body, and then across the midline, just as it does in a robo mutant. In the comm gain-of-function, the pCC fascicle freely crosses and midline and forms the same circles or whirls as it does in the robo loss-offunction.

Overexpression of Comm Leads to Reduced Levels of Robo Protein. Having established that the comm overexpression generates a bona fide robo-like axon guidance phenotype, we next examined Robo expression in these embryos using the anti-Robo MAb 13C9. The sca-GAL4 driver begins driving expression in the neuroepithelium before axon outgrowth (-stage 9) has begun and switches off by stage 13; sca-GAL4 does not express in the epidermis. In wild type embryos, the pattern of Robo protein expression begins in the neuroepithelium, as well in some lateral epidermal stripes, but is conspicuously absent from the midline region. In comm gain-of-function embryos, Robo expression in the neuroepithelium is greatly reduced or absent, while the epidermal expression outside the nervous system is maintained. This same pattern can be observed around the time when the first growth cones are extending. In wild type embryos during stages 12 and 13, no Robo is seen at the midline, but there is a high level of Robo expression on ipsilaterally projecting growth cones such as pCC and a significant level throughout the neuroepithelium. In contrast, WO 99/25833 PCT/US98/24327 in comm gain-of-function embryos, the pCC growth cone lacks Robo protein and the neuroepithelium expresses greatly reduced levels of Robo.

The dramatic reduction in the levels of Robo were observed until about stage 14, coincident with the sca-GAL4 driver ceasing expression. In the sca-GAL4; UAS-robo embryos, Robo protein begins to accumulate throughout the CNS after stage 14, reaching significant levels (but still below wild type) by stage 16. Interestingly, in these transgenic embryos, although we observe some Robo-positive axons in the commissures at later stages, Robo expression remains higher in longitudinal tracts. We interpret the Robo-positive axons in the commissures as later axons following misguided pioneer axons; fasciculation with the pioneers allows these Robo-positive axons to cross the midline in spite of modest levels of Robo.

The elav-GAL4 line also expresses pan-neurally but only in post-mitotic neurons; it begins driving expression ofUAS transgenes during stage 12 and remains expressed throughout the rest of embryogenesis. Ectopic expression of comm by elav-GAL4 led to a less severe version of the robo phenotype. We interpret this weaker phenotype as being due to either a weaker overall level of Comm expression or because increased Comm initiates after the pioneers have already established the initial pathways. In addition, since sca-GAL4 drives expression in midline glia, the source of normal comm expression, while elav-GAL4 does not drive expression in the midline glia, the possibility exists that the less severe phenotype of the elav transgene is due to this lack of midline comm expression.

To address this issue, we attempted to increase the level of Comm specifically at the midline using multiple GAL4 lines, including sim-GAL4, slit-GAL4, F63-GAL4, and p52A- GAL4, all of which express at the midline during the period of commissure formation. When UAS-comm was expressed by any of these four lines, only very weak BP102 phenotypes were observed, although because most of these inserts are homozygous lethal, we have not been able to easily increase the dosage with these lines to comparable levels as with the sca-GAL4 line. None of these gain-of-function phenotypes was as strong as that observed with the sca- GAL4 line. We also cannot rule out that these differences in the strength of the gain-offunction phenotypes using different GAL4 lines do not reflect differences in timing, levels of expression, or location of expression within the CNS.

We conclude that the normal function of comm is to down-regulate the low level of WO 99/25833 PCT/US98/24327 Robo expression present on commissural axons, thereby allowing them to cross the midline.

Increasing levels of Comm in the CNS lead to more severe robo-like phenotypes, indicating a dosage sensitivity. This sensitivity to dosage is also reflected in the behavior ofAp axons in robo heterozygotes, thus showing a parallel dosage sensitivity by either decreasing Robo or increasing Comm.

These results indicate that control of Robo expression is complex and highly regulated from transcription to translation to post-translational. We show that there is an inverse correlation between Comm expression and Robo expression. In wild type embryos, Comm is expressed at the midline, and Robo expression is very low on commissural axons crossing the midline. In comm hypomorphic mutant embryos, those few axons that do cross the midline now express higher levels of Robo protein. In comm gain-of-function embryos (using transgenic constructs that drive over- and ectopic expression of comm), the overall levels of Robo are dramatically decreased wherever increased Comm expression coincides with Robo expression. Furthermore, using certain GAL4 lines that drive transient comm expression, we observe that once Comm disappears in older embryos, Robo protein expression begins to increase towards its normal levels. Thus, Comm down-regulates Robo expression in a very tight fashion.

Only a small amount of Comm is normally expressed at the midline. The midline also expresses high levels of a putative repellent that is the ligand for the Robo receptor. Growth cones that express high levels of Robo, such as ipsilaterally projecting growth cones from the outset or commissural growth cones once they cross the midline, are relatively immune to significant down-regulation by the normally low levels ofmidline Comm and thus are prevented from crossing the midline. Only abnormally high levels of Comm (using transgenes that drive overexpression) are sufficient to down-regulate this Robo expression to a level that allows these growth cones to cross the midline. In contrast, growth cones that normally express lower levels of Robo those commissural growth cones that cross the midline in the presence of Comm) are highly sensitive to Comm, in that the normal low levels of Comm can further reduce their levels of Robo and thus allow them to cross the midline. In the absence of Comm, these growth cones can not cross the midline, due to their low levels of Robo; in the robo; comm double mutant they all freely cross.

Genetic Stocks. All robo alleles were isolated on chromosomes deficient for Fasciclin WO 99/25833 PCT/US98/24327 III as described in Seeger et al., 1993. The robo phenotype is independent of the absence of FasIII Protein Immunocytochemistry. Immunocytochemistry was performed as described by Patel (1994). For anti-Robo staining, MAb 13C9 was diluted 1:10 in PBS with 0.1% Tween- 20, and the embryos were fixed and cracked so as to minimize exposure to methanol. The presence of triton and storage of embryos in methanol were both found to destroy the activity of MAb 13C9. For anti-Connectin staining with MAb C1.427, the embryos were fixed in 3.7% formaldehyde/PEM buffer (100mM PIPES, 2mM EGTA, ImM MgSO 4 C1.427 was diluted 1:10 in PBS with 0.1% Triton. The apterous-tau-lacZ embryos were hand devitellinized and dissected on poly-lysine coated slides and subsequently fixed for minutes with 3.7% formaldehyde; rabbit anti-P-galactosidase (Cappell) was used at 1:10,000 and biotinylated anti-rabbit secondary was used at 1:1000 and enhanced with the Vectastain Elite ABC kit (Vector Laboratories).

Transformation of Drosophila, robo rescue and overexpression. The comm cDNA was inserted as a 1.7kb XhoI-Xbal fragment into the Xhol and Xba sites of pUAST (Brand and Perrimon, 1993). Transformant lines were generated and mapped by standard procedures.

References Brand, A. H. and Perrimon, N. (1993) Development 118, 401-415.

Hidalgo, and Brand, A.H. (1997) Development 124, 3253-3262.

Kidd, Brose, Mitchell, Fetter, Tessier-Lavigne, Goodman, and Tear, G. (1997). Roundabout controls axon crossing of the CNS midline and defines a new subfamily of evolutionarily conserved guidance receptors. Cell, in review.

Klimbt, Jacobs, J. and Goodman, C. S. (1991) Cell 64, 801-815.

Lundgren, et al. (1995) Development 121, 1769-1773.

Mayer, U. and Niisslein-Volhard, C. (1988) Genes Dev. 2, 1496 1511.

Meadows, et al. (1994) J. Cell Sci. 107, 321-328.

Nose, Mahajan, V. and Goodman, C. S. (1992) Cell 70, 553-567.

Patel, N. H. (1994) In "Methods in Cell Biology, Vol 44. Drosophila melanogaster: Practical Uses in Cell Biology" S. B. Goldstein and E. Fyrberg, eds) Academic Press, New York.

Sambrook, Fritsch, E. and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual (Cold Spring Harbor, New York: Cold Spring Harbor Laboratory).

Seeger, Tear, Ferres-Marco, D. and Goodman C.S. (1993) Neuron 10, 409 426.

Tear et al. (1996) Neuron 16, 501 514.

All references, including any patents or patent applications, cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. The discussion of the references states what their authors assert, and the applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art, in Australia or in any other country.

All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

24 C 6 oo• C /f SP 3001 SEP 2001 EDITORIAL NOTE NO.14094/99 Sequence listing pages 1-25 is to be inserted after the description and before the claims.

WO 99/25833 PCT/US98/24327 SEQUENCE LISTING GENERAL INFORMATION: APPLICANT: Goodman, Corey S.

Kidd, Thomas Russel, Claire Tear, Guy Mitchell, Kevin (ii) TITLE OF INVENTION: Methods for Modulating Nerve Cell Function (iii) NUMBER OF SEQUENCES: 14 (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: SCIENCE TECHNOLOGY LAW GROUP STREET: 75 DENISE DRIVE CITY: HILLSBOROUGH STATE: CALIFORNIA COUNTRY: USA ZIP: 94010 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: OSMAN, RICHARD A REGISTRATION NUMBER: 36,627 REFERENCE/DOCKET NUMBER: B98-006 (ix) TELECOMMUNICATION INFORMATION: TELEPHONE: (650) 343-4341 TELEFAX: (650) 343-4342 INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 4188 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:

ATGCATCCCA

CCATCTCGCA

GTGGCCAGCA

CATCCCACGG

GGCAAGCCGG

AAGAAATCGC

GGCAAGAAGG

GCCGTTAGTC

CCCAAAGACA

GGCATTCCAG

GCCATGTCGT

AGCAATGTGG

ACCCGCGAGA

CCCAAGGATC

TGCATCCCGA

GTCGGAGCAG

ATGGCCTGCC

ATCTGGTCGT

AACCCACCAT

ACCGCGTCCA

AGCAGGACGG

GCCATGCCTC

CGCGAGTGGC

AGCCAACGCT

TTGGCGCCAG

AGCCCATTGA

GCAGCTATGC

AGGTGATGCT

AAACCACGCC

CAGGATGTGG

AGCAGTCAGA

TAAGAAGAAT

TGAGTGGTTT

GTTCAAGGAC

CGGAGAGTAC

CCTCCAGATA

CAAAGGCGAG

GATTTGGATA

CTCCCGCGTT

TGAGGGCAAC

CAAGCTGATT

CTACGGCCAG

ATCGCCCGGA

CTCCTGCCCG

GGCCAGTACC

GAACCCGCCA

AAGGATGGCG

GGCGCCCTCT

TGGTGCGTGG

GCTGTTTTGC

ACGGCTCTGC

AAGGACGGCG

CGAATTGTGG

TACAAGTGCA

GTCCAGGTCA

ACAGCCACTT

GCACGAGCAC

CCTGGCTGCT

AATCGCCACG

CGCTCAACTG

AACCCGTCAG

TCTTTTACAG

CCAAGAACCG

GCGACGATTT

TGGAGTGTGG

TTCCCTTGGA

ACGGTGGCAA

TTGCCCAGAA

AACCATACTT

TCCACTGCTC

CACTAATAAC

CCTCGTCCTG

TATCATCGAG

CAAAGTGGAG

CACCAACGAA

GACAATGCAA

AGTGGGCCAG

TCGCGTGGAG

GCCGCCCAAA

CGACCTGAAA

CCTGCTGATC

TCTGGTAGGC

TATGAAGGAG

AGTGGGCGGT

120 180 240 300 360 420 480 540 600 660 720 780 840 SUBSTITUTE SHEET (RULE 26) WO 99/25833 WO 9925833PCTIUS98/24327

GATCCGCCGC

CGAATCCTTC

ACCTATGTCT

GTCCACGCTC

GTTGTCCAAC

GAAGGAGTAT

GATGGAACTC

GCTTTCAGTG

GAGCGTCCAC

GTTGCTACTT

GATGGACATG

GTCGATGACC

GGAGAAACTT

CGGGCAGCTG

AGTCGCACCA

CCAATCATTG

GCTGCCCATC

TATGTGTTCC

AATGTTATTA

GCTCGAACTT

AGTGCCGTTA

CTGCGCATAC

ATGGATGCCT

TTCTTCCTAA

CTCACCTATG

CAAACAGCCG

TATGGCTACA

CTTAATGCTA

GTGAGGTTGA

TTCATGGACC

GGGCGACATG

ATTAATCCCA

CTGGTCTGCA

AAGCGCAAGC

ATAACCGCAT

CGAACTGCCG

AACAGCAGTC

ACCCGTAACC

GCCACCACTA

AGTGCCGATA

CCAGCGGTTC

TCAGAGTTTC

CAAGGATCTC

AATCAAAGCA

GGAGTATCGC

TCGGCAGTGG

CCACAGTTAC

CTGCCATTTG

CGATGTGCCC

CAACCCCCAC

CACCCGATGC

GATCACTCGA

AGTGCCAAAC

GAGAGCGAGA

TGCAATAGCT

CGAAAGTGTT

ACGACGAGAA

GCGAGGCACA

CGCCGAACTT

TACCTTGCAT

CCACTCTTAT

TGCAGATTAC

TAGTCGATTC

CTCCGATTAT

TACCCTGTCG

CCGTACAAGC

TTCAACTAAG

CCTGGGCTGC

ATCCTAGCAC

GCATTAGTCT

GATACACTGT

GAGTCGGCGA

TAGTTAGAGC

AAACCATTGA

TGCTGACAGG

GACTTGAGTG

ACTATAAGGA

CTGCAGAATC

CACCCTTTTT

AAGATGTTCC

GTTGGGTGCG

AGATTGAGGT

CCACCACATC

ACTCCTTTAC

CCACCCATCA

AGGGACAGGA

CCACTCATA.A

TCGTTCTTCT

ATCAAATGAC

TAAATATCAA

ATACTGACAA

AATCCAACTA

TTACCACCTT

TGATCATTGG

AGGACTCGGG

CTGTTGTCAA

TACCCCCGCC

CTGAATCTTC

TTCTGAACGC

CCCAATATGC

CTGGCGGCAC

CGGCCTACTT

CCACACAGCG

AAAGCCGCGC

CGCCAGTTCC

ACCCGACCTC

GGAGCTCGCA

AACGCGGAGG

ACGAGAACAT

CCCGCGAGGG

GTGGAAAAAG

AAGTTTAGAG

CAACAATGTC

TACGAAAAGA

GGCCTCCGGA

GTTCCCAAAT

GGATGTGCGG

CTCTACAGTA

TCAAATCGGA

GGCCACTGGA

GGGCAATCGA

TGACTCTGGT

CACACTAACG

TTATCCTGCT

TCGTTGGGCT

AGAGTACTTC

CACTCAAGTC

TGAGAATACT

GGCAGATTTC

AAAGTCGGTG

GATGCTCCAC

TGCCAGTGTA

GTTTGTGGTG

TGAGACAATT

CTCCGCACCA

TTGGACTCCG

CAGCGCCGGT

TGTGCTCCTA

CAAGGCAGGA

TGTGCATCCG

TCTCACGTAT

AAAGACCACT

AGTCCTGGTT

CAAGGAATTG

TAGCAAAGAG

AGACTCAGGA

CAATAACTCC

CTACAATTGT

TACCTCTTCC

AACTCATTCG

ATCCAACTAT

GCCAGAACAC

GCGGAAGAGC

ATCCATACAC

TGTCGCCTGT

CCAGAACCGC

TGCCACCACG

TCATGCAGCC

CTGCAACAGC

CGTACCCGAG

CTCCAACCAC

GAGTCACAAG

ACACCACCGT

GCTGGCGGAG

GAGGAGGGCA

ATATCCAACA

GGTCAGATCA

CCCAGTAACA

AACCCTCCGC

AGTTCGCACG

CAGGAAGACG

CGGGTTTTCC

CCTGCCAATC

AATCCCAGTC

TACAGCATCA

ACCTACACCT

GTGGAAAAAC

CCTCCAGGAA

AAAAGCCAAG

AGTCCGGATC

ACTATCTCGG

CAGGGTATTT

GATGCAGCTT

GAGCTAATAG

GTGAGCGCTG

CCATCCGCAC

GGAAACCTTA

GAAGGACAGC

CCGGATAACA

CCACCCTCCC

AACACCATGA

AATAACCTAA

GATGGACCTT

CCACGGGCAC

CATAACAATG

GACTACCTAT

ATTTCGGCGG

GGTCACTTAA

AGCCTTTGGA

TTAAGCGAAT

GATGGAGGAA

CGCAAGAGCC

AGTGAGACCT

CCCTATTCTG

CTTCAGTATC

CCACCTCCGT

TCCAAAAGCG

AGCAGCTCCT

CCACCGGAAA

TATCAGATAA

GGTCCAGGAG

AGCGAGTACC

TGCGATGCCT

GGCTGGTACC

CAGATCTACC

CGGCAGCTGC

CGACGAGCCC

TACGAGCAGC

ATATTCCGGT

TAACGCCCAC

GCGCTAGGGC

AGAAAGTGGG

CGTCTGTATT

GAAGGCAGTA

AAGGCTACTA

TGCAAGTCAG

AAACACTGCC

CCCGTATCAA

TCCAAGGAAG

GCACTGCATC

CCGGTTCTAC

CACCTAAAGT

AGAAACCCGG

TGCAAACTGG

GTCTCACTCC

CTGTGCCTTC

CTGCCAATGA

ATGCCTCGGC

ATGAGAAATA

AGTATCACTC

AGAAGTACAC

CCAGTAACTC

TTCAGATTGG

AGCACCACAA

AGGTGCTGGC

CCACCGGAGC

ACTCCAAACC

ATCCAAGCGG

GCAACATACC

CTGGACCGTG

CTATTTCGAT

GTGTGGTCAG

TAGACCATCA

CGAAGCTACT

CCGATTATGC

CCGATAATCC

GCACCAAGAC

ACGCATTTGC

CGGTTGAACC

CTTCTACCTA

CAGGTTCCGG

CGGGCGGCTT

ACGTTTATAG

CGCCCACAAA

GAGCTGTACC

AGGCTGGACT

TGGCCACACC

AACCGGTGCA

AGTGCTCCTC

AGCTCGAGGA

CGGTGGTGCA

GCCAGTACAC

GTCCAGAGCG

CGATGAGGGC

TTCTCTTATA

ACTAAATGGG

CTGGACCAAG

TGTGGCTGCC

TGTGTGTTCC

CTCGGTAGAC

CAAGGGATCA

GTGGTTCCAC

CTCACTGAGA

TGGCGAACGA

ATCTCTTCAC

CCTGAATGTC

AGCTGTGGGC

TTGGATTGTG

TGGCACTTCG

CGGCTTATCA

TTTGTCAGCA

TATCAATGCT

CGTAGAGGGC

GATCACTGTT

CAAGTATGAG

CAAGACAGCC

CATGTACAAC

TGGCAATTTG

CAATATGACT

TGTGTACAGC

GATATCACTA

CACCCATGAT

ACCTGGCGAC

GCTAATGGTG

GGTCTACTTC

TGACAACGAA

TCGTGGATGG

ATCCCACGTT

AGAAGTTGAC

CACGCCGTAC

AACATCTATA

CGGTCAGGTG

GATCAACTGG

TGGATACGCA

CATTTCTACA

TTCAGCTTGG

CAATCCGCTG

CCAACATCCG

ACCCAACCAC

GAATGCAGCG

CTCGCCCATG

TCCCAATAGC

CGAGTGCTCG

GCACGGCAGC

GCCGTGCATG

CAGCGATTGC

900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 3000 3060 3120 3180 3240 3300 3360 3420 3480 3540 3600 3660 3720 3780 3840 3900 3960 4020 4080 4140 CGACACCTGC TCCTGCAGCG AGGGATCCTG TCTTTACGCC SUBSTITUTE SHEET (RULE 26) WO 99/25833 PTU9/42 PCT[US98/24327 GAGGCGGGCG AGCCGGCGCC TCGTCAAATG ACTGCTAAGA ACACCTAA INFORMATION FOR SEQ ID NO:2: Wi SEQUENCE CHARACTERISTICS: LENGTH: 1395 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Met His Pro Met His Pro Giu Asn His Ala Ile Ala Arg Ser Thr Ser 4188 Thr Pro Val1 Leu Gly Ser Leu Giu His 145 Pro Gly Gly Arg Pro 225 Thr Phe Thr Lys Asp 305 Thr Ala Asn Trp Gly Val Pro Asn Phe 115 Trp, Ser Asp Pro Pro 195 Arg Asp Glu Lys His 275 Glu Lys Val1 Leu Asn Leu Gin Lys Glu Giu 100 Tyr Cys Leu Thr Lys 180 Leu Ile Glu Ser Giu 260 Cys Glu Ser Cys Ile Ser Leu Gin Asn 70 Thr Lys Thr Ala Ile 150 Val Ile Asp Asp Asn 230 Tyr Lys Val1 Asn Glu 310 Ala His Ser Leu 40 Pro Pro Giu His Gin 120 Asn Val Lys Glu Lys 200 Gly Lys Lys Gin Gly 280 Pro Ser Asn Pro Arg 25 Val1 Arg Ala Trp Arg 105 Gly Arg Leu Gly Pro 185 Ala Asn Cys Leu Val1 265 Asp Val1 Asn Asn Pro 345 Val Ser Ser Ala Ser Ile Ile Thr Leu 75 Phe Lys Val Gin Lys Lys Val Gly Arg Asp 155 Giu Thr 170 Thr Leu Met Ser Leu Leu Ile Ala 235 Ile Val 250 Met Leu Pro Pro Ser Arg Ile Thr 315 Val Gly 330 Asn Phe Val Gin Arg Asn Glu Asn Asp Phe Giu Gin 140 Asp Ala Ile Phe Ile 220 Gin Gin Tyr Pro Ala 300 Pro Gin Thr Leu Met Gly His Cys Gly Lys Gin 125 Ala Phe Leu Trp Gly 205 Ser Asn Val1 Gly Lys 285 Arg Thr Ile Lys Pro Trp Leu Pro Lys Glu Asp 110 Asp Val Arg Leu Ile 190 Ala Asn Leu Lys Gin 270 Val Ile Asp Ser Arg 350 Cys Leu Pro Thr Val Pro Gly Gly Ser Val Giu 175 Lys Ser Val Val1 Pro 255 Thr Leu Leu Giu Ala 335 Pro Met Leu Ala Asp Giu Val1 Ala Gly Arg Giu 160 Cys Asp Ser Giu Gly 240 Tyr Ala Trp His Gly 320 Arg Ser Ala 340 Asn Lys Lys Val Gly Leu Asn Gly SUBSTITUTE SHEET (RULE 26) WO 99/25833 PTU9/42 PCT/TJS98/24327 Ser Thr 385 Asp Tyr Pile Ile Pro 465 Asp Ser Thr Leu Pro 545 Ser Giy Asp Gin Vai 625 Asn Asp Ile Leu Tyr 705 Met Thr Gin Aia Trp 785 Giy 370 Leu Giy Vali Leu Giy 450 Cys Giy Ser Cys Thr 530 Ser Arg Aia Leu Vai 610 Arg Vali Leu Asp His 690 Lys Asp Lys Pro Pro 770 Val1 355 Asn Met Thr Cys Gin 435 Pro Arg His Leu Thr 515 Val1 Thr Thr Vali Gin 595 Thr Aia Ile Ser Aia 675 Val Asp Aila Tyr Ser 755 Pro Arg Pro Pro Gin 405 Aia Ser Asn Thr Val1 485 Vali Ser Lys Pro Ile 565 Pro Giy Ser Asn Thr 645 Ala Ala Aia Ser Ala 725 Phe Ser Asn Thr Pro Asn 390 Ile Phe Ser Gin Gly 470 Gin Asp Giy Pro Al a 550 Ser Ile Trp Giy Thr 630 Ile Arg Ile Asp Val 710 Glu Phe Lys Ile Pro 790 Ser 375 Ser Thr Ser Vai Thr 455 Asn Ala Asp Giu Giy 535 Pro Leu Ile Ile Leu 615 Gin Glu Thr Asn Giu 695 Pro Ser Leu Thr Gin 775 Pro 360 Vali Ser Asp Vali Asp 440 Leu Pro Gly Leu Arg 520 Ser Pro Arg Gly Val 600 Thr Giy Ala Leu Ala 680 Lys Ser Phe Thr Ala 760 Ile Pro Phe His Val1 Val1 425 Giu Pro Ser Asn Gin 505 Gly Thr Gly Trp Tyr 585 Ala Pro Ile Asp Leu 665 Ser Tyr Ala Val1 Pro 745 Leu Giy Ser Trp Gly Arg 410 Asp Arg Lys Pro Arg 490 Leu Glu Ser Thr Ala 570 Thr Ala Gly Ser Phe 650 Thr Al a Val Gin Val1 730 Phe Thr Met Gin 365 Glu Tyr Asp Thr Pro 445 Val1 Lys Ile Ser Trp 525 Arg Val1 Gin Tyr Val 605 Tyr Ser Ala Ser Leu 685 Leu Ser Leu Thr Asp 765 Gin Asn Gly Val1 Giu Val1 430 Ile Ala Trp Ile Giy 510 Al a Al a Leu Glu Phe 590 Gly Val1 Gly Ser Val1 670 Giu Arg Ile Lys Ile 750 Vali Thr Gly Ser Ala 400 Tyr Val1 Gin Leu His 480 Gly Tyr Thr Asp Val 560 Pro Pro Thr Leu Ser 640 Asn Leu Met His Val1 720 Tyr Gly Ser Gly Leu 800 SUBSTITUTE SHEET (RULE 26) WO 99/25833 PCT/US98/24327 Tyr Gly Tyr Lys Ile Glu Val Ser Ala Gly Asn Thr Met Lys Val Leu 805 810 815 Ala Asn Met Thr Leu Asn Ala Thr Thr Thr Ser Val Leu Leu Asn Asn 820 825 830 Leu Thr Thr Gly Ala Val Tyr Ser Val Arg Leu Asn Ser Phe Thr Lys 835 840 845 Ala Gly Asp Gly Pro Tyr Ser Lys Pro Ile Ser Leu Phe Met Asp Pro 850 855 860 Thr His His Val His Pro Pro Arg Ala His Pro Ser Gly Thr His Asp 865 870 875 880 Gly Arg His Glu Gly Gin Asp Leu Thr Tyr His Asn Asn Gly Asn Ile 885 890 895 Pro Pro Gly Asp Ile Asn Pro Thr Thr His Lys Lys Thr Thr Asp Tyr 900 905 910 Leu Ser Gly Pro Trp Leu Met Val Leu Val Cys Ile Val Leu Leu Val 915 920 925 Leu Val Ile Ser Ala Ala Ile Ser Met Val Tyr Phe Lys Arg Lys His 930 935 940 Gin Met Thr Lys Glu Leu Gly His Leu Ser Val Val Ser Asp Asn Glu 945 950 955 960 Ile Thr Ala Leu Asn Ile Asn Ser Lys Glu Ser Leu Trp Ile Asp His 965 970 975 His Arg Gly Trp Arg Thr Ala Asp Thr Asp Lys Asp Ser Gly Leu Ser 980 985 990 Glu Ser Lys Leu Leu Ser His Val Asn Ser Ser Gin Ser Asn Tyr Asn 995 1000 1005 Asn Ser Asp Gly Gly Thr Asp Tyr Ala Glu Val Asp Thr Arg Asn Leu 1010 1015 1020 Thr Thr Phe Tyr Asn Cys Arg Lys Ser Pro Asp Asn Pro Thr Pro Tyr 1025 1030 1035 1040 Ala Thr Thr Met Ile Ile Gly Thr Ser Ser Ser Glu Thr Cys Thr Lys 1045 1050 1055 Thr Thr Ser Ile Ser Ala Asp Lys Asp Ser Gly Thr His Ser Pro Tyr 1060 1065 1070 Ser Asp Ala Phe Ala Gly Gin Val Pro Ala Val Pro Val Val Lys Ser 1075 1080 1085 Asn Tyr Leu Gin Tyr Pro Val Glu Pro Ile Asn Trp Ser Glu Phe Leu 1090 1095 1100 Pro Pro Pro Pro Glu His Pro Pro Pro Ser Ser Thr Tyr Gly Tyr Ala 1105 1110 1115 1120 Gin Gly Ser Pro Glu Ser Ser Arg Lys Ser Ser Lys Ser Ala Gly Ser 1125 1130 1135 Gly Ile Ser Thr Asn Gin Ser Ile Leu Asn Ala Ser Ile His Ser Ser 1140 1145 1150 Ser Ser Gly Gly Phe Ser Ala Trp Gly Val Ser Pro Gin Tyr Ala Val 1155 1160 1165 Ala Cys Pro Pro Glu Asn Val Tyr Ser Asn Pro Leu Ser Ala Val Ala 1170 1175 1180 Gly Gly Thr Gin Asn Arg Tyr Gin Ile Thr Pro Thr Asn Gin His Pro 1185 1190 1195 1200 Pro Gin Leu Pro Ala Tyr Phe Ala Thr Thr Gly Pro Gly Gly Ala Val 1205 1210 1215 Pro Pro Asn His Leu Pro Phe Ala Thr Gin Arg His Ala Ala Ser Glu 1220 1225 1230 Tyr Gin Ala Gly Leu Asn Ala Ala Arg Cys Ala Gin Ser Arg Ala Cys SUBSTITUTE SHEET (RULE 26) WO 99/25833 PCT/US98/24327 1235 1240 1245 Asn Ser Cys Asp Ala Leu Ala Thr Pro Ser Pro Met Gin Pro Pro Pro 1250 1255 1260 Pro Val Pro Val Pro Giu Gly Trp Tyr Gin Pro Val His Pro Asn Ser 1265 1270 1275 1280 His Pro Met His Pro Thr Ser Ser Asn His Gin Ile Tyr Gin Cys Ser 1285 1290 1295 Ser Glu Cys Ser Asp His Ser Arg Ser Ser Gin Ser His Lys Arg Gin 1300 1305 1310 Leu Gin Leu Giu Giu His Gly Ser Ser Ala Lys Gin Arg Gly Gly His 1315 1320 1325 His Arg Arg Arg Ala Pro Val Val Gin Pro Cys Met Giu Ser Glu Asn 1330 1335 1340 Glu Asn Met Leu Ala Giu Tyr Glu Gin Arg Gin Tyr Thr Ser Asp Cys 1345 1350 1355 1360 Cys Asn Ser Ser Arg Giu Gly Asp Thr Cys Ser Cys Ser Giu Gly Ser 1365 1370 1375 Cys Leu Tyr Ala Glu Ala Gly Glu Pro Ala Pro Arg Gin Met Thr Ala 1380 1385 1390 Lys Asn Thr 1395 INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 4146 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

GGTGAAAATC

TTTACGTTTA

GGTCGCGAAC

TTCTTTCTCA

GCCAAAAACG

CGCGACGAAT

ATGGAATGCG

CAGACCCTGA

ATCCAGGAAG

GGCACCCGGG

GGACCCCAGA

GGCGATCCCC

CGTAAGTTTT

CTGAAGCTGG

GCGGTGGGCG

ATACGCCCCA

AATGGACATC

CCCGGCTATC

TCGATAGCTC

GCCGTGGGCA

CCGCCGATTA

CTGCCATGCC

CCCATCGATG

TCGGATCTTC

GGAAAATCCT

CACGCATCAT

ATTGCCAGGC

TGAAGACGGA

AGGTTATCCA

AGTTTGGAGT

TCCGTTTGGA

GTGCCCCCCG

ATCTTGTCGG

CCCGCCAATC

AGTCGGCCAC

ATCAGACGGC

TGCCTGATGT

CTTGGCTTCA

ACGACGTTAC

GCATCACGGC

AGAATCAGCT

CCCGACCAAC

GGGATGGCCG

GATTTGCCCG

GCGTCAGCAG

TCGAACAGGG

GAACTCTGGG

TGCAGGAGCA

AGCGCCACGA

CTTGGAGTGG

CGAGCATCCC

CGAGGGCAAT

TACGGGTTCG

CTCACGTAGA

GGCACGGTCC

GCCGGCAAAT

AGGATCTCCG

GAACAAGCGG

GGACGACGGA

CGCTTTTCTT

GGTGGTGGGC

CCTGTGGCGA

TTCAGCTTCA

TCTGGAGGAC

CACTGGCATC

GGTGGAGATC

GCTCTACTGG

CATGGAAGTG

TGAGGATTCC

TCGGACTGTG

GCCCGTGAAT

CACTCCAGTG

CGAGCGGCGG

GGATGAAGGC

TTACCTTCGT

ATGGACACGA

CCAACACCAA

CATCGCATAA

GAGAGCGATG

AGGAATGCAA

ACCCGCGTGG

GAGCCGCAAA

ATTCGCATTG

CGCTACCAGT

AAAGTGCATG

AGCTCGGTGG

CGCACTGCCT

GGTCGTGTGC

ATGGGCGAGT

CTCACCGTTC

GGTGATGAAG

TCGGTGGAGG

ACCCTGACGC

GGAAAGGTGG

GTCAGTGTGG

CAAACGTTGC

CCACAGGTCT

AATCTTTCGG

TTGTACACCT

CTGGACACCC

CGGTGCCAAA

CCATTCAATG

TGCTGCCCGC

CGGGCACTTA

CGTTGCAAGT

CCCAAGGCGA

TCTCGTGGCG

TCGACGGTGG

GTGTGGTC.A

TACGTCCATT

TCTTCCAGTG

CCGGCGGCAA

ACGTACTTGA

ACACTTGCGA

ACGCTCCCCC

TGCTGTTCGA

GCAACAGCTC

CCGAGGGGCG

TCACTTGCAA

ATACGCAATT

CCGTTAAATC

CTTGGTACCT

ACGCTGGAGC

GCGTGGCCAG

CGACAAATCC

AAATGATCCA

GTTTAAGGAC

CGGGGGTCTA

CTGGTGCGAG

GGCAGTTCTC

GGTGGCCCTG

CAAGAACGGC

CAATCTGGCC

GAATGTGGTT

CCTCATCCGA

CCGCATCGGA

TATGCCACTG

GGACCGCAGT

GGCGGACAAT

CAAATTTGTG

GTGCCAAGCG

CCTGCTGCTC

CTCGGTGCTC

CGCCCTGAAC

CGAGCTGCCA

AATTGTGGTT

GGATGGCATA

CTTAACCATT

CAATCGCAAC

GAATATCAAG

120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 SUBSTITUTE SHEET (RULE 26) WO 99/25833 PTU9/42 PCTIUS98/24327

TTCTTCAGAG

GAGAAGGGCG

AGTCTGGTGG

GTGGGCACTA

TACTTCTTTC

GAACCCATTA

GCCAGTCTGC

AGCATGAAAC

GCGAGACAGT

CCGCTGCTGG

TCGACAAAGC

GGAGGAGCTC

GGCGCCTCAT

GTGCCATTTT

GAAGATGTTC

GTCTTCCTCA

CATGTTATAG

ACCATCGATG

ACCGTGGGCG

TTGCGTTTGG

AACGATGTGC

ATGCTGTCCT

GCCCTAAATA

GCGCGCAATG

TCGCCCGGCG

TGCGGTGCCC

GGCAGCGGTG

CAGCGGTACG

GGCAAGGCAC

TCTTCGATCC

CCAGTGCCCG

CAGCAGCAAC

CTGCCACCCA

ACGGGTCCTT

CACATCCACA

GCCAAGCAGT

CCCAACTGCA

AAGAACCAGC

GACTCGGGCT

CACGCGCTGT

GTGGACGAGC

CCGGCGGAAG

GGCCAGGAAT

GAACGGAGCC

GTCTGA

CCCCAGAACT

AAA.ATTCGGT

GCTATGTAAT

GGGTGCAAA

TAATTCGAGC

CGGTGGGAAC

TGTCCGGAGA

TCACCTGGCA

TGCCAAATCC

GCTCTACATC

CAAATATTGC

CGACCCCACT

CCTGCACCAT

ACAAATCCGT

CCTCTGAGGC

AATGGAAGGC

TCCGAGGTAT

CCGCTTCGCC

TGGCGGCCGG

ATCCCATCAC

TGACGCAGCC

TTGGCGCAAT

CAATGCGTGG,

GAA.ACGGCTA

GCGACTCGCT

CCGGTTCTCC

CCAGCGGCGG

TGGGCGAGTA

CCAGCGAGTA

TGAGTCCCCA

GCTATGGGCT

AGGCGCAGCA

GCAACATCTA

CGCGCTCTGT

TCACCGAGAA

CCTCGCCGAT

GCATCGGCAG

AGAATCTCCT

GCGGTGGATC

ACCACACGGC

AGCAGCCTCC

GTCACCTGCA

GCATCAAGGA

TCCTCAGCAA

TTCCACCTAC

GACTCTCAGC

CGAGATGTTT

TACCACGTTT

CGAGAACTCC

GCGCTACTTC

TGTTGTGGAG

GATCATCAAT

AATAGTCAAC

CACATCCGCA

AGCTGCCGGC

GAACACCAAG,

CACCGGGCTC

CGAGGGCAAG

ACCATATGGA

ACCAGAACTC

TGACACTGCC

TACTCTGGTT

AAATAACGCT

AAAGCGACTC

CTGGTTCATA

GGTCTTTGTG

CAATCACACG

CTGGCTGGAC

GGAGATGCAA

GGCCGGCGGT

CGATGACATT

CTCCAACATA

TGGTCGGCAT

CCAGCAGCAA

CCAGCGCCCA

GACGCACCAG

CCAGCAGATG

CTACTCTGAG

CAAGCTGAGC

ATCCTCGCAG

GGAAAGTGCC

GGATCTCGAC

TCCCTCCCCG

GGATGGGGAT

ACAGCAGCAG

GTCCTGGCGG

ACCCAGCGAG

CTCGGGTAGC

CCAGGGCCGC

TGGACGAGGA

GGCAAAAACG

ACCCAAACGG

CATGGCTTAT

AATAGTGGTC

CTGAGCAACG

GGCAAATACG

AATCCGGCGC

TCCGCATCCG

AAACGTGATG

TATCGCATGC

GTCCAGTACA

CCGTCGAATT

ATGGAGGCTC

AAGGATCGGC

CACAATTTCT

TTGGCCAATC

GGAGTTGGTC

GATCCGTTCA

ATACTCCTGG

AAGCGCAAGC

AGCGACGTGC

TCCTCCACCG

AAGGATCACA.

GGCACCTCTT

CATGGAGGAC

CCGACCGACT

GGCAACGCCT

CAGCAGCAGC

ATGCACCCAC

CAACACCAGG

TCCACCACCA

CAGTATTACT

AACTGCCACA

TTCGCCAGCG

CGCTTCAAGG

GGCTCCTCGA

ATGGCCATGC

CTGGACGACA

CAGCAGCTGA

AATCAGAGCA

TTGATCTACG

GGCACCAGCA

CAGGAAAACC

GCAACAAGGT

AAACGGATGG

GTCTGCTGCC

CACTGCCCAG

TGGATCTGAG

CCAGTGTGGT

TCGAGGGCTT

CCGTTACTAG

CCTCGGCATC

GGGAGACAAA

TAACGATTCT

CGCTGTATGA

CGCGCATCGC

TGCTGTTGAA

ATGGTGTTCT

CACGCATTTT

TCACCGAAGG

CTTATTGTGT

TCAATCAGCG

GCGCCATCCT

ACATGATGAT

TCAAAATGCC

GCGGAATGGT

TCGCCGACTA

CCGGTGGATC

ACGGCAGCGA

ATGCAGAGGT

CCCCGGCCCC

AGCCGCGTTA

ACTACCAGCA

CTCTCCAGCA

GCGAGATATA

ACCCCAAGGA

CCTATGAGGC

TGAGGCGGCA

TGCTAAACAC

TGTGCTACAA

TGATGTCGCA

TGGAACGACT

TTCCCCTGGT

CGCGGAGCAG

CTCCGGGAAG

GCCAGCCAGC

GCAAATGGTG

GGGCGGCTCC

CTGGGTGGCT

GGGTGTGAAT

TCCGATGTCG

CGAGGCTCGT

GGACTCCACT

CTATGTCTAT

CAATACCAAT

GGCATTGATT

CCAGAGTGGA

CAATGGCGGT

ATTTTTCATC

TCGCACCCTT

CTCCTCCGCG

CTTGAACTAT

GACAAATGTC

CGTCATGTAC

CCCAGCTACT

GGACCATGTT

GGCCGTTCTT

GAAGCAGTCG

GAGTCTATCG

GTGGCGTCCC

TGCGCCGGTC

CGGTGGCGCG

ACGCAATCAG

GTCCAGTTTT

TTATGCCACC

TCAACAGCGA

GCAGCAGCAT

GCACCAGCAA

CCCCACGAAC

CAAGCAGAGA

GGCTCCTGGC

GCAGCTGCCG

GGATCAGGGC

CGGTCTGGCA

CGAGGACGAG

GTACGTCAAG

CCCACAGCAT

TCGGAAGAAC

CGTGGCCAGC

TGGCCACAAT

1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 3000 3060 3120 3180 3240 3300 3360 3420 3480 3540 3600 3660 3720 3780 3840 3900 3960 4020 4080 4140 4146 INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 1381 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: Gly Glu Asn Pro Arg Ile Ile Glu His Pro Met Asp Thr Thr Val Pro SUBSTITUTE SHEET (RULE 26) WO 99/25833 WO 9925833PCTIUS98/24327 1 Lys Pro Gly Val1 Ala Val1 Val1 Ser Leu 145 Ile Lys His Val1 Pro 225 Arg Giu Giu Gly Asn 305 Asn Ser Thr Asp Val 385 Pro Ser Val Asn Thr Ser Ile Lys Ala Ala Pro 130 Val Gin Asn Val1 Gly 210 Asp Lys Asp Tyr Ile 290 Gin Giy Leu Pro Ser 370 Ser Pro Ile Ser Asp Ile His His Asn Vali Gin 115 Giu Giy Giu Val1 Arg 195 Ser Vai Phe Arg Thr 275 Leu Leu His Leu Glu 355 Gly Ser Ile Vai Trp, 435 Pro Gin Arg Ser Giu Leu i00 Giy Pro Asn Aia Val 180 Pro Ser Leu Ser Ser 260 Cys Thr Val1 Pro Leu 340 Giy Lys Arg Ile Val1 420 Tyr Phe Trp Ile Arg Phe Arg Glu Gin Lys Arg 165 Giy Phe Vali Trp Trp 245 Leu Glu Vai Giu Arg 325 Pro Arg Vali Thr Giu 405 Leu Leu Thr Phe Phe Lys Met Leu 55 Arg Giu 70 Giy Val Asp Giu Val Aia Ile Ser 135 Arg Ile 150 Gin Ser Thr Arg Leu Ile Val Phe 215 Arg Arg 230 Leu His Lys Leu Ala Asp His Ala 295 Ile Gly 310 Pro Thr Gly Tyr Ser Val Val Thr 375 Val Val 390 Gin Giy Pro Cys Asp Gly Asn Cys 25 Asp Gly 40 Pro Ala Ser Asp Ala Arg Phe Arg 105 Leu Met 120 Trp Arg Arg Ile Asp Asp Giu Ser 185 Arg Gly 200 Gin Cys Thr Ala Ser Ala Asp Asp 265 Asn Ala 280 Pro Pro Asp Giu Leu Tyr Arg Asp 345 Leu Ser 360 Cys Asn Ser Val Pro Val Arg Thr 425 Ile Pro 440 10 Gin Arg Gly Aia Ser 90 Leu Giu Lys Val Giy 170 Ala Pro Arg Ser Ser 250 Vai Val Lys Val Trp 330 Giy Ile Ala Asp Asn 410 Leu Ile Ala Glu Gly Gly 75 Arg Giu Cys Asn Asp 155 Arg Thr Gin Ile Giy 235 Gly Thr Giy Phe Leu 315 Ser Arg Al a Leu Thr 395 Gin Gly Asp Giu Giy Leu Lys Leu Phe Thr Tyr Asn Ala Pro Ala Gly Ala 125 Gly Gin 140 Gly Gly Tyr Gin Ala Phe Asn Gin 205 Gly Gly 220 Gly Asn Arg Val Leu Glu Gly Ile 285 Val Ile 300 Phe Giu Val Glu Met Giu Arg Phe 365 Asn Ala 380 Gin Phe Thr Leu Thr Pro Val Gin 445 Asn Thr Phe Trp Thr Asn 110 Pro Thr Asn Cys Leu 190 Thr Asp Met His Asp 270 Thr Arg Cys Gly Val1 350 Ala Val1 Giu Pro Val1 430.

Glu Pro Asp Leu Cys Leu Thr Arg Leu Leu Val 175 Lys Ala Pro Pro Val1 255 Met Ala Pro Gin Asn 335 Thr Arg Gly Leu Val 415 Pro His Thr Thr Lys Giu Gin Arg Gly Asn Ala 160 Val1 Val1 Val1 Leu Leu 240 Leu Gly Thr Lys Ala 320 Ser Leu Giu Ser Pro 400 Lys Gin Giu SUBSTITUTE SHEET (RULE 26) WO 99/25833 WO 9925833PCTIUS98/24327 Arg Arg Asn Leu Ser Asp Ala Gly Ala Leu Thr Ile Ser Asp Leu Gin 450 455 460 Arg 465 Gly Pro Pro Leu Tyr 545 Val1 Pro Leu Tyr Ser 625 Ser Phe Ala Ser Asn 705 Gly Leu Tyr Gly Ser 785 Val Leu Phe Leu Ala 865 Leu His Lys Asn Pro Ser 530 Val Gly Gly Ser Phe 610 Gly Met Tyr Pro Al a 690 Ile Gly Asn Thr Lys 770 Giu Phe Leu Ser Val 850 Ala Arg Giu Ser Ile Gly 515 Trp Ile Thr Val Leu 595 Asn Asp Lys Val Val1 675 Ser Ala Ala Gly Leu 755 Pro Ala Leu Asn Arg 835 Leu Gly Leu Asp Ser Lys 500 Lys Thr Glu Arg Asn 580 Pro Ser Val1 Leu Tyr 660 Thr Ala Ala Pro Gly 740 Tyr Ser Pro Lys Tyr 820 Ile Ala Asn Asp Glu Trp 485 Phe Pro Arg Met Val1 565 Tyr Ser Gly Val Thr 645 Ala Ser Ser Ala Thr 725 Gly Glu Asn Tyr Trp 805 His Leu Asn Asn Pro Gly 470 Ser Phe Gin Ser Phe 550 Gln Phe Pro Leu Giu 630 Trp Arg Asn Ala Gly 710 Pro Ala Phe Ser Gly 790 Lys Val1 Thr Leu Al a 870 Ile Leu Giy Arg Met Asn 535 Gly Asn Phe Met Asp 615 Leu Gin Gin Thr Ser 695 Lys Leu Ser Phe Arg 775 Met Ala Ile Asn Thr 855 Gly Thr Tyr Tyr Ala Val1 520 Lys Lys Thr Leu Ser 600 Leu Ser Ile Leu Asn 680 Ala Arg Asn Ser Ile 760 Ile Glu Pro Val1 Val1 840 Giu Val1 Lys Thr Leu Pro 505 Glu Val1 Asn Thr Ile 585 Giu Ser Asn Ile Pro 665 Pro Ser Asp Thr Cys 745 Val Ala Ala Glu Arg 825 Thr Gly Gly Arg Cys Arg 490 Glu Lys Gly Glu Phe 570 Arg Pro Glu Ala Asn 650 Asn Leu Ala Gly Lys 730 Thr Pro Arg Leu Leu 810 Gly Ile Val1 Pro Leu Val 475 Leu Leu Gly Gly Thr 555 Thr Ala Ile Ala Ser 635 Gly Pro Leu Leu Glu 715 Tyr Ile Phe Thr Leu 795 Lys Ile Asp Met Tyr 875 Asp Ala Asp Ser Giu Ser 540 Asp Gln Glu Thr Arg 620 Val1 Lys Ile Gly Ile 700 Thr Arg Thr Tyr Leu 780 Leu Asp Asp Ala Tyr 860 Cys Pro Ser Thr Thr Asn 525 Ser Gly Thr Asn Val 605 Al a Val1 Tyr Val Ser 685 Ser Asn Met Gly Lys 765 Glu Asn Arg Thr Ala 845 Thr Val1 Phe Asn Pro Tyr 510 Ser Leu Trp Gly Ser 590 Giy Ser Asp Val1 Asn 670 Thr Thr Gin Leu Leu 750 Ser Asp Ser His Ala 830 Ser Val1 Pro Ile Arg Thr 495 Pro Val1 Val1 Val1 Leu 575 His Thr Leu Ser Glu 655 Asn Ser Lys Ser Thr 735 Val1 Val1 Val1 Ser Gly 815 His Pro Gly Ala Asn Asn 480 Asn Gly Thr Gly Ala 560 Leu Gly Arg Leu Thr 640 Gly Pro Thr Pro Gly 720 Ile Gin Glu Pro Ala 800 Val1 Asn Thr Val1 Thr 880 Gin SUBSTITUTE SHEET (RULE 26) WO 99/25833 PCTIUS9824327 885 890 895 Arg Asp His Val Asn Asp Val Leu Thr Gin Pro Trp Phe Ile Ile Leu 900 905 910 Leu Gly Ala Ile Leu Ala Val Leu Met Leu Ser Phe Gly Ala Met Val 915 920 925 Phe Val Lys Arg Lys His Met Met Met Lys Gin Ser Ala Leu Asn Thr 930 935 940 Met Arg Gly Asn His Thr Ser Asp Val Leu Lys Met Pro Ser Leu Ser 945 .950 955 960 Ala Arg Asn Gly Asn Gly Tyr Trp Leu Asp Ser Ser Thr Gly Gly Met 965 970 975 Val Trp Arg Pro Ser Pro Gly Gly Asp Ser Leu Glu Met Gin Lys Asp 980 985 990 His Ile Ala Asp Tyr Ala Pro Val Cys Gly Ala Pro Gly Ser Pro Ala 995 1000 1005 Gly Gly Gly Thr Ser Ser Gly Gly Ser Gly Gly Ala Gly Ser Gly Ala 1010 1015 1020 Ser-Gly Gly Asp Asp Ile His Gly Gly His Gly Ser Glu Arg Asn Gin 1025 1030 1035 1040 Gin Arg Tyr Val Gly Glu Tyr Ser Asn Ile Pro Thr Asp Tyr Ala Glu 1045 1050 1055 Val Ser Ser Phe Gly Lys Ala Pro Ser Glu Tyr Gly Arg His Gly Asn 1060 1065 1070 Ala Ser Pro Ala Pro Tyr Ala Thr Ser Ser Ile Leu Ser Pro His Gin 1075 1080 1085 Gin Gin Gin Gin Gin Gin Pro Arg Tyr Gin Gin Arg Pro Val Pro Gly 1090 1095 1100 Tyr Gly Leu Gin Arg Pro Met His Pro His Tyr Gin Gin Gin Gin His 1105 1110 1115 1120 Gin Gin Gin Gin Ala Gin Gin Thr His Gin Gin His Gin Ala Leu Gin 1125 1130 1135 Gin His Gin Gin Leu Pro Pro Ser Asn Ile Tyr Gin Gin Met Ser Thr 1140 1145 1150 Thr Ser Glu Ile Tyr Pro Thr Asn Thr Gly Pro Ser Arg Ser Val Tyr 1155 1160 1165 Ser Glu Gin Tyr Tyr Tyr Pro Lys Asp Lys Gin Arg His Ile His Ile 1170 1175 1180 Thr Glu Asn Lys Leu Ser Asn Cys His Thr Tyr Glu Ala Ala Pro Gly 1185 1190 1195 1200 Ala Lys Gin Ser Ser Pro Ile Ser Ser Gin Phe Ala Ser Val Arg Arg 1205 1210 1215 Gin Gin Leu Pro Pro Asn Cys Ser Ile Gly Arg Glu Ser Ala Arg Phe 1220 1225 1230 Lys Val Leu Asn Thr Asp Gin Gly Lys Asn Gin Gin Asn Leu Leu Asp 1235 1240 1245 Leu Asp Gly Ser Ser Met Cys Tyr Asn Gly Leu Ala Asp Ser Gly Cys 1250 1255 1260 Gly Gly Ser Pro Ser Pro Met Ala Met Leu Met Ser His Glu Asp Glu 1265 1270 1275 1280 His Ala Leu Tyr His Thr Ala Asp Gly Asp Leu Asp Asp Met Glu Arg 1285 1290 1295 Leu Tyr Val Lys Val Asp Glu Gin Gin Pro Pro Gin Gin Gin Gin Gin 1300 1305 1310 Leu Ile Pro Leu Val Pro Gin His Pro Ala Glu Gly His Leu Gin Ser 1315 1320 1325 SUBSTITUTE SHEET (RULE 26) WO 99/25833 PTU9142 PCT/US98/24327 Trp Arg Asn Gin Ser Thr Arg Ser Ser Arg Lys Asn Gly Gin Giu Cys 1330 1335 1340 Ile Lys Glu Pro Ser Glu Leu Ile Tyr Ala Pro Giy Ser Val Ala Ser 1345 1350 1355 1360 Glu Arg Ser Leu Leu Ser Asn Ser Gly Ser Giy Thr Ser Ser Gin Pro 1365 1370 1375 Ala Gly His Asn Val 1380 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 3894 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID

ATGTACTATC

AAAATTCCTA

GTATCTAGGG

ACATGGTACA

ATTGTTCTCG

GACAGCGATG

AACGAAGGAT

GTTCAGGCTC

GAGCCGGTTG

TACACTCTAC

ACTTATCAGT

AGTGTCTTTG

GCCGCAGTGC

CGCAAAAATG

AGAATCGAAA

GCGGGAACTC

AAACCAGCAG

GGTCAACCGA

AGTTATGTGT

GAAGTTCGTC

TCGTTGAGCA

AAGAGCAAAA

TCAGCCGTGA

AATCAGACCC

ACTCCAGGAA

AGTCAACATT

TACACTTGCA

GAAGATCACA

TCTCCAACGC

GCTCCCTCCA

GACCTCGGAC

AAGGGTCTGA

ATTGGAACGC

GCGCTTTCTG

CAACTCATAA

AAGAAGAGGA

AGAACCAATG

TCAAATTTAA

TAGGTTTTTA

ATGCCTCAAA

GATCGCCAGC

AGGATGGACA

ACACGGGATC

CGGGAGCGTA

CGTTAAAATT

TTGGTGGAGA

TGAGCTGGCG

ACTCTGACGG

GTGTTGCCAA

AGAAACCAAA,

TGTTTGATTG

AGCCGATGCC

GAGTTCAACC

TTGAAGCATC

ACCAGTCAGT

GTCCCGCCTA

CCGCTGATGG

AAGTTGATGA

AGGCAGCTTT

TGGGCAAACA

CCGGAAACAC

TTATGGTTGG

TATCATGGCT

CAACGGGAAG

TTGCGAAGAA

CTAGCAATGC

AACCCATTAT

CATCTGGCGC

AGACGTGGTT

AACCATCTCA

CGAGTGTGTC

ACAGAACAA~

AACTCGAGGA

AACTTGAAGA

ATAATCAATA

TGCCATTCAC

CCACACTCAC

TCTCGCTCCC

AACCCTCAAC

GCCCGTAATC

CCTGTTTCTT

CTATTGTGTG

GGCGATGCTT

GATGGCCGTT

GAAAGACGAC

AAACCTCATC

CAACATGGTC

GTTTGAGCAA

TCGTGTGACT

AGTTACACGT

ATCAGACGAA

TGCACATCTT

TCCAGCTGGA

TTTTTGGAGC

TAGAACGAAA

GGGAGCTTAT

GAAAGCAACA

GAAACAAAAA

ACCCGCCAAA

ATCATCAGCC

CAGGGATGGG

TCTACATATT

CGAGGATGGA

ACAATTTGTT

TGTCAATGTC

AGGACCAATC

TAACATTCCA

CTCGTATATG

GTCGGCTCTC

AATGGACATG

AGTGAAGACT

GCTGATTGAT

CGTGAATGTG

CAACTATGAG

ACACACACAC

GTGATAATCG

TGTGGTGCAA

ACGAATAAGG

CTGAAAGTGA

GCCAGCAACG

CGCGAAGACT

CTGGAATGCA

AAAGAGCTCC

ATTGATCCGG

GGAGAACGGG

GAACCCAAGG

GGAGATCCTC

GCATACATTG

GGTGAATACG

CGTGTCCAGG

GGCACGGCAA

AAGGAAGGCC

GTTTCACCAA

GTGTGCGCTG

TTTGAAACCA

AATGTTCAAT

CCACCACCAA

ATCCTTCCAT

CTACCTATTG

GCCGATTTAA

GAGTCAACAT

CGGATGCCGG

ACTGATACCG

ACTGGTTATA

GACTACGTGG

TTTGTGATTC

GTTACCACTA

GCCATCGCTG

ATTAATTCTA

GGTTACTACA

ACCAGCCCTA

TTTTTCGTGA

ACACATACAT

AACATCCCAT

AGCCATCTAC

AGCAAGTGAA

ATAGTGGAAA

AGCACGGAGA

TTCGAGTTCG

GTCCGCCACG

GAATTCAAGA

TCGATCGAAG

TGTCCAATCC

ACATGACGGT

AACCACAAAT

CCAAGGATAA

TTTGCTATGC

CACCTCCATC

CTTTTGAATG

AACAGGATCT

CTGGAACATT

GAATGAACTC

AAGGCCGTGT

CAATTATCAA

CAATCGAGCA

GTCAGGCTAG

ACATTACAGA

AGAAACCTGA

GGTCGGCATC

ATCCATCGAA

AAGTAGAGCT

TCATTCAGTA

CATCTACTGA

GAGCAGAAAA

GCAAGCCAGC

AGAAGAGACT

CGGCCGTTCG

TCAAGTGGAG

GCACCGAAAA

TTCCTTATCA

AAATTTTGAT

CGATGTGGTG

CGCCAAAATC

CAGCCACCGG

AAACGGAAAA

AGTGAAGTCG

GCCAAGAACA

TGGATTCCCG

CATGCCACGA

CGATTCTGGT

CGCAAGATTG

CGACGTCGGA

TACGTGGAA

TCGGGGGTTG

ACGAAATCCA

CTTCCAGACA

CACCTTGGTC

TCTTTTCCCA.

GACAATTGAG

GGCAGGAAGC

CCAAAAAAAA

ATATTTAATT

TGGTCATCAA

CGGAAAACCA

TAGTCGTATC

CACCGGAGTT

TCTGACTGTT

CTTCCCGTCT

CCACTGGAAT

CTACAGTCCA

ATATAGAATA

TGAGAAAGGT

AGCTCAAGTT

CACTTCGGAA

TTTGTTCTGG

AGGGCCTCCA

CTATGTTGTT

TTCCGGAGTT

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 SUBSTITUTE SHEET (RULE 26) WO 99/25833 PTU9/42 PCT/US98/24327

CATAGTATTC

TCATTGCCAC

TGGAAAGCAC

GTTGGTCAAG

ACTCTGTTCC

GGTGGAGTTG

AAACACCTTG

CATGTTCCTG

TATTGTTACT

AATGATGGAA

CAGAATCCAA

CTCTATTCGC

ACGATGCACA

GGTA.ATGCGA

ACCACAACAC

GCGCCAGCAA

ACCGCTGGAA

GGACTCCATC

AGCTATGTTC

CGGAGAACAC

CCACCACCTG

ACTCCACGAG

AATGCAAGGC

GACGACGATA

GACGGGGAGA

ATGGGTATCT

CGATTCCGGT

ATGGAGCACC

CAGAGGATGT

CAAAAGCCGA

CGCCCAACAA

ATTTAGTGAC

GAGTCTCACA

CTGCTCAACA

TGATTGTCAT

GGAGGAATAG

GTGTTCATAT

TGTACAACAC

TGACACCAAA

GACCAGGATC

TGTCTACTTT

TGGTCCTGTC

TGCCAACAAA

GACGATCTCG

ACCGGACTAG

AGCTTCACTC

CACCGAATAA

GAGGGCACGT

AAGACACCTA

CAACGAGTCG

GTCAGCGGTC

ACTCTGAAGG

CTGCAAGTAC

CAATTCCACG

GAGTAATTCC

GCGA.ATCCGT

CGGCATCAAC

CAATCGGAAC

TGGAATGACG

TGGAACGAGT

AGAAAACGAA

TGTTGCAATT

CAGAAACAGT

GGCTTCGA.AT

TGCTGGA.AGA

TGCGCAAGAC

CGAGCATCAC

TTATGGAAAC

GAACCAACAA

TCCCGTGCC.A

ATCGAGCCGT

CGGAAGTCAA

ATCCGATGGA

GACTCTGATG

TTATGACACA

CGATTCGGTC

GAATCGGAAT

TTCGTTGATG

AGACGTTCCG

GCTGGCTCAT

TAACAATGGA

ATGGACGTGT

ATGCTCAACC

GGAATTCTCA

ATCACTACAA

TATAAAATTC

GAAGTCATCA

TCATTTTTGT

CTGATTATTT

GATGGAAAGG

AATCTTTGGG

ATGACTATGA

TTTTTCAACA

TATCATTATG

CAATATCACG

CCAGCTTGGC

CCAGAGCCAC

GCATCCGATG

CGGTCGGATA

ACTGGTAGTA

GACTTTATTC

GCAACTAGGC

AGTGACGGAG

TTGGGAGGAA

ATGGACGATG

CGTGGAGGTG

AGTTGTTACG

ATCGTCACAC

TGACCGCCGA

TGACCACTCT

AAGGATTCCA

ACGAGAGAGC

GTGTAGCGGC

TGAATCAAGA

ATGGGCTGAT

TCGTAGTCAT

ATCGAAGTTT

ATGTTGCACA

ACAATAGAAA

ATTGTGATGA

CTCAACTGAC

ATGATCCATC

TCAATGACAA

CGGCGCGATA

GGAGAGGAAC

GTCCACCTCA

GTAAGGAAAG

CGCCACCACC

GTCAGTTGAA

CTTTTGCTCG

GGCCGCTGAA

ATGGTGGATC

TTAGAAAAGC

GGACAAACGG

AAGAACAAAC

AGCTCCACCT

TCGTATCTCT

AATTGTTATT

TGCCAGTGTT

TAGAAGCAAT

CACGCTGGAA

CAATAAATCT

CATTATAGCC

TATAAAGATC

AAATCCGAAT

TGGCCAGGCT

CTACAGTGGA

TGGCGGACCT

TCCATATGCC

AATGCTTCGC

TGCAGATCAT

TCTGAATGGC

CACAGATGTG

AACTGGGGAG

TTCCAATCCA

TCGTGGAAGT

GGTTGATGTG

AGGGAAACGA

TTCTGAAGCT

AGTTCCTCGA

CACTGCTCAA

TTGA

2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 3000 3060 3120 3180 3240 3300 3360 3420 3480 3540 3600 3660 3720 3780 3840 3894 INFORMATION FOR SEQ ID NO:6: Wi SEQUENCE CHARACTERISTICS: LENGTH: 1297 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: Met 1 Ile Ile Tyr Tyr Leu Asn Phe Asp Glu His Pro Gly 5 Lys Phe Tyr His Thr Thr His Thr His Thr Tyr Ile Pro Asn Ala 25 Val1 Asn Leu Ala Ile Asp Val Leu Asn Cys Val Ser Ser Arg Gly Pro Val Ile Pro Ala Thr Trp Tyr Lys Gly Ala Lys Asp Gly Pro 55 Thr Thr Ala Lys Ile Val1 Gin Pro Val Asn Lys Glu Asn Ser His Ile Arg Val Leu Asp Thr Asp Ser Leu Phe Lys Val Asn Ser Gly Ala Ser Lys Asn Gly Asn Glu His 115 Met Leu Arg Lys 100 Gly Ser Asp Ala Gly 105 Asn Tyr Tyr Cys Val Giu Val Lys Ser 120 Val1 Glu Gly Ser 110 Lys Leu Ala Gin Ala Leu Glu Asp Phe 130 Gly Gly Arg 135 Leu Arg Pro Arg Thr 140 Glu Met Ala Val Glu Cys Ser Pro Pro Arg Gly Ph-e Pro SUBSTITUTE SHEET (RULE 26) WO 99/25833 WO 9925833PCT/US98/24327 145 Giu Asp Pro Met Lys 225 Ala Ile Ile Asp Giu 305 Lys Cys Gly Thr Val1 385 Ser Val1 Gin Ala Met 465 Thr Asp Leu Asp Ser 545 Ser Leu Pro Met Val1 Val1 210 Pro Ala Thr Ala Glu 290 Ala Pro Thr Gin Lys 370 Asp Leu Gin Ser Lys 450 Val Pro Ser Lys Gly 530 Asn Pro His Val Pro Asp 195 Giy Lys Val1 Trp Lys 275 Gly Ser Ala Leu Gin 355 Val1 Giu Ser Lys Ile 435 Pro Gly Gly Arg Lys 515 Giu Ala Thr Trp, Val1 Arg 180 Arg Giu Phe Leu Lys 260 Asp Giu Ala Asp Val 340 Asp Ser Gly Lys Lys 420 Ile Pro Ser Ile Ile 500 Pro Ser Gin Gin Asn 580 Ser 165 Tyr Ser Arg Giu Phe 245 Arg Asn Tyr His Gin 325 Gly Leu Pro Al a Ala 405 Lys Lys Pro Ser Ser 485 Ser Asp Thr Phe Pro 565 Ala 150 Trp Thr Asp Val1 Gin 230 Asp Lys Arg Val Leu 310 Ser Gin Leu Thr Tyr 390 Ala Ser Tyr Thr Ala 470 Trp Gin Thr Trp Val 550 Ile Pro Arg Leu Ser Ser 215 Glu Cys Asn Gly Cys 295 Arg Val1 Pro Phe Gly 375 Val1 Leu Lys Leu Ile 455 Ile Leu His Gly Ser 535 Arg Ile Ser Lys His Giy 200 Asn Pro Arg Giu Leu 280 Tyr Val1 Pro Ser Pro 360 Thr Cys Lys Met Ile 440 Giu Leu Arg Ser Val 520 Ala Met Val1 Thr Asp Ser 185 Thr Pro Lys Val1 Pro 265 Arg Ala Gin Ala Pro 345 Ser Leu Ala Ala Gly 425 Ser His Pro Asp Thr 505 Tyr Ser Pro Asn Ser 585 Giu Asn Cys Leu 220 Thr Asp Val1 Arg Pro 300 Pro Thr Phe Ser Giu 380 Asn Giu Lys Thr Gin 460 Ala Pro Leu Ile Val1 540 Ser Asp Gly Arg Ile 190 Ala Val1 Asp Gin Arg 270 Gin Giy Phe Thr Ser 350 Asp Val Ala Lys Lys 430 Asn Gin Gly Asp Ile 510 Lys Asp Phe Giu Ile 590 Ile 175 Ile Asn Phe Val1 Pro 255 Ala Pro Thr Gin Phe 335 Lys Gly Arg Gly Gly 415 Asn Thr Thr Lys Ile 495 Ala Asn His Pro Val1 575 Thr 160 Gin Asp Asn Glu Gly 240 Gin Tyr Ser Leu Thr 320 Glu Glu Arg Gin Ser 400 Arg Val Pro Leu Pro 480 Thr Asp Glu Thr Ser 560 Giu Gly SUBSTITUTE SHEET (RULE 26) WO 99/25833 PCT/US98/24327 Tyr lie Ile Gin Tyr Tyr Ser Pro Asp Leu Gly Gin Thr Trp Phe Asn 595 600 605 Ile Pro 625 Ile Ala Ala Lys Leu 705 Arg Asn Val Asn Glu 785 Trp Gin Thr Met Val 865 Lys Ile Ile Asn Val 945 Gin Asn Asn His Pro 610 Ser Gly Ala Glu Thr 690 Glu Thr Tyr Ile Ser 770 Asp Lys Ile Asn Thr 850 Ser His Asn Phe Ser 930 His Asn Gly Asn His Asp His Thr Gin Lys 675 Ile Glu Asn Val Pro 755 Met Val Ala Val Glu 835 Tyr His Leu Lys Val 915 Asp Met Pro Gin Cys 995 Tyr Tyr Ser Pro Val 660 Arg Asn Leu Asp Val 740 Tyr Asp Arg Pro Ile 820 Arg Lys Gly Ala Ser 900 Val Gly Ala Met Ala 980 Asp His Val Tyr Ser 645 Ala Leu Ser Ile Asn 725 Ser His Val Ile Lys 805 Val Ala Ile Thr Ala 885 His Ile Lys Ser Tyr 965 Leu Asp Tyr Ala Met 630 Val Leu Thr Thr Asp 710 Gin Asn Ser Leu Arg 790 Ala Gly Ala Arg Ser 870 Gin Val Ile Asp Asn 950 Asn Tyr Tyr Ala Ser 615 Phe Ser Ser Ser Ala 695 Gly Tyr Leu Gly Thr 775 Met Asp Gin Ser Val 855 Glu Gin Pro Ile Arg 935 Asn Thr Ser Ser Gin Thr Glu Val Ile Ser Ala Asp Lys 665 Glu Gin 680 Val Arg Tyr Tyr Val Asn Met Pro 745 Val His 760 Ala Glu Leu Asn Gly Ile Ala Pro 825 Val Thr 840 Ala Ala Val Ile Glu Asn Val Ile 905 Ala Tyr 920 Ser Phe Leu Trp Ala Gly Leu Thr 985 Gly Thr 1000 Leu Thr Tyr Arg Leu 650 Asn Leu Leu Ile Val 730 Phe Ser Ala Leu Asn 810 Asn Leu Arg Met Glu 890 Val Cys Ile Asp Arg 970 Pro Met Gly Arg Ala 635 Val Lys Ile Phe Lys 715 Thr Thr Ile Pro Thr 795 Gly Asn Phe Ser Asn 875 Ser Ile Tyr Lys Val 955 Met Asn His Gly Ile 620 Glu Thr Met Lys Trp 700 Trp Ser Asn His Pro 780 Thr Ile Asn His Asn 860 Gin Phe Val Trp Ile 940 Ala Thr Ala Arg Pro Gly Glu Ser Met 670 Glu Lys Gly Ser Glu 750 Ala Leu Arg Lys Asn 830 Val Gly Thr Tyr Ile 910 Asn Asp Asn Lys Gly 640 Pro Ile Val Lys Pro 720 Glu Phe Ser Pro Ser 800 Phe Thr Gly Gly Glu 880 Leu Ile Arg Ser Asn 960 Arg Phe Glu Met Met Asn Gin Asp 990 Pro Gly 1005 Gly Asn 1010 1015 1020 Ser Thr Phe Tyr Gly Asn Gin Tyr His Asp Asp Pro Ser Pro Tyr Ala SUBSTITUTE SHEET (RULE 26) WO 99/25833 PCT/US98/24327 1025 1030 1035 1040 Thr Thr Thr Leu Val Leu Ser Asn Gin Gin Pro Ala Trp Leu Asn Asp 1045 1050 1055 Lys Met Leu Arg Ala Pro Ala Met Pro Thr Asn Pro Val Pro Pro Glu 1060 1065 1070 Pro Pro Ala Arg Tyr Ala Asp His Thr Ala Gly Arg Arg Ser Arg Ser 1075 1080 1085 Ser Arg Ala Ser Asp Gly Arg Gly Thr Leu Asn Gly Gly Leu His His 1090 1095 1100 Arg Thr Ser Gly Ser Gin Arg Ser Asp Ser Pro Pro His Thr Asp Val 1105 1110 1115 1120 Ser Tyr Val Gin Leu His Ser Ser Asp Gly Thr Gly Ser Ser Lys Glu 1125 1130 1135 Arg Thr Gly Glu Arg Arg Thr Pro Pro Asn Lys Thr Leu Met Asp Phe 1140 1145 1150 Ile Pro Pro Pro Pro Ser Asn Pro Pro Pro Pro Gly Gly His Val Tyr 1155 1160 1165 Asp Thr Ala Thr Arg Arg Gin Leu Asn Arg Gly Ser Thr Pro Arg Glu 1170 1175 1180 Asp Thr Tyr Asp Ser Val Ser Asp Gly Ala Phe Ala Arg Val Asp Val 1185 1190 1195 1200 Asn Ala Arg Pro Thr Ser Arg Asn Arg Asn Leu Gly Gly Arg Pro Leu 1205 1210 1215 Lys Gly Lys Arg Asp Asp Asp Ser Gin Arg Ser Ser Leu Met Met Asp 1220 1225 1230 Asp Asp Gly Gly Ser Ser Giu Ala Asp Gly Glu Asn Ser Glu Gly Asp 1235 1240 1245 Val Pro Arg Gly Gly Val Arg Lys Ala Val Pro Arg Met Gly Ile Ser 1250 1255 1260 Ala Ser Thr Leu Ala His Ser Cys Tyr Gly Thr Asn Gly Thr Ala Gin 1265 1270 1275 1280 Arg Phe Arg Ser Ile Pro Arg Asn Asn Gly Ile Val Thr Gin Glu Gin 1285 1290 1295 Thr INFORMATION FOR SEQ ID NO:7: SEQUENCE CHARACTERISTICS: LENGTH: 4956 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:

ATGAAATGGA

CACCTGTTTC

GGGACGCCAA

CTTCGTCAGG

AAAGGAGAAC

TGGTACAAAG

TTGCTGCCGA

GATGAAGGAG

GCATCGCTGG

GTTGCAGTAG

ACCATTTCAT

CGAGGAGGAA

AACATGTTCC

TGGCCCAGCT

TCCCCACCTC

AAGATTTTCC

CTGCAACTTT

GGGGAGAGAG

GTGGATCTTT

TCTATGTCTG

AAGTAGCCAT

GAGAGCCTGC

GGAAGAAAGA

AGCTCATGAT

TTTTTTGGTC

TATTCCAGAC

TGATAACGAT

ACCTCGCATT

GAACTGCAAA

AGTGGAGACA

ATTTTTCTTA

TGTAGCAAGG

ACTTCGGGAT

AGTAATGGAA

TGGCTCTCCA

CACTTACACC

ATGATATCAC

CCTGAAGATG

GACAATTCGC

GTTGAACACC

GCTGAAGGCC

GACAAAGATG

CGTATAGTAC

AATTACCTTG

GACTTCAGAC

TGCCAACCTC

CTGGATGATA

CGTAAAAGTG

TCCTCAGCTT

TAGAGAGGGG

TGGGCTATAC

CTTCAGACCT

GCCCCACACC

ACCCTCGCTC

ATGGACGGAA

GAGAGGCTGT

AAAACCCTTC

CACGAGGCCA

AAGATGAAAG

ACGCTGGCAA

ATCCCCAAAT

GAACGACCAC

AGGCTCCCGT

GATTGTCTCA

CACTATTGAA

ACACCGAATG

AAGTAGACCT

GAGCCACAAT

GGATGTCATG

TCCTGAGCCC

AATAACTATA

ATATGTTTGT

120 180 240 300 360 420 480 540 600 660 720 SUBSTITUTE SHEET (RULE 26) WO 99/25833 WO 9925833PCT/US98/24327

GTTGGTACCA

AGACCATCAT

TTTAAATGTG

GAGCTGCCCA

ACAGCTGGTG

GCATCTGCTA

GTTGTTGCTT

GCTATTTTCT

TCATCCAGCC

TCTGATGTTG

GCATATTTGG

CCTGTGAATC

AGTCCAGTGC

CGAATCAAAC

GGTCGGTACA

GAAGTTCAAG

CCTAGTGCCC

TGGCAACCAA

CATGCATCTG

ATTAAAGGAC

GGAATTAGTG

AGTCAGGGGG

CACAACCCCA

TCTCAGTATA

TCAGACTGGT

CTCAGAAAGG

GCAGATAGTG

GGTGTAACTG

CCTCCAGAAG

GAAACTCGAT

TTTCTTGTTC

GGGGTAAAGA

GAGGACCAAG

GCAGGTATTG

CACCGCAAGA

TTTACCTTCA

AGGCCTGGAC

CCTAATACTG

AACAGCGACA

AACCAACTGG

GTGGACCTTA

GGGCGTTTTG

TCAAACCTCA

CCACTGGGAC

AAGCTGAACA

TCATACGACC

GGGAGTCAGG

AACTGGGCAG

GAGTACAACA

GCAAGGATGT

CCCCCTGTTC

GCCACTCTGA

GAGACTGGCC

CCACCACGGC

GATATGGATA

ATATGGTTGG

TTGTGAAGAG

AGGCCCGAGG

AATCCAGATA

ACATGGGTTC

CTCTGACTGT

TGGGACGGAC

GGAGGAGAGA

GATTTTCAGT

GTTATTACAT

AAGTTACAGA

AGACTGTAGC

CCACCATTCT

AGTTGGAGAA

CCTGCATTGC

AATTTGGAGT

CATCAAAACC

ATTTGAATTC

GTAGCAGCTG

TCAAACCTAA

ATCCAAGCCA

TGGACCACAA

CCGTCCTTTC

TACAAGGATA

TAGTTTTTGA

GAGTCAACTA

AAATCAAGTT

TATCCAAGAA

ACACTCAAAA

ACCACATCAA

CTGGAATCCG

GTGAGCCTCA

TCAGCCTCGC

GAGCAGCCTG

AGAGAAACGG

CACCAACAGT

TTCTCAACAT

GCAACAACCA

GCAACCTCAC

ATAACAAACA

GTAACAAAAT

TCAATCCATC

GCAACAACAT

AGCAGAAACA

AAGATTATCG

AGAACACAGG

GGCACAAGAA

ACCTGCTTCC

TTTCTGTAGA

ATTTGCAACA

GGGGAGCAGC

CTCCCTCCCC

ACATGCAGCA

CGATCTCCCC

CGGATGCGCC

GGAACGTGAG

ACCCAGTAAC

TGACCCTGTA

TGAAATCCGA

ATACACTTGT

TCAAGAACCT

TGTAACTTTT

AGGGAGTCAG

CTCCCAGACT

CTGCCAGACT

TGTGATTGCA

CGTGGATGGC

GTGGAGAAAG

TGGAGTACTG

ATCAACCCCC

TCCAGTTCAG

TGAAGTGACA

AGGAGCAACT

GCAGACCGTA

TGCAATTTAC

AATATCAGAT

GCAGGTCCAG

TTCCTCTTCC

TAAAATTCTC

AGTGAGGACG

TGAAATTAAG

TGCCAAAACC

TGATGGAAAC

TGGAATGGTC

CAAAACAGTG

ATACAGTGTG

GTTCATCCAG

TCAGCAGATT

TTGGATCATC

ACTTACTAGT

AACTTACCAG

CAGTGAACCT

CAATGACTGC

TACCTACAGT

AACAAATCTG

CAATGAGATG

AGGGCAGCCT

GAACAATGGC

AGAAGTGGCA

AGCAAATGAC

AGGATCCTAC

AGGGGCAAGA

TCCTCCCCCA

TGAAAGCTAT

AGATGAATTA

TTCTTCTCCA

ACAGGAAGAA

CCAGCCCGAC

TCCACATACC

AGAAGAGGAA

AGTGAAGTAG

TTGGCAGTAA

CCTACAGTAC

GATGATCATA

GTTGCAGAAA

CCACATTTTG

CAGTGTGAAG

AATCTACTTT

GGCGACCTCA

TTAAATGTTG

GATCGGCCTC

ACTTTCGTCC

GATGGAGTCC

CAGATCCGAT

AGTGGTGAAG

CCTCCAAGAC

GATGTCAGCA

CCAACATCTT

GCAGAGAATG

CTTTTCCTTG

CCAGTGAAAA

AGAGAGCTGG

ATCGAAGTGC

TATCGGCCAT

CCAGCCAAAA

GCTCGCCCTT

CTGGAAGAAG

GGAACTGCAA

CAAGAGTATA

GATGGTTCCA

GAAGTGGCAG

CTGGATGCCC

TCAGATGTGG

CTCATGGTCT

ACCTACGCGG

AGAGGAGGCG

GCCGCGCAGC

TCCATCAGCT

CGCCCAGCTG

ATGCTCCCTG

AAAACCTTCA

ACTCCTTACG

AGCGGGGACT

CCAGTTCAGT

ACAGTTCCTC

AACAGCTCAG

ACACCCAAGG

GCACATCCTC

GACCAAGAAA

GAAGAGGAGG

GCTGCCGTGT

CTCCAGCCCA

AGGAGACGGC

TATGGCTACA

GAAGACGAAG

CCGAGCTGAC

CTGTGGATGA

GATGGAGGAA

CCTTGAAAAT

ATATGGTGGG

TTGTGAAACC

CAACCGGAAA

TCTCATATCA

CAATTACTAA

CTGGAAGCAT

CCCCAGT-TAT

TCAGCTGTGT

TCGTTTCAAC

ATGCTAAGCT

CAACATGGAG

CTACTGACCC

GAAATACAGT

ATATTATAGA

TGAAAACAGA

TGAGGGCAGC

CACAAGATGT

GAAATGCTGT

ACTGGACAGT

CTGGAGCCAA

ACAGTGTGGT

TTTTTAATGA

CACCCAGTGC

TTCTAGTTAG

AGGTTTGGTG

CCTTTTCCGT

CCAGCACTGG

ATGGAAACCC

TGAAGCAGCC

TCAGCATCTG

GTATCAGAAA

AAGCTGTCAG

CATGGCTGGC

GCTGCACGGC

ATTGTATAGC

AGTCAACTGT

ATAGCCCAA

CCACCACTCA

CTGGCGAGAA

ACAACATCGT

CAACTATCCC

ACCGGGGCAG

TACCAAAACA

CTCCACACAG

TGCCATGTCC

AAGATGAACG

CCTATAGCCA

TGTTACAGGA

AGCCTGTGAG

TTTCAGGACC

CCGACATGGA

TGTCTTAGAG

CAGTGCAGAA

AGATGATGGA

TAGGAAGGTG

CAAAGCTGAA

CCGTGACCAG

TCCTCAACCA

ACCACCACAG

TGTCCAGCGA

CATCACAAAG

TCGACAAGGT

GGCCACAGGC

CCAAGACTCT

GGGTGATACT

TGCTTACATT

AAATTTAATC

CACATTATCG

AGCCTTCAGC

AACATCTGCC

TAATGCATAT

CCTACCAACA

TCTGCACCTC

AGATCAACAG

CCACGGAGAA

AATCCCTGAT

ATTTCAAGGA

CCCACCCCAA

TTGGCAGCCA

TCTGGGCAAT

GGTCATTCCC

GGCTGGGTCT

TGTGTCACCT

GGCCTTCATA

GCTTTATCGA

AGTCCCGTCT

CAGTGGAGGG

AGACACGTGG

AGGCAATGGA

AAATTATAAC

TTATGGTGAT

TCTGAAGGAT

GCTCATCCAG

GCACTGGAAA

GGAGCAAAAC

ATACAACCAA

TAGTACATCT

GGGTGGCATG

CAATAGCGAA

CGTGCCACCA

AGGCCCCACT

TCAGTCCACT

TTGTCCAGAG

TCCTCCTCCA

CCTGGTCTCA

GGTAGCCAAG

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 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 3000 3060 3120 3180 3240 3300 3360 3420 3480 3540 3600 3660 3720 3780 3840 3900 3960 4020 SUBSTITUTE SHEET (RULE 26) WO 99/25833 WO 9925833PCT/US98/24327

ATGCAAACCA

GACCTGGAGA

GACAACATTT

GCTGACTTTG

CGGCAAATGC

ACAAGTCCCG

GCCAAGAAAC

CCACCACCTC

CTGGAAGTAC

TCATCAGACA

GTTGACATGC

AAAGGACGTG,

CAAGAGGATA

CCCAGTTCCT

AATGTAGGTC

GAAGGCTTTT

GCTCTGTCAC!

CCAGCGGACG

CCCAGGCAGT

AGGATGCTGC

TGTCTACAGA

TGAAACACCA

CTGTGCCGCC

GACCTGTAGT

GAAAAGGAAG,

GAACAAATCC

GAAACAAGGC

TTCTACCTTA

CAAGCTCAAT

GAAGAAATAT

GTTACGTGGG

GGGGTCCATG

CTCCAGTGTT

CGCAGCAGCG

TGGCCGTCGA

CAGCAACATG

GCCAGGACAT

ACCTGC.TATA

GGTGCCAAAA

CAGTTACAAG

AGGTGATCCC

AGCAAAACGA

TTGTAGACCT

GTCATCAAGA

T.GCAGAAATG

CTTGAGCAGA

ATCAACGGCT

AGTTCTTCGG

GCAGAGTATG

CATTTTCATG

AGTGCCGCCG

CTGCGCAGAG

AAGTCACCTA

CTCCCTTCTA

GGGAGAGAAG

AGAGAAGCAC

GACCTTCCAC

ACTTTTCCAA

GGATCAGGAA

CAGGTACTTG

CACCTGCCTC

GGGGCTCAGC

ACGGCTCCTT

CTGGTCTGAA

CGTCTCAGTG

TAATGCAGAA

AAACCTACAC

CTGCCCAATC

TGGATGCAAG

TGTTGGATGG

AGGAACAGCA

CAGCAAAGAC

CATCAAATAA

GCAGACAAAG

GAGGATATGA

CAGTGTTGGG

CTCAGAGGAG,

TTTCACTGAT

AGTAGCACGA

CCCTAGGCCC

AACCAGACCA

AGATGATCTT

CAAGACACAG

AACAGACAGA

AAGACAGGTT

AAATGACGGG

TCATCTCATC

TCCCAGAGAT

AGAACAAGCA

AAGAGGAGAA

4080 4140 4200 4260 4320 4380 4440 4500 4560 4620 4680 4740 4800 4860 4920 4956 GATAATAATG AAGAATTAGA GGAAACTGAA AGCTGA INFORMATION FOR SEQ ID NO:8: SEQUENCE CHARACTERISTICS: LENGTH: 1651 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: Met Lys Trp Lys His Val Pro Phe Leu Val Met Ile Ser Leu 1 Leu 10 Gin Leu Ser Ser Pro Asn His Leu Phe Leu Ala Gly Leu Ile Pro Asp Val Giu Asn Asp Asp Arg Gly Asn Asp His Thr Pro Ile Pro Leu Asp Pro Glu Thr Ser Asp Arg Gin Giu Asn Ser Leu Asp Phe Gly Tyr Thr Gly Ser 55 Val Glu His Pro Ser 75 Arg Asp Pro Pro Arg Ile Leu Ile Val Lys Ser Gly Giu Pro Thr Leu Asn Cys Ala Giu Gly Arg Pro Thr Pro Thr Ile Asp Asp Pro Phe Leu Arg Giu 100 Arg Tyr Lys Gly Arg Val Giu Ser His Arg Met 120 Arg Leu Pro Ser Thr Asp Lys 110 Ser Leu Phe Glu Gly Val Ile Val His 130 Tyr Val Gly 135 Asn Lys Ser Arg Pro 140 Ala Cys Val Ala 145 Ala Arg 150 Ala Tyr Leu Gly Val Ser His Ser Leu Giu Ile Leu Arg Phe Arg Gin Asn Pro 175 Ser Asp Val Pro Pro Arg 195 Ser Pro Leu Met 180 Gly Ala Val Gly Ala Val Met His Pro Glu Pro 200 Giu Ile Ser Trp Lys 205 Arg Glu Cys Gin 190 Lys Asp Gly Gly Giy Lys Asp Asp Lys 210 Leu Met 225 Asp 215 Arg Arg Ile Thr Ile Thr Tyr Lys Ser Asp Ala 235 Lys Tyr Val Cys 240 SUBSTITUTE SHEET (RULE 26) WO 99/25833 PCT/US98/24327 Val Gly Thr Asn Met Val Gly Glu Arg Glu Ser Glu Val Ala Glu Leu 245 250 255 Thr Val Leu Glu Arg Pro Ser Phe Val Lys Arg Pro Ser Asn Leu Ala 260 265 270 Val Thr Val Asp Asp Ser Ala Glu Phe Lys Cys Glu Ala Arg Gly Asp 275 280 285 Pro Val Pro Thr Val Arg Trp Arg Lys Asp Asp Gly Glu Leu Pro Lys 290 295 300 Ser Arg Tyr Glu Ile Arg Asp Asp His Thr Leu Lys Ile Arg Lys Val 305 310 315 320 Thr Ala Gly Asp Met Gly Ser Tyr Thr Cys Val Ala Glu Asn Met Val 325 330 335 Gly Lys Ala Glu Ala Ser Ala Thr Leu Thr Val Gin Glu Pro Pro His 340 345 350 Phe Val Val Lys Pro Arg Asp Gin Val Val Ala Leu Gly Arg Thr Val 355 360 365 Thr Phe Gin Cys Glu Ala Thr Gly Asn Pro Gin Pro Ala Ile Phe Trp 370 375 380 Arg Arg Glu Gly Ser Gin Asn Leu Leu Phe Ser Tyr Gin Pro Pro Gin 385 390 395 400 Ser Ser Ser Arg Phe Ser Val Ser Gin Thr Gly Asp Leu Thr Ile Thr 405 410 415 Asn Val Gin Arg Ser Asp Val Gly Tyr Tyr Ile Cys Gin Thr Leu Asn 420 425 430 Val Ala Gly Ser Ile Ile Thr Lys Ala Tyr Leu Glu Val Thr Asp Val 435 440 445 Ile Ala Asp Arg Pro Pro Pro Val Ile Arg Gin Gly Pro Val Asn Gin 450 455 460 Thr Val Ala Val Asp Gly Thr Phe Val Leu Ser Cys Val Ala Thr Gly 465 470 475 480 Ser Pro Val Pro Thr Ile Leu Trp Arg Lys Asp Gly Val Leu Val Ser 485 490 495 Thr Gin Asp Ser Arg Ile Lys Gin Leu Glu Asn Gly Val Leu Gin Ile 500 505 510 Arg Tyr Ala Lys Leu Gly Asp Thr Gly Arg Tyr Thr Cys Ile Ala Ser 515 520 525 Thr Pro Ser Gly Glu Ala Thr Trp Ser Ala Tyr Ile Glu Val Gin Glu 530 535 540 Phe Gly Val Pro Val Gin Pro Pro Arg Pro Thr Asp Pro Asn Leu Ile 545 550 555 560 Pro Ser Ala Pro Ser Lys Pro Glu Val Thr Asp Val Ser Arg Asn Thr 565 570 575 Val Thr Leu Ser Trp Gin Pro Asn Leu Asn Ser Gly Ala Thr Pro Thr 580 585 590 Ser Tyr Ile Ile Glu Ala Phe Ser His Ala Ser Gly Ser Ser Trp Gin 595 600 605 Thr Val Ala Glu Asn Val Lys Thr Glu Thr Ser Ala Ile Lys Gly Leu 610 615 620 Lys Pro Asn Ala Ile Tyr Leu Phe Leu Val Arg Ala Ala Asn Ala Tyr 625 630 635 640 Gly Ile Ser Asp Pro Ser Gin Ile Ser Asp Pro Val Lys Thr Gin Asp 645 650 655 Val Leu Pro Thr Ser Gin Gly Val Asp His Lys Gin Val Gln Arg Glu 660 665 670 Leu Gly Asn Ala Val Leu His Leu His Asn Pro Thr Val Leu Ser Ser 18 SUBSTITUTE SHEET (RULE 26) WO 99/25833 PCTIS98/24327 675 680 685 Ser Ser Ile Glu Val His Trp Thr Val Asp Gin Gin Ser Gin Tyr Ile 690 695 700 Gin Gly Tyr Lys Ile Leu Tyr Arg Pro Ser Gly Ala Asn His Gly Glu 705 710 715 720 Ser Asp Trp Leu Val Phe Glu Val Arg Thr Pro Ala Lys Asn Ser Val 725 730 735 Val Ile Pro Asp Leu Arg Lys Gly Val Asn Tyr Glu Ile Lys Ala Arg 740 745 750 Pro Phe Phe Asn Glu Phe Gin Gly Ala Asp Ser Glu Ile Lys Phe Ala 755 760 765 Lys Thr Leu Glu Glu Ala Pro Ser Ala Pro Pro Gin Gly Val Thr Val 770 775 780 Ser Lys Asn Asp Gly Asn Gly Thr Ala Ile Leu Val Ser Trp Gin Pro 785 790 795 800 Pro Pro Glu Asp Thr Gin Asn Gly Met Val Gin Glu Tyr Lys Val Trp 805 810 815 Cys Leu Gly Asn Glu Thr Arg Tyr His Ile Asn Lys Thr Val Asp Gly 820 825 830 Ser Thr Phe Ser Val Val Ile Pro Phe Leu Val Pro Gly Ile Arg Tyr 835 840 845 Ser Val Glu Val Ala Ala Ser Thr Gly Ala Gly Ser Gly Val Lys Ser 850 855 860 Glu Pro Gin Phe Ile Gin Leu Asp Ala His Gly Asn Pro Val Ser Pro 865 870 875 880 Glu Asp Gin Val Ser Leu Ala Gin Gin Ile Ser Asp Val Val Lys Gin 885 890 895 Pro Ala Phe Ile Ala Gly Ile Gly Ala Ala Cys Trp Ile Ile Leu Met 900 905 910 Val Phe Ser Ile Trp Leu Tyr Arg His Arg Lys Lys Arg Asn Gly Leu 915 920 925 Thr Ser Thr Tyr Ala Gly Ile Arg Lys Val Pro Ser Phe Thr Phe Thr 930 935 940 Pro Thr Val Thr Tyr Gin Arg Gly Gly Glu Ala Val Ser Ser Gly Gly 945 950 955 960 Arg Pro Gly Leu Leu Asn Ile Ser Glu Pro Ala Ala Gin Pro Trp Leu 965 970 975 Ala Asp Thr Trp Pro Asn Thr Gly Asn Asn His Asn Asp Cys Ser Ile 980 985 990 Ser Cys Cys Thr Ala Gly Asn Gly Asn Ser Asp Ser Asn Leu Thr Thr 995 1000 1005 Tyr Ser Arg Pro Ala Asp Cys Ile Ala Asn Tyr Asn Asn Gin Leu Asp 1010 1015 1020 Asn Lys Gin Thr Asn Leu Met Leu Pro Glu Ser Thr Val Tyr Gly Asp 1025 1030 1035 1040 Val Asp Leu Ser Asn Lys Ile Asn Glu Met Lys Thr Phe Asn Ser Pro 1045 1050 1055 Asn Leu Lys Asp Gly Arg Phe Val Asn Pro Ser Gly Gin Pro Thr Pro 1060 1065 1070 Tyr Ala Thr Thr Gin Leu Ile Gin Ser Asn Leu Ser Asn Asn Met Asn 1075 1080 1085 Asn Gly Ser Gly Asp Ser Gly Glu Lys His Trp Lys Pro Leu Gly Gin 1090 1095 1100 Gin Lys Gin Glu Val Ala Pro Val Gin Tyr Asn Ile Val Glu Gin Asn 1105 1110 1115 1120 19 SUBSTITUTE SHEET (RULE 26) WO 99/25833 PCT/US98/24327 Lys Leu Asn Lys Asp Tyr Arg Ala Asn Asp Thr Val Pro Pro Thr Ile 1125 1130 1135 Pro Tyr Asn Gin Ser Tyr Asp Gin Asn Thr Gly Gly Ser Tyr Asn Ser 1140 1145 1150 Ser Asp Arg Gly Ser Ser Thr Ser Gly Ser Gin Gly His Lys Lys Gly 1155 1160 1165 Ala Arg Thr Pro Lys Val Pro Lys Gin Gly Gly Met Asn Trp Ala Asp 1170 1175 1180 Leu Leu Pro Pro Pro Pro Ala His Pro Pro Pro His Ser Asn Ser Glu 1185 1190 1195 1200 Glu Tyr Asn Ile Ser Val Asp Glu Ser Tyr Asp Gin Glu Met Pro Cys 1205 1210 1215 Pro Val Pro Pro Ala Arg Met Tyr Leu Gin Gin Asp Glu Leu Glu Glu 1220 1225 1230 Glu Glu Asp Glu Arg Gly Pro Thr Pro Pro Val Arg Gly Ala Ala Ser 1235 1240 1245 Ser Pro Ala Ala Val Ser Tyr Ser His Gin Ser Thr Ala Thr Leu Thr 1250 1255 1260 Pro Ser Pro Gin Glu Glu Leu Gin Pro Met Leu Gin Asp Cys Pro Glu 1265 1270 1275 1280 Glu Thr Gly His Met Gin His Gin Pro Asp Arg Arg Arg Gin Pro Val 1285 1290 1295 Ser Pro Pro Pro Pro Pro Arg Pro Ile Ser Pro Pro His Thr Tyr Gly 1300 1305 1310 Tyr Ile Ser Gly Pro Leu Val Ser Asp Met Asp Thr Asp Ala Pro Glu 1315 1320 1325 Glu Glu Glu Asp Glu Ala Asp Met Glu Val Ala Lys Met Gin Thr Arg 1330 1335 1340 Arg Leu Leu Leu Arg Gly Leu Glu Gin Thr Pro Ala Ser Ser Val Gly 1345 1350 1355 1360 Asp Leu Glu Ser Ser Val Thr Gly Ser Met Ile Asn Gly Trp Gly Ser 1365 1370 1375 Ala Ser Glu Glu Asp Asn Ile Ser Ser Gly Arg Ser Ser Val Ser Ser 1380 1385 1390 Ser Asp Gly Ser Phe Phe Thr Asp Ala Asp Phe Ala Gin Ala Val Ala 1395 1400 1405 Ala Ala Ala Glu Tyr Ala Gly Leu Lys Val Ala Arg Arg Gin Met Gin 1410 1415 1420 Asp Ala Ala Gly Arg Arg His Phe His Ala Ser Gin Cys Pro Arg Pro 1425 1430 1435 1440 Thr Ser Pro Val Ser Thr Asp Ser Asn Met Ser Ala Ala Val Met Gin 1445 1450 1455 Lys Thr Arg Pro Ala Lys Lys Leu Lys His Gin Pro Gly His Leu Arg 1460 1465 1470 Arg Glu Thr Tyr Thr Asp Asp Leu Pro Pro Pro Pro Val Pro Pro Pro 1475 1480 1485 Ala Ile Lys Ser Pro Thr Ala Gin Ser Lys Thr Gin Leu Glu Val Arg 1490 1495 1500 Pro Val Val Val Pro Lys Leu Pro Ser Met Asp Ala Arg Thr Asp Arg 1505 1510 1515 1520 Ser Ser Asp Arg Lys Gly Ser Ser Tyr Lys Gly Arg Glu Val Leu Asp 1525 1530 1535 Gly Arg Gin Val Val Asp Met Arg Thr Asn Pro Gly Asp Pro Arg Glu 1540 1545 1550 Ala Gin Glu Gin Gin Asn Asp Gly Lys Gly Arg Gly Asn Lys Ala Ala SUBSTITUTE SHEET (RULE 26) WO 99/25833 PCTIUS98/24327 Lys Arg Asp Leu Pro Pro Ala Lys Thr His Leu Ile Gin Glu Asp Ile 1570 1575 1580 Leu Pro Tyr Cys Arg Pro Thr Phe Pro Thr Ser Asn Asn Pro Arg Asp 1585 1590 1595 1600 Pro Ser Ser Ser Ser Ser Met Ser Ser Arg Gly Ser Giy Ser Arg Gin 1605 1610 1615 Arg Giu Gin Ala Asn Val Gly Arg Arg Asn Ile Ala Giu Met Gin Val 1620 1625 1630 Leu Gly Giy Tyr Giu Arg Gly Giu Asp Asn Asn Giu Giu Leu Giu Glu 1635 1640 1645 Thr Giu Ser 1650 INFORMATION FOR SEQ ID NO:9: SEQUENCE CHARACTERISTICS: LENGTH: 1300 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: miscfeature LOCATION: 855..1187 OTHER INFORMATION: /note= "N signifies gap in sequence (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:

CAGATTGTTG

CCAGCTGTTT

CAGCCCAACA

CGTTCCGACG

AAAGCTCAAC

GGCCCAGCCA

GGTGATCCTC

CCAAGAGCAA

ACTGGCACTT

CTGGATGTGA

GGGCCACCAT

CAGCCAGGTA

TCAGTGAGCA

AGAGGACTGC

TCAGTGACCC

CCTCCCCCCC

CAAGAAAATG

CACCAAGGTC

CGAGGCGTGG

CCATCCCCAC

AAGACCTCGA

AAGTCAGAGG

CTCAAGGTCG

TTTGGCAGAA

GTAGATGCTC

CGGGTTACTA

TGGAGGTTAC

ACCAAACGCT

TTCCTGTAAT

CAATTCAAGA

ATACTTGTGT

CAGAGTCTGG

CCAAACCGCA

CCCCTGGAAC

ACAGCTGGCA

GGCCCAATAC

AAGTNAAACC

CAGTCCAGCC

GCTATGACAG

TGGAAGATGA

CTTCTTCTCC

GGGAAGAGAT

CCTACCAGCC

CCTCGGCCCA

AACAGTGACA

AGAAGGCAGC

AGTGTCACCA

CATCTGCCAG

TGATGTTTTG

GGCAGTGGAT

TAGCTGGTTA

GCAAGGCACA

GGCTACAAGT

AGCAACAATC

AGTCACTGAT

CCTTCCAGCA

GACCGTGGCA

AATCTACTTA

ACAGAAAAAC

CCTTCCTGGC

TGATAGCTGG

ACTGGAAGAA

TGCTATCTCC

GCAACCCATG

CATTTTCTAC

CTAAAAAACA

TTTCCCTGTG

CAGAACCTAC

ACTGGAGACC

GCTTTAACTG

ACAGATAGAC

GGTACAGCGT

AAGGAGGGAT

CTGCAGATTA

TCAAGTGGAG

AGTAAAAACT

GTTACTAAGA

AGTGCATATA

AACCATGTAA

TTCATGGTCA

AATGGATCCA

ACGGAGCTGG

TGCCCACCAT

GATGATGATA

TTTGGACAGC

CTGCAGGCTT

TGACAGTAAC

CAAGGGAGGG

AAACTAAAGG

TTTTCCCAAA

TCACAATCAC

TGGCAGGAAG

CTCCACCTAT

TACTGAAATG

TTACTTTTCC

AGAATTTACG

AGGCTTCCTG

ATGATTTAAG

ACAGTGTCAC

TCATTGAGGC

AGACCACCCT

GAGCGATCAA

CTTGGGCCAA

AACACTATGC

TGCCAGTACA

GGGTCCCAAC

AGTCCACTGC

CACCTNTTTA

ACCAGTGCAG

AAACCCACAG

CCAACCCCAG

CAACATTCAA

CATTTTAGCA

AATTCTACAA

TAAAGCCACT

GGGTAGAGAT

GATTTCTGAT

GAGTGCAGTG

TGACCTGCCA

CTTGTCCTGG

TTTCAGCCAA

CTATACTGTA

CCCCAAGGTY

TGTCCCTCTA

AGTGGAACAA

AACTTACTTA

ACCTCCTGTT

AACTCTTACT

CCTCCTCTCA

CCCTGAGTCA

120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1300 INFORMATION FOR SEQ ID NO:l0: SEQUENCE CHARACTERISTICS: LENGTH: 434 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear 21 SUBSTITUTE SHEET (RULE 26) WO 99/25833 PCT/US98/24327 (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Modified-site LOCATION: 285..396 OTHER INFORMATION: /note= "Xaa signifies gap in sequence" (xi) SEQUENCE DESCRIPTION: SEQ ID Gin Ile Val Ala Gin Gly Arg Thr Val Thr Phe Pro Cys Glu Thr Lys 1 5 10 Gly Asn Pro Gin Pro Ala Val Phe Trp Gin Lys Glu Gly Ser Gin Asn 20 25 Leu Leu Phe Pro Asn Gin Pro Gin Gin Pro Asn Ser Arg Cys Ser Val 40 Ser Pro Thr Gly Asp Leu Thr Ile Thr Asn Ile Gin Arg Ser Asp Ala 55 Gly Tyr Tyr Ile Cys Gin Ala Leu Thr Val Ala Gly Ser Ile Leu Ala 70 75 Lys Ala Gin Leu Glu Val Thr Asp Val Leu Thr Asp Arg Pro Pro Pro 90 Ile Ile Leu Gin Gly Pro Ala Asn Gin Thr Leu Ala Val Asp Gly Thr 100 105 110 Ala Leu Leu Lys Cys Lys Ala Thr Gly Asp Pro Leu Pro Val Ile Ser 115 120 125 Trp Leu Lys Glu Gly Phe Thr Phe Pro Gly Arg Asp Pro Arg Ala Thr 130 135 140 Ile Gin Glu Gin Gly Thr Leu Gin Ile Lys Asn Leu Arg Ile Ser Asp 145 150 155 160 Thr Gly Thr Tyr Thr Cys Val Ala Thr Ser Ser Ser Gly Glu Ala Ser 165 170 175 Trp Ser Ala Val Leu Asp Val Thr Glu Ser Gly Ala Thr Ile Ser Lys 180 185 190 Asn Tyr Asp Leu Ser Asp Leu Pro Gly Pro Pro Ser Lys Pro Gin Val 195 200 205 Thr Asp Val Thr Lys Asn Ser Val Thr Leu Ser Trp Gin Pro Gly Thr 210 215 220 Pro Gly Thr Leu Pro Ala Ser Ala Tyr Ile Ile Glu Ala Phe Ser Gin 225 230 235 240 Ser Val Ser Asn Ser Trp Gin Thr Val Ala Asn His Val Lys Thr Thr 245 250 255 Leu Tyr Thr Val Arg Gly Leu Arg Pro Asn Thr Ile Tyr Leu Phe Met 260 265 270 Val Arg Ala Ile Asn Pro Lys Val Ser Val Thr Gin Xaa Lys Pro Gin 275 280 285 Lys Asn Asn Gly Ser Thr Trp Ala Asn Val Pro Leu Pro Pro Pro Pro 290 295 300 Val Gin Pro Leu Pro Gly Thr Glu Leu Glu His Tyr Ala Val Glu Gin 305 310 315 320 Gin Glu Asn Gly Tyr Asp Ser Asp Ser Trp Cys Pro Pro Leu Pro Val 325 330 335 Gin Thr Tyr Leu His Gin Gly Leu Glu Asp Glu Leu Glu Glu Asp Asp 340 345 350 Asp Arg Val Pro Thr Pro Pro Val Arg Gly Val Ala Ser Ser Pro Ala 355 360 365 Ile Ser Phe Gly Gin Gin Ser Thr Ala Thr Leu Thr Pro Ser Pro Arg 370 375 380 Glu Glu Met Gin Pro Met Leu Gin Ala Ser Pro Xaa Phe Thr Ser Ser 22 SUBSTITUTE SHEET (RULE 26) WO 99/25833 PCTIUS98/24327 385 390 395 400 Gin Arg Pro Arg Pro Thr Ser Pro Phe Ser Thr Asp Ser Asn Thr Ser 405 410 415 Ala Ala Leu Ser Gin Ser Gin Arg Pro Arg Pro Thr Lys Lys His Lys 420 425 430 Gly Gly INFORMATION FOR SEQ ID NO:11: SEQUENCE CHARACTERISTICS: LENGTH: 444 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:

GCCCAGGCAG

CAAGATGCTG

GTGTCCACAG

CAGAAACACC

CCAGTGCCAC

CGGCCTGTCA

AGAAAAGGAG

CGAACAAATC

TTGCTGCAGC

CTGGCCGCCG

ACAGCAACAT

AGCCAGGACA

CACCTGCTAT

TGGTGCCAAA

GCAGTTACAA

CAAGTGACCC

TGCGGAGTAT

CCACTTCCAT

GAGTGCTGTT

TCTGCGCAGG

AAAATCGCCC

ACTCGCGTCT

GGGGAGAGAA

CAGA

GCGGGCCTGA

GCCTCTCAGT

GTGATCCAGA

GAAGCCTACG

ACTGTCCAGT

ATAGAAGCAA

GCTCTGGATG

AAGTGGCTCG

GCCCAAGGCC

AAGCCAGACC

CAGATGATCT

CCAAGGCACA

GGACAGATAG

GAAGACAAGT

CCGCCAAATG

CACGAGTCCT

CGCCAAGAAG

TCCACCCCCT

GCTGGAGGTA

ATCGTCAGAC

CACTGACCTG

INFORMATION FOR SEQ ID NO:12: SEQUENCE CHARACTERISTICS: LENGTH: 148 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: Ala Gin Ala Val Ala Ala Ala Ala Glu Tyr Arg Ala Gly Leu Lys Val Ala Arg Arg Gin Gin Cys Pro Met Arg Gin Asp Ala Ala Gly 25 Val Arg His Phe Pro Thr Ser Pro 40 Arg Ser Thr Asp Ser Gin His Ala Ser Asn Met Ser Lys His Gin Ala Val Val Ile Pro Gly His Leu Pro Val Pro Pro Glu Val Gin Lys Arg Arg 70 Pro Ala Arg Pro Arg Ser Asp Gly Ala 55 Glu Pro Ala Lys Ala Tyr Ala Asp Thr Lys Asp Gin Leu Ile Lys Ser Val Met Val 105 Ser Asp Arg Pro 90 Pro Val Gin Leu Pro Pro Lys Leu Ala 100 Asp Ser Lys Ala Ser Ile Glu 110 Tyr Lys Gly Thr Asn Pro Ala Arg Thr Lys Gly Gly S 50 Arg Glu Ala Leu 130 Ser Asp Pro Arg 145 Arg 135 Val Thr Asp Leu 140 INFORMATION FOR SEQ ID NO:13: SEQUENCE CHARACTERISTICS: SUBSTITUTE SHEET (RULE 26) WO 99/25833 WO 9925833PCT/US98/24327 LENGTH: 1781 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:

TGAGTGCCTC

TGGCAAATTT

CAGCTAGCAG

ATAGAAACTC

CAAACGATTT

CGCAAGCAGA

ACCACGGATT

ATATCCGCGC

CAGATCACCT

TTCAACGCCC

AGCGGCTGGT

GATCTGGCCG

ATCTTCGGGG

TCGCTGGTCA

CGCACTTGGA

TACTCCTCGC

CATCCACATC

ACAATCGCCA

GCCTACGGCA

ATTTCCAATT

GCAGCGGCAG

CCATCCTCTT

CCAAGTGGGC

GCAGTGGCAG

TTGGTCGTTG

GTCAGTCGAG

TGCATCGTCT

TCTCCCTCCC

TTACGCAAGA

AGCCCTAATA

GCCCACGACT

CCCGCATGGC

TCAGTCAACT

TACGGCAAAT

GTTTACGTGT

AAATAAAGTT

ATCCAACGGT

CGGCCAGCAG

TCGAGATACC

TGCTCCAGAG

CTCCGGATGG

ACTTGCGGGA

ATGCCGACAT

TTCTGTCGCT

AAAAATGCA.A

ACCTGGTTGG

ATCGGAGCAG

CCGGATTGCC

TGAAGTTCGT

GGGAGAAGCA

TGGAGGAGGA

CGGAATCCAG

GGCAGGATGA

TAGCACAAAG

TTGTTGCCGC

GGTTTAGGGC

GCTA.ATGTTG

TCTAAGAAAA

CTGAGTATTA

TCTAAGCGAG

CACTCAAGAG

ATGGCATTCT

TATAGCCGCT

AAAAAAACCC

CGTGACGCGA

TACAAATTAA

GGAGACCACC

CATGAGTCCC

CAGTGCCCAC

GATTGGCAAC

CAGCATCAGC

TAGGTCCGAG

GCATGTGATC

GGTCTTCATC

TAAGGACATA

TTGCGATGCC

CGAGGCTGGG

CAGCTACGAT

TTATCCCTCG

GGAGCCGCTG

TAAAGCCGAC

CAACTTGGCC

GGAGGTGGAT

TTTACAGCCG

GTGATTGCCC

CGGGAGTAGC

TTTGCCCCAT

TTTAGTTTTG

TTTATTGTAC

AGTTGAAGAA

CGAGTGAATC

CAGCGACCCG

CCAGCGAACG

GAAATCGAAC

GTGTGCCAGC

AATAAATCAA

ACAACTGCCG

GCTGCAATTG

GATCTGCGAC

GCAGCGGTGT

ACCGAGCAGC

GAATCGGGCG

ATCAATTATC

CTCTTCAGCT

CGTGCCCAGA

AGCAGACTGC

TTCTACCAAA

GAGGCACTGC

TTGGCGGCCG

AATAAGCTGC

TCCTCATCGT

ACAGCAACGC

AATTCAGATT

GCGGCGCCTG

ACCCAGGGGT

AGCCCAATTG

GTAATAACAT

GTTTTTAGTT

TATGTATAAA

AAAATATAAA

GAGAAAATGA

ATGTAGGTAT

GACGCACCGA

CCATGACTTA

AGTTGTGTGC

AGGCAATCAC

AAGAGCTCTA

CCGAGCACCT

ACATCGACGC

CCTACGATCC

TCTCCAAGTC

AATCCTCGTG

TGTGGATCGG

GCTACTTCTA

TCCATGCGGC

TGTTGCACCA

TCGAATCGCC

ATCATCAGCC

TGCATCATCA

AAAAGTGCAA

CCACGTCTGC

CTGCCATTTG

CCGATTCCGC

CCGACGATGA

CAGAGGTCAG

CTTAGTCACT

ATATCTCCGA

ATCTGTTATA

ACCGAATTGT

A

GTGAGTGGAA

TATGCTCTGC

AAATCGAGCT

TATATAGAGC

GTGTGCCAGA

CATGATTAGC

TGCGGAGTAC

GCAGCAGAAT

CCTCAACTCC

CGCTCCACCC

GGTGGTTCTG

GTGGAGCCAG

AGTGGTCAGC

CAGGAAGTTC

CAGTGACTCG

GCAGATGCAG

GCCCTGCTAC

TCGGCACTTT

TCACCATTGC

GCTGTCAGCG

ATCCGCGTCG

CATTAACATG

AATTGCAGTT

TTGCGCCTCA

GGAATAAGGG

TGTAAATAGT

GAATGTACCC

TATTATTGTA

CTATCGATTA

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 1781 INFORMATION FOR SEQ ID NO:i4: Wi SEQUENCE CHARACTERISTICS: LENGTH: 370 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:i4: Met Ile Ser Thr Thr Asp Tyr Pro Thr Val Glu Thr Thr 1 5 10 Glu Glu Leu Tyr Ala Glu Tyr Ile Ser Ala Pro Ala Ser 25 Pro Ala Ala Ile Ala Giu His Leu Gin Gin Asn Gin Ile 40 Ile Pro Ser Ala His Asp Leu Arg His Ile Asp Ala Leu 55 Asn Ala Leu Leu Gin Arg Ile Gly Asn Ala Ala Val Ser 70 75 Thr Thr Ala Ser Met Ser Thr Phe Giu Asn Ser Phe Tyr Asp Pro SUBSTITUTE SHEET (RULE 26) WO 99/25833 WO 9925833PCTIUS98/24327 Ala Pro Pro Ser Gly Trp Ser Pro Asp Gly Ser Ile Ser Thr Giu Gin 90 Leu Giu Asp Leu 145 Arg Ile Ala Ser Ile 225 Arg Val Leu Glu Ser 305 Ile Ser Ser Giu Met 130 Val Lys His Ser Ser 210 Ala His His Asn Asp 290 Ser Asn Asp Lys Ser 115 His Ile Phe Ala Arg 195 Giu Thr Phe His Lys 275 Lys Ser Met Ser Ser 100 Gly Val Leu Arg Ala 180 Leu Ala Gly Ala His 260 Leu Ala Giu Pro Ala 340 Val Ser Ile Leu 150 Trp Asp Leu Phe Pro 230 Gly- His Lys Ser Ser 310 Gly Ala Leu Ser Asn 135 Val Lys Ser His Tyr 215 Ser Met- Cys Cys Ser 295 Asn Arg Val Asp Trp 120 Tyr Phe Lys Tyr Gin 200 Gin Tyr Lys Ile Lys 280 Ser Leu Gin Ala Leu 105 Trp Leu Ile Cys Ser 185 Gin Ile Asp Phe- Ser 265 Leu Ser Ala Asp Val 345 Al a Ser Trp Leu Asn 170 Ser Met Glu Glu Va..

250 Asn Ser Thr Thr Glu 330 Ala Asp Gin Ile Phe 155 Lys His Gin Ser Ala 235 Tyr Trp Ala Ser Al a 315 Glu Val1 Arg Phe 125 Val1 Cys Ile Val Pro 205 Pro His Ser Lys Ala 285 Ser Pro Asp Gin Arg Asp Ser Phe Ala 175 Cys His Tyr Gin Ala 255 Giu Val Ser Ile Ser 335 Leu Ser Ala Ser Tyr 160 Gin Asp Arg Thr Pro 240 Ala Pro Giu Pro Cys 320 Asp Gin Pro Ala Ala Pro Ala Asp Asp Asp Cys Ala Ser Leu Val Val Val Val 355 Ala Ala 370 360 365 SUBSTITUTE SHEET (RULE 26)

Claims (11)

1. A method of promoting axon outgrowth of a human neuron determined to have expressed on its surface an amount of a natural human Robo and to be subject to an axon outgrowth repulsion mediated by the Robo, said method comprising the step of contacting the neuron with an amount of a natural Comm polypeptide sufficient to reduce the amount of Robo expressed on the neuron, wherein the Comm is in an amount and under said conditions sufficient to reduce the amount of Robo expressed on the neuron, wherein the Comm is provided to the neuron exogenously in a pharmaceutically acceptable composition and whereby the amount of Robo expressed on the surface is reduced, thereby reducing the outgrowth repulsion mediated by the Robo.

2. A method according to claim 1, wherein the Robo consists of SEQ ID NO:8. S

3. A method according to claim 1 or claim 2, wherein the SComm polypeptide consists of SEQ ID NO:14.

4. A method according to any one of claims 1 to 3, wherein the Comm polypeptide is encoded by a natural sequence nucleic acid comprising SEQ ID NO:13 or a fragment thereof at least 36 nucleotides in length.

5. A method according to any one of claims 1 to 4, S 30 wherein the Comm polypeptide is encoded by a natural sequence nucleic acid comprising SEQ ID NO:13 or a ooo fragment thereof at least 72 nucleotides in length.

6. A method according to any one of claims 1 to wherein the Comm polypeptide is encoded by a natural sequence nucleic acid comprising SEQ ID NO:13 or a fragment thereof at least 144 nucleotides in length. \\melbf iles\hme\Pcabral\Keep\peci\ 1409 .99.doc 1/02/02

7. A method according to any one of claims 1 to 6 wherein the neuron is in vitro.

8. A method according to any one of claims 1 to 6, wherein the neuron is in situ.

9. A method according to any of claims 1 to 6 or 8, wherein the neuron is in situ and the composition comprises fibers coated, embedded or derivatized with the Comm polypeptide.

A method according to any one of claims 1 to 6 or 8 wherein the neuron is in situ and the composition comprises fibers coated, embedded or derivatized with the Comm peptide.

11. A method according to claim 1, substantially as herein described with reference to the written 20 description. .Dated this 1st day of February 2002 25 THE REGENTS OF THE UNIVERSITY OF CALIFORNIA By their Patent Attorneys S• GRIFFITH HACK SFellows Institute of Patent and Trade Mark Attorneys of Australia *26 \\melb_tiles\home$\Pcabral\Keep\speci\14094.99.doc 1/02/02