CA2229126A1 - Opioid antagonists and methods of their use - Google Patents

CA2229126A1 - Opioid antagonists and methods of their use - Google Patents.

Classifications. C — CHEMISTRY; METALLURGY C07 — ORGANIC CHEMISTRY C07K — PEPTIDES C07K14/00 — Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof C07K14/435 — Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans C07K14/665 — Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans derived from pro-opiomelanocortin, pro-enkephalin or pro-dynorphin A — HUMAN NECESSITIES A61 — MEDICAL OR VETERINARY SCIENCE; HYGIENE A61K — PREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES A61K38/00 — Medicinal preparations containing peptides. Description. W O 97/07212 PCT. US96/13305 OPIOID ANTAGONISTS AND METHODS OF TE. _ .lion was made at least in part with. -,--l support under National Tnctitlltes of Health grant ROl MH48991. The gv. may have eertain rights to this hl. BACKGROUND OF THE INVENIION Field of the I./ This invention relates to. from mqmmqliqn species and ligands specific for such u. , which are active in the. .. of opioid action. Specifirq-lly. the invention relates to the isolation of an e-.. 1. peptide ligand speeifie for a novel .. -.. liqn reeeptor, the 10. .. of its anti-opioid properties, and use of the ligand for reversing physiologic effects of opiates such as. -;--- The invention also relates to the co. u.:lion of analogues, d. ,.ivalivt:s and peptide. of this c.-dcg_.lu. nmqliqn reeeptor ligand, and their use as opiate .,e. Sperifirqlly provided is a mqmm li. uc opioid reeeptor ligand, synthetic c. l. s and analogues thereof, and methods of making and using 1 5 sueh ligands. P. The use (and abuse) of opiates, sueh as opium and lllul. hille, have been known sinee antiquity (reviewed in B-u. ,hl, 1993, Proe. Natl. Aead. Sci. USA 90: 5391-5393). Since the n;. 1. eentury, chemical. ion and synthesis of _any u-u- hillc analogues have 2 0 been achieved in an effort to diseover a collllvuul.d having the qnqlgecir effeets of llwl. Jhillc but lacking its addietive potential. These efforts have heretofore been m,C. h The biology behind the reasons for the. lpecir nd addietive properties of .llo-, lhillc and ",o" l-i.. uu. ds was first. y of endogenous morphine-like cclu. uullds termed rn' linc (see DiChara & North, 1992, Trends in Pl-al---acol. Sci. 13: 185-2 5 193 for review). .yillg this finding of an endogenous opioid was the biorhrmir. levidence for a family of related but distinet opiate . c. "s, each of which displays a unique pl, l profile of response to opiate agonists and. onictC (see McKnight & Rees, 1991, N_ulu. for review). To date, four distinet opiate. have been by their l. e the 3 0 K, and o , (the o reeeptor has been. I to be a non-opioid reeeptor. ;livily with some opioid agonists). Thus""-""" are known in the art, and some of these proteins have been isolated 1. lly and their c. g genes have been recently cloned using genetic r.,g;. . - -;. g means. Kieffer et al., 1992, Proc. Natl. Acad. Sci. USA 89: 12048-12052 .l; A the isolation of a cDNA copy of the mouse. -opioid receptor by GA. lci7.7ion cloning. Evans et al., 1992, Science. : 1952-1955 AicrlnseA the isolation of a cDNA copy of the mouse. -opioid receptor by. W O 97/07212 PCT. US96/13305 Chen et al., 1993, Molec. pl. ol 44: 8-12 11iCrl'JSP. d the isolation of a cDNA copy of the rat. -opioid receptor. Yasuda a al., 1993, Proc. Natl. Acad. Sci. USA 90: 6736-6740. IOSP(l the isolation of a cDNA copy of each of the mouse K- and. -opioid receptor. Bzdega et al., 1993, Proc. Natl. Acad. Sci. USA 90: 9305-9309 disclose the isolation and. Iocation of the. -opioid receptor in the mouse. The present Ill. Ul 7 have cloned, tA. n receptor gene, d in co-owned and co-pending U.S. Patent. ppli-qtil n No. 08/149,093, filed November 8, 1993, which is hereby hlUjl. . cc in its entirety. 10 SpP. 1 therein are nucleic acids. nrorline the novel. ue this receptor gene, cells cU. ,iaillg the novel receptor gene, and methods for making and using such nucleic acids, as well as cc,n.Llu-; . and cells for opioid. n and novel drug screening. The nucleic acid sequence of the gene and the deduced amino acid sequence of the cognate receptor protein were 15 also. licrlosed in this prior application. The receptor gene was previously referred to in the co-pending case as an MSOR (mPthqrlonP specihc opioid receptor) gene because the receptor bound m. lorlP with a high affinity. Such high affLnity co.. l.e ;r.- ;Iy for mPthq. although this term is not intended to imply that only mPth. ionP binds to the receptor. Indeed, a. s shown herein, an 2 0 L. ls ligand of the present invention binds to the same receptor with startlingly high affinity (producing a physiologic response when. lP or nanomole dose i.c.v.). Now that the. ns ligand has been discovered, and tliC. in co-owned and co-pending U.S. Patent Application No. 08/514,451 filed August 11, l99S, the MSOR terminology is no longer needed. The ligand has been named orphanin FQ (OFQ) from the initial and terminal amino acids of the .I;crlo. " e (F for phenylalanine and Q for gl--lP---;-, ). Hence the receptor will now be referred to as the orphanin FQ receptor (OFQR), which is an anti-opioid receptor (AOR). A great adv. e in efforts for developing novel p7j. uLlulJic drugs which e2certtheir activity ( .ise) via binding to ", "", , opioid or anti-opioid 1C. lul7 3 0 would be to identify the. Iigand(s) which bind to such l. tUl 7. Certain such opioid ligands have been isolated in the prior art, in h. e the peptides c-, . (see Jaffe and Martin, 1990, "Opioid. n and Gilman, eds., The Ph. i.;al Basis of T>.u,. UWll Press, Inc.: New York), Chapter 21, p. 491-531). Although opiates are powerfu Ml. iC agents, their Ih. c is sllffiriPntly low to cause many p-ubi' in their clinical use. Such. lubl include stupor, l. that may be ulld. . of an acute surgical e.ll. or other opiates can W O 97/07212 . PCTrUS96/13305 also develop, and may be difficult for a clinician to. gnnce It has been l. l to reverse opiate effects is clinically useful in these. --either for reducing physiologic effects of the opiate (ulldc;,i, g a willldlawal aylld.ullle to flf. rltlirtinn In spite of the ;.. of opioid allLaE,oni. , progress has only slowly been made in d. of this class of drugs. The best known such. onict is , an Ol. that resembles oAy..lu. h 30 minutes. They were then weighed and given an sc injection of saline (Sal; 10 15 ml/mg) or nql. nP (NAL; 1 mg/kg and 10 mg/kg), or handled but given no injection (None). T.----.. Ai. " mice were lightly '-lpsthpti7p-A with halothane and given and icv injection of vehicle (VEH; 2.5. 1 of artificial CSF) or 2.5 nmole OFQ in 2.5. 1 vehicle (OFQ-2.5). A separate group of Sal-treated mice received no icv injection (None). Between 5 and 10 minutes later, all mice were given an L. uriFal (ip) injection of 0.9% acetic acid and placed back in 2 0 their observation. t 30 minutes, the number of Ah. minql constrictions (L,uE; s of the torso with a cn.

concave archirig of the back) were counted and recorded. Four mice in separate cl. were observed sim. cly by a single e. r blind to drug a. ntlitinn ANOVA revealed a signifir. s. The results are shown in Fig. 13B. The decrea. sed number of constrictions in VEH25 mice is believed to be due to SIA, which was reversed by NAL and OFQ-2.5. The 2.5 nmolc icv dose of OFQ was pha. rql1y 5llffiriPnt to inhibit the. n lgPsic effect of morphine within 30 minutes after the OFQ was. - g 3 0 Reversal of. .al Latency OFQ reverses the. r actions of ulul. Lillc, which are TnPAi. tPA by the opioid SIA 5llh5tr. 1 of their baseline ll. ilivily. It pluduccd a profound. that peaked at 30 minutes post-injection (Fig. 14) and lasted about 2 hours. But OFQ reversed this. lg 3 5 in a dose. manner (at doses of 2.5-25 mnole). Swiss-Webster mice of both se. ces (N=6-ll per group) were tested for baseline n. ilivily on the 49. cample 7), injected s.c. with ulu- Jhillc sulfate (MOR; 5 mg/kg), and then retested 20 min later to establish the r. Of W O 97/07212 PCT. Ciq Analgesic mice (those with TW latencies of at least double their baseline; all e. cept 3) were then lightly. P, injected i.c.v. with vehicle (Veh; 2.5. 1 artificial CSF) or OFQ (2.5, 5, 10 or 25 nmole in 2.5. 1 vehicle), and retested for TW latencies 10, 25, 40 and 100 min later by an. blind to OFQ dose. The 25 mnol OFQ dose 5 produced marked atonia and flaccid paralysis in up to 50% of mice. These side-effects should not, however, be conO;d. ;d a cullruulld of the present data because mice injected with this high OFQ dose actually reacted faster to the thermal ctjmnlllc Fig. 14 shows time-course data d. h;--e qnqlgPciq and its dose- l reversal by four doses of OFQ. Symbols l. SEM) TW latencies from 10 49. C water at each time point. A linear. c,oa;ùll analysis was used to calculate the dose of OFQ causing a 50%;l-I-;I,;l;on of the effect of S mg/kg u.ul. lPtPrminPd that this ED50 was 7.5 mnol in a 2.5. 1 volume. The ED50 is the dose required to produce a response of 50% of ma. t inhibition of qn. lgPciq would be a 50% inhibition of opiate qnql. eciq The ED50 would be a ph. lly sllffiri-ont amount of OFQ to achieve signifirqnt inhibition. W O 97/07212 PCTrUS96/13305 EXA. IPLE 10 A'' ' of p'- -L. , the ability of OFQ to r.. ,;. opioid effects other than. , studied here was the ability 5 to induce hypoth. in rodents at high doses. The DBA/2J mice strain was used to study. n of hypot-hrnni. because of its known robust LylJo. c of DBA/2J mice (15-30 g; The Jackson LalJula. cly; N = 7-8 per group) was assessed ;. ly before and 30 min after systemic. aiion of saline (Sal; 10ml/kg) or. UlC (MOR; 20 mg/kg, i.p.). I.. ' ,r, mice were injected i.c.v. with vehicle (Veh; 2.5. 1 artificial CSF) or 5 or 10 nmole OFQ in 2.5. 1 vehicle (OFQ-5 and OFQ-10), and retested 15, 30 and 60 min later. As shown in Fig. 15, there was a 6. C decrease in body t. ,AaLulci produced byMOR, in addition to a transient Ly. ia produced by halothane. The body. I. c of saline-treated mice l-,lllaulCd constant at about 38. ccept for a brief hypothermic episode from 15 e. , OFQ itself did not affect body tC,Ilp., alulc. However, both doses of OFQ. y reversed 20 mg/kg MOR 1. at 60-90 minutes post-MOR injection (F. 33=44.94,p 5 nmole p, l atonia in some Auol. nAPnt mice that may have hindered jumping behavior. Withdrawal jumping lJ. d by OFQ is shown in Fig. 16. Bars l. mean (iSEM) number of withdrawal jumps in the 20-min testing period. Ratios above the error bars describe the number of animals e. chibiting jumping behavior relative to the total number tested. The wilL L. d by i.c.v. OFQ was uuc. luivocal, albeit modest in c. d by systemic n. (mP (NAL). Other withdrawal :.yulpt. g wet-dog shakes and ptosis, were observed in mice treated with OFQ, but not in mice merely given injectionC of vehicle. Pl. l of withdrawal shall be defined as increasing total withdrawal jumps in the protocol of E. of Affinity of OFQ R. PcPrtor Ag -The peptide of the present invention does not bind with high srecifi. that lack of receptor gl,e. ;ly of ligand-receptor binding can be A. hiuc was used as a c. to assay receptor binding affinity for OFQ. ML- t' d CHO cells ((LC-7) were used at a. -CHO cells were rinsed three times with 50 mM Tris-HCI buffer (pH 7.7), h. , A in the same buffer using a glass-Teflon h.. Jg. ;,. A at 42,000 g at 4. C for 15 minutes. The pellet was 35 . in S0 mM Tris-HCI buffer and ;--- --1, t- for one hour. Following pre-i.-- vb. ;I-eA above and the pellet was stored at 70. W O 97/07212 PCTf. S96/13305 The pellet was thawed, washed with 50 mM Tris-HCl buffer and the holll. ,nàle was Cf ntrifilgPA as above. The pellet was. A in the binding buffer and the protein cu. n was adjusted to 100. i by a standard curve A. say using bovine serum albutnin as the standard. In equilihrillm co. l. "-G-I,hi--e was utilized as an. , and OFQ was used as a displacer. In addition, DAMGO, U50488H and DPDPE were also used as a di. , K and receptor e,. sed CHO cells in these assays. Binding reactions were con. lcted in an inrllh. ti-m tube with 50 mM Tris-HCl buffer (pH 7.7) in the presence of l0 .. cP inhibitor bestatin at 25. C for 60 minutes in a total volume of 1 ml using a final [3H].li. ,.l jnn of 0.36 nM. All of the tubes CJ. cudiull and [3H]di. ulphhle. A ligand of interest was also added to each .. alaliOIl. A subset of the tubes cf nt. inPd 10 micromolar nalo. cone as the ligand of interest, to permit fl. l iu,. of non-specific binding. Another subset of the tubes. ;';nf'd OFQ in the presence of the [3H]di. rPnnPnt of OFQ from each class of receptor by the. .-G-l!Li--e displacer. Another subset of tubes cu..l; ;nr. DPDPE, while yet another subset cu. 1 U50488H as the ligand of interest. Each of the ligands of interest was present in a cu-, .-l-aLion from zero in order of. IF Up to 100 micromolar. The c. .lG",hi"e displacer was varied, in 2 0 different sets of tubes, across the range shown in Fig. 17. At the end of the 60 minutes period of inrllh. d byvacuum filtration using a Brandel CellHarvester over polyethylenimine (0.5%)-soaked GF/B filters. The Cell Harvester has a manifold that 5imlllt. nPo-lcly emptied out all of the reaction tubes in a controlled manner and deposits the contents on a filter disk that c. Junds to each 2 5 tube, washed 2 times with 2 ml each of ice. old 50 mM Tri-HCl buffer, and the contents again. g filter disk. Filter disks were placed in minivials, allowed to elute overnight in EcoLume 5rintill. n fluid (ICN, Costa Mesa, CA), and then counted in a Rerkm. The resulting data were ladioac.

counts, which were proportional to percent total 3 0 bound di. Iiy:rl-l The percent bound ligand of interest (e.g., OFQ) was inversely. al to the amount of l. livily detected. As shown in Fig. 17, the percent of total OFQ at the. was stable over a broad range of di. -I-,-linnc The DAMGO, DPDPE and U50488H ligands (known to bind to the. ) were, however, cu. f. A 3 5 For each set of ligand. l studies, the results obtained with the 5rintilP-tinn counter were analyzed using an LRM co.. xl with a GraphPad Inplot (from GraphPad, Inc., of San Diego, CA) software program. This program p. a W O 97/0721Z PCT. oion fit of a logistic equation to the data, and. luduced the graphs shown in Fig. 17. Using this data, the program further cc,u, tci, IC50 (the cu. 1irm of unl-qh ligand, such as OFQ) that inhibits 50% of the radioactively labeled ligand binding, such as 5 [3H]di. Liue). IC5o and Kj are related by the equation Kj = IC50/(l +[L]/KD) where [L] is the cnl. ion of labeled ligand dip,." l,inP, and K" is defined as the. qfion constant for [3H]di. o data are shown in the following Table 1: 10 Affinity of opioid receptor agonists and OFQ at mu, delta and kaPPa , d in CHO cells C. -site DAMGO 2.0i0.1 DPDPE 0.59iO.01 U50488H 1.6i0.2 OrphanFQ 2083 i432 2246i339 754i89 *IC50 values were derived from co. ,.lls using an opioid. Data are me. n SE values derived from three inA- IJP - ;. The ligand of interest has a low IC50 ( c 100nM) if it binds with high affinity to the receptor. All ligands of interest bound with high affinity to one of the opioid , , eAcept for OFQ which had an 2 5 IC. o greater 2000 at. , and 754 at the K site. These values show that OFQ does not ;ri. q-lly binding to a receptor shall mean having an affinity GA. ed by an IC50 of less than about 100nm. In p- , the affinity is 0.1-lOnm, preferably less than lnm, and most p.ef. .al,ly less than 0.1nm. F ,'e 13 The affinity of any of the peptides of the present invention can be. by an affinity study as d- ;hetl in EAample 12. Hence, an OFQ analogue with amino acid. for the OFQ of EAample 12. An OFQR l._-, r. ,d CHO cell 3 5 m. JlCooillg cell 1ll. s of EAample 12 to d- -r affinity of the peptide analogue to the OFQ receptor. This provides a W O 97/07212 PCT. lLlllg method for fl- t. ;-lg whether the peptide binds Cpcrifirqlly (Ki or IC50 s 100nm) to the OFQR. Pl4 Protocol for CL' g OFQ Anti-Opioid A O. .. , of the OFQ/OFQR system will have C. I clinical utility. Although the --.1. 1 will not have Z'''tqlg,PQi. ' actions itself, it is eApected to be effective as an adjunct to lllu. ;--g with the body's hu.. ;r anti-opioid 10 ",. .1. pected to allow use of lower ll.. "1. lir doses of Llul. Lhle to achieve greater: nqlgPcir effects. Lower doses of lllol. Lille have the adVallla, ;c of producing fewer Dide-erf. and will also help avoid development of tolerance or addiction that can be. l following prolonged use of high doses of lllul. lC. The OFQR qntqgoniQ-t is also eApected to lessen the signs and symptoms of 15 opiate wi. lldlawal in addicts. Use of the qntagftnict in this manner has been made possible by the present hl. llulD' rerognition t_at the OFQ/OFQR i. mediates an anti-opioid effect. Although a method of using an anti-opioid _- has not been known to the art, oligomlrleotifl. Ps have previously been reported that. l.;,, the effect of OFQ. See Meunier et al., Nat. re 377:532-535 (1995), which is hl. Pc 20 reported in that paper were - l;flPc to translated regions of the OFQR. Antisense ctligftnllrlp. tiflp mASt25,9] was the 17-mer 3'-GGAGAAAGGACGGGGTA-5' yle .y to bases 9-25 of the tranCI. PA region of the mouse ORL, mRNA. Controlmissense oligo-- s the 17-mer 3'-GGAGAAGGGGCGCGGCA-S' / to bases 9-25 of the. region of the human ORL, mRNA. The 25 OI;g. -d in sterile physiological saline at the final con. . Male Swiss CDl mice (20-25g; Charles River) were manually injected with 101*1 of solution directly into the lateral brain ventrical every day for 4 coll. DDiOn of the receptor was inhibited by the .''.I;C_. Clfolidpc Other oli. ul ;-lf s of similar length could be mapped to bases 9-25 (using the genetic. ode of Table 3 0 1) to provide similar. ftnictc are used in the present method to treat an opiate withdrawal s. d with a sequence that is s. nti tlly hnmnlngollc to the OFQ amino acid s. "'f, but with one or more amino acid D--l. l;l--linnc made. The D, f (or other olieo. litlr) is then screened using the cAMP 35 a. CHO cells. Peptides that do not inhibit cAMP Prr. inn (using the assay. cample 5) are then selected as a partial agonist that acts as an atll. .6ûlliDI. The selected peptides obtained by this straighlrul-.a d assay are then. .,d to animals (as in EAample 11) to t. ion of opate withdrawal, as evidenced by l. ;liun in withdrawal jumping co", to animals not l. ;ûuioL. Synthetic peptide col.lbiud. ul;al libraries may also be screened to find an agonist- ,onict peptide that does not inhibit cAMP qrcllm. lo,,oi assay. Screening is 5 p. ,.rululcd using the terhniqllPs d. i in Houghten et al., and use of synthetic peptide couul,i. tvlial libraries for basic research and drug diaL. y, Nature 354:84-86, iUWl. ,ucc. The soluble, nu"Ou. o,l bound peptide libraries appear useful in virtuatly all in vitro and in vivo assays, as reported by Ostresh, "Libraries from libraries: tend the range and , of c_emical 1 0 diversity, n PNAS. The s. Orul use of these libraries has been reported for the d. .ll of receptor-active opioid peptides. See Dooley, "An All D-Ar. uno Opioid Peptide with Central Analgesic Activity from a COUU1JiU. IiaI Library," Science 266:2019-2022, 1994, which is illC. hniqlnPs are tested for iut. ,ucc with opiate 15 wiLll. l signs, as in E. ample 8. It should be nnrlPrstnod that the foregoing disclosure. c;,. c certain specific Pmho. iimPntc of the iu. e.lliUU and that all mnrlifi(qtionc or alt. O equivatent thereto are within the spirit and scûpe of the i" .,liu" as set forth in the. W O 97/07212 PCT. US96/13305 SEQUENCE LISTING (1) GENERAL INFORMATION (i) APPLICANTS: Grisel, Judith E. Grandy, David K. Mogil, Jeffrey S. Bunzow, James R. Civelli, Olivier RPincrh. i Rainer Klaus Nothacker, Hans-Peter Mtm. hJames (ii)TITLE OF INVENTION: Opioid. and Methods of Their Use (iii) NUMBER OF SEQUENCES: 10 (iv)CORRESPONDENCE ADDRESS: (A) ADDRESSEE: Klarquist Sp. n Campbell Leigh & Whinston LLP (B) STREET: 121 S.W. Salmon, Suite 1600 (C) CITY: Portland (D) STATE: Oregon 2 0 (E) COUNTRY: USA (F) ZIP: 97204 (v)COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy Disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: PatentIn Release #1.0, Ve. sion WP5.1 ASCII te. ct (vi)CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: (B) FILING DATE: 3 0 (C) CLASSIFICATION: (vii) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: 08/533,058 (B) FILING DATE: 3 November 1995 (C) APPLICATION NUMBER: 08/514,541 (D) FILING DATE: 11 August 1995 (viii) ATTORNEY/AGENT INFORMATION (A) NAME: Richard J. Polley, Esq. (B) REGISTRATION NUMBER: 28,107 (C) REFERENCE/DOCKET NUMBER: 899-45532/RJP (i:c) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: (503) 226-7391 (B) TELEFAX: (503) 228-9446 -W O 97/07212 PCTrUS96/13305 (2) INFORMATION FOR SEQ ID NO: 1 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 31 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: (A) DESCRIPTION: DNA (genonuc) ( ci) SEQUENCE DESCRIPTION: SEQ ID NO: 1: (3) INFORMATION FOR SEQ ID NO: 2 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: (A) DESCRIPTION: DNA (genomic) 2 0 ( ci) SEQUENCE DESCRIPTION: SEQ ID NO:2: (4) INFORMATION FOR SEQ ID NO: 3 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1452 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: 3 0 (A) DESCRIPTION: cDNA (i. ) FEATURE: (A) NAME/KEY: S'UTR (B) LOCATION: 1. .181 (i. c) FEATURE: 3 5 (A) NAME/KEY: CDS (B) LOCATION: 182..1282 (i. c) FEATURE: (A) NAME/KEY: 3'UTR -W O 97/07212 PCT. B) LOCATION: 1283..1452 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: CACTGTGCTC CTGCCTGCCC GC. lGC TAAGCATTGG GGTCTATTTT GCGCCCAGCT120 TCTGAAGAGG. lGC CGTTGGAGGA ACTGTACTGA GTGGCTTTGC AGGGTGACAG180 Met Glu Ser Leu Phe Pro Ala Pro Tyr Trp Glu Val Leu His Gly 1 5 lO 15 Ser His Phe Gln Gly Asn Leu Ser Leu Leu Asn Glu Thr Val Pro His His Leu Leu Leu Asn Ala Ser His Ser Ala Phe Leu Pro Leu Gly Leu Lys Val Thr Ile Val Gly Leu Ile Leu Ala Val Cys Ile Gly Gly Leu Leu Gly Asn Cys Leu Val Met Tyr Val Ile Leu Arg Thr Pro Lys Met Lys Thr Ala Thr Asn Ile Tyr Ile Phe Asn Leu Ala Leu Ala Asp Thr CTG GTC TTG CTA ACA CTG CCC TTC CAG GGC ACA GAC ATC CTA CTG GGC 514. 5 Leu Val Leu Leu Thr Leu Pro Phe Gln Gly Thr Asp Ile Leu Leu Gly lO0 105 llO. Phe Trp Pro Phe Gly Lys Ala Leu Cys Lys Thr Val Ile Ala Ile Asp Tyr Tyr Asn Met Phe Thr Ser Thr Phe Thr Leu Thr Ala Met Ser Val Asp Arg Tyr Val Ala Ile Cys His Pro Ile Arg Ala Leu Asp Val Arg ACA TCC AGC A. A GCC CAG GCT GTT AAT GTG GCC ATA TGG GCC CTG GCT 706 Thr Ser Ser Lys Ala Gln Ala Val Asn Val Ala Ile Trp Ala Leu Ala TCA GTG GTT GGT GTT CCT GTT GCC ATC ATG GGT TCA GCA CAA GTG GAA 754. 0 Ser Val Val Gly Val Pro Val Ala Ile Met Gly Ser Ala Gln Val Glu Asp Glu Glu Ile Glu Cy8 Leu Val Glu Ile Pro Ala Pro Gln Asp Tyr Trp Gly Pro Val Phe Ala Ile Cy5 Ile Phe Leu Phe Ser Phe Ile Ile Pro Val Leu Ile Ile Ser Val Cy8 Tyr Ser Leu Met Ile Arg Arg Leu Arg Gly Val Arg Leu Leu Ser Gly Ser Arg Glu Lys Asp Arg Asn Leu Arg Arg Ile Thr Arg Leu Val Leu Val Val Val Ala Val Phe Val Gly -W O 97/07212 PCT. Trp Thr Pro Val Gln Val Phe Val Leu Val Gln Gly Leu Gly Val 5 Gln Pro Gly Ser Glu Thr Ala Val Ala Ile Leu Arg Phe Cy5 Thr Ala Leu Gly Tyr Val Asn Ser Cys Leu Asn Pro Ile Leu Tyr Ala Phe Leu Asp Glu Asn Phe Lys Ala Cy5 Phe Arg Lys Phe Cy8 Cys Ala Ser Ser CTG CAC CGG GAG ATG CAG GTT TCT GAT CGT GTG CGG ACG ATT GCC A. G 1234 Leu His Arg Glu Met Gln Val Ser Asp Arg Val Arg Thr Ile Ala Lys Asp Val Gly Leu Gly Cys Lys Thr Ser Glu Thr Val Pro Arg Pro Ala TGACTAGGCG TGGACCTGCC CATGGTGCCT GTCAGCCCAC AGAGCCCATC CTACACCCAI. 342 2 0 CACGGAGCTC ACACAGGTCA CTG. LA GGTTGACCCT GAACCTTGAG CATCTGGAG¢402 CTTGAATGGC l. G GATCAGGATG CTCAGTCCTA GAGGAAGACC 1452 (5) INFORMATION FOR SEQ ID NO: 4 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 367 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: (A) DESCRIPTION: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: Met Glu Ser Leu Phe Pro Ala Pro Tyr Trp Glu Val Leu His Gly Ser His Phe Gln Gly Asn Leu Ser Leu Leu Asn Glu Thr Val Pro His His 3 5 Leu Leu Leu Asn Ala Ser His Ser Ala Phe Leu Pro Leu Gly Leu Lys Val Thr Ile Val Gly Leu Ile Leu Ala Val Cys Ile Gly Gly Leu Leu Gly Asn Cys Leu Val Met Tyr Val Ile Leu Arg Thr Pro Lys Met Lys Thr Ala Thr Asn Ile Tyr Ile Phe Asn Leu Ala Leu Ala Asp Thr Leu _ CA 02229l26 l998-02-09 Val Leu Leu Thr Leu Pro Phe Gln Gly Thr Asp Ile Leu Leu Gly Phe Trp Pro Phe Gly Lys Ala Leu Cys Lys Thr Val Ile Ala Ile Asp Tyr Tyr Asn Met Phe Thr Ser Thr Phe Thr Leu Thr Ala Met Ser Val Asp Arg Tyr Val Ala Ile Cys His Pro Ile Arg Ala Leu Asp Val Arg Thr Ser Ser Lys Ala Gln Ala Val Asn Val Ala Ile Trp Ala Leu Ala Ser Val Val Gly Val Pro Val Ala Ile Met Gly Ser Ala Gln Val Glu Asp Glu Glu Ile Glu Cys Leu Val Glu Ile Pro Ala Pro Gln Asp Tyr Trp Gly Pro Val Phe Ala Ile Cys Ile Phe Leu Phe Ser Phe Ile Ile Pro Val Leu Ile Ile Ser Val Cys Tyr Ser Leu Met Ile Arg Arg Leu Arg Gly Val Arg Leu Leu Ser Gly Ser Arg Glu Lys Asp Arg Asn Leu Arg Arg Ile Thr Arg Leu Val Leu Val Val Val Ala Val Phe Val Gly Cys Trp Thr Pro Val Gln Val Phe Val Leu Val Gln Gly Leu Gly Val Gln Pro Gly Ser Glu Thr Ala Val Ala Ile Leu Arg Phe Cys Thr Ala Leu Gly Tyr Val Asn Ser Cys Leu Asn Pro Ile Leu Tyr Ala Phe Leu Asp Glu Asn Phe Lys Ala Cys Phe Arg Lys Phe Cys Cys Ala Ser Ser Leu His Arg Glu Met Gln Val Ser Asp Arg Val Arg Thr Ile Ala Lys Asp Val Gly Leu Gly Cys Lys Thr Ser Glu Thr Val Pro Arg Pro Ala (6) INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 17 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: (A) DESCRIPTION: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: Phe Gly Gly Phe Thr Gly Ala Arg Lys Ser Ala Arg Lys Leu Ala Asn Gln (7) INFORMATION FOR SEQ ID NO: 6: (i) SEQUENCE CHARACTERISTICS: _ CA 02229l26 l998-02-09 W O 97/07212 PCT. US96/13305 (A) LENGTH: 17 amino acids (B) TYPE: amino acid (D) TOPOLOGY: l inear (ii) MOLECULE TYPE: (A) DESCRIPTION: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: Phe Gly Gly Phe Thr Gly Ala Arg Lys Ser Ala Arg Lys Tyr Ala Asn 1 5 lO 15 Gln (8) INFORMATION FOR SEQ ID NO. 7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 17 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO. 7 Tyr Gly Gly Phe Leu Arg Arg Ile Arg Pro Lys Leu Ly. Trp Asp Asn Gln 1 5 lO 15 (9) INFORMATION FOR SEQ ID NO. 8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 16 amino acids (B) TYPE: amino acid (D) TOPOLOGY: l inear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO. 8 Tyr Gly Gly Phe Met Thr Ser Glu Lys Ser Gln Thr Pro Leu Val Thr 1 5 lO 15 (10) INFORMATION FOR SEQ ID NO: 9 (i) SEQUENCE CHARACTERISTICS: (A) L. : 13 amino acids (B) TYPE: amino acid (D) TOPOLOGY: l inear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO. 9 Tyr Gly Gly Phe Leu Arg Arg Gln Phe Ly. Val Val Thr (11) INFORMATION FOR SEQ ID NO: 10 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 5 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO. 10 Tyr Gly Gly Phe Leu.