CA2265476A1 - Conjugates useful in the treatment of prostate cancer - Google Patents

Abstract

Chemical conjugates which comprise oligopeptides, having amino acid sequences that are selectively proteolytically cleaved by free prostate specific antigen (PSA), hydrophilic oligopeptide blocking groups and known cytotoxic agents are disclosed. Such conjugates are useful in the treatment of prostatic cancer and benign prostatic hypertrophy (BPH).

Description

?WO 9811065115202530.. ,......,............._......._....._ ._. .CA 02265476 l999-03- 10PCTIUS97/16087TITLE OF THE INVENTIONCONJUGATES USEFUL IN THE TREATMENT OFPROSTATE CANCERBACKGROUND OF THE INVENTIONIn 1994 cancer of the prostate gland is expected to bediagnosed in 200,000 men in the U.S. and 38,000 American males willdie from this disease (Gamick, M.B. (1994). The Dilemmas of ProstateCancer. Scientific American, April:72-81). Thus, prostate cancer is themost frequently diagnosed malignancy (other than that of the skin) inU.S. men and the second leading cause of cancer-related deaths (behindlung cancer) in that group.Prostate specific Antigen (PSA) is a single chain 33 l<Daglycoprotein that is produced almost exclusively by the human prostateepithelium and occurs at levels of 0.5 to 2.0 mg/ml in human seminal?uid (Nadji, M., Taber, S.Z., Castro, A., et al. (1981) Cancer48:l229; Papsidero, L., Kuriyama, M., Wang, M., et al. (1981). JNCI66:37; Qui, S.D., Young, C.Y.F., Bihartz, D.L., et al. (1990), J. Urol.14421550; Wang, M.C., Valenzuela, L.A., Murphy, G.P., et al. (1979).Invest. Urol. 172159). The single carbohydrate unit is attached atasparagine residue number 45 and accounts for 2 to 3 l<Da of the totalmolecular mass. PSA is a protease with chymotrypsin-like specificity(Christensson, A., Laurell, C.B., Lilja, H. (1990). Eur. J. Biochem.1942755-763). It has been shown that PSA is mainly responsible fordissolution of the gel structure formed at ejaculation by proteolysis ofthe major proteins in the sperm entrapping gel, Semenogelin I andSemenogelin II, and fibronectin (Lilja, H. (1985). J . Clin. Invest.76:l899; Lilja, H., Oldbring, J., Rannevik, G., et al. (1987). J. Clin.Invest. 802281; McGee, R.S., Herr, J.C. (1988). Biol. Reprod. 392499).The PSA mediated proteolysis of the gel-forming proteins generatesseveral soluble Semenogelin I and Semenogelin II fragments and solublefibronectin fragments with liquefaction of the ejaculate and release ofprogressively motile sperrnatoza (Lilja, H.. Laurell, C.B. (1984).Scand. J. Clin. Lab. Invest. 44:447; McGee, R.S., Herr, J.C. (1987).?1015202530W0 98/10651CA 02265476 l999-03- 10PCTlUS97/ 16087-2-Biol. Reprod. 372431). Furthennore, PSA may proteolytically degradeIGFBP—3 (insulin-like growth factor binding protein 3) allowing IGF tostimulate specifically the growth of PSA secreting cells (Cohen et al..(1992) J. Clin. Endo. & Meta. 75:1046—l053).PSA complexed to alpha 1 — antichymotrypsin is thepredominant molecular form of serum PSA and may account for up to95% of the detected serum PSA (Christensson, A., Bjork, T., Nilsson,0., et al. (1993). J. Urol. 1502100-105; Lilja, H., Christensson, A.,Dahlén, U. (1991). Clin. Chem. 37:1618—1625; Stenman, U.H.,Leinoven, J., Alfthan, H., et al. (1991). Cancer Res. 512222-226). Theprostatic tissue (normal, benign hyperplastic. or malignant tissue) isimplicated to predominantly release the mature, enzymatically activeform of PSA, as this form is required for complex formation with alpha1 — antichymotrypsin (Mast, A.E., Enghild, J.J., Pizzo, S.V., et al.(1991). Biochemistry 30:1723-1730; Perlmutter, D.H., Glover, G.I.,Rivetna, M., et al. (1990). Proc. Natl. Acad. Sci. USA 873753-3757).Therefore, in the microenvironment of prostatic PSA secreting cells thePSA is believed to be processed and secreted in its mature enzymaticallyactive form not complexed to any inhibitory molecule. PSA also formsstable complexes with alpha 2 — macroglobulin, but as this results inencapsulation of PSA and complete loss of the PSA epitopes, the in vivosignificance of this complex formation is unclear. A free,noncomplexed form of PSA constitutes a minor fraction of the serumPSA (Christensson, A., Bjork, T., Nilsson, O., et al. (1993). J. Urol.l50:100-105; Lilja, H., Christensson, A., Dahlén, U. (1991). Clin.Chem. 37:l618—1625). The size of this form of serum PSA is similar tothat of PSA in seminal ?uid (Lilja, H., Christensson, A., Dahlén, U.(1991). Clin. Chem. 37:l618—1625) but it is yet unknown as to whetherthe free form of serum PSA may be a zymogen; an internally cleaved,inactive form of mature PSA; or PSA manifesting enzyme activity.However, it seems unlikely that the free form of serum PSA manifestsenzyme activity, since there is considerable (100 to 1000 fold) molarexcess of both unreacted alpha 1 - antichymotrypsin and alpha 2 -macroglobulin in serum as compared with the detected serum levels of?W0 98/ 1065110202530CA 02265476 l999-03- 10PCT/US97/ 16087-3-the free 33 kDa form of PSA (Christensson, A., Bjork, T., Nilsson, 0.,et al. (1993). J. Urol. 150:l00-105; Lilja, 11., Christensson, A.,Dahlén, U. (1991). Clin. Chem. 3711618-1625).Serum measurements of PSA are useful for monitoring thetreatment of adenocarcinoma of the prostate (Duffy, M.S. (1989). Ann.Clin. Biochem. 26:379—387; Brawer, M.K. and Lange, RH. (1989).Urol. Suppl. 5:11-16; l-lam, M. and Kimura, H. (1989). J. Lab. Clin.Med. 1 132541-548), although above normal serum concentrations ofPSA have also been reported in benign prostatic hyperplasia andsubsequent to surgical trauma of the prostate (Lilja, H., Christensson,A., Dahlén, U. (1991). Clin. Chem. 3721618-1625). Prostate. —metastases are also known to secrete immunologically reactive PSAsince serum PSA is detectable at high levels in prostatectomized patientsshowing widespread metatstatic prostate cancer (Ford, T.F., Butcher,D.N., Masters, R.W., et al. (1985). Brit. 1. Urology 57:50-55).Therefore, a cytotoxic compound that could be activated by theproteolytic activity of PSA should be prostate cell specific as well asspecific for PSA secreting prostate metastases.It is the object of this invention to provide a novel anti-cancer composition useful for the treatment of prostate cancer whichcomprises oligopeptides having solubility augmenting oligopeptideblocking groups in conjugation with a cytotoxic agent.Another object of this invention is to provide a method oftreating prostate cancer which comprises administration of the novelanti-cancer composition.SUMMARY OF THE INVENTIONChemical conjugates which comprise oligopeptides, havingamino acid sequences that are selectively proteolytically cleaved by freeprostate specific antigen (PSA), hydrophilic oligopeptide blockinggroups and known cytotoxic agents are disclosed. Such conjugates areuseful in the treatment of prostatic cancer and benign prostatichypertrophy (BPH).?WO 981106511015202530CA 02265476 l999-03- 10PCT/US97/16087-4-DETAILED DESCRIPTION OF THE INVENTIONThe instant invention relates to novel anti-cancercompositions useful for the treatment of prostate cancer. Suchcompositions comprise the oligopeptides covalently bonded directly, orthrough a chemical linker, to a cytotoxic agent. The oligopeptides arechosen from oligomers that are selectively recognized by the freeprostate specific antigen (PSA) and are capable of being proteolyticallycleaved by the enzymatic activity of the free prostate specific antigen.Such a combination of an oligopeptide and cytotoxic agent may betermed a conjugate.The conjugates of the instant invention are furthercharacterized by having a hydrophilic blocking group at the N—terminusof the oligopeptide which contributes to the aqueous solubility of theconjugate. Examples of such hydrophilic blocking groups include butare not limited to hydroxylated and polyhydroxylated alkanoyl moietiesand alkanoyl moieties that incorporate ether functionalities.Ideally, the cytotoxic activity of the cytotoxic agent isgreatly reduced or absent when the oligopeptide containing the PSAproteolytic cleavage site is bonded directly, or through a chemicallinker, to the cytotoxic agent and is intact. Also ideally, the cytotoxicactivity of the cytotoxic agent increases significantly or returns to theactivity of the umnodified cytotoxic agent upon proteolytic cleavage ofthe attached oligopeptide at the cleavage site.Furthermore, it is preferred that the oligopeptide is selectedfrom oligopeptides that are not cleaved or are cleaved at a much slowerrate in the presence of non—PSA proteolytic enzymes when compared tothe cleavage of the oligopeptides in the presence of free enzymaticallyactive PSA.For the reasons above, it is desireable for the oligopeptideto comprise a short peptide sequence, preferably less than ten aminoacids. Most preferably the oligopeptide comprises seven or feweramino acids. Because the conjugate preferably comprises a short aminoacid sequence, the solubility of the conjugate may be in?uenced to agreater extent by the generally hydrophobic character of the cytotoxic?WO 98110651l015202530CA 02265476 l999-03- 10PCT/US97/ 16087_5..agent component. Therefore, the hydrophilic blocking groups of theinstant conjugates are selected to offset or diminish such a hydrophobiccontribution by the cytotoxic agent.While it is not necessary for practicing this aspect of theinvention, a preferred embodiment of this invention is a conjugatewherein the oligopeptide, and the chemical linker if present, aredetached from the cytotoxic agent by the proteolytic activity of the freePSA and any other native proteolytic enzymes present in the tissueproximity, thereby releasing unmodified cytotoxic agent into thephysiological environment at the place of proteolytic cleavage.Phannaceutically acceptable salts of the conjugates are also included.It is understood that the oligopeptide that is conjugated tothe cytotoxic agent, whether through a direct covalent bond or througha chemical linker, does not need to be the oligopeptide that has thegreatest recognition by free PSA and is most readily proteolyticallycleaved by free PSA. Thus, the oligopeptide that is selected forincorporation in such an anti-cancer composition will be chosen both forits selective, proteolytic cleavage by free PSA and for the cytotoxicactivity of the cytotoxic agent—proteolytic residue conjugate (or, in whatis felt to be an ideal situation, the unmodified cytotoxic agent) whichresults from such a cleavage. The tenn "selective" as used in connectionwith the proteolytic PSA cleavage means a greater rate of cleavage of anoligopeptide component of the instant invention by free PSA relative tocleavage of an oligopeptide which comprises a random sequence ofamino acids. Therefore, oligopeptide component of the instantinvention is a prefered substrate of free PSA. The term "selective" alsoindicates that the oligopeptide is proteolytically cleaved by free PSAbetween two specific amino acids in the oligopeptide.The oligopeptide components of the instant invention areselectively recognized by the free prostate specific antigen (PSA) andare capable of being proteolytically cleaved by the enzymatic activity ofthe free prostate specific antigen. Such oligopeptides comprise anoligomer selected from:?10152030W0 98/10651CA 02265476 l999-03- 10PCT/US97I16087_ 6 -a) AsnLysIleSerTyrGln|Ser (SEQ.ID.NO.: 1),b) LysIleSerTyrGln|Ser (SEQ.ID.NO.: 2),C) AsnLysIleSerTyrTyr|Ser (SEQ.ID.NO.: 3),d) AsnLysAlaSerTyrGln|Ser (SEQ.ID.NO.: 4),e) SerTyrG1n|SerSer (SEQ.ID.NO.: 5);f) LysTyrGln|SerSer (SEQ.ID.NO.: 6);g) hArgTyrGln|SerSer (SEQ.ID.NO.: 7);h) hArgChaGln|SerSer (SEQ.ID.NO.: 8);i) TyrGln|SerSer (SEQ.ID.NO.: 9);j) TyrGln|SerLeu (SEQ.ID.NO.: 10);k) TyrGln|SerN1e (SEQ.ID.NO.: ll);1) ChgGln|SerLeu (SEQ.ID.NO.: 12);m) ChgGln|SerN1e (SEQ.ID.NO.: 13);wherein hArg is homoarginine, Cha is cyclohexylalanine and Chg iscyclohexylglycine.In an embodiment of the instant invention, the oligopeptidecomprises an oligomer that is selected from:a) AsnLysIleSerTyrG1n|SerSer (SEQ.ID.NO.: 14),?W0 98/10651202530CA 02265476 l999-03- 10PCTIUS97/16087-7-b) AsnLysIleSerTyrGln|SerAla (SEQ.ID.NO.: 15),V c) AlaAsnLysIleSerTyrTyr|Ser (SEQ.ID.NO.: 16),d) AlaAsnLysAlaSerTyrGln|Ser (SEQ.ID.NO.: 17),e) SerTyrGln|SerSerThr (SEQ.ID..NO.: 18),f) SerTyrGln|SerSerSer (SEQ.ID.NO.: 19),g) LysTyrGlnlSerSerSer (SEQ.ID.NO.: 20),h) hArgTyrG1n|SerSerSer (SEQ.ID.NO.: 21),i) SerTyrGln|SerSerLeu (SEQ.ID.NO.: 22);j) SerTyrGln|SerLeu (SEQ.ID.NO.: 23);k) SerChgGln|SerLeu (SEQ.ID.N().: 24);l) hArgChgGln|SerLeu (SEQ.ID.NO.: 25); andm) hArgTyrGln|SerLeu (SEQ.ID.NO.: 26).In a more preferred embodiment of the instant invention,the oligopeptide comprises an oligomer selected from:G1yG1uAsnG1yValGlnLysAspValSerGlnArgSerIleTyr|SerGlnThrGlu(SEQ.ID.NO.: 27),AlaSerTyrG1n|SerSerLeu (SEQ.ID.NO.: 28);SerhArgChgGln|SerLeu (SEQ.ID.NO.: 29);?WO 98/1065115202530CA 02265476 l999-03- 10PCT/U S97! 16087_ 3 _hArgSerSerTyrGln|SerNle (SEQ.ID.NO.: 30);hArgAlaSerChgG1n|SerLeu (SEQ.ID.NO.: 31);hArgSerSerTyrG1n|SerLeu (SEQ.ID.NO.: 32);hArgSerSerChg|SerLeu (SEQ.ID.NO.: 33);SerhArgChgGlnlSerLeu (SEQ.ID.NO.: 34);hArgTyrGln|SerLeu (SEQ.ID.NO.: 35);hArgSerSerChgGln|SerLeu (SEQ.ID.NO.: 36);SerhArgTyrGln|SerLeu (SEQ.ID.NO.: 37);SerSerTyrG1n|SerLeu (SEQ.ID.NO.: 38);SerSerSerChgGln|SerLeu (SEQ.ID.NO.: 39);3PAL—SerSerChgG1n|SerLeu (SEQ.ID.NO.: 40);SerSerChgGln|SerLeu (SEQ.ID.NO.: 41);SerSerSerChgGln|Ser(dLeu) (SEQ.ID.NO.: 42);SerSerSerChgG1n|SerVa1 (SEQ.ID.NO.: 43);ProSerSerChgG1n|SerVal (SEQ.ID.NO.: 44);GlySerSerChgGln|SerLeu (SEQ.ID.NO.: 45);hSerSerSerChgG1nlSerLeu (SEQ.ID.NO.: 46);?1015202530W0 98/ 10651CA 02265476 l999-03- 10PCTIUS97/16087-9-hArgSerSerChgGln|SerNle (SEQ.ID.NO.: 47);hArgTyrGln|SerSerSerLeu (SEQ.ID.NO.: 55);LysTyrGln|SerSerSerLeu (SEQ.ID.NO.: 56);SerTyrGln|SerSerSerLeu (SEQ.ID.NO.: 57);SerSerChgGln-Ser(dLeu) (SEQ.ID.NO.: 58); and3PAL—SerSerChgGln-Ser(dLeu) (SEQ.ID.NO.: 59); andAlaSerChgGln-SerLeu (SEQ.ID.NO.:: 60).The phrase "oligomers that comprise an amino acidsequence" as used hereinabove, and elsewhere in the DetailedDescription of the Invention, describes oligomers of from about 3to about 100 amino acids residues which include in their amino acidsequence the specific amino acid sequence decribed and which aretherefore proteolytically cleaved within the amino acid sequencedescribed by free PSA. Preferably, the oligomer is from 5 to 10amino acid residues. Thus, for example, the following oligomer:hArgSerAlaChgGln|SerLeu (SEQ.ID.NO.: 48);comprises the amino acid sequence:ChgGln|SerLeu (SEQ.ID.NO.: 12); and would therefore comewithin the instant invention. It is understood that such oligomersdo not include semenogelin I and semenogelin II.A person of ordinary skill in the peptide chemistry artwould readily appreciate that certain amino acids in a biologically activeoligopeptide may be replaced by other homologous, isosteric and/orisoelectronic amino acids wherein the biological activity of the originaloligopeptide has been conserved in the modified oligopeptide. Certain?1015W0 98l10651CA 02265476 l999-03- 10PCT/US97ll6087-10-unnatural and modified natural amino acids may also be utilized toreplace the corresponding natural amino acid in the oligopeptides ofthe instant invention. Thus, for example, tyrosine may be replaced by3-iodotyrosine, 2-methyltyrosine, 3-fluorotyrosine, 3—methyltyrosineand the like. Further for example, lysine may be replaced withN'—(2-imidazo1yl)lysine and the like. The following list of aminoacid replacements is meant to be illustrative and is not limiting:Original Amino AcidAlaArgAsnAspGluGlnGlyIleLeuLysMetOmithinePheSerTh rTrpTyrValReplacement Amino Acidg s1GlyLys, OmithineGlnGluAspAsnAlaVal, Leu, Met, Nlelle, Val, Met, NleArg, OmithineLeu, lle, Nle, ValLys, ArgTyr, TrpThrSerPhe, TyrPhe, TrpLeu, Ile, Met, NleThus, for example, the following oligopeptides may besynthesized by techniques well known to persons of ordinary skill in theart and would be expected to be proteolytically cleaved by free PSA:AsnArgIleSerTyrGln|SerAsnLysValSerTyrGln|SerAsnLysMetSerTyrG1n|SerSer(SEQ.ID.NO.: 49)(SEQ.ID.NO.: 50)(SEQ.ID.NO.: 51)?1015202530CA 02265476 l999-03- 10W0 98/10651 PCT/US97/16087-11-AsnLysLeuSerTyrGln |SerSerAsnLysIleSerTyrGln|SerGlnLysIleSerTyrG1n|SerSer(SEQ.ID.NO.: 52)(SEQ.IlD.NO.: 53)(SEQ.ID.NO.: 54),The inclusion of the symbol "|" within an amino acidsequence indicates the point within that sequence where the oligopeptideis proteolytically cleaved by free PSA.The compounds of the present invention may haveasymmetric centers and occur as racernates, racemic mixtures, and asindividual diastereomers, with all poss:ible isomers, including opticalisomers, being included in the present invention. Unless otherwisespecified, named amino acids are understood to have the natural "L"stereoconfigurationThe following abbreviations are utilized in the specificationand figures to denote the indicated amino acids and moieties:hR or hArg: homoargininehY or hTyr: homotyrosineCha: cyclohexylalanineAmf: 4—aminomethylphenylalanineDPL: 2-(4,6—dimethylpyrimidinyl)lysine(imidazolyl)K: N’-(2—imidazolyl)lysineMe2PO3-Y: O-dimethylphosphotyrosineO—Me-Y: O-methyltyrosineTIC: tetrahydro-3-isoquinoline carboxylic acidDAP: l,3—diaminopr0paneTFA: tri?uoroacetic acidAA: acetic acid3PAL 3-pyridyl-alanineThe conjugates of the instant invention comprise oligomerswherein the N-terminus amino acid is modified with a hydrophilicblocking group. Such blocking groups are chosen based upon thepresence of hydrophilic functionality. The presence of the hydrophilic?l0152025WO 98/10651CA 02265476 l999-03- 10PCT/US97/16087-12-functionality distinquishes the instant conjugates from conjugatespreviously disclosed that also had N—terrninus blocking groups. Suchblocking of the terminal amino group may also reduce or eliminate theenzymatic degradation of such peptidyl therapeutic agents by the actionof exogenous amino peptidases which are present in the blood plasmaof warm blooded animals. Blocking groups that increase the hydro-philicity of the conjugates and therefore increase the aqueous solubilityof the conjugates include but are not limited to hydroylated alkanoyl,polyhydroxylated alkanoyl, polyethylene glycol, glycosylates, sugarsand crown ethers.Preferably the blocking group is selected froma)oHO Y3 nH‘ R2b)O ,H3C/ O (1LuP 0wherein:R1 and R2 are independently selected from:a) hydrogen,b) unsubstituted or substituted aryl, unsubstituted orsubstituted heterocycle, C3-C10 cycloalkyl, C2-C6 alkenyl,C2-C6 alkynyl, halogen, C1—C6 perfluoroalkyl, R120-,R3C(O)NR3-, (R3)2NC(O)~, R32N-C(NR3)-, R4S(O)mNH,CN, N02, R3C(O)-, N3, -N(R3)2, or R4OC(O)NR3-,c) unsubstituted C 1 —C6 alkyl,d) substituted C 1-C6 alkyl wherein the substituent on thesubstituted C 1-C5 alkyl is selected from unsubstituted or?W0 98/106511015202530CA 02265476 l999-03- 10PCT/US97Il6087-13-substituted aryl, unsubstituted or substituted heterocyclic,c3—c1o cycloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, R30-,R4S(O)mNH, R3C(O)NR3-, (R3)2NC(O)—, R32N—C(NR3)-,CN, R3C(O)-, N3, -N(R3')2, and R4OC(O)-NR3-; orR1 and R2 are combined to form — (CH2)_q — wherein one of thecarbon atoms is optionally replaced by a moiety selected from: O,s(o)m, ~NC(O)-, NH and -N(COR4)— ;R3 is selected from: hydrogen, aryl, substituted aryl. heterocycle,substituted heterocycle, C1-C6 alkyl and C3—C10cycloalkyl;R4 is selected from: aryl, substituted aryl, heterocycle, substitutedheterocycle, C1-C6 alkyl and C3—C10 cycloalkyl;m is O, l or 2;n is l, 2, 3 or 4;p is zero or an integer between 1 and 100; andq is O or 1, provided that if p is zero, q is 1; ands is 3, 4 or 5.The conjugates of the present invention may haveasymmetric centers and occur as racemates, racemic mixtures, and asindividual diastereomers, with all possible isomers, including opticalisomers, being included in the present invention. When any variable(e.g. aryl, heterocycle, R3 etc.) occurs more than one time in anyconstituent, its definition on each occuirence is independent of everyother occurence. For example, HO(CR1R2)2- represents HOCHQCHZ-,HOCH2CH(OH)-, HOCH(CH3)Cl~I(OH)—, etc. Also, combinations ofsubstituents and/or variables are permissible only if such combinationsresult in stable compounds.As used herein, "alkyl" and the alkyl portion of aralkyl andsimilar terms, is intended to include both branched and straight—chain?W0 98/ 1065110I5202530CA 02265476 l999-03- 10PCT/US97/16087-14-saturated aliphatic hydrocarbon groups having the specified number ofcarbon atoms; "a1koxy" represents an alkyl group of indicated numberof carbon atoms attached through an oxygen bridge.As used herein, "cycloa1kyl" is intended to include non-aromatic cyclic hydrocarbon groups having the specified number ofcarbon atoms. Examples of cycloalkyl groups include cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl and the like."Alkenyl" groups include those groups having the specifiednumber of carbon atoms and having one or several double bonds.Examples of alkenyl groups include vinyl, allyl, isopropenyl, pentenyl,hexenyl, heptenyl, cyclopropenyl, cyclobutenyl, cyclopentenyl,cyclohexenyl, 1-propenyl, 2-butenyl, 2-methyl-2—butenyl, isoprenyl,famesyl, geranyl, geranylgeranyl and the like."Alkynyl" groups include those groups having the specifiednumber of carbon atoms and having one triple bonds. Examples ofalkynyl groups include acetylene, 2—butynyl, 2—pentynyl, 3—pentynyl andthe like."Halogen" or "halo" as used herein means ?uoro, chloro,bromo and iodo.As used herein, "aryl," and the aryl portion of aralkyl andaroyl, is intended to mean any stable monocyclic or bicyclic carbon ringof up to 7 members in each ring, wherein at least one ring is aromatic.Examples of such aryl elements include phenyl, naphthyl,tetrahydronaphthyl, indanyl, biphenyl, phenanthryl, anthryl oracenaphthyl.The term heterocycle or heterocyclic, as used herein,represents a stable 5- to 7—membered monocyclic or stable 8- tol1—membered bicyclic heterocyclic ring which is either saturatedor unsaturated, and which consists of carbon atoms and from one tofour heteroatoms selected from the group consisting of N, O, andS, and including any bicyclic group in which any of the above-definedheterocyclic rings is fused to a benzene ring. The heterocyclic ringmay be attached at any heteroatom or carbon atom which results in thecreation of a stable structure. Examples of such heterocyclic elements?WO 98/10651IO152025CA 02265476 l999-03- 10PCT/U S97/ 16087-15-include, but are not limited to, azepinyll, benzimidazolyl, benzisoxazolyl,benzofurazanyl, benzopyranyl, benzothiopyranyl, benzofuryl,benzothiazolyl, benzothienyl, benzoxazolyl, chromanyl, cinnolinyl,dihydrobenzofuryl, dihydrobenzothienyl, dihydrobenzothiopyranyl,dihydrobenzothiopyranyl sulfone, furyl, imidazolidinyl, imidazolinyl,imidazolyl, indolinyl, indolyl, isochromanyl, isoindolinyl, isoquinolinyl,isothiazolidinyl, isothiazolyl, isothiazolidinyl, morpholinyl,naphthyridinyl, oxadiazolyl, 2—oxoazepinyl, oxazolyl, 2-oxopiperazinyl,2—oxopiperdiny1, 2-oxopyrrolidinyl, piperidyl, piperazinyl, pyridyl,pyrazinyl, pyrazolidinyl, pyrazolyl, pyridazinyl, pyrimidinyl,pyrrolidinyl, pyrrolyl, quinazolinyl, quinolinyl, quinoxalinyl, «tetrahydrofuryl, tetrahydroisoquinolinjyl, tetrahydroquinolinyl,thiamorpholinyl, thiamorpholinyl sulfoxide, thiazolyl, thiazolinyl,thienofuryl, thienothienyl, and thienyl.As used herein in the tenns "substituted C1-8 alkyl","substituted aryl" and "substituted heterocycle" include moietiescontaining from 1 to 3 substituents in '(lLddltlOIl to the point of attachmentto the rest of the compound. Such additional substituents are selectedfrom F, Cl, Br, CF3, NH2, N(C1-C6 alkyl)2, N02, CN, (C1-C6alkyl)O—, —OH, (C1-C6 alkyl)S(0)m—, (C1-C6 alkyl)C(O)NH—,H2N-C(NI-l)-, (C1-C6 alkyl)C(O)-, (C1-C6 alkyl)OC(O)-, N3,(C1-C6 alkyl)OC(O)NH— and C1-C2() alkyl.When R1 and R2 are combined to form — (CH2)s -, thecyclic moieties and heteroatom—contain.ing cyclic moieties so definedinclude, but are not limited to:?CA 02265476 l999-03- 10W0 98/ 10651 PCT/US97/16087_ 16 _ 2? ..”."’~ V‘ -75% O,4 N40 NO H I0 con‘1015As used herein, the term "PEG" represents certainpolyethylene glycol containing substituents having the designatednumber of ethyleneoxy subunits. Thus the term PEG(2) representsO ,H3C\O/\/ \/\O/Kn/‘IL.Oand the term PEG(6) representsH3C\O/\/O\/\O/\/O\/\O/\/O\/\O/?n/‘lgQAs used herein, the term "(d)(2,3—dihydroxypropionyl)"represents the following structure:OHHO\/'\n/.1;0As used herein, the term "(2R,3S) 2,3,4-trihydroxybutanoyl" represents the following structure:9H0Ho\/\.)J\f€HO?W0 98/106511015202530CA 02265476 l999-03- 10PCT/US97l16087-17-Because the conjugates of the invention can be used formodifying a given biological response.. cytotoxic agent is not to beconstrued as limited to classical chemical therapeutic agents. Forexample, the cytotoxic agent may be a protein or polypeptide possessinga desired biological activity. Such proteins may include, for example,a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheriatoxin; a protein such as tumor necrosis factor, on-interferon, [3-interferon, nerve growth factor, platelet derived growth factor, tissueplasminogen activator; or, biological response modifiers such as, forexample, lymphokines, interleukin-l ("IL-1"), interleukin—2 ("IL-2"),interleukin-6 ("IL-6"), granulocyte macrophage colony stimulatingfactor ("GM-CSF"), granulocyte colony stimulating factor ("G—CSF"),or other growth factors.The preferred cytotoxic agents include, in general,alkylating agents, antiproliferative agents, tubulin binding agents andthe like. Preferred classes of cytotoxic agents include, for example,the anthracycline family of drugs, the vinca drugs, the mitomycins,the bleomycins, the cytotoxic nucleosides, the taxanes, the pteridinefamily of drugs, diynenes and the podophyllotoxins. Particularlyuseful members of those classes include, for example, doxorubicin,carrninomycin, daunorubicin, aminopterin, methotrexate, methopterin,dichloro-methotrexate, mitomycin C, porfiromycin, 5-fluorouracil,6-mercaptopurine, cytosine arabinoside, podophyllotoxin, or podo-phyllotoxin derivatives such as etoposide or etoposide phosphate,melphalan, vinblastine, vincristine, leurosidine, vindesine, leurosine,taxol and the like. Other useful cytotoxic agents include estramustine,cisplatin and cyclophosphamide. One skilled in the art may makechemical modifications to the desired cytotoxic agent in order to makereactions of that compound more convenient for purposes of preparingconjugates of the invention.A highly preferred group of cytotoxic agents for thepresent invention include drugs of the following formulae:?CA 02265476 l999-03- 10W0 98/10651 PCT/US97I16087-13-THE METHOTREXATE GROUP OF FORMULAU):H2N N N R8 COR9W/’ ’ I “.7 IN\ \N N CONHCHCHZCHQCOZHR12 R8(1)5 in whichR12 is amino or hydroxy;R7 is hydrogen or methyl;R8 is hydrogen, fluoro, chloro, bromo or iodo;10 R9 is hydroxy or a moiety which completes a salt of thecarboxylic acid;THE MITOMYCIN GROUP OF FORMULA (2):oHEN CHZOCONH2| OCH3H30 NO N‘ R10(2)15 in whichR10 is hydrogen or methyl;?CA 02265476 l999-03- 10W0 98/ 10651 PCT/U S97/ 16087-19-THE BLEOMYCIN GROUP OF FORMULA 132CONH2 NH2N -H CONH2 0\ 11i O HO CH3:;} o N? ‘RH2~*?Y O N fm N?CH N 0 VI\3 H N CH3 8H HO CH30 NI IHO NJ0HOOH O OH“O o\ OHCONH2(3)5 in which R11 is hydroxy, amino, C1-C3 alkylamino,di(C1-C3 alkyl)amin0, C4-C6 polymethylene amino,NH+ II-NHCHZCHQCHQS-CH3 ; or -NHCHZCHQCHZCHZNH-C—NH22ICH310 MELPHALAN OF FORMULA (4):HO2—CH-CH2 N(CH2CH2Cl);2lNH2(4)?CA 02265476 l999-03- 10W0 98/10651 PCT/US97/16087-20-6—MERCAPTOPURINE OF FORMULA 5 I HN’ NI >KN ,;(5)5 A CYTOSINE ARABINOSIDE OF FORMULA (6):NH2I \“N/KOHOH2C\/O 'W\OHHO(5)THE PODOPHYLLOTOXINS OF FORMULA(7):10R14R13%O0%HO OOH?CA 02265476 l999-03- 10W0 98/ 10651 PCT/US97l16087in whichR13 is hydrogen or methyl;R14 is methyl or thienyl;or a phosphate salt thereof; 5THE VINCA ALKALOID GROUP OF DRUGS OF FORMULA (8):R16N ‘AR17I 1 ",3 .H ECH3O10in whichR15 is H, CH3 or CHO; when R17 and R18 are taken singly;R18 is H, and one of R16 and R17 is ethyl and the other is Hor OH; when R17 and R13 are taken together with the15 carbons to which they are attached, they form anoxirane ring in which case R16 is ethyl;R19 is hydrogen, (C1-C3 alkyl)-CO, or chlorosubstituted(C1-C3 alkyl)—CO;?CA 02265476 l999-03- 10W0 98/ 10651 PCT/US97/16087-22-DIFLUORONUCLEOSIDES OF FORMULA (9J:R2‘ 0 CHZOHF OH(9)5in whichR21 is a base of one of the formulae:0 O R2322 CH=CHR24“N R “N I N> ”I%A ' /K ¢L\ /Q N H2N N N\ 0 f!“INH2 NH2N/ R22 A I K I N)O N N \i10 in whichR22 is hydrogen, methyl, bromo, ?uoro, chloro or iodo;R23 is —OH or —NH2;R24 is hydrogen, bromo, chloro or iodo;or,15?CA 02265476 l999-03- 10WO 98/10651 PCTIUS97/16087-23-THE ANTHRACYCLINES ANTIBIOTICS OF FORMULA (10): CH3 0R5 RCR5(10)5whereinRa is -CH3, -CH2OH, -CH20C0(CH2)3CH3, or—CH2OCOCH(OC2H5)2;10 Rbis -OCH3,-OH or-H;RC is —NH2, -NHCOCF3, 4—morph0linyI, 3—cyan0—4-morpholinyl, 1-piperidinyl, 4-methoxy—l—piperidinyI,benzylamine, dibenzylaimine, cyanomethylamine, orI-cyano-2-methoxyethyl amine;15 R5 is -OH —OTHP or —H; andR6 is -OH or -H provided thatR6 is not -OH when R5 is -OH or —OTHP.?CA 02265476 l999-03- 10W0 98/ 10651 PCT/US97/16087-24-ESTRAMUSTINE g l l 1(ClCH2CH2)2NCOO(ll)5 CYCLOPHOSPHAMIDEU2)O\ ,N(CH2CH2C|)2'.°\okm12The most highly preferred drugs are the anthracycline10 antiobiotic agents of Formula (10), described previously. One skilledin the art understands that this structural formula includes compoundswhich are drugs, or are derivatives of drugs, which have acquired inthe art different generic or trivial names. Table l, which follows.represents a number of anthracycline drugs and their generic or trivial15 names and which are especially preferred for use in the presentinvention.?CA 02265476 l999-03- 10PCT/US97/ 16087W0 98/10651C_o_£_..EOxO_u ._O..w QEMC o>cwE8_m cu m_ ..C_o%EmCUN..D:_o5_:o:E..w _£ 0E5: u>:mEo:m cm 2 ..:_o>Eo::mw._mI I0 MLUOUIZI QIFO NIZI I NIZIO IO NIZI I0 NIZI IO NIZI I0 NIZI I0 NIZI I0 NIZUM WM own::MIUOmzuomIUOmIUOII0mIUOmT.U©MIUDamomEumamuvouomzuIONEU:Om:UIONIUEuEumazmuoiuooomzurdmmumummm mm-m<ampEoE:._omoS.oEE_muEoE_:mEE9mEoEc:moEu.5:._o$w)E_.U_D5_DKO3I . .J it 1m?o??ocsmv.U::omEoU?101520W0 98/ 10651CA 02265476 l999-03- 10PCT/US97/16087-26-Of the compounds shown in Table 1, the most highlypreferred cytotoxic agents are doxorubicin, vinblastine and desacetyl-vinblastine. Doxorubicin (also referred to herein as "DOX") is thatanthracycline of Formula (10) in which R3 is -CHQOH, RC is —OCH3,R4 is —NH2, R5 is -OH, and R6 is —H.The blocked oligopeptide-cytotoxic agent conjugate of theinstant invention wherein the cytotoxic agent is the preferred cytotoxicagent doxorubicin may be described by the general formula 1 below: NH O/ AROH oligopeptideC-terminus \N-terminuswherein:oligopeptide is an oligopeptide which is selectively recognizedby the free prostate specific antigen (PSA) and is capable of beingproteolytically cleaved by the enzymatic activity of the free prostatespecific antigen, and wherein the C-terminus carbonyl is covalentlybound to the amine of doxorubicin and the N-terminus amine iscovalently bound to the carbonyl of the blocking group;R is selected from?CA 02265476 l999-03- 10W0 98Il065l PCT/US97/ 16087-27-R1 and R2 are independently selected from: hydrogen. OH, C1-C6alkyl, C1 -C6 alkoxy, C1-C6 aralkyl and aryl;n is 1, 2, 3 or 4;10 p is zero or an integer between 1 and 100;q is 0 or 1, provided that if p is zero, q is l;or the pharmaceutically acceptable salt thereof.15 In a preferred embodiment of the oligopeptide—cytotoxicagent conjugate:R is selected from20 a)HO 3'R‘ R2?CA 02265476 l999-03- 10W0 98/10651 PCT/US97/16087_ 23 _b)C)5b)10 R1 and R2 are independently selected from: hydrogen, C1-C6 alkyl and1520aryl;n is 1, 2, 3 or 4;n’ is 0,l,2or3;p is zero or an integer between 1 and 14;q is 0 or 1, provided that if p is zero, q is 1;or the pharmaceutically acceptable salt thereof.The following compounds are specific examples of theoligopeptide—cytotoxic agent conjugate of the instant invention:?CA 02265476 l999-03- 10WO 98110651 PCT/US97/16087 /CH3 03 NH/OH Xwherein X is:OHO\/U\SerSerSerChgGlnSerLeu-—§- (SEQ.|D.NO.: 61),OHO\)J\ SerSerChgGInSerLeu—§— (sEQ_1D_No,; 52),O,0 0H30 \/\O/\/ \/u\ SerSerSerChgGlnSerLeu-—§—(SEQ.|D.NO.: 63).or the pharmaceutically acceptable salt: thereof.Further examples of conjugates of an oligopeptide anddoxorubicin wherein the N -terminus of the oligopeptide is blocked by ahydrophilic moiety and the C—te1minus of the oligopeptide is attached tothe doxorubicin at the 3'—amine are as follows:?WO 981106512030CA 02265476 l999-03- 10PCT/US97/ 16087-30-2-hydroxyacetyl—hArgSerSe1'TyrG1n-SerNle—DOX (3') (SEQ.ID.NO.:64)2-hydroxyacetyl—hArgSerSe1‘ChgGln—SerN1e-DOX (3') (SEQ.ID.NO.:65)2—hydroxyacetyl—SerhArgChgGln—SerLeu~DOX (3') (SEQ.ID.NO.: 66)2-hydroxyacetyI-hArgSerSerChgGln—SerLeu-DOX (3') (SEQ.ID.NO.:67)2—hydroxyacetyl—hArgAlaSerChgG1n—SerLeu-DOX(3') (SEQ.ID.NO.:68)(d) 2,3—dihydroxypr0pionyl-SerhArgChgGln—SerLeu-DOX (3')(SEQ.ID.NO.: 69) .(l) 2,3—dihydroxypropionyl-SerhArgChgGln—SerLeu-DOX (3')(SEQ.ID.NO.: 70)PEG(2)-SerhArgChgGIn-SerLeu—DOX (3') (SEQ.ID.NO.: 71)PEG(2)-hArgChgG1n—SerLeu—DOX (3') (SEQ.ID.NO.: 72)(2R,3S) 2,3,4—trihydroxybutanoyl-hArgChgGln—SerLeu—DOX (3')(SEQ.ID.NO.: 73)PEG(2)—SerhArgTyrGln-SerLeu—DOX(3') (SEQ.ID.NO.: 74)PEG(2)—hArgTyrG1n—SerSerSerLeu—DOX (3') (SEQ.ID.NO.: 75)PEG(2)—LysTyrG1n—SerSerSerLeu—DOX (3') (SEQ.ID.NO.: 76)2-hydroxyacetyl—hArgSerSerTyrGln-SerLeu-DOX (3') (SEQ.ID.NO.:77)(l)(2,3-dihydroxypropionyl)hArgSerSerChgGlnSerLeu—DOX (3')(SEQ.ID.NO.: 78)PEG(2)-hArgSerSerChgGln-SerLeu—DOX (3') (SEQ.ID.NO.: 79)2—hydroxyacety1—SerTyrGIn-SerSerSerLeu—DOX (3') (SEQ.ID.NO.:80)PEG(16)-SerhArgTyrGln—SerLeu-DOX (3') (SEQ.ID.NO.: 81)(2R,3S) 2,3,4—trihydr0xybutanoyI—SerhArgChgGln—SerLeu—DOX (3')(SEQ.ID.NO.: 82)PEG(2)—SerhArgTy1Gln-SerLeu-DOX (3') (SEQ.ID.NO.: 83)(d)(2,3—dihydroxypropionyl)-hArgSerSerChgGln-SerLeu-DOX(3')(SEQ.ID.NO.: 84)?W0 98I1065115202530CA 02265476 l999-03- 10PCT/U S97! 16087-31-(l)(2,3-dihydroxypropionyl)SerSerSerChgGln-Ser(dLeu)—DOX (3')(SEQ.ID.NO.: 85)(d)(2,3—dihydroxypropionyl)SerSerSerChgGln-SerLeu—DOX (3')(SEQ.ID.NO.: 86)(l)(2,3—dihydroxypropionyl)SerSerSerC'hgGln—SerLeu-DOX (3')(SEQ.ID.NO.: 87)(l)(2,3—dihydroxypropionyl)SerSerChgGln-Ser(dLeu)—DOX (3')(SEQ.ID.NO.: 88)(d)(2,3-dihydroxypropionyl)SerSerChgGln-SerLeu-DOX (3')(SEQ.ID.NO.: 89)PEG(2)—SerSerChgGln-Ser(dLeu)-DOX (3') (SEQ.ID.NO.: 90)PEG(2)SerSerChgGln—SerLeu-DOX (3') (SEQ.ID.NO.: 91)PEG(2)—SerSerSerChgGln-Ser(dLeu)—DOX (3') (SEQ.ID.NO.: 92)(2,3—dihydroxypropionyl)—3PAL—SerSerChgGln—Ser(dLeu)-DOX (3')(SEQ.ID.NO.: 93)(d)(2,3—dihydroxypropionyl)-3PAL—SerSerChgGln—SerLeu—DOX (3')(SEQ.ID.NO.: 94)(l)(2,3-dihydroxypropionyl)—SerSerChgGln—SerLeu—DOX (3')(SEQ.ID.NO.: 95)(2,3-dihydroxypropionyl)-hSerSerSerC'hgGln-SerLeu-DOX (3')(SEQ.ID.NO.: 96)PEG(2)-AlaSerChgGIn-SerLeu-DOX (3') (SEQ.ID.NO.: 97)PEG(6)-SerSerChgGln—SerLeu—DOX (3') (SEQ.ID.NO.: 98)PEG(6)—SerSerSerChgGln-SerLeu-DOX (3') (SEQ.ID.NO.: 99)PEG(6)-AlaSerChgGln-SerLeu-DOX (3') (SEQ.ID.NO.: 100)PEG(4)-3PALSerSerChgGln—SerLeu—DOX (3') (SEQ.ID.NO.: 101)or the phannaceutically acceptable salt thereof.The oligopeptide-cytotoxic: agent conjugate of the instantinvention wherein the cytotoxic agent is the preferred cytotoxic agentvinblastine or desacetylvinblastine may be described by the generalformula II below:?1CA 02265476 l999-03- 10W0 98ll065l PCT/US97/16087 XL — oligopeptide - Rwherein:oligopeptide is an oligopeptide which is specifically recognizedby the free prostate specific antigen (PSA) and is capable of beingproteolytically cleaved by the enzymatic activity of the free prostatespecific antigen;XL is - NH — (Cl-I2)r - NH —R is selected froma)HO 3?CA 02265476 l999-03- 10WO 98110651 PCT/US97I16087-33-R1 and R2 are independently selected from: hydrogen, OH, C1-C6alkyl, C1-C6 alkoxy, C1-C6 aralkyl and aryl;R19 is hydrogen, (C1-C3 alkyl)—CO, or chlorosubstituted5 (C1-C3 alkyl)—CO;n is l, 2, 3 or 4;p is zero or an integer between 1 and 100;q is O or 1, provided that if p is zero, q is 1;10r is l,2,3,4or5,or the pharmaceutically acceptable salt thereof.15 The another embodiment of the oligopeptide—cytotoxicagent conjugate of the instant invention wherein the cytotoxic agent isthe preferred cytotoxic agent vinblastine or desacetylvinblastine may bedescribed by the general formula III below: C:O -/oligopeptide - NRdReI” N-terminusC—terminus20 wherein:?101520WO 98/10651CA 02265476 l999-03- 10PCT/US97/16087-34-oligopeptide is an oligopeptide which is specifically recognizedby the free prostate specific antigen (PSA) and is capable of beingproteolytically cleaved by the enzymatic activity of the free prostatespecific antigen,Rd and Re are independently selected from: hydrogen,C1-C5—alkyl, -C1-C6-alkyl-OH, —C1—C()—alkyl-di—OH,—C1-C6—a|kyl—tri-OH ando/H30 <\/\oi®q\S,s\provided that at least one Rd and R9 are not hydrogen orC1-C6-alkyl, orRd and Re are combined to form a -CH2CH2OCH2Cl-12- diradical;R19 is hydrogen, (C1-C3 alkyl)-CO, or chlorosubstituted(C1-C3 alkyl)-CO;zero or an integer between 1 and 100;0 or 1, provided that if p is zero, q is l;pisqisThe following compounds are specific examples of theoligopeptide—desacetylvinblastine conjugate of the instant invention:?CA 02265476 l999-03- 10W0 98/10651 PCT/US97/16087 HO\/KSerSerSerChgGi|n—SerLeu—-— NH(SEQ.ID.NO~.: 61),OH“EN ‘tIIHI !".1 .H 2CH3O SerSerSerChgGln-SerLeu-—tr)!‘(SEQ.lD.NO.: 102),?W0 98/106511015202530CA 02265476 l999-03- 10PCT/US97/16087_ 36 _or the pharmaceutically acceptable salt thereof.The oligopeptides, peptide subunits and peptide derivatives(also termed "peptides") of the present invention can be synthesizedfrom their constituent amino acids by conventional peptide synthesistechniques, preferably by solid—phase technology. The peptides are thenpurified by reverse-phase high performance liquid chromatography(HPLC).Standard methods of peptide synthesis are disclosed, forexample, in the following works: Schroeder et al., "The Peptides",Vol. 1, Academic Press 1965; Bodansky et al., "Peptide Synthesis",Interscience Publishers, 1966; McOmie (ed.) "Protective Groups inOrganic Chemistry", Plenum Press, 1973; Barany et al., "The Peptides:Analysis, Synthesis, Biology" _2_, Chapter 1, Academic Press, l980, andStewart er al., "Solid Phase Peptide Synthesis", Second Edition, PierceChemical Company, 1984. The teachings of these works are herebyincorporated by reference.The pharrnaceutically acceptable salts of the compoundsof this invention include the conventional non—toxic salts of thecompounds of this invention as formed, e.g., from non—toxicinorganic or organic acids. For example, such conventionalnon-toxic salts include those derived from inorganic acids such ashydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitricand the like: and the salts prepared from organic acids such asacetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric,citric, ascorbic, pamoic, maleic, hydroxymaleic, phenyl—acetic,glutamic, benzoic, salicylic, sulfanilic, 2-acetoxy-benzoic, fumaric,toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic,isethionic, trifluoroacetic and the like.The conjugates of the instant invention which comprisethe oligopeptide containing the PSA cleavage site and a cytotoxic agentmay similarly be synthesized by techniques well known in the medicinalchemistry art. For example, a free amine moiety on the cytotoxic agentmay be covalently attached to the oligopeptide at the carboxyl terminus?1015202530W0 98/ 10651. ...c......_............ M- .CA 02265476 l999-03- 10PCT/US97/ 16087-37-such that an amide bond is formed. Similarly, an amide bond may beformed by covalently coupling an amine moiety of the oligopeptide anda carboxyl moiety of the cytotoxic agent. For these purposes a reagentsuch as 2-(lH—benzotriazol—1-yl)—l,3,3—tetramethyluronium hexafluoro-phosphate (known as HBTU) and 1-hyroxybenzotriazole hydrate (knownas HOBT), dicyclohexyl- carbodiimide (DCC), N-ethyl—N-(3—dimethyl—aminopropyl)- carbodiimide (EDC), diphenylphosphorylazide (DPPA),benzotriazol—1—yl—oxy-tris-(dimethylamino)phosphonium hexafluoro-phosphate (BOP) and the like, used in combination or singularly, maybe utilized.Furthermore, the instant conjugate may be formed by anon-peptidyl bond between the PSA cleavage site and a cytotoxic agent.For example, the cytotoxic agent may be covalently attached to thecarboxyl terminus of the oligopeptide via a hydroxyl moiety on thecytotoxic agent, thereby forming an ester linkage. For this purpose areagent such as a combination of HBTU and HOBT, a combination ofBOP and imidazole, a combination of DCC and DMAP, and the likemay be utilized. The carboxylic acid may also be activated by formingthe nitro-phenyl ester or the like and reacted in the presence of DBU(l ,8—diazabicyclo[5,4,0]undec—7-ene.The instant conjugate may also be formed by attachmentof the oligopeptide to the cytotoxic agent via a linker unit. Such linkerunits include, for example, a biscarbonyl alkyl diradical whereby anamine moiety on the cytotoxic agent is connected with the linker unitto form an amide bond and the amino terminus of the oligopeptide isconnected with the other end of the linlker unit also forming an amidebond. Conversely, a diaminoalkyl diradical linker unit, whereby acarbonyl moiety on the cyctotoxic agent is covalently attacted to one ofthe amines of the linker unit while the other amine of the linker unit iscovalently attached to the C terminus of the oligopeptide, may also beuselful. Other such linker units which are stable to the physiologicalenvironment when not in the presence of free PSA, but are cleavableupon the cleavage of the PSA proteolytic cleavage site, are alsoenvisioned. Furthermore, linker units may be utilized that, upon?W0 98/ 106511015202530CA 02265476 l999-03- 10PCT/US97/ 16087-38-cleavage of the PSA proteolytic cleavage site, remain attached to thecytotoxic agent but do not significantly decrease the cytotoxic activityof such a post-cleavage cytotoxic agent derivative when compared withan unmodified cytotoxic agent.One skilled in the art understands that in the synthesis ofcompounds of the invention, one may need to protect various reactivefunctionalities on the starting compounds and intermediates while adesired reaction is carried out on other portions of the molecule. Afterthe desired reactions are complete, or at any desired time, nonnallysuch protecting groups will be removed by, for example, hydrolyticor hydrogenolytic means. Such protection and deprotection steps areconventional in organic chemistry. One skilled in the art is referred toProtective Groups in Organic Chemistrv, McOmie, ed., Plenum Press,NY, NY (1973); and, Protective Groups in Organic Svnthesis, Greene,ed., John Wiley & Sons, NY, NY (1981) for the teaching of protectivegroups which may be useful in the preparation of compounds of thepresent invention.By way of example only, useful amino-protecting groupsmay include, for example, C1-C10 alkanoyl groups such as forrnyl,acetyl, dichloroacetyl, propionyl, hexanoyl, 3,3—diethylhexanoyl,y—chlorobutryl, and the like; C1—C1() alkoxycarbonyl and C5—C15aryloxycarbonyl groups such as tert—butoxycarbonyl,benzyloxycarbonyl, allyloxycarbonyl, 4—nitrobenzyloxycarbonyl,fluorenylmethyloxycarbony1 and cinnamoyloxycarbonyl; halo-(C1—C10)-alkoxycarbonyl such as 2,2,2-trichloroethoxycarbonyl; andC1-C15 arylalkyl and alkenyl group such as benzyl, phenethyl, allyl,trityl, and the like. Other commonly used amino-protecting groups arethose in the fonn of enamines prepared with B-keto-esters such asmethyl or ethyl acetoacetate.Useful carboxy-protecting groups may include, forexample, C1-C1() alkyl groups such as methyl, tert-butyl, decyl; halo-C1—C10 alkyl such as 2,2,2—trichloroethyl, and 2—iodoethyl; C5—C15arylalkyl such as benzyl, 4—methoxybenzyl, 4-nitrobenzyl, triphenyl—methyl, diphenyl—methyl; C1-C10 alkanoyloxymethyl such as acetoxy—?W0 98/1015511015CA 02265476 l999-03- 10PCT/US97/ 16087-39-methyl, propionoxymethyl and the like; and groups such as phenacyl,4-halophenacyl, allyl, dimethylallyl, tri--(C1-C3 alkyl)silyl, such astrimethylsilyl, [3-p—toluenesulfonylethyl, [3-p—nitrophenyl—thioethyl,2,4,6-trimethylbenzyl, B—methylthioethyl, phthalimidomethyl, 2,4-dinitro—phenylsulphenyl, 2—nitrobenzhydryl and related groups.Similarly, useful hydroxy protecting groups may include,for example, the formyl group, the chloroacetyl group, the benzylgroup, the benzhydryl group, the trityl group, the 4-nitrobenzyl group,the trimethylsilyl group, the phenacyl group, the tert—butyl group, themethoxymethyl group, the tetrahydropyranyl group, and the like.With respect to the preferred embodiment of an oligo—peptide combined with the anthracycline antibiotic doxombicin, thefollowing Reaction Schemes illustrate the synthsis of the conjugates ofthe instant invention.?1CA 02265476 l999-03- 10W0 98/ 10651 PCT/US97/16087-40-REACTION SCHEME I CH3O OH 5dox12CH3 0N\HOH ‘-———:—]oligopeptide --I GI . . ICH2O —l ohgopeptxde-1 G O G is a hydrophilic N-terminusCH3 13 blocking groupOH?CA 02265476 l999-03- 10W0 98/10651 PCT/US97/ 16087-41-REACTION SCHEME HHN-- oli o e3tide—GO OH I, 9 p I I CHZOHOHCH3O O OH OO 14OH?1CA 02265476 l999-03- 10W0 98/10651 PCT/US97/16087-42-REACTION SCHEME Ill mac 0 OHS (CH2)2CO2H\ “YG‘ is a hydrophilic C—terminalQ 15 moietyOH?CA 02265476 l999-03- 10W0 98/10651 PCT/US97I 16087-43 .-REACTION SCHEME IV OH1 1 ‘HZNHN-—| oligopeptide T GH O 16OH?51lCA 02265476 l999-03- 10W0 98/10651 PCT/US97ll6087-44-REACTION SCHEME V OH H N | . |2 \n/\*l:'J—| oligopeptlde-I GO OHReaction Scheme VI illustrates preparation of conjugatesof the oligopeptides of the instant invention and the vinca alkaloidcytotoxic agent vinblastine wherein the attachment of vinblastine is at?CA 02265476 l999-03- 10W0 98/111651 PCT/US97/ 16087-45-the C-tenninus of the oligopeptide. The use of the 1,3-diaminopropanelinker is illustrative only; other spacer units between the carbonyl ofvinblastine and the C—terrninus of the oliigopeptide are also envisioned.Furthermore, Scheme VI illustrates a synthesis of conjugates wherein5 the C-4-position hydroxy moiety is reacetylated following the additionof the linker unit. Applicants have discovered that the desacetylvinblastine conjugate is also efficacious and may be prepared byeliminating the steps shown in Reaction Scheme VI of protecting theprimary amine of the linker and reacting the intermediate with acetic10 anhydride, followed by deprotection of the amine. Conjugation of theoligopeptide at other positions and functional groups of vinblastine maybe readily accomplished by one of ordinary skill in the art and is also. expected to provide compounds useful :in the treatment of prostatecancer.15?CA 02265476 l999-03- 10WO 98110651 PCT/US97/ 16087- 46 _REACTION SCHEME VIOH“EtN ."/H HZN ‘ CHZCHQCH2 ' NH22. BOC-Cl 1. Ac2O, pyridine2. aq. HCI H NH - BOC ?CA 02265476 l999-03- 10W0 98/101551 PCT/US97l16087-47-REACTION SCHEME VI (Continued)(3— oligopeptide Oéx N/-\/\ N/ oligopeptide —GH H5 The oligopeptide-cytotoxic agent conjugates of theinvention are administered to the patient in the form of a pharmaceuticalcomposition which comprises a conjugate of of the instant invention anda pharmaceutically acceptable carrier, excipient or diluent therefor.As used, "pharmaceutically acceptable" refers to those agents which10 are useful in the treatment or diagnosis of a warm—blooded animalincluding, for example, a human, equine, procine, bovine, murine,canine, feline, or other mammal, as well as an avian or other warrn—blooded animal. The preferred mode of administration is parenterally,particularly by the intravenous, intramuscular, subcutaneous,?WO 9811065110152025301CA 02265476 l999-03- 10PCTIU S97/16087-43-intraperitoneal, or intralymphatic route. Such formulations can beprepared using carriers, diluents or excipients familiar to one skilledin the art. In this regard, gee, _e_,g. Remington's PharmaceuticalSciences, 16th ed., l980, Mack Publishing Company, edited by Osole_t a_l. Such compositions may include proteins, such as serum proteins,for example, human serum albumin, buffers or buffering substancessuch as phosphates, other salts, or electrolytes, and the like. Suitablediluents may include, for example, sterile water, isotonic saline, diluteaqueous dextrose, a polyhydric alcohol or mixtures of such alcohols, forexample, glycerin, propylene glycol, polyethylene glycol and the like.The compositions may contain preservatives such as phenethyl alcohol,methyl and propyl parabens, thimerosal, and the like. If desired, thecomposition can include about 0.05 to about .20 percent by weight ofan antioxidant such as sodium metabisulfite or sodium bisulfite.As used herein, the term "composition" is intended toencompass a product comprising the specified ingredients in the specificamounts, as well as any product which results, directly or indirectly,from combination of the specific ingredients in the specified amounts.For intravenous administration, the composition preferablywill be prepared so that the amount administered to the patient will befrom about .01 to about 1 g of the conjugate. Preferably, the amountadministered will be in the range of about .2 g to about 1 g of theconjugate. The conjugates of the invention are effective over a widedosage range depending on factors such as the disease state to be treatedor the biological effect to be modified, the manner in which theconjugate is administered, the age, weight and condition of the patient aswell as other factors to be determined by the treating physician. Thus,the amount administered to any given patient must be determined on anindividual basis.One skilled in the art will appreciate that although specificreagents and reaction conditions are outlined in the following examples,modification can be made which are meant to be encompassed by thespirit and scope of the invention. The following preparations andexamples, therefore, are provided to further illustrate the invention,?WO 98/10651101520CA 02265476 l999-03- 10PCTIUS97/16087- 49 _and are not limiting.EXAMPL_l;§EXAMPL 1Preparation of Oligopeptides which Comprise the PSA MediatedCleamge SiteBlocked oligopeptides were prepared by solid-phasesynthesis, using a double coupling protocol for the introduction ofamino acids on the Applied Biosystems model 430A automatedpeptide synthesizer. Deprotection and removal of the oligopeptide fromthe resin support were achieved by treatment with liquid hydrofluoricacid. The oligopeptides were purified by preparative high pressureliquid chromatography on reverse phase C18 silica columns using anaqueous 0.1% trifluoroacetic acid/acetonitrile gradient. Identity andhomogeneity of the oligopeptides were confirmed by amino acidcomposition analysis, high pressure liquid chromatography, and fastatom bombardment mass spectral analysis. The oligopeptides that wereprepared by this method are shown in Table 2.?CA02265476 1999-03-10WO 98/10651 PCT/US97/16087- 50 _TAB LE 2SEQ.|D.NO. P ID P - Time to 50% Substrate_Cleavage by? 1 o 3 Ac-ANKASYQ-SL-acid 1351 0 4 Ac-ANKASYQ-SL-acid 2201 O5 Ac-hR(CHA)Q-SNle-acid 200 (PS)1 0 6 Ac~ShFiYQ-SN|e-acid 25 (PS)1 O 7 Ac-ShRChgQ-SNIe~acid INSOLUBLE1 0 8 Ac-hRSSYQ-SN|e-acid 25 (PS)1 0 9 AchRSSChgQ-SL—acid 120(45*)6 6 2-hydroxyacetyl-ShRChgQ—SL-acid 120(30‘)1 1 O Ac-hF1SSYQ—SNle-acid 25 (PS)5 4 2-hydroxyacetyl-hRSSYQ-SN|e~acid 451 1 1 Ac-hRASChgQ-SL-acid 505 8 2-hydroxyacetyl-hRASChgQ—SL-acid 705 4 2-hydroxyacetyl-hFiSSYQ-SL-acid 35 (PP)6 7 2—hydroxyacetyI—hRSSChgSL-acid (PP)6 9 2,3-dihydroxypropionylShRChgQ-SL—acid 757 0 2(8)-2,3—dihydroxy propionyIShRChgQSL-acid 35‘1 1 2 2-hydroxyacetylhRYQ-SL-acid 105‘2 9 ShRChgQ-SL -acid 4 HOUR = 8%7 1 PEG(2)-S-hFlChgQ—SL-acid 301 1 3 PEG(1)—ShF1ChgQ—SL-acid 120'7 8 2(S)2,3-dihydroxypropionly-hRSSChgQ-SL~acid 25'7 9 PEG(2)-hRSSChgQ—SL-acid 40‘7 4 PEG(2)—ShRYQ—SL-acid 35'1 1 4 PEG(1)-hRSSChgQ-SL-acid 301 1 5 PEG(1)-ShRYQ-SL-acid 901 1 6 PEG(15)-ShRYQ—SL-acid 408 1 PEG(16)-ShFlYQ—SL-acid 401 1 7 PEG(1?)-ShRYQ-SL~acid 558 2 (2R,3S) 2,3,4—trihydroxybutanoyl-ShRChgQ-SL-acid 908 3 2,3-dihydroxypropionyl-hRSSChgQ-SL-acid 501 1 8 PEG(2)-SSYQ—SL-acid 1501 1 9 PEG(14)ShRYQ-SL-acid 401 2 O PEG(18)ShRYQ~SL—acid 40?WO 98110651 PCT/US97/16087- 51. -TABLE 2 gcontinuedlSEQ.ID.NO. Time to 50% Substrate.Cleavage byI : I PE 5 it I.1 21 PEG(19)ShFlYQ-SL-acid 609 4 (d)2.3—dihydroxypropionyl-3PAL-SSChgQSL-acid 806 3 PEG(2)SSSChgQ-SL-acid 1501 O1 PEG(2)-3PAL—SSChgQ-SL-acid 808 7 (|)2,3-dihydroxypropionyl-SSSChgQ-SL-acid 806 1 (OH-Ac)SSSChgQ—SL-acid 1201 2 2 (1)2,3-dihydroxypropionyl-SSChgQ-SL—acid 1808 7 (I)2,3—dihydroxypropionyl—SSSChgQ-SL-acid 1 101 2 3 (|)2.3—dihydroxypropiony|-3PAL»-SSChgQ—SL-acid 701 24 2.3-dihydroxypropiony|—SSSChgQ—SL-acid 1201 2 5 2,3-dihydroxypropionyl-ShIRYQ-SL-acid 356 2 2-hydroxyacetyl-SSChgQ-SL-acid 1801 2 5 2,3—dihydroxypropionyl-ShRYQ-SL-acid 501 01 PEG(4)-B—PAL-SSChgQ-SL~acid 601 2 5 Ac-SSSChgQ-Svr-acid1 2 7 Ac-PSSChgQ-SV»-acid1 2 8 2,3—dihydroxypropiony|-GSSChgQ-SL-acid 1609 6 2,3—dihydroxypropiony|-hSSSChgQ-SL-acid 160EXAMPLE 251015CA 02265476 l999-03- 10Assessment of the Recognition of Oligopeptides by Free PSAThe oligopeptides prepared as described in Example 1were individually dissolved in PSA digestion buffer (.12 mMtris(hydroxymethyl)—aminomethane pH8.0, 25 mM NaCl, 0.5 mMCaCl2) and the solution added to PSA at a molar ration of 100 to l.The reaction is quenched after various reaction times by the additionof tri?uoroacetic acid (TFA) to a final 1% (volume/volume). Thequenched reaction was analyzed by HPLC on a reversed-phase C18column using an aqueous 0.l%TFA/acetonitrile gradient. The results ofthe assessment are shown in Table 2. Table 2 shows the amount of time(in minutes) required for 50% cleavage of the noted oligopeptides withenzymatically active free PSA. Oligopeptides containing free amine?1015202530W0 98/10651CA 02265476 l999-03- 10PCT/US97/16087-52-moieties (ie. comprising hArg, Om, Lys and or 3PAL) were tested asTFA salts. All other oligopeptides were tested as neutral compounds.EXAMPLE 3Preparation of N-(2-I-Iydroxyacetyl)—Ser-Ser-Ser-Chg—Gln-Ser-Leu—Dox(3-3L2—HO—Ac-Ser(Bzl)-Ser(Bzl)—Ser(Bzl)-Chg-Gln-Ser—Leu-PAM Resin (3-1).Starting with 0.5 mmol (O.67g) Boc—Leu-PAMresin (Applied Biosystems Inc. - ABI), the protected peptide wassynthesized on a 430A ABI peptide synthesizer. The protocol used a4 fold excess (2 mmol) of each of the following protected amino acids:Boc-Ser(OBzl), Boc—Gln, Boc-Chg. Coupling was achieved using DCCand HOBT activation in methyl—2—pyrrolidinone. Removal of the Bocgroup was performed using 50% TFA in methylene chloride and theTFA salt neutralized with diisopropylethylamine. 2-Hydroxyacetic acidwas used for the introduction of the N terminal blocking group, whichwas also carried out on the peptide synthesizer. At the completion ofthe synthesis, the peptide resin was dried to provide the title resin-peptide conjugate.Step A:2-HO-Ac—Ser—Ser—Ser-Chg-Ser-Leu-OH (3-2).The protected peptide resin (3-1), 1.2 g, was treated withHF (15 ml) for lhr at 0°C in the presence of anisole (1.5 ml). Afterevaporation of the HF, the residue was washed with ether 3 times,and extracted with 20% HOAC. The crude peptide products fromthe HF—cleavage after lyophilization were purified by preparatory HPLCon a Delta-Pak C18 column with 0.1% trifluoroacetic acid —aqueousacetonitrile solvent systems using l00—70% O.1%TFA—H20, 60minlinear gradient. Fractions containing product of at least 99% (HPLC)purity were combined to provide the title blocked peptide.Step B:?202530CA 02265476 l999-03- 10W0 98/10651 PCTIUS97/16087_ 53 _FABMS: 804.85Peptide Content: l.03NMOle/mg.HPLC: 99% pure @214, retention times: 1 1.16 min, (VydacC18, gradient of 95%A/B to 50%VA/B over 30 min,A=0.1%TFA-H20, B=0.1%TFA—CH3CN)§t_e_:p_C_: 2—HO-Ac—Ser-Ser-Ser—Chp:—Ser-Leu-Dox (3-3)A solution of 241 mg (0.30 mmol) of OH-Ac-Ser-Ser-Ser-Chg-Gln-Leu-OH (3-2) in 3.0 ml anhyd. N-methyl pyrrolidine (NMP)(or DMF), 46 mg (0.30 mmol) of HOBT, 63 mg (0.33 mmol) of EDC ,46 mg (0.09 mmol) of doxorubicin was added and pH was adjusted withdiisopropylethylamine (DIEA) to pH 8.5. The solution was stirred at0°C for llhrs., and then reaction was quenched by H4‘. The organicsolvent was removed under reduced pressure and the residue was dilutedwith 15ml of water, and purified by preparative HPLC using a NH4Ac(4g/4L)—CH3CN gradient, ie. 95-50%/X, 60min. Lyophilization of purefractions gave a red powder. The red powder was dissolved in distil.H20, filtered, and lyophilized to provide the title conjugate (1-3).ES+ + NH4+ : 1347.61Peptide Content: 541.72 NMOle/mg.HPLC: 99% pure @214, retention times: 20.8 min, (VydacC18, gradient of 95%A/B to 50%A/B over 30 min,A=0.1%TFA-H20, B=0.1%TFA, CH3CN)EXAMPl;l_3_4Preparation of N-[2-{ 2-(2—methoxyeth,oxy)ethoxy } acetyl]-Ser-Ser-Ser-Chg-Gln—Ser—Leu-DoxThe title conjugate was prepared in the manner described inExample 3, but substituting 2-{2—(2—methoxyethoxy)ethoxylacetic acidfor 2-hydroxyacetic acid in Step A.?CA 02265476 l999-03- 10W0 98/10651 PCT/US97/16087_ 54 _ES+ + NI-14+ : 1450.72Peptide Content: 534.36 NMOle/mg.HPLC: 99% pure @214, retention times: 21.99 min, (VydacC18, gradient of 95%A/B to 50%A/B over 30 min,5 A=0.l%TFA-H20, B=0.1%TFA, CH3CN)l015202530EXAMPLE 5Preparation of N-2(R)-2,3-dihydroxypropionyl—Ser-Ser-Ser—Chg—Gln-Ser—Leu—Dox (5-3)N—2(R)—2,3—dihydroxypropionyl-Ser(Bzl)—Ser(Bzl)—Ser(Bzl)-Ch2—Gln—Ser—Leu—PAM Resin (5-1).Starting with 0.5 mmol (0.67g) Boc—Leu-PAM resin,the protected peptide was synthesized on a 430A ABI peptidesynthesizer. The protocol used a 4 fold excess (2 mmol) of each of thefollowing protected amino acids: Boc—Ser(OBzl), Boc-Gln and Boc-Chg.Coupling was achieved using DCC and HOBT activation in methyl-2-pyrrolidinone. Removal of the Boc group was performed using50% TFA in methylene chloride and the TFA salt neutralized withdiisopropylethylamine. D—Glyceric acid, which was converted fromD-Glyceric acid calcium salt, was used for the introduction of the Nterminal blocking group. At the completion of the synthesis, thepeptide resin was dried to provide the title resin—peptide conjugate.Step A:N—2(R)-2,3—dihydroxypropionyl—Ser-Ser-Ser-Chg-Gln-Ser-Leu—Dox (5-3)The title conjugate was prepared in the manner describedin Example 3, Steps B and C, but substituting the resin peptide conjugate5-1 for the resin—peptide conjugate used in Example 3, Step B.Step B:1377.55620.85 NMOle/Ihg.ES'*‘ + NH4+ :Peptide Content:?CA 02265476 l999-03- 10WO 98/10651 PCT/US97/16087_ 55 _HPLC: 99% pure @214, retention times: 20.71 min, (VydacC13, gradient of 95%A/B to 50%A/B over 30 min,A=0.1%TFA-I-120, .B=O.1%TFA, CH3CN)5 EXAMPLQ20Preparation of N-2(S)—2,3—dihydroxypiropionyl—Ser-Ser—Ser-Chg—GIn-Ser-Leu-DoxThe title conjugate was prepared in the manner describedin Example 5, but substituting L-glyceric acid for D-glyceric acid inStep A.ES+ + NH4+ 2 1377.62Peptide Content: 641.59 NMOle/mg.HPLC: 99% pure @214, retention times: 20.57 min, (VydacC18, gradient of 95%A/B to 50%A/B over 30 min,A=0.1%TFA-H20, B=0.1%TFA, CH3CN)Table 3 shows other blocked peptide-doxorubicin conjugates that wereprepared by the procedures described in Examples 3-6, but utilizing theappropriate amino acid residues and blocking group acylation.?CA 02265476 l999-03- 10WO 98110651 PCT/US97/16087_ 56 -TAB LE 3SEQ, P D TID - Time to 50% Substrate ~1D_,L\JQ Cleavage by[gas P§A (Min)6 4 2-hydroxyacety1-HomoRSSYO-SNIe-DOX (3') 60‘6 6 2-hydroxyacetyI—SHomoRChgQ-SL-DOX (3') 156 7 2-hydroxyacetyl-HomoRSSChgQ-SL~DOX (3') 126 8 2-hydroxyacetyl-HomoRASChgQ-SL-DOX (3') 106 9 (d)2,3-dihydroxypropionyl—SHomoRChgQ-SL—DOX (3') 657 0 (l)2,3—dihydroxypropionyl-SHomoRChgQ-SL-DOX (3') 157 1 PEG(2)-SHomoRChgO-SL-DOX (3') 257 2 PEG(2)-HomoRChgQ—SL-DOX (3') 4 HOUR = 12%7 3 (2R,3S) 2,3,4-1rihydroxybutanoy|—HomoRChgO-SL—DOX (3') 4 HOUR = 0%7 4 PEG(2)—SHomoRYQ-SL—DOX(3') 357 5 PEG(2)—HomoRYO-SSSL—DOX (3') 4 HOUR = 40% (PS)7 6 PEG(2)—KYQ—SSSL—DOX (3') 4 HOUR = 20% (PS)7 7 2-hydroxyacetyl-HomoRSSYQ-SL—DOX (3') 16 (PS)7 8 (l)2.3-dihydroxypropiony|HomoRSSChgQSL—DOX (3') 127 9 PEG(2)-HomoRSSChgQ-SL-DOX (3') 118 0 2-hydroxyacety|-SYQ—SSSL-DOX (3') (PS)8 1 PEG(16)—SHomoRYQ-SL-DOX (3') 658 2 (2R,3S) 2.3,4-trihydroxybutanoyl-SHomoRChgO-SL-DOX (3') 458 3 PEG(2)-SHomoRYQ-SL-DOX (3') 608 4 (d)2,3-dihydroxypropiony|-HomoRSSChgQSL-DOX(3’) 128 5 (|)2.3-dihydroxypropionylSSSChgO-S(dL)-DOX (3') 1808 6 (d)2,3-dihydroxypropionylSSSChgQ-SL—DOX (3') 5587 (|)2,3—dihydroxypropiony|SSSChgO—SL—DOX (3') 2588 (I)2,3-dihydroxypropiony|SSChgQ-S(dL)-DOX (3') 3 HOUR = 22%89 (d)2,3-dihydroxypropionylSSChgQ-SL-DOX (3') 12091 PEG(2)SSChgO-SL-DOX (3') 9092 PEG(2)-SSSChgQ—S(dL)-DOX (3') 3 HOURS = 46%63 PEG(2)—SSSChgQ-SL-DOX (3') 6094 (d)2,3-dihydroxypropionyl-3PALSSChgQ-SL-DOX (3').AcOH 12 (PS)95 (1)2,3—dihydroxypropionyl-SSChgQ—SL-DOX (3') 2561 2-hydroxyacety|—SSSChgQ-SL—DOX (3') 2596 2,3-dihydroxypropionyl-HomoSSSChgQ-SL-DOX (3') 359 7 PEG(2)-ASChgQ-SL—DOX (3') 4598 PEG(6)-ASChgO—SL-DOX (3') 16062 2-hydroxyacety|—SSChgQ-SL—DOX (3') 45?W0 98/106511015202530CA 02265476 l999-03- 10PCT/US97/16087- 57 -EXAMPL_l_3__ZAssessment of the Recognition of Oligopeptide-Doxorubicin Conjugatesbv Free PSAThe conjugates prepared as described in Examples3-6 were individually dissolved in PSA digestion buffer (50 mMtris(hydroxymethyl)—aminomethane pH7.4, 140 mM NaCl) and thesolution added to PSA at a molar ration of 100 to 1. The reaction isquenched after various reaction times by the addition of tri?uoroaceticacid (TFA) to a final 1% (volume/volume). The quenched reaction wasanalyzed by HPLC on a reversed—phase C18 column using an aqueous0.l%TFA/acetonitrile gradient. The results of the assessment are shownin Table 3. Table 3 shows the amount of time (in minutes) required for50% cleavage of the noted oligopeptide:-cytotoxic agent conjugates withenzymatically active free PSA. If no salt is indicated for the conjugate,the free conjugate was tested. An alternative PSA digestion buffer(12 mM tris(hydroxymethyl)—aminomethane pH8.0, 25 mM NaCl,0.5 mM CaCl2) was utilized in the assessment of the 2-hydroxyacetyl-hArgSerSerTyrGln—SerNle-DOX (3') (SEQ.ID.NO.: 30) conjugate.EXAMPLE 8In vitro Assay of Cvtotoxicitv of Peotidvl Derivatives of DoxorubicinThe cytotoxicities of the cleaveable oligopeptide—doxorubicin conjugates, prepared as described in Examples 3-6, againsta line of cells which is known to be killed by unmodified doxorubicinwas assessed with an Alamar Blue assay. Specifically, cell culturesof LNCap prostate tumor cells or DuPRO cells in 96 well plates wasdiluted with medium containing various concentrations of a givenconjugate (final plate well volume of 2'.O0ul). The cells were incubatedfor 3 days at 37°C, 20ul of Alamar Blue is added to the assay well. Thecells were further incubated and the assay plates were read on a EL—3lOELISA reader at the dual wavelengths of 570 and 600 nm at 4 and 7hours after addition of Alamar Blue. Relative percentage viability at the?W0 98/10651CA 02265476 l999-03- 10-58-PCT/US97/16087various concentration of conjugate tested was then calculated versuscontrol (no conjugate) cultures. Results of this assay are shown in Table4. If no salt is indicated, the free conjugate was tested.TABLE 4S_E_Q_. E D DE-D X N E LNCaP Cell Kill inlE..N_O_. 72 HRS, { 43 HRS }H. 50 I “J6 4 2-hydroxyacetyl-hFlSSYQ—SNle-DOX (3') 3.6 (DuPRO > 100)6 6 2-hydroxyacetyl-ShFlChgQ—SL-DOX (3') 5.1 (DUPRO > 100)6 7 2-hydroxyacetyl—hFlSSChgQ-SL—DOX (3') 5.5 (DuPRO > 100)6 8 2-hydroxyacetyl—hRASChgQ-SL—DOX (3') 7.9 (DuPRO > 100) (PS)6 9 (d)2,3—dihydroxypropionyl-ShRChgQ—SL-DOX (3') 5.8 (DUPRO > 100) n=27 0 (1)2,3-dihydroxypropionyl—ShFlChgQ-SL-DOX (3') 9.4 (DuPRO > 100) n = 27 1 PEG(2)-ShRChgQ-SL-DOX (3') 8.1 (DUPRO > 100)7 2 PEG(2)-hFlChgQ-SL-DOX (3') INSOLUBLE7 3 (2R,3S) 2.3,4—trihydroxybutanoyl—hFlChgQ—SL-DOX (3') PS7 4 PEG(2)-ShRYQ-SL-DOX(3') 4.5 (DuPRO > 100)7 5 PEG(2)—hFlYQ-SSSL-DOX (3') 14 (DuPRO > 100) (PS)7 6 PEG(2)-KYQ-SSSL—DOX (3') 12.8 (DUPRO > 100) (PS)7 7 2-hydroxyacetyl-hRSSYQ-SL—DOX (3') 13.6 (DuPRO > 100) (PS)7 8 (|)2,3-dihydroxypropionylh?SSChgQSL-DOX (3') 7.5 (DuPRO > 100)7 9 PEG(2)-hRSSChgQ-SL-DOX (3') 5.7 (DuPRO > 100)8 0 2-hydroxyacetyl-SYQ—SSSL-DOX (3') 18.8 (DuPRO = 50) (PS)8 1 PEG(16)-ShRYQ-SL-DOX (3') 45 (DuPFlO = 100)8 2 (2R,3S) 2,3.4—trihydroxybutanoyl-ShFtChgQ-SL-DOX (3') 14.1 (DUPRO > 100)8 3 PEG(2)—ShRYQ-SL—DOX (3') 34 (DuPRO = 100) n=28 4 (d)2.3—dihydroxypropionyl—hRSSChgQSL-DOX(3') 7.7 (DUPFIO >100) n = 28 5 (l)2,3-dihydroxypropiony|SSSChgQ-S(dL)—DOX (3') 91 (DUPRO > 100)8 6 (d)2,3—dihydroxypropionyISSSChgQ-SL-DOX (3') 5.8 (DUPRO > 100) n = 387 (I)2,3-dihydroxyproplony|SSSChgQ-SL-DOX (3') 5.5 (DUPRO > 100)88 (l)2,3-dihydroxypropionylSSChgQ-S(dL)-DOX (3') > 100 (DUPRO > 100)89 (d)2,3-dihydroxypropionylSSChgO-SL-DOX (3') 9.1 (DUPRO > 100)91 PEG(2)SSChgQ-SL-DOX (3') 8.8 (DuPRO > 100)63 PEG(2)-SSSChgQ-SL-DOX (3') 10 (DuPRO > 100) n=294 (d)2,3-dihydroxypropiony|—3PAL-SSChgQ-SL-DOX (3'). 5.5 (DUPRO > 100)AcOH95 (1)2,3—dihydroxypropionyl-SSChgQ—SL-DOX (3') 13 (DUPRO > 100) n = 261 2-hydroxyacety|-SSSChgQ—SL-DOX (3') 7.2 (DUPRO > 100) n = 396 2,3-dihydroxypropionyl—hSSSChgQ-SL-DOX (3') 5.1 (DUPRO = 90)97 PEG(2)-ASChgQ-SL-DOX (3') 5.6 (DuPFlO = 100) n=298 PEG(6)-ASChgQ—SL-DOX (3') 12 (DUPRO = 100)62 2-hydroxyacetyl—SSChgQ-SL-DOX (3') 4.8 (DuPRO > 100)?W0 98/106511015202530CA 02265476 l999-03- 10PCT/US97/ 16087-59..EXAMPLILQIn vivo Efficacv of Peotidvl —Cvtotoxic Agent ConiugatesLNCaP.FGC or DuPRO—l cells are trypsinized,resuspended in the growth medium and centifuged for 6 mins. at200xg. The cells are resuspended in serum-free Ot—MEM and counted.The appropriate volume of this solution containing the desired numberof cells is then transferred to a conical centrifuge tube, centrifuged asbefore and resuspended in the appropriate volume of a cold 1:1 mixtureof ot—MEM-Matrigel. The suspension is kept on ice until the animalsare inoculated.Harlan Sprague Dawley male nude mice (10-12 weeksold) are restrained without anesthesia and are inoculated with 0.5 mLof cell suspension on the left flank by subcutaneous injection using a22G needle. Mice are either given approximately 5x105 DuPRO cellsor l.5xl07 LNCaP.FGC cells.Following inoculation with the tumor cells the mice aretreated under one of two protocols:Protocol A:One day after cell inoculation the animals are dosed witha 0.1-0.5 mL volume of test conjugate, doxorubicin or vehicle control(sterile water). Dosages of the conjugate and doxorubicin are initiallythe maximum non-lethal amount, but may be subsequently titratedlower. Identical doses are administered at 24 hour intervals for 5 days.After 10 days, blood samples are removed from the mice and the semmlevel of PSA is determined. Similar serum PSA levels are determined at5-10 day intervals. At the end of 5.5 weeks the mice are sacrificed andweights of any tumors present are measured and serum PSA againdetermined.The animals‘ weights are determined at the beginning andend of the assay.?1015202530W0 98/10651CA 02265476 l999-03- 10PCT/U S97/ 16087-60-Protocol B:Ten days after cell inoculation,blood samples are removedfrom the animals and serum levels of PSA are determined. Animals arethen grouped according to their PSA serum levels. At 14-15 days aftercell inoculation, the animals are dosed with a 0.1-0.5 mL volume of testconjugate, doxorubicin or vehicle control (sterile water). Dosages ofthe conjugate and doxorubicin are initially the maximum non-lethalamount, but may be subsequently titrated lower. Identical doses areadministered at 24 hour intervals for 5 days. Serum PSA levels aredetermined at 5-10 day intervals. At the end of 5.5 weeks the mice aresacrificed, weights of any tumors present are measured and serum PSAagain determined. The animals’ weights are determined at the beginningand end of the assay.EXAMPLE 10In vitro determination of proteolytic cleavage of conjugates byendogenous non-PSA DroteasesStep A: Preparation of proteolytic tissue extractsAll procedures are carried out at 4°C. Appropriateanimals are sacrificed and the relevant tissues are isolated and storedin liquid nitrogen. The frozen tissue is pulverized using a mortarand pestle and the pulverized tissue is transfered to a Potter—Elvejehhomogenizer and 2 volumes of Buffer A (50 mM Tris containing 1.15%KCl, pH 7.5) are added. The tissue is then disrupted with 20 strokesusing first a lose fitting and then a tight fitting pestle. The homogenateis centrifuged at 10,000 x g in a swinging bucket rotor (HB4-5), thepellet is discarded and the re—supematant centrifuged at 100,000 x g(Ti 70). The supernatant (cytosol)is saved.The pellet is respuspended in Buffer B (10 mM EDTAcontaining 1.15% KCI, pH 7.5) using the same volume used in step?W0 98/1065]101520CA 02265476 l999-03- 10PCT/U S9'7l 16087-61-as used above with Buffer A. The suspension is homogenized in adounce homogenizer and the solution centrifuged at l00,000x g. Thesupernatant is discarded and the pellet resuspended in Buffer C (10 mMpotassium phosphate buffer containing0.25 M sucrose, pH 7.4), using1/2 the volume used above, and homogenized with a douncehomogenizer.Protein content of the two solutions (cytosol andmembrane) is determine using the Bradford assay. Assay aliquots arethen removed andfrozen in liquid N2. The aliquots are stored at —700C.Proteolytic cleavage assayFor each time point, 20 microgram of peptide—doxorubicinconjugate and 150 micrograms of tissue protein, prepared as describedin Step A and as determined by Bradford in reaction buffer are placedin solution of final volume of 200 microliters in buffer (50 mM TRIS,140 mM NaCl, pH 7.2). Assay reactions are run for O, 30, 60, 120, and180 minutes and are then quenched with 9 microliters of 0.1 M ZnCl2Step B:and immediately placed in boiling water for 90 seconds. Reactionproducts are analyzed by HPLC using a VYDAC C18 15 cm column inwater / acetonitrile (5% to 50% acetonitrile over 30 minutes).?1CA 02265476 1999-03-l0W0 98/10651 PCT/US97/16087_ 62 _SEQUENCE LISTING5 (1) GENERAL INFORMATION(1) APPLICANT: FENG, DONG-MEIGARSKY, VICTOR, M.JONES, RAYMOND, E.10 OLIFF, ALLEN, I.WAI, JENNY, M.1520253035404550(ii) TITLE OF THE INVENTION: CONJUGATES USEFUL IN THETREATMENTOF PROSTATE CANCER(iii) NUMBER OF SEQUENCES: 128CORRESPONDENCE ADDRESS:(A) ADDRESSEE: Merck & Co.,(B) STREET: P.O. Box 2000,(C) CITY: Rahway(D) STATE: NJ(E) COUNTRY: USAF) ZIP: 07065-0900Inc.126 E. Lincoln Ave.(V) COMPUTER READABLE FORM:(A) MEDIUM TYPE: Diskette(B) COMPUTER: IBM Compatible(C) OPERATING SYSTEM: DOS(D) SOFTWARE: FastSEQ for Windows Version 2.0(Vi) CURRENT APPLICATION DATA:(A) APPLICATION NUMBER:(B) FILING DATE:(C) CLASSIFICATION:(Vii) PRIOR APPLICATION DATA:(A) APPLICATION NUMBER: 60,026,015(B) FILING DATE: 09—DEC-1996(viii) ATTORNEY/AGENT INFORMATION:(A) NAME: Muthard, David A(B) REGISTRATION NUMBER: 35,297(C) REFERENCE/DOCKET NUMBER: 19784Y) TELECOMMUNICATION INFORMATION:A) TELEPHONE: 908-594-3903B) TELEFAX: 908-594-4720C) TELEX:?W0 98/1065]102025303540455055CA 02265476 1999-03-10- 63:-(2) INFORMATION FOR SEQ ID NO:l:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 7 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(Xi) SEQUENCE DESCRIPTION: SEQ ID N021:Asn Lys Ile Ser Tyr Gln Ser15(2) INFORMATION FOR SEQ ID NO:2:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 6 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:Lys Ile Ser Tyr Gln Ser1S(2) INFORMATION FOR SEQ ID NO:3:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 7 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:Asn Lys Ile Ser Tyr Tyr Ser15(2) INFORMATION FOR SEQ ID NO:4:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 7 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:PCT/US97/16087?WO 98/1065110152025303540455055CA 02265476 1999-03-10PCT/US97/16087-64-Asn Lys Ala Ser Tyr Gln Ser1 S(2) INFORMATION FOR SEQ ID NO:5:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 5 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:Ser Tyr Gln Ser Ser1 5(2) INFORMATION FOR SEQ ID NO:6:(i) SEQUENCE CHARACTERISTICS:( ) LENGTH: 5 amino acids( ) TYPE: amino acid( ) STRANDEDNESS: single( ) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:Lys Tyr Gln Ser Ser1 S(2) INFORMATION FOR SEQ ID NO:7:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 5 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(ix) FEATURE:(A) NAME/KEY: Other(B) LOCATION: l...1(D) OTHER INFORMATION: Homoarginine(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:Xaa Tyr Gln Ser Ser1 5(2) INFORMATION FOR SEQ ID NO:8:?CA 02265476 1999-03-10W0 98/ 1065110152025303540455055- 55 _(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 5 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(ix) FEATURE:(A) NAME/KEY: Other(B) LOCATION: l...l(D) OTHER INFORMATION: Homoarginine(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:Xaa Cys Gln Ser Ser1 S(2) INFORMATION FOR SEQ ID NO:9:SEQUENCE CHARACTERISTICS:) LENGTH: 4 amino acids) TYPE: amino acid) STRANDEDNESS: single) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:Tyr Gln Ser Ser1(2) INFORMATION FOR SEQ ID NO:lO:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 4 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:l0:Tyr Gln Ser Leu1(2) INFORMATION FOR SEQ ID NO:ll:(i SEQUENCE CHARACTERISTICS:) LENGTH: 4 amino acids) TYPE: amino acid))STRANDEDNESS: single)(A(B(C(D TOPOLOGY: linearPCT/US97ll6087?WO 9811065110152025303540455055CA 02265476 1999-03-10PCT/US97/16087- 66 _(ii) MOLECULE TYPE: peptide(ix) FEATURE:(A) NAME/KEY: Other(B) LOCATION: 4...4(D) OTHER INFORMATION: Norleucine(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:ll:Tyr Gln Ser Leu1(2) INFORMATION FOR SEQ ID NO:l2:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 4 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(ix) FEATURE:(A) NAME/KEY: Other(B) LOCATION: l...l(D) OTHER INFORMATION: Cyclohexylglycine(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:l2:Xaa Gln Ser Leu1(2) INFORMATION FOR SEQ ID NO:l3:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 4 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(ix) FEATURE:(A) NAME/KEY: Other(B) LOCATION: l...l(D) OTHER INFORMATION: Cyclohexylglycine(A) NAME/KEY: otherB) LOCATION: 4...4(D) OTHER INFORMATION: Norleucine(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:l3:Xaa Gln Ser Leu1?10152025303540455055W0 98/1065!(2)CA 02265476 1999-03-10PCT/US97l 16087-67- -INFORMATION FOR SEQ ID NO:l4:(i) SEQUENCE CHARACTERISTICS:(A(B(C(D))))LENGTH: 8 amino acidsTYPE: amino acidSTRANDEDNESS: singleTOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi)SEQUENCE DESCRIPTION: SEQ ID NO:14:Asn Lys Ile Ser Tyr Gln Ser Ser1(2)(i)(A(B(C(DS))))5INFORMATION FOR SEQ ID NO:l5:EQUENCE CHARACTERISTICS:LENGTH: 8 amino acidsTYPE: amino acidSTRANDEDNESS: singleTOPOLOGY: linear(ii) MOLECULE TYPE: peptide(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:l5:Asn Lys Ile Ser Tyr Gln Ser Ala1(2)(i)(A(B(C(DS))))5INFORMATION FOR SEQ ID NO:16:EQUENCE CHARACTERISTICS:LENGTH: 8 amino acidsTYPE: amino acidSTRANDEDNESS: singleTOPOLOGY: linear(ii) MOLECULE TYPE: peptide(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:Ala Asn Lys Ile Ser Tyr Tyr Ser15(2) INFORMATION FOR SEQ ID NO:l7:(i)S(A)(B)EQUENCE CHARACTERISTICS:LENGTH: 8 amino acidsTYPE: amino acid(c) STRANDEDNESS: single(D) TOPOLOGY: linear(ii)(xi)MOLECULE TYPE: peptideSEQUENCE DESCRIPTION: SEQ ID NO:l7:?W0 98/1065]10152025303540455055CA 02265476 1999-03-10PCT/US97/16087_6g_Ala Asn Lys Ala Ser Tyr Gln Ser1 5(2) INFORMATION FOR SEQ ID NO:l8:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 6 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l8:Ser Tyr Gln Ser Ser Thr1 5(2) INFORMATION FOR SEQ ID NO:l9:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 6 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l9:Ser Tyr Gln Ser Ser Ser1 S(2) INFORMATION FOR SEQ ID NO:20:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 6 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:Lys Tyr Gln Ser Ser Ser1 5(2) INFORMATION FOR SEQ ID NO:2l:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 6 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear?W0 98/1065110Xaa Tyr Gln Ser Ser152025CA 02265476 1999-03-10PCTIUS97/16087-69»-(ii) MOLECULE TYPE: peptide(ix) FEATURE:(A)(B)(D)NAME/KEY: OtherLOCATION: l...1OTHER INFORMATION: Homoarginine(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:1(2)(i) S(A)(B)(C)(D)(ii)(xi)Ser5INFORMATION FOR SEQ ID NO:22:EQUENCE CHARACTERISTICS:LENGTH: 6 amino acidsTYPE: amino acidSTRANDEDNESS: singleTOPOLOGY: linearMOLECULE TYPE: peptideSEQUENCE DESCRIPTION: SEQ ID NO:22:Ser Tyr Gln Ser Ser Leu3035401(2)(i)(A)(B)(C)(D)5INFORMATION FOR SEQ ID NO:23:SEQUENCE CHARACTERISTICS:LENGTH: 5 amino acidsTYPE: amino acidSTRANDEDNESS: singleTOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi)SEQUENCE DESCRIPTION: SEQ ID NO:23:Ser Tyr Gln Ser Leu4550551(2)5INFORMATION FOR SEQ ID NO:24:(i) SEQUENCE CHARACTERISTICS:(A)(B)(C)(D)(ii)LENGTH: 5 amino acidsTYPE: amino acidSTRANDEDNESS: singleTOPOLOGY: linearMOLECULE TYPE: peptide(ix) FEATURE:(A)(B)NAME/KEY: OtherLOCATION: 2...2?10152025303540455055W0 98/10651CA 02265476 1999-03-10PCTlUS97/ 16087-70- -(D) OTHER INFORMATION: Cyclohexylglycine(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:Ser Xaa Gln Ser Leu1 S(2) INFORMATION FOR SEQ ID NO:25:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 5 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(ix) FEATURE:(A) NAME/KEY: Other(B) LOCATION: l...lOTHER INFORMATION: Homoarginine(A) NAME/KEY: Other(B) LOCATION: 2...2(D) OTHER INFORMATION: Cyclohexylglycine(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:Xaa Xaa Gln Ser Leu1 S(2) INFORMATION FOR SEQ ID NO:26:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 5 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: Single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(ix) FEATURE:(A) NAME/KEY: Other(B) LOCATION: l...l(D) OTHER INFORMATION: Homoarginine(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:Xaa Tyr Gln Ser Leu1 5(2) INFORMATION FOR SEQ ID NO:27:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 19 amino acids(B) TYPE: amino acid?WO 98/10651101520253040455055(C)(D)CA 02265476 1999-03-10PCT/US97/16087- 7] _STRANDEDNESS:TOPOLOGY:singlelinear(ii) MOLECULE TYPE: peptide(xi)Gly Glu Asn1Gln Thr Glu(2)(ii)SEQUENCE DESCRIPTION: SEQ ID NO:27:Gly Val Gln Lys Asp Val Ser Gln Arg Ser Ile Tyr Ser5 10 15INFORMATION FOR SEQ ID NO:28:EQUENCE CHARACTERISTICS:LENGTH: 7 amino acidsTYPE: amino acidSTRANDEDNESS: singleTOPOLOGY: linearMOLECULE TYPE: peptide(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:Ala Ser Tyr1(2)Gln Ser Ser Leu5INFORMATION FOR SEQ ID NO:29:(i) SEQUENCE CHARACTERISTICS:(A)(B)(C)(D)LENGTH: 6 amino acidsTYPE: amino acidSTRANDEDNESS: singleTOPOLOGY: linear(ii) MOLECULE TYPE: peptide(ix) FEATURE:(A)(B)(D)(A)(B)(D)(xi)Ser Xaa Xaa1(2)(i)(A)(B)NAME/KEY: otherLOCATION: 2...2OTHER INFORMATION: HomoarginineNAME/KEY: OtherLOCATION: 3...3OTHER INFORMATION: CyclohexylglycineSEQUENCE DESCRIPTION: SEQ ID NO:29:Gln Ser Leu5INFORMATION FOR SEQ ID NO:30:SEQUENCE CHARACTERISTICS:LENGTH:TYPE:7 amino acidsamino acid, ......._..—-—-——u-—..........-._.. .. ,?10152025303540455055W0 98/10651CA 02265476 1999-03-10PCTIUS97Il6087- 72 _(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(ix) FEATURE:(A) NAME/KEY: Other(B) LOCATION: l...l(D) OTHER INFORMATION: Homoarginine(A) NAME/KEY: Other(B) LOCATION: 7...7(D) OTHER INFORMATION: Norleucine(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:Xaa Ser Ser Tyr Gln Ser Leu1 5(2) INFORMATION FOR SEQ ID NO:3l:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 7 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(ix) FEATURE:(A) NAME/KEY: Other(B) LOCATION: l...l(D) OTHER INFORMATION: Homoarginine(A) NAME/KEY: Other(B) LOCATION: 4 ..4(D) OTHER INFORMATION: Cyclohexylglycine(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:Xaa Ala Ser Xaa Gln Ser Leu1 S(2) INFORMATION FOR SEQ ID NO:32:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 7 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(ix) FEATURE:(A) NAME/KEY: Other(B) LOCATION: l...l?CA 02265476 1999-03-10W0 98/10651 PCT/US97ll6087- 73 -(D) OTHER INFORMATION: Homoarginine(xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:5 Xaa Ser Ser Tyr Gln Ser Leu10LII2025303540455055l S(2) INFORMATION FOR SEQ ID NO:33:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 6 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii)(ix)MOLECULE TYPE: peptideFEATURE:(A)(B)(D)NAME/KEY: OtherLOCATION: 1...lOTHER INFORMATION: Homoarginine(A)(B)(D)NAME/KEY: OtherLOCATION: 4...4OTHER INFORMATION: Cyclohexylglycine(xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:Xaa Ser Ser Xaa Ser Leu1 S(2) INFORMATION FOR SEQ ID NO:34:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 6 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(ix) FEATURE:(A) NAME/KEY: Other(B) LOCATION: 2...2(D) OTHER INFORMATION: Homuarginine(A) NAME/KEY: Other(B) LOCATION: 3...3(D) OTHER INFORMATION: Cyclohexylglycine(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:Ser Xaa Xaa Gln Ser Leu1 5(2) INFORMATION FOR SEQ ID NO:3S:?W0 98/1065110152025303540455055CA 02265476 1999-03-10PC1VUS97??087- 74 -(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 5 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(ix) FEATURE:(A) NAME/KEY: Other(B) LOCATION: l...l(D) OTHER INFORMATION: Homoarginine(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:Xaa Tyr Gln Ser Leu1 5(2) INFORMATION FOR SEQ ID NO:36:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 7 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(ix) FEATURE:(A) NAME/KEY: Other(B) LOCATION: l...l(D) OTHER INFORMATION: Homoarginine(A) NAME/KEY: Other(B) LOCATION: 4...4(D) OTHER INFORMATION: Cyclohexylglycine(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:Xaa Ser Ser Xaa Gln Ser Leu1 S(2) INFORMATION FOR SEQ ID NO:37:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 6 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(ix) FEATURE:(A) NAME/KEY: Other(B) LOCATION: 2...2?W0 98/1065!10152025303540455055CA 02265476 1999-03-10_ 75 _(D) OTHER INFORMATION: Homoarginine(xi)SEQUENCE DESCRIPTION: SEQ ID NO:37:Ser Xaa Tyr Gln Ser Leu15(2) INFORMATION FOR SEQ ID NO:38:(i) SEQUENCE CHARACTERISTICS:(A)(B)(C)(D)(ii)(xi)LENGTH: 6 amino acidsTYPE: amino acidSTRANDEDNESS: singleTOPOLOGY: linearMOLECULE TYPE: peptideSEQUENCE DESCRIPTION: SEQ ID NO:38:Ser Ser Tyr Gln Ser Leu15(2) INFORMATION FOR SEQ ID NO:39:(i) SEQUENCE CHARACTERISTICS:(A)(B)(C)(D)(ii)(ix)LENGTH: 7 amino acidsTYPE: amino acidSTRANDEDNESS: singleTOPOLOGY: linearMOLECULE TYPE: peptideFEATURE:(A) NAME/KEY: Other(B) LOCATION: 4...4(D) OTHER INFORMATION: Cyclohexylglycine(Xi)SEQUENCE DESCRIPTION: SEQ ID NO:39:Ser Ser Ser Xaa Gln Ser Leul5(2) INFORMATION FOR SEQ ID NO:40:(i) SEQUENCE CHARACTERISTICS:(A)(B)(C)(D)(ii)(ix)LENGTH: 7 amino acidsTYPE: amino acidSTRANDEDNESS: singleTOPOLOGY: linearMOLECULE TYPE: peptideFEATURE:(A) NAME/KEY: Other(B) LOCATION: l...l(D) OTHER INFORMATION:3-PyridylalaninePCT/US97/16087?CA 02265476 1999-03-10W0 98/ 10651 PCT/US97/16087- 76 _(A) NAME/KEY: Other(B) LOCATION: 4...4(D) OTHER INFORMATION: Cyclohexylglycine10152025303540455055(xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:Xaa Ser Ser Xaa Gln Ser Leu1 S(2) INFORMATION FOR SEQ ID NO:4l:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 6 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(ix) FEATURE:(A) NAME/KEY: Other(B) LOCATION: 3...3(D) OTHER INFORMATION: Cyclohexylglycine(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:4l:Ser Ser Xaa Gln Ser Leu1 5(2) INFORMATION FOR SEQ ID NO:42:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 7 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(ix) FEATURE:(A) NAME/KEY: other(B) LOCATION: 3...3(D) OTHER INFORMATION: Cyclohexylglycine(A) NAME/KEY: Other(B) LOCATION: 7...7(D) OTHER INFORMATION: Leucine with UnnaturalStereoconfiguration(xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:Ser Ser Ser Xaa Gln Ser Xaal S?W0 98/10651I0152025303540455055CA 02265476 1999-03-10PC'I‘IUS97I16087- 77 _(2) INFORMATION FOR SEQ ID NO:43:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 7 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(ix) FEATURE:(A) NAME/KEY: other(B) LOCATION: 4...4(D) OTHER INFORMATION: Cyclohexylglycine(xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:Ser Ser Ser Xaa Gln Ser Val1 S(2) INFORMATION FOR SEQ ID NO:44:SEQUENCE CHARACTERISTICS:) LENGTH: 7 amino acids) TYPE: amino acid) STRANDEDNESS: single) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(ix) FEATURE:(A) NAME/KEY: Other(B) LOCATION: 4...4(D) OTHER INFORMATION: Cyclohexylglycine(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:Pro Ser Ser Xaa Gln Ser Val1 5(2) INFORMATION FOR SEQ ID NO:45:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 7 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(ix) FEATURE:(A) NAME/KEY: Other(B) LOCATION: 4...4(D) OTHER INFORMATION: Cyclohexylglycine(xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:?W0 98/1065110152025303540455055CA 02265476 1999-03-10PCT/US97I 16087_ 78 -Gly Ser Ser Xaa Gln Ser Leu1 5(2) INFORMATION FOR SEQ ID NO:46:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 7 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(ix) FEATURE:(A) NAME/KEY: otherB LOCATION: l...lOTHER INFORMATION: Homoserine(A) NAME/KEY: Other(B) LOCATION: 4...4(D) OTHER INFORMATION: Cyclohexylglycine(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:Xaa Ser Ser Xaa Gln Ser Leu1 5(2) INFORMATION FOR SEQ ID NO:47:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 7 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(ix) FEATURE:(A) NAME/KEY: Other(B) LOCATION: l...l(D) OTHER INFORMATION: Homoarginine(A) NAME/KEY: Other(B) LOCATION: 4...4(D) OTHER INFORMATION: Cyclohexylglycine(A) NAME/KEY: Other) LOCATION: 7...7) OTHER INFORMATION: Norleucine(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:Xaa Ser Ser Xaa Gln Ser Leu1 5?W0 98Il065l10152025303540455055(2)CA 02265476 1999-03-10PCTIUS97Il6087-79- .INFORMATION FOR SEQ ID NO:48:(i) SEQUENCE CHARACTERISTICS:(A)(B)(C)(D)(ii)(ix)(A)(B)(D)(A)(B)LENGTH: 7 amino acidsTYPE: amino acidSTRANDEDNESS: singleTOPOLOGY: linearMOLECULE TYPE: peptideFEATURE:NAME/KEY: OtherLOCATION: 1...1OTHER INFORMATION: HomoarginineNAME/KEY: OtherLOCATION: 4...4(D) OTHER INFORMATION: Cyclohexylglycine(xi)SEQUENCE DESCRIPTION: SEQ ID NO:48:Xaa Ser Ala Xaa Gln Ser Leu1(2)5INFORMATION FOR SEQ ID NO:49:(i) SEQUENCE CHARACTERISTICS:(A)(B)(C)(D)(ii)(xi)LENGTH: 7 amino acidsTYPE: amino acidSTRANDEDNESS: singleTOPOLOGY: linearMOLECULE TYPE: peptideSEQUENCE DESCRIPTION: SEQ ID NO:49:Asn Arg Ile Ser Tyr Gln Ser1S(2) INFORMATION FOR SEQ ID NO:50:(i)(A)(B)(C)(D)(ii)(xi)SEQUENCE CHARACTERISTICS:LENGTH: 7 amino acidsTYPE: amino acidSTRANDEDNESS : s ingleTOPOLOGY: linearMOLECULE TYPE: peptideSEQUENCE DESCRIPTION: SEQ ID NO:50:Asn Lys Val Ser Tyr Gln Ser1S?W0 98/10651WU2025303540455055CA 02265476 1999-03-10PCTlUS97Il6087_ 80 _(2) INFORMATION FOR SEQ ID NO:5l:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 8 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:5l:Asn Lys Met Ser Tyr Gln Ser Ser1 S(2) INFORMATION FOR SEQ ID NO:52:(i SEQUENCE CHARACTERISTICS:) LENGTH: 8 amino acids) TYPE: amino acid) STRANDEDNESS: single) TOPOLOGY: linear6853“(ii) MOLECULE TYPE: peptide(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:Asn Lys Leu Ser Tyr Gln Ser Ser1 S(2) INFORMATION FOR SEQ ID NO:53:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 7 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:Asn Lys Ile Ser Tyr Gln Ser1 5(2) INFORMATION FOR SEQ ID NO:54:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 8 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: Single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:?CA 02265476 1999-03-10WO 98110651 PCTIUS97ll6087_ 81 _Gln Lys Ile Ser Tyr Gln Ser Ser1 55 (2) INFORMATION FOR SEQ ID NO:SS:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 7 amino acids(B) TYPE: amino acid10 (C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(ix) FEATURE:15(A) NAME/KEY: Other(B) LOCATION: l...l(D) OTHER INFORMATION: Homoarginine20 (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:S5:Xaa Tyr Gln Ser Ser Ser Leu1 525 (2) INFORMATION FOR SEQ ID NO:56:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 7 amino acids(B) TYPE: amino acid30 (C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide35 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:Lys Tyr Gln Ser Ser Ser Leu1 540 (2) INFORMATION FOR SEQ ID NO:57:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 7 amino acids(B) TYPE: amino acid45 (C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide50 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:S7:Ser Tyr Gln Ser Ser Ser Leu1 S55 (2) INFORMATION FOR SEQ ID NO:58:?IO152025303540455055CA 02265476 1999-03-10W0 98/ 10651 PCT/US97l16087-82- .(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 6 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(ix) FEATURE:(A)(B)(D)NAME/KEY: OtherLOCATION: 3. .3OTHER INFORMATION: Cyclohexylglycine(A) NAME/KEY: Other(B) LOCATION: 6...6(D) OTHER INFORMATION: Leucine with UnnaturalStereoconfiguration(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:S8:Ser Ser Xaa Gln Ser Xaa1 5(2) INFORMATION FOR SEQ ID NO:59:SEQUENCE CHARACTERISTICS:)(A) LENGTH: 7 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(ix) FEATURE:(A) NAME/KEY: Other(B) LOCATION: l...l(D) OTHER INFORMATION: Pyridylalanine(A) NAME/KEY: Other(B) LOCATION: 4...4(D) OTHER INFORMATION: Cyclohexylglycine(A) NAME/KEY: Other(B) LOCATION: 7...7(D) OTHER INFORMATION: Leucine with UnnaturalStereoconfiguration(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:59:Xaa Ser Ser Xaa Gln Ser Xaa1 5(2) INFORMATION FOR SEQ ID NO:60:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 6 amino acids?CA 02265476 1999-03-10W0 98/10651 PCT/US97/ 16087-33-(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear5 (ii) MOLECULE TYPE: peptide(ix) FEATURE:(A) NAME/KEY: Other(B) LOCATION: 3...310 (D) OTHER INFORMATION: Cyclohexylglycine(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:60:Ala Ser Xaa Gln Ser Leu15 1 5(2) INFORMATION FOR SEQ ID NO:6l:(i) SEQUENCE CHARACTERISTICS:20 (A) LENGTH: 7 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear25 (ii) MOLECULE TYPE: peptide(ix) FEATURE:(A) NAME/KEY: OtherB LOCATION: 1...130 (D) OTHER INFORMATION: N-(2—Hydroxyacetyl)Serine(A) NAME/KEY: Other(B) LOCATION: 4...4(D) OTHER INFORMATION: Cyclohexylglycine35(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6l:Xaa Ser Ser Xaa Gln Ser Leu1 540(2) INFORMATION FOR SEQ ID NO:62:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 6 amino acids45 (B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide50 (ix) FEATURE:(A) NAME/KEY: Other(B) LOCATION: l...l(D) OTHER INFORMATION: N-(2-Hydroxyacetyl)Serine55(A) NAME/KEY: other?W0 98/ 1065110152025303540455055CA 02265476 1999-03-10PCTlUS97ll6087-84-(B) LOCATION: 3...3(D) OTHER INFORMATION: Cyclohexylglycine(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:62:Xaa Ser Xaa Gln Ser Leu1 5(2) INFORMATION FOR SEQ ID NO:63:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 7 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(ix) FEATURE:(A) NAME/KEY: Other(B) LOCATION: l...l(D) OTHER INFORMATION: N—(PEG-2)Serine(A) NAME/KEY: Other(B) LOCATION: 4...4(D) OTHER INFORMATION: Cyclohexylglycine(xi) SEQUENCE DESCRIPTION: SEQ ID NO:63:Xaa Ser Ser Xaa Gln Ser Leu1 5(2) INFORMATION FOR SEQ ID NO:64:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 7 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(ix) FEATURE:(A) NAME/KEY: Other(B) LOCATION: 1...l(D) OTHER INFORMATION: N-(2—Hydroxyacetyl)Homoarginine(A) NAME/KEY: Other(B) LOCATION: 7...7(D) OTHER INFORMATION: Norleucine(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:64:Xaa Ser Ser Tyr Gln Ser Leu1 5?WO 98/1065110152025(2)CA 02265476 1999-03-10PCT lUS97Il6087-85-INFORMATION FOR SEQ ID NO:65:(i) SEQUENCE CHARACTERISTICS:(A)(B)(C)(D)LENGTH: 7 amino acidsTYPE: amino acidSTRANDEDNESS: singleTOPOLOGY: linear(ii) MOLECULE TYPE: peptide(ix) FEATURE:(A)(B)(D)(A)(B)(D)(A)(B)(D)NAME/KEY: OtherLOCATION: l...1OTHER INFORMATION: N-(2"Hydroxyacetyl)HomoarginineNAME/KEY: OtherLOCATION: 4...4OTHER INFORMATION: CyclohexylglycineNAME/KEY: OtherLOCATION: 7...7OTHER INFORMATION: Norleucine(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:65:Xaa Ser Ser Xaa Gln Ser Leu13035404550(2)5INFORMATION FOR SEQ ID NO:66:(i) SEQUENCE CHARACTERISTICS:(A)(B)(C)(D)LENGTH: 6 amino acidsTYPE: amino acidSTRANDEDNESS: singleTOPOLOGY: linear(ii) MOLECULE TYPE: peptide(ix) FEATURE:(A)(B)(D)(A)(B)(D)(A)(B)(D)(xi)NAME/KEY: otherLOCATION: 1...1OTHER INFORMATION: N-(2—Hydroxyacetyl)SerineNAME/KEY: OtherLOCATION: 2...2OTHER INFORMATION: HomoarginineNAME/KEY: OtherLOCATION: 3...3OTHER INFORMATION: CyclohexylglycineSEQUENCE DESCRIPTION: SEQ ID NO:66:Xaa Xaa Xaa Gln Ser Leu55 15?W0 98/ 10651101520253035404550551CA 02265476 1999-03-10PCT/US97Il6087_ 86 _(2) INFORMATION FOR SEQ ID NO:67:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 7 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(ix) FEATURE:(A) NAME/KEY: Other(B) LOCATION: l...l(D) OTHER INFORMATION: N-(2—Hydroxyacetyl)Homoarginine(A) NAME/KEY: Other(B) LOCATION: 4...4(D) OTHER INFORMATION: Cyclohexylglycine(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:67:Xaa Ser Ser Xaa Gln Ser Leu1 5(2) INFORMATION FOR SEQ ID NO:68:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 7 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(ix) FEATURE:(A) NAME/KEY: other(8) LOCATION: l...l(D) OTHER INFORMATION: N-(2-Hydroxyacetyl)Homoarginine(A) NAME/KEY: other(B) LOCATION: 4...4(D) OTHER INFORMATION: Cyclohexylglycine(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:68:Xaa Ala Ser Xaa Gln Ser Leu1 S(2) INFORMATION FOR SEQ ID NO:69:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 6 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear?W0 98/ 1065110152025303540455055CA 02265476 1999-03-10PCTlUS97l 16087-37-(ii) MOLECULE TYPE: peptide(ix) FEATURE:(A) NAME/KEY: Other(B) LOCATION: l...l(D) OTHER INFORMATION: N-((d)-2,3~Dihydroxypropionyl)Serine(A) NAME/KEY: Other(B) LOCATION: 2...2(D) OTHER INFORMATION: Homoarginine(A) NAME/KEY: Other(B) LOCATION: 3...3(D) OTHER INFORMATION: Cyclohexylglycine(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:69:Xaa Xaa Xaa Gln Ser Leu1 S(2) INFORMATION FOR SEQ ID NO:70:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 6 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(ix) FEATURE:(A) NAME/KEY: Other(B) LOCATION: l...l(D) OTHER INFORMATION: N—((1)-2,3-Dihydroxypropionyl)Serine(A) NAME/KEY: other(B) LOCATION: 2...2(D) OTHER INFORMATION: Homoarginine(A) NAME/KEY: Other(B) LOCATION: 3...3(D) OTHER INFORMATION: Cyclohexylglycine(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:70:Xaa Xaa Xaa Gln ser Leu1 S(2) INFORMATION FOR SEQ ID NO:71:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 6 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear?10152025303540455055WO 98110651CA 02265476 1999-03-10PCTIUS97/16087-gg-(ii) MOLECULE TYPE: peptide(ix) FEATURE:(A) NAME/KEY: Other(B) LOCATION: l...l(D) OTHER INFORMATION: N-(PEG—2)Serine(A) NAME/KEY: Other(B) LOCATION: 2...2(D) OTHER INFORMATION: Homoarginine(A) NAME/KEY: Other(B) LOCATION: 3...3OTHER INFORMATION: Cyclohexylglycine(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:71:Xaa Xaa Xaa Gln Ser Leu1 S(2) INFORMATION FOR SEQ ID NO:72:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 5 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(ix) FEATURE:(A) NAME/KEY: Other(B) LOCATION: I...lD) OTHER INFORMATION: N—(PEG—2)Homoarginine(A) NAME/KEY: Other(B) LOCATION: 2...2(D) OTHER INFORMATION: Cyclohexylglycine(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:72:Xaa Xaa Gln Ser Leu1 5(2) INFORMATION FOR SEQ ID NO:73:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 5 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(ix) FEATURE:(A) NAME/KEY: Other?CA 02265476 1999-03-10W0 98/ 10651 PCT/U S97] 16087-39..(B) LOCATION: l...l(D) OTHER INFORMATION: N-((2R,3S)—2,3,4-Trihydroxybutanoyl)Homoarginine5 (A) NAME/KEY: other(B) LOCATION: 2...2(D) OTHER INFORMATION: Cyclohexylglycine(xi)SEQUENCE DESCRIPTION: SEQ ID NO:73:10Xaa Xaa Gln Ser Leu1 5(2) INFORMATION FOR SEQ ID NO:74:15(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 6 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: Single20 (D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(ix) FEATURE:25 (A) NAME/KEY: Other(B) LOCATION: 1...1(D) OTHER INFORMATION: N—(PEG—2)Serine(A) NAME/KEY: other30 (B) LOCATION: 2...2(D) OTHER INFORMATION: Homoarginine(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:74:35 Xaa Xaa Tyr Gln Ser Leu1 5(2) INFORMATION FOR SEQ ID NO:75:40 (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 7 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear45(ii) MOLECULE TYPE: peptide(ix) FEATURE:(A) NAME/KEY: other50 (B) LOCATION: 1...1(D) OTHER INFORMATION: N—(PEG—2)Homoarginine(xi) SEQUENCE DESCRIPTION: sao ID NO:75:55 Xaa Tyr Gln Ser Ser Ser Leu1 S?W0 98ll065l10152025303540455055CA 02265476 1999-03-10PCT/US97ll6087_ 90 _(2) INFORMATION FOR SEQ ID NO:76:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 7 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(ix) FEATURE:(A) NAME/KEY: Other(B) LOCATION: l...1(D) OTHER INFORMATION: N—(PEG—2)Lysine(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:76:Xaa Tyr Gln Ser Ser Ser Leu1 5(2) INFORMATION FOR SEQ ID NO:77:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 7 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(ix) FEATURE:(A) NAME/KEY: Other(B) LOCATION: l...l(D) OTHER INFORMATION: N-(2—Hydroxyacetyl)Homoarginine(xi) SEQUENCE DESCRIPTION: SEQ ID NO:77:Xaa Ser Ser Tyr Gln Ser Leu1 5(2) INFORMATION FOR SEQ ID NO:78:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 7 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(ix) FEATURE:(A) NAME/KEY: Other(B) LOCATION: l...l(D) OTHER INFORMATION: N-((1)-2,3-Dihydroxypropionyl)Homoarginine?W0 98/1065110152025303540455055(A)(B)(D)CA 02265476 1999-03-10PCT/U S97/ 16087-91-NAME/KEY: OtherLOCATION: 4...4OTHER INFORMATION: Cyclohexylglycine(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:78:Xaa Ser Ser1(2)Xaa Gln Ser Leu5INFORMATION FOR SEQ ID NO:79:(i) SEQUENCE CHARACTERISTICS:(A)(B)(C)(D)LENGTH: 7 amino acidsTYPE: amino acidSTRANDEDNESS: singleTOPOLOGY: linear(ii) MOLECULE TYPE: peptide(ix) FEATURE:(A)(B)(D)635$NAME/KEY: OtherLOCATION: l...lOTHER INFORMATION: N-(PEG—2)HomoarginineNAME/KEY: OtherLOCATION: 4...4OTHER INFORMATION: Cyclohexylglycine(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:79:Xaa Ser Ser1(2)Xaa Gln Ser Leu5INFORMATION FOR SEQ ID NO:80:(i) SEQUENCE CHARACTERISTICS:(A)(B)(C)(D)LENGTH: 7 amino acidsTYPE: amino acidSTRANDEDNESS: singleTOPOLOGY: linear(ii) MOLECULE TYPE: peptide(ix) FEATURE:(A)(B)(D)NAME/KEY: OtherLOCATION: l...lOTHER INFORMATION: N-(2-Hydroxyacetyl)Serine(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:80:Xaa Tyr Glnl(2)Ser Ser Ser Leu5INFORMATION FOR SEQ ID NO:8l:?101520253040455055CA 02265476 1999-03-10W0 98/10651-92-(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 6 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(ix) FEATURE:(A) NAME/KEY: OtherB) LOCATION: 1...l(D) OTHER INFORMATION: N—(PEG—16)Serine(A) NAME/KEY: OtherB) LOCATION: 2...2(D) OTHER INFORMATION: Homoarginine(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:81:Xaa Xaa Tyr Gln Ser Leu1 5(2) INFORMATION FOR SEQ ID NO:82:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 6 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: Single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(ix) FEATURE:( ) NAME/KEY: Other( ) LOCATION: l...l( ) OTHER INFORMATION: N—((2R,3S)—2,3,4—Trihydroxybutanoyl)SerineABD(A) NAME/KEY: Other(B) LOCATION: 2...2(D) OTHER INFORMATION: Homoarginine(A) NAME/KEY: Other(B) LOCATION: 3...3(D) OTHER INFORMATION: Cyclohexylglycine(xi) SEQUENCE DESCRIPTION: SEQ ID NO:82:Xaa Xaa Xaa Gln Ser Leu1 5(2) INFORMATION FOR SEQ ID NO:83:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 6 amino acids(B) TYPE: amino acidPCT/U S97/ 16087?))CA 02265476 1999-03-10PCT/US97/16087_ 93 -STRANDEDNESS: singleTOPOLOGY: linear(ii) MOLECULE TYPE: peptide(A)(B)(D)(A)(B)(D)wo 98/10651(c(D5 (ix)1015 (xi)2025303540455055FEATURE:NAME/KEY: OtherLOCATION: l...1OTHER INFORMATION: N-(PEG—2)SerineNAME/KEY: OtherLOCATION: 2...2OTHER INFORMATION: HomoarginineSEQUENCE DESCRIPTION: SEQ ID NO:83:Xaa Xaa Tyr Gln Ser Leu1(2)(i)(A(B(C(DS))))5INFORMATION FOR SEQ ID NO:84:EQUENCE CHARACTERISTICS:LENGTH: 7 amino acidsTYPE: amino acidSTRANDEDNESS: singleTOPOLOGY: linear(ii) MOLECULE TYPE: peptide(ix)(A)(B)(D)Dihydroxypropionyl)HomoarginineFEATURE:NAME/KEY: otherLOCATION: 1...lOTHER INFORMATION: N—(d)~2,3-(A) NAME/KEY: Other(B) LOCATION: 4...4(D) OTHER INFORMATION: Cyclohexylglycine(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:84:Xaa Ser Ser Xaa Gln Ser Leu1(2)(i)(B(C(D5INFORMATION FOR SEQ ID NO:85:SEQUENCE CHARACTERISTICS:(A))))LENGTH: 7 amino acidsTYPE: amino acidSTRANDEDNESS: singleTOPOLOGY: linear(ii) MOLECULE TYPE: peptide(ix)FEATURE:(A) NAME/KEY: Other?CA 02265476 1999-03-10W0 98/10651 PCT/US97/16087_ 94 _ .(B) LOCATION: 1...1(D) OTHER INFORMATION: N-((1)-2,3—Dihydroxypropionyl)Serine(A) NAME/KEY: Other5 (B) LOCATION: 4...4(D) OTHER INFORMATION: Cyclohexylglycine(A) NAME/KEY: Other(B) LOCATION: 7...710 (D) OTHER INFORMATION: Leucine with Unnatural1520303540455055Stereoconfiguration(xi) SEQUENCE DESCRIPTION: SEQ ID NO:85:Xaa Ser Ser Xaa Gln Ser Xaa1 5(2) INFORMATION FOR SEQ ID NO:86:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 7 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(ix) FEATURE:(A) NAME/KEY: Other(B) LOCATION: l...l(D) OTHER INFORMATION: N—((d)-2,3—Dihydroxypropionyl)Serine(A) NAME/KEY: Other(B) LOCATION: 4...4(D) OTHER INFORMATION: Cyclohexylglycine(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:86:Xaa Ser Ser Xaa Gln Ser Leu1 5(2) INFORMATION FOR SEQ ID NO:87:SEQUENCE CHARACTERISTICS:)(A) LENGTH: 7 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(ix) FEATURE:(A) NAME/KEY: Other(B) LOCATION: l...l(D) OTHER INFORMATION: N—((1)-2,3—Dihydroxypropionyl)Serine?W0 98/1065!10152025303540455055(A)(B)(D)(xi)Xaa Ser Ser1(2)(i)(A)(B)(C)(D)(ii)CA 02265476 1999-03-10PCTIU S97] 16087_ 95 -NAME/KEY: OtherLOCATION: 4...4OTHER INFORMATION: CyclohexylglycineSEQUENCE DESCRIPTION: SEQ ID NO:87:Xaa Gln Ser Leu5INFORMATION FOR SEQ ID NO:88:SEQUENCE CHARACTERISTICS:LENGTH: 6 amino acidsTYPE: amino acidSTRANDEDNESS: singleTOPOLOGY: linearMOLECULE TYPE: peptide(ix) FEATURE:(A) NAME/KEY: Other(B) LOCATION: l...l(D) OTHER INFORMATION: N—((1)-2,3—Dihydroxypropionyl)Serine(A) NAME/KEY: other(B) LOCATION: 3...}(D) OTHER INFORMATION: Cyclohexylglycine(A) NAME/KEY: Other(B) LOCATION: 6...6(D) OTHER INFORMATION: Leucine with UnnaturalStereoconfiguration(xi)Xaa Ser Xaa1(2)SEQUENCE DESCRIPTION: SEQ ID NO:88:Gln Ser Xaa5INFORMATION FOR SEQ ID NO:89:(i) SEQUENCE CHARACTERISTICS:(A)(B)(C)(D)LENGTH: 6 amino acidsTYPE: amino acidSTRANDEDNESS: singleTOPOLOGY: linear(ii) MOLECULE TYPE: peptide(ix) FEATURE:(A)(B)(D). ..I.~_ 2.---A- - --~~NAME/KEY: OtherLOCATION: 1...lOTHER INFORMATION: N—((d)-2,3-Dihydroxypropionyl)SerineNAME/KEY: OtherLOCATION: 3...3OTHER INFORMATION: Cyclohexylglycine?10152025303540455055W0 98/10651CA 02265476 1999-03-10PCT/US97/16087- 96 _(Xi) SEQUENCE,DESCRIPTION: SEQ ID NO:89:Xaa Ser Xaa Gln Ser Leu1 S(2) INFORMATION FOR SEQ ID NO:90:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 6 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(ix) FEATURE:(A) NAME/KEY: Other(B) LOCATION: l...1(D) OTHER INFORMATION: N—(PEG—2)Serine(A) NAME/KEY: Other(B) LOCATION: 3...3(D) OTHER INFORMATION: Cyclohexylglycine(A) NAME/KEY: Other(B) LOCATION: 6...6(D) OTHER INFORMATION:StereoconfigurationLeucine with Unnatural(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:90:Xaa Ser Xaa Gln Ser Xaa1 S(2) INFORMATION FOR SEQ ID NO:9l:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 6 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(ix) FEATURE:(A) NAME/KEY: OtherLOCATION: l...l(D) OTHER INFORMATION: N~(PEG—2)Serine(A) NAME/KEY: other(B) LOCATION: 3...3(D) OTHER INFORMATION: Cyclohexylglycine(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:9l:?10152025303540455055W0 98/ 10651Xaa Ser Xaal(2)CA 02265476 1999-03-10PCT/US97/ 16087- 97 _Gln Ser Leu5INFORMATION FOR SEQ ID NO:92:(i) SEQUENCE CHARACTERISTICS:(A)(B)(C)(D)LENGTH: 7 amino acidsTYPE: amino acidSTRANDEDNESS: singleTOPOLOGY: linear(ii) MOLECULE TYPE: peptide(ix) FEATURE:(A)(B)(D)(A)(B)(D)NAME/KEY: OtherLOCATION: l...lOTHER INFORMATION: N-(PEG—2)SerineNAME/KEY: otherLOCATION: 4...4OTHER INFORMATION: CyclohexylglycineNAME/KEY: otherLOCATION: 7...7OTHER INFORMATION: Leucine with UnnaturalStereoconfiguration(xi) SEQUENCE DESCRIPTION: SEQ ID NO:92:Xaa Ser Ser1(2)(i) S(A)(B)(C)(D)Xaa Gln Ser Xaa5INFORMATION FOR SEQ ID NO:93:EQUENCE CHARACTERISTICS:LENGTH: 7 amino acidsTYPE: amino acidSTRANDEDNESS: singleTOPOLOGY: linear(ii) MOLECULE TYPE: peptide(ix) FEATURE:(A) NAME/KEY: other(B) LOCATION: l...l(D) OTHER INFORMATION: N-(2,3-Dihydroxypropionyl)-3-Pyridylalanine(A) NAME/KEY: Other(B) LOCATION: 4...4(D) OTHER INFORMATION: Cyclohexylglycine(A) NAME/KEY: Other(B) LOCATION: 7...7(D) OTHER INFORMATION: Leucine with UnnaturalStereoconfiguration?10152025303540455055W0 98/10651CA 02265476 1999-03-10PCT/US97/16087-98-(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:93:Xaa Ser Ser Xaa Gln Ser Xaa1 5(2) INFORMATION FOR SEQ ID NO:94:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 7 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(ix) FEATURE:(A) NAME/KEY: Other(B) LOCATION: l...l(D) OTHER INFORMATION: N-((d)—2,3—Dihydroxypropionyl)-3-Pyridylalanine(A) NAME/KEY: Other(B) LOCATION: 4...4(D) OTHER INFORMATION: Cyclohexylglycine(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:94:Xaa Ser Ser Xaa Gln Ser Leu1 5(2) INFORMATION FOR SEQ ID NO:9S:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 6 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(ix) FEATURE:(A) NAME/KEY: other(B) LOCATION: l...l(D) OTHER INFORMATION: N-((1)-2,3—Dihydroxypropiony1)Serine(A) NAME/KEY: Other(B) LOCATION: 4...4(D) OTHER INFORMATION: Cyclohexylglycine(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:95:Xaa Ser Xaa Gln Ser Leu1 S?10152025303540455055W0 98/10651CA 02265476 1999-03-10PCT/US97/ 16087-99..(2) INFORMATION FOR SEQ ID NO:96:SEQUENCE CHARACTERISTICS:) LENGTH: 7 amino acids) TYPE: amino acid) STRANDEDNESS: single) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(ix) FEATURE:(A) NAME/KEY: Other(B) LOCATION: l...l(D) OTHER INFORMATION: N—(2,3—Dihydroxypropionyl)Homoserine(A) NAME/KEY: Other(B) LOCATION: 4...4(D) OTHER INFORMATION: Cyclohexylglycine(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:96:Xaa Ser Ser Xaa Gln Ser Leu1 5(2) INFORMATION FOR SEQ ID NO:97:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 6 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(ix) FEATURE:(A) NAME/KEY: Other(B) LOCATION: l...l(D) OTHER INFORMATION: N-(PEG-2)Alanine(A) NAME/KEY: Other(B) LOCATION: 3...3(D) OTHER INFORMATION: Cyclohexylglycine(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:97:Xaa Ser Xaa Gln Ser Leu1 5(2) INFORMATION FOR SEQ ID NO:98:(i SEQUENCE CHARACTERISTICS:) LENGTH: 6 amino acids) TYPE: amino acid))STRANDEDNESS: single)(A(B(C(D TOPOLOGY: linear?W0 98Il065l101520303540455055CA 02265476 1999-03-10PC T/U S97/ 16087— 100 —(ii) MOLECULE TYPE: peptide(ix) FEATURE:(A) NAME/KEY: Other(B) LOCATION: 1...l(D) OTHER INFORMATION: N-(PEG—6)Serine(A) NAME/KEY: Other(B) LOCATION: 3...3(D) OTHER INFORMATION: Cyclohexylglycine(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:98:Xaa Ser Xaa Gln Ser Leu1 S(2) INFORMATION FOR SEQ ID NO:99:EQUENCE CHARACTERISTICS:LENGTH: 7 amino acidsTYPE: amino acidSTRANDEDNESS: SingleTOPOLOGY: linear(i)((((U 0 m >\./\/s/xzm(ii) MOLECULE TYPE: peptide(ix) FEATURE:A NAME/KEY: Other(B) LOCATION: l...lD) OTHER INFORMATION: N—(PEG—6)Serine(A) NAME/KEY: Other(B) LOCATION: 4 ..4(D) OTHER INFORMATION: Cyclohexylglycine(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:99:Xaa Ser Ser Xaa Gln Ser Leu1 S(2) INFORMATION FOR SEQ ID NO:l0O:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 6 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(ix) FEATURE:(A) NAME/KEY: Other(B) LOCATION: l...l(D) OTHER INFORMATION: N-(PEG-6)Alanine(A) NAME/KEY: Other?CA 02265476 1999-03-10W0 98/ 10651 PCT/US97/ 16087— 101 -(B) LOCATION: 3...3(D) OTHER INFORMATION: Cyclohexylglycine(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l00:Xaa Ser Xaa Gln Ser Leu1 510 (2) INFORMATION FOR SEQ ID NO:lO1:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 7 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single15 (D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(ix) FEATURE:20 (A) NAME/KEY: Other(B) LOCATION: 1...1D) OTHER INFORMATION: N-(PEG—4)—3-Pyridylalanine(A) NAME/KEY: OtherB LOCATION: 4...4(D) OTHER INFORMATION: Cyclohexylglycine25(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:10l:30 Xaa Ser Ser Xaa Gln Ser Leu1 5(2) INFORMATION FOR SEQ ID N02102:35 (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 7 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear40(ii) MOLECULE TYPE: peptide(ix) FEATURE:(A) NAME/KEY: Other45 (B) LOCATION: 4...4(D) OTHER INFORMATION: Cyclohexylglycine(A) NAME/KEY: Other(B) LOCATION: 7...750 (D) OTHER INFORMATION: Leucine—2—Hydroxyethylamine(xi) SEQUENCE DESCRIPTION: seq ID N02102:Ser Ser Ser Xaa Gln Ser Xaa55 1 5?10152025303540455055WO 98/10651CA 02265476 1999-03-10PCT/US97/16087- 102 -(2) INFORMATION FOR SEQ ID NO:103:SEQUENCE CHARACTERISTICS:) LENGTH: 9 amino acids) TYPE: amino acid) STRANDEDNESS: single) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(ix) FEATURE:(A) NAME/KEY: Other(B) LOCATION: l...l(D) OTHER INFORMATION: N—Acetylalanine(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:l03:Xaa Arg Lys Ala Ser Tyr Gln Ser Leu1 5(2) INFORMATION FOR SEQ ID NO:lO4:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 9 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(ix) FEATURE:(A) NAME/KEY: Other(B) LOCATION: l...1(D) OTHER INFORMATION: N—Acetylalanine(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l04:Xaa Arg Lys Ala Ser Tyr Gln ser Leu1 S(2) INFORMATION FOR SEQ ID NO:l05:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 5 amino acids(B) TYPE: amino acid(C) ATRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(ix) FEATURE:(A) NAME/KEY: OtherB) LOCATION: l...1(D) OTHER INFORMATION: N—Acetylhomoarginine(A) NAME/KEY: Other?CA 02265476 1999-03-10W0 98/10651 PCT/US97/16087— 103 —LOCATION: 2...2OTHER INFORMATION: Cyclohexylalanine(A) NAME/KEY: Other5 (B) LOCATION: 5...5(D) OTHER INFORMATION: Norieucine(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:l05:10152025303540455055.:............_.................,..... ..................,~...—.-...Xaa Xaa Gln Ser Leu1 S(2) INFORMATION FOR SEQ ID NO:106:(i SEQUENCE CHARACTERISTICS:) LENGTH: 6 amino acids) TYPE: amino acid))STRANDEDNESS: singleTOPOLOGY: linear,\,\,\,\~.»ABCD(ii) MOLECULE TYPE: peptide(ix) FEATURE:(A) NAME/KEY: Other(B) LOCATION: l...l(D) OTHER INFORMATION: N—Acetylserine(A) NAME/KEY: Other(B) LOCATION: 2...2(D) OTHER INFORMATION: Homoarginine(A) NAME/KEY: Other(B) LOCATION: 6...6(D) OTHER INFORMATION: Norleucine(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:l06:Xaa Xaa Tyr Gln Ser Leu1 S(2) INFORMATION FOR SEQ ID NO:107:(i SEQUENCE CHARACTERISTICS:) LENGTH: 6 amino acids) TYPE: amino acid) STRANDEDNESS: single))(((( TOPOLOGY: linearUOt1Z13>'(ii) MOLECULE TYPE: peptide(ix) FEATURE:(A) NAME/KEY: Other(B) LOCATION: 2...2(D) QTHER INFORMATION: Homoarginine(A) NAME/KEY: Other?101520253040455055CA 02265476 1999-03-10W0 98/10651 PCTlUS97/ 16087- 104 —(B) LOCATION: 3...3(D) OTHER INFORMATION: Cyclohexylglycine(A) NAME/KEY: Other(B) LOCATION: 6...6(D) OTHER INFORMATION: Norleucine(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:lO7:Ser Xaa Xaa Gln Ser Leu1 S(2) INFORMATION FOR SEQ ID NO:108:(i) SEQUENCE CHARACTERISTICS:( ) LENGTH: 7 amino acids( ) TYPE: amino acid( ) STRANDEDNESS: single( ) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(ix) FEATURE:(A) NAME/KEY: Other(B) LOCATION: l...I(D) OTHER INFORMATION: N—Acetylhomoarginine(A) NAME/KEY: Other(B) LOCATION: 7...7(D) OTHER INFORMATION: Norleucine(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:108:Xaa Ser Ser Tyr Gln Ser LeuI S(2) INFORMATION FOR SEQ ID NO:lO9:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 7 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(ix) FEATURE:(A) NAME/KEY: Other(B) LOCATION: l...l(D) OTHER INFORMATION: N-Acetylhomoarginine(A) NAME/KEY: Other) LOCATION: 4...4) OTHER INFORMATION: Cyclohexylglycine(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:lO9:?W0 98/10651CA 02265476 1999-03-10PCT/U S97/ 16087Xaa Ser Ser Xaa Gln Ser Leu101520l(2)(i)(A)(B)(C)(D)(ii)5INFORMATION FOR SEQ ID NO:l10:SEQUENCE CHARACTERISTICS:LENGTH: 7 amino acidsTYPE: amino acidSTRANDEDNESS: singleTOPOLOGY: linearMOLECULE TYPE: peptide(ix) FEATURE:(A)(B)(D)(xi)SEQUENCE DESCRIPTION: SEQNAME/KEY: OtherLOCATION: 1...lOTHER INFORMATION: N—AcetylhomoarginineNAME/KEY: OtherLOCATION: 7...7OTHER INFORMATION: NorleucineID NO:ll0:Xaa Ser Ser Tyr Gln Ser Leu303540455055l(2)(i)(A)(B)(C)(D)(ii)5INFORMATION FOR SEQ ID NO:lll:SEQUENCE CHARACTERISTICS:LENGTH: 7 amino acidsTYPE: amino acidSTRANDEDNESS: singleTOPOLOGY: linearMOLECULE TYPE: peptide(ix) FEATURE:(A) NAME/KEY: other(B) LOCATION: l...1(D) OTHER INFORMATION: N~Acetylhomoarginine(A) NAME/KEY: Other(B) LOCATION: 4...4(D) OTHER INFORMATION: Cyclohexylglycine(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:l1l:l(2)(i)(A)Xaa Ala Ser Xaa Gln Ser Leu5INFORMATION FOR SEQ ID N02112:SEQUENCE CHARACTERISTICS:LENGTH: 5 amino acids?CA 02265476 1999-03-10WO 98110651 PCT/US97/16087— 106 — -(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear5 (ii) MOLECULE TYPE: peptide10152025303540455055(ix) FEATURE:(A) NAME/KEY: Other(B) LOCATION: 1...1(D) OTHER INFORMATION: N-(2—Hydroxyacetyl)Homoarginine(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:l12:Xaa Tyr Gln Ser Leu1 5(2) INFORMATION FOR SEQ ID NO:1l3:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 6 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(ix) FEATURE:(A) NAME/KEY: Other(B) LOCATION: 1...l(D) OTHER INFORMATION: N—(PEG—l)Serine(A) NAME/KEY: Other(B) LOCATION: 2...2(D) OTHER INFORMATION: Homoarginine(A) NAME/KEY: Other(B) LOCATION: 3...3(D) OTHER INFORMATION: Cyclohexylglycine(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:ll3:Xaa Xaa Xaa Gln Ser Leu1 S(2) INFORMATION FOR SEQ ID NO:ll4:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 7 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(ix) FEATURE:(A) NAME/KEY: other?10152025303540455055W0 98/ 10651(B)(D)(A)(B)(D)(xi)SEQUENCE DESCRIPTION:CA 02265476 1999-03-10PCTIU S97] 16087— 107 -LOCATION: l...lOTHER INFORMATION: N'(PEG—1)HomoarginineNAME/KEY: OtherLOCATION: 4...4OTHER INFORMATION: CyclohexylglycineSEQ ID NO:l14:Xaa Ser Ser Xaa Gln Ser Leu1(2)(i) S(A)(B)(C)(D)8INFORMATION FOR SEQ ID NO:ll5:EQUENCE CHARACTERISTICS:LENGTH:TYPE:STRANDEDNESS:TOPOLOGY:6 amino acidsamino acidsinglelinear(ii) MOLECULE TYPE: peptide(ix) FEATURE:(A) NAME/KEY: Other(8) LOCATION: l...l(D) OTHER INFORMATION: N-(PEG—l)Serine(A) NAME/KEY: Other(B) LOCATION: 2...2(D) OTHER INFORMATION: Homoarginine(Xi) SEQUENCE DESCRIPTION: SEQ ID N02115:Xaa Xaa Tyr Gln Ser Leu'1(2)(i)(A)(B)(C)(D)(ii)5INFORMATION FOR SEQ ID NO:ll6:SEQUENCE CHARACTERISTICS:LENGTH:TYPE:STRANDEDNESS:TOPOLOGY:6 amino acidsamino acidsinglelinearMOLECULE TYPE: peptide(ix) FEATURE:(xi)NAME/KEY: OtherLOCATION: l...lOTHER INFORMATION: N—(PEG—l5)SerineNAME/KEY: OtherLOCATION: 2...2OTHER INFORMATION: HomoarginineSEQUENCE DESCRIPTION: SEQ ID N01116:?W0 98/10651LII1520303540455055CA 02265476 1999-03-10PCT/US97/ 16087-108-Xaa Xaa Tyr Gln Ser Leu1 S(2) INFORMATION FOR SEQ ID NO:ll7:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 6 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(ix) FEATURE:(A) NAME/KEY: Other(B) LOCATION: l...l(D) OTHER INFORMATION: N~(PEG—17)Serine(A) NAME/KEY: Other(B) LOCATION: 2...2(D) OTHER INFORMATION: Homoarginine(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:l17:Xaa Xaa Tyr Gln Ser Leu1 S(2) INFORMATION FOR SEQ ID NO:l18:SEQUENCE CHARACTERISTICS:) LENGTH: 6 amino acids) TYPE: amino acid) STRANDEDNESS: single) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(ix) FEATURE:(A) NAME/KEY: Other(B) LOCATION: l...1(D) OTHER INFORMATION: N-(PEG—2)Serine(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:ll8:Xaa Ser Tyr Gln Ser Leu1 5(2) INFORMATION FOR SEQ ID NO:l19:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 6 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear?10152025303540455055WO 98110651CA 02265476 1999-03-10PCT/U S97/ 16087-109-(ii) MOLECULE TYPE: peptide(ix) FEATURE:(A) NAME/KEY: Other(B) LOCATION: 1...1(D) OTHER INFORMATION: N-(PEG—14)Serine(A) NAME/KEY: Other(B) LOCATION: 2...2(D) OTHER INFORMATION: Homoarginine(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:ll9:Xaa Xaa Tyr Gln Ser Leu1 S(2) INFORMATION FOR SEQ ID NO:l20:SEQUENCE CHARACTERISTICS:) LENGTH: 6 amino acids) TYPE: amino acid) STRANDEDNESS: single)(1)(((( TOPOLOGY: linearUOCUIV(ii) MOLECULE TYPE: peptide(ix) FEATURE:(A) NAME/KEY: OtherB) LOCATION: 1...1(D) OTHER INFORMATION: N-(PEG—18)Serine(A) NAME/KEY: Other(B) LOCATION: 2...2(D) OTHER INFORMATION: Homoarginine(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:l20:Xaa Xaa Tyr Gln Ser Leu1 S(2) INFORMATION FOR SEQ ID NO:12l:SEQUENCE CHARACTERISTICS:) LENGTH: 6 amino acids) TYPE: amino acid) STRANDEDNESS: single) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(ix) FEATURE:(A) NAME/KEY: Other(B) LOCATION: 1...l(D) OTHER INFORMATION: N-(PEG—19)Serine(A) NAME/KEY: Other?CA 02265476 1999-03-10W0 98/10651 PCT/US97/16087- 1 10 —(B) LOCATION: 2...2(D) OTHER INFORMATION: Homoarginine(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:121:10152025303540455055Xaa Xaa Tyr Gln Ser Leu1 S(2) INFORMATION FOR SEQ ID NO:122:U)(i .EQUENCE CHARACTERISTICS:LENGTH: 6 amino acidsTYPE: amino acidSTRANDEDNESS: single)(A(B(C(D TOPOLOGY: linearVa,‘/C,(ii) MOLECULE TYPE: peptide(ix) FEATURE:(A NAME/KEY: Other(8) LOCATION: l...1(D) OTHER INFORMATION: N—((1)-2,3—Dihydroxypropionyl)SerineNAME/KEY: Other(B) LOCATION: 3...3(D) OTHER INFORMATION: Cyclohexylglycine(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:l22:Xaa Ser Xaa Gln Ser Leu1 S(2) INFORMATION FOR SEQ ID NO:l23:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 7 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(ix) FEATURE:(A) NAME/KEY: Other(B) LOCATION: l...1(D) OTHER INFORMATION: N—((l)-2,3-Dihydroxypropionyl)-3-Pyridylalanine(A) NAME/KEY: Other(B) LOCATION: 4...4(D) OTHER INFORMATION: Cyclohexylglycine(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l23:Xaa Ser Ser Xaa Gln Ser Leu1 5?W0 98/1065]10152025303540455055(2)(i)(A)(B)(C)(D)(ii)(ix)(xi)Xaa Ser Ser1(2)(i)(A)(B)(C)(D)(ii)SEQUENCE DESCRIPTION:CA 02265476 1999-03-10PCT/U S97] 16087- 111 —INFORMATION FOR SEQ ID NO:l24:SEQUENCE CHARACTERISTICS:LENGTH: 7 amino acidsTYPE: amino acidSTRANDEDNESS: singleTOPOLOGY: linearMOLECULE TYPE: peptideFEATURE:NAME/KEY: OtherLOCATION: l...lOTHER INFORMATION: N-(2,3—Dihydroxypropionyl)SerineNAME/KEY: OtherLOCATION: 4...4OTHER INFORMATION: CyclohexylglycineSEQ ID NO:l24:Xaa Gln Ser Leu5INFORMATION FOR SEQ ID NO:l25:SEQUENCE CHARACTERISTICS:LENGTH: 6 amino acidsTYPE: amino acidSTRANDEDNESS: singleTOPOLOGY: linearMOLECULE TYPE: peptide(ix) FEATURE:(A) NAME/KEY: Other(B) LOCATION: l...l(D) OTHER INFORMATION: N—(2,3-Dihydroxypropionyl)Serine(A) NAME/KEY: Other(B) LOCATION: 2...2(D) OTHER INFORMATION: Homoarginine(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l2S:Xaa Xaa Tyr1(2)(iSSEEVS))))Gln Ser Leu5INFORMATION FOR SEQ ID NO:l26:EQUENCE CHARACTERISTICS:LENGTH: 7 amino acidsTYPE: amino acidSTRANDEDNESS: singleTOPOLOGY: linear?CA 02265476 1999-03-10W0 98/10651 PCT/US97/16087- 112 -(ii) MOLECULE TYPE: peptide(ix) FEATURE:5 (A) NAME/KEY: Other(B) LOCATION: l...l(D) OTHER INFORMATION: N—acetylserine(A) NAME/KEY: other10 (B) LOCATION: 4...4(D) OTHER INFORMATION: Cyclohexylglycine(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:126:15 Xaa Ser Ser Xaa Gln Ser Val20304050551 5(2) INFORMATION FOR SEQ ID NO:l27:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 7 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(ix) FEATURE:(A) NAME/KEY: OtherB) LOCATION: l...l(D) OTHER INFORMATION: N—Acetylproline(A) NAME/KEY: Other(B) LOCATION: 4...4(D) OTHER INFORMATION: Cyclohexylglycine(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:l27:Xaa Ser Ser Xaa Gln Ser Vall S(2) INFORMATION FOR SEQ ID NO:l28:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 7 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(ix) FEATURE:(A) NAME/KEY: other(B) LOCATION: 1...l(D) OTHER INFORMATION: N-(2,3—Dihydroxypropionyl)Glycine?CA 02265476 1999-03-10W0 98/ 10651 PCT/US97/ 16087-113-(A) NAME/KEY: other(B) LOCATION: 4...4(D) OTHER INFORMATION: Cyclohexylglycine5 (Xi) SEQUENCE DESCRIPTION: SEQ ID N02128:Xaa Set Set Xaa Gln Ser Leu1 510

Claims (33)

. -114-WHAT IS CLAIMED IS:

1. A conjugate which is useful for the treatment of prostate cancer which comprises a cytotoxic agent attached to a oligopeptide, wherein the oliopeptide comprises a sequence of amino acids that is selectively proteolytically cleaved by free prostate specific antigen, wherein the means of attachment is a covalent bond or through a chemical linker and wherein the point of attachment on the oligopeptide is at the C-terminus, and which further comprises a hydrophilic blocking group at the N-terminus of the oligopeptide, or the pharmaceutically acceptable salt thereof.

2. The conjugate according to Claim 1 wherein the cytotoxic agent is a member of a class of cytotoxic agents selected from the following classes:
a) anthracycline family of drugs, b) the vinca alkaloid drugs, c) the mitomycins, d) the bleomycins, e) the cytotoxic nucleosides, f) the pteridine family of drugs, g) diynenes, h) estramustine, i) cyclophosphamide, j) the taxanes and k) the podophyllotoxins, or the pharmaceutically acceptable salt thereof.

3. The conjugate according to Claim 2 wherein the cytotoxic agent is selected from the following cytotoxic agents:
a) doxorubicin, b) carminomycin, c) daunorubicin, d) aminopterin, e) methotrexate, f) methopterin, g) dichloro-methotrexate, h) mitomycin C, i) porfiromycin, j) 5-fluorouracil, k) 6-mercaptopurine, I) cytosine arabinoside, m) podophyllotoxin, n) etoposide, o) etoposide phosphate, p) melphalan, q) vinblastine, r) vincristine, s) leurosidine, t) vindesine, u) estramustine, v) cisplatin, w) cyclophosphamide, x) taxol, and y) leurosine, or the pharmaceutically acceptable salt thereof.

4. The conjugate according to Claim 2 wherein the cytotoxic agent is selected from doxorubicin and vinblastine or a cytotoxic derivative thereof.

5. The conjugate according to Claim 2 wherein the cytotoxic agent is doxorubicin or a cylotoxic derivative thereof.

6. The conjugate according to Claim 1 wherein the oligopeptide comprises an oligomer selected from:
a) AsnLyslleSerTyrGln¦Ser (SEQ.ID.NO.: 1), b) LysIleSerTyrGln¦Ser (SEQ.ID.NO.: 2), c) AsnLysIleSerTyrTyr¦Ser (SEQ.ID.NO.: 3), d) AsnLysAlaSerTyrGln¦Ser (SEQ.ID.NO.: 4), e) SerTyrGln¦SerSer (SEQ.ID.NO.: 5);
f) LysTyrGln¦SerSer (SEQ.ID.NO.: 6);
g) hArgTyrGln¦SerSer (SEQ.ID.NO.: 7);
h) hArgChaGln¦SerSer (SEQ.ID.NO.: 8);
I) TyrGln¦SerSer (SEQ.ID.NO.: 9);
j) TyrGln¦SerLeu (SEQ.ID.NO.: 10);
k) TyrGln¦SerNle (SEQ.ID.NO.: 11);
I) ChgGln¦SerLeu (SEQ.ID.NO.: 12); and m) ChgGln¦SerNle (SEQ.ID.NO.: 13).

7. The conjugate according to Claim I wherein the oligopeptide comprises an oligomer selected from:
a) AsnLysIleSerTyrGln¦SerSer (SEQ.ID.NO.: 14), b) AsnLysIleSerTyrGln¦SerAla (SEQ.ID.NO.: 15), c) AlaAsnLysIleSerTyrTyr¦Ser (SEQ.ID.NO.: 16), d) AlaAsnLysAlaSerTyrGln¦Ser (SEQ.ID.NO.: 17), e) SerTyrGln¦SerSerThr (SEQ.ID.NO.: 18), f) SerTyrGln¦SerSerSer (SEQ.lD.NO.: 19), g) LysTyrGln¦SerSerSer (SEQ.ID.NO.: 20), h) hArgTyrGln¦SerSerSer (SEQ.ID.NO.: 21), i) SerTyrGln¦SerSerLeu (SEQ.IDl.NO.: 22);
j) SerTyrGln¦SerLeu (SEQ.ID.NO.: 23);
k) SerChgGln¦SerLeu (SEQ.ID.NO.: 24);
I) hArgChgGln¦SerLeu (SEQ.ID.NO.: 25); and m) hArgTyrGln¦SerLeu (SEQ.ID.NO.: 26).

8. The conjugate according to Claim 1 wherein the oligopeptide comprises an oligomer selected from:

GlyGluAsnGlyValGlnLysAspValSerGlnArgSerIleTyr¦SerGlnThrGlu (SEQ.ID.NO.: 27), AlaSerTyrGln¦SerSerLeu (SEQ.ID.NO.: 28);

SerhArgChgGln¦SerLeu (SEQ.ID.NO.: 29);

hArgSerSerTyrGln¦SerNle (SEQ.ID.NO.: 30);
hArgAlaSerChgGln¦SerLeu (SEQ.ID.NO. : 31 );
hArgSerSerTyrGln¦SerLeu (SEQ.ID.NO.: 32);
hArgSerSerChg¦SerLeu (SEQ.ID.NO.: 33);
SerhArgChgGln¦SerLeu (SEQ.ID.NO.: 34);
hArgTyrGln¦SerLeu (SEQ.ID.NO.: 35);
hArgSerSerChgGln¦SerLeu (SEQ.ID.NO.: 36);
SerhArgTyrGln¦SerLeu (SEQ.ID.NO.: 37);
SerSerTyrGln¦SerLeu (SEQ.ID.NO.: 38);
SerSerSerChgGln¦SerLeu (SEQ.ID.NO.: 39);
3PAL-SerSerChgGln¦SerLeu (SEQ.ID.NO.: 40);
SerSerChgGln¦SerLeu (SEQ.ID.NO.: 41 );
SerSerSerChgGln¦Ser(dLeu) (SEQ.ID.NO.: 42);
SerSerSerChgGln¦SerVal (SEQ.ID.NO.: 43);
ProSerSerChgGln¦SerVal (SEQ.ID.NO.: 44);
GlySerSerChgGln¦SerLeu (SEQ.ID.NO.: 45);
hSerSerSerChgGln¦SerLeu (SEQ.ID.NO.: 46);

hArgSerSerChgGln¦SerNle (SEQ.ID.NO.: 47);
hArgTyrGln¦SerSerSerLeu (SEQ.ID.I'~O.: 55);
LysTyrGln¦SerSerSerLeu (SEQ.ID.NO.: 56);
SerTyrGln¦SerSerSerLeu (SEQ.ID.NO.: 57);
SerSerChgGln-Ser(dLeu) (SEQ.ID.NO).: 58); and 3PAL-SerSerChgGln-Ser(dLeu) (SEQ.ID.NO.: 59); and AlaSerChgGln-SerLeu (SEQ.ID.NO.: 60).

9. The conjugate according to Claim 1 wherein the hydrophilic blocking group is selected from:
a) and b) wherein:
R1 and 1?2 are independently selected from:
a) hydrogen, b) unsubstituted or substituted aryl, unsubstituted or substituted heterocycle, C3-C10 cycloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, halogen, C1-c6 perfluoroalkyl, R12O-, R3C(O)NR3-, (R3)2NC(O)-, R32N-C(NR3)-, R4S(O)mNH, CN, NO2, R3C(O)-, N3, -N(R3)2, or R4OC(O)NR3-, c) unsubstituted C1 -C6 alkyl, d) substituted C1-C6 alkyl wherein the substituent on the substituted C1-C6 alkyl is selected from unsubstituted or substituted aryl, unsubstituted or substituted heterocyclic, C3-C10 cycloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, R3O-, R4S(O)mNH, R3C(O)NR3-, (R3)2NC(O)-, R32N-C(NR3)-, CN, R3C(O)-, N3, -N(R3)2, and R4OC(O)-NR3-; or R1 and R2 are combined to form - (CH2)s - wherein one of the carbon atoms is optionally replaced by a moiety selected from:
O, S(O)m, -NC(O)-, NH and -N(COR10)-;

R3 is selected from: hydrogen, aryl, substituted aryl, heterocycle, substituted heterocycle, C1-C6 alkyl and C3-C10 cycloalkyl;

R4 is selected from: aryl, substituted aryl, heterocycle, substituted heterocycle, C1 -C6 alkyl and C3-C10 cycloalkyl;
m is 0, 1 or 2;
n is 1, 2, 3 or 4;
p is zero or an integer between 1 and 100; and q is 0 or 1, provided that if p is zero, q is 1; and s is 3, 4 or 5.

10. A conjugate which is useful for the treatment of prostate cancer of the formula 1:

wherein:
oligopeptide is an oligopeptide which is selectively recognized by the free prostate specific antigen (PSA) and is capable of being proteolytically cleaved by the enzymatic activity of the free prostate specific antigen, and wherein the C-terminus carbonyl is covalently bound to the amine of doxorubicin and the N-terminus amine is covalently bound to the carbonyl of the blocking group;
R is selected from a) b) R1 and R2 are independently selected from: hydrogen, OH, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 aralkyl and aryl;

n is 1, 2, 3 or 4;
p is zero or an integer between 1 and 100;
y is 0 or 1, provided that if p is zero, y is 1;
or the pharmaceutically acceptable salt thereof.

11. The conjugate according to Claim 10 wherein:
R is selected from C) b) R1 and R2 are independently selected from: hydrogen, C1-C6 alkyl and aryl;

n is 1, 2, 3 or 4;
n' is 0, 1, 2 or 3;
p is zero or an integer between 1 and 14;
q is 0 or 1, provided that if p is zero, q is 1;
or the pharmaceutically acceptable salt thereof.

12. The conjugate according to Claim 10 wherein:
oligopeptide is an oligomer that comprises an amino acid sequence selected from:
a) AsnLysIleSerTyrGln¦Ser (SEQ.ID.NO.:1), b) LysIleSerTyrGln¦Ser (SEQ.ID.NO.:2), c) AsnLysIleSerTyrTyr¦Ser (SEQ.ID.NO.:3), d) AsnLysAlaSerTyrGln¦Ser (SEQ.ID.NO.:4), e) SerTyrGln¦SerSer (SEQ.ID.NO.:5);
f) LysTyrGln¦SerSer (SEQ.ID.NO.:6);
g) hArgTyrGln¦SerSer (SEQ.ID.NO.:7);
h) hArgChaGln¦SerSer (SEQ.ID.NO.:8);

i) TyrGln¦SerSer (SEQ.ID.NO.:9);
j) TyrGln¦SerLeu (SEQ.ID.NO.:10);
k) TyrGln¦SerNle (SEQ.ID.NO.:11);
l) ChgGln¦SerLeu (SEQ.ID.NO.:12);
m) ChgGln¦SerNle (SEQ.ID.NO.:13);
or an optical isomer or pharmaceutically acceptable salt thereof.

13. The conjugate according to Claim 10 wherein:
oligopeptide is an oligomer that comprises an amino acid sequence selected from:
GlyGluAsnGlyValGlnLysAspValSerGlnArgSerIleTyr¦SerGlnThrGlu (SEQ.ID.NO.:27), AlaSerTyrGln¦SerSerLeu (SEQ.ID.NO.:28);
SerhArgChgGln¦SerLeu (SEQ.ID.NO.:29);
hArgSerSerTyrGln¦SerNle (SEQ.ID.NO.:30);
hArgAlaSerChgGln¦SerLeu (SEQ.ID.NO.:31);
hArgSerSerTyrGln¦SerLeu (SEQ.ID.NO.:32);
hArgSerSerChg¦SerLeu (SEQ.ID.NO.:33);
SerhArgChgGln¦SerLeu (SEQ.ID.NO.:34);

hArgTyrGln¦SerLeu (SEQ.ID.NO.:35);
hArgSerSerChgGln¦SerLeu (SEQ.ID.NO.:36);
SerhArgTyrGln¦SerLeu (SEQ.ID.NO.:37);
SerSerTyrGln¦SerLeu (SEQ.ID.NO.:38);
SerSerSerChgGln¦SerLeu (SEQ.ID.NO.:39);
3PAL-SerSerChgGln¦SerLeu (SEQ.ID.NO.:40);
SerSerChgGln¦SerLeu (SEQ.ID.NO.:41);
SerSerSerChgGln¦Ser(dLeu) (SEQ.ID.NO.:42);
SerSerSerChgGln¦SerVal (SEQ.ID.NO.:43);
ProSerSerChgGln¦SerVal (SEQ.ID.NO.:44);
GlySerSerChgGln¦SerLeu (SEQ.ID NO.:45);
hSerSerSerChgGln¦SerLeu (SEQ.ID.NO.:46);
hArgSerSerChgGln¦SerNle (SEQ.ID.NO.:47);
hArgTyrGln¦SerSerSerLeu (SEQ.ID.NO.:55);
LysTyrGln¦SerSerSerLeu (SEQ.ID.NO.:56);
SerTyrGln¦SerSerSerLeu (SEQ.ID.NO.:57);
SerSerChgGln-Ser(dLeu) (SEQ.ID.NO.:58); and 3PAL-SerSerChgGln-Ser(dLeu) (SEQ.ID.NO.:59); and AlaSerChgGln-SerLeu (SEQ.ID.NO.:60).

or an optical isomer or pharmaceutically acceptable salt thereof.

14. The conjugate according to Claim 10 which is selected from:

wherein X is:

(SEQ.ID.NO.:61), (SEQ.ID.NO.:62), (SEQ.ID.NO.:63), or an optical isomer or pharmaceutically acceptable salt thereof.

15. The conjugate according to Claim 10 which is selected from:

2-hydroxyacetyl-hArgSerSerTyrGln-SerNle-DOX (3') (SEQ.ID.NO.:
64) 2-hydroxyacetyl-hArgSerSerChgGln-SerNle-DOX (3') (SEQ.ID.NO.:
65) 2-hydroxyacetyl-SerhArgChgGln-SerLeu-DOX (3') (SEQ.ID.NO.:66) 2-hydroxyacetyl-hArgSerSerChgGln-SerLeu-DOX (3') (SEQ.ID.NO.:
67) 2-hydroxyacetyl-hArgAlaSerChgGln-SerLeu-DOX (3') (SEQ.ID.NO.:
68) (d) 2,3-dihydroxypropionyl-SerhArgChgGln-SerLeu-DOX (3') (SEQ.ID.NO.:69) (1) 2,3-dihydroxypropionyl-SerhArgChgGln-SerLeu-DOX (3') (SEQ.ID.NO.:70) PEG(2)-SerhArgChgGln-SerLeu-DOX (3') (SEQ.ID.NO.:71) PEG(2)-hArgChgGln-SerLeu-DOX (3') (SEQ.ID.NO.:72) (2R,3S) 2,3,4-trihydroxybutanoyl-hArgChgGln-SerLeu-DOX (3') (SEQ.ID.NO.:73) PEG(2)-SerhArgTyrGln-SerLeu-DOX(3') (SEQ.ID.NO.:74) PEG(2)-hArgTyrGln-SerSerSerLeu-DOX (3') (SEQ.ID.NO.:75) PEG(2)-LysTyrGln-SerSerSerLeu-DOX (3') (SEQ.ID.NO.:76) 2-hydroxyacetyl-hArgSerSerTyrGln-SerLeu-DOX (3') (SEQ.ID.NO.:
77) (1)(2,3-dihydroxypropionyl)hArgSerSerChgGlnSerLeu-DOX (3') (SEQ.ID.NO.:78) PEG(2)-hArgSerSerChgGln-SerLeu-DOX (3') (SEQ.ID.NO.:79) 2-hydroxyacetyl-SerTyrGln-SerSerSerLeu-DOX (3') (SEQ.ID.NO.:
80) PEG(16)-SerhArgTyrGln-SerLeu-DOX (3') (SEQ.ID.NO.:81) (2R,3S) 2,3,4-trihydroxybutanoyl-SerhArgChgGln-SerLeu-DOX (3') (SEQ.ID.NO.:82) PEG(2)-SerhArgTyrGln-SerLeu-DOX (3') (SEQ.ID.NO.:83) (d)(2,3-dihydroxypropionyl)-hArgSerSerChgGln-SerLeu-DOX(3') (SEQ.ID.NO.:84) (1)(2,3-dihydroxypropionyl)SerSerSerChgGln-Ser(dLeu)-DOX (3') (SEQ.ID.NO.:85) (d)(2,3-dihydroxypropionyl)SerSerSerChgGln-SerLeu-DOX (3') (SEQ.ID.NO.:86) (1)(2,3-dihydroxypropionyl)SerSerSerChgGln-SerLeu-DOX (3') (SEQ.ID.NO.:87) (1)(2,3-dihydroxypropionyl)SerSerChgGln-Ser(dLeu)-DOX (3') (SEQ.ID.NO.:88) (d)(2,3-dihydroxypropionyl)SerSerChgGln-SerLeu-DOX (3') (SEQ.ID.NO.:89) PEG(2)SerSerChgGln-SerLeu-DOX (3') (SEQ.ID.NO.:91) (d)(2,3-dihydroxypropionyl)-3PAL-SerSerChgGln-SerLeu-DOX (3') (SEQ.ID.NO.:94) (1)(2,3-dihydroxypropionyl)-SerSerChgGln-SerLeu-DOX (3') (SEQ.ID.NO.:95) (2,3-dihydroxypropionyl)-hSerSerSerChgGln-SerLeu-DOX (3') (SEQ.ID.NO.:96) PEG(2)-AlaSerChgGln-SerLeu-DOX (3') (SEQ.ID.NO.:97) PEG(6)-SerSerChgGln-SerLeu-DOX (3') (SEQ.ID.NO.:98) PEG(6)-SerSerSerChgGln-SerLeu-DOX (3') (SEQ.ID.NO.:99) PEG(6)-AlaSerChgGln-SerLeu-DOX (3') (SEQ.ID.NO.:100) PEG(4)-3PALSerSerChgGln-SerLeu-DOX (3') (SEQ.ID.NO.:101) or an optical isomer or pharmaceutically acceptable salt thereof.

16. The conjugate according to Claim 1 of the formula II:

wherein:

oligopeptide is an oligopeptide which is specifically recognized by the free prostate specific antigen (PSA) and is capable of being proteolytically cleaved by the enzymatic activity of the free prostate specific antigen and wherein the point of attachment of the oligopeptide to XL is at the C-terminus;
XL is -NH-(CH2)r-NH-R is selected from a) b) R1 and R2 are independently selected from: hydrogen, OH, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 aralkyl and aryl;

n is 1, 2, 3 or 4;
p is zero or an integer between 1 and 100;
q is 0 or 1, provided that if p is zero, q is 1;
r is 1, 2, 3, 4 or 5, or a pharmaceutically acceptable salt thereof.

17. The conjugate according to Claim 16 which is:

or a pharmaceutically acceptable salt or optical isomer thereof.

18. A conjugate of the formula III:

wherein:

oligopeptide is an oligopeptide which is specifically recognized by the free prostate specific antigen (PSA) and is capable of being proteolytically cleaved by the enzymatic activity of the free prostate specific antigen, R d and R e are independently selected from: hydrogen, C1-C6-alkyl, -C1-C6-alkyl-OH, -C1-C6-alkyl-di-OH, -C1-C6-alkyl-tri-OH and provided that at least one R d and R e are not hydrogen or C1-C6-alkyl, or R d and R e are combined to form a -CH2CH2OCH2CH2- diradical;

p is zero or an integer between 1 and 100;
q is 0 or 1, provided that if p is zero, q is 1;

or a pharmaceutically acceptable salt thereof.

19. The conjugate according to Claim 18 which is:

or a pharmaceutically acceptable salt or optical isomer thereof.

20. A pharmaceutical composition comprising a pharmaceutical carrier, and dispersed therein, a therapeutically effective amount of a compound of Claim 1.

21. A pharmaceutical composition comprising a pharmaceutical carrier, and dispersed therein, a therapeutically effective amount of a compound of Claim 10.

22. A pharmaceutical composition comprising a pharmaceutical carrier, and dispersed therein, a therapeutically effective amount of a compound of Claim 14.

23. A pharmaceutical composition comprising a pharmaceutical carrier, and dispersed therein, a therapeutically effective amount of a compound of Claim 15.

24. A method for treating prostate cancer which comprises administering to a mammal in need thereof a therapeutically effective amount of a composition of Claim 20.

25. A method for treating prostate cancer which comprises administering to a mammal in need thereof a therapeutically effective amount of a composition of Claim 21.

26. A method for treating prostate cancer which comprises administering to a mammal in need thereof a therapeutically effective amount of a composition of Claim 22.

27. A method for treating prostate cancer which comprises administering to a mammal in need thereof a therapeutically effective amount of a composition of Claim 23.

28. A method for treating benign prostatic hyperplasia which comprises administering to a mammal in need thereof a therapeutically effective amount of a composition of Claim 20.

29. A method for treating benign prostatic hyperplasia which comprises administering to a mammal in need thereof a therapeutically effective amount of a composition of Claim 21.

30. A method for treating benign prostatic hyperplasia which comprises administering to a mammal in need thereof a therapeutically effective amount of a composition of Claim 22.

31. A method for treating benign prostatic hyperplasia which comprises administering to a mammal in need thereof a therapeutically effective amount of a composition of Claim 23.

32. A pharmaceutical composition made by combining the compound of Claim 1 and a pharmaceutically acceptable carrier.

33. A process for making a pharmaceutical composition comprising combining a compound of Claim 1 and a pharmaceutically acceptable carrier.