CA2248550A1 - Cytokine designated lerk-8 - Google Patents

Abstract

The invention is directed to a protein designated Lerk-8, DNA encoding the Lerk-8, and host cells transformed with Lerk-8 DNA. Antibodies that are immunoreactive with Lerk-8 are also provided. The Lerk-8 protein binds to the cell surface receptors known as elk and hek.

Description

Wo 97/36919 PCTIUS97/04533 TITI.F.

RA(~K(~,R()lJNI) OF THF. ~NVF.l~TI()N

~leills known as the ,~,ceplol tyrosine kinases have an in~in~ic kinase activity that is activated upon ligand bin-ling This class of pl~t~ins is cha~?~t~.;7~d by conserved ~l.u~Lu,~al rnotifs within the catalytic dcm~in~ (Hanks et al., Science, 242:42, 1988) and can be subdivided into families based on ~lluelul~l r~ s of the regions N-terminal to the catalytic domain.
The eph family of receptors, named after the first memh~r i~o1~ted (Hirai et al., Science 238:1717, 1987) is the largest ~ul~r~u~ily of receptor tyrosine kinases. Among the of this family are chicken cek4 (Sa~adi et al. New Biol. 3:769, 1991) and cekS
(Pasquale, E.B., Cell Regulanon 2:523, 1991); murine mek4 (Sa.iJadi et al., supra), bsk (Zhou et al., J. Neurosci. Res., 37:129, 1994), nuk (~enkem-oyer et al., Oncogene 9:1001, 1994), and sek (Gilardi-Hebenstreit et al., Oncogene 7:2499, 1992); rat elk (Letwin et al., Oncogene 3:621, 1988; Lhotak et al., Mol. Cell. Biol. 11:2496, 1991), eek (Chan et al., Oncogene 6:1057, 1991), ehk-1 and ehk-2 (Maisonpierre et al., Oncogene 8:3277, 1993): and human hek (Boyd et al., J. Biol. Chem., 267:3262, 1992; Wicks et al., PNAS USA, 89:1611, 1992), hek2 (Bohme et al., Oncogene 8:2857, 1993), eck (~ inrlb~rg et al. Mol. Cell. Biol. 10:6316, 1990), and erk (Chan et al., supra).
The ~ cins of this ~ubr~l~ly are related not only in their cytoplasmic dc,lnains, but also in their extracell~ r domains, which are 41 to 68% i~l~ntiral Interestingly, the tissue dis~ibutions of these various l~el)tol~ are diverse. Recall~e many eph-related receptor tyrosine kinases are primarily expressed in the brain, it has been postul~te~l that these receptors and their ligands rpay be involved in the growth, dirr~ iation, and survival of neurons.
Those ligands that have been i~n~ife~l for the ~c~ or tyrosine kinases are a diverse group of proteins that affect the growth, differentiation, and survival of cells e~.~,ssing the receptors. Certain ligands have been found to bind to more than one - 35 receptor of the eph family. Ex~ll~les are the ligands for hek and elk that are described below.
Tclentifi~ tion Of ~ itiQn~l ligands for hek and elk that may exist would prove useful in investigating the nature of cellular processes reg~ ted by signaling through these l'eCeplO~. If enh~-ce ~ l or inhibition of a particular biological signal meAi~teA through these receptors is desired, it is advantageous to identify each of the proteins that may play a role in tr~n~uction of such cign~ls Further, it is known that certain ~r~ teins can bind to Iccep~ ~ without initi~ting signal tr~nc~luction, including interleukin-l receptor ~nt~gonict protein (Fic~nberg et al., Nature 343:341, 1990; ~nnum et al., Nature 343:336, 1990;
and Carter et al., Nature 344:633, 1990). ~lenhfi~tion of additional proteins that bind hek or eL~c is also desirable in order to d~ lf.ne wl~ eY any of such proteins functions as an antagonist.

~IJMMARY OF THF ~l~VF.NTION
The present invention is dircct~ to a novel cytokine ~l~ci~n~t~ Lerk-8. Lerk-8 binds to the cell surface ~ known as hek and elk, which are m~ e.~ of the above-described ephlelk farnily of l.,ceplul tyrosine kin~ces Purified Lerk-8 proteins are provided herein, along with isolated DNAs encoding Lerk-8, eA~ >n vectors co~ ,lising the Lerk-8 DNA, and host cells tran~rolllled with the e~r~ssion vectors. Processes for producing Lerk-8 include culturing such L~ rolll-ed host cells under conrlitionc that ~lulllOlt eA~,lc;ssion of Lerk-8 polypeptides, and recovering the Lerk-8. The invention also enc~ cses antibodies that are directed against Lerk-8.
nF.TATl.F.O l)F.~(~R~PTION OF THF. Tl~VF.l~TION
A novel cytokine desi~t~d Lerk-8 is provided herein. This cytokine binds to the ~,C~,~)lOI tyrosine kinases known as elk and hek.
The present invention encomr~cses DNA encoding Lerk-8, expression vectors comprising the Lerk-8 DNA, and host cells transformed with the eA~Iession vectors. A
method for pro~ucing Lerk-8 polypeptides comrrices culturing the transformed host cells under co~ ;ons conducive to eAplcssion of Lerk-8, and recovering the expressed Lerk-8.
Purified Lerk-8 polypeptides in both soluble and me.~ ane-bound form are disclosed.
Lerk-8 polypep~i-les or ;~ -ogenic fraem~nt~ thereof may be employed as ogens to gelle.ale antibodies that are ;~ v~-o.~active therewith. In one emk)-lim~nt of the invention, the antibodies are monoclonal antibodies.
A cDNA enro~ing human Lerk-8 was icol~~ as described in c~ 1. The nucleotide sequence of this Lerk-8 cDNA is plcsenled in SEQ ID NO: 1, and the amino acid sequence enrQded thereby is presented in SEQ ID NO:2. This Lerk-8 protein comprises an N-terrninal signal peptide (amino acids -27 to -1), an extracellular domain (amino acids 1 to 197), a llansnlelllblane region (amino acids 198 to 224), and a cytoplasmic domain (amino acids 225 to 313).
The c~lr~ trA molecular weight of the mature human Lerk-8 protein (amino acids 1to 313 of SEQ ID NO:2) is about 33 kilc~ ltons, and the isoelectric point (pI) is 8.46. One WO 97/36919 PCTAUSg7/04533 en.h4l;n~ of the present invention thus is dil~t~t to a pu~irlcd human Lerk-8 p otein chara~ 3d by a calculated ~le- nl~r weight of about 33 ~il~qltl-n~ and a pI of 8.46, wlle,~,n the N-t~nninsl amino acid s~ re of a mature form Of the protein is Leu-Ser-Leu-Glu-A~Val-Tyr-TIp-Asn-Ser-Ala-Asn- (amino acids 1-12 of SEQ ID NO:2). The S calculated nY~ n1qr weight is based on the -l~c~lar weight of a protein having the s~ ;r~ amino acid s~.,~-re, exclusive of any glyco~.rlalion. The skilled artisan will recognize tnat glycosylatod forms of the protein will have a higher nY~leculsr weight.
Lerk-8 fra~ment~ e.g., frae~nt~ that retain the ability to bind hek or elk, are l,lv.rided as well. Examples of such fraem~nts are soluble Lerk-8 polypeptides.
The present invention provides both cell membrane-bound and soluble (se~et~l) forms of Lerk-8. Soluble Lerk-8 polypeptiAes include the ~ or-binding domain of a Lerk-8, but lack the llnr,~"~ n,h,ane region that would cause retention of the polypeptide on a oell ~~mbl~ne. In one embodiment, a soluble Lerk-8 comprises the entire eytr~çe~ r domain (e.g., amino acids 1 to 197 of the human Lerk-8 of SE Q ID N 0:2). In another t~ tive~ the soluble ~ol~ ide is a fragment of the Lerk-8 extr~elllll~r domain that retains the ability to bind elk or hek. The portion of the extra(~elhll~r domain believed to be mostim~l~.lforn,ce~ .rbindingin~lude~ amino acids 1 to 142 of SEQ ID N 0:2. The .n~ c" of the extrPre~ r domain (amino acids 143 to 197) conSl es a spacer region.
F.~ es of soluble human Lerk-8 polypeptides include, but are not limited to, 20 polypeptides L~ cat~,d at the C-terminus so that the C-t~qnnin~l amino acid is any of the cisi~lues bel~ en or incl~l~ling the residues at positions 142 and 197 of SEQ ID N0:2. In other words, such soluble Lerk-8 polypeptides cc,...~ e amino acids 1 to y of SEQ ID
N0:2, wL~ y is any integer from 142 through 197.
Soluble Lerk-8 may be ir~entifi~l (and distinguished from its non-soluble 25 ~ e-bound COUlllCl~ S) by s~ ~.ng intact cells ~plessing a Lerk-8 polypeptide from the culture me~ m, e.g., by centrifugation, and assaying the nr~ium (supernatant) for thc ~s~ncc of the desired protein. The ~r~sellce of Lerk-8 in the .n~1;u~ in~ a~es that the protein was seclet~d from the cells and thus is a soluble form of the desired protein.
Soluble forms of Lerk-8 possess certain advantages over the mwll~ e-bound 30 forrn of the protein. Purification of the protein from reco~"b~nant host cells is facilitated, since the soluble proteins are secreted from the cells. Further, soluble proteins are generally more suitable for certain applic~tion~, e.g., for intravenous ~rimini~tration.
When initially e~ ,ssed within a host cell, soluble Lerk-8 polypeptides advantagcously c.>mprise the native signal peptide or one of the heterologous leader or 35 signal peptides described below that is functional within the host cells employed. l~Q1~ted DNA sequences encoding soluble Lerk-8 proteins are encompassed by the present invention.

T~nrqtPA Lerk-8, in~ f1ine soluble polypeptidçs, may be ~ d by any of a nu,lll~, of conventic~n-q~ h.~ es A DNA secluPnce ç ~ e a tn-n~tPd Lerk-8 may be~I,f~r.,.~llysy..~ fs;~usingknowntecl.~ ues. DNA L~ f..~ alsomaybe~l~)ducedby ;c1;on endo..~lckA~e Ai~-stiQn of a full length cloned DNA ~~uer~e, and icolqtP~ by 5 cl~ll~hul~,sis on agarose gels. O~ n~ çotif1es that l~onsl.ucl the 5' or 3'-terminus of a DNA f~emPnt to a desired point may be utili7~d Linkers co..~ g restriction c~ O~ k~ cleavage site(s) may be employed to insert the desired DNA fraemPnt into an ;on vector. The well known polymerase chain l~ion (PCR) procedure also may be employed to amplify a DNA rla~ ,n~ encoflin~ a particular protein fragTnPnt Primers 10 that define the desired termini of the DNA fnq.~mPn~ are employed in the PCR. As a further qltP,rnqtive, known mutag~n~Pcic teçhn;ques may be employed to insert a stop codon at a desired point, e.g., ;~""~ ly dow,lsl~.l of the codon for the last amino acid of the ~c~l~,r-binding domqin An eA~ ,ssed sequence tag (EST) contqin~ regions of identity with SEQ ID NO:l (see e~ ,lc 1). The COIll~)Ut~ q~t-q-hqnk record for this EST (accession no. H10006) senls a DNA sequence 454 nucleotides in length When the EST H10006 sequence is aligned with SEQ ID NO:l, regions of identity are found bclwee.l nucleotirles 663 and 1118 of SEQ ID NO:l. Certain of the nucleotides in EST H10006 are unidentifi~l (i.e., are ~le~ q-~ed "N" in the r1At,q,~hAnk record h-ecause their identity was unknown). The EST
20 se~uence contains inserted nucleotides not found in the cc..~onding positions in SEQ ID
NO: l, as well as ~kletion.~ and ~ ~AI~-hes when cc,lllpalf d to SEQ ID NO: l .
No reading frarne is irlentifif~ in the rlqtAhvqnk file for the EST, and the s-~uence lacks an i~ ;At;on codon. Further, the ahove-mentioned insertions and d-letion~ would cause shifts in the reading frarne, con~p~d to the reading frame of the Lerk-8 sequence of 25 SEQ ID NO: 1. However, even if a reading frame had heen ~,lufi~lqte<l and identified, and n..f,~ made for the inserted, delet~l and llniden~ified nucleotides, a translate of EST
H10006 would lack one of the four cysteine residues that are conserved in the other Lerk proteins (dcs~;libGd below), and would lack other conserved residues as well. The four conserved ~;y~ les are believed to be i~ for the ~iope. ly of binding to elk and hek.
Other pl.leins that bind to both hek and elk have been discovered, and are flçsi~nAt~A Lerk-l through Lerk-7 (ligands of the eph-_elated _inases). Lerks 2 and 5 are type 1 IIA~S~e~hI~nC proteins (as is Lerk-8), while ~erks 1, 3, 4, 6, and 7 are anchored to the cell .~.~I..h~u~e by GPI linkage. The percent identity of the amino acid sequences of these six proteins ranges from about 24 to 59%, and the proteins each have four conserved 35 cy~ine resid-les.
Holzman et al. (Mol. Cell. Biol. 10:5830, 1990) reported the cloning of cDNA fora protein called B61. The ability of B61 to bind to eLk and to hek was discovered WO 97/36919 PCTtUS97tO4533 subs~suently, and the B61 protein was given the ~ltern~tive desi~ tign Lerk-1 (Be.~L ...~nn et al., EMBO J. 13:3757, 1994). B61 has also been ~'.,~,t~d to be a ligand for the above-~esc~ l ,~eplor tyrosine kinase known as eck (Bardey et al., Nah~re 368:558, 1994~.
Lerk-2, also known as elk }igand, is describe~l in PCT arplir~tinn WO 94/11384.
S Lerk-3 and Lerk-4, also known as hek li~n-l~, are both ~s~ri~e~7 in PCT ~Mlif ~ti-)n WO
95/06065, Lerk-5 is tlGs~1 in WO 96/01839, Lerk-6 in WO 96tlO911, and Lerk-7 in WO 96/17925.
The percent identity of the human Lerk-8 amino acid se~,e-~ce of SEQ ID NO:2 with the full length amino acid se~en~e of various other proteins is as follows, ~l,~
10 "h" lepl~s~nls human, "m" ~ senls mouse, and "r" ~ ,se.-t~ rat.:

h Lerk-1 25.14 h Lerk-2 40.80 r Lerk~2 39.69 m Lerk-2 40.00 h Lerk-3 24.88 h Lerk-4 25.41 h Lerk-5 41.23 m Lerk-5 42.07 m Lerk-6 26.18 h Lerk-7 24.88 As used herein, the term "Lerk-8" refers to a genus of polypeptides that are ~bs~ lly homologous to the human Lerk-8 protein ~lescri~l in r~h~l~plc 1. The 25 polypeptides preferably comprise an amino acid sequence that is at least 80% iden~i~ and more pl~f~,.~ly at least 90% i(len~i~l, to the amino acid sequence of SEQ lD NO:2, as further described below. The Lerk-8 polypeptides are capable of binding to the above-d~srribe~ lec~tcl~ rlesign~te~ hek and elk. Certain uses of Lerk-8 flow from this ability to bind to elk or hek, as described in more detail below. Human Lerk-8 nucleic acids and 30 ~lut~lns are within the scope of the present invention, as are Lerk-8 nucleic acids and proteins derived from other ""-. ""~ n species that include but are not limited to murine, bovine, porcine, equine, or various primate species.
Due to dhe known de~,~,n~, ~y of the genetic code, wllclGill more than one codoncan encode the same amino acid, a DNA sequence can vary from that shown in SEQ ID
35 NO:1 and s~ll encode a Lerk-8 protein having the amino acid sequence of SEQ ID NO:2.
Such variant DNA sequences may result from silent mutations (e.g., occurring during PCR
amplification), or may be the product of delibel~tte mutagenesis of a native sequence. The present invention thus provides i~o1~t~1 DNA se~ nres ~1~ ~1 from native Lerk-8 DNA
sc~jucl~ce,s (e.g., cDNA comrri~ing the nucl~ooti(le se~ nce pl~s_nlcd in SEQ ID NO:1) and DNA that is dee~ ...dte as a result of the genetic code to a native Lerk-8 DNA
Sc~lu~ Ct.
S The Lerk-8 polypeptides provided herein include variants of native Lerk-8polypeptides that retain a l -lc~gie~l activity of a native Lerk-8. Such variants include ol~l)~ des that are subst~nti~lly homologous to native Lerk-8, but which have an amino acid ~ ce Lrr~ from that of a native Lerk-8 ~ se of one or more ~1eletion~, ins.,llions or substi-lltion~. Likewise, the Lerk-8 en~ol;n~ DNAs of the present invention include variants that differ from a native Lerk-8 DNA se~uen~e because of one or more dclGI;o--~, insertions or substitutions, but that encode a biologically active Lerk-8 polypeptide. The term "biologically active" as it refers to Lerk-8, in-lic~tes that the Lerk-8 is capable of binding to hek or to elk.
The variant DNA or amino acid sequences preferably are at least 80% iriçntic~l to a native Lerk-8 sequ~nce, most plc~lably at least 90% identir~l The percent identity may be d~,t~ ...;ne~l for example, by com~ing sequence inro,...-~;on using the GAP C<~ UICI
prograrn, version 6.0 described by Dcve,~ et al. (Nucl. Acids Res. 12:387, 1984) and available from the University of Wisconsin Genetics C'o~ , Group (UWGCG). The ~l~ f~ di default p~i~i..~,te.~ for the GAP program include: (1) a unary co..~ ison matrix 20 (con~ g a value of 1 for identities and 0 for non-i-l~ntities) for nucleotides, and the weighted co...~ ;con matriix of Gribskov and Burgess, Nucl. Acids Res. 14:6745, 1986, as desc-.;lxcl by Schwartz and Dayhoff, eds., Atlas of Protein Sequence and Structure, National BiomeAi~l Research Foundation, pp. 353-358, 1979; (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap; and (3) no penalty for 25 end gaps.
~ clr1itinn~l embo~1im~nt~ of variant amino acid sequcnces are those COml)lk.~ng conservative s~lbstinltion~ m~ning that one or more amino acid residues of a native Lerk-8 is ~ ced by a dirr~ residue, but that the conservatively ~.ub..~ led Lerk-8 polypeptide retains a desired biological activity of the native protein (e.g., the ability to bind eLk or hek).
30 FY~mrles of conservative substitutions include sllbstitntion of residues that do not alter the secon-l~. y or tertiary structure of the protein.
A given amino acid may be replaced by a residue having similar physic)ch~ icdl chara~ ,lislics. FY~mrle5 of conservative ~.ubslilulions include substitution of one plirh~ti~
residue for another, such as Ile, Val, Leu, or Ala for one another, or substitutions of one 35 polar residue for another, such as between Lys and Arg; Glu and Asp; or Gln and Asn.
Other conservative substitutions, e.g., involving substitutions of entire regions having sirnilar hydrophobicity characteri~ti~s, are well known.

The invention further incl~ es Lerk-8 polypepti~le~ with or will~ul associated native-pattern glycosylation. Lerk-8 eA~ ,ssGd in yeast or m~smmAlisn eA~l~ssion systems (e.g., COS-7 cells) may be sinilar to or ~i~ifir-s-ntly d;fr~n~ from a native Lerk-8 pol~plide in molecular weight and ~ly~;osylation pattern, de~rl;ng upon the choice of S eA~ ~ss;on system. E~ siol~ of Lerk-8 polypeptides in ~.srt~ri~l e~iessiol1 systems, such as E. coli, provides non-glycosylated mrl~c1-1es N-glycosylation sites can be ,-~;r.~ to preclude glycosylation, allowing c~ ion of a more h~ oge,~eous, ~~luced c&lJGhyd~dte analog in ~,A"",~lisn and yeast c~ s;~ion systems. N-glycosylation sites in eukaryotic pol~ les are chsr~çt~ri7~d by 10 an amino acid triplet Asn-X-Y, ~.L~,~ X is any amino acid except Pro and Y is Ser or Thr. The human L~rk-8 protein of SEQ ID NO:2 co.~.. ;~,s one such triplet. at amino acids 183-185 of SEQ ID NO:2~ A~y~u~iale substitutions, ~rlition~ or ~rletiQn~ to the nUClff)ti ~e se~uence encoding these triplets will result in prevention of ~ çlln~nt of carbohydrate residues to the Asn side chain. ~ ol~ of a single nucleotide, chosen so that Asn is replaced by a dirre~ amino acid, for example, is suffi~ient to inactivate an N-glycosylation site~ Known procedures for inactivating N-glycosylation sites in proteins include those described in U.S. Patent 5,071,972 and EP 276,846.
In another example of variants, sequen~e~ encoding Cys residues that are not ec~.~ for biol~gir~l activity can be altered to cause the Cys residues to be deleted or replaced with other amino acids, ~ ing formation of incorrect intr~ cnl~r ~ nlfi(le bridges upon renaturation. Cysteine residues coll~onLng to the four cysteines that are conserved among the Lerk proteins are found at positions 35, 65, 77, and 129 of SEQ ID
NO:2. These four ~y~ines desirably remain unaltered in Lerk-8 variants.
Other variants are prepared by m~lifir~tion of acljacenl dibasic amino acid residues to ~nh~nce expression in yeast systems in which KEX2 protease activity is present. EP
212,914 discloses the use of site-specific mllt~gen~sis to inactivate KEX2 protease ce~ g sites in a protein. KEX2 protease processing sites are inactivated by deleting~
adding or sub~ ;.-g residues to alter Arg-Arg, Arg-Lys, and Lys-Arg pairs to eliminate the oc~ ce of these ~ c~ .t basic residues. The human Lerk-8 contains such ~r~ e.~l basic residue pairs at amino acids 13-14, 63-64, 151-152, 225-226, 226-227, and 227-228 of SEQ ID NO:2. Lys-Lys pairings are cr.n~i~lerably less susceptible to KEX2 cleavage, and conversion of Arg-Lys or Lys-Arg to Lys-Lys ~ se~ a conservative and preferred a~p.uach to inactivating KEX2 sites.
Naturally occurring Lerk-8 variants are also encomp~secl by the invention.
- 35 Examples of such variants are proteins that result from alternate rnRNA splicing events or from proteolytic cleavage of the Lerk-8 protein. Alt~rn~t~ splicing of mlRNA may, for eY~rnrle, yield a L.ul.~aled but biologically active Lerk-8 protein, such as a naturally WO 97/36919 PCT/US97/04~33 oc~...;..~ soluble fcrrn of the protein. Va~iations ~ttrib~ 'e to proteolysis include, for example, dirr~l~,ncGs in the N- or C t~mini upon ~ pl~ssion in Lrr~,.cllt types of host cells, due to p¢oteolytic removal of one or more t~nnin~l amino acids from the Lerk-8 protein (gçn~rally from 1-5 tçrminql amino acids). Thus, Lerk-8 ~IGt~lS in which the N ~5 residue is any of amino acids 1 to 5 of SEQ ID NO:2, and the C t' ----;--~l residue is any of amino acids 308 to 313 of SEQ ID NO:2 are ~;ircdlly provided herein. For solubleLerk-8, the C t~ ....;.~ql residue may be any of amino acids 192 to 197 of SEQ ID NO:2.
Lerk-8 proteins in which lirr~"~,ncGs from the amino acid se~lu~nce of SEQ ID NO:2 are atlributable to genedc poly.l~l~hism (allelic variadon arnong individuals producing the 10 protein) are also col.t~ dt~ herein.
One i~ol~ted Lerk-8 cDNA included a single nuclc~lide s~b~ n when CC~lll~l with the cDNA described in example 1. The variant Lerk-8 DNA se~lucnce differs from the DNA sequence ~.~senlcd in SEQ ID NO:1, in that the nucleodde at position 1370 of the variant is cytosine (C), ra~her than the ~ -c (G) found at that position in SEQ ID NO: 1.
15 In the amino acid sequence of this Lerk-8 protein, the residue at posidon 298 is leucine.
Regarding the Çc).~,going (li~cu~sion of the signal pepdde and various domains of Lerk-8 protein, the skilled ardsan will recognize that the above-described boundaries of such regions of the protein are al~p~ i--late. The boundaries of the tr~n~.,.k...hl~le region (which may be predicted by using cc....~ plugl~ulls available for that purpose) may 20 differ from those described above. Thus, soluble Lerk-8 polypepddes in which the C-tçrrnin.lc of the extra~e~ r domain differs from the residue so i(1çntifisd above are col~ ,plated herein. As another illustradon, cleavage of a signal pepdde can occur at sites other than those ~l~liclt~d by co.~ .,tt. program. Further, it is recognized that a protein pl~ation can comylisc a llli,~lul~; of protein mole~nles having lirrtlcnt N-terrnin~l amino 25 acids, due to cleavage of the signal pepdde at more than one site.
~ o-~-p~ . analysis of the hwnan Lerk-8 protein in~ atçs that cleavage of the signal pepdde is most likely to occur after amino acid -1 of SEQ ID NO:2. Four ~ rn~tive signal pepdde cleavage sites plcdicL~d by Cf~ . program are (in ~çsce.~ g order of likelihood) located after ~csidues 3, -5, 2, and -2 of SEQ ID NO:2. Thus, mature human 30 Lerk-8 polyy~p~ides in which the N t"-"-;n~l amino acid is s~lçcted from the residues at positions 4, -4, 3, and -1 are provided herein, in addition to the embo~lim~n~ in which the N t~ . ..,;.,~l amino acid is the residue at position 1.
Variants and derivadves of native Lerk-8 pl~t~ins may be pl~al~d by mutation of nucleodde s~uçnces encoding native Lerk-8 polypepddes. Mu~ions can be introduced at 3~ particular loci by synthe~i7in~ oligonucleoddes co..~ a mutant sequence, flanked by restriction sites enabling ligadon to fr~ nt~ of the nadve sequence. Following ligation, the resulting recon~llu~;lcd sequence encodes an analog having the desired arnino acid wo 97136919 PCT/USg7/04~33 insertion, ~ub~ ;on, or deletion. Alternatively, olig~ ;de~ ~t~l site-specific g~--c~iC pl~)cedw~,s can be employed to introduce a desired muta~tiQn Methods for making such altP~r.atif)nc include those disclosed by Walder et al. (Gene 42:133, 1986);
Bauer et al. (Gene 37:73, 1985); Craik (BioTechniques, January 1985, 12- 19); Smith et al.
5 (GeneticEngineering:Principlesand Methods, Plenum Press, 1981); Kunkel (Proc. Narl.
Acad. Sci. USA 82:488, 1985); Kunkel et al. (Methods in Enzymol. 154:367, 1987); and U.S. Patent Nos. 4,518,584 and 4,737,462.
Lerk-8 may be moflifi~1 to create Lerk-8 derivatives by forming covalent or a~ a~re conjugates with other chemical moieties, such as glycosyl groups, lipids, 10 phGspk~;, acetyl groups and the like. Covalent derivatives of Lerk-8 rnay be ~lepa,~d by linking the chemical moieties to filn~ti~nal groups on Lerk-8 amino acid side chains or at the N t~lllinus or C-termin~c of a Lerk-8 polypeptide or the extracelllllar domain thereof.
Other derivatives of Lerk-8 within the scope of this invention include covalent or ag~ e conjugates of Lerk-8 polypeptides with other proteins or polypeptides, such as 15 by synthesis in lecc ~llbinallt culture as N-terminal or C-terminal fusions.
Lerk-8 polypeptide fusions can comprise peptides added to fa~iIitat~ purifica-tion and i~ientifi~ation of Lerk-8. Such peptides include, for examrle, poly-His or the ~ntig,-nic idçn~ific~tion peptides described in U.S. Patent No. 5,011,912 and in Hopp et al., BiolTechnology 6:1204, 1988. One such peptide is the Flag~ peptide, Asp-Tyr-Lys-Asp-20 Asp-Asp-Asp-Lys (SEQ ID NO:3), which is highly ~ntigenic and provides an epitope reversibly bound by a specific monoclonal antibody, enabling rapid assay and facile p~lrifi~tiQn of expressed recomhin~nt protein. A murine hyhridom~ design~ted 4E11 produces a monoclonal antihody that binds the Flag6~ peptide in the pl~,sellce of certain divalent metal cations, as described in U.S. Patent 5,011,912, hereby illCu~ led by 25 reference. The 4Ell hybridoma cell line has been deposited with the A~ ica~l Type Culture Cnllection under accession no. HB 9259. Monoclonal ~ntibofiies that bind the Flag E9 peptide are available from F~ctm~n Kodak Co., Scientifi~ Im~ing Systems Division, New Haven, Co~necticu~
Lerk-8 ~ tt;ins (incl---ling fr~gm~ntc and variants) may be tested for the ability to 30 bind hek or elk in any suitable assay. Biolog~c~l activity of a Lerk-8 variant rnay be d~t~ cA, for example, by assaying for the variant's ability to con~ with a native Lerk-8 for binding to hek or elk (i.e. cc,l~eLi~ e binding assays).
Co~ eLiLi~e binding assays can be l)~,lrolllled following conventional m~th~lology.
g~ntc that may be employed in co..~l.e~ /e binding assays include radiolabeled, soluble 35 Lerk-8 and intact hek/elk-tiA~ ,s~ing cells. For example, radio!~kele~l soluble native Lerk-8 can be used to cc,~ cle with a soluble Lerk-8 variant for binding to cell surface-bound hek or elk. Instead of intact cells, one could substihlte a soluble hek/Fc or eLk/Fc fusion protein CA 02248550 l998-09-l4 bound to a solid phase thrvugh the j"h~ of Prvtein A or Protein G (on the solid phase) with the Fc ~iety. Chromalv~al)hy col1lmnc that contain Protein A and Protein G
include those available from P1.,.. r~i~ Biotech, Inc., Piscataway, NJ. Another type of cr..~~ e binding assay utilizes r: ~lirt~qhPl~ soluble hek orelk, such as a soluble hek/Fc S or elk/Fc fusion protein, and intact cells e~ Gssing Lerk-8. In yet another alternative, a Lerk-8 may be assayed for the ability to cO~ h. with one of the other Lerk p~olel.~s (Lerks 1 through 7, described above) for binding to elk or hek. ~ tive results can be obLained by c~ e autvradiographic plate binding assays, while Sc~ plots (Scatchar~, Ann. N.Y. Acad. Sci. 5l:660, 1949) may be utilized to g~ ,.ale qll~ntit~tive results.
It is ~ s ''- that the Lerk-8 of the present invention will bind to other lec~tul~ of the eph farnily (see the bac~vund section). Such binding can be analyzed using a suitable assay analogous to those ~lesçtibe~l above.
Uses of Lerk-8 that flow frvm the ability to bind elk and hek inch1de~ but are not lin~ted to, the following. Lerk-8 finds use as a protein purification reagent. Lerk-8 15 polypeptides may be s~ h~ to a solid support m~tPri~1 and used to purify hek or elk proteins by affinity cLo1llatography. In particular emkY1im~ntc~ Lerk-8 fr~grnP-ntc or fusion l,rol~ nc (e.g., Lerk-8/~c fusions) co~ ing the receptor-binding domain of Lerk-8 are ~ he~l to a solid support by conventi--n~1 procedures. As one eY~mp1e, chl~ at~,graphy colnmnc con~ining functional groups that will react with fi1nctiQn~1 20 groups on amino acid side chains of proteins are available (Ph~ ni~ Biotech, Inc., Piscataway, NJ). Lerk-81Fc fusion proteins can be ~tt~eh~l to Protein A- or Protein G-c~ nt i11;~g cl~matography columns through interaction with the Fc moiety.
Lerk-8 pl~,~;ns also find use in purifying or idcnliryillg cells that express hek or elk on the cell surface. The Lerk-8 (or fragment or fusion thereof) is bound to a solid phase 25 such as a column cl,-.~ tography matrix or a similar suitable substrate. For example, m~71çtit~ mi~l~,~l,c~,s can be coated with Lerk-8 and held in an incuh~tion vessel through a magneti~ field. Suspensions of cell I~ Ul~S col~ .;ng hekJeLk-t;~ssing ceUs are conl;..,t~ with the solid phase having Lerk-8 thereon. Cells e..~ ssi~lg hek or elk on the cell surface bind to the fixed Lerk-8, and unbound cells then are washed away.
~1t~n~tiWly, ,lu~ s of cells s~ ,ecl~1 of c~ inil~g hekleLk+ cells first can be ill~"~ d with biotinylated Lerk-8. Inruh~tion periods are typicaUy at least one hour in duration to ensure s11fficient binding to hek/elk. The resulting n~ then is passed through a column packed with avidin-coated beads, whereby the high affinity of biotin for avidin provides the binding of the cell to the beads. Procedures for using avidin-coated beads are known (see ~e.~,nso1l, et al. J. Cell. Biochem., lOD:239, 1986). Washing of unbound m~ 1 and the release of the bound cells is ~lîo.llled using convention~1mçthofl~

The thus-puliLed cell pop~ sti~n then may be used in vanous in vitro studies or in vivo p.-~cclulcs, e.g., to repopllls-te tissues in a mammal. To i1lu~trste7 neural cells e~ ,sil~g eLk may be isolated by the foleg~ g pl'Oc~lUl~, then a lmini~t~red to a "~
~ rrl;~d with a ne~1e~ tive disorder. Hek+ cells include certain h-nl~rni~ cells 5 (identified below). ~ ste~1 leukem a cells can be used in studies of the effects of various drugs on the cells, for example.
To identify ~.1iti.~ 1 types of cells that express hek or elk on the cell surface, Lerk-8 can be c~ ~ju~;. t~ to a ~ le moiety such as a ra~ionucli-l~. As one eY-s-mplc, ralt ~ elin~ with 125I can be p~,lr~.l,.led by any of several standard n~tho~l~lcOies that 10 yield a r.~ ;o~ 25I-Lerk-8 -~e~ e labeled to high specific activity. Other ~letet~
moieties include C.~rlllCS that can catalyze a colv. ;~ .h ;r or ~luololll~ic reaction. Cells to be tested for hek/elk-e~ ssion are ccntnctP.~l with labeled Lerk-8. After incubation, labeled Lerk-8 is removed and the presence or absence of the ~etect~hle moiety on the cells is ~lct~ ed Lerk-8 proteins also find use in .u~ g the bi--lo~r~l activity of elk or hek proteins in terms of their binding affinity for Lerk-8. Lerk-8 proteins thus may be employed by those col~lucling "quality a~.~.ul~lce" studies, e.g., to l,loni~(,l shelf life and stability of elk or hek protein under dirr~,le"~ conditions. To illustrate, Lerk-8 may be ellll)lo~c~ in a binding affinity study to Illea~7ulG the biological activity of an elk protein that has been stored at dirr~ e~ atul~s~ or produced in dirr~G,~ cell types. Lerk-8 also may be used to det~lllli.~e whether biological activity is retained after mrAifieation of an elk or hek protein (e.g., che.n:~31 modific~tion, tnlnr~tirJn~ mutation, etc.). The binding affinity of the m~ified elk protein for Lerk-8 is coll~ d to that of an ~mm~lified elk protein to detect any adverse impact of the moflifir~tion~ on biological activity of elk.
Likewise, the bic~o~r~l activity of a hek protein can be ~sessed using Lerk-8. The bi~lo~r~l activity of an elk or hek protein thus can be asc~ ne~ before it is used in a ~,seal~;h study, for eY~mrle Lerk-8 polypeptides also find use as carriers for delivering agents ~tt~hPA thereto to cells bearing the elk or hek oell surface receptor. Expression of hek antigen has been l~,pc~lLd for certain leukemic cell lines, incl~-ling the human T-cell le~ Pmi~ cell lines d~Psign~t~ JM and HSB-2 and the human pre-B cell le~ pmi~ cell line de~ign~t-PA LK63 (Wicks et al., Proc. Natl. Aca~'. Sci. USA, 89:1611, 1992; Boyd et al., J. Biol. Chem.
267:3262, 1992. Lerk-8 proteins thus can be used to del*er tli~gnostir~ or ll,e.~ ~ic agents to these cells (or to other cell types found to express hek or eLk on the cell surface) in ~ 35 in vitro or in vivo pl~CC~ S.
One P,Y~mrlP of such use is to expose a hek+ le~ Prni~ cell line to a th~
agent/Lerk-8 conjugate to assess whether the agent exhibits cytotoxicity toward the leuke~ic cells. A n~ of d~rr~ a~ c agents attached to Lerk-8 may be ;-IGlud~l in an assay to detect and compare the ~ UAic effect of the agents on the leukemic cells. Lerk-8/ li~nostic agent conjugates may be employed to detect the ~.Gs~,nce of hek~
cells in vitro or in vivo.
S De~ctable (.i;~........ ~l;r) and ~ m;r agents that may be ~ ~1 to a Lerk-8 ~ly~tide inrlude, but are not limited to, drugs, to~ns, radiom~cli~les~ chlvmol)hores, en~ymes that catalyze a col~ h;c or fluulull~hic l~ n, and the like, with the particular agent being chosen acco~ g to the i~ ed ~ FY~mrl~s of drugs include those used in treating various forms of cancer, e.g., nillu~en Illusku~s such as L-10 ~hcn~lalanine nitrogen mustard or c~clo~h~ le, inte~alating agents such as cis-amino-1ir~1Oroplatinum, L~ abolites such as 5-lluu~ul~cil, vinca ~ loi~l~ such as vincristine, and antibiotics such as bleomycin, doxorubicin, daunorubicin, and derivatives thereof. Among the toxins are ricin, abrin, rlirhth~n~ toxin, Pseudornonas aeruginosa eYçl~Ein A, ribosomal inactivating proteins, mycoto~ins such as trichothecenes, and 15 derivatives and fr~ nt~ (e.g., single chains) thereof. Radion-l~lides suitable for tliaenosti~ use include, but are not limited to, 123I, 13lI, 99mTC, lllIn, and 76Br.
UC1;~eS ~ui~ble for tL~I~ulic use inrlude, but are not limited to, 131I~ 211At, 77Br, 186Re 188Re, 212pb, 212gi, 109pd, 64CU, and 67Cu.
Such agents may be attached to the Lerk-8 by any suitable conventional procedure.
20 Lerk-8, being a protein, comprises functional groups on amino acid side chains that can be reacted with rl,-,clim-~l groups on a desired agent to fomm covalent bonds, for ex~mple Altematively, dhe protein or agent may be derivatized to gcn~lale or attach a desired reactive fimrtion~l group. The deriv~ti7~tion may involve dl~hll~enl of one of the bifunctional CO~ g l~agenls available for ~tt~hing various molecules to ~ leins (Pierce Chemical 25 Company, Rockrold, Illinois). A n.~ of techniques for r~linl~heling proteins are known. Ra~lionuclide metals may be ~t~ çd to Lerk-8 by using a suitable bifunctional ch.o1~ting agent, for çx~mple Conjugalt;s Co~ Jlisillg Lerk-8 and a suitable ~ nostir or ~ a~ulic agent (prer~,ably covalendy linked) are thus plepa,~d. The conjugates are ~1mini~t~red or 30 otherwise employed in an amount appropliatt; for the particular applir~tinn Another use of the Lerk-8 of the present invention is as a research tool for studying the role that Lerk-8, in conjunclion with elk or hek, may play in growth or dirr~,r~,ntialion of cells bearing the elk or hek l~cel,lol. The Lerk-8 polypeptides of the present invention also may be employed in in vitro assays for ~i~tecticn of elk or Lerk-8 or the interactions 35 thereof. Likewise, Lerk-8 finds use in assays for hek or the interaction of Lerk-8 with hek.
The possibility that hek plays a role in tnmnrigçnçci~ has been suggested (Boyd et al., su~ra).

The Lerk-8 DNA and pol~p~;dfs of the present invention may be used in lo~l& n~ for any ~lis~ er n~A;r-~1 (directly or ind~lly) by defective, or amounts of, Lerk-8. Lerk-8 polypepti(les may be :~lminict~e~cd to a ",~."",~l hrll;rt~ with such a disorder. Alternatively, a gene therapy approach may be taken.
S Dis~lo~ . herein of native Lerk-8 .""~ se~lu~,nces permits the rlete~ion of defective Lerk-8 genes, and the replacement thereof with normal Lerk 8 e-n~ofling genes. Defective genes may be rk t~-te~l in in vitro ~ ~ostic assays, and by c~....~r- ;cion of a native Lerk-8 I. ,c~ t;~e s~ufn~ ;~lo3e~ herein with that of a Lerk-8 gene derived from a person su~p~t~ of ha~ g a defect in this gene.
As ~ cll~sed above, when various rat tissues were analyzed for elk mRNA, b were de~ctfYl only in brain and testis (Lhotak et al., su~ra). Expression of rec~: rs for Lerk proteins on neural tissues has led to investigation of the roles that Lerk proteins may play in development or legencl~lion of the nervous system. Lerk-7 has been l~,t~d to be involved in _xon ~ An~e and axon bundle formation (Winslow et al., lS Neuron 14:973-981, 1995; Drescher et al., Cell 82:359-370, 1995). The Lerk-8 of the present invention may be employed in studies of the effects of binding of Lerk-8 to l~,C~l~ on neural tissue. The role that Lerk-8 may play in inducing or regulating plVCe,SSeS s~csociAted with the nervous system can be investigated. Lerk-8 may be a~l...;-.i~i~,~id in vivo to regulate or p~ lOtc development of the nervous system.
Certain of the above-described Lerk proteins have been l~ cd to possess n~,~lule.;li~e ~ ies, e.g., to protect hi~)~ocallll,al neurons against gl~
meAi~t~d excitotoxicity. The involvement of an eYcitotoyic c~,lllpollelll in a number of disorders of the neural system has been est~bli~he~d. Re;~yonsi~reness to gl..~ ; is a normal fullclioll in the developing and mature central nervous system (CNS). In ~ ition 25 to its noImal role in excitatory synaptic tr~n~mi~ion and plasticity, however, ~I.-I;..,.~te can also mediate or otherwise participate in a number of CNS dysfunctional states, inrlu~ling but not limited to ~ 1'S ~ e~ce~ Huntington's Disease, Parkinsonism, stroke (i~l.~-..;~), epilepsy, and AIDS-related d~ (reviewed in Meldrum and Ga~ w~iLe, Trends Pharmacol. Sci. 11:379, 1990; Choi, J. Neurosci. 10:2493, 1990; Lipton et al., Neuron 7:111, 1991; and Andersson et al., Eur. J. Neurosci. 3:66, 1991).
The Lerk-8 polypeptides provided herein find use in a method for treating disorders of neural tissue, involving c~nt~cting the neural tissue with Lerk-8. Such disorders include injury to neural tissue, or neurologic ~lise~ses, either chronic or acute. FY~ )1eS of such disorders include, but are not limited to, the above-described conditions involving ~ 35 disfunction of the CNS. Lerk-8 may be aAmini~t--red to a .,~.,.".~l, including a human, arrect~l with such a condition.

.... ..

Certain of the Lerk proteins have been found to ~,lumoLe ~qngio~enesic~ Lerk-8 likewise may find use in plO~luLillg angiogene~i~, which can be be,nefir~l for wound h~lqlin~, ~tim~ tin~ neo~A~~ of grafted tissues, or in treating any con~litiQn in which in~ ased ~qngiog~nesi~ is desired.
Co~osiLions c~.. l.. ;~;.-g an effective amount of a Lerk-8 polypeptide of the present invention, in cr.-~-h;..q~ with other co...~ t,~ such as a physiologically acceptable ~ uen~ carrier, or excipient, are provided herein. Lerk-8 can be rc.. ~
~co~ to known ",~ used to prepare ph~qrmq~euti~lly useful co~ os;~;ons. Lerk-8 can be co.-~hil-ed in ~/1.,,;,~l,,,~;, either as the sole active material or with other known 10 active mqt~ri~ql~ with ph~ e.l~ic?l1y suitable tlihl~nt~ (e.g., saline, Tris-HCl, acetate, and l)hu~ e buffered solutions), ,~ s~ a~i~es (e.g., ~I,illlclusal, benzyl alcohol, parabens), ermll~ifi~rs, solubilizers, adjuvants and/or carriers. Suitable f~ rmtll~tions for pk~ e~l;c~l colll~silions include those described in Remington's Pharrtmre~q~n~
Sciences, 16th ed. 1980, Mack p~ shing ~omp~ny, Easton, PA.
In ~drlition, such c<lllpo~itions can contain Lerk-8 comrl~Y~(I with polyethylene glycol (PEG), metal ions, or incul~uldted into polyrneric con,l)ounds such as polyacetic acid, polyglycolic acid, hydrogels, dextran, etc., or inc~ ,ul~led into liposomes, microçmnlsionc, micelles, llnilqnn~ r or mllltilq-nellqr vesicles, erythrocyte ghosts or spheroblasts. Such compositions will influence the physical state, solubility, stability, rate 20 of in vivo release, and rate of in vivo clearance of Lerk-8, and are thus chosen according to the int~nAecl appli~qtion Lerk-8 also can be conjugated to antibodies di~ t;d against tissue-specific receptors, ligands or antigens, or coupled to ligands of tissue-specfflc plol~. Lerk-8 ~AlJIessed on the surface of a cell may find use, as well.
Such c~.--po~;l;. ns may contain a Lerk-8 polypeptide in any forrn described herein, 25 such as native ~lole;ns, variants, derivatives, oligomers, and b ~log~-~q-lly active fr~gmentc.
In one e~ the co...l.osil;on comprises a soluble Lerk-8 polypeptide, preferably an oli~oln~r co.~ h~g soluble Lerk-8 polypeptides.
Lerk-8 can be aAministe.ed in any suitable manner, e.g., topically, parenterally, or by inhqlqtion The term ~pa~ dl" inc!ll~1ec injection, e.g., by subcutaneous, 30 intravenous, or intrqmuccul-qr routes, also including 1~.~ ~ a~lministration, e.g., at a site of diseaseorinjury. Sllct~in~releasefrom ;~ 9~ is also co~-le...pl-q-t~ One skilled in the p4,1inenl art will l~co~ , that suitable dosages will vary, de~nding upon such factors as d~e nature of the disorder to be tréated, the patient's body weight, age, and gene~l concli~n, and the route of qt1minictration. Prelimin~ry doses can be dct~,.l"illcd according 35 to animal tests, and the scaling of dosages for human a~lminictration are ~,ru~ ed acc~.,ding to art-acce,.)tcd practices.

... .. . . .... .. ... . . .

01;~ Forms of T ~ 8 r,.co~r~c~ by the present invention are olig~mprs that contain Lerk-8 poly~l;Aes. Lerk-8 oligclmers may be in the form of covalently-linked or non-covalently-linked dimers, trimers, or higher oligomers.
One e~ of the invention is dil~t~d to olignm~rs cnmpriQ;ng ml~ltiple Le.rk-8 pol~ ;de~ joined via covalent or non-covalent ;~ ;on~ ~Iween peptide moieties fused to the Lerk-8 polypeptiA~o-s. Such peplides may be peptide linkers (spacers), or ~ Ae s that have the ~u~ ly of IJlO"~'JI;~g ~ligom~i7~til~n T ulcine zippers and certain pol~ iAes derived from ~ntiboAi~s are among the ~pl ;~es that can l"ulllut~
10 oli~,n-~ t;oi- of Leric-8 ~l~p~ es ~ttl ~hed thereto, as d~s~ - ;hecl in more detail below.
In particular e.l~b~l;..~ t~, the oligomers co~r~e from two to four Lerk-8 polypeptides The Lerk-8 moieties of the oligomer may be soluble polypeptides, asdescribed above.
As one alternative, a Lerk-8 oligomer is ~l~al~d using polypeptides denved from ;.. ~ oglobulins. F~ alion of fusion proteins Cf~ .ing certain heterologous poly~ll;fles fused to various portions of antibody-derived polypeptides (including the Fc domain) has been described, e.g., by .A~hk.-n~7.i et al. (PNAS USA 88:10535, 1991); Byrn et al. (Nature 344:677, 1990); and Hollenbaugh and Aruffo ("Construction of Tmml-noglobulin Fusion Proteins", in Current Protocols in Immunology, Suppl. 4, pages 20 10.19.1 - 10.19.11, 1992).
One embo~l;...- .~l of the present invention is directed to a Lerk-8 dirner cu~llyli~ g two fusion proteins created by fusing Lerk-8 to the Fc region of an antibody. The Fc pol~ idc preferably is fused to the C ~....;..~.~ of a soluble Lerk-8. A gene fusion en~ g the Lerk-8/Fc fusion protein is inserted into an ~ululJliate t;~ ssion vector.
25 Lerk-8/Fc fusion plv~il~s are t;~ ..Sed in host cells transforrned with the recomhin~nt e~ ,ss;on vector, and allowed to ~semhle much like antibody molecules, whereuponin~l~;h~ll tii~ulfide bonds form ~L~.~,en the Fc mr i~ties to yield divalent Lerk-8.
Provided herein are fusion ~ eins comrri~ing a Lerk-8 polypep~ide fused to an Fcpol~plidf. derived from an antibody. DNA ~nco 1;,.~ such fusion proteins, as well as 30 dimers CO,~ ing two fusion proteins joined via ~ ulfi-le bonds ~ve en the Fc moieties thereof, are also provided. The term "Fc polypeptide" as used herein includes native and mutein forms of polypep~i(les derived from the Fc region of an antibody. Tl~ cal~d forms of such polypeptides co~ g the hinge region that pl~lllfi~S dilll~ ion are also included One suitable Fc polypeptide, described in PCT application WO 93/10151, is a 35 single chain polypepdde e~f.~tl;ng from the N-terminal hinge region to the nadve C-t~ of the Fc region of a human IgG1 antibody. Another useful Fc polypepdde is the Fc mutein ~J~feS~ih~ in U.S. Patent 5,457,035 and in Baum et al., (EMBO J. 13:3992-4001, 1994). The amino acid sequence of this mutein is irl~.ntir~l to that of the native Fc SC'1~J ~ e ~S~ in WO 93/10151, except that arnino acid 19 has been changed from Leu to Ala, amino acid 20 has been changed from Leu to Glu, and amino acid 22 has been ch~ng~ from Gly to Ala. The mutein .oYhibitc reduced affinity for Fc receptors.
S In other c .. bal;.. ~ , Lerk-8 may be sl~bst1t lt~ for the variable portion of an antibody heavy or light chain. If fusion proteins are made with both heavy and light chains of an antibody, it is possible to form a Lerk-8 oligomer with as many as four Lerk-8 extracellular regions.
.Alt~m~tively, the r)li~omer is a fusion protein cnmrricing mlllti,1~ Lerk-8 10 polypeptides, with or without peptide linkers (spacer ~lides). Among the suitable peptide linkers are those described in U.S. Patents 4,751,180 and 4,935,233, which are hereby incc,~ t~d by reference. A DNA sequence encoding a desired peptide linker may be inserted between, and in the same reading frarne as, the DNA sequences encoding Lerk-8, using any suitable conventional technique. For example, a ch~om~ ly syntheci7ed 15 oligonllçleotide e"coding the linker may be ligated ~.~n sequences encoding Lerk-8. In one emk~im~n~, a fusion protein co,ll~,lises from two to four soluble Lerk-8 polypepti-les, sepal~led by peptide linkers.
Another method for preparing oligomeric Lerk-8 involves use of a leucine zipper.~ etlcin~ zipper dom~inc are peptides that l)~nlole oligu~ a~ion of the proteins in which 20 they are found. T euçine zippers were ~rigin~lly identifi~l in several DNA-binding proteins (I ~n(lc~hlll7 et al., Science 240:1759, 1988), and have since been found in a variety of dirr~.~in~ proteins. Among the known leucine zippers are naturally occurring peptides and derivatives thereof that dimerize or Ll;...~ . ;7e. Examples of leucine zipper dc....~ suitable for prlYlu~ing soluble oligomeric proteins are ~Psçribed in PCT appli~tion WO 94/10308, 25 the leucine zipper derived from lung ~lrn~ t protein D (SPD) rl~s~ribe~l in Hoppe et al.
(FEBSLetters 344:191, 1994) and U.S. Patent ~Mli~tion serial no. 08/446,922, hereby ~ldt~d by reference. Recc?mhin~nt fusion proteins comrri~ing a soluble Lerk-8 polypeptide fused to a leucine zipper peptide are t;*,l~i.sed in suitable host cells, and the soluble oligomeric Lerk-8 that forms is recovered f~m the culture Sll~rn~t~nt Oligomeric Lerk-8 has the plv~ y of bivalent, trivalent, etc. binding sites for elk or hek. The above-described fusion proteins comprising Fc mrie i~S (and oligomers formed Ih~lGflvlll) offer the advantage of facile purification by affinity chromatography over Prvtein A or Protein G columns.

Expression Systems Suitable host cells for ~A~ ,s~ion of Lerk-8 polypeptides include prokaryotes, yeast or higher eukaryotic cells. A~l.lv~)liate cloning and expression vectors for use with bactcrial, fungal, yeast, and ~ n.~ n cellular hosts are ~i~SÇri~, for example, in Pouwels et al. Cloning Vectors: A I~bora~ory Maru~al, Elsevier, New York, (1985).
Cell-free translation ~.~St~ lS could also be employed to produce Lerk-8 polypeptides using RNAs derived from DNA constructs r~ nse~l herein.
S The cA~ ssion vector may include DNA enro~ling a signal or leader peptide fused to the N t~ . Illil~ll~ of a Lerk-8 poly~ e The signal or leader peptide co-tran~l~ti~n~lly or post-tr~n~l~ti~n~lly directs transfer of the Lerk-8 from its site of ~yl~lhesis to a site inside or outside of the cell 11~ or cell wall. The signal or leader peptide is cleaved from the mature Lerk-8 polypeptide. The choice of signal or leader peptide is depçn(lçnt on the type of host cell that is to be employed.
Suitable prokaryotic host cells for ~ Ç(.~ n include, for example, E. coli, ~nr~ subtilis, Salmonella typhimurium, and various other species within the genera Pseudomonas, Streptomyces, and Staphylococcus. In a prokaryotic host cell, such as E.
coli, a Lerk-8 polypeptide may include an N-terminal m.othionine residue to f~cilit~t.o e~lession of the l~c~lllb;n~nt polypeptide in the prokaryotic host cell. The N-terminal Met may be cleaved from the expressed recol.lb~ant Lerk-8 polypeptide.
Lerk-8 polypeptides may be e~ ssed in yeast host cells, preferably from the genus Saccharomyces (e.g., S. cerevisiae). Other genera of yeast, such as Pichia, K.
Iactis or Kluyveromyces, may also be employed. Yeast vectors may contain an origin of repli~ n sequence from a 2~ yeast plasmid, an autonomously replir~ting sequence (ARS), a ~ ol~,. region, sequences for polyadenylation, sequences for transcription termin~ti~n, and a sele~t~hle marker gene.
Suitable ~ tel sequences for yeast vectors include, among others, promoters for metallothion~in, 3-pho~hoglycerate kinase (I~ et al., J. Biol. Chem. 255:2073, 1980) or other glycolytic enzymes (Hess et al., J. Adv. Enzyme Reg. 7:149, 1968; and Holland et al., Biochem. 17:4900, 1978), such as enol~ce, glyceraldehyde-3-phosphate dehydrogenase, hexcl-in~e, pyruvate decarboxylase, phosphofructokin~e, glucose-6-ph~sph~ts isom~,.ase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosph~te isomerase, phosphoglucose isomerase, and glucokin~e Another ~lt~n~tive is the glucose-repressible ADH2 ~ "~olel described by Russell et al. (J. Biol. Chem. 258:2674, 1982) and Beier et al. (Nanlre 300:724, lg82). Other suitable vectors and ~lolll~tel~ for use in yeast e~ ssion are further desc rihecl in ~ An, EPA-73,657 or in Fleer et. al., Gene, 107:285-195 (1991); and van den Berg et. al., BiolTechnology, 8:135-139 (1990).
Shuttle vectors re~ le in both yeast and E. coli may be constructed by inserting DNA
se~llenees from pBR322 for selection and re~ tion in E. coli (Ampr gene and origin of replication) into the above-described yeast vectors.

A s..it~hle leader se4llence (e.g. the a-factor leader of Saccharomyces) may be e~b~cd to direct s~;le~ion of the Lerk-8 polypeptide from yeast cells. The a-factor leader sequ~nee is generally ins~ .~n the IJ,ulllbt~ se~lu~.nce and the structural genesequence. See, e.g., Kurjan et al., Cell 30:933, 1982; Bitter et al., Proc. Natl. Acad. Sci.
USA 81:5330, 1984; U. S. Patent 4,546,082; and EP 324,274. Other leader sequences suitable for facilitating secretion of l~hius~ polypep~i-les from yeast hosts are known to those of skill in the art. A leader sequence may be m Y1ifi~A near its 3' end to contain one or rnore l~ licl;on sites. This will f~ilit~ fusion of the leader se~uence to the ~lluc~ l gene.
Yeast tr~n~f~rrnation protocols are known to those of skill in the art. One suchprotocol is described by Hinnen et al., Proc. Natl. Acad. Sci. USA 75: 1929, 1978. The Hinnen et al. protocol selects for Trp+ tran~r~ in a selective m~jllm, wherein the selective ~n~li~ consists of 0.67% yeast nitrogen base, 0.5% c~mino acids, 2%
glucose, 10 llg/ml adenine and 20 llg/rnl uracil.
Yeast host cells ~ srûlmed by vectors con~ n~ ADH2 promoter sequence may be grown for inducing e~ ssion in a "rich" .~e~l;.. An exzmrle of a rich meAillm is one cnl-s~ -g of 1% yeast extract, 2% peptone, and 1% glucose suppk .. -t~ with 80 llglml a~1enine and 80 ~lg/ml uracil. De.~ ession of the ADH2 plulllol~. occurs when glucose is eYh~-lct~ from the meAillm ~l~mm~ n or insect host cell culture systems could also be employed to express l~.Col-llJ;n"nt Lerk-8 polypeptides. Baculovirus systems for production of heterologous proteins in insect cells are reviewed by Luckow and Sull~ , BiolTechnology 6:47 (1988). Established cell lines of l~ n origin also may be employed. FYzmrl~s of suitable m nm~lizn host cell lines include the COS-7 line of monkey kidney cells (ATCC
CRL 1651; Gluzman et al., Cell 23: 175, 1981), L cells, C127 cel}s, 3T3 cells (ATCC CCL
163), Chinese hz...~t. . ovary (CHO) cells, HeLa cells, the BHK (ATCC CRL 10) cell line, and the CV-l/EBNA-1 cell line (ATCC CRL 10478) derived from the African green monkey kidney cell line CV1 (ATCC CCL 70) as described by McMahan et al. (EMBO J.
10: 2821, 1991).
Tr~s~ ional and tr~n~la~ionAl control sequences for Illh~ Ali~n host cell e~ssion vectors may be excised from viral genomes. Commonly used ~r~lllot~l s~uences and enh~nce~ sequences are derived from Polyoma virus, Adenovirus 2, Simian Virus 40 (SV40), and human cytomegalovirus. DNA sequences derived from the SV40 viral genome, for e.c~mple, SV40 origin, early and late pl~lllotel, enhancer, splice, and polyadenylation sites may be used to provide other genetic Plf n~ for expression of a ~llu~ilul~l gene sequence in a n~ n host cell. Vi~al early and late plonlot~ls are particularly useful because both are easily obtained from a viral genome as a fragment .

which may also contain a viral origin of ~ n (Fiers et al., Nature 273:113, 1978).
Smaller or larger SV40 L~C~ .n!j may also be used, provided the a~ uA;~ lely 250 bp 5~ re ~ l;ng from the Hind III site toward the Bgl I site located in the SV40 viral origin of .~,~lic&lion site is i~ ud~d S Examples ûf eA~I~ssion vectors for use in .. ----~ n host cells are those cûr.;,hu~t~d as rlisclose~l by Okayama and Berg (Mol. Cell. Biol. 3:280, 1983). A useful system for stable high level ~;Apl~s~ion of ..~ n cDNAs in C127 murine ~
~ iPl cells can be constructed su~ lly as ries~ihe~ by Cosman et al. (Mol.
Immunol. 23:935, 1986). A useful high eA~I~,ssion vector, PMLSV N1/N4, ~lescnbe~l by Co~m~n a al., Nature 312:768, 1984 has been depo~ir~ as ATCC 39890. Other ~;A~I~s~;on vectors suitable for use in ~ n host cells are pDC201 (Sims et al., Science 241:585, 1988), pDC302 (Mûsley et al., Cel~, 59:335, 1989), pDC406 (McMahan et al., EMBO J. 10:2821, 1991), HAV-EO (Dower et al., J. I~nunol. 142:4314, 1989), and the vectors described in EP-A-0367566 and WO 91/18982. Further alternatives are 15 vectors derived fro n retroviruses.
In place of the native signal sequence, a heterologous signal sequlence may be added, such as the signal sequence for IL-7 described in United States Patent 4,965,195;
the signal sequence for lL-2 receptor described in Cosman et al., Nature 312:768 (1984);
the IL-4 ~~,ceplc signal peptide described in EP 367,566; the type I IL-1 receptor signal 20 peptide described in U.S. Patent 4,968,607; and the type II IL-1 receptor signal peptide described in EP 460,846.

Lerk-8 Protein pl~rifif ~til~n Lerk-8 polypeptides of the present invention may be produced by recrmhin~nt 25 e~ ssion systems as described above, or purified from naturally occurring cells. One process for producing Lerk-8 comprises culturing a host cell transformed with ane~ ,ssion vector comprising a I)NA sequ~-nce that encodçs Lerk-8 under ccn-litionc ..rr.. ie"l to plul.-ole expression of Lerk-8. Lerk-8 is then recovered from the culture l~. A;l-... or cell extracts, depen-ling upon the ~y.~,ssion system employed and whether the 30 Lerk-8 is secreted from the cells. In one em~lim~n~, a human Lerk-8 protein comprises the amino acid sequence of the protein that is e~pl~ssed by host cells transformed with an eAyl~ssion vector con~ining the Lerk-8 cDNA found in strain ATCC 97441.
As is known to the skilled artisan, procedures for purifying a reconlbil~ protein will vary according to such factors as the type of host cells employed and whether or not 3~ the ~o.~ protein is secreted into the culture me~ m Other conci<1erations include the types of co~ that are to be removed, which may vary acco~ g to the particular host cells employed to express the desired protein.

Por e~ le, when e~pl~ssion ~ s that secrete the lcco~b;nA~ protein are c,u~lo.,cd, the culture medium first may be co~c~ t~ using a cc~ .. ially available protein cc-~c~ ;on filter, for e~cA-.~l le, an Amicon or Millipore Pellicon ultrafiltration unit. Following the conce~ .Lion step, the co,-c~ ate can be applied to a pl~nficqtion S ma~ix such as a gel filtration matrix. Alt4l..a~i~,ely, an anion e~ell~ ~g,. resin can be e..~ cd, for e~.n.ple, a matrix or ~ t~ having pendant diethy~ ;n~lllyl (DEAE) groups. The l..--hi~s can be acryl- mi~ie, agarose, deYtrq-n, c~ 10se or other support m,qteriql~ cu.l~....only employed in protein p~lrifi.-qtion~ ~lt~mA~ively, a cation exchqnge step c. n be employed. Sl~ cation e---h-~E.r.~ include various in~oluble ~ ;CeS
10 COI~ e sul~op-ul)yl or c.ul~c~u~ yl groups. Sulr~lu~yl groups are ~fell~d. Inq.~ itiol-, one or more .~ phase high P~ C liquid Cbl~J~ IO~Ia~hY (RP-HPLC) steps employing hydrophobic RP-HPLC media, (e.g., silica gel having ~~ t methyl or other ~,lirhAtir groups) can be employed. Some or all of the fc,.~going purification steps, in various combinations, may be employed to provide a purified Lerk-8 15 protein.
A further alternative is affinity cluu.llatoE,-dphy, employing a cL,u.naLographymatrix co~ ;..g hek, elk, or an antibody that binds Lerk-8. The Lerk-8 polypeptides can be recovered from an affinity column using conv~l.l;ol-Al techniques, (e.g., elution in a high salt buffer), then dialyzed into a lower salt buffer for use.
Recc~ nt protein produced in b~AtPriAl culture can be i~olAItt~A by initial disruption of the host cells, centrifugation, extraction from cell pellets if an in~o~ e polypeptide, or from the supçrn~A~t~A~nt fluid if a soluble polypeptide, followed by one or more conc~ Lion, salting-out, ion eYchAnge, affinity puriflr-Ati~n or size exclusion el,-u..~tography steps. Finally, RP-HPLC can be employed for final pmifir,tion steps.
Microbial cells can be disrupted by any convenient method, inrlll~1ing freeze-thaw cycling, soni( A~tioJl~ mPçh~Anic~l disruption, or use of cell lysing agents.
In yeast host cells, Lerk-8 is preferably e~ cssed as a secreted polypeptide, tosimplify purification. Rec~ h;n7 n~ polypeptides secreted from a yeast host cellf~ ;on can be purified by me~ho ls analogous to those disclosed by Urdal et al. (J.
Chrornatog. 296:171, 1984). Urdal et al. describe two sequential, reversed-phase HPLC
steps for p~lrifir~tiQn of recombin~nt human IL-2 on a p~ ti~e HPLC column.
The desired degree of purity depends on the intPn-led use of the protein. A
relatively high degree of purity is desired when the protein is to be a~ h, ~;d in vivo, for ex~mple. Advantageously, Lerk-8 polypeptides are purified such that no protein bands 35 co~;",onding to other (non-Lerk-8) proteins are ~iPtpct~l= upon analysis by SDS-poly~lamide gel electrophoresis (SDS-PAGE). It will be recognized by one skilled in the ~~ enl field that multiple bands cû~ onding to Lerk-8 protein may be vim~li7-PA by .... . . .. ..

SDS-PAGE, due to ~ ..,n~al glycosylation, diLr~n~al post-tPn~l~tinn~ >ces..;..g,and the like, as ~ cll~se~ above. Lerk-8 most preferably is purified to s~lb~
ho....~ e ly, as i~ t~ by a single protein band upon analysis by SDS-PAGE. The protein band may be vic~li7~l by silver st~ining~ Coomassie blue staining, or (if the 5 protein is radiolabeled) by autoradiography.

Nucleic ~ and Uses Thereof The present ill~.l~ion provides isolated Lerk-8 nucleic acids useful in the p,~l.l~;~;on of Lerk-8 poly~p!i~les, as ~iiccll~s~l above. Such nucleic acids ine~ le, but are 10 not limited to, the human Lerk-8 DNA of SEQ ID NO:1, in both single-stranded and double-stranded form, as well as the RNA complernent thereof. Lerk-8 DNA of the present i~vell~ion includes, for ex~mrlç7 cDNA, genomic DNA, chemically synth~ i7f~
DNA, DNA ~mplifiç~l by PCR, and comhin~tior-~ thereof. Genomic DNA may be isolated by convçntion~l lech~ ues using the cDNA isolated in ~ le 1, or a suitable fragment 15 thereof, as a probe.
Particular e...ho~ nl~ of Lerk-8-enro~lin~ DNAs include a DNA co..~ -g ~ el~o~ s 398 to 1420 of SEQ ID NO:1 (fncoding full length human Lerk-8, inrl~l~ling the N-~....: .~l signal peptide) and a DNA con~l;sillg nucleotides 479 to 1420 of SEQ ID
NO:l (en~o~ing full length mature human Lerk-8). Particular embo~l;..u .l!i of DNA
en~f~ g a soluble human Lerk-8 are a DNA comprising nucleotides 398 to 1069 of SEQ
ID NO:l (c nco.l;.~g the signal peptide and extr~ce!l~ domain) or comrri.~ing nucleotides 479 to 1069 of SEQ ID NO: 1 (encoding the extln~çll..l~r domain).
The present invention further provides fragments of Lerk-8 l~ucle~lide sequences.
Such fr~grn-ont~ desirably comprise at least about 17 contiguous nurleotiflf s of a Lerk-8 25 DNA sequence, e.g., at least 17 c~nsec~ltive nucleotides of the human Lerk-8 s~uence ~l~sent~ in SEQ ID NO:l. DNA and RNA conlplç...en of said fragrnl~nt~ are provided herein, along with both single-stranded and double-stranded forms of the Lerk-8 DNA.
Arnong the uses of such Lerk-8 nucleic acid fra~m~nt~ is use as a probe. Such probes may be employed in cross-species hybri-li7~tion procedures to isolate Lerk-8 DNA
30 from i~dil;l;o~l ",-- ""~ n species. As one e~ le, a probe cc,l~ onding to the extr~e~ r domain of a Lerk-8 may be employed. The probes also find use in detecting the presence of Lerk-8 nucleic acids in in vitro assays and in such procedures as Northern and Southern blots. Cell types ~ ssing Lerk-8 can be identifierl Such procedures are well known, and the skilled artisan can choose a probe of suitable length, depending on the 35 particular int~onrled application. In particular embo-lim~ntc, Lerk-8 nucleic acid mnlecllles c. mrrise at least 30 contiguous nucleotides of the DNA sequence of SEQ ID NO: 1, or the DNA or RNA complement thereof. The probes may be labeled (e.g., with 32p) by co~ 1 tccl~ ucs.
8 nucleic acid r~a~ cnt~ also find use as ~ , e.g., in poly~nerase chain C~ (PCR). S' and 3' p~ els coll.,~nding to the termini of a desired Lerk-8 DNA
S (e.g., a DNA en~ g a soluble Lerk-8) are e.lllJlo~,d in i~olq~ing and ~~ yi~lg the DNA, using con~e .~ gl PCR techniqu~,s Other useful r.~.D..t~ of the Lerk-8 nucleic acids include qnti~en~e or sense cs cc....~ g a single-stranded nucleic acid ~u~,~ce (either RNA or DNA) capable of binding to target Lerk-8 mRNA (sense) or Lerk-8 DNA (~nti~-n~e) sequences.
0 ~nt;~l~G or sense oligQnllcl~otides, acc~ di~lg to the present invention, conlr i~e a r.,~g.~ t of the coding region of Lerk-8 cDNA. Such a fragment generally comprises at least about 14 nucleotides, preferably from about 14 to about 30 nucleotides. The ability to derive an ~ ;cen~e or a sense oligonucleotide, based upon a cDNA sequence encoding a given protein is described in, for ex~rnrle, Stein and Cohen (Cancer Res. 48:2659, 1988) and van der Krol et al. (BioTechniques 6:958, 1988).
Binding of ~ntisçn~e or sense oligonuc!eotides to target nucleic acid sequences results in the fc~lmaLion of duplexes that block Lla-~sclil,Lion or tr~n~l~tion of the target sçquenre by one of several means, inclnfling enh~n~ed degradation of the dnrleYes, Ul~ ~lllf,naLiol- of transcription or translation, or by other means. The ~nti~çn~e 20 oligonucleotides thus may be used to block e~iession of Lerk-8 proteins. .Anti~çn~e or sense oligonucleotides further comprise oligonucleotides having modified sugar-phospho~liest~r backbones (or other sugar linkages, such as those described in WO91/06629) and wherein such sugar link~ges are resistant to endogenous nucleases.
Such oligo.~.~cle~l;des with resistant sugar link~g~s are stable in vivo (i.e., capable of 25 resisting enzymatic degradation) but retain sequence specificity to be able to bind to target nuc1eotirle sequences.
Other examples of sense or ~nti~nce oligonucl~oti~1es include those o~igon-lc leoti-les which are covalently linked to organic moieties, such as those described in WO 90/10448, and other moieties that increases affinity of the oligonucleotide for a target 30 nucleic acid sequence, such as poly-L-lysine). Further still, interc~l~ting agents, such as çlliph--ine, and aLIcylating agents or metal complexes may be al~l ed to sense or ~n~i~e~n~e oligonucleoti~les to modify binding specificitiec of the antisense or sense oligonucleotide for the target nucleotide seguence.
~ çQ~e or sense oligonucleotides may be introduced into a cell cont~inin~ the 35 target nucleic acid sequence by any gene transfer method, including, for example, CaPO4-mediated DNA transfection, elecllv~la~ion, or by using gene ll~n~r~,l vectors such as Epstein-Barr virus. In a ~Ic~ ,d procedure, an ~ntisen~e or sense oligonucleotide is WO 97/36919 rCTtUS97t04533 .,-~.~d into a suitable retroviral vector. A oell co~ ;..;ng the target nucleic acid S¢~U~ CC
is contacted with the l~..,lhi.~A-.î retroviral vector, either in vivo or ex vivo. Suitable retroviral vectors inrlufle~ but are not limited to, those derived from the murine retrovirus M-MuLV, N2 (a 1el1O~US derived from M-MuLV), or the double copy vectors desi~n~t~?A
S DCISA, DCTSB and DCT5C (see WO 90/13641).
Sense or Anti~nce oligonlle~ ,s also may be i~ Aluced into a ccll CO~lA~ing the target l.~lcolide s~ n~e by l¢, ... ~ of a conjugate with a ligand binding molecule, as ~Ic,~" il~d in WO 91/04753. Suitable ligand binding -'-cl~lP-s in~lu¢lp~ but are not limited to, cell surface receptors, growth factors, other cytokines, or other ligands that bind to cell surface l~ce~ . Preferably, conjugation of the ligand binding -'e !llP does not _L~t~tislly interfere with the ability of the ligand binding '~:~le to bind to its cc~ .ponding lecule or receptor, or block entry of the sense or ~nti~nse oligonl~¢~ idc or its conjugated version into the cell.
Alternatively, a sense or an ~nti~en~e olig~ lc~.o~;-le may be introduced into a cell co.~-inin~ the target nucleic acid se~u¢nce by formation of an oligonucleotide-lipid conlrl~Y~ as d~s~libed in WO 90/10448. The sense or ~nti~en~e oligonucleotide-lipid complex is preferably disso~,iated within the cell by an endogenous lipase.

At~libodies .Antiho 1i~s that are ;~ v.~a~;~i.re with Lerk-8 polypeptides are provided herein.
Such antibodies ~e¢.ific~lly bind Lerk-8, in that the antibodies bind to Lerk-8 via the antigen-binding sites of the antibody (as opposed to non-specific binding).
Polyclonal and monoclonal antibodies may be ~el,~ucd by conventional techniques.See, for example, Monoclonal Antibodies, ~Iybridomas: A New Dimension in Biological Analyses, Kennet et al. (eds.), Plenum Press, New York (1980); and Antibodies: ALoboratory Manual, Harlow and Land (eds.), Cold Spring Harbor Labc~lat(,ly Press, Cold Spring Harbor, NY, (1988). Production of mon~ clonal antibodies directed against Lerk-8 is fur~er illu~tldted in r~ e 3.
Antigen-binding fr~r tc of such antibodies, which may be produced by 30 convel;o--Al tPçhni~ues, are also encoll~p~cse~3 by the present invention. Examples of such r.ilg"~ include, but are not limited to, Fab, F(ab'), and F(ab')2 frgmçntc Antibody fr.q.~mrnt~ and derivatives produced by genetic çn~h-f~ . ;ng techniques are also provided.
The mnnoclnnql ntibalies of the present invention include cl~;ll,...;r antibodies, e.g., I.J ..~n;;f~ versions of murine monoc~nAl antibodies. Such h~....AI~i7e~1 antibodies - 35 may be ~,cp~ed by known techniques, and offer the advantage of reduced immlmogenicity when the qnt~ ies are ~l1mini~trred to humans. In one embolimr.lt a i-...nAni7~
~mxll~nAI antibody comprises the variable region of a murine antibody (or just the antigen WO 97t36919 PCTtUS97/04533 binding site thereof) and a col.s~ region derived from a human antibody. ~lt~,rn~tively, a l.---.. .;.d antibody fragment rnay co..~ e the antigen binding site of a murinemonoclc-n~l antibody and a variable region fragment (Lacking the antigen-binding site) derived from a human antibody. ~cdul.,s for the ~ el;on of cl,;..~ .;c and further S e~ noclo~ ~;es include those desc~iheA in 12iec~ et al. (Nature 332:323, 1988), Liu et al. (PNAS 84:3439, 1987), LalTick et al. (Bio/Techno~ogy 7:934, 1989), and Winter and Harris (TIPS 14: 139, May, 1993).
Among the uses of the antibodies are use in assays to detect the ~ sel ce of Lerk-8 polypepti~les, either in vitro or in vivo. The ~ ;es also may be employed in ~ufirying 10 Lerk-8 proteins by immlmo~rr~ y chrc ~--~o~rhy.
Those antibodies that ~ ;n~lly can block binding of Lerk-8 to l~cepu.l~ (e.g., elk or hek) may be used to inhibit a biological activity .~ ed by the binding of Lerk-8 to the l~pl~,.s. Such an antibody may be employed in an in vitro procedure, or a~lmini~tered in vivo to inhibit a Lerk-8-m~ te~ biological activity. Disorders m~oAi~terl or exd~lb~.L~d 15 (directly or indirectly) by the binding of Lerk-8 to cell surface receptors are thus treated.
Pha~ ~eutic~l coml)o~itions co~ -g an antibody that is directed against Lerk-8, and a suit~ble, diluent, excipient, or carrier, are provided herein. Suitable cc,.lll)onenls of such compositions are as described above for ccnnposiLions containing Lerk-8 proteins.
Also provided herein are conjugates comprising a detect~ (e.g., diagnostic) or 20 the~d~;uLic agent, ~tt ~hed to an antibody directed against Lerk-8. Ex~mples of such agents are pl~,s~nted above. The conjugates find use in in vitro or in vivo procedures.The following examples are provided to further illustrate particular embo~limen~ of the invention, and are not to be construed as limiting the scope of the present invention.

FXAMPI,F 1: (~lo~i-u of H--mqn l ~rl~-8 ~I)NA
A cDNA çn-~otling a human Lerk-8 of the present invention was i~ol~ted by the following procedure. A search of the GenBanlc sequ-,nce ~t~h~nk (tfasta), using amino acid sequences of Lerk-2 and Lerk-5 as search terms, ~ ntifi~d an EST (~CeS5;C!n no.
H10006) eYhibitin~ ~ignific~nt homology. Using the reading frames of Lerks 2 and 5 as 30 guides (to adjust for frarne shifts and stop codons that would result from the insertions and deletions in the EST, cclllpd,ed to the Lerk-2 and Lerk-5 sequences), a translate of the EST
was eluc~ te~l ~lignm~nt of this translate of the EST with Lerk-2 and Lerk-5 amino acid sequences revealed se~ ence identity of about 50% with both Lerk-2 and Lerk-5, in the overlapping regions. The second, third, and fourth cysteines that are conserved in Lerks 1-35 7 were identifi~d in the EST translate.
Oligonucleotides based on the EST were synthe~i7~A for use as 5' and 3' primers ina polymerase chain reaction (PCR). The primers defined the termini of a l lObp internal CA 02248550 l998-09-l4 fragment of the EST. DNA from human cDNA li~ i.os in a phage A vector was used as the ~ , in the PCR DNA frqgn~nt~ of the e~l~e~t~ size (110bp) were qnlrlifie~A from three of the cDNA librqries~ which were derived from fetal brain, dermal fibroblast, and l,anc,ealic turnor.
The same two ol;~ les used as primers in the PCR were end labeled with 32p for use as probes. The human dermal fibroblast cDNA library was s~ ,ene~d with the probes by allowing hyl";-l;7~ ?n at 63~C, followed by washing at 63~C in lx SSC. One clone, de~igrqt~A A1 was i~olq~ted The coding region of this clone c~ sl,onA~ to l.ucle{?l;~Aes 500 to 1420 of SEQ lD NO:1, which encode amino acids 8 through 313 of SEQ II) NO:2.
A L,~ of this clone was ~mrlified by PCR, l~~-le~ and used as a probe in s.;l~eni~g the hurnan fetal brain cDNA library (hybri~li7~tion at 63~C, followed by washing at 63~C in lx SSC). Three hybridizing clones were isolated, and the DNA sequences were ~le t~ -- ...il~ One clone, desi~n;~-ed A2, included a full length coding region.
The nucleo~i~e se~uen~e of the hurnan Lerk-8 cDNA of clone ~2, and the amino acid se4~ ce el-co~l~ thereby, are pl~esenled in SEQ ID NO:l and SEQ ID NO:2, l~i,~;~i./ely. The human Lerk-8 protein of SEQ ID NO:2 compri~es an N-terminal signal peptide (amino acids -27 to -1), an e~c~cçll~ r domain (amino acids 1 to 197), ae region (arnino acids 198 to 224), and a cytoplasmic domain (amino acids 225 to 313).
S~mples of a cell Iysate con~ining a recombinant phage vector (~gtlO cont~ining the human Lerk-8 cDNA of clone ~2 inserted into the EcoRI restriction site of the vector) were depos;lc~l with the ~ . Type Culture Collection, Rockville, Maryland. The s~nples were deposited on February 14, 1996, under the terms of the Budapest Treaty, and were ~si~n5r1 ~cession nu.n~l ATCC 97441.

FXAMPl F 2: Rir~ tu~y The binding of Lerk-8 to elk or hek can be :~csessed in any convf ~.lio,~l binding assay. One suitable procedure is as follows.
A DNA and çncorl~d amino acid sequçnce for rat elk cDNA is disclosed in Lhotak et al. (Mol. Cell. Biol. 11:2496, 1991), hereby incoll)ol~led by reference. The rat elk protein has a S38 amino acid extr~ellul~r domain, a 25 amino acid tr~n~ - . Ibl ~e domain, and a 419 amino acid cytoplasmic dom~in A DNA and enrode~l amino acid se~uence for human hek cDNA is plc;sellled in Wicks et al. (Proc. Natl. Acad. Sci. USA, 89:1611, 1992), incc,~ ed herein by ler~ "ce. This hek protein comprises (from N- to C-terminus) a 521 amino acid extracellular ~omqin~ a 24 amino acid ~ k.a~e rlomqin, and a 418 amino acid ic domqin.
I~Fc-..nhit--..l soluble elk/Fc and he~/Fc fusion prot~eins are ~a~cd by any suitable " e.g., as d~ ed in PCT al~plic~ - WO 96/01839, hereby incc,ll,u,~d by S lc,f~l~ce. The elk/Fc and hek/Fc fusion ~ ins are pllrifi~ by affinity chromatography, using a protein A se~h..lu~ col-.mn.
Cells e~l~i,sl.~g ,~..lh~ nt Le.rk-8 on the cell surface are IJl~alcd. Lerk-8 DNA
maybeal~q~l;rl~byPCR. The~ employedinthePCR are s~lectP~l to define the termini of the coding region of the Le.rk-8 DNA, and also add an Xho I restriction site at the 10 5' end and a Not I site at the 3' end of the ~mrlifie~l DNA.
The PCR reaction plc~duc~ are Aig~s~ with Xho I and Not I and inserted into an c~ s~ioll vector cleaved with Sal I (which is co-npqti~1c with Xho I) and Not I. The expression vector, designated pDC410, is a .. ;.. ~ c~ s~ion vector that also replicates in E. coli, and is similar to pDC406 (McMahan et al., EMBO J. 10:2821, 1991).
The pDC410 mlll, '~ cloning site (mcs) differs from that of pDC406 in that it conlains a~ ;o~ul reshi~ n sites and three stop codons (one in each reading frame). A T7 pol~ ~e ~lu~ ~. dowl,sllealll of the mcs f~ it~tes se~uencing of DNA inserted into the mcs. In a~ tion~ the EBV origin of le~licalion is replaced by DNA encoding the SV40 large T antigen (driven from an SV40 promoter) in pDC410.
CV1-EBNA-1 cells in 10 cm2 dishes are llan:>çe~iled with the l~c,.. ki.~fll~le~ ,ssion vector co~ ing Lerk-8 DNA. The CV-1/EBNA-1 cell line (ATCC CRL
10478) cc.l.~ ely eAplesses EBV nuclear antigen-l driven from the CMV ;~n..lf.l;~t~o-early e-~hA~e. /~ bt~,r. CVl-EBNA-1 was derived from the African Green Monkey kidney cell line CV-l (ATCC CCL 70), as deswibed by McMahan et al. (EMBO J.
25 10:2821, 1991).
The ~ r~t~,d cells are cultured for 24 hours, and the cells in each dish then are sp~it into a 24-well plate. After culhlring an nd~litinn~l 48 hours, the transfected cells (about 4 x 104 cells/well) are washed with BM-NFDM, which is binding ..~l;,.... (RPMI 1640 conl~ 25 mg/ml bovine serum albumin, 2 mg/ml sodium azide, 20 mM Hepes pH 7.2) 30 to which 50 mglml nonfat dry milk has been added. The cells then are inr~u~terl for 1 hour at 37-C with various cQr~centrations of the above-clesç~ibe~ eL~/Fc fusion protein or hek/Fc fusion protdn. Cells then are washed and incubated with a constant salwaling concentTation of a 125I-mouse anti-human IgG in binding Ill~]iwll, with gentle ~gjt~hon for 1 hour at 37-C. After extensive washing, cells are released via trypsinization.
The mouse anti-human IgG employed above is directed against the Fc region of human IgG and can be obtained from Jackson ~n~ nu~se~;h Labolal~lies7 Inc., WestGrove, PA. The antibody is radioic~inate~ using the standard chloramine-T method. The antibody will bind to the Fc portion of any elk/Fc or hek/Fc fusion protein that has bound to the cells. In all assays, non-s~e~;r~c binding of 125I-anLibo.ly is assayed in the absence of elk/Fc (or hek/Fc), as well as in the p.~,sence of elk/Fc (or hek/Fc) and a 200-fold molar excess of l~nl~beled mouse anti-human IgG antibody.
Cell-bound 125I " ltibody iS quantified on a Pa~ i Aut~ count~". Affinity calculations (.Sc~ .1, Ann. N.Y. Acad. Sci. S1:660, 1949) are generated on RS/1 (BBN
Software, RQston, MA) run on a Microvax c~ ,ute F.XAMPl,~ ~: M~L~ ~ I,~rl~.~
This example illustrates a method for plGpalil~g .~.o~ ntibo~1ie~ that bind Lerk-8. S~;t~le j""~ O~ c that may be employed in g_-~w~ing such ~ntiwies include, but are not limited to, purified Lerk-8 protein or an ;.".. nr)g~ni~ fragment thereof such as the extr~ce~ r domain, or fusion proteins co..~ ;..g Lerk-8 (e.g., a soluble Lerk-8tFc fusion protein).
Purified Lerk-8 can be used to g~ ~ monoclonal antibodies ;.. ~-c.l~active using conventional techniques such as those described in U.S. Patent 4,411,993. Briefly, mice are in~ ;7.d with Lerk-8 imm..nogen e.mnlcifie~ in complete Freund's adjuvant, and inje~t~1 in amou~lls ranging from 10-100 ~lg subcu~neoucly or inllalk~;loO~ y. Ten to twelve days later, the immllni7~ animals are boosted with 20 ~1~ 1 Lerk-8 em~llcifie~l in incomplete Freund's adjuvant. Mice are p~rinrlicAlly bo~st~d Ih~ ~t~,. on a weekly to bi-weekly ;."",...~;,,.tinn sch~PAI-lP Serum sAm~les are p - ;Orli(~ally tA~en by retro-orbital ble~lin~ or tail-tip exci~ion to test for Lerk-8 AntiboAi~s by dot blot assay, ELISA (Enzyme-Linked ~mmllnosorbent Assay) or inhibition of hek or elk binAin~
Following Aetection of an a~ yl;ate antibody titer, positive ~nim~lc are provided one last intravenous inje~tion of Lerk-8 in saline. Three to four days later, the AnimAls are ificeA spleen cells harvested, and spleen cells are fused to a murine myeloma cell line, e.g., NS1 orpreferably P3x63Ag8.653 (ATCC CRL 1580). Fusions ~ne~le hybridom cells, which are plated in mllltiple microtiter plates in a HAT (hypoxAnthine, al-~ino~t~
30 and thymidine) selective ~le~ n~ to inhibit proliferation of non-fused cells, myeloma hybrids, and spleen cell hybrids.
The hy~rirlomq cel~s are sc,l~ned by ELISA for reactivity against purified Lerk-8 by r~rtq~tions of the techniques disclosed in Engvall et al., Irnmunochem. 8:871, 1971 and in U.S. Patent 4,703,004. A pl~ ,d screening technique is the antibody capture 35 techni~lue described in Bec~ n et al., (J. ~mmunol. 144:4212, 1990) Positive hyl,.;dc....A cells can be injected in~ o,~FAlly into syngeneic BALB/c mice to produce ascites cm.~ g high concen~ ons of anti-Lerk-8 monoclonal antibodies. Alternatively, ...... .....

CA 02248550 l998-09-l4 h~,;.10..,~ cells can be grown in vitro in flasks or roller bottles by various techniques.
~ol-oclo~ antibodies produced in mouse ascites can be purified by ~ ;.. sulfate n, followed by gel exclusion ch~ alography. ~lt~ hvely, affinity clu.~ ~graphy based upon binding of antibody to Protein A or Protein G can also be S used, as can affinity .,h.~....st~.~,hy based upon binding to Le~-8.

CA 02248~0 1998-09-14 SEQUENCE LISTING

~1) GENERAL INFORMATION:
~i) APPLICANT: Cerretti, Douglas P.
(ii) TITLE OF INVENTION: Cytokine Designated Lerk-8 (iii) NUMBER OF SEQUENCES: 3 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Kathryn A. Anderson, Immunex Corporation (B) STREET: 51 Univer~ity Street (C) CITY: Seattle (D) STATE: WA
(E) COUNTRY: USA
(F) ZIP: 98101 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: Apple Power Macintosh (C) OPERATING SYSTEM: Apple Operating System 7.5.3 (D) SOFTWARE: Microsoft Word for Power Macintosh 6Ø1 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: --to be assigned--(B) FILING DATE: l9-MAR-1997 (C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Anderson, Kathryn A.
(B) REGISTRATION NUMBER: 32,172 tC) REFERENCE/DOCKET NUMBER: 2839-WO
~ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (206) 587-0430 (B) TELEFAX: (206) 233-0644 (C) TELEX: 756822 (2) INFORMATION FOR SEQ ID NO:l:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1708 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO

(vii) IMMEDIATE SOURCE:
- (B) CLONE: huLerk8 CA 02248~0 l998-09-l4 WO 97/36919 PCT~US97/04533 (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 398..1420 (ix) FEATURE:
(A) NAME/REY: mat_peptide (B) LOCATION: 479..1417 (ix~ FEATURE:
(A) NAME/KEY: ~ig_peptide (B) LOCATION: 398...478 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:

CAGTCCCCCA CTTAGGCGGG CT~.ACAGATC CCGGGGTGCT GGCGCGTGGG CCGGGGGCGC 180 Met Gly Pro Pro His Ser Gly Pro Gly Gly Val Arg Val Gly Ala Leu Leu Leu Leu Gly Val Leu Gly Leu Val Ser Gly Leu Ser Leu Glu Pro Val Tyr Trp Asn Ser Ala Asn Lys Arg Phe Gln Ala Glu Gly Gly Tyr Val Leu Tyr Pro Gln Ile Gly Asp Arg Leu Asp Leu Leu Cys Pro Arg Ala Arg Pro Pro Gly Pro His Ser Ser Pro Asn Tyr Glu Phe Tyr Lys Leu Tyr Leu Val Gly Gly Ala Gln Gly Arg Arg Cys Glu Ala Pro Pro Ala Pro Asn Leu Leu Leu - Thr Cys Asp Arg Pro Asp Leu Asp Leu Arg Phe Thr Ile Lys Phe Gln CA 02248~0 l998-09-l4 W O 97/36919 PCTrUS97/04533 Glu Tyr Ser Pro Asn Leu Trp Gly His Glu Phe Arg Ser His His Asp Tyr Tyr Ile Ile Ala Thr Ser Asp Gly Thr Arg Glu Gly Leu Glu Ser Leu Gln Gly Gly Val Cys Leu Thr Arg Gly Met Lys Val Leu Leu Arg Val Gly Gln Ser Pro Arg Gly Gly Ala Val Pro Arg Lys Pro Val Ser Glu Met PrO Met Glu Arg Asp Arg Gly Ala Ala His Ser Leu Glu Pro Gly Lys Glu Asn Leu Pro Gly Asp Pro Thr Ser Asn Ala Thr Ser Arg Gly Ala Glu Gly Pro Leu Pro Pro Pro Ser Met Pro Ala Val Ala Gly Ala Ala Gly Gly Leu Ala Leu Leu Leu Leu Gly Val Ala Gly Ala Gly Gly Ala Met Cys Trp Arg Arg Arg Arg Ala Lys Pro Ser Glu Ser Arg His Pro Gly Pro Gly Ser Phe Gly Arg Gly Gly Ser Leu.Gly Leu Gly Gly Gly Gly Gly Met Gly Pro Arg Glu Ala Glu Pro Gly Glu Leu Gly Ile Ala Leu Arg Gly Gly Gly Ala Ala Asp Pro Pro Phe Cys Pro His Tyr Glu Lys Val Ser Gly Asp Tyr Gly His Pro Val Tyr Ile Val Gln Asp Gly Pro Pro Gln Ser Pro Pro Asn Ile Tyr Tyr Lys Val *

TTCClGG1lrl GAGGGACACC TCTAACATCT CGGCCCCCTG TGCCCCCCCA GCCCCTTCAC 1540 CA 02248~0 1998-09-14 W O97/36919 PCT~US97tO4533 TCCTCCCGGC TG~l~lCClC GTCTCCACTT TTAGGATTCC TTAGGATTCC CACTGCCCCA 1600 .-CClGCCC TCCCGTTTGG CCATGGGTGC CCCCCTCTGT CTCAGTGTCC CTGGATCCTT 1660 ~CC~rGGG GAGGGGCACA GGCTCAGCCT CCTCTCTGAC CATGCCGG 1708 ~2) INFORMATION FOR SEQ ID NO:2:
~i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 341 amino acids ~B) TYPE: amino acid ~D) TOPOLOGY: linear ~ii), MOLECULE TYPE: protein txi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Met Gly Pro Pro His Ser Gly Pro Gly Gly Val Arg Val Gly Ala Leu Leu Leu Leu Gly Val Leu Gly Leu Val Ser Gly Leu Ser Leu Glu Pro Val Tyr Trp Asn Ser Ala Asn Lys Arg Phe Gln Ala Glu Gly Gly Tyr Val Leu Tyr Pro Gln Ile Gly Asp Arg Leu Asp Leu Leu Cys Pro Arg Ala Arg Pro Pro Gly Pro His Ser Ser Pro Asn Tyr Glu Phe Tyr Lys Leu Tyr Leu Val Gly Gly Ala Gln Gly Arg Arg Cys Glu Ala Pro Pro Ala Pro Asn Leu Leu Leu Thr Cys Asp Arg Pro Asp Leu Asp Leu Arg Phe Thr Ile Lys Phe Gln Glu Tyr Ser Pro Asn Leu Trp Gly His Glu Phe Arg Ser His His Asp Tyr Tyr Ile Ile Ala Thr Ser Asp Gly Thr Arg Glu Gly Leu Glu Ser Leu Gln Gly Gly Val Cys Leu Thr Arg Gly Met Lys Val Leu Leu Arg Val Gly Gln Ser Pro Arg Gly Gly Ala Val }35 140 145 Pro Arg Lys Pro Val Ser Glu Met Pro Met Glu Arg Asp Arg Gly Ala Ala His Ser Leu Glu Pro Gly Lys Glu Asn Leu Pro Gly Asp Pro Thr Ser Asn Ala Thr Ser Arg Gly Ala Glu Gly Pro Leu Pro Pro Pro Ser CA 02248~0 1998-09-14 W O 97/36919 PCTrUS97/04533 Met Pro Ala Val Ala Gly Ala Ala Gly Gly Leu Ala Leu Leu Leu Leu Gly Val Ala Gly Ala Gly Gly Ala Met Cys Trp Arg Arg Arg Arg Ala Lys Pro Ser Glu Ser Arg His Pro Gly Pro Gly Ser Phe Gly Arg Gly Gly Ser Leu Gly Leu Gly Gly Gly Gly Gly Met Gly Pro Arg Glu Ala Glu Pro Gly Glu Leu Gly Ile Ala Leu Arg Gly Gly Gly Ala Ala Asp Pro Pro Phe Cys Pro His Tyr Glu Lys Val Ser Gly Asp Tyr Gly His Pro Val Tyr Ile Val Gln Asp Gly Pro Pro Gln Ser Pro Pro Asn Ile Tyr Tyr Lys Val *

(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO

(vii) IMMEDIATE SOURCE:
(B) CLONE: FLAG peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
Asp Tyr Lys Asp Asp Asp Asp Lys

Claims (27)

What is claimed is:

1. An isolated DNA encoding a Lerk-8 polypeptide that binds hek or elk, wherein said Lerk-8 polypeptide comprises an amino acid sequence that is at least 80% identical to a sequence selected from the group consisting of residues -27 to 313 of SEQ ID
NO:2 and residues 1 to 313 of SEQ ID NO:2.

2. A DNA of claim 1, wherein said Lerk-8 polypeptide comprises an amino acid sequence that is at least 90% identical to a sequence selected from the group consisting of residues -27 to 313 of SEQ ID NO:2 and residues 1 to 313 of SEQ IDNO:2.

3. A DNA of claim 2, wherein said Lerk-8 polypeptide comprises an amino acid sequence selected from the group consisting of residues -27 to 313 of SEQ ID
NO:2 and residues 1 to 313 of SEQ ID NO:2.

4. A DNA of claim 1 or 2, wherein said Lerk-8 polypeptide is naturally occurring.

5. An isolated DNA encoding a mature human Lerk-8 polypeptide that binds hek or elk, wherein said Lerk-8 polypeptide is characterized by:
a) a calculated molecular weight of about 33 kilodaltons;
b) an isoelectric point (pI) of about 8.46; and c) an N-terminal amino acid sequence Leu-Ser-Leu-Glu-Pro-Val-Tyr-Trp-Asn-Ser-Ala-Asn-(amino acids 1-12 of SEQ ID NO:2).

6. An isolated DNA encoding a soluble Lerk-8 polypeptide that binds hek or elk, wherein said Lerk-8 polypeptide comprises an amino acid sequence that is at least 80% identical to a sequence selected from the group consisting of residues -27 to x of SEQ ID NO:2 and residues 1 to x of SEQ ID NO:2, wherein x represents an integer from 142 to 197, inclusive.

7. A DNA of claim 6, wherein said soluble Lerk-8 polypeptide comprises an amino acid sequence selected from the group consisting of residues -27 to x of SEQ ID
NO:2 and residues 1 to x of SEQ ID NO:2, wherein x represents an integer from 142 to 197, inclusive.

8. An isolated DNA encoding a Lerk-8 polypeptide selected from the group consisting of:
a) the human Lerk-8 polypeptide of SEQ ID NO:2; and b) a fragment of the polypeptide of (a), wherein said fragment is capable of binding elk or hek.

9. A DNA of claim 8, wherein said fragment is a soluble fragment.

10. An expression vector comprising a DNA of claim 1, 2, 3, 4, 5, 6, 7, 8, or 9.

11. A host cell transformed with an expression vector of claim 10.

12. A process for producing a Lerk-8 polypeptide, comprising culturing a host cell of claim 11 under conditions that promote expression of the Lerk-8 polypeptide, and recovering the Lerk-8 polypeptide.

13. A purified Lerk-8 polypeptide, wherein said polypeptide is encoded by a DNA
according to claim 1, 2, 3, 4, 5, 6, 7, 8, or 9.

14. A purified Lerk-8 polypeptide comprising an amino acid sequence that is at least 80%
identical to the sequence of residues 1 to 313 of SEQ ID NO:2.

15. A Lerk-8 polypeptide of claim 14, comprising an amino acid sequence that is at least 90% identical to the sequence of residues 1 to 313 of SEQ ID NO:2.

16. A Lerk-8 polypeptide of claim 15, comprising the amino acid sequence of residues 1 to 313 of SEQ ID NO:2.

17. A Lerk-8 polypeptide of claim 15, wherein said Lerk-8 comprises amino acids 1 through 297 and 299 through 313 of SEQ ID NO:2, wherein the residue at position 298 is leucine.

18. A purified human Lerk-8 protein that binds hek or elk, wherein a mature form of said protein is characterized by:
a) a calculated molecular weight of about 33 kilodaltons;
b) an isoelectric point (pI) of about 8.46; and c) an N-terminal amino acid sequence Leu-Ser-Leu-Glu-Pro-Val-Tyr-Trp-Asn-Ser-Ala-Asn-(amino acids 1-12 of SEQ ID NO:2).

19. A purified soluble Lerk-8 polypeptide that binds hek or elk, wherein said Lerk-8 polypeptide comprises an amino acid sequence that is at least 80% identical to the sequence of residues 1 to x of SEQ ID NO:2, wherein x represents an integer from 142 to 197, inclusive.

20. A soluble Lerk-8 polypeptide of claim 19, wherein said Lerk-8 comprises the sequence of residues 1 to x of SEQ ID NO:2, wherein x represents an integer from 142 to 197, inclusive.

21. A purified Lerk-8 polypeptide selected from the group consisting of:
a) the human Lerk-8 polypeptide of SEQ ID NO:2; and b) a fragment of the polypeptide of (a), wherein said fragment is capable of binding elk or hek.

22. A Lerk-8 polypeptide of claim 21, wherein said fragment is a soluble fragment.

23. An oligomer comprising from two to four Lerk-8 polypeptides of claim 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22.

24. An oligomer of claim 23, wherein said oligomer is a dimer comprising two soluble Lerk-8/Fc fusion proteins.

25. A pharmaceutical composition comprising a Lerk-8 polypeptide or oligomer of claim 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25, and a suitable diluent, excipient, or carrier.

26. An antibody that is directed against a Lerk-8 polypeptide of claim 13, 14, 15, 16,17, 18,19,20,21, or 22.

27. An antibody according to claim 26, wherein the antibody is a monoclonal antibody.