CA2243984A1 - Neural cell adhesion molecule splicing variants - Google Patents

Abstract

NrCAMvar polypeptides and polynucleotides and methods for producing such polypeptides by recombinant techniques are disclosed. Also disclosed are methods for utilizing NrCAMvar polypeptides and polynucleotides in the design of protocols for the treatment of diabetes, obesity and cancer, among others, and diagnostic assays for such conditions.

Description

2 PCT~GB98/0043 NEURAL CELL AOHESION MOLECULE SP~ICING VARIANTS

~IELD OF INVENTION
This invention relntes to newly i~lPnlifiPd splice-varinnt polynucleotides, polypeptides enroded by them and to the use of such polynucleotides and polypeptides, and to their prod~ction More particulnrly, the polynucleotides and polypeptides of the present invention relnte to the cell ~if n m(~ P family, hereinafter referred to as NrCAMvar.
The invention also relates to inhihiling or activating the action of such polynn~lPoti(1Ps and polypeptides.

BACKGROUND OF THE INVENTION
The NgCAM-related cell adhesion molecule NrCAM, also called bravo, was first i~lPntifiPd and ch~ctpriced in chick by Grumet et al.,(l991). Although the .sPqnPnr~ of rat NrCAM is not p~lblichp~l~ it has been cloned and se~uenced (Davis and Bennett, 1994).
This cell surfnce glyco~ lein is n member of the immunoglobulin ~Ig) ~ ~, r~."ily, and is very simi}ar in structure to chick NgCAM, hum~n and mouse L1 and chick ~ r~s~ilLEnch consists of six Ig dom~inc, five fibronectin type IIl-like repeats, a tr~ncmPml~nP
domain and an intr~elllllnr region. These neural cell surface proteins play a critical role ill nervous system developmPnt Studies from Bennett et al., (Bennett and Gilligan, 1993:
Davis and Bennett, 1994) suggested that these molec-llPc, inf ~ n~ chick and rat NrCAM, have ankyrin binding activity s--, gesting thnt they mny be i,..po ~t in membrane-cyt~-.ckPlpt~l connections in brain. A role for NrCAM in the in vivo g~lid~n~e of chick commiccl~r~l neurones has been idenlifi~Pd and distinguished from thnt of NgCAM (Stoeckli 25 and I ~ntlmPsse~, 1995). Chick NrCAM in floor plnte cells together with axonin-1 on commiCC -rnl growth cones is ecg~nti~l for nccurate p:lthfin(lin~ at the midline whereas NgCAM is required for f:-ccirlll~tiQn of the commicc~-al nPIlrit~s As well as intPr:~ting with axonin- I, NrCAM cnn also bind nt the cell surface with Fl 1, another member of the Ig s--E~erf~mily (Morales et al., 1993).
Recently a highly conserved humnn homologue to chick NrCAM wns c~Ps~-ihPd (Lane et al., (~P~m ~C 35 (3), 456465 (1996)) with 82% nmino acid identity to the chick protein. The tr~ncme~ ne and intr~cellulnr dom~ins of hum~n NrCAM nre 100%
irlPntiC~I to the chick homologue while percent identities for individunl extrncellulnr (lom~inc vnry from 66% for IgVI to 93% for IgIV. Lnne et al .i~lentifiPd two alternntively 35 spliced exons, AE12 ~nrotling n 12-amino-~cid section 5' to FNIII-5. ~nd AE93 encoding the 93-nmino-ncids coLlesl>o.lding to the w~lole of FNIII-S ~Figure 2). Four different isoforms were found: with both AE12 nnd AE93, with only AE12 or AE93, and without _ W O 98/36062 PCT~GB98~00434 either AE12 or AE93. In addition to AE12 and AE93, two more splice variants have been i~lentifiPd in chick, AE19 and AE10. AEI9 encodes a l9-amino-acid section between IgII
and IgIII while AE10 is a 10 amino-acid section between IgVI and FNIII-I (Grumet et al., 1991). Using human NrCAM probes, Lane et al. observed one major RNA band of 5 -7.0kb in multiple brain tissues including amygd~a, caudate nucleus, corpus cnlk-sllm, hippocnmrus, hypoth~lnm--s, s~hctnnti:~ nigra, subth~l~mir nucleus, and thnlnmll$ In chick, the same size of RNA W'dS found in brain tissue but not in embryo heart, gizzard or liver on Northern blots.
This indicates that these cell nrlh~i~n n~l~llP~c have an Pct~hlich~ ~ in 10 v~,a~e dcv~ and are c~nc~ently c~n~ htPs for ~ ;c targets. Clearly there is a need for i(lPntifirnti~n and ch~rartf~ri7:lti()n of furthP~ "P~ and variants, inrln-iing splice variants, of the cell nrlhpsir~n n~t~-lP famtly which can play a role in preventing, ~..~1;. ,- i.fi..
a~ c~ g dysfunctions or diseases.

In one aspect, the invention relates to NrCAMvar polypeptidec and 1~l~ "l m~t~ ls and methods for their prn~lrtion Another aspect of the illvt~ ion relates to methods for using such NrCAMvar polypeptides and polymlrl~xi lPc Such uses include the ~of diabetes, obesity and cancer, arnong others. In still another aspect, the invention relates to 20 methods to identify agonists and antagonists using the msltpri~lc provided by the invention, and treating conditions associated with NrCAMvar imh~l~nr~ with the i~iPntifiP~ coll~l~
inrlnrling diabetes, obesity and cancer. Yet another aspect of the invention relates to ~ gnnctic assays for ~etpcting diseases ~ccori~t~ with il~L"u~liate NrCAMvar activity or levels, inr]n~1ing diabetes, obesity and cancer.
I~RIEF DESCRIPI ION OF THE DRAWINGS
Figure 1 shows the ml~lPotj-le and deduced amino acid seqnpn~e of a hum~n NrCAMvar; SEQ ID NOS: 1 and 2, respectively.
Figure 2 shows a c~," ~ .on of Ihe sequences of hurnan NrCAMv~r of the precent 30 invention and hum~n and chic~ NrCAM cDNAs.

DESCRIErrION OF TIIE INVENTION
Deffnitions The following definitions are provided to fanilit:lt~ undersr~n~ing of certain terrns 35 used frequently hcrein.

"NrCAMvar'- refers, among others, generally to a polypeptide having the amino acid sequenre set forth in SEQ ID NO:2 or an allelic variant thereof.
"NrCAMvar activity or NrCAMvar polypeptide activ}ty't or "biological activity ofthe NrCAMvar or NrCAMvar polypeptide" refers to the metabolic or phy~c~ oglc~l S filnrtion of said NrCAMvar inrh~ n~ similar activities or improved activities or these activities with decreased undesirable side-effects. Also included are nn~i~pnic and immunog~nic activities of said NrCAMvar.
"NrCAMvar gene" refers to a polynucleotide having the nucleotide sequence set forth in SEQ ID NO: 1 or allelic variants thercof and/or their comr1Prnl~nts.
"Antibodies" as used herein includes polyclonal and m~-no-~lonal antibodies, chimeric, single chain, and hllm~ni7Pd antibodies, as well as Fab fragments, inr~ n~ the products of an Fab or other imml~n~globulin expression library.
"Isolated" means altered "by the hand of man" from the natural state. If an "isolated" composition or s~bstnnre OCCUB in nature, it has been changed or removed from 15 its original ~ ."ent, or both. For exinmr~le, a polynucleotide or a polypeptide naturally present in a living animal is not "isolated," but the same polym~cleotirle or polypeptide separated from the coe~rictin~ mntPrinl$ of it~s natural state is "isolated", as the terrn is employed herein.
''polynllrl~otide~ generally refers to any polyribonllrl~offde or 20 polydeoxribon--rlf~o~ P~ which may be nnm~li fied RNA or DNA or mor~ifiPd RNA or DNA. "Polynucleotides" include, without limit~tinn single- and double-str:ln~lPd DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-st~n(~f~d RNA, and RNA that is mixture of single- and double-stranded regions, hybrid m~leclllpc comprising DNA and RNA th~t may be single-stranded or, more typically, double-25 stranded or a mixture of single- and double-str~nded regions. In addition, "polynucleotide"
refers to triple-stranded regions comrricin~ RNA or DNA or both RNA and DNA. Theterm polyn-~rleoticle also includes DNAs or RNAs contninin~, one or more modifiPd bases and DNAs or RNAs with backbones modified for stability or for other reasons.
"Modiffed" bases include, for example, tritylated bases and unusual bases such as inosine.
30 A variety of modifications have been made to DNA and RNA; thus, "polyn~cl~oQticle"
e"ll,.. ces ~-hemicinlly, enzymatically or metabolically modified forms of polynucleotides as typically found in nature, as well as the chernical forms of DNA and RNA l h i-- <~ of viruses and cells. "Polynucleotide" ~so embraces relatively short polynucleotides, often referred to as oligonucleotides.
"Polypeptide" refers to any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or mo~i fif~d peptide bonds, i.e., peptide isosteres.

WO 98/36062 PCT~GB98~W434 "Polypeptide" refers to both short ch~ins, c~)m monly referred to as peptides, oligopeptides or oligomers, and to longer chains, generally referred to as proteins. Polypeptides may cont~in amino acids other than the 20 gene-encQded amino acids. "Polypeptides" include amino ~cid sequences modifiecl either by natur~l processes, such as posttr~n~l~tion~l S procPccin~, or by chemical m ~i fic~tion techniques which are well known in the art. Such mo li fisn~iQnc are well desrribe~ in b~sic texts and in more detailed monographs, as well as in a voluminous l~se~.;h literature. Moriifir~tionc can occur anywhere in a polypeptide, the peptide backbone, the arnino acid side-chains and the arnino orcarboxyl ter~uni. It will be appreciated that tne sarne type of motiifisi~tion may be present in the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modific~tions. Polypeptides may be branched as a result of ubiq litinntion, and they may be cyclic, with or without brnnshinP Cyclic, branched and branched cyclic polypeptides may result from posttranslation natural processes or may be made by synthetic meth~s Mr~ificntions include acetylation, acylation, ADP-ribosylation, amidation, cov31ent nft~rhmf~nt of flavin, cov~ent ~ttnrhml~nt of a heme moiety, covalent ~ rhment of a nucleotide or nucleotide derivative, covalent ~tti~rhm~nt of a lipid or lipid derivative, covalent nttnrhmPnt of ph--~hrltidylin~-cit~-l, cross-linking, cyrli~nti~n, ~lisnlfide bond forrnation, demethylation, formation of covalent cross-links, f~-rmntion of cystine, formation of pyro~l~-t~mnt~, formylation, gamma-carboxylation, glycosylation, GPI anchor forrnation, hydroxylation, iodination, methylation, myristoylation, oxi-lntion, proteolytic processing, phosphorylation, prenylation, rnremi7ntton, selenoylation, sulfation, transfer-RNA mP~ tf~d addition of arnino acids to proteins such as arginylation, and ubiquitination. See, for inct~nce, PROTEINS -STRUCTURE AND MO~ECULAR PROPER l l ~ S, 2nd E~., T. E. Creighton, W. H.
Elt;e,llan and Company, New York, 1993 and Wold, F., p~ nc1~ti~n~1 Protein M~ific~ionc- Perspectives and PlosLJe~, pgs. 1-12 in POSTTRANSLATIONAL
COVALENT MODIFICATION OF PROTEINS, B. C. Johnson, Ed., P cnrlemic Press, New York, 1983; Seifter et al., "Analysis for protein modifications and nonprotein cofactors", Meth En~mol (1990) 182:626-646 and Rattan et al., "Protein Synthesis:
Posttranslational Modifications and Aging", Ann NYAcad Sci (1992) 663:48-62.
"Variant" as the term is used herein, is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide respectively, but retains essenti~l properties. A typical variant of a polynucleotide differs in nucleotide seql~nre from another, reference polynucieotide. Ch~nges in the nucleotide sequence of the variant may 35 or may not alter the amino acid sPq~ n~e of a polypeptide encoded by the ,ere~e~ce polynurleotide Nucleotide changes m~y result in amino acid ~ul~ iQm, additions, _ WO 98/36062 PCT/GB98~00434 rlPletionc, fusions and truncations in the polypeptide encoded by the reference seq~lPnne, as --ccPd below. A typic~ variant of ~ polypeptide differs in amino acid sequence from another, ~ef~ ce polypeptide. Generally, differences are limited so that the se(luPn~ PS of the reference polypeptide and the vari~nt are closely simi3ar overall and. in many regions, 5 icl~.ntir 71 A variant and reference polypeptide may differ in amino acid sequ~pn~e by one or more ~ onc~ 3dditions, deletions in any comhin~tion A sl-hstit~lt~Pd or inserted amino acid residue may or may not be one encoded by the genetic code. A variant of a polynucleotide or polypeptide may be a naturnlly occ~ ng such as an allelic variant, or it may be a variant th~t is not known to occur naturally. Non-n~turally occllrrin~ variants of 10 polym~ PotidPc and polypeptides may be m~de by mnt~enPcictenhni(l~es or by direct synthesis.
"Identity" is a me~ure of the identity of nucleotide ~eqllenl~ps or ar~uno acid sPquen~nPC In general. the cpquen~ps are aligned so th~t the highest order match is obt~ined "Identity" per se has an ~rt-recognized m~ nin~ and can be calculated using 15 pllhliChPd ~erhni~ Pc See, e.g.: (COMPUTAT~ONAL MOLECULAR BlOLOGY, Lesk, A.M., ed.. Oxford University Press, New York, 1988; BIOCOMPUTING:
INFO~MATICS AND GENOME PROJECTS, Smith, D.W., ed., ~ cmic Press, New York, 1993; COMPUTER ANALYSIS OF S~QUENCE DATA, PART I, Griffin, A.M., and Griffin, H.G., eds., ~umr~ns Press, New Jersey, 1994; SEQUENCE ANALYSIS IN
20 MOLECIJLAR BIOLOGY, von Heinje, G., ~n~ emi~ Press, 1987; and SEQUENCE
ANALYSIS PRIMER, Gribskov, M. and D~v~ , J., eds., M Stockton Press, New York, 1991). While there exist ~ number of methods to measure identity between two polynucleotide or polypeptide sequences, the term "identity" is well known to skilled artisans (C~llo, H., ~nd Lipton, D., SMM J Applied Math (1988) 48: 1073). Methods 2~ commonly employed to determine identity or similarity between two sequen~ec include, but are not limited to, those disclosed in Guide to Huge Computers, M~rtin J. Bishop, ed., Ac~lemic Press, San Diego, 1994, and C~rillo, H., and Lipton, D., SIAM JApplied Math (1988) 48:1073. Methods to determine identity and simil~rity are codified in cn~progr~ms. Preferred computer progr m methods to ~et~ninl~ identity and similarity 30 between two sequences include, but are not limited to, GCS ~ g.~n package (Devereux, J., et al., Nucleic Acids Researcll ( 1984) 12(1):387), BLASTP, BLASTN, FASTA
(Atschul, S.F. et al., J Molec Biol ( 1990) 215:403).

The invention ~iC~l~es ~ new splice vari:mt of N~CAM (termed NrCAMvar) which 35 c~ ,l" lc~ the AE10EC2 spL~t?n(~p~ which is ~bsent in the pnhlichP~ hum~n NrCAM sP~TtIPnre (Lane et al., 1996) but which is present in the chick se~-e~ ddition the NrCAMvar does not have the AEIOKI s~len~ which is present in the Lane et a~ human sPfrlPnr~ (Figure 2).
NrCAMvar is expressed at high levels in the br~in, pancreas and adrenal catex and at lower levels in placenta, adrenal medlllla, thyroid and ~estis. The pllhli~h~ human NrCAM, however, appears not to be e,~ ed in the pancreas.
s Polypeptides of the Invention In one aspect, the present invention relates to novel NrCAMvar polypeptides. TheNrCAMvar polypeptides include the polypeptide of SEQ ID NO:2; as well as polypeptides co~l ~p~ icin~ the amino acid sequence of SEQ ID NO: 2. r~Ç~ably NrCAMvar polypeptide 10 exhibit at le st one ' ~l~jr~l activity of NrCAMvar.
The NrCAMvar polypeptides may be in the forrn of the "mature ' protein or may be a part of a larger protein such as a fusion protein. It is often advantageous to include an ac~ tion~1 amino acid sPql~Pnce which cont~ins secretory or leademserlnencPc, pro-se~lmPnres, s~PqnPnrPs which aid in p~rification such as multiple hictiAinp residues, or an 15 ~Aflitjf~n~l sequPnl e for stability during recombinant procl~lctil n Bi-~l~ir~lly active fragments of the NrCAMvar polypeptides are also inrll-~ed in the invention. A fragment is a polypeptide having an amino acid selr ~P~ - ~ that entirely is the same as part, but not all, of the amino acid s~P~ P~ of the aro~ np~i NrCAMvar polypeptides.
As with NrCAMvar polypeptides, fragments may be "free-~t~nt11n~," or cn~ -- i~d within a 20 larger polypeptide of which they form a part or region, most preferably as a single co"l i....~.
region.
P~f~led fragments include. for PY~mplP truncation polypeptides having ~e amin~
acid ~IPnr~ of NrCAMvar polypeptides, except for deletion of a cnntinnol~s series of residues that includes the amino terminus, or a c~)ntim-r,~-c series of residues tl at includes the 25 carboxyl terminus or deletion of two cn- ~l i. ~,,--c series of residues, one inrlu~in~ the amino terrninus and one inrln~ling the carboxyl terminus. Also pl~rtl~ are Ç~ ~ char~ri7P~I
by structural or fimrtion~l aUlibuL~s such as fragments that ~ ~mLI~ ; alpha-helix and alpha-helix forming regions, beta-sheet and l~ta-sheet-fo~ning regions, turn and turn-forming regions, coil and coil-forrning regions, hydrophilic regions, hydrophobic regions, alpha ~mrhir~thic 30 regions, beta ~mrhir~thic regions, ffexible regions, surface-forming regions, substrate binding region, and high ~n~ig~ ~ index regions. Bi~logir,: lly active fragments are those that mediate NrCAMvar activity, inrlll-finsJ those with a similar activity or an improved activity, ~ with a d~sed undecirable activity. Also includcd are those th~t are ~nti~nic or immnn~ger:~ in an animal, especiaLly in a humarL
Preferably, all of these polyp ptide fragments retain the hio~c~ir~l activity of the NrCAMvar, inrlu(ling antigenic activity. Variants of the defined 5~1~' ~8'~- and fragments also WO 98~6062 PCT/GB98/UO434 fo~n p~rt of the present invention. Preferred variants are those that vary from the referents by conservative an~ino acid s~lbctih-tir,nc -- i.e., those that s~l~.ctit~lte ~ residue with another of like rh:lr~r~Pri.ctirc Typic~ such.snh.ctihltirnc are arnong Ala, Val, Leu and ne; among Ser and Thr; among the acidic residues Asp and Glu; among Asn and Gln; and among the b~sic residue~s Lys and Arg; or aromatic residuec Phe and Tyr.
The NrCAMvar polypeptides of the invention can be p.~t d in any suit~ble rnanner.
Such polypeptides include isolated naturally or~lrring polypeptide~s, ~ - -bi n ~ y produced polypeptides, synthPtic~ily produced polypeptides, or polypeptides produced by a cnlol-in~tim of these methods. Me~s for plep~i.lg such polypeptidec are well u~:,~d in the ar~
Poly~ cl~lides of the Invention Ar~her aspect of the invention relates to NrCAMvar polyr~rl~XkiP-S NrCAMvar polym~rl~tid~s include isolated polynllr~ k!~s which encode the NrCAMvar polypeptides and fr:l~nPntc, and polynurlP~tidPc closely related thereto. More specifically, NrCAMvar 15 polynucleotide of the invention include a polr--rl~ti-ie co-.~lisillg the mlrl~i~ s~lP~e set forth in SEQ ID NO: 1 ~nro~inE a NrCAMvar polypeptide of SEQ ID NO: 2, and polyn--rlff}ti~c having the particular se~ "nP of SEQ ID NO:l. Also included under NrCAMvar polynucleotides ~re a nucleotide sequence which has sllffiri~nt identity to a nucleotide se~uenre c.,~ d in SEQ ID NO: 1 to hybddize under cnnr1itinn~ useable for 20 ~mrlifir~tjon or for use as a probe or marker. The invention also provides polynllcl~potid~ps which are co- F y to such NrCAMvar polyn--rl~oo~ Pe NrCAMvar of the invention is structurally related to other proteins of the cell a~irm mnlealles, as shown by the results of sc~ uPnring the cDNA Pnroding human NrCAMvar. The cDNA s~l~ncP, contains an open reading frame Pnro~ling a polypeptide of 1304 arnino acids.
25 Arnino acid of sequence of Figure 1 (SEQ ID NO:2) has about >99% identity (using BlastP) in 1299 arnino acid residues with Human NrCAM (Lane, RP et al, ~: 35 (3), 456465 (1996)). ~llrlPofi<lP s~ Pnr~ of Figure 1 (SEQ ID NO:1) has about ~gg~ identity (usir~
BlastN) in 3897 nucleotide residues with Human NrCAM (Genomics 35 (3), 456-465 (1996)).
Figure 2 shows the splice variant AElOK
Ooe polymlrlrofic~ of the present invention encoding NrCAMvar may be obtained using standard cloning and ~ ng, from a cDNA library derived from mRNA in cells of hu~r~n adrenal using the expressed sequence tag (EST) analysis (Adams, M.D., et aL
Science (1991) 252:1651-1656; Adams, M.D. et al., Nature, (1992) 355:632-634;
Adams, M.D., et aL, Nature (1995) 377 Supp:3-174). Polynucleotides of the invention 35 can also be obtained from natural sources such as genomic DNA libralies or can be syn~heci7ed using well known and commercially available te~ es The nucleotide .cPr~ nrP encoding NrCAMvnr polypeptide of SEQ ID N0:2 may be idPntir~l over its entire length to thc coding seqllenr,e set forLh in Figure 1 (SEQ ID
N0: 1), or may be a degenerate form of this nucleotide sequence encoding the polypepdde of SEQ ID N0:2, or may he high y i~lPn~ic~l to a nl~rlPoti(l~P sequence that encodes the 5 polypeptide of SEQ ID N0:2.
When the polynucleotides of the invention ~re used for the recl mhin~nt prod~lctio of NrCAMvar polypeptide, the polym-r7PotirlP may include the coding ~l~e~ for the m3ture polypeptide or a fi~nPnt thereof, by itself; the coding ~1"~ for the rnature polypeptide or ~agrnent in reading frarne with othcr coding se~7lenrp~t such as those ~orltn~ a 10 leader or secretory sP~lPnrP a pre-, or pro- or prepro- protein ~ P~ P, f~ other fusion pepdde portions. For PYnm?7e> a rnarker serl~lpnre which f:-rilit~tPs pllrifir~tion of the fused polypeptide can 7.~e encoded In cert~in preferred en~ii..le,.ls of this aspect of the invention, the rnarAcer .sefrlPnrP. is a hexa-histidine pepdde, as provided in the pQE vec70r (Qiagen, Inc.~ ar~ flP.cfTi7.
in Gentz et al., Proc Na~l Acad Sc~ USA (1989) 86:821-824, or is an HA tag. The 15 polym1rlP~i~ may also c07~ain non-coding 5' and 3' S~lPnr,P-s~ such as l~ Cr. ;1~1, non-trnnslated ~l"~ s, sp7icing and polyadenylntion sign~s, ribosorne binding sit~s and ~I~P.
th~t st~bilize m~N~
Further AJ- ;:r~P~ eml}odiments are polyn~ Potid~ enrc~in~ NrCAMvar variants ~ )- - q~l icP the ami~ acid s~P~ pl ~ee NrcAMv~ polypeptide of Flgure 1 (SEQ ID N0:2) in 20 which several, 5- 10, 1 -5, 1-3, 1 -2 or 1 amino acid residues are ~ ~, deleted ~r added, in any cc~- - -l-; " ~ l ir~n The present invention further relates to polynncl~tirlP.c that hybrid~e to the herein above~rri~ Sf~-f. nrPc In this regard the present invention especially relatf~s to polyn~-rl~i-~s which hybridize under stringent Cf~n~iitif)nc tO the herein above~l~ ~ il~
25 polymlrlP~ c. As herein used, the terrn "s."ngf l-l rnn~litif~n$" means hybri~li7~tion will occur or~y if there is at least 95% ~nd preferably nt le st 97% identity between the ~l~PnrP~j.
polynnrlf,o~ Pc of the invention, which are identica.l o~ snffirif--ntly identical to a mlr~ .SP~ ..rP, cont: inP~ in SEQ ID N0 1, may be used as hyhridi7~ti~n probes for cDNA and genomic DNA, to isolate full-length cDNAs and genornic clor~s f~nr~ing 30 NrCAMvar polypeptide and to isolate cDNA and genornic clones of other genes that have a high 5~~ll If ~)r~ similarity to the NrCAMvar gene. Such hyhri~ii7~tion l~-l .r .l.-f~ are hlown to those of sldll in the art. Typically these mlrlfY-tirle sf~lrncP-~ are 70% 1dPntir~l, preferably 80%
i~Pntir:ll, more preferably 90% identic~ to that of the referen~. The probes generally will ~;oll~lise at least 15 nnrlfx-ti(~ Preferably, such probes will have at least 30 m-rl~ti~Pc and 35 rr~y have at least 50 nllrl~Qti~lf~ ParticulQrly preferred probes will range between 30 and 50 mlrlPQti~.

W O 98/36062 PCT/~B98/00434 In one embodi~nt, to obtain a polym~nl~i~ encoding NrCAMvar C~ ;~; the steps of S~ L~ g an appl~-i~t~ library und~r stingen~ hybr~ 7~tic)n c~nt1itinn.c with a laWed probe having the SEQ ID NO: 1 or a ~agn~nt thereof, and isolating full-length cDNA and genomic clones cnnt~inin~ said polyn~rl~ti~lP S~l~n( P~ Such hyhr~ 7~tit~n i ' q~ are well known to those of sldll in the art Stringent hyhridi7.~ n cf)nrli~ n~ are as defined above or alternatively c ~n~lition~ under overr~ight in(~nb~til~n at 42~C n a solution ~ g 50%
forrns~m: lP, SxSSC (lSOmM N~Cl, l5rnM tri~inm citrate), 50 mM sodium ~
(pH7.6), Sx De~ aL~ ~; solution, 10 % dext~an sulfate, and 20 11~i~L~L~~ tUL~, sheared salrnon sperrn DNA, followed by washing the filters in 0. lx SSC at about 65~C.
~0 The polyn~rl~ticlPc and polypeptides of the present irlvention rnay be employed as research reagents and mnterinls for discovery of lL~LIl e..~ and ~ rc to animal ar~d hum~n disease.

Vectors, Host Cells, Expression The present invention also relates to vectors which c~ "l,l ic~ a polynnf l~i~ or polynnrl~otir~ of the present invention, and host cells which are ~rnPtir~lly e,~ d with vectors of the invention and to the production of polypeptides of the invention by 1~ b~
t~ , Cell-free trnnCl~tit~n systems can also be employed to produce such proteins using RNAs derived from the DNA C~ LU-,~ of the present ill~, ILiolL
For ~ nnt pro~-cti~-n, host cells can be gen~ti~nlly e.~u~d to il~
expression systerns or portions thereof for polymlnlPofi~s of the present invention. ~ Lliu~
of pdymlrl~i~s into host cells can be effected by methods dpcrrihed in many standard laboratory mnm lnls, such as Davis et aL, BASIC M~THODS IN MOLECULAR BIOLOGY
(1986) and S~-~ WI~ et al., MOLECULAR CLON NG: A lABORATORYMANUAL, 2nd Ed, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989) such as calciurn p~rhnt~ trnncf~i~n~ DEAE~extr~n mPAintPd ~i~n~r~;li~n~ tranSVeCtiO~ ~i"j~io", catior~c lipid-n~~ r~;~ n, el~;~ puLalion, trnns~lc~ion, scrape }oading, ballistic introduction or inf~,tion R~l~c:"~tive ~ r-s of a~-~,p.iate hosts include bacterial cells, such as ~ cccci, staphylococci, E coli, S~reptomyces and Bacillus s~tilis ce~ls; fungal cells, such as yeast cells and Asper~illus cells; insect cells such as Drosophila S2 and Spodoptera S f9 cells; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, 293 and Bowes m~l~n~ m~
cells; and plant cells.
A great variety of e.~ ion systems can be used. Such systems include, among 35 others, ~h.~ ~o, nl, ~ic~mnl ~nd virus derived systems, e.g., vectors derived from bacterial pl~cm ~c, from blctrri~hn~, from tr~sposons, from yeast episo--les, from insertion ~ mPntc, WO 98/36062 PCT/GB98~ 34 .

from ye st ~ U11~6011~1 PlPmPnts, from viruses such as baculûviruses, papova viruses, such as SV40, vaccinia viruses, adenovin~ses, fowl pox viruses, pseu~bies viruses ar~retroviruses, and vec~rs der.ived from fOln~ ion~ thereof. such as those derived from plasmid and b~rtPrioFh~ genetic P~PmPntc, such as cosmids and rh~l~Pmi~. The ex~ ~o,l systems may S con~in con~rol regiûns that regul~te as well as eng~,~ eAI~I~o~L ~enerally, any system ~
vect(}r st~itable t~ m:~int lin propagate or express polyn-lrl~tic~ to produce a polypepTide in a ho~st m~y be used. The ~,u~ te n--rlPoti-le se~lPnr~ m~y be inserted into an ~ l~o~l syst~m by any of a vatiety of well-known and routine i ' ~nP5, such as. for PY~mrTP~ th~e set forth in Sambrook et al.. MOLFCULAR CLONING, A lABORATORYMANUAL (sl~pra).
For secretion of the tr~nsl~tP~ protein into th~ lumen of the Pn~l~mir retirlllnm, into the p~rirl~mic space or into t~ extracellular en~,~u~ L, app.op~iate secretion signals may be .-~ ted into the desired polypeptide. These sign~s may be Pnh~n~lc to the polypeptide or they may be heterologous signals.
If the NrCAMvar polypeptide is to be e~cp-~ed for use in s~eel~lg assays. generally, 15 it is preferred that the polypeptide be produced at the surface of the cell. In this event. the cells may be harvested prior to use in the screening assay. If NrCAMvar polypeptide is secreted into the medium. the medium c~m be recovered in order to recover and purify the polypeptide; if produced intr~nPlllllnrly, the cells must first be Iysed before the polypeptide is recovered.
20 NrCAMvar polypeptides can be recovered and punfied from Ir~ n, n~ cell cultures by well-known meth~ds i n~ mm~ninm sulfdte or eth3nol p, ~ ;~ " " acid eY~ion, anion or cation PY~h~ng~ oll~Lu~.~hy, phc~rh~llnl~P chromdtography, h~
,hL'u~l~L~ hy, affinity chromatography, hydroxylapatite Llln~ pl Iy and lectin onLat ~grlphy. Most prefer:lbly, high per r( .. . "~ n~ liquid ~;h~u~ pl-y is employed for p~lrific~ )n Well hlown tPrhnirIu~ for refolding proteins may be employed to It:~,Gl~dte active cu--r~4-ndLion when the polypeptide is denatured dllring isolation and or pllrifin~tion Dia~nostic Assays This invention also relates to the use of NrCAMvar polym~l~ti~ for use as ~ gn{~c~ic reagcnts. Detection of a mutated forrn of NrCAMvar gene ~ tf-~l with a dysfi-n~ion will provide a ~i~gn(~s~ic tool that can add to or define a ~ gn~cic of a disease or jllcC~L)ti~iTity to a disease which rcsults from under-expression, over-expression or altered G,~.l~sion of NrCAMvar. Individuals carrying mllr:-tionc in the NrCAMvar gene may be detcctcd at the DNA level by a variety of t~hni~
Nucleic acids for ~ onnci~ may be obtained from ~ subject's ce~ls. such as from blood, urine, s~liv~. tissue biopsy or ~utopsy material. The geno-m--ic DNA rnay be used directly for cll~tP~tion or rnay be ~mrlifiP~ enzymatic~lly by using PCR ~ other ~mrlifie~tifm tP-~hn;~ Ps prior to an~ysLc. RNA or cDNA may Ico be usedin s~ r f~shiorL Deletions and insertions c~n be c1etectP~i by a change in si~ of the ~mrlifipd product in cn-nl~n~ ,.. to the noIIr~l genotype. Point mnt~it)nC c~n be icipntifipcl by h~blidi~il g ~mrlifiPA DNA to labeled S NrCAMvnrml~lP~i~eS~ P~ f~ Perfëctly m tchedcP~Irn~Pcc~nbe~ g-~icl-~ifrom micmn~ lplexes by RNase ~iigP~ Ctinn or by dif~erencec in melting lr~ hl~ . DNA
c~P~upnre lirrPl~--~ m~y also be d_tected by alL~;~l~u~ in le~ ~l~c; mobility of DNA
i~agmentc in geLs, with or withollt ~P.~nhl,;,~g agenLc, c~ by dir~ DNA s~l~Pn. i~g See, e.g., Myers et al., Sc~ence (1985) 230:1242. Sequence changes at spe~fic kY~ti~)nc may also be 10 revealed by nuclease protection assays, such as RNase and S1 L).~Li~.. or the chemical cleavage method See Cotton et al., Proc Na~l Acad Sci USA (1985) 85: 4397~1401. In another embodiment, an ~rray of oligonucleotides probes co~ h~ NrCAMvar nucleotide sequenre or ftagments thereof can he c~n~LIu~;~ed to conduct effi~ient screening of e.g., genetic mutntions. Arrny terhnology methods are well known and have general 15 ap~lir~lhility and can be used to address a vanety of questions ln molec~ r genetics inclurling gene expression, genetic linkage, and genetic variability. (See for eY~mp M.Chee et al., Science, Vol 274, pp 610-613 (1996)).
The ~ ~stic assays offer a process for rli~gn~cin~ or ~lrlf ....;- ~;~g a s~crlopfi}~ility to Diabetes, obesity and cancer through c~PtPcti-n of ~---~ fi (--~ in the NrCAMvar gene by the 20 methods ~1~. . ;l~i In nddition, Dinbetes, obesity and cancer, cnn be ~ ~l by methods cr,mrricing ~etermining from a sample derived from a subject an ~ not~n~11y decreased or increased level of NrCAMvnr polypeptide or NrCAMvar mRNA. Decreased or in~n!~cPd expression can be measured at the RNA level using any of the mpthn~is well known in the art for the 25 clll~ntit:~tion of polynnrleotides, such as, for e~mple. PCR, RT-PCR, RNase protPction, Northern blotting and other hybridizntion m.oth~i.c Assay i ql~Ps that can be used to P leve~s of a protein, such as nn NrCAMvar polypeptide. in a sample derived from a host are well-known to those of skill in the ar~ Such assay methods include r~ mmllnr~Cc~ys, ~ e-binding assnys, Western Blot analysis and ELISA assays.
Ch~o~ J..~ Assays The nucleoade serl~pn~pc of th~ present invention are also valuable for ~ xo~
i~lPntifir~tion The sequence is c~ific~ny targeted to and can hybridize with a p~li~,u loc~aon on an individual human ~ "n-~so- - ~ The m~pping of re~evant s~~ ~ t35 .,h.-,--,-~sn--~P-~ according to the present invention is an irnpor~nt first step in c~l~lating those "c~ vith gene ~sso~i ~t-Pd disease. Once a se~uPn~e has been mappe~i to a pre~ise so"~ location, the physical position of the sPf~l~nre on the ~ ~( " ~ can be correlnted with genetic m lp dnta. Such dntn are found, for ~ mr~, in V. ~cKIlsirl~
li~n TnhPrit:lnre in M~n (avnilable on line through Johns ~opldns University Welch Medical Library). The rel~io~chir~ bet veen genes and dise~ses t}~t have been m pped to the S sarne .:hlol~ 1 regIon are then idPntifi~ through linlcage annlysis (c~ nre of physically adjncent genes).
The di~ell~ in the cDNA or genornic sequence between affected and l~n~ffec~d individualS cnn also be determined. If a mutation is observed in some or all of the affected individualS but not in ~ny normal individuals, then the mutation is likely to be the causatiYe 10 agent of the disease.

~ ntiho~lif~c The polypeptides of the invention or their fr~gments or analogs thereof, or cells eALll~sing them can also be used as imml-n~g~onc to produce ~ntih~ ~ i.. l.. ~il;c for the 15 NrCAMvar polypeptides. The term "l"""",~ .ric" means that the antibodies havesl-hs~n~ l greater affinity for the polypeptides of the invention than their affinity for other related polyE~-L,li les in the prior art.
An~ibodies g~ ~1 against the NrCAMvar polypeptides c~n be obtained by a~ 1" ,; "~ the polypeptides ~ epitope-bearing fr~7~rruntc, analogs or cells to an arlimal, 20 preferably a 1~, th- 111 I'A n, using routine pl~ls. For ~l~a~n of m~n~10n~1 ~ILi~ " , any e which provides :~ntiho~ oc produced by cnntin~ c ceLI line cultures can he used FY:lmp1es include the hybridomn t~hn;lnç ~Kohler, G. and Milstein, C., Nature (1975) 256:495-497), the triornn tPnhn;1uç the human B~ell hybri~mn tP~ ' , (Kozbor et al., Imrmmology Today (1983) 4:72) and the EBV-hybridomn tf~l ' , ~ (Cole et al., 25 MONOCLONAL ANTIBODIES AND CANCER T~RAPY, pp. 77-96, Alan R. Liss, Inc"
1985).
Te~ lU~ for the production of single chain n~Lil~ " (U.S. Patent No. 4,946,77~) can also be adnpted to produce single chain nntibodies to polypeptides of this invellliOIL Also, ~1 ~sS ~ ~ rnice, or other ol~nl.isll~innlurTing other m~mm 71c, rnay be used to express 30 1~ ";,P~ 7ntiho~
The above ~l~snrihed ~nriho~liPc mny be employed to isolate or to identify clones eAyl~ing the polypeptide o~ to purify the polypeptides by affinity ~ ull~L~Iy.
Arti~" agninst NrCAMv~r polypeptides may also be ernployed to treat DJnbetes, obesity and cancer, among others.
Vaccines -Another aspect of the invention relates to a method for in~lllring an immlln--logical responce in a m~tmmnl which comprises inocll1nting the mnmm~l with NrCAMvar polypeptide, or a fragment thereof, adequate to produce antibody and/or T cell immune response to protect said animal from Diabetes, obesity and cancer, among others. Yet S another aspect of the invention relates to a method of indllring immtln~ ktgical response in a mnmmnl which comprises, delivering NrCAMvar polypeptide via a vector directingexpression of NrCAMvar pol,vnucleotide rn vivo in order to induce such an immllnl~lc~gical onse to produce antibody to protect said animal from dicences Further aspect of the invention relates to an immllnolQgicaVvaccine form--1nti~l10 (conl~osilion) which, when introduced into a m~tmmnli~n host, induces an immllnf)logical response in that mnmm~l to a NrCAMvar polypeptide wherein the composition cnmrticps a NrCAMvar polypeptide or NrCAMv~r gene. The vaccine ff~r~mlllntion may further comprice a suitable carrier. Since NrCAMvar polypeptide may be br~oken down in the stomnrh, it is preferably ~-lminictPred p~ y (inr~ in~ S~ ti~n~oous, il,l...-----c~ r, 15 intravenous, intradermal etc. injection). Formulations suitable for parenteral ndminictt~tion include aqueous and non-aqueous sterile injection solutions which may contain anti-- xidnntc, buffers. bn~teriQstntc and solutes which render the f~rm~ tion instonic with the blood of the reripie~t; and aqueous and non-aqueous steri~e cl.sren.cionc which may include s~cpes~ing agents or thinl~Pnin~ agents. The for~mlll~tirJnc may be 20 presented in uniWose or multi-dose c~ u.;.~ , for example, sealed ampoules and vials and may be stored in a freeze-dried condition requiring only the addition of the sterile liquid carrier immt~rlintely prior to use. The vaccine form--l~tion may also }nclude adjuvant systems for enhnnrinP the immunogenicity of the formlllntion, such as oil-in water systems and other systems known in the art. The dosage will depend on the specific activity of the 25 vaccine and can be readily deterrnined by routine ex~rimrntintion S~b .U~I~ Assays The NrCAMvar polypeptide of the present invention may be employed in a s.l~-l--lg process for compounds which activate (agonists) or inhibit activation of (antagonists, or 30 ~:-wise called illhil~iLul~) the NrCAMv~r polypeptide of the present invention. Thus, polypeptides of the invention may also be used to ~ssess identify agonist or nntn"onictc from~ for rYnmrle cells, cell-free p~alaLions, chemic~ libraries, and natural product mixtures. These agonists or nn~ngonistc may be natural substrates, ligands, I~Ol:~, etc., as the case may be, of the polypeptide of the present invention; or m~y be structur~ or filnrti~nnl mimetirc of the 35 polypeptide of the present invention. See Coligan et al., Current Protocols i/z Imml nology 1(2):Chapter S (1991).

NrCAMvar polypeptide~s are ~lh~ it~ls in the m~mm~ n ho~st and are .~o~ le for m~any 1- n~icnl filnrtion$~ inrl~l~in~ n~ny p:~th~l~it~c A~c~ lh~y, it is de~sirou~s to find compounds and drugs which Stim~T:~te NrCAMvar polypeptide on the one hand and which can inhibit the function of NrCAMvar polypeptide on the other h~r~ In general, ago~Lts are S em~loyed for ll~ilpeulic and prophylactic ~ul~ses for such c~n~Titi~n~ as Diabetes, obesity and cancer. Antagonist~s may be employed for a variety of th~ltic and prophylactic ~s for such cnn~ nc a~s Diabetes, obesity ar~ cancer.
In general, such s~l~e ~lg pl~lUl~S t~y involve using ap~ ia~ cells which express the NrCAMvar polypeptide or respond to NrCAMvar polypeptide of the present 10 invention. Such celLs include celLs from m~mmnlc, yeast, Drosophila or ~ coli. Cells which express the NrCAMvar polypeptide (or cell Illel~ e c~-nt~inin~ the expressed polypeptide) or respond to NrCAMvar polypeptide are then cl nn~cted with a test ~ d to observe binding, c~r stimnlS~tif n or inhihiti~n of a fi1nnti~mn1 re ponce. The ability of the ceIls which were r-~ntslrt~Pd with the -~ s~ s~ coll~~ is cc~ s~- ~d with ~he sslrne cellS which were rlot 15 COIIIS~ ~P~forNrCAMvaraCtivity.
The asslys mly simply test binding of a cS~ntli~nt~ compound wherein adherence to the cells bearing the Nt~AMvlr polypeptide is detected by means of a label direct~y or indirectly slcco~isltPd with the cs~n~ te colllpound or in an assay involving cou,peLilioil with a labeled cou~etilul. Further, these assays m~y test whether the c~n~i(lS t~ compound 20 results in a signS~l gçnPr~tPrl by activsltion of the NrCAMvlr polypeptide, using ~1etPrtir,n systems ap~luL~liSIte to the cells bearing the NrCAMvlr polypeptide. ~nhibit~t~s of activltion are generally assayed in the L,l~:sence of a known agonist and the effect on activ~tion by the agonist by the presence of the ns~n~ s~t~ compound is observed. Stands~rd methods for con-lucting such screening assays are well understood in the art.
F~s~mp1~c of potenti~ NrCAMvslr polypeptide s~ntsi~nist~ include s~nffhQ~ti~ps or, in sorne cases, oligo~ tf~ ~ or proteitls which slre closely rel~ted to the ligands, ~uL~ t~, ~tUL~, etc., as the case may be, of the NrCAMvar polypeptide, e.g., a fragment of the ligands, snl ~ lr~c, lecel~luls, or sm~ll mn~ os which bind to the polypeLide of the present invention but do not elicit a response, so that the actlvity of the pc)lypeptide is prevented Prophylactic and Th~- d~t:U~iC Methods This invenLion provides methods of treating an ahncrm~ con iitionc related to bo~h an excess of ~nd insuffiril-n~ amounts of NrCAMvar polypeptide activity, inn1~ ing diabetes, obe~sity and cancer.
If the acLivity of N~{~AMvar polypepLide is in excess, sever~l approaches are available.
One ~pproach ~;""'1" i~t~ ~ n ~IPI il-P to a subjcct an inhibitor compound (:~nt:lgt~nic~) as .

herein~bove llf?s~ril~ along with ~ ph~ np~lti~ ~lly ~- CPpt~hlo c3rrier in an amount effective to inhibit activation by blocking binding of ligands to the NrCAMvar polypeptide. or by g a second signal, and thereby alleviating the abno~mal cnn~itton In ano~er approach, soluble forms of NrCAMvar polypeptides still capable of S binding the ligand in competi~ion with endogenous NrCAMvar polypeptide may be ~lminict~-red. Typic~ embodiments of such col-lpe~to,~ comprise fi~m~nts of the NrCAMvar polypeptide.
In still another approach, expression of the gene encoding endogenous NrCAMvar polypeptide can be inhi~ited using expression hk}cl~ing techniqllec. Known such t~fhni~lues 10 involve the use of ~n~i~en~e sequences, either intern~lly ~PnP~tPd or sep~rately minictPred. See, for PY~mrle O't:~onnor, J Neurochem (1991) 56:560 in Qligodeoxynucleotides as Antisense Inhibitors of Gene Expression. CRC Press, Boca Raton, F~ (1988). Altern~tively, oligonucleotides which form triple helices with the gene can be supplicd. See, for PY~mrle, Lee et al., Nucleic Acids Res (1979) 6:3073; Cooney 1~ et al., Science (1988) 241:456; Dervan er al., Science (1991) 251:1360. These oligomers can be ~r~mini~tered per se or the relevant oligomers c~n be e.~ e~ed in vivo.
Fa~ tre~ting a~n~m~1 cnn~liti~nc relat~d to an under~ iol~ of NrCAMvar and its activi~.y, several a~u~;l~ are also available. One approach ~ ,.. ~ ~ a~ t,o a subject a ~ lly effec~ive amount of a c~ which activates NrCAMvar 20 polypeptide, i.e.. an agonist as ~.. . ;h~l above, in ~ ,-~,~ iml with a ph~rm~r~ltir~lly acceptable carrier, to thereby alleviate the 3hn~m~ iliolL Alternatively, gene therapy rnay be employed to effect the en~lo~ru)l-~ production of NrCAMv~r by the relevant cells in the subject. For pYi~mrle, a polymlnl~ti~ of the invention may be engu~ d for expression in a reF~lici~tinn defective retroviral vector, as ~icc~csefl above. The retroviral expression Cs.)l~ UI.;I
25 may then be isolat~d and introduced into a p~c~ g~ing cell ~ d~ with a retrovira~l plasmid vector ~u-~ RNA ~nrc~in~ a polypeptide of the present invention such t'natt the p?r'~gin~
cell now produce~s inf~ti~lc viral p~rticles c~ -i"i,-g the gene of intere~st. These producer celLs rnay be ~ d to a subject for ~ ~gi"~P~ i-,g cells in vivo and ~..L),~.~.i~,n of the polypeptide in vivo. For overview of gene ther~py, see Cnapter 20, Gene 777~rapy and other A~r~le~ r 30 Genetic~based Therapeutic Approaclles, (~7lKl references cited therein) in Human Molecul~r Grnrtirs, T Str tcnan and A P Read, BIOS Scientific pnhlic'n~ Ltd (1996).

Fo~ ;on and Atlminict~ttion Peptide~s, such ats the soluble forTn of NrCAMvar polypeptides, atni agonists and 35 ~ntatgonist peptides o~ smnll rn ~ s~ may be ft~rrntll~t~ in c~ " ~ with a suitable phar r ~rPl~tirr~l c~ier. Such forrmll~tionc ~ ,~.ise a the..~r,~ lly effective arroLtnt of the .

polypeptif~ or CO~ and a ph~nn~c~utic~llya~xpt~ carrier f~ ~Y~, ~ - Such carriers ~nclude but are not limitf d to, saline, buffered salirle, dextrose, w~ter, glycerol, eth~nol, and f~ ""1~ nc thereof. Forrnnl~tit-n should suit the m~de of ~-h~Iin ~1- ..ti~n,andis well wi~in the skill of the art. The i~lV~ n furthcr relatcs to ph~rrn~ lti~-~l packs and kits ~""1" i~
5 one or more conti~in~rs filled with one or more of the ingredien~s of the a~f. r--~ iO,u.
compf~sitions of the invention.
Polypeptides and other compounds of the present lnvention may be employed alone or in a njllnf tion with other compounds, such as ~ pcuLiC ~l~
Preferred forrns of systemic ~-1- "i.~ if ~n of the ph~ ~-tif i~l co ,4~iLions include 10 inj~tion. typically by intravenous inj~,~ n Other injection routes, such as s~ c, u~ SC~ r, or i~ f"~Pnl, can be used. Alternative m~ns for systemic ~t(l"~ , i.ti~n include tr~ ;- "~ ~c~ and ~ cl lpl l n ~ - ..tion using p~ such aS bile salts or fusidic acids or other detergents. In addiaon, if properly formt~ tf~d in enteric f.)r f.. u, f~m~ tifmc~ oral ~ l. ,,tion rnay also be possible. A~ fL,~ io n of these C~ -~15 rnay also be topical and/or ky~slli7~1, in the forrn of salves, pastes, gels and the like.
The dosage r~nge ref~ired depends on the choice of pepade, the route of a~l",i,~ tifl-,thenatureoftherf~....,.li~ti~n,thenatureofthesub3ect~sfr~ inn,andthe j~r1~nt of the attf~n-1ing L" ;~ in.~f . . Suitable dosagPs, however, are in the range of O. l-100 ,ug/kg of subject Wide ~,~iati~ in the needed dosage, however, are to be PY~tf~j in view of 20 the variety of cc,n~u~b available and the differing f~ffi~Pn~iPC of various routes of :-- I".i":ii, ~tiC"L For PYnmrl~, oral ~ ation would be f~ ~ to require higher dosages than a~ ;,tic n by intravenous injPrti(m Variations in these dosage levels can be adjusted using stand~rd Pmpiri~ ~l routines for ~)~imi751tinll, as is we}l In u Irl ~lod in the art.
Polypepades used in ~ ,~..~ can also be ge-~ ~ted ~~ u h 1~1 n ~1 ICIy in the subject, in 25 trentment m~1~litiPs often referred to as "gene therapy~ as d~PSCrihPd above. Thus, f~x PY:~mpl~P, cells from a subject rnay be engine~ ~d with a polymlrl~P~ti IP~ such as a DNA or RNA, to encode a polypepade ex vivo, and for Py~mrle by the use of a retroviral plasmid vector. The cells are then introduced into the subj~c~.

30 FY~mr~
The ~Y~mr'Ps below are carned out using st~rd i ~ , ~, which are well known and rouane to those of sldll in the ~rt, exccpt where otherwise ~-, il~i in detail. The ~ l"~
illustrate, but do not limit the inventioL

35 F~Y~mple I

Cloning of human NrCAMvar eDNA
The HGS EST dntnhnse was sereened using the chiek NrCAM sequPnre and three HGS
EST clones (EST99669, EST237133, and EST373834) were obtained. EST99669 and EST237133 clones were from human adren~l cDNA library while EST373834 was from S human striatum cDNA library. cDNA elones EST237133 and EST373834 eonta~ned several ~eoRI or Eco RVX~Io I fragments, suggesting that inserts from several different genes may be present. Only those fragments eont~ining the EST sequenee homologous to NrCAM were subcloned and used for further ehar~ctpri~cn~ion. These clones were end sequeneed and used as probes labelled with [a-3ZPldCTP (,AmPr.ch~m) to screen a human fetal brain (20-wk) Agtl 1 cDNA library (Clontech). Four positive eDNA elones were isolated and the inserts were cloned into pBluescript l~ln$mi~1c (Maniatis et al., 1982).
1itionnl sections of the gene were isolated using gene-specifie primers to amplify eDNA
from the human fetal brain Mar~thonTM eDNA (Clonteeh) and the Agtl 1 fetal braineDNA library. Al~ sequeneing was performed on an ABI373 sequencer using the ABI
P~ISMTM dye terminator eyele sequeneing ready reaetion kit. Sequenees were ~ccPmhled using the Wiseonsin GCG package.

Co--lpalisol1 of both DNA and amino acid seq~enr,Ps for human NrCAMvar (SEQ ID
NO:1) and the ~!uhli.ch~od NrCAM seq~Pnre (Lane et al.) revealed that they were >99%
20 identir~1 in ove~la~ing regions. The DNA se~lPnre of human NrCAMvar of SEQ IDNO:1 is 77.1% i-~Pntirnl to that of ehiek gene while the arnino aeid sP~IIP~rP (SEQ ID
NO:2~ is 80% jrientienl Evidenee for alternative splieing of AEI9, AE12 and AE93, was htnin~d either through seq~eneing of cDNA clones or PCR products from human cDNAand in addition two novel regions, AElOK and AEIOL were found to be differentially 25 absent in the present (AElOK) versus Lanes (AElOL) sPq~1enre (see Figure 2).

a) ~xon structure:
EST99669 w~s found to contain a eontiguous genomie s~ enre not homologous to anyse.luen~,e of the chick gene. E~cnminn~iQn of this seq~enee revealed a splice donor 30 cc,l~se~ s sequence. This probably eorresponds to an intron:exon bound;lry represented in eDNA due to innomrlete mRNA processing. A donor and an acceptor were present in EST373~34. In addition to alternatively splieed regions AE12 and AE93, identified by Lane et al. (1996), AEI9 was also found to be absent from some cDNA fragments obtained from human fet~l brain cDNA. In ~d-iition, two novel alternative}y splieed 35 regions, encoding 10-amino-acid sections, were i~entified (see Figure 2). AE93 was W O 98/36062 PCT~GB98~W434 absent in EST237133 which was from hum~n adrenal cDNA library while both AE12 and AE93 were obscrved in PCR products from human adult brain cDNA.

bJ Results from Northern blots:
S A mRNA band of ~7.0kb was observed for hum~n brain, placenta, pancreas, adrenal medull~ and cortex, thyroid, and testis tissues. The results also showed that this gene is highly e~ ed in brain, pancreas, and adren~l cortex tissues (the levels of rnRNA on the blots used are controlled at Clon~Prh and sarnples are tested for their integrity by hyhrirlic:~tion With an actin gene probe).
cJ Chromosomal loca~ tion:
To obtain precise k?~ iQn of the human NrCAM IQCUS, primers from an intron (sbpl2) and from ~n exon (sbpl3) were c~Pci&nPd according to the se~lence of EST373834. These primers produced a PCR product of 214bp, and were used to screen 15 for the presence of this gene in the ~PnPhri~l~e 4 radiation hybrid panel. The res~lts were analysed by the WlGCR cYr nm~nt:ll m~rpin~ server and are shown in Table 1. These data place the human NrCAM gene on the long arrn of chromosomp 7 at 7q21-22 between D7S666 and D7S658.

20 Table 1: Data vectors obtained from testing GenP~rkl~e 4 ~adiation Hydrids panel using primers sbpl2 and 13.

25 NrCAM 00000 00100 10001 00010 00000 10110 00000 00000 01100 00000 Each digit corresponds to one of 93 ceil lines in the radia~ion hybrid panel. 0 and I
/ ~, esent negative and positcve PCR assays respectively. 2 shows that the assay was contradictory between duplicate experiments or was untested W O 98J36062 PCT~GB98/00434 .

Since panc,eatic fimction is intim:l~ely involved in the dev~loElmf~nt of diabetes, NrCAMvar becomes a t~rget m(~lecllle in the management of this disease. This suggestion is supported by the genomic mapping data. A locus for non-insulin~ependent diabetes mellitus (NIDDM), also c~lled Type II ~ het~, has been mapped to the same region of 5 ~ 7 as NrCAMvar (Prochazka, 1995).

CA 02243984 l998-07-27 SEQUENCE LISTING

INFORMATION FOR SEQ ID NO:1:
s (i) SEQUENCE C~ARACTERISTICS:
(A) LENGTH: 3997 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii~ MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:

3s GAGAGGATGG GACCTTGATC TGCAGAGCTA ATGGCAACCC CAAACCCAGA ATTAGCTGGT 1200 SUBSTITUTE SHEEr (RULE!26) - CA 02243984 l998-07-27 CGTGTGTGGC CAACACCACT CTGGACAGCG TCTCCGCCAG CGCTGTGCTT AGC~ 1920 ~5 ACCACCAAAC TGAAGTTTCT GGAACACAGA CCACAGCCCA GCTGAAGCTG TCTCCTTACG 2160 3s ACTTTTATGT TGAATATGGT GTAGCAGGCA GCAAAGAAGA ATGGAGAAAA GAAATTGTAA 3360 ATGGTTCTCG GAGCTTCTTT GGGTTAAAGG GTCTAATGCC AGGAACAGCA TACAAGTTTC 3q20 SUBSTITUTE SHEET (RULE 26) A 02243984 l998-07-27 W O 98/36062 PC~/GB98~00434 CTCCTGTCAA CGCCATGAAT TC~'l"l"l'~'l"l"l' AATCATAGAA CTTGATTCCG ATGATGTCTT 3960 INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1304 amino acids (B) TYPE: amino acid ~C) STRANDEDNESS: slngle (D) TOPOLOGY: linear ( ii ) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID No:2:

Met Gln Leu Lys Ile Met Pro Lys Lys Lys Arg Leu Ser Ala Gly Arg Val Pro Leu Ile Leu Phe Leu Cys Gln Met Ile Ser Ala Leu Glu Val Pro Leu Asp Pro Lys Leu Leu Glu Asp Leu Val Gln Pro Pro Thr Ile Thr Gln Gln Ser Pro Lys Asp Tyr Ile Ile Asp Pro Arg Glu Asn Ilc Val Ile Gln Cys Glu Ala Lys Gly Lys Pro Pro Pro Ser Phe Ser Trp Thr Arg Asn Gly Thr His Phe Asp Ile Asp Lys Asp Pro Leu Val Thr Met Lys Pro Gly Thr Gly Thr Leu Ile Ile Asn Ile Met Ser Glu Gly - SUBSTITI~TE SHEET (RULE 26) CA 02243984 l998-07-27 W O 98l36062 PCT/GB98/00434 Lys Ala Glu Thr Tyr Glu Gly VaL Tyr Gln Cys Thr Ala Arg Asn Glu Arg Gly Ala Ala Val Ser Asn Asn Ile Val Val Arg Pro Ser Arg Ser Pro Leu Trp Thr Lys Glu Lys Leu Glu Pro Ile Thr Leu Gln Ser Gly Gln ser Leu Val Leu Pro Cys Arg Pro Pro Ile Gly Leu Pro Pro Pro 0 Ile Ile Phe Trp Met Asp Asn Ser Phe Gln Arg Leu Pro Gln Ser Glu Arg Val Ser Gln Gly Leu Asn Gly Asp Leu Tyr Phe Ser Asn Val Leu Pro Glu Asp Thr Arg Glu Asp Tyr Ile Cys Tyr Ala Arg Phe Asn His Thr Gln Thr Ile Gln Gln Lys Gln Pro Ile Ser Val Lys Val Ile Ser Val Asp Glu Leu Asn Asp Thr Ile Ala Ala Asn Leu Ser Asp Thr Glu Phe Tyr Gly Ala Lys Ser Ser Arg Glu Arg Pro Pro Thr Phe Leu Thr Pro Glu Gly Asn Ala Ser Asn Lys Glu Glu Leu Arg Gly Asn Val Leu Ser Leu Glu Cys Ile Ala Glu Gly Leu Pro Thr Pro Ile Ile Tyr Trp Ala Lys Glu Asp Gly Met Leu Pro Lys Asn Arg Thr Val Tyr Lys Asn Phe Glu Lys Thr Leu Gln Ile Ile His Val Ser Glu Ala Asp Ser Gly Asn Tyr Gln Cys Ile Ala Lys Asn Ala Leu Gly Ala Ile His His Thr Ile Ser Val Arg Val Lys Ala Ala Pro Tyr Trp Ile Thr Ala Pro Gln Asn Leu Val Leu Ser Pro Gly Glu Asp Gly Thr Leu Ile Cys Arg Ala Asn Gly Asn Pro Lys Pro Arg Ile Ser Trp Leu Thr Asn Gly Val Pro .

Ile Glu Ile Ala Pro Asp Asp Pro Ser Arg Lys Ile Asp Gly Asp Thr Ile Ile Phe Ser Asn Val Gln Glu Arg Ser Ser Ala Val Tyr Gln Cys 420 425 q30 Asn Ala Ser Asn Glu Tyr Gly Tyr Leu Leu Ala Asn Ala Phe Val Asn Val Leu Ala Glu Pro Pro Arg Ile Leu Thr Pro Ala Asn Thr Leu Tyr 0 Gln Val Ile Ala Asn Arg Pro Ala Leu Leu Asp Cys Ala Phe Phe Gly Ser Pro Leu Pro Thr Ile Gln Trp Phe Lys Gly Ala Lys Gly Ser Ala Leu His Glu Asp Ile Tyr Val Leu His Glu Asn Gly Thr Leu Glu Ile Pro Val Ala Gln Lys Asp Ser Thr Gly Thr Tyr Thr Cys Val Ala Arg Asn Lys Leu Gly Met Ala Lys Asn Glu Val His Leu Glu Ile Lys Asp Pro Thr Trp Ile Val Lys Gln Pro Glu Tyr Ala Val Val Gln Arg Gly 545 s50 555 560 Ser Met Val Ser Phe Glu Cys Lys Val Lys His Asp His Thr Leu Ser Leu Thr Val Leu Trp Leu Lys Asp Asn Arg Glu Leu Pro Ser Asp Glu Arg Phe Thr Val Asp Lys Asp His Leu ,Val Val Ala Asp Val Ser Asp 595 600 . 605 Asp Asp Ser Gly Thr Tyr Thr Cys Val Ala Asn Thr Thr Leu Asp Ser Val Ser Ala Ser Ala Val Leu Ser Val Val Ala Pro Thr Pro Thr Pro Ala Pro Val Tyr Asp Val Pro Asn Pro Pro Leu Asp Leu Glu Leu Thr Asp Gln Leu Asp Lys Ser Val Gln Leu Ser Trp Thr Pro Gly Asp Asp Asn Asn Ser Pro Ile Thr Thr Ile His Asp Glu Tyr Glu Asp Ala Met W O 98l3606Z PCT/GB98/00434 His Lys Pro Gly Leu Trp His His Gln Thr Glu Val Ser Gly Thr Gln Thr Thr Ala Gln Leu Lys Leu Ser Pro Tyr Val Asn Tyr Ser Phe Arg 705 ~10 715 720 Val Met Ala Val Asn Ser Ile Gly Lys Ser Leu Pro Ser Glu Ala Ser Glu Gln Tyr Leu Thr Lys Ala Ser Glu Pro Asp Lys Asn Pro Thr Ala 0 Val Glu Gly Leu Gly Ser Glu Pro Asp Asn Leu Val Ile Thr Trp Lys Pro Leu Asn Gly Phe Glu Phe Asn Gly Pro Gly Leu Gln Tyr Lys Val Ser Trp Arg Gln Lys Val Gly Asp Asp Glu Trp Thr Ser Val Val Val Ala Asn Val Ser Lys Tyr Ile Val Ser Gly Thr Pro Thr Phe Val Pro Tyr Leu Ile Lys Val Gln Ala Leu Asn Asp Met Gly Phe Ala Pro Glu Pro Ala Val Val Met Gly His Ser Gly Glu Asp Leu Pro Met Val Ala Pro Gly Asn Val Arg Val Asn Val Val Asn Ser Thr Leu Ala Glu Val His Trp Asp Pro Val Pro Leu Lys Ser Ile Arg Gly His Leu Gln Gly Tyr Arg Ile Tyr Tyr Trp Lys Thr Gln Ser Ser Ser Lys Arg Asn Arg 885 . 890 895 Arg His Ile Glu Lys Lys Ile Leu Thr Phe Gln Gly Ser Lys Thr His Gly Met Leu Pro Gly Leu Glu Pro Phe Ser His Tyr Thr Leu Asn Val Arg Val Val Asn Gly Lys Gly Glu Gly Pro Ala Ser Pro Asp Arg Val Phe Asn Thr Pro Glu Gly Val Pro Ser Val Pro Ser Ser Leu Lys Ile Val Asn Pro Thr Leu Asp Ser Leu Thr Leu Glu Trp Asp Pro Pro Ser CA 02243984 l998-07-27 His Pro Asn Gly Ile Leu Thr Glu Tyr Thr LeU Lys Tyr Gln Pro Ile 980 985 9g0 Asn Asn Thr His Glu ~eu Gly Pro Leu Val Asp Leu Lys Ile Pro Ala Asn Lys Thr Arg Trp Thr Leu Lys Asn Leu Asn Phe Thr Thr Arg Tyr Lys Phe Tyr Phe Tyr Ala Gln Thr Ser Ala Gly Ser Gly Ser Gln Ile 0 Thr Glu Glu Ala Val Thr Thr Val Asp Glu Ala Gly Ile Leu Pro Pro Asp Val Gly Ala Gly Lys Val Gln Ala Val Asn Pro Arg Ile Ser Asn Leu Thr Ala Ala Ala Ala GlU Thr Tyr Ala Asn Ile Ser Trp Glu Tyr Glu Gly Pro Glu Tyr Ala Asn Phe Tyr Val Glu Tyr Gly Val Ala Gly Ser Lys Glu Glu Trp Arg Lys Glu Ile Val Asn Gly Ser Arg Ser Phe Phe Gly Leu Lys Gly Leu MeC Pro Gly Thr Ala Tyr Lys Phe Arg Val Gly Ala Val Gly Gly Pro Arg Phe Val Ser Ser Glu Gly Val Phe Glu Thr Gly Pro Ala Met Ala Ser Arg Gln Val Asp Ile Ala Thr Gln Gly Trp Phe Ile Gly Leu ~eC Cys Ala Val Ala Leu Leu Ile Leu Ile Leu Leu Ile Val Cys Phe Ile Arg Arg Asn Lys Gly Gly Lys Tyr Pro Val Lys Glu Lys Glu Asp Ala His Ala Asp Pro Glu Ile Gln Pro Met Lys Glu Asp Asp Gly Thr Phe Gly Glu Tyr Ser Asp Ala Glu Asp His Lys Pro Leu Lys Lys Gly Ser Arg Thr Pro Ser Asp Arg Thr Val Lys Lys GlU Asp Ser Asp Asp Ser Leu Leu Asp Tyr Gly Glu Gly Val Asn Gly - CA 02243984 l998-07-27 Gln Phe Asn G~u Asp Gly Ser Phe Ile Gly Gln Tyr Ser Gly Lys Lys Glu Lys Glu Pro Ala Glu Gly Asn Glu Ser Ser Glu Ala Pro Ser Pro 1285 12gO 1295 Val Asn Ala Uet Asn Ser Phe Val

Claims (25)

What is claimed is:

1. An isolated polynucleotide comprising a nucleotide sequence encoding the NrCAMvar polypeptide of SEQ ID:NO2; or a nucleotide sequence complementary to said nucleotide sequence.

2. The polynucleotide of claim 1 which is DNA or RNA.

3. The polynucleotide of claim 2 wherein said nucleotide sequence comprises the NrCAMvar polypeptide encoding sequence contained in SEQ ID:NO2.

4. The polynucleotide of SEQ ID NO: 1.

5. A polynucleotide probe or primer comprising at least 15 contiguous nucleotides of the polynucleotide of claim 3.

6. A DNA or RNA molecule comprising an expression system, wherein said expression system is capable of producing a NrCAMvar polypeptide comprising an amino acid sequence of SEQ ID:NO2 when said expression system is present in a compatible host cell.

7. A host cell comprising the expression system of claim 7.

8. A process for producing a NrCAMvar polypeptide comprising culturing a host of claim 7 and under conditions sufficient for the production of said polypeptide.

9. The process of claim 8 which further includes recovering the polypeptide from the culture.

10. A process for producing a cell which produces a NrCAMvar polypeptide thereof comprising transforming or transfecting a host cell with the expression system of claim 6 such that the host cell, under appropriate culture conditions, produces a NrCAMvar polypeptide.

11. Cells produced by the process of claim 10.

12. A polypeptide which comprises the amino acid sequence of SEQ ID
NO:2.

13. The polypeptide encoded in SEQ ID:NO2.

14. A NrCAMvar polypeptide prepared by the method of claim 9.

15. An antibody immunospecific for the NrCAMvar polypeptide of claim 12.

16. A method for the treatment of a subject in need of enhanced NrCAMvar polypeptide activity comprising:
(a) administering to the subject a therapeutically effective amount of an agonist to said polypeptide; and/or (b) providing to the subject NrCAMvar polynucleotide in a form so as to effect production of said polypeptide activity in vivo.

17. A method for the treatment of a subject having need to inhibit NrCAMvar polypeptide activity comprising:
(a) administering to the subject a therapeutically effective amount of an antagonist to said polypeptide; and/or (b) administering to the subject a nucleic acid molecule that inhibits the expression of the nucleotide sequence encoding said polypeptide; and/or (c) administering to the subject a therapeutically effective amount of a polypeptide that competes with said polypeptide for its ligand, substrate, or receptor.

18. A process for diagnosing a disease or a susceptibility to a disease in a subject related to expression or activity of NrCAMvar polypeptide in a subject comprising:
(a) determining the presence or absence of a mutation in the nucleotide sequence encoding said NrCAMvar polypeptide in the genome of said subject; and/or (b) analyzing for the presence or amount of the NrCAMvar polypeptide expression in a sample derived from said subject.

19. A method for identifying compounds which inhibit (antagonize) or agonize the NrCAMvar polypeptide which comprises:

(a) contacting a candidate compound with cells which express the NrCAMvar polypeptide (or cell membrane expressing NrCAMvar polypeptide) or respond to NrCAMvar polypeptide; and (b) observing the binding, or stimulation or inhibition of a functional response; or comparing the ability of the cells (or cell membrane) which were contacted with the candidate compounds with the same cells which were not contacted for NrCAMvar polypeptide activity.

20. An agonist identified by the method of claim 19.

21. An antagonist if identified by the method of claim 19.

22. A polynucleotide consisting essentially of a DNA sequence obtainable by screening an appropriate library containing the NrCAMvar gene under stringent hybridization conditions with a probe having the sequence of SEQ ID:NO 1 or a fragment thereof; and isolating said DNA sequence.

23. A polypeptide obtainable by expressing a nucleotide sequence comprising that of SEQ ID NO: 1

24. A method for the treatment of diabetes, obesity or cancer which comprises administering to the subject a therapeutically effective amount of a modulator of NrCAMvar polypeptide activity.

25. A process according to claim 20 for diagnosing presence of or susceptibility to diabetes, obesity or cancer.