Note: Descriptions are shown in the official language in which they were submitted.
CA 02224799 1997-12-16
W O 97/00952 PCT~US96/10618
HUhL~N EDG-2 ~r~R HOMOLOG
TECHNICAL FIELD
The present invention is in the field of ",olec-~'qr biology; more particularly the
5 present invention desc,iL,es a nucleic acid sequence and an amino acid sequence for a novel
human EDG-2 receptor homolog.
BACKGROUND ART
The EDG-2 ,eceptor is a putative G-protein coupled seven transmembrane ,eceptur
(T7G) which was initially cloned from sheep mRNA (GenBank U18405; Masana Ml et al
(1994) unpublished). Human edg-1 is cG"""only grouped with orphan ~eceptur:, because its
endogenous ligand is not known (Hla T and Maciag T (1990) J Biol Chem 265:9308-13).
Several T7G receptor:, have been classified as orphan rec~ tors; they include LCR-1 from
brain, the mas oncogene ~csoc:~ted with ~p d~ lllo d ca~c;nG",a, RDC-1 known from several
major organs and R334 from rat brain and testis. In some of these cases a ligand was initially
1 5 proposed and has since been discounted. The orphan rece~Jtors vary in number of amino acids
in i"sles Jl~ weight, in glycosylation sites, and p~esence and number of dis~ ide bonds (Watson
S and Arkinstall S (1994) The G P,ùtei, Linked Receptor Facts Book, Acade",:c Press San
Diego CA).
They are ho. wcr related to other T7Gs by their seven hydu~phob ~ dolllai.ls which
20 span the plasma ",e"ll,,dne and form a bundle of antiparallel a helices. These transmel"l,rdne
seg",ent~ (TMS) are des;y"ated by roman numerals l-VII and account for structural and
functional features of the receptor. In most cases the bundle of helices forms a binding pocket;
ho.vever when the binding site must acco"""oda~e more bulky " e'ecu~es the extracellular N-
terminal segment or one or more of the three extracellular loops participate in binding and in
25 s~hsequent induction of con(or",ational change in intracellular portions of the rece~,lor. The
activated receptor, in turn, interacts with an intracs"u'qr G-protein cou.~'ex which mediates
further intr~ro ller signalling activities generally the production of second messengers such
as cyclic AMP (cAMP), phospholipase C inositol l,i~,hosphate or ion channel proteins.
T7G receptors are e~Jressed and activated during numerous de\relop",e,ltal and disease
30 processes Ide"lilicdlion of a novel T7G receptor provides the opportunity to diagnose or
intervene in such processes and the receptor can be used in screening assays to identify
physic'~g;cal or pharmaceutical molecules which trigger prolong or inhibit its activity.
DISCLOSURE OF THE INVENTION
The subject invention provides a unique nucleotide sequence which encodes a novel
35 human EDG-2 receptor homolog (HEDG). The cDNA herein des;y"ated hedg was identified and
cloned using Incyte Clone No. 80853 from a rheume ~id synovium cDNA library.
CA 02224799 1997-12-16
WO 97/00952 PCTrUS96/10618
The invention relates to the use of nucleic acid and amino acid sequences ot HEDG, or its
variants, in the diagnosis or treatment of activated, inflamed or .~ a~cd cells and/or tissues
associ-l.sd with its eA~ression. Aspects of the invention include the ant snse DNA of hedg;
cloning or e~ression vectors containing hedg; host cells or orga";~,."s l,ans~or",ed with
S ex~ ,ession vectors containing hedg; a method for the production and recovery of purified HEDG
from host cells; and purified protein, HEDG, which can be used to identify inhibitors for the
downregulation of signal transcluction involving HEDG
BRIEF DESCRIPTION OF DRAWINGS
Figures 1 A and 1 B shows the alignment of the nucleic acid sequence (coding region of SEQ
0 ID NO: 1 ) and amino acid sequence (SEQ ID NO:2) for HEDG. The alignment of the sequences was
produced using MacDNAsis suttw~re (Hitachi Software Engineering Co Ud)
Figure 2 displays the alignment of HEDG with sheep EDG-2 (U18405; SEQ ID NO:3) and
human EDG-1 (Gl 119130; SEQ ID NO:4) receplur:i. Note the conserved Arg36 and Ser37
cleavage site cl,a,d-,1a~islic of these T7G ",~!e ~ s. Sequences for Fig. 2 were aligned using the
15 multisequence alignment prog,a", of DNAStar software (DNAStar Inc, Madison Wl).
MODES FOR CARRYING OUT THE INVENTION
As used herein and des;yl ,dled by the upper case abbreviation, HEDG, refers to an EDG2
receplor homolog in either naturally occurring or synthetic form and active ~dylllent~ thereof
which have the amino acid sequence of SEQ ID NO:2. In one embodiment, the polypeptide HEDG is
20 encoded by mRNAs tldns.;,il,ed from the cDNA, as des;ylldled by the lower case abbreviation,
hedg, of SEQ ID NO:1.
The novel human edg-2 ~eceplor ho",~'cg, HEDG, which is the subject of this patent
a~F' - ~n, was discovered among the partial cDNA sequences (Incyte Clone 80853) e)-~,,essed
in a rheumatoid synovium library. It is more distantly hG",ologous to human edg-1 which was
25 cloned from human vascular endull,vli- I cells and exl,ressed in epithelioid cells, fibroblasts,
",elanG.;ytes, and vascular smooth muscle cells.
An "oligonucleotide" is a stretch of nu~'eGItide residues which has a sufficient number of
bases to be used as an n' j ."er, amplimer or probe in a polymerase chain reaction (PCR).
Oligonu,vl~-otides are p~epar~d from geno",ic or cDNA sequence and are used to amplify, reveal or
30 confirm the presence of a similar DNA or RNA in a particular cell or tissue. Oligonucleolides or
oligomers cG",prise portions of a DNA sequence having at least about 10 nuclcotides and as many
as about 35 nucleotides, preferably about 25 nucleotides.
~ Probes" may be derived from naturally occurring or recombinant single- or double-
stranded nucleic acids or be cl,e", -lly synthesized. They are useful in detecting the presence
35 of identical or similar sequences.
CA 02224799 l997-l2-l6
W O 97/00952 PCTrUS96/10618
A "portion" or "fragment" of a polynucleotide or nucleic acid cor",i~lises all or any part
of the nuc'sQtide sequence having fewer nuc'e~t ie~ than about 6 kb, pre~erdbly fewer than
about 1 kb which can be used as a probe. Such probes may be labelled with reporter ",ole~ ~'es
usingSnick l.ansldlion, Klenow fill-in reaction, PCR or other methods well known in the art.
5 After pretesting to opli",i~e reaction conditions and to eliminate false positives, nucleic acid
probes may be used in Southern, northern or in ~1~ hyl"idi,dlions to determine v,/l,t,ll,er DNA
or RNA encoding HEDG is present in a cell type, tissue, or organ.
"Reporter" molecules are those radionuclides, enzymes, fluorescent,
cl-ell 'urn )escenl, or chromogenic agents which associa~e with, ~e t 'I ~'n the presence of, and
10 may allow qua"lificalion of a particular nu~'eo~;de or amino acid sequence.
"Recombinant nu~'8~: 'e variants" encoding HEDG may be synthesized by making use of
the "redundancy" in the genetic code. Various codon substitutions, such as the silent changes
which~roduce specific reslriction sites or codon usage-specific mutations, may be introduced
to opli-";~e cloning into a plasmid or viral vector or eA~,ression in a particular prokaryotic or
15 eukaryotic host system, respectively.
"Chimeric" "~oleu~'~s may be constructed by introducing all or part of the nu:'~clide
sequence of this invention into a vector containing additional nucleic acid sequence which might
be e~l~e~ted to change any one (or more than one) of the l~ ;"g HEDG chdrd~:teri:,lics:
cellular location, distribution, ligand-binding affinities, interchain affinities,
20 degradation/turnover rate, signalling, etc.
UActive" refers to those forms, ~,ag",enl:" or domains of any HEDG polypeptide which
retain the biologic and/or antigenic activities of any naturally occurring HEDG."Naturally occurring HEDG" refers to a polypeptide produced by cells which have not
been genetically engineered and specifically conte",plates various polypeptides arising from
25 post-translational ."o~ic~lions of the polypeptide including but not limited to acetylation.
carboxylation, glycosylation, phosphorylation, lipidation and acylation.
"Derivative" refers to those polypeptides which have been cl,e"~ "y i"odi~ied by suct~
tecl,r,,!es as ubiquitination, labelling (see above), pegylation (derivatization with
polyethylene glycol), and chemical insertion or substitution of amino acids such as or"itl,
30 which do not normally occur in human proteins.
"RecG",b. ,ant polypeptide variant" refers to any polypeptide which differs fromnaturally occurring HEDG by amino acid i"se,lions, deletions and/or suhstitutions, created
using lecG",b: ,anl DNA techniques. Guidance in determining which amino acid residues may be
rep'~ced added or deleted without ::~hC''.'~ 19 activities of interest may be found by companng
35 the sequence of HEDG with that of related polypeptides and ",il, "i~i"g the number of amino ac~d
CA 02224799 1997-12-16
PCT~US96/10618
W O 97/00952
sequence cl ,anges made in highly conserved regions.
Amino acid "substitutions" are conservative in nature when they result from replac;"g
one amino acid with another having similar structural and/or che",: -' prope,lies such as the
replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, or a
5 11"~or,i"e with a serine.
"Insel1ions" or "deletions" are typically in the range of about 1 to 5 amino acids. The
variation allowed may be ex,ueri",el)lally determined by producing the peptide s~",1i,e1ically or
by sy~1e,na1ically making i"se,lions deletions, or s~hstitutions of nuc'se1 'es in the hedg
sequence using recG" b:. ,a"1 DNA techrl les
A "signal or leader sequence" can be used when desired, to direct the polypeptide
through a ",e",brdne of a cell. Such a sequence may be naturally present on the poly~,eptides of
the present invention or provided from he1ele'~3cus sources by rec~r"5:.,d,lt DNA techn- les
An "~':gopert ~e" is a short stretch of amino acid residues and may be eA~.ressed from an
oligonuc'e~t:d6. It may be functionally equivalent to and the same length as (or cons;de,ably
15 shorter than) a fl~g",en1 " "portion " or "seg",en1 of a polypeptide. Such sequences co.,.p,ise
a stretch of amino acid residues of at least about 5 amino acids and often about 17 or more amino
acids, typically at least about 9 to 13 amino acids and of sufficient length to display biologic
and/or antigen-o activity.
~Inhibitor" is any sul~tance which retards or prevents a chemical or physiological
20 reaction or response. Common inhibitors include but are not limited to a"li~ense ",ole~'es
antibodies, and a"tdgon ~
~ Standard" eAvression is a quantitative or qualitative measurement for cGr"pari~on. It
is based on a s~ ,1icAIly appropriale number of normal sar" ~ les and is created to use as a basis
of c~l"~,arison when performing diag"o-~1ic assays running clinical trials, or f~IIDW;II9 patient
25 treatment profiles.
~ UAnimal" as used herein may be defined to include human dGme:,1ic (cats dogs, etc.)
agricultural (cows horses, sheep, etc) or test species (mouse, rat, rabbit etc).The present invention provides a nucleotide sequence uniquely identifying a novel seven
t,d,.~",en,b,ane receptor human EDG-2 or HEDG. Because HEDGis specifically ex~ ssed in
30 i"fla",ed rheumatoid synovium the nucleic acids (hedg) polypeptides (HEDG) and an1ibod es to
HEDGafe:useful in diagnostic assays which survey for increased recept~ production.
Excessive eA~ression of HEDGis likely to be Acsoci~ted with the activation of T Iymphocytes and
other cells which respond to i"flal"",ation and can result in the production of abundant
p,u1eases and other ",o'ecu'es which can lead to tissue damage or destruction. Therefore a
35 diaylloalic test for excessive ex~,ression of HEDG can accelertl1e diag"ûsis and proper 1,ed1",en1
CA 02224799 1997-12-16
W O 97/00952 PCT~US96/10618
of abnormal conditions caused by viral bacterial or fungal infections; allergic (esponses;
mechanical injury .,ssoci~led with trauma; hereditary d;se-~ges; Iy,."~h~."a or carcinoma; or
other con.litions which activate the genes of Iymphoid tissues.
The nu e cllide sequences encoding HEDG (or their complement) have numerous
5 a~p' -~ -ns in techr,.,~es known to those skilled in the art of ".~lecu'qr biology. These
techl, ~_es include use as hybri~ alion probes, use in the construction of oligo."e,:, for PCR,
use for ~;I"ul"osG",e and gene ~apF Ig, use in the ,~co",b: ,ant production of HEDG, and use in
gene,dlion of anliaense DNA or RNA, their che" :--1 analogs and the like. Uses of nu~ e: ~ es
enco~ ~9 HEDG di ~ losed herein are eAelllplary of known techr, les and are not i"tended to limit
10 their use in any techr,:, le known to a person of ordinary skill in the art. Fu.ll,er",ore, the
nucleotide sequences ~; losed herein may be used in ",e ec~l'qr biology techniques that have not
yet been dcveloped, provided the new teel---:, les rely on pfope,lies of nu: ect de sequences that
are currently known, e.g., the triplet genetic code, specific base pair interactions, etc.
It will be apprec;ated by those skilled in the art that as a result of the degeneracy of the
15 genetic code a multitude of HEDG-encoding nu e.,t ~e sequences may be produced. Some of these
will only bear minimal hG".o!agy to the nuc eot;~e sequence of the known and naturally
occurring HEDG. The invention has spec;f;c-Ally cGnle",plated each and every possible variation
of nu c'e~t; 'e sequence that could be made by sele 1i"g CGIIIl )ations based on possible codon
che ~es. These combinations are made in accG,ddnce with the aldnddrd triplet genetic code as
20 applied to the nu: e~t de sequence of naturally occurring hedg, and all such varialions are to be
considered as being :,~ecificP~y d; ~losed
Although the nu~ ~Gt;~e sequences which encode HEDG its derivatives or its variants are
pr~ferdbly capable of hybridi~i,.g to the nu~ e t; e sequence of the naturally occurring hedg
under sl-i..gent conditions, it may be advdn'~geous to produce nu 'e t:de sequences encoding
25 HEDG or its derivatives possesC;"g a subsldntially different codon usage. Codons can be s~le. t~d
to increase the rate at which eA~,ression of the peptide occurs in a particular prokaryotic or
eukaryotic eA~,re:ssion host in accG,dance with the frequency with which particular codons are
utilized by the host. Other reasons for suL,~lantially altering the nu eo: ~:e sequence enco~ ,9
HEDG and/or its derivatives without altering the encoded aa sequence include the production of
30 RNA transcripts having more desirable properties such as a greater half-life than transcripts
produced from the naturally occurring sequence.
Nu- -_'ide sequences encoding HEDG may be joined to a variety of other nucleotide
- sequences by means of well est-h' ~hed recG",bina"l ~A-techniques (Sambrook J et al ~1989)
Alo e ~'qr Cloning: A Laboratory Manual Cold Spring Harbor Laboratory Cold Spring Harbor
- 35 NY; or Ausubel FM et al (1989) Current Protocols in Molecular Biology John Wiley & Sons
CA 02224799 1997-12-16
Wo 97/00952 PCr/US96/10618
New York City). Useful nucleotide sequences for joining to hedg include an asso,l",e"t of cloning
vectorssuchasplas", ~ cos",.~ lambdaphagederivatives phage-.. " andthelike. Vectors
of interest include e~,rassion vectors replication vectors, probe gene,dlion vectors,
sequencing vectors etc. In general vectors of interest may contain an origin of l~p -n
5 functional in at least one o,yani~"" convenient resl~i ;tion endonucle~ce sensitive sites and
select~ e markers for one or more host cell systems.
Another aspect of the subject invention is to provide for hedg-specific hyLridi dlion
probes capable of hyL,ricli ing with naturally occurring nu~leotide sequences encoding HEDG.
Such probes may also be used for the d~leclion of similar T7G encoding sequences and should
10 preferably contain at least 50% of the nu: ~Dt;~es from the hedg sequence. The hyL,.idi ut;on
probes of the subject invention may be derived from the nu 'eo::de sequence p,ese,ltad as SEQ ID
NO:1 o~ from geno,.. ~ sequences including pror"ùter, enhancer~ or introns of the native gene.
I IyL.ri li ation probes may be labeled by a variety of reporter molecu es using tecl,n ,Les well
known in the art.
PCR as desc,ibed US Patent Nos. 4 683195; 4,800195; and 4,965 188 provides
additional uses for oligonu 'e - es based upon the nu~ le lide sequence which encodes HEDG.
Such probes used in PCR may be of recombinant origin, chemically s~"ltl,esi ad or a mixture of
both. O: ~ ~ ~-al~ may co-,.prise discrete nu~ . t 'e sequences employed under opli..,i ed
conditions for ide,llificalion of hedg in specific tissues or didyllGslic use. The same two
20 o gs l,e,:" a nested set of Q ~ I,ers or even a degene,dte pool of o i~ n-er:. may be e" r!cycd
under less sl,i"genl con litions for identification of closely related DNAs or RNAs.
Other means of producing specific hyl,ridi dtion probes for hedg include the cloning of
nucleic acid sequences encoding HEDG or HEDG derivatives into vectors for the production of
mRNA probes. Such vectors are known in the art are co"""e, ially available and may be used
25 to sy"ll,esi e RNA probes in vitro by means of the addition of the appr-,priate RNA polymerase
as T7 or SP6 RNA polymerase and the apprupriala reporter mo e~ ~ -s.
It is possible to produce a DNA sequence or portions thereof entirely by synthetic
chemistry. After synthesis, the nucleic acid sequence can be inserted into any of the many
available DNA vectors and their ,especli./e host cells using techn es which are well known
30 the art. Moreover synthetic chemistry may be used to introduce mutations into the nucle.L~
sequence. Alte",ataly, a portion of sequence in which a mutation is desired can be synthesized
and reco",~..,ed with longer portion of an existing genomic or reco",~:nant sequence.
The nucleotide sequence for hedg can be used in an assayto detect i"fla"""dlion or disease
~csoc;aled with abnor",al levels of HEDG expression. The cDNA can be labeled by methods known
35 in the,~art added to a fluid cell or tissue sample from a patient and incubated under h),bridi i.)s
CA 02224799 1997-12-16
W O 97100952 PCTAUS96/10618
cohdilions. After an inc~h~tion period the sample is washed with a cG",paliLle fluid which
optionally cGhl~i ls a reporter ",ole u~e. After the cG",palible fluid is rinsed off the reporter
",~!e-ll'e is qua,ltitdted and cG",par~d with a sldndard as previously defined. If kinase
expression is siy"i~icanlly di~erenl from standard e~-u,ession the assay indicates
i"~la"""ation or disease.
The nu~'eotide sequence for hedg can be used to construct hyl"idi~dlion probes for
r"_rr: ,g the native gene. The gene may be mapped to a particular ch~",oso",e or to a specific
region of a cl"u",osG",e using well known ll~c_pr .Ig techn ~ues. These le.:l",' ~es include in~
hyL,ridi~dlion of chrc",oso",al spreads (Verma et al (1988) Human Ch,vr,,osu,,,es; A Manual of
Basic Techn:~ues Pe,yalllon Press New York City), flow-sorted cl,ro",osG",al prepa,dtions, or
artificial cl,fo",osG",e constructions such as yeast artificial chr~",osG",es (YACs), bacterial
artificial chr~",osG",es (BACs) bacterial P1 constructions or single cl,,u~osGme cDNA
libraries.
In ~i~ h),l,ridi alion of cl,ru",osG",al preparations and physical ll-~Pr -l9 techh, les
such as linkage analysis using es~ ed chr~",osol"al markers are invaluable in exten' ,9
genetic maps. EAdlll 'es of genetic map data can be found in the yearly genome issue of Science
(eg 1994 265:1981f). Often locating a gene on the cl,,u,,,osG,,,e of another ",a",r"-' -, species
may reveal ~csû~ 'ed markers which can be used to help identify the an-'cgous human
cl "~o" ,os~" ,e.
2û New n~'eotide sequences can be assiylled to chlcJIllosGh~al subregions by physical
mapping. The " ~p~' ,g of new genes or nuc'e~t'~'s sequences provide useful landmarks for
inve:,ligelora searching for disease genes using positional cloning or other gene discovery
tecl,., ~ les Once a disease or syndrome such as ataxia telangiectasia (AT), has been crudely
localized by genetic linkage to a particular genor, e region, for ex~l"ple AT to 11q22-23 (Gatti
et al (1988) Nature 336:577-580), any sequences mapping to that area may ~epresen~ or
reveal genes for further invesligAIion. The nu~'eotide sequence of the subject invention may
also be used to detect differences in gene sequence between normal and carrier or affected
individuals.
I~L~C' ~t ie sequences encoding hedg may be used to produce a purified oligo- orpolypeptide using well known methods of recGI"b ,anl DNA technology. Goeddel (1990 Gene
E~uression Tecl",~!cgy Methods and Enzymology Vol 185 Acade",'~ Press San Diego CA) is
one among many publications which teach eAuression of an isolated nuclcotide sequence. The
oligopeptide may be expressed in a variety of host cells etther prokaryotic or eukaryotic. Host
cells may be from the same species from which the nucleotide sequence was derived or from a
dfflerent species. Advantages of producing an oligonucleotide by reccmbinant DNA technology
CA 02224799 l997-l2-l6
WO 97/00952 PCT~US96tlO618
include obtaining ~dequ~te amounts of the protein for pu,ilicalion and the availability of
simplified pu,ilicalion procedures.
Cells l-ilnslu-,,,ed with DNA encoding HEDG may be cultured under conditions suitable
for the exuression of T7Gs, their e~lr~ce" llar, I,c~ns,,,e,,,brane or intracellular domains and
5 recovery of such peptides from cell culture. HEDG (or any of its domains) produced by a
recG",' ,anl cell may be secreted or may be conlai"ed intracellularly depending on the
particular genetic construction used. In general it is more convenient to prepare recG" ~. ~ar,l
proteins in secreted form. Pu~ilicalion steps vary with the production process and the
particular protein produced. Often an ol.gop~ plide can be produced from a ch' "eric nu 'eJt'~e
10 sequence. This is acco",F'i~ hed by ligating the nuc'~otides from hedg or a desired portion of the
polypeptide to a nucleic acid sequence encoding a polypeptide domain which will facilitate
protein purification (Kroll DJ et al (1993) DNA Cell Biol 12:441-53).
In addition to recoll b'..a"l production l,.lg",enls of HEDG may be produced by direct
peptide synthesis using solid-phase techniques (eg Stewart et al (1969) Solid-Phase Peptide
15 Synthesis WH Freeman Co, San Eranc;~co CA; Merrifield J(1963) J Am Chem Soc
85:2149-2154. Automated synthesis may be achieved for e~alllrle~ using Applied Biosystems
431A Peptide Synthesizer (Foster City CA) in accGrJance with the instructions provided by the
manufacturer. Additionally, a particular portion of HEDG may be mutated during direct
ay,lthesis and co",'~:.,ed with other parts of the peptide using che" ' -' IllelhOds.
~ WEDG for antibody induction does not require biological activity; ho.. ever, the protein
must be a"ligen:~ Fe~Jtides used to induce specific antibodies may have an aa sequence
consi li"g of at least five aa, p,eferably at least 10 aa. They should mimic a portion of the aa
sequence of the protein and may contain the entire aa sequence of a small naturally occurring
"~o'e~u'e such as HEDG. An a"tigen c portion of HEDG may be fused to another protein such as
2s keyhole limpet hemocyanin, and the cl ' "eric " oleou'e used for antibody production.
Antibodies specific for HEDG may be produced by inoc~ tion of an app,upriale animal
with the polypeptide or an anliyen-c I~dylllenl. An antibody is specific for HEDG if it is produced
against an epitope of the polypeptide and binds to at least part of the natural or reco",~.,anl
protein. Antibody production includes not only the stimulation of an immune response by
30 injection into animals but also analogous processes such as the production of synthetic
a"lil,o~'es the screening of recoi"binant immunoglobulin libraries for specific-binding
".o'ecu'es (eg Orlandi R et al (1989) PNAS 86:3833-3837, or Huse WD et al (1989)Science 256:1275-1281) or the in vitro stimulation of Iymphocyte populations. Current
technology (Winter G and Milstein C (1991) Nature 349:293-299) provides for a number of
35 highly specific binding reagents based on the p~i"e;~le~ of antibody formation. These techniques
CA 02224799 1997-12-16
W O 97/00952 PCTrUS96/10618
may be adapted to produce ",Q 6~u es which spe~ifically bind HEDGs.
An additional embodiment of the subject invention is the use of HEDG specific antibodies
inhibitors leceptor:, or their analogs as bioactive agents to treat i,lfla,.,..,alion or disease
including but not limited to viral bacterial or fungal infections; allergic responses;
5 mechanical injury associaled with trauma; hereditary ~ise~ses; I~""pl,G,--a or caK;.,o",a; or
other conditions which activate the genes of Iymphoid tissues.
Bioactive co",posi~ions co"",,isi"g agonists antagonists teceplors or inhibitors of
HEDG may be administered in a suitable therapeutic dose determined by any of several
mell,odo'r ss including clinical studies on mammalian species to determine maximal t~ le
10 dose and on normal human subjects to determine safe dose. Additionally the bioactive agent may
be CG""~ : -ed with a variety of well ~s - shted compounds or CG"~pO .itions which enllance
stability or pharrn~rol~ properties such as half-life. It is co"ler"~,ldled that the
II,erapeutic bioactive co",position may be delivered by intravenous infusion into the
b ~ a", or any other effective means which could be used for treating prcLI~ "s involving
15 excessive Iyl"phG. ~te and leukocyte lldllici~illg.
Rheumatoid arthritis is currently evaluated on the basis of swelling re~ponse toNSAlDs x-rays, etc. HEDG is most likely ex~,,essed on the surface of the liiJn)blas-~ T and B
Iy."~)hoc~tes monocyte/macrophages or mast cells which co",p,ise the cells of the i"ll~",ed
synovium. Once a~eq~te sldndar-l~ are Pst- hed an assay for the abnG""al ex~ress;on of
20 HEDG ?s-a viable diag"o:,lic tool for assess;"g the extent that RA has p,oy,~ssed. The eA~,ession
of HEDG in. a sustained i"fl~"""alory tes~onse makes it a valuable II,e,~peutic target for
screening drug libraries. Inhibitors of HEDG are useful for controlling signal transductiQn and
signaling cAccades in cells of the rheumatoid synovium.
The e.~",~!es below are provided to illustrate the subject invention. These ex~lll, !es are~5 provided by way of illustration and are not included for the purpose of limiting the in
vention.
INDUSTRIAL APPLICABILITY
Isolation of mRNA and Construction of the cDNA Library
The hedg sequence of this app ~ ~n was first ide"lilied in Incyte Clone No. 80853
among the sequences cGI"prising the rheumatoid synovium library. Rheumatoid synovial tissue
30 was obtained from the hip joint removed from a 68 year old female with erosive nodular
rheumatoid arthritis. The tissue was frozen ground to powder in a mortar and pestle and Iysed
i"""edialely in buffer conlai~ ,g guanidinium isothiocyanate. Lysis was followed by several
phenol-chloroform extractions and ethanol precipitations. Poly-A+ mRNA was isoiated using
biotinylated oligo d(T) and streptavidin coupled to paramagnetic particles (Poly(A) Tract
35 Isolation System Promega Madison Wl).
CA 02224799 1997-12-16
WO 97/00952 PcrluS96/10618
Using this Poly-A+ mRNA a custom cDNA library was constructed by Stlalagene (La
Jolla CA). Synthesis of cDNA was primed with oligo d(T), and adapter oligonu~'e~:- 'es were
ligated onto the cDNA ".~ ec~'es enabling them to be inserted into the Uni-ZAPTM vector system
(Stratagene). Alternative unidi.ectional vectors might include, but are not limited to, pcDNAI
(Invitrogen, San Diego CA) and pSHlox-1 (Novagen Madison Wl).
I l l~olqtion of cDNA Clones
The phage", d forms of individual cDNA clones were obtained by the in yivo excision
process, in which the host bacterial strain was co-infected with both the library phage and an
f1 helper phage. Polypeptides or enzymes derived from both the library-containing phage and
the helper phage nicked the DNA initiated new DNA synthesis from defined sequences on the
target E)NA, and created a smaller single sllanded circular phage", d DNA ",~!e~ ~'e that
included all DNA sequences of the pBluescript phage",:d and the cDNA insert. The phage" ~ DNA
was released from the cells, purified, and used to reinfect fresh host cells (SOLRTM, St,atagel)e)
where double-stranded DNA was produced.
DNA was purified using the QIAWELL-8 Plasmid Pu,i~ica~ion System (QIAGEN Inc
Chals~r,o,ll, CA) an anion-eAchange resin system with EMPORET~ ",e",brane tecl,nQ!agy (3M
Minneap~' - MN). The DNA was eluted from the pu,i~icalion resin and p,epared for DNA
sequencing and other analytical manipulations.
111 Sequencing of cDNA Clones
The cDNA inserts from random isolates of the rheumatoid synovium library were
sequenced in part. Methods for DNA sequencing are well known in the art. Conventional
enzymatic methods e" '~ycd DNA polymerase Klenow ~ld9lllel~ SEQUENASE~) (US Biocl,e", --
Corp Cleveland OH) or Taq polymerase to extend DNA chains from an oligonu 'est,~'e primer
annealed to the DNA template of interest. Methods have been developed for the use of both
single- and double-stranded templates. The chain termination reaction products were
electrophoresed on urea-acrylamide gels and detected either by autoradiog,aphy (for
radionuclide-labeled precursors) or by fluorescence (for fluorescent-labeled precursors).
Recent improvements in mechar,i~ed reaction preparation sequencing and analysis using the
fluorescent detection method have permitted e~ ansion in the number of sequences that are
determined per day using machines such as the Catalyst 800 and the Applied Biosystems 377 or
373 DNA sequencers.
V Homology Searching of cDNA Clones and Deduced Ploteir.s
Each sequence so obtained was cc l l l~al ed to sequences In GenBank using a search
algG,ill"" developed by Applied Biosystems and incorporated into the INHERITT~ 670 Sequence
Analysis System. In this algorithm Pattern Specification Language (developed by TRW Inc Los
CA 02224799 1997-12-16
W O 97/00952 PCTAUS96/10618
Angeles CA) was used to determine regions of ho",o!agy. The three palalllale~s that detemline
how the sequence comparisons run were window size window offset, and error i !erdnce. Using
a co",b.nalion of these three pa,d",~tera the DNA d~t~h~-ce was sear~;l,ed for sequences
containing regions of hor"o!cyy to the query sequence and the a~ ,u~,iale sequences were
5 scored with an initial value. Suhsequently these hG"ia'Dgous regions were examined using dot
matrix holllQl-lgy plots to distinguish regions of hGIIlolcgy from chance ",dt~;l,es.
~ Smith-~haterman alig"",er,ls were used to display the results of the hG":~!agy search.
..
Peptide and protein sequence homologies were asce,ldi"ed using the INHERITT~ 670Sequence Analysis System in a way similar to that used in DNA sequence homologies. Pattem
10 Specifi~ion Language and para",a~er w;.ldo.~j were used to search protein d~'Ahs-ces for
sequences containing regions of ho",o!cgy which were scored with an initial value. Dot-matrix
hGlllo'Dy~/ plots were examined to distinguish regions of significant hGIll~lo~y from chance
",dtches.
Altematively BLAST which stands for Basic Local Alignment Search Tool is used tosearch for local sequence alignments (Altschul SF (1993) J Mol Evol 36:290-300; Altschul
SF et al (1990) J Mol Biol 215:403-10). BLAST produces aligl""e"ta of both nucle,tide and
aa sequences to determine sequence similarity. Because of the local nature of the alig"",e"ta,
BLAST is espec;a~y useful in d~ter"~ ~ ,9 exact ",atcl,es or in iderllify;.,g hGII~D'~ jS. Wher~as it
is ideal for ",alcl,es which do not contain gaps, it is i"appropriale for performing motif-style
20 searching. The fundd,nental unit of BLAST algorithm output is the High-scoring Segment Pair
(HSP).
An HSP consists of two sequence fragments of arbitrary but equal lengths whose
~' S ,",ent is locally maximal and for which the r' 3 ,r"enl score meets or eYceeds a II"esho'd or
cutoff score set by the user. The BLAST approach is to look for HSPs t;et~- een a query sequence
25 and a ~ hA-ce sequence, to evaluate the sl~ lic~l s;yll 'i~-nce of any ",atcl,es found and to
report only those ",dlcl)es which satisfy the user sele_ted II,iesho'd of siy"iticance. The
parameter E le '' hes the sl;~tisliG~lly sigr,iricanl II,rasho'd for lepo,ti"g rl~t~h~ce sequence
",d~ches. E is i"t~",r~led as the upper bound of the ex~,ectad frequency of chance occurrence of
an HSP (or set of HSPs) within the context of the entire d~t~hase search. Any d ~l~h~se sequence
30 whose match satisfies E is reported in the program output.
V Identification, Full Length Cloning, Sequencing and Translation
Analysis of INHERITTM results from randomly picked and sequenced portions of clones
trom the rheumatoid synovium library identified Incyte 8~853 as a hGIllc'Dg of sheep EDG-2
recept,or. The partial sequence displayed 92.6% identity with nucle~,lide sequence of ~ccession
U18405 in GenBank (Masana Ml et al supra). The cr)NA insert CGIll,uiisillg Incyte 80853 was
CA 02224799 1997-12-16
Wo 97/00952 PCr/US96/10618
fully sequenced and used as the basis for cloning the full length cDNA.
The cDNA was extended to full length using a modified XL-PCR (Perkin Elmer)
procedure as di~ losed in Patent Arp ;~-tion Serial No 08/487,112 filed 7 June 1995 and the
rheumatoid synovium cDNA library as a te",plale. Primers were desiylled based on known
5 sequence; one primer was s~",ll,esi~ed to initiate extension in the a,~ti~en~e direction (XLR =
TCATCTTGATCCCCATCCC; i I C I G) and the other to extend sequence in the sense direction (XLF =
AGTCTCCGAGTATTGGG l ~;~; l G l G). The primers allowed the sequence to be eAtended 'outward"
generali"g a".r ~,ns containing new ullh.lo.~,l nuc s.,~;de sequence for the gene of interest. The
primers were designed using Oligo 4.0 (National ''ios. ences Inc, Plymouth MN) to be 22-30
1 o nu e~t;des in length, to have a GC content of 50% or more, and to anneal to the target sequence
at te",pe,dlures about 68~-72~ C. Any stretch of nu~1e ~;des which would result in hairpin
structures and primer-primer dimerizations were avoided.
By fe'l~.; ,g the instructions for the XL-PCR kit and thoroughly mixing the enzyme and
reaction mix high fidelity amplification is obtained. Beginning with 25 pMol of each primer
5 and the lecG"""ended conce,lt~dtions of all other col-,pone--ls of the kit PCR was pe-h""ed
using the Peltier Thermal Cycler (PTC200; MJ Research Wale,l- .~., MA) and the f~ll J,;.,g
parameters:
Step 1 94~ C for 60 sec (initial denaturation)
Step 2 94~ C for 15 sec
Step 3 65~ C for 1 min
Step 4 68~ C for 7 min
Step 5 Repeat step 2-4 for 15 ad.litional cycles
Step 6 94~ C for 15 sec
Step 7 65~ C for 1 min
Step 8 68~ C for 7 min + 15 sec/cycle
Step 9 Repeat step 6-8 for 11 additional cycles
Step 10 72~ C for 8 min
Step 11 4~ C (and holding)
At the end of 28 cycles 50 ~l of the reaction mix was removed; and the remaining30 reactioi~ rriix was run for an additional 10 cycles as outlined below:
Step i 94~ C for 15 sec
Step 2 65~ C for 1 min
Step 3 68~ C for (10 min + 15 sec)/cycle
Step 4 Repeat step 1-3 for 9 additional cycles
Step 5 72~ C for 10 min
A 5-10 1ll aliquot of the reaction mixture was analyzed by ele-;t,ophoresis on a low
concer.l.dtion (about 0.6-0.8%) agarose mini-gel to determine which reactions were
successful in extending the sequence. Although all extensions potentially contain a full length
gene some of the largest products or bands were selected and cut out of the gel. Further
40 pu-ificalion involved using a col"",ercial gel exl.aclion method such as QlAQuickTM (QIAGEN
CA 02224799 1997-12-16
W O 97/00952 PCT~US96/10618
Inc). After recovery of the DNA, Klenow enzyme was used to trim single-all~nded, nuc'eQti~e
ove,l,angs creating blunt ends which facilitated ~.'!S -n and cloning.
After ethanol preGipitation, the products were redissolved in 13 1ll of ligation buffer.
Then, 1111 T4-DNA ligase (15 units) and 1~L1 T4 polynucleotide kinase were added, and the
5 mixture was incuhated at room tempet~ure for 2-3 hc~urs or o~ernight at 16~ C. Co~ ,el~--l ~.
coli cells (in 40 1ll of appropriate media) were transformed with 3 1ll of ligation mixture and
cultured in 80 1ll of SOC medium (Sambrook J et al, supra). After incuhation for one hour at
37~ C, the whole tran~ r",dlion mixture was plated on Luria Bertani (LB)-agar (Salllbreok J
et al, supra) containing carbenicillin at 25 mg/L (2xCarb). The fo'la~ ;.,9 day, 12 co'~n es
were ,dndo",ly picked from each plate and cultured in 150 ~11 of liquid LB/2xCarb medium
placed in an individual well of an appropridle, co",.llercially-available, sterile 96-well
microtiter plate. The fo'lDwing day, 5 111 of each overnight culture was l-dnsler-ed into a non-
sterile 96-well plate and after dilution 1:10 with water, 5 ~LI of each sample was llansfer.~d
into a PCR array.
For PCR ar. r' 'ica~ion, 15 ~LI of conce.. ~,dled PCR reaction mix (1.33X) containing
0.75 units of Taq polymerase, a vector primer and one or both of the gene specific primers used
for the extension reaction were added to each well. Amplification was pe.lu...,ed using the
follcwi.,g conditions:
Step 1 94~ C for 60 sec
Step 2 94~ C for 20 sec
Step 3 55~ C for 30 sec
Step 4 72~ C for 90 sec
Step 5 Repeat steps 2-4 for an additional 29 cycles
Step 6 72~ C for 180 sec
Step 7 4~ C (and holding)
Aliquots of the PCR reactions were run on agarose gels together with molecular weight
markers. The sizes of the PCR products were co",pared to the original partial cDNAs, and
app.op!iate clones were s~lecled, ligated into plasmid and sequenced.
The nuc ' o ~;d o and aa sequences for human HEDG are shown in Figure 1. The coding
region of hedg begins at nucleotide 309 and ends at nu:'e~';de 1403 of SEQ ID NO:1. When the
three pcss:b'e l,anslalions of HEDG were searched against protein rl~t~h~ceS such as Swissrlol
and PIR, no exact Illat.;l,es were found. Figure 2 shows the comparison between the amino acid
sequences of HEDG, sheep EDG-2 (U18405) and human EDG-1 (Gl 119130).
V I Antisense analysis
Knowledge of the correct, co",~ ? cDNA sequence of HEDG enables its use as a tool for
antisense technology in the invealig~tion of gene function. Oligonucleotides, cDNA or genomic
fragments cG~prisi~g the antisense strand of hedg are used either in vitro or in vivo to inhibit
e,.~,ression of the mRNA. Such tecl"~Q'cgy is now well known in the art, and antisense molecu'es
CA 02224799 1997-12-16
WO 97/00952 PCr/US96tlO618
can be designed at various locations along the nucleotide sequences. By treatment of cells or
whole test animals with such anlisense sequences, the gene of interest is effectively tumed off.
Frequently, the function of the gene is asceildi-,ed by observing behavior at the intracellular,
cellular, tissue or oryanislllal level (eg, lethality, loss of differentiated function, cllanges in
5 mor~,holoyy~ etc).
In addition to using sequences constructed to interrupt lldns.;,i~.lion of a particular open
reading frame, modifications of gene ex~,ression is obtained by designing al~tiaense sequences to
intron regions, pru,.,oter/enhancer elements, or even to trans-acting regulatory genes.
Similarly, inhibition is achieved using Hogeboom base-pairing m~thedo'cJy, aiso known as
10 "triple helix" base pairing.
V 11 E~,r~ssion of HEDG
E~ ssion of hedg is accG", I hed by subcloning the cDNAs into app~opriale e,.~,r~ ssion
vectors and l,ana~e~ti,.g the vectors into ar~'cgous e,~.ression hosts. In this particular case,
the cloning vector previously used for the generdlion of the cDNA library also provides for
15 direct ex~ ssion of hedg sequences in E. coli. U~,sl,ea", of the cloning site, this vector contains
a pru..,ùler for 13-g~'~to-c;~i~ce, f~"Dwed by sequence containing the amino-terminal Met and
the suhsequent 7 residues of ~-g~ ;t"sidase. Illlll,ed;alely f.l' .\;,lg these eight residues is an
engineered ba-;teriophage promoter useful for artificial priming and l,dns~ ,lion and fo
providing a number of unique endonucle~~e resl,i~;tion sites for cloning.
Induction of the isol~ d l,a"s~t:cted bacterial strain with IPTG using sldndard ul~tllods
produces a fusion protein cor,e:.ponding to the first seven residues of 13-gala~losid~ce, about 15
residues of "linker", and the peptide encoded within the cDNA. Since cDNA clone inserts are
gene,dled by an esse"lially random process, there is one chance in three that the included cDNA
will lie in the correct frame for proper llanslalion. It the cDNA is not in the proper reading
frame, it is obtained by deletion or i,lse,lion of the apprupridle number ot bases using well
known ,llell,ods including in vitro muldgenesis, digeslion with exonuc'-~e lll or mung bean
nuc'es~e, or the inclusion of an oligonucleotide linker of appropridle length.
The hedg cDNA is shuttled into other vectors known to be useful for exl,ression of protein
in specific hosts. Oligonucleotide primers containing cloning sites as well as a segment of DNA
(about 25 bases) sufficient to hybridize to stretches at both ends of the target cDNA is
synthesized cllell -~y by standard Ill~lhods. These primers are then used to amplify the
desired gene segment by PCR. The resulting gene segment is digesled with appfop,idle
lesl,i~tion enzymes under standard conditions and isolated by gel electrophoresis. Alternately,
similar gene sey,llenl~ are produced by digeslion of the cDNA with appr~,priale restriction
enzymes. Using appropridle primers, seylllenl~ of codin-l sequence from more than one gene are
CA 02224799 1997-12-16
WO 97/00952 PcrluS96/10618
ligated tog~tl,er and cloned in appropridle vectors. It is possible to opli",i~e e~,ression by
construction of such chimeric sequences.
Suitable e~uression hosts for such chimeric ",~le les include, but are not limited to,
mammalian cells such as Chinese I la",:,ter Ovary (CHO) and human 293 cells insect cells such
..
5 as Sf9 cells yeast cells such as Saccharomyces cerevisiae. and bacteria such as ~ colt. For
each of these cell systems a useful e,~l,,ession vector also includes an origin of r~ 1' n to
allow prop~g~tion in bacteria and a select~'e marker such as the B-lactd",ase antibiotic
resistance gene to allow plasmid selection in bacteria. In addition, the vector may include a
second sele~ -' e marker such as the neomycin phosphol,dnslerase gene to allow s~l&ction in
10 I~dn:,~tcted eukaryotic host cells. Vectors for use in eukaryotic eA~,,ession hosts require RNA
p,ocessi"g el~ ."e"t~ such as 3' polyadenylation sequences if such are not part of the cDNA of
interest.
Ad ' 'icnally, the vector cor,lzi.,s pro",oters or enhancers which increase genee,~.,ession. Such p,u",-~ter:, are host specific and include MMTV SV40 and metallothionine
15 pru",ote,:, for CHO cells; trp lac tac and T7 pro",o~ers for bacterial hosts; and alpha factor
alcohol oxidase and PGH pro",otera for yeast. Transc,i~lion enhancer:" such as the rous
sarcG",a virus enhancer are used in mammalian host cells. Once ho",ogeneous cultures of
~eco" bi ,dnt cells are obtained through sldnddld culture ",etl,ods, large quantities of
reco".'~:.,a,ltly produced HEDG are recovered from the conditioned medium and analyzed using
20 ch,ol,,atoyldphic methods known in the art.
V 111 Isolation of necG,~ h~ant HEDG
HEDG is eA~,essed as a ch' "eric protein with one or more additional polypeptidedomair~s added to facilitate protein purification. Such p~ icdtion facilitating dol"ai"s include
but arè'not limited to metal cl,eldli"g peptides such as histidine-tr~",lophan modules that allow
25 pu.ification on i"""obili~ed metals protein A domains that allow pu.ilication on immobilized
immunoglobulin and the domain utilized in the FLAGS extension/affinity purification system
(Immunex Corp Seattle WA). The inclusion of a cleavable linker sequence such as Factor XA or
ente'~ol~i"ase (Invit,ogen) between the pu,ilication domain and the HEDG sequence is useful to
facilitate e~ression of HEDG.
30 I X Testing of Chimeric T7Gs
Functional chimeric T7Gs are constructed by combining the extr~cellu' r receptive
sequences of a new isoform with the trans",e",brdne and intracellular segments of a known
isoform for test purposes. This concept was de",onsl,ated by Kobilka et al (1988 Science
240:1310-1316) who created a series of chimeric a2-132 adrenergic receptors (AR) by
35 inse,li"g prog,essively greater amounts of a2-AR transmembrane sequence into 132-AR. The
CA 02224799 1997-12-16
W O 97/00952 PCTrUS96/10618
binding activity of known agonists changed as the l"~leu~ e shifted from having more a2 than 132
conformation and inten"edidle constructs de",on:il,dted mixed specificity. The spe~ ifi~ for
binding ar,lagon;sls ho.~ever cGr,el~led with the source of the domain Vll. The i",,~o,ld,)ce of
T7G domain Vll for ligand recGg" ~ian was also found in chimeras utilizing two yeast a-factor
~eceptol~ and is significant because the yeast receplors are cla~ ied as ",: ~e -neous
,eceptol:,. Thus functional role of specific domains appears to be preserved throughout the
T7G family regardless of category .
In parallel fashion internal se~""e"ta or cytoplasmic domains trom a particuîar
isoform are exchal-ged with the anAIogo~s domains of a known T7G and used to identify the
structural deten"i"ar,l:j responsible for coupling the receptors to trimeric G-proteins
(Dohlman et al (1991) Annu Rev Biochem 60:653-88). A chimeric receptor in which
domains V Vl and the intracellular connecting loop from B2-AR were suhstit~ted into a2-AR
was shown to bind ligands with a2-AR speci~ie;ty but to stimulate adenylate cyclase in the
manner of B2-AR. This de",on~l,dtes that for ad~ene,!Jic-type rece~,~ura G-protein
recoy" ~icn is present in domains V and Vl and their connecti"g loop. The OppG~;~' situation was
predicted and observed for a chimera in which the V->VI loop from a1-AR replaced the
cGrlespori li.,g domain on B2-AR and the resulting lece~Jtor bound ligands with B2-AR
spec;f;~;ily and activated G-protein-",edia~ed phosphatidylinositol turnover in the a1-AR
manner. Finally, chimeras constructed from muscarin ,eceptùrs also de",onsl,dted that V-
>Vl loop is the major determinant for -spe- if i~ity of G-protein activity (Bolander FF supra).
Chimeric or modified T7Gs containing suhstitutions in the eAl,_~e ular and
lldnslllelllbldne regions have shown that these portions of the (ecep~or determine ligand binding
specificity. For eAdlllr!e two Ser residues conserved in domain V of all adlene,y;c and D
catecholamine T7G feceptù,~ are necessary for potent agonist activity. These serines are
believed to form hydrogen bonds with the cdtecllol moiety of the agonists within the T7G bindmg
site. Similarly an Asp residue present in domain lll of all T7Gs which bind b ageni~ amines
believed to form an ion pair with the ligand amine group in the T7G binding site.
Functional cloned T7Gs are ex~ ressed in heler. ~gous e,~ ssion systems and their
b-o~~ir-' activity ~csessed (eg Marullo et al (1988) Proc Natl Acad Sci 85:7551-55; King ~t
al (1990) Science 250:121-23). One heterologous system introduces genes tor a ",a"""a'~
T7G and a mammalian G-protein into yeast cells. The 17G is shown to have applopriate ligand
specificity and affinity and trigger appropriate biological activation--growth arrest and
morphological changes--of the yeast cells.
An altemate procedure for testing chimeric receptors is based on the procedure
utilizing the P2U purinergic receptor (P2U) as published by Erb et al (1993 Proc Natl Acad
CA 02224799 1997-12-16
WO 97/00952 PCT/US96/10618
Sci 90:10449-53). Function is easily tested in cultured K562 human leukemia cells because
these cells lack P2U receptor:,. K562 cells are transfected with ex,uression vectors COnla;~l 19
either normal or ch ..eric P2U and loaded with fura-a. fluorescent probe for Ca++. Activation
of properly assembled and functional P2U receptors with extracellular UTP or ATP mobilizes
5 intracellular Ca++ which reacts with fura-a and is measured spectrofluoru...el.ically.
As with the T7G receptors above, chimeric genes are created by combining sequences
for extracellu' r receptive segments of any newly discovered T7G polypeptide with the
nucleotides for the transmembrane and intracellular seg---enls of the known P2U ".ole~u'e.
Bathing the transfected K562 cells in ",:~ o.~ells containing appropriale ligands triggers
1 0 binding and fluofescerlt activity defining ~ C~ol~ of the 17G ".ole ~'e. Once ligand and function
are es'-'' ~hed, the P2U system is useful for defining antagonists or inhibitors which block
binding and prevent such fluoresce.,t reactions.
X Production of HEDG Specific Antibo~ies
Two app-oacl)es are utilized to raise a,ltil,od s to HEDG, and each app-oach is useful for
1 5 generaling either polyclonal or ,.,onoclonal antibodies. In one appruach, denatured protein from
reverse phase HPLC sepa-alion is oblai.-ed in quantities up to 75 mg. This denatured protein is
used to immunize mice or rabbits using alandard p-otocols; about 100 ~- U9ldlllS are ~deq~te
for immu.,i~dlion of a mouse, while up to 1 mg might be used to immunize a rabbit. For
identifying mouse hy~,ido",as, the denatured protein is radioiodinated and used to screen
20 potential murine B-cell hyl,.idG,..as for those which produce antibody. This procedure requires
only small quantities of protein, such that 20 mg is sufficient for labeling and screening of
several thousand clones.
In the second app-uach, the amino acid sequence of an app-op,iate HEDG domain, as
deduced from l.dnslalion of the cDNA, is analyzed to deter--,;.,e regions of high antigen ~ity.
25 Oligopeptides col"~uris;ng appropriate hyd~ophilic regions, as illustrated in Figure 3, are
sy,ltl,esked and used in suitable immu"i~dlion protocols to raise antibodies. Analysis to select
app.op~idle epit~pes is described by Ausubel FM et al (supra). The optimal amino acid
sequences for immunization are usually at the C-terminus, the N-terminus and those
intervening, hy~,uph"ic regions of the polypeptide which are likely to be exposed to the
30 external en~i,or""ent when the protein is in its natural conformation.
Typically, selecled peptides, about 15 residues in length, are synthesized using an
Applied Biosystems Peptide Synthesizer Model 431A using fmoc-chemistry and coupled to
keyhole limpet hemocyanin (KLH; Sigma, St Louis MO) by reaction with M-maleimidoben-
zoyl-N- hydroxysucci"i",ide ester (MBS; Ausubel FM et al, supra). If necess~ry, a cysteine is
CA 02224799 1997-12-16
WO 97/00952 PCr/US96/10618
introduced at the N-terminus of the peptide to permit coupling to KLH. Rabbits are immunized
with the peptide-KLH co",, 'ex in Colll~'Ete Freund's adjuvant. The resulting antisera are tested
for antipeptide activity by binding the peptide to plastic, b'c-k ,9 with 1% bovine serum
albumin, reacting with antisera, washing and reacting with labeled (radioactive or
5 fluorescent), affinity purified, specific goat anti-rabbit IgG.
Hy~ridG",as are prepared and screened using -~;ldnda,d tecl)l,.~es Hybrid6i"as of
interest are dete~;t~Pd by screening with labeled HEDG to identify those fusions producing the
",onoclonal antibody with the desired specili~,ity. In a typical protocol, wells of plates (FAST;
Becton-Dickinson, Palo Alto CA) are coated during incubation with affinity purified, specific
10 rabbit anti-mouse (or suitable antispe~ ~s lg) ar,li6c~ ~s at 10 mg/ml. The coated wells are
blocked with 1% BSA, washed and inc~h-tPd with su,uerlldldrlt~ from hybrido",as. After washing
the wells are inc~hAted with labeled HEDG at 1 mg/ml. Supe",dla"ts with specific antibodies
bind more labeled HEDG than is dcte- ble in the background. Then clones producing specific
a,l~ibod s are eA~.anded and subjected to two cycles of cloning at limiting dilution. Cloned
15 hybrido",as are injected into pristane-treated mice to produce ascites, and ",onoclonal antibody
is purified from mouse ascitic fluid by affinity ~h~o~dluy,aphy on Protein A. I\lonoclonal
antibodies with a~i"ilies of at least 10B M-1, preferably 109to 101~ or al.ùnger, are typically
made by slandd,d procedures as des~,ibed in Harlow and Lane (1988) Antibodies: A Laboratory
Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY; and in Goding (1986)
20 ~lcnoclonal Antibodies: Principles and Practice, Acadel,. ~ Press, New York City, both
incorporated herein by l~lerence.
X I Diagnostic Test Using HEDG Specific Antibodies
Particular HEDG antibodies are useful for im,e:,liydlillg signal transduction and the
diag"osis of infectious or hereditary condi~ions which are cha,acte,i~ed by differences in the
25 amount or distribution of HEDG or do~.lal.~dll, products of an active signaling c~q-~ctqde. Since
HEDG was found in a human rheumatoid library, it appears to be upreg~'qtPd in cell types
mainly involved in immune protection or defense.
Diaylloalic tests for HEDG include Ill~:lhods utilizing antibody and a label to detect HEDG in
human body fluids, membranes, cells, tissues or extracts of such. The polyl e~Jtides and
30 a~ .od e5 of the present invention are used with or without ",odi~icalion. Frequently, the
polypeptides and antibodies are labeled by joining them, either covalently or noncovalently,
with a substance which provides for a detectable signal. A wide variety of labels and con,ug~tion
techniques are known and have been reported extensively in both the sc;c.,li~ic and patent
literature. Suitable labels include radionuclides, enzymes, substrates, cofactors, inhibitors,
35 fluorescer,l agents, chemiluminescent agents, chromogenic agents, magnetic particles and the
CA 02224799 1997-12-16
PC rluS96/10618
WO 97/00952
like. Paten~ teaching the use of such labels include US Patent Nos. 3,817,837; 3,850,752;
3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241. Also, recG"lb nanl
immunoglobulins may be produced as shown in US Patent No. 4,816,567, inco,,uo,aled herein
by reference.
A variety of prutocols for measuring soluble or membrane-bound HEDG, using either
polyclonal or monoclonal a,.~ odies specific for the protein, are known in the art. Examples
include enzymc I l~ed immu"osorbenl assay (ELISA), r~dic..ll",l",oassay (RIA) and
fluo,~scenl activated cell sorting (FACS). A two-site ."onoclonal-based immu"oassay utilizing
monoclonal antibodies reactive to two non-inle,~ring ~eF t~Fes on HEDG is prefer,ed, but a
10 cG""~t:tili~/e binding assay may be tll~ yed. These assays are desu,il,ed, among other places, in
~ 'd~t, DE et al (1983, J Exp Med 158:1211f).
X l l Purification of Native HEDG Using Specific Antibodies
Native or recGIllb..~a~ll HEDG is purified by immunoaffinity chrorllaloy,dphy using
afl!;L''"?S specific for HEDG. In general, an immu"odlfi,l ~y column is constructed by
covalently coupling the anti-TRH antibody to an activated ch,o",ato~a,dph c resin.
Polyclonal immunoglobulins are prepared from immune sera either by precipitationwith a"""on-~m sulfate or by pu,ilicdlion on immobilized Protein A (Pl,ar",adcia LKB
Biotecl,no'a~y, Piscala.:ay NJ). Likewise""onGclonal antibodies are prepared from mouse
ascites fluid by a"""on-~m sulfate precipitation or cl,,u,,,alog,dphy on immobilized Protein A.
Partially purified immunoglobulin is covalently alldcl-ed to a cl,~o,,,alug,dphio resin such as
CnBr-activated Sepharuse (Pharmacia LKB Biole.:l,r,a!c=,y). The antibody is coupled to the
resin, the resin is b'oo'.o~l and the derivative resin is washed according to the manufacturer's
instructions.
Such immunoaffinity columns are utilized in the pl"ilicalion of HEDG by preparing a
fraction from cells containing HEDG in a soluble form. This prepdrdtion is derived by
so' ~i~ir;~ion of whole cells or of a subcellular fraction obtained via differenlial centrifugation
(with or without addition of detergent) or by other .ll~thods well known in the art.
Alte"ldli~ly, soluble HEDG containing a signal sequence is secreted in useful quantity into the
medium in which the cells are grown.
A soluble HEDG-containing preparation is passed over the immunoaffinity column, and
the column is washed under conditions that allow the preferential absc,rbance of HEDG (eg, high
ionic strength buffers in the presence of detergent). Then, the column is eluted under
condilions that disrupt antibody/protein binding (eg, a buffer of pH 2-3 or a high concentration
of a chaotrope such as urea or thiocyanate ion), and HEDG is collected.
3 5 X l l l Drug Screening
CA 02224799 1997-12-16
WO 97/00952 PCr/USs6/10618
This invention is particularly useful for screening therapeutic compounds by using
HEDG or binding fragments thereof in any of a variety of drug screening techniques. The
polypeptide or fragment e",F'cycd in such a test is either free in solution affixed to a solid
support borne on a cell surface or located i,lt,_~e" Il-rly. One method of drug screening
utilizes eukaryotic or prokaryotic host cells which are stably l,ansfGr",ed with recG",~i.,ar,l
nucleic acids ex~"essi"g the polypeptide or l~aylllent. Drugs are screened against such
transformed cells in competitive binding assays. Such cells either in viable or fixed form are
used for sldndard binding assays. One measures for example the fo""ation of cGr, ~' ~s
between HEDG and the agent being tested. Alternatively one examines the diminution in
cGrll~ ' : f~""alion between HEDG and a receptur caused by the agent being tested.
Thus the present invention provides ",ell,ods of screening for drugs or any other agents
which affect signal tran-~duction. These ",etl,ods well known in the art cG""~ise cGnla.;ti"g
such an agent with HEDG polypeptide or a ~,ag",ent thereof and assaying (i) for the plesence of
a co" r -:: between the agent and the HEDG polypeptide or f,dg",ent or (ii) for the p(esence of a
CGIIIrE:: between the HEDG polypeptide or fragment and the cell. In such competitive binding
assays the HEDG polypeptide or f-ày",enl is typically labeled. After suitable incuh~tion free
HEDG polypeptide or fla9"~ent is separated from that present in bound form and the amount of
free or uncomplexed label is a measure of the ability of the particular agent to bind to HEDG or
to interfere with the HEDG and agent co",r' :
Another tecl,r'~ e for drug screening provides high throughput screening for compounds
having suitable binding affinity to the HEDG poly~eptides and is des~,il,ed in detail in European
Patent Applicalion 84/03564 published on September 13 1984 i"cGrl.G,dted herein by
r~fe,ence. Briefly stated large numbers of different small peptide test compounds are
s~ l,esi~ed on a solid substrate such as plastic pins or some other surface. The peptide test
2 5 compounds are reacted with HEDG polypeptide and washed. Bound HEDG polypeptide is then
delecled by ",ell,ocls well known in the art. Purified HEDG are also coated directly onto plates
for use in the dlo~e",erllioned drug screening techn.~ es. In addition non-neulrdli~i"g
antiL- "ss are used to capture the peptide and i"""obili~e it on the solid support.
This invention also cor,l~",plates the use of co"",~lilive drug screen ~9 assays in which
neutralizing antibodies capable of binding HEDG specifically compete with a test compound for
binding to HEDG polypeptides or fragments thereof. In this manner the a"lil,od;es are used to
detect the presence of any peptide which shares one or more anligen'~ determinants with HEDG.
XIV Rational Drug Design
The goal of rational drug design is to produce structural analogs of biologically active
polypeptides of interest or of small molecules with which they interact agonists antagonists
CA 02224799 1997-12-16
W O 97/00952 PCTrUS96/10618
or inhibitors. Any of these exdmr'es are used to fashion drugs which are more active or stable
forms of the polypeptide or which enl~ance or interfere with the function of a polypeptide iD
vivo (eg Hodgson J (1991) Bio/Technology 9:19-21, incorporated herein by reference).
In one approach, the three-dimensional structure of a protein of interest, or of a
s protein-inhibitor co",rle ~, is determined by x-ray crys~allo~raphy, by computer rr~odeUng or,
most typically, by a co"~bina~ion of the two approaches. Both the shape and charges of the
polypeptide must be ascertained to elucidate the structure and to determine active site(s) of the
molecule. Less often, useful i~fum~dlion regarding the structure of a polypeptide is gained by
modeling based on the structure of holl ~'~gous proteins. In both cases, relevant structural
0 infor",alion is used to design efficient inhibitors. Useful exdl", 'es of rational drug design
includes ",~'e~ J1es which have improved activity or stability as shown by Braxton S and Wells
JA (1992, Biochemistry 31:7796- 7801) or which act as inhibitors, agoni~l~, or antagonists
of native peptides as shown by Athauda SB et al (1993 J Biochem 113:742-46), i"cGr~,o,dted
herein by ,eference.
1 5 It is also possible to isolate a target-specific antibody, select~d by functional assay, as
described above, and then to solve its crystal structure. This app,oach, in p,i", ~e, yields a
phammacore upon which slJhse~uent drug design is based. It is possible to bypass protein
crystallography altogether by generating anti-idiotypic an~iLQ~'Es (anti-ids) to a functional,
pharmacologically active antibody. As a mirror image of a mirror image, the binding site of the
20 anti-ids is e~l~e~t d to be an analog of the original recq~or. The anti-id is then used to identify
and isolate peptides from banks of che" ' ally or biologically produced p~"ides. The isolated
pepticles then act as the phar",aco,t:.
By virtue of the present invention, sufficient amount of polypeptide are made available
to perform such analytical studies as X-ray crys'-'lc3 dphy. In addition, knowledge of the
25 HEDG amino acid sequence provided herein provides guidance to those e",r'~;"g computer
r"ode'' ~9 tecl", ~ es in place of or in addition to x-ray cry ' 'lcyl~phy.
XV Ide.,tilic..lion of Other ~e..bers of the Signal Transduction Complex
The inventive purified HEDGis a research tool for ide"lifica~ion, characterization and
purification of interacting G or other signal transduction pathway proteins. P~ artive lab~
30 are il,cG"~ordled into a selected HEDG domain by various methods known in the art and used ID
vitro to capture interacting mcle ~l~s. A prefer,~d method involves labeling the primar~
amino groups in HEDG with 1251 Bolton-Hunter reagent (Bolton, AE and Hunter, WM (1973)
Biochem J 133: 529). This reagent has been used to label various molecules without
concomitant loss of biological activity (Hebert CA et al (1991) J Biol Chem 266: 18989;
3~ McColl S et al (1993) J Immunol 150:4550-4555).
CA 02224799 1997-12-16
W O 97100952 PCTAJS96/10618
Labeled HEDG is useful as a reagent for the pu,ificalion of ".e!e ~'es with which it
interacts. In one embodiment of affinity pu,ificalion""e",b,dne-bound HEDG is covalently
coupled to a chlo",dlog,dphy column. Cell-free extract derived from synovial cells or putative
target cells is passed over the column, and molecules with appfop,iate affinity bind to HEDG.
5 HEDG-complex is recovered from the column, and the HEDG-binding ligand d;ia.ssoci~ted and
subjected to N-terminal protein sequencing. This aa sequence is then used to identify the
captured molecule or to design degenerate oligonu-,leotide probes for cloning the relevant gene
from an appropriate cDNA library.
In an alter"ale method, antibodies are raised against HEDG, spe~;fic~-lly l"onoclonal
10 ar,lil,cdies. The ",onoclonal dr,~ od eE are screened to identify those which inhibit the binding of
labeled HEDG. These ",onoclonal antibodies are then used therapeutically.
XVI Use and Administration of Antibodies, Inhibitors, or Antagonists
Antibodies, inhibitors, or antagonists of HEDG (or other t,eatt"ents to limit signal
transduction, LST) provide different effects when adminhtered II,e,apeutically. LSTs are
15 formulated in a nontoxic, inert, pharmaceutically ~reept-~le aqueous carrier medium
preferably at a pH of about 5 to 8, more preferdbly 6 to 8, although pH may vary according to
the characteristics of the antibody, inhibitor, or antagonist being formulated and the condition
to be treated. Cha,a.;t~,ialics of LSTs include solubility of the ",e'e ~'e!, half-life and
anligen ,, Iy/immullogen Iy. These and other characterialics aid in defining an effective
20 carrier. Native human proteins are pref~:r.ed as LSTs, but organic or s~ntl~elic mclec~'es
resulting from drug screens are equally effective in particular situations.
LSTs are delivered by known routes of adl"i.)i~l-dlion including but not limited to topical
creams and gels; transmucosal spray and aerosol; t,ansde""al patch and bandage; i",ect-~'e,
intravenous and lavage formulations; and orally ad",..,;slered liquids and pills particularly
formulated to resist stomach acid and enzymes. The particular formulation, exact dosage, and
route of admini~l-dlion is determined by the attending physician and varies according to each
specific situation.
Such determinations are made by considering multiple varia~'es such as the cond,~ion to
be treated, the LST to be administered, and the pharmacokinetic profile of a particular LST.
Additional factors which are taken into account include severity of the disease state, patient's
age, weight, gender and diet, time and frequency of LST ad". ~;~I.ation, possible cG",b. ,ation
with other drugs, reaction sensitivities, and tolerance/response to therapy. Long acting LST
fommulations might be administered every 3 to 4 days, every week, or once every two weeks
de~,en ' ,9 on half-life and clearance rate of the particular LST.
3s Normal dosage amounts vary from 0.1 to 100,000 micrograms, up to a total dose of
CA 02224799 1997-12-16
W O 97/00952 PCT~US96/10618
about 1 g, depeh ,9 upon the route of ad" ,;;,l-alion. ~ u -nce as to particular dosages and
methods of delivery is provided in the literature; see US Patent Nos. 4 657,760; 5 206 344;
or 5,225,212. Those skilled in the art employ different formulations for different LSTs.
Adm ,i~l,dlion to cells such as nerve cells necessit~tPs delivery in a manner diflere,lt from that
5 to other cells such as vascular endoll,c'-' cells.
It is cor,le",plated that abno""al signal transduction, trauma, or ~1;seqRes which trigger
HEDG activity are treatable with LSTs. These condilions or ~ise~ces are ~specifically diay"osed
by the tests ~iiscussed above and such testing should be pe,l~r",ed in sus~-e~lPd cases of viral,
bacterial or fungal infections; allergic responses; mechanical injury ~sso.:-lPd with trauma;
10 hereditary dise~-~es; Iy",pho",a or carcinoma; or other conditions which activate the genes of
Iymphoid tissues.
All pub ~ ns and patents mentioned in the above -specific~t:on are herein i"cGr~Grdted
by reference. Various ".~ ~s and variations of the desc,iL,ed method and system of the
invention will be apparent to those skilled in the art without departing from the scope and
15 spirit of the invention. Although the invention has been described in conne~ion with specific
preler,ed embodiments, it should be understood that the invention as claimed should not be
unduly limited to such specific e",bod,."ents. Indeed various modifications of the above-
desc,iL,ed modes for carrying out the invention which are obvious to those skilled in the field of
",olecu' r biology or related fields are intended to be within the scope of the f~"~wi.,g claims.
CA 02224799 l997-l2-l6
WO 97/00952 PCl'tUS96tlO618
s~:uu~N~ LISTING
(1) GENERAL INFORMATION
(i) APPLICANT: Coleman, Roger
Guegler, Karl J.
Au-Young, Janice
B~n~' -n, Olga
Seilhamer, Jeffrey J.
(ii) TITLE OF THE lNV NllON: A NOVEL HUMAN EDG-2 R~ '1'OK HOMOLOG
(iii) NU.MBER OF ~UU~N~:S: 6
(iv) CORRES~ONv N~ nnR~.~S:
(A) ADDRESSEE: Incyte Pharmaceuticals, Inc.
(B) STREET: 3174 Porter Drive
(C) CITY: Palo Alto
(D) STATE: CA
(E) COUN1KY: USA
(F) ZIP: 94304
(v) COM~U~:K READABLE FORM:
(A) MEDIUM TYPE: Diskette
(B) C~ U1~:K: IBM Compatible
(C) OPERATING SYSTEM: DOS
(D) SOFTWARE: FastSEQ Version 1.5
(vi) ~uKR~ APPLICATION DATA:
(A) APPLICATION NUMBER: TO BE ASSIGNED
(B) FILING DATE: 20-JUN-1996
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: 60/000,352
(B) FILING DATE: 20-JUN-1995
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: 08/567,817
(B) FILING DATE: 06-DEC-1995
(viii) Al~OKN~Y/AGENT INFORMATION:
(A) NAME: Glaister, Debra J.
(B) REGISTRATION NU.MBER: 33,888
(C) REFERENCE/DOCKET NUMBER: PF-0042 PCT
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 415-855-0555
(B) TELEFAX: 415-845-4166
(C) TELEX:
(2) INFORMATION FOR SEQ ID NO:1:
QU~NU~: CHARACTERISTICS:
(A) LENGTH: 1875 base pairs
(B) TYPE: nucleic acid
(C) sTR~Nn~nN~s single
24
CA 02224799 l997-l2-l6
WO 97/00952 PCI'/US96/10618
(D) TOPOLOGY linear
(li) MOLECULE TYPE cDNA
(vii) INMEDIA$E SOURCE
(A) nrRR~Ry Rheumatoid Synovium
(B) CLONE 80853
(xi) S~Qu~N~ DESCRIPTION SEQ ID NO 1
GGCAGGTACG GCCGGATTCC CGGGTCGACC ACGCGTCCGC TCTCAAGGGA ACAG~lC~lG 60
CCCAGGTCTG TGGGTACTCA GCATGGATAT CA~l~lCC-l GTGAGTGATG GGAAAGAACT 120
AGCAGAGGTG GACGTCTGAT TTATGAAGCT CCCCATCCAC CTATCTGAGT ACCTGACTTC 180
TCAGGACTGA CACCTACAGC ATCAGGTACA CAG~rl-lCC TAGCATGACT TCGATCTGAT 240
CACCACACAA GAAAATTTGT CTCCCGTAGT TCTGGGGCGT GTTCACCACC TACAACCACA 300
GAGCl~lCAT GG~rGC~ATC TCTACTTCCA lCCCl~ AAT TTCACAGCCC CAGTTCACAG 360
CCATGAATGA ACCACAGTGC TTCTACAACG AGTCCATTGC ~lu~l.llAT AACCGAAGTG 420
GAAAGCATCT TGCCACAGAA TGGAACACAG TCAr-rAAr-CT GGTGATGGGA CTTGGAATCA 480
~i~lll~lAT CTTCATCATG TTGGCCAACC TAl ~Gl-AT GGTGGCAATC TATGTCAACC 540
GCCGCTTCCA rlllCClATT TATTACCTAA TGGCTAATCT GG~l~ GCA GA l ~ lG 600
~l~GGll~GC CTACTTCTAT CTCATGTTCA ACACAGGACC CAATACTCGG AGACTGACTG 660
TTAGCACATG G~rC~llC~l CAGGGCCTCA TTGACACCAG CCTGACGGCA r~ ~l~GCCA 720
ACTTACTGGC TATTGCAATC GAGAGGCACA TTACG~rl~ll CCGCATGCAG CTCCACACAC 780
GGATGAGCAA CCGGCG~rA ~l~ lCA ll~lG~lCAT CTGGACTATG GCCATCGTTA 840
~l~G~rGClAT ACCCAGTGTG GG~rGGAACT GTATCTGTGA TATTGAAAAT TGTTCCAACA 90O
TGGCACCCCT CTACAGTGAC TCTTACTTAG r~rl~ aGGC CATTTTCAAC ll~l~ACcT 960
ll~l~lAAT G~rwll~lC TATGCTCACA ~r~r GGCTA ~l~ CGCCAG AGGACTATGA 1020
GAA~r~lCG GCATAGTTCT GGACCCCGGC GGAATCGGGA TACCATGATG A~l~ll~lGA 1080
AGA~r~l~l CArl~l~-ll GGGGG~ A TCATCTGCTG GA~.C~r~GA ~r~l~llll~l~ 1140
TACTTCTAGA C~r~ ~l~l CCACAGTGCG AC~lG~lGGC CTATGAGAAA ll-llC~lC 1200
C~ll~Cl~A ATTCAACTCT GCCATGAACC CCATCATTTA CTCCTACCGT GACAAAGAAA 1260
TGAGCGCCAC CTTTAGACAG Al~l. ~ GCCAGCGCAG TGAGAACCCC ACCGCCCCCA 1320
CAGAAGGCTC AGACCG~lCG G~- C~ CCC TCAArrArAC CATCTTGGCT GGAGTTCACA 1380
GCAATGACCA Cl~ ~ll TAr~AAcGr~A~A ACTGAGATGA GGAACCAGCC ~lC~....lr 1440
GrAGr~ATA~A CAAGC~lCCC CCTACCCAAT TGCCAGGGCA AGG~aGG~a TrArArArGA 1500
GAAAAGTCAA CTCATGTACT TAAACACTAA CCAATGACAG TAl l~lrCC TGGACCCCAC 1560
AAGACTTGAT ATATATTGAA AATTAGCTTA TGTGACAACC CTCATCTTGA TCCCCATCCC 1620
TTCTGAAAGT AGGAAGTTGG AG-r-llGCA ATGGAATTCA AGAACAGACT CTGGAGTGTC 1680
CATTTAGACT ACACTAACTA GACTTTTAAA AGAlrll~lG l~l~lG CAAGTCAGAA 1740
TAAATTCTGG CTAGTTGAAT CCACAACTTC ATTTATATAC AGGCTTCCCT TTTTTATTTT 1800
TAAAGGATAC GTTTCACTTA ATAAACACGT TTATGCCTAA AAAAAAAAAA AAAAAAAAAA 1860
AAAAAAAAAA AAAAC 1875
(2) INFORMATION FOR SEQ ID NO 2
(i) S~UU~NC~ CHARACTERISTICS
(A) LENGTH 364 amino acids
(B) TYPE amino acid
(C) ST~A~D~n~S single
(D) TOPOLOGY linear
(ii) NOLECULE TYPE peptide
(vii) IN~EDIATE SOURCE
(A) LIBRARY Rheumatoid Synovium
(B) CLONE 80853
(xi) S~U~N~ DESCRIPTION SEQ ID NO 2
Met Ala Ala Ile Ser Thr Ser Ile Pro Val Ile Ser Gln Pro Gln Phe
CA 02224799 l997-l2-l6
WO 97/00952 PCT/US96/10618
~hr Ala Met Asn Glu Pro Gln Cys Phe Tyr Asn Glu Ser Ile Ala Phe
Phe Tyr Asn Arg Ser Gly Lys His Leu Ala Thr Glu Trp Asn Thr Val
Ser Lys Leu Val Met Gly Leu Gly Ile Thr Val Cys Ile Phe Ile Net
Leu Ala Asn Leu Leu Val Met Val Ala Ile Tyr Val Asn Arg Arg Phe
80~is Phe Pro Ile Tyr Tyr Leu Met Ala Asn Leu Ala Ala Ala Asp Phe
95~he Ala Gly Leu Ala Tyr Phe Tyr Leu Met Phe Asn Thr Gly Pro Asn
100 105 110
Thr Arg Arg Leu Thr Val Ser Thr Trp Leu Leu Arg Gln Gly Leu Ile
115 120 125
Asp Thr Ser Leu Thr Ala Ser Val Ala Asn Leu Leu Ala Ile Ala Ile
130 135 140
Glu Arg His Ile Thr Val Phe Arg Met Gln Leu His Thr Arg Met Ser
145 150 155 160~sn Arg Arg Val Val Val Val Ile Val Val Ile Trp Thr Met Ala Ile
165 170 175~al Met Gly Ala Ile Pro Ser Val Gly Trp Asn Cys Ile Cys Asp Ile
180 185 190
Glu Asn Cys Ser Asn Met Ala Pro Leu Tyr Ser Asp Ser Tyr Leu Val
195 200 ' 205
Phe Trp Ala Ile Phe Asn Leu Val Thr Phe Val Val Met Val Val Leu
210 215 220
Tyr Ala His Ile Phe Gly Tyr Val Arg Gln Arg Thr Met Arg Met Ser
225 230 235 240~rg His Ser Ser Gly Pro Arg Arg Asn Arg Asp Thr Met Met Ser Leu
245 250 255~eu Lys Thr Val Val Ile Val Leu Gly Gly Phe Ile Ile Cys Trp Thr
260 265 270
Pro Gly Leu Val Leu Leu Leu Leu Asp Val Cys Cys Pro Gln Cys Asp
275 280 285
Val Leu Ala Tyr Glu Lys Phe Phe Leu Leu Leu Ala Glu Phe Asn Ser
290 295 300
Ala Met Asn Pro Ile Ile Tyr Ser Tyr Arg Asp Lys Glu Met Ser Ala
305 310 315 320~hr Phe Arg Gln Ile Leu Cys Cys Gln Arg Ser Glu Asn Pro Thr Ala
325 330 335~ro Thr Glu Gly Ser Asp Arg Ser Ala Ser Ser Leu Asn His Thr Ile
340 345 350
Leu Ala Gly Val His Ser Asn Asp His Ser Val Val
355 360
(2~ INFORMATION FOR SEQ ID NO:3:
QU~NC~: CHARACTERISTICS:
(A) LENGTH: 393 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(vii) IMMEDIATE SOURCE:
(A) LIBRARY: GenBank
(B) CLONE:U18405
(xi) S~:~u~: DESCRIPTION: SEQ ID NO:3:
CA 02224799 1997-12-16
WO 97/00952 PCT/US96/10618
~et Ala Ala Ala Ser Thr Ser Ser Pro Val Val Ser Gln Pro Gln Phe
15~hr Ala Met Asn Glu Pro Gln Cys Phe Tyr Asn Glu Ser Ile Ala Phe
Phe Tyr Asn Arg Ser Gly Lys Tyr Leu Ala Thr Glu Trp Asn Thr Val
Ser Lys Leu Val Met Gly Leu Gly Ile Thr Val Cys Ile Phe Ile Met
Leu Ala Asn Leu Leu Val Met Val Ala Ile Tyr Val Asn Arg Arg Phe
80~is Phe Pro Ile Tyr Tyr Leu Met Ala Asn Leu Ala Ala Ala Asp Phe
95~he Ala Gly Leu Ala Tyr Phe Tyr Leu Met Phe Asn Thr Gly Pro Asn
100 105 110
Thr Arg Arg Leu Thr Val Ser Thr Trp Leu Leu Arg Gln Gly Leu Ile
115 120 125
Asp Thr Thr Val Thr Ala Ser Val Ala Asn Leu Leu Ala Ile Ala Ile
130 135 140
Glu Arg His Ile Thr Val Phe Arg Met Gln Leu His Thr Arg Met Ser
145 150 155 160~sn Arg Arg Val Val Val Val Ile Val Val Ile Trp Thr Met Ala Ile
165 170 175~al Met Gly Ala Ile Pro Ser Val Gly Trp Asn Cys Ile Cys Asp Ile
180 185 190
Glu Asn Cys Ser Asn Met Ala Pro Leu Tyr Ser ASp Ser Tyr Leu Val
195 200 205
Phe Trp Ala Ile Phe Asn Leu Val Thr Phe Val Val Met Val Val Leu
210 215 220
Tyr Ala His Ile Phe Gly Tyr Val Arg Gln Arg Thr Met Arg Met Ser
225 230 235 240~rg His Ser Ser Gly Pro Arg Arg Asn Arg Asp Thr Met Met Ser Leu
245 250 255~eu Lys Thr Val Val Ile Val Leu Gly Ala Phe Ile Ile Cys Trp Thr
260 265 270
Pro Gly Leu Val Leu Leu Leu Leu Asp Val Cys Cys Pro Gln Cys Asp
275 280 285
Val Leu Ala Tyr Glu Lys Phe Phe Leu Leu Leu Ala Glu Phe Asn Ser
290 295 300
Ala Met Asn Pro Ile Ile Tyr Ser Tyr Arg Asp Lys Glu Met Ser Ala
305 310 315 320~hr Phe Arg Gln Ile Leu Cys Cys Gln Arg Ser Glu Asn Thr Ser Gly
325 330 335~ro Thr Glu Gly Ser Asp Arg Ser Ala Ser Ser Leu Asn His Thr Ile
340 345 350
Leu Ala Gly Val His Ser Asn Asp His Ser Val Phe Arg Lys Glu Thr
355 360 365
Lys Met Arg Gly Gly His His Leu Leu Arg Asp Glu Gln Pro Pro Pro
370 375 380
Pro Glu Arg Pro Gly Gln Gly Arg Val
385 390
(2) INFORMATION FOR SEQ ID NO: 4:
( i ) S~:Qu~;NCE CHARACTERISTICS:
(A) LENGTH: 381 amino acids
(B) TYPE: amino acid
( C ) STRANDEDNESS: s ingl e
( D) TOPOLOGY: linear
( ii ) MOLECULE TYPE: peptide
CA 02224799 1997-12-16
W O 97/00952 PCTrUS96tlO618
(vii) INNEDIATE SOURCE:
(A) LIBRARY: GenBank
(B) CLONE: 119130
(xi~ ~;uu~;NC:E DESCRIPTION: SEQ ID NO:4:
~et Gly Pro Thr Ser Val Pro Leu Val Lys Ala His Arg Ser Ser Val
15~er Asp Tyr Val Asn Tyr Asp Ile Ile Val Arg His Tyr Asn Tyr Thr
Gly Lys Leu Asn Ile Ser Ala Asp Lys Glu Asn Ser Ile Lys Leu Thr
Ser Val Val Phe Ile Leu Ile Cys Cys Phe Ile Ile Leu Glu Asn Ile
Phe Val Leu Leu Thr Ile Trp Lys Thr Lys Lys Phe His Arg Pro Met
80~yr Tyr Phe Ile Gly Asn Leu Ala Leu Ser Asp Leu Leu Ala Gly Val
95~la Tyr Thr Ala Asn Leu Leu Leu Ser Gly Ala Thr Thr Tyr Lys Leu
100 105 110
Thr Pro Ala Gln Trp Phe Leu Arg Glu Gly Ser Met Phe Val Ala Leu
115 120 125
Ser Ala Ser Val Phe Ser Leu Leu Ala Ile Ala Ile Glu Arg Tyr Ile
130 135 140
Thr Met Leu Lys Net Lys Leu His Asn Gly Ser Asn Asn Phe Arg Leu
145 150 155 160~he Leu Leu Ile Ser Ala Cys Trp Val Ile Ser Leu Ile Leu Gly Gly
165 170 175~eu Pro Ile Met Gly Trp Asn Cys Ile Ser Ala Leu Ser Ser Cys Ser
180 185 190
Thr Val Leu Pro Leu Tyr His Lys His Tyr Ile Leu Phe Cys Thr Thr
195 200 205
Val Phe Thr Leu Leu Leu Leu Ser Ile Val Ile Leu Tyr Cys Arg Ile
210 215 220
Tyr Ser Leu Val Arg Thr Arg Ser Arg Arg Leu Thr Phe Arg Lys Asn
225 230 235 240~le Ser Lys Ala Ser Arg Ser Ser Glu Asn Val Ala Leu Leu Lys Thr
245 250 255~al Ile Ile Val Leu Ser Val Phe Ile Ala Cys Trp Ala Pro Leu Phe
260 265 270
Ile Leu Leu Leu Leu Asp Val Gly Cys Lys Val Lys Thr Cys Asp Ile
275 280 285
Leu Phe Arg Ala Glu Tyr Phe Leu Val Leu Ala Val Leu Asn Ser Gly
290 295 300
Thr Asn Pro Ile Ile Tyr Thr Leu Thr Asn Lys Glu Met Arg Arg Ala
305 310 315 320~he Ile Arg Ile Met Ser Cys Cys Lys Cys Pro Ser Gly Asp Ser Ala
325 330 335~ly Lys Phe Lys Arg Pro Ile Ile Ala Gly Met Glu Phe Ser Arg Ser
3g0 3g5 350
Lys Ser Asp Asn Ser Ser His Pro Gln Lys Asp Glu Gly Asp Asn Pro
355 360 365
Glu Thr Ile Met Ser Ser Gly Asn Val Asn Ser Ser Ser
370 375 380 .,
(2) lN~ OR~IATION FOR SEQ ID NO: 5:
i ) SEQu~ CHMACTERISTICS:
(A) LENGTH: 24 base pairs
~B~ TYPE: nucleic acid
28
CA 02224799 1997-12-16
W O 97/009~2 PCTrUS96/10618
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(vii) IMMEDIATE SOURCE:
(A) Oligomer R
(Xi) ~:yU~N~'~: DESCRIPTION: SEQ ID NO:5:
TCATCTTGAT CCCCATCCCT TCTG 24
(2) INFORMATION FOR SEQ ID NO:6:
yu N~: CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STR~N~ N~.sS single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(vii) IMMEDIATE SOURCE:
(A) Oligomer F
(xi) ~UU~NC~ DESCRIPTION: SEQ ID NO:6:
A~~ CCGAG TA~G~CC TGTG 24
