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W 096/29409 1 PCTrUS96/04037
RAGE TUMORRE~lECIlON ANTIGENS
Field of t~e I~ tiUII
This invention relates to mlcleic acid me~ 11~ which code for tumor rejection antigens and
~ ereof. The tu~nor rejection antigen ~ are pr~sed, i~er alia, into at least one t~nor
rejection antigen that is presented by HLA m~ ~ The nucleic acid m~l~ll~, proteins coded for by such
me'~clll~ andp~ptides de~ived Ll~ iulll, as well as rela~ed antibodies and ~;yLuLuxic 1Y111~110UYL~S, are usefi~l,
interaZia, in.1;~ and1~ contexts.
R~ ,. u.~.-d of the ~ tiUII
The process by ~ich the ~ I immune syst~n ~eco~i7Pe and ~acts to foreign or alien
m~t~ is ~)j "p An i" ~ facet ofthe systern is the T cell l~nse. T cells can reco~i7P and
interact with other oells via cell sufiâce r~ s on the other cells of peptides and mt)lt~~ s referred to as
15 humanleukocyteantigens("HLA")ormajorl,;-l~""l~l;l,;l;ly ,'~--~("MHCs"). Thepeptidesare
derived from larger m~lP~ t~; which are ~l~ c~d by the cells ~ich also present the ~A/MHC mcl~ lP
See Male et al., Advanced Tmrnlmt)lo~ (JP. Lipincott Company, 1987), especially chapters ~10. The
;"t~ 1;0l~ of T cells and cr p l~ ~ of ~A/peptide is rP~ tt~1, requinng a specific T cell for a specific
~" ,~ of an HLA mt lP~lllt and a peptide. If a specific T cell is not prese~nt, there is no T cell L~
20 even if its partner ~ is present Similarly, there is no response if the specific c~ mplPx is absent, buttlle
T cell is present The "~ " ~ is involved in the immune system's s~~ ~ to foreign " ,~ , in
;llllllllllpp~tht~ ip~andinl~ul~ce~ arilllll~lllll~lil;~
The " IPrl ,~ ", by which T cells ~i7P alien " ~i~t - ;~1~ also has been implir~tPA in cancer. A
number of cytolytic T lymphocyte (CTL) clones directed against ~ntologous mt 1~"("~ have been ~le~li~l.
25 In some ;"~ r~C~, the antigens recogni7P~l by these clones have been rl l~ 1 In PCT aprlir~ion
PCT/US92/04354, pllhlichPA onNovember 26, 1992, the "MAGE" family, atumor specific family of genes,
is ~ r~ The ~ r products ofthese genes are processed into peptides which, in turn, are expressed
on cell sln f~r~ This can lead to lysis ofthe turnor cells by specific CTLs. The genes are said to code for
~ "tumorrejection antigen~ i" or "TRAP" ml~lP~lllPs, and the peptides derived IL~;l~ulll are referred to
30 as "tumor rejection antigens" or "TRAs". See TraversaIi et al., Tmmlm~ ~PnPtics 35: 145 (1992), van der
Bruggenetal.,Science254: 1643~1991),forfurther;l~r""~1;ononthisfamilyofgenes. Also,seeU.S.
PatentNo. 5,342,774.
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In U.S. Patent 5,405,940, MAGE ~ f C are taught which are ~ lL~d by the ~A-A1
m~lf~l llf Given the known ~ifir.ity of particular pephdes for parhcular HLA m~ cl 11~, one should
expect a particular peptide to bind one HLA mf~ llf but not others. This is,, --~. I~lL, because different
individuals possess different HLA phenotypes. As a result, while i~lf ntifi~ ~tion of a par~icular peptide as
being a partner for a specific HLA mol~clll~ has tli~gnnsti~. and tl If'~ A~ ;r r~mifir~ti~n~, these are only
relevant for individuals with that particular HLA phenotype. There is a need for further work in the area. A
because cellular ~hn~rm~litif~ are not restricted to one parhcular HLA phenotype, and targeted therapy
requires some knowledge ofthe phenotype ofthe ~hnt~rm~l cells at issue.
It also was discovered that a MAGE expression product is processed to a second TRA. This second
10 TRA is presented by ~A-C clone 10 m~l ~c~ c Therefore, a given TRAP can yield a plurality of TRAs.
In PCT W094/14459, pllhli~h~:l July 7,1994, tyrosinase is clf ~rihe~l as a tumor rejechon anhgen
~;uL~ol. This ~c .ce ~ o~C that a m~lf clllf which is produced by some normal cells (e.g
melanocytes), is processed in tumor cells to yield a turnor rejechon anhgen that is presented by ~A-A2
m~l~,lllf~,
In PCT W094/21126, published S~l~ 29,1994, a second TRA, not derived from tyrosinase is
taughtto be presented by HLA-A2 m~ The TRA is derived from a TRAP~ but is coded for by a
non-MAGE gene. This tli~l-~sllre shows that a particular HLA moltoclll~ may present TRAs derived from
different sources.
In PCT W095/00159, p~lhlieh~ January 5 1995~ an unrelated tumor rejechon antigen precursor~ the
20 so called "BAGE" precursor, is described. TRAs are derived from the TRAP and also are described. They
form complexes with MHC moleclll~ ~A-C-Clone 10.
In PCT W095/03422, published February 2~ 1995~ another unrelated tumor rejection antigen
~l~;UI~l, the so called "GAGE" precursor~ is described. The GAGE precursor is not related to the BAGE or
the MAGE family.
The work which is presented by the papers, patents and patent appli~ it nc described above deaL for
the most part, with the MAGE family of g~enes, the BAGE gene and the GAGE gene. These genes are
expressed in a number oftumors but are cr-" ,p]~t~ly silent in normal tissues except testis. None is expressed
in renal carcinoma
It now has been discovered that another gene family~ the "RAGE" genes~ encodes additional tumor
rejection antigens and precursors thereof. The RAGE genes do not show homology to the MAGE falnily of
genes, to the BAGE gene or the GAGE gene. The RAGE genes are expressed in renal tumor cells, but not in
normal renal cells. The RAGE genes are also expressed in cer~in othertumor cell types.
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_ ~ _
The invention is elal~l~ d upon in the ~ osllre which follows.
S,~ sl .~ of the ~ tiUII
The invention provides isolated nucleic acid m~l-c~ c~ expression vectors cnnt~inin,, those
s m~lle~,lll~s and host cells (.~ rr~ A with those molecules. The invention also provides isolated proteins and
peptides~ and antibodies to those proteirls and pcptides. Kits cnnt~inin,o the foregoing mnl~lllec ~ if inn~11y
are provided. The foregoing can ~e used in the ~ .,nn~ic or trea~nent of c~n~liti~-n~ cl~ r" ,r~l by the
expression of a RAGE TRA or TRAP.
According to one as~ect ofthe invention~ an isolated polypeptide is provided. It includes at least the
l O amino acid seq~Pnre of SEQ.ID.NO.40 and is a RAGE TRA. In preferred embo(lim~.nt~, the isolated
polypeptide includes at least the amino acid s~ll~nr~ of SEQ.IDNO. 43. In other embodiments the isolated
polypeptide rnay consist ec~Pn~ ly of or rnay even be only the amino acid seqll~nre of SEQ.IDNO.40 or
SEQ.ID.NO.43.
Acc~ , to another a~ect ofthe invention, an isolated nucleic acid mnlPclll~ is provided. The
l 5 mnlecllle encodes a polypeptide selected from the group con~i~tin~ of SEQ.ID.NO.40 and SEQ.IDNO.43.
The isolated nucleic acid can include SEQ.ID.NO.44 and ~l~r~ldl,ly includes SEQ.ID.NO.45. In other
embodiments the isolated nucleic: acid may consist ~P.nti~lly of or may even be only SEQ.IDNOs.44 or 45
According to another as ~ect ofthe inventiorL an isolated nucleic acid ml~le~ is provided which
hybridizes under ~nn,,ent c~n-lition~ to a molecule c~ c;~1 " ,o of the nucleic acid se~ on~e of SEQ.ID.NO. l .
20 SEQ.ID.NO.4, SEQ.IDNO.6, S!EQ.IDNO.lO~ SEQ.ID.NO.l2~ SEQ.ID. NO.13. SEQ.IDNO.14,
SEQ.ID.NO.15~ SEQ.ID.NO.17. SEQ.ID.NO.23. and/or SEQ.IDNO.35. and which codes for RAGE TRAs
or TRAPs. with the proviso that the isolated nucleic acid mt~ does not code for a MAGE. GAGE or
BAGE TRA or TRAP. In prefe:rred embo-limPnt~ the isolated nucleic acid molecule is an rnRNA mnJec
or a cDNA m~lPclllP In one emibodiment, the isolated nucleic acid molecule is cl mrlPmPnt~ry to
25 nucleotides selected from the ~,roup COI l~;~i;l Ig, of 204 to 326 of SEQ.IDN0.1~ 313 to 399 of SEQ.IDN0.1.
1to6650fSEQ.ID.N0.1,27:3to4490fSEQ.ID.N0.12.217to2760fSEQ.ID.N0.12,185to2470f
SEQ.ID.N0.13 and 269 to 832 of SEQ.ID.N0.14. In another embodiment the isolated nucleic acid consists
P~.nli~lly of SEQ.IDN0.1, SEQ.IDN0.4. SEQ.ID. N0.6. SEQ.IDN0.10. SEQ.ID.N0.12. SEQ.ID.
N0.13, SEQ.ID.N0.14, SEQ.II)N0.15. SEQ.ID.N0.17. SEQ.IDN0.23. SEQ.ID.N0.35. SEQ.IDN0.44
30 and/or SEQ.IDN0.45.
According to another a~ect of the invention~ expression vectors and host cells c ~ l l l 1; ~ l; l Ig those
expression vectors are provided. The expression vectors include any one or more ofthe isolated nucleic acid
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m~~lec~ s ~lt?srrihel above. In one embodiment, the expression vector ~. "l" ;~s the isolated nucleic acid of
SEQ.IDNOs.44 or 45. Other expression vectors a~l~ling to the invention include the isolated nucleic acids
dr~~ ed above and anucleic acid which codes for an ~A molr~~ le which can present the TRAs ofthe
invention to cytolytic T cells. One r~x~mrlr~ is HLA-B7. The host oells may endogenously express the HLA
5 ~ lr~ such as HLA-B7.
According to another aspect ofthe invention, isolated nucleic acid m~ ,l~clll~s that are unique
r.~ ",~ of SEQ.IDNO.l, SEQ.IDNO.12, SEQ.ID.NO.13 or SEQ.ID.NO.14 ortheirc~ ""l,~ Mts are
provided. Such unique r.-~ . lrl ~l ~i are used to identify or to selectively amplify the nucleic acids ~1r~c~ ril~
above. When the unique r~g.~ are used for identifying expression ofthe above nucleic acids. the unique
o fi~,mr~.nts pl~ dbly are between 200 and 1310 nucleotides in length, 200 and 1234 nucleotides in len~L
200 and 2050 nucleotides in length or 200 and 1167 nucleotides in length. When the unique fi~mr~nts are
used to amplif~ the above-described nucleic acid m~l~lll,s, the unique fi~mr~nts are between 12 and 32
nucleotides in length. It will be recoOr~ized that ~mrlifi.~~fi~n procedures are not exclusive of procedures that
rnight be used to identify a nucleic acid m-~lr~Clllr~
According to another aspect ofthe invention, kits for (lctr~stinv- the presence of expression of a TRA
or TRAP are provided. Such kits employ t~.vo or more ofthe above{lr~s' rihe~l molcc. llr~s isolated in separate
c ~ ;- Ir~i and p~~.k~1 in a single pZ~~k:l~_,r In one such kit~ a pair of:~nnrlifi~~~tion prirners are provided~
each of tbe pair ~ J i . ,g cs~nti~l ly of a 12-32 in length nucleotide contiguous se~~m~nt of SEQ.IDNO. 1 or
the c~-mr~ .nt thereof, SEQ.ID.NO.12 or the crmrl--nPnt thereof, SEQ.ID.NO.13 orthe c mplt-mt~nt
thereof, or SEQ.ID.NO.14 orthe comrl~.m~nt thereof~ and wherein the contiguous se~o n~ntc are non-
O~/r~ 10~ Preferably the ~mrlifir~til~n prirnes are PCR prirners. wherein one of the primers is a
cr~nti~lous segmt-nt of the Watson strand and another of the prirners is the c(lmpl~mPnt of a contiguous
s~om~ont of Crick strand. In certain embodiments, prLrnes are constructed and ~t~n~ to selectively arnplify
and/or identify only one of the RAGE family, such as only RAGE I or a portion of only RAGE l . etc. For
~s example. one of the pair can be c~nti~ us in RAGE 1 genes and allelic variants thereofbut not contiguous in
RAGE '~, 3 or 4 genes. More ~rifir~lly as an exarnple . a first primer can be a nucleic acid umcictina
P~Pnti~lly of any one of SEQ.I~N-Os.50-57~ and a second prirner can consist ~ nti~lly of a 1~-3~ in len th
nucleotide contiguous segrnent of SEQ.ID.NO. 1 t or the c mplPm~nt thereof. ~ ~n~lina upon the choice of
the first prirner.
Another kit according to the invention is an expression kit co. "I" ;~ , a separate portion ofthe
isolated nucleic acid mol~c~ which codes for a RAGE TE<AP~ or a m~lec lk- in~ l(linL a RAGE TR~ and
an EILA ~l~S~llLillg mnl~clll~? that forrns a c~mpl~x with that TRA and that ~il ;" "ll~ c a cytolytic T cell
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W096129409 5 PCT~US96/04037
. One such kit includes a mlcleic acid which codes for the peptide of SEQ.ID.NO.40 or
SEQ.lD.NO.43 and a nucleic acid nt~'~cllle which codes for HLA-B7. Another kit accc,ldil~g to the
invention is an expression kit c " ~ ~l "; ~; "g a separate portion of the isolated nucleic acid m~lPcl llP which
hybridizesunder~ c~nnt1itinnctoam~'-clllP~~ l;"~ofthenucleicacidsequPMreofSEQ.IDNO.1.
s SEQ.IDNO.4, SEQ.ID.NO.6, SEQ.ID.NO.10, SEQ.ID.12, SEQ.ID.13, SEQ.ID.14, SEQ.ID.15~ SEQ.ID.17
SEQ.ID.23 and/or SEQ.ID.35 and ~;vhich codes for a RAGE TRAP, and a nucleic acid moleclllP which codes
for HLA-B7.
According to another aspect ofthe invention isolated TRAPs coded for by the above molecules and
useful fi-,t, mt~ntc thereof also are provided. Antibodies to such mnlecl llf'S and to complexes of HLA and
l o RAGE TRAs also are provided.
According to another aspect ofthe irLvention. m~-thf~ for tli~n~in~ a disorder ~'.h~ rd~~~ by
expression of a RAGE TRAP are provided. One method involves a RAGE TR~P which is processed to a
RAGE derived TRA that forms a complex with HLA m- k c~ The method involves ~ g ahi~ l sarnple isolated from a subject with an agent that binds the complex and then ~lr~ ;l l;l ,g binding
between the cc-lFl - ~ and the agent as a ~ rl ~ ion of the disorder. In one embodiment, the method
binding of the agent to a complex of RAGE TRA and HLA-B7. In this embodiment, the RAGE
TRA can be selected from the grou~p c~n~i~ting ofthe peptide of SEQ.IDNO.40 and the peptide of
SEQ.ID.NO.43. Another method i]~volves c~ a a biological sarnple isolated from a subject with an
agent that is specific for a RAGE mlcleic acid or an expression product thereof. Int~ rt~nn between the agent
andthe nucleic acid or expressionproduct thereofthen is ~lrL~ 1 intPr~r.ti~n being indicative ofthe
disorder. The agent may be a nucle.ic acid which hybridizes under ~l l ;l l~,rl 1l cr,n~litl'rJn~ to a m~lec.lllP
cr~n.~ictin,, ofthe nucleic acid sequence selected from the group conci~tin,a of SEQ.ID.NO.l . SEQ.IDNO.4.
SEQ.IDNO.6, SEQ.ID.NO.10. SEQ.ID.NO.12. SEQ.ID. NO.13, SEQ.ID.NO.14. SEQ.IDNO.15.
SEQ.IDNO.17, SEQ.ID.NO.23 and SEQ.ID.NO.35~ and which codes for a TRAP. with the proviso that the
isolated nucleic acid mol~c3ll.o does not code for a MAGE. GAGE~ or BAGE TRAP. Another method
involves cont~.ting a biological sarnple isolated from a subject with an agent that is specific for a RAGE
tumorrejectionanti_,enpeptideandthen-lr(~"-.;ll;l.ginteractionbetweenthepeptideandt'ne~entasa
on ofthe disorder In one embodiment! the peptide is selected from the roup con~i~ing of
SEQ.IDNO.40 and SEQ.ID.NO.43.
According to another a~t ofthe invention~ an isolated hiolo~ir~ dlion is provided. The
ylc~ n consists ~ Mti~lly of cytolytic T cells specific for complexes of an HLA molecule and a RAGE
TRA. In one embodiment, the cytolytic T cells are specific for complexes of an HLA-B7 m~lh c~lle and the
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--6-
TRA. In this embodiment, the antigen can be a peptide selected from the group c~ l ,x;~l l", ofthe peptide of
SEQ.ID.NO.40 and the peptide of SEQ.ID.NO.43.
Another aspect of the invention thus involves a method for enriehino selectively a population of T
cells with cytolytic T oells specific for complexes of an HLA m~ clllP and a RAGE TRA. The method
5 involves c~nt~rtin,, an isolated population of T oells ~ 0 cytolytic T cell ~l~;ulxol~ with an agent
resulting in ~lr.,s~ n of a cvlll~ of a RAGE TRA and E~A ~l~llLillg mr' ~c lle, in an amount
sllffiri~nt to selectively enrich the isolated population of T cells with said cytolytic T cells. In one preferred
Pmhotlim~nt the HLA molerlll~ is E~A-B7 and the RAGE TRA is selected from the group conxixtino of a
peptidec~nxixtin~,oftheaminoacidsofsEQ.ID.No.4oandapeptidec~ ;xl;~goftheaminoacidsof
10 SEQ.ID.NO.43.
Still another aspect ofthe invention involves methods for treahng a subject with a disorder
d by expression of a RAGE TRA or TRAP. One such method involves ~,1" ,;, I;~lrl ;1 Ig to a
subject in need of such l~ lll an effective amount of an a=,ent which er~iches selectively in the subject the
ce of complexes of E~A and RAGE IE~ resulting in cytolytic T cell l~unse reactive with such
complexes, s~ffiriPnt to ~n~Pliorate the disorder. Such agents include the RAGE TRAPs and recolll~il~lL
cells ~ lC~illg c~mplDx~s of the HLA and RAGE TRA. In one embodiment~ such agents include cells
c~ lg a complex of HLA-B7 and a peptide co~ l; "a of the peptide of SEQ.ID.NO.40 0r the peptide of
SEQ.ID.NO.43. Another method involves all, 1 ~ ;"o to a subject in need of suchtreatment an arnount of
~t~log~lus cytolytic T cells snfficipnt to ~mPli~ r~tP the disorder. wherein the ~lltologous cytolytic T cells are
20 specific for complexes of an ~A m~rllle and a RAGE TRA.
In c nnPcti/-n with any isolated nucleic acid encoding a TRAP or TRA as ~lP~rrihe(l above~ the
invention also Pmhr~r~c ~iPg~"e~ i llr nucleic acids that differ from the isolated nucleic acid in codon sequence
only due to the cle~enrr~r.y of the genetic code or c~ nPnt~ of any of the foregoing nucleic acids.
The invention also Pmhr~rPs fimrti~n~l variants and equivalents of all ofthe molecules described
2~ above.
The invention also involves the discovery and i~olation of TRAPs and TRAs which are expressed in
tumor cells, particularly in renal tumor cells. and not expressed in normal renal cells. Prior to the present
invention. TRAPs and/or TRAs ofthe type described herein were not known and itlPntifi~d for renal
carrin~m~, despite the knowledge and identity of other TRAPs and TRAs for numerous other cell types.
30 S~l.li~ill, ly~ the RAGE-1 gene ofthe present invention was expressed in only 1 of 57 renal carrinom~c and
the best ~ntioenir peptide for a particular cytotoxic T cell clone was discovered to be a ~er~mpn not the usual
n- n~m Pr peptide. Thus, ~ldillg to another aspect of the invention, the invention provides TRAPs. TRAs
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and nucleic acids coding for TRAPs and TRAs, which are not MAGE. BAGE AND GAGE TRAPs. TRAs
and nucleic acids, which are expressed in turnor ceUs, particularly in renal tumor ceils but not in norm3al renal
cells,andareob~ lebyaproc~ssc(-".l";~
isolating renal turnor cells from apafient,
i~l~tin,, lymphocytes from the patient,
C.~ the rerlal tumor cells withthe lyrnphocytes in vitro,
i~Qls~tina a cytotoxic T ce:U clone among the lymphocytes reactive with the ren 1 fumor ceUs~
,~lc~ lg arl expression library from rmRNA of the renal tumor oells,
~;l~llillg the expression library with the cytotoxic T cell clone for a library member reactive wif~h the
I o cytotoxic T oeU clone,
i~ol~tina said libraly member reactive with the cytotoxic T cell clone7 and
s~l~n~in~ the r~al cell nucleic acid in said library m~ . said renal ceU nucleic acid encoding the
TRAP (orportionthe~eof);..~ l.g the TE~A. The invention also provides sequ~nrPc having h~-m~ ay to
such nucleic acids and coding for renal ~SSo~ /t~Cl TRAPs and TRAs. ~serlllPnrPs which hybridize under
15 !~1l;ll~lll c--nl1ifi~n~tosuchnucleicacidsandcodingforrenal~cc;o(~i~f~TRApsandTRAs~ct>mrlt~m~nt~
unique fi~omPnt~ and ~1P~I~ ofthe foregoing, the TRAPs and TRAs thernselves, as well as fimrtion~l
variants and equivalents of any of the r~l~uoillg, all of which can be cor~sidered to be RAGE nucleic acids~
TRAPs and TRAs.
The invention also provi~1es agents that selectively enrich in a subject the ~l~sell~:e of complexes of
20 HLA/RAGE TRAs for use as a rn~ir~m~nt Such agents include but are not lirnited to. RAGE TRAs
andlor RAGE TRAPs; ,~ ." ,h;"~"l cells ~Ayl~illg RAGE TRAs and/or RAGE TRAPs and also
~X~ illg ~ lU~ HLA molecules. l~ l,i"~ or not. and fimr~fi~n~l variants and equivalents offlle
foreg$oing. Specific examples include the RAGE TRA of SEQ.IDNO. 43; any f~ment ofthe RAGE
TRAP of SEQ.ID.NO 5 inrhl~lirla the TRA of SEQ.ID.NO. 43: the RAGE TRAP of SEQ IDNO. 5:
25 recomhin~nt cells expressing the TRA of SEQ.ID.NO. 43 and HLA-B7; and/or any other RAGE TRA.
RAGE TRAP or fi~nrfion~ grnont thereof and/or cells expressing such molecules.
The invention also provides agents that selectively enrich in a subject the presence of complexes of
ELAIRAGE TRAs in the m~ml1'~rhlre of a medicament for treating cancer. Sucll agents include. but are not
limited toS RAGE TRAs and/or RAGE TRAPs? reco, l 1l )il 1i11 1l cells expressing RAGE TRAs and/or RAGE
30 TRAPs and also ~X~ g a~ ~A ml~koculo~ recomhin~nt or not: and fimrtion~l variallts and
equivalents ofthe foregoing. Specific examples include the RAGE TRA of SEQ.ID.NO. 43s any fi~omrnt of
the RAGE TRAP of SEQ.IDNO. 5 inrl~lrling the TRA of SEQ.IDNO. 43; the RAGE TRAP of
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SEQ.ID.NO. S; recombinant cells ~ Sillg the TRA of SEQ.ID.NO. 43 and ~A-B7; and/or any other
RAGETRA,RAGETRAPorfimrtil-n~ mpntthereofandlorcells~x~l~lllgsuchm~lpclllpc
The invention also provides cytotoxic T cells specific for complexes of HLA and RAGE TRA for
use as a mP~1ir~nnP.nt One nl nlimitin~ IC is autologous cytotoxic T cells specific for tumor cells
5 e~y~essi~lg c~-mrl.~ c of HLA-B7 and RAGE TRA.
The invention also provides cytotoxic T cells specific for c~ mplP~Pc of HLA and RAGE TRA in the
",;t",lr~ l"~ofamPrir~nnPntfortreatingcancer. Onen~nlimhingexarnpleisautologouscytotoxicTcells
specific for tumor cells ~ lg c~mr~ Pc of HLA-B7 and RAGE TRA.
The invention also provides rh~rm~ellti~ lion~ c-~"l~;";,-g any one or more ofthe
10 ~n~ ",~"~ laboveorth~ughout~e.crerifi~ti--n. Suchrh~rmzlrelltir~ nscaninclude
rh~rm~relltir~lly acc~l.~lc diluent carriers or ~xririPntc
These and other objects ofthe invention will be described in fuIther detail in c-~nnP~tion with the
detailed ~Psrrirtir,n ofthe invention.
BnefDes~ l. of the D~.. ;.. ~ ~
Figure 1 is a graph showing the levels ofturnornecrosis factorproduced when CTL Clone 263/17 is
comhin~r with COS cells ~ncfP~tPrl with HLA-B7 cDNA and a cDNA encoding a RAGE TRAP.
Figure2isa srh~orn~ticl~ ~"~ n oftheRAGE-l.2~3and4cDNAs. Closedblackboxes
indicate the different ORF in each ofthe three reading frarnes. Shaded areas in the RAGE-2, 3 and 4 cDNAs
l~ tsr~lr-nrPcthatare ull,~ dtotheRAGE-l sequence, i"rl~fl;~o twoinsertions. TheS'teIrninal
~lurl ~r~ ob~ined by PCR is inrlir~tPA with dashed boxes. The 3' end ofthis PCR serlnP.nre is identical to
the o~ lg 5' end sequences ofthe RAGE-2, 3 and 4 cDNAs. The ~ntigPni~ peptide encoded by
RAGE-1 is ;"(~ lrfl
Figure 3 is a graph showing the levels oftumor necrosis fàctor produced whel1 CTL Clone 263/17 is
comhin~-A. with COS cells Ll~ll Ixr~ r~1 with HLA-B7 cDNA and a cDNA encoding a RAGE TRAP or a
minigP.nr- encoding ORF2 of a RAGE TRAP.
Fi=ure 4 is a raph rlP.t~ilino the levels oftumor necrosis factor produced when CTL Clone 263/17 is
comhinPcl ~ith peptide fi~amPnt~ ofthe TRAP encoded by 0RF2 ofthe RAGE ~ene and COS cells
l,~,,~rrl;lrA withHLA-B7.
Figure 5 is a graph rlr~pi('tillg the lytic activity of CTL clone 267/17 against HLA-B7- LB~3-EBV B
cells pulsed with increasing ~nc~llL~Lions ofthe peptides inrlll(ling a RAGE TRA.
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WO 96J29409 9 PCT~US~S~0 1~37
Bnef D~ iOII of the ~qn~
SEQ.ID.NO.1 is the mlrlP~ti~ sPJlupnre ofthe RAGE-1 cDNA.
SEQ.ID.NO.2 is open reading frame 1 (ORF1) ofthe cDNA of SEQ.ID.NO.l .
~ SEQ.ID.NO.3 is the ~ r~lr~ ~rnino ~cid sP~uPnre of SEQ.ID.NO.2.
SEQ.ID.NO.4 is open reading frame 2 (ORF2) ofthe cDNA of SEQ.ID.NO.l .
SEQ.ID.NO.5 is the tr~n~lslt~ amino ~id sequence of SEQ.ID.NO.4.
SEQ.IDNO.6 is open reading frarne 3 (ORF3) ofthe cDNA of SEQ.ID.NO.1.
SEQ.ID.NO.7 is the tr~n~lslt~A amino acid sP~uPnre of SEQ.ID.NO.6.
SEQ.ID.NO.8 is open reading frame 4 (ORF4) ofthe cDNA of SEQ.ID.NO.1.
I o SEQ.ID.NO.9 is the tr~n~lslt~A arr~ino ~cid seql-Pnr~ of SEQ.IDNO.8.
SEQ.ID.NO. 10 is open rea~ing frame 5 (ORF5) of the cDNA of SEQ.IDNO. 1.
SEQ.ID.NO.11 is the LlA~ rS~ nOICidSP~q~1P.nre of SEQ.IDNO.10.
SEQ.ID.NO.12 is the mlrl~oti-lP ~u~llce ofthe RAGE-2 cDNA.
SEQ.ID.NO.13 is the nucleotide seqll~nre ofthe RAGE-3 cDNA.
1~ SEQ.IDNO.14 is the nucl~otide ~l~ ofthe RAGE4 cDNA.
SEQ.IDNO.15 is open reading f~me 2' (ORF7') ofthe cDNA of SEQ.ID.NO.12.
SEQ.ID.NO.16 isthetr~n~lsltPd ~ino acid se~uPn~e of SEQ.IDNO.15.
SEQ.ID.NO.17 is open reading frarne 3' (ORF3') ofthe cDNA of SEQ.ID.NO.12.
SEQ.ID.NO.18 is the tr~ncht~PA c~nino acid sequence of SEQ.ID.NO.17.
SEQ.ID.NO.19 is open rea~ing fraIne 4 (ORF4) ofthe cDNA of SEQ.IDNO.12.
SEQ.ID.NO.20 is the tr~n~ ~1 amino acid sP~lPn~e of SEQ.ID.NO.19.
SEQ.ID.NO.21 is open re~ding frame 5 (ORF5 ) ofthe cDNA of SEQ.ID.NO.12.
SEQ.ID.NO.Z is the tr~n~l~tPIl amino lcid sequPnre of SEQ.IDNO.21.
SEQ.ID.NO._3 is open reading frarne 6 (ORF6) ofthe cDNA of SEQ.ID.NO.13.
SEQ.IDNO.24 is the Tr~n~l~t~PA amino acid sequence of SEQ.ID.NO.23.
SEQ.ID.NO.25 is open reading ~ame 2' (ORF2') of the cDNA of SEQ.IDNO. 13.
SEQ.IDNO.'76 is the l~an~l~terl amino acid sequence of SEQ.IDNO.25.
SEQ.IDNO.77 is open reading f~me 3' (ORF3') ofthe cDNA of SEQ.IDNO.13.
SEQ.ID.NO.28 is the 1,~"~ ~ amino acid s~qllPn~e of SEQ.IDNO.27.
SEQ.IDNO.'79 is open reading frame 4 (ORF4) of ~e cDNA of SEQ.ID.NO.13.
SEQ.ID.NO.30 is the lr~n~l~t~l arnino acid seqllrnre of SEQ.ID.NO.'79.
SEQ.ID.NO.3 1 is open re~ling frame 5 (ORF5) ofthe cDNA of SEQ.ID.NO.13.
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WO 96/29409 10 PCT~US96/04037
SEQ.IDNO.32 is the tr~n~l~t~1 amino acid s~~ ; of SEQ.ID.NO.3 1.
SEQ.ID.NO.33 is open reading frarne 2' (ORF2') ofthe cDNA of SEQ.ID.NO.14.
SEQ.ID.NO.34 is the l,~"~ l amino acid serlu~nr~ of SEQ.ID.NO.33.
SEQ.IDNO.35 is open reading frarne 3"(0RF3") ofthe cDNA of SEQ.IDNO.14.
SEQ.ID.NO.36 is the translated amino acid sequ~nr~ of SEQ.ID.NO.35.
SEQ.ID.NO.37 is open r~ading frarne 4' (ORF4') ofthe cDNA of SEQ.ID.NO.14.
SEQ.ID.NO.38 is the ~ 1 arnino acid ~ nre of SEQ.ID.NO.37.
SEQ.ID.NO.39 is the ~lotl~m~r peptide c nt~ining the RAGE turnor rejection antigen m~ntinnPd in
c- nn~c,ti ~n with Figure 4.
SEQ.ID.NO.40 is a l~ fi~gmPnt (arnino acids 1-9) ofthe peptide described in SEQ.ID.NO.39.
SEQ.ID.NO.41 is a l-.~ rn~,nt (amino acids 2-10) ofthe peptide des~nhed in
SEQ.ID.NO.39.
SEQ.ID.NO.42 is a ~ "~ rl-,~ 1lr~,l (arnino acids 3-l l) ofthe peptide ~ 1 in
SEQ.ID.NO.39.
SEQ.ID.NO.43 is a decarner fi~gmPnt (amino acids 1 -10) of the peptide described in
SEQ.ID.NO.39.
SEQ.ID.NO.44 is the nucleotide .s~l l~n~e of a DNA which encodes the peptide of SEQ.IDNO.40.
SEQ.IDNO.45 is the nucleotide sequence of a DNA which encodes the peptide of SEQ.IDNO.43.
SEQ.IDNO.46 is a sense prirner used in PCR tests for expression ofthe RAGE TRAP.SEQ.ID.NO.47 is an ~"l i~, .~ prirner used in PCR tests for expression of the RAGE TRAP~
c mmon to all RAGE genes tested.
SEQ.ID.NO.48 is an ~ ~ prirner~ specific for RAGE-l, used in PCRtests for expression ofthe
RAGE-l TRAP gene.
SEQ.ID.NO.49 ~ the region of RAGE genes flanking tlle insertion point of ORF2. with the
25 insertion ~lPci$n~t~1 by N.
SEQ.ID.NOs.50-57 are PCR prirners useful in itlPntifi~ti(~n of RAGE 1.
Detailed Des~ Jlioll of the I~ lllioll -
An antigen recogni7~d on a renal cell carcinoma by autologous CTL restricted by HLA-B7 is
encoded by a previously unknown gene. This gene is silent in all normal tissues (in~ ling testis). except for
retina and it is ex~ d in several turnor samples.
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W 096/29409 -11- PCT/US~Gr~ 7
EXAMPLE 1: Description of an anh-rerlal cell carcinoma CTL clone of patient
LE921 1
Tumor line LE921 1-RCC is a renal cell carcinoma line derived from a tumor sample of a female
~ 5 patient r~ned LE9211. A sample thereofwas irr~ t~rl so as to render it non-proliferative. These irr~ tl~d
cells were then used to isolate cytolytic T cell clones ("CTLs") specific thereto.
A sample of ~ ,,,1 blood rn~n~ ml~ r cells ("PBMCs") was taken from patient LE 9211. and
~nt~.t~d to the in~ t~A c~ uma cells. Af~er 14 days, the rnixture was observedfor lysis ofthe
C~ ulll~ cells, which in~ t~d that CTLs specific for a complex of peptide and HLA molecule presented
lo by the c~ ullla cells were present in the sample.
The lysis assay employed was a chrornium release assay followin~ Herin et al.~ Int. J. Cancer 39:390-
396 (1987). ~he assay, however, is briefiy described herein. The target carcinoma cells were grown in vitro.
and then ~ "~l~A at 107 cells/rnl in Dulbecco's Modified Eagles Medium (DMEM) supl l~om~nt~ with
30% FCS. and in~llh~t~l for 4~ mimltes at 37~C with ~00 ~Ci/rnl of Na(5'Cr)04. Labeled cells were washed
three tirnes with DMEM. These we]e then r~l~M~ in DMEM supr-lPm~-nt~d with 10 rnM Hepes and
10% fetal calfserum (FCS), after which 100 ,ul aliquots col l~ a 103 cells were di~LIil.ul~d into 96 well
microplates. Samples of lymphocytes were added in 100 ~11 ofthe same m~ m and assays were carried out
in ~ pli~t~ Plates were c~ntrifilaed for 4 mirLUteS at lOOg and in~ h~t~d for four hours at 37~C in a 8% C0,
zitmt~sl~h~re.
Plates were cPntnfi-ged agairl~ and 100 ~l aliquots of s~ ~L~lL were collected and counted.
Percen~e of 5'Crrelease was calcu].ated as follows:
%siCrrelease=(ER-SR)x 100
(MR-SR)
where ER is observed, ~-Yllrl ;11 Irl l1-~l 5'Crrele~. SR is spontaneous release measured by inrllh~in_ 10~ labeled
cells in 200 ~11 of medium alone. and MR is mziximnm release. obtained by adding 100 111 0.3% Triton X-100
to target cells.
Those m~ nf)mlrlrzir blood samples which showed high CTL activitv were expanded and cloned ~ia
,o lirniting dilution, and were screened aaairL using the same mPth~ology. A first CTL clone was then isolated.
The clone is referred to as 263/17 l~ l . A second CTL clone~ 361A/17. was obtained similarlv from ~le
sarne experirnent and was used as described further below when CTL 263117 failed to grow in~lPfinitle~ .
CTL clones 263/17 and 361A/17 were capable of lysing spP~ifi~z~lly the autologous tumor cells and not
NK-target K562 cells. NK - target K562 cells are available from the ATCC. Rockville. Maryland.
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wo96/29409 - 12- Pcrlu~ 37
CTL clone 263/17 produced TNF when stim~ ~d with the autologous tumor cells. To identify the HLA
molecllle that presented the antigen to CTL clone 263/17, inhihition exl~, ;" ~"l~i were c_r.ried out where the
production of TNF WaS tested in the ~ ce of m~n~AlonAl antibodies directed against HLA molecules or
against CD4/CD8 accessory m~l ~cl llPS Four mnn~lon,ll, ntiho~liPA~ were found to i~Lhibit the production of TNF
5 by CTL 263/17: (1) m~n~l~n~l _ntibody W6/32, which is directed against all HLA class I molecules (Parham
et al., 1979, J. Tmmlmnl, 123:342), (2) antibody Bl .23.2 which recognizes HLA-B and C mnlPclllPs (Rebai et
al., 1983, Tissue Antigens, 22:107); (3) antibody ME-l which .s~ifi~lly reco~i7Ps HLA-B7 (Ellis et aL 1982.
Hurn. Tmmlm~-l 5:49); and (4) antibody B9.4.1 against CD8. No inhihition was found with antibodies directed
against HLA Class II DR mol~clllPs (L243: T ~mpson et al.. 1980. J. Tmmlm~ 125:293). against HLA-A3
10 (GAPA 3: Berger et al1982 Hyhri~l~m~ 1:87) or aga]nst CD4 (13B.8.82). The cr)nrlllsi-)n was that CTL
263/17 was ofthe CD8 type, and recogni7~ an antigen ~lcs~llL~d by HLA-B7.
To define the tumor ~ y ofthis CTL clone, nomlal kidney cells derived from another pa~ient which
are also HLA-B7 (PTEC-HLA-B7 cells) were tested. These cells derive from the proximal tubular epith~lil-m
which is the site of origin of renal cell ~"l~n,a PTEC-HLA-B7 cells were not Iysed by the CTL~ ~sll,,gt-sting
that the antigen is ~ific~lly expressed on tumors.
Renal cell ~-,illullla line MZ-1851, which is derived from another HLA-B7 patient. was also Iysed by
the CTL, showing that the antigen is shared by independent turnûrs.
EXA~LE 7: Isolation of a cDNA clûne that directs the expressiûn ofthe antigen reço~ni7t~ci bv CTL
263/17
A. cDNA library
RNA was isolated from LE-921 l-RCC and poly-A RNA was purified by oligo-dT binding. cDNA
~s was prepared by reverse l~,."~(-"~ n with an oligo-dT primer cont~ining aNot I site. followed by second
strand synthesis (Sl ~ ;l It Choice Systern. BRL. Life Technologies). The cDNA was then ligated to a
BstXI adaptor, digested with Not I, si7e-fractionated (Sephacryl S-500 HR coll-rnn~ BRL Life Technologies)
and cloned unidirectionally into the BstXI and Not I sites of pcDNA-I-Arnp (Invitrogen). The recombinant
plasrnid was then el~ d into DH5a E. coli bacteria. 1500 pools of 100 reco~ bacteria ~ere
30 ~mplifiPcl and plad DNA of each pool was extracted by allcaline lysis~ potassium acetate precipitation and
phenol exh~fi~n
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B. Transfection of COS cells
Plasmid DNA from the different pools was co-~ Xr~ d into COS cells with 60 ng ofthe HLA-B7
cDNA (cloned by PCR from the cD~A of another HLA-B7 patient and inserted into plasmid vector
~ pcDSRalpha). The l,-~."xr~;l;on was made in flllrlir~tr wells. Briefiy, samples of COS-7 cells were seeded, at
5 15,000 cells/well into tissue culture flat bottommicrowells, in DMEM sllrplPm~nt~l with 10% fetal calf
serum. The cells were inr.llh~t~d ovemight at 37~C. medium was removed and then replaced by 50 !ll/well of
DMEM mt-Aillm ~."I~;";",, 10% Nu-Serum (Collaborative Research, Bedford, MA)~ 40011g/ml DEAE-
dextran, and 100 IlM chloroquine, plus 100 ng ofthe pl~cmi~l~ Following four hours of inNlh~ n at 37~C.
the m~lillm was removed, and repla~,ed by 50 !11 Of PBS cv"l~;";" 10% dirnethyl sulfoxide (DMSO). This
I o m~1illm was removed after two mimltes and replaced by 200 ~11 of DMEM snrplf mrntrd with 10% FCS.
Following this change in m~ lm, COS cells were i~ r~l for 2448 hours at 37~C. The
~ "~ir~ "l~; then were screened with CTL 263/17. A~Ler fiIst removing the m~ lm~ 3ooo cTL 263/l7 cells
wereaddedtoeachwellinl00ll1Oflrn~lillmc~"l1~"l;"g2~U/rnlL-2. TheamountofTNFpresentinthe
sll~n~t~nt was then measured by tes~ng its cytotoxicity for WEH[ 164.13 cells. Most pools gave a TNF
;onbelow10pg/ml. Twopools(1157and1319)gavehigher~l)r~ )n.~inbothofthe
(ll,l,lif~l~wells(24to37pg/rnl). Thebacteriaofpool 1319wereclonedand 1200cloneswereobtained.
Their plasmid DNA was extracted and t~n~f~r. t~l into COS cells with HLA-B7. The LL-~L~r~;~ll~ were
screened with CTL 263/17. One cDNA clone (9H3) gave a hi~h TNF production by CTL 263/17. Fip,ure 1
shows the result obtained when this cDNA (60 r~) was ~ r~L~d into COS cells with the HLA-B7 cDNA
20 (60 ng) and screened with CTL 263/17.
This cDNA also was stably ~ r~ ~ into LB23-SAR cells. an HLA-B7 sarcorna line. The lysis test
then was ~;l~Ullll~l with CTL clone 361A/17. which recognizes the sarne antigen as CTL clone 263/17.
These stably ~n~fectPd cells were recognized in the sarne manner as the COS-HLA-B7-cDNA 9H3 cells.
~5 EXAMPLE 3: SeqllPnre of cDNA 9H3
cDNA clone 9H3 is 1130 bp long. This cDNA was not coml let~ because its size was smallerthal1
that of an rnRNA observed on aNo]thern blot (1.6kb). The 5' end ofthe cDNA was cloned by RACE-PCR
andtheentiresequencewas cr,nfinnr(l ThisentiresequenceisshownasSEQ.IDNO.l. ACI mr~n~onwitl
30 the seq~lPnrr~ reported in rl~t~h~nk~ showed at the 3' end a high homology with a short sequence of 235 bp
called "expressed sequence tag". whose fi~nction is unknown (I). and at the 5' end a lirnited homology (75%
in a stretch of 95 bases) withthe i1"~ ~, strand oftwo hurnan r.n~ n~-us retroviruses called RTVL-H~
andRGH~(~ 3).
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W 096/29409 -14- rCTrUS96/04037
The gene was called RAGE, for Renal tumor AntiGEn.
The sequence contains five open reading frames~ ORF 1: 99 base pairs rn.~o~1ing a protein of 32
residues; ORF2: 123 base pairs r nr~in~ a protein of 40 residues; OR~3: 87 base pai~ encoding a protein of
28 residues; ORF4: 288 base pairs Pn.~~ing a protein of 95 residues, and ORF5: 222 base pairs rnr~ling a
5 protein of 73 residues. (SEQ.ID.NOs.2, 4, 6, 8 and 10. respectively). SEQ.ID.NO.4 codes for the TRAP
from which the Anti~enir peptide reactive with CTL 263/17 (as an HLA-B7/peptide complex) is derived.
EXAMPLE 4: kl~ntifit~, tinn of Additional RAGE Genes
Thisexample~l~rihçsthei-l~ntifi.~,tinnofthreea(lt1itinnAlRAGEgenesalldthecl~"";,.~liQnthatonl~;
1 o the RAGE gene i~lrntifi~l in the above examples, now ~i,a ~~~1 RAGE-1. encodes a RAGE TRA reactive with
CTL 263/17.
A probe was ~ d from RAGE-1 cDNA and used to screen a LE9211-RCC cDNA librar~ for
l(1ition~l RAGE genes. Three cDNAs homologous to RAGE. labeled RAGE-2 (SEQ.IDNO.12) RAGE-3
(SEQ.ID.NO.13) and RAGE-4 (SEQ.ID.NO.14), were i~o!ated. The RAGE-2,3 and 4 genes were seq~rnred
15 by standard mPth~ l, ofthe mlrl~ti~ u~n~ ofthese RAGE cDNAs with the RAGE-1 cDNA
showed that truncated and novel open reading ~ames (ORFs) were present in the newly i~lrntifi~cl RAGE
cDNAs. RAGE-2, RAGE-3 and R ~GE4 c-)nt~in~cl an insel~ of 37 bp at position 249 of RAGE1 (within the
seq~lPnr~ c~ .li,.g to ORF~ (SFQ.TlD.NO.4) of RAGE-1). For the RAGE-2 cDNA! c~" ~ ;nl~ with the
cosmid seq-~rnre int1it~t~d that this insertion c(J~ -ds to the beginnina of an exon. Its absence from the
~o RAGE-1 cDNA might result from the use of ~n altern~tive downstream acceptor site. In ~ litinn~ RAGE-2. 3
and 4 differ from RAGE-1 in lacking a nucleotidc at posilion 192 of RAGE-1. These changes cianifir~ntl~
modifythe ORFs of RAGE-2,3 and 4 that are hl)mnlogous to ORF~ and 3 of RAGE-1. In ~ lition. RAGE-3
has another insertion of 47 bp at the 5' end. Except for these di~l~llC~. the RAGE-1 ~ 2 and 3 sequences are
i~lrntir~l
~5 RAGE~ is about 800 bp longer than the other RAGE cDNAs. Its 5' se~urn(~e is identical to that of
RAGE-2. but from position 434 to the poly-A tail. the RAGE-4 sequence differs totally ~om the other RAGE
cDNAs. The RAGE~ cDNA was shown not to be rhimrri~ The starhng position of the 3' unrelated sequence
cul~ ds to an exon-intron boundry in the RAGE genomic seqllrnre~ and the 3' unrelated seq~-rnre was
present in the 3' end of the RAGE gene. Therefore. the RAGE4 cDNA appears to result from difrtl~llLial
30 splicing ofthe RAGE-2 gene. The srhPm~tic ~ligllmrnt of ~e four cDNAs is shown in Fig. 2. There are 17
ORFs in the four RAGE cDNAs. Of these 17.10 are diffèrent. The ORFs are as follows:
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w096129409 - 15- PcT~Tssb/04o37
Gene ORF N~ r~t~ No.SEQ.II).NO.
RAGE-1 ORF1 173-271 2
ORF2 204 326 4
ORF3 313-399 6
ORF4 323-610 8
ORF5 ~14'1665 10
RAGE-2 ORE;2' 217-276 15
ORF3' 273-449 17
ORF4 373-660 19
ORF5 494715 21
RAGE-3 ORF6 185 247 23
ORF2' 274333 25
ORF3' 330--506 27
ORF4 43~717 29
ORF5 ~51--772 31
RAGE~ ORF2' 213 272 33
ORF3" 269-832 35
ORF4' 369-557 37
The RAGE-2, RAGE-3 and RAGE-4 cDNAs were cloned into expression pl~crni~l~ by art-sTandard
25 procedures and ~ rn l~l as described with HLA-B7 into COS-7 cells to (1rl~" "" ,P if these cDNAs also
encoded the antigen reco,,ni7PA by CTL 263/17. Parallel conTrol e~rim~nts with the RAGE cD~TA (now
referred to as RAGE-l) and with LE,921 l-RCC cells were also p.orf~mlPA
Tnl~3lh~ti~n of LE921 1-RCC cells or C0S-7 cells cotr~n~f~ctPd with RAGE-l and HLA-B7 with CTL
263/17 stron_ly induced release of ~NF by CTL 263/17. Col~ r~ ;on of RAGE-2. RAGE-3 or RAGE ~'
30 and HLA-B7 did not elicit TNF release. Therefore~ only RAGE-l was able to transfer expression ofthe
antigen recognized by CTL 263/17.
EXAMPLE 5: Ident;fication of ORF C""l~;";"(J RAGE turnor rejection antigen
The 37 bp insertion in RAOE,-2~ 3 and 4 caused ~ e Irl ~ n of ORF2 in these three gJel1es.
35 It was r~-~cont~l therefore, that the ~nti<)P1lic peptide reco~i7P(l by CTL 263/17 was encoded by the 3' end of
ORF~. To test this hypothesis. the I)NA sequences cvl,~nding to ORF2 of RAGE 1 and ORF2' of
RAGE-2 and RAGE-3 were cloned into an expression vector and ll-,lll'ir~ d into COS-7 cells with
HLA-B7 as (leC~.rihe~l above. As positive controls, the RAGE-l cDNA was cv~ sr~;~d with HLA-B7 into
COS-7 cells or LE291 1-RCC cells ~vvere used. These ~ rr,,~",~ or LE291 l-RCC cells were used to
provoke release of TNF from CTL :263/17 cells. Arnong the ORF LI~Lsr~ . only the ORF'7 from
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w096/29409 - 16- Pcr/u:, -/0~037
RAGE-1 ~.lcr~!irlllly ~ 11~1 TNF release from CTL 263/17 cells (Fig.3). This ex~"llcl,l confirmPcl
that the RAGE ~nti~;Pnic peptide rcco~ni7~1 by CTL 263/17 cells was encoded by the 3' end of ORF2 of
RAGE-l.
EXAMPLE 6: I-lPntifir~tinn of RAGE b~nor rejection anhgen peptide
Synthetic peptides cc llr~l nl~l;l Ig to the 3' end of RAGE-l ORF2 were synthP.~i7~A and tested for
stim~ hnn of TNF release from CTL 263/17 cells COS-7 cells were tr~n~fPct~P"l with HLA-B7 as described
above and a synthetic peptide c0~ 3lldillg to a 3' portion of ORF2 was added to the culture. CTL 263/17
cells were added and the production of TNF was measured afcer 18 hours (Fig.4). Peptide
0 SPSSNRIR~TST (SEQ.ID.NO.39) PffiriPntly shm~ t~l the release of TNF from CTL 263/17. Sinoe
peptides presented by HLA class I mnlPclllP~ are usually 9 amino acids in length, we tested nnn~mPric
peptides (SEQ.ID.NOs.40, 41 and 42) derived from the (1c~Pc~mPnc peptide (SEQ.ID.NO.39) previously
used to shmnl~tP TNF release from CTL 263/17 oells. The results ofthese PxrPrimPntC are shown in Fig. 4.
One ofthese peptides (SPSSNRIRN, SEQ.ID.NO.40) was recogr~ed by CTL 263/17, but to a far lesser
extent than the do lPrz~mPric~ peptide, which slla~e~tPcl that the nnn~mPr (SEQ.IDNO.40) was not the optimal
peptide. The ~lPr~mPrir, peptide (SPSSNRIRNT, SEQ.ID.NO.43) was very Pffic~iPntly recogr~i7ed by CTL
263/17.
EXAMPLE 7: Activitv of RAGE turnor rejection anti_en nnn~mPr and decamer peptides
This example shows the ability ofthe RAGE TRA peptide to induce lysis of ~A-B7-ex~ sillg cells
and the relative Pffiripnrip~ ofthe ~n~ and decarnerpeptides.
Non~mPri~. and ~lPc~mPric RAGE peptides (SEQ.ID.NOs.40 and 43. respectively) were tested for the
ability to induce cell lysis of HLA-B7- LB23-EBV B cells by CTL 263/17 cells in a dose ~ ;yOnSe assa~.
Lyophili7ed peptides were dissolved at 20 mg/rnl in DMSO? then diluted to 2 mg/rnl in 10mM acetic acid
~5 and stored at -80~C. Target cells, HLA-B7 EBV-I~"!iro, " ,Pfl lymphoblastoid cells (LB23-EBV cells). were
labeled with s'Cr as described above, for 1 hour at 37~C followed by extensive washing to remove
urlin~l~u~dL~l label. LB23-EBV cells were then in-~3lh~tPrl in 9~well microplates inthe ~ lce of various
c~ r"~ n~ of peptides for 30 rninutes at 37~C. CTL~63/17 were then added in an equal volume of
mediumatan~;Lul.L~lratioofl0:1. Chromium-~lreleasewasmeasuredaf~er4hours. Fig.~shows
,o the results ofthe dose response assay. Halfmaximal lysis of LB~3-EBV cells was induced at a Con~Pnh~tion
of 30 ng/ml SPSSNRIRNT peptide (SEQ.ID.NO.43).
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EX.9MPLE 8: Expression of RAGE-1 ~ene
The expression of RAGE was tested by PCR using the following primers:
SEQ.ID.NO.46
- GTG TCT CCT TCG TCT CTA CTA (sense primer)
5SEQ.IDNO.47
- GGT GTG CCG ATG ACA TCG (~ i~"~P primer comm~n to all RAGE ~rJ,enes)
SEQ.IDNO.48
- GAG GTA TTC CTG ATC CTG (;~ ;c~ r primer specific for RAGE-1)
Fi~ total RNA was taken fiom the parhcular sample, using art recogni~ed techni~ues. This RNA~ was used to prepare cDNA. The protocol used to make the cDNA involved comhinin~ 4 ~11 o
f 5x reverse
wcbuffer, 1 ,ulofeachdNTP(103n~,2~Llofdithio~3reitol(100rnM),2!l1OfdT-15prirner(20
IlM), 0.5 ~11 of RNasin (40 units/lll). and 1 ,ul of M-MLV reverse l~ c~ (200 units/~ll). Next. 6.5 111 of
lr~ 3~- RNA (1 ,ug/3~5 ,ul water~ or 2 ~g total template RNA) was added. The total volume ofthe mix~ure
was 20 IlL This was mixed and in~l llr~~1 at 42~C for 60 mimlt~c, after which it was chilled on ice. A total of~5 80 ,ul of water was then added, to 100 !11 total. This mixture was stored
at -20~C until used in PCR.
The 3~,ents for PCR in~ A
- 5 microliterc of lOx ]3ynaZy3ne buffer
- 20 pmoles of each p3imer
5 n~nnmnlPc of each dNTP
- 1 unit of polyrn~n7in~ er~ne "DynazYrne" (2 ur~its/~l)
- 5 ~11 of cDNA (~~ to 100 ng total RNA)
- water to a final volume of 50 !11
The mixture was c~-mhinrA and layered with one drop of mineral oil. The mixture was LL~ ,d to
athermocycler block, preheated to 94~C, and ~qmrlifi~ti~n was catTied out for one cycle of 15 min at 94~C.
2~ followed by 33 cycles of:
- 1 min. at 94~C
- 2 3nin. at 56~C or 60~C (see below)
- 3 min. at 72~C
A f~l~l extension step of 15 min. was then performed at 72~C. Expression of all RAGE ~enes was tested by
30 PCR ~mplifi~tic)n with pan-RAOE, sense (SEQ.ID.NO.46) and ~ntic~nce (SEQ.ID.NO.47) primers usin~ an
~nnP~lin,~ step of 60~C for 2 mimlt~ s Expression of only RAGE-l gene was tested by PCR ~mplific~ti~n
with pan-RAGE sense (SEQ.ID.NO.46) and RAGE-1-specific ;.~ c~ (SEQ.ID.NO.48) prirners using an
CA 02211448 1997-07-25
W 096/29409 -18- PCTrU5~6/01~37
~. " ,~,l i "~ step of 56~C for 2 mim ltPc The PCR product of l 94 base pairs (general to all RAGE genes tested)
and 239 base pairs ~specific for RAGE-1 genes) were vi~l-li7P~l on an agarose gel (1.5%) ~."~,",i"g
ethi~illm bromide.
The gene was found to be tumor-specific. The gene was silent in all normal tissues tested, except for
retina In particular, the gene was silent in adrenals~ bladder, bone marrow, braiIL breast, cerebellum~ colon~
hear~ kidney, liver, lung, mPl,n~c.ytes, muscle, nevus, ovary, pl~Pnt,, prostate. skin, splenocytes, .ctom,th~
testis, thymocytes, uterus and healing wounds. The gene, however, was found to be expressed in a variety of
tumor cell lines and tumor tissue samples (Table 1). It is also expressed in some other turnors which are not
listed here. although not frequently.
Table 1. E~ m of R9GE-1 Gene in Tumor ~S-~rtr'
r~l Type N~ ' of Tumors E~. e~
ALL RAGE RAGE-1
15TumorSamples
Renal carcinoma 2/57 1/57
Sar~omas 5/25 3125
Bladder tumors superficial -0/29 0/29
infilt~tinf~-3/37 3/37
~rl"~ ,,c primary lesions -2/60 2/60
m~,~ 8/177 6/177
20 Head and neck tumors 2/50 1/50
Md,,,,,~u~r Cill~illUlllaS 3/128 1/128
Prostatic c~:l,,u" as 0/22 0/22
Colorectal carcinomas 0/48 0/48
T ellkPmi~ 0/19 0/19
2~ Lung carcinomas (NSCLC') 0/59 0/59
(SCLC)
0/5 0/5
MesothPli~m~ 1/3 0/3
Brain tumors 0/11 0/11
Oesoph~e tumors 0/7 0/7
30 Ovalian turnors 0/3 0/3
Tumor Cell Lines
Renal c~"lu",a 8/19 7/19
Bladder tumors 3l3 3l3
3~ MesothPlil m,~ 11/19 8/19
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Headandneckturnors 3/7 1/7
Sarcomas 2/6 1/6
M~lsin--msis 11/78 7/78
Colorectal C~ 1/17 ~ 1/17
s Lung ~.,illoll~s (NSCL(~) 0/2 0/2
(SCLC)
0/26 0/26
T ~ k~.misis/Ly~ .nll~s 0/11 0/11
Brain tumors 0/1 0/1
Gas~ic tumors 0/2 0/2
lo
' NSCLC: non-small cell l -ung C~
The ~lcO ~ xt~ es sho~ the isc,lsitic)n of a nucleic acid molecule which codes for a TR~P. This
TRAP coding molecule, however~ is not hf m~logous with any ofthe previously rlis~Jose1 coding sequences
5 des,cribed in the lcr~ c -~es set forth s~7ra. Hence, one a~pect of the invention is an isolated nucleic acid
mol~culP which includes all or a unique portion ofthe nucleotide serlu~-n~e set fordl in SEQ.ID.NO.17
SEQ.IDNO.4. SEQ.ID.NO.6 or SEQ.ID.NO.10. It is also expected that antigens derived from other RAGE
ORFs encoded by SEQ.ID.NOs. lr2,~ 13 and 14 rnay be recognized cytolytic T lyrnphocyte clones otherthan
CTL263/17. Thus, the invention in smother spect involves any one or more of the RAGE family of genes,
20 in~.lurlin~ isolated unique portions tkereof such as portions ~nr~lin~ APs and TRAs~ RAGE TRAPs and
TRAs derived l~~lcrlolll and all of the ~lislc~n~lstic and therapeutic m~isllitiPs relating thereto. The foregoing
seqll~n~eS are not MAGE, BAGE or GAGE sequences. as will be seen by c~ a them to the MAGE~
BAGE or GAGE ~l~ Ir~ ~ P~ described in the lcLcllcc~.
Also a part of the invention .3re those nucleic acid sequences which also code for a non-MAGE. noll-
~5 BAGE and non-GAGE turnor rejection antigen precursor but which hybridi_e to a nucleic acid molecule
co~ ~x;i~ J. ofthe above ~ s~ril~l nucleotide ~lu~ . under strin~ nt conditions. The terrn ".strina,~.nt
c nrliti~ ns" as used herein refers to pararneters with which the art is familiar. More specifically. strin~ent
c-)n-litinn~ as used herein. refers to hyhricli7~tion at 65~C in hvbridi_ation buffer (3.5 x SSC. 0.02% Ficoll.
0.02% Polyvinyl pyrolidone 0.02% Bovine Serum Albumin. ~mM NaH,PO, (pH7). 0.5% SDS. 2mM
30 EDTA). SSC is 0.15M Sodium Chloride/0.lSM Sodium Citrate. pH 7: SDS is Sodium dodecyl Sulphate:
and EDTA is Ethylene diamine tetra acetic acid. After hyhri~i7~tion the " Irlnl~l ~."P upon wl~ich the DNA is
~rcll~ is washed at 2xSSC at room Irl I Iprl ~ c and then at 0.1xSSC/0.1xSDS at 65~C.
There are other c ~nrlition~ reagents. and so forth which can be used~ which result in the same degree
of 51rin~.nry The ski~led artisan ~;vill be familiar with such c--n-1iti~n~ and thus they are not ~ en here. The
CA 022ll448 l997-07-25
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skilled artisan also is familiar with the mPth~rlok-gy for s~ g cells, ~l~rtldl~ly cancer cells. for expression
of such m~ l-clllPc which then are routinely isolated, followed by i~l~ti~ n of the ~~ ;lll nucleic acid.
In ~el~ll,llg for RAGE family " ~" -l~ i, a Southern blot may be pPrform~d using the foregoing
con-1ition.~, together with a 3'P probe. After w~LIl~g the mPmhr~nP to which the DNA was finally
s ll~ sr~"~d, the mPmhr~nP can he placed against X-ray filrn to detect the r~ .tive signal.
The invention thus provides isolated unique f~mPntc of SEQ.ID.NO.1 or its complPnnPnt A unique
r, ~ ", is one that is a 'signature' for RAGE genes. It, for example, is long enough to assure that its precise
seqllPn~e is not found in mnl~culPs outside ofthe RAGE farnily as defined by clairn 23. Preferred unique
fr~gmPnts are those found only in ORF2 or its comrl~-nPnt Unique fr~,amPnt~ can be used as probes in
10 Southern blot assays to identify RAGE family mpmhp~ ~ ;l la those expressing oRF2 or can be used in
~ )lir~ion assays such as those employing PCR As known to those skilled in the al~ large probes such as
200 bp or more are ~l~r~ll~ for certain uses such as Southern blots. while srnaller fr~amPnt~ will be preferred
for uses such as PCR. As will be recog,ni7P~l by those skilled in the art. the si~e of a ur~ique fr~amPnt will
depend upon its conservency in the genetic code. Thus, some regions of SEQ.ID.NO.1. SEQ.ID.NO.12,
15 SEQ.IDNO.13 and SEQ.ID.NO.14 will require longer sPamPntc to be unique while others will require only
shortse~mPnt~,typicallybetween 12and32bp(e.g. 12, 13,14,15, 16, 17. 18, 19,20,21,22. 73.24,25,26,
27, 28,29,30,31 and/or 32 bases long). Virtually any segment of SEQ.ID.NO.1 that is 18 or more
nucleotides in length will be unique. Those skilled in the art are well versed in methods for sPIPcting such
ces, typically on the basis ofthe ability ofthe unique fi~;rn~nt to selectively ~ hn~lisll the sequence
~o of interest from n- nf~nnily ~ " ,l~, ~ A ~ ofthe sequence ofthe fr~g n~nt to those on known data
bases typically is all that is ~ece~y, although in vih~o c~ ,-- r, I l l l;lI~Il y hyhricli7~ti~ n and sequencing analysis
may be ~ . r(,. " ~P.1
For any pair of PCR primers col~L~ d and ~ngecl to selectively arnplify RAGE-l~ a RAGE-l
specific primer may be used. Such a primer is a contiguous stretch of RAGE-l which hybridizes to both
~s sides ofthe insertion point in 0RF2 which is altered by the insertion of additional nucleotides in other RAGE
genes. Such a specific primer would fully hybridi_e to a COl1tigUOUS stretch of nucleotides onlv in RAGE-l.
but would hybridi_e only in patt to RAGEgenes that do not share ORF ''. For efficient PCR priming and
RAGEli~lf.ntifi~tion the RAGEl specific pnrner should be cons~ucted and arranged so it does not
hybridize effici~ntly at its 3' end to RAGE genes other than RAGEl.To~(~c-)mrlish this. the primer can be
30 ~i~Ps~nbe~l as having two ends: a 5' endthat is contiguous with and c~ mrlemPnt~ly to one side ofthe insertion
point joined directly to a 3' end that is contiguous with and c~mpl~rnpnt~7 to the opposite side ofthe insertion
point. By making the 5' end ofthe primer ~lh~t~nti~lly longerthan the 3' end~ and by making the 3' end short
CA 02211448 1997-07-25
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(i.e. 14 nucleotides), then the kinetics of hyhri~li7Atinn will strongly favor hybnd~ation at the 5' end. In this
in~t~nrP 3' initiated PCR extension v~ill occur only when both the 5' and 3' ends hybridize to the nucleic acid.
i.e. only when ORF 2 is present wi~out an insert.
RAGE-1 specific prirners. as ~1P~Tihe~1 above, may be flr~ to prime DNA synthesis on either
strand ofthe DNA heli~ ~nl~l herein as the Watson or the Crick s~ands. The se~ onre in RAGE 1
which flanks the insertion point, is 5'-CAAACANGGATCA-3' (SEQ.ID.NO.49, Watson strand~ N is a
m~ eoti~l~ insert). A RAGE-1 speci Eic prirner dP~ion~d to ~ clliially arnplify the Watson strand of
RAGE-1 typically would c~ i~ ] 2 and preferably 1~ ormore nucleotides ary to the nucleotides ofthe
Watson strand 3' to the insertion pOillt. The lr~ ;l l;l Ig portion ofthe pnmer would be one to four nucleotides
o long and would be cl mrl~m.ont~Ty to the ~1~ er 5' to the insertion point. Such a prirner would be perfectly
compl~ ntdl~ and contiguous with its c~mrl~m~nt in RAGE-1. The 3' end ofthe prirner would hybridize
to its cnmrlem~nt in the Watson s~nd and initiate ~Ytton~inn In RAGE genes other than RAGE-1~ the
inse~tion of ~ l ,el -" ,l~l , ,rnt~Ty nucleotides at the irlsertion point of ORF2 would ~L,l,~ 11y elimin~te
hyhrifli7~ti~n ofthe 3' end ofthe RA.GE-l specific primer to the Watson shand 5' ofthe insert. The mi~m~tr.l
15 gen~t~l at the 3' end of the primer when hybridi_ed to RAGE genes. other ~an RAGE-1. would preclude
efficient ~mrlifir~tion ofthose genes. Exemplary prLrners consist ~.nti~lly ofthe follow~n~ nrec
wherein N is ~ro, one or more contiguous nucleotides on the ~ l;dL~ Watson or Crick strands:
5'-NTATTCCTGATCCT-3'(SEQ.IDNO. 50);
20 5'-NTATTCCTGATCCTG-3'(SEQ.ID.NO. 51);
5'-NTATTCCTGATCCTGT-3'(SE',Q.ID.NO. 52),
5'-NTATTCCTGATCCTGTT-3'(SEQ.ID.NO. 53);
S'-NCAAGTTCAAACAG-3'(SEQ.IDNO. 54),
5'-NCAAGTTCAAACAGG-3'(SE,Q.ID.NO. 55),
2~ 5'-NCAAGTTCAAACAGGA-3'(SEQ.IDNO. 56) and
5'-NCAAGTTCAAACAGGAT-3'(SEQ.IDNO. 57).
The expression of RAGE-I may also be detected by PCR using prLmers which initiate extension on
opposite sides of the insertion point. Analysis of arnplifir~linn products can rli~ting~lich RAGE-1
30 ~mrlifir~tic~n products from non-R~GE-1 amrlifir~ion products by the length ofthe ~mplific~tion products.
Because the RAGE-1 gene does not contain the insert present in other RAGE genes, ~mplifir~tion products
derived from ~AGE-1 will be shoIterthan amplifir~ n products derived from other RAGE genes (by about
CA 02211448 1997-07-25
W 096/29409 -22- PCTrUS96/04037
37 base pairs). This ~ ce may be tliChn~liehPCl readily using standard mPtho~lc inthe aTt. ~ ition~l
mPth~lc which can (1ictin~ h nucleotide sP~ ~PnrPt~ of snhst~nti~l homology, such as ligase chain rection
('~CR'~ and other mPthr~c will be ~cllL to skilled artisans. RAGE 2~ 3 and 4 specific primers rnay be
prepared in a like marmer.
The invention also includes the use of nucleic acid .C~llPn~Pti which include altemative codons that
encode the same amino acid residues as encoded by the RAGE genes. For Px~mpl~ as tliC.~lr s~d above in
Exarnple 7~ a (l~mPric peptide SPSSNRIRNT (SEQ.ID.NO.43) is a RAGE tumor rejection antigen. The
serine residues (amino acids No. 1, 3 and 4 of SEQ.ID.NO.40) for exarnple~ are encoded by the codons TCA.
AGT and TCA, respectively. In addition to TCA and AGT, serine amino acid residues may also be encoded
10 by the codons TCC, TCG, TCT and AGC. Each ofthe six codons is equivalent forthe pulposes of Pncor1ina
a serine residue. Thus, it will be a~ to one of ordinary skill in The art that any of the serine-encoding
nucleotide triplets may be employed to direct the protein synthesis ~ ;, in vitro or in vivo~ to incorporate
a serine residue. Similarly, nucleotide ~~ e triplets which encode other arnino acid residues c(,l ~ i"a a
RAGE tumor rejection antigen include: CCA? CCC. CCG and CCT (proline codons)? CGA. CGC. CGG.
CGT, AGA and AGG (arginine codons); ACA, ACC, ACG and ACT (threonine codons); AAC and AAT
nP codons); and ATA, ATC and ATT (ic~lelT(inP codons). Other arnino acid residues may be
encoded similarly by multiple ml~ ootirle .~qnPnrP~ Thus. the invention Pmhr~cPc ~PgJ~-, Ir~ nucleic acids
that differ from the biologically isolated nucleic acids in codon sequence due to the (lPgPnPr~cy of the enetic
code.
The ~ lcs above also show the isolation of peptides which are RAGE TRAs. These PxPmpl~ry
peptides are processed tr~ncl~h~n products of the nucleic acids of SEQ.IDNO. 1. As such it will be
a~,ul~;d~d by one of ordina~ skill in the art that the h~nCl~hon products from which a RAGE TRA is
processed to a final forrn for l~lrSrl ~ 1 ;on may be of any length or sequence so long as they Pn~mr~s~ the
RAGE TRA. As ~lr~ lrA in the examples above~ peptides or proteins as small as 9. 10~ or 1~ amino
acids and as large as the amino acid s~llPn~e encoded by ORF1 are ~ id~ly processed. presented by
HLA-B7 and recogni7~d by CTL263/17. The peptide of SEQ.ID.NO.23 may have one. two. three. four. five.
six. seven. eight. nine. ten, or more amino acids added to either or both ends. The antigPnic portion of such a
peptide is cleaved out under physiological çontiitinn~ for presentation by HLA class I molecnlt~ ~
The amino acid sequence of proteins and peptides from which RAGE TRAs are derived may be of
natural or non-natural origiIL that is, they may cc" "l " ,~ a natural RAGE TRAP molecule or mav comprice a
mo lifi~cl sequence as long as the amino acid sequence retainc the tumor rejection antigen sequPnce
reçogni7P~ by the CTL when presented on the surface of a cell. For example~ RAGE tumor rejection
CA 02211448 1997-07-25
W 096/29409 -23- PCT~US~ 4037
antigens in this context may be fusion proteins of a R~GE turnor rejection anti~en and unrelated amino acid
s~ .nrPc~ the ~ "~ rtl polypeptide of ORF2 ofthe RAGE-1 gene, synthetic peptides of amino acid
~l~lrl ~ shown in SEQ.ID.NOs.39, 40 and 43, labeled peptides, peptides isolated from patients with renal
cell carcinoma, peptides isolated frorn cultured cells which express RAGE-l, peptides coupled to n~n~r~
5 mc1~~ s for exarnple in certain drug delive~ systems and other m~]~111ec which include the amino acid
seq~l~n~e of SEQ.ID.NO.40.
It will also be seen from the ex~n~lcs that the invention ~mhr~Pc the use ofthe sequences in
expression vecto~, as well ac to L~ sr~;l host cells and cell lines. be these prokaryotic (e.g., E. colz~, or
euk~yotic (e.g., CHO cells. COS cells, yeast expression systems and lt; C~ Ih; ~ l1 baculovims expression in
o insect cells). The expression vectors require that the ~~ cllL sequ~on~P . i.ethose described sup~a~ be
operably linked to apromoter. As it has been found that human HLA-B7 presentc a TRA derived from these
genes, the expression vector may also include a nucleic acid sequence coding for HLA-B7. In a ~ tion
where the vector contains hoth coding seqn~M~c, it can he used to l~ lsr~;l a cell which does not no~nally
express either one. The TRAP or TRA coding sequence may be used alone. when. e.g. the host cell already
1~ ~x.~ ~sHLA-B7. Of course, there is no lirnit on the paTticular host cell which can be used. As the vectors
which contain the two coding s~nf nrP~ may be used in HLA-B7 ~lcsclllillg cells if desired. and the nucleic
acid coding for the TRAP or TRA can be used in host cells which do not express ~A-B7.
The invention also r~ ; so called expression kits. which allow the artisan to prepare a desired
~A~lc~ion vector or vectors. Such ~xpression kits include at least separate portions of at least two ofthe
previously tiicc11~cerl m~tt-n~lc Ottler Ct~nl~llclll~ may be added~ as desired.To ~licting71i~h the nucleic acid molecules and the TRAPs ofthe invention from the previously
des~;l;bed MAGE farnily, BAGE gene and GAGE gene, the invention shall be referred to as the RAGE
farnily of genes and TRAPs. Hence" whenever "RAGE" is used herein~ ~ific~lly excluded are MAGE~
BAGE and GAGE genes~ gene praducts~ TRAPs and TRAs.
2s The invention as described herein has a number of uses~ some of which are described herein. Firs~ the
invention permits the artisanto ~ c~nose a disorder ('1,;.,~ d by expression ofthe TRAP. These methods
involvecl~L~""il,;"gexpressionoflhel~APgene~and/orTRAsdelivedtherefrom~suchasaTRApresellted
by ~A-B7. In the former situation. such ~ " ,i, ~ ionc can be canied out via any standard nucleic acid
(lt-lrl 1 l l;l ~ ;on assay~ in~ ing the polymerase chain reaction~ or assaying widl labeled hybridization probes.
In the latter si~ ion~ assaying with binding partnes for complexes of TRA and HLA~ sucll as antibodies~ is
especially preferred. An alternate method for tlt~lrl I l~il ,,~lion iS a TNF release assay~ ofthe type described
s~pra.
CA 022ll448 l997-07-25
W 096/29409 -24- PCT/U~G~0~037
The i~l~tion ofthe TRAP gene also makes it possible to isolate the TRAP mnlec3lle itself, and/or
TRAs derived ~lcl~r,ulll, ~eri~lly TRAP and/or TRA molt~c.nl~s c~ the amino acid se~ln~nrpc
coded for by SEQ.ID.NO.1 or4. Other TRAPs or TRAs encoded by SEQ.ID.NOs. 1, 12~ 13 and 14 and
recognized by other CTL clones andtor~ s~ d by otherHLA mnl~lll~c rnay be isolated by the procedures
detailed herein. CIhere are numerous HLA molecules known to those skilled in the art, in~hlrlin$~ but not
lirnited to, those encoded by HLA-A, HLA-B, HLA-C, HLA-E, HLA-F and HLA-G genes.) A variety of
mPthn-1nlogiPc well-known to the skilled rr~rti~inn~r can be utilized to obtain isolated TRAP mnlrc~ c
andtor TRAs derived ~ . The protein may be purified from cells which naturally produce the protein.
Alternatively, an expression vector may be introduced into cells to cause production ofthe protein. In another
10 method, rnRNA ~ may be micl~ lje~ d or otherwise introduced into cells to cause production ofthe
encoded protein. Tr~ml~inn of mRNA in cell-free extracts such as the reticulocyte lysate system also may be
usedtoproduceprotein. Peptidesc~",l";~;"gTRAsoftheinventionmayalsobe synthrci~fd invih~o. Those
skilled in the art also can readily follow known mt~thn lc for isolating proteins in order to obtain isolated
TRAP and/or TRAs derived th~ lll. These include. but are not limited to. immunochr~mnto~rhy.
HPLC, size-exclusion cl~.,ln~.~rhy, ion-exchange cl.l.""~ hy and imrnune-affinity cl~ ~rhy.
These isolated m-ll~nl~c when processed and presented as the TRA~ or as complexes of TRA and HLA.
such as HLA-B7, may be c~ mhin~1 with m~t~i~l~ such as adjuvants to produce vaccines useful in treating
di~ld~lj rh~ rd by expression ofthe TRAP mn~x~ In ~-klitinn vaccines can be prepared from cells
which present the TRA/HLA complexes on their surface~ such as non-proliferative cancer cells. non-
20 proliferative l[," ,~r~ etcetera. In all cases where cells are used as a vaccine. these can be cells transfectedwith coding sequences for one or both ofthe con~ necessary to provoke a CT
L response. or be cells
whichalreadyexpressbothmnl~ cwithouttheneedforl,~"~r~-i;nn Vaccinesalso~.,rl,",~ naked
DNA or RNA! encoding a RAGE TRA or ~l~;U~ thereof. which may be produced in vitro and
~l",;",~ dviainjection~particlebombardment~nasal~rir~tionandothermethods. Vaccinesofthe"naked
nucleic acid" type have been ~1Pnn~n~t~ to provoke an imm~-n~ ir~ in~ lin~r CJenPr~ti~n of
CTLs specific forthe peptide encoded by the naked nucleic acid (Science 259:1745-1748. 1993).
The TRAP molecule. its associated TRAs. as well as complexes of TR~ and HLA. mav be used to
produce antibodies. using standard techniques well known to the art. Standard l~r~lcllce works settin~ forth
the general l" "~ s of antibody production include Catty. D., Antibodies. A Practical Approach Vol. 1.
30 IRL Press, W~ DC (1988); Klein. J., Tmmlmt~lo~v: The Science of Cell-Non-Cell Di~i- l ;" ,i, ,~l ion.
John Wiley and Sons~ New Yor~; (1982), Kennett, R.~ et al., Monoclonal Antibodies~ Hvhri~l~ m~ A New
Dirnension In Biological Analvses, Plenum Press~ New York (1980); Campbell. A., Monoclonal Antibodv
CA 02211448 1997-07-25
W 096/29409 -25- PCTAU~9~ 37
Technologv. in Laboratorv Techniques and Bio( l Ir~ / and MolecularBiologv. Vol. 13 (Burdon, R et al.
EDS.), Elsevier Al-~L~ (1984), and EiserL HN., Microbiologv, third editiorL Davis, B.D. et al. EDS.
(Harper & Rowe, Phil~ lphi~ (1980).
- The antibodies ofthe present invention thus are ~ d by any of a vanety of mPthnrls in~lmling
~(l" ,;, .~ , ;"g proteirL fi~mPntc of pn~teirL cells e~ g the protein or fi~nPntc thereofand the like to an
animal to induce polyclonal antibodies. The production of monoclonal antibodies is according to techniques
well known in the arL As detailed herein, such antibodies may be used for example to identify tissues
expressrng protein or to purify protein. Antibodies also may be coupled to specific labeling agents for
imaging or to ~ntihlmnragents, inrllltlin~ but not limitedto, melhotlexal~, r~tlioio~lin~tP~l compounds, toxins
I o such as ricir~ other cytostatic or cytolytic drugs. and so forth. Antibodies ~lC~ according to the invention
also ~l~,f~ ly are specific for the TRA/HLA c~ es ~lPc-~rihetl herein.
When "disorder" is used herein, it refers to any pathological cl n~lition where the tumor rejection
antigen ~l~;Ul~l is ~l~d. An exarnple of such a disorder is cancer renal cell carcinoma in particular.
Some tl~ approaches based upon the ~li$rl.lslme are premised on a response by a subject's
immune system, leading to lysis of TRA ~ illg cells, such as HLA-B7 cells. One such a~ acll is the
~(l.";"~ nof~lltrl~ llcCTLsspecifictothecomplextoasubjectwith~hn~rrn~lcellsofthephenotype
at issue. It is within the skill ofthe artisan to develop such CTLs in vitro. Generally. a sarnple of cells taken
from a subject, such as blood cells~ are ~ r(l with a oell ~ lg the complex and capable of provoking
CTLs to proliferate. The target cell can be a ~ "~r~;l;" ,l such as a COS cell ofthe type rlPs~rihe-l supra.
These L~r~ present the desir~d complex oftheir surface and~ when comhinPd with a CTL of interest.
lr its prolif~r~ti~n COS cells, such as those used herein are widely available. ac are other suitable host
cells. Specific production of a CTL cIone has been ~ s~ihPd above. The clonally expanded autologous
CTLs then are ~ ilr cd to the subject. Other CTLs specific to RAGE-l and CTLs specific to RAGE
TRAs encoded by RAGE-2, 3, or 4 rnay be isolated and ~ lr~cd by similar methods.Todetailatl-r~ mPth d~-looy.referredtoasadoptivetransfer(Greenberg.J.Tmmlmol. 136(5):
1917 (1986); Riddel etal.. Science257: 238 (7-10-92)- Lynchetal. Eur. J. Tmmlm~-l 21: 1403-1410 (1991):
Kast et al.. Cell 59: 603-614 (11-17-89)). cells IJlCS~ illg the desired complex are comhinPd v~ith CTLs
leading to pr~-lifPr~ti~-n ofthe CTLs specific thereto. The pr lifPr~t~l CTLs are then a~l" lil li~lrl~ll to a subject
with a cellular ~1 " ,. " " ,~lity which is ch~ tPti7Pcl by certain of the ~hn~rm~l cells ~.]lC~'7~11LiLl(J the particulal-
30 complex. The CTLs then lyse tlle ~hnorm~l cells, thereby achieving the desired 11Ir~ uli~ goal.
The foregoing therapy assumes that at least some ofthe subject's ~hn~rm~l cells present the relevant
HLA~rRA c- m~ . This can be ~lrlrl 1 l l;l IPl1 very easily, as the art is very familiar with methods for
CA 022ll448 l997-07-25
W 096/29409 -26- PCTnUS9f'~1~37
identi ~ring cells which present a particular HLA molecule~ as well as how to identify cells ~ A~ iillg DNA of
the pertinent ~llPn( PC~ in this case a RAGE sequence. Once cells ~ illg the relevant ~ ~ are
i(lPntifi~1 viathe foregoing screening m~th~ol--gy, they canbe cr~mhinPcl with a sample from apatient,
where the sarnple contains CTLs. If the c-)mplPx ~ llillg cells are lysed by the rnixed CTL sarnple, then it
s can be ~ISSl lm~1 that a RAGE derived, TRA is being ~ d, and the subject is an ~ n~ tP for
the th~relltic approaches set forth supra.
Adoptive transfer is not the only form oftherapy that is available in accol~L~lce with the invention.
CTLs can also be provoked in vivo, using anumber of approaches. One approach~ i.e., the use of non-
proliferative cells expressing the c~mplt-x, has been elal~oldL~d upon supra. The cells used in this approach
10 rnay be those that norm~lly express the coll~ ~ such as irr~rli, ted tumor cells or cells ~ r(l with one or
both ofthe genes necessary for ~ lion ofthe complex. Chen et al., Proc. Natl. Acad. Sci. USA 88: 110-
114 (January, 1991) ex~ "l,lirrs this approach showing the use of ~--, "~r~l~ cells ~:A~ lg HPVE7
peptides in a ~ ;c regime. Various cell types may be used. Similarly~ vectors ca~ying one or both of
the genes of interest may be used. Vi~l or bacterial vectors are especially prefe~red. For e~m~le nucleic
15 acids which encode aRAGE TRA may be operably linked to promoter and r"lli1"~f . seq~lPn~Ps which direct
expresion ofthe RAGE T~A in certain tissues or cell types. The nucleic acid may be "lcol~ldl~d into an
expression vector. Expression vectors may be unrnodified exll~dclllumosomal nucleic acids, pl"smitls or virral
~. ,"" ~PS constructed or modified to enable insertion of exogenous nucleic acids~ such as those encodine
RAGE TRAs. Nucleic acids encoding a RAGE TRA also may be inserted into a retroviral ~ennmP thereby
~7o ~rilit~1tin~~ integration ofthe nucleic acid into the genome ofthe tar~et tissue or cell type. In these systems. the
gene of interest is calried by a microol~s.lL e.g.~ a Vaccinia virus~ retrovirus or the bacteria BCG and the
m~tPri,lls de facto "infect" host cells. The cells which result present the complex of interest, and are
reco~i7~1 by autologous CTLs. which then proliferate.
A similar effect can be achieved by c ol "l ,; "; "~ the TRAP or a .stim~ tory fi~c~mP.nt thereof with an
adjuvant to facilitate l~lcr~ lion into ~A-B7 plcs~ g cells in vivo. The TRAP is processed to yield the
peptide partner ofthe ~A molecule while the TRA is presented without the need for further processine.
Generally~ subjects can receive an intr~tlPrmsll injection o~ an effèctive arnount ofthe RAGE TRAP. and/or
Tl~As derived lhclc~ l. Initial doses can be followed by booster doses~ following immlmi7~ttion protocols
standard in the art.
,0 As part ofthe ;" " "ll";~l;nn protocols~ stlhst~tnrPs which potentiate the immune response may be
~l" ,;l l;x~ d with nucleic acid or peptide components of a cancer vaccine. Such immlmP response
~"li~l;"~ conl~o~d may be cl~ssifip~l as either adjuvants or c~ytokines. Adjuvants rnay enhance the
CA 02211448 1997-07-25
W 096129409 -27- PCTAUS9~CI.~7
immlm~ logir~ se by providir.lg a reservoir of anhgen ( e~rell~ rly or within ma~;lu~ s),
activating macrophages and ~ specific sets of lymphocytes. Adjuvar.~ts of many kinds are well
known in ~e art; specific examples include MPL (Smit-hKline Beecham)~ a c nn~;ent~.r obtained aPLer
~ p~ifif~hon and acid hydrolysis of Salmonella minnesota Re 595 lipopolys~crh~ri~1e, QS21 (~mithKlin~
s Beecharn), a pure QA-21 sapor~in p~lrified ~om Ouillja saponaria extract~ and various water-in-oil ~.mlllcicmc
~lc~ ;l from hio~ hle oils such as squalene andlor tocopherol. Cytokines are also usefuL in
v~r.in~tion protocols as a result of lymphocyte shm-ll~t ly properhes. Many cytokines useful for such
puIposeswillbeknowntooneofordina~yskillintheart inrlll-lin~intt-rl~llkin-12(IL-12)whichhasbeen
shown to enhance the protective effects of vaccines (Science 268: 1432-1434. 1995).
When~l",il,;~lr~d,the ~ ,compositionsofthepresentinventionare~.l",;";~lr,edin
.l,i1""~ tir~11y,1~cept-,lhle~ 1ionc Such~ )n~mayroutinelycontainrh,lrrn"relltir~lly
~c~t~l-le c~,, Irrl ,~ nc of salt, bul~fering a~ents, preservatives. c~mp,ltihle catTiers, suprlemPnt-lry immune
;l 1~ agents such as adjuvants and cytokines and optionally other thP~~ ltic a=,entc.
The l,lcl~"llion~ ofthe inven~on are ,~ lr,~d in effective amounts. An effective amount is th~t
5 amount of a ph"rm"r~lltir~ rl~ n that alone, or together with fi~ther doses. slimlll~trc the desired
~ . Int-he case oftrea~ng cancer, t-he desired ~c~l~vl~ is inhihiting the pro_ression ofthe cancer. This
may involve only slowing the proglession ofthe disease ~ ul~ily. althouch more preferably. it involves
halting the progression of the disease p~. " ,~, Irl ~lly. This can be monitored by routine methods or can be
mol~ lcd according to ~ gn~tic methods ofthe invention ~ rnc~rd herein.
Where it is desired to stimlll~ an irnmune response using a thP~pelltir composition ofthe invention.
t-his may involve the s~iml-l, ti~ n of a humoral antibody lC::i~lllS~ resulhng in an increase in antibody titer in
semm, a clonal expansion of cytotoxic lymphocytes. or some other ~ ir~hl~ imml]nolngic ~ ol~e. It is
helieved that doses of immlm-)gen~ rancing from one nano ram/kilo ~am to 100 miligr~m~iloar~m~
depending upon the mode of ~ ion~ would be effective. The preferred range is believed to be
2~ bet~,veen 500 n~n~ ~m~ and 500 micrograms per kilogr~m The absolute amount will depend upon a variety
offactors. inr-ln~in,a, the material selected for ~ l;on~ wl1etl1er ~e ~ ,-,,lion is in single or multiple
doses. and individual patient 1"~ lt Irl ~i incln-lin, age. physical con~iti~n size~ weight. and the stage of the
~ disease. These factors are well known to those of ordinary skill in the art and can be addressed with no more
t-han rouhne exp~rim~nt~*-)n
C~er aspects ofthe invention will be clear to the skilled altisan and need not be repeated here.
The terms and expressions which have been employed are used as terrns of ~ rrirti~n and not of
limit~ti~ n. and there is no int~.nti~n in the use of such terms and expressions of excluding any equivalents of
CA 02211448 1997-07-25
W 096/29409 -28- PCTrUS96/04037
the features shown and ~lP~rihe~ or portions thereof. it being recog~fized that vatious m~ifir~tit~n~ are
possible within the scope ofthe invention.
R~f~
s 1) Gleser,L.,andSwaroop,A. 1992. E~ ~d~lllP~-retagsandchrom-s m~l lor~li7~ti- nofcDNA
clones from a s~lhtr~rt~l retinal pigment epith~linm library. G~nomir~ 13.873-876.
2) Mager, D.. and I;l~;lll~l, J.D. 1987. Human ~n~1ogen~us retrovirus-like genome with Type C pol
~nt~nrf?~ and gasg s~ nrp~ relatedto hum~n T-cell lymphotropic viruses. J. Virol. 61~ 40604066.
3) ~rose, Y., T~k~ t~ ~, M.. Ha~ada, F. 1993 . Presence of env genes in m~mhr~; of the RTVL-H
lo family of hurnan rn~ n~ nc retrovirus-like ~l~nrntc Virology 192, 52-61.
CA 02211448 1997-07-25
W 096129409 29 PCT~U5~5
,~P~lPn~P Listing
~ (1) GENERAL INFORMATION:
s
(i) APPLIC~NT:
(A) NAME: LUDW:[G IN~ l'Ul~ FOR CANCER RESEARCH
~B) ~l~l:1345 Avenue o~ the Americas
(C) CITY: New ~.~ork
(D) STATE: NEW YORK
(E) COUNTRY: ~ E~ STATES OF AMERIC~
(F) ZIP: 10105
(i) APPLICANT:
(A) NAME: LEIDEN UNIVERSITY
(B) ~'lX~'l': Stationsweg 46
(C) CITY: Leiden
(E) COUNTRY: TE~E NETEERL~NDS
~F) ZIP: 2312 l~V
(ii) TITLE OF INVENT:CON: RAGE TUMOR REJECTION ANTIGENS
(iii) N~MBER OF SEQU~OE S: 57
(iv) CORRESPONDEN OE ADDRESS:
(A) ADDRESSEE: WOLF, GREENFIELD & SACKS, P.C.
(B) ~l~Ll~: 600 ATL~NTIC AVENUE
(C) CITY: BOSTt)N
(D) STATE: MASS~AC~U~ lS
(E) COUNTRY: ~ITED STATES OF AMERICA
(F) ZIP: 02210
CA 022ll448 l997-07-25
W096/29409 30 PCTrUS96104037
(v) COMPUTER RE~DABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTW~RE: Pat~ntIn Release #1.0, Version #1.25
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CL~SSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION N~MBER: US 08/401,015
(B) FILING DATE: 21-M~R-1995
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION N~MBER: US 08/530,569
(B) FILING DATE: 20-SEP-1995
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: GATES, EDWARD R.
(B) REGISTRATION NUMBER: 31,616
(C) ~K~OE /DOCKET NUMBER: L0461/7002WO
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 617-720-3500
(B) TELEFAX: 617-720-2441
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
CA 022ll448 l997-07-25
W 096l29409 -31- PCT/Ub,G~4037
(A) LENGTH: 1311 base pairs
(B) I YPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: ],inear
(ii) MOLECU~E T~PE: cDNA
(iii) H~PUl'~'l'lCAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOUR OE :
(A) ORGANISM: Homo sapiens
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
GGTGAGCAGC CA~AGCAGGC ATCCCCGCAG TTGACTTGCC ACCAAGGGAA TGTGGGTGAA 60
TGACCAAGGC AGGCATCCTC GC~GTGATCA GACACCAATG GAGTGTGGGT GAATAATCAG 120
GCAGGCATCC CCGCAGTGAT TA~ACACCAA GAGAAGACTA TTCCTGAGTC TGTGACTGGT 180
GCTGGAGll~l TGAGTCCACA G~TA~AATGT GTCTCCTTCG TCTCTACTAG AGAGGAA~A 240
GAACTGGAAT TGGAAGAACA G~AGACTGA AGGGTAGCAA GAGAGGCTGG AGAAGAGAGT 300
GAA~AGACCG CTTACCTGAT Tl'GAAATTGT CTGCAGCCCC TCTITCCTGG AGTA~ATG~A 360
CTGGACCA~A TCTCA~AAAA TCCACGATGT CATCGGCACA CCCGCTCAGA AGATCCTCAC 420
CAAGTTCA~A CAGGATCAGG A~TACCTCTA CTAACAACCA A'l-l-l'GTCCCC ACAATGCCTC 480
CA 02211448 1997-07-25
W096/29409 -32- PCTnUS96/04037
lC~lC~lGC ACGC~ATGGT GGCCTATGAT CCCGATGAGA GAATCGCCGC CCACCAGGCC 540
CTGCAGCACC CCTA~'llCCA AG~ACAGAGA AACA~lCC~l' A~AGCAAGAG GAGGACCGTC 600
CCAAGAGACG AGGACCGGCC TATGTCATGG AACTGCCCAA ACTA~AGCTT TCGGGAGTGG 660
TCAGACTGTC GT~'l'lACTCC AGCCCCACGC TGCA~lC~l GCTTGGATCT GG~AC~AATG 720
GAAGAGTGCC GGTGCTGAGA C~l-l~AAGT GCATCCCTGC GAGC~AG~AG ACAGATCCGC 780
AGAAGGACCT TAAGCCTGCC CCGCAGCAGT GTCGCCTGCC CACCATAGTG CGGA~AGGCG 840
GA~GATAACT GAGCAGCACC GTCGTCTCGA ~'l'l'~AGGC AACACC~AGC CCGACCGGGC 90O
CAGGCCTGGG TGATCTGCTG CTGAGACGCC ACGGAGGGCT GGGGATGCGC CTGCGTCCGT 960
TTCGCGCTGG CC~CTCT GGGTGCTGCC CTGCGCCCTG CCGCACCCGC GGCCCGCGCA 1020
GCTGCCTAGG ATGTTCTGGG CTAATATACT TGTAA~ACCA CCGCATTCTA GG~llll~ll 1080
TCA'l'l'llCGT TAAGAAl'l-l'G GGGCAGGAAA TA~'l'l'l'~'l'~A ~'l'l'l'~'l'ATAT G~ATCA~AAC 1140
A~ACGAGCAG GCA~ l~l GAl~l~ll~G GC~l~ AAGGTGGGTT CTGC~l~lCC 1200
~5 CTTCCCAGCG CTG~ l~A GTCGTGGAGC GCCATCATGT C'l'l'ACCAGTG ACGCTGCTGA 1260
CACCCCTGAC llllATTA~A GAATA~GCTG TCGTTAAAAA AAAPAAAAAA A 1311
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQ~EN OE CH~RACTERISTICS:
CA 02211448 1997-07-25
W O 96129409 33 PCT/U~3G~1~37
(A) LENGTH: 99 base pairs
(B) TYPE: nucleic acid
(C) STR~NDEDNESS: single
(D) TOPOLOGY: 1;n~r
s
(ii) MOLECULE TYPE: cDNA
(iii) HYP~'l~ lC~L: ~FO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(vi) ORIGINAL SOUROE.:
IS (A) ORGANISM: Homo sapiens
(ix) FE~TURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..99
(xi) SEQUENCE DESCRïPTION: SEQ ID NO:2:
ATG A~C TGG ACC A~A TCT C~A A~A ATC CAC GAT GTC ATC GGC ACA CCC 48
Met Asn Trp Thr Lys Ser Gln Lys Ile His Asp Val Ile Gly Thr Pro
1 5 10 15
GCT CAG AAG ATC CTC ACC A~G TTC A~A CAG GAT CAG G~A TAC CTC TAC 96
Ala Gln Lys Ile Leu Thr Lys Phe Lys Gln Asp Gln Glu Tyr Leu Tyr
20 25 30
TAA 99
CA 02211448 1997-07-25
W096/29409 34 PCTrUS96/04037
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CH~RACTERISTICS:
(A) LENGTH: 32 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: l;n~r
(ii) MOLECULE TYPE: protein
(xi) SEQUEN OE DESCRIPTION: SEQ ID NO:3:
Met Asn Trp Thr Lys Ser Gln Lys Ile His Asp Val Ile Gly Thr Pro
l 5 l0 15
Ala Gln Lys Ile Leu Thr Lys Phe Lys Gln Asp Gln Glu Tyr Leu Tyr
20 25 30
~0 (2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUEN OE CHARACTERISTICS:
(A) LENGTH: 123 base pairs
(B) TYPE: nucleic acid
~5 (C) STR~NDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYP~'l'~'l'lC~L: NO
(iv) ANTI-SENSE: NO
CA 02211448 1997-07-25
W 096129409 35 PCT/u~ 7
(v) FRAGMENT TYPE: 'Lnternal
- (vi) ORIGINAL SOURCE
(A) ORGANISM: ~Iomo sapiens
(ix) FEATURE:
(A) NAME/KEY: ~_DS
(B) LOCATION: 1. 123
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
ATG TCA TCG GCA CAC CCG CTC AGA AGA TCC TCA CCA AGT TCA A~C AGG 48
15 Met Ser Ser Ala His Pro :Leu Arg Arg Ser Ser Pro Ser Ser Asn Arg
1 5 10 15
ATC AGG AAT ACC TCT ACT AAC AAC C~A 'l~ GTC CCC ACA ATG CCT CTC 96
Ile Arg Asn Thr Ser Thr Asn Asn Gln Phe Val Pro Thr Met Pro ~eu
20 25 30
CCT CCT GCA CGC AAT GGT GGC CTA TGA 123
Pro Pro Ala Arg Asn Gly Gly Leu
35 40
2~
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUEN OE CH~XACTERISTICS:
(A) LENGTH: 40 amino acids
(B) TYPE: amino acid
(D) TOPOLC~: line~r
CA 02211448 1997-07-25
W096/29409 -36- PCTrUS~'01C37
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
Met Ser Ser Ala His Pro Leu Arg Arg Ser Ser Pro Ser Ser Asn Arg
l 5 l0 15
Ile Arg Asn Thr Ser Thr Asn Asn Gln Phe Val Pro Thr Met Pro Leu
Pro Pro Ala Arg Asn Gly Gly Leu
35 40
15 (2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUEN OE CHAR~CTERISTICS:
(A) L NGTH: 87 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
~5 (iii) HYP~ CAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(vi) ORIGINAL SOUR OE :
(A) ORGANISM: Homo sapiens
CA 02211448 1997-07-25
W 096129409 37 PCT/US,''~037
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..87
(xi) SEQUENOE DESCR]:PTION: SEQ ID NO:6:
ATG GTG GCC TAT GAT CCC GAT GAG AGA ATC GCC GCC CAC CAG GCC CTG 48
Met Val Ala Tyr Asp Pro Asp Glu Arg Ile Ala Ala His Gln Ala Leu
1 5 10 15
CAG CAC CCC TAC TTC CAA GAA CAG AGA AAC AGT CCC TAA 87
Gln His Pro Tyr Phe Gln Glu Gln Arg Asn Ser Pro
20 25
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUEN OE CHA'RA~ ISTICS:
(A) LENGTH: 28 amino acids
(B) TYPE: amino acid
(D) TOPOLO~Y: 1 i n~.~r
(ii) MOLECULE TYPE: protein
~5
(xi) SEQUEN OE DESCRIPTION: SEQ ID NO:7:
Met Val Ala Tyr Asp PrG Asp Glu Arg Ile Ala Ala His Gln Ala Leu
1 5 10 15
Gln His Pro Tyr Phe Gln Glu Gln Arg Asn Ser Pro
20 25
CA 02211448 1997-07-25
W O 96/29409 - 38 - PC~rrUS96/04037
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARA~-l~KISTICS:
(A) LENGTH: 288 base pairs
(B) TYPE: nucleic acid
(C) STR~NDEDNESS: single
(D) TOPOLOGY: 1 i n~r
(ii) MOLECULE TYPE: cDNA
(iii) HYPO~ CAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Homo sapiens
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..288
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
ATG ATC CCG ATG AGA GAA TCG CCG CCC ACC AGG CCC TGC AGC ACC CCT 48
Met Ile Pro Met Arg Glu Ser Pro Pro Thr Arg Pro Cys Ser Thr Pro
1 5 10 15
ACT TCC AAG AAC AGA G~A ACA GTC CCT AAA GCA AGA GGA GGA CCG TCC 96
CA 02211448 1997-07-25
W 096/29409 39_ PCTrUSg6/04037
Thr Ser Lys Asn Arg Glu Thr Val Pro Lys Ala Arg Gly Gly Pro Ser
CAA GAG ACG AGG ACC GGC CTA TGT CAT GC-A ACT GCC C~A ACT A~A GCT 144
Gln Glu Thr Arg Thr Gly Leu Cys His Gly Thr Ala Gln Thr Lys Ala
TTC GGG AGT GGT CAG ACT GTC GTC TTA CTC CAG CCC CAC GCT GCA GTC 192
Phe Gly Ser Gly Gln Thr Val Val Leu Leu Gln Pro His Ala Ala Val
1050 55 60
CGT GCT TGG ATC TGG AAC A~A TGG AAG AGT GCC GGT GCT GAG ACC CTT 240
Arg Ala Trp Ile Trp Asn Lys Trp Lys Ser Ala Gly Ala Glu Thr Leu
65 70 75 80
GAA GTG CAT CCC TGC GAG CAA G~A GAC AGA TCC GCA GAA G~A CCT TA~ 288
Glu Val His Pro Cys Glu Gln Glu Asp Arg Ser Ala Glu Gly Pro
85 90 95
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CH~RA~ ISTICS:
(A) LENGTH: 95 amino acids
(B) TYPE: c~mino acid
(D) TOPOLOGY: linear
~ (ii) MOLEC~LE TYPE: protein
(xi) SEQUEN OE DESCRIPTION: SEQ ID NO:9:
CA 022ll448 l997-07-25
W096/29409 40 PCTrUS96/04037
Met Ile Pro Met Arg Glu Ser Pro Pro Thr Arg Pro Cys Ser Thr Pro
1 5 10 15
Thr Ser Lys Asn Arg Glu Thr Val Pro Lys Ala Arg Gly Gly Pro Ser
Gln Glu Thr Arg Thr Gly Leu Cys His Gly Thr Ala Gln Thr Lys Ala
Phe Gly Ser Gly Gln Thr Val Val Leu Leu Gln Pro His Ala Ala Val
Arg Ala Trp Ile Trp Asn Lys Trp Lys Ser Ala Gly Ala Glu Thr Leu
1~ 65 70 75 80
Glu Val His Pro Cys Glu Gln Glu Asp Arg Ser Ala Glu Gly Pro
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CH~RA~l~RISTICS:
(A) LENGTH: 222 base pairs
25 (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: l; n~r
(ii) MOLECULE TYPE: cDNA
(iii) HYPO'l'~LllCAL: NO
CA 022ll448 l997-07-25
W096/29409 41 PCT~US96104~37
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
S (vi) ORIGINAL SOURCE:
(A) ORG~NISM: Homo sapiens
(ix) F~ATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1.... 222
(xi) SEQUENOE DESCRIPTION: SEQ ID NO:10:
15 ATG G~A CTG CCC A~A CTA AAG Cl-l TCG GGA GTG GTC AGA CTG TCG TC~ 48
Met Glu Leu Pro Lys Leu Lys Leu Ser Gly Val Val Arg Leu Ser Ser
1 5 10 15
TAC TCC AGC CCC ACG CTG CAG TCC GTG CTT G~A TCT GGA ACA AAT GGA 96
20 Tyr Ser Ser Pro Thr Leu Gln Ser ~al Leu Gly Ser Gly Thr Asn Gly
20 25 30
AGA GTG CCG GTG CTG AGA.CCC TTG AAG TGC ATC CCT GCG AGC AAG AAG 144
Arg Val Pro Val Leu Arg Pro Leu Lys Cys Ile Pro Ala Ser Lys Lys
35 40 45
ACA GAT CCG CAG AAG GAC' CTT AAG CCT GCC CCG CAG CAG TGT CGC CTG 192
Thr Asp Pro Gln Lys Asp Leu Lys Pro Ala Pro Gln Gln Cys Arg Leu
50 55 60
CCC ACC ATA GTG CGG ALA GGC GGA AGA TAA 222
CA 022ll448 l997-07-25
W096/29409 -42- PCTrU596/0l--7
Pro Thr Ile Val Arg Lys Gly Gly Arg
(2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUEN OE CH~RACl~ISTICS:
(A) LENGTH: 73 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: li n~r
(ii) MOLECULE TYPE: protein
(xi) SEQUEN OE DESCRIPTION: SEQ ID NO:11:
Met Glu Leu Pro Lys Leu Lys Leu Ser Gly Val Val Arg Leu Ser Ser
1 5 10 15
Tyr Ser Ser Pro Thr Leu Gln Ser Val Leu Gly Ser Gly Thr Asn Gly
20 25 30
Arg Val Pro Val Leu Arg Pro Leu Lys Cys Ile Pro Ala Ser Lys Lys
~5 Thr Asp Pro Gln Lys Asp Leu Lys Pro Ala Pro Gln Gln Cys Arg Leu
50 55 60
Pro Thr Ile Val Arg Lys Gly Gly Arg
(2) INFORMATION FOR SEQ ID NO:12:
CA 022ll448 l997-07-2~
~os6l2s40s 43 PCT~u~ 0l-7
(i) SEQUENCE CHARAC'l'~RISTICS:
(A) LENGTH: 1168 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
S (D) TOPOLOGY: 1 in~r
(ii) MOLECULE TYPE: cDNA
(iii) HYP~~ CAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
IS (vi) ORIGIN~L SOUROE':
(A) ORGANISM: Homo sapiens
(xi) SEQUENCE DESCRI:PTION: SEQ ID NO:12:
CTGTGACTGG TGCTGGAGTT Tl'GAGTCCAC AGATA~ATG TGTCTCCTTC GTCTCTACTA 60
GAGAG~AA~A AGAACTGGAA Tl'GG~AG~AC AGGGAGACTG AAGGGTAGCA AGAGAGGCTG 120
~5 GAGAAGAGAG TGA~AGACC G(TTACCTGA ~ lGAAATTG TCTGCAGCCC ~ CCTG 180
GAGTA~ATGA ACTGGACCAA ATCTCAAAAA TCCACGATGT CATCGGCACA CCCGCTCAGA 240
AGATCCTCAC C~AGTTCA~A C~GTCGAGAG CTATGAAlll TGAllllCCT lllAAAAAGG 300
GATCAGGAAT ACCTCTACTA ACAACC~ATT TGTCCCCACA ATGCCTCTCC CTCCTGCACG 360
CA 022ll448 l997-07-25
W096/29409 44 PCTnUS96/04037
C~A~ l~GC CTATGATCCC GATGAGAGAA TCGCCGCCCA CCAGGCCCTG CAGCACCCCT 420
A~l-lCC~AGA ACAC-AGA~AC AGTCCCTA~A GCAAGAGGAG GACCGTCCCA AGAGACGAGG 480
ACCGZCCTAT GTCATGG~AC TGCCCA~ACT A~AG~-l-llCG GGA~l~'l~A GA~l~l~lC 540
TTACTCCAGC CCCACGCTGC A~l~l~CT TGGATCTGGA ACAAATGZAA GAGTGCCGGT 600
GCTGAGACCC TTG~AGTGCA 'l~C~'lGCGAG CAAGAAGACA GATCCGCAGA AGGAC~l'l~A 660
GCCTGCCCCG CAGCAGTGTC GCCTGCCCAC CATAGTGCGG A~AGGCGGAA GATAACTGAG 720
CAGCACCGTC ~'l'~'l'~ACTT CGZAGZC~AC ACCAAGCCCG ACCGGGCCAG GC~l~l~A 780
TCTGCTGCTG AGACGCCACG GAGGGCTGGG GATGCGCCTG CGTCCGlllC GCGCTGGCCG 840
GGGCTCTGGG TGCTGCCCTG CGCCCTGCCG CACCCGCGGC CCGCGCAGCT GCCTAGGATG 90O
TTCTGGGCTA ATATACTTGT A~ACCACCG CATTCTAGGG TTTT~'l'l'l'CA TTTTCGTTAA 960
G~ATTTGGGG CAGGAAATAC ll'l'~l~ACTT TGTATATG~A TCAAAACA~A CGAGCAGGCA 1020
TTTCTGTGAT ~l~ll~GGCG l~'l'l~AAG GTGG~ l~ C~'l'~lCC~ll' CCCAGCGCTG 1080
CTGGTCAGTC GTGGAGCGCC ATC~l'~'l'~'l-l' ACCAGTGACG CTGCTGACAC CCCTGA.~'ll-l 1140
TATTA~AG~A TAAG~l~l~G TTA~AAAA 1168
(2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUEN OE CH~RACTERISTICS:
CA 022ll448 l997-07-25
W O 96/29409 45 PCTÇUS9''01~7
(A) LENGTH: 1235 base pairs
(B) TYPE: nuc:Leic acid
(C) STRANDEDNF,SS: single
(D) TOPOLOGY: l' n~r
s
(ii) MOLECULE TYPE: cDNA
(iii) HYP~'l'~'l'lCAL: I~O
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(vi) ORIGINAL SOURC]3:
(A) ORGANISM: Homo sapiens
(xi) SEQUEN OE DESCRIPTION: SEQ ID NO:13:
20 A~ ~AGT CTGTGACTGG T~CTG5AGTT TTGAGTCCAC AGATA~AATG TGTCTCCTTC 60
GTCTCTACTA GAGAG5A~A AI~AACTG5AA TTG5AAG~AC AGGGAGACTG AAGGGTAGCA 120
AGAGAG5CTG GAGAAGAGAG T(~A~AAGACC GCTTACCTGA lllGA~ATTG ATGGTGGCGT 180
2~
G55AATGAAG AATGTGATAT ACAl~lll~G A~l~l~llCT GCAGCCCCTC ~ CCTG5AG 240
TA~ATGAACT G5ACCAAATC T~AAAATCC ACGATGTCAT CGGCACACCC GCTCAG~AGA 300
'l'C~'l'~ACCAA GTTCAAACAG TCGAGAGCTA TGAAllllGA TTTTC~llll A~A~AG5GAT 360
CAGGAATACC TCTACTAACA ACCAATTTGT CCCCAC~ATG CCTCTCCCTC CTGCACGCAA 420
CA 022ll448 l997-07-25
W096/29409 -46- PCTrUS9G~01037
~l~l~CCTA TGATCCCGAT GAGAGAATCG CCGCCCACCA GGCCCTGCAG CACCCCTACT 480
TCCAAGAACA GAGA~ACAGT CCCTA~AGCA AGAGGAGGAC C~'l'CC~AAGA GACGAGGACC 540
GGCCTATGTC ATGGAACTGC CCA~ACTA~A G~l-l-lCGGGA GTGGTCAGAC 'l'~'l'C~l~l-l~ 600
CTCCAGCCCC ACGCTGCAGT CCGTGCTTGG ATCTGGAACA AATGGAAGAG TGCCGGTGCT 660
GAGACC'~'l-l~ AAGTGCATCC CTGC'GAGC~A GAAGACAGAT CCGCAGAAGG ACCTTAAGCC 720
TGCCCCGCAG CA~l~l'~GCC TGCCCACCAT AGTGCGGAAA GGCGGAAGAT AACTGAGCAG 780
CA~'l'G~'l'~ TCGA~ l'C~ AGGCAACACC AAGCCCGACC G-GCCAGGCC TGGGTGATCT 840
IS GCTGCTGAGA CGCCACGGAG GGCTa3aGAT GCGCCTGCGT CCGll-lCGCG CTGG~ G 90O
CTCTGCGTGC TGCCCTGCGC CCTGCCGCAC CCGC'GGCCCG CGCAGCTGCC TAGGATGTTC 960
TGGGCTAATA TACTTGTA~A ACCACCGCAT TCTAGGGTTT T~'l'l'l'CAl-ll TCGTTAAGAA 1020
TTTGGGGCAG GA~ATA~l'l-l GTAA~l-l-l~l ATATGAATCA A~ACAAACGA GCAGGCA'l-l-l 1080
CTGTGATGTG TTGGGCGTGG TTGGAAGGTG GGTTCTGCGT GTCCCTTCCC AGCGCTGCTG 1140
GTCAGTCGTG GAGCGCCATC ATGTCllACC AGTGACGCTG CTGACACCCC TGA~TTTTAT 1200
TA~AGAATAA GCTGTCGTTA CAGTATTGCA AAAAA 1235
(2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUEN OE CHARA~'l'~KISTICS:
CA 022ll448 l997-07-25
W096129409 PCT/u~
-47-
(A) LENGTH: 2051 base pairs
(B) TYPE: m ~ c acid
(C) STRANDEDI~ESS: single
- (D) TOPOLOGY: l; n~i~
s
(ii) MOLEC~LE TYPEo cDNA
(iii) HYPOl'~llCAL: NO
(iv) ~NTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Homo sapiens
(xi) SEQUEN OE DESC~IPTION: SEQ ID NO:14:
20 GA~ l~CT GGA~llll~A GTCCACAGAT A~AA'l'~'l'~l'~ TCCl-l'CGTCT CTACTAGAGA 60
G5AAAAAGAA CTGGAATTGG AAGAACAGGG AGACTGAAGG GTAGC~AGAG AGGCTGGAGA 120
AGAGAGTGAA AAGACCGCl'l'ACCTGA'l'l'l'G A~ATTC-TCTG CAGCCCCTCT TTCCTG5AGT 180
AAATG~ACTG GACCAAATCT CAA~AATCCA CGATGTCATC GGCACACCCG CTCAGAAGAT 240
CCTCACCAAG TTCAAACAGT CGAGAGCTAT GAA'lll~lGAT l'l'l'C~l~ lA AAAAGGGATC 300
AG5AATACCT CTACTAACAA CCA~l'l'l'~'l'C CCCACAATGC CTCTCCCTCC TGCACGC~AT 360
GGTGGCCTAT GATCCCGATG A'JAGAATCGC CGCCCACCAG GCCCTGCAGC ACCCCTACTT 420
=
CA 022ll448 l997-07-2s
W096/29409 -48- PCTrUS9G~'~1037
CCAAG~ACAG AG~ACCCAGA ACGG~AGCGA GGATGAAGGC CTCAGCCGTC ~LC~lCCCCA 480
TTCA~ACACG TTCATCCCTC AA~l'~l'GC TGAGCACCTG CATGCTGCCC GGCCGCAGTG 540
TCACC~llCT 'l'~'l'~'l~AGCC TACCCTCATC CACCCACCTC ACCCTCCTGA C~llA~AG~A 600
GACACCGGGC AGAAGCACAG GGGAGCCCAG TCACACCCCA CACTGGCGGG GGCAGGCCTT 660
GCAGGGAG~A GCAGTAAGCA GCC~l~l~ TCAGCCA m CCATCTGGCA CTCAGACGTG 720
"
CA~'l'~'l'l'C~ TGTGACAGGC GGCAGCAGTG CGACCGTGAC CTCCCATCTG CTCTGCTGTC 780
CCCACACCTG CGGTGCAGCC AGCCTGCCAC AAGGCAGCTA GAGTCCAGCT AGACCCACCC 840
CTGGCACGGC CGA~'l'~l'l'C CTGG~'ll~l'l' CTGGGCCT~A TCCCCGTGCA TTCTCCAACG 900
CCAG~AGTGT ~AGA~AGTGC AAGGC~AC~A GTGAG~AGAG CA~ACCCA~A TCGTACCAGG 960
G~AGCTAGTC TTTCCAGGGC ACCTGAGTGA GGGCATGACC AGC~llGACG CTGCCTCGCT 1020
~0
ACCATCTGCC CAGGGCCTGC TG~ATGCTTG AGTCCATGGT GACA~l~lG GG~ACAGTTA 1080
CGAGGCAGTT AGAllll~A AGTCATGTTG GCCCACTTGG CTACAGAGCA GTCTTAG~A 1140
~5 CAGCACCATA AA~ATA~AGA CTTATTCCTG ACACACATGC ATCTAGAGTA AACTGGGGCG 1200
TATCTGACAG CGTTAGTACA GTGATGGCCA AATGCA~ACT GCATTCCAGA ACCAGCG~AG 1260
GGTGACAGAC TGGGCTGAGG CAGAGCTAGG ACTAACCATC TCGAGTGATG CCATCTCGGG 1320
GCC~ACA~A ~l-lll~GACA CGGCTGGATC ATCTGACC~A ACTGCTC~AA TCT~lACACA 1380
CA 02211448 1997-07-25
W 096/29409 4 PCTAUS~C'0'~7
9 _
ATTATTGTCC TGGTATTA~A CTTTCACCTG CCACTTCCA~ CA~AC~GGAG AC~G~ATAAG 1440
GAGATGACCA GGAAGATGGC TGGATTA~GA ATTCTAGACT TGGCCGGGTG Cw TGGCTCA 1500
CACCrGTAAT CCrAGCATCT TGGGAG~CTG AGGCAGGAGG ATCG~llGAG CCCAGAG'l'l'l' 1560
GAGACCAGCC TAGGC~ACAT AAGGAGACCC CATCTCTACA A~ATATCA~A AAATTACCCA 1620
GGTAT wT w CACACACGTG 'rGATTCCAGC TACTCGGGAG GCTGAC-AT w GA wATCACT 1680
TGAACCCA w A wll w J~C 'rAC~ATGAGC TATGATCGCA CCA~llCATT CCAGCCTwA 1740
TGACAGAAGA CTCACTCCAT ~GTTCATGGC CCC~'l'~ATCC AGAGTCCCTG CTGGCGCCTT 1800
CGAGTGGGGC AGGCTGAGAA CTCAAGCTGT AACTAATGTC TCCTCCGAAG A~AACTAAAC 1860
CGAGG~CTGA GCrGATGTGA AGllllCCGT GGCTGCATTC ATACAAATGG TGA~AATGTA 1920
GCATACCTCC CCTCAAAAGC CTGA~AGTAA AGACATGCCC CCAATTTA~T GTGATG~ATT 1980
~0
AGAGA~ATAG Gl~llCAGACA CTTCATGGTT TA~AGTCTCA CA~AATA~AG ~ l'C~AAGG 2040
P22~ LAA A 2051
(2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARA~'l'~KISTICS:
(A) LENGTH: 60 base pairs
(B) TYPE: n~cleic acid
(C) STRANDEDNESS: single
(D) TOPOL~GS~r: 1 i n~r
CA 022ll44s l997-07-25
W096/29409 50 PCTrUS96/04037
(ii) MOLECULE TYPE: cDNA
(iii) HYPOl~llCAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(vi) ORIGINAL SOUR OE :
(A) ORGANISM: Homo sapiens
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..60
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
ATG TCA TCG GCA CAC CCG CTC AGA AGA TCC TCA CCA AGT TCA AAC AGT 48
Met Ser Ser Ala His Pro Leu Arg Arg Ser Ser Pro Ser Ser Asn Ser
1 5 10 15
CGA GAG CTA TGA 60
Arg Glu ~eu
(2) INFORMATION FOR SEQ ID NO:16:
(i) SEQUEN OE CH~RACTERISTICS:
(A) LENGTH: 19 amino acids
(B) TYPE: amino acid
CA 022ll448 l997-07-25
W 096l29409 -51- PCTAUS96104037
(D) TOPOI~X~Y: linear
(ii) MO~ECULE T~'E: protein
(xi) ~U~N~'~ DESCRIPTION: SEQ ID N0:16:
Met Ser Ser Ala His Pro Leu Arg Arg Ser Ser Pro Ser Ser Asn Ser
1 5 10 15
Arg Glu Leu
(2) INFORMATION FOR SEQ ID NO:17:
(i) SEQUEN OE CH~ CTERISTICS:
(A) LENGTH: ~77 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY:: linear
(ii) MOLECU~E TYPE: cDNA
(iii) HYP~l~llCAL: NO
~5
(iv) ANTI-SENSE: N0
- (v) FRAGMENT TYPE- internal
(vi) ORIGINAL SO~R(_E:
(A) ORG~NISM: Homo sapiens
CA 022ll448 l997-07-25
W096/29409 52 PCTrUS96/~1~37
(ix) FEATURE:
(A) N~ME/KEY: CDS
(B) LOCATION: 1..177
(xi) SEQUEN OE DESCRIPTION: SEQ ID NO:17:
ATG AAT '11'1' GAT '11'1' CCT TTT A~A AAG GGA TCA GGA ATA CCT CTA CTA 48
Met Asn Phe Asp Phe Pro Phe Lys Lys Gly Ser Gly Ile Pro Leu Leu
1 5 10 15
ACA ACC A~T TTG TCC CCA CAA TGC CTC TCC CTC CTG CAC GCA ATG GTG 96
Thr Thr Asn Leu Ser Pro Gln Cys Leu Ser Leu Leu His Ala Met Val
20 25 30
1~
GCC TAT GAT CCC GAT GAG AGA ATC GCC GCC CAC CAG GCC CTG CAG CAC 144
Ala Tyr Asp Pro Asp Glu Arg Ile Ala Ala His Gln Ala Leu Gln His
35 40 45
~0 CCC TAC TTC C~A G~A CAG AGA AAC AGT CCC TAA 177
Pro Tyr Phe Gln Glu Gln Arg Asn Ser Pro
50 55
~5 (2) INFORMATION FOR SEQ ID NO:18:
(i) SEQUENOE CHARACTERISTICS:
(A) LENGTH: 58 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: 1- ne~r
(ii) MOLEC~LE TYPE: protein
CA 022ll448 l997-07-25
WO96l29409 53 PCT~US9C~ t~ /
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:
Met Asn Phe Asp Phe Pro Phe Lys Lys Gly Ser Gly Ile Pro Leu Leu
1 5 10 15
~ Thr Thr Asn Leu Ser Pro Gln Cys Leu Ser Leu Leu His Ala Met Val
Ala Tyr Asp Pro Asp Glu Arg Ile Ala Ala His Gln Ala Leu Gln His
35 40 45
Pro Tyr Phe Gln Glu G1n Arg Asn Ser Pro
50 55
(2) INFORMATION FOR SEQ ID NO:19:
(i) SEQUENCE CH~ ISTICS:
(A) LENGTH: :288 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDI.~ESS: single
(D) TOPOL~GY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYP~l'~l'lC~L: NO
~ (iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(vi) ORIGINAL SOURCE:
CA 022ll448 l997-07-25
W 096/29409 54 PCTrUS96/01C-7
(A) ORGANISM: Homo sapiens
( ix) FEATURE:
(A) N~ME/~Y: CDS
s (B) LOCATION: 1. . 288
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:
l 0 ATG ATC CCG ATG AGA GAA TCG CCG CCC ACC AGG CCC TGC AGC ACC CCT 4 8
Met Ile Pro Met Arg Glu Ser Pro Pro Thr Arg Pro Cys Ser Thr Pro
5 10 15
ACT TCC A~G AAC AGA GAA ACA GTC CCT A~A GCA AGA GGA GGA CCG TCC 96
Thr Ser Lys Asn Arg Glu Thr Val Pro Lys Ala Arg Gly Gly Pro Ser
20 25 30
CAA GAG ACG AGG ACC GGC CTA TGT CAT G(~A ACT GCC C~A ACT A~A GCT 144
Gln Glu Thr Arg Thr Gly Leu Cys His Gly Thr Ala Gln Thr Lys Ala
35 40 45
TTC GGG AGT GGT CAG ACT GTC GTC TTA CTC CAG CCC CAC GCT GCA GTC 192
Phe Gly Ser Gly Gln Thr Val Val Leu Leu Gln Pro His Ala Ala Val
50 55 60
CGT GCT TGG ATC TGG AAC A~A TGG A~G AGT GCC GGT GCT GAG ACC CTT 240
Arg Ala Trp Ile Trp Asn Lys Trp Lys Ser Ala Gly Ala Glu Thr Leu
65 70 75 80
,0 G~A GTG CAT CCC TGC GAG C~A GAA GAC AGA TCC GCA G~A GGA CCT TAA 288
Glu Val His Pro Cys Glu Gln Glu Asp Arg Ser Ala Glu Gly Pro
CA 022ll448 l997-07-25
W 096129409 55 PCT/US~01b_/
(2) INFORM~TION FOR SEQ ID NO:20:
(i) SEQUEN OE C~R~CTERISTICS:
S (A) LENG~I: 95 amino acids
(B) TYPE: amino acid
(D) TOPOL(X~Y: 1i n~r
(ii) MOLEC~LE T~?E: protein
(xi) SEQ~EN OE DESCRIPTION: SEQ ID NO:20:
Met Ile Pro Met Arg Glu Ser Pro Pro Thr Arg Pro Cys Ser Thr Pro
1 5 10 15
Thr Ser Lys Asn Arg Glu Thr Val Pro Lys Ala Arg Gly Gly Pro Ser
Gln Glu Thr Arg Thr Gly Leu Cys His Gly Thr Ala Gln Thr Lys Ala
35 40 45
Phe Gly Ser Gly Gln Thr Val Val Leu Leu Gln Pro His Ala Ala Val
Arg Ala Trp Ile Trp Asn Lys Trp Lys Ser Ala Gly Ala Glu Thr Leu
65 70 75 80
Glu Val His Pro Cys Glu Gln Glu Asp Arg Ser Ala Glu Gly Pro
(2) INFORMATION FOR SE~ ID NO:21:
CA 022ll448 l997-07-25
W096/29409 56 PCTrUS96/04037
(i) SEQUEN OE CH~RA~l~KISTICS:
(A) LENGTH: 222 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: l; n~r
(ii) MOLECULE TYPE: cDNA
(iii) HYPOl~'l'lCAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
15 (vi) ORIGINAL SOUR OE :
(A) ORGANISM: Homo sapiens
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1.... 222
(~i) SEQUEN OE DESCRIPTION: SEQ ID NO:21:
25 ATG G~A CTG CCC A~A CTA AAG CTT TCG G~A GTG GTC AGA CTG TCG TCT 48
Met Glu Leu Pro Lys Leu Lys Leu Ser Gly Val Val Arg Leu Ser Ser
1 5 10 15
TAC TCC AGC CCC ACG CTG CAG TCC GTG ~ GGA TCT GGA ACA AAT GGA 96
30 Tyr Ser Ser Pro Thr Leu Gln Ser Val Leu Gly Ser Gly Thr Asn Gly
CA 02211448 1997-07-25
W O9612940g 57 PCTfUS96/04037
AGA GTG CCG GTG CTG AGA CCC ITG AAG TGC ATC CCT GCG AGC AAG AAG 144
Arg Val Pro Val Leu Arg Pro Leu Lys Cys Ile Pro Ala Ser Lys Lys
35 40 45
S ACA GAT CCG Q G AAG GAC ~rl AAG CCT GCC CCG CAG CAG TGT CGC CTG 192
Thr Asp Pro Gln Lys Asp Leu Lys Pro Ala Pro Gln Gln Cys Arg Leu
50 55 60
CCC ACC ATA GTG CGG A~. GGC GGA AGA TA~ 222
10 Pro Thr Ile Val Arg Lys Gly Gly Arg
65 70
( 2 ) INFORM;~TION FOR SEQ ID NO : 22:
(i) SEQUENOE CHARA~KISTICS:
(A) LENGTH: 73 amino acids
(B ) TYPE: amino acid
(D) TOPOLO~: 1 ln~;~
( ii ) MOLEC~LE TYPE: protein
(xi ) SEQUENOE DESCRIPTION: SEQ ID NO: 22:
~5 Met Glu Leu Pro Lys Leu Lys Leu Ser Gly Val Val Arg Leu Ser Ser
Tyr Ser Ser Pro Thr Leu Gln Ser Val Leu Gly Ser Gly Thr Asn Gly
,0
Arg Val Pro Val Leu Arg Pro Leu Lys Cys Ile Pro Ala Ser Lys Lys
CA 02211448 1997-07-25
W096/29409 58 PCTrUS96/04037
Thr Asp Pro Gln Lys Asp Leu Lys Pro Ala Pro Gln Gln Cys Arg Leu
50 55 60
Pro Thr Ile Val Arg Lys Gly Gly Arg
65 70
(2) INFORMATION FOR SEQ ID NO:23:
(i) SEQUEN OE CHARA~l~KISTICS:
(A) LENGTH: 63 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: 1 ln~r
(ii) MOLECULE TYPE: cDNA
(iii) HYP~l~llC~L: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(vi) ORIGINAL SOUROE:
(A) ORG~NISM: Homo sapiens
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: l..63
(xi) SEQUEN OE DESCRIPTION: SEQ ID NO:23:
CA 02211448 1997-07-25
W 096/29409 59 PCTrUS9~J'~ 7
ATG AAG AAT GTG ATA TAC ATC ~ GGA GTC TGT TCT GCA GCC CCT Cll 48
Met Lys Asn Val Ile Tyr Ile Phe Gly Val Cys Ser Ala Ala Pro Leu
1 5 10 15
5 TCC TGG AGT A~A TGA 63
Ser Trp Ser Lys
l0 (2) INFORMATION FOR SEQ ID NO:24:
(i) SEQ~EN OE CHP.RACTERISTICS:
(A) LENGT~: 20 amino acids
(B) TYPE: amino acid
(D) TOPOLC~ n~r
(ii) MOLECULE TYE'E: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:
Met Lys Asn Val Ile T~- Ile Phe Gly Val Cys Ser Ala Ala Pro Leu
1 5 10 15
Ser Trp Ser Lys
(2) INFORMATION FOR SEQ ID NO:25:
(i) SEQUENCE CHP~ ~KISTICS:
(A) LENGTH: 60 base pairs
(B) TYPE: nucleic acid
CA 022ll448 l997-07-25
W 096/Z9409 60 PCTIU~3GJ01037
(C) STRANDEDNESS: single
(D) TOPOLOGY: 1 ln~r
(ii) MOLECULE TYPE: cDNA
s
(iii) HYPOl~llCAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(vi) ORIGINAL SOUR OE:
(A) ORGANISM: Homo sapiens
1~ (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..60
(xi) SEQUEN OE DESCRIPTION: SEQ ID NO:25:
ATG TCA TCG GCA CAC CCG CTC AGA AGA TCC TCA CCA AGT TCA A~C AGT 48
Met Ser Ser Ala His Pro Leu Arg Arg Ser Ser Pro Ser Ser Asn Ser
1 5 10 15
2~
CGA GAG CTA TGA 60
Arg Glu Leu
,0
(2) INFORMATION FOR SEQ ID NO:26:
CA 022ll448 l997-07-25
W096/29409 -61- PCTrU$9G,!~1t~7
(i) SEQUENOE C~RA~l~KISTICS:
(A) LENGTEI: 19 amino acids
(B ) TYPE: amino acid
(D) TOPO~X~Y: linear
(ii) MOLEC~LE TYPE: protein
(xi) SEQUENOE DESCRIPTION: SEQ ID NO:26:
Met Ser Ser Ala His Pro Leu Arg Arg Ser Ser Pro Ser Ser Asn Ser
1 5 10 15
Arg Glu Leu
1~
(2) INFORMATION FOR SE52 ID NO:27:
(i) SEQUENCE CH~ ~-l'~KISTICS:
(A) LENGTH: ~77 base pairs
(B) TYPE: nucleic acid
(C) STR~NDED~ESS: single
(D) TOPOLOGY: linear
2~ (ii) MOLECULE TYPE.: cDNA
(iii) HYP~l~l'lCAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE.: internal
CA 02211448 1997-07-25
W096/29409 -62- PCTrUS96/04037
(vi) ORIGINAL SOUR OE:
(A) ORG~NISM: Homo sapiens
(ix) FTATURE:
(A) N~ME/KEY: CDS
(B) LOCATION: 1..177
(xi) SEQUEN OE DESCRIPTION: SEQ ID NO:27:
ATG AAT '111~ GAT ~1~11 CCT TTT AAA AAG GGA TCA GGA ATA CCT CTA CTA 48
Met Asn Phe Asp Phe Pro Phe Lys Lys Gly Ser Gly Ile Pro Leu Leu
1 5 10 15
15 ACA ACC AAT TTG TCC CCA C~A TGC CTC TCC CTC CTG CAC GCA ATG GTG 96
Thr Thr Asn Leu Ser Pro Gln Cys Leu Ser Leu Leu His Ala Met Val
20 25 30
GCC TAT GAT CCC GAT GAG AGA ATC GCC GCC CAC CAG GCC CTG CAG CAC 144
20 Ala Tyr Asp Pro Asp Glu Arg Ile Ala Ala His Gln Ala Leu Gln His
35 40 45
CCC TAC TTC CAA G~A CAG AGA AAC AGT CCC TAA 177
Pro Tyr Phe Gln Glu Gln Arg Asn Ser Pro
50 55
(2) INFORMATION FOR SEQ ID NO:28:
,0 (i) SEQUEN OE CH~RA~'l'~ISTICS:
(A) LENGTH: 58 amino acids
(B) TYPE: amino acid
CA 02211448 1997-07-25
W 096/29409 -63- PCT~US~C~a~37
(D) TOPOLCX~Y: l; n~r
(ii) MOLECULE IYE'E: protein
(xi) SEQUEN OE DE',CRIPTION: SEQ ID NO:28:
Met Asn Phe Asp Phe Pro Phe Lys Lys Gly Ser Gly Ile Pro Leu Leu
1 5 10 15
~0 Thr Thr Asn Leu Ser Pro Gln Cys ~eu Ser Leu Leu His Ala Met Val
Ala Tyr Asp Pro Asp Glu Arg Ile Ala Ala His Gln Ala Leu Gln His
35 40 45
Pro Tyr Phe Gln Glu Gln Arg Asn Ser Pro
50 55
(2) INFORMATION FOR SEQ ID NO:29:
(i) SEQUENCE CH~RA~l~RISTICS:
(A) LENGTH: :288 base pairs
(B) TYPE: nucleic acid
~5 (C) STRANDED]~ESS: single
(D) TOPOLOGY: linear
(ii) MOLEC~LE TYPE: cDNA
(iii) HYP~~ CAL: NO
(iv) ANTI-SENSE: NO
CA 02211448 1997-07-25
W O 96/29409 -64- PCTrUS96/04037
(v) FRAGMENT TYPE: internal
(vi) ORIGINAL SOUR OE :
(A) ORG~NISM: Homo sapiens
s
(ix) FEATURE:
(A) N~ME/KEY: CDS
(B) LOCATION: 1..288
(xi) SEQUEN OE DESCRIPTION: SEQ ID NO:29:
ATG ATC CCG ATG AGA GAA TCG CCG CCC ACC AGG CCC TGC AGC ACC CCT 48
Met Ile Pro Met Arg Glu Ser Pro Pro Thr Arg Pro Cys Ser Thr Pro
1 5 10 15
ACT TCC AAG AAC AGA G~A ACA GTC CCT A~A GCA AGA GGA GGA CCG TCC 96
Thr Ser Lys Asn Arg Glu Thr Val Pro Lys Ala Arg Gly Gly Pro Ser
20 25 30
C~A GAG ACG AGG ACC GGC CTA TGT CAT GGA ACT GCC C~A ACT A~A GCT 144
Gln Glu Thr Arg Thr Gly Leu Cys His Gly Thr Ala Gln Thr Lys Ala
35 40 45
25 TTC GGG AGT GGT CAG ACT GTC GTC TTA CTC CAG CCC CAC GCT GCA GTC 192
Phe Gly Ser Gly Gln Thr Val Val Leu Leu Gln Pro His Ala Ala Val
50 55 60
CGT GCT TGG ATC TGG AAC A~A TGG AAG AGT GCC GGT GCT GAG ACC CTT 240
,0 Arg Ala Trp Ile Trp Asn Lys Trp Lys Ser Ala Gly Ala Glu Thr Leu
CA 02211448 1997-07-25
W 096129409 65 PCTAUS~G/01~7
GAA GTG CAT CCC TGC GAG C~A GAA GAC AGA TCC GCA GA~ GG~ CCT TAA 288
Glu Val His Pro Cys Glu Gln Glu Asp Arg Ser Ala Glu Gly Pro
85 90 95
(2) INFORMATION FOR SEQ ID NO:30:
(i) SEQUENCE CH~ ~KIsTIcs:
(A) LENGTH: 95 amino acids
(B) TYPE: amino acid
(D) TOPOL~Y: lin~r
(ii) MOLECULE TYPE: protein
(xi) SEQUEN OE DES~RIPTION: SEQ ID NO:30:
Met Ile Pro Met Arg Glu Ser Pro Pro Thr Arg Pro Cys Ser Thr Pro
1 5 10 15
Thr Ser Lys Asn Arg Glu Thr Val Pro Lys Ala Arg Gly Gly Pro Ser
Gln Glu Thr Arg Thr Gly Leu Cys His Gly Thr Ala Gln Thr Lys Ala
Phe Gly Ser Gly Gln Thr Val Val Leu Leu Gln Pro His Ala Ala Val
Arg Ala Trp Ile Trp Asn Lys Trp Lys Ser Ala Gly Ala Glu Thr Leu
30 65 70 75 80
CA 02211448 1997-07-25
W 096/29409 -66- PCTAUS96/04037
Glu Val His Pro Cys Glu Gln Glu Asp Arg Ser Ala Glu Gly Pro
(2) INFORMATION FOR SEQ ID NO:31:
(i) SEQUEN OE CHARACTERISTICS:
(A) LENGTH: 222 base pairs
(B) TYPE: nucleic acid
(C) STR~NDEDNESS: single
(D) TOPOLOGY: 1 i ne~r
( ii ) ~T .F.~ .~. TYPE: cDNA
(iii) HYP~~ C~L: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(vi) ORIGINAL SOURCE:
(A) ORG~NISM: Homo sapiens
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOC~TION: 1222
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:
CA 02211448 1997-07-25
W V96129409 67 r~l/u~c,~ lo ~
ATG G~A CTG CCC A~A CTA AAG ~l~l' TCG GGA GTG GTC AGA CTG TCG TCT 48
Met Glu Leu Pro Lys LRU ~ys Leu Ser Gly Val Val Arg ~eu Ser Ser
1 5 10 15
-
S TAC TCC AGC CCC ACG CTG CAG TCC GTG CTT G~A TCT G5A ACA AAT G5A 96
Tyr Ser Ser Pro Thr Leu Gln Ser Val Leu Gly Ser Gly Thr Asn Gly
20 25 30
AGA GTG CCG GTG CTG AGA CCC TTG AAG TGC ATC CCT GCG AGC AAG AAG 144
10 Arg Val Pro Val Leu Arg Pro Leu Lys Cys Ile Pro Ala Ser ~ys ~ys
35 40 45
ACA GAT CCG CAG A~G GAC CTT AAG CCT GCC CCG CAG CAG TGT CGC CTG 192
Thr Asp Pro Gln Lys Asp Leu Lys Pro Ala Pro Gln Gln Cys Arg Leu
l~ 50 55 60
CCC ACC ATA GTG CGG A~A GGC GGA AGA TAA 222
Pro Thr Ile Val Arg ~ys Gly Gly Arg
65 70
(2) INFORMATION FOR SEQ ID NO:32:
(i) SEQUENOE C ~AC~ERISTICS:
~5 (A) LENGTH: 73 amino acids
(B) TYPE: amino acid
(D) TOPOL~Y: linear
(ii) MO~ECU~E TYPE: protein
(xi) SEQ~ENOE DES(_RIPTION: SEQ ID NO:32:
CA 02211448 1997-07-25
W 096/29409 -68- PCTrUS95J-~1D_7
Met Glu Leu Pro Lys Leu Lys Leu Ser Gly Val Val Arg Leu Ser Ser
1 5 10 15
Tyr Ser Ser Pro Thr Leu Gln Ser Val Leu Gly Ser Gly Thr Asn Gly
20 25 30
Arg Val Pro Val Leu Arg Pro Leu Lys Cys Ile Pro Ala Ser Lys hys
Thr Asp Pro Gln Lys Asp heu Lys Pro Ala Pro Gln Gln Cys Arg Leu
50 55 60
Pro Thr Ile Val Arg Lys Gly Gly Arg
65 70
1~
(2) INFORMATION FOR SEQ ID NO:33:
(i) SEQUENOE CHARA~ISTICS:
(A) LENGTH: 60 base pairs
(B) TYPE: nucleic acid
(C) STR~NDEDNESS: single
(D) TOPOLOGY: lin~r
(ii) MOLECU~E TYPE: cDNA
(iii) HYPOl~!lCAL: NO
(iv) ANTI-SENSE: NO
~,0
(v) FRAGMENT TYPE: internal
CA 022ll448 l997-07-25
W096/29409 69 PCT~US96/04037
(vi) ORIGINA~ SOUROE :
(A) ORG~NISM: Homo sapiens
(ix) FEATURE:
(A) N~ME/KEY: CDS
(B) LOCATION: 1..60
(xi) SEQUEN OE DESCRIPTION: SEQ ID NO:33:
ATG TCA TCG GCA CAC CCG CTC AGA AGA TCC TCA CCA AGT TCA AAC AGT 48
Met Ser Ser Ala His Pro ~eu Arg Arg Ser Ser Pro Ser Ser Asn Ser
1 5 10 15
IS CGA GAG CTA TGA 60
Arg Glu Leu
20 (2) INFORMATION FOR SEQ ID NO:34:
(i) SEQUENOE CH~R~CTERISTICS:
(A) LENGTH: 19 am~no acids
(B) TYPE: amino acid
(D) TOPOLOGY: 1 in~r
(ii) MOLECULE TYPE: protein
(xi) SEQUENOE DESCRIPTION: SEQ ID NO:34:
CA 02211448 1997-07-25
W096/29409 70 PCT/ub~6/04037
Met Ser Ser Ala His Pro Leu Arg Arg Ser Ser Pro Ser Ser Asn Ser
l 5 l0 15
Arg Glu Leu
(2) INFORMATION FOR SEQ ID NO:35:
IO (i) SEQUEN OE CHARACTERISTICS:
(A) LENGTH: 564 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: 1i n~r
(ii) MOLECU.LE TYPE: cDNA
(iii) HYP~ C~L: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: i nt~rn~ 1
(vi) ORIGINAL SOUR OE :
(A) ORG~NISM: Homo sapiens
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: l..564
(xi) SEQUEN OE DESCRIPTION: SEQ ID NO:35:
CA 02211448 1997-07-25
W O 96129409 -71- PCT~US96104037
ATG A~T l l 1 GAT 'l l l' CCT 1 1 1 A;~A A~G GGA TCA G5A ATA CCT CTA CTA 48
Met Asn Phe Asp Phe Pro Phe Lys Lys Gly Ser Gly Ile Pro Leu Leu
5 10 15
S ACA ACC AAT TTG TCC CC~. CA~ TGC CTC TCC CTC CTG CAC GCA ATG GTG 96
Thr Thr Asn Leu Ser Prc Gln Cys Leu Ser Leu Leu His Ala Met Val
20 25 30
GCC TAT GAT CCC GAT GAG AGA ATC GCC GCC CAC CAG GCC CTG C~G CAC 144
Ala Tyr Asp Pro Asp Glu Arg Ile Ala Ala His Gln Ala Leu Gln His
35 40 45
CCC TAC TTC C~A G~A CAG AGA ACC CAG A~C Gr-'A AGC GAG GAT G~A GGC 192
Pro Tyr Phe Gln Glu Gln Arg Thr Gln Asn Gly Ser Glu Asp Glu Gly
50 55 60
CTC AGC CGT CCT CCT CCC CAT TCA A~C ACG TTC ATC CCT C~A CCC TCT 240
Leu Ser Arg Pro Pro Pro His Ser Asn Thr Phe Ile Pro Gln Pro Ser
65 70 75 80
~0
GCT GAG CAC CTG CAT GC.r GCC CGG CCG CAG TGT CAC CCT TCT TGT GTG 288
Ala Glu His Leu His Ala Ala Arg Pro Gln Cys His Pro Ser Cys Val
85 90 95
'~ AGC CTA CCC TCA TCC ACC CAC CTC ACC CTC CTG ACC TTA AAG AAG ACA 336
Ser Leu Pro Ser Ser Thr His Leu Thr Leu Leu Thr Leu Lys Lys Thr
100 105 110
CCG GGC AGA AGC ACA GGG GAG CCC AGT CAC ACC CCA CAC TGG CGG GGG 384
Pro Gly Arg Ser Thr Gly Glu Pro Ser His Thr Pro His Trp Arg Gly
115 120 125
CA 022ll448 l997-07-25
W 096/29409 7~ PCT/u~9Gl~o1~37
CAG GCC TTG CAG GGA GAA GCA GTA AGC AGC CAT CTC CAT CAG CCA TTT 432
Gln Ala Leu Gln Gly Glu Ala Val Ser Ser His Leu His Gln Pro Phe
130 135 140
5 CCA TCT GCC ACT CAG ACG TGC ACG TCT TCG TGT GAC AGG CGG CAG CAG 480
Pro Ser Gly Thr Gln Thr Cys Thr Ser Ser Cys Asp Arg Arg Gln Gln
145 150 155 160
TGC GAC CGT GAC CTC CCA TCT GCT CTG CTG TCC CCA CAC CTG CGG TGC 528
10 Cys Asp Arg Asp Leu Pro Ser Ala Leu Leu Ser Pro His Leu Arg Cys
165 170 175
AGC CAG CCT GCC ACA AGG CAG CTA GAG TCC AGC TAG 564
Ser Gln Pro Ala Thr Arg Gln Leu Glu Ser Ser
180 185
(2) INFORMATION FOR SEQ ID NO:36:
(i) SEQUENCE CH~RA~l~RISTICS:
(A) LENGTH: 187 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: l; n~r
(ii) MOLECULE TYPE: protein
(xi) SEQUEN OE DESCRIPTION: SEQ ID NO:36:
Met Asn Phe Asp Phe Pro Phe Lys Lys Gly Ser Gly Ile Pro Leu Leu
30 1 5 10 15
CA 02211448 1997-07-25
WO 96129409 73 _ PCT/~7S96~04037
Thr Thr Asn Leu Ser Pro Gln Cys Leu Ser Leu Leu His Ala Met Val
20 25 30
Ala Tyr Asp Pro Asp Glu Arg Ile Ala Ala His Gln Ala Leu Gln His
535 40 45
Pro Tyr Phe Gln Glu Gln Arg Thr Gln Asn Gly Ser Glu Asp Glu Gly
Leu Ser Arg Pro Pro Pro His Ser Asn Thr Phe Ile Pro Gln Pro Ser
65 70 75 80
Ala Glu His Leu His Ala Ala Arg Pro Gln Cys His Pro Ser Cys Val
85 90 95
Ser Leu Pro Ser Ser Thr His Leu Thr Leu Leu Thr Leu Lys Lys Thr
100 105 110
Pro Gly Arg Ser Thr Gly Glu Pro Ser His Thr Pro His Trp Arg Gly
20115 120 125
Gln Ala Leu Gln Gly Glu Ala Val Ser Ser His Leu His Gln Pro Phe
130 135 140
Pro Ser Gly Thr Gln Th-r Cys Thr Ser Ser Cys Asp Arg Arg Gln Gln
145 153 155 160
Cys Asp Arg Asp Leu Pro Ser Ala Leu Leu Ser Pro His Leu Arg Cys
165 170 175
Ser Gln Pro Ala Thr Arg Gln Leu Glu Ser Ser
180 185
CA 02211448 1997-07-25
W 096129409 PCTrUS~61~1~37
-74-
(2) INFORMATION FOR SEQ ID NO:37:
(i) SEQUENOE CHARACTERISTICS:
(A) LENGTH: 189 base pairs
S (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOL3GY: l; n~r
(ii) MOLEC~LE TYPE: cDNA
(iii) HYPOl~llCAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Homo sapiens
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..189
(xi) SEQUENOE DESCRIPTION: SEQ ID NO:37:
ATG ATC CCG ATG AGA GAA TCG CCG CCC ACC AGG CCC TGC AGC ACC CCT 8
Met Ile Pro Met Arg Glu Ser Pro Pro Thr Arg Pro Cys Ser Thr Pro
1 5 10 15
CA 02211448 1997-07-25
W 096/29409 75 PCTrUS~5.'a~C37
ACT TCC AAG AAC AGA G~A CCC AGA ACG GAA GCG AGG ATG AAG GCC TCA 96
Thr Ser Lys Asn Arg Glu Pro Arg Thr Glu Ala Arg Met Lys Ala Ser
20 25 30
5 GCC GTC CTC CTC CCC ATT C~A ACA CGT TCA TCC CTC A~C CCT CTG CTG 144
Ala Val Leu Leu Pro Ile Gln Thr Arg Ser Ser Leu Asn Pro Leu Leu
35 40 45
AGC ACC TGC ATG CTG CCC GGC CGC AGT GTC ACC CTT CIT GTG TGA 189
10 Ser Thr Cys Met Leu Pro Gly Arg Ser Val Thr Leu Leu Val
50 55 60
(2) INFORMATION FOR SEQ ID NO:38:
1~
(i) SEQUENOE CH~R~CTERISTICS:
(A) LENGTH:: 62 amino acids
(B) TYPE: amino acid
(D) TOPOLC~: l; n~r
(ii) MOLECULE TYPE: protein
(iii) HYPO~ C~L: NO
(xi) SEQ~ENCE DESCRIPTION: SEQ ID NO:38:
Met Ile Pro Met Arg Glu Ser Pro Pro Thr Arg Pro Cys Ser Thr Pro
1 5 10 15
,0 Thr Ser Lys Asn Arg Glu Pro Arg Thr Glu Ala Arg Met ~ys Ala Ser
CA 02211448 1997-07-25
W 096/29409 -76- PCTrU5g6/010~7
Ala Val Leu Leu Pro Ile Gln Thr Arg Ser Ser Leu Asn Pro Leu Leu
Ser Thr Cys Met Leu Pro Gly Arg Ser Val Thr Leu Leu Val
50 55 60
(2) INFORMATION FOR SEQ ID NO:39:
IO (i) SEQUEN OE CH~RA~l'~KISTICS:
(A) LENGTH: 12 amino acids
(B) TYPE: amino acid
(C) STR~NDEDNESS: single
(D) TOPOLOGY: 1;ne~r
(ii) MOLECULE TYPE: peptide
(iii) HYPOl~l~lCAL: NO
(v) FRAGMENT TYPE: internal
(vi) ORIGINAL SOUROE :
(A) ORGANISM: Homo sapiens
(xi) SEQUEN OE DESCRIPTION: SEQ ID NO:39:
Ser Pro Ser Ser Asn Arg Ile Arg Asn Thr Ser Thr
1 5 10
aO
(2) INFORMATION FOR SEQ ID NO:40:
CA 02211448 1997-07-25
W 096/29409 77 PCTrUS96~4037
(i) SEQUEN OE CH~RA.~l~ISTICS:
(A) LENGT~: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
S (D) TOPOLOGY: 1inP~r
(ii) MOLEC'ULE TYPE: peptide
(iii) HYPO~ CAL: NO
(v) FRAGMENT TYPE: internal
(vi) ORIGINAL SOURCE:
(A) ORG~NISM: Homo sapiens
(xi) SEQUENOE DESCF'IPTION: SEQ ID NO: A O:
Ser Pro Ser Ser Asn Arg Ile Arg Asn
1 5
(2) INFORMATION FOR SEQ ID NO:41:
(i) SEQUEN OE CH~ CTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDED~~SS: single
(D) TOPOLOGY: l; n~r
,0 (ii) MOLECULE TYPE: peptide
(iii) HYPOl~ llCAL: NO
CA 022ll448 l997-07-25
W096l29409 -78- PCT/u~3G~ 37
(v) FRAGMENT TYPE: internal
(vi) ORIGINAL SOUR OE :
(A) ORGANISM: Homo sapiens
(xi) SEQUEN OE DESCRIPTION: SEQ ID NO:41:
Pro Ser Ser Asn Arg Ile Arg Asn Thr
l 5
(2) INFORMATION FOR SEQ ID NO:42:
(i) SEQUEN OE CHARA~'l~ISTICS:
IS (A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: 1 i n~r
(ii) MOLECULE TYPE: peptide
(iii) HYP~~ C~L: NO
(v) FRAGMENT TYPE: internal
(vi) ORIGINAL SOUROE :
(A) ORGANISM: Homo sapiens
(xi) SEQUEN OE DESCRIPTION: SEQ ID NO:42:
Ser Ser Asn Arg Ile Arg Asn Thr Ser
1 5
CA 022ll448 l997-07-25
W 096129409 79 PCT~U~9''01~37
(2) INFORMATION FOR SEQ ID NO:43:
(i) SEQUENOE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: ami:no acid
(C) STE~NDEDNESS: single
(D) TOPOLOGY: 1i n~r
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(v) FRAGMENT TYPE: lnternal
(vi) ORIGINAL SOUR OE :
(A) ORG~NISM: Homo sapiens
(xi) SEQUEN OE DESCE~IPTION: SEQ ID NO:43:
Ser Pro Ser Ser A~,n Arg Ile Arg Asn Thr
1 5 10
(2) INFORMATION FOR SEQ ID NO:44:
~5
(i) SEQUENOE CH~ CTERISTICS:
(A) LENGTH: ~7 base pairs
(B) TYPE: nucleic acid
(C) STE~NDED:NESS: single
(D) TOPOLOGY: li ne~r
(ii) MOLECULE TYPE: cDNA
CA 02211448 1997-07-25
W 096/29409 80 PCT/u~ 1o~7
(iii) HYPO'l'~l'lCAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(vi) ORIGINAL SO~R OE :
(A) ORGANISM: Homo sapiens
(xi) SEQ~EN OE DESCRIPTION: SEQ ID NO:44:
TCACCAAGTT C~AACAGC~T CAGG~AT 27
(2) INFORMATION FOR SEQ ID NO:45:
(i) SEQ~EN OE CHARAw ~KISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: l;~Y
(ii) MOLECULE TYPE: cDNA
(iii) HYP~l~llCAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(vi) ORIGINAL SO~R OE :
(A) ORGANISM: Homo sapiens
CA 02211448 1997-07-25
W096/29409 -81- PCTrUS9G/01~7
(xi) SEQUEN OE DESCRIPTION: SEQ ID NO:45:
TCACCAAGTT CAAACAGGAT C~G~AATACC 30
(2) INFORMATION FOR SEQ ID NO:46:
(i) SEQUENOE CH~F~CTERISTICS:
(A) LENGTH: 21 base pairs
10(B) TYPE: nucleic acid
(C) STRANDED~ESS: single
(D) TOPOLOGY :: l; n~r-
(ii) MOLECULE TYPE:. cDNA
(iii) HYPOl'~llCAL: NO
(iv) ANTI-SENSE: NO
(v) FR~GMENT TYPE: internal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Homo sapiens
(xi) SEQUENCE DESCRIPTIO~: SEQ ID NO:46:
~ lCCll~ CGTCTCTACT A 21
(2) INFORMATION FOR SEQ ID NO:47:
(i) SEQUENCE CH~CTERISTICS:
CA 022ll448 l997-07-25
W096/29409 -82- PCTrUS96104037
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STR~NDEDNESS: single
(D) TOPOLOGY: l; nP~r
s
(ii) MOLECU.LE TYPE: cDNA
(iii) HYP~l~llCAL: NO
(iv) ANTI-SENSE: YES
(v) FRAGMENT TYPE: internal
(vi) ORIGINAL SOUR OE :
(A) ORG~NISM: Homo sapiens
(xi) SEQUEN OE DESCRIPTION: SEQ ID NO:47:
20 W l~l~CCGA TGACATCG 18
(2) INFORMATION FOR SEQ ID NO:48:
(i) SEQUEN OE CH~RA~-l'~KISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: 1; n~r
(ii) MOLECU~E TYPE: cDNA
(iii) HYPOl~llCAL: NO
CA 02211448 1997-07-25
W 096/294U9 83 PCTAUS9C/~
(iv) ANTI-SENSE: YES
(v) FRAGMENT TYPE: internal
S (vi) ORIGIN~L SOURCE:
(A) ORG~NISM: Homo sapiens
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:
GAGGTATTCC TGATCCTG 18
(2) INFORMATION FOR SE~~ ID NO:49:
(i) SEQ~ENCE CH~RACTERISTICS:
(A) LENGTH: 13 base pairs
(B) TYPE: nucleic acid
(C) STRANDED~ESS: single
(D) TOPOLOGY: 1 in~r
(ii) MOLEC~LE TYPE: cDNA
(iii) HYPO~ CAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(vi) ORIGINAL SOUROE :
(A) ORGANISM: Homo sapiens
CA 02211448 1997-07-25
W O 96/29409 - 84 - P{~rrJS96104037
(xi) SEQU~ DESCRIPTION: SEQ ID NO:49:
CAAAQ NGGA TCA 13
(2) INFORMATION FOR SEQ ID NO:50:
(i) SEQUEN OE CH~RACTERISTICS:
(A) LENGTH: 14 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: sin~le
(D) TOPOLOGY: li ne~r
(ii) MOLECULE TYPE: cDNA
(iii) HYPOl~ lCAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(vi) ORIGINAL SOUR OE :
(A) ORGANISM: Homo sapiens
(xi) SEQUE.N OE DESCRIPTION: SEQ ID NO:50:
NTATTCCTGA TCCT 1
(2) INFORMATION FOR SEQ ID NO:51:
(i) SEQUEN OE CH~RACTERISTICS:
(A) LENGTH: 15 base pairs
CA 02211448 1997-07-25
W 096/29409 -85- PCT/U~/n10~7
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: l;ne~r
-
S (ii) MOLECULE TYPE: cDNA
(iii) HYPO'l'~'l'lCAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Homo sapiens
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:
NTATTCCTGA TCCTG 15
(2) INFORMATION FOR SEQ ID NO:52:
(i) SEQUENCE CH~RA~lERISTICS:
(A) LENGTH: 16 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: li n~r
(ii) MOLECULE TYPE: cDN~
,0
(iii) HYPOl~llCAL: NO
CA 022ll448 l997-07-25
W096t29409 -86- PCTtUS~6/011-7
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
S (vi) ORIGINAL SOUR OE :
(A) ORG~NISM: Homo sapiens
(xi) SEQUEN OE DESCRIPTION: SEQ ID NO:52:
NTAll~l~A lG~l~l' 16
(2) INFORMATION FOR SEQ ID NO:53:
(i) SEQu~ CH~RACTERISTICS:
(A) LENGTH: 17 base pairs
(B) TYPE: nucleic acid
(C) STR~NDEDNESS: single
(D) TOPO~OGY: 1 ln~r
(ii) MOLECULE TYPE: cDNA
(iii) HYPO~ CAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(vi) ORIGINAL SOUR OE :
(A) ORG~NISM: Homo sapiens
CA 02211448 1997-07-25
W 096129409 -87- pCT~US96/04037
(xi) SEQUENOE DESC~'IPTION: SEQ ID NO:53:
NTATTCCTGA 'l'C~ l' 17
(2) INFORMATION FOR SEQ ID NO:54:
(i) ~Qu~ CH~ CTERISTICS:
(A) LENGTH: ]4 base pairs
(B) TYPE: nucleic acid
(C) STRANDED~ESS: single
(D) I3POLOGY: lin~r
(ii) MOLECULE TYPE: cDNA
(iii) H~O~ CAL: NO
(iv) ANTI-SENSE: YES
(v) FRAGMENT TYPE~ internal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Homo sapiens
(xi) SEQUENOE DESCRIPTION: SEQ ID NO:54:
NC~AGTTC~A ACAG 14
(2) INFORMATION FOR SEQ ID NO:55:
(i) SEQUEN OE CH~ l~KISTICS:
(A) LENGTH: :L5 base pairs
CA 02211448 1997-07-25
W096/29409 -88- PCTrUS96104037
(B) TYPE: nucleic acid
(C) STR~NDEDNESS: single
(D) TOPOLOGY: li n~r
(ii) MOLECULE TYPE: cDNA
(iii) HYPO~ CAL: NO
(iv) ~NTI-SENSE: YES
(v) FRAGMENT TYPE: intPrn~l
(vi) ORIGINAL SOUR OE :
(A) ORG~NISM: Homo sapiens
1~
(xi) SEQUENOE DESCRIPTION: SEQ ID NO:55:
NCAAGTTC~A ACAGG 15
(2) INFORMATION FOR SEQ ID NO:56:
(i) SEQUEN OE CHARACTERISTICS:
(A) LENGT~: 16 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: 1 in~r
(ii) MOLECULE TYPE: cDNA
,0
(ili) HYP~ CAL: NO
CA 022ll448 l997-07-25
w ~96l29409 89 PCT/US9~0l~37
(iv) ANTI-SENSE: YES
(v) FRAGMENT TYPE: ;nt~rn~l
.,
S (vi) ORIGINAL SOUR OE:
(A) ORGANISM: Homo sapiens
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:
l0 NCAAGITCAA ACAGGA 16
(2) INFORMATION FOR SEQ ID NO:57:
(i) SEQUENOE CH~RACTERISTICS:
(A) LENGTH: 17 base pairs
(B) TYPE: nu.cleic acid
(C) STRANDEDNESS: single
(D) TOPOLCGY: 1 in~r
(ii) MOLECULE TYPE': cDNA
~0
(iii) HYP~l~l'lCAL: NO
(iv) ANTI-SENSE: Y'ES
(v) FRAGMENT TYPE: internal
(vi) ORIGINAL SOUR OE :
(A) ORGANISM: Homo sapiens
(xi) SEQUEN OE DESC~IPTION: SEQ ID NO:57:
N Q AGTTCAA ACAGGAT 17
