Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
CA 02258507 1998-12-14
~ WO 97/48807 PCT/US96/10561
Chemokine ,B-15
The present invention relates to a human CC çh~rnf~kine protein (i.e.,
- a cytokine having the first two of its four cysteine residues ~j~c~nt as infiir;~ted
5 by "CC") and to polynucleotides encoding this protein.
BACKGI~OUND OF THE INVENTION
The discovery of IL-8, in 1987, revea}ed the ~yict~once of a novel class
of small cytokines, now called rh~mol~in~ that are widely studied because of their
ability to activate leukocytes and their polenLial role as metli~tQrs of i- .n~ ;nns.
A number of different human chemokines have been identified after IL-8, by
cloning or biochPrniç~l purification and arnino acid seqllçnring All have four
15 conserved cysteines that form characteristic dislllfi~le bonds, a short amino-
terminal and a longer carboxy-terminal seq~lenre Two subfa nilies are
tin~ hed by the arrangement of the first two cysteines, which are either
se~led by one amino acid (CXC rh~molrin~) or are .q~ (CC chemokines.).
Ch~mnl-in~ cDNAs typically encode proteins of 92-99 amino ac;ds in length that
20 are secreted after cleavage of a leader sequence of 20-25 amino acids. Modeling
on the basis of the NMR-derived structure of IL-8 suggests that CXC and CC
chemokines are folded in a similar manner.
The first human CC chemokine was identified by di~el enlial
hybri~i7~tion cloning and was termed LD78 (Obaru, K. Fukuda, M., Maeda, S.
and Shim~ K. (1986) J. Biochem. (Tolyo) 99, 885-894.) Several cDNA
isoforms of a closely related human chemokine, Act-2, were later described
~Miller, M.D. and Krangel, M.S. (1992) Crit. Rev. Immunol. 12, 17-46), and two
similar ploteil~s, macrophage infl~mm~tory protein la (~P-la) and MIP-1~,
were purified forrn the culture me~ m of lipopolysaccharide (LPS)-stim~ ted
mouse macrophages (VVolpe, S.D., Davatelis, G. Sherry, B. et al. (1988) J. Exp.
Med. 167, 570-581). On the basis of more than 70% amino acid identity, the
murine and human proteins are considered as homologs, and the terms human
-
CA 02258507 1998-12-14
~ WO 97/48807 PCT/US96/10561
MIP-la and M~-1~ are commonly used instead of LD78 and Act-2. The best
charqct~-n7~ CC rhemo1~ine is monocyte chRmot~rtic protein 1 (MCP-1), which
was p~rifie~ and cloned ~om ~L~;le~lL sources (Miller, M.D. and Krangel, M.S.
(l99V Cnt. Rev. Tmml-nnl. 12, 1746; Yoshtm~re, T. I~obincon, E.A. Tanaka, S.
Appella, E. and Leonardo, E. J. (1989) J. Tmml~nol. 142, 1956-1962; ~rzltcushimsl
K., Larsen, C.G., DuBois, G.C. and Oppenheim~ J.J. (1989) J. Exp. Med. 169,
1485-1490). Other CC rhemclt~inç~ 309 ~Miller, M.D., Hata, S., De Waal
Malafyt, R. and Krangel~ M.S. (1989) J. Tmm--nol. 143, 2907-29163, RANTES
(Schall, T. J. Jongstra, J., Dyer, B. J. et al. (1988) J. Tmm--nnl. 141, 1018-1025)
and HC14 (Chang, H. C., Hsu, F., Freeman, G. J., G~iffin, J.D. and Reinherz, E.
L. {1989) Int. Tmmllnnl. 1, 388-397), were purified or cloned as products of
activated T cells. HC14, termed MCP-2, was also isolated from osteos~ collla cell
cultures (VanDamme, J. Proost, P., Lenaerts, J-P. and Opd~n~kker, G. (1992) J.
Exp. Med. 176, 59-65), along with a novel CC chemokine, MCP-3, which was
subseq~Pntly cloned and ~ e ,sed (Minty, A. Chalon, P. Guillemot, J. C. et al.
(1993) Eur. Cytokine Netw. 4, 99-110; Opclen~kker, G. Froyen, G Fiten, P.,
Proost, P. and Van Damme, J.(1993) Biochem. Biophys. Res. Commun. 1991,
535-542). These CC chemokines share a sequence identif~ with MCP-l of
between 29 and 71% (MCP-2 and MCP-3 have 62-71% identity with MCP-1).
MCP-l, the prototype of the CC chemokine sub-family, is ç~pmot~tic
for monocytes but not for neutrophils (Yosl.;...~e, T. Robinso4 E. A. Tanaka~
S. Appella, E. and Leonardo, E. J. (1989? J. Tmmlln~)l. 142, 1956-1962;
~A~ h;...~ K., Larsen, C. G., DuBois, G. C. and O~ p~ ; .., J. J. (1989) J. Exp.Med. 169, 1485-1490) and was initially considered to be a co-n~el~alL of IL-8.
25 Tn~lee~l~ monocytes respond to all CC r.hpmokinp~ as judged from stim~
dependent ~Ca2+~i rh~n~Ps (Miller, M. D. and Krangel, M. S. (1992) Crit. Rev.
Tmm--nol 12, 17 '16; Bioscho~, S.C., Krieger, M. Brunner, T. et al. (1993) Eur.
J. ~mmlmnl 23, 761-767; McColl, S. R., ~f~hich~ M., Levasseur, S., Noete, K.
and Schall, T. J. (1993) J. Tmmlmol 150, 45504560). MCP-l, MCP-2 and MCP-
30 3 induce monocyte infiltration on intradermal injection into rats and rabbits (VanDamme, J. Proost, P., Lenaerts, J-P. and Opdens~kk~r, G. (i992) J. Exp. Med.
176, 59-65; Zacha, C. O. C., Anderson, A. O., Tho...l soll, H. L. et al. (1990) J.
CA 02258507 1998-12-14
- WO 97/48807 PCTIUS96/10561 - -
Exp. Med. 171, 2177-2182), and MCP-1 also elicits in monocytes a Ict~ oly
burst (h~iller, M. D. and Krangel, M. S. (1992) Crit. Rev. Tmmllnol 12, 17-46)
and the ~ ~;oll of B2 integrins (Jiang, Y., Beller, D. I., Frendl, G. and Graves,
D. T. (1992) J. Tmmllnol. 148, 2423-2428).
While the view that CXC ~hPmol~inps act on n~ op~ s and CC
chemokines act on monocytes app~e;-lLly remains valid, recent studies have
revealed that CC ~hPmnlinRc have a much wider range of ~ ~'c g c~l activities since
they can also activate some Iymphocytes and, in particular, basophil and eosinophil
leukocytes. Thus, there is a contim~inp~ need in the art for i~o!~tin,~ novel CC
~h~mf~kine~
SUMMAR Y OF THE INVENTION
The present invention provides isolated nucleic acid molecules
comprising a polynucleotide encoding a human fhPmolcinP~ ~-15 (CK,B-15)
polypeptide having the amino acid sequenr~e in Figure 1 [SEQ ID NO:2] or the
amino acid seq~Pnce encoded by the cDNA clone deposited in a bacterial host as
ATCC Deposit Number 97519 on April 25, 1996. The nucleotide seqllPnre
determined by seq~U~nrinf~ the deposited CK,B-15 cDNA clone, which is shown in
Figure 1 ~SEQ ID NO: 1], cont~in~ an open reading frame encoding a polypeptide
of 149 amino acid residues in~ ling an initiation codon at positions 1-3, a leader
seq~nce of about 20 amino acid residues and a ded~lced molec~ r weight of
about 16 kDa. The 129 amino acid sequence of the predicted mature CK~-15
protein is shown in Figure 1 (last 129 residues) and in SEQ ID NO:2 (from amino
acid residue 1 to residue 129).
Thus, one aspect of the invention provides ieol~ted nucleic acid
m~ c~ a polynl~ otide having a nucleotide sequence selected from
the group CQ~ g of: (a) a nucleotide sequence encoding the chemokine ,B- 15
~ 30 polypeptide having the complete amino acid sequence in SEQ ID NO:2; (b) a
nllclP~oti~p~ S~qop~nr~e Pnr~riir~ the mature chemokine ~-15 pol~,~Lide having the
amino acid sequen~e at positions 1-129 in SEQ ID NO:2; (c) a nucleotide
CA 02258507 l998- l2- l4
- WO 97/48807 PCT/US96/10561 - -
sequence ~,ncod..,x the ~hemnl ine ,B-15 polypeptide having the w ~ ~ e amino
acid seqll~nce ~ncoded by the cDNA clone co--l~,ed in ATCC Deposit No.
97519; (d) a mlrleoti-le sequence encoding the mature ch~m-kin~ ,B-15
polypeptide having the amino acid sequence encoded by the cDNA clone
S co~ ed in ATCC Deposit No. 97519; and (e) a nu~leotir~e se~llen~e
comrlPm~nt~ry to any of the n~~Cleotirle sequences in (a3, (b), (c) or (d) above.
r~ bly, the nucleic acid molecule will encode the mature polypeptide in SEQ
ID:2 or encoded by the above-described deposited cDNA.
Further embod;~ e of the invention include ;-CQ!~ted nucleic acid
10 molecules that comprise a polynucleotide having a nllrleotirle sequ~nce at least
90% id~ntir~l and more preferably at least 95%, 97%, 98% or 99% id~nti~ ~l to
any of the nucleotide sequences in (a), ~b), (c), (d) or (e) above, or a
polynucleotide which hybridizes under stringent hybri-li7~tiQn conditions to a
polynllrl~oti~le having a nucleotide sequence identic~1 to a nucleotide seq~nce in
15 (a), (b), (c), (d) or (e), above. The polynl-cleotirle which hybrizes does not
hybridize under ~Il,-,genl hybrirli7~tioll conditions to a polynucleotide having a
nucleotide sequ~nce CQno~ ;ug of only A residues or of only T rR~ -ee An
~d~ on~l nudeic acid embodiment of the invention relates to an icQl~ted nucleic
acid mr~ lle COIll~ g a polynucleotide which f~nc~P,s the amino acid seq~n~e
20 of an epitope-bearing portion of a çhPmokine ,B-15 polypeptide having an amino
acid sequ~nce in (a), (b), (c) or (d), above.
The present invention also relates to . ~cc,mbinanl vectors which include
the ;~Q~ted nucleic acid molecules of the present invention and to host cells
co~ the reco-..l)inant vectors, as well as to methods of making such
vectors and host cells and for using them for production of CK~-15
polypeptides or peptides by recombinant techniques.
The invention further provides an isolated chemokine ,B-I 5 po!ypeptide
having amino acid sequ~nce selected from the group conci~ting of: (a) the amino
acid s~uence ofthe ch~mo~ine ~B-l~ polypeptide having the co~plete 149 arnino
acid sequ~nce inc1u(1ing the leader sequ~nce shown in Figure 1 ~SEQ ID NO:2];
(b) the amino acid sequence of the mature chemokine ,B-15 polypeptide ~without
the leader) having the amino acid sequence at positions 1-129 in SEQ ID NO:2;
CA 022~8~07 1998-12-14
Wo 97/48807 pcTluss6/lo56l -
(c) the amino acid sequence of the chemokine ~-15 polypeptide having the
co , '~ ~e amino acid sequence in~ ling the leader encoded by the cDNA clone
co.-lh;~.cd in ATCC Deposit No.97519; and ~d) the amino acid sequ~nce ofthe
mature l~h~mQ'-in~ ,B-15 polypeptide having the amino acid seq~n~e encoded by
S the cDNA c1One ~- ~ d in ATCC Deposit No.97519. The polypeptides of the
present invention also include polypeptides having an amino acid seql~nçe with
at least 90% ~ ily, more preferably at least 95% similarity to those described
in (a), (b), (c) or (d) above, as well as polypeptides having an amino acid se~ en.~e
at least 80% i(l~ntic~l~ more preferably at least 90% ir~Pntic~l~ and still more preferably 95~/O, 97%,98% or 99% idçntic~l to those above.
An ~d~ition~l embodiment of this aspect of the invention relates to a
peptide or polypeptide which has the amino acid sequ~nce of an epitope-bearing
portion of a Gh~o-mnbin~ 15 polypeptide having an amino acid sequence desc~ ed
in (a), (b), (c) or (d), above. Peptides or polypeptides having the amino acid
15 sequence of an epitope-bearing portion of a ~,h.omnl ine ,B-15 polypeptide ofthe
invention include portions of such polypeptides with at least six or seven,
p-~t;-~l>ly at least nine, and more ylert;l~ly at least about 30 amino acids to about
50 arnino scids, ~lthou~h epitope-bearing polypeptides of any length up to and
in~ the entire amino acid sequence of a polypeptide of the invention
20 described above also are in~ ded in the invention. In another embodiment the
invention provides an isolated antibody that binds spe~ifi~ ~lly to a çh~mct-ine ~-15
polypeptide having an amino acid sequence described in (a), (b), (c) or (d) above.
The present inventors have discovered that CKB-15 is t;~yressed only
in tissue ofthe thymus. Figure 3. For a number of thymus disorders, si~,--ir.c~.lly
25 higher or lower levels of CK,B-15 gene expression can be c~etectecl in thyrnus tissue
or bodily fluids (e.g., serum, plasma, urine, synovial fluid or spinal fluid) taken
from an individual having such a disorder, relative to a"standard" CK,B-15 gene
~yr~i~ Oll Ievel, i.e., the CK~-15 expression level in thymu~ tissue or bodily fluids
from an individual not having the thymus disorder. Thus, the invention provides
30 a d;a~-o~ie method usefill during diagnosis of a thymus disorder, which involves
(a) assaying ~hPmokine ~B-15 gene ~ - t;s~ion level in cells or body fluid of that
individual; (b) co--~ya~ng that çh~mokine ~-15 gene c.~ ession ievel with a
CA 02258507 1998-12-14
~ W097/48807 ' rC~/US96/10561
hern~line ~-15 gene e,~ ,;s:,;on level, whereby an illc~ se or decrease
in the assayed chen~n1-ine ~-15 gene e,~ ;s~ion level co,l~ ed to the standard
~AI3r~,ssion level is indicat;ve of a thymus disorder. An ~ lition~l aspect of the
invention is related to a method for ~ ,.l of an individual in need of an
5 increasedlevelof ~h.9mot~ine,B-15 activityinthebody COIlly~ lg~ le~
to such an indivdual a composition comprising an icslqted chemokine ~-15
polypeptide of the invention.
BRIEFDESCRIPTION OF THE DR,4 WINGS
Figure I shows the nucleotide [S~Q ID NO: 1] and dech-ced amino acid
[SEQ n~ NO:2] sequences ofthe complete chemokine ,B-15 protein det~ ed
by se~uenrin~ of the DNA clone cc~tlt~ined in ATCC Deposit No. 97519. The
15 protein has a leader sequence of about 20 amino acid residues (underlined) and a
d~ ced molec~lT~r weight of about 16 kDa. The amino acid seqll~?nce of the
pl~ Led mature CK,B-15 protein is shown in Figure 1 (last 129 amino acids) and
in SEQ ID NO:2 (from amino acid residue 1 to residue 129).
Figure 2 shows the regions of similarity between the amino acid
20 seql~nces of the CK~B-15 protein and the mouse macrophage infl~ oly
protein-related protein 2 (M~P-2) [SEQ ID NO:3].
Figure 3 shows a Northern blot ~say for ~ .l t;SSiOII of mRNA from the
CK~3-15 gene in various human tissues. The panel labeled '~ k;X~2" shows
hybri~i7~tion to the CK~-15 cDNA probe which that labeled "ACT~' shows
25 hybridization of a cDNA encoding actin whic.h serves as a positive control
in~lic~tir~s~ the presence of intact RNA in each sample.
CA 02258507 1998-12-14
~ WO 97/48807 PCT/US96/10561
~7
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides j~ol~ed nucleic wid molccllles
comprising a polym~ eoti-le f "'o.~ g the rh~.m~in9 13-15 (CK~-15) protein
5 having the amino acid sequence shown in Figure 1 [SEQ ID NO:2] which was
d~Lc;.l...l.ed by sequ~nrin~ a cloned cDNA. CK,13-15 is a novel member ofthe ,B-chemokine subfamily (CC) whose genes are on human chromnsome 17 and on
mouse chromnsome 11 (Wilson, S.D., etal., J. Exp. Med. 171:1301(1990) and
Modi, W.S., et al., Hum. Genet. 84:185 (1990)). The CK,13-15 protein of the
10 plesen~ invention shares sequence homology with the mouse macrophage
infls.. , 1c.,y protein-related protein 2 (MMRP-2) (Figure 2) [SEQ ID NO:3].
The nucleotide sequence shown in Figure 1 tSEQ ID NO:l~ was obtained by
seq~lPn-~in~ the ~l~l~X~2 cDNA clone encoding a CK~-15 polypeptide, which
was deposited on April 25, 1996 at the American Type Culture Colle~,Lioll, 12301Park Lawn Dr;ve, Rockville, Maryland 20852, and given ~cce~;on number 97519.
The deposited clone is co..~ .ed in the pBluescript SK(-) plasmid (Str?~t~ne,
LaJolla, CA).
CA 02258507 1998-12-14
- WO 97/48807 PCT/US96/10561
Nucleic Acid Mole~ ,s
Unless otherwise inrlic~te~l, all nl~cleotide sequP-nre,s det~rmine~ by
sequ~nring a DNA molecule herein were dete n-ined using an JIO~ e(~ DNA
S ~ll~Pnr~r (such as the Model 373 from Applied Biosy~lelns, Inc.), and all amino
acid se~Pnr,ç,s of polypeptides encocled by DNA molccul~,c dete~ ed herein
were p~ ;lèd by 1,,~ ;on of aDNA sequ~nre d~L~ -d as above. Therero~t;,
as is known in the art for any DNA seqU~nce determined by this automated
~l~luach, any nurl~ti~le sequen~e dt;lelll~ined herein may contain a some errors.
10 Nucl~ti~le s~u~n~c del~ d by allto-m~tinn are typically at least about 90~/0
;CAI~ more typically at least about 95% to at least about 99.9% i~1.sntic~1 to the
actual nucleotide sequence of the sequ~n~ed DNA ml-~er3l1e The actual sequence
can be rnore precisely determined by other approaches in~lu-lin~ manual DNA
seqU~nrin~ methods well known in the art. As is also known in the art, a single
15 insertion or deletion in ~ d~L~..~,ined nucleotide sequence co-l"~ d to the actual
sequPnce will cause a frame shift in translation of the nucleotide sequçnce suchthat the predicted amino acid sequence encoded by a deLe II--l-ed nucleotide
sequence will be comp'e~.oly di~e.ent from the arnino acid sequence actually
~nrodecl by the sequenced DNA molecule, be~ at the point of such an
20 insertion or deletion.
Unless otherwise in~lic~t~ each "nucleotide seqU~nce" set forth herein
is p-ti~-Led as a sequence of deoxyribonucleotides (abbreviated A, G, C and T).
However, by "nucleotide seq~1~n~e" of a nucleic acid molecllle. or polynucleotide
is in~çn~erl for a DNA molecule or polynucleotide, a sequence of
25 deo~yl il,onucleotides, and for an R~A molecule or polynucleotide, the
col~ JOndi~lg s~uen-~e of ribonucleotides (A, G, C and U) where each thymidine
deoxynucleotide (T~ in the specified deoxynucleotide sequ~nce in is replaced by
the ribon-~ oti~e uridine (U). For ;..~ re c;nce to an RNA molecule having
the s~llçnce of SEQ ID NO: 1 set forth using deoxyribonucleotide abbreviations
30 is int~n~lefl to indic~te an RNA molecule having a seqllen~e in which each
deoxynucleoti-le A, G or C-of SEQ ID NO:1 has been replaced by the
co..esl,onding ribonucleotide A~ G or C, and each deoxynucleotide T has been
CA 02258507 1998-12-14
- WO 97/48807 PCT/US96/10561
repl~-e~l by a ribomlcleoti~le U.
Using the ;..r~.l"lAl;on provided herein, such as the mlrleotirle sequPnre
in Figure 1, a nucleic acid molec~lle of the present invention encoding a CK~-15poly~eplidc may be obl;~ ed using standard cloning and scfcening procedures,
S such as those for cloning cDNAs using mRNA as starting material. Illustrative of
~ the inventionJ the nucleic acid mt~lec~le described in Figure 1 [SEQ ID NO:l] was
discovered in a cDNA library derived from human thymus tissue. The dete, .~ ed
nucleotide sequçnce of the CK,B- 15 cDNA of Figure 1 co~llains an open reading
frame ~nro~lins~ a protein of 149 amino acid residues with an ;llili~lion codon at
pO~itiOllS 1-3 ofthe nuc1eotide sequence shown in Figure 1 [SEQ ID NO. 1], and
a predicted leader seql~enre of about 20 amino acid rç~;rlu~ and a cled~lced
molecular weight of about 16 kDa. The amino acid sequence of the predicted
mature CK~-15 protein is shown in Figure 1 [SEQ ID NO: 1] from amino acid
residue 21 to residue 149. The CK~-15 protein shown in Figure 1 [S~Q ID
NO:2] is about 34% identical and about 53% similar to MMRP2 (Figure 2). As
one of c,ld;-l~uy sl~ll would a~pi ~;ale, due to the possibilities of seq~lenring errors
dicc~lcc~d above, as well as the variability of cleavage sites for leaders in .lirre,el,l
known l,r~l~,s, the actual CK~-15 polypeptide encoded by the deposited cDNA
co",~-ises about 149 amino acids, but may be anywhere in the range of 142-154
20 amino acids; and the actual leader sequ~nre ofthis protein is about 20 amino acids,
but may be anywhere in the range of about 15 to about 25 amino acids
As ~ , nucleic acid molecules of the present invention may be in
the form of RNA, such as rrRNA, or in the form of DNA, inC~ ng, for ~ c~,
cDNA and genomic DNA obtained by cloning or produced ~ ly. The
25 DNA rnay be dol ' 'e-stranded or single-stranded. Single-stranded DNA or RNA
may be the coding strand, also known as the sense strand, or it may be the non-
coding strand, also ltirelled to as the anti-sense strand.
By "isolated" nucleic acid molecule(s) is in~P!nr~ed a nucleic acid
molecule, DNA or RNA, which has been removed from its native en~,h onme..l
30 For e~ plf .c;con-bina ,L DNA molec~lles contained in a vector are consideredisolated for the purposes of the present invention. Further eA~ !es of icol~ted
DNA mo~e ~leC include recombinant DNA molecules ~ ed in heterologous
-
CA 02258507 1998-12-14
WO 97/48807 PCT/US96/10561
/c~
host cells or purified (partially or subs~nti~lly) DNA mslec~llss in solution.
T.eol~ted RNA ml~lec llPS include in vivo or in vifro RNA l~ sc-ipls of the DNA
ofthe present invention. ~ol~ted Nucliec acid molcr,l-le5 acco~ g to
the present invention further include such 5nolec~ s produced synsheti~
Isolated nuc1eic acid molecules of the present invention include DNA
molecules colll~ in~, an open reading frame (ORF) with an initiation codon at
p;~ I;O,..c 1-3 ofthe mlr~ ti-le seq~l~nre shown in Figure I tSEQ ID NO:l]; DNA
mol- ' - g cO~-Iplisillg the coding sequence for the mature CK,B-l 5 protein shown
inFigure 1 (last 129 amino acids) and SEQ ID NO:2 (residues 1-129); and DNA
10 ~ e,S which Cf)...~ e a sequence s~-bst~nti~lly di~elenl from those describedabove but which, due to the degenel~y ofthe genetic code, still encode the CK~-
15 protein. Of course, the genetic code is well known in the art. Thus, it wouldbe routine for one skilled in the art to generate the degenerate variants described
above.
In another aspect, the invention provides isolated nucleic acid molec~ s
~n~,otlin~ the CKB-15 polypeptide having an aniino acid seq~n~e encoded by the
cDNA clone cr)nt~inecl in the plasmid deposited as ATCC Deposit No. 97519 on
April 25, 1996. Preferably, this nucleic acid molecllle will encode the mature
polypeptide ~ncoded by the above-described deposited cDNA clone. The
20 invention further provides an isolated nucleic acid molecule having the nucleotide
sequence shown in Figure 1 [SEQ ID NO:l] or the nucleotide sequ.sn~e ofthe
CK,B-15 cDNA co~ d in the above-described deposited clone, or nucleic acid
m~ having a s~lP.n~e compl/ . ~ y to one of the above sequences. Such
isolated molecules, particularly DNA molecules, are useful as probes for gene
25 ' ~ g by in situ hybridization with chromosomes and for ~l~lec' ;~ c,~,res~ion
ofthe CK~-15 gene in human tissue, for in.~t~nc~, by Northern blot analysis. As
des~;,;l,cd in detail below below, detectin~E~ altered CK~-15 gene e,.~-res~ion in
certain tissues or bodily fluids is indicative of thyînus disorders.
In another aspect, the invention provides an i~ol~ted nucieic acid
30 molecl-1e cO---~,.isillg a polyml~leoti~le which hybridizes under ~-i..gelll
hybridization con~litions to a portion of the polynucleotide in a nucleic acid
mo~ e ofthe invention described above, for in.~n~; the cDNA done co,lL~incd
CA 02258507 l998- l2- l4
~ WO 97/48807 PCT/US96/10561
in ATCC Deposit 97519. By ";,~ ge"L hybri~li7~tion con~1itiQncll is ir~tf~n~ed
o~ at 42~C in a ssl~ltion COIIIpliSIl-g. SO% rO~ 9 5x SSC
(150 mMNaCI, 15mM trico~ium citrate), 50 mM sodium phosph~te (pH7.6), 5x
De ~' dl's s~' , 10% dextran sulfate, and 20 llg/ml d~ aluled~ sheared salmon
5 sperm DNA, followed by washing the filters in 0. lx SSC ât about 65~C. By â
polym~ kol;de which hybridizes to a "portion" of a polynucleotide is intf~nf~ d a
pol~ ict~4~ e (either DNA or RNA) hybridizing to at least about 15 nucleotides
(nt), and more preferably at least about 20 nt, still more plerel~ly at least about
30 nt, and even more pl1rel~1y about 30-70 nt ofthe l-re..,nce polynucleotide.
These are useful as ~ ;c probes and primers as fliscl lcsefl above and in more
detail below.
Of course, polynucleotides hybridizing to a larger portion of the
,nce polyn~lrl~ti(le (e.g., the deposited cDNA clone), for i~ r,f~, â portion
50-750 nt in length, or even to the entire length of the rGrelc,lce polynucleotide,
also useful as probes according to the present invention, as are polynucleotidescorresponding to most, if not all, of the nucleotide sequenr,e of the deposited
cDNA or the n~lcleoti~e sequence as shown in Figure 1 [SEQ ID NO:l]. By a
portion of a polymlrleotirle of "at least 20 nt in length," for ~Y~mplç, is int~n~led
20 or more ~--I;g~lu~lc nucleotides from the nucleotide sec~ence of the IGÇ~lt--ce
polynucleoti~le~ (e.g., the deposited cDNA or the nucleotide sequence as shown
in Figure 1 [SEQ ID NO: 1]). As in~ te~l such portions are useful diagnosticallyeither as a probe according to conventional DNA hybridization techniques or as
:i for ~mplifi~tion of a target sequence by the polymerase chain reaction
~PCR), as desribed, for in~ )ce, in Molec2~1ar Clonin$ A Laborato7y Manual,
2nd. edition, edited by Sambrook J., Fritsch, E. F. and lU~nis~tiC, T., (1989), Cold
Spring Harbor Laboratory Press, the entire disclosure of which is hereby
i.~col~ol~led herein by IGrerence.
Since a CK~-15 cDNA clone has been deposited and its deter nined
nucleotide sequence is provided in Figure 1 [SEQ ~ NO: 1], generating
polynucleotides which hybridize to a portion of the CK,(~-15 cDNA molecule
would be routine to the skilled artisan. For example, restriction endom~ ce
cleavage or shearing by sonication of the CK~B-15 cDNA clone could easily be
CA 02258507 1998-12-14
- WO 97/48807 PCT/IJS96/10561
' /~
used to ~,.,nclale DNA portions of various sizes which are polynucleotides that
hybridize to a portion of the CK,B-15 cDNA molecule. Alternatively, the
hybridizing polynucleotides of the present invention could be generated
synthP,tir~lly acco~ g to known techniques. Of course, a polynucleotide which
5 hyl~id;~ only to a poly A sequçnr,e ~such as the 3 ' terminal poly(A) tract of the
CK~-15 cDNAshowninFigure 1 [SEQIDNO:1]), ortoa~ ..P~1Z-,y stretch
of T (or U) resides, would not be inrlu(led in a polynucleotide of the inventionused to hybridize to a portion of a nucleic acid of the invention, since such a
polynucleotide would hybridize to any nucleic acid mnlf~cllle contain a poly (A)stretch or the cc p~~ np~nt thereof (e.g., practially any double-standed cDNA
clone).
As in~ic~te~l nucleic acid molecules of the present invention which
encode the CE~-15 protein polypeptide may inrlude, but are not limited to those
encoding the amino acid seq~lP nre of the mature polypeptide, by itself; the coding
1~ sequPnce for the mature polypeptide and additional ~eql~p~nce~ such as those
P.ncol ' lv the about 20 amino acid leader or secretory seq~P nce, such as a pre-, or
pro- or prepro- protein seq~lP,nr,P; the coding sequence of the mature polypeptide,
with or without the aforPmP~nti~ned additional coding sequences, to~t4~ with
Pd~itir~nS-l non-coding seq~lP~n~çe incln~lin~? for e~mplP, but not lirnited to introns
and non-coding 5' and 3' sequences, such as the transcribed, non-L.~ ed
s~ ~Pn~Pe that play a role in ll~s~;.ipLion, mRNA plQCf,,~,';.-g - in~ in~ splicing
and polyadenylation signals, for P~x~mple - ribosome binding and stability of
mRNA; an additional coding seq~lence which codes for additional amino acids,
such as those which provide a(1~ition~l fimction~litie,s Thus, the seq~en~e
2~ Pl-co.l~ o. the polypeptide may be fused to a marker sequence, such as a seq~ence
encoding a peptide which f~cilit~tes purification of the fused polypeptide. In
certain p- t r~ d embodiments of this aspect of the invention, the marker amino
acid ~u~nce is a hexa-hieti-lit e peptide, such as the tag provided in a pQE vector
(Qiagen, Inc.), among others, many of which are comm~rcially available. As
described in Gentz et a1. (1989) Proc. Natl. Acad. Sci., USA 86:821-824, for
~, hexa-l~ . .f provides for convenient purifiç~tion of the fusion protein.
The "HA" tag is another peptide useful for purification which cc>-~ onds to an
CA 02258507 1998-12-14
- WO 97/48807 PCT/US96/10561
~3
epitope derived from the infl-lç.n7~ hem~gf~ tinin protein, which has been
desc.il,ed by Wilson et al., Cell 37: 767 (1984).
The present invention further relates to variants of the nucleic acid
f~ ~ ofthe present invention, which encode portions, analogs or de~ dti~es
5 of the CK~-15 protein. Variants may occur naturally, such as a natura1 allelic- variant. By an "allelic variant" is intPnted one of several alternate forms of a gene
oc~ ,..,g a given locus on a chromosome of an C~ Q' ~~ Genes II, Lewin, ed.
Non-naturally occurring variants may be produced using art-known m~lts-~f nf cicter.hn:q~e
Such variants include those produced by nucleotide sub.stit.~tinnQ
deletions or ~d~1itione The s~1bstit~tions, deletions or additions may involve one
or more mlcle~tidpe The variants may be altered in coding or non-coding regions
or both. Alterations in the coding regions may produce conservative or non-
conservative amino acid snhstit~tinne~ dPletione or ~drlitionQ Especially plert;.,ed
15 among these are silent Ql~bstitl-tione, ~ itionc~ and deletions, which do not alter the
pr ~e~ lies and activities of the CK,B-15 protein or portions thereof. A1so especially
pler~l~d in this regard are conservative substitutions. Most highly p~ d are
nucleic acid -' ~ llPc f nr.o-ling the mature CK,B-15 protein having the amino acid
sequçnce shown in Figure 1 [SEQ ID NO:2~ or the mature CK~-15 amino acid
20 seq-~f~ nce encoded by the deposited cDNA clone.
Further embo-limPnts of the invention include isolated nucleic acid
s cc,ln~Jli~g a polynucleotide ha~ng a nucleotide sequence at least 90%
idP.ntic~l, and more preferably at least 95%, 97%, 98% or 99% i~çntic~l to (a) anucleotide spllupnre encoding the full-length rhpmokine ~-15 polypeptide having
25 the c~.- . ' amino acid seq~Pn~e in SEQ ID NO:2 incl~l~in~ the predicted leader
seq~çnce; (b) a nucleotide sequence encoding the mature chemokine 13-15
polypeptide (full-length polypeptide with the !eader removed) having the amino
acid seq~lPnce at positions 1-129 in SEQ ID NO:2; (c) a nucleotide sequçn~e
f ~f'o~ , the full-length chemokine ,B-15 polypeptide having the complete amino
30 acid s~u~.nre inr.~ the leader f n~ode(~ by the cDNA clone contained in ATCC
Deposit No. 97519; (d) a nucleotide sequence encoding the mature chemokine ,B-
15 polypeptide having the amino acid sequence encoded by the cDNA clone
CA 02258507 1998-12-14
- WO 97/48807 - PCT/US96/10561
14
co~ e~ in ATCC Deposit No. 97519; or (e) a nucleotide sequ~nr
CO , I ~ ' ry to any of the nucleotide sequences in (a), (b), (c) or (d).
~3y a pol~ r~!;~1e having a nucleotide sequ~r,e at least, for ~ mple,
95% "id~ntiç~t" to a ~c;fel ,nce nucleotide sequ~nre ~nCorling a chemokine ,B-15S poly~eplide is int~nrled that the nucleotide sequpnce of the polynucleotide is
ntir~l to the reference sequence except that the polynucleotide sequ~nre may
include up to five point mllt~tio~e per each 100 nucleotides of the reference
m~r4~!oti-1e se~u~nce encoding the chemokine ,B-15 polypeptide. In other words,
to obtain a poly~l~cleoti~e having a nucleotide sequence at least 95% idçntic~l to
a rt~ cG nucleotide sequence, up to 5% of the nucleotides in the lerGl~nce
seq~nre may be deleted or s~bstihlted with another m-cleoti~1ç; or a ~-un~l)er of
nucleotides up to 5% of the total nucleotides in the l~,fc;r~llce se~uen~e may be
inserted into the ref~;r~nce sequence. These mllt~tione ofthe reference eeq~1~nrmay occur at the 5' or 3 ' tern~inal positions of the reference nucleotide sequence
or anywhere b~Lwet;l- those ter ninal positions, interspersed either individually
among nucleotides in the ,c~ ce sequence or in one or rnore contiguous groups
within the l~f~ ;nce sequ~nre.
As a practical matter, whether any particular nucleic acid molecule is at
least 90%, 95%, 97%, 98% or 99% idl~.ntis~l to, for ;.~ nc~, the n~ e
~e~ql~.onr~ shown in Figure 1 or to the mlrl~Qtitlf~e sequence of the deposited cDNA
clone can be cl~ eA COIIV~ ;on~lly using known computer programs such as
the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix,
~J,~n-~ti~e Computer Group, University Research Parlc, 575 Science Drive,
Madison, WI 53711. 13estfit uses the local homology algorithm of Smith and
Ws~ (Advances in Applied ~th-o.m~tics ~: 482-489, 1981) to find the best
se~n~qnt of homology between two seq~ lçnces When using Bestfit or any other
sequence ~ ..l program to determine whether a particular sequPnce is, for
c~; 95% id~nti~l to a ~~ ce sequ~onre according to the present invention,
the phl~l~t;L~.~ are set~ of course, such that the percentage of identity is c~lc~ ted
30 over the fi~ll length ofthe Ief~ nce m~t leoti~le sequ~once and that gaps in homology
of up to 5% of the total number of nucleotides in the .~lence sequence are
allowed.
CA 022~8~07 1998- 12- 14
~ WO 97/48807 pcTluss6llos6l
The present applic~tic!n is directed to nucleic acid molcc~ c at least
9O~/O, 95%, 97%, 98% or 99% identic~l- to the nucleic acid se~ re shown in
Figure 1 [SEQ ID NO: 1] or to the nucfeic acid seqllence of the deposited cDNA,
"..,..~,e~,li.re of wL~,Ll.er they encode a polypeptide having CK,B-15 activity. This
is be~ .se, even where a particular nucleic acid molec~lle does not encode a
polypeptide having CK,B- 15 activity, one of skill in the art would still know how
to use the nucleic acid molecllle~ for inct~nce, as a hybri~ ion probe or a
poly~ ,.ase chain ~~lion (PCR) primer. Uses ofthe nucleic acid m~ les of the
present invention that do not encode a polypeptde having CK,B-15 activity include,
inter alia, (1) i~ ting the CK,B-15 gene or allelic variants thereof in a cDNA
library; (2) in situ hybri(li7~tion (e.g., "~1~ ) to metaphase chromosomal spreads
to provide precise chromosomal location of the CK,B-15 gene as described in
Verma e~ al., Human Chro~2osom~: a Manual of Basic Techniques, Pe. alllOIl
Press, New York (1988); and Northem Blot analysis for lletecting CK~- 15 mRNA
~ sD;on in speciffc tissues (e.g., thymus tissue).
Preferred, however, are nucleic acid molcc~ c having se~luenr,~c at least
90%, 95%, 97%, 98% or 99% i~~ntif;~l to the nucleic acid seqllence shown in
Figure 1 [SEQ ID NO: 1] or to the nucleic acid sequence of the deposited cDNA
which do, in fact, encode a polypeptide having CK,13-15 protein activity. By "a
polypeptide having CK,B-15 activity" is intrn~le~ polypeptides ~ ~;Ling activitysimilar, but not l-~c~ss~ily iflenti~l, to an activity ofthe CK~-15 protein ofthe
invention (either the full-lenght protein or, plere,ably, the mature protein) asmeasured in a particular biological assay. Like other CC cy~Q~cinPc CK~-15
exhibits activity on monocytes, lyrnphocytes and neutrophils. However, un}ike
other known CC cytokin~c, CK,B-15 has been shown to be c ~ lesscd only in the
thymus. Th~t;ruit;, CK~-15 is particularly active in modlll~ting activities of cells
in the thymus, particularly early thymocytes. For example, stimlll3tion of earlythymocyte proliferation by CK,B-15 is assayed in a standard proliferation assay
(see, forinct~nc~, Spits et al. (1987) J. Tmmllnt~l. 139:1142; Dalloul et al. (1989)
Eur. J. Tmtmlnol 19:1985; Murphy et al., (1992) Ped. Res. 32:269; Ruggiero et
al, (1996) J. Tmmllnol. 156:3737). Briefiy, the assay invovles purifir~tion of
thymo~ytes from human thymus, plating them in media with or without CK,B-15,
CA 02258507 l998- l2- l4
- WO 97/48807 PCT/US96/10561
/~
and dt~t~l.ni~ ,g the change with elapsed time in the rate of proliferation or the
number of cells con~ ed to control cultures, by convrntion~l means
R~pr,~f~ ve cell lines could also be imployed in such assaS~s.
CK~-15 also tne~ tes the d;~e~ ialion of i~ LLy~ic T cell
5 p~ecu~;~ol~ into mature T-lymphocytes which are either at,B+ or y/~+ T cell
Lor ly~ o~iyl~s (as defined in Barcena et al. (1990) J. Exp. Med. 172:439)
This effect is .,.~1: ~1~ by mod~ ting (either inf7~r.in,~ or il-h;l,;~ ) the apoptosis
of sp~ fir, subsets of thymocytes within the thymus or by directly in~l1cing thedirrt;-~ ;o~- of a specific subset. In ~dflition CK~B-15 also directs the homing of
the ;.. ~ .. e Iymphocyte precursor to the thyumus for proper ln~.lu.aLion. This
activity is de~ol~ ted by in vitro chemotaxis assays using primary proge~
or , t;p. ~se~ e cell lines. CK~B- 15 also metii~t~s proper T-lymphocyte
maturation via the thynuc epith~ cells, for ~ le, by providing a co-
~l;"" ~l~loly signal for proliferation or di~. clllialion~ as shown by various in vitro
15 assays for human thyrno~yte proliferation or dirr~G.lliation (Ru~giero et al. ~1996
J. ~mmlmol 156:3737; Barcena et al. (1990) J. Exp. Med. 172:439; Singer et al.
(1990) J. Tmm~-nr l 144:2931).
The CK~-15 protein of the present invention also modulates colony
r~,l...n~;o~l of bone ..-~-uw progenitor cells, as does the macrophage infl~
20 protein related protein-2 ~RP-2). An in vifro colony forming assay for
p the extent of ~ ' ' on of myeloid progenitor cells is described in Youn
et al., The Journal of ~mmunology 155:2661-2667 (1995). Briefly, the assay
involves collectillg human or mouse bone marrow cells and plating the same on
agar, adding one or more growth factors and either (1) llnn~;led host cell-
25 supel..ala..l CQ~ g CK~-15 protein (or a c~nf7id~te polypeptide) or (2)
no~ n~r~ ed host cell-s.lpe .laLa..L control, and measuring the effect on colonyformation by murine and human CFlJ-granulocyte-macrophages (C~IJ-GM), by
human burst-ro--,--ng unit-erythroid (BFIJ-E), or by human CFU granulocyte-
elyLllloid-macrophage-meg~k~ryocyte (CFIJ-GEMM~.
CK~B-15 protein modulates early thymocyte proliferation and
difrt;.~ iaLion in a dose-dependent manner in the above-described assays. Thus,
"a polypeptide having CK~-15 protein activity" incl~ldes polypeptides that also
CA 022~8~07 1998-12-14
~ WO 97/488û7 J ,~ PCT/US96/10561
exhibit any of the same thymcoyte mod~ tin~ activities in the above-described
assays in a dose-dependent manner. Although the degree of dose- dependent
activity need not be id~ntic~l to that of the CK,B-15 protein, plt;re-~bly, "a
polypeptide having CK,B-15 protein activity" will exhibit s~ s~ ly similar dose-5 depP~n~l~nce in a given sctivity as conl~aled to the. CK,B-15 protein (i.e., the
t~ e polypeptide will exhibit greater activity or not more than about tenfold
less and, prer~.dl)ly, not more than about twofold less activity relative to the~,f~ ce CK~-15 protein).
Of course, due to the degeneracy of the genetic code, one of ordinary
10 skill in the art will imm~ tely recogni7e that a large number ofthe nucleic acid
molecules having a seql-~nce at least 90%, 95%, 97%, 98%, or 99% identic~l to
the nucleic acid sequence of the deposited cDNA or the nucleic acid seq~ence
shown in FiBre 1 ~SEO ID NO:I} will encode a polypeptide "having CKB-15
protein activity." In fact, since degenerate variants of these nucleotide sequences
15 all encode the same polypeptide, this will be clear to the skilled artisan even
without l~elrullllillg the above described co-llpa~ison assay. It will be further
l~O~;'~ in the art that, for such nucleic acid molecules that are not degellel~le
variants, a reasonable number will also encode a polypeptide having CK~-15
protein activity. This is because the skilled artisan is fully aware of amino acid
20 substit~ltinnc that are either less likely or not likely to significantly effect protein
function (e.g., repl ~c ing one aliphatic amino acid with a second aliphatic amino
acid).
For example, ~ nc e concerning how to make phenotypically silent
amino acid substitions is provided in Bowie, J. U., et al., "Deciphel;ng the
25 Mcc~e in Protein Sequences: Tolerance to Amino Acid Substitutions," Science
247:1306-1310 (1990), wherein the authors int1ic~te that there are two main
a~pl u&c,lles for studying the tolerance of an amino acid sequence to change. The
first m~othod relies on the process of evolution, in which mllt~tir~nC are either
accepted or rejected by natural selection. The second approach uses genetic
30 ~ P to introduce amino acid changes at specific positions of a cloned gene
- and sql~tinnc or screens to identify sequences that ".~ functionality. As the
authors state, these studies have revealed that ploteins are ~ Jlisill~ly tolerant of
CA 02258507 1998-12-14
~ WO 97/48807 PCTIUS96/10561
amino acid s~lbstitutiQns~ The authors fi~rther il rlir~te which amino acid r.hsln~,c~
are lilcely to be pe~ in ~ e at a certain position of the protein. For ~ c, most
buried amino acid residues require nonpolar side chains, whereas few features ofsurface side chains are generally conserved. Other such phenotypically silent
5 ~ ;Qn~ are described in Bowie, J.IJ., et al., supra, and the references cited
therein.
Vec~ors and Host Cells
The present i,.~.. l on also relates to vectors which include the icol~ted
DNA molecules of the present invention, host cells which are ~ y
nee,~d with the l~con-l)il-al-l vectors, and the production of CK,B-15
polypeptides or portions thereof by reco,l,billallt techn~ es
R~co...l~ constructs may be introduced into host cells using well
known te~hn;~uPc such as infection, tr~nc~uction~ re~liQn, transvection,
eleclloporation and l-~n~r~ ,-alion. The vector may be, for e~."F'~, a phage,
plasmid, viral or retroviral vector. Retroviral vectors may be rerlic?~tion
~"~ or replication defective. In the latter case, viral propagation generally
will occur only in co,nl,len~nting host cells.
The polynucleotides may be30ined to a vector c~ a select~1-le
marker for propagation in a host. Generally, a plasmid vector is introduced in apl~CilJ;~ ; such as a calcium phosphate pleci~ilale, or in a CO111~leA with a
chal~,ed lipid. If the vector is a virus, it may be p~r~ged in vitro using an
app.opliate p~ g cell line and then tr~n~dure(l into host cells.
Prerel.t;d are vectors co~llpli~ g cis-acting control regions to the
polynu~ e of interest. Al)pr~,p-iale tr~ns-acting factors may be supplied by thehost, supplied by a conlrlçm~ntin~ vector or supplied by the vector itself upon
introduction into the host.
~ certain preIelTed embo-lim~ntc in this regard, the vectors provide for
specific e~p-t;~iol~, which may be in~illcible and~or cell type-specific. Particularty
pr~.Gd among such vectors are-those irl~luc~ e by environm~nt~l factors that areeasy to manipulate, such as telllp~ re and nutrient additives.
CA 02258507 1998-12-14
- WO 97/48807 PCT/US96/10561
~5
~ p~;Oll vectors useful in the present invention include chromosom~1-
, episomal- and virus-derived vectors, e.g., vectors derived from bacterial
pl~en~ ~e, bacteriophage, yeast epieompe~ yeast chromosom~l plompnte, viruses
such as baculoviruses, papova viruses, vaccinia viruses, adenoviruses, fowl pox
5 viruses, pseudo. ~bies viruses and retroviruses, and vectors derived from
c >...hi~ ne thereof, such as coemirle and ph~gPm;r~e
The DNA insert shou1d be operatively linked to an ~p~ ,.iale
pro,..oter, such as the phage lambda PL promoter, the E. coli lac, trp and tac
p.~,l..oLe ~, the SV40 early and late promoters and promoters of retroviral LTRs,
10 to name a few. Other suitable promoters will be known to the skilled artisan. The
expression constructs will further contain sites for transcription
initi~tion,te,--- ~l~1ion and, in the transcribed region, a ribosome binding site for
tr~nC1~ion. The coding portion of the mature l-~s.i-i~ls ~,Ap-essed by the
constructs will include a translation initi~ting AUG at the bex;.~ g and a
15 te-...;..i1~;nn codon app-~"~-ialely positioned at the end ofthe polypeptide to be
As i".~ ~ the ~les:i.on vectors will pl-erel~bly include at least one
se1eç~ble marker. Such ~-lalht;l~ include dihydrofolate reduct~ee or neo~"ycill
or el~k~yoLic cell culture and tetracycline or ~mpirillin ~ e genes
20 for5;..1~ in E.coli andother b~ct~on~ Re~lesen~ e~,.~.lplesofapplopliale
hosts include bacterial cells, such as E. coli, Streptomyces and Salmonella
~,.,,..~, ..~". cells; fungal cells, such as yeast cells; insect cells such as Drosophila
S2 and Spodoptera Sf~ cells; animal cells such as CHO, COS and Bowes
l"f~ n".~ cells; and plant cells. Appropriate culture media and conditions for the
25 above-desc-i~ed host cells are known in the art.
Among vectors plere..ed for use in bacteria include pA2, pQE70,
pQE60 and pQE-9, available from Qiagen; pBS vectors, Phagescript vectors,
Bluescript vectors, pNH8A, pNH1 6a, pNHl 8A, pNH46A, available from
Sl-s~ ; and ptrc99a, pKK~ 3, pKK233-3, pDRS40, pRIT5 available from
30 Pl.~...a~ia. Among l,-ere..t:d eukaryotic vectors are pWLNEO, pSV2CAT,
pOG44, pXI 1 and pSG available from Stratagene; and pSVK3, pBPV, pMSG and
pSVL available from Pharmacia. Other suitable vectors will be readily appdl en~
CA 02258507 1998-12-14
- WO 97/48807 PCT/US96/10561
2Q
to the skilled artisan.
Among known bacterial promoters suitable for use in the present
invention include the E. coli lacI and ~acZ promoters, the T3 and T7 promoters,
the .~Pf p~ -oL~l-, the lambda PR and PL promoters and the bp promoter
S Suitable eukaryotic promoters inciude the CMV iln~e~liA~e early promoter, the
HSV Lhy,ll,line kinase promoter, the early and late SV40 promoters, the
pr~,,m(,l~.~ of retroviral LTRs, such as those ofthe Rous sareon,a virus (RSV), and
metaUothionçin promoters, such as the mouse mPt~ll~hion~in-I p-u-.,oter.
Introrl-~Gtion of the construct into the host cell can be eff~te-l by
10 Ggl~ m pho~ h~e Ir~ r~ n DEAE-dextran me~ ted ~ r~;Qn cAtiolli
lipid-...~d;~led ll;~n~re.;~ , electroporation, tr~n~d~lction, in~ection or other
m~th~ Such mPth~ are described in many standard laboratory mAm~ , such
as Davis et al., BASIC METHODS IN MOI,ECULAR BIOLOGY, (1986).
T.~s.,.i~,lion of the DNA encoding the polypeptides of the present
15 invention by higher t:uk~yuLes may be il~ sed by il~selL~, an ~nh~nr,er seqllçnr
into the vector. F.~ rf~b are cis-acting elP~m~nt~ of DNA, usually about from 10to 300 bp that act to ,ll~,r~se transcriptional activity of a promoter in a given host
cell-type. Examples of el-l-Al-ce. b include the SV40 ~nh~nrer, whlch is located on
the late side of the replication ûrigin at bp 100 to 270, the uylulllegalovirus early
20 pl ullwLe~ h~r~i, the polyoma e~ ~h~ l~r~. on the late side of the rçrlirDtiQn origin,
and adenovirus enh~ncçrs.
For secretion of the tr~n~l~te(l protein into the lu nen of the endoplasmic
reticlllllm~ into the pwi~laslnic space or into the extr~c~llnl~r environl.,t;nL,
a~plo~ Le secretion signals may be incorporated into the ~,A~J.essed polypeptide.
25 The signals may be endogenous to the polypeptide or they may be heterologous
signals.
The polypeptide may be ~ylc:~sed in a modified form, such as a fusion
protein, and may include not only secretion signals, but also ~ liti~n~l
h~iel~lo~,o--~ fim~tion~l regions. Thus, for inct~nce~ a region of additional amino
30 acids, particularly chalged amino acids, may be added to the N-terminus of the
polypeptide to improve stability and percietence in the~host cell, during
purification, or during subsequent h~n-lling and storage. Also, peptide moieties
CA 02258507 1998-12-14
- WO 97/48807 PCT/US96/10561 - -
may be added to the polypeptide to fs~r,ilit~te pUrifiC&~tioll
The CK,~-15 protein can be recovered and purified from reco.-lbin~.L
cell cultures by well-known meth~ 5 inr,l-lding ~.. o~ m sulfate or ethanol
precipitation, acid extraction, anion or cation ~S ~ e cluo,..~lography,
5 phosph~ crlltllQse ~ omalography, hydrophobic interaction cillum~Lography~
affir~ity ~,hlo... i~Qgraphy, hydroxylapatite cL~ tography and lectin
chlo.~ Q~raphy. Most preferably, high pe-rc,.-~ ce liquid clro,--alography
("HPLC") is employed for purification. Polypeptides of the present
invention include naturally purified products, products of r~l~~m C~I ~y~lLLc~iC10 plu~lu~s, and products produced by reco-,ll----~l~ techniqu~s from a prokaryotic
or ~yOlic host, inr~ ing, for ~ lç; bacterial, yeast, higher plant, insect and
",5""",~ cells. Dt;~~~ .g upon the host employed in a rec~ An~ production
procedure, the polypeptides of the present invention may be glycosylated or may
be non-glycosylated. In ~k~itioll, polypeptides of the invention may also include
15 an initial mor1ified m.~thionine residue, in some cases as a result of host-...e~ ted
processes.
CK,¢I S Polypeptides and Pep~ides
The invention further provides an isolated CK,B-15 polypeptide having
the amino acid sequence ~ncQded by the deposited cDNA, or the amino acid
seqll~nce in Figure 1 ~SEQ ID NO:2], or a peptide or polypeptide comprising a
portion of the above polypeptides. The terms "peptide" and "oligopeptide" are
con~;drred synonymous (as is commonly recognized) and each term can be used
interrh~n~ bly as the context l~luhes to indicate a chain of at least to arnino
acids coupled by peptidyl link~gçs The word "polypeptide" is used herein for
chains cc...~ g more than ten amino acid rçsiclues All oligopeptide and
polypeptide formulas or sequences herein are written from left to right and in the
direction from amino terminus to carboxy terminus.
It will be reco l~ed in the art that some amino acid sequence ofthe
CK~-15 polypeptide can be varied without significant effect ofthe structure or
~;.nl l;nl~ ofthe protein. If such di~t;,~ues in sequçnr.e are contemrl~te~l~ it should
CA 02258507 1998-12-14
~ W097l48807 PCT/US96/lOS61
D a
be rçmemhPred that there will be critical areas on the protein which deLe~ .c
activity. In general, it is possible to replace residues which form the tertiary~Ll~lclult;, provided that residues pe,rol"lillg a similar filnction are used. In other
inct~n~;eC~, the type of residue may be comr1et~ly ~l,impo,la,-L if the alteration
5 occurs at a non-critical region of the protein.
Thus, the invention further in~ludes variations of the CK,B-15
polypeptide which show s~ lA~ 1 CK~-15 polypeptide activity or which include
regions of CK,B-15 protein such as the protein portions riiccusse(l below. Such
mut~nt~ include deletions, insertions, inversions, repeats, and type s.,l.~ ;Qns10 (for eAal,~l~, s~ one hydrophilic residue for another, but not stronglyhy~llophilic for strongly hydr~Jphobic as a rule). Small changes or such "neutral"
arnino acid s~lbstitutiQnc ~,vill generally have little effect on activity.
Typical1y seen as conservative substitutions are the rerl~c~ ..lx one
for another, among the ~iirh~tic arnino acids Ala, Val, Leu and Ile; inLelcllal~ge of
15 the hydroxyl residues Ser and Th~, exchange ofthe acidic residues Asp and Glu,
substitllti~ n bt;lweel~ the arnide residues Asn and Gln, ~Y~h~r~e of the basic
residues Lys and Arg and repl~cen~ontc among the aromatic residues Phe, Tyr.
As i~ Aled in detail above, further ~~ nce conc~ which arnino
acid çh~r~eS are lilcely to be phenotypically silent (i.e., are not likely to have a
20 significant deleterious effect on a function) can be found in Bowie, J.U., et al.,
"Deciphering the Message in Protein Seq~lçnce~s: Tolerance to Amino Acid
SUbstit~ti~ n~," Science 247:1306-1310 ~1990).
The polypeptides of the present invention are prert;, ~hly provided in an
isolated form, and pl~r~l~hly are sul,,~ lly purified. A reComhin~ntly produced
25 version ofthe CK~-15 polypeptide can be subst~nti~ily purified by the one-step
method des~;-il,ed in Smith and Johnson, Gene 67:3140 (1988).
The polypeptides of the present invention include the polypeptide
encoded by the deposited cDNA inr1l1r1in5~ the leader, the mature polypeptide
Pl~-,ode l by the dP~po~it~A the cDNA minus the leader (i.e., the mature protein), the
30 polypeptide of SEQ ID NO:2 inrlut1in~ the leader, the polypeptide of SEQ ID
NO:2 minus the leader, as well as polypeptides which have at lèast 90% ~imi1s-rity,
more p.~rt;~ at least 95% ~ l;kuiL~, and still more p-~r~bly at least 97%, 98%
CA 02258507 1998-12-14
- W O 97/48807 PCT~US96110561
~3
or 99% ~ il&lily to those described above. Further poly~ Ps of the present
i~velllion include polypepti~P-s at least 80% i~Pnti~l, more ~l_fG~bly at least 90%
or 95% identir~l, still more p-c:r~ bly at least 97%, 98% or 99% id~ntir~l to the
.
poly~JIide Pnc4ded by the deposited cDNA, to the polypeptide of SEQ ID NO:2,
5 and also include portions of such polypeptides with at least 30 arnino acids and
- more preferably at least 50 arnino acids.
By "% ~imi~ ty~ for two polypeptides is intP.nded a similarity score
produced by coll~ing the amino acid sequences ofthe two po}ypeptides using
the Bestfit program (Wiscon~i" Sequçnce Analysis Package, Version 8 for Unix,
10 Genetics Comp~ltPr Group, University Research Park, 575 Science Drive,
Madison, WI 53711~ and the default settingc for de~e-l-".h"~g, similarity. Bestfit
uses the local homology algolillllll of Smith and Waterman (Advances in Applied
M~ ;.,S2:482-489,l981) to find the best se~tnpnt of ~ l~ily between two
seqllPnces,
By a polypeptide having an amino acid sequP-nce at least, for; , 'e,
95% "idP'ntir~l" to a ~c;r~ ce amino acid sequence of a chemokine ,B-15
poly~Lide is intrn~iç~l that the amino acid seqllrnr,e of the polypeptide is idPntiC~l
to the rerer~"ce sç~ ,ce except that the polypeptide sequ~pn~e may include up tofive amino acid alterations per each 100 amino acids of the reference amino acidof the chpmokine ,B-15 polypeptide. In other words, to obtain a polypeptide
having an amino acid sequçnce at least 95% idçntir~l to a .~r~;lence amino acid
sequPnrP; up to 5% of the amino acid residues in the reference sequPnce may be
deleted or ~ .led with another amino acid, or a number of amino acids up to
5% ofthe total amino acid residues in the reference sequence may be inserted into
the ,er~l1ce sP~nPnr~e These alterations ofthe reference sequence may occur at
the amino or carboxy ~e",~ al positions of the reference amino acid sequence or
~ywlle~ ~ between those terrninal po~itir~n~ interspersed either individually among
s in the ,~;r~,~,ce sequPnce or in one or more cQnti~Qll~ groups within the
lef~.lce sequPnre.
As a practical matter, whether any particular polypeptide is at least
90%, 95%, 97%, 98% or 99% identical to, for in~t~ncP., the amino acid sequence
shown in Figure 1 tSEQ ID NO:2] or to the amino acid sequence encoded by
CA 02258507 1998-12-14
~ WO 97/48807 PCT/US96/10561
a~
o~ ,rl cDNA done çan be d~le~ ed conv~ntion~lly using known co~ ul~,
programs such the Bestfit program ~ CQl~ Seq~lon~e Analysis F'acl~ge,
Version 8 for Unix, Genetics C~o-,~uler Group, University Research Park 575
Science Drive, M~Aicon WI 53711. When using Bestfit or any other sequ~nce
5 ~li~m~nt program to determine whether a particular sequ~nce is, for in~t~n~
9s% id~nti~l to a rer~;;r~"lce seqU~nce according to the present invention, the
parameters are set, of course, such that the pel cenl&ge of identity is c~lc~
over the fi~ll length of the reference amino acid sequence and that gaps in
h~lm~ y of up to 5% ofthe total number of amino acid residues in the It r~ "lce
10 se~ n~e are allowed.
As des~lil ed in detail below, the polypeptides of the present invention
can be used to raise polyclonal and monoc1onal antibodies, which are useful in
~ .o~l;c assays for cletecti~ CK,B-15 protein e,~ ion as desc-;l ed below or
as ~ o~ t~ and antagonists capable of çnh~n~ing or inhibiting CK~-15 protein
15 function. Further, such polypeptides can be used in the yeast two-hybrid system
to "capture" CK~-15 protein binding plul~:L~, which are also Ç~n~ te agonist andantagonist accol-Llg to the present invention. The yeast two hybrid system is
described in Fields and Song, Na~ure 340:245-246 (1989).
In another aspect, the invention provides a peptide or polypeptide
20 COl~pli~ g an epitope-bearing portion of a polypeptide of the invention. The
epitope of this polypeptide portion is an immllnogenic or ~ntig~n;c epitope of apoly~lide ofthe invention. An "immllnogenic epitope" is defined as a part of a
protein that elicits an antibody .t::~onse when the whole protein is the imm~mo~en
These ;~ ogenic epitopes are believed to be confined to a few loci on the
25 mf~CCIlle On the other hand, a region of a protein mr lecllle to which an antibody
can bind is defined as an "antigenic epitope." The number of immlmogenic
ep;lQpe..S of a protein generally is less than the number of ~nti~nic epiLopes. See,
for in.~t~nc~, Geysen, H. M., Meloen, R. H. and Barteling, S. J. (1984) Use of
peptide s~ esis to probe viral ~nti~n~ for epitopes to a resolution of a single
amino acid. Proc. Natl. Acad. Sci. USA 81:3998-4002.
As to the se~ection of peptides or polypeptides bearing an ~ntig~n:c
epitope ~l.e., that contain a region of a protein molecule to which an antibody can
CA 02258507 1998-12-14
- WO 97/48807 PCT/US96110S61 - -
~3~
bind), it is well known in that art that relatively short synthetic peptides that mirnic
part of a protein s~ e are routinely capable of eliciting an antiserum that reacts
with the partially ",;.":~.L ~d protein. See, for il~xl~nce~ tcliff~p~ J. G., Shinn~
T. M., Green, N. and Learner, R. A. (1983) Antibodies that react with
predetermined sites on proteins. Science 219:660-666. Peptides capable of
-' ., protein-reactive sera are frequently r~esenled in the p~ Uy sequPnce
of a protein, can be characterized by a set of simple chem;c~l rules, and are
c~ ..P~l neither to imm-lnodominant regions of intact proteins (i.e., immlmogenic
epitopes) nor to the amino or carboxyl terminals. Peptides that are c~ ,lely
10 h~rdlo~ and those of six or fewer residues general1y are inPffective at in-~u-~.ing
antibodies that bind to the mimicked protein; longer, so}uble peptides, especially
those co~ P proline rçcidlles usually are effective. Sutcliffe et al., supra, at661. For inet~nf ~o~ 18 of 20 peptides deeigned according to these ~li~lPlin~os7CO~ 8-39 residues covering 75% of the sequ~nre of the influen7~ virus
15 hçm~ tinin HAl polypeptide chain, induced antibodies that reacted with the
HAl protein or intact virus; and 12/12 peptides from the MuLV polymerase and
18/18 from the rabies glycoprotein in(1uced antibodies that plec;~ ed the
respective plvl~ls.
~ Anti~n:, epitope-bearing peptides and polypeptides of the invention are
20 thc~cr~le useful to raise antibodies, in~lllrling monoclonal antibodies, that bind
speçific~lly to a polypeptide of the invention. Thus, a high proportion of
hybridomas obt~in~d by fusion of spleen cells from donors immuni7~d with an
antigen epitope-bearing peptide generally secrete antibody reactive with the native
protein. .S~tcliffe et al., supra, at 663. The antibodies raised by ~ntig~onic epitope-
25 bearing peptides or polypeptides are useful to detect the mim;~ d protein, ands~ntiho~;~s to .liÇrelcllL peptides may be used for tracking the fate of various regions
of a protein ~ or which undergoes posttr~n~l~tion processing. The peptides
and anti-peptide antibodies may be used in a variety of qualitative or q~ eassays for the mimicked protein, for inct~nce in coll.~,cLiLion assays since it has
30 been shown that even short peptides (e.g., about 9 arnino acids) can bind anddisplace the iarger peptides in immm~opl~ iL~Lion assays. ~ See, for i~ n~
Wllson, I. A., Nirnan, H. L., ~ought.on, R. A., Cherenson, A. R., Connolly, M. L.
CA 02258507 1998-12-14
~ WO 97/48807 PCT/US96/lOS61 - -
2G~
and Lerner, R. A. (1984~ The structure of an ~nti~Pnic dele-",ill~,l in a protein.
Cell 37:767-778 at 777. The anti-peptide antibodies of the invention also are
useful for purific~tion of the mimi-~Pd protein, for in~t~n~e~ by adsorption
chlo.~ ography using methods well known in the art.
Antigenic epitope-bearing peptides and polypeptides of the invention
desi~ne~ accord"~g to the above ~lidPlinPs preferably contain a seq~lenr,e of atIeast seven, more p~ ~ly at least nine and most ~ rt;lably ~.lweell about 15 to
about 30 amino acids cont~ined within the amino acid seq~lence of a polypeptide
ofthe invention. However, peptides or polypeptides con~p.;sil~3 a larger portion10 of an amino acid sequence of a polypeptide ofthe invention, co~ about 30
to about 50 amino acids, or any length up to and inc~l-ding the entire an~ino acid
sequence of a polypeptide of the invention, also are conci~çred epitope-bearing
peptides or polypeptides of the invention and a}so are useful for inrlucing
antibodies that react with the mimi~Pd protein. Plert:l~bly, the amino acid
se lllPnce ofthe epitope-bearing peptide is sPl~cted to provide bul,sla"l;al solubility
in ~q~ solvents ~I.e., the sequence in~l~ldes relatively hydrophilic residues and
highly hy(ll .,phob ~ sequences are preferably avoided); and sequences co. .~ g
proline residues are particularly preferred.
The epitope-bearing peptides and polypeptides of the invention may be
produced by any conventional means for malcing peptides or polypeptides
inelu-1in~ lt;colllb;nall~ means using nucleic acid molecules ofthe invention. For
in.cts-nrl9, a short epitope-bearing arnino acid sequence may be filsed to a larger
polypeptide which acts as a carrier during recombinant production and
purific~tion as well as during immllni7~tion to produce anti-peptide antibodies.Epitope-bearing peptides also may be synthPsi7ed using known methods of
chemical synthesis. For in.ct~nce, Houghten has descri~ed a simple method for
synthesis of large numbers of peptides, such as 10-20 mg of 248 different 13
residue p~Lides repl ese,~ single amino acid variants of a se~npnt of the EIAl
polypeptide which were p.epaled and characterized (by ELISA-type binding
studies) in less than four weeks. Hol~gh~Pn, R. A. (1985) General method for
the rapid solid-phase synthesis of large numbers of peptides: ~,ecir,cily of antigen-
antibody il,Len~lion at the level of individual amino acids. Proc. Natl. Acad. Sci.
CA 02258507 1998-12-14
- WO 97/48807 PCT/US96/10561
d~t7
USA 82:5131-5135. l~his ".Sim--lt~neoue Multiple Peptide Sy"lhes;s (SMPS)"
process is further described in U.S. Patent No. 4,631,211 to Houghten et al.
(1986). In this procedure the individual resins for the solid-phase ~"l}-cs;s ofvarious pc~,lides are eQ~ in se~ le solvent-p~ ~~bl~ ~ nçl ~tc en~bling the
5 optimal use ofthe many id~ntif~l repetitive steps involved in solid-phase mPthnrie
A co ,'~: Iy manual procedure allows 500-1000 or more syntheses to be
con~ucte~ eim-llt~nPo-lely. ~o~ghtP!n et al., supra, at 5134.
Epitope-bearing peptides and polypeptides of the invention are used to
induce ~ otlies according to methnrls well known in the art. See, for inetAn~
10 Sutcliffe et al., s~pra; Wilson et 1., supra; Chow, M., Yabrov, R., Bittle, J.,
Hogel, J. and R~ltimnre, D., Proc. Natl. Acad. Sci. USA 82:910-914; and Bittle,
F. J., Fry, C. M., Rowlands, D. J., Brown, F., Bittle, J. L., ~Ioughten, R. A. and
Lerner, R. A. (1985) J. gen. Virol. 66:2347-2354. Generally, animals may be
;.. -.:~ with free peptide; however, anti-peptide antibody titer may be boosted
15 by coupling ofthe peptide to a maclo,llolecular carrie~, such as keyhole limpet
hc -~ (KLE3) or tetanus toxoid. l~or inet~nce~ peptides coll~A~ g cysteine
may be cD L~ ' to carrier using a linker such as m-m~ midQbenzoyl-N-
hydroxy~ de ester (~S), while other peptides may be collFI~ to carrier
using a more general linking agent such as glutaraldehyde. Animals such as
20 rabbits, rats and mice are immlmi7~.d with either free or carrier-coupled peptides,
for ;~ - r,, by i~,LI~e~iLoneal andlor intradermal injection of Pml~leione col~l Ai..;-~g
about 100 ~lg peptide or carrier protein and Freund's adjuvant. Several booster
~ ~c ~ may be needed, for i~ ~, at intervals of about two weeks, to provide
a useful titer of anti-peptide antibody which can be detecte~, for ~Y~mple, by
25 ELISA assay using free peptide adsorbed to a solid surface. The titer of anti-
peptide antibodies in serum from an imml~ni7ed animal may be increased by
selection of anti-peptide antibodies, for in.~t~neÇ, by adsorption to the peptide on
a solid ~UppOl L and elution of the selected antibodies accol di-~g to methods well
known in the art.
T.---.. --l~o~l- ~ epitope-bearing peptides ofthe invention, i.e., those parts
of a protein that elicit an antibody response when the whole protein is the
immllnogen, are identifie~l accoldillg to methods known in the art. For i~ cc~,
CA 02258507 1998-12-14
- WO 97/48807 PCT/US96110561
Geysen et al., 1984, s2~pra, di~clc~s~ a procedure for rapid concurrent ~.,Lhesis on
solid supports of hundreds of peptides of sufflcient purity to react in an enzyme-
linlced ;.. -~os~sll,e.-l assay. Interaction of s~ .PSi~ed peptides with antibodies
is then easily d~,tGeled without removing them from the support. In this matmer
a peptide bearing an imm~nogenic epitope of a desired protein rnay be idpntifi~
routinely by one of ordinary skill in the art. For in~t~ncp~ the imml.n.-logically
illlpol l~lL epitope in the coat protein of foot-and-mouth disease virus was located
by Geysen et al. with a resolution of seven amino acids by synthesis of an
ov~ia~p~g set of all 208 pos~ible he~ ides covering the entire 213 amino acid
10 seq~ence ofthe protein. Then, a complete repl~cement set of peptides in whichall 20 amino acids were substituted in turn at every position within the epitopewere syr~tllp~ and the particular amino acids conre-,ing specificity for the
reaction with antibody were determined. Thus, peptide analogs of the epitope-
bearing peptides of the invention can be made routinely by this method. U.S
Patent No. 4,708,781 to Geysen (1987) further desc,ibes this method of
identifying a peptide bearing an immllnogenic epitope of a desired protein.
FurtherstiU, U.S. Patent No. 5,194,392 to Geysen (199Q) describes a
general mf~thod of ~~tstecting or deterrnining the seqll~nce of molu ..~ (amino
acids or other compounds) which is a topological equivalent ofthe epitope (i.e.,20 a "r ~ c") which is comple~ . y to a particular paratope (antigen binding
site) of an antibody of interest. More generally, U.S. Patent No. 4,433,092 to
Geysen (1989) desc.ibes a method of detectin~ or detern~ining a sequenre of
nn.. .t.. ~ which is a topog~ c~l equivalent of a ligand which is comple- ~*-~ yto the ligand binding site of a particular l~tol of interest. Similarly, U.S. Patent
2~ No. 5,480,971 to Ho~ht~n, R. A. et al. (1996) on Perallylated Oligopeptide
Mixtures ~ ,tos~ linear Cl-C,-allyl peralkylated oligopeptides and sets and
libraries of such pept~ as well as mtothods for using such oligopeptide sets andlibraries for d~le~ g the sequence of a peralkylated oligopeptide that
p~ ially binds to an acceptor mf 'cc -~e of interest. Thus, non-peptide analogs
30 of the epitope-bearing peptides of the invention also can be made routinely by
these methods.
The entire disclosure of each document cited in this section on
CA 022~8~07 1998-12-14
~ Wo 97/48807 PCT/US96/10561
a9
"Polypeptides snd Peptides" is hereby incorporated herein by ..,f~,rcnce
Thymus-Re~ote~1 D~sorder Diagnosis
The present inventors have discovered that CK13-15 is eA~"essed only
in thymus tissue. For a number of thyrnus-related disorders, subst~nti~lly altered
(increased or decl~sed) levels of CK,B-15 gene e~,lession can be detected in
thymus tissue or other cells or bodily fluids (e.g., sera, plasma, urine, synovial fluid
or spinal fluid) taken from an individual having such a disorder, relative to a
"~ d~u-l ' CK,B-15 gene eAI,Iession level, that is, the CK,B-15 eA~,cs,ion level in
thymus tissue or bodily fluids from an individual not having the thymus disorder.
Thus, the invention provides a diagnostic method useful during ~liagno~i~ of a
thymus disorder, which involves measuring the e A~les~ion level of the gene
encoding the CK,B-15 protein in thymus tissue or other cells or body fiuid from an
individual and con~ g the measured gene ~ ession level with a standard
CK,B-15 gene ~,A~lession level, whc~clly an increase or decrease in the gene
e A~ression level compaed to the standard is indicative of a thymus disc,lder.
By individual is intpn~led m~mm~ n individuals, prerel2.bly hllrns-n~
By "measuring the ~ "e.,.,ion level of the gene encoding the CK~-l S protein" isintenrled q~ it~tively or qu~ l;vely measuring or e~ the level of the
CK~-15 protein or the level of the mRNA encoding the CK13- 15 protein in a firstbiological sample either directly (e.g., by determining or estim~ting absolute
protein level or rnRNA level) or relatively (e.g., by comparing to the CK~-15
protein level or m~NA level in a second biological sample). P,crel~bly, the CK,B-
15 prote;n level or mRNA level in the first biological sarnple is measured or
ed and compared to a standard CK,B-15 protein level or mRNA level, the
d~l being taken from a second biological sample obtained from an individual
not having the disorder or being de~ by averaging levels from a population
of individuals not having a disorder of the thymus. As will be appreciated in the
art, once a ~ d~.l CK,B-15 protein level or mRNA level is known, it can be used
repe~te-1ly as a standard for co"lp~ison.
By "biological sample" is int~n~ed any biological sample obt~ined from
CA 02258507 1998-12-14
- WO 97/48807 PCT/US96/10561
~7~
an individual, body fluid, cell line, tissue culture, or other source which conL~ls
CK,B-15 protein or mRNA. As indicated, biological s~mplçs inc}ude body fluids
ch as sera, p1asma, urine, synovial fluid and spinal fluid) which contain secr~led
mature CK13-15 protein, thymus tissue, and other tissue sources found to express5 CK~3-15 or a CK~-15 lece~,lur. Methods for obt~ininP~ tissue biopsies and bodyfluids from ...~ are well known in the art. Where the biological sample is to
include rnRNA, a tissue biopsy is the prert;" ed source.
The present invention is useful for diagnosis or tre;ltm~nt of various
thyrnus-related disorders in n~ lc, prefel~ly hllm~n~ Such disorders include
10 the following tumors and cancers, hypoactivity, hy~ ity, atrophy,
e~ l~,~,...c: ,1 of the thymus, and the like. Other disorders include disregulation of
T-lymphocyte selection or activity and would include but not be limited to
disorders involving ~toimml~nity~ arthritis, lellkP-mi~c~ Iymphomas,
immlln~s~lpple",;on, sepsis, would h~iing, acute and chronic infl*.. ~;on, cell
mP~ t.o,d i~ y~ humor immlmity, THl/TH2 imh~l~nce~ and the like.
Total cellular RNA can be isolated from a biologiç~l sample using any
suitable technique such as the single-step ~l~ni~inil~m-thiocyanate-phen
chloroforrn method described in Chomç7ynski and Sacchi, Anal. Biochem.
162:156-159 (1987). Levels of mRNA encoding the CK13-15 protein are then
assayed using any approp- iate method. These include Northern blot analysis, S 1mlrlea~e ~ pping, the polymerase chain reaction (PCR), reverse l,~nsc~ ion in
coml)il.aLion with the polymerase chain reaction (RT-PCR), and reverse
scli~lion in coll.t,inalion with the ligase chain reaction {RT-LCR).
Northern blot analysis can be pelroll-led as des~;lil.ed in Harada et al.,
Cell 63:303-312 (1990). Briefly, total RNA is p,el)~t;d from a biological sampleas df ~;. ;beA above. For the Northern blot, the RNA is del~lul t;d in an ~pl~l Upl iale
buffier (such as glyoxal/dimethyl sulfoxide/sodium phosrh~te buffer~, subjected to
agarose gel el~~ phoresis~ and ll~ r~;llt;d onto a nitrocell--lose filter. After the
RNAs have been linked to the filter by a W linker, the filter is prehybridized in a
30 solution cc~ .;~ fol~ ide, SSC, Denh~J~'s solution, denatured salmon
spenn, SDS, and sodium phosph~te buffer. CK~ 15 protein cDNA labeled
ac~lding to any appropriate method (such as the 32P-multiprimed DNA labeling
CA 02258507 1998-12-14
- WO 97/48807 PCT/US96/10561
3~
system ~A...~ I,~.,)) is used as probe. After hybri~ tion overnight, the filter is
washed and exposed to x-ray film. cDNA for use as probe accordi-lg to the
present invention is described in the sections above and will prere.~ly at least 15
bp in length
S Sl llla~illg can be pelru-lned as described in Fu3ita et al., Cell 49:357-
367 (1987). To prepare probe DNA for use in S 1 Illapping, the sense strand of
above~ A1 cDNA is used as a t~ e to s~ p~ e labeled ~ e~qe DNA.
The ~ ;sel~e DNA can then be tiiPP~sted using an app.up.i~le restriction
~n~on~ Irlez~cs to gene ~.~e further DNA probes of a desired length. Such ~ ~ ~l ;se~-~e
probes are useful for vi~ i7ing protected bands coll~onding to the target
nRNA (i.e., mRNA enroding the CK~-15 protein). Northern blot analysis can be
pe.r~,l.--ed as described above.
P~ert:.~bly, levels of mRNA encoding the CK~-15 protein are assayed
using the RT-PCR method described in Makino ef al., Technique 2:295-301
( 1990). By this methorl, the radioactivities of the "~mr~ir,on.~" in the
polyacrylamide gel bands are linearly related to the initial concé--L-~lion of the
target mRNA. Briefly, this method involves adding total RNA i~Q!~ted ~om a
biological sample in a reaction mixture co~ p a RT primer and ap~,up-iale
buffer. APter i~ b~ g for primer ~nne~ling, the mixture can be s~lpp!~ nted
with a RT buffer, dNTPs, DTT, RNase inhibitor and reverse Llalls.,l;~se. After
inrl lb~tion to achieve reverse ~ s~ ion of the RNA, the RT products are then
sub3ect to PCR using labeled primers. Alternatively, rather than labeling the
p.im~"s, a labeled dNTP can be incl~fled in the PCR reaction mixture. PCR
;Oll can be Iré~rc~ned in a DNA thermal cycler accor~ing to conve ILional
t~ es. After a suitable number of rounds to achieve ~mrlifir~tion, the PCR
reaction mixture is el~iLIuphG-c;sed on a polyacrylamide gel. After drying the gel,
the ~ ~ ity of the app. Opl iaLe bands (corresponding to the mRNA encoding
the CK,B-15 protein) is 4ua~ ed using an im~ in~ analyzer. RT and PCR
reaction ingredients and conditions, reagent and gel concentrations, and labeling
methods are well known in the art. Variations on the RT-PCR method will be
~ppalellL to the skilled artisan.
Any set of oligonucleotide primers which will amplify reverse
CA 02258507 1998-12-14
- WO 97148807 PCT/US96/10561
3~
sclil~d target tnRNA can be used and can be de-cig1led as desc,il,cid in the
sectiorl~ above.
Assaying CK,B-15 protein levels in a biological sample can occur using
any art-known method. Plerellt;d for assaying CKp-15 protein levels in a
5 biolo~c~l sample are antibody-based technifluçc For eY~mple, CK~-15 protein
~ ,;on in tissues can be studied with Ç~ ;c~l immlmnhictological n~-~.thoflc
In these, the specific recognition is provided by the plilll~y antibody (polyclonal
or mnnnrl~n~l) but the seC.~ y detection system can utilize fluorescenl~ el~yllle,
or other conjugated secondary antibodies. As a result, an immunohi~tQlogical
0 ~jlAi~ of tissue section for pathological c~ ;on is obL~ined. Tissues can
also be ~A~ ed~ e.g., with urea and neutral detergent, for the liberation of CK~-
15 protein for Western-blot or dot/slot assay (J~lk~nrn, M., et aL, J. Cell BioL101:976-985 (1985~; J~lk~nPn, M., et al., J. Cell . BioL 105:3087-3096 (1987)).
In this technique, which is based on the use of c~tiQnic solid phases, q~ntit~tiQn
15 of CK~-15 protein can be accompli~hed using i~ ted CK~-15 protein as a
d~d. This terhni~lue can also be applied to body fluids. With these s~ c,
a molar cf~ ion of CK~-15 protein will aid to set standard values of CE~,13- 15
protein content for di~el elll body fluids, like serum, plasma, urine, synovial fluid,
spinal fluid, etc. The norma} appe~ ce of CK,13-15 protein ~mount~ can then be
20 set using values from healthy individuals, which can be colllpal ed to those obtained
from a test subject.
Other antibody-based methods useful for detectin~ C~K13-15 protein
levels include immlmo~s~ys~ such as the enz)nne linked imml-nrJsQrbent assay
(ELISA) and the l, /l~ ""-mo~ y ~RIA). For; . '-, CK13-15 protein-specific
25 mnnnrlon~ il.odies can be used both as an immlmo~fliolb~llL and as an enzyme-labeled probe to detect and quantif~ the CK~-15 protèin. The amount of CK,B-15
protein present in the sample can be calculated by ler~l-,nce to the amount present
in a standard pl~p~Lion using a linear regression con~puLer algorithm. Such an
ELISA for ~ . a tumor antigen is described in Iacobelli e~ al., Breast Cancer
Research ond T~uh.. _,.l 11:19-30 (1988~. In another ELISA assay, two distinctspecific monoclonal antibodies can be used to detect CK~-15 protein in a body
fluid. In this assay, one of the antibodies is used as the imm~mo~sQrbent and the
CA 02258507 l998- l2- l4
- WO 97/48807 PCT/US96/10561
other as the enzyme-labeled probe.
The above techniques may be cnr~dl~cted ~ nti~lly as a "one-step" or
"two-step" assay. The "one-step" assay involves coînt~rti~ CK~-15 protein with
immobilized antibody and, without washing, cont~ctin~ the ~,~lu-G with the
S labeled antibody. The "two-step" assay involves washing before cont~rti~ the
~ub~lulG with the labeled antibody. Other conventional m~thnds may also be
employed as .sl~it~hle It is usually desirable to immobilize one cc,l,lpollel-l ofthe
assay system on a support, thereby allowing other components of the system to bebrought into contact with the component and readily removed from the sample.
.S~it~ble enzyme labels inrlu(le~ for ~x~mpl~ those from the oxidase
group, which catalyze the production of hydrogen peroxide by reacting with
substrate. Glucose oxidase is particularly pr~fel I t;d as it has good stability and its
substrate (glucose) is readily available. Activity of an oxidase label may be assayed
by measuring the concentration of hydrogen peroxide formed by the enzyme-
labeled antibody/substrate reaction. Besides enzymes, other suitable labels include
radioisotopecs, such as iodine (l25I, l2lI), carbon (I~C), sulfur (35S), tritium (3H~,
indium ("2In), and tec~ eli~lm (99mTc), and fluorescent labels, such as fluole~ccin
and rho-l~tnine~ and biotin.
In ~d~iition to assaying CK,B-15 protein levels in a biolo~c~l sample
obtained from an individual, CK,B-15 protein can also be de~ected in vivo by
im~S~in,~ Antibody labels or markers for in vivo im~ in~ of CK~-15 protein
include those detect~ble by X-radiography, N~ or ESR. For X-radiography,
s -it~hle labels include radioisotopes such as barium or cesium, which emit
detectable ra~ tion but are not overtly harmful to the sub3ect. Suitable il~hcl~for NMR and ESR include those with a detect~hle characteristic spin, such as
deuterium, which may be incorporated into the antibody by labeling of nutrients
for the relevant hybridoma.
A CK~-15 protein-specific antibody or antibody portion which has been
- labeled with an ~pr~ Le dc~e~blG im~ging moiety, such as a radioisotope (for
; ~ ~e7 13~ 2In, 99mTc), a radio-opaque substance, or a material detect~ble by
nuclear m~,~n~tic leSOnallCe, iS introduced (for example, palc,llelally,
~hc lt~npou~ly or il~h~cliloneally) into the n.~-.. ~l to be .oY~mined for a thymus
-
CA 02258507 1998-12-14
-WO 97/48807 PCT/US96/10561
3~
disorder. It will be understood in the art that the size of the subject and the
im~ging system used will determine the quantity of im~in~ moieties needed to
produce t~ o.ctic images. In the case of a radioisotope moiety, for a human
subject, the quantity of r~ ctivity injected will normally range from about 5 to5 20 m~ cllries of 99n'Tc. The labeled antibody or antibody portion will then
~.lerel~..lially aec~m~ te at the loc~tiorl of cells which contain CKB-15 protein.
In vivo tumor im~in~ iS described in S. W. Burchiel ef al.,
"Tmmllnoph~l..Acokinetir.s of Radiolabeled Antibodies and Their Portions"
~Chapter 13 in Tumor I~ . The Radiochemical Detec~ion of Cancer, eds.,
10 S. W. Burchiel and B. A. Rhodes, Masson Publishing Inc. (1982)).
CK13-15-protein specific antibodies for use in the present invention can
be raised against the intact CK,B-15 protein or an ~ntigçnic polypeptide portionthereof, which may presented together with a carrier protein, such as an albun~in,
to an animal system (such as rabbit or mouse) or, if it is long enough (at least15 about 25 amino acids), without a carrier.
As used herein, the term "antibody" (Ab) or 'lmono~ antibody"
(Mab) is meant to include intact molecules as well as antibody portions (such as,
for . p'e Fab and F(ab')2 portions~ which are capable of specifically binding toCK~-15 protein. Fab and F(ab')2 portions lack the Fc portion of intact antibody,20 clear more rapidly ~om the circulation, and may have less non-specific tissuebinding of an intact antibody (Wahl et al., J Nucl. Mecl 24:316-325 (1983)).
Thus, these portions are pl ~rel I ed.
The antibodies of the present invention may be plepalcd by any of a
variety of metho~ls For example, cells c~.yles~ g the CK~-15 protein or an
25 ~ ~ - portion thereof can be ~iminiqtered to an animal in order to induce theproduction of sera co..l~;..;..~ polyclonal antibodies. In a pl~r~llcd method, aprepa.~lion of CK,B-15 protein is prepared and pur;fied as described above to
render it ~ b~lnl~l;Ally free of natural co~ln~ ; Such a p,~a~lion is then
introduced into an animal in order to produce polyclonal antisera of greater
30 specific activity.
In the most p-~rt:llc;d method, the antibodies ofthe present invention are
monoclonal antibodies (or CK~-15 protein binding portions thereof). Such
CA 022~8~07 1998-12-14
~ W~ 97/48807 PCT/US96/10561 -
3S---
monnl~tc~n~l ~nt bc '- - - can be p~ e~ Gd using hybridoma te~hnnl~gy (Koh1er et al.,
Na~re 256:495 (1975); Kohler et al., Eur. J. ImmunoL 6:511 (1976); Kohler et
al., Eur. ~ ~ .. ~1. 6:292 (1976); TT;.~.. ~.lil~ e~al., In: M~ noclonalAntibodies
and T-Cell Hybr~ 7~, Elsevier, N.Y., pp. 563-681 (1981)). In general, such
S procedures involve immllni~ing an animal (preferably a mouse) with a CK~-15
protein antigen or, more plerel'ably~ with a CK~-15 protein t,A~le~hl~ cell.
Suitable cells can be recognized by their capacity to bind anti-CK~-15 protein
antibody. Such cel}s may be cultured in any suitable tissue culture m~-lillm;
however, it is plerel~ble to culture cells in Earle's modified Eagle's ...e~
10 supple-.~ ed with 10% fetal bovine serum (inactivated at about 56~C), and
suppl~ n~nted with about 10 ~lg/l of non.?~nti~l amino acids, about 1,000 U/ml
of p~ ~ '1in and about 100 ,ug/ml of ~lleptolllycin~ The splenocytes of such mice
are extracted and fused with a suitable myeloma cell line. Any suitable myeloma
cell line may be employed in accordance with the present invention; however, it is
15 preferable to employ the parent myeloma cell line (SP2O), available from the
Arnerican Type Culture Collection, Rockville, Maryland. After fusion, the
rçslllting hybridoma cells are selectively ~--~ i"e(l in HAT me~ m~ and then
cloned ~oy limiting dilution as described by Wands et al. ~G~stroenterolof~y 80:225-
232 (1981)). The hybridoma cells obtained through such a selection are then
20 assayed to identify clones which secrete antibodies capable of binding the CKB- 15
~ntigt~.n
Alternatively, additional antibodies capable of binding to the CK,B-~5
protein antigen may be produced in a two-step procedure through the use of anti-idiotypic antibodies. Such a method makes use of the fact that antibodies are
25 IL~;II~I~eS 9~ ;g~ , and therefore it is possible to- obtain an antibody which binds
to a second antibody. In accordance with this method, CK,B-15 protein specific
antibodies are used to immllni7e an animal, preferably a mouse. The splenocytes
of such an animal are then used to produce hybridoma cells, and the hybridoma
cells are s.,reened to identify clones which produce an antibody whose ability to
30 bind to the CK,B-15 protein-specific antibody can be blo~ d by the CK~-15
protein antigen. Such antibodies co--lp-ise anti-idiotypic antibodies to the CK,~-15
protein-specific antibody and can be used to immllni7e an animal to induce
CA 02258507 1998-12-14
- WO 97/48807 PCT/US96/10561
36
formation offurther CK,B-15 protein-specific antibodies.
It will be a~ ,ialed that Fab and F~ab')2 and other portions of the
antibodies of the present invention may be used accol.li.~g to the mP.tho-l~
disclosed herein. Such portions are typically produced by proteoiytic cleavage,
S using c;l~y~eS such as papai~ (to produce Fab portions) or pepsin (to produce
F~ab')2 portions). Alternatively, CK~-15 protein-binding portions can be produced
through the appl;c~tiQn of recomhin~nt DNA technology or through synthetic
r3~ 1, y.
Where in vivo im~ging iS used to detect e--h~ .cd levels of CK,~-15
10 protein for ~I ~gnr~c ~ in h~ ne, it may be preferable to use ' ~ ed" chirneric
mnnnrl~ln~l antibodies. Such antibodies can be produced using genetic constructsderived from hybridoma cells producing the monoclonal antibodies described
above. M~th~tle for pro(l~-ctng chimeric antibodies are known in the art. See, for
review, Morrison, Science 229:1202 (1985); Oi e~ al., BioTec*niques 4:214
(1986); Cabillyetal., U.S. PatentNo. 4,816,567; Ts~n~ chi etal., EP 171496;
Morrisonetal., EP 173494; Neubergeretal., WO.8601533; Robinson etal., WO
8702671; Boulianne et al., Nature 312:643 (1984); Neuberger et al., Nature
314:268 (1985).
Further suitable labels for the CK,B- 15 protein-specific antibodies of the
20 present invention are provided below. Examples of suitable enzyme labels include
malate dehydro~n~eP; staphylococcal n~lrle~e., delta-5-steroid isonlel~se, yeast-
alcohol dehydrogenase, alpha-glycerol phr eph~te dehydrogenase, triose phosphateiso~ se, peroxidase, alkaline phosphatase, aspar~in~e~ glucose ~riA~ee~ beta-
~ lc~ rihsn-lr,le~e~., ureaee, c~t~l~ee~ glucose-6-phosphate dehydrogenase,
25 ~ co~mylase, and acetylcholine esterase.
F . ' of suitable r~iisieotopic labels include 3H, IllIn, l251, l3'I,32p,
S, C, Cr,57To,58Co,59Fe,75Se, l52Eu,9~Y 67Cu 2~7Ci 2llAt 2l2pb 47SC 109Pd
etc. IllIn is a plt;rellt;d isotope where in vivo im~gin~ is used s}nce its avoids the
problem of dehalogenation of the '2sI or l3~I-labeled monoclonal antibody by the30 liver. In ~ ition, this r~ionllcleQtide has a more favorable gamrna e~ .;o
energy for im~ing (Perkins et al., Eur. J. Nucl. Mecl. 10:2g6-301 (1985);
Carasquillo etal., J. Nz~cl. Mecl 28:281-287 (1987)). For example, IllIn coupled
CA 02258507 1998-12-14
- WO 97/48807 PCT/US96110561 . - -
J~7
to mr n~lon~l ~ntiho~ with 1-~'-isothiocyanatobenzyl)-DPTA has shown little
uptake in non-tumorous tissues, particularly the liver, and Ih~role ~nh~nr~es
spe~ 'y oftumor lor~ti7~ti~m (h ~teb~n e~ al., J. Nucl. Med. 28:861-870 (1987)).
F ~ of suitable non-radioactive ~-st ~ Iabe}s inc}ude '5'Gd, 55~,
l62Dy, 52Tr, and 56Fe.
F.Y~mples of suitable fluolescenl }abels include an l52Eu }abel, a
nuo~ .n label, an isothiocyanate label, a rhodamine label, a phycoerythrin label,
a phycocyanin label, an allophycocyanin label, an o-phth~kl~hyde label, and a
fiuo.~,sc~ e Iabel
Examples of suitable toxin labels include dirhthetia toxin, ricin, and
cholera toxin.
Examples of ~.h~milllmin~scent labels include a luminal label, an
isoluminal label, an aromatic acridinium ester label, an imi~l~7Ole label, an
acridinium salt label, an oxalate ester label, a luciferin label, a luciferase label, and
an aequorin label.
F~mpt~s of nuclear m~gnetic resonance co~ ~sLil~g agents include
heavy metal nuclei such as Gd, Mn, and Fe.
Typical tec' ~, ~es for binding the above-described labels to antibodies
are provided by Kennedy et al. ~CIin. Chim. Acta 70:1-31 (1976)), and Schurs et
al. (Clin. Chim. Acta 81:1-40 (1977)). Coupling terhniq~çs mentioned in the
latter are the glutaraldehyde methorl~ the periodate method, the ~lim~l~imide
m~th~-1, the m-m~ b~n7~1-N-hydroxy-s~ccinimide ester method, all of which
methods are incorporated by reference herein.
Chromosome Assays
The nucleic acid m~ lec~ of the present invention are also valuable for
chromnsome identification. The se~uence is spe~ific~lly targeted to and can
hybridize with a particular location on an individual human chromosome.
Moreover, there is a current need for identifying particular sites on the
chromosom~ Few chromosome marking reagents based on actual sequence data
CA 02258507 1998-12-14
WO 97/48807 PCT/US96/10561 - -
3Y'
(repeat poly,.lu,~,l,.~lls) are pl~elltly available for marking cl~lo.--osornA~ tocation.
The n-&p~ g of DNAs to chromosomes according to the present invention is an
i.npo.lallL first step in co..-,laL.Ilg those sequences wth genes Aesociqted with
disease.
S In certain plt;re"ed emboclimente in this regard, the cDNA herein
osed is used to clone genomic DNA of a CK~- l 5 protein gene. This can be
~ccomr1iehed using a variety of well known techniq~es and libraries, which
generally are available col,lmelc;ally. The genomic DNA then is used for in situcll.. Illosû---e ll.~pll.~, using well known techniques for this purpose. Typically,
10 in ~ccor~ ce with routine procedures for chromosome ,.,AI.pi~ some triat and
error may be neC~ Y to identify a genomic probe that gives a good in situ
hybri-1i7Ation signal.
In some cases, in addition, sequ~?nres can be mapped to chromosomes
by ~I~,p7~illg PCR primers (I l~f~l~bly 15-25 bp) from the cDNA.C~ lel
15 analysis ofthe 3' un~ ed region ofthe gene is used to rapidly select primers
that do not span more than one exon in the geno~ c DNA, thus coml-!icqtinE the
AntrlifirAtion process. These primers are then used for PCR scl~;elfillg of somatic
cell hybr~ds C~ individual human chromosomes. Only those hyblids
CO~ ;n~ the human gene corresponding to the primer will yield an amplified
20 portion.
PCR ~ pin~ of somatic cell hybrids is a rapid procedure for aeeigning
a l~uL~;ulal DNA to a particular chromnsom~ Using the present invention with thesame oligonucleotide primers, sublocAli7qtion can be achieved with panels of
portions from specific chromosomes or pools of large ~enomic clones in an
25 ~nqlog~u~ manner. Other mapping strategies that can similarly be used to map to
its chromosome include in situ hybrirli7qti~ n, ples-,lc;e~ , with labeled flow-sorted
chlu.~ and pr~.s.ole.,tion by hybridization to construct chromosome specific-
cDNA lit,l~ies.
Fluorescence in sit?~ hybridization ( FISH ) of a cDNA clone to a
30 met~qphq~e chro~osom~q-l spread can be used to provide a precise chromosomal
location in one step. This technique can be used with probes ~om the cDNA as
short as ~0 or 60 bp. For a review of this technique, see Verma et al., HUMAN
CA 02258507 l998- l2- l4
~ WO 97/48807 PCTIUS96/10561
~S
CHROMOSOMES: A MANUAL OF BASIC TEC~IQUES, P~ on Press,
New York (1988).
Once a ~upnc~ has been mapped to a precise ~ o...oso~ 1 locaffon,
the ~ ,;cal position ofthe sequ~nee on the chromosome can be co..~laled with
5 genetic map data. Such data are found, for ~ . Ie, in V. McKusick,
MENDELIAN INEIERITANCE IN MAN, available on-line lLluugll Johns
Hopkins University, Welch Medical Library. The r~l~tionchirl between genes and
~iice~es that have been mapped to the same cl--.~l..oso...~l region are then
id~ntified through linkage analysis (coinheritance of physicalty ~ .nt genes).
}O Next, it is necec~.y to determine the di~elences in the cDNA or
~nom;C sequence b~;lwt;ell ~ ~clecl and unaffected individuals. If a mutation isobserved in some or all of the ~rrecled individuals but not in any normal
individuats, then the mutation is likely to be the causative agent of the disease.
With current resolution of physical ~-.apping and genetic l--appi,-~,
te~hn:quPc~ a cDNA p.~cisely localized to a chromosomal region ~ccori~ed ~,vith
the disease could be one of bet ,veen 50 and 500 potentiat causative genes. (This
s~c.c ~meS 1 mf~.~h~ce mapping resol~1tion and one gene per 20 kb~.
CA 02258507 1998-12-14
- WO 97/48807 PCT/US96/10561 --
~/c,
Tr~..h.~e,.l of Thymus-R~lnf~Disorders
As noted above, unlike other known CC cytol~inPs, CK~3-lS has been
shown to be e,~.re~d only in the thymus. Therefore, CKp-15 is particularly
5 active in mf)f~ ti~g activities of monocytes in the thymus, particularly those of
early thymocytes, such as the activities described above in relation to the
description of a "polypeptide having CK~-lS activity." Given the thymocyte
activities modlll~ted by CK,B-lS, it is readily a~palenl that a s-lbst~nti~1ly altered
t ased or de~;l~) level of e~ ion of CK~3-l S in an individual colllpaled
10 to the standard or "norrnal" }evel produces pathological conflition.~ such as those
des~ ed above in relation to diagnosis of thymus-related disolde.~. It will alsobe apprtcialed by one of ordil1~y skill that, since the CK~-lS protein of the
invention is Ll~n~ ed with a leader peptide s~it~l~le for secretion of the mature
protein from the cells which express CK,13-15, when CK,B-15 protein (particularly
15 the mature form) is added from an exogenous source to cells, tissues or the body
of an individual, the protein will exert its modul~ting activities on any of its target
cells of that individual. Thf l t:r.l c, it will be appreciated that con-lition~ caused by
a decrease in the standard or normal level of CK~3-lS activity in an individual,particularly disol .lel:i of the thymus, can be treated be ~ alioll of CK~3-15
20 protein. Thus, the invention also provides a method of ll~ "l of an individual
inneedofanil..,l~a3~1evel of CK,B-lS activitycomprising ~ln~;n;~lelingto such
an individual a pharm~ceutical composition COlllpli~illg an amount of an i~ol~ted
CK~-15 polypeptide ofthe invention, particularly a mature form ofthe CK,B-15
protein ofthe invention, effective to increase the CK,B-15 activity level in such an
25 individual.
In addition, since the CK~3-15 protein suppresses myeloid cell growth
when ~f7mini~tered to an individual, the invention provides m~ thod~ for
~u~ myeloid cel p~ elalion in an individual, which involve ~fl~ hlg
a myelo~u~ e amount of CK~3-15 either alone or together with one or more
30 chemokines s~lected from the group co~ g of Macrophage Tnfl~ o,y
Protein-la ~-la), Macrophage T..n~ ory Protein-2a ~IIP-2a), Platelet
Factor 4 (PF4), Interleu ~in-8 (rL-8), Macrophage Chel..ola~ic and Activating
CA 022~8~07 1998-12-14
- WO 97/48807 PCT/US96/10561
Y/
Factor ~MCAF~, and Macloph~ge T~ nly Protein-Related Protein-2 ~MRP-
2). The myelosupprcssi~e con~posi~ions of the present invention thus provide
myeloprotecli~re effects and are useful in co~ nction with thc . - . s that have an
adverse affect on myeloid cells. This is because the myelosu~ e~
S comrosit;nnc of the present invention place myeloid cells in a slow-cycling state
~ thereby providing protP~tinn against cell damage caused by, for ~"~"ple, radiation
therapy or ch~mo1lle~lJy using cell-cycle active drugs, such as cytosine
arabinoside and h~ yul ~,a.
The myelosu~plcssi~e pharm~ce~ltis~l compositiQnc of the present
invention are also useful in the tre~tmP!nt of leuk~mi~ which causes a
Lyyc~ }iferative myeloid cell state. Thus, the invention further provides methods
for treating lenk~mi~, which involve ~dminict~ring to a lel-kP!mi~ patient a
myelosl".pres~;~re amount of CK,13-15 either alone or together with one or more
~h~.mokines s~lected from the group consisting of MIP-la, MIP-2cc, PF4, IL-8,
MCAF, and MRP-2.
By "~upp~ ,"g myeloid cell proliferation" is intPn~led decreasing the
cell 1~ uLrt;~ ~lion of myeloid cells and/or increasing the percenLage of myeloid cells
in the slow-cycling phase. As above, by "individual" is int~nrled ~-A~ n
individuals, preferably hllm~nc Pr~incllb~tiQn of the myelosupplessi~e
compositions of the present invention with ~cetonitrile (ACN) cignifi~slntly
çnh~nees the specific activity of these chemokines for suppression of myeloid
progenitor cells. Thus, plerelal~ly, prior to ~lminictration, the myelosuppresive
compositions ofthe present invention are plt;L-ealed with ACN as described in
~r~ ey-erH. E., etal., Ann-HemafoL 71(5,~:235-46(1995) and PCT Pub}ication
WO 94/13321, the entire fliC~los~res of which are hereby incorporated herein by
c, ~"ce.
The myelosu~pressi.~e compositions of the present invention may be
used in co~ with a variety of cl-~ Lllerapeutic agents inr.lllrling alkylating
agents such as nitrogen mustards, ethylP!nimin~ lyll~P~ P!c alkyl
30 ~ r~ n-LIu~.luul~ and llia~ene~ ntimet~bolites such as folic acid analogs,
y ' '~ine zm~lo~s, in particular fluorouracil and cytosine arabinoside, and purine
gnglr~gs natural products such as vinca alkaloids, epipodophyllotoxins, antibiotics,
-
CA 02258507 1998-12-14
- WO 97/48807 PCT/US96/10561 - -
enzymes and biolc~c~l response mor~ifiers; and m;~cçll~nçous products such as
plz-tin-lnt coor~in~tion complexes, anthr~c~ne~1ionr~ s~s~ e~ urea such as
Ly~ yul~, methyl Iryd~ e derivatives, and adrenocorticoid ~u~plcss~l
(~h~r~ ~G-Iticagentscanben~tn~ yl~ edatknownconc~ 1~alions
5 a~.l;.* to known l ~ The my~ s~ e cc,nlpGs.lions of the present
invention can be co-~rl~ ed with a chemother~GuLic agent, or ~ 1 ed
S~ GIY~ either before or after chemotherapeutic ~ 1 ation
Certain rhemokines~ such as MIP-lB, M~-2B and GRO-a, inhibit (at
least partially block) the myeloid su~ Gs~ e affects of the myelo~upp.e~ e
10 compositions of the present invention Thus, in a filrther emborlim~nt the
invention provides m~tho~ls for inhibiting myelosuppression, which involves
slr~m;~istl~ring an effective amount of a mye}osupp- G~ re inh;t-i~Qr selected from
the group co .~ of MIP-113, MIP-2B and GRO-a to a ,n~ l previously
~osed to the myelos~lppl esive agent CK13- l S either alone or together with one15 or more of M~-la, MIP-2a, PF4, IL 8, MCAF, and MRP-2
One of ordinary skill will appreciate that effective ~m~lnt~ of the
CK13- l S polypeptides for treating an individual in need of an increased level of
CK,B-lS activity ~in~ ling amounts of CK~-lS polypeptides effective for
my~los -rpression with or without myelosupp- es~i~Je agents or myelo~u~p~ e
20 inhibitors) can be determined empirically for each con~lition where ~ ;n~lionof CK~-lS is jn~lic~te~l The polypeptide having CK~-15 activity my be
~tlmini~t~red in pharm~ce~lti~l compositions in co..-bi~l~Lion with one or more
pharn ace~tif~tly acceptable excipients It will be understood that, when
s~3mini~t~red to a human patient, the total daily usage of the pharm~reutir9l
25 CO..~pO~ ;~;nn~ of the present invention will be de~ ed by the ~ff~nt1ing phy:jic;a~l
within the scope of sound medical jud~m~nt The specific l~ ~e~llically t;neclivedose level for any particular patient will depend upon a variety of factors in~ t~ ing
the type and degree of the response to be achieved; the specific composition an
other agent, if any, employed; the age, body weight, general health, sex and diet
30 of the patient; the time of ~rlmini~tration~ route of ~ lion, and rate of
excretion of the composition; the duration of the llr~ l, drugs (such as a
chemotherapeutic agent) used in combillalion or coin~ nt~l with the specific
CA 02258507 1998- 12- 14
- WO 97/48807 PCT/US96110561 - -
~3
composition; and like f~ctors well known in the 2ne-2ir,,s 1 arts.
For; . '-, ~tief~rt~!ry results are obtained by oral ~dminietration of
a polypeptide having CK~- 15 activity in doss ges on the order of from 0.05 to 10
mg/kg/day, p~cr~ ~ly 0.1 to 7.5 mg/kg/day, more preferably 0. ~ to 2 mg/kg/day,
5 ~A~ d once or, in divided doses, 2 to 4 times per day. On ~ c~ ion
~ parenterally, for ~oys2mple by i.v. drip or infusio~, doss~g~s on the order of from
0.01 to 5 n~ y, p.~;rt;.~ly 0.05 to 1.0 mg/kg/day and more plere;,~bly 0.1 to1.0 mgtkgtday can be used. Suitable daily dos-s~g~s for patients are thus on theorder offrom 2.5 to 500 mg p.o., prere~~1y 5 to 250 mg p.o., more preferably 5
to 100 mg p.o., or on the order offrom 0.5 to 250 mg i.v., plere ~bly 2.5 to 125mg i.v. and more preferably 2.5 to 50 mg i.v.
Do~l~, may also be arranged in a patient specific manner to provide
a predetenmined conr~-ntration of an CK~- 15 activity in the blood, as determined
by an RIA technique, for inetsnce Thus patient doss~ging may be A.~ etecl to
~ evc regular on-going trough blood levels, as measured by RIA, on the order
offrom 50 to 1000 ng/ml, preferably 150 to 500 ng/ml.
Pl~"--.sc~ l;rAI compositions of the invention may be ~miniet~red
orally, recta11y, pa e~ lly, intracietem~lly, intravaginally, L~l.~e.iloneally,
topicslly (as by powders, o;~ ; drops or transdermal patch), bucally, or as an
orat or nasal spray. By ",ol~ ~AC~t;~11Y acceptable carrier" is meant a non-toxic
solid, s~mien1it1 or liquid filler, diluent, ~ 7p~ ting material or formulation
auxiliary of any type. The term "parenteral" as used herein refers to modes of
iel.alion which include intravenous, intrAm-lec--lAr, intraperitoneal,
intrasternal, subcuts~neous and i I~l~Licular injection and infiJsiQn
Pharm~ceutic~l compoeitione of the present invention for pale .1.,.~1
tinn can co---l-- ;ee ph"..~.AC~ tic ~lly acceptable sterile aqueo~e or nnn~ eQus
solutions, dispersions, s lep~oneions or emulsions as well as sterile powders for
1~ c(~ tion into sterile inject~hle soh-tione or dispersions just prior to use.
F.Y~nnrl~s of suitable aqueous and non~tlueQus carriers, ~ ente solvents or
30 vehicles include water, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the likç), carboxymethylceuulose and suitable n~iAI~reSthereof, vegetable oils (such as olive oil), and injectable organic esters such as
CA 02258507 1998-12-14
-- W~ 97/48807 PCTIUS96/10561
y~f
ethyl oleate. Proper fluidity can be ~n~;..l,.;nç~i, for ~ - ~f...j,lr., by the use of coating
materials such as le~ithin, by the .,"~ ce of the required particle size in the
case of dis~ ;OIIS, and by the use of surf~ct~nt.~
The co~ oY;I;on~ ofthe present invention may also contain adjuvants
5 such as pl_se. v~ es, wellillg agents, emulsifying agents, and Jis~ ing agents.
Prevention of the action of microor~ni~m~ may be ensured by the inc~ ;on of
various ~.1;1 acle~ial and ~ntifi~np;~l agents, for ~Y~mple, ~ , chlorob~lt~phenol sorbic acid, and the like. It may also be desirable to include isolol c agents
such ais sugars, sodium chloride, and the like. Prolonged abso-~,Lon of the
10 inje ~ pha~ s-ceutic~l form may be brought about by the il~,lu~;oll of agents which delay abso~plion such as ~ min-lm monostearate and gelatin.
In some cases, in order to prolong the effect of the pha~n~ceutic~l
co..l~osilion, it is desirable to slow the absorption of the drug from s~1b.;~ nP,o~ls
or ~Il.i.. ~c~ r injection. This may be accomrlich~d by the use of a li~uid
s~sp~n~ion of crystalline or amorphous material with poor water solubility. The
rate of absorption of the drug then depends upon its rate of dissolution which, in
turn, may depend upon crystal size and crystalline form. Alternatively, delayed
abso,~lioll of a p~c;l-Lt;l~lly ~f1mini~t~red drug form is accomrli~hed by dissolving
or suspending the drug in an oil vehicle.
T~ 'e depot forms are made by r~ ing microencapsuled In~l~ices
of the drug in biodegradable polymers such as polylactide-polyglycolide.
Depending upon the ratio of drug to polymer and the nature of the particular
polymer employed, the rate of drug release can be controlled. Fy~mples of other
biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot
25 ije~le fi,.l.~ ;ol-s are also prepared by ellLI~ Jing the drug in liposomes or
rnicroemlllQ;ons which are co...p~l;hle with body tissues.
The injectable formulations can be sterilized, for example, by filtration
through a bacterial-ret~inin~ filter, or by incorporating sterilizing agents in the
~orm of sterile solid compositions which can be dissolved or dispersed in sterile
30 water or other sterile injectable me(lium just prior to use.
Solid dosage forms fpr oral a~mini~tration include capsules, tablets,
pills, powders, and granules. In such solid dosage forms, the active compounds
CA 02258507 1998-12-14
- WO 97/48807 PCT/US96/10561
y~
are mixed w th at least one item pl~ ce~lti~ y acceptable eYrip:~~t or carrier
such as sodium citrate or dir,~~lri-lm phosphate and/or a) fillers or ~Yt~nrl~rs such
as Dl~.,l.~s, lactose, sucrose, gII1CQSe~ m~nnitQl and silicic acid, b) binders such as,
for rY mpl~, carbo~y.ll~tl.ylcerulose, ,A~lginA~t~s~ gelatin, polyvh-yl~yl,oli~lon~
S sucrose, and acacia, c) hllmect~~nt~ such as glycerol, d) rii~inte~.~ali..~g agents such
as agar-agar, ç,~l.~;-lm c&.l,onale, potato or tapioca starch, alginic acid, certain
c.~tes, and sodium c~l,onate, e) solution rela-ding agents such as l)~;n, f)-
absol~,lion accelc~ such as quaternary 7~~ --o~ --- CQl~ , g) wetting
agents such as, for ~ , cetyl alcoho1 and glycerol monostc,~rate, h)al)so.l,_nl~
10 such as kaolin and b~ntonite clay, and I) lubricants such as talc, c~ir;~m stearate,
~..a~.~ - -lm stearate, solid polyethylene glycols, sodium lauryl sulfate, and ~~ lu-~,s
thereof. In the case of ~~rsl~ , tablets and pills, the dosage form may also
CGI-IIJ- ise buffering agents.
Solid compositions of a similar type may also be employed as fillers in
15 soft and hard filled gelatin c~rsllles using such excipients as lactose or milk sugar
as well as high moleclll~r weight polyethylene glycols and the like.
The solid dosage forms oftablets, dragees, c~rslll~s, pills, and granules
can be ple~ t;d with coa~ g.~; and shells such as enteric coatings and other
co~ well known in the pharm~ce~ti~l formlll~ting art. They may optionally
20 contain opacifying agents and can also be of a composition that they release the
active ingredient(s) only, or plerele.,Lially, in a certain part ofthe ~ e~ i tract,
optionally, in a delayed manner. Examples of embedding compositions which can
be used include polymeric s~bst~nces and waxes.
The active compounds can also be in micro-enc~rs-~l~ted form, if
25 apl)lop.;~e, with one or more ofthe above-mentioned ~
Liquid dosage forms for oral ~mini~tration include pharrn~ce~ti~5-11y
~IC'-r '-le r~ml~ ,S~ ltion~ sr-~n~i-)n~ syrupsandelixirs. Inadditiontothe
active co~ ounds, the liquid dosage forms may contain inert diluents col..,.lollly
used in the art such as, for ~Y~mrle~ water or other solvents, sol~lbiii7ins~ agents
30 and ~m ll~ifir~rs such as ethyl alcohol, isoptopyl alcohol, ethyl c~l,onale, ethyl
ncet~te, ben_yl alcohol, benzyl b~ n7o~te~ propylene glycol, 1,3 butylene glycol,
d;.. w~ oils (in particular, cottonseed, groundnut, corn, germ, olive,
CA 02258507 1998-12-14
- WO 97/48807 Y6 PCT/US96/10561
castor, and sesame oils), glycerol, tGL. ~IJydl urL~ ryl alcohol, polyethylene glycols
and fatty acid esters of soll i~an, and mixtures thereof.
Besides inert d~ ntc, the ora1 compositinns can also include adjuvants
such as wetting agents, emulsifying and suspending agents, ~vet;lf ~ n~vo~i~Jg,
5 and p~, ru~ ng agents.
S~-srenQ;~nc in addition to the active compounds, may contain
s~ ,en~ agents as, for ex~mrlç ethoxylated isostearyl ~lcohnl~
polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, ~lumin~ltn
metahydroxide, bentonite, agar-agar, and tr~qg~nth and mixtures thereof.
The active po1ypeptide can also be ~ ;cl~ed in the form of
LPOSJ~ S~ As is known in the art, liposomes are generally derived from
rhocrhn~ ' or other lipid ~~ ces T i~OSQ~e~ are fiormed by mono- or multi-
l~mellslr hydrated liquid crystals that are dispersed in an ~q~eous ..Lc~ Any
non-toxic, physiologically acceptable and metabolizable lipid capable of ru~ ng
liposomes can be used. The present compositions in liposome form can contS~in
AitiQn to the agent or inhibitor, stabilizers, preservatives, eYcip ~ntc, and the
like. The p-ere--c;d lipids are the phospholipids and the phosphatidyl cholates
, both natural and synthetic. Methods to form liposomes are known in
the art. See, for ~Y~mpl~, Prescott, Ed., Mef*ods in CeUB ology, Volume XlV,
~c~d~mic Press, New York, N.Y. (1976~, p. 33 etse~.
Having generally decen~çd the islvention, the same will be more readily
u..dc;l~Lood by .~;rt;.ence to the following examples, which are provided by way of
i~lustration and are not in~nr1ed as limiting
E~AMPLES
~- , Ic 1: F, cD~..on and Purzfcation of CK~I~ ~n E. col~
The DNA seq~lçn~e encoding the mature CK,B-15 protein in the
de~o~iled cDNA clone was amplified using PCR oligonucleotide primers specific
to the amino terminal sequences ofthe CK~-15 protein and to vector sequ~nce~s
CA 022=,8=,07 1998-12-14
~ W O 97/48807 PCTrUS96/10561
Y7
3' to the gene. Additional n~ eotides cQ~ p re.st~ on sites to f~r.ilh~te
cloning were added to the 5' and 3' sequences re~,e.;Li~tely.
The 5' oliPon~rteotide primer had the sequence 5' GCC ( rTC GAC
GTC CAC ACC CAA GGT GTC 3' lSEQ ID NO:4] cc...~ the underlined
S SaU restriction site, which encodes 18 nucleotides ofthe CK,B-15 protein coding
- sequence in Figure 1 [SEQ ID NO:1] beEinnin~ imm~ tf~ly after the signal
peptide.
The 3' primer had the sequence 5' GCC TCT AGA GGA GCC CAG
AAA TGA GCC GGC 3' [SEQ ID NO:5; co.~ the unde.l,l1ed XbaI
10 restriction site followed by 21 nucleotides comrl~ y to the last 21
nucleotides ~ eJ;~(ely after the CK,B-15 protein coding sequ~nce in Figure 1.
The restriction sites were convenient to restriction enzyme sites in the
bacterial cA~,~ssion vector pD10 (pQE9), which were used for b~ct~
eA~l~;oll in these . I ' (Qiagen, Inc. 9259 Eton Avenue, CLdl:~wo~ ll" CA,
91311). [pD10]pQE9 encodes ~mpieillin antibiotic r~qi~t~n~e ("Ampr") and
COI~IainS a bacterial origin of replication ("ori"), an IPTG indudble promoter, a
ribosome binding site ("RBS"), a 6-His tag and restriction enzyme sites.
The ~mplifiç~l CK13-15 protein DNA and the vector pQE9 both were
rli~e.sted with SalI and XbaI and the ~liEeeted DNAs were then ligated together.20 InSGILion ofthe CK,B-15 protein DNA into the restricted pQE9 vector placed the
CK,B-15 protein coding region duw"~le~ll of and operably linked to the vector's
IPTG-in-lurible promoter and in-frame with an i~ g AUG app,.,p,iately
po~itiQned for tr~nCl~tion of CK,B-15 protein.
The ligation mixture was l~ru~ ed into competent E. coli cells using
25 standard procedures. Such procedures are described in Sambroolc et al.,
MOLECULAR CLONlNG: A LABORATORY MANUAL., 2nd Ed.; Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989). E. coli strain
M15/rep4, ~..~ E mllltirle copies ofthe pi~emid pREP4, which e,.l,-Gsscs lac
lG~l~sor and confers l~lly~,ul rPeiet~n~e ("Kanr"), was used in carrying out the30 illu~ e ~ 'e described here. This straîn, which is only one of many that are
sllit~le for CA~G~ g CK~-15 protein, is available col"-~e~cially from Qiagen.
Tran~r~"",an~s were identi~ied by their ability to grow on LB plates in
CA 02258507 1998-12-14
- WO 97l48807 PCT/US96/10561
Y8'
the presence of Amr;rjllin and k~ula~ ,h~. Plasmid DNA was i~ stecl ~om
f~ L~ ?~ ~ and the identity ofthe cloned DNA was cci- .r....~d by restriction
allaly~s.
Clones cn..ln....~ the desired constructs were gro ~m overnight ("O/N")
S in liquid culture in L13 media suppl~m~nted with both Amri~.illin (100 ~glml) and
kanamycin (25 llg/ml).
The OtN culture is used to inoculate a large culture, at a AillltiQn of
a~plv~ ly 1:100 to 1:250. The cells aregrown to an optical density at 600nm
("OD600") of between 0.4 and 0.6. Isopropyl-B-D-thiogala-;lG~,y~ oside
10 ("lE'TG") is then added to a final concentration of 1 mM to induce l-~s~ lionfrom lac r~lessor sensitive promoters, by inactivating the lacI ~~,pl~ssor. Cells
subsequently are ;..~ ed further for 3 to 4 hours. Cells then are harvested by
c~ntrifilg7.tion and disrupted, by standard methods. Tn~ n bodies are purified
from the di..,~led cells using routine collection techni~ s, and protein ~s
15 solubili7ed from the inclusion bodies into 8M urea. The 8M urea sol~tiQn
C~J~ P the so' ~ i7P~1 protein is passed over a PD- 10 column in 2X phosphate-
l~u~ercd saline ("PBS"), thereby removing the ure~ I -Ct~Al~gil~g the buffer andI t;r~- ~ g the protein. The protein is purified by a iùrther step of cl~ u~ ography
to remove endotoxin. Then, it is sterile filtered. The sterile filtered protein
20 p. el,al~lion is stored in 2X PBS at a concentrAtion of 95 ~/ml.
Analysis ofthe p.epal~Lion by standard methods of polyacrylamide gel
el~L,..~llole .;;, reveAls that the pr~al~lion contains about 95% monomer CK,B-l~
protein having the expected mnlec~ r weight of al.~,ro~ tcl~ 16.7 kDa.
F~- , 'e 2~ o~ing and Ex~ression of CK/~15 protein in a Baculovirus
.on System
The cDNA sequence encoding the full length CK,~-15 protein in the
30 deposited done is amplified using PCR oligonucleotide primers col-es~otlding to
the 5' and 3' sequences of the gene:
The 5' primer has the sequence 5' GCC TCT AGA GCC ATC ATG
CA 02258507 1998-12-14
- WO 97/48807 PCTtUS96/10561
~S
AAC CTG TGG CTC CTG GCC 3' [SEQ ID NO:6] co..~ g the underlined
XbaI restriction ~,.~y-~-e site followed by 21 bases of the seq~lence of CK,B-15protein in Figure 1. Inserted into an eA~fession vector, as desc;~ ed below, the 5'
end of the ~mplified fragment çncorling CK,13-15 provides an Pfficient signal
S peptide. An çffic;~nt signal for initiation of translation in eukaryotic cells, as
described by Kozak, M., J. Mol. Biol. 196: 947-950 (1987) is app~o~ ly
located in the vector portion of the construct.
The 3' primer has the sequence 5' GCC TCT AGA GGA GCC CAG
AAA TGA CCC GGC 3' [SEQ lD NO:7] cc~ .;..g the underlined XbaI
restriction site followed by nucleotides complçmpnt~ry to the last 21 nucleotides
ofthe CK,B-15 coding sequence set out in Figure 1.
The arnplified fragrnent is isolated from a 1% agarose gel using a
comrnercially available kit ("Geneclean," BIO 101 Inc., La Jolla, Ca.). The
fragrnent then is rfigloet~l with XbaI and again is purified on a 1% agarose gel. This
fragment is desi n~ted herein F2.
The vector pA2-GP is used to express the CK~-15 protein in the
bacu10virus cA~JIG~ ion system, using standard methods, as described in .~llmm.srs
et al, A MANUAL OF METHODS FOR BACULOVIRUS VECTORS AND
INSECT CFT T CULTURE pRocFnuREs~ Texas Agricultural ExpelimGIiLal
Station Bulletin No. 1555 (1987). This c,~l~lession vector collt~in.e the strongpolyl.el-in plu~oLer of the Au~ographa californica nuclear polyhedrosis virus
(AcMNPV) followed by convenient restriction sites. The signal peptide of
AcMNPV gp67, inclu(lin~ the N-terrninal methionin~, is located just u~ l of
a Bani~II site. The polyadenylation site of the simian virus 40 ("SV40") is used for
efficient polyadenylation. For an easy selection of recolllbin~lL virus the beta-
~os;ds~ce gene from E. coli is inserted in the same orientation as the
polyhedrin promoter and is followed by the polyadenylation signal of the
polyhedrin gene. The polyhedrin sequ~nces are flanked at both sides by viral
seqU~nr~c for cell-mrAi~ted hnmnlogouc IGcombillation with wild-type viral DNA
to generate viable virus that express the cloned poiynucleotide.
Many other baculovirus vectors could be used in place of pA2-GP, such
as pAc373, pVL941 and pAcIMl provided, as those of skill readily will
CA 02258507 1998-12-14
- WO 97148807 PCT/US96/10561
S-
~app~ le, that construction provides app-up,i~Lely located signals for
ion, 1.~ oll, tr~ffi~in~ and the like, such as an in-frarne AUG and a
signa1 peptide, as ,~u.,~. Such vectors are described in Luckow et al., Virology170: 31-39, among others.
The plasmid is f1i~cted with the restriction enzyme ~oaI and then is
dephospho~laled using calf int~ctin~l phssph~t~ee using routine procedures
known in the art. The DNA is then icsl~ted from a 1% agarose gel using a
y available kit (~ n-~le~ BIO 101 Lnc., La Jolla, Ca.). This vector
DNA is ~eci~n~ted herein "V2".
Fr~nPnt F2 and the dephocrholylated plasmid V2 are ~igated together
with T4 DNA ligase. E coli HB101 cells are transformed with ligation mix and
spread on culture plates. Bacteria are j(1entified that contain the plasmid with the
human CK,B- 15 gene by digesting DNA from individual colonies using XbaI and
then analyzing the ~ ctinn product by gel electrophoresis. The sequ~nce of the
15 cloned fragment is co~ .,cd by DNA seql~çnçin~ This plasmid is ~esi~n~ted
herein pBacCK,B-15.
5 ~lg of the plasmid pBacCK~-15 is co-Llal,srt:c~ed with 1.0 ~lg of a
coi-....~.,;ally available linea~ized baculovirus DNA ("BaculoGoldTM baculovirusDNA", rl~..~ e-. San Diego, CA.), using the lipofection method desc.il~ed by
Felgner et aL, Proc. Natl. Acad. Sci. USA 84: 7413-7417 (1987). 1,ug of
BaculoGoldTM virus DNA and S ~g of the plasmid pBacCK,B-15 are rnixed in a
sterile well of a microtiter plate co~ 50 ~11 of serum-free Grace's ~eJ;u~--
(Ufe Terhnolo~os Inc., Gaithersburg, MD). Afterwards 10 ~11 Lipofectin plus 90
~ll Grace's mer1illm are added, mixed and incub~ted for 15 n-in~ltes at room
1el.~e ~re. Then the transfection mixture is added drop-wise to Sf9 insect cells(ATCC CRL 1711) seeded in a 35 mm tissue culture plate with I ml Grace's
. without serum. The plate is rocked back and forth to mix the newly added
solution. The plate is then in~llb~ted for 5 hours at 27~C. Aflter 5 hours the
~"...~r~;,;.~,. soll.tio~ is removed from the plate and I ml of Grace's insect merii~-m
30 ~iUp~ tÇCI with 10% fetal calf serum is added. The plate is put back into an
in~llh7~t~r and cultivation is contjnlled at 27~C for four days.
After four days the supelll~Lanl is collected and a plaque assay is
CA 02258507 1998-12-14
- W.O 97/48807 PCT/US96/10561
p~. r"....~ as ~ ;I,e~ by S~"~"'ltll ~ and Smith, cited above. An agarose gel with
"Blue Gal" (Life Technologies Inc., Gaithersburg) is used to allow easy
idP-ntifirRtion and i~sl~tion of gal ~ hlg clones, which produce blue-stained
rl~q~es (A detailed des~ lion of a "plaque assay" of this type can also be foundS in the user's guide for insect cell culture and baculovirology distributed by Life
~ Terhnolo~P,~ Inc., Gaithersburg, page 9-10).
Four days after serial dilution, the virus is added to the cells. A~er
applop,i~Le incub~R~tion~ blue stained plaques are picked with the tip of an
Eppendorf pipette. The agar cQl~l;.il.;..g the recol~b;.~,,..l viruses is then
l~ in an Eppendorftube co~ g 200 111 of Grace's me~ m The agar
is removed by a brief centrifugation and the ~upe~ LallL col~l Ri~ the
.C~.,.I.;.~..l baculovirus is used to infect Sf~ cells seèded in 35 mm dishes. Four
days later the ~ l R~ of these culture dishes are harvested and then they are
stored at 4~C. A clone co..l~ properly inserted hESSB I, II and m is
15 id~ ~ byDNAanalysis inrl~ing restriction "~appil1g and sequenrin~ This is
~lç~ ted herein as V-CK~B-15.
Sf9 cells are grown in Grace's mef~ m su~plr ~ ed with 10% heat-
inactivated FBS. The cells are infected with the ,t:col.-b;~ .l baculovirus V-CK,B-
15 at a ml11tiplirity of infection ("MOI") of about 2 (about 1 to about 3~. Six
20 hours later the m~ m is removed and is replaced with SF900 II m~ lm minus
' '~n;n~ and cysteine (available from Life Technologies Inc., Gaithersburg). 42
hours later, S ,uCi of 35s-mpthiolline and S IlCi 35S-cysteine (available from
A.l,e,~l,&"l) are added. The cells are further incl-b~ted for 16 hours and then they
are h~ ve~ed by centrifugation, Iysed and the labeled. ~r~L~ s are vi~ li7P,d by25 SDS-PAGE and autoradiography.
F-- ~rl~3: E~cpression in M~7mmn~inn Cells fCOS)
The eA~ ion pl~mir1, pCK,B-15 HA, is made by cloning a cDNA
~.nr,s~ CK~3-15 into the eA,~,ies~;on vector pcDNAI/Amp (whtch can be obt~inpd
from Invitrogen, Inc.).
CA 02258507 1998-12-14
- WO 97/48807 PCT/US96/10561 - -
The c A~ ,s:iion vector pcDNAVamp con~ains: (1) an E.coli origin of
.~.p'ics l;on effective for prop~g~tion in E. coli and other prokaryotic cells; (2) an
gene for selectic~n of pl~mid-co~ prokaryotic cells; (3)
an SV40 origin of replir.~tion for propagation in eukaryotic cells; (4) a CMV
S l~lulnoter, a polylinker, an SV40 intron, and a polyadenylation signal &.,~lged so
that a cDNA conveniently can be placed under e A~ulcssion control of the CMV
promoter and operably linked to the SV40 intron and the polyadenylation signal
by means of restriction sites in the polylinker.
A DNA L~.lenl Pnro~fing the entire CK~-15 precursor and an HA tag
10 fi~sed in f ame to its 3' end is cloned into the polylinker region of the vector so that
reco~ .;.. n~ protein e A~.les~ioll is directed by the CMV promoter. The HA tag
co-.e:,~ol~ds to an epitope derived from the inflllPn7~ h~m~llltinin protein
described by Wilson et al., Cell 37: 767 (1984). The fusion ofthe HA tag to the
target protein allows easy detection of the reco,..l~hlalll protein with an antibody
15 that recognizes the HA epitope.
The plasmid construction strategy is as follows.
The CK~-15 cDNA of the deposited clone is ~mptified using primers
that contain convenient restriction sites, much as described above ~ ding the
construction of e~lt;ssion vectors for e,.~-ession of CK,B-15 in E. coli. To
20 ~~ te ~ n and ~,h~ ~el ~ion of the e~l c:s~ed CK,13- 15, one
ofthe prirners col~ a h~m~ggllltinin tag ("HA tag") as desl,lil,ed above.
.S~ ~~e prirners include that following, which are used in this PY~mpl~
The S' prirner, col.~ ;..g the underlined HindIII site, an AUG start codon and 6 codons of the 5' coding region has the following sequence:
5' GCG A ~G Cl'r ATG AAC CTCi TGG CTC CTG GCC 3' ~SEQ ID NO:8~.
The 3' primer, co..~ the underlined XhoI site, a stop codon, 9
codons ~I-e~e&f~er forming the h~m~gl~tinin HA tag, and 22 bp of 3' coding
seq-~nce (at the 3' end) has the following sequence:
S' GCG CTC GAG TCA AGC GTA GTC TGG GAC GTC GTA TGG GTA
30 CAG TCC TGA ATT AGC TGA TAT C 3' [SEQ ID NO:9].
The PCR amplified DNA fragment and the vector, pcDNAI/Amp, are
CA 02258507 1998-12-14
- WO 97/48807 PCT/US96/10561
f3
Ai~ted with Hindm and XhoI and then ligated. The ligation ~ Lult; is
,~..cr. ~"~F,~ into E;. coli strain SURE (available from Str~t~e~ne Cloning Systems,
11099 North Torrey Pines Road, La Jolla, CA 92037) the transformed culture is
plated on ~mrjr~ in media plates which then are ;I~c~ to allow growth of
S ~ t;D;Dt~l~ ~ nlc - . s Plasmid DNA is i~ol~ted from r~ s;DL~ll colonies and
.ed by restriction analysis and gel sizing for the pl~s~nce of the CB,B-15-
en~o~in~ fra~n~nt
For ~ Jr~Dion of ~ CK,B-15, COS cells are tr~n~fected with
an ~lwDion vector, as described above, using DEAE-DEXI~AN, as described,
10 for i~ ;.m~., in Sambrook et al., MOLECULAR CLONING: A LABORATORY
MANUAL, Cold Spring Laboratory Press, Cold Spring Harbor, New York
(1989). Cells are inc~b~ted under conditions for t:,~ple~slon of CK~-15 by the
vector.
Expression of the CK~- 15 HA fusion protein is detected by
15 r~ '*ellin~ and immunopleci~ ;on, using methods described in, for example
Harlow et al., ANTIBODIES: A LABORATORY MANUAL, 2nd Ed.; Cold
Spring Harbor L~ulaLul y Press, Cold Spring Harbor, New York (1988~. To this
end, two days after tr~n~f~ction, the cells are labeled by incllb~tion in media
c~"~ 35S-cysteine for 8 hours. The cells and the rnedia are cnllecte~l~ and the
20 cells are washed and the Iysed with detergent-co..~ g RIPA buffer: 150 mM
NaCI, 1% NP~0, 0.1% SDS, 1% NP~0, 0.5% DOC, 50 mM TRIS, pH 7.5, as
described by Wilson et al. cited above. Proteins are p-~cipilal~d from the cell
Iysate and from the culture media using an HA-specific monoclonal antibody. The
led proteins then are analyzed by SDS-PAGE gels and autoradiography.
25 An ~,lesD;on product of the expected size is seen in the cell Iysate, which is not
seen in negative controls.
CA 02258507 l998- l2- l4
- W097/48807 PCT/US96/10561 --
~y
F~ ~ le 4: Tissue ~.h.bution of CK~lS protein ! A r~;on
Northern blot analysis was carried out to t~ e the levels of
~iAyr~ssion of CK,B-15 protein in human tissues, using methods desclibed by,
S among others, Sambrook et al, cited above. PolyA+ was purchased form Clontech
(1020 East Meadow Circle, Palo Alto, CA 94303).
About 1 ~g of PolyA' RNA was size resolved by ele.;l.ophol~i,is
through a 1% agarose gel under strongly ~hqn~tllrin~ c~ n~hions RNA was blotted
from the gel onto a nylon filter, and the filter then was prep~Gd for hybri~1i7~tion
to a ~letect~hly labeled polymlc~eoti~le probe.
As a probe to detect rnRNA that encodes CK~-15 protein, the ~ntiee.~lce
strand of the coding region of the cDNA insert in the deposited clone was labeled
to a high specific activity. The cDNA was labeled by primer PYt~n.eion, using the
Prime-It kit, available from St~ e The reaction was carried out using 50 ng
of the cDNA, following the standard reaction plulocol as reco~ ded by the
supplier. The labeled polynucleotide was purified away from other labeled
reaction components by column chromatography using a Select-G-50 column,
obtained from S-Prime - 3-Prime, Inc. of 5603 Arapahoe Road, Boulder, CO
80303.
The labeled probe was hybridized to the filter, at a concentration of
1,000,000 cprn/rnl, as described in Kreider et al., Molecular and Cellular Biology,
Sept. 1990, pp. 4846-4853. Thereafter the probe solution was drained and the
filter was washed twice at room temperature and twice at 65 ~C with 0.1 x SSC,
0.1% SDS. The filter then was then dried and exposed to film at -70~C overnight
with an intensifying screen. The results of a typical Nothern blot using the CK,B-
15 cDNA probe are shown in Figure 3.
F~~2r~o 5: Gene tkerapeuhc ~, ~i, ,.on of human CKJJ-lS protein
Fibroblasts are obtained from a subject by skin biopsy. The resulting
tissue is placed in tissue-culture me~ium and separated into small pieces. Smailchunks of the tissue are placed on a wet surface of a tissue culture flask,
CA 02258507 1998-12-14
- WO 97/48807 PCT/US96/10561
a~r~.Y;~Al~ly ten pieces are placed in each flask. The flask is turned upside down,
closed tight and left at room temperature overnight. After 24 hours at room
temperature, the ilask is inverted - the chunks of tissue remain f~xed to the bottom
of the flask - and fresh media is added (e.g., Ham's F12 media, with 10% ~BS,
5 peni~ in and ~ll~lolllycil~). The tissue is then inc~b~ted at 37~C for
appro~,llalely one week. At this time, fresh media is added and sl~bseq~-ent~y
rhA~ every several days. Af~er an ad-liti( n~l two weelcs in culture, a monolayer
of ~b~ubla~t~ e-nt;~ S. The monolayer is trS/~ ~t;d and sca1ed into larger flasks.
A vector for gene therapy is digested with r~ostriction enzymes for
10 cloning a portion to be ~A~ -essed. The di~sted vector is treated with calf intestin~l
phosph~t~e to prevent self-ligation. The dephosphorylated, linear vector is
fractionated on an agarose gel and purified.
CK,B-15 protein cDNA capable of c,~lessiilg active CK,B-15 protein,
is i~te l The ends of the portion are modified, if necç~ry, for cloning into the15 vector. For i~ n~e, S" overh~n~ing may be treated with DNA polymerase to
create blunt ends. 3' overh~nging ends may be removed using Sl n~ e
Linkers may be ligated to blunt ends with T4 DNA ligase.
Equal ~ ntiti~s of the Moloney murine le -k~mi~ virus tinear backbone
and the CK~-15 protein portion are mixed together and joined using T4 DNA
20 ligase. The ligation mixture is used to t,~.~rolll- E coli and the bacteria are then
plated onto agar-co~ k~~ ycill. Kanamycin phenotype and restriction
analysis confirm that the vector has the pl .~pe- Iy inserted gene.
P~cl~gin~ cells are grown in tissue culture to conflllent density in
Dulbecco's Modi_ed Eagles Medium (DMEM) with 10% calf serum (CS),
25 p.onicillin and streptomycin. The vector co.~ the CK~-15 protein gene is
introduced into the p~ ginE~ cells by standard te~hniq~les Infectious viral
p&~ ,les co..~ g the CK,B-15 protein gene are collected from the p~ gin~
cells, which now are called producer cells.
Fresh media is added to the producer cells, and after an approp.iale
30 ;...~ ;on period media is harvested from the plates of confll~nt producer cells.
The media, cQ.~ the infectiQII~ viral particles, is filtered through a Millipore
filter to remove det~h~cl producer cells. The filtered media then is used to i~fect
CA 02258507 1998-12-14
- WO 97/48807 PCT/US96/10561 - -
Sk,
ffbroblast celts. Media is removed from a sub-conflllent plate of fibroblasts and
quict~y r~,placed with the filtered media. Polybrene (Atdrich) may be inc~ ed inthe media to fPc~ te tr~neduc~ion After app~op-idle inrl~b~tion, the media is
removed and . t"l. ced with fresh media. If the titer of virus is high, then virtualty
5 all ~r~l&,ts wilt be infected and no selection is required. If the titer is low, then
it is l~F.,C.~ to use a retroviral vector that has a s~tect~hle marker, such as neo
or his, to select out trQned~lced cells for eYp~neion
T ~ ru,-l,ed fibroblasts then may be injected into rats, either alone or
after having been grown to cnnfl~1~nce on microcarrier beads, such as cytodex 3
10 beads. The injected fibroblasts produce CK,B-15 protein product, and the
biological actions of the protein are conveyed'to'the' liost.
It witl be clear that the invention may be practiced othe~wise than as
particularly described in the fol egoing description and examples.
1~ Numerous modifications and variations of the present invention are
pc ~ in light of the above tePr.hinge and, therefore, are within the scope of the
appended claims.
The rlieclosl-res of atl patents, patent applications, and publications
~ere~led to herein are hereby incorporated by reference.
CA 022~8~07 l998-l2-l4
~ W O 97/48807 ~ 7 PCT~US96/1056l ~
SEQUENCE LISTING
(1~ GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: Human Genome Sciences, Inc
(B) STREET: 9410 Key West Avenue
(C) CITY: Rockville
(D) STATE: Maryland
(E) COUNTRY: USA
(F) POSTAL CODE (ZIP): 20850-3338
(G) TELEPHONE: 301-309-8504
(H) TELEFAX: 301-309-8512
(i) APPLICANT:
(A) NAME: Wei, Ying-Fei
(B) STREET: 13524 Straw Bale Lane
(C) CITY: Darnestown
(D) STATE: Maryland
(E) COUNTRY: USA
(F) POSTAL CODE (ZIP): 20878
(i) APPLICANT:
(A) NAME: Kreider~ Brent
(B) STREET: 13014 Praine Knoll Court
(C) CITY: Germantown
(D) STATE: Maryland
(E) COUNTRY: USA
(F) POSTAL CODE (ZIP): 20874
~i) APPLICANT:
(A) NAME: Rosen, Craig
(B) STREET: 22400 Rolling lIill Road
(C) CITY: Laytonsville
(D) STATE: Maryland
(E) COUNTRY: USA
(F) POSTAL CODE (ZIP): 20882
(ii) TITLE OF INVENTION: CHEMOKINE BETA 15
(iii) NUMBER OF SEQUENCES: 9
(iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1 0, Version ~1.30 (EPO)
(v) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: (To Be Advised)
(B) FILING DATE: 17-JUN-1996
CA 022~8~07 1998-12-14
~ WO 97/48807 ~ PCTAUS96/10561
(vii) CORRESPONDENCE ADDRBSS:
(A) ADDRESSEE: STERNE, KESSLER, GOLDSTEIN & FOX P L.L.C.
(B) STREET: 1100 NEW YORK AVENUE, SUITE 600
(C) CITY: WA~ lN-~ 1 ON
(D) STATE: DC
(E) C~UN'1'~Y: USA
(F) ZIP: 20005-3934
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Goldstein, Jorge A
(B) REGISTRATION NUMBER: 29,021
(C) REFERENCE/DOCKET NUMBER: 1488.042PC00
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 989 ~ase pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 88..534
(ix) FEATURE:
(A) NAME/KEY: sig_peptide
(B) LOCATION: 88..147
(ix) FEATURE:
(A) NAME/KEY: mat_peptide
(B) LOCATION: 148..534
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
CCGGCGGGCA TCAGCTCCCT TGACCCAGTG GATATCGGTG GCCCCGTTAT TCGTCCAGGT 60
~CCCAGGGAG GAGGACCCGC CTGCAGC ATG AAC CTG TGG CTC CTG GCC TGC 111
Met Asn Leu Trp Leu Leu Ala Cys
-20 -15
CTG GTG GCC GGC TTC CTG GGA GCC TGG GCC CCC GCT GTC CAC ACC CAA
159
Leu Val Ala Gly Phe Leu Gly Ala Trp Ala Pro Ala Val ~is T~Lr Gln
-10 -5
GGT GTC TTT GAG GAC TGC TGC CTG GCC TAC CAC TAC CCC ATT GGG TGG
207
CA 022~8~07 1998-12-14
- WO 97/48807 PCT/US96/10561
~ - 5
Gly Val Phe Glu Asp Cys Cys Leu Ala Tyr His Tyr Pro Ile Gly Trp
GCT GTG CTC CGG CGC GCC TGG ACT TAC CGG ATC CAG GAG GTG AGC GGG
255~la Val Leu Arg Arg Ala Trp Thr Tyr Arg Ile Gln Glu Val Ser Gly
AGC TGC AAT CTG CCT GCT GCG ATA TTC TAC CTC CCC AAG AGA CAC AGG
303~er Cys Asn Leu Pro Ala Ala Ile Phe Tyr Leu Pro Lys Arg His Arg
AAG GTG TGT GGG AAC CCC A~A AGC AGG GAG GTG CAG AGA GCC ATG AAG
351
Lys Val Cys Gly Asn Pro Lys Ser Arg Glu Val Gln Arg Ala Met Lys
CTC CTG GAT GCT CGA AAT AAG GTT TTT GCA AAG CTC CAC CAC AAC ACG
399
Leu Leu Asp Ala Arg Asn Lys Val Phe Ala Lys Leu His His Asn Thr
CAG ACC TTC CA~ GGC CCT CAT GCT GTA A~G AAG TTG AGT TCT GGA AAC
447
Gln Thr Phe Gln Gly Pro His Ala Val Lys Lys Leu Ser Ser Gly Asn
100
TCC AAG TTA TCA TCG TCC A~G TTT AGC AAT CCC ATC AGC AGC AGC AAG
495~er Lys Leu Ser Ser Ser Lys Phe Ser Asn Pro Ile Ser Ser Ser Lys
105 110 115
AGG AAT GTC TCC CTC CTG ATA TCA GCT AAT TCA GGA CTG TGAGCCGGCT
544
Arg Asn Val Ser Leu Leu Ile Ser Ala Asn Ser Gly Leu
120 125
CATTTCTGGG CTCCATCGGC ACAGGAGGGC CGGATCTTTC TCCGATAAAA CCGTCGCCCT 604
ACAGACCCAG CTGTCCCCAC GC~l~l~l~l TTTGGGTCAA GTCTTAATCC CTGCACCTGA 664
GTTGGTCCTC CCTCTGCACC CCCACCACCT CCTGCCCGTC TGGCAACTGG A~AGAGGGAG 724
TTGGCCTGAT TTTAAGCCTT TTGCCGCTCC GGGGACCAGC AGCAATCCTG GGCAGCCAGT 784
GGCTCTTGTA GAGAAGACTT AGGATACCTC TCTCACTTTC TGTTTCTTGC CGTCCACCCC 844
GGGCCATGCC AGTGTGTCCC TCTGGGTCCC TCCAAAACTC TGGTCAGTTC AAGGATGCCC 904
CTCCCAGGCT ATGCTTTTCT ATAACTTTTA AATA~ACCTT GGGGGGTGAT GGAGTCA~AA 964
AA~}~U~AAa A2~U~AAAA A~AAA 989
= ~
CA 02258507 1998-12-14
- WO 97/48807 PCT/US96/10561
<o C'
(2) INFO.~MATION FOR SEQ ID NO:2:
(i) SEQUENCE C~ARACTERISTICS:
(A) LENGTH: 149 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Met Asn Leu Trp Leu Leu Ala Cys Leu Val Ala Gly Phe Leu Gly Ala
-20 -15 -10 -5
~rp Ala Pro Ala Val His Thr Gln Gly Val Phe Glu Asp Cys Cys Leu
1 5 10
Ala Tyr His Tyr Pro Ile Gly Trp Ala Val Leu Arg Arg Ala Trp Thr
Tyr Arg Ile Gln Glu Val Ser Gly Ser Cys Asn Leu Pro Ala Ala Ile
Phe Tyr Leu Pro Lys Arg His Arg ~ys Val Cys Gly Asn Pro Lys Ser
~rg Glu Val Gln Arg Ala Met Lys Leu Leu Asp Ala Arg Asn Lys Val
~he Ala Lys Leu His His Asn Thr Gln Thr Phe Gln Gly Pro His Ala
go
Val Lys Lys Leu Ser Ser Gly Asn Ser Lys Leu Ser Ser Ser Lys Phe
100 105
Ser Asn Pro Ile Ser Ser Ser Lys Arg Asn Val Ser Leu Leu Ile Ser
110 . 115 120
Ala Asn Ser Gly Leu
125
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 95 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
CA 022~8~07 1998-12-14
- WO 97/48807 ~e ~ PCT/US96/10561 - -
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
Ala Thr Glu Thr Lys Glu Val Gln Ser Ser ~eu Lys Ala Gln Gln Gly
1 5 10 15
Leu Glu Ile Glu Met Phe His Met Gly Phe Gln Asp Ser Ser Asp Cys
Cys Leu Ser Tyr Asn Ser Arg Ile Gln Cys Ser Arg Phe Ile Gly Tyr
Phe Pro Ile Ser Gly Gly Cys Thr Arg Pro Gly Ile Ile Phe Ile Ser
Lys Arg Gly Phe Gln Val Cys Ala Asn Pro Ser Asp Arg Arg Val Gln
65 70 75 80
Arg Cys Arg Leu Glu Gln Asn Ser Gln Pro Arg Thr Tyr Lys Gln
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE C~ARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
~C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
GCCGTCGACG TCCACACCCA AGGTGTC 27
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
GCCTCTAGAG GAGCCCAGAA ATGAGCCGGC 30
CA 022~8~07 l998- l2- l4
- WO 97/48807 PCT/US96/10561
62
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
GCCTCTAGAG CCATCATGAA CCTGTGGCTC CTGGCC 36
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: ~ingle
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
GCCTCTAGAG GAGCCCAGAA ATGACCCGGC 30
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGT~: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
GCGAAGCTTA TGAACCTGTG GCTCCTGGCC - 30
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE C~ARACTERISTICS:
CA 022S8S07 l998-l2-14
~ W 097/48807 PCTrUS96/10561
(A) LENGTH: 61 base pairs 63
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
GCGCTCGAGT CAAGCGTAGT CTGGGACGTC GTATGGGTAC AGTCCTGAAT TAGCTGATAT 6C
C 61
CA 02258507 l998-l2-l4
~ W O 97/48807 PCT~US96/10~61
(~?Y
INDIC~TIONS RE ~ 'rING TO A DEl'OSI'r~D MICROO~G~NISM
(PCT RuIc13~is)
~The indioatjo.~c made hclow rclate IO the microor~anism referred to in the dcs~iplion
on pa~e 3 7 linc 18
1~ IDENTIFICATION OF DEPOSIT ~urthcr deposils are idenlified on an ~dr~ition~l sheet
Name o~ depositary instilulion
AMERICAN T~PE CULTURE C~T.r,~.~TT~
Address of depositary instilutiOn ~includ~n~g posla/ codc nnd coun~)
12 301 Parklawn Drive
Rockville, Maryland 20852
United States of America
D;~le of deposit ~cccssion ~lumbcr
Al?ril 25, 1996 ATCc 97519
G ADDITIONAL INDICATIONS (IcaYcblank if nol oppl;cablcJ Tbis information is ~ n~in~d on an add~ ' sbeet
HTSEX82 CDN2.
In respect o~ those designations in which a European Patent is sought a sample
of the deposited microorganism will be made avai~able until the publicati~n of
the mention of the grant o~ the European patent or until the date on which the
applicat~n has been refused or withdrawn or is deemed to be withdrawn, only by
the issue o~ such a sample to an expert nnm;n~ted by the person r~questing the
sample (Rule 28(4) EPC).
D. DESIGNATED STATES FOR W HICIIINDICATIONS A~E ~ADE(~Ih~ arcnr~f~ralld~i~cdSta~)
E. SEPAI~ATE FURNTS~IING OF INI)ICATIONS (Icavc blank if no~ applicablc)
'rbelndiCatiOnSIisledbelOWWillbesubmi~ledtothclnlernaliollalBureaulaler(sp~cifylhc8cncralnatu~coft~c; ~' ~.' G8'., ~4cc~rion
N~mbcrofDcposil )
~or receivin~ Office use only ~or International Bureau use only
~/nlis shcel was receivcd with tbe intcrnalional a~plication ~ Illis shcet was reccived by the Intcrnational Burcau on-
J~uthoriz~ uthori~cd officcr
i:orn~ I~C~ t>/134 (Illly 19~12)
