Note: Descriptions are shown in the official language in which they were submitted.
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--1--
METHODS FOR SELECTIVELY K~T ,~,TNG OR Il~l~ll~l l l~G
THE GROWTH OF CELLS EXPRESSING THE WAF1 GENE
.
Field of the Invention
The present invention relates to the selective killing or to the selective inhibition of the
growth of cells. More particularly, the present invention relates to methods and compositions
useful in killing or inhibiting the growth of cells expressing the WAF 1 gene. The present
invention has particular utility in the field of cancer therapy.
Ba~k~round of the Invention
In recent years, much progress has been made toward underst~n(1ing the cell cycle of
eukaryotic cells. In brief, the cell cycle consists of four phases: (1) the mitotic phase, M, in
which a cell with duplicated genetic m~teri~l undergoes mitosis to produce ~ ghtel cells, (2) a
first gap phase, Gl, during which the cell grows and is, generally, metabolically active, (3) a
synthesis phase, S, during which the cell dllplic~tes its genetic material, and (4) a second gap
phase, G2, in which the cell prepares for mitosis and, perhaps, another cell cycle. The control or
regulation of the cell cycle is a complex process involving dozens of intracellular and
extracellular signals which appear to act at particular "checkpoints" at the transitions between
~lirr~cllL stages of the cell cycle. Amongst the known components of cell cycle regulation are
the families of cyclins, cyclin-dependent kinases (CDKs), and cyclin-dependent kinase inhibitors
(CDIs). Although the details of cell cycle control remain to be resolved, it is clear that the
lc;s~ion or inhibition of these molecules, as well as their stoichiometries, play a major role in
controlling cell proliferation, dirrel~ iation and death (see, e.g., Hunter and Pines (1994) Cell
2s 78:539-542).
At the same time, it has become clear that many of these same regulatory components of
the norrnal cell cycle also play a role in oncogenesis. See, e.g.. Hunter and Pines (1994) Cell
79:573-582. The p53 "tumor suppressor" gene product, for example, not only has been linked to
Gl arrest but p53 mutants have been linked to a variety of cancers (see, e.g., Vogelstein, B. and
~ 30 Kinzler, K.W. (1992) Cell 70:523-526, Fisher, D.E. (1994) Cell 78:539-542). This gene appears
to play a role in the normal cellular response to DNA damage in m~mmz~ n cells such that,
when DNA damage has occurred, the p53 gene is expressed and the cell either arrests at the G,
stage of the cell cycle or enters the apoptotic cell death pathway. Heritable defects in p53 are
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believed to be causally involved with oncogenesis either by allowing cells with damaged DNA to
replicate and pass on the damaged genetic m~tPri~l or by ~levellLi~lg darnaged cells from
undergoing apoptosis. See, e.g., Csmm~n, et al. (1995) Genes & Devel. 9:600-611. The p53
protein is now known to be a transcriptional regulator and several genes have been i(lentified
s with putative p53-binding sites (El-Deiry et al. (1993) Cell 75:817-825).
WAF 1 is a CDI which appears to be involved in the arrest of the cell cycle at acheckpoint in Gl. Because of the varying means by which it has been i~lentifie-l, this CDI has
been known by a variety of names in the recent l i I P, ~I . . . e: WAF 1 (Wild-type p53 activated
1~gment 1; El-Deiry, et al. (1993) Cell 75:817-825), Cipl (CDK-interacting protein l; Harper, et
10 al. (1993) Cell 75:805-816), SDIl (Sen~scPnt cell-derived inhibitor l; Noda, et al. (1994) Exp.
Cell Res. 211: 90-98) and p21 (21,000 Da cyclin-dependent kinase inhibitor protein; see, e.g.,
Jiang, et al. (1995) Oncogene l Q: 1855-1864). For con~ tPnc y, this CDI will he~ arlel be
referred to as WAFl irrespective ofthe nomencl~tllre used in any references cited.
Harper et al. (1993) disclosed the nucleic acid and arnino acid sequences of a human
lS WAFl gene and protein. These researchers found that WAFl is a potent inhibitor of a variety of
cyclin-CDK complexPs and can inhibit the phosphorylation of the retinobl~t~-m~ gene product
(Rb). Inhibition of the CDKs and hypophosphorylation of Rb can lead to cell cycle arrest in Gl.
Consonant with this, Harper et al. found that WAF 1 caused a dose-dependent in vitro decrease in
human fibroblasts in the S phase ofthe cell cycle (implying WAFl-dependent Gl arrest) and
20 conclude that loss of WAFl function might contribute to cell proliferation even in the presence
of negative growth signals.
El-Deiry et al. (1993) also disclosed the nucleic acid and amino acid sequences of a
human WAFl gene. They noted that the gene included an upstream pS3-binding site and,
consonant with this, found that WAFl was strongly induced both by pS3 and by W irradiation
2s (which also in~ cç~ pS3). In addition, these authors found that WAFl expression inhibited the
growth of a variety of human tumor lines in vitro. These results, and others, suggested that
WAFl might be a downstream mediator of Gl arrest controlled by the pS3 tumor suppressor.
Curiously, the introduction of WAFl-s~nti~n~e had no effect on cell growth. Hence, WAFl is
not required for normal cell proliferation. Furthermore, in the absence of wild-type pS3,
30 induction of Gl arrest by serum starvation or trç~tmcnt with mimosine did not induce WAFl.
This suggested that WAF1 ~;xples~ion might be one of a series of mech~ni~m~ by which cells
car~y out Gl arrest. Despite these complications, these authors conclude that the "identification
-
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of WAF 1 and its regulatory regioi~ potentially provides==a- novel drug discovery approach:
compounds that activate expression of WAF 1 might bypass the p53 defect in tumors with
endogenous p53 mutation" (emI~h~ added).
Sheikh et al. investig~t~l the regulation of WAF1 eA~les~ion in human breast carcinomas
s (Sheikh et al. (1994) Onco~ene 9:3407-3415). Cells expressing wild-type p53 were found to
c~ n~ ; v~;ly express WAF 1 at levels 26-33 fold higher than p53 I l l ~ ; In addition,
exogenous mutant p53 (Val-143) counteracted the positive transcriptional effect of endogenous
wild-type p53. Nonetheless, these authors found that WAF1 could be in~ ced both by p53-
independent as well as p53-dependent sign~lin~ palhwdy~. In particular, they found that the
0 DNA--l~nn~in~ agent etoposide and serum starvation could induce both WAF1 expression and
growth arrest even in p53 mutant cell lines.
Recently, Jiang et al. (1995) found that WAFl is dirr~,~Glllially expressed during growth,
dirr~ ltiation and progression in human melanoma cells. Using subtractive cDNA
hybricli7~tions and anti-p53 and anti-WAF1 antibodies, these authors found (1) lower levels of
5 WAF1 expression in prolir~dLhlg and metastatic human melanomas as compared to normal or
immortalized melanocytes and (2) an increase in WAF1 protein levels in melanoma cells after
induction of growth arrest and t~nnin~l diLrelcllliation (using recombinant human fibroblast ~-
illlelre~ (IFN-,B) and mezerein to induce t~nnin~l diLr~..,.lliation). In addition, levels of p53
and WAF1 were found to be inversely correlated during growth arrest and dirrel~llLiation of
human melanoma cells, indicating that WAF 1 induction may occur independent of p53
t;A~lGs:iion. In light of these and other results, Jiang et al. suggest that agents that can increase
WAF 1 expression may prove beneficial in metastatic melanoma therapy by directly inducing an
irreversible loss of proliferative capacity and t~?nnin~l cell dirrt;l~llLiation.
Using a WAF1-~nti~n~e expression vector, N~k~ni~hi et al. invçsti~ted the effect of
2s WAF1 inhibition on normal human fibroblasts grown to Go arrest in vitro (N~k~ni~hi et al.
(1995) Proc. Natl. Acad. Sci. (USA) 92:4352-4356). These authors found higher levels of
WAF1 protein in cells arrested in Go than in mitogen-stimnl~ted cells in early S phase. In
addition, N~k~ni~hi et al. found that expression of WAFl-antisense RNA caused cells arrested in
Go to resume the cell cycle and proliferate.
The role of WAF 1 in the t~rrnin:~l dirrele~lliation and concomitant cell cycle arrest of
normal tissue was investigated by Parker et al. (1995) Science 267:1024-1027. Using in situ
hybridization with a WAF1 probe, Parker et al. studied the tissue-specific pattern of WAF1
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--4--
expression in mouse embryos at varying stages of development. The ~x~l~;s~ion of WAF 1 was
found to correspond to the presence of post-mitotic, t~rrnin~lly dirr~ te~l cells (e.g., muscle,
neurons). The pattern of WAF 1 ~X~l ession did not, however, correspond to that of p53
~;x~l~;ssion and mouse embryos lacking the p53 gene showed normal WAFI expression and
embryogenesis. Similarly, WAF1 t;x~l~;s~iion in adult mice was found to be loc~li7~1 to
t~-rmin~lly dirr~L . . I .~t~l tissues and to be unaltered in p53-knockouts. Thus, these authors
concluded that WAFl has a role in normal termin~l dirr~ lliation and embryogenesis but that
WAF1 induction is not dependent upon p53 induction.
Another example of p53-independent in~l~lc fion of WAF 1 was demon~tr~t~c~ in
10 embryonic fibroblasts from p53 knock-out mice (Michieli et al. (1994) Cancer Res. 54:3391-
3395). Michieli et al. found that serum, platelet-derived growth factor (PDGF), fibroblast growth
factor (FGF) and epidermal growth factor (EGF) were able to induce WAF1 in p53-deficient as
well as normal cells. In contrast, y-irr~ tion was able to induce WAF1 in normal cells but not
in cells lacking wild-type p53. These authors conclude that WAFl may be in~luce~l either by a
p53-dependent pathway activated by DNA damage or by a p53-independent ~Lllw~y activated
by mitogens. The fact that WAF1 is in~ ce~l by mitogens is curious in light of its al~pal~llL role
in G~ arrest and suggests that WAFl may have an unknown function independent of G~ arrest.
Many additional reports in the last several years have supported the hypothesis that
WAFl is an important element in regulating the cell cycle transition from G~ to S phase and that
20 WAFl can be in~ c e~l by a p53-dependent p~Lllw~y activated by DNA damage as well as p53-
independent ~Lhw~y~ activated by cell dirrt;l~llLiation signals. In addition, to those discussed
above, p53-independent inducers of WAF1 have now been shown to include transforming
growth factor 13 (TGFO in keratinocytes (Datto et al. (1995) Proc. Natl. Acad. Sci. (USA~
92:5545-5549); 12-0-tetradecanoyl phorbol-13-acetate (TPA), 1,25-dihydroxyviL~llill D3 (Vit
2s D3), retinoic acid (RA) and dimethyl-sulfoxide (DMSO) in promyelocytic lellkemi~ cells (~iang
et al. (1994) Oncogene 9:3397-3406); and MyoD in skeletal muscle (Halevy et al. (1995) Science
267:1018-1021).
Summarv of the Invention
The present invention provides methods of killing or inhibiting the growth of cells in
which the WAF1 gene is being expressed. The methods comprise the ~mini~tration a WAF1
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W O 97/03681 PCTAUS96/11886
inhibitor to cells in which a WAFl-dependent pathway has been in~ ecl and wherein said
inhibitor is ~flmini.~tered in an amount sufficient to kill or inhibit the growth of said cells.
e In one set of embo-1iment~, the inhibitor comprises a WAFl-antisense oligonucleotide.
In particular, the oligonucleotide may be a modified oligonucleotide in which the backbone
s linkages, termini or bases have been modified to increase re~i.et~nce to degradation or to increase
binding affinity. Particularly ~l~rell~d modified oligonucleotides are those c~ a plurality
of phosphorothioate linkages. In all embo~lim~nt~ in which the WAF 1 inhibitor is an ~nti.cen.~e
oligonucleotide, it is preferred that the oligonucleotide is selected from the group con~i~ting of
(a) oligonucleotides comprising at least 10 consecutive bases from the WAFl sequence disclosed
0 as SEQ ID NO.: 1, and (b) oligonucleotides capable of hybridizing to the complements of the
oligonucleotides of (a) under physiological conditions. In another set of embolliment~, the
inhibitor is a vector which expresses a WAFl~ nti.~en~e oligonucleotide.
In an ~lt~rn~tive set of embo-liment~, the inhibitor is an inhibitor of WAFl gene
transcription. In another set of embo-liment~, the inhihitor is an inhibitor of WAFl protein
activity such as an intracellular anti-WAFl antibody or a fragment of either WAFl or a CDK
which competitively inhibits the formation of complexes between the WAF 1 protein and
endogenous CDKs. Other examples are described below.
In further embo~liment.~, the method may include subjecting the cells to a trç~tment which
induces a WAF 1 -dependent pathway. This pathway may be a pS3-dependent pathway and the
treatment may be one which induces pS3 gene ~x~l~s~ion. Such trç~tment.~ may include X-
irradiation, y-irradiation, UV-irradiation, ~lmini.ctering to the cells an alkylating agent,
~rlmini~t~ring to the cells cisplatin, ~lmini.~t~ring to the cells bleomycin, doxorubicin,
mini~tering to the cells 5-fluorouracil, ~lmini.~tering to the cells geni~tein, ~fimini~tering to the
cells hydrogen peroxide, or ~cimini~tering to the cells methylmethane sulfonate. Alternatively.
2s the ~dLllw~y may be a p53-independent pathway. For such pathways, the treatment may include
mini.~t~rin~ to the cells differentiation-inducing agents or inhibitors of DNA synthesi.~ Such
treatments may include ~clmini.~tering to the cells a ph~n~eutical composition selected from
the group con~ ting of PDGF, FGF, EGF, NGF, ~-interferon, TGF~, TPA, Vit D3, RA. DMSO,
MyoD, IL2, rapamycin, aphidicolin, etoposide, methuL~ , cytosine arabinoside, 6-thioguanine, 6-mel~;a~L~ ine.
For all of the above-described embodiments, the method is particularly intended for use
with cells which abnormally proliferate, and in a particularly important embodiment with cancer
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cells in a human host. Pler~ ed cancer cells include neurobl~etom~ melanoma, epithelioma,
fibrobl~t~m~ carcinnm~ lellkemi~ and myeloma cells.
Thus, the present invention provides a method of treating a human patient havingcancerous cells in which a WAF 1 -dependent pathway has been in~1~1ce-1 comprieing
5 zl~minietering a WAF1 inhibitor to the patient in an amount sufficient to kill or inhibit the growth
of the cells. The WAFl inhibitor may be any of those described above. In addition, the
tre~tment may also include subjecting the patient to a tre~tment which induces a WAF 1-
dependent ~dlllwdy. This additional tre~tmenf may include any of those described above. In
particular, the additional treatment may include radiation or chemotherapy therapy which induces
0 DNA damage in the cells or which in~ cçe growth arrest or dirrt:l~llLiation of the cells. As in all
embo-1iment~ described above, the ~l~r~lled WAFl inhibitor a WAFl-:~ntieenee oligonucleotide
and, preferably, a modified WAF 1 -antisense oligonucleotide with a plurality of phosphorothioate
link~gçs
The invention further involves use of the foregoing compositions in the pl~dldlion of
5 medic~mente and in particular the ~lc~dLion of mediç~ment~ for treating ~hn/~ l cell
proliferation such as cancer.
Detailed De~ .I,lion of the Invention
The present invention depends, in part, upon the surprising discovery that the selective
20 inhibition of the ~lession of WAFl in cells in which a WAFl-dependent pdLllw~y has been
in~11lre~ does not lead to cell proliferation but, rather, to cell death. This result is particularly
surprising in that WAF 1 is believed to play a role in the Gl arrest and/or t~rmin~l ~lirr~ Liation
of cells and, therefore, it has previously been proposed that inducing the WAF 1 gene or
otherwise increasing the levels of the WAF 1 protein might be a useful means of controlling the
2s growth of cell lines in vitro and, more important, tumor cells in vivo. The present invention, in
contrast, provides methods of killing or inhibiting the growth of cells by inhibiting transcription
of the WAF 1 gene, translation of the WAF 1 mRNA transcript, or activity of the WAF 1 protein.
Without being bound to any particular theory of the invention, applicants believe that conditions
or trç~tment~ which result in the induction of WAFl also induce other components of a Gl arrest
30 or dirr~ lLiation pathway. Although WAFl is only one component in these "WAFl-dependent"
~dlhwdy~, it appears e~sçnti~l Therefore, when WAFl is inhibited after induction of a WAF1-
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-7-
dependent p~lhw~y~ it is believed that the cells cannot complete the Gl arrest or dir~elcllLiation
p~Lhw~y~ and, in.etç~-l, initiate programmed cell death or apoptosis.
Clearly, the present invention is useful only with cells in which the WAF 1 gene is being
expressed. Thus, in some embo-limenf~, the target cells of the invention may, without prior
5 tre~nent already be expressing WAFl . In other embotliment~, however, the methods of the
invention include an additional treatment which induces the expression of a WAF 1 -dependent
pathway. Such additional tre~tment will typically comprise radiation or chemotherapies.
Definitions:
lo In order to more clearly and concisely describe the subject matter of the present
invention, the following definitions are provided for specific terms used in the claims appended
hereto:
WAFl. As used herein, the abbreviation "WAF1" means the human cyclin-dependent
lcinase inhibitor gene described in the various references cited herein and denoted as "WAFl,"
15 "Cipl ," "CIPl ," "SDIl," or "p21." A cDNA to one allele of WAFl was disclosed in Harper et
al. (1993) and El-Deiry et al. (1993). In addition, one WAFl allele and the corresponding
protein are disclosed herein as SEQ. ID NO.: 1 and SEQ ID NO.: 2, respectively. The translation
initiation codon of this cDNA is found at base positions 76-78 and the stop codon is at positions
568-570, defining an open reading frame of 492 bases. As will be obvious to one of ordin~y
20 skill in the art, other functional alleles of WAF 1 are likely to exist in the human population and
are embraced by the abbreviation "WAFl" as used herein.
WAFl-dependent pathway. As used herein, the term "WAFl-dependent p~lhw~y" means
a biochemical ~lhw~y in human cells in which ~x~ ion of the WAFl gene is in~11lçe~ Such a
pathway may require induction of WAF 1 expression by the pS3 tumor suppressor protein, in
25 which case the pathway is said to be "p53-dependent." Alternatively. the pathway may not
require induction of WAFl expression by the p53 tumor suppressor protein, in which case the
p~l~lw~y is said to be 'p53-independent."
WAF 1 inhibitor. As used herein, the terrn "WAF 1 inhibitor" means a compound which,
when present in a cell, inhibits the transcription of the WAF 1 gene, translation of the WAF 1
30 mRNA transcript, or activity ofthe WAF1 protein product. Examples include WAF1-~nti~l~n~e,
antibodies or fr~P~ment~ of antibodies which act intracellularly against the WAF1 protein or
WAFl-cyclin-CDK complexes, fragments of CDKs which would act as competitive inhibitors of
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WAF1 interaction with endogenous CDKs, small molecule inhibitors such as OK-1035[Biochemical and Biophysical Research Coll..llullications 221, 207-212 (1996)] and ribozymes
which inhibit WAFl c~lc~ion.
WAFl-z~nti.ct-nse oli~onucleotide. As used herein, the term "WAFl-antisense
oligonucleotide" or "WAF 1 -antisense" describes an oligonucleotide that is an
oligoribonucleotide, oligodeoxyribonucleotide or a modified oligonucleotide which hybridizes
under physiological conditions to a WAFl mRNA transcript or to WAFl DNA and, thereby, acts
as a WAFl inhibitor. The ~nti~n~e molecule, of course, is constructed and arranged so as to
L~,r~,,c with transcription or translation of WAFl upon hybridization with the target. Those
o skilled in the art will recognize that the exact length of the ~ntieen~e oligonucleotide and its
degree of complement~rity will depend upon the specific target selected, including the sequence
of the target and the particular bases which comprise that sequence. It is ~lcr~ cd that the
~nti~f~n~e oligonucleotide be selected so as to hybridize selectively with the target under
physiological conditions, i.e., to hybridize ~u~s~ lly more with the target sequence than with
any other sequence in the target cell under physiological conditions.
I. Induction of WAFl-Dependent P~Lhw~y~
The pS3 protein is a potent inducer of WAF1 cx~lcs~ion. The.crulc, pS3 or inducers of
pS3 may be used to induce WAF 1 -dependent p~lhw~y~ A variety of pS3 inducers are known in
the art and, because of pS3's activity as a tutnor ~u~lcs~or, are already in use in the field of
cancer therapy. Plcr~,llcd pS3 inducers are DNA--l~m~in~ radiation (e.g., X-rays, y-rays, W),
DNA-~ m~ging compounds (e.g., alkylating agents such as nitrogen mustard, chlorambucil,
melph~ n, cyclopho~ph~mitle, bllclllf~n, and nitrosureas (BCNU, CCNU, methyl-CCNU); as
well as etoposide, cisplatin, bleomycin, doxorubicin, S-fluorouracil, genistein, hydrogen
peroxide, and methylmethane sulfonate).
In addition, as noted above, WAF1 has been shown to be in~lcecl by pS3-independent
hw~ys. Additional l~lcrtllcd inducers of WAF1 therefore include WAFl-inducing growth
factors and diLrclcllliation factors such as PDGF, FGF, EGF, nerve growth factor ~NGF), 13-
hllclr~ )n, TGF,B, TPA, Vit D3, RA, DMSO, MyoD and IL2, rapamycin and inhibitors of DNA
synthesis such as aphidicolin, methotrexate, cytosine arabinoside, 6-thioguanine, 6-
30 mclc~lo~ e.
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_g
II. Inhibition of WAF I Expression
The present invention depends, in part, upon the discovery that the selective inhibition of
the t;~e~ion of WAFl in cells in which a WAFl-dependent p~lhw~y has been in~ e(l leads to
the inhibition of cell growth and/or cell death. Thus, the present invention requires that the
5 targeted cells be subject to conflicting conditions: conditions in~ c.ing WAFl-dependent G,
arrest or dirr~lc.-liation and conditions under which WAFl expression is inhibited.
In most ~lert,led embo(1iment~, the WAFl-inhibiting conditions comprise trez~tment with
WAFl-antisense oligonucleotides. Based upon SEQ. ID NO.: 1, one of skill in the art can easily
choose and synthe~i7~ any of a number of a~ ul"iate antisense molecules. In order to be
lo sufficiently selective and potent for WAFl inhibition, such WAFl-~nti~en~e oligonucleotides
should comprise at least 10 bases and, more preferably, at least 15 bases. Typically, zlntieçn~e
molecules are between 15 and 32 bases. Most preferably, the ~nti~en~e oligonucleotides
c- mpri~e 18-20 bases. Although oligonucleotides may be chosen which are ~ntiS~n~e to any
region of the WAFl gene or mRNA transcript, in ~l~r~l.ed embo-liment~ the ~nti~n~e
15 oligonucleotides correspond to the N-termin~l or, more preferably, tr~n~l~tion initiation region of
the WAFl mRNA or to mRNA splicing sites. In addition, WAFl ~nti~n~e may, preferably, be
targeted to sites in which mRNA secondary structure is not expected (see, e.g., Sainio et al.
(1994) Cell. Mol. Neurobiol. 14(5):439-457) and at which proteins are not expected to bind.
As will be obvious to one of oldin~u y skill in the art, the WAFl-inhibiting antisense
20 oligonucleotides of the present invention need not be perfectly complement~ry to the WAF1
gene or mRNA transcript in order to be effective. Rather, some degree of mi~m~tches will be
acceptable if the antisense oligonucleotide is of sufficient length. In all cases, however, the
oligonucleotides should have sufficient length and complement~rity so as to selectively hybridize
to a WAFl transcript under physiological conditions. Preferably, of course, mi~m~telle~ are
25 absent or minim~l In addition, although it is not recommended, the WAFl-~nti~en~e
oligonucleotides may have one or more non-complementary sequences of bases inserted into an
otherwise complementary WAFl-antisense oligonucleotide sequence. Such non-complementary
sequences may "loop" out of a duplex formed by a WAFl transcript and the bases fl~nkin~; the
non-complementary region. Therefore, the entire oligonucleotide may retain an inhibitory effect
30 despite an ~ Lly low percentage of complement~rity.
The WAF 1 -antisense oligonucleotides of the invention may be composed of
deoxyribonucleotides, ribonucleotides, or any combination thereof. The 5' end of one nucleotide
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W O 97/03681 PCT~US96/11886
-10-
and the 3' end of another nucleotide may be covalently linked, as in natural systems, via a
phosphodiester internllcleotide linkage. These oligonucleotides may be prepared by aIt
recognized methods such as phosph~ramidate, H-phosphonate chemi~try, or
methylphosphor~mi-l~te ch.omi~try (see, e.g., Uhlm~nn et al. (1990) Chem. Rev. 90:543-584;
5 Agrawal (ed.) Meth. Mol. Biol., Humana Press, Totowa, NJ (1993) Vol. 20; and U.S. Patent No.
5,149,798) which may be carried out m~nllally or by an automated synth~ei7~r (reviewed in
Agrawal et al. (1992) Trends Biotechnol. 10:152-158).
The WAFl -antisense oligonucleotides of the invention also may include modified
oligonucleotides. That is, the oligonucleotides may be modified in a number of ways which do
0 not conl~,lumise their ability to hybridize to nucleotide sequences contained within the
tran~crirtion initiation region or coding region ofthe WAFl gene. The term "modified
oligonucleotide" as used herein describes an oligonucleotide in which at least two of its
nucleotides are covalently linked via a synthetic linkage, i.e., a linkage other than a
phosphodiester linkage between the S' end of one nucleotide and the 3' end of another nucleotide.
5 The most p~r~felled synthetic linkages are phc-sph~rothioate linkages. Additional ~ f~ d
synthetic linkages include alkylphosphonates, phosphorodithioates, phosphate esters,
aL~ylphosph~-nothio~t~, phosphor~mitlates, carbamate~ carbonates, phosphate triest~r
~cet~mid~tr-7 and carboxymethyl esters. Oligonucleotides with these linkages or other
modifications can be prepared according to known methods (see, e.g., Agrawal and Goodchild
20 (1987) Tetrahedron Lett. 28:3539-3542; Agrawal et al. (1988) Proc. Natl. Acad. Sci. (USA)
85:7079-7083; Uhlmann et al. (1990) Chem. ~ev. 90:534-583, Agrawal et al. (1992) Trends
Biotechnol. 10:152-158; Agrawal (ed.) Meth. Mol. Biol.. Humana Press, Totowa, NJ (1993) Vol.
20).
The term "modified oligonucleotide" also encomp~e~ oligonucleotides with a modified
25 base and/or sugar. For example, modified oligonucleotides include oligonucleotides having the
sugars at the most 3' and/or most 5' positions attached to chemical groups other than a hydroxyl
group at the 3' position and other than a phosphate group at the 5' position. Other modified
ribonucleotide-co..l~;..i..g oligonucleotides may include a 2'-O-alkylated ribose group such as a
2'-O-methylated ribose, or oligonucleotides with arabinose instead of ribose. In addition,
30 unoxidized or partially oxidized oligonucleotides having a substitution in one nonbridging
oxygen per nucleotide in the molecule are also considered to be modified oligonucleotides.
CA 02227370 1998-01-20
W O 97/03681 PCTrUS96/11886
-11-
Such modifications may be at some or all of the int~rmll~leoside linkages, at either or both
ends of the oligonucleotide, and/or in the interior of the molecule (reviewed in Agrawal et al.
(1992) Trends Biotechnol. 10:152-158 and Agrawal (ed.) Meth. Mol. ~iol.. Hllm~n~ Press,
Totowa, NJ (1993) Vol. 20). Also considered as modified oligonucleotides are oligonucleotides
s having nuclease resi~t~nce-conferring bulky substi~lent~ at their 3' and/or 5' end(s) and/or
various other structural modifications not found in vivo without human intervention. Other
modifications include additions to the int~rnll~leoside phosphate linkages, such as cholesteryl or
mine compounds with varying numbers of carbon residues between the amino groups and
t~rmin~l ribose.
o The inhibition of WAFl ~ es~ion need not be accomplished by means of a WAFl-
s~nti~ence oligonucleotide. Rather, inhibitors of WAFl transcription or WAFl protein activity
also may be employed to the same effect. For example, antibodies or fr~gment~ of antibodies
which act intracellularly against the WAFl protein or WAFl-cyclin-CDK complexes, fr~gment~
of CDKs or recombinant genes encoding frQgment~ of CDK which would act as competitive
inhibitors of WAFl interaction with endogenous CDKs, and ribozymes which inhibit WAFl
t~ ion.
Without being bound to any particular theory of the invention, it is believed that
tre~tment~ which result in the induction of WAFl also induce other components of a G, arrest or
diLf~;lellLiation ~aLllw~y. Although WAFl is only one component in these "WAFl-dependent"
~LLhw~y~, it appears essential. When WAFl is inhibited, it is believed that the cells cannot
complete the G~ arrest or dir~lGllLiation ~Lllw~y~ and, instead, initiate programmed cell death.
As a general rule, therefore, the WAF 1 -inhibiting conditions must be more specific to WAF 1
than the WAF1-inducing contlition~ so that the WAFl-dependent pathway is induced while
WAFl itself is inhibited. Thus, the WAFl-inclllcing conditions and WAF1-inhibiting conditions
2s should not act at the same point or level in the WAFl-dependent ~Lhw~y. For example,
simultaneous ~lmini~tration of the p53 protein (as the WAFl-inducing condition) and
~imini~tration of anti-p53 antibodies (as the WAFl-inhibiting condition) would be ineffective
because the conditions would largely counteract each other at the same level. In contrast,
induction of the p53 gene (as the WAF 1 -inducing condition) and ~-lmini~tration of WAF 1
antisense oligonucleotides (as the WAFl-inhibiting condition) would be effective because p53 is
a pleiotropic inducer and the WAF 1 -antisense oligonucleotides would not inhibit all components
of the WAF 1 -dependent pathway.
CA 02227370 1998-01-20
W O 97/03681 PCT~US96/11886 -12-
III. Methods of Treatment in Cancer Therapy
The methods of the present invention are particularly well suited for use in the field of
cancer therapy. Because current radiation and chemotherapy methods typically involve
tre~tmente which cause DNA damage and/or induce t~rrninzll ~lirr~lcllLiation of tumor cells and/or
5 inhibit proliferation of tumor cells, these trç~tment~, in many cases, already induce expression of
the WAF1 gene. Therefore, by combining these tre~tmente with a-lminietr~tion of a WAFl-
inhibitor, a more effective means of killing or inhibiting the grow~ of tumor cells is provided.
In one set of plc;r~ d embodiments, a cancer patient is treated with ionizing radiation
(e.g., X-rays or y-rays) or an agent (e.g.,doxorubicin) which causes DNA damage, induces pS3
10 expression and, thereby, induces a p53-depen~lent, WAF1-dependent pathway toward cell cycle
arrest. At the same time or shortly thereafter, a WAFl inhibitor is a-lminietered to the patient.
This ~1mini~tr~tion may be oral, intravenous, p~uc;llL~ ~dl, cutaneous or subcutaneous. The
~ 1minietr~tion also may be localized to the region of the tumor by injection to or perfusion of the
tumor site. Preferably, the WAFl inhibitor is WAFl-~ntieçnee ~-1miniet~red in a
15 ph~rrn~eutically acceptable carrier or a recombinant vector with a WAFl-~ntie-onee gene which
expresses a WAFl-antisense oligonucleotide.
In another series of embo~limente~ a cancer patient is treated with compounds which
induce expression of a pS3-independent, WAF 1 -dependent p~Lhw~y. These embot1imente may
be of particular importance because pS3 mutations are associated with many cancers and mutant
20 pS3 proteins may fail to induce WAF1 expression. In plcr~ d embo-limente, the patient has
been treated with an agent that induces pS3-independent G~ arrest or .lirreL~llLiation of the tumor
cells and at the same time, or shortly thereafter, is arlminietered a WAFl inhibitor. A~,plo~liate
non-proliferation and differentiation agents are well known in the art and vary according to
tumor type. For example, ~I~-interferon and mezerein may be a~lminietered to melanoma patients,
25 or TGF,~ may be ~lmini~tered to any of a number of dirrefellL types of cancer patients in order to
induce a WAF 1 -dependent pathway. A WAF 1 inhibitor, as described above, may beeimnlt~neously or subsequently a~lmini etered to kill or further inhibit the growth of the tumor
cells.
The cancer may be virtually any cancer, including, but not limited to, brain cancer
30 including glioblastoma and medulloblastoma, breast cancer, cervical cancer, colon cancer,
en~ metrial cancer, liver cancer, lung cancer, oral cancer, prostate cancer, sarcomas, skin cancer,
and renal cancer.
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W O 97/03681 -13- PCTAUS96/11886
WAFl-antisense oligonucleotides or other WAFl inhibitors may be fl-1mini~tered as part
of a rh~rm~rentical composition. Such a rh~rm~relltical composition may include the WAFl
inhibitor in combination with any standard physiologically and/or rh~rm~r,elltically acceptable
carriers which are known in the art. The term "ph~rm~reutically acceptable" means a non-toxic
s m:~teri~l that does not ill~clr~;;rc with the effectiveness of the biological activity of the active
ingredients. The term "physiologically acceptable" refers to a non-toxic material that is
c~,lll~dlible with a biological system such as a cell, cell culture, tissue, or org~ni~m The
characteristics of the carrier will depend on the route of ~q~minictration. Physiologically and
rh~rm~reufically acceptable carriers include diluents, fillers, salts, buffers, stabilizers,
o solubilizers, and other m~teri~l~ which are well known in the art. The rh~rm~r,elltical
composition of the invention may also contain other active factors and/or agents which inhibit
WAFl expression or otherwise inhibit cell growth or increase cell death. Such additional factors
andlor agents may be included in the rh~rm~reutical composition to produce a synergistic effect
or to . . . i . .i . . . i~ side-effects caused by the WAF 1 inhibitor of the invention.
The phz~rm~relltical composition of the invention may be in the form of a liposome in
which WAFl-~nti~n~e oligonucleotides are combined, in addition to other ph~n~reutically
acceptable carriers, with ~mphir~thic agents such as lipids which exist in aggregated form as
micelles, insoluble monolayers, liquid crystals, or l~mell~r layers which are in aqueous solution.
Suitable lipids for liposomal formulation include, without limitation, monoglycerides,
20 diglycerides, sulfatides, lysolecithin, phospholipids, saponin, bile acids, and the like. PlcpaldLion
of such liposomal formulations is within the level of skill in the art, as disclosed, for example, in
U.S. Patent No. 4,235,871,U.S. Patent No. 4,501,728, U.S. Patent No. 4,837,028, and U.S.
Patent No. 4,737,323.
The ph~rm~ceutical composition of the invention may further include compounds such as
25 cyclodextrins and the like which enhance delivery of oligonucleotides into cells, as described by
Zhao et al. (in press). When the composition is not ~tlmini~tPred systemically but, rather, is
injected at the site of the target cells, cationic dcLelgcllls (e.g. Lipofectin) may be added to
enhance uptake.
When a therapeutically effective amount of a WAFl inhibitor is ~-lmini~tered orally, the
30 inhibitor will be in the form of a tablet, capsule, powder, solution or elixir. When ~tlmini~tered
in tablet form, the phzlrm~reutical composition of the invention may additionally contain a solid
carrier such as a gelatin or an adjuvant. The tablet, capsule, and powder may contain from about
CA 02227370 1998-01-20
W O 97/03681 PCTAUS96/11886
-14-
5 to 95% of a WAF 1 -antisense oligonucleotide and preferably from about 25 to 90% of the
oligonucleotide. When iq-lmini~tered in liquid form, a liquid carrier such as water, petroleum,
oils of animal or plant origin such as peanut oil, miner~l oil, soybean oil, sesame oil, or synthetic
oils may be added. The liquid form of the ph~rmz~rell~ical composition may further contain
s physiological saline solution, dextrose or other saccharide solution, or glycols such as ethylene
glycol, propylene glycol or polyethylene glycol. When ~f~mini~tered in liquid form, the
ph~rm~celltical composition may contain from about 0.5 to 90% by weight of a WAFl-antisense
oligonucleotide and preferably from about 1 to 50% of the oligonucleotide.
When a thcl~euLically effective amount of a WAFl inhibitor is ~-imini~t~-red by
0 hlLIdVCllOUS, cutaneous or subcutaneous injection, the inhibitor will be in the form of a pyrogen-
free, ~c~ dlly acceptable aqueous solution. The ~ lion of such ~ clllcl~lly acceptable
solutions, having due regard to pH, isotonicity, stability, and the like, is within the skill in the art.
A ~l~fc.led ph~rm~el~tical composition for illLl~VClloUS, cutaneous, or subcutaneous injection
should contain, in addition to the WAF 1 inhibitor, an isotonic vehicle such as Sodium Chloride
15 Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection,
T ~ct~tç~l Ringer's Injection, or another vehicle as known in the art. The ph~rm~relltical
composition of the present invention may also contain stabilizers, preservatives, buffers,
antioxidants, or other additives known to those of skill in the art.
In pler~lled embodiments, when the target cells are readily ~ccç~ihle, sl~lmini~tration of
20 WAFl -~nti~en~e oligonucleotides is localized to the region of the targeted cells in order to
m;lxi~ .e the delivery of the WAF 1 -~nti.~n~ç, minimi~(~ WAFl inhibition in non-target cells,
and . . . i .~ e the amount of WAF l-antisense needed per tre~tment Thus, in one plefellèd
embodiment, ~imini~tration is by direct injection at or perfusion of the site of the targeted cells,
such as a tumor. Al~ ively, the WAFl-~nti~Pn~e oligonucleotides may be adhered to small
25 particles (e.g.,microscopic gold beads) which are impelled through the membranes of the target
cells (see, e.g., U.S. Pat. No. 5,149,655).
In another series of embo-liment~, a recombinant gene is constructed which encodes a
WAFl-~nti~çn~e oligonucleotide and this gene is introduced within the targeted cells on a vector.
Such a WAF1-~nti~çn~e gene may, for example, consist of the normal WAFl sequence, or a
30 subset of the normal WAFl sequence, operably joined in reverse orientation to a promoter
region. An operable WAFl-~nti~n~e gene may be introduced on an integration vector or may be
introduced on an ~ s:jion vector. In order to be most effective, it is ~ler~lled that the WAFl-
CA 02227370 l99X-01-20
W O 97/03681 PCT~US96111886
-15-
~ntisen~e sequences be operably joined to a strong eukaryotic promoter which is inducible or
constitutively e~pressed.
In all of the above-described methods of tre~tment, the WAF1 inhibitors are ~flministered
in therapeutically effective amounts. In addition, in those methods in which the patient also is
5 subjected to trç~tment that induces a WAF1-dependent pathway, these tre~tm~nts are also
~1ministered in therapeutically effective amounts. As used herein, the term "therapeutically
effective amount" means the total amount of each active component of the ph~rm~elltical
composition or method that is sufficient to show a meaningful patient benefit, i.e., the killing or
inhibition of the growth of the target cells. When applied to an individual active ingredient,
1 o ~ mini.stered alone, the term refers to that ingredient alone. When applied to a combination, the
term refers to combined amounts of the active ingredients that result in the therapeutic effect,
whether iq-lmini.stered in combination, serially or simultaneously.
The amount of WAF 1 inhibitor in the ph~rm~ceutical composition of the present
invention will depend not only upon the potency of the inhibitor but also upon the nature and
15 St;~ y of the condition being treated, and on the nature of prior tre~tments which the patent has
undergone. Ultimately, the ~ttt-n(lin~ physician will decide the amount of WAF 1 inhibitor with
which to treat each individual patient. Initially, the :~tt~n(lin~ physician will ~-lminister low
doses of the inhibitor and observe the patient's response. Larger doses of a WAF 1 inhibitor may
be ~tiministered until the optimal therapeutic effect is obtained for the patient, and at that point
20 the dosage is not increased further. In l,lcfe.led embodiments, the WAF1 inhibitor is a WAFl-
~nti~onse oligonucleotide and it is contemplated that the various ph~rm~ceutical compositions
used to practice the method of the present invention should contain about 1.0 llg to about 100 mg
of oligonucleotide per kg body weight.
The duration of intravenous therapy using the ph~rm~reutical composition of the present
25 invention will vary, depending on the severity of the disease being treated and the condition and
potential idiosyncratic response of each individual patient. It is contemplated that the duration of
each application of a WAFl-~ntisense oligonucleotide will be in the range of 12 to 24 hours of
continuous intravenous ~-lministration. Ultimately the attending physician will decide on the
a~l~l;ate duration of intravenous therapy using the ph~rm~celltical composition of the present
30 invention.
CA 02227370 1998-01-20
W O 97/03681 PCT~US96/11886
-16-
E2~ ..lal Examples
The efficacy of the present invention was demon~tr~qte~i in vitro on a neuroblastoma cell
line using conditions known to inhibit cell proliferation and to induce dirr~lcllliation as the
WAF1-in~ .in~ conditions and using tre~trn~?nt with WAF1-~nti~Pn~e oligonucleotides as
5 WAF 1 -inhibiting conditions. As described above, this technique did not lead to cell proliferation
but, on the contrary, increased cell death. SH-SY5Y (Biedler, J. et al. (1978) Cancer Res.
38:3751-3757), a neuroblastoma cell line, was used as a model for neuronal tr. ..~i..~l
~lirr~ liation (LoPresti P. et al., (1992) Cell Growth Diff. 3:627-635; Poluha, W. et al., (1995)
Qncogene 10:185-189). These cells express low levels of both the low-affinity nerve growth
0 factor receptor (LNGFR) and the TrkA NGF receptor (Baker, D. et al., (1989)49:4142-4146,
Poluha, W. et al., (1995) Onco~ene 10:185-189). SH-SY5Y cells treated with nerve growth
factor (NGF) and aphidicolin, a specific and reversible inhibitor of DNA polymerases (oc and ~),
cease cell proliferation and extend long neurites (Jensen, L. (1987) Dev. Biol. 120:56-64;
LoPresti, P. et al., (1992) Cell Growth Diff. 3:627-635). Thus, "aphidicolin + NGF" is a WAF1-
15 in~ in~ tre~tment for these cells. The dirrele..1i~te~l cells require NGF for survival and, in thepresence of NGF, are stable for 4-6 weeks. These cells express neuronal m~rker.~ and resembl
e
~y~ hPtic neurons. In co~tr~ct~ NGF alone does not stop cell proliferation and in-l~ces only
slight neurite extension (Chen, J. et al., (1990) (~ell Growth Diff. 1:79-85; Sonnenfeld and Ishii,
(1982) J Neurosci. Res. 8:375-391). Treatment with aphidicolin does not induce neurite
20 extension, and the cells resume prolifer~tion, following removal of aphidicolin.
Cells were m~int:~inP-l in RPMI 1640 medium supplementç~l with 10% heat-inactivated
fetal bovine serum, 2 mM ?~ e and 100 ,ug/ml of g~llL~llicin. For dirr~lc;llliation studies,
cells were plated on Primaria (Falcon Plastics) dishes or flasks and were treated with 100 ng/ml
of NGF (2.5 S; Bioproducts for Science) and/or 0.3~1M of aphidicolin. Fresh
25 (aphidicolin+NGF)-cont~inin~ mediurn was added every 2-3 days.
To verify the role of WAF1 in neuroblastic dirr~ lliation, expression of the mRNA for
WAF 1 was ~ ~sesse~l by Northern blotting. Expression of the WAF 1 mRNA transcript was up-
regulated following tre~tment for 1 hr with aphidicolin+NGF and further increased as the
aphidicolin+NGF tre~tnnent progressed. On day 6, the cells were changed from
30 aphidicolin+NGF medium to NGF-cont~ining medium. Despite the removal of aphidicolin from
the medium, expression of WAF 1 transcripts was slightly greater on day 14 than on day 6.
Treatrnent with aphidicolin alone also induced WAF1 expression, but following removal on day
CA 02227370 1998-01-20
W O 97/03681 -17- PCT~US96/11886
6 of aphidicolin from the medium, ~ re~ion greatly declined. Tre~tment with NGF alone did
not induce expression of WAF 1. Ethidium bromide staining of RNA prior to transfer, as well as
rehybridization of filters with a ,~-actin probe confirmed that the RNA was intact. These studies
d~mf)n~trated that sustained G~le:~iion of WAFl mRNA is specifically associated with tçrmin~l
5 ~lirr~;lcllliation of SH-SY5Y cells.
Using Western blotting, we then f1et~ rmin~d whether the level ofthe WAF1 protein was
elevated. Treakments of SH-SY5Y cells with aphidicolin+NGF enhanced levels of WAF1 (20-
fold). Expression of WAF 1 protein persisted following removal of aphidicolin from the medium
but at slightly lower levels. Tr~tm~nt of cells with aphidicolin alone up-regulated the WAF 1
10 (14-fold) level. Following removal of aphidicolin from the merlillm, G~ s~ion returned to
starting levels. Treakment of cells with NGF alone did not induce expression of WAF 1.
As a WAF1-inhibitor, an ~nti~n~e oligonucleotide with phosphorothioate linkages was
employed. In addition, to enhance entry of the oligonucleotide into cells, the cationic detergent
Lipofectin was used (Quatkone, A. et al., (1995) Biochemica 1:25-29; Wagner, R. (1994) Nature
15 372:333-335). The low concenkation of d~el~ ~:lll used in the experiment~ had no effect on cell
viability or dirrelc"Liation.
Phosphorothioate oligonucleotides (100 ~lM) and Lipofectin (1 mg/ml, a 1:1 (w/w)lni2s~G of N-(1-(2,3-dioleyloxy)propyl)-N,N,N-kimethylammonium chloride and dioleoyl
phosphatdylethanolamine (Gibco)) were incubated at 37~C for 15 min. The oligonucleotide-
20 detergent mix was diluted with serum-cont~ining medium and added to the SH-SY5Y cells. In
most cases, the dilution was 1: 100 giving a final oligonucleotide concentration of 1 ~lM. Fresh
oligonucleotide-c.."l~;";..~ medium was added to the cells each day. A WAF1-antisense
oligonucleotide was employed which is complement~ry to the region around the translational
start site (5'-TCC CCA GCC GGT TCT GAC AT-3' from Oligos, Etc.). For controls, an 18-mer
Control-1 (5'-TGG ATC CGA CAT GTC AGA-3') and an ~nti~en~e oligonucleotide directed
against M. tuberculosis (5'-CGC TTC ATC CTG CCG TGT CGG-3'), Control-2, were
employed. Expression in the of WAFl-in~ e~l cells was reduced by the ~nti~en~e
oligonucleotide but not by two control oligonucleotides. As judged by Western blotting, the
~n~ n~e oligonucleotide, but not the control, decreased WAF1 expression by 2.0-2.5 fold.
The ~nti~(?n.~e oligonucleotide had no apparent effect on the morphology or proliferation
of control cells. However, cells treated with both WAF 1 -inducing and WAF 1 -inhibiting
conditions di~ ted into neuronal cells but the number of live cells per dish was much less
CA 02227370 1998-01-20
W O 97/03681 PCT~US96/11886
-18-
than for cells treated with only WAF l -in~ in~ con~1iti- n~- Cells were stained with Hoechst
33342 to assay for apoptotic bodies which are char~t~-ri~tic of programmed cell death (Gregory,
C. et al., (1991) Nature 349:612-614). In four independent experiment.c, the percentage of cells
treated with both WAF1-inclllcing and WAFl-inhibiting conditions which exhibited apoptotic
s bodies was roughly twice that for cells treated with only inducing conditions or for cells treated
with the inducing conditions and control oligonucleotides.
-
CA 02227370 l998-0l-20
WO97/03681 PCT~US96/11886
-19-
~U~N~ LISTING
(l) GENERAL INFORMATION:
, ~
(i) APPLIC~NT:
(A) NAME: WOR OESTER FOUNDATION FOR BIOMEDICAL RESE~RCH, INC.
(B) ~ K~h~ 222 MAPLE AVENUE
(C) CITY: SHREWSBURY
(D) STATE: M~SSA~U~'l'l'~
(E) ~VUN'l'~Y: UNIl~V STATES OF AMERICA
(F) ZIP: 01545
(ii) TITLE OF IN~FNTION: METHODS AND PR~Vu~l~ FOR SF~T .T~cTIvELy
KILLrNG OR INHIBITING THE GROWTH OF ~~T T L~ ~X~K~SING THE
W~Fl GENE
(iii) NUMBER OF SEQUEN OES: 2
(iv) CORRESPONDEN OE ADDRESS:
(A) ADDRESSEE: WOLF, ~K~N~ n & SACKS, P.C.
(B) ~'l'K~h'l': 600 ATL~NTIC AVENUE
(C) CITY: BOSTON
(D) STATE: M~
(E) ~UN'l'KY: USA
(F) ZIP: 02210
,~
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPu~l~: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #l.O, Version #l.25
CA 02227370 l998-0l-20
WO97/03681 PCT~US96/11886
-20-
(vi) CUF~NT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(vii) PRIOR APPLIC~TION DATA:
(A) APPLICATION NUMBER: US 60/001,248
(B) FILING DATE: 20-JULY-1995
(viii) ATTORNEY/AG~wT INFORMATION:
(A) NAME: GATES, EDW~RD R.
(B) REGISTRATION NUMBER: 31,616
(C) K~-~K~OE/DOCKET NUMBER: W0461/7028WO
(ix) TELECOMMUNICATION INFORMATION:
(A) TEL~N~: 617-720-3500
(B) T~.TEFAX: 617-720-2441
(2) INFORMATION FOR SEQ ID NO:1:
(i) SE~U~ CHARACTERISTICS:
(A) LENGTH: 2121 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: l, n~
(ii) MOLECULE TYPE: cDNA
(iii) H~ CAL: NO
(iv) ANTI-SENSE: NO
CA 02227370 1998-01-20
WO97/03681 PCT~US96/11886
-21-
(vi) ORIGINAL SOUROE:
(A) ORGANISM: HOMO SAPIENS
,,
(ix) FEAT~JRE:
(A) NAME/KEY: CDS
(B) LOcATION: 76..570
(xi) ~U~N~ DESCRIPTION: SEQ ID NO:l:
GCCGAAGTCA ~ll~C~ll~l~ GAGCCGGAGC TaGGC30G3~ TTCGCCGAGG CACCGAGGCA 60
CTCAGAGGAG GCGCC ATG TCA G~A CCG GCT GGG GAT GTC CGT CAG AAC CCA lll
Met Ser Glu Pro Ala Gly Asp Val Arg Gln Asn Pro
l 5 lO
TGC GGC AGC AAG GCC TGC CGC CGC CTC TTC GGC CCA GTG GAC AGC GAG 159
Cys Gly Ser Lys Ala Cys Arg Arg Leu Phe Gly Pro Val Asp Ser Glu
15 20 25
CAG CTG AGC CGC GAC TGT GAT GCG CTA ATG GCG GGC TGC ATC CAG GAG 207
Gln Leu Ser Arg Asp Cys Asp Ala Leu Met Ala Gly Cys Ile Gln Glu
30 35 40
25 GCC CGT GAG CGA TGG AAC TTC GAC ~ GTC ACC GAG ACA CCA CTG GAG 255
Ala Arg Glu Arg Trp Asn Phe Asp Phe Val Thr Glu Thr Pro Leu Glu
45 50 55 60
GGT GAC TTC GCC TGG GAG CGT GTG CGG GGC CTT GGC CTG CCC AAG CTC 303
30 Gly Asp Phe Ala Trp Glu Arg Val Arg Gly Leu Gly Leu Pro Lys Leu
CA 02227370 l998-0l-20
WO97/03681 PCT~US96/11886
-22-
TAC Cll' CCC ACG GGG CCC CGG C!SA GGC CGG GAT GAG TTG GGA GGA GGC 351
Tyr Leu Pro Thr Gly Pro Arg Arg Gly Arg Asp Glu Leu Gly Gly Gly
5 AGG CGG CCT GGC ACC TCA CCT GCT CTG CTG CAG GGG ACA GCA GAG GAA 399
Arg Arg Pro Gly Thr Ser Pro Ala Leu Leu Gln Gly Thr Ala Glu Glu
100 105
GAC CAT GTG GAC CTG TCA CTG TCT TGT ACC CIT GTG CCT CGC TCA GGG 447
Asp His Val Asp Leu Ser Leu Ser Cys Thr Leu Val Pro Arg Ser Gly
110 115 120
GAG CAG GCT GAA GGG TCC CCA GGT GSA CCT GGA GAC TCT C~G GGT CGA 495
Glu Gln Ala Glu Gly Ser Pro Gly Gly Pro Gly Asp Ser Gln Gly Arg
125 130 135 140
A~A CGG CGG CAG ACC AGC ATG ACA GAT TTC TAC CAC TCC A~A CGC CGG 543
Lys Arg Arg Gln Thr Ser Met Thr Asp Phe Tyr His Ser Lys Arg Arg
145 150 155
CTG ATC TTC TCC AAG AGG AAG CCC TAATCCGCCC ACAGGAAGCC TGCA~lC~l~ 597
Leu Ile Phe Ser Lys Arg Lys Pro
160 165
25 G~AGCGCGAG GGCCTCAAAG GCCCGCTCTA CA~ l~C CTTAGTCTCA ~ 657
CTTAATTATT A'l'l~ l~'l'l'l' TAATTTA~AC ACCTCCTCAT GTACATACCC l~G~CCCC 717
CTG~C~C~A GC~l~l~CA TTAGAATTAT TTA~ACAA~A ACTAGGCGGT T&~ATGAGAG 777
GTTCCTAAGA GTa{ra3GCA lllllAl~ ATGAAATACT ATl~l~A~AGCC 'l'C~'l'~ATCCC 837
CA 02227370 l998-0l-20
WO97/03681 -23- PCT~US96/11886
~l~'l-l'~'l'C'~'l''lll'~l'~l~'l' CCCGGAGGTT ~l~GCCG GCTTCATGCC AGCTACTTCC 897
~ TCCTCCCCAC 'l'l~l~CTG W'l~'lACCC TCTGGAGGGG 'l~'l~l'C~'l' TCCCATC'GCT 957
S GTCACAGGCG GTTATGA~AT TCA~C'~'l-l''l~'~'l~AC'AC TCAGACCTGA A'l-l~lll-llC 1017
ATTTGAGA~G TA~ACAGATG GCACTTTGAA GGGGCCTCAC CGA~l~G~ CATCATCAAA 1077
A~'l'l'l'w AG T~'~C~'1'~'ACC lC~lw ~AGG 'll~AGGG TGACCCTGAA GTGAGCACAG 1137
CCTAGG ~ TG AG~'l~ w AC CTGGTACCCT CCTGGCTCTT GATAC'~C'CC'C' lW ~l~ll~l 1197
GAAGGCAGGG GGAA W'l~ GTACTGGAGC AGACCACCCC GCCTGCCCTC ATGGC~C'~lC' 1257
TGACCTGCAC TGGGGAGCCC GTCTCAGTGT TGAG~llll C~l~lll~ W'C~C~'l~'l'A 1317
~llll~AGG AGCCCCAGCT TA~'l-l'~l'l' CTCCAGCTGG GCTCTGCAAT 'l'C~C~l~-l~C 1377
'l~'l~'l'C~l CC~C~ll~'l'C 'l'l'l'~'C~'l'l'~'A GTAC~l~l~ ATGCTCCAGG TGGCTCTGAG 1437
~ 'l~C~l~lCC CACCCCCACC CCCAGCTCAA TGGACTGG~A GGGGAAGGGA CACACAAGAA 1497
GAAGGGCACC CTAGTTCTAC CTCAGGCAGC TCAAGCAGCG ACCGCCCCCT CCTCTAGCTG 1557
TGGGGGTGAG ~'l~C~ATGT GGTGGCACAG GCCCCCITGA GTGGGGTTAT ~'l'~'l'~'l'~'l'l'A 1617
GGGGTATATG ATGGGG5AGT AGA'l~'l'l'l~l' AG5AGGGAGA CACTGGCCCC TCA~ATC'GTC 1677
CAGC'GACCTT CCTCATCCAC CCCATCCCTC CCCAGTTCAT TGCACTTTGA TTAGCAGCGG 1737
AACAAGGAGT CAGACATTTT AAGA'l~l~ CAGTAGAGGC TATGGACAGG GCATGCCACG 1797
CA 02227370 l998-0l-20
WO97/03681 PCT~US96/11886
-24-
~ l'CATA TGGGGC~GG3 AGTA~ l~G~A CT~ACGTTGA GCCC~l~AG 1857
GCACTGAAGT GCTTAGTGTA CTTGGAGTAT ~ l~A CCCCA~ACAC CTTCCAGCTC 1917
CTGTAACATA CTGGCCTGGA ~ l'C TCGGCTCCCC A~l~l~lC~ C~lll 1977
CTCCACCTAG A~'l~'lA~ACC TCTCGAGGGC AG~GACCACA C~l~lAcTG 'l-l'~l~l~l~CT 2037
TTCACAGCTC CTCCCACAAT GCT'G~ATATA CAGCAGGTGC TCAATAAATG All~llAGTG 2097
ACTTTA~AAA AP2vA~AaAA AA~A 2121
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENOE CH~RACTERISTICS:
(A) LENGTH: 164 amino acids
(B) TYPE: amino acid
(D) TOPOLCX~Y: 1 i n~;~
(ii) MOLEC~LE TYPE: protein
(xi) SEQUENOE DESCRIPTION: SEQ ID NO:2:
Met Ser Glu Pro Ala Gly Asp Val Arg Gln Asn Pro Cys Gly Ser Lys
1 5 10 15
Ala Cys Arg Arg Leu Phe Gly Pro Val Asp Ser Glu Gln Leu Ser Arg
20 25 30
Asp Cys Asp Ala Leu Met ~la Gly Cys Ile Gln Glu Ala Arg Glu Arg
CA 02227370 l998-0l-20
WO97/03681 PCT~US96/11886
-25-
Trp Asn Phe Asp Phe Val Thr Glu Thr Pro Leu Glu Gly Asp Phe Ala
Trp Glu Arg Val Arg Gly Leu Gly Leu Pro Lys Leu Tyr heu Pro Thr
Gly Pro Arg Arg Gly Arg Asp Glu Leu Gly Gly Gly Arg Arg Pro Gly
85 90 95
Thr Ser Pro Ala Leu Leu Gln Gly Thr Ala Glu Glu Asp His Val Asp
100 105 110
15 Leu Ser Leu Ser Cys Thr Leu Val Pro Arg Ser Gly Glu Gln Ala Glu
115 120 125
Gly Ser Pro Gly Gly Pro Gly Asp Ser Gln Gly Arg Lys Arg Arg Gln
130 135 140
Thr Ser Met Thr Asp Phe l~r His Ser Lys Arg Arg Leu Ile Phe Ser
145 150 155 160
Lys Arg Lys Pro
