Note: Descriptions are shown in the official language in which they were submitted.
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flt3 LiQaad Chimeric Proteins
. The present application is a Continuation-in-Part of
08/837,026 filed April 11, 1997 which is incorporated
herein by reference.
FIELD OF THE INVENTION
The present invention relates to chimeric
proteins or multi-functional hematopoietic receptor
agonists, comprising a human flt3 agonist. These
chimera proteins retain one or more activities of
native flt3 ligand and the other component of the
chimera protein. The chimera protein may also show
improved hematopoietic cell-stimulating activity or
an activity not seen for flt3 ligand and the other
factor when co-administered. The chimera protein may
also show an improved activity profile which may
include reduction of undesirable biological
activities associated with native flt3 ligand and/or
have improved physical properties which may include
increased solubility, stability and refold
efficiency.
BACKGROUND OF THE INVENTION
Colony stimulating factors which stimulate the
differentiation and/or proliferation of bone marrow
cells have generated much interest because of their
therapeutic potential for restoring depressed levels
" of hematopoietic stem cell-derived cells. Colony
stimulating factors in both human and murine systems
have been identified and distinguished according to
their activities. For example, granulocyte-CSF (G-
CSF) and macrophage-CSF (M-CSF) stimulate the in
vitro formation of neutrophilic granulocyte and
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macrophage colonies, respectively while GM-CSF and
interl,eukin-3 (IL-3) have broader activities and
stimulate the formation of both macrophage,
neutrophilic and eosinophilic granulocyte colonies.
Certain factors such as flt3 ligand are able to
predominately affect stem cells.
Tyrosine kinase receptors are growth factor
receptors that regulate the proliferation and
differentiation of a number of cell. Certain tyrosine
kinase receptors function within the hematopoietic
system. Flt3 (Roseate et al., Oncogene, 6:1641-1650,
1991) and flk-2 (Matthews et al., Cell, 65:1143-1152,
1991) are forms of a tyrosine kinase receptor that is
related to c-fms and c-kit receptors. The flk-2 and
flt3 receptors are similar in amino acid sequence and
vary at two amino acid residues in the extracellular
domain and diverge in a 31 amino acid segment located
near the C-terminus.
Flt3 ligand is a hematopoietic growth factor
which has the property of being able to regulate the
growth and differentiation of hematopoietic
progenitor and stem cells. Because of its ability to
support the growth and proliferation of progenitor
cells, flt3 receptor agonists have potential for
therapeutic use in treating hematopoietic disorders
such as aplastic anemia and myelodysplastic
syndromes. Additionally, flt3 receptor agonists will
be useful in restoring hematopoietic cells to normal
amounts in those cases where the number of cells has
been reduced due to diseases or to therapeutic
treatments such as radiation and chemotherapy.
WO 94/28391 discloses the native flt3 ligand
protein sequence and a cDNA sequence encoding the
flt3 ligand, methods of expressing flt3 ligand in a
host cell transfected with the cDNA and methods of
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3
treating patients with a hematopoietic disorder using
flt3 7,igand.
US Patent No. 5,554,512 is directed to human
S flt3 ligand as an isolated protein, DNA encoding the
flt3 ligand, host cells transfected with cDNAs
encoding flt3 ligand and methods for treating
patients with flt3 ligand.
WO 94/26891 provides mammalian flt3 ligands,
including an isolate that has an insertion of 29
amino acids, and fragments there of.
The human blood-forming (hematopoietic) system
replaces a variety of white blood cells (including
neutrophils, macrophages, and basophils/mast cells),
red blood cells (erythrocytes) and clot-forming cells
(megakaryocytes/platelets). The hematopoietic
systems of the average male has been estimated to
produce on the order of 4.5 x 1011 granulocytes and
erythrocytes every year, which is equivalent to an
annual replacement of total body weight (Dexter et
al., BioEssays, 2;154-158, 1985).
U.S. Patent 4,999,291 discloses DNA and methods
for making G-CSF the disclosure of which is
incorporated herein by reference in it entirety.
U.S. Patent 4,810,643 relates to DNA and methods
of making G-CSF and Cys to Ser substitution variants
of G-CSF.
Kuga et al. (Biochem. + Biophys. Res. Comm.
159:103-111, 1988) made a series of G-CSF variants to
partially define the structure-function relationship.
Kuga et al. found that internal and C-terminal
deletions abolished activity, while N-terminal
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deletions of up to 11 amino acids and amino acid
substitutions at positions 1, 2 and 3 were active.
Watanabe et al. (Anal. Biochem. 195:38-44, 1991)
made a variant to study G-CSF receptor binding in
which amino acids 1 and 3 were changed to Tyr for
radioiodination of the protein. Watanabe et al. found
this Tyrl, Tyr3 G-CSF variant to be active.
Erythropoietin is a naturally-occurring
glycoprotein hormone with a molecular weight that was
first reported to be approximately 39,000 daltons (T.
Miyaki et al., J. Biol. Chem. 252:5558-5564 (1977)).
The mature hormone is 166 amino acids long and the
"prepro" form of the hormone, with its leader
peptide, is 193 amino acids long (F. Lin, U.S. Patent
No. 4,703,008). The mature hormone has a molecular
weight, calculated from its amino acid sequence, of
18,399 daltons (K. Jacobs et al., Nature 313:806-810
(1985); J. K. Browne et al., Cold Spring Harbor Symp.
Quant. Biol. 5:1693-702 (1986).
The first mutant erythropoietins (i.e.,
erythropoietin analogs), prepared by making amino
acid substitutions and deletions, have demonstrated
reduced or unimproved activity. As described in U.S.
Patent N0. 4,703,008, replacement of the tyrosine
residues at positions 15, 40 and 145 with
phenylalanine residues, replacement of the cysteine
residue at position 7 with an histidine, substitution
of the proline at position 2 with an asparagine,
deletion of residues 2-6, deletion of residues 163-
166, and deletion of residues 27-55 does not result
in an apparent increase in biological activity. The
Cys~-to-Hiss mutation eliminates biological activity.
A series of mutant erythropoietins with a single
amino acid substitution at asparagine residues 24, 38
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-
or 83 show severely reduced activity (substitution at
position 24) or exhibit rapid intracellular
degradation and apparent lack of secretion
(substitution at residue 38 or 183). Elimination of
5 the O-linked glycosylation site at serine126 results
in rapid degradation or lack of secretion of the
erythropoietin analog (S. Dube et al., J. Biol. Chem.
33:17516-17521 (1988). These authors conclude that
glycosylation sites at residues 38, 83 and 126 are
required for proper secretion and that glycosylation
sites located at residues 24 and 38 may be involved
in the biological activity of mature erythropoietin.
Deglycosylated erythropoietin is fully active in
in vitro bioassays (M. S. Dorsdal et al.,
Endocrinology 116:2293-2299 (1985); U.S. Patent No.
4,703,008; E. Tsuda et al., Eur J. Biochem.
266:20434-20439 (1991). However, glycosylation of
erythropoietin is widely accepted to play a critical
role in the in vivo activity of the hormone (P. H..
Lowy et al., Nature 185:102-105 (1960); E. Goldwasser
and C. K. H.. Kung, Ann. N.Y. Acad. Science 149:49-53
(1968); W. A. Lukowsky and R. H.. Painter, Can. J.
Biochem. :909-917 (1972); D.W. Briggs et al., Amer.
J. Phys. 201:1385-1388 (1974); J.C. Schooley, Exp.
Hematol. 13:994-998; N. Imai et al., Eur. J. Biochem.
194:457-462 (1990); M.S. Dordal et al., Endocrinology
116:2293-2299 (1985); E. Tsuda et al., Eur. J.
Biochem. 188:405-411 (1990); U.S. Patent No.
4,703,008; J.K. Brown et al., Cold Spring Harbor
Symposia on Quant. Biol. 51:693-702 (1986); and K.
Yamaguchi et al., J. Biol. Chem. 266:20434-20439
(1991). The lack if in vivo biological activity of
deglycosylated analogs of erythropoietin is
attributed to a rapid clearance of the deglycosylated
hormone from the circulation of treated animals.
This view is supported by direct comparison of the
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6
plasma half-life of glycosylated and deglycosylated
erythz:opoietin (J. C. Spivak and B.B. Hoyans, Blood
73:90-99 (1989), and M.N. Fukuda, et al., Blood -
73:84-89 (1989).
Oligonucleotide-directed mutagenesis of
erythropoietin glycosylation sites has effectively
probed the function of glycosylation but has failed,
as yet, to provide insight into an effective strategy
for significantly improving the characteristics of
the hormone for therapeutic applications.
A series of single amino acid substitution or
deletion mutants have been constructed of
erythropoietin, involving amino acid residues 15, 24,
49, 76, 78, 83, 143, 145, 160, 162, 163, 164, 165 and
166. In these mutants are altered the carboxy
terminus, the glycosylation sites, and the tyrosine
residues of erythropoietin. The mutants have been
administered to animals while monitoring hemoglobin,
hematocrit and reticulocyte levels (EP No. 0 409
113). While many of these mutants retain in vivo
biological activity, none show a significant increase
in their ability to raise hemoglobin, hematocrit or
reticulocyte (the immediate precursor of an
erythrocyte) levels when compared to native
erythropoietin.
Another set of mutants has been constructed to
probe the function of residues 99-119 (domain 1) and
residues 111-129 (domain 2) (Y. Chern et al., Eur. J.
Biochem. 202:225-230 (1991)). The domain 1 mutants
are rapidly degraded and inactive in an in vitro
bioassay while the domain 2 mutants, at best, retain
in vitro activity. These mutants also show no
enhanced in vivo biological activity as compared to
wild-type, human erythropoietin. These authors
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conclude that residues 99-119 play a critical role in
the structure of erythropoietin.
The human erythropoietin molecule contains two
disulfide bridges, one linking the cysteine residues
at positions 7 and 161, and a second connecting
cysteines at positions 29 and 33 (P.H. Lai et al., J.
Biol. Chem. 261:3116-3121 (1986)). Oligonucleotide-
directed mutagenesis has been used to probe the
function of the disulfide bridge linking cysteines 29
and 33 in human erythropoietin. The cysteine at
position 33 has been converted to a proline residue,
which, mimics the structure of murine erythropoietin
at this residue. The resulting mutant has greatly
reduced in vitro activity. The loss of activity is so
severe that the authors conclude that the disulfide
bridge between residues 29 and 33 is essential for
erythropoietin function (F.K. Lin, Molecular and
Cellular Aspects of Erythropoietin and
Erythropoiesis, pp. 23-36, ed. I.N. Rich, Springer-
Verlag, Berlin (1987)).
U.S. Patent No. 4,703,008 by Lin, F-K.
(hereinafter referred to as "the '008 patent")
speculates about a wide variety of modifications of
EPO, including addition, deletion, and substitution
analogs of EPO. The '008 patent does not indicate
that any of the suggested modifications would
increase biological activity per se, although it is
stated that deletion of glycosylation sites might
increase the activity of EPO produced in yeast (See
the '008 patent at column 37, lines 25-28). Also,
the '008 patent speculates that EPO analogs which
. have one or more tyrosine residues replaced with
phenylalanine may exhibit an increased or decreased
receptor binding affinity.
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Australian Patent Application No. AU-A-59145/90
by Fibi, M et al. also discusses a number of modified
EPO proteins (EPO muteins). It is generally
speculated that the alteration of amino acids 10-55,
70-85, and 130-166 of EPO. In particular, additions
of positively charged basic amino acids in the
carboxyl terminal region are purported to increase
the biological activity of EPO.
U.S. Patent No. 4,835,260 by Shoemaker, C.B.
discusses modified EPO proteins with amino acid
substitutions of the methionine at position 54 and
asparagine at position 38. Such EPO muteins are
thought to have improved stability but are not
proposed to exhibit any increase in biological
activity relative to wild type EPO.
WO 91/05867 discloses analogs of human
erythropoietin having a greater number of sites for
carbohydrate attachment than human erythropoietin,
such as EPO (Asn6g), EPO {Asn125, Ser127), EPO
(Thr125), and EPO (Pro124, Thr125),
WO 94 /24160 discloses erythropoietin muteins
which have enhanced activity, specifically amino acid
substitutions at positions 20, 49, 73, 140, 143, 146,
147 and 154.
WO 94/25055 discloses erythropoietin analogs,
including EPO (X33, Cys139, des-Arg166) and EPO
(Cys139, des-Arg166).
Stem cell factor has the ability to stimulate
growth of early hematopoietic progenitors which are
capable of maturing to erythroid, megakaryocyte,
granulocyte, lymphocyte and macrophage cells. Stem
cell factor treatment of mammals results in absolute
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9
increases in hematopoietic cells of both the myeloid
and l~,rmphoid cells .
EP 0 423 980 discloses novel stem cell factor
(SCF) polypeptides including SCFl'laa, SCF1-ls', SCF1-lso
SCF1-isi, SCFl-isz, SCFl'isa, SCFi-iss, SCFl-ia3, SCFl'las~ SCFl'
iaa, SCFl'ies, SCFl-zzo, SCFl-zaa ,
U.S. 4,877,729 and U.S. 4,959,455 disclose human
IL-3 and gibbon IL-3 cDNAs and the protein sequences
for which they code. The hIL-3 disclosed has serine
rather than proline at position 8 in the protein
sequence.
International Patent Application (PCT) WO
88/00598 discloses gibbon- and human-like IL-3. The
hIL-3 contains a Sera -> ProB replacement.
Suggestions are made to replace Cys by Ser, thereby
breaking the disulfide bridge, and to replace one or
more amino acids at the glycosylation sites.
U.S. 4,810,643 discloses the DNA sequence
encoding human G-CSF.
WO 91/02754 discloses a fusion protein comprised
of GM-CSF and IL-3 which has increased biological
activity compared to GM-CSF or IL-3 alone. Also
disclosed are nonglycosylated IL-3 and GM-CSF analog
proteins as components of the multi-functional
hematopoietic receptor agonist.
WO 92/04455 discloses fusion proteins composed
of IL-3 fused to a lymphokine selected from the group
consisting of IL-3, IL-6, IL-7, IL-9, IL-11, EPO and
G-CSF.
. WO 95/21197 and WO 95/21254 disclose fusion
proteins capable of broad multi-functional
hematopoietic properties.
GB 2,285,446 relates to the c-mpl ligand
(thrombopoietin) and various forms of thrombopoietin
which are shown to influence the replication,
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- 10
differentiation and maturation of megakaryocytes and
megakaryocytes progenitors which may be used for the
treatment of thrombocytopenia.
EP 675,201 A1 relates to the c-mpl ligand
(Megakaryocyte growth and development factor (MGDF),
allelic variations of c-mpl ligand and c-mpl ligand
attached to water soluble polymers such as
polyethylene glycol.
WO 95/21920 provides the murine and human c-mpl
ligand and polypeptide fragments thereof. The
proteins are useful for in vivo and ex vivo therapy
for stimulating platelet production.
Summary of the Invention
The present invention encompasses recombinant
chimeric proteins comprising a flt3 agonist and
another factor. The other factor may be a colony
stimulating factor (CSF), cytokine, lymphokine,
interleukin, hematopoietic growth factor which
include but are not limited to GM-CSF, c-mpl ligand
(also known as TPO or MGDF), M-CSF, erythropoietin
(EPO), IL-1, IL-4, IL-2, IL-3, IL-5, IL 6, IL-7, IL-
8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, LIF, flt3
ligand, human growth hormone, B-cell growth factor,
B-cell differentiation factor, eosinophil
differentiation factor, stem cell factor (SCF) also
known as steel factor or c-kit ligand, stem cell
growth factor (SCGF)(Hiraoka, A. et al. Proc. Natl.
Acad. Sci USA 94:7577-7582, 1997) and Stromal Cell-
derived Factor 1 (SDF-1) (Bleul, C.C. et al., J.
Exp. Med 184:1101-1109, 1996) , (herein collectively
referred to as "hematopoietic growth factors". The
chimera proteins can also be co-administered or
sequentially administered with one or more additional
colony stimulating factor(s), cytokine(s),
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lymphokine(s), interleukin(s), hematopoietic growth
factox(s) which include but are not limited to GM-
CSF, c-mpl ligand (also known as TPO or MGDF), M-CSF,
erythropoietin (EPO), IL-1, IL-4, IL-2, IL-3, IL-5,
IL 6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13,
IL-15, LIF, human growth hormone, B-cell growth
factor, B-cell differentiation factor, eosinophil
differentiation factor, stem cell factor (SCF) also
known as steel factor or c-kit ligand, SCGF and SDF-1
(herein collectively referred to "hematopoietic
growth factors". These co-administered mixtures may
be characterized by having the usual activity of both
of the peptides or the mixture may be further
characterized by having a biological or physiological
activity greater than simply the additive function of
the presence of the G-CSF receptor agonists or the
second hematopoietic growth factor alone. The
chimeric protein may also provide an enhanced effect
on the activity or an activity different from that
expected by the presence of the flt3 ligand or the
second colony stimulating factor. The chimeric
protein may also have an improved activity profile
which may include reduction of undesirable biological
activities associated with native human flt3.
Detailed Description of the Invention
The present invention encompasses multi-
functional hematopoietic receptor agonists or
chimeric proteins formed from covalently linked
polypeptides, each of which may act through a
different and specific cell receptor to initiate
complementary biological activities. Hematopoiesis
requires a complex series of cellular events in which
stem cells generate continuously into large
populations of maturing cells in all major lineages.
There are currently at least 20 known regulators with
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hematopoietic proliferative activity. Most of these
proli~erative regulators can only stimulate one or
another type of colony formation in vitro, the
precise pattern of colony formation stimulated by
each regulator is quite distinctive. No two
regulators stimulate exactly the same pattern of
colony formation, as evaluated by colony numbers or,
more importantly, by the lineage and maturation
pattern of the cells making up the developing
colonies. Proliferative responses can most readily be
analyzed in simplified in vitro culture systems.
Three quite different parameters can be
distinguished: alteration in colony size, alteration
in colony numbers and cell lineage. Two or more
factors may act on the progenitor cell, inducing the
formation of larger number of progeny thereby
increasing the colony size. Two or more factors may
allow increased number of progenitor cells to
proliferate either because distinct subsets of
progenitors cells exist that respond exclusively to
one factor or because some progenitors require
stimulation by two or more factors before being able
to respond. Activation of additional receptors on a
cell by the use of two or more factors is likely to
enhance the mitotic signal because of coalescence of
initially differing signal pathways into a common
final pathway reaching the nucleus (Metcalf, Nature
339:27, 1989). Other mechanisms could explain
synergy. For example, if one signaling pathway is
limited by an intermediate activation of an
additional signaling pathway which is caused by a
second factor, then this may result in a super
additive response. In some cases, activation of one
receptor type can induce an enhanced expression of
other receptors (Metcalf, Blood 82:3515-3523, 1993).
Two or more factors may result in a different pattern
of cell lineages than from a single factor. The use
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of multi-functional hematopoietic receptor agonists
may have a potential clinical advantage resulting
from a proliferative response that is not possible by
any single factor.
The receptors of hematopoietic and other growth
factors can be grouped into two distinct families of
related proteins: (1) tyrosine kinase receptors,
including those for epidermal growth factor, M-CSF
(Sherr, Blood 75:1, 1990) and SCF (Yarden et al.,
EMBO J. 6:3341, 1987): and (2) hematopoietic
receptors, not containing a tyrosine kinase domain,
but exhibiting obvious homology in their
extracellular domain (Bazan, PNAS USA 87:6934-6938,
1990). Included in this latter group are
erythropoietin (EPO) (D'Andrea et al., Cell 57:277,
1989), GM-CSF (Gearing et al., EMBO J. 8:3667, 1989),
IL-3 (Kitamura et al., Cell 66:1165, 1991), G-CSF
(Fukunaga et al., J. eio. Chem. 265:14008-15, 1990),
IL-4 (Harada et al., PNAS USA 87:857, 1990), IL-5
(Takaki et al., E1~0 J. 9:4367, 1990), IL-6 (Yamasaki
et al., Science 241:825, 1988), IL-7 (Goodwin et al.,
Cell 60:941-51, 1990), LIF (Gearing et al., E1~0 J.
10:2839, 1991) and IL-2 (Cosman et al. , Mo1-Immunol.
23: 935-94, 1986). Most of the latter group of
receptors exists in a high-affinity form as
heterodimers. After ligand binding, the specific a-
chains become associated with at least one other
receptor chain (~i-chain, y-chain). Many of these
factors share a common receptor subunit. The a-chains
for GM-CSF, IL-3 and IL-5 share the same (3-chain
(Kitamura et al., Cell 66:1165, 1991), Takaki et al.,
EM80 J. 10:2833-8, 1991) and receptor complexes for
IL-6, LIF and IL-11 share a common (3-chain (gp130)
(Taga et al., Cell 58:573-81, 1989; Gearing et al.,
Science 255:1434-7, 1992). The receptor complexes of
IL-2, IL-4, IL-7, IL-9 and IL-15 share a common y-
chain (Kondo et al., Science 262:1874, 1993; Russell
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14
et al., Science 266: 1042-1045, 1993; Noguchi et
al., Science 262:1877, 1993; Giri et al., ~O J.
13:2822-2830, 1994).
The use of a multiply acting hematopoietic
factor may also have a potential advantage by
reducing the demands placed on factor-producing cells
and their induction systems. If there are limitations
in the ability of a cell to produce a factor, then by
lowering the required concentrations of each of the
factors, and using them in combination may usefully
reduce demands on the factor-producing cells. The use
of a multiply acting hematopoietic factor may lower
the amount of the factors that would be needed,
probably reducing the likelihood of adverse side-
effects.
Novel compounds of this invention are
represented by a formula selected from the group
consisting of:
R1-L1-R2, R2-L1-R1, R1-R2, and R2-R1
TnThere R1 is a flt3 agonist and R2 is a
hematopoietic growth factor. Preferably R2 is a
hematopoietic growth factor with a different but
complementary activity than R1. By complementary
activity is meant activity which enhances or changes
the response to another cell modulator. The R1
polypeptide is joined either directly or through a
linker segment to the R2 polypeptide. The term
"directly" defines multi-functional hematopoietic
receptor agonists in which the polypeptides are
joined without a peptide linker. Thus L1 represents a
chemical bond or polypeptide segment to which both R1
and R2 are joined in frame, most commonly L1 is a
linear peptide to which R1 and R2 are joined by amide
bonds linking the carboxy terminus of R1 to the amino
terminus of L1 and carboxy terminus of L1 to the
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amino terminus of R2. By "joined in frame" is meant
that there is no translation termination or
disruption between the reading frames of the DNA
encoding R1 and R2.
A non-exclusive list of other growth factors,
i.e. colony stimulating factors (CSFs), are
cytokines, lymphokines, interleukins, hematopoietic
growth factors which can be joined to R1 include GM-
CSF, G-CSF, c-mpl ligand (also known as TPO or MGDF),
M-CSF, erythropoietin (EPO), IL-1, IL-4, IL-2, IL-3,
IL-5, IL 6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12,
IL-13, IL-15, LIF, flt3 ligand, human growth hormone,
B-cell growth factor, B-cell differentiation factor,
eosinophil differentiation factor, stem cell factor
(SCF) also known as steel factor or c-kit ligand IL-
12. Additionally, this invention encompasses the use
of modified R1 or R2 molecules or mutated or modified
DNA sequences encoding these R1 or R2 molecules. A
"c-mpl ligand variant" is defined an c-mpl ligand
molecule which has amino acid substitutions and/or
portions of c-mpl ligand deleted, disclosed in United
States Application Serial Number 08/383,035 as well
as other variants known in the art. A "G-CSF variant"
is defined an G-CSF molecule which has amino acid
substitutions and/or portions of G-CSF deleted, as
disclosed herein, as well as other variants known in
the art. Preferably, R2 is G-CSF, GM-CSF, c-mpl
ligand or EPO.
The linking group (L1) is generally a
. polypeptide of between 1 and 500 amino acids in
length. The linkers joining the two molecules are
preferably designed to (1) allow the two molecules to
fold and act independently of each other, (2) not
have a propensity for developing an ordered secondary
structure which could interfere with the functional
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domains of the two proteins, (3) have minimal
hydrophobic characteristics which could interact with
the functional protein domains and (4) provide steric
separation of R1 and R2 such that R1 and R2 could
interact simultaneously with their corresponding
receptors on a single cell. Typically surface amino
acids in flexible protein regions include Gly, Asn
and Ser. Virtually any permutation of amino acid
sequences containing Gly, Asn and Ser would be
expected to satisfy the above criteria for a linker
sequence. Other neutral amino acids, such as Thr and
Ala, may also be used in the linker sequence.
Additional amino acids may also be included in the
4linkers due to the addition of unique restriction
sites in the linker sequence to facilitate
construction of the multi-functional hematopoietic
receptor agonists.
Preferred L1 linkers of the present invention
include sequences selected from the group of
formulas:
(Gly3Ser)n (SEQ ID N0:1), (Gly4Ser)n (SEQ ID N0:2),
(GlySSer)n (SEQ ID N0:3), (GlynSer)n (SEQ ID N0:4) or
(AlaGlySer)n (SEQ ID N0:5), where n is an integer
(collectively referred to herein as "GlySer"
linkers).
One example of a highly-flexible linker is the
glycine and serine-rich spacer region present within
the pIII protein of the filamentous bacteriophages,
e.g. bacteriophages M13 or fd (Schaller et al., PNAS
USA 72: 737-741, 1975). This region provides a long,
flexible spacer region between two domains of the
pIII surface protein. The spacer region consists of
the amino acid sequence:
3 5 GlyGlyGlySerGlyGlyGlySerGlyGlyGlySerGluGlyGlyGlySerGlu
GlyGlyGlySerGluGlyGlyGlySerGluGlyGlyGlySerGlyGlyGlySer
(sEQ ID N0:6).
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'the present invention also includes linkers in
which an endopeptidase recognition sequence is
included. Such a cleavage site may be valuable to
separate the individual components of the multi-
functional hematopoietic receptor agonist to
determine if they are properly folded and active in
vitro. Examples of various endopeptidases include,
but are not limited to, plasmin, enterokinase,
kallikrein, urokinase, tissue plasminogen activator,
clostripain, chymosin, collagenase, Russell's viper
venom protease, postproline cleavage enzyme, V8
protease, Thrombin and factor Xa.
Peptide linker segments from the hinge region of
heavy chain immunoglobulins IgG, IgA, IgM, IgD or IgE
provide an angular relationship between the attached
polypeptides. Especially useful are those hinge
regions where the cysteines are replaced with
serines. Preferred linkers of the present invention
include sequences derived from murine IgG gamma 2b
hinge region in which the cysteines have been changed
to serines (Bell et al. US Patent 4,936,233). These
linkers may also include an endopeptidase cleavage
site. Examples of such linkers include the following
sequences:
IleSerGluProSerGlyProIleSerThrIleAsnProSerProProSerLys
GluSerHisLysSerPro ( SEQ ID NO : 7 ) , and
3 0 IleGluGlyArgIleSerGluProSerGlyProIleSerThrIleAsnProSer
ProProSerLysGluSerHisLysSerPro (SEQ ID N0:8) (COllectlVely
referred to herein as "IgG2b" linkers).
The present invention is, however, not limited
by the form, size or number of linker sequences
employed and the only requirement of the linker is
that functionally it does not interfere with the
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folding and function of the individual molecules of
the mqlti-functional hematopoietic receptor agonist.
Hematopoietic growth factors can be
characterized by their ability to stimulate colony
formation by human hematopoietic progenitor cells.
The colonies formed include erythroid, granulocyte,
megakaryocyte, granulocytic macrophages and mixtures
thereof. Many of the hematopoietic growth factors
have demonstrated the ability to restore bone marrow
function and peripheral blood cell populations to
therapeutically beneficial levels in studies
performed initially in primates and subsequently in
humans. Many or all of these biological activities
of hematopoietic growth factors involve signal
transduction and high affinity receptor binding.
Multi-functional hematopoietic receptor agonists of
the present invention may exhibit useful properties
such as having similar or greater biological activity
when compared to a single factor or by having
improved half-life or decreased adverse side effects,
or a combination of these properties.
Multi-functional hematopoietic receptor agonists
which have little or no agonist activity maybe useful
as antagonists, as antigens for the production of
antibodies for use in immunology or immunotherapy, as
genetic probes or as intermediates used to construct
other useful hIL-3 muteins.
The present invention also includes the DNA
sequences which code for the multi-functional
hematopoietic receptor agonist proteins, DNA
sequences which are substantially similar and perform
substantially the same function, and DNA sequences
which differ from the DNAs encoding the multi-
functional hematopoietic receptor agonists of the
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invention only due to the degeneracy of the genetic
code. ~rlso included in the present invention are the
. oligonucleotide intermediates used to construct the
mutant DNAs and the polypeptides coded for by these
oligonucleotides.
Genetic engineering techniques now standard in
the art (United States Patent 4,935,233 and Sambrook
et al., "Molecular Cloning A Laboratory Manual", Cold
Spring Harbor Laboratory, 1989) may be used in the
construction of the DNA sequences encoding flt3
ligand, EPO, G-CSF, GM-CSF, other hematopoietic
growth factors and the chimeric proteins of the
present invention. One such method is cassette
mutagenesis (Wells et al., Gene 34:315-323, 1985) in
which a portion of the coding sequence in a plasmid
is replaced with synthetic oligonucleotides that
encode the desired amino acid substitutions in a
portion of the gene between two restriction sites.
Pairs of complementary synthetic oligonucleotides
encoding the desired gene can be made and annealed to
each other. The DNA sequence of the oligonucleotide
would encode sequence for amino acids of desired gene
with the exception of those substituted and/or
deleted from the sequence.
Plasmid DNA can be treated with the chosen
restriction endonucleases then ligated to the
annealed oligonucleotides. The ligated mixtures can
be used to transform competent bacterial cells such
as E. coli strain JM101 resistance to an appropriate
antibiotic. Single colonies can be picked and the
plasmid DNA examined by restriction analysis and/or
DNA sequencing to identify plasmids with the desired
genes.
Cloning of the DNA sequences of the novel
multifunctional hematopoietic agonists wherein at
least one of the with the DNA sequence of the other
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hematopoietic growth factor may be accomplished by
the use of intermediate vectors. Alternatively one
gene can be cloned directly into a vector containing
the other gene. Linkers and adapters can be used for
joining the DNA sequences, as well as replacing lost
sequences, where a restriction site was internal to
the region of interest. Thus genetic material (DNA)
encoding one polypeptide, peptide linker, and the
other polypeptide is inserted into a suitable
expression vector which is used to transform
bacteria, yeast, insect cells or mammalian cells. The
transformed organism is grown and the protein
isolated by standard techniques. The resulting
product is therefore a new protein which has a
hematopoietic growth factor joined by a linker region
to a second colony stimulating factor.
Another aspect of the present invention provides
plasmid DNA vectors for use in the expression of
these novel multi-functional hematopoietic receptor
agonists. These vectors contain the novel DNA
sequences described above which code for the novel
polypeptides of the invention. Appropriate vectors
which can transform microorganisms capable of
expressing the multi-functional hematopoietic
receptor agonists include expression vectors
comprising nucleotide sequences coding for the multi-
functional hematopoietic receptor agonists joined to
transcriptional and translational regulatory
sequences which are selected according to the host
cells used.
Vectors incorporating modified sequences as
described above are included in the present invention
and are useful in the production of the multi-
functional hematopoietic receptor agonist
polypeptides. The vector employed in the method also
contains selected regulatory sequences in operative
association with the DNA coding sequences of the
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invention and which are capable of directing the
replication and expression thereof in selected host
cells.
As another aspect of the present invention,
there is provided a method for producing the novel
multi-functional hematopoietic receptor agonists.
The method of the present invention involves
culturing suitable cells or cell line, which has been
transformed with a vector containing a DNA sequence
coding for expression of a novel multi-functional
hematopoietic receptor agonist. Suitable cells or
cell lines may be bacterial cells. For example, the
various strains of E. coli are well-known as host
cells in the field of biotechnology. Examples of
such strains include E. coli strains JM101 (Yanish-
Perron et al. Gene 33: 103-119, 1985) and MON105
(Obukowicz et al., Applied Environmental Microbiology
58: 1511-1523, 1992). Also included in the present
invention is the expression of the multi-functional
hematopoietic receptor agonist protein utilizing a
chromosomal expression vector for E. coli based on
the bacteriophage Mu (Weinberg et al., Gene 126: 25-
33, 1993). Various strains of B. subtilis may also
be employed in this method. Many strains of yeast
cells known to those skilled in the art are also
available as host cells for expression of the
polypeptides of the present invention. When
expressed in the E. coli cytoplasm, the gene encoding
the multi-functional hematopoietic receptor agonists
of the present invention may also be constructed such
that at the 5' end of the gene codons are added to
encode Met 2-Ala 1- or Met 1 at the N-terminus of the
protein. The N termini of proteins made in the
cytoplasm of E. coli are affected by post-
translational processing by methionine aminopeptidase
(Ben Bassat et al., J. Bac. 169:751-757, 1987) and
possibly by other peptidases so that upon expression
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the methionine is cleaved off the N-terminus. The
multi-functional hematopoietic receptor agonists of
the present invention may include multi-functional
hematopoietic receptor agonist polypeptides having
Met 1, Ala 1 or Met 2-Ala 1 at the N-terminus. These
mutant multi-functional hematopoietic receptor
agonists may also be expressed in E. coli by fusing a
secretion signal peptide to the N-terminus. This
signal peptide is cleaved from the polypeptide as
part of the secretion process.
Also suitable for use in the present invention
are mammalian cells, such as Chinese hamster ovary
cells (CHO). General methods for expression of
foreign genes in mammalian cells are reviewed in
Kaufman, R. J., 1987) Genetic Engineering, Principles
and Methods, Vol. 9, J. K. Setlow, editor, Plenum
Press, New York. An expression vector is constructed
in which a strong promoter capable of functioning in
mammalian cells drives transcription of a eukaryotic
secretion signal peptide coding region, which is
translationally joined to the coding region for the
multi-functional hematopoietic receptor agonist. For
example, plasmids such as pcDNA I/Neo, pRc/RSV, and
pRc/CMV (obtained from Invitrogen Corp., San Diego,
California) can be used. The eukaryotic secretion
signal peptide coding region can be from the gene
itself or it can be from another secreted mammalian
protein (Bayne, M. L. et al., Proc. Natl. Acad. Sci.
USA 84: 2638-2642, 1987). After construction of the
vector containing the gene, the vector DNA is
transfected into mammalian cells. Such cells can be,
for example, the COS7, HeLa, BHK, CHO, or mouse L
lines. The cells can be cultured, for example, in
DMEM media (JRH Scientific). The polypeptide
secreted into the media can be recovered by standard
biochemical approaches following transient expression
for 24 - 72 hours after transfection of the cells or
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23 -
after establishment of stable cell lines following
selection for antibiotic resistance. The selection
of suitable mammalian host cells and methods for
transformation, culture, amplification, screening and
product production and purification are known in the
art. See, e.g., Gething and Sambrook, Nature,
293:620-625, 1981), or alternatively, Kaufman et al,
Mol. Cell. Biol., 5(7):1750-1759, 1985) or Howley
et al., U.S. Pat. No. 4,419,446. Another suitable
mammalian cell line is the monkey COS-1 cell line. A
similarly useful mammalian cell line is the CV-1 cell
line.
Where desired, insect cells may be utilized as
host cells in the method of the present invention.
See, e.g., Miller et al., Genetic Engineering, 8:277-
298 (Plenum Press 1986) and references cited therein.
In addition, general methods for expression of
foreign genes in insect cells using Baculovirus
vectors are described in: Summers, M. D. and Smith,
G. E., 1987) - A manual of methods for Baculovirus
vectors and insect cell culture procedures, Texas
Agricultural Experiment Station Bulletin No. 1555.
An expression vector is constructed comprising a
Baculovirus transfer vector, in which a strong
Baculovirus promoter (such as the polyhedron
promoter) drives transcription of a eukaryotic
secretion signal peptide coding region, which is
translationally joined to the coding region for the
multi-functional hematopoietic receptor agonist
polypeptide. For example, the plasmid pVL1392
(obtained from Invitrogen Corp., San Diego,
California) can be used. After construction of the
vector carrying the gene encoding the multi-
functional hematopoietic receptor agonist
polypeptide, two micrograms of this DNA is co-
transfected with one microgram of Baculovirus DNA
(see Summers & Smith, 1987) into insect cells, strain
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SF9. Pure recombinant Baculovirus carrying the
multi-functional hematopoietic receptor agonist is
used to infect cells cultured, for example, in Excell
401 serum-free medium (JRH Biosciences, Lenexa,
Kansas). The multi-functional hematopoietic receptor
agonist secreted into the medium can be recovered by
standard biochemical approaches. Supernatants from
mammalian or insect cells expressing the multi-
functional hematopoietic receptor agonist protein can
be first concentrated using any of a number of
commercial concentration units.
The multi-functional hematopoietic receptor
agonists of the present invention may be useful in
the treatment of diseases characterized by decreased
levels of either myeloid, erythroid, lymphoid, or
megakaryocyte cells of the hematopoietic system or
combinations thereof. In addition, they may be used
to activate mature myeloid and/or lymphoid cells.
Among conditions susceptible to treatment with the
polypeptides of the present invention is leukopenia,
a reduction in the number of circulating leukocytes
(white cells) in the peripheral blood. Leukopenia
may be induced by exposure to certain viruses or to
radiation. It is often a side effect of various
forms of cancer therapy, e.g., exposure to
chemotherapeutic drugs, radiation and of infection or
hemorrhage. Therapeutic treatment of leukopenia with
these multi-functional hematopoietic receptor
agonists of the present invention may avoid
undesirable side effects caused by treatment with
presently available drugs.
The multi-functional hematopoietic receptor
agonists of the present invention may be useful in
the treatment of neutropenia and, for example, in the
treatment of such conditions as aplastic anemia,
cyclic neutropenia, idiopathic neutropenia, Chediak-
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Higashi syndrome, systemic lupus erythematosus (SLEy,
leukemia, myelodysplastic syndrome and myelofibrosis.
- The multi-functional hematopoietic receptor
agonist of the present invention may be useful in the
treatment or prevention of thrombocytopenia.
Currently the only therapy for thrombocytopenia is
platelet transfusion which are costly and carry the
significant risks of infection (HIV, HBV) and
alloimunization. The multi-functional hematopoietic
receptor agonist may alleviate or diminish the need
for platelet transfusion. Severe thrombocytopenia may
result from genetic defects such as Fanconi's Anemia,
Wiscott-Aldrich, or May Hegglin syndromes. Acquired
thrombocytopenia may result from auto- or allo-
antibodies as in Immune Thrombocytopenia Purpura,
Systemic Lupus Erythromatosis, hemolytic anemia, or
fetal maternal incompatibility. In addition,
splenomegaly, disseminated intravascular coagulation,
thrombotic thrombocytopenic purpura, infection or
prosthetic heart valves may result in
thrombocytopenia. Severe thrombocytopenia may also
result from chemotherapy and/or radiation therapy or
cancer. Thrombocytopenia may also result from marrow
invasion by carcinoma, lymphoma, leukemia or
fibrosis.
The multi-functional hematopoietic receptor
agonists of the present invention may be useful in
the mobilization of hematopoietic progenitors and
stem cells in peripheral blood. Peripheral blood
derived progenitors have been shown to be effective
in reconstituting patients in the setting of
autologous marrow transplantation. Hematopoietic
growth factors including G-CSF and GM-CSF have been
shown to enhance the number of circulating
progenitors and stem cells in the peripheral blood.
This has simplified the procedure for peripheral stem
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cell collection and dramatically decreased the cost
of the procedure by decreasing the number of pheresis
required. The multi-functional hematopoietic receptor
agonist may be useful in mobilization of stem cells
and further enhance the efficacy of peripheral stem
cell transplantation.
The multi-functional hematopoietic receptor
agonists of the present invention may also be useful
in the ex vivo expansion of hematopoietic progenitors
and stem cells. Colony stimulating factors (CSFs),
such as hIL-3, have been administered alone, co-
administered with other CSFs, or in combination with
bone marrow transplants subsequent to high dose
chemotherapy to treat the neutropenia and
thrombocytopenia which are often the result of such
treatment. However the period of severe neutropenia
and thrombocytopenia may not be totally eliminated.
The myeloid lineage, which is comprised of monocytes
(macrophages), granulocytes (including neutrophils)
and megakaryocytes, is critical in preventing
infections and bleeding which can be life-
threatening. Neutropenia and thrombocytopenia may
also be the result of disease, genetic disorders,
drugs, toxins, radiation and many therapeutic
treatments such as conventional oncology therapy.
Bone marrow transplants have been used to treat
this patient population. However, several problems
are associated with the use of bone marrow to
reconstitute a compromised hematopoietic system
including: 1) the number of stem cells in bone
marrow, spleen, or peripheral blood is limited, 2)
Graft Versus Host Disease, 3) graft rejection and 4)
possible contamination with tumor cells. Stem cells
make up a very small percentage of the nucleated
cells in the bone marrow, spleen and peripheral
blood. It is clear that a dose response exists such
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that a greater number of stem cells will enhance
hematopoietic recovery. Therefore, the in vitro
expansion of stem cells should enhance hematopoietic
recovery and patient survival. Bone marrow from an
allogeneic donor has been used to provide bone marrow
for transplant. However, Graft Versus Host Disease
and graft rejection limit bone marrow transplantation
even in recipients with HLA-matched sibling donors.
An alternative to allogeneic bone marrow transplants
is autologous bone marrow transplants. In autalogous
bone marrow transplants, some of the patient's own
marrow is harvested prior to myeloablative therapy,
e.g. high dose chemotherapy, and is transplanted back
into the patient afterwards. Autologous transplants
eliminate the risk of Graft Versus Host Disease and
graft rejection. However, autologous bone marrow
transplants still present problems in terms of the
limited number of stems cells in the marrow and
possible contamination with tumor cells. The limited
number of stem cells may be overcome by ex-vivo
expansion of the stem cells. In addition, stem cells
can be specifically isolated, based on the presence
of specific surface antigens such as CD34+ in order
to decrease tumor cell contamination of the marrow
graft.
The following patents contain further details on
separating stem cells, CD34+ cells, culturing the
cells with hematopoietic factors, the use of the
cells for the treatment of patients with
hematopoietic disorders and the use of hematopoietic
factors for cell expansion and gene therapy.
5,061,620 relates to compositions comprising human
hematopoietic stem cells provided by separating the
stem cells from dedicated cells.
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5,199,942 describes a method for autologous
hematppoietic cell transplantation comprising: (1)
obtaining hematopoietic progenitor cells from a
patient; (2) ex-vivo expansion of cells with a growth
factor selected from the group consisting of IL-3,
flt3 ligand, c-kit ligand, GM-CSF, IL-1, GM-CSF/IL-3
chimera protein and combinations thereof; (3)
administering cellular preparation to a patient.
5,240,856 relates to a cell separator that includes
an apparatus for automatically controlling the cell
separation process.
WO 91/16116 describes devices and methods for
selectively isolating and separating target cells
from a mixture of cells.
WO 91/18972 describes methods for in vitro culturing
of bone marrow, by incubating suspension of bone
marrow cells, using a hollow fiber bioreactor.
WO 92/18615 relates to a process for maintaining and
expanding bone marrow cells, in a culture medium
containing specific mixtures of cytokines, for use in
transplants.
WO 93/08268 describes a method for selectively
expanding stem cells, comprising the steps of (a)
separating CD34+ stem cells from other cells and (b)
incubating the separated cells in a selective medium,
such that the stem cells are selectively expanded.
WO 93/18136 describes a process for in vitro support
of mammalian cells derived from peripheral blood.
WO 93/18648 relates to a composition comprising human
neutrophil precursor cells with a high content of
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29 -
myeloblasts and promyelocytes for treating genetic or
acquired neutropenia.
WO 94/08039 describes a method of enrichment for
human hematopoietic stem cells by selection for cells
which express c-kit protein.
WO 94/11493 describes a stem cell population that are
CD34+ and small in size, which are isolated using a
counterflow elutriation method.
w0 94/27698 relates to a method combining
immunoaffinity separation and continuous flow
centrifugal separation for the selective separation
of a nucleated heterogeneous cell population from a
heterogeneous cell mixture.
WO 94/25848 describes a cell separation apparatus for
collection and manipulation of target cells.
The long term culturing of highly enriched CD34+
precursors of hematopoietic progenitor cells from
human bone marrow in cultures containing IL-la, IL-3,
IL-6 or GM-CSF is discussed in Brandt et al J. Clin.
Invest. 86:932-941, 1990).
One aspect of the present invention provides a method
for selective ex-vivo expansion of stem cells. The
term "stem cell" refers to the totipotent
hematopoietic stem cells as well as early precursors
and progenitor cells which can be isolated from bone
marrow, spleen or peripheral blood. The term
"expansion" refers to the differentiation and
proliferation of the cells. The present invention
provides a method for selective ex-vivo expansion of
stem cells, comprising the steps of: (a) separating
stem cells from other cells, (b) culturing said
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separated stem cells with a selective media which
contains multi-functional hematopoietic receptor
agonist proteins) and (c) harvesting said stems
cells. Stem cells, as well as committed progenitor
cells destined to become neutrophils, erythrocytes,
platelets, etc. may be distinguished from most other
cells by the presence or absence of particular
progenitor marker antigens, such as CD34, that are
present on the surface of these cells and/or by
morphological characteristics. The phenotype for a
highly enriched human stem cell fraction is reported
as CD34+, Thy-1+ and lin-, but it is to be understood
that the present invention is not limited to the
expansion of this stem cell population. The CD34+
enriched human stem cell fraction can be separated by
a number of reported methods, including affinity
columns or beads, magnetic beads or flow cytometry
using antibodies directed to surface antigens such as
the CD34+. Further, physical separation methods such
as counterflow elutriation may be used to enrich
hematopoietic progenitors. The CD34+ progenitors are
heterogeneous, and may be divided into several sub-
populations characterized by the presence or absence
of co-expression of different lineage associated cell
surface associated molecules. The most immature
progenitor cells do not express any known lineage
associated markers, such as HLA-DR or CD38, but they
may express CD90(thy-1). Other surface antigens such
as CD33, CD38, CD41, CD71, HLA-DR or c-kit can also
be used to selectively isolate hematopoietic
progenitors. The separated cells can be incubated in
selected medium in a culture flask, sterile bag or in
hollow fibers. Various colony stimulating factors may
be utilized in order to selectively expand cells.
Representative factors that have been utilized for
ex-vivo expansion of bone marrow include, c-kit
ligand, IL-3, G-CSF, GM-CSF, IL-1, IL-6, IL-11, flt3
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ligand or combinations thereof. The proliferation of
the stem cells can be monitored by enumerating the
number of stem cells and other cells, by standard
techniques (e.g, hemacytometer, CFU, LTCIC) or by
flow cytometry prior and subsequent to incubation.
Several methods for ex-vivo expansion of stem
cells have been reported utilizing a number of
selection methods and expansion using various colony
stimulating factors including c-kit ligand (Brandt et
al., Blood 83:1507-1514 [1994], McKenna et al., Blood
86:3413-3420 [1995]), IL-3 (Brandt et al., Blood
83:1507-1514 [1994], Sato et al., Blood 82:3600-3609
[1993]), G-CSF (Sato et al., Blood 82:3600-3609
[1993]), GM-CSF (Sato et al., Blood 82:3600-3609
[1993]), IL-1 (Muench et al., Blood 81:3463-3473
[1993]), IL-6 (Sato et al., Blood 82:3600-3609
[1993]), IL-11 (Lemoli et al., Fxp. Hem. 21:1668-1672
[1993], Sato et al., Blood 82:3600-3609 [1993]), flt3
ligand (McKenna et al., Blood 86:3413 3420 [1995])
and/or combinations thereof (Brandt et al., Blood
83:1507 1514 [1994], Haylock et al., Blood 80:1405-
1412 [1992], Koller et al., Biotechnology 11:358-363
[1993], (Lemoli et al., Exp. Hem. 21:1668-1672
[1993]), McKenna et al., Blood 86:3413-3420 [1995],
Muench et al., Blood 81:3463-3473 [1993], Patchen et
al., Biotherapy 7:13-26 [1994], Sato et al., Blood
82:3600-3609 [1993], Smith et al., Exp. Hem. 21:870-
877 [1993], Steen et al., Stem Cells 12:214-224
[1994], Tsujino et al., Exp. Hem. 21:1379-1386
[1993]). Among the individual colony stimulating
factors, hIL-3 has been shown to be one of the most
potent in expanding peripheral blood CD34+ cells
(Sato et al., Blood 82:3600-3609 [1993], Kobayashi et
al., Blood 73:1836-1841 [1989]). However, no single
factor has been shown to be as effective as the
combination of multiple factors. The present
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32
invention provides methods for ex vivo expansion that
utilise multi-functional hematopoietic receptor
agonists that are more effective than a single factor
alone.
Another aspect of the invention provides methods
of sustaining and/or expanding hematopoietic
precursor cells which includes inoculating the cells
into a culture vessel which contains a culture medium
that has been conditioned by exposure to a stromal
cell line such as HS-5 (WO 96/02662, Roecklein and
Torok-Strob, Blood 85:997-1105, 1995) that has been
supplemented with a multi-functional hematopoietic
receptor agonist of the present invention.
Another projected clinical use of growth factors
has been in the in vitro activation of hematopoietic
progenitors and stem cells for gene therapy. Due to
the long life-span of hematopoietic progenitor cells
and the distribution of their daughter cells
throughout the entire body, hematopoietic progenitor
cells are good candidates for ex vivo gene
transfection. In order to have the gene of interest
incorporated into the genome of the hematopoietic
progenitor or stem cell one needs to stimulate cell
division and DNA replication. Hematopoietic stem
cells cycle at a very low frequency which means that
growth factors may be useful to promote gene
transduction and thereby enhance the clinical
prospects for gene therapy. Potential applications of
gene therapy (review Crystal, Science 270:404-410
[1995]) include; 1) the treatment of many congenital
metabolic disorders and immunodeficiencies (Kay and
Woo, Trends Genet. 10:253-257 [1994]), 2)
neurological disorders (Friedmann, Trends Genet.
10:210-214 (1994]), 3) cancer (Culver and Blaese,
Trends Genet. 10:174-178 [1994]) and 4) infectious
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diseases (Gilboa and Smith, Trends Genet. 10:139-144
[1994]).
There are a variety of methods, known to those
with skill in the art, for introducing genetic
S material into a host cell. A number of vectors, both
viral and non-viral have been developed for
transferring therapeutic genes into primary cells.
Viral based vectors include; 1) replication deficient
recombinant retrovirus (Boris-Lawrie and Temin, Curr.
Opin. Genet. Dev. 3:102-109 [1993], Boris-Lawrie and
Temin, Annal. New York Acad. Sci. 716:59-71 [1994],
Miller, Current Top. Microbiol. Immunol. 158:1-24
[1992]) and replication-deficient recombinant
adenovirus (Berkner, BioTechniques 6:616-629 [1988],
Berkner, Current Top. Microbiol. Imniunol. 158:39-66
[1992], Brody and Crystal, Arsnal. New York Acad. Sci.
716:90-103 [1994]). Non-viral based vectors include
protein/DNA complexes (Cristiano et al., PNAS USA.
90:2122-2126 [1993], Curiel et al., PNAS USA 88:8850-
8854 [1991], Curiel, Annal. New York Acad. Sci.
716:36-58 [1994]), electroporation and liposome
mediated delivery such as cationic liposomes (Farhood
et al., Annal. New York Acad. Sci. 716:23-35 [1994]).
The present invention provides an improvement to
the existing methods of expanding hematopoietic
cells, which new genetic material has been
introduced, in that it provides methods utilizing
multi-functional hematopoietic receptor agonist
proteins that have improved biological activity,
including an activity not seen by any single colony
stimulation factor.
Many drugs may cause bone marrow suppression or
hematopoietic deficiencies. Examples of such drugs
are AZT, DDI, alkylating agents and anti-metabolites
used in chemotherapy, antibiotics such as
chloramphenicol, penicillin, gancyclovir, daunomycin
and sulfa drugs, phenothiazones, tranquilizers such
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34 -
as meprobamate, analgesics such as aminopyrine and
dipyrpne, anti-convulsants such as phenytoin or
carbamazepine, antithyroids such as propylthiouracil
and methimazole and diuretics. The multi-functional
hematopoietic receptor agonists of the present
invention may be useful in preventing or treating the
bone marrow suppression or hematopoietic deficiencies
which often occur in patients treated with these
drugs.
Hematopoietic deficiencies may also occur as a
result of viral, microbial or parasitic infections
and as a result of treatment for renal disease or
renal failure, e.g., dialysis. The multi-functional
hematopoietic receptor agonists of the present
invention may be useful in treating such
hematopoietic deficiencies.
Various immunodeficiencies, e.g., in T and/or B
lymphocytes, or immune disorders, e.g., rheumatoid
arthritis, may also be beneficially affected by
treatment with the multi-functional hematopoietic
receptor agonists of the present invention.
Immunodeficiencies may be the result of viral
infections, e.g., HTLVI, HTLVII, HTLVIII, severe
exposure to radiation, cancer therapy or the result
of other medical treatment. The multi-functional
hematopoietic receptor agonists of the present
invention may also be employed, alone or in
combination with other colony stimulating factors, in
the treatment of other blood cell deficiencies,
including thrombocytopenia (platelet deficiency), or
anemia. Other uses for these novel polypeptides are
the in vivo and ex vivo treatment of patients
recovering from bone marrow transplants, and in the
development of monoclonal and polyclonal antibodies
generated by standard methods for diagnostic or
therapeutic use.
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Other aspects of the present invention are
methods and therapeutic compositions for treating the
conditions referred to above. Such compositions
comprise a therapeutically effective amount of one or
more of the mufti-functional hematopoietic receptor
agonists of the present invention in a mixture with a
pharmaceutically acceptable carrier. This
composition can be administered either parenterally,
intravenously or subcutaneously. When administered,
the therapeutic composition for use in this invention
is preferably in the form of a pyrogen-free,
parenterally acceptable aqueous solution. The
preparation of such a parenterally acceptable protein
solution, having due regard to pH, isotonicity,
stability and the like, is within the skill of the
art. The treatment of hematopoietic deficiency may
include administration of a pharmaceutical
composition containing the mufti-functional
hematopoietic receptor agonists to a patient. The
mufti-functional hematopoietic receptor agonists of
the present invention may also be useful for the
activation and amplification of hematopoietic
precursor cells by treating these cells in vitro with
the mufti-functional hematopoietic receptor agonist
proteins of the present invention prior to injecting
the cells into a patient.
The dosage regimen involved in a method for
treating the above-described conditions will be
determined by the attending physician considering
various factors which modify the action of drugs,
e.g., the condition, body weight, sex and diet of the
patient, the severity of any infection, time of
administration and other clinical factors.
Generally, a daily regimen may be in the range of 0.2
- 150 ug/kg of mufti-functional hematopoietic
receptor agonist protein per kilogram of body weight.
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Dosages would be adjusted relative to the activity of
a given multi-functional hematopoietic receptor
agonist protein and it would not be unreasonable to
note that dosage regimens may include doses as low as
0.1 microgram and as high as 1 milligram per kilogram
of body weight per day. In addition, there may exist
specific circumstances where dosages of multi-
functional hematopoietic receptor agonist would be
adjusted higher or lower than the range of 0.2 - 150
micrograms per kilogram of body weight. These
include co-administration with other hematopoietic
growth factors or IL-3 variants or growth factors;
co-administration with chemotherapeutic drugs and/or
radiation; the use of glycosylated mufti-functional
hematopoietic receptor agonist protein; and various
patient-related issues mentioned earlier in this
section. As indicated above, the therapeutic method
and compositions may also include co-administration
or sequential administration other hematopoietic
growth factors. A non-exclusive list of other
appropriate hematopoietic growth factors, colony
stimulating factors (CSFs), cytokines, lymphokines,
and interleukins for simultaneous or serial co-
administration with the chimeric proteins of the
present invention includes GM-CSF, G-CSF, G-CSF
Serb , c-mpl ligand (also known as TPO or MGDF), M-
CSF, erythropoietin (EPO), IL-1, IL-4, IL-2, IL-3,
IL-3 variant, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10,
IL-11, IL-12, IL-13, IL-15, IL-16, LIF, B-cell growth
factor, B-cell differentiation factor and eosinophil
differentiation factor, stem cell factor (SCF) also
known as steel factor or c-kit ligand, SCSF, SDF-1 or
combinations thereof. "hIL-3 variant" is defined as a
hIL-3 molecule which has amino acid substitutions
and/or portions of hIL-3 deleted as disclosed in WO
94/12638, WO 94/12639 and WO 95/00646, as well as
other variants known in the art. The dosage recited
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above would be adjusted to compensate for such
additional components in the therapeutic composition.
Progress of the treated patient can be monitored by
periodic assessment of the hematological profile,
e.g., differential cell count and the like.
Determination of activity of the chimeric roteins
Biological activity of the multi-functional
hematopoietic receptor agonist proteins of the
present invention can be determined by DNA synthesis
in factor-dependent cell lines or by counting the
colony forming units in an in vitro bone marrow
assay. The chimeric proteins may be assayed by a
number of in vitro and in vivo models known to those
skilled in the art. Examples of such assays include
put are not limited to:
Methylcellulose Assay
This assay reflects the ability of colony stimulating
factors to stimulate normal bone marrow cells to
produce different types of hematopoietic colonies in
vitro (Bradley et al., Aust. Exp Biol. Sci. 44:287-
300, 1966), Pluznik et al., J. Cell Comp. Physio
66:319-324, 1965).
Methods
Approximately 30 mL of fresh, normal, healthy bone
marrow aspirate are obtained from individuals
following informed consent. Under sterile conditions
samples are diluted 1:5 with a 1X PBS (#14040.059
Life Technologies, Gaithersburg, MD.) solution in a
50 mL conical tube (#25339-50 Corning, Corning MD).
Ficoll (Histopaque 1077 Sigma H-8889) is layered
under the diluted sample and centrifuged, 300 x g for
30 min. The mononuclear cell band is removed and
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washed two times in 1X PBS and once with 1~ BSA PBS
(Cell~ro Co., Bothel, WA). Mononuclear cells are
counted and CD34+ cells are selected using the
Ceprate LC (CD34) Kit (CellPro Co., Bothel, WA)
column. This fractionation is performed since all
stem and progenitor cells within the bone marrow
display CD34 surface antigen.
Cultures are set up in triplicate with a final volume
of 1.0 mL in a 35 X 10 mm petri dish (Nunc#174926).
Culture medium is purchased from Terry Fox Labs.
(HCC-4230 medium (Terry Fox Labs, Vancouver, B.C.,
Canada) and erythropoietin (Amgen, Thousand Oaks,
CA.) is added to the culture media. 3,000-10,000
CD34+ cells are added per dish. Recombinant IL-3,
purified from mammalian cells or E. coli, and multi-
functional hematopoietic receptor agonist proteins,
in conditioned media from transfected mammalian cells
or purified from conditioned media from transfected
mammalian cells or E. coli, are added to give final
concentrations ranging from .001 nM to 10 nM.
Recombinant hIL-3, GM-CSF, c-mpl ligand and multi-
functional hematopoietic receptor agonist are
supplied in house. G-CSF (Neupogen) is from Amgen
(Thousand Oaks Calf.). Cultures are resuspended using
a 3cc syringe and 1.0 mL is dispensed per dish.
Control (baseline response) cultures received no
colony stimulating factors. Positive control
cultures received conditioned media (PHA stimulated
human cells: Terry Fox Lab. H2400). Cultures are
incubated at 37°C, 5~ C02 in humidified air.
Hematopoietic colonies which are defined as greater
than 50 cells are counted on the day of peak response
(days 10-11) using a Nikon inverted phase microscope
with a 40x objective combination. Groups of cells
containing fewer than 50 cells are referred to as
clusters. Alternatively colonies can be identified by
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spreading the colonies on a slide and stained or they
can be picked, resuspended and spun onto cytospin
slides for staining.
Human Cord Blood Hemopoietic Growth Factor Assays
Bone marrow cells are traditionally used for in vitro
assays of hematopoietic colony stimulating factor
(CSF) activity. However, human bone marrow is not
always available, and there is considerable
variability between donors. Umbilical cord blood is
comparable to bone marrow as a source of
hematopoietic stem cells and progenitors (Bro~neyer
et al., PNAS USA 89:4109-113, 1992; Mayani et al.,
Blood 81:3252-3258, 1993). In contrast to bone
marrow, cord blood is more readily available on a
regular basis. There is also a potential to reduce
assay variability by pooling cells obtained fresh
from several donors, or to create a bank of
cryopreserved cells for this purpose. By modifying
the culture conditions, and/or analyzing for lineage
specific markers, it is be possible to assay
specifically for granulocyte / macrophage colonies
(CFU-GM), for megakaryocyte CSF activity, or for high
proliferative potential colony forming cell (HPP-CFC)
activity.
Methods
Mononuclear cells (MNC) are isolated from cord blood
within 24 hr. of collection, using a standard density
gradient (1.077 g/mL Histopaque). Cord blood MNC have
been further enriched for stem cells and progenitors
by several procedures, including immunomagnetic
selection for CD14-, CD34+ cells; panning for SBA-,
CD34+ fraction using coated flasks from Applied
Immune Science (Santa Clara, CA); and CD34+
selection using a CellPro (Bothell, WA) avidin
column. Either freshly isolated or cryopreserved
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CD34+ cell enriched fractions are used for the assay.
Duplicate cultures for each serial dilution of sample
(concentration range from 1 pM to 1204 pM) are
prepared with 1x104 cells in lml of 0.9~
5 methycellulose containing medium without additional
growth factors (Methocult H4230 from Stem Cell
Technologies, Vancouver, BC.). In some experiments,
Methocult H4330 containing erythropoietin (EPO) was
used instead of Methocult H4230, or Stem Cell Factor
10 (SCF), 50 ng/mL (Biosource International, Camarillo,
CA) was added. After culturing for 7-9 days, colonies
containing >30 cells are counted. In order to rule
out subjective bias in scoring, assays are scored
blind.
AML Proliferation Assay for Bioactive Human
Interleukin-3
The factor-dependent cell line AML 193 was
obtained from the American Type Culture Collection
(ATCC, Rockville, MD). This cell line, established
from a patient with acute myelogenous leukemia, is a
growth factor dependent cell line which displayed
enhanced growth in GM-CSF supplemented medium
(Lange, B., et al., Blood 70: 192, 1987; Valtieri,
M., et al., J. Immunol. 138:4042, 198?). The ability
of AML 193 cells to proliferate in the presence of
human IL-3 has also been documented. (Santoli, D.,
et al., J. Immunol. 139: 348, 1987). A cell line
variant was used, AML 193 1.3, which was adapted for
long term growth in IL-3 by washing out the growth
factors and starving the cytokine dependent AML 193
cells for growth factors for 24 hours. The cells are
then replated at 1x105 cells/well in a 24 well plate
in media containing 100 U/mL IL-3. It took
approximately 2 months for the cells to grow rapidly
in IL-3. These cells are maintained as AML 193 1.3
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thereafter by supplementing tissue culture medium
(see below) with human IL-3.
AML 193 1.3 cells are washed 6 times in cold
Hanks balanced salt solution (HBSS, Gibco, Grand
Island, NY) by centrifuging cell suspensions at 250 x
g for 10 minutes followed by decantation of the
supernatant. Pelleted cells are resuspended in HBSS
and the procedure is repeated until six wash cycles
are completed. Cells washed six times by this
procedure are resuspended in tissue culture medium at
a density ranging from 2 x 105 to 5 x 105 viable
cells/mL. This medium is prepared by supplementing
Iscove's modified Dulbecco's Medium (IMDM, Hazelton,
Lenexa, KS) with albumin, transferrin, lipids and 2-
mercaptoethanol. Bovine albumin (Boehringer-
Mannheim, Indianapolis, IN) is added at 500 ug/mL;
human transferrin (Boehringer-Mannheim, Indianapolis,
IN) is added at 100 ug/mL; soybean lipid (Boehringer-
Mannheim, Indianapolis, IN) is added at 50 ug/mL; and
2-mercaptoethanol (Sigma, St. Louis, MO) is added at
5 x 10 5 M.
Serial dilutions of human interleukin-3 or
multi-functional chimeric hematopoietic receptor
agonist proteins are made in triplicate series in
tissue culture medium supplemented as stated above in
96 well Costar 3596 tissue culture plates. Each well
contained 50 ul of medium containing interleukin-3 or
multi-functional chimeric hematopoietic receptor
agonist proteins once serial dilutions are completed.
Control wells contained tissue culture medium alone
(negative control). AML 193 1.3 cell suspensions
prepared as above are added to each well by pipetting
50 ul (2.5 x 104 cells) into each well. Tissue
culture plates are incubated at 37°C with 5~ C02 in
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humidified air for 3 days. On day 3, 0.5 uCi 3H-
thymidine (2 Ci/mM, New England Nuclear, Boston, MA)
is added in 50 ul of tissue culture medium. Cultures
are incubated at 37°C with 5~ C02 in humidified air
for 18-24 hours. Cellular DNA is harvested onto
glass filter mats (Pharmacia LKB, Gaithersburg, MD)
using a TOMTEC cell harvester (TOMTEC, Orange, CT)
which utilized a water wash cycle followed by a 70~
ethanol wash cycle. Filter mats are allowed to air
dry and then placed into sample bags to which
scintillation fluid (Scintiverse II, Fisher
Scientific, St. Louis, MO or BetaPlate Scintillation
Fluid, Pharmacia LKB, Gaithersburg, MD) is added.
Beta emissions of samples from individual tissue
culture wells are counted in a LKB BetaPlate model
1205 scintillation counter (Pharmacia LKB,
Gaithersburg, MD) and data is expressed as counts per
minute of 3H-thymidine incorporated into cells from
each tissue culture well. Activity of each human
interleukin-3 preparation or multi-functional
chimeric hematopoietic receptor agonist protein
preparation is quantitated by measuring cell
proliferation (3H-thymidine incorporation) induced by
graded concentrations of interleukin-3 or multi-
functional chimeric hematopoietic receptor agonist.
Typically, concentration ranges from 0.05 pM - 105 pM
are quantitated in these assays. Activity is
determined by measuring the dose of interleukin-3 or
multi-functional chimeric hematopoietic receptor
agonist protein which provides 50~ of maximal
proliferation (ECSO = 0.5 x (maximum average counts
per minute of 3H-thymidine incorporated per well
among triplicate cultures of all concentrations of
interleukin-3 tested - background proliferation
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measured by 3H-thymidine incorporation observed in
triplicate cultures lacking interleukin-3). This ECSo
value is also equivalent to 1 unit of bioactivity.
Every assay is performed with native interleukin-3 as
a reference standard so that relative activity levels
could be assigned.
Typically, the multi-functional chimeric
hematopoietic receptor agonist proteins were tested
in a concentration range of 2000 pM to 0.06 pM
titrated in serial 2 fold dilutions.
Activity for each sample was determined by the
concentration which gave 50~ of the maximal response
by fitting a four-parameter logistic model to the
data. It was observed that the upper plateau
(maximal response) for the sample and the standard
with which it was compared did not differ. Therefore
relative potency calculation for each sample was
determined from EC50 estimations for the sample and
the standard as indicated above. AML 193.1.3 cells
proliferate in response to hIL-3, hGM-CSF and hG-CSF.
TF1 c-mpl ligand dependent proliferation assay
The c-mpl ligand proliferative activity can be
assayed using a subclone of the pluripotential human
cell line TF1 (Kitamura et al., J. Cell Physiol
140:323-334. [1989]). TF1 cells are maintained in h-
IL3 (100 U/mL?. To establish a sub-clone responsive
to c-mpl ligand, cells are maintained in passage
media containing 10$ supernatant from BHK cells
transfected with the gene expressing the 1-153 form
of c-mpl ligand (pMON26448). Most of the cells die,
but a subset of cells survive. After dilution
cloning, a c-mpl ligand responsive clone is selected,
and these cells are split into passage media to a
density of 0.3 x 106 cells/mL the day prior to assay
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set-up. Passage media for these cells is the
following: RPMI 1640 (Gibco), 10~ FBS (Harlan, Lot
#91206), 10$ c-mpl ligand supernatant from
transfected BHK cells, 1 mM sodium pyruvate (Gibco),
2 mM glutamine (Gibco), and 100 ug/mL penicillin-
streptomycin (Gibco). The next day, cells are
harvested and washed twice in RPMI or IMDM media with
a final wash in the ATL, or assay media. ATL medium
consists of the following: IMDM (Gibco), 500 ug/mL of
bovine serum albumin, 100 ug/mL of human transferrin,
50 ug/mL soybean lipids, 4 x 10-8M beta-
mercaptoethanol and 2 mL of A9909 (Sigma, antibiotic
solution) per 1000 mL of ATL. Cells are diluted in
assay media to a final density of 0.25 x 106 cells/mL
in a 96-well low evaporation plate (Costar) to a
final volume of 50 ul. Transient supernatants
(conditioned media) from transfected clones are added
at a volume of 50 ul as duplicate samples at a final
concentration of 50~ and diluted three-fold to a
final dilution of 1.8~. Triplicate samples of a dose
curve of IL-3 variant pMON13288 starting at 1 ng/mL
and diluted using three-fold dilutions to 0.0014ng/mL
is included as a positive control. Plates are
incubated at 5~ C02 and 37° C. At day six of
culture, the plate is pulsed with 0.5 Ci of 3H/well
(NEN) in a volume of 20 ul/well and allowed to
incubate at 5~ C02 and 37° C for four hours. The
plate is harvested and counted on a Betaplate
counter.
MUTZ-2 Cell Proliferation Assay
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A cell line such as MUTZ-2, which is a human myeloid
leukemia cell line (German Collection of
Microorganisms and Cell Cultures, DSM ACC 271), can
be used to determine the cell proliferative activity
of flt3 receptor agonists. MUTZ-2 cultures are
maintained with recombinant native flt3 ligand (20-
100ng/mL) in the growth medium. Eighteen hours prior
to assay set-up, MUTZ-2 cells are washed in IMDM
medium (Gibco) three times and are resuspended in
IMDM medium alone at a concentration of 0.5-0.7 x
10E6 cells/mL and incubated at 37°C and 5~C02 to
starve the cells of flt3 ligand. The day of the
assay, standards and flt3 receptor agonists are
diluted to two fold above desired final concentration
in assay media in sterile tissue culture treated 96
well plates. Flt3 receptor agonists and standards are
tested in triplicate. 50u1 of assay media is loaded
into all wells except row A. 75u1 of the flt3
receptor agonists or standards are added to row A and
25u1 taken from that row and serial dilutions (1:3)
performed on the rest of the plate (rows B through
G). Row H remains as a media only control. The
starved MUTZ-2 cells are washed two times in IMDM
medium and resuspended in 50u1 assay media. 50u1 of
cells are added to each well resulting in a final
concentration of 0.25 x 10E6cells/mL. Assay plates
containing cells are incubated at 37°C and 5~C02 for
44hrs. Each well is then pulsed with luCi/well of
tritiated thymidine in a volume of 20u1 for four
hours. Plates are then harvested and counted.
Other in vitro cell based proliferation assays
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Other in vitro cell based assays, known to those
skilled in the art, may also be useful to determine
the activity of the multi-functional chimeric
hematopoietic receptor agonists depending on the
factors that comprise the molecule in a similar
manner as described in the AML 193.1.3 cell
proliferation assay. The following are examples of
other useful assays.
TF1 proliferation assay: TF1 is a pluripotential
human cell line (Kitamura et al., J. Cell Physiol
140:323-334. [1989]) that responds to hIL-3.
32D proliferation assay: 32D is a murine IL-3
dependent cell line which does not respond to human
IL-3 but does respond to human G-CSF which is not
species restricted.
Baf/3 proliferation assay: Baf/3 is a murine IL-3
dependent cell line which does not respond to human
IL-3, human flt3 ligand or human c-mpl ligand but
does respond to human G-CSF which is not species
restricted.
T1165 proliferation assay: T1165 cells are a IL-6
dependent murine cell line (Nordan et al., 1986)
which respond to IL-6 and IL-11.
Human Plasma Clot meg-CSF Assay: Used to assay
megakaryocyte colony formation activity (Mazur et
al., 1981).
Transfected cell lines:
Cell lines such as the murine Baf/3 cell line
can be transfected with a hematopoietic growth factor
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receptor, such as the human G-CSF receptor or human
c-mpl receptor, which the cell line does not have.
These transfected cell lines can be used to determine
the activity of the ligand for which the receptor has
been transfected into the cell line.
One such transfected Baf/3 cell line was
made by cloning the cDNA encoding c-mpl from a
library made from a c-mpl responsive cell line and
cloned into the multiple cloning site of the plasmid
pcDNA3 (Invitrogen, San Diego Ca.). Baf/3 cells were
transfected with the plasmid via electroporation. The
cells were grown under 6418 selection in the presence
of mouse IL-3 in Wehi conditioned media. Clones were
established through limited dilution.
In a similar manner the human G-CSF receptor can
be transfected into the Baf/3 cell line and used to
determine the bioactivity of the multi-functional
chimeric hematopoietic receptor agonists.
Analysis of c-mpl ligand proliferative activity
Methods
1. Bone marrow proliferation assay
a. CD34+ Cell Purification:
Bone marrow aspirates (15-20 mL) were obtained
from normal allogeneic marrow donors after informed
consent. Cells were diluted 1:3 in phosphate
buffered saline (PBS, Gibco-BRL), 30 mL were layered
over I5 mL Histopaque-1077 (Sigma) and centrifuged
for 30 minutes at 300 RCF. The mononuclear interface
layer was collected and washed in PBS. CD34+ cells
were enriched from the mononuclear cell preparation
using an affinity column per manufacturers
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48 '
instructions (CellPro, Inc., Bothell WA). After
enrichment, the purity of CD34+ cells was 70~ on
average as determined by using flow cytometric
analysis using anti-CD34 monoclonal antibody
conjugated to fluorescein and anti-CD38 conjugated
to phycoerythrin (Becton Dickinson, San Jose CA).
Cells were resuspended at 40,000 cells/mL in X-
Vivo 10 media (Bio-Whittaker, Walkersville, MD) and 1
mL was plated in 12-well tissue culture plates
(Costar). The growth factor rhIL-3 was added at 100
ng/mL (pMON5873) was added to some wells. hIL3
variants were used at 10 ng/mL to 100 ng/mL.
Conditioned media from BHK cells transfected with
plasmid encoding c-mpl ligand or multi-functional
chimeric hematopoietic receptor agonists were tested
by addition of 100 ul of supernatant added to 1 mL
cultures (approximately a 10~ dilution). Cells were
incubated at 37°C for 8-14 days at 5~ C02 in a 37°C
humidified incubator.
b. Cell Harvest and Analysis:
At the end of the culture period a total cell
count was obtained for each condition. For
fluorescence analysis and ploidy determination cells
were washed in megakaryocyte buffer (MK buffer, 13.6
mM sodium citrate, 1 mM theophylline, 2.2 dun PGE1, 11
mM glucose, 3~ w/v BSA, in PBS, pH 7.4,) (Tourer et
al., Blood 70: 1735-1742, 1987) resuspended in 500 ul
of MK buffer containing anti-CD4la FITC antibody
{1:200, AMAC, Westbrook, ME) and washed in MK buffer.
For DNA analysis cells were permeablized in MK buffer
containing 0.5~ Tween 20 (Fisher, Fair Lawn NJ)for 20
min. on ice followed by fixation in 0.5~ Tween-20 and
1~ paraformaldehyde (Fisher Chemical) for 30 minutes
followed by incubation in propidium iodide
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(Calbiochem , La Jolla Ca) (50 ug/mL) with RNAase
(400 U/mL) in 55~ v/v MK buffer (200mOsm) for 1-2
hours on ice. Cells were analyzed on a FACScan or
Vantage flow cytometer (Becton Dickinson, San Jose,
CA). Green fluorescence (CD4la-FITC) was collected
along with linear and log signals for red
fluorescence (PI) to determine DNA ploidy. All cells
were collected to determine the percent of cells that
were CD41+. Data analysis was performed using
software by LYSIS (Becton Dickinson, San Jose, CA).
Percent of cells expressing the CD41 antigen was
obtained from flow cytometry analysis(Percent).
Absolute (Abs) number of CD41+ cells/mL was
calculated by: (Abs)=(Cell Count)*(Percent)/100.
2. Megakaryocyte fibrin clot assay.
CD34+ enriched population were isolated as described
above. Cells were suspended at 25,000 cells/mL with
or without cytokine(s) in a media consisting of a
base Iscoves IMDM media supplemented with 0.3~ BSA,
0.4mg/mL apo-transferrin, 6.67uM FeCl2, 25ug/mL
CaCl2, 25ug/mL L-asparagine, 500ug/mL e-amino-n-
caproic acid and penicillin/streptomycin. Prior to
plating into 35mm plates, thrombin was added (0.25
Units/mL) to initiate clot formation. Cells were
incubated at 37°C for 13 days at 5~ C02 in a 37°C
humidified incubator.
At the end of the culture period plates were fixed
with methanol:acetone (1:3), air dried and stored at
-200C until staining. A peroxidase
immunocytochemistry staining procedure was used
(Zymed, Histostain-SP. San Francisco, CA) using a
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cocktail of primary monoclonal antibodies consisting
of anti-CD4la, CD42 and CD61. Colonies were counted'
after staining and classified as negative, CFU-MK
(small colonies, 1-2 foci and less that approx. 25
cells), BFU-MK (large, multi-foci colonies with > 25
cells) or mixed colonies (mixture of both positive
and negative cells.
EXAMPLES 1 & 2
Construction of an expression vectors,
pMON32364 and pMON32377, comprising a DNA sequence
encoding a multi-functional receptor agonist
comprising an IL-3 (15-125) variant joined via the
IgG2b linker to a flt-3 (1-134) ligand and an IL-3
(15-125) variant joined via the IgG2b linker to a
flt-3 (1-139) ligand, respectively. Plasmids,
pMON32364 and pMON32377, were assembled by cloning
gel-purified NcoI/HindIII DNA fragments from
pMON30237 SEQ ID N0:53 and pMON30238 SEQ ID N0:54,
containing the flt-3 (1-134) ligand and flt-3 (1-
139) into vector, pMON30311 (derivative of
pMON13058 - W095/21254), DNA digested with
AfIIII/HindIII and SAP-treated (pMON30311 is a
BHK-specific vector containing NcoI-IL-3 receptor
agonist-IgG2B-AfIIII-HindIII as an insert) using
standard ligation conditions. The ligation
mixtures were used to transform competent DHSa
cells (Gibco BRL cat #18265-017) following the
manufacturer's recommended protocol, and vector
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DNA was isolated from ampicillin-resistant
colonies. The DNA sequence of resulting genes (SEQ
ID N0:21 and SEQ ID N0:22 respectively) were
determined by automated fluorescent DNA sequencing
on an ABI 373/377 DNA sequencer (Perkin Elmer ABI)
using Sequencher (Gene Codes) software. The
resulting vectors, pMON32364 and pMON32377,
encodes the proteins of SEQ ID N0:42 and SEQ ID
N0:43 respectively.
EXAMPLES 3 & 4
Construction of an expression vectors,
pMON30247 and pMON30246, comprising a DNA sequence
encoding a multi-functional receptor agonist
comprising an IL-3 (15-125) variant joined via the
IgG2b linker to a flt-3 (1-134) ligand and an IL-3
(15-125) variant joined via the GlySer linker to a
flt-3 (1-134) ligand, respectively. Plasmids,
pMON30246 and pMON30247, were constructed by
cloning the gel purified NcoI/AfIIII restriction
fragment from pMON30244 (GlySer linker) SEQ ID
N0:65 and pMON30245 (IgG2B linker) SEQ ID N0:66
respectively, into vector, pMON30237, digested
with NcoI (which contains hFlt3L 1-134) as
described in Examples 1 & 2. The DNA sequence of
resulting genes, SEQ ID N0:13 and SEQ ID N0:14,
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encodes the proteins of SEQ ID N0:42 and SEQ ID
N0:43, respectively.
EXAMPLES 5 & 6
Construction of an expression vectors,
pMON30249 and pMON30248, comprising a DNA sequence
encoding a multi-functional receptor agonist
comprising an IL-3 (15-125) variant joined via the
IgG2b linker to a flt-3 (1-139) ligand and an IL-3
(15-125) variant joined via the GlySer linker to a
flt-3 (1-139) ligand, respectively. Plasmids,
pMON30248 and pMON30249, were constructed by
cloning the gei purified NcoI/AfIIII restriction
fragment from pMON30244 (GlySer linker) and
pMON30245 (IgG2B linker) respectively, into
vector, pMON30238, digested with Ncol (which
contains hFlt3L 1-139) as described in Examples 1
& 2. The DNA sequence of resulting genes, SEQ ID
N0:15 and SEQ ID N0:16, encodes the proteins of
SEQ ID N0:36 and SEQ ID N0:37, respectively.
EXAMPLES 7 & 8
Construction of an expression vectors,
pMON32392 and pMON32393, comprising a DNA sequence
encoding a multi-functional receptor agonist
comprising a flt3 (1-134) ligand joined via the
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IgG2b linker to an IL-3 (15-125) variant and a
flt-3~ (1-139) ligand joined via the IgG2b linker
to an IL-3 (15-125) variant, respectively.
Plasmids, pMON32392 and pMON32393, were
constructed by Polymerase Chain Reaction (PCR)
methods. Plasmid, pMON30237 and pMON30238, DNA was
used as the template in the PCR reaction with
primer pairs N-term SEQ ID N0:29/134rev SEQ ID
N0:30 and N-term SEQ ID N0:29/139rev SEQ ID N0:31,
respectively, to introduce an in-frame SnaBI
restriction site at the C-terminus. Standard PCR
reaction mixtures were set up using an Invitrogen
PCR Optimizer kit (Invitrogen). Amplification
cycle conditions were as follows: seven cycles of
94°C, one minute, 65°C, two minutes, and 72°C 2
1/2 minutes; followed by ten cycles of 94°C, one
minute, 70°C, two minutes, and 72°C 2 1/2
minutes. The product of the PCR reactions were
purified using the Wizard PCR Purification kit
(Promega), and eluted in 50 ~.1 dH20. 20 ~l of each
purified PCR product were digested in 50 ~tl
reaction mixture volumes with 10U each of Ncol and
SnaBI for 90 minutes at 37°C. One ~,g of vector,
pMON26431 (derivative of pMON13061 - W095/21254),
was digested with 7.5U each of NcoI and SnaBI in a
20 ~1 reaction volume for 90 minutes at 37°C,
followed by the addition of 1U shrimp alkaline
phosphatase. The reaction was incubated an
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additional 10 minutes at 37°C, and both inserts
and vector were gel purified as described
previously. Ligation times and temperatures were
modified to include incubation at 16°C for 3 hour,
followed by 2 hour at ambient temperature.
Transformations and DNA sequence confirmation were
done as described in Examples 1 & 2. The DNA
sequence of resulting genes, SEQ ID N0:23 and SEQ
ID N0:24, encodes the proteins of SEQ ID N0:44 and
SEQ ID N0:45, respectively.
r«..,...~ r., "
Construction of an expression vector,
pMON30328, comprising a DNA sequence encoding a
multi-functional receptor agonist comprising a
flt3 (1-134) ligand joined via the IgG2b linker to
a G-CSF receptor agonist. Plasmid, pMON30328, was
constructed by subcloning the gel-purified
NcoI/HindIII restriction fragment from pMON30237
into plasmid, pMON30309 (derivative of pMON13149 -
W095/21254), digested with AfIIIT/HindIII
(contains G-CSF/IgG2b-AfIIII/HindIII) as described
in Examples 1 & 2. The DNA sequence of resulting
gene, SEQ ID N0:50, encodes the protein of SEQ ID
N0:60.
EXAMPLE 10
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Construction of an expression vector,
pMON30329, comprising a DNA sequence encoding a
multi-functional receptor agonist comprising a G-
CSF receptor agonist joined via the IgG2b linker
to a flt3 (1-139) ligand. Piasmid, pMON30329, was
constructed by subcloning the gel-purified
NcoI/HindIII restriction fragment from pMON30238
into plasmid pMON30309 digested with
AflIII/HindIII (which contains G-CSF/IgG2b-
AfIIII/HindIII) as described in Examples 1 & 2.
The DNA sequence of resulting gene, SEQ ID N0:17,
encodes the protein of SEQ ID N0:38.
EXAMPLE 11
Construction of an expression vector,
pMON32175, comprising a DNA sequence encoding a
multi-functional receptor agonist comprising a
flt3 (1-139) ligand joined via the IgG2b linker to
a G-CSF receptor agonist. Plasmid, pMON32175, was
constructed by subcloning the gel-purified
NcoI/SnaBI restriction fragment from pMON32393
into pMON26430 (derivative of pMON13060 -
W095/21254) digested with NcoI/SnaBI as described
in Examples 1 & 2. The DNA sequence of resulting
gene, SEQ ID N0:19, encodes the protein of SEQ ID
N0:40.
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EXAMPLE 12
Construction of an expression vector,
pMON32191, comprising a DNA sequence encoding a
multi-functional receptor agonist comprising a
flt3 (1-139) ligand joined via the IgG2b linker to
a G-CSF receptor agonist. pMON32191 was assembled
by subcloning the gel-purified NcoI/SnaBI
restriction fragment from pMON32393 SEQ ID N0:58
into plasmid pMON31123 digested with NcoI/SnaBI
(which contains the GlySer/G-CSF moiety) as
described in Examples 1 & 2. The DNA sequence of
resulting gene, SEQ ID N0:20, encodes the protein
of SEQ ID N0:41.
EXAMPLE 13
Construction of an expression vector,
pMON35767, comprising a DNA sequence encoding a
multi-functional receptor agonist comprising a
flt3 (1-139) ligand joined via the IgG2b linker to
a G-CSF receptor agonist. Plasmid, pMON35767, was
constructed by subcloning the gel-purified
NcoI/HindIII restriction fragment from pMON32191
SEQ ID N0:20 into the BHK expression vector
pMON3934, which is a derivative of pMON3359.
pMON3359 is a pUCl8-based vector containing a
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mammalian expression cassette. The cassette
includes a herpes simplex viral promoter IE110 (-
800 to +120) followed by a modified human IL-3
signal peptide sequence and an SV40 late poly-
adenylation (poly-A) signal which has been
subcloned into the pUCl8 polylinker (See
Hippenmeyer et al., Bio/Technology, 1993, pp.1037-
1041). The DNA sequence of resulting gene, SEQ ID
N0:20, encodes the protein of SEQ ID N0:41.
EXAMPLE 14
Construction of an expression vector,
pMON32173, comprising a DNA sequence encoding a
multi-functional receptor agonist comprising a
flt3 (1-139) ligand joined via the IgG2b linker to
a flt3 (1-139) ligand. Plasmid, pMON32173, was
constructed by subcloning the gel-purified --130bp
NcoI/Sacl restriction fragment from pMON32342 SEQ
ID N0:52 and the -290bp Sacl/SnaBI restriction
fragment from pMON32393 into plasmid pMON30329
digested with Ncol/SnaBI as described in Examples
1 & 2. The DNA sequence of resulting gene, SEQ ID
N0:18, encodes the protein of SEQ ID N0:39.
EXAMPLE 15
Construction of an expression vector, pMON45419,
comprising a DNA sequence encoding a multi-functional
receptor agonist comprising a c-mpl (1-153) ligand
joined via the IgG2b linker to a flt3 (1-139) ligand.
Plasmid, pMON45419, was constructed by subcloning the
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NcoI/SnaBI restriction fragment from pMON26474
(derivative of pMON26472 - W095/21254) into plasmid,
pMON32173 SEQ ID N0:56, digested with NcoI/SnaBI as
described in Examples 1 & 2. The DNA sequence of
resulting gene, SEQ ID N0:25, encodes the protein of
SEQ ID N0:46.
EXAMPLE 16
Construction of an expression vector, pMON45420,
comprising a DNA sequence encoding a multi-functional
receptor agonist comprising a flt3 (1-139) ligand
joined via the IgG2b linker to a c-mpl (1-153)
ligand. Plasmid, pMON45420 (derivative of pMON26471 -
W095/21254) was assembled by subcloning the
NcoI/SnaBI restriction fragment from pMON32191 into
plasmid, pMON26473, digested with NcoI/SnaBI as
described in Examples 1 & 2. The DNA sequence of
resulting gene, SEQ ID N0:26, encodes the protein of
SEQ ID N0:47.
EXAMPLE 17
Construction of plasmid pMON46408 which encodes a
mufti-functional receptor agonist comprising EPO
joined via a Gly Ser linker to a fit3 (1-139) ligand
Plasmid pMON46408 was constructed in a two step
cloning procedure. First, an intermediate plasmid,
pMON46406, was constructed. This plasmid encodes the
human EPO sequence joined to a GlySer linker sequence
containing the restriction enzyme sites AfIIII and
HindIII. The following three DNA fragments were
ligated together to form plasmid pMON46406:
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59
1. A 480 by NcoI-StuI fragment encoding EPO
except for the terminal 6 amino acids
2. The annealed oligonucleotides epostu-xma.seq
SEQ ID NO: 32 and epostu-xma.rev SEQ ID N0:33
which comprises a Stul-XmaI fragment encoding
the terminal 6 amino acids of EPO and a portion
of the GlySer polypeptide linker sequence
3. The 3,052 by NcoI-XmaI vector fragment of
plasmid pMON13180
The ligation mixture was used to transform competent
MON105 cells and transformants were selected on LB
Amp plates. Colonies were picked and analyzed by DNA
sequencing analysis to identify a correct clone. A
correct clone was assigned pMON46406.
In order to construct pMON46408, plasmid pMON46406
was digested with AflIII and HindIII and the
vector portion was purified. This was ligated
with the 423 by Ncol-HindIII fragment of plasmid
pMON32342 SEQ ID N0:52, which encodes the flt-3
(1-139) ligand. The ligated ligation mixture was
used to transform competent MON105 cells and
transformants were selected on LB Amp plates.
Colonies were picked and analyzed by DNA
sequencing analysis to identify a correct clone.
A correct clone was assigned pMON46408. The DNA
sequence of resulting gene, SEQ ID N0:28, encodes
the protein of SEQ ID N0:49.
EXAMPLE 18
Determination of bioactivity of selected chimera
proteins
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Selected chimeras of the present invention were
assayed in a Baf3 cell line transfected with the
flt3/flk2 receptor (Baf3/flt3) to determine flt3
ligand bioactivity.
Table 1
Baf3/flt3 assay
pMON30249 Comparable to native flt3 ligand alone
pMON32173 Comparable to native flt3 ligand alone
pMON32392 Comparable to native flt3 ligand alone
pMON32393 Comparable to native flt3 ligand alone
pMON32364 Comparable to native flt3 ligand alone
pMON32377 Comparable to native flt3 ligand alone
Additional details about recombinant DNA methods
which may be used to create the variants, express
them in bacteria, mammalian cells or insect cells,
purification and refold of the desired proteins and
assays for determining the bioactvity of the proteins
may be found in WO 95/00646, WO 94/12639, WO
94/12638, wo 95/20976, w0 95/21197, w0 95/20977, wo
95/21254 and US 08/383,035 which are hereby
incorporated by reference in their entirety.
Further details known to those skilled in the
art may be found in T. Maniatis, et al., Molecular
Cloning, A Laboratory Manual, Cold Spring Harbor
Laboratory, 1982) and references cited therein,
incorporated herein by reference; and in J. Sambrook,
et al., Molecular Cloning, A Laboratory Manual, 2nd
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edition, Cold Spring Harbor Laboratory, 1989) and
references cited therein, incorporated herein by
reference. Protein purification methods know to those
skilled in the art are described in detail in Methods
in Enzymology, Volume 182 'Guide to Protein
Purification' edited by Murray Deutscher, Academic
Press, San Diego, CA (1990).
All references, patents or applications cited
herein are incorporated by reference in their
entirety as if written herein.
Various other examples will be apparent to the
person skilled in the art after reading the present
disclosure without departing from the spirit and
scope of the invention. It is intended that all such
other examples be included within the scope of the
appended claims.
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SEQUENCE LISTING
(1) GENERAL INFORMATION
(i) APPLICANT: G. D. Searle Corporate Patent Department
(ii) TITLE OF THE INVENTION: Flt3 Ligand Chimeric
Protein
{iii) NUMBER OF SEQUENCES: 65
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: G. D. Searle Corporate Patent Department
(B) STREET: P.O. Box 55110
(C) CITY: Chicago
(D) STATE: IL
(E) COUNTRY: USA
(F) ZIP: 60680
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Diskette
(B) COMPUTER: IBM Compatible
(C) OPERATING SYSTEM: DOS
(D) SOFTWARE: FastSEQ for Windows Version 2.0
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE: 10-APR-1998
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: 08/837,026
(B) FILING DATE: 11-APR-1997
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Bennett, Dennis A
(B) REGISTRATION NUMBER: 34.547
(C) REFERENCE/DOCKET NUMBER: C-3018/1/PCT
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 314-737-6986
(B) TELEFAX: 314-737-6972
(C) TELEX:
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
Gly Gly Gly Ser
1
(2) INFORMATION FOR SEQ ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:
Gly Gly Gly Gly Ser
I 5
(2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:
Gly Gly Gly Gly Gly Ser
1 5
(2) INFORMATION FOR SEQ ID N0:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:
Gly Ser
1
(2) INFORMATION FOR SEQ ID N0:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
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(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:5:
Ala Gly Ser
1
(2) INFORMATION FOR SEQ ID N0:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:6:
Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Glu Gly Gly Gly
1 S 10 15
Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser
20 25 30
Gly Gly Gly Ser
(2) INFORMATION FOR SEQ ID N0:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:7:
Ile Ser Glu Pro Ser Gly Pro Ile Ser Thr Ile Asn Pro Ser Pro Pro
1 5 10 15
Ser Lys Glu Ser His Lys Ser Pro
(2) INFORMATION FOR SEQ ID N0:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi} SEQUENCE DESCRIPTION: SEQ ID N0:8:
Ile Glu Gly Arg Ile Ser Glu Pro Ser Gly Pro Ile Ser Thr Ile Asn
1 5 10 15
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Pro Ser Pro Pro Ser Lys Glu Ser His Lys Ser Pro
20 25
(2) INFORMATION FOR SEQ ID N0:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 349 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:9:
Ala Thr Gln Asp Cys Ser Phe Gln His Ser Pro Ile Ser Ser Asp Phe
1 5 10 15
Ala Val Lys Ile Arg Glu Leu Ser Asp Tyr Leu Leu Gln Asg Tyr Pro
20 25 30
Val Thr Val Ala Ser Asn Leu Gln Asp Glu Glu Leu Cys Gly Gly Leu
35 40 45
Trp Arg Leu Val Leu Ala Gln Arg Trp Met Glu Arg Leu Lys Thr Val
50 55 60
Ala Gly Ser Lys Met Gln Gly Leu Leu Glu Arg Val Asn Thr Glu Ile
65 70 75 g0
His Phe Val Thr Lys Cys Ala Phe Gln Pro Pro Pro Ser Cys Leu Arg
85 90 95
Phe Val Gln Thr Asn Ile Ser Arg Leu Leu Gln Glu Thr Ser Glu Gln
100 105 110
Leu Val Ala Leu Lys Pro Trp Ile Thr Arg Gln Asn Phe Ser Arg Cys
115 120 125
Leu Glu Leu Gln Cys Gln Pro Asp Ser Ser Thr Leu Tyr Val Glu Gly
130 135 140
Gly Gly Gly Ser Pro Gly Glu Pro Ser Gly Pro Ile Ser Thr Ile Asn
145 150 155 160
Pro Ser Pro Pro Ser Lys Glu Ser His Lys Ser Pro Asn Met Ala Thr
165 170 175
Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu Leu Lys Ser
180 185 190
Leu Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala Ala Leu Gln Glu
195 200 205
Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu Val Leu
210 215 220
Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser Ser Cys Pro
225 230 235 240
Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser Gln Leu His Ser Gly
245 250 255
Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu Gly Ile Ser Pro
260 265 270
Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val Ala Asp Phe
275 280 285
Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met Ala Pro Ala
290 295 300
Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala Ser Ala Phe Gln
305 310 315 320
Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gln Ser Phe Leu
325 330 335
Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln Pro
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340 345
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1047 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:10:
GCCACCCAGG ACTGCTCCTT TCAACACAGC CCCATCTCCT CCGACTTCGC TGTCAAAATC
CGTGAGCTGT CTGACTACCT GCTTCAAGAT TACCCAGTCA CCGTGGCCTC CAACCTGCAG
120
GACGAGGAGC TCTGCGGGGG CCTCTGGCGG CTGGTCCTGG CACAGCGCTG GATGGAGCGG
180
CTCAAGACTG TCGCTGGGTC CAAGATGCAA GGCTTGCTGG AGCGCGTGAA CACGGAGATA
240
CACTTTGTCA CCAAATGTGC CTTTCAGCCC CCCCCCAGCT GTCTTCGCTT CGTCCAGACC
300
AACATCTCCC GCCTCCTGCA GGAGACCTCC GAGCAGCTGG TGGCGCTGAA GCCATGGATC
360
ACTCGCCAGA ACTTCTCCCG GTGCCTGGAG CTGCAGTGTC AGCCCGACTC CTCAACCCTG
420
TACGTAGAGG GCGGTGGAGG CTCCCCGGGT GAACCGTCTG GTCCAATCTC TACTATCAAC
480
CCGTCTCCTC CGTCTAAAGA ATCTCATAAA TCTCCAAACA TGGCTACACC ATTGGGCCCT
540
GCCAGCTCCC TGCCCCAGAG CTTCCTGCTC AAGTCTTTAG AGCAAGTGAG AAAGATCCAG
600
GGCGATGGCG CAGCGCTCCA GGAGAAGCTG TGTGCCACCT ACAAGCTGTG CCACCCCGAG
660
GAGCTGGTGC TGCTCGGACA CTCTCTGGGC ATCCCCTGGG CTCCCCTGAG CTCCTGCCCC
720
AGCCAGGCCC TGCAGCTGGC AGGCTGCTTG AGCCAACTCC ATAGCGGCCT TTTCCTCTAC
780
CAGGGGCTCC TGCAGGCCCT GGAAGGGATA TCCCCCGAGT TGGGTCCCAC CTTGGACACA
840
CTGCAGCTGG ACGTCGCCGA CTTTGCCACC ACCATCTGGC AGCAGATGGA AGAACTGGGA
900
ATGGCCCCTG CCCTGCAGCC CACCCAGGGT GCCATGCCGG CCTTCGCCTC TGCTTTCCAG
960
CGCCGGGCAG GAGGGGTCCT GGTTGCTAGC CATCTGCAGA GCTTCCTGGA GGTGTCGTAC
1020
CGCGTTCTAC GCCACCTTGC GCAGCCC
1047
(2) INFORMATION FOR SEQ ID N0:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 349 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
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(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:11:
Ala Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu Leu
1 5 10 15
Lys Ser Leu Glu Gin Val Arg Lys Ile Gln Gly Asp Gly Ala Ala Leu
20 25 30
Gln Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu
35 40 45
Val Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser Ser
50 55 60
Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser Gln Leu His
65 70 75 g0
Ser Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu Gly Ile
85 90 95
Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val Ala
100 105 110
Asp Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met Ala
115 120 125
Pro Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala Ser Ala
130 135 140
Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gln Ser
145 150 155 160
Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln Pro Tyr
165 170 175
Val Glu Gly Gly Gly Gly Ser Pro Gly Glu Pro Ser Gly Pro Ile Ser
180 185 190
Thr Ile Asn Pro Ser Pro Pro Ser Lys Glu Ser His Lys Ser Pro Asn
195 200 205
Met Ala Thr Gln Asp Cys Ser Phe Gln His Ser Pro Ile Ser Ser Asp
210 215 220
Phe Ala VaI Lys Ile Arg Glu Leu Ser Asp Tyr Leu Leu Gln Asp Tyr
225 230 235 240
Pro Val Thr Val Ala Ser Asn Leu Gln Asp Glu Glu Leu Cys Gly Gly
245 250 255
Leu Trp Arg Leu Val Leu Ala Gln Arg Trp Met Glu Arg Leu Lys Thr
260 265 270
Val Ala Gly Ser Lys Met Gln Gly Leu Leu Glu Arg Val Asn Thr Glu
275 280 285
Ile His Phe Val Thr Lys Cys Ala Phe Gln Pro Pro Pro Ser Cys Leu
290 295 300
Arg Phe Val Gln Thr Asn Ile Ser Arg Leu Leu Gln Glu Thr Ser Glu
305 310 315 320
Gln Leu Val Ala Leu Lys Pro Trp Ile Thr Arg Gln Asn Phe Ser Arg
325 330 335
Cys Leu Glu Leu Gln Cys Gln Pro Asp Ser Ser Thr Leu
340 345
(2) INFORMATION FOR SEQ ID N0:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1047 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
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(xi) SEQUENCE DESCRIPTION: SEQ ID N0:12:
GCTACACCAT TGGGCCCTGC CAGCTCCCTG CCCCAGAGCT TCCTGCTCAA GTCTTTAGAG
CAAGTGAGAA 'AGATCCAGGG CGATGGCGCA GCGCTCCAGG AGAAGCTGTG TGCCACCTAC
120
AAGCTGTGCC ACCCCGAGGA GCTGGTGCTG CTCGGACACT CTCTGGGCAT CCCCTGGGCT
180
CCCCTGAGCT CCTGCCCCAG CCAGGCCCTG CAGCTGGCAG GCTGCTTGAG CCAACTCCAT
240
AGCGGCCTTT TCCTCTACCA GGGGCTCCTG CAGGCCCTGG AAGGGATATC CCCCGAGTTG
300
GGTCCCACCT TGGACACACT GCAGCTGGAC GTCGCCGACT TTGCCACCAC CATCTGGCAG
360
CAGATGGAAG AACTGGGAAT GGCCCCTGCC CTGCAGCCCA CCCAGGGTGC CATGCCGGCC
420
TTCGCCTCTG CTTTCCAGCG CCGGGCAGGA GGGGTCCTGG TTGCTAGCCA TCTGCAGAGC
480
TTCCTGGAGG TGTCGTACCG CGTTCTACGC CACCTTGCGC AGCCCTACGT AGAGGGCGGT
540
GGAGGCTCCC CGGGTGAACC GTCTGGTCCA ATCTCTACTA TCAACCCGTC TCCTCCGTCT
600
AAAGAATCTC ATAAATCTCC AAACATGGCT ACCCAGGACT GCTCCTTCCA ACACAGCCCC
660
ATCTCCTCCG ACTTCGCTGT CAAAATCCGT GAGCTGTCTG ACTACCTGCT TCAAGATTAC
720
CCAGTCACCG TGGCCTCCAA CCTGCAGGAC GAGGAGCTCT GCGGGGGCCT CTGGCGGCTG
780
GTCCTGGCAC AGCGCTGGAT GGAGCGGCTC AAGACTGTCG CTGGGTCCAA GATGCAAGGC
840
TTGCTGGAGC GCGTGAACAC GGAGATACAC TTTGTCACCA AATGTGCCTT TCAGCCCCCC
900
CCCAGCTGTC TTCGCTTCGT CCAGACCAAC ATCTCCCGCC TCCTGCAGGA GACCTCCGAG
960
CAGCTGGTGG CGCTGAAGCC CTGGATCACT CGCCAGAACT TCTCCCGGTG CCTGGAGCTG
1020
CAGTGTCAGC CCGACTCCTC AACCCTG
1047
(2) INFORMATION FOR SEQ ID N0:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 798 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:13:
GCTAACTGCT CTATAATGAT CGATGAAATT ATACATCACT TAAAGAGACC ACCTGCACCT
TTGCTGGACC CGAACAACCT CAATGACGAA GACGTCTCTA TCCTGATGGA CCGAAACCTT
120
CGACTTCCAA ACCTGGAGAG CTTCGTAAGG GCTGTCAAGA ACTTAGAAAA TGCATCAGGT
180
ATTGAGGCAA TTCTTCGTAA TCTCCAACCA TGTCTGCCCT CTGCCACGGC CGCACCCTCT
240
CA 02284127 1999-09-10
WO 98/46750 69 PCT/US98/07511
CGACATCCAA TCATCATCAA GGCAGGTGAC TGGCAAGAAT TCCGGGAAAA ACTGACGTTC
300
TATCTGGTTA CCCTTGAGCA AGCGCAGGAA CAACAGTACG TAGAGGGCGG TGGAGGCTCC
360
CCGGGTGGTG GTTCTGGCGG CGGCTCCAAC ATGGCCACCC AGGACTGCTC CTTCCAACAC
420
AGCCCCATCT CCTCCGACTT CGCTGTCAAA ATCCGTGAGC TGTCTGACTA CCTGCTTCAA
480
GATTACCCAG TCACCGTGGC CTCCAACCTG CAGGACGAGG AGCTCTGCGG GGCGCTCTGG
540
CGGCTGGTCC TGGCACAGCG CTGGATGGAG CGGCTCAAGA CTGTCGCTGG GTCCAAGATG
600
CAAGGCTTGC TGGAGCGCGT GAACACGGAG ATACACTTTG TCACCAAATG TGCCTTTCAG
660
CCCCCCCCCA GCTGTCTTCG CTTCGTCCAG ACCAACATCT CCCGCCTCCT GCAGGAGACC
720
TCCGAGCAGC TGGTGGCGCT GAAGCCCTGG ATCACTCGCC AGAACTTCTC CCGGTGCCTG
780
GAGCTGCAGT GTCAGCCC
798
(2) INFORMATION FOR SEQ ID N0:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 843 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:14:
GCTAACTGCT CTATAATGAT CGATGAAATT ATACATCACT TAAAGAGACC ACCTGCACCT
TTGCTGGACC CGAACAACCT CAATGACGAA GACGTCTCTA TCCTGATGGA CCGAAACCTT
120
CGACTTCCAA ACCTGGAGAG CTTCGTAAGG GCTGTCAAGA ACTTAGAAAA TGCATCAGGT
180
ATTGAGGCAA TTCTTCGTAA TCTCCAACCA TGTCTGCCCT CTGCCACGGC CGCACCCTCT
240
CGACATCCAA TCATCATCAA GGCAGGTGAC TGGCAAGAAT TCCGGGAAAA ACTGACGTTC
300
TATCTGGTTA CCCTTGAGCA AGCGCAGGAA CAACAGTACG TAGAGGGCGG TGGAGGCTCC
360
CCGGGTGAAC CGTCTGGTCC AATCTCTACT ATCAACCCGT CTCCTCCGTC TAAAGAATCT
420
CATAAATCTC CAAACATGGC CACCCAGGAC TGCTCCTTCC AACACAGCCC CATCTCCTCC
480
GACTTCGCTG TCAAAATCCG TGAGCTGTCT GACTACCTGC TTCAAGATTA CCCAGTCACC
540
GTGGCCTCCA ACCTGCAGGA CGAGGAGCTC TGCGGGGCGC TCTGGCGGCT GGTCCTGGCA
600
CAGCGCTGGA TGGAGCGGCT CAAGACTGTC GCTGGGTCCA AGATGCAAGG CTTGCTGGAG
660
CGCGTGAACA CGGAGATACA CTTTGTCACC AAATGTGCCT TTCAGCCCCC CCCCAGCTGT
720
CTTCGCTTCG TCCAGACCAA CATCTCCCGC CTCCTGCAGG AGACCTCCGA GCAGCTGGTG
780
CA 02284127 1999-09-10
WO 98/46750 ~0 PCT/US98/07511
GCGCTGAAGC CCTGGATCAC TCGCCAGAAC TTCTCCCGGT GCCTGGAGCT GCAGTGTCAG
840
CCC
843
(2) INFORMATION FOR SEQ ID N0:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 813 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:15:
GCTAACTGCT CTATAATGAT CGATGAAATT ATACATCACT TAAAGAGACC ACCTGCACCT
TTGCTGGACC CGAACAACCT CAATGACGAA GACGTCTCTA TCCTGATGGA CCGAAACCTT
120
CGACTTCCAA ACCTGGAGAG CTTCGTAAGG GCTGTCAAGA ACTTAGAAAA TGCATCAGGT
180
ATTGAGGCAA TTCTTCGTAA TCTCCAACCA TGTCTGCCCT CTGCCACGGC CGCACCCTCT
240
CGACATCCAA TCATCATCAA GGCAGGTGAC TGGCAAGAAT TCCGGGAAAA ACTGACGTTC
300
TATCTGGTTA CCCTTGAGCA AGCGCAGGAA CAACAGTACG TAGAGGGCGG TGGAGGCTCC
360
CCGGGTGGTG GTTCTGGCGG CGGCTCCAAC ATGGCCACCC AGGACTGCTC CTTCCAACAC
420
AGCCCCATCT CCTCCGACTT CGCTGTCAAA ATCCGTGAGC TGTCTGACTA CCTGCTTCAA
480
GATTACCCAG TCACCGTGGC CTCCAACCTG CAGGACGAGG AGCTCTGCGG GGCGCTCTGG
540
CGGCTGGTCC TGGCACAGCG CTGGATGGAG CGGCTCAAGA CTGTCGCTGG GTCCAAGATG
600
CAAGGCTTGC TGGAGCGCGT GAACACGGAG ATACACTTTG TCACCAAATG TGCCTTTCAG
660
CCCCCCCCCA GCTGTCTTCG CTTCGTCCAG ACCAACATCT CCCGCCTCCT GCAGGAGACC
720
TCCGAGCAGC TGGTGGCGCT GAAGCCCTGG ATCACTCGCC AGAACTTCTC CCGGTGCCTG
780
GAGCTGCAGT GTCAGCCCGA CTCCTCAACC CTG
813
(2) INFORMATION FOR SEQ ID N0:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 858 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:16:
GCTAACTGCT CTATAATGAT CGATGAAATT ATACATCACT TAAAGAGACC ACCTGCACCT
CA 02284127 1999-09-10
WO 98/46750 ~ 1 PCT/US98/07511
TTGCTGGACC CGAACAACCT CAATGACGAA GACGTCTCTA TCCTGATGGA CCGAAACCTT
120
CGACTTCCAA ACCTGGAGAG CTTCGTAAGG GCTGTCAAGA ACTTAGAAAA TGCATCAGGT
180
ATTGAGGCAA'TTCTTCGTAA TCTCCAACCA TGTCTGCCCT CTGCCACGGC CGCACCCTCT
240
CGACATCCAA TCATCATCAA GGCAGGTGAC TGGCAAGAAT TCCGGGAAAA ACTGACGTTC
300
TATCTGGTTA CCCTTGAGCA AGCGCAGGAA CAACAGTACG TAGAGGGCGG TGGAGGCTCC
360
CCGGGTGAAC CGTCTGGTCC AATCTCTACT ATCAACCCGT CTCCTCCGTC TAAAGAATCT
420
CATAAATCTC CAAACATGGC CACCCAGGAC TGCTCCTTCC AACACAGCCC CATCTCCTCC
480
GACTTCGCTG TCAAAATCCG TGAGCTGTCT GACTACCTGC TTCAAGATTA CCCAGTCACC
540
GTGGCCTCCA ACCTGCAGGA CGAGGAGCTC TGCGGGGCGC TCTGGCGGCT GGTCCTGGCA
600
CAGCGCTGGA TGGAGCGGCT CAAGACTGTC GCTGGGTCCA AGATGCAAGG CTTGCTGGAG
660
CGCGTGAACA CGGAGATACA CTTTGTCACC AAATGTGCCT TTCAGCCCCC CCCCAGCTGT
720
CTTCGCTTCG TCCAGACCAA CATCTCCCGC CTCCTGCAGG AGACCTCCGA GCAGCTGGTG
780
GCGCTGAAGC CCTGGATCAC TCGCCAGAAC TTCTCCCGGT GCCTGGAGCT GCAGTGTCAG
840
CCCGACTCCT CAACCCTG
858
(2) INFORMATION FOR SEQ ID N0:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1047 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:17:
GCTACACCAT TGGGCCCTGC CAGCTCCCTG CCCCAGAGCT TCCTGCTCAA GTCTTTAGAG
CAAGTGAGAA AGATCCAGGG CGATGGCGCA GCGCTCCAGG AGAAGCTGTG TGCCACCTAC
120
AAGCTGTGCC ACCCCGAGGA GCTGGTGCTG CTCGGACACT CTCTGGGCAT CCCCTGGGCT
180
CCCCTGAGCT CCTGCCCCAG CCAGGCCCTG CAGCTGGCAG GCTGCTTGAG CCAACTCCAT
240
AGCGGCCTTT TCCTCTACCA GGGGCTCCTG CAGGCCCTGG AAGGGATATC CCCCGAGTTG
300
GGTCCCACCT TGGACACACT GCAGCTGGAC GTCGCCGACT TTGCCACCAC CATCTGGCAG
360
CAGATGGAAG AACTGGGAAT GGCCCCTGCC CTGCAGCCCA CCCAGGGTGC CATGCCGGCC
420
TTCGCCTCTG CTTTCCAGCG CCGGGCAGGA GGGGTCCTGG TTGCTAGCCA TCTGCAGAGC
480
TTCCTGGAGG TGTCGTACCG CGTTCTACGC CACCTTGCGC AGCCCTACGT AGAGGGCGGT
540
CA 02284127 1999-09-10
WO 98/46750 ~2 PCT/US98/07511
GGAGGCTCCC CGGGTGAACC GTCTGGTCCA ATCTCTACTA TCAACCCGTC TCCTCCGTCT
600
AAAGAATCTC ATAAATCTCC AAACATGGCC ACCCAGGACT GCTCCTTCCA ACACAGCCCC
660
ATCTCCTCCG 'ACTTCGCTGT CAAAATCCGT GAGCTGTCTG ACTACCTGCT TCAAGATTAC
720
CCAGTCACCG TGGCCTCCAA CCTGCAGGAC GAGGAGCTCT GCGGGGGCCT CTGGCGGCTG
780
GTCCTGGCAC AGCGCTGGAT GGAGCGGCTC AAGACTGTCG CTGGGTCCAA GATGCAAGGC
840
TTGCTGGAGC GCGTGAACAC GGAGATACAC TTTGTCACCA AATGTGCCTT TCAGCCCCCC
900
CCCAGCTGTC TTCGCTTCGT CCAGACCAAC ATCTCCCGCC TCCTGCAGGA GACCTCCGAG
960
CAGCTGGTGG CGCTGAAGCC CTGGATCACT CGCCAGAACT TCTCCCGGTG CCTGGAGCTG
1020
CAGTGTCAGC CCGACTCCTC AACCCTG
1047
(2) INFORMATION FOR SEQ ID N0:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 942 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:18:
GCCACTCAGG ACTGCTCTTT TCAACACAGC CCCATCTCCT CCGACTTCGC TGTCAAAATC
CGTGAGCTGT CTGACTACCT GCTTCAAGAT TACCCAGTCA CCGTGGCCTC CAACCTGCAG
120
GACGAGGAGC TCTGCGGGGG CCTCTGGCGG CTGGTCCTGG CACAGCGCTG GATGGAGCGG
180
CTCAAGACTG TCGCTGGGTC CAAGATGCAA GGCTTGCTGG AGCGCGTGAA CACGGAGATA
240
CACTTTGTCA CCAAATGTGC CTTTCAGCCC CCCCCCAGCT GTCTTCGCTT CGTCCAGACC
300
AACATCTCCC GCCTCCTGCA GGAGACCTCC GAGCAGCTGG TGGCGCTGAA GCCCTGGATC
360
ACTCGCCAGA ACTTCTCCCG GTGCCTGGAG CTGCAGTGTC AGCCCGACTC CTCAACCCTG
420
TACGTAGAGG GCGGTGGAGG CTCCCCGGGT GAACCGTCTG GTCCAATCTC TACTATCAAC
480
CCGTCTCCTC CGTCTAAAGA ATCTCATAAA TCTCCAAACA TGGCTACCCA GGACTGCTCC
540
TTCCAACACA GCCCCATCTC CTCCGACTTC GCTGTCAAAA TCCGTGAGCT GTCTGACTAC
600
CTGCTTCAAG ATTACCCAGT CACCGTGGCC TCCAACCTGC AGGACGAGGA GCTCTGCGGG
660
GGCCTCTGGC GGCTGGTCCT GGCACAGCGC TGGATGGAGC GGCTCAAGAC TGTCGCTGGG
720
TCCAAGATGC AAGGCTTGCT GGAGCGCGTG AACACGGAGA TACACTTTGT CACCAAATGT
780
GCCTTTCAGC CCCCCCCCAG CTGTCTTCGC TTCGTCCAGA CCAACATCTC CCGCCTCCTG
840
CA 02284127 1999-09-10
WO 98146750 73 PCT/US98/07511
CAGGAGACCT CCGAGCAGCT GGTGGCGCTG AAGCCCTGGA TCACTCGCCA GAACTTCTCC
900
CGGTGCCTGG AGCTGCAGTG TCAGCCCGAC TCCTCAACCC TG
942
(2) INFORMATION FOR SEQ ID N0:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1047 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:19:
GCCACCCAGG ACTGCTCCTT TCAACACAGC CCCATCTCCT CCGACTTCGC TGTCAAAATC
CGTGAGCTGT CTGACTACCT GCTTCAAGAT TACCCAGTCA CCGTGGCCTC CAACCTGCAG
120
GACGAGGAGC TCTGCGGGGG CCTCTGGCGG CTGGTCCTGG CACAGCGCTG GATGGAGCGG
180
CTCAAGACTG TCGCTGGGTC CAAGATGCAA GGCTTGCTGG AGCGCGTGAA CACGGAGATA
240
CACTTTGTCA CCAAATGTGC CTTTCAGCCC CCCCCCAGCT GTCTTCGCTT CGTCCAGACC
300
AACATCTCCC GCCTCCTGCA GGAGACCTCC GAGCAGCTGG TGGCGCTGAA GCCATGGATC
360
ACTCGCCAGA ACTTCTCCCG GTGCCTGGAG CTGCAGTGTC AGCCCGACTC CTCAACCCTG
420
TACGTAGAGG GCGGTGGAGG CTCCCCGGGT GAACCGTCTG GTCCAATCTC TACTATCAAC
480
CCGTCTCCTC CGTCTAAAGA ATCTCATAAA TCTCCAAACA TGGCTACACC ATTAGGCCCT
540
GCCAGCTCCC TGCCCCAGAG CTTCCTGCTC AAGTGCTTAG AGCAAGTGAG GAAGATCCAG
600
GGCGATGGCG CAGCGCTCCA GGAGAAGCTG TGTGCCACCT ACAAGCTGTG CCACCCCGAG
660
GAGCTGGTGC TGCTCGGACA CTCTCTGGGC ATCCCCTGGG CTCCCCTGAG CTCCTGCCCC
720
AGCCAGGCCC TGCAGCTGGC AGGCTGCTTG AGCCAACTCC ATAGCGGCCT TTTCCTCTAC
780
CAGGGGCTCC TGCAGGCCCT GGAAGGGATA TCCCCCGAGT TGGGTCCCAC CTTGGACACA
840
CTGCAGCTGG ACGTCGCCGA CTTTGCCACC ACCATCTGGC AGCAGATGGA AGAACTGGGA
900
ATGGCCCCTG CCCTGCAGCC CACCCAGGGT GCCATGCCGG CCTTCGCCTC TGCTTTCCAG
960
CGCCGGGCAG GAGGGGTCCT GGTTGCTAGC CATCTGCAGA GCTTCCTGGA GGTGTCGTAC
1020
CGCGTTTTAC GCCACCTTGC GCAGCCC
1047
(2) INFORMATION FOR SEQ ID N0:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1003 base pairs
(B) TYPE: nucleic acid
CA 02284127 1999-09-10
WO 98/46750 ~4 PCT/US98/07511
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi)'SEQUENCE DESCRIPTION: SEQ ID N0:20:
GGCCACTCAG GACTGCTCTT TTCAACACAG CCCCATCTCC TCCGACTTCG CTGTCAAAAT
CCGTGAGCTG TCTGACTACC TGCTTCAAGA TTACCCAGTC ACCGTGGCCT CCAACCTGCA
120
GGACGAGGAG CTCTGCGGGG GCCTCTGGCG GCTGGTCCTG GCACAGCGCT GGATGGAGCG
180
GCTCAAGACT GTCGCTGGGT CCAAGATGCA AGGCTTGCTG GAGCGCGTGA ACACGGAGAT
240
ACACTTTGTC ACCAAATGTG CCTTTCAGCC CCCCCCCAGC TGTCTTCGCT TCGTCCAGAC
300
CAACATCTCC CGCCTCCTGC AGGAGACCTC CGAGCAGCTG GTGGCGCTGA AGCCCTGGAT
360
CACTCGCCAG AACTTCTCCC GGTGCCTGGA GCTGCAGTGT CAGCCCGACT CCTCAACCCT
420
GTACGTAGAG GGCGGTGGAG GCTCCCCGGG TGGTGGTTCT GGCGGCGGCT CCAACATGGC
480
TACACCATTG GGCCCTGCCA GCTCCCTGCC CCAGAGCTTC CTGCTCAAGT CTTTAGAGCA
540
AGTGAGAAAG ATCCAGGGCG ATGGCGCAGC GCTCCAGGAG AAGCTGTGTG CCACCTACAA
600
GCTGTGCCAC CCCGAGGAGC TGGTGCTGCT CGGACACTCT CTGGGCATCC CCTGGGCTCC
660
CCTGAGCTCC TGCCCCAGCC AGGCCCTGCA GCTGGCAGGC TGCTTGAGCC AACTCCATAG
720
CGGCCTTTTC CTCTACCAGG GGCTCCTGCA GGCCCTGGAA GGGATATCCC CCGAGTTGGG
780
TCCCACCTTG GACACACTGC AGCTGGACGT CGCCGACTTT GCCACCACCA TCTGGCAGCA
840
GATGGAAGAA CTGGGAATGG CCCCTGCCCT GCAGCCCACC CAGGGTGCCA TGCCGGCCTT
900
CGCCTCTGCT TTCCAGCGCC GGGCAGGAGG GGTCCTGGTT GCTAGCCATC TGCAGAGCTT
960
CCTGGAGGTG TCGTACCGCG TTCTACGCCA CCTTGCGCAG CCG
1003
(2) INFORMATION FOR SEQ ID N0:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 843 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:21:
GCTAACTGCT CTATAATGAT CGATGAAATT ATACATCACT TAAAGAGACC ACCTAACCCT
TTGCTGGACC CGAACAACCT CAATTCTGAA GACATGGATA TCCTGATGGA ACGAAACCTT
120
CGAACTCCAA ACCTGCTCGC ATTCGTAAGG GCTGTCAAGC ACTTAGAAAA TGCATCAGGT
180
CA 02284127 1999-09-10
WO 98/46750 ~5 PGT/US98/07511
ATTGAGGCAA TTCTTCGTAA TCTCCAACCA TGTCTGCCCT CTGCCACGGC CGCACCCTCT
240
CGACATCCAA TCATCATCAA GGCAGGTGAC TGGCAAGAAT TCCGGGAAAA ACTGACGTTC
300
TATCTGGTTA'CCCTTGAGCA AGCGCAGGAA CAACAGTACG TAGAGGGCGG TGGAGGCTCC
360
CCGGGTGAAC CGTCTGGTCC AATCTCTACT ATCAACCCGT CTCCTCCGTC TAAAGAATCT
420
CATAAATCTC CAAACATGGC GACTCAGGAC TGTTCTTTCC AACACAGCCC CATCTCCTCC
480
GACTTCGCTG TCAAAATCCG TGAGCTGTCT GACTACCTGC TTCAAGATTA CCCAGTCACC
540
GTGGCCTCCA ACCTGCAGGA CGAGGAGCTC TGCGGGGGCC TCTGGCGGCT GGTCCTGGCA
600
CAGCGCTGGA TGGAGCGGCT CAAGACTGTC GCTGGGTCCA AGATGCAAGG CTTGCTGGAG
660
CGCGTGAACA CGGAGATACA CTTTGTCACC AAATGTGCCT TTCAGCCCCC CCCCAGCTGT
720
CTTCGCTTCG TCCAGACCAA CATCTCCCGC CTCCTGCAGG AGACCTCCGA GCAGCTGGTG
780
GCGCTGAAGC CCTGGATCAC TCGCCAGAAC TTCTCCCGGT GCCTGGAGCT GCAGTGTCAG
840
CCC
843
(2) INFORMATION FOR SEQ ID N0:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 858 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:22:
GCTAACTGCT CTATAATGAT CGATGAAATT ATACATCACT TAAAGAGACC ACCTAACCCT
TTGCTGGACC CGAACAACCT CAATTCTGAA GACATGGATA TCCTGATGGA ACGAAACCTT
120
CGAACTCCAA ACCTGCTCGC ATTCGTAAGG GCTGTCAAGC ACTTAGAAAA TGCATCAGGT
180
ATTGAGGCAA TTCTTCGTAA TCTCCAACCA TGTCTGCCCT CTGCCACGGC CGCACCCTCT
240
CGACATCCAA TCATCATCAA GGCAGGTGAC TGGCAAGAAT TCCGGGAAAA ACTGACGTTC
300
TATCTGGTTA CCCTTGAGCA AGCGCAGGAA CAACAGTACG TAGAGGGCGG TGGAGGCTCC
360
CCGGGTGAAC CGTCTGGTCC AATCTCTACT ATCAACCCGT CTCCTCCGTC TAAAGAATCT
420
CATAAATCTC CAAACATGGC AACCCAGGAC TGCTCTTTTC AACACAGCCC CATCTCCTCC
480
GACTTCGCTG TCAAAATCCG TGAGCTGTCT GACTACCTGC TTCAAGATTA CCCAGTCACC
540
GTGGCCTCCA ACCTGCAGGA CGAGGAGCTC TGCGGGGGCC TCTGGCGGCT GGTCCTGGCA
600
CAGCGCTGGA TGGAGCGGCT CAAGACTGTC GCTGGGTCCA AGATGCAAGG CTTGCTGGAG
660
CA 02284127 1999-09-10
WO 98/46750 ~6 PCT/US98/07511
CGCGTGAACA CGGAGATACA CTTTGTCACC AAATGTGCCT TTCAGCCCCC CCCCAGCTGT
720
CTTCGCTTCG TCCAGACCAA CATCTCCCGC CTCCTGCAGG AGACCTCCGA GCAGCTGGTG
780
GCGCTGAAGC 'CCTGGATCAC TCGCCAGAAC TTCTCCCGGT GCCTGGAGCT GCAGTGTCAG
840
CCCGACTCCT CAACCCTG
858
(2) INFORMATION FOR SEQ ID N0:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 843 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:23:
GCCACTCAGG ACTGCTCCTT CCAACACAGC CCCATCTCCT CCGACTTCGC TGTCAAAATC
CGTGAGCTGT CTGACTACCT GCTTCAAGAT TACCCAGTCA CCGTGGCCTC CAACCTGCAG
120
GACGAGGAGC TCTGCGGGGG CCTCTGGCGG CTGGTCCTGG CACAGCGCTG GATGGAGCGG
180
CTCAAGACTG TCGCTGGGTC CAAGATGCAA GGCTTGCTGG AGCGCGTGAA CACGGAGATA
240
CACTTTGTCA CCAAATGTGC CTTTCAGCCC CCCCCCAGCT GTCTTCGCTT CGTCCAGACC
300
AACATCTCCC GCCTCCTGCA GGAGACCTCC GAGCAGCTGG TGGCGCTGAA GCCCTGGATC
360
ACTCGCCAGA ACTTCTCCCG GTGCCTGGAG CTGCAGTGTC AGCCCTACGT AGAGGGCGGT
420
GGAGGCTCCC CGGGTGAACC GTCTGGTCCA ATCTCTACTA TCAACCCGTC TCCTCCGTCT
480
AAAGAATCTC ATAAATCTCC AAACATGGCT AACTGCTCTA TAATGATCGA TGAAATTATA
540
CATCACTTAA AGAGACCACC TAACCCTTTG CTGGACCCGA ACAACCTCAA TTCTGAAGAC
600
ATGGATATCC TGATGGAACG AAACCTTCGA ACTCCAAACC TGCTCGCATT CGTAAGGGCT
660
GTCAAGCACT TAGAAAATGC ATCAGGTATT GAGGCAATTC TTCGTAATCT CCAACCATGT
720
CTGCCCTCTG CCACGGCCGC ACCCTCTCGA CATCCAATCA TCATCAAGGC AGGTGACTGG
780
CAAGAATTCC GGGAAAAACT GACGTTCTAT CTGGTTACCC TTGAGCAAGC GCAGGAACAA
840
CAG
843
(2) INFORMATION FOR SEQ ID N0:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 858 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
CA 02284127 1999-09-10
WO 98/46750 ~~ PGT/US98/07511
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:24:
GCCACCCAGG ACTGCTCCTT CCAACACAGC CCCATCTCCT CCGACTTCGC TGTCAAAATC
CGTGAGCTGT CTGACTACCT GCTTCAAGAT TACCCAGTCA CCGTGGCCTC CAACCTGCAG
120
GACGAGGAGC TCTGCGGGGG CCTCTGGCGG CTGGTCCTGG CACAGCGCTG GATGGAGCGG
180
CTCAAGACTG TCGCTGGGTC CAAGATGCAA GGCTTGCTGG AGCGCGTGAA CACGGAGATA
240
CACTTTGTCA CCAAATGTGC CTTTCAGCCC CCCCCCAGCT GTCTTCGCTT CGTCCAGACC
300
AACATCTCCC GCCTCCTGCA GGAGACCTCC GAGCAGCTGG TGGCGCTGAA GCCCTGGATC
360
ACTCGCCAGA ACTTCTCCCG GTGCCTGGAG CTGCAGTGTC AGCCCGACTC CTCAACCCTG
420
TACGTAGAGG GCGGTGGAGG CTCCCCGGGT GAACCGTCTG GTCCAATCTC TACTATCAAC
480
CCGTCTCCTC CGTCTAAAGA ATCTCATAAA TCTCCAAACA TGGCTAACTG CTCTATAATG
540
ATCGATGAAA TTATACATCA CTTAAAGAGA CCACCTAACC CTTTGCTGGA CCCGAACAAC
600
CTCAATTCTG AAGACATGGA TATCCTGATG GAACGAAACC TTCGAACTCC AAACCTGCTC
660
GCATTCGTAA GGGCTGTCAA GCACTTAGAA AATGCATCAG GTATTGAGGC AATTCTTCGT
720
AATCTCCAAC CATGTCTGCC CTCTGCCACG GCCGCACCCT CTCGACATCC AATCATCATC
780
AAGGCAGGTG ACTGGCAAGA ATTCCGGGAA AAACTGACGT TCTATCTGGT TACCCTTGAG
840
CAAGCGCAGG AACAACAG
858
(2) INFORMATION FOR SEQ ID N0:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 939 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:25:
GCCACTCAGG ACTGCTCTTT TCAACACAGC CCCATCTCCT CCGACTTCGC TGTCAAAATC
CGTGAGCTGT CTGACTACCT GCTTCAAGAT TACCCAGTCA CCGTGGCCTC CAACCTGCAG
120
GACGAGGAGC TCTGCGGGGG CCTCTGGCGG CTGGTCCTGG CACAGCGCTG GATGGAGCGG
180
CTCAAGACTG TCGCTGGGTC CAAGATGCAA GGCTTGCTGG AGCGCGTGAA CACGGAGATA
240
CACTTTGTCA CCAAATGTGC CTTTCAGCCC CCCCCCAGCT GTCTTCGCTT CGTCCAGACC
300
AACATCTCCC GCCTCCTGCA GGAGACCTCC GAGCAGCTGG TGGCGCTGAA GCCCTGGATC
360
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ACTCGCCAGA ACTTCTCCCG GTGCCTGGAG CTGCAGTGTC AGCCCGACTC CTCAACCCTG
420
TACGTAGAGG GCGGTGGAGG CTCCCCGGGT GGTGGTTCTG GCGGCGGCTC CAACATGGCG
480
TCTCCGGCGC'CGCCTGCTTG TGACCTCCGA GTCCTCAGTA AACTGCTTCG TGACTCCCAT
540
GTCCTTCACA GCAGACTGAG CCAGTGCCCA GAGGTTCACC CTTTGCCTAC ACCTGTCCTG
600
CTGCCTGCTG TGGACTTTAG CTTGGGAGAA TGGAAAACCC AGATGGAGGA GACCAAGGCA
660
CAGGACATTC TGGGAGCAGT GACCCTTCTG CTGGAGGGAG TGATGGCAGC ACGGGGACAA
720
CTGGGACCCA CTTGCCTCTC ATCCCTCCTG GGGCAGCTTT CTGGACAGGT CCGTCTCCTC
780
CTTGGGGCCC TGCAGAGCCT CCTTGGAACC CAGCTTCCTC CACAGGGCAG GACCACAGCT
840
CACAAGGATC CCAATGCCAT CTTCCTGAGC TTCCAACACC TGCTCCGAGG AAAGGTGCGT
900
TTCCTGATGC TTGTAGGAGG GTCCACCCTC TGCGTCAGG
939
(2) INFORMATION FOR SEQ ID N0:26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 996 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:26:
GCGTCCCCAG CTCCACCTGC TTGTGACCTC CGAGTCCTCA GTAAACTGCT TCGTGACTCC
CATGTCCTTC ACAGCAGACT GAGCCAGTGC CCAGAGGTTC ACCCTTTGCC TACACCTGTC
120
CTGCTGCCTG CTGTGGACTT TAGCTTGGGA GAATGGAAAA CCCAGATGGA GGAGACCAAG
180
GCACAGGACA TTCTGGGAGC AGTGACCCTT CTGCTGGAGG GAGTGATGGC AGCACGGGGA
240
CAACTGGGAC CCACTTGCCT CTCATCCCTC CTGGGGCAGC TTTCTGGACA GGTCCGTCTC
300
CTCCTTGGGG CCCTGCAGAG CCTCCTTGGA ACCCAGCTTC CTCCACAGGG CAGGACCACA
360
GCTCACAAGG ATCCCAATGC CATCTTCCTG AGCTTCCAAC ACCTGCTCCG AGGAAAGGTG
420
CGTTTCCTGA TGCTTGTAGG AGGGTCCACC CTCTGCGTCA GGGAATTCCA TGCATACGTA
480
GAGGGCGGTG GAGGCTCCCC GGGTGAACCG TCTGGTCCAA TCTCTACTAT CAACCCGTCT
540
CCTCCGTCTA AAGAATCTCA TAAATCTCCA AACATGGCTA CCCAGGACTG CTCCTTCCAA
600
CACAGCCCCA TCTCCTCCGA CTTCGCTGTC AAAATCCGTG AGCTGTCTGA CTACCTGCTT
660
CAAGATTACC CAGTCACCGT GGCCTCCAAC CTGCAGGACG AGGAGCTCTG CGGGGGCCTC
720
TGGCGGCTGG TCCTGGCACA GCGCTGGATG GAGCGGCTCA AGACTGTCGC TGGGTCCAAG
780
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ATGCAAGGCT TGCTGGAGCG CGTGAACACG GAGATACACT TTGTCACCAA ATGTGCCTTT
840
CAGCCCCCCC CCAGCTGTCT TCGCTTCGTC CAGACCAACA TCTCCCGCCT CCTGCAGGAG
900
ACCTCCGAGC'AGCTGGTGGC GCTGAAGCCC TGGATCACTC GCCAGAACTT CTCCCGGTGC
960
CTGGAGCTGC AGTGTCAGCC CGACTCCTCA ACCCTG
996
(2) INFORMATION FOR SEQ ID N0:27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1020 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:27:
GCCACTCAGG ACTGCTCTTT TCAACACAGC CCCATCTCCT CCGACTTCGC TGTCAAAATC
CGTGAGCTGT CTGACTACCT GCTTCAAGAT TACCCAGTCA CCGTGGCCTC CAACCTGCAG
120
GACGAGGAGC TCTGCGGGGG CCTCTGGCGG CTGGTCCTGG CACAGCGCTG GATGGAGCGG
180
CTCAAGACTG TCGCTGGGTC CAAGATGCAA GGCTTGCTGG AGCGCGTGAA CACGGAGATA
240
CACTTTGTCA CCAAATGTGC CTTTCAGCCC CCCCCCAGCT GTCTTCGCTT CGTCCAGACC
300
AACATCTCCC GCCTCCTGCA GGAGACCTCC GAGCAGCTGG TGGCGCTGAA GCCCTGGATC
360
ACTCGCCAGA ACTTCTCCCG GTGCCTGGAG CTGCAGTGTC AGCCCGACTC CTCAACCCTG
420
TACGTAGAGG GCGGTGGAGG CTCCCCGGGT GAACCGTCTG GTCCAATCTC TACTATCAAC
480
CCGTCTCCTC CGTCTAAAGA ATCTCATAAA TCTCCAAACA TGGCCCCACC ACGCCTCATC
540
TGTGACAGCC GAGTCCTGGA GAGGTACCTC ~'TGGAGGCCA AGGAGGCCGA GAATATCACG
600
ACGGGCTGTG CTGAACACTG CAGCTTGAAT GAGAATATCA CTGTCCCAGA CACCAAAGTT
660
AATTTCTATG CCTGGAAGAG GATGGAGGTC GGGCAGCAGG CCGTAGAAGT CTGGCAGGGC
720
CTGGCCCTGC TGTCGGAAGC TGTCCTGCGG GGCCAGGCCC TGTTGGTCAA CTCTTCCCAG
?80
CCGTGGGAGC CCCTGCAGCT GCATGTGGAT AAAGCCGTCA GTGGCCTTCG CAGCCTCACC
840
ACTCTGCTTC GGGCTCTGCG AGCCCAGAAG GAAGCCATCT CCCCTCCAGA TGCGGCCTCA
900
GCTGCTCCAC TCCGAACAAT CACTGCTGAC ACTTTCCGCA AACTCTTCCG AGTCTACTCC
960
AATTTCCTCC GGGGAAAGCT GAAGCTGTAC ACAGGGGAGG CCTGCAGGAC AGGGGACAGA
1020
(2) INFORMATION FOR SEQ ID N0:28:
(i) SEQUENCE CHARACTERISTICS:
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WO 98/46750 g0 PGT/US98/07511
(A) LENGTH: 975 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:28:
GCCCCACCAC GCCTCATCTG TGACAGCCGA GTCCTGGAGA GGTACCTCTT GGAGGCCAAG
GAGGCCGAGA ATATCACGAC GGGCTGTGCT GAACACTGCA GCTTGAATGA GAATATCACT
120
GTCCCAGACA CCAAAGTTAA TTTCTATGCC TGGAAGAGGA TGGAGGTCGG GCAGCAGGCC
180
GTAGAAGTCT GGCAGGGCCT GGCCCTGCTG TCGGAAGCTG TCCTGCGGGG CCAGGCCCTG
240
TTGGTCAACT CTTCCCAGCC GTGGGAGCCC CTGCAGCTGC ATGTGGATAA AGCCGTCAGT
300
GGCCTTCGCA GCCTCACCAC TCTGCTTCGG GCTCTGCGAG CCCAGAAGGA AGCCATCTCC
360
CCTCCAGATG CGGCCTCAGC TGCTCCACTC CGAACAATCA CTGCTGACAC TTTCCGCAAA
420
CTCTTCCGAG TCTACTCCAA TTTCCTCCGG GGAAAGCTGA AGCTGTACAC AGGGGAGGCC
480
TGCAGGACAG GGGACAGATA CGTAGAGGGC GGTGGAGGCT CCCCGGGTGG TGGTTCTGGC
540
GGCGGCTCCA ACATGGCCAC TCAGGACTGC TCTTTTCAAC ACAGCCCCAT CTCCTCCGAC
600
TTCGCTGTCA AAATCCGTGA GCTGTCTGAC TACCTGCTTC AAGATTACCC AGTCACCGTG
660
GCCTCCAACC TGCAGGACGA GGAGCTCTGC GGGGGCCTCT GGCGGCTGGT CCTGGCACAG
720
CGCTGGATGG AGCGGCTCAA GACTGTCGCT GGGTCCAAGA TGCAAGGCTT GCTGGAGCGC
780
GTGAACACGG AGATACACTT TGTCACCAAA TGTGCCTTTC AGCCCCCCCC CAGCTGTCTT
840
CGCTTCGTCC AGACCAACAT CTCCCGCCTC CTGCAGGAGA CCTCCGAGCA GCTGGTGGCG
900
CTGAAGCCCT GGATCACTCG CCAGAACTTC TCCCGGTGCC TGGAGCTGCA GTGTCAGCCC
960
GACTCCTCAA CCCTG
975
(2) INFORMATION FOR SEQ ID N0:29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:29:
GTAGTCCATG GCCACCCAGG ACTGCTCC
28
(2) INFORMATION FOR SEQ ID N0:30:
CA 02284127 1999-09-10
wo 9s~~w~so 81 rcrms9sro~si ~
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:30:
GCATTACGTA GGGCTGACAC TGCAGCTCCA G
31
(2) INFORMATION FOR SEQ ID N0:31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:31:
GCATTACGTA CAGGGTTGAG GAGTCGGGCT G
31
(2) INFORMATION FOR SEQ ID N0:32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 44 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:32:
CCTGCAGGAC AGGGGACAGA TACGTAGAGG GCGGTGGAGG CTCC
44
(2) INFORMATION FOR SEQ ID N0:33:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 48 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:33:
CCGGGGAGCC TCCACCGCCC TCTACGTATC TGTCCCCTGT CCTGCAGG
48
(2) INFORMATION FOR SEQ ID N0:34:
CA 02284127 1999-09-10
WO 98/46750 82 PC'T/US98/07511
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 266 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: None
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:34:
Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His His Leu Lys Arg
1 5 10 15
Pro Pro Ala Pro Leu Leu Asp Pro Asn Asn Leu Asn Asp Glu Asp Val
20 25 30
Ser Ile Leu Met Asp Arg Asn Leu Arg Leu Pro Asn Leu Glu Ser Phe
35 40 45
Val Arg Ala Val Lys Asn Leu Glu Asn Ala Ser Gly Ile Glu Ala Ile
50 55 60
Leu Arg Asn Leu Gln Pro Cys Leu Pro Ser Ala Thr Ala Ala Pro Ser
65 70 75 80
Arg His Pro Ile Ile Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg Glu
85 90 95
Lys Leu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu Gln Gln
100 105 110
Tyr Val Glu Gly Gly Gly Gly Ser Pro Gly Gly Gly Ser Gly Gly Gly
115 120 125
Ser Asn Met Ala Thr Gln Asp Cys Ser Phe Gln His Ser Pro Ile Ser
130 135 140
Ser Asp Phe Ala Val Lys Ile Arg Glu Leu Ser Asp Tyr Leu Leu Gln
145 150 155 160
Asp Tyr Pro Val Thr Val Ala Ser Asn Leu Gln Asp Glu Glu Leu Cys
165 170 175
Gly Ala Leu Trp Arg Leu Val Leu Ala Gln Arg Trp Met Glu Arg Leu
180 185 190
Lys Thr Val Ala Gly Ser Lys Met Gln Gly Leu Leu Glu Arg Val Asn
195 200 205
Thr Glu Ile His Phe Val Thr Lys Cys Ala Phe Gln Pro Pro Pro Ser
210 215 220
Cys Leu Arg Phe Val Gln Thr Asn Ile Ser Arg Leu Leu Gln Glu Thr
225 230 235 240
Ser Glu Gln Leu Val Ala Leu Lys Pro Trp Ile Thr Arg Gln Asn Phe
245 250 255
Ser Arg Cys Leu Glu Leu Gln Cys Gln Pro
260 265
(2) INFORMATION FOR SEQ ID N0:35:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 281 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: None
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:35:
Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His His Leu Lys Arg
CA 02284127 1999-09-10
WO 98/46750 83 PCT/IJS98/07511
1 5 10 15
Pro Pro Ala Pro Leu Leu Asp Pro Asn Asn Leu Asn Asp Glu Asp Val
20 25 30
Ser Ile Leu Met Asp Arg Asn Leu Arg Leu Pro Asn Leu Glu Ser Phe
35~ 40 45
Val Arg Ala Val Lys Asn Leu Glu Asn Ala Ser Gly Ile Glu Ala Ile
50 55 60
Leu Arg Asn Leu Gln Pro Cys Leu Pro Ser Ala Thr Ala Ala Pro Ser
65 70 75 80
Arg His Pro Ile Ile Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg Glu
85 90 95
Lys Leu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu Gln Gln
100 105 110
Tyr Val Glu Gly Gly Gly Gly Ser Pro Gly Glu Pro Ser Gly Pro Ile
115 120 125
Ser Thr Ile Asn Pro Ser Pro Pro Ser Lys Glu Ser His Lys Ser Pro
130 135 140
Asn Met Ala Thr Gln Asp Cys Ser Phe Gln His Ser Pro Ile Ser Ser
145 150 155 160
Asp Phe Ala Val Lys Ile Arg Glu Leu Ser Asp Tyr Leu Leu Gln Asp
165 170 175
Tyr Pro Val Thr Val Ala Ser Asn Leu Gln Asp Glu Glu Leu Cys Gly
180 i85 190
Ala Leu Trp Arg Leu Val Leu Ala Gln Arg Trp Met Glu Arg Leu Lys
195 200 205
Thr Val Ala Gly Ser Lys Met Gln Gly Leu Leu Glu Arg Val Asn Thr
210 215 220
Glu Ile His Phe Val Thr Lys Cys Ala Phe Gln Pro Pro Pro Ser Cys
225 230 235 240
Leu Arg Phe Val Gln Thr Asn Ile Ser Arg Leu Leu Gln Glu Thr Ser
245 250 255
Glu Gln Leu Val Ala Leu Lys Pro Trp Ile Thr Arg Gln Asn Phe Ser
260 265 270
Arg Cys Leu Glu Leu Gln Cys Gln Pro
275 280
(2) INFORMATION FOR SEQ ID N0:36:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 271 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: None
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:36:
Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His His Leu Lys Arg
1 5 10 I5
Pro Pro Ala Pro Leu Leu Asp Pro Asn Asn Leu Asn Asp Glu Asp Val
20 25 30
Ser Ile Leu Met Asp Arg Asn Leu Arg Leu Pro Asn Leu Glu Ser Phe
35 40 45
Val Arg Ala Val Lys Asn Leu Glu Asn Ala Ser Gly Ile Glu Ala Ile
50 55 60
Leu Arg Asn Leu Gln Pro Cys Leu Pro Ser Ala Thr Ala Ala Pro Ser
65 70 75 g0
CA 02284127 1999-09-10
WO 98/46750 g4 PCT/US98/07511
Arg His Pro Ile Ile Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg Glu
85 90 95
Lys Leu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu Gln Gln
100 105 110
Tyr Val Glti Gly Gly Gly Gly Ser Pro Gly Gly Gly Ser Gly Gly Gly
115 120 125
Ser Asn Met Ala Thr Gln Asp Cys Ser Phe Gln His Ser Pro Ile Ser
130 135 140
Ser Asp Phe Ala Val Lys Ile Arg Glu Leu Ser Asp Tyr Leu Leu Gln
145 150 155 160
Asp Tyr Pro Val Thr Val Ala Ser Asn Leu Gln Asp Glu Glu Leu Cys
165 170 175
Gly Ala Leu Trp Arg Leu Val Leu Ala Gln Arg Trp Met Glu Arg Leu
180 185 190
Lys Thr Val Ala Gly Ser Lys Met Gln Gly Leu Leu Glu Arg Val Asn
195 200 205
Thr Glu Ile His Phe Val Thr Lys Cys Ala Phe Gln Pro Pro Pro Ser
210 215 220
Cys Leu Arg Phe Val Gln Thr Asn Ile Ser Arg Leu Leu Gln Glu Thr
225 230 235 240
Ser Glu Gln Leu Val Ala Leu Lys Pro Trp Ile Thr Arg Gln Asn Phe
245 250 255
Ser Arg Cys Leu Glu Leu Gln Cys Gln Pro Asp Ser Ser Thr Leu
260 265 270
(2) INFORMATION FOR SEQ ID N0:37:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 286 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: None
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:37:
Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His His Leu Lys Arg
1 5 10 15
Pro Pro Ala Pro Leu Leu Asp Pro Asn Asn Leu Asn Asp Glu Asp Val
20 25 30
Ser Ile Leu Met Asp Arg Asn Leu Arg Leu Pro Asn Leu Glu Ser Phe
35 40 45
Val Arg Ala Val Lys Asn Leu Glu Asn Ala Ser Gly Ile Glu Ala Ile
50 55 60
Leu Arg Asn Leu Gln Pro Cys Leu Pro Ser Ala Thr Ala Ala Pro Ser
65 70 75 80
Arg His Pro Ile Ile Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg Glu
85 90 95
Lys Leu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu Gln Gln
100 105 110
Tyr Val Glu Gly Gly Gly Gly Ser Pro Gly Glu Pro Ser Gly Pro Ile
115 120 125
Ser Thr Ile Asn Pro Ser Pro Pro Ser Lys Glu Ser His Lys Ser Pro
130 135 140
Asn Met Ala Thr Gln Asp Cys Ser Phe Gln His Ser Pro Ile Ser Ser
145 150 155 150
Asp Phe Ala Val Lys Ile Arg Glu Leu Ser Asp Tyr Leu Leu Gln Asp
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165 170 175
Tyr Pro Val Thr Val Ala Ser Asn Leu Gln Asp Glu Glu Leu Cys Gly
180 185 190
Ala Leu Trp Arg Leu Val Leu Ala Gln Arg Trp Met Glu Arg Leu Lys
195 200 205
Thr Val Ala Gly Ser Lys Met Gln Gly Leu Leu Glu Arg Val Asn Thr
210 215 220
Glu Ile His Phe Val Thr Lys Cys Ala Phe Gln Pro Pro Pro Ser Cys
225 230 235 240
Leu Arg Phe Val Gln Thr Asn Ile Ser Arg Leu Leu Gln Glu Thr Ser
245 250 255
Glu Gln Leu Val Ala Leu Lys Pro Trp Ile Thr Arg Gln Asn Phe Ser
260 265 270
Arg Cys Leu Glu Leu Gln Cys Gln Pro Asp Ser Ser Thr Leu
275 280 285
{2) INFORMATION FOR SEQ ID N0:38:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 349 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: None
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:38:
Ala Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu Leu
1 5 10 15
Lys Ser Leu Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala Ala Leu
20 25 30
Gln Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu
35 40 45
Val Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser Ser
50 55 60
Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser Gln Leu His
65 70 75 80
Ser Gly Leu Phe Leu Tyr Gln Giy Leu Leu Gln Ala Leu Glu Gly Ile
85 90 95
Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val Ala
100 105 110
Asp Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met Ala
115 120 125
Pro Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala Ser Ala
130 135 140
Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gln Ser
145 150 155 160
Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln Pro Tyr
165 170 175
Val Glu Gly Gly Gly Gly Ser Pro Gly Glu Pro Ser Gly Pro Ile Ser
180 185 190
Thr Ile Asn Pro Ser Pro Pro Ser Lys Glu Ser His Lys Ser Pro Asn
195 200 205
Met Ala Thr Gln Asp Cys Ser Phe Gln His Ser Pro Ile Ser Ser Asp
210 215 220
Phe Ala Val Lys Ile Arg Glu Leu Ser Asp Tyr Leu Leu Gln Asp Tyr
225 230 235 240
CA 02284127 1999-09-10
WO 98146750 8b PCT/US98/0'7511
Pro Val Thr Val Ala Ser Asn Leu Gln Asp Glu Glu Leu Cys Gly Gly
245 250 255
Leu Trp Arg Leu Val Leu Ala Gln Arg Trp Met Glu Arg Leu Lys Thr
260 265 270
Val Ala Gly Ser Lys Met Gln Gly Leu Leu Glu Arg Val Asn Thr Glu
275 280 285
Ile His Phe Val Thr Lys Cys Ala Phe Gln~Pro Pro Pro Ser Cys Leu
290 295 300
Arg Phe Val Gln Thr Asn Ile Ser Arg Leu Leu Gln Glu Thr Ser Glu
305 310 315 320
Gln Leu Val Ala Leu Lys Pro Trp Ile Thr Arg Gln Asn Phe Ser Arg
325 330 335
Cys Leu Glu Leu Gln Cys Gln Pro Asp Ser Ser Thr Leu
340 345
(2) INFORMATION FOR SEQ ID N0:39:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 314 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: None
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:39:
Ala Thr Gln Asp Cys Ser Phe Gln His Ser Pro Ile Ser Ser Asp Phe
1 5 10 15
Ala Val Lys Ile Arg Glu Leu Ser Asp Tyr Leu Leu Gln Asp Tyr Pro
20 25 30
Val Thr Val Ala Ser Asn Leu Gln Asp Glu Glu Leu Cys Gly Gly Leu
35 40 45
Trp Arg Leu Val Leu Ala Gln Arg Trp Met Glu Arg Leu Lys Thr Val
50 55 60
Ala Gly Ser Lys Met Gln Gly Leu Leu Glu Arg Val Asn Thr Glu Ile
65 70 75 80
His Phe Val Thr Lys Cys Ala Phe Gln Pro Pro Pro Ser Cys Leu Arg
85 90 95
Phe Val Gln Thr Asn Ile Ser Arg Leu Leu Gln Glu Thr Ser Glu Gln
100 105 110
Leu Val Ala Leu Lys Pro Trp Ile Thr Arg Gln Asn Phe Ser Arg Cys
115 120 125
Leu Glu Leu Gln Cys Gln Pro Asp Ser Ser Thr Leu Tyr Val Glu Gly
130 135 140
Gly Gly Gly Ser Pro Gly Glu Pro Ser Gly Pro Ile Ser Thr Ile Asn
145 150 155 160
Pro Ser Pro Pro Ser Lys Glu Ser His Lys Ser Pro Asn Met Ala Thr
165 170 175
Gln Asp Cys Ser Phe Gln His Ser Pro Ile Ser Ser Asp Phe Ala Val
180 185 190
Lys Ile Arg Glu Leu Ser Asp Tyr Leu Leu Gln Asp Tyr Pro Val Thr
195 200 205
Val Ala Ser Asn Leu Gln Asp Glu Glu Leu Cys Gly Gly Leu Trp Arg
210 215 220
Leu Val Leu Ala Gln Arg Trp Met Glu Arg Leu Lys Thr Val Ala Gly
225 230 235 240
Ser Lys Met Gln Gly Leu Leu Glu Arg Val Asn Thr Glu Ile His Phe
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245 250 255
Val Thr Lys Cys Ala Phe Gln Pro Pro Pro Ser Cys Leu Arg Phe Val
260 265 270
Gln Thr Asn Ile Ser Arg Leu Leu Gln Glu Thr Ser Glu Gln Leu Val
275 280 285
Ala Leu Lys Pro Trp Ile Thr Arg Gln Asn Phe Ser Arg Cys Leu Glu
290 295 300
Leu Gln Cys Gln Pro Asp Ser Ser Thr Leu
305 310
(2) INFORMATION FOR SEQ ID N0:40:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 349 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: None
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:40:
Ala Thr Gln Asp Cys Ser Phe Gln His Ser Pro Ile Ser Ser Asp Phe
1 5 10 15
Ala Val Lys Ile Arg Glu Leu Ser Asp Tyr Leu Leu Gln Asp Tyr Pro
20 25 30
Val Thr Val Ala Ser Asn Leu Gln Asp Glu Glu Leu Cys Gly Gly Leu
35 40 45
Trp Arg Leu Val Leu Ala Gln Arg Trp Met Glu Arg Leu Lys Thr Val
50 55 60
Ala Gly Ser Lys Met Gln Gly Leu Leu Glu Arg Val Asn Thr Glu Ile
65 70 75 80
His Phe Val Thr Lys Cys Ala Phe Gln Pro Pro Pro Ser Cys Leu Arg
85 90 95
Phe Val Gln Thr Asn Ile Ser Arg Leu Leu Gln Glu Thr Ser Glu Gln
100 105 110
Leu Val Ala Leu Lys Pro Trp Ile Thr Arg Gln Asn Phe Ser Arg Cys
115 120 125
Leu Glu Leu Gln Cys Gln Pro Asp Ser Ser Thr Leu Tyr Val Glu Gly
130 135 140
Gly Gly Gly Ser Pro Gly Glu Pro Ser Gly Pro Ile Ser Thr Ile Asn
145 150 155 160
Pro Ser Pro Pro Ser Lys Glu Ser His Lys Ser Pro Asn Met Ala Thr
165 170 175
Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu Leu Lys Cys
180 I85 190
Leu Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala Ala Leu Gln Glu
195 200 205
Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu Val Leu
210 215 220
Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser Ser Cys Pro
225 230 235 240
Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser Gln Leu His Ser Gly
245 250 255
Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu Gly Ile Ser Pro
260 265 270
Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val Ala Asp Phe
275 280 285
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Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met Ala Pro Ala
290 295 300
Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala Ser Ala Phe Gln
305 310 315 320
Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gln Ser Phe Leu
325 330 335
Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln Pro
340 345
(2) INFORMATION FOR SEQ ID N0:41:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 334 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: None
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:41:
Ala Thr Gln Asp Cys Ser Phe Gln His Ser Pro Ile Ser Ser Asp Phe
1 5 10 15
Ala Val Lys Ile Arg Glu Leu Ser Asp Tyr Leu Leu Gln Asp Tyr Pro
20 25 30
Val Thr Val Ala Ser Asn Leu Gln Asp Glu Glu Leu Cys Gly Gly Leu
35 40 45
Trp Arg Leu Val Leu Ala Gln Arg Trp Met Glu Arg Leu Lys Thr Val
50 55 60
Ala Gly Ser Lys Met Gln Gly Leu Leu Glu Arg Val Asn Thr Glu Ile
65 70 75 80
His Phe Val Thr Lys Cys Ala Phe Gln Pro Pro Pro Ser Cys Leu Arg
85 90 95
Phe Val Gln Thr Asn Ile Ser Arg Leu Leu Gln Glu Thr Ser Glu Gln
100 105 110
Leu Val Ala Leu Lys Pro Trp Ile Thr Arg Gln Asn Phe Ser Arg Cys
115 120 125
Leu Glu Leu Gln Cys Gln Pro Asp Ser Ser Thr Leu Tyr Val Glu Gly
130 135 140
Gly Gly Gly Ser Pro Gly Gly Gly Ser Gly Gly Gly Ser Asn Met Ala
145 150 155 160
Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu Leu Lys
165 170 175
Ser Leu Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala Ala Leu Gln
180 185 190
Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu Val
195 200 205
Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser Ser Cys
210 215 220
Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser Gln Leu His Ser
225 230 235 240
Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu Gly Ile Ser
245 250 255
Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val Ala Asp
260 265 270
Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met Ala Pro
275 280 285
Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala Ser Ala Phe
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290 295 300
Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gln Ser Phe
305 310 315 320
Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln Pro
325 330
(2} INFORMATION FOR SEQ ID N0:42:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 281 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: None
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:42:
Ala Asn Cys Ser IIe Met Ile Asp Glu Ile Ile His His Leu Lys Arg
1 5 10 15
Pro Pro Asn Pro Leu Leu Asp Pro Asn Asn Leu Asn Ser Glu Asp Met
20 25 30
Asp Ile Leu Met Glu Arg Asn Leu Arg Thr Pro Asn Leu Leu Ala Phe
35 40 45
Val Arg Ala Val Lys His Leu Glu Asn Ala Ser Gly Ile Glu Ala Ile
50 55 60
Leu Arg Asn Leu Gln Pro Cys Leu Pro Ser Ala Thr Ala Ala Pro Ser
65 70 75 80
Arg His Pro Ile Ile Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg Glu
85 90 95
Lys Leu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu Gln Gln
100 105 110
Tyr Val Glu Gly Gly Gly Gly Ser Pro Gly Glu Pro Ser Gly Pro Ile
115 120 125
Ser Thr Ile Asn Pro Ser Pro Pro Ser Lys Glu Ser His Lys Ser Pro
130 135 140
Asn Met Ala Thr Gln Asp Cys Ser Phe Gln His Ser Pro Ile Ser Ser
145 150 155 160
Asp Phe Ala Val Lys Ile Arg Glu Leu Ser Asp Tyr Leu Leu Gln Asp
165 170 175
Tyr Pro Val Thr Val Ala Ser Asn Leu Gln Asp Glu Glu Leu Cys Gly
180 185 190
Gly Leu Trp Arg Leu Val Leu Ala Gln Arg Trp Met Glu Arg Leu Lys
195 200 205
Thr Val Ala Gly Ser Lys Met Gln Gly Leu Leu Glu Arg Val Asn Thr
210 215 220
Glu Ile His Phe Val Thr Lys Cys Ala Phe Gln Pro Pro Pro Ser Cys
225 230 235 240
Leu Arg Phe Val Gln Thr Asn Ile Ser Arg Leu Leu Gln Glu Thr Ser
245 250 255
Glu Gln Leu Val Ala Leu Lys Pro Trp Ile Thr Arg Gln Asn Phe Ser
260 265 270
Arg Cys Leu Glu Leu Gln Cys Gln Pro
275 280
(2) INFORMATION FOR SEQ ID N0:43:
(i) SEQUENCE CHARACTERISTICS:
CA 02284127 1999-09-10
WO 98/~t6750 90 PCT/US98/07511
(A) LENGTH: 286 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: None
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:43:
Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His His Leu Lys Arg
1 5 10 15
Pro Pro Asn Pro Leu Leu Asp Pro Asn Asn Leu Asn Ser Glu Asp Met
20 25 30
Asp Ile Leu Met Glu Arg Asn Leu Arg Thr Pro Asn Leu Leu Ala Phe
35 40 45
Val Arg Ala Val Lys His Leu Glu Asn Ala Ser Gly Ile Glu Ala Ile
50 55 60
Leu Arg Asn Leu Gln Pro Cys Leu Pro Ser Ala Thr Ala Ala Pro Ser
65 70 75 80
Arg His Pro Ile Ile Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg Glu
85 90 95
Lys Leu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu Gln Gln
100 105 110
Tyr Val Glu Gly Gly Gly Gly Ser Pro Gly Glu Pro Ser Gly Pro Ile
115 120 125
Ser Thr Ile Asn Pro Ser Pro Pro Ser Lys Glu Ser His Lys Ser Pro
130 135 140
Asn Met Ala Thr Gln Asp Cys Ser Phe Gln His Ser Pro Ile Ser Ser
145 150 155 160
Asp Phe Ala Val Lys Ile Arg Glu Leu Ser Asp Tyr Leu Leu Gln Asp
165 170 175
Tyr Pro Vai Thr Val Ala Ser Asn Leu Gln Asp Glu Glu Leu Cys Gly
180 185 190
Gly Leu Trp Arg Leu Val Leu Ala Gln Arg Trp Met Glu Arg Leu Lys
195 200 205
Thr Val Ala Gly Ser Lys Met Gln Gly Leu Leu Glu Arg Val Asn Thr
210 215 220
Glu Ile His Phe Val Thr Lys Cys Ala Phe Gln Pro Pro Pro Ser Cys
225 230 235 240
Leu Arg Phe Val Gln Thr Asn Ile Ser Arg Leu Leu Gln Glu Thr Ser
245 250 255
Glu Gln Leu Val Ala Leu Lys Pro Trp Ile Thr Arg Gln Asn Phe Ser
260 265 270
Arg Cys Leu Glu Leu Gln Cys Gln Pro Asp Ser Ser Thr Leu
275 280 285
(2) INFORMATION FOR SEQ ID N0:44:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 281 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: None
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:44:
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Ala Thr Gln Asp Cys Ser Phe Gln His Ser Pro Ile Ser Ser Asp Phe
1 5 10 15
Ala Val Lys Ile Arg Glu Leu Ser Asp Tyr Leu Leu Gln Asp Tyr Pro
20 25 30
Val Thr Val Ala Ser Asn Leu Gln Asp Glu Glu Leu Cys Gly Gly Leu
35 40 45
Trp Arg Leu Val Leu Ala Gln Arg Trp Met Glu Arg Leu Lys Thr Val
50 55 60
Ala Gly Ser Lys Met Gln Gly Leu Leu Glu Arg Val Asn Thr Glu Ile
65 70 75 80
His Phe Val Thr Lys Cys Ala Phe Gln Pro Pro Pro Ser Cys Leu Arg
85 90 95
Phe Val Gln Thr Asn Ile Ser Arg Leu Leu Gln Glu Thr Ser Glu Gln
100 105 110
Leu Val Ala Leu Lys Pro Trp Ile Thr Arg Gln Asn Phe Ser Arg Cys
115 120 125
Leu Glu Leu Gln Cys Gln Pro Tyr Val Glu Gly Gly Gly Gly Ser Pro
230 135 140
Gly Glu Pro Ser Gly Pro Ile Ser Thr Ile Asn Pro Ser Pro Pro Ser
145 150 155 160
Lys Glu Ser His Lys Ser Pro Asn Met Ala Asn Cys Ser Ile Met Ile
165 170 175
Asp Glu Ile Ile His His Leu Lys Arg Pro Pro Asn Pro Leu Leu Asp
180 185 190
Pro Asn Asn Leu Asn Ser Glu Asp Met Asp Ile Leu Met Glu Arg Asn
195 200 205
Leu Arg Thr Pro Asn Leu Leu Ala Phe Val Arg Ala Val Lys His Leu
210 215 220
Glu Asn Ala Ser Gly Ile Glu Ala Ile Leu Arg Asn Leu Gln Pro Cys
225 230 235 240
Leu Pro Ser Ala Thr Ala Ala Pro Ser Arg His Pro Ile Ile Ile Lys
245 250 255
Ala Gly Asp Trp Gln Glu Phe Arg Glu Lys Leu Thr Phe Tyr Leu Val
260 265 270
Thr Leu Glu Gln Ala Gln Glu Gln Gln
275 280
(2) INFORMATION FOR SEQ ID N0:45:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 286 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: None
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:45:
Ala Thr Gln Asp Cys Ser Phe Gln His Ser Pro Ile Ser Ser Asp Phe
1 5 10 15
Ala Val Lys Ile Arg Glu Leu Ser Asp Tyr Leu Leu Gln Asp Tyr Pro
20 25 30
Val Thr Val Ala Ser Asn Leu Gln Asp Glu Glu Leu Cys Gly Gly Leu
35 40 45
Trp Arg Leu Val Leu Ala Gln Arg Trp Met Glu Arg Leu Lys Thr Val
50 55 60
Ala Gly Ser Lys Met Gln Gly Leu Leu Glu Arg Val Asn Thr Glu Ile
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65 70 75 80
His Phe Val Thr Lys Cys Ala Phe Gln Pro Pro Pro Ser Cys Leu Arg
85 90 95
Phe Val Gln Thr Asn Ile Ser Arg Leu Leu Gln Glu Thr Ser Glu Gln
100 105 110
Leu Val Ala Leu Lys Pro Trp Ile Thr Arg Gln Asn Phe Ser Arg Cys
115 120 125
Leu Glu Leu Gln Cys Gln Pro Asp Ser Ser Thr Leu Tyr Val Glu Gly
130 135 140
Gly Gly Gly Ser Pro Gly Glu Pro Ser Gly Pro Ile Ser Thr Ile Asn
145 150 155 160
Pro Ser Pro Pro Ser Lys Glu Ser His Lys Ser Pro Asn Met Ala Asn
165 170 175
Cys Ser Ile Met Ile Asp Glu Ile Ile His His Leu Lys Arg Pro Pro
180 185 190
Asn Pro Leu Leu Asp Pro Asn Asn Leu Asn Ser Glu Asp Met Asp Ile
195 200 205
Leu Met Glu Arg Asn Leu Arg Thr Pro Asn Leu Leu Ala Phe Val Arg
210 215 220
Ala Val Lys His Leu Glu Asn Ala Ser Gly Ile Glu Ala Ile Leu Arg
225 230 235 240
Asn Leu Gln Pro Cys Leu Pro Ser Ala Thr Ala Ala Pro Ser Arg His
245 250 255
Pro Ile Ile Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg Glu Lys Leu
260 265 270
Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu Gln Gln
275 280 285
(2) INFORMATION FOR SEQ ID N0:46:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 313 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: None
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:46:
Ala Thr Gln Asp Cys Ser Phe Gln His Ser Pro Ile Ser Ser Asp Phe
1 5 10 15
Ala Val Lys Ile Arg Glu Leu Ser Asp Tyr Leu Leu Gln Asp Tyr Pro
20 25 30
Val Thr Val Ala Ser Asn Leu Gln Asp Glu Glu Leu Cys Gly Gly Leu
35 40 45
Trp Arg Leu Val Leu Ala Gln Arg Trp Met Glu Arg Leu Lys Thr Val
50 55 60
Ala Gly Ser Lys Met Gln Gly Leu Leu Glu Arg Val Asn Thr Glu Ile
65 70 75 80
His Phe Val Thr Lys Cys Ala Phe Gln Pro Pro Pro Ser Cys Leu Arg
85 90 95
Phe Val Gln Thr Asn Ile Ser Arg Leu Leu Gln Glu Thr Ser Glu Gln
100 105 110
Leu VaI Ala Leu Lys Pro Trp Ile Thr Arg Gln Asn Phe Ser Arg Cys
115 120 125
Leu Glu Leu Gln Cys Gln Pro Asp Ser Ser Thr Leu Tyr Val Glu Gly
130 135 140
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Gly Gly Gly Ser Pro Gly Gly Gly Ser Gly Gly Gly Ser Asn Met Ala
145 150 155 160
Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu Leu
165 170 175
Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gln Cys Pro Glu Val
180 185 190
His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp Phe Ser Leu
195 200 205
Gly Glu Trp Lys Thr Gln Met Glu Glu Thr Lys Ala Gln Asp Ile Leu
210 215 220
Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met Ala Ala Arg Gly Gln
225 230 235 240
Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gln Leu Ser Gly Gln
245 250 255
Val Arg Leu Leu Leu Gly Ala Leu Gln Ser Leu Leu Gly Thr Gln Leu
260 265 270
Pro Pro Gln Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe
275 280 285
Leu Ser Phe Gln His Leu Leu Arg Gly Lys Val Arg Phe Leu Met Leu
290 295 300
Val Gly Gly Ser Thr Leu Cys Val Arg
305 310
(2) INFORMATION FOR SEQ ID N0:47:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 332 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: None
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:47:
Ala Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu
1 5 10 15
Leu Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gln Cys Pro Glu
20 25 30
Val His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp Phe Ser
35 40 45
Leu Gly Glu Trp Lys Thr Gln Met Glu Glu Thr Lys Ala Gln Asp Ile
50 55 60
Leu Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met Ala Ala Arg Gly
65 70 75 80
Gln Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gln Leu Ser Gly
85 90 95
Gln Val Arg Leu Leu Leu Gly Ala Leu Gln Ser Leu Leu Gly Thr Gln
100 105 110
Leu Pro Pro Gln Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile
115 120 125
Phe Leu Ser Phe Gln His Leu Leu Arg Gly Lys Val Arg Phe Leu Met
130 135 140
Leu Val Gly Gly Ser Thr Leu Cys Val Arg Glu Phe His Ala Tyr Val
145 150 155 160
Glu Gly Gly Gly Gly Ser Pro Gly Glu Pro Ser Gly Pro Ile Ser Thr
165 170 175
Ile Asn Pro Ser Pro Pro Ser Lys Glu Ser His Lys Ser Pro Asn Met
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180 185 190
Ala Thr Gln Asp Cys Ser Phe Gln His Ser Pro Ile Ser Ser Asp Phe
195 200 205
Ala Val Lys Ile Arg Glu Leu Ser Asp Tyr Leu Leu Gln Asp Tyr Pro
210 ~ 215 220
Val Thr Val Ala Ser Asn Leu Gln Asp Glu Glu Leu Cys Gly Gly Leu
225 230 235 240
Trp Arg Leu Val Leu Ala Gln Arg Trp Met Glu Arg Leu Lys Thr Val
245 250 255
Ala Gly Ser Lys Met Gln Gly Leu Leu Glu Arg Val Asn Thr Glu Ile
260 265 270
His Phe Val Thr Lys Cys Ala Phe Gln Pro Pro Pro Ser Cys Leu Arg
275 280 285
Phe Val Gln Thr Asn Ile Ser Arg Leu Leu Gln Glu Thr Ser Glu Gln
290 295 300
Leu Val Ala Leu Lys Pro Trp Ile Thr Arg Gln Asn Phe Ser Arg Cys
305 310 315 320
Leu Glu Leu Gln Cys Gln Pro Asp Ser Ser Thr Leu
325 330
(2) INFORMATION FOR SEQ ID N0:48:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 340 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: None
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:48:
Ala Thr Gln Asp Cys Ser Phe Gln His Ser Pro Ile Ser Ser Asp Phe
1 5 10 15
Ala Val Lys Ile Arg Glu Leu Ser Asp Tyr Leu Leu Gln Asp Tyr Pro
20 25 30
Val Thr Val Ala Ser Asn Leu Gln Asp Glu Glu Leu Cys Gly Gly Leu
35 40 45
Trp Arg Leu Val Leu Ala Gln Arg Trp Met Glu Arg Leu Lys Thr Val
50 55 60
Ala Gly Ser Lys Met Gln Gly Leu Leu Glu Arg Val Asn Thr Glu Ile
65 70 75 80
His Phe Val Thr Lys Cys Ala Phe Gln Pro Pro Pro Ser Cys Leu Arg
85 90 95
Phe Val Gln Thr Asn Ile Ser Arg Leu Leu Gln Glu Thr Ser Glu Gln
100 105 110
Leu Val Ala Leu Lys Pro Trp Ile Thr Arg Gln Asn Phe Ser Arg Cys
115 120 125
Leu Glu Leu Gln Cys Gln Pro Asp Ser Ser Thr Leu Tyr Val Glu Gly
130 135 140
Gly Gly Gly Ser Pro Gly Glu Pro Ser Gly Pro Ile Ser Thr Ile Asn
145 150 155 160
Pro Ser Pro Pro Ser Lys Glu Ser His Lys Ser Pro Asn Met Ala Pro
165 170 175
Pro Arg Leu Ile Cys Asp Ser Arg Val Leu GIu Arg Tyr Leu Leu Glu
180 185 190
Ala Lys Glu Ala Glu Asn Ile Thr Thr Gly Cys Ala Glu His Cys Ser
195 200 205
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Leu Asn Glu Asn Ile Thr Val Pro Asp Thr Lys Val Asn Phe Tyr Ala
210 215 220
Trp Lys Arg Met Glu Val Gly Gln Gln Ala Val Glu Val Trp Gln Gly
225 230 235 240
Leu Ala Leu Leu Ser Glu Ala Val Leu Arg Gly Gln Ala Leu Leu Val
245 250 255
Asn Ser Ser Gln Pro Trp Glu Pro Leu Gln Leu His Val Asp Lys Ala
260 265 270
Val Ser Gly Leu Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu Arg Ala
275 280 285
Gln Lys Glu Ala Ile Ser Pro Pro Asp Ala Ala Ser Ala Ala Pro Leu
290 295 300
Arg Thr Ile Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg Val Tyr Ser
305 310 315 320
Asn Phe Leu Arg Gly Lys Leu Lys Leu Tyr Thr Gly Glu Ala Cys Arg
325 330 335
Thr Gly Asp Arg
340
(2) INFORMATION FOR SEQ ID N0:49:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 325 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: None
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:49:
Ala Pro Pro Arg Leu Ile Cys Asp Ser Arg Val Leu Glu Arg Tyr Leu
10 15
Leu Glu Ala Lys Glu Ala Glu Asn Ile Thr Thr Gly Cys Ala Glu His
20 25 30
Cys Ser Leu Asn Glu Asn Ile Thr Val Pro Asp Thr Lys Val Asn Phe
35 40 45
Tyr Ala Trp Lys Arg Met Glu Val Gly Gln Gln Ala Val Glu Val Trp
50 55 60
Gln Gly Leu Ala Leu Leu Ser Glu Ala Val Leu Arg Gly Gln Ala Leu
65 70 75 80
Leu Val Asn Ser Ser Gln Pro Trp Glu Pro Leu Gln Leu His Val Asp
85 90 95
Lys Ala Val Ser Gly Leu Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu
100 105 110
Arg Ala Gln Lys Glu Ala Ile Ser Pro Pro Asp Ala Ala Ser Ala Ala
115 120 125
Pro Leu Arg Thr Ile Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg Val
130 135 140
Tyr Ser Asn Phe Leu Arg Gly Lys Leu Lys Leu Tyr Thr Gly Glu Ala
145 150 155 160
Cys Arg Thr Gly Asp Arg Tyr Val Glu Gly Gly Gly Gly Ser Pro Gly
165 170 175
Gly Gly Ser Gly Gly Gly Ser Asn Met Ala Thr Gln Asp Cys Ser Phe
180 185 190
Gln His Ser Pro Ile Ser Ser Asp Phe Ala Val Lys Ile Arg Glu Leu
195 200 205
Ser Asp Tyr Leu Leu Gln Asp Tyr Pro Val Thr Val Ala Ser Asn Leu
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210 215 220
Gln Asp Glu Glu Leu Cys Gly Gly Leu Trp Arg Leu Val Leu Ala Gln
225 230 235 240
Arg Trp Met Glu Arg Leu Lys Thr Val Ala Gly Ser Lys Met Gln Gly
245 250 255
Leu Leu Glu Arg Val Asn Thr Glu Ile His Phe Val Thr Lys Cys Ala
260 265 270
Phe Gln Pro Pro Pro Ser Cys Leu Arg Phe Val Gln Thr Asn Ile Ser
275 280 285
Arg Leu Leu Gln Glu Thr Ser Glu Gln Leu Val Ala Leu Lys Pro Trp
290 295 300
Ile Thr Arg Gln Asn Phe Ser Arg Cys Leu Glu Leu Gln Cys Gln Pro
305 310 315 320
Asp Ser Ser Thr Leu
325
(2) INFORMATION FOR SEQ ID N0:50:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1032 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:50:
GCTACACCAT TGGGCCCTGC CAGCTCCCTG CCCCAGAGCT TCCTGCTCAA GTCTTTAGAG
CAAGTGAGAA AGATCCAGGG CGATGGCGCA GCGCTCCAGG AGAAGCTGTG TGCCACCTAC
120
AAGCTGTGCC ACCCCGAGGA GCTGGTGCTG CTCGGACACT CTCTGGGCAT CCCCTGGGCT
180
CCCCTGAGCT CCTGCCCCAG CCAGGCCCTG CAGCTGGCAG GCTGCTTGAG CCAACTCCAT
240
AGCGGCCTTT TCCTCTACCA GGGGCTCCTG CAGGCCCTGG AAGGGATATC CCCCGAGTTG
300
GGTCCCACCT TGGACACACT GCAGCTGGAC GTCGCCGACT TTGCCACCAC CATCTGGCAG
360
CAGATGGAAG AACTGGGAAT GGCCCCTGCC CTGCAGCCCA CCCAGGGTGC CATGCCGGCC
420
TTCGCCTCTG CTTTCCAGCG CCGGGCAGGA GGGGTCCTGG TTGCTAGCCA TCTGCAGAGC
480
TTCCTGGAGG TGTCGTACCG CGTTCTACGC CACCTTGCGC AGCCCTACGT AGAGGGCGGT
540
GGAGGCTCCC CGGGTGAACC GTCTGGTCCA ATCTCTACTA TCAACCCGTC TCCTCCGTCT
600
AAAGAATCTC ATAAATCTCC AAACATGGCC ACCCAGGACT GCTCCTTCCA ACACAGCCCC
660
ATCTCCTCCG ACTTCGCTGT CAAAATCCGT GAGCTGTCTG ACTACCTGCT TCAAGATTAC
720
CCAGTCACCG TGGCCTCCAA CCTGCAGGAC GAGGAGCTCT GCGGGGGCCT CTGGCGGCTG
780
GTCCTGGCAC AGCGCTGGAT GGAGCGGCTC AAGACTGTCG CTGGGTCCAA GATGCAAGGC
840
TTGCTGGAGC GCGTGAACAC GGAGATACAC TTTGTCACCA AATGTGCCTT TCAGCCCCCC
900
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CCCAGCTGTC TTCGCTTCGT CCAGACCAAC ATCTCCCGCC TCCTGCAGGA GACCTCCGAG
960
CAGCTGGTGG CGCTGAAGCC CTGGATCACT CGCCAGAACT TCTCCCGGTG CCTGGAGCTG
1020
CAGTGTCAGC ' CC
1032
(2) INFORMATION FOR SEQ ID N0:51:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1005 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:51:
ATGGCCACTC AGGACTGCTC TTTTCAACAC AGCCCCATCT CCTCCGACTT CGCTGTCAAA
ATCCGTGAGC TGTCTGACTA CCTGCTTCAA GATTACCCAG TCACCGTGGC CTCCAACCTG
120
CAGGACGAGG AGCTCTGCGG GGGCCTCTGG CGGCTGGTCC TGGCACAGCG CTGGATGGAG
180
CGGCTCAAGA CTGTCGCTGG GTCCAAGATG CAAGGCTTGC TGGAGCGCGT GAACACGGAG
240
ATACACTTTG TCACCAAATG TGCCTTTCAG CCCCCCCCCA GCTGTCTTCG CTTCGTCCAG
300
ACCAACATCT CCCGCCTCCT GCAGGAGACC TCCGAGCAGC TGGTGGCGCT GAAGCCCTGG
360
ATCACTCGCC AGAACTTCTC CCGGTGCCTG GAGCTGCAGT GTCAGCCCGA CTCCTCAACC
420
CTGTACGTAG AGGGCGGTGG AGGCTCCCCG GGTGGTGGTT CTGGCGGCGG CTCCAACATG
480
GCTACACCAT TGGGCCCTGC CAGCTCCCTG CCCCAGAGCT TCCTGCTCAA GTCTTTAGAG
540
CAAGTGAGAA AGATCCAGGG CGATGGCGCA GCGCTCCAGG AGAAGCTGTG TGCCACCTAC
600
AAGCTGTGCC ACCCCGAGGA GCTGGTGCTG CTCGGACACT CTCTGGGCAT CCCCTGGGCT
660
CCCCTGAGCT CCTGCCCCAG CCAGGCCCTG CAGCTGGCAG GCTGCTTGAG CCAACTCCAT
720
AGCGGCCTTT TCCTCTACCA GGGGCTCCTG CAGGCCCTGG AAGGGATATC CCCCGAGTTG
780
GGTCCCACCT TGGACACACT GCAGCTGGAC GTCGCCGACT TTGCCACCAC CATCTGGCAG
840
CAGATGGAAG AACTGGGAAT GGCCCCTGCC CTGCAGCCCA CCCAGGGTGC CATGCCGGCC
900
TTCGCCTCTG CTTTCCAGCG CCGGGCAGGA GGGGTCCTGG TTGCTAGCCA TCTGCAGAGC
960
TTCCTGGAGG TGTCGTACCG CGTTCTACGC CACCTTGCGC AGCCG
1005
(2) INFORMATION FOR SEQ ID N0:52:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 420 base pairs
(B) TYPE: nucleic acid
I
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(C) STRANDEDNESS: single
(D} TOPOLOGY: linear
{xi)'SEQUENCE DESCRIPTION: SEQ ID N0:52:
GCCACTCAGG ACTGCTCTTT TCAACACAGC CCCATCTCCT CCGACTTCGC TGTCAAAATC
CGTGAGCTGT CTGACTACCT GCTTCAAGAT TACCCAGTCA CCGTGGCCTC CAACCTGCAG
120
GACGAGGAGC TCTGCGGGGG CCTCTGGCGG CTGGTCCTGG CACAGCGCTG GATGGAGCGG
180
CTCAAGACTG TCGCTGGGTC CAAGATGCAA GGCTTGCTGG AGCGCGTGAA CACGGAGATA
240
CACTTTGTCA CCAAATGTGC CTTTCAGCCC CCCCCCAGCT GTCTTCGCTT CGTCCAGACC
300
AACATCTCCC GCCTCCTGCA GGAGACCTCC GAGCAGCTGG TGGCGCTGAA GCCCTGGATC
360
ACTCGCCAGA ACTTCTCCCG GTGCCTGGAG CTGCAGTGTC AGCCCGACTC CTCAACCCTG
420
(2) INFORMATION FOR SEQ ID N0:53:
{i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 405 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:53:
GCCACCCAGG ACTGCTCCTT CCAACACAGC CCCATCTCCT CCGACTTCGC TGTCAAAATC
CGTGAGCTGT CTGACTACCT GCTTCAAGAT TACCCAGTCA CCGTGGCCTC CAACCTGCAG
120
GACGAGGAGC TCTGCGGGGC GCTCTGGCGG CTGGTCCTGG CACAGCGCTG GATGGAGCGG
180
CTCAAGACTG TCGCTGGGTC CAAGATGCAA GGCTTGCTGG AGCGCGTGAA CACGGAGATA
240
CACTTTGTCA CCAAATGTGC CTTTCAGCCC CCCCCCAGCT GTCTTCGCTT CGTCCAGACC
300
AACATCTCCC GCCTCCTGCA GGAGACCTCC GAGCAGCTGG TGGCGCTGAA GCCCTGGATC
360
ACTCGCCAGA ACTTCTCCCG GTGCCTGGAG CTGCAGTGTC AGCCC
405
(2) INFORMATION FOR SEQ ID N0:54:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 420 base pairs
(B} TYPE: nucleic acid
(C} STRANDEDNESS: single
{D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:54:
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GCCACCCAGG ACTGCTCCTT CCAACACAGC CCCATCTCCT CCGACTTCGC TGTCAAAATC
CGTGAGCTGT CTGACTACCT GCTTCAAGAT TACCCAGTCA CCGTGGCCTC CAACCTGCAG
120
GACGAGGAGC'TCTGCGGGGG CCTCTGGCGG CTGGTCCTGG CACAGCGCTG GATGGAGCGG
180
CTCAAGACTG TCGCTGGGTC CAAGATGCAA GGCTTGCTGG AGCGCGTGAA CACGGAGATA
240
CACTTTGTCA CCAAATGTGC CTTTCAGCCC CCCCCCAGCT GTCTTCGCTT CGTCCAGACC
300
AACATCTCCC GCCTCCTGCA GGAGACCTCC GAGCAGCTGG TGGCGCTGAA GCCCTGGATC
360
ACTCGCCAGA ACTTCTCCCG GTGCCTGGAG CTGCAGTGTC AGCCCGACTC CTCAACCCTG
420
(2) INFORMATION FOR SEQ ID N0:55:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 420 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:55:
GCCACTCAGG ACTGCTCTTT TCAACACAGC CCCATCTCCT CCGACTTCGC TGTCAAAATC
CGTGAGCTGT CTGACTACCT GCTTCAAGAT TACCCAGTCA CCGTGGCCTC CAACCTGCAG
120
GACGAGGAGC TCTGCGGGGG CCTCTGGCGG CTGGTCCTGG CACAGCGCTG GATGGAGCGG
180
CTCAAGACTG TCGCTGGGTC CAAGATGCAA GGCTTGCTGG AGCGCGTGAA CACGGAGATA
240
CACTTTGTCA CCAAATGTGC CTTTCAGCCC CCCCCCAGCT GTCTTCGCTT CGTCCAGACC
300
AACATCTCCC GCCTCCTGCA GGAGACCTCC GAGCAGCTGG TGGCGCTGAA GCCCTGGATC
360
ACTCGCCAGA ACTTCTCCCG GTGCCTGGAG CTGCAGTGTC AGCCCGACTC CTCAACCCTG
420
(2) INFORMATION FOR SEQ ID N0:56:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 942 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:56:
GCCACTCAGG ACTGCTCTTT TCAACACAGC CCCATCTCCT CCGACTTCGC TGTCAAAATC
CGTGAGCTGT CTGACTACCT GCTTCAAGAT TACCCAGTCA CCGTGGCCTC CAACCTGCAG
120
GACGAGGAGC TCTGCGGGGG CCTCTGGCGG CTGGTCCTGG CACAGCGCTG GATGGAGCGG
180
CA 02284127 1999-09-10
WO 98/46750 i~~ PCTNS98/0'1511
CTCAAGACTG TCGCTGGGTC CAAGATGCAA GGCTTGCTGG AGCGCGTGAA CACGGAGATA
240
CACTTTGTCA CCAAATGTGC CTTTCAGCCC CCCCCCAGCT GTCTTCGCTT CGTCCAGACC
300
AACATCTCCC~GCCTCCTGCA GGAGACCTCC GAGCAGCTGG TGGCGCTGAA GCCCTGGATC
360
ACTCGCCAGA ACTTCTCCCG GTGCCTGGAG CTGCAGTGTC AGCCCGACTC CTCAACCCTG
420
TACGTAGAGG GCGGTGGAGG CTCCCCGGGT GAACCGTCTG GTCCAATCTC TACTATCAAC
480
CCGTCTCCTC CGTCTAAAGA ATCTCATAAA TCTCCAAACA TGGCTACCCA GGACTGCTCC
540
TTCCAACACA GCCCCATCTC CTCCGACTTC GCTGTCAAAA TCCGTGAGCT GTCTGACTAC
600
CTGCTTCAAG ATTACCCAGT CACCGTGGCC TCCAACCTGC AGGACGAGGA GCTCTGCGGG
660
GGCCTCTGGC GGCTGGTCCT GGCACAGCGC TGGATGGAGC GGCTCAAGAC TGTCGCTGGG
720
TCCAAGATGC AAGGCTTGCT GGAGCGCGTG AACACGGAGA TACACTTTGT CACCAAATGT
780
GCCTTTCAGC CCCCCCCCAG CTGTCTTCGC TTCGTCCAGA CCAACATCTC CCGCCTCCTG
840
CAGGAGACCT CCGAGCAGCT GGTGGCGCTG AAGCCCTGGA TCACTCGCCA GAACTTCTCC
900
CGGTGCCTGG AGCTGCAGTG TCAGCCCGAC TCCTCAACCC TG
942
(2) INFORMATION FOR SEQ ID N0:57:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1003 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:57:
GGCCACTCAG GACTGCTCTT TTCAACACAG CCCCATCTCC TCCGACTTCG CTGTCAAAAT
CCGTGAGCTG TCTGACTACC TGCTTCAAGA TTACCCAGTC ACCGTGGCCT CCAACCTGCA
120
GGACGAGGAG CTCTGCGGGG GCCTCTGGCG GCTGGTCCTG GCACAGCGCT GGATGGAGCG
180
GCTCAAGACT GTCGCTGGGT CCAAGATGCA AGGCTTGCTG GAGCGCGTGA ACACGGAGAT
240
ACACTTTGTC ACCAAATGTG CCTTTCAGCC CCCCCCCAGC TGTCTTCGCT TCGTCCAGAC
300
CAACATCTCC CGCCTCCTGC AGGAGACCTC CGAGCAGCTG GTGGCGCTGA AGCCCTGGAT
360
CACTCGCCAG AACTTCTCCC GGTGCCTGGA GCTGCAGTGT CAGCCCGACT CCTCAACCCT
420
GTACGTAGAG GGCGGTGGAG GCTCCCCGGG TGGTGGTTCT GGCGGCGGCT CCAACATGGC
480
TACACCATTG GGCCCTGCCA GCTCCCTGCC CCAGAGCTTC CTGCTCAAGT CTTTAGAGCA
540
AGTGAGAAAG ATCCAGGGCG ATGGCGCAGC GCTCCAGGAG AAGCTGTGTG CCACCTACAA
600
CA 02284127 1999-09-10
WO 98/46750 ifl~ PCT/US98/07511
GCTGTGCCAC CCCGAGGAGC TGGTGCTGCT CGGACACTCT CTGGGCATCC CCTGGGCTCC
660
CCTGAGCTCC TGCCCCAGCC AGGCCCTGCA GCTGGCAGGC TGCTTGAGCC AACTCCATAG
720
CGGCCTTTTC'CTCTACCAGG GGCTCCTGCA GGCCCTGGAA GGGATATCCC CCGAGTTGGG
780
TCCCACCTTG GACACACTGC AGCTGGACGT CGCCGACTTT GCCACCACCA TCTGGCAGCA
840
GATGGAAGAA CTGGGAATGG CCCCTGCCCT GCAGCCCACC CAGGGTGCCA TGCCGGCCTT
900
CGCCTCTGCT TTCCAGCGCC GGGCAGGAGG GGTCCTGGTT GCTAGCCATC TGCAGAGCTT
960
CCTGGAGGTG TCGTACCGCG TTCTACGCCA CCTTGCGCAG CCG
1003
(2) INFORMATION FOR SEQ ID N0:58:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 858 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:58:
GCCACCCAGG ACTGCTCCTT CCAACACAGC CCCATCTCCT CCGACTTCGC TGTCAAAATC
CGTGAGCTGT CTGACTACCT GCTTCAAGAT TACCCAGTCA CCGTGGCCTC CAACCTGCAG
120
GACGAGGAGC TCTGCGGGGG CCTCTGGCGG CTGGTCCTGG CACAGCGCTG GATGGAGCGG
180
CTCAAGACTG TCGCTGGGTC CAAGATGCAA GGCTTGCTGG AGCGCGTGAA CACGGAGATA
240
CACTTTGTCA CCAAATGTGC CTTTCAGCCC CCCCCCAGCT GTCTTCGCTT CGTCCAGACC
300
AACATCTCCC GCCTCCTGCA GGAGACCTCC GAGCAGCTGG TGGCGCTGAA GCCCTGGATC
360
ACTCGCCAGA ACTTCTCCCG GTGCCTGGAG CTGCAGTGTC AGCCCGACTC CTCAACCCTG
420
TACGTAGAGG GCGGTGGAGG CTCCCCGGGT GAACCGTCTG GTCCAATCTC TACTATCAAC
480
CCGTCTCCTC CGTCTAAAGA ATCTCATAAA TCTCCAAACA TGGCTAACTG CTCTATAATG
540
ATCGATGAAA TTATACATCA CTTAAAGAGA CCACCTAACC CTTTGCTGGA CCCGAACAAC
600
CTCAATTCTG AAGACATGGA TATCCTGATG GAACGAAACC TTCGAACTCC AAACCTGCTC
660
GCATTCGTAA GGGCTGTCAA GCACTTAGAA AATGCATCAG GTATTGAGGC AATTCTTCGT
720
AATCTCCAAC CATGTCTGCC CTCTGCCACG GCCGCACCCT CTCGACATCC AATCATCATC
780
AAGGCAGGTG ACTGGCAAGA ATTCCGGGAA AAACTGACGT TCTATCTGGT TACCCTTGAG
840
CAAGCGCAGG AACAACAG
858
(2) INFORMATION FOR SEQ ID N0:59:
CA 02284127 1999-09-10
WO 98/46750 102 PCT/US98/07511
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 402 base pairs
(B) TYPE: nucleic acid
(C)' STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:59:
ATGGCTCCAA TGACTCAGAC TACTTCTCTT AAGACTTCTT GGGTTAACTG CTCTAACATG
ATCGATGAAA TTATAACACA CTTAAAGCAG CCACCTTTGC CTTTGCTGGA CTTCAACAAC
120
CTCAATGGGG AAGACCAAGA CATTCTGATG GAAAATAACC TTCGAAGGCC AAACCTGGAG
180
GCATTCAACA GGGCTGTCAA GAGTTTACAG AATGCATCAG CAATTGAGAG CATTCTTAAA
240
AATCTCCTGC CATGTCTGCC CCTGGCCACG GCCGCACCCA CGCGACATCC AATCCATATC
300
AAGGACGGTG ACTGGAATGA ATTCCGTCGT AAACTGACCT TCTATCTGAA AACCTTGGAG
360
AACGCGCAGG CTCAACAGAC CACTCTGTCG CTAGCGATCT TT
402
(2) INFORMATION FOR SEQ ID N0:60:
(i) SEQUENCE CHARACTERISTICS:
(A} LENGTH: 344 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: None
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:60:
Ala Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu Leu
1 5 10 15
Lys Ser Leu Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala Ala Leu
20 25 30
Gln Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu
35 40 45
Val Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser Ser
50 55 60
Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser Gln Leu His
70 75 80
Ser Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu Gly Ile
85 90 95
Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val Ala
100 105 110
Asp Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met Ala
115 120 125
Pro Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala Ser Ala
130 135 140
Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gln Ser
145 150 155 160
CA 02284127 1999-09-10
WO 98/46750 103 PCTNS98/07511
Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln Pro Tyr
165 170 175
Val Glu Gly Gly Gly Gly Ser Pro Gly Glu Pro Ser Gly Pro Ile Ser
180 185 190
Thr Ile Asn Pro Ser Pro Pro Ser Lys Glu Ser His Lys Ser Pro Asn
195 200 205
Met Ala Thr Gln Asp Cys Ser Phe Gln His Ser Pro IIe Ser Ser Asp
210 215 220
Phe Ala Val Lys Ile Arg Glu Leu Ser Asp Tyr Leu Leu Gln Asp Tyr
225 230 235 240
Pro Val Thr Val Ala Ser Asn Leu Gln Asp Glu Glu Leu Cys Gly Gly
245 250 255
Leu Trp Arg Leu Val Leu Ala Gln Arg Trp Met Glu Arg Leu Lys Thr
260 265 270
Val Ala Gly Ser Lys Met Gln Gly Leu Leu Glu Arg Val Asn Thr Glu
275 280 285
Ile His Phe Val Thr Lys Cys Ala Phe Gln Pro Pro Pro Ser Cys Leu
290 295 300
Arg Phe Val Gin Thr Asn Ile Ser Arg Leu Leu Gln Glu Thr Ser Glu
305 310 315 320
Gln Leu Val Ala Leu Lys Pro Trp Ile Thr Arg Gln Asn Phe Ser Arg
325 330 335
Cys Leu Glu Leu Gln Cys Gln Pro
340
(2) INFORMATION FOR SEQ ID N0:61:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 133 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: None
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:61:
Pro Met Thr Gln Thr Thr Ser Leu Lys Thr Ser Trp Val Asn Cys Ser
1 5 10 15
Asn Met Ile Asp Glu Ile Ile Thr His Leu Lys Gln Pro Pro Leu Pro
20 25 30
Leu Leu Asp Phe Asn Asn Leu Asn Gly Glu Asp Gln Asp Ile Leu Met
35 40 45
Glu Asn Asn Leu Arg Arg Pro Asn Leu Glu Ala Phe Asn Arg Ala Val
50 55 60
Lys Ser Leu Gln Asn Ala Ser Ala Ile Glu Ser Ile Leu Lys Asn Leu
65 70 75 BO
Leu Pro Cys Leu Pro Leu Ala Thr Ala Ala Pro Thr Arg His Pro Ile
85 90 ~ 95
His Ile Lys Asp Gly Asp Trp Asn Gly Ile Phe Arg Arg Lys Leu Thr
100 105 110
Phe Tyr Leu Lys Thr Leu Glu Asn Ala Gln Ala Gln Gln Thr Thr Leu
125 120 125
Ser Leu Ala Ile Phe
130
(2) INFORMATION FOR SEQ ID N0:62:
CA 02284127 1999-09-10
WO 98/46750 1~ PCT/US98/07511
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 287 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D)' TOPOLOGY: linear
(ii) MOLECULE TYPE: None
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:62:
Pro Met Thr Gln Thr Thr Ser Leu Lys Thr Ser Trp Val Asn Cys Ser
1 5 10 15
Asn Met Ile Asp Glu Ile Ile Thr His Leu Lys Gln Pro Pro Leu Pro
20 25 30
Leu Leu Asp Phe Asn Asn Leu Asn Gly Glu Asp Gln Asp Ile Leu Met
35 40 45
Glu Asn Asn Leu Arg Arg Pro Asn Leu Glu Ala Phe Asn Arg Ala Val
50 55 60
Lys Ser Leu Gln Asn Ala Ser Ala Ile Glu Ser Ile Leu Lys Asn Leu
65 70 75 80
Leu Pro Cys Leu Pro Leu Ala Thr Ala Ala Pro Thr Arg His Pro Ile
85 90 95
His Ile Lys Asp Gly Asp Trp Asn Gly Ile Phe Arg Arg Lys Leu Thr
100 105 110
Phe Tyr Leu Lys Thr Leu Glu Asn Ala Gln Ala Gln Gln Thr Thr Leu
115 120 125
Ser Leu Ala Ile Phe Tyr Val Glu Gly Gly Gly Gly Ser Pro Gly Gly
130 135 140
Gly Ser Gly Gly Gly Ser Asn Met Ala Thr Gln Asp Cys Ser Phe Gln
145 150 155 160
His Ser Pro Ile Ser Ser Asp Phe Ala Val Lys Ile Arg Glu Leu Ser
165 170 175
Asp Tyr Leu Leu Gln Asp Tyr Pro Val Thr Val Ala Ser Asn Leu Gln
180 185 190
Asp Glu Glu Leu Cys Gly Ala Leu Trp Arg Leu Val Leu Ala Gln Arg
195 200 205
Trp Met Glu Arg Leu Lys Thr Val Ala Gly Ser Lys Met Gln Gly Leu
210 215 220
Leu Glu Arg Val Asn Thr Glu Ile His Phe Val Thr Lys Cys Ala Phe
225 230 235 240
Gln Pro Pro Pro Ser Cys Leu Arg Phe Val Gln Thr Asn Ile Ser Arg
245 250 255
Leu Leu Gln Glu Thr Ser Glu Gln Leu Val Ala Leu Lys Pro Trp Ile
260 265 270
Thr Arg Gln Asn Phe Ser Arg Cys Leu Glu Leu Gln Cys Gln Pro
275 280 285
(2) INFORMATION FOR SEQ ID N0:63:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 302 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: None
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:63:
CA 02284127 1999-09-10
WO 98/46750 105 PCT/US98/07511
Pro Met Thr Gln Thr Thr Ser Leu Lys Thr Ser Trp Val Asn Cys Ser
1 5 10 15
Asn Met Ile Asp Glu Ile Ile Thr His Leu Lys Gln Pro Pro Leu Pro
' 20 25 30
Leu Leu Asp Phe Asn Asn Leu Asn Gly Glu Asp Gln Asp Ile Leu Met
35 40 45
Glu Asn Asn Leu Arg Arg Pro Asn Leu Glu Ala Phe Asn Arg Ala Val
50 55 60
Lys Ser Leu Gln Asn Ala Ser Ala Ile Glu Ser Ile Leu Lys Asn Leu
65 70 75 80
Leu Pro Cys Leu Pro Leu Ala Thr Ala Ala Pro Thr Arg His Pro Ile
85 90 95
His Ile Lys Asp Gly Asp Trp Asn Gly Ile Phe Arg Arg Lys Leu Thr
100 105 110
Phe Tyr Leu Lys Thr Leu Glu Asn Ala Gln Ala Gln Gln Thr Thr Leu
115 120 125
Ser Leu Ala Ile Phe Tyr Val Glu Gly Gly Gly Gly Ser Pro Gly Glu
130 135 140
Pro Ser Gly Pro Ile Ser Thr Ile Asn Pro Ser Pro Pro Ser Lys Glu
145 150 155 160
Ser His Lys Ser Pro Asn Met Ala Thr Gln Asp Cys Ser Phe Gln His
165 170 175
Ser Pro Ile Ser Ser Asp Phe Ala Val Lys Ile Arg Glu Leu Ser Asp
180 185 190
Tyr Leu Leu Gln Asp Tyr Pro Val Thr Val Ala Ser Asn Leu Gln Asp
195 200 205
Glu Glu Leu Cys Gly Ala Leu Trp Arg Leu Val Leu Ala Gln Arg Trp
210 215 220
Met Glu Arg Leu Lys Thr Val Ala Gly Ser Lys Met Gln Gly Leu Leu
225 230 235 240
Glu Arg Val Asn Thr Glu Ile His Phe Val Thr Lys Cys Ala Phe Gln
245 250 255
Pro Pro Pro Ser Cys Leu Arg Phe Val Gln Thr Asn Ile Ser Arg Leu
260 265 270
Leu Gln Glu Thr Ser Glu Gln Leu Val Ala Leu Lys Pro Trp Ile Thr
275 280 285
Arg Gln Asn Phe Ser Arg Cys Leu Glu Leu Gln Cys Gln Pro
290 295 300
(2} INFORMATION FOR SEQ ID N0:64:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 407 base pairs
(B} TYPE: nucleic acid
(C} STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:64:
CCATGGCTAA CTGCTCTATA ATGATCGATG AAATTATACA TCACTTAAAG AGACCACCTG
CACCTTTGCT GGACCCGAAC AACCTCAATG ACGAAGACGT CTCTATCCTG ATGGATCGAA
120
ACCTTCGACT TCCAAACCTG GAGAGCTTCG TAAGGGCTGT CAAGAACTTA GAAAATGCAT
180
CA 02284127 1999-09-10
WO 98/46750 106 PCT/US98/07511
CAGGTATTGA GGCAATTCTT CGTAATCTCC AACCATGTCT GCCCTCTGCC ACGGCCGCAC
240
CCTCTCGACA TCCAATCATC ATCAAGGCAG GTGACTGGCA AGAATTCCGG GAAAAACTGA
300
CGTTCTATCT GGTTACCCTT GAGCAAGCGC AGGAACAACA GTACGTAGAG GGCGGTGGAG
360
GCTCCCCGGG TGGTGGTTCT GGCGGCGGCT CCAACATGTA AGGTACC
407
(2) INFORMATION FOR SEQ ID N0:65:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 452 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii} MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:65:
CCATGGCTAA CTGCTCTATA ATGATCGATG AAATTATACA TCACTTAAAG AGACCACCTG
CACCTTTGCT GGACCCGAAC AACCTCAATG ACGAAGACGT CTCTATCCTG ATGGATCGAA
120
ACCTTCGACT TCCAAACCTG GAGAGCTTCG TAAGGGCTGT CAAGAACTTA GAAAATGCAT
180
CAGGTATTGA GGCAATTCTT CGTAATCTCC AACCATGTCT GCCCTCTGCC ACGGCCGCAC
240
CCTCTCGACA TCCAATCATC ATCAAGGCAG GTGACTGGCA AGAATTCCGG GAAAAACTGA
300
CGTTCTATCT GGTTACCCTT GAGCAAGCGC AGGAACAACA GTACGTAGAG GGCGGTGGAG
360
GCTCCCCGGG TGAACCGTCT GGTCCAATCT CTACTATCAA CCCGTCTCCT CCGTCTAAAG
420
AATCTCATAA ATCTCCAAAC ATGTAAGGTA CC
452
